Dendritic cells lose ability to present protein antigen after stimulating antigen-specific T cell responses, despite upregulation of MHC class II expression.

Immature dendritic cells (DC) take up, process and present protein antigens; mature DC are specialized for stimulating primary T cell responses with increased expression of MHC class II and co-stimulatory molecules, but are incapable of processing and presenting soluble protein. The current study examined whether maturation of DC is triggered by T cell recognition of antigens presented by immature DC. Human DC derived from CD34+ progenitor cells by culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-6 (IL-6) in serum-free medium could prime naive CD4+ T cells to keyhole limpet hemocyanin (KLH) and ovalbumin (OVA). The cultured DC retained the ability to prime T cells to native protein for at least 15 days. To test for changes in DC function after participation in an immune response, DC were co-cultured with either allogeneic or autologous CD4+ T cells. DC co-cultured with autologous T cells retained the ability to prime T cells to intact protein antigens. By contrast, DC which had previously stimulated an allogeneic T cell response lost ability to prime T cells to soluble proteins. However, such <> induced a MLR and stimulated peptide-specific primary CD4+ T cell responses. This indicated that <> did not die or lose the ability to prime, but lost the ability to process and present subsequent antigens. Following participation in T cell activation, DC increased surface expression of MHC class II, co-stimulatory molecules CD40 and B7.2, and the intercellular adhesion molecule-1 (ICAM-1). In addition, our data suggest that interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) are involved in this T cell-mediated DC maturation.

Granulocyte-macrophage colony-stimulating factor: an effective adjuvant for protein and peptide-based vaccines.

The current studies evaluate granulocyte-macrophage colony-stimulating factor (GM-CSF) as a vaccine adjuvant. An important issue for developing vaccine therapy for human malignancy is identifying adjuvants that can elicit T-cell responses to proteins and peptides derived from "self" tumor antigens. GM-CSF, in vitro, stimulates the growth of antigen-presenting cells such as dendritic cells and macrophages. Initial experiments examined whether GM-CSF injected into the skin of rats could affect the number or character of antigen presenting cells, measured as class II major histocompatability complex expressing cells, in lymph nodes draining the injection site. Intradermal (id) inoculation of GM-CSF every 24 hours for a total of five inoculations resulted in an increase of class II+ fluorescing cells that peaked at the fourth inoculation. Subcutaneous (sq) inoculation resulted in an increase of class II+ fluorescing cells that peaked following the second inoculation, then decreased over time. Using this schema for "conditioning" the inoculation site, GM-CSF was administered id or sq for five injections and a foreign antigen, tetanus toxoid (tt), was given at the beginning or the end of the immunization cycle. Id immunization was more effective than sq at eliciting tt specific immunity. In addition, GM-CSF id, administered as a single dose with antigen, compared favorably with complete Freund's adjuvant (CFA) and alum in eliciting tt specific antibody and cellular immunity. We have shown that immunity to rat neu (c-erbB-2) protein, an oncogenic self protein, can be generated in rats by immunization with peptides derived from the normal rat neu sequence plus CFA. The current study demonstrates that rat neu peptides inoculated with GM-CSF could elicit a strong delayed type hypersensitivity reaction (DTH) response, whereas peptides alone were non-immunogenic. GM-CSF was as effective as CFA in generating rat neu specific DTH responses after immunization with a neu peptide based vaccine. Soluble GM-CSF is a potent adjuvant for the generation of immune responses to foreign proteins as well as peptides derived from a self tumor antigen.

Peptide-based, but not whole protein, vaccines elicit immunity to HER-2/neu, oncogenic self-protein.

