Evaluation of transcription factors and cytokine expressions of T‐cell subsets in CD19 deficiency and their possible relationship with autoimmune disease

CD19 deficiency is a rare, predominantly antibody deficiency, and there are few studies showing that it can be seen in autoimmune diseases. The aim of study was evaluated to transcription factor and cytokine expressions of helper T (Th)‐cell subsets in CD19 deficiency and the possible mechanism role of this factor expression in autoimmune disease. Transcription factor and cytokine expressions of Th1, Th2, Th17, and regulatory T (Treg) cells were investigated by real‐time polymerase chain reaction (qPCR) method. In the study, in the patient/control comparison, transcription factor and cytokine expressions of Th1 (T‐bet, STAT1, and STAT4) were found to be significantly downregulated, but IFN‐γ was significantly upregulated in patients. Th2 factor GATA3, STAT6, IL‐4, and IL‐5 were significantly downregulated. For Th17, RORγt was downregulated while IL‐22 was upregulated. In the heterozygous/control comparison, there was no significant change in gene expressions other than IL‐5. T‐bet, STAT1, GATA3, IL‐4, RORγt, FoxP3, and TGF‐β were significantly downregulated in the patient/heterozygous comparison. It was revealed for the first time that the expression of the transcription factors and cytokines in CD19 deficiency. These findings might be showing the predominance of Th1 factors and suppressed Treg factors which could be related with autoimmunity in CD19 deficiency.

CD19 deficiency due to CD19 gene mutation is a common variable immunodeficiency (CVID) that is in the group of "predominantly antibody deficiencies" in "inborn errors of immunity (IEI)" classification [1,2].CD19 deficiency was first identified in 2006 by our group in four cases with susceptibility to infection, hypogammaglobulinemia, and normal mature B-cell count in peripheral blood [3].To date, 11 cases of CD19 deficiency have been reported in the literature.Although frequent infection is a common finding in CD19 deficiency, it has been reported to be seen in autoimmune diseases in some patients [2,4,5].However, there is few studies in the literature on the mechanisms of autoimmune diseases in CD19 deficiency.Regulatory T cells (Treg) are cells that maintain immune tolerance and function in the prevention of autoimmune diseases and are characterized by the expression of CD4, CD25, and forkhead box P3 (FoxP3) [6].Treg cells perform their immune homeostasis function by acting on other T helper (Th) cells, Th1, Th2, and Th17.Functioning of these cells depends on phosphorylation of signal transducers and activators of transcription (STAT) and subsequent cytokine release resulting from activation of transcription factors.
We aimed to assess transcription factors and cytokines related to Treg, Th1, Th2, and Th17 subsets to investigate potential autoimmune mechanisms in CD19 deficiency when systemic lupus erythematosus (SLE) was observed in one of our patients during follow-up (index case).

Study design and patients
The study was carried out in the Department of Pediatric Immunology and Allergy in January 2023.Three previously described patients diagnosed with CD19 deficiency (c.973_973insA; p.Arg325AlafsX4; GenBank: AH005421.2) [3,[7][8][9].All of the patients were from different families and there was consanguinity between the patients' parents.The study included a total of six heterozygous (carrier) genotype individuals for the relevant mutation: the mother, father, and two siblings of one patient (P3) (four in total), the child and partner of the other patient (P2) (two in total) (Fig. S1).The study flow chart was shown in Fig. 1.For Th1; interferon gamma (IFN-c), T-box transcription factors (T-bet), STAT1, STAT4, for Th2; interleukin (IL)-4, IL-5, IL-13, STAT6, GATA binding protein 3 (GATA3), for Th17; IL-17, IL-21, IL-22, IL-6, retinoic acid receptor-related orphan nuclear receptor gamma (RORct), STAT3, for Treg; IL-10, transforming growth factor-beta (TGF-b), FoxP3, and STAT5 expressions were evaluated by real-time polymerase chain reaction (qPCR).It was ensured that the patient, heterozygous, and controls did not have any infection at the time of the study.Two-milliliter blood samples were taken from all patients, heterozygous, and healthy controls into K3-EDTA tubes for relevant gene expression analyses.Flow cytometric and qPCR analyses were performed in triplicate.Expression results of Th-cell subsets were compared in three different groups, including patient, heterozygous, and control individuals (patient/control, heterozygous/control, and heterozygous/patient).The studies reported herein were approved by Institutional Review Board (confirmation number 13216).Written informed consent was obtained from participants.

