The receptor PD-1 controls follicular regulatory T cells in the lymph nodes and blood.

CD4(+)CXCR5(+)Foxp3(+) follicular regulatory T cells (T(FR) cells) inhibit humoral immunity mediated by CD4(+)CXCR5(+)Foxp3(-) follicular helper T cells (T(FH) cells). Although the inhibitory receptor PD-1 is expressed by both cell types, its role in the differentiation of T(FR) cells is unknown. Here we found that mice deficient in PD-1 and its ligand PD-L1 had a greater abundance of T(FR) cells in the lymph nodes and that those T(FR) cells had enhanced suppressive ability. We also found substantial populations of T(FR) cells in mouse blood and demonstrated that T(FR) cells in the blood homed to lymph nodes and potently inhibited T(FH) cells in vivo. T(FR) cells in the blood required signaling via the costimulatory receptors CD28 and ICOS but were inhibited by PD-1 and PD-L1. Our findings demonstrate mechanisms by which the PD-1 pathway regulates antibody production and help reconcile inconsistencies surrounding the role of this pathway in humoral immunity.

Correlations of the expression of γδ T cells and their co-stimulatory molecules TIGIT, PD-1, ICOS and BTLA with PR and PIBF in the peripheral blood and decidual tissues of women with unexplained recurrent spontaneous abortion.

Semi-allogeneic embryos are not rejected by the maternal immune system due to maternal-fetal immune tolerance. Progesterone (P) receptor (PR)-expressing γδ T cells are present in healthy pregnant women. In the presence of P, these cells secrete an immunomodulatory protein called progesterone-induced blocking factor (PIBF), which can facilitate immune escape and is important in preventing embryonic rejection. This work investigated the correlations of the expression of γδ T cells and their co-stimulatory molecules T cell immunoglobulin and ITIM domain (TIGIT), programmed cell death 1 (PD-1), inducible co-stimulator (ICOS) and B and T lymphocyte attenuator (BTLA) with progesterone receptor (PR) and progesterone-induced blocking factor (PIBF) in peripheral blood and decidual tissue in women with unexplained recurrent spontaneous abortion (URSA) and normal pregnant (NP) women. We confirmed that γδ T cell proportions and PIBF expression in the peripheral blood and decidua of URSA women decreased significantly, while PR expression in decidua decreased. However, TIGIT, PD-1, ICOS and BTLA expression in γδ T cells in peripheral blood did not change, while TIGIT and PD-1 expression in γδ T cells in decidua increased significantly. Under the action of PHA-P (10 µg/ml), co-blocking of TIGIT (15 µg/ml) and PD-1 (10 µg/ml) antibodies further induced γδ T cell proliferation, but PIBF levels in the culture medium supernatant did not change. At 10-10 M P, γδ T cells proliferated significantly, and PIBF concentrations in the culture medium supernatant increased. γδ T cells co-cultured with P, TIGIT and PD-1 blocking antibodies showed the most significant proliferation, and PIBF concentrations in the culture medium supernatant were the highest. These results confirm that P is necessary for PIBF production. The TIGIT and PD-1 pathways participate in γδ T cell proliferation and activation and PIBF expression and play important roles in maintaining pregnancy.

Induction of peripheral blood T follicular helper cells expressing ICOS correlates with antibody response to hepatitis B vaccination.

T follicular helper (TFH) cells, a critical subset of CD4+ T cells, provide help to B cells during the procession of the humoral immune response in the germinal center (GC) and extrafollicular sites. CXCR5+ CD4+ T cells in human circulating blood, referred to herein as peripheral TFH (pTFH) cells, share phenotypes and functional properties with TFH cells in GC. Hepatitis B vaccine protects about 60% of the chronic hepatitis C patients from hepatitis B. The immunological bases that lead to the induction of protective antibody response is not well understood. In the present study, the pTFH cells subsets were determined in 18 healthy controls (anti-HBs ≥ 100 mIU/mL; HC), 21 nonresponders (anti-HBs < 10 mIU/mL; NR), and 23 weak responders (10 mIU/mL ≤ anti-HBs < 100 mIU/mL; WR) of chronic hepatitis patients upon routine hepatitis B vaccination. Though the frequency of the pTFH cell was equivalent in HC, WR, and NR, ICOS+ pTFH cells in HC underwent expansion with increased IL-21 secretion and production of serum anti-HBs response at 4 weeks after a full course of hepatitis B vaccination. These changes were not shown in both NR and WR. Analysis of ICOS+ pTFH cells represents a novel cellular determinant of the hepatitis B vaccine-induced humoral immune response, which may have relevance for design of hepatitis B vaccine.

