IMMUNE REGULATION AND TOLERANCE II

T.L. Sumpter¹, H. Turnquist², A.W. Thomson^3
1Surgery, Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh/PA/UNITED STATES OF AMERICA, ²Surgery, Thomas Starzl Transplantation Institute, Pittsburgh/PA/UNITED STATES OF AMERICA, ³, Thomas Starzl Transplantation Institute, Pittsburgh/PA/UNITED STATES OF AMERICA

Body: Introduction: The inherent tolerogenic properties of the liver are thought to involve organ-specific mechanisms for controlling maturation of APC, particularly dendritic cells (DC). Hepatic DC have reduced allostimulatory capacity compared to their counterparts in primary lymphoid organs such as the spleen. Furthermore, hepatic DC are phenotypically immature, IL-10^hi-IL-12^lo and poor inducers of allogeneic T cell responses. The molecular mechanisms mediating this difference are unresolved, though the Signal Transducer Activating T Cells (STAT)-3 has been implicated in maintaining liver DC in this immature state. The balance between STAT3 and STAT1 activation in DC represents the state of cell activation, with high levels of activated STAT3 found in immature or ‘tolerogenic’ DC and high levels of activated STAT1 exhibited by mature or ‘immunogenic’ DC. Hepatic murine myeloid (m) DC express the membrane adaptor DNAX-activating protein of 12kDa (DAP12). DAP12 dampens co-stimulatory molecule and inflammatory cytokine expression in bone marrow derived-DC. We hypothesized that DAP12 achieves a similar role in regulating the maturation of hepatic DC. Methods: DAP12 function was evaluated in magnetically purified C57BL/6 hepatic DC (CD11c⁺CD11b⁺NK1.1^-B220^-) using DAP12 siRNA. Subsequent analysis was done by flow cytometry and ELISA. Results: DAP12-silenced liver DC increased CD80, CD86, B7-H1 and B7-H2 expression, but retained low levels of MHC class II, ICOSL, B7-H3 and B7-H4 compared to DC transfected with negative control siRNA. Liver DC with reduced DAP12 expression exhibited enhanced T cell stimulatory capacity in allogeneic CFSE-dilution MLR. Loss of DAP12 elevated IL-12p70, TNFa and IL-6 secretion but diminished IL-10 secretion. The loss of IL-10 suggested changes in activation of STAT3. Phospho-flow cytometry was used to evaluate STAT3 activation in liver or control, splenic DC. Phenotypic evaluation of the STAT3 positive cells revealed phospho-STAT3 only in the mature (CD11c⁺CD80⁺ or CD11c⁺CD86⁺) DC. Transfection with DAP12 siRNA reduced phospho-STAT3 in the CD80⁺ liver DC compared to control transfected liver DC. Intriguingly, STAT3 activation was unchanged in splenic DC transfected with DAP12 siRNA compared with those transfected with negative control siRNA. The level of STAT1 phosphorylation in liver DC with reduced DAP12 expression remained unchanged. Conclusions: We propose that DAP12 negatively regulates liver DC maturation by enhancing STAT3, but not STAT1 activation. These data reveal, for the first time, a tissue-specific role for DAP12 in regulation of hepatic DC maturity. Understanding DAP12 regulation and elucidation of agonist molecules upstream of DAP12 in liver DC will yield novel therapeutic targets to promote DC-based tolerance for transplant recipients.

Disclosure: All authors have declared no conflicts of interest.