Tumor-Associated Lymphatic Vessels Upregulate PDL1 to Inhibit T-Cell Activation

Abstract

Tumor-associated lymphatic vessels (LVs) play multiple roles during tumor progression, including promotion of metastasis and regulation of antitumor immune responses by delivering antigen from the tumor bed to draining lymph nodes (LNs). Under steady-state conditions, LN resident lymphatic endothelial cells (LECs) have been found to maintain peripheral tolerance by directly inhibiting autoreactive T-cells. Similarly, tumor-associated lymphatic endothelium has been suggested to reduce antitumor T-cell responses, but the mechanisms that mediate this effect have not been clarified. Using two distinct experimental tumor models, we found that tumor-associated LVs gain expression of the T-cell inhibitory molecule PDL1, similar to LN resident LECs, whereas tumor-associated blood vessels downregulate PDL1. The observed lymphatic upregulation of PDL1 was likely due to IFN-g released by stromal cells in the tumor microenvironment. Furthermore, we found that blocking PDL1 results in increased T-cell stimulation by antigen-presenting LECs in vitro. Taken together, our data suggest that peripheral, tumor-associated lymphatic endothelium contributes to T-cell inhibition, by a mechanism similar to peripheral tolerance maintenance described for LN resident LECs. These findings may have clinical implications for cancer therapy, as lymphatic expression of PDL1 could represent a new biomarker to select patients for immunotherapy with PD1 or PDL1 inhibitors.

Keywords: PD1; T-cell exhaustion; abortive proliferation; immune checkpoint; lymph node; peripheral tolerance; tumor vasculature; tumor-induced immunosuppression.

Figure 1

PDL1 is expressed in tumor-associated lymphatic vessels (LVs). (A) Representative images of LVs (stained for LYVE-1, red) in control (Ctr) back skin (C57BL/6 background, top row) and B16F10-VEGFC melanoma (bottom row) co-stained for PDL1 (green). (B) Quantification of PDL1 staining intensity within the LYVE-1+ area of LVs in control back skin (N = 98 vessels from 10 individual mice), in the inner tumor mass of B16F10-VEGFC tumors (N = 17 vessels from six individual mice) and in the tumor periphery (N = 34 vessels from seven individual mice). (C) Similar to the B16F10-VEGFC model, PDL1 staining in LYVE-1-positive LVs was absent in the abdominal skin of BALB/c mice (top row) but was observed in 4T1 breast cancer-associated LVs (bottom row). (D) Quantification of PDL1 staining intensity within the LYVE-1+ area of LVs in control abdominal skin (N = 42 vessels from five individual mice), in the inner tumor mass of 4T1 tumors (N = 40 vessels from six individual mice), and in the tumor periphery (N = 35 vessels from five individual mice).

Figure 2

PDL1 is upregulated in tumor-associated lymphatic vessels but down-regulated in tumor-associated blood vessels. (A) Example FACS plots to illustrate the gating strategy used to identify lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BECs) in skin samples and tumors. Left panel: total endothelial cells (ECs) were identified as CD31+CD45− cells in a control skin sample (pre-gated for living singlets). Right panel: LECs were differentiated from BECs by staining for podoplanin (PDPN). (B) Example histogram of PDL1 staining intensity measured by FACS in LECs of control (Ctr) back skin and B16F10-VEGFC tumors. (C) Quantification of staining intensity in control (Ctr) back skin and B16F10-VEGFC-associated LECs (N = 5 mice/group). (D) Example histogram of PDL1 staining intensity measured by FACS in LECs of control abdominal skin and 4T1 tumors. (E) Quantification of staining intensity in control abdominal skin and 4T1-associated LECs (N = 4 mice/group; one out of two experiments with similar results is shown). (F,G) Quantification of PDL1 staining intensity in BECs of B16F10-VEGFC tumors [(F), N = 5 mice/group) and of 4T1 tumors [(G), N = 4 mice/group; one out of two experiments with similar results is shown].

Figure 3

PDL1 is constitutively expressed in lymph node (LN) lymphatic endothelial cells (LECs) and is not affected by the presence of an upstream tumor. (A) Example FACS plots of a LN (pre-gated for living singlets). CD45− LN stromal cells (left, LNSC) were separated into podoplanin (PDPN)+/CD31− follicular reticular cells (FRCs), PDPN+/CD31+ LECs, PDPN−/CD31+ BECs, and PDPN−/CD31− “double negative” (DN) cells (right panel). (B–E) Quantification of PDL1 staining intensity by FACS in LN LECs (B), BECs (C), FRCs (D), and DNs (E) in B16F10-VEGFC draining inguinal LNs (left panels, N = 5 mice/group) and 4T1 draining inguinal LNs (right panels, N = 4 mice/group; one out of two experiments with similar results is shown), compared to control (Ctr) LNs in naive mice. LECs expressed the highest levels of PDL1. Only minor changes in PDL1 expression levels of BECs and FRCs in 4T1 draining LNs were observed.

Figure 4

Lymphatic endothelial cells (LECs) upregulate PDL1 in response to IFN-g. (A) Immortalized mouse LECs (imLECs) were treated with VEGF-A (20 ng/ml), VEGF-C (200 ng/ml), TNF-a (40 ng/ml), or IFN-g (100 ng/ml). Expression of PDL1 was assessed by qPCR after 6, 24, and 48 h. Incubation with IFN-g resulted in a significant upregulation of PDL1 mRNA (pooled data of three individual experiments are shown). (B) Example histogram of surface PDL1 expression assessed by FACS in imLECs treated with TNF-a or IFN-g or not (Ctr). (C) Quantification of surface PDL1 in untreated (Ctr), TNF-a treated, and IFN-g treated imLECs (N = 3). (D) qPCR showing PDL1 expression upon imLEC treatment with tumor cell conditioned media (Cond med) derived from B16F10-VEGFC or 4T1 cells, compared to control media (Ctr med) (pooled data of three individual experiments are shown). (E) Example histograms of surface PDL1 expression assessed by FACS in imLECs treated with B16F10-VEGFC (left) or 4T1 cell conditioned medium (right). (F) Quantification of surface PDL1 expression in imLECs treated with control (Ctr med) or tumor cell conditioned media (Cond med) (N = 3). (G,H) qPCR data showing IFN-g expression in B16F10-VEGFC (G) and 4T1 tumor tissue (H) compared to control tissue (back skin resp. abdominal skin) (N = 7–9 for B16F10-VEGFC and 4–7 for 4T1).

Figure 5

T-cells interact with tumor-associated lymphatic vessels. Representative images of CD8+ and CD4+ T-cells (green) interacting with LYVE-1+ lymphatic endothelial cells (red) in B16F10-VEGFC melanoma (A) and 4T1 breast cancer (B). T-cells interacting with the abluminal (arrows) and the luminal (arrowheads) surface of the lymphatic endothelium were observed in both tumor models.

Figure 6

PDL1 blockade increases antigen-specific stimulation of CD8+ T-cells by lymphatic endothelial cells (LECs). (A) PDL1 surface expression on immortalized mouse LECs (imLECs) cocultured with OT-1 CD8+ T-cells and pulsed with SIINFEKL peptide or not (Control) was determined by FACS (N = 3). (B) Representative FACS plots of CD25 and IFN-g expression by OT-1 CD8+ T-cells (pre-gated for living singlets) after coculture with control or SIINFEKL-pulsed imLECs, in the presence of PDL1-blocking antibodies or control IgG. (C,D) Quantification of CD25+ OT-1 cells (C) and IFN-g+ OT-1 cells (D) by FACS gated as in panel (B) (N = 3).