Aberrant populations of circulating T follicular helper cells and regulatory B cells underlying idiopathic pulmonary fibrosis.

BACKGROUND: T follicular helper (Tfh) cells have been identified as a new category of helper T cells, which express CXCR5 on their surface and induce the production of antigen-specific antibodies. Many investigations have found morbid proliferation and/or activation of Tfh cells in systemic autoimmune and allergic diseases. It is also known that Tfh cells are regulated by regulatory B (Breg) cells in the deteriorating such diseases. Recently, CXCL13, a ligand of CXCR5, has been reported to increase in the peripheral blood and lungs of patients with idiopathic pulmonary fibrosis (IPF). This study aimed to investigate the involvement of Tfh cells and Breg cells in IPF. METHODS: Peripheral blood samples were obtained from 18 patients with IPF. We isolated heparinized peripheral blood mononuclear cells and investigated the proportions of Breg cells, Tfh cells, PD-1+ICOS+ Tfh cells (activated form of Tfh cells), and the Tfh-cell subsets by flow cytometry. These cell profiles were compared with those of 21 healthy controls. Furthermore, we investigated the correlations between profiles of lymphocytes and lung physiology. RESULTS: The median proportions of Tfh cells per total CD4+ T cells and of PD-1+ICOS+ proportion of Tfh cells per total Tfh cells was significantly more in the IPF patients (20.4 and 5.2%, respectively) compared with healthy controls (15.4 and 2.1%, respectively; p = 0.042 and p = 0.004, respectively). The proportion of Tfh2 cells per total Tfh cells was significantly higher and the proportion of Tfh17 was smaller in the IPF patients than healthy controls. The percentage of Breg cells to total B cells was significantly decreased in the IPF patients (median, 8.5%) compared with that in the controls (median, 19.7%; p < 0.001). The proportion of Breg cells was positively correlated with the annual relative change in diffusing capacity of the lungs for carbon monoxide in the IPF patients (r = 0.583, p = 0.018). CONCLUSION: Proliferation and activation of Tfh cells and a decrease in Breg cells were observed in the peripheral blood of patients with IPF. The profile of the Tfh-cell subset also changed. Specific humoral immunity aberration would likely underlie complicated pathophysiology of IPF.

Virus-Specific CD4+ T Cells Have Functional and Phenotypic Characteristics of Follicular T-Helper Cells in Patients With Acute and Chronic HCV Infections.

BACKGROUND & AIMS: Follicular T-helper (Tfh) cells contribute to pathogen-specific antibody responses by providing maturation signals to B cells. In mice with viral infections, virus-specific Tfh cells expand and are required to contain the infection. However, less is known about human virus-specific Tfh cells or their functions during infection. We investigated whether virus-specific CD4+ T cells from patients with hepatitis C virus (HCV) infection had phenotypic or functional features of Tfh cells and contribute to the production of HCV-specific antibodies. METHODS: We collected blood samples from patients with acute and chronic HCV infection and healthy individuals (controls). We performed MHC class II tetramer analyses, assays to detect intracellular cytokines in response to HCV exposure, and analyses to quantify HCV-specific antibodies. In addition, we collected liver tissues from patients with chronic HCV infection or nonviral liver disease to analyze markers of Tfh cells. RESULTS: HCV-specific CD4+ T cells from patients with acute HCV infection expressed markers of Tfh cells and secreted interleukin 21 in response to HCV exposure. Longitudinal analyses of HCV-specific T-cell responses and antibody responses showed an association between expression of inducible T-cell co-stimulator and induction of virus-specific antibodies in patients with acute HCV infection. Markers of Tfh cells were barely detectable in the peripheral blood samples from patients with chronic HCV infection, but were detected in liver tissues. CONCLUSIONS: Virus-specific Tfh cells can be detected in blood samples from patients with acute HCV infection; inducible T-cell co-stimulator expression correlates with production of HCV-specific antibodies. In patients with chronic infection, Tfh cells seem to disappear from the blood but are detectable in the liver.

High frequencies of circulating Tfh-Th17 cells in myasthenia gravis patients.

