Cell-Penetrating Anti-Protein Kinase C Theta Antibodies Act Intracellularly to Generate Stable, Highly Suppressive Regulatory T Cells

E Ilker Ozay; Sudarvili Shanthalingam; Heather L Sherman; Joe A Torres; Barbara A Osborne; Gregory N Tew; Lisa M Minter

doi:10.1016/j.ymthe.2020.05.020

. 2020 May 23;28(9):1987–2006. doi: 10.1016/j.ymthe.2020.05.020

Cell-Penetrating Anti-Protein Kinase C Theta Antibodies Act Intracellularly to Generate Stable, Highly Suppressive Regulatory T Cells

E Ilker Ozay ^1,⁴, Sudarvili Shanthalingam ², Heather L Sherman ¹, Joe A Torres ¹, Barbara A Osborne ^1,², Gregory N Tew ^1,^2,³, Lisa M Minter ^1,^2,^∗

PMCID: PMC7474270 PMID: 32492367

Abstract

Regulatory T cells maintain immunological tolerance and dampen inflammatory responses. Administering regulatory T cells can prevent the immune-mediated tissue destruction of graft-versus-host disease, which frequently accompanies hematopoietic stem cell transfer. Neutralizing the T cell-specific kinase, protein kinase C theta, which promotes T cell effector functions and represses regulatory T cell differentiation, augments regulatory T cell immunosuppression and stability. We used a synthetic, cell-penetrating peptide mimic to deliver antibodies recognizing protein kinase C theta into primary human CD4 T cells. When differentiated ex vivo into induced regulatory T cells, treated cells expressed elevated levels of the regulatory T cell transcriptional regulator forkhead box P3, the surface-bound immune checkpoint receptor programmed death receptor-1, and pro-inflammatory interferon gamma, previously ascribed to a specific population of stable, highly suppressive human induced regulatory T cells. The in vitro suppressive capacity of these induced regulatory T cells was 10-fold greater than that of T cells differentiated without antibody delivery. When administered at the time of graft-versus-host disease induction, using a humanized mouse model, antibody-treated regulatory T cells were superior to non-treated T cells in attenuating lethal outcomes. This antibody delivery approach may overcome obstacles currently encountered using patient-derived regulatory T cells as a cell-based therapy for immune modulation.

Keywords: cell-penetrating peptide mimics, intracellular antibody delivery, PKCθ, induced regulatory T cell, FOXP3, graft-versus-host disease, cell-based therapy

Graphical Abstract

Using cell-penetrating peptide mimics to deliver polyclonal anti-pPKCθ antibodies into human CD4 T cells enhances their ex vivo differentiation into a unique regulatory T cell population with increased in vitro and in vivo suppressive functions.

Introduction

Naive CD4 T cells differentiate into unique T helper (Th) subsets in response to specific signals generated in peripheral tissues. Regulatory T cells (Tregs) are a subset of differentiated Th cells that function to mitigate immune responses and maintain immunological tolerance.¹ In humans, Tregs are characterized in vivo as CD4⁺CD25⁺CD127⁻FOXP3⁺ cells, and are consistently suppressive across species and in multiple disease models.2, 3, 4, 5, 6, 7, 8 Treg function is critical for attenuating autoimmune responses, controlling tumor and microbial immunity, preventing graft rejection in mice and humans, and suppressing the immune-mediated tissue destruction of graft-versus-host disease (GvHD), which frequently accompanies hematopoietic stem cell transplantation (HSCT).9, 10, 11, 12, 13 In autoimmune conditions, Treg function can be negatively regulated by the inflammatory cytokine milieu.¹⁴ Appropriate migration to secondary lymphoid organs and subsequent expansion are necessary prior to Treg trafficking to sites of inflammation where they exert their suppressive functions in vivo.¹⁵

The immunological synapse (IS) is a supramolecular signaling complex that coalesces in an ordered fashion at the contact point between naive T cells and antigen-loaded dendritic cells (DCs). Following stimulation through the T cell receptor (TCR), the T cell-specific kinase protein kinase C theta (PKCθ) is phosphorylated at threonine 538 and translocates to the centermost region of the IS. Here, it links activation signals from the TCR with costimulatory signals provided by CD28, culminating in transcription of immune-responsive genes.16, 17, 18 Interestingly, PKCθ is the only PKC isoform recruited to the center of the IS. However, unlike in effector T cells, in Tregs PKCθ is sequestered away from the central domain of the IS.¹⁹^,²⁰ Through this differential positioning in the IS, PKCθ promotes activation of effector T cell functions at the expense of Treg programs.21, 22, 23 Inhibiting the actions of PKCθ with small-molecule inhibitors or using small interfering RNA (siRNA) approaches enhances the suppressive capacity of Tregs, restores impaired function of Tregs from rheumatoid arthritis patients, and blocks the autoimmune response in a mouse model of colitis.²⁴ Therefore, attenuating PKCθ activity in Tregs may be a valuable component in Treg adoptive immunotherapy when used to treat autoimmune conditions or GvHD.²⁵

Initial evidence shows that PKCθ is required in fully functional mature, but not immature, T cell responses by bridging stimuli received through the TCR to downstream gene transcription, including those generated by nuclear factor-κB (NF-κB), nuclear factor of activated T cells (NFAT), and activator protein 1 (AP1) transcriptional regulators.¹⁸^,²⁶ More recent work demonstrated that immune cells from PKCθ-deficient mice, transplanted together with T cell-depleted bone marrow (BM) stem cells from wild-type mice, did not induce GvHD in recipients. This is in contrast to the majority of recipient mice that died from GvHD when wild-type immune cells were transferred together with BM stem cells.²⁷ PKCθ-deficient immune cells did not confer GvHD. They did, however, retain their ability to protect recipient mice from bacterial and viral infections, as well as mediate immune depletion of residual leukemia cells. These and follow-up studies reinforce the notion that inhibiting PKCθ activity during BM transplantation may constitute a beneficial approach to limiting the severity of GvHD, while maintaining important anti-tumor surveillance.

Effectively and specifically blocking PKCθ function is challenging, due to the high structural homology it shares with eight, more broadly expressed family members. As a result, many existing small-molecule PKCθ inhibitors have toxic or off-target effects and show suboptimal penetration into T cells.²⁸ In contrast, using antibodies to modulate cell surface receptor function, either positively or negatively, is now a widely accepted immunotherapeutic approach. However, because of its intracellular residence PKCθ is not a suitable candidate for antibody-targeting strategies. Recently, we developed and reported on a successful and highly specific strategy for routine and effective intracellular antibody delivery using cell-penetrating peptide mimics (CPPMs) and demonstrated its powerful application by targeting a phosphorylated threonine residue (Thr538) of activated PKCθ (phosphorylated PKCθ [pPKCθ]) in the context of T cell immunomodulation.²⁹ Manipulating primary T cells ex vivo using intracellular anti-pPKCθ delivery constrained pPKCθ in the cytosol and reduced the capacity of these cells to adopt a pro-inflammatory Th type 1 (Th1) cell fate.²⁹

Naive CD4 T cells can be induced to differentiate ex vivo into Tregs (iTregs). In an allogeneic mouse model of BM transplantation, adoptively transferring iTregs provided beneficial relief from disease by suppressing immune-mediated, acute GvHD.³⁰ This approach has now entered the clinic where cellular immunotherapy using adoptively transferred iTregs is recognized as a feasible and efficacious option to treat immune-mediated conditions.⁹ The first in-human trial of adoptive iTreg therapy delivered encouraging outcomes for preventing GvHD associated with allogeneic HSCT, and it offers great promise for treating immune-mediated diseases and allograft rejection.30, 31, 32

Herein, we report that ex vivo CPPM delivery of an antibody specific for pPKCθ enhances the differentiation and expansion of iTregs in culture. Moreover, anti-pPKCθ-treated iTregs exhibited increased suppressive properties in vitro, as characterized by increased surface expression of the co-inhibitory receptor programmed cell death 1 (PD-1). Anti-pPKCθ iTregs could be detected in vivo up to 17 days after their administration into recipient mice and, compared to control iTregs, were highly efficacious in preventing GvHD in a humanized mouse model. Therefore, CPPM delivery of anti-pPKCθ into human CD4 T cells represents an approach that overcomes obstacles associated with ex vivo expansion and sustained in vivo stability, and it provides a powerful, reproducible, and effective means of generating iTregs for therapeutic application.

Results

Intracellular Anti-pPKCθ Delivery Prevents Nuclear Accumulation of pPKCθ in iTregs

In fully activated CD4 T cells, PKCθ is phosphorylated on Thr538 by germinal center kinase-like kinase (GLK) downstream of co-stimulatory signals provided by CD28 engagement on the cell surface.³³ This activated form of PKCθ is important for negatively regulating Treg function, and it may mediate these effects through an AKT/Foxo1/3 pathway.³⁴ Additionally, PKCθ in Tregs is sequestered away from the IS, suggesting that Thr538 both phosphorylation and recruitment to the IS may be important for PKCθ to exert its inhibitory actions on iTreg formation. We previously demonstrated we could utilize a synthetic CPPM to achieve highly efficient intracellular antibody delivery into human primary T cells ex vivo.²⁹ Non-covalently complexing the CPPM, comprised of 13 U of phenyl-containing moiety and 5 U of diguanidine moiety, with anti-pPKCθ reduced nuclear translocation of pPKCθ, attenuated downstream signaling and compromised Th1 differentiation.²⁹ In the present study, we predicted that delivering anti-pPKCθ prior to iTreg polarization would similarly alter pPKCθ function to enhance iTreg differentiation. To confirm intracellular antibody uptake, we isolated CD4 T cells from human peripheral blood mononuclear cells (hPBMCs) and incubated them with CPPM-immunoglobulin G (IgG) only or CPPM-anti-pPKCθ, followed by staining with a fluorescently conjugated secondary antibody. We found that the delivery efficiency of CPPM-anti-pPKCθ (or CPPM-anti-rabbit IgG as a non-specific control) into CD4 T cells was approximately 90%, with high intracellular delivery detected for both antibodies (Figure S1A). Incubating CD4 T cells with CPPM alone, or with uncomplexed anti-pPKCθ, showed no detectable uptake of anti-pPKCθ (Figure S1A). We subsequently incubated CD4 T cells with anti-pPKCθ only, CPPM only, CPPM-anti-IgG only, DMSO only (as a vehicle control for CPPM-antibody complexing), or CPPM-anti-pPKCθ and then differentiated the cells into iTregs over 5 days of culture using commercially available iTreg-polarizing reagents (Figure 1A). We deviated from the manufacturer’s directions, which suggested stimulating cells only with anti-CD3, by also cross-linking the CD28 receptor to provide the co-stimulatory signals needed to phosphorylate PKCθ on Thr538 and, thus, generate the target epitope recognized by anti-pPKCθ. At the end of the 5-day differentiation period, the percent of CD4⁺CD25⁺FOXP3⁺ iTregs was greater than 90% regardless of pre-treatment conditions (Figure S1B). Human CD4 T cells did not tolerate CPPM-IgG delivery well, showing increased levels of cell death by the end of the differentiation period. Therefore, based on the nearly identical staining patterns of CD25, CD127, and FOXP3 exhibited by DMSO-treated (DMSO-iTregs) and CPPM-anti-pPKCθ-treated (anti-pPKCθ-iTregs) iTregs (Figure S1C), we chose these two populations to further compare differences in iTreg functions in vitro and in vivo.

We used flow cytometry to quantify total pPKCθ in stimulated DMSO- and anti-pPKCθ-treated conventional T cells (Tconvs) and in iTregs. We observed similar percentages of pPKCθ-positive cells in Tconv and iTreg populations (Figure 1B). However, when we assessed the abundance of pPKCθ on a per cell basis, as measured by median fluorescence intensity (MFI; Figure 1C), we found that anti-pPKCθ-iTregs expressed lower levels of pPKCθ, compared to DMSO-iTregs. Although it did not differ significantly in iTregs, regardless of pre-treatment, PRKCQ gene expression was significantly reduced in anti-pPKCθ-Tconvs (Figure 1D). These data are consistent with a previous report in which we describe that anti-pPKCθ delivery into T cells, prior to their in vitro differentiation into Th1 cells, diminishes PKCθ activity, including driving its own expression.²⁹ PKCθ can function both in the nucleus and the cytosol;³⁵ therefore, we further quantified PKCθ cytoplasmic and nuclear distribution in Tconvs and iTregs. We detected strong cytosolic expression of the 82-kDa protein, as expected, in Tconvs and in DMSO-iTregs. Consistent with the flow cytometric assessment of pPKCθ, there was much less of this isoform in the cytosol of anti-PKCθ-iTregs (Figure 1E). We next used imaging flow cytometry to quantify the abundance of pPKCθ in the nucleus of iTregs. Nuclear pPKCθ protein was significantly reduced in anti-pPKCθ-iTregs, compared to DMSO-iTregs (Figure 1F). Collectively, these data demonstrate that CPPM-mediated delivery of anti-pPKCθ into CD4 T cells alters the cellular distribution of pPKCθ within ex vivo-differentiated iTregs.

Ex Vivo CPPM-Anti-pPKCθ Delivery Generates a Unique Population of CD4⁺CD25^highFOXP3^high iTregs That also Produces Interferon γ (IFNγ)

Reduced nuclear accumulation of PKCθ in anti-pPKCθ-iTregs is consistent with the reported nuclear role for PKCθ in pro-inflammatory gene regulation in human CD4 T cells.³⁵ In complementary studies, Zanin-Zhorov et al.²¹ provided compelling evidence that using a small-molecule inhibitor to block PKCθ activity in CD4 T cells enhanced their regulatory phenotype. However, cell toxicity and off-target effects constitute two major obstacles to using small-molecule inhibitors to intervene in signaling pathways. We predicted that inhibiting the actions of PKCθ using anti-pPKCθ, delivered ex vivo into the cytosol of human CD4 T cells, would enhance the ex vivo differentiation of functional, stable iTregs. High surface expression of CD25, the high-affinity subunit of the interleukin 2 (IL-2) receptor, is a hallmark of Tregs. More than 90% of differentiated iTregs exhibited high surface CD25, including those treated with CPPM-anti-pPKCθ (Figure 2A). On a per cell basis, the concentration of surface CD25 on anti-pPKCθ-iTregs was increased more than that expressed on DMSO-iTregs or on Tconvs (Figure 2B). We further stratified iTregs phenotypically using flow cytometry to quantify the percent of CD4⁺CD25^high cells that also expressed the signature iTreg master transcriptional regulator, FOXP3. When we applied this gating strategy, we found anti-pPKCθ delivery enhanced the percentage of FOXP3-expressing CD4⁺CD25^high cells following iTreg polarization (Figures 2C and 2D). In comparison, nearly 50% of CD4⁺CD25^high DMSO-iTregs remained FOXP3⁻ (Figure 2D). We also assessed whether FOXP3 abundance varied between treatments. In parallel to increased CD25 expression on anti-pPKCθ-iTregs, we observed significantly more FOXP3 protein in anti-pPKCθ-iTregs, compared to DMSO-iTregs (Figure 2E). This was notable, because FOXP3 gene expression in iTregs did not differ between the treatments (Figure 2F) and suggests that anti-pPKCθ treatment may act to increase the stability of FOXP3.

