Figure S1: CCR6 and CCL20 expression is increased in multiple tumors.

Figure S4: Ccr6-/- Tregs have reduced glycolysis compared to WT Tregs.

Sensitivity analysis for handling residual CAR19 qPCR data below limit of quantification (LOQ) in leukapheresis material

The impact of chimeric antigen receptor (CAR)-T cell expansion and persistence on clinical outcomes and treatment-related morbidity in patients with relapsed/refractory multiple myeloma (RRMM) remains incompletely defined, in part due to limited availability of standardized CAR-T cell quantification assays. We evaluated CAR-T cell kinetics and their association with efficacy and toxicity in RRMM patients treated with idecabtagene vicleucel (ide-cel) or ciltacabtagene autoleucel (cilta-cel). Using a uniform flow cytometry-based platform (N=90; cilta-cel, n=54; ide-cel, n=36), we observed significantly greater CAR-T cell expansion with cilta-cel than with ide-cel (median 106 vs 49 cells/uL). Peak CAR-T cell expansion was associated with clinical response in the ide-cel cohort but not with cilta-cel, where rapid and excessive expansion was instead associated with an increased risk of delayed neurotoxicities (DNTs), a complication with potential long-term functional consequences (median peak 1,009 vs 96 cells/uL). To identify clinically accessible biomarkers of CAR-T cell expansion, we analyzed absolute lymphocyte count (ALC) as a surrogate biomarker in a larger multicenter cohort (N=532; cilta-cel, n=256; ide-cel, n=276). Higher peak ALC was significantly associated with the development of DNTs, particularly Parkinsonism after cilta-cel. A peak ALC ³3000/uL - or ³2500/uL following a daily twofold increase - predicted elevated DNT risk (sensitivity 81%, specificity 59%). Together, these findings delineate distinct expansion-toxicity relationships in cilta-cel and ide-cel therapy, establish ALC as a practical, uniformly available surrogate for CAR-T cell expansion, and define quantitative thresholds that may enable early recognition of patients at risk for DNT, informing preemptive strategies to mitigate morbidity following cilta-cel.

Supplemental Figure 2 shows lab values over time (A) and at peak/nadir (B) in grade 2+ vs no IECHS

CD39 expression on CD4+ and CD8+ CAR22, stratified by median expression levels (High/Low) or using a 20% cutoff, and its correlation with CAR22 expansion as measured by flow cytometry and qPCR, treatment response, and survival outcomes. (A) The CD39 expression level on CD4+ or CD8+ CAR22 in the product, stratified by median (> median or < median) or using a 20% cutoff, shows no significant difference in CAR22 maximal expansion (CMax) measured by flow cytometry in peripheral blood (PB) after infusion. (B) CD39 expression on CD4+ or CD8+ CAR22, stratified by median, shows no significant difference in CAR22 area under the curve from day 0 to 28 (AUC0-28) post-infusion as measured by flow cytometry in PB. (C) CD39 expression on CD4+ or CD8+ CAR22, stratified by median, shows no significant difference in CAR22 CMax and AUC0-28 post-infusion as measured by qPCR in PB. (D) CD39 expression on CD4+ or CD8+ CAR22, stratified by median, shows no significant difference in complete response rate, progression-free survival, or overall survival. Dotted lines represent the 95% confidence interval.

