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. Author manuscript; available in PMC: 2023 Mar 1.
Published in final edited form as: Am J Transplant. 2021 Nov 8;22(3):717–730. doi: 10.1111/ajt.16870

Dual JAK2/Aurora kinase A inhibition prevents human skin graft rejection by allo-inactivation and ILC2-mediated tissue repair

Kelly Walton 1, Kirsti Walker 2, Megan Riddle 2, Brent H Koehn 2, Jordan Reff 3, Elizabeth M Sagatys 3,4, Michael A Linden 5, Joseph Pidala 3,6,7, Jongphil Kim 8, Marie C Lee 6,9, John V Kiluk 6,9, Jane Yuet Ching Hui 10, Sang Y Yun 6,11, Yan Xing 2, Heather Stefanski 2, Harshani R Lawrence 5,11, Nicholas J Lawrence 5,11, Jakub Tolar 2, Claudio Anasetti 3,6,7, Bruce R Blazar 2, Said M Sebti 12, Brian C Betts 1
PMCID: PMC8897228  NIHMSID: NIHMS1749783  PMID: 34668635

Abstract

Prevention of allograft rejection often requires lifelong immune suppression, risking broad impairment of host immunity. Nonselective inhibition of host T cell function increases recipient risk of opportunistic infections and secondary malignancies. Here we demonstrate that AJI-100, a dual inhibitor of JAK2 and Aurora kinase A, ameliorates skin graft rejection by human T cells and provides durable allo-inactivation. AJI-100 significantly reduces the frequency of skin-homing CLA+ donor T cells, limiting allograft invasion and tissue destruction by T effectors. AJI-100 also suppresses pathogenic Th1 and Th17 cells in the spleen yet spares beneficial regulatory T cells (Tregs). We show dual JAK2/Aurora kinase A blockade enhances human type 2 innate lymphoid cell (ILC2) responses, which are capable of tissue repair. ILC2 differentiation mediated by GATA3 requires STAT5 phosphorylation (pSTAT5) but is opposed by STAT3. Further, we demonstrate that Aurora kinase A activation correlates with low pSTAT5 in ILC2s. Importantly, AJI-100 maintains pSTAT5 levels in ILC2s by blocking Aurora kinase A and reduces interference by STAT3. Therefore, combined JAK2/Aurora kinase A inhibition is an innovative strategy to merge immune suppression with tissue repair after transplantation.

Introduction

Rejection prophylaxis after solid organ transplantation is based on broadly suppressive calcineurin-inhibitors (CNI)1–3 or corticosteroids that fail to tolerize host responses, block T cell receptor (TCR) activity, and impair beneficial Tregs4–6. Lifelong immune suppression is typically required and places patients at risk of opportunistic infections, secondary malignancies, and graft loss due to insufficient control of host anti-donor immunity3,7–9. These challenges are of importance in vascular composite tissue allotransplantation (VCA), as the allograft contains large portions of antigenic skin1,2. VCA surgically replaces damaged body structures in their entirety; by using cadaveric hand, limb, ventral wall, and face grafts1,2. While VCA offers clear cosmetic and functional benefits, maintaining allotolerance is a challenge1,2. Thus, strategies to selectively impair host alloreactivity, without compromising normal T cell effector function or toleragenic Tregs is needed in VCA and solid organ transplantation.

We have shown that JAK2 inhibition alone, delays but does not eliminate skin graft rejection10. JAK2 blockade also reduces acute graft-versus-host disease (GVHD), a donor-mediated alloresponse after allogeneic hematopoietic cell transplantation (allo-HCT)10,11. JAK2 is required for receptor signal transduction of proinflammatory cytokines, including IL-6, IL-12, IL-23, and interferons10–15. Pan-JAK inhibitors that target JAK2 or JAK3 plus JAK1 are indeed immune suppressive16–18. However, by blocking JAK1 and related common gamma chain receptor cytokine signaling, key effector functions by cytotoxic T lymphocytes and Tregs are jeopardized10,11. This was demonstrated by the phase 2b trial of tofacitinib (JAK1/3 inhibitor) versus cyclosporine as rejection prophylaxis for renal allografts. While tofacitinib met the endpoint of noninferiority, patients receiving the JAK1/3 inhibitor experienced greater infectious complications compared to cyclosporine19. We have shown that JAK2 inhibition with pacritinib suppresses alloreactive T cells and spares anti-viral and anti-tumor cytotoxic T lymphocytes (CTL), natural killer (NK) cells, and Tregs10,11,20. Pacritinib inhibits JAK2 without suppressing JAK1 and downstream pSTAT5 in response to common gamma chain receptor cytokines, as required by CTLs, NK cells, and Tregs alike10.

We have demonstrated that combined JAK2 and Aurora kinase A blockade synergistically suppresses alloreactive human T cells13. The dual inhibitor of JAK2/Aurora kinase A, AJI-100, reduces xenogeneic GVHD yet spares Tregs and anti-tumor CTLs13. This is distinct from JAK2 or Aurora kinase A inhibition alone, which delays but does not prevent GVHD13. The combination targets T cell activation by impairing costimulation via Aurora kinase A (signal 2) and JAK2 cytokine signal transduction by IL-6 (signal 3)13. Yet, T cell receptor signaling (signal 1) is left unperturbed13. Thus, CTLs and Tregs receive pSTAT5 while pH3ser10 (target of Aurora kinase A) and pSTAT3 (target of JAK2) are inhibited, respectively13.

