Detection of Vaccinia-specific CD8⁺ cells producing GzB post Vaccinia reimmunization. Three healthy volunteers who had been vaccinated in childhood were bled prior to revaccination with Dryvax^® (day 0), and on days 14 and 105 after the revaccination. Ex vivo IFN-γ (a, b) or GzB (a, c) ELISPOT assays were performed with 400,000 PBMC per well, using Vaccinia virus (Vaccinia WR/ABI at 1 moi) for recall. For each time point and donor the mean spot numbers for triplicate wells are shown and connected by a line. (a) Original well images are shown for one of the donors, with analytes and time points specified.

CD8⁺ memory cells that do not secrete GzB ex vivo start secreting it 2 - 4 days after restimulation. CD8⁺ cells and B cells were isolated from PBMC of healthy donors and cultured with an individual CEF peptide that induced an ex vivo IFN-γ response (here: EBV BMLF 1 259) or an A-2-restricted HIV control peptide (RT346-354 LAI – this peptide did not induce an ex vivo IFN-γ response). After culture for the specified duration in a tissue culture plate, the cells were harvested, counted, and cell numbers adjusted before transfer into IFN-γ or GzB pre-coated ELISPOT plates, followed by a 24 h single color ELISPOT test. The results for the IFN-γ assay correspond to the y-axis on the left, those of the GzB assay to the y-axis on the right. Mean and SD for duplicate wells are shown. Results are shown for one antigenic peptide (here: EBV BMLF 1 259) in one donor and are fully representative of six donors tested.

CD8⁺ cell-derived IFN-γ spots are smaller than CD4⁺ T cell-derived IFN-γ spots. (a) Spot numbers obtained for PBMC or purified CD8⁺ cells were normalized for 100,000 cells and corresponding data points are connected. (b) CEF-peptide induced IFN-γ spots are CD8⁺ cell derived while protein-antigen-induced IFN-γ spots are produced by CD4⁺ cells. PBMC, CD8⁺ and CD4⁺ enriched cells were tested for either mumps protein (solid bars) or EBV BMLF 1 259 peptide (open bars). Cell numbers of CD8⁺ and CD4⁺ cells were selected as specified in Materials and Methods matching the frequency of these cells in the PBMC. (c) Mean IFN-γ spot size induced by CEF peptides in PBMC vs. purified CD8⁺ cells. (d) Mean IFN-γ spot size produced by CD8⁺ and CD4⁺ cells. IFN-γ spot size distributions induced by the individual CEF peptides and the protein antigens mumps or candida were compared. The bars show the mean ± SD of spot sizes established in 18 donors for each purified cell type as indicated on the x-axis. A representative example of CEF-peptide-induced CD8⁺ cell derived IFN-γ spots is shown (e). Spot size distribution for such CD8 cell-derived spots from triplicate wells is shown in (g). A representative example of protein antigen-induced CD4 cell derived IFN-γ spots is shown in (f). The spot size distribution for triplicate wells of CD4 cell derived spots is shown in (h).

The cytokine signature of EBV- and CMV-specific CD8⁺ memory cells at isolation from healthy donors. Twenty nine donors were screened for CEF peptide reactivity. Of these subjects, donors were selected that are HLA-A2 positive and that showed an IFN-γ recall response to EBV BMLF1 259 and CMV pp 65 495, as specified by the solid circle or the solid square, respectively. (a) The peptide-induced secretion of an extended panel of cytokines (IL-2, TNF-α, GM-CSF, IL-4, IL-5, IL-10) and GzB was measured in ex vivo ELISPOT assays in freshly isolated PBMC. The medium subtracted mean spot number is shown for all cytokines. The number of donors tested for each cytokine and peptide was between 4 and 12, as specified. (b) The cytokine signature of EBV BMLF1 259 and CMV pp 65 495-specific purified CD8⁺ cells. CD8⁺ cells and B cells were purified by negative selection as specified in Materials and Methods. One hundred thousand CD8⁺ cells were plated with 50,000 B cells. The peptides are specified by the symbols as in Fig. 1a. (c) Purified CD8⁺ cells and B cells were stimulated with PHA. The legend to (a) and (b) applies.

HIV peptides induce GzB secretion in PBMC of HIV-infected donors. GzB and IFN-γ secretion by CD8⁺ cells is dissociated in these donors. (a and b), PBMC were obtained from 7 HIV-infected individuals and tested in 24 h ex vivo IFN-γ (a) and GzB (b) ELISPOT assays. A peptide library covering HIV-1 gag peptides 1 – 480 was used for recall. The peptides were 20-mers and walked the sequence of gag in steps of 5 a.a. They were tested individually (as specified by the peptide serial numbers on the x-axis), in duplicate well. The means of the duplicates are shown. (c and d) Because 20 a.a. long peptides can induce CD4⁺ cells in addition to CD8⁺ cells, PBMC of HIV-infected subjects were CD4 cell-depleted before testing with the HIV peptide library in IFN-γ (c) and GzB (d) ELISPOT assays. Since all HIV⁺ donors had known low CD4⁺ cell count (approx. 150 CD4⁺ cells/µl), similar numbers of CD8⁺ T cells were used for antigen stimulation in both the total (a and b) and the CD4⁺ depleted (c and d) cell populations. Selected peptides were tested that previously scored positive/negative. An asterisk highlights peptides that either induced IFN-γ or GzB only.