Higher Frequency of Circulating PD-1high CXCR5+CD4+ Tfh Cells in Patients with Chronic Schistosomiasis

The current knowledge of immunological responses to schistosomiasis is insufficient for the development of vaccine and therapies. The role of T follicular helper (Tfh) cells in schistosome infections is not fully defined. The frequency of circulating Tfh cells and serum cytokine levels were analyzed in 11 patients with chronic schistosomiasis and 10 healthy controls (HC), who reside in an endemic area for Schistosomiasis japonicum. Significantly higher frequencies of circulating CXCR5⁺ CD4⁺ Tfh cells and higher expression levels of ICOS and PD-1 in CXCR5⁺ CD4⁺ Tfh cells were observed in patients with chronic schistosomiasis compared to HC. The levels of IL-21 in serum and the expression of IL-21 mRNA were higher in chronic schistosomiasis patients than in HC. Moreover, the frequency of circulating PD-1^high CXCR5⁺ CD4⁺ Tfh cells positively correlated with the levels of IL-21 in serum from patients with chronic schistosomiasis. A positive correlation was also found between the frequency of PD-1^high CXCR5⁺ CD4⁺ Tfh cells and the levels of soluble egg antigen (SEA)-specific antibodies in serum samples from the patient group. Our study is the first regarding Tfh cells in chronic human schistosomiasis and the finding indicate that PD-1^high CXCR5⁺ CD4⁺Tfh cells might play an important role in the production of specific antibodies in schistosomiasis. This study contributes to the understanding of immune response to schistosomiasis and may provide helpful support in vaccine development.     

PD-1对伯氏疟原虫感染小鼠体液免疫应答的影响

利用伯氏疟原虫Plasmodium berghei ANKA(P.b ANKA)感染BALB/c小鼠,PD-1单抗阻断后,流式细胞术检测脾脏浆细胞、滤泡辅助性T细胞(Tfh)数量。qRT-PCR检测IL-21、IL-10和IL-6 mRNA水平,ELISA检测血清抗体,以探讨程序性死亡受体-1(programmed cell death-1, PD-1)在疟原虫初次感染中对体液免疫应答的影响。结果发现,PD-1单抗阻断加速了P.b ANKA感染小鼠的死亡。与对照组相比,PD-1阻断组感染后第12天短寿浆细胞(CD138~+CD44~+)数量明显降低(P0.05),长寿浆细胞(CD138~+CD44~-、CD138~-CD44~+)和Tfh(CD4~+CXCR5~+)细胞数量无差异性改变,脾细胞IL-21的mRNA水平明显下降(P0.05),血清抗裂殖子表面蛋白(merozoite surface protein, MSP)-1特异性IgG无明显改变。P.b ANKA感染中PD-1通路可能通过影响Tfh分泌IL-21进而干扰浆细胞数量影响体液免疫应答。     

Regulation of Autoimmune Germinal Center Reactions in Lupus-Prone BXD2 Mice by Follicular Helper T Cells

BXD2 mice spontaneously develop autoantibodies and subsequent glomerulonephritis, offering a useful animal model to study autoimmune lupus. Although initial studies showed a critical contribution of IL-17 and Th17 cells in mediating autoimmune B cell responses in BXD2 mice, the role of follicular helper T (Tfh) cells remains incompletely understood. We found that both the frequency of Th17 cells and the levels of IL-17 in circulation in BXD2 mice were comparable to those of wild-type. By contrast, the frequency of PD-1⁺CXCR5⁺ Tfh cells was significantly increased in BXD2 mice compared with wild-type mice, while the frequency of PD-1⁺CXCR5⁺Foxp3⁺ follicular regulatory T (Tfr) cells was reduced in the former group. The frequency of Tfh cells rather than that of Th17 cells was positively correlated with the frequency of germinal center B cells as well as the levels of autoantibodies to dsDNA. More importantly, CXCR5⁺ CD4⁺ T cells isolated from BXD2 mice induced the production of IgG from naïve B cells in an IL-21-dependent manner, while CCR6⁺ CD4⁺ T cells failed to do so. These results together demonstrate that Tfh cells rather than Th17 cells contribute to the autoimmune germinal center reactions in BXD2 mice.     

