Human regulatory T (T reg ) cells are essential for immune homeostasis. The transcription factor (TF) FOXP3 maintains T reg cell identity, yet the complete set of key TFs that control T reg cell gene expression remains unknown. Here, we used pooled and arrayed Cas9 ribonucleoprotein (RNP) screens to identify TFs that regulate critical proteins in primary human T reg cells under basal and pro-inflammatory conditions. We then generated 54,424 single-cell transcriptomes from T reg cells subjected to genetic perturbations and cytokine stimulation, which revealed distinct gene networks individually regulated by FOXP3 and PRDM1, in addition to a network co-regulated by FOXO1 and IRF4. We also discovered that HIVEP2, not previously implicated in T reg cell function, co-regulates another gene network with SATB1 and is important for T reg cell-mediated immunosuppression. By integrating CRISPR screens and scRNA-seq profiling, we have uncovered novel transcriptional regulators and downstream gene networks in human T reg cells that could be targeted for immunotherapies.

Infusion of regulatory T cells (Tregs) engineered with a chimeric antigen receptor (CAR) targeting donor-derived human leukocyte antigen (HLA) is a promising strategy to promote transplant tolerance. Here, we describe an anti-HLA-A2 CAR (A2-CAR) generated by grafting the complementarity-determining regions (CDRs) of a human monoclonal anti-HLA-A2 antibody into the framework regions of the Herceptin 4D5 single-chain variable fragment and fusing it with a CD28-ζ signaling domain. The CDR-grafted A2-CAR maintained the specificity of the original antibody. We then generated HLA-A2 mono-specific human CAR Tregs either by deleting the endogenous T-cell receptor (TCR) via CRISPR/Cas9 and introducing the A2-CAR using lentiviral transduction or by directly integrating the CAR construct into the TCR alpha constant locus using homology-directed repair. These A2-CAR+TCRdeficient human Tregs maintained both Treg phenotype and function in vitro. Moreover, they selectively accumulated in HLA-A2-expressing islets transplanted from either HLA-A2 transgenic mice or deceased human donors. A2-CAR+TCRdeficient Tregs did not impair the function of these HLA-A2+ islets, whereas similarly engineered A2-CAR+TCRdeficientCD4+ conventional T cells rejected the islets in less than 2 weeks. A2-CAR+TCRdeficient Tregs delayed graft-versus-host disease only in the presence of HLA-A2, expressed either by co-transferred peripheral blood mononuclear cells or by the recipient mice. Altogether, we demonstrate that genome-engineered mono-antigen-specific A2-CAR Tregs localize to HLA-A2-expressing grafts and exhibit antigen-dependent in vivo suppression, independent of TCR expression. These approaches may be applied towards developing precision Treg cell therapies for transplant tolerance.

We encapsulated cyclosporine A (CsA) in poly(ethylene glycol)-b-poly(d,l-lactide-co-glycolide) (PEG-PLGA) nanoparticles (NPs) by nanoprecipitation of CsA and PEG-PLGA. The resulting CsA/PEG-PLGA-NPs were <100 nm in diameter with a narrow particle size distribution. The NP size could be controlled by tuning the polymer concentration, solvent, or water/solvent ratio during formulation. The PEGylated NPs maintained non-aggregated in salt solution. Solid NPs lyoprotected with bovine serum albumin were prepared for the convenience of storage and transportation. The release kinetics of CsA (55.6% released on Day 1) showed potential for maintaining therapeutic CsA concentrations in vivo. In T-cell assays, both free CsA and CsA/PEG-PLGA-NPs suppressed T-cell proliferation and production of inflammatory cytokines dose dependently. In a mixed lymphocyte reaction assay, the IC50 values for free CsA and CsA/PEG-PLGA-NPs were found to be 30 and 35 ng/mL, respectively. This nanoparticulate CsA delivery technology constitutes a strong basis for future targeted delivery of immunosuppressive drugs with improved efficiency and potentially reduced toxicity.

Regulatory T-cells (Tregs) play a pivotal role in the maintenance of immune tolerance and hold great promise as cell therapy for a variety of immune-mediated diseases. However, the cellular mechanisms that regulate Treg maintenance and homeostasis have yet to be fully explored. While Tregs express Granzyme-B (GrB) to suppress effector T-cells via direct-killing, the mechanisms by which they protect themselves from GrB-mediated self-inflicted damage are unknown. We show, for the first time, that both iTregs and nTregs increase their intracellular expression of GrB and its endogenous inhibitor, Serine Protease Inhibitor-6 (Spi6) upon activation. Sub-cellular fractionation and measurement of GrB activity in the cytoplasm of Tregs show that activated Spi6−/− Tregs had significantly higher cytoplasmic GrB activity. We observed an increase in GrB-mediated apoptosis in Spi6−/− nTregs and impaired suppression of alloreactive T-cells in vitro. Spi6−/− Tregs were rescued from apoptosis by the addition of a GrB inhibitor (Z-AAD-CMK) in vitro. Furthermore, adoptive transfer experiments showed that Spi6−/− nTregs were less effective than WT nTregs in suppressing Graft-versus-host-disease (GVHD) due to their impaired survival, as shown in our in vivo bioluminescence imaging. Finally, Spi6-deficient recipients rejected MHC class II-mismatch heart allografts at a much faster rate and showed a higher rate of apoptosis among Tregs, as compared to WT recipients. Our data demonstrate, for the first time, a novel role for Spi6 in Treg homeostasis by protecting activated Tregs from GrB-mediated injury. These data could have significant clinical implications for Treg-based therapy in immune-mediated diseases.

