Memory T Cells Constitute a Subset of the Human CD8⁺CD45RA⁺ Pool with Distinct Phenotypic and Migratory Characteristics

CD8⁺CD45RA⁺ tetramer-binding cells express high levels of LFA-1. PBMC from an HLA-A2/B8 individual seropositive for EBV were screened by four-color flow cytometry to assess the phenotype of RAK-B8 tetramer-binding T cells. Cells were stained with a panel of surface marker Abs on FITC, tetramer PE, CD8 ECD, and CD45RA PE-Cy5. Cells were gated to determine the expression of each marker in the indicated populations. Filled areas of histograms indicate the higher-expressing population where a biphasic distribution was observed within the CD8⁺CD45RA⁺ population. Both CD11a and CD18 (the α- and β-chains of LFA-1, respectively) showed an absolute relationship with tetramer-binding cells. These results were consistent in 18 individuals tested using EBV tetramers GLC-A2, YVL-A2, and RAK-B8 or CMV tetramers NLV-A2 and TPR-B7.

Stability of the CD8⁺CD45RA⁺LFA-1^high phenotype on tetramer-bearing cells. a, Cryopreserved PBMC samples from adult patients with EBV-associated IM collected at intervals after infection were stained using CD11a FITC, tetramer PE, CD8 ECD, and CD45RA PE-Cy5 for four-color flow cytometry. The expression of CD11a was examined in the indicated cell populations. All tetramer-binding cells were in the high level population of CD11a expression at diagnosis and 1 and 10 years after infection. Results are from one HLA-B8 individual using RAK-B8 tetramer and are representative of three individuals tested. Filled areas of histograms indicate the CD11a^high population. Results for CD18 expression were identical with those for CD11a in each case. b, T cells isolated from umbilical veins were stained using CD11a FITC, CD8 ECD, and CD45RA PE-Cy5. The CD8⁺CD45RA⁺ fraction showed no cells with high-level expression of CD11a or CD18. Filled areas of histograms indicate the CD11a^high population. Representative of three subjects. c, Purified adult CD8⁺ T cell subsets (>95% purity) and whole cord blood mononuclear cells were labeled with a PNA probe specific for telomere repeat sequences and analyzed by flow cytomery. PNA fluorescence in adult CD8⁺CD45RA⁺CD11a^high cells was significantly less intense than in the CD8⁺CD45RA⁺CD11a^low cells but was indistinguishable from primed CD8⁺CD45RO⁺ cells. Data are representative of three experiments.

Chemokine receptor expression by CD8⁺ T cell subsets. PBMC from healthy adults were stained for flow cytometry with a panel of antichemokine receptor Abs labeled with FITC, CD11a PE, CD8 ECD, and CD45RA PE-Cy5. Expression of chemokine receptors was examined in CD8⁺CD45RA⁺CD11a^high, CD8⁺CD45RA⁺CD11a^low, and CD8⁺CD45RO⁺ cells. Expression of chemokine receptors by CD45RA⁺CD11a^high memory cells closely paralleled that of primed rather than naive CD8⁺ (CD45RA⁺CD11a^low) cells. Representative of five experiments.

Comparison of chemokine receptor expression by tetramer-binding CD45RA⁺ T cells and CD45RA⁺CD11a^high cells. PBMC from EBV-seropositive subjects were screened for chemokine receptor expression as described in Fig. 4. In parallel experiments, CD11a PE was replaced by the appropriate PE tetramer. Tetramer-binding CD8⁺CD45RA⁺ T cells show the same profile of chemokine receptor expression as CD8⁺CD45RA⁺CD11a^high cells. Representative of three experiments.

CD8⁺CD45RA⁺ memory cells migrate similarly to primed T cells. a, PBL from adult donors were depleted of adherent cells by culture on human serum-coated plates and enriched for CD8⁺ lymphocytes (60–80% CD8⁺) by immunomagnetic bead depletion using the Ab cocktail described in Materials and Methods. Overnight migration through 6.5-mm diameter, 5.0-μm Transwells was measured in the presence and absence of chemokines. Cells were resuspended from the Transwell chambers, accurately counted by flow cytometry, and phenotyped by staining with CD11a FITC, CD45RA PE, and CD8 Tricolor. Specific migration of the indicated cell populations was determined by subtracting the values obtained in the absence of chemokine. In vitro migration of CD45RA⁺CD11a^high memory cells closely paralleled that of primed rather than naive CD8⁺ cells. Mean ± SD of three experiments. b, Cells freshly isolated from blood, lymph node, and liver tissue of a single individual were stained with CCR7 FITC, CD11a PE, CD8 ECD, and CD45RA PE-Cy5 for four-color flow cytometry to determine the distribution of CD8⁺CD45RA⁺ subpopulations. All histograms were gated to include only CD8⁺CD45RA⁺ cells. The reciprocal distribution of CD11a and CCR7 most effectively discriminates naive CD8⁺CD45RA⁺CD11a^low cells from memory CD8⁺CD45RA⁺CD11a^high cells. One of eight individuals tested.