ADAM10 Regulates Transcription Factor Expression Required for Plasma Cell Function

A disintegrin and metalloprotease 10 (ADAM10) is a key regulator of cellular processes by shedding extracellular domains of transmembrane proteins. We have previously demonstrated that deletion of B cell expressed ADAM10 results in changes in lymphoid tissue architecture and impaired germinal center (GC) formation. In this study, mice were generated in which ADAM10 is deleted in B cells following class switch recombination (ADAM10Δ/ΔIgG1-cre+/−mice). Despite normal GC formation, antibody responses were impaired in ADAM10Δ/ΔIgG1-cre+/− mice, implicating ADAM10 in post-GC and extrafollicular B cell terminal differentiation. Surprisingly, plasma cell (PC) numbers were normal in ADAM10Δ/ΔIgG1-cre+/− mice when compared to controls. However, PCs isolated from ADAM10Δ/ΔIgG1-cre+/− mice exhibited decreased expression of transcription factors important for PC function: Prdm1, Xbp1 and Irf4.Bcl6 is a GC transcriptional repressor that inhibits the PC transcriptional program and thus must be downregulated for PC differentiation to occur. Bcl6 expression was increased in PCs isolated from ADAM10Δ/ΔIgG1-cre+/− mice at both the mRNA and protein level. These results demonstrate that ADAM10 is required for proper transcription factor expression in PCs and thus, for normal PC function

A Novel STAT3 Mutation in a Qatari Patient With Hyper-IgE Syndrome

Autosomal dominant hyper-IgE syndrome caused by mutations in the transcription factor STAT3 (AD-HIES) is characterized by a collection of immunologic and non-immune features including eczema, recurrent infections, elevated IgE levels, and connective tissue anomalies. We report the case of a Qatari child with a history of recurrent staphylococcal skin infections since infancy, who was found to have a novel, de novo mutation in STAT3 (c.1934T>A, p.L645Q). The absence of mucocutaneous candidiasis and undetectable IgE levels until the age of 7 years prolonged the time to molecular confirmation of the cause for the patient's immune deficiency. STAT3 p.L645Q was found to have decreased transcriptional capacity. The patient also had low levels of Th17 cells and STAT3 phosphorylation was impaired in patient-derived cells. Nearly 100 unique mutations in STAT3 have been reported in association with AD-HIES

Genetic errors of immunity distinguish pediatric non-malignant lymphoproliferative disorders

Background Pediatric non-malignant lymphoproliferative disorders (PLPD) are clinically and genetically heterogeneous. Long-standing immune dysregulation and lymphoproliferation in children may be life-threatening, and a paucity of data exists to guide evaluation and treatment of children with PLPD. Objective The primary objective of this study was to ascertain the spectrum of genomic immunologic defects in PLPD. Secondary objectives included characterization of clinical outcomes and associations between genetic diagnoses and those outcomes. Methods PLPD was defined by persistent lymphadenopathy, lymph organ involvement, or lymphocytic infiltration for more than 3 months, with or without chronic or significant EBV infection. Fifty-one subjects from 47 different families with PLPD were analyzed using whole exome sequencing (WES). Results WES identified likely genetic errors of immunity in 51% to 62% of families (53% to 65% of affected children). Presence of a genetic etiology was associated with younger age and hemophagocytic lymphohistiocytosis. Ten-year survival for the cohort was 72.4%, and patients with viable genetic diagnoses had a higher survival rate (82%) compared to children without a genetic explanation (48%, p = 0.03). Survival outcomes for individuals with EBV-associated disease and no genetic explanation were particularly worse than outcomes for subjects with EBV-associated disease and a genetic explanation (17% vs. 90%; p = 0.002). Ascertainment of a molecular diagnosis provided targetable treatment options for up to 18 individuals and led to active management changes for 12 patients. Conclusion PLPD therefore defines children with high risk for mortality, and WES informs clinical risks and therapeutic opportunities for this diagnosis

Fyn kinase is required for optimal humoral responses.

The generation of antigen-specific antibodies and the development of immunological memory require collaboration between B and T cells. T cell-secreted IL-4 is important for B cell survival, isotype switch to IgG1 and IgE, affinity maturation, and the development of germinal centers (GC). Fyn, a member of the Src family tyrosine kinase, is widely expressed in many cell types, including lymphocytes. This kinase is known to interact with both the B cell and T cell receptor (BCR and TCR, respectively). While Fyn deletion does not impair the development of immature T cells and B cells, TCR signaling is altered in mature T cells. The current study demonstrates that Fyn deficient (KO) B cells have impaired IL-4 signaling. Fyn KO mice displayed low basal levels of IgG1, IgE and IgG2c, and delayed antigen-specific IgG1 and IgG2b production, with a dramatic decrease in antigen-specific IgG2c following immunization with a T-dependent antigen. Defects in antibody production correlated with significantly reduced numbers of GC B cells, follicular T helper cells (TFH), and splenic plasma cells (PC). Taken together, our data demonstrate that Fyn kinase is required for optimal humoral responses

Fyn deficient mice have impaired germinal center formation and reduced T_FH numbers.

<p>Mice were immunized with NP-KLH emulsified in alum and 14 days post-immunization GC formation and T_FH frequency were assessed by flow cytometry. (A) Representative dot plot for GC B cells (gated on B220⁺ cells). (B) Quantification of GC B cells. (C) Representative dot plot for T_FH cells (gated on CD4⁺ cells) (D) Quantification of T_FH cells. Bars represent the mean ± SE of 6 mice per group. Data represent results obtained in at least two independent experiments. (**p<0.01, ***p<0.001).</p

Plasma Cells from ADAM10^Δ/ΔIgG1-cre^+/− mice have altered gene expression.

<p>ADAM10^Δ/ΔIgG1-cre^+/− and controls were immunized with NP-KLH emulsified in alum. Twenty-one days following immunization, splenic PCs were isolated via magnetic bead isolation. mRNA was isolated and (A) <i>Xbp1</i> (B) <i>Prdm1</i>, (C) <i>Irf4</i> and (D) <i>Bcl6</i> message levels were determined by qPCR. (E) The ratio of <i>Prdm1</i> to <i>Bcl6</i> was calculated. Bars represent the mean ± SE of 3 independent studies; cells from 3 mice from each genotype pooled in each study. (*p<0.05, **p<0.01, ***p<0.001).</p

Model.

<p>Wild type plasma cells express higher levels of Blimp1, IRF4 and XBP1, while Bcl6 is repressed. This allows for antibody secretion. In the case of ADAM10^Δ/ΔIgG1-cre^+/− mice, Bcl6 levels are higher than seen in wild type. Moreover, Blimp1, IRF4 and XBP1 expression are decreased, leading to impaired antibody secretion.</p

Fyn KO B cells have impaired STAT3 and STAT5 activation upon IL-4 stimulation.

<p>WT and Fyn KO naive B cells were isolated from mice and stimulated with IL-4 (30 ng/ml) at 37°C for indicated times. Cells were lysed and phosphorylated forms of STAT3 (pY705 and pS727), STAT5 (pY694) and STAT6 (pY641) were assessed by western blot. Non-phosphorylated proteins were used as loading controls. Representative image of 3 independent experiments.</p