Identification of an MSI-H Tumor-Specific Cytotoxic T Cell Epitope Generated by the (−1) Frame of U79260(FTO)

Microsatellite instability (MSI-H) induced by defects of the DNA mismatch repair system results in insertion or deletion of single nucleotides at short repetitive DNA sequences. About 15% of sporadic and approximately 90% of hereditary nonpolyposis colorectal cancers display MSI-H. When affecting coding regions, MSI-H results in frameshift mutations and expression of corresponding frameshift peptides (FSPs). Functional tumor promoting relevance has been demonstrated for a growing number of genes frequently hit by MSI-H. Contrary, immune reactions against FSPs are involved in the immune surveillance of MSI-H cancers. Here, we provide conclusive data that the (−1) frame of U79260(FTO) encodes an HLA-A0201-restricted cytotoxic T cell epitope (FSP11; TLSPGWSAV). T cells specific for FSP11 efficiently recognized HLA-A0201(pos) tumor cells harboring the mutated reading frame. Considering the exceptionally high mutation rate of U79260(FTO) in MSI-H colorectal carcinoma (81.8%), this recommends that FSP11 be a component of future vaccines

An MSI Tumor Specific Frameshift Mutation in a Coding Microsatellite of MSH3 Encodes for HLA-A0201-Restricted CD8+ Cytotoxic T Cell Epitopes

BACKGROUND: Microsatellite instability (MSI) resulting from inactivation of the DNA mismatch repair system (MMR) characterizes a highly immunological subtype of colorectal carcinomas. Those tumors express multiple frameshift-mutated proteins which present a unique pool of tumor-specific antigens. The DNA MMR protein MSH3 is frequently mutated in MSI(+) colorectal tumors, thus making it an attractive candidate for T cell-based immunotherapies. METHODOLOGY/PRINCIPAL FINDINGS: FSP-specific CD8(+) T cells were generated from a healthy donor using reverse immunology. Those T cells specifically recognized T2 cells sensitized with the respective peptides. Specific recognition and killing of MSI(+) colorectal carcinoma cells harbouring the mutated reading frame was observed. The results obtained with T cell bulk cultures could be reproduced with T cell clones obtained from the same cultures. Blocking experiments (using antibodies and cold target inhibition) confirmed peptide as well as HLA-A0201-specificity. CONCLUSIONS: We identified two novel HLA-A0201-restricted cytotoxic T cell epitopes derived from a (-1) frameshift mutation of a coding A(8) tract within the MSH3 gene. These were (386)-FLLALWECSL (FSP18) and (387)-LLALWECSL (FSP19) as well as (403)-IVSRTLLLV (FSP23) and (402)-LIVSRTLLLV (FSP31), respectively. These results suggest that MSH3(-1) represents another promising MSI(+)-induced target antigen. By identifying two distinct epitopes within MSH3(-1), the sustained immunogenicity of the frameshift mutated sequence was confirmed. Our data therefore encourage further exploitation of MSH3 as a piece for peptide-based vaccines either for therapeutic or--even more important--preventive purposes

Ex-vivo Clonally Expanded B Lymphocytes Infiltrating Colorectal Carcinoma Are of Mature Immunophenotype and Produce Functional IgG

Background: Tumor infiltrating B cells (TiBc) have not yet been investigated in detail. This may at least in part be due to technical difficulties. Here we describe a straightforward and reproducible method to isolate and culture TiBc from primary colorectal carcinomas (CRC). Methods/Results: TiBc cultures were generated by Epstein-Barr virus (EBV) immortalization. With this method, monoclonal TiBc cultures were obtained for 14/19 CRCs. As assessed by flow cytometry and ELISA, TiBc showed an activated immunophenotype (CD23 +, CD80 +) and produced immunoglobulin (Ig; IgG secretion in 55 % of the cultures). In functional in vitro analysis, most of the IgGs specifically bound to allogeneic CRC target cells. These data suggest that TiBc are antigenexperienced and thus may exhibit functionality in situ. Additionally, mini-cultures generated from 12 further CRCs revealed TiBc outgrowth exclusively in the presence of EBV. Conclusion: In summary, this simple method provides a cellular tool and our data set the stage for analysing the bivalent role of TiBc; being antigen-presenting cells on the one hand and tumor-specific antibody producers on the other. Additionally, the generation of long-term TiBc cultures and their monoclonal Ig may serve to identify novel tumor-specifi

Multidrug-resistance proteins are weak tumor associated antigens for colorectal carcinoma

