Figure 1.
Clonally expanded memory CD4+ T cells target an immunodominant region of influenza H1-HA.(A) Memory CD4+ Tcm, Tem, and cTfh cell subsets were isolated from blood samples of donor HD1 6 and 12 mo after Inflexal V vaccination. Cells were labeled with CFSE and stimulated with the Inflexal V vaccine in the presence of autologous monocytes. Shown is the CFSE profile and the percentage of proliferating CFSElo cells on day 6 in the 12-mo sample. Percentage of CFSElo in the 6-mo sample was 38% (Tcm), 65% Tem, and 57% cTfh. (B) The Inflexal V–reactive CFSElo T cells were sorted, relabeled with CFSE, and stimulated with recombinant H1-HA in the presence of autologous monocytes. After 5 d, CFSElo proliferating T cells were sorted and cloned by limiting dilution. Shown is the experiment performed with the 12-mo sample; comparable results were obtained with the 6-mo sample. (C) A total of 456 H1-HA–specific CD4+ T cell clones were isolated from the Tcm, Tem, and cTfh cultures based on the proliferative response (stimulation index ≥3) to a pool of overlapping peptides spanning the entire H1-HA sequence. Proliferation was assessed on day 3 after a 16-h pulse with [3H]thymidine and expressed as counts per minute. The data are representative of at least two independent experiments. (D) Epitope mapping of the 456 H1-HA–specific T cell clones. Epitopes were identified by screening the T cell clones with the individual H1-HA peptides in at least three independent experiments, with consistent results. The x axis indicates H1-HA amino acid sequence; each color-coded segment represents the sequence recognized by individual clones isolated from Tcm (black), Tem (green), or cTfh (red) cultures. The numbers of H1-HA–reactive T cell clones isolated from each subset are reported. The chart at the bottom indicates HA1 and HA2 domains colored in blue and red, respectively (FP, fusion peptide; TM, transmembrane). (E) Rearranged TCR Vβ sequences of H1-HA–specific T cell clones were determined by RT-PCR followed by Sanger sequencing. Shown in the pie charts are the repertoires of rearranged TCR Vβ sequences of T cell clones recognizing immunodominant H1-HA401–420 and H1-HA411–430 epitopes. Each slice of the chart indicates a different TCR Vβ clonotype (H1-HA401–420, n = 16; H1-HA411–430, n = 39); the number of sister clones bearing the same TCR Vβ sequence are reported for each slice. The total number of clones sequenced is reported at the center. (F and G) To evaluate the clonal expansion of H1-HA–reactive memory T cell clones, TCR Vβ deep sequencing was performed on Inflexal V–reactive CFSElo Tcm, Tem, and cTfh cells and on ex vivo total Tcm, Tem, and cTfh cells isolated from the same blood samples and immediately sequenced. Comparison of TCR Vβ clonotype frequency distribution of total (x axis) and Inflexal V–reactive cells (y axis) is shown in F. Dots outside the dashed lines represent clonotypes that were found in only one of the two samples and that were assigned an arbitrary frequency value for graphical purposes. (G) The frequency distribution of TCR Vβ clonotypes from ex vivo total Tcm, Tem, and cTfh cells isolated from a blood sample of donor HD1 obtained 48 mo after the 2013/14 Inflexal V vaccination. TCR Vβ clonotypes recognizing the immunodominant H1-HA401–420 or H1-HA411–430 peptides found in any of the samples analyzed are colored in green and orange, respectively.

Clonally expanded memory CD4+ T cells target an immunodominant region of influenza H1-HA. (A) Memory CD4+ Tcm, Tem, and cTfh cell subsets were isolated from blood samples of donor HD1 6 and 12 mo after Inflexal V vaccination. Cells were labeled with CFSE and stimulated with the Inflexal V vaccine in the presence of autologous monocytes. Shown is the CFSE profile and the percentage of proliferating CFSElo cells on day 6 in the 12-mo sample. Percentage of CFSElo in the 6-mo sample was 38% (Tcm), 65% Tem, and 57% cTfh. (B) The Inflexal V–reactive CFSElo T cells were sorted, relabeled with CFSE, and stimulated with recombinant H1-HA in the presence of autologous monocytes. After 5 d, CFSElo proliferating T cells were sorted and cloned by limiting dilution. Shown is the experiment performed with the 12-mo sample; comparable results were obtained with the 6-mo sample. (C) A total of 456 H1-HA–specific CD4+ T cell clones were isolated from the Tcm, Tem, and cTfh cultures based on the proliferative response (stimulation index ≥3) to a pool of overlapping peptides spanning the entire H1-HA sequence. Proliferation was assessed on day 3 after a 16-h pulse with [3H]thymidine and expressed as counts per minute. The data are representative of at least two independent experiments. (D) Epitope mapping of the 456 H1-HA–specific T cell clones. Epitopes were identified by screening the T cell clones with the individual H1-HA peptides in at least three independent experiments, with consistent results. The x axis indicates H1-HA amino acid sequence; each color-coded segment represents the sequence recognized by individual clones isolated from Tcm (black), Tem (green), or cTfh (red) cultures. The numbers of H1-HA–reactive T cell clones isolated from each subset are reported. The chart at the bottom indicates HA1 and HA2 domains colored in blue and red, respectively (FP, fusion peptide; TM, transmembrane). (E) Rearranged TCR Vβ sequences of H1-HA–specific T cell clones were determined by RT-PCR followed by Sanger sequencing. Shown in the pie charts are the repertoires of rearranged TCR Vβ sequences of T cell clones recognizing immunodominant H1-HA401–420 and H1-HA411–430 epitopes. Each slice of the chart indicates a different TCR Vβ clonotype (H1-HA401–420, n = 16; H1-HA411–430, n = 39); the number of sister clones bearing the same TCR Vβ sequence are reported for each slice. The total number of clones sequenced is reported at the center. (F and G) To evaluate the clonal expansion of H1-HA–reactive memory T cell clones, TCR Vβ deep sequencing was performed on Inflexal V–reactive CFSElo Tcm, Tem, and cTfh cells and on ex vivo total Tcm, Tem, and cTfh cells isolated from the same blood samples and immediately sequenced. Comparison of TCR Vβ clonotype frequency distribution of total (x axis) and Inflexal V–reactive cells (y axis) is shown in F. Dots outside the dashed lines represent clonotypes that were found in only one of the two samples and that were assigned an arbitrary frequency value for graphical purposes. (G) The frequency distribution of TCR Vβ clonotypes from ex vivo total Tcm, Tem, and cTfh cells isolated from a blood sample of donor HD1 obtained 48 mo after the 2013/14 Inflexal V vaccination. TCR Vβ clonotypes recognizing the immunodominant H1-HA401–420 or H1-HA411–430 peptides found in any of the samples analyzed are colored in green and orange, respectively.

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