CIITA-induced MHC class II expression in tumor cells: a novel universal strategy of anti-tumor vaccination.

Priming and activation of CD4+ T helper (TH) cells against tumor associated antigens can be achieved after their recognition on antigen presenting cells (APC) only within the context of MHC class II (MHC-II) molecules. We previously reported successful triggering of TH-specific long lasting anti-tumor immune response in H-2d haplotype BALB/c mice using tumor cells genetically modified to express endogenous MHC-II genes (I-A and I-E) after transfection with CIITA (MHC-II transactivator). Now, we investigated the pertinence of this approach in H-2b haplotype C57BL/6 mice that only express I-A molecules due to a defect in their I-E\u3b1 gene. MC38 colon carcinoma cells of the H-2b haplotype were stably transfected with CIITA. Selected MHC-II positive clones were injected into C57BL/6 mice. Complete rejection or significant growth retardation as compared to MHC-II negative parental tumor was obtained. Subsequent challenge of the protected mice with parental tumor proved that the CIITA-transfected tumor engendered efficient anti-tumor vaccination. Then, adoptive cell transfer from the vaccinated mice to na\uefve recipients demonstrated that CD4+ TH cells orchestrate the anti-tumor protection. Finally, the use of CD11c.DTR transgenic mice in which conditional deletion of dendritic cells (DC) can be performed, and the use of Liposomal Clodronate as a depletion agent for macrophages, proved that CIITA-driven MHC-II positive tumor cells act as surrogate APC for priming and activating CD4+ TH cells, without the need of either DC or macrophages. These results demonstrate the validity of CIITA-driven MHC-II+ tumor cells as anti-tumor vaccination tool in mouse models of different haplotypes. Moreover, they prove that expression of a single MHC-II restriction element in tumor cells is sufficient to trigger anti-tumor CD4+ TH cells and, more importantly, demonstrate for the first time that CIITA-driven MHC-II expressing tumor cells can act as professional APCs in vivo to prime na\uefve tumor-specific CD4+ TH cells

CIITA dependent MHC class II IA expression in tumor cells triggers CD4 T cell protective and long lasting antitumor immunity

We previously demonstrated the generation of a CD4 T cell-specific long lasting anti-tumor immune response in the H-2d BALB/c mouse model by using tumor cells that have been genetically modified with CIITA to express MHC-II I-E and I-A molecules. We have now investigated the pertinence of this approach in the H-2b C57BL/6 mouse model despite the defect in their I-E\u3b1 gene and thus the lack of expression of I-E subset. To this purpose we injected in vivo the CIITA-driven I-A-only MHC-II-positive LLC (lewis Lung Carcinoma) and MC38 colon carcinoma in the C57BL/6 mice and their growth rate along with the recipient's immune response were analyzed. The CIITA-transfected, MHC-II-positive tumor cells were either completely rejected or showed a significant growth retardation compared to the MHC-II-negative parental tumor. The protected mice were re-injected with the parental tumors and a complete rejection was obtained proving that a specific long lasting immune response was triggered by the CIITA-transfected tumors. Furthermore, total splenocytes or purified CD4+, CD8+ T cells and B cells were transferred from the vaccinated recipients to na\uefve recipients that were co-injected with the parental tumor cells and the results showed that CD4+ TH cells were the main effectors of the immune response against the tumor cells. Interestingly, similar results were obtained in C57BL/6-DOG transgenic mice whose dendritic cells could be conditionally ablated after administration of Diphteria toxin. These results demonstrate the validity of triggering a specific, long-lasting anti-tumor immune response using CIITA-driven MHC class II positive tumor cells of different MHC haplotype as stimulators. Importantly, the results establish that expression of a single MHC-II restriction element, I-A, in tumor cells is sufficient to trigger CD4+ TH cell protective immune response, strongly suggesting that the relevant tumor-associated antigenic repertoire can be displayed without the need of the I-E restriction element. Finally, these results strongly suggest that CIITA-modified tumor cells can act as antigen presenting cells in vivo to prime na\uefve CD4+ TH cells against tumor antigens. Abstract nr A014

Tumor Immunology meets Immunology: Modified cancer cells as professional APC for priming na\uc3\uafve tumor-specific CD4+ T cells

