cTfr Cells Research Articles

#2240 The complex fluctuation of circulating T follicular cells in patients with lupus nephritis (LN)

Abstract Background and Aims Follicular T (TF) cells constitute a subgroup of cells recently discovered, located in the germinal center (GC) of lymphoid follicles, where they mediate B lymphocyte development, maturation, and antibody production. Meanwhile, T cells with similar markers have also been detected in the peripheral blood, considered as a circulating memory compartment with unknown origin or function. TF cells seem to be implicated in the pathogenesis of systemic lupus erythematosus (SLE) and lupus nephritis (LN) by assisting the selection of high-affinity B cells in germinal centers; nonetheless, research about their circulating regimen is lacking. Herein, we describe the population of circulating TF cells in patients with LN and their association with disease hallmarks. Method Flow cytometry was performed to identify cTF (CD4+CD45RA-CXCR5+) cells and their subsets, defined as cTF1 (CD4+CD45RA-CXCR5+CXCR3+CCR6-), cTF2 (CD4+CD45RA-CXCR5+CXCR3-CCR6-), cTF17 (CD4+CD45RA-CXCR5+CXCR3-CCR6+), and activated-cTF, cTF-ICOS+ (CD4+CD45RA-CXCR5+ICOS+), as well as cTF-Regulatory (cTFR) (CD4+CD45RA-CXCR5+CD127-CD25+FOXP3+). Peripheral blood was collected from 25 LN patients (LN) and 25 healthy controls (HC) of similar age. Results Here are presented preliminary results analyzed in 15 LN patients (age = 38 ± 8yrs) and compared to 9 HC (age = 31.5 ± 7yrs). Although our findings were not significant in total and the percentage and total number of cTFH cells were similar between LN and HC [7.1 (0.6-22.4)% and 52.57 (3.38-412.7)cells/μL, vs. 6.6 (1.9-12.1)% and 45.65 (13.3-69.6)cells/μL, respectively], the fluctuation of the different subsets is interesting. cTF-ICOS+, cTF1, cTF2, and also surprisingly cTFR cells were upregulated in LN compared to HC [0.9 (0-5.2) vs 0.51 (0-1.45)cells/μL, 9.3 (0.2-31.6) vs. 3.02 (0.4-23.08)cells/μL, 10.89 (0-47.41) vs 3.37 (0.46-39.01)cells/μL, and 0.21 (0-12) vs 0.12 (0-1.53) cells/μL, respectively], while the cTF17 compartment was condensed [5.36 (0.79-213.8) vs 8.37 (0.22-29.2)cells/μL, respectively]. Clinical and laboratory markers of the disease were then assessed, resulting in a strong positive correlation of the SLEDAI score with the percentage of cTF2 and cTFR (r = 0.643 and 0.706, respectively) and of C3 levels with cTF percentage (r = 0.663) and cTF-ICOS (+) number (r = 0.719) while the correlation was negative between C4 levels with the percentage of cTF1 (r = −0.577) and cTF2 (r = −582) and between ANA antibody levels and cTF-ICOS (+) cells (r = −657). The analysis did not show any association between the anti-dsDNA antibody levels and the understudied subpopulations. Conclusion cTF cells tend towards 1 and 2 rather than the 17 phenotype in LN. From the various subsets, cTF2 appear to serve as a possible monitoring marker. Certainly, the complexity of the disease imposes further investigation.

