The demonstration that LEC express PTA and function as tolerance-inducing APC in LN was the culmination of our work over several years to understand tolerance to PTA expressed in both melanocytes and melanomas, termed melanocyte differentiation antigens, which are targets in both autoimmune vitiligo and melanoma immunotherapy. Using a model system based on recognition of one such melanocyte differentiation antigen, tyrosinase, we showed that tyrosinase-specific CD8 T-cells do not undergo central tolerance in the thymus [25
]. In addition, under steady state conditions, DC in LN do not present tyrosinase. Instead, tolerance to tyrosinase is strictly due to direct expression of tyrosinase mRNA and display of tyrosinase antigen by MHC I molecules on LEC [12
]. This antigen presentation leads to activation and initial proliferation of tyrosinase-specific CD8 T-cells, but these cells undergo apoptosis and deletion rather than accumulating [12
While this process of abortive proliferation has been shown in many models of CD8 deletional tolerance, the mechanisms involved in driving this outcome have been somewhat unclear. Some previous work had established that peripheral tolerance could be induced by antigen engagement in the absence of costimulation [27
], while other studies pointed to the engagement of inhibitory molecules [32
]. While investigating the mechanisms involved in LEC-induced deletion, we found that both of these processes were involved and interdependent [26
]. LEC do not express any of the costimulatory molecules that normally drive immunogenic accumulation of activated T-cells, such as CD80, CD86, OX40L, 4-1BBL, or CD70. However, they express multiple ligands that can activate inhibitory pathways, and express a particularly high level of PD-L1. Indeed, deletion of tyrosinase-specific CD8 T-cells is strictly dependent on engagement of the PD-1/PD-L1 pathway [26
]. However, it is antigen activation in the absence of costimulation that leads to rapid, high-level upregulation of PD-1 on tyrosinase-specific CD8 T-cells, which is required for deletion to occur. This can be overcome by administration of exogenous anti-4-1BB costimulation. Importantly, tyrosinase-specific CD8 T-cells that are rescued from LEC-mediated deletion gain effector function and induce autoimmune vitiligo. Given that LEC express multiple PTA, these results suggest that impairment of LEC-induced tolerance could have a role in the induction of numerous autoimmune disorders. While there is considerable interest in PD-1/PD-L1 as a mechanism to suppress pathology in peripheral tissue and in the genesis of clonal exhaustion in tumors [37
], these results establish a central role of this pathway in peripheral tolerance induction.
LEC that have acquired soluble OVA cross-present it to CD8 T cells in a TAP1-dependent manner, and in vitro
, this leads to decreased OT-I IFN? and IL-2 production and increased expression of Annexin V, PD-1, CD80, CTLA-4 [21
]. Cross-presentation of tumor antigens by LEC from the tumor draining LN also leads to increased Annexin V staining on co-cultured tumor-specific CD8 T-cells ex vivo
]. This demonstrates that cross-presentation of soluble antigens by LEC in vitro
leads to dysfunctional T-cell activation, similar to the phenotype seen when LEC directly present the PTA tyrosinase in vivo
]. This suggests that under steady-state conditions, the constellation of inhibitory molecules expressed by LEC combined with their lack of costimulatory molecules predisposes LEC to induce dysfunctional T-cell activation, regardless of the source of the antigen. This raises important questions about whether the tolerance-inducing state of LEC can be modulated, such as during infection when the LEC may acquire foreign antigens draining through the lymphatics.
T-cell tolerance can take many forms, including deletion, anergy, or Treg
induction. The form of tolerance induced has been shown to depend on several factors, including TCR avidity, availability of costimulatory or inhibitory pathways, and the cytokine environment [40
]. mTEC, DC, and eTAC have all been shown to induce multiple forms of tolerance [2
], while to date LEC have only been shown to induce deletion in vivo
]. A question of some interest is whether LEC can also induce other forms of tolerance, based on the molecules they express or the particular microenvironmental niche in the LN that they occupy.