Subset Of CD4 Research Articles

Increased interleukin-9 and Th9 cells in patients with refractory Graves' disease and interleukin-9 polymorphisms are associated with autoimmune thyroid diseases.

Autoimmune thyroid diseases (AITDs) are prevalent disorders, primarily encompassing Graves' disease (GD) and Hashimoto's thyroiditis (HT). Despite their common occurrence, the etiology of AITDs remains elusive. Th9 cells, a new subset of CD4+T cells with immunomodulatory properties, have been linked to the development of various autoimmune diseases. However, research on the role of Th9 cells in AITDs is limited. We investigated the expression of Th9 cells,their functional cytokine IL-9, and transcription factor IRF4 in peripheral blood mononuclear cells (PBMCs) and plasma of AITD patients and healthy controls. Additionally, we explored the genetic association between four loci polymorphisms (rs31564, rs2069879, rs1859430, and rs2069868) of the IL-9 gene and AITDs. We reported, for the first time, that refractory GD patients exhibited elevated mRNA levels of IL-9 and IRF4 in PBMCs, increased IL-9 protein levels in plasma, and a higher proportion of Th9 cells in peripheral blood when compared to normal controls. Furthermore, human recombinant IL-9 protein was found to enhance IFN-g secretion in PBMCs from both GD patients and normal controls. At the genetic association level, after adjusting for age and sex, the rs2069879 polymorphism exhibited a significant association with AITDs under an additive model (P<0.001, OR= 0.05, 95% CI=0.03-0.08). Our results reveal that Th9 cells may exert a pivotal role in the pathogenesis and progression of refractory GD and HT, and IL-9 holds promise as a novel therapeutic target for the management of AITDs.

Abstract 4991: Radiotherapy-induced immunological and therapeutic response depends on stem-like TCF-1+PD-1+CD8+ T-cells

Abstract Radiotherapy is a frequently used treatment for cancer. Recent data, including ours, showed that radiotherapy promotes an inflammatory response in the tumor that supports tumor-specific immunity, and in fact, the efficacy of radiotherapy in pre-clinical models depends on CD8+ T-cells. According to our preliminary data, the response to radiotherapy is independent of de novo recruited CD8+ T-cells, suggesting that radiation drives differentiation and proliferation of preexisting intratumoral CD8+ T-cells. Intratumoral CD8+ T-cells are heterogeneous concerning phenotype and function. Under conditions of chronic stimulation by antigen, a subset of CD8+ T-cells expressing PD-1 and the transcription factor TCF-1 was described. These so-called stem-like CD8+ T-cells, which give rise to effector cells, were shown to be essential for the clinical response to PD-1 blockade. How radiotherapy influences the different CD8+ T-cell subsets in the tumor microenvironment has not been comprehensively investigated. We propose that the efficacy of radiotherapy depends on the presence of stem-like CD8+ T-cells, presumably by giving rise to effector cells. To address this, we selectively depleted TCF-1+ cells from tumor-bearing mice immediately before radiotherapy. Subsequently, we correlated tumor regression with the immunological response using single-cell analyses of intratumoral CD8+ T-cells. In the absence of TCF-1+ cells, we found a reduced efficacy and reduced number of intratumoral effector cells after radiotherapy. We currently analyze our single-cell RNA-sequencing data and expect to discover novel radiotherapy-induced pathways in CD8+ T-cells that contribute to therapeutic efficacy. Citation Format: Hakan Köksal, Michael Herbst, Nicola Marti, Maries van den Broek. Radiotherapy-induced immunological and therapeutic response depends on stem-like TCF-1+PD-1+CD8+ T-cells [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 4991.

Abstract 18: The polyamine-hypusine axis is a promising avenue to improve T-cell metabolism and persistence for adoptive cell therapy

