Essential role for Bclaf1 in lung development and immune system function.

Bcl-2 associated factor 1 (Bclaf1) is a nuclear protein that was originally identified in a screen of proteins that interact with the adenoviral bcl-2 homolog E1B19K. Overexpression of Bclaf1 was shown to result in apoptosis and transcriptional repression that was reversible in the presence of Bcl-2 or Bcl-x(L). Furthermore, antiapoptotic members, but not proapoptotic members of the Bcl-2 protein family, were shown to interact with Bclaf1 and prevent its localization to the nucleus. Bclaf1 has also recently been identified as a binding partner for Emerin, a nuclear membrane protein that is mutated in X-linked recessive Emery-Dreifuss muscular dystrophy. To ascertain the in vivo function of Bclaf1, we have generated mice that carry a targeted mutation of the bclaf1 locus. In this study, we show that Bclaf1 is required for proper spatial and temporal organization of smooth muscle lineage during the saccular stage of lung development. We also show that Bclaf1 is dispensable for thymocyte development but is essential for peripheral T-cell homeostasis. Despite its postulated role as a proapoptotic protein, Bclaf1-deficient cells did not show any defect in cell death linked to development or after exposure to various apoptotic stimuli. Our findings show a critical role for Bclaf1 in developmental processes independent of apoptosis.

Caspase-12 compensates for lack of caspase-2 and caspase-3 in female germ cells.

Previously, we analyzed mice lacking either caspase-2 or caspase-3 and documented a role for caspase-2 in developmental and chemotherapy-induced apoptosis of oocytes. Those data also revealed dispensability of caspase-3, although we found this caspase critical for ovarian granulosa cell death. Because of the mutual interdependence of germ cells and granulosa cells, herein we generated caspase-2 and -3 double-mutant (DKO) mice to evaluate how these two caspases functionally relate to each other in orchestrating oocyte apoptosis. No difference was observed in the rate of spontaneous oocyte apoptosis between DKO and wildtype (WT) females. In contrast, the oocytes from DKO females were more susceptible to apoptosis induced by DNA damaging agents, compared with oocytes from WT females. This increased sensitivity to death of DKO oocytes appears to be a specific response to DNA damage, and it was associated with a compensatory upregulation of caspase-12. Interestingly, DKO oocytes were more resistant to apoptosis induced by methotrexate (MTX) than WT oocytes. These results revealed that in female germ cells, insults that directly interfere with their metabolic status (e.g. MTX) require caspase-2 and caspase-3 as obligatory executioners of the ensuing cell death cascade. However, when DNA damage is involved, and in the absence of caspase-2 and -3, caspase-12 becomes upregulated and mediates apoptosis in oocytes.

Fine characterization of childhood and adult acute lymphoblastic leukemia (ALL) by a proB and preB surrogate light chain-specific mAb and a proposal for a new B cell ALL classification.

The expression of the surrogate light chain (psiL) - made of the lambda-like (or lambda5) and the VpreB proteins - is a B cell-specific maturation marker. Using an anti-human VpreB mAb (4G7), we recently identified in human normal bone marrows, proB and preB cells that express the psiH-psiL proB (proBCR) and the mu-psiL preB (preBCR) receptors, respectively. In the present study, FACS and biochemical analysis confirm the broad proB and preB reactivity of the 4G7 mAb that contrasts with the narrow specificity of other available anti-psiL reagents for preB cells. This mAb was used to explore intracytoplasmic and cell surface expression of the VpreB protein on a series of 92 precursor B cell ALLs (from 40 child and 52 adult patients), in combination with 24 other mAbs. The major result concerns the identification within proB (or BI) and common (or BII) ALLs, of proBCR and proBCR+ ALLs that express the VpreB in the cytoplasm or at the cell surface, respectively. The percentage of ALLs within these two VpreB sub-groups differ considerably between the ALL origin. In the pediatric series, ALLs present in the majority a proBCR+ phenotype whereas we observed a proBCR+ phenotype for adult ALLs. Based on VpreB expression, and in combination with other published data, we propose a refined classification for precursor B cell ALLs.

