Neural-net-based cell deconvolution from DNA methylation reveals tumor microenvironment associated with cancer prognosis.

DNA methylation is a pivotal epigenetic modification that defines cellular identity. While cell deconvolution utilizing this information is considered useful for clinical practice, current methods for deconvolution are limited in their accuracy and resolution. In this study, we collected DNA methylation data from 945 human samples derived from various tissues and tumor-infiltrating immune cells and trained a neural network model with them. The model, termed MEnet, predicted abundance of cell population together with the detailed immune cell status from bulk DNA methylation data, and showed consistency to those of flow cytometry and histochemistry. MEnet was superior to the existing methods in the accuracy, speed, and detectable cell diversity, and could be applicable for peripheral blood, tumors, cell-free DNA, and formalin-fixed paraffin-embedded sections. Furthermore, by applying MEnet to 72 intrahepatic cholangiocarcinoma samples, we identified immune cell profiles associated with cancer prognosis. We believe that cell deconvolution by MEnet has the potential for use in clinical settings.

Regulatory T Cells for Control of Autoimmunity.

Regulatory T (Treg) cells, which specifically express the master transcription factor FoxP3, are indispensable for the maintenance of immunological self-tolerance and homeostasis. Their functional or numerical anomalies can be causative of autoimmune and other inflammatory diseases. Recent advances in the research of the cellular and molecular basis of how Treg cells develop, exert suppression, and maintain their function have enabled devising various ways for controlling physiological and pathological immune responses by targeting Treg cells. It is now envisaged that Treg cells as a "living drug" are able to achieve antigen-specific immune suppression of various immune responses and reestablish immunological self-tolerance in the clinic.

Single-cell transcriptome landscape of circulating CD4+ T cell populations in autoimmune diseases.

CD4+ T cells are key mediators of various autoimmune diseases; however, their role in disease progression remains unclear due to cellular heterogeneity. Here, we evaluated CD4+ T cell subpopulations using decomposition-based transcriptome characterization and canonical clustering strategies. This approach identified 12 independent gene programs governing whole CD4+ T cell heterogeneity, which can explain the ambiguity of canonical clustering. In addition, we performed a meta-analysis using public single-cell datasets of over 1.8 million peripheral CD4+ T cells from 953 individuals by projecting cells onto the reference and cataloging cell frequency and qualitative alterations of the populations in 20 diseases. The analyses revealed that the 12 transcriptional programs were useful in characterizing each autoimmune disease and predicting its clinical status. Moreover, genetic variants associated with autoimmune diseases showed disease-specific enrichment within the 12 gene programs. The results collectively provide a landscape of single-cell transcriptomes of CD4+ T cell subpopulations involved in autoimmune disease.

Downregulation of TCF7 and LEF1 is a key determinant of tumor-infiltrating regulatory T-cell function.

Forkhead box P3 (Foxp3)-expressing regulatory T (Treg) cells play essential roles in immune homeostasis but also contribute to establish a favorable environment for tumor growth by suppressing anti-tumor immune responses. It is thus necessary to specifically target tumor-infiltrating Treg cells to minimize effects on immune homeostasis in cancer immunotherapy. However, molecular features that distinguish tumor-infiltrating Treg cells from those in secondary lymphoid organs remain unknown. Here we characterize distinct features of tumor-infiltrating Treg cells by global analyses of the transcriptome and chromatin landscape. They exhibited activated phenotypes with enhanced Foxp3-dependent transcriptional regulation, yet being distinct from activated Treg cells in secondary lymphoid organs. Such differences may be attributed to the extensive clonal expansion of tumor-infiltrating Treg cells. Moreover, we found that TCF7 and LEF1 were specifically downregulated in tumor-infiltrating Treg cells both in mice and humans. These factors and Foxp3 co-occupied Treg suppressive function-related gene loci in secondary lymphoid organ Treg cells, whereas the absence of TCF7 and LEF1 accompanied altered gene expression and chromatin status at these gene loci in tumor-infiltrating Treg cells. Functionally, overexpression of TCF7 and LEF1 in Treg cells inhibited the enhancement of Treg suppressive function upon activation. Our results thus show the downregulation of TCF7 and LEF1 as markers of highly suppressive Treg cells in tumors and suggest that their absence controls the augmentation of Treg suppressive function in tumors. These molecules may be potential targets for novel cancer immunotherapy with minimum effects on immune homeostasis.

