Conservation of Epstein-Barr virus cytotoxic T-cell epitopes in posttransplant lymphomas: implications for immune therapy

Abstract

Posttransplant lymphoproliferative disease can be treated by the infusion of Epstein-Barr virus-specific cytotoxic T lymphocytes, which were raised against lymphocytes immortalized with a laboratory strain of Epstein-Barr virus (B95.8). Whether the immunodominant epitopes in B95.8 are shared in virus from tumors will affect the general applicability of this therapy. We have characterized the viral strain and the sequence of commonly recognized cytotoxic T-lymphocyte epitopes in 25 posttransplant lymphoproliferative disease specimens from 19 patients. Type A virus was present in 24 of 25 specimens. No variation in two LMP2A epitopes and a few variations mostly outside the targeted epitopes or silent in three EBNA3C epitopes were found, with one variation (Arg to Lys) detected in an EBNA3C epitope in 12 of 24 tumors. However, cytotoxic T lymphocytes to B95-8-derived EBNA3C peptides specifically lysed both B95-8 and the Lys-variant peptide-loaded target cells, although with less efficiency. These results suggest that adoptive immunotherapy using cytotoxic T lymphocytes expanded with B95.8 stimulators or vaccine strategies using B95.8-derived sequence will generally target Epstein-Barr virus strains present in posttransplant lymphoproliferative disease tumors.

Figure 1.

PCR typing for EBV subtypes. A 153-bp band for type A EBV and a 246-bp band for type B virus are shown. Only one PTLD tumor (case 2) is associated with type B EBV infection.

Figure 2.

Sequencing for EBNA3C CTL epitopes. Lane 1, case 8; lane 2, case 2, type B EBV; lane 3, case 3a; lane 4, case 3b. The variations between type A and B EBV are shown by thick arrows including one missense mutation (A to T) within a CTL epitope (filled thick arrow). Two mutations (one silent C to T, one missense G to A) among type A EBV isolates are shown by thin arrows.

Figure 3.

Direct PCR sequencing analyses of EBV CTL epitopes in PTLD. A: The HLA A2.1-restricted EBNA3C epitope (underlined) is overlapped with the HLA B44 epitope (italics). B: The HLA A2.1-restricted LMP2A epitope (underlined) is overlapped with the HLA A24.2 epitope (italics). Tumor samples 3a, 3b, 3c, 4a, 4b, 7a, 7b, 7c, 9a, or 9b are from a single patient at different times.

Figure 4.

Cloning sequencing for EBV CTL epitopes in PTLD patients (cases 3, 4, 7, and 9) with multiple tumors at different times. A: The EBNA3C epitopes. B: The LMP2A epitope. The bolded variations were only detected by cloning sequencing, but the possibility that these changes represent PCR artifacts cannot be excluded.

Figure 5.

CTL killing of B95.8-derived peptide (Arg peptide) (left) and Lys-variant peptide (right). CTL clones were raised against these peptide-loaded autologous PBMCs, and tested for CTL recognition to peptide-loaded autologous PHA blasts labeled with ⁵¹CrO₄. Arg peptide, Lys peptide, and ctrl peptide are autologous PHA blasts loaded with different peptides.