Mixed lymphocyte culture (MLC) response was measured with the use of Ki-67 monoclonal antibody and responding cells were verified by CD3 and CD56 surface markers staining. Stimulator cells were discriminated from responder cells on the basis of forward and side scatter. Allogeneic, but not autologous MLC had Ki-67+ responder cells in lymphocyte gate at the end of the culture. In allogeneic MLC T cells and natural killer (NK) cells were in a similar proportion Ki-67+ (mean ± SD: 59.25% ± 9.72% versus 61.75% ± 13.2%). Ki-67+ NK cells had higher CD56 mean fluorescence intensity than those lacking Ki-67 (745 ± 357 versus 196 ± 56p < 0.0001). NK cells contribution to responding lymphocytes was positively correlated with the percentage of Ki67+ cells in NK population by the end of the culture (r = 0.74, p = 0.002). NK cells response in MLC increased upon supplementation of the culture medium with human recombintant interleukin-2 (IL-2). Responder cells from single individual were tested with 8 Bw4+ and 8 Bw4- as well as with 9 CNK1+ and 9 CNK1- stimulators. In IL-2 supplemented MLC killer inhibitory receptor expressing cells expanded when ligands for this receptor were absent in stimulating population. Consequently, stimulator cells lacking Bw4 promoted NKB1 + cells expansion (7.2% ± 3% versus 3.6% ± 1%, p = 0.0031), whereas HLA-C NK1 negative stimulators promoted CD158a+ cells expansion (9.6% ± 4.8% versus 6% ± 2.6%, p = 0.0385).
Bibliographical noteFunding Information:
The Polish State Scientific Committee Grant No 4P05B07719 supported this work.
- Cellular immunology
- Killer inhibitory receptors
- Mixed lymphocyte culture
- NK cells