Contributions of PD-L1 reverse signaling to dendritic cell trafficking

Abstract

Programmed Death-1 (PD-1)/Programmed Death-Ligand 1 (PD-L1) interactions are critical for dampening the immune response to both self and foreign antigens. The signaling of PD-L1 via its cytoplasmic domain, rather than through its interactions with PD-1 via the extracellular domain, has been termed PD-L1 reverse signaling. While this signaling is beneficial for cancer progression, little is understood about the consequences of PD-L1 reverse signaling in immune cells that express PD-L1 at steady state or in response to infection. Loss of PD-L1 during infection leads to unchecked T cell proliferation and increased autoimmune T cell responses. While the T cell intrinsic role of PD-1 for inhibiting T cell responses has been well explored, little to no effort has been directed at investigating the consequences of PD-L1 reverse signaling on the DCs interacting with PD-1+ T cells. We recently reported a defect in dendritic cell trafficking from the skin to the draining lymph node following immunization or infection in the absence of PD-L1. We demonstrated that a region within the cytoplasmic tail was responsible for the defect in dendritic cell trafficking. Here, we review the processes involved in dendritic cell trafficking and highlight what we know about PD-L1 expression, PD-L1 post-translational modifications, PD-L1 intracellular interactions and PD-L1 extracellular interactions.

Graphical abstract

PD-L1 forward/reverse signals influence cellular functions. PD-L1 can be expressed by cancer cells to prevent T cell activity or to promote cancer cell survival via PD-L1 reverse signaling. PD-L1 can be expressed by many cells in chronic infection/autoimmune disease and promote T cell exhaustion/dysfunction and is highly expressed by dendritic cells during infection, promoting dendritic cell migration from the skin to the lymph node via reverse signaling. Created with BioRender.com

PD-L1 interacts with PD-1 to inhibit the T cell response.

Our current understanding of PD-L1/PD-1 signaling comes largely from normal regulation of the immune response. During infection PD-1 is upregulated and remains high on effector T cells while PD-L1 is upregulated on macrophages and dendritic cells. PD-1 and PD-L1 interactions have been demonstrated to be critically important for mitigating T cell responses and inducing immune cell tolerance [1–13]. This interaction has been well documented to down modulate the expression of the T cell receptor (TCR) [14] and the cell cycle to dampen the T cell response via direct PD-1 signaling into the T cell [15–17]. In instances of cancer or chronic infection PD-1 and PD-L1 expression is often dysregulated resulting in immune dysfunction and chronic exhaustion [15–21]. As such, PD-1, PD-L1 and PD-L2 are designated as part of an immune checkpoint pathway which limits T cell responses. High expression of PD-L1 has been observed in tumors and demonstrates the immune inhibitory microenviroment within the tumor. PD-1 and PD-L1 have been targeted extensively with immunotherapies [22]. These immunotherapies are designed to block PD-1/PD-L1 interactions to promote T cell responses against the tumor and have been successful in patients.

Programmed death ligand 1 (PD-L1) reverse signaling.

Many of the studies described in the above section have documented the importance of PD-L1 as the ligand for PD-1 and demonstrated the signaling events within T cells that are mediated by this interaction. However, over 10 years ago it was demonstrated that PD-L1 reverse signaling could act as a molecular shield against cytotoxic T cells in vitro [23]. This study showed that removal of the cytoplasmic domain of PD-L1 in target cells left them vulnerable to killing by CD8 T cells [23]. Following this finding, several investigators began to identify targets of PD-L1 reverse signaling in cancer cells [24]. In these studies, PD-L1 was shown to mediate the epithelial to mesenchymal transition through the RAS/ERK/MEK pathway [25], AKT phosphorylation was demonstrated in PD-L1^hi compared to PD-L1^lo melanoma cells during autophagy [26] and more recently STAT3 phosphorylation in the context of type 1 interferon (IFN) was shown to protect melanoma cells from apoptosis [27]. In glioblastoma multiforme (GBM), PD-L1, via its cytoplasmic domain, was shown to interact with H-RAS [25]. This interaction resulted in MAPK activation and lead to enhanced GBM motility and invasion [25]. This study provided the first evidence that loss of PD-L1 expression by a cancer cell could not only protect the cell from death, but could also promote cellular movement and metastasis. Thus far, PD-L1 reverse signaling has largely been studied in cancer cells and has significantly improved our understanding of how the intracellular domain of PD-L1 can impact cellular responses. In recent work performed by our lab, we demonstrated that normal lymphatic endothelial cell (LEC) expression of PD-L1 also promotes LEC survival during viral infection or polyI:C injection [28]. We went on to investigate the requirement for PD-L1 reverse signaling in dendritic cells and the involvement of PD-L1 in dendritic cell (DC) migration from the skin to the draining lymph node (LN) in the setting of viral infection or polyI:C injection [29]. In this review we will focus less on the implications of PD-L1 expression by cancer cells (reviewed in [24]) and instead focus on what is and is unknown about how PD-L1 reverse signaling and PD-L1 interactions may affect dendritic cell migration and subsequent T cell activation.

