Perforin-dependent elimination of dendritic cells regulates the expansion of antigen-specific CD8+ T cells in vivo

Abstract

The lifespan and survival of dendritic cells (DC) in vivo are potentially critical to the expansion of T cell immune responses. We have previously reported that DC loaded with specific antigen are rapidly eliminated by cytotoxic T lymphocytes (CTL) in vivo, but the site, mechanism, and consequences of DC elimination were not defined. In this article we show that DC elimination in vivo occurs in a perforin-dependent manner and does not require IFN-gamma or the presence of CD4(+)CD25(+) regulatory T cells. Most importantly, failure to eliminate DC had profound consequences on the CTL immune response. Perforin-deficient mice showed a progressive increase in the numbers of antigen-specific CD8(+) T cells after repeated immunizations with DC. In contrast, in control mice the number of antigen-specific CD8(+) T cells did not notably increase with repeated immunizations. Lastly, we also show that CTL-mediated elimination of DC occurs in peripheral tissues but not in the lymph node. Our data suggest that CTL act as "gatekeepers" that control access of antigen-loaded DC into the lymph node, thereby preventing continued expansion of antigen-specific T cells.

Fig. 1.

Antigen-loaded DC are eliminated in tissues of DC-immunized mice and are sensitive to CTL killing in vitro. Groups of CD45-congenic mice were immunized s.c. in the flank with LPS-treated congenic DC as indicated. Seven days later mice were injected intradermally in the ear with equal numbers of C57BL/6 DC labeled with CFSE or CMTMR and loaded with gp33 or left untreated, respectively. After 48 h mice were killed, and ear cell suspensions were prepared, stained with CD45.2-specific antibodies, and examined for the presence of fluorescent donor DC by FACS. Bars indicate the average proportion of gp33-loaded DC (DC⁺) remaining in tissue, as a percent of the coinjected control DC (DC^–), +SD for four samples.

Fig. 2.

Antigen-loaded DC survive in lymph nodes and induce proliferation of specific CTL adoptively transferred i.v. (A) Groups of C57BL/6 mice were injected s.c. in both forelimbs with mixtures of fluorescent target DC as described in the legend to Fig. 1. The same mice were also injected i.v. with 10 million in vitro-activated CTL or with no CTL as indicated on the left. CTL were injected either 24 h before or 24 h after the DC, to allow DC time to reach the lymph node. Mice were killed 24 h or 48 h after the last injection as indicated, and the presence of labeled DC in lymph nodes was determined. Bars indicate the average proportion of gp33-loaded DC (DC⁺) remaining in lymph nodes, expressed as a percentage of control DC (DC^–), + SD, for groups of five mice. (B) Groups of CD45-congenic mice were injected with CD45-congenic, gp33-loaded DC in the forelimb (Right) or left untreated (Left). One day later both groups were injected i.v. with 10 million CFSE-labeled, in vitro-activated CTL. T cell proliferation in the draining lymph node was examined 72 h after CTL transfer. Results show total, live-gated lymph node cells from individual mice that were representative of groups of three.

Fig. 3.

Antigen-loaded DC are not eliminated in PKO mice. Groups of C57BL/6 and PKO mice were immunized by s.c. injection of gp33-loaded DC in the flank. One week later all mice were injected in both forelimbs with mixtures of fluorescent target DC as described in the legend to Fig. 1 and killed 24 or 96 h later as indicated. (A) Representative FACS profiles from C57BL/6 (Left) and PKO (Right) mice killed at 96 h. (B) Total number of DC recovered from the lymph nodes of C57BL/6 or PKO mice. (C) Average proportion of gp33-loaded DC (DC⁺) remaining in lymph nodes, expressed as percentage of control DC (DC^–), + SD for groups of three mice.

Fig. 4.

IFN-γ and CD4⁺CD25⁺ T_reg are not required for the elimination of DC in vivo. (A) Groups of C57BL/6 (Left) and IFN-γ^–/– (Right) mice were immunized with DC only or DC loaded with gp33 peptide, by s.c. injection into the flank. Seven days later all mice received CFSE-labeled DC loaded with gp33 peptide by s.c. injection into each forelimb. Seventy-two hours later the draining lymph nodes were harvested and the percent DC was determined by FACS staining. Bars show the percentages of CFSE⁺ DC in lymph nodes; average + SD for groups of three mice are shown. (B) Groups of C57BL/6 mice were injected with 100 μg of the anti-CD25 antibody PC61 (Right) or left untreated (Left) and immunized with the indicated DC the next day. Target fluorescent DC were injected on day 7. Bars indicate the average proportion of gp33-loaded DC (DC⁺) remaining in lymph node, expressed as a percent of control DC (DC^–), + SD, for groups of four mice.

Fig. 5.

Multiple DC immunizations lead to increased CD8⁺ T cell expansion in PKO mice compared with C57BL/6. Groups of C57BL/6 mice or PKO mice were injected with TCR transgenic T cells from L318 or PKO-L318 mice, respectively, and repeatedly immunized s.c. with gp33-loaded DC or DC only at the times indicated by arrows. Immunizing DC were activated by plastic adherence (A and C) or treatment with 100 ng/ml LPS (B). (A) The percentage of Vα2⁺Vβ8⁺ T cells in peripheral blood was determined by FACS analysis at different times after immunization. Data points represent the average + SD for groups of three mice. (B) The percentage of Vα2⁺Vβ8⁺ T cells in lymph nodes and spleen was determined in C57BL/6 (Left) and PKO (Right) mice 14 days after the fourth DC immunization. Bars represent the average + SD for groups of five (DC + gp33) or three (DC only) mice. (C) Survival of gp33-loaded DC in multiply immunized mice. Seven days after the third DC immunization, mice were injected in both forelimbs with mixtures of fluorescent target DC as described in the legend to Fig. 1. The proportion of gp33-loaded DC (DC⁺) remaining in lymph node, expressed as a percent of the coinjected control DC (DC^–), was determined 40 h after injection. Bars represent average + SD for groups of three mice. Data in A and C refer to the same experiment. Data in B are from a similar but independent experiment that gave comparable results.