A role for the transcriptional repressor Blimp-1 in CD8(+) T cell exhaustion during chronic viral infection

Abstract

T cell exhaustion is common during chronic infections and can prevent optimal immunity. Although recent studies have demonstrated the importance of inhibitory receptors and other pathways in T cell exhaustion, the underlying transcriptional mechanisms are unknown. Here, we define a role for the transcription factor Blimp-1 in CD8(+) T cell exhaustion during chronic viral infection. Blimp-1 repressed key aspects of normal memory CD8(+) T cell differentiation and promoted high expression of inhibitory receptors during chronic infection. These cardinal features of CD8(+) T cell exhaustion were corrected by conditionally deleting Blimp-1. Although high expression of Blimp-1 fostered aspects of CD8(+) T cell exhaustion, haploinsufficiency indicated that moderate Blimp-1 expression sustained some effector function during chronic viral infection. Thus, we identify Blimp-1 as a transcriptional regulator of CD8(+) T cell exhaustion during chronic viral infection and propose that Blimp-1 acts as a transcriptional rheostat balancing effector function and T cell exhaustion.

Figure 1. Blimp-1 was highly expressed in exhausted antigen-specific CD8 T cells during chronic viral infection

A) Blimp-1 message by qPCR. DbGP33+CD8 T cells were sorted by FACS from LCMV Arm (acute) or clone 13 (chronic) infected mice at the indicated d.p.i. Naïve controls were CD44^lo CD8 T cells sorted from Arm immune. Error bars show range of expression in triplicate wells. B) Blimp-1 YFP reporter expression in CD8 T cells on d8 p.i. with LCMV Arm or clone 13. Plots are gated on total CD8 T cells. C) Kinetics of Blimp-1 YFP reporter expression after LCMV Arm or clone 13 infection. Histograms are gated on DbGP33+ CD8 T cells from the blood of LCMV Arm (shaded) or LCMV clone 13 (open) infection. Data points in graph show Blimp-1 YFP reporter MFI of DbGP33+ CD8 T cells from LCMV Arm (black circles) or LCMV clone 13 (white circles) at the indicated d.p.i.. Error bars are standard error of the mean (SEM). N=2−3 mice per time point. D) Blimp-1 YFP reporter MFI in tissues 30 d.p.i. with LCMV Arm or clone 13. Dot plot is gated on total CD8 T cells. Histograms are gated on DbGP33+ CD8 T cells from LCMV Arm (shaded) or LCMV clone (open) infection. Open red histograms show CD44^lo CD8 T cells from LCMV Arm infection.

Figure 2. CD8 T cell responses in Blimp-1 conditional knockout mice during chronic viral infection

A) Efficiency of granzyme B-Cre-mediated gene deletion. Rosa26-f/stop/f-YFP × gzmB-Cre mice or wt littermates were infected with LCMV clone 13. CD8 T cells were analyzed for YFP expression at d3 (blood) and d6 (spleen) p.i. Plots are gated on total CD8 T cells and histograms are gated on DbGP33+ CD8 T cells. Numbers above gates in the histograms indicate the percentage of DbGP33+ CD8 T cells that are YFP+. B) Viral loads in multiple tissues in wt and CKO mice. CKO mice and wt littermates were infected with LCMV clone 13 and analyzed at d30 p.i. unless otherwise noted. Viral titers were measured by plaque assay at the indicated time points in the serum and tissues. Data is representative of four independent experiments. Error bars in serum titers represent SEM. N=3−10 mice per time point. C) Absolute number of antigen-specific CD8 T cells in wt and CKO mice. Frequency of DbGP33+ CD8 T cells was measured in the blood at indicated time points. Absolute number of tetramer+ CD8 T cells was measured in each tissue at d30 pi. Numbers above spleen graph indicate fold increase of CKO over wt for each tetramer+ population. Error bars represent SEM, and n=4−8 for each tissue. Data represents three independent experiments. D) Phenotype of wt and CKO antigen-specific CD8 T cells. Plots are gated on total CD8 T cells in the spleen. Numbers in lefthand plots show percentage of total CD8 T cells that are DbGP33+. Numbers in center and righthand plots indicate the percentage of DbGP33+ CD8 T cells that are positive for each marker. Graphs represent two independent experiments, Arm indicates marker expression for memory CD8 T cells from Arm immune mice (∼d30 p.i.). Error bars are SEM. For CD127, *p=0.04, **p=0.002 by Student's t-test. E) Effector function of wt and CKO antigen-specific CD8 T cells compared to memory CD8 T cells (d30+ Arm immune). Splenocytes were stimulated with GP33 peptide for 5 hrs. Plots are gated on CD8 T cells. Numbers in each plot show the percent of IFNγ producing CD8 T cells that also produced the second indicated cytokine. Numbers are graphed to the right.

