Purpose. It is well recognised that the efficacy of anti-CD20 antibody (Ab)-based immunotherapeutic strategies largely relies on the ability to induce ADCC by means of the aggregation of the FcgammaRIIIA, CD16, on NK cells. We address the impact of receptor aggregation by anti-CD20 Ab-opsonised targets on CD16 dynamics and hence on NK cytotoxicity. Methods. The chimeric Rituximab and the human Ofatu- mumab anti-CD20 Abs were used to opsonise the human CD20+ Raji cell line or primary B-CLL cells. Primary cultured NK cells derived from healthy donors or B-CLL patients were allow to interact with strepta- vidin-loaded opsonised targets for 30 min to 12 hours, magnetically iso- lated and tested for phenotypic, functional and biochemical analysis. Result and Discussion. We show that opsonised target-experienced NK cells undergo a progressive CD16 down-modulation. Unexpectedly, the sustained CD16 stimulation induces a major and persistent impairment of the spontaneous cytotoxic response against a panel of sensitive tar- gets attributable to the cross-tolerance of several unrelated NK activa- tion receptors including NKG2D, DNAM-1 NKp46 and 2B4. Notably, also in autologous setting, NK cell stimulation with Rituximab- opsonised B-CLL cells induces a marked CD16 down-modulation asso- ciated to a significant impairment of the cytolytic potential. NK hypore- sponsive status correlates to CD16 down-modulation, but is largely independent from the exhaustion of cytolytic mediators or from the down-modulation of activation receptor expression. Moreover, the abil- ity to form conjugates with sensitive targets and to polarise lytic gran- ule at immune synapse is preserved; by contrast, degranulation is impaired. We are investigating the molecular mechanisms by which CD16 partecipates in spontaneous cytotoxicity, in particular the sig- nalling network involved in the control of granule exocytosis. Conclu- sions. We propose a new mechanism of exhaustion of cytotoxic effec- tors that may impact on the immunocompetence of therapeutic-Ab treated patients.
A sustained CD16 aggregation induced by therapeutic antibody-opsonised targets impairs cytotoxic responses in human NK cells
CAPUANO, CRISTINA;
2014-01-01
Abstract
Purpose. It is well recognised that the efficacy of anti-CD20 antibody (Ab)-based immunotherapeutic strategies largely relies on the ability to induce ADCC by means of the aggregation of the FcgammaRIIIA, CD16, on NK cells. We address the impact of receptor aggregation by anti-CD20 Ab-opsonised targets on CD16 dynamics and hence on NK cytotoxicity. Methods. The chimeric Rituximab and the human Ofatu- mumab anti-CD20 Abs were used to opsonise the human CD20+ Raji cell line or primary B-CLL cells. Primary cultured NK cells derived from healthy donors or B-CLL patients were allow to interact with strepta- vidin-loaded opsonised targets for 30 min to 12 hours, magnetically iso- lated and tested for phenotypic, functional and biochemical analysis. Result and Discussion. We show that opsonised target-experienced NK cells undergo a progressive CD16 down-modulation. Unexpectedly, the sustained CD16 stimulation induces a major and persistent impairment of the spontaneous cytotoxic response against a panel of sensitive tar- gets attributable to the cross-tolerance of several unrelated NK activa- tion receptors including NKG2D, DNAM-1 NKp46 and 2B4. Notably, also in autologous setting, NK cell stimulation with Rituximab- opsonised B-CLL cells induces a marked CD16 down-modulation asso- ciated to a significant impairment of the cytolytic potential. NK hypore- sponsive status correlates to CD16 down-modulation, but is largely independent from the exhaustion of cytolytic mediators or from the down-modulation of activation receptor expression. Moreover, the abil- ity to form conjugates with sensitive targets and to polarise lytic gran- ule at immune synapse is preserved; by contrast, degranulation is impaired. We are investigating the molecular mechanisms by which CD16 partecipates in spontaneous cytotoxicity, in particular the sig- nalling network involved in the control of granule exocytosis. Conclu- sions. We propose a new mechanism of exhaustion of cytotoxic effec- tors that may impact on the immunocompetence of therapeutic-Ab treated patients.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
