Isolation of a kidney-specific peptide recognized by alloreactive HLA- A3-restricted human CTL

• Published in: The Journal of immunology (1950) Vol. 154; no. 8; pp. 3880 - 3887
• Main Authors: Poindexter, NJ, Naziruddin, B, McCourt, DW, Mohanakumar, T
• Format: Journal Article
• Language: English
• Published: United States Am Assoc Immnol 15.04.1995
• Subjects: Amino Acid Sequence; Autoantigens - immunology; Cytotoxicity, Immunologic; Epitopes; HLA-A3 Antigen - immunology; Humans; In Vitro Techniques; Isoantigens - isolation & purification; Kidney - immunology; Molecular Sequence Data; Oligopeptides - chemistry; Oligopeptides - immunology; T-Lymphocytes, Cytotoxic - immunology
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.154.8.3880

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.