Treg Responses Research Articles

PD-1 or CTLA-4 blockade promotes CD86-driven Treg responses upon radiotherapy of lymphocyte-depleted cancer in mice.

Radiotherapy (RT) is considered immunogenic, but clinical data demonstrating RT-induced T cell priming are scarce. Here, we show in a mouse tumor model representative of human lymphocyte-depleted cancer that RT enhanced spontaneous priming of thymus-derived (FOXP3+Helios+) Tregs by the tumor. These Tregs acquired an effector phenotype, populated the tumor, and impeded tumor control by a simultaneous, RT-induced CD8+ cytotoxic T cell (CTL) response. Combination of RT with CTLA-4 or PD-1 blockade, which enables CD28 costimulation, further increased this Treg response and failed to improve tumor control. We discovered that upon RT, the CD28 ligands CD86 and CD80 differentially affected the Treg response. CD86, but not CD80, blockade prevented the effector Treg response, enriched the tumor-draining lymph node migratory conventional DCs that were positive for PD-L1 and CD80 (PD-L1+CD80+), and promoted CTL priming. Blockade of CD86 alone or in combination with PD-1 enhanced intratumoral CTL accumulation, and the combination significantly increased RT-induced tumor regression and OS. We advise that combining RT with PD-1 and/or CTLA-4 blockade may be counterproductive in lymphocyte-depleted cancers, since these interventions drive Treg responses in this context. However, combining RT with CD86 blockade may promote the control of such tumors by enabling a CTL response.

Immunomodulation of susceptibility to pneumococcal pneumonia infection in mouse lungs exposed to carbon nanoparticles via dysregulation of innate and adaptive immune responses

Carbon nanotubes (CNTs) are emerging pollutants of occupational and environmental health concern. While toxicological mechanisms of CNTs are emerging, there is paucity of information on their modulatory effects on susceptibility to infections. Here, we investigated cellular and molecular events underlying the effect of multi-walled CNT (MWCNT) exposure on susceptibility to Streptococcus pneumoniae infection in our 28-day sub-chronic exposure mouse model. Data indicated reduced phagocytic function in alveolar macrophages (AMs) from MWCNT-exposed lungs evidenced by lower pathogen uptake in 1-h infection assay. At 24-h post-infection, intracellular pathogen count in exposed AMs showed 2.5 times higher net increase (2-fold in vehicle- versus 5-fold in MWCNT-treated), indicating a greater rate of intracellular multiplication and/or survival due to MWCNT exposure. AMs from MWCNT-exposed lungs exhibited downregulation of pathogen-uptake receptors CD163, Phosphatidyl-serine receptor (Ptdsr), and Macrophage scavenger receptors class A type 1 (Msr1) and type 2 (MSr2). In whole lung, MWCNT exposure shifted the macrophage polarization state towards the immunosuppressive phenotype M2b and increased the CD11c+ dendritic cell population required to activate the adaptive immune response. Notably, the MWCNT pre-exposure dysregulated T-cell immunity, evidenced by diminished CD4 and Th17 response, and exacerbated Th1 and Treg responses (skewed Th17/Treg ratio), thereby favoring the pneumococcal infection. Overall, these findings indicated that MWCNT exposure compromises both innate and adaptive immunity leading to diminished host lung defense against pneumonia infection. To our knowledge, this is the first report on an immunomodulatory role of CNT pre-exposure on pneumococcal infection susceptibility due to dysregulation of both innate and adaptive immunity targets.

