Ly-6I, a new member of the murine Ly-6 superfamily with a distinct pattern of expression.

A new member of the mouse Ly-6SF, designated Ly-6I, has been isolated as a gene homologous to a segment of the Ly-6C gene. A single allelic difference in the mature protein sequence was identified, which is similar to other Ly-6SF members. Ly-6I mRNA has been detected in a wide range of tissues and cell lines, and a rabbit polyclonal Ab has been used to determine that Ly-6I protein is present at a low constitutive level on cell lines from several different lineages. In contrast to Ly-6C and Ly-6A/E, the Ly-6I gene is only weakly responsive to IFNs. Expression in vivo is most abundant on bone marrow populations and is coexpressed with Ly-6C on granulocytes and macrophages. However, Ly-6I is also expressed on immature B cell populations that do not express Ly-6C. Expression on mature B cells in spleen is uniformly low. Similarly, Ly-6I is expressed on TCRlow/int, but not TCRhigh, thymocytes. Ly-6I is re-expressed on Ly-6Chigh T cells in the periphery. Thus, Ly-6I may be a useful marker to define maturation stages of both T and B lymphocytes as well as subsets of monocytes and granulocytes.

Rejection of an MHC class II negative tumor following induction of murine syngeneic graft-versus-host disease.

Cyclosporin A (CsA) has been used clinically to induce graft-versus-host disease following autologous bone marrow transplantation in an attempt to destroy residual leukemia cells and reduce relapse. To analyze the antitumor potential of murine syngeneic graft-versus-host disease (SGVHD), C3H/HeN mice were lethally irradiated, reconstituted with T cell-depleted syngeneic bone marrow (ATBM) and treated with CsA for 21 days. Graft-versus-leukemia activity was assessed by challenging groups of olive oil-treated control ATBM (OO-ATBM) and CsA-treated (CsA-ATBM) mice 1 week after CsA therapy with graded doses of the syngeneic 38C13 B cell lymphoma. Following CsA treatment, up to 70% of CsA-ATBM developed SGVHD and more than 70% of the animals injected with 500 38C13 cells exhibited long-term survival (MST >80 days). In contrast, none of the OO-ATBM control mice developed SGVHD, and more than 75% of these mice died following injection of 500 38C13 tumor cells (MST = 34 days). Long-term survivors were not resistant to tumor challenge suggesting that tumor-specific immunity did not develop. Finally, class II negative 38C13 cells cultured in IL-4 or IL-10 were not inducible for MHC class II molecules, demonstrating that class II-independent antitumor mechanisms exist in SGVHD mice.

Structure/function characterization of porcine CD59: expression, chromosomal mapping, complement-inhibition, and costimulatory activity.

BACKGROUND: Complement regulatory proteins have become important targets to potentially modulate inflammatory reactions or transplant rejection. Since pig into human xenotransplantation could potentially overcome the enormous shortage of donor organs and tissues, characterization of porcine complement regulatory proteins is critical. METHODS: The porcine CD59 cDNA has been isolated from porcine aortic endothelial cells and its structure determined. In addition, a molecular genetic analysis of the gene and its transcriptional properties and a functional analysis have been performed utilizing the transfected cDNA. RESULTS: The most prominent mRNA species is 1.8 kilobases but cloned reverse transcriptase polymerase chain reaction products suggest that multiple polyadenylation sites are utilized. Gene mapping was performed utilizing a polymorphism identified in the 3' UT, and the gene was localized to within 3 cM of follicle-stimulating hormone, beta polypeptide in the middle of the chromosome 2 linkage map. RNA expression was equivalent in endothelial, kidney, and testis cell lines. Comparisons have been made with CD59 sequences from other species to identify possible important domains of the protein. The cDNA has been utilized to express an epitope-tagged or wild-type protein either transiently on COS-7 cells or stably in Chinese hamster ovary cells. The porcine CD59 protein effectively inhibited the antibody-mediated lytic activity of both porcine and human complement. In contrast to human CD59, porcine CD59 is incapable of providing costimulation to human T cells. CONCLUSIONS: These data suggest that overexpression of porcine CD59 might be more effective than human CD59 in prolonging xenograft survival with transgenic pig organs because of reduced immunoreactivity.

In vivo reactivity of T cell clones isolated from mice with syngeneic graft-versus-host disease.

Syngeneic graft-versus-host disease (SGVHD) has been shown to occur in murine syngeneic radiation bone marrow chimeras following a short course of cyclosporine. To analyze the effector mechanisms present in diseased animals, four T cell clones (1D5, 1D8, 1C10, 2D8) were isolated from the spleens of C3H/HeN mice late in the disease course by cloning on irradiated syngeneic spleen cells. These clones were CD4+, alpha beta TCR+ and responded to I-Kk in vitro. In addition to I-Ek reactivity, three of the clones exhibited crossreactivity with the superantigen MIs 1a (mtv 7). Clones 1D5 and 1C10 were found to express TCR V beta chains (V beta 4 and V beta 8.1, respectively), which are normally present in the T cell repertoire of C3H/HeN mice. All SGVHD clones were found to be autoreactive in that they responded to syngeneic stimulator cells in the absence of xenogeneic serum proteins. To test in vivo activity, the 1D5 SGVHD clone was injected into the hind footpad of mice where it was shown to induce footpad swelling in a cell dose-dependent, I-Ek-specific manner in sublethally irradiated, but not normal mice. Histological analysis indicated that the clone induced dermal and subcutaneous edema that correlated directly with injection of 1D5 and not the control clone. Preliminary experiments suggested that the other three autoreactive clones behaved in a similar manner. These data are consistent with the involvement of a self-class II-specific CD4+ T cell in murine SGVHD.