In healthy carriers of human cytomegalovirus (HCMV), the virus-specific memory CD8 ؉ T-cell population is often dominated by CD28 ؊ CD45RA hi cells that exhibit direct ex vivo cytotoxicity but whose capacity for proliferation and generation of further memory cells has been questioned. We show that when highly purified CD28 ؊ CD45RA hi CD8 ؉ T cells are stimulated with viral peptide presented by autologous monocytes, the virusspecific T cells show early up-regulation of CD137 (4-1BB) and CD278 (ICOS), reexpress CD28, and proliferate with similarly high cloning efficiency in limiting dilution analysis as CD28 ؉ CD45RO hi cells or CD28 ؊ CD45RO hi cells. Using peptidepulsed autologous fibroblasts transfected with individual costimulatory ligands as antigen presenting cells, we showed CD137L to be a key costimulatory ligand for proliferation of CD28 ؊ CD45RA hi CD8 ؉ T cells and not CD80, CD86, or CD275 (ICOSL). Therefore, CD28 ؊ CD45RA hi CD8 ؉ T cells were not terminally differentiated but required a specific costimulatory signal for proliferation. IntroductionUnderstanding the heterogeneity of human antigen-experienced CD8 ϩ T cells has advanced by studying combinations of different phenotypic cell surface molecules. Sallusto et al 1 subdivided human antigen-experienced CD8 ϩ T cells on the basis of chemokine receptor CCR7 expression into CCR7 ϩ "central memory" cells capable of homing to secondary lymphoid organs and CCR7 Ϫ "effector memory" cells. The low molecular weight isoform of the common leukocyte antigen CD45RO 2 is expressed by antigenexperienced CD28 ϩ CD45RO hi cells, most of which express CCR7, and by CD28 Ϫ CD45RO hi cells, most of which lack CCR7. The high molecular weight isoform CD45RA is expressed by naive CD28 ϩ CD45RA hi cells that express CCR7. CD28 Ϫ CD45RA hi cells have been termed "effector memory RA" (T EMRA ) or "revertant memory" cells because of their re-expression of CD45RA. [3][4][5] On the basis of expression of costimulatory molecules CD28 and CD27, human antigen-experienced CD8 ϩ T cells have also been subdivided into early CD28 ϩ CD27 ϩ cells (most of which express CD45RO and CCR7), intermediate CD28 Ϫ CD27 ϩ and late CD28 Ϫ CD27 Ϫ cells (most of which lack CCR7). In association with different human persistent virus infections, the phenotypes of circulating virus-specific CD8 ϩ T cells in long-term virus carriers vary according to the virus. 6,7 It appears that the phenotype of virus-specific CD8 ϩ T cells may be associated with the site(s) in which virus antigen is expressed. Thus, in carriers of Epstein-Barr virus (EBV), CD8 ϩ T cells directed against the EBV latencyassociated antigens expressed in infected B cells are predominantly CD28 ϩ CD45RO hi cells, whereas CD8 ϩ T cells directed against EBV lytic antigens expressed in pharyngeal epithelial cells are predominantly CD28 Ϫ CD45RA hi cells. 8 The ␤ herpesvirus human cytomegalovirus (HCMV) establishes life-long infection with latency in cells of the myeloid lineage. 9 The CD8 ϩ T-cell response to HCMV in immunocompetent sub...

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