Phenotypically defined subpopulations of circulating follicular helper T cells in common variable immunodeficiency

Abstract Background Common variable immunodeficiency (CVID) is characterized by low immunoglobulin G and IgA/IgM, decreased switched memory B cells, impaired response to vaccine, and an increased susceptibility to infections and autoimmunity. TFH cells play an important role in germinal center reaction where it supports isotype switching, somatic hypermutation, generation of memory B cells, and differentiation of B cells to plasma cells. The objective was to study the distribution of three subsets of TFH cells and their relationship with autoimmune diseases associated with CVID. Methods TFH cells have been divided into TFH1 (interleukin 21 [IL‐21] and interferon γ), TFH2 (IL‐21 and IL‐4), and TFH17 (IL‐21 and IL‐17) cells. Mononuclear cells from 25 patients with CVID and age and gender‐matched controls were stained with various monoclonal antibodies (anti‐CD4 APC, anti‐CXCR5 FITC, anti‐CCR6 PerCP, and anti‐CXCR3 PE) and isotype controls and analyzed for TFH1 (CD4+CXCR5+CXCR3+CCR6−), TFH2 (CD4+CXCR5+CXCR3−CCR6−), and TFH17 (CD4+CXCR5+CXCR3−CCR6+) cells by multicolor flow cytometry. Twenty thousand cells were acquired and analyzed by FlowJo software. Statistical analysis of comparison of patients and healthy controls was performed by paired t test using PRISM 7 software. Results TFH2 and TFH17 cells subpopulations of TFH cells were significantly decreased (P < .003 and P < .006, respectively) in CVID as compared with controls. No significant difference was observed in any of TFH cell subpopulations between CVID with and those without autoimmunity group. Conclusion Alterations in TFH cell subpopulation may play a role in defects in B cell compartment in CVID.


| INTRODUCTION
Common variable immunodeficiency (CVID) is heterogeneous and most common primary immunodeficiency disease in adults characterized by low serum immunoglobulins immunoglobulin G (IgG), IgA, and/or IgM, impaired specific antibody response to vaccines, and increased susceptibility to recurrent infections. [1][2][3][4][5] In addition, patients with CVID have increased prevalence of allergic, autoimmune, and granulomatous disorders, and malignancy, the majority being lymphoreticular malignancy. [5][6][7][8][9][10] A number of gene mutations have been reported in CVID; however, they account for less than 20% of CVID patients. [11][12][13] Therefore, in majority of patients with CVID cause(s) is unknown. The predominant defects appear to be in the B cell compartment including impaired immunoglobulin isotype switching and differentiation of B cells into plasma cells despite normal number of B cells; postgerminal center B cells are defective and switched memory B cells are reduced. [14][15][16] The follicular helper (T FH ) cells are major CD4 + T helper subset that are essential for B cell differentiation into immunoglobulin producing plasma cells, and for the generation of memory B cells in the germinal center (GC). 17,18 GCs are primary sites for class-switched recombination and affinity maturation. T FH cells regulate GC formation, and selection of high-affinity antibodyproducing B cells and support isotype class switching. 19,20 An increased cT FH cells response in the GC is associated with the expansion of low affinity and autoreactive B cells. 21,22 T FH cells are characterized by the expression of CXCR5 and transcription factor B cell lymphoma 6 (Bcl6), and production of their signature cytokine, the interleukin 21 (IL-21). 23-25 CXCR5 plays and important role in the migration of B cells to germinal follicles to support immunoglobulin production. 26 Although T FH cells are predominantly found in lymph nodes and spleen, a small proportion of these cells are also found in the circulation. Vella et al 27 compared T FH cells from lymph nodes, thoracic duct lymph, and blood and showed that they share TCR clonotype, phenotype, and transcriptional signature, and therefore cT FH represents T FH cells in GC.
A role of T FH cells in antibody-mediated autoimmune disease has been established in both mice and humans. 21,22 Because T FH cells play a role in class switching and autoimmunity, and an observed deficiency of switched memory B cells and increased autoimmunity in CVID, we evaluated cT FH 1, cT FH 2, and cT FH 17 cells in CVID patients and examined their relationship with autoimmune diseases associated with CVID.

| Subjects
A total of 25 patients (seven men and 18 women, aged 15-82 years) with CVID and 25 healthy controls (13 men and 12 women, aged, 20-67 years) were enrolled in the study. Pan American and ESID Criteria were used to diagnose CVID patients. 1 Clinical and immunological features of these patients have been published. 29 All patients were receiving immunoglobulin replacement treatment. Blood samples were drawn at trough level. The Institutional Review Board committee (human research), University of California at Irvine approved this study protocol. Written and signed informed consent was obtained from all subjects.

| Immunophenotyping
Ten ml of heparinized blood was diluted with Hank's buffered salt solution (HBSS). Mononuclear cells (MNC) were separated by Ficoll-Hypaque density gradient using lymphocyte separation medium. Cells were suspended in HBSS and used for immunophenotyping. Cells were incubated with different monoclonal antibodies and isotype controls (below) for 30 minutes on ice in the dark. Cells were washed and cT FH 1, cT FH 2, and cT FH 17 analyses were performed by multicolor flow cytometry (FACSCelesta; Becton-Dickinson, San Jose, CA). Twenty thousand cells were acquired and analyzed by FlowJo software (Treestar Inc., Ashland, OR).
Statistical analysis of comparison of patients and healthy controls was performed by paired t test for equality of means using PRISM 7 software.