Role of the frequency of blood CD4(+) CXCR5(+) CCR6(+) T cells in autoimmunity in patients with Sjögren's syndrome

The blood CD4⁺ CXCR5⁺ T cells, known as “circulating” Tfh, have been shown to efficiently induce naïve B cells to produce immunoglobulin. They play an important role in certain autoimmune diseases. In the present study, we show for the first time that the frequency of CD4⁺ CXCR5⁺ T cells is increased in pSS patients and positively correlated with autoantibodies in the blood. The concentration of Th17-like subsets (CD4⁺ CXCR5⁺ CCR6⁺) in pSS patients was found to be significantly higher than in healthy controls. Functional assays showed that activated Th17-like subtypes in the blood display the key features of Tfh cells, including invariably coexpressed PD-1, ICOS, CD40L and IL-21. Th17 subsets were found to highly express Bcl-6 protein and Th1 and Th2 were not. Bcl-6 is believed to be a master transforming factor for Tfh cell differentiation and facilitate B cell proliferation and somatic hypermutation within the germinal center. These data indicate that Th17 subsets of CD4⁺ CXCR5⁺ T cells in the blood may participate in the antibody-related immune responses and that high frequency of CD4⁺ CXCR5⁺ CCR6⁺ Tfh cells in blood may be suitable biomarkers for the evaluation of the active immune stage of pSS patients. It might provide insights into the pathogenesis and perhaps help researchers identify novel therapeutic targets for pSS.     

Inhibition of Increased Circulating Tfh Cell by Anti-CD20 Monoclonal Antibody in Patients with Type 1 Diabetes

Objectives

Follicular helper T (Tfh) cells exert an important role in autoimmune diseases. Whether it might be involved in type 1 diabetes (T1D) is unknown. Our aim was to investigate the role of Tfh cells in patients with T1D and the effect of anti-CD20 monoclonal antibody (rituximab) on Tfh cells from T1D patients.

Patients and Methods

Fifty-four patients with T1D and 37 healthy controls were enrolled in the current study. 20 of those patients were treated with rituximab. The frequencies of circulating CD4⁺CXCR5⁺ICOS⁺T cells were analyzed by flow cytometry. The serum autoantibodies were detected by radioligand assay. The levels of IL-21, IL-6 and BCL-6 were assessed using ELISA and/or real-time PCR.

Results

Increased frequencies of circulating Tfh cells together with enhanced expression of IL-21 were detected in patients. The correlation between the frequencies of circulating Tfh cells and the serum autoantibodies or C-peptide level was comfirmed. After rituximab therapy, follow-up analysis demonstrated that the frequencies of circulating Tfh cell and serum IA2A were decreased. The levels of IL-21, IL-6 and Bcl-6 mRNA were decreased after treatment. Furthermore, beta cell function in 10 of 20 patients was improved.

Conclusions

These data indicate Tfh cells may participate in the T1D-relatede immune responses and B cells might play a role in the development of Tfh responses in the disease progression.     

Inhibition of Aberrant Circulating Tfh Cell Proportions by Corticosteroids in Patients with Systemic Lupus Erythematosus

Objective

To observe the proportion of peripheral T follicular helper (Tfh) cells in patients with systemic lupus erythematosus (SLE) and to assess the role of steroids on Tfh cells from SLE patients.

Methods

Peripheral blood mononuclear cells (PBMCs) from 42 SLE patients and 22 matched healthy subjects were collected to assess proportions of circulating CXCR5⁺PD1⁺/CD4⁺ T cells (Tfh), CD4⁺CCR6⁺ T cells (Th17-like) and CD19⁺CD138⁺ plasma cells by flow cytometry. 8 of the patients had their blood redrawn within one week after receiving methylprednisolone pulse treatment. Disease activity was evaluated by SLE disease activity index. To test the effect of IL-21 and corticosteroids on Tfh cells in vitro, PBMCs harvested from another 15 SLE patients were cultured with medium, IL-21, or IL-21+ dexamethasone for 24 hours and 72 hours. PBMCs from an independent 23 SLE patients were cultured with different concentrations of dexamethasone for 24 hours.