Recent studies show that the frequencies of circulating follicullar helper T (cTfh) cells are significantly higher in myasthenia gravis (MG) patients compared with healthy controls (HC). And, they are positively correlated with levels of serum anti-acetylcholine receptor antibody (anti-AchR Ab). It is unclear whether cTfh cell subset frequencies are altered and what role they play in MG patients. In order to clarify this, we examined the frequencies of cTfh cell counterparts, their subsets, and circulating plasmablasts in MG patients by flow cytometry. We determined the concentrations of serum anti-AChR Ab by enzyme-linked immunosorbent assay (ELISA). We assayed the function of cTfh cell subsets by flow cytometry and real-time polymerase chain reaction (RT-PCR). We found higher frequencies of cTfh cell counterparts, cTfh-Th17 cells, and plasmablasts in MG patients compared with HC. The frequencies of cTfh cell counterparts and cTfh-Th17 cells were positively correlated with the frequencies of plasmablasts and the concentrations of anti-AChR Ab in MG patients. Functional assays showed that activated cTfh-Th17 cells highly expressed key molecular features of Tfh cells including ICOS, PD-1, and IL-21. Results indicate that, just like cTfh cell counterparts, cTfh-Th17 cells may play a role in the immunopathogenesis and the production of anti-AChR Ab of MG.

Increased numbers of circulating ICOS⁺ follicular helper T and CD38⁺ plasma cells in patients with newly diagnosed primary biliary cirrhosis.

BACKGROUND: Aberrant activation of follicular helper T (TFH) and B cells is associated with the development of autoimmune diseases. However, little is known about the potential role of these cells in the development of primary biliary cirrhosis (PBC). AIM: This study aimed at characterizing the numbers of different subsets of circulating Tfh and B cells as well as evaluating their potential association with the levels of immunoglobulins and autoantibodies in newly diagnosed PBC patients. METHODS: The numbers of circulating CD27(+), CD38(+), CD86(+) and CD95(+) B cells as well as inducible T cell costimulator (ICOS)(+) and programmed death-1 (PD-1)(+), IL-21(+) TFH cells were examined in 58 patients with newly diagnosed PBC and 30 matched healthy controls (HCs). RESULTS: The numbers of circulating CD38(+)CD19(+), CD86(+)CD19(+), and CD95(+)CD19(+) B cells; CD3(+)CD4(+)CXCR5(+)ICOS(+) and CD3(+)CD4(+)CXCR5(+)PD-1(+) Tfh cells; and the levels of serum IL-21 in the PBC patients were significantly greater, but the numbers of CD27(+)CD19(+) B cells were significantly less than those in the HCs (p < 0.05). The numbers of CD3(+)CD4(+)CXCR5(+)ICOS(+) Tfh cells were positively correlated with the numbers of CD38(+)CD19(+) and CD86(+)CD38(+)CD19(+) B cells and the levels of serum anti-mitochondrial antibodies against M2 antigen (AMA-M2), AMA and immunolgubin M (IgM) in the PBC patients. The levels of serum IL-21 were positively correlated with the levels of serum AMA-M2, AMA, IgG and IgM, but negatively with the numbers of CD27(+)CD19(+) B cells in the PBC patients. CONCLUSIONS: Increased numbers of circulating ICOS(+) and IL-21(+) Tfh and CD38(+) plasma cells may be exhibited by patients with recent diagnoses of PBC.

Augmented ICOS expression in patients with early diffuse cutaneous systemic sclerosis.

OBJECTIVE: Inducible costimulator (ICOS), expressed on activated T cells, and its ligand, ICOS ligand (ICOSL), expressed on antigen-presenting cells, have been considered a single receptor-ligand pair. Here we investigated the expression of ICOS and ICOSL in patients with SSc. METHODS: ICOS expression on peripheral blood T cells, and ICOSL expression on B cells and macrophages was determined by flow cytometry. Expression of ICOS and ICOSL was assessed by immunohistological staining and real-time PCR of lesional skin. RESULTS: ICOS expression levels were specifically increased on both peripheral blood memory T cells and Tregs from early dcSSc patients compared with those from healthy controls. Mean ICOSL expression on B cells or macrophages was comparable between SSc patients and healthy controls. ICOS-expressing T cells, ICOSL-expressing macrophages and mRNA levels of ICOS and ICOSL were increased in the lesional skin of patients with early dcSSc. In vitro ICOS costimulation enhanced production of IFN-γ, IL-4 and IL-17A from T cells in SSc patients vs normal controls. Soluble ICOS levels were significantly increased in SSc patients and were negatively associated with the presence of ACAs and positively associated with CRP values. Serum levels of soluble ICOS were more closely associated with clinical features compared with levels of soluble IL-2 receptor. CONCLUSION: Augmented ICOS signalling may contribute to the pathogenesis of SSc during early progressive disease. Soluble ICOS levels may be used as a serum marker for the activity and severity of SSc.

