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Identification of major histocompatibility complex (MHC)-associated peptides recognized by T-lymphocytes is a crucial prerequisite for the detection and manipulation of specific immune responses in cancer, viral infections, and autoimmune diseases. Unfortunately immunogenic peptides are less abundant species present in highly complex mixtures of MHC-extracted material. Most peptide identification strategies use microcapillary LC coupled to nano-ESI MS/MS in a challenging on-line approach. Alternatively MALDI PSD analysis has been applied for this purpose. We report here on the first off-line combination of nanoscale (nano) LC and MALDI TOF/TOF MS/MS for the identification of naturally processed MHC peptide ligands. These peptides were acid-eluted from human leukocyte antigen (HLA)-A2, HLA-A3, and HLA-B/-C complexes separately isolated from a renal cell carcinoma cell lysate using HLA allele-specific antibodies. After reversed-phase HPLC, peptides were further fractionated via nano-LC. This additional separation step provided a substantial increase in the number of detectable candidate species within the complex peptide pools. MALDI MS/MS analysis on nano-LC-separated material was then sufficiently sensitive to rapidly identify more than 30 novel HLA-presented peptide ligands. Peptide sequences contained perfect anchor amino acid residues described previously for HLA-A2, HLA-A3, and HLA-B7. The most promising candidate for a T-cell epitope is an HLA-B7-binding nonamer peptide derived from the tumor-associated gene NY-BR-16. To demonstrate the sensitivity of our approach we characterized peptides binding to HLA-C molecules that are usually expressed at the cell surface at approximately only 10% the levels of HLA-A or HLA-B. In fact, multiple renal cell carcinoma peptides were identified that contained anchor amino acid residues of HLA-Cw5 and HLA-Cw7. We conclude that the nano-LC MALDI MS/MS approach is a sensitive tool for the rapid and automated identification of MHC-associated tumor peptides.

Allogeneic cell therapy as a means to break immunotolerance to solid tumors is increasingly used for cancer treatment. To investigate cellular alloimmune responses in a human tumor model, primary cultures were established from renal cell carcinoma (RCC) tissues of 56 patients. In three patients with stable RCC line and human leukocyte antigen (HLA)-identical sibling donor available, allogeneic and autologous RCC reactivities were compared using mixed lymphocyte/tumor cell cultures (MLTC). Responding lymphocytes were exclusively CD8+ T cells, whereas CD4+ T cells or natural killer cells were never observed. Sibling MLTC populations showed higher proliferative and cytolytic antitumor responses compared with their autologous counterparts. The allo-MLTC responders originated from the CD8+ CD62L(high)+ peripheral blood subpopulation containing naive precursor and central memory T cells. Limiting dilution cloning failed to establish CTL clones from autologous MLTCs or tumor-infiltrating lymphocytes. In contrast, a broad panel of RCC-reactive CTL clones was expanded from each allogeneic MLTC. These sibling CTL clones either recognized exclusively the original RCC tumor line or cross-reacted with nonmalignant kidney cells of patient origin. A minority of CTL clones also recognized patient-derived hematopoietic cells or other allogeneic tumor targets. The MHC-restricting alleles for RCC-reactive sibling CTL clones included HLA-A2, HLA-A3, HLA-A11, HLA-A24, and HLA-B7. In one sibling donor-RCC pair, strongly proliferative CD3+CD16+CD57+ CTL clones with non-HLA-restricted antitumor reactivity were established. Our results show superior tumor-reactive CD8 responses of matched allogeneic compared with autologous T cells. These data encourage the generation of antitumor T-cell products from HLA-identical siblings and their potential use in adoptive immunotherapy of metastatic RCC patients. (Cancer Res 2006; 66(23): 11447-54)

Although current allo-transplantation therapy can induce considerable graft-versus-tumor (GvT) effects in RCC patients, it is also accompanied by severe, even life-threatening side effects, mainly due to graft-versus-host disease (GvHD). Efforts aiming to improve the specificity and efficiency of allogeneic cell therapy in this disease (e.g. specific donor lymphocyte infusion or vaccination) will certainly benefit from the identification of potential anti-tumor effector mechanisms and their corresponding target structures. We recently demonstrated that RCC-reactive cytotoxic T-lymphocyte (CTL) clones can be isolated from peripheral blood of healthy donors matched with the RCC stimulators for HLA-class I. These CTL were found to recognize a broad panel of RCC antigens with restricted or ubiquitous tissue expression (Doerrschuck A, et al., Blood July 1, 2004, Epub). We now extended our analyses on peripheral blood mononuclear cells (PBMC) of further HLA-matched healthy sibling (1) and unrelated individuals (4) and compared these results with available autologous patient PBMC. While mixed lymphocyte/tumor-cell culture (MLTC) responders derived from allogeneic donors showed a robust antigen-dependent proliferation over several weeks, a weak if any proliferative response was seen with autologous MLTC populations. By analysing the fine specificity of MLTC-derived clonal CTL the majority of allogeneic effectors recognized exclusively their RCC stimulators, but not corresponding lymphoblastoid-cell lines or natural killer target K562. These CTL were restricted by various HLA-A, -B or -C molecules. We further isolated CTL clones that exhibit an extraordinary strong recognition of RCC and various epithelial tumor-cell lines. Antibody blocking experiments provided clear evidence that these CTL are restricted by a not yet defined HLA-Ib molecule and, simultaneously, by a NKG2D-dependent mechanism. Other rapidly proliferating CD3+ CD8+ CTL clones were obtained that showed a non-HLA-restricted reactivity against RCC and a minor but consistent reactivity against targets with low or absent HLA-class I expression (e.g. K562). In conclusion, our results demonstrate that a heterogeneous panel of RCC-reactive HLA-Ia/Ib-restricted CTL can be isolated from PBMC of HLA-class I-matched healthy individuals. Alternatively, CTL recognizing RCC in a non-HLA restricted manner can be obtained. Our observations might reflect the superior ability to activate and expand RCC-directed T cells from PBMC of allogeneic healthy donors compared to the autologous setting. At this point, we cannot conclude whether these various CTL populations contribute to effective anti-RCC immune responses occurring in vivo. Answering this question will certainly require to identify further CTL-defined target structures at the molecular level. This would allow us to analyse the expression of candidate antigens and the frequency of specific CTL in RCC patients after allogeneic blood stem-cell transplantation, and to correlate these findings with clinical GvT and GvH events.