Figure 3. Blimp-1 expression correlated with expression of inhibitory receptors on antigen-specific CD8 T cells during chronic viral infection

A) Blimp-1 mRNA in PD-1^hi and PD-1^int subsets of exhausted CD8 T cells. Plot is gated on wt CD8 T cells from spleen at d30 post LCMV clone 13 infection. For qPCR, PD-1^hi and PD-1^int DbGP33+ CD8 T cells were sorted from the spleens of LCMV clone 13 infected wt mice. Graph shows fold increase in Blimp-1 expression over naïve CD44^lo CD8+ T cells sorted from LCMV Arm immune mice. Error bars show range of expression in triplicate wells. B) Blimp-1 YFP reporter expression in inhibitory receptor hi versus lo subsets of exhausted CD8 T cells. Blimp-1 YFP reporter mice were infected with LCMV clone 13 and YFP expression was analyzed in the spleen at 30 days p.i.. Histograms are gated on DbGP33+ CD8 T cells that are inhibitory receptor^lo (shaded) or inhibitory receptor^hi (open). Numbers in gray show YFP MFI of shaded histograms, numbers in black represent YFP MFI of open histograms. C) Correlation of Blimp-1 expression and number of inhibitory markers expressed. Blimp-1 YFP reporter mice were infected with LCMV clone 13 and inhibitory receptor expression was determined on DbGP33+ CD8 T cells from the spleen at d30 p.i. Boolean gating established the populations that expressed a combination of 3, 2, 1 or no inhibitory markers and Blimp-1 YFP MFI was determined for each subgroup. Graph represents two independent experiments. D) Inhibitory receptor expression on antigen-specific CD8 T cells from wt and CKO mice. CKO and wt littermates were infected with LCMV clone 13 and analyzed on d30 p.i. for inhibitory marker expression in the spleen. Plots are gated on total CD8 T cells. Histograms are gated on DbGP33+ CD8 T cells. Numbers above the gates indicate the percentage of DbGP33+ CD8 T cells in each gate. Numbers in red are the MFI of each marker. Pie charts represent DbGP33+ CD8 T cell populations from wt and CKO mice. Each colored slice indicates the fraction of DbGP33+ CD8 T cells that expresses a combination of 4, 3, 2, 1 or no inhibitory receptors. E) Inhibitory receptor expression in total CD44^hi CD8 T cells in wt and CKO mice 30 days p.i. Histograms are gated on CD44^hi CD8 T cells from wt (shaded) or CKO (open) mice. Open red histograms represent CD44^lo CD8 T cells from wt mice.

Figure 4. Blimp-1 haploinsufficiency in antigen-specific CD8 T cells leads to rapid viral control

A) Viral titers in CKO, het and wt littermates d60 post LCMV clone 13 infection. Viral load was determined in the serum on the indicated days and in the tissues on d60 p.i. by plaque assay. Lines on the graphs represent limit of detection. Viral titers are representative of three independent experiments, n=3−17. For kidney titers, wt vs. het *p=0.03, wt vs. CKO **p=0.003, het vs. CKO **p=0.002 by Student's t-test. B) PD-1 expression on antigen-specific CD8 T cells from CKO, het and wt mice. PD-1 MFI was measured on DbGP33+ CD8 T cells from the blood at d15 p.i.. Data is representative of three experiments. Wt vs. het **p=0.007, wt vs. CKO *p=0.01 by Student's t-test. C) Representative plots of PD-1 expression at d15 p.i. in CKO, het and wt mice. Plots are gated on total CD8 T cells from the blood. Numbers in red indicate the PD-1 MFI of the DbGP33+ CD8 T cells.