Results

Compared to normal controls, percentages of circulating Tfh cells, but not Th17 cells, were elevated in SLE patients and correlated with disease activity. Proportions of Tfh cells in SLE patients were positively correlated with those of plasma cells and serum levels of antinuclear antibodies. After methylprednisolone pulse treatment, both percentages and absolute numbers of circulating Tfh cells were significantly decreased. In vitro cultures showed an increase of Tfh cell proportion after IL-21 stimulation that was totally abolished by the addition of dexamethasone. Both 0.5 and 1 µM dexamethasone decreased Tfh cells dose dependently (overall p = 0.013).

Conclusions

We demonstrated that elevated circulating Tfh cell proportions in SLE patients correlated with their disease activities, and circulating levels of plasma cells and ANA. Corticosteroids treatment down-regulated aberrant circulating Tfh cell proportions both in vivo and in vitro, making Tfh cells a new treatment target for SLE patients.     

Circulating CXCR5+CD4+ T cells assist in the survival and growth of primary diffuse large B cell lymphoma cells through interleukin 10 pathway

Diffuse large B cell lymphoma (DLBCL) is a common and aggressive cancer caused by the malignant transformation of B cells. Although it has been established that the follicular helper T (Tfh) cells play a central role in B cell development, little information is available on their involvement in DLBCL pathogenesis. We studied the role of the peripheral Tfh equivalent, the CXCR5⁺ CD4⁺ T cells, in DLBCL. Data showed that compared to CXCR5^- CD4⁺ T cells, CXCR5⁺ CD4⁺ T cells were significantly more effective at promoting the proliferation as well as inhibiting the apoptosis of primary autologous DLBCL tumor cells. Surprisingly, we found that at equal cell numbers, CXCR5⁺ CD4⁺ T cells in DLBCL patients secreted significantly less interleukin (IL)-21 than CXCR5^- CD4⁺ T cells, while the level of IL-10 secretion was significant elevated in the CXCR5⁺ compartment compared to the CXCR5^- compartment. Neutralization of IL-10 in the primary DLBCL-CXCR5⁺ CD4⁺ T cell coculture compromised the CXCR5⁺ CD4⁺ T cell-mediated pro-tumor effects, in a manner that was dependent on the concentration of anti-IL-10 antibodies. The CXCR5⁺ compartment also contained significantly lower frequencies of cytotoxic CD4⁺ T cells than the CXCR5^- compartment. In conclusion, our investigations discovered a previously unknown pro-tumor role of CXCR5-expressing circulating CD4⁺ T cells, which assisted the survival and proliferation of primary DLBCL cells through IL-10.     

Rhesus Macaque Lymph Node PD-1hiCD4+ T Cells Express High Levels of CXCR5 and IL-21 and Display a CCR7loICOS+Bcl6+ T-Follicular Helper (Tfh) Cell Phenotype

CD4 T follicular helper (Tfh) cells play a unique and essential role in the generation of B cell responses in the lymph node microenvironment. Here we sought to determine if differential expression of PD-1 could be used to delineate Tfh cells in rhesus macaque lymph nodes (LN). CD3⁺CD4⁺ T cells were found to harbor a unique subset of cells that expressed the Program death-1 (PD-1) receptor at significantly high levels that were enriched in the LN compartment as compared to peripheral blood. The LN CD4⁺PD1^hi T cells expressed a predominantly CD28⁺CD95⁺ central memory phenotype and were CCR7^loICOS^hiBcl6^hi. Additionally, CD4⁺PD1^hi T cells preferentially expressed high levels of CXCR5 and IL-21 and significantly correlated with Bcl6⁺Ki-67⁺ IgG⁺ B cells. As Bcl6 is primarily expressed by proliferating B cells within active germinal centers, our results suggest that LN CD4⁺PD1^hi T cells likely localize to active GC regions, a characteristic that is attributable to Tfh cells. Overall, our findings suggest that high levels of PD-1 expression on CD4⁺ T cells in LN of rhesus macaques can serve as a valuable marker to identify Tfh cells and has implications for studying the role of Tfh cells in Human immunodeficiency virus (HIV), Simian immunodeficiency virus (SIV) and other infectious diseases that use the rhesus macaque model.     

