Treg-Resistant Cytotoxic CD4+ T Cells Dictate T Helper Cells in Their Vicinity: TH17 Skewing and Modulation of Proliferation

Cytotoxic CD4⁺ T cells (CD4 CTL) are terminally differentiated T helper cells that contribute to autoimmune diseases, such as multiple sclerosis. We developed a novel triple co-culture transwell assay to study mutual interactions between CD4 CTL, conventional TH cells, and regulatory T cells (Tregs) simultaneously. We show that, while CD4 CTL are resistant to suppression by Tregs in vitro, the conditioned medium of CD4 CTL accentuates the suppressive phenotype of Tregs by upregulating IL-10, Granzyme B, CTLA-4, and PD-1. We demonstrate that CD4 CTL conditioned medium skews memory TH cells to a TH17 phenotype, suggesting that the CD4 CTL induce bystander polarization. In our triple co-culture assay, the CD4 CTL secretome promotes the proliferation of TH cells, even in the presence of Tregs. However, when cell−cell contact is established between CD4 CTL and TH cells, the proliferation of TH cells is no longer increased and Treg-mediated suppression is restored. Taken together, our results suggest that when TH cells acquire cytotoxic properties, these Treg-resistant CD4 CTL affect the proliferation and phenotype of conventional TH cells in their vicinity. By creating such a pro-inflammatory microenvironment, CD4 CTL may favor their own persistence and expansion, and that of other potentially pathogenic TH cells, thereby contributing to pathogenic responses in autoimmune disorders.     

CD85k Contributes to Regulatory T Cell Function in Chronic Viral Infections

Regulatory T cells (Tregs) prevent excessive immune responses and limit immune pathology upon infections. To fulfill this role in different immune environments elicited by different types of pathogens, Tregs undergo functional specialization into distinct subsets. During acute type 1 immune responses, type 1 Tregs are induced and recruited to the site of ongoing Th1 responses to efficiently control Th1 responses. However, whether a similar specialization process also takes place following chronic infections is still unknown. In this study, we investigated Treg specialization in persistent viral infections using lymphocytic choriomeningitis virus (LCMV) and murine cytomegalovirus (MCMV) infection as models for chronic and latent infections, respectively. We identify CD85k as a Th1-specific co-inhibitory receptor with sustained expression in persistent viral infections and show that recombinant CD85k inhibits LCMV-specific effector T cells. Furthermore, expression of the CD85k ligand ALCAM is induced on LCMV-specific and exhausted T cells during chronic LCMV infection. Finally, we demonstrate that type 1 Tregs arising during chronic LCMV infection suppress Th1 effector cells in an ALCAM-dependent manner. These results extend the current knowledge of Treg specialization from acute to persistent viral infections and reveal an important functional role of CD85k in Treg-mediated suppression of type 1 immunity.     

Regulatory T Cells Exhibit Interleukin-33-Dependent Migratory Behavior during Skin Barrier Disruption

Regulatory T cells (Tregs) suppress immune responses and maintain immunological self-tolerance and homeostasis. We currently investigated relationships between skin barrier condition and Treg behavior using skin barrier-disrupted mice. Skin barrier disruption was induced by repeated topical application of 4% sodium dodecyl sulfate (SDS) on mice. The number of CD4⁺ forkhead box protein P3 (Foxp3)⁺ Tregs was higher in 4% SDS-treated skins than in controls. This increasing was correlated with the degree of acanthosis. The numbers of interleukin (IL)-10⁺ and transforming growth factor (TGF)-β⁺ Tregs also increased in 4% SDS-treated skins. Localization of IL-33 in keratinocytes shifted from nucleus to cytoplasm after skin barrier disruption. Notably, IL-33 promoted the migration of Tregs in chemotaxis assay. The skin infiltration of Tregs was cancelled in IL-33 neutralizing antibody-treated mice and IL-33 knockout mice. Thus, keratinocyte-derived IL-33 may induce Treg migration into barrier-disrupted skin to control the phase transition between healthy and inflammatory conditions.     

