Quantification of HLA class I molecules on renal cell carcinoma using Edman degradation

<p>Abstract</p> <p>Background</p> <p>Unimpaired HLA class I antigen presentation is a prerequisite for the recognition of tumor cells by cytotoxic T lymphocytes and thus essential for the success of anticancer immunotherapeutic concepts. Several approaches have been taken in the immunotherapy of metastatic renal cell carcinoma (RCC), however of limited success. HLA loss or down-regulation have often been reported and might interfere with immunotherapeutic approaches aimed at the recognition of HLA-presented peptides.</p> <p>Methods</p> <p>We employed a quantitative method of molecular analysis for the comparison of HLA amounts on primary tumor, normal kidney and metastases of RCC, using Edman degradation. We analyzed a series of 47 RCC samples including corresponding renal parenchyma, local lymph node metastases and distant metastases.</p> <p>Results</p> <p>Results of quantitative Edman degradation revealed significantly higher HLA yields on primary tumor and metastases compared to normal kidney tissue. This effect was shown not to result from infiltrating immune cells, since tumor-infiltrating lymphocytes had no influence on the overall HLA recovery from tumor tissue. Unexpectedly, we found a higher amount of HLA class I molecules on distant metastases compared to local lymph node metastases.</p> <p>Conclusion</p> <p>Edman degradation allows the direct quantitative comparison of HLA class I protein expression by tumor or normal tissue and metastases of RCC patients. Our results raise hopes for improving the success and effectiveness of future immunotherapeutic concepts for metastatic RCC.</p

Study of the role of functional variants of SLC22A4, RUNX1 and SUMO4 in systemic lupus erythematosus

Background: Functional polymorphisms of the solute carrier family 22, member 4 (SLC22A4), runt related transcription factor 1 (RUNX1) and small ubiquitin- like modifier 4 (SUMO4) genes have been shown to be associated with several autoimmune diseases. Objective: To test the possible role of these variants in susceptibility to or severity of systemic lupus erythematosus (SLE), on the basis that common genetic bases are shared by autoimmune disorders. Methods: 597 SLE patients and 987 healthy controls of white Spanish origin were studied. Two additional cohorts of 228 SLE patients from Sweden and 122 SLE patients from Colombia were included. A case control association study was carried out with six single nucleotide polymorphisms (SNP) spanning the SLC22A4 gene, one SNP in RUNX1 gene, and one additional SNP in SUM04 gene. Results: No significant differences were observed between SLE patients and healthy controls when comparing the distribution of the genotypes or alleles of any of the SLC22A4, RUNX1, or SUMO4 polymorphisms tested. Significant differences were found in the distribution of the SUMO4 genotypes and alleles among SLE patients with and without nephritis, but after multiple testing correction, the significance of the association was lost. The association of SUMO4 with nephritis could not be verified in two independent SLE cohorts from Sweden and Colombia. Conclusions: These results suggest that the SLC22A4, RUNX1, and SUMO4 polymorphisms analysed do not play a role in the susceptibility to or severity of SLE

A mutated HLA-A*0101 allele in the colorectal cell line HCA-7.

The colorectal cell line HCA-7 expresses surface human leucocyte antigen-A*0201 (HLA-A*0201), but lacks expression of HLA-A*0101 whilst the normal B-cell line (EVA-1224), derived from the same individual, expresses both surface HLA-A1 and HLA-A2. Amplification refractory mutation system-polymerase chain reaction analysis, using sequence-specific primers, suggested that HCA-7 has a mutation in a 7 base pair (bp) cytosine repeat sequence located at the beginning of Exon 4 (bp 621-627). Cloning and sequencing revealed HCA-7 to have eight cytosine residues in this repeat sequence. In contrast, EVA-1224 contained only 7 cytosines. Analysis of the mRNA for HLA-A*010 using reverse trancriptase-polymerase chain reaction (RT-PCR), with an allele-specific 5' primer in exon 2 (bp 253-271) and a series of 3' primers in exons 3, 4 and 7 and in the 3'untranslated region, revealed that HCA-7 contained a shortened message terminating in the region of the exon 3/4 boundary. The insertion of an extra cytosine in this region, which is only two bases from the exon 3/4 splice site, is presumed to lead to a splicing defect between exons 3 and 4 resulting in the lack of expression of a functional HLA-A*0101 product. HCA-7 is mismatch repair (MMR) defective due to lack of expression of hMLH1 resulting from hypermethylation of the promoter region. The consequential increase in errors in single-nucleotide repeat stretches of DNA can account for the HLA-A*0101 mutation. This has probably then been selected for in the tumour to enable escape from immune attack against an HLA-A*0101-restricted tumour-specific determinant that has also arisen as a result of the tumour being MMR defective