We now apply this strategy to our human skin xenograft model to investigate the effect of AJI-100 as rejection prophylaxis after tissue transplantation. Given that JAK2 or Aurora kinase A blockade alone are insufficient to fully control alloreactive human T cells10,13, we hypothesized that combined JAK2/Aurora kinase A inhibition would improve allograft survival. We now show AJI-100 significantly reduces skin graft rejection. Dual JAK2/Aurora kinase A blockade supports potent ILC2s that are involved in tolerance, tissue regeneration, and repair21–26. We found that Aurora kinase A activation correlates with low pSTAT5 in ILC2s. pSTAT5 regulates GATA3 expression and is required for ILC2 development27,28. Conversely, pSTAT3 can oppose pSTAT5 binding to GATA329,30. AJI-100 maintains adequate pSTAT5 levels in ILC2s by blocking Aurora kinase A and reduces interference by STAT3. Thus, AJI-100 offers the combined benefits of selective immune suppression and tissue repair after transplant.

Methods

Key resources and extended methods are provided as Supporting Information.

Experimental Model and Subject Details

Human PBMCs were acquired from consented, healthy donors at Memorial Blood Center (Minneapolis, Minnesota) and isolated immune cells were cultured at 37°C and 5% CO2 in complete RPMI media supplemented with 10% heat-inactivated pooled human serum (GeminiBio) plus 1% Penicillin/Streptomycin. Human skin was acquired from consented mastectomy patients, using nondiagnostic cutaneous tissue.

Mice

NOD-scid gamma-deficient (NSG) mice (female or male, ages 6–24 weeks old) were used. All mice were treated in adherence to the NIH Guide for the Care and Use of Laboratory Animals and the research protocol was approved by the Institutional Animal Care and Use Committee.

Xenogeneic human skin transplantation

NSG mice received a 1cm2 human skin graft from non-diagnostic mastectomy tissue as described (MCC 17634, an IRB-approved protocol)10,31. Skin graft rejection was induced 30 days later with an inoculum of 5×106 human PBMCs (allogeneic to the skin donor). Where indicated, the PBMCs were Treg-depleted using CD25 microbeads. Mice received AJI-100 (50mg/kg i.p. once daily) or vehicle13 for 14 days, starting on the day of PBMC injection. The grafts were monitored daily for signs of rejection. During select experiments, mice were humanely euthanized on day +21 and the recipient spleens and skin grafts were harvested for analysis. Skin rejection scoring was performed by a blinded, expert pathologist according to standard criteria32. Processed spleens cells were phenotyped by flow cytometry to identify human Th1, Th2, Th17, Treg, and ILC subsets. Skin grafts were prepared, stained (Treg: CD4+/Foxp3+, Th1: CD4+/T-bet+, Th2: CD4+/GATA3+, ILC2: CD4−, GATA3+), and imaged as previously described for IHC (Vista, CA, USA) analysis33.

ILC cultures and in vitro experiments

Human PBMCs were T cell depleted with CD3 microbeads to enrich for ILC2s and then cultured with IL-7 and IL-33 (both 10ng/ml) at 5×105 cells/1ml of complete RPMI media supplemented with 10% human, heat-inactivated, pooled serum in 24-well plates. As indicated, DMSO, TG101348 (350nM, ), alisertib (1.75μM), a combination of both inhibitors, or AJI-100 (750nM) were added once on day 013. The cells were incubated for 7 days, with cytokines replenished on days +3 and +5. The cells were then harvested for phenotypic analysis by flow cytometry (ILC1: lineage−, CD45+, CD127+, CD294−, CD117−; ILC2: lineage−, CD45+, CD127+, CD294+, CD161+; ILC3: lineage−, CD45+, CD127+, CD294−, CD117+)22,24,34. Where indicated, autologous human Tregs (isolated by magnetic bead separation) were cultured with ILC2 cultures (supplemented with IL-7 10ng/ml and IL-33 10 ng/ml) at a 1:1 ratio of Tregs to CD3-depleted PBMCs. Supernatants from ILC2 cultures were collected to quantify amphiregulin and IL-13 levels by ELISA. For phospho-p38 experiments, the ILC2s or Tregs were stimulated with IL-33 (10 ng/ml) for 25 minutes. For pSTAT3, pSTAT5, and pH3ser10 experiments, the ILC2-enriched cells were stimulated with IL2 plus IL-7 (10 ng/ml) for 25 minutes in the presence of AJI-100 or DMSO. Phosphoprotein staining was performed as described10,11.

Scratch Assay

To perform the scratch test assay, a monolayer of human fibroblasts (ATCC, 1×105) were grown in triplicate in flat-bottom 96-well plates for 24 hours. A uniform scratch (width = 750μm) was made using a 96-pin WoundMaker (Essen Bioscience). Supernatants from ILC2 cultures (100μl) were pipetted over the scratch after a single wash with PBS. The plate was incubated at 37°C for up to 4 days and imaged at 3-hour intervals using an IncuCyte ZOOM microscope (Essen Bioscience) to document wound closure.

