Virus-specific CD8+ T cells upregulate programmed death-1 expression during acute friend retrovirus infection but are highly cytotoxic and control virus replication

Abstract

It was recently reported that inhibitory molecules such as programmed death-1 (PD-1) were upregulated on CD8(+) T cells during acute Friend retrovirus infection and that the cells were prematurely exhausted and dysfunctional in vitro. The current study confirms that most activated CD8(+) T cells upregulated expression of PD-1 during acute infection and revealed a dichotomy of function between PD-1(hi) and PD-1(lo) subsets. More PD-1(lo) cells produced antiviral cytokines such as IFN-γ and TNF-α, whereas more PD-1(hi) cells displayed characteristics of cytotoxic effectors such as production of granzymes and surface expression of CD107a. Importantly, CD8(+) T cells mediated rapid in vivo cytotoxicity and were critical for control of acute Friend virus replication. Thus, direct ex vivo analyses and in vivo experiments revealed high CD8(+) T cell functionality and indicate that PD-1 expression during acute infection is not a marker of T cell exhaustion.

Figure 1. Expression of PD-1 on activated CD8⁺ T cells during acute FV infection

CD8⁺ T cells from FV infected B6 mice were stained for the activation-associated glycoform of CD43 and PD-1 to detect the expression of PD-1 on the total population of activated CD8⁺ T cells at different time points after FV infection. CD8⁺ T cells positive for CD43 during acute FV infection expressed several other effector T cell markers, like CD44 and CD69, and were negative for CD62L (data not shown (23)). A. A representative dot plot shows how gated CD8 T cells were stained for the activation-associated glycoform of CD43 and PD-1 to analyze the two different populations of activated CD8⁺ T cells. B. The percentages of PD-1 positive CD8⁺ T cells expressing the activation marker CD43 (black bar) or were CD43 negative (white bar) are shown for a group of 6 – 10 mice on day 10 post FV infection. Data were pooled from two independent experiments with similar results. Each column represents the mean percentage plus SEM. Statistically significant differences between the groups are indicated by a P value. C. Kinetic analysis of the expansion of activated (CD43⁺) CD8⁺ T cells and their relative expression of PD-1 (hi=high; lo=low). Each column represents mean numbers of CD43⁺ CD8⁺ T cells per one million nucleated cells for a group of 6 – 10 mice. Black bars show the numbers of PD-1^hi CD43⁺ CD8⁺ cells plus SEM and white bars show numbers of PD-1^lo CD8⁺ CD43⁺ T cells plus SEM. Data were pooled from two independent experiments with similar results. The kinetics of the relative virus titer is indicated by a dotted line. D. The percentages of D^bGagL class I tetramer reactive virus-specific CD8⁺ T cells, which were PD-1^hi (black bar) or PD-1^lo (white bar) are shown for a group of 5 – 10 mice on day 10 post FV infection. Data were pooled from two independent experiments with similar results. Each column represents the mean percentage plus SEM of CD8⁺ tetramer⁺ T cells.

Figure 2. Phenotype of activated PD-1^hi and PD-1^lo CD8⁺ T cells from acutely FV infected mice

C57BL/6 mice were infected with FV and CD8⁺ T cells were harvested at 10 dpi. A. Multi-parameter flow cytometry was used to compare activated (CD43⁺) PD-1^hi and PD-1^lo CD8⁺ T cells for expression of CD44, CD122, CD62L, CD127, and intracellular Bcl-2. Staining with control antibody and staining of CD8⁺ T cells from naive mice are shown as negative controls. Dual staining of gated CD8⁺ CD43⁺ T cells for CD127 and Bcl-2 is shown in a representative dot plot (numbers represent the percentage of double positive cells). The expression of the inhibitory receptors LAG-3, BTLA, 2B4, and Tim-3 on activated PD-1^lo or PD-1^hi CD8⁺ T cells was analyzed. 6 – 9 mice per group were analyzed in three independent experiments. Representative histogram or dot plots and the mean fluorescence intensities plus SEM for each group are shown. Significant differences between the two cell populations were found for P< .01: CD122 and CD127; P< .001: Bcl-2; P< .0001: LAG-3, BTLA, 2B4, and Tim-3.

