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. 2007 Oct 15;179(8):4988-95.
doi: 10.4049/jimmunol.179.8.4988.

The surprising kinetics of the T cell response to live antigenic cells

Aaron J Tyznik  1 , Michael J Bevan
Affiliations

The surprising kinetics of the T cell response to live antigenic cells

Aaron J Tyznik et al. J Immunol. .

Abstract

Cooperation between CD4(+) and CD8(+) T cells is required for the proper development of primary effector and memory CD8(+) T cells following immunization with noninflammatory immunogens. In this study, we characterized murine CD4(+) and CD8(+) T cell responses to male-specific minor histocompatibility (HY) Ags following injection of live male cells into females of the same strain. Male cells are rejected 10-12 days after transfer, coinciding with the expansion and effector function of CD8(+) CTLs to two H-2D(b)-restricted epitopes. Although anti-HY CD4(+) T cell responses are readily detectable day 5 posttransfer, CD8(+) responses are undetectable until day 10. The early CD4(+) response is not dependent on direct presentation of Ag by donor male cells, but depends on presentation of the male cells by recipient APC. The CD4(+) T cell response is required for the priming of CD8(+) T cell effector responses and rejection of HY-incompatible cells. Unexpectedly, HY-specific CD4(+) T cells are also capable of efficiently lysing target cells in vivo. The delay in the CD8(+) T cell response can be largely abrogated by depleting T cells from the male inoculum, and donor male CD8(+) T cells in particular suppress host anti-HY CD8(+) responses. These data demonstrate dramatic differences in host T cell responses to noninflammatory Ags compared with responses to pathogens. We explain the delayed CD8(+) response by proposing that there is a balance between cross-presentation of Ag by helper cell-licensed dendritic cells, on the one hand, and veto suppression by live male lymphocytes on the other.

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Conflict of interest statement

Disclosures

The authors have no financial conflict of interest.

