PD-1 Limits IL-2 Production and Thymic Regulatory T Cell Development.

Inhibitory proteins, such as programmed cell death protein 1 (PD-1), have been studied extensively in peripheral T cell responses to foreign Ags, self-Ags, and neoantigens. Notably, these proteins are first expressed during T cell development in the thymus. Reports suggest that PD-1 limits regulatory T cell (Treg) development, but the mechanism by which PD-1 exerts this function remains unknown. The present study expands the evaluation of murine PD-1 and its ligands in the thymus, demonstrating that some of the highest expressers of PD-1 and programmed death-ligand 1 are agonist selected cells. Surprisingly, we reveal a selective role for PD-1 in regulating the developmental niche only for Tregs because other agonist selected cell populations, such as NK T cells, remain unchanged. We also ruled out PD-1 as a regulator of proliferation or cell death of agonist selected Tregs and further demonstrated that PD-1-deficient Tregs have reduced TCR signaling. Unexpectedly, the data suggest that PD-1-deficient thymocytes produce elevated levels of IL-2, a Treg niche-limiting cytokine. Collectively, these data suggest a novel role for PD-1 in regulating IL-2 production and the concurrent agonist selection of murine thymic Tregs. This observation has implications for the use of checkpoint blockade in the context of cancer and infection.

Universal recording of immune cell interactions in vivo.

Immune cells rely on transient physical interactions with other immune and non-immune populations to regulate their function1. To study these 'kiss-and-run' interactions directly in vivo, we previously developed LIPSTIC (labelling immune partnerships by SorTagging intercellular contacts)2, an approach that uses enzymatic transfer of a labelled substrate between the molecular partners CD40L and CD40 to label interacting cells. Reliance on this pathway limited the use of LIPSTIC to measuring interactions between CD4+ T helper cells and antigen-presenting cells, however. Here we report the development of a universal version of LIPSTIC (uLIPSTIC), which can record physical interactions both among immune cells and between immune and non-immune populations irrespective of the receptors and ligands involved. We show that uLIPSTIC can be used, among other things, to monitor the priming of CD8+ T cells by dendritic cells, reveal the steady-state cellular partners of regulatory T cells and identify germinal centre-resident T follicular helper cells on the basis of their ability to interact cognately with germinal centre B cells. By coupling uLIPSTIC with single-cell transcriptomics, we build a catalogue of the immune populations that physically interact with intestinal epithelial cells at the steady state and profile the evolution of the interactome of lymphocytic choriomeningitis virus-specific CD8+ T cells in multiple organs following systemic infection. Thus, uLIPSTIC provides a broadly useful technology for measuring and understanding cell-cell interactions across multiple biological systems.

Anti-TIGIT antibody improves PD-L1 blockade through myeloid and Treg cells.

Tiragolumab, an anti-TIGIT antibody with an active IgG1κ Fc, demonstrated improved outcomes in the phase 2 CITYSCAPE trial (ClinicalTrials.gov: NCT03563716 ) when combined with atezolizumab (anti-PD-L1) versus atezolizumab alone1. However, there remains little consensus on the mechanism(s) of response with this combination2. Here we find that a high baseline of intratumoural macrophages and regulatory T cells is associated with better outcomes in patients treated with atezolizumab plus tiragolumab but not with atezolizumab alone. Serum sample analysis revealed that macrophage activation is associated with a clinical benefit in patients who received the combination treatment. In mouse tumour models, tiragolumab surrogate antibodies inflamed tumour-associated macrophages, monocytes and dendritic cells through Fcγ receptors (FcγR), in turn driving anti-tumour CD8+ T cells from an exhausted effector-like state to a more memory-like state. These results reveal a mechanism of action through which TIGIT checkpoint inhibitors can remodel immunosuppressive tumour microenvironments, and suggest that FcγR engagement is an important consideration in anti-TIGIT antibody development.

CTLA-4 antibody-drug conjugate reveals autologous destruction of B-lymphocytes associated with regulatory T cell impairment.

