Immunophenotyping of putative human B1 B cells in healthy controls and common variable immunodeficiency (CVID) patients

B1 B cells represent a unique subset of B lymphocytes distinct from conventional B2 B cells, and are important in the production of natural antibodies. A potential human homologue of murine B1 cells was defined recently as a CD20⁺CD27⁺CD43⁺ cell. Common variable immunodeficiency (CVID) is a group of heterogeneous conditions linked by symptomatic primary antibody failure. In this preliminary report, we examined the potential clinical utility of introducing CD20⁺CD27⁺CD43⁺ B1 cell immunophenotyping as a routine assay in a diagnostic clinical laboratory. Using a whole blood assay, putative B1 B cells in healthy controls and in CVID patients were measured. Peripheral blood from 33 healthy donors and 16 CVID patients were stained with relevant monoclonal antibodies and underwent flow cytometric evaluation. We established a rapid, whole blood flow cytometric assay to investigate putative human B1 B cells. Examination of CD20⁺CD27⁺CD43⁺ cells is complicated by CD3⁺CD27⁺CD43^hi T cell contamination, even when using stringent CD20 gating. These can be excluded by gating on CD27⁺CD43^lo–int B cells. Although proportions of CD20⁺CD27^–CD43^lo–int cells within B cells in CVID patients were decreased by 50% compared to controls (P < 0·01), this was not significant when measured as a percentage of all CD27⁺ B cells (P = 0·78). Immunophenotypic overlap of this subset with other innate-like B cells described recently in humans is limited. We have shown that putative B1 B cell immunophenotyping can be performed rapidly and reliably using whole blood. CD20⁺CD27⁺CD43^lo–int cells may represent a distinct B1 cell subset within CD27⁺ B cells. CVID patients were not significantly different from healthy controls when existing CD27⁺ B cell deficiencies were taken into account.     

L-selectin in patients with common variable immunodeficiency (CVID): a comparative study with normal individuals

L-selectin is one of the key members of the selectin family of adhesion molecules and initiates leucocyte attachment to specialized high endothelial venules. The shed form, which retains functional activity, can be detected in biological fluids and is increased in diseases of many kinds. In the present study, we investigated L-selectin expression on leucocytes and measured the soluble form in the plasma of healthy individuals and patients with CVID. A significant loss of L-selectin expression is found on CVID B cells, which is marked by the presence of a substantial population of L-selectin-negative B cells in the peripheral blood of some CVID patients. On CD4⁺ T cells, the loss in L-selectin expression affects mostly the CD45RO⁺ population. Peripheral blood leucocytes other than lymphocytes express L-selectin molecule normally. Moreover, soluble L-selectin was detected in significantly increased levels in CVID plasma compared with healthy controls. Our data suggest that the loss of L-selectin expressed by lymphocytes may be due to increased or aberrant lymphocyte activation in CVID patients who remain immunodeficient, and down-regulation of L-selectin from these lymphocytes may significantly contribute to the elevated levels of soluble L-selectin in the plasma, which may in turn affect further lymphocyte trafficking.     

Lymphocyte characteristics in children with common variable immunodeficiency

The diagnosis of common variable immunodeficiency (CVID) is reserved for patients who suffer from undefined B cell dysfunction. Division of the CVID population into subgroups enables research for underlying disease causes. We studied clinical features and lymphocyte characteristics in 38 children with CVID and compared them to 30 children with less severe antibody deficiencies (e.g. specific antibody deficiency combined with IgG subclass deficiency) and with 65 pediatric controls. Most pediatric immune phenotypes were comparable to adult CVID phenotypes, including a selective increase in newly formed B cells and a decrease in memory B cells and CD4⁺ T cells. Eighteen percent of pediatric patients had a mutation in the TNFRSF13B gene, which requires further investigation. Finally, pediatric patients with decreased class-switched memory B cells had significantly more complications.     