*Ex Vivo* CPPM-Anti-pPKCθ Delivery Generates a Unique Population of CD4⁺CD25^highFOXP3^high iTregs That Also Produce IFNγ

(A and B) Percentage of total CD4⁺CD25⁺ T cells (A) and MFI and representative histograms of CD25 expression within CD4⁺CD25⁺ T cell gate of non-differentiated and iTreg-differentiated cells (B). (C) Representative FACS clouds showing percent of CD25⁺FOXP3⁺ cells within CD4⁺CD25⁺ T cell gate. (D) Percent FOXP3⁺ and FOXP3⁻ cells within the CD4⁺CD25⁺ T cell gate. (E) Fold increase in FOXP3 MFI and representative histogram in non-differentiated and iTreg-differentiated cells. (F) *FOXP3* expression, shown relative to untreated, non-differentiated T cells. (G and H) Gene expression, percent positive, MFI, and representative histograms of (G) PD-1- and (H) IFNγ-expressing iTregs differentiated without or with CPPM-anti-pPKCθ are shown. Data represent the mean ± SEM of three independent experiments. ∗p < 0.05, **p < 0.01, by unpaired, two-tailed Student’s t test.

Co-inhibitory receptor-ligand engagement increases cell-cell contact time and leads to decreased cytokine production by effector T cells.³⁶ However, these receptors are effective in cell-cell suppression only when they are expressed on the cell surface.37, 38, 39 For instance, freshly isolated, naturally occurring (n)Tregs retain PD-1 in intracellular compartments and upon TCR signaling will translocate PD-1 to the cell surface and, subsequently, become more suppressive.³⁹ We detected higher levels of PDCD1, as well as higher surface PD-1 expression, in anti-pPKCθ-iTregs (Figure 2G). Of note, we observed that ex vivo-generated anti-pPKCθ-iTregs also expressed increased amounts of IFNG transcripts and produced more IFNγ than did DMSO-iTregs (Figure 2H), which is consistent with unique populations of highly suppressive Tregs previously described.⁴⁰^,⁴¹ These results demonstrate that when peripheral CD4 T cells are activated ex vivo, using methods that mimic physiological activation and co-stimulatory signals, and then cultured with a defined polarizing cocktail, these cells can be successfully differentiated into iTregs. However, delivering anti-pPKCθ across the cell membrane prior to differentiating CD4 T cells generated a greater percentage of CD4⁺CD25^highFOXP3^high iTregs, increased the concentration of FOXP3 and surface PD-1 expressed by these iTregs, and induced IFNγ production.

Anti-pPKCθ-iTregs Exhibit Superior Suppressive Capabilities In Vitro

Tregs function to suppress the activation of nearby T cells, through mechanisms that are both direct, i.e., cell-cell contact, and indirect, i.e., release of anti-inflammatory cytokines.⁴² Tregs that are defined by the CD4⁺CD25^highFOXP3^high phenotype, and which were increased in the iTreg population following anti-pPKCθ delivery, are presumed to possess potent suppressive capabilities. However, an iTreg phenotype alone does not demonstrate functional suppression. Therefore, we utilized a standard in vitro suppression assay to determine whether anti-pPKCθ delivery generated iTregs with superior suppressive activity, compared to their DMSO-iTreg counterparts. We activated hPBMCs, mimicking physiological conditions by stimulating them with soluble anti-CD3 and anti-CD28. We labeled these responder T cells (T_resps) with the vital dye UltraGreen and mixed them in culture at three different ratios with ex vivo-differentiated suppressor iTregs (T_supps). We used a second vital dye, Red650, which emits fluorescence at a longer wavelength, to label the T_supps (Figure 3A). We used flow cytometry to track the proliferative responses of T_resps and T_supps at the end of the 4-day suppression assay. When we cultured anti-pPKCθ-iTregs with responder cells at a 1:10 ratio, and the suppression we observed was as strong as when DMSO-treated iTregs were mixed with responder cells at a 1:1 ratio, suggesting that anti-pPKCθ-iTregs potently suppress T cell proliferation in standard suppression assays (Figure 3B). Furthermore, anti-pPKCθ-iTregs proliferated in culture more extensively than did DMSO-iTregs, as indicated by the loss of Red650 fluorescence (Figure 3C). This was also reflected in the overall percentages of iTregs at the end of the co-culture period, when we detected significantly higher percentages of CD4⁺CD25^highFOXP3^high cells in co-cultures containing anti-pPKCθ-iTregs compared to those with DMSO-iTregs (Figure 3D).

Following activation, both CD4⁺CD25⁻ Tconvs and CD4⁺CD25⁺ iTregs increase expression of FOXP3. However, FOXP3 in Tconvs remains mostly in the cytosol, whereas in iTregs, FOXP3 is localized primarily to the nucleus.⁴³ Furthermore, when a mutant, nuclear-translocating form of FOXP3 was expressed in Jurkat T cells, it endowed these transfected cells with suppressive capabilities.⁴³ To ask whether there were differences in the cellular distribution of FOXP3 in DMSO-Tregs and anti-pPKCθ-iTregs, we again used imaging flow cytometry to assess FOXP3 localization. As indicated by the increased positive nuclear similarity score, we detected significantly more nuclear FOXP3 in anti-pPKCθ-iTregs than in DMSO-iTregs, consistent with their enhanced suppressive capacity (Figure 3E). PD-1 expression in Tregs has been associated with increased FOXP3 stability,⁴² and IFNγ signaling upregulates the co-inhibitory ligand for PD-1, PD-L1, on activated T cells.⁴² Thus, it is likely that anti-pPKCθ delivery modulates several key iTreg signaling pathways to convey superior in vitro suppression.

Adoptively Transferring Anti-pPKCθ-iTregs Attenuates Disease and Prolongs Survival in a Humanized Mouse Model of GvHD

Using Treg adoptive immunotherapy in mouse models of GvHD represents a viable strategy for understanding T cell biology, as well as Treg-mediated suppression.⁴⁴ iTreg abundance correlated with attenuated GvHD severity and increased long-term graft tolerance, without the need for drug-induced immunosuppression.³²^,⁴⁵ Therefore, we assessed the translational potential of anti-pPKCθ-iTregs using a pre-clinical mouse model of acute GvHD.²⁹ In this humanized mouse model, PBMCs (graft) are transferred, via the tail vein, into lightly irradiated recipient (host) NOD-scid IL2rγ^null (NSG) mice. The BM is the target of immune-mediated destruction, with symptoms peaking approximately 17 days after PBMCs are transferred. This model is uniformly fatal, and mice succumb to lethal BM failure approximately 3 weeks after disease induction.²⁹ To test the translational potential of administering anti-pPKCθ-treated iTregs as a cell-based prophylaxis for GvHD, we differentiated iTregs from a single donor ex vivo without or with anti-pPKCθ delivery. We used PBMCs from the same donor to induce GvHD in host mice (Figure 4A). Our rationale for this approach stems from the understanding that immune-competent T cells residing in the graft are activated and expand in the host as the result of conditioning regimens that produce a pro-inflammatory setting. We reasoned that if peripheral blood CD4 T cells from the donor were differentiated into iTregs ex vivo, before administering to the host at the time of BM transplantation, they would prevent expansion of alloreactive T cells in the host and attenuate acute GvHD. To test this hypothesis, we transferred ex vivo-differentiated iTregs, together with disease-inducing PBMCs, at a ratio of 1:3 into NSG mice. We chose this “responder/suppressor” ratio guided by our in vitro suppression results (Figure 3). On day 17, we analyzed BM, peripheral blood, and spleens of diseased animals to evaluate the efficacy of iTreg treatment.

To confirm equivalent disease induction across all of the cohorts, we evaluated the extent to which the transferred PBMCs expanded in vivo. We collected samples from the peripheral blood and spleens from mice with GvHD that received no iTregs, that received DMSO-iTregs, or that received anti-pPKCθ-iTregs, and evaluated the percentage of cells that expressed the human leukocyte antigen CD45 as a measure of cellular expansion after transfer. The percentages of human CD45⁺ cells detected in the peripheral blood and spleens of GvHD mice did not differ greatly, regardless of whether they also received iTregs, and the overall cellularity of the spleens also appeared similar when stained with hematoxylin and eosin (Figures S2A and S2B), indicating comparable disease induction across all treatment cohorts. When we refined our analyses of cells by subsets and noted fewer circulating CD4 T cells in mice treated with anti-pPKCθ-iTregs, but otherwise there were no significant differences in the percentages of human CD4 or CD8 T cells between treated or untreated animals (Figure S2C).

Hematopoiesis in the BM gives rise to circulating white and red blood cells. Pancytopenia is a hallmark of the GvH-mediated BM failure that accompanies this model. Transferring DMSO-iTregs when GvHD was induced afforded discernable protection to circulating white and red blood cells, and this protection was significantly enhanced in mice that received anti-pPKCθ-iTregs (Figure 4B). Similarly, when we evaluated BM cellularity, either by counting nucleated cells in the BM or by histological assessment (Figure 4C), it was evident that administering anti-pPKCθ-iTregs at the time of disease induction provided robust protection of the BM compartment. This was likely due to differences in the percentages, as well as in absolute numbers, of PBMCs that had infiltrated the BM by day 17 (Figure 4D). However, the distribution of T cells recruited to the BM by this time was similar in mice that received iTregs, regardless of how these cells were differentiated prior to infusion (Figure 4E). These results led us to conclude that treating mice with anti-pPKCθ-iTregs may alter the kinetics of T cell migration into the BM, rather than the percentages of BM-infiltrating CD4 and CD8 T cells.

We next evaluated whether administering iTregs attenuated disease severity in a humanized mouse model of GvHD. Treating diseased mice with DMSO-iTregs reduced their cumulative GvHD clinical score, and nearly doubled their median survival time, compared to untreated mice with GvHD (38 days versus 21 days, respectively; Figures 4F and 4G). More remarkable was the impact on GvHD that co-administering anti-pPKCθ-iTregs had in this model. As a cohort, mice that received anti-pPKCθ-iTregs had less severe symptoms of disease, as compared to mice that received DMSO-iTregs or no treatment, respectively (Figure 4F). Predictably, mice that exhibited less severe symptoms also lived longer. The median length of survival for mice treated with anti-pPKCθ-iTregs was twice that of DMSO-iTreg-treated mice, and nearly 4-fold that of untreated mice (78 days versus 38 days versus 21 days, respectively; Figure 4G). Quite unexpectedly, one of the five mice treated with anti-pPKCθ-iTregs exhibited full rescue from lethal GvHD, surviving a full 100 days after disease induction. Overall, these in vivo results demonstrated that anti-pPKCθ-iTregs were highly efficacious as a cell-based therapy in a humanized mouse model of GvHD, when infused at the time of disease induction.

Ex Vivo-Generated Anti-pPKCθ-iTregs Exhibit a Stable, Unique FOXP3^highPD-1⁺IFNγ^high Phenotype In Vivo

In this pre-clinical model of GvHD, BM destruction is mediated by Th1 responses resulting from an imbalanced Th1/Th2 response.²⁹ When we measured circulating cytokines in mice treated with anti-pPKCθ-iTregs, we detected significantly increased IL-2 and IL-10, both of which enhance the immunosuppressive functions of Tregs in vivo. Signature Th2 cytokines, IL-4 and IL-13, which can counteract Th1 responses, were also increased in the plasma of anti-pPKCθ-iTreg-treated mice (Figure 5A), suggesting that anti-pPKCθ-iTregs alter Th cell responses in vivo.

Administering anti-pPKCθ-iTregs proved superior in reducing the severity of GvHD and extending survival in the humanized mouse model. Accumulating evidence suggests that in vitro-expanded Tregs have unstable FOXP3 expression and may lose their suppressive properties when encountering proinflammatory conditions, such as those that characterize the GvHD milieu in vivo.⁴⁶ Therefore, we sought to better evaluate the in vivo stability of anti-pPKCθ-iTregs, compared to iTregs generated without anti-pPKCθ delivery. We identified iTregs based on CD4, CD25, and FOXP3 expression, as described previously,³²^,⁴⁵ and used magnetic beads to sort iTreg (CD4⁺CD127⁻CD25⁺) populations recovered from the BM, peripheral blood, and spleens of iTreg-treated mice on day 17 after GvHD induction. We noted significantly higher percentages, as well as total numbers, of CD4⁺CD25⁺FOXP3⁺ iTregs in the BM of mice that received anti-pPKCθ-iTregs, compared to animals that received DMSO-iTregs (Figures 5B and 5C). The percentage of FOXP3^high cells among BM-infiltrating iTregs was also significantly greater in mice that received anti-pPKCθ-iTregs (Figure 5D). Furthermore, FOXP3 expression in anti-pPKCθ-iTregs isolated from the BM was greater than its expression in DMSO-iTregs, although it did not reach significance (Figure 5D). The percentages of iTregs in peripheral blood and spleen did not differ greatly between treatments (Figures S3A and S4A). However, the FOXP3^high iTreg population was consistently and significantly greater in peripheral blood and spleens of anti-pPKCθ-iTreg-treated mice (Figures S3B and S4B). When we further analyzed the CD4⁺CD25⁺FOXP3⁺ iTreg population recovered from the BM of mice treated with anti-pPKCθ-iTregs, we found a significantly lower percentage of cells that stained positively for pPKCθ, compared to mice treated with DMSO-iTregs (Figure 5E). In addition, pPKCθ^low iTregs were significantly higher in peripheral blood, but not in spleen, of anti-pPKCθ-iTreg-treated mice (Figures S3C and S4C). Consistent with decreased percentages of pPKCθ-expressing Tregs in the BM, we also noted that BM-infiltrating anti-pPKCθ-iTregs tended to express less total pPKCθ, as indicated by a higher percentage of pPKCθ^low iTregs, as well as less nuclear pPKCθ, than did DMSO-iTregs, although these data did not reach statistical significance (Figures 5E and 5F). We did not observe differences in pPKCθ nuclear localization among iTregs circulating in the peripheral blood or that had trafficked to the spleen (Figures S3D and S4D).

