The shared tumor-specific antigen encoded by mouse gene P1A is a target not only for cytolytic T lymphocytes but also for tumor rejection.

A number of human tumor antigens have been characterized recently using cytolytic T lymphocytes (CTL) as screening tools. Some of them are encoded by MAGE-type genes, which are silent in normal tissues except in male germ cells, but are activated in a variety of tumors. These tumor-specific shared antigens appear to be promising targets for cancer immunotherapy. However, the choice of these antigens as targets has been questioned because of the lack of direct evidence that in vivo responses against such antigens can lead to tumor rejection. The antigen encoded by the mouse gene P1A represents the only available animal model system for MAGE-type tumor antigens. We show here that mice immunized by injection of L1210 leukemia cells expressing P1A and B7-1 (L1210.P1A.B7-1) are efficiently protected against a challenge with a lethal dose of mastocytoma P815 tumor cells, which express P1A. Mice immunized with L1210 cells expressing B7-1 but not P1A were not protected. Furthermore, we observed that P1A-transgenic mice, which are tolerant to P1A, were not protected after immunization with L1210.P1A.B7-1. These results demonstrate that the immune response to P1A is the major component of the tumor rejection response observed in normal mice, and support the use of tumor-specific shared antigens as targets for the immunotherapy of human cancer.

Contribution of donor-specific antibodies to acute allograft rejection: evidence from B cell-deficient mice.

BACKGROUND: The role of T lymphocytes in acute allograft rejection is well established. The involvement of B lymphocytes in this process, however, is more controversial. A series of reports showed that mice without a functional B-cell compartment rejected allografts with the same kinetics as control animals. In rats, however, alloantibodies were found to play a decisive role in allograft rejection. To provide an explanation for the discrepant results, we readdressed the role of B cells and antibodies in mice with disrupted immunoglobulin mu chain genes. The use of cyclosporine (CsA), which strongly suppresses T cells, allowed us to focus specifically on the function of B cells. METHODS: C57BL/6 mice rendered B cell deficient by targeted disruption of the immunoglobulin mu chain gene (referred to as microMT/microMT mice) and microMT/+ control mice with one functional mu chain were heterotopically transplanted with fully MHC-disparate BALB/c hearts. CsA was administered subcutaneously by Alzet osmotic pumps. Normal and immune serum specific for donor hearts was given to assess the role of antibodies in the rejection process. RESULTS: Both B cell-deficient microMT/microMT and heterozygous microMT/+ mice were found to reject transplanted hearts within a similar period of time. In contrast, when T cells were partially suppressed with CsA, graft survival was significantly prolonged in microMT/microMT mice as compared with heterozygous controls. Passive transfer of donor-specific immune serum, obtained from microMT/+ animals rejecting allogeneic hearts, to CsA-treated microMT/microMT mice significantly accelerated allograft rejection as opposed to recipients treated with normal serum. CONCLUSIONS: B lymphocytes and antibodies play an important role in acute allograft rejection particularly when the dominant T-cell compartment is partially suppressed.

A mutated HLA-A2 molecule recognized by autologous cytotoxic T lymphocytes on a human renal cell carcinoma.

Many human tumor cells have been shown to express antigens that are recognized by autologous cytotoxic T lymphocytes (CTL) and the molecular nature of a number of melanoma antigens has been defined recently. Here we describe the characterization of an antigen recognized on a renal cell carcinoma by autologous CTL clones. This antigen is encoded by the HLA-A2 gene present in the tumor cells. The sequence of this gene differs from the HLA-A2 sequence found in autologous peripheral blood lymphocytes by a point mutation that results in an arginine to isoleucine exchange at residue 170, which is located on the alpha-helix of the alpha 2 domain. Transfection experiments with the normal and mutated HLA-A2 cDNA demonstrated that this amino acid replacement was responsible for the recognition of the HLA-A2 molecule expressed on the tumor cells. The mutant HLA-A2 gene was also detected in the original tumor tissue from the patient, excluding the possibility that the mutation had appeared in vitro. Thus, HLA class I molecules carrying a tumor-specific mutation can be involved in the recognition of tumor cells by autologous CTL.

T cell development and repertoire of mice expressing a single T cell receptor alpha chain.

We examined T cell development and T cell repertoire in transgenic mice expressing a single T cell receptor (TCR) alpha chain derived from the H-2Db-lymphocytic choriomeningitis virus (LCMV)-specific cytolytic T lymphocyte (CTL) clone P14. To generate these alpha P14 mice, mice transgenic for the P14 TCR alpha chain were backcrossed to TCR alpha-deficient mice. Thymi from alpha P14 mice exhibited a marked decrease of mature CD4+8- and CD8+4- single-positive thymocytes comparable to thymi from TCR alpha-deficient mice. Correspondingly, the number of peripheral T cells was reduced in the CD4 (tenfold) and in the CD8 (twofold) subsets when compared to normal mice. T cells from alpha P14 mice generated a primary anti-LCMV CTL response when stimulated in vitro with LCMV in contrast to normal mice which require priming in vivo; elimination of LCMV in vivo was, however, not improved. Flow cytometric analysis of T cells with V beta-specific antibodies showed a diverse endogenous TCR V beta repertoire. Functional analysis of the T cell repertoire, however, revealed a strongly reduced (30-fold) allogeneic and the absence of a vesicular stomatitis virus-specific CTL response and an impaired ability to provide T cell help for antibody isotype switching. Thus, T cell selection in the thymus was impaired and the T cell repertoire was limited in mice expressing only one type of TCR alpha chain.

Regulation of RAG-1 and CD69 expression in the thymus during positive and negative selection.

