Figure 4.
Figure 4. Clonal expansions and cytokine production following in vivo antigenic challenge of long-term tolerant B6 mice and their graft-rejecting controls. (A) Ex vivo data. Freshly isolated splenocytes from graft-rejected mice have a higher frequency of HYDbUty tetramer–positive CD8+ T cells and HY peptide–specific IFN-γ–producing cells than their tolerant counterparts. Seven PBS-treated graft-rejected mice (○), and 6 mice pretreated with 3 × 100 μg HYAbDby that retained syngeneic male skin grafts for more than 400 days (▵), were boosted intraperitoneally with 5 × 106 male splenocytes. Seven days later spleen cell suspensions from individual animals were analyzed by FACS for percentage of HYDbUty tetramer–positive CD8+ T cells (i-ii) and by ELISPOT for IFN-γ production in response to irradiated female or male B6 splenocytes alone, or irradiated female splenocytes pulsed with HYDbUty, HYDbSmcy, or HYAbDby peptide. The results are expressed as spots/2 × 105 cells (iii-iv). (B) After in vitro restimulation. Splenic cultures from graft-rejected, in vivo–boosted mice show large clonal expansions of HYDbUty tetramer–positive CD8+ T cells following in vitro restimulation, whereas responses of tolerant mice are lower; intracellular IFN-γ and IL-10 cytokine responses of T cells from the in vitro cultures from tolerant mice are also diminished. Splenocytes from the rejected (•) or tolerant (▴) mice, shown in panels Ai-ii, were incubated with irradiated B6 male spleen cells and IL-2 for 7 days. The cultured cells were analyzed by FACS and the results shown as a percentage of HYDbUty tetramer–positive CD8+ T cells (i-ii). The cultured cells were also analyzed for intracellular cytokine production after restimulation with unpulsed (○, ▵), or HYDbUty and HYAbDby peptide–pulsed APCs (•, ▴) for 6 hours in the presence of monensin. The cells were stained with anti-CD8 or anti-CD4, fixed, permeabilized, and then stained with anti–IFN-γ or anti–IL-10. Results are shown as the percentage of IFN-γ (iii-iv) or IL-10–producing cells (v-vi) in the CD8+ (iii,v) or CD4+ T-cell population (iv,vi). Note: the y-axes of panels iii to vi differ to facilitate identification of individual mice.

Clonal expansions and cytokine production following in vivo antigenic challenge of long-term tolerant B6 mice and their graft-rejecting controls. (A) Ex vivo data. Freshly isolated splenocytes from graft-rejected mice have a higher frequency of HYDbUty tetramer–positive CD8+ T cells and HY peptide–specific IFN-γ–producing cells than their tolerant counterparts. Seven PBS-treated graft-rejected mice (○), and 6 mice pretreated with 3 × 100 μg HYAbDby that retained syngeneic male skin grafts for more than 400 days (▵), were boosted intraperitoneally with 5 × 106 male splenocytes. Seven days later spleen cell suspensions from individual animals were analyzed by FACS for percentage of HYDbUty tetramer–positive CD8+ T cells (i-ii) and by ELISPOT for IFN-γ production in response to irradiated female or male B6 splenocytes alone, or irradiated female splenocytes pulsed with HYDbUty, HYDbSmcy, or HYAbDby peptide. The results are expressed as spots/2 × 105 cells (iii-iv). (B) After in vitro restimulation. Splenic cultures from graft-rejected, in vivo–boosted mice show large clonal expansions of HYDbUty tetramer–positive CD8+ T cells following in vitro restimulation, whereas responses of tolerant mice are lower; intracellular IFN-γ and IL-10 cytokine responses of T cells from the in vitro cultures from tolerant mice are also diminished. Splenocytes from the rejected (•) or tolerant (▴) mice, shown in panels Ai-ii, were incubated with irradiated B6 male spleen cells and IL-2 for 7 days. The cultured cells were analyzed by FACS and the results shown as a percentage of HYDbUty tetramer–positive CD8+ T cells (i-ii). The cultured cells were also analyzed for intracellular cytokine production after restimulation with unpulsed (○, ▵), or HYDbUty and HYAbDby peptide–pulsed APCs (•, ▴) for 6 hours in the presence of monensin. The cells were stained with anti-CD8 or anti-CD4, fixed, permeabilized, and then stained with anti–IFN-γ or anti–IL-10. Results are shown as the percentage of IFN-γ (iii-iv) or IL-10–producing cells (v-vi) in the CD8+ (iii,v) or CD4+ T-cell population (iv,vi). Note: the y-axes of panels iii to vi differ to facilitate identification of individual mice.

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