Abstract Immunocompetent models present additional challenges for CAR T therapy due to heterogeneous antigen expression and intact host immunity, making them a clinically relevant setting for evaluating therapeutic robustness. B7H3-targeted CAR T cell therapy has shown promise in some preclinical solid tumors, but its efficacy and ability to induce durable immunity in immunocompetent ovarian cancer models remain largely unexplored. Here, we evaluated the efficacy of a novel murine specific B7H3 CAR construct across multiple syngeneic immunocompetent ovarian cancer models with distinct genetic backgrounds and B7H3 antigen expression profiles. ID8 cell lines bearing Trp53-/- (homologous recombination (HR) proficient) and Trp53-/-Brca2-/- (HR deficient) mutations with heterogeneous B7H3 expression, and BPPNM (Brca1-/- Pten-/- Trp53-/-R172H Nf1-/- MycOE) cell lines and homogenous B7H3 expression were used to establish intraperitoneal tumors in immunocompetent C57BL/6 mice. Treatment with a single intraperitoneal dose of B7H3 CAR T cells resulted in significant tumor regression in all models tested, with varying degrees of response and increased survival in all 3 tumor models. Our findings lead us to speculate that heterogeneity in B7H3 antigen expression contributed to differential treatment outcomes. Notably, in a fallopian tube-derived BPPNM model, B7H3 CAR T cell therapy resulted in a complete responder that remained tumor-free following two tumor re-challenges, demonstrating durable anti-tumor immunity. To assess whether this protection was transferable, splenocytes from the complete responder were adoptively transferred into tumor-bearing recipient mice, resulting in pronounced tumor suppression and suggesting the presence of functional tumor-immunity. In summary, these results demonstrate that the murine specific B7H3 CAR T cells are active in immunocompetent ovarian cancer models and can induce systemic, long-lasting anti-tumor responses. Antigen heterogeneity remains a barrier to uniform efficacy, highlighting the need for future combination strategies to enhance consistency and durability of B7H3 CAR T cell responses. Citation Format: Thu Huyen Pham, Elizabeth Allen, Supreeti Tallapragada, Justine Chan, Naiara Martinez Velez, Elena Sotillo, Crystal L. Mackall, Oliver Dorigo. B7H3 CAR T cell therapy across immunocompetent ovarian cancer models with evidence of transferable anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB140.

Comparison of CAR19 transgene level, CAR22 product characteristic and cell kinetics across different CAR19 co-stimulatory domain

There is limited systemic data on the dynamics of BCMA-target antigen expression with BCMA CAR-T at relapse. We analyzed 76 patients receiving standard-of-care BCMA-directed CAR-T who underwent real-time BCMA expression evaluation at baseline (n = 50), relapse (6), or both (20) using flow cytometry (FC) and/or immunohistochemistry (IHC). BCMA was universally expressed at baseline with significant heterogeneity in expression level. No concordance was seen between FC and IHC in categorizing high vs. low expression (Spearman: 0.07, Cohen kappa: 0). Plasma cell BCMA expression by FC correlated with clinical outcomes, whereas IHC did not. High BCMA expression by FC was associated with increased likelihood for VGPR/CR (p = 0.007) and longer time to progression (p = 0.005), including the ciltacabtagene autoleucel cohort (median: 23.0 vs. 7.7 months, p = 0.02). Relapsed patients retained BCMA expression by FC, though 29% (5/16) had BCMA loss by IHC, with 4/5 showing concurrent positive BCMA expression by FC. BCMA expression at relapse by FC was significantly lower than baseline (p = 0.04); downregulation (≥25% decrease) occurred in 50% (8/16) with paired samples. Higher BCMA expression by FC correlated with higher likelihood of deep, durable responses following BCMA-directed CAR-T. While BCMA loss is rare, decreased expression is common at relapse, with implications for sequencing BCMA-directed therapies.

Immune effector cell-associated hemophagocytic lymphohistiocytosis-like syndrome (IEC-HS) is a poorly characterized inflammatory toxicity of chimeric antigen receptor (CAR) T cells with high risk of mortality. In this study, we describe IEC-HS manifestations in patients with large B-cell lymphoma and B-cell acute lymphoblastic leukemia after CD22-directed CAR T cells. IEC-HS occurred in 19 of 54 patients (35%), including 11 grade 1 and 8 grade 2 or higher. IEC-HS was associated with higher nonrelapse mortality (NRM) yet lower relapse rates. CAR T-cell expansion in peripheral blood was significantly associated with IEC-HS severity. Cytokine profiling identified 41 cytokines primarily related to the IFNγ, TNFα, and IL1 families that significantly correlated with IEC-HS severity. We developed a parsimonious model composed of IFNγ, IL10, and IL1RA that correlated with grade 2+ IEC-HS on day 14, outperforming the full signature (AUC 0.93 vs. 0.75, P = 0.038). Thus, a cytokine signature with potential prognostic utility helps distinguish IEC-HS from inflammatory toxicities with overlapping symptoms. SIGNIFICANCE:IEC-HS is a serious inflammatory toxicity of CAR T cells. We demonstrate that IEC-HS after CD22-directed CAR T-cell therapy is associated with lower rates of relapse yet higher NRM. CAR T-cell expansion and a 41-cytokine signature are associated with IEC-HS, and a simplified signature of IFNγ, IL10, and IL1RA precedes severe disease. See related commentary by Rocco and Shah, p. 163.