Figure 5. Blimp-1 regulates cytoxicity in antigen-specific CD8 T cells during chronic viral infection

CKO, het and wt littermates were infected with LCMV clone 13 and analyzed at d30 p.i. unless otherwise noted. A) Function of CD8 T cells from CKO, het and wt mice. Splenocytes were stimulated with GP33 peptide for 5 hrs and the percentage of GP33-specific IFNγ+ CD8 T cells that also produced the second indicated cytokine was determined by ICS. Data are representative of two independent experiments, n=4−6. Error bars are SEM. B) Splenocytes from wt, conditional het, and CKO mice were stimulated for 5 hrs with GP33 peptide and degranulation based on CD107 staining was determined. Plots are gated on total CD8 T cells. Numbers in plots indicate MFI of IFNγ. Graph shows the number of DbGP33+ CD8 T cells that are also CD107+. Data represents two independent experiments. Error bars are SEM. C) Granzyme B expression in CD8 T cells from CKO, het or wt mice. Dot plots are gated on total CD8 T cells from the spleen and histograms are gated on DbGP33+ CD8 T cells. Numbers above the gates indicate the percentage of DbGP33+ CD8 T cells that express granzyme B. D) Specific killing by CD8 T cells from CKO, het and wt mice as determined by an in vitro killing assay on d8 and d30 p.i. with LCMV clone 13. Equal numbers of DbGP33+ CD8 T cells were plated with GP33 peptide labeled target cells at a 2:1 ratio. After 16−20 hrs, specific lysis was calculated. Error bars are SEM.

Figure 6. The role Blimp-1 in CD8 T cells during chronic LCMV infection was cell-intrinsic

A) Experimental design for the generation of mixed BM chimeras reconstituted with prdm1^f/f × gzmB-Cre (part B-E) or prdm1^f/f × CD4-Cre (part F) BM. B) Phenotype of CD8 T cells from mixed BM chimeras. Leftmost plot is gated on total CD8 T cells from the spleen. Top row of plots is gated on Ly5.2+ CD8 T cells (CKO) and bottom row is gated on Ly5.2- CD8 T cells (wt). Numbers in plots indicate the percentage of DbGP33+ CD8 T cells positive for each marker. Numbers are graphed to the right. Data are representative of two experiments, n=4. Error bars are SEM. For CD127, **p=0.005 and for CD62L, *p=0.01 by Student's t-test. C) Effector function of CD8 T cells from mixed BM chimeras. Splenocytes were stimulated with GP33 peptide for 5 hrs. Top row of plots is gated on CKO and bottom row is gated on wt CD8 T cells. Numbers in plots indicate the percentage of IFNγ+ CD8 T cells that also produce a second cytokine. D) Granzyme B expression in CD8 T cells from mixed BM chimeras. Plots are gated on CD8 T cells from the spleen. Histograms are gated on DbGP33+ CD8 T cells. Top row is CKO and bottom row is wt. Numbers above gates show percentage of DbGP33+ CD8 T cells that express granzyme B. E) Inhibitory receptor expression on CD8 T cells from mixed BM chimeras. Histograms are gated on wt DbGP33+ CD8 T cells (shaded) or CKO DbGP33+ CD8 T cells (open). Pie charts show either wt or CKO DbGP33+ CD8 T cells. Colored slices indicate the fraction of the total population that expressed a combination of 4, 3, 2, 1, or no inhibitory markers. F) Inhibitory receptor expression on CD8 T cells from mixed BM chimeras reconstituted with prdm1^f/f × CD4-Cre and wt bone marrow. Histograms are gated on wt DbGP33+ CD8 T cells (shaded) or open CKO DbGP33+ CD8 T cells (open). Pie charts show either wt or CKO DbGP33+ CD8 T cells.