CMV-Specific Cytokine-Induced Killer Cells Comprise Concomitant Cytotoxicity Against Leukemia and Cytomegalovirus

Introduction

Infection is one of the main causes of mortality and morbidity after allogeneic stem cell transplantation, especially in patients who received T cell depleted haploidentical stem cells. Reactivation or de novo infection of cytomegalovirus (CMV) is amongst the most frequent complications and occur due to a lack of virus-specific T cells post-transplant. Pre-emptive immunotherapy may support both reconstitution of viral specific responses on one hand and may prevent impending leukemic relapse on the other hand. Therefore we established a protocol to generate CMV-specific cytokine-induced killer cells (CIK_pp65) with dual cytolytic function against CMV and AML.

Protocol

CIK cells were generated in vitro from peripheral blood mononuclear cells (PBMC) of CMV-seropositive healthy donors using IFN-γ, activating monoclonal anti-CD3 antibody (MAb), interleukin (IL)-2 and IL-15. An additional single stimulation with human CMVpp65 protein was adequate to increase the amount of cytotoxic CMV-specificcells within CIK cells up to 23%. In total the CMVpp65 stimulation resulted in up to 11.0-fold increased frequency of CMV-dextramer⁺CD8⁺cells after 15 days of expansion (n=12).

Results

Cytotoxicity

Next we investigated cell-mediated cytotoxicity against leukemic cell lines THP-1 and K562, pp65 loaded cell line T2 and CMV-infected primary fibroblasts. CIK cell cytotoxicity is described as mediated by activating NK-cell receptor NKG2D. This receptor was blocked in order to determine the specific MHC-mediated cytotoxicity in experiments targeting pp65 loaded cells. The lysis of pp65 loaded cells by CIK_pp65 cells was significant higher as compared to conventional CIK cells (effector to target cell ratio of 5:1, 39.9±21.6% to 13.6±10.6%, P<0.01). CIK_pp65 cells also induced high cytotoxicity in infected fibroblasts (up to 55%, 10:1 E:T ratio). The anti-leukemic effect was retained in CIK_pp65 cells. CIK_pp65 cells revealed a mean cytotoxicity of 71.5%, 60.7% and 37.8% against THP-1 and 55.0%, 50.0%, 20.5% against K562 in 40:1, 20:1 and 5:1 E:T ratio, respectively. In contrast, the reactivity against allogeneic PBMC remained low (18% lysis, 40:1 E:T ratio) and allogeneic mock-infected fibroblasts were not lysed at all. This clearly indicates towards the low alloreactive potential of CIK_pp65 cells.

Phenotype

Furthermore we characterized subpopulation and memory phenotype of CIK_pp65 cells in detailed flow cytometric analyses and examined the cytokine secretion pattern by cytometric bead array. After expansion the population mainly consisted of a CD3⁺CD56^- T cell (77.6±4.5%) and CD3⁺CD56⁺ T-NK cell phenotype (20.0±12.6%). The T-NK cells additionally co-expressed high amounts of CD8 cytotoxic antigen (63.8±16.8%). Interestingly, the T-NK cell compartment contained higher amounts CMV-specific CD8⁺ cells (mean 5.5%) than the T cell compartment (mean 1.3%). Expression of activating NKG2D and CD25 receptor was strongly positive in both cell fractions. Remarkably, almost 30% of T-NK cells expressed γδ⁺ T cell receptor, whereas T cells only expressed 4.5% of this receptor type. The cytotoxic T cells within the CIK_pp65 cells consisted of a mixed naïve (CD45RA⁺CD62L⁺), central memory (CD45RO⁺CD62L⁺) and effector memory (CD45RO⁺CD62L^-) phenotype, the cytotoxic T-NK cells mainly of effector memory and EMRA (CD45RA⁺CD62L^-) phenotype. Cytokine secretion (granzyme B, IFN-γ, MIP-1α, TNF-α, Fas-L, IP-10, IL-10, IL-6 and IL-4) were measured during the expansion period and cytotoxic assays and resulting data confirmed the cytotoxic nature of the cells and indicated towards a mainly T_H1 cell type character.

Conclusion

In conclusion CIK_pp65 cells can easily be generated from donor PBMC and might represent advantage to conventional CIK cells. Our pre-clinical data demonstrate the concomitant cytotoxicity of generated cells against leukemia cells and CMV, as well as low alloreactivity and limited risk to induce GvHD. Therefore CIK_pp65 cells may represent an effective tool for pre-emptive immunotherapy in patients which have both an apparent risk of CMV reactivation and leukemic relapse after allogeneic stem cell transplantation.