MS is an autoimmune disease in which immune-mediated attack of the brain and spinal cord results in inflammation and demyelination of the central nervous system (CNS). Studies of experimental autoimmune encephalomyelitis (EAE), a murine model of MS that recapitulates a number of immunopathological processes found in human MS, reveal an important role for CD4+
T cells in disease pathogenesis. While a number of studies have shown decreased Treg function in relapsing-remitting MS, Viglietta et al.
) first reported a functional defect in CD4+
T cells isolated from peripheral blood of MS patients. When tested with in vitro
suppression assays, CD4+
Tregs were less effective at suppressing the proliferation and IFN-γ production by effector T cells from either autologous or healthy individuals in the presence of lower concentrations of anti-CD3. The interpretation of many human Treg studies is technically limited by the lack of Treg-specific surface markers. Isolation of CD4+
cells not only results in an enriched population of Tregs but also may include effector T cells as well. To avoid this complication, Michel et al.
) isolated CD4+
T cells that expressed only low levels of the IL-7Rα chain (CD127) from MS patients and healthy controls. Foxp3 interacts with the CD127 promoter and likely represses its protein expression, resulting in an inverse correlation between Foxp3 and CD127 expression (158
). These CD4+
Tregs represented a more pure population of regulatory cells that were more suppressive than their CD4+
). Hence, unlike previous reports, Tregs from MS patients, selected on the basis of CD127low
expression, exhibit suppressive functions that are comparable to healthy individuals. These data suggest that previous findings of reduced Treg function in MS patients may be due, in part, to CD4+
effector cells within the unfractionated CD4+
pool. However, it remains unclear whether peripheral blood CD4+
Tregs are functionally similar to CD4+
Tregs present in the CNS during inflammation.
Studies of Treg cell function in EAE, reveal critical roles for Tregs in the prevention and amelioration of CNS inflammation by limiting encephalitogenic T-cell expansion and function. Adoptive transfer of CD4+
Tregs decreased inflammation of the CNS and conferred substantial protection from clinical EAE (161
). Depletion of Tregs with an anti-CD25 monoclonal antibody (PC61) rendered naturally resistant B10.S mice susceptible to proteolipid protein (PLP139–151
)-induced EAE (162
). Furthermore, Treg depletion prior to disease induction facilitated the expansion of PLP139–151
-reactive cells, enhanced proinflammatory cytokine production (IL-6, IL-17 and IFN-γ), and increased disease severity in the otherwise resistant mice (163
). These studies convincingly demonstrated that Tregs are instrumental in providing a protective barrier from immune-mediated tissue damage, thus setting an activation-threshold for autoimmune disease onset.
During EAE, an accumulation of Tregs in the CNS has consistently been seen during disease resolution (129
). McGeachy et al
) demonstrated central roles for Tregs during the induction and effector phases of EAE. Low numbers of adoptively transferred Tregs protect recipient mice from disease induction, whereas depletion of Tregs prevents recovery from EAE and further exacerbates disease progression (165
). Moreover, CNS-derived Tregs were capable of suppressing both naive CD4+
cells and CNS-derived CD4+
cells. However, the interpretation of the functional capacity of CNS-derived Tregs is limited by their lack of antigen specificity and also by the phenotype of CNS-derived CD4+
T cells. As T cells generally upregulate CD25 upon activation, the isolation of CD4+
cells from the CNS as putative effector cells may not accurately represent an encephalitogenic effector population from an inflamed CNS. In contrast, using Foxp3-GFP reporter mice and MOG-tetramers to further define antigen-specific Tregs and T effector cells, Korn et al.
) demonstrated that although Tregs are critical for regulating naïve MOG-specific T cells, they may not be as effective at regulating MOG-reactive T effectors within the target site. Tregs are often tested ex vivo
against non-encephalitogenic effector cells. Korn et al
) suggest that antigen-specific encephalitogenic effector cells derived from sites of inflammation are distinct and respond differently to Tregs than non-encephalitogenic effector T cells. When CNS-infiltrating tetramer-positive Tregs were combined with MOG tetramer-reactive T effector cells, Tregs could no longer maintain suppressive function, at least in part owing to the production of pro-inflammatory cytokines (IL-6 and TNF-α) that may overwhelm the suppressive capacity of Tregs (128
). Hence, the total milieu in which ‘Treg meets T-effector meets cytokines’, affects disease resolution. How inhibitory cytokines and molecules alter this balance remains to be investigated.