Identification of the novel FOXP3-dependent Treg cell transcription factor MEOX1 by high-dimensional analysis of human CD4+ T cells.

CD4+ T cells play a central role in the adaptive immune response through their capacity to activate, support and control other immune cells. Although these cells have become the focus of intense research, a comprehensive understanding of the underlying regulatory networks that orchestrate CD4+ T cell function and activation is still incomplete. Here, we analyzed a large transcriptomic dataset consisting of 48 different human CD4+ T cell conditions. By performing reverse network engineering, we identified six common denominators of CD4+ T cell functionality (CREB1, E2F3, AHR, STAT1, NFAT5 and NFATC3). Moreover, we also analyzed condition-specific genes which led us to the identification of the transcription factor MEOX1 in Treg cells. Expression of MEOX1 was comparable to FOXP3 in Treg cells and can be upregulated by IL-2. Epigenetic analyses revealed a permissive epigenetic landscape for MEOX1 solely in Treg cells. Knockdown of MEOX1 in Treg cells revealed a profound impact on downstream gene expression programs and Treg cell suppressive capacity. These findings in the context of CD4+ T cells contribute to a better understanding of the transcriptional networks and biological mechanisms controlling CD4+ T cell functionality, which opens new avenues for future therapeutic strategies.

Treg-based immunotherapy for antigen-specific immune suppression and stable tolerance induction: a perspective.

FoxP3-expressing regulatory T cells (Tregs), whether naturally generated in the immune system or unnaturally induced from conventional T cells (Tconvs) in the laboratory, have much therapeutic value in treating immunological diseases and establishing transplantation tolerance. Natural Tregs (nTregs) can be selectively expanded in vivo by administration of low-dose IL-2 or IL-2 muteins for immune suppression. For adoptive Treg cell therapy, nTregs can be expanded in vitro by strong antigenic stimulation in the presence of IL-2. Synthetic receptors such as CAR can be expressed in nTregs to equip them with a particular target specificity for suppression. In addition, antigen-specific Tconvs can be converted in vitro to functionally stable Treg-like cells by a combination of antigenic stimulation, FoxP3 induction, and establishment of the Treg-type epigenome. This review discusses current and prospective strategies for Treg-based immune suppression and the issues to be resolved for achieving stable antigen-specific immune suppression and tolerance induction in the clinic by targeting Tregs.

Regulatory T cells in autoimmune kidney diseases and transplantation.

Regulatory T (Treg) cells that express the transcription factor forkhead box protein P3 (FOXP3) are naturally present in the immune system and have roles in the maintenance of immunological self-tolerance and immune system and tissue homeostasis. Treg cells suppress T cell activation, expansion and effector functions by various mechanisms, particularly by controlling the functions of antigen-presenting cells. They can also contribute to tissue repair by suppressing inflammation and facilitating tissue regeneration, for example, via the production of growth factors and the promotion of stem cell differentiation and proliferation. Monogenic anomalies of Treg cells and genetic variations of Treg cell functional molecules can cause or predispose patients to the development of autoimmune diseases and other inflammatory disorders, including kidney diseases. Treg cells can potentially be utilized or targeted to treat immunological diseases and establish transplantation tolerance, for example, by expanding natural Treg cells in vivo using IL-2 or small molecules or by expanding them in vitro for adoptive Treg cell therapy. Efforts are also being made to convert antigen-specific conventional T cells into Treg cells and to generate chimeric antigen receptor Treg cells from natural Treg cells for adoptive Treg cell therapies with the aim of achieving antigen-specific immune suppression and tolerance in the clinic.

