T-bet overexpression enhances CAR T cell effector functions and antigen sensitivity

Abstract

Background

T cells modified to express a chimeric antigen receptor (CAR) are successful against B-lineage malignancies but fail to induce durable remissions in up to half of patients and have shown limited efficacy against other types of cancer. Strategies to improve CAR T cell potency and responses to low antigen densities without inducing CAR T cell dysfunction or limiting persistence are necessary to expand durability of remissions.

Methods

We overexpressed T-bet in human and mouse CAR T cells to mimic exposure to signal 3 cytokines during T cell priming to promote T helper cell 1 (Th1) polarization of CD4+CAR T cells with the goal of enhancing antitumor activity. Using human CAR T cells and xenograft models we interrogated the impact of T-bet overexpression on CAR T cell antitumor activity in vitro and in vivo. We also used a syngeneic murine CAR T cell model to study the impact of T-bet overexpression on long-term persistence and secondary responses to tumor rechallenge.

Results

T-bet overexpression reduced expression of the Th2 cytokine interleukin 4 and promoted polyfunctional production of Th1-associated cytokines in response to CAR stimulation. T-bet overexpression enhanced some effector functions in vitro but did not improve CAR T cell-mediated control of leukemia expressing high levels of antigen in vivo. T-bet overexpression also improved effector function of murine CD19 CAR T cells with no impairment to the persistence or ability of persistent CAR T cells to re-expand and clear a secondary leukemia challenge. Finally, T-bet overexpression promoted enhanced in vitro function against leukemia expressing low levels of CD19, which translated to improved control of CD19lo leukemia in vivo by human C19 CAR T cells containing a 4-1BB costimulatory domain.

Conclusions

Together, our data demonstrate that T-bet overexpression induces a reduction in Th2 cytokine production, an increase in polyfunctional Th1 cytokine production and enhances 4-1BB CAR T cell activity against cancers expressing low levels of target antigen without promoting a loss in functional CAR T cell persistence.

Correspondence to Dr Terry J Fry; [email protected]

WHAT IS ALREADY KNOWN ON THIS TOPIC

• Chimeric antigen receptor (CAR) T cell therapies induce remissions in a high percentage of patients, but many of those patients relapse within 2 years of treatment. Relapses after CAR T cell therapy commonly include modulation of target antigen levels to below the threshold for optimal CAR T cell responses.

WHAT THIS STUDY ADDS

• T-bet overexpression modulates CAR T cell effector functions, increasing the potency and responsiveness to low-antigen densities in 4-1BB costimulated CAR T cells without inhibiting their ability to persist.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

• T-bet overexpression may be a strategy to enhance potency or antigen sensitivity of 4-1BB-costimulated CAR T cells.

Introduction

Chimeric antigen receptor (CAR) T cell therapies have demonstrated high rates of initial success against B-cell malignancies but fail to induce durable remissions in a high percentage of patients and have shown limited success at treating other cancers. Successful therapeutic outcomes require CAR T cells to proliferate and acquire effector functions to clear tumors without driving terminal effector differentiation or exhaustion that limits functional persistence. However, these two aspects of T cell biology tend to oppose each other,¹ as demonstrated for CAR T cells where more potent effector functions are often associated with limited persistence.^{2 3} Antigen-positive relapses occur due to a lack of CAR T cell persistence or lack of potency^{4 5} which can be exacerbated by a reduction in antigen expression levels below the threshold necessary for optimal CAR T cell activation.^6–10 Importantly, CAR sensitivity has been shown to be inferior to the sensitivity of T cells responding via the T cell receptor (TCR).^{11 12} Modifications to CAR designs have been shown to enhance sensitivity but are typically associated with an increase in signal strength which would be predicted to impair persistence.^{13 14} Therefore, strategies to enhance potency, especially in the context of low target antigen levels, without compromising persistence have the potential to improve long-term outcomes.

During a primary immune response to infection, conventional T cells rely on signals 1 and 2 through their TCR and costimulatory receptors, respectively, to become fully activated and require signal 3 from cytokines for differentiation and expansion.¹⁵ Response to infections that rely on CD8 T cells for clearance (viruses or intracellular bacteria) relies on a type 1 immune response orchestrated by T-bet,¹⁶ responses to large extracellular pathogens rely on a type 2 immune response directed by GATA3,¹⁷ and responses to fungal infections or extracellular bacteria generate type 17 responses coordinated by RORγt.¹⁸ T helper cell 1 (Th1) responses which are associated with secretion of interferon gamma (IFNγ), TNF-α, and interleukin (IL)-2, are generally optimal for antitumor responses.¹⁹

CAR T cell products are manufactured from T cells isolated from peripheral blood that are typically activated through CD3 and CD28 in the presence of cytokines such as IL-2, IL-7 and/or IL-15, bypassing the coordination of inflammatory lineage-directing signals from responding immune cells that would occur during conventional T cell priming. This has the potential to impact both CD4 CAR T cell (CAR4) subset differentiation as well as the effector functions acquired by CD8 CAR T cells (CAR8).²⁰ We sought to enhance the functional characteristics of the CAR T cell response by introducing a transgene driving expression of lineage-specifying transcription factors that are normally upregulated in response to signal 3 cytokines. T-bet overexpression enhanced CAR T cell degranulation, cytokine production and cytotoxicity, leading to enhanced antitumor responses. In a syngeneic immune-competent mouse model, T-bet overexpression did not impair the ability of CAR T cells to persist and respond to a secondary leukemia challenge. Finally, CAR T cells overexpressing T-bet mount a superior response to leukemia cells expressing low levels of target antigen, highlighting a role for T-bet in enhancing effector functions and antigen sensitivity of CAR T cells.

