CD4 T cell depletion increases memory differentiation of endogenous and CAR T cells and enhances the efficacy of Super2 and IL-33-armored CAR T cells against solid tumors

Abstract

Background

Responsiveness to chimeric antigen receptor (CAR) T cell therapy correlates with CAR T cell expansion and persistence in vivo. Multiple strategies improve persistence by increasing stem-like properties or sustaining CAR T cell activity with combination therapies. Here, we describe the intrinsic ability of CAR T cells to differentiate into memory T cells, the effect of cytokine armoring, and neoadjuvant CD4 depletion therapy on CAR and tumor-specific endogenous memory T cells.

Methods

TRP1-specific or NKG2D CAR T cells alone or with Super2+IL-33 (S233) armoring and/or CD4 depletion were evaluated in immunocompetent B16F10 melanoma or MC38 colon cell carcinoma models without preconditioning. We characterized CAR and endogenous tumor-specific memory T cell precursors, establishment of circulating (T_CIRC) and resident (T_RM) memory T cell subsets, and ability to protect against secondary tumors.

Results

TRP1-specific or NKG2D CAR T cells had no effect on primary tumor growth in immunocompetent mice unless they were combined with S233 armoring or CD4 depletion. Unarmored CAR T cells expressed a stem-like phenotype in the tumor-draining lymph node and differentiated into CAR T_CIRC memory cells in lymphoid organs and CAR T_RM cells in the skin. In contrast, S233-armored CAR T cells exhibited an activated effector phenotype and differentiated inefficiently into CAR effector and central memory T cells. Combining CD4 therapy with unarmored CAR T cells increased CAR T_CIRC and T_RM memory T cells. Either CD4 depletion therapy or S233-armored CAR T cells induced activation of tumor-specific endogenous T cells that differentiated into both T_CIRC and T_RM memory T cells. CD4 depletion and S233-armored CAR T cell combination therapy synergized to increase endogenous memory T cells.

Conclusions

Unarmored TRP-1-specific or NKG2D CAR T cells have intrinsic stem-like properties and differentiate into memory T cell subsets but are non-protective against primary or secondary tumors. S233 cytokine armoring alone or with CD4 depletion improved effector responses but limited CAR memory T cell generation. S233-armored CAR T cells or CD4 depletion therapy induced endogenous tumor-specific T_CIRC and T_RM T cells, but the combination potentiated endogenous memory T cell generation and resulted in improved protection against B16F10 rechallenge.

WHAT IS ALREADY KNOWN ON THIS TOPIC

• Chimeric antigen receptor (CAR) T cell persistence is positively associated with improved clinical responses and durable remissions. Strategies to extend persistence include armoring CAR T cells with homeostatic cytokines but the effect of armored cytokines on the differentiation of CAR effector versus memory T cells and their impact on the primary and memory antitumor response remains unclear.

WHAT THIS STUDY ADDS

• This study uses the B16F10 and MC38 immunocompetent solid tumor models to show that TRP1-specific and NKG2D CAR T cells have an inherent potential to differentiate into CAR memory T cells that are potentiated by anti-CD4 depleting monoclonal antibody therapy. Yet, these CAR T cells exhibited limited effector activity and no efficacy against either primary or secondary tumor challenges. Armoring CAR T cells with interleukin (IL)-2 superkine and IL-33 endowed CAR T cells with high effector activity but limited their differentiation into CAR memory T cells; however, in combination with CD4 depletion, it induced the priming and differentiation of endogenous tumor-specific CD8 T cells into broadly distributed memory T cells and resulted in improved protection against secondary tumors.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

• Optimal CAR T cell engineering strategies should consider balancing CAR T cell effector function, induction of CAR memory T cell differentiation, and activation of endogenous T cells to produce immediate and durable tumor protection.

Introduction

Adoptively transferred chimeric antigen receptor (CAR) T cells are remarkably effective against advanced hematological tumors, mediating complete remission in >50% of some patients.¹ However, CAR T-cell therapies have limited success against solid tumors due to the immunosuppressive tumor microenvironment (TME). To improve efficacy, CAR T cells can be “armored” by ectopically expressing growth factors, such as interleukin (IL)-15, to promote their expansion and survival within the TME.² Alternatively, “arming” CAR T cells with inflammatory cytokines, IL-12 or IL-18, activates host T cells to promote epitope spreading.3,7 We recently armored and armed CAR T cells with an IL-2 superkine (Super2) and tissue alarmin IL-33 (hereafter, S233). These cytokines act synergistically and, through TCR or with multiple CAR constructs, promote regression of primary and metastatic solid tumors including melanoma without the need for pre-conditioning treatments.^{8 9} S233 expression increases CAR T-cell expansion and tumor infiltration but also triggers epitope spreading by activating tumor-specific endogenous T cells.⁸

However, the ability of CAR T cells to provide durable antitumor immunity remains an open question for both liquid and solid tumors. Analysis of two long-term survivors who received CD19-specific CAR T cells shows decade-long persistence of CAR T cells, leukemia regression, and loss of normal B cells.¹⁰ CAR T cell expansion and persistence are linked to clinical response and progression-free survival^{10 11} and are positively associated with phenotypic or transcriptional features of memory T cells in the manufactured CAR T-cell product.^{12 13} These findings have spurred the field to optimize in-vitro manufacturing conditions to increase memory T-cell potential within the CAR T-cell product.14,16 These strategies have largely succeeded in improving CAR T cell-mediated control of primary tumor growth.^{14 15} But the potential of CAR T cells to differentiate in vivo into memory T cell subsets post-treatment and the effect of various armored cytokines and combination therapies on CAR and tumor-specific endogenous memory T cells are largely unknown.