Chimeric antigen receptor (CAR) T-cell therapy holds great promise for patients with cancer, and the identification of predictive biomarkers is crucial in finding new ways to guide therapy. Major challenges to the application of informatics and machine learning in CAR T-cell therapy include limited sample sizes and non-uniformity in data generation across cancer indications and trials. Here we took a global, pan-haematologic cancer approach, analysing 256 patients across 5 cancer types and 13 clinical trials. We generated data using a framework that included pre-infusion clinical features, over 2 million apheresis T cells analysed by flow cytometry using 17 unique markers, ex vivo T-cell expansion during CAR T-cell manufacture, more than 90,000 measurements of 30 serum markers and serial tracking of circulating CAR T cells using qPCR. From this data resource, we demonstrate the potential of pan-cancer predictive biomarkers that capture generalizable characteristics of treatment response and non-response in CAR T-cell therapy.

Background aims Broader access to autologous chimeric antigen receptor (CAR) T-cell therapy is limited by costly, device-bound manufacturing workflows and reliance on dedicated instrumentation for discrete unit operations, including T-cell enrichment and culture. These constraints affect academic, decentralized and high-throughput centralized manufacturing programs. We developed a closed-system CAR T-cell manufacturing workflow centered on a gas-permeable G-Rex bioreactor platform that enables direct in-vessel CD4⁺/CD8⁺ T-cell enrichment, static viral transduction and expansion without requiring a separate standalone enrichment device or spinoculation hardware. Methods Key unit operations of a G-Rex-based GD2 CAR T-cell manufacturing process were optimized, including RetroNectin-mediated static retroviral transduction, coating concentration and timing, and media-exchange strategy. T-cell phenotype and functional potency were characterized longitudinally throughout the 7-day culture period. The optimized workflow was translated to a manufacturing-scale G-Rex 100M-CS process incorporating a newly developed, previously unpublished method for direct in-vessel CD4⁺/CD8⁺ magnetic enrichment using detachable beads and a prototype SepaRex magnetic base. At-scale products from three healthy donors were assessed for enrichment performance, transduction efficiency, expansion, viability, phenotype, cytokine secretion and tumor-cell killing, and benchmarked against historical products manufactured using a clinically validated CliniMACS Prodigy-based GD2 CAR T-cell process. Results Retronectin-coated G-Rex cultures supported efficient static retroviral transduction without spinoculation, and media exchange between 24 and 96 hours post-transduction did not significantly affect final yield or viability. Direct in-vessel CD4⁺/CD8⁺ enrichment achieved 61.6 ± 8.6% recovery and 87.2 ± 7.9% purity. At manufacturing scale, the integrated workflow generated GD2 CAR T cells with 62.3 ± 5.1% transduction efficiency, 26.8 ± 4.7-fold expansion, >94% viability and >96% CD3⁺ T-cell purity. Final products retained central memory features, secreted cytokines and mediated cytotoxicity against GD2-expressing tumor targets, with manufacturing performance comparable to the historical Prodigy-based process. Conclusions This closed, scalable and cost-conscious G-Rex-based workflow integrates T-cell enrichment, activation, static viral transduction and expansion within a single bioreactor-centered process. By eliminating dedicated enrichment instrumentation and reducing reliance on highly automated, device-bound culture systems, this bioreactor-centered platform may support flexible CAR T-cell manufacturing across academic, decentralized, and high-throughput centralized settings.

The CAR T-cell maximal expansion (CMax) and area under the curve from day 0 to 28 (AUC0-28) for CAR19 and CAR22 following CAR19 or CAR22 infusion in the same patient. (A) Analysis by flow cytometry. (B) Analysis by qPCR assay. No statistical comparison was performed due to the limited number of patients.

Figure S3: Ccr6-/- Tregs are transcriptionally and metabolically distinct from WT Tregs.