Circulating CXCR5+CD4+ T Follicular-Like Helper Cell and Memory B Cell Responses to Human Papillomavirus Vaccines

Through the interaction of T follicular helper (Tfh) cells and B cells, efficacious vaccines can generate high-affinity, pathogen-neutralizing antibodies, and memory B cells. Using CXCR5, CXCR3, CCR6, CCR7, PD1, and ICOS as markers, Tfh-like cells can be identified in the circulation and be classified into three functionally distinct subsets that are PD1⁺ICOS⁺, PD1⁺ ICOS^-, or PD1^-ICOS^-. We used these markers to identify different subsets of CXCR5⁺CD4⁺ Tfh-like cells in response to highly immunogenic and efficacious vaccines for human papillomaviruses (HPV): Cervarix and Gardasil. In this small study, we used PBMC samples from 11 Gardasil recipients, and 8 Cervarix recipients from the Vaccine Research Center 902 Study to examine the induction of circulating Tfh-like cells and IgD^-CD38^HiCD27⁺ memory B cells by flow cytometry. PD1⁺ICOS⁺ CXCR3⁺CCR6^-CXCR5⁺CD4⁺ (Tfh1-like) cells were induced and peaked on Day (D) 7 post-first vaccination, but not as much on D7 post-third vaccination. We also observed a trend toward increase in PD1⁺ICOS⁺ CXCR3^-CCR6^-CXCR5⁺CD4⁺ (Tfh2-like) cells for both vaccines, and PD1⁺ICOS⁺ CXCR3^-CCR6⁺CXCR5⁺CD4⁺ (Tfh17-like) subset was induced by Cervarix post-first vaccination. There were also minimal changes in the other cellular subsets. In addition, Cervarix recipients had more memory B cells post-first vaccination than did Gardasil recipients at D14 and D30. We found frequencies of memory B cells at D30 correlated with anti-HPV16 and 18 antibody titers from D30, and the induction levels of memory B cells at D30 and PD1⁺ICOS⁺Tfh1-like cells at D7 post-first vaccination correlated for Cervarix. Our study showed that induction of circulating CXCR5⁺CD4⁺ Tfh-like subsets can be detected following immunization with HPV vaccines, and potentially be useful as a marker of immunogenicity of vaccines. However, further investigations should be extended to different cohorts with larger sample size to better understand the functions of these T cells, as well as their relationship with B cells and antibodies.     

The effect of aging on the frequency,phenotype and cytokine production of human blood CD4 + CXCR5 + T follicular helper cells: comparison of aged and young subjects

Background

T cell-dependent B-cell responses decline with age, indicating declined cognate helper activity of aged CD4?+?T cells for B cells. However, the mechanisms remain unclear. T follicular helper (Tfh) cells, a novel T helper subset, play an essential role in helping B cells differentiation into long-lived plasma cells in germinal center (GC) or short-lived plasma cells. In the present study, we proposed that there might existe changes of proportion, phenotype or cytokine production of blood Tfh cells in healthy elderly individuals compared with healthy young individuals.

Results

The results showed that frequencies of aged blood CXCR5?+?CD4?+?Tfh cells increased compared with young subjects. Both aged and young blood CXCR5?+?CD4?+?Tfh cells constitutively expressed CD45RO, CCR7 and CD28, and few of these cells expressed CD69 or HLA-DR, which indicated that they were resting memory cells. There was no significant difference of IL-21 frequency production by aged blood CXCR5?+?CD4?+?Tfh determined by FACS compared with young individuals, however, aged PBMCs produced significantly higher levels of IL-21 evaluated by ELISA. Furthermore, there were no significant differences of percentages of IFN-γ, IL-4, IL-17 or IL-22 production by aged Tfh cells compared with their counterparts of young individuals respectively. However, frequencies of IL-17+ cells within aged CD4?+?CXCR5-T cells were markedly lower than in the young individuals. Furthermore we observed different frequencies of IFN-γ, IL-17, IL-4 or IL-22 production by Tfh or by CD4?+?CXCR5- cells in aged and young subjects respectively.

Conclusions

Our data demonstrated that the frequencies of blood memory CXCR5?+?CD4?+?Tfh cells increased in the elderly population. There were similar frequencies of Th characterized cytokine production such as IL-21, IFN-γ, IL-4, IL-17 or IL-22 in aged and young Tfh cells. However, aged PBMCs produced a significantly higher amount of IL-21 compare to young subjects.

