Summary The polyclonal cytokine‐induced killer (CIK) cells exhibit potent cytotoxicity against a variety of tumour cells including autologous and allogeneic acute myeloid leukaemic (AML) targets. At maturity, three lymphocyte subsets: CD3− CD56+, CD3+ CD56− and CD3+ CD56+, constitute the bulk of the CIK cell culture. The CD3− CD56+ subset behaves like classical natural killer (NK) cells where cytotoxicity is potentiated by blocking the human leucocyte antigen Class I molecules in the AML targets. Both the CD3+ CD56+ and CD3+ CD56− subsets, though known to kill autologous and allogeneic targets to a comparable degree and therefore non‐major histocompatibility complex (MHC)‐restricted, nevertheless require the presence of the MHC molecule on the target, which interacts with their CD3–T‐cell receptor complex. Although CIK cells are often termed ‘NK‐like’ T cells, we have demonstrated that the well‐characterized NK receptors KIR, NKG2C/E, NKG2D and DNAM‐1 are not involved in the process of AML recognition for the CD3+ CD56− and CD3+ CD56+ subsets. The CD3+ CD56+ and CD3+ CD56− subsets express a polyclonal and comparable TCRVβ repertoire in a Gaussian distribution. The CD3+ CD56+ subset kills AML targets more efficiently than its CD3+ CD56− counterpart because of the presence of a higher proportion of CD8+ cells. The CD3+ CD56+ subset comprise more terminally differentiated late effector T cells that bear the CD27+ CD28− or CD27− CD28− phenotype, with a higher granzyme A content. In comparison, the phenotype of the CD3+ CD56− subset is consistent with early effector T cells that are CD27+ CD28+ and CD62L+, known to be less cytotoxic but possess greater proliferative potential.

Central memory T lymphocytes were reported to develop after acute but not chronic infection, which prompted this feasibility study on generating long-term CD8 T cells ex vivo, by applying a culture condition that simulates an acute infection. During 35 d of culture, naive T cells (CD45RA+, CD127+, CCR7+, CD62L+, CXCR3+) first developed into effector T cells (CD45RA+/−, CD127+/−, CCR7+/−, CD62L+, CXCR3+), followed by three intermediate stages: intermediate T cells 1 (CD45RO+, CD127+/−, CCR7+, CD62L+, CXCR3+), intermediate T cells 2 (CD45RO+, CD127−, CCR7−, CD62L+, CXCR3+), and intermediate T cells 3 (CD45RO+/−, CD127+, CCR7+, CD62L−, CXCR3+) before reverting to stable CD45RA+ central memory T cells (CD45RA+, CD127+, CCR7+, CD62L+, CXCR3+). If both anti-CD3 and the inflammatory milieu persisted beyond day 10, intermediate T cells 2 gradually developed into effector memory T cells (CD45RO+, CD127−, CCR7−, CD62L−, CXCR3+). Furthermore, intermediate T cells 2 or effector memory T cells, when cultured in persistent inflammatory cytokines devoid of anti-CD3, were converted to central memory T cells (CD45RO+, CCR7+, CD62L+). Overall, these results support ex vivo memory-like T lymphocyte production and favor a developmental pathway including both divergent and linear relationships.

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.