**4. Discussion**

Using flow cytometry, we determined the lymphocyte profiles of four patient groups: CVID-C, CVID-OI, SLE, and HCs.

In our study, patients with a complicated CVID phenotype had low proportions of Tregs and NK cells. Decreased proportions of Tregs in CVID were first reported by Fevang et al. [36]. They included 26 patients diagnosed with CVID according to the WHO classification. In CVID patients, they found significantly reduced expression of the transcription factor FoxP3 in CD3+ cells and a decreased proportion of T CD4+CD25highFoxP3+ cells in the CD4(+) population, as measured by flow cytometry. The lowest proportion of Tregs was found in patients with CVID and splenomegaly (spleen size > 13 cm on ultrasound examination). Treg proportions correlated negatively with neopterin levels as a marker of chronic inflammation [36]. Kofod-Olsen et al. in a study of 26 patients with CVID, demonstrated an association between decreased levels of Tregs and autoimmune phenomena [37]. Moreover, in patients with CVID, the functions of Tregs were disturbed [36,38]. One study that included 20 children with CVID (mean age 173 months) found no difference in Tregs between CVID cases and healthy controls [39]. This suggests different etiopathologies of childhood vs. adult-onset CVID.

We found low NK cells in the CVID-C group (median, 54 cells/μL). Similarly, patients with CVID from the French registry who had a severe reduction of NK cells (<50/μL at study inclusion) presented with a complicated phenotype [40]. Therefore, our results support the finding that low NK cell numbers can be a biomarker of complicated CVILD [41,42].

We found that patients with CVID-C had reduced Th17 proportions. Th17 cell involvement in CVID has not been well studied [18,43]. Barbosa et al. were the first to evaluate circulating Th17 cells in 30 patients with CVID [43]. They found a significant reduction in Th17 cells in CVID patients who had less than 2% smB cells and more than 10% of cd21low B cells. This suggested that there is a link between B cell maturation disturbances and maintenance of Th17 cells [43]. Edwards et al. found low Th17 numbers in patients with predominantly deficient antibodies and noninfectious complications [18].

Bregs play a critical role in immune homeostasis and tolerance. Despite extensive efforts to phenotypically characterize Bregs, we still lack a definitive set of phenotypic markers or a signature transcriptional regulator (equivalent to FoxP3 Tregs) that enables us to comprehensibly identify Bregs [44]. In our study, Bregs were identified as CD19+ CD5+ CD1dhigh. CD1d is a major phenotypic marker highly expressed in many Breg cells, and it may play a crucial role in Breg-cell-mediated suppression [45]. The upregulation of CD1d on B cells is associated with B-cell-mediated protection against inflammation [44,46]. In our study, we observed a tendency for decreased Bregs in CVID-C patients; however, this was not statistically significant. Yesillic et al. found significantly lower proportions of Breg cells in 25 adult patients with CVID [47]. On the other hand, Kofod-Olsen et al. showed that an elevated frequency of pro-B10 cells in CVID correlated with autoimmunity and splenomegaly [37]. In another study that included 42 adult patients with CVID, the Breg number was low, and their function was disturbed [48]. Although 40% of patients in that study had autoimmune symptoms, there was no correlation between the reduction of Breg cells and autoimmunity. Discrepancies among the studies might be a result of different methodologies of Breg assessment and differences in the incidence and types of autoimmunity in published cohorts.

T cell subsets in our CVID-C patients were characterized by low naive CD4+T cells, low RTE CD4+ T cells, low naïve CD8+T cells, and low RTE CD8+T cells. These results illustrate that profound T cell pool abnormalities are part of the picture of complicated CVID. Signs of senescence in T cell maturation have been reported in CVID-C patients. Stuchlý et al. hypothesized that in CVID with autoimmune thrombocytopenia, naive CD4+ T cell pool depletion occurred and increased the likelihood of promoting autoreactive T cells to memory stages concurrently with B cell activation [49]. Activated B cells, which cannot progress through germinal center reactions, fail to produce isotype-switched antibodies [50].

To better understand autoimmunity, we compared the cytometry results between CVID and SLE patients, as a prototypic autoimmune disease. Patients with CVID and SLE both had lymphopenia. We found that only a more detailed analysis of peripheral lymphocyte subset counts showed differences; specifically, smB cells and plasmablasts were reduced in CVID, which is in agreement with other studies [30]. Therefore, low smB cells detected in patients with autoimmunity should increase the awareness of immunodeficiency. In a recently published study, retrospective evaluation of available peripheral lymphocyte subset counts revealed lower proportions of class-switched memory B cells [9] in patients with inflammatory rheumatic diseases. These patients also presented with mutations in PID genes and were subsequently revealed to have hypogammaglobulinemia.

