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.

Tyrosine phosphorylation of the Wiskott-Aldrich syndrome protein by Lyn and Btk is regulated by CDC42.

The Wiskott-Aldrich syndrome (WAS) is a rare immunodeficiency disease affecting mainly platelets and lymphocytes. Here, we show that the WAS gene product, WASp, is tyrosine phosphorylated upon aggregation of the high affinity IgE receptor (Fc epsilonRI) at the surface of RBL-2H3 rat tumor mast cells. Lyn and the Bruton's tyrosine kinase (Btk), two protein tyrosine kinases involved in Fc epsilonRI-signaling phosphorylate WASp and interact with WASp in vivo. Interestingly, expression of a GTPase defective mutant form of CDC42, that interacts with WASp, is accompanied by a substantial increase in WASp tyrosine phosphorylation. This study suggests that activated CDC42 recruits WASp to the plasma membrane where it becomes phosphorylated by Lyn and Btk. We conclude that WASp represents a connection between protein tyrosine kinase signaling pathways and CDC42 function in cytoskeleton and cell growth regulation in hematopoietic cells.

Construction of temperature and Zn-dependent human stromal cell lines that amplify hematopoietic precursors from cord blood CD34+ cells.

Forty-five human stromal cell lines were established from long-term bone marrow cultures transformed with a new vector, pNu MTSVts, which contains the Zn-inducible metallothionein promoter and the temperature-dependent SV40 T antigen from SV40 A58 mutant. Six of these cell lines were studied because of their growth capacity. All cell lines differed with respect to growth potential, expression of cell surface markers, and cytokine transcripts. Major histocompatibility complex (MHC) class I, CD29, CD49d, and CD51 were present on all stromal cell lines, MHC class II and CD34 were consistently absent, and CD11a (LFA-1), CD18 (ICAM-1R), CD54 (ICAM-1), CD58 (LFA-3) CD56 (N-CAM), CD106 (V-CAM), laminin, and collagen IV were diversely expressed. All cell lines contained interleukin (IL)-1alpha, IL-1beta, IL-2, IL-5, and macrophage colony-stimulating factor (M-CSF) transcripts, whereas granulocyte M-CSF, TNFalpha, IL-3, IL-4, and IL-7 were diversely expressed. The most characteristic feature of these cells was their varying capacity to expand cord blood CD34+ cells. One of these stromal cell lines ensured more than twofold expansion of the initial CD34+CD10-CD19- population in the first 2 weeks. Differentiation toward the B cell lineage was limited, producing only very small numbers of CD19+ cells after 6 weeks of culture.

The SH3 domain of Bruton's tyrosine kinase interacts with Vav, Sam68 and EWS.

Bruton tyrosine kinase (BTK) is a cytoplasmic protein tyrosine kinase which controls crucial steps of differentiation of B lymphocytes. Mutations affecting either the PH, SH3, SH2 or kinase domain of BTK all give rise to X linked agammaglobulinaemia (XLA) in humans. In this study, the authors report that the BTK-SH3 domain binds to a set of proteins expressed in pro-B, pre-B and B cell lines. Three of them were characterized as Vav, Sam68 and EWS. The authors show that a Pro-->Leu substitution in a region of the SH3 domain, which is deleted in an XLA patient, is sufficient to abolish BTK-SH3 binding potential. The authors also report that several of the BTK-SH3 binding proteins, including Sam68, EWS and Vav, are tyrosine phosphorylated in conditions that also promote BTK kinase activity. For EWS and Sam68 this tyrosine phosphorylation was cell cycle dependent.

A non-XLA primary deficiency causes the earliest known defect of B cell differentiation in humans: a comparison with an XLA case.