Germline CTLA-4 deficiency causes severe autoimmune diseases characterized by dysregulation of Foxp3+ Tregs, hyper-activation of effector memory T cells, and variable forms autoimmune cytopenia including gradual loss of B cells. Cancer patients with severe immune-related adverse events (irAE) after receiving anti-CTLA-4/PD-1 combination immunotherapy also have markedly reduced peripheral B cells. The immunological basis for B cell loss remains unexplained. Here, we probe the decline of B cells in human CTLA-4 knock-in mice by using anti-human CTLA-4 antibody Ipilimumab conjugated to a drug payload emtansine (Anti-CTLA-4 ADC). The anti-CTLA-4 ADC-treated mice have T cell hyper-proliferation and their differentiation into effector cells which results in B cell depletion. B cell depletion is mediated by both CD4 and CD8 T cells and at least partially rescued by anti-TNF-alpha antibody. These data revealed an unexpected antagonism between T and B cells and the importance of regulatory T cells in preserving B cells.

A molecular signature for IL-10-producing Th1 cells in protozoan parasitic diseases.

Control of visceral leishmaniasis (VL) depends on proinflammatory Th1 cells that activate infected tissue macrophages to kill resident intracellular parasites. However, proinflammatory cytokines produced by Th1 cells can damage tissues and require tight regulation. Th1 cell IL-10 production is an important cell-autologous mechanism to prevent such damage. However, IL-10-producing Th1 (type 1 regulatory; Tr1) cells can also delay control of parasites and the generation of immunity following drug treatment or vaccination. To identify molecules to target in order to alter the balance between Th1 and Tr1 cells for improved antiparasitic immunity, we compared the molecular and phenotypic profiles of Th1 and Tr1 cells in experimental VL caused by Leishmania donovani infection of C57BL/6J mice. We also identified a shared Tr1 cell protozoan signature by comparing the transcriptional profiles of Tr1 cells from mice with experimental VL and malaria. We identified LAG3 as an important coinhibitory receptor in patients with VL and experimental VL, and we reveal tissue-specific heterogeneity of coinhibitory receptor expression by Tr1 cells. We also discovered a role for the transcription factor Pbx1 in suppressing CD4+ T cell cytokine production. This work provides insights into the development and function of CD4+ T cells during protozoan parasitic infections and identifies key immunoregulatory molecules.

STING activation promotes autologous type I interferon-dependent development of type 1 regulatory T cells during malaria.

The development of highly effective malaria vaccines and improvement of drug-treatment protocols to boost antiparasitic immunity are critical for malaria elimination. However, the rapid establishment of parasite-specific immune regulatory networks following exposure to malaria parasites hampers these efforts. Here, we identified stimulator of interferon genes (STING) as a critical mediator of type I interferon production by CD4+ T cells during blood-stage Plasmodium falciparum infection. The activation of STING in CD4+ T cells by cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) stimulated IFNB gene transcription, which promoted development of IL-10- and IFN-γ-coproducing CD4+ T (type I regulatory [Tr1]) cells. The critical role for type I IFN signaling for Tr1 cell development was confirmed in vivo using a preclinical malaria model. CD4+ T cell sensitivity to STING phosphorylation was increased in healthy volunteers following P. falciparum infection, particularly in Tr1 cells. These findings identified STING expressed by CD4+ T cells as an important mediator of type I IFN production and Tr1 cell development and activation during malaria.

Reproductive outcomes after pregnancy-induced displacement of preexisting microchimeric cells.

Pregnancy confers partner-specific protection against complications in future pregnancy that parallel persistence of fetal microchimeric cells (FMcs) in mothers after parturition. We show that preexisting FMcs become displaced by new FMcs during pregnancy and that FMc tonic stimulation is essential for expansion of protective fetal-specific forkhead box P3 (FOXP3)-positive regulatory T cells (Treg cells). Maternal microchimeric cells and accumulation of Treg cells with noninherited maternal antigen (NIMA) specificity are similarly overturned in daughters after pregnancy, highlighting a fixed microchimeric cell niche. Whereas NIMA-specific tolerance is functionally erased by pregnancy, partner-specific resiliency against pregnancy complications persists in mothers despite paternity changes in intervening pregnancy. Persistent fetal tolerance reflects FOXP3 expression plasticity, which allows mothers to more durably remember their babies, whereas daughters forget their mothers with new pregnancy-imprinted immunological memories.

