Hodkin Reed-Sternberg cells actively polarize monocytes towards immunosuppressive macrophages
Hodgkin Reed-Sternberg (HRS) cells actively reshape the tumor microenvironment (TME) to promote tumor survival. Recent in vitro studies show that supernatants from Hodgkin Lymphoma (HL) cell lines drive polarization of tumor-associated macrophages (TAMs) toward an M2-like phenotype, marked by CD163 and PD-L1 expression. However, it remains unclear whether this polarization repurpose TAMs to support tumor progression. We investigated whether HL cell lines directly polarize monocytes and macrophages to support tumor survival. As a secondary objective, we evaluated whether TAMs expressing PD-L1 influence responses to PD-1 blockade.
We stimulated human-isolated monocytes from healthy donors with HL cell lines L428 and SUPHD1. After six days, we collected cells and supernatants to assess M2 polarization by measuring surface marker expression, gene upregulation, and cytokine production. We then incorporated these stimulated monocytes into an in vitro model to examine their impact on PD-1 blockade: peripheral blood mononuclear cells (PBMCs) co-cultured with HL cells and monocytes were treated with a PD-1 inhibitor, and immune activation cytokines were measured.
HL cells induced strong polarization of monocytes and macrophages toward an immunosuppressive M2-like phenotype. Stimulation with HL cell lines induced CD163 expression on average to 39% (L428) and 32% (SUPHD1), versus 2% in unstimulated monocytes. PD-L1 expression increased on average to 78% (L428) and 86% (SUPHD1), compared to 7% in unstimulated cells. Stimulated monocytes upregulated CCL13 and MRC1 by at least 3-fold on average, confirming differentiation into monocyte-derived macrophages (MDMs) with M2-like phenotype. MDMs showed increased secretion of CCL17 and CCL22, consistent with an M2a phenotype. Specifically, CCL17 levels averaged 4117 pg/mL (L428) and 880 pg/mL (SUPHD1), while CCL22 levels averaged 3643 pg/mL (L428) and 3007 pg/mL (SUPHD1). In the PD-1 blockade model, PBMCs from healthy donors co-cultured with L428 (PD-L1 negative) and MDMs (PD-L1 positive) exhibited a modest 1.2-fold increase in the levels of IL-2 and IFNγ after PD-1 inhibition across three donors.
Our data demonstrate that HL cells primarily polarize human monocytes into M2a macrophages, as confirmed by a multiparameter panel. These findings suggest that TAMs display an M2a phenotype in HL and might specifically contribute to tumor cell migration, invasion, and growth. Finally, we provide experimental evidence that PD-L1 expression on M2a MDMs can modulate immune responses to PD-1 blockade, supporting the development of rationale strategies with PD-1 blockade for HL patients.