| cT FH subpopulations in CVID
CXCR5 + CD4 cT FH are further subdivided by the expression of CXCR3 and CCR6 and cytokines they produce into T FH 1, T FH 2, and T FH 17 cells. 28 MNC were incubated with panel of monoclonal antibodies defining T FH 1, T FH 2, and T FH 17 cells and isotype controls and analyzed using multicolor flow cytometry. Cumulative data from 25 patients with CVID and healthy controls are shown in Figure 1. cT FH 2 and cT FH 17 cells were significantly decreased in CVID patients when compared to controls (P < .003, P < .006, respectively). cT FH 1 cells were comparable between two groups (P < .802). controls to defined T FH 1, T FH 2, and T FH 17 subsets of follicular helper T cells and analyzed with multicolor flow cytometry using FACSCelesta. Data are expressed as mean ± SD. Statistical analysis was performed with GraphPad Prism version 8.4.3 for Windows (GraphPad Software, San Diego, CA).
3.2 | cT FH subpopulations in CVID with and without autoimmunity cT FH cells play a role in autoimmunity and autoimmune diseases. 21,22,30 Therefore, we analyzed our data for the presence and absence of autoimmunity in CVID. Data are shown in Figure 2. cT FH 17 cells tended to be higher in CVID patients with autoimmunity as compared with those without autoimmunity. However, we observed no significant difference in cT FH 1, cT FH 2, and cT FH 17 cells between CVID patients with or without autoimmune disease (P > .754, P > .177, P > .230, respectively). There were only seven of 25 CVID patients with autoimmune disease.
Bossaller et al 34 and Grimbacher et al 35 reported decreased proportions of CXCR5 + CD4 + cT FH cells in CVID patients with inducible T cell costimulator (ICOS) deficiency. Cunill et al 14 observed increased CD4 + CXCR5 + cT FH cells in CVID as compared with controls; however, these differences were observed only between CVID with lowswitched B cells (smB − ) vs normal controls. Coraglia et al 36 reported no difference in CD4 + CXCR5 + cT FH cells that expressed IL-10, IL-21, or IL-4 between CVID with and without autoimmune diseases as compared with controls. However, they observed increased proportions of PD1 + CCR7 + T FH in CVID with autoimmune diseases as compared with CVID without autoimmune diseases and controls. Cunill et al 14 when used expression of CXCR3 and CCR6 to define cT FH 1, cT FH 2, and cT FH 17, observed increased cT FH 1 cells, and decreased T FH 17 cells in CVID with low-switched memory B cells as compared with CVID with normal switched memory B cells and healthy controls. No difference was observed in T FH 2 cells. Unger et al 37 also observed increased T FH 1 and decreased T FH 17 cells in CVID patient. Increased T FH 1 cells were observed in patients with autoimmune manifestations and strongest shift in T FH 1 cells was observed in CVID with increased CD21 low B cells. Turpin et al 38 reported higher proportions of cT FH 1, cT FH 17 and low cT FH 2 in CVID patients than control subjects. Increased IFN-γ-producing T FH 1 cells in CVID were observed in CVID with noninfectious manifestations. However, Le Coz et al 39 did not observed increased IFNγ producing T FH cells in CVID. They observed increased IL-21 producing T FH cells and imbalance in T FH 1 /T FH 2 to T FH 17. We observed significantly decreased cT FH 2 in CVID that is in agreement with report by Turpin et al. 39 Our observations of decreased T FH 17 cells in CVID are in agreement with reports of Cunill et al 14 and Unger et al. 37 However, similar to Le Coz et al, 39 we did not observed any significant difference in T FH 1 cells in CVID. Our results are different from those of increased T FH 1 cells reported by Cunill et al 14 and Unger et al. 35 However, we did not analyze our data in relation to switched B cells. The role of T FH 1 cells in the pathogenesis of CVID is questionable. Desjardins et al 40 demonstrated that an addition of exogenous IFNγ to cultures of B cells had no effect on B cells from CVID patients. We did not observed significant difference in any of subsets of cT FH cells between CVID patients with and without autoimmune disease. In various autoimmune diseases including SLE, IgG4-related diseases, Sjogren's syndrome, rheumatoid arthritis, myasthenia F I G U R E 2 T FH cell subsets relations to autoimmune diseases in CVID. T FH 1, T FH 2, and T FH 17 subsets and T FH 1/T FH 17 ratio were compared for CVID patients with autoimmune diseases (n = 7) and without autoimmune diseases (n = 18). CVID, common variable immunodeficiency gravis, autoimmune thyroid disease, different patterns in cT FH cell subsets have been reported (reviewed in Ueno 31 ]. Therefore, type of autoimmune diseases associated with CVID as well difference in characterization of CVID may explain discrepancy among various studies. Furthermore, we need to consider a role of regulatory lymphocytes in autoimmunity associated with CVID. We have reported decreased proportion of CD4 + Treg, CD8 + Treg, and Breg cells in CVID patients. 29 More recently, cT FR has been shown to regulate GC reaction at multiple levels. [41][42][43] cT FR regulate proliferation and cytokine production, as well as B cell proliferation and immunoglobulin production. [43][44][45] Cunill et al 14  However, they demonstrated that a combination of IL-21, IL-4, and anti-CD40 induced class-witched recombination and differentiation of B cells to immunoglobulin secreting cells in CVID. IL-21R/IL-4 double deficient mice exhibit a CVID phenotype with low IgG and IgA and normal IgM, suggesting a critical role of IL-21, that is produced by cT FH cells, in regulating immunoglobulin isotype switch. 47 In summary, a decreased in T FH cell subsets may play a role in poor GC reactions including decreased isotype switching, impaired affinity maturation, generation of memory B cells, and B cell differentiation to plasma cells that are characteristics of CVID. To understand the pathogenesis of defects in B cell compartment and autoimmune and inflammatory manifestations, further comprehensive studies of all phenotypic and functionally defined subsets cT FH cells, including cT FR in homogenously subclassified groups of CVID patients are needed.