Memory T cells play a crucial role in long-term immunity against pathogens and cancer due to their heightened antigen sensitivity and ability to mount a rapid and robust response to secondary antigen exposure.¹⁷ Memory T cell subsets are broadly categorized into circulating (T_CIRC) and tissue-resident memory (T_RM) T cells based on differential migratory and functional capacities.¹⁷ T_CIRC cells include effector memory T (T_EM) cells that patrol in and out of tissues and possess high effector potential, as well as central memory (T_CM) cells that traffic among lymphoid organs and have a long lifespan and high proliferative potential. T_RM cells reside in non-lymphoid tissues, constitutively express effector molecules, and serve as the initial line of defense against secondary antigen encounters in peripheral tissues.¹⁷ In the context of cancer, the persistence of tumor-specific memory T cells enables ongoing immune surveillance to prevent tumor recurrence. Transcriptional signatures of CD8 memory T cells, particularly T_RM cells in solid tumors are associated with increased immunotherapy responsiveness and improved clinical outcomes.18,20 We previously reported that melanoma-specific CD8 T_RM cells are established in the skin and lymph node following neo-adjuvant CD4 depletion therapy and are required for durable antitumor immunity at primary and metastatic sites.^{21 22}

Recent mechanistic studies demonstrated that neo-adjuvant CD4 depletion therapy improves B16F10 melanoma-specific memory T cell differentiation by eliminating inhibitory conventional CD4 T cells.²³ Type I regulatory T (Tr1) cells that arise in response to high-dose neoantigen vaccination have also been reported to limit CD8-mediated tumor immunity.²⁴ Immunotherapies that broadly deplete CD4 T cells improve antitumor responses against late-stage solid tumors in mice and patients.^{25 26} Together, these findings led us to assess the impact of CD4 depletion therapy on the ability of CAR T cells and endogenous T cells to differentiate into memory T cells. We found that unarmored CAR T cells differentiated in vivo into CAR T_CIRC and T_RM cells but were ineffective against both primary and secondary B16F10 tumors. In contrast, S233-armored CAR T cells, which mount a robust primary response, differentiated into a limited number of CAR memory T cells that were restricted to the T_CIRC compartment. As previously reported, CD4 depletion therapy induced endogenous tumor-specific T_CIRC and T_RM T cells.²¹ CD4 depletion increased CAR memory T cells but did not improve protection against secondary tumors. However, combining CD4 depletion therapy with S233-armored CAR T cells significantly improved the formation of endogenous tumor-specific T_CIRC and T_RM cells and delayed or prevented secondary tumor growth even when administered at a lethal dose. These results highlight the need to consider the balance between CAR effector function and memory differentiation as well as the importance of activating endogenous T cells to provide both immediate and durable antitumor protection.

Methods

Mice

Eight- to twelve-week-old female C57BL/6 (B6), CD45.1 B6 congenic, and Thy1.1+ Pmel-1 TCR transgenic (JAX stock #005023) mice were purchased from Charles River or Jackson laboratories and housed under SPF conditions in the Dartmouth College vivarium and monitored by veterinarian staff. Mice were euthanized if they had trouble moving, breathing, eating, or drinking, lost >15% of body weight, or when the tumor reached 15 mm in diameter. No adverse events were detected. ARRIVE1 reporting guidelines were used for reporting.²⁷

Cell lines

B16F10 (CRL-6475) and HEK 293T (CRL-1168) cell lines were purchased from ATCC. B16F10 and HEK 293T cells were cultured in RPMI-1640 (Cytiva SH30255.01) and Dulbecco’s modified Eagle (Cytiva SH30243.02) media supplemented with 10% heat-inactive FBS (Cytiva SH30071.02), respectively. Splenocytes and primary T cells were cultured in complete RPMI (cRPMI: RPMI-1640, 10% heat-inactive FBS, 10 mmol/L HEPES (Gibco 15630080), 100 μmol/L non-essential amino acids (Gibco 11140050), 1 mmol/L sodium pyruvate (Gibco 11360070), 100 U/mL penicillin (Gibco 15140122), 100 mg/mL streptomycin (Gibco 15140122), and 55 μmol/L of 2-mercaptoethanol (Gibco 21985023)) with 25 U/mL of recombinant human IL-2 (NCI-Frederick BRB Repository).

CAR and cytokine constructs

The TA99 CAR and Super2+IL-33 MSCV-based retroviral constructs are described in Brog et al and Zhang et al.^{8 28}

Retroviral packaging and transfection

HEK 293T cells passaged one to two times at 60% confluency in the absence of Pen-Strep were seeded onto 10 cm culture dishes at a density of 2.0×10⁶ cells per dish and cultured for ~36 hours (80%–90% confluency). One hour before transfection, cells were provided with fresh media. DNA transfections were performed using polyethylenimine (PEI). Briefly, 5 µg of CAR or cytokine plasmid was combined with 5 µg of pCL-Eco packaging plasmid (Addgene 12371) in 600 µL of OptiMEM (Gibco 31985062). To this mixture, 30 µL of PEI (1 µg/µL) was added and incubated for 10 min at room temperature prior to dropwise addition to HEK 293T cells. The media was replaced with 5 mL of fresh media including Pen-Strep 18 hours post-transfection. Viral supernatants were harvested at 48 and 72 hours post-transfection, filtered through a 0.45 µm filter, and used immediately or stored at −80°C.

CAR T cell generation

Splenocytes from CD45.1⁺ B6 mice were activated using Concanavalin A (2.5 µg/mL; Sigma C5275) in cRPMI media. Following 24 hours, activated T cells were retrovirally transduced to express Super2 and IL-33 by seeding 8.0×10⁶ cells per well in a 24-well plate, resuspension in viral supernatant and polybrene (1 mg/mL; Sigma 107689), followed by spinoculation at 1500×g for 90 min at 37°C. The viral supernatant was subsequently removed, and cells were maintained in culture at a concentration of 10⁶ cells/mL in cRPMI with 25 U/mL recombinant human IL-2. A second transduction to express TA99 (anti-TRP1) or NKG2D CAR was performed 24 hours later using the same protocol. Flow cytometry analysis was conducted 72 hours post-transduction, and transduction efficiency was evaluated prior to CAR T cell adoptive transfer.