     

Impaired Antibody Response to Influenza Vaccine in HIV-Infected and Uninfected Aging Women Is Associated with Immune Activation and Inflammation

Background

Aging and HIV infection are independently associated with excessive immune activation and impaired immune responses to vaccines, but their relationships have not been examined.

Methods

For selecting an aging population we enrolled 28 post-menopausal women including 12 healthy volunteers and 16 HIV-infected women on antiretroviral treatment with <100 HIV RNA copies/ml. Antibody titers to trivalent influenza vaccination given during the 2011-2012 season were determined before and 4 weeks after vaccination.

Results

Seroprotective influenza antibody titers (≥1:40) were observed in 31% HIV⁺ and 58% HIV-uninfected women pre-vaccination. Following vaccination, magnitude of antibody responses and frequency of seroprotection were lower in HIV⁺ (75%) than in HIV^– (91%) women. Plasma IL-21, the signature cytokine of T follicular helper cells (Tfh), and CD4 T cell IL-21R were upregulated with seroconversion (≥4 fold increase in antibody titer). Post-vaccine antibody responses were inversely correlated with pre-vaccination plasma TNFα levels and with activated CD4 T cells, including activated peripheral (p)Tfh. Plasma TNFα levels were correlated with activated pTfh cells (r=0.48, p=0.02), and inversely with the post-vaccination levels of plasma IL-21 (r=-0.53, p=0.02). In vitro TNFα blockade improved the ability of CD4 T cells to produce IL-21 and of B cells to secrete immunoglobulins, and addition of exogenous IL-21 to cell cultures enhanced B cell function. Higher frequencies of activated and exhausted CD8 T and B cells were noted in HIV⁺ women, but these markers did not show a correlation with antibody responses.

Conclusions

In aging HIV-infected and uninfected women, activated CD4 and pTfh cells may compromise influenza vaccine-induced antibody response, for which a mechanism of TNFα-mediated impairment of pTfh-induced IL-21 secretion is postulated. Interventions aimed at reducing chronic inflammation and immune activation in aging, HIV-infected patients may improve their response to vaccines.     

Decreased Frequencies of Circulating CD4+ T Follicular Helper Cells Associated with Diminished Plasma IL-21 in Active Pulmonary Tuberculosis

Background

Circulating T follicular helper (Tfh) cells represent a distinct subset of CD4⁺ T cells and are important in immunity to infections. Although they have been shown to play a role in experimental models of tuberculosis infection, their role in human tuberculosis remains unexplored.

Aims/Methodology

To determine the distribution of circulating Tfh cells in human TB, we measured the frequencies of Tfh cells ex vivo and following TB - antigen or polyclonal stimulation in pulmonary TB (PTB; n = 30) and latent TB (LTB; n = 20) individuals, using the markers CXCR5, PD-1 and ICOS.

Results

We found that both ex vivo and TB - antigen induced frequencies of Tfh cell subsets was significantly lower in PTB compared to LTB individuals. Similarly, antigen induced frequencies of Tfh cells expressing IL-21 was also significantly lower in PTB individuals and this was reflected in diminished circulating levels of IL-21 and IFNγ. This was not accompanied by diminished frequencies of activated or memory B cell subsets. Finally, the diminution in frequency of Tfh cells in PTB individuals was dependent on IL-10, CTLA-4 and PD-L1 in vitro.

Conclusions

Thus, PTB is characterized by adiminution in the frequency of Tfh cell subsets.     