Results

T-bet overexpression skews CAR T cells towards Th1-associated cytokine production

We hypothesized that due to a lack of lineage-directing cytokines during activation and manufacturing, CAR4s may not differentiate into canonical T cell subsets. We observed both IFNγ and IL-4 production from CD19-directed human CAR4s after stimulation (figure 1A), in agreement with previous reports.²¹ To attempt to enhance Th1-associated cytokine production from CAR T cells, we transduced T-bet with a truncated EGFR into human T cells along with either a CD19-directed CD28-costimulated or 4-1BB-costimulated CAR (1928 or 19BB) (figure 1B and C). CAR T cells transduced with T-bet (Tbet-CAR T cells) expressed T-bet at levels approximately 2.5× higher than control CAR T cells (figure 1D).

Figure 1. T-bet overexpression promotes Th1-associated cytokine production in CAR T cells. (A) Expression of IFN[gamma] and IL-4 in 1928 and 19BB CAR T cells after 6-hour stimulation with PMA+ionomycin (B) Schematic of CAR, T-bet overexpression, and EGFR control constructs. All T cells were cotransduced with either the 1928 or 19BB CAR, plus either T-bet-EGFR or EGFR. (C) Representative transduction efficiencies (D) T-bet GMFI in CAR T cells. Mock T cells are gated on NGFR - EGFR - cells, and CAR T cells are gated on NGFR + EGFR + T cells. (E) Expression of IFN[gamma] and IL-4 in T-bet + CAR4s (NGFR + EGFR + ) and T-bet- CAR4s (NGFR + EGFR - ) after 6-hour stimulation with PMA+ionomycin. Lines connect CAR T cells made from the same donor. (F-K) Cytokine production of NGFR + EGFR + T cells from (F-H) 1928 and (I-K) 19BB CAR4s and CAR8s after 15 hours co-culture with NALM6. Lines connect CAR T cells made from the same donor. (L-M) Distribution of the number of cells making zero, one, two, or three of the cytokines I IFN[gamma], TNF-[alpha], and IL-2 for (L) 1928 and (M) 19BB CAR T cells. All data points were generated from two to three independent donors, each collected in triplicate. Statistics are nested t-tests (E-K) or two-way ANOVA with Sidak’s multiple comparisons tests (L-M). Data represent mean+/-SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; CAR, chimeric antigen receptor; IFN[gamma], (interferon gamma); IL, interleukin; ns, not significant; Th1, T helper cell 1.

When stimulated with Phorbol Myristate Acetate (PMA)/ionomycin, fewer Tbet-CAR T cells produced IL-4 (figure 1E) relative to controls. After stimulation with NALM6, a higher proportion of Tbet-CAR T cells produced IFNγ, IL-2 and TNF-α in 1928 and 19BB CAR T cells (figure 1F–K), in agreement with previous reports.²² Polyfunctional cytokine secretion from T cells is associated with improved control of infection and improved CAR T cell outcomes.^23–25 T-bet overexpression increased the proportion of 1928 CAR8s and 19BB CAR4s and CAR8s producing all three cytokines (figure 1L and M). These results demonstrate that T-bet overexpression increases Th1-associated cytokine production from human CAR T cells costimulated with either CD28 or 41BB.

T-bet overexpression enhances in vitro CAR T cell effector functions but does not improve potency against high-antigen-expressing leukemia

T-bet plays a central role in determining the differentiation trajectory of T cells into effector cells.^{26 27} Thus, we next looked at the impact of T-bet overexpression on other effector functions. T-bet-overexpressing 1928 CAR4s and CAR8s both degranulated at a higher frequency after co-culture with NALM6. Similar proportions of Tbet-19BB CAR T cells degranulated relative to controls (figure 2A and B). Proliferation in response to NALM6 was similar in Tbet-CAR T cells relative to controls (figure 2C and D). 1928 but not 19BB Tbet-CAR T cells mediated improved in vitro killing of NALM6 (figure 2E and F). Thus, T-bet overexpression has a modest impact on in vitro effector function. Interestingly, this was more pronounced in CD28-costimulated CARs.

Figure 2. T-bet enhances CAR T cell effector functions but not in vivo efficacy. (A) CD107a expression on NGFR + EGFR + 1928 CAR4s and CAR8s after 6-hour co-culture with NALM6. Lines connect CAR T cells manufactured from the same donor. (B) CD107a expression on NGFR + EGFR + 19BB CAR4s and CAR8s after 6-hour co-culture with NALM6. Lines connect CAR T cells manufactured from the same donor. (C) Percent of 1928 NGFR + EGFR + CAR T cells in the starting culture that went into divisions as measured by CellTrace Violet dye dilution after 72-hour co-culture with NALM6. (D) Percent of 19BB NGFR + EGFR + CAR T cells in the starting culture that went into divisions as measured by CellTrace Violet dye dilution after 72-hour co-culture with NALM6. (E) Representative specific lysis curve of NALM6 leukemia cells after 15-hour co-culture with 1928 CAR T cells. (F) Representative specific lysis curve of NALM6 leukemia cells after 15-hour co-culture with 1928 CAR T cells. (G-J) NSG mice were injected with 1×10 6 NALM6 on day -4 and treated with 1×10 6 1928 CAR+T cells on day 0. Cell dose was determined based on NGFR expression. (G) Representative IVIS images from one experimental replicate (H) Flux data as measured by IVIS imaging for each individual mouse pooled from experiments using two different T cell donors (I) Flux at day 11 and day 18 after CAR T cell transfer. (J) Survival of mice after CAR T cell transfer pooled from experiments using two different T cell donors. (K-N) NSG mice were injected with 1×10 6 NALM6 on day -4 and treated with 1×10 6 19BB CAR+T cells on day 0. Cell dose was determined based on NGFR expression. (K) Representative IVIS images from one experimental replicate (L) Flux data as measured by IVIS imaging for each individual mouse pooled from experiments using two different T cell donors (M) Flux at day 11 and day 18 after CAR T cell transfer. (N) Survival of mice after CAR T cell transfer pooled from experiments using two different T cell donors. Experiments contain data from three independent donors each collected in triplicate except for survival curves and flux data (H-J, L-N) which are pooled from two independent experiments with 4-5 mice per group per experiment each using CAR T cells manufactured from two different donors. n=10 mice total in each group. Statistical analyses are nested t-tests (A-D), two-way ANOVA with Sidak’s multiple comparisons tests (E-F), Mann-Whitney tests (I, M) and Log-rank Mantel-Cox tests (J, N). Data represent mean+/-SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; CAR, chimeric antigen receptor; E:T, effector to target cell ratio; IVIS, In Vivo Imaging System; ns, not significant.