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.

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.

Skartsis N, Martinez L, Duque JC, Tabbara M, Velazquez OC, Asif A, Andreopoulos F, Salman LH, Vazquez-Padron RI. c-Kit signaling determines neointimal hyperplasia in arteriovenous fistulae. Am J Physiol Renal Physiol 307: F1095-F1104, 2014. First published September 3, 2014 doi:10.1152/ajprenal.00292.2014.-Stenosis of arteriovenous (A-V) fistulae secondary to neointimal hyperplasia (NIH) compromises dialysis delivery, which worsens patients' quality of life and increases medical costs associated with the maintenance of vascular accesses. In the present study, we evaluated the role of the receptor tyrosine kinase c-Kit in A-V fistula neointima formation. Initially, c-Kit was found in the neointima and adventitia of human brachiobasilic fistulae, whereas it was barely detectable in control veins harvested at the time of access creation. Using the rat A-V fistula model to study venous vascular remodeling, we analyzed the spatial and temporal pattern of c-Kit expression in the fistula wall. Interestingly, c-Kit immunoreactivity increased with time after anastomosis, which concurred with the accumulation of cells in the venous intima. In addition, c-Kit expression in A-V fistulae was positively altered by chronic kidney failure conditions. Both blockade of c-Kit with imatinib mesylate (Gleevec) and inhibition of stem cell factor production with a specific short hairpin RNA prevented NIH in the outflow vein of experimental fistulae. In agreement with these data, impaired c-Kit activity compromised the development of NIH in A-V fistulae created in c-Kit W/Wv mutant mice. These results suggest that targeting of the c-Kit signaling pathway may be an effective approach to prevent postoperative NIH in A-V fistulae. arteriovenous fistula; hemodialysis; neointima THE ARTERIOVENOUS (A-V) fistula is the preferred type of vascular access for hemodialysis patients (29). It achieves higher patency rates, has fewer complications than synthetic grafts (12,29,31), and has a lower risk of infections than central venous catheters (17,29). Despite its advantages, A-V fistulae frequently fail to mature or become dysfunctional after successful dialysis sessions, and this occurs primarily due to the development of neointimal hyperplasia (NIH) within the A-V fistula circuit (2, 5, 39). Stenosed A-V fistulae can sometimes be salvaged through angioplasty or surgical interventions, but these attempts often result in restenosis or additional complications (32). Therefore, there is an unmet medical need for treatments that prevent NIH and improve A-V fistula function. To this end, it is necessary to better define the factors underlying the pathological remodeling of the A-V fistula wall.A-V fistula maturation is a dynamic vascular process with multiple factors contributing to the development of NIH. These include vein configuration (22) In the present study, we investigated the role of c-Kit in the pathological remodeling of A-V fistulae. We show that activation of the c-Kit signaling pathway in adventitial and neointimal cells precedes arteri...

The exact origin of the neointimal cells in arteriovenous fistulae (AVFs) has not been conclusively established. This study attempts to elucidate the anatomical source of neointimal cells in AVFs. Experimental fistulas were created in Lewis wild type (WT) and transgenic rats that constitutively expressed the Green Fluorescent Protein (GFP) in all tissues. AVFs were created by anastomosing the left renal vein to the abdominal aorta after unilateral nephrectomy. Three potential sources (bone marrow, feeding artery and the fistula vein itself) were examined. The contribution of bone marrow (BM) derived cells to the AVF neointima was examined in lethally irradiated WT rats that had been rescued with GFP BM cells. Neointimal cells in these chimeric rats were mostly GFP negative indicating the non-BM origin of those cells. Then, the contribution of arterial cells to the AVF neointima was assessed in a fistula made with a GFP aorta that had been implanted orthotopically into a WT rat. In this model, most of the neointimal cells were also GFP negative demonstrating that AVF neointimal cells are not derived from the feeding artery cells. Finally to study local resident cells contribution to the formation of neointimal lesions, a composite fistula was created by interposing a GFP vein between the renal vein and the aorta in a WT recipient rat. GFP neointimal cells were only found in the transplanted vein. This study suggests that neointimal cells originate from the local resident cells of the AVF.