HER-2/neu, an overexpressed oncogenic protein, has been proposed as a human cancer vaccine target. HER-2/neu is a "self" protein, however, and methods of vaccine strategies that would be effective in immunizing patients to a "self" tumor Ag have not been established. Many of the tumor Ags defined in humans are nonmutated self proteins, e.g., MAGE, and overcoming tolerance may be key in the generation of effective anti-tumor immunity. One theory states that tolerance to self proteins is directed only to dominant epitopes of proteins and not to every portion of the protein. Accordingly, tolerance can be circumvented by immunization to peptide fragments, but not whole protein. The studies outlined here demonstrate rat neu-specific immunity could be elicited in rats by vaccination with immunogenic rat neu peptides, but not by immunization with the intact protein. A rat model was used since rat neu protein is 89% homologous to human HER-2/neu protein and has a similar tissue distribution and level of expression. Rats were immunized with groups of peptides derived from the amino acid sequence of the intracellular domain or extracellular domain of rat neu protein and both groups developed CD4+ T cell immunity and Ab immunity to rat neu peptides and protein. Animals immunized in a similar fashion with intact purified rat neu protein did not develop Ab or T cell immunity to rat neu. Furthermore, rats that developed neu-specific immunity showed no histopathologic evidence of autoimmunity directed against organs expressing basal levels of rat neu protein. These studies suggest an immunization strategy that might be effective in human cancer vaccines targeting self tumor Ag.

T cells receptor beta-chain repertoires are nonrandomly selected in responses to HLA-DR1.

T cell receptor (TCR) beta-chain usage by HLA-DR1 alloreactive T cell lines was examined to determine whether common TCR gene segments were used preferentially. We have demonstrated previously that a DR1-specific, human renal allograft-derived T cell line and replicate, anti-DR1 mixed lymphocyte reactions (MLR), established from an unrelated responder/stimulator pair, were selected for T cells expressing V beta 8. In this report V beta 8+ beta-chains from these T cell lines were sequenced to assess clonality and determine the contribution made by the beta-chain junctional regions. All 11 V beta 8+ cDNA clones sequenced from the allograft-derived T cell lines used J beta 2.7 and C beta 2 and had identical junctions, indicating the presence of a predominant V beta 8+ clone. All seven V beta 8+ sequences from the first anti-DR1 MLR and eight of the nine fron the second also used J beta 2.7 and C beta 2 were identical to one another, indicating that a common V beta 8+ clone was selected in these replicate cultures. The sequences of the predominant V beta 8+ beta-chains from the allograft-derived T-cell line and the MLR differed by only 10 nucleotides and four amino acids at the VDJ beta junction. To determine the reproducibility of TCR V beta selection in responses to DR1, additional MLR were established by pairing three different DR1+ stimulators with the same responder. The TCR repertoires of the resulting DR1-specific cell lines were examined. A preference was seen for utilization for certain homologous TCR V beta segments. The data suggest that particular TCR V beta or V beta/J beta combinations may be selected in alloresponses as evidenced either utilization of highly similar beta-chains or homologous V beta segments.

Immunity to the HER-2/neu oncogenic protein.

The study of oncogenic viruses led to the discovery that transforming retroviruses contain oncogenes homologous with and/or derived from cellular proto-oncogenes. In humans malignant transformation is often the result of the activation of proto-oncogenes. Normal proto-oncogenes can be activated to transforming proto-oncogenes by a variety of mechanisms including point mutation, translocation and amplification. Development of successful strategies for the immunotherapy of human cancers is an area of intense investigation. Part of the problem in developing cancer-specific immunotherapy has been the lack of well-defined tumour antigens. Our laboratory has focused on the question of whether oncogenic proteins expressed by transforming proto-oncogenes can serve as targets for immune attack. Some patients with HER-2/Neu-positive breast cancer have an existent immune response to the HER-2/neu protein with no clinical signs of autoimmunity, supporting the idea that overexpressed oncogenic proteins can be targeted in therapy without fear of destructive autoimmunity. The identification of candidate cytotoxic T lymphocyte epitopes might allow the generation of tumour-specific cytotoxic T lymphocytes for use in therapy and identify potential epitopes for peptide vaccines.

Variables affecting the T cell receptor V beta repertoire heterogeneity of T cells infiltrating human renal allografts.

