The human PD-1 gene: complete cDNA, genomic organization, and developmentally regulated expression in B cell progenitors.

We report the complete cDNA sequence and the genomic structure of the human PD-1 homologue. An analysis of the expression pattern of the human PD-1 gene (hPD-1) and the murine PD-1 gene (mPD-1) in developing bone marrow B-lineage cells was also undertaken. The full length hPD-1 cDNA is 2106 nucleotides long and encodes a predicted protein of 288 amino acid residues. The hPD-1 and mPD-1 genes share 70% homology at the nucleotide level and 60% homology at the amino acid level. Four potential sites for N-linked glycosylation are conserved, as are a stretch of amino acids between two cysteine residues resembling a V-set immunoglobulin domain, and another region containing a motif similar to an immunoreceptor tyrosine-based inhibitory motif. Isolation of the genomic locus of the hPD-1 gene reveals that the gene is composed of five exons located on human chromosome 2 at band q37. The 5' flanking region lacks TATA and CAAT cis-acting elements, but includes a number of potential transcription factor binding sites and a dominant transcription start site. The mPD-1 gene was preferentially expressed in pro-B cells from murine adult bone marrow. Although hPD-1 was not preferentially expressed in pro-B cells from human fetal bone marrow, treatment of isolated pro-B cells with interleukin-7 resulted in a dramatic increase in expression. These data suggest that PD-1 may play a role in B-cell differentiation during the pro-B cell stage.

Ig D(H) gene segment transcription and rearrangement before surface expression of the pan-B-cell marker CD19 in normal human bone marrow.

The onset of IgH transcription and rearrangement is a defining characteristic of the progenitor population in which B-lineage commitment occurs. These features were used to better define the earliest stage of B-cell commitment in humans and to determine if these stages differ as a function of human ontogeny. Fetal and adult bone marrow mononuclear cells were sorted into B-lineage subpopulations on the basis of surface expression of the stem cell marker CD34, the pan-B-cell marker CD19, and IgM and analyzed for transcription and rearrangement of the IgH locus. The locus was found to be transcriptionally active before surface expression of CD19, as indicated by the presence of germline I mu, C mu, and D(H)Q52 transcripts in the CD34+ CD19- subpopulation. Transcripts from IgH alleles that had undergone DJC mu rearrangements were also detected in the CD34+ CD19- subpopulation. Within this subpopulation, low levels of DXP-containing DJC mu transcripts were detected in both fetal and adult cells. Although D(H)Q52 DJC mu transcripts were abundant in fetal CD34+ CD19- cells, they were not detected in cells of the same phenotype derived from adult bone marrow. In both fetus and adult, V(H)3-and V(H)6-containing VDJC mu transcripts were detected only in the CD19+ subpopulations. These data indicate that transcription of D(H)Q52-J(H) and DXP-J(H) rearrangements differs during fetal and adult B lymphopoiesis. Moreover, in both fetus and adult, transcription of unrearranged components of the IgH locus and DJ rearrangements can proceed before the surface expression of CD19.

The J chain gene is transcribed during B and T lymphopoiesis in humans.

In mice and chickens, J chain appears to be expressed only in activated B cells and plasma cells. In humans, studies based mainly on transformed cells suggest that J chain expression may initiate during earlier stages in B lineage differentiation. In the present study, we isolated a series of hematopoietic subpopulations from human fetal and adult tissues by immunofluorescence cell sorting and examined each subpopulation for J chain expression by reverse transcriptase-PCR. In fetal and adult bone marrow, J chain transcripts were detected at all stages of B lineage differentiation, including the progenitor (CD34+/CD19-) and pro-B (CD34+/CD19+) cell subpopulations. J chain mRNA was also detected during fetal thymocyte development: double negative (CD4-/CD8-) through single positive (CD4+ or CD8+) cell subpopulations. The J chain message was not detected in peripheral CD3+ T cells, CD14+ monocytes, and CD56+ NK cells from either fetal or adult samples. The nucleotide sequence of J chain PCR products from CD34+/CD19- bone marrow progenitors and CD4+/CD8- thymocytes proved identical to the previously reported sequence of functionally spliced J chain mRNA. These results suggest that the J chain gene is transcriptionally active during early stages of both B cell and T cell differentiation, before the expression of their respective Ag receptors.

B cells are generated throughout life in humans.

This analysis of B cell development as a function of age reveals a relatively widespread distribution of progenitor B (pro-B), pre-B, and B cells in fetal tissues, and thus supports the idea of a multifocal origin of B lineage cells during embryonic development. From mid-gestation onward, the bone marrow is the major site of B cell generation in humans. A relatively constant ratio of bone marrow precursors to B cells of immature phenotype (CD24highCD10+CD20lowIgD-) is maintained from mid-gestation through the eighth decade of life. The persistence of recombinase gene activity in pro-B cells further attests the sustained production of B cells in bone marrow. Interestingly, a subpopulation of B cells with mature phenotype (CD24lowCD10-CD20highIgD+) accumulates in the bone marrow during childhood, and this becomes the predominant B cell subpopulation in adult bone marrow. This mature population of bone marrow B cells may represent a subpopulation of recirculating B cells that have undergone selection in the periphery.