One means by which iTregs can suppress activated T cells is through cell contact-dependent mechanisms, specifically by upregulating the immune-inhibitory receptor PD-1, which will engage its cognate ligand, PD-L1, on activated T cells.⁴⁷ Percentages of PD-1-expressing iTregs in BM were similar in mice regardless of iTreg treatment (Figure 5G). Both the percentage of PD-1^high-expressing iTregs, as well as total intracellular and surface-expressed PD-1 (MFI), trended higher in anti-pPKCθ-iTregs, and we could detect a population of PD-1^high-expressing anti-pPKCθ-iTregs that was not present within the DMSO-iTreg population (Figure 5G). We did not observe differences in PD-1^high iTregs isolated from peripheral blood or spleen of mice from either cohort (Figures S3E and S4E).

We also investigated changes in gene expression in iTregs. Specifically, there were significantly higher levels of PDCD1 and IFNG transcripts in anti-pPKCθ-iTregs isolated from the BM, compared to DMSO-iTregs (Figures 5H and 5I, respectively). These findings led us to evaluate plasma levels of IFNγ in iTreg-treated mice. In contrast to DMSO-iTreg-treated mice, we measured significantly greater levels of circulating IFNγ in mice that received anti-pPKCθ-iTregs (Figure 5J). This contrasted with splenic iTregs, which showed no difference in PDCD1 and significantly lower IFNG mRNA levels (Figures S4F and S4G). Altogether, these results indicate that compared to DMSO-iTregs, and even up to 17 days after in vivo administration, anti-pPKCθ-iTregs display an increased ability to traffic to sites of inflammation (here the BM) and express high levels of PD-1, and they can produce large quantities of IFNγ, suggesting that the combination of these unique characteristics contributes to reducing disease severity.

Ex Vivo-Generated Anti-pPKCθ-iTregs Show Increased PD-1 Co-localization with PKCθ and NFATc1

Consistent with reports in the literature, we observed increased surface PD-1 on anti-pPKCθ-iTregs, compared to DMSO-iTregs, and this correlated with their higher expression of PDCD1 (Figure 2G). Anti-pPKCθ-iTregs also expressed more IFNγ than did DMSO-iTregs (Figure 2H). Therefore, we sought to further characterize DMSO-iTregs and anti-pPKCθ-iTregs to increase our understanding of their functional differences. We used imaging flow cytometry to ask whether surface PD-1 association with other signaling proteins differed between DMSO-iTregs and anti-pPKCθ-iTregs differentiated ex vivo. We detected higher PD-1-pPKCθ co-localization in anti-pPKCθ-iTregs (Figure 6A), even though pPKCθ is significantly reduced in these cells (Figure 2), suggesting that the much of the pPKCθ that is expressed in anti-pPKCθ-iTregs may be associated with surface PD-1. Downstream of CD28 signaling, NFATc1 is dephosphorylated on specific residues by the calcium-dependent phosphatase calcineurin, mediated by the enzymatic activity of the calcineurin B (CnB) subunit.⁴⁸ Dephosphorylating NFATc1 unmasks a nuclear localization signal that allows its nuclear import. Once in the nucleus, NFATc1 differentially associates with various transcription partners to regulate context-specific gene expression, including IFNG, FOXP3, and PDCD1.49, 50, 51 Compared to DMSO-iTregs, we found that PD-1 colocalized with NFATc1 to a much greater extent in anti-pPKCθ-iTregs (Figure 6B). As a result, nuclear NFATc1 levels were significantly reduced in anti-pPKCθ-iTregs, although NFATc1 cytosolic levels were comparable regardless of iTreg treatment (Figure 6C). As with NFATc1, cytosolic CnB levels were comparable in DMSO-iTregs and anti-pPKCθ-iTregs, but DMSO-iTregs expressed significantly more nuclear CnB (Figure 6D). We determined that CnB functioned similarly in DMSO-iTregs and anti-pPKCθ-iTregs, since treating either with the CnB inhibitor, tacrolimus, equivalently reduced nuclear NFATc1 localization and concomitantly increased its cytosolic levels (Figure 6C). We also noted that tacrolimus treatment decreased the amount of nuclear CnB in DMSO-iTregs suggesting, perhaps, that NFATc1 and CnB are both imported into the nucleus of DMSO-iTregs (Figure 6D). This does not appear to be the case in anti-pPKCθ-iTregs, and it is possible that the increased association of NFATc1 with PD-1 in these cells makes it less accessible to dephosphorylation by CnB. Nuclear CnB has not been previously reported in T cells, and additional investigation is needed to determine whether CnB acts similarly in the nucleus to maintain a pool of dephosphorylated and, therefore, nuclear-resident NFATc1 in DMSO-iTregs.

NFATc1 and CnB Subcellular Localization in iTregs Is Associated with Signaling through IFNγ and PD-1

We next investigated whether NFATc1 or CnB localization was linked to PD-1 or IFNγ signaling in ex vivo-generated iTregs. During differentiation, we treated DMSO-iTregs and anti-pPKCθ-iTregs with antibodies to neutralize either IFNγ- or PD-1-initiated signaling, then used imaging flow cytometry to ask how modulating these cell-extrinsic signals affected NFATc1 and CnB localization. In DMSO-iTregs, both anti-IFNγ and anti-PD-1 treatment reduced nuclear NFATc1, while nuclear CnB was reduced following anti-IFNγ treatment only (Figures 7A and 7C). In stark contrast, in anti-pPKCθ-treated iTregs, only anti-IFNγ treatment increased nuclear NFATc1, while higher amounts of nuclear CnB were detected following anti-IFNγ or anti-PD-1 treatment (Figures 7B and 7D). These results clearly indicate that signaling downstream of PD-1 and IFNγ converges at the level of NFATc1 and CnB subcellular localization in iTregs, and this is further modulated when iTregs are treated with anti-pPKCθ during ex vivo differentiation.

NFATc1 and Calcineurin B Subcellular Localization in iTregs Is Associated with Signaling through IFNγ and PD-1

(A–D) Percent of cells positive for nuclear and cytosolic (A and B) NFATc1 and (C and D) calcineurin B, together with representative nuclear localization score distributions, quantification of nuclear localization similarity scores, and representative images, as determined by AMNIS imaging flow cytometry analysis of 1,000 iTregs differentiated without or with CPPM-anti-pPKCθ. (A and C) DMSO-iTregs and (A–D) anti-pPKCθ-iTregs were treated with or without anti-IFNγ or anti-PD-1 during iTreg differentiation. Data represent the mean ± SEM of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, by unpaired, two-tailed Student’s t test.

Discussion

In this study, we demonstrate that targeting pPKCθ using a cell-penetrating antibody enhanced ex vivo iTreg differentiation and expansion. Anti-pPKCθ-treated iTregs showed increased expression of FOXP3, the co-inhibitory receptor PD-1, and IFNγ. iTregs differentiated in the presence of anti-pPKCθ displayed a stable phenotype in vivo when examined 17 days after transfer into mice, using a humanized mouse model of GvHD, and were highly efficacious in attenuating symptoms and prolonging survival.

Cell-based therapies are being actively investigated within the developing field of personalized medicine, the use of which can minimize side effects and provide long-term management of immunological diseases.⁴⁴^,⁵² T cells are highly specific, adaptable, “smart” therapeutic agents that selectively target tissues by tuning their activities in response to inflammatory microenvironments. Freshly isolated Tregs, administered together with BM allograft, can ameliorate GvHD and enhance HST engraftment.⁵³^,⁵⁴ Following in vivo administration, Tregs can exert their suppressive function by inhibiting effector T cell activation and altering cytokine production and migration. Tregs can downregulate DC maturation in a cell contact-dependent manner, inhibit monocyte and macrophage survival through Fas-FasL signaling, and constrain neutrophil activity by promoting their apoptosis.55, 56, 57, 58 Recent studies showed that, rather than isolating nTregs from patients and expanding them in culture, ex vivo conversion of CD4⁺CD25⁻ T cells into iTregs by culturing them with polarizing factors such as transforming growth factor β (TGF-β), IL-2, all-trans retinoic acid, DNA methyltransferase (DNMT) inhibitors, histone deacetylase (HDAC) inhibitors, butyrate, and/or rapamycin conveys greater immunosuppressive functions, making iTregs more attractive for immunotherapy.⁵⁹^,⁶⁰ In our study, we found that intracellular anti-PKCθ delivery, coupled with a standard differentiation protocol and anti-CD3 plus anti-CD28 stimulation, enhanced ex vivo iTreg differentiation. This strategy appears to be highly efficient for ex vivo iTreg generation, since anti-PKCθ-treated iTregs expressed higher levels of nuclear FOXP3, surface PD-1, and IFNγ, compared to DMSO-treated iTregs.

Additional obstacles to successful Treg-based immunotherapy involve maintaining Treg stability and reducing plasticity by sustaining FOXP3 expression, as well as proper trafficking to target organs, following adoptive transfer in vivo.¹^,⁶¹^,⁶² In our study, we utilized a humanized mouse model of GvHD that targets the BM for immune-mediated destruction. Our results demonstrated that mice treated with anti-pPKCθ-iTregs had significantly more FOXP3^high-expressing iTregs in the BM and experienced a greater reduction in the severity of GvHD, compared to mice treated with DMSO-iTregs. Furthermore, anti-pPKCθ-iTregs recovered from the BM of treated mice displayed an immunophenotype very similar to ex vivo-differentiated anti-pPKCθ-iTregs, suggesting these cells remain stable in vivo, even several weeks after transfer.

PKCθ has been implicated as a driver protein of aberrant T cell activation in the context of GvHD progression.²⁷^,²⁹ PKCθ is phosphorylated on Thr538 to complete its activation and facilitate its translocation both to the IS and to the nucleus.¹⁹^,³⁵^,⁶³ Studies suggest that inhibiting PKCθ may constitute an effective therapy for T cell-mediated diseases,²²^,²⁵^,²⁶^,⁶⁴ and inhibiting PKCθ function in Tregs enhances their suppressive function both in vitro and in vivo.²¹^,²³ However, these studies utilized small-molecule inhibitors that lacked the ability to specifically target PKCθ actions, potentially affecting closely related PKC family members, such as PKCα and PKCδ. Considering the greater specificity of antibody binding, we showed that we could modify PKCθ function in iTregs, using a highly specific cell-penetrating antibody-delivery strategy.

Molecular mechanisms that link reduced PKCθ signaling with transcriptional changes, as well as co-inhibitory receptor expression on the cell surface, remain to be fully elucidated. We observed near complete loss of nuclear pPKCθ and significantly reduced cytosolic anti-pPKCθ-iTregs. Studies suggest that PKCθ can be found in close proximity to PD-1 in the cytosol.⁶⁵^,⁶⁶ Indeed, PKCθ-PD1 co-localization was increased in anti-pPKCθ-iTregs, compared to DMSO-iTregs, potentially stabilizing PD-1 and enhancing iTreg PD-1-PD-L1-mediated suppression, which has been shown to be an important mechanism by which Tregs negatively regulate the immune response.⁶⁷

Interestingly, anti-pPKCθ-iTregs exhibited high IFNγ expression both in vitro and in vivo. The actions of IFNγ in immune responses and inflammatory processes are paradoxical. IFNγ can facilitate Th1 differentiation and T cell migration to sites of inflammation and initiate proinflammatory signaling events. However, IFNγ-producing Th1 cells can also trigger the immune system to initiate control mechanisms.⁶⁸ T cells require three signals for full activation: signal 1 is received through the TCR, signal 2 is a co-stimulatory signal (i.e., though the CD28 co-receptor), while signal 3 is mediated by cytokine receptors.⁶⁹ When naive T cells encounter elevated IFNγ, in the absence of signaling through the TCR and CD28, activation of multiple cellular and molecular events leads to peripheral conversion of CD4⁺CD25⁻ T cells to CD4⁺CD25⁺ Tregs, to regulate overt inflammation and suppress autoimmune responses.⁶⁸ It has been argued that the source of IFNγ is critical to driving either pro-inflammatory or anti-inflammatory responses in GvHD.⁷⁰^,⁷¹ However, functional consequences of IFNγ production by Tregs in GvHD remain unexplored. It has been suggested that IFNγ produced by Tregs is advantageous in preventing allogeneic skin graft rejection.⁴⁰^,⁴¹ Moreover, allogeneic donor FOXP3-expressing Tregs appeared to express IFNγ upon bone marrow transplantation (BMT) and prevented the development of lethal GvHD.⁵³^,⁵⁴ However, donor Tregs treated with neutralizing anti-IFNγ monoclonal antibody or Tregs from IFNγ-knockout donor mice failed to prevent the lethal GvHD.⁴⁰^,⁷⁰ Furthermore, STAT1, an important mediator in IFNγ signaling, was found to be critical to the induction of CD4⁺CD25⁺ Tregs.⁷² Anti-pPKCθ delivery into iTregs leads to increased IFNγ. This finding is consistent with recent studies that showed perturbing the CARMA1-Bcl10-MALT-1 (CBM) complex in Tregs generates IFNγ-producing Tregs.⁷³^,⁷⁴ We previously showed that PKCθ interacts with the CBM complex.⁷⁵ Furthermore, delivering anti-pPKCθ into CD4 T cells prior to in vitro Th1 polarization reduces phosphorylation of the PKCθ substrate, CARMA1.²⁹ Thus, it is possible that in anti-pPKCθ-iTregs, we are similarly perturbing the CBM complex, leading to increased IFNγ production by these cells. Additional studies are needed to fully elucidate the mechanism driving this phenomenon. Anti-pPKCθ-iTregs produced significantly more IFNγ in vitro and in vivo, resulting in higher levels of IFNγ in the plasma from the mice that received anti-pPKCθ-iTregs. These findings, coupled with the presence of more CD4⁺CD25⁺FOXP3⁺ iTregs in the BM of anti-pPKCθ-iTreg-treated mice, led us to conclude that IFNγ may act as both an extrinsic and intrinsic factor to promote iTreg differentiation and enhance their suppressive function in this humanized mouse model of GvHD.

