ROLE OF THE MUCIN P-SELECTIN GLYCOPROTEIN LIGAND-1 IN THE CONTROL OF IMMUNE SUPPRESSION EXERTED BY REGULATORY T CELLS

Mucin P-selectin glycoprotein ligand-1 (PSGL-1) binds L-, E- and P-selectin and controls leukocyte trafficking under physiological and pathological conditions. Whereas PSGL-1 role is emerging in T cell homeostasis, its involvement in the induction of autoimmune diseases is not clear. In this PhD project, we studied the role of the mucin PSGL-1 in the induction and development of experimental autoimmune encephalomyelitis (EAE), an autoimmune and inflammatory pathology of the central nervous system (CNS), used as experimental model for human multiple sclerosis. Here we show that active EAE induced with myelin olygodendrocyte glycoprotein (MOG)35-55 peptide was more severe in PSGL-1-/- mice, compared to WT (wild-type) animals. In addition, MOG35-55-specific T cells produced from PSGL-1 deficient mice transferred a significantly more severe disease than WT cells in a passive-transfer model of EAE, suggesting a role for PSGL-1 in regulatory mechanisms during EAE. In particular, we focused our attention on CD4+CD25+Foxp3+ regulatory T (Treg) cells, which are known to regulate autoimmune pathologies development and are crucial in controlling EAE severity. We found that PSGL-1 deficient Tregs, compared to WT Tregs, were not able to suppress EAE when injected in the pre-clinical phase of the disease (day +7 post-immunization). Moreover, in vitro proliferation assays shown that PSGL-1-/- Tregs have a reduced ability to suppress CD4+CD25- T cells proliferation. These results suggested us a dual effect of PSGL-1 deficiency on Treg functionality in the pre-clinical phase of EAE: 1. a possible deficit in Treg trafficking into the inflamed CNS during EAE development; 2. a reduced suppression by Tregs of in vivo encephalitogenic T cells activation in draining lymph nodes of EAE mice. Based on these hypotheses, we developed our project with two aims: AIM 1: ANALYSIS OF Treg TRAFFICKING IN THE CNS DURING EAE. Intravital microscopy experiments showed that PSGL-1 deficient Tregs display a dramatic decrease of adhesive interactions in inflamed brain pial vessels, compared to WT Tregs. By performing in vivo migration assays in the inflamed CNS, we observed that regulatory T cells preferentially migrate in the CNS in the pre-clinical phase of the disease (day +7 post-immunization), compared to the disease peak (day +14 post-immunization), and Tregs from PSGL-1-/- mice present a strongly reduced migration capacity into the inflamed CNS in the pre-clinical phase of EAE, when compared to WT Tregs. Our results also demonstrated that PSGL-1 is functionally active on naïve Tregs, compared to naïve CD4+CD25- T cells, suggesting a crucial role for PSGL-1 in Treg homeostasis and trafficking in the immune system. AIM 2: ANALYSIS OF Treg-MEDIATED SUPPRESSION OF ANTIGEN-SPECIFIC T CELL ACTIVATION IN DRAINING LYMPH NODES OF EAE MICE. By using two-photon laser microscopy, we studied MOG35-55-specific T cell behaviour in draining lymph nodes of EAE mice, in the presence or absence of exogenous WT or PSGL-1-/- Tregs. We analyzed two different time points: an “early” pre-clinical phase of EAE (day +1 post-immunization) and a “late” pre-clinical phase of EAE (day +7 post-immunization). In the early pre-clinical phase of EAE (day +1 post-immunization), as previously shown, we found that MOG35-55-specific T cells showed a significant increase in their motility and decrease of the arrest coefficient in the presence of WT Tregs in draining lymph nodes of EAE mice, due to reduced contact times with antigen-presenting dendritic cells. At this time point, also PSGL-1 deficient Tregs efficiently modulated encephalitogenic T cells behaviour. On the contrary, PSGL-1-/- Tregs failed to modulate the motility behaviour and proliferation of MOG35-55-specific T cells during the late (day +7 post-immunization) pre-clinical phase of the disease, while exogenous WT Tregs still affect MOG35-55-specific T cells motility. In conclusion, our data demonstrate that PSGL-1 has a key role in CD4+CD25+Foxp3+ Treg migration in the CNS during the pre-clinical phase of EAE; moreover, PSGL-1 expression is necessary for an efficient suppression of late antigen-dependent T cell activation exerted by Tregs in the draining lymph nodes of EAE mice. Overall, our results demonstrate a previously unknown key role for PSGL-1 in Treg functionality in a model of autoimmune disease.