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Page 284 Cencioni. Neuroimmunol Neuroinflammation 2020;7:277-90 I http://dx.doi.org/10.20517/2347-8659.2020.18

mice. The authors used a hypomethylating agent 5-aza-2’-deoxycytidine, which reduces methylation in a
“CPG” island located near the transcription site of Cd274 gene. The reduced methylation favours the gene
transcription and upregulation of PDL-1 in dendritic cells. This effect was observed in DC isolated from
EAE mice pre-treated with hypomethylating agent and in vitro treatment of bone marrow dendritic cells
with the hypomethylating agent. Furthermore, DC isolated from EAE mice pre-treated with 5-aza or bone
marrow dendritic cells treated in vitro with 5-aza suppressed proliferation and release of inflammatory
cytokines such as IL-17 and TNFa when the DC were co-cultured with CD4 T cells isolated from EAE
mice. In addition, an inhibition of EAE was observed in mice pre-treated with 5-aza before EAE induction.
An increase of PDL-1 and PDL-2 was detected in DC isolated from EAE mice in accordance with the in
vitro results. Moreover, blocking of PDL-1 but not PDL-2 exacerbated the EAE symptoms, confirming that
[95]
the link of PDL-1 but not PDL-2 with PD-1 is relevant in the suppression of T cell function by DC .

PD-1 depletion was also applied in mice immunised with a peptide of myelin oligodendrocyte glycoprotein
[62]
as adjuvant to develop EAE . After PD-1 depletion, the mice recovered from EAE with a clinical score
of one at the end of the experiments compared with the control mice that showed a score four without
+
recovery. Depletion of PD-1 reduced the fractions of PD-1 CD4 and PD-1 CD8 T cells but not B cells in
+
+
+
the CNS as compared with the controls. Besides, PD-1 depletion did not alter the ability of the treated mice
to mount an immune response. The baseline number of PD-1 cells in the blood and peripheral lymphoid
+
[62]
organs was low, confirming that PD-1 is expressed on autoreactive cells infiltrating target organs .
In view of publications giving PD-1 a crucial role in protection against autoimmunity in human and animal
[96]
models, Jang et al. investigated how PD-1 controls the activation and accumulation of autoreactive
T cells, by constraining them in anergic state. PD-1 is one of the checkpoint inhibitors investigated in
self-tolerance and discussed previously in CD8 T cells. Self-reactive cells were deleted during thymic
[97]
development and, of those that survived thymic deletion, only 10%-25% preferentially differentiated into
[96]
immune suppressive regulatory T cells (Tregs) [98,99] . Jiang et al. demonstrated that PD-1 is required in
[96]
culling endogenous peripheral high-affinity autoreactive CD4 T cells and protect against autoimmunity .
The tracking of endogenous autoreactive CD4 T cells showed that more than 90% of autoreactive CD4 T
[96]
cells remained FOXp3- effectors and were not regulatory T cell precursors, despite the high TCR affinity .
Instead, self-reactive CD4 T cells acquired cell-intrinsic tolerance through the expression of the immune
[96]
checkpoint molecule PD-1 . Monitoring the progeny of individual autoreactive CD4 T cell clones showed
that the clones with the greatest expansion burst size and highest TCR affinity expressed high levels of PD-1
[96]
and the affinity for the self-antigen induces the expression of PD-1 and the absence of PD-1 converts this
signal when priming with consequent cell activation. A similar mechanism was described to induce the
peripheral CD8 T tolerance in vivo. The peripheral CD8 T tolerance is induced by resting dendritic cells
and depends on activation of PD-1 and CTLA-4 pathways [100] .

Several studies have analysed the gene expression and protein levels of PD-1 and PDL-1 in MS, focusing
on delineating any correlation with disease susceptibility or risk of progression in MS. PD-1 gene
polymorphism has been investigated in MS, and the PD 1.3 SNP has been reported to correlate with
progression of the disease, demonstrating that human polymorphisms that reduce PD-1 activity increase
the risk of disease. Furthermore, a significant reduction in PD-1 expression was observed in patients
with mutation as compared with donors with wild-type phenotype. Furthermore, patients bearing the
mutual allele showed a lower suppression of IFNg-producing CD4 T cells after aCD3-PD-1-microbead
stimulation compared with healthy donors [101] . In addition, PD-1 and PDL-1 expression in peripheral
blood mononuclear cells reduced in a cohort of patients with MS as compared with healthy donors [102] .
The association of three PD-1 SNPs, namely PD-1.3, PD-1.5 and PD-1.9, with MS and disease outcome
were investigated in a cohort of 203 patients with a diagnosis of relapsing-remitting and secondary-
progressive MS showing any association with MS risk [103] . The expression of inhibitory receptor genes,