Phosphorylated STAT3 and PD‐1 regulate IL‐17 production and IL‐23 receptor expression in Mycobacterium tuberculosis infection

We studied the factors that regulate IL‐23 receptor expression and IL‐17 production in human tuberculosis infection. Mycobacterium tuberculosis (M. tb)‐stimulated CD4⁺ T cells from tuberculosis patients secreted less IL‐17 than did CD4⁺ T cells from healthy tuberculin reactors (PPD⁺). M. tb‐cultured monocytes from tuberculosis patients and PPD⁺ donors expressed equal amounts of IL‐23p19 mRNA and protein, suggesting that reduced IL‐23 production is not responsible for decreased IL‐17 production by tuberculosis patients. Freshly isolated and M. tb‐stimulated CD4⁺ T cells from tuberculosis patients had reduced IL‐23 receptor and phosphorylated STAT3 (pSTAT3) expression, compared with cells from PPD⁺ donors. STAT3 siRNA reduced IL‐23 receptor expression and IL‐17 production by CD4⁺ T cells from PPD⁺ donors. Tuberculosis patients had increased numbers of PD‐1⁺ T cells compared with healthy PPD⁺ individuals. Anti‐PD‐1 antibody enhanced pSTAT3 and IL‐23R expression and IL‐17 production by M. tb‐cultured CD4⁺ T cells of tuberculosis patients. Anti‐tuberculosis therapy decreased PD‐1 expression, increased IL‐17 and IFN‐γ production and pSTAT3 and IL‐23R expression. These findings demonstrate that increased PD‐1 expression and decreased pSTAT3 expression reduce IL‐23 receptor expression and IL‐17 production by CD4⁺ T cells of tuberculosis patients.     

Loss of regulatory characteristics in CD4+CD25+/hi T cells induced by impaired transforming growth factor beta secretion in pneumoconiosis

Pneumoconiosis is caused by the accumulation of airborne dust in the lung, which stimulates a progressive inflammatory response that ultimately results in lung fibrosis and respiratory failure. It is possible that regulatory cells in the immune system could function to suppress inflammation and possibly slow or reverse disease progression. However, results in this study suggest that in pneumoconiosis patients, the regulatory T cells (Tregs) and B cells are functionally impaired. First, we found that pneumoconiosis patients presented an upregulation of CD4⁺CD25⁺ T cells compared to controls, whereas the CD4⁺CD25⁺ and CD4⁺CD25^hi T cells were enriched with Th1‐ and Th17‐like cells but not Foxp3‐expressing Treg cells and evidenced by significantly higher T‐bet, interferon (IFN)‐γ, and interleukin (IL)‐17 expression but lower Foxp3 and transforming growth factor (TGF)‐β expression. Regarding the CD4⁺CD25^hi T‐cell subset, the frequency of this cell type in pneumoconiosis patients was significantly reduced compared to controls, together with a reduction in Foxp3 and TGF‐β and an enrichment in T‐bet, RORγt, IFN‐γ, and IL‐17. This skewing toward Th1 and Th17 types of inflammation could be driven by monocytes and B cells, since after depleting CD14⁺ monocytes and CD19⁺ B cells, the levels of IFN‐γ and IL‐17 were significantly decreased. Whole peripheral blood mononuclear cells and isolated monocytes and B cells in pneumoconiosis patients also presented reduced capacity of TGF‐β secretion. Furthermore, monocytes and B cells from pneumoconiosis patients presented reduced capacity in inducing Foxp3 upregulation, a function that could be rescued by exogenous TGF‐β. Together, these data indicated a potential pathway for the progression of pneumoconiosis through a loss of Foxp3⁺ Treg cells associated with impaired TGF‐β secretion.     