RNA isolation, cDNA synthesis, and qPCR analysis
First of all, primers were designed for Th1, Th2, Th17, and Treg cell transcription factors and cytokines using IDT PrimerQuest (Table S1).
RNA isolation, PBMC were suspended in 500 lL of QIAzol (Qiagen, India).Phase separation was performed using chloroform, the RNA was precipitated and dissolved in DNase/RNase-free water after being washed with 70% ethanol.The RNA concentrations were then determined, and cDNA synthesis was initiated using the cDNA Synthesis Kit with RNase Inh (High Capacity from A.B.T TM , Turkey).
cDNA synthesis was performed in accordance with the procedure using the cDNA Synthesis Kit with RNase Inh (High Capacity, A.B.T TM , Turkey) with ProFlex 3 9 32 well PCR system (Thermo Fisher Scientific Inc., Waltham, MA, USA).Gene expressions of Th1, Th2, Th17, and Treg cell transcription factors and cytokines were determined by qPCR.
qPCR analysis was performed using SYBRGreen master mix (Hibrigen, 29 SYBR Green Master Mix, Turkey) with QuantStudio 3 qPCR system (Thermo Fisher Scientific Inc., Waltham, MA, USA).A final reaction volume of 10 lL was used for each gene, containing 5 lL of 29 SYBR Green master mix, 5 pMol of forward and reverse primer, and 2 lL of cDNA.The PCR profile included an initial denaturation step at 95 °C for 10 s, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/ extension at 57 °C for 30 s.

Statistical analysis
For normalization GAPDH housekeeping gene was used.Comparative Livak's 2-DDCT method was used to calculate relative gene expression [10].The comparisons of groups (patients/control, patients/carrier, and carrier/control) were analyzed with the Statistical Package for the Social Sciences software, version 21 (IBM SPSS Corp., Armonk, NY, USA) using the Student's t-test.After normalization, DCt values were used in the analyses.Tests were considered a baseline significance level of p < 0.05.Used VolcaNoseR to generate volcano plots that show only unique and/or upregulated features [11].

Demographic characteristics of patients
The diagnosis age of patients (P1, P2, and P3) were 8, 10, and 6 years, respectively.The current ages of the patients (P1, P2, and P3) were 31, 21, and 8 years respectively, and the M/F ratio of patients was 1/2.The flow cytometric evaluation of peripheral blood lymphocytes revealed that CD3+ total T, CD4+ helper T, CD8+ cytotoxic T, and CD3-CD16+ CD56+ natural killer (NK) cell numbers were found to be normal but there was no CD19 expression on CD20+ B lymphocyte (Table 1).CD4+ FoxP3+ Treg cell mean fluorescence intensity (MFI) value of the patients was 2565 AE 846 and 4458 AE 837 in the control (p = 0.001) [12].Clinical features on admission of patients were recurrent sinopulmonary infections.As a result of heterozygous/control comparison, IL-5 (2.87-fold; p = 0.050) was downregulated.There was no significant difference in transcription factors and cytokine expressions of other Th2, Th1, Th17, and Treg cells (Fig. 2B).

