Utility of peripheral blood B cell subsets analysis in common variable immunodeficiency

Comparison of clinical and immunological features and mortality in common variable immunodeficiency and agammaglobulinemia patients

BackgroundInfections caused by bacteria or viruses are frequent in common variable immunodeficiency (CVID) patients due to antibody deficiencies, which may be associated with altered T cell function. CVID patients are frequently in contact with pathogen-associated molecular patterns (PAMPs), leading to the activation of innate immunity through Toll-like receptors (TLR) affecting T cell activation. We evaluated the effect of TLR activation on T cells in CVID patients undergoing intravenous immunoglobulin (IVIg) replacement using synthetic ligands.MethodsExpression of exhaustion, activation and maturation markers on T cells from peripheral blood as well as regulatory T cells and follicular T cells in peripheral blood mononuclear cells (PBMCs) from CVID and healthy individuals were evaluated by flow cytometry. PBMCs cultured with TLR agonists were assessed for intracellular IFN-γ, TNF, IL-10, IL-17a or IL-22 secretion as monofunctional or polyfunctional T cells (simultaneous cytokine secretion) by flow cytometry.ResultsWe found increased expression of the exhaustion marker PD-1 on effector memory CD4+ T cells (CD45RA−CCR7−) in the peripheral blood and increased expression of CD38 in terminally differentiated CD8+ T cells (CD45RA+CCR7−). Furthermore, a decreased frequency of naïve regulatory T cells (CD45RA+Foxp3low), but not of activated regulatory T cells (CD45RA−Foxp3high) was detected in CVID patients with splenomegaly, the non-infectious manifestation in this CVID cohort (43.7 %). Moreover, the frequency of peripheral blood follicular helper T cells (CD3+CD4+CXCR5+PD-1+ICOS+) was similar between the CVID and control groups. Upon in vitro TLR3 activation, a decreased frequency of CD8+ T cells secreting IFN-γ, IL-17a or IL-22 was detected in the CVID group compared to the control group. However, a TLR7/TLR8 agonist and staphylococcal enterotoxin B induced an increased Th22/Tc22 (IL-22+, IFN-γ−, IL-17a−) response in CVID patients. Both TLR2 and TLR7/8/CL097 activation induced an increased response of CD4+ T cells secreting three cytokines (IL-17a, IL-22 and TNF)in CVID patients, whereas CD8+ T cells were unresponsive to these stimuli.ConclusionThe data show that despite the unresponsive profile of CD8+ T cells to TLR activation, CD4+ T cells and Tc22/Th22 cells are responsive, suggesting that activation of innate immunity by TLRs could be a strategy to stimulate CD4+ T cells in CVID.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0900-2) contains supplementary material, which is available to authorized users.

The work presented in the first section of this thesis describes how a novel homozygous P440S mutation in LCK results in decreased LCK protein expression and defective TCR signaling. The severe signaling defect impedes thymocyte development, which results in T cell lymphopenia. We find P440S Treg development and function to be compromised, however the loss of T cell function is only partial in conventional T cells. This residual activity in mutant conventional T cells drives the development of intestinal inflammation in P440S mice. In contrast, Lck knock out mice do not develop any intestinal inflammation, despite sharing similar central and peripheral T cell defects. These results indicate that a complete loss of Lck expression and function results in only a state of immunodeficiency, whereas the residual mutant Lck expression and function produces immunodeficiency with immune dysregulation. In the second section of this thesis, we describe our efforts to identify perturbations in both immune cell subsets and antigen receptor signal transduction within a subset of common variable immunodeficiency (CVID) patients with granulomatous lymphocytic interstitial lung disease (GLILD). We determined that CVID patients, regardless of GLILD status, demonstrate increased frequency of HLADR+CD4+ T cells, CD57+CD8+ T cells, and CD21lo B cells when compared to healthy controls. However, CVID patients with GLILD demonstrated discordant antigen receptor signaling events within these subsets of activated lymphocytes. These findings suggest that while increased frequencies of activated lymphocyte subsets may be a common finding in CVID and CVID/GLILD, the downstream cellular function is only disturbed in CVID/GLILD. Our findings provide a cohesive picture of the phenotypic and signaling differences amongst healthy vs. CVID vs. CVID/GLILD individuals, setting up a foundation to identify specific pathways that may be aberrantly signaling in CVID/GLILD patients to better guide personalized therapy.