In vivo mouse experiments

B6 mice were shaved and then inoculated intradermally with 300,000 B16F10 tumor cells. Tumors were measured every other day using calipers starting on day 5 or 6 after tumor establishment, and the tumor volume was calculated by multiplying length, width, and height. On day 8 post-tumor inoculation, mice were randomized into groups and treated in random order with 7×10⁶ CAR T cells or S233-armored CAR T cells via intravenous tail vein injections. For CD4 depletion therapy, mice were injected intraperitoneally with 250 µg per dose of anti-mouse CD4 antibody (mAb clone GK1.5; BioXcell Cat: #BE0003-1) on days 5 and 11 after tumor inoculation. For ICB therapy, anti-mouse PD-L1 (B7-H1 mAB clone 10F.9G2; BioXcel Cat: #BE0101) was injected at a dose of 200 µg intraperitoneally on days 7, 9, and 11 post-tumor injections. Anti-mouse CTLA-4 (CD152 mAB clone 9H10; BioXcell Cat: #BE0131) was injected at a dose of 100 µg intraperitoneally on days 4, 7, and 10 post-tumor injections. Anti-mouse VISTA was kindly provided by Randolph J. Noelle (mAB clone 13F3.2E9; Lot #BP-2874-019-6) and injected at a dose of 300 μg intraperitoneally every other day from D2 to D12 post-tumor inoculation. Live mouse imaging using IVIS was conducted as previously detailed in our prior work.⁸ Tumor growth delay was calculated based on the day on which tumor volume surpassed 500 mm³ using the following formula: % Delay=(treated (T)−control non-treated (C))/C)×100%. For analysis of memory T cells, tumors were surgically resected, and wounds were closed using skin clips on day 18 as previously described.²⁹ No mice were excluded from the analysis.

Tissue harvest and processing

Spleen, tumor-draining lymph node (TDLN), and skin were harvested on Day 15 post-tumor inoculation to characterize effector T cells and 30+ days post-surgery (day 48+ post-tumor inoculation) to characterize memory T cells. Spleen and TDLN were mashed through a 70 µm strainer, and red blood cells in the spleen were lysed. The fatty layer was gently removed from the skin and then the skin was minced and incubated in glass vials containing 15 mL of collagenase solution (930 U/mL collagenase IV (Worthington # CLS-4/LS004188) in RPMI-1640, 5% FBS, 2 mM MgCl₂, 2 mM CaCl₂, 1% HG solution (3% L-glutamine B-grade (CALBIOCHEM #3521), 12% HEPES (FISHER #BP310-500)) for 30 min at 37°C while stirring at 350 rpm. The skin was subsequently mechanically dissociated twice using a gentleMacs Dissociator (Miltenyi Biotec) using m_spleen_0.1.01 setting and then filtered through a 70 µM filter two times followed by a 50 µM filter (Amazon: ASIN # B081S71FTN).

Flow cytometry

Single-cell suspensions were stained with viability dye for 10 min at RT, incubated with Fc block for 10 min, and then antibodies specific for cell surface receptors for 45 min at RT. All antibodies are listed in online supplemental table 1. Cells were run on a Cytek Aurora spectral cytometer. Data were analyzed using Flowjo V.10.10.0.

Statistical analyses

Preliminary data suggested that effect sizes between points will be approximately 1.7, which ensures a power of 80% with a sample size of 5 per group and a type-I error of α=0.05. For survival studies, 5–8 mice/group were used. All in vivo mouse studies consisted of 5–8 mice per group, and experiments were repeated two to four times. Data from replicate experiments were combined for statistical analysis, except when stated otherwise in the figure legend. One- or two-way analysis of variance was employed to identify differences between CAR T-cell experimental groups using GraphPad Prism. All data were analyzed and are depicted as means, with error bars representing SD.

Results

CD4 depletion therapy but not ICB immunotherapy increased CAR T cell efficacy against B16F10 melanoma

We previously reported that second-generation anti-TRP1 (TA99) CAR T cells had no therapeutic effect as a single-line therapy in the immunocompetent, non-immunogenic B16F10 melanoma model without preconditioning regimens.⁸ Armoring TA99 CAR T cells with Super2 and IL-33 cytokines, however, significantly prolonged overall survival but only delayed tumor growth by approximately 1 week (figure 1A–D). Using live luminescence imaging to track CAR T cell localization and abundance, we found that the loss of S233-armored CAR T-cell signal coincided with the loss of tumor control (online supplemental figure 1). This led us to determine whether inhibitory receptors limit CAR T cell responses. However, there was no difference in B16F10 tumor growth or overall survival when S233-armored CAR T cells were combined with anti-PD-L1, -CTLA-4, or -VISTA blockade (online supplemental figure 2). While CD4 T cells can enhance antitumor immunity, immunosuppressive subsets including Tregs and Tr1 cells can limit activation of tumor-specific CD8 T cells.^{23 24} To determine whether CD4 T cells limited CAR T cell responses, we treated B16F10 tumor-bearing mice with anti-CD4 antibody 3 days prior to and following transfer of unarmored TA99 or S233-armored TA99 CAR T cells (figure 1A). Consistent with previous reports, the depletion of CD4 T cells delayed tumor growth by 27% compared with untreated mice (no treatment, NT) (figure 1B,C). In contrast, the transfer of unarmored TA99 CAR T cells with or without CD4 depletion had no additional effect on tumor growth in comparison to NT or anti-CD4 treated mice, respectively. S233-armored CAR T cells delayed tumor growth by 44%, similar to or modestly better than CD4 depletion alone (figure 1B,C). The combination of S233-armored CAR T cells and CD4 depletion yielded the best tumor control, delaying tumor growth by 140% and enhancing overall survival compared with other groups (figure 1B–D), highlighting the importance of eliminating the suppressive CD4 T cells in improving CAR T cell efficacy.

