Treg Suppressive Function Research Articles

OP0316 EMERGING BEST-IN-CLASS IL-2 VARIANT HIGHLIGHTS TREG-DIRECTED THERAPY FOR AUTOIMMUNE DISEASE

Background:Impairment or deficiency of regulatory T cells (Treg) is associated with chronic inflammation and autoimmune diseases. Interleukin 2 (IL-2) is a cytokine indispensable for Treg expansion and immunosuppressive function. However, expansion of cytotoxic effector T (Teff) and NK cells and the associated vascular leakage side effect limit the use of IL-2 in autoimmune diseases [1].Objectives:Cugene developed a long-acting IL-2 variant with high Treg specificity and low toxicity to restore immune homeostasis and self-tolerance, and potentially cure autoimmune and inflammatory diseases.Methods:IL-2 variants were generated based on the quaternary structure of IL-2 and IL-2Rαβγ (alpha, beta, gamma) complex. Biological activity was determined by examining differential signaling activity in induction of STAT5 phosphorylation in defined lymphocyte populations of human PBMC using flow cytometry. Binding activity was evaluated by ELISA. Pharmacokinetics, pharmacodynamics, safety and tolerability were assessed in mice and cynomolgus monkeys. Treg suppressive function was determinedin vivo/ex vivo,and anti-inflammatory and anti-antibody production efficacy were determined in delayed-type hypersensitivity (DTH) and T-cell-dependent antibody response (TDAR) models.Results:Structure-based rational design and activity-guided fine-tuning generated an optimized IL-2 variant, CUG252. It demonstrated a strong and near wild-type IL-2 ability to stimulate STAT5 phosphorylation in IL-2Rαβγ dominant Treg cells but abolished activities in IL-2Rβγ dominant effector CD4, CD8 and NK cells. This was a result of biased binding activity to IL-2Rα while dramatically attenuated binding to IL-2Rβγ complex. In mice and monkeys, administration of CUG252 resulted in dose-dependent increases in Treg proliferation and expansion by more than 10- and 30-fold, respectively, with largely abolished activities in CD4+ T conventional, cytotoxic CD8+ Teff and NK cells. The ratio of Treg/Teff cells achieved was as high as 0.4 in mice and 1.2 in monkeys. Both CD4+ and CD8+ Tregs were expanded with preferential increases in memory over naïve subsets. A substantial increase in Treg-suppressive capacity over T effector cells was corroborated by enhanced expression of functional and inhibitory markers, including CD25, Foxp3, PD-1, CTLA-4, Tim3 and ICOS. In DTH and TDAR models, CUG252 strongly inhibited antigen-driven inflammation, B cell maturation, and antibody production. The sustained PK/PD profile supports monthly dosing or better in humans. CUG252 was well-tolerated and no changes in body weight, body temperature, clinical pathology or signs of vascular leakage were observed. Moreover, CUG252 demonstrated superior manufacturability.Conclusion:CUG252 demonstrates an emerging best-in-class profile among IL-2 variants. It displayed exquisite Treg-selectivity while retaining potency comparable to wild-type IL-2. It showed strong anti-inflammatory and anti-antibody production efficacy with significantly improved therapeutic index and manufacturability. Its favorable drug-like property and robust preclinical efficacy warrant further evaluation in patients with a variety of inflammation and autoimmune diseases.