Dermal dendritic cell (DC) migration through lymphatics.

Upon infection, tissue resident DCs must leave the tissue and migrate to the LN for initiation of the adaptive immune response. Disruption of E-cadherin junctions between DCs and keratinocytes, DC release of matrix metalloproteinases (MMP)2 and 9, downregulation of tissue inhibitors of metalloproteinases (TIMP) [30–32] and digestion of large hyaluronan (HA) non-sulfated glycosaminoglycans are required for tissue release [33] (Figure 1). These processes occur concurrently with the upregulation of CC chemokine receptor (CCR)7 and major histocompatibility complex (MHC) Class II [34]. As the DCs migrate through the extracellular matrix (ECM) via “amoeboid movement” to the lymphatic capillaries, the DCs are guided by CC chemokine ligand (CCL)21 secreted from the lymphatics [35, 36]. Once the DCs reach the lymphatic capillaries they enter at specialized transmigratory cups coated with intracellular adhesion molecule 1 (ICAM1), vascular cellular adhesion molecule 1 (VCAM1) and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) where local release of CCL21 facilitates DC transit [37–40](Figure 1). The DCs are guided by lymphatic derived chemotactic CCL21 gradients, until they reach the downstream lymphatic collector where they are propelled to the lymph node by lymphatic flow [41]. Once in the LN the conventional DC subsets, cDC1 and cDC2 are further guided by CCL19 and CCL21 expressed by the fibroblastic reticular network [42–44] (Figure 1). As such, XCR1+ cDC1s express the most CCR7 and travel the furthest to interact with CD8 T cells deep in the cortex [45] while XCR1− CD11b+ cDC2s remain near the B cell follicles and interact predominantly with CD4+ T cells [46]. The migration of antigen bearing dermal DCs from the dermis through the lymphatics is required to prime naïve T cells [47–51]. This is specifically important in response to localized skin infections like Leishmania [52], bacterial infections like methicillin-resistant Staphylococcus aureus (MRSA)[53] and pox viruses[54] which require DC trafficking to prime naïve T cells in the lymph node [47]. Indeed, DC migration to the LN is of critical importance for immune system activation in response to most bacterial infections (400-2000nm) as they are too large to pass through the lymphatic capillaries [49]. However, protein immunization (30-50kD) and virus given subcutaneously [55] pass through the lymphatics directly to the LN resulting in antigen presentation by LN resident cDCs [49]. DC migration is also required to interact with antigen bearing LECs in the subcapsular sinus of the LN [56, 57] where the process of antigen exchange occurs for the benefit of protective immunity [56, 57].

Figure 1. Dendritic cell migration from the skin or tissue to the draining lymph node.