Figure 1. Effects of unarmored and S233-armored chimeric antigen receptor (CAR) T cells on B16F10 tumor growth and survival in the presence or absence of CD4 T cells. (A) Experimental schematic. TA99 CAR T cells with or without Super2 IL-33 (S233) armor (7×10⁶) were transferred into B16F10 tumor-bearing mice (3×10⁵) on day eight post-tumor inoculation. Some groups received 200 µg αCD4 depleting antibody on days 5 and 11. (B) Individual and average tumor volumes measured every other day starting day six post-tumor inoculation. Data are plotted as mean+SD. Two-way analysis of variance analysis with Tukey post-hoc test. ****p<0.0001. (C) Tumor growth delay resulting from CAR T cell therapy. (D) Kaplan-Meier curve depicting survival probability and analyzed using the log-rank test. **p<0.01. (B–D) Data are from 1 of 2 representative experiments with 3–6 biological replicates per experiment.

S233-armored CAR T cells exhibit a highly activated effector phenotype compared with more stem-like unarmored CAR T cells

When used in a neoadjuvant setting, depletion of CD4 T cells prior to B16F10 surgical resection leads to the differentiation of tumor-specific memory T cells that provide protection against B16F10 re-challenge.²¹ This prompted us to determine the potential of TA99 CAR T cells to adopt a memory precursor phenotype 1-week post-transfer with or without CD4 depletion (figure 2A). Unarmored and S233-armored TA99 CAR T cells were found in the TDLN in similar overall numbers and largely maintained an MPEC phenotype defined by CD127⁺KLRG1⁻ expression, comparable to their phenotype prior to transfer (figure 2B–D, online supplemental figure 3A,B). CD4 depletion did not significantly affect CAR T cell numbers in the TDLN but resulted in a small decrease in MPEC proportions of S233-armored TA99 CAR T cells (figure 2D).

Figure 2. Unarmored TA99 chimeric antigen receptor (CAR) T cells resemble memory precursor effector cells (MPECs) while S233-armored TA99 CAR T cells appear hyperactivated. (A) Experimental schematic. Tumor-draining lymph nodes (TDLNs) were collected on day 15 post-tumor inoculation. (B–C) Flow cytometric analysis of CD45.1⁺ CAR T cells. (B) Representative flow plots and (C) Counts of CD45.1⁺ CAR T cells, gated on CD8⁺CD44⁺. (D) Representative flow plots and proportions of CD45.1⁺ CAR T cell MPECs (defined by CD127⁺KLRG1⁻), gated on CD8⁺CD44⁺CD45.1⁺. (E–J) Representative plots and mean fluorescence intensity (MFI) of CAR MPECs in the TDLN at D15, gated on CD8⁺CD44⁺CD45.1⁺, (E) CD127, (F) CD62L, (G) CD44, (H) CD69, (I) LAG-3, and (J) PD-1 and Tim-3 expression. Data are from 1 of 2–4 representative experiments with 4–6 biological replicates per experiment and are plotted as mean+SD. Ordinary one-way analysis of variance with Holm-Šídák test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

Unarmored TA99 CAR T cells expressed elevated stemness factors, including higher TCF-1 expression prior to adoptive transfer (online supplemental figure 3C) and higher expression of CD127 and CD62L, which are associated with CAR T cell persistence and proliferative capacity,^{30 31} in the TDLN 1 week after transfer (figure 2E,F). In contrast, S233-armored TA99 CAR T cells expressed lower CD127 and CD62L levels, particularly when combined with CD4 depletion (figure 2E,F). Additionally, S233-armored TA99 CAR T cells appeared more activated and expressed higher CD44 and CD69 compared with unarmored CAR T cells in the TDLN or CAR T cells prior to transfer (figure 2G,H, online supplemental figure 3B,C).

Unarmored and S233-armored TA99 CAR T cells were also abundant in the spleen of all groups, with unarmored TA99 CAR T cells present in similar MPEC proportions compared with TDLN (online supplemental figure 4A–C). However, there was a significant decrease in splenic S233-armored TA99 CAR T MPECs. In combination with CD4 depletion, we observed an increase in CD127⁻KLRG1⁺ SLEC proportions, suggesting that systemically distributed S233-armored CAR T cells are hyperactivated and are more differentiated terminal effector cells (online supplemental figure 4A–C).

PD-1 is rapidly induced following T cell activation³² and was found to be equally expressed in unarmored and S233-armored TA99 CAR T cells prior to transfer (online supplemental figure 3D). One week post-transfer, unarmored TA99 CAR T cells in the TDLN and spleen expressed little to no PD-1, and while they were abundant in the tumor, remained PD-1^low, consistent with reduced T cell activity (figure 2J,I, online supplemental figure 4D–F). In contrast, S233-armored TA99 CAR T cells in the TDLN and spleen expressed higher proportions of PD-1⁺Tim3⁻ cells, indicating that S233-armoring supported an activated CAR T cell phenotype (figure 2J–I, online supplemental figure 4D). CD4 depletion further increased the PD-1+Tim3⁻ subset in S233-armored TA99 CAR T cells (figure 2J–I, online supplemental figure 4D), suggesting that host or CAR CD4 T cells limit the activation of S233-armored CAR CD8 T cells.