Treg suppressor function within allografts is required for tolerance

Abstract The role of regulatory CD4+Foxp3+ T cells (Treg) suppressor function within allografts in tolerance remain unclear. To directly address this, we first used a model where immune reactions are restrained to the graft only. Effector T cells (Teff) alone, or with Treg (2–3×106 each) were transferred to B6 mice lacking secondary lymphoid organs (SLO−; splenectomized LTβR−/−) 5 days after islet allograft (Balb/c). Adoptive transfer of Teff alone induced acute rejection (10d MST). In contrast, Teff + Treg co-transfers significantly prolonged graft survival, with 65% of recipients surviving long-term (>100d MST). Interestingly, Treg did not affect Teff proliferation or accumulation within allografts, but significantly reduced MHC-II expression on DCs and IFN-γ in Teff. Secondly, given that S1PR1 signaling is necessary for lymphocyte exit from SLO, we prevented Treg migration to allografts during tolerance by removing S1PR1 expression in endogenous Treg specifically. Tamoxifen-fed B6.Foxp3EGFP-Cre/Ert2.S1PR1fl/fl (S1PR1KO-Treg) or B6.S1PR1fl/fl (WT-Treg) mice received Balb/c islets. Untreated mice rejected allografts acutely (18d MST). Interestingly, while anti-CD45RB treatment induced tolerance in WT-Treg control mice (95d MST), all S1PR1KO-Treg mice rejected their graft (28d MST; p<0.05 vs WT-Treg). Also, these results correlated with a 25% decrease in Treg infiltrating islet allografts and with a 22% increase in Treg in lymph nodes on day 10 in S1PR1KO-Treg + anti-CD45RB compared to WT-Treg + anti-CD45RB recipients. Overall, our data demonstrate that Treg can suppress rejection by Teff within allografts without prior activation in SLO, and suggest that Treg suppressor function within allografts is required for tolerance.

Suppressive Mechanisms Induced by Tregs in Celiac Disease

CD is a systemic immune-mediated disorder caused by the dietary gluten in individuals who are genetically susceptible to the disease. In fact, CD is a T cell-mediated immune disease in which gluten-derived peptides activate the lamina propria CD4+ Teff cells, and these T-cell subsets can cause the intestinal tissue damages. Also, there are additional subsets of CD4+ T cells with suppressor functions. These subsets express the master transcription factor, FOXP3, and include Tr1 cells and CD4+CD25+ Tregs, which are the main population involved in maintaining the peripheral tolerance, preventing the autoimmune diseases and limiting the chronic inflammatory diseases such as CD. The suppressive function of Tregs is important to maintain the immune homeostasis. This paper examined the features and the basic mechanisms used by Tregs to mediate the suppression in CD.

CTLA-4-dependent trogocytosis promotes the interactions between Tregs and antigen-presenting cells

Abstract Regulatory T cells (Tregs) are responsible for the maintenance of immune homeostasis. They express the coinhibitory receptor CTLA-4 in high quantities, whose loss causes severe systemic autoinflammation in both mice and humans. CTLA-4 limits the expression of costimulatory CD80/CD86 proteins by antigen-presenting cells (APCs). However, the details of this mechanism are not fully understood. When we analyzed in vitro cocultures of CD80- or CD86-GFP expressing murine dendritic cells (DCs) with ex vivo sorted Tregs, we found that mutant Tregs, whose CTLA-4 lacked the cytoplasmic tail necessary for its endocytosis function, could still uptake CD80- or CD86-GFP from DCs via CTLA-4-dependent trogocytosis. Moreover, the specific CD80/86 uptake by CTLA-4, also facilitated the indirect transfer of membrane lipid particles and other surface proteins. Both flow cytometry and confocal microscopy revealed that immune synapse formation between Tregs and APCs was enhanced depending on CTLA-4 expression by Tregs. Confocal microscopy also demonstrated that uptaken CD80/CD86-GFP proteins colocalized with LFA-1 capping, an indicator of stable immune synapses. Furthermore, in vivo delivery of CTLA-4 WT and knockout (KO) Tregs into CD45.1+RAG2 KO mice showed that transfer of congenic CD45.1 protein from host cells to donor Tregs was significantly diminished in CTLA-4 KO Tregs compared to WT Tregs. The tail portion of CTLA-4 was also found to be dispensable for CD80/86 downregulation and suppressive functions of Tregs. In conclusion, CTLA-4-promoted immune synapse formation provides a stable platform between Tregs and APCs, which results in both depletion of CD80/CD86 via trogocytosis and indirect uptake of other surface proteins from APCs.