Amyloid-β specific regulatory T cells attenuate Alzheimer’s disease pathobiology in APP/PS1 mice

BackgroundRegulatory T cells (Tregs) maintain immune tolerance. While Treg-mediated neuroprotective activities are now well-accepted, the lack of defined antigen specificity limits their therapeutic potential. This is notable for neurodegenerative diseases where cell access to injured brain regions is required for disease-specific therapeutic targeting and improved outcomes. To address this need, amyloid-beta (Aβ) antigen specificity was conferred to Treg responses by engineering the T cell receptor (TCR) specific for Aβ (TCRAβ). The TCRAb were developed from disease-specific T cell effector (Teff) clones. The ability of Tregs expressing a transgenic TCRAβ (TCRAβ -Tregs) to reduce Aβ burden, transform effector to regulatory cells, and reverse disease-associated neurotoxicity proved beneficial in an animal model of Alzheimer’s disease.MethodsTCRAβ -Tregs were generated by CRISPR-Cas9 knockout of endogenous TCR and consequent incorporation of the transgenic TCRAb identified from Aβ reactive Teff monoclones. Antigen specificity was confirmed by MHC-Aβ-tetramer staining. Adoptive transfer of TCRAβ-Tregs to mice expressing a chimeric mouse-human amyloid precursor protein and a mutant human presenilin-1 followed measured behavior, immune, and immunohistochemical outcomes.ResultsTCRAβ-Tregs expressed an Aβ-specific TCR. Adoptive transfer of TCRAβ-Tregs led to sustained immune suppression, reduced microglial reaction, and amyloid loads. 18F-fluorodeoxyglucose radiolabeled TCRAβ-Treg homed to the brain facilitating antigen specificity. Reduction in amyloid load was associated with improved cognitive functions.ConclusionsTCRAβ-Tregs reduced amyloid burden, restored brain homeostasis, and improved learning and memory, supporting the increased therapeutic benefit of antigen specific Treg immunotherapy for AD.Graphical

The dual role of regulatory T cells in hepatitis B virus infection and related hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major contributor to cancer-related deaths worldwide. Hepatitis B virus (HBV) infection is a major etiologic factor leading to HCC. While there have been significant advancements in controlling HBV replication, achieving a complete cure for HBV-related HCC (HBV-HCC) remains an intricate challenge. HBV persistence is attributed to a myriad of mechanisms, encompassing both innate and adaptive immune responses. Regulatory T cells (Tregs) are pivotal in upholding immune tolerance and modulating excessive immune activation. During HBV infection, Tregs mediate specific T cell suppression, thereby contributing to both persistent infection and the mitigation of liver inflammatory responses. Studies have demonstrated an augmented expression of circulating and intrahepatic Tregs in HBV-HCC, which correlates with impaired CD8+ T cell function. Consequently, Tregs play a dual role in the context of HBV infection and the progression of HBV-HCC. In this comprehensive review, we discuss pertinent studies concerning Tregs in HBV infection, HBV-related cirrhosis and HCC. Furthermore, we summarize Treg responses to antiviral therapy and provide Treg-targeted therapies specific to HBV and HCC.

Commensal bacteria promote type I interferon signaling to maintain immune tolerance in mice.

Type I interferons (IFNs) exert a broad range of biological effects important in coordinating immune responses, which have classically been studied in the context of pathogen clearance. Yet, whether immunomodulatory bacteria operate through IFN pathways to support intestinal immune tolerance remains elusive. Here, we reveal that the commensal bacterium, Bacteroides fragilis, utilizes canonical antiviral pathways to modulate intestinal dendritic cells (DCs) and regulatory T cell (Treg) responses. Specifically, IFN signaling is required for commensal-induced tolerance as IFNAR1-deficient DCs display blunted IL-10 and IL-27 production in response to B. fragilis. We further establish that IFN-driven IL-27 in DCs is critical in shaping the ensuing Foxp3+ Treg via IL-27Rα signaling. Consistent with these findings, single-cell RNA sequencing of gut Tregs demonstrated that colonization with B. fragilis promotes a distinct IFN gene signature in Foxp3+ Tregs during intestinal inflammation. Altogether, our findings demonstrate a critical role of commensal-mediated immune tolerance via tonic type I IFN signaling.

