Epitope Mapping of VZV-specific skin TRM

T cell lines were generated from skin biopsies taken from rash site and contralateral (uninvolved) site skin of persons recovered from herpes zoster at days 45, 90, and 360 from rash onset. The cell lines were screened for reactivity to all VZV open-reading frame (ORF) products using high throughput assays designed to activate and measure responses of only CD8 T cells or only CD4 T cells. Artificial antigen presenting cells expressing a single HLA class I molecule from a study subject and a single VZV ORF was used to activate CD8 T cells. Secreted IFNgamma was measured (data represent OD450) in culture supernatants using ELISA. For CD4 T cells, crude proteins made by in vitro transcription translation (IVTT) was used as antigen. T cell proliferation was measured using 3H-thymidine incorporation assay (data represent CPM).

Proteins that generated positive responses were mapped to epitope containing proteins using B-LCL as antigen presenting cells. Further studies using pan-specific HLA-DR, HLA-DP, or HLA-DQ blocking antibodies were done to identify the restricting HLA locus of the CD4 T cell response. More detailed studies confirmed the restricting HLA molecule through using artificial antigen presenting cells expressing single HLA class I (for CD8 T cells) or class II (for CD4 T cells). IFNg ELISA (OD450) was used to measure T cell activation.