Conventional CD4 Research Articles

The role of iTr35 cells in the inflammatory response and fibrosis progression of systemic sclerosis.

To evaluate the role of induced immunosuppressive T regulatory (iTr) 35 cells in SSc-related inflammation and fibrosis. Sixty-eight SSc patients were enrolled in this study. Subsets of iTr35 and Tr1 were measured by flow cytometry. IL-35 and IL-10 levels were measured using ELISA. Expressions of iTr35, Tr1, fibrosis-related genes and proteins associated with signalling pathways were determined using immunofluorescence, western blot and immunohistochemistry assays. In peripheral blood, the proportions of the iTr35 cells were higher and Tr1 cells were lower than the control group. Similarly, IL-35 expression was increased, while IL-10 levels were decreased. In fibroblasts from skin tissue, the expression levels of EBI3, IL-12Ap35, Foxp3 and IL-10 were decreased, but collagen I, TGF-β, alpha smooth muscle actin (α-SMA) and fibronectin levels were increased. Phosphorylated STAT3/6 were increased, but iTr35 and Tr1 cell levels were significantly decreased. When CD4+ cells were incubated with both recombinant human (rh)IL-35 and rhIL-10, the cell numbers of iTr35 and Tr1 were greater than the same type of cells treated with rhIL-35 or rhIL-10 alone. However, the viability of conventional CD4+ T cells was decreased by gradually increasing iTr35 cells. Moreover, iTr35 cells affected α-SMA expression through the STAT3/6 signalling pathway. Both iTr35 and Tr1 cells are involved in SSc-related inflammation and fibrosis. IL-35 can induce iTr35 cells, showing a synergistic effect with IL-10. We also found that iTr35 cells can inhibit T cell proliferation and differentiation via the STAT3/6 signalling pathway, thereby causing fibrosis.

Post-transplant cyclophosphamide prevents xenogeneic graft-versus-host disease while depleting proliferating regulatory T cells

Graft-versus-host disease (GVHD) remains a serious limitation of allogeneic hematopoietic cell transplantation (allo-HCT). While post-transplant administration of cyclophosphamide (PTCy) is increasingly used as GVHD prophylaxis, its precise mechanisms of action and its impact on graft-versus-leukemia effects have remained debated. Here, we studied the mechanisms of xenogeneic GVHD (xGVHD) prevention by PTCy in different humanized mouse models. We observed that PTCy attenuated xGVHD. Using flow cytometry and single-cell RNA-sequencing, we demonstrated that PTCy depleted proliferative CD8+ and conventional CD4+ Tcells but also proliferative regulatory Tcells (Treg). Further, T-cell receptor β variable region sequencing (TCRVB) analyses demonstrated that highly xenoreactive T-cell clones were depleted by PTCy. Although Treg frequencies were significantly higher in PTCy-treated than in control mice on day 21, xGVHD attenuation by PTCy was not abrogated by Treg depletion. Finally, we observed that PTCy did not abrogate graft-versus-leukemia effects.

Insulin Receptor Substrate 1 Signaling Inhibits Foxp3 Expression and Suppressive Functions in Treg Cells through the mTORC1 Pathway.

Regulatory T (Treg) cells play an important role in immune homeostasis by inhibiting cells within the innate and adaptive immune systems; therefore, the stability and immunosuppressive function of Treg cells need to be maintained. In this study, we found that the expression of insulin receptor substrate 1 (IRS1) by Treg cells was lower than that by conventional CD4 T cells. IRS1-overexpressing Treg cells showed the downregulated expression of FOXP3, as well as Treg signature markers CD25 and CTLA4. IRS1-overexpressing Treg cells also showed diminished immunosuppressive functions in an in vitro suppression assay. Moreover, IRS1-overexpressing Treg cells were unable to suppress the pathogenic effects of conventional T cells in a transfer-induced colitis model. IRS1 activated the mTORC1 signaling pathway, a negative regulator of Treg cells. Moreover, IRS1 destabilized Treg cells by upregulating the expression of IFN-γ and Glut1. Thus, IRS1 acts as a negative regulator of Treg cells by downregulating the expression of FOXP3 and disrupting stability.