Galectin-9 Induced Myeloid Suppressor Cells Expand Regulatory T Cells in an IL-10-Dependent Manner in CVB3-Induced Acute Myocarditis

The objective of the study was to explore the effects of galectin-9 on myeloid suppressor cells in Coxsackievirus B3 (CVB3)-induced myocarditis and the possible mechanisms involved. For this purpose, BALB/c male mice were infected with CVB3 on day 0 and then received intraperitoneal (IP) administration of recombinant galectin-9 or phosphate-buffered saline (PBS) daily from day 3 to day 7. The phenotypes and functions of myeloid suppressor cells were evaluated. The role and mechanism of myeloid suppressor cells and subsets in CVB3-induced myocarditis in vitro were explored. We found that galectin-9 remarkably increased the frequencies of CD11b⁺Gr-1⁺ cells in the cardiac tissue and spleen with myocarditis. Ly-6G⁺ cells were decreased and Ly-6C⁺ cells were increased in galectin-9-treated mice. In addition, CD11b⁺Gr-1⁺ cells were highly effective in suppressing CD4⁺ T cells. Moreover, our data demonstrate that CD11b⁺Gr-1⁺ cells are capable of expanding regulatory T cells (Tregs) from a preexisting population of natural Tregs, which depends on IL-10 but not TGF-β. Our results indicate that galectin-9 therapy may represent a useful approach to ameliorate CVB3-induced myocarditis.     

GMI,an Immunomodulatory Peptide from Ganoderma microsporum,Restrains Periprosthetic Joint Infections via Modulating the Functions of Myeloid-Derived Suppressor Cells and Effector T Cells

Periprosthetic joint infections (PJIs) caused by Staphylococcus aureus infection are difficult to treat due to antibiotic resistance. It is known that the biofilms from methicillin-resistant S. aureus (MRSA) promote expansion of myeloid-derived suppressor cells (MDSCs) to suppress T-cell proliferation and benefit bacterial infections. This study finds that GMI, a fungal immunomodulatory peptide isolated from Ganoderma microsporum, suppresses MDSC expansion to promote the proliferation of cytotoxic T cells. The enhancement is likely attributed to increased expression of IL-6 and TNF-α and reduction in ROS expression. Similar beneficial effects of GMI on the suppression of MDSC expansion and IL-6 expression are also observed in the whole blood and reduces the accumulation of MDSCs in the infected bone region in a mouse PJI infection model. This study shows that GMI is potentially useful for treating S. aureus-induced PJIs.     

Deregulated Expression of Immune Checkpoints on Circulating CD4 T Cells May Complicate Clinical Outcome and Response to Treatment with Checkpoint Inhibitors in Multiple Myeloma Patients

Unlike solid-tumor patients, a disappointingly small subset of multiple myeloma (MM) patients treated with checkpoint inhibitors derive clinical benefits, suggesting differential participation of inhibitory receptors involved in the development of T-cell-mediated immunosuppression. In fact, T cells in MM patients have recently been shown to display features of immunosenescence and exhaustion involved in immune response inhibition. Therefore, we aimed to identify the dominant inhibitory pathway in MM patients to achieve its effective control by therapeutic interventions. By flow cytometry, we examined peripheral blood (PB) CD4 T cell characteristics assigned to senescence or exhaustion, considering PD-1, CTLA-4, and BTLA checkpoint expression, as well as secretory effector function, i.e., capacity for IFN-γ and IL-17 secretion. Analyses were performed in a total of 40 active myeloma patients (newly diagnosed and treated) and 20 healthy controls. At the single-cell level, we found a loss of studied checkpoints’ expression on MM CD4 T cells (both effector (Teff) and regulatory (Treg) cells) primarily at diagnosis; the checkpoint deficit in MM relapse was not significant. Nonetheless, PD-1 was the only checkpoint distributed on an increased proportion of T cells in all MM patients irrespective of disease phase, and its expression on CD4 Teff cells correlated with adverse clinical courses. Among patients, the relative defect in secretory effector function of CD4 T cells was more pronounced at myeloma relapse (as seen in declined Th1/Treg and Th17/Treg cell rates). Although the contribution of PD-1 to MM clinical outcomes is suggestive, our study clearly indicated that the inappropriate expression of immune checkpoints (associated with dysfunctionality of CD4 T cells and disease clinical phase) might be responsible for the sub-optimal clinical response to therapeutic checkpoint inhibitors in MM.     

