Block In B-cell Development Research Articles

Characterizing A Novel Variant in ABCB1 in a Family with Activated PI3K Delta Syndrome-like Presentation

IntroductionActivated PI3K delta syndrome (APDS) is a rare inborn error of immunity (IEI) due to a heterozygous gain-of-function mutation in the PIK3CD or PIK3R1 genes. APDS patients have high variability in clinical manifestations including recurrent sinopulmonary infections, lymphoproliferation, and susceptibility to CMV and/or EBV viremia. APDS is characterized by a block in B-cell development to follicular stage. Therefore, increase in transitional compartment is almost exclusively seen in untreated APDS patients. We describe a family that present with APDS-like clinical and laboratory findings, while harboring a novel variant in ATP binding cassette subfamily B member 1 (ABCB1), which is involved in adenosine salvage and mTORC1 pathways. MethodsRetrospective chart review was performed for clinical and laboratory data. Extensive immune phenotyping was obtained. Genetic evaluation included targeted immune panel and whole exome sequencing. Mitotracker-green detection by flow cytometry was used to determine progression to follicular B cell stage. Case PresentationIndex case is a 14-year-old female with a complex presentation of infectious and non-infectious complications notable for lifelong hypogammaglobulinemia with poor vaccine response, multiple intubations for upper respiratory illnesses, bronchiectasis, and short stature. Patient had 3 other siblings who also had similar presentation with varied severity, including features of short stature, recurrent infections, alopecia universalis and immune dysregulation. Three siblings required immunoglobulin replacement therapy. Extensive immune-phenotyping revealed significant expansion of CD24hiCD38hi transitional and CD19hiCD21lo B-cells, with decrease in follicular and memory B-cell compartment in all. Although clinical and lab findings prompted APDS diagnosis, repeated targeted genetic panel testing did not show variants in PIK3CD or PIK3R1. Consequent whole exome sequencing revealed a candidate variant in ABCB1 p.Ala1187Thr that was shared among all. Interestingly, surface expression of ABCB1 was significantly elevated in patients compared to healthy controls. Initial functional testing showed disruption in mitochondrial efflux, further supporting the block from transitional to follicular stage. Discussion/ConclusionsDetermining diagnostic approaches in APDS-like disorders liked to ABCB1 variant is challenging, especially when pathogenesis is yet to be determined. Although further functional testing of ABCB1 and its potential role in PI3K signaling is needed, ABCB1 gene may play an important role in APDS pathology.

Disease-associated mutations in topoisomerase IIβ result in defective NK cells

Hoffman syndrome is a syndromic, inborn error of immunity due to autosomal-dominant mutations in TOP2B, an essential gene required to alleviate topological stress during DNA replication and gene transcription. Although mutations identified in patients lead to a block in B-cell development and the absence of circulating B cells, an effect on natural killer (NK) cells was not previously examined. We sought to determine whether disease-associated mutations in TOP2B impact NK-cell development and function. Using a knockin murine model and patient-derived induced pluripotent stem cells (iPSCs), we investigated NK-cell development in mouse bone marrow and spleen, and performed immunophenotyping by flow cytometry, gene expression, and functional assessment of cytotoxic activity in murine NK cells, and human IPSC-derived NK cells. Mature NK cells were reduced in the periphery of TOP2B knockin mice consistent with patient reports, with reduced cytotoxicity toward target cell lines. IPSCs were successfully derived from patients with Hoffman syndrome, but under optimal conditions showed reduced cytotoxicity compared with iPSC-derived NK cells from healthy controls. Hoffman syndrome-associated mutations in TOP2B impact NK-cell development and function in murine and human models.

DOCK2 Knockdown Mitigates Disease Progression in Murine Models Harboring a FLT3/ITD Mutation