Rapamycin Controls Lymphoproliferation and Reverses T-Cell Responses in a Patient with a Novel STIM1 Loss-of-Function Deletion

PurposeDeficiency of stromal interaction molecule 1 (STIM1) results in combined immunodeficiency accompanied by extra-immunological findings like enamel defects and myopathy. We here studied a patient with a STIM1 loss-of-function mutation who presented with severe lymphoproliferation. We sought to explore the efficacy of the mTOR inhibitor rapamycin in controlling disease manifestations and reversing aberrant T-cell subsets and functions, which has never been used previously in this disorder.MethodsClinical findings of the patient were collected over time. We performed immunological evaluations before and after initiation of rapamycin treatment, including detailed lymphocyte subset analyses, alterations in frequencies of circulating T follicular helper (cTFH) and regulatory T (Treg) cells and their subtypes as well as T cell activation and proliferation capacities.ResultsA novel homozygous exon 2 deletion in STIM1 was detected in a 3-year-old girl with severe lymphoproliferation, recurrent infections, myopathy, iris hypoplasia, and enamel hypoplasia. Lymphoproliferation was associated with severe T-cell infiltrates. The deletion resulted in a complete loss of protein expression, associated with a lack of store-operated calcium entry response, defective T-cell activation, proliferation, and cytokine production. Interestingly, patient blood contained fewer cTFH and increased circulating follicular regulatory (cTFR) cells. Abnormal skewing towards TH2-like responses in certain T-cell subpopulations like cTFH, non-cTFH memory T-helper, and Treg cells was associated with increased eosinophil numbers and serum IgE levels. Treatment with rapamycin controlled lymphoproliferation, improved T-cell activation and proliferation capacities, reversed T-cell responses, and repressed high IgE levels and eosinophilia.ConclusionsThis study enhances our understanding of STIM1 deficiency by uncovering additional abnormal T-cell responses, and reveals for the first time the potential therapeutic utility of rapamycin for this disorder.

Circulating T follicular helper 2 cells, T follicular regulatory cells and regulatory B cells are effective biomarkers for predicting the response to house dust mite sublingual immunotherapy in patients with allergic respiratory diseases.

The relationships between T follicular helper (Tfh) cells and antigen-specific immunoglobulins (sIgs) in patients with allergic respiratory diseases who are receiving antigen immunotherapy (AIT) have not been fully clarified. Therefore, we started to perform house dust mite sublingual immunotherapy (HDM-SLIT) for 20 patients with atopic asthma comorbid with allergic rhinitis (AA+AR) who were already receiving ordinary treatments including inhaled corticosteroid (ICS). We examined percentages of circulating T follicular helper (cTfh) and regulatory (cTfr) cells and percentages of circulating regulatory T (cTreg) and B (cBreg) cells by FACS and we examined levels of Der-p/f sIgs by ELISA. Based on the symptom score (asthma control questionnaire: ACQ) and medication score ((global initiative for asthma: GINA) treatment step score) in patients with AA, the patients were divided into responders and non-responders. The percentage of cTfh2 cells significantly decreased and the percentage of cTfh1 cells significantly increased within the first year. Der-p/f sIgEs decreased after a transient elevation at 3 months in both groups. Notably, the percentage of cTfh2 cells and the ratio of cTfh2/cBreg cells and Der-p/f sIgEs greatly decreased in responders from 6 months to 12 months. The percentages of cTfr and cTreg cells showed significant negative correlations with the percentage of cTfh2 cells. The percentage of IL-4+ cTfh cells were significantly decreased and the percentage of IFN-γ+ cTfh cells were increased before treatment to 24 months in 6 patients examined (4 responders and 2 non-responders). We performed multi plelogistic regression analysis based on these results, the ratios of cTfh2/cTfr cells and cTfh2/cBreg cells at the start of therapy were statistically effective biomarkers for predicting the response to HDM-SLIT in patients with AA+AR.