We next evaluated Tbet-CAR T cells in immunodeficient mice engrafted with NALM6, a setting where clearance of tumor is dependent on both effector function and capacity for expansion. When treating with a stress dose of bulk CAR T cells consisting of CAR4s and CAR8s, overexpression of T-bet had a negative impact on in vivo leukemia control and survival in 1928 CAR T cells (figure 2G–J) and no impact on leukemia control or survival in 19BB CAR T cells (figure 2K–N). Collectively, these data demonstrate that overexpression of T-bet can skew CAR T cells towards Th1 cytokines but with minimal overall effect on activity against leukemia expressing high antigen levels.

Murine T-bet overexpressing CAR T cells show Th1 skewing and enhanced in vitro functions

Increased potency regulated by T-bet can be associated with terminal effector differentiation.²⁸ Thus, we hypothesized that T-bet could induce differentiation into short-lived effectors at the expense of persistence leading to inability to control leukemia long-term. We used a syngeneic mouse model to test this hypothesis in an immune-competent setting where xenoreactivity would not be a confounding factor in the assessment of response durability. To first determine whether T-bet overexpression had similar impacts on murine CAR T cells as observed in human CAR T cells, we cotransduced T-bet into murine T cells with a murine CD19-targeting CAR²⁸ with a CD28 costimulatory domain (figure 3A and B). As with human Tbet-CAR T cells, murine Tbet-overexpressing CAR4s produced IL-4 at a lower frequency and IFNγ at a higher frequency when stimulated with the B6-derived B-ALL line E2A-PBX relative to controls with no impact on IFNγ in Tbet-overexpressing CAR8s (figure 3C–E). Similarly, T-bet enhanced degranulation of CAR4s but not CAR8s (figure 3F). We used several different constructs to overexpress RORγt in murine CAR T cells resulting in RORγt expression at 2.5×–7× over controls (online supplemental figure S1A). When RORγt was overexpressed at a sufficient level, CAR4s produced IL-17 (online supplemental figure S1B–I). Interestingly, we observed that RORγt-containing transgene expression did not track with functional readouts in vitro as most cells that were stimulated by CD19 on target cells did not express EGFR, suggesting EGFR under-reported CAR expression in this construct (online supplemental figure S1J).

Figure 3. T-bet overexpressing CAR T cells mediate tumor clearance with no clear deficit in the formation of memory precursors. (A) Schematic of murine CAR and T-bet constructs. (B) T cells were isolated from naïve splenocytes and cotransduced with a murine CAR and T-bet-GFP. (C-E) T cells were co-cultured with E2A-PBX for 6 hours. (C) IL-4 production from murine CAR4s as detected by intracellular cytokine staining. (D) IFN[gamma] production from CAR4s and CAR8s as detected by intracellular cytokine staining. (F) CD107a expression on CAR T cells after 4-hour co-culture with E2A-PBX. (G) Quantification of E2A-PBX in the tibia of leukemia-bearing mice 10 days after treatment with 3×10 5 CAR+T cells. (H) Schematic of tri-cistronic CAR constructs. (I) Representative CAR expression on T cells. (J) T-bet GMFI on CAR+T cells. (K-U) C57Bl6/J CD45.1 hosts were engrafted with 1×10 6 E2A-PBX leukemia cells on day -4, sublethally irradiated on day -1, and given 1×10 6 CAR T cells on day 0. Bone marrow was harvested at days 4 and 11 after CAR T cell transfer. (K) Staining of endogenous B cells (left gate) and E2A-PBX leukemia cells (right gate) in the bone marrow. (L) Number of CAR4s per tibia. (M) Number of CAR8s per tibia. (N) FOXO1 GMFI on CAR+T cells. (O) TCF1 GMFI on CAR+T cells. (P) Expression of CD122 on CAR+T cells. (Q) Expression of IL7R[alpha] and KLRG1 on CAR+T cells. (R) Quantification of IL7R[alpha]+ CAR T cells. (S) Quantification of KLRG1+CAR T cells. (T) Expression of CD62L CAR+T cells. (U) Eomes expression on CAR+T cells. Statistics are t-tests (D-G) or Welch’s t-tests (K-U). Data are pooled from two to three independent experiments with 4-5 mice per group per experiment. Each data point represents one replicate for in vitro studies (D-G) or one mouse for in vivo studies (K-U). Data represent mean+/-SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. CAR, chimeric antigen receptor; GMFI, geometric mean fluorescence intensity; IFN[gamma], interferon gamma; IL, interleukin; ns, not significant; PD1, programmed cell death protein-1.

