Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection

Abstract

T cell exhaustion often occurs during chronic infection and prevents optimal viral control. The molecular pathways involved in T cell exhaustion remain poorly understood. Here we show that exhausted CD8+ T cells are subject to complex layers of negative regulation resulting from the coexpression of multiple inhibitory receptors. Exhausted CD8+ T cells expressed up to seven inhibitory receptors. Coexpression of multiple distinct inhibitory receptors was associated with greater T cell exhaustion and more severe infection. Regulation of T cell exhaustion by various inhibitory pathways was nonredundant, as blockade of the T cell inhibitory receptors PD-1 and LAG-3 simultaneously and synergistically improved T cell responses and diminished viral load in vivo. Thus, CD8+ T cell responses during chronic viral infections are regulated by complex patterns of coexpressed inhibitory receptors.

Figure 1. Memory and exhausted CD8⁺ T cells express multiple inhibitory receptors

(a) Nearest neighbor analysis using gene expression profiles from. Numbers indicate the pearson correlation coefficient with numbers closer to 1 representing better correlation with the pattern of PDCD1 expression. Superscripted letters indicate nearest (^a) to furthest (^b) neighbor for this set of genes. (b) The expression of eleven inhibitory receptors on H-2D^bgp33⁺ CD8⁺ splenocytes from LCMV Arm and clone 13 mice (d30p.i.) was examined by flow cytometry by single marker staining in 4 color panels. Naive CD8⁺ T cells were total CD44^Lo CD8⁺ cells from LCMV Arm immune mice. Mean fluorescence intensity is displayed for each population. Stains are representative of 3-5 mice each from at least 3 independent experiments.

Figure 2. Four types of LCMV infection

(a) C57BL/6 mice were infected with LCMV Arm, T1B, or clone 13. Also a group of mice was depleted of CD4 T cells with an injection of 200 μg of anti-CD4 (GK1.5) one day prior and one day after infection with LCMV clone 13 (anti-CD4 plus clone 13). Serum was collected from mice longitudinally and serum viral titers were determined by plaque assay. n=5 per time point. (b) Spleens were harvested from LCMV infected mice d3 p.i. and viral load was determined by plaque assay. n=4. (c,d) The frequency of H-2D^bgp33⁺ CD8⁺ T cells was monitored in the peripheral blood by flow cytometry and plotted over time. Numbers indicate the percent of CD8⁺ T cells that are tetramer⁺. (e) The expression of CD62L and CD127 was monitored on H-2D^bgp33⁺ CD8⁺ T cells by flow cytometry. Data from (d and e) represent n=5 per time point and are representative of 2 independent experiments.

Figure 3. Influence of the severity of infection on inhibitory receptor expression and CD8⁺ T cell function

(a) The expression of four of the exhaustion associated inhibitory receptors was examined on H-2D^bgp33 CD8⁺ splenocytes from mice infected with Arm, T1B, clone 13 or anti-CD4 plus clone 13 on d30 p.i. by multiparameter flow cytometry. The numbers indicate the percent of tetramer⁺ CD8⁺ T cells expressing the indicated receptor. Similar inhibitory receptor expression profiles were also observed on H-2D^bgp276 tetramer⁺ CD8 T cells (data not shown) (b) ICS was performed on splenocytes from the four infection groups on d30 p.i using gp33-41 peptide. IFN-γ and TNF co-production was examined by flow cytometry. Numbers indicate the percent of IFN-γ⁺ CD8⁺ T cells also producing TNF. (c) The mean fluorescence intensity of inhibitory receptor expression was determined for each receptor during each infection. (d) MFI of IFN-γ for 3-5 mice per infection. All data are representative of three independent experiments with 3-5 mice per experiment.

Figure 4. Concurrent expression of multiple inhibitory receptors increased with viral load and correlated with decreased functionality

The simultaneous expression of multiple inhibitory receptors (PD-1, LAG-3, 2B4, and CD160) on H-2D^bgp33⁺ CD8⁺ splenocytes from the four infection groups was examined on d8 and d30 p.i. using Boolean gating analysis. (a) Individual populations were grouped according to the total number of inhibitory receptors expressed. (b) Relative abundance of each possible individual inhibitory receptor expression profile. (c) Intracellular cytokine staining with IFN-γ and TNF using gp33-41 peptide stimulation of exhausted CD8⁺ T cells (d30p.i. LCMV clone 13). ICS was done in conjunction with staining for 4 inhibitory receptors. Note that the expression of the inhibitory receptors examined here does not change during the 5 hr ICS stimulation (data not shown). After Boolean gating, the percentage of gp33 responding cells that produced both IFN-γ and TNF was determined and individual populations were grouped according to the total number of inhibitory receptors expressed. All data are representative of three independent experiments with 3-5 mice per experiment.

Figure 5. PD-L1 plus LAG-3 blockade during chronic LCMV infection enhances antiviral CD8⁺ T cell responses

(a) C57BL/6 mice were CD4 depleted and infected with LCMV clone 13. On D28 groups of mice were treated with either an isotype control antibody, anti-AG3, anti-PD-L1, or both anti-LAG3 and anti-PD-L1 every third day for two weeks (n=5-7 per group). (b) Staining with H-2D^bgp276 tetramer on CD8⁺ splenocytes after two weeks of treatment. Numbers indicate the percent of CD8⁺ T cells that were tetramer⁺. (c) Total number of H-2D^bgp276⁺ CD8⁺ T cells in the spleen after treatment. Note that similar results were observed for H-2D^bnp396, and H-2D^bgp33 tetramer staining (data not shown). (d) Total number of degranulating (CD107a⁺) and/or IFN-γ producing CD8⁺ splenocytes in response to peptide stimulation after two weeks of treatment. (e) Staining with H-2D^bgp276 tetramer on CD8⁺ PBMCs after two weeks of treatment. Numbers indicate the percent of CD8⁺ T cells that were tetramer⁺. (f) Frequency of H-2D^bgp276⁺ CD8⁺ T cells per 10⁶ PBMCs before an after treatment. All data are representative of three independent experiments with 5-7 mice per experiment.

Figure 6. Improved function of exhausted CD8⁺ T cells following dual LAG-3 plus PD-L1 blockade

(a) Percent of H-2D^bgp276 CD8⁺ splenocytes that produced IFN-γ in response to gp276 peptide stimulation. (b,c) CD107a degranulation assay performed in conjunction with ICS for IFN-γ with gp276 peptide stimulation. The percentage of responding cells that both produced IFN-γ and degranulated is indicated. (d,e) Intracellular staining for Ki67 in H-2D^bgp276⁺ CD8⁺ splenocytes. The percentage of H-2D^bgp276⁺ CD8⁺ splenocytes expressing Ki67 is indicated. A similar trend was observed by BrdU incorporation (data not shown). All data are representative of three independent experiments with 5-7 mice per experiment.

Figure 7. Increased viral control with dual PD-L1 plus LAG3 blockade

(a) Longitudinal analysis of viral load in the serum. (b) Viral load in the spleen, liver, kidney, and brain. Data are representative of three independent experiments with 5-7 mice per experiment.