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Regulatory T cells (Tregs) promote immune homeostasis by maintaining self-tolerance and regulating inflammatory responses. Under certain inflammatory conditions, Tregs can lose their lineage stability and function. Previous studies have reported that ex vivo exposure to retinoic acid (RA) enhances Treg function and stability. However, it is unknown how RA receptor signaling in Tregs influences these processes in vivo. Herein, we employed mouse models in which RA signaling is silenced by the expression of the dominant negative receptor (DN) RARα in all T cells. Despite the fact that DNRARα conventional T cells are hypofunctional, Tregs had increased CD25 expression, STAT5 pathway activation, mTORC1 signaling and supersuppressor function. Furthermore, DNRARα Tregs had increased inhibitory molecule expression, amino acid transporter expression, and metabolic fitness and decreased antiapoptotic proteins. Supersuppressor function was observed when wild-type mice were treated with a pharmacologic pan-RAR antagonist. Unexpectedly, Treg-specific expression of DNRARα resulted in distinct phenotypes, such that a single allele of DNRARα in Tregs heightened their suppressive function, and biallelic expression led to loss of suppression and autoimmunity. The loss of Treg function was not cell intrinsic, as Tregs that developed in a noninflammatory milieu in chimeric mice reconstituted with DNRARα and wild-type bone marrow maintained the enhanced suppressive capacity. Fate mapping suggested that maintaining Treg stability in an inflammatory milieu requires RA signaling. Our findings indicate that RA signaling acts as a rheostat to balance Treg function in inflammatory and noninflammatory conditions in a dose-dependent manner.

Current Food and Drug Administration (FDA)-approved CD19-specific chimeric antigen receptor (CAR) T-cell therapies for B-cell malignancies are constitutively active and while efficacious, can cause morbidity and mortality. Their toxicities might be reduced if CAR T-cell activity was regulatable rather than constitutive. To test this, we compared the efficacies and morbidities of constitutively active (conventional) and regulatable (switchable) CAR (sCAR) T-cells specific for human CD19 (huCD19) in an immune-competent huCD19+transgenic mouse model.Conventional CAR (CAR19) and sCAR T-cells were generated by retrovirally transducing C57BL/6 (B6) congenic T-cells with constructs encoding antibody-derived single chain Fv (sFv) fragments specific for huCD19 or a peptide neoepitope (PNE), respectively. Transduced T-cells were adoptively transferred into huCD19 transgenic hemizygous (huCD19Tg/0) B6 mice; healthy B-cells in these mice expressedhuCD19Tg. Prior to transfer, recipients were treated with a lymphodepleting dose of cyclophosphamide to enhance T-cell engraftment. In tumor therapy experiments, CAR19 or sCAR T-cells were adoptively transferred intohuCD19Tg/0mice bearing a syngeneic B-cell lymphoma engineered to express huCD19. To regulate sCAR T cell function, a switch protein was generated that contained the sCAR-specific PNE genetically fused to an anti-huCD19 Fab fragment. Recipients of sCAR T-cells were injected with the switch to link sCAR effector with huCD19+target cells. Mice were monitored for survival, tumor burden (where appropriate), morbidity (as measured by weight loss and clinical scores), and peripheral blood lymphocyte frequency.CAR19 and sCAR T-cells functioned comparably regarding in vivo expansion and B-cell depletion. However, sCAR T-cells were better tolerated as evidenced by the recipients’ enhanced survival, reduced weight loss, and improved clinical scores. Discontinuing switch administration allowed healthy B-cell frequencies to return to pretreatment levels.In our mouse model, sCAR T-cells killed huCD19+healthy and malignant B-cells and were better tolerated than CAR19 cells. Our data suggest sCAR might be clinically superior to the current FDA-approved therapies for B-cell lymphomas due to the reduced acute and chronic morbidities and mortality, lower incidence and severity of side effects, and B-cell reconstitution on cessation of switch administration.

Regulatory T-cells (Treg) are critical for the maintenance of immune homeostasis and tolerance induction. While the immunosuppressive mechanisms of Treg have been extensively investigated for decades, the mechanisms responsible for Treg cytotoxicity and their therapeutic potential in regulating immune responses have been incompletely explored and exploited. Conventional cytotoxic T effector cells (Teffs) are known to be important for adaptive immune responses, particularly in the settings of viral infections and cancer. CD4+ and CD8+ Treg subsets may also share similar cytotoxic properties with conventional Teffs. Cytotoxic effector Treg (cyTreg) are a heterogeneous population in the periphery that retain the capacity to suppress T-cell proliferation and activation, induce cellular apoptosis, and migrate to tissues to ensure immune homeostasis. The latter can occur through several cytolytic mechanisms, including the Granzyme/Perforin and Fas/FasL signaling pathways. This review focuses on the current knowledge and recent advances in our understanding of cyTreg and their potential application in the treatment of human disease, particularly Graft-versus-Host Disease (GVHD).

