Treg Cell Phenotype Research Articles

Phenotypes of regulatory T cells in different stages of COPD

BackgroundPrevious studies have shown that failure to control inflammatory processes mediated by regulatory T (Treg) cells contributes to chronic obstructive pulmonary disease (COPD) development and progression. The activity of Treg cells depends on their phenotypic characteristics: resting Treg (rTreg, CD3+CD4+CD25+FOXP3+CD25++CD45RA+) and activated Treg (aTreg, CD3+CD4+CD25+FOXP3+CD25+++CD45RA−) cells exhibit immunosuppressive activity, while cytokine-secreting T cells (FrIII, CD3+CD4+CD25+FOXP3+CD25++CD45RA−) exhibit proinflammatory activity. Previous findings have shown an increased density of cytokine-secreting T cells in COPD patients experiencing exacerbation. However, the methods for evaluating COPD under stable conditions are lacking. AimTo evaluate Treg cell phenotypes in patients with different stages of COPD under stable conditions. MethodsPeripheral blood mononuclear cells (PBMCs) were isolated from non-obstructed smokers and ex-smokers (NOS group, n = 19) and COPD patients at different stages (COPD I-II group, n = 25; COPD III-IV group, n = 25). The phenotypic characteristics of Treg cells and Th17 cells and their respective intracellular cytokines were analyzed by flow cytometry. ResultsBoth obstructed groups showed an increase in the proportion of rTregs, while the COPD III-IV group showed additional increases in total Treg and Th17 cells and in IL-10+ cells. There was an increase in proinflammatory mediators (CD3+CD4+IL-17+ cells; CD3+CD4+RORγt+ cells) in the COPD I-II group. In contrast, the NOS group demonstrated high proportions of proinflammatory Treg cells and proinflammatory CD8+ T cells (CD3+CD8+IL-17+). ConclusionDespite the increase in both total Treg cells and the rTreg phenotype from the early stages of COPD, there was a decrease in cells expressing IL-10, suggesting a failure in controlling the inflammatory process. These events precede the progression of the inflammatory process mediated by Th17 cells.

HSP90β shapes the fate of Th17 cells with the help of glycolysis-controlled methylation modification.

Ulcerative colitis (UC) is a refractory inflammatory disease associated with immune dysregulation. Elevated levels of heat shock protein (HSP) 90 in the β but not α subtype were positively associated with disease status in UC patients. This study validated the possibility that pharmacological inhibition or reduction of HSP90β would alleviate colitis, induced by dextran sulfate sodium, in mice and elucidated its mechanisms. Histopathological and biochemical analysis assessed disease severity, and bioinformatics and correlation analysis explained the association between the many immune cells and HSP90β. Flow cytometry was used to analyse the homeostasis and transdifferentiation of Th17 and Treg cells. In vitro inhibition and adoptive transfer assays were used to investigate functions of the phenotypically transformed Th17 cells. Metabolomic analysis, DNA methylation detection and chromatin immunoprecipitation were used to explore these mechanisms. The selective pharmacological inhibitor (HSP90βi) and shHSP90β significantly mitigated UC in mice and promoted transformation of Th17 to Treg cell phenotype, via Foxp3 transcription. The phenotypically-transformed Th17 cells by HSP90βi or shHSP90β were able to inhibit lymphocyte proliferation and colitis in mice. HSP90βi and shHSP90β selectively weakened glycolysis by stopping the direct association of HSP90β and GLUT1, the key glucose transporter, to accelerate ubiquitination degradation of GLUT1, and enhance the methylation of Foxp3 CNS2 region. Then, the mediator path was identified as the "lactate-STAT5-TET2" cascade. HSP90β shapes the fate of Th17 cells via glycolysis-controlled methylation modification to affect UC progression, which provides a new therapeutic target for UC.

Genetic screening to identify novel targets enhancing regulatory T-cell (Treg) phenotype.

