The Effects of IL-2 Concentration and Adding Manner on Proliferation and Function of CIKs

Background: Cytokine-induced killer cells (CIKs) adoptive cell transfer (ACT) is a common 11 malignant tumor treatment method. Interleukin-2 (IL-2) is one of essential cytokines for CIKs 12 culture. In most studies, only the effect of IL-2 concentration on function of CIKs was studied, but 13 the difference between multifarious adding manner of IL-2 was not explored. 14 Methods: This study established a novel sequential adding manner of IL-2. Different concentration 15 of IL-2 was added in different CIKs induction phase. Then, proliferation ability of CIKs was 16 evaluated using cell proliferation curves, immune phenotype was analyzed by flow cytometry 17 (FCM), IFN- γ secretion ability and cytotoxicity were detected by ELISA Kit and CCK-8 Kit 18 respectively. Multiple comparison tests were conducted between each group to compare function of 19 CIKs in 12 experimental groups. 20 Results: As the IL-2 concentration increased, the number of CIKs continued to increase in each 21 group, but its function was not positively related with its number: CD3+ CD56+ subpopulation ratio, 22 INF- γ secretion ability and cytotoxicity presented irregular changes. During quiescent phase and 23 logarithmic growth phase, adding 300 and 1000 U/mL IL-2 respectively could obtain amounts of 24 powerful CIKs (CD3+ CD56+ subpopulation ratio: 40.9%, INF- γ secretion ability : 542pg/mL, 25 cytotoxicity: 40:1, 74.22). 26 Conclusions: Different concentration of IL-2 had a greater influence on biological function of CIKs 27 in different growth phase, it was better to add IL-2 sequentially in quiescent phase and exponential 28 growth phase of CIKs.


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Interleukin 2 (IL-2) is one of immune system signaling molecule, and initially named T cell 43 growth factor (TCGF) [8,9]. IL-2 plays an important role in promoting T cell proliferation and 4 function [10], also it was essential cytokines for CIKs growth. IL-2R on the surface of activated NK 45 cells and T cells is the receptor of IL-2, formed by up to three subunits (α, β, and γ). The 46 high-affinity interaction of IL-2 and IL-2R promote the activation of cell main signaling pathways 47 and play a vital role in promoting the proliferation and function of NK cells [10][11][12]. Therefore, 48 concentration and adding manner of IL-2 directly affect the quality of CIKs, and then affect its 49 clinical efficacy. At present, in the cultivation of CIKs, IL-2 concentration and adding manner had 50 no uniform standard. In this study, we performed a novel sequential addition: to add IL-2 with 51 different concentration in different CIKs growth phase (quiescent phase and exponential growth 52 phase). There are two primary aims: (1) to explore relationship between concentration, adding 53 manner of IL-2 and CIKs function; (2) to establish a new culture system in order to obtain CIKs 54 with the best proliferation capacity, activity and cytotoxicity.

CIKs induction 57
CIKs ACT was approved by the Institutional Review Board of Lanzhou University Second Hospital.

Proliferation of CIKs 67
Cell morphology was observed regularly, and the survival rate of CIKs was calculated by 68 trypan-blue exclusion. Then, cell proliferation curves were drawn based on cell counting 69 (proliferation fold = number of cells after proliferation / number of cells before proliferation).

IFN-γ secretion ability of CIKs 76
The expression levels of INF-γ were determined by ELISA (Proteintech): CIKs at day 16 from each 77 group were harvested (1×10^6 CFU/mL). 100μL of sample was added into each well coated with 78 antibody, and the plate was incubated at 37℃ for 120 min; by washing 4 times, 100μL of detection 79 antibody-biotinylated was added into each well, then the plate was incubated at 37℃ for 60min; 80 after washing, streptavidin-HRP solution (100μL per well) was added followed by incubation for 81 30mins; washing again, TMB substrate was added, and plates was sited in dark room for 15mins; 82 stop solution was added and optical density (OD) value was detected at 450nm.

Proliferation of CIKs 100
The viability of CIKs in each group was greater than 95%. In all groups, proliferation ability of 101 CIKs enhanced with the concentration of IL-2 increased; high concentration IL-2 groups showed a 102 better growth tendency, and its proliferation fold could reach 157.54 in day16 (Fig.1, Table 1).

Cytotoxicity of CIKs 115
In each group (Fig. 4), the lowest cytotoxicity appeared at E:T=10:1; after that, cytotoxicity 116 increased as E:T raised, and it reached the highest at 40:1, but gradually decreased at 80:1 and 160:1.

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On the other hand, with a constant E:T (Fig. 5), group A3 showed a better cytotoxicity, then we 125 compared group A3 with the others: (1) when E:T=10:1, there was a high significant difference 126 between A3 and other groups (P<0.01); (2) at 20:1, no difference was found between group A3 and 127 A1, A2, but high significant difference with the others (P<0.01); (3) at 40:1 and 80:1, no significant 128 difference was found between the group A3 and A2, and high significant differences with the others 129 (P<0.01); (4) at 160:1, no significant difference was found between group A3 and A1, A2, A4, 130 however high significant differences with the others (P<0.01).

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In this study, we added IL-2 sequentially during CIKs culture, our research demonstrated hat:

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(1) IL-2 concentration was proportional to CIKs proliferation capacity, however, these CIKs did not    Fig.4 The cytotoxicity of CIKs in different groups 293 6. Fig.5   P>0.05 was considered to be not a significant difference; : Dunnett's multiple comparisons test was performed between group A3 and other groups.  The cytotoxicity of CIKs in different groups Figure 5 The cytotoxicity of CIKs at different effector-target ratio *: P<0.05 was considered as signi cant difference; **: P<0.01 was considered as high signi cant difference; ns: P>0.05 was considered to be not a signi cant difference; : Dunnett's multiple comparisons test was performed between group A3 and other groups.