Sitagliptin Induces Tolerogenic Human Dendritic Cells

Sitagliptin, an anti-diabetic drug, is a dipeptidyl peptidase (DPP)-4/CD26 inhibitor with additional anti-inflammatory and immunomodulatory properties. In this study, we investigated for the first time the effect of sitagliptin on the differentiation and functions of human dendritic cells generated from monocytes (MoDCs) for 4 days using the standard GM-CSF/IL-4 procedure. LPS/IFN-γ treatment for an additional 24 h was used for maturation induction of MoDCs. Sitagliptin was added at the highest non-cytotoxic concentration (500 µg/mL) either at the beginning (sita 0d protocol) or after MoDC differentiation (sita 4d protocol). Sitagliptin impaired differentiation and maturation of MoDCs as judged with the lower expression of CD40, CD83, CD86, NLRP3, and HLA-DR, retention of CD14 expression, and inhibited production of IL-β, IL-12p70, IL-23, and IL-27. In contrast, the expression of CD26, tolerogenic DC markers (ILT4 and IDO1), and production of immunoregulatory cytokines (IL-10 and TGF-β) were increased. Generally, the sita 0d protocol was more efficient. Sitagliptin-treated MoDCs were poorer allostimulators of T-cells in MoDC/T-cell co-culture and inhibited Th1 and Th17 but augmented Th2 and Treg responses. Tolerogenic properties of sitagliptin-treated MoDCs were additionally confirmed by an increased frequency of CD4+CD25+CD127- FoxP3+ Tregs and Tr1 cells (CD4+IL-10+FoxP3-) in MoDC/T-cell co-culture. The differentiation of IL-10+ and TGF-β+ Tregs depended on the sitagliptin protocol used. A Western blot analysis showed that sitagliptin inhibited p65 expression of NF-kB and p38MAPK during the maturation of MoDCs. In conclusion, sitagliptin induces differentiation of tolerogenic DCs, and the effect is important when considering sitagliptin for treating autoimmune diseases and allotransplant rejection.

OC 69.2 Costimulatory Domains of Second- and Third-Generation CAR-Tregs affect the Type of FVIII-Specific Treg Responses and Might be able to Stabilize the Treg Phenotype

OC 69.2 Costimulatory Domains of Second- and Third-Generation CAR-Tregs affect the Type of FVIII-Specific Treg Responses and Might be able to Stabilize the Treg Phenotype

Appendectomy Mitigates Coxsackievirus B3-Induced Viral Myocarditis.

Appendix has a distinct abundance of lymphatic cells and serves as a reservoir of microbiota which helps to replenish the large intestine with healthy flora. And it is the primary site of IgA induction, which shapes the composition of the intestinal microbiota. Recent population-based cohort studies report that appendectomy is associated with an increased risk of acute myocardial infarction and ischemic heart disease. Here, whether appendectomy has an effect on the occurrence and development of coxsackievirus B3 (CVB3)-induced viral myocarditis is studied. 103 TCID50 CVB3 was inoculated i.p. into appendectomized and sham-operated mice. RNA levels of viral load and pro-inflammatory cytokines in the hearts and the intestine were detected by RT-PCR. Compared to sham-operated mice, appendectomized mice exhibited attenuated cardiac inflammation and improved cardiac function, which is associated with a systemic reduced viral load. Appendectomized mice also displayed a reduction in cardiac neutrophil and macrophage infiltration and pro-inflammatory cytokine production. Mechanistically, we found that CVB3 induced an early and potent IL-10 production in the cecal patch at 2 days post infection. Appendectomy significantly decreased intestinal IL-10 and IL-10+ CD4+ Treg frequency which led to a marked increase in intestinal (primary entry site for CVB3) anti-viral IFN-γ+ CD4+ T and IFN-γ+ CD8+ T response and viral restriction, eventually resulting in improved myocarditis. Our results suggest that appendix modulates cardiac infection and inflammation through regulating intestinal IL-10+ Treg response.