The transcription factor NFATc1 functions to link extracellular stimuli to gene transcription, through its association with other transcriptional partners, including but not limited to AP-1, NF-κB, and the various Th master transcriptional regulators. Through selective pairing, NFATc1 can regulate both pro-inflammatory, i.e., cytokine production, and anti-inflammatory, i.e., PD-1 expression, genetic programs.⁴⁹^,⁵¹ To generate anti-pPKCθ-iTregs, we provided co-stimulation though CD28 in the context of iTreg differentiation, together with anti-pPKCθ delivery. Therefore, it is entirely possible that we have altered NFATc1 availability for binding to its nuclear partners, thus generating unique regulation of iTreg genes, including regulation of FOXP3, PD-1, and IFNγ. Our data showing increased co-localization between NFATc1 and PD-1 in anti-pPKθ-iTregs supports this notion, in that its increased association with PD-1 may physically prevent CnB from accessing and dephosphorylating NFATc1, thus impeding its nuclear translocation. However, additional studies are needed to confirm this model.

Dephosphorylation by CnB is necessary to facilitate NFATc1 nuclear translocation.⁷⁶ Once in the nucleus it may be re-phosphorylated by one of several kinases, including GSK3β and casein kinase 1/2, promoting its export to the cytosol.⁷⁷ Compared to anti-pPKCθ-iTregs, DMSO-iTregs expressed higher amounts of nuclear NFATc1, and of nuclear CnB as well. This raises the intriguing possibility that a pool of nuclear CnB may function to keep NFATc1 dephosphorylated and, thus, nuclear-resident, in DMSO-iTregs. Reduced PKCθ activity is known to enhance GSK3β function.⁷⁸ Thus, it is equally intriguing to speculate that GSK3β may be acting to re-phosphorylate nuclear NFATc1 in anti-pPKCθ-iTregs to facilitate its nuclear export, accounting for the reduced amount of nuclear NFATc1 expressed in these cells. Further investigation would be required to determine whether this is, indeed, a mechanism that supports our observations.

Our results, together with supporting evidence from the literature, demonstrate that modulating PKCθ function in iTregs reprograms their cell fate and enhances their immunosuppressive capacity both in vitro and in vivo. Intracellular anti-pPKCθ delivery using cell-penetrating peptide mimics is a promising strategy to fine-tune iTreg differentiation to favor generating and expanding a unique and highly suppressive population, characterized by increased expression of FOXP3, PD-1, and IFNγ. More importantly, using a humanized model of GvHD, we show that adoptively transferring anti-pPKCθ-iTregs into mice is highly efficacious in attenuating the severity of GvHD in vivo when transferred at the time of disease induction. In humans, this would correlate to giving anti-pPKCθ-iTregs at the time of HSCT, with the prediction that the transferred iTregs would effectively suppress the rapidly expanding donor T cell population within the stem cell graft. Our data suggest that anti-pPKCθ-iTregs, generated from PBMCs of the stem cell donor, may have prophylactic value when given to recipients at the time of HSCT. However, note that all animal models have limitations, and extrapolating results from murine models to humans must be done with care. Further study is needed to answer remaining questions regarding optimal ex vivo differentiation conditions, optimal concentration and timing of doses, and the potential need for adjuvant immunosuppressive therapy that will enhance and not counteract iTreg administration. Nonetheless, our study constitutes a compelling argument to further explore the use of CPPM-antibody delivery as a means of programming immune cells ex vivo to generate readily available, stable, efficacious, and personalized cell-based therapeutics.

Materials and Methods

Animals

All animal studies were approved by, and conducted under oversight of, the Institutional Animal Care and Use Committee of the University of Massachusetts, Amherst. Seven-week-old female NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice were purchased from The Jackson Laboratories (Bar Harbor, ME, USA). Mice were rested 1 week prior to use, housed under pathogen-free conditions in micro-isolator cages, and received acidified water (pH 3.0) supplemented with trimethoprim + sulfamethoxazole throughout the duration of the experimental procedures.

Antibodies and Reagents

Antibodies used in this study were as follows: CD3ε (clone UCHT1), CD28 (clone CD28.2), CD4 (clone RPA-T4), CD8 (clone RPA-T8), CD25 (clone BC96), CD25 (clone BC96), CD25 (clone BC96), CD45RA (clone HI100), CD45RO (clone UCHL1), CD127 (clone A019D5), CD127 (clone A019D5), PD-1 (clone EH12.2H7), and NFATc1 (clone7A6), all from BioLegend (San Diego, CA, USA); CD4/CD8 cocktail (clones RPA-T4 and RPA-T8) and human CD45 (clone 2D1) from eBioscience (Santa Clara, CA, USA); mouse CD45 (clone 30-F11) and IFNγ (clone B27) from BD Biosciences (Billerica, MA, USA); histone H3 (clone 96C10), pPKCθ (Thr538), and total PKCθ (clone E1I7Y) from Cell Signaling Technology (Danvers, MA, USA); anti-PP2B-B1/2 (CnB; clone D-1) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); α-Tubulin (clone B-5-1-2) from Sigma-Aldrich (St. Louis, MO, USA); and pPKCθ (Thr538; clone F4H4L1) and F(ab′)₂-goat anti-rabbit IgG (H+L) secondary antibody (Qdot625, polyclonal) from Life Technologies (Carlsbad, CA, USA). For nuclear staining, DRAQ5™ was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Live/dead staining was performed utilizing either Zombie Aqua or Zombie Violet fixable viability kit (BioLegend). For in vitro suppression assay, cells were tracked using labeling with CytoTell UltraGreen or CytoTell Red650 (AAT Bioquest, Sunnyvale, CA, USA). Tacrolimus was obtained from Thermo Fisher Scientific (Agawam, MA, USA).

Human iTreg Differentiation Coupled with Intracellular CPPM-Anti-pPKCθ Delivery

1 μM CPPM (P₁₃D₅) and 25 nM of anti-pPKCθ (Thr538, clone F4H4L1) were complexed in PBS (phosphate-buffered saline, pH 7.2) at a 40:1 ratio (CPPM/anti-pPKCθ) for 30 min at room temperature (RT). CD4 T cells were isolated from human PBMCs (STEMCELL Technologies, Vancouver, BC, Canada) using a MojoSort human T cell isolation kit (BioLegend). Isolated human CD4 T cells were treated with the CPPM-antibody complex for 4 h at 37°C or DMSO as vehicle control. Cells were harvested and washed with PBS, then washed twice with 20 U/mL heparin in PBS for 5 min on ice to remove cell surface-bound complexes. For iTreg differentiation, a CellXVivo human Treg differentiation kit (R&D Systems, Minneapolis, MN, USA) was used and iTreg differentiation media were prepared using X-VIVO 15 chemically defined, serum-free hematopoietic cell medium according to the manufacturer’s instructions. Treated cell pellets were resuspended in iTreg differentiation media and seeded onto wells of a 12-well tissue culture plate pre-coated with 5 μg/mL anti-CD3ε plus 2.5 μg/mL anti-CD28, and stimulated for 5 days at 37°C.

Immunoblotting

iTregs were harvested on day 5 of differentiation. Nuclear and cytosolic extracts were prepared using NE-PER nuclear and cytosolic extraction kit (Thermo Scientific). 1× SDS Laemmli buffer was added to samples, which were separated on 8% gels using SDS-PAGE. Blots were probed with anti-pPKCθ (Thr538) and anti-total PKCθ, then re-probed using anti-α-tubulin and anti-histone H3 as cytosolic and nuclear loading controls, respectively.

Protein Subcellular Localization Using AMNIS Imaging Flow Cytometry

For ex vivo analysis, iTregs were harvested on day 5 of differentiation. For in vivo analysis, BM, spleen, and peripheral blood were collected on day 17 and single-cell suspensions were prepared from each sample. Cells were surface stained for CD4 and CD25. Each sample was then fixed and permeabilized according to the manufacturer’s directions using the Foxp3 staining buffer kit (BD Biosciences) and stained for FOXP3, NFATc1, CnB, and pPKCθ (Thr538) followed by Qdot625-labeled secondary antibody. Nuclei were stained using the cell-permeable DRAQ5 fluorescent probe (Thermo Fisher Scientific). Cells were visualized and quantified using an ImageStream^X Mk II imaging flow cytometer (EMD Millipore, Billerica, MA, USA). Subcellular localization and nuclear similarity scores for FOXP3, pPKCθ (Thr538), NFATc1, and CnB proteins were determined using the nuclear localization wizard and the IDEAS software following masking of nuclear and non-nuclear regions to quantify proteins localized out of and within the nucleus, respectively.

In Vitro Suppression Assay

On day 0, human CD4 T cells were plated onto anti-CD3+anti-CD28-coated wells and differentiated for 5 days in iTreg differentiation media. On day 5, iTregs (suppressors) were loaded with the cell tracker dye Red650 (allophycocyanin [APC] fluorescence). Total hPBMCs (responders) were thawed and stimulated with soluble anti-CD3+anti-CD28 and crosslinked using mouse IgG. Responder cells were then loaded with a different cell tracker dye, UltraGreen (fluorescein isothiocyanate [FITC] fluorescence). Responder cells were seeded onto tissue culture plates and suppressors were added to responders at the indicated ratio. Cells were co-cultured for 4 days and proliferation of responder and suppressor cells was determined. Percent suppression was calculated as follows: Suppression (%) = 100% − FITC-negative cells (%).

Surface versus Intracellular Expression of Co-inhibitory Receptors

iTregs were harvested on day 5 of differentiation. Cells were stained with Zombie Aqua viability dye. For surface-only expression, cells were directly stained using PD-1. For surface+intracellular expression, cells were surface stained, then fixed and permeabilized according to the manufacturer’s directions using the Foxp3 staining buffer kit, followed by staining with anti-PD-1. Samples were acquired using a BD LSRFortessa flow cytometer (Becton Dickinson) and MFIs were calculated. For intracellular-only expression, MFI of surface staining was subtracted from MFI of surface+intracellular staining.

Quantitative Real-Time PCR

Total RNA was isolated from samples using a Quick-RNA isolation kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s protocol. 1 μg of total RNA was reverse-transcribed to cDNA using dNTP (2′-deoxynucleoside 5′-triphosphate) (New England Biolabs, Ipswich, MA, USA), Moloney murine leukemia virus (M-MuLV) reverse transcription buffer (New England Biolabs), oligo(dT) (Promega, Madison, WI, USA), RNase inhibitor (Promega), and M-MuLV reverse transcriptase (New England Biolabs) on a Mastercycler gradient thermal cycler (Eppendorf, Hamburg, Germany). Quantitative real-time PCR primers used in this study are listed in Table S1. Quantitative real-time PCR was performed in duplicate with 2× SYBR Green qPCR master mix (BioTool, Houston, TX, USA) using the RealPlex2 system (Eppendorf). Quantitative real-time PCR conditions were as follows: 95°C for 1 min, 95°C for 25 s, 62°C for 25 s (40 cycles), 95°C for 1 min, 62°C for 1 min, and 95°C for 30 s. Relative gene expression was determined using the ΔΔCt method. The results are presented as fold gene expression normalized to the housekeeping gene β-actin and relative to Tconv+DMSO samples for ex vivo experiments and relative to naive+DMSO for in vivo experiments.

In Vivo Suppression Analysis Using Adoptive Transfer of iTregs in a Humanized GvHD Model

CD4 T cells were isolated from healthy donor hPBMCs, subsequently treated with the CPPM-anti-pPKCθ complex, and differentiated for 5 days into iTregs, as described. On day 4, total hPBMCs from the same donor were thawed and rested overnight in fresh RPMI 1640 complete media (10% fetal bovine serum, 100 U/mL penicillin-streptomycin, 1 mM sodium pyruvate, 2 mM l-glutamine) at 37°C in 5% CO₂ incubator. On day 5, NOD.Cg-Prkdc^scid Il2rg^tm1WjI/SzJ (NSG) mice were conditioned with 2 Gy of total body irradiation using a ¹³⁷Cs source and then rested for 4–6 h. 10 × 10⁶ hPBMCs were mixed with 3.3 × 10⁶ iTregs and adoptively transferred into irradiated NSG mice via the tail vein. Body weight and disease symptoms were observed daily. On day 17, some animals were sacrificed for tissue analysis. After CO₂ asphyxiation, peripheral blood was obtained using cardiac puncture. Sterna and spleens were collected for histology. BM cells were recovered from the tibias and femurs of both legs by flushing the bones with complete RPMI 1640 media. Splenocytes were isolated by manipulation through a 40-μm filter. Red blood cells were lysed in ACK lysis buffer, and the remaining white blood cells were enumerated using trypan blue exclusion. White and red cell counts were performed using a scil Vet abc hematology analyzer (scil Animal Care, Gurnee, IL, USA). BM, spleen, and peripheral blood were assessed for percent engraftment of hPBMCs [% positive human CD45/(% positive human CD45 cells + % positive mouse CD45 cells)] and infiltration of human CD4 and CD8 T cells. Human CD4 T cells were also analyzed for CD25, CD127, FOXP3, PD-1, and pPKCθ (Thr538) expression.

GvHD Clinical Scoring

GvHD severity was assessed using a standardized scoring system, as previously described,³⁶ which included five different criteria (weight loss, posture, activity, fur texture, and skin integrity). Mice were weighed, evaluated daily, and graded from 0 (least severe) to 2 (most severe) for each criterion, beginning day 12 after disease induction. Daily clinical scores were generated by adding the grades for the five criteria. When a clinical score of 8 was reached, mice were removed from the study and humanely euthanized. The day animals were from the study was recorded as the day of lethal GvHD induction.

