Viral replication and associated weight loss are not affected by CD28. A, Weight loss was measured at the indicated days p.i. as described in Materials and Methods. Each line represents an individual mouse. B, Virus titers in the lung and spleen were measured at the indicated time points. Representative data from two independent experiments consisting of four to eight mice per group are shown.

Kinetics of the CD8⁺ T cell response against i.n. VV-WR infection in CD28^−/− mice. A and B, The B8R-specific primary CD8⁺ T cell response in the lung and spleen was quantified by tetramer staining at 10 days p.i. A, Representative plots gated on CD8⁺ cells are shown. Numbers indicate percentage of B8R tetramer⁺ CD8⁺ T cells. B, Total numbers of B8R-specific CD8⁺ T cells in each organ are graphed. C–E, Kinetics of the CD8⁺ T cell responses in the peripheral blood upon VV-WR infection. The percentages of CD8⁺ (C), CD62L^lowCD8⁺ (D), and B8R tetramer⁺CD8⁺ (E) were measured at the indicated time points. F and G, B8R-specific memory CD8⁺ T cells in the lung and spleen were measured by tetramer staining at 66 days p.i. F, Percentages of B8R tetramer⁺CD8⁺ T cells are graphed. Each dot represents an individual mouse. G, Total number of B8R-specific CD8⁺ T cells in each organ. Representative data from two independent experiments consisting of four mice per group are shown. Error bars and numbers in parentheses indicate SD. *, p < 0.05; **, p < 0.01.

CD28 costimulation is required both during the priming and recall phase of VV-specific memory CD8⁺ T cells to maximize the recall response. A, CD28 costimulation during the primary response is required for an optimal VV-specific recall response. In brief, 1.5 × 10⁶ purified CD8⁺ T cells from spleens of VV-WR-infected B6 or CD80/CD86^−/− mice (number of B8R-specific memory CD8⁺ T cells determined by tetramer staining) were transferred into naive B6-CD45.1⁺ mice. One day posttransfer, mice were challenged with 2 × 10⁶ PFU of VV-WR i.p. and expansion of B8R-specific CD8⁺ T cells in the spleen was enumerated by tetramer staining 5 days later. B, CD28 costimulation during challenge maximizes the recall response. In brief, 4 × 10⁶ purified CD8⁺ T cells from spleens of VV-WR-infected B6-CD45.1⁺ mice (number of B8R-specific memory CD8⁺ T cells determined by tetramer staining) were transferred into naive B6 or CD80/CD86^−/− mice. One day posttransfer, mice were challenged with 2 × 10⁶ PFU of VV-WR i.p. and expansion of B8R-specific CD8⁺ T cells in the spleen was enumerated by tetramer staining 5 days later. Experimental schemes are shown above, and representative data from two individual experiments are shown. ○, B6→B6; ●, CD80/CD86^−/− →B6 (A) or B6→CD80/CD86^−/− (B). Each set of connected circles represent individual mice, and numbers indicate average fold expansion of transferred memory cells after 5 days in each group, calculated as written in Materials and Methods. *, p < 0.05.

VV-specific memory CD8⁺ T cells exhibit an altered phenotype and do not produce IL-2 in the absence of CD28 costimulation. A, CD122 and CD27 expression on B8R-specific memory CD8⁺ T cells in various organs was analyzed by tetramer staining at day 66 (lung) and day 90 (spleen and BM) p.i. Representative plots gated on CD8⁺ B8R tetramer⁺ cells are shown. Numbers above indicate percentage of cells staining positive for the respective markers, and numbers below in parentheses represent the mean fluorescence intensity. B, Cytokine production by B8Rspecific memory CD8⁺ T cells was measured on day 66 (lung) and day 131 (spleen and BM) p.i. by intracellular cytokine staining following stimulation by B8R peptide. C, Cytotoxicity was assessed by an in vivo cytotoxicity assay as described in Materials and Methods. Representative data from two independent experiments consisting of three or four mice per group are shown. *, p < 0.05; **, p < 0.01.