People with Primary Progressive Multiple Sclerosis Have a Lower Number of Central Memory T Cells and HLA-DR+ Tregs.

The importance of circulating immune cells to primary progressive multiple sclerosis (PPMS) pathophysiology is still controversial because most immunotherapies were shown to be ineffective in treating people with PPMS (pwPPMS). Yet, although controversial, data exist describing peripheral immune system alterations in pwPPMS. This study aims to investigate which alterations might be present in pwPPMS free of disease-modifying drugs (DMD) in comparison to age- and sex-matched healthy controls. A multicentric cross-sectional study was performed using 23 pwPPMS and 23 healthy controls. The phenotype of conventional CD4+ and CD8+ T cells, regulatory T cells (Tregs), B cells, natural killer (NK) T cells and NK cells was assessed. Lower numbers of central memory CD4+ and CD8+ T cells and activated HLA-DR+ Tregs were observed in pwPPMS. Regarding NK and NKT cells, pwPPMS presented higher percentages of CD56dimCD57+ NK cells expressing NKp46 and of NKT cells expressing KIR2DL2/3 and NKp30. Higher disease severity scores and an increasing time since diagnosis was correlated with lower numbers of inhibitory NK cells subsets. Our findings contribute to reinforcing the hypotheses that alterations in peripheral immune cells are present in pwPPMS and that changes in NK cell populations are the strongest correlate of disease severity.

Regulatory T cells suppress the formation of potent KLRK1 and IL-7R expressing effector CD8 T cells by limiting IL-2.

Regulatory T cells (Tregs) are indispensable for maintaining self-tolerance by suppressing conventional T cells. On the other hand, Tregs promote tumor growth by inhibiting anticancer immunity. In this study, we identified that Tregs increase the quorum of self-reactive CD8+ T cells required for the induction of experimental autoimmune diabetes in mice. Their major suppression mechanism is limiting available IL-2, an essential T-cell cytokine. Specifically, Tregs inhibit the formation of a previously uncharacterized subset of antigen-stimulated KLRK1+ IL-7R+ (KILR) CD8+ effector T cells, which are distinct from conventional effector CD8+ T cells. KILR CD8+ T cells show superior cell-killing abilities in vivo. The administration of agonistic IL-2 immunocomplexes phenocopies the absence of Tregs, i.e., it induces KILR CD8+ T cells, promotes autoimmunity, and enhances antitumor responses in mice. Counterparts of KILR CD8+ T cells were found in the human blood, revealing them as a potential target for immunotherapy.

Impact of SIV infection on mycobacterial lipid-reactive T cell responses in Bacillus Calmette-Guérin (BCG) inoculated macaques.

Although BCG vaccine protects infants from tuberculosis (TB), it has limited efficacy in adults against pulmonary TB. Further, HIV coinfection significantly increases the risk of developing active TB. In the lack of defined correlates of protection in TB disease, it is essential to explore immune responses beyond conventional CD4 T cells to gain a better understanding of the mechanisms of TB immunity. Here, we evaluated unconventional lipid-reactive T cell responses in cynomolgus macaques following aerosol BCG inoculation and examined the impact of subsequent SIV infection on these responses. Immune responses to cellular lipids of M. bovis and M. tuberculosis were examined ex vivo in peripheral blood and bronchioalveolar lavage (BAL). Prior to BCG inoculation, innate-like IFN-γ responses to mycobacterial lipids were observed in T cells. Aerosol BCG exposure induced an early increase in frequencies of BAL γδT cells, a dominant subset of lipid-reactive T cells, along with enhanced IL-7R and CXCR3 expression. Further, BCG exposure stimulated greater IFN-γ responses to mycobacterial lipids in peripheral blood and BAL, suggesting the induction of systemic and local Th1-type response in lipid-reactive T cells. Subsequent SIV infection resulted in a significant loss of IL-7R expression on blood and BAL γδT cells. Additionally, IFN-γ responses of mycobacterial lipid-reactive T cells in BAL fluid were significantly lower in SIV-infected macaques, while perforin production was maintained through chronic SIV infection. Overall, these data suggest that despite SIV-induced decline in IL-7R expression and IFN-γ production by mycobacterial lipid-reactive T cells, their cytolytic potential is maintained. A deeper understanding of anti-mycobacterial lipid-reactive T cell functions may inform novel approaches to enhance TB control in individuals with or without HIV infection.

