Novel cancer cellular therapy approaches involving long-term ex vivo IL-2 stimulated highly cytotoxic natural killer (NK) cells are emerging. However, adhesion properties of such NK cells are not very well understood. Herein, we describe the novel observation of permanently activated alphaLbeta2 integrin leukocyte function-associated antigen (LFA)-1 adhesion receptor in long-term IL-2 activated NK cells and the permanent NK cell lines KHYG-1 and NK-92. We show that such cytokine activated NK effectors constitutively adhered to the LFA-1-ligand ICAM-1, whereas binding to the lower affinity ligand ICAM-3 required additional exogenous activating conditions. The results demonstrate an extended conformation and an intermediate affinity state for the LFA-1 population expressed by the NK cells. Interestingly, adhesion to ICAM-1 or K562 induced pronounced cell spreading in KHYG-1, but not in NK-92, and partially in long-term IL-2 stimulated primary NK cells. It is conceivable that such differential adhesion characteristics may impact motility potential of such NK effectors with relevance to clinical tumor targeting. KHYG-1 could be a useful model in planning future targeted therapeutic approaches involving NK effectors with augmented functions.
Introduction: Cytokine-induced killer ( CIK) cells are polyclonal non-MHC restricted T cells with potent cytotoxicity against acute myeloid leukemia (AML) cells in vitro. We have established culture of CIK cells with GMP compliance and infused into patients with various haematological malignancies. These include group 1, as adjuvant therapy post autologous transplant for acute myeloid leukaemia (AML), group 2, in untreated disease and group 3, for relapse post allogeneic transplant (NCT 00460694, NCT 00394381). Patients, Methods and Results: A total of 39 CIK cultures was produced over a 2 year period which resulted in 65 infusions given to 21 patients. We have demonstrated that it is feasible to expand CIK in large scale culture from both patients and allogeneic stem cell donors. The CD3+CD56+ subset expanded a median of 42.7 fold from 1.3% (0.2–5.3%) to 31.1% (10.4–76.9%) post culture for CIK derived from patients’s leukapheresis product, which is comparable with that derived from healthy haemopoietic stem cell donors. The cytotoxicity of these CIK against a panel of allogeneic AML targets showed variable potency (0% to 69%), with a median of 38%. Self limiting fever was the only infusion related side effect. Patients In group 1 received an autologous transplant as consolidation for AML followed by adjuvant infusions of CIK cells., These were successfully cultured for 9 of 11 patients and infused into all 9 in aliquots of between 1–4 infusions. Follow up is short and a comparison against historical autologous transplant results are similar. Group 2 consisted of patients with overt leukemia who have failed or are unfit for chemotherapy. All 4 had CIK cells successfully cultured from a product containing variable % of leukemic cells, 3 of the patients who survived to receive CIK infusion did not have any response. However one of them had an incidental regression of basal cell carcinoma after 2 infusions. In group 3, 6 patients who relapsed after an allogeneic transplant received allogeneic CIK after failing donor lymphocyte infusions (DLI). Another 3 patients had CIK generated from their own leukapheresis product due to donor unavailability (1 post cord blood and 2 post MUD transplant). Amongst the 8 patients who have received CIK infusion, two with overt refractory relapse (1 AML and 1 Hodgkin’s disease) did not respond to 3 and 4 infusions respectively. Four patients (2 AML and 2 ALL) had CIK infusion post salvage chemotherapy and therefore remission could not be entirely attributed to CIK infusion. Two patients had measurable responses attributable solely to CIK infusion. One was a post-transplant relapsed T cell ALL refractory to 5 different salvage regimen and repeated DLI. A marrow remission was achieved after one further Gemcitabin/Mitoxantrone salvage chemotherapy followed by CIK infusion. Marrow remission was maintained for 10 months with 4–6 weekly infusion of CIK while extramedullary (EM) disease progressed, suggesting control of marrow leukemia by CIK while leukemia evolution manifested at EM sites, known to be less susceptible to immunotherapy. A second patient had refractory Hodgkin’s disease in the lungs and vertebrae. A partial reduction in the size of lung nodules was achieved after the second CIK infusion but this was not sustainable. The dose of allo- CIK ranged from 10 – 200 million CD34/kg given as a step-wise increment for each patient. Three patients developed acute GVHD, one grade II liver, one grade II skin andgut, and a third patient had grade I upper gut GVHD, at doses of 50, 100 and 10 million CD3/kg respectively. All responded promptly to prednisolone at 1mg/kg. Conclusion: We have shown that CIK infusion is feasible and safe in both the autologous and allogeneic setting, and GVHD that occurs is easily controlled. It is unlikely that CIK is effective against a large tumour burden. Its efficacy as an adjuvant therapy to eradicate minimal residual disease requires larger patient numbers and longer follow up. For allogeneic transplant, CIK culture has an additional advantage of expanding unrelated donor cells where availability is a problem by harvesting donor cells from patients for expansion. Further numbers are needed to compare against unmanipulated DLI in terms of efficacy and reduced GVHD severity.