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm characterized by clonal expansion of myeloid blasts in the bone marrow and other tissues. The FMS-like tyrosine kinase-3 (FLT3) receptor gene is the most commonly mutated gene in AML, and patients who harbor a FLT3/ITD mutation have a relatively poor prognosis.Previous work in the laboratory has demonstrated that FLT3/ITD interacts with Dedicator of Cytokinesis 2 (DOCK2), which is a guanine nucleotide exchange factor for Rac GTPases. Expression of DOCK2 is limited to hematopoietic cells, and it is also expressed in the leukemic blasts of patients with FLT3/ITD AML. Knockdown (KD) of DOCK2 results in reduced Rac1 activity, and leads to decreased survival of leukemic cells that carry a FLT3/ITD mutation. This effect is seen in both in vitro studies and a mouse xenograft model, and is enhanced by treatment with cytarabine. Initial studies have shown that the anti-leukemic effect of DOCK2 KD may, in part, be due to reduced expression of both FLT3 and key DNA Damage Response (DDR) factors. In order to further investigate the relationship between DOCK2 and FLT3/ITD in hematopoietic neoplasms, we utilized mice that harbor a spontaneous Dock2 mutation (Dock2/Hsd). This mutation results in a substantial reduction in Dock2 mRNA and protein expression in hematolymphoid cells.Flt3/ITD mice develop a myeloproliferative neoplasm (MPN) that includes greatly increased spleen size. In contrast, the spleens of progeny of Flt3/ITD mice bred with Dock2/Hsd mice were greatly reduced in size. Flow cytometric analysis of the bone marrow also demonstrated that DOCK2 KD results in at least a partial reversal of the Flt3/ITD phenotype. Flt3/ITD mice characteristically demonstrate an increased fraction of immature hematopoietic stem/progenitor cells (Lin-Sca-1+c-KIT+ cells or LSKs); however, the fraction of LSKs in Flt3/ITD-Dock2/Hsd mice is similar to that seen in the marrow of WT mice. While the Flt3/ITD-Dock2/Hsd mice continued to show a disruption in the early to late Pro-B transition similar to that described in Flt3/ITD mice, the Flt3/ITD-Dock2/Hsd mice had a small but significant increase in the late Pre-B cell population, suggesting a less stringent block in B-cell development. Additionally, as compared with Flt3/ITD mice, the bone marrow of Flt3/ITD-Dock2/Hsd mice exhibited a decreased fraction of Mac1+Gr-1+ myeloid cells with a concomitant increase in Ter119+CD41- erythroid precursors, to levels that are indistinguishable from WT mice. These findings indicate that DOCK2 KD mitigates the myeloproliferative phenotype of Flt3/ITD mice.Since experiments using FLT3/ITD cell lines have suggested that DOCK2 KD may act by reducing expression of the FLT3 protein itself and also by inhibiting the DNA Damage Response, we evaluated these factors in the Flt3/ITD-Dock2/Hsd mice. Quantitative PCR of mouse whole bone marrow revealed that the mRNA levels of the Flt3 transcription regulators Meis1 and c-Myb were decreased in the Flt3/ITD-Dock2/Hsd mice, compared with Flt3/ITD mice, and the expression of Flt3 itself was markedly decreased as well. Moreover, reduced expression of key DDR factors including Chk1, Wee1, Rad51, Pim1, MSH2, MSH6 and MLH1 was also observed in Flt3/ITD-Dock2/Hsd mice.Finally, to assess the role of DOCK2 in leukemogenesis and disease progression, the Dock2/Hsd mutation was introduced into a murine AML model that carries both Flt3/ITD and the Nup98-HoxD13 (NHD13) fusion gene. The Flt3/ITD-NHD13 mice develop AML with 100% penetrance and short latency; however, the expression of Dock2/Hsd in Flt3/ITD-NHD13 mice extended the median survival of these mice from approximately 110 days to over 155 days (and counting).These findings suggest that DOCK2 may cooperate with FLT3/ITD and play an important role in disease progression of FLT3/ITD leukemia. Thus, pharmacologic inhibition of DOCK2 may provide a novel and promising therapeutic target for FLT3/ITD AML. Additionally, since DOCK2 KD results in decreased expression of DDR factors, the addition of DDR inhibitors to conventional chemotherapy may be useful in the treatment of FLT3/ITD AML. DisclosuresNo relevant conflicts of interest to declare.

Lack of CD45 in FLT3-ITD mice results in a myeloproliferative phenotype, cortical porosity, and ectopic bone formation.

The receptor tyrosine kinase FLT3 is expressed in myeloid and lymphoid progenitor cells. Activating mutations in FLT3 occur in 25-30% of acute myeloid leukaemia (AML) patients. Most common are internal tandem duplications of sequence (ITD) leading to constitutive FLT3-ITD kinase activity with an altered signalling quality promoting leukaemic cell transformation. Here, we observed the attenuating role of the receptor-like protein tyrosine phosphatase (RPTP) CD45/Ptprc in FLT3 signalling in vivo. Low level expression of this abundant RPTP correlates with a poor prognosis of FLT3-ITD-positive AML patients. To get a further insight into the regulatory role of Ptprc in FLT3-ITD activity in vivo, Ptprc knock-out mice were bred with FLT3-ITD knock-in mice. Inactivation of the Ptprc gene in FLT3-ITD mice resulted in a drastically shortened life span and development of severe monocytosis, a block in B-cell development and anaemia. The myeloproliferative phenotype was associated with extramedullary haematopoiesis, splenohepatomegaly and severe alterations of organ structures. The phenotypic alterations were associated with increased transforming signalling of FLT3-ITD, including activation of its downstream target STAT5. These data reveal the capacity of Ptprc for the regulation of FLT3-ITD signalling activity in vivo. In addition, histopathology and computed tomography (CT) revealed an unexpected bone phenotype; the FLT3-ITD Ptprc-/- mice, but none of the controls, showed pronounced alterations in bone morphology and, in part, apparent features of osteoporosis. In the spleen, ectopic bone formation was observed. The observed bone phenotypes suggest a previously unappreciated capacity of FLT3-ITD (and presumably FLT3) to regulate bone development/remodelling, which is under negative control of CD45/Ptprc.