Nrf2 activation improves experimental rheumatoid arthritis.

Rheumatoid arthritis is a systemic autoimmune disease with pain and functional disorder of joints. Multiple strategies toward treatment of the rheumatoid arthritis are operating, while there are concerns of serious adverse effects of the therapeutic drugs. Here, we show that activation of Nrf2 (Nuclear factor erythroid 2-related factor 2) efficiently improves arthritis of SKG mice, which develop T cell-mediated autoimmune arthritis by zymosan A injection. We found that genetic Nrf2 activation by knockdown of Keap1 (Kelch-like ECH-associated protein 1), a negative regulator of Nrf2, repressed arthritis by inhibiting the expression of pro-inflammatory cytokines and inducing the expression of antioxidant enzymes in SKG mice. In addition, oral administration of CDDO-Im, a representative chemical inducer of Nrf2, had effects of both prevention and treatment toward arthritis of SKG mice in an Nrf2-dependent manner. We also found that Nrf2 activation through myeloid-cell lineage-specific Keap1 disruption did not achieve significant improvement in the arthritis of SKG mice. In contrast, expressions of pro-inflammatory cytokine genes were decreased, and those of antioxidant enzyme genes were increased in fibroblast-like synoviocytes (FLS) isolated from SKG mouse. Our results thus demonstrate that Nrf2 activation exerts marked anti-arthritis effects in the SKG experimental rheumatoid arthritis model mice, supporting the contention that the Nrf2 activation is a new therapeutic strategy for the rheumatoid arthritis.

A genetic method specifically delineates Th1-type Treg cells and their roles in tumor immunity.

Regulatory T (Treg) cells expressing the transcription factor (TF) Foxp3 also express other TFs shared by T helper (Th) subsets under certain conditions. Here, to determine the roles of T-bet-expressing Treg cells, we generate a mouse strain, called VeDTR, in which T-bet/Foxp3 double-positive cells are engineered to be specifically labeled and depleted by a combination of Cre- and Flp-recombinase-dependent gene expression control. Characterization of T-bet+Foxp3+ cells using VeDTR mice reveals high resistance under oxidative stress, which is involved in accumulation of T-bet+Foxp3+ cells in tumor tissues. Moreover, short-term depletion of T-bet+Foxp3+ cells leads to anti-tumor immunity but not autoimmunity, whereas that of whole Treg cells does both. Although ablation of T-bet+Foxp3+ cells during Toxoplasma infection slightly enhances Th1 immune responses, it does not affect the course of the infection. Collectively, the intersectional genetic method reveals the specific roles of T-bet+Foxp3+ cells in suppressing tumor immunity.

Tumor-infiltrating regulatory T cells as targets of cancer immunotherapy.

Regulatory T cells (Tregs) are abundant in tumor tissues, raising a question of whether immunosuppressive tumor-infiltrating Tregs (TI-Tregs) can be selectively depleted or functionally attenuated to evoke effective anti-tumor immune responses by conventional T cells (Tconvs), without perturbing Treg-dependent immune homeostasis in healthy organs and causing autoimmunity. Here, we review current cancer immunotherapy strategies, including immune checkpoint blockade (ICB) antibodies against CTLA-4 and PD-1 and discuss their effects on TI-Tregs. We also discuss approaches that exploit differentially regulated molecules on the cell surface (e.g., CTLA-4) and intracellularly (e.g., T cell receptor signaling molecules) between TI-Tregs and Tconvs as well as their dependence on cytokines (e.g., IL-2) and metabolites (e.g., lactate). We envisage that targeting TI-Tregs could be effective as a monotherapy and/or when combined with ICB antibodies.

A sex-biased imbalance between Tfr, Tph, and atypical B cells determines antibody responses in COVID-19 patients.

