Fig 2: HVEM is expressed at the surface of melanoma cells and shows a coincident expression pattern with BTLA expressed by CD8+ TILs.(a) Fresh melanoma metastases were analyzed by flow cytometry and the percentage of melanoma cells expressing HVEM, BTLA, LIGHT, CD160, and PD-L1 is shown. Respectively for each parameter, 15, 9, 6, 12, and 9 melanoma metastases were analyzed and the median with 95% IC is shown. (b) Each stacked bar plot represents a tumor sample for which the melanoma cell surface expression of HVEM and PD-L1 had been analyzed. 8.5–92.3% of cells were HVEM+PD-L1−, 0.1–15.3% HVEM+PD-L1+, while 0% were HVEM−PD-L1+. (c) Two representative examples of melanoma metastases analyzed for HVEM and PD-L1 co-expression and isotype controls are shown. In the example shown on the left panel, the few PD-L1+ cells were all HVEM+, whereas the majority of cells were HVEM+PD-L1–. In the example shown in the right panel, all the melanoma cells were HVEM+PD-L1–. (d) The TILs found in fresh melanoma metastases were analyzed by flow cytometry and the percentage of CD8+ TILs expressing BTLA, LIGHT, and CD160 is shown. Eight, 12, and 19 melanoma metastases were respectively analyzed and the median with 95% IC is shown. (e) A frozen section of a melanoma sample was stained for CD8 (green), BTLA (red), Melan-A (white), and HVEM (blue). The yellow box shows CD8+BTLA+ double-stained cells. The blue box shows Melan-A+HVEM+ cells. The merged image showing quadruple staining highlights the vicinity of CD8+BTLA+ cells and Melan-A+HVEM+ cells.

Fig 3: Expression of HVEM and microphthalmia-associated transcription factor (MITF) correlate strongly.(a) The strong correlation existing in the range of expression of HVEM (TNFRSF14) and MITF is specific to skin melanoma compared to other cancer cell lines of the Cancer Cell Line Encyclopedia (CCLE). (b) HVEM and MITF strongly correlated at the metastatic (distant + lymph node) stage on TCGA data (p = 2.282e-13) and not at the primary stage (p = .7703). The red dots represent the mean of MITF-low and MITF-high groups. The SEM for the MITF-low group and MITF-high group was 0.4473 and 0.0114, respectively, at the primary stage, and 0.0339 and 0.0041 at the metastatic stage, respectively. (c, d, e) Two melanoma cell lines, SKMEL28 and MeWo, and one short-term culture isolated from patient (C.09.10) were transfected with control siRNA (siCtl) or two different MITF siRNAs (siMITF1, siMITF2). MITF and HVEM expression was quantified by qPCR. Results are mean±SD of two experiments carried out in triplicate for MeWo, and one experiment carried out in triplicate for SKMEL28 and C.09.10. A two-tailed paired t-test was used for comparison between control and target siRNA (*: p < .05, **: p < .01, ***: p < .001, ****: p < .0001)ADC: Adenocarcinoma, NSCLC: Non-Small Cell Lung Carcinoma, SCC: Squamous Cell Carcinoma, TCC: Transitional Cell Carcinoma, UADT: Upper-aero-digestive tract.

Fig 4: The genes co-expressed with HVEM are distinct from those associated with a T cell-inflamed signature and correspond to genes involved in melanoma aggressiveness.(a) Unsupervised hierarchical clustering of the TCGA skin cutaneous melanoma dataset identified genes whose expression is co-regulated with HVEM (TNFRSF14) expression. This allowed definition of an “HVEM signature” (see top of heatmap) that comprises genes involved in melanoma proliferation and aggressiveness. This analysis also demonstrated that HVEM neither correlates with an IFN? signature as PD-L1 does, nor links to a T cell-inflamed pattern (see T Cell Infl at bottom of heatmap). Gene expressions were log2 transformed and median centered. (b) HVEM expression is not linked to BRAF, NRAS, or c-KIT mutational status in melanoma metastases. Analysis of BRAF, NRAS, and c-KIT hotspot mutations among 13 melanoma samples identified by flow cytometry as HVEM+ found expected percentages of samples with BRAF V600 mutations, NRAS Q61 mutations, and wild-type BRAF, NRAS, and c-KIT.

Fig 5: HVEM expression scoring.(a) Our developed HVEM expression score (see Materials and Methods) revealed the broad and heterogeneous pattern of HVEM expression at the surface of melanoma cells. (b) Representative section of four melanoma metastases (a–d; magnification: X200) and of a normal skin control (e; magnification: X100) stained for HVEM. Four representative TMA spots were taken from each melanoma metastases sample (melanoma cells identified with black triangles) and each was ranked as 0: negative, absence of signal (panel a), 1: weak and inferior to the level of HVEM signal on lymphocytes (*) (panel b); 2: intermediate signal, equal to the level of HVEM signal on lymphocytes (panel c), and 3: intense signal, superior to the level of HVEM signal on lymphocytes (panel d). The sum of the intensities of the four TMA spots provided a score of 0–12. Note that in the normal skin section shown in panel e, HVEM is only expressed by sebaceous glands. (c) Distribution of high and low HVEM expression scores among melanoma metastatic sites assessed by immunohistochemistry.