Pathological role of LncRNAs in immune-related disease via regulation of T regulatory cells.

Human regulatory T cells (Tregs) are essential in pathogenesis of several diseases such as autoimmune diseases and cancers, and their imbalances may be promoting factor in these disorders. The development of the proinflammatory T cell subset TH17 and its balance with the generation of regulatory T cells (Treg) is linked to autoimmune disease and cancers. Long non-coding RNAs (lncRNAs) have recently emerged as powerful regulatory molecules in a variety of diseases and can regulate the expression of significant genes at multiple levels through epigenetic regulation and by modulating transcription, post-transcriptional processes, translation, and protein modification. They may interact with a wide range of molecules, including DNA, RNA, and proteins, and have a complex structural makeup. LncRNAs are implicated in a range of illnesses due to their regulatory impact on a variety of biological processes such as cell proliferation, apoptosis, and differentiation. In this regard, a prominent example is lncRNA NEAT1 which several studies have performed to determine its role in the differentiation of immune cells. Many other lncRNAs have been linked to Treg cell differentiation in the context of immune cell differentiation. In this study, we review recent research on the various roles of lncRNAs in differentiation of Treg cell and regulation of the Th17/Treg balance in autoimmune diseases and tumors in which T regs play an important role.

Mapping the T cell repertoire to a complex gut bacterial community.

Certain bacterial strains from the microbiome induce a potent, antigen-specific T cell response1-5. However, the specificity of microbiome-induced T cells has not been explored at the strain level across the gut community. Here, we colonize germ-free mice with complex defined communities (roughly 100 bacterial strains) and profile T cell responses to each strain. The pattern of responses suggests that many T cells in the gut repertoire recognize several bacterial strains from the community. We constructed T cell hybridomas from 92 T cell receptor (TCR) clonotypes; by screening every strain in the community against each hybridoma, we find that nearly all the bacteria-specific TCRs show a one-to-many TCR-to-strain relationship, including 13 abundant TCR clonotypes that each recognize 18 Firmicutes. By screening three pooled bacterial genomic libraries, we discover that these 13 clonotypes share a single target: a conserved substrate-binding protein from an ATP-binding cassette transport system. Peripheral regulatory T cells and T helper 17 cells specific for an epitope from this protein are abundant in community-colonized and specific pathogen-free mice. Our work reveals that T cell recognition of commensals is focused on widely conserved, highly expressed cell-surface antigens, opening the door to new therapeutic strategies in which colonist-specific immune responses are rationally altered or redirected.

Brachytherapy for targeting the immune system in cervical cancer patients.

BACKGROUND: New combinations based on standard therapeutic modalities and immunotherapy require understanding the immunomodulatory properties of traditional treatments. The objective was to evaluate the impact of brachytherapy (BT) on the immune system of cervical cancer and to identify the best modality, High-dose-rate brachytherapy (HDR-BT) vs. Pulsed-dose-rate (PDR-BT), to target it. METHODS: Nineteen patients enrolled in a prospective study received chemoradiation (CRT) and subsequently HDR-BT or PDR-BT. Peripheral blood samples were obtained for immunophenotyping analysis by flow-cytometry before CRT, BT, and two and four weeks after BT. The Friedman one-way ANOVA, Conover post hoc test, and the Wilcoxon signed-rank test were used to compare changes in cell populations at different periods, perform multiple pairwise comparisons and assess differences between treatment groups (PDR and HDR). RESULTS: Natural killer cells (NKs) were the best target for BT. Patients receiving HDR-BT achieved significantly higher values ​​and longer time of the CD56dimCD16 + NK cells with greater cytotoxic capacity than the PDR-BT group, which presented their highest elevation of CD56-CD16 + NK cells. Furthermore, both BT modalities were associated with an increase in myeloid-derived suppressor cells (MDSCs), related to a worse clinical prognosis. However, there was a decrease in the percentage of CD4 + CD25 + Foxp3 + CD45RA + regulatory T cells (Tregs) in patients receiving HDR-BT, although there were no significant differences between BT. CONCLUSIONS: Immune biomarkers are important predictive determinants in cervical cancer. Higher cytotoxic NK cells and a trend toward lower values of Tregs might support the use of HDR-BT to the detriment of PDR-BT and help develop effective combinations with immunotherapy.

