Jiyang Wang

A critical role for DNA end-joining proteins in both lymphogenesis and neurogenesis

XRCC4 was identified via a complementation cloning method that employed an ionizing radiation (IR)-sensitive hamster cell line. By gene-targeted mutation, we show that XRCC4 deficiency in primary murine cells causes growth defects, premature senescence, IR sensitivity, and inability to support V(D)J recombination. In mice, XRCC4 deficiency causes late embryonic lethality accompanied by defective lymphogenesis and defective neurogenesis manifested by extensive apoptotic death of newly generated postmitotic neuronal cells. We find similar neuronal developmental defects in embryos that lack DNA ligase IV, an XRCC4-associated protein. Our findings demonstrate that differentiating lymphocytes and neurons strictly require the XRCC4 and DNA ligase IV end-joining proteins and point to the general stage of neuronal development in which these proteins are necessary.

RAG2 :GFP KNOCKIN MICE REVEAL NOVEL ASPECTS OF RAG2 EXPRESSION IN PRIMARY AND PERIPHERAL LYMPHOID TISSUES

We generated mice in which a functional RAG2:GFP fusion gene is knocked in to the endogenous RAG2 locus. In bone marrow and thymus, RAG2:GFP expression occurs in appropriate stages of developing B and T cells as well as in immature bone marrow IgM+ B cells. RAG2:GFP also is expressed in IgD+ B cells following cross-linking of IgM on immature IgM+ IgD+ B cells generated in vitro. RAG2:GFP expression is undetectable in most immature splenic B cells; however, in young RAG2:GFP mice, there are substantial numbers of splenic RAG2:GFP+ cells that mostly resemble pre-B cells. The latter population decreases in size with age but reappears following immunization of older RAG2:GFP mice. We discuss the implications of these findings for current models of receptor assembly and diversification.

Resident bone marrow macrophages produce type 1 interferons that can selectively inhibit interleukin-7-driven growth of B lineage cells

Type 1 interferons alpha and beta are found to be potent inhibitors of IL-7-induced growth of early B lineage cells, while having no effect on cell growth induced by IL-2, IL-3, IL-4, or autogenous factors. The combination of IL-7 and interferons alpha/beta induces bcl-2 down-regulation and cell death by apoptosis. These conclusions were derived initially from experiments employing exogenous cytokines, but functional type 1 interferons are also shown to be produced by resident bone marrow macrophages. As physiological modulators of IL-7-driven proliferation and cell survival, interferons alpha/beta may cooperate with other homeostatic factors to maintain the balanced production of normal B lineage cells.

INTERFERONS ALPHA /BETA INHIBIT IL-7-INDUCED PROLIFERATION OF CD4- CD8- CD3- CD44+ CD25+ THYMOCYTES, BUT DO NOT INHIBIT THAT OF CD4- CD8- CD3- CD44- CD25- THYMOCYTES

Type 1 interferons (IFN‐α/β) have recently been shown to inhibit interleukin‐7 (IL‐7)‐induced growth and survival of early B‐lineage cells. The CD3− CD4− CD8− (triple negative; TN) thymocytes from normal mice strongly proliferated upon stimulation with IL‐7 in suspension culture. Such an IL‐7‐induced proliferation was suppressed by the addition of IFN‐α/β, but a fraction of the TN thymocytes still showed proliferation. The IL‐7‐induced growth of TN thymocytes from scid mice, which lack the CD44− CD25− subpopulation, was completely inhibited by the addition of IFN‐α/β. The IL‐7 induced proliferation of CD4− CD8− thymocytes from T‐cell receptor (TCR) transgenic mice, the majority of which are CD3+ CD44− CD25−, was resistant to IFN‐α/β‐mediated suppression. In fetal thymus organ cultures (FTOC), the addition of IL‐7 greatly increased the population of CD4− CD8− CD44+ CD25+ thymocytes and IFN‐α/β inhibited this IL‐7‐driven expansion. In contrast, the addition of IL‐7 markedly decreased the percentages of CD4− CD8− CD3− CD44− CD25− cells, and IFN‐α/β reversed the effect and increased the subpopulations of CD44− CD25+ and CD44− CD25.− Finally, IFN‐β mRNA was found to be expressed in the thymus. The data suggest that type 1 interferons inhibit IL‐7‐driven proliferation of TN thymocytes, but do not block the normal differentiation process.