Magnetic Sorting of In Vivo iTregs for mRNA Analysis

BM and spleens were collected from NSG mice 17 days after GvHD induction. BM cells were recovered from the tibias and femurs. Splenocytes were isolated by manipulation through a 40-μm filter. Red blood cells were lysed in ACK lysis buffer, and the remaining white blood cells were enumerated using trypan blue exclusion. Cells were incubated with human CD4 T lymphocyte enrichment cocktail (BD Biosciences) followed by incubation with BD IMag streptavidin particles plus (BD Biosciences) to deplete the non-CD4 T cell fraction. Biotinylated anti-CD127 antibody and biotinylated anti-CD25 antibody, followed by an incubation with BD IMag streptavidin particles plus, were applied sequentially to obtain the iTreg fraction (CD127⁻CD25⁺) and naive T cell fraction (CD127⁺CD25⁻). Total RNA was obtained from isolated cells as described.

Histology

Sterna and spleens harvested on day 17 were fixed overnight in 10% neutral buffered formalin (NBF) (VWR, Radnor, PA, USA), decalcified 48 h (Cal-Rite; Richard Allen Scientific, San Diego, CA, USA), preserved in 70% ethanol at 4°C until processed, paraffin embedded, sectioned, and stained with hematoxylin and eosin.

LEGENDPlex Bead-Based Immunoassay

Peripheral blood for cytokine analysis was obtained in heparin-coated syringes from animals via cardiac puncture, immediately after humane euthanasia on day 17. The LEGENDPlex Human Th1/Th2 panel (8-plex; BioLegend) was used to determine the level of IFNγ, IL-2, IL-4, and IL-10. Data were acquired using a BD LSRFortessa flow cytometer and analyzed using LEGENDPlex software, version 7.0 (BioLegend).

Statistical Analysis

The results shown are the mean ± SEM; all ex vivo experimental replicates were repeated at least three times. All in vivo experimental replicates were repeated in three separate experiments. An unpaired, two-tailed Student’s t test using Prism 5 (GraphPad, San Diego, CA, USA) was used for statistical comparison of two groups, with Welch’s correction applied when variances were significantly different. Survival benefit was determined using Kaplan-Meier analysis with an applied log rank test. p values of ≤0.05 were considered significantly different.

Author Contributions

Conceptualizations: E.I.O. and L.M.M. Methodology: E.I.O., S.S., and L.M.M. Investigations: E.I.O., S.S., H.L.S., and J.A.T. Writing – Original Draft: E.I.O. and L.M.M. Writing – Review & Editing: E.I.O., B.A.O., and L.M.M. Project Administration: G.N.T. and L.M.M. Funding Acquisition: B.A.O. and L.M.M. Supervision: B.A.O., G.N.T., and L.M.M.

Conflicts of Interest

The authors declare no competing interests.

Acknowledgments

The authors thank A.S. Burnside, Director of the Flow Cytometry Core Facility at the Institute for Applied Life Sciences, University of Massachusetts Amherst, Amherst, MA, for guidance, the University of Massachusetts Amherst Animal Care staff for excellent care of research animals, and R.A. Goldsby for critical assessment of the manuscript. This work was supported in part by the National Institutes of Health in the form of a Fellowship from the University of Massachusetts to H.L.S. as part of the Biotechnology Training Program (National Research Service Award T GM108556), by the National Institutes of Health (NIH 5P01CA16600 to B.A.O.), and by the Department of Defense (W81XWH1910540 to L.M.M.).

Footnotes

Supplemental Information can be found online at https://doi.org/10.1016/j.ymthe.2020.05.020.

