Calibration of CAR activation potential directs alternative T cell fates and therapeutic potency

Abstract

Chimeric antigen receptors (CARs) are synthetic receptors that target and reprogram T cells to acquire augmented antitumor properties¹. CD19-specific CARs that comprise CD28 and CD3ζ signaling motifs² have induced remarkable responses in patients with refractory leukemia^3-5 and lymphoma⁶ and were recently approved by the US Food and Drug Administration⁷. These CARs program highly performing effector functions that mediate potent tumor elimination^4,8 despite the limited persistence they confer on T cells^3-6,8. Extending their functional persistence without compromising their potency should improve current CAR therapies. Strong T cell activation drives exhaustion^9,10, which may be accentuated by the redundancy of CD28 and CD3ζ signaling^11,12 as well as the spatiotemporal constraints imparted by the structure of second-generation CARs². Thus, we hypothesized that calibrating the activation potential of CD28-based CARs would differentially reprogram T cell function and differentiation. Here, we show that CARs encoding a single immunoreceptor tyrosine-based activation motif direct T cells to different fates by balancing effector and memory programs, thereby yielding CAR designs with enhanced therapeutic profiles.

Extended Data Fig. 1 ∣. Impact of ITAM-mutated 1928ζ CARs on T cell function in vitro, T cell differentiation and antitumor activity in vivo.

a, Cytotoxic activity as determined by 4-h ⁵¹Cr release assay 1 week after expansion of effector cells on irradiated 3T3-CD19 (data are shown as means of n = 2 independent experiments performed in triplicates). b, Cumulative cell counts of indicated CAR T cells upon weekly stimulation with CD19⁺ target cells (n = 3 independent experiments). All data are means ± s.e.m. P values were calculated with two-tailed paired Student′s t-test. c, NALM6-bearing mice were treated with 5 × 10⁴ CAR⁺ T cells. Kaplan–Meier analysis of survival comparing the in vivo efficacy of wild-type 1928ζ or indicated 1928ζ mutants (n = 10 mice, pooled data from two independent experiments). Control (Ctl) refers to untreated mice (n = 6). P value was determined by a one-sided log-rank Mantel–Cox test. d, Phenotype of CAR T cells as demonstrated by percentage of central memory (CD62L⁺CD45RA⁻) and effector memory (CD62L⁻CD45RA⁻) CD4⁺CAR T cells 48 h upon second stimulation with CD19⁺ target cells. Two-tailed paired Student′s t-test was performed, data represent means ± s.e.m. of n = 4 independent experiments. e, NALM6-bearing mice were treated with 5 × 10⁴ CAR T cells and euthanized at day 10 after infusion; bone marrow CAR T cells were analyzed by FACS. Representative flow cytometric analysis of phenotype for indicated CAR T cells as determined by CD62L/CD45RA expression, gated on CAR⁺CD4⁺ T cells. Representative of 5 mice per group in at least n = 2 independent experiments with similar results.

Extended Data Fig. 2 ∣. Analysis of effector function in 1928ζ mutants compared to wild-type 1928ζ.

a, Cytotoxic activity of 1928ζ mutants compared to wild-type 1928ζ using an 18-h bioluminescence assay with FFL-expressing NALM6 cells as targets. Experiments were performed 1 week after expansion of effector cells on CD19⁺ target cells. Data are means ± s.e.m. (n = 4 independent experiments performed in triplicates). *P < 0.05 (2¹: P = 0.0273, 2⁻¹: P = 0.0387, 2⁻²: P = 0.0125), **P = 0.0018 as calculated by two-tailed paired Student′s t-test of average of triplicates. b,c, Granzyme B (GrB) expression (n = 4 independent experiments) on CD8⁺ CAR T cells (b) and cytokine secretion (c) of CD4⁺ and CD8⁺ CAR T cells upon 2nd stimulation with CD19-expressing target cells. All data are means ± s.e.m. (IFNγ and IL2, n = 4; TNFα, n = 5 independent experiments). Unstimulated (Unstim.) wild-type 1928ζ cells were used as control. Significant differences compared to 1928ζ were determined by two-tailed paired Student′s t-test.

