Programmed death 1 regulates development of central memory CD8 T cells after acute viral infection

Abstract

The T cell response possesses a number of inhibitory receptors to regulate the extent of the antiviral response and prevent immune pathology. These receptors are generally transiently upregulated during an effector response and then downregulated during memory. Some inhibitory receptors, such as programmed death 1 (PD-1) and LAG-3, were shown to be aberrantly upregulated during memory to chronic lymphocytic choriomeningitis virus infection, limiting functional capabilities. However, little is known about the impact of inhibitory receptors on memory development during a normal CD8 T cell response to acute virus infection. Our previous data showed that PD-1 is aberrantly upregulated during a secondary response by memory CD8 T cells that were generated without CD4 T cell help. Therefore, we examined the role of PD-1 in memory differentiation during acute vaccinia virus infection in intact mice. In the absence of PD-1, the primary and memory CD8 T cell responses were enhanced. Moreover, there were distinct phenotypic and functional changes in the memory PD-1(-/-) CD8 T cells. Higher levels of CD62L, CD27, and CCR7 were detected; cells produced more IL-2 and made an enhanced secondary response. These changes indicate a skewing of the memory population toward the central memory phenotype in the absence of PD-1 signaling.

FIGURE 1

Significantly increased Ag-specific CD8 T cell response to VV-WR in the absence of PD-1 signaling in the PD1^−/− model. PD1^−/− and WT mice were infected with 1000 PFU of VV-WR i.n. Ag-specific responses were measured by staining with the B8R tetramer and αCD8 Ab at days 14, 21, and 30 postinfection in the blood (n = 4 per group). Data are representative of three experiments. *p < 0.01, **p < 0.001.

FIGURE 2

Robust recall response in the absence of PD-1 signaling in the PD1^−/− model. CD8⁺ cells were purified from spleens of WT and PD^−/− mice at day 71 postinfection and transferred to naive C57BL/6 recipients by i.v. injection. The number of Ag-specific PD^−/− or WT cells was determined using B8R tetramer staining and was normalized prior to adoptive transfer. In replicate experiments, 5–10 × 10³ B8R tetramer⁺ CD8 T cells were adoptively transferred. The recipients of WT or PD1^−/− B8R tetramer⁺ CD8 T cells received equal numbers of Ag-specific cells. The naive hosts were challenged with 2 × 10⁶ VV-WR (i.p.) and monitored 6 d postinfection for the recall response. Ag-specific cell numbers at day 6 were determined by multiplying the total cell number of the specific donor population by the percentage of CD8⁺ cells and the percentage of B8R⁺/CD8⁺ cells. Each point represents data from an individual mouse. Data are representative of two experiments. The t test was performed, giving a p value < 0.0001.

FIGURE 3

Increased IL-2 production by Ag-specific memory CD8 T cells in the absence of PD-1 signaling in the PD1^−/− model. Spleen cells were isolated from PD1^−/− and WT mice at 71 d postinfection and incubated for 5 h with or without B8R peptide in the presence of brefeldin A. CD8 T cells were stained to measure the production of IFN-γ (A) and IL-2 (B) in the IFN-γ–producing CD8⁺ T cell population. A, Representative dot plots of WT and PD1^−/− CD8 T cells. The numbers are the percentage of IFN-γ produced by the CD8 population. Data are shown graphically (lower panel). The background was subtracted using the no-peptide control values. B, Representative dot plots of WT and PD1^−/− CD8 T cells. The numbers are the percentage of IL-2 produced in the IFN-γ⁺ CD8 population. Data are shown graphically (lower panel). Data are representative of three experiments. *p < 0.05, Student t test. NP, no peptide stimulation; WP, with peptide stimulation.

FIGURE 4

Increased Ag-specific CD8 response in the absence of PD-1 signaling in the BMC model. PD1^−/− and WT B6 BM were mixed at a 1:1 ratio and adoptively transferred i.v. into Ly5.2 hosts that were lethally irradiated. Fifty days later, reconstitution of the immune system was confirmed, and only mice that had an ~1:1 reconstitution of WT/PD1^−/− were used in the experiment. Mice were infected with 1000 PFU of VV-WR i.n., and their Ag-specific response was determined using B8R tetramer staining. WT B6 and PD1^−/− CD8 T cells were tracked using the CD45.2 congenic marker. A, Representative plots of the CD8 response at 10 d postinfection. The numbers show the percentage of B8R tetramer⁺ CD8⁺ T cells. B, Representative plots of the CD8 response during the contraction phase at 21 d postinfection. C, Representative plots of the CD8 response at a memory time point at 56 d postinfection. D, Graph showing the percentage of B8R tetramer⁺ CD8⁺ T cells at all time points tested. E, Graph showing the number of B8R tetramer⁺ CD8⁺ T cells at all time points tested. Each point represents data from an individual mouse; data are representative of four experiments. *p < 0.05, **p < 0.005; paired t test.