CD36-BATF2\MYB Axis Predicts Anti-PD-1 Immunotherapy Response in Gastric Cancer.

Despite the utilization of anti-PD-1 therapy in gastric cancer (GC), the absence of a reliable predictive biomarker continues to pose a challenge. In this study, we utilized bioinformatic analysis and immunohistochemistry to develop a prediction model for activated CD4+ memory T cells, considering both mRNA and protein levels. An elevation of activated CD4+ memory T cells in GC was noted, which exhibited a strong association with the patients' overall survival. By utilizing WGCNA and DEG analysis, we discovered that BATF2, MYB, and CD36 are genes that exhibit differential expression and are linked to activated CD4+ memory T cells. Afterwards, a forecast model was built utilizing Stepwise regression and immunohistochemistry relying on the three genes. The model's high-risk score showed significant associations with a suppressive immune microenvironment. Moreover, our model exhibited encouraging prognostic value and superior performance in predicting response to immune checkpoint blockade therapy compared with the conventional CD8+PD-L1 model. In terms of mechanism, CD36 could function as a receptor upstream that identifies Helicobacter pylori and fatty acids. This recognition then results in the reduction of the BATF2-MYB protein complex and subsequent alterations in the transcription of genes associated with classical T cell activation. As a result, the activation state of CD4+ memory T cells is ultimately suppressed. The CD36-BATF2/MYB signature serves as a robust predictor of anti-PD-1 immunotherapy response in GC.

Interleukin 35: protective role and mechanism in type 1 diabetes.

Interleukin 35 (IL-35), a cytokine secreted by regulatory T (Treg) cells from the differentiation of conventional CD4+ T cells, is a member of the IL-12 family. The IL-12 family of cytokines exhibits an anti-inflammatory property. IL-35 has recently been shown to influence the immune modulation in various diseases, including inflammatory bowel disease, Graves' disease, rheumatoid arthritis, colitis, psoriasis, and type 1 diabetes (T1D). T1D is an immune-related disease caused by destruction of pancreatic β cells, characterized by an absolute lack of insulin. Recently, studies have suggested that protective effects of IL-35 work by improving blood glucose levels and preventing an attack of inflammatory factors on the islets. The protective mechanism may be closely related to the anti-inflammatory properties of IL-35, which include regulating macrophage phenotype, suppressing T cell proliferation, decreasing the differentiation of Th17 cells, increasing the Treg cell population, and inducing IL-35-producing regulatory T cells (iTr35). Here, we review the protective effects and mechanisms of action of IL-35 in T1D.

Single-Cell RNA Sequencing Maps Immune Cell Heterogeneity in Mice with Allogeneic Cardiac Transplantation

Objective: Immune cells play important roles in mediating allograft rejection and tolerance after cardiac transplantation. However, immune cell heterogeneity at the single-cell level, and how immune cell states shape transplantation immunity, remain incompletely characterized. Methods: We performed single-cell RNA sequencing (scRNA-seq) on immune cells in LNs from a mouse syngeneic and allogeneic cardiac transplantation model. Nine T cell clusters were identified through unsupervised analysis. Pathway enrichment analysis was used to explore the functional differences among cell subpopulations and to characterize the metabolic heterogeneity of T cells. Results: We comprehensively determined the transcriptional landscape of immune cells, particularly T cells, and their metabolic transcriptomes in LNs during mouse cardiac transplantation. On the basis of molecular and functional properties, we also identified T cell types associated with transplantation-associated immune processes, including cytotoxic CD8+ T cells, activated conventional CD4+ T cells, and dysfunctional Tregs. We further elucidated the contribution of JunB to the induction of Th17 cell differentiation and restriction of Treg development, and identified that HIF-1a participates in T cell metabolism and function. Conclusions: We present the first systematic single-cell analysis of transcriptional variation within the T cell population, providing new insights for the development of novel therapeutic targets for allograft rejection.

