Dendritic epidermal gamma/delta T cells (DETC) activated in vivo proliferate in vitro in response to Mycobacterium leprae antigens

Background gamma/delta T-cell receptor (TCR)+ dendritic epidermal T cells ( DETC) are part of a primitive defense system in the skin; they are capable of responding only to a limited number of antigens. The aim of the present study was to test whether DETC can proliferate in vitro in response to anti gens of Mycobacterium leprae. Methods DETC were obtained from CBA mouse ear skin by trypsinization and Hi stopaque gradient centrifugation. The resulting epidermal cell suspension c ontained up to 20% DETC, as analyzed by the fluorescence activated cell sor ter (FACS) after staining with anti-Thy-1 or anti-gamma/delta TCR monoclona l antibodies (mAbs). The freshly isolated cells, or DETC cultured up to 4 w eeks with interleukin-2 (IL-2), were exposed in vitro for up to 6 days to v arying doses of the following M. leprae antigens: (1) integral (live) M. le prae bacilli; (2) Dharmendra antigen; and (3) PGL-1 (phenolic glycolipid of M. leprae). The DETC response was assessed by tritiated thymidine (H-3-TdR ) incorporation. Results The freshly isolated DETC, or DETC cultured up to 4 weeks with IL-2 , did not respond significantly to any of the M. leprae antigens, although at the same time they were able to respond vigorously to concanavalin A (Co n A), as positive control. If, however, DETC were isolated from skin, paint ed 7 days before with croton oil (10 mu L/cm(2) to cause irritant dermatiti s, they were able to respond to all M. leprae antigens by a 3-4-fold incras e in the H-3-TdR uptake. The most effective stimulator was a 1 : 1 mixture of Dharmendra and PGL-1 (0.01 mu g/mL), which was as effective as in-fold h igher doses of either antigen alone. Cell counts confirmed that increased D NA synthesis was associated with cell proliferation. Experiments employing alpha/beta-TCR CBA murine spleen cells and epidermal cell suspension treate d with anti-gamma/delta or anti alpha/beta mAbs+C' proved that only the gam ma/delta DETC were the responder cells to M. leprae antigens. Conclusions The results suggest that activation of DETC in vivo may make th em responsive to M. leprae antigens. A significant increase in the number o f class II major histocompatibility complex (MHC) positive, nondendritic ce lls was observed in the croton oil-treated epidermis. We hypothesize that c roson oil-induced upregulation of class II MHC expression, which endows epi dermal cells with antigen-presenting capabilities, might be an important fa ctor in vivo in delivering an immunogenic signal to resident DETC in the sk in.