ABSTRACT
Cytotoxicity assays with patient peripheral blood mononuclear cell (PBMC)-derived natural killer (NK) cells are useful in evaluating the innate immunity of patients with cancer. However, the size of the NK cell population in PBMC preparations may have significant effects on the assay outcome. Therefore, the present study examined the effect of NK cell frequency in a cytotoxicity system to investigate NK cell immunity in post-surgical colorectal cancer patients. For this, hemacytotoxicity was assessed using PBMC preparations, and lymphocyte subset populations were analyzed in samples obtained from 47 patients and 45 healthy volunteers. In addition, a new theoretical parameter, the 'NK lytic index', was termed to represent the per-cell cytotoxicity and compensate for the NK cell frequency effect during PBMC preparations. Notably, the patterns of hemacytotoxicity and NK lytic index did not coincide in follow-up studies with consecutive patients following surgical intervention. In addition, it was determined that NK cell NKG2D expression influences NK lytic index, but not hemacytotoxicity. Transforming growth factor (TGF)-ß-bound lymphocytes influenced hemacytotoxicity and NK lytic index. These findings indicate that total cell activity (hemacytotoxicity) is not a sum of per-cell activities (NK lytic indexes), suggesting that clinicians should employ NK lytic index in addition to hemacytotoxicity in order to precisely determine how to enhance NK cell immunity in patients with cancer, either focusing on recovering the number of NK cells or boosting NK cell activity in single cell levels, or both.
ABSTRACT
Flow cytometric immunophenotyping of peripheral blood lymphocyte subsets is a powerful tool for evaluating cellular immunity and monitoring immune-mediated diseases. The numbers and proportions of blood lymphocyte subsets are influenced by factors such as gender, age, ethnicity, and lifestyle. This study aimed to establish reference ranges for peripheral blood lymphocyte subsets in a healthy Korean population. Blood samples from 294 healthy adults were collected. Lymphocyte subsets were analyzed using a single-platform method with a flow cytometer; white blood cells and lymphocytes were analyzed using an automated hematology analyzer. The mean value of the white blood cell count was 5,665 cells/µl, and the mean values of the subtype counts (percentages) were as follows: lymphocytes, 1,928 cells/µl (35.08%); CD3(+) cells, 1,305 cells/µl (67.53%); CD3(+)CD4(+) cells, 787 cells/µl (40.55%); CD3(+)CD8(+) cells, 479 cells/µl (25.23%); CD3(-)CD19(+) cells, 203 cells/µl (10.43%); and CD3(-)CD56(+) cells, 300 cells/µl (15.63%). Additionally, the CD4(+)/CD8(+) ratio was 1.81. In this study, gender and age significantly influenced blood lymphocyte subsets. Our results demonstrate that, as with other populations, a healthy Korean population has its own, region-specific, lymphocyte subset reference ranges.
ABSTRACT
PURPOSE: This study aimed to assess the cytolytic activity and the phenotype of circulating blood immune cells in cancer patients by using a simple preparation of peripheral blood mononuclear cells (PBMCs). METHODS: Peripheral blood was obtained from 94 diagnosed colorectal cancer (CRC) patients and 112 healthy donors. PBMCs were cocultured with K562 cells for 2 hours and lactate dehydrogenase released from the dead K562 cells was measured by using a spectrophotometer. Meanwhile, PBMCs were stained with fluorescence conjugated monoclonal antibodies (mAbs) and analyzed by flow cytometry. RESULTS: The cytolytic activity of PBMCs were significantly different between CRC patient and healthy groups (8.82% ± 3.84% vs. 17.51% ± 8.57%; P < 0.001). However, no significant difference in the cytolytic activity was observed after surgery in the CRC patient group (before surgery, 8.82% ± 3.84% vs. after surgery, 9.95% ± 4.94%; P = 0.326). In addition, the percentage of peripheral blood natural killer cells was significantly higher in the preoperative patient group than in the healthy group (19.97% ± 11.51% vs. 15.60% ± 5.77%, P = 0.041). In contrast, the percentage of peripheral blood lymphocytes was lower in the preoperative patient group than in the healthy group (28.41% ± 8.31% vs. 36.4% ± 8.6%, P < 0.001). CONCLUSION: These results demonstrate that circulating blood immune cells of CRC patients are functionally impaired and undergo an immunophenotypic perturbation, and show that a simple preparation of PBMCs can be useful to evaluate cellular immunity in cancer.