Activation of CRLF2/IL7RA Signaling in Normal Human Cord Blood Hematopoietic Progenitors Induces Philadelphia-like B-Cell Precursor Pre-Leukemia and Leukemia In Vivo

Philadelphia-like (Ph-like) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a subgroup of BCP-ALL with an expression pattern similar to BCR-ABL+ BCP-ALL that is associated with poor prognosis.Aberrant expression of CRLF2 in BCP-ALL constitutes the majority of Ph-like BCP-ALL cases. CRLF2 is a receptor subunit that together with the IL7RA subunit comprises the receptor of the proinflammatory cytokine TSLP. Though activation of the IL7R pathway is commonly associated with T-cell malignancies, we previously described IL7RA-activating mutations in BCP-ALL predominantly in the context of CRLF2 aberrant expression (Shochat C. et al. J. Exp. Med. 2011).Here we aimed to test the role of aberrations in CRLF2 and IL7RA in the development of Ph-Like BCP-ALL. Both CRLF2 and TSLP differ extensively between mice and human in amino acid sequence and in lineage expression pattern; loss-of-function germline mutations in IL7RA are associated with lack of B and T cells in mice but with lack of only T cells in humans. Hence, we chose to test the hypothesis that activation of CRLF2/IL7RA contributes to the development of Ph-like BCP-ALL in the context of human lymphopoiesis by using a human xenograft system.To aberrantly activate TSLP/IL7 signaling, we transduced cord-blood (CB) CD34+ hematopoietic progenitors with a set of lentiviral vectors carrying CRLF2 and/or IL7RA [(wild type (IL7RAwt) or IL7R bearing an activating mutation (IL7RAins)] under a B-cell promoter/enhancer (to accentuate B-cell lineage expression). The backbone vector (BB) expressing GFP was used as a control. Transduced CB cells were transplanted into NOD/LtSz-scid IL2Rγnull (NSG) mice and engrafted cells were analyzed 24-30 weeks after transplantation. To test for self-renewal capacity, BM cells from primary engrafted mice were serially transplanted into secondary recipients and the occurrence of human engraftment was tested 24-30 weeks after transplantation.Enforced expression of activated IL7RA with or without CRLF2 led to a significant block in B-cell development at the B-cell progenitor stage (CD19+CD10+sIgM-) in vivo resembling the differentiation stage of leukemic cells (figure Bi). Repertoire sequencing of CD10+CD19+-transduced cells that were sorted from BM of transplanted mice revealed a significantly higher population of DJ-rearranged cells in the CRLF2-IL7RAins-transduced population than in BB-transduced cells (mean ratio of DJ/total rearrangement: BB:0.35+/-0.024, CRLF2-IL7RAins:0.76+/- 0.07, p=0.039, n=3 paired cord blood), in agreement with the early differentiation block phenotype measured by immunophenotyping. These cells furthermore exhibited a Ph-like gene expression pattern when compared to BB-transduced cells in gene set enrichment analysis.Overexpression of IL7RA alone significantly enhanced the early-B fraction (CD19+CD10+CD34+) in the BM of transplanted mice (figure Bii). Additionally, aberrant expression of IL7RA enhanced self-renewal capacity as was evident by an increased ability of the transduced cells to engraft in secondary recipients (number of mice with detectable human engraftment out of secondary transplanted mice: BB:0/6, CRLF2-IL7RAwt:0/3, CRLF2-IL7RAinst:0/3, IL7RAwt-GFP:3/6, IL7RAins-GFP:5/8).Notably, in one case, secondary transplantation of IL7RAins-transduced CB triggered the development of acute BCP-ALL. The leukemic cells (CD19+CD10+CD34+sIgM-) were clonal as validated by V(D)J rearrangement (figure Cii), had the ability to further propagate in serial transplantations and gained secondary Ph-like BCP-ALL-characteristic chromosomal deletions in the short arm of chromosome 9 (in the region including the genes for CDKN2A/B, PAX5 and JAK2) and the short arm of chromosome 7 (the region including IKZF1) (figure Ciii).These results support the hypothesis that aberrant activation of the CRLF2/IL7RA pathway in human B-cell lineage progenitors creates a pre-leukemic state by arresting differentiation of B-cell progenitors, instating Ph-like expression pattern and inducing self-renewal. This is the first model of de novo Ph-like BCP-ALL development from normal human hematopoietic progenitors in vivo. Additionally, we present here a first direct in vivo demonstration of a role for IL7 in human B-cell development. DisclosuresNo relevant conflicts of interest to declare.