We report a detailed comparison of B cell defects in two patients, one XLA and one non-XLA. Both had severe agammaglobulinemia with a total absence of CD19+ cells in the periphery. In the non-XLA case, CD19 expression was also highly impaired in the bone marrow, resulting in the absence of both B and preB compartments. Early proB cells were present since CD34+CD10+ and some CD19+CD10+ mostly CD34+ were identified, although diminished. By contrast, in the XLA patient the CD34+CD19+ proB cells were increased whereas the CD34-CD19+ preB cell population was low. Semi-quantitative RT-PCR analysis performed on mononuclear bone marrow cells from the non-XLA patient indicated that lambda-like, VpreB, Rag-1, Rag-2 and TdT transcripts expressed during proB cell stages were found at normal levels whereas E2A, CD10, Syk, Pax-5, CD19, Ig alpha, Ig beta, VH-C mu and V kappa-C kappa transcripts characteristic of later stages were severely depressed. By contrast in the XLA patient most of these transcripts were observed in normal amounts. The phenotype of the non-XLA patient resembles that of Pax-5 or Ig beta knock-out mice, but since the coding sequence of both cDNAs were shown to be normal, the blockage might rather result from an altered regulation of one of these genes or from defect of other genes. All these data indicate that the non-XLA patient suffers from a new genetic defect that results in an arrest of differentiation within the proB cell compartment, before the onset of Ig gene rearrangements. From all agammaglobulinemias reported so far, including XLA cases and those resulting from C mu gene defects, the non-XLA patient exhibits the earliest blockage in the B cell differentiation pathway.

Human thymic B cells largely overexpress the VH4 Ig gene family. A possible role in the control of tolerance in situ?

Human thymus contains a small population of B cells, a fraction of which expresses CD2 and/or CD5, as revealed by FACS analysis. Human thymic B cells enriched on CD19 panning revealed a constant co-expression of CD19 and Ig light chains. In the absence of detectable B precursors, these cells had reached at least the stage of IgM+ immature B lymphocytes. Analysis of Ig transcripts by PCR amplification first revealed a strong bias in favor of the VH4 and sequencing of 45 VH4-D-J cDNA clones isolated from two thymuses indicated that this thymic repertoire significantly differed from that of peripheral blood lymphocytes. Most genes of the VH4 family were used with various D-J combinations and a relatively high frequency of somatic mutations. This repertoire thus appears clearly selected in the thymus. This is of particular interest since the VH4 gene family is frequently encountered in autoimmunity a situation in which the number of thymic B cells is largely increased. They could play a major role in the control of tolerance in situ.

A human non-XLA immunodeficiency disease characterized by blockage of B cell development at an early proB cell stage.

We report a detailed analysis of a B cell defect affecting a patient girl born from first cousin parents, characterized by a severe non-X-linked agammaglobulinemia with a total absence of CD19- cells in the periphery. In the bone marrow, CD19 expression was also highly impaired, resulting in the absence of both B and preB compartments. By contrast, CD34+CD10+, CD34psiL+, and some CD19+CD10+ mostly CD34+ early proB cells were present, although diminished. Semiquantitative RT-PCR analysis performed on mononuclear bone marrow cells indicated that lambda-like, VpreB, Rag-1, Rag-2, and TdT transcripts expressed during proB cell stages were found at normal levels whereas E2A, CD10, Syk, Pax-5, CD19, Igalpha, Igbeta, VH-Cmu, and Vkappa-Ckappa transcripts characteristic of later stages were severely depressed. This phenotype resembles that of Pax-5 knock-out mice, but since the coding sequence of the patient Pax-5 cDNA was shown to be normal, the defect might rather result from an altered regulation of this gene. All these data indicate that the patient suffers from a new genetic defect that results in an arrest of differentiation within the proB cell compartment, i.e., earlier than X-linked agammaglobulinemia, before the onset of Ig gene rearrangements.

Cell surface expression of surrogate light chain (psi L) in the absence of mu on human pro-B cell lines and normal pro-B cells.

Surrogate light chains (psi L) encoded by lambda-like (lambda 5) and VpreB genes play a critical role in controlling the early steps of B cell differentiation. We prepared new anti-VpreB monoclonal antibodies (mAb) (3C7/6F6) which preferentially recognize the VpreB epitope at the cell surface of human cell lines that do not express the mu chain. These mAb provide the first characterization of human pro-B cell lines expressing surface psi L. We demonstrate that surface psi L expression is considerably enhanced upon interleukin-7 stimulation and that the psi L complex is formed independently of the Ig alpha/Ig beta heterodimer. Finally, using these antibodies, we confirm the existence of a normal pro-B cell population in human adult bone marrow. These cells are CD34+ CD38+ psi L+, do or do not express CD19, CD10, or both epitopes, and may represent the earliest cell population committed to B cell differentiation.

The VDJ repertoire expressed in human preB cells reflects the selection of bona fide heavy chains.