Figure 3. Functional properties of activated PD-1^hi and PD-1^lo CD8⁺ T cells during acute FV infection

C57BL/6 mice were infected with FV and CD8⁺ T cells were harvested at 10 dpi. Multi-parameter flow cytometry was used to compare activated (CD43⁺) PD-1^hi and PD-1^lo CD8⁺ T cells for intracellular production of the cytokines IFNγ, TNFα, and IL-2 and the production of granzymes or the expression of the degranulation marker CD107a. A. Mean percentages plus SEM of CD43⁺ CD8⁺ T cells producing IFNγ, TNFα, or IL-2 are shown. B. Mean percentages plus SEM of individual CD43⁺ CD8⁺ T cells producing no, 1, 2, or 3 different cytokines at the same time. The figure shows PD-1^hi (left) versus PD-1^lo (right) cells using data pooled from two independent experiments (n=8) with similar results. C. Mean percentages plus SEM of activated PD-1^hi and PD-1^lo CD8⁺ T cells expressing granzyme A, granzyme B or CD107a. D. Mean percentages plus SEM of PD-1^hi or PD-1^lo tetramer⁺ CD8⁺ T cells expressing the degranulation marker CD107a. Each column represents the mean percentage plus SEM for a group of 5 – 10 mice. Data were pooled from two independent experiments with similar results. Differences were analyzed by the unpaired t-test. Statistically significant differences between the groups are indicated by P values. E. Representative histograms showing killing of target cells loaded with the FV D^bGagL peptide (CFSE high; +peptide) or control cells without peptide (CFSE low; -peptide) in spleens of naïve or FV infected mice with an H-2^b/b (C57BL/6) or H-2^a/b ((B10.A x A.BY)F1) genetic background. Numbers in the figure indicate the mean percentage of killing for the whole group. Data from one representative mouse of a group of 7 mice are shown. Two independent experiments with similar results were performed.

Figure 4. Inhibitory signal blockade during acute FV infection

Mice were treated with blocking antibodies at the time of infection and every other day for a total of 5 injections. A. CD8⁺ splenocytes from naïve mice (gray bar) or mice treated with anti-PDL-1 (black bars) or isotype-matched control IgG (white bars) at 10 days post-infection were analyzed by flow cytometry for FVgagL tetramer binding. Gated CD8⁺ tetramer⁺ T cells were analyzed for B. Intracellular expression of granzyme B and C. surface expression of CD107a. Bars depict the mean fluorescent intensity (MFI). D. An in vivo cytotoxicity assay was performed using adoptive transfer of CellTrace Violet labeled control splenocytes and CFSE-labeled FVgagL peptide loaded targets into the anti-PDL-1 or control IgG treated mice at 10 dpi. Two hours after transfer, the spleens were analyzed by flow cytometry for the percentage of target cell killing. Data are combined from two independent experiments with similar results and mean values with standard deviation are shown (n=8 mice per group). E. Spleen infectious centers at 10dpi from mice treated as indicated. All columns of data were compared to the Ig control using a one-way ANOVA with Tukey’s post test. All columns were significantly different (P < .0001) except the control versus anti-PDL-1 (n=8 for the control and anti-PDL-1 groups and n=4 for anti-Tim-3 and dual treatment groups).

Figure 5. CD8⁺ T cell depletion during acute FV infection

Mice were depleted of CD8⁺ T cells as described the methods section and remained depleted to less than 1% of splenocytes in through 14 dpi. Data on viral loads are combined from two separate experiments. The dotted line represents the detection limit of the assay. A. Infectious centers at 7 dpi. Differences between the groups were statistically significant (P= .0192). B. Infectious centers at 14 dpi. Differences between the groups were statistically significant (P< .0001).