Expression of interleukin (IL)‐19 and IL‐24 in inflammatory bowel disease patients: a cross‐sectional study

Interleukin (IL)‐19 and IL‐24 belong to the IL‐20 subfamily, and are involved in host defence against bacteria and fungi, tissue remodelling and wound healing. Nevertheless, no previous studies have explored their expression in Mexican mestizo patients with inflammatory bowel disease (IBD). The aim of the study was to characterize and to enumerate peripheral and tissue IL‐19‐ and IL‐24‐producing cells, as well as gene expression in patients with IBD with regard to its clinical activity. We studied a total of 77 patients with ulcerative colitis (UC), 36 Crohn's disease (CD) and 33 patients as control group (without endoscopic evidence of intestinal inflammation). Gene expression was measured by real‐time–polymerase chain reaction (RT–PCR). Protein expression was detected in biopsies by immunohistochemistry and in freshly isolated peripheral blood mononuclear cells by flow cytometry. IL‐19 and IL‐24 gene expression was elevated significantly in patients with active IBD versus the inactive disease and non‐inflammatory control groups (P < 0·05). However, IL‐19‐ and IL‐24‐producing cells were only increased in active CD versus active UC and non‐inflammatory tissues (P < 0·05). IL‐19 was produced conspicuously by circulating B cells and monocytes in patients with inactive disease (P < 0·05). Conversely, IL‐24 was noticeably synthesized by peripheral B cells, CD4⁺ T cells, CD8⁺ T cells and monocytes in patients with active disease. In conclusion, IL‐19‐ and IL‐24‐producing cells in active CD patients were increased compared with active UC and non‐inflammatory tissues. These cytokines could significantly shape and differentiate inflammatory process, severity and tolerance loss between UC and CD pathophysiology.     

Periodontitis‐associated pathogens P. gingivalis and A. actinomycetemcomitans activate human CD14+ monocytes leading to enhanced Th17/IL‐17 responses

The Th17/IL‐17 pathway is implicated in the pathogenesis of periodontitis (PD), however the mechanisms are not fully understood. We investigated the mechanism by which the periodontal pathogens Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa) promote a Th17/IL‐17 response in vitro, and studied IL‐17⁺ CD4⁺ T‐cell frequencies in gingival tissue and peripheral blood from patients with PD versus periodontally healthy controls. Addition of Pg or Aa to monocyte/CD4⁺ T‐cell co‐cultures promoted a Th17/IL‐17 response in vitro in a dose‐ and time‐dependent manner. Pg or Aa stimulation of monocytes resulted in increased CD40, CD54 and HLA‐DR expression, and enhanced TNF‐α, IL‐1β, IL‐6 and IL‐23 production. Mechanistically, IL‐17 production in Pg‐stimulated co‐cultures was partially dependent on IL‐1β, IL‐23 and TLR2/TLR4 signalling. Increased frequencies of IL‐17⁺ cells were observed in gingival tissue from patients with PD compared to healthy subjects. No differences were observed in IL‐17⁺ CD4⁺ T‐cell frequencies in peripheral blood. In vitro, Pg induced significantly higher IL‐17 production in anti‐CD3 mAb‐stimulated monocyte/CD4⁺ T‐cell co‐cultures from patients with PD compared to healthy controls. Our data suggest that periodontal pathogens can activate monocytes, resulting in increased IL‐17 production by human CD4⁺ T cells, a process that appears enhanced in patients with PD.     

Glatiramer acetate attenuates the pro‐migratory profile of adhesion molecules on various immune cell subsets in multiple sclerosis

An altered expression pattern of adhesion molecules (AM) on the surface of immune cells is a premise for their extravasation into the central nervous system (CNS) and the formation of acute brain lesions in multiple sclerosis (MS). We evaluated the impact of glatiramer acetate (GA) on cell‐bound and soluble AM in the peripheral blood of patients with relapsing–remitting MS (RRMS). Fifteen patients treated de novo with GA were studied on four occasions over a period of 12 months. Surface levels of intracellular cell adhesion molecule (ICAM)‐1, ICAM‐3, lymphocyte function‐associated antigen (LFA)‐1 and very late activation antigen (VLA)‐4 were assessed in T cells (CD3⁺CD8⁺, CD3⁺CD4⁺), B cells, natural killer (NK) cells, natural killer T cells (NK T) and monocytes by five‐colour flow cytometry. Soluble E‐selectin, ICAM‐1, ICAM‐3, platelet endothelial cell adhesion molecule (PECAM)‐1, P‐selectin and vascular cell adhesion molecule (VCAM)‐1 were determined with a fluorescent bead‐based immunoassay. The pro‐migratory pattern in RRMS was verified by comparison with healthy controls and was characterized by up‐regulation of LFA‐1 (CD3⁺CD4⁺ T cells, B cells), VLA‐4 (CD3⁺CD8⁺ T cells, NK cells), ICAM‐1 (B cells) and ICAM‐3 (NK cells). Effects of GA treatment were most pronounced after 6 months and included attenuated levels of LFA‐1 (CD3⁺CD4⁺) and VLA‐4 (CD3⁺CD4⁺, CD3⁺CD8⁺, NK, NK T, monocytes). Further effects included lowering of ICAM‐1 and ICAM‐3 levels in almost all immune cell subsets. Soluble AM levels in RRMS did not differ from healthy controls and remained unaltered after GA treatment. The deregulated pro‐migratory expression profile of cell‐bound AM is altered by GA treatment. While this alteration may contribute to the beneficial action of the drug, the protracted development and unselective changes indicate more secondary immune regulatory phenomena related to these effects.     

