Dendritic cells maximize the memory CD8 T cell response to infection

Abstract

Costimulatory signals from dendritic cells (DCs) are required for naive T cells to respond to antigenic stimulation. To what extent DCs reactivate memory T cells during recall responses is not known. Here, an in vivo depletion system has been used to analyze the role of DCs in reactivating CD8 memory T cells during recall responses to three different microbial infections. We show a profound decrease in the numbers of responding memory CD8 T cells in both lymphoid and nonlymphoid tissues during the recall responses to infection with vesicular stomatitis virus, Listeria monocytogenes (Lm), or influenza virus. These data show that interaction with DCs is a major mechanism driving T cell reactivation in vivo, even during a tissue-specific infection of the respiratory tract.

Figure 1. DCs Are Ablated in Lymphoid and Nonlymphoid Organs after DT Treatment

DTR BM chimeras were injected with two doses of either 4 ng/g body weight DT or PBS 3 days apart and 24 hr later analyzed to determine the efficacy of DC ablation. Pooled samples of liver, peripheral LN, and LP from the two groups of mice were used to gain enough numbers of DCs for analysis. DCs were identified as MHC II⁺ CD11c⁺ GFP⁺ by flow cytometry, and the plots shown are gated on MHC class II⁺ cells. DC numbers in all organs tested were generally depleted by ~90% after treatment. The total number of DCs is shown in each plot. Four mice were used per group and the experiment repeated twice. Data shown is representative of one experiment.

Figure 2. DCs Are Required for the Initiation of an Anti-Lm-Ova Recall Response

DTR or wild-type (wt) bone marrow-reconstituted mice were treated 4 and 1 days before infection with DT to ablate DCs in the DTR mice. Ly5.2⁺ ova-specific CD8⁺ memory cells were transferred into both groups 1 day before the mice were infected i.v. with 10³ cfu Lm-ova. DT was administered every 3 days, and the mice were analyzed 6 days after infection for Ly5.2⁺ ova-specific CD8⁺ T cells. (A) The proportion of Ly5.2⁺ ova⁺ cells was decreased in all organs analyzed. The numbers in the upper left corner of each panel represent percent of ova tet⁺ LFA⁺ cells among the donor CD8⁺ T cells. (B) The total number of Ly5.2⁺ ova-specific cells is dramatically less in the absence of DCs in all organs and was calculated from the proportion of tet⁺ cells and total cellularity of each organ. The data represent a total of six mice per group from two individual experiments. All values are means ± SEM.

Figure 3. DCs Are Required for the Initiation of an Anti-VSV Recall Response

DTR or wt bone marrow-reconstituted mice were treated after the protocol in Figure 2 except the mice were infected i.v. with 10⁵ pfu VSV. DT was administered every 3 days, and the mice were analyzed 6 days after infection for 5.2⁺ N tet⁺ CD8⁺ T cells. (A) The proportion of Ly5.2⁺ N tet⁺ cells is decreased in all organs analyzed to varying degrees. The numbers in the upper left corner of each panel represent percent of N tet⁺ LFA⁺ cells among the donor CD8⁺ T cells. (B) The total number of Ly5.2⁺ N tet⁺ cells was calculated from the proportion of tet⁺ cells and total cellularity of each organ. The data is from one experiment of three mice per group, and the values are means ± SEM. The experiment was repeated twice with similar results.

Figure 4. CD8 Memory T Cells Respond to Peptide-Loaded DCs in DC-Depleted Mice

VSV-specific CD8 memory T cells (50,000) were transferred to the indicated chimeras, and all were treated with DT on the day of transfer. One day later, 5 × 10⁵ purified splenic DCs (either coated with the VSV N peptide or not) were transferred i.v. 6 days later, the response in the lung (shown here) and the spleen (data not shown) was measured by flow cytometry using an N peptide H-2K^b tetramer. Data shown is gated on CD8⁺ T cells. Responses in the spleen, though less robust, were similar between groups.

Figure 5. Partial DC Requirement for the Initiation of an Anti-Influenza Recall Response

DTR or wt bone marrow-reconstituted mice were treated following the protocol in Figure 2 except the mice were infected i.n. with 10³ pfu WSN-ova. DT was administered every 3 days, and the mice were analyzed 8 days after infection for Ly5.2⁺ ova-specific CD8⁺ T cells. (A) The proportion of Ly5.2⁺ ova⁺ cells is decreased in all organs analyzed with the exception of the MedLN. The numbers in the upper left corner of each panel represent percent of ova tet⁺ LFA⁺ cells among the donor CD8⁺ T cells. (B) The total number of Ly5.2⁺ ova⁺ cells was calculated from the proportion of tet⁺ cells and total cellularity of each organ,. The data represents five mice (wt) or six mice (DTR) from two individual experiments. All values are means ± SEM.

Figure 6. The Reduced Recall Response to Influenza Is Delayed in the Absence of DCs

(A) The total cellularity of the lungs, BAL fluid, and spleen is equal in the DTR and wt mice, 11 days after infection. The cellularity of the MedLN is still dramatically reduced at this time point. (B) Despite the equal total cellularity in the BAL fluid and spleen, there is still a reduced number of ova⁺ CD8⁺ cells in these organs. By day 11, the number of ova⁺ CD8⁺ cells in the lungs of wt and DTR mice are comparable. The data represent six mice (wt) or four mice (DTR) from two individual experiments. All values are means ± SEM.

Figure 7. DCs Are Required for More than 48 hr to Generate a Full Recall Response

(A) Ly5.2+ ova -specific CD8⁺ memory cells were transferred into both groups of mice 1 day before the mice were infected i.v. with 10⁵ pfu VSV-ova. A single dose of DT was administered at either day 0, 1, or 2 of infection and CD8⁺ Ly5.2⁺ ova-tet⁺ cells in the spleens were quantitated six days after infection. The percent decrease was calculated based on total tet⁺ cell numbers in each group. The data represent four mice in each group from two individual experiments. (B) CFSE-labeled Ly5.2⁺ VSV N protein-specific CD8⁺ memory cells were transferred into the indicated chimeras 1 day before the mice were infected i.v. with 10⁵ pfu VSV. A single dose of DT was administered at either day 0, 1, 3, or 5, and CD8⁺ Ly5.2⁺ N tet⁺ cells in the spleens were quantitated six days after infection. Values represent total tet⁺ cells from three mice per group. (C) CFSE analysis of the mice in (B) treated at day 0 with DT. Analysis is of gated Ly5.2⁺ CD8⁺ tet⁺ cells. The total number of CFSE-high cells is indicated.