Genome-wide CRISPR screens revealed a reciprocal role of SWI/SNF complexes in controlling regulatory T cell function

Abstract Regulatory T cells (Tregs) are a specialized immune population that act to suppress overactive inflammatory response and maintain immune homeostasis. There is of great interest in harnessing Treg for controlling autoimmune disease or boosting anti-tumor immunity. Since the transcription factor, Forkhead box P3 (Foxp3), is known to control Treg development and function, understanding the regulation of Foxp3 could potentially lead to the development of therapeutics for treating autoimmune diseases and cancer. Here we performed a genome-wide CRISPR/Cas9 knockout screen to identify the regulators of Foxp3 in mouse primary Tregs. The results showed that Foxp3 regulators are highly enriched in genes encoding chromatin regulators, such as SAGA and SWI/SNF complexes. Among the SWI/SNF complexes, the GBAF complex promoted Foxp3 expression, whereas the PBAF complex repressed its expression. Functional genomic studies revealed that both GBAF and PBAF co-localized with Foxp3 in genome-wide binding sites. Targeting of GBAF reduced Foxp3 binding and altered expression a subset of Foxp3 target genes, indicating that GBAF actively participates in the Foxp3-dependent transcriptional program. Importantly, Treg suppressor function can be manipulated by targeting GBAF or PBAF, demonstrating in in vitro suppression assay, as well as mouse in autoimmune diseases and tumor model. Collectively, we provide an unbiased analysis of genes and networks regulating Foxp3, and reveal SWI/SNF complexes, GBAF and PBAF, as novel targets that could be exploited to manipulate Treg function for therapeutics of autoimmune diseases and cancer.

Critical roles of TIGIT+ human regulatory B cells in the suppression of antibody-mediated allograft rejection

Abstract TIGIT (T cell immunoreceptor with Ig and ITIM domains) is a co-inhibitory receptor, highly expressed by Tregs, and contributes to the suppressive function of Tregs by limiting pro-inflammatory Th1 and Th17 cell responses. Herein, we report that subsets of human regulatory B cells (Bregs) express surface TIGIT and can thus play a critical role in immune regulation. Using data from human B cell surface arrays and RNA-seq, we identified two major TIGIT+ human Bregs subsets, exhibiting CD24hiCD27+CD39hiIgD+IgMhiCD1chi marginal zone (MZ)-like and CD24hiCD27+CD39+IgD−IgM+CD1c+ memory Bregs. We further demonstrated that TIGIT+ Bregs were also capable of suppressing immune responses by their ability to express not only IL-10 and PD-L1, but also granzyme B and TGFb1. In addition, TIGIT+ Bregs can also suppress immune responses by altering dendritic cell functions. In line with these observations, liver allograft recipients with donor-specific alloantibody (DSA) had significantly lower numbers of TIGIT+ Bregs than those without DSA. Moreover, the frequency of TIGIT+ Bregs correlated with that of CD4+CD25+CD127lo Tregs, while it inversely correlated with the frequency of follicular helper T cells. We thus concluded that TIGIT+ Bregs play critical roles in immune regulation in liver allograft recipients.

Blimp1 suppresses proliferation in human regulatory T cells as a potential homeostasis regulator

Abstract B lymphocyte-induced maturation protein 1 (Blimp1), encoded by the PRDM1 gene, is a multifunctional transcriptional regulator that has established roles in mouse T cells. However, the mechanisms underlying its function in the proliferation, suppressive function, and homeostasis of human regulatory T cells (Tregs) are not known. To directly evaluate the role of Blimp1 in human Tregs, Blimp1α, the active isoform of Blimp1 that has dominant expression, was knocked out by Crispr-Cas9 using dual-targeting sgRNAs. High knockout efficiency was verified at DNA, RNA, and protein level by T7E1, RT-PCR, and Western blot analyses, respectively. Knockout of Blimp1α in cultured human Tregs resulted in increased cell number, upregulated Ki67 expression, and enhanced [3H]thymidine incorporation, consistent with a normal inhibitory role of Blimp1α in the proliferation of human Tregs. Compared with control cells, Blimp1α-knockout Tregs showed high CD25 expression and enhanced tyrosine phosphorylation of STAT5 (pSTAT5), suggesting a potential role of Blimp1 in regulation of IL-2R signaling. In vitro suppression assay showed that knockout of Blimp1α slightly decreased the suppressive function of Tregs. Knockout of Blimp1α also increased the expressions of Tigit and CD62L, but decreased the expressions of Foxp3, CD73, and Bcl-2, suggesting that Blimp1α may contribute to maintaining Treg identity. Collectively, our results are consistent with a model whereby Blimp1α regulates homeostasis of human Tregs by constraining proliferation in part by limiting IL-2R signaling while promoting Treg function and identity.

