Reverted exhaustion phenotype of circulating lymphocytes

While classical Hodgkin lymphoma (HL) is highly susceptible to anti-programmed death protein 1 (PD1) antibodies, the exact modes of action remain controversial. To elucidate the circulating lymphocyte phenotype and systemic effects during anti-PD1 1st-line HL treatment we applied multicolor flow cytometry, FluoroSpot and NanoString to sequential samples of 81 HL patients from the NIVAHL trial (NCT03004833) compared to healthy controls. HL patients showed a decreased CD4 T-cell fraction, a higher percentage of effector-memory T cells and higher expression of activation markers at baseline. Strikingly, and in contrast to solid cancers, expression for 10 out of 16 analyzed co-inhibitory molecules on T cells (e.g., PD1, LAG3, Tim3) was higher in HL. Overall, we observed a sustained decrease of the exhausted T-cell phenotype during anti-PD1 treatment. FluoroSpot of 42.3% of patients revealed T-cell responses against ≥1 of five analyzed tumor-associated antigens.

Fig: A Lymphocyte subsets in peripheral blood mononuclear cells (PBMCs) of Hodgkin lymphoma (HL) patients before therapy (BT, n = 72) and healthy donor PBMCs (HC, n = 20) were analyzed by flow cytometry. B Differences in T-cell activation (CD69+%Tcells, CD25+%Tcells, and CD62L+%Tcells) and T-cell differentiation (CCR7CD45RA effector memory (EM) T cells) of HL patients before therapy (BT) and healthy control PBMCs (HC). C Differences in B-cell activation (CD86+%Bcells) and B-cell differentiation (plasmablasts%Bcells) of HL patients before therapy (BT) and healthy control PBMCs (HC). D Immune-regulatory molecule expression was determined by flow cytometry. Significant up/downregulation of co-inhibitory and co-stimulatory molecule expression on T cells, CD4 T cells, CD8 T cells, B cells, and NK cells of HL patients before therapy (n = 72) compared to the mean expression obtained from healthy control PBMCs (n = 20). Significant differences calculated by unpaired, two-tailed Mann–Whitney test are indicated by asterisks. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. When appropriate, mean ± SD is indicated.

Importantly, these responses were more frequently observed in samples from patients with early excellent response to anti-PD1 therapy. In summary, an initially exhausted lymphocyte phenotype rapidly reverted during anti-PD1 1st-line treatment. The frequently observed IFN-y responses against shared tumor-associated antigens indicate T-cell-mediated cytotoxicity and could represent an important resource for immune monitoring and cellular therapy of HL.

Garcia-Marquez, M.A., Thelen, M., Reinke, S. et al. Reverted exhaustion phenotype of circulating lymphocytes as immune correlate of anti-PD1 first-line treatment in Hodgkin lymphoma. Leukemia (2021).

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