Inhibition of antigen-induced proliferation ofT cells from radiation- induced bone marrow chimeras

Chimeric BIO.A T cells that had matured in a (BlO.A x B10.Q)F1 environment acquired the ability to respond to poly(Glu'Lys35Phe9) (GL4), an antigen to which the BlO.A mouse is a nonresponder. The response of the chimeric BlO.A T cells was initiated by.GL4 on responder BlO.Q antigen-presenting cells (APC) but not by GL4W on nonresponder BlO.A APC. Similarly, chimeric BlO.Q T cells that had matured in a (BlO.A x BlO.Q)F, environment acquired the ability to respond to poly(Glu'Ala'Tyr'0) (GAT) when the antigen was presented on responder BIO.A APC, but not when GAT was presented on nonresponder BlO.Q APC. No syngeneic haplotype preference was observed for either antigen. These interactions between H-2 nonidentical T cells and APC were inhibited by anti-H-2 antisera and a monoclonal anti-Ia antibody directed against the APC but not by such antibodies when they were directed against the T cell. These data suggest that, when they develop in a responder chimeric environment, genotypic nonresponder T cells become responders by acquiring receptors that allow them to recognize responder I region products on the surface of APC. Furthermore, the data demonstrate that the site of action of the blocking effects of the anti-Ia antibodies is the APC, thus providing strong evidence in support of the idea that Ia antigens on APC are the Ir gene products. One approach to understanding the mechanism of immune response (Ir) gene function has been to determine the cell(s) in which the genes are expressed. Experiments with T cells from F1 (responder x nonresponder) guinea pigs (1) and mice (2), in general, have shown that Ir gene-controlled responses can be initiated by antigen-pulsed responder antigen-presenting cells (APC) but not by antigen-pulsedsnonresponder APC. These findings suggested that one cell type that must express responder Ir gene products is the APC. Furthermore, the interaction between F1 T cell and responder APC could be inhibited by anti-Ia antisera specific for the Ia molecules encoded in the same I subregion in which the Ir gene for the antigen mapped (3,4). Experiments with radiation-induced bone marrow chimeras in general have confirmed the idea that the APC must be of responder genotype to generate Ir gene-controlled responses (5-8). In addition, such chimeras have allowed the analysis of the requirements for Ir gene expression at the T-cell level in the absence of allogeneic effects. Chimeric nonresponder T cells which have matured in a responder environment become phenotypic responders, suggesting that the nonresponder T cell is not defective simply because it does not possess the responder Jr gene. In-an effort to learn more about the nature ofthe phenotypic alteration ofnonresponder T cells in the responder environment, we made chimeras of the type B10.A + B10.Q (B10.A x BIO.Q)Fl. B10.A mice are responders to poly(Glu6OAlaTyrl0) (GAT) and nonresponders to poly(Glu'Lys35 Phe9) (GLO). The B1O.Q mice are responders to GLOW and nonresponders to GAT. B10.A + B1O.Q -(BIO.A X BLO.Q)F1 chimeras were primed with GLUT and GAT, and the repertoire of each donor type T-cell population was assayed by killing the other donor type cells with anti-H-2 antisera and complement and adding back to the culture irradiated APC of the killed donor type. Such studies revealed that the chimeric nonresponder T cell becomes a phenotypic responder by learning to recognize responder major histocompatibility complex (MHC) products on the APC. This interaction could be inhibited by anti-Ia antisera specific for the APC, including a monoclonal antibody, but not by anti-Ia antisera specific for the T cell. These experiments provide another argument in support of the idea that the Ia antigens on APC are products of the Ir gene. MATERIALS AND METHODS Animals. B1O.Q and (B10.A X B10.Q)F1 animals were bred in our own animal colony from stocks originally supplied by J. H. Stimpfling (McLaughlin Research Institute, Great Falls, MT). B10.A mice were purchased from The Jackson Laboratory (Bar Harbor, ME). B1O.T(6R) mice were bred from stocks originally supplied by David Sachs (Transplantation Biology Section, Immunology Branch, National Cancer Institute, Bethesda, MD). Chimeras. Radiation-induced bone marrow chimeras were made as described (7). Briefly, (B1O.A X B10.Q)F1 mice were given 925-975 R (0.24-25 C/kg) from a heavily filtered x-ray source and reconstituted on the same day with 107 T