Abstract There are significant barriers to the application of Adoptive Cell Therapy (ACT) to many cancer types. One of these barriers is a result of poor metabolic fitness of T cells, leading to poor proliferation, persistence, and dampened anti-tumor function. Leveraging T-cell metabolism to improve their function is becoming increasingly important to make significant strides in immunotherapy. Stat5a has been shown to be a master regulator of energy and metabolism in T cells, highlighting it as a promising avenue to improve T-cell metabolism for ACT. We have established that the expression of Constitutively Active Signal Transducer and Activator of Transcription 5A (CASTAT5) on tumor-specific CD4+ T cells improves their persistence, polyfunctionality, and anti-tumor effects in a mouse model. This study further examines Stat5a and its downstream metabolic targets in the context of ACT. We carried out scRNAseq analysis and found that Stat5a directly interacts with genes involved in polyamine metabolism. More specifically, Stat5a activation increases the expression of ornithine decarboxylase (Odc1), spermidine synthase (Srm), deoxyhypusine synthase (Dhps), and deoxyhypusine hydroxylase (Dohh) in a subset of CD4+ T cells expressing CASTAT5. Cleavage Under Targets and Tagmentation (Cut&amp;Tag) analysis of histone H3K27ac also demonstrated increased H3K27ac at promoter and enhancer regions of polyamine pathway genes in CASTAT5 compared to control CD4+ T cells. To further examine the function of Stat5a, the murine pro-B cell line, Ba/F3, is being used as a model to study Stat5a-mediated signaling. We have found that upon withdrawal of IL-3 from Ba/F3 cells, Stat5a activation is significantly downregulated. Our scRNA-seq-based metabolic flux estimation showed a significant reduction in metabolic fluxes and related genes, including polyamines, following IL-3 withdrawal. Correspondingly, Stat5a ChIP-Seq and Cut&amp;Tag analyses of H3K27ac revealed Stat5a binding to active enhancer regions in the presence of IL-3 that are lost following its withdrawal. Untargeted metabolomics confirmed the marked Stat5a-mediated reduction in metabolite abundance after IL-3 deprivation, in a time-dependent manner. The polyamine-hypusine axis revealed in our studies is a promising avenue to improve T-cell function. This axis has been shown to regulate T-cell function, and potentially modulate mitochondrial respiration and T-cell activation. Hence, our studies have identified polyamine supplementation and gene modulation as promising strategies to improve the metabolism of Adoptive T cells in a Stat5a-mediated manner. Further studies will evaluate the effectiveness of this avenue in improving T-cell function, improving their efficacy for ACT, and improving patient outcomes. Citation Format: Mercy Kehinde-Ige, Ogacheko Okoko, Gang Zhou, Huidong Shi. The polyamine-hypusine axis is a promising avenue to improve T-cell metabolism and persistence for adoptive cell therapy [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 18.

Abstract 6424: A patient centric full spectrum flow cytometry assay for comprehensive phenotyping of CAR T cells in clinical trials

Abstract Purpose While there is a strong desire to fully understand next generation CAR-T product characteristics in clinical trials, limited amounts of blood specimens are available as the enrolled patients are very sick and lymphodepleted. To address this high unmet need, we developed a single tube 20-color full spectrum flow cytometry assay for an in-depth characterization of CAR-T cells that support fulfilment of five key clinical trial endpoints, namely, manufacturing success (fitness and memory state), pharmacokinetics, response or resistance (ICIs), efficacy (gross tumor burden) and safety (B cell regeneration) for use across a broad range of CAR-T therapies and clinical indications. Study Design: The single tube 20-parameter full spectrum flow cytometry (FC) assay was designed to include CD45, HLA-DR, CD3, CD4, CD8, CD56 as backbone markers, CD45RA, CCR7, CD95, CD25 and CD127 antibodies allowed for the identification of all major subsets of CD4 and CD8 T cells. CXCR3, CD38, PD1, LAG3, and TIM3 were used for identification of activation/exhaustion phenotypes, while CD20 and HLA-DR were useful for identification of B cells and monocytes respectively. Additionally, we left two parameters (PE and AF647) open for customized CAR-T detection reagents or other customized markers. This assay was developed using Cytek® Northern Lights™ and frozen PBMCs. Results This robust 20-color full spectrum FC assay allowed a thorough analysis of the major T cell populations cells. FMO (Fluorescence-Minus-One) control studies confirmed the specificity of the antibodies and of the panel in identifying Naïve, EMRA, CM, EM, Th17, T stem cell memory and regulatory T cells with minimum background on all parameters. This method showed to be highly reproducible and can be adapted for use with many CAR-T detection reagents. Conclusion In summary, we developed a robust 20-parameter multiparameter full spectrum flow cytometry assay focused on different populations of T cells in a single tube. The inclusion of drop ins for prospective CAR-T cell detection reagents allows for easy integration of future advances in CAR-T cell research. This universal panel shows great potential for usage across CAR-T clinical trials, as it serves as a valuable tool understand product fitness, PK, biomarker monitoring and disease burden simultaneously. Citation Format: Keegan Vaughan, Selvam Muthusamy, Coral Zhao, Austin Carbajal, Charlie Fu, Naveen Dakappagari, Zeni Alfonso. A patient centric full spectrum flow cytometry assay for comprehensive phenotyping of CAR T cells in clinical trials [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 6424.

Abstract 6738: Overlapping and distinct mechanisms of effective neoantigen cancer vaccines and immune checkpoint therapy