O005 Perturbations in gut microbial metabolites promotes a pro-inflammatory state increasing risk of acute rejection post renal transplantation

Abstract Introduction Emerging evidence suggests that the gastrointestinal microbiota-immune axis impacts on extra-intestinal health. We aim to identify the influence of microbial metabolites on recipient's immunity; testing the hypothesis that reduced availability of bacterial-derived metabolites associated with immunoregulation e.g., short chain fatty acids (SCFAs) and tryptophan-derivatives promote a pro-inflammatory state increasing risk of acute rejection (AR). Methods Ninety recipients and 21 live-donors were recruited with urine, stool and blood samples collected at baseline and up to 12-months after surgery. Flow cytometry was used to assess for circulating subpopulations of CD19+ B-cells and CD4+ T cells (cTFH - CD3+CD4+CD45RA-CXCR5+ and cTFR -CD3+CD4+CXCR5+FoxP3+). Faecal SCFAs and indole derivatives were identified by mass spectroscopy and high-performance liquid chromatography. Results Patients with AR had an almost 10-fold reduction of FoxP3+ cTFR cells after transplantation compared to baseline (p=0.03). There were lower frequencies of cTFR cells at 3-months when compared to matched recipients without AR (0.009%±0.014% vs 0.10%±0.12%; p=0.01). In AR, there was a trend for higher frequencies of plasmablasts, resting memory B-cells and TFH cells at baseline, with fewer transitional B-cells at 3-months. Despite tryptophan availability increasing after transplantation, patients with AR displayed reduced levels of SCFAs at 1-month. Conclusion Complex changes in microbial metabolites exist after transplantation that may influence the balance of cTFH vs cTFR cells. Despite increase in dietary tryptophan, recipients with AR may harbour gut microbes that are unable to metabolise tryptophan into immune-regulatory metabolites, which predisposes a pro-inflammatory immune state.

Altered Circulating Follicular T Helper Cell Subsets and Follicular T Regulatory Cells Are Indicators of a Derailed B Cell Response in Lupus, Which Could Be Modified by Targeting IL-21R.

Systemic lupus erythematosus (SLE) is characterized by the breakdown of self-tolerance, the production of high-affinity pathogenic autoantibodies and derailed B cell responses, which indicates the importance of central players, such as follicular T helper (TFH) subsets and follicular T regulatory (TFR) cells, in the pathomechanism of the disease. In this study, we aimed to analyze the distribution of the circulating counterparts of these cells and their association with disease characteristics and B cell disproportions in SLE. We found that the increased percentage of activated circulating TFH (cTFH) and cTFR cells was more pronounced in cutaneous lupus; however, among cTFH subsets, the frequency of cTFH17 cells was decreased in patients with lupus nephritis. Furthermore, the decreased proportion of cTFH17 cells was associated with low complement C4 levels and high disease activity scores. We also investigated whether the blocking of the IL-21 receptor (IL-21R) with an anti-IL-21R monoclonal antibody inhibits the B cell response, since IL-21 primarily produced by TFH cells potentially promotes humoral immunity. We observed that anti-IL-21R inhibited plasmablast generation and immunoglobulin production. Our study demonstrated that, besides cTFR/cTFH imbalance, cTFH17 cells play a crucial role in SLE pathogenesis, and modulating cTFH-B cell interaction through the IL-21/IL-21R pathway may be a promising therapeutic strategy to suppress the pathological B cell response.

Altered distributions in circulating follicular helper and follicular regulatory T cells accountable for imbalanced cytokine production in multiple sclerosis.

Follicular T helper (Tfh) and regulatory (Tfr) cells are distinct subsets of CD4+ T lymphocytes, regulating humoral immune responses in the germinal center. It is widely accepted that dysregulated Tfh and Tfr cells are associated with autoimmunity. In this study, we evaluated the frequencies of circulating chemokine receptor (CXCR)5+ programmed cell death 1 (PD-1+ ) Tfh (cTfh) and CXCR5+ PD-1+ forkhead box protein 3 (FoxP3+ ) CD25+ Tfr (cTfr) cells, and their corresponding cytokines from the peripheral blood mononuclear cells of 28patients with relapsing-remitting multiple sclerosis (MS) and 16 age- and sex-matched healthy controls (HC). Subsets of cTfh cells by Th1- and Th17-related surface markers (CXCR3 and CCR6) were also evaluated. We found that the frequency of cTfh cells was significantly higher in MS patients compared to that of HC. Conversely, the frequency of cTfr cells was lower in MS patients than that of HC. Interleukin (IL)-21-producing cTfh cells were significantly increased in MS patients, while IL-10-secreting cTfr cells were lower in MS patients compared to levels in HC. Among cTfh cells, cTfh17.1 cells were the major subtypes that were significantly increased in MS patients compared to HC, with the frequency of IL-21-secreting cells being the highest. These results suggest that an imbalanced distribution of cTfh and cTfr exist in MS patients, which contributes to the reciprocally altered IL-21 and IL-10 production.

