Susan E. Stepp

2B4 Acts As a Non-Major Histocompatibility Complex Binding Inhibitory Receptor on Mouse Natural Killer Cells

Natural killer (NK) cells are critical in the immune response to tumor cells, virally infected cells, and bone marrow allografts. 2B4 (CD244) is expressed on all NK cells and the ligand for 2B4, CD48, is expressed on hematopoietic cells. Cross-linking 2B4 on NK cells with anti-2B4 monoclonal antibody leads to NK cell activation in vitro. Therefore, 2B4 is considered to be an activating receptor. Surprisingly, we have found, using antibody-blocking and 2B4-deficient NK cells, that NK lysis of CD48+ tumor and allogeneic targets is inhibited by 2B4 ligation. Interferon γ production by NK cells is also inhibited. Using a peritoneal tumor clearance assay, it was found that 2B4−/− mice have increased clearance of CD48+ tumor cells in vivo. Retroviral transduction of 2B4 was sufficient to restore inhibition in 2B4−/− primary NK cells. It was found that although mature NK cells express SH2D1A, in vitro–derived NK cells do not. However, both populations are inhibited by 2B4 ligation. This indicates that 2B4 inhibitory signaling occurs regardless of the presence of SH2D1A. These findings reveal a novel role for 2B4 as a non–major histocompatibility complex binding negative regulator of NK cells.

2B4 (CD244) and CS1: novel members of the CD2 subset of the immunoglobulin superfamily molecules expressed on natural killer cells and other leukocytes

Summary: 2B4 is a member of the CD2 subset of the immunoglobulin superfamily molecules expressed on natural killer (NK) cells and other leukocytes. It is the high affinity ligand for CD48. Engagement of 2B4 on NK‐cell surfaces with specific antibodies or CD48 can trigger cell‐mediated cytotoxicity, interferon‐γ secretion, phosphoinositol turnover and NK‐cell invasiveness. The function of 2B4 in CD8+ T cells and myeloid cells remains unknown. The cytoplasmic domain of 2B4 contains unique tyrosine motifs (TxYxxV/I) that associate with src homology 2 domain‐containing protein or signaling lymphocyte activation molecule (SLAM)‐associated protein, whose mutation is the underlying genetic defect in the X‐linked lymphoproliferative disease (XLPD). Impaired signaling via 2B4 and SLAM is implicated in the immunopathogenesis of XLPD. CS1 is a novel member of the CD2 subset that contains two of the unique tyrosine motifs present in 2B4 and SLAM. Signaling through 2B4, CS1 and other members of the CD2 subset may play a major role in the regulation of NK cells and other leukocyte functions.

Perforin: more than just an effector molecule

Abstract Perforin mediates the destruction of virus-infected or transformed cells. However, the finding that perforin deficiency is the underlying cause of familial hemophagocytic lymphohistiocytosis indicates that it also regulates the immune response.

Virus-specific CD8 T cells in peripheral tissues are more resistant to apoptosis than those in lymphoid organs.

CD8 T cells persist at high frequencies in peripheral organs after resolution of an immune response, and their presence in the periphery is important for resistance to secondary challenge. We show here that LCMV-specific T cells in peripheral tissue (peritoneal cavity, lung, fat pads) reacted much less with the apoptotic marker Annexin-V than those in spleen and lymph nodes. This was not due to a TCR-based selection. In comparison to lymphoid tissue, T cells in the periphery expressed lower levels of Fas and Fas ligand and were resistant to activation-induced cell death in vitro. This may contribute to the survival of nondividing peripheral memory T cells, enabling them to efficiently function without being driven into apoptosis.

Gene structure of the murine NK cell receptor 2B4: presence of two alternatively spliced isoforms with distinct cytoplasmic domains

The NK cell receptor 2B4 is expressed on the surface of all murine NK cells and a subset of T cells. Ligation of 2B4 with monoclonal antibodies increases target cell lysis and IFN‐γ production. 2B4 is the high‐affinity counter‐receptor for CD48 in mice and humans. 2B4‐L is a member of the CD2 subgroup of the immunoglobulin supergene family, which includes CD48, LFA‐3, CD84, Ly9 and SLAM. Here we describe 2B4‐S, a second 2B4 isoform, and the genomic structure of the 2B4 gene. 2B4‐S is identical to the 5′ end of 2B4‐L, differing only at the 3′ end, corresponding to a portion of the cytoplasmic domain and the 3′ untranslated sequence. Both 2B4‐L and 2B4‐S are expressed on IL‐2‐activated NK cells. The genomic clone of 2B4 reveals that the two cDNA clones are products of alternative splicing. Since they differ only in a portion of the cytoplasmic domain, it is likely that they transduce different signals.

Dynamics of Ly49 expressing cytotoxic lymphocyte subsets in response to virus infection

Viral infections induce first a loss and then an increase in natural killer (NK) and CD8(+) T cells. NK cells expressing Ly49G2 were selectively expanded by several viruses and poly I:C. CD8(+) T cells expressing Ly49G2 were selectively expanded by poly I:C and participated in the antigen-specific response to lymphocytic choriomeningitis virus.

Molecular characterization of the rat NK cell receptor 2B4

2B4 (CD244) is a cell surface glycoprotein of the immunoglobulin superfamily involved in the regulation of natural killer and T lymphocyte function. It is the high affinity counter-receptor for CD48. In mouse and human NK cells, crosslinking of 2B4 with a specific monoclonal antibody or with CD48 can trigger cell-mediated cytotoxicity, IFN-gamma secretion, phosphoinositol turnover and NK cell invasiveness. Recent reports of defective 2B4 signaling and NK cell function in X-linked lymphoproliferative syndrome suggest that this may contribute to the progression of this human disease. Here we describe the molecular characterization of the rat 2B4 gene. The cDNA encodes a protein of 395 amino acid residues that contain two Ig domains in the extracellular region and three unique tyrosine motifs (TxYxxV/I/A) in the cytoplasmic region. The predicted protein has 81 and 68% similarity with mouse 2B4 and human 2B4, respectively. Additionally, it has 94 and 89% similarity at the protein level with the recently reported rat 2B4 related genes, r2B4R-tm and r2B4R-se respectively. Northern blot analysis indicated the presence of multiple transcripts in rat LAK cells and RNK-16 cells. Immunoprecipitation and deglycosylation studies showed that rat 2B4 is glycosylated to similar extent as that of mouse and human 2B4. The cloning of r2B4 in the light of the availability of rat NK cell lines should facilitate in vitro and in vivo experiments to decipher the functional role of 2B4 in NK cell biology.

Tumor viral escape from inhibited T cells

Functionally active inhibitory receptors that impart negative signals have been found recently on T cells. One such inhibitory receptor, CD94-NKG2A, can induce cytolytic anergy in CTLs specific for polyomavirus.

B cell memory: sapping the T cell.

Efficiently defending a host from repeat invasions by the same pathogen requires a lasting B-cell response. The identification of a molecule that keeps B cells prepared may help to explain some immune disorders and lead to improved vaccines.