A substantial proportion of common variable immunodeficiency (CVID) patients has duodenal inflammation of largely unknown etiology. However, because of its histologic similarities with celiac disease, gluten sensitivity has been proposed as a potential mechanism. We aimed to elucidate the role of the duodenal microenvironment in the pathogenesis of duodenal inflammation in CVID by investigating the transcriptional, proteomic, and microbial signatures of duodenal biopsy samples in CVID. DNA, total RNA, and protein were isolated from snap-frozen pieces of duodenal biopsy samples from CVID (with and without duodenal inflammation), healthy controls, and patients with celiac disease (untreated). RNA sequencing, mass spectrometry-based proteomics, and 16S ribosomal DNA sequencing (bacteria) were then performed. CVID separated from controls in regulation of transcriptional response to lipopolysaccharide and cellular immune responses. These differences were independent of mucosal inflammation. Instead, CVID patients with duodenal inflammation displayed alterations in transcription of genes involved in response to viral infections. Four proteins were differently regulated between CVID patients and healthy controls-DBNL, TRMT11, GCHFR, and IGHA2-independent of duodenal inflammation. Despite similar histology, there were major differences in CVID with duodenal inflammation and celiac disease both at the RNA and protein level. No significant difference was observed in the bacterial gut microbial signature between CVID, celiac, and healthy controls. Our findings suggest the existence of altered functions of the duodenal epithelium, particularly in response to lipopolysaccharide and viruses. The latter finding was related to duodenal inflammation, suggesting that viruses, not gluten sensitivity, could be related to duodenal inflammation in CVID.

Perturbations of the CD8+ T-cell repertoire in CVID patients with complications

Subclinical infection and dosing in primary immunodeficiencies.

Common variable immunodeficiency (CVID) patients have markedly decreased immune response to vaccinations. In this study we evaluated humoral and T cell-mediated responses against severe acute respiratory syndrome coronavirus-2 (SARS-Cov-2) with additional flow cytometric changes in CVID patients receiving booster vaccination with BNT162b2 after two ChAdOx1 nCoV-19. The BNT162b2 vaccine raised the anti-spike protein S immunoglobulin G over the cut-off value from 70% to 83% in CVID, anti-neutralizing antibody had been raised over a cut-off value from 70% to 80% but levels after boosting were significantly less in both tests than in healthy controls (*p=0.02; **p=0.009 respectively). Anti-SARS-CoV-2 immunoglobulin A became less positive in CVID after boosting, but the difference was not significant. The cumulative interferon-γ positive T cell response by ELISpot was over the cut-off value in 53% of the tested individuals and raised to 83% after boosting. This and flow cytometric control of cumulative CD4+ and CD8+ virus-specific T cell absolute counts in CVID were also statistically not different from healthy individuals after boosting. Additional flow cytometric measures for CD45+ lymphocytes, CD3+, and CD19+ cells have not shown significant differences from controls except for lower CD4+T cell counts at both time points (**p=0.003; **p=0.002), in parallel CD4+ virus-specific T-cell ratio was significantly lower in CVID patients at the first time point (*p: 0.03). After boosting, in more than 33% of both CVID patients and also in their healthy controls we detected a decrease in absolute CD45+, CD3+, CD3+CD4+, and CD3+CD8+, CD19+, and CD16+56+ cell counts. CD16+CD56+ cell counts were significantly lower compared to controls before and after boosting (*p=0.02, *p=0.02). CVID patients receiving immunosuppressive therapy throughout the previous year or autologous stem cell transplantation two years before vaccination had worse responses in anti-spike, anti-neutralizing antibody, CD3+CD4+T, CD19+ B, and natural killer cell counts than the whole CVID group. Vaccinations had few side effects. Based on these data, CVID patients receiving booster vaccination with BNT162b2 after two ChadOx1 can effectively elevate the levels of protection against COVID-19 infection, but the duration of the immune response together with COVID-19 morbidity data needs further investigation among these patients.

