Introduction Despite advances in Hodgkin lymphoma (HL) treatment, effective strategies for relapsed/refractory (R/R) HL, especially after PD-1 blockade failure, remain a critical need. Monocyte-driven inflammation and dysfunctional CD4⁺ T cells have been implicated in resistance to PD-1 inhibitors (Paczkowska et al., Nat Commun, 2024). Based on this, we hypothesized that HDAC inhibitors (HDACi) may restore PD-1 sensitivity by enhancing antigen presentation and promoting a tumor microenvironment conducive to anti-tumor immunity. In the present study, we leveraged single-cell combined indexing of transcriptome and epitopes (CITE)-sequencing and high-resolution spatial transcriptomics to characterize the circulating and microenvironmental features associated with response to combined HDACi and PD-1 blockade in R/R HL.

Methods Our group recently completed a phase II trial evaluating entinostat plus pembrolizumab in R/R HL (Stuver et al., ASH 2024). To investigate mechanisms of response and resistance, we analyzed 20 longitudinally collected peripheral blood samples from 9 patients and 6 paired (pre- and on-treatment) tumor biopsies using single-cell RNA, TCR, and surface epitope profiling (Single Cell Immune Profiling, 10x Genomics) and spatial transcriptomics (Visium HD, 10x Genomics). Cell types in spatial data were annotated using an optimal transport-based approach leveraging a published HL reference atlas (Stewart et al., Blood, 2023). Cell-cell communication analysis was conducted to identify ligand-receptor interactions influencing treatment sensitivity and resistance.

Results 39 patients were enrolled, including 22 (56%) who had previously failed anti-PD1 therapy. Among 38 evaluable patients, complete and objective response rates were 47% and 63%, respectively. Peripheral blood analysis was performed on 9 patients (median age, 36 [21–77]) with samples collected at Cycle 1 Day 1, Cycle 2 Day 1, and, if applicable, at progression. Of these, 5 (55%) achieved complete remission, 1 (11%) partial remission, and 3 (34%) experienced progression.

After filtering, 68,988 cells were analyzed: 37,710 monocytes and 21,013 T cells. We identified 11 T cell subsets, including CD8⁺ and CD4⁺ T cells exhibiting a stress-associated transcriptional program (RUNX1, TOX, tyrosine phosphatases). Myeloid profiling revealed enrichment of inflammatory classical monocytes (IL-1β, NLRP3, NF-κB targets) in patients who had failed PD-1 monotherapy (p < 0.0001 for IL1B in monocytes).

Longitudinal profiling showed that entinostat plus pembrolizumab upregulated antigen presentation genes (B2M, HLA-DRA, HLA-A, HLA-B, CIITA, CD74) in both responders and non-responders. Resistance was associated with persistent inflammatory monocytes, reduced clonal expansion of effector CD8⁺ T cells (6.97% vs. 24% in responders), and a skewed CD4⁺ T cell compartment marked by elevated frequencies of stressed and Treg CD4⁺ cells (LFC > 5, p < 0.05). These findings suggest that inflammatory monocytes may suppress anti-tumor immunity by impairing antigen-dependent CD8⁺ T cell priming and expansion.

Spatial profiling confirmed that treatment resistance was associated with tumor infiltration by IL1B⁺ monocytes, T follicular helper (Tfh) and Treg cells, highlighting immunosuppressive monocyte-CD4+ T cell niches as key drivers of resistance. Furthermore, we identified the CCL17–CCR4 axis as a central mediator of immune evasion and resistance to PD1 blockade in HL. Specifically, sustained CCL17 expression by local networks of HRS cells and inflammatory monocytes was found to recruit Tfh and Treg cells, thereby preventing CD8⁺ T cell surveillance. Co-expression of STAT6 and CCL17 was observed in all pre-treatment samples but persisted only in non-responder patients on-treatment. These findings suggest that HDAC inhibition may overcome resistance by suppressing STAT6 activity in HRS cells, reducing CCL17 expression, disrupting the monocyte-HRS feedback loop, and restoring effective CD8⁺ T cell-mediated tumor immunity.

Conclusion:

Our integrative single-cell and spatial analysis reveals that adaptive immune networks and localized immunosuppressive niches critically shape the response to checkpoint blockade in Hodgkin lymphoma. From these findings, the spatial organization of the tumor microenvironment emerges as a critical determinant of therapeutic sensitivity and a promising target for intervention.

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2025
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