PD-1 and its ligands are expressed on a variety of cell types within the CNS. PD-1 is constitutively expressed in retinal neurons of naive mice, while PD-L1 and PD-L2 are found on retinal neurons under inflammation conditions (168
). Similarly, during active EAE, meningial infiltrates express PD-1, PD-L1, and PD-L2 (38
), suggesting a role for the PD-1 pathway in regulating inflammatory processes within the CNS. PD-L1 is also expressed on astrocytes and vascular endothelial cells and is induced by IL-12 on CD11b+
APCs in mice with EAE (169
). This PD-L1 expression is due to IFN-γ, since it is abrogated in IFN-γdeficient mice. Studies with IL-12p35 deficient mice, demonstrated that IL-12 suppresses EAE in C57BL/6 mice (170
). Furthermore, microglial cell expression of PD-L1 is regulated by IFN-γ (171
). Together, these data implicate the PD-L1:PD-1 pathway as central to disease progression. In addition, studies of German MS patients revealed a mutation found in the enhancer element of the PD-1 gene that affects Runx1 binding and results in a decreased ability of PD-1 to inhibit IFN-γ production. As Runx1 augments Foxp3 expression, this mutation may also result in decreased Foxp3 expression by Tregs (133
Initial studies describing PD-1 and PD-L2 as critical to EAE pathogenesis used neutralizing antibodies specific for PD-1 (J43) or PD-L2 (TY25) monoclonal antibodies during disease induction (172
). Salama et al.
) showed that PD-L1 or PD-L2 blockade led to the expansion of MOG-reactive T cells, increased lymphocytic infiltration of the CNS, and ultimately, accelerated disease onset and severity. Studies with PD-1 and PD-L-deficient mice indicate that PD-1 and PD-L1, but not PD-L2, are predominantly responsible for regulating disease severity (173
). PD-1 and PD-L1-deficient mice may have intrinsic deficits in regulation. Restimulation of T cells from PD1−−
with active disease resulted in copius production of IL-17 and IFN-γ as compared to WT mice, suggesting that T cells with deficient PD-1 signaling may be preferentially polarized toward effector T-cell differentiation (174
A number of reports have indicated a role for regulatory T cells in EAE. Adoptive transfer studies have identified critical functions for PD-L1 on both the transferred T cells and in the recipient animal (173
). As these studies were done with CD4+
enriched cells, it is likely that regulatory populations expressing PD-L1 may be directly converting or suppressing effector CD4+
T cells via PD-1 ligation, since PD-1 and PD-L1 are highly expressed on Tregs (60
). Moreover, host expression of PD-L1 may contribute to the peripheral conversion of transferred T cells toward an iTreg phenotype, a notion supported by the observation that vascular endothelial expression of PD-L1 may induce Treg cell populations (155
). More direct support for a role of the PD-1:PD-L1 pathway in iTreg induction came from studies in EAE identifying that PD-1 is directly modulated by pertussis toxin (PT) administration (175
). Pertussis toxin was originally thought to increase the permeability of the blood brain barrier prior to disease onset in EAE (176
). PT was also shown to induce P-selectin on pial vessels and enhance adhesion of activated T cells (178
). Importantly, PT administration decreased the frequency and function of Tregs and increased Th1 and Th2 responses (180
). Wang et al.
) showed PD-1-deficient mice given MOG/CFA without pertussis toxin develop fulminant disease, owing to a reduced iTreg frequency. This is in marked contrast to WT mice which do not develop EAE in the absence of PT. Splenocytes from WT mice immunized with MOG/CFA in the absence of PT had a two- to three-fold increase in Foxp3+
Treg frequency compared with PD-1-deficient mice. In vitro
conversion of CD4+
T cells into iTregs and their subsequent suppressive activity was PD-1 dependent. Although pertussis treatment does not alter the expression of the Treg phenotypic markers CD45RB, CD103, GITR, and CTLA-4, PT directly downregulates PD-1 on Tregs, underscoring a central role for PD-1 in iTreg development and supplying a mechanism for the immunological adjuvancy of PT in multiple strains of mice (175
These numerous studies clearly demonstrate that the interplay between the PD-1 pathway and Tregs is instrumental in regulating inflammation within the CNS. That neurons express PD-L1 (and PD-L2) upon inflammation and produce TGF-β is intriguing in light of evidence describing that encephalitogenic T cells are converted to iTregs upon neuronal encounter (166
). When neurons isolated from 7-day-old mice were co-cultured with encephalitogenic T cells, this resulted in an outgrowth of a CD4+
T-cell population that expressed Foxp3, CD25, TGF-β1, and CTLA-4 and could suppress encephalitogenic T cells and inhibit EAE. It is thus plausible that encephalitogenic effectors T cells encounter neurons and stimulate their PD-L1 expression. Neuronal PD-L1 then drives iTreg conversion from effector T cells, effectively restraining CNS inflammation and spreading infectious tolerance.