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Related post: break-through with the development of a new method for cloning antigen specific, proliferating T lymphocytes directly from primed murine lymph nodes. The T lymphocytes are stimulated in vitro with antigen for three 22-74 ZOl-AI-00147-05 LI days and then cloned in soft agar in the presence of antigen. After 5 days the colonies are picked with the aid of a stereomicroscope and the cells ex- panded in liquid culture using antigen, irradiated spleen cells and a growth factor derived from Concanavalin A stimulated splenocytes. Recloning in the absence of this factor was performed to ensure clonal ity Generic Compazine of the cells. The clonal precursor frequency was low (1 to 2 x 10" ) and showed the expected dependence on Ir gene status i.e. cells from low responder strains yielded fewer colonies than cells from high responder or F, strains. All the clones were antigen specific and in particular clones derived from DNP-OVA immunized mice were carrier specific, i.e., they could be stimulated with the ovalbumin (OVA)carrier alone but Purchase Compazine not with the dinitrophenyl hapten (DNP) on another carrier such as DNP-GLA. However, the clone was capable of detecting the hapten Buy Compazine since a 3-fold higher concentration of OVA was required to give an equivalent stimulation to that of DNP-OVA. In addition to their specific antigen, the cells also required that the irradiated spleens be histo- compatible at the I-A subregion in order to stimulate T cells to proliferate. The fact that a clone of T cells was I-A restricted in its antigen recognition demonstrated for the first time that suppressor T-cell function cannot account for the phenomenon of major histocompatibility complex (MHC) restriction in cell interactions. In deciphering the mechanism of action of Ir gene products, it was deemed necessary to investigate the types of antigenic determinants Buy Compazine Online recognized by T lymphocytes to ascertain whether the repertoire of receptors on these cells was any different from that of B lymphocytes. To facilitate such experiments we turned to the small globular proteins as antigens, because they provided us with probes for which we possessed a thorough understand- ing of the amino acid sequence and a precise knowledge of the three- dimensional structure. Thus, we could localize the antigenic determinants by Order Compazine Online cross-stimulation with species variants and enzymatic or chemical clea- vage fragments of the molecules. For the last few years we have focused on the T-lymphocyte proliferative response of BIO. A and B10.S(9R) mice to pigeon cytochrome c^. This response appears to be directed at a single determinant comprised of three amino acid residues from different parts of the molecule: the isoleucine at position 3, the glutamine at position 100 and the lysine at position 104. These three residues lie Purchase Compazine Online next to each other on the outer surface of the back Order Compazine face of the molecule. Although these results suggest that T cells recognize three dimensional determinants, cyanogen bromide cleavage fragments of the molecule could stimulate the T cells primed to the whole molecule. In fact, one fragment, residues 81-104, containing only two of the three Compazine Online critical amino acids and lacking any of the a helical content this segment possesses in the native structure, stimulated better than the whole molecule on a molar basis. Furthermore, this fragment was immunogenic and the response was under the control of two MHC-1 inked Ir genes, just as was the response to the whole protein. These results have raised the possibility that T-cell receptors recognize anigen fragments on the surface of macrophages rather than intact molecules. Mixing experiments demonstrated that many of the responding T cells were recognizing the two critical amino acids found in the Cheap Compazine fragment as part of the same determinant. This result demonstrated for the first time that two Ir genes could control the immune response to a single antigenic determinant. Finally, antigen- 22-75 ZOl-AI-00147-05 LI presentation experiments showed that both Ir gene products must be expressed in the same presenting cell in order to generate an immune response to pigeon cytochrome c^. Significance to Biomedical Research Our attempts to understand the mechanism of T-lymphocyte activation are of fundamental importance to a basic understanding of how the immune system functions. Since this system plays a critical role in the bodies defense mechanisms against infectious diseases, tumors and transplanted tissues, achieving our goal would hopefully provide major insights into how to manipulate the immune system for the benefit of the patient. PUBLICATIONS : Solinger, A.M., Ultee, M.E., Margoliash, E., and Schwartz, R.H.: T-lymphocyte response to cytochrome c_. I. Demonstration of a T-cell heteroclitic pro- liferative response and identification of a topographic antigenic determinant on pigeon cytochrome c^ whose immune recognition requires two complementing major histocompatibility complex-linked immune response genes. J. Exp. Med . 150: 838-848, 1979. Ultee, M.E., Margoliash, E., Lipkowski, A., Flouret, G., Solinger, A.M., Lebwohl, D. , Matis, L.A., Chen, C. and Schwartz, R.H.: The T-lymphocyte response to cytochrome c^. II. Molecular characterization of a pigeon cyto- chrome c^ determinant recognized by proliferating T lymphocytes of the BIO. A mouse. Molecular Immunol . In press. Schwartz, R.H.: Presentation of Soluble Protein and Synthetic Antigens. In Manual of Macrophage Methodology : Collection, Characterization and Function . H.B. Herscowitz, H.T. Holden, J. A. Bellanti, and A. Ghaffar (Eds.). Marcel Dekker, Inc., New York. pp. 454-470. 1980. Sredni, B. , Tse, H.Y., and Schwartz, R.H.: Direct cloning and extended culture of antigen-specific MHC-restricted, proliferating T lymphocytes. Nature 283: 581-583, 1980. Tse, H.Y., Schwartz, R.H., and Paul, W.E. Cell-cell interactions in the T cell proliferative response. I. Analysis of the cell types involved and evidence for nonspecific T cell recruitment. J. Immunol . In press. Schwartz, R.H., Kim, K.J., Asofsky, R. and Paul, W.E.: Antigen-presenting lymphoid tumors in the murinerT lymphocyte proliferative response to DNP- ovalbumin and poly (Glu Lys Buy Cheap Compazine Phe ). In Macrophage Regulation of Immunity . E.R. Unanue and A.S. Rosenthal (Eds.) Academic Press, New York. pp. 277-284, 1980.
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