Biology and clinical relevance of follicular cytotoxic T cells.

Follicular cytotoxic T (Tfc) cells are a newly identified subset of CD8+ T cells enriched in B cell follicles and their surroundings, which integrate multiple functions such as killing, memory, supporting and regulation. Tfc cells share similarities with follicular helper T (Tfh) cells, conventional cytotoxic CD8+ T (Tc cells)cells and follicular regulatory T (Tfr) cells, while they express distinct transcription factors, phenotype, and perform different functions. With the participation of cytokines and cell-cell interactions, Tfc cells modulate Tfh cells and B cells and play an essential role in regulating the humoral immunity. Furthermore, Tfc cells have been found to change in their frequencies and functions during the occurrence and progression of chronic infections, immune-mediated diseases and cancers. Strategies targeting Tfc cells are under investigations, bringing novel insights into control of these diseases. We summarize the characteristics of Tfc cells, and introduce the roles and potential targeting modalities of Tfc cells in different diseases.

Mapping single-cell transcriptomes in the intra-tumoral and associated territories of kidney cancer.

Tumor behavior is intricately dependent on the oncogenic properties of cancer cells and their multi-cellular interactions. To understand these dependencies within the wider microenvironment, we studied over 270,000 single-cell transcriptomes and 100 microdissected whole exomes from 12 patients with kidney tumors, prior to validation using spatial transcriptomics. Tissues were sampled from multiple regions of the tumor core, the tumor-normal interface, normal surrounding tissues, and peripheral blood. We find that the tissue-type location of CD8+ Tcell clonotypes largely defines their exhaustion state with intra-tumoral spatial heterogeneity that is not well explained by somatic heterogeneity. De novo mutation calling from single-cell RNA-sequencing data allows us to broadly infer the clonality of stromal cells and lineage-trace myeloid cell development. We report six conserved meta-programs that distinguish tumor cell function, and find an epithelial-mesenchymal transition meta-program highly enriched at the tumor-normal interface that co-localizes with IL1B-expressing macrophages, offering a potential therapeutic target.

Neoadjuvant chemotherapy drives intratumoral T cells toward a proinflammatory profile in pancreatic cancer.

BACKGROUNDPancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. At diagnosis, only 20% of patients with PDAC are eligible for primary resection. Neoadjuvant chemotherapy can enable surgical resection in 30%-40% of patients with locally advanced and borderline resectable PDAC. The effects of neoadjuvant chemotherapy on the cytokine production of tumor-infiltrating T cells are unknown in PDAC.METHODSWe performed multiplex immunofluorescence to investigate T cell infiltration in 91 patients with PDAC. Using flow cytometry, we analyzed tumor and matched blood samples from 71 patients with PDAC and determined the frequencies of T cell subsets and their cytokine profiles. Both cohorts included patients who underwent primary resection and patients who received neoadjuvant chemotherapy followed by surgical resection.RESULTSIn human PDAC, T cells were particularly enriched within the tumor stroma. Neoadjuvant chemotherapy markedly enhanced T cell density within the ductal area of the tumor. Whereas infiltration of cytotoxic CD8+ T cells was unaffected by neoadjuvant chemotherapy, the frequency of conventional CD4+ T cells was increased, and the proportion of Tregs was reduced in the pancreatic tumor microenvironment after neoadjuvant treatment. Moreover, neoadjuvant chemotherapy increased the production of proinflammatory cytokines by tumor-infiltrating T cells, with enhanced TNF-α and IL-2 and reduced IL-4 and IL-10 expression.CONCLUSIONNeoadjuvant chemotherapy drives intratumoral T cells toward a proinflammatory profile. Combinational treatment strategies incorporating immunotherapy in neoadjuvant regimens may unleash more effective antitumor responses and improve prognosis of pancreatic cancer.FUNDINGThis work was supported by the Jung Foundation for Science and Research, the Monika Kutzner Foundation, the German Research Foundation (SE2980/5-1), the German Cancer Consortium, and the Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden.