The Imbalance of Circulating Follicular T Helper Cell Subsets in Primary Sjögren's Syndrome Associates With Serological Alterations and Abnormal B-Cell Distribution.

Since B-cell hyperactivity and pathologic antibody response are key features in the immunopathogenesis of primary Sjögren’s syndrome (pSS), the role of follicular T helper (TFH) cells as efficient helpers in the survival and differentiation of B cells has emerged. Our aim was to investigate whether a change in the balance of circulating (c)TFH subsets and follicular regulatory T (TFR) cells could affect the distribution of B cells in pSS. Peripheral blood of 38 pSS patients and 27 healthy controls was assessed for the frequencies of cTFH cell subsets, TFR cells, and certain B cell subpopulations by multicolor flow cytometry. Serological parameters, including anti-SSA, anti-SSB autoantibodies, immunoglobulin, and immune complex titers were determined as part of the routine diagnostic evaluation. Patients with pSS showed a significant increase in activated cTFH cell proportions, which was associated with serological results. Frequencies of cTFH subsets were unchanged in pSS patients compared to healthy controls. The percentages and number of cTFR cells exhibited a significant increase in autoantibody positive patients compared to patients with seronegative pSS. The proportions of transitional and naïve B cells were significantly increased, whereas subsets of memory B cells were significantly decreased and correlated with autoantibody production. Functional analysis revealed that the simultaneous blockade of cTFH and B cell interaction with anti-IL-21 and anti-CD40 antibodies decreased the production of IgM and IgG. Imbalance in TFH subsets and TFR cells indicates an ongoing over-activated humoral immune response, which contributes to the characteristic serological manifestations and the pathogenesis of pSS.

Human pregnancy levels of estrogen and progesterone contribute to humoral immunity by activating TFH /B cell axis.

Circulating TFH (cTFH ) cells express CXCR5, PD-1, and, when activated, ICOS, and release IL-21. According to the production of IFN-γ, IL-4, and IL-17 and expression of FoxP3, these cells are also classified as cTFH 1, cTFH 2, cTFH 17, and cTFR cells, respectively. This CD4+ T-cell subset is pivotal to efficient humoral immunity, and pregnancy appears to favor IgG production. Here, not only pregnancy amplified the in vivo production of anti-HBsAg IgG in HBV immunized women, but the frequency of cTFH cells was directly correlated with estradiol levels. In vitro, pregnancy-related dose of 17-β-estradiol (E2) directly increased the percentage of different cTFH subsets. While E2 and progesterone (P4) increased the proportion of differentiated TFH cells derived from naïve CD4+ T-cells, only E2 amplified the release of IL-21 in those cell cultures. In addition, E2 and P4 increased the proportion of memory B cells and plasma cells, respectively. In SEB-activated B/TFH cell co-cultures, E2, in the presence of P4, increased the production of total IgG. Finally, among the hormones, P4 was stronger in upregulating the percentage of IL-10+ TFR cells. Collectively, our findings suggested that E2 and P4 cooperate in the humoral immune response by favoring the expansion of different cTFH and B cell subsets.

Imbalance of Circulating Tfh/Tfr Cells in Patients With Parkinson's Disease.