New Flow Cytometric Methods for Monitoring STAT5 Signaling Reveal Responses to SARS-CoV-2 Antigen-Specific Stimulation in FOXP3+ Regulatory T Cells also in Patients with Advanced Chronic Lymphocytic Leukemia

Increased frequency of CD4+CD25+ regulatory T-cells (Treg) has been associated with disease progression in chronic lymphocytic leukemia (CLL). Flow cytometric methods, which allow for the simultaneous analysis of their specific transcription factor Foxp3 and activated STAT proteins, together with proliferation can help to elucidate the signaling mechanisms driving Treg expansion and suppression of FOXP3- conventional CD4+T-cells (Tcon). Herein, we first report a novel approach in which STAT5 phosphorylation (pSTAT5) and proliferation (BrdU-FITC incorporation) could be analyzed specifically in FOXP3+ and FOXP3- responding cells after CD3/CD28 stimulation. The addition of magnetically purified CD4+CD25+ T-cells from healthy donors to cocultured autologous CD4+CD25- T-cells resulted in suppression of Tcon cell cycle progression accompanied by a decrease in pSTAT5. Next, a method using imaging flow cytometry is presented for the detection of cytokine-dependent pSTAT5 nuclear translocation in FOXP3-expressing cells. Finally, we discuss our experimental data obtained by combining Treg pSTAT5 analysis and antigen-specific stimulation with SARS-CoV-2 antigens. Applying these methods on samples from patients revealed Treg responses to antigen-specific stimulation and significantly higher basal pSTAT5 in CLL patients treated with immunochemotherapy. Thus, we speculate that through the use of this pharmacodynamic tool, the efficacy of immunosuppressive drugs and their possible off-target effects can be assessed.

A single-dose, tolerizing vaccine induces antigen-specific regulatory T cells and durable remission while preserving healthy immunity in a mouse model of Multiple sclerosis

Abstract Multiple sclerosis (MS) is an inflammatory disorder that occurs when autoreactive lymphocytes mistakenly attack myelin, resulting in motor deficits and cognitive deterioration. Current drugs are non-curative and require life-long treatment that make compliance challenging, and existing therapeutics can leave patients vulnerable to opportunistic infections. An experimental approach to overcome these hurdles is to drive long-lasting, antigen-specific regulatory T cells (Tregs) that dampen responses to autoantigens. Here, we co-deliver antigen and rapamycin in micron-sized, diffusion-limited, polymer microparticles (MPs) directly to lymph nodes (LNs) where immune responses are orchestrated. Using adoptive transfer of transgenic T cells, we show that MPs generate antigen-specific Tregs in response to two different MP-derived peptides – evidence that MPs do not interfere with induction of peripheral tolerance in LNs. In experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, single-dose treatment demonstrates potent, long-lasting efficacy (up to three months) following a single treatment. Tolerizing effects are observed during chronic disease – the most challenging for treatment regimens. Efficacy is accompanied by reduced demyelination in spinal cords of treated mice, the cause of paralysis in EAE and MS. Thus, antigen-specific reprogramming of Tregs in LNs reshapes inflammation in distal tissues without need for systemic or repeated dosing. Importantly, MP-treated EAE mice mount healthy immune responses to vaccine challenge. This work advances a strategy to locally reprogram antigen-specific, tolerogenic responses that preserve healthy immunity – a hurdle faced by current MS therapeutics. Supported by grants from NIH (R01 AI169686).

Complete tolerogenic response adjuvant stimulates Treg response to immunization

Abstract We identified the lowest composition necessary to generate a vaccination adjuvant that promotes a Treg response following immunization in mice and named it “complete tolerogenic adjuvant.” This novel adjuvant may allow us to utilize the well-established “antigen plus adjuvant” method of vaccination to induce Treg cell-mediated antigen-specific immunosuppression. The minimal composition is dexamethasone, rapamycin, and monophosphoryl lipid A with the following mass ratios: 8:20:3. We have demonstrated why immunosuppressive and immunogenic substances are both required for the formation of genuine adjuvants for Treg cells by dissecting the roles of each of these components during vaccination. Currently, research is ongoing in our group to begin to assess the usefulness of this adjuvant for antigen immunization in murine models of human autoimmune/inflammatory diseases. Specifically, we are evaluating the tolerogenic efficacy of this adjuvant for vaccination regimen targeting the apolipoprotein B antigen, using the ApoE−/− mouse model of atherosclerosis. These studies are expected to help drive the development of further, perhaps more effective, complete tolerogenic adjuvants that could be used to create a plethora of new, novel vaccines for treating immunological illnesses. Master program and NIH grant

