T Cells Control Chemokine Secretion by Keratinocytes

By co-culturing murine keratinocytes with splenic T cells we show here that T cells affect the synthesis and secretion of chemokines by keratinocytes. Overall design: Primary murine keratinocytes were isolated from tails of adult mice. Upon preparation of skin, skin pieces were incubated O/N in dispase solution at 4oC followed by a short digestion in trypsin and incubation for 6 h in S-MEM medium containing 1.3 mM Ca++. After 6 h cells were replenished with fresh medium with keratinocyte SFM medium containing 0.06 mM Ca++ for further culture. All cell splittings were performed with TrypsinLE. Splenic T cells were isolated using mouse CD8 microbeads kit and cultured in X-VIVOTM 15 medium. Cells were pre-activated by CD3/CD28 (5 g CD3 and 2µg CD28) for 1 d. CD8+ T cells were co-cultivated with keratinocytes for 1h. The T cells were washed off from the keratinocytes. Both types of cells were separatedly collected, and the RNA sequenced. 2 parallel experiments.