A novel approach to identify antigens recognized by CD4 T cells using complement-opsonized bacteria expressing a cDNA library.

In patients with hematological malignancies receiving HLA-matched stem cell transplantation, T cells specific for minor histocompatibility antigens play a major role in graft rejection, induction of graft-versus-host disease and beneficial graft-versus-leukemia reactivity. Several human minor histocompatibility antigens recognized by T cells have been identified, but only two are presented by HLA class II molecules. In search of an efficient approach to identify antigenic peptides processed through the HLA class II pathway, we constructed a cDNA library in bacteria that were induced to express proteins. Bacteria were opsonized with complement to enforce receptor-mediated uptake by Epstein-Barr virus immortalized B cells that were subsequently used as antigen-presenting cells. This approach was validated with an HLA class II-restricted antigen encoded by gene DBY. We were able to identify bacteria expressing DBY diluted into a 300-fold excess of bacteria expressing a nonrelevant gene. Screening of a bacterial library using a DBY-specific CD4 T cell clone resulted in the isolation of several DBY cDNAs. We propose this strategy for a rapid identification of HLA class II-restricted antigenic peptides recognized by CD4 T cells.

A MAGE-1 peptide recognized on HLA-DR15 by CD4(+) T cells.

Antigens encoded by MAGE genes and recognized by T cells are of interest for cancer immunotherapy because of their strict tumoral specificity and because they are shared by many tumors. Several MAGE-1 peptide that are recognized by CD8(+) cytolytic T lymphocytes have been used in therapeutic vaccination trials. To obtain anti-tumor immune response, vaccines combining peptides recognized by CD8(+) and peptides recognized by CD4(+) T cells might be optimal. We focused therefore on the identification of MAGE peptides recognized by CD4(+) T cells. We report here the identification of MAGE-1 epitope EYVIKVSARVRF, which is presented to CD4(+) T lymphocytes by HLA-DR15. This HLA allele is present in 29 % of Asians and 17 % of Caucasians.

A MAGE-A3 peptide presented by HLA-DP4 is recognized on tumor cells by CD4+ cytolytic T lymphocytes.

Antigens encoded by MAGE-A3 and recognized by T cells are interesting targets for tumor immunotherapy because they are strictly tumor specific and shared by many tumors of various histological types. A number of MAGE-A3 antigenic peptides presented by HLA class I molecules have been used in clinical trials, and regressions of melanoma metastasis have been observed. We report here the identification of a MAGE-A3 epitope, TQHFVQENYLEY, presented to CD4+ T lymphocytes by HLA-DP4 molecules, which are expressed in approximately 76% of Caucasians. This new epitope may be useful both for therapeutic vaccination and for the evaluation of the immune response in cancer patients. Interest ingly, the CD4+ T cells lysed HLA-DP4 tumor cells expressing MAGE-A3, indicating that this epitope, in contrast to other class-II MAGE-A3 epitopes, is presented at the surface of tumor cells. The study of this disparity in the presentation of two epitopes from the same protein may lead to a better understanding of the endogenous class II presentation pathway.

A MAGE-A4 peptide presented by HLA-A2 is recognized by cytolytic T lymphocytes.

The MAGE-encoded antigens that are recognized by cytolytic T lymphocytes (CTL) are shared by many tumors and are strictly tumor specific. Clinical trials involving therapeutic vaccination of cancer patients with MAGE antigenic peptides or proteins are in progress. To increase the range of patients eligible for therapy with peptides, it is important to identify additional MAGE epitopes. We have used a method to identify CTL epitopes, which selects naturally processed peptides. CD8(+) T cells, obtained from individuals without cancer, were stimulated with autologous dendritic cells infected with a recombinant adenovirus containing the MAGE-A4 coding sequence. Responder cell microcultures that specifically lysed autologous EBV-transformed B cells infected with vaccinia-MAGE-A4 were cloned using autologous stimulator cells infected with a Yersinia enterocolitica carrying the MAGE-A4 sequence. An anti-MAGE-A4 CTL clone was obtained and the epitope was found to be decapeptide GVYDGREHTV (amino acids 230-239) presented by HLA-A2 molecules. The CTL clone lysed HLA-A2 tumor cells expressing MAGE-A4. This is the first reported antigenic peptide encoded by MAGE-A4. It may be valuable for cancer immunotherapy because MAGE-A4 is expressed in 51% of lung carcinomas and 63% of esophageal carcinomas, whereas about 50% of Caucasians and Asians express HLA-A2.

