Altered leukocyte subsets and immune proteome indicate proinflammatory mechanisms in mastocytosis.

BACKGROUND: Indolent systemic mastocytosis (ISM) is characterized by pathologic accumulation of mast cells. The mechanism behind its phenotypic heterogeneity is not well understood. Interaction of mast cells with other immune cells might cause systemic inflammation and thereby associated symptoms. OBJECTIVE: We investigated peripheral leukocyte compartments and serum immune proteome in ISM. METHODS: Peripheral blood leukocyte phenotyping using flow cytometry in a cohort of 18 adults with ISM and 12 healthy controls. Targeted proteomics was performed to measure 169 proteins associated with inflammation on serum of another 20 ISM patients and 20 healthy controls. RESULTS: Proportions of plasmacytoid dendritic cells and monocytes were significantly decreased while TH2 cells were increased in peripheral blood of ISM patients. Furthermore, a shift from naive to memory T cells was observed. Hierarchical clustering of the serum proteome revealed 2 distinct subgroups within ISM patients. In subgroup A (n = 8), 62 proteins were significantly overexpressed, whereas those of subgroup B (n = 12) were comparable to healthy controls. Patients in subgroup A displayed upregulated signaling pathways downstream of Toll-like receptor 4, TNF-α, and IFN-γ. Fatigue was more often present in subgroup A compared to B (75% vs 33% respectively, P = .06). CONCLUSIONS: Altered distribution of leukocyte subsets and a proinflammatory proteome were observed in subsequent 2 cohorts of ISM patients. We hypothesize that neoplastic mast cells recruit and activate plasmacytoid dendritic cells, monocytes, and T cells, leading to a vicious cycle of inflammation.

Comel-Netherton syndrome: A local skin barrier defect in the absence of an underlying systemic immunodeficiency.

BACKGROUND: Comel-Netherton syndrome (NS) is a rare autosomal disease, characterized by severe skin disease, hair shaft defects, atopic diathesis, and increased susceptibility for skin infections. Since patients with NS suffer from recurrent infections, it has been hypothesized that an underlying immunodeficiency attributes to this. Here, we studied clinical and immunological characteristics of the cohort of NS patients in the Netherlands in order to identify whether potential immunodeficiencies result in the increased risk of infectious complications. METHODS: Phenotypes were scored for severity of skin condition, specific hair shaft defects, atopy, and recurrent infections. Patients' blood samples were collected for quantification of serum immunoglobulin (Ig) levels, specific antibodies against Streptococcus pneumoniae, and allergen-specific IgE, as well as detailed immunophenotyping of blood leukocyte and lymphocyte subsets by flow cytometry. RESULTS: A total of 14 patients were included with age range 3-46 years and varying degrees of skin involvement. All patients presented with atopic symptoms (food allergy, n = 13; hay fever, n = 10; asthma, n = 7). Recurrent skin infections were common, particularly in childhood (n = 12). Low levels of specific antibodies against S pneumoniae were found in 10 of 11 evaluated patients. Detailed immunological analysis was performed on 9 adult patients. Absolute numbers of lymphocyte subsets and serum immunoglobulin levels were all within normal ranges. CONCLUSION: Multidisciplinary evaluation of our national cohort showed no evidence for a severe, clinically relevant systemic immunodeficiency. Therefore, we conclude that in Dutch NS patients the increased risk of infections most likely results from the skin barrier disruption and that increased allergen penetration predisposes to allergic sensitization.

Induction of IgG2 and IgG4 B-cell memory following sublingual immunotherapy for ryegrass pollen allergy.

BACKGROUND: While treatment for atopic rhinitis is aimed mostly to relieve symptoms, only allergen-specific immunotherapy (AIT) is targeted to modify the natural history of allergic diseases. This results in sustained clinical tolerance, even when treatment has stopped. The immunomodulatory effects of AIT are attributed mainly to increased regulatory T-cell function and increased allergen-specific IgG4 , yet little is known about the effect on the memory B-cell compartment. OBJECTIVE: We aimed to examine the effects of AIT on the IgE- and IgG subclass-expressing memory B cells. METHODS: We recruited 29 patients with atopic seasonal rhinoconjunctivitis and performed a longitudinal analysis of the peripheral immune compartment before, during, and after sublingual immunotherapy (SLIT) for allergy to temperate grass pollen, predominantly to ryegrass pollen (RGP; Lolium perenne). Using flow cytometry on peripheral blood mononuclear cells and serum immunoassays, we analyzed the effects of a 4 months preseasonal treatment regimen comprising two or three courses in consecutive years on circulating IgE+ and IgG+ memory B cells and allergen-specific Ig levels. RESULTS: SLIT increased RGP-specific serum IgG2 and IgG4 , as well as the frequencies of IgG2+ and IgG4+ memory B cells, whereas no effect was observed on the IgE+ memory B-cell compartment. Furthermore, SLIT enhanced proportions of regulatory T cells specific to RGP. These changes were associated with clinical improvement. CONCLUSION: Our data provide evidence for immunological effects of SLIT on B-cell memory. Skewing responses toward IgG2 and IgG4 subclasses might be a mechanism to suppress IgE-mediated allergic responses.