Sex-biased humoral immune responses to COVID-19 patients have been observed, but the cellular basis for this is not understood. Using single-cell proteomics by mass cytometry, we find disrupted regulation of humoral immunity in COVID-19 patients, with a sex-biased loss of circulating follicular regulatory T cells (cTfr) at a significantly greater rate in male patients. In addition, a male sex-associated cellular network of T-peripheral helper, plasma blasts, proliferating and extrafollicular/atypical CD11c+ memory B cells was strongly positively correlated with neutralizing antibody concentrations and negatively correlated with cTfr frequency. These results suggest that sex-specific differences to the balance of cTfr and a network of extrafollicular antibody production-associated cell types may be a key factor in the altered humoral immune responses between male and female COVID-19 patients.

Mature dendritic cells enriched in regulatory molecules may control regulatory T cells and the prognosis of head and neck cancer.

We previously reported that regulatory T (Treg) cells expressing CTLA-4 on the cell surface are abundant in head and neck squamous cell carcinoma (HNSCC). The role of expanded Treg cells in the tumor microenvironment of HNSCC remains unclear. In this study, we reveal that the tumor microenvironment of HNSCC is characterized by the high expression of genes related to Treg cells, dendritic cells (DCs), and interleukin (IL)-17-related molecules. Increased expression of IL17A, IL17F, or IL23A contributes to a favorable prognosis of HNSCC. In the tumor microenvironment of HNSCC, IL23A and IL12B are expressed in mature dendritic cells enriched in regulatory molecules (mregDCs). The mregDCs in HNSCC are a migratory and mature phenotype; their signature genes strongly correlate with Treg signature genes in HNSCC. We also observed that IL17A was highly expressed in Th17 cells and exhausted CD8+ T cells in HNSCC. These data suggest that mregDCs in HNSCC may contribute to the prognosis by balancing Treg cells and effector T cells that produce IL-17. Targeting mregDCs may be a novel strategy for developing new immune therapies against HNSCC.

Construction of a T cell receptor signaling range for spontaneous development of autoimmune disease.

Thymic selection and peripheral activation of conventional T (Tconv) and regulatory T (Treg) cells depend on TCR signaling, whose anomalies are causative of autoimmunity. Here, we expressed in normal mice mutated ZAP-70 molecules with different affinities for the CD3 chains, or wild type ZAP-70 at graded expression levels under tetracycline-inducible control. Both manipulations reduced TCR signaling intensity to various extents and thereby rendered those normally deleted self-reactive thymocytes to become positively selected and form a highly autoimmune TCR repertoire. The signal reduction more profoundly affected Treg development and function because their TCR signaling was further attenuated by Foxp3 that physiologically repressed the expression of TCR-proximal signaling molecules, including ZAP-70, upon TCR stimulation. Consequently, the TCR signaling intensity reduced to a critical range generated pathogenic autoimmune Tconv cells and concurrently impaired Treg development/function, leading to spontaneous occurrence of autoimmune/inflammatory diseases, such as autoimmune arthritis and inflammatory bowel disease. These results provide a general model of how altered TCR signaling evokes autoimmune disease.

Protocol to evaluate cell lineage stability of mouse natural and induced regulatory T cells using bisulfite sequencing.

The establishment of regulatory T cells (Treg)-specific demethylation regions (TSDRs) is essential for the Treg-lineage stability. Here, we present a protocol using bisulfite sequencing to assess Treg-lineage stability. The protocol describes the isolation of lymphocytes and DNA extraction, followed by bisulfite conversion in unmethylated CpG DNA, bisulfite PCR and cloning, and sequencing to define the TSDR methylation. This protocol uses lymph nodes and spleen tissues and can be adapted to assess the methylation status of Tregs in other tissue types.

Myasthenia gravis-specific aberrant neuromuscular gene expression by medullary thymic epithelial cells in thymoma.