Expression and Function of Fas during Differentiation and Activation of B Cells

Fas (Apo-1, CD95) cell surface antigen belongs to the tumor necrosis factor receptor family and mediates apoptosis of a variety of cell types, including lymphocytes, after ligation with Fas ligand (FasL). Recent studies on the role of Fas/FasL interaction in the immune responses strongly suggest the relevance of dysregulation in Fas-mediated apoptosis as a cause of autoimmune disorders. While Fas is not an essential molecule in the elimination or functional inactivation (anergy) of autoreactive B cells, it is indispensable to the maintenance of peripheral tolerance and prevention of autoimmunity. Studies in the past few years have begun to reveal the mechanism by which susceptibility to Fas-mediated apoptosis in B cells is regulated to allow antigen-specific B cells survive and differentiate and to eliminate nonspecifically activated, potentially selfreactive B cells.

Appearance of the LAT protein at an early stage of B-cell development and its possible role

The linker protein LAT is expressed mainly in T and natural killer (NK) cells. LAT‐deficient mice have an arrest of intrathymic T‐cell development at the CD4+ CD8+ stage and lack mature T cells in the periphery. However, no gross abnormality in development and function of the B and NK cells has been described. Here we report that LAT is expressed in mouse progenitor B (pro‐B) and precursor B (pre‐B) cells, but not in immature or mature B cells. LAT in pre‐B cells becomes tyrosine phosphorylated upon cross‐linking of the pre‐B‐cell receptor (pre‐BCR) by anti‐µ antibody. Incubation of 1xN/2b (mouse pre‐B‐cell line) cells or bone marrow cells from µMT/µMT mice, which lack B cells after the small pre‐B‐cell stage, with anti‐Igβ antibody resulted in the downregulation of LAT expression. Transgenic mice which expressed LAT protein in B‐lineage cells showed an increased proportion of pro‐ and large pre‐B cells in the bone marrow and a remarkable reduction in the numbers of mature B cells in peripheral lymphoid tissues. Collectively, the present results indicate that LAT is expressed in the cells at the early stages of B‐lineage development, but is absent in immature and mature B cells. LAT may play a crucial role in the negative regulation of B‐cell development at the transition from pre‐B to mature B‐cell stages, and signal(s) via the pre‐BCR may extinguish LAT expression, thus allowing pre‐B‐cell differentiation towards the mature B‐cell stage.

Regulation of bcl-xL expression and Fas susceptibility in mouse B cells by CD40 ligation, surface IgM crosslinking and IL-4.

CD40 is one of the key molecules involved in the survival, growth and differentiation of B lymphocytes. In contrast, Fas (Apo-1, CD95) mediates apoptosis of a variety of cell types, including lymphocytes. Recent studies have found that Fas expression on mouse B cells could be strongly induced by CD40 ligation, a helper T cell-derived signal. Here, evidence is provided that CD40 ligation induced two distinct signals: one leading to the upregulation of Fas and the other leading to the enhanced Fas susceptibility. B lymphoma cell lines, CH31 and WEHI279, expressed Fas on cell surfaces, but were resistant to anti-Fas antibody (Ab) induced apoptosis. Treatment with CD40 ligand (CD40L), however, greatly enhanced Fas susceptibility of these cells. Similarly, normal splenic B cells became highly susceptible to Fas-mediated apoptosis following prolonged signaling through CD40. While CD40 ligation enhanced Fas-mediated apoptosis, stimulation with anti-IgM and IL-4 partially protected CD40L-activated B cells from Fas-mediated apoptosis. It was found that bcl-xL gene expression in normal splenic B cells was induced drastically by treatment with anti-IgM and IL-4, but not CD40L. By contrast, the expression of bcl-2 or bax was not significantly affected by these treatments. Moreover, in three of the four B lymphoma cell lines tested, Fas susceptibility correlated with the status of bcl-xL expression. The data suggest that an increase in bcl-xL expression may protect B cells from Fas-mediated apoptosis.

