Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distinct stages of functional impairment

Abstract

Chronic viral infections often result in ineffective CD8 T-cell responses due to functional exhaustion or physical deletion of virus-specific T cells. However, how persisting virus impacts various CD8 T-cell effector functions and influences other aspects of CD8 T-cell dynamics, such as immunodominance and tissue distribution, remains largely unknown. Using different strains of lymphocytic choriomeningitis virus (LCMV), we compared responses to the same CD8 T-cell epitopes during acute or chronic infection. Persistent infection led to a disruption of the normal immunodominance hierarchy of CD8 T-cell responses seen following acute infection and dramatically altered the tissue distribution of LCMV-specific CD8 T cells in lymphoid and nonlymphoid tissues. Most importantly, CD8 T-cell functional impairment occurred in a hierarchical fashion in chronically infected mice. Production of interleukin 2 and the ability to lyse target cells in vitro were the first functions compromised, followed by the ability to make tumor necrosis factor alpha, while gamma interferon production was most resistant to functional exhaustion. Antigen appeared to be the driving force for this loss of function, since a strong correlation existed between the viral load and the level of exhaustion. Further, epitopes presented at higher levels in vivo resulted in physical deletion, while those presented at lower levels induced functional exhaustion. A model is proposed in which antigen levels drive the hierarchical loss of different CD8 T-cell effector functions during chronic infection, leading to distinct stages of functional impairment and eventually to physical deletion of virus-specific T cells. These results have implications for the study of human chronic infections, where similar T-cell deletion and functional dysregulation has been observed.

Figures

FIG. 1.

Viral kinetics during acute and chronic LCMV infection. Panels A to E show the titer of infectious virus in the indicated tissues at various times p.i. with either LCMV Armstrong (open symbols) or LCMV Cl-13 (filled symbols). (A) Titers in serum, expressed in PFU per milliliter. (B and C) Titers in the spleen, brain, liver, and kidney, respectively, expressed as PFU per gram of tissue. Data are the average for two to eight mice/time point.

FIG. 2.

Chronic LCMV infection alters CD8 T-cell immunodominance. (A) Responses to four LCMV T-cell epitopes were determined using MHC tetramer staining. The number of CD8 T cells/spleen specific for the Db/NP396, Db/GP33, Kb/GP34, or Db/GP276 epitope is plotted over time following infection with LCMV Armstrong (open symbols) or LCMV Cl-13 (closed symbols). Data are the average for two to eight mice/time point. The dotted line indicates the level of detection based on tetramer staining of splenocytes from naive mice. (B) The percent of CD8 T cells staining positive for each of the four MHC class I tetramers is shown for a representative LCMV Armstrong-immune (around day 400 p.i.) mouse (top) and a representative LCMV Cl-13-infected (day 60 p.i.) mouse (bottom). Fluorescence-activated cell sorter plots are gated on CD8+ T cells, and the number indicates the percent of CD8 T cells staining with the indicated tetramer. It should be noted that the Db/GP33 and Kb/GP34 tetramers stain distinct nonoverlapping CD8 T-cell populations (data not shown).

FIG. 3.

Chronic LCMV infection substantially alters the tissue distribution of virus-specific CD8 T cells. (A) Lymphocytes were isolated from four lymphoid tissues (spleen, PBMC, LN, and bone marrow [BM]) and three nonlymphoid tissues (liver, lung, and brain) of LCMV Armstrong-immune mice (around day 400 p.i.; similar results were observed at numerous time points between days 30 and 400; data not shown) or LCMV Cl-13-chronically infected mice (day 25 p.i.; similar results were observed at days 30, 60 and 70 p.i.; data not shown) and stained with MHC tetramers of Db/GP33 (upper two rows) and Db/GP276 (lower two rows). All plots are gated on CD8+ cells, and the numbers indicate the percentages of CD8 T cells staining with the indicated tetramers. (B) The number of Db/GP33-positive (left panel) or Db/GP276-positive (right panel) CD8 T cells in the liver was determined over time by MHC tetramer staining following acute LCMV Armstrong infection (open symbols) or during chronic LCMV Cl-13 infection (filled symbols). (C) The number of Db/GP33- and Db/GP276-specific CD8 T cells in the bone marrow was determined at day 60 p.i. for either LCMV Armstrong or Cl-13. The data indicate the number of cells in the total bone marrow calculated as described in Materials and Methods. A similar trend was observed at day 30 p.i. (D) CD44 expression on Db/GP33 (left panel) or Db/GP276 (right panel) tetramer-positive cells from LCMV Armstrong-immune (around day 400 p.i.) mice (gray histogram) or LCMV Cl-13-infected (day 25 p.i.) mice (open histogram). Histograms are gated on CD8+/tetramer-positive cells from the spleen. Similar results were observed for all tissues examined (data not shown and see panel A).