Aberrant immune responses, such as high Tfollicular helper (Tfh) and germinal center B cells (GCB) in cGVHD inducing antibody (Ab) secretion with tissue deposition and fibrosis, require distinct metabolic reprogramming to sustain pathogenesis. Leucine, isoleucine, and valine are essential BCAAs that signal via mTORC1 to support glycolysis and protein synthesis. Alternatively, BCAAs can be oxidized to metabolites that directly enter the tricarboxylic acid cycle or become substrates for gluconeogenesis and ketogenesis. Branched chain ketoacid dehydrogenase kinase (BDK) is a negative regulator of BCAA metabolism, decreasing downstream oxidation and increasing mTORC1 and glycolysis. We sought to determine whether increasing BCAA oxidation could be exploited to inhibit cGVHD. BDK is inhibitable by 3,6-Dichlorobenzo[β] thiophene-2-carboxylic acid (BT2), increasing BCAA oxidation and decreasing T effector (Teff) activation. In a multiorgan cGVHD model with bronchiolitis obliterans, mice that received BDK knockout vs wildtype donor T cells had improved survival and pulmonary function with decreased GCB frequencies (p-value 0.005) and a trend toward decreased Tfh/Tfollicular regulatory ratios. An in vitro assay measuring Tfh-mediated B cell Ab production showed that BT2 downregulates IgG class switching, which is key for cGVHD pathogenesis. BT2-treated T effectors but not T regulatory cells (Treg) exhibited increased oxidative phosphorylation (OXPHOS). Therefore, limiting BCAAs inhibits Teff switch from a highly glycolytic state to OXPHOS. In contrast, BCAAs are not required for Treg energy production or survival. These data suggest that augmenting BCAA oxidation could be a new therapeutic approach for treating cGVHD. Supported by NIH P01 AI056299

Despite graft-versus-host disease (GVHD) prophylactic agents, the success and wider utilization of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is limited by GVHD. Increasing donor graft regulatory T cell (Treg):effector T cell (Teff) ratios can substantially reduce GVHD in cancer patients, but pre-HSCT conditioning regimens and GVHD create a challenging inflammatory environment for Treg stability, persistence, and function. Metabolism plays a crucial role in T cell and Treg differentiation, and development of effector function. Although glycolysis is a main driver of allogeneic T cell-driven GVHD, oxidative phosphorylation is a main driver of Treg suppressor function. This review focuses on recent advances in our understanding of Treg metabolism in the context of GVHD, and discusses potential therapeutic applications of Tregs in the prevention or treatment of GVHD in cancer patients.

Damage to the gastrointestinal (GI) tract following hematopoietic stem cell transplant (HSCT) has been shown to drive the development of acute graft-versus-host disease (aGVHD). We have demonstrated clinically that infusing regulatory T-cells (Treg) during HSCT reduces aGVHD risk while supporting immune recovery; however, high Treg doses were necessary. Here we demonstrate that by promoting Treg homing to the GI tract early post-HSCT, aGVHD severity was reduced. Ex vivo expanded murine Treg were retrovirally transduced to overexpress the orphan chemoattractant receptor GPR15 for colon specific T-cell homing. Despite no change in Treg phenotype or in vitro suppressive function when administered to HSCT recipient mice at the time of transplantation, mice that received GPR15+ Treg exhibited significantly reduced aGVHD severity and prolonged survival compared to control Treg mice (p = 0.0035). To confirm that this resulted from an increased Treg accumulation in the GI tissues, we then assessed the frequency and retention of GPR15+ Treg in mice post-HSCT by in vivo and ex vivo bioluminescent imaging as well as flow analysis. From these data, we observed not only superior localization of GPR15+ Treg within GI tissues, but also significantly reduced inflammation and tissue damage in GPR15+ Treg recipient mice early post-HSCT. Our data provides evidence that deliberate targeting of ex vivo expanded Treg to the GI tract during HSCT significantly reduces injury to the GI tract and reduces aGVHD severity by simulating an increase in Treg dose in vivo. These data provide a rational for the future development of clinical Treg products for increased control of GI aGVHD following HSCT.