Abstract Tregs possess immunosuppressive properties that can restore and maintain immune homeostasis via various suppressive mechanisms. A marker of Tregs is the transcription factor Forkhead box P3 (FoxP3), which regulates Treg development and function. Autoimmune patients have shown dysfunction or reduction in FoxP3+ Tregs, highlighting the critical role of these cells in preventing pathogenic immune responses. There is great interest in developing medical interventions against autoimmune diseases by enhancing Treg function and/or numbers. Currently, a major limitation to generate efficient Treg therapies is the lack of selective targets as starting points for drug discovery. To identify novel Treg targets, Evotec developed a CRISPR/Cas9-based arrayed screening platform using human CD4+ T-cells. About 8,000 genes were screened with a FACS read-out and the effect of gene knockout (KO) on the frequency of FoxP3+ Tregs was assessed. We identified ~450 targets where the KO led to an increase of FoxP3+ cells within the CD4+ T-cell compartment, indicating their potential for drug development starting points. In addition to FoxP3, the effect of KO on CD4+ conventional as well as Treg viability, proliferation and markers such as, CTLA-4, PD-1 or CD25 was monitored. The hits are being confirmed and the impact of the target KO on Treg function and phenotype assessed in a panel of in vitro assays. The goal is to identify novel Treg targeting product candidates to treat autoimmune diseases.

Abstract 4512: Discovery of highly potent, selective, and orally bioavailable IKZF2 degrader and its anti-tumor activity in syngeneic mouse models

Abstract Molecular glue degraders (MGDs) are emerging as innovative therapeutic modalities, owing to their efficacy in targeting previously undruggable targets. MGDs induce close proximity between the target protein and E3 ligase, resulting in the degradation of the target protein. In the wake of the remarkable success of PD-1/PD-L1 inhibitors, various approaches to modulate the activity of regulatory T cells (Tregs) activity have been explored. Tregs represent the primary immune suppressor and significantly impede antitumor immune responses. IKZF2, a marker of stable suppressive Treg, is essential for maintaining the stable Treg cell phenotype. Depletion of IKZF2 can induce a transition from Treg to effector T cell (Teff) phenotypes, thereby enhancing anti-tumor responses. Leveraging an Immunomodulatory drug (IMiD), Novartis has developed the IKZF2 degrader DKY709, which is currently undergoing Phase I clinical trials, both as a monotherapy and in combination with a PD-1 immune checkpoint inhibitor. In this presentation, we present the preclinical results of the best-in-class IKZF2 degrader, PRT-101. PRT-101 elicits rapid and robust degradation of IKZF2, demonstrating subnanomolar DC50 and achieving 100% Dmax in a proteasome- and CRBN-dependent manner. However, PRT-101 does not induce the degradation of well-known neosubstrates, including IKZF1, IKZF3, SALL4, and CK1a. Global proteomic analysis reveals that PRT-101 exclusively facilitates IKZF2 degradation. The degradation of IKZF2 by PRT-101results in an increase in IL-2 secretion, a marker of T effector function. Furthermore, oral administration of PRT-101 exhibits excellent pharmacokinetics, concurrent with IKZF2 degradation, yielding superior anti-tumor effects compared to DKY709 in the MC38 mouse syngeneic model. In conclusion, our findings underscore the potential of the novel IKZF2 degrader as a promising immuno-oncology target for the treatment of solid tumors. Citation Format: Seulki Park, Jeong Hee Moon, Gaseul Lee, Hyun Jin Kim, Jeong-Hoon Kim, Jong Yeon Hwang. Discovery of highly potent, selective, and orally bioavailable IKZF2 degrader and its anti-tumor activity in syngeneic mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4512.

Treg cells as a protective factor for Hashimoto`s thyroiditis: a Mendelian randomization study.

Hashimoto's thyroiditis (HT), a chronic autoimmune disorder impacting thyroid function, is a growing public health concern. The relationship between Treg cells and HT has been extensively studied, with Treg cells considered crucial in suppressing HT progression. However, these studies have mainly been observational, limiting our understanding of Treg cells' impact on HT risk. Leveraging large datasets, we utilized Mendelian randomization (MR) analysis to examine the causal association between Treg cell biomarkers and HT, providing additional validation for these relationships. Comprehensive two-sample Mendelian randomization analysis was performed to determine the causal association between Treg cells signatures and HT in this study. Based on publicly available genetic data, we explored causal associations between 165 Treg cells signatures and HT risk. The European cohort study has identified five Treg cell phenotypes that causally protect against HT risk. Resting Treg %CD4 (OR = 0.975, 95% CI = 0.954~0.998, P = 0.030); CD4 on resting Treg (OR = 0.938, 95% CI = 0.882~0.997, P = 0.041; CD28- CD8dim %CD8dim (OR = 0.983, 95% CI = 0.969~0.998, P = 0.030); CD25 on CD39+ resting Treg (OR = 0.926, 95% CI = 0.864~0.991, P = 0.026); 5) CD28 on activated & secreting Treg (OR = 0.969, 95% CI = 0.942~0.996, P = 0.025). The Asian cohort study has identified four Treg cell phenotypes negatively correlated with the risk of HT. CD25hi %T cell (OR = 0.635, 95% CI = 0.473~852, P = 0.002); CD4 Treg %CD4 (OR = 0.829, 95% CI = 0.687~1.000, P = 0.050); CD127-CD8br %T cell (OR = 0.463, 95% CI =0.311~0.687, P< 0.001); CD3 on resting Treg (OR = 0.786, 95% CI = 0.621~0.994, P = 0.044). Our study has demonstrated the close connection between Treg cells and HT by genetic means, thus providing foundational basis for future research.