MTORC1 plays an important role in osteoblastic regulation of B-lymphopoiesis

Skeletal osteoblasts are important regulators of B-lymphopoiesis, serving as a rich source of factors such as CXCL12 and IL-7 which are crucial for B-cell development. Recent studies from our laboratory and others have shown that deletion of Rptor, a unique component of the mTORC1 nutrient-sensing complex, early in the osteoblast lineage development results in defective bone development in mice. In this study, we now demonstrate that mTORC1 signalling in pre-osteoblasts is required for normal B-lymphocyte development in mice. Targeted deletion of Rptor in osterix-expressing pre-osteoblasts (Rptorob−/−) leads to a significant reduction in the number of B-cells in the bone marrow, peripheral blood and spleen at 4 and 12 weeks of age. Rptorob−/− mice also exhibit a significant reduction in pre-B and immature B-cells in the BM, indicative of a block in B-cell development from the pro-B to pre-B cell stage. Circulating levels of IL-7 and CXCL12 are also significantly reduced in Rptorob−/− mice. Importantly, whilst Rptor-deficient osteoblasts are unable to support HSC differentiation to B-cells in co-culture, this can be rescued by the addition of exogenous IL-7 and CXCL12. Collectively, these findings demonstrate that mTORC1 plays an important role in extrinsic osteoblastic regulation of B-cell development.

Immunoglobulin knockout chickens via efficient homologous recombination in primordial germ cells

Gene targeting by homologous recombination or by sequence-specific nucleases allows the precise modification of genomes and genes to elucidate their functions. Although gene targeting has been used extensively to modify the genomes of mammals, fish, and amphibians, a targeting technology has not been available for the avian genome. Many of the principles of humoral immunity were discovered in chickens, yet the lack of gene targeting technologies in birds has limited biomedical research using this species. Here we describe targeting the joining (J) gene segment of the chicken Ig heavy chain gene by homologous recombination in primordial germ cells to establish fully transgenic chickens carrying the knockout. In homozygous knockouts, Ig heavy chain production is eliminated, and no antibody response is elicited on immunization. Migration of B-lineage precursors into the bursa of Fabricius is unaffected, whereas development into mature B cells and migration from the bursa are blocked in the mutants. Other cell types in the immune system appear normal. Chickens lacking the peripheral B-cell population will provide a unique experimental model to study avian immune responses to infectious disease. More generally, gene targeting in avian primordial germ cells will foster advances in diverse fields of biomedical research such as virology, stem cells, and developmental biology, and provide unique approaches in biotechnology, particularly in the field of antibody discovery.

Aldehyde dehydrogenases are regulators of hematopoietic stem cell numbers and B-cell development

High levels of the aldehyde dehydrogenase isoform ALDH1A1 are expressed in hematopoietic stem cells (HSCs); however, its importance in these cells remains unclear. Consistent with an earlier report, we find that loss of ALDH1A1 does not affect HSCs. Intriguingly, however, we find that ALDH1A1 deficiency is associated with increased expression of the ALDH3A1 isoform, suggesting its potential to compensate for ALDH1A1. Mice deficient in ALDH3A1 have a block in B-cell development as well as abnormalities in cell cycling, intracellular signaling, and gene expression. Early B cells from these mice exhibit excess reactive oxygen species and reduced metabolism of reactive aldehydes. Mice deficient in both ALDH3A1 and ALDH1A1 have reduced numbers of HSCs as well as aberrant cell cycle distribution, increased reactive oxygen species levels, p38 mitogen-activated protein kinase activity and sensitivity to DNA damage. These findings demonstrate that ALDH3A1 can compensate for ALDH1A1 in bone marrow and is important in B-cell development, both ALDH1A1 and 3A1 are important in HSC biology; and these effects may be due, in part, to changes in metabolism of reactive oxygen species and reactive aldehydes.

