Interference with immunoglobulin (Ig)alpha immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation modulates or blocks B cell development, depending on the availability of an Igbeta cytoplasmic tail.

To determine the function of immunoglobulin (Ig)alpha immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation, we generated mice in which Igalpha ITAM tyrosines were replaced by phenylalanines (Igalpha(FF/FF)). Igalpha(FF/FF) mice had a specific reduction of B1 and marginal zone B cells, whereas B2 cell development appeared to be normal, except that lambda1 light chain usage was increased. The mutants responded less efficiently to T cell-dependent antigens, whereas T cell-independent responses were unaffected. Upon B cell receptor ligation, the cells exhibited heightened calcium flux, weaker Lyn and Syk tyrosine phosphorylation, and phosphorylation of Igalpha non-ITAM tyrosines. Strikingly, when the Igalpha ITAM mutation was combined with a truncation of Igbeta, B cell development was completely blocked at the pro-B cell stage, indicating a crucial role of ITAM phosphorylation in B cell development.

B cell development is arrested at the immature B cell stage in mice carrying a mutation in the cytoplasmic domain of immunoglobulin beta.

The B cell receptor (BCR) regulates B cell development and function through immunoglobulin (Ig)alpha and Ig beta, a pair of membrane-bound Ig superfamily proteins, each of which contains a single cytoplasmic immunoreceptor tyrosine activation motif (ITAM). To determine the function of Ig beta, we produced mice that carry a deletion of the cytoplasmic domain of Ig beta (Ig beta Delta C mice) and compared them to mice that carry a similar mutation in Ig alpha (MB1 Delta C, herein referred to as Ig alpha Delta C mice). Ig beta Delta C mice differ from Ig alpha Delta C mice in that they show little impairment in early B cell development and they produce immature B cells that respond normally to BCR cross-linking as determined by Ca(2+) flux. However, Ig beta Delta C B cells are arrested at the immature stage of B cell development in the bone marrow and die by apoptosis. We conclude that the cytoplasmic domain Ig beta is required for B cell development beyond the immature B cell stage and that Ig alpha and Ig beta have distinct biologic activities in vivo.

Isolation of murine natural killer cells.

This unit describes the isolation of natural killer (NK) cells from mouse spleen. The basic protocol describes a method for preparing a highly purified NK cell population from mouse spleen by cytotoxic depletion of contaminating cells with selected monoclonal antibodies (MAbs), complement lysis, and density-gradient centrifugation to eliminate dead cells. The advantage of this negative selection process is that the NK cells are not coated with antibody and, therefore, are not at risk of activation by antibody cross-linking. Purity can then be assessed by cell surface phenotype.

OCA-B integrates B cell antigen receptor-, CD40L- and IL 4-mediated signals for the germinal center pathway of B cell development.

Many of the key decisions in lymphocyte differentiation and activation are dependent on integration of antigen receptor and co-receptor signals. Although there is significant understanding of these receptors and their signaling pathways, little is known about the molecular requirements for signal integration at the level of activation of gene expression. Here we show that in primary B cells, expression of the B-cell specific transcription coactivator OCA-B (also known as OBF-1 or Bob-1) is regulated synergistically by the B-cell antigen receptor, CD40L and interleukin signaling pathways. Consistent with the requirement for multiple T cell-dependent signals to induce OCA-B, we find that OCA-B protein is highly expressed in germinal center B cells. Accordingly, germinal center formation is blocked completely in the absence of OCA-B expression in B cells, whereas the helper functions of OCA-B-deficient T cells are indistinguishable from controls. The requirement for OCA-B expression in B cells is germinal center specific since the development of primary B cell follicles, the marginal zone and plasma cells are all intact. Thus, OCA-B is the first example of a transcriptional coactivator that is both synergistically induced by and required for integration of signals that mediate cell fate decisions.

Natural killer cell proliferation induced by anti-NK1.1 and IL-2.

NKR-P1 molecules are involved in natural killing of certain tumour targets. Indeed, the NK1.1 (NKR-P1C) molecule is the most specific serological marker on murine NK cells in C57BL/6 mice. Previous studies of NKR-P1 have indicated that anti-NKR-P1 mAb induced NK cells to kill otherwise insensitive targets, NK cell phosphoinositol turnover and Ca++ flux but it was not previously known if all NK cells were activated. In this study we report that immobilized anti-NK1.1 also specifically induced proliferation as measured by thymidine incorporation. The response required low doses of IL-2 for a synergistic effect. Cells stimulated with anti-NK1.1 + IL-2 displayed characteristic cytolytic activity against a NK-sensitive tumour target, YAC-1. However, anti-NK1.1-stimulated cells displayed delayed proliferation kinetics, heterogeneity of the expression of the very early antigen marker, CD69, and altered expression of the Ly-49 family members when compared to NK cells activated by high concentrations of IL-2. Taken together, these data demonstrate that immobilized anti-NK1.1 triggers only a subpopulation of NK cells.

Ku80 is required for immunoglobulin isotype switching.

