Page 201 - Haematologica Vol. 110 - January 2025

LETTER TO THE EDITOR
Hodgkin/Reed-Sternberg cells induce GPNMB expression and release from macrophages to suppress T-cell responses to the Epstein-Barr virus-encoded LMP2A protein
Classical Hodgkin lymphoma (cHL) is characterized by the presence of Hodgkin-Reed-Sternberg (HRS) cells surround- ed by a prominent inflammatory tumor microenvironment (TME). Although the TME is thought to prevent immune rec- ognition of HRS cells by tumor-specific T cells, the mech- anisms responsible are poorly understood. Here, we show that tumor-associated macrophages (TAM) in the TME of cHL strongly express glycoprotein non-metastatic B (GPNMB). Co-culture with cHL cell lines induced the M2 polarization of macrophages, which was accompanied by increased sur- face expression of GPNMB and its release as a soluble form. Importantly, soluble recombinant GPNMB (rGPNMB) inhibited CD8+ T-cell recognition of Epstein-Barr virus (EBV)-derived tumor epitopes in cHL cells, suggesting that inhibiting GPNMB in the cHL TME could enhance anti-tumor immune responses. cHL is characterized by single malignant HRS cells sur- rounded by a pro-inflammatory tumor microenvironment (TME) that supports HRS cell survival, growth and immune escape. The EBV genome is present in HRS cells in 30-50% of cHL and expresses the immunologically subdominant EBV latent proteins, Epstein-Barr nuclear antigen 1 and latent membrane proteins, LMP1 and LMP2A.1 Cells expressing LMP1 and LMP2A are sensitive to lysis by EBV-specific cytotoxic CD8+ T cells in vitro.1,2 Moreover, EBV-specific CD8+ T cells have been shown to be present in the cHL TME.3 These data suggest that immune suppressive mechanisms operate in the TME of EBV+ cHL.
Macrophages are broadly classified into M1 (classically-ac- tivated) and M2 (alternatively-activated) macrophages de- pending on their anti/pro-inflammatory properties and their polarization fluctuates in response to different stimuli/signals received from their environment. Tumor-associated macro- phages (TAM) infiltrating cHL tissues have M2-like charac- teristics and a higher frequency of these cells is associated with inferior survival of cHL patients.4 Multiplex immunofluo- rescence has revealed that the majority of PD-L1-expressing cells in the TME of cHL are macrophages, which are in close proximity to PD-1-expressing CD4+ T cells, suggesting that macrophages contribute to the dysregulation of anti-tumor T-cell responses in cHL.5 Importantly, this dysfunctional T-cell phenotype is reversible, as evidenced by the success of PD-1 blockade therapy in cHL patients with relapsed or refractory disease.6
GPNMB, also known as DC-HIL receptor, is a transmembrane protein that is known to be overexpressed in numerous can- cer types, and in some of these has been shown to promote a more metastatic phenotype.7 GPNMB was also shown to
function as a novel immune checkpoint that can bind to its ligand, syndecan-4, and inhibit T-cell activation.8-12 Accord- ingly, blocking GPNMB was shown to exacerbate autoimmune responses, inhibit wound healing, and potentiate anti-tumor immunity in melanoma-bearing hosts.10-14 The secreted form of GPNMB (sGPNMB) has also been shown to be functional and can exclude T cells from pre metastatic niches to promote tumor progression and reduce the trans-endothelial migration of T cells.15 Moreover, elevated plasma levels of sGPNMB are associated with resistance to PDL-1 inhibitor monotherapy in patients with advanced non-small cell lung carcinoma.16 Notably, GPNMB expression was shown to define a subset of mononuclear phagocytes associated with inferior survival of patients with colorectal cancer.17 In glioblastoma, these GPNMB-expressing macrophages were unable to activate T cells.9 In this study, we have explored the expression and po- tential role of GPNMB as a novel immune checkpoint in cHL. We first investigated GPNMB expression in 86 cases of histologically confirmed cHL of known EBV status using GPNMB-specific antibodies. The cases were obtained with ethical approval from West Midlands - Black Country Re- search Ethics Committee, UK (REC:16/WM/0037, IRAS project ID:181189). Immunohistochemistry revealed the expression of GPNMB in morphologically characteristic TAM in all cases, but only rarely in HRS cells (Figure 1A). There were no sig- nificant differences in the number of GPNMB-positive cells between cases based on subtype, age, EBV status or CD8 counts (data not shown). GPNMB expression was associated with a shorter progression-free survival and overall survival although these differences were only of borderline signifi- cance (Online Supplementary Figure S1A). We also interrogated our unpublished Nanostring GeoMx data which revealed an inverse correlation between PD-L1 expression and GPNMB expression in the macrophage-enriched regions of interest (Online Supplementary Figure S1B). As expected, macrophages were also positive for GPNMB in normal tonsil (Figure 1A). Using multiplex immunofluorescence, we confirmed that in most cases GPNMB was expressed in CD68-positive TAM but not in CD30-positive tumor cells. We also observed very low or undetectable levels of GPNMB expression (compared to germinal center [GC] B cells) in a panel of cHL cell lines using reverse transcription quantitative real-time polymerase chain reaction (data not shown), further supporting the observation that HRS cells do not generally express GPNMB.
We reasoned that HRS cells might mediate the high-level expression of GPNMB observed in TAM. To test this, we ex- posed M1 and M2 macrophages to cHL-derived conditioned
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