Cutting edge: CD40-CD40 ligand pathway plays a critical CD8-intrinsic and -extrinsic role during rescue of exhausted CD8 T cells

Abstract

CD8 exhaustion mediated by an inhibitory programmed death-1-programmed death ligand-1 (PD-L1) pathway occurs in several chronic infections, including toxoplasmosis. Although blockade of the programmed death-1-PD-L1 pathway revives this response, the role of costimulatory receptors involved in this rescue has not been ascertained in any model of CD8 exhaustion. This report demonstrates that one such costimulatory pathway, CD40-CD40L, plays a critical role during rescue of exhausted CD8 T cells. Blockade of this pathway abrogates the ameliorative effects of anti-PD-L1 treatment on CD8 T cells. Additionally, we demonstrate in an infectious disease model that CD8-intrinsic CD40 signaling is important for optimal CD8 polyfunctionality, proliferation, T-bet upregulation, and IL-21 signaling, albeit in the context of CD8 rescue. The critical role of CD40 during the rescue of exhausted CD8 T cells may provide a rational basis for designing novel therapeutic vaccination approaches.

Figure 1

High expression of CD40 on CD8 T cells in αPD-L1 treated mice. A, Chronically infected mice were treated with αPD-L1 for 2 weeks and then sacrificed at week 7 pi. CD40 expression was analyzed on splenic CD8 T cells. Dotted line represents background fluorescence throughout. B, represents PD-1 expression on CD40⁺ and CD40⁻ splenic CD8 T cells in αPD-L1 treated mice. C, Splenic CD8 T cells from control and αPD-L1 treated mice were evaluated for CD40L expression. The data represent 3 experiments with at least 4 mice per group.

Figure 2

Blockade of CD40-CD40L pathway abrogates αPD-L1 mediated rescue of CD8 T cells. A, αPD-L1 or αCD40L or both were administered to chronically infected mice for 2 weeks and then sacrificed at week 7 pi. Absolute number of CD8 T cells was evaluated in spleen and brain. CD40 (B) and Ki-67 (C) expression was assessed in these mice by flow cytometry. D, IFNγ and Gzb production by CD8 T cells was evaluated in splenocytes or brain cells from infected mice in presence of TLA. E, Splenic CD8 T cells purified from in vivo antibody treated animals were evaluated for T-bet (top panel) and Eomes (bottom panel) after 6h in vitro stimulation with agonistic CD40 or control antibody. The data represent 2 experiments with at least 4 mice per group. Error bars represent standard deviation throughout.

Figure 3

CD40 plays a CD8 intrinsic role during rescue of exhausted CD8 T cells. Chronically infected mixed BM chimeras were treated with αPD-L1 for 2 weeks and analyzed at week 7 pi. A and B, Relative abundance of WT (CD90.1) and KO (CD90.2) CD8 T cells evaluated in spleen and brain by flow cytometry is presented as representative flow plots (A) or bar graphs (B). C, represents frequency of WT and KO splenic CD8 T cells in control chimeras expressing PD-1. D and E, The percentage of proliferating congenic splenic CD8 T cells was assessed by intracellular staining for Ki-67. Flow plots in red denote KO CD8 T cells throughout. Data is presented as histograms (D) or bar graphs (E). F and G, Histograms (F) and bar graphs (G) depict active caspase expressing CD8 T cells, evaluated after incubating splenocytes from chimeras at 37°C for 5h. H, IFNγ, Gzb and TNFα production by congenic CD8 T cells were evaluated in splenocytes or brain cells from infected mice in presence of TLA. I, represents the percentage of bifunctional (top panel) and trifunctional (bottom panel) CD8 T cells in spleen and brain. Results are representative of 2 experiments with at least 3 mice per group.

Figure 4

CD40 plays a strictly T cell intrinsic role in mediating IL-21 and IL-21R expression on T cells. A, IL-21R expression was measured in CD40⁺ and CD40⁻ splenic and brain CD8 T cells from αPD-L1 treated C57BL/6 mice. Congenic splenic CD8 (B and C) and CD4 (D and E) T cells in control and αPD-L1 treated BM chimeras were assessed for IL-21R expression. Data is presented as histograms (B and D) or bar graphs (C and E). F and G, Flow plots (F) and bar graphs (G) depict the percentage of IL-21 producing splenic CD4 T cells in these chimeras. The data represent 2 experiments with 3 mice per group.