Marcela Vlkova

Age dependency and mutual relations in T and B lymphocyte abnormalities in common variable immunodeficiency patients

Common variable immunodeficiency (CVID) is primary hypogammaglobulinaemia with an unknown aetiopathogenesis. Although various abnormalities of T and B cells have been described, their pathogenetic roles are unclear. We determined T and B lymphocyte subsets known to be abnormal in CVID in order to disclose possible relations between numerical abnormalities in those cells. Markers associated with B cell development (CD21, CD27, IgM, IgD) were determined on B lymphocytes (CD19+); T lymphocyte development (CD45RA, CD45RO, CD62L) and activation markers (CD25, CD27, CD28, CD29, CD38, CD57, HLA‐DR) were determined on CD4+ and CD8+ T lymphocytes in 42 CVID patients and in 33 healthy controls. Abnormalities in CD4+ T lymphocyte activation markers (increase in CD29, HLA‐DR, CD45RO, decrease in CD27, CD62L, CD45RA) were observed particularly in patients with a decreased number of memory (CD27+) and mature (CD21+) B cells (group Ia according to the Freiburg groups classification), while abnormalities observed in CD8+ cells (increase in CD27 and CD28 and decrease in HLA‐DR, CD57 and CD38) did not depend upon grouping patients together according to B lymphocyte developmental subpopulations. We observed correlations between immature B cells (IgM+ CD21–) and expression of CD27, CD62L, CD45RA, CD45RO and HLA‐DR on CD4+ T cells in CVID patients but not in the control group. The expression of CD27 and CD45RA on CD4+ T lymphocytes, such as the percentage of IgD+ CD27– and IgD+ CD27+ cells in B lymphocytes, showed age dependency to be more significant than in the control group. Our study demonstrates that T and B lymphocyte abnormalities in CVID are partially related to each other. Some of those abnormalities are not definite, but may evolve with age of the patient.

Antibody forming cells and plasmablasts in peripheral blood in CVID patients after vaccination.

Common variable immunodeficiency (CVID), the most frequent primary antibody disorder, is characterized by hypogammaglobulinaemia and impaired antibody production. Poor vaccination response is essential for the diagnosis of CVID. Their under laying defects remain to be elucidated. Routine determination of antibody production in serum from CVID patients after vaccination and investigation of B cell function in vivo is complicated due to substitution therapy. Therefore we investigated antibody production on the B-cell level by ELISPOT and characterized changes in B-cell subpopulations in CVID patients, including plasmablasts, in peripheral blood by flow cytometry after vaccination for specification of the diagnosis. Thirty-seven CVID patients and eighty healthy volunteers were immunized with tetanus toxoid and pneumococcal polysaccharide vaccines. Specific antibody levels and B cell subpopulations were measured before vaccination and on day 7 after vaccination by ELISPOT assay and flow cytometry respectively. Of the thirty-seven well defined CVID patients studied, thirty lacked detectable spot forming cells producing specific IgG, IgA or IgM antibodies against employed vaccines and seven had only weak responses compared to controls. In the control group, an increase in circulating plasmablasts on day 7 post immunization corresponded with the appearance of antibody forming cells. In contrast, CVID patients failed to increase plasmablasts significantly in peripheral blood after antigen challenge. Our findings indicate that CVID patients have a block in terminal B-cell differentiation and that flow based assessment of plasmablasts in peripheral blood after vaccination serves as a surrogate diagnostic marker for assessing in vivo antibody responses in patients suspected to have CVID.

T and B lymphocyte subpopulations and activation/differentiation markers in patients with selective IgA deficiency

Selective deficiency of immunoglobulin A (IgAD) and common variable immunodeficiency (CVID) are genetically closely related diseases, both of unknown pathogenesis. A plethora of abnormalities in lymphocyte subpopulations and expression of activation markers were repeatedly documented in CVID patients, while almost no data are available about lymphocyte subpopulations in IgAD patients. We determined basic lymphocyte subpopulations and those subpopulations that were reported to be abnormal in CVID patients (CD25, human leucocyte antigen (HLA)‐DR CD45RA, CD45RO, CD27, CD28 and CD29 on both CD4+ and CD8+ cells, CD57 and CD38 on CD8+ cells, CD21, CD27, IgM, IgD on B lymphocytes) in 85 patients with IgAD, 47 patients with CVID and in 65 healthy controls. Statistical analysis was performed by the Mann–Whitney U‐test; significant P‐values were determined by means of Bonferonis correction. Our results showed an increase in the relative number of CD8+ cells and a decrease in the absolute number of CD4+ cells compared to healthy people, but similar abnormalities in CVID patients were much more expressed. IgAD patients had significantly decreased expression of HLA‐DR and increased expression of CD25 on CD4+ lymphocytes, also CD29 expression was decreased on CD8+ cells, while other activation/differentiation markers on T cells (including the expression of CD45RA and CD45RO antigens) were not changed. There were no statistically significant abnormalities in B lymphocyte developmental stages in IgAD patients compared to healthy controls. Our observation showed that the majority of T and B lymphocyte subpopulation abnormalities described previously in CVID are not present in IgAD patients.