Upon infection with a pathogen dendritic cells (DC) become activated and must leave the tissue in order to present antigens to naïve T cells within the draining lymph node (LN) . DC migration from the skin requires the release of dermal DCs from the extracellular matrix (ECM) via matrix metalloproteinases (MMP) [30–32] and hyaluronidase (HA) [33]. The DCs upregulate the chemokine receptor CCR7 and migrate toward the chemokine ligand CCL21 produced by the lymphatic capillaries comprised of oak leaf shaped LECs with highly permeable button like junctions [35, 36]. Upon reaching the lymphatic capillaries the DCs enter at transmigratory cups via interactions with integrins an cellular adhesion molecules such as ICAM1 and VCAM1. DC entry also requires interactions with LYVE-1 expressed by the lymphatic endothelial cells via interaction with Hyaluronan on the DC [37–40]. Once the DCs have squeezed through the lymphatic capillaries via actin myosin contractility they crawl along the luminal surface of the lymphatic capillaries until they enter the collecting vessels where they are propelled by lymphatic flow. Entry into the LN cortex where the T cells reside involves the expression of CCL19 and CCL21 by the fibroblastic reticular cell network [42–44].

DC activation and PD-L1.

The process of DC migration described above occurs following immunization or infection and occurs concurrently with DC activation [58, 59]. This activation includes, in addition to the upregulation of CCR7 and MHC class II, upregulation of CD80, CD86 and CD40 [34]. Upregulation of the activation markers and PD-L1 expression on DCs can result of IFN receptor signaling [60–62]. Migratory DCs present antigens acquired in the tissue providing at least three signals to initiate a productive T cell response. These three signals include recognition of peptide MHC by the T cell receptor, CD28-CD80/86 (B7.1/2) interactions between the T cell and DC, and cytokine production by the DC (IL-12, CD70) [63–65]. PD-L1 (B7-H1) and other B7-H family members B7-H3 and B7-H4 act as co-inhibitory receptors and unlike CD80/86 (B7.1/2), B7-H family members can impede T cell responses [66, 67]. As such, loss of PD-L1 during infection leads to unchecked T cell proliferation and increased autoimmune T cell responses [15–17]. Whether PD-L1 intracellular signaling in DCs also affects T cell responses has not been well explored.

PD-L1 expression in the steady state is limited to cDC2s while PD-L1 expression is induced on both cDC1 and cDC2s [60] during infection as a consequence of induction of IFNα/β, IFNγ or tumor necrosis factor (TNF)α. As a result of IFN receptor engagement, IRF1 and STAT3 can localize to the PD-L1 promoter [61, 62, 68] (Figure 2). PD-L1 upregulation occurs within hours in response to lipopolysaccharide (LPS) or polyI:C on DCs [29, 60]. Why PD-L1 upregulation by DCs occurs early on has been elusive, however recent reports have demonstrated that CD80 interactions with PD-L1 prevent PD-1 access to PD-L1 [9, 12, 13, 69]. These reports highlight how CD80 - PD-L1 extracellular interactions could block PD-L1 interaction with PD-1 to promote T cell activation. Further, these studies demonstrated that in the absence of DC activation, and thus in the absence of CD80, PD-L1 - PD-1 interactions took place and T cells were de-activated. Interestingly, cell types that do not express CD80 also upregulate PD-L1 in response to IFNα/β, IFNγ, and TNFα [3, 28, 70]. One cell type of particular interest to DC activation and migration is the lymphatic endothelial cell (LEC). As mentioned above DC migration from the skin to the draining lymph node must occur through the lymphatic vasculature which is comprised of LECs. Following immune activation lymphatic vessels must expand and remodel to allow for DC entry and trafficking into the draining lymph node [71, 72]. LECs immediately and dramatically upregulate PD-L1 as a result of type 1 and type 2 IFN signaling [3, 28, 70]. How PD-L1 upregulation by LECs during infection or immunization may influence lymphatic remodeling and DC migration is not yet understood. The requirement for PD-L1 upregulation on cell types that interact with DCs may be an important path of investigation to truly understand the consequences of PD-L1 reverse signaling during the initial stages of an immune response.

Figure 2. Dendritic cell signaling events during activation.

Upon reception of type 1 IFNs dendritic cells will upregulate the transcription of CCR7, CD80, CD86, MHC Class II and PD-L1 [58, 59] [34]. Activation of PD-L1 has been demonstrated to be through STAT3 and IRF1 [61, 62]. The upregulation of CD80, PD-L1 and CCR7 lead to chemokine receptor signaling events. CCR7, a G-protein coupled receptor, releases the Gα and Gβγ subunits of the large G-proteins. Gα activation leads to phospholipase C activity and ERK phosphorylation. PLC activity also results in calcium flux and and actin remodeling [78]. All of these activities are required for DC migration and are impeded by the loss of cytoplasmic PD-L1 [29]. How PD-L1 impedes PLC activity is unknown. The extracellular interaction of PD-L1 with CD80 could be an important extracellular interaction required for these migratory signaling events.