DISCUSSION
CD19 deficiency is a disease characterized by impaired B cell number and function, and it is seen that there is no change in total T cells.In our study, in which molecular subsets of T cells and the connection of these cells with possible autoimmune mechanisms were interpreted, changes in T-cell subsets were shown.
Differentiation stages of CD4+ Th cells can be distinguished by their gene expression provided by a number of transcription factors.With the effect of IL-12 in the microenvironment, CD4+ Th cells differentiate into Th1 cells via STAT1, STAT4, and T-bet.Th1 cells are cells that take part in the fight against viral infection [13].When the Th1 cell were compared with the patient/control, it was determined that IL-12 expression was upregulated, STAT1, STAT4, and T-bet expressions were downregulated.Conversely, IFN-c expressions were found to be upregulated.Interestingly, it seems surprising that transcription factor expressions and STAT1 and STAT4 expressions are downregulated and IFN-c expressions are upregulated.In a study by Matsushita et al. in CD19À/À mice, they found increased IFN-c expression similar to our findings [14].In general, the mechanisms by which CD19 regulates the production of both IFN-c and other cytokines are still unclear.In addition, downregulation of Th1 cytokine and transcription factor expressions in the patient/control comparison, it was thought that the upregulation detected in IFN-c expression was caused by NK cells.Because IL-12 expression was also significantly upregulated in the patient/control comparison.In fact, in the presence of IL-12 in the medium, CD4+ Th cells are expected to differentiate towards Th1.Considering the antigen-presenting properties of B cells [15], it also indicates that CD19 deficiency impairs the antigen-presenting properties of B cells.However, there is also evidence in the literature that the absence/lowness of CD19 expression causes expression loss by affecting STAT1 functions [16].In addition, STAT1 was downregulated in the patient/control comparison, while it was unchanged expression in the heterozygous/control comparison.heterozygous genotypes supports the literature on Su et al. [16].Although the primary function of B cells is to produce antibodies, they also act as antigen-presenting cells for T cells [17].B and T-cell interactions promote Th2 rather than Th1 differentiation [14].In our study, it was shown that Th2 differentiation were impaired.In the patient/control comparison, GATA3 and STAT6 expression were significantly downregulated, while the heterozygous/control comparison was shown that expression of GATA3 and STAT6 were unchanged.In addition, Th2 cytokines expressions (IL-4 and IL-5) of patients were also significantly downregulated compared to the control.The increased level of IFN-c in the microenvironment inhibits Th2 cell differentiation [18].It was thought that the downregulation of the transcription factor and cytokines of the Th2 cell detected in our study was due to increased IFN-c expression, and this information was found to be compatible with the literature.