Donor-specific, alloreactive T cell lines may be grown from cells infiltrating human renal allografts. These T cell lines utilize restricted T cell receptor (TCR) beta-chain variable (V beta) gene repertoires, although long-term culture appears to be necessary for restriction to be observed. This study was undertaken to determine the effects of potential selective pressures on the TCR repertoires of allograft-infiltrating cells. TCR V beta repertoires of 30 allograft-derived T cell populations, cultured for defined, short time periods, were examined using polymerase chain reaction. When first derived, V beta repertoires of graft-infiltrating T cells were as heterogeneous as those of peripheral blood lymphocytes (PBL). There was no relationship between the length of time an allograft was in situ or the extent of HLA mismatch and repertoire heterogeneity. Repertoire restriction was positively correlated with the length of time cells were cultured in vitro. Long-term, alloreactive mixed lymphocyte reactions (MLR), established from normal, unsensitized PBL, also demonstrated V beta repertoire restriction during expansion in vitro. Restricted alloreactive populations emerged much more slowly from the MLR than from the allograft-derived cultures, however, implying that graft infiltrates contain previously activated populations of T cells. This observation, taken together with the fact that long-term, graft-derived cell lines maintain donor specificity, suggests that functional subsets must be allowed to emerge from heterogeneous infiltrates before TCR repertoire may be correlated with alloreactivity and/or graft rejection.

T cell receptor V beta gene usage in HLA-DR1-reactive human T cell populations. The predominance of V beta 8.

The functional specificity and T cell receptor (TCR) V beta gene expression of three class II HLA-DR1-reactive human T cell populations were examined. WP2, a renal allograft-derived, long-term CD4+ T cell line, was specifically cytotoxic for DR1, one of the mismatched antigens present on the allograft. Initial studies of WP2 using six TCR V beta-specific mAb revealed a predominance of T cells expressing a member of the V beta 8 gene family. A smaller, yet significant, number of cells expressed TCR using V beta 5.1. Semiquantitative V beta-specific polymerase chain reaction (PCR) analyses of RNA derived from this T cell line confirmed the presence of V beta 8 and V beta 5.1 messages. The PCR signal for V beta 8 was the strongest, followed by those for V beta 4 and V beta 5. An earlier WP2 culture had a very similar PCR profile, with a dominant signal for V beta 8, although signals for V beta 4 and V beta 5 were considerably lower. Previous PCR analyses of eight other renal allograft-derived, long-term T cell lines, grown under identical in vitro conditions, revealed no other example of predominant usage of V beta 8. We established two replicate long-term, anti-DR1 mixed lymphocyte reactions using PBL from two unrelated normal donors as responder and stimulator. The MLR were given alloantigen every 10 days, and RNA was obtained from the cultured cells immediately prior to each stimulation. PCR analyses of RNA taken at 10-day intervals over a total of 60-70 days indicated that, although the MLR were initially quite heterogeneous with regard to V beta message expression, by the end of the fourth or fifth Ag cycle the predominant PCR signals observed in both MLR were for V beta 8. These results suggest that T cells using V beta 8 gene-encoded segments as part of their TCR may have a selective advantage in responses to DR1.

T-cell receptor gene usage and expression in renal allograft-derived T-cell lines.

Southern blot analyses indicate that the T-cell receptors of alloreactive T-cell lines derived from needle biopsies of human kidney allografts are selected based on beta-chain usage. In order to examine this selection at the level of T-cell-receptor expression, we have generated monoclonal antibodies directed toward the T-cell-receptors of three allograft-derived T-cell lines, MH3, WP3, and EH3. Monoclonal antibodies have been isolated which appear to react specifically with each of these three T-cell lines. One anti-MH3 antibody precipitates a molecule from the surface of MH3 cells that comigrates with the alpha/beta TcR on a polyacrylamide gel. Ten WP3-reactive monoclonal antibodies were identified which cause a modulation of CD3 from the surface of WP3 T cells, although none as yet precipitates a molecule from lysates of surface 125I-labeled WP3 cells. Since Northern blot analysis of EH3 RNA has revealed that a member of the V beta 6 gene family is expressed by this T-cell line, we are attempting to identify a monoclonal antibody reactive with this V beta 6 gene product.