Immune response and treatment targets of chronic hepatitis B virus infection: innate and adaptive immunity.

Chronic hepatitis B virus (HBV) infection is a major global public health risk that threatens human life and health, although the number of vaccinated people has increased. The clinical outcome of HBV infection depends on the complex interplay between viral replication and the host immune response. Innate immunity plays an important role in the early stages of the disease but retains no long-term immune memory. However, HBV evades detection by the host innate immune system through stealth. Therefore, adaptive immunity involving T and B cells is crucial for controlling and clearing HBV infections that lead to liver inflammation and damage. The persistence of HBV leads to immune tolerance owing to immune cell dysfunction, T cell exhaustion, and an increase in suppressor cells and cytokines. Although significant progress has been made in HBV treatment in recent years, the balance between immune tolerance, immune activation, inflammation, and fibrosis in chronic hepatitis B remains unknown, making a functional cure difficult to achieve. Therefore, this review focuses on the important cells involved in the innate and adaptive immunity of chronic hepatitis B that target the host immune system and identifies treatment strategies.

Co-transplantation of autologous Treg cells in a cell therapy for Parkinson's disease.

The specific loss of midbrain dopamine neurons (mDANs) causes major motor dysfunction in Parkinson's disease, which makes cell replacement a promising therapeutic approach1-4. However, poor survival of grafted mDANs remains an obstacle to successful clinical outcomes5-8. Here we show that the surgical procedure itself (referred to here as 'needle trauma') triggers a profound host response that is characterized by acute neuroinflammation, robust infiltration of peripheral immune cells and brain cell death. When midbrain dopamine (mDA) cells derived from human induced pluripotent stem (iPS) cells were transplanted into the rodent striatum, less than 10% of implanted tyrosine hydroxylase (TH)+ mDANs survived at two weeks after transplantation. By contrast, TH- grafted cells mostly survived. Notably, transplantation of autologous regulatory T (Treg) cells greatly modified the response to needle trauma, suppressing acute neuroinflammation and immune cell infiltration. Furthermore, intra-striatal co-transplantation of Treg cells and human-iPS-cell-derived mDA cells significantly protected grafted mDANs from needle-trauma-associated death and improved therapeutic outcomes in rodent models of Parkinson's disease with 6-hydroxydopamine lesions. Co-transplantation with Treg cells also suppressed the undesirable proliferation of TH- grafted cells, resulting in more compact grafts with a higher proportion and higher absolute numbers of TH+ neurons. Together, these data emphasize the importance of the initial inflammatory response to surgical injury in the differential survival of cellular components of the graft, and suggest that co-transplanting autologous Treg cells effectively reduces the needle-trauma-induced death of mDANs, providing a potential strategy to achieve better clinical outcomes for cell therapy in Parkinson's disease.

Th1-like Treg cells are dressed to suppress anti-tumor immunity.

Mechanisms of Th1-like Treg suppression are unknown in cancer. Two studies in Immunity by Ayala et al. and Zagorulya et al. demonstrate that Th1-like Treg cells interact with type 1 dendritic cells in tumors and draining lymph nodes to potently suppress anti-tumor immunity.

Differentiation, regulation and function of regulatory T cells in non-lymphoid tissues and tumors.