Measurement of peripheral B cell subpopulations in common variable immunodeficiency (CVID) using a whole blood method

Recent reports have described reduced populations of CD27+ memory B cells and increased percentages of undifferentiated B cells in peripheral blood of patients with common variable immunodeficiency (CVID). This work has prompted two attempts to classify CVID based on rapid flow cytometric quantification of peripheral blood memory B cells and immature B cells. Evidence to support the hypothesis that such in vitro B cell classification systems correlate with clinical subtypes of CVID is being sought. For the classification to be useful in routine diagnosis, it is important that the flow cytometric method can be used without prior separation of peripheral blood mononuclear cells (PBMC). We have examined 23 CVID patients and 24 controls, using both PBMC and whole blood, and find an excellent correlation between these methods. The reproducibility of the method was excellent. We classified the CVID patients by all three of the existing classifications, including secretion of immunoglobulin by B cells in vitro as described by Bryant, as well as the more recent flow cytometric classification methods. Only one patient changed classification as a result of using whole blood.     

Two novel cationic staphylococcal proteins induce IL-2 secretion, proliferation and immunoglobulin synthesis in peripheral blood mononuclear cells (PBMC) of both healthy controls and patients with common variable immunodeficiency (CVID).

Two cationic proteins, a neutral phosphatase (NP-tase) and a 70-kD protein (p70) were isolated from Staphylococcus aureus by ion exchange chromatography. We compared their properties to those of the well established B cell mitogen of whole, fixed Staph. aureus strain Cowan I cells (SAC). Both purified proteins were able to induce immunoglobulin synthesis in PBMC cultures of healthy donors. NP-tase and p70 also induced immunoglobulin synthesis of PBMC from those patients with CVID who were also responsive to SAC plus IL-2 stimulation. Immunoglobulin synthesis in response to NP-tase and to p70 was time- and dose-dependent and could be inhibited by addition of specific antibodies against the proteins. In contrast to SAC, no addition of exogenous IL-2 was necessary to obtain maximal immunoglobulin synthesis induced by NP-tase or p70. However, neither protein was able to induce immunoglobulin synthesis in B cell-enriched cultures. High amounts of IL-2 were found in supernatants of PBMC from healthy donors following stimulation with low concentrations of NP-tase or p70, and this was associated with vigorous lymphocyte proliferation. Both proteins behave like typical antigens, and not like lectins or superantigens, since an NP-tase-stimulated T cell line showed an antigen-specific, MHC-restricted secondary response. In addition, no preferential T cell receptor V beta chain usage was found with eight V beta-specific MoAb. It is likely that the two proteins induce antigen-specific T cell activation, which is then followed by polyclonal activation of B cells via CD40 receptors and cytokine release.     

Granulomatous-lymphocytic interstitial lung disease (GLILD) in common variable immunodeficiency (CVID)

Infectious complications of the lung occur quite frequently in patients with common variable immunodeficiency (CVID), a clinical syndrome that represents a primary immunodeficiency. However, there appears to be noninfectious pulmonary complications in association with CVID as well, and recently the term granulomatous-lymphocytic interstitial lung disease (GLILD) has been created to describe these noninfectious, diffuse lung disease complications that develop in CVID patients. They exhibit both granulomatous and lymphoproliferative histologic patterns, consisting of lymphocytic interstitial pneumonia (LIP), follicular bronchiolitis, and lymphoid hyperplasia. There are many unanswered questions surrounding this relatively unstudied entity. In an attempt to answer some of these questions, this review discusses in detail pathologic and clinical features of GLILD and its proposed pathogenesis with a particular attention to potential role of human herpesvirus 8 (HHV-8). Lastly, therapeutic approach is discussed to generate novel treatment strategy to better care for a subgroup of CVID patients afflicted with this entity.     

