The tissue sections were blocked and permeabilized with blocking solution containing rabbit serum and 0

The tissue sections were blocked and permeabilized with blocking solution containing rabbit serum and 0.1% Triton X-100. RAG2, AID, or UNG) required for generating antibody diversity and, therefore, cannot make antibodies. Additionally, transgenic mice that express a reporter fluorescent protein under the control of Ig1 constant region fail to express transcripts in DRG sensory NPB neurons. Furthermore, neural sequestration of antibodies occurs in mice rendered deficient in neuronal knock out (KO) or MT mice]. Finally, adoptive transfer of KO mice revealed that antibody sequestration was observed in DRG sensory neurons of chimeric mice with WT BM but not with afferent lymphatic channels, and the trafficking into lymph nodes of intracellular antigen in antigen-presenting cells (APCs). Significant changes in lymph node architecture, inhibition NPB of lymphocyte egress, and expansion of the lymph node stroma occur following antigen-mediated activation of intranodal immune responses. We recently discovered that neural signals provide an essential mechanism in lymph nodes to retain antigen and lymphocytes (2). Lymph nodes are innervated by both sensory (afferent) and motor (efferent) neurons, with abundant innervation of the APCs in the T-cell zone, subsinoidal layer, and cortical extrafollicular zones (3, 4). The neuronal density within the lymph nodes is dynamic, expanding significantly following antigenic stimulation of an early lymphocyte response (5, 6). In na?ve mice exposed to the antigen keyhole limpet hemocyanin (KLH) in the hind paw for the first time, we observed that KLH flowed rapidly from the popliteal lymph node (adjacent to the injection site) to the sciatic lymph node by traveling up the lymphatic chain. Surprisingly, prior exposure to KLH significantly impaired the flow of KLH from the popliteal to the sciatic lymph node during subsequent antigen exposure. This restriction of antigen flow from distal to proximal lymph nodes was antigen-specific and required functioning sensory neural signals, because inhibiting neural signals to the lymph node region restored antigen flow in immunized animals (2). The neural mechanism NPB that restricted antigen flow is mediated by a NES subset of NaV1.8+ sensory neurons, which include nociceptors, because antigen failed to accumulate in the distal lymph node of animals rendered deficient in NaV1.8+ neurons (2). Early work had previously established that immune complexes can interact Fc receptors expressed on sensory neurons to induce intracellular signaling mechanisms NPB that require transient receptor potential canonical 3 (TRPC3) channel and the Syk-PLC-IP3 pathway (7, 8) NPB that mediates the release of calcitonin gene-related peptide (CGRP) and substance P (9). Immunohistochemistry using labeled antigen revealed colocalization of labeled antigen and FcRI receptors in neurons at the site of antigen injection in the paw (2). Thus together, these studies implicate sensory neurons in regulating antigen trafficking during immunization through a pathway that requires NaV1.8 and FcR. As we continued to explore the role of sensory neurons in immunization, we were very surprised to observe that sensory neurons obtained from the dorsal root ganglia (DRGs) of immunized animals contained abundant levels of antigen-specific antibodies. While others have reported the localization of antibodies to neurons in brain, to our knowledge, the localization of antibodies to sensory neurons in peripheral tissues has not been previously described. expression has been identified in brain neurons, but convincing evidence that neurons can synthesize antigen-specific antibodies is lacking. Accordingly, here we used molecular genetic analyses, and transgenic and chimeric mice to show for the.