CD38 Correlates with an Immunosuppressive Treg Phenotype in Lupus-Prone Mice

CD38 is a transmembrane glycoprotein expressed by T-cells. It has been reported that patients with systemic lupus erythematosus (SLE) showed increased CD38⁺CD25⁺ T-cells correlating with immune activation and clinical signs. Contrariwise, CD38 deficiency in murine models has shown enhanced autoimmunity development. Recent studies have suggested that CD38⁺ regulatory T-cells are more suppressive than CD38⁻ regulatory T-cells. Thus, we have suggested that CD38 overexpression in SLE patients could play a role in regulating immune activation cells instead of enhancing it. This study found a correlation between CD38 with FoxP3 expression and immunosuppressive molecules (CD69, IL-10, CTLA-4, and PD-1) in T-cells from lupus-prone mice (B6.MRL-Fas^lpr/J). Additionally, B6.MRL-Fas^lpr/J mice showed a decreased proportion of CD38⁺ Treg cells regarding wild-type mice (WT). Furthermore, Regulatory T-Cells (Treg cells) from CD38-/- mice showed impairment in expressing immunosuppressive molecules and proliferation after stimulation through the T-cell receptor (TCR). Finally, we demonstrated an increased ratio of IFN-γ/IL-10 secretion in CD38-/- splenocytes stimulated with anti-CD3 compared with the WT. Altogether, our data suggest that CD38 represents an element in maintaining activated and proliferative Treg cells. Consequently, CD38 could have a crucial role in immune tolerance, preventing SLE development through Treg cells.     

CD4+ T-Cell Plasticity in Non-Infectious Retinal Inflammatory Disease

Non-infectious uveitis (NIU) is a potentially sight-threatening disease. Effector CD4⁺ T cells, especially interferon-γ-(IFNγ) producing Th1 cells and interleukin-17-(IL-17) producing Th17 cells, are the major immunopathogenic cells, as demonstrated by adoptive transfer of disease in a model of experimental autoimmune uveitis (EAU). CD4⁺FoxP3⁺CD25⁺ regulatory T cells (Tregs) were known to suppress function of effector CD4⁺ T cells and contribute to resolution of disease. It has been recently reported that some CD4⁺ T-cell subsets demonstrate shared phenotypes with another CD4⁺ T-cell subset, offering the potential for dual function. For example, Th17/Th1 (co-expressing IFNγ and IL-17) cells and Th17/Treg (co-expressing IL-17 and FoxP3) cells have been identified in NIU and EAU. In this review, we have investigated the evidence as to whether these ‘plastic CD4⁺ T cells’ are functionally active in uveitis. We conclude that Th17/Th1 cells are generated locally, are resistant to the immunosuppressive effects of steroids, and contribute to early development of EAU. Th17/Treg cells produce IL-17, not IL-10, and act similar to Th17 cells. These cells were considered pathogenic in uveitis. Future studies are needed to better clarify their function, and in the future, these cell subsets may in need to be taken into consideration for designing treatment strategies for disease.     

shRNA-Mediated XRCC2 Gene Knockdown Efficiently Sensitizes Colon Tumor Cells to X-ray Irradiation in Vitro and in Vivo

Colon cancer is one of the most common tumors of the digestive tract. Resistance to ionizing radiation (IR) decreased therapeutic efficiency in these patients’ radiotherapy. XRCC2 is the key protein of DNA homologous recombination repair, and its high expression is associated with enhanced resistance to DNA damage induced by IR. Here, we investigated the effect of XRCC2 silencing on colon tumor cells’ growth and sensitivity to X-radiation in vitro and in vivo. Colon tumor cells (T84 cell line) were cultivated in vitro and tumors originated from the cell line were propagated as xenografts in nude mice. The suppression of XRCC2 expression was achieved by using vector-based short hairpin RNA (shRNA) in T84 cells. We found that the knockdown of XRCC2 expression effectively decreased T84 cellular proliferation and colony formation, and led to cell apoptosis and cell cycle arrested in G2/M phase induced by X-radiation in vitro. In addition, tumor xenograft studies suggested that XRCC2 silencing inhibited tumorigenicity after radiation treatment in vivo. Our data suggest that the suppression of XRCC2 expression rendered colon tumor cells more sensitive to radiation therapy in vitro and in vivo, implying XRCC2 as a promising therapeutic target for the treatment of radioresistant human colon cancer.     

