Xianliang Rui

Tissue-specific T cell immune responses play a critical role in maintaining organ health but can also drive immune pathology during both autoimmunity and alloimmunity. The mechanisms controlling intratissue T cell programming remain unclear. Here, we leveraged a nonhuman primate model of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation to probe the biological underpinnings of tissue-specific alloimmune disease using a comprehensive systems immunology approach including multiparameter flow cytometry, population-based transcriptional profiling, and multiplexed single-cell RNA sequencing and TCR sequencing. Transcriptional profiling revealed substantial biological differences between T cells infiltrating the lung and liver during aGVHD. These included enrichment for transcriptional pathways controlling extracellular matrix remodeling and chemotaxis in the lung and enrichment for transcriptional pathways linked to nucleic acid metabolism and proliferation in the liver. Single-cell RNA sequencing and TCR sequencing substantiated divergent organ-specific transcriptional programing of tissue-infiltrating T cells, which was linked to clonal expansion, with expanded clones progressively enriched for C-X3-C motif chemokine receptor 1 ( CX3CR1 )–expressing CD8 effector T cells in the lung and eomesodermin ( EOMES )–expressing CD8 effector-memory T cells in the liver. This divergent evolution of T cells was maintained even for T cells sharing the same TCRs, indicating its independence from antigen specificity. Together, these results provide insights into the role that tissue microenvironment–derived signals play in local T cell transcriptional programming during alloimmune-mediated clonal expansion and suggest potential opportunities to develop tissue-specific therapeutics to curtail pathogenic immunity after transplant.

Regulatory T cells (T _regs ) make major contributions to immune homeostasis. Because T _reg dysfunction can lead to both allo- and autoimmunity, there is interest in correcting these disorders through T _reg adoptive transfer. Two of the central challenges in clinically deploying T _reg cellular therapies are ensuring phenotypic stability and maximizing potency. Here, we describe an approach to address both issues through the creation of OX40 ligand (OX40L)–specific chimeric antigen receptor (CAR)–T _regs under the control of a synthetic forkhead box P3 ( FOXP3 ) promoter. The creation of these CAR-T _regs enabled selective T _reg stimulation by engagement of OX40L, a key activation antigen in alloimmunity, including both graft-versus-host disease and solid organ transplant rejection, and autoimmunity, including rheumatoid arthritis, systemic sclerosis, and systemic lupus erythematosus. We demonstrated that OX40L–CAR-T _regs were robustly activated in the presence of OX40L-expressing cells, leading to up-regulation of T _reg suppressive proteins without induction of proinflammatory cytokine production. Compared with control T _regs , OX40L–CAR-T _regs more potently suppressed alloreactive T cell proliferation in vitro and were directly inhibitory toward activated monocyte-derived dendritic cells (DCs). We identified trogocytosis as one of the central mechanisms by which these CAR-T _regs effectively decrease extracellular display of OX40L, resulting in decreased DC stimulatory capacity. OX40L–CAR-T _regs demonstrated an enhanced ability to control xenogeneic graft-versus-host disease compared with control T _regs without abolishing the graft-versus-leukemia effect. These results suggest that OX40L–CAR-T _regs may have wide applicability as a potent cellular therapy to control both allo- and autoimmune diseases.

Chimeric antigen receptor (CAR) T cells hold promise as a therapy for B-cell–derived malignancies, and despite their impressive initial response rates, a significant proportion of patients ultimately experience relapse. Although recent studies have explored the mechanisms of in vivo CAR T-cell function, little is understood about the activation of surrounding CARneg bystander T cells and their potential to enhance tumor responses. We performed single-cell RNA sequencing on nonhuman primate (NHP) and patient-derived T cells to identify the phenotypic and transcriptomic hallmarks of bystander activation of CARneg T cells following B-cell–targeted CAR T-cell therapy. Using a highly translatable CD20 CAR NHP model, we observed a distinct population of activated CD8+ CARneg T cells emerging during CAR T-cell expansion. These bystander CD8+ CARneg T cells exhibited a unique transcriptional signature with upregulation of natural killer-cell markers (KIR3DL2, CD160, and KLRD1), chemokines, and chemokine receptors (CCL5, XCL1, and CCR9), and downregulation of naïve T-cell-associated genes (SELL and CD28). A transcriptionally similar population was identified in patients after a tisagenlecleucel infusion. Mechanistic studies revealed that interleukin-2 (IL-2) and IL-15 exposure induced bystander-like CD8+ T cells in a dose-dependent manner. In vitro activated and patient-derived T cells with a bystander phenotype efficiently killed leukemic cells through a T-cell receptor–independent mechanism. Collectively, to our knowledge, these data provide the first comprehensive identification and profiling of CARneg bystander CD8+ T cells following B-cell–targeting CAR T-cell therapy and suggest a novel mechanism through which CAR T-cell infusion might trigger enhanced antileukemic responses. Patient samples were obtained from the trial #NCT03369353, registered at www.ClinicalTrials.gov.

There is growing appreciation for the emergence of CAR ^neg bystander T cells after CAR-T cell infusion. However, their phenotypic and transcriptomic hallmarks and mechanisms of activation remain uncertain. We performed single-cell RNA-Seq (scRNA-Seq) on non-human primate (NHP) and patient-derived T cells to interrogate CAR ^neg T cells following B cell targeted CAR-T cell therapy. In a NHP model, we observed a distinct population of activated CD8+ CAR ^neg T cells emerging during CAR-T cell expansion. These bystander CD8+ CAR ^neg T cells exhibited a unique transcriptional signature with upregulation of NK-cell markers ( KIR3DL2, CD160, KLRD1 ), chemokines and chemokine receptors ( CCL5, XCL1, CCR9 ), and downregulation of naive T cell-associated genes ( SELL, CD28 ). A transcriptionally similar population was identified in patients following Tisangelecleucel infusion. Mechanistic studies revealed that IL-2 and IL-15 exposure induced bystander-like CD8+ T cells. These T cells efficiently killed leukemic cells through a TCR-independent mechanism. Together, these data identify bystander CD8+ T cells as a novel mechanism by which CAR-T cell infusion can induce further anti-leukemic activity, measurable in both NHP and in patients. Statement of Significance: We have deeply interrogated CAR ^neg bystander CD8+ T cells during CAR-T cell expansion in non­human primates and patients receiving Tisangelecleucel to identify the unique transcriptomic signature defining these cells, and to determine that IL-2-and IL-15-induced cytotoxic bystander T cells are capable of killing in a TCR-independent manner. These data highlight the potential of bystander T cells for leukemia control and provide a critical foundation for their future analysis.