PD-L1 promotes dendritic cell migration.

As noted above, PD-L1 has been demonstrated in a glioblastoma multiforme cell line to increase migration and pseudopodia length compared to short hairpin (sh)-PD-L1 cells where PD-L1 expression was minimal [25]. The expression of PD-L1 was important for ERK phosphorylation as they showed decreased ERK phosphorylation in the sh-PD-L1 cells[25]. As dendritic cells express relatively high levels of PD-L1 which is dramatically upregulated following immunization or infection [29, 60] we investigated whether dendritic cell migration may also be affected by PD-L1 expression. Using both bone marrow chimeras to eliminate hematopoietic but not stromal PD-L1 expression followed by FITC painting or in vivo DC transfer assays we determined that loss of PD-L1 expression by DCs resulted in decreased DC migration from the skin to the draining lymph node [29]. This loss of DC migration in the absence of PD-L1 occurred in response to polyI:C and listeria monocytogenes, but was not affected by contact hypersensitivity [29]. To investigate whether the intracellular domain or the extracellular domain was contributing to defects in DC migration we created a mouse model in which just three residues, TSS, of the cytoplasmic domain were mutated to alanine [29]. The extracellular domain of PD-L1 remained intact and compared to the Pdl1^−/− DCs, the cytoplasmic mutant Pdl1^CyMt mouse had WT levels of CD80 and PD-L1 expression following polyI:C injection [29]. We showed that the Pdl1^CyMt mice phenocopied the defect in DC migration we showed in the Pdl1^−/− mice and again confirmed this defect was DC intrinsic as bone marrow derived DCs were unable to migrate in vitro or in vivo. While this was the first time that PD-L1 reverse signaling in DCs was demonstrated to have an effect on DC migration, many others had demonstrated significant impacts on T cell responses caused by PD-L1 forward signaling [1–13]. We demonstrated no differences in T cell priming in the Pdl1^CyMt mouse compared to WT except in infections where DC migration was required [29]. The defect in DC migration was caused by the loss of chemokine receptor signaling. The exact mechanism of how PD-L1 reverse signaling in dendritic cells results in lost chemokine receptor signaling or why DC migration is only impeded following interferon inducing stimuli (polyI:C, LPS, listeria, but not contact hypersensitivity) is not yet resolved, however, several considerations are outlined below.

Cytokine signaling and PD-L1.

Type 1 IFN induction in DCs occurs following viral infection, listeria infection and vaccination with several different adjuvants that signal through TLR3, 4, 7, 9, among others. Type 1 IFN signals are received via the IFNαβ receptor which results in activation of JAK1 and TYK2 causing phosphorylation of the receptor (reviewed in [73]). Phosphorylation of the IFNαβR recruits and activates STAT1 via phosphorylation and the subsequent upregulation of interferon regulatory factor (IRF) and interferon stimulated genes (ISG) [73]. To prevent over activation of an IFN response, STAT3 activation, also a result of IFN receptor signaling, triggers the transcription of Suppressor of Cytokine Signaling (SOCS) proteins. Of the SOCS proteins, SOCS3 is upregulated by IL-10 while SOCS1 becomes upregulated by IL-6 [74]. Induction of the SOCS proteins results in a positive feedback loop, causing increased expression of IL-6 or IL-10 while decreasing IFNαβR expression [74]. Increased IL-6 and IL-10 expression both lead to STAT3 phosphorylation. In Pdl1^−/− mice, mice with a cytoplasmic mutation in PD-L1, or cells lacking PD-L1; STAT3, but not STAT1 phosphorylation is significantly increased compared to WT as a result of type 1 IFN receptor signaling [27, 29]. The mechanism behind this difference in STAT3 phosphorylation in the absence of PD-L1 reverse signaling is not understood. Furthermore, whether these differences in STAT3 phosphorylation are a result of increased IL6 or IL10 expression or secretion, as a result of PD-L1 reverse signaling, remains to be investigated.