Reduced Dendritic Cells Expressing CD200R1 in Children with Inflammatory Bowel Disease: Correlation with Th17 and Regulatory T Cells

Loss of tolerance of the adaptive immune system towards indigenous flora contributes to the development of inflammatory bowel diseases (IBD). Defects in dendritic cell (DC)-mediated innate and adoptive immune responses are conceivable. The aim of this study was to investigate the expression of the inhibitory molecules CD200R1 and their ligand CD200 on DCs, to clarify the role of the DCs in the pathogenesis of IBD. Thirty-seven pediatric IBD patients (23 with Crohn’s disease (CD) and 14 with ulcerative colitis (UC)) with mean age 13.25 ± 2.9 years were included. Fourteen age-matched healthy pediatric volunteers (five males and nine females) served as a control group (HC). The percentage of CD11c⁺ myeloid dendritic cells (mDCs) and CD123⁺ plasmacytoid DCs (pDCs) expressing CD200R1 and CD200 were evaluated in peripheral blood using flow cytometry and were correlated with routine biochemical, serological markers, serum levels of cytokines and with the percentages of circulating regulatory T cells (Treg) and CD4⁺ producing IL-17 (Th17). IBD patients showed a significant decrease in the percentage of pDCs and mDCs expressing CD200R1 compared to that of HC. Patients with UC showed increased expressions of the CD200 molecule on pDCs as compared to HC. DCs expressing CD200R1 were found to be correlated positively with Treg and negatively with TH17 and erythrocyte sedimentation rate (ESR). Our findings suggest that IBD is associated with dysregulation in the CD200R1/CD200 axis and that the decrease in DCs expressing CD200R1 may contribute to the imbalance of Th17 and Treg cells and in the pathogenesis of IBD.     

Imbalance between Th17 and Regulatory T-Cells in Sarcoidosis

Sarcoidosis is a systemic granulomatous disease, which is thought to result from an aberrant immune response. CD4⁺ T lymphocytes play an important role in the development of granulomas. Previously, the immunopathogenesis of sarcoidosis was focused on Th1/Th2 disturbances. The aim of this study was to evaluate the balance between newer CD4⁺ T lymphocytes, i.e., Treg and Th17 cells. In our studies, a decrease in Treg cells and an increase in Th17 cells were observed in the peripheral blood and BALF of sarcoidosis patients. A significant increase in the Th17/Treg cell ratio was observed in sarcoidosis patients. After treatment with prednisone, the expression of Foxp3 mRNA was elevated in the peripheral blood, and expression of (ROR)γt mRNA showed a downward trend. These findings suggest that sarcoidosis is associated with an imbalance between Th17 and Treg cells in peripheral blood and BALF. Therefore, targeting the cytokines that affect the Th17/Treg ratio could provide a new promising therapy for pulmonary sarcoidosis.     

The Many Faces of CD4+ T Cells: Immunological and Structural Characteristics

As a major arm of the cellular immune response, CD4⁺ T cells are important in the control and clearance of infections. Primarily described as helpers, CD4⁺ T cells play an integral role in the development and activation of B cells and CD8⁺ T cells. CD4⁺ T cells are incredibly heterogeneous, and can be divided into six main lineages based on distinct profiles, namely T helper 1, 2, 17 and 22 (Th1, Th2, Th17, Th22), regulatory T cells (Treg) and T follicular helper cells (Tfh). Recent advances in structural biology have allowed for a detailed characterisation of the molecular mechanisms that drive CD4⁺ T cell recognition. In this review, we discuss the defining features of the main human CD4⁺ T cell lineages and their role in immunity, as well as their structural characteristics underlying their detection of pathogens.     

Reduced Immunosenescence of Peripheral Blood T Cells in Parkinson’s Disease with CMV Infection Background

Immunosenescence is a process of remodeling the immune system under the influence of chronic inflammation during aging. Parkinson’s disease (PD) is a common age-associated neurodegenerative disorder and is frequently accompanied by neuroinflammation. On the other hand, cytomegalovirus (CMV), one of the most spread infections in humans, may induce chronic inflammation which contributes to immunosenescence, differentiation and the inflation of T cells and NK cells. Currently, there is no clear understanding of immunosenescence severity in PD patients infected with CMV. In this study, we analyzed differentiation stages and immunosenescence characteristics of T cells and NK cells in 31 patients with mild and moderate PD severity, 33 age-matched and 30 young healthy donors. The PD patients were 100% CMV-seropositive compared to 76% age-matched and 73% young CMV-infected healthy donors. The proportion of effector memory T cells re-expressing CD45RA, CD57⁺CD56⁻ T cells and CD57⁺CD56⁺ T cells was significantly reduced in PD patients compared with CMV-seropositive age-matched healthy individuals. The CD57⁺CD56⁻ T cell proportion in PD patients was similar to that of CMV-seropositive young healthy donors. Thus, PD is characterized by reduced peripheral blood T cell immunosenescence, even against the background of CMV infection.     

