Following D⁺/R^- alloSCT, HCMV pp65-specific CD8⁺ T cells were undetectable in HCMV-seronegative recipient of alloSC transplant 07, but an immunodominant HCMV-specific donor CD8⁺ T-cell clone was detectable in recipient 08 who developed severe GVHD. (A) Gated on CD3⁺ T cells. MHC class I HCMV pp65_417-426 tetramer⁺ CD8⁺ T cells were present in donor 07 PBMCs and allograft but were undetectable in recipient 07 at 30 months after alloSCT. (B) With the use of clonotypic probing, the Vβ14⁺ donor CD8⁺ T-cell clone specific for HCMV pp65_417-426 (clone 7.1) was not detectable in any of the sequential peripheral blood samples taken from recipient 07 following alloSCT. Weekly blood tests for HCMV DNA were consistently negative. In the same donor-recipient pair, the Vβ14⁺ donor CD8⁺ T-cell clone specific for EBV EBNA3C_880-891 (clone 7.2) was detected in recipient PBMCs at all time points tested following alloSCT. In the controls, the HCMV⁺and EBV⁺-labeled lanes refer to the positive control cDNA used from clones 7.1 and 7.2, respectively. (C) The Vβ13.1⁺ donor CD8+ T-cell clone specific for HCMV pp65_495-503 (clone 8.1) was detectable in sequential peripheral blood samples taken from recipient 08 after alloSCT. Recipient 08 had severe GVHD and died at 6 months after alloSCT. Weekly blood tests for HCMV DNA were negative.