Genetic insights into the relationship between immune cell characteristics and ischemic stroke: A bidirectional Mendelian randomization study.

Ischemic stroke, a major contributor to global disability and mortality, is underpinned by intricate pathophysiological mechanisms, notably neuroinflammation and immune cell dynamics. Prior research has identified a nuanced and often paradoxical link between immune cell phenotypes and ischemic stroke susceptibility. The aim of this study was to elucidate the potential causal links between the median fluorescence intensity (MFI) and morphological parameters (MP) of 731 immune cell types and ischemic stroke risk. By analyzing extensive genetic datasets, we conducted comprehensive Mendelian randomization (MR) analyses to discern the genetic correlations between diverse immune cell attributes (MFI and MP) and ischemic stroke risk. Our study identified key immune cell signatures linked to ischemic stroke risk. Both B cells and T cells, among other immune cell types, have a bidirectional influence on stroke risk. Notably, the regulatory T-cell phenotype demonstrates significant neuroprotective properties, with all odds ratio (OR) values and confidence intervals (CIs) being less than 1. Furthermore, CD39 phenotype immune cells, particularly CD39+ CD8+ T cells (inverse variance weighting [IVW] OR 0.92, 95% CI 0.87-0.97; p = 0.002) and CD39+ activated CD4 regulatory T cells (IVW OR 0.93, 95% CI 0.90-0.97; p < 0.001), show notable neuroprotection against ischemic stroke. This investigation provides new genetic insights into the interplay between various immune cells and ischemic stroke, underscoring the complex role of immune processes in stroke pathogenesis. These findings lay a foundation for future research, which may confirm and expand upon these links, potentially leading to innovative immune-targeted therapies for stroke prevention and management.

Immunophenotyping with high-dimensional flow cytometry identifies Treg cell subsets associated with recurrence in papillary thyroid carcinoma.

The activation of Treg cell subsets is critical for the prognosis of tumor patients; however, their heterogeneity and disease association in papillary thyroid carcinoma (PTC) need further investigation. We performed high-dimensional flow cytometry for immunophenotyping on thyroid tissues and matched peripheral blood samples from patients with multinodular goiters or PTC. We analyzed CD4+ T cell and Treg cell phenotypes and compared the recurrence-free survival of PTC patients with different Treg cell subset characteristics using TCGA. Furthermore, PTC recurrent and non-recurrent group were compared by multiplex immunohistochemistry. High-dimensional flow cytometry and bioinformatics analysis revealed an enrichment of Tregs in tumors compared with multinodular goiters and peripheral blood specimens. Moreover, effector Tregs (e-Tregs) as well as FOXP3+ non-Tregs were enriched in tumor samples, and the expression of CD39, PD-1, and CD103 increased on tumor Tregs. TCGA data analysis showed that individuals with CD39hi PD-1loCD103loe-Treghi and CD39loPD-1loCD103hie-Treghi expression patterns had a high recurrence rate. According to the multiplex immunohistochemistry and analysis, compared with non-recurrent group, the proportion of high recurrence rate effector Treg clusters (CD39+PD-1-CD103- plus CD39-PD-1-CD103+) was increased in recurrent patients. Overall, our results highlight the potential of e-Treg subsets as future immunotherapy targets for PTC recurrence.