Transgene IL-6 Enhances DC-Stimulated CTL Responses by Counteracting CD4+25+Foxp3+ Regulatory T Cell Suppression via IL-6-Induced Foxp3 Downregulation

Dendritic cells (DCs), the most potent antigen-presenting cells have been extensively applied in clinical trials for evaluation of antitumor immunity. However, the efficacy of DC-mediated cancer vaccines is still limited as they are unable to sufficiently break the immune tolerance. In this study, we constructed a recombinant adenoviral vector (AdV_IL-6) expressing IL-6, and generated IL-6 transgene-engineered DC vaccine (DC_OVA/IL-6) by transfection of murine bone marrow-derived ovalbumin (OVA)-pulsed DCs (DC_OVA) with AdV_IL-6. We then assessed DC_OVA/IL-6-stimulated cytotoxic T-lymphocyte (CTL) responses and antitumor immunity in OVA-specific animal tumor model. We demonstrate that DC_OVA/IL-6 vaccine up-regulates expression of DC maturation markers, secretes transgene-encoded IL-6, and more efficiently stimulates OVA-specific CTL responses and therapeutic immunity against OVA-expressing B16 melanoma BL6-10_OVA in vivo than the control DC_OVA/Null vaccine. Moreover, DC_OVA/IL-6-stimulated CTL responses were relatively maintained in mice with transfer of CD4⁺25⁺Foxp3⁺ Tr-cells, but significantly reduced when treated with anti-IL-6 antibody. In addition, we demonstrate that IL-6 down-regulates Foxp3-expression of CD4⁺25⁺Foxp3⁺ Tr-cells in vitro. Taken together, our results demonstrate that AdV-mediated IL-6 transgene-engineered DC vaccine stimulates potent CTL responses and antitumor immunity by counteracting CD4⁺25⁺ Tr immunosuppression via IL-6-induced Foxp3 down-regulation. Thus, IL-6 may be a good candidate for engineering DCs for cancer immunotherapy.     

Expression of PD-1 Molecule on Regulatory T Lymphocytes in Patients with Insulin-Dependent Diabetes Mellitus

Type 1 diabetes is caused by autoreactive T cells that destroy pancreatic beta cells. Animal models suggested that a CD4⁺CD25⁺ population has a regulatory function capable of preventing activation and effector functions of autoreactive T cells. However, the role of CD4⁺CD25^high T cells in autoimmunity and their molecular mechanisms remain the subject of investigation. We therefore evaluated T regulatory cell frequencies and their PD-1 expression in the peripheral blood of long-standing diabetics under basal conditions and after CD3/CD28 stimulation. Under basal conditions, the percentages of T regulatory cells were significantly higher while that of T effector cells were significantly lower in patients than in controls. The ratio of regulatory to effector T cells was higher in patients than that in controls, suggesting that T regulatory cells were functional in patients. Percentages of total PD-1⁺, PD-1^low and PD-1^high expressing T regulatory cells did not change in patients and in controls. After stimulation, a defect in T regulatory cell proliferation was observed in diabetics and the percentages of total PD-1⁺, PD-1^low and PD-1^high expressing cells were lower in patients. Our data suggest a defective activation of T regulatory cells in long-standing diabetics due to a lower expression of PD-1 on their surface.     

Myeloid-Derived Suppressor Cells Gain Suppressive Function during Neonatal Bacterial Sepsis

Neonates are at an increased risk of an infectious disease. This is consistent with an increased abundance of myeloid-derived suppressor cells (MDSCs) compared with older children and adults. Using a murine model of neonatal bacterial sepsis, we demonstrate that MDSCs modulate their activity during an infection to enhance immune suppressive functions. A gene expression analysis shows that MDSCs increased NOS2, Arg-1 and IL-27p28 expression in vitro and in vivo in response to Escherichia coli O1:K1:H7 and this is regulated at the level of the gene expression. Changes in the effector gene expression are consistent with increased enzymatic activity and cytokine secretion. The neonatal MDSCs express toll-like receptor (TLR) 2, 4 and 5 capable of recognizing pathogen-associated molecular patterns (PAMPS) on E. coli. However, a variable level of effector expression was achieved in response to LPS, peptidoglycan or flagellin. Individual bacterial PAMPs did not stimulate the expression of Arg-l and IL-27p28 equivalently to E. coli. However, the upregulation of NOS2 was achieved in response to LPS, peptidoglycan and flagella. The increased immune suppressive profile translated to an enhanced suppression of CD4+ T cell proliferation. Collectively, these findings increase our understanding of the dynamic nature of MDSC activity and suggest that these cells abundant in early life can acquire activity during an infection that suppresses protective immunity.