Identification of five MAGE-A1 epitopes recognized by cytolytic T lymphocytes obtained by in vitro stimulation with dendritic cells transduced with MAGE-A1.

MAGE genes are expressed by many human tumors of different histological types but not by normal cells, except for male germline cells. The Ags encoded by MAGE genes and recognized by T cells are therefore strictly tumor-specific. Clinical trials involving therapeutic vaccination of cancer patients with MAGE antigenic peptides or proteins are in progress. To increase the range of patients eligible for therapy with peptides, it is important to identify additional MAGE epitopes recognized by CTL. Candidate peptides known to bind to a given HLA have been used to stimulate T lymphocytes in vitro. In some instances, CTL clones directed against these synthetic peptides have been obtained, but these clones often failed to recognize tumor cells expressing the relevant gene. Therefore, we designed a method to identify CTL epitopes that selects naturally processed peptides. Monocyte-derived dendritic cells infected with a recombinant canarypoxvirus (ALVAC) containing the entire MAGE-A1 gene were used to stimulate CD8+ T lymphocytes from the blood of individuals without cancer. Responder cell microcultures that specifically lysed autologous cells expressing MAGE-A1 were cloned using autologous stimulator cells either transduced with a retrovirus coding for MAGE-A1 or infected with recombinant Yersinia-MAGE-A1 bacteria. The CTL clones were tested for their ability to lyse autologous cells loaded with each of a set of overlapping MAGE-A1 peptides. This strategy led to the identification of five new MAGE-A1 epitopes recognized by CTL clones on HLA-A3, -A28, -B53, -Cw2, and -Cw3 molecules. All of these CTL clones recognized target cells expressing gene MAGE-A1.

Identification of MAGE-3 epitopes presented by HLA-DR molecules to CD4(+) T lymphocytes.

MAGE-type genes are expressed by many tumors of different histological types and not by normal cells, except for male germline cells, which do not express major histocompatibility complex (MHC) molecules. Therefore, the antigens encoded by MAGE-type genes are strictly tumor specific and common to many tumors. We describe here the identification of the first MAGE-encoded epitopes presented by histocompatibility leukocyte antigen (HLA) class II molecules to CD4(+) T lymphocytes. Monocyte-derived dendritic cells were loaded with a MAGE-3 recombinant protein and used to stimulate autologous CD4(+) T cells. We isolated CD4(+) T cell clones that recognized two different MAGE-3 epitopes, MAGE-3114-127 and MAGE-3121-134, both presented by the HLA-DR13 molecule, which is expressed in 20% of Caucasians. The second epitope is also encoded by MAGE-1, -2, and -6. Our procedure should be applicable to other proteins for the identification of new tumor-specific antigens presented by HLA class II molecules. The knowledge of such antigens will be useful for evaluation of the immune response of cancer patients immunized with proteins or with recombinant viruses carrying entire genes coding for tumor antigens. The use of antigenic peptides presented by class II in addition to peptides presented by class I may also improve the efficacy of therapeutic antitumor vaccination.

Estimation of the frequencies of anti-MAGE-3 cytolytic T-lymphocyte precursors in blood from individuals without cancer.

Attempts to detect a cytolytic T-lymphocyte (CTL) response in melanoma patients vaccinated with MAGE-3 peptides have been negative so far, even though some tumor regressions have been observed. The detection of such responses may require very sensitive detection assays for CTL precursors. To this end, we set up a method whereby a large number of CD8+ T-cell microcultures are stimulated with autologous antigen-presenting cells incubated with a peptide, in the presence of interleukin (IL)-6 and IL-12 during the first week, and IL-2 and IL-7 from the second week. We report here that not only monocyte-derived dendritic cells but also activated T cells incubated with the MAGE-3 antigenic peptide presented by HLA-A2 were effective in activating specific CTL precursors present in the blood of individuals without cancer. These precursors were detected in the CD8+ CD45RA+ subpopulation of T cells. Among the CD8+ T-lymphocyte population of blood donors, the frequency of CTL precursors specific for the MAGE-3.A2 antigen ranged from 4 to 17 x 10(-7). For the MAGE-3 antigenic peptide presented by HLA-A1, this frequency ranged from 0.4 to 3 x 10(-7). Knowing that several parameters of this procedure still have to be optimized, we will begin to use it to evaluate the CTL precursor frequencies of cancer patients before and after injection of MAGE peptides.