Regulatory B cells in CVID patients fail to suppress multifunctional IFN-γ+TNF-α+CD4+ T cells differentiation

Common variable immunodeficiency (CVID) refers to primary hypogammaglobulinemia with unknown pathogenesis. Although there is evidence for intrinsic B cell defects in some CVID patient groups, various abnormalities in cytokine production by T cells in CVID patients are frequently observed. Here, we demonstrate a relationship in the production of pro-inflammatory Th1 cytokines and regulatory B cells producing IL-10 between CVID patients and healthy controls. We describe CD19(+)CD24(hi)CD38(hi)IL-10(+) regulatory B cells generated after T cell stimulation of human peripheral blood lymphocytes ex vivo are able to suppress IFN-γ(+)TNF-α(+) producing CD4(+) T cells. This process is impaired in CVID patients, who present with both low numbers of CD19(+)CD24(hi)CD38(hi)IL-10(+) B cells and increased numbers of IFN-γ(+)TNF-α(+)CD4(+) T cells. Disruption of the regulatory B cell response to T cell stimulation explains the excessive T cell activation regarded as an immunoregulatory abnormality that is a frequent finding in CVID patients.

Characterization of Lymphocyte Subsets in Patients with Common Variable Immunodeficiency Reveals Subsets of Naive Human B Cells Marked by CD24 Expression

Increased proportions of naive B cell subset and B cells defined as CD27negCD21negCD38neg are frequently found in patients with common variable immunodeficiency (CVID) syndrome. Current methods of polychromatic flow cytometry and PCR-based detection of κ deletion excision circles allow for fine definitions and replication history mapping of infrequent B cell subsets. We have analyzed B cells from 48 patients with CVID and 49 healthy controls to examine phenotype, frequency, and proliferation history of naive B cell subsets. Consistent with previous studies, we have described two groups of patients with normal (CVID-21norm) or increased (CVID-21lo) proportions of CD27negCD21negCD38neg B cells. Upon further analyses, we found two discrete subpopulations of this subset based on the expression of CD24. The B cell subsets showed a markedly increased proliferation in CVID-21lo patients as compared with healthy controls, suggesting developmental arrest rather than increased bone marrow output. Furthermore, when we analyzed CD21pos naive B cells, we found two different subpopulations based on IgM and CD24 expression. They correspond to follicular (FO) I and FO II cells previously described in mice. FO I subset is significantly underrepresented in CVID-21lo patients. A comparison of the replication history of naive B cell subsets in CVID patients and healthy controls implies refined naive B cell developmental scheme, in which human transitional B cells develop into FO II and FO I. We propose that the CD27negCD21negCD38neg B cells increased in some of the CVID patients originate from the two FO subsets after loss of CD21 expression.

B-lymphocyte Subpopulations in Patients with Selective IgA Deficiency

IntroductionSelective deficiency IgA (IgAD) is the most common primary abnormality of immunoglobulin production with unknown pathophysiology. It is genetically related to common variable immunodeficiency (CVID), where besides IgA also IgG and frequently IgM serum levels are decreased. In this study we focused on determination of B-lymphocyte developmental stages and searching for similarities between CVID and IgAD.Materials and MethodsUsing flow cytometry we determined major lymphocyte subpopulations and B-lymphocyte subsets: naïve (CD27-IgD+), marginal zone cells (CD27+IgD+), class-switched memory cells (CD27+IgD-), “double-negative” B cells (CD27-IgD-), transitional cells (IgM++CD38++), plasmablasts (CD38+++IgM+ or IgM-), and CD21lowCD38low cells in 80 patients with IgAD, 48 patients with CVID, and 80 control persons.ResultsCompared to healthy controls, a decrease in the absolute number and frequency of CD4+ cells (both < 0.001) was observed in IgAD patients. A decrease in the frequency of switched memory cells (P < 0.001), transitional cells (P = 0.035) as well as plasmablasts (P < 0.001) and an increase in the CD21lowCD38low subset (P = 0.007) was observed in IgAD patients compared to control persons. No significant differences were observed in the remaining B-cell developmental subsets. A decrease in CD27+IgD- (<0.4% of peripheral blood lymphocytes), frequently observed in CVID patients and also previously reported in IgAD, was found in only five patients (6%) with IgAD, two of them being first-degree relatives of CVID patients.ConclusionOur results show a decrease of terminally differentiated B-lymphocyte subsets in patients with IgAD, similar as previously found in patients with CVID, but these results are less expressed than in CVID patients.