Prophylactic and therapeutic use of synthetically glycosylated antigens to limit allergic airway disease

Abstract Asthma is a disease involving chronic airway inflammation affecting more than 300 million people worldwide. There is currently no cure for asthma. While effective, corticosteroids cause broad immune suppression and long-term debilitating disease. Novel immune modulatory therapeutics leave patients vulnerable to infections. Therefore, there is a current need for new therapeutics that target the underlying causes of the disease without triggering detrimental side effects. Through synthetic glycosylation, our platform targets hepatic antigen presenting cells and leverages the liver’s tolerance-inducing mechanisms to limit inflammatory T cell responses, while promoting regulatory T cell (Treg) responses, thus preventing broad immunosuppression. In a model of experimental allergic airway inflammation, prophylactic treatment with synthetically mannosylated OVA (pMan OVA) ameliorated the allergic response to inhaled OVA. Our treatment reduced lung and airway eosinophilia, systemic levels of IgE and OVA-specific IgG1, and airway mucus. Furthermore, pMan OVAtreatment reduced OVA specific lung T H2 cells, and exhibited reduced production of IL-4, IL-5 and IL-13 upon antigen specific restimulation. Treatment with pMan OVAwas accompanied by induction of OVA-specific Tregs, which negatively correlated with features of allergic airway disease. In previously sensitized mice, therapeutic intervention with pMan OVAreduced airway and lung eosinophilia, B cell and CD4 +T cell inflammation. Altogether, these data suggest a potential role for prophylactic and therapeutic intervention with synthetically glycosylated allergens in ameliorating disease severity in a model of allergic airway inflammation.

Selective inhibition of estrogen receptor alpha restores regulatory T-cell function in autoimmune hepatitis

Abstract Dysfunction in regulatory T-cells (Tregs) is a hallmark of autoimmune hepatitis (AIH), a serious hepatopathy often leading to end-stage liver damage and transplantation, if not treated. Treg impairment in AIH has been linked to defective response to aryl hydrocarbon receptor (AhR) activation. AhR acts as a modulator of toxin and adaptive immunity. We have previously shown that altered response of Tregs to AhR activation derives from heightened levels of estrogen receptor alpha (Erα), an AhR non-canonical binding partner. In this study we investigated whether Erα selective antagonism modulates Treg function and response to AhR in AIH. Experiments were performed on immunomagnetically purified Tregs, exposed to the Erα-specific antagonist methyl-piperidinopyrazole (MPP), in combination with or without unconjugated bilirubin (UCB), a known AhR endogenous ligand. In healthy controls (HC), UCB boosted Treg ability to suppress T-responder cell proliferation, and this was further increased when UCB was combined with MPP. In AIH, addition of MPP significantly ameliorated Treg ability to suppress, when exposed to UCB. Stimulation of Tregs with UCB and MPP boosted CYP1A1, CD39 and IL-10 levels both in AIH and HC. Analysis of Treg transcriptome indicated that treatment of Tregs with MPP increased the levels of CD73, an ectoenzyme that works in tandem with CD39, to ultimately generate adenosine. In conclusion, selective inhibition of Erα restores Treg ability to respond to AhR resulting in increased CD39 levels and ameliorated suppressive function in AIH. This could provide a new therapeutic strategy to boost and maintain immunotolerance in AIH. Supported by grants from NIH (R01 DK124408, R01 DK108894)

Inhibition of distinct glycolytic enzymes produces differential effects on CD4 T cell function