Quantitative differences in CD45 expression unmask functions for CD45 in B-cell development, tolerance, and survival

The receptor-like tyrosine phosphatase CD45 positively regulates antigen receptor signaling by dephosphorylating the inhibitory tyrosine of the src family kinases. CD45-deficient mice fail to fully unmask the role of CD45 in B cells because of the expression of a partially redundant tyrosine phosphatase, CD148. However, mice that are doubly deficient in CD45 and CD148 exhibit a very early block in B-cell development, thereby obscuring later roles for CD45. To overcome these limitations, here we take advantage of an allelic series of mice in which CD45 expression is titrated broadly (0-180%). Although high expression of CD45 inhibits T-cell receptor (TCR) signaling, we show that CD45 plays a purely positive regulatory role during B-cell receptor (BCR) signaling. In concert with exaggerated BCR signaling, increasing CD45 expression drives enhanced receptor editing in the bone marrow and profound loss of follicular and marginal zone B cells in the spleen. In the context of the IgHEL/sHEL model of B-cell tolerance, such high CD45 expression transforms anergy into deletion. Unexpectedly, elimination of the autoantigen sHEL in this model system in order to block clonal deletion fails to rescue survival of mature B cells. Rather, high CD45 expression reduces B-cell activating factor receptor (BAFFR) expression and inhibits B-cell activating factor (BAFF)-induced B-cell survival in a cell-intrinsic manner. Taken together, our findings reveal how CD45 function diverges in T cells and B cells, as well as how autoreactive B cells are censored as they transit development.

T and B lymphocyte abnormalities in bone marrow biopsies of common variable immunodeficiency

AbstractIn common variable immunodeficiency (CVID) defects in early stages of B-cell development, bone marrow (BM) plasma cells and T lymphocytes have not been studied systematically. Here we report the first morphologic and flow cytometric study of B- and T-cell populations in CVID BM biopsies and aspirates. Whereas the hematopoietic compartment showed no major lineage abnormalities, analysis of the lymphoid compartment exhibited major pathologic alterations. In 94% of the patients, BM plasma cells were either absent or significantly reduced and correlated with serum immunoglobulin G levels. Biopsies from CVID patients had significantly more diffuse and nodular CD3+ T lymphocyte infiltrates than biopsies from controls. These infiltrates correlated with autoimmune cytopenia but not with other clinical symptoms or with disease duration and peripheral B-cell counts. Nodular T-cell infiltrates correlated significantly with circulating CD4+CD45R0+ memory T cells, elevated soluble IL2-receptor and neopterin serum levels indicating an activated T-cell compartment in most patients. Nine of 25 patients had a partial block in B-cell development at the pre-B-I to pre-B-II stage. Because the developmental block correlates with lower transitional and mature B-cell counts in the periphery, we propose that these patients might form a new subgroup of CVID patients.

Constitutive reductions in mTOR alter cell size, immune cell development, and antibody production

AbstractMammalian TOR (mTOR) regulates cell growth, proliferation, and migration. Because mTOR knock-outs are embryonic lethal, we generated a viable hypomorphic mouse by neo-insertion that partially disrupts mTOR transcription and creates a potential physiologic model of mTORC1/TORC2 inhibition. Homozygous knock-in mice exhibited reductions in body, organ, and cell size. Although reductions in most organ sizes were proportional to decreased body weight, spleens were disproportionately smaller. Decreases in the total number of T cells, particularly memory cells, and reduced responses to chemokines suggested alterations in T-cell homing/homeostasis. T-cell receptor-stimulated T cells proliferated less, produced lower cytokine levels, and expressed FoxP3. Decreased neutrophil numbers were also observed in the spleen, despite normal development and migration in the bone marrow. However, B-cell effects were most pronounced, with a partial block in B-cell development in the bone marrow, altered splenic populations, and decreases in proliferation, antibody production, and migration to chemokines. Moreover, increased AKTSer473 phosphorylation was observed in activated B cells, reminiscent of cancers treated with rapamycin, and was reduced by a DNA-pk inhibitor. Thus, mTOR is required for the maturation and differentiation of multiple immune cell lineages. These mice provide a novel platform for studying the consequences of constitutively reduced mTORC1/TORC2 activity.

Immunologic messenger molecules: Cytokines, interferons, and chemokines

Cytokines and chemokines are secreted proteins involved in numerous aspects of cell growth, differentiation, and activation. A prominent feature of these molecules is their effect on the immune system with regard to cell trafficking and development of immune tissue and organs. The nature of an immune response determines which cytokines are produced and ultimately whether the response is cytotoxic, humoral, cell mediated, or allergic. For this chapter, cytokines are grouped according to those that are predominantly antigen-presenting cell or T lymphocyte derived; that mediate cytotoxic, humoral, cell mediated, and allergic immunity; or that are immunosuppressive. A discussion of chemokine function and their role in cell trafficking and disease follows.