Background: Follicular helper T (Tfh) cells and follicular regulatory T (Tfr) cells are essential for B cell differentiation, germinal center formation, and humoral immune responses. Immunity and inflammation have been thought to be involved in Parkinson's disease (PD). In this study, we aimed to identify whether circulating Tfh and Tfr (cTfh and cTfr) cells contribute to PD.Methods: Thirty-nine PD patients and 26 health controls (HCs) were enrolled. The numbers of cTfh (CD4+CXCR5+PD-1+) cells and cTfr (CD4+CXCR5+CD25hiCD127low) cells were analyzed via flow cytometry. The serum concentrations of interleukin (IL)-4, IL-10, IL-21, and transforming growth factor (TGF)-β were examined by cytometric bead array.Results: The percentage of cTfh cells among CD4+ T cells in PD patients was significantly higher than that in HCs [3.68% (2.64–5.70%) vs. 1.94% (1.32%−2.99%), P < 0.001], while the percentage of cTfr cells among CD4+ T cells in PD patients was slight decreased but without significance [1.05% (0.62–1.54%) vs. 1.3% (0.63–1.90%), P > 0.05]. The percentage of CD19+ B cells in peripheral blood mononuclear cells was significantly lower in PD patients than in HCs [5.35% (4.13–9.38%) vs. 8.68% (5.61–12.93%), P = 0.014]. The serum concentrations of IL-4, IL-10, IL-21, and TGF-β in PD patients did not differ significantly from those in HCs (P > 0.05). There was a positive trend of the correlation between the number of cTfh and the serum IL-4 concentrations in PD patients (P = 0.032, r = 0.353). There was a positive trend of the correlation between the number of cTfr and the serum IL-10 concentrations in PD patients (P = 0.047, r = 0.328), A positive trend of the correlation were found for the serum concentration of IL-21 with H-Y stage (r = 0.356, P = 0.026) and UPDRS-III score (r = 0.347, P = 0.030).Conclusions: These results indicate that an imbalance of cTfh and cTfr cells may be involved in the chronic progression of PD, and IL-21 may be a biomarker for monitoring the severity of this disease.

Immunosuppression Has Long-Lasting Effects on Circulating Follicular Regulatory T Cells in Kidney Transplant Recipients.

Background: FoxP3+ follicular regulatory T cells (Tfr) have been identified as the cell population controlling T follicular helper (Tfh) cells and B cells which, are both involved in effector immune responses against transplanted tissue.Methods: To understand the biology of Tfr cells in kidney transplant patients treated with tacrolimus and mycophenolate mofetil (MMF) combination immunosuppression, we measured circulating (c)Tfh and cTfr cells in peripheral blood by flow cytometry in n = 211 kidney transplant recipients. At the time of measurement patients were 5–7 years after transplantation. Of this cohort of patients, 23.2% (49/211) had been previously treated for rejection. Median time after anti-rejection therapy was 4.9 years (range 0.4–7 years). Age and gender matched healthy individuals served as controls.Results: While the absolute numbers of cTfh cells were comparable between kidney transplant recipients and healthy controls, the numbers of cTfr cells were 46% lower in immunosuppressed recipients (p < 0.001). More importantly, in transplanted patients, the ratio of cTfr to cTfh was decreased (median; 0.10 vs. 0.06), indicating a disruption of the balance between cTfr and cTfh cells. This shifted balance was observed for both non-rejectors and rejectors. Previous pulse methylprednisolone or combined pulse methylprednisolone + intravenous immunoglobulin anti-rejection therapy led to a non-significant 30.6% (median) and 51.2% (median) drop in cTfr cells, respectively when compared to cTfr cell numbers in transplant patients who did not receive anti-rejection therapy. A history of alemtuzumab therapy did lead to a significant decrease in cTfr cells of 85.8% (median) compared with patients not treated with anti-rejection therapy (p < 0.0001). No association with tacrolimus or MMF pre-dose concentrations was found.Conclusion: This cross-sectional study reveals that anti-rejection therapy with alemtuzumab significantly lowers the number of cTfr cells in kidney transplant recipients. The observed profound effects by these agents might dysregulate cTfr functions.

