Unexpected Abundance of HLA Class II Presented Peptides in Primary Renal Cell Carcinomas

Abstract

Purpose: To elicit a long-lasting antitumor immune response, CD8+ and CD4 + Tcells should be activated. We attempted to isolate HLA-DR^presented peptides directly from dissected solid tumors, in particular from renal cell carcinoma, to identify MHC class II ligands from tumorassociated antigens (TAA) for their use in peptide-based immunotherapy. Experimental Design: Tumor specimens were analyzed by immunohistochemical staining for their HLA class II expression. HLA class II peptides were subsequently isolated and identified by mass spectrometry. Gene expression analysis was done to detect genes overexpressed in tumor tissue. Peptides from identifiedTAAs were used to induce peptide-specific CD4 + T-cell responses in healthy donors and in tumor patients. Results: In the absence of inflammation, expression of MHC class II molecules is mainly restricted to cells of the immune system. To our surprise, we were able to isolate and characterize hundreds of class II peptides directly from primary dissected solid tumors, especially from renal cell carcinomas, and from colorectal carcinomas and transitional cell carcinomas. Infiltrating leukocytes expressed MHC class II molecules and tumor cells, very likely under the influence of IFNg. Our list of identified peptides contains ligands from several TAAs, including insulin-like growth factor binding protein 3 and matrix metalloproteinase 7. The latter bound promiscuously to HLA-DR molecules and were able to elicit CD4 + T-cell responses. Conclusions: Thus, our direct approach will rapidly expand the limited number of T-helper epitopes fromTAAs for their use in clinical vaccination protocols. CD4+ helper T cells play an important role in orchestrating the effector function of antitumor T-cell responses (1), and for this reason, the identification of CD4 + T-cell epitopes derived from tumor-associated antigens (TAA) has recently been a major focus of attention (2, 3). Even in the absence of CTL effector cells, helper T cells in the mouse can inhibit tumor angiogenesis via IFNg (4) and counteract tumor progression via the induction of an antibody response (5). In contrast to HLA class I ligands, only a small number of class II ligands of TAA has been described. Because HLA class II molecules are constitutively presented on cells of the immune system alone (6), the possibility of isolating class II peptides directly from primary tumors as opposed to class I ligands (7) has not been considered viable. Therefore, numerous strategies to target antigens into the class II processing pathway of antigenpresenting cells have been described (e.g., the incubation of antigen-presenting cells with the antigen of interest to enable it to be taken up, processed, and presented; ref. 8).To identify HLA class II ligands from TAA for their use in peptide-based immunotherapy, we attempted to isolate HLA-DR -presented peptides directly from dissected solid tumors, in particular from renal cell carcinoma (RCC). Even if the majority of tumor cells were class II negative, with state-of-the-ar...

Thyroglobulin Peptides Associate In Vivo to HLA-DR in Autoimmune Thyroid Glands

“…Later, 55 peptides were sequenced from 9 human intestine samples from patients with inflammatory bowel disease (13). Recently, a large number of HLA class II-associated peptides have been identified from primary renal carcinoma samples (14).…”

“…Peptide sequences were obtained by electrospray tandem MS using an LCQ ion trap mass spectrometer (Thermo Fisher Scientific) with a nanospray electrospray ionization (nESI) source (nESI-ion trap MS (ITMS)/MS from Protana A/S, Odense, Denmark), by a MALDI-TOF/TOF in a mass spectrometer (AB 4700 mass . Expression scores are based on immunofluorescence staining on cryostat sections as follows: ϩ, positive staining in Ͻ10% follicles (focal); ϩϩ, 10 -50% positive follicles (multifocal); ϩϩϩ, Ͼ50% positive follicles (generalized) (14).…”

High DGK-α and Disabled MAPK Pathways Cause Dysfunction of Human Tumor-Infiltrating CD8+ T Cells That Is Reversible by Pharmacologic Intervention

“…The most common ones observed in many tumors, such as lack of infiltration with cytotoxic lymphocytes or loss of MHC class I, do not explain the immune escape of human RCC satisfactorily: RCC tissues are generally strongly infiltrated by various types of immune cells that are considered to be associated with an effective immune response, notably dendritic cells, NK cells, and CD8 + T lymphocytes expressing MHC class I-restricted tumor-reactive TCRs (5-7, 11, 12). Moreover, renal tumors express MHC class I and class II molecules on the cell surface, and T cell epitopes are eluted from tumor tissues (13,14), indicating good Ag processing and presentation in situ. However, earlier studies analyzing NK cells (11) and T cells of RCC showed that infiltrating cytotoxic lymphocytes are functionally inactive but are able to gain function when cultured ex vivo (4,5,15), suggesting that inhibitory mechanisms act at the tumor site.…”

Harnessing the Immune System to Fight Cancer: The Promise of Genetic Cancer Vaccines

“…In this respect promising results in phase II studies have been obtained by Immatics (www.immatics.com) . This technology consists in the vaccination of patients with multiple tumour-associated peptides (TUMAPs) that can be isolated from tumour specimens and identified by mass spectrometry (Dengjel et al, 2006). The most advanced product, IMA901, a combination of several TUMAPs for the treatment of renal cell carcinoma, completed a Europe-wide multi-center Phase II clinical trial and has recently commenced a Phase III trial.…”