T-bet overexpressing CAR T cells mediate comparable expansion and clearance of high antigen-expressing leukemia

When transferred into E2A-PBX leukemia-bearing mice, which express high levels of CD19, T-bet CAR T cells mediated equivalent clearance of leukemia compared with mice receiving control CAR T cells (figure 3G), consistent with human Tbet-CAR T cells. To test the effect of T-bet on CAR T cell differentiation along the memory/effector axis in vivo, we generated a homogeneous starting population with concordance of CAR and T-bet expression using a multicistronic construct containing T-bet followed by a CD19 CAR with an EGFR reporter (Tbet-CAR) (figure 3H). Tbet-CAR T cells expressed CAR at a similar level to T cells with CAR alone (figure 3I) and approximately 3.5 times more T-bet than T cells expressing CAR alone (figure 3J). Tbet-CAR T or control CAR T cells generated from CD45.2 T cells were adoptively transferred into E2A-PBX (CD45.2) leukemia-bearing syngeneic CD45.1 mice. Bone marrow was analyzed at day 4 post-CAR T cell transfer, representing the time of peak CAR T cell expansion, and day 11, after antigen clearance and CAR T cell contraction. By day 4 post-CAR T cell transfer, both CARs cleared endogenous B cells and leukemia, maintained through day 11 (figure 3K). At day 4 post-CAR T cell transfer, Tbet-CAR4s expanded to a greater extent than control CAR4s, maintained through day 11, with no difference in the number of CAR8s (figure 3L and M). Interestingly, CAR and RORγt transgene expression was not detected following adoptive transfer of RORγt-overexpressing CAR T cells into leukemia-bearing mice despite sustained clearance of leukemia and persistent B cell aplasia indicating the presence of functional CAR T cells while suggesting that RORγt plays a role in repressing expression of genes encoded by the transgene by an unknown mechanism (online supplemental figure S1K–Q).

Th1 overexpressing does not impair CAR T cell memory precursor formation

The transcription factors FOXO1 and TCF1 are both important for the establishment of long-lived memory T cell populations and regulate the expression of other memory-associated markers.²⁹ T-bet overexpression did not impact the number of FOXO1+CAR T cells (figure 3N) or the amount of TCF1 expressed (figure 3O). IL-7 and IL-15 are necessary for the maintenance of memory T cell populations.^{30 31} By day 11 CD122, which encodes the IL-15Rβ chain also used by IL-2, was expressed at higher levels on Tbet-CAR T cells (figure 3P), suggesting an increased ability to respond to survival signals mediated by IL-15. In contrast, fewer Tbet-CAR T cells expressed IL7Rα, suggesting a decreased ability to respond to IL-7-mediated survival signals (figure 3Q and R). However, Tbet-CAR T cells did not display a higher proportion of KLRG1+effector cells, suggesting Tbet-CAR T cells are not more skewed towards short-lived effector cells (figure 3Q and S). CD62L, a marker of central memory T cells,³² was expressed on a higher proportion of Tbet-CAR T cells at day 4, but this difference was lost by day 11 (figure 3T). The transcription factor Eomes, along with T-bet, controls the differentiation of T cells into SLECs or MPECs, with higher amounts of Eomes associated with the formation of MPECs and higher amounts of T-bet associated with the formation of SLECs.^{27 33} A smaller proportion of Tbet-CAR T cells expressed Eomes than control CAR T cells (figure 3U). Overall, Tbet-CAR T cells do not display obvious deficits in the expression of transcription factors that control T cell persistence and capacity for self-renewal.

T-bet overexpression does not prevent long-term persistence or functional memory responses

We next looked at the ability of Tbet-CAR T cells to persist well beyond tumor clearance. To reduce the impact of B cell precursors in the bone marrow after leukemia clearance on CAR T cell persistence, Rag-deficient mice were engrafted with E2A-PBX leukemia and treated with Tbet-CAR or control CAR T cells (figure 4A). By 24 days post CAR T cell transfer, mice treated with either Tbet-CAR or control CAR T cells had cleared leukemia (figure 4B), whereas all mice treated with mock T cells had succumbed to leukemia by 13 days post CAR T cell transfer (data not shown). Tbet-CAR T cells persisted equally to control CAR T cells at day 24 (figure 4C and D). As in wild-type (WT) hosts (figure 3J), Tbet-CAR and control CAR T cells expressed similar levels of FOXO1 (figure 4E). However, consistent with day 11 post-transfer in WT recipients (figure 3), fewer Tbet-CAR T cells expressed Eomes and IL7Rα, with comparable CD62L expression and higher expression of CD122 (figure 4F–J). Long-term survival of mice treated with Tbet-CAR T cells was no different compared with receiving control CAR T cells (figure 4K). This demonstrates that T-bet does not impair functional CAR T cell persistence beyond clearance of leukemia.

Figure 4. T-bet overexpression does not hinder CAR T cell responses to a secondary leukemia challenge. (A) Experimental setup. Rag KO mice were engrafted with 1×10 6 E2A-PBX on day -4 and treated with 2×10 6 CAR+T cells on day 0. Bone marrow was harvested and analyzed by flow cytometry 24 days after CAR T cell transfer. (B) Presence of E2A-PBX in the bone marrow and quantification of the number of E2A-PBX per tibia. (C) Quantification of CAR4s per tibia, as detected by EGFR expression. (D) Quantification of CAR8s per tibia, as detected by EGFR expression. (E) FOXO1 expression on CAR T cells. (F) Eomes expression on CAR T cells. (G) Expression of IL7R[alpha] and CD62L on CAR T cells. (H) Quantification of IL7R[alpha]+ CAR T cells. (I) Quantification of CD62L+CAR T cells. (J) CD122 expression on CAR T cells. (K) Survival of CAR T cell-treated mice after CAR T cell transfer. (L) Experimental setup. Rag KO mice were engrafted with 1×10 6 E2A-PBX on day -4 and given 2×10 6 CAR+T cells on day 0. On day 26 after CAR T cell transfer, mice were rechallenged with 2.5×10 6 E2A-PBX. Bone marrow was harvested and analyzed by flow cytometry 10 days after rechallenge. (M) Presence of E2A-PBX in the bone marrow. (N) Quantification of CAR4s per tibia, as detected by EGFR expression. (O) Quantification of CAR8s per tibia, as detected by EGFR expression. (P) Expression of FOXO1 on CAR T cells. (Q) Expression of TCF1 on CAR T cells. (R) Expression of IL7R[alpha] and CD62L on CAR T cells. (S) Quantification of IL7R[alpha]+ CAR T cells. (T) Quantification of CD62L+CAR T cells. (U) Expression of Eomes on CAR T cells. (V) Proportion of CAR T cells that are PD1+TIM3+. (W) Survival of CAR-treated mice after leukemia rechallenge. Data are combined from two independent experiments with 4-5 mice per group per experiment. Total n=10 mice for CAR group and n=9 mice for Tbet-CAR group. Statistical analyses are Mann-Whitney tests (B-F, H-J, M-Q, S-V) or Log-rank Mantel-Cox tests (K, W). Data represent mean+/-SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. BM, bone marrow; CAR, chimeric antigen receptor; GMFI, geometric mean fluorescence intensity; IL, interleukin; ns, not significant; PD1, programmed cell death protein-1.