Figures

Figure 1
Figure 1
Primary CD8+ T cell responses to HY coincide with the clearance of male cells. Female mice were injected with 2 × 107 live male splenocytes and survival of donor cells and host CD8+ T cell responses were followed. At the indicated time points, splenocytes were stained directly ex vivo with H-2Db/Smcy-specific tetramers or stimulated in vitro with Uty, Smcy, or no peptide and stained for levels of intracellular IFN-γ. Numbers indicate the percentage of IFN-γ+ cells in the CD8+ population. A, Response of host CD8+ T cells to Uty or Smcy peptides in the spleen. B, HY-specific CD8+ T cells measured by intracellular IFN-γ production as well as H-2Db/Smcy-specific tetramer staining. Results are representative of two independent experiments. C, Total HY-specific CD8+ T cell responses were followed for 4 wk postimmunization with male cells. The values represent the combined response to the dominant epitopes Uty and Smcy measured by ICS. Error bars, SEM. Results are representative of six independent experiments. D, Survival of donor male or female UbC-GFP-transgenic cells in the spleen of host mice. Error bars, SEM (n = 3 for each time point).
Figure 2
Figure 2
In vivo cytolytic activity 15 days after primary immunization with male splenocytes. A, Equal numbers of Ly5.1 CFSEhigh HY peptide-pulsed female splenocytes and CFSElow control peptide-pulsed female splenocytes were injected i.v. into Ly5.2 C57BL/6 female mice (naive or day 15 postimmunization). In vivo cytotoxicity was assessed 8 h later by flow cytometry. Histograms are gated on Ly5.1+ splenocytes in recipient mice. Numbers at the top of each plot represent the percentage of CFSElow or CFSEhigh cells of the total Ly5.1+ donor cells recovered. Representative plots of Uty-pulsed (top panels) or Smcy-pulsed target cells (bottom panels). B, Cumulative data from three mice per group from two independent experiments representing Uty- or Smcy-pulsed target cells. C, Kinetics of cytolytic activity toward male splenocytes expressing endogenous levels of male Ag. Equal numbers of Ly5.1 CFSEhigh male and CFSElow female splenocytes were coinjected into Ly5.2 naive or day 15 immune female mice. In vivo cytotoxicity was assessed 24, 48, and 72 h after adoptive transfer by flow cytometry. Histograms are gated on Ly5.1+ donor cells recovered in the spleen of host mice. D, Cumulative data from two independent experiments of three mice per group at each indicated time point.
Figure 3
Figure 3
Primary CD4+ T cell responses to HY are rapid compared with the CD8+ T cell response. Female mice were injected with 2 × 107 male splenocytes and host CD4+ T cell responses were followed. At the indicated time points, splenocytes were harvested and stimulated in vitro for 4 h with Dby or no peptide and stained for intracellular IFN-γ and IL-2 accumulation. A, Intracellular IFN-γ staining in the CD4+ T cell population following in vitro peptide stimulation at the indicated times postimmunization. Numbers indicate the percentage of CD4+ T cells producing IFN-γ. Results are representative of six separate experiments of two to three mice per time point. B, Percentage of Dby-specific CD4+ T cells producing IFN-γ and IL-2 at the indicated time points. Numbers in parentheses indicate the percentage of IFN-γ+, CD4+ T cells producing IL-2. C, Kinetics of the Dby-specific CD4+ T cell response. Values represent percentage of IFN-γ+ cell in the CD4+ T cell population. Results are representative of six separate experiments of two to three mice per time point. Error bars, SEM.
Figure 4
Figure 4
Direct Ag presentation by donor male cells is not necessary for the rapid Dby-specific T cell response, but host CD4+ T cell responses are necessary for rejection of male cells and development of CD8+ effector T cell responses. A, Female mice received 2 × 107 live male MHC class II-deficient splenocytes and CD4+ and CD8+ T cell responses were monitored. Values represent the combined percentage of IFN-γ+ cells in the CD8+ T cell population responding to Uty and Smcy or the percentage of Dby-specific cells in the CD4+ T cell population. Results are representative of three separate experiments with two mice per time point. B, MHC class II-deficient female mice received 2 × 107 B6 male splenocytes. Response of host CD8+ T cells to Uty or Smcy peptides in the spleen were analyzed at various time points. Numbers indicate the percentage of IFN-γ+ cells in the CD8+ population.
Figure 5
Figure 5
MHC class II-restricted cytolytic activity following immunization with male cells is detectable 9 days postimmunization. A, Top panels, equal numbers of Ly5.1 CFSEhigh Dby peptide-pulsed and CFSElow control peptide-pulsed female splenocytes were injected i.v. into Ly5.2 naive or day 9 immune female mice. In vivo cytotoxicity was assessed in the lymph nodes 48 h after transfer. Representative histograms are gated on Ly5.1+MHC class IIhigh donor cells. Numbers at the top of each plot represent the percentage of CFSElow or CFSEhigh cells of the total Ly5.1+MHC class IIhigh donor cells recovered. A, Lower panels, equal numbers of Ly5.1 β2m−/− CFSEhigh male and CFSElow female splenocytes were coinjected into Ly5.2 naive or day 9 immune NK-depleted female mice. In vivo cytotoxicity was assessed in the lymph nodes 48 h after transfer. Representative histograms are gated on Ly5.1+MHC class IIhigh donor cells. Numbers at the top of each plot represent the percentage of CFSElow or CFSEhigh cells of the total Ly5.1+MHC class IIhigh donor cells recovered. B, Cumulative data from three independent experiments of three mice per group. Error bars, SEM.
Figure 6
Figure 6
CD8+ T cell responses to HY are accelerated when the immunogen is depleted of T cells. Female mice were injected with 2 × 107 male intact or T subset-depleted splenocytes and host CD8+ T cell responses were followed. Host mice were sacrificed at days 9, 12, and 15 postimmunization and splenocytes were assessed for intracellular IFN-γ production. A, Representative flow plots displaying CD8+ T cell response to Smcy peptide in the spleen following immunization with total, T-depleted, CD8-depleted, or CD4 depleted male splenocytes. Numbers indicate the percentage of IFN-γ+ cells in the CD8+ population. B, Total HY-specific CD8+ T cell responses to intact and T subset-depleted splenocytes as indicated. Values represent the combined response to Uty and Smcy peptides in each mouse at the indicated time points. Values plotted are the mean of two to three mice per time point from a minimum of four independent experiments. Statistically significant differences using the equal variable Student t test are indicated with * (p ≤ 0.0005) or ** (p ≤ 0.03). C, CD8+ T cell response to HY Ags was suppressed with increasing numbers of male donor cells. Female mice received 1 × 107 (■), 2 × 107 (formula image), or 4 × 107 (□) male UbC-GFP splenocytes and host T cell responses were measured at days 9, 12, and 15. Numbers represent the combined CD8+ T cell responses to Uty246–254 and Smcy738–746 measured by intracellular IFN-γ production. Error bars, SEM (n = 4 mice for each group) and are representative of two independent experiments. Statistically significant differences using Student's t test are indicated with * (p ≤ 0.02).
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