Glucose promotes Treg differentiation and suppresses intestinal inflammation

Abstract Glucose, the most abundant monosaccharide, has been characterized as the most important source of energy in human body, which is also considered as a potential risk factor in varies of diseases. However, the roles of glucose in regulation of intestinal T cell responses and inflammation are still controversial. In this report, we investigated whether and how glucose regulates the CD4+ T cell responses in intestinal inflammation. We treated Rag−/− mice which were transferred with CBir1 CD4+ T cells, which are specific for immunodominant microbiota antigen CBir1 flagellin, with drinking water with or without 0.6% or 6% (w/v) glucose for two weeks. We found that 6%, but not 0.6%, glucose in drinking water promoted regulatory T cells (Treg), but not Th1 and Th17 cells, differentiation in the mesenteric lymph nodes (MLN). In vivo administration of glucose to WT mice at the dose of 6%, but not 0.6%, also increased Treg cells in MLN. Within in vitro assay, glucose promoted Treg differentiation in a dose-dependent manner from 4500ug/ml to 15000ug/ml, but inhibited Treg differentiation with the dose higher than 15000ug/ml. Moreover, glucose-treated T cells cultured under Treg conditions showed enhanced suppressive activity toward naïve T cell proliferation. Mechanistically, glucose upregulated the expression of EGFR, Areg and Gzmb, the genes associated with regulation of Treg suppressive function. Furthermore, glucose at the dose of 6% alleviated the colitis in RAG−/− mice induced by CBir1 T cells, and increased Treg, but not Th1 and Th17, cells in MLN. Collectively, our study indicates that glucose promotes Treg differentiation and function under certain conditions, which plays an important role in intestinal homeostasis.

Divergent roles of retinoic acid signaling in T regulatory cell function

Abstract Regulatory T cells (Tregs) modulate immune responses to maintain immune homeostasis. Retinoic acid (RA) signaling promotes Treg generation and stability. Paradoxically, we observed that dominant negative RA receptor α expressed only in donor T cells (DNRARαfl/flCD4Cre) dampened inflammation and graft-versus-host disease (GVHD), while increasing Tregs. We found that diminishing RA signaling significantly enhanced their in vitro suppressive capacity. In vivo the adoptive transfer of DNRARαfl/flCD4Cre Tregs at the time of transplant was significantly (p<0.01) more effective than wild type (WT) controls in ameliorating GVHD. Mechanistically, enhanced suppression was associated with elevated expression of suppressive (CTLA-4, GITR, CD39) and fitness (CD25, pSTAT5) markers. To evaluate the intrinsic requirement of RA signaling in Tregs, we generated transgenic mice with conditional DNRARα expression in Tregs (DNRARαfl/fl x Foxp3YFPCre). Surprisingly, the extent of ablation of RA signaling resulted in divergent phenotypes. Heterozygous ablation (DNRARαfl/wt) was permissive of enhanced Treg suppression. In striking contrast and unexpectedly, homozygous ablation (DNRARαfl/fl) impaired Treg suppressive function and caused multi-organ (lung, liver) autoimmunity in >90% of females and males by 3 months of age. Poor suppression is not cell intrinsic as DNRARαfl/fl Tregs isolated from mixed bone marrow chimeras of congenic WT and DNRARαfl/fl x Foxp3YFP Cre had enhanced function. Our studies indicate that RA signaling in Tregs can be targeted to prevent autoimmunity by enhancing Treg suppression or alternatively, promote antitumor responses by reducing Treg function.