Tumor-infiltrating dendritic cells are defective in their antigen-presenting function and inducible B7 expression in rats.

Tumors are tolerated by the immune system notwithstanding the expression of tumor-associated antigens. PROb tumor cells, derived from a rat colon carcinoma, are rejected by tumor-immune hosts but give rise to progressive tumors in naive hosts. Paradoxically, these tumors are heavily infiltrated by dendritic cells that express MHC class II and ICAM-1. These tumor-infiltrating dendritic cells (TiDCs) could be expected to process and present to T cells the antigens released by the adjacent tumor cells. Indeed, we report here that TiDCs, compared with splenic dendritic cells, are poor stimulators of primary allogeneic T-cell proliferation and cytokine [interleukin-2 (IL-2) and interferon-gamma] production. Most of them (89-97%) do not express B7, an essential co-stimulatory signal for T cells, even after a culture period allowing B7 up-regulation on epidermal Langerhans cells. GM-CSF in association with tumor necrosis factor-alpha or IL-4, or cell-associated CD40-ligand, all known to be potent stimulators of B7 expression on other dendritic cells, did not restore B7 expression by TiDCs. After a first exposure to TiDCs, allogeneic T-cell response to a second challenge to splenic dendritic cells was decreased. The failure of most dendritic cells infiltrating PROb tumors to express B7, even after stimulation, may contribute to their poor capacity to stimulate T cells and could play a role in the immune tolerance allowing tumor growth.

Inflammatory cells infiltrating human colorectal carcinomas express HLA class II but not B7-1 and B7-2 costimulatory molecules of the T-cell activation.

Colon cancer cells express potentially immunogenic proteins but are not rejected by the immune system. To induce an effective immune response, antigenic peptides have to be presented to T lymphocytes by professional antigen-presenting cells in association with HLA class II molecules. Antigen-presenting cells also have to express B7 family molecules, B7-1 and B7-2, which deliver the costimulatory signals that are required to prevent T cell anergy. We studied B7-1 and B7-2 expression by the antigen-presenting cells that infiltrate colorectal cancer stroma. In 25 samples of colorectal carcinomas, a panel of monoclonal antibodies was used to label macrophages, dendritic cells, and T lymphocytes that infiltrate the tumor stroma and the morphologically normal distant mucosa. The expression of HLA class II and B7 molecules involved in T-cell activation was studied using specific monoclonal antibodies. Biopsy pieces from two patients with active Crohn's disease were used as controls. All of the samples were heavily infiltrated by macrophages and/or dendritic cells that strongly expressed HLA class II molecules. In contrast, antibodies to B7-1 and/or B7-2 stained no cells in 16 of the 25 samples of colorectal tumors and less than 1% of the inflammatory cells that infiltrated tumor stroma of the other nine tumor samples. B7 molecules were also poorly expressed by rare cells in the lamina propria of the morphologically normal colorectal mucosa. In contrast, many inflammatory cells that infiltrated the two Crohn's disease samples strongly expressed B7-1 and B7-2, especially in the granulomas. We conclude that most HLA class II+ inflammatory cells that infiltrate colorectal cancers do not express the B7-1 and B7-2 costimulatory molecules. This defect may contribute to the failure of the immune system to recognize tumor cells as antigenic.

T-Cell co-stimulation by the CD28 ligand B7 is involved in the immune response leading to rejection of a spontaneously regressive tumor.

Cell variants from experimental tumors may lose their tumorigenicity or give rise to tumors that regress after a short period of progression in immunocompetent syngeneic animals. Rejection of these tumor cells is often T-cell-dependent. It has recently been reported that, besides the specific signal delivered through the clonogenic receptor, T-cell activation requires a co-stimulatory signal, delivered through its CD28 receptor by B7-1 and/or B7-2 molecules expressed at the surface of the antigen-presenting cells. CTLA4Ig, a fusion molecule that specifically inhibits B7-1 and B7-2 binding to their receptors of T cells, was used to investigate the role of B7 in the spontaneous regression of the tumors induced in syngeneic rats by REGb cells, a regressor cell line established from a chemically induced colon carcinoma. When rats received either 1 or 3 CTLA4Ig injections, REGb tumors grew 3 or 7 times larger than in control animals, respectively. However, in most animals, single or repeated CTLA4Ig injections delayed rather than suppressed REGb tumor rejection. Antibodies to CTLA4Ig appeared in treated rats and could explain this transient effect. Neither REGb cells nor freshly isolated MHC class-II+ antigen-presenting cells infiltrating REGb tumors expressed B7, establishing that the target of CTLA4Ig was not located inside the tumor. In contrast, MHC class-II+ B7+ accessory cells were found in the tissue, rather than the tumor itself, was the site of tumor-antigen presentation to tumor-specific T cells. These results establish the role of B7/CD28 co-stimulation pathway in the control of a spontaneously regressive tumor.