Supplemental Information

Document S1. Figures S1–S4 and Table 1

mmc1.pdf^{(872KB, pdf)}

Document S2. Article plus Supplemental Information

mmc2.pdf^{(6MB, pdf)}

References

1.Ohkura N., Kitagawa Y., Sakaguchi S. Development and maintenance of regulatory T cells. Immunity. 2013;38:414–423. doi: 10.1016/j.immuni.2013.03.002. [DOI] [PubMed] [Google Scholar]
2.Hori S., Nomura T., Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science. 2003;299:1057–1061. doi: 10.1126/science.1079490. [DOI] [PubMed] [Google Scholar]
3.Khattri R., Cox T., Yasayko S.A., Ramsdell F. An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat. Immunol. 2003;4:337–342. doi: 10.1038/ni909. [DOI] [PubMed] [Google Scholar]
4.Sakaguchi S., Miyara M., Costantino C.M., Hafler D.A. FOXP3+ regulatory T cells in the human immune system. Nat. Rev. Immunol. 2010;10:490–500. doi: 10.1038/nri2785. [DOI] [PubMed] [Google Scholar]
5.Brunkow M.E., Jeffery E.W., Hjerrild K.A., Paeper B., Clark L.B., Yasayko S.A., Wilkinson J.E., Galas D., Ziegler S.F., Ramsdell F. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat. Genet. 2001;27:68–73. doi: 10.1038/83784. [DOI] [PubMed] [Google Scholar]
6.Fontenot J.D., Rasmussen J.P., Williams L.M., Dooley J.L., Farr A.G., Rudensky A.Y. Regulatory T cell lineage specification by the forkhead transcription factor Foxp3. Immunity. 2005;22:329–341. doi: 10.1016/j.immuni.2005.01.016. [DOI] [PubMed] [Google Scholar]
7.Liu W., Putnam A.L., Xu-Yu Z., Szot G.L., Lee M.R., Zhu S., Gottlieb P.A., Kapranov P., Gingeras T.R., Fazekas de St Groth B. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J. Exp. Med. 2006;203:1701–1711. doi: 10.1084/jem.20060772. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Simonetta F., Chiali A., Cordier C., Urrutia A., Girault I., Bloquet S., Tanchot C., Bourgeois C. Increased CD127 expression on activated FOXP3+CD4+ regulatory T cells. Eur. J. Immunol. 2010;40:2528–2538. doi: 10.1002/eji.201040531. [DOI] [PubMed] [Google Scholar]
9.Ganguly S., Ross D.B., Panoskaltsis-Mortari A., Kanakry C.G., Blazar B.R., Levy R.B., Luznik L. Donor CD4+ Foxp3+ regulatory T cells are necessary for posttransplantation cyclophosphamide-mediated protection against GVHD in mice. Blood. 2014;124:2131–2141. doi: 10.1182/blood-2013-10-525873. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Lee E.S., Lim J.-Y., Im K.-I., Kim N., Nam Y.-S., Jeon Y.-W., Cho S.-G. Adoptive transfer of Treg cells combined with mesenchymal stem cells facilitates repopulation of endogenous Treg cells in a murine acute GVHD model. PLoS ONE. 2015;10:e0138846. doi: 10.1371/journal.pone.0138846. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Komanduri K.V., Champlin R.E. Can Treg therapy prevent GVHD? Blood. 2011;117:751–752. doi: 10.1182/blood-2010-11-317305. [DOI] [PubMed] [Google Scholar]
12.Lu S.Y., Huang X.J., Liu K.Y., Liu D.H., Xu L.P. High frequency of CD4+ CD25− CD69+ T cells is correlated with a low risk of acute graft-versus-host disease in allotransplants. Clin. Transplant. 2012;26:E158–E167. doi: 10.1111/j.1399-0012.2012.01630.x. [DOI] [PubMed] [Google Scholar]
13.Pankratz S., Bittner S., Herrmann A.M., Schuhmann M.K., Ruck T., Meuth S.G., Wiendl H. Human CD4+ HLA-G+ regulatory T cells are potent suppressors of graft-versus-host disease in vivo. FASEB J. 2014;28:3435–3445. doi: 10.1096/fj.14-251074. [DOI] [PubMed] [Google Scholar]
14.Shevach E.M. Mechanisms of Foxp3+ T regulatory cell-mediated suppression. Immunity. 2009;30:636–645. doi: 10.1016/j.immuni.2009.04.010. [DOI] [PubMed] [Google Scholar]
15.Nguyen V.H., Zeiser R., Dasilva D.L., Chang D.S., Beilhack A., Contag C.H., Negrin R.S. In vivo dynamics of regulatory T-cell trafficking and survival predict effective strategies to control graft-versus-host disease following allogeneic transplantation. Blood. 2007;109:2649–2656. doi: 10.1182/blood-2006-08-044529. [DOI] [PubMed] [Google Scholar]
16.Isakov N., Altman A. Protein kinase Cθ in T cell activation. Annu. Rev. Immunol. 2002;20:761–794. doi: 10.1146/annurev.immunol.20.100301.064807. [DOI] [PubMed] [Google Scholar]
17.Gupta S., Manicassamy S., Vasu C., Kumar A., Shang W., Sun Z. Differential requirement of PKC-θ in the development and function of natural regulatory T cells. Mol. Immunol. 2008;46:213–224. doi: 10.1016/j.molimm.2008.08.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Sun Z., Arendt C.W., Ellmeier W., Schaeffer E.M., Sunshine M.J., Gandhi L., Annes J., Petrzilka D., Kupfer A., Schwartzberg P.L., Littman D.R. PKC-θ is required for TCR-induced NF-κB activation in mature but not immature T lymphocytes. Nature. 2000;404:402–407. doi: 10.1038/35006090. [DOI] [PubMed] [Google Scholar]
19.Isakov N., Altman A. PKC-theta-mediated signal delivery from the TCR/CD28 surface receptors. Front. Immunol. 2012;3:273. doi: 10.3389/fimmu.2012.00273. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Sumoza-Toledo A., Eaton A.D., Sarukhan A. Regulatory T cells inhibit protein kinase Cθ recruitment to the immune synapse of naive T cells with the same antigen specificity. J. Immunol. 2006;176:5779–5787. doi: 10.4049/jimmunol.176.10.5779. [DOI] [PubMed] [Google Scholar]
21.Zanin-Zhorov A., Ding Y., Kumari S., Attur M., Hippen K.L., Brown M., Blazar B.R., Abramson S.B., Lafaille J.J., Dustin M.L. Protein kinase C-θ mediates negative feedback on regulatory T cell function. Science. 2010;328:372–376. doi: 10.1126/science.1186068. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Zhang E.Y., Kong K.F., Altman A. The yin and yang of protein kinase C-theta (PKCθ): a novel drug target for selective immunosuppression. Adv. Pharmacol. 2013;66:267–312. doi: 10.1016/B978-0-12-404717-4.00006-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Roybal K.T., Wülfing C. Inhibiting the inhibitor of the inhibitor: blocking PKC-θ to enhance regulatory T cell function. Sci. Signal. 2010;3:pe24. doi: 10.1126/scisignal.3132pe24. [DOI] [PubMed] [Google Scholar]
24.Boschelli D.H. Small molecule inhibitors of PKCθ as potential antiinflammatory therapeutics. Curr. Top. Med. Chem. 2009;9:640–654. doi: 10.2174/156802609789007372. [DOI] [PubMed] [Google Scholar]
25.Sun Z. Intervention of PKC-θ as an immunosuppressive regimen. Front. Immunol. 2012;3:225. doi: 10.3389/fimmu.2012.00225. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Isakov N. PKCθ is a key regulator of T-cell behavior and a drug target for T cell- mediated diseases. J. Clin. Cell. Immunol. 2012;S12:008. [Google Scholar]
27.Valenzuela J.O., Iclozan C., Hossain M.S., Prlic M., Hopewell E., Bronk C.C., Wang J., Celis E., Engelman R.W., Blazar B.R. PKCθ is required for alloreactivity and GVHD but not for immune responses toward leukemia and infection in mice. J. Clin. Invest. 2009;119:3774–3786. doi: 10.1172/JCI39692. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Mochly-Rosen D., Das K., Grimes K.V. Protein kinase C, an elusive therapeutic target? Nat. Rev. Drug Discov. 2012;11:937–957. doi: 10.1038/nrd3871. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Ozay E.I., Gonzalez-Perez G., Torres J.A., Vijayaraghavan J., Lawlor R., Sherman H.L., Garrigan D.T., Jr., Burnside A.S., Osborne B.A., Tew G.N., Minter L.M. Intracellular delivery of Anti-pPKCθ (Thr538) via protein transduction domain mimics for immunomodulation. Mol. Ther. 2016;24:2118–2130. doi: 10.1038/mt.2016.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Joffre O., Gorsse N., Romagnoli P., Hudrisier D., van Meerwijk J.P.M. Induction of antigen-specific tolerance to bone marrow allografts with CD4+CD25+ T lymphocytes. Blood. 2004;103:4216–4221. doi: 10.1182/blood-2004-01-0005. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Taylor P.A., Panoskaltsis-Mortari A., Swedin J.M., Lucas P.J., Gress R.E., Levine B.L., June C.H., Serody J.S., Blazar B.R. L-Selectinhi but not the L-selectinlo CD4+25+ T-regulatory cells are potent inhibitors of GVHD and BM graft rejection. Blood. 2004;104:3804–3812. doi: 10.1182/blood-2004-05-1850. [DOI] [PubMed] [Google Scholar]
32.Trzonkowski P., Bieniaszewska M., Juścińska J., Dobyszuk A., Krzystyniak A., Marek N., Myśliwska J., Hellmann A. First-in-man clinical results of the treatment of patients with graft versus host disease with human ex vivo expanded CD4+CD25+CD127− T regulatory cells. Clin. Immunol. 2009;133:22–26. doi: 10.1016/j.clim.2009.06.001. [DOI] [PubMed] [Google Scholar]
33.Chuang H.C., Lan J.L., Chen D.Y., Yang C.Y., Chen Y.M., Li J.P., Huang C.Y., Liu P.E., Wang X., Tan T.H. The kinase GLK controls autoimmunity and NF-κB signaling by activating the kinase PKC-θ in T cells. Nat. Immunol. 2011;12:1113–1118. doi: 10.1038/ni.2121. [DOI] [PubMed] [Google Scholar]
34.Du X., Shi H., Li J., Dong Y., Liang J., Ye J., Kong S., Zhang S., Zhong T., Yuan Z. Mst1/Mst2 regulate development and function of regulatory T cells through modulation of Foxo1/Foxo3 stability in autoimmune disease. J. Immunol. 2014;192:1525–1535. doi: 10.4049/jimmunol.1301060. [DOI] [PubMed] [Google Scholar]
35.Sutcliffe E.L., Bunting K.L., He Y.Q., Li J., Phetsouphanh C., Seddiki N., Zafar A., Hindmarsh E.J., Parish C.R., Kelleher A.D. Chromatin-associated protein kinase C-θ regulates an inducible gene expression program and microRNAs in human T lymphocytes. Mol. Cell. 2011;41:704–719. doi: 10.1016/j.molcel.2011.02.030. [DOI] [PubMed] [Google Scholar]
36.Thaventhiran T., Sethu S., Yeang H.X.A., Al-Huseini L., Hamdam J., Sathish J.G. T cell co-inhibitory receptors-functions and signalling mechanisms. J. Clin. Cell. Immunol. 2013;S12:004. [Google Scholar]
37.Wang X.B., Zheng C.Y., Giscombe R., Lefvert A.K. Regulation of surface and intracellular expression of CTLA-4 on human peripheral T cells. Scand. J. Immunol. 2001;54:453–458. doi: 10.1046/j.1365-3083.2001.00985.x. [DOI] [PubMed] [Google Scholar]
38.Woo S.R., Li N., Bruno T.C., Forbes K., Brown S., Workman C., Drake C.G., Vignali D.A. Differential subcellular localization of the regulatory T-cell protein LAG-3 and the coreceptor CD4. Eur. J. Immunol. 2010;40:1768–1777. doi: 10.1002/eji.200939874. [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Raimondi G., Shufesky W.J., Tokita D., Morelli A.E., Thomson A.W. Regulated compartmentalization of programmed cell death-1 discriminates CD4+CD25+ resting regulatory T cells from activated T cells. J. Immunol. 2006;176:2808–2816. doi: 10.4049/jimmunol.176.5.2808. [DOI] [PubMed] [Google Scholar]
40.Koenecke C., Lee C.-W., Thamm K., Föhse L., Schafferus M., Mittrücker H.-W., Floess S., Huehn J., Ganser A., Förster R., Prinz I. IFN-γ production by allogeneic Foxp3+ regulatory T cells is essential for preventing experimental graft-versus-host disease. J. Immunol. 2012;189:2890–2896. doi: 10.4049/jimmunol.1200413. [DOI] [PubMed] [Google Scholar]
41.Sawitzki B., Kingsley C.I., Oliveira V., Karim M., Herber M., Wood K.J. IFN-γ production by alloantigen-reactive regulatory T cells is important for their regulatory function in vivo. J. Exp. Med. 2005;201:1925–1935. doi: 10.1084/jem.20050419. [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Francisco L.M., Sage P.T., Sharpe A.H. The PD-1 pathway in tolerance and autoimmunity. Immunol. Rev. 2010;236:219–242. doi: 10.1111/j.1600-065X.2010.00923.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Magg T., Mannert J., Ellwart J.W., Schmid I., Albert M.H. Subcellular localization of FOXP3 in human regulatory and nonregulatory T cells. Eur. J. Immunol. 2012;42:1627–1638. doi: 10.1002/eji.201141838. [DOI] [PubMed] [Google Scholar]
44.Hahn S.A., Bellinghausen I., Trinschek B., Becker C. Translating Treg therapy in humanized mice. Front. Immunol. 2015;6:623. doi: 10.3389/fimmu.2015.00623. [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Fang Z., Hua Z., Changying L., Jianmin W., Chengjuan L., Kangli X., Jing C. High level of CD4+CD25+CD127− Treg cells in donor graft is associated with a low risk of aGVHD after allo-HSCT for children with hematologic malignancies. J. Cell Sci. Ther. 2013;4:148. [Google Scholar]
46.Beres A.J., Drobyski W.R. The role of regulatory T cells in the biology of graft versus host disease. Front. Immunol. 2013;4:163. doi: 10.3389/fimmu.2013.00163. [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Kinter A.L., Godbout E.J., McNally J.P., Sereti I., Roby G.A., O’Shea M.A., Fauci A.S. The common γ-chain cytokines IL-2, IL-7, IL-15, and IL-21 induce the expression of programmed death-1 and its ligands. J. Immunol. 2008;181:6738–6746. doi: 10.4049/jimmunol.181.10.6738. [DOI] [PubMed] [Google Scholar]
48.Hogan P.G., Chen L., Nardone J., Rao A. Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev. 2003;17:2205–2232. doi: 10.1101/gad.1102703. [DOI] [PubMed] [Google Scholar]
49.Kiani A., García-Cózar F.J., Habermann I., Laforsch S., Aebischer T., Ehninger G., Rao A. Regulation of interferon-γ gene expression by nuclear factor of activated T cells. Blood. 2001;98:1480–1488. doi: 10.1182/blood.v98.5.1480. [DOI] [PubMed] [Google Scholar]
50.Vaeth M., Schliesser U., Müller G., Reissig S., Satoh K., Tuettenberg A., Jonuleit H., Waisman A., Müller M.R., Serfling E. Dependence on nuclear factor of activated T-cells (NFAT) levels discriminates conventional T cells from Foxp3+ regulatory T cells. Proc. Natl. Acad. Sci. USA. 2012;109:16258–16263. doi: 10.1073/pnas.1203870109. [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Bally A.P.R., Austin J.W., Boss J.M. Genetic and epigenetic regulation of PD-1 expression. J. Immunol. 2016;196:2431–2437. doi: 10.4049/jimmunol.1502643. [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Fishbach M.A., Bluestone J.A., Lim W.A. Cell-based therapeutics: the next pillar of medicine. Sci. Transl. Med. 2013;5:179ps7. doi: 10.1126/scitranslmed.3005568. [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Taylor P.A., Lees C.J., Blazar B.R. The infusion of ex vivo activated and expanded CD4+CD25+ immune regulatory cells inhibits graft-versus-host disease lethality. Blood. 2002;99:3493–3499. doi: 10.1182/blood.v99.10.3493. [DOI] [PubMed] [Google Scholar]
54.Hoffmann P., Ermann J., Edinger M., Fathman C.G., Strober S. Donor-type CD4+CD25+ regulatory T cells suppress lethal acute graft-versus-host disease after allogeneic bone marrow transplantation. J. Exp. Med. 2002;196:389–399. doi: 10.1084/jem.20020399. [DOI] [PMC free article] [PubMed] [Google Scholar]
55.Misra N., Bayry J., Lacroix-Desmazes S., Kazatchkine M.D., Kaveri S.V. Cutting edge: human CD4+CD25+ T cells restrain the maturation and antigen-presenting function of dendritic cells. J. Immunol. 2004;172:4676–4680. doi: 10.4049/jimmunol.172.8.4676. [DOI] [PubMed] [Google Scholar]
56.Taams L.S., van Amelsfort J.M., Tiemessen M.M., Jacobs K.M., de Jong E.C., Akbar A.N., Bijlsma J.W., Lafeber F.P. Modulation of monocyte/macrophage function by human CD4+CD25+ regulatory T cells. Hum. Immunol. 2005;66:222–230. doi: 10.1016/j.humimm.2004.12.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Venet F., Pachot A., Debard A.L., Bohe J., Bienvenu J., Lepape A., Powell W.S., Monneret G. Human CD4+CD25+ regulatory T lymphocytes inhibit lipopolysaccharide-induced monocyte survival through a Fas/Fas ligand-dependent mechanism. J. Immunol. 2006;177:6540–6547. doi: 10.4049/jimmunol.177.9.6540. [DOI] [PubMed] [Google Scholar]
58.Lewkowicz P., Lewkowicz N., Sasiak A., Tchórzewski H. Lipopolysaccharide-activated CD4+CD25+ T regulatory cells inhibit neutrophil function and promote their apoptosis and death. J. Immunol. 2006;177:7155–7163. doi: 10.4049/jimmunol.177.10.7155. [DOI] [PubMed] [Google Scholar]
59.Lu L., Lan Q., Li Z., Zhou X., Gu J., Li Q., Wang J., Chen M., Liu Y., Shen Y. Critical role of all-trans retinoic acid in stabilizing human natural regulatory T cells under inflammatory conditions. Proc. Natl. Acad. Sci. USA. 2014;111:E3432–E3440. doi: 10.1073/pnas.1408780111. [DOI] [PMC free article] [PubMed] [Google Scholar]
60.Schmidt A., Éliás S., Joshi R.N., Tegnér J. In vitro differentiation of human CD4+FOXP3+ induced regulatory T cells (iTregs) from naïve CD4+ T cells using a TGF-β-containing protocol. J. Vis. Exp. 2016;30:55015. doi: 10.3791/55015. [DOI] [PMC free article] [PubMed] [Google Scholar]
61.Li X., Liang Y., LeBlanc M., Benner C., Zheng Y. Function of a Foxp3 cis-element in protecting regulatory T cell identity. Cell. 2014;158:734–748. doi: 10.1016/j.cell.2014.07.030. [DOI] [PMC free article] [PubMed] [Google Scholar]
62.Booth N.J., McQuaid A.J., Sobande T., Kissane S., Agius E., Jackson S.E., Salmon M., Falciani F., Yong K., Rustin M.H. Different proliferative potential and migratory characteristics of human CD4+ regulatory T cells that express either CD45RA or CD45RO. J. Immunol. 2010;184:4317–4326. doi: 10.4049/jimmunol.0903781. [DOI] [PubMed] [Google Scholar]
63.Bi K., Tanaka Y., Coudronniere N., Sugie K., Hong S., van Stipdonk M.J., Altman A. Antigen-induced translocation of PKC-theta to membrane rafts is required for T cell activation. Nat. Immunol. 2001;2:556–563. doi: 10.1038/88765. [DOI] [PubMed] [Google Scholar]
64.Chand S., Mehta N., Bahia M.S., Dixit A., Silakari O. Protein kinase C-θ inhibitors: a novel therapy for inflammatory disorders. Curr. Pharm. Des. 2012;18:4725–4746. doi: 10.2174/138161212802651625. [DOI] [PubMed] [Google Scholar]
65.Yokosuka T., Takamatsu M., Kobayashi-Imanishi W., Hashimoto-Tane A., Azuma M., Saito T. Programmed cell death 1 forms negative costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2. J. Exp. Med. 2012;209:1201–1217. doi: 10.1084/jem.20112741. [DOI] [PMC free article] [PubMed] [Google Scholar]
66.Sheppard K.A., Fitz L.J., Lee J.M., Benander C., George J.A., Wooters J., Qiu Y., Jussif J.M., Carter L.L., Wood C.R., Chaudhary D. PD-1 inhibits T-cell receptor induced phosphorylation of the ZAP70/CD3ζ signalosome and downstream signaling to PKCθ. FEBS Lett. 2004;574:37–41. doi: 10.1016/j.febslet.2004.07.083. [DOI] [PubMed] [Google Scholar]
67.Gianchecchi E., Fierabracci A. Inhibitory receptors and pathways of lymphocytes: The role of PD-1 in Treg development and their involvement in autoimmunity onset and cancer progression. Front. Immunol. 2018;9:2374. doi: 10.3389/fimmu.2018.02374. [DOI] [PMC free article] [PubMed] [Google Scholar]
68.Wang Z., Hong J., Sun W., Xu G., Li N., Chen X., Liu A., Xu L., Sun B., Zhang J.Z. Role of IFN-γ in induction of Foxp3 and conversion of CD4+ CD25− T cells to CD4+ Tregs. J. Clin. Invest. 2006;116:2434–2441. doi: 10.1172/JCI25826. [DOI] [PMC free article] [PubMed] [Google Scholar]
69.Corthay A. A three-cell model for activation of naïve T helper cells. Scand. J. Immunol. 2006;64:93–96. doi: 10.1111/j.1365-3083.2006.01782.x. [DOI] [PubMed] [Google Scholar]
70.Wood K.J., Sawitzki B. Interferon γ: a crucial role in the function of induced regulatory T cells in vivo. Trends Immunol. 2006;27:183–187. doi: 10.1016/j.it.2006.02.008. [DOI] [PubMed] [Google Scholar]
71.Lu Y., Waller E.K. Dichotomous role of interferon-γ in allogeneic bone marrow transplant. Biol. Blood Marrow Transplant. 2009;15:1347–1353. doi: 10.1016/j.bbmt.2009.07.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
72.Nishibori T., Tanabe Y., Su L., David M. Impaired development of CD4+ CD25+ regulatory T cells in the absence of STAT1: increased susceptibility to autoimmune disease. J. Exp. Med. 2004;199:25–34. doi: 10.1084/jem.20020509. [DOI] [PMC free article] [PubMed] [Google Scholar]
73.Yang D., Zhao X., Lin X. Bcl10 is required for the development of suppressive function of Foxp3+ regulatory T cells. Cell. Mol. Immunol. 2019 doi: 10.1038/s41423-019-0297-y. Published online October 8, 2019, 1038/s41423-019-0297-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
74.Di Pilato M., Kim E.Y., Cadilha B.L., Prüßmann J.N., Nasrallah M.N., Seruggia D., Usmani S.M., Misale S., Zappulli V., Carrizosa E. Targeting the CBM complex causes Treg cells to prime tumours for immune checkpoint therapy. Nature. 2019;570:112–116. doi: 10.1038/s41586-019-1215-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
75.Shin H.M., Tilahun M.E., Cho O.H., Chandiran K., Kuksin C.A., Keerthivasan S., Fauq A.H., Miele L., Thome M., Osborne B.A., Minter L.M. NOTCH1 can initiate NF-κB activation via cytosolic interactions with components of the T cell signalosome. Front. Immunol. 2014;26:249. doi: 10.3389/fimmu.2014.00249. [DOI] [PMC free article] [PubMed] [Google Scholar]
76.Hogan P.G. Calcium-NFAT transcriptional signalling in T cell activation and T cell exhaustion. Cell Calcium. 2017;63:66–69. doi: 10.1016/j.ceca.2017.01.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
77.Shen T., Cseresnyés Z., Liu Y., Randall W.R., Schneider M.F. Regulation of the nuclear export of the transcription factor NFATc1 by protein kinases after slow fibre type electrical stimulation of adult mouse skeletal muscle fibres. J. Physiol. 2007;579:535–551. doi: 10.1113/jphysiol.2006.120048. [DOI] [PMC free article] [PubMed] [Google Scholar]
78.Goode N., Hughes K., Woodgett J.R., Parker P.J. Differential regulation of glycogen synthase kinase-3β by protein kinase C isotypes. J. Biol. Chem. 1992;267:16878–16882. [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S4 and Table 1

mmc1.pdf^{(872KB, pdf)}

Document S2. Article plus Supplemental Information

mmc2.pdf^{(6MB, pdf)}

[bib1] 1.Ohkura N., Kitagawa Y., Sakaguchi S. Development and maintenance of regulatory T cells. Immunity. 2013;38:414–423. doi: 10.1016/j.immuni.2013.03.002. [DOI] [PubMed] [Google Scholar]