Thymus-derived regulatory T cells (tTreg) have been used as a cellular therapeutic to prevent or treat autoimmunity, graft rejection, and graft versus host disease (GVHD) in humans. Metabolic pathways affect tTreg differentiation, expansion and suppressive function. Recent experiments challenge the initial paradigm that tTregs depend on fatty acid oxidation (FAO) and glycolysis. Murine studies also show that central memory and effector tTreg have distinct metabolic profiles, but human studies to date use pooled populations of tTreg. The goal of the current study was to identify and compare the metabolic pathways critical for naïve and memory human tTreg, and to determine whether these pathways differ from naïve human CD4 T cells. We found that memory tTreg have higher rates of glycolysis and oxidative phosphorylation (OXPHOS) than naïve tTreg, both at rest and after stimulation. Activated naïve and memory tTreg had high basal OXPHOS, but neither glucose nor FAO were major substrates. Consistent with this, culture in low glucose did not affect expansion of either naïve or memory tTreg, but greatly decreased that of CD4 Teff. Extracellular lactate increased tTreg basal OCR and, in contrast to the lactate-dependent inhibition of CD4 T cells, enhanced tTreg expansion. This is consistent with differential expression of a class of lactate transporters (monocarboxylate transporters, or MCT) where Treg have increased expression of MCT1/2, which favor import whereas Teff have increased expression of MCT4, which favors export. These data demonstrate that naïve and memory tTreg should be analyzed separately, and provide a molecular insight into why tTreg, but not Teff, function in a low glucose, high lactate environment.

GVHD remains the primary complication of allogeneic hematopoietic stem cell transplantation. Considerable interest exists in understanding how metabolism affects immune cell function. We show targeting FAS represents an attractive strategy to ameliorate cGVHD. Acetyl-CoA carboxylase 1 (ACC1) catalyzes the first step of FAS, and can be pharmacologically inhibited with either Soraphen A or 5-(Tetradecyloxy)-2-furoic acid (TOFA). FAS is critical for T cell effector responses, a finding we confirmed using an in vivo multiorgan system cGVHD model with bronchiolitis obliterans, a model dependent on germinal centers (GC), IgG2c secretion, lung deposition and fibrosis. Compared to wild type T cells, cGVHD mice receiving CD4CreACC1fl/fl allogeneic T cells had reduced cGVHD severity assessed by pulmonary function test and immune phenotyping. As thymic regulatory T cell (tTreg) infusion can prevent and treat cGVHD, we examined tTreg FAS needs. Inhibiting FAS in sorted tTreg via drug (Soraphen A; TOFA) or genetic (FoxP3CreACC1fl/fl) strategies increased oxidative and glycolytic metabolism measured by Seahorse assay, that correlated with superior in vitro suppressor function. These findings were extended in vivo where transfer of FoxP3CreACC1fl/fl tTreg, at day 28 following disease establishment, decreased cGVHD severity. Daily treatment of cGVHD mice from day 28 to day 56 with TOFA reversed cGVHD. An in vitro assay in which T follicular regulatory cells suppress T follicular helper and GC B cell class switching showed that inhibiting tTreg FAS can increase their suppression of GC responses. These data provide evidence that inhibiting FAS can prevent or treat cGVHD and enhance Treg function, providing a new approach to ameliorate cGVHD.

Background aims Chimeric antigen receptors (CARs) offer great potential toward a functional cure of human immunodeficiency virus (HIV) infection. To achieve the necessary long-term virus suppression, we believe that CARs must be designed for optimal potency and anti-HIV specificity, and also for minimal probability of virus escape and CAR immunogenicity. CARs containing antibody-based motifs are problematic in the latter regard due to epitope mutation and anti-idiotypic immune responses against the variable regions. Methods We designed bispecific CARs, each containing a segment of human CD4 linked to the carbohydrate recognition domain of a human C-type lectin. These CARs target two independent regions on HIV-1 gp120 that presumably must be conserved on clinically significant virus variants (i.e., the primary receptor binding site and the dense oligomannose patch). Functionality and specificity of these bispecific CARs were analyzed in assays of CAR-T cell activation and spreading HIV-1 suppression. Results T cells expressing a CD4-dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DCSIGN) CAR displayed robust stimulation upon encounter with Env-expressing targets, but negligible activity against intercellular adhesion molecule (ICAM)-2 and ICAM-3, the natural dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin ligands. Moreover, the presence of the lectin moiety prevented the CD4 from acting as an entry receptor on CCR5-expressing cells, including CD8+ T cells. However, in HIV suppression assays, the CD4-DCSIGN CAR and the related CD4-liver/lymph node-specific intercellular adhesion molecule-3-grabbing non-integrin CAR displayed only minimally increased potency compared with the CD4 CAR against some HIV-1 isolates and reduced potency against others. By contrast, the CD4-langerin and CD4-mannose binding lectin (MBL) CARs uniformly displayed enhanced potency compared with the CD4 CAR against all the genetically diverse HIV-1 isolates examined. Further experimental data, coupled with known biological features, suggest particular advantages of the CD4-MBL CAR. Discussion These studies highlight features of bispecific CD4-lectin CARs that achieve potency enhancement by targeting two distinct highly conserved Env determinants while lacking immunogenicity-prone antibody-based motifs.