Dysregulation of toll‐like receptor (TLR) 2 expression on monocytes and upregulation of the frequency of T cells expressing TLR2 in patients with chronic hepatitis C virus infection

Toll‐like receptors (TLRs) initiate inflammatory responses that may play a role in disease progression in patients infected with hepatitis C virus (HCV). TLR2 and TLR4 surface expression were assessed on CD14⁺ monocytes, CD4⁺ and CD8⁺ T cells in treatment naïve patients with chronic HCV infection with fibrosis, without fibrosis, co‐infected with human immunodeficiency virus (HIV), and in healthy controls. Increased expression of TLR2 was found on monocytes in HCV‐infected patients with fibrosis (p < 0.01), co‐infected with HIV (p = 0.03), and possibly in patients without fibrosis (p = 0.07) when compared to controls. TLR2 positive CD4⁺ and CD8⁺ T cells were upregulated in patients with fibrosis only (p < 0.01). However, expression of TLR2 was not associated with T cell activation. TLR4 expression was similar in patients and healthy controls. In conclusion, TLR2 expression on monocytes and the frequency of T cells expressing TLR2 may contribute to disease progression in chronic HCV infection.     

Comprehensive T‐cell immunophenotyping and next‐generation sequencing of human papillomavirus (HPV)‐positive and HPV‐negative head and neck squamous cell carcinomas

The success of programmed cell death 1 (PD‐1) inhibition in achieving a clinical response in a subset of head and neck squamous cell carcinoma (HNSCC) patients emphasizes the need to better understand the immunobiology of HNSCC. Immunophenotyping was performed for 30 HCSCC patients [16 human papillomavirus (HPV)‐positive; 14 HPV‐negative] on matched tissue from the primary tumour site, locally metastatic cervical lymph nodes (LNs), uninvolved local cervical LNs, and peripheral blood. CD4⁺ and CD8⁺ T‐cell lymphocytes obtained from tissue were analysed for expression levels of the inhibitory receptors PD‐1, TIM‐3 and CTLA‐4. Next‐generation sequencing of the T‐cell receptor (TCR) β chain was performed on patients (n = 9) to determine receptor repertoire diversity and for clonality analysis. HPV‐negative HNSCC patients, particularly those with stage IV disease, had significantly higher proportions of CD8⁺ T cells expressing CTLA‐4 in tumour tissue (P = 0.0013) and in peripheral blood (P = 0.0344) than HPV‐positive patients, as well as higher expression levels of TIM‐3⁺PD‐1⁺ CD8⁺ T cells (P = 0.0072) than controls. For all patients, PD‐1 expression on CD8⁺ T cells – particularly in HPV‐negative HNSCC cases – strongly correlated (r = 0.63, P = 0.013) with tumour size at the primary site. The top CD8⁺ TCR clones from tumour tissue significantly overlapped with circulating peripheral blood TCR clones (r = 0.946), and HPV‐positive patients had frequently expanded TCR clones that were more hydrophobic – and potentially more immunogenic – than those from HPV‐negative patients. Collectively, our findings demonstrate, for the first time, that high‐stage HPV‐negative HNSCC patients with primary tumours at different sites in the head and neck have elevated peripheral CTLA‐4⁺CD8⁺ T‐cell levels, that tumour‐familiar CD8⁺ T cells are detectable in peripheral blood from HNSCC patients, and that TCRs from HPV‐positive HNSCC patients potentially recognize distinctly immunogenic cognate antigens. However, our findings are preliminary, and need to be further confirmed in a larger patient cohort; also, how these factors affect patient response to immunotherapy needs to be determined. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Oestrogen up‐regulates interleukin‐21 production by CD4+ T lymphocytes in patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease in which abnormal immune responses are mediated by tissue‐binding autoantibodies and immune complex deposition. Because most SLE patients are women of child‐bearing age, oestrogen has been suggested to play an important role in SLE pathogenesis. One proposed role is to induce B‐cell activation, culminating in increased autoantibody production. Interleukin‐21 (IL‐21) has been shown to be crucial in the differentiation of activated B cells into plasma cells. We therefore hypothesized that oestrogen up‐regulates IL‐21 production and induces subsequent B‐cell activation in SLE patients. Peripheral blood was obtained from 22 SLE patients and 16 healthy controls. Expression levels of IL‐21 and its receptor in serum, peripheral blood mononuclear cells, and CD4⁺ T cells were higher in SLE patients than in healthy controls. Exposure of CD4⁺ T cells from SLE patients to 17β‐oestradiol led to a dose‐ and time‐dependent increase in IL‐21 expression, which was abolished in the presence of mitogen‐activated protein kinase (MAPK) (MAPK kinase, p38, Jun N‐terminal kinase) inhibitors. B cells from healthy controls showed increased antibody production when they were co‐cultured with oestrogen‐treated CD4⁺ T cells from SLE patients. Treatment with IL‐21 antibody abrogated the increased antibody production of the co‐culture systems. This study revealed the association between oestrogen and IL‐21 in SLE patients. Oestrogen up‐regulates IL‐21 expression of CD4⁺ T cells via MAPK‐dependent pathways in SLE patients, which in turn induces increased antibody production by B cells.     