[bib2] 2.Hori S., Nomura T., Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science. 2003;299:1057–1061. doi: 10.1126/science.1079490. [DOI] [PubMed] [Google Scholar]

[bib3] 3.Khattri R., Cox T., Yasayko S.A., Ramsdell F. An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat. Immunol. 2003;4:337–342. doi: 10.1038/ni909. [DOI] [PubMed] [Google Scholar]

[bib4] 4.Sakaguchi S., Miyara M., Costantino C.M., Hafler D.A. FOXP3+ regulatory T cells in the human immune system. Nat. Rev. Immunol. 2010;10:490–500. doi: 10.1038/nri2785. [DOI] [PubMed] [Google Scholar]

[bib5] 5.Brunkow M.E., Jeffery E.W., Hjerrild K.A., Paeper B., Clark L.B., Yasayko S.A., Wilkinson J.E., Galas D., Ziegler S.F., Ramsdell F. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat. Genet. 2001;27:68–73. doi: 10.1038/83784. [DOI] [PubMed] [Google Scholar]

[bib6] 6.Fontenot J.D., Rasmussen J.P., Williams L.M., Dooley J.L., Farr A.G., Rudensky A.Y. Regulatory T cell lineage specification by the forkhead transcription factor Foxp3. Immunity. 2005;22:329–341. doi: 10.1016/j.immuni.2005.01.016. [DOI] [PubMed] [Google Scholar]

[bib7] 7.Liu W., Putnam A.L., Xu-Yu Z., Szot G.L., Lee M.R., Zhu S., Gottlieb P.A., Kapranov P., Gingeras T.R., Fazekas de St Groth B. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J. Exp. Med. 2006;203:1701–1711. doi: 10.1084/jem.20060772. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib8] 8.Simonetta F., Chiali A., Cordier C., Urrutia A., Girault I., Bloquet S., Tanchot C., Bourgeois C. Increased CD127 expression on activated FOXP3+CD4+ regulatory T cells. Eur. J. Immunol. 2010;40:2528–2538. doi: 10.1002/eji.201040531. [DOI] [PubMed] [Google Scholar]

[bib9] 9.Ganguly S., Ross D.B., Panoskaltsis-Mortari A., Kanakry C.G., Blazar B.R., Levy R.B., Luznik L. Donor CD4+ Foxp3+ regulatory T cells are necessary for posttransplantation cyclophosphamide-mediated protection against GVHD in mice. Blood. 2014;124:2131–2141. doi: 10.1182/blood-2013-10-525873. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib10] 10.Lee E.S., Lim J.-Y., Im K.-I., Kim N., Nam Y.-S., Jeon Y.-W., Cho S.-G. Adoptive transfer of Treg cells combined with mesenchymal stem cells facilitates repopulation of endogenous Treg cells in a murine acute GVHD model. PLoS ONE. 2015;10:e0138846. doi: 10.1371/journal.pone.0138846. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib11] 11.Komanduri K.V., Champlin R.E. Can Treg therapy prevent GVHD? Blood. 2011;117:751–752. doi: 10.1182/blood-2010-11-317305. [DOI] [PubMed] [Google Scholar]

[bib12] 12.Lu S.Y., Huang X.J., Liu K.Y., Liu D.H., Xu L.P. High frequency of CD4+ CD25− CD69+ T cells is correlated with a low risk of acute graft-versus-host disease in allotransplants. Clin. Transplant. 2012;26:E158–E167. doi: 10.1111/j.1399-0012.2012.01630.x. [DOI] [PubMed] [Google Scholar]

[bib13] 13.Pankratz S., Bittner S., Herrmann A.M., Schuhmann M.K., Ruck T., Meuth S.G., Wiendl H. Human CD4+ HLA-G+ regulatory T cells are potent suppressors of graft-versus-host disease in vivo. FASEB J. 2014;28:3435–3445. doi: 10.1096/fj.14-251074. [DOI] [PubMed] [Google Scholar]

[bib14] 14.Shevach E.M. Mechanisms of Foxp3+ T regulatory cell-mediated suppression. Immunity. 2009;30:636–645. doi: 10.1016/j.immuni.2009.04.010. [DOI] [PubMed] [Google Scholar]

[bib15] 15.Nguyen V.H., Zeiser R., Dasilva D.L., Chang D.S., Beilhack A., Contag C.H., Negrin R.S. In vivo dynamics of regulatory T-cell trafficking and survival predict effective strategies to control graft-versus-host disease following allogeneic transplantation. Blood. 2007;109:2649–2656. doi: 10.1182/blood-2006-08-044529. [DOI] [PubMed] [Google Scholar]

[bib16] 16.Isakov N., Altman A. Protein kinase Cθ in T cell activation. Annu. Rev. Immunol. 2002;20:761–794. doi: 10.1146/annurev.immunol.20.100301.064807. [DOI] [PubMed] [Google Scholar]

[bib17] 17.Gupta S., Manicassamy S., Vasu C., Kumar A., Shang W., Sun Z. Differential requirement of PKC-θ in the development and function of natural regulatory T cells. Mol. Immunol. 2008;46:213–224. doi: 10.1016/j.molimm.2008.08.275. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib18] 18.Sun Z., Arendt C.W., Ellmeier W., Schaeffer E.M., Sunshine M.J., Gandhi L., Annes J., Petrzilka D., Kupfer A., Schwartzberg P.L., Littman D.R. PKC-θ is required for TCR-induced NF-κB activation in mature but not immature T lymphocytes. Nature. 2000;404:402–407. doi: 10.1038/35006090. [DOI] [PubMed] [Google Scholar]

[bib19] 19.Isakov N., Altman A. PKC-theta-mediated signal delivery from the TCR/CD28 surface receptors. Front. Immunol. 2012;3:273. doi: 10.3389/fimmu.2012.00273. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib20] 20.Sumoza-Toledo A., Eaton A.D., Sarukhan A. Regulatory T cells inhibit protein kinase Cθ recruitment to the immune synapse of naive T cells with the same antigen specificity. J. Immunol. 2006;176:5779–5787. doi: 10.4049/jimmunol.176.10.5779. [DOI] [PubMed] [Google Scholar]

[bib21] 21.Zanin-Zhorov A., Ding Y., Kumari S., Attur M., Hippen K.L., Brown M., Blazar B.R., Abramson S.B., Lafaille J.J., Dustin M.L. Protein kinase C-θ mediates negative feedback on regulatory T cell function. Science. 2010;328:372–376. doi: 10.1126/science.1186068. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib22] 22.Zhang E.Y., Kong K.F., Altman A. The yin and yang of protein kinase C-theta (PKCθ): a novel drug target for selective immunosuppression. Adv. Pharmacol. 2013;66:267–312. doi: 10.1016/B978-0-12-404717-4.00006-8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib23] 23.Roybal K.T., Wülfing C. Inhibiting the inhibitor of the inhibitor: blocking PKC-θ to enhance regulatory T cell function. Sci. Signal. 2010;3:pe24. doi: 10.1126/scisignal.3132pe24. [DOI] [PubMed] [Google Scholar]

[bib24] 24.Boschelli D.H. Small molecule inhibitors of PKCθ as potential antiinflammatory therapeutics. Curr. Top. Med. Chem. 2009;9:640–654. doi: 10.2174/156802609789007372. [DOI] [PubMed] [Google Scholar]

[bib25] 25.Sun Z. Intervention of PKC-θ as an immunosuppressive regimen. Front. Immunol. 2012;3:225. doi: 10.3389/fimmu.2012.00225. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib26] 26.Isakov N. PKCθ is a key regulator of T-cell behavior and a drug target for T cell- mediated diseases. J. Clin. Cell. Immunol. 2012;S12:008. [Google Scholar]

[bib27] 27.Valenzuela J.O., Iclozan C., Hossain M.S., Prlic M., Hopewell E., Bronk C.C., Wang J., Celis E., Engelman R.W., Blazar B.R. PKCθ is required for alloreactivity and GVHD but not for immune responses toward leukemia and infection in mice. J. Clin. Invest. 2009;119:3774–3786. doi: 10.1172/JCI39692. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib28] 28.Mochly-Rosen D., Das K., Grimes K.V. Protein kinase C, an elusive therapeutic target? Nat. Rev. Drug Discov. 2012;11:937–957. doi: 10.1038/nrd3871. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib29] 29.Ozay E.I., Gonzalez-Perez G., Torres J.A., Vijayaraghavan J., Lawlor R., Sherman H.L., Garrigan D.T., Jr., Burnside A.S., Osborne B.A., Tew G.N., Minter L.M. Intracellular delivery of Anti-pPKCθ (Thr538) via protein transduction domain mimics for immunomodulation. Mol. Ther. 2016;24:2118–2130. doi: 10.1038/mt.2016.177. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib30] 30.Joffre O., Gorsse N., Romagnoli P., Hudrisier D., van Meerwijk J.P.M. Induction of antigen-specific tolerance to bone marrow allografts with CD4+CD25+ T lymphocytes. Blood. 2004;103:4216–4221. doi: 10.1182/blood-2004-01-0005. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib31] 31.Taylor P.A., Panoskaltsis-Mortari A., Swedin J.M., Lucas P.J., Gress R.E., Levine B.L., June C.H., Serody J.S., Blazar B.R. L-Selectinhi but not the L-selectinlo CD4+25+ T-regulatory cells are potent inhibitors of GVHD and BM graft rejection. Blood. 2004;104:3804–3812. doi: 10.1182/blood-2004-05-1850. [DOI] [PubMed] [Google Scholar]

[bib32] 32.Trzonkowski P., Bieniaszewska M., Juścińska J., Dobyszuk A., Krzystyniak A., Marek N., Myśliwska J., Hellmann A. First-in-man clinical results of the treatment of patients with graft versus host disease with human ex vivo expanded CD4+CD25+CD127− T regulatory cells. Clin. Immunol. 2009;133:22–26. doi: 10.1016/j.clim.2009.06.001. [DOI] [PubMed] [Google Scholar]

[bib33] 33.Chuang H.C., Lan J.L., Chen D.Y., Yang C.Y., Chen Y.M., Li J.P., Huang C.Y., Liu P.E., Wang X., Tan T.H. The kinase GLK controls autoimmunity and NF-κB signaling by activating the kinase PKC-θ in T cells. Nat. Immunol. 2011;12:1113–1118. doi: 10.1038/ni.2121. [DOI] [PubMed] [Google Scholar]

[bib34] 34.Du X., Shi H., Li J., Dong Y., Liang J., Ye J., Kong S., Zhang S., Zhong T., Yuan Z. Mst1/Mst2 regulate development and function of regulatory T cells through modulation of Foxo1/Foxo3 stability in autoimmune disease. J. Immunol. 2014;192:1525–1535. doi: 10.4049/jimmunol.1301060. [DOI] [PubMed] [Google Scholar]

[bib35] 35.Sutcliffe E.L., Bunting K.L., He Y.Q., Li J., Phetsouphanh C., Seddiki N., Zafar A., Hindmarsh E.J., Parish C.R., Kelleher A.D. Chromatin-associated protein kinase C-θ regulates an inducible gene expression program and microRNAs in human T lymphocytes. Mol. Cell. 2011;41:704–719. doi: 10.1016/j.molcel.2011.02.030. [DOI] [PubMed] [Google Scholar]

[bib36] 36.Thaventhiran T., Sethu S., Yeang H.X.A., Al-Huseini L., Hamdam J., Sathish J.G. T cell co-inhibitory receptors-functions and signalling mechanisms. J. Clin. Cell. Immunol. 2013;S12:004. [Google Scholar]

[bib37] 37.Wang X.B., Zheng C.Y., Giscombe R., Lefvert A.K. Regulation of surface and intracellular expression of CTLA-4 on human peripheral T cells. Scand. J. Immunol. 2001;54:453–458. doi: 10.1046/j.1365-3083.2001.00985.x. [DOI] [PubMed] [Google Scholar]

[bib38] 38.Woo S.R., Li N., Bruno T.C., Forbes K., Brown S., Workman C., Drake C.G., Vignali D.A. Differential subcellular localization of the regulatory T-cell protein LAG-3 and the coreceptor CD4. Eur. J. Immunol. 2010;40:1768–1777. doi: 10.1002/eji.200939874. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib39] 39.Raimondi G., Shufesky W.J., Tokita D., Morelli A.E., Thomson A.W. Regulated compartmentalization of programmed cell death-1 discriminates CD4+CD25+ resting regulatory T cells from activated T cells. J. Immunol. 2006;176:2808–2816. doi: 10.4049/jimmunol.176.5.2808. [DOI] [PubMed] [Google Scholar]

[bib40] 40.Koenecke C., Lee C.-W., Thamm K., Föhse L., Schafferus M., Mittrücker H.-W., Floess S., Huehn J., Ganser A., Förster R., Prinz I. IFN-γ production by allogeneic Foxp3+ regulatory T cells is essential for preventing experimental graft-versus-host disease. J. Immunol. 2012;189:2890–2896. doi: 10.4049/jimmunol.1200413. [DOI] [PubMed] [Google Scholar]

[bib41] 41.Sawitzki B., Kingsley C.I., Oliveira V., Karim M., Herber M., Wood K.J. IFN-γ production by alloantigen-reactive regulatory T cells is important for their regulatory function in vivo. J. Exp. Med. 2005;201:1925–1935. doi: 10.1084/jem.20050419. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib42] 42.Francisco L.M., Sage P.T., Sharpe A.H. The PD-1 pathway in tolerance and autoimmunity. Immunol. Rev. 2010;236:219–242. doi: 10.1111/j.1600-065X.2010.00923.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib43] 43.Magg T., Mannert J., Ellwart J.W., Schmid I., Albert M.H. Subcellular localization of FOXP3 in human regulatory and nonregulatory T cells. Eur. J. Immunol. 2012;42:1627–1638. doi: 10.1002/eji.201141838. [DOI] [PubMed] [Google Scholar]