No change in STAT1 expression in individuals with
CD4+ Th cells differentiate in the direction of Th2 with the effect of IL-4, STAT6, GATA3 and the cells release IL-4, IL-5, and IL-13 cytokines Red dots: increased significant expression; Blue dots: decreased significant expression; Gray dots: insignificant change expression/unchanged.For volcano plots, the right side of the "0" point on the fold-change (Log 2 ) axis represents increased expression and the left side represents decreased expression.The value of "1.3"on the significance (ÀLog 10 ) axis indicates p < 0.05 at the top and p > 0.05 at the bottom.)[19].IL-4has been shown to play a vital role in Treg cell development and function [20].Studies in IL-4À/À mice have reported that the inhibition function of Treg cells against autoimmune antigens is reduced compared to wild-type mice [21].In a study conducted in mice with GATA3 deletion, the master transcription factor of Th2 cell, it was reported that symptoms of autoimmune splenomegaly and lymphadenopathy developed at 16 weeks of age [22,23].It has been reported that deletion in GATA3 also causes decreased expression of FoxP3 [24].In our study, it was shown that FoxP3 expression was significantly downregulated in the patient/control comparison, besides it was unchanged in the heterozygous/control comparison.In addition, the MFI rate in Treg cells was significantly lower in patients compared to controls, according to flow cytometric analysis.Studies indicate that along with the loss of FoxP3 expression, Treg cells become autoimmune effector cells [6,25,26].Although there are few studies in the literature that Treg functions are regulated by Th2 cell transcription factor and cytokines, there is no study on CD19 deficiency.In our study, it was thought that downregulation of GATA3, STAT6, IL-4, and IL-5expressions together with decreased Treg function could pave the way for autoimmunity in CD19 deficiency.Moreover, in patients with CD19 deficiency, a major defect occurs in the Th2 cells, and accordingly, it is thought that autoimmune diseases occur as a result of low FoxP3 expression.
Decrease in the number of Treg cells and/or dysfunction cause loss of immune tolerance and the development of autoimmune diseases [6].In fact, Treg cells act not alone but through cytokines secreted by other Th-cell subsets.IL-4 produced by Th2 cells and IL-17 produced by Th17 cells have been shown to inhibit Treg cell development and function [27].Our study supports the literature finding that IL-4 and IL-17 expression is downregulated in patients and reveals for the first time the predisposition of autoimmune diseases seen in CD19 deficiency.
RORct, the major transcription factor for Th17 cells, is induced by TGF-b [6,28].This information indicates that TGF-b has a link in the differentiation of Treg and Th17 cells.In our study, RORct and STAT3 expressions were significantly downregulated in the patient/control comparison.Moreover, RORct and STAT3 expressions was unchanged in the heterozygous/ control comparison.In addition, Th17 cells have been shown to play a role in some autoimmune diseases (uveitis).In the study, it was thought that IFN-c suppressed Th17 responses, which contributed to the development of autoimmunity [29].In our study, although there was increased IFN-c expression, there was no significant change in Th17 cell-derived IL-17 expression.Therefore, although there is still an unexplained situation in these cells due to CD19 deficiency, it is thought that Th-cell subgroups contribute to the autoimmune situation that occurs in patients at different rates.Although the contribution of these interactions to autoimmunity is still controversial, CD19 deficiency partially impairs Th17 cell responses.
The interaction between B and CD4+ Th cell is important for optimal immune responses in preventing the development of autoimmune diseases [30].Antigen-presenting activated B cells provide differentiation and proliferation of CD4+ Th cells.There is no study in the literature that includes CD19 deficiency and Th cells.Although IL-10 regulates Treg development and activity, IL-10 secreted by B cells (especially regulatory B cells) has been shown to inhibit Th1 and Th17 responses [31][32][33].The patient, who was diagnosed with SLE and followed up with CD19 deficiency, presented to us for the first time with hematuria.The hematuria finding regressed during routine intravenous immunoglobulin (IVIg) treatment.However, he was diagnosed with SLE upon discontinuing IVIg treatment voluntarily.In other words, in a period that can be called a late period, the autoimmune disease clinic fully manifested itself.
There are a few major limitations of the study, one of which is the few of patients and the other is the functional analysis of T-cell subsets in which changes are detected.
In conclusion, the change in Th-cell subsets due in CD19 deficiency was demonstrated for the first time at the molecular level.In addition, in this study, possible mechanisms for the interaction between Th-cell subsets and Treg cells were tried to be explained.We think that other T-cell subsets, especially Treg, are different in CD19 deficiency and may contribute to the risk of autoimmunity.

Ó
2023 The Authors.APMIS published by John Wiley & Sons Ltd on behalf of Scandinavian Societies for Pathology, Medical Microbiology and Immunology.123 CD19 DEFICIENCY AND HELPER T-CELL SUBSETS Transcription factor and cytokine analysis of Th subsets Transcription factor and cytokine expression results of Th1, Th2, Th17, and Treg cells were compared as patient/control, heterozygous/control, and heterozygous/patient.As a result of patient/control comparison, T-bet (5.20-fold; p = 0.024), which is the major transcription factor of Th1 cell, and STAT1 (3.77-fold;

Fig. 2 .
Fig. 2. Changes in the expression of transcription factor and cytokines of Th-cell subsets (A, B, and C show the volcano plots of the comparisons.D is a heat map plot of comparisons).(A) Patients/control comparison; (B) Heterozygous/control comparison; (C) Patients/heterozygous comparison.Red dots: increased significant expression; Blue dots: decreased significant expression; Gray dots: insignificant change expression/unchanged.For volcano plots, the right side of the "0" point on the fold-change (Log 2 ) axis represents increased expression and the left side represents decreased expression.The value of "1.3"on the significance (ÀLog 10 ) axis indicates p < 0.05 at the top and p > 0.05 at the bottom.)

Table 1 .
Laboratory features of the patients 2 Below normal value.Ó 2023 The Authors.APMIS published by John Wiley & Sons Ltd on behalf of Scandinavian Societies for Pathology, Medical Microbiology and