Purification of a nuclear trans-acting factor involved in the regulated transcription of a human immunoglobulin heavy chain gene

The expression of the rearranged human immunoglobulin gamma 1 heavy chain gene (HIG1) was shown to be induced through its enhancer by the positive regulatory trans-acting factor(s) that was contained only in cells of B lineage. The trans-acting factors were purified from mouse myeloma NS1 cells, and HIG1-inducing activity was found mainly in fractions of molecular weight 53-127 kd and in a fraction eluted from a heparin-Sepharose column with 0.5 M KCI. This semipurified fraction contained proteins binding to the conserved octamer sequence, ATGCAAAT, in the promoter region, as well as to sequences in the enhancer region. The 0.5 M KCI eluates from a heparin-Sepharose column were applied to a DNA affinity column of synthetic oligonucleotides of the octamer sequence and the sequence TATTTTAGGAAGCAAA in the HpaII-BgIII region of the HIG1 gene enhancer. The protein eluted from the enhancer sequence-specific DNA affinity column showed a strong inducing activity for the HIG1 gene, and the molecular weight of a predominant protein was 96 kd.

The enigmatic role of glutamyl aminopeptidase (BP‐1/6C3 antigen) in immune system development

Summary: The murine B‐lymphocyte differentiation antigen BP‐1 /6C3, a homodimeric integral membrane protein composed of M, 140,000 sub‐units, has been identified as glutamyl aminopeptidase (EAR EC 3,4. 11.7). This ecto‐enzyme cleaves acidic amino acid residues from the amino terminal of polypeptide substrates such as angiotensin II and cholecystoki‐nin‐8. Although BP‐1/6C3/EAP is expressed by cells in many tissues, among hematopoietic cell lineages this ecto‐enzyme is restricted to immature B‐lineage cells where its expression is upregulated by interleukin‐7 and viral transformation, BP‐1/6C3/EAP thus serves as a valuable marker of progression along the B‐cell differentiation pathway, but a corresponding biological role has not yet been established.

Proliferation of CD3+ B220− single‐positive normal T cells was suppressed in B‐cell‐deficient lpr mice

It is known that lpr mice develop systemic lymphadenopathy and lupus erythematosus‐like autoimmune disease that are associated with the accumulation of CD4− CD8− (double‐negative; DN) CD3+ B220+ abnormal T cells as well as normal mature CD4+ or CD8+ single‐positive (SP) CD3+ T cells. In order to clarify the role of B cells in the lymphoproliferation and autoimmunity of lpr mice, we created B‐cell‐deficient C57BL/6 (B6) lpr mice (B6lpr/lprμMT/μMT) by crossing B6lpr/lpr mice with B6μMT/μMT mice in which the B‐cell development was arrested at pre‐B stage owing to a targeted disruption of the immunoglobulin μ heavy‐chain gene locus. In the B‐cell‐deficient B6‐lpr mice, both lymphadenopathy and splenomegaly were markedly suppressed. Although the accumulation of both CD3+ B220− SP normal T cells and CD3+ B220+ DN abnormal T cells was inhibited in the B‐cell‐deficient lpr mice, the decrease in numbers of CD3+ B220− SP normal T cells occurred more strikingly than that of the CD3+ B220+ DN abnormal T cells. Glomerulonephritis did not develop in the B‐cell‐deficient lpr mice over 40 weeks. The present results indicate that the B cells thus play a crucial role in the extensive proliferation of normal CD3+ B220− mature SP T cells rather than the accumulation of abnormal DN T cells.