Background: Costimulatory blockade with anti-CD40L mAb plus donor specific splenocyte transfusion (DST) induces alloantigen specific tolerance. Previously we showed that stromal cell interactions with leukocytes, and stromal cell signaling in the lymph node (LN), were required during tolerance induction. In particular, the fibroblastic reticular cell (FRC) stromal subset was required for proper LN structure and function. Here we tested the hypothesis that FRC directly respond to DST and anti-CD40L mAb to regulate the tolerogenic LN response.

Methods: C57BL/6 mice were tolerized with DST plus anti-CD40L mAb or immunized with DST only. After 6 to 24 hours whole LNs were analyzed by immunohistochemistry (IHC); or FRC were flow sorted and analyzed by qRT-PCR. NaÏve FRC were flow sorted and activated with various stimuli (i.e. interferon gamma (IFNΓ), tumor necrosis factor alpha (TNFΑ), LPS, polyinosine-polycytidylic acid (poly I:C), and agonistic anti-CD40 mAb). Changes in gene expression of immunomodulatory molecules were analyzed by qRT-PCR.

Results: FRC responded rapidly in vivo to allogeneic DST immunization by producing inflammatory cytokines and chemokines such as IL-6, CXCL2, CXCL9, CXCL11, CCL5, and CCL21. Importantly, FRC also up regulated a basal low-level expression of CD40. FRC responded to tolerance induction with anti-CD40L mAb by producing suppressive of molecules, such as PD-L1 and IDO. An increase in PD-L1 was also detected by IHC in tolerized LN. In vitro, naÏve FRC responded directly to various stimuli by producing inflammatory or immunoregulatory molecules. CD40 was significantly up regulated by inflammatory stimuli, and FRC were directly activated by agonistic anti-CD40 mAb.

Conclusion: FRC respond rapidly to allogeneic DST stimulation by producing inflammatory molecules, which contribute to immunity and rejection. FRC respond differently to DST and anti-CD40L mAb by producing suppressive immunomodulatory molecules, which contribute to tolerance. The mechanism of the FRC response involves CD40, so that naÏve FRC express a low level of CD40, which is up regulated by allogeneic stimulation and various inflammatory stimuli, especially by the agonistic CD40 mAb. Up regulation of CD40 facilitates FRC communication with T cells via CD40-CD40L, thereby altering FRC intrinsic gene expression of immune regulatory molecules. Since blockade of this interaction induces tolerance, the identification of FRC-T cell CD40-CD40L interaction represents a new locus for tolerance induction.

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