Abstract
Natural killer (NK) cells are increasingly recognized as an important component in the graft versus leukemia response following allogeneic hematopoietic stem cell transplantation. Immunotherapeutic strategies aim to promote NK cell activity however, the presence of regulatory T cells (Tregs) which inhibit effector immune responses pose a potential challenge to the efficacy of such regimens. We have previously shown that ‘in-vitro’ culture of AML cells transduced with a self-inactivating lentivirus (LV) encoding CD80 (B7.1) and IL2 enhance allogeneic (allo) and autologous (auto) T cell proliferation and cytotoxicity. The effect on NK cell activity and Tregs has not previously been studied and is of particular importance as IL2 stimulates NK cell and Treg activity. Peripheral blood mononuclear cells (PBMCs) from healthy donors (allo) or AML patients (auto) were cultured for 7 days ‘in-vitro’ with either unmodified or LV-CD80/IL2 AMLs. The number of NK cells (CD3−CD56+) and Tregs (CD3+CD4+CD25highFoxp3+) was examined by multi-color flow cytometry. We observe an increase in the number of NK cells (p<0.001) with an increase in the expression of the activation receptors NKp30, NKp44, CD244, CD25, CD69 and HLA-DR following allo culture with LV-CD80/IL2 AML compared with unmodified AML. Autologous culture provides a weaker stimulus ‘in-vitro’ however, a higher number of NK cells (p=0.002) and a consistent increased expression of the activation receptors NKp30, NKp44, NKp46, NKG2D, NKG2C and CD69, as well as up regulation of the cytolytic marker CD107a was detected following auto stimulation with LV-CD80/IL2 AML compared with unmodified AML. Up regulation of CD107a was also observed in allo cultures stimulated with both unmodified and LV-CD80/IL2 AML cells. In contrast, a consistent increase in the number of Tregs was observed following allo (p=0.043) but not auto (p=0.515) LV-CD80/IL2 AML culture. Foxp3 may be unregulated on activated CD4+ T cells therefore the number of CD3+CD4+CD25highFoxp3+CD27+ Tregs was also examined. An increase in the number of CD27+ Tregs was observed following allo (p=0.017) but not auto (p=0.807) LV-CD80/IL2 AML cell culture. A standard 51Cr release assay was used to examine cytotoxicity against primary unmodified AMLs on days 0 and 7 following LV-CD80/IL2 AML cell culture. Tregs are capable of suppressing CD4+ and CD8+ T cell and NK cell cytotoxicity, therefore lysis of unmodified AMLs was initially examined using whole PBMCs as effectors. Even in the presence of Tregs an increase in lysis of allo unmodified AMLs was observed: 2.2% day 0, 4.6% following culture with unmodified AMLs; 20.4% following LV-CD80/IL2 AML cell culture. Importantly, an increase in lysis of auto AML was also detected: 0% day 0, 2.1% unmodified AML culture, 16% LV-CD80/IL2 AML culture. The ratio of Tregs to effector T cells is important for the suppressive function of Tregs. The number of Tregs in the cytotoxicity assays is likely to be lower than that required for a significant suppressive effect to be observed. We next examined the cytotoxicity of NK cells using K562 and unmodified AMLs as targets. NK cells were negatively isolated on days 0 and 7 following either unmodified AML or LV-CD80/IL2 AML cell culture and used as effectors in a 51Cr release assay. In keeping with the changes in NK cell activation receptor expression, we demonstrate a significant increase in NK cell cytotoxicity against both K562 and primary unmodified AMLs. Lysis of K562 increased from 46.7% on day 0 to 90.4% after LV-CD80/IL2 stimulation. Importantly, an increase in lysis of both allo and auto unmodified AMLs was detected following LV-CD80/IL2 AML cell culture. Lysis of allo AMLs increased from a median of 11.8% on day 0, 8.7% following culture with unmodified AML to 20.1% following LV-CD80/IL2 AML cell culture using a low effector: target ratio of just 5:1. Importantly, an increase in lysis of auto AML from 0.4% on day 0, 2.1% with unmodified AML cells to 21.5% following LV-CD80/IL2 AML stimulation was observed. LV-CD80/IL2 AML cells enhance NK cell activation and cytotoxicity against allo and auto unmodified AMLs. Furthermore, cytotoxicity is enhanced even in the presence of Tregs with an increase in Tregs only observed following allo culture. Vaccination of patients with LV-CD80/IL2 AML cells therefore represents a potential strategy to promote T and NK cell cytotoxicity and enhance anti-leukemia immune responses in patients with AML.
Enhancing NK cell-mediated cytotoxicity to cisplatin-resistant lung cancer cells via MEK/Erk signaling inhibition