Ubiquitin-specific peptidase 18 regulates the differentiation and function of Treg cells

Ubiquitin-specific peptidase 18 (USP18) plays an important role in the development of CD11b+ dendritic cells (DCs) and Th17 cells, however, its role in the differentiation of other T cell subsets, especially in regulatory T (Treg) cells, is unknown. In our study, we used Usp18 KO mice to study the loss of USP18 on the impact of Treg cell differentiation and function. We found that USP18 deficiency upregulates the differentiation of Treg cells, which may lead to disrupted homeostasis of peripheral T cells, and downregulates INF-γ, IL-2, IL-17A producing CD4+ T cells and INF-γ producing CD8+ T cells. Mechanistically, we also found that the upregulation of Tregs is due to elevated expression of CD25 in Usp18 KO mice. Finally, we found that the suppressive function of Usp18 KO Tregs is downregulated. Altogether, our study was the first to identify the role of USP18 in Tregs differentiation and its suppressive function, which may provide a new reference for the treatment of Treg function in many autoimmune diseases, and USP18 can be used as a new therapeutic target for precise medical treatment.

Lipid restriction enhances suppression

Immunometabolism Regulatory T cells (Tregs) suppress immune responses to maintain tolerance and limit autoimmunity. Because lipid metabolism is crucial for the activity of Tregs, Field et al. explored the role of the lipid chaperone fatty-acid binding protein 5 (FABP5) in mouse and human Treg function. Targeting FABP5 through either genetic or pharmacological means caused mitochondrial dysfunction, which depressed oxidative phosphorylation and promoted a switch to glycolysis. FABP5 inhibition enhanced the suppressive activity of Tregs through a mechanism involving mitochondrial DNA release and subsequent cGAS-STING–dependent type I interferon signaling. The researchers found that the lipid-restrictive nature of the tumor microenvironment influenced Fabp5 gene expression and facilitated Treg suppressor function. Cell Metab. 31 , 422 (2019).

Interleukin-27 Enforces Regulatory T Cell Functions to Prevent Graft-versus-Host Disease.

Graft-versus-host disease (GvHD) remains a significant complication of allogeneic hematopoietic cell transplantation (HCT), associated with significant morbidity and mortality. GvHD is characterized by dysregulated immune responses and resulting tissue damage of target organs. Recent investigations have focused on Foxp3+ regulatory T cells (Tregs) as a therapeutic tool, based on its regulatory functions in GvHD pathogenesis and their instrumental role in mitigating GvHD severity while preserving graft-versus-leukemia (GvL) activity. There are several challenges to its clinical application, including their paucity, impaired suppressive activity, and instability in vivo. Herein, we report that IL-27 pre-stimulation enhances suppressive functions of both mouse and human Tregs. In a complete MHC mismatched murine bone marrow transplant model, IL-27 pre-stimulated polyclonal iTregs diminish acute (a)GvHD lethality, while preserving the GvL effect. Allo-antigen specificity further improves suppressive functions when combined with IL-27 pre-stimulation. In a xenogeneic (human to mouse) GvHD model, IL-27 pre-stimulated human iTregs are superior in protecting recipients from GvHD. Lastly, we compared gene expression profiles of circulating Tregs isolated from HCT recipients with and without aGvHD and found that Tregs from aGvHD patients express distinct gene signatures enriched in immune activation and inflammation. Therefore, these results highlight a novel function of IL-27 in enforcing Treg functions to prevent aGvHD mediated lethality, proposing the hypothesis that dysregulated Treg functions may account for the potential mechanisms underlying GvHD development.