FIGURE 5

Robust recall response in the absence of PD-1 signaling in the BMC model. CD8⁺ cells were purified from spleens of chimeric mice at day 60 postinfection and transferred into Thy1.1⁺ naive recipients. The number of Ag-specific PD1^−/− or WT cells was determined using CD45.2 as a congenic marker and B8R tetramer staining, prior to adoptive transfer. We adoptively transferred 1–2 × 10⁶ CD8 T cells containing 5–10 × 10³ B8R tetramer⁺ WT and PD-1^−/− CD8 T cells. To measure the magnitude of the recall response, the naive hosts were challenged with 2 × 10⁶ VV-WR i.p. and monitored 6 d postinfection. The number of Ag-specific cells of WT or PD1^−/− origin was determined by multiplying the total cell number by the percentage of CD8 T cells and the percentage of B8R⁺/CD8 T cells and the percentage of CD45.2⁻ or CD45.2⁺ cells, respectively. A t test was performed, giving a p value of 0.01. Each point represents data from an individual mouse, and data are representative of two experiments.

FIGURE 6

Increased cytokine production by Ag-specific memory CD8 T cells in the absence of PD-1 signaling in the BMC model. Spleen cells from chimeric mice were incubated for 5 h with or without B8R peptide in the presence of brefeldin A. IFN-γ (A) or IL-2 (B) (in the IFN-γ⁺ CD8 T cells) production was measured with intracellular cytokine staining, and cells were stained for the congenic marker CD45.2 to determine PD-1 status. A, Representative dot plots of WT and PD-1^−/− CD8 T cells. The numbers are the percentages of IFN-γ–producing cells in the CD8 population in the WT (CD45.2⁻) or PD-1^−/− (CD45.2⁺) compartment. The data are presented graphically (lower panel). The background was subtracted using the no-peptide control values. B, Representative dot plots of WT and PD1^−/− CD8 T cells. The numbers are the percentage of IL-2 produced in the IFN-γ–producing CD8 population in the WT (CD45.2⁻) or PD-1^−/− (CD45.2⁺) compartment. A graphical representation of the data is shown (lower panel). Each point represents data from an individual mouse, and data are representative of three experiments. *p < 0.01, paired t test. NP, no peptide stimulation; WP, with peptide stimulation.

FIGURE 7

Early skewing toward a central memory surface marker phenotype. Mixed BMC mice were infected with VV-WR (1000 PFU i.n.). Twenty-one days postinfection, spleen cells were stained with B8R tetramer plus the activation/memory markers indicated. A, Percentage of CD27^+ve cells among the Ag-specific CD8 population. B, Fluorescence intensity per cell of CD27 staining in the Ag-specific CD8 populations. C, Percentage of CD62L^hi cells among the Ag-specific CD8 population. D, Percentage of KLRG1⁺ cells among the Ag-specific CD8 population. Each pair of points represents data from an individual chimeric mouse; data are representative of three experiments. *p < 0.05, **p < 0.005; paired t test.

FIGURE 8

Early Ag-specific WT CD8s have more effector memory phenotype CD8 T cells compared with PD1^−/− CD8 T cells. Mixed BMC mice were infected with VV-WR (1000 PFU i.n.). Twenty-one days postinfection, B8R tetramer staining was performed on spleen cells together with staining for CCR7 and CD62L. A, CD62L and CCR7 double-positive population stained to indicate T_cm Ag-specific CD8 T cells. B, CD62L and CCR7 double-negative cells stained to indicate T_em Ag-specific CD8 T cells. Each pair of points represents data from an individual chimeric mouse; data are representative of two experiments. ***p < 0.0001, paired t test.

FIGURE 9

Memory Ag-specific WT CD8 T cells have more T_em phenotype CD8 T cells compared with PD1^−/− CD8 T cells. Chimeric mice were infected with VV-WR (1000 PFU i.n.). Spleen cells were stained with B8R tetramer plus αCD27 Ab 60 d postinfection. A, The percentage of CD27^+ve cells among the Ag-specific CD8 population. B, Fluorescence intensity per cell for CD27 staining in the Ag-specific CD8 populations. Each pair of points represents data from an individual chimeric mouse, and data are representative of two experiments. *p < 0.01, paired t test.

FIGURE 10

Memory Ag-specific PD1^−/− CD8 T cells preferentially home to lymphoid organs. A, Mixed BMC mice were infected with VV-WR (1000 PFU i.n.). Fifty-six days postinfection, virus-specific CD8 T cells were measured in the organs indicated using B8R tetramer. B, PD1^−/− and WT mice were infected with VV-WR (1000 PFU i.n.). Sixty days postinfection, B8R-specific CD8 T cells were identified using B8R tetramer in the organs shown. Each point represents data from an individual mouse; data are representative of two experiments. *p < 0.05, **p < 0.001. L-NODE, inguinal lymph node. ●, PD1^−/−; ○, WT.