Phenotypic analysis of T follicular helper and T follicular regulatory cells in primary selective IgM deficiency

Selective IgM deficiency (SIgMD) is a rare immunodeficiency characterized by serum IgM below two standard of mean, and normal IgG and IgA levels. Both in human and mice with selective IgM deficiency, germinal centers cells are decreased. The development of germinal center and humoral immunity are regulated in part by follicular helper T (TFH) and follicular regulatory T (TFR) cells. However, the analysis of circulating TFH (cTFH) and TFR (cTFR) cells in the pathogenesis of SIgMD has not been explored. We observed lower percentage of cTFR cells in SIgMD patients than in control group. However, we did not observe any significant difference in the percentage of cTFH cells and their subsets between both experimental groups. When data were analyzed according to specific antibody response to pneumococcal polysaccharide, we observed a higher percentage of cTFH cells in SIgMD patients with specific antibody deficiency than in SIgMD patients with normal specific antibody response. Our results suggest that cTFH cells and their subsets are preserved in SIgMD patients. However, the role of lower percentage of cTFR cells in the pathogenesis of this immunodeficiency is not clear.

Altered ratio of circulating follicular regulatory T cells and follicular helper T cells during primary EBV infection

Follicular help T cells (Tfh) play an important role in the activation and differentiation of B cells, while follicular regulatory T cells (Tfr) control Tfh and resulting humoral immune responses. Accumulating evidence has demonstrated that the dysregulation of Tfr contributed to the pathogenesis of infectious diseases. However, the role of Tfr in Epstein–Barr virus (EBV) infection remains lacking. Fifty-five EBV-infected infectious mononucleosis (IM) patients and 21 healthy individuals (HIs) were recruited in the study. We investigated the number of Tfr (FoxP3+CXCR5+PD-1+CD4+) and Tfh (FoxP3−CXCR5+PD-1+CD4+) of peripheral blood in IM patients at diagnosis (D0) and day 15 after diagnosis (D15) via multicolor flow cytometry. Results revealed that circulating Tfh (cTfh) and Tfr (cTfr) of IM at D0 were both increased compared to HIs, and cTfr began to decline and return to normal at D15, while cTfh was still higher than those of HIs. More interestingly, the cTfr/cTfh ratio of IM at D0 and D15 was lower than that of HIs, suggesting that the balance between cTfh and cTfr was disturbed during primary EBV infection. Correlation analysis showed a positive correlation between cTfr with CD19+IgD+CD27− naive B cells, CD19+IgD−CD27hi plasmablasts or CD19+CD24hiCD27hi B cells. Moreover, both cTfr and the cTfr/cTfh ratio of IM at D0 were negatively correlated with EBV DNA virus load. These results indicate that an imbalance of cTfr and cTfh cells may be involved in the immunopathogenesis of EBV-infected IM patients and may provide novel strategies for controlling EBV-related disease.

Increased circulating Tfh to Tfr ratio in chronic renal allograft dysfunction: a pilot study

BackgroundT follicular helper (Tfh) cells play a control role in contribution of B cell differentiation and antibody production. T follicular regulatory (Tfr) cells inhibit Tfh-B cell interaction.MethodsTo identify whether circulating Tfh (cTfh) and Tfr (cTfr) cells contribute to chronic renal allograft dysfunction (CAD), 67 kidney transplant recipients (34 recipients with CAD, 33 recipients with stable function) were enrolled. The frequency of cTfh and cTfr cells, the level of serum CXCL13 were measured.ResultsThe frequency of cTfr cells in CAD group was significantly lower than that in stable group (0.31% vs 0.68%, P = 0.002). The cTfh to cTfr ratio in CAD group was significantly higher than that in stable group (55.4 vs 25.3, P = 0.013). Serum CXCL13 in CAD group was significantly higher than stable group (30.4 vs 21.9 ng/ml, P = 0.025). After linear regression analysis, the cTfh to cTfr ratio was an independent risk factor for estimated glomerular filtration rate (eGFR) in recipients (standardized coefficient = − 0.420, P = 0.012). After logistic regression analysis, the cTfh to cTfr ratio was an independent risk factor for CAD (OR = 1.043, 95%CI = 1.004–1.085, P = 0.031).ConclusionThe imbalance between cTfh and cTfr cells contribute to the development of CAD.