Abstract The goal of cancer vaccines and immune checkpoint therapy (ICT) is to expand and sustain T cells with enhanced anti-tumor capabilities. Here, we asked whether these distinct immunotherapies utilize similar cellular and functional mechanisms. We used multiple approaches to compare effective therapeutic mutant neoantigen (NeoAg) cancer vaccines with αPD-1, αCTLA-4, or αPD-1 and αCTLA-4 ICT in preclinical BrafV600EPten−/−Cdkn2a−/− melanoma models. Synthetic long peptide (SLP) MHC-I NeoAg vaccines induced a more than 3-fold expansion of proliferating intratumoral NeoAg-specific CD8 T cells that were functional despite high expression of PD-1 and TIM-3. NeoAg vaccines required not only CD8, but also CD4 T cells for efficacy. αCTLA-4 and/or αPD-1 ICT also increased the frequency and effector capabilities of CD8 T cells but to much less of an extent than NeoAg vaccines. These included NeoAg-specific CD8 T cells that displayed reduced expression of PD-1 and TIM-3. αCTLA-4 treated tumors displayed robust induction of ICOS+ Th1-like CD4 T cells expressing Bhlhe40 that resembled Th1-like CD4 T cells described in αCTLA-4-treated patients and when αCTLA-4 was combined with αPD-1, a small subset of Th2-like CD4 T cells was observed. Remarkably, we noted divergent effects on certain subsets of intratumoral macrophage populations induced by NeoAg vaccines as compared to ICT. Although effective NeoAg vaccines expanded M1-like iNOS+ macrophages with ICT also following this same pattern, NeoAg vaccines expanded rather than suppressed (as observed with ICT) distinct subpopulations of M2-like CX3CR1+ CD206+ macrophages. CODEX spatial imaging using a 46-marker panel revealed tumors from mice treated with NeoAg vaccines displayed concentrations of CD8 T cells expressing functional markers and the proliferation marker Ki67, with mice treated with αCTLA-4 or αPD-1 displaying both Ki67+ conventional CD4 and CD8 T cells within interior regions of tumor. Interestingly, these regions contained prominent blood vessels, along with MHC-II+ dendritic cells and iNOS+ macrophages. Our work shows that NeoAg vaccines lead to distinct, as well as overlapping alterations when compared to ICT. These distinct alterations provided a rationale for combination therapies that we validated in both our melanoma model, as well as the MC38 tumor model, where delayed treatment with NeoAg vaccines combined with αCTLA-4 or αPD-1 yielded anti-tumor immunity that was superior to combination αPD-1 and αCTLA-4. These findings suggest that NeoAg vaccines combined with single-agent ICT may be an effective therapy with potentially less toxicity than αPD-1/αCTLA-4 combination ICT. Further, we hypothesize that immunotherapies that deplete CX3CR1+ CD206+ macrophages may synergize with NeoAg cancer vaccines, which we are currently assessing. Citation Format: Sunita Keshari, Alexander S. Shavkunov, Qi Miao, Akata Saha, Charmelle D. Williams, Josué E. Pineda, Kenneth Hu, Kristen E. Pauken, Ken Chen, Matthew M. Gubin. Overlapping and distinct mechanisms of effective neoantigen cancer vaccines and immune checkpoint therapy [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 6738.

Abstract 6537: A novel CCR4 antagonist induces potent anti-tumor response through inhibition of Treg migration into the tumor microenvironment

Abstract Regulatory T cells (Tregs), an immunosuppressive subset of CD4+ T cells, play a vital role in maintaining immune homeostasis by regulating response to self antigens and maintaining tolerance. On the contrary, Tregs infiltrating the tumor microenvironment (TME) impede effective anti-tumor immunity by inhibiting tumor-specific T cell responses and generating a suppressive TME, thus promoting tumor progression. Clinically, Tregs have been proven to play a significant role in resistance to immune checkpoint inhibitors (ICIs) leading to lower response rates. Hence, targeting Tregs would lead to activation of tumor-specific effector T cells and improved long-term anti-tumor immune responses with ICI treatment. CCR4, a chemokine receptor highly expressed on Tregs, spearhead the migration of Tregs and their accumulation in the TME, by responding to the chemokines CCL22 and CCL17 secreted by the inflamed cells in the tumor. CCR4 expression is associated with poor prognosis in various solid tumors. This warrants the therapeutic targeting of CCR4-expressing Tregs to enable effective and durable anti-tumor immune responses. We sought to discover and develop novel small molecule antagonists of CCR4 for use in cancer therapy. Multiple series of CCR4 antagonists were identified by iterative medicinal chemistry efforts and SAR based approach. These compounds were optimized towards attaining required potency, acceptable physicochemical properties, high selectivity and desirable pharmacokinetic profile to achieve anti-tumor activity. Aurigene’s CCR4 antagonists exhibited potent inhibition of CCR4 activity in the reporter-based β-arrestin recruitment assay. This translated well into potent inhibition of CCL22-mediated chemotaxis of CCR4-expressing Tregs in an in vitro cell migration assay. Furthemore, the lead compounds showed desirable drug-like properties and excellent pharmacokinetic exposure in rodents. In an ongoing study in a syngeneic mice tumor model, one of the lead compounds is being evaluated for impact on Treg infiltration, CD8:Treg ratio in the tumor and efficacy. In summary, we have identified novel CCR4 antagonists with desirable drug-like properties that are being evaluated further to identify a clinical candidate. Citation Format: Rashmi Nair, Chandrasekhar Abbineni, Krishna Chaitanya Talluri, Amit A Dhudashiya, Aravind AB, NaveenR Kumar, Ramya R, D Balasubramanyam, Pathur Obanna, Rabin Madhaiyan, DS Samiulla, Girish Daginakatte, Kishore Narayanan, Kavitha Nellore, Shekar Chelur, Susanta Samajdar, Murali Ramachandra. A novel CCR4 antagonist induces potent anti-tumor response through inhibition of Treg migration into the tumor microenvironment [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 6537.

