Characterization of complementary determinant region 3δ in human MutS homologue 2-specific γδ T cells.

γδT cells function as sentinels in early host responses to infections and malignancies. Previously, we found ectopically expressed human MutS homologue 2 (hMSH2), recognized by γδT cells, triggered a γδT cell-mediated cytolysis to tumor cells. However, the characteristics of hMSH2-specific γδ Τ cells are not fully understood. In this study, we investigated the complementary determinant region (CDR) 3δ diversity of hMSH2-specific γδ T cells. We found that the CDR3δ sequences of hMSH2-specific γδ T cells displayed limited diversity, while the length and germline gene usage showed no differences compared with whole CDR3δ immune repertoire. There are more hydrophilic amino acids in P/N insert of hMSH2-specific γδ T cells including the more conserved amino acid at the position 97. Our results offer clues to understanding antigen recognition pattern of γδ T cells to stress-induced hMSH2 of tumor cells and also the mechanism of γδT cell-mediated tumor immune surveillance.

Autoantibodies to DNA mismatch repair enzymes in polymyositis/dermatomyositis and other autoimmune diseases: a possible marker of favorable prognosis.

Objective. Myositis-specific autoantibodies(MSAs) are useful tools for identifying clinical subsets of patients with idiopathic inflammatory myopathies(IIMs). There have been few reports on antibodies to some DNA mismatch repair enzymes (MMREs) inpatients with IIMs. This study was undertaken to determine the frequencies and clinical associations of antibodies to 7 types of MMREs (MLH1, MLH3, MSH2,MSH3, MSH6, PMS1, and PMS2) in patients with IIMs and other systemic autoimmune diseases.Methods. Clinical data and serum samples were collected from 239 Japanese patients with IIMs (147 with adult dermatomyositis, 13 with juvenile dermatomyositis,57 with polymyositis, and 22 with myositis overlap syndrome). One hundred patients with other diseases, including 40 with systemic lupus erythematosus(SLE), were assessed as disease controls. The presence of anti-MMRE antibodies in serum was examined by immunoprecipitation, enzyme-linked immunosorbenassay, and immunoprecipitation/Western blotting.Results. Anti-MMRE antibodies were found in 1 patients with IIMs and 3 patients with SLE. They were restricted to MLH1, PMS1, MSH2, and PMS2, with simultaneous positivity for more than one of these antibodies occurring in some patients. Nine IIM patients with anti-MMREs also had other MSAs and their associated clinical features. All patients with anti-MMREs were still living at the time of the present analysis.Conclusion. Anti-MMRE antibodies, which often co exist with other MSAs, may be serologic markers forgood prognosis in IIMs.

Frameshift-derived neoantigens constitute immunotherapeutic targets for patients with microsatellite-instable haematological malignancies: frameshift peptides for treating MSI+ blood cancers.

PURPOSE: Microsatellite instability (MSI) resulting from loss of functional DNA mismatch repair was recently found in various haematological disorders. In coding sequences, MSI leads to frameshift mutations (FSMs) and the production of C-terminally altered proteins which are foreign to the immune system. Here, we wondered whether these frame-shifted peptide (FSP) sequences represent tumour-specific antigens also for MSI(+) leukaemia and lymphomas (L/L). MATERIAL AND METHODS: A total of 33 coding region microsatellites were examined in MSI(+) L/L cell lines for the presence of FSMs. Thereafter, recognition of MSI(+) cells by established FSP-specific CD8(+) T cell lines was quantified using interferon (IFN)-γ enzyme-linked immunospot (ELISpot) assays. In each experiment, MSI(+) L/L cell lines and T2 targets exogenously loaded with the cognate peptide (=internal control) were employed. Supplementary, lytic activity towards tumour cells was analysed by standard chromium release assay ((51)Cr). RESULTS: Mutational profiling of 33 coding microsatellite loci in nine MSI(+) L/L cell lines revealed instability in at least nine microsatellites. In each cell line, a distinct mutational profile was observed. Only three of the 33 loci were stable. FSP-specific and human leukocyte antigen-A2 (HLA-A2)-restricted T cells specifically recognised MSI(+) L/L cells endogenously expressing TGFßRII(-1), Caspase 5 (-1) and MSH3 (-1) in ELISpot assays. Moreover, specific killing of Caspase 5 (-1) and MSH3 (-1) expressing L/L cell lines was achieved in functional cytotoxicity assays. CONCLUSION: Data presented here expand the importance of FSPs as shared and general tumour-specific antigens. Consequently, they open new avenues for specific immunotherapies not only for solid but also for MSI(+) haematological malignancies.