We next treated leukemia-bearing mice with Tbet-CAR or control CAR T cells followed by rechallenge with E2A-PBX at 26 days after CAR T cell transfer (figure 4L). By 13 days post-rechallenge, neither group of mice had detectable E2A-PBX present in the bone marrow (figure 4M) and Tbet-CAR and control CAR T cells were present in equivalent numbers overall in the bone marrow (figure 4N and O). The expression of memory-associated markers TCF1, FOXO1, CD62L, CD122, and IL7Rα on CAR T cells was equivalent between groups (figure 4P–T). Only Eomes was differentially expressed between groups and remained expressed in a higher number of control CAR T cells than Tbet-CAR T cells (figure 4U). The fraction of PD1+TIM3+ CAR T cells was no different in Tbet-CAR T cells than in control CAR T cells (figure 4V), which together with the ability to clear the leukemia rechallenge suggests T-bet overexpression does not drive exhaustion of CAR T cells. There was no difference in the survival of mice treated with control CAR or Tbet-CAR T cells after rechallenge (figure 4W). Taken together, this indicates that, despite some impact on phenotype, T-bet overexpression does not impair functional CAR persistence.

T-bet overexpression improves CAR T cell potency against low-antigen leukemia

We next hypothesized T-bet’s inability to improve in vivo CAR T cell responses (figures 2 and 4D, E) may be masked by high signal strength in the setting of high antigen. Thus, we next tested the impact of T-bet overexpression on human CAR T cell responses against NALM6 with variable CD19 on the surface (approximately 16,500 and 2,200 CD19 molecules per cell, compared with 37,000 on WT NALM6) (figure 5A). T-bet overexpression increased the proportion of CAR T cells producing IFNγ, IL-2 or TNF-α (figure 5B and C). In 1928 Tbet-CAR T cells, this was associated with increased production of each of the three cytokines (online supplemental figure S2A–C), while in 19BB CAR T cells, this was primarily associated with increased proportions of cells producing IFNγ and TNF-α but not IL-2 (online supplemental figure S2D–F). T-bet overexpression promoted increased degranulation of both 1928 and 19BB CAR4s and CAR8s against CD19mid and CD19lo clones (figure 5D and E). Furthermore, an increased proportion of 1928 and 19BB Tbet-CAR T cells divided in response to CD19lo leukemia (figure 5F and G). Together, our data suggest T-bet overexpression can recruit a higher proportion of CAR T cells into the pool of functional responders against low antigen-expressing tumors.

Figure 5. T-bet overexpression enhances responses against CD19lo leukemia. (A) CD19 was knocked out of NALM6 leukemia and reintroduced to generate NALM6 lines with varying CD19 antigen levels. (A) Expression and quantification of CD19 molecules/cell on NALM6 cell lines. (B-C) Quantification of the proportion of (B) 1928 and (C) 19BB NGFR+EGFR+ CAR T cells producing at least one of the cytokines IFN[gamma], TNF-[alpha], and IL-2 after 15-hour co-culture with NALM6 by intracellular cytokine staining. (D-E) Quantification of the proportion of (D)1928 and (E)19BB NGFR+EGFR+ CAR T cells expressing CD107a after a 4-hour co-culture with NALM6. (F-G) Quantification of the precursor frequency of (F)1928 and (G)19BB NGFR+EGFR+ CAR T cells that went into divisions as determined by CellTrace Violet staining after a 72-hour co-culture with NALM6. (H-K) NSG mice were injected with 1×10 6 CD19lo NALM6 on day -4 and treated with 1×10 6 1928 CAR+T cells on day 0. Cell dose was determined based on NGFR expression. (H) Representative IVIS images from one experimental replicate (I) Flux data as measured by IVIS imaging for each individual mouse pooled from experiments using two different T cell donors. (J) Quantification of flux at day 11 after CAR T cell transfer. (K) Survival of mice after CAR T cell transfer pooled from experiments using two different T cell donors. (L-O) NSG mice were injected with 1×10 6 CD19lo NALM6 on day -4 and treated with 1×10 6 19BB CAR+T cells on Day 0. Cell dose was determined based on NGFR expression. (L) Representative IVIS images from one experimental replicate. (M) Flux data as measured by IVIS imaging for each individual mouse pooled from experiments using two different T cell donors. (N) Flux at day 11 and day 18 after CAR T cell transfer. (O) Survival of mice after CAR T cell transfer pooled from experiments using two different T cell donors. Survival curves and flux data are pooled from two independent experiments with a total of n=10 mice per condition. Statistical analyses are two-way ANOVA with Sidak’s multiple comparisons tests (B-G) and each data point consists of CAR T cells generated from three separate donors, with data collected in triplicate. Statistical analyses of flux data are Mann-Whitney tests (J, M). Statistical analyses of survival experiments are log-rank Mantel-Cox tests (K,O). Data represent mean+/-SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; CAR, chimeric antigen receptor; IFN[gamma], interferon gamma; IL, interleukin; IVIS, In Vivo Imaging System; ns, not significant.