Absence of memory B cells in patients with common variable immunodeficiency

The molecular basis of common variable immunodeficiency (CVID) is unknown. To assess humoral immunity in CVID, we selected 24 patients with early or late onset of disease. X-linked agammaglobulinemia (XLA), X-linked hyper-IgM syndrome (XHIM), and non-XHIM were excluded based on clinical phenotype, assessment of the immune response, presence of Bruton's tyrosine kinase (Btk) in monocytes or platelets, and normal expression of CD40 ligand by activated T cells. The number of circulating B cells was within the normal range or reduced. IgD(-) CD27(+) memory B cells were markedly reduced or absent in all 24 patients and IgD(+) CD27(+) B cells were diminished in 8 patients. Circulating B cells from all 6 patients examined, including CVID patients with IgD(+) CD27(+) cells, failed to undergo somatic hypermutation in immunoglobulin-variable (V)-region genes, similar to cord blood B cells. B cells from CVID patients produced IgM and IgG, but not IgA upon the engagement of Ig receptor and CD40 in the presence of IL-2 and IL-10. B cells from all but 5 patients secreted IgE when stimulated by CD40 crosslinking in the presence of IL-4. The observation of defective memory B cells with abnormal cell marker expression and function demonstrates that naive CVID B cells including those expressing IgD(+) CD27(+), in analogy to cord blood and hyper-IgM syndrome B cells, may be responsible for their failure to differentiate into plasma cells and to produce high-affinity antibodies of different isotypes.     

T cell phenotypes in patients with common variable immunodeficiency disorders: associations with clinical phenotypes in comparison with other groups with recurrent infections

Common variable immunodeficiency disorders (CVID) are a group of heterogeneous conditions that have in common primary failure of B cell function, although numerous T cell abnormalities have been described, including reduced proliferative response and reduced regulatory T cells. This study compared the T cell phenotype of CVID patients subdivided into clinical phenotypes as well as patients with partial antibody deficiencies [immunoglobulin (Ig)G subclass deficiency and selective IgA deficiency], X-linked agammaglobulinaemia (XLA) and healthy and disease controls. Absolute numbers of T cell subpopulations were measured by four-colour flow cytometry: naive T cells, central and effector memory and terminally differentiated (TEM) T cells, using CD45RA and CCR7 expression. Early, intermediate and late differentiation status of T cells was measured by CD27/CD28 expression. Putative follicular T cells, recent thymic emigrants and regulatory T cells were also assessed. Significant reduction in naive CD4 T cells, with reduced total CD4 and recent thymic emigrant numbers, was observed in CVID patients, most pronounced in those with autoimmune cytopenias or polyclonal lymphoproliferation. These findings suggest a lack of replenishment by new thymically derived cells. CD8 naive T cells were reduced in CVID patients, most significantly in the autoimmune cytopenia subgroup. There was a reduction in early differentiated CD4 and CD8 T cells and increased CD8 TEM in the CVID patients, particularly autoimmune cytopenia and polyclonal lymphoproliferation subgroups, suggesting a more activated T cell phenotype, due perhaps to an antigen-driven process. XLA patients had significantly reduced putative follicular T cells, which may depend on B cells for survival, while no significant alterations were observed in the T cells of those with IgG subclass deficiency or selective IgA deficiency.     

Antigen presentation by common variable immunodeficiency (CVID) B cells and monocytes is unimpaired

CVID is a primary immunodeficiency syndrome comprising a heterogeneous group of patients with hypogammaglobulinaemia and defective formation of specific antibodies. Previous studies demonstrated defective T cell responsiveness to antigen in a major subgroup of patients. In the present study we investigated the capacity of peripheral blood monocytes and Epstein–Barr virus (EBV)-transformed B cell lines from seven patients with CVID, including two patients expressing an extended MHC haplotype described to be associated with CVID, to present antigen (Tet. Tox.) to CD4⁺ antigen-specific T cell lines from healthy controls. The results presented show an unimpaired capacity of peripheral blood monocytes to present antigen in all patients studied. In addition, the present study demonstrates for the first time that CVID B cells function normally as antigen-presenting cells (APC). These findings indicate that expression of a certain MHC phenotype in CVID is not associated with a defect in the presentation of recall antigen by monocytes and B cells. Based on these studies, uptake, processing and re-expression of recall antigen in association with MHC class II molecules on the APC surface are functional and there is no indication for structural abnormalities of the MHC class II molecules expressed by the patients studied that could be essential for their function in antigen binding and presentation.     