Statistical Analysis

The log-rank test was used to analyze skin graft survival. The Mann-Whitney test was used for comparisons of independent data. Paired comparisons were determined with the paired t test. ANOVA was used for group comparisons, including a Sidak’s or Dunn’s post-test for correction of multiple comparisons. Values shown in bar graphs represent mean ± SEM. A two-sided P value <0.05 was considered statistically significant.

Results

The JAK2/Aurora kinase A inhibitor, AJI-100, significantly reduces skin graft rejection by allogeneic human T cells

Using an established xenogeneic model10,31, AJI-100 significantly reduced skin graft rejection by allogeneic, human T cells, compared to vehicle controls (Figure 1A, B). The dose of AJI-100 was based on the ability of 50mg/kg to suppress JAK2 and Aurora kinase A activity in human T cells from our prior studies in xenogeneic GVHD13. Skin grafts from AJI-100-treated mice also demonstrated significantly less microscopic tissue rejection at day +21, as evaluated by a blinded, expert pathologist (Figure 1C, D)32. In vitro studies support that the immune suppressive effect of AJI-100 is durable (Supplemental Figure 1A–D), as monocyte-derived dendritic cell (moDC)-stimulated T cells treated with AJI-100 during primary cultures exhibited allo-inactivation during rechallenge with fresh donor moDCs in the absence of AJI-100 (Supplemental Figure 1A,B). Moreover, the allo-inactivation mediated by AJI-100 was antigen specific, as AJI-100-treated T cells still responded to third party allogeneic moDCs (Supplemental Figure 1C). Lasting antigen specific allo-inactivation by AJI-100 was selective as conventional CD4+ and CD8+ T cells were impaired, but CD4+ Tregs retained proliferative responses toward alloantigen during fresh moDC restimulation (Supplemental Figure 1A,B). AJI-100 (750 nM) was nontoxic toward allo-stimulated T cells (Supplemental Figure 1E).

Figure 1: The JAK2/Aurora kinase A inhibitor, AJI-100, significantly reduces skin graft rejection by allogeneic human T cells.

Figure 1:

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NSG mice received a 1cm2 split thickness human skin graft, followed by 5×106 human PBMCs i.p. (allogeneic to the skin) 30 days later. AJI-100 50mg/kg or vehicle was given i.p. daily from days 0 to +14. (A) Graph shows AJI-100 ameliorates human skin graft rejection compared to vehicle controls, log-rank test. (B) Graph shows the mean % rejection ± SEM per graft for each experimental condition, Mann-Whitney. (C) Histologic representations of the skin grafts uniformly harvested on day +21 demonstrate that AJI-100 reduces rejection severity of the graft. * = lymphocytic infiltration. ^ = spongiosis. # = interface dermatitis. (D) Bar graph shows skin graft rejection scores at day +21 among vehicle- and AJI-100-treated mice, Mann-Whitney. Pooled data from n=3 experiments, up to 11 mice per experimental condition. *P<.05, **P=.001–.01, and ***P=.0001–.001.

AJI-100 reduces CD4+ T cell inflammation in human skin grafts

Given that AJI-100 improves allograft survival, we investigated the effect of dual JAK2/Aurora kinase A inhibition on tissue inflammation and graft-resident T cells. Mice were transplanted with human skin grafts, and recipients were euthanized 21 days after receiving the inoculum of allogeneic human PBMCs. Immunohistochemistry (IHC) evaluated skin graft inflammation and tissue-resident T cell populations. Skin inflammation manifests as basal epidermal keratinocyte proliferation, as measured by Ki-67 expression35. AJI-100 reduced keratinocyte hyperproliferation in the skin grafts, compared to vehicle-treated mice (Figure 2A). Moreover, human CD4+ T cell infiltrates in the skin grafts were significantly decreased by AJI-100 (Figure 2A, B). Th1 and Th2 cells are implicated in allograft rejection36. While the amount of tissue-resident Th1 cells (CD4+-red, T-bet+-brown) seemed reduced with AJI-100, the difference did not meet statistical significance (Figure 2A,C). However, human Th2 cells (CD4+-red, GATA3+-brown) in the skin grafts were significantly inhibited by AJI-100 (Figure 2A,D). Though human Tregs are known to suppress alloreactive T cells37,38, it is unknown if Tregs mediate immune suppression within target-organs or remotely. Here we observed that Tregs (CD4+-red, Foxp3+-brown) within the human skin grafts were significantly reduced among AJI-100-treated mice (Figure 2A,E). The reduction in skin-infiltrating CD8+ T cells by AJI-100 was not statistically significant (Supplemental Figure 2). Consistent with our prior report in GVHD13, AJI-100 significantly reduced the frequency of pathogenic Th1 and Th17 cells in recipient spleens yet spared human CD4+ Tregs, Th2 cells, and overall CD4+ T cell viability (Supplemental Figure 3A–H). AJI-100 significantly reduced the absolute numbers of spleen-resident Th1 cells, while the decrease in total Th17 cells was not statistically significant (Supplemental Figure 3I,J). Further, the absolute numbers of Tregs and Th2 cells in the spleen were similar among AJI-100 and vehicle treated mice (Supplemental Figure 3K,L). Cutaneous lymphocyte-associated antigen receptor (CLA) mediates skin-homing by responder T cells39,40. AJI-100 significantly reduced the amount of CLA+ CD4+ T cells, including Th1, Th2, and Treg subsets (Supplemental Figure 4A,B). Thus, we surmise that the reduction in tissue-infiltration by CD4+ T cell subsets is primarily mediated by reduced skin homing among AJI-100 exposed lymphocytes.