Abstract 2716: The O-glycan epitope targeting anti-human carcinoma monoclonal antibody (mAb) NEO-201 can also target human regulatory T cells (Tregs)

Abstract Introduction:NEO-201 is a humanized IgG1 mAb that targets Core 1 and/or extended Core 1 O-glycans expressed by several human solid and blood tumors, as well as neutrophils, but it does not bind to most normal tissues and human immune cell subsets (B cells, CD4+ T cells, CD8+ T cells, NK cells, monocytes). Functional analysis revealed that NEO-201 mediates the killing of its target cells through ADCC and CDC. Previous study has shown that approximately 4.6% of CD4+ T cells express NEO-201 target antigen and that those CD4+ T cells have Tregs phenotype. In addition, in a recent published phase 1 study we observed that NEO-201 binds to CD4+/CD25+/, CD127−/, Foxp3+/, CD15s+ cells from peripheral blood mononuclear cells (PBMCs) from cancer patients with solid tumors. The purpose of this study was to further characterize the phenotype of the specific subset of CD4+ T cells expressing the NEO-201 target antigen. Experimental Design: Human PBMCs were obtained from 7 healthy donors (HD) collected at Osaka University and 6 cancer patients from our ongoing phase II clinical trial (Clinical Trial NCT03476681) evaluating the efficiency of the combination of NEO-201 with pembrolizumab in adults with solid tumors resistant to checkpoint inhibitors. Phenotypic analysis was performed by flow cytometry using NEO-201 and antibodies CD3, CD4, CD25, CD45RA, FoxP3, and CD15s. The same gating strategy was applied for both normal donors and patients to evaluate which fraction of CD4+ T cells is recognized by NEO-201. Fraction (Fr) 1 is naïve Treg, Fr ll is effector Treg, Fr lll is non-Treg, Fr lV is effector CD4 T cells and Fr V is naïve CD4 T cells. Results: Flow cytometry analysis of PBMCs revealed that NEO-201 recognizes naïve Tregs (nTregs: CD3+/CD4+/CD45RA+/Foxp3low cells) while it does not bind to effector Tregs (eTregs: CD3+/CD4+/CD45RA−/Foxp3high cells) in both HD and cancer patients. The % of nTregs in cancer patients was higher than HD. Preliminary results from the ongoing Phase II clinical trial showed that subjects with durable SD had a reduction of circulating nTregs after treatment with NEO-201 compared to baseline levels. Conclusions: NEO-201 binds to human Tregs with significantly higher % of binding to Fr l and a greater % of Tregs expressing NEO-201 target antigen in cancer patients compared to HD. It is conceivable that naïve Tregs in cancer patients express high levels of Core 1 O-glycans. Furthermore, when subjected to TCR stimulation, naïve Tregs undergo proliferation and differentiate into eTregs. eTregs can then infiltrate into tumor microenvironment (TME). These data suggest that depletion of circulating nTregs in cancer patients after NEO-201 treatment may prevent the differentiation of nTregs into eTregs and their accumulation in the TME. This study suggests the potential use of NEO-201 to reduce the Treg-mediated suppression of anticancer immunity. Citation Format: Atsushi Tanaka, Massimo Fantini, Philip M. Arlen, Christina M. Annunziata, Kwong Y. Tsang, Shimon Sakaguchi. The O-glycan epitope targeting anti-human carcinoma monoclonal antibody (mAb) NEO-201 can also target human regulatory T cells (Tregs) [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 2716.

Identification of CREB5 as a prognostic and immunotherapeutic biomarker in glioma through multi-omics pan-cancer analysis

BackgroundThe functional relevance of cyclic adenosine monophosphate (cAMP)-response element-binding protein 5 (CREB5) in cancers remains elusive, despite its significance as a member of the CREB family. The current research aims to explore the role of CREB5 in multiple cancers. MethodsPan-cancer analysis was performed to explore the expression patterns, prognostic value, mutational landscape as well as single-cell omic, immunologic, and drug sensitivity profiles of CREB5. Furthermore, we incorporated five distinct machine learning algorithms and determined that the least absolute shrinkage and selection operator-COX (LASSO-COX) algorithm, which exhibited the highest C index, was the optimal selection. Subsequently, we constructed a prognostic model centered around CREB5-associated genes. To elucidate the biological function of CREB5 in glioma cells, several assays including cell counting kit-8 (CCK-8), wound healing, transwell, flow cytometric were performed. ResultsCREB5 was overexpressed in pan-cancer and was linked to unfavorable prognosis, particularly in glioma. Furthermore, genetic alterations were determined in various types of cancer, and modifications in the CREB5 gene were linked to the prognosis. The single-cell omics and enrichment analyses showed that CREB5 was predominantly expressed in malignant glioma cells and was critically involved in the regulation of various oncogenic processes. Elevated levels of CREB5 were strongly linked with the infiltration of cancer-associated fibroblasts and the Th1 subset of CD4+ T cells. The validated CREB5-associated prognostic model reliably predicted the prognosis and drug response of glioma patients. The in vitro experiments showed that CREB5 promoted glioma cell proliferation, invasion, migration, and gap phase 2/mitotic (G2/M) phase arrest and recruited M2 macrophages into glioma cells. ConclusionCREB5 has the potential to act as an oncogene and a biological marker in multiple cancers, particularly glioma.

