I used culture of PBMC supernatant to detect drug-restricted cytotoxic protein of granzyme B. The samples were from PBMC of (1) patients of drug remission, (2) patients of drug tolerant, and (3) healthy donor.

The scheme of culture was as follows: Briefly, PBMC was isolated using Ficoll and final conc. of cell was set up of 5x105 cells/well in 24-well plate for total volume of 1ml. IL-2 as growth factor added along with stimulant (in day 0); the cultures incubated for 24 to 144 hours. Group of culture divided into two for every sample, control (no stimulant) and treatment.

I noticed that conc. of cytotoxic proteins in control of the three groups of sample unexpectedly increased by time. Likewise, the conc. form (2) was highest in both group of culture followed by (1) and (3).

Given PBMC response of the drug is HLA- and CDR3-restricted, why did conc. in patients of drug tolerant, and healthy donor increase over time? Any thoughts are welcome and are very appreciated.

Thanks!