cell-depleted bone marrow cells from one or both parents. T cell-depleted bone marrow was obtained by treating donor mice with anti-thymocyte antiserum and cortisone in vivo and rabbit antimouse brain plus guinea pig complement in vitro. The chimeras were used no sooner than 3 months after irradiation. Spleen cells from individual chimeras were H-2 typed before use and all were found to be entirely ofdonor origin. All B10.A + B10.Q -* (B10.A X B1O.Q)Fl chimeras were found to be balanced mixtures ofboth donors (33-67%). Antigens and Immunization. GL46 (originally purchased from Miles-Yeda, Rehovot, Israel) was the generous gift ofAlan Rosenthal (Merck, Rahway, NJ). GAT (lot 6) was purchased from Miles-Yeda. Both antigens were emulsified in complete Freund's adjuvant containing 1 mg of Mycobacterium tuberculosis strain H37Ra (Difco) per ml and administered to mice in Abbreviations: GL4b, poly(Glu56Lys35Phe9); GAT, poly(Glu"OAWaTyr'l); APC, antigen-presenting cells; Ir gene, immune response gene; PPD, purified-protein derivative ofMycobacterium tuberculosis; MHC, major histocompatibility complex. 514 The publication costs ofthis articlewere defrayed in part bypage charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 78 (1981) 515 both hind footpads and the base of the tail at a total dose of 30 ,ug per mouse. Both antigens were used in culture at a final concentration of 100 ug/ml. Purified-protein derivative of Mycobacterium tuberculosis (PPD) (Connaught Medical Research Laboratory, Willowdale, ON) was used in culture at 20 ,ug/ml. When single parent-* FI chimeras were immunized, 108 T celldepleted F. bone marrow and spleen cells were given intravenously as a source of the other parental APC. These cells were killed with appropriate antisera and complement before assay. Antisera. (A/J x BI0.A)F1 anti-B10.Q (anti-Q) and (A.TH x B1O.Q)Fl anti-B10.A (anti-A) antisera were made by repeated intraperitoneal injection ofthe F1 mice with 107 B10.Q or B10.A spleen cells (9). The anti-Q antiserum, which has specificity for the entire H-2 region ofthe q haplotype, had a plateau cytotoxic titer for B10.Q lymph node and spleen cells of 1:512. The antiA antiserum, which has specificity for the K, I, and S (but not D) regions of the a haplotype, had a plateau cytotoxic titer for B10.A lymph node and spleen cells of 1:512. Both antisera specifically inhibited T-cell proliferative responses of the strain against which they were reactive at a concentration in culture of 1% (vol/vol). The anti-Q antiserum had no effect on B10.A Tcell proliferation at 1% concentration, and the anti-A antiserum had no effect on B10.Q T-cell proliferation at 1% concentration. A.TH anti-A.TL (no.2081 from David Sachs), which has specificity for Ik and Sk, inhibited B10.A T-cell proliferation at a concentration of1% and had no effect on B10.QT-cell proliferation, presumably reflecting an absence of significant reactivity against Ia.3, a determinant shared by B10.A and B1O.Q. This result was also confirmed by cytotoxicity testing on B10.Q spleen cells. A monoclonal antibody with specificity for Ia. 17 was produced from cells of the hybridoma designated 10-2.16 (10) which had been passaged in vitro in modified Eagle's medium supplemented with 10% fetal calf serum after being obtained from the Salk Institute (La Jolla, CA). The cell culture supernatant possessing a plateau cytotoxic titer of 1:256 against B10.A spleen cells was the generous gift of Richard Hodes (Immunology Branch, National Cancer Institute). T-Cell Proliferation and Blocking Studies. B10.A + B10.Q (B10.A x B10.Q)F, chimeras that had been immunized 8 days previously were sacrificed by cervical dislocation and their popliteal, inguinal, and para-aortic lymph nodes were harvested. Lymph node T cells eluted from nylon wool columns (11) were treated with either anti-A or anti-Q antiserum plus rabbit complement in a two-stage cytotoxicity procedure. Those cells remaining after treatment with anti-Q plus complement were designated "chimeric B1O.A" cells and those remaining after treatment with anti-A plus complement were designated "chimeric B1O.Q" cells. Chimeric lymph node cells (4 x 105) were plated in 96-well flat-bottomed microtiter plates along with soluble antigen and 108 irradiated (2000 R) spleen cells from either parent. Each culture was pulsed with 1 ,Ci (1 Ci = 3.7 X 1O0 becquerels) of[3H]thymidine (6.7 Ci/mmol; New England Nuclear) on day 4, and incorporation was measured 18 hr later. The cpm data are expressed as arithmetic mean