NK-клетки, предобработанные эстриолом и клетками комменсальной флоры, регулируют созревание Treg и Th17 из CD4+ лимфоцитов у больных рассеянным склерозом и здоровых доноров

Multiple sclerosis (MS) is an inflammatory chronic disease of the central nervous system, one of the pathogenetic mechanisms of which is an immune-mediated attack to the myelin sheath of nerve axons under the control of autoreactive T cells. NK cells, one of subtypes of ILC cells, have the ability to regulate autoimmune mechanisms through the production of cytokines or cytotoxic effects directly against effector cells, such as autoreactive T cells or antigen-presenting cells. At the same time, they can also lyse microglial, astrocyte, and oligodendrocyte cells, indicating a dual function of NK cells in MS. The influence of the pregnancy hormone estriol (E3) and commensal microflora on the ability of NK cells to modulate the level of T regulatory (Treg) and IL-17-producing (Th17) lymphocytes from patients with multiple sclerosis (MS) in comparison with healthy donors was studied. To activate NK cells, Ecsherichia coli K12 and Lactobacillus plantarum 8R-A3 strains were used. The phenotype of Treg and Th17 cells was assessed by flow cytometry using monoclonal antibody staining. It was found that NK cells incubated with E3 and E. coli stimulate the formation of Tregs, and pretreatment of NK cells with L. plantarum inhibits the formation of Th17 from CD4+ lymphocytes. In general, regulatory lymphocytes from MS patients turned out to be more sensitive to the influence of NK cells than cells from healthy donors.

Reproductive Immunology and Embryo Acceptance

Pregnancy is a test of the fundamental laws of transplantation immunology - the coexistence of genetically disparate tissues without host immune rejection. Advances over recent decades show that implantation and placental development depend on an active state of maternal immune tolerance, and breakdown of tolerance is a key contributor to infertility and recurrent pregnancy loss. Tolerance in pregnancy depends on regulatory T (Treg) cells, which are critical to inhibit effector immunity, constrain inflammation, and support maternal vascular adaptations that allow placental access to maternal nutrient supply. Treg cell defects are implicated in disorders of embryo implantation and placental development, but the origins of Treg cell dysfunction are unknown. In recent studies, we have comprehensively analysed the phenotypes and transcriptional profile of peripheral blood Treg cells in individuals with early pregnancy failure (recurrent miscarriage and recurrent implantation failure). Compared to fertile women, women with early pregnancy failure have fewer total Treg cells and altered Treg cell phenotypes, accompanied by lower Treg:Th1 and Treg:Th17 ratios. RNAseq demonstrates an aberrant Treg cell gene expression profile in early pregnancy failure, with upregulation of pro-inflammatory genes including CSF2, IL4, IL17A, IL21, and IFNG. Our data indicate that Treg cell defects in women with early pregnancy failure can arise due to loss of lineage fidelity associated with impaired FOXP3 regulation. Factors including metabolic function, luteal phase progesterone, and male partner seminal fluid components are all implicated in causing Treg cell deficit. Treg cells may be a useful target for therapeutic interventions to improve uterine receptivity, but this will depend on developing diagnostic tests that are informative. Moreover, safe, and effective interventions to modulate these cells are in their infancy, and personalized approaches matched to specific diagnostic criteria will be needed.

CD8+ Treg cells play a role in the obesity-associated insulin resistance

Obesity-related chronic low-grade inflammation may trigger insulin resistance and type 2 diabetes (T2D) development. Cells with regulatory phenotype have been shown to be reduced during obesity, especially CD4+ Treg cells. However, little is known about the CD8+ Treg cells. Therefore, we aim to characterize the CD8+ Treg cells in human peripheral blood and adipose tissue, specifically, to address the effect of obesity and insulin resistance in this regulatory immune cell population. A group of 42 participants with obesity (OB group) were recruited. Fourteen of them were evaluated pre- and post-bariatric surgery. A group of age- and sex-matched healthy volunteers (n = 12) was also recruited (nOB group). CD8+ Treg cell quantification and phenotype were evaluated by flow cytometry, in peripheral blood (PB), subcutaneous (SAT), and visceral adipose tissues (VAT). The OB group displayed a higher percentage of CD8+ Treg cells in PB, compared to the nOB. In addition, they were preferentially polarized into Tc1- and Tc1/17-like CD8+ Treg cells, compared to nOB. Moreover, SAT displayed the highest content of CD8+ Tregs infiltrated, compared to PB or VAT, while CD8+ Tregs infiltrating VAT displayed a higher percentage of cells with Tc1-like phenotype. Participants with pre-diabetes displayed a reduced percentage of TIM-3+CD8+ Tregs in circulation, and PD-1+CD8+ Tregs infiltrated in the VAT. An increase in the percentage of circulating Tc1-like CD8+ Treg cells expressing PD-1 was observed post-surgery.In conclusion, obesity induces significant alterations in CD8+ Treg cells, affecting their percentage and phenotype, as well as the expression of important immune regulatory molecules.