Background: KHYG-1 and NK-92 are highly cytotoxic IL-2 dependent NK cell lines. NK cytotoxicity is regulated through an array of receptors with activating and inhibitory functions for spontaneous elimination of pathogen infected or tumor cells. Important roles have been described for the leukocyte function-associated antigen (LFA)- 1 adhesion receptor (αLβ2) in NK cytotoxicity. In T cells, the integrin LFA-1 occurs in a non-functional, bent form and requires activation, through divalent cations e.g., for ligand binding. In marked contrast, IL-2 stimulated NK cells could directly engage the LFA-1 ligand intercellular adhesion molecules (ICAM)-1, thereby inducing conjugate formation, granule polarization, degranulation, and tumor cell lysis (Barber DF et al., 2004. JI 173:3653–9). Strikingly, in the NK cell line KHYG-1 granules were constitutively polarized (clustered around the MTOC; Suck G et al., 2006. Int Immunol 18:1347–54) and LFA-1 downstream signaling molecules, the spleen tyrosine kinase (Syk) and extracellular signal-regulated MAP kinase (ERK) constitutively phosphorylated (Suck G et al., 2005. Exp Hem. 33:1160–71). These previous findings prompted us to investigate LFA-1 activation state and functional involvement in cytotoxicity in KHYG-1 and NK- 92. Methods: Adhesion assays were performed on immobilized ICAMs and % adherence determined in a fluorescence plate reader (Tang XY et al., 2008 JI, 180:4793–804). LFA-1 was activated with Mg/EGTA (5 mM MgCl2 and 1.5 mM EGTA), monoclonal antibody (mAb) KIM185 (10 mg/mL) or both; LFA-1 mAb, clone MHM24, was used for blocking studies, 1–10 mg/ml and mAb KIM127 for immunoprecipitation; cytotoxicity, conjugate formation, and degranulation (CD107a) were measured by Flow cytometry and cell morphology imaged by phase contrast (Olympus, 1X70) or confocal microscopy (Zeiss, LSM510 Meta/Nikon A1). Excel software was employed for statistical analyses. Results: In cell binding assays KHYG-1 and NK-92 showed constitutive adhesion to the LFA1-1 ligand ICAM-1, 61% +/− 5.8% SD and 55% +/− 7.5% SD, respectively. However, only 22% NK-92 and 10.5% KHYG-1 cells adhered to the lower affinity ligand ICAM-3 and activation with Mg/EGTA or KIM185 was required to increase binding to 73% in KHYG- 1 and to 62% in NK-92. Immunoprecipitation experiments with the activation reporter mAb KIM127 revealed an activated extended LFA-1 conformation in both cell lines. Together these results suggested an intermediate affinity activation state for the LFA- 1 population in KHYG-1 and NK-92. Despite such similarities, ICAM-1 engagement triggered pronounced cell spreading in KHYG-1, similar to phorbol ester stimulated T cells, but not in NK-92 or primary NK cells. It is conceivable that the capacity to undergo cytoskeletal remodeling in KHYG-1 may be impaired as potentially also reflected in the constitutively polarized granule state. In addition, LFA-1 blocking studies with LFA- 1 antibody MHM24 did not inhibit target conjugate formation in KHYG-1, compared to partial inhibition in NK-92, and cytotoxicity against K562 was only about 40% diminished in KHYG-1, compared to almost complete abrogation (maximum 98%) in NK-92. Nevertheless, overall cytolytic potential against K562 was comparable among the two cell lines, which implicated important functions for other receptors at least in KHYG-1. Conclusion: Results suggested a constitutively activated intermediate affinity state for LFA-1 in KHYG-1 and NK-92. It is conceivable that a similar activated state occurs in primary NK cells. Interestingly, although cytoskeletal dysfunction was indicated in KHYG-1, cytotoxicity of the cell line was unaffected, rendering it a valuable model for the study of alternative pathways.
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