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328 Promoting NK survival and function within the tumor microenvironment
  1. Matthew Dysthe,
  2. Ishwar Navin,
  3. Corrine Baumgartner,
  4. Stephanie Fetzko,
  5. Cliona Rooney and
  6. Robin Parihar
  1. Baylor College of Medicine, Houston, TX, USA


Background The efficacy of CAR-NK cellular therapies in solid tumors is limited by insufficient survival and expansion within the suppressive tumor microenvironment (TME). To augment CAR-NK survival and anti-tumor function, CAR-NK cells are typically administered with exogenous cytokines or other signal modifiers that converge on signal transducer and activator of transcription-5 (STAT5) activation, an essential signaling node for NK survival and function.1 However, systemic administration of these cytokines or signal modifiers is associated with toxicity,2 unintended bolstering of TME components,3 and CAR-NK rejection.4 Thus, novel strategies to enhance CAR-NK function within the TME are needed. To promote CAR-NK survival and function in the TME while bypassing the need for exogenous signal dependency, we genetically modified GD2-specific CAR-NK cells to express a constitutively active IL-7 receptor complex, termed C7R (C7R.GD2-NK), that intrinsically confers persistent STAT5 activity within NK cells.

Methods To mimic challenges of the TME, we created an in vitro TME model by preconditioning CD14+ monocytes with CHLA255 neuroblastoma cells for 72 hours, converting them to an inhibitory M2-TAM phenotype expressing suppressive cytokines and ligands. 4 days after the addition of the C7R.GD2-NK cells to this immunosuppressive milieu, we assessed NK survival, expansion, cytokine secretion, and anti-tumor function.

Results Resting C7R.GD2-NK cells exhibited 3.4-fold (± 0.7; n=4 donors, p<0.005) higher phosphorylated STAT5 (pSTAT5) compared to resting GD2-NK cells. C7R.GD2-NK cells alone in long-term culture expanded for 14 days without exogenous cytokine support but not indefinitely. In short-term co-cultures with GD2+ CHLA255 cells, C7R.GD2-NK demonstrated similar IFNγ levels as GD2-NK stimulated with exogenous IL-2 or IL-15 (mean% GD2-NK IFNy+ of 26.1, 21.2, and 22.4, respectively). In the in vitro TME model, C7R.GD2-NK cells expanded less than IL-15- or IL-2-stimulated GD2-NK cells (mean fold expansions of 2.8-, 4.5-, and 4.3-fold, respectively) but demonstrated similar anti-tumor activity. Furthermore, cell culture supernatants from C7R.GD2-NK treated conditions exhibited similar levels of NK effector cytokines IFNγ, TNF-α, and GM-CSF to exogenous cytokine-stimulated GD2-NK cells. Ongoing experiments in an in vivo TME xenograft model will assess the benefit of C7R in CAR-NK activity.

Conclusions C7R drives STAT5 activity in GD2 CAR-NK cells within TME conditions without the need for exogenous cytokines. The NK-intrinsic STAT5 signal is sufficient to promote NK survival, expansion, and function within the TME, potentially without the side effects of exogenous cytokines or cytokine modulators. Thus, C7R may be a viable alternative therapeutic strategy to augment CAR-NK efficacy in solid tumors.


  1. Gotthardt D, Sexl V. STATs in NK cells: The good, the bad, and the ugly. Front Immunol. 2017; 7:694

  2. Dutcher JP, Schwartzentruber DJ, Kaufman HL, Agarwala SS, Tarhini AA, Lowder JN, Atkins MB. High dose interleukin-2 (Aldesleukin) – expert consensus on best management practices-2014. J Immunother Cancer. 2014; 2:26

  3. Nelson B. IL-2, Regulatory T Cells, and Tolerance. J Immunol. 2004; 172:3983-3988

  4. Berrien-Elliot M, et al. Systemic IL-15 promotes allogeneic cell rejection in patients treated with natural killer cell adoptive therapy. Blood. 2022; 139:1177-1183

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