Kinetics of cytotoxic activity of effector and memory gp33-specific T cells. Cytotoxic activity of spleen effector and memory CD8⁺ T cells was analyzed by ⁵¹Cr release assay. EL4 cells, pulsed with gp33 peptide or unpulsed, were used as target cells. Specific lysis was determined at the indicated time points of incubation. E/T ratios indicate the ratio between gp33 tetramer positive T cells and target cells. One of four similar experiments is shown.

Cell surface expression of CD107a, intracellular IFNγ production, and grB content was analyzed at the indicated time points in effector and memory (d50) gp33-specific CD8⁺ T cells in the presence or absence of CHX. Plots are gated on CD8⁺ gp33 tetramer⁺ T cells. One of two equivalent experiments is shown.

Kinetics of degranulation, IFNγ production, and cytotoxicity of gp33-specific central and effector memory T cells. (A) Tetramer staining, cell surface CD107a expression, and intracellular IFNγ production was analyzed at the indicated time points in sorted CD62L positive and negative cells from LCMV memory mice (day 50). The top row shows staining of CD62L⁺ T cells (day 50) for CD8 and gp33 tetramer after the indicated time points of stimulation. The two bottom rows show staining for cell surface CD107 and intracellular IFNγ. Plots are gated on CD8⁺ gp33 tetramer⁺ T cells. One of three similar experiments is shown. (B) Cytotoxic activity of sorted CD62L positive and CD62L negative spleen cells of memory mice (day 50) was analyzed by ⁵¹Cr release assay. EL4 cells, pulsed with gp33 peptide or unpulsed were used as target cells. Specific lysis was determined at the indicated time points of incubation. E/T ratios indicate the ratio between gp33 tetramer positive T cells and target cells. The graphs on the right side show the percentage of grB⁺CD107⁻ and CD107a⁺ cells of CD8⁺tet⁺ T cells at the indicated time points after stimulation. One of three similar experiments is shown. (C) Percentage of grB⁺ cells of CD8⁺ tet⁺ cells. grB positive cells were quantified before antigen stimulation amongst CD8⁺tet⁺ T cells of d8 effector mice, of memory mice (days 35 and 50), of sorted CD62L⁺ and CD62L⁻ memory cells (days 30, 50, and 115), and of naive TCR transgenic mice.

In vivo cytotoxicity of adoptively transfused gp33-specific central and effector memory cells. (A) CD62L positive and negative CD8 T cells were purified from d35 LCMV memory mice. (left) gp33 tetramer staining: numbers indicate the percentage of CD8⁺ tet⁺ T cells. (right) CD62L expression on tetramer-gated CD8⁺ T cells. (B) Target cell numbers in LN 4 h after transfer. 3 × 10⁵ CD62L positive or CD62L negative CD8⁺tet⁺ T cells were transferred into naive recipient mice followed by transfer of 5 × 10⁶ gp33-pulsed and mock-pulsed CFSE/PKH26 stained target cells 3 h later. CFSE^low cells were pulsed with gp33 peptide and CFSE^high cells were mock pulsed. 4 h after target cell transfer, the ratio of CFSE^low/high target cells was analyzed in the LN. One representative staining of each group is shown. Cells are gated on PKH26 postive cells. Numbers indicate the percentage of specific target cell elimination. (C) Specific target cell elimination (a) 4 h after target cell transfer into recipients having been transfused with equal numbers of CD62L positive of CD62L negative LCMV memory T cells. (b) Percentage of CD8⁺ tet⁺ T cells 4 h after target cell transfer. (c) Percentage of PKH26⁺ target cells 4 h after target cell transfer. (d) E/T ratio (E = percentage of tetramer⁺ lymphocytes; T = percentage of PKH26⁺ lymphocytes) 4 h after target cell transfer.

Kinetics of cell surface expression of CD107 and production of intracellular IFNγ. Spleen cells of acutely infected mice (“Effector,” day 8 after i.v. infection with 200 pfu LCMV WE) or memory mice (50 d after i.v. infection with 200 pfu LCMV WE) were incubated with 1 μg/ml gp33 peptide. At the indicated time points, surface expression of CD107a and the intracellular content of IFNγ was analyzed by flow cytometry. Plots are gated on CD8⁺ gp33 tetramer⁺ T cells. One representative staining of at least six different experiments is shown.

Kinetics degranulation and grB content of naive, effector, and memory gp33-specific T cells. (A) Spleen cells from memory (d50), acutely infected, or naive P14 transgenic mice were stimulated with 1 μg/ml gp33 peptide. At the indicated time points, cell surface expression of CD107a and intracellular grB content was analyzed by flow cytometry. Plots are gated on CD8⁺ gp33 tetramer⁺ T cells. One of three similar experiments is shown. (B) Acquisition of cell surface CD107 and changes in grB content in stimulated effector CD8⁺ T cells. The plots 1–4 show the percentage of CD8⁺ tet⁺ T cells present in the four quadrants (1, CD107⁺grB⁻; 2, CD107⁺grB⁺; 3, CD107⁻grB⁻; and 4, CD107⁻grB⁺) after different time points of stimulation.

Degranulation and surface expression of CD62L and CD49d of central and effector gp33-specific memory T cells. (A) CD62L positive and CD62L negative cells were sorted from the spleen of LCMV memory mice (day 50) and stimulated with 1 μg/ml gp33 peptide. After the indicated time points, cell surface expression of CD107a and CD62L was analyzed by flow cytometry. Plots are gated on CD8⁺ gp33 tetramer⁺ T cells. One of three similar experiments is shown. (B) CD62L positive and CD62L negative cells were sorted from the spleen of LCMV memory mice (day 50) and stimulated with 1 μg/ml gp33 peptide. After the indicated time points, cell surface expression of CD107a and CD49d was analyzed by flow cytometry. Plots are either gated on CD8⁺ gp33 tetramer⁺ T cells or on CD8⁺ T cells. One of three similar experiments is shown. (C) Cell surface versus intracellular staining of CD107a. CD62L positive and CD62L negative cells were sorted from the spleen of LCMV memory mice (day 50). Sorted memory cells and day 8 effector cells were stained extracellularly or intracellularly for CD107a. Plots are gated on CD8⁺ T cells. One of two similar experiments is shown.

In vivo cytotoxicity of acutely LCMV-infected mice and memory mice. (A) gp33 peptide–loaded and unloaded target cells were adoptively transferred into acutely infected or memory recipient mice. Percentage of specific target cell elimination was analyzed in the blood at the indicated time points. (B) Specific target cell elimination was quantified in blood, spleen, and LN at 2 and 6 h after adoptive transfer of target cells. Numbers indicate the percentage of gp33 tetramer positive lymphocytes in the respective organs. (C) E/T ratio in blood, spleen, and LN at 2 and 6 h after adoptive transfer of target cells. Effector cells were gp33 tetramer positive lymphocytes. (D) Percentage of CD8⁺gp33 tetramer⁺CD62L⁺ cells in blood, spleen, and LN at 2 and 6 h after adoptive transfer of target cells. One of two similar experiments is shown.