Interestingly, several reports have demonstrated that type 1 IFN can inhibit DC migration from the skin to the draining LN [75–77]. In one of these reports IFNβ treatment decreased DC migration as a result of decreased CCR7 and MMP9 expression [76]. The decrease in CCR7 and MMP9 expression was a result of STAT1 upregulation by IFNβ [76]. As PD-L1 had been shown to be protective against IFNβ induced cell death it was possible that PD-L1 could act to modulate IFNβ regulation of CCR7 expression. In the absence of PD-L1 no differences in CCR7 expression were observed following polyI:C injection in vivo or after LPS stimulation in vitro when DC migration was impeded [29]. Together this predicts that PD-L1 could act as a rheostat for either type 1 IFN signaling or STAT signaling. This rheostat activity could be direct or via manipulation of the expression of other cytokines that downregulate IFN signaling, such as IL-6 or IL-10, as described above. Further investigation as to how loss of migration could occur revealed that downstream chemokine receptor signaling in the Pdl1^−/− or Pdl1^CyMt mouse was impeded, in contrast to S1P1R [29]. Chemokine receptors are G protein coupled receptors that release and activate Gα and Gβγ subunits upon ligation. Activation and release of the Gα subunit causes PLC activation and downstream MAPK activity, calcium flux and actin polymerization while Gβγ activation and release leads to PI3K activity and changes in cell viability [78] (Figure 2). Mutation of the TSS region of the cytoplasmic domain of PD-L1 led to decreased chemokine receptor mediated PLC activation, ERK phosphorylation and actin polymerization [29]. How exactly PD-L1 promotes chemokine signaling following DC activation and migration by polyI:C compared to contact hypersensitivity is still unclear. However, it remains a possibility that the modifications within the cytoplasmic tail of PD-L1 could contribute to stabilization of the chemokine receptor, the Gα protein or PLC via direct or indirect interactions at the membrane (Figure 2). Further, PD-L1 may regulate type 1 IFN receptor signaling thereby changing the cytokine milieu and modulating the DC chemokine receptor signaling events. Finally, while CCR7 surface and transcript expression was normal in the absence of PD-L1 reverse signaling it is possible that CCR7 localization is impaired. Thus, understanding what interacts with PD-L1, and what modifications occur within the cytoplasmic domain, in different cell types will be critical to our understanding of PD-L1 reverse signaling.

PD-L1 modifications and the cytoplasmic domain.

The murine cytoplasmic region of PD-L1 spans 30 amino acids from arginine (R) 271 to threonine (T) 290. The murine cytoplasmic domain is similar to the human with a lysine residue found at amino acid 280, immediately following a threonine-serine-serine (TSS) motif found at amino acids 277-279. PD-L1 stabilization is regulated by a cysteine residue, 5 amino acids upstream of the TSS motif in the cytoplasmic region of PD-L1, that is palmitoylated and anchored to the membrane [79] (Figure 3). Indeed, loss of this cysteine residue along with the 7 amino acids before it mimics loss of PD-L1 in response to type 1 IFN sensitivity in melanoma cells [27]. Additionally, PD-L1 mono- and poly-Ubiquitination occurs both before and after epidermal growth factor (EGF) stimulation. Predicted ubiquitination sites are at amino acids lysine (K) K178 and K281 in human PD-L1 [80]. Only residue K281 of the predicted ubiquitination sites in human PD-L1 resides in the cytoplasmic domain (Figure 3). Other modifications such as phosphorylation (T80,S184), acetylation and glycosylation (asparagine (N) 35, N192, N200, N219) have been shown, but primarily occur within the extracellular region of PD-L1 [81]. Intriguingly, phosphorylation of residue S284 in humans, homologous to murine S279, has been detected in several mass spectrometry studies in cancer cells and cancer cell lines [82–87] (Figure 3). While the implications of this phosphorylation have not been readily addressed, mutation of the TSS region in mouse has severe implications for DC migration while maintaining protein stability and expression [29]. Deletion of the DTSSK residues in melanoma cells appears to make them less sensitive to type 1 interferon induced cell death, however this mutation includes the ubiquitinated lysine reside which could have additional effects [27]. These findings implicate phosphorylation and/or ubiquitination of PD-L1 cytoplasmic residues in the regulation of motility and potentially survivability. Intriguingly, PD-L1 has been demonstrated to interact with RAS to induce ERK and MAPK signaling events in GBM cells[25]. It is still unclear which region or if the cytoplasmic domain of PD-L1 interacts with RAS in GBM cells or if PD-L1 interacts with RAS in dendritic cells. However, loss of the TSS domain significantly impeded ERK phosphorylation in dendritic cells [29]. Therefore, it seems likely that the short cytoplasmic domain of PD-L1 may signal via either or both post-translational modifications and/or direct protein-protein interactions.

