CIITA-Transduced Glioblastoma Cells Uncover a Rich Repertoire of Clinically Relevant Tumor-Associated HLA-II Antigens.

CD4+ T cell responses are crucial for inducing and maintaining effective anticancer immunity, and the identification of human leukocyte antigen class II (HLA-II) cancer-specific epitopes is key to the development of potent cancer immunotherapies. In many tumor types, and especially in glioblastoma (GBM), HLA-II complexes are hardly ever naturally expressed. Hence, little is known about immunogenic HLA-II epitopes in GBM. With stable expression of the class II major histocompatibility complex transactivator (CIITA) coupled to a detailed and sensitive mass spectrometry-based immunopeptidomics analysis, we here uncovered a remarkable breadth of the HLA-ligandome in HROG02, HROG17, and RA GBM cell lines. The effect of CIITA expression on the induction of the HLA-II presentation machinery was striking in each of the three cell lines, and it was significantly higher compared with interferon gamma (IFNÉ£) treatment. In total, we identified 16,123 unique HLA-I peptides and 32,690 unique HLA-II peptides. In order to genuinely define the identified peptides as true HLA ligands, we carefully characterized their association with the different HLA allotypes. In addition, we identified 138 and 279 HLA-I and HLA-II ligands, respectively, most of which are novel in GBM, derived from known GBM-associated tumor antigens that have been used as source proteins for a variety of GBM vaccines. Our data further indicate that CIITA-expressing GBM cells acquired an antigen presenting cell-like phenotype as we found that they directly present external proteins as HLA-II ligands. Not only that CIITA-expressing GBM cells are attractive models for antigen discovery endeavors, but also such engineered cells have great therapeutic potential through massive presentation of a diverse antigenic repertoire.

Restriction factors in human retrovirus infections and the unprecedented case of CIITA as link of intrinsic and adaptive immunity against HTLV-1.

BACKGROUND: Immunity against pathogens evolved through complex mechanisms that only for sake of simplicity are defined as innate immunity and adaptive immunity. Indeed innate and adaptive immunity are strongly intertwined each other during evolution. The complexity is further increased by intrinsic mechanisms of immunity that rely on the action of intracellular molecules defined as restriction factors (RFs) that, particularly in virus infections, counteract the action of pathogen gene products acting at different steps of virus life cycle. MAIN BODY AND CONCLUSION: Here we provide an overview on the nature and the mode of action of restriction factors involved in retrovirus infection, particularly Human T Leukemia/Lymphoma Virus 1 (HTLV-1) infection. As it has been extensively studied by our group, special emphasis is given to the involvement of the MHC class II transactivator CIITA discovered in our laboratory as regulator of adaptive immunity and subsequently as restriction factor against HIV-1 and HTLV-1, a unique example of dual function linking adaptive and intrinsic immunity during evolution. We describe the multiple molecular mechanisms through which CIITA exerts its restriction on retroviruses. Of relevance, we review the unprecedented findings pointing to a concerted action of several restriction factors such as CIITA, TRIM22 and TRIM19/PML in synergizing against retroviral replication. Finally, as CIITA profoundly affects HTLV-1 replication by interacting and inhibiting the function of HTLV-1 Tax-1 molecule, the major viral product associated to the virus oncogenicity, we also put forward the hypothesis of CIITA as counteractor of HTLV-1-mediated cancer initiation.

CIITA-Driven MHC Class II Expressing Tumor Cells as Antigen Presenting Cell Performers: Toward the Construction of an Optimal Anti-tumor Vaccine.

Construction of an optimal vaccine against tumors relies on the availability of appropriate tumor-specific antigens capable to stimulate CD4+ T helper cells (TH) and CD8+ cytolytic T cells (CTL). CTL are considered the major effectors of the anti-tumor adaptive immune response as they recognize antigens presented on MHC class I (MHC-I) molecules usually expressed in all cells and thus also in tumors. However, attempts to translate in clinics vaccination protocols based only on tumor-specific MHC-I-bound peptides have resulted in very limited, if any, success. We believe failure was mostly due to inadequate triggering of the TH arm of adaptive immunity, as TH cells are necessary to trigger and maintain the proliferation of all the immune effector cells required to eliminate tumor cells. In this review, we focus on a novel strategy of anti-tumor vaccination established in our laboratory and based on the persistent expression of MHC class II (MHC-II) molecules in tumor cells. MHC-II are the restricting elements of TH recognition. They are usually not expressed in solid tumors. By genetically modifying tumor cells of distinct histological origin with the MHC-II transactivator CIITA, the physiological controller of MHC-II gene expression discovered in our laboratory, stable expression of all MHC class II genes was obtained. This resulted in tumor rejection or strong retardation of tumor growth in vivo in mice, mediated primarily by tumor-specific TH cells as assessed by both depletion and adoptive cell transfer experiments. Importantly these findings led us to apply this methodology to human settings for the purification of MHC-II-bound tumor specific peptides directly from tumor cells, specifically from hepatocarcinomas, and the construction of a multi-peptide (MHC-II and MHC-I specific) immunotherapeutic vaccine. Additionally, our approach unveiled a noticeable exception to the dogma that dendritic cells are the sole professional antigen presenting cells (APC) capable to prime naïve TH cells, because CIITA-dependent MHC-II expressing tumor cells could also perform this function. Thus, our approach has served not only to select the most appropriate tumor specific peptides to activate the key lymphocytes triggering the anti-tumor effector functions but also to increase our knowledge of intimate mechanisms governing basic immunological processes.

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.

Tumor Immunology meets Immunology: Modified cancer cells as professional APC for priming naïve 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.