Increased Tim-3 expression on peripheral lymphocytes from patients with rheumatoid arthritis negatively correlates with disease activity

Tim-3 has been reported as an important regulatory molecule and plays a pivotal role in several autoimmunity diseases. Here, we demonstrated the increased expression of Tim-3 on peripheral CD4⁺ T, CD8⁺ T, NKT cells and monocytes from RA patients compared to those from healthy controls. Percentage of Tim-3⁺ cells in peripheral blood mononuclear cells (PBMCs) showed an inverse correlation with disease activity score 28 (DAS28) and plasma TNF-α level. Similar negative correlations were found between disease activity and Tim-3 levels on CD4⁺ T, CD8⁺ T and NKT cells. Consistently, Tim-3 expression on CD3⁺ T cells was further increased in patients with disease remission after treatment. Tim-3 expression on CD8⁺ T and NKT cells negatively correlates with plasma TNF-α. Our results suggest that Tim-3 might participate in the proceeding of RA by its negative regulation on various T cell subsets. Tim-3 might be a potential new marker for assessing severity of RA.     

Patients with Wegener’s granulomatosis demonstrate a relative deficiency and functional impairment of T‐regulatory cells

An increased proportion of CD4⁺ CD25⁺ T cells has been reported in Wegener’s granulomatosis (WG) and may represent an accumulation of regulatory T cells (Treg). CD25 is also expressed on recently activated effector T cells. We have determined the relative proportion of these subsets in a large patient cohort. The fraction of Treg in peripheral blood mononuclear cells from patients and healthy controls was determined by assessment of Foxp3 expression on CD4⁺ CD25⁺ T cells. The functional activity of Treg was determined by their ability to suppress proliferation and cytokine production in response to proteinase‐3. Although WG patients demonstrated an increased fraction of CD4⁺ CD25⁺ T cells, the percentage of Foxp3‐positive cells was decreased. In addition, the percentage of Treg was inversely related to the rate of disease relapse. CD4⁺ CD25^hi T cells were able to suppress T‐cell proliferation to proteinase‐3 in healthy controls and anti‐neutrophil cytoplasm antibody (ANCA)‐ negative patients (at time of sampling) but not in ANCA‐positive patients. In patients with active disease, an increased proportion of CD4⁺ Foxp3⁺ cells was associated with a more rapid disease remission. Patients with WG demonstrate abnormalities in the number and function of Treg and this is most pronounced in those with most active disease. This information is of value in understanding the pathogenesis and potential treatment of this disease.     