Figure 2: AJI-100 reduces CD4+ T cell inflammation in human skin grafts.

Figure 2:

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Human skin grafts were harvested from transplanted NSG mice 21 days after allogeneic PBMC (5×106) injection. Mice were treated with AJI-100 (50 mg/kg) or vehicle control from days 0 to +14. (A) Representative images show IHC staining for basal keratinocyte Ki-67 (purple line delineates dermal/epidermal junction), Th1 cells (CD4+-red, T-bet+-brown), Th2 cells (CD4+-red, GATA3+-brown), and Tregs (CD4+-red, Foxp3+-brown). Bar graphs show the mean ± SEM of (B) total # skin invasive CD4+ cells, (C) %CD4+, T-Bet+ Th1 cells, (D) %CD4+, GATA3+ Th2 cells, and (E) %CD4+, Foxp3+ Tregs at day +21, ANOVA. Pooled data from n=3 experiments, up to 11 mice per experimental condition. *P<.05. NS = not significant.

Combined inhibition of JAK2 and Aurora kinase A supports human ILC2s

ILC2s are involved in tissue repair and facilitate immune tolerance after transplantation23–26. To investigate the effect of JAK2 and/or Aurora kinase A inhibition on human ILC2s, PBMCs were first T cell depleted and the medium was supplemented with IL-7 and IL-33 to support ILC2 development. Alisertib (Aurora kinase A inhibitor, 1.75μM), TG101348 (JAK2 inhibitor, 350nM), a combination of TG101348 plus alisertib, AJI-100 (dual inhibitor, 750nM) or DMSO vehicle (<0.01%) was added once on day 0 of a 7-day culture. The concentrations of alisertib and TG101348 were based on our reported synergy experiments, and result in comparable suppression of pSTAT3 (target of JAK2) and histone 3 serine 10 (H3ser10, target of Aurora kinase A) as AJI-10013. ILC2s were identified phenotypically as lineage−, CD45+, CD127+, CD294+, CD161+22,34. While Aurora kinase A inhibition with alisertib did not enhance ILC2 development, JAK2 blockade with TG101348 increased the amount of human ILC2s in vitro (Figure 3A). Greater frequencies of ILC2s were achieved with combined JAK2/Aurora kinase A inhibition, with a robust effect produced by AJI-100 (Figure 3A). ILC1s are implicated in tissue rejection, while ILC3s are exceedingly rare but may support tolerance27,41. AJI-100 significantly increased the absolute numbers of ILC2s and reduced numbers of pathogenic ILC1s41,42 (lineage−, CD127+, T-bet+). ILC3s (lineage−, CD127+, RORγt+) were limited in quantity altogether, but further reduced by AJI-100 (Figure 3B–G). Though AJI-100 did not enhance the proliferative capacity of ILC2s compared to controls (Figure 3E), the frequency of Ki-67+ ILC1s and ILC3s were significantly decreased (Figure 3F,G).

Figure 3: Combined inhibition of JAK2 and Aurora kinase A supports human ILC2s.

Figure 3:

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Human PBMCs were T cell depleted to enrich for ILC2s and cultured with IL-7 and IL-33 for 7 days. DMSO, alisertib (Aurora kinase A inhibitor, 1.75μM), TG101348 (JAK2 inhibitor, 350nM), a combination of both inhibitors, or AJI-100 (JAK2/Aurora kinase A inhibitor, 750nM) were added once on day 0. The cells were then harvested and ILC2s were identified phenotypically as lineage−, CD45+, CD127+, CD294+, CD161+. (A) Representative contour plots from one of two independent experiments are shown. Graphs show the absolute numbers of B) ILC2s, C) ILC1s (CD127+, T-bet+), and D) ILC3s (CD127+, RORγ+) cells after treatment with AJI-100 or DMSO, paired t test. Bar graphs show the frequency of Ki-67+ E) ILC2s, F) ILC1s, and G) ILC3s after treatment with AJI-100 or DMSO for 7 days, paired t test. n=5–7 independent experiments. (H) Enriched ILC2s and cultured with IL-7 and IL-33 for 7 days. The cells were then harvested and serum-starved for 4 hours to quench protein-phosphorylation; in the presence of AJI-100 (750 nM) or DMSO. Bar graph shows the %pSTAT3+ (target of JAK2), %pSTAT5+ (required for ILC2 development), or %pH3ser10+ (target of Aurora kinase A), CD294+ ILC2s after a 25-minute stimulation with IL-2 plus IL-7 or anacardic acid (Aurora kinase A activator, 50μM), in the presence of AJI-100 or DMSO, ANOVA. Replicate means ± SEM from 3 independent experiments are shown. (I) Graph shows the Pearson correlation coefficient of pSTAT5 and pH3ser10 MFI in cytokine activated ILC2s normalized to unstimulated controls from 3 independent experiments. J,K) Bar graph and histogram show the MFI of GATA3 among polarized ILC2s treated with AJI-100 or DMSO, paired t test. *P<.05, **P=.001–.01, and ****P=<.0001. NS = not significant.