Although recent therapeutic approaches have revitalized the enthusiasm of the immunological way to combat cancer, still the comprehension of immunity against tumors is largely incomplete. Due to their specific function, CD8+ T cells with cytolytic activity (CTL) have attracted the attention of most investigators because CTL are considered the main effectors against tumor cells. Nevertheless, CTL activity and persistence is largely dependent on the action of CD4+ T helper cells (TH). Thus establishment of tumor-specific TH cell response is key to the optimal response against cancer. Here we describe emerging new strategies to increase the TH cell recognition of tumor antigens. In particular, we review recent data indicating that tumor cells themselves can act as surrogate antigen presenting cells for triggering TH response and how these findings can help in constructing immunotherapeutic protocols for anti-cancer vaccine development

CIITA-driven MHC class ii expressing tumor cells can efficiently prime naive CD4+ TH cells in vivo and vaccinate the host against parental MHC-II-negative tumor cells

Our previous studies showed that non-immunogenic H-2d tumor cells of distinct epithelial histotypes can become highly immunogenic, induce a protective CD4+ T cell response and vaccinate the animals against parental MHC-II-negative cells if they are rendered MHC class II-positive by stable transfection with the Air1-encoded MHC-II transcriptional activator CIITA. These studies did not establish, however, whether tumor immunity was the consequence of a direct priming of naive CD4+ T lymphocytes by CIITA-driven MHC-II-expressing tumor cells or by MHC-II-tumor antigen complexes engulfed by dendritic cells (DC) and exposed on the surface of these professional antigen presenting cells (APC). In the present investigation, we provide definitive evidence that CIITAtumor cells are the crucial APC in vivo for CD4+ T cell priming. By using a transgenic H-2b mouse model, the CD11c.DTR C57BL/6 mice, in which DC can be functionally deleted by administration of diphteria toxin, we show that CIITA-tumor cells of two distinct histotypes can be rejected or strongly retarded in their growth in DC-deleted mice. To rule out that in absence of DC, other professional APC could prime naive CD4+ T cells, we deleted the macrophages in CD11c.DTR C57BL/6 mice by administration of liposome Clodronate and still obtained rejection or strong retardation in tumor growth of CIITA-tumor cells. Our results challenge the diffuse belief that non-professional APC cannot efficiently prime naive T cells in vivo. Moreover, the demonstration of the general validity of our approach in different genetic backgrounds may open a way for new strategies of antitumor treatment in clinical settings

CIITA-related block of HLA class II expression, upregulation of HLA class I, and heterogeneous expression of immune checkpoints in hepatocarcinomas: implications for new therapeutic approaches

Hepatocellular carcinoma (HCC) is the second cause of death for cancer worldwide, justifying the urgent need for novel therapeutic approaches. Immunotherapeutic strategies based on triggering and/or rescuing tumor antigen-specific T cells may be promising particularly if combined together. As preliminary step toward this goal, we have investigated the expression of antigen presenting molecules (HLA class I and class II) and immune checkpoints (PD-1 and PD-L1) in 43 HCC samples from distinct patients and in HCC cell lines. While normal hepatocytes did not express HLA class I and II, HCC cells strongly upregulated HLA class I while remaining negative for HLA class II. The absence of HLA class II expression in HCC cell lines correlated with lack of expression of the HLA class II transactivator, CIITA, which could not be rescued even after interferon-gamma treatment. This was due to high methylation levels of interferon-gamma-sensitive CIITA promoter IV strongly suggesting a biologically relevant developmental silencing of HLA-II expression in liver cell lineage. HCC tumor tissues showed a variable degree of leukocyte infiltration. Infiltrating lymphocytes expressed PD-1, while PD-L1 was expressed in cells with monocyte-macrophage morphology mostly localized at the tumor margin, but not in tumor cells. De novo expression of HLA class I, instrumental for presenting tumor antigens to cytotoxic T lymphocytes, and the correct characterization of the cells expressing checkpoint inhibitors in the tumor tissue should be the ground for setting novel strategies of combined approaches of immunotherapy in HCC based on tumor peptide vaccines and anti-checkpoint inhibitor antibodies