Tumor-derived exosomes induce initial activation by exosomal CD19 antigen but impair the function of CD19-specific CAR T-cells via TGF-β signaling.

Tumor-derived exosomes (TEXs) enriched in immune suppressive molecules predominantly drive T-cell dysfunction and impair antitumor immunity. Chimeric antigen receptor (CAR) T-cell therapy has emerged as a promising treatment for refractory and relapsed hematological malignancies, but whether lymphoma TEXs have the same impact on CAR T-cell remains unclear. Here, we demonstrated that B-cell lymphoma-derived exosomes induce the initial activation of CD19-CAR T-cells upon stimulation with exosomal CD19. However, lymphoma TEXs might subsequently induce CAR T-cell apoptosis and impair the tumor cytotoxicity of the cells because of the upregulated expression of the inhibitory receptors PD-1, TIM3, and LAG3 upon prolonged exposure. Similar results were observed in the CAR T-cells exposed to plasma exosomes from patients with lymphoma. More importantly, single-cell RNA sequencing revealed that CAR T-cells typically showed differentiated phenotypes and regulatory T-cell (Treg) phenotype conversion. By blocking transforming growth factor β (TGF-β)-Smad3 signaling with TGF-β inhibitor LY2109761, the negative effects of TEXs on Treg conversion, terminal differentiation, and immune checkpoint expression were rescued. Collectively, although TEXs lead to the initial activation of CAR T-cells, the effect of TEXs suppressed CAR T-cells, which can be rescued by LY2109761. A treatment regimen combining CAR T-cell therapy and TGF-β inhibitors might be a novel therapeutic strategy for refractory and relapsed B-cell lymphoma.

Anti-IL-6R prevents experimental type 1 diabetes

Abstract Type 1 diabetes (T1D) is an autoimmune disease, where the insulin producing β cells are damaged by immune attacks leading to a failure of blood glucose control. The anti-inflammatory cytokine IL-35 has been shown to prevent and reverse T1D in mouse models. IL-6 signals through a homodimer of gp130 which is one of the receptors of IL-35. We thus hypothesized that blocking IL-6 signaling would facilitate IL-35 signaling and prevent T1D. We used multiple low dose streptozotocin (MLDSTZ) to induce T1D in male CD-1 mice and treated them with anti-IL-6R to study the immune response. We found that anti-IL-6R treatment prevented MLDSTZ mice from developing hyperglycemia, and preserved the ability of β cells to produce insulin. We then used flow cytometry to investigate the T cell and B cell response on day 13 after the first injection of MLDSTZ. The proportions of Treg cells were not altered by anti-IL-6R treatment in MLDSTZ treated mice, but the treatment elevated the proportions of IL-35 producing Treg cells in the spleen and thymus. It also decreased IFN-γ production in CD4, CD8, Treg and B cells in the spleen. Furthermore, anti-IL-6R treatment only tended to increase IL-17 production in CD4, CD8 and Treg cells. Altogether, our data demonstrate that anti-IL-6R treatment prevents T1D in MLDSTZ mice by maintaining the Treg cell phenotype and by reducing IFN-γ production by T and B cells.

