*2.3. Flow Cytometry Analysis*

All blood samples were drawn during routine visits. If the CVID patients were on immunoglobulin replacement therapy (IgRT), blood samples were drawn before the day of IgG infusion according to the national regulations for treatment reimbursement.

Lymphocyte subset percentages were determined according to literature [15,35] by flow cytometry using a panel of monoclonal antibodies using FACS Canto II BD flow cytometry (Becton Dickinson (BD) Biosciences, Franklin Lakes, NJ, USA). Subsequently, all eight-color surface staining panels for the basic subpopulation of lymphocytes were evaluated as follows: CD4 FITC, CD3 PerCP-Cy5-5, CD19 PE-Cy7, CD8 APC, CD16 APC-H7, and CD45 V500 (BD Biosciences).

B cell subpopulations were defined using the following antibodies: IgD PE, CD27 PerCP-Cy5-5, CD19 PE-Cy7, IgM APC, CD38 APC-H7, CD21 V450, and CD45 V500 (BD Biosciences).

CD4 T cell and CD8 maturation were defined using CD4 FITC, CD196 PE, CD197 PerCP-Cy5-5, CD45RO PE-Cy7, CD45RA APC, CD3 APC-H7, CD8 V450, and CD45 V500 (BD Biosciences) antibodies.

For Th17 cells, CD4 FITC, CD196 PE, CD45RO PE-Cy7, and CD45 V500 (BD Biosciences) antibodies were used.

Recent thymic emigrant (RTE) CD4+ or CD8+ cells were analyzed using CD4 FITC, CD62L PE, CD31 PerCP-Cy5-5, CD45RO PE-Cy7, CD45 RA APC, CD3 APC-H7, and CD8 V450 CD45 V500 (BD Biosciences) antibodies.

For Tregs, we used CD127 FITC, CD4 PerCP-Cy5-5, CD25 APC, CD3 APC-H7, CD45 V500 (BD Biosciences) antibodies, and for regulatory B cells (Bregs), CD1d PE, CD19 PE-Cy7, CD5 APC, and CD45 V500 (BD Biosciences) antibodies were used.

After surface staining for 15 min at 21 ◦C in the dark, erythrocytes were lysed with 2 mL of BD Pharm Lyse buffer (BD Biosciences) for 10 min. Following centrifugation and washing with Cell Wash buffer (BD Biosciences), the mixture was stored in the dark for analysis by flow cytometry within 2 h. Data were analyzed with DIVA Analysis software (version 8.0.1, BD Biosciences, San Jose, CA 95131 USA) and Infinicyt 1.8 Flow Cytometry (Cytognos, Salamanca, Spain).

Lymphocyte counts were obtained using a SYSMEX XN-1500 (Sysmex Corp., Kobe, Japan) hematological analyzer.

Internal quality control was performed daily by checking the optical detector, aligning lasers, and fluid systems using CS&T IVD Beads BD FACS Diva (BD Biosciences), San Jose, CA 95131 USA, respectively, according to the manufacturers' guidelines. Internal reference values of lymphocyte counts and proportions are presented in Supplementary Materials (Tables S1–S3).

We distinguished the following subpopulations in B cell maturation:


We distinguished the following subpopulations in T cell CD4+ or CD8+ maturation:


We distinguished the following other lymphocytes subpopulations:


Representative B and T lymphocyte maturation gating strategies in patients are presented in Figures S1–S3 (Supplementary Materials).

#### *2.4. Statistical Analysis*

All statistical analyses were performed using Statistica® software (version 13.0; TIBCO Software, Palo Alto, CA, USA). Statistical significance was set at *p* < 0.05. The results are expressed as medians (Q1–Q3) of the lymphocyte populations. For group comparisons, the Kruskal–Wallis, analysis of variance (ANOVA), and post hoc analysis tests were used.

#### **3. Results**

#### *3.1. The Clinical Characteristics of Patients*

Among the 33 patients with CVID, 14 were women and 19 were men, and the overall mean age at the time of blood sampling was 37.75 years (min–max: 21–66 years). Among the 9 patients with CVID-OI, 4 (44%) were men, and among 24 patients with CVID-C, 16 (66%) were men. The mean age was 37.0 years (±17.2) and 38.4 years (±12.4) in patients with CVID-OI and CVID-C, respectively. By the time of blood sampling, two patients were receiving low-dose prednisolone (5 mg/day), and one was being treated with methotrexate and etanercept due to psoriatic arthritis.

All CVID patients had a positive history of increased susceptibility to infections; however, only 9 of 33 (27%) presented with a clinical phenotype limited to infections. The clinical characteristics of the patients are summarized in Table 1. All 17 SLE patients were female, mean age 43.05 years (min–max 18–60). The SLE patients had low or mild disease activity according to the Systemic Lupus Erythematosus Disease Activity Index 2000 SLEDAI2K (mean value 3.4). Sixteen patients with SLE were treated with antimalarials, and ten patients received prednisolone (5–15 mg/day).


