ZFH-2 is required for Drosophila ovarian follicle development and is expressed at the band/interband boundaries of polytene chromosomes.

The transcription factor ZFH-2 has well-documented roles in Drosophila neurogenesis and other developmental processes. Here we provide the first evidence that ZFH-2 has a role in oogenesis. We demonstrate that ZFH-2 is expressed in the wild-type ovary and that a loss of zfh-2 function produces a mutant ovary phenotype where egg chambers are reduced in number and fused. We also show that a loss of zfh-2 function can suppress a daughterless loss-of-function ovary phenotype suggesting a possible genetic relationship between these two genes in the ovary. We also show that ZFH-2 is located at the boundary between bands and interbands on polytene chromosomes and that at a subset of these sites ZFH-2 colocalizes with the insulator/promoter cofactor CP190.

Dynamic Immune Phenotypes of B and T Helper Cells Mark Distinct Stages of T1D Progression.

Multiple studies of B- and T-cell compartments and their response to stimuli demonstrate alterations in established type 1 diabetes (T1D). Yet it is not known whether these alterations reflect immune mechanisms that initiate islet autoimmunity, promote disease progression, or are secondary to disease. To address these questions, we used samples from the TrialNet Pathway to Prevention study to investigate T-cell responses to interleukin (IL)-2 and regulatory T cell-mediated suppression, the composition of the B-cell compartment, and B-cell responses to B-cell receptor and IL-21 receptor engagement. These studies revealed stage-dependent T- and B-cell functional and immune phenotypes; namely, early features that differentiate autoantibody-positive at-risk first-degree relatives (FDRs) from autoantibody-negative FDRs and persisted through clinical diagnosis; late features that arose at or near T1D diagnosis; and dynamic features that were enhanced early and blunted at later disease stages, indicating evolving responses along the continuum of T1D. We further explored how these specific phenotypes are influenced by therapeutic interventions. Our integrated studies provide unique insights into stable and dynamic stage-specific immune states and define novel immune phenotypes of potential clinical relevance.

Altered BCR and TLR signals promote enhanced positive selection of autoreactive transitional B cells in Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disorder frequently associated with systemic autoimmunity, including autoantibody-mediated cytopenias. WAS protein (WASp)-deficient B cells have increased B cell receptor (BCR) and Toll-like receptor (TLR) signaling, suggesting that these pathways might impact establishment of the mature, naive BCR repertoire. To directly investigate this possibility, we evaluated naive B cell specificity and composition in WASp-deficient mice and WAS subjects (n = 12). High-throughput sequencing and single-cell cloning analysis of the BCR repertoire revealed altered heavy chain usage and enrichment for low-affinity self-reactive specificities in murine marginal zone and human naive B cells. Although negative selection mechanisms including deletion, anergy, and receptor editing were relatively unperturbed, WASp-deficient transitional B cells showed enhanced proliferation in vivo mediated by antigen- and Myd88-dependent signals. Finally, using both BCR sequencing and cell surface analysis with a monoclonal antibody recognizing an intrinsically autoreactive heavy chain, we show enrichment in self-reactive cells specifically at the transitional to naive mature B cell stage in WAS subjects. Our combined data support a model wherein modest alterations in B cell-intrinsic, BCR, and TLR signals in WAS, and likely other autoimmune disorders, are sufficient to alter B cell tolerance via positive selection of self-reactive transitional B cells.

Altered B cell homeostasis is associated with type I diabetes and carriers of the PTPN22 allelic variant.

The PTPN22 genetic variant 1858T, encoding Lyp620W, is associated with multiple autoimmune disorders for which the production of autoantibodies is a common feature, suggesting a loss of B cell tolerance. Lyp620W results in blunted BCR signaling in memory B cells. Because BCR signal strength is tightly coupled to central and peripheral tolerance, we examined whether Lyp620W impacts peripheral B cell homeostasis in healthy individuals heterozygous for the PTPN221858T variant. We found that these subjects display alterations in the composition of the B cell pool that include specific expansion of the transitional and anergic IgD(+)IgM(-)CD27(-) B cell subsets. The PTPN22 1858T variant was further associated with significantly diminished BCR signaling and a resistance to apoptosis in both transitional and naive B cells. Strikingly, parallel changes in both BCR signaling and composition of B cell compartment were observed in type 1 diabetic subjects, irrespective of PTPN22 genotype, revealing a novel immune phenotype and likely shared mechanisms leading to a loss of B cell tolerance. Our combined findings suggest that Lyp620W-mediated effects, due in part to the altered BCR signaling threshold, contribute to breakdown of peripheral tolerance and the entry of autoreactive B cells into the naive B cell compartment.

Aberrant underexpression of CD81 in precursor B-cell acute lymphoblastic leukemia: utility in detection of minimal residual disease by flow cytometry.

We studied CD81 expression by flow cytometry (FC) on benign precursor B cells (hematogones) and leukemic blasts in precursor B-cell acute lymphoblastic leukemia (pre-B-ALL) and established its usefulness in minimal residual disease (MRD) assays. Hematogones showed uniformly bright CD81 expression. In 98 pre-B-ALLs at diagnosis or overt relapse, 80 (82%) showed aberrantly decreased CD81 intensity. We used hematogones in 139 MRD- specimens to set a lower threshold for normal CD81 expression. In 133 specimens positive for residual pre-B-ALL, 87.2% showed increased CD81-dim immature B cells (>10%) and/or a discrete cluster of CD81-dim cells in a background of hematogones. Only 1 of 139 MRD- specimens showed more than 10% CD81-dim cells. Decreased CD81 expression was maintained in 91% of aberrant cases analyzed before and after chemotherapy. Decreased CD81 expression is a sensitive and specific marker for residual pre-B-ALL, even in a background of hematogones, making CD81 a useful addition to a panel for MRD detection by FC.

Immunophenotypic study of basophils by multiparameter flow cytometry.

CONTEXT: The immunophenotypic profile of basophils is not yet fully established, and the immunophenotypic changes in chronic myelogenous leukemia are not fully characterized. OBJECTIVE: To establish a comprehensive immunophenotypic spectrum of normal basophils and to assess the range of immunophenotypic aberrations of basophils in chronic myelogenous leukemia. DESIGN: Using 4-color flow cytometry, we compared the immunophenotypic profile of basophils in peripheral blood or bone marrow samples from 20 patients with no evidence of neoplasia to basophils from 15 patients with chronic myelogenous leukemia. RESULTS: Basophils in control cases were all positive for CD9, CD13, CD22, CD25 (dim), CD33, CD36, CD38 (bright), CD45 (dimmer than lymphocytes and brighter than myeloblasts), and CD123 (bright), and were negative for CD19, CD34, CD64, CD117, and HLA-DR. Basophils in all chronic myelogenous leukemia patients possessed 1 to 5 immunophenotypic aberrancies. The most common aberrancies were underexpression of CD38, followed by aberrant expression of CD64 and underexpression of CD123. CD34 and CD117 were present in cases with basophilic precursors. Myeloblasts showed a distinct immunophenotypic profile, as they typically expressed CD34 and CD117, showed dimmer expression (compared with basophils) of CD38, CD45, and CD123, and lacked expression of CD22. CONCLUSIONS: Flow cytometric immunophenotyping can identify immunophenotypic aberrations of basophils in chronic myelogenous leukemia, and discriminate basophils from myeloblasts.