Common variable immunodeficiency in adults: current diagnostic protocol and laboratory measures

Common variable immunodeficiency (CVID) is one of the most common symptomatic primary immune defects in adults. Unfortunately, the diagnosis of CVID is often delayed because of its variable presentations and manifestations. Sincere efforts from experts in the field of primary immunodeficiency disorders all over the world have made us wiser regarding the clinical features, associated complications and long-term outcome of the disease. However, a systematic compilation of diagnostic protocols and laboratory measures is desirable. The present review highlights in brief the immunological and molecular defects characterizing adult CVID along with a comprehensive discussion on currently followed laboratory criteria and methods used to identify these cases. The article may benefit the clinicians, immunologists, infectious disease specialists, pulmonary medicine specialists, gastroenterologists, dermatologists and other physicians who are involved in the team management of the patients with CVID.     

A novel compound heterozygous TACI mutation in an autosomal recessive common variable immunodeficiency (CVID) family

Common variable immunodeficiency (CVID) is a primary immune disorder characterized by low immunoglobulin serum levels and increased susceptibility to infections. Underlying genetic causes are only known in less than 15% of patients and encompass mutations in the genes encoding for ICOS, TACI, BAFF-R, CD19, CD20, CD81 and MSH5. TACI is the most frequently mutated gene among CVID patients. We report on two pediatric Italian male siblings with hypogammaglobulinemia and recurrent respiratory and gastrointestinal infections in association with a novel compound heterozygous TACI mutation. Both patients carry the I87N/C104R mutation that has not been reported yet. This results in aberrant TACI expression and abrogates APRIL binding on EBV B cells. This study identifies a novel combined mutation in TNFRSF13B increasing the spectrum of TACI mutations associated with CVID.     

CD95 expression and function on lymphocyte subpopulations in common variable immunodeficiency (CVID); related to increased apoptosis.

Apoptosis is now recognized as a central process of development and disease, and it has been proposed as one of the mechanisms that may account for the lymphopenia seen in some diseases. In this study we measured spontaneous apoptosis and CD95 expression on different cell subpopulations from CVID patients, using flow cytometric techniques. We divided our patients into two groups according to their CD4+ and CD4+CD45RA+ cell counts. Our results clearly show increased spontaneous apoptosis and CD95 expression on the CD4+ and CD4+CD45RA+ subsets from lymphopenic CVID patients compared with normal subjects and disease controls. Interestingly, our lymphopenic CVID patients presented a profound reduction in absolute counts, mainly affecting the CD4+CD45RA+ subpopulation. We also found a statistically significant direct correlation between absolute numbers of CD4+CD45RA+ T cells and spontaneous apoptosis on the same subset in CVID patients, but attempts to induce CD95-mediated apoptosis were unsuccessful despite increased CD95 expression on CD4+ T cells. These findings suggest that apoptosis could be one of the mechanisms implicated in the significant lymphopenia present in these patients.     

Low ficolin-2 levels in common variable immunodeficiency patients with bronchiectasis

Common variable immunodeficiency (CVID) encompasses a heterogeneous group of antibody deficiencies characterized by susceptibility to recurrent infections and sequelae, including bronchiectasis. We investigated the relevance of the lectin complement pathway in CVID patients by analysing ficolin-2 and ficolin-3 serum levels and genotyping single nucleotide polymorphisms (SNPs) in the FCN2 and FCN3 genes. Our results show that ficolin-2 levels in CVID patients are significantly lower (P < 0·0001) than in controls. The lowest ficolin-2 levels are found in CVID patients with bronchiectasis (P = 0·0004) and autoimmunity (P = 0·04). Although serum levels of ficolin-3 were similar in CVID patients and controls, CVID patients with bronchiectasis again showed lower levels when compared to controls (P = 0·0001). Analysis of single nucleotide polymorphisms in the FCN2 gene confirmed known influences on ficolin-2 serum levels, but did not support a genetic basis for the observed ficolin-2 deficiency in CVID. We found that CVID patients with bronchiectasis have very low levels of ficolin-2. The reason for the deficiency of ficolin-2 in CVID and any possible causal relationship is currently unknown. However, as bronchiectasis is a very important factor for morbidity and mortality in CVID, ficolin-2 could also serve as biomarker for monitoring disease complications such as bronchiectasis.     