DOCK11 deficiency in patients with X-linked actinopathy and autoimmunity

AbstractDedicator of cytokinesis (DOCK) proteins play a central role in actin cytoskeleton regulation. This is highlighted by the DOCK2 and DOCK8 deficiencies leading to actinopathies and immune deficiencies. DOCK8 and DOCK11 activate CDC42, a Rho–guanosine triphosphate hydrolases involved in actin cytoskeleton dynamics, among many cellular functions. The role of DOCK11 in human immune disease has been long suspected but, to the best of our knowledge, has never been described to date. We studied 8 male patients, from 7 unrelated families, with hemizygous DOCK11 missense variants leading to reduced DOCK11 expression. The patients were presenting with early-onset autoimmunity, including cytopenia, systemic lupus erythematosus, skin, and digestive manifestations. Patients' platelets exhibited abnormal ultrastructural morphology and spreading as well as impaired CDC42 activity. In vitro activated T cells and B-lymphoblastoid cell lines from patients exhibited aberrant protrusions and abnormal migration speed in confined channels concomitant with altered actin polymerization during migration. Knock down of DOCK11 recapitulated these abnormal cellular phenotypes in monocytes-derived dendritic cells and primary activated T cells from healthy controls. Lastly, in line with the patients’ autoimmune manifestations, we also observed abnormal regulatory T-cell (Treg) phenotype with profoundly reduced FOXP3 and IKZF2 expression. Moreover, we found reduced T-cell proliferation and impaired STAT5B phosphorylation upon interleukin-2 stimulation of the patients’ lymphocytes. In conclusion, DOCK11 deficiency is a new X-linked immune-related actinopathy leading to impaired CDC42 activity and STAT5 activation, and is associated with abnormal actin cytoskeleton remodeling as well as Treg phenotype, culminating in immune dysregulation and severe early-onset autoimmunity.

Long-term glucocorticoid exposure persistently impairs CD4+ T cell biology by epigenetically modulating the mTORC1 pathway

Conventional glucocorticoid (GC) treatment has a long-term influence on T-cell immunity, resulting in an increased risk of opportunistic infection after drug withdrawal. The underlying mechanisms remain ambiguous. This study demonstrated that long-term GC treatment induced persistent lymphopenia in patients with primary glomerular disease. GCs continuously suppressed the proportion of CD4+ T cells even after the daily dose was tapered down to the physiologic equivalences, leading to a significant decline of the CD4/CD8 ratio. Meanwhile, GCs impaired CD4+ T cell biology, leading to enhanced apoptotic cell death, reduced proliferative capacity, downregulated pro-inflammatory genes, and upregulated immunoregulatory genes. Specifically, GCs altered FOXP3 expression pattern in CD4+ T cells and favored their acquisition of an active T regulatory (Treg) cell phenotype with enhanced IL-10 production upon stimulation. Mechanistically, GCs tampered with the transcriptional regulation of mechanistic target of rapamycin complex 1 (mTORC1) pathway, resulting in an inhibitory impact on the signaling activity. Targeting mTORC1 signaling by siRNAs could sufficiently modify the viability of GC-exposed CD4+ T cells. By high-throughput sequencing of genome-wide DNA methylation and mRNA, we further uncovered a causal relationship between the altered DNA methylation level and transcription activity in a subset of mTORC1 pathway genes in long-term GC exposure. Taken together, this study reveals a novel regulation of mTORC1 signaling, which might dominate the long-term influence of GC on CD4+ T cell biology in a dose-independent manner.

A SARS-CoV-2-specific CAR-T-cell model identifies felodipine, fasudil, imatinib, and caspofungin as potential treatments for lethal COVID-19

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced cytokine storm is closely associated with coronavirus disease 2019 (COVID-19) severity and lethality. However, drugs that are effective against inflammation to treat lethal COVID-19 are still urgently needed. Here, we constructed a SARS-CoV-2 spike protein-specific CAR, and human T cells infected with this CAR (SARS-CoV-2-S CAR-T) and stimulated with spike protein mimicked the T-cell responses seen in COVID-19 patients, causing cytokine storm and displaying a distinct memory, exhausted, and regulatory T-cell phenotype. THP1 remarkably augmented cytokine release in SARS-CoV-2-S CAR-T cells when they were in coculture. Based on this “two-cell” (CAR-T and THP1 cells) model, we screened an FDA-approved drug library and found that felodipine, fasudil, imatinib, and caspofungin were effective in suppressing the release of cytokines, which was likely due to their ability to suppress the NF-κB pathway in vitro. Felodipine, fasudil, imatinib, and caspofungin were further demonstrated, although to different extents, to attenuate lethal inflammation, ameliorate severe pneumonia, and prevent mortality in a SARS-CoV-2-infected Syrian hamster model, which were also linked to their suppressive role in inflammation. In summary, we established a SARS-CoV-2-specific CAR-T-cell model that can be utilized as a tool for anti-inflammatory drug screening in a fast and high-throughput manner. The drugs identified herein have great potential for early treatment to prevent COVID-19 patients from cytokine storm-induced lethality in the clinic because they are safe, inexpensive, and easily accessible for immediate use in most countries.

