Search

Regulatory T cells (Tregs) make major contributions to immune homeostasis. Because Treg dysfunction can lead to both allo- and autoimmunity, there is interest in correcting these disorders through Treg adoptive transfer. Two of the central challenges in clinically deploying Treg 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)–Tregs under the control of a synthetic forkhead box P3 (FOXP3) promoter. The creation of these CAR-Tregs enabled selective Treg 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-Tregs were robustly activated in the presence of OX40L-expressing cells, leading to up-regulation of Treg suppressive proteins without induction of proinflammatory cytokine production. Compared with control Tregs, OX40L–CAR-Tregs 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-Tregs effectively decrease extracellular display of OX40L, resulting in decreased DC stimulatory capacity. OX40L–CAR-Tregs demonstrated an enhanced ability to control xenogeneic graft-versus-host disease compared with control Tregs without abolishing the graft-versus-leukemia effect. These results suggest that OX40L–CAR-Tregs may have wide applicability as a potent cellular therapy to control both allo- and autoimmune diseases. Editor's summary: Regulatory T cell (Treg) therapies have emerged as a promising approach for diseases ranging from autoimmunity to transplantation, but delivery of a mixed pool of Tregs has not proven consistently successful. To make an improved Treg-based therapy, Rui et al. developed OX40L–CAR-Tregs, where expression of an OX40 ligand (OX40L)–specific chimeric antigen receptor (CAR) is expressed only in Tregs that have turned on the master Treg transcription factor FoxP3. The authors showed that OX40L–CAR-Tregs outperformed control, standard Tregs in their ability to suppress effector T cells, in part through their ability to strip OX40L from the surface of antigen-presenting cells. In vivo, OX40L–CAR-Tregs were able to suppress development of graft-versus-host disease without an obvious detrimental effect on the antileukemia response, supporting further development of this potent cellular therapy. —Courtney Malo [ABSTRACT FROM AUTHOR]

Notch signaling promotes T cell pathogenicity and graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) in mice, with a dominant role for the Delta-like Notch ligand DLL4. To assess whether Notch's effects are evolutionarily conserved and to identify the mechanisms of Notch signaling inhibition, we studied antibody-mediated DLL4 blockade in a nonhuman primate (NHP) model similar to human allo-HCT. Short-term DLL4 blockade improved posttransplant survival with durable protection from gastrointestinal GVHD in particular. Unlike prior immunosuppressive strategies tested in the NHP GVHD model, anti-DLL4 interfered with a T cell transcriptional program associated with intestinal infiltration. In cross-species investigations, Notch inhibition decreased surface abundance of the gut-homing integrin α4β7 in conventional T cells while preserving α4β7 in regulatory T cells, with findings suggesting increased β1 competition for α4 binding in conventional T cells. Secondary lymphoid organ fibroblastic reticular cells emerged as the critical cellular source of Delta-like Notch ligands for Notch-mediated up-regulation of α4β7 integrin in T cells after allo-HCT. Together, DLL4-Notch blockade decreased effector T cell infiltration into the gut, with increased regulatory to conventional T cell ratios early after allo-HCT. Our results identify a conserved, biologically unique, and targetable role of DLL4-Notch signaling in intestinal GVHD. Editor's summary: Acute graft-versus-host disease (aGVHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation, and additional treatment strategies are urgently needed. Here, Tkachev et al. moved one candidate treatment, blockade of the Delta-like Notch ligand DLL4, closer to the clinic. DLL4 blockade has previously been shown to control aGVHD in mice, and here the authors found that that efficacy translated into nonhuman primate models. The authors also identified the mechanism by which the treatment may specifically control intestinal aGVHD; DLL4 blockade decreased effector T cell infiltration into the gut, thereby increasing the relative frequency of regulatory T cells. These studies highlight the clinical potential for targeting DLL4-Notch signaling for patients with aGVHD. —Courtney Malo [ABSTRACT FROM AUTHOR]

