Dysregulation of Interleukin (IL)-18 Gene Expression and Protein Production in Basal and Activated Cord Blood (CB) vs. Peripheral Blood (PB) Mononuclear Cells and Differential Effects of IL-18 in CB vs. PB T and NK Cell Immunity.

IL-12 and IL-15 are critically important cytokines that regulate the activation of T-cell lymphocyte and NK cell subsets. We have previously demonstrated reduced IL-12 & IL-15 gene and protein expression between activated CB vs. PB mononuclear cells (MNC) (Lee/Cairo, Blood; 1996 & Qian/Cairo, Blood; 1997). IL-18 has been shown to enhance T-cell cytolytic activity & provide antitumor immunity (Osaki et al, J Immunol 1998). In the present study, we compared IL-18 gene expression, protein production and IL-18 mRNA transcript half-life in CB vs. APB MNC and the effects of IL-18 on IFN-γ protein production from CB vs. APB MNC and ex-vivo expansion and activation of fresh CB MNC upon stimulation with IL-12 + IL-2+anti-CD3 ±IL-18. MNC were isolated from CB and APB by Ficoll gradient centrifugation and cultured in RPMI for 2 hours before the addition of staphylococcus aureus enterotoxin (SEB). 24 hours after SEB, the supernatant was measured for IL-18 by ELISA. Furthermore, basal and activated MNC IL-18 mRNA expression was measured by qRT-PCR in APB & CB. APB & CB IL-18 mRNA Actinomycin D half-life studies were performed as previously described (Lee/Cairo, Blood, 1996). IFN-γ production from IL-18 ± IL-12 activated CB vs. APB MNC was measured by ELISA at 48 hours. For ex-vivo expansion, non-adherent CB MNC were cultured for 48 hours with either AIM-V media or AIM-V + IL-2 (5ng/mL) + IL-12 (10ng/mL) + anti-CD3 (50ng/mL) ± escalating doses of IL-18 (1, 10, or 100ng/mL). We demonstrated that the constitutive levels of IL-18 secreted by APB MNC were significantly higher than CB MNC (16.09±6.09 vs. <10 pg/ml, not detectable, p<0.05). SEB dramatically increased IL-18 levels in APB > CB MNC (20hrs) (628.41±34.08 vs. 358.23±9.82 pg/ml, p<0.05). IL-18 mRNA and protein levels were significantly lower in CB compared to APB MNC (2hrs) (0.329 ± 0.131 vs.1.482 ± 0.505, p<0.05). IL-18 mRNA half life was significantly shorter in CB vs. APB MNC (3.281±0.222 vs. 4.967±0.411 min, p<0.05). IL-18 independently or synergistically with IL-12 induced IFN-γ production from both APB and CB MNC (313.67±83.18 vs. 50.00±6.66 pg/ml, p<0.05). Lymphocyte subset expansion of CD8⁺/25^+, CD4⁺/25⁺ and CD16⁺/56⁺ was significantly increased with IL-12 +IL-2+anti-CD3 and IL-18(10ng/ml) compared to media alone (CD8⁺/25⁺: 31±4.9 vs. 0.25±0.8%, p<0.001; CD4⁺/25⁺: 61±9 vs. 2.6±0.7%, p<0.001; CD3⁻/CD16⁺/56⁺: 58±11.2 vs. 16.5±5.5%, p<0.001, respectively). Furthermore, there was also significant increase in CD16⁺/56⁺ subset cultured in IL-2, IL-12, anti-CD3 vs. IL-2, IL-12, anti-CD3 and IL-18 at 1, 10 and 100ng/ml (16.07±0.87 vs.39.38±9.92 vs.58.27±11.17 vs.66.71±6.25%, p<0.001). There was also significant increase in NK cytotoxicity with IL-2, IL-12, anti-CD3 vs. IL-2, IL-12, anti-CD3 and IL-18 (10ng/ml) (43.1±3.2 vs. 91.5±2.42; p<0.01). These results suggest a significant decrease in IL-18 mRNA expression and protein production in activated CB vs. APB MNC, which was in part secondary to increased degradation of CB IL-18 mRNA. IL-18 in combination with IL-12, IL-2 and anti-CD3 showed enhanced CB NK expansion and cytotoxicity. These results may have important implications in immune reconstitution and graft versus tumor activity following UCBT.