Regulatory T Cells Overexpressing Peli1 Show Better Efficacy in Repairing Ovarian Endocrine Function in Autoimmune Premature Ovarian Insufficiency

Regulatory T (Treg) cell dysfunction is involved in the pathogenesis of autoimmune premature ovarian insufficiency (POI). Adoptive transfer of Treg cells has been shown to be effective in the treatment of autoimmune POI in mice. However, the therapeutic effect of Treg cell therapy is limited because the phenotype and function of Treg cells is not properly maintained when they are reinfused in an inflammatory environment. Therefore, enhancing the function of Treg cells using genetic engineering is of great significance for improving the efficacy of Treg cells in the treatment of immune diseases. In this study, we investigated the role of the E3 ubiquitinated ligase Pellino 1 (Peli1) in the proliferation and immunosuppressive function of Treg cells and the therapeutic effect of Treg cells overexpressing Peli1 on autoimmune POI. The results showed that the overexpression of Peli1 promoted cell proliferation and enhanced the immunosuppressive function of Treg cells in vitro. After the adoptive transfer of Treg cells overexpressing Peli1 in autoimmune POI mice, the apoptosis rate of ovarian granulosa cells declined. The levels of the inflammatory inhibitors interleukin 10 and transforming growth factor-β as well as the ovarian hormone estradiol were elevated. The number of primordial, primary, secondary, and mature follicles was restored to a certain extent compared with those in control subjects. These results revealed that the adoptive transfer of Treg cells overexpressing Peli1 promoted its efficacy against zona pellucida protein 3 peptide-induced POI, which provides new insights into the treatment of autoimmune POI.

Disordered T cell-B cell interactions in autoantibody-positive inflammatory arthritis.

T peripheral helper (Tph) cells, identified in the synovium of adults with seropositive rheumatoid arthritis, drive B cell maturation and antibody production in non-lymphoid tissues. We sought to determine if similarly dysregulated T cell-B cell interactions underlie another form of inflammatory arthritis, juvenile oligoarthritis (oligo JIA). Clonally expanded Tph cells able to promote B cell antibody production preferentially accumulated in the synovial fluid (SF) of oligo JIA patients with antinuclear antibodies (ANA) compared to autoantibody-negative patients. Single-cell transcriptomics enabled further definition of the Tph gene signature in inflamed tissues and showed that Tph cells from ANA-positive patients upregulated genes associated with B cell help to a greater extent than patients without autoantibodies. T cells that co-expressed regulatory T and B cell-help factors were identified. The phenotype of these Tph-like Treg cells suggests an ability to restrain T cell-B cell interactions in tissues. Our findings support the central role of disordered T cell-help to B cells in autoantibody-positive arthritides.

Glucocorticoid regulation of the mTORC1 pathway modulates CD4+ T cell responses during infection.

Conventional glucocorticoid (GC) treatment poses significant risks for opportunistic infections due to its suppressive impact on CD4+ T cells. This study aimed to explore the mechanisms by which GCs modulate the functionality of CD4+ Tcells during infection. We consistently measured FOXP3, inflammatory cytokines and phospho-S6 ribosomal protein levels in CD4+ T cells from patients undergoing conventional GC treatment. Using Foxp3EGFP animals, we investigated the dynamicactivation of the mechanistic target of rapamycin complex 1 (mTORC1) pathway and its correlation with the immunoregulatory function of CD4+ T cells under the influence of GCs. GCs dynamically altered the expression pattern of FOXP3 in CD4+ T cells, promoting their acquisition of an active T regulatory (Treg) cell phenotype upon stimulation. Mechanistically, GCs undermined the kinetics of the mTORC1 pathway, which was closely correlated with phenotype conversion and functional properties of CD4+ T cells. Dynamic activation of the mTORC1 signaling modified the GC-dampened immunoregulatory capacity of CD4+ T cells by phenotypically and functionally bolstering the FOXP3+ Treg cells. Interventions targeting the mTORC1 pathway effectively modulated the GC-dampened immunoregulatory capacity of CD4+ T cells. These findings highlight a novel mTORC1-mediated mechanism underlying CD4+ T cell immunity in the context of conventional GC treatment.