Aberrant production of soluble inducible T cell co­stimulator and soluble programmed cell death protein 1 in patients with chronic hepatitis B.

Previous studies demonstrated that immune dysregulation is an important cause of hepatitis B virus (HBV)­mediated liver damage. Co­stimulators including programmed cell death protein 1 (PD­1) and inducible T cell co­stimulator (ICOS) are involved in the pathogenesis of HBV. In the present study, the serum levels of soluble (s)PD­1 and sICOS in patients with chronic HBV infections, were investigated, and the association between sPD­1 and sICOS levels and liver injury degree was investigated. Serum sPD­1 and sICOS levels were increased in the HBV­patient group particularly in the HBV external core antigen positive group. In the immune clearance group, sPD­1 and sICOS were increased compared with the tolerance group. Furthermore, the relative mRNA expression levels were also increased in patients with HBV. However there was no correlation between sPD­1 and sICOS levels and HBV antibodies or PD­1/ICOS mRNA copies. The altered sPD­1 and sICOS serum levels in the different HBV groups may reflect the dysregulation of T cell activation, and may be associated with the HBV pathological process.

Circulating CCR7+ICOS+ Memory T Follicular Helper Cells in Patients with Multiple Sclerosis.

OBJECTIVE: This study is aimed at examining the potential roles of circulating memory T follicular helper (Tfh) cells in patients with multiple sclerosis (MS). METHODS: The numbers of different subsets of circulating memory Tfh cells in 25 patients with relapsed MS before and after treatment as well as 14 healthy controls (HC) were examined by flow cytometry. The levels of plasma IL-21 in all patients and cerebrospinal fluid (CSF) IL-21 in some MS patients and controls with non-inflammatory neuronal diseases (NND) were measured by ELISA. RESULTS: In comparison with that in the HC, the numbers of circulating CD3+CD4+CXCR5+CD45RA-, ICOS+, CCR7+ and CCR7+ICOS+ memory Tfh cells and the levels of plasma IL-21 significantly increased in MS patients, but significantly decreased in the patients with complete remission (CR). The levels of CSF IL-21 were significantly higher in the MS patients than that in the NND patients. The numbers of CCR7+ICOS+ memory Tfh cells were positively correlated with the EDSS scores, the levels of plasma and CSF IL-21, IgG, MBP-Ab or MOG-Ab. CONCLUSIONS: Our findings indicated that circulating memory Tfh cells, especially CCR7+ICOS+ memory Tfh cells, may be associated with the relapse of MS and may serve as a new therapeutic target.

Immunoregulatory molecules in patients with gestational diabetes mellitus.

Induction of maternal-fetal immune tolerance is essential for the development of normal pregnancy. Impaired expression of costimulatory molecules may lead to intense inflammatory reaction, a mechanism involved in the pathophysiology of gestational diabetes mellitus (GDM). The aim of this study was to investigate whether immunoregulatory molecules are involved in the physiopathology of GDM. This case-control study included 30 healthy pregnant women and 20 GDM patients. Flow cytometry was used to assess peripheral blood T subpopulations (CD4(+) and CD8(+)), the expression of immunoregulatory molecules (CD28, ICOS, CTLA-4, and PD-1) and activation markers (CD69 and HLA-DR). Compared to healthy women, GDM patients had a significantly higher frequency of CD4(+)CD69(+) and CD8(+)CD69(+) T cells; only patients with insulin-treated GDM had increased numbers of CD4(+)HLA-DR(+) T cells. We also observed significantly higher percentages of CD4(+)CD28(+)HLA-DR(+), CD3(+)CD4(+)ICOS(+), CD3(+)CD4(+)PD-1(+), CD8(+)CD28(+)CD69(+), CD8(+)CD28(+)HLA-DR(+), CD8(+)CTLA-4(+)HLA-DR(+), and CD3(+)CD8(+)ICOS(+) T cells and lower frequency of CD3(+)CD4(+)CTLA-4(+), CD3(+)CD8(+)CTLA-4(+), and CD8(+)ICOS(+)HLA-DR(+) T cells in GDM patients compared to healthy pregnant women. This first study assessing costimulatory molecules in GDM patients shows that these patients have exacerbated markers of T cell activation along with CTLA-4 deficiency, findings that indicate that the maternal-fetal tolerance is compromised in these patients.