Ectopic expression of human MutS homologue 2 on renal carcinoma cells is induced by oxidative stress with interleukin-18 promotion via p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) signaling pathways.

Human MutS homologue 2 (hMSH2), a crucial element of the highly conserved DNA mismatch repair system, maintains genetic stability in the nucleus of normal cells. Our previous studies indicate that hMSH2 is ectopically expressed on the surface of epithelial tumor cells and recognized by both T cell receptor γδ (TCRγδ) and natural killer group 2 member D (NKG2D) on Vδ2 T cells. Ectopically expressed hMSH2 could trigger a γδ T cell-mediated cytolysis. In this study, we showed that oxidative stress induced ectopic expression of hMSH2 on human renal carcinoma cells. Under oxidative stress, both p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) pathways have been confirmed to mediate the ectopic expression of hMSH2 through the apoptosis-signaling kinase 1 (ASK1) upstream and activating transcription factor 3 (ATF3) downstream of both pathways. Moreover, renal carcinoma cell-derived interleukin (IL)-18 in oxidative stress was a prominent stimulator for ectopically induced expression of hMSH2, which was promoted by interferon (IFN)-γ as well. Finally, oxidative stress or pretreatment with IL-18 and IFN-γ enhanced γδ T cell-mediated cytolysis of renal carcinoma cells. Our results not only establish a mechanism of ectopic hMSH2 expression in tumor cells but also find a biological linkage between ectopic expression of hMSH2 and activation of γδ T cells in stressful conditions. Because γδ T cells play an important role in the early stage of innate anti-tumor response, γδ T cell activation triggered by ectopically expressed hMSH2 may be an important event in immunosurveillance for carcinogenesis.

Ectopically expressed human tumor biomarker MutS homologue 2 is a novel endogenous ligand that is recognized by human γδ T cells to induce innate anti-tumor/virus immunity.

Human (h) MutS homologue 2, a nuclear protein, is a critical element of the DNA mismatch repair system. Our previous studies suggest that hMSH2 might be a protein ligand for TCRγδ. Here, we show that hMSH2 is ectopically expressed on a large panel of epithelial tumor cells. We found that hMSH2 interacts with both TCRγδ and NKG2D and contributes to Vδ2 T cell-mediated cytolysis of tumor cells. Moreover, recombinant human MSH2 protein stimulates the proliferation and IFN-γ secretion of Vδ2 T cells in vitro. Finally, hMSH2 expression is induced on the cell surface of Epstein-Barr virus-transformed lymphoblastoid cell lines, and the induction increases the sensitivity of these lymphoblastoid cell lines to γδ T cell-mediated cytolysis. Our data suggest that hMSH2 functions as a tumor-associated or virus infection-related antigen recognized by both Vδ2 TCR and NKG2D, and it plays a role in eliciting the immune responses of γδ T cells against tumor- and virus-infected cells. The recognition of ectopic surface-expressing endogenous antigen by TCRγδ and NKG2D may be an important mechanism of innate immune response to carcinogenesis and viral infection.

Combined deficiency of MSH2 and Sµ region abolishes class switch recombination.

Class switch recombination (CSR) is mediated by G-rich tandem repeated sequences termed switch regions. Transcription of switch regions generates single-stranded R loops that provide substrates for activation-induced cytidine deaminase. Mice deficient in MSH2 have a mild defect in CSR and analysis of their switch junctions has led to a model in which MSH2 is more critical for switch recombination events outside than within the tandem repeats. It is also known that deletion of the whole Sµ region severely impairs but does not abrogate CSR despite the lack of detectable R loops. Here, we demonstrate that deficiency of both MSH2 and the Sµ region completely abolishes CSR and that the abrogation occurs at the genomic level. This finding further supports the crucial role of MSH2 outside the tandem repeats. It also indicates that during CSR, MSH2 has access to activation-induced cytidine deaminase targets in R-loop-deficient Iµ-Cµ sequences rarely used in CSR, suggesting an MSH2-dependent DNA processing activity at the Iµ exon that may decrease with transcription elongation across the Sµ region.

