Ex vivo phenotype and frequency of influenza virus-specific CD4 memory T cells

Abstract

Recent advances in class II tetramer staining technology have allowed reliable direct ex vivo visualization of antigen-specific CD4 T cells. In order to define the frequency and phenotype of a prototype response to a nonpersistent pathogen, we have used such techniques to analyze influenza virus-specific memory CD4 T cells directly from blood. These responses are stably detectable ex vivo at low frequencies (range, 0.00012 to 0.0061% of CD4 T cells) and display a distinct "central memory" CD62L(+) phenotype.

FIG. 1.

Frequencies of circulating DR1-HA tetramer-positive cells ex vivo. (A) Example of ex vivo DR1-HA tetramer staining of CD4 T cells before and after magnetic bead enrichment for PE-positive cells. PBMCs were stained with PE-conjugated DR1-HA tetramer, APC-conjugated anti-CD4 antibody, PerCP-conjugated anti-CD14 and -CD19 antibodies to exclude monocytes and B cells, respectively, and Via-Probe to exclude dead cells. Cells were incubated with anti-PE microbeads, and PE-positive cells were selected (enriched) on a magnetic column. Plots are gated on CD4⁺ CD14⁻ CD19⁻ Via-Probe⁻ cells. The plot on the left shows DR1-HA tetramer staining of CD4⁺ cells before enrichment, and the plot on the right shows the same cell population after enrichment. The frequency of DR1-HA tetramer-positive cells after enrichment was calculated by dividing the number of CD4⁺ tetramer⁺ cells after enrichment by the input number of CD4 cells. In this example, the input number of CD4 cells was 759,340 and the output number of CD4⁺ DR1-HA⁺ cells was 46. (B) Reproducibility of the frequency estimations of DR1-HA tetramer-positive cells in six healthy DR1-positive individuals from a single time point. Between two and eight replicate tetramer staining assays were performed on a single PBMC collection from each subject. The frequency of tetramer-positive cells was analyzed as described above. The horizontal bar represents the mean tetramer-positive frequency for each individual. (C) Longitudinal analysis of DR1-HA tetramer-positive cells in two DR1-positive individuals. Four PBMC samples were obtained over a 12-month period and analyzed as described above. The mean frequency of DR1-HA tetramer-positive cells at each time point is plotted for subjects 1 and 4.

FIG. 2.

Ex vivo phenotypic and functional analysis of DR1-HA tetramer-positive cells. (A) Example of phenotypic analysis on DR1-HA tetramer-positive cells for six markers pertaining to memory status of CD4⁺ T cells. Fresh PBMCs were stained as described for Fig. 1A with the addition of an FITC-conjugated antibody for either CD25, CD27, CD28, CD45RA, CD62L, or CCR7. Plots are shown after enrichment for PE-positive cells and are gated on CD4⁺ CD14⁻ CD19⁻ Via-Probe⁻ events as described for Fig. 1. The frequency shown indicates the percentage of CD4⁺ DR1-HA tetramer⁺ cells positive for each marker. These results were obtained from subjects 1 and 4. (B) Summary of phenotypic data for four DR1-positive individuals. Individual data points were obtained as described above. (C) Ex vivo functional analysis of HA-specific CD4 cells. The plot on the left indicates enrichment for DR1-HA tetramer-positive cells from subject 6; the frequency of tetramer-positive cells in this subject was 0.0018% of CD4 T cells. PBMCs from the same time point were stimulated with the DR1-HA peptide (aa 306 to 318) for 6 h at 37°C, followed by enrichment for IFN-γ- and IL-2-positive cells with the IL-2 (PE) and IFN-γ (APC) secretion assay cell enrichment and detection kits according to the instructions of the manufacturer (Miltenyi Biotec). The percentages shown indicate the relative proportions of CD4 cells producing IFN-γ and/or IL-2 following stimulation with the HA peptide; background levels of nonspecific cytokine production have been subtracted. The total frequency of cytokine-positive cells after subtraction of background in this subject was 0.0017%. The frequencies of IFN-γ single-positive, IFN-γ IL-2 double-positive, and IL-2 single-positive cells were 0.00018, 0.0013, and 0.00021%, respectively, of CD4 cells.