The Association of Circulating T Follicular Helper Cells and Regulatory Cells with Acute Myeloid Leukemia Patients

Objective: This study aims to investigate the association of circulating T follicular helper (cTfh) cells and T follicular regulatory (cTfr) cells with acute myeloid leukemia (AML) patients. Methods: A total of 22 newly diagnosed, untreated AML patients as well as 26 healthy controls were enrolled. Percentages of cTfh and cTfr cells were detected using flow cytometry. Results: Compared to healthy controls, a significantly higher percentage of cTfr cells was observed in AML patients (4.10 ± 11.18 vs. 0.63 ± 0.38%) (p < 0.05). In addition, a significantly lower cTfh/cTfr ratio was found in the AML patients’ group when compared to the control group (9.04 ± 9.19 vs. 11.66 ± 5.68) (p < 0.05). A lower level of plasma IL-2 and TGF-β1 was found in AML patients. Based on the complete remission (CR) response after one cycle of inductive chemotherapy, patients were divided into two groups at sample collection: AML with and without CR. Significantly lower percentages of cTfr cells and a higher cTfh/cTfr ratio were found in the group of AML patients with CR than in the AML patients without CR. Conclusion: There was a significantly higher percentage of cTfr cells in AML patients. cTfr cells may have a potential association with the pathogenesis of AML patients.

Alemtuzumab Treatment Leads to Delayed Recovery of T Follicular Regulatory Cells, and May Therefore Predispose Patients to de novo Donor-Specific Antibody Formation

Background - T follicular helper (Tfh) and regulatory (Tfr) cells are key players in the formation of long-lived antibody responses. Their circulating counterparts, cTfh and cTfr, are often used as biomarkers because longitudinal sampling of secondary lymphoid tissues is unfeasible in clinical studies. This is the first study to track cTfh and cTfr cells following therapeutic lymphocyte depletion with alemtuzumab, an anti-CD52 monoclonal antibody, to infer the influence of this treatment on the germinal centre response. Methods - Samples from 61 transplant recipients were taken at the time of transplant, and regular intervals post-transplant for both flow-cytometric immunophenotyping, and Luminex-based donor-specific antibody (DSA) assessment. Results - Patients treated with alemtuzumab (42/61, 69%) had a significantly lower ratio of cTfr to cTfh at all time points post-transplant. We found that, despite a high proportion of Tregs in the recovering cell population, cTfr cells did not repopulate in alemtuzumab treated patients, while cTfh cells reconstituted to higher than pre-transplant levels over the 2-year follow-up. Conclusions - Alemtuzumab has been used as first-line induction immunosuppression and treatment for steroid-resistant rejection in transplantation; our data suggests alemtuzumab-treated patients have a lower cTfr to cTfh ratio even 2 years post-transplant, and may therefore be at higher risk of de novo DSA formation.

IL-10+ T follicular regulatory cells are associated with the pathogenesis of IgG4-related disease