Regulatory T cells (Tregs) play a substantial role in inhibiting excessive immune response. A large number of studies have focused on the tissue homeostasis maintenance and remodeling characteristics of Tregs in non-lymphoid tissues, such as the skin, colon, lung, brain, muscle, and adipose tissues. Herein, we overview the kinetics of Treg migration to non-lymphoid tissues and adaptation to the specific tissue microenvironment through the development of tissue-specific chemokine receptors, transcription factors, and phenotypes. Additionally, tumor-infiltrating Tregs (Ti-Tregs) play an important role in tumor generation and immunotherapy resistance. The phenotypes of Ti-Tregs are related to the histological location of the tumor and there is a large overlap between the transcripts of Ti-Tregs and those of tissue-specific Tregs. We recapitulate the molecular underpinnings of tissue-specific Tregs, which might shed new light on Treg-based therapeutic targets and biomarkers for inflammatory diseases and cancer.

SARS-CoV2 infection induce miR-155 expression and skewed Th17/Treg balance by changing SOCS1 level: A clinical study.

BACKGROUND: One of the regulators in severe acute respiratory syndrome coronavirus2 (SARS-CoV2) infection is miRNAs. In COVID-19 patients, immunological responses to SARS-CoV2 infection may be impacted by miR-155, a miRNA associated to inflammation. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMCs) of 50 confirmed COVID-19 patients /Healthy Controls (HCs) was isolated by Ficoll. The frequency of T helper 17 and regulatory T cells was analyzed by flowcytometry. The RNA was extracted from each sample and after synthesis of c-DNA, the relative expression of miR-155, suppressor of cytokine signaling (SOCS-1), Signal transducer and activator of transcription 3(STAT3), and Fork Head Box Protein 3 (FoxP3) was evaluated by real-time PCR. The protein level of STAT3, FoxP3 and RORγT in the isolated PBMCs measured by western blotting. The serum level of IL-10, TGF-ß, IL-17 and IL21 was assessed by ELISA method. RESULTS: The population of Th17 cells showed a significant rise, whereas Treg cells reduced in COVID-19 cases. The master transcription factor of Treg (FoxP3) and Th17 (RORγT) relative expression showed the same pattern as flowcytometry. STAT3 level of expression at RNA and protein level increased in COVID-19 cases. FOXP3 and SOCS-1 proteins were down-regulated. The relative expression of miR-155, up-regulated in PBMC of COVID-19 patients and revealed a negative correlation with SOCS-1. The serum cytokine profile showed a reduction in TGF-ß, on the other hand an increase was seen in IL-17, IL-21 and IL-10 in COVID-19 cases toward control group. CONCLUSION: Based on the studies conducted in this field, it can be suggested that Th17/Treg in covid-19 patients can be affected by miR-155 and it can be considered a valuable diagnostic and prognostic factor in this disease.

Improvement of Islet Engrafts via Treg Induction Using Immunomodulating Polymeric Tolerogenic Microparticles.

Type 1 diabetes (T1D) is a life-threatening condition for which islet transplantation offers a way to extend longevity and vastly improve quality of life, but the degree and duration of success can vary greatly due to the patient's protective immunity against foreign material. The field is in need of cellular engineering modalities to promote a localized, tolerogenic environment to protect transplanted islet tissue. Artificial antigen-presenting cells (aAPCs) can be designed exogenously to mimic immune cells, such as dendritic cells, and administered to patients, allowing greater control over T cell differentiation. As regulatory T cell (Treg) modulation can reduce the activity of cytotoxic T-effector populations, this strategy can be used to promote immune acceptance of both biomaterials and cellular transplants, such as islets. A new class of poly(lactic-co-glycolic acid) (PLGA) and PLGA/PBAE-blend aAPCs containing transforming growth factor beta and conjugated with anti-CD3 and anti-CD28 antibodies, called tolerogenic aAPCs (TolAPCs), are specifically designed to generate a tolerogenic response by inducing Tregs. We characterized TolAPCs' physical and chemical properties via advanced particle imaging and sizing modalities and investigated their impact on the local and systemic immune system across BALB/c and C57BL/6 mouse strains as well as healthy male and female mice via histologic, gene expression, and immunofluorescence staining methods. Strain-specific differences were observed, whereas sex made no difference in the TolAPC response. TolAPCs stimulated the expansion of FOXP3+ Tregs and provided islet cell protection, maintaining improved glucose-stimulated insulin secretion in vitro when co-cultured with cytotoxic CD8+ T cells. We also explored the ability of this TolAPC platform to promote tolerance in a streptozotocin-induced murine T1D C57BL/6 mouse model. We achieved partial islet protection over the first few days following co-injection with PLGA/PBAE TolAPCs; however, grafts failed soon thereafter. Analysis of the local injection site demonstrated that other immune cell types, including APCs and cytotoxic natural killer cells, increased in the islet injection site. While we aimed to promote a localized tolerogenic microenvironment in vivo using biodegradable TolAPCs to induce Tregs and extend islet transplant durability, further TolAPC improvements will be required to both elongate efficacy and control additional immune cell responders.

