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Cbl-b silenced human NK cells respond stronger to cytokine stimulation
  1. Guenther Lametschwandtner1,
  2. Monika Sachet2,
  3. Isabella Haslinger1,
  4. Hannes Mühleisen1 and
  5. Hans Loibner1
  1. Aff1 Apeiron Biologics Vienna Austria
  2. Aff2 grid.22937.3d0000000092598492Department of SurgeryMedical University Vienna Vienna Austria

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Meeting abstracts

The E3 ubiquitin ligase cbl-b has been identified as an important gatekeeper limiting T cell activation and more recently also NK cell activation. Hence, cbl-b deficient NK cells displayed stronger anti-tumor responses and this has been linked to a key role of cbl-b for modulation of TAM-receptors. However, in T cells cbl-b has been described to interact with different key regulators of T cell receptor and costimulatory signaling pathways. We have therefore investigated, whether abrogation of cbl-b function in human NK cells mediates increased responsiveness to cytokine stimulation.

Primary human NK cells were isolated and cbl-b silenced by electroporation with siRNA directed against cbl-b. Cbl-b silenced NK cells reacted stronger to tumor cell contact and this response was synergistically enhanced by cytokine stimulation with IL-2 and IL-12. Moreover, stimulation of cbl-b silenced NK cells with cytokines in the absence of tumor cells, either with IL-2 and IL-12 or IL-2 alone led to enhanced activation of NK cells, resulting in increased secretion of effector cytokines like IFN-g and TNF-a and upregulation of the activation marker CD69. Similar results were obtained for stimulation of cbl-b silenced T cells with other key cytokines of innate immune responses, particularly type I Interferons like IFN-a and IFN-b. These data demonstrate that the proposed central role of cbl-b as a negative regulator of various signaling pathways, including Akt, also applies to NK cells.

Together, these results show that interfering with cbl-b function in human NK cells enables synergistic responses to key cytokines of innate and adaptive immune responses. Thus, targeting cbl-b in the context of tumor-immune therapy should not be confined to the T cell compartment, but include NK cells as well. This can be achieved in the context of adoptive cell therapies, when autologous patient PBMCs are silenced for cbl-b ex vivo and retransferred afterwards. Such a protocol was established and is currently tested in a Phase I trial at Wake Forest University. The observed synergism of cbl-b silencing and NK cell stimulation by cytokines like IL-2 could be of particular relevance when tumor-reacting cbl-b silenced T cells infiltrate the tumor and secrete enhanced amounts of IL-2. In addition, it also provides a rationale for combinations of cbl-b targeting approaches with local application of cytokines at the tumor site (e.g. intratumoral injection of IL-2).