Lung CCR6−CXCR3− type 2 helper T cells as an indicator of progressive fibrosing interstitial lung diseases

Progressive fibrosing interstitial lung diseases (PF-ILDs) have a poor prognosis and may be resistant to corticosteroids and/or immunosuppressants, but antifibrotic therapies such as nintedanib and pirfenidone have been shown to slow the deterioration of lung function. The aim of this study was to identify the characteristic cellular profile of bronchoalveolar lavage fluid at diagnostic bronchoscopy for predicting PF-ILDs, defined as fibrotic diseases on chest high-resolution computed tomography with more than a 5% relative decline in the percent predicted value of forced vital capacity (FVC) over 6 months. The proportions of inflammatory cells, CCR6−CXCR3− T helper type 2 (Th2) cells among conventional CD4+ T cells in bronchoalveolar lavage fluid (BALF) and peripheral blood, were measured by flowcytometry. The proportion of lymphocytes in BALF was significantly higher in non-PF-ILD patients than in PF-ILD patients. The proportion of Th2 cells in BALF, but not in peripheral blood, was significantly higher in PF-ILD patients than in non-PF-ILD patients. Multivariate analysis showed that a greater population of Th2 cells in BALF was the only indicator for PF-ILDs. An increased proportion of Th2 cells in BALF is associated with greater deterioration of lung function in fibrotic interstitial lung diseases.

BATF epigenetically and transcriptionally controls the activation program of regulatory T cells in human tumors.

Regulatory T (Treg) cells suppress effective antitumor immunity in tumor-bearing hosts, thereby becoming promising targets in cancer immunotherapy. Despite the importance of Treg cells in tumor immunity, little is known about their differentiation process and epigenetic profiles in the tumor microenvironment (TME). Here, we showed that Treg cells in the TME of human lung cancers harbored a completely different open chromatin profile compared with CD8+ T cells, conventional CD4+ T cells in the TME, and peripheral Treg cells. The integrative sequencing analyses including ATAC, single-cell RNA, and single-cell ATAC sequencing revealed that BATF, IRF4, NF-κB, and NR4A were important transcription factors for Treg cell differentiation in the TME. In particular, BATF was identified as a key regulator, which leveraged Treg cell differentiation through epigenetically controlling activation-associated gene expression, resulting in the robustness of Treg cells in the TME. The single-cell sequencing approaches also revealed that tissue-resident and tumor-infiltrating Treg cells followed a common pathway for differentiation and activation in a BATF-dependent manner heading toward Treg cells with the most differentiated and activated phenotypes in tissues and tumors. BATF deficiency in Treg cells remarkably inhibited tumor growth, and high BATF expression was associated with poor prognosis in lung cancer, kidney cancer, and melanoma. These findings indicate one of the specific chromatin remodeling and differentiation programs of Treg cells in the TME, which can be applied in the development of Treg cell-targeted therapies.

Tetramer-aided sorting and single-cell RNA sequencing facilitate transcriptional profiling of antigen-specific CD8+ T cells