Autoimmune enteropathy (AIE) and common variable immunodeficiency (CVID) can both manifest as chronic diarrhea and small intestinal villous atrophy, making their differentiation challenging. To explore the similarities and differences in the clinical manifestations, laboratory tests, pathological features, and long-term prognoses between these two diseases. This retrospective study included 26 AIE patients and 29 CVID patients with gastrointestinal (GI) involvement who were admitted to our center from June 2012 to May 2024, with all their medical records reviewed. Differences between the two diseases were evaluated via statistical tests. Compared with CVID patients, AIE patients experienced a shorter duration of severe diarrhea, greater weight loss, and more severe hypoalbuminemia and electrolyte imbalances. Furthermore, CVID patients exhibited a notable history of recurrent respiratory infections; significantly lower serum levels of IgG, IgM, and IgA; a marked decrease in B-cell and CD4 + T-cell counts; and a significant inversion of the CD4 + /CD8 + ratio within peripheral blood lymphocyte subsets. Endoscopically, AIE patients are more likely to present with active inflammatory changes, such as erosions and hyperemia. On the basis of histopathological analysis of 23 AIE patients and 24 CVID patients, AIE patients presented with reduced goblet and Paneth cells, pronounced neutrophilic infiltration, and more frequent apoptotic bodies, while CVID patients demonstrated reduced plasma cells and deep crypt lymphocytosis. The diagnostic efficiency of the five pathological items in the duodenum (AUC 0.937), which includes goblet cell and Paneth cell reduction, was greater than that of the four-item combination (AUC 0.622). Long-term follow-up indicated that patients with both conditions were prone to diarrhea relapse, and CVID patients showed a slightly longer median relapse-free survival than did AIE patients. Although AIE patients and CVID patients share many similarities, they exhibit significant differences. A thorough medical history, laboratory tests, and endoscopic and histopathological results provide compelling evidence for their differential diagnosis. Key Points • Both AIE and CVID with gastrointestinal involvement are immune-mediated diseases characterized by chronic diarrhea and small intestinal villi atrophy, making clinical diagnosis difficult. • AIE and CVID have different characteristics that can be used to distinguish them, especially pathological findings.

Somatic Mutations in T Cells As Possible Regulators of Immunodeficiency

Objectives: Common variable immunodeficiency (CVID) patients experience clinical manifestations rather than recurrent respiratory infections including autoimmunity, enteropathy, and lymphoproliferation. We evaluated the correlation of lymphocyte subpopulations with such manifestations. Methods: Twenty-six genetically unsolved CVID patients were subdivided into four phenotypes: infection only (IO), autoimmunity (AI), chronic enteropathy (CE), and lymphoproliferative disorders (LP) and examined for lymphocyte subsets by flow cytometry and TCD4+ proliferation by Carboxyfluorescein succinimidyl ester (CFSE) test. Results: We detected reduced memory B and increased total, effector memory (EM), cytotoxic, and activated TCD8+ in IO, AI and CE, decreased plasmablasts, total and naive TCD4+, Regulatory TCD4+ (Treg) and naive TCD8+ in IO and CE, elevated CD21low B and terminally differentiated effector memory (TEMRA) TCD8+ in IO and AI, increased helper T (Th2) and Th17 in IO, decreased Th1 in AI and defective total and naive B and central memory (CM) TCD4+ in CE. IO showed reduced TCD4+ proliferation response. Conclusions: In genetically unsolved CVID patients, increased Th2 and Th17 and reduced Treg is associated with IO, increased CD21low B and TEMRA TCD8+ and reduced Th1 is contributed to AI and reduced total and naive B, CM TCD4+ and naive TCD8+ and expanded total TCD8+ is correlated with CE.