Successful interaction of the T cell receptor (TCR) with major histocompatibility complex (MHC) molecules during thymic selection down-regulates the expression of the recombination activating genes (RAG)-1 and -2 in cortical thymocytes and thereby prevents further endogenous TCR alpha-chain gene rearrangements (Borgulya, P., Kishi, H., Uematsu, Y. and von Boehmer, H., Cell. 1992. 69: 529-537; Brändle, D., Müller, C., Rülicke, T., Hengartner, H. and Pircher, H., Proc. Natl. Acad. Sci. USA 1992. 89: 9529-9533). To address the question whether down-regulation of RAG-1 activity represents an irreversible process we have blocked TCR-MHC interactions of thymocytes with thymic stromal cells. Firstly, transgenic (Tg) mice expressing a virus-specific MHC class I (H-2Db)-restricted TCR were injected with anti-Db or anti-CD8 monoclonal antibodies and RAG-1 expression was examined by in situ hybridization on thymus sections. The results show that cortical thymocytes up-regulated RAG-1 expression within 24 h after antibody administration. Secondly, immature thymocytes from TCR Tg mice were released from the thymic microenvironment and cultured in vitro for 14 h in single-cell suspension. The amount of RAG-1 mRNA was increased sixfold in cultured cells when compared to freshly isolated thymocytes. In addition, we show that immature thymocytes from TCR transgenic mice bearing non-selective MHC molecules (H-2d) down-regulated RAG-1 expression after antigen-induced TCR engagement. Cytofluorometric analysis further revealed that surface expression of CD69 on immature thymocytes inversely correlated with RAG-1 expression during positive and negative selection processes.

Separation of thymic education from antigen presenting functions of major histocompatibility complex class I molecules.

Participation of transmembrane (TM) and glycosyl-phosphatidylinositol (GPI) anchored H-2Db molecules in antigen presentation and thymic selection events was investigated using transgenic mice. Both GPI-Db and TM-Db can efficiently present H-Y antigen, influenza and lymphocytic choriomeningitis virus (LCMV) peptides to primed cytotoxic, H-2Db-restricted T cells. Transgenic mice expressing GPI-Db, although unable to reject TM-Db skin grafts, nevertheless generate secondary CTL responses which can lyse TM-Db-bearing targets, indicating that GPI-Db mice fail to delete all TM-Db-reactive T cells. Furthermore, double-transgenic mice bearing GPI-Db and a T-cell receptor (TcR) for H-2Db+LCMV do not positively select receptor positive, CD8+CD4- T cells. This paradoxical behaviour of GPI-Db molecules suggests that the structural requirements for antigen presentation and thymic selection by class I molecules are different and may explain why GPI-linked class I molecules, such as Qa-2, do not appear to function as restriction elements in vivo.

Engagement of the T-cell receptor during positive selection in the thymus down-regulates RAG-1 expression.

We have examined the expression of the recombination activating gene RAG-1 by in situ hybridization to thymi from mice bearing transgenes for the T-cell receptor (TCR) alpha chain, TCR beta chain, or both TCR alpha and beta chains. RAG-1 transcription was found in the thymic cortex of transgenic mice carrying a single TCR alpha- or TCR beta-chain transgene, comparable to normal mice. However, RAG-1 transcription was strikingly reduced in the thymic cortex from transgenic mice carrying both TCR alpha- and beta-chain genes and expressing major histocompatibility complex (MHC) class I (H-2b) molecules necessary for positive selection of the transgenic TCR. In contrast, thymi of transgenic mice also carrying both TCR alpha- and beta-chain genes but expressing MHC molecules (H-2d) that did not positively select the transgenic TCR displayed high levels of RAG-1 transcription. The low thymic RAG-1 expression coincided with high transgenic TCR alpha-chain surface expression and with inhibition of endogenous TCR alpha-chain rearrangement. Our findings suggest that binding of the TCR to self MHC molecules during positive selection down-regulates RAG-1 transcription in cortical thymocytes and thereby prevents further TCR alpha-chain rearrangements.

Involvement of both T cell receptor V alpha and V beta variable region domains and alpha chain junctional region in viral antigen recognition.

We have studied the lymphocytic choriomeningitis virus (LCMV)-specific cytotoxic T cell response in transgenic mice expressing either the T cell receptor (TcR) alpha (V alpha 2/J alpha TA31) or the corresponding TcR beta (V beta 8.1/D beta/J beta 2.4) chain originally isolated from the LCMV glycoprotein specific (residues 32-42), H-2Db-restricted T cell clone P14. The expression of single transgenic TcR chains did not influence the corresponding endogenous TcR V gene usage in unstimulated T cells indicating that one particular TcR alpha or beta chain can randomly pair with different V beta or V alpha chains without any obvious bias. However, upon infection with LCMV, reactive cytotoxic T lymphocytes (CTL) from P14 beta-transgenic mice were predominantly V alpha 2+ whereas CTL from P14 alpha-transgenic mice preferentially expressed V beta 8.1 and unexpectedly also V beta 8.3 (but not V beta 8.2). Correspondingly, the LCMV-specific CTL response in both alpha and beta TcR-transgenic mice was strongly biased to the original P14 T cell epitope (LCMV glycoprotein residues 32-42). Sequence analysis of a large panel of LCMV-reactive "half-transgenic" TcR from P14 single receptor chain-transgenic mice revealed a highly conserved VJ alpha and a more diverse VDJ beta junctional region. This report demonstrates that the antigen specificity of the studied TcR depends on the specific combination of both TcR alpha and beta chains which implies that amino acids located in the TcR V alpha and V beta segments as well as in the junctional region are involved in binding of the viral antigenic fragment.