We next evaluated the impact of T-bet overexpression on CAR T cell efficacy against CD19lo leukemia in vivo. At a stress dose of bulk T cells containing CAR4s and CAR8s, T-bet overexpression did not impact 1928 CAR T cell-mediated clearance of CD19lo leukemia (figure 5H–J) or long-term survival (figure 5K). However, 19BB Tbet-CAR T cells demonstrated superior tumor clearance against CD19lo leukemia compared with controls (figure 5L–N), leading to prolonged survival (figure 5O) which has been shown to have weaker signaling and inferior antigen sensitivity compared with CD28-costimulated CAR T cells.^{2 34} In line with the ability of mice to clear leukemia rechallenge, the lack of a survival deficit suggests T-bet overexpression does not promote CAR T cell dysfunction.

Discussion

Despite the success of CAR T cells, strategies to prevent relapses and to broaden the applicability of CAR T cell therapies against tumors other than B cell-lineage malignancies are both challenges. Relapses with sustained antigen expression as well as those associated with tumor expressing decreased antigen density are common in B lineage malignancies.³⁵ Thus, improving CAR T cell potency, especially in settings of low antigen, has the potential to improve the durability of CAR T cell-induced remissions. The conditions in which CAR T cells are manufactured, including the cytokine milieu, impact the phenotype and functionality of CAR T cells.²⁰ CAR T cells are generally manufactured using CD3 activation, generally with a costimulatory signal, in a non-antigen-specific manner with IL-2, IL-15, and/or IL-7, bypassing the T cells’ interaction with APCs and the cytokine milieu conventional T cells experience on activation in vivo during which contextual cytokine signals instruct CD4 T cell differentiation. Interestingly, in both human and murine CAR T cells, we and others observed populations of CAR4s that simultaneously produce both IFNγ and IL-4²¹ further underscoring that CAR4s may not differentiate into canonical CD4 T cell subsets.

Recent work has demonstrated a negative role of IL-4 in CAR T cell function, identifying IL-4 signaling as an inducer of CAR T cell dysfunction, and IL-4 neutralization as a strategy to improve CAR T cell persistence.³⁶ In line with other reports,^{21 36} we observed IL-4 production from CAR4s on stimulation. Furthermore, Th1-polarized T cells have been shown to demonstrate lineage plasticity and potential conversion when later exposed to Th2-polarizing conditions and high levels of T-bet were necessary to maintain the Th1 phenotype when exposed to IL-4.³⁷ In this study, we sought to enhance the commitment of CAR4s to a Th1-like phenotype and mitigate potential conversion to a Th2-like phenotype and to enhance effector functions of CD8+CAR T cells (CAR8s) by overexpressing T-bet in CAR T cells.³⁷ In addition to a reduction of IL-4 produced by CAR4s, we observed greater Th1 effector functions from CAR4s overexpressing T-bet, including increased IFNγ, IL-2, and TNF-α, production, increased degranulation, and increased proliferation, especially against low-antigen-expressing leukemia. The increased CAR4 proliferation translated in vivo into a syngeneic model, where we saw increased expansion of Tbet-CAR4s. These results demonstrate the ability to modulate CAR T cell cytokine production to more closely mirror canonical CD4 T cell subsets via overexpression of lineage-specifying transcription factors.

We chose to overexpress T-bet in CAR8s as well, as signal 3 cytokines IL-12 and type I IFN, which upregulate T-bet expression, promote optimal CD8 T cell proliferation, cytotoxicity, and improve antigen sensitivity.^38–40 We did not observe increased degranulation or proliferation of CAR8s in response to high-antigen leukemia, possibly due to an already large proportion of T cells responding. However, T-bet overexpression improved CAR8 cytokine production and polyfunctionality against WT leukemia, and improved CAR8 cytokine production and polyfunctionality, degranulation, and proliferation in response to low-antigen leukemia.

The fate decisions of CD8 T cells along the terminally differentiated effector/long-lived memory cell axis are canonically understood to depend on the balance of T-bet and Eomes with higher amounts of T-bet leading to a terminal effector state and more Eomes leading to memory cell formation.^{27 41} This led us to hypothesize that T-bet overexpression may be improving effector functions by promoting terminal effector differentiation at the expense of memory formation, leading to reduced CAR T cell persistence and failure to control leukemia long-term. However, in a syngeneic model we demonstrate that Tbet-CAR T cells displayed equivalent functional persistence to mediate long-term survival. While we did not test all characteristics of T cell memory such as dependence on IL-7 or IL-15 for survival, Tbet-CAR T cells were able to re-expand and clear a leukemia rechallenge and mediate long-term survival of the rechallenged mice. This demonstrates that T-bet overexpression did not drive differentiation of CAR T cells solely into short-lived terminal effector cells, and that Tbet-CAR T cells were able to adopt characteristics of memory T cells. These data support the growing body of evidence that CAR T cell fate decisions along the memory/effector axis may not be regulated in the same way as in conventional T cells responding to infection or vaccination.

The level of T-bet expressed is similar between 1928-Tbet and 19BB CAR T cells and does not account for the difference in efficacy observed with different costimulatory domains. It is established that there is a “Goldilocks zone” of signal intensity that promotes optimal T cell antitumor activity.⁴² CD28-costimulated CAR T cells are known to signal with a higher intensity and more rapid kinetics than their 4-1BB-costimulated counterparts.² In settings of high signal intensity, such as a 1928 CAR against WT leukemia which expresses high levels of antigen, T-bet overexpression reduces overall efficacy in vivo. In settings of moderate signal intensity, such as 1928 CAR T cells against CD19lo leukemia or 19BB CAR T cells against WT leukemia, overall efficacy is not impacted. In settings of low CAR signal intensity, such as a 19BB CAR T cell against CD19lo leukemia, overall efficacy is improved. The enhancement of most in vitro effector functions with T-bet overexpression seen in 1928 and 19BB CAR T cells in short-term assays, paired with the observed results of our in vivo experiments, suggests a model in which T-bet overexpression increases signal intensity through the CAR, improving the efficacy of CAR T cells that receive a low-intensity signal and decreasing the efficacy of CAR T cells that receive a high-intensity signal. This model accounts for the T-bet-mediated differences in in vivo efficacy observed in 1928 and 19BB CAR T cells, demonstrating that T-bet overexpression is beneficial in the settings of low CAR signal intensity, and therefore may be an effective strategy to improve the efficacy of 4-1BB CAR T cells which have been shown to have reduced antigen sensitivity compared with CD28-costimulated CARs.