NLR、25-（OH）D3、IL-6、PCT与重症肺炎支原体肺炎患儿免疫功能和预后不良的关系研究

摘要 目的：探讨中性粒细胞与淋巴细胞比值（NLR）、25-羟维生素D3 [25-（OH）D3]、白细胞介素-6（IL-6）、降钙素原（PCT）与重症肺炎支原体肺炎（MMP）患儿免疫功能和预后不良的关系。方法：选取2019年2月至2021年12月我院收治的106例重症MMP患儿作为重症组,同期收治的101例轻症MMP患儿（轻症组）作为对照。检测外周血中性粒细胞计数、淋巴细胞计数、T淋巴细胞亚群以及血清25-（OH）D3、IL-6、PCT水平,计算NLR。分析NLR、25-（OH）D3、IL-6、PCT与T淋巴细胞亚群的相关性。重症MMP患儿治疗后随访半年,根据重症MMP患儿的预后情况分为预后良好组（75例）和预后不良组（31例）,多因素Logistic回归分析影响重症MMP患儿预后的因素。结果：重症组NLR、IL-6、PCT水平,CD8⁺高于轻症组（P＜0.05）,25-（OH）D3水平、CD3⁺、CD4⁺、CD4⁺/CD8⁺低于轻症组（P＜0.05）。NLR、IL-6、PCT水平与CD3⁺、CD4⁺、CD4⁺/CD8⁺呈负相关（P＜0.05）,与CD8⁺呈正相关（P＜0.05）;25-（OH）D3与CD3⁺、CD4⁺、CD4⁺/CD8⁺呈正相关（P＜0.05）,与CD8⁺呈负相关（P＜0.05）。多因素Logistic回归分析显示：肺大片实变影、NLR（较高）、IL-6（较高）、PCT（较高）是重症MPP患儿预后不良的危险因素（P＜0.05）,25-（OH）D3（较高）是保护因素（P＜0.05）。结论：重症MMP患儿NLR、IL-6、PCT水平升高,25-（OH）D3水平降低,且与细胞免疫功能低下以及预后不良有关,检测NLR、IL-6、PCT、25-（OH）D3有助于评估重症MMP患儿的预后。     

Altered chemokine receptor profile on circulating leukocytes in human heart failure

Chemokines and their receptors have been implicated in the pathogenesis of different forms of heart failure (HF). We examined CC-and CXC-chemokine receptor expression in fresh peripheral blood leukocyte populations from 24 end-stage HF patients consisting of coronary artery disease (CAD; n=6) and hypertrophic cardiomyopathy (HCM; n=7) or idiopathic dilated cardiomyopathy (IDCM; n=8) or valvular disease (VD; n=3) and compared the data with 18 healthy controls. Levels of CCR1, 2, 3, 4, 5, and 7, and CXCR1, 2, 3, and 4 were measured by flow cytometry, and the expression profile was assessed as molecules of equivalent soluble fluorochrome units as well as frequency (percentage) of CD3⁺, CD4⁺, and CD8⁺ T cells and monocytes or granulocytes. Frequency of CD3⁺ CXCR4⁺, CD3⁺ CXCR1⁺, and CD3⁺ CXCR3⁺ cells was significantly increased in HF patients, whereas only CCR7 and CXCR4 expression levels were elevated on CD3⁺ cells. Both CD4⁺ CXCR4⁺ and CD8⁺ CXCR4⁺ cell frequencies were significantly increased irrespective of cardiac disease etiology. Elevated CCR7 expression was less pronounced on CD4⁺ than CD8⁺ cells in patients with CAD and IDCM. Expression of CXCR4 on CD8⁺ cells was upregulated substantially, regardless of the cause of disease. CD8⁺ CXCR1⁺ and CD8⁺ CXCR3⁺ but not CD4⁺ CXCR1⁺ or CD4⁺ CXCR3⁺ cells were increased in the HF patients with IDCM and CAD, respectively. Expression of CXCR1 or CXCR3 on both CD4⁺ and CD8⁺ cells did not differ in all the groups. For monocytes, frequency of CD14⁺ CCR1⁺ and CD14⁺ CCR2⁺ cells was significantly decreased in CAD patients, whereas, increase in CD14⁺ CXCR4⁺ cell frequency was accompanied with elevated CXCR4 expression. On granulocytes, CXCR1 and CXCR2 receptors were downregulated in all patients, compared with controls. Our results suggest that the altered expression profile of CC- and CXC-chemokine receptors on circulating leukocyte populations involves enhanced activation of the immune system, perhaps as part of the pathogenic mechanisms in HF. Modulation of the chemokine network could offer interesting novel therapeutic modalities for end-stage HF.     

A regulatory effect of IL-21 on T follicular helper-like cell and B cell in rheumatoid arthritis

Introduction

Interleukin (IL)-21 is a member of type I cytokine family. Recent studies indicate that IL-21 can promote T follicular helper (Tfh) cell differentiation and survival, a specialized T cell subset which provides help for B cell. It can also regulate the activation, proliferation and differentiation of human B cell and immunoglobulin (Ig) production as well as isotype switching of plasma cell. Rheumatoid arthritis (RA) is characterized by auto-antibodies overproduction such as rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) antibody, suggesting a pivotal role of Tfh cell and B cell in the pathogenesis of RA. This study aimed to investigate whether IL-21 had a regulatory effect on Tfh cell and B cell in RA.