In early steps of B cell differentiation, mu chains are transiently expressed in association with a surrogate light chain (psi L) composed of the lambda-like and VpreB monomorphic polypeptides, thus forming a putative preB receptor. Using a monoclonal anti-VpreB antibody, preB cells were isolated from two adult human bone marrow samples and their VDJ repertoire analyzed at the transcription level. All VH families were identified and further analysis focused on VH3 sequence analysis of 37 distinct VDJ cDNA clones. The VH3 genes expressed in the two bone marrow samples were also encountered in fetal liver and adult peripheral blood lymphocytes with a roughly similar contribution of 3.30, 3.23, 3.9 and 3.53. The characteristic features of the preB repertoire as compared to the activation B repertoire include the quasi absence of somatic mutations, limited N diversity and a shorter third complementarity-determining region (CDR3). It also significantly differs from the fetal repertoire, which makes higher usage of DQ52 and has CDR3 of even shorter lengths. The almost constant presence of glycine residues in the CDR3 and predominance of JH4 with a low level of DQ52 DH usage, suggest that preB cell clones are submitted to an initial selective pressure which should be antigen independent. The bona fide heavy chains would be merely selected for their ability to interact with the surrogate light chains, thus shaping the repertoire that will be co-expressed with immunoglobulin light chains in IgM molecules.

Internalization of B cell and pre-B cell receptors is regulated by tyrosine kinase and phosphatase activities.

Prior to the expression of the B cell antigen receptor, the mu heavy chain associates with two non-polymorphic polypeptides, lambda like and VpreB, which form a pseudo-light chain complex in pre-B cells and pre-B cell lines. Surface expression of the so-called pre-B cell receptor (pre-BCR) occurs only in the presence of Ig alpha and Ig beta, known to be involved both in B cell antigen receptor (BCR) signaling and trafficking. Although the pre-BCR organization is consistent with an efficient transport to the cell surface, most of the newly synthesized receptor remains within the cells, and so far, no data are available concerning the rate of exit from the endoplasmic reticulum. Using the human pre-B cell line Nalm-6, we found that only a small fraction (2%) of newly synthesized pre-BCR is transported to the cell surface within 4-6 h after synthesis, where it is constitutively re-internalized. Membrane Ig-heavy chain cross-linking induced internalization of surface pre-BCR within a few minutes, and the mechanisms underlying endocytosis were analyzed by immunofluorescence and confocal microscopy. Preincubation of the cells with either genistein or orthovanadate, which inhibit, respectively, tyrosine kinases and tyrosine phosphatases, blocked pre-BCR internalization in a dose-dependent manner, indicating that both activities are required for endocytosis. BCR internalization was also inhibited in a reversible manner by the drugs. In contrast, neither drug affected the size of the steady-state pool of internalized transferrin receptors. Thus, our data show that tyrosine phosphorylation and dephosphorylation are both required for cross-linking-induced pre-BCR and BCR internalization.

Fetal versus adult PreB or B cells: the human VH repertoire.

At the preB stage, when only the IGH locus has rearranged, mu chains become expressed in association with the psi L chains, lambda-like and VpreB, thus forming the preB receptor. By the use of a monoclonal anti VpreB antibody, preB cells were isolated from two adult bone marrow samples, and the VH repertoire was analyzed and compared to fetal, XLA (X-linked agammaglobulinemia), and adult B repertoires. Most VH genes identified were also expressed in fetal liver, XLA bone marrow, and adult PBLs, with similar predominant usage of certain germline genes. Multiple D/D fusions, limited N diversity, and preferential use of JH4 with a low level of DQ52 usage were also identified. Few mutations could be observed, not specifically localized in CDR regions, that could be interpreted as not positively selected. Conversely, a shorter length of CDR3 appeared to be the hallmark of the preB step. Thus, the association of psi L chains with mu does not bring about a bias in the VH gene usage, but a first selection on the CDR3 region could be the result of recognition by given autoantigens or ligands different for preB cells and B cells.

The human pre-B cell receptor: structural constraints for a tentative model of the pseudo-light (psi L) chain.

