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Ly9 is a mouse cell membrane antigen found on all lymphocytes and coded for by a gene that maps to chromosome 1. We previously described the isolation and characterization of a full-length cDNA clone for mouse Ly9. Using cross-species hybridization we isolated cDNA clones encoding the human homologue Humly9. Analysis of the predicted protein sequence suggests that the extra-cellular portion of the Humly9 molecules is composed of four Ig-like domains: a V domain (V) without disulphide bonds and a truncated C2 domain (tC2) with two disulphide bonds, a second V domain without disulphide bonds and a second tC2 with two disulphide bonds, i.e., as V-tC2-V-tC2. The gene encoding Humly9 was mapped to chromosome 1 by analysis of human/hamster hybrids, and more specifically to the 1q22 region by in situ hybridization. The protein sequence data support the view that Humly9 belongs to the immunoglobulin-superfamily subgroup which includes CD48, CD2, and LFA-3.

We describe the production and characterization of a mouse mAb, S-450-33.2, recognizing the Ly-9.2 specificity. This mAb was used to purify Ly-9 molecules from lymphoid cell lines, and the amino-terminal amino acids were determined. The mAb was also used in a eukaryotic expression system, to isolate cDNA clones encoding Ly-9. Analysis of RNA showed that Ly-9 expression is lymphocyte specific, as determined by the presence of a single hybridizing 2.4-kb species found only in lymphoid cells. Genomic DNA analysis indicated that Ly-9 is encoded by a single-copy gene of 10 to 15 kb. The predicted polypeptide belongs to the Ig superfamily of cell surface molecules with four extracellular Ig-like domains, i.e., a non-disulfide-bonded V domain, a truncated C2 domain with two disulfide bonds, a second non-disulfide-bonded V domain, and a truncated C2 domain with two disulfide bonds (V-C2-V-C2). The sequence data also support the view that Ly-9 belongs to the subgroup of the Ig superfamily that includes Bcm-1, CD2, and LFA-3.

HuLy-m3 is an Mr 47,000 pan-leukocyte antigen detected by the monoclonal antibody (mAb) 5-4.8. This report describes the isolation and analysis of a cDNA clone encoding HuLy-m3. Serological analysis demonstrated that antibodies of the CD48 cluster also reacted with transfected cells expressing HuLy-m3. The DNA sequence of the clone suggests linkage to the cell membrane through a glycosyl phosphatidylinositol tail and this was verified experimentally. Sequence similarity with the human B-cell activation antigen Blast-1 was noted.

Nude (athymic) or anti-lymphocyte serum-treated mice have absent delayed graft rejection due to impaired T-cell responses. Nonetheless, these mice can reject skin grafts, acutely, when treated with anti-H-2 antibody and additional complement. Resolution of the components in the H-2 antisera, by either absorption or by selective production of antisera of restricted specificity has demonstrated that anti-H-2K or H-2D antiser are graft destructive, and as shown elsewhere, are nonenhancing. By contrast, anti-Ia sera are not capable of causing allograft destruction even when used in extremely high doses and were previously noted to be enhancing. The mechanism of such differential effects is not apparent, but the findings are clearly of importance to transplantation in man, where sera reacting specifically with B cells may also be enhancing and nondestructive.

The alloantigenic phenotype of 21 radiation-induced thymomas is described. Monoclonal antibodies and conventional antisera, absorbed to remove contaminating antibodies, were used to test the thymomas directly for the presence of H-2, Ia, Ly-1, Ly-2, Ly-4, Ly-5, Ly-6, Ly-9, Ly-15, Thy-1, TL, and Qa-2 antigens, and for surface immunoglobulin. The phenotypes obtained by direct tests were also confirmed by absorption studies. The tumors were all of T-cell origin (Thy-1+Ig-) and showed a restricted heterogeneity of their cell surface phenotype, concordant with the known alloantigenic phenotypes of functional T-cells. Thus the thymomas could be classified into 7 groups based on the differing expressions of Ia, Ly-1, Ly-2, Ly-4, and Ly-6 specificities; all tumors were H-2+, Ly-5+, Ly-9+, and Ly-15+. Thus a wide variety of phenotypically diverse tumors could be detected; if these represent the clonal expansion of different functional subsets, they could provide a valuable set of functional T-cells.

Enhancement of skin grafts was previously shown to be possible when there were antigenic differences arising from the @K end" but not the @D end" of the H-2 complex. Subsequent studies demonstrated that anti-Ia antibodies in the anti-K end sera were responsible for graft prolongation and not the anti-H-2K antibodies and that D end antisera were not enhancing as they lacked Ia antibody activity. The studies reported herein have extended these findings by two separate approaches. First, by using appropriate congenic and recombinant strains, enhancement by D end antigenic differences could be observed, provided that Ia antibodies were also present. In this way, H-2D and H-2K alloantigens are similar and both require additional I region differences to enable skin graft enhancement to be observed. Second, H-2Kb, H-2Dd, and H-2Ld mutants were examined and putative antisera used to attempt to prolong graft survival in these unique coisogenic strains. In no case was graft survival significantly prolonged, even in the presence of demonstrable antibodies. Therefore, in two different approaches, wherein single gene products were examined (derived by mutation and by recombination), enhancement of skin allografts could not be observed, unless additional Ia alloantigenic differences were also present.

Antigen stimulation in mice such as occurs with the rejection of an allogeneic tumor graft caused a substantial rise in serum glycolipid Ia levels. However, mice bearing a measurable syngeneic tumor had no detectable Ia antigens in their sera; this observation was made in several different strains of inbred mice with 5 different tumors. In each instance the serum Ia level fell as the tumor grew progressively, reached zero at about the time the tumor was visible, and remained at this zero level until the mouse died. Similar results were found in humans: Tumor-bearing patients had markedly suppressed serum Ia levels. The mechanism of the fall in serum Ia glycolipid levels is not known, but the measurement of the serum Ia glycolipid content appears to reflect the level of activation of the immune system, and the suppression of serum glycolipid Ia levels found in tumor-bearing mice and patients may have important clinical implications.

Based on graft rejection in C57BL/6 and B10.A(4R), but not in B10.A mice, skin graft rejection and delayed-type hypersensitivity (DTH) responses to the male HY antigen were considered to be under the control of the "IBb" gene in the mouse H-2 complex. These two phenomena were re-examined in the B6.C-H-2bm12 mutant strain [mutation in the A beta gene in IA leading to an alteration in Iab serologically detected specificities and the inability to generate cytotoxic T (Tc) cells to H-Y]. In this study the bm12 mutant was shown to produce weak DTH responses to H-Y. By contrast, bm12 female mice were unable to reject male skin grafts unless they had received prior footpad priming of male spleen cells, when graft rejection occurred, albeit slowly. In C57BL/6 mice the response to the HY antigen therefore appears to be solely under the control of the IAb gene. In other strains, response/nonresponse is presumably dictated by the ability of IA/IE interactions to produce T-helper responses.