Immunohistochemistry for PMS2 and MSH6 alone can replace a four antibody panel for mismatch repair deficiency screening in colorectal adenocarcinoma.

AIMS: Currently, testing for mismatch repair deficiency in colorectal cancers is initiated by performing immunohistochemistry with four antibodies (MLH1, PMS2, MSH2 and MSH6). If any one of these stains is negative the tumour is considered microsatellite unstable and, if clinical circumstances warrant it, the patient is offered genetic testing for Lynch's syndrome. Due to the binding properties of the mismatch repair heterodimer complexes, gene mutation and loss of MLH1 and MSH2 invariably result in the degradation of PMS2 and MSH6, respectively, but the converse is not true. We propose that staining for PMS2 and MSH6 alone will be sufficient to detect all cases of mismatch repair deficiency and should replace routine screening with all four antibodies. METHODS: The electronic database of the department of Anatomical Pathology, Royal North Shore Hospital, Sydney, Australia, was searched for all colorectal carcinomas on which a four panel immunohistochemical microsatellite instability screen was performed. An audit of the slides for concordant loss of MLH1-PMS2 and MSH2-MSH6 was then undertaken. Unusual or discordant cases were reviewed and, in some cases, re-stained to confirm the staining pattern. RESULTS: Of 344 cases of colorectal cancer which underwent four antibody immunohistochemistry, 104 displayed loss of at least one mismatch repair protein. Of these, 100 showed concordant mismatch repair loss (i.e., loss of MLH1 and PMS2 or loss of MSH2 and MSH6). The four discordant cases comprised two single negative cases (1 MSH6 negative/MSH2 positive case, 1 PMS2 negative/MLH1 positive) and two triple negative (both MLH1/PMS2/MSH6 negative). The microsatellite instability (MSI) group showed a relatively high median age (69.3 years) due to the departmental policy of testing all cases with possible MSI morphology regardless of age. CONCLUSIONS: The sensitivity and specificity of a two panel test comprised of PMS2 and MSH6, compared to a four panel test, is 100%. No false negatives or positives were identified. We conclude that the two panel test should replace a four panel protocol for immunohistochemical screening for mismatch repair deficiency.

Dependence of nucleotide substitutions on Ung2, Msh2, and PCNA-Ub during somatic hypermutation.

During somatic hypermutation (SHM), B cells introduce mutations into their immunoglobulin genes to generate high affinity antibodies. Current models suggest a separation in the generation of G/C transversions by the Ung2-dependent pathway and the generation of A/T mutations by the Msh2/ubiquitinated proliferating cell nuclear antigen (PCNA-Ub)-dependent pathway. It is currently unknown whether these pathways compete to initiate mutagenesis and whether PCNA-Ub functions downstream of Ung2. Furthermore, these models do not explain why mice lacking Msh2 have a more than twofold reduction in the total mutation frequency. Our data indicate that PCNA-Ub is required for A/T mutagenesis downstream of both Msh2 and Ung2. Furthermore, we provide evidence that both pathways are noncompetitive to initiate mutagenesis and even collaborate to generate half of all G/C transversions. These findings significantly add to our understanding of SHM and necessitate an update of present SHM models.

Differential expression of hMLH1 in sporadic human colorectal cancer tumors and distant metastases.