In human pre-B cells, the mu chain is associated with a surrogate light chain composed of the lambda-like and Vpre-B gene products. This pre-B cell receptor presumably triggers early steps of B cell differentiation, We have determined the NH2-terminal amino acid sequence of the lambda-like chain, showing that the mature chain results from the cleavage of a leader segment of 44 residues, leaving a polypeptide of 169 amino acids having partial features of the Ig light chain domains, with the exception of the first 50 amino acid NH2-terminal region. We have completed the nucleotide sequence of the Vpre-B gene, which appears to contain 126 residues in its mature form of which the 24 COOH-terminal portion was not Ig-related. Analysis of transfectants has provided direct evidence that lambda-like and Vpre-B chains assemble together even in the absence of heavy chain, prompting the search for a structural basis of this interaction. Comparison with the domain organization of the regular Ig lambda chain suggests that most of the psi L chain can be accommodated within a CL-VL-like structure, with an extra "subdomain" contributed by the non-Ig-like portions of both the lambda-like and Vpre-B polypeptides.

Activation of src family kinases in human pre-B cells by IL-7.

IL-7 was identified originally as a specific pre-B cell growth factor. We have investigated its signal transduction mechanism by using the human pre-B cell line Nalm-6, and have found that it stimulates tyrosine phosphorylation of various proteins: pp27, pp43, pp54, pp64, pp78, pp90, pp105, and pp120. Antiphosphotyrosine immunoprecipitates from IL-7-stimulated Nalm-6 showed two major proteins of M(r) = 60,000 and 55,000, capable of autophosphorylation. Autophosphorylation was maximal 10 min after the cells were challenged with the cytokine. Antiphosphotyrosine immunoprecipitates from IL-7-stimulated cells also increased tyrosine phosphorylation of the exogenously added substrate histone H2B. Furthermore, by using a polyclonal anti-IL-7 receptor (IL-7R) Ab in Western blotting analysis, we observed that antiphosphotyrosine immunoprecipitates were associated with the IL-7R in a transient manner. These data indicate that the IL-7R associates with tyrosine-phosphorylated proteins as its amino acid sequence is devoid of a putative site of tyrosine phosphorylation. These results were confirmed as several 32P-labeled proteins were visualized after immunoprecipitation by using anti-IL-7R Ab. Anti-IL-7R immunoprecipitates from IL-7-stimulated cells revealed a unique band of M(r) = 60,000 associated with the receptor able to autophosphorylate in the presence of ATP and Mn2+. Hence, we identified p59fyn and p53/56lyn to be stimulated by IL-7. In contrast to p53/56lyn, p59fyn was found to be associated constitutively with the cloned IL-7R. These data emphasize the role of the src family in hematopoiesis.

Interleukin-7 regulates c-myc expression in murine T cells and thymocytes: a role for tyrosine kinase(s) and calcium mobilization.

Interleukin-7 (IL-7) was originally identified as a pre-B cell growth factor whose proliferating activity has been extended to numerous target cells including T lymphocytes. We investigated c-myc mRNA expression, an oncogene associated with proliferation, in the murine T cell line D10 G4.1 and freshly isolated thymocytes since both target cells proliferate in response to IL-7. We find that blockade of the tyrosine kinase pathway by genistein, a potent tyrosine kinase inhibitor, inhibits both IL-7-dependent D10 G4.1 cell proliferation and c-myc mRNA expression which appears to involve de novo mRNA synthesis and to be under the control of short-lived protein repressor(s). We have also examined possible signal transduction pathways which might regulate c-myc mRNA expression in the murine T cell line. IL-7 biological activity is not affected by stimulation of the protein kinase C pathway by phorbol esters. Thus, IL-7 regulates c-myc mRNA expression in a protein kinase C-independent manner and these data are strengthened by protein kinase C depletion which does not modify IL-7 c-myc mRNA responsiveness. In contrast and independent of protein kinase C activation, intracellular calcium mobilization by means of ionomycin reduces IL-7 induction of c-myc mRNA expression and may represent a physiological mechanism whereby IL-7 bioactivity is regulated. The activity of IL-7 on c-myc mRNA expression has been extended to freshly isolated thymocytes and we find a synergistic effect of IL-7 with concanavalin A. Taken together our results illuminate the molecular mechanism of IL-7 c-myc induction in the T lineage by ascribing a role for tyrosine kinase and increase in intracellular calcium in both IL-7 induced gene induction and cell proliferation.

Discrete early pro-B and pre-B stages in normal human bone marrow as defined by surface pseudo-light chain expression.