Deficiency of chemokine receptors CCR1, CCR5 AND CX3CR1 causes defective osteoclast differentiation and defective bone remodeling

oc/oc fetal liver cells after conditioning with irradiation or busulfan. Here, we hypothesized that transplantation without prior conditioning could reverse osteopetrosis and rescue oc/oc mice. Oc/oc and control mice were injected iv at day 1 after birth with normal or lineage depleted (lin−) BM cells (1 or 5×106). More than 85% of oc/ oc mice injected with 5×106 lin-cells survived long term (up to a year), while the other oc/oc groups died at 3–4 w. Engraftment, as determined by % donor cells in peripheral blood (PB) was 4–5% at 3, 6, 12 and 20 w. Several mice were sacrificed at 3–4 w for thorough analysis. Histopathology of femurs revealed 40–50% reversal of osteopetrosis at 3 w but almost complete reversal at 4 w. Micro-CT of femurs and calvariae confirmed these findings. BM cells were harvested and in vitro differentiated to osteoclasts. While the number of osteoclasts formed from BM cells of control mice and oc/oc mice was the same, bone resorption did not occur by cells generated from oc/oc mice. Osteoclasts from transplanted oc/oc mice resorbed bone although it was only 5% as compared to the controls. Confocal microscopy revealed that resorption was only associated with osteoclasts which were at least partially derived from fusion of donor cells in oc/oc mice. Lineage analysis of BM, PB and spleen did not show any evidence for selective recruitment of cells to the osteoclast lineage. Analysis of PB at 9 months revealed that the block in B-cell development in oc/oc mice was partially reversed. Assessment of vision with a visual drum tracking test showed that blindness was prevented in transplanted oc/oc mice. Our findings are of biological significance as they show that coupling of bone resorption and formation can be shifted by only a small fraction of normal cells in this severe disease model. Furthermore, they are clinically significant and encouraging for the continued development of stem cell based gene therapy of IMO. Conflict of interest: None declared.

Autosomal recessive agammaglobulinemia: Novel insights from mutations in Ig-beta

Agammaglobulinemia is a rare primary immuno-deficiency characterized by an early block of B-cell development in the bone marrow resulting in the absence of peripheral B cells and low/absent immunoglobulin serum levels. Mutations in the Bruton tyrosine kinase and in components of the pre-B-cell receptor (pre-BCR), such as mu heavy chain, surrogate light chain, and Igalpha have been found in 85% to 90% of patients affected by this disease. Here we review the recent advances in the characterization of molecular defects underlying an early block in B-cell development, focusing on the novel finding of the first two patients with agammaglobulinemia caused by mutations in Igbeta, the transmembrane protein that associates with Igalpha as part of the pre-BCR complex. Characterization of novel genetic defects involving components of the pre-BCR is crucial for a better understanding of the biology of early B-cell development and may have therapeutic and prognostic implications.

Donor T Cells from B Cell Deficient Mice Inhibit B Cell Development in Normal Recipients after Hematopoietic Cell Transplantation.

Allogeneic hematopoietic cell transplant (HCT) recipients often exhibit B cell (BC) lymphopenia due, in part, to graft-versus-host-disease (GVHD). Here, we studied the impact of donor T cells (TC) on BC deficiency post minor antigen-mismatched HCT. Following lethal irradiation, BALB.B mice were given FACS purified hematopoietic stem cells (HSC: cKIT+Thy1.1loLin-Sca-1+) alone, with whole splenocytes (SP), CD4 or CD8 TC from minor antigen-mismatched C57BL/6 (B6) mice. Chimerism analyses were performed on day (d) 30, 60, and 90. When pure HSC were transplanted, BCs reconstituted promptly (median 33% of lymphocytes [d30]; 61% [d60]; 74% [d90]), whereas TC engraftment was retarded and did not achieve full donor chimerism. Addition of SP or CD4 TCs, or to a lesser degree CD8 TCs, delayed BC reconstitution, with extremely low percentages of BCs beyond d60. This BC suppression correlated with the degree of acute GVHD, and BC numbers increased with recovery from GVHD. Additionally, this BC suppression was in stark contrast to TC development, with TC transfer resulting in early conversion to full donor chimerism. To test if previous events in the donor sensitize TCs against BC features (e.g. minor antigens), thereby promoting anti-BC cytotoxicity post-HCT, TCs from B6 muMT mice were co-transplanted with HSC. muMT mice are devoid of mature BCs because they lack the mu chain; consequently, their TCs were not exposed to BCs prior to transfer. Remarkably, BC engraftment was completely prevented through d90. TCs regenerated faster, but the vast majority originated from spleen and not HSC. To differentiate this lack of BC engraftment from GVHD-associated, alloreactive BC lymphopenia, syngenic B6 recipients were used. Again, initially complete blockade of BC engraftment was observed, although this suppression was overcome earlier post-HCT as compared to the minor-mismatched pair (median % BC d60: 'HSC only' recipients 52%; +CD4 17%; +CD8 48%). To clarify if this phenomenon was a purely cytotoxic reaction of muMT TC against BCs, we used WT B6 HSC +/− SP as donors and lethally or sublethally irradiated muMT mice as recipients. All groups, including sublethally irradiated animals, where host muMT TC were still present, engrafted BCs making a direct anti-BC cytotoxicity unlikely as the sole cause of the BC inhibition. FACS analysis of bone marrow was used to assess the developmental stages of BCs (Hardy fractions (Fr.) A-F) and revealed GVHD recipients with peripheral B lymphopenia have a shift of B220+ cells from more mature Fr. D-F to immature Fr. A-C stages and a lower proportion of IgM expressing BC. Recipients of the muMT TCs showed, in addition to a shift to more immature stages, a clear block in BC development with an absent switch to the expression of IgM (stage D to E)(Fig. 1). In conclusion, muMT TCs are capable of blocking BC maturation when transferred into WT mice, suggesting defective TC activity in muMT animals necessary for the co-development of both BCs and TCs. Furthermore, this study provides evidence that mature TCs are capable of interfering with BC regeneration post-HCT. Hence, our HCT combinations using WT and muMT B6 mice provide a powerful tool to study the role of TC function in the process of donor BC development post-HCT.

