Association Between Cytometric Biomarkers, Clinical Phenotype, and Complications of Common Variable Immunodeficiency

Abstract

Background: Common variable immunodeficiency (CVID) is a heterogeneous group of immune disorders. The patients are classified according to the clinical manifestation with the infection-only phenotype (CVID_inf) and CVID with immune dysregulation (CVID_id).

Methods: We performed a retrospective clinical analysis of 64 CVID patients (34 males, 53.13%; mean age: 41.4 years; SD: ±21.4 years). We divided the patients into subgroups according to the clinical manifestation (CVID_inf and CVID_id) and according to B cell phenotypic profiling after performing flow cytometry with the use of the EUROclass classification. We compared clinical manifestations, selected laboratory parameters, and therapy in these groups. All CVID_id patients were tested after the manifestation of complications associated with immune dysregulation and in eight patients during the immunosuppressive treatment (systemic corticosteroids and hydroxychloroquine).

Results: Two-thirds of patients in our cohort had symptoms resulting from immune dysregulation. Almost half of the patients had autoimmune complications. A higher proportion of marginal zone B cells was associated with autoimmune complications. A lower percentage of naïve B cells was connected to autoimmunity, whereas a lower proportion of transitional B cells was associated with rheumatic diseases and splenomegaly. Patients with lymphadenopathy had a higher percentage of double-negative T cells and a lower percentage of switched memory B cells. We performed molecular-genetic testing in 28% (n = 17) of patients and found a causal pathogenic variant in 23.5% (n = 4) of this group.

Conclusion: Based on our results, there is an association between specific cytometric parameters, clinical phenotype, and complications of CVID. The use of the subpopulations of B cells can be helpful in the diagnosis of these specific clinical complications in CVID patients and could help to personalise the therapeutic approach.

Keywords: b cell phenotypic profiling; common variable immunodeficiency; immune dysregulation; marginal zone b cells; transitional b cells.

Figure 1. Age distribution of our CVID cohort.

CVID: common variable immunodeficiency.

Figure 2. Hierarchical gating strategy used for immunophenotyping of B lymphocytes in patients with CVID.

(A) Distribution based on side scatter analysis and expression of CD19. The population of all B lymphocytes expressing the CD19 marker is located in red square. (B) Distribution according to expression of IgD and CD27, naive CD19+IgD+CD27- (upper left quadrant), non-switched memory/marginal zone-like B lymphocytes CD19+IgD+CD27+ (upper right quadrant), and class-switched memory CD19+IgD-CD27+ (lower right quadrant). (C) Distribution according to the expression of CD38 and CD21. The population of activated B lymphocytes was determined as cells with low expression of CD21 and CD38 (lower left quadrant). (D) Distribution according to the expression of IgM and CD38, high expression of IgM and CD38 is characteristic for transitional B cells (upper green square), and lymphocytes expressing CD19 and CD38 with low expression of IgM were considered to be plasmablasts (lower green square). CVID: common variable immunodeficiency.

Figure 3. Division of the patients according to the EUROclass classification.

The distribution of patients according to the EUROclass classification after the examination of B cell phenotyping by flow cytometry. The EUROclass classification separates patients according to the proportion of B cells, switched memory B cells, transitional B cells, and CD21^low B cells - nearly absent B cells (B-, <1%), the proportion > 1% B cells (B+), severely reduced switched memory B cells (SmB-, <2%), the proportion > 2% of SmB (SmB+), the expansion of transitional B cells (Tr^high, >9%), without the expansion of transitional B cells (Tr^norm, <9%), and the expansion of CD21^low B cells (CD21^low, >10%), without the expansion of CD21^low B cells (CD21^norm, <10%) [9]. Group B+SmB+CD21^norm had 20 patients (32.8%), group B+SmB+CD21^low had 18 patients (29.5%), group B⁺SmB⁻CD21^normTr^norm was observed in nine patients (18%), group B⁻ had six patients (10%), group B⁺SmB⁻CD21ˡᵒʷTr^norm had five patients (8%), and group B⁺SmB⁻CD21^normTrʰⁱᵍʰ was observed in three patients (5%). We did not identify any patient from class B+SmB-CD21ˡᵒʷTrʰⁱᵍʰ.

Figure 4. Complications of CVID patients.

The first number above each column represents the number of patients with specific complications, and the second number represents the percentage of the cohort total. CVID: common variable immunodeficiency.

Figure 5. Venn diagram of autoimmune complications.

Numbers in areas of the Venn diagram indicate the number of patients with a specific combination of complications.

Figure 6. Proportion of selected flow cytometry parameters according to the presence of complications.

Groups of CVID patients with (Yes) and without (No) specific complications are compared according to the following: (A) the percentage of marginal zone B cells (autoimmune complications, p = 0.001); (B) the proportion of naïve B cells (autoimmune complications, p = 0.01); (C) the proportion of marginal zone B cells (rheumatic disease, p = 0.013); (D) transitional B cells (rheumatic disease, p = 0.007); (E) transitional B cells (splenomegaly, p = 0.046); (F) switched memory B cells (lymphadenopathy, p = 0.034); and (G) the percentage of CD3+CD4-CD8-TCRα/β (lymphadenopathy, p = 0.029). Mann-Whitney U test was used in graphs A-F. χ² test was used in graph G (comparison of the group of patients with >2.7% of double-negative T cells (DNT) cells in peripheral blood to patients with <2.7%). If a p-value is less than 0.05, it is flagged with one star (*). If a p-value is less than 0.01, it is flagged with two stars (**). If a p-value is less than 0.001, it is flagged with three stars (***). CVID: common variable immunodeficiency.