Infection of Epstein–Barr Virus is Associated with the Decrease of Helios+FoxP3+Regulatory T Cells in Active Ulcerative Colitis Patients

ABSTRACT Background: Loss of immune homeostasis to enteric pathogens is considered to be involved in the pathogenesis of ulcerative colitis (UC), and regulatory T cells (Tregs) are key for this immune homeostasis. Helios exhibits an important effect on regulating the suppressive function of Tregs. Epstein–Barr virus (EBV) is more commonly detected in UC. However, whether there is an association between EBV infection and Helios+Tregs and its impact on disease activity of UC remain unclear. We aimed to explore the clinical significance of Helios+Tregs and their potential association with EBV infection in UC. Methods: Seventy-six UC patients and 38 controls were consecutively enrolled. Helios+FoxP3+Tregs were analyzed using flow cytometry and compared among groups. Eight active UC patients treated with 5-aminosalicylic acid were followed up. Correlation analyses were conducted between Helios+FoxP3+Tregs and disease activity indicators. In addition, EBV viral loads in the mucosal lesion were quantified in active UC by quantitative polymerase chain reaction and were comprehensively analyzed in subgroups of different disease severity, and their associations with Helios+FoxP3+Tregs were also analyzed. Results: Helios+FoxP3+Tregs were significantly decreased in active UC and were inversely correlated with serum C-reactive protein and Mayo score. Moreover, we observed the recovery of Helios+FoxP3+Tregs in followed-up active UC achieving remission after treatment. EBV loads were higher in active UC, and levels of Helios+FoxP3+Tregs in the EBV-positive subgroup were lower than the EBV-negative subgroup in moderate and severe active patients. Most importantly, we found that Helios+FoxP3+Tregs were significantly negatively correlated with EBV viral loads. Conclusion: Helios+FoxP3+Tregs are likely to play a pivotal role in disease activity of UC and may be influenced by EBV infection.

Dysregulation of Glutamate Transport Enhances Treg Function That Promotes VEGF Blockade Resistance in Glioblastoma.

Anti-VEGF therapy prolongs recurrence-free survival in patients with glioblastoma but does not improve overall survival. To address this discrepancy, we investigated immunologic resistance mechanisms to anti-VEGF therapy in glioma models. A screening of immune-associated alterations in tumors after anti-VEGF treatment revealed a dose-dependent upregulation of regulatory T-cell (Treg) signature genes. Enhanced numbers of Tregs were observed in spleens of tumor-bearing mice and later in tumors after anti-VEGF treatment. Elimination of Tregs with CD25 blockade before anti-VEGF treatment restored IFNγ production from CD8+ T cells and improved antitumor response from anti-VEGF therapy. The treated tumors overexpressed the glutamate/cystine antiporter SLC7A11/xCT that led to elevated extracellular glutamate in these tumors. Glutamate promoted Treg proliferation, activation, suppressive function, and metabotropic glutamate receptor 1 (mGlutR1) expression. We propose that VEGF blockade coupled with glioma-derived glutamate induces systemic and intratumoral immunosuppression by promoting Treg overrepresentation and function, which can be pre-emptively overcome through Treg depletion for enhanced antitumor effects. SIGNIFICANCE: Resistance to VEGF therapy in glioblastoma is driven by upregulation of Tregs, combined blockade of VEGF, and Tregs may provide an additive antitumor effect for treating glioblastoma.

Hybrid spherical nucleotide nanoparticles can enhance the synergistic anti-tumor effect of CTLA-4 and PD-1 blockades.

Combined blockades of CTLA-4 and PD-1 can yield better overall complementary clinical outcomes than individual blockades, but the response rates are still relatively low. To investigate the anti-tumor effects of various combined strategies, we designed various spherical nucleotide nanoparticles (SNPs) loaded with CTLA-4 aptamer (cSNPs), PD-1 siRNA (pSNPs) or both (hybrid SNPs, or hSNPs). The results demonstrated that hSNPs could promote significantly stronger anti-tumor immune responses in a nonredundant fashion than the mixture of pSNPs and cSNPs (pSNPs & cSNPs). We reasoned that this is because all individual immune cells could receive both CTLA-4 and PD-1 blockades when they engulfed hSNPs, but it is much less likely that individual immune cells could receive both CTLA-4 and PD-1 blockades as many of them may not take both pSNPs and cSNPS from pSNPs & cSNPs. Further results revealed that the synergistic immune stimulatory effects of CTLA-4 and PD-1 blockades in the form of hSNPs were at least partly through regulating the immune suppressive function of both Tregs and TIM3+ exhausted-like CD8 T cells and allowing effector T cells to expand. This mechanism is not identical to earlier reported mechanisms of CTLA-4 and PD-1 blockades.