<p>Abstract</p> <p>Background</p> <p>Multidrug resistance (MDR) is a clinically, highly relevant phenomenon. Under chemotherapy many tumors show an increasing resistance towards the applied substance(s) and to a certain extent also towards other agents. An important molecular cause of this phenomenon is an increased expression of transporter proteins. The functional relationship between high expression levels and chemotherapy resistance makes these MDR and MRP (MDR related protein) proteins to interesting therapeutic targets. We here wanted to systematically analyze, whether these proteins are tumor specific antigens which could be targeted immunologically.</p> <p>Results</p> <p>Using the reverse immunology approach, 30 HLA-A2.1 restricted MDR and MRP derived peptides (MDP) were selected. Stimulated T cell lines grew well and mainly contained activated CD8⁺cells. Peptide specificity and HLA-A2.1 restriction were proven in IFN-γ-ELISpot analyses and in cytotoxicity tests against MDP loaded target cells for a total of twelve peptides derived from MDR-1, MDR-3, MRP-1, MRP-2, MRP-3 and MRP-5. Of note, two of these epitopes are shared between MDR-1 and MDR-3 as well as MRP-2 and MRP-3. However, comparably weak cytotoxic activities were additionally observed against HLA-A2.1⁺tumor cells even after upregulation of MDR protein expression by <it>in vitro </it>chemotherapy.</p> <p>Conclusions</p> <p>Taken together, these data demonstrate that human T cells can be sensitised towards MDPs and hence, there is no absolute immunological tolerance. However, our data also hint towards rather low endogenous tumor cell processing and presentation of MDPs in the context of HLA-A2.1 molecules. Consequently, we conclude that MDR and MRP proteins must be considered as weak tumor specific antigens-at least for colorectal carcinoma. Their direct contribution to therapy-failure implies however, that it is worth to further pursue this approach.</p

Cryopreservation of human colorectal carcinomas prior to xenografting

<p>Abstract</p> <p>Background</p> <p>Molecular heterogeneity of colorectal carcinoma (CRC) is well recognized, forming the rationale for molecular tests required before administration of some of the novel targeted therapies that now are rapidly entering the clinics. For clinical research at least, but possibly even for future individualized tumor treatment on a routine basis, propagation of patients' CRC tissue may be highly desirable for detailed molecular, biochemical or functional analyses. However, complex logistics requiring close liaison between surgery, pathology, laboratory researchers and animal care facilities are a major drawback in this. We here describe and evaluate a very simple cryopreservation procedure for colorectal carcinoma tissue prior to xenografting that will considerably reduce this logistic complexity.</p> <p>Methods</p> <p>Fourty-eight CRC collected ad hoc were xenografted subcutaneously into immunodeficient mice either fresh from surgery (N = 23) or after cryopreservation (N = 31; up to 643 days).</p> <p>Results</p> <p>Take rates after cryopreservation were satisfactory (71%) though somewhat lower than with tumor tissues fresh from surgery (74%), but this difference was not statistically significant. Re-transplantation of cryopreserved established xenografts (N = 11) was always successful. Of note, in this series, all of the major molecular types of CRC were xenografted successfully, even after cryopreservation.</p> <p>Conclusions</p> <p>Our procedure facilitates collection, long-time storage and propagation of clinical CRC specimens (even from different centres) for (pre)clinical studies of novel therapies or for basic research.</p

Combinations of TLR Ligands: A Promising Approach in Cancer Immunotherapy

Toll-like receptors (TLRs), a family of pattern recognition receptors recognizing molecules expressed by pathogens, are typically expressed by immune cells. However, several recent studies revealed functional TLR expression also on tumor cells. Their expression is a two-sided coin for tumor cells. Not only tumor-promoting effects of TLR ligands are described but also direct oncopathic and immunostimulatory effects. To clarify TLRs’ role in colorectal cancer (CRC), we tested the impact of the TLR ligands LPS, Poly I:C, R848, and Taxol on primary human CRC cell lines (HROC40, HROC60, and HROC69) in vitro and in vivo (CT26). Taxol, not only a potent tumor-apoptosis-inducing, but also TLR4-activating chemotherapeutic compound, inhibited growth and viability of all cell lines, whereas the remaining TLR ligands had only marginal effects (R848 > LPS > Poly I:C). Combinations of the substances here did not improve the results, whereas antitumoral effects were dramatically boosted when human lymphocytes were added. Here, combining the TLR ligands often diminished antitumoral effects. In vivo, best tumor growth control was achieved by the combination of Taxol and R848. However, when combined with LPS, Taxol accelerated tumor growth. These data generally prove the potential of TLR ligands to control tumor growth and activate immune cells, but they also demonstrate the importance of choosing the right combinations

Optimized creation of glioblastoma patient derived xenografts for use in preclinical studies

Additional file 1. Sequences of oligonucleotides and probes.Nucleotide sequences and labeling of all oligonucleotides and probes used in this study