SIVSing through GVHD: Currently, there are few ways to track leukocyte movements in vivo. Potter and colleagues developed a method to track leukocyte migration in vivo called serial intravascular staining or SIVS. The authors infused differently labeled antibodies at different time points to reveal distinct leukocyte population kinetics in healthy macaques and those infected with Mycobacterium tuberculosis. Tkachev et al. then applied SIVS to shed light on donor T cell trafficking in macaques that developed acute graft-versus-host disease (GVHD) after transplant. They found that donor cytotoxic CD8+ T cells infiltrated the gastrointestinal tract and developed the transcriptional signature of tissue-resident memory T cells. SIVS will be useful for revealing leukocyte trafficking kinetics not only in GVHD and infection but in a variety of other diseases as well. Organ infiltration by donor T cells is critical to the development of acute graft-versus-host disease (aGVHD) in recipients after allogeneic hematopoietic stem cell transplant (allo-HCT). However, deconvoluting the transcriptional programs of newly recruited donor T cells from those of tissue-resident T cells in aGVHD target organs remains a challenge. Here, we combined the serial intravascular staining technique with single-cell RNA sequencing to dissect the tightly connected processes by which donor T cells initially infiltrate tissues and then establish a pathogenic tissue residency program in a rhesus macaque allo-HCT model that develops aGVHD. Our results enabled creation of a spatiotemporal map of the transcriptional programs controlling donor CD8+ T cell infiltration into the primary aGVHD target organ, the gastrointestinal (GI) tract. We identified the large and small intestines as the only two sites demonstrating allo-specific, rather than lymphodepletion-driven, T cell infiltration. GI-infiltrating donor CD8+ T cells demonstrated a highly activated, cytotoxic phenotype while simultaneously developing a canonical tissue-resident memory T cell (TRM) transcriptional signature driven by interleukin-15 (IL-15)/IL-21 signaling. We found expression of a cluster of genes directly associated with tissue invasiveness, including those encoding adhesion molecules (ITGB2), specific chemokines (CCL3 and CCL4L1) and chemokine receptors (CD74), as well as multiple cytoskeletal proteins. This tissue invasion transcriptional signature was validated by its ability to discriminate the CD8+ T cell transcriptome of patients with GI aGVHD from those of GVHD-free patients. These results provide insights into the mechanisms controlling tissue occupancy of target organs by pathogenic donor CD8+ TRM cells during aGVHD in primate transplant recipients. [ABSTRACT FROM AUTHOR]

Exhausted T cells in cancer and chronic viral infection express distinctive patterns of genes, including sustained expression of programmed cell death protein 1 (PD-1). However, the regulation of gene expression in exhausted T cells is poorly understood. Here, we define the accessible chromatin landscape in exhausted CD8+ T cells and show that it is distinct from functional memory CD8+ T cells. Exhausted CD8+ T cells in humans and a mouse model of chronic viral infection acquire a state-specific epigenetic landscape organized into functional modules of enhancers. Genome editing shows that PD-1 expression is regulated in part by an exhaustion-specific enhancer that contains essential RAR, T-bet, and Sox3 motifs. Functional enhancer maps may offer targets for genome editing that alter gene expression preferentially in exhausted CD8+ T cells. [ABSTRACT FROM AUTHOR]

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.

Organ infiltration by donor T cells is critical to the development of acute graft-versus-host disease (aGVHD) in recipients after allogeneic hematopoietic stem cell transplant (allo-HCT). However, deconvoluting the transcriptional programs of newly recruited donor T cells from those of tissue-resident T cells in aGVHD target organs remains a challenge. Here, we combined the serial intravascular staining technique with single-cell RNA sequencing to dissect the tightly connected processes by which donor T cells initially infiltrate tissues and then establish a pathogenic tissue residency program in a rhesus macaque allo-HCT model that develops aGVHD. Our results enabled creation of a spatiotemporal map of the transcriptional programs controlling donor CD8 + T cell infiltration into the primary aGVHD target organ, the gastrointestinal (GI) tract. We identified the large and small intestines as the only two sites demonstrating allo-specific, rather than lymphodepletion-driven, T cell infiltration. GI-infiltrating donor CD8 + T cells demonstrated a highly activated, cytotoxic phenotype while simultaneously developing a canonical tissue-resident memory T cell (T RM ) transcriptional signature driven by interleukin-15 (IL-15)/IL-21 signaling. We found expression of a cluster of genes directly associated with tissue invasiveness, including those encoding adhesion molecules ( ITGB2 ), specific chemokines ( CCL3 and CCL4L1 ) and chemokine receptors ( CD74 ), as well as multiple cytoskeletal proteins. This tissue invasion transcriptional signature was validated by its ability to discriminate the CD8 + T cell transcriptome of patients with GI aGVHD from those of GVHD-free patients. These results provide insights into the mechanisms controlling tissue occupancy of target organs by pathogenic donor CD8 + T RM cells during aGVHD in primate transplant recipients., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)