A Neuropilin-1 Antagonist Exerts Antitumor Immunity by Inhibiting the Suppressive Function of Intratumoral Regulatory T Cells.

Regulatory T cells (Treg) are targeted for cancer immunotherapy because they suppress antitumor immunity. Although the importance of neuropilin-1 (NRP1) in the stability and function of intratumoral Tregs is well-documented, targeting of NRP1+ Tregs for anticancer immunotherapy has not been well explored. Here, we found that an NRP1 antagonist [Fc(AAG)-TPP11], generated by fusion of the NRP1-specific binding peptide TPP11 with the C-terminus of an effector function-deficient immunoglobulin Fc(AAG) variant, inhibits intratumoral NRP1+ Treg function and stability. Fc(AAG)-TPP11 triggered the internalization of NRP1, reducing its surface expression on Tregs and thereby inhibiting the suppressive function of Tregs. In two murine syngeneic tumor models, Fc(AAG)-TPP11 retarded tumor growth, comparable with a Treg-depleting anti-CTLA-4 antibody, without noticeable toxicity. Fc(AAG)-TPP11 inhibited NRP1-dependent Treg function, inducing unstable intratumoral Tregs, with reduced expression of Foxp3 and enhanced production of IFNγ, which subsequently increased the functionality and frequency of intratumoral CD8+ T cells. We also observed selective expression of NRP1 on Tregs isolated from human tumors, but not from the blood of healthy donors and patients with cancer, as well as ex vivo inhibition of intratumoral NRP1+ Treg function by Fc(AAG)-TPP11. Our results suggest that the NRP1 antagonist Fc(AAG)-TPP11 has therapeutic potential for the inhibition of intratumoral NRP1+ Tregs with limited unfavorable effects on peripheral Tregs.

Mitochondrial Integrity Regulated by Lipid Metabolism Is a Cell-Intrinsic Checkpoint for Treg Suppressive Function

SummaryRegulatory T cells (Tregs) subdue immune responses. Central to Treg activation are changes in lipid metabolism that support their survival and function. Fatty acid binding proteins (FABPs) are a family of lipid chaperones required to facilitate uptake and intracellular lipid trafficking. One family member, FABP5, is expressed in T cells, but its function remains unclear. We show that in Tregs, genetic or pharmacologic inhibition of FABP5 function causes mitochondrial changes underscored by decreased OXPHOS, impaired lipid metabolism, and loss of cristae structure. FABP5 inhibition in Tregs triggers mtDNA release and consequent cGAS-STING-dependent type I IFN signaling, which induces heightened production of the regulatory cytokine IL-10 and promotes Treg suppressive activity. We find evidence of this pathway, along with correlative mitochondrial changes in tumor infiltrating Tregs, which may underlie enhanced immunosuppression in the tumor microenvironment. Together, our data reveal that FABP5 is a gatekeeper of mitochondrial integrity that modulates Treg function.