Extended Data Fig. 3 ∣. Impact of ITAM location within 1928ζ CARs on T cell function and therapeutic potency.

a, Cytotoxic activity as determined by 4-h ⁵¹Cr release assay 1 week after expansion of effector cells on irradiated 3T3-CD19 (data are means of n = 2 independent experiments performed in triplicates). b, Cumulative cell counts of indicated CAR T cells upon weekly stimulation with CD19⁺ target cells (n = 3 independent experiments). All data are means ± s.e.m.; P values were calculated with two-tailed paired Student′s t-test. c, Cytotoxic activity of D12 and D23 compared to wild-type 1928ζ as determined by 18-h bioluminescence assay with FFL-expressing NALM6 cells as targets. Experiments were performed 1 week after expansion of effector cells on CD19⁺ target cells. Data are means ± s.e.m. (n = 4 independent experiments performed in triplicates). P value was calculated by two-tailed paired Student′s t-test of average of triplicates and showed no significant difference (P> 0.05) between D12/D23 and wild-type 1928ζ for all E/T ratios. d, NALM6-bearing mice were treated with 5 × 10⁴ CAR T cells. Kaplan–Meier analysis of survival of mice treated with wild-type 1928ζ or indicated 1928ζ mutants (n = 10 mice per group). Control refers to untreated mice (n = 6). P value was calculated by a one-sided log-rank Mantel–Cox test.

Extended Data Fig. 4 ∣. Influence of ITAM location within 1928ζ CARs on effector function in vitro.

a, Granzyme B (GrB) expression on CD8⁺ CAR T cells (n = 5 independent experiments). b, Cytokine secretion of CD4⁺ and CD8⁺ CAR T cells upon second stimulation with CD19-expressing target cells. Unstimulated wild-type 1928ζ cells were used as control. All data are means ± s.e.m. (IFNγ and IL2, n = 4; TNFα, n = 5 independent experiments). Each individual symbol indicates one sample. Significant differences compared to 1928ζ were determined by two-tailed paired Student′s t-test.

Extended Data Fig. 5 ∣. T cell differentiation and effector function of TRAC-encoded 1928ζ mutants.

NALM6-bearing mice were treated with 1 × 10⁵ CAR T cells and euthanized at day 17 after infusion. Bone marrow and spleen CAR T cells were analyzed and counted by FACS. a, Histogram and flow cytometric analysis of CAR expression 4 d after CAR gene integration into the TRAC locus. Representative of four independent experiments with similar results. b, Cell numbers of CD4⁺ and CD8⁺ CAR T cells, c, Percentage of CD8⁺ T_CM (CD62L⁺CD45RA⁻) and flow cytometric analysis of CD62L/CD45RA expression on bone marrow CD8⁺CAR T cells (representative of n = 5 mice per group in one independent experiment). d, Ratio of CAR⁺IL7R⁺ to tumor cells and exemplary flow cytometric analysis of IL7R⁺ CAR T cells in the bone marrow of mice. e, Enumeration of CAR T cells in the spleen of mice. In b, c, d and e all data are means ± s.e.m., two-tailed Mann–Whitney analysis was performed, n = 5 mice per group. f, Cytotoxic activity of TRAC-1XX, TRAC-XX3 and wild-type TRAC-1928ζ (18-h bioluminescence assay with FFL-expressing NALM6 as targets). Experiments were performed 4 d post transduction, 1 week and 3 weeks after expansion with weekly CD19 antigen stimulations. Symbols demonstrate means of triplicates (one representative donor).

Extended Data Fig. 6 ∣. In vivo T cell exhaustion of TRAC-1928ζ mutants compared to wild-type TRAC-1928ζ.

a, NALM6-bearing mice were treated with 1 × 10⁵ CAR T cells and euthanized at day 17 after infusion. FACS analysis of expression of exhaustion markers on CAR⁺ T cells, representative of n = 5 mice per group in one independent experiment. b–g, NALM6-bearing mice were treated with 1 × 10⁵ TRAC-edited naive T cells. 16 (b–c) and 36 (e,g) d after CAR administration, TRAC-1928ζ and TRAC-1XX cells from bone marrow and spleen were exposed to ex vivo stimulation with NALM6 or PMA/Ionomycin (Iono). Cytokine and granzyme B (GrB)/ CD107a expression on CAR T cells as demonstrated by percentage of expression and flow cytometric analyses, representative for n = 3 mice in two independent experiments (b) and for n = 3 replicates (g). Expression of exhaustion markers PD1⁺LAG3⁺ on CAR T cells (d) and cytotoxic activity (f) of TRAC-1XX (day 36) after 10 h of co-culture with NALM6. All data are means ± s.e.m., n = 3 mice per group

Extended Data Fig. 7 ∣. T cell memory formation in TRAC-1XX compared to wild-type TRAC-1928ζ.

