Abstract P135

Immunophenotyping of CD8+ T cells from classical Hodgkin Lymphoma patients treated with PD-1 blockade

Introduction: Classical Hodgkin lymphoma (cHL) features rare Hodgkin–Reed–Sternberg (HRS) cells within a dense, dysfunctional immune infiltrate. While PD-1 blockade helps some relapsed cHL patients, durable responses are uncommon. In solid tumors, progression from intermediate to terminal T-cell exhaustion drives resistance to PD-1 blockade. We evaluated T-cell exhaustion states in relapsed cHL.

Methods: We performed longitudinal single-cell RNA sequencing (scRNA-seq) of PBMCs from one patient during PD-1 inhibitor (PD1i) therapy. T-cell clones in blood and tissue were tracked by TCR sequencing. Gene expression was analyzed using Seurat and assessed for memory stem-like and terminal effector-like signatures. Findings were validated at the protein level by flow cytometry in 11 relapsed HL patients (pre- and post-PD1i) and 6 healthy age-matched controls, and by PhenoCycler imaging of 24 tissue biopsies (13 diagnostic, 11 post-PD1i).

Results: scRNA-seq identified 25,268 cells, 10,593 were T cells. Markers defined nine PBMC clusters, with key transcriptional changes in T cells. TCR analysis detected clonotypes in 39.5% of T cells, with overlap between scRNA-seq, bulk PBMCs, and tumor tissue. At relapse, CD8+ T cells expanded, inverting the CD4+/CD8+ ratio. Longitudinal analysis of gene expression networks showed a shift from TCF1+ precursor to CX3CR1+ T-bet+ intermediate exhausted states, coinciding with relapse and expansion of a dominant CD8+ clonotype. Flow cytometry of PBMC confirmed enrichment of CD8+ CX3CR1+ T-bet+ cells in relapsed cHL, mainly PD1i non-responders, compared to controls (p = 7.3 × 10−3). Healthy controls had more CD8+ TCF1+ CD127+ stem-like cells compared to relapse cHL patients (p = 1.07 × 10−2). Spatial imaging of cHL tissue post PD1i had increased CX3CR1, PD-1, and T-bet expression in CD8+ T cells, while diagnostic cHL tissue had lower CX3CR1 and higher TCF7 (p = 2.44 × 10−4). Thus, diagnostic cHL T cells, in blood and tissue, are mainly stem-like, while relapsed HL contains more intermediate/terminally exhausted T cells, likely contributing to poor PD1i responses.

Conclusions: Multiomic analyses show CD8+ T-cell states change dynamically during PD1i in cHL. Durable responses likely depend on maintaining progenitor-exhausted T cells, while relapse involves the accumulation of intermediate-exhausted CD8+ T cells. This may explain better PD-1 inhibitor responses in frontline versus later lines of therapy.