Abstract Introduction: When T cells are activated, they upregulate glycolysis and take on a requisite Warburg phenotype. In this study, we evaluated the effect of inhibiting two distinct glycolytic enzymes, GAPDH and PGAM, on CD4 T cell differentiation. Methods/Results: Using the GAPDH inhibitor heptelidic acid, we found that GAPDH inhibition produces a potent anti-inflammatory phenotype in Th1 cells, significantly reducing IFNγ expression. We further showed that GAPDH inhibition produces the anti-inflammatory metabolite methylglyoxal, which is necessary for its anti-inflammatory effect. GAPDH inhibition also produced anti-inflammatory effects in vivo, as heptelidic acid significantly reduced disease severity and altered immune subsets in a therapeutic treatment paradigm in the MOG 35–55EAE model of multiple sclerosis. Furthermore, we showed that GAPDH inhibition enhances Treg polarization, while inhibiting PGAM, a downstream glycolytic enzyme, potently blocked Treg polarization. Additionally, we found that inhibition of PGAM increases serine biosynthesis and subsequently alters 1-carbon metabolism. We also found that PGAM gene expression is associated with Treg differentiation and response to immunotherapy in lung cancer patients. Conclusion: Overall, our in vitroand in vivoresults indicate that targeting the glycolytic enzyme GAPDH produces an anti-inflammatory phenotype via the methylglyoxal pathway, while targeting the downstream enzyme PGAM produces an opposite effect via regulation of serine synthesis. Our findings suggest that the functional consequences of glycolysis inhibition depend on the specific enzymes targeted, which represent promising novel therapeutic targets for immunological disease. This work was supported by NIH/NINDS grant K08NS104266 and Conrad N. Hilton Foundation Marilyn Hilton Bridging Award for Physician Scientists grant 17316 to MDK. WHG was supported by NIH MSTP Grant T32 GM136577 and the American Association of Immunologists’ Careers in Immunology Fellowship Program.

Dietary L-tryptophan determines the number of colonic regulatory T-cells and susceptibility to colitis via GPR15

Abstract Environmental factors play a prominent role in the onset of immunological disorders such as ulcerative colitis, but the mechanisms are unclear. Diet is a major environmental factor, mostly known to influence the gut microbiota, which in turn affects the host immune system. Here, we demonstrated the microbiota-independent effect of the ubiquitous dietary component, L-tryptophan (L-Trp) on mucosal immune responses. We discovered that the amount of ingested L-Trp determines the number of colonic CD4 +T-cells through the aryl hydrocarbon receptor (AhR), which triggers the direct transcriptional activation of the colon T-cell homing receptor, GPR15. However, the default pathway connecting L-Trp, AhR, GPR15, and T-cells is microbiota-independent and involves host IDO1 and IDO2 enzymes, selectively increasing GPR15 +Tregs. Consequently, two weeks of L-Trp supplementation nearly doubled the colonic GPR15 +Tregs and significantly decreased the future risk of colitis. Our extensive, in vitro screening of AhR ligands suggests that AhR ligand-specific variations in target gene expression may account for the Treg-selective response to L-Trp. We also identified benzo[a]pyrene, a compound enriched in cigarette smoke and grilled meat, as an AHR agonist that strongly promotes GPR15+FOXP3+ Treg generation. Thus, our findings uncover mechanisms by which routine dietary and behavioral practices have a significant impact on colonic Treg responses, suggesting a potential non- invasive therapeutic approach for colitis. Supported by grant from NIH (R01AI141787, R21AI142318, T32 AI134646, 5P30CA056036-22) Supported by grant from CCFA (Career Development Award 329388)

Presentation of Human Neural Stem Cell Antigens Drives Regulatory T Cell Induction.

Transplantation of human neural stem cells (hNSCs) is a promising regenerative therapy to promote remyelination in patients with multiple sclerosis (MS). Transplantation of hNSCs has been shown to increase the number of CD4+CD25+Foxp3+ T regulatory cells (Tregs) in the spinal cords of murine models of MS, which is correlated with a strong localized remyelination response. However, the mechanisms by which hNSC transplantation leads to an increase in Tregs in the CNS remains unclear. We report that hNSCs drive the conversion of T conventional (Tconv) cells into Tregs invitro. Conversion of Tconv cells is Ag driven and fails to occur in the absence of TCR stimulation by cognate antigenic self-peptides. Furthermore, CNS Ags are sufficient to drive this conversion in the absence of hNSCs invitro and invivo. Importantly, only Ags presented in the thymus during T cell selection drive this Treg response. In this study, we investigate the mechanisms by which hNSC Ags drive the conversion of Tconv cells into Tregs and may provide key insight needed for the development of MS therapies.