NALM6-bearing mice were treated with 1 × 10⁵ or 5 × 10⁵ TRAC-edited naive T cells. a–c, 16 and 36 d after administration of 1 × 10⁵ TRAC-1928ζ and TRAC-1XX, CARs were isolated from bone marrow and spleen. a–b, Cell number of CAR T cells (a), central memory (T_CM: CD62L⁺CD45RA⁻), effector (T_EFF: CD62L⁻CD45RA⁺) and IL7R-expressing bone marrow CAR T cells (b). All data are means ± s.e.m., n = 3 mice per group. c, Representative flow cytometric analysis of CD62L/CD45RA expression on TRAC-1928ζ and TRAC-1XX bone marrow CAR T cells at day 36 in one independent experiment (n = 3 mice per group). d–g, NALM6-bearing mice were treated with 5 × 10⁵ TRAC-edited naive T cells and were either rechallenged with NALM6 cells (n = 5 mice per group) or no further rechallenge with tumor was performed (TRAC-1928ζ, n = 6 mice; TRAC-1XX, n = 7 mice). d,e, Cell number of total CAR T cells (d), T_CM, T_EFF and IL7R⁺ CAR T cells (e) in the spleen of treated mice 63 d post CAR administration (rechallenge: TRAC-1928ζ, n = 4 mice; TRAC-1XX, n = 5 mice. No rechallenge, n = 5 mice per group). All data are means ± s.e.m.; a two-tailed unpaired Student's t-test was used for statistical analysis. f, FACS analysis of IL7R⁺, CD62L⁺ and CD45RA⁺ expression on TRAC-1928ζ and TRAC-1XX CAR T cells at day 63 post CAR infusion (representative for at least n = 3 mice per group in one independent experiment). g, Expression of exhaustion markers PD1⁺TIM3⁺LAG3⁺ on CAR T cells derived from the spleen (rechallenge: TRAC-1928ζ, n = 4 mice; TRAC-1XX: n = 5 mice. No rechallenge, n = 5 mice per group). All data are means ± s.e.m.; P value was determined by a two-tailed unpaired Student's t-test.

Extended Data Fig. 8 ∣. Transcriptional profiles of TRAC-encoded 1928ζ mutants and sorted control T cells.

a, Principal component analysis (PCA) of global transcriptional profiles of CD8⁺ TRAC-1XX, TRAC-XX3 and TRAC-1928ζ after stimulation with CD19 target cells (left) and of sorted control T cell subsets (right): T_N, T_SCM and T_EFF. Experiment was performed in technical triplicates for each CAR construct and in six replicates for each control subset. b, Representative GSEA enrichment plot (GSE10239), demonstrating downregulation of memory- relative to effector-related genes and naive- relative to effector-related genes in 1928ζ versus 1XX and in 1928ζ versus XX3 (n = 3 mice per group). c, Heat map of 900 differentially expressed genes among CD8⁺ T cell subsets as described by Gattinoni et al (left) compared to differential gene expression of sorted control T cell subsets (T_EFF, T_SCM and T_N).