Vpre-B and lambda-like genes are selectively expressed in B cell precursors and encode polypeptide chains associated in a mu-pseudo light chain (mu-psi L) complex which is thought to regulate some early steps of B cell differentiation. We have generated anti-Vpre-B monoclonal antibodies which allowed us to identify different steps of differentiation from the pro-B to the immature B cells by following surface expression of Vpre-B, mu and light chains in normal adult human bone marrow. Already present at the surface of a small fraction of B cell progenitors (CD34+/CD19+) the Vpre-B molecule was consistently found coexpressed with CD19 and was also found with the sequentially occurring CD10, CD20, CD21, CD22 and CD5 markers. Three discrete cell types were identified: (i) a subpopulation expressing Vpre-B without mu and which represented an early stage of differentiation, (ii) a minor subpopulation co-expressing Vpre-B and mu without the conventional light chains and (iii) a major subpopulation co-expressing Vpre-B, mu and kappa or lambda chains, considered an intermediate pre-B/B stage. The presence of the psi L chain in various cell subpopulations, in possible association with discrete molecules and/or different contexts, suggests its involvement at several steps of early B cell differentiation.

Reconstitution of human immunoglobulin VH repertoire after bone marrow transplantation mimics B-cell ontogeny.

Reconstitution of the human Ig repertoire after allogeneic and autologous bone marrow transplantation (BMT) was followed by analysis of the expression of the six VH families using in situ hybridization on BM cells at 30, 60, and 90 days postgraft. Our results indicate that during the early post-transplantation period, the expressed repertoire of VH is dramatically affected: VH3, the major expressed family in adult B cells, is decreased two- to threefold and is compensated by transient overexpression of the other families, especially VH4, VH5, and VH6. Similar results were observed in allogeneic and autologous grafted patients. Kinetics of reconstitution mimics normal repertoire development described in ontogeny, although normalization, as compared with the adult pattern, may take as long as 1 year.

Structure, biosynthesis, and transduction properties of the human mu-psi L complex: similar behavior of preB and intermediate preB-B cells in transducing ability.

In human preB cells, the mu chain is associated with a surrogate light chain composed of the VpreB and lambda-like gene products. Using anti-peptide antibodies directed against VpreB and lambda-like epitopes, we identified the discrete components of the mu-psi L (pseudo-light) chain complex in various preB cell lines, and in intermediate preB-B cells that co-expressed the psi L and the kappa chain. The lambda-like gene product was identified as a single band at 20 kDa, disulfide linked to the mu chain. VpreB was detected at 16 kDa and, depending upon the cell lines, an isoform of this polypeptide was also present at 15 kDa. In addition, lambda-like--VpreB chain complexes not associated with mu were identified both in cell lysates and culture supernatants. Pulse-chase experiments indicated that VpreB was transiently associated with two new polypeptides of molecular weights 17.5 and 36 kDa. Expression of mu-psi L and co-expression of mu-psi L and microL at the surface of preB and intermediate preB-B cells respectively was detected by cytofluorimetry. The signal transduction ability of the complex in both types of cells was shown by measuring the calcium mobilization and the phosphorylation of tyrosyl residues upon stimulation by anti-mu. Signal events were similar in both cases, but differed from those induced in a mature B cell line. This points to a definite function of the preB cell receptor and suggests that the intermediate preB-B cell line still lacks some molecular components that condition initiation of a mature B cell transduction signal.

Bone marrow cells in X-linked agammaglobulinemia express pre-B-specific genes (lambda-like and V pre-B) and present immunoglobulin V-D-J gene usage strongly biased to a fetal-like repertoire.

Expression of Ig and Ig-related genes has been studied in bone marrow cells from two patients with severe form of X-linked agammaglobulinemia (XLA). Phenotypic analysis revealed the presence of pre-B cells, in the absence of mature B cell markers. The pre-B-specific genes, lambda-like and V pre-B, were normally transcribed. Sequence analysis of 48 distinct V-D-J cDNA clones directly derived from XLA bone marrow cells indicated that they had characteristics of an early fetal pre-B repertoire. All VH families were identified, with a strong bias in the gene usage: a few VH genes were largely overexpressed, either germline or slightly mutated; most genes had been located 3' of the VH locus and were also used in fetal liver (8-13 wk of gestation). Short D regions, (resulting from D-D fusion, making usage of all D genes in both orientations with utilization of the three reading frames), restricted N diversity, and a fetal JH usage pattern were also observed. Taken together, our data suggest that the XLA defect does not alter V-D-J rearrangements nor the expression of mu, lambda-like, and V pre-B transcripts and most likely results in a poor efficiency of some critical steps of the B cell maturation.