A limitation of CB as a source for ACI is the limited number of immunoeffector T and NK cell subsets. IL-15 is an important lymphokine regulating NK activity, T-cell proliferation and CTL activities. We have previously demonstrated reduced IL-15 mRNA expression and protein production from CB which may in part contribute to the immaturity of CB cellular immunity (Qian/Cairo et al, Blood 1997). NK cells are characterized by NK receptors which exist in both activatory and inhibitory forms (Moretta et al, Ann Rev Immun 2001). We have previously demonstrated the ex vivo expansion, maturation and activation of fresh CB derived T lymphocytes with anti-CD3, IL-2, IL-12 and IL-7 (Robinson/Cairo et al, Exp Hem 2001). For this study, we compared the expansion, proliferation, activation and survival of CB mononuclear cells (MNC) with IL-2, anti-CD3, FLT-3 ± IL-15. CB MNC were isolated by Ficoll density centrifugation and incubated overnight in AIM-V media. The non-adherent MNC fraction was cultured in AIM-V media alone and with IL-2 (5ng/ml), anti-CD3 (50ng/ml), FLT-3 (50ng/ml) and IL-15 (10 &100ng/ml) for 48 hrs. Cell proliferation was determined by the colorimetric assay system using WST-1. Expression of T & NK cell subsets were analyzed by three color flow cytometry using CD3, CD8, CD4, CD25, CD45RO, CD16, CD56, CD158a, CD158b, NKB1, CD94 & NKG2A monoclonal antibodies. Apoptosis markers were determined by measuring Annexin-V & PI by flow cytometry. Cytotoxicity was determined by europium release assay using K562 & Daudi target cells at 20:1 E:T ratio. Expansion of lymphocyte subsets of CD4+/25+, CD8+/25+, CD3+/45RO+ & CD94/NKG2A was significantly increased with IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) compared to IL-2, anti-CD3, FLT-3 and compared to media (CD4+/25+: 67.2±1.2 vs 39.19±5.3 vs 3.54±0.3%, p<0.01; CD8+/25+: 57.8±8.6 vs 16.94±0.3 vs 0.99±0.4%, p<0.01; CD3+/45RO+: 77.13±1.0 vs 29.96±1.9 vs 6.69±0.3%, p<0.001; CD94/NKG2A: 3.4±0.68 vs 0.29±0.092 vs 0.23±0.09%, p<0.01, respectively). We also noted a significant increase in CD3−/16+/56+ with IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) compared to media alone (p<0.01). There was no significant increase in expansion of CD3+/16+/56+, CD3−/56+/158a, CD3−/56+/158b or CD3−/56+/NKB1 in various culture conditions described above. IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) resulted in significant improvement in cell survival compared to media alone (Annexin-V staining) (3.3±0.26 vs 15.51±3.3%, p<0.05). We also noted increase in cell proliferation with IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) as compared to IL-2, anti-CD3, FLT-3 vs. media alone (1.11±0.09 vs 0.82±0.01 vs 0.69±0.03 OD, p<0.05). We also noted significant increase in cytotoxicity with IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) as compared to IL-2, anti-CD3, FLT-3 vs media alone (K562–57.81±8.1 vs 17.5±1.4 vs 2.68±0.6%, p<0.01; Daudi-79.8±1.7 vs 23.69±1.5 vs 6.38±0.6, p<0.001) The combination of IL-2 + anti-CD3 + FLT-3 + IL-15 appears to enhance expansion and cytotoxicity of CB CTL and NK subsets in short term culture in part secondary to inhibition of apoptosis. Future studies are warranted to determine if the combination of anti-CD3 + IL-2 + FLT-3 + IL-15 can enhance CB CTL and NK cells for ACI to induce GVHM effects following UCBT.
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