Lentinan confers protection against type 1 diabetes by inducing regulatory T cell in spontaneous non-obese diabetic mice.

BACKGROUND: Lentinan (LNT) is a complex fungal component that possesses effective antitumor and immunostimulating properties. However, there is a paucity of studies regarding the effects and mechanisms of LNT on type 1 diabetes. OBJECTIVE: In the current study, we investigated whether an intraperitoneal injection of LNT can diminish the risk of developing type 1 diabetes (T1D) in non-obese diabetic (NOD) mice and further examined possible mechanisms of LNT's effects. METHODS: Pre-diabetic female NOD mice 8 weeks of age, NOD mice with 140-160 mg/dL, 200-230 mg/dL or 350-450 mg/dL blood glucose levels were randomly divided into two groups and intraperitoneally injected with 5 mg/kg LNT or PBS every other day. Then, blood sugar levels, pancreas slices, spleen, PnLN and pancreas cells from treatment mice were examined. RESULTS: Our results demonstrated that low-dosage injections (5 mg/kg) of LNT significantly suppressed immunopathology in mice with autoimmune diabetes but increased the Foxp3+ regulatory T cells (Treg cells) proportion in mice. LNT treatment induced the production of Tregs in the spleen and PnLN cells of NOD mice in vitro. Furthermore, the adoptive transfer of Treg cells extracted from LNT-treated NOD mice confirmed that LNT induced Treg function in vivo and revealed an enhanced suppressive capacity as compared to the Tregs isolated from the control group. CONCLUSION: LNT was capable of stimulating the production of Treg cells from naive CD4 + T cells, which implies that LNT exhibits therapeutic values as a tolerogenic adjuvant and may be used to reverse hyperglycaemia in the early and late stages of T1D.

Neoadjuvant immune checkpoint blockade triggers persistent and systemic Treg activation which blunts therapeutic efficacy against metastatic spread of breast tumors.

The clinical successes of immune checkpoint blockade (ICB) in advanced cancer patients have recently spurred the clinical implementation of ICB in the neoadjuvant and perioperative setting. However, how neoadjuvant ICB therapy affects the systemic immune landscape and metastatic spread remains to be established. Tumors promote both local and systemic expansion of regulatory T cells (Tregs), which are key orchestrators of tumor-induced immunosuppression, contributing to immune evasion, tumor progression and metastasis. Tregs express inhibitory immune checkpoint molecules and thus may be unintended targets for ICB therapy counteracting its efficacy. Using ICB-refractory models of spontaneous primary and metastatic breast cancer that recapitulate the poor ICB response of breast cancer patients, we observed that combined anti-PD-1 and anti-CTLA-4 therapy inadvertently promotes proliferation and activation of Tregs in the tumor, tumor-draining lymph node and circulation. Also in breast cancer patients, Treg levels were elevated upon ICB. Depletion of Tregs during neoadjuvant ICB in tumor-bearing mice not only reshaped the intratumoral immune landscape into a state favorable for ICB response but also induced profound and persistent alterations in systemic immunity, characterized by elevated CD8+ T cells and NK cells and durable T cell activation that was maintained after treatment cessation. While depletion of Tregs in combination with neoadjuvant ICB did not inhibit primary tumor growth, it prolonged metastasis-related survival driven predominantly by CD8+ T cells. This study demonstrates that neoadjuvant ICB therapy of breast cancer can be empowered by simultaneous targeting of Tregs, extending metastasis-related survival, independent of a primary tumor response.

