Isolation of a kidney-specific peptide recognized by alloreactive HLA-A3-restricted human CTL.
The molecular nature of tissue-specific Ags involved in MHC-restricted CTL responses is as yet undefined. To determine the specificity of these peptides, their function, and their possible relationship to allograft rejection, we have utilized human kidney-specific CD8+ CTL clones to screen reversed-phase HPLC (RP-HPLC)-separated self peptides presented by allo-class I molecules. One of these clones is HLA-A3-restricted and the other HLA-B62-restricted, lysing human kidney cell lines but not MHC identical B lymphoblastoid cells which express the appropriate HLA molecules. We have identified a biologically active RP-HPLC fraction containing self peptides eluted from affinity-purified MHC molecules from HLA-A3+ kidney. This peptide is not expressed in HLA-A3+ spleen. Similarly, a HLA-B62-associated peptide fraction was identified in kidney but not in spleen using the HLA-B62-restricted CTL clone. Sequence analysis of the biologically active fraction from HLA-A3 kidney revealed multiple peptides. Because of the ambiguity of the peptide sequence, a mixed peptide library corresponding to this sequence was synthesized that included the HLA-A3 binding motif. The biologically active peptide library was RP-HPLC fractionated and the fraction containing HLA-A3-restricted CTL activity was sequenced. The resulting sequence of the alloreactive HLA-A3-restricted peptide epitope is GPPGVTIVK. By using this unique strategy, we describe the successful isolation and sequencing of an antigenic peptide that is recognized by a human alloreactive kidney-specific CTL clone.
Poindexter
N J
NJ
Department of Surgery, Washington University School of Medicine, St. Louis, MO 63110, USA.
Naziruddin
B
B
McCourt
D W
DW
Mohanakumar
T
T
D000595:Amino Acid Sequence; D001324:Autoantigens / Q000276:immunology; D003602:Cytotoxicity, Immunologic; D000939:Epitopes; D015791:HLA-A3 Antigen / Q000276:immunology*; D006801:Humans; D066298:In Vitro Techniques; D007519:Isoantigens / Q000302:isolation & purification*; D007668:Kidney / Q000276:immunology*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D001324:Autoantigens; D000939:Epitopes; D015791:HLA-A3 Antigen; D007519:Isoantigens; D009842:Oligopeptides