FIG. 3.

Chronic LCMV infection substantially alters the tissue distribution of virus-specific CD8 T cells. (A) Lymphocytes were isolated from four lymphoid tissues (spleen, PBMC, LN, and bone marrow [BM]) and three nonlymphoid tissues (liver, lung, and brain) of LCMV Armstrong-immune mice (around day 400 p.i.; similar results were observed at numerous time points between days 30 and 400; data not shown) or LCMV Cl-13-chronically infected mice (day 25 p.i.; similar results were observed at days 30, 60 and 70 p.i.; data not shown) and stained with MHC tetramers of Db/GP33 (upper two rows) and Db/GP276 (lower two rows). All plots are gated on CD8+ cells, and the numbers indicate the percentages of CD8 T cells staining with the indicated tetramers. (B) The number of Db/GP33-positive (left panel) or Db/GP276-positive (right panel) CD8 T cells in the liver was determined over time by MHC tetramer staining following acute LCMV Armstrong infection (open symbols) or during chronic LCMV Cl-13 infection (filled symbols). (C) The number of Db/GP33- and Db/GP276-specific CD8 T cells in the bone marrow was determined at day 60 p.i. for either LCMV Armstrong or Cl-13. The data indicate the number of cells in the total bone marrow calculated as described in Materials and Methods. A similar trend was observed at day 30 p.i. (D) CD44 expression on Db/GP33 (left panel) or Db/GP276 (right panel) tetramer-positive cells from LCMV Armstrong-immune (around day 400 p.i.) mice (gray histogram) or LCMV Cl-13-infected (day 25 p.i.) mice (open histogram). Histograms are gated on CD8+/tetramer-positive cells from the spleen. Similar results were observed for all tissues examined (data not shown and see panel A).

FIG. 3.

Chronic LCMV infection substantially alters the tissue distribution of virus-specific CD8 T cells. (A) Lymphocytes were isolated from four lymphoid tissues (spleen, PBMC, LN, and bone marrow [BM]) and three nonlymphoid tissues (liver, lung, and brain) of LCMV Armstrong-immune mice (around day 400 p.i.; similar results were observed at numerous time points between days 30 and 400; data not shown) or LCMV Cl-13-chronically infected mice (day 25 p.i.; similar results were observed at days 30, 60 and 70 p.i.; data not shown) and stained with MHC tetramers of Db/GP33 (upper two rows) and Db/GP276 (lower two rows). All plots are gated on CD8+ cells, and the numbers indicate the percentages of CD8 T cells staining with the indicated tetramers. (B) The number of Db/GP33-positive (left panel) or Db/GP276-positive (right panel) CD8 T cells in the liver was determined over time by MHC tetramer staining following acute LCMV Armstrong infection (open symbols) or during chronic LCMV Cl-13 infection (filled symbols). (C) The number of Db/GP33- and Db/GP276-specific CD8 T cells in the bone marrow was determined at day 60 p.i. for either LCMV Armstrong or Cl-13. The data indicate the number of cells in the total bone marrow calculated as described in Materials and Methods. A similar trend was observed at day 30 p.i. (D) CD44 expression on Db/GP33 (left panel) or Db/GP276 (right panel) tetramer-positive cells from LCMV Armstrong-immune (around day 400 p.i.) mice (gray histogram) or LCMV Cl-13-infected (day 25 p.i.) mice (open histogram). Histograms are gated on CD8+/tetramer-positive cells from the spleen. Similar results were observed for all tissues examined (data not shown and see panel A).

FIG. 3.