Effect of soluble inducible costimulator level and its polymorphisms on age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly population. Evidence has shown that the human immune system may play critical roles in this disease. Inducible costimulator (ICOS) promotes T-cell activation, differentiation, and T:B-cell interactions. The aim of the study was to understand the effect of ICOS on the development of AMD from genetic polymorphism perspective and serum level perspective. Two ICOS polymorphisms, rs10183087A/C and rs10932037C/T, were tested in 223 AMD cases and 262 healthy controls. The serum level of soluble ICOS (sICOS) was compared among subjects with different genotypes, as well as between AMD patients and controls. Data showed that prevalence of rs10183087CC genotype was significantly increased in AMD than in controls (p=0.001). Function analysis revealed that subjects carrying rs10183087CC genotype had higher serum levels of sICOS than those with AA or AC genotypes (p<0.05). When we compared serum levels of sICOS between cases and controls, results showed that AMD patients had significantly increased sICOS levels than healthy donors (p<0.05). Also, wet type cases were observed to have higher sICOS levels than cases with dry type (p<0.05). These data suggested ICOS polymorphism could affect the susceptibility to AMD by elevating protein expression, and serum levels of sICOS may be closed correlated with the development and progression of this disease.

Increased production of soluble inducible costimulator in patients with diffuse cutaneous systemic sclerosis.

Inducible costimulator (ICOS) is crucial for T cell proliferation, production of various cytokines, and T cell-dependent B-cell responses. To determine the serum soluble ICOS (sICOS) level and its association with clinical parameters in patients with systemic sclerosis (SSc), serum sICOS level was examined by enzyme-linked immunosorbent assay in 38 patients with SSc and 24 healthy individuals. The expression of ICOS and ICOS ligand in skin was examined immunohistochemically. There was no significant difference in serum sICOS level between patients with SSc and healthy individuals. Patients with diffuse cutaneous SSc had higher levels of sICOS than those with limited cutaneous SSc (P < 0.05) or healthy individuals (P < 0.05). Serum sICOS level correlated positively with the severity of skin sclerosis. Patients with SSc and elevated sICOS level more often had interstitial lung disease and decreased vital capacity than those with normal sICOS level. The serum sICOS level was significantly greater in patients with early phase SSc than those with late phase SSc. ICOS and ICOS ligand immunostaining were observed on infiltrating dermal mononuclear cells in lesional skin tissue. These results suggest that the serum level of sICOS is increased in patients with diffuse cutaneous SSc and correlates with the severity and activity of skin sclerosis and interstitial lung disease. ICOS may contribute to the development of SSc. In addition, measurement of serum sICOS level in patients with early SSc may offer an important means for further evaluation of SSc disease severity.

Involvement of inducible costimulator- and interleukin 10-positive regulatory T cells in the development of IgG4-related autoimmune pancreatitis.

OBJECTIVES: Immunoglobulin G4 (IgG4)-related autoimmune pancreatitis (AIP) is a new clinical entity of pancreatic disorder. There are immunologic and histological abnormalities, including increased serum IgG4 levels and the infiltration of IgG4-positive plasmacytes. However, the role of IgG4 is unclear. Recently, regulatory T cells (Tregs) were reported to contribute to the development of various autoimmune diseases as well as in B-cell shifting to IgG4-producing plasmacytes. We studied Tregs in the pancreas and peripheral blood. METHODS: We recruited 44 patients with IgG4-related AIP. For comparison, we recruited 37 patients with other pancreatic diseases and 27 healthy subjects as controls. We studied infiltrating cells in the pancreas by immunohistochemistry and analyzed inducible costimulator-positive Tregs and interleukin 10-positive Tregs in the peripheral blood by flow cytometry. RESULTS: The ratio of Foxp3-positive cells to infiltrated mononuclear cells (Foxp3/Mono) in AIP patients was significantly higher than in patients with alcoholic chronic pancreatitis. In AIP, Foxp3/Mono and IgG4/Mono were positively correlated. Inducible costimulator-positive Tregs were significantly higher in AIP patients than in the patients with other pancreatic diseases and the healthy control group. Interleukin 10-positive Tregs were significantly higher in AIP patients than in the healthy control group. CONCLUSIONS: Increased quantities of inducible costimulator-positive Tregs may influence IgG4 production in IgG4-related AIP.