Th2-skewed peripheral T-helper cells drive B-cells in allergic bronchopulmonary aspergillosis.

Patients with allergic bronchopulmonary aspergillosis (ABPA) suffer from repeated exacerbations. The involvement of T-cell subsets remains unclear. We enrolled ABPA patients, asthma patients and healthy controls. T-helper type 1 (Th1), 2 (Th2) and 17 (Th17) cells, regulatory T-cells (Treg) and interleukin (IL)-21+CD4+T-cells in total or sorted subsets of peripheral blood mononuclear cells and ABPA bronchoalveolar lavage fluid (BALF) were analysed using flow cytometry. RNA sequencing of subsets of CD4+T-cells was done in exacerbated ABPA patients and healthy controls. Antibodies of T-/B-cell co-cultures in vitro were measured. ABPA patients had increased Th2 cells, similar numbers of Treg cells and decreased circulating Th1 and Th17 cells. IL-5+IL-13+IL-21+CD4+T-cells were rarely detected in healthy controls, but significantly elevated in the blood of ABPA patients, especially the exacerbated ones. We found that IL-5+IL-13+IL-21+CD4+T-cells were mainly peripheral T-helper (Tph) cells (PD-1+CXCR5-), which also presented in the BALF of ABPA patients. The proportions of circulating Tph cells were similar among ABPA patients, asthma patients and healthy controls, while IL-5+IL-13+IL-21+ Tph cells significantly increased in ABPA patients. Transcriptome data showed that Tph cells of ABPA patients were Th2-skewed and exhibited signatures of follicular T-helper cells. When co-cultured in vitro, Tph cells of ABPA patients induced the differentiation of autologous B-cells into plasmablasts and significantly enhanced the production of IgE. We identified a distinctly elevated population of circulating Th2-skewed Tph cells that induced the production of IgE in ABPA patients. It may be a biomarker and therapeutic target for ABPA.

Investigating Vα7.2+/CD161- T Cell and MAIT Cell Profiles Using Flow Cytometry in Healthy Subjects and Subjects with Atopic Dermatitis.

This study investigates the roles of mucosal-associated invariant T (MAIT) cells and Vα7.2+/CD161- T cells in skin diseases, focusing on atopic dermatitis. MAIT cells, crucial for bridging innate and adaptive immunity, were analyzed alongside Vα7.2+/CD161- T cells in peripheral blood samples from 14 atopic dermatitis patients and 10 healthy controls. Flow cytometry and machine learning algorithms were employed for a comprehensive analysis. The results indicate a significant decrease in MAIT cells and CD69 subsets in atopic dermatitis, coupled with elevated CD38 and polyfunctional MAIT cells producing TNFα and Granzyme B (TNFα+/GzB+). Vα7.2+/CD161- T cells in atopic dermatitis exhibited a decrease in CD8 and IFNγ-producing subsets but an increase in CD38 activated and IL-22-producing subsets. These results highlight the distinctive features of MAIT cells and Vα7.2+/CD161- T cells and their different roles in the pathogenesis of atopic dermatitis and provide insights into their potential roles in immune-mediated skin diseases.

Integration Analysis of Single-Cell Multi-Omics Reveals Prostate Cancer Heterogeneity.

Prostate cancer (PCa) is an extensive heterogeneous disease with a complex cellular ecosystem in the tumor microenvironment (TME). However, the manner in which heterogeneity is shaped by tumors and stromal cells, or vice versa, remains poorly understood. In this study, single-cell RNA sequencing, spatial transcriptomics, and bulk ATAC-sequence are integrated from a series of patients with PCa and healthy controls. A stemness subset of club cells marked with SOX9highARlow expression is identified, which is markedly enriched after neoadjuvant androgen-deprivation therapy (ADT). Furthermore, a subset of CD8+CXCR6+ T cells that function as effector T cells is markedly reduced in patients with malignant PCa. For spatial transcriptome analysis, machine learning and computational intelligence are comprehensively utilized to identify the cellular diversity of prostate cancer cells and cell-cell communication in situ. Macrophage and neutrophil state transitions along the trajectory of cancer progression are also examined. Finally, the immunosuppressive microenvironment in advanced PCa is found to be associated with the infiltration of regulatory T cells (Tregs), potentially induced by an FAP+ fibroblast subset. In summary, the cellular heterogeneity is delineated in the stage-specific PCa microenvironment at single-cell resolution, uncovering their reciprocal crosstalk with disease progression, which can be helpful in promoting PCa diagnosis and therapy.