Exactly how T-bet modulates CAR T cell responsiveness to low levels of antigen requires further study. One hypothesis involves the integrins LFA-1 and ICAM-1, expressed by T cells and NALM6, respectively. CAR T cells are less efficient than conventional T cells at exploiting accessory receptors but increasing the LFA1-ICAM1 adhesion axis improved antigen sensitivity of CAR T cells.¹¹ T-bet promotes the expression of LFA-1 on T cells, potentially increasing T cell binding to ICAM-1 on leukemia cells, thus helping to stabilize the T cell:leukemia cell interaction, increasing the duration of CAR signaling, amplifying the signaling intensity and resulting in activation of a higher proportion of CAR T cells.

In summary, this work demonstrates that T-bet overexpression in CAR T cells is a viable strategy to enhance type 1 while reducing type 2 immune responses in CAR T cells. It also demonstrates a role for T-bet in enhancing CAR T cell effector functions without detriment to the ability of T-bet-overexpressing CAR T cells to persist and respond to a secondary leukemia challenge. Most importantly, our work implicates T-bet as playing a role in antigen sensitivity, and T-bet overexpression may be a viable strategy to enhance 4-1BB CAR T cell responses against tumor antigens expressed at low levels and prevent outgrowth of antigen-low tumor cells.

Methods

Mouse strains

SJL-Ptprca Pepcb/BoyJ (“B6-CD45.1,” Strain #:002014), B6.129S7-Rag1tm1Mom/J (“Rag1^KO,” Strain #:002216), C57BL/6J mice (“B6,” Strain #:000664), and C57BL/6Tg(Nr4a1-EGFP/cre)820Khog/J (“NUR77-GFP reporter mice”, Strain #:016617) were obtained from The Jackson Laboratory. All experiments were performed using female mice. NSG mice (NOD-scid-gamma, NOD.Cg-Prkdc^scidIl2rgtm1Wjl/SzJ; Strain #: 005557) were obtained from The Jackson Laboratory. All mice were bred and/or maintained in the animal facility at University of Colorado Anschutz Medical Campus.

Cell lines

The E2A-PBX pre-B-ALL line was generated in the lab as previously described.⁴³ CD19− E2aPbx variants were made by CRISPR/CAS9–mediated mutation of the CD19 locus. All murine cell lines and primary mouse T cells were cultured in complete mouse media consisting of Roswell Park Memorial Institute (RPMI) 1640 with 10% heat-inactivated fetal calf serum, 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin/streptomycin, 1% L-glutamine (Invitrogen), and 1% HEPES buffer (Sigma-Aldrich) and 1 mL of 2-mercaptoethanol (Sigma-Aldrich). The parental NALM6 cell line was obtained from American Type Culture Collection and was transduced to express GFP and luciferase. NALM6 were cultured in R10 (RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, 100 U/mL streptomycin and 2 mM GlutaMAX).

Human CAR constructs

The second generation 1928 CAR consists of the FMC63 ScFv, cd28 hinge and transmembrane domain and the signaling domains of CD28 and CD3ζ, followed by a 2A sequence and a truncated non-signaling NGFR. The second generation 19BB CAR consists of the FMC63 ScFv, CD8α hinge and transmembrane domain and the signaling domains of 4-1BB and CD3ζ, followed by a 2A ribosomal skip sequence and a truncated non-signaling NGFR. The T-bet overexpression construct consists of T-bet followed by a 2A ribosomal skip site and a truncated non-signaling EGFR. The EGFR construct does not contain a ribosomal skip site.

Human CD19 CAR T cell generation

Healthy human donor whole blood obtained from the Children’s Hospital Colorado Blood Donor Center, under an institutional board-approved protocol. Peripheral blood mononuclear cells were isolated using Lymphocyte Separation Medium (Corning) according to manufacturer protocol. T cells were then purified using an EasySep Human T Cell Isolation Kit (STEMCELL) and cryopreserved in 90% heat-inactivated FBS and 10% dimethyl sulfoxide (DMSO). Lentiviral vectors encoding CD19 CAR constructs were generated by transient transfection of the Lenti-X viral producer cell line with the CAR constructs, as well as lentiviral packaging and envelope plasmids (pMDLg/pRRE, pMD.2G, and pRSV-Rev, all obtained from Addgene) using Lipofectamine-3000 (Thermo Fisher) in Opti-MEM (Gibco). Media was removed and replaced 6 hours after transfection. Supernatant containing lentiviral vector was harvested 24 and 52 hours post-transfection and pooled and spun at 3,000 rotations per minute (RPM) for 10 min to remove cell debris and frozen at −80°C for later use. Healthy human T cells were thawed on day 0 and resuspended at 1e6/mL in human T cell expansion media (hTCEM: made up of AIM V medium (Gibco), supplemented with 5% heat-inactivated FBS, 2 mmol/L L-glutamine, 10 mmol/L HEPES), and 40 IU/mL recombinant human IL-2 (R&D Systems), and activated for 48 hours with CD3/CD28 Human T Activator Dynabeads (Gibco) at a cell:bead ratio of 1:3. On day 2, T cells were transduced with lentivirus in the presence of 10 ug/mL protamine sulfate (Sigma) and 40 IU/mL IL-2 (R&D Systems), spinning for 2 hours at 1,000×G. On day 3, CD3/CD28 Human T activator microbeads were removed using a magnetic rack and T cells were resuspended at 0.5e6/mL in hTCEM with 100 IU/mL recombinant human IL-2. T cells were expanded for 5–6 more days, with new media and cytokines added every other day. Following expansion, the transduction efficiency of the CD19-CAR was evaluated by flow cytometry staining with NGFR-AF647 (BioLegend) and CAR T cells were cryopreserved or used immediately for in vitro or in vivo assays.

