Continuous recruitment of naive T cells contributes to heterogeneity of antiviral CD8 T cells during persistent infection

Abstract

Numerous microbes establish persistent infections, accompanied by antigen-specific CD8 T cell activation. Pathogen-specific T cells in chronically infected hosts are often phenotypically and functionally variable, as well as distinct from T cells responding to nonpersistent infections; this phenotypic heterogeneity has been attributed to an ongoing reencounter with antigen. Paradoxically, maintenance of memory CD8 T cells to acutely resolved infections is antigen independent, whereas there is a dependence on antigen for T cell survival in chronically infected hosts. Using two chronic viral infections, we demonstrate that new naive antigen-specific CD8 T cells are primed after the acute phase of infection. These newly recruited T cells are phenotypically distinct from those primed earlier. Long-lived antiviral CD8 T cells are defective in self-renewal, and lack of thymic output results in the decline of virus-specific CD8 T cells, indicating that newly generated T cells preserve antiviral CD8 T cell populations during chronic infection. These findings reveal a novel role for antigen in maintaining virus-specific CD8 T cells during persistent infection and provide insight toward understanding T cell differentiation in chronic infection.

Figure 1.

Visualizing PyV infection. (A) Splenic PyV DNA levels ± SEM over time. (B) Number of splenic D^b-LT359 tetramer⁺ CD8 T cells ± SD over time. (C) Splenic CD8 T cell responses 246 d after infection. Plots are gated on CD8 T cells, and values reflect the percentage of tetramer⁺ cells in each quadrant (n = 3–9 mice).

Figure 2.

Loss of PyV-specific CD8 T cells in an antigen-bearing host. (A) Experimental setup. (B) Host mice were bled at day 4 after transfer to establish a base level of D^b-LT359 tetramer⁺ cells of donor and host origin. PBLs were monitored over time, and values indicate the percentage of tetramer⁺ cells ± SEM of either host or donor origin normalized for input at day 4 after transfer (n = 4 mice). Two independent experiments were performed. The donor tetramer⁺ cell frequencies varied from 0.3–0.6% of CD8 T cells at day 4, and host tetramer⁺ frequencies varied from 1.3-3.8% of CD8 T cells. (C) Transferred PyV-specific cells were monitored over time for CFSE fluorescence. Plots are gated on donor tetramer⁺ cells. Values indicate the percentage of cells within the marked regions. (D) CFSE- labeled memory P14 cells were transferred to naive congenic mice (n = 3) or congenic mice infected by PyV 200 d earlier (n = 4). Spleens of secondary hosts were analyzed 36 d later. Plots are gated on P14 cells.

Figure 3.

Priming of new, naive antiviral CD8 T cells during persistent infection. (A) Partial bone marrow chimerism protocol. (B) Mice infected by PyV 84 d earlier were either injected with splenocytes or bone marrow cells or treated with busulfan and injected with bone marrow cells the next day (n = 3–8 mice). Spleens were analyzed 42 d later and assessed for chimerism. Each plot contains 10⁵ events, and values represent the percentage of lymphocytes within the indicated regions. (C) Mice infected by PyV 115 d earlier were treated with busulfan and injected with bone marrow cells as in A. Organs were analyzed for D^b-LT359 tetramer⁺ CD8 T cells 51 d after cell transfer. Plots are gated on donor or host CD8 T cells, and values indicate the percentage of tetramer⁺ cells. 10 and 8% of tetramer⁺ cells are of donor origin in the spleen and lung, respectively. (D) Mice infected by LCMV clone 13 120 d earlier were treated with busulfan and received bone marrow cells as in A. Organs were analyzed for D^b-gp276 tetramer⁺ CD8 T cells 48 d after cell transfer (n = 6). Plots are gated on host or donor CD8 T cells, and values represent the percentage of CD8 T cells that are tetramer⁺. 7.5 and 9.4% of tetramer⁺ cells are of donor origin in the liver and lung, respectively. (E) Naive mice were thymectomized (thymx; n = 8) or sham operated (sham; n = 6). 45 d after PyV infection, spleens were analyzed for tetramer⁺ cells. The experiment was repeated twice with a combined p-value of 0.04.

Figure 4.

Dynamic phenotype of PyV-specific CD8 T cells. Mice infected by PyV 35 (A) or 95 (C) d earlier received busulfan and bone marrow cells as in Fig. 3 A. Organs were analyzed for tetramer⁺ CD8 T cells 45 (A) or 140 (C) d after cell transfer. Plots are gated on tetramer⁺ cells. Host cells (black), donor cells (red), and host CD44^lo CD8 T cells (blue) are shown. In A, 6–7% of tetramer⁺ cells are of donor origin; in C, 5–6% of tetramer⁺ cells are of donor origin (n = 3–8 mice). SPL, spleen; LG, lung. (B) CD27 and CD62L expression on splenic tetramer⁺ cells over time. Plots are gated on CD8 T cells, and values in plots indicate the percentage of tetramer⁺ cells that express high levels of either CD27 or CD62L. The plot for CD62L at day 246 is the same as in Fig. 1 C (n = 3–12 mice per time point).