The significant reduction in pSTAT3+ and pH3ser10+ ILC2s by AJI-100 provides further evidence that concurrent JAK2/Aurora kinase A inhibition supports ILC2 responses (Figure 3H, Supplemental Figure 5). Conversely, anacardic acid, an activator of Aurora kinase A43, significantly increased pH3ser10+ ILC2s (Figure 3H) and this correlated with a reduction in pSTAT5 (Figure 3I, Supplemental Figure 5). Human ILC2s require pSTAT5 for GATA3-mediated development, and pSTAT3 is known to interfere with this differentiation cue28,29. Dual JAK2/Aurora kinase A blockade maintained robust expression of GATA3 in ILC2s (Figure 3J,K). We surmise AJI-100 supports human ILC2s by favoring a beneficial ratio of required pSTAT5 to inhibitory pSTAT3 rather than increased proliferation.

Dual JAK2/Aurora kinase A inhibition polarizes human ILC2 production of amphiregulin

ILC2s mediate tissue repair via amphiregulin, an epidermal growth factor-like molecule23,26. To test their capacity for tissue repair, supernatants from polarized ILC2s treated with AJI-100 or vehicle were added to human fibroblast sheets after sustaining a uniform, linear, mechanical scratch. Supernatants from AJI-100-treated cultures significantly hastened wound healing as demonstrated by more rapid return of tissue confluence and wound closure compared to vehicle controls (Figure 4A,B). The supernatants from AJI-100-treated ILC2 cultures contained significantly greater concentrations of amphiregulin and lacked pro-fibrotic IL-13 compared to DMSO-treated controls (Figure 4C,D). KLRG1 expression indicates the capacity of ILC2s to produce IL-1329. Consistent with this phenotypic impact on ILC2 function, we confirmed that AJI-100 reduced KLRG1 among treated ILC2s (Figure 4E,F). These data are clinically relevant as IL-13 is associated with tissue fibrosis29, a hallmark of chronic GVHD and allograft rejection; yet amphiregulin is beneficial in orchestrating tissue repair23,26. Thus, AJI-100 has the potential to polarize ILC2-mediated tissue regeneration over pathogenic tissue fibrosis and sclerosis.

Figure 4: Dual JAK2/Aurora kinase A inhibition polarizes human ILC2 production of amphiregulin.

Figure 4:

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Uniform cuts were made upon human fibroblast monolayers and then cultured with supernatants from AJI-100- or DMSO-treated ILC2 cultures. (A) Graph shows the relative wound healing density (replicate means + SEM) for monolayers cultured with AJI-100- or DMSO-treated supernatants, ANOVA. (B) Representative images at 51 hours depict the degree of wound closure for cut fibroblast monolayers cultured with AJI-100- or DMSO-supernatants. Dotted lines demark borders of the cut. One of two independent experiments is shown. Bar graph shows the amount of (C) amphiregulin and (D) IL-13 (replicate means ± SEM) measured in the supernatants of ILC2 cultures treated with AJI-100 or DMSO after 7 days, paired t test. One of two independent experiments is shown. E) Bar graph and F) representative contour plots show the %KLRG1+ ILC2s after 7 days of culture in IL-7 and IL-33-supplemented medium, treated with AJI-100 or DMSO, paired t test. *P<.05.

Dual JAK2/Aurora kinase A inhibition with AJI-100 increases the proportion of human ILC2 populations in vivo

To evaluate the effect of JAK2/Aurora kinase A inhibition on ILC2s in vivo, recipient NSG mice bearing human skin and allogeneic PBMCs were treated with AJI-100 (50mg/kg) or vehicle and euthanized on day +21. Within the spleen, mice treated with AJI-100 demonstrated a significant increase in the frequency of human ILC2s (lineage−, CD45+, CD127+, CD294+, CD161+) (Figure 5A). Pathogenic ILC1s (lineage−, CD45+, CD127+, CD294−, CD117−)24 were significantly reduced, while the decrease in ILC3s (lineage−, CD45+, CD127+, CD294−, CD117+)24 was not statistically significant (Figure 5B,C). There was a trend toward increased absolute numbers of human ILC2s in the spleens of mice treated with AJI-100, though it did not reach statistical significance (Figure 5D). Absolute numbers of ILC1s were significantly reduced by AJI-100, while the reduction of ILC3s was not statistically significant (Figure 5E,F). Overall, the proportion of beneficial ILC2s to ILC1s and ILC3s in the recipient spleen was significantly increased by AJI-100 (Figure 5G). The relative amount of human ILC2s (CD4−, GATA3+) (Figure 5H), but not amphiregulin (Supplemental Figure 6), in the skin grafts was significantly greater with AJI-100.

Figure 5: Dual JAK2/Aurora kinase A inhibition with AJI-100 favors human ILC2 populations in vivo.