[Dendritic cells and immune function in cancer]. / Cellules dendritiques et fonction immune dans les cancers.

In tumor cells, abnormal proteins expression results from DNA mutations or fusion associated with carcinogenesis or tumor progression. Those abnormal, often clearly defined proteins should be recognized by the immune system and induce an immune response leading to tumor rejection. Actually, most tumors escape the immune response through a specific tolerance, able to suppress or to modify the immune response against tumor associated antigens. Factors which contribute to tumor immunological escape are not elucidated, but could involve a defect in tumor-antigen presentation to the host immune system. An effective immune response against tumor requires tumor-associated antigens to be processed into immunogenic peptides which are presented to T lymphocytes in association with MHC molecules. T-cell fonctional activation requires also a costimulatory signal delivered to the CD28 receptor on T cells by the B7 family of molecules expressed by the antigen-presenting cells. Most tumor cells express MHC class I molecules, a minority also express MHC class II molecules and only a few lymphoma have been reported to express B7. So, tumor cells are not able to present efficiently their specific antigens to competent T cells. Most tumors are yet infiltrated by inflammatory cells, some of them possessing the capacity to process tumor antigens and to present them to competent T cells, either inside the tumor itself, or after migration into the draining lymph nodes. Among antigen-presenting cells, dendritic cells, unlike B lymphocytes and macrophages, are the only cells able to stimulate naive T lymphocytes. They present effectively antigens in situ and stimulate naive and memory T lymphocytes into secondary lymphoid organs. Actually, dendritic cells are supposed to take place in the antitumor immune response, and dendritic cells infiltration inside numerous neoplasms is often associated to an immune response against tumor. However, many questions still underline the failure to recognize stimuli involved in the mobilization (and the retention?) of dendritic cells inside tumor, or which incite them to migrate out of it to ensure their antigen presenting cell function effectively. The secretion of immunosuppressive factors like IL-10, either by tumor cells and by tumor-infiltrating leukocytes represents one of the mechanisms involved in the modulation of the antigen-presenting cell function and in tumor immunological escape. Recent works were undertaken to increase tumor cells immunogenicity. B7.1 molecule transfection allows tumor cells to present directly their antigens and leads to their eradication in vivo. Those results suggest that tumor-antigens presentation is limited in tumor-bearing hosts.

Surface phenotype and functions of tumor-infiltrating dendritic cells: CD8 expression by a cell subpopulation.

Although the function and significance of tumor-infiltrating dendritic cells (TIDC) in the immune response to tumor have never been clearly demonstrated, their location suggests that they play a critical role in the presentation of tumor antigen to specific T cells. We studied the morphological and functional characteristics of interstitial dendritic cells (DC) located inside tumors obtained by injection of cancer cells into syngeneic rats. Single and double immunostaining of tumor sections revealed a dense network of cells which expressed class II major histocompatibility complex (MHC II) molecules. Cell morphology and surface markers were characteristic of DC populations in other tissues. These DC were in close contact with tumor cells and increased in number as the tumor grew larger. Unexpectedly, a subpopulation of morphologically characteristic TIDC expressed both CD8 and MHC II molecules. TIDC were purified from tumors by gradient centrifugation and immunobeads and characterized by morphology, ultrastructural study and surface markers studied by flow cytometry. TIDC were negative for the CD5 molecule (a pan T cell marker), and were not labeled with 3.2.3 monoclonal antibody (mAb) (an NK cell marker) or with Ki-M2R mAb (a macrophage marker). A subpopulation of TIDC expressed the CD8 molecule, confirming the in situ results. TIDC expressed high levels of class I and class II MHC molecules and the adhesion molecule ICAM-1. This expression is compatible with effective antigen presenting function. Purified TIDC triggered rapid and high levels of proliferation of tumor-immune T cells in vitro, demonstrating the potential of these cells to constitutively process and present tumor-associated antigens.