Podoplanin-rich stromal networks induce dendritic cell motility via activation of the C-type lectin receptor CLEC-2.

To initiate adaptive immunity, dendritic cells (DCs) move from parenchymal tissues to lymphoid organs by migrating along stromal scaffolds that display the glycoprotein podoplanin (PDPN). PDPN is expressed by lymphatic endothelial and fibroblastic reticular cells and promotes blood-lymph separation during development by activating the C-type lectin receptor, CLEC-2, on platelets. Here, we describe a role for CLEC-2 in the morphodynamic behavior and motility of DCs. CLEC-2 deficiency in DCs impaired their entry into lymphatics and trafficking to and within lymph nodes, thereby reducing T cell priming. CLEC-2 engagement of PDPN was necessary for DCs to spread and migrate along stromal surfaces and sufficient to induce membrane protrusions. CLEC-2 activation triggered cell spreading via downregulation of RhoA activity and myosin light-chain phosphorylation and triggered F-actin-rich protrusions via Vav signaling and Rac1 activation. Thus, activation of CLEC-2 by PDPN rearranges the actin cytoskeleton in DCs to promote efficient motility along stromal surfaces.

Intestinal tolerance is converted to autoimmune enteritis upon PD-1 ligand blockade.

The B7 family member programmed death-1 ligand (PD-L1) has been shown to play an inhibitory role in the regulation of T cell responses in several organs. However, the role of PD-L1 in regulating tolerance to self-Ags of the small intestine has not been previously addressed. In this study, we investigated the role of PD-L1 in CD8(+) T cell tolerance to an intestinal epithelium-specific Ag using the iFABP-tOVA transgenic mouse model, in which OVA is expressed as a self-Ag throughout the small intestine. Using adoptive transfer of naive OVA-specific CD8(+) T cells, we show that loss of PD-1:PD-L1 signaling, by either Ab-mediated PD-L1 blockade or transfer of PD-1(-/-) T cells, leads to considerable expansion of OVA-specific CD8(+) T cells and their differentiation into effector cells capable of producing proinflammatory cytokines. A fatal CD8(+) T cell-mediated inflammatory response develops rapidly against the small bowel causing destruction of the epithelial barrier, severe blunting of intestinal villi, and recruitment and activation of myeloid cells. This response is highly specific because immune destruction selectively targets the small intestine but not other organs. Collectively, these results indicate that loss of the PD-1:PD-L1 inhibitory pathway breaks CD8(+) T cell tolerance to intestinal self-Ag, thus leading to severe enteric autoimmunity.

Peripheral tolerance induction by lymph node stroma.

In this review we have highlighted the role of LNSCs in the regulation of CD8+ T cell immune responses in peripheral lymph nodes, thereby adding another layer of protection, in addition to the role of resting DCs, against autoimmunity. LNSCs have recently been implicated in the induction of peripheral CD8+ T cell tolerance due to their ability to endogenously express, process, and present PTAs. Furthermore, LNSCs express surface molecules, such as MHC class II and PD-L1, similar to those expressed by mTECs in the thymus and APCs. For future studies it will be important to address some of the new questions that have emerged with respect to the biology and function of LNSCs. Further work will help us to (1) dissect the specific roles that DCs and LNSCs have in the induction and maintenance of tolerance to intestinal antigens, (2) gain a more in-depth understanding of the molecular mechanisms underlying self-tolerance induction by LNSCs and the impact of inflammation on this function, (3) evaluate the relationship of LNSCs to the FRN, and (4) determine if the APC function of LNSCs extends to the acquisition and presentation of exogenous antigens. Finally, it is important to mention that so far the studies done on LNSCs have focused on their role in CD8+ T cell tolerance. At the moment, we do not know if presentation of PTAs by LNSCs can also induce tolerance of CD4+ T cells. Based on the finding that LNSCs express MHC class II (I-A(b)) molecules it is possible that they may present self-antigens to CD4+ T cells and induce tolerance. However, this has yet to be elucidated.

Lymphoid organ-resident dendritic cells exhibit unique transcriptional fingerprints based on subset and site.

Lymphoid organ-resident DC subsets are thought to play unique roles in determining the fate of T cell responses. Recent studies focusing on a single lymphoid organ identified molecular pathways that are differentially operative in each DC subset and led to the assumption that a given DC subset would more or less exhibit the same genomic and functional profiles throughout the body. Whether the local milieu in different anatomical sites can also influence the transcriptome of DC subsets has remained largely unexplored. Here, we interrogated the transcriptional relationships between lymphoid organ-resident DC subsets from spleen, gut- and skin-draining lymph nodes, and thymus of C57BL/6 mice. For this purpose, major resident DC subsets including CD4 and CD8 DCs were sorted at high purity and gene expression profiles were compared using microarray analysis. This investigation revealed that lymphoid organ-resident DC subsets exhibit divergent genomic programs across lymphoid organs. Interestingly, we also found that transcriptional and biochemical properties of a given DC subset can differ between lymphoid organs for lymphoid organ-resident DC subsets, but not plasmacytoid DCs, suggesting that determinants of the tissue milieu program resident DCs for essential site-specific functions.

Lymph node fibroblastic reticular cells directly present peripheral tissue antigen under steady-state and inflammatory conditions.

Lymph node stromal cells (LNSCs) can induce potent, antigen-specific T cell tolerance under steady-state conditions. Although expression of various peripheral tissue-restricted antigens (PTAs) and presentation to naive CD8+ T cells has been demonstrated, the stromal subsets responsible have not been identified. We report that fibroblastic reticular cells (FRCs), which reside in the T cell zone of the LN, ectopically express and directly present a model PTA to naive T cells, inducing their proliferation. However, we found that no single LNSC subset was responsible for PTA expression; rather, each subset had its own characteristic antigen display. Studies to date have concentrated on PTA presentation under steady-state conditions; however, because LNs are frequently inflammatory sites, we assessed whether inflammation altered stromal cell-T cell interactions. Strikingly, FRCs showed reduced stimulation of T cells after Toll-like receptor 3 ligation. We also characterize an LNSC subset expressing the highest levels of autoimmune regulator, which responds potently to bystander inflammation by up-regulating PTA expression. Collectively, these data show that diverse stromal cell types have evolved to constitutively express PTAs, and that exposure to viral products alters the interaction between T cells and LNSCs.

Unconventional antigen-presenting cells in the induction of peripheral CD8(+) T cell tolerance.

Bone marrow-derived APCs are considered the predominant cell type involved in the induction and maintenance of T cell tolerance in vivo. In the periphery, cross-presentation of self-antigens by DCs, in particular, CD8alpha(+) DCs, has been the most discussed mechanism underlying the induction of CD8(+) T cell tolerance against self. However, nonhematopoietic APCs in the liver, skin, parenchymal tissues, and lymph nodes can also present self- and exogenous antigens to CD8(+) T cells under steady-state conditions. Although far surpassed by their DC counterparts in their ability to stimulate T cell responses, these unconventional APCs have been shown to play a role in the induction, maintenance, and regulation of peripheral CD8(+) T cell tolerance by a multitude of mechanisms. In this review, we will discuss the different nonhematopoietic cells that have been shown to present tissue-specific or exogenous antigens to naïve CD8(+) T cells, thereby contributing to the regulation of T cell responses in the periphery.