Methods

Serum IL-21 concentrations were measured by ELISA. The correlations between serum IL-21 levels and clinical features of RA patients were analyzed by Spearman''s rank test. The percentages of Tfh-like cells, IL-21 receptor (R) expression on Tfh-like cells and B cells in peripheral blood (PB) were analyzed by flow cytometry. Peripheral blood mononuclear cells (PBMC) were stimulated by rIL-21 (100 ng/ml) in the presence or absence of anti-CD40 and/or anti-IgM, and changes of IL-21R, activation-associated surface markers (CD25, CD69 and CD40), the proliferation, apoptosis and differentiation of B cells were analyzed by flow cytometry. Production of IgG and IgM in the culture supernatants was determined by ELISA.

Results

The results showed that the serum IL-21 levels in RA patients were significantly higher than that of healthy controls (HC). IL-21 concentrations were positively correlated with 28-joint count disease activity score (DAS28) and anti-CCP antibody in RA patients with high IL-21 levels. Furthermore, the frequencies of peripheral CXCR5⁺PD-1⁺CD4⁺ Tfh-like cells markedly increased in RA patients and the percentages of Tfh-like cells were positively correlated with DAS28 and anti-CCP antibody levels. Moreover, elevated IL-21 levels were also correlated with the frequencies of Tfh-like cells. IL-21R expression on both Tfh-like cells and B cells were significantly enhanced in RA patients. In cultures vitro, exogenous IL-21 upregulated IL-21R expression and activation-associated surface markers on B cells and promoted more B cell proliferation in RA than in HC. This IL-21-mediated effect could be reversed by IL-21R-specific neutralizing antibody. Importantly, IL-21 promoted more differentiation of B cell into plasmablast and higher levels of IgG and IgM production in RA than in HC.

Conclusions

Increased serum IL-21 levels in RA patients correlate with DAS28, anti-CCP antibody and frequencies of Tfh-like cells. IL-21 supports B cell activation, proliferation and antibody secretion via IL-21R pathway. Thus, IL-21 may be involved in the pathogenesis of RA and antagonizing IL-21 could be a novel strategy for the therapy of RA.     

Enhancing Specific-Antibody Production to the ragB Vaccine with GITRL That Expand Tfh,IFN-γ+ T Cells and Attenuates Porphyromonas gingivalis Infection in Mice

The outer membrane protein RagB is one of the major virulence factors of the periodontal pathogen Porphyromonas gingivalis (P. gingivalis). In order to induce protective immune response against P. gingivalis infection, an mGITRL gene-linked ragB DNA vaccine (pIRES-ragB-mGITRL ) was constructed. Six-week-old female BALB/c mice were immunized with pIRES-ragB-mGITRL through intramuscular injection and then challenged by subcutaneous injection in the abdomen with P. gingivalis. RagB-specific antibody-forming cells were evaluated by an Enzyme-linked immunosorbent spot, and specific antibody was determined by enzyme-linked immunosorbent assay. In addition, the frequencies of Tfh and IFN-γ⁺ T cells in spleen were measured using flow cytometer, and the levels of IL-21 and IFN-γ mRNA or proteins were detected by real time RT-PCR or ELISA. The data showed that the mGITRL-linked ragB DNA vaccine induced higher levels of RagB-specific IgG in serum and RagB-specific antibody-forming cells in spleen. The frequencies of Tfh and IFN-γ⁺ T cells were obviously expanded in mice immunized by pIRES-ragB-mGITRL compared with other groups (pIRES or pIRES-ragB ). The levels of Tfh and IFN-γ⁺ T cells associated cytokines were also significantly increased in pIRES-ragB-mGITRL group. Therefore, the mice immunized with ragB plus mGITRL showed the stronger resistant to P. gingivalis infection and a significant reduction of the lesion size caused by P. gingivalis infection comparing with other groups. Taken together, our findings demonstrated that intramuscular injection of DNA vaccine ragB together with mGITRL induced protective immune response dramatically by increasing Tfh and IFN-γ⁺ T cells and antibody production to P. gingivalis.