Immunophenotyping of CD8+ T Cells in Classical Hodgkin Lymphoma Reveals a Shift Toward Intermediate Exhaustion Following PD-1 Blockade

Abstract The immune microenvironment of classical Hodgkin lymphoma (cHL) is characterized by rare Hodgkin–Reed-Sternberg (HRS) cells surrounded by dysfunctional lymphocytes. HRS cells exploit the PD-L1/PD-1 axis to suppress antitumor immunity. Although PD1 blockade is effective in some relapsed cHL patients, the mechanisms of resistance remain unclear. We investigated T-cell dynamics in cHL using whole-exome sequencing (WES) of plasma circulating tumor DNA and longitudinal single-cell RNA and T-cell receptor sequencing of peripheral blood lymphocytes from a relapsed patient before and after anti-PD1 therapy. Findings were validated by flow cytometry in 11 relapsed cHL patients (pre- and post anti-PD1) and 6 healthy age-matched controls, and by PhenoCycler imaging of 18 tissue biopsies (13 diagnostic, 5 post anti-PD1). WES identified two somatic mutations at relapse—MEX3B-Lys201* and TNFRSF10A-Tyr409His—potentially contributing to immune escape. Immune profiling revealed clonal expansion of CD8⁺ T cells at relapse and a shift from precursor-like (TCF1⁺) to intermediately exhausted (CX3CR1⁺) phenotypes. This pattern was confirmed in other relapsed cHL patients, in whom TCF1⁺ populations diminished, and CX3CR1⁺ T-bet⁺ populations became dominant among those progressing on anti-PD1 therapy. In post anti-PD1 treatment tissue biopsies, CD8⁺ T cells exhibited increased levels of exhaustion markers (TCF1⁻, CX3CR1⁺), a phenotype not observed in diagnostic cHL or healthy donors. These results suggest that durable anti-PD1 responses depend on the preservation of progenitor-exhausted T cells, whereas relapse post anti-PD1 is marked by the accumulation of intermediately exhausted clones. Strategies to prevent T-cell exhaustion or combine anti-PD1 with approaches targeting antigen burden may help overcome resistance.