CD28 costimulation is required for secondary expansion of VV-specific memory CD8⁺ T cells. A and B, VV-WR-infected C57BL/6 or CD28^−/− mice were challenged on day 125 p.i. with B8R peptide plus LPS i.v. and expansion of B8R-specific CD8⁺ T cells in the spleen was measured by tetramer staining 6 days later. A, Representative plots gated on CD8⁺ cells are shown and numbers indicate percentages of B8R tetramer⁺CD8⁺ T cells (±SD). B, Total number of B8R-specific CD8⁺ T cells in the spleen 6 days later. Error bars indicate SD. C and D, 2.5 × 10⁶ purified CD8⁺ T cells from spleens of VV-WR-infected B6 or CD28^−/− mice (number of B8R-specific memory CD8⁺ T cells determined by tetramer staining) were transferred into naive B6-CD45.1⁺ mice. One day posttransfer, mice were challenged with 2 × 10⁶ PFU of VV-WR i.p., and expansion of B8R-specific CD8⁺ T cells in the spleen was enumerated by tetramer staining 5 days later. Experimental scheme (C, above), gating (C, below), and total number of memory cells before and after challenge (D) are shown. D,○, B6→B6; ●, CD28^−/− →B6. Each set of connected dots represent individual mice, and numbers indicate average fold expansion of transferred memory cells after 5 days in each group, calculated as written in Materials and Methods. **, p < 0.01.

Recall responses in CD28-deficient mice are restored by IL-2 signaling during recall but not priming. A and B, C57BL/6 or CD28^−/− mice were infected i.n. with VV-WR and were treated daily with R-IgG or 50 μg of anti-IL-2 plus 1.5 μg of mIL-2 (IL-2 immune complex (IC), see Materials and Methods) during priming from days 0 to 6. After 2 mo, 2.8 × 10⁶ purified CD8⁺ T cells from each group (number of B8R-specific memory CD8⁺ T cells determined by tetramer staining) were transferred into naive B6-CD45.1⁺ mice. One day posttransfer, mice were challenged with VV-WR i.p., and expansion of B8R-specific CD8⁺ T cells in the spleen were enumerated by tetramer staining 5 days postchallenge. A, Experimental scheme. B, Total numbers of B8R-specific CD8⁺ memory cells before and after challenge are shown. Donor groups are indicated below. C and D, Experiment similar to A and B was conducted, except R-IgG or IL-2 immune complex was given daily during the recall response. C, Experimental scheme. D, Total numbers of B8R-specific CD8⁺ memory cells before and after challenge are shown. Each set of connected circles represent individual mice, and numbers indicate average fold expansion of transferred memory cells after 5 days in each group, calculated as indicated in Materials and Methods. **, p < 0.01.

Costimulatory requirements for recall responses of memory CD8⁺ T cells during a low-level persistent infection. Naive C57BL/6, MHV-68-infected C57BL/6, or CD28^−/− mice were challenged with 2 × 10⁶ PFU of VV-ORF6 i.p. and were analyzed for ORF6-specific CD8⁺ T cell expansion in the spleens by tetramer staining 5 days postchallenge. Representative plots gated on CD8⁺ cells (A) and total number of ORF6-specific CD8⁺ T cells (B) are shown. Numbers indicate percentage of ORF6–tetramer⁺CD8⁺ T cells (±SD). Representative data from two independent experiments consisting of three or four mice per group are shown. C and D, CD28 costimulation is required both during the priming and recall phase for maximal recall responses of MHV-68-specific CD8⁺ T cells. C, In brief, 2 × 10⁶ purified CD8⁺ T cells from spleens of MHV-68-infected B6 or CD80/CD86^−/− mice (number of ORF6-specific memory CD8⁺ T cells determined by tetramer staining) were transferred into naive B6-CD45.1⁺ mice. One day posttransfer, mice were challenged with 2 × 10⁶ PFU of VV-ORF6 i.p. and expansion of B8R-specific CD8⁺ T cells in the spleen were enumerated by tetramer staining 5 days later. D, In brief, 8 × 10⁶ purified CD8⁺ T cells from spleens of MHV-68-infected B6-CD45.1⁺ mice (number of ORF6-specific memory CD8⁺ T cells determined by tetramer staining) were transferred into naive B6 or CD80/CD86^−/− mice. One day posttransfer, mice were challenged with 2 × 10⁶ PFU of VV-ORF6 i.p. and expansion of ORF6-specific CD8⁺ T cells in the spleen were enumerated by tetramer staining 5 days later. Representative data from two individual experiments are shown. ○, B6→B6; ●, CD80/CD86^−/−→B6 (C) or B6→CD80/CD86^−/− (D). Each set of connected circles represent individual mice, and numbers indicate average fold expansion of transferred memory cells after 5 days in each group, calculated as written in Materials and Methods. *, p < 0.05; **, p < 0.01.