Antiviral CD8⁺ T cells express high levels of PD1 during GVHD, but can be expanded ex vivo. BALB/c (non-GVHD) and DBA/2 (GVHD) were transplanted with BALB/c BM (5 × 10⁶) and CD3⁺ T (2.5 × 10⁶) cells. Mice were infected with 5 × 10³ pfu MCMV 4 weeks after transplantation, and spleen cells were harvested on day 6 after MCMV infection. (A-C) The expression of PD1 was determined for CD8⁺ T cells in conjunction with IE1-specific tetramer binding, and compared with uninfected control mice. (A) Representative plots depicting the expression of PD1 and IE1-tetramer binding by splenic CD8⁺ T cells. (B) The mean fluorescence for PD1 expression by CD8⁺ T cells and (C) IE1-specific CD8⁺ T cells. (D-E) CD8⁺ T cells were purified from the spleens of infected GVHD mice and cultured for 5 days with DCs and IE1 peptide, with and without 10 ng/mL IL-2. (D) Representative plots depicting IE1⁺ CD8⁺ T cells. (E) The number of IE1-specific CD8⁺ T cells recovered after coculture with DCs. (A-C) n = 4 mice per group, representative of at least 3 independent experiments; (D-E) data are from 1 experiment with a pool of 6 mice. *P = .01-.05, **P = .001-.01, ***P = .0001-.001. BMDC, DCs prepared in vitro after culturing BM from uninfected BALB/c with Flt3 ligand for 11 days; cDC, conventional DCs enriched from uninfected BALB/c spleens.