**Table 1.** The clinical characteristics of patients with CVID and SLE.

CVID: Common variable immunodeficiency, SLE: systemic lupus erythematosus, SLEDAI2K: Systemic Lupus Erythematosus Disease Activity Index 2000.

#### *3.2. Peripheral Main Lymphocyte Subsets, Tregs, Bregs, and Th17 Cells*

The proportion of total lymphocytes was low in the CVID-C and SLE groups. It differed significantly from that of the HC group. CVID-OI patients had all lymphocyte proportions similar to those of HC.

The proportion of T lymphocytes was the lowest in CVID-C and differed significantly from that in HCs. In SLE patients, T lymphocytes were also significantly lower than in the HC group. The proportion of T lymphocytes in CVID-OI was similar to that in HC. The results are summarized in Table 2.

**Table 2.** Differences in the median of lymphocyte subpopulations and absolute numbers between (a) the CVID with complicated phenotype (CVID-C) group, (b) CVID phenotype limited to only infections (CVID-OI group), (c) patients with systemic lupus erythematosus (SLE), and (d) healthy controls (HCs). Data expressed as median (Q1–Q3). \* *p* < 0.05. ANOVA: analysis of variance, Post Hoc: post hoc analysis tests.


Among the T lymphocytes, the proportion of CD4+ T cells was significantly reduced in patients with CVID-C, CVID-OI, and SLE compared to that in HC. There were no differences in the proportion of CD8+ T cells between the disease groups and HC. The CD4/CD8 ratio was reduced in CVID-C and CVID-OI compared with that in HCs.

The proportion of circulating B cells was reduced in CVID-C and differed significantly from that in HC and SLE.

The NK cell proportion was marked decreased in CVID-C patients compared to HC patients.

The proportion of Th17 cells was reduced in CVID-C and SLE and differed significantly from that in HCs. The Th17 cell counts were the lowest in SLE (Figure 1).

**Figure 1.** Differences in the median percentages of lymphocyte subpopulations between the CVID with complicated phenotype (CVID-C) group, the CVID phenotype limited to only infections (CVID-OI), patients with systemic lupus erythematosus (SLE), and healthy controls (HCs). Data expressed as median (Q1–Q3), \* *p* < 0.05.

Treg counts were low in CVID-C and SLE patients and differed significantly from HCs. Breg counts were lowest in CVID-C patients, but no significant differences were noted between the disease groups and the HC group (Figure 2). Treg counts were low in CVID-C and SLE patients and differed significantly from HCs.

**Figure 2.** Differences in the median percentages of T regulatory cells (Tregs) and B regulatory cells (Bregs) between the CVID with complicated phenotype (CVID-C) group, CVID phenotype limited to only infections (CVID-OI group), patients with systemic lupus erythematosus (SLE), and healthy controls (HCs). Data are expressed as median (Q1–Q3) \* *p* < 0.05.

Considering the absolute numbers of the above-mentioned lymphocyte populations, the same trends were observed. A difference was observed only in the CD8 lymphocytes of SLE patients, which were significantly less than in the HCs (Table 2).

## *3.3. B Lymphocyte Maturation*

Analysis of the maturation of B-lymphocytes showed that the proportion of transitional B cells was highest in CVID-OI and differed significantly from that in HCs. The frequency of nonswitched memory B cells was the highest in patients with CVID-C. The difference in nonswitched memory B cell counts was significant between the CVID-C and SLE groups and between the CVID-C and HC groups. Class-switched memory B cell percentages were low in CVID-C and CVID-OI. The difference was significant in comparison with the percentage of smB cells in HCs and SLE. The proportion of CD21low B cells was higher in CVID-C and CVID-OI than in HCs. Plasmablasts were significantly higher in SLE patients than in CVID-C and CVID-OI patients (Table 3, Figure 3). The proportions of B lymphocyte maturation for each patient with CVID-C, CVID-OI, SLE, and HC are presented on heat maps in Figure 4.

**Figure 3.** Differences in the proportions of B lymphocyte maturation between the CVID with complicated phenotype (CVID-C) group, CVID phenotype limited to only infections (CVID-OI group), patients with systemic lupus erythematosus (SLE), and healthy controls (HCs). Data expressed as median (Q1–Q3), \* *p* < 0.05.

**Table 3.** Differences in the proportions of B lymphocyte maturation between (a) the CVID with complicated phenotype (CVID-C) group, (b) CVID phenotype limited to only infections (CVID-OI group), (c) patients with systemic lupus erythematosus (SLE), and (d) healthy controls (HCs). Data expressed as median (Q1–Q3). \* *p* < 0.05. ANOVA: analysis of variance, Post Hoc: post hoc analysis tests.


**Figure 4.** The proportions of B lymphocyte maturation for each patient with CVID with complicated phenotype (CVID-C) group, CVID phenotype limited to only infections (CVID-OI group), patients with SLE, and healthy controls (HCs). Data expressed as a median percentage (%).