Type I interferons augment regulatory T cell polarization in concert with ancillary cytokine signals.

In the transplant community, research efforts exploring endogenous alternatives to inducing tolerogenic allo-specific immune responses are much needed. In this regard, CD4 + FoxP3+ regulatory T cells (Tregs) are appealing candidates due to their intrinsic natural immunosuppressive qualities. To date, various homeostatic factors that dictate Treg survival and fitness have been elucidated, particularly the non-redundant roles of antigenic CD3ζ/T-cell-receptor, co-stimulatory CD28, and cytokine interleukin (IL-)2 dependent signaling. Many of the additional biological signals that affect Tregs remain to be elucidated, however, especially in the transplant context. Previously, we demonstrated an unexpected link between type I interferons (IFNs) and Tregs in models of multiple myeloma (MM)-where MM plasmacytes escaped immunological surveillance by enhancing type I IFN signaling and precipitating upregulated Treg responses that could be overturned with specific knockdown of type I IFN signaling. Here, we elaborated on these findings by assessing the role of type I IFN signaling (IFN-α and -β) on Treg homeostasis within an alloimmune context. Specifically, we studied the induction of Tregs from naïve CD4 T cells. Using in vitro and in vivo models of murine skin allotransplantation, we found that type I IFN indeed spatiotemporally enhanced the polarization of naïve CD4 T cells into FoxP3+ Tregs. Notably, however, this effect was not independent of, and rather co-dependent on, ancillary cytokine signals including IL-2. These findings provide evidence for the relevance of type I IFN pathway in modulating FoxP3+ Treg responses and, by extension, stipulate an additional means of facilitating Treg fitness via type I IFNs.

Abstract 6414: Concomitant anti-tumor immunity and checkpoint blockade responses are restored by depletion of CD62L expressing cells

Abstract Studies have shown the importance of CD62L in leukocyte trafficking. However, the significance of CD62L-expressing cells in concomitant anti-tumor immunity has not been investigated. Using a model of post-surgical tumor immunity, we show that in vivo depletion of CD62L-expressing cells before or immediately after surgical tumor resection enabled the development of anti-tumor concomitant responses against poorly immunogenic B16 (melanoma) or MTE-Ras (head & neck cancer) tumor cells. Anti-CD62L mAb administration depleted the cellular immune components that express CD62L, including NK, NKT, Mo, Ma, B, and T cells. Within the T cell compartment, naïve T cells (CD44lo CD62Lhi), effector (CD44hi CD62Llo), memory (CD44hi CD62Lhi) and regulatory T cells (Treg, CD4 CD25 Foxp3) were also depleted, while effector CD62L-negative T cells were spared in the TDLN. Tumor immunity did not occur in the absence of a primary tumor (sham surgery group) or after CD8 depletion. Whereas anti-CTLA-4 or anti-PD-1 mAbs failed as monotherapies, their administration during CD62L depletion restored their anti-tumor properties. The unified global gene expression analysis of the TDLN shows the significant enrichment of biological processes involved in the CD8 T cell effector phase, IL-2/IL-15, TNF-α, IFN-γ, IFN-α, IL-6, responses, mitotic cell division, glycolysis, and acetylation. Also, genes related to Treg responses were downregulated. These data point to a process in which depletion of CD62L cells in tumor-bearing hosts transforms the TDLN into a highly active immunological site in which tumor-reactive effector CD8 T cells (CD62L-negative) actively divide in the absence of Treg, accompanied by a cytokine response, glucose consumption, and acetylation of transcriptional factors and histones. These results identify an unrecognized role of CD62L-expressing cells in regulating concomitant anti-tumor immunity and restoring anti-tumor responses by checkpoint blockade inhibitors. Our study provides a new framework that could help understand how anti-tumor responses are limited. Citation Format: Brianna Burke, Lourdes Plaza-Rojas, Cynthia Perez, Elena Kostenko, Anna Austin, Justin Boucher, Kushal Prajapati, Michael Delos Reyes, Christine Chung, Jose-Alejandro Guevara-Patino. Concomitant anti-tumor immunity and checkpoint blockade responses are restored by depletion of CD62L expressing cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6414.