Chronic LCMV infection substantially alters the tissue distribution of virus-specific CD8 T cells. (A) Lymphocytes were isolated from four lymphoid tissues (spleen, PBMC, LN, and bone marrow [BM]) and three nonlymphoid tissues (liver, lung, and brain) of LCMV Armstrong-immune mice (around day 400 p.i.; similar results were observed at numerous time points between days 30 and 400; data not shown) or LCMV Cl-13-chronically infected mice (day 25 p.i.; similar results were observed at days 30, 60 and 70 p.i.; data not shown) and stained with MHC tetramers of Db/GP33 (upper two rows) and Db/GP276 (lower two rows). All plots are gated on CD8+ cells, and the numbers indicate the percentages of CD8 T cells staining with the indicated tetramers. (B) The number of Db/GP33-positive (left panel) or Db/GP276-positive (right panel) CD8 T cells in the liver was determined over time by MHC tetramer staining following acute LCMV Armstrong infection (open symbols) or during chronic LCMV Cl-13 infection (filled symbols). (C) The number of Db/GP33- and Db/GP276-specific CD8 T cells in the bone marrow was determined at day 60 p.i. for either LCMV Armstrong or Cl-13. The data indicate the number of cells in the total bone marrow calculated as described in Materials and Methods. A similar trend was observed at day 30 p.i. (D) CD44 expression on Db/GP33 (left panel) or Db/GP276 (right panel) tetramer-positive cells from LCMV Armstrong-immune (around day 400 p.i.) mice (gray histogram) or LCMV Cl-13-infected (day 25 p.i.) mice (open histogram). Histograms are gated on CD8+/tetramer-positive cells from the spleen. Similar results were observed for all tissues examined (data not shown and see panel A).

FIG. 4.

Progressive loss of cytokine production during chronic LCMV Cl-13 infection. (A) The fraction of tetramer-positive cells able to produce IFN-γ (top row) was determined for the NP396-, GP33-, and GP276-specific populations at the indicated times p.i. with LCMV Cl-13 (Chronic) or Armstrong (Acute). The percentage of the tetramer-positive population able to produce TNF-α (middle row) or IL-2 (bottom row) was determined using three-color intracellular cytokine staining. Asterisks indicate too few cells available for analysis as a result of deletion. Open bars indicate LCMV Armstrong infection, while the black bars indicate LCMV Cl-13 infection. The data represent the average and standard deviation for three to six mice/time point. (B) An example of fully functional GP33-specific memory CD8 T cells from an LCMV Armstrong-immune mouse (top row) and three examples of different states of functional exhaustion of GP33-specific T cells during LCMV Cl-13 infection are shown. The percentage in the left column indicates the percentage of CD8 T cells that is Db/GP33 tetramer-positive. The percentage in the second, third, or fourth column indicates the percentage of tetramer-positive cells that is IFN-γ+, TNF-α+, or IL-2+, respectively. Plots for tetramer and IFN-γ staining are gated on all lymphocytes. Plots for TNF-α and IL-2 are gated on CD8 T cells. Similar results were observed for the GP276 response (data not shown). (C) Lymphocytes from the PBMC (upper panels) or intrahepatic lymphocytes (lower panels) of LCMV Cl-13-infected mice (chronic; day 35 p.i. for PBMC, day 60 p.i. for liver) were examined for functional exhaustion. The left columns show the percent Db/GP33 tetramer-positive cells (numbers indicate percentages of CD8 T cells). Production of IFN-γ (gated on all lymphocytes) and TNF-α (gated on CD8 T cells) is shown in the middle and right columns, respectively. Numbers indicate the percentage of tetramer-positive cells making cytokine. No IL-2-producing cells were detected from CD8 T cells from chronically infected mice (data not shown). Similar results were observed at other time points and for GP276-specific responses (data not shown). Note that the GP33 peptide used for intracellular cytokine staining stimulates both the Db/GP33 and Kb/GP34 response. Therefore, the percent functional of tetramer-positive cells shown in panels A, B, and C was determined using both DbGP33 and KbGP34 tetramers. As shown in Fig. 2, the Kb/GP34 becomes negligible by 2 to 3 weeks p.i. Therefore, only the Db/GP33 tetramer stain is shown. No specific staining was observed when using isotype control antibodies.

FIG. 4.

