B-2 lymphocytes and the balance of pro and anti-inflammatory cytokines in infectious and autoimmune phenotypes of common variable immune deficiency

Introduction

Common variable immune deficiency (CVID) is the most common type of primary immunodeficiency (PID) with impaired antibody production [1]. The main laboratory diagnostic characteristic of CVID is severe hypogammaglobulinemia, while increased susceptibility to infections is its typical clinical feature [2]. Meanwhile, infectious manifestations may not always determine the leading clinical presentations. This is stipulated by the heterogeneity of the mechanisms of development of hypogammaglobulinemia in CVID. There is no single genetic disorder that specifies this variant of inborn errors in the immune system: a genetically monogenic cause of CVID is found in only 10–20% of patients [3]. Impaired antibody production can occur due to defects in the cooperation of T and B lymphocytes during the formation of an immunological synapse, defects in the own signaling pathways of B cells or regulatory components of the immune response. Clinically, this variant of immune dysfunction can exhibit itself as lymphoproliferative, gastrointestinal, and autoimmune disorders. A systematic analysis of the Web of Science, Scopus, and PubMed databases conducted by the study team from the earliest available date to February 2020 showed that autoimmune manifestations accounted for 29.8% (95% CI: 26.4–33.3; I2 = 82.8%) of all patients with a proven CVID diagnosis. The prevalence of hematologic autoimmune diseases, autoimmune gastrointestinal disorders, autoimmune rheumatologic diseases, autoimmune skin diseases, and autoimmune endocrinopathy in patients with CVID was 18.9%, 11.5%, 6.4%, 5.9%, and 2.5%, respectively [4]. In some patients with CVID, autoimmune manifestations may be the only clinical sign of the disease, which introduces additional difficulties in diagnosing the root cause of the disorders and, subsequently, the therapy efficacy [5][6]. In most cases, patients with CVID have a combination of infectious syndrome and organ-specific inflammatory processes. The immune dysregulation underlying the non-infectious manifestations of CVID is difficult to identify in terms of generally accepted diagnostic criteria for this PID variant [7]. However, the development of such kind of diagnostic tests is necessary for the adequate treatment of immune-mediated inflammatory complications in CVID. The prospects in achieving this destination imply the identification of systemic biomarkers among which the B lymphocytes as the final stage determining antibody synthesis can play a significant role [8][9]. Another important biomarker in this aspect is the cytokine spectrum that reflects the involvement of the regulatory mechanisms of the immune response in the pathogenesis of various CVID phenotypes [10][11][12].

The purpose of the study was the comparative characterization of the subpopulation composition of B-lymphocytes and the cytokine spectrum in peripheral blood at infectious and non-infectious manifestations of CVID.

Materials and methods

The results of the examination of 10 people (5 women, 5 men, 32–65 years old) with a CVID diagnosis are presented in this study. The diagnosis was made in accordance with the clinical recommendations of the Russian Association of Allergists and Clinical Immunologists (RAACI) and the criteria of the European Scientific Society of Primary Immunodeficiencies (www.esid.org) for the diagnosis of patients with primary humoral immunodeficiencies. In six patients, the main clinical manifestation was related to an infectious phenotype of the disease with insults of the bronchi, lungs, frontal and maxillary sinuses. In four patients, non-infectious manifestations predominated including enteropathy, which upon further examination was classified as Crohn's disease (1 individual), hemolytic anemia (1 individual), and autoimmune hepatitis (2 individuals). All patients received replacement therapy with intravenous immunoglobulins (IVIG) at a dose of 0.4 g/kg monthly. This paper presents the results of immunological testing obtained during the screening period before the next IVIG transfusion. The comparison group consisted of 10 practically healthy blood donors aged 20–32 years. Voluntary informed consent to conduct research was obtained from all participants.

The levels of cytokines IL-4, IL-10, IL-17, TNF-α, IFN-ɣ in blood serum were determined by ELISA using diagnostic test systems produced by Vector-Best ZAO (Russia). Phenotypic characterization of peripheral blood B cells was carried out using flow cytometry (Cytomics FC 500, USA). When analyzing the parameters of B-lymphocytes, the relative content (in relation to the total number of peripheral blood lymphocytes) of B-2 lymphocytes (CD3^–CD19⁺CD5^–) and their subpopulations was assessed, including naive B-lymphocytes (CD19⁺CD27^–), switched memory B-cells (CD19⁺CD27⁺IgD^–IgM^–), and unswitched memory B cells (CD19⁺CD27⁺IgD⁺IgM⁺). For this purpose, suitably matched monoclonal antibodies with a set of different color fluorophores produced by Beckman Coulter (USA) were used.