Treatment with rapamycin can restore regulatory T-cell function in IPEX patients

Immune-dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a lethal disease caused by mutations in a transcription factor critical for the function of thymus-derived regulatory T (Treg) cells (ie, FOXP3), resulting in impaired Treg function and autoimmunity. At present, hematopoietic stem cell transplantation is the therapy of choice for patients with IPEX syndrome. If not available, multiple immunosuppressive regimens have been used with poor disease-free survival at long-term follow-up. Rapamycin has been shown to suppress peripheral T cells while sparing Treg cells expressing wild-type FOXP3, thereby proving beneficial in the clinical setting of immune dysregulation. However, the mechanisms of immunosuppression selective to Treg cells in patients with IPEX syndrome are unclear. We sought to determine the cellular and molecular basis of the clinical benefit observed under rapamycin treatment in 6 patients with IPEX syndrome with different FOXP3 mutations. Phenotype and function of FOXP3-mutated Treg cells from rapamycin-treated patients with IPEX syndrome were tested by flow cytometry and invitro suppression assays, and the gene expression profile of rapamycin-conditioned Treg cells by droplet-digital PCR. Clinical and histologic improvements in patients correlated with partially restored Treg function, independent of FOXP3 expression or Treg frequency. Expression of TNF-receptor-superfamily-member 18 (TNFRSF18, glucocorticoid-induced TNF-receptor-related) and EBV-induced-3 (EBI3, an IL-35 subunit) in patients' Treg cells increased during treatment as compared with that of Treg cells from untreated healthy subjects. Furthermore inhibition of glucocorticoid-induced TNF-receptor-related and Ebi3 partially reverted invitro suppression by invivo rapamycin-conditioned Treg cells. Rapamycin is able to affect Treg suppressive function via a FOXP3-independent mechanism, thus sustaining the clinical improvement observed in patients with IPEX syndrome under rapamycin treatment.

BAFF gene silencing attenuates allergic airway inflammation by promoting the generation of Tregs via activating pro-Treg cytokines

AimsAllergic airway inflammation is one of the major pathological events involved in asthma, and dysregulation of regulatory T cells (Treg) plays a crucial role in the development of allergic airway inflammation. Here, we attempted to investigate the regulatory effects of B cell-activating factor (BAFF) on Tregs in allergic airway inflammation. Main methodsBAFF expression was analyzed by ELISA, quantitative reverse transcription PCR (RT-PCR) and Western blot assays. The levels of IL-4, TGF-β, IL-2, and IL-10 were tested using ELISA kits. Flow cytometry was conducted to analyze the populations of CTLA4+ Foxp3+ Tregs. Key findingsBAFF was found to be aberrantly expressed in sputum and lungs in patients with asthma as well as OVA sensitized mice. BAFF silencing by lentiviral BAFF shRNA reduced the number of eosinophils and levels of IL-4 in the BAL fluid, as well as the Fizz1 expression in the lungs of OVA mice. Additionally, the population of CTLA4+ Foxp3+ Tregs were significantly decreased in OVA mice and had a negative correlation to BAFF levels in asthmatic patients and OVA mice. BAFF silencing in vivo increased levels of CTLA4+ Foxp3+ Tregs and the secretion of IL-10, and improved the regulatory phenotype and suppressor function of Tregs in vitro. Furthermore, BAFF can affect Tregs generation by regulating the production of the pro-Treg cytokines IL-2 and TGF-β. SignificanceBAFF has an inhibitory effect on the generation and suppressor function of Tregs by affecting pro-Tregs cytokines, thereby contributing to the development of allergic airway inflammation.

Dendritic Cells Control Regulatory T Cell Function Required for Maintenance of Intestinal Tissue Homeostasis.

Dendritic cells (DCs) together with regulatory T cells (Tregs) are essential mediators of immune homeostasis. Disruption of function or frequency of either cell type can lead to fatal autoimmunity. We previously described that mice constitutively lacking DCs (∆DC) develop autoimmunity characterized by reduced body weight, autoantibodies, and pronounced intestinal inflammation. In this study, we show that lack of DCs leads to an altered gene expression profile in peripheral but not thymic Tregs with increased expression of inhibitory receptors. The suppressive function of Tregs from ΔDC mice was impaired in T cell cocultures. In a model of transfer colitis, Tregs from ∆DC mice were only functional in the presence of DCs in recipient mice. Lack of MHC class II on DCs also resulted in upregulation of inhibitory receptors on Tregs, reduced body weight, and elevated serum IgA levels. Further analysis of the IgA response revealed an expansion of IgA+ germinal center B cells and plasma cells in mesenteric lymph nodes and more IgA-coated commensal bacteria in feces of ∆DC mice. Thus, we show a critical role for DCs to establish intestinal homeostasis by regulating Treg function for prevention of spontaneous inflammation.