HETEROGENEITY OF NK AND NKT LYMPHOCYTE POPULATIONS IN HEALTHY DONORS

Natural killer(NK) and  NKT lymphocytes are important components of innate immunity, and compose a first-line defense against cancer. These populations are characterized by high heterogeneity and are divided into several subpopulations, by differences in functional activity as well as CD56  and CD16  expression. Studying  heterogeneity for these  lymphocyte populations in healthy  donors  is important, due  to imbalance between  different  lymphocyte subsets in cancer patients. Changes in the ratio of these subpopulations may be of prognostic and clinical  significance in malignant diseases. The present  study was conducted with peripheral blood  lymphocytes in 50 healthy  donors. When  analysing  population of NK  lymphocytes we have identified 18.0±11.3% of antigen-positive cells, their fluctuations ranged  from 7.6 to 29.2%, whereas average number of cells with  CD3^-CD56⁺ and  CD3^-CD16⁺   phenotypes was equal  to 16,2±8.1%, and  11,0±6.7%, respectively. The  subpopulation analysis  showed  that  the  primary  pool  of NK  cells  was presented by CD56^dimCD16^dim cells  by  52.3±19.9 percent.  We  detected  minor   subpopulations,  e.g.,  CD56^dimCD16^bright,  CD56^-CD16⁺, CD56^brightCD16^- (0.3±0.2%, 1.7±0.9%, and  1.3±0.6%,  respectively). Search  for  intracellular perforin has revealed  that the number of CD56⁺Perf⁺ cells comprized 25.1±14.8%, CD16⁺Perf⁺, 23.8±16.0%. Cytometric analysis showed that perforin is found, predominantly, in CD56^dimCD16^dim NK lymphocytes, whereas the cells with CD56^dimCD16^bright, CD56-CD16⁺, CD56^brightCD16^- immunophenotypes did not produce perforin. For the first time, we have discovered a subpopulation of NK cells with the СD56dimCD16dim immunophenotype that did not contain intracellular perforin (2.0%).  The NKT cell population with СD3⁺CD16/СD56⁺  phenotype was detected in 7.1% (25% – 3.45; 75% – 8.75) antigen-positive cells, within a range of 2.5 to 11.9%. Analysis with a combination of monoclonal antibodies CD3/CD56/CD16 has shown that the number of CD3⁺ CD56⁺ cells was 4.33% (25% – 2.25; 75% – 7.3), whereas the number of CD3⁺CD16⁺ was 3.087% (25% – 0.9; 75% – 6.2). These  data  demonstrate that  the differences in results  between  the CD3/CD16/CD56, and  CD3/CD16 test systems are statistically significant  (p < 0.05). It was shown that the primary-pool NKT-cells are CD56⁺CD16^- cells, whose number is about 5.45% (25% – 2.95; 75% – 7.3) among  total CD3⁺ lymphocyte population. Two minor subpopulations were also detected which differed in expression  of CD56 and CD16 antigens. Hence, the level of CD56^-CD16⁺ cells was 3% (25% – 0.25; 75% – 3.05),  and the number of CD56⁺CD16⁺ was equal to 0.67% (25% – 0.25; 75% – 0.9). Hence, the observed wide phenotypic diversity of NK and NKT-cells reflects their  ability  to exert  various  functional activities.  This  study, showing  high  heterogeneity of NK  and  NKT lymphocytes, may serve as a basis for the study of imbalances between  different  subpopulations of these cells in cancer patients.

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