HMGB1 Promotes the Differentiation of Th17 via Up‐Regulating TLR2 and IL‐23 of CD14+ Monocytes from Patients with Rheumatoid Arthritis

High‐mobility group box 1 (HMGB1) is a non‐histone nuclear protein that is released extracellulary and has been implicated in autoimmune disease. Toll‐like receptor 2 (TLR2) signalling is thought to be essential for the inflammatory response and for immune disorders. In recent studies, enhanced HMGB1 and TLR2 expressions have been found in rheumatoid arthritis (RA), respectively. The aim of this study is to explore whether HMGB1 stimulation can up‐regulate the expression of TLR2 on CD14⁺ monocytes from patients with RA and to clarify the subsequent events involving Th17 cells and Th17 cell‐associated cytokine changes. Our results showed that the frequency of CD14⁺ cells in peripheral blood mononuclear cell (PBMC) was obviously increased, and enhanced expression of TLR2 on CD14⁺ monocytes was also found in patients with RA, compared with healthy controls with statistical significance (P < 0.001). In addition, the levels of IL‐17, IL‐23 and IL‐6 in supernatants from cultured monocytes from patients and in patient’s plasma were increased, and NF‐κB, the downstream target of TLR2, also showed a marked elevation after monocytes were stimulated by HMGB1. This implies that the enhanced TLR2 pathway and Th17 cell polarization may be due to HMGB1 stimulation in rheumatoid arthritis.     

Direct cloning and tetramer staining to measure the frequency of intestinal gluten‐reactive T cells in celiac disease

Knowledge of the frequency of disease‐driving CD4⁺ T cells in lesions of chronic human inflammatory diseases is limited. In celiac disease (CD), intestinal gluten‐reactive CD4⁺ T cells, which recognize gluten peptides only in the context of the disease‐associated HLA‐DQ molecules, are key pathogenic players. By cloning CD4⁺ T cells directly from intestinal biopsies of CD patients, we found that 0.5–1.8% of CD4⁺ T cells were gluten reactive. About half of the gluten‐reactive T cells were specific for either the immuno‐dominant DQ2.5‐glia‐α1a or DQ2.5‐glia‐α2 epitopes, suggesting that direct visualization of gluten‐specific T cells could be possible. Assessed by flow cytometry, tetramer‐positive T cells were present in 10/10 untreated CD patients with a frequency of 0.1–1.2% of CD4⁺ T cells. Gluten‐specific T cells were also detectable in most treated CD patients (7/10). Moreover, the frequency of gluten‐specific T cells correlated with the degree of histological damage in the gut mucosa as scored by Marsh‐grading, and also with serum IgA anti‐transglutaminase 2 antibody levels. Tetramer staining of gluten‐reactive T cells in biopsy material is a useful tool for future studies of such cells in CD and could also potentially serve as a diagnostic supplement in selected cases.     

Emerging role of stem cell memory‐like T cell in immune thrombocytopenia

Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by autoantibody‐mediated platelet destruction. Multiple factors have been implicated in ITP pathogenesis, including T‐lymphocyte dysfunctions. Increasing studies have indicated that stem cell memory‐like T cell (TSCM) plays an important role in the development of multiple autoimmune diseases. This study aimed to explore the clinical correlation between the TSCM subset and ITP. The percentages of peripheral blood naïve T cells (TNs), TSCMs, central memory T cells (TCMs), effector memory T cells (TEMs) and effector T cells (TEs) among CD4⁺ and CD8⁺ T cells in 20 ITP patients before and after treatment were detected using flow cytometry. Our results showed that the percentages of peripheral blood CD4⁺ and CD8⁺ T cells in ITP patients were imbalanced. The percentage of CD8⁺ TSCMs in peripheral blood before treatment in ITP patients was significantly higher than that in healthy controls, whereas the percentages of the other T cell subsets did not exhibit significant differences. Our study further analysed the correlation between the change in the percentage of CD8⁺ TSCMs and the treatment efficacy. The results showed that the percentage of peripheral blood CD8⁺ TSCMs in ITP patients after glucocorticoid treatment significantly decreased and the changes of the percentages of CD8⁺ TSCMs before and after treatment in complete response (CR) and response (R) patients were obvious. Our finding showed that the imbalance of the percentage of CD8⁺ TSCMs might be involved in the development of ITP and might serve as a novel indicator of efficacy.