Somatic defects in the mismatch repair system constitute an important pathway in colorectal carcinogenesis. We have examined the expression of mismatch repair proteins in sporadic stage IV colorectal tumors and their derived metastases. Sporadic tumors were further examined for differences in expression between the tumor transition zone and the invasive front. Expression of hMSH2, hMLH1, and hPMS2 was screened immunohistochemically in 92 stage IV tumors and derived liver metastases. In cases with loss of mismatch repair protein expression, lymph node metastases were also examined. Clinicopathological parameters and Ki-67 staining indexes were evaluated and compared. Four tumors displayed a complete loss of hMLH1/hPMS2 expression at the transition zone; however, three of these expressed both proteins at the invasive front and in liver and lymph node metastases. A further four were predominantly hMLH1/hPMS2 negative at the transition zone, but with distinct subclones of hMLH1/hPMS2-expressing cells at the transition zone. All of these tumors expressed hMLH1/hPMS2 at the invasive front and in liver metastases, with three also expressing hMLH/hPMS2 in lymph node metastases. No significant difference in the proliferative index was observed for the hMLH1/hPMS2-compromised group. In stage IV tumors re-expression of hMLH1/hPMS2 occurred, leading to different patterns of expression within the primary tumor and between tumor and metastases. This may have functional importance for the chemosensitivity of metastases compared to the primary tumor.

Identification of human T cell receptor gammadelta-recognized epitopes/proteins via CDR3delta peptide-based immunobiochemical strategy.

Human T lymphocytes, bearing T cell receptor (TCR) gammadelta, play an important role in anti-tumor/microbe immune responses. However, few tumor antigens recognized by TCRgammadelta have been defined so far. To investigate antigenic epitopes/proteins recognized by gammadeltaT cells, we have established a new immunobiochemical strategy that uses complementarity-determining region 3 of TCR delta chain (CDR3delta) peptide-mediated epitope/protein-binding assays. CDR3delta peptides synthesized using the CDR3 region in TCR Vdelta2 chain were validated for their binding specificity to target cells or tissues. These CDR3delta peptides were then employed as probes to pan putative epitopes in a 12-mer random peptide phage-displayed library and to identify putative protein ligands within tumor protein extracts by affinity chromatography and liquid chromatography/electrospray ionization-tandem mass spectrometry analysis. As a result, we have identified nine peptides and two proteins for TCRgammadelta, including human mutS homolog 2 (hMSH2) and heat shock protein (HSP) 60. All nine tested epitope peptides not only bind to gammadeltaT cells but also functionally activate gammadeltaT cells in vitro. Identification of HSP60 confirms the validity of this method as HSP60 is an identified ligand for TCRgammadelta. We show that hMSH2 is expressed on the surface of SKOV3 tumor cells, and cytotoxicity of Vdelta2 gammadeltaT cells to SKOV3 cells was blocked by anti-hMSH2 antibody, suggesting that hMSH2 may be a new ligand for TCRgammadelta. Taken together, our findings provide a novel immunobiochemical strategy to identify epitopes/proteins recognized by gammadeltaT cells.

Optimization of antibodies for detection of the mismatch repair proteins MLH1, MSH2, MSH6, and PMS2 using a biotin-free visualization system.

Testing for microsatellite instability (MSI) has become an important step in the planning of therapeutic and follow-up procedures for patients with colorectal cancer, both as a prognostic marker and as a screening tool for hereditary non-polyposis colorectal cancer. Today the gold standard for MSI testing is based on the polymerase chain reaction. Immunohistochemistry may represent an alternative or complement to molecular MSI testing. Antibodies against the protein products of the most commonly affected mismatch repair genes (hMLH1, hMSH2, hMSH6, and hPMS2) have been available for some time now. However, the quality of the primary antibody and optimization of the antigen retrieval methods are essential to get reproducible results. The aim of the present study was to test and optimize a panel of antibodies against the mismatch repair proteins MLH1, MSH2, MSH6, and PMS2 using biotin-free, polymer-based visualization systems. The antibodies were tested on multitissue blocks containing normal tissue and tumor tissue from patients with known microsatellite-stable and microsatellite-instable tumors. For all four antibody groups, the chosen clones gave specific and reproducible staining. Furthermore, with the PowerVision+ detection system, the influence of endogenous biotin was eliminated, the incubation time with the primary antibody was significantly reduced, and the primary antibody could be further diluted. The authors found that immunohistochemistry may provide a cost-effective and time-saving complement to the molecular MSI analysis, and using the PowerVision+ detection system has greatly decreased the turnaround time as well as reduced the cost of immunohistochemistry in the authors' laboratory.