IgG4-related disease (IgG4-RD) is a chronic fibroinflammatory disease characterized by elevation of serum IgG4 level as well as infiltration of IgG4+ plasma cells in various affected organs. The etiology of IgG4-RD is still not fully understood. Since IgG4-RD is more prevalent in the elderly, aging in itself is considered to be an important risk factor of IgG4-RD. However, the relationship between the pathogenesis of IgG4-RD and immunosenescence remains unknown. To clarify age-related features underlying IgG4-RD, we focused on T follicular regulatory (Tfr) cells, which share forkhead box P3 with regulatory T cells, since the percentage of Tfr cells is known to depend on age. Studies of blood specimens from patients with IgG4-RD and from healthy volunteers demonstrated a marked elevation of circulating Tfr (cTfr) cells in patients with IgG4-RD. Moreover, the percentage of cTfr cells was significantly correlated with various clinical parameters including the level of serum IgG4 and the number of involved organs in IgG4-RD patients. The percentages of tonsillar and blood Tfr cells were increased with aging in healthy volunteers, whereas the suppressive effect of cTfr cells on B cell function in elderly subjects was impaired in comparison with that in young subjects due to a defect in the production of a regulatory cytokine, IL-10. Given that the number of IL-10-producing cTfr cells in IgG4-RD patients was markedly increased compared with that in healthy elderly subjects, these findings suggest that an abnormal aging process of Tfr cells may be related to the pathogenesis of IgG4-RD.

Decreased expression of circulating Aire and increased Tfh/Tfr cells in myasthenia gravis patients.

Myasthenia gravis (MG) is a rare prototypical autoimmune disorder caused by antibodies (Ab) against postsynaptic membrane proteins. Most reports have investigated the role of autoimmune regulator gene (Aire) in thymic tissue in machianism of MG initiation. So far, the expression of Aire in human peripheral blood cells (we call it circulating Aire expression in the following passage) has not been reported. Herein, we explore the expression of Aire in peripharal blood, circulating T-follicular helper (cTfh) and T-follicular regulatory (cTfr) cells in MG patients. In our research, we found that the acetylcholine receptor (AChR) Ab level is higher in generalized MG (GMG) than that in ocular MG (OMG). Compared with the control group (CG), lower expression of Aire was found in MG patients, especially in GMG. The ratio of Tfh/Tfr was higher in GMG patients, and then in the OMG patients, and lowest in CG. All these differences above were statistically significant. Negative relation was discovered between expression of Aire in circulating blood and ratio of Tfh/Tfr, so did it exist between Aire expression and the severity of MG. Meanwhile, positive relation was discovered between ratio of Tfh/Tfr and the severity of MG. However, no significant relation was manifested in our study between the subset age of MG and Aire level. Overall, these findings imply circulating Aire might play a role in the imbalance of cTfh and cTfr cells and participate in the pathogenesis of MG.

The Role of Circulating T Follicular Helper Cells and Regulatory Cells in Non-Small Cell Lung Cancer Patients.

T follicular helper (Tfh) cells and T follicular regulatory (Tfr) cells are identified as the new subset of immune cells. This study aims to investigate the role of circulating Tfh cells (cTfh) and Tfr (cTfr) cells in the pathogenesis of non-small cell lung cancer (NSCLC). A total of 27 NSCLC patients and 19 age and sex-matched healthy controls were enrolled. The percentage of cTfh and cTfr was detected by flow cytometric analysis. Compared to healthy controls, a significantly higher percentage of both cTfh and cTfr cells were observed in NSCLC patients (for cTfh, 18.88%±16.84% versus 5.98%±3.70%, P<0.01; for cTfr, 2.67%±2.20% versus 1.14%±0.76%, P<0.01). Furthermore, there was a positive correlation between cTfh/cTfr ratio and age in NSCLC patients (P<0.05). When taking age 60 as a cut-off, the percentage of both cTfh cells and cTfr cells were higher in older patients than younger patients. Moreover, our data showed there was lower percentage of cTfh cells in NSCLC patients with early stage disease (I and II) (12.10%±12.22%) than that in advanced stage disease (III and IV) (30.41%±17.87%) (P<0.01). However, no significant relationship was observed between cTfr cells and clinical stage in NSCLC patients. A higher percentage of cTfh cells was observed in patients with squamous cell carcinoma compared with adenocarcinoma (31.70%±20.73% versus. 13.48%±11.78%, P<0.05). Taken together, there was significantly higher percentage of cTfh and cTfr cells in NSCLC patients. cTfh and cTfr cells might play an important role in the pathogenesis of NSCLC patients.