Immunoglobulin recombinase gene activity is modulated reciprocally by interleukin 7 and CD19 in B cell progenitors.

Bone marrow stromal cells promote B cell development involving recombinase gene-directed rearrangement of the immunoglobulin genes. We observed that the stromal cell-derived cytokine interleukin 7 (IL-7) enhances the expression of CD19 molecules on progenitor B-lineage cells in human bone marrow samples and downregulates the expression of terminal deoxynucleotidyl transferase (TdT) and the recombinase-activating genes RAG-1 and RAG-2. Initiation of the TdT downregulation on the first day of treatment, CD19 upregulation during the second day, and RAG-1 and RAG-2 downmodulation during the third day implied a cascade of IL-7 effects. While CD19 ligation by divalent antibodies had no direct effect on TdT or RAG gene expression, CD19 cross-linkage complete blocked the IL-7 downregulation of RAG expression without affecting the earlier TdT response. These results suggest that signals generated through CD19 and the IL-7 receptor could modulate immunoglobulin gene rearrangement and repertoire diversification during the early stages of B cell differentiation.

Identification of the TCL1 gene involved in T-cell malignancies.

The TCL1 locus on chromosome 14q32.1 is frequently involved in chromosomal translocations and inversions with one of the T-cell receptor loci in human T-cell leukemias and lymphomas. The chromosome 14 region translocated or rearranged involves approximately 350 kb of DNA at chromosome band 14q32.1. Within this region we have identified a gene coding for a 1.3-kb transcript, expressed only in restricted subsets of cells within the lymphoid lineage and expressed at high levels in leukemic cells carrying a t(14;14)(q11;q32) chromosome translocation or a inv(14)(q11;q32) chromosome inversion. The cognate cDNA sequence reveals an open reading frame of 342 nt encoding a protein of 14 kDa. The TCL1 gene sequence, which, to our knowledge, shows no sequence homology with other human genes, is preferentially expressed early in T- and B-lymphocyte differentiation.

Differential roles of stromal cells, interleukin-7, and kit-ligand in the regulation of B lymphopoiesis.

Newly formed B lymphocytes are a population of rapidly renewed cells in the bone marrow of mammals and their steady state production presumably depends on a cascade of regulatory cells and cytokines. Although considerable information has been forthcoming about the role of interleukin-7 (IL-7) in potentiating pre-B-cell proliferation, few studies have addressed the possibility that multiple cytokines are involved in the progression of early events in cellular differentiation and proliferation in this hematopoietic lineage. Our laboratory previously described pre-B-cell differentiation mediated by the bone marrow stromal cell line S17. In this study, we further delineate the role of stromal cells in differentiation and proliferation of pre-B cells. These experiments show that the stromal cell line S17 potentiates the proliferative effect of IL-7 on B-lineage cells and that this S17-derived potentiator can be replaced with recombinant kit-ligand (KL). Our results further show that pre-B-cell formation from B220-, Ig- progenitor cells and expression of mu heavy chain of immunoglobulin is uniquely dependent on the presence of S17 stromal cells and cannot be reproduced with IL-7, KL, or costimulation with both IL-7 and KL. These data contribute to a rapidly evolving model of stromal cell regulation of B-cell production in the marrow and suggest unique roles for IL-7, KL, and as yet uncharacterized stromal cell-derived lymphokines in this process.

Bone marrow stromal cell regulation of B lymphopoiesis: interleukin-1 (IL-1) and IL-4 regulate stromal cell support of pre-B cell production in vitro.

Bone marrow stromal cells appear to be key regulatory elements in hematopoiesis and lymphopoiesis. These stromal cells respond to cytokine exposure and alter their pattern of hematopoietic growth factor production, suggesting a degree of functional plasticity. We examined the effect of two cytokines, interleukin-1 (IL-1) and IL-4, on stromal cell regulation of pre-B cell generation using the bone marrow stromal cell line, S17. Neither lymphokine potentiated pre-B cell generation in the absence of stromal cells. However, addition of either 10 U/mL rIL-1 alpha or 50 U/mL rIL-4 to cultures of bone marrow cells containing S17 cells dramatically suppressed subsequent pre-B cell formation. Preculture of S17 stromal cells with either rIL-1 or rIL-4 completely abrogated their ability to support pre-B cell generation in subsequent coculture with freshly explanted bone marrow cells. Conditioned medium from IL-1- or IL-4-treated S17 cells also suppressed pre-B-cell generation in culture. Although it is not yet known which induced stromal cell factors are responsible for failure of pre-B-cell generation in treated cultures, these data do clearly demonstrate that local levels of IL-1 and IL-4 in the hematopoietic microenvironment may play a significant role in regulation of bone marrow stromal cell function. These data also demonstrate that fibroblastic stromal cells are primary target cells that respond to cytokine concentration and affect lymphopoietic cell development.