Classical Hodgkin lymphoma (cHL) features rare Hodgkin/Reed–Sternberg (HRS) cells within an immune-dense microenvironment, yet refractory disease persists in a subset of patients. This thesis interrogates how PD-1/PD-L1 signaling suppresses CD8⁺ T-cell cytotoxic immunity by integrating tumor-intrinsic and microenvironmental mechanisms. We combined mechanistic co-cultures, PLA coupled to multiplex immunofluorescence (PLA/mIF) on FFPE tissues, and RNA-seq of tumor-educated macrophages to link cellular mechanism to tissue context. Using mechanistic co-cultures of antigen-specific CD8⁺ T cells with HRS cells, we show that MHC-I–intact HRS cells directly restrain CD8⁺ effector function through PD-1/PD-L1 engagement; both PD-1 blockade and HRS PD-L1 knockout enhanced CD8⁺ activation and killing. In complementary systems incorporating tumor-associated macrophages (TAMs), PD-L1⁺ macrophages suppressed CD8⁺ T-cell activity via PD-1, and this inhibition was partially reversed by PD-1 blockade. Proximity ligation assay combined with multiplex immunofluorescence on FFPE sections confirmed in situ PD-1/PD-L1 engagement at two interfaces: CD8⁺ - HRS, and CD8⁺-TAMs contacts. Notably, EBV-positive tumors exhibited higher interaction rates across both interfaces, consistent with preserved MHC-I and enriched myeloid niches. PLA/IF further demonstrated that in tumors with limited CD8–HRS contact, CD8⁺ T cells continued to form PD-1/PD-L1 interactions with macrophages, indicating that TAMs can function as alternative checkpoint partners when direct tumor engagement is reduced. Together, these data support a multilayered model in which HRS cells initiate checkpoint-mediated suppression that TAMs amplify and stabilize. Long-term co-cultures of HRS cells with PD-1–expressing cells revealed no sustained proliferative or survival advantage from PD-L1 ligation under nutrient-replete conditions, suggesting context dependence of PD-L1 reverse signaling. Finally, RNA-seq of macrophages exposed to HRS-conditioned media showed M2-like polarization with increased mediators such as VSIG4, MMP9, and PDGF ligands, reinforcing checkpoint-mediated suppression and impairing T-cell function. Together, these data support a layered model of immune evasion in cHL: HRS cells can initiate PD-1/PD-L1–dependent inhibition where MHC-I is intact, while M2-like TAMs act as compensatory/alternative partners that sustain suppression when direct CD8–HRS engagement is limited. This framework explains the variable efficacy of PD-1 blockade and supports macrophage-targeted therapies for more durable responses.
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