Lorraine Flaherty

Identification of a new CML target antigen controlled by a gene associated with theQa-2 locus

BALB/cBy anti-BALB/cJ spleen cells were tested in a secondary cellmediated lympholysis assay. The effector cells generated displayed a positive cytotoxic effect against Con A lymphoblasts from only those strains that were typed serologically as having theQa-2a allele. Confirmation that the target antigen is controlled by a locus closely associated with or identical toQa-2 was obtained by the findings that target cells from B6.K2 (Qa-2a,Qa-3a) mice were lysed by the effector cells, while those from theQa-2, 3 congenic strain B6.K1 (Qa-2b,Qa-3b) were not. The fact that target cells from aQa-2-positive/Qa-3-negative strain (DBA/1,Qa-2ai,Qa-3b) were killed indicates that the target antigen is controlled, at least in part, by theQa-2 locus, not the Qa-3.There is no observedH-2 genetic restriction for this cytotoxic effect, since target cells which have theQa-2a allele but differ from the stimulator cells at theH-2K, D, andI regions were lysed efficiently.

Histoincompatibilities Found Between Congenic Strains Which Differ At Loci Determining Differentiation Antigens

The study of mouse congenic strains, which differ at loci determining differentiation antigens, has led to the discovery of six previously unreported histocompatibility loci— H(Ly-1), H(Ly-2-N8), H(Ly-2-N16), H(Ly-2, Ly-3), H(Ea-2), and H(Tla). Each of these loci determines skin graft rejection and most have been shown to determine tumor graft rejection as well. Two of these loci, H(Ly-2-N16) and H(Ea-2), were detectable only after preimmunization procedures were employed. A synergistic interaction was found between two of the loci, where preimmunization with both an H(Ly-2-N8)- and H(Ly-2-N16)-incompatible graft was necessary in order for animals to respond to an H(Ly-2-N16) difference alone. H(Ly-1) and H(Tla) caused unusual rejection patterns. First skin grafts were often rejected where later ones were accepted. The H(Tla) locus should be very close to the H-2 complex of the mouse and is an example of a histocompatibility gene whose presence is not revealed by hemagglutination or cytotoxicity tests.

Contamination of Ia antiserum A.TL anti-A.TH with antibodies related to theT1a region

It has been shown previously that the T l a region encodes antigens expressed on LNCs, two of which we have provisionally designated Qa-1 (Stanton and Boyse 1976) and Qa-2 (Flaherty 1976). These are not the classically defined TL antigens which are expressed on thymocytes of some mouse strains and some T-cell leukemias of all strains (Boyse et al. 1965). Antibody to Qa-1 antigen was found as a contaminant of antisera used to define the classical TL antigens, leading us to question whether other antisera made in mouse strains differing in their T l a regions might also contain these or other e T l a region antibodies. Two antisera used to define Ia antigens are A. TL anti-A. T H (cda S) and A. T H anti-A. TL (cdak), because prior to the discovery of Qa-1 and Qa-2 it was believed that the only relevant difference between the A.TH and A.TL mouse strains was in the I region of the MHC (David et al. 1973). However, the T l a region of A.TL is derived from DBA/2 (TL.2) and that of A.TH from A (TL. 1, 2, 3) (Klein 1973, Frelinger et al. 1974). Thus we investigated the possibility that these antisera might contain c~Tla region antibodies against antigens expressed on LNCs. LNCs from mice aged five to six weeks were tested in cytotoxicity assays by either oneor two-stage tests. The antisera used were: ( B 6 x A T l a b) antiA S L 1 (c~TLi); A T l a b anti-A spleen and LNC (~TLii) (Komuro et al. 1973); A . T L a n t i A . T H spleen and LNC; A. T H an t i -A .TL spleen and LNC. Selected rabbit serum absorbed with mouse thymus, spleens, and LNCs in the presence of EDTA (Boyse et al. 1970) was used in the one-stage test at a dilution of 1:4 (final dilution 1:12) as a source of complement. For the two-stage reaction, the cells were first incubated with antiserum i~ the cold for 20 minutes, and then washed with medium 199 and resuspended in 100 lal of rabbit serum (diluted 1 : 14), not absorbed, but rigorously selected for high complement activity

Biochemical Demonstration. of Anti-Qa Activity in an H-2.28 Public Specificity Antiserum*

The Qa-2,3 region I codes for a molecule weighing approximately 45,000 daltons which is associated with f12 microglobulin (Michaelson et al. 1977). Although this molecule resembles D and K gene products of the H-2 region (Nathenson et al. 1974), the Qa-2,3 locus maps outside the limits of the H-2 region conventionally defined by H-2K and H-2D (Flaherty 1976). Since most of the 14-2 congenic strains that have been examined have the Qa-Tla haplotype of the donor H-2 region rather than that of the background strain (Frelinger et al. 1974), it is to be expected that some H-2 antisera may recognize products of the Qa region, just as they may include TL antibodies (Flaherty et aL 1977). One of us (L.F.) has pointed out that the strain distribution of the H-2.28 specificity when coded by the D end of H-2 corresponds closely with the Qa-2+,3 § phenotype (Flaherty et aL 1978), and thus some H-2.28 antisera may contain cr-Qa-2,3. One such antiserum, D28 (Table 1), was in fact shown in the cytotoxicity assay to identify Qa as well as H-2 antigens (Flaherty et al. 1978). Here we describe the demonstration of a-Qa activity in D28 antiserum by biochemical methods, as seen by the ability of this antiserum to precipitate Qa molecules from detergent-solubilized material. Cells from spleen or lymph nodes were prepared and radioiodinated as described previously (Michaelson et al. 1977) and then lysed in phosphate-buffered saline (PBS) containing 0.5% Nonidet P-40 (NP-40; Shell Corporation, New York). The lysates were clarified at 1200 g. Supernatants were then dialyzed at 4 ~ C against PBS. Lysates were depleted of B-cell Ig by treatment with rabbit a-mouse Ig serum and goat cr-rabbit Ig serum. Alloantigen was then precipitated with alloantiserum followed by goat c~-mouse Ig serum. Lysate so treated can be used again for