CD4⁺ and CD8⁺ T cell responses to recombinant rhLCV lytic infection antigens by IFN-γ ELISPOT. rhLCV-specific T cell lines were derived from 13 naturally and 2 experimentally (141-97 and 144-97) rhLCV-infected rhesus macaques. Antigen-specific T cell responses were measured by IFN-γ ELISPOT of T cell lines stimulated with rVV-infected, autologous baLCV-LCL for 16 h. Numbers of spot forming cells (SFC) per million cells after subtraction of background values with control rVV infection are reported for positive results, and undetectable responses of <50 SFC are blank. Responses were assigned to CD4⁺ T cells if SFC were reduced to <50 SFC cells after CD4⁺ T cell depletion (white boxes), or CD8⁺ T cells if SFC were equal or increased after CD4⁺ T cell depletion (black boxes). Results that failed to fulfill these criteria after CD4⁺ T cell depletion were interpreted as indeterminate results (range, 15% to 90% of original ELISPOT response after CD4⁺ T cell depletion) and are shown as gray boxes. Animals with T cell lines studied by ICS are indicated by an asterisk. Indeterminate responses confirmed or not confirmed as CD8⁺ T cell responses by ICS are shown as gray boxes with black or white triangles, respectively. Naturally infected animals are listed in order of increasing age at time of study, and experimentally infected animals were studied 7 years after inoculation.

CD4⁺ and CD8⁺ T cell responses to recombinant EBV late lytic infection antigens by IFN-γ ELISPOT. EBV-specific T cell lines were derived from five seropositive adult humans by repetitive stimulation with irradiated, autologous EBV-LCL. Antigen-specific T cell responses were measured by IFN-γ ELISPOT of T cell lines stimulated with rVV-infected PBMC for 16 h. Numbers of spot forming cells (SFC) per million cells after subtraction of control values from vector control rVV-infected PBMC are reported for positive results, and responses of <50 SFC are blank. Responses were assigned to CD4⁺ T cells if SFC were reduced to <50 SFC cells after CD4⁺ T cell depletion (white boxes) or to CD8⁺ T cells if SFC stayed the same or increased after CD4⁺ T cell depletion (black boxes). Indeterminate results (range, 57% to 94% of original ELISPOT response after CD4⁺ T cell depletion) are shown as gray boxes. All indeterminate responses were confirmed as CD8⁺ T cell responses by ICS (data not shown), indicated by black triangles.

Intracellular cytokine staining of T cell lines stimulated with recombinant lytic infection antigens. T cell lines were exposed to rhLCV-LCL as a positive control, autologous, vector control rVV-infected baLCV-LCL as a negative control, or baLCV-LCL infected with rVV expressing individual lytic infection antigens at a ratio of 10:1 for 12 h. Cells were surface stained for CD3 and CD8, permeabilized, and stained for intracellular IFN-γ and TNF-α as previously described (5). At least 10⁶ events were collected with a FACSCalibur flow cytometer (BD) and analyzed by FlowJo software (Treestar). (A) Representative ICS analysis for Mm406-97 T cell line for IFN-γ (top row) and TNF-α (bottom row). Samples were gated on CD3⁺ cells, and plots show cytokine (x axis) versus CD8⁺ T cells (y axis). The percentage of cytokine positive CD8⁺ T cells for each analysis is shown at the top right. (B) Summary of ICS responses in rhLCV-specific T cell lines from 10 animals. Animals with indeterminate ELISPOT responses against E or L antigens are identified with a superscript letter. The percentage of cytokine-positive CD8⁺ T cells is shown for each test performed (blank, not done) with positive ICS responses (>2-fold increase over controls) shown in black boxes and negative ICS responses (<2-fold increase over control) shown in white boxes.