Myasthenia gravis (MG) is a neurological disease caused by autoantibodies against neuromuscular-associated proteins. While MG frequently develops in thymoma patients, the etiologic factors for MG are not well understood. Here, by constructing a comprehensive atlas of thymoma using bulk and single-cell RNA-sequencing, we identify ectopic expression of neuromuscular molecules in MG-type thymoma. These molecules are found within a distinct subpopulation of medullary thymic epithelial cells (mTECs), which we name neuromuscular mTECs (nmTECs). MG-thymoma also exhibits microenvironments dedicated to autoantibody production, including ectopic germinal center formation, T follicular helper cell accumulation, and type 2 conventional dendritic cell migration. Cell-cell interaction analysis also predicts the interaction between nmTECs and T/B cells via CXCL12-CXCR4. The enrichment of nmTECs presenting neuromuscular molecules within MG-thymoma is further confirmed immunohistochemically and by cellular composition estimation from the MG-thymoma transcriptome. Altogether, this study suggests that nmTECs have a significant function in MG pathogenesis via ectopic expression of neuromuscular molecules.

PGE2-EP2/EP4 signaling elicits immunosuppression by driving the mregDC-Treg axis in inflammatory tumor microenvironment.

Active inflammation generally promotes immune activation. However, in the tumor microenvironment (TME), active inflammation occurs in parallel with immunosuppression, and both contribute to tumor growth. Why inflammation does not lead to immune activation in TME remains unclear. In this study, using the immune checkpoint inhibitor-insensitive mouse cancer model and single-cell RNA sequencing, we show that PGE2-EP2/EP4 signaling simultaneously promotes active inflammation by inducing expression of the NF-κB genes in myeloid cells and elicits immunosuppression by driving the mregDC (mature DC enriched in immunoregulatory molecules)-Treg (regulatory T cell) axis for Treg recruitment and activation in the tumor. Importantly, the EP2/EP4 expression level is strongly correlated with the gene signatures of both active inflammation and the mregDC-Treg axis and has significant prognosis value in various human cancers. Thus, PGE2-EP2/EP4 signaling functions as the key regulatory node linking active inflammation and immunosuppression in TME, which can be targeted by EP2 and EP4 antagonists for cancer therapeutics.

The ratio of CD8 + lymphocytes to tumor-infiltrating suppressive FOXP3 + effector regulatory T cells is associated with treatment response in invasive breast cancer.

PURPOSE: FOXP3 + and CD8 + are recognized markers of tumor-infiltrating lymphocytes (TILs) for breast cancer. FOXP3 + TILs are composed of effector Tregs (eTregs) and other subpopulations that are classified by their differences in suppressive function. In this prospective study, we evaluated Treg subpopulations and CD8 + TILs in breast cancer. METHODS: 84 patients with breast cancer were enrolled. Fresh TILs were extracted andTregs were classified into eTregs (CD4+FOXP3highCD45RA-), other FOXP3+ Treg subsets (naïve and non-Tregs), and total CD8+CD4- TILs using flow cytometry. The suppression strength of each Treg subpopulation was analyzed. The association between TIL subpopulations, clinicopathological characteristics, and response to chemotherapy was evaluated. RESULTS: The mean CD8/eTreg ratio value was 7.86 (interquartile range: 4.08-12.80). The proliferation function of eTregs was significantly suppressed compared with that of the other subpopulations (proliferation rates: control: 89.3%, + naiiveTreg: 64.2%, + non-Treg: 78.2% vs eTreg 1.93%; all P < 0.05). The patients with high with a high CD8 + /eTreg ratio achieved excellent pathological complete response (pCR) rate of neoadjuvant chemotherapy (90.2%) and the CD8/eTreg ratio were independent predictive factors for pCR (odds ratio:18.7(confidence interval 1.25-279) P < 0.05). A detailed assessment of the CD8/eTreg ratio for each patient who underwent NAC revealed that high CD8/eTreg ratio showed a significantly higher pCR rate compared to patients with a low CD8/FOXP3 ratio (39.6% vs 13.3, P < 0.05) in triple negative subtype patients with stromal TILs < 50%. CONCLUSIONS: A high CD8/eTreg ratio enhances pCR rate in patients with invasive breast cancer.

