PD-L1 but not PD-L2 is required for E2-induced protection against severe EAE in IL-10 deficient mice.
Initial studies were carried out in WT and IL-10-KO female mice receiving E2 pellets seven days prior to the induction of EAE and further treatments on days 1, 7 & 14 post-EAE induction with Vehicle (Veh) or antibodies to PD-L1 (α-PD-L1), PD-L2 (α-PD-L2) or their respective isotype controls (α-ISO). Mice were scored for 21 days post-immunization to determine if neutralization of PD-L1 or PD-L2 abrogated E2-induced protection against EAE in IL-10-KO vs. WT mice. As is shown in Fig. 1a, treatment of E2-primed WT mice with Veh, α-PD-L1 or α-ISO resulted in near complete protection of all treatment groups over the 21-day scoring period. In contrast, treatment of E2-primed IL-10-KO mice with Veh or α-ISO resulted in moderate to low clinical signs of EAE (consistent with lack of IL-10 mediated suppression), whereas treatment with α-PD-L1 rapidly reversed the E2-mediated protection beginning on Day 13 and resulting in significantly more severe clinical EAE over days 17–21 than E2-primed WT mice treated with α-PD-L1 as well as E2-primed IL-10-KO mice treated with Veh- or α-ISO (Figs. 1b and 1c). In a parallel experiment shown in Fig. 2, E2-primed WT mice treated with Veh, α-ISO and α-PD-L2 again showed near complete protection against EAE over a 21 day scoring period, whereas E2-primed IL-10-KO mice treated with Veh or α-ISO developed moderate to background levels of EAE as above. However, unlike E2-primed IL-10-KO mice treated with α-PD-L1, those treated with α-PD-L2 did not exceed the EAE background levels observed in the Veh- or α-ISO-treated groups (Fig. 2b and 2c).
These data demonstrated that functional PD-L1 but not PD-L2 is required to sustain E2-induced protection against EAE in mice lacking IL-10. We thus focused our subsequent studies on α-PD-L1 treated E2-primed vs. Sham primed IL-10-KO mice in order to clearly identify E2-associated differences compared to E2 untreated control mice over a 21-day observation period. Female IL-10-KO mice were primed with E2 pellets or sham treatment seven days prior to EAE induction and subsequently treated on days 1, 7 and 14 with Veh, α-PD-L1 or α-ISO after immunization to induce EAE as above. As shown in Fig. 3a, sham-primed IL-10-KO mice treated with Veh or α-ISO developed typically severe EAE over the 21-day observation period (peak scores ~ 4.5–5.0) with a transient reduction in EAE scores in the α-PD-L1 treatment group on days 13–15 post-induction. In contrast, E2-primed IL-10-KO mice treated with α-PD-L1 developed severe EAE in real time beginning on Day 15 (concurrent with the third α-PD-L1 injection) indistinguishable from all of the Sham pre-treatment groups by Day 18 after disease induction but significantly greater than E2-primed IL-10-KO mice treated with Veh or α-ISO (Figs. 3b and 3c). To validate effects of α-PD-L1 treatment, our FACS evaluation of IL-10-KO spleen cells documented an E2-dependent reduction of PD-L1+ cells in the E2 vs. Sham Veh, α-ISO and α-PD-L1 treatment groups (Fig. 3d). These results clearly demonstrate that PD-L1 is a critical E2-induced compensatory component required for inhibition of EAE in IL-10-KO mice.
Treatment with α-PD-L1 increased percentages of CD11b + CD45 hi cells in the brain and CD11b+, CD4 + and CD19 + cells in the spleen of E2-primed IL-10-KO mice concordant with increasing EAE severity over time.
Local activation of resident microglia, recruitment of infiltrating macrophages into the CNS and enhancement of T and B-cells numbers all can enhance EAE disease severity (Wiedrick et al. 2021). FACS analysis demonstrated that α-PD-L1 treatment of E2-primed IL-10-KO mice (that resulted in increased EAE severity) resulted in an increased percentage of activated CD11b+CD45hi but not resting CD11b+CD45low microglia/macrophages in the brain (Fig. 4a & 4b) and a parallel increase in CD11b+ cells in the spleen (Fig. 4c) with no other changes observed in the indicated control groups. Moreover, there were significantly increased percentages of CD4+ T-cells and CD19+ B-cells in the spleens of E2-primed IL-10-KO α-PD-L1 treated mice vs. E2-primed IL-10-KO Veh and α-ISO treated mice (Figs. 4d & 4e). These findings support the contention that loss of PD-L1 mediated E2-associated protection against EAE in IL-10-KO mice is strongly reflected by increased numbers of activated macrophages, T-cells and B-cells in brain and spleen.
Increased percentage of CD74 + cells in brain and spleen mice upon treatment with α-PD-L1 reflects loss of E2-mediated protection against EAE in IL-10-KO mice.
Upregulation of CD74 in lymphoid tissues and CNS is an indicator of increased inflammation and EAE disease severity induced through the MIF/CD74 axis (Benedek et al. 2013). As shown in Fig. 5a, FACS analysis revealed ~ 20% CD74+ cells in brains of Sham Veh and Sham ISO treated mice that developed severe clinical EAE, but a significantly lower percentage of CD74+ cells (~ 3%, p < 0.01) in E2-protected Veh and α-ISO treated groups that developed only mild EAE. In contrast, both Sham- and E2-primed groups treated with α-PD-L1 had equivalent ~ 20% levels of CD74+ cells and severe EAE, clearly demonstrating that the increase in CD74+ cells after α-PD-L1 treatment reflected the loss of E2 protection against EAE. A similar pattern of responses was observed in the spleen although the inhibitory effect of E2 on CD74 expression in the Veh and ISO control groups was less pronounced (Fig. 5b).
Increased E2-dependent expression of CD73 + cells in spleens of IL-10-KO Veh and α-ISO mice is further enhanced after α-PD-L1 treatment of both Sham- and E2-primed treatment groups.
CD73 is an IL-10 independent immune regulatory receptor expressed on Bregs and other cells that mediates immunosuppression by upregulating adenosine production (Kaku et al. 2014). Our previous study demonstrated that CD73 expression was increased on both Sham- and E2-primed splenic Breg cells from IL-10-KO vs. WT mice and that there was a significant increase in CD73 expression in spinal cords of E2-primed vs. Sham-primed IL-10-KO mice. Here, we further demonstrate that in the spleen, increased CD73 expression in IL-10-KO mice is greater in E2- vs. Sham-primed groups and that even higher expression occurs in both groups after treatment with α-PD-L1 (Fig. 6). This result confirms that increased CD73 expression in IL-10-KO mice is enhanced after E2 priming and further shows that this increase could be further enhanced after antibody neutralization of PD-L1.