Immunophenotypical alterations in a subset of patients with common variable immunodeficiency (CVID).

We investigated the expression of surface molecules on lymphocytes from 20 patients with CVID and 40 healthy subjects. Lymphocytes were analysed by dual colour flow cytometry. We identified a subset of patients (8 of 20) characterized by low CD4/CD8 ratio (less than 1.1), expansion of T cells co-expressing the activation marker HLA-DR and significant increase in CD8+ T cells co-expressing CD57. Expression of the adhesion molecules LFA-3 (CD58) and ICAM-1 (CD54) was significantly increased in this subgroup. In addition, within the CD4+ T cells the percentage of CD29+ (memory) cells was increased, while the CD45RA and LAM-1 (Leu-8) antigens were depressed. These results indicate that in a subgroup of CVID patients T cells are activated in vivo and the CD57+CD8+ lymphocyte subpopulation, supposed to comprise functional suppressor T cells, is expanded. We suggest a chronic viral infection in these patients, but it is not clear whether this is primary or secondary to the underlying defect.     

Impaired up-regulation of CD70 and CD86 in naive (CD27-) B cells from patients with common variable immunodeficiency (CVID)

CVID is characterized by reduced serum levels of all switched immunoglobulin isotypes (IgG, IgA, IgE) predisposing patients to recurrent infections of their respiratory and gastrointestinal tract. Correspondingly, most CVID patients exhibit a severely decreased proportion of class switched memory B cells (CD19+CD27+IgD-IgM-IgG+ or IgA+) in their peripheral blood (CVID type I). We previously identified a subgroup of CVID patients showing a significantly reduced expression of CD86 and CD137 following activation in vitro of PBMC or purified B cells (CD19+) with anti-IgM plus IL-2. Here we extend our previous studies by asking whether highly purified, cell-sorted naive B cells show already an expression defect of B cell surface molecules relevant in activation (CD39, CD69), differentiation (CD24, CD27, CD38) or T-B interaction (CD25, CD70, CD86). We stimulated cell-sorted, naive B cells (CD19+CD27-IgM+IgDhighIgG-IgA-) from 10 CVID patients and 10 healthy controls for 4 days with anti-IgM plus IL-2 in the absence or presence of autologous CD4+ T cells and measured the expression of the referred surface molecules. Based on reduced or normal numbers of switched memory B cells the CVID patients had previously been classified into eight type I patients and two type II patients, respectively. Interestingly, only the molecules CD25, CD70 and CD86, all relevant in cognate T-B interaction, showed a significantly lower expression in naive B cells from CVID patients compared to controls. While coculture with autologous CD4+ T cells normalized the CD25 expression, CD70 and CD86 expression remained subnormal, notably in the eight CVID patients of type I. These findings strongly suggest an intrinsic signalling or expression defect for CD70/CD86 at the level of naive B cells in type I CVID patients.     

Anti-tumour necrosis factor-alpha therapy for severe enteropathy in patients with common variable immunodeficiency (CVID)

We present three common variable immunodeficiency (CVID) patients with severe inflammatory bowel disease of unknown aetiology, resistant to steroid treatment, treated with infliximab. After exclusion of any infection, infliximab was given at a dose of 5 mg/kg every 4 weeks for a 3 month induction followed by every 4-8 weeks depending on clinical response. Two of these patients had predominantly small bowel disease; they both showed clinical response to infliximab with weight gain and improvement of quality of life scores. The third patient had large bowel involvement with profuse watery diarrhea; this patient improved dramatically within 48 hours of having infliximab treatment. All three patients have been maintained on infliximab treatment for between 5 and 53 months (mean 37 months) with no evidence of increased susceptibility to infections in the patients with small bowel disease, although the third patient developed two urinary tract infections and a herpes zoster infection following therapy. This is the first small case series to show that infliximab is a useful addition to current therapy in this rare group of patients with potentially life threatening enteritis.