Figure 5:

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NSG mice were transplanted with human skin grafts and allogeneic PBMCs (5×106) as described. Recipient mice were treated with AJI-100 (50 mg/kg) or vehicle from days 0 to +14. The host spleens and human skin grafts were harvested on day +21. Bar graphs show the percentage (A-C) and absolute numbers (D–F) of human ILC2s (lineage−, CD45+, CD127+, CD294+, CD161+), ILC1s (lineage−, CD45+, CD127+, CD294−, CD117−), and ILC3s (lineage−, CD45+, CD127+, CD294−, CD117+) in the recipient spleens, Mann-Whitney. ILCs are expressed as proportions of CD127+ cells. (G) Bar graph shows the ratio of ILC2s to ILC1s and ILC3s in the recipient spleen, Mann-Whitney. (H) Bar graph demonstrates the fold change in skin-resident ILC2s (CD4−, GATA3+) relative to no PBMC controls using the calculation: (GATA3+, CD4− cells/HPF vehicle versus AJI-100)/(GATA3+, CD4− cells/HPF untreated controls), Mann-Whitney. Pooled data from n=5 experiments, up to 17 mice per experimental condition. *P<.05, ***P=.0001–.001, ****P=<.0001. NS = not significant.

Human Tregs compete against ILC2s for IL-33

IL-33 is a key growth factor for ILC2s22 and supports Treg suppressive potency44. IL-33 receptor signaling requires MyD88, but not JAK2 or Aurora kinase A45. To investigate the relationship between human Tregs and ILC2s, polarized ILC2 were cultured with purified peripheral Tregs (pTreg) at a ratio of 1:1. The medium was supplemented with IL-7, with or without IL-33. AJI-100 or DMSO was added once on day 0 of culture. After 7 days, the cells were harvested and the amount of ILC2s were determined by flow cytometry. Like our prior experiments, AJI-100 with IL-33 significantly increased the proportion of human ILC2s (Figure 6A). The addition of human pTregs significantly curtailed ILC2 development despite the addition of IL-33 and AJI-100 (Figure 6A). IL-33 signals through the ST2 receptor via phospho-p38(34). We discovered that purified pTregs are highly responsive to IL-33, with significantly more pTregs expressing phospho-p38 after IL-33 stimulation compared to purified ILC2s (Figure 6B,C). When pTregs were co-cultured with ILC2s at a 1:1 ratio, pTregs maintained their competitive advantage over ILC2s, with significantly more pTregs expressing phospho-p38 in response to IL-33 (Figure 6B,C). This provides evidence that human pTregs are more efficient at IL-33 signal transduction, compared to ILC2s. We went on to confirm that significantly more human pTregs express ST2 compared to ILC2s, and that pTreg surface expression of ST2 was much denser (Figure 6D–F), supporting the capacity for enhanced IL-33 signal transduction by pTregs. Consistent with these findings, the relative amount of ILC2s in mice bearing ST2-deficient Tregs was significantly greater than mice lacking the ST2 receptor (Supplemental Figure 7). This provides direct evidence that Tregs have a competitive advantage over ILC2s in vivo, and this relationship is mediated by ST2 expression.

Figure 6: Human Tregs compete against ILC2s for IL-33.

Figure 6:

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A) Human PBMCs were T cell depleted to enrich for ILC2s and cultured with IL-7 plus AJI-100 (750nM) or DMSO for 7 days. The medium was supplemented with or without IL-33 (10 ng/ml), and autologous peripheral Tregs (isolated by magnetic bead purification) were added as indicated (CD3− PBMC to Treg ratio = 1:1). Bar graph shows the fold change in ILC2s (mean ± SEM, normalized to CD3-depleted) recovered among each condition after 7 days of culture. Replicate means from 3 independent experiments are shown, ANOVA. B, C) Bar graph and representative histograms show the percentage (mean ± SEM) of magnetic bead purified human ILC2s or Tregs expressing phospho-p38 at baseline or after stimulation with IL-33 (25 minutes) cultured separately or together (1:1 ratio), ANOVA. Replicate means from 3 independent experiments are shown. D–F) Bar graphs (mean ± SEM) and representative contour plots show the amount of ST2 expression among purified human ILC2s and peripheral Tregs, paired t test. Replicate means from 3 independent experiments are shown. **P=.001–.01, ***P=.0001–.001, ****P<.0001.

Treg-depletion plus AJI-100 improves the frequency of human ILC2 without limiting Treg induction in vivo

We went on to further characterize human Treg:ILC2 interactions in vivo. NSG mice bearing human skin grafts were transplanted with an inoculum of Treg-depleted (99.9% Treg-free)46, human PBMCs (allogeneic to the skin donor)10. The mice were then treated with AJI-100 or vehicle as described. At day +21, the mice were euthanized, and the skin grafts and spleens were harvested for analysis. Treg-depletion plus AJI-100 significantly increased the frequency of human ILC2s in the recipient spleens (Figure 7A). AJI-100 reduced ILC1s and ILC3s, though only significantly effecting the latter population of cells in these Treg-depleted PBMC experiments (Figure 7B,C). The ratio of ILC2s to ILC1s and ILC3s in the recipient spleen was robust and significantly increased by AJI-100 (Figure 7D). Despite Treg-depletion of the transplanted PBMC product, induced peripheral Tregs were significantly increased in vivo with AJI-100 (Figure 7E). Treg-depletion plus AJI-100 significantly prevented allograft rejection, where the degree of tissue damage was negligible compared to vehicle-controls (Figure 7F,G).