SAP-expressing T peripheral helper cells identify systemic lupus erythematosus patients with lupus nephritis.

T follicular (TFH) and peripheral helper (TPH) cells have been increasingly recognized as a pathogenic subset of CD4 T cells in systemic lupus erythematosus (SLE). The SLAM Associated Protein (SAP) regulates TFH and TPH function by binding to the co-stimulatory signaling lymphocyte activation molecule family (SLAMF) receptors that mediate T cell - B cell interactions. SAP and SLAMF are critical for TPH-dependent B cell maturation into autoantibody-producing plasma cells that characterize SLE pathogenesis. We hypothesized that SAP-expressing TPH cells are involved in the pathogenesis of lupus nephritis (LN). Peripheral blood mononuclear cells (PBMC) were isolated using density gradient separation from whole blood. Cells were stained for cell surface markers, followed by permeabilization and staining of intracellular SAP for spectral flow cytometry analysis. We also analyzed SAP expression from renal infiltrating LN T cells using the available single-cell RNA sequencing (scRNA seq) Accelerated Medicines Partnership (AMP) SLE dataset. PBMC from 30 patients with SLE (34 ± 10 years old, 83% female), including 10 patients with LN, were analyzed. We found an increase in total SAP-positive CD4 and CD8 T cells in SLE compared with controls (55.5 ± 2.6 vs. 41.3 ± 3.4, p=0.007, and 52.5 ± 3.0 vs. 39.2 ± 2.8, p=0.007 respectively). In CD4 T cells, the highest SAP expression was in the TPH subset. The frequency of SAP+TPH in circulation correlated with disease activity; SLE patients with renal disease had higher levels of circulating SAP+TPH that remained significant after adjusting for age, sex, race, low complements, and elevated anti-dsDNA (p=0.014). scRNA-seq data of renal infiltrating T cells in LN identified SAP expression to localize to the TFH-like CD4 cluster and GZMK+ CD8 cluster. Increased SAP expression in LN was associated with the differential expression of SLAMF3 and SLAMF7 and granzyme K and EOMES. The existence of two predominant SAP-expressing subsets, the TFH-like CD4 T cells, and GZMK+ effector CD8 T cells, was verified using scRNA-seq data from a human transcriptomic atlas of fifteen major organs. The expansion of SAP-expressing T helper cells was associated with LN in our cohort and verified using scRNA-seq data of renal infiltrating T cells. Improved SLAM and SAP signaling understanding can identify new therapeutic targets in LN.

TIGIT acts as an immune checkpoint upon inhibition of PD1 signaling in autoimmune diabetes.

Chronic activation of self-reactive T cells with beta cell antigens results in the upregulation of immune checkpoint molecules that keep self-reactive T cells under control and delay beta cell destruction in autoimmune diabetes. Inhibiting PD1/PD-L1 signaling results in autoimmune diabetes in mice and humans with pre-existing autoimmunity against beta cells. However, it is not known if other immune checkpoint molecules, such as TIGIT, can also negatively regulate self-reactive T cells. TIGIT negatively regulates the CD226 costimulatory pathway, T-cell receptor (TCR) signaling, and hence T-cell function. The phenotype and function of TIGIT expressing islet infiltrating T cells was studied in non-obese diabetic (NOD) mice using flow cytometry and single cell RNA sequencing. To determine if TIGIT restrains self-reactive T cells, we used a TIGIT blocking antibody alone or in combination with anti-PDL1 antibody. We show that TIGIT is highly expressed on activated islet infiltrating T cells in NOD mice. We identified a subset of stem-like memory CD8+ T cells expressing multiple immune checkpoints including TIGIT, PD1 and the transcription factor EOMES, which is linked to dysfunctional CD8+ T cells. A known ligand for TIGIT, CD155 was expressed on beta cells and islet infiltrating dendritic cells. However, despite TIGIT and its ligand being expressed, islet infiltrating PD1+TIGIT+CD8+ T cells were functional. Inhibiting TIGIT in NOD mice did not result in exacerbated autoimmune diabetes while inhibiting PD1-PDL1 resulted in rapid autoimmune diabetes, indicating that TIGIT does not restrain islet infiltrating T cells in autoimmune diabetes to the same degree as PD1. Partial inhibition of PD1-PDL1 in combination with TIGIT inhibition resulted in rapid diabetes in NOD mice. These results suggest that TIGIT and PD1 act in synergy as immune checkpoints when PD1 signaling is partially impaired. Beta cell specific stem-like memory T cells retain their functionality despite expressing multiple immune checkpoints and TIGIT is below PD1 in the hierarchy of immune checkpoints in autoimmune diabetes.