We sought to endow T cell autonomous regulation of cell surface protein expression by exploiting the conditional proteolytic activity of ADAM17 following T cell activation. Screening of canonical ADAM17 substrates yielded a minimal 15-aa CD62L-derived motif that confers rapid and reversible cleavage of a receptor following T cell activation—termed activation-induced release (AIR). Embedding AIR into tonic-signaling CARs reduced basal CAR expression proportional to the degree of tonic signaling induced, curtailing exhaustion and improving antitumor potency. In non-tonic signaling CARs, AIR decreased activation-induced cell death and enhanced T cell expansion after stimulation. AIR’s modularity supports higher-order logic-gating; AIR-regulated peptide masks enable antigen-dependent unmasking of an EGFR-targeting CAR. Finally, CRISPR knockin of AIR into endogenous FAS or TGFBR2 endowed them with activation-induced shedding, which enhanced tumor clearance while preserving signaling in non-activating conditions. AIR is a compact switch that provides fast, autonomous regulation of surface proteins for next-generation cell therapies.

Cell cycle (CC) dynamics are reflected in diverse T cell processes such as TCR activation, expansion, contraction, differentiation, senescence, anergy, and exhaustion; linking CC behaviors to functional and dysfunctional T cell states in development and disease. Progression through CC checkpoints is also tightly linked to cell fate decisions across development. Yet, much remains unknown about the connection between CC sensing and T cell differentiation programs. To disentangle the relationship across T cell state, time-since-activation, receptor signaling, division, and CC, we leverage high-throughput single-cell mass cytometry for parallel measurement of these diverse biological states. By modulating CC progression and receptor signaling with inhibitors as well as tonic signaling Chimeric Antigen Receptor (CAR) models of T cell exhaustion, we reveal that earlier G1/S CC programs crosstalk with receptor signaling to control T cell fate, and that exhaustion programs are downstream to aberrant, S-G2 phase CC arrest signatures in tonic CAR signaling in vitro , in situ, and in vivo across human cancers in association with CD8 T-lymphocyte dysfunction.

Test characteristics of cytokine signatures.

Cytokine signaling is critical to the function of natural immune cells and engineered immune cell therapies such as chimeric antigen receptor (CAR) T cells. It remains unclear how the limited set of signal transducers and activators of transcription (STATs) and other proteinsa activated by these cytokine receptors can encode the observed diversity of immune cell phenotypes. To understand how signaling downstream of cytokines control immune cell phenotype, we sought to map the structure of Janus kinase (JAK)/STAT signaling domains to cell signaling and resulting CAR T cell function. We recombined 14 signaling motifs to construct a library of ∼30,000 constitutively active synthetic cytokine receptors (SCRs) with intracellular domains composed of novel signaling motif combinations that activate different signaling cascades. We experimentally tested ∼450 SCRs which generated a range of CAR T cell memory, cytotoxicity, and proliferation. SCRs with pSTAT1 and 3 signaling generated effector memory CAR T cells, while SCRs with strong pSTAT5 generated effector CAR T cells with potent anti-tumor activity, measured by flow cytometry and phosphoproteomics. Subsequent kinase substrate enrichment analysis (KSEA) identified key differences of kinases associated with cell signaling networks, such as CDKs that are correlated with high proliferative profile of effector CAR T cells. To map the structure-signaling-phenotype landscape we trained models to predict signaling and CAR T cell phenotype that result from varied motif combinations. From neural network predictions we identified features, including strong STAT5 and Shc signaling, that promote unsafe autonomous CAR T cell proliferation. Models also revealed a trade-off between memory and cytotoxicity, with a Pareto front encoded by a continuous change in signaling. These results demonstrate that recombination of a limited set of signaling motifs creates a continuous spectrum of signaling that encodes a corresponding spectrum of cell phenotype. This work synthetically expands the combinatorial space of JAK/STAT signaling and provides a foundation for rational design of CAR T cells with improved cytotoxicity, memory, and safety profiles. Wansang Cho, Jenny Y. Liu, Alex N. Beckett, Erin Craig, Dain R. Brademan, Judith C. Lunger, Peng Xu, Katie Ho, Ethan E. Chen, Antonio Salcido-Alcantar, Lucas E. Sant'Anna, Kamal Obbad, Nakoa Po, Sophia Joy Ong, Elena Sotillo, Robert Tibshirani, Ruth Huttenhain, Crystal L. Mackall, Kyle G. Daniels. Programmable JAK/STAT signaling drives CAR T cells to enhanced functional states [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr A013.