Significance of regulatory T cells in cancer immunology and immunotherapy.

Immunosuppression in the tumour microenvironment (TME) attenuates antitumor immunity, consequently hindering protective immunosurveillance and preventing effective antitumor immunity induced by cancer immunotherapy. Multiple mechanisms including immune checkpoint molecules, such as CTLA-4, PD-1, and LAG-3, and immunosuppressive cells are involved in the immunosuppression in the TME. Regulatory T (Treg) cells, a population of immunosuppressive cells, play an important role in inhibiting antitumor immunity. Therefore, Treg cells in the TME correlate with an unfavourable prognosis in various cancer types. Thus, Treg cell is considered to become a promising target for cancer immunotherapy. Elucidating Treg cell functions in cancer patients is therefore crucial for developing optimal Treg cell-targeted immunotherapy. Here, we describe Treg cell functions and phenotypes in the TME from the perspective of Treg cell-targeted immunotherapy.

Abstract B37: PLX-4107, a selective IKZF2 degrader, reprograms suppressive regulatory T cells and demonstrates anti-tumor activity

Abstract Immune checkpoint therapy has demonstrated durable clinical responses in multiple solid tumor types. Reduced clinical response to checkpoint therapy has been linked to the presence of potent immunosuppressive regulatory T cells (Tregs) within the tumor microenvironment that contribute to tumor immune evasion. The transcription factor Helios (IKZF2) is a marker of highly suppressive Tregs and is required to maintain a stable, suppressive Treg cell phenotype in the inflammatory tumor microenvironment. Depletion of IKZF2 in Tregs results in both loss of suppressive activity and conversion of Tregs into effector-like T cells, leading to anti-tumor immunity. Targeted protein degradation using the endogenous Ubiquitin Proteasome System (UPS) has enabled targeting undruggable proteins, such as IKZF2, that have no known small molecule binding pocket. We have designed small molecules that promote a novel interaction between IKZF2 with the E3 ubiquitin ligase substrate receptor, Cereblon, leading to proximity induced protein degradation. PLX-4107 is a novel molecular glue that is a highly selective, potent, and rapid degrader of IKZF2 via the redirection of the E3 substrate receptor, Cereblon. Degradation of IKZF2 by PLX-4107 is blocked in the presence of proteasome and neddylation inhibitors as well as a Cereblon knock-out cell line, confirming that degradation is mediated by the UPS and specifically through the involvement of Cereblon. Proteome-wide analysis demonstrated that PLX-4107 selectively depletes IKZF2 protein levels without degrading other known Cereblon neo-substrates. In vitro, PLX-4107 mediated degradation of IKZF2 resulted in conversion of suppressive Tregs into CD4+ effector-like T cells, coupled with an increased production of the effector cytokines IL2 and IFNg. Oral administration of PLX-4107 to cynomolgus monkeys demonstrated sustained pharmacodynamic response, persistent depletion of IKZF2, and reprogramming of Tregs, consistent with the catalytic mechanism of protein degradation. In vivo, T cell expansion studies showed that administration of PLX-4107 decreased both Treg CD25 expression and proliferation, along with increased activation of CD8+ T effector cells. PLX-4107 was evaluated in in vivo xenograft efficacy studies and demonstrated dose dependent single agent anti-tumor activity that was dependent on the presence of T cells. In addition, co-administration of PLX-4107 and anti-PD-1 antibody Pembrolizumab resulted in tumor growth inhibition and significant combination benefit. PLX-4107 is a novel molecular glue that selectively degrades the undruggable transcription factor, IKZF2. PLX-4107 mediated IKZF2 degradation results in conversion of Tregs to an effector-like T cell phenotype, single agent antitumor activity and the ability to enhance the efficacy of immune checkpoint therapy in vivo. Citation Format: Peggy A Thompson, Pengyu Yang, Linette Yang, Susan Song, Yujun Huang, Xiaoming Li, Alejandro Dearie, Stephen Chien, Mary E Spalding, Gabrielle Blanco, Elizabeth Daniele, Julia Toth, Aleksandar Jamborcic, Gregory Parker, Simon Bailey. PLX-4107, a selective IKZF2 degrader, reprograms suppressive regulatory T cells and demonstrates anti-tumor activity [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B37.