Evaluation of the Functional Landscape of Systemic Immunity in Classical Hodgkin Using a Novel Single Cell Platform (Isolight)

Background: Classical Hodgkin lymphoma (HL) has a unique histopathology, with rare malignant Hodgkin/Reed Sternberg (HRS) cells surrounded by a strong inflammatory cellular component in the tumor microenvironment (TME). Although extensive studies describe the interdependence of the HRS cells and the TME, the impact of HL on systemic immunity has not been well described. Here, we develop a new approach, employing a recently commercialized single cell cytokine secretion platform (IsoLight) to assess, precisely and comprehensively, the function of peripheral blood mononuclear cells (PBMCs) in HL patients.

Methods: Cells were selected from 4 HL patients: 2 newly diagnosed who had a complete response (CR) to therapy standard first line therapy, and 2 relapsed patients who progressed on second line chemotherapy (PD). Cryopreserved PBMCs from a pre-treatment and post-treatment time point for each patient were thawed, rested overnight, stimulated with PMA/ionomycin and loaded into the IsoLight single cell cytokine secretion system. IsoLight captures single cells in microwells; as cytokines are secreted, they are bound by antibodies lining the microwell cover. Bound cytokines are then revealed by fluorescent secondary antibodies and photos are taken at various time points to assess fluorescence intensity, which corresponds to the relative amount of each cytokine secreted. Twenty thousand cells can be assayed per sample simultaneously.

Results: The percentage of cytokine-secreting cells varied dramatically by donor (12%-48%), with monofunctional cells making only TNFa, MIP1b, or IL-15 dominating the functional landscape. Polyfunctional cells, capable of making three or more cytokines simultaneously represented only 0.1-7% of the cells in each sample, but there were more of these cells, and each secreted higher levels of cytokines, in individuals who responded to therapy with a CR. Responders also secreted higher levels of IL2, Perforin, IL4, IL12, MIP1a, and TNFb (p values ranging from 0.005 to 0.03), and lower levels of IL9 and IL22 (p=0.0028 and 0.021, respectively), compared to non-responders at diagnosis. Responders lost expression of IL4, IL7, and MIP1a over the course of treatment (pre- vs post-treatment, p=0.01 to 0.05), while non-responders gained cells that expressed IL4, IL5, IL10, IL17, and TNFb from diagnosis to end of treatment (p=0.001 to 0.05).

Conclusion: This work represents an important methodological advance in immune monitoring for hematologic malignancies. Single cell cytokine secretion technology measures more cytokines simultaneously than flow cytometry, providing a sample-sparing and comprehensive overview of the functional landscape of immune cells in a patient. Moreover, the technology provides cell-by-cell information about cytokine secretion, unlike Luminex. Our work represents the first application of this technology to HL, which we use to define, for the first time, the particular combinations of 32 cytokines that can be secreted by individual immune cells. We also identify candidate cytokines whose frequency at diagnosis may predict treatment outcome, and reveal changes in cytokine levels over treatment time that may distinguish patients destined to relapse. Immunotherapy may impact PBMC function differently, this may partially explain the high efficacy of this therapy in the relapsed population. The impact of immunotherapy on cytokine levels is currently under investigation by our group in a larger study. Other important questions which are under investigation include the impact of prior chemotherapy on cytokine profiles in relapsed patients, and whether certain cytokines which increase during treatment may be a surrogage for tumor bulk in patients with PD. Cytokines elevated in patients with poor responses to treatment include IL9, IL10, IL17, and IL22, which may present attractive drug targets if validated in our larger ongoing follow-up study.