THU0017 The Role of HLA DQ2 and DQ8 in Dissecting Celiac-Like Disease in Common Variable Immunodeficiency

Background: Common variable immunodeficiency (CVID) is one of the most prevalent symptomatic primary immunodeficiencies (PIDs), which manifest a wide clinical variability such as gastrointestinal (GI) disorder.Patients and Methods: A total of 240 patients with CVID were enrolled in this study. The patients were evaluated for demographic data, clinical manifestations, and immunologic profile.Results: In demographic data, the frequency of consanguinity and mortality rate were higher in patients with GI manifestation than those without GI manifestation. History of GI manifestations was evident in 147 patients (61.3%). The most common GI manifestation in patients with CVID was chronic diarrhea (29.6%). The prevalence of GI disease was 59 of 102 (57.8%) in female patients and 88 of 138 (63.8%) in male patients. The frequency of recurrent infection was higher in patients with GI manifestation than in those without GI manifestation. Also, CVID patients with GI manifestations had lower WBC and CD4+ T cells than patients without GI manifestations.Conclusion: CVID patients are at increased risk of infectious conditions in the GI tract; hence GI manifestations are one of the most important presentations in CVID patients which can appear as a first manifestation or appear during the course of disease.

Common variable immunodeficiency (CVID) is the most common primary immunodeficiency. Prevalence varies greatly between countries and studies. Most diagnostic criteria include hypogammaglobulinemia and impaired vaccine response. To evaluate the minimum prevalence as well as the clinical and immunological phenotypes of CVID in Southern Finland. We performed a cross-sectional study to assess all adult CVID patients followed up in three hospital districts in Southern and South-Eastern Finland between April 2007 and August 2015. CVID diagnosis was based, with a minor modification, on the ESID/PAGID criteria for primary CVID. Antipolysaccharide responses to Pneumovax® were defined as impaired only if 50% or more of the serotypes did not reach a level of 0.35 µg/mL after vaccination. We further characterized the patients' B cell phenotypes and complications associated with CVID. In total, 9 patients were excluded due to potential secondary causes before diagnosis. ESID/PAGID criteria were met by 132 patients (males 52%), of whom, 106 had "probable" and 26 "possible CVID." Based on the population statistics in the three hospital districts, the minimum adult prevalence per 100,000 inhabitants in Finland for all CVID ("probable CVID," respectively) patients was 6.9 (5.5). In the highest prevalence district (Helsinki and Uusimaa), the prevalence was 7.7 (6.1). CVID patients suffer from frequent complications. Ten patients died during follow-up. Of probable CVID patients, 73% had more than one clinical phenotype. Intriguingly, gradual B cell loss from peripheral blood during follow-up was seen in as many as 16% of "probable CVID" patients. Patients with possible CVID displayed somewhat milder clinical and laboratory phenotypes than probable CVID patients. We also confirm that large granular lymphocyte lymphoproliferation is a CVID-associated complication. The prevalence of CVID in Finland appears the highest recorded, likely reflecting the genetic isolation and potential founder effects in the Finnish population. Studies to discover potential gene variants responsible for the high prevalence in Finland thus seem warranted. Increased awareness of CVID among physicians would likely lead to earlier diagnosis and improved quality of care.

Increased Prevalence of Neoplasms In Patients Diagnosed with Common Variable Immunodeficiency.

All patients with congenital X-linked agammaglobulinemia (XLA) and the majority of patients with common variable immunodeficiency (CVI) have reduced ecto-5′-nucleotidase (ecto-5′-NT) activity in their peripheral blood lymphocytes (1–3). Ecto-5′-NT deficiency in XLA and CVI patients may be partially explained by an absence of mature B lymphocytes. However, the majority of these patients also have decreased ecto-5′-NT activity in total T cell preparations. We have previously demonstrated that T? (suppressor) cells have only one third the ecto-5′-NT activity of Tμ (helper) cells (4); however, little is known regarding ecto-5′-NT activity in T cell subsets as defined by the monoclonal antibodies OKT4 (helper/inducer) and OKT8(cytotoxic/suppressor). Since many agammaglobulinemia patients have defects in T cell regulation of immunoglobulin biosynthesis, the experiments described here were designed to determine: (1) whether OKT4+ and OKT8+ cells isolated from normal individuals have different levels of ecto-5′-NT activity, and (2) whether low T cell ecto-5′-NT activity in XLA and CVI patients can be explained by altered proportions of subsets of regulatory T cells.