GPR41 and GPR43 regulate CD8+ T cell priming during herpes simplex virus type 1 infection.

Naïve CD8+ T cells need to undergo a complex and coordinated differentiation program to gain the capacity to control virus infections. This not only involves the acquisition of effector functions, but also regulates the development of a subset of effector CD8+ T cells into long-lived and protective memory cells. Microbiota-derived metabolites have recently gained interest for their influence on T cells, but much remains unclear about their role in CD8+ T cell differentiation. In this study, we investigated the role of the G protein-coupled receptors (GPR)41 and GPR43 that can bind microbiota-derived short chain fatty acids (SCFAs) in CD8+ T cell priming following epicutaneous herpes simplex virus type 1 (HSV-1) infection. We found that HSV-specific CD8+ T cells in GPR41/43-deficient mice were impaired in the antigen-elicited production of interferon-gamma (IFN-γ), tumour necrosis factor-alpha (TNF-α), granzyme B and perforin, and failed to differentiate effectively into memory precursors. The defect in controlling HSV-1 at the site of infection could be restored when GPR41 and GPR43 were expressed exclusively by HSV-specific CD8+ T cells. Our findings therefore highlight roles for GPR41 and GPR43 in CD8+ T cell differentiation, emphasising the importance of metabolite sensing in fine-tuning anti-viral CD8+ T cell priming.

Loss of chromosome Y in regulatory T cells

BackgroundMosaic loss of chromosome Y (LOY) in leukocytes is the most prevalent somatic aneuploidy in aging humans. Men with LOY have increased risks of all-cause mortality and the major causes of death, including many forms of cancer. It has been suggested that the association between LOY and disease risk depends on what type of leukocyte is affected with Y loss, with prostate cancer patients showing higher levels of LOY in CD4 + T lymphocytes. In previous studies, Y loss has however been observed at relatively low levels in this cell type. This motivated us to investigate whether specific subsets of CD4 + T lymphocytes are particularly affected by LOY. Publicly available, T lymphocyte enriched, single-cell RNA sequencing datasets from patients with liver, lung or colorectal cancer were used to study how LOY affects different subtypes of T lymphocyte. To validate the observations from the public data, we also generated a single-cell RNA sequencing dataset comprised of 23 PBMC samples and 32 CD4 + T lymphocytes enriched samples.ResultsRegulatory T cells had significantly more LOY than any other studied T lymphocytes subtype. Furthermore, LOY in regulatory T cells increased the ratio of regulatory T cells compared with other T lymphocyte subtypes, indicating an effect of Y loss on lymphocyte differentiation. This was supported by developmental trajectory analysis of CD4 + T lymphocytes culminating in the regulatory T cells cluster most heavily affected by LOY. Finally, we identify dysregulation of 465 genes in regulatory T cells with Y loss, many involved in the immunosuppressive functions and development of regulatory T cells.ConclusionsHere, we show that regulatory T cells are particularly affected by Y loss, resulting in an increased fraction of regulatory T cells and dysregulated immune functions. Considering that regulatory T cells plays a critical role in the process of immunosuppression; this enrichment for regulatory T cells with LOY might contribute to the increased risk for cancer observed among men with Y loss in leukocytes.

Exhausted Tumor-infiltrating CD39+CD103+ CD8+ T Cells Unveil Potential for Increased Survival in Human Pancreatic Cancer.

Patients with pancreatic cancer with a high proportion of CD39+CD103+ CD8+ T cells exhibiting a tumor-reactive phenotype have improved survival rates, suggesting their potential utility in selecting candidates for immunotherapy trials.

Low-dose Interleukin-2 Therapy: Fine-tuning Treg in Solid Organ Transplantation?

Regulatory T cells (Treg), a subset of CD4 + T cells, are potent regulators of immune reactions, which have been shown to be a promising therapeutic alternative to toxic immunosuppressive drugs. Data support the utility of Treg in managing immunopathologies, including solid organ transplant rejection, graft-versus-host disease, and autoimmune disorders. Notably, reports suggest that interleukin-2 (IL-2) is critical to survival of Treg, which constitutively express high levels of CD25, that is, the IL-2 receptor α-chain, and are exquisitely sensitive to IL-2, even at very low concentrations in contrast to effector T cells, which only upregulate IL-2 receptor α-chain on activation. This has led to the notion of using low doses of exogenous IL-2 therapeutically to modulate the immune system, specifically Treg numbers and function. Here, we summarize developments of clinical experience with low-dose IL-2 (LD-IL-2) as a therapeutic agent. So far, no clinical data are available to support the therapeutic use of LD-IL-2 therapy in the solid organ transplant setting. For the latter, fine-tuning by biotechnological approaches may be needed because of the narrow therapeutic window and off-target effects of LD-IL-2 therapy and so to realize the therapeutic potential of this molecule.