Isotype switching is the DNA recombination mechanism by which antibody genes diversify immunoglobulin effector functions. In contrast to V(D)J recombination, which is mediated by RAG1, RAG2 and DNA double-stranded break (DSB) repair proteins, little is known about the mechanism of switching. We have investigated the role of DNA DSB repair in switch recombination in mice that are unable to repair DSBs due to a deficiency in Ku80 (Ku80(-/-)). B-cell development is arrested at the pro-B cell stage in Ku80(-/-) mice because of abnormalities in V(D)J recombination, and there are no mature B cells. To reconstitute the B-cell compartment in Ku80(-/-) mice, pre-rearranged VB1-8 DJH2 (mu i) and V3-83JK2 (kappa i) genes were introduced into the Ku80(-/-) background (Ku80(-/-)mu i/+kappa i/+). Ku80(-/-)mu i/+ kappai/+ mice develop mature mIgM+ B cells that respond normally to lipopolysaccharide (LPS) or LPS plus interleukin-4 (IL-4) by producing specific germline Ig constant region transcripts and by forming switch region-specific DSBs. However, Ku80(-/-)mu i/+kappa i/+ B cells are unable to produce immunoglobulins of secondary isotypes, and fail to complete switch recombination. Thus, Ku80 is essential for switch recombination in vivo, suggesting a significant overlap between the molecular machinery that mediates DNA DSB repair, V(D)J recombination and isotype switching.

TRAF2 is essential for JNK but not NF-kappaB activation and regulates lymphocyte proliferation and survival.

TRAF2 is believed to mediate the activation of NF-kappaB and JNK induced by the tumor necrosis factor receptor (TNFR) superfamily, which elicits pleiotropic responses in lymphocytes. We have investigated the physiological roles of TRAF2 in these processes by expressing a lymphocyte-specific dominant negative form of TRAF2, thereby blocking this protein's effector function. We find that the TNFR superfamily signals require TRAF2 for activation of JNK but not NF-kappaB. In addition, we show that TRAF2 induces NF-kappaB-independent antiapoptotic pathways during TNF-induced apoptosis. Inhibition of TRAF2 leads to splenomegaly, lymphadenopathy, and an increased number of B cells. These findings indicate that TRAF2 is involved in the regulation of lymphocyte function and growth in vivo.

Cellular mechanisms involved in protection and recovery from influenza virus infection in immunodeficient mice.

We investigated the role of different lymphocyte subpopulations in the host defense reaction against influenza virus infection, taking advantage of various immunodeficient mouse strains. Whereas, following immunization, wild-type animals showed complete protection against challenge with a lethal dose of A/PR8/34 (PR8) virus, mice that lack both B and T cells but not NK cells (namely, scid and RAG2(-/-) mice) did not display any protective effect in similar conditions. By contrast, J(H)D(-/-) mice devoid of B cells and immunized with virus showed a protective response after challenge with a lethal dose. The immunized J(H)D(-/-) mice that survived completely recovered from the influenza virus infection. Immunized J(H)D(-/+) mice exhibited a more complete protection, suggesting the role of specific antibodies in resistance to infection. To assess the role of natural immunity in the host defense against influenza virus, we carried out experiments with scid mice challenged with lower but still lethal doses of PR8 virus. While an increased NK activity and an increased number of NK1.1+ cells in lungs of scid mice infected with PR8 virus were noted, in vivo depletion of the NK1.1+ cells did not affect the overall survival of the mice. Our results show that specific T cells mediate protection and recovery of J(H)D(-/-) mice immunized with live virus and challenged with lethal doses of influenza virus.

Host MHC class I molecules modulate in vivo expression of a NK cell receptor.

Target cell expression of certain MHC class I molecules correlates with resistance to lysis by NK cells. To explain this correlation, one hypothesis states that NK cells may possess two types of receptors; one may activate NK cells whereas another, specific for target cell MHC class I molecules, may inhibit natural killing by transducing negative signals. The cell surface molecule, Ly-49, is expressed on an NK cell subpopulation (15% to 20%) in spleens from C57BL/6 (H-2b) mice. Previously, we showed that lysis by Ly-49+ IL-2-activated NK cells was globally inhibited when targets expressed either H-2Dd or an H-2k-class I molecule, consistent with the hypothesis that Ly-49 is an inhibitory NK cell receptor that engages these MHC class I molecules. We now have determined the influence of specific host MHC class I molecules on Ly-49 expression. In two-color flow cytometric examination of splenic cells, Ly-49+ NK1.1+ cells were undetectable in MHC-congenic strains expressing Dd or Dk, in C57BL/6 mice transgenic for membrane-bound Dd, and in B10.D2dm1 mice. These data establish that Dd itself is sufficient for this effect and suggest that Ly-49 engages Dd-alpha 1/alpha 2 domains. Cross-linking of Ly-49 with membrane-bound Dd may be required because Ly-49+ NK1.1+ cells were readily detectable in C57BL/6 strains transgenic for soluble forms of Dd. To examine whether this effect could be the result of down-regulation of Ly-49 expression or negative selection of Ly-49+ cells, we determined Ly-49 expression on highly purified, freshly isolated NK cell populations (> 90% NK1.1+ CD3- cells). These experiments demonstrated that Ly-49+ cells were present in normal numbers but that Ly-49 expression was markedly decreased in congenic mice expressing H-2Dd or Dk, and in the strain transgenic for membrane-bound H-2Dd. Thus, expression of a putative MHC class I-specific NK cell receptor is modulated by its apparent interaction with alpha 1/alpha 2 domains of host MHC class I molecules.