Combined deficiencies in Bruton tyrosine kinase and phospholipase Cγ2 arrest B-cell development at a pre-BCR+ stage

AbstractBruton tyrosine kinase (Btk) and phospholipase Cγ2 (PLCγ2) are 2 key molecules involved in B-cell receptor (BCR) signaling. Biochemical studies have placed them in a linear signaling pathway, with Btk acting upstream of PLCγ2. Consistent with this, mice lacking either molecule display a leaky but similar block in B-cell development. Here, we generated Btk−/−PLCγ2−/− mice and showed that combined deficiencies in Btk and PLCγ2 severely arrested B lymphopoiesis at the large pre–B-cell stage. In contrast to either single mutant, Btk−/−PLCγ2−/− pre–B cells expressed high levels of pre-BCR on their cell surfaces and exhibited reduced immunoglobulin light chain gene rearrangements. Pre-BCR–induced calcium signaling was also drastically compromised in Btk−/−PLCγ2−/− pre–B cells compared with wild-type and single-mutant cells. Interestingly, immunoglobulin heavy chain allelic exclusion remained intact in the absence of Btk and PLCγ2. Overall, our results suggest that Btk and PLCγ2 have combinatorial roles in regulating pre–B cell differentiation.

Mutations in the B-Cell Transcription Factor PAX5 Found in B-Progenitor Acute Lymphoblastic Leukemia Impair Normal PAX5 Activity.

Using genome-wide profiling of DNA copy number abnormalities using high-resolution single nucleotide polymorphism arrays, we recently identified a high frequency of genomic aberrations involving the PAX5 gene in pediatric B-progenitor ALL. PAX5 is a critical transcriptional regulator of B lymphocyte commitment and differentiation. Mutations, including partial tandem duplication, complete and focal deletions, point mutations in the DNA-binding or transactivation domain, and three translocations that encode PAX5 fusion proteins were observed in 31.7% of B-ALL. The PAX5 deletions were mono-allelic and resulted in either loss of the entire gene, or the deletion of only a subset of the exons leading to the production of PAX5 proteins that lacked the DNA-binding paired domain (exons 2–4) and/or the transcriptional activation domain (exons 7–10). In murine systems, the complete absence of PAX5 results in the arrest of B-cell development at the pro-B-cell stage prior to immunoglobulin heavy chain rearrangement, whereas haploinsufficiency leads to a partial block in B-cell development. Importantly, in the primary leukemia samples, the mono-allelic loss of PAX5 was associated with reduced expression of PAX5 by flow cytometry and quantitative RT-PCR, suggesting that haploinsufficiency contributes to the block in differentiation characteristic of B-progenitor ALL. To determine if the other identified PAX5 mutations result in hypomorphic alleles, we analyzed the DNA-binding and transcriptional activity of the encoded proteins. DNA-binding activity was assessed by electrophoretic mobility gel-shift assays using a labeled oligonucleotide probes from the promoters of the PAX5 target genes CD19 and CD79A (mb-1), and transcriptional activity was assessed by a luciferase-based reporter assays using the PAX5-dependent reporter plasmid, luc-CD19. Analysis was performed on the paired-domain mutants P80R and P34Q, the focal deletions Δe2-5, Δe2-6, Δe2-7, Δe2-8, and Δe6-8, and the PAX5-ETV6 and PAX5-FOXP1 translocation-encoded fusion proteins. As expected, DNA-binding was abrogated in deletion mutants that lacked the paired domain (Δe2-5, Δe2-6, Δe2-7, Δe2-8). In contrast, the PAX5 Δe6-8, which retains the paired DNA binding domain but lacks a significant portion of the transcriptional regulatory domain, had normal DNA binding activity. Importantly, the paired domain point mutants impaired DNA-binding in a promoter specific manner, with P80R having a marked reduction in binding to both the CD19 and mb-1 promoters, whereas P34Q showed reduced binding only to the mb-1 promoter. Surprisingly, the PAX5-ETV6 and the PAX5-FOXP1 translocations had markedly reduced DNA-binding activity despite retention of the PAX5 paired domain. As expected each of the mutants with impaired or absent DNA-binding activity were found to have markedly reduced transcriptional activity when compared to wild type PAX5. Similarly, those mutants with altered or deleted transcriptional activation domains had reduced transcriptional activity, as did the two PAX5 translocation-encoded fusion proteins (PAX5-ETV6 and PAX5-FOXP1). Moreover, transfection of increasing amounts of PAX5-ETV6 or PAX5-FOXP1 together with a fixed amount of wild type PAX5 revealed that the fusion proteins competitively inhibit the transcriptional activation of wild type PAX5. Taken together, these data indicate that the identified PAX5 mutations impair DNA-binding and/or transcriptional activity. This loss of normal PAX5 function in turn would contribute to the observed arrest in B-cell development seen in ALL.