Chronic T cell activation induces expression of additional inhibitory receptors including Tim-3, LAG-3 and TIGIT, and co-expression of PD-1 and Tim-3 is often used to identify dysfunctional or exhausted T cells in tumors.³³ A small proportion of S233-armored CAR T cells expressed LAG-3 and co-expressed PD-1 and Tim-3 in the TDLN and spleen, but PD-1⁺Tim-3⁺ exhausted cells were readily detected in B16F10 tumors (figure 2J–I, online supplemental figure 4D,F). CD4 depletion had little effect on PD-1, Tim-3, and LAG-3 expression in unarmored TA99 CAR T cells. CD4 depletion cooperated with S233-armoring to increase PD-1⁺Tim-3⁺ CAR T cells in the TDLN and spleen but did not further increase exhausted cells within B16F10 tumors (figure 2J–I, online supplemental figure 4D,F).

To determine whether CD4 depletion and/or S233-armoring has similar effects on CAR memory precursor and activation phenotypes in another CAR T cell solid cancer model, we evaluated NKG2D CAR T cells in the immunocompetent MC38 colon cell carcinoma model. Using a similar treatment scheme as B16F10, we adoptively transferred 7×10⁶ NKG2D CAR T cells into C57BL/6 mice 8 days after intradermally injection of 10⁶ MC38 cells with or without CD4 depletion (figure 3A). Unarmored and S233-armored NKG2D CAR T cells were found in similar frequencies (figure 3B,C). Like TA99 CAR T cells in the B16F10 model, while unarmored and S233-armored NKG2D CAR T cells largely exhibited an MPEC phenotype across all groups, S233-armoring resulted in reduced CD127 expression levels (figure 3C,D). Unarmored NKG2D CAR T cells expressed higher CD127, lower activation markers, CD44 and CD69, and did not express PD-1 or other inhibitory receptors, Tim-3, LAG-3, and TIGIT (figure 3D–I). In contrast, S233-armored NKG2D CAR T cells expressed higher activation markers, had substantial PD-1⁺Tim3⁻ and PD-1⁺Tim3⁺ populations, and expressed high levels of LAG-3 and TIGIT (figure 3E–I). CD4 depletion had little effect on tumor control in the MC38 immunogenic model or expression of activation markers in NKG2D CAR T cells except it further increased PD-1⁺Tim3⁻ cells without increasing PD-1⁺Tim3⁺ cells (online supplemental figure 5A, figure 3G). Like TA99 CAR T cells, unarmored splenic NKG2D CAR T cells were overwhelmingly the MPEC phenotype while S233-armored NKG2D CAR T cells had an increase in SLEC phenotype cells (online supplemental figure 5B,C). Unarmored NKG2D CAR T cells in the tumor lacked PD-1 expression unlike S233-armored NKG2D CAR T cells, which were either PD-1⁺Tim3⁻ or PD-1⁺Tim3⁺ cells and expressed high levels of LAG-3 and TIGIT (online supplemental figure 5D–G). CD4 depletion did not alter MPEC, activation, or exhaustion phenotypes of either unarmored or S233-armored CAR T cells (online supplemental figure 5B–G).

Figure 3. Characterization of chimeric antigen receptor (CAR) memory precursor effector cell phenotypes from the tumor-draining lymph node (TDLN) in another model system (MC38). (A) Experimental schematic. TDLN were collected on day 15 post-tumor inoculation. (B–C) Flow cytometric analysis of CD45.1⁺ CAR T cells. (B) Representative flow plots and proportions of CD45.1⁺ NKG2D CAR T cells, gated on CD8⁺CD44⁺. (C) Representative flow plots and proportions of CD45.1⁺ NKG2D CAR T cells. (D–F) Representative histograms and mean fluorescence intensity (MFI) of NKG2D CAR memory precursor effector cells in the TDLN at D15, gated on CD8⁺CD44⁺CD45.1⁺, (D) CD127, (E) CD44, (F) CD69, (G) PD-1 and Tim-3, (H) LAG-3 and TIGIT expression. Data are from 1 of 2 representative experiments with 4–6 biological replicates per experiment and are plotted as mean+SD. Ordinary one-way analysis of variance with Holm-Šídák test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

Collectively, these data indicate that unarmored CAR T cells preferentially express memory-associated but not activation markers in the spleen, TDLN, and tumor, while S233-armoring promotes an activated memory precursor phenotype in the TDLN and increased differentiation into terminal effector cells in the spleen and tumor. CD4 depletion had little effect on CAR T cell proportions in lymphoid organs but led to a reduction in tumor-infiltrating CAR T cells in both B16F10 and MC38 models. Yet CD4 depletion augmented the S233-armored CAR T cell-dependent tumor growth delay of B16F10, a non-immunogenic tumor but had little effect on MC38 (figure 2, onlinesupplemental figures 46). In the immunogenic tumor MC38, CD4 depletion had little effect on CAR effector T cell phenotypes or tumor growth kinetics (figure 3, online supplemental figure 5).

CD4 depletion increased CAR T cell persistence and in combination with S233-armored CAR T cells promoted durable tumor immunity

To determine whether CAR memory T cell precursors differentiate into memory cells, we resected primary tumors on day 18, and allowed mice to recover (figure 4A,B). Continued luminescence imaging to track CAR T cell dynamics revealed that unarmored TA99 CAR T cells persisted at a low level for up to 14 days post-surgery while the originally more abundant S233-armored TA99 CAR T cells dropped quickly following tumor surgery (figure 4C,D). CD4 depletion increased the overall magnitude of unarmored TA99 CAR T cells but not S233-armored TA99 CAR T cells post-surgery. To determine whether treated mice exhibited durable antitumor immunity, we re-challenged mice with a lethal dose of B16F10 cells at least 30 days post-surgery (figure 4E). Mice originally treated with unarmored TA99 CAR T cells showed no delay in secondary tumor growth compared with naïve mice (figure 4F,G). Partial protection was observed in a subset of mice originally treated with unarmored TA99 CAR T cells combined with CD4 depletion or S233-armored TA99 CAR T cells (figure 4F,G). Mice originally treated with S233-armored TA99 CAR T cells and CD4 depletion exhibited the most significant delay in secondary tumor growth (figure 4F,G). Thus, combining S233-armored TA99 CAR T cells with CD4 depletion promotes the most durable antitumor immunity in response to secondary B16F10 tumor re-challenge without further immunotherapy.