Figure 7: Treg-depletion plus AJI-100 improves the frequency of human ILC2 without limiting Treg induction in vivo.

Figure 7:

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NSG mice received a 1cm2 human skin graft, followed by 5×106 human, Treg-depleted PBMCs (allogeneic to the skin graft) 30 days later. AJI-100 50mg/kg or vehicle was given daily from days 0 to +14. Bar graphs show the percentage of human (A) ILC2s (lineage−, CD45+, CD127+, CD294+, CD161+), (B) ILC1s (lineage−, CD45+, CD127+, CD294−, CD117−), and (C) ILC3s (lineage−, CD45+, CD127+, CD294−, CD117+) at day +21 in the recipient spleens of AJI-100- and vehicle-treated mice, Mann-Whitney. ILCs are expressed as proportions of CD127+ cells. (D) Bar graph shows the proportion of ILC2s to ILC1s and ILC3s in the recipient spleen, Mann-Whitney. (E) Bar graph demonstrates the frequency of CD4+ Tregs in the recipient spleens at day +21, Mann-Whitney. (F) Bar graph shows skin graft rejection scores at day +21 among vehicle- and AJI-100-treated mice after Treg-depletion of the PBMCs, Mann-Whitney. (G) Representative H&E sections show the amount of skin rejection at day +21 among AJI-100- or vehicle-treated mice after receiving allogeneic, Treg-depleted PBMCs. * = lymphocytic infiltration. ^ = spongiosis. # = interface dermatitis, + = exocytosis. Pooled data from n=2 experiments, up to 8 mice per experimental condition. *P<.05 and **P=.001–.01. NS = not significant.

Discussion

Here we demonstrate that concurrent JAK2/Aurora kinase A inhibition prevents skin graft rejection and provides durable allo-inactivation. AJI-100 significantly reduced the amount of pathogenic Th2 cells36,47 in the human skin grafts, Th1 cells in the periphery, and curtailed T cell skin-homing39,40 by limiting CLA expression. We use a well-established human skin xenograft model10,35,48,49 that permits the study of human T cells in vivo. However, limitations of the model include impaired host adaptive and innate immunity, concurrent xeno- and alloantigen within the system, and potential alterations in human T cell homing and/or persistence. We have shown JAK2 or Aurora kinase A inhibitors delayed but did not eliminate xenogeneic GVHD13 or tissue rejection10 caused by human T cells. Alternatively, combined blockade of JAK2 and Aurora kinase A is synergistic in suppressing alloreactivity, and that our dual inhibitor, AJI-100, significantly prevented xenogeneic GVHD while maintaining donor anti-tumor immunity13. The threshold to achieve immune tolerance after solid organ transplantation or VCA is high, where many patients require lifelong immune suppression41. Therefore, it is clinically relevant that AJI-100 can improve the survival of highly antigenic human skin grafts.

We now show AJI-100 facilitates the in vitro expansion of potent human ILC2s that secrete significant amounts of amphiregulin implicated in tissue repair22,44,45. ILC2s orchestrate tissue regeneration in the skin, lung, and gastrointestinal tract23. Conversely, ILC2s also mediate pathogenic tissue fibrosis via IL-13, as observed in idiopathic pulmonary fibrosis and systemic sclerosis50,51. Concurrent JAK2/Aurora kinase A inhibition directs human ILC2s to produce beneficial amphiregulin but not IL-13. Tissue fibrosis is a hallmark of chronic GVHD and solid organ rejection, and may be amenable to AJI-10052. Amphiregulin was not increased in vivo by AJI-100. However, tissue injury, Tregs, amphiregulin, and keratinocyte proliferation were increased among vehicle-treated skin grafts. Tregs reflexively produce excess amphiregulin as a late countermeasure to inflammation, as observed in acute GVHD53–56. Excess amphiregulin can paradoxically induce keratinocyte hyperproliferation57. Thus, reduced allo-immune graft inflammation by AJI-100 is consistent with the relative increase in ILC2s, decreased Tregs, and intermediate amphiregulin in the skin grafts. ILC2s may suppress inflammation with mechanism beyond amphiregulin, like IL-953,58.

A key discovery is that human Tregs outcompete ILC2s for IL-33. IL-33 is required by ILC2s to mediate skin repair21,53. Compared to ILC2s, peripheral Tregs exhibit dense surface expression and activity of ST2, the IL-33 receptor. Though Tregs are a cornerstone of allotolerance, we show peripheral Tregs present early after transplant may oppose critical ILC2-mediated tissue repair. While Treg-depletion eliminates competition for IL-33, dual JAK2/Aurora kinase A blockade produces a high ratio of pSTAT5 to pSTAT3, which is required for ILC2 differentiation27,28. We have demonstrated that AJI-100 supports the differentiation of potent iTregs through a similar mechanism favoring pSTAT5 stabilization of Foxp313. AJI-100 facilitated robust Treg induction when pTregs were removed from the PBMCs to hasten ILC2 recovery. Moreover, mice treated with Treg-depletion plus AJI-100 exhibited negligible rejection. These experiments characterize an important and previously unrecognized relationship between Tregs and ILC2s. Improved understanding of Treg:ILC2 interactions could have implications in the design and use of Treg or ILC2 adoptive cellular therapy.