Autosomal primary immunodeficiencies affecting human bone marrow B-cell differentiation.

Since the initial report of X-linked agammaglobulinemia by Bruton, numerous autosomal primary immune deficiencies affecting early B-cell differentiation have been described in humans. The identification of these autosomal mutations has been facilitated by phenotype comparison with knockout mice. In mice, defects in B-cell development have been observed after disruption of genes encoding transcription factors, the interleukin-7 pathways as well as structural or signaling components of the pre-B-cell receptor. In general, the phenotypes of primary immune deficiencies in humans correlate with those observed in mutant mice, validating the use of the mouse model approach. In addition, we report a follow-up analysis of an autosomal primary deficiency in a young female patient born from consanguinous parents and characterized by the absence of pre-B and B-cell compartments. The patient's gene defect was identified as a cytosine insertion at the beginning of the CH1 exon of the Ig(mu) gene, resulting in a stop codon at position 48 and the absence of Ig(mu) chain expression. The precise phenotype of this patient is compared to other autosomal primary immunodeficiencies affecting humans and mice.

The human (PsiL+mu-) proB complex: cell surface expression and biochemical structure of a putative transducing receptor.

The surrogate light chain (PsiL) associates with mu and Igalpha-Igbeta chains to form the preB-cell receptor that plays a critical role in early B-cell differentiation. Discrepancies exist in human concerning the existence of PsiL+mu- proB cells and the biochemical structure of such a proB-cell complex remains elusive. Among new antihuman VpreB monoclonal antibodies (MoAbs), 5 of the gamma kappa isotype bound to recombinant and native VpreB protein with high affinity. They recognized 4 discrete epitopes, upon which 2 were in the extra-loop fragment. Such MoAbs detected the PsiL at the cell surface of either preB or on both proB and preB cells. The previously reported SLC1/SLC2 MoAbs recognize a conformational epitope specific for the mu/PsiL association in accordance with their preB-cell reactivity. Using the proB/preB 4G7 MoAb, PsiL cell surface expression was detected on normal bone marrow, not only on CD34(-)CD19(+) preB but also on CD34(+)CD19(+) proB cells. Futhermore, this MoAb identified PsiL+mu- fresh proB leukemic cells of the TEL/AML1 type. Biochemical studies showed that, at the proB stage, the PsiL is associated noncovalently with two proteins of 105 and 130 kD. Triggering of this complex induces intracellular Ca2+ flux, suggesting that the PsiL may be involved in a new receptor at this early step of the B-cell differentiation.

Mu-surrogate light chain physicochemical interactions of the human preB cell receptor: implications for VH repertoire selection and cell signaling at the preB cell stage.

The surrogate light chain (SL) composed of the A-like and VpreB polypeptides is organized as two Ig domains and an extra-loop structure. It associates to the mu-chain in preB cells. We have produced human VpreB, SL, two Fdmu (VH-CH1), and the two corresponding Fab-like (Fdmu-SL) recombinant proteins in baculovirus. The correctness of the general conformation of the proteins was assessed by epitope mapping and affinity measurements using a new batch of anti-VpreB mAbs. Plasmon resonance analysis showed that both VpreB and the entire SL associated with the Fdmu fragments, with Kd values of 3x10(-8) M for VpreB-Fdmu and of 10(-9) to 10(-10) M, depending upon the V(H), for SL-Fdmu. These results indicate that the A-like chain, in addition to be covalently bound to the Cmu1 domain, also interacts with the VH domain. Therefore, a dual role of the SL emerges: 1) interaction of the C-domain of A-like would release the mu-chain from its interaction with binding protein in the endoplasmic reticulum, and 2) interaction of a part of A-like and most of VpreB would bind to VH, ensuring a "quality control" of the native heavy chain that represents the first step of selection of the B cell repertoire. We also demonstrated that two Fab-like fragments did not interact with each other, suggesting that activation of the cell surface preB receptor does not involve aggregation neither in cis nor in trans of the Fab-like structures.