Statistical processing of the results was carried out using the STATISTICA 10.0 software (USA). The results obtained were described by calculating the median (Me) and interquantile range of values in the form of the 25th and 75th percentiles, which is presented in the text as Me [LQ; UQ]. The analyzed indicators did not have a normal distribution; the Shapiro-Wilko test was used. Comparative analysis of groups was performed using the Wilcoxon test. Differences were considered statistically significant at p<0.05.

Results

Phenotypic analysis of the subpopulation composition of peripheral blood B lymphocytes is presented in Table 1. The data obtained show that both groups of patients do not differ from each other in the total amount of circulating B-2 cells, as well as from the control comparison group. Meanwhile, the relative amount of memory B cells in all patients with CVID is reduced in comparison with the control parameters of practically healthy people. More specifically, the proportion of memory B cells in patients with infectious CVID manifestation is less (12% of all B-2 lymphocytes) when compared to patients with non-infectious manifestation (14% of B-2 cells). It should be noted that in the healthy comparison group, the corresponding indicator accounts for 30%. Further analysis of the surface receptors of B-cells, which characterize the ability of B-lymphocytes to synthesize various classes of immunoglobulins, reveals that in patients with infectious CVID manifestations, the proportion of switched memory B-lymphocytes in the total pool of B-2 cells is greater (2.3%) than in the group with autoimmune manifestations (1.4%). Respectively, patients with autoimmune CVID manifestations have more unswitched memory B-lymphocytes than patients with infectious disease manifestations.

The results of the comparative analysis of peripheral blood cytokine levels are presented in Table 2. These data show that in patients with infectious CVID manifestations relative to the comparison group of healthy donors, the levels of IFN-ɣ and TNF-α are increased two-fold but there are no differences in the serum IL-17 level. Meanwhile, in autoimmune manifestations, the contents of all the three cytokines mentioned above are increased in relation to the comparison control parameters; in particular, the level of TNF-α is threefold greater, whereas the IL-17 and IFN-ɣ are almost an order of magnitude higher. In addition, the absence of differences between the patient groups is revealed by the serum levels of IL-4 and IL-10, whose values are within the reference rates regardless of the type of clinical CVID manifestations.

Discussion

It is worth noting that the disruption of the maturation processes of memory B-lymphocytes is one of the typical signs of CVID; however, it does not manifest itself in 100% of cases of diagnosed disease [13]. The present study confirmed that all examined patients with CVID were characterized by a decrease in memory B cells relative to the parameters of practically healthy donors. However, despite a certain resemblance of detected changes in the two patient groups, differences in the clinical exhibition of primary hypogammaglobulinemia correspond to the degree and quality of the identified changes in B-cell phenotypes. Indicatively, in patients with autoimmune CVID manifestations, which have a greater amount of memory B cells compared to patients with infectious CVID manifestations, there are fewer B-2 lymphocytes switched to synthesize specific immunoglobulins of various classes. It is important to emphasize that similar trends were noted by researchers when analyzing the variability of the subpopulation composition of B cells in patients with autoimmune thrombocytopenia [7], which is the most common variant of autoimmune disorder in CVID. This study demonstrated quantitative changes in different mediator contents, responsible for pro-inflammatory reaction and the modulating immune response, which is consistent with the information available in the scientific databases [14]. From the presented results, it follows that manifestations of cytokine dysregulation in immune processes are detected regardless of the clinical type of CVID manifestation. However, the qualitative and quantitative characteristics of changes in the cytokine spectrum differ depending on the clinical type of CVID manifestation. In particular, the unifying feature is an increase in the contents of the leading pro-inflammatory mediator TNF-α as well as IFN-ɣ, which is the regulator of the Th-1 variant of the immune response. The differences between the groups of patients with CVID are due to the fact that in the autoimmune phenotype, the degree of activation of IFN-ɣ and TNF-α is greater than in the other group; in addition, only this clinical phenotype of CVID is characterized by an excess of Th-17 cytokine levels in the profile. In other words, these pro-inflammatory mediators specify the differences between groups of patients with CVID, namely their predominance in autoimmune phenotype.

Conclusions

The autoimmune phenotype of CVID is characterized by a greater amount of circulating memory B cells and a smaller proportion of switched memory B cells than in the infectious type of the disease manifestations.

In the autoimmune phenotype of CVID, the spectrum and contents of proinflammatory cytokines are greater than in the infectious phenotype.

The most dynamic changes in the two clinical phenotypes of CVID are associated with the production of the Th-1 cytokine of the IFN-γ profile, with the degree of production being more pronounced in the autoimmune variant.

The mediator Th-17 variant of the IL-17 immune response exceeds the value of the comparison group of practically healthy donors only in the autoimmune phenotype of CVID.