Progressive loss of cytokine production during chronic LCMV Cl-13 infection. (A) The fraction of tetramer-positive cells able to produce IFN-γ (top row) was determined for the NP396-, GP33-, and GP276-specific populations at the indicated times p.i. with LCMV Cl-13 (Chronic) or Armstrong (Acute). The percentage of the tetramer-positive population able to produce TNF-α (middle row) or IL-2 (bottom row) was determined using three-color intracellular cytokine staining. Asterisks indicate too few cells available for analysis as a result of deletion. Open bars indicate LCMV Armstrong infection, while the black bars indicate LCMV Cl-13 infection. The data represent the average and standard deviation for three to six mice/time point. (B) An example of fully functional GP33-specific memory CD8 T cells from an LCMV Armstrong-immune mouse (top row) and three examples of different states of functional exhaustion of GP33-specific T cells during LCMV Cl-13 infection are shown. The percentage in the left column indicates the percentage of CD8 T cells that is Db/GP33 tetramer-positive. The percentage in the second, third, or fourth column indicates the percentage of tetramer-positive cells that is IFN-γ+, TNF-α+, or IL-2+, respectively. Plots for tetramer and IFN-γ staining are gated on all lymphocytes. Plots for TNF-α and IL-2 are gated on CD8 T cells. Similar results were observed for the GP276 response (data not shown). (C) Lymphocytes from the PBMC (upper panels) or intrahepatic lymphocytes (lower panels) of LCMV Cl-13-infected mice (chronic; day 35 p.i. for PBMC, day 60 p.i. for liver) were examined for functional exhaustion. The left columns show the percent Db/GP33 tetramer-positive cells (numbers indicate percentages of CD8 T cells). Production of IFN-γ (gated on all lymphocytes) and TNF-α (gated on CD8 T cells) is shown in the middle and right columns, respectively. Numbers indicate the percentage of tetramer-positive cells making cytokine. No IL-2-producing cells were detected from CD8 T cells from chronically infected mice (data not shown). Similar results were observed at other time points and for GP276-specific responses (data not shown). Note that the GP33 peptide used for intracellular cytokine staining stimulates both the Db/GP33 and Kb/GP34 response. Therefore, the percent functional of tetramer-positive cells shown in panels A, B, and C was determined using both DbGP33 and KbGP34 tetramers. As shown in Fig. 2, the Kb/GP34 becomes negligible by 2 to 3 weeks p.i. Therefore, only the Db/GP33 tetramer stain is shown. No specific staining was observed when using isotype control antibodies.

FIG. 4.

Progressive loss of cytokine production during chronic LCMV Cl-13 infection. (A) The fraction of tetramer-positive cells able to produce IFN-γ (top row) was determined for the NP396-, GP33-, and GP276-specific populations at the indicated times p.i. with LCMV Cl-13 (Chronic) or Armstrong (Acute). The percentage of the tetramer-positive population able to produce TNF-α (middle row) or IL-2 (bottom row) was determined using three-color intracellular cytokine staining. Asterisks indicate too few cells available for analysis as a result of deletion. Open bars indicate LCMV Armstrong infection, while the black bars indicate LCMV Cl-13 infection. The data represent the average and standard deviation for three to six mice/time point. (B) An example of fully functional GP33-specific memory CD8 T cells from an LCMV Armstrong-immune mouse (top row) and three examples of different states of functional exhaustion of GP33-specific T cells during LCMV Cl-13 infection are shown. The percentage in the left column indicates the percentage of CD8 T cells that is Db/GP33 tetramer-positive. The percentage in the second, third, or fourth column indicates the percentage of tetramer-positive cells that is IFN-γ+, TNF-α+, or IL-2+, respectively. Plots for tetramer and IFN-γ staining are gated on all lymphocytes. Plots for TNF-α and IL-2 are gated on CD8 T cells. Similar results were observed for the GP276 response (data not shown). (C) Lymphocytes from the PBMC (upper panels) or intrahepatic lymphocytes (lower panels) of LCMV Cl-13-infected mice (chronic; day 35 p.i. for PBMC, day 60 p.i. for liver) were examined for functional exhaustion. The left columns show the percent Db/GP33 tetramer-positive cells (numbers indicate percentages of CD8 T cells). Production of IFN-γ (gated on all lymphocytes) and TNF-α (gated on CD8 T cells) is shown in the middle and right columns, respectively. Numbers indicate the percentage of tetramer-positive cells making cytokine. No IL-2-producing cells were detected from CD8 T cells from chronically infected mice (data not shown). Similar results were observed at other time points and for GP276-specific responses (data not shown). Note that the GP33 peptide used for intracellular cytokine staining stimulates both the Db/GP33 and Kb/GP34 response. Therefore, the percent functional of tetramer-positive cells shown in panels A, B, and C was determined using both DbGP33 and KbGP34 tetramers. As shown in Fig. 2, the Kb/GP34 becomes negligible by 2 to 3 weeks p.i. Therefore, only the Db/GP33 tetramer stain is shown. No specific staining was observed when using isotype control antibodies.

FIG. 5.