Extended Data Fig. 9 ∣. Impact of CD3ζ ITAM mutations in TRAC-1928ζ on T cell differentiation state and effector profile.

a, GSEA of a signature of the top 200 genes upregulated in exhausted CD8 T cells relative to naive or memory CD8 T cells as derived from GSE41867, demonstrating enrichment of exhaustion signature in TRAC-1928ζ versus TRAC-1XX or TRAC-XX3 and in the sorted control T_EFF versus T_N and T_SCM. Experiment was performed in technical triplicates for each CAR construct and in six replicates for each control subset. b, Gene ontology analysis demonstrating significantly enriched gene sets associated with inflammation, cytokine and chemokine signaling in 1928ζ versus XX3, 1XX versus XX3 and 1928ζ versus 1XX (n = 3). Transcriptional analysis was performed after CAR gene integration into the TRAC locus of naive T cells and stimulation with CD19⁺ target cells. Results are shown in order of significance as −log₁₀ (corrected P value). P values were determined by a one-tailed Fisher′s exact test and the Benjamini–Hochberg method was used to correct for multiple hypotheses testing. c, Heat map of selected differentially expressed genes between CAR constructs related to inflammation, cytokine and chemokine activity. d, Flow cytometric analysis of T cell differentiation state on CD8⁺CAR T cells after stimulation with CD19 antigen (representative for n = 2 independent experiments with similar results).

Extended Data Fig. 10 ∣. Gating strategy to analyze CAR T cells obtained from bone marrow of treated mice.

a,b Representative flow cytometric analysis of TRAC-1928ζ (a) compared to TRAC-1XX (b) on day 17 post CAR infusion. Placement of gating was based on FMO controls.

Fig. 1 ∣. 1928ζ ITAMs differentially regulate CAR T cell potency.

a, Cytoplasmic regions of wild-type and mutated 1928ζ CARs. The ζ chain of the 1928ζ CAR was mutated in one or two of its three ζ signaling domains, named ITAM1, ITAM2, and ITAM3, from a membrane-proximal to a membrane-distal direction. In 1XX, X2X, XX3, and X23 CARs, the two tyrosines (Y) in the respective ITAM are point-mutated to two phenylalanines (F) for the indicated ITAMs. b, Flow cytometric analysis showing expression levels of CAR and LNGFR for the constructs depicted in a. Data are representative of at least five independent experiments with similar results. Untransduced T (UT) cells were used as the control. c–e, Nalm6-bearing mice were treated with 5 × 10⁴ CAR⁺ T cells. c, Tumor burden (average radiance) of mice is shown, comparing the in vivo efficacy of wild-type 1928ζ, 1XX, X2X, XX3, and X23 (n = 10 mice per group, results were pooled from two independent experiments). Control refers to untreated mice (n = 6). d, Number of CAR T cells in the bone marrow of mice 17 d post-infusion (results were pooled from two independent experiments, n = 10 mice per group). e, Phenotype of CAR T cells in the bone marrow of mice 10 d after CAR infusion, as demonstrated by the percentage of T_CM and T_EFF cells. Representative results of two independent experiments are shown (n = 5 mice per group). All data are mean ± s.e.m. In d and e, P values were determined by two-tailed Mann–Whitney U-tests. See also Extended Data Figs. 1 and 2.

Fig. 2 ∣. ITAM position within 1928ζ CARs determines antitumor efficacy.

a, Cytoplasmic domains of 1928ζ CARs with deletions in the CD3ζ chain. In D12, deletion mutations remove ITAM1 and ITAM2, while ITAM2 and ITAM3 are removed in D23. b, Flow cytometric analysis showing the expression levels of CAR and LNGFR for the indicated constructs. Data are representative of at least five independent experiments with similar results. UT, untransduced T cells were used as the control. c–e, Nalm6-bearing mice were treated with 5 × 10⁴ CAR⁺ T cells. c, Tumor burden (average radiance) of mice treated with wild-type 1928ζ, D12, or D23 (wild-type 1928ζ and D23: n = 10; D12: n = 9; pooled data from two independent experiments). d, Number of CAR T cells in the bone marrow of mice 17 d post-infusion (results were pooled from two independent experiments, n = 10 mice per group). e, Phenotype of CAR T cells in the bone marrow of mice 10 d after CAR infusion, as demonstrated by the percentage of T_CM and T_EFF cells (pooled data from two independent experiments, n = 10 mice per group). All data are mean ± s.e.m. In d and e, P values were determined by two-tailed Mann–Whitney U-tests. See also Extended Data Figs. 3 and 4.