Figure 3. PD-L1 domains.

A. Upon activation DCs upregulate CD80 and PD-L1 [34]. The interaction between PD-L1 and CD80 requires Y56 on PD-L1 and L107 on CD80 and does not impede the interaction between CD80 and the CDR3 region of CD28, but does prevent PD-L1 – PD-1 interactions [9, 12, 13, 69]. This is likely because CD80 and PD-1 interactions with PD-L1 require the Y56 residue of PD-L1 where mutation of PD-1 residues K78, I126 and E136 blocks interaction of PD-1 with PD-L1. Interaction of CD28 with CD80 leads to CD80 reverse signaling that causes increased IL-6 and IFNg production [93]. Stabilization of PD-L1 requires palmitoylation of the cysteine residue [79] within the cytoplasmic tail while actin polymerization and signaling events downstream of chemokine receptors that result in cellular migration require the TSS motif [29]. Phosphorylation and Ubiquitination are likely on the serine and lysine residues nearby, but their function is unknown [79]. The TSS region may also inhibit the RMLDVEFLKC domain to prevent STAT3 activation [27]. B. During de-activation of the DC CD80 becomes down regulated and PD-L1 is now allowed to interact with PD-1 [9, 12, 13, 69]. Whether PD-1 – PD-L1 interactions result in PD-L1 reverse signaling events is unknown. PD-1 ligation to PD-L1 results in downregulation of the T cell receptor and apoptosis.

PD-L1 extracellular interactions and reverse signaling.

Like CD80 (B7.1) and CD86 (B7.2), PD-L1 (B7-H1) is a B7 family member belonging to the immunoglobulin superfamily containing both IgC-like and IgV-like domains. These family members have limited homology and diverge dramatically in their cytoplasmic regions [88]. While CD80 and CD86 interact with CD28, CTLA4 and ICOS, PD-L1 interacts with PD-1 via PD-1 residues K78, I126 and E136 [89] and CD80 via residue L107 [9] and not CD28, CTLA4 or ICOS [90]. PD-L1 interactions with PD-1 are most well described to occur in trans with T cells, however, PD-L1 can also interact with PD-1 expressed on the DC in cis [12]. CD80 interactions with PD-L1 were demonstrated to only occur in cis on DCs via residue Y56 on PD-L1 and residue L107 on CD80 [9] (Figure 2,3). Evidence for this was provided several years ago now when CD80-PD-L1 interactions were evaluated by biacore analysis. This study found that only when CD80 was immobilized to a plate with a flexible linker was PD-L1 able to bind to CD80, demonstrating that PD-L1 interaction with CD80 likely did not occur between two cells [91]. Interestingly, reverse signaling through CD80 and CD86 as a result of CD28 interactions at the CDR3 region of CD28 [92] can lead to upregulation of interleukin (IL)-6 and IFNγ [93] to promote T cell responses (Figure 3). Whether PD-L1 interactions with CD80 also encourage the upregulation of IL-6 and IFNγ is still unknown. However, based on the similarity between B7 family members it is not surprising that cytoplasmic signaling events occur with any or all members. Still, it remains undefined whether interactions with the extracellular domain of PD-L1 can influence intracellular signaling events within dendritic cells. Indeed, we and others have demonstrated that loss of PD-L1 significantly impedes CD80 upregulation by dendritic cells [29, 94]. This upregulation appears to be dependent on the extracellular interaction of CD80 with PD-L1 rather than the intracellular interactions as mutation of a TSS motif within the cytoplasmic domain of PD-L1 does not impede CD80 expression or upregulation, but does impair DC migration [29]. Whether this loss of DC migration is related to IL6 or IFNγ mis-regulation is still unknown, but an intriguing path to pursue based on recent findings that IL6 interactions are necessary for DC migration [95].