WESTERN DIET DEPLETES SMALL INTESTINAL INTRAEPITHELIAL LYMPHOCYTES VIA FXR-INTERFERON PATHWAY

Abstract BACKGROUND The prevalence of obesity has continued to increase over the past several decades. Understanding how diet-induced obesity modulates mucosal immunity is of clinical relevance. We previously showed that consumption of a high fat, high sugar “Western” diet (WD) reduces the density and function of small intestinal Paneth cells, a small intestinal epithelial cell type with innate immune function. We hypothesized that obesity could also result in repressed adaptive immunity. METHODS Small intestine samples from non-inflammatory bowel disease (IBD) subjects who underwent resection were used for CD3 immunohistochemistry (IHC), and the CD3+ intraepithelial lymphocytes (IEL) densities were correlated with the patients’ body mass index (BMI). Mice were fed with either standard diet (SD) or WD for various durations, and IELs were isolated for flow cytometry analysis. RESULTS In non-IBD subjects, high BMI correlated with reduced IEL density. We recapitulated this in wild type (WT) mice fed with WD. A 4-week WD consumption was able to deplete IEL (including major subsets CD4+, CD8+, CD8α+8β-CD4+, and γδCD8+ T cells) but not splenic lymphocytes, and the effect was reversible after another 4 weeks of SD washout. ob/ob mice that spontaneously developed obesity while fed with SD did not show IEL depletion, suggesting that the quality, not the quantity of the diet is key to this phenotype. Importantly, WD-associated IEL depletion was not dependent on the presence of gut microbiota, as WD-fed germ-free mice also showed IEL depletion. We previously showed that a key molecular pathway activated by WD in the ileum is the FXR pathway. Phagocyte-specific Fxr conditional knockout (cKO) mice were protected from WD-mediated IEL depletion, suggesting that FXR activation in the phagocytes is critical in mediating this phenotype. Activated FXR signaling stimulated phagocytes to secrete type I interferon (IFN), and inhibition of type I IFN signaling within the phagocytes (type I IFN monoclonal antibody treatment and Stat1 cKO mice) prevented WD-mediated IEL loss. CONCLUSION WD consumption depletes IEL by a FXR-IFN pathway mediated by gut phagocytes. These findings have significant clinical implications in understanding the pathogenesis of gut inflammatory disorders such as IBD.

CD8+ T Cell Biology in Cytokine Storm Syndromes.

Familial forms of hemophagocytic lymphohistiocytosis (HLH) are caused by loss-of-function mutations in genes encoding perforin as well as those required for release of perforin-containing cytotoxic granule constituent. Perforin is expressed by subsets of CD8+ T cells and NK cells, representing lymphocytes that share mechanism of target cell killing yet display distinct modes of target cell recognition. Here, we highlight recent findings concerning the genetics of familial HLH that implicate CD8+ T cells in the pathogenesis of HLH and discuss mechanistic insights from animal models as well as patients that reveal how CD8+ T cells may contribute to or drive disease, at least in part through release of IFN-γ. Intriguingly, CD8+ T cells and NK cells may act differentially in severe hyperinflammatory diseases such as HLH. We also discuss how CD8+ T cells may promote or drive pathology in other cytokine release syndromes (CSS). Moreover, we review the molecular mechanisms underpinning CD8+ T cell-mediated lymphocyte cytotoxicity, key to the development of familial HLH. Together, recent insights to the pathophysiology of CSS in general and HLH in particular are providing promising new therapeutic targets.

Optimization and intra-assay validation of a multiparametric flow cytometric test for monitoring circulating TREGs.

Multiparametric flow cytometry (MFC) represents an essential tool for immune monitoring, and validation of MFC panels is a fundamental prerequisite in routine laboratory settings as well as for translational and clinical research purposes. Regulatory T cells (TREGs) constitute a subset of CD4+ effector T cells that modulate the immune response in numerous settings, including autoimmune disease, allergy, microbial infection, tumor immunity, transplantation, and more. These cells comprise a small fraction of total CD4+ T cells in human peripheral blood and mouse spleen. In oncology, TREG cells are highly relevant, as they are involved in the suppression of the anti-tumor response in many types of cancer, to the extent that the first immune checkpoint inhibitor approved for clinical use in humans was a monoclonal antibody directed against CTLA-4, a molecule functionally associated with TREGs. Due to all these factors, robust assays are mandatory to accurately determine TREG cell frequency and function. Here, we describe the validation of an 8-color flow-cytometry protocol for TREG detection and analysis in a real-world laboratory scenario. The entire process includes the workflow plan and the standard operating procedure resembling each phase, from the panel design to the staining, acquisition, and analysis steps. Validation is planned to be performed in replicates on fresh whole blood samples derived from multiple healthy subjects. The analytical validity of the TREG cell assay is ensured by testing the intra-assay accuracy. The detailed procedure for the entire process is accompanied by important troubleshooting suggestions and other useful tips.