TET2-mediated upregulation of 5-hydroxymethylcytosine in LRRC39 promoter promotes Th1 response in association with downregulated Treg response in Vogt-Koyanagi-Harada disease

DNA 5-Hydroxymethylcytosine (5-hmC), an oxidative reaction mediated by the ten-eleven translocation (TET) family, has been reported to play an essential role in the progression of auto-inflammatory and autoimmune diseases. By far, little is known about the effect of DNA 5-hmC and the TET family on the development of Vogt–Koyanagi–Harada (VKH) disease. In this study, we discovered that the global DNA 5-hmC level and the TET activity were elevated in association with the up-regulated expression of TET2 at both mRNA and protein levels in CD4+T cells from active VKH patients compared to healthy controls. Integrated analysis of DNA 5-hmC pattern and transcription profile of CD4+ T cells revealed that 6 candidate target genes were involved in the development of VKH disease. The promoter 5-hmC and mRNA levels of leucine rich repeat containing 39 (LRRC39) were verified to be elevated in active VKH patients. Functional experiments showed that TET2 could up-regulate LRRC39 mRNA expression by increasing the promoter 5-hmC level of LRRC39 in CD4+ T cells from active VKH patients. Up-regulated LRRC39 expression could increase the frequencies of IFN-γ+ and IL-17+ CD4+ T cells as well as the secretions of IFN-γ and IL-17 in association with the decreased frequency of CD4+CD25+FOXP3+ regulatory T (Treg) cells and the reduced production of IL-10. Additionally, restoration of LRRC39 rescued TET2-silencing-mediated reduced frequency of IFN-γ+ CD4+ T cells and increased frequency of CD4+CD25+FOXP3+ Treg cells. Collectively, our study reveals a novel axis, the TET2–5-hmC-LRRC39–Th1/Treg responses axis, in the pathogenesis of VKH and provides a potential target for further investigation into the epigenetic therapy of this disease.

Identification of Tregs-Related Genes with Molecular Patterns in Patients with Systemic Sclerosis Related to ILD

Systemic Sclerosis (SSc) is an autoimmune disease that is characterized by vasculopathy, digital ulcers, Raynaud's phenomenon, renal failure, pulmonary arterial hypertension, and fibrosis. Regulatory T (Treg) cell subsets have recently been found to play crucial roles in SSc with interstitial lung disease (ILD) pathogenesis. This study investigates the molecular mechanism of Treg-related genes in SSc patients through bioinformatic analyses. The GSE181228 dataset of SSc was used in this study. CIBERSORT was used for assessing the category and proportions of immune cells in SSc. Random forest and least absolute shrinkage and selection operator (LASSO) regression analysis were used to select the hub Treg-related genes. Through bioinformatic analyses, LIPN and CLEC4D were selected as hub Treg-regulated genes. The diagnostic power of the two genes separately for SSc was 0.824 and 0.826. LIPN was associated with the pathway of aminoacyl-tRNA biosynthesis, Primary immunodeficiency, DNA replication, etc. The expression of CLEC4D was associated with the pathway of Neutrophil extracellular trap formation, PPAR signaling pathway, Staphylococcus aureus infection, Systemic lupus erythematosus, TNF signaling pathway, and Toll-like receptor signaling pathway. Through bioinformatic analyses, we identified two Treg-related hub genes (LIPN, CLEC4D) that are mainly involved in the immune response and metabolism of Tregs in SSc with ILD. Moreover, our findings may provide the potential for studying the molecular mechanism of SSc with ILD.