The expansion of the CD21low B cell compartment and increased plasmablasts are expected findings in autoimmune diseases. This was not the case in the SLE patients in our study. Moreover, they had the lowest Th17 percentages. This can be explained by the low disease activity in our patients [50]. However, Treg imbalance persists despite low SLE activity and might be a further attractive therapeutic goal [51].

Our study has some limitations. First, the sample size of each group was relatively small. Due to the limited sample size, we were not able to assess the relationships between specific clinical symptoms and lymphocyte subsets. However, the limited number of CVID patients is to be expected as it is a rare disease, and our sample size is similar to that of other single-center studies. On the other hand, we collected extensive data from each patient and only included patients with a confirmed diagnosis.

In conclusion, we confirmed that patients with CVID and autoimmune phenomena had reduced Treg, Th17, and NK cells.

Future studies are needed to confirm whether reductions of Treg, Th17, and NK cells might be a biomarker of more complicated CVID cases. Nevertheless, class-switched memory B cells can help distinguish patients with different causes of autoimmunity. Our results suggest that T and B cell maturation analyses should be performed routinely in clinical practice.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/jcm10153356/s1, Table S1: Internal laboratory reference values of lymphocyte subpopulations and absolute numbers, Table S2: Internal laboratory reference values if the proportions of B lymphocyte maturation, Table S3: Internal laboratory reference values if the proportions of T lymphocyte maturation, Figure S1: Representative gating strategy of main lymphocytes subpopulation and T regulatory (Tregs), B regulatory cells (B regs) and Th17 cells in study group, Figure S2: Representative B lymphocytes maturation gating strategy in study group. (A) B lymphocytes gating strategy: FSC-A vs. FSC-H plot: Gating the cells that have an equal area and height, thus removing clumps (greater FSC-A relative to FSC-H) and debris (very low FSC), CD45 vs. SSC-A plot: Selection of lymphocytes based on their SSC/CD45+ properties, CD19 vs. SSC-A plot: Selection of lymphocytes B based on their SSC/CD19+ properties. (B) Representative dot plots of each maturation B subsets cells: transitional B cells (orange), naïve B cells (blue), non-switched memory B cells (red), class switched memory B cells (yellow), CD21low cells (purple) and plasmablasts (green) based on their IgD/IgM, IgD/CD38, CD21/CD38, IgM/CD38, CD27/CD21 properties (phenotypes of all cells described in section: material and method), Figure S3: Representative T lymphocytes maturation gating strategy in study group. (A) T lymphocytes gating strategy: FSC-A vs. FSC-H plot: Gating the cells that have an equal area and height, thus removing clumps (greater FSC-A relative to FSC-H) and debris (very low FSC), CD45 vs. SSC-A plot: Selection of lymphocytes based on their SSC/CD45+ properties, CD3 vs. SSC-A plot: Selection of lymphocytes T based on their SSC/CD3+ properties. CD4 vs. CD8 plot: Selection of lymphocytes T CD4+ (pink) and CD8+ (yellow) based on their CD4+ or CD8+ properties. (B) Representative dot plots of each maturation T CD4+ subsets cells: recent thymic emigrants T CD4+ cells, naïve CD4+ T cells, effector CD4+ T cells, central memory CD4+ T cells and effector memory CD4+ T cells based on CD197/CD45RO, CD62L/CD45RA, CD45RA/CD31 properties (phenotypes of all cells described in section: material and method). (C) Representative dot plots of each maturation T CD8+ subsets cells: recent thymic emigrants T CD8+ cells, naïve

CD8+ T cells, effector CD8+ T cells, central memory CD8+ T cells and effector memory CD8+ T cells based on their CD197/CD45RO, CD62L/CD45RA, CD45RA/CD31 properties (phenotypes of all cells described in section: material and method).

**Author Contributions:** Conceptualization, E.W.-S., E.R. and I.K.; methodology, E.W.-S., E.R. and I.K.; formal analysis, E.W.-S., E.R., I.K. and K.J.-R.; investigation, E.W.-S., E.R., I.K., M.K. and D.S.; resources, E.W.-S., E.R., I.K., M.K. and D.S.; data curation, E.W.-S. and E.R.; writing—original draft, E.W.-S.; writing—review and editing, E.W.-S., E.R., I.K. and K.J.-R.; visualization, E.W.-S., I.K. and E.R.; supervision, E.W.-S., E.R. and K.J.-R.; project administration, E.W.-S. and K.J.-R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study protocol was approved by the Bioethics Committee of the Military Institute of Medicine (approval no. 7/WIM/2020). All patients were informed in detail orally about the course, aims, and scope of this research. Blood sampling was limited to routine assessments.

**Informed Consent Statement:** Separate written consent for blood sampling and review of records were not required by the IRB due to the retrospective nature of this study. All patient data were confidential, and the study procedures complied with the Declaration of Helsinki.

**Data Availability Statement:** Data supporting reported results can be made available by request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.
