Epicutaneous Immunization with Protein Antigen in the Presence of TLR4 Ligand Induces TCRαβ+CD4+ T Contrasuppressor Cells That Reverse Skin-Induced Suppression of Th1-Mediated Contact Sensitivity

Abstract

Our previous work showed that epicutaneous (EC) immunization of mice with different protein Ags applied on the skin in the form of a patch induces a state of subsequent Ag-nonspecific unresponsiveness due to suppressor CD4+8+ T cells (Ts) that inhibit Th1-mediated contact sensitivity (CS) reactions via released TGF-β. In the present work we show that EC immunization with Ag together with the TLR4 ligand LPS induced cells that could prevent suppression by the Ag-nonspecific Ts. These up-regulatory cells, called contrasuppressor T cells (Tcs), belong to a population of Ag-specific TCRαβ CD4+ lymphocytes and are different from Th1 CD4+ cells that mediate the CS reaction. Experiments using knockout mice showed that EC induced contrasuppression is MyD88, INF-γ, and IL-12 dependent, whereas IL-6 is not involved in this phenomenon. Additional experiments with anti-IFN-γ mAb showed that IFN-γ is required for induction of Tcs cells but does not play a crucial role in the effector phase of contrasuppression. Additionally, treatment of CS effector cells with rIL-12 makes them resistant to EC induced suppression without affecting Ts cells, whereas IL-12 neutralization in vitro abrogates contrasuppression. These data show that IL-12 is indeed involved in the effector phase of EC induced contrasuppression and that this cytokine does not act directly on Ts cells. The mechanism of action of Tcs protects Th1 effector cells mediating CS from the nonspecific Ts, leaving suppression to other Ags intact. Ts and Tcs cells do not influence each other and can be induced simultaneously in the same animal.

Partial depletion of natural gut flora by antibiotic aggravates collagen induced arthritis (CIA) in mice

“…Cells were distributed in triplicate wells in flat 24 well Falcon plates. After 48 h culture supernatants were collected and then tested for cytokine concentration [21,25].…”

Epicutaneous immunization with protein antigen TNP-Ig alleviates TNBS-induced colitis in mice

“…Cells were distributed in triplicate wells in flat 24 well Falcon plates. After 48 h, culture supernatants were collected and then tested for cytokine concentration [16].…”

Complementary methods for contact hypersensitivity (CHS) evaluation in mice

“…Five days later inguinal and axillary lymph nodes were isolated and 3 × 10 5 LNC were cultured in 96-U bottom microtiter plates in triplicate in the presence of three fold dilutions of DNP-BSA in 200 μl of RPMI 1640 containing 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 25 mM Hepes, 5×10 −5 M 2-mercaptoethanol and 10% fetal calf serum for 48 h prior to the addition of [ 3 H]-thymidine for an additional 18 h. [ 3 H]-thymidine incorporation was determined by β liquid scintillation counting (Ptak et al, 2009). Results are presented as mean±SE.…”