Figure 4. Unarmored and S233-armored chimeric antigen receptor (CAR) T cell expansion kinetics and antitumor response to B16F10 tumor rechallenge. (A) Experimental schematic. (B) Tumor weights on the day of surgery. Data are from 13 to 24 total mice per group, combining four independent experiments. (C) Representative luminescence images and (D) quantification of TA99 CAR T cells (7×10⁶) with or without S233-armoring transferred into B16F10 tumor-bearing mice on day eight post-tumor inoculation. Imaging was performed on days 15, 21, 27, and 33. Data are from 1 of 2 representative experiments with 5–8 biological replicates per experiment and analyzed using ordinary two-way analysis of variance with the Tukey test. *p<0.05 for TA99 CAR +αCD4 versus S233 CAR +αCD4 at all time points. (E) Experimental schematic. B16F10 tumor-bearing mice were treated as previously described. Tumors were then resected on day 18 post-tumor inoculation, and mice were re-challenged with 3×10⁵ B16F10 on day 30+ post-surgery (48+ post-initial tumor inoculation). (F) Individual tumor volumes following B16F10 re-challenge. (G) Quantification of tumor growth delay following B16F10 rechallenge. Data are from three combined experiments with 8–19 total mice per group. (B, G) All data are plotted as mean+SD and analyzed using ordinary one-way analysis of variance with Holm-Šídák test. ****p<0.0001, ***p<0.001, **p<0.01.

CD4 depletion increases CAR memory T cell differentiation

Next, we evaluated CAR memory T cells in the TDLN, spleen, and skin at least 30 days after tumor resection surgery (figure 5A). Unarmored TA99 CAR memory T cells were found in the TDLN and spleen at low numbers; however, they were significantly increased when mice were also treated with CD4 depletion (figure 5B,C, online supplemental figure 6A). In contrast, S233-armored TA99 CAR T cells were largely absent at a memory timepoint in the TDLN and spleen unless CD4 T cells were depleted (figure 5B,C, online supplemental figure 6A). Unarmored NKG2D CAR memory T cells also developed in the immunogenic MC38 model and enhanced by CD4 depletion (online supplemental figure 6B). While CD4 depletion did not improve the ability of S233-armored NKG2D CAR T cells to delay MC38 tumor growth, it was able to increase S233-armored NKG2D memory CAR T cells (onlinesupplemental figures 5A 6B). We phenotyped CAR memory T cells by flow cytometry and found that most CAR memory T cells in the TDLN preferentially differentiated into CD69⁻CD62L⁺ T_CM cells irrespective of S233 armoring or CD4 depletion (figure 5D,E). A small proportion of CAR memory T cells from CD4-depleted mice were CD69⁺CD103⁺, characteristic of tissue-resident memory cells (figure 5D–G). The proportion of CAR T_RM cells did not differ in the presence or absence of S233 armoring (figure 5F,G). Unarmored TA99 CAR memory T cells were found in the skin of treated mice, and CD4 depletion increased their frequency (figure 5H,I). All CAR memory T cells in the skin were CD69⁺CD103⁺ T_RM cells (figure 5J,K). In contrast, few S233-armored TA99 CAR T cells populated the skin, irrespective of CD4 depletion (figure 5H–J). Together, these data show that unarmored CAR T cells have the capacity to form circulating and resident T cell memory and that their numbers are increased in the absence of CD4 T cells. S233-armoring impedes their ability to efficiently form memory, particularly in the skin.

Figure 5. Phenotypic characterization of chimeric antigen receptor (CAR) memory T cell subsets in the tumor-draining lymph nodes (TDLN) and skin. (A) Experimental schematic. B16F10 tumors were resected on day 18 post-tumor inoculation, and TDLN, spleen, and skin were harvested at least 30 days post-surgery (D48+ post-tumor inoculation). (B–G) Flow cytometric analysis of CD45.1⁺ CAR T cells in the TDLN on day 30+ post-surgery. (B) Representative flow plots and (C) counts of CD45.1⁺ CAR T cells, gated on CD8⁺CD44⁺. (D) Representative flow plots and (E) frequency of CD45.1⁺ CAR T cell memory subsets, gated on CD8⁺CD44⁺CD45.1⁺. (F) Representative flow plots and (G) frequency of CAR T cell expression of CD69 and CD103, gated on CD8⁺CD44⁺CD45.1⁺CD62L⁻CD69⁺. Data are from three combined experiments with 8–27 total mice per group for TDLN. (H–K) Flow cytometric analysis of CD45.1⁺ CAR T cells in the skin on day 30+ post-surgery. (H) Representative flow plots and (I) frequency of CD45.1⁺ CAR T cells, gated on CD8⁺CD44⁺. (J) CD69 and CD103 expression and (K) frequency of CD69⁺CD103⁺ CAR T cells in the skin, gated on CD8⁺CD44⁺CD45.1⁺. Data are from two combined experiments with 4–16 total mice per group for skin. Data are plotted as mean+SD and analyzed using ordinary one-way analysis of variance with Holm-Šídák test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

S233-armored CAR T cells in combination with CD4 depletion enhanced antigen-specific endogenous T_RM cells in the TDLN and skin

Mice treated with CD4 depletion therapy and S233-armored CAR T cells exhibited the most significant protection against B16F10 rechallenge (figure 4F,G) despite the limited generation of CAR memory T cells (figure 5). This led us to examine endogenous tumor-specific memory T cells. Both CD4 depletion and cytokine armoring can activate endogenous tumor-specific T cells.^{3 29} We previously reported that S233-armored CAR T cell treatment increased the number of tumor-specific endogenous effector T cells identified by TRP2-tetramer (TRP2^Tet+) staining within the tumor.⁸ Endogenous TRP2^Tet+ T cells expressing MPEC markers were observed on day 15 following either S233-armored CAR T cell treatment or CD4 depletion (online supplemental figure 9). We next quantified and characterized TRP2^Tet+ memory T cells (figure 6A). TRP2^Tet+ memory T cells were absent from the TDLNs without CD4 depletion (figure 6B,C). However, S233-armored CAR T cell treatment in combination with CD4 depletion significantly increased the overall frequency and absolute numbers of TRP2^Tet+ T cells compared with CD4 depletion alone or in combination with unarmored TA99 CAR T cells (figure 6B,C). Greater than 60% of TRP2⁺ memory T cells in the TDLN expressed tissue residence markers, CD69 and CD103 (figure 6E,F).