Chronic immune activation in common variable immunodeficiency (CVID) is associated with elevated serum levels of soluble CD14 and CD25 but not endotoxaemia

Common variable immunodeficiency (CVID), the most frequent symptomatic immunoglobulin primary immunodeficiency, is associated with chronic T cell activation and reduced frequency of CD4+ T cells. The underlying cause of immune activation in CVID is unknown. Microbial translocation indicated by elevated serum levels of lipopolysaccharide and soluble CD14 (sCD14) has been linked previously to systemic immune activation in human immunodeficiency virus/acquired immune deficiency syndrome (HIV‐1/AIDS), alcoholic cirrhosis and other conditions. To address the mechanisms of chronic immune activation in CVID, we performed a detailed analysis of immune cell populations and serum levels of sCD14, soluble CD25 (sCD25), lipopolysaccharide and markers of liver function in 35 patients with CVID, 53 patients with selective immunoglobulin (Ig)A deficiency (IgAD) and 63 control healthy subjects. In CVID subjects, the concentration of serum sCD14 was increased significantly and correlated with the level of sCD25, C‐reactive protein and the extent of T cell activation. Importantly, no increase in serum lipopolysaccharide concentration was observed in patients with CVID or IgAD. Collectively, the data presented suggest that chronic T cell activation in CVID is associated with elevated levels of sCD14 and sCD25, but not with systemic endotoxaemia, and suggest involvement of lipopolysaccharide‐independent mechanisms of induction of sCD14 production.

Altered Serum Cytokine Signature in Common Variable Immunodeficiency

PurposeCommon variable immunodeficiency (CVID) is the most frequent form of primary symptomatic hypogammaglobulinemia. CVID patients display a number of abnormalities in lymphocyte subpopulations including chronic T-cell activation and decreased numbers of circulating CD4+ T cells and NK cells. We and others have recently shown that CVID is associated with increased concentration of soluble CD14 (sCD14) and other factors indicating limited microbial translocation.MethodsTo address the mechanisms of chronic immune activation in CVID, we performed a detailed analysis of cytokine serum levels in 36 patients with CVID, 52 patients with selective IgA deficiency (IgAD), and 56 healthy volunteers.ResultsWe show that CVID is associated with elevated serum levels of CXCL-10/IP-10, IL-1R antagonist, TNF-α, IL-10, IL-12 (p40), CCL-2/MCP-1, G-CSF, and CCL-11/eotaxin. The detected cytokine signature is consistent with an ongoing activation of cells of myeloid lineage. In contrast, the levels of cytokines typically produced by CD4+ T helper cells of Th1 (IFN-γ, IL-2), Th2 (IL-9, IL-13), and Th17 (IL-17) subtypes were suppressed in CVID patients compared to healthy donors.ConclusionsPresented data suggest that the altered cytokine profile observed in patients with CVID may be attributed to the activation of monocyte-macrophage and granulocyte lineages, possibly driven by the translocation of bacterial components across the gastrointestinal or respiratory tracts mucosal barrier.

Early manifestation and recognition of C2 complement deficiency in the form of pyogenic infection in infancy

Objective:  Although frequently asymptomatic, C2 complement component deficiency may lead to severe pyogenic infections or lupus‐like illness. In the present report, we describe infectious manifestations in infancy and childhood in our C2‐deficient patients.

Selective IgM Deficiency: Clinical and Laboratory Features of 17 Patients and a Review of the Literature

PurposePrimary selective IgM deficiency (sIgMD) is a primary immunodeficiency with unclear pathogenesis and a low number of published cases.MethodsWe reviewed clinical and laboratory manifestations of 17 sIgMD patients. Serum IgM, IgG, and its subclasses, IgA, IgE, antibodies against tetanus toxoid, pneumococcal polysaccharides and Haemophilus influenzae type b, isohemagglutinins, and T and B lymphocyte subsets, expressions of IgM on B cells and B lymphocyte production of IgM were compared with previously reported case reports and a small series of patients, which included 81 subjects in total.ResultsWe found that some patients in our cohort (OC) and published cases (PC) had increased IgE levels (OC 7/15; PC 21/37), decreased IgG4 levels (OC 5/14), very low titers of isohemagglutinins (OC 8/8; PC 18/21), increased transitional B cell counts (OC 8/9), decreased marginal zone B cell counts (OC 8/9), and increased 21low B cell counts (OC 7/9). Compared with the PC (20/20), only two of five OC patients showed very low or undetectable production of IgM after stimulation. A majority of the patients had normal antibody production to protein and polysaccharide antigens, basic lymphocyte subset counts, and expression of surface IgM molecules on B cells.ConclusionsLow IgM levels are associated with various immunopathological disorders; however, pathogenic mechanisms leading to decreased IgM serum level in selective IgM deficiency remain unclear. Moreover, it is difficult to elucidate how strong these associations are and if these immunopathological conditions are primary or secondary.