Generation of CD19^Lo NALM6 leukemia cell lines

Details regarding the generation of CD19^Lo NALM6 clones are available in online supplemental methods.

Mouse CAR constructs and transcription factors

The CD19 mouse CAR has been previously described⁴⁴ and consists of the 1D3 ScFv, a CD28 hinge/transmembrane domain, and the signaling domains of CD28 and CD3ζ followed by a 2A ribosomal skip site and a truncated non-signaling human EGFR. The generation of T-bet-IRES-GFP has been described previously.¹⁶ A codon-optimized murine T-bet sequence codon-optimized murine was ordered from Thermo Fisher GeneArt and cloned into the MSCV backbone to generate tricistronic CAR constructs. A codon-optimized murine T-bet sequence was ordered from Thermo Fisher GeneArt and cloned into the MSCV backbone to generate tricistronic CAR constructs. MIGR-RORγt was a gift from Dan Littman (Addgene plasmid # 24069; http://n2t.net/addgene:24069; RRID:Addgene_24069). A codon-optimized murine RORγt sequence was ordered from Thermo Fisher GeneArt and cloned into the MSCV backbone to generate tricistronic CAR constructs.

Generation of murine CD19 CAR T cells and syngeneic in vivo studies

Murine CAR T cells were generated and tested in vivo as previously described.^{44 45} Additional details are available in online supplemental methods.

Flow cytometry

Flow cytometry was performed using an LSRFortessa X-20 flow cytometer (BD Biosciences) and analyzed using FlowJo (BD Biosciences). Antibodies and dyes used in staining are listed in the supplemental methods (online supplemental tables 1 and 2). Intracellular flow cytometry staining was performed using the Transcription Factor Buffer Set (BD Biosciences) for in vitro and ex vivo staining of transcription factors according to the manufacturer’s protocol, or Cytofix/Cytoperm Fixation/Permeablization Kit (BD Biosciences) for intracellular cytokine staining (ICCS).

CD107a degranulation assays

Degranulation assays were performed using a 1:1 effector to target cell ratio (E:T) with 1e5 of each cell type in a 96-well round-bottom plate. Degranulation assays incubated for 6 hours (murine CAR T cells) or 4 hours (human CAR T cells) in the presence of 2 uM monensin and 1 uL of CD107a antibody. After incubation, cells were stained for surface markers and analyzed by flow cytometry.

ICCS assays

ICCS assays were performed using a 1:2 (murine CAR T cells) or 1:1 (human CAR T cells) E:T with 1–2×10⁵ of each cell type in a 96-well round-bottom plate. Cells were incubated for 6 hours (murine CAR T cells) or 15 hours (human CAR T cells) in the presence of 1 uM monensin and 2.5 µM Brefeldin A. After incubation, cells were stained for surface markers, fixed and permeabilized using Cytofix/Cytoperm buffer set (BD Biosciences), stained for cytokines, and analyzed by flow cytometry.

Cytotoxicity assays

Cytotoxicity assays were performed by adding 5×10⁴ luciferase-expressing NALM6 and the correct number of CAR+T cells for the desired E:T ratio in a total volume of 200 µl R10 to each well of a 96-well white plate. Cells were incubated for 15 hours. After incubation, D-luciferin was added to each well and luminescence signal was detected by a Tecan microplate reader. The percent specific lysis per well was calculated by determining the percent of luminescence lost in each condition compared with NALM6 only controls.

Proliferation assays

T cells were labeled with a 2.5 µM CellTrace Violet solution according to manufacturer’s protocol. 2×10⁴ CAR+T cells and 2×10⁴ NALM6 cells were added to a 96-well plate in a total volume of 200 ul and incubated for 72 hours. After incubation, cells were collected and stained for surface markers and analyzed by flow cytometry. The percent of cells divided was calculated as previously described.⁴⁶

Xenograft survival studies

Female NSG mice aged 6–23 weeks old were injected intravenously with 1×10⁶ luciferase+NALM6 cells on day −4. Leukemia progression was monitored by bioluminescent imaging using a Xenogen In Vivo Imaging System (IVIS) after intraperitoneal injection of firefly luciferin (PerkinElmer). Survival was calculated based on predefined endpoints of leukemia or xenogeneic graft versus host disease. Further details are provided in online supplemental methods.

Statistical analysis

All statistics were calculated using GraphPad Prism. Data are reported as mean±SE of the mean. Statistical tests for each experiment are described in figure legends.

We would like to thank the CU Anschutz OLAR and the animal facility for their support. Portions of this work have been presented previously (Cimons, 2023 #314; Cimons, 2023 #313).

Data availability statement

Data are available upon reasonable request. All data relevant to the study are included in the article or uploaded as supplementary information.

Ethics statements

Patient consent for publication

Not applicable.

Ethics approval

All experiments were performed in compliance with the study protocol approved by University of Colorado Anschutz Medical Campus Institutional Animal Care and Use Committee (IACUC, protocol no: 00751).

Footnote

Contributors JMC conceptualized the studies, performed experiments and data analysis and wrote the paper. KRD conceptualized the studies, generated reagents, and provided expertise related to the mouse CAR and leukemia models. SDB performed experiments. MCY designed and generated DNA constructs. AJN performed experiments. AMR-R performed experiments. EK conceptualized the studies and provided expertise related to the mouse CAR and leukemia models. TJF conceptualized, supervised and acquired funding for the studies and wrote the paper. TJF is the guarantor of this manuscript.

Funding This work was funded by the University of Colorado School of Medicine, and by Hyundai Hope on Wheels.

Competing interests TJF is currently a consultant at Sana Biotechnology.

Provenance and peer review Not commissioned; externally peer reviewed.

Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

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