[bib44] 44.Hahn S.A., Bellinghausen I., Trinschek B., Becker C. Translating Treg therapy in humanized mice. Front. Immunol. 2015;6:623. doi: 10.3389/fimmu.2015.00623. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib45] 45.Fang Z., Hua Z., Changying L., Jianmin W., Chengjuan L., Kangli X., Jing C. High level of CD4+CD25+CD127− Treg cells in donor graft is associated with a low risk of aGVHD after allo-HSCT for children with hematologic malignancies. J. Cell Sci. Ther. 2013;4:148. [Google Scholar]

[bib46] 46.Beres A.J., Drobyski W.R. The role of regulatory T cells in the biology of graft versus host disease. Front. Immunol. 2013;4:163. doi: 10.3389/fimmu.2013.00163. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib47] 47.Kinter A.L., Godbout E.J., McNally J.P., Sereti I., Roby G.A., O’Shea M.A., Fauci A.S. The common γ-chain cytokines IL-2, IL-7, IL-15, and IL-21 induce the expression of programmed death-1 and its ligands. J. Immunol. 2008;181:6738–6746. doi: 10.4049/jimmunol.181.10.6738. [DOI] [PubMed] [Google Scholar]

[bib48] 48.Hogan P.G., Chen L., Nardone J., Rao A. Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev. 2003;17:2205–2232. doi: 10.1101/gad.1102703. [DOI] [PubMed] [Google Scholar]

[bib49] 49.Kiani A., García-Cózar F.J., Habermann I., Laforsch S., Aebischer T., Ehninger G., Rao A. Regulation of interferon-γ gene expression by nuclear factor of activated T cells. Blood. 2001;98:1480–1488. doi: 10.1182/blood.v98.5.1480. [DOI] [PubMed] [Google Scholar]

[bib50] 50.Vaeth M., Schliesser U., Müller G., Reissig S., Satoh K., Tuettenberg A., Jonuleit H., Waisman A., Müller M.R., Serfling E. Dependence on nuclear factor of activated T-cells (NFAT) levels discriminates conventional T cells from Foxp3+ regulatory T cells. Proc. Natl. Acad. Sci. USA. 2012;109:16258–16263. doi: 10.1073/pnas.1203870109. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib51] 51.Bally A.P.R., Austin J.W., Boss J.M. Genetic and epigenetic regulation of PD-1 expression. J. Immunol. 2016;196:2431–2437. doi: 10.4049/jimmunol.1502643. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib52] 52.Fishbach M.A., Bluestone J.A., Lim W.A. Cell-based therapeutics: the next pillar of medicine. Sci. Transl. Med. 2013;5:179ps7. doi: 10.1126/scitranslmed.3005568. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib53] 53.Taylor P.A., Lees C.J., Blazar B.R. The infusion of ex vivo activated and expanded CD4+CD25+ immune regulatory cells inhibits graft-versus-host disease lethality. Blood. 2002;99:3493–3499. doi: 10.1182/blood.v99.10.3493. [DOI] [PubMed] [Google Scholar]

[bib54] 54.Hoffmann P., Ermann J., Edinger M., Fathman C.G., Strober S. Donor-type CD4+CD25+ regulatory T cells suppress lethal acute graft-versus-host disease after allogeneic bone marrow transplantation. J. Exp. Med. 2002;196:389–399. doi: 10.1084/jem.20020399. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib55] 55.Misra N., Bayry J., Lacroix-Desmazes S., Kazatchkine M.D., Kaveri S.V. Cutting edge: human CD4+CD25+ T cells restrain the maturation and antigen-presenting function of dendritic cells. J. Immunol. 2004;172:4676–4680. doi: 10.4049/jimmunol.172.8.4676. [DOI] [PubMed] [Google Scholar]

[bib56] 56.Taams L.S., van Amelsfort J.M., Tiemessen M.M., Jacobs K.M., de Jong E.C., Akbar A.N., Bijlsma J.W., Lafeber F.P. Modulation of monocyte/macrophage function by human CD4+CD25+ regulatory T cells. Hum. Immunol. 2005;66:222–230. doi: 10.1016/j.humimm.2004.12.006. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib57] 57.Venet F., Pachot A., Debard A.L., Bohe J., Bienvenu J., Lepape A., Powell W.S., Monneret G. Human CD4+CD25+ regulatory T lymphocytes inhibit lipopolysaccharide-induced monocyte survival through a Fas/Fas ligand-dependent mechanism. J. Immunol. 2006;177:6540–6547. doi: 10.4049/jimmunol.177.9.6540. [DOI] [PubMed] [Google Scholar]

[bib58] 58.Lewkowicz P., Lewkowicz N., Sasiak A., Tchórzewski H. Lipopolysaccharide-activated CD4+CD25+ T regulatory cells inhibit neutrophil function and promote their apoptosis and death. J. Immunol. 2006;177:7155–7163. doi: 10.4049/jimmunol.177.10.7155. [DOI] [PubMed] [Google Scholar]

[bib59] 59.Lu L., Lan Q., Li Z., Zhou X., Gu J., Li Q., Wang J., Chen M., Liu Y., Shen Y. Critical role of all-trans retinoic acid in stabilizing human natural regulatory T cells under inflammatory conditions. Proc. Natl. Acad. Sci. USA. 2014;111:E3432–E3440. doi: 10.1073/pnas.1408780111. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib60] 60.Schmidt A., Éliás S., Joshi R.N., Tegnér J. In vitro differentiation of human CD4+FOXP3+ induced regulatory T cells (iTregs) from naïve CD4+ T cells using a TGF-β-containing protocol. J. Vis. Exp. 2016;30:55015. doi: 10.3791/55015. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib61] 61.Li X., Liang Y., LeBlanc M., Benner C., Zheng Y. Function of a Foxp3 cis-element in protecting regulatory T cell identity. Cell. 2014;158:734–748. doi: 10.1016/j.cell.2014.07.030. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib62] 62.Booth N.J., McQuaid A.J., Sobande T., Kissane S., Agius E., Jackson S.E., Salmon M., Falciani F., Yong K., Rustin M.H. Different proliferative potential and migratory characteristics of human CD4+ regulatory T cells that express either CD45RA or CD45RO. J. Immunol. 2010;184:4317–4326. doi: 10.4049/jimmunol.0903781. [DOI] [PubMed] [Google Scholar]

[bib63] 63.Bi K., Tanaka Y., Coudronniere N., Sugie K., Hong S., van Stipdonk M.J., Altman A. Antigen-induced translocation of PKC-theta to membrane rafts is required for T cell activation. Nat. Immunol. 2001;2:556–563. doi: 10.1038/88765. [DOI] [PubMed] [Google Scholar]

[bib64] 64.Chand S., Mehta N., Bahia M.S., Dixit A., Silakari O. Protein kinase C-θ inhibitors: a novel therapy for inflammatory disorders. Curr. Pharm. Des. 2012;18:4725–4746. doi: 10.2174/138161212802651625. [DOI] [PubMed] [Google Scholar]

[bib65] 65.Yokosuka T., Takamatsu M., Kobayashi-Imanishi W., Hashimoto-Tane A., Azuma M., Saito T. Programmed cell death 1 forms negative costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2. J. Exp. Med. 2012;209:1201–1217. doi: 10.1084/jem.20112741. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib66] 66.Sheppard K.A., Fitz L.J., Lee J.M., Benander C., George J.A., Wooters J., Qiu Y., Jussif J.M., Carter L.L., Wood C.R., Chaudhary D. PD-1 inhibits T-cell receptor induced phosphorylation of the ZAP70/CD3ζ signalosome and downstream signaling to PKCθ. FEBS Lett. 2004;574:37–41. doi: 10.1016/j.febslet.2004.07.083. [DOI] [PubMed] [Google Scholar]

[bib67] 67.Gianchecchi E., Fierabracci A. Inhibitory receptors and pathways of lymphocytes: The role of PD-1 in Treg development and their involvement in autoimmunity onset and cancer progression. Front. Immunol. 2018;9:2374. doi: 10.3389/fimmu.2018.02374. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib68] 68.Wang Z., Hong J., Sun W., Xu G., Li N., Chen X., Liu A., Xu L., Sun B., Zhang J.Z. Role of IFN-γ in induction of Foxp3 and conversion of CD4+ CD25− T cells to CD4+ Tregs. J. Clin. Invest. 2006;116:2434–2441. doi: 10.1172/JCI25826. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib69] 69.Corthay A. A three-cell model for activation of naïve T helper cells. Scand. J. Immunol. 2006;64:93–96. doi: 10.1111/j.1365-3083.2006.01782.x. [DOI] [PubMed] [Google Scholar]

[bib70] 70.Wood K.J., Sawitzki B. Interferon γ: a crucial role in the function of induced regulatory T cells in vivo. Trends Immunol. 2006;27:183–187. doi: 10.1016/j.it.2006.02.008. [DOI] [PubMed] [Google Scholar]

[bib71] 71.Lu Y., Waller E.K. Dichotomous role of interferon-γ in allogeneic bone marrow transplant. Biol. Blood Marrow Transplant. 2009;15:1347–1353. doi: 10.1016/j.bbmt.2009.07.015. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib72] 72.Nishibori T., Tanabe Y., Su L., David M. Impaired development of CD4+ CD25+ regulatory T cells in the absence of STAT1: increased susceptibility to autoimmune disease. J. Exp. Med. 2004;199:25–34. doi: 10.1084/jem.20020509. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib73] 73.Yang D., Zhao X., Lin X. Bcl10 is required for the development of suppressive function of Foxp3+ regulatory T cells. Cell. Mol. Immunol. 2019 doi: 10.1038/s41423-019-0297-y. Published online October 8, 2019, 1038/s41423-019-0297-y. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib74] 74.Di Pilato M., Kim E.Y., Cadilha B.L., Prüßmann J.N., Nasrallah M.N., Seruggia D., Usmani S.M., Misale S., Zappulli V., Carrizosa E. Targeting the CBM complex causes Treg cells to prime tumours for immune checkpoint therapy. Nature. 2019;570:112–116. doi: 10.1038/s41586-019-1215-2. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib75] 75.Shin H.M., Tilahun M.E., Cho O.H., Chandiran K., Kuksin C.A., Keerthivasan S., Fauq A.H., Miele L., Thome M., Osborne B.A., Minter L.M. NOTCH1 can initiate NF-κB activation via cytosolic interactions with components of the T cell signalosome. Front. Immunol. 2014;26:249. doi: 10.3389/fimmu.2014.00249. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib76] 76.Hogan P.G. Calcium-NFAT transcriptional signalling in T cell activation and T cell exhaustion. Cell Calcium. 2017;63:66–69. doi: 10.1016/j.ceca.2017.01.014. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib77] 77.Shen T., Cseresnyés Z., Liu Y., Randall W.R., Schneider M.F. Regulation of the nuclear export of the transcription factor NFATc1 by protein kinases after slow fibre type electrical stimulation of adult mouse skeletal muscle fibres. J. Physiol. 2007;579:535–551. doi: 10.1113/jphysiol.2006.120048. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib78] 78.Goode N., Hughes K., Woodgett J.R., Parker P.J. Differential regulation of glycogen synthase kinase-3β by protein kinase C isotypes. J. Biol. Chem. 1992;267:16878–16882. [PubMed] [Google Scholar]

PERMALINK

Cell-Penetrating Anti-Protein Kinase C Theta Antibodies Act Intracellularly to Generate Stable, Highly Suppressive Regulatory T Cells

E Ilker Ozay

Sudarvili Shanthalingam

Heather L Sherman

Joe A Torres

Barbara A Osborne

Gregory N Tew

Lisa M Minter

Abstract

Graphical Abstract

Introduction

Results

Intracellular Anti-pPKCθ Delivery Prevents Nuclear Accumulation of pPKCθ in iTregs

Figure 1.

Ex Vivo CPPM-Anti-pPKCθ Delivery Generates a Unique Population of CD4+CD25highFOXP3high iTregs That also Produces Interferon γ (IFNγ)

Figure 2.

Anti-pPKCθ-iTregs Exhibit Superior Suppressive Capabilities In Vitro

Figure 3.

Adoptively Transferring Anti-pPKCθ-iTregs Attenuates Disease and Prolongs Survival in a Humanized Mouse Model of GvHD

Figure 4.

Ex Vivo-Generated Anti-pPKCθ-iTregs Exhibit a Stable, Unique FOXP3highPD-1+IFNγhigh Phenotype In Vivo

Figure 5.

Ex Vivo-Generated Anti-pPKCθ-iTregs Show Increased PD-1 Co-localization with PKCθ and NFATc1

Figure 6.

NFATc1 and CnB Subcellular Localization in iTregs Is Associated with Signaling through IFNγ and PD-1

Figure 7.

Discussion

Materials and Methods

Animals

Antibodies and Reagents

Human iTreg Differentiation Coupled with Intracellular CPPM-Anti-pPKCθ Delivery

Immunoblotting

Protein Subcellular Localization Using AMNIS Imaging Flow Cytometry

In Vitro Suppression Assay

Surface versus Intracellular Expression of Co-inhibitory Receptors

Quantitative Real-Time PCR

In Vivo Suppression Analysis Using Adoptive Transfer of iTregs in a Humanized GvHD Model

GvHD Clinical Scoring

Magnetic Sorting of In Vivo iTregs for mRNA Analysis

Histology

LEGENDPlex Bead-Based Immunoassay

Statistical Analysis

Author Contributions

Conflicts of Interest

Acknowledgments

Footnotes

Supplemental Information

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases

Ex Vivo CPPM-Anti-pPKCθ Delivery Generates a Unique Population of CD4⁺CD25^highFOXP3^high iTregs That also Produces Interferon γ (IFNγ)

Ex Vivo-Generated Anti-pPKCθ-iTregs Exhibit a Stable, Unique FOXP3^highPD-1⁺IFNγ^high Phenotype In Vivo