As first-line therapy for non-Hodgkin's lymphoma (NHL), patients receive combined chemo- and immunotherapy, including the anti-CD20 antibody rituximab. A main action of rituximab is linking NK cells via CD16a receptors to B-cells via CD20, resulting in antibody-dependent cell-mediated cytotoxicity (ADCC). Unfortunately, approximately 30-50 % of patients treated with first-line therapy relapse. Therefore, it is of great interest to better understand the mechanisms of therapy in detail to minimize relapse rate. ADCC of NK cells has been well studied. In addition to NK cells, a small population of T cells also expresses CD16 on their surface and may thus play a role for therapy. So far, the phenotype and function of these mostly unconventional CD16⁺ T cells have not been well characterized.
 
We sorted CD16⁺ T cells from peripheral blood mononuclear cells of healthy donors and examined their cytotoxic function using a time-resolved calcein-based and a caspase-dependent GFP-RFP-FRET assay, which allows to distinguish between necrotic and apoptotic cell death at single cell level. We developed three antibody panels for flow cytometry to characterize CD16⁺ T cells as mucosal-associated invariant T cells (MAIT; Vα7.2⁺, CD161^high), invariant natural T cells (iNKT; Vα24⁺) and γδ T cells (TCR δ1/2⁺).
 
Preliminary results from eight donors show that 32-94 % of the sorted CD16⁺ T cells are γδ T cells. In most cases, these are TCR δ2⁺ T cells. Moreover, the analysis also showed a positive correlation between ADCC and the proportion of CD4^- CD8^- γδ T cells. ADCC mainly leads to apoptotic (and not necrotic) killing of the B-cell line TMD8. These results suggest that CD16⁺ T cells are capable of mediating rituximab-dependent and caspase-dependent cytotoxicity and that these are mainly CD4^- CD8^- and express γδ TCR.