BackgroundRecent advances in single-cell technologies and an improved understanding of tumor antigens have empowered researchers to investigate tumor antigen-specific CD8+ T cells at the single-cell level. Peptide-MHC I tetramers are often utilized to enrich antigen-specific CD8+ T cells, which however, introduces the undesired risk of altering their clonal distribution or their transcriptional state. This study addresses the feasibility of utilizing tetramers to enrich antigen-specific CD8+ T cells for single-cell analysis. MethodsHLA-A*02:01-restricted human cytomegalovirus (CMV) pp65 peptide-specific CD8+ T cells were used as a model for analyzing antigen-specific CD8+ T cells. Single-cell RNA sequencing and TCR sequencing were performed to compare the frequency and gene expression profile of pp65-specific TCR clones between tetramer-sorted, unstimulated- and tetramer-stimulated total CD8+ T cells. ResultsThe relative frequency of pp65-specific TCR clones and their transcriptional profile remained largely unchanged following tetramer-based sorting. In contrast, tetramer-mediated stimulation of CD8+ T cells resulted in significant gene expression changes in pp65-specific CD8+ T cells. An Antigen-Specific Response (ASR) gene signature was derived from tetramer-stimulated pp65-specific CD8+ T cells. The ASR signature had a predictive value and was significantly associated with progression free survival in lung cancer patients treated with anti-PD-L1, anti-VEGF, chemotherapy combination (NCT02366143). The predictive power of the ASR signature was independent of the conventional CD8 effector signature. ConclusionsOur findings validate the approach of enriching antigen-specific CD8+ T cells through tetramer-aided Fluorescence-Activated Cell Sorting (FACS) sorting for single-cell analysis and also identifies an ASR gene signature that has value in predicting response to cancer immunotherapy.

Engineering Human MAIT Cells with Chimeric Antigen Receptors for Cancer Immunotherapy.

Engineering immune cells with chimeric Ag receptors (CARs) is a promising technology in cancer immunotherapy. Besides classical cytotoxic CD8+ T cells, innate cell types such as NK cells have also been used to generate CAR-T or CAR-NK cells. In this study, we devised an approach to program a nonclassical cytotoxic T cell subset called mucosal-associated invariant T (MAIT) cells into effective CAR-T cells against B cell lymphoma and breast cancer cells. Accordingly, we expressed anti-CD19 and anti-Her2 CARs in activated primary human MAIT cells and CD8+ T cells, expanded them in vitro, and compared their cytotoxicity against tumor cell targets. We show upon activation through CARs that CAR-MAIT cells exhibit high levels of cytotoxicity toward target cells, comparable to CD8+ CAR-T cells, but interestingly expressed lower levels of IFN-γ than conventional CAR CD8+ T cells. Additionally, in the presence of vitamin B2 metabolite 5-ARU (5-amino-4-d-ribitylaminouracil dihydrochloride), which is a conserved compound that activates MAIT cells through MHC class I-related (MR1) protein, MAIT cells killed MR1-expressing target breast cancer and B cell lymphoma cell lines in a dose-dependent manner. Thus, MAIT cells can be genetically edited as CAR-T cells or mobilized and expanded by MR1 ligands as an off-the-shelf novel approach to cell-based cancer immunotherapy strategies while being comparable to conventional methods in effectivity.

Prognostic value of FOXP3+ regulatory T cells for patients with locally advanced oropharyngeal squamous cell carcinoma.

BackgroundOropharyngeal squamous cell carcinoma (OPSCC) is the most common neoplasm originating at the base of the tongue or in the tonsils or soft palate. In this study, we investigated the prognostic value of FOXP3+ regulatory T cells in OPSCC.MethodsTumor tissues of patients with locally advanced OPSCC were analyzed using quantitative multiplex immunohistochemistry. Staining of CD8+ T cells, conventional CD4+FOXP3- T cells (Tconv cells), CD4+FOXP3+ regulatory T cells (Treg cells), CD20+ B cells, and CD68+ macrophages was performed, and cell density was evaluated in both the tumor and its stroma.ResultsAmong the 71 patients included in this study, males constituted 93.0% of the cohort, and the median age was 59 years (range: 42–80 years). A total of 56 patients (78.9%) had a smoking history, and 53 (74.6%) patients were positive for human papillomavirus (HPV). The most frequent site of OPSCC was the tonsils (70.4%), followed by the base of the tongue (25.4%). The proportion of Treg cells was lower in the tumors of patients with HPV than in those of patients without HPV. Patients with OPSCC whose tumor Treg cell levels were above the median had longer relapse-free survival (RFS) periods than those with tumor Treg cell levels below the median (HR, 0.12; 95% CI, 0.03–0.46; p = 0.02). Our multivariate analysis identified high Treg levels (HR, 0.13; 95% CI, 0.02–1.00; p = 0.05) as an RFS factor that predicted a good prognosis.ConclusionsOur results demonstrated that high Treg cell density in locally advanced OPSCC tumors was correlated with longer RFS.