CCR8-targeted specific depletion of clonally expanded Treg cells in tumor tissues evokes potent tumor immunity with long-lasting memory.

Foxp3-expressing CD25+CD4+ regulatory T cells (Tregs) are abundant in tumor tissues. Here, hypothesizing that tumor Tregs would clonally expand after they are activated by tumor-associated antigens to suppress antitumor immune responses, we performed single-cell analysis on tumor Tregs to characterize them by T cell receptor clonotype and gene-expression profiles. We found that multiclonal Tregs present in tumor tissues predominantly expressed the chemokine receptor CCR8. In mice and humans, CCR8+ Tregs constituted 30 to 80% of tumor Tregs in various cancers and less than 10% of Tregs in other tissues, whereas most tumor-infiltrating conventional T cells (Tconvs) were CCR8- CCR8+ tumor Tregs were highly differentiated and functionally stable. Administration of cell-depleting anti-CCR8 monoclonal antibodies (mAbs) indeed selectively eliminated multiclonal tumor Tregs, leading to cure of established tumors in mice. The treatment resulted in the expansion of CD8+ effector Tconvs, including tumor antigen-specific ones, that were more activated and less exhausted than those induced by PD-1 immune checkpoint blockade. Anti-CCR8 mAb treatment also evoked strong secondary immune responses against the same tumor cell line inoculated several months after tumor eradication, indicating that elimination of tumor-reactive multiclonal Tregs was sufficient to induce memory-type tumor-specific effector Tconvs. Despite induction of such potent tumor immunity, anti-CCR8 mAb treatment elicited minimal autoimmunity in mice, contrasting with systemic Treg depletion, which eradicated tumors but induced severe autoimmune disease. Thus, specific removal of clonally expanding Tregs in tumor tissues for a limited period by cell-depleting anti-CCR8 mAb treatment can generate potent tumor immunity with long-lasting memory and without deleterious autoimmunity.

Effect of Impaired T Cell Receptor Signaling on the Gut Microbiota in a Mouse Model of Systemic Autoimmunity.

OBJECTIVE: T cell receptor (TCR) signaling abnormalities and gut dysbiosis are thought to be involved in the development of systemic lupus erythematosus (SLE). However, it is not known whether these mechanisms are interrelated. This study was undertaken to explore the impact of defective TCR signaling on microbiota-driven immune responses and the consequent triggering of systemic autoimmunity. METHODS: The responses of B6SKG mice harboring a mutation in ZAP-70 leading to spontaneous development of SLE were evaluated under specific pathogen-free (SPF) and germ-free (GF) conditions. The gut microbiome was analyzed using 16S ribosomal RNA sequencing. Secretory IgA production in the gut and follicular helper T (Tfh) cell development in the spleen and Peyer's patches were analyzed. Interleukin-17 (IL-17)-deficient mice and segmented filamentous bacteria (SFB)-specific TCR-transgenic mice were used to examine the role of IL-17 and thymic selection. RESULTS: SLE development in B6SKG mice was significantly more attenuated under GF conditions than under SPF conditions. The gut microbiota in B6SKG mice was altered, which was associated with the expansion of SFB and consequent development of SLE by driving Th17 cell differentiation, which was in turn blunted by IL-17 deficiency. Notably, although systemic Tfh development and autoantibody IgG response were enhanced, local gut Tfh and IgA responses were impaired. Moreover, experiments in SFB-specific TCR-transgenic mice revealed that this differential response was caused by altered thymic selection of self- and microbiota-reactive TCR because of defective TCR signaling. CONCLUSION: Our findings indicate that defective TCR signaling alters the gut microbiota and promotes systemic autoimmunity by driving Th17 cell differentiation.