Figure 6. Phenotypic characterization of endogenous tumor-specific memory T cell subsets in the tumor-draining lymph nodes (TDLN) and skin. (A) Experimental schematic. B16F10 tumors were resected on day 18 post-tumor inoculation, and TDLN and skin were harvested at least 30 days post-surgery (D48+ post-tumor inoculation). (B–F) Flow cytometric analysis of endogenous TRP2-tetramer⁺ CD8 T cells in the TDLN on day 30+ post-surgery. (B) Representative flow plots and (C) counts of TRP2-Tet⁺ CD8 T cells, gated on CD45.1⁻CD8⁺CD44⁺. (D) Representative flow plots of TRP2-Tet⁺ CD8 memory T cell subsets, gated on CD45.1⁻CD8⁺CD44⁺. (E) Representative flow plots and (F) frequency of TRP2-Tet⁺ T cell expression of CD69 and CD103, gated on CD8⁺CD44⁺CD45.1⁻CD62L⁻CD69⁺. Data are from three combined experiments with 8–27 total mice per group for TDLN. (G–I) Flow cytometric analysis of TRP2-tetramer⁺ T cells in the skin on day 30+ post-surgery. (G) Representative flow plots and (H) frequency of TRP2-Tet⁺ T cells, gated on CD8⁺CD44⁺CD45.1⁻. (I) Frequency of TRP2-Tet⁺ T_RM cells in the skin, gated on CD8⁺CD44⁺CD45.1⁻CD103⁺CD69⁺. (J–K) Melanoma-associated vitiligo (MAV) phenotype and prevalence. (J) Representative images of CD4 depletion and S233-armored chimeric antigen receptor (CAR) T cell treated mice. (K) Quantification of MAV prevalence. Data are from two combined experiments with 4–16 total mice per group for skin. Data are plotted as mean+SD and analyzed using ordinary one-way analysis of variance with Holm-Šídák test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

Like the TDLN, CD4 depletion alone or in combination with TA99 CAR T cells induced TRP2⁺ T cells in the skin. However, CD4 depletion combined with S233-armored CAR T cells further increased skin TRP2^Tet+ T cells (figure 6G,H). Skin TRP2^Tet+ T cells were predominantly CD69⁺CD103⁺ T_RM cells (figure 6I). B16F10 tumor-bearing mice treated with neoadjuvant CD4 depletion therapy results in melanoma-associated vitiligo (MAV) in 60%–70% of treated mice.²⁹ MAV-affected mice are protected against secondary tumors, consistent with the improved overall survival observed for MAV-affected melanoma patients, highlighting the significance of T_RM cells in durability tumor immunity.^{21 34} We found that combining CD4 depletion with S233-armored CAR T cells resulted in 100% MAV prevalence and substantially increased T_RM numbers in the skin and TDLN (figure 6J,K). Together these data indicate that combination therapies that include depletion of CD4 T cells and S233-armored CAR T cells can improve durable antitumor immunity via induction of tumor-specific tissue-resident memory.

Discussion

Our work demonstrates that both unarmored and armored CAR T cells possess the ability to persist and develop into multiple memory T cell subsets. While unarmored CAR T cells generated dysfunctional effector responses to solid tumors, they efficiently differentiated into T_CIRC and T_RM cells in the lymphoid and tissue compartments, yet they remained dysfunctional. IL-2 superkine and IL-33 armoring improved CAR T cell efficacy to primary tumors and increased effector markers on CAR memory T cell precursors in the SLOs but limited their differentiation into memory T cells. CD4 depletion increased the magnitude of CAR memory T cells irrespective of armoring; however, S233 armoring prevented the memory differentiation of CAR T cells in the skin. As observed with IL-12- or IL-18-armored CAR T cells,^{3 35} S233-armored CAR T cells triggered epitope spreading and activation of endogenous T cells.⁸ Combining CD4 depletion with S233-armored CAR T cells potentiated the development of endogenous tumor-specific memory T cells, particularly T_RM cells, and resulted in optimal protection against secondary tumors.

Engineering CAR T cells to express cytokines can influence their intrinsic state by increasing their proliferation, effector activity, and survival. Armoring CAR T cells with IL-18 increases CAR T cell proliferation in vitro and in vivo.³ IL-12-armored CAR T cells exhibit significantly enhanced IFNγ and TNFα production, cytotoxic capacity in vitro, and resistance to apoptosis in vivo in the ID8 ovarian tumor model.³⁶ We previously reported that S233-armored CAR T cells increased their in vivo expansion and tumor infiltration.⁸ We now demonstrate that S233 armoring results in a highly activated CAR effector population that includes both memory precursors and terminal effectors expressing higher CD69 and PD-1 levels. PD-1 expression can mark activated T cells with increased functionality and enhanced capability to recognize tumor-specific neoantigens and eliminate autologous tumors.^{37 38} This is consistent with S233 armoring promoting a heightened effector phenotype capable of contributing to the improved tumor immunity observed. However, S233-armored CAR T cells also expressed inhibitory receptors associated with exhaustion and a terminally differentiated population in the spleen and tumor, which was amplified by CD4 T cell depletion. S233-armored CAR T cell memory precursors also did not differentiate efficiently into CAR memory T cells. Given the multiple parallels between IL-12-, IL-18- and S233-armored CAR T cells, we propose that while these cytokines can enhance CAR T cell effector phenotypes to improve antitumor activity, they can also result in decreased differentiation into CAR memory T cells, thereby impairing long-lasting CAR T cell-dependent antitumor protection. It will be important to directly evaluate the effect of IL-12 and IL-18 armoring on CAR effector versus memory T cell potential to better predict their effect on short- and long-term patient outcomes.

