Background: In classical Hodgkin lymphoma (cHL), the rare malignant Hodgkin Reed-Sternberg (HRS) cells are surrounded by an inflammatory infiltrate. Yet, the host anti-tumor immune response is ineffective. HRS cells have multifaceted mechanisms to evade the immune system including 9p24.1/PD-L1/PD-L2 genetic alterations leading to overexpression of PD-1 ligands and subsequent T cell exhaustion, aberrant antigen presentation and modulation of the tumor microenvironment (TME).

The clinical success of PD-1 blockade in cHL suggests the TME contains reversibly exhausted T-effectors (Teff). Paradoxically, durable responses are observed in patients with β2M/MHC class I loss on HRS cells, raising the possibility of non-CD8+ mediated mechanisms of efficacy of PD-1 blockade. For this reason, we sought to characterize HRS cells and the surrounding TME.

Methods: Using CyTOF technology, we evaluated 7 primary cHL suspensions and 10 reactive lymphoid tissue (RLT) samples at the single-cell protein level. We designed a custom panel of 39 isotope-conjugated antibodies. A combination of surface and intracellular markers distinguish T cell subsets according to lineage, differentiation, polarization, activation and exhaustion. Additional markers were incorporated to identify B cells, NK cells and macrophages. HRS cells were defined by CD15/CD30/Pax5 positivity. Inclusion of β2M and MHC class I allowed assessment of antigen presentation on HRS cells.

The data was acquired on a Helios CyTOF and analyzed using a fast k-weighted nearest neighbor algorithm, X-shift. X-shift clustered cells with phenotypic similarities together. Then, samples were separated into cHL and RLT and the contribution of a sample to a given cluster was quantified.

Results: Comparison of viable cell suspensions from RLT and cHL revealed loss of naïve T-cells and skewing towards differentiation of Teff in both CD4+ and CD8+ subsets in cHLs. This prompted a second X-shift analysis focused on CD3+ cells, which highlighted salient differences between cHL and RLT within the CD4+ subset. In cHL, we found expansion of Teff and regulatory T cells (Treg) with a reduction of follicular helper T cells. Furthermore, both Treg and Teff populations were largely Th1 (T-bet+/CCR5+) polarized. Evaluation of PD-1 expression showed Tregs had little/no PD-1 while Teff had intermediate/high expression. Hence, Tregs retain functionality in contrast to Teff, which are exhausted, providing two mechanisms of immunosuppression.

Manual gating identified HRS cells with a characteristic phenotype: CD15, CD30, Pax5, rosetted by CD4+ T cells. Importantly, we found loss or decrease of β2M and MHC class I in 5/7 cases.

Conclusions: The TME in cHL is CD4+ T cell rich with frequent loss of MHC class I on HRS cells. Differential PD-1 expression results in functional CD4+ Tregs and exhausted Teff, a synergistic bases for the observed immunosuppression in cHL.

Citation Format: Fathima Z. Cader, Ron C. Schackmann, Xihao Hu, Kirsty Wienand, Robert A. Redd, Bjoern Chapuy, Jing Ouyang, Nicole E. Paul, Evisa Gjini, Mikel Lipschitz, Laura M. Selfors, Philippe Armand, David Wu, Jonathan R. Fromm, Donna Neuberg, Xiaole S. Liu, Scott J. Rodig, Margaret A. Shipp. Single-cell mass cytometry of classical Hodgkin lymphoma defines an exhausted and immunosuppressive microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5675.