Yani Peng's research while affiliated with UCSF University of California, San Francisco and other places

The present disclosure relates to chimeric-antigen-receptor regulatory T cells (CAR-Tregs) that include an exogenous nucleic acid encoding a chimeric antigen receptor (CAR) comprising the complementarity determining regions (CDRs) of a human anti-HLA-A2 antibody grafted onto an antibody scaffold, a hinge domain, a transmembrane domain; and an intracellular domain, where the CAR-Treg expresses the CAR on the cell surface. Vectors and methods of making and using such CAR-Tregs are disclosed as well.

Promoting immune tolerance to transplanted organs can minimize the amount of immunosuppressive drugs that patients need to take, reducing lifetime risks of mortality and morbidity. Regulatory T cells (Tregs) are essential for immune tolerance, and preclinical studies have shown their therapeutic efficacy in inducing transplantation tolerance. Here, we report the results of a phase 1/2 trial (ARTEMIS, NCT02474199) of autologous donor alloantigen-reactive Treg (darTreg) therapy in individuals 2 to 6 years after receiving a living donor liver transplant. The primary efficacy endpoint was calcineurin inhibitor dose reduction by 75% with stable liver function tests for at least 12 weeks. Among 10 individuals who initiated immunosuppression withdrawal, 1 experienced rejection before planned darTreg infusion, 5 received darTregs, and 4 were not infused because of failure to manufacture the minimal infusible dose of 100 × 106 cells. darTreg infusion was not associated with adverse events. Two darTreg-infused participants reached the primary endpoint, but an insufficient number of recipients were treated for assessing the efficacy of darTregs. Mechanistic studies revealed generalized Treg activation, senescence, and selective reduction of donor reactivity after liver transplantation. Overall, the ARTEMIS trial features a design concept for evaluating the efficacy of Treg therapy in transplantation. The mechanistic insight gained from the study may help guide the design of future trials.

Alloantigen‐specific regulatory T cell (Treg) therapy is a promising approach for suppressing alloimmune responses and minimizing immunosuppression after solid organ transplantation. Chimeric antigen receptor (CAR) targeting donor alloantigens can confer donor reactivity to Tregs. However, CAR Treg therapy has not been evaluated in vascularized transplant or multi‐MHC mismatched models. Here, we evaluated the ability of CAR Tregs targeting HLA‐A2 (A2‐CAR) to prolong survival of heterotopic heart transplants in mice. After verifying the in vitro activation, proliferation, and enhanced suppression of A2‐CAR Tregs in the presence of A2‐antigen, we analyzed the in vivo function of Tregs in C57BL/6 (B6) mice receiving A2‐expressing heart allografts. A2‐CAR Treg infusion increased the median survival of grafts from B6.HLA‐A2 transgenic donors from 23 days to 99 days, whereas median survival with polyclonal Treg infusion was 35 days. In a more stringent model of haplo‐mismatched hearts from BALB/cxB6.HLA‐A2 F1 donors, A2‐CAR Tregs slightly increased median graft survival from 11 days to 14 days, which was further extended to >100 days when combined with a 9‐day course of rapamycin treatment. These findings demonstrate the efficacy of CAR Tregs, alone or in combination with immunosuppressive agents, toward protecting vascularized grafts in fully immunocompetent recipients.

Treg therapies are being tested in clinical trials in transplantation and autoimmune diseases, however, the impact of inflammation on Tregs remains controversial. We challenged human Tregs ex-vivo with pro-inflammatory cytokines IL-6 and TNFα and observed greatly enhanced proliferation stimulated by anti-CD3 and anti-CD28 (aCD3/28) beads or CD28 superagonist (CD28SA). The cytokine-exposed Tregs maintained high expression of FOXP3 and HELIOS, demethylated FOXP3 enhancer, and low IFNγ, IL-4, and IL-17 secretion. Blocking TNF receptor using etanercept or deletion of TNF receptor 2 using CRISPR/Cas9 blunted Treg proliferation and attenuated FOXP3 and HELIOS expression. These results prompted us to consider using CD28SA together with IL-6 and TNFα without aCD3/28 beads (beadless) as an alternative protocol for therapeutic Treg manufacturing. Metabolomics profiling revealed more active glycolysis and oxidative phosphorylation, increased energy production, and higher antioxidant potential during beadless Treg expansion. Finally, beadless expanded Tregs maintained suppressive functions in vitro and in vivo. These results demonstrate that human Tregs positively respond to proinflammatory cytokines with enhanced proliferation without compromising their lineage identity or function. This property can be harnessed for therapeutic Treg manufacturing.