Balancing effector and memory T cell potential in adoptive cell therapy presents a significant obstacle for engineering effective tumor immunity since the signals driving terminal and memory cell fate are generally diametrically opposing. For example, IL-2 and IL-15 preferentially promote effector and memory differentiation, respectively.³⁹ Thus one strategy for increasing memory differentiation is to replace Super2 in the S233-armored CAR T cells with a different IL-2 mutein H9T, which activates STAT5 with similar kinetics as IL-15, to increase memory T cell generation while retaining some of the proliferative effects of IL-2.⁴⁰ Alternatively, different CAR costimulatory domains can impart distinct functional properties and differentiation potential. Second-generation CAR constructs that include the CD28 costimulatory domain enhance proliferation and cytokine production, while those with the 4-1BB domain induce prolonged persistence.⁴¹ Thus, incorporating a mixed population of CAR T cells expressing either CD28 or 4-1BB CARs could capitalize on both aspects of enhanced proliferation and prolonged persistence. Regardless of the approach, engineering CAR T cells with a balance of effector and memory potential will be important to ensure immediate and durable tumor immunity.

Cytokines can also reshape the TME and boost the endogenous antitumor immune response. Armoring CAR T cells with IL-12 or IL-18 can recruit a diverse array of antitumor immune cells, including M1 macrophages, dendritic cells, and NK cells, induce expansion of endogenous CD8 T cells, and reduce the presence of MDSCs and Tregs.3,7 Using single-cell RNA sequencing analysis, we previously showed that S233-armored CAR T cells shift tumor-associated macrophages (TAMs) from an M2 suppressive phenotype to an M1 phenotype.⁸ This is likely through the direct or indirect activity of the pleiotropic cytokine IL-33, which can support pro- and anti-inflammatory properties of alternatively activated macrophages and other immune cells implicated in CAR T cell persistence.42,44 However, IL-33 expression without Super2 in TA99 CAR T cells had little effect on TAM or MDSC surface marker phenotypes (onlinesupplemental figures 8 9). Further studies that identify the cells activated by Super2 and IL-33 and the mechanisms responsible for altering the TME are required to fully understand how Super2 and IL-33 synergize to promote robust antitumor responses.

Conventional CD4 T cells have divergent roles in tumor immunity that vary depending on context. CD4 T cells can express cytotoxic effectors and have the capacity to directly kill tumor cells.⁴⁵ Numerous studies describe the ability of CD4 helper T cells to promote CD8 T cell priming and effector activity.⁴⁶ Yet, conventional CD4 T cells can limit the priming of tumor-specific CD8 T cells by type 1 conventional dendritic cells (cDC1).²³ Cancer vaccines that avoid induction of Tr1 cells, which kill cDC1s, result in more effective antitumor efficacy.²⁴ Thus, CD4 depletion therapies that improve antitumor immunity against solid tumors in mice and patients^{25 26} likely act by eliminating inhibitory conventional and regulatory CD4 T cell subsets. The ability of CD4 depletion to further improve the ability of S233-armored CAR T cells to delay B16F10 but not MC38 tumor growth suggests that not all tumors are limited by Tr1 cells. Whether differences in tumor immunogenicity contribute to the divergent effect of CD4 depletion remains to be addressed.

Importantly, CD4 depletion promotes the differentiation of tumor-specific CD8 T cells into both T_CIRC and T_RM subsets.^{29 47 48} The presence of T_RM cells in melanoma patients has been linked to improved survival rates and tumor rejection.^{22 34 49} T_RM cells are associated with the prevalence of MAV in patients and mice.²¹ Neoadjuvant CD4 depletion results in 60%–70% MAV prevalence in the B16F10 tumor model.²⁹ Combining S233-armored CAR T cells with CD4 depletion increased MAV prevalence to 100%. While CD4 depletion and S233-armored CAR T cells enhanced endogenous T_CIRC memory T cells, they synergized to increase CD8 T_RM cells in the skin, indicating that they may act through distinct mechanisms to promote T_RM cell establishment. On rechallenge with a lethal dose of B16F10 at a memory timepoint, we observed a significant delay and some complete responses against secondary tumors. These findings are significant as they demonstrate that despite the inability of S233-armored CAR T cells to efficiently form CAR memory T cells, they can direct endogenous T cell differentiation towards T_CIRC and T_RM cells following the elimination of immunosuppressive CD4 T cells. The collaboration between multiple memory T cell subsets is important in improving durable antitumor immunity.^{34 50}

Our findings highlight the complex relationship between CAR T cells and endogenous immune cells. They underscore the importance of neutralizing the suppressive TME, especially inhibitory CD4 T cells, to increase CAR T cell-intrinsic cytotoxicity and to enable armored CAR T cells to activate endogenous tumor-specific lymphocytes. The strong positive correlation between memory T cell signatures within either tumors or lymphoid organs and improved overall survival rates and patient prognosis highlights the importance of balancing effector and memory potential to ensure protective immediate and long-lasting antitumor responses.¹⁸ Further studies to identify the relative contributions of CAR T cells versus endogenous T cells in adoptive cell therapies will inform future priorities for adoptive T cell engineering.

Data availability statement

Data are available upon reasonable request.