Single-cell CAR T atlas reveals type 2 function in 8-year leukaemia remission.

Despite a high response rate in chimeric antigen receptor (CAR) T cell therapy for acute lymphocytic leukaemia (ALL)1-3, approximately 50% of patients relapse within the first year4-6, representing an urgent question to address in the next stage of cellular immunotherapy. Here, to investigate the molecular determinants of ultralong CAR T cell persistence, we obtained a single-cell multi-omics atlas from 695,819 pre-infusion CAR T cells at the basal level or after CAR-specific stimulation from 82 paediatric patients with ALL enrolled in the first two CAR T ALL clinical trials and 6 healthy donors. We identified that elevated type 2 functionality in CAR T infusion products is significantly associated with patients maintaining a median B cell aplasia duration of 8.4 years. Analysis of ligand-receptor interactions revealed that type 2 cells regulate a dysfunctional subset to maintain whole-population homeostasis, and the addition of IL-4 during antigen-specific activation alleviates CAR T cell dysfunction while enhancing fitness at both transcriptomic and epigenomic levels. Serial proteomic profiling of sera after treatment revealed a higher level of circulating type 2 cytokines in 5-year or 8-year relapse-free responders. In a leukaemic mouse model, type 2high CAR T cell products demonstrated superior expansion and antitumour activity, particularly after leukaemia rechallenge. Restoring antitumour efficacy in type 2low CAR T cells was attainable by enhancing their type 2 functionality, either through incorporating IL-4 into the manufacturing process or by priming manufactured CAR T products with IL-4 before infusion. Our findings provide insights into the mediators of durable CAR T therapy response and suggest potential therapeutic strategies to sustain long-term remission by boosting type 2 functionality in CAR T cells.

FOXO1 is a master regulator of memory programming in CAR T cells.

A major limitation of chimeric antigen receptor (CAR) T cell therapies is the poor persistence of these cells in vivo1. The expression of memory-associated genes in CAR T cells is linked to their long-term persistence in patients and clinical efficacy2-6, suggesting that memory programs may underpin durable CAR T cell function. Here we show that the transcription factor FOXO1 is responsible for promoting memory and restraining exhaustion in human CAR T cells. Pharmacological inhibition or gene editing of endogenous FOXO1 diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype and impaired the antitumour activity of CAR T cells. Overexpression of FOXO1 induced a gene-expression program consistent with T cell memory and increased chromatin accessibility at FOXO1-binding motifs. CAR T cells that overexpressed FOXO1 retained their function, memory potential and metabolic fitness in settings of chronic stimulation, and exhibited enhanced persistence and tumour control in vivo. By contrast, overexpression of TCF1 (encoded by TCF7) did not enforce canonical memory programs or enhance the potency of CAR T cells. Notably, FOXO1 activity correlated with positive clinical outcomes of patients treated with CAR T cells or tumour-infiltrating lymphocytes, underscoring the clinical relevance of FOXO1 in cancer immunotherapy. Our results show that overexpressing FOXO1 can increase the antitumour activity of human CAR T cells, and highlight memory reprogramming as a broadly applicable approach for optimizing therapeutic T cell states.

Transplantation for immune dysregulatory disorders: current themes and future expectations.

PURPOSE OF REVIEW: Primary immune regulatory disorders (PIRDs) are an increasing indication for hematopoietic stem cell transplant (HCT) in pediatric patients. Here, we provide an updated overview of HCT for PIRDs, and discuss future avenues for improvement in outcomes. RECENT FINDINGS: There are now more than 50 described monogenic PIRDs, which impact all aspects of immune tolerance, regulation, and suppression. Disease characteristics are highly variable, and HCT remains the only option for cure. We review advances in targeted therapies for individual PIRDs, which have significantly improved outcomes and the ability to safely bridge to transplant. Additionally, advances in GVHD prevention, graft manipulation, personalized conditioning regimens, and supportive care have all increased survival after HCT. The high inflammatory state increases the risk of nonengraftment, rejection, and autologous reconstitution. Therapy to reduce the inflammatory state may further improve outcomes. In addition, although younger patients with fewer comorbidities have better outcomes, the clinical courses of these diseases may be extremely variable thereby complicating the decision to proceed to HCT. SUMMARY: HCT for PIRDs is a growing consideration in cell therapy. Yet, there remain significant gaps in our understanding of which patients this curative therapy could benefit the most. Here, we review the current data supporting HCT for PIRDs as well as areas for future improvement.