Expansion of blood IgG4+ B, TH2, and regulatory T cells in patients with IgG4-related disease.

BACKGROUND: IgG4-related disease (IgG4-RD) is a systemic fibroinflammatory condition affecting various organs and has a diverse clinical presentation. Fibrosis and accumulation of IgG4+ plasma cells in tissue are hallmarks of the disease, and IgG4-RD is associated with increased IgG4 serum levels. However, disease pathogenesis is still unclear, and these cellular and molecular parameters are neither sensitive nor specific for the diagnosis of IgG4-RD. OBJECTIVE: Here we sought to develop a flow cytometric gating strategy to reliably identify blood IgG4+ B cells to study their cellular and molecular characteristics and investigate their contribution in disease pathogenesis. METHODS: Sixteen patients with histologically confirmed IgG4-RD, 11 patients with sarcoidosis, and 30 healthy subjects were included for 11-color flow cytometric analysis of peripheral blood for IgG4-expressing B cells and TH subsets. In addition, detailed analysis of activation markers and chemokine receptors was performed on IgG4-expressing B cells, and IgG4 transcripts were analyzed for somatic hypermutations. RESULTS: Cellular and molecular analyses revealed increased numbers of blood IgG4+ memory B cells in patients with IgG4-RD. These cells showed reduced expression of CD27 and CXCR5 and increased signs of antibody maturation. Furthermore, patients with IgG4-RD, but not patients with sarcoidosis, had increased numbers of circulating plasmablasts and CD21low B cells, as well as TH2 and regulatory T cells, indicating a common disease pathogenesis in patients with IgG4-RD. CONCLUSION: These results provide new insights into the dysregulated IgG4 response in patients with IgG4-RD. A specific "peripheral lymphocyte signature" observed in patients with IgG4-RD, could support diagnosis and treatment monitoring.

Human IgE(+) B cells are derived from T cell-dependent and T cell-independent pathways.

BACKGROUND: The prevalence of IgE-mediated diseases has been increasing worldwide, yet IgE-expressing B cells are poorly characterized, mainly because of their scarcity and low membrane IgE levels. OBJECTIVE: We sought to study the immunobiology of human IgE-expressing B cells in healthy subjects and patients with allergic disease. METHODS: We used a stepwise approach for flow cytometric detection and purification of human IgE-expressing B cells in control subjects, CD40 ligand-deficient patients, and patients with atopic dermatitis. Molecular analysis of replication histories, somatic hypermutation (SHM), and immunoglobulin class-switching was performed. RESULTS: Using multicolor flow cytometry, we reliably detected IgE-expressing plasma cells and 2 IgE-expressing memory B-cell subsets. These IgE-expressing cells showed molecular and phenotypic signs of antigen responses. The replication history and SHM levels of IgE(+) plasma cells and CD27(+)IgE(+) memory B cells fitted with a germinal center (GC)-dependent pathway, often through an IgG intermediate, as evidenced from Sγ remnants in Sµ-Sε switch regions. CD27(-)IgE(+) cells showed limited proliferation and SHM and were present in CD40 ligand-deficient patients, indicating a GC-independent origin. Patients with atopic dermatitis had normal numbers of blood IgE(+) plasma cells and CD27(+)IgE(+) memory B cells but increased numbers of CD27(-)IgE(+) memory B cells with high SHM loads compared with those seen in healthy control subjects and patients with psoriasis. CONCLUSIONS: We delineated GC-dependent and GC-independent IgE(+) B-cell responses in healthy subjects and indicated involvement of the GC-independent pathway in a human IgE-mediated disease. These findings provide new insights into the pathogenesis of IgE-mediated diseases and might contribute to accurate monitoring of IgE(+) B cells in patients with severe disease undergoing anti-IgE treatment.

Alterations in peripheral blood B cell subsets and dynamics of B cell responses during human schistosomiasis.

Antibody responses are thought to play an important role in control of Schistosoma infections, yet little is known about the phenotype and function of B cells in human schistosomiasis. We set out to characterize B cell subsets and B cell responses to B cell receptor and Toll-like receptor 9 stimulation in Gabonese schoolchildren with Schistosoma haematobium infection. Frequencies of memory B cell (MBC) subsets were increased, whereas naive B cell frequencies were reduced in the schistosome-infected group. At the functional level, isolated B cells from schistosome-infected children showed higher expression of the activation marker CD23 upon stimulation, but lower proliferation and TNF-α production. Importantly, 6-months after 3 rounds of praziquantel treatment, frequencies of naive B cells were increased, MBC frequencies were decreased and with the exception of TNF-α production, B cell responsiveness was restored to what was seen in uninfected children. These data show that S. haematobium infection leads to significant changes in the B cell compartment, both at the phenotypic and functional level.