Functional Analysis of Foxp3 and Its Mutants by Retroviral Transduction of Murine Primary CD4+ T Cells.

The transcription factor Foxp3/FOXP3 orchestrates regulatory T (Treg) cell development and function by interacting with numerous target genes and partner proteins. Functional analysis of naturally occurring or engineered Foxp3/FOXP3 mutations has provided important insights into how the complex Foxp3/FOXP3-centered molecular network operates. Here, we describe detailed protocols for retroviral transduction of murine primary conventional CD4+ T cells to determine the impacts of Foxp3 mutations on the Treg-cell-like phenotype and function conferred by Foxp3.

Adoptive Transfer and Bone Marrow Chimera Models to Analyze Treg Function and Differentiation.

Cellular adoptive transfer and mixed bone marrow chimera are cornerstone experimental tools for immuno-biology. Here we describe protocols for adoptive transfer and bone marrow chimera to address the effect of a specific mutation on T regulatory cell (Treg) function and differentiation, respectively. Treg function can be quantitatively measured by analyzing the expansion of conventional CD4 T cells and their differentiation into helper cells. The quantitative measure of Treg differentiation is addressed by analyzing the number and phenotype of Foxp3-expressing cells. The use of congenic markers is instrumental for these approaches.

Distepharinamide, a novel dimeric proaporphine alkaloid from Diploclisia glaucescens, inhibits the differentiation and proliferative expansion of CD4+Foxp3+ regulatory T cells

BackgroundCD4+Foxp3+ regulatory T cells (Tregs) represent the primary cellular mechanism of tumor immune evasion. Elimination of Treg activity by the pharmacological agent may enhance anti‐tumor immune responses. However, Treg-eliminating agents, especially those with small molecules, are rarely reported. PurposeTo identify small molecule inhibitors of Treg cells from natural products. MethodsCompounds from Diploclisia glaucescens were isolated by column chromatography, and structures were identified by spectroscopic evidence and quantum calculations. The tet-On system for Foxp3-GFP expression in Jurkat T cells was generated to screen Treg inhibitors based on Foxp3 expression. The effect of the compound on TNF-induced proliferative expansion of naturally occurring Tregs (nTregs) and TGF-β-induced generation of Tregs (iTregs) from naive CD4+ Tcells was further examined. ResultsA novel dimeric proaporphine alkaloid, designated as distepharinamide (DSA) with a symmetric structure isolated from the stems of D. glaucescens, restrained the doxycycline (Doxy)-induced Foxp3-tGFP expression, decreased the half-life of Foxp3 mRNA as well as reduced the mRNA levels of chemokine receptors (CCR4, CCR8 and CCR10) in Jurkat T cells with inducible Foxp3-tGFP expression. In lymphocytes or purified Tregs from wild-type C57BL/6 mice or from C57BL/6-Tg(Foxp3-DTR/EGFP)23.2Spar/Mmjax mice, DSA markedly inhibited TNF-induced proliferative expansion of Tregs present in the unfractionated CD4+ T cells, accompanied by the down-regulation of TNFR2, CD25 and CTLA4 expression on Tregs. Furthermore, DSA potently inhibited TGF-β-induced differentiation of Foxp3-expressing iTregs. Importantly, the expression of Foxp3 mRNA by both nTregs and iTregs was decreased by DSA treatment. Nevertheless, DSA at the same concentrations did not inhibit the proliferation of conventional CD4+ and CD8+ T cells stimulated by anti-CD3/CD28 antibodies. ConclusionDSA, a novel dimeric proaporphine alkaloid, potently inhibited the expansion of nTregs and generation of iTregs. Therefore, DSA or its analogs may merit further investigation as novel immunotherapeutic agents.