As PD-L1-CD80 interactions occur in cis and block PD-L1-PD-1 interactions [9, 12, 13, 69] it is possible that upregulation of CD80 and PD-L1 on DCs could mediate the signaling events that occur downstream of PD-L1 (Figure 3). While extracellular regions of PD-L1 and CD80 interact, it has yet to be shown whether intracellular interactions between PD-L1 and CD80 occur [91]. If interactions between PD-L1 and CD80 are necessary for PD-L1 or CD80 reverse signaling, then inhibiting this interaction would impede PD-L1 or CD80 reverse signaling. If this was the case, PD-L1 antibodies, which block both PD-1 and CD80 interactions with PD-L1, would have significant consequences for T cell responses that require DC migration. However, it has been demonstrated that blocking PD-L1 interactions enhance T cell responses by maintaining T cell activation [5, 96, 97]. It remains to be seen whether blocking PD-L1 - CD80 interactions influence DC migration. However, in the event that CD80 – PD-L1 interactions are important for DC migration this could confound the observed T cell responses in the presence of PD-L1 blocking antibodies. Finally, what, if any, role PD-L1 reverse signaling has in memory T cell responses or during chronic infection is completely unexplored. Future studies will be important to parse out differences in PD-L1 forward and reverse signaling and could be performed using genetic mouse models where either the extracellular or intracellular domain of PD-L1 or the binding domains found within each are mutated.

Conclusions.

PD-L1, a relatively well-known transmembrane protein, has a complex array of functions that modulate immune responses. The hijacking of this protein by cancer cells thus has implications in different cellular functions from increasing cell viability to increasing migration and invasion. However, the function of PD-L1 reverse signaling in cells that constitutively express PD-L1 and dramatically upregulate PD-L1 during activation [28, 29, 60, 70] is not well understood. Our understanding of what PD-L1 interacts with and the post-translational modifications that occur during dendritic cell activation, aside from interactions with PD-1 and CD80 is insufficient. Therefore, to move this field forward, significant work must be done to evaluate both intracellular and extracellular events controlled by PD-L1, particularly in situations where blocking interactions are involved.

Abbreviations:

PD-L1

programmed death ligand 1

PD-1

programmed death 1

TCR-T

cell receptor

DC

dendritic cell

cDC

conventional dendritic cell

LEC

lymphatic endothelial cell

RAS

rat sarcoma

ERK

extracellular signal related kinase

MEK

MAPK/ERK kinase

AKT

Ak strain transforming

STAT

signal transducer and activator or transcription

IFN

interferon

GBM

glioblastoma multiforme

MAPK

mitogen activated protein kinase

LN

lymph node

MMP

matrix metalloproteinase

CCR-C

C chemokine receptor

ECM

extracellular matrix

HA

hyaluronic acid

ICAM

intercellular adhesion molecule

VCAM

vascular cell adhesion molecule

Lyve

lymphatic vessel endothelial hyaluronan receptor

CCL-C

C chemokine ligad

XCR1

X-C motif chemokine receptor

MHC

major histocompatibility complex

IRF

interferon regulatory factor

WT

wild-type

S1P1R

sphingosine-1-phosphate receptor 1

PI3K

phosphatidylinositol-3 kinase

PLC

phospholipase C

TNF

tumor necrosis factor

FITC

fluorescein isothiocyanate

EGF

epidermal growth factor

Ig

immunoglobulin

IL-6

interleukin 6

CDR3

complementarity-determining regions 3

CTLA4

cytotoxic T-lymphocyte-associated protein 4

ICOS

inducible T-cell costimulator

CyMt

cytoplasmic mutant