Functional studies of BCL11A: characterization of the conserved BCL11A-XL splice variant and its interaction with BCL6 in nuclear paraspeckles of germinal center B cells

BackgroundChromosomal aberrations of BCL11A at 2p16.1 have been reported in a variety of B-cell malignancies and its deficiency in mice leads to a profound block in B-cell development.ResultsAlternative pre-mRNA splicing of BCL11A produces multiple isoforms sharing a common N-terminus. The most abundant isoform we have identified in human lymphoid samples is BCL11A-XL, the longest transcript produced at this locus, and here we report the conservation of this major isoform and its functional characterization. We show that BCL11A-XL is a DNA-sequence-specific transcriptional repressor that associates with itself and with other BCL11A isoforms, as well as with the BCL6 proto-oncogene. Western blot data for BCL11A-XL expression coupled with data previously published for BCL6 indicates that these genes are expressed abundantly in germinal-center-derived B cells but that expression is extinguished upon terminal differentiation to the plasma cell stage. Although BCL11A-XL/BCL6 interaction can modulate BCL6 DNA binding in vitro, their heteromeric association does not alter the homomeric transcriptional properties of either on model reporter activity. BCL11A-XL partitions into the nuclear matrix and colocalizes with BCL6 in nuclear paraspeckles.ConclusionWe propose that the conserved N-terminus of BCL11A defines a superfamily of C2HC zinc-finger transcription factors involved in hematopoietic malignancies.

Overexpression of HES-1 is not sufficient to impose T-cell differentiation on human hematopoietic stem cells

AbstractBy retroviral overexpression of the Notch-1 intracellular domain (ICN) in human CD34+ hematopoietic stem cells (HSCs), we have shown previously that Notch-1 signaling promotes the T-cell fate and inhibits the monocyte and B-cell fate in several in vitro and in vivo differentiation assays. Here, we investigated whether the effects of constitutively active Notch-1 can be mimicked by overexpression of its downstream target gene HES1. Upon HES-1 retroviral transduction, human CD34+ stem cells had a different outcome in the differentiation assays as compared to ICN-transduced cells. Although HES-1 induced a partial block in B-cell development, it did not inhibit monocyte development and did not promote T/NK-cell-lineage differentiation. On the contrary, a higher percentage of HES-1-transduced stem cells remained CD34+. These experiments indicate that HES-1 alone is not able to substitute for Notch-1 signaling to induce T-cell differentiation of human CD34+ hematopoietic stem cells.

Molecular basis of common variable immunodeficiency

Common variable immunodeficiency (CVID) is the most prevalent human primary immunodeficiency requiring medical attention. Until recently, the only known genetic defect specific to CVID was the inducible costimulatory receptor (ICOS) deficiency, which accounts for less than 1% of the patients. Recently, mutations in the TNF receptor family member transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), which mediates isotype switching in B cells, were found to be present in 10% to 20% of patients with CVID. Mutations in TACI were also found in relatives of patients with CVID who had IgA deficiency (IgAD), as well as in a patient with isolated IgAD. In the majority of patients described to date, only one TACI allele is mutated, showing an autosomal dominant transmission of the disease. B cells from individuals with TACI mutations did not produce IgG and IgA in response to the TACI ligand a proliferation-inducing ligand (APRIL), probably reflecting impaired isotype switching. These results suggest that TACI mutations can lead to CVID and IgAD.