Role of thymic output in regulating CD8 T-cell homeostasis during acute and chronic viral infection

Abstract

Although it is well documented that CD8 T cells play a critical role in controlling chronic viral infections, the mechanisms underlying the regulation of CD8 T-cell responses are not well understood. Using the mouse model of an acute and chronic lymphocytic choriomeningitis virus (LCMV) infection, we have examined the relative importance of peripheral T cells and thymic emigrants in the elicitation and maintenance of CD8 T-cell responses. Virus-specific CD8 T-cell responses were compared between mice that were either sham thymectomized or thymectomized (Thx) at approximately 6 weeks of age. In an acute LCMV infection, thymic deficiency did not affect either the primary expansion of CD8 T cells or the proliferative renewal and maintenance of virus-specific lymphoid and nonlymphoid memory CD8 T cells. Following a chronic LCMV infection, in Thx mice, although the initial expansion of CD8 T cells was normal, the contraction phase of the CD8 T-cell response was exaggerated, which led to a transient but striking CD8 T-cell deficit on day 30 postinfection. However, the virus-specific CD8 T-cell response in Thx mice rebounded quickly and was maintained at normal levels thereafter, which indicated that the peripheral T-cell repertoire is quite robust and capable of sustaining an effective CD8 T-cell response in the absence of thymic output during a chronic LCMV infection. Taken together, these findings should further our understanding of the regulation of CD8 T-cell homeostasis in acute and chronic viral infections and might have implications in the development of immunotherapy.

FIG. 1.

Effect of thymectomy on virus-specific CD8 T-cell responses during an acute LCMV infection. Groups of SThx and Thx mice were infected with LCMV-Arm. On the eighth day after infection, the number of CD8 T cells specific to the CTL epitopes NP396, GP33, and GP276 was determined by staining splenocytes with anti-CD8, anti-CD44, and MHC-I tetramers. The dot plots in panel A are gated on total CD8 T cells, and the numbers are the percentages of tetramer-binding CD8 T cells of total splenocytes or total CD8 T cells (parentheses). The data in panel B represent absolute numbers of epitope-specific CD8 T cells in the spleen. The data are the means of three mice/group ± standard deviations (SD) and are representative of two independent experiments.

FIG. 2.

Effect of thymic deficiency on CD8 T-cell memory in an acute viral infection. Groups of SThx and Thx mice were infected with LCMV-Arm, and on day 40 postinfection, the number of CD8 T cells in the spleen that are specific to the indicated epitopes was determined by staining with anti-CD8, anti-CD44, and MHC-I tetramers. The data in panel A are the total number of naive (CD8⁺ CD44^lo) and activated/memory (CD8⁺ CD44^hi) CD8 T cells in the spleen. The data in panel B are the total numbers of epitope-specific CD8 T cells in the spleen. The data shown are the means of three mice/group ± SD and are representative of two independent experiments.

FIG. 3.

Effect of thymic deficiency on the proliferative renewal of LCMV-specific memory CD8 T cells following an acute viral infection. About 200 days after infection with LCMV-Arm, groups of SThx and Thx mice were treated with BrdU for 8 days in drinking water. At the end of BrdU pulse, splenocytes or mononuclear cells isolated from the liver were stained with anti-CD8, anti-CD44, MHC-I tetramers, and anti-BrdU antibodies and analyzed by flow cytometry. The histograms are gated on the indicated subsets of CD8 T cells, and the numbers are the percentages of BrdU-positive cells among the gated population. The data are the means of three mice/group ± SD.

FIG. 4.

CD8 T-cell responses to a chronic LCMV infection in thymectomized mice. Groups of SThx and Thx mice were infected with LCMV-clone 13, and the number of virus-specific CD8 T cells in the spleen was determined by staining with anti-CD8, anti-CD44, and MHC-I tetramers. The dot plots represent staining for LCMV-specific CD8 T cells on day 8 postinfection, and the numbers are the percentages of epitope-specific CD8 T cells among splenocytes or total CD8 T cells (parentheses). Data in the bottom panel are the total number of CD8 T cells in the spleen that are specific to the indicated epitopes on various days after infection. Each data point represents the cell numbers of an individual mouse.

FIG. 5.

Proliferation of virus-specific CD8 T cells in thymectomized mice during a chronic LCMV infection. SThx and Thx mice were infected with LCMV-clone 13. Between days 15 and 25 and 30 to 40 after infection, mice were treated with BrdU in drinking water. At the end of each pulse, splenocytes were stained with anti-CD8, MHC-I tetramers, and anti-BrdU and analyzed by flow cytometry. The histograms showing BrdU staining are gated on tetramer-binding CD8 T cells. The numbers are the percentages of BrdU-positive cells among the tetramer-binding CD8 T cells ± SD (3 mice/group).

FIG. 6.

Effect of thymectomy on viral clearance during a chronic LCMV infection. Following LCMV-clone 13 infection, the viral titers in the liver and lung of sham-thymectomized (▵) and thymectomized (•) mice were quantitated by a plaque assay using Vero cells. Each data point represents the viral titer of an individual mouse.

FIG. 7.

In vivo cytotoxic activity in thymectomized mice in a chronic LCMV infection. On days 8, 21, and 45 after infection with LCMV-clone 13, the cytotoxic activity in the spleens of SThx and Thx mice was assessed in vivo as described in Materials and Methods. Uninfected and LCMV-Arm-infected C57BL/6 mice were used as negative and positive controls, respectively. The numbers represent percent specific cytotoxicity ± SD, and the data are the means of four mice/group.

FIG. 8.

Virus-specific IFN-γ-producing CD8 T cells in the spleen of thymectomized mice during a chronic LCMV infection. (A) On the indicated days after LCMV-clone 13 infection, the number of epitope-specific IFN-γ-producing CD8 T cells in the spleen of SThx (▵) and Thx (•) mice was quantitated ex vivo by intracellular staining. Each data point represents the total number of epitope-specific IFN-γ-producing CD8 T-cell numbers in an individual mouse. (B) On days 8 and 30 after LCMV-clone 13 infection, the number of GP33-specific CD8 T cells in the spleen was enumerated by tetramer staining and intracellular IFN-γ staining. The data are the percentages of GP33-specific tetramer-binding CD8 T cells that failed to produce IFN-γ. Each data point represents the percentage of dysfunctional GP33-specific CD8 T cells in individual mice.

FIG. 9.

Protective immunity against rechallenge in LCMV-immune SThx and Thx mice. Approximately 45 days after LCMV-clone 13 infection, Thx and SThx mice were rechallenged with LCMV-clone 13. Naive SThx and Thx mice were infected as controls. Five days after rechallenge, the viral titers in the liver were quantitated by plaque assay (A), and the numbers of LCMV-specific CD8 T cells in the spleen were enumerated by intracellular cytokine staining (B). The plotted data are derived from individual mice.