Memory CD8+ T cells are gatekeepers of the lymph node draining the site of viral infection

Abstract

It is uncertain how immunity protects against systemic viral diseases. Here, we demonstrate that in the absence of persistent virus, not only antibodies but also recall responses by long-lived memory CD8(+) T cells prevent mousepox, a disease caused by ectromelia virus, a close relative of the virus of human smallpox. Moreover, we show that to protect, recall CD8(+) T cells directly kill targets in the lymph node draining the primary site of infection thus curbing systemic viral spread. Therefore, our work provides the basis for a model where lymph nodes are not just organs where lymphocytes become activated and proliferate but also the sites where a major fight against virus spread takes place.

Fig. 1.

Long-lived memory CD8⁺ T cells prevent mousepox. (A) Weight loss. BALB/c mice were treated as indicated and at different times posttreatment were infected in the footpad with ECTV. The body weight was determined over the following 4 weeks. Data are expressed as percentage average of initial weight ±SEM. All groups consisted of five mice except the VACV-treated group, which consisted of eight mice. In the group of mice treated with naïve CD8⁺ T cells, only one mouse survived past day 13 p.i. Experiments were repeated three times, except the challenge after 4 months post memory CD8⁺ T cell inoculation, which was performed once. (B) Histopathology. Mice were treated as indicated and 1 day later were infected with ECTV in the footpad. Seven days p.i., mice were killed, and organs were collected and processed for histopathology. Microphotographs representative of three mice. (Magnification of hematoxylin/eosin stains: spleen, ×10; liver, ×24.) Stippled lines demarcate the sites of liver necrosis.

Fig. 2.

Ab and memory CD8⁺ T cells protect from mousepox and death but only in immunocompetent mice. (A) Memory CD8⁺ T cells do not protect BALB/c mice unable to generate antibodies. Groups of four or five BALB/c or B cell KO mice were treated as indicated and challenged with ECTV in the footpad. Survival was monitored for 4 weeks. The experiment is representative of two. Data for B cell KO mice treated with memory CD8⁺ T cells correspond to a separate experiment where untreated B cell KO mice also died on day 6, and all BALB/c that received memory CD8⁺ T cells survived. (B) Antibodies do not protect mice unable to generate CD8⁺ T cell responses. Groups of five BALB/c mice were treated as indicated and infected with ECTV in the footpad. Survival was monitored for 4 weeks. The experiment shown is representative of three.

Fig. 3.

Memory CD8⁺ T cells reduce virus spread to vital organs. (A) Virus titers. BALB/c mice were transferred with memory or naïve CD8⁺ T cells or immunized with VACV. One day after adoptive cell transfer or 2 months after VACV immunization, mice were infected in the footpad with ECTV. One, 3, 4, and 7 days p.i., mice were killed and virus titers were determined in the indicated organs. Data corresponds to four or five mice per group ±SEM, and most of the time points were repeated at least twice. DL, detection limit. (B and C) Memory CD8⁺ T cells respond rapidly in the D-LN. Thy1 KO mice were adoptively transferred with CFSE-labeled CD8⁺ T cells from naïve or memory (VACV-immune) BALB/c mice. Animals were challenged or not with ECTV in the footpad and donor (Thy1⁺) CD8⁺ T cell responses were determined in D-LN, ND-LN, and spleen by intracellular IFN-γ and GzB staining at different times p.i. CFSE dilution into daughter cells was used to determine proliferation of donor-derived CD8⁺ T cells. Stains are as indicated. Data correspond to pools of LN or spleens from two to five mice. In B, both dot plots correspond to D-LN from mice that received memory CD8⁺ T cells and are gated on donor-derived Thy1⁺ cells. (D) In vivo killing of antigen-bearing targets occurs earlier in the D-LN. BALB/c mice were adoptively transferred with memory CD8⁺ T cells and infected or not with ECTV. Three or 4 days p.i., mice were inoculated i.v. with control and antigen-loaded targets consisting of a 1:1 mixture of BALB/c splenocytes labeled with 0.8 μM CFSE (control targets, CFSE^low) or pulsed with a pool of three peptides corresponding to three ECTV CD8⁺ T cell determinants and labeled with 4 μM CFSE (antigen-loaded targets, CFSE^high). Mice were killed 150 min after target inoculation, and the proportion of CFSE^low and CFSE^high cells was determined by FACS in D-LN and spleen. Histograms are gated on CFSE-positive cells. Numbers indicate percentage of specific killing of CFSE^high cells calculated as detailed in Materials and Methods. Data correspond to pools of three mice. The experiment was repeated four times with identical results. (E) Increased virus titers in spleen and liver after removal of the D-LN. Mice were subjected to surgery to remove the left popliteal LN (filled bars). Control mice were similarly operated, but the LN was not removed (open bars). Three days after surgery, mice received 3 × 10⁶ memory CD8⁺ T cells i.v. and the next day were infected with ECTV in the left footpad. Virus titers were determined in spleen and liver 4 days p.i. Data correspond to five mice per group.