ILC2 deficiency is associated with an increased incidence of acute GVHD24. Recently, adoptive transfer of ILC2s was shown to reduce lethal GVHD in rodents26. ILC2s lacking amphiregulin lost this protective effect26. ILC2s also suppress alloreactive Th1 and Th17 cells26. Beyond GVHD, ILC2s prevent rejection of islet cell allografts in immunocompetent, C57BL/6 mice25. ILC2s are also implicated in routing T cell tolerance toward solid tumors via a PD-1 dependent mechanism59,60. Therefore, ILC2s are clearly involved in tolerance induction. Conversely, ILC1s are implicated in autoimmune colitis and multiple sclerosis, and contribute to tissue rejection via INFγ and TNFα42. Thus, enhancing the balance of ILC2s to ILC1s with AJI-100 is advantageous in the transplant setting. ILC3s are rare post-transplant but may support tolerance like ILC2s24,42. AJI-100 had little impact on ILC3s among hosts transplant with Treg-replete PBMCs.

While AJI-100 reduced skin graft rejection and preserved the frequency and absolute number of human Tregs in the spleen after transplantation with Treg-replete PBMCs, skin-resident Tregs were significantly decreased compared to vehicle controls. This is consistent with known Treg homing patterns that favor lymphoid structures61, and prior data showing that GVHD target-organ Treg content does not correlate with immune tolerance33,62. The reduced amount of Tregs in the skin grafts may also be due to earlier and/or more rapid trafficking kinetics of AJI-100 exposed Tregs. Alternatively, AJI-100 may dampen Treg homing to the skin by limiting CLA expression. However, AJI-100 enhances Treg potency13 and circulating Tregs can still mediate suppression remotely33,62. Our present work in skin graft rejection is consistent with our findings in GVHD prevention, where AJI-100 reduces alloreactivity, preserves Tregs, supports Treg induction, and suppresses key alloimmune effectors like Th1 cells13.

Dual JAK2/Aurora kinase A inhibition provides an innovative platform to promote durable immune tolerance, ILC2 reconstitution, and selective suppression of alloreactive T cells. Insights from this present work are expected to inform novel VCA and solid organ rejection prophylaxis strategies, and also impact GVHD prevention as well. Supporting ILC2 populations in vivo with AJI-100 could prove to be more economical and efficient than ex vivo expansion and adoptive transfer of ILC2s. Rejection prophylaxis concurrently targeting JAK2 and Aurora kinase A signal transduction merits further investigation.

Supplementary Material

supinfo

Acknowledgments:

The Flow Cytometry, USF Comparative Medicine and Vivarium, Analytic Microscopy, Biostatistics, and Tissue Cores at Moffitt/USF were utilized in completing this work. The core facilities are supported partially by the Moffitt Cancer Center Support Grant, P30-CA076292. The University of Minnesota Flow Cytometry Resource was also used to complete this work.

Funding:

This work was supported by W81XWH-15-RTR-IDA (to B.C.B.) and R01 HL133823 (to B.C.B.); R50 CA211447 (to H.R.L.); and R01 HL11879 (to B.R.B.); R01 HL56067 (to B.R.B.); R37 AI34495 (to B.R.B.), and LLS Translational Research Grant 6462-15 (to B.R.B.).

Abbreviations:

allo-HCT

allogeneic hematopoietic cell transplantation

CNI

calcineurin-inhibitors

CTL

cytotoxic T lymphocytes

GVHD

graft-versus-host disease

ILC2

human type 2 innate lymphoid cell

IHC

Immunohistochemistry

moDC

monocyte-derived dendritic cell

NK

natural killer

NSG

NOD-scid gamma-deficient

pTreg

peripheral Tregs

Tregs

regulatory T cells

pSTAT5

STAT5 phosphorylation

VCA

vascular composite tissue allotransplantation

Footnotes

Disclosure:

The authors of this manuscript have conflicts of interest to disclose as described by the American Journal of Transplantation. S.M.S., N.J.L., and H.R.L. have a patent (US9249124) associated with the synthesis and use of AJI-100. B.C.B., S.M.S., N.J.L., H.R.L., J.P., and C.A. hold a provisional patent regarding the use of AJI-100 in the prevention and treatment of GVHD. Neither the inventors nor their institutions have received payment related to claims described in the patent. The other authors have no conflicts of interest to disclose.

Supporting Information

Additional supporting information may be found online in the Supporting Information section at the end of the article.

Data Availability Statement:

Information and reagent requests may be directed to Brian C. Betts, MD (bett0121@umn.edu). Fulfillment of reagent requests will require a signed and executed material transfer agreement.

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Associated Data

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

Supplementary Materials

supinfo
NIHMS1749783-supplement-supinfo.pdf (1.1MB, pdf)

Data Availability Statement

Information and reagent requests may be directed to Brian C. Betts, MD (bett0121@umn.edu). Fulfillment of reagent requests will require a signed and executed material transfer agreement.

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