Treg therapy is being tested in clinical trials in transplantation and autoimmune diseases, however, the impact of inflammation on Tregs is unclear. In this study, we challenged human Tregs ex-vivo with pro-inflammatory cytokines, TNFa and IL-6. These cytokines enhanced Treg proliferation induced by anti-CD3 and anti-CD28 or CD28 superagonist (CD28SA) while maintaining high expression of FOXP3 and HELIOS, demethylated FOXP3 enhancer, and low expression of cytokines IFNg, IL-4 and IL-17. Blocking TNF receptor signaling using etanercept or deletion of TNF receptor 2 using CRISPR/Cas9 blunted Treg proliferation and attenuated FOXP3 and HELIOS expression, revealing the importance of TNFR2 signaling in Treg proliferation and lineage stability. The robust proliferation induced by CD28SA with IL-6 and TNFa may be adopted for the expansion of therapeutic Tregs. Metabolomics analysis showed that Tregs expanded with CD28SA plus cytokines had more active glycolysis and oxidative phosphorylation, increased energy production, and higher antioxidant potential. Finally, CD28SA plus cytokine-expanded Tregs had comparable suppressive activity in vitro and in vivo in a humanized mouse model of graft-versus-host-disease when compared to Tregs expanded using the conventional protocol. These results demonstrate that human Tregs positively respond to proinflammatory cytokines with enhanced proliferation without compromising their lineage identity or function.

Naturally occurring cases of monogenic type 1 diabetes (T1D) help establish direct mechanisms driving this complex autoimmune disease. A recently identified de novo germline gain-of-function (GOF) mutation in the transcriptional regulator STAT3 was found to cause neonatal T1D. We engineered a novel knock-in mouse incorporating this highly diabetogenic human STAT3 mutation (K392R) and found that these mice recapitulated the human autoimmune diabetes phenotype. Paired single-cell TCR and RNA sequencing revealed that STAT3-GOF drives proliferation and clonal expansion of effector CD8+ cells that resist terminal exhaustion. Single-cell ATAC-seq showed that these effector T cells are epigenetically distinct and have differential chromatin architecture induced by STAT3-GOF. Analysis of islet TCR clonotypes revealed a CD8+ cell reacting against known antigen IGRP, and STAT3-GOF in an IGRP-reactive TCR transgenic model demonstrated that STAT3-GOF intrinsic to CD8+ cells is sufficient to accelerate diabetes onset. Altogether, these findings reveal a diabetogenic CD8+ T cell response that is restrained in the presence of normal STAT3 activity and drives diabetes pathogenesis.

Naturally occurring cases of monogenic type 1 diabetes (T1D) provide rare opportunities to establish direct mechanisms that cause this complex autoimmune disease. A recently identified de novo germline gain-of-function (GOF) mutation in the transcriptional regulator signal transducer and activator of transcription 3 (STAT3) was shown to cause neonatal T1D at birth. To investigate the role of STAT3 hyperactivity in T1D, we engineered a novel knock-in (KI) mouse incorporating this highly diabetogenic human mutation (K392R) in the STAT3 gene. These mice developed accelerated diabetes with severe insulitis and insulin autoantibodies, thereby recapitulating the human autoimmune diabetes phenotype. Paired T cell receptor (TCR) and transcriptome (RNA) sequencing in single cells revealed that STAT3-GOF drives the proliferation and clonal expansion of highly cytotoxic effector CD8+ T cells that are resistant to terminal exhaustion. Single-cell ATAC-seq showed that these effector T cells are epigenetically distinct and revealed differential chromatin architecture induced by STAT3-GOF. Analysis of islet TCR clonotypes revealed an effector CD8+ T cell reacting against the known antigen IGRP, and STAT3-GOF in an IGRP-reactive TCR transgenic model demonstrated that STAT3-GOF intrinsic to CD8+ T cells is sufficient to accelerate diabetes onset. Taken together, these findings reveal a diabetogenic CD8+ T cell response that is restrained in the presence of normal STAT3 activity and drives diabetes pathogenesis.