T Cell Subsets and the Expression of Related MicroRNAs in Patients with Recurrent Early Pregnancy Loss.

This study explored the role of T cell subsets and the expression of related microRNAs in patients with recurrent early pregnancy loss (EPL). Fifty patients with EPL loss between May 2018 and May 2021 were randomly selected as the EPL group, and 50 pregnant women with normal pregnancies or normal delivery outcomes were randomly selected as the control group. The expression levels of T cell subset-related markers and T cell subset-related miRNAs, in addition to the frequencies of T cell subsets, in peripheral blood of the two groups were analyzed. In terms of T cell-related markers, the results showed that the expression levels of the transcriptional regulator TBX-21 (T-bet) and interferon regulatory factor 4 (IRF4) were significantly upregulated in peripheral blood of the patients in the EPL group (P < 0.05), whereas the expression levels of GATA binding protein 3 (GATA3) and glucocorticoid-induced tumor necrosis factor receptor (GITR) were significantly downregulated (P < 0.05). In the EPL group, the expression of mir-106b, mir-93, and mir-25 was upregulated (1.51 ± 0.129, 1.43 ± 0.132, and 1.73 ± 0.156, respectively) in regulatory T (Treg) cell-related T cell subsets, whereas the expression of miR-146a and miR-155 was downregulated (P < 0.05). The frequencies of Treg and exhausted T cells in the EPL group were significantly lower than those in the control group (P < 0.05). The cell frequencies of T helper 17 (Th17) cells and exhausted Treg cells in the EPL group were significantly higher than those in the control group (P < 0.05). In conclusion, immune cells and associated miRNA profiles can be used as prognostic biomarkers for the treatment of human reproductive disorders, such as EPL.

Tumor-associated macrophages and Tregs influence and represent immune cell infiltration of muscle-invasive bladder cancer and predict prognosis.

INTRODUCTION AND OBJECTIVE: Muscle-invasive urothelial bladder cancer (MIBC) is associated with limited response rates to systemic therapy, risk of recurrence and death. Tumor infiltrating immune cells have been associated with outcome and response to chemo-and immunotherapy in MIBC. We aimed to profile the immune cells in the tumor microenvironment (TME) to predict prognosis in MIBC and responses to adjuvant chemotherapy. METHODS: We performed multiplex immunohistochemistry (IHC) profiling and quantification of immune and stromal cells (CD3, CD4, CD8, CD163, FoxP3, PD-1, and CD45, Vimentin, αSMA, PD-L1, Pan-Cytokeratin, Ki67) in 101 patients with MIBC receiving radical cystectomy. We used uni- and multivariate survival analyses to identify cell types predicting prognosis. Samples were subdivided using K-means clustering for Treg and macrophage infiltration resulting in 3 clusters, Cluster 1: Treg high, cluster 2: macrophage high, cluster 3: Treg and macrophage low. Routine CD68 and CD163 IHC were analyzed with QuPath in an extended cohort of 141 MIBC. RESULTS: High concentrations of macrophages were associated with increased risk of death (HR 10.9, 95% CI 2.8-40.5; p < 0.001) and high concentrations of Tregs were associated with decreased risk of death (HR 0.1, 95% CI 0.01-0.7; p = 0.03) in the multivariate Cox-regression model adjusting for adjuvant chemotherapy, tumor and lymph node stage. Patients in the macrophage rich cluster (2) showed the worst OS with and without adjuvant chemotherapy. The Treg rich cluster (1) showed high levels of effector and proliferating immune cells and had the best survival. Cluster 1 and 2 both were rich in PD-1 and PD-L1 expression on tumor and immune cells. CONCLUSION: Treg and macrophage concentrations in MIBC are independent predictors of prognosis and are important players in the TME. Standard IHC with CD163 for macrophages is feasible to predict prognosis but validation to use immune-cell infiltration, especially to predict response to systemic therapies, is required.