In vitro cytotoxicity is impaired during chronic infection. Splenocytes isolated from LCMV Armstrong- or LCMV Cl-13-infected mice at day 8 p.i. were tested for cytotoxicity in a 5-h 51Cr release assay ex vivo. To accurately compare cytotoxicity from LCMV Armstrong- and LCMV Cl-13-infected mice, the effector/target cell (E:T) ratios were adjusted for the individual Db/NP396, Db/GP33 plus Kb/GP34, and Db/GP276 tetramer-positive populations in the first, second, and third rows, respectively. Since the GP33 peptide stimulates both the Db/GP33 and Kb/GP34 populations, the numbers of Db/GP33 and Kb/GP34 tetramer-positive cells were combined to obtain an accurate E:T ratio for GP33-pulsed targets. Targets were peptide-pulsed (top three rows) or LCMV-infected (bottom row) MC57 fibroblasts. E:T ratios for LCMV-infected targets indicate total splenocytes to targets (bottom row). Filled symbols represent peptide-coated or LCMV-infected targets, while open symbols are unpulsed, uninfected targets.

FIG. 6.

Correlation of antigen load to functional impairment of virus-specific CD8 T cells. (A) Virus in serum was quantitated by plaque assay, and the level of virus was plotted against cytokine responses. Top row, the percentage of tetramer-positive (tetramer+) cells producing IFN-γ in chronically infected mice (1 to 2 months p.i.) is plotted against viral load. Middle and bottom rows, the percentage of tetramer-positive cells that can synthesize TNF-α or IL-2 at day 15 after Cl-13 infection is plotted against the viral load. Lines indicate the linear regression best fit. (B) Splenocytes from uninfected mice (Naive Sp; left column), LCMV Cl-13-infected mice (day 30 p.i.; D30 Cl-13 Sp, middle column), or LCMV carrier mice (Carrier Sp, right column) were depleted of CD8 T cells and used as APC to stimulate CFSE-labeled, purified memory CD8 T cells from LCMV Armstrong-immune mice (>30 days p.i.). Proliferation was assessed after 60 h of coincubation by determining loss of CFSE fluorescence in the four tetramer-positive populations indicated on the left. Similar results were observed when APC were not depleted of CD8 T cells (data not shown).

FIG. 6.

Correlation of antigen load to functional impairment of virus-specific CD8 T cells. (A) Virus in serum was quantitated by plaque assay, and the level of virus was plotted against cytokine responses. Top row, the percentage of tetramer-positive (tetramer+) cells producing IFN-γ in chronically infected mice (1 to 2 months p.i.) is plotted against viral load. Middle and bottom rows, the percentage of tetramer-positive cells that can synthesize TNF-α or IL-2 at day 15 after Cl-13 infection is plotted against the viral load. Lines indicate the linear regression best fit. (B) Splenocytes from uninfected mice (Naive Sp; left column), LCMV Cl-13-infected mice (day 30 p.i.; D30 Cl-13 Sp, middle column), or LCMV carrier mice (Carrier Sp, right column) were depleted of CD8 T cells and used as APC to stimulate CFSE-labeled, purified memory CD8 T cells from LCMV Armstrong-immune mice (>30 days p.i.). Proliferation was assessed after 60 h of coincubation by determining loss of CFSE fluorescence in the four tetramer-positive populations indicated on the left. Similar results were observed when APC were not depleted of CD8 T cells (data not shown).

FIG. 7.

Model for hierarchical loss of T-cell function during chronic viral infection. Memory T cells that persist in the absence of antigen following an acute infection are fully functional and capable of immediate synthesis of IFN-γ, TNF-α, and IL-2 upon antigen reencounter (top). Persisting virus results in CD8 T cells with various levels of function (bottom). Functional T cells can coexist with virus if antigen encounters are infrequent. Partial Exhaustion I represents the stage in which IL-2 and TNF-α are impaired even while CD8 T cells maintain the ability to produce IFN-γ. Partial Exhaustion II represents a stage where IFN-γ production also becomes impaired. At this point some functional cells can be detected by IFN-γ production, but many cells are exhausted. Full Exhaustion is the complete loss of all effector functions including IL-2, TNF-α, and IFN-γ production. Finally, deletion of epitope-specific CD8 T cells can result if epitope presentation to T cells is high and/or sustained. This hierarchical loss of function is dramatically influenced by antigen such that in the presence of low levels of virus, T cells maintain greater functional capacity, but as viral load increases, effector functions are progressively lost. Ultimately, a high antigen load leads to the physical deletion of specific T cells.