LC MALDI-TOF MS/MS and LC ESI FTMS analyses of HLA-B27 associated peptides isolated from peripheral blood cells.

Peptides eluted from peripheral blood cells of HLA-B*2705 healthy donor were analyzed by LC MALDI MS/MS and LC ESI FTMS techniques. The sequences of 92 peptide ligands identified from one healthy blood donor by LC MALDI-TOF MS/MS were compared with those previously published from in vitro long-term cell cultures available in SYFPEITHI database and splenocytes. It was found that 18 sequences confirmed within 1ppm mass error by LC ESI FTMS were already described and 3 of them matched with those previously reported from HLA-B*2705 splenocytes. Another 38 sequences validated within the same mass error were not found in SYFPEITHI database and are identified here for the first time. Finally, 36 sequences (5 sequences already published in SYFPEITHI database) were evaluated by LC MALDI-TOF MS/MS but no matches in the list of monoisotopic masses obtained from LC ESI FTMS were found.

Comparison of amino acid compositions of peptides eluted from HLA-B27 molecules of healthy individuals and patients with ankylosing spondylitis.

HLA-B27 is a relative risk factor for ankylosing spondylitis (AS) and is present in about 10% in European populations but in 95% of AS patients. Various data suggest that the HLA-B27 molecule itself could be the strongest risk factor, but there is no explanation for this association. To define differential antigen presenting features of HLA-B27 in healthy individuals and AS patients, a question that cannot be addressed by biochemical studies on cell lines, the HLA-B27 protein was purified from peripheral blood lymphocytes of AS patients and healthy controls and pool sequencing of the bound peptides was performed. Results show that peptides are rich in proline (Pro) and the content of arginine (Arg) is much lower in comparison with sequences listed in the register of peptides eluted from cell cultures. Statistically significant differences were detected in frequencies of a subset of amino acids, predominantly at positions in the middle of the peptides. The frequency of Glu was increased and Gln was decreased in peptides from AS patients. Detailed analysis of purity of the immunoisolated HLA molecules excluded that the peptides might originate from any co-purified HLA molecules other than B27. We conclude that statistically significant increase in the Glu/Gln ratio of peptides from AS patients, consistent with increased deamidation in vivo, may account for differential antigenicity of HLA-B27 in patients. Source protein(s) of deamidated peptides remain unknown.

Peptides eluted from HLA-B27 of human splenocytes and blood cells reveal a similar but partially different profile compared to in vitro grown cell lines.

The sequences and profiles of peptides which bind to HLA-B*2705 splenocytes and peripheral blood cells were compared with those previously published from in vitro long-term cell cultures. B*2705 peptide profile analysed by solid-phase Edman degradation and 15 individual peptide sequences determined by LC-MS/MS were partially similar to those defined from in vitro long-term cell cultures. Arg at P2 was found in 11 of 15 sequenced peptides (73.3%). This value is lower in comparison with other published data. Two sequences were matching to unknown proteins, which displayed similarity with myosin. These are first data on peptide sequences isolated directly from HLA-B27 molecules without prior in vitro propagation of the cells.

Antibodies to HLA class II antigens as a risk factor for acute rejection of the allogeneic kindey.

We have investigated the association between the presence of antibodies to HLA class II antigens and the development of acute and chronic rejection after kidney transplantation. Sera from seventy-one patients before, shortly (2 weeks), and in the period between 8 and 22 months after transplantation were analyzed by the standard complement-dependent cytotoxicity (CDC) test, ELISA-LATM, and LAT tests. Absence of antibodies to HLA class II antigens before and shortly after transplantation was associated with a lower incidence of rejection episodes in the first post-transplant year. Donor-specific class II antibodies could not be detected by the ELISA-LAT test and there was no statistically significant difference in serum creatinine levels between the antibody-positive and antibody-negative patient groups two years after transplantation. Our study suggests that anti-HLA class II antibodies represent a risk factor for the development of acute immunological complications during the first year after transplantation.

Clinical relevance of antibodies to HLA antigens undetectable by the standard complement-dependent cytotoxicity test.

Recent literary data suggest that antibodies to HLA antigens undetectable by the standard complement-dependent cytotoxicity test may cause not only chronic, but also acute immunological complications after kidney transplantation. The aim of this study was to investigate the significance of non-cytotoxic antibodies to HLA antigens for the development of immunological complications and a worse graft prognosis after first kidney transplantation. Sera before and early after transplantation from 120 first kidney recipients were analyzed by flow cytometry (FCXM), ELISA and the standard complement-dependent cytotoxicity (CDC) test. Pre-transplant FCXM negativity was related to a lower incidence of rejection episodes in the first post-transplant year ( P<0.01). A significant association between acute rejection and the presence of antibodies to HLA class II antigens before and after transplantation was also found ( P<0.05). Our study supports the findings of other centers of the detrimental role to the kidney graft played by anti-HLA antibodies undetectable by the classical CDC test.