Background: Costimulatory blockade with anti-CD40L mAb plus donor specific splenocyte transfusion (DST) induces alloantigen specific tolerance. Tolerance depends on proper cell interactions and signaling in the lymph node (LN). Lymphotoxins (LTs) are TNF family cytokines important for LN organization. LTs are expressed on activated lymphocytes and interact with the receptors LTΒR, HVEM, and TNFR on various cells such as DC, monocytes and stromal cells. Fibroblastic reticular cells (FRC), a LN stromal cell type, determine LN structure, the integrity of which depends on LTΒR signaling. We hypothesized that LTΒR signaling regulates the function of FRC required for proper LN structure and tolerance.

Methods: Vascularized cardiac transplants were performed from BALB/c donors to C57BL/6 recipients. Mice were tolerized by administration of DST on day -7 and anti-CD40L mAb on days -7, -4, 0 and +4. Mice were treated with control IgG or blocking LTΒRIg. LNs were analyzed by immunohistochemistry (IHC). FRC were flow sorted and analyzed by flow cytometry and qRT-PCR. NaÏve FRC were flow sorted and cultured with CD4+ T cells, which were activated with anti-CD3 and anti-CD28, along with control IgG or LTΒRIg in vitro.

Results: FRC responded to tolerogen rapidly in vivo, and within 6 to 12 hours expressed suppressive molecules such as PD-L1 and IDO. LTΒRIg reversed these responses and inhibited iNOS expression in the LN. In vitro LTΒRIg inhibited FRC PD-L1 expression. LTΒRIg also induced higher mRNA expression of the inflammatory chemokines, CXCL1, CXCL2, CXCL12, CCL5, CCL21, and IL-6 in FRC up to 5 days after LTΒRIg treatment, when compared to controls. Expression of these inflammatory molecules was commensurate with fibrosis and inflammation in allografts caused by LTΒRIg treatment.

Conclusions: FRC undergo acute changes after tolerance induction, so that the tolerogenic environment is rapidly established, and LTΑΒ-LTΒR interactions are required for tolerance. Blocking LTΒR signaling during tolerance induction down regulated FRC immune regulatory molecules, and increased expression of FRC inflammatory chemokines and cytokines. These observations show that LTΒR signaling regulates the communication between lymphocytes and FRC, which is required for tolerance by rapidly altering expression of inflammatory and immune regulatory molecules. Thus, blocking LTΒR signaling during tolerance induction channeled FRC responses towards an inflammatory and away from a tolerogenic phenotype.

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