Results
Subset distributions differ in CMV, EBV, FLU, and HIV-1–specific CD8+ T cellsModels of virus infection were used to confirm that combined staining for these markers distinguishes the effector, memory, and recently activated subsets and that the maturation state can provide a measure of the degree of control over the virus. We evaluated the distribution of antigen-specific T cells across the various maturation states in the context of several common viral infections. FLU represents a resolved infection in which virus is no longer present. EBV and CMV represent persistent, well-controlled infections. In the case of EBV, antigen burden is expected to be moderate because the virus can propagate without the need to replicate. In contrast, CMV represents a persistent infection with ongoing replication in the host. In addition, we also studied a series of HIV-1–infected patients who were chosen to reflect a spectrum of viral control.
These phenotypes and functional assignments are based on the collective experimental data found elsewhere. Combined CD45RA and CD27 staining distinguishes terminally differentiated and intermediate effector cells from acute, intermediate memory, and memory T cells. For cells that are CD27+, CD45RA, and CD28 staining is then used to distinguish the acute, intermediate memory, and memory subsets. In nonhuman primate immunization studies, we have observed that tetramer-positive T cells sometimes pass through the RA+27+28− stage before regaining CD28 expression (data not shown), and this is the basis for describing the RA+27+28− cells as an intermediate stage of maturation toward the memory subset.
Subset distributions for CMV-, EBV-, and FLU-specific responses from healthy donors and for HIV-1–specific responses in patients with PVLs >1000 copies/mL were determined by staining PBMC with the various combinations of tetramer and cell surface markers.
The distribution for the different virus-specific T cells was remarkably similar among different donors. For FLU and EBV, the tetramer-positive cells were >85% CD27+, which made determination of the subset distribution on the basis of the combined stains straightforward. The results from 7 donors indicate that FLU-specific CD8+ T cells have matured primarily to memory. On average, 79% of the FLU tetramer-positive cells were found to be memory, 59% of these were RA−27+28+, and 20% had reverted to CD45RA expression and were RA+27+28+. Compared with that of FLU, EBV-specific T cells had a more heterogenous subset distribution. For 10 donors, the tetramer-positive cells were primarily memory (on average 55%), with the rest being distributed between the acute stage and a stage that is apparently an intermediate between the acute and memory stages of maturation. In the case of CMV, where the cells are mixed for CD27 expression, the subset distribution is determined by accounting for the CD27− cells in the CD45RA and CD28 subsets (see Subjects, Materials, and Methods). CMV-specific T cells had the most heterogenous distribution of the 3 viruses, but terminally differentiated effectors predominated (CD45RA+CD27−CD28−). In contrast to well controlled infections, in which the majority of T cells have matured past the acute stage, HIV-1–specific T cells from donors with PVLs >1000 copies/mL are of a phenotype that is associated with acute-phase expansions. For 8 donors, 60%–90% (mean, 76%) of the tetramer-positive cells remained in the RA−27+28− subset.