Fig. 3 ∣. TRAC-1XX augments T cell potency by decreasing T cell exhaustion and developing into long-lived memory T cells with effective recall responses.

a–d, Nalm6-bearing mice were treated with 1 × 10⁵ or 5 × 10⁵ TRAC-CAR T cells. a, Kaplan–Meier analysis of survival of mice treated with 1 × 10⁵ TRAC-1XX or TRAC-XX3 compared with TRAC-1928ζ (TRAC-1928ζ and TRAC-XX3: n = 5 mice per group; TRAC-1XX: n = 7). Control refers to untreated mice (n = 3). P values were determined by a one-sided log-rank Mantel–Cox test. b, Kaplan–Meier analysis of survival of mice treated with 5 × 10⁵ (n = 5 mice per group) or 1 × 10⁵ (TRAC-1928ζ: n = 10 mice, TRAC-1XX: n = 5 mice) TRAC-CAR T cells. P values were determined by a one-sided log-rank Mantel–Cox test. c,d, The number of cells (c) and expression of the exhaustion markers PD1⁺TIM3⁺LAG3⁺ on bone marrow CAR T cells (d) were determined for TRAC-1XX and TRAC-XX3 and compared with TRAC-1928ζ. Data are shown as mean ± s.e.m., and each symbol denotes an individual mouse (n = 5 mice per group). P values were determined by two-tailed Mann–Whitney U-tests. e–h, Nalm6-bearing mice were treated with 5 × 10⁵ TRAC-edited naive T cells and were rechallenged with Nalm6 cells (n = 5 mice per group), as indicated by the arrows. No further rechallenge with tumor was performed for the controls (TRAC-1928ζ: n = 6 mice; TRAC-1XX: n = 7 mice). e, Tumor burden (average radiance) of mice, comparing the in vivo efficacy of TRAC-1928ζ and TRAC-1XX following tumor rechallenge versus no further rechallenge. All data are mean ± s.e.m. A two-tailed unpaired Student's t-test was used for the statistical analysis of tumor burden at day 59 post-CAR administration (TRAC-1928ζ: n = 4; TRAC-1XX: n = 5). f,g, The number of total CAR T cells (f), T_CM, T_EFF, and IL7R⁺ CAR T cells (g) in the bone marrow of treated mice 63 d post-CAR administration (rechallenge: TRAC-1928ζ: n = 4 mice, TRAC-1XX: n = 5 mice; no rechallenge: n = 5 mice per group). All data are mean ± s.e.m.; a two-tailed unpaired Student's t-test was used for the statistical analysis. h, Expression of the exhaustion markers PD1⁺TIM3⁺LAG3⁺ on bone marrow CAR T cells. All data are mean ± s.e.m. P values were determined by a two-tailed unpaired Student's t-test. See also Extended Data Figs. 5, 6, and 7.

Fig. 4 ∣. CD3ζ ITAM mutations in 1928ζ CARs establish distinct transcriptional signatures.

Gene expression profiles of CD8⁺ TRAC-1928ζ, TRAC-1XX, and TRAC-XX3 CAR T cells (initially sorted naive T cells) 24 h post stimulation with CD19⁺ target cells. a, Normalized enrichment score of significantly up- or downregulated gene sets in 1928ζ versus 1XX and 1928ζ versus XX3 (n = 3 replicates per group) as determined by GSEA using the MSigDB C7 gene ontology sets. For all pathways, the false discovery rate (FDR) q ≤ 0.02. GSE datasets are indicated in parentheses. stim, stimulated. b, Differentially expressed genes (FDR q < 0.05) between sorted effector and naive/memory T cells (left) (n = 6 replicates per group) and heat map demonstrating the expression profiles of the same genes for CAR T cells (n = 3 replicates per group). TF, transcription factor. c, Normalized enrichment score of significantly enriched gene sets (FDR q ≤ 0.03) related to phenotypic and functional T cell features comparing TRAC-1928ζ versus TRAC-XX3, TRAC-1928ζ versus TRAC-1XX, and TRAC-1XX versus TRAC-XX3 as identified with GSEA analysis (n = 3 replicates per group). JAK–STAT, Janus kinase-signal transducer and activator of transcription. d, Impact of defined CD3ζ ITAM mutations in 1928ζ CAR T cells on effector- and memory-associated T cell attributes. 1XX CAR T cells display balanced effector and memory traits. See also Extended Data Figs. 8 and 9.