The Current State of Chimeric Antigen Receptor T Cell Therapy for B Lymphoblastic Leukemia.

Over the past decade, CAR T cell therapy has transformed the treatment of relapsed or refractory B-ALL in children and adults. CD19-directed CAR T cells can induce complete remissions in a large majority of patients with B-ALL, and up to half of these patients will go on to maintain durable remissions. However, significant challenges remain for patients who relapse or do not respond. This review will discuss the history of CAR T cell therapy for B-ALL, the treatment considerations for CAR T cell recipients, and current clinical trials and future directions for CAR T cell therapy in B-ALL.

The pseudokinase Trib1 regulates the transition of exhausted T cells to a KLR + CD8 + effector state and its deletion improves checkpoint blockade.

T cell exhaustion (T EX ) impairs the ability of T cells to clear chronic infection or cancer. While exhausted T cells are hypofunctional, some exhausted T cells retain effector gene signatures, a feature that is associated with expression of KLRs (killer lectin-like receptors). Although KLR + T cells may improve control of chronic antigen, the signaling molecules regulating this population are poorly understood. Using scRNA-seq, flow cytometry, RNA velocity, and scTCR-seq, we demonstrate that deleting the pseudokinase Trib1 shifts T EX towards CX3CR1 + intermediates (T INT ) with robust enrichment of KLR + CD8 + T cells (T KLR ) via clonal T cell expansion. These changes are associated with globally increased KLR gene expression throughout the exhaustion program. Further, Trib1 loss augments anti-PD-L1 blockade to improve viral clearance by expanding the T KLR population. Together, these data identify Trib1 as an important regulator of T cell exhaustion whose targeting enhances the KLR + effector state and improves the response to checkpoint inhibitor therapy.

The pseudokinase Trib1 regulates the transition of exhausted T cells to a KLR+ CD8+ effector state, and its deletion improves checkpoint blockade.

CD8+ T cell exhaustion (TEX) impairs the ability of T cells to clear chronic infection or cancer. While TEX are hypofunctional, some TEX retain effector gene signatures, a feature associated with killer lectin-like receptor (KLR) expression. Although KLR+ TEX (TKLR) may improve control of chronic antigen, the signaling molecules regulating this population are poorly understood. Using single-cell RNA sequencing (scRNA-seq), flow cytometry, RNA velocity, and single-cell T cell receptor sequencing (scTCR-seq), we demonstrate that deleting the pseudokinase Trib1 shifts TEX toward CX3CR1+ intermediates with robust enrichment of TKLR via clonal T cell expansion. Adoptive transfer studies demonstrate this shift toward CD8+ TKLR in Trib1-deficient cells is CD8 intrinsic, while CD4-depletion studies demonstrate CD4+ T cells are required for improved viral control in Trib1 conditional knockout mice. Further, Trib1 loss augments anti-programmed death-ligand 1 (PD-L1) blockade to improve viral clearance. These data identify Trib1 as an important regulator of CD8+ TEX whose targeting enhances the TKLR effector state and improves checkpoint inhibitor therapy.

Trib1 regulates T cell differentiation during chronic infection by restraining the effector program.

In chronic infections, the immune response fails to control virus, leading to persistent antigen stimulation and the progressive development of T cell exhaustion. T cell effector differentiation is poorly understood in the context of exhaustion, but targeting effector programs may provide new strategies for reinvigorating T cell function. We identified Tribbles pseudokinase 1 (Trib1) as a central regulator of antiviral T cell immunity, where loss of Trib1 led to a sustained enrichment of effector-like KLRG1+ T cells, enhanced function, and improved viral control. Single-cell profiling revealed that Trib1 restrains a population of KLRG1+ effector CD8 T cells that is transcriptionally distinct from exhausted cells. Mechanistically, we identified an interaction between Trib1 and the T cell receptor (TCR) signaling activator, MALT1, which disrupted MALT1 signaling complexes. These data identify Trib1 as a negative regulator of TCR signaling and downstream function, and reveal a link between Trib1 and effector versus exhausted T cell differentiation that can be targeted to improve antiviral immunity.