Effective induction of naive and recall T-cell responses by targeting antigen to human dendritic cells via a humanized anti-DC-SIGN antibody.

Current dendritic cell (DC)-based vaccines are based on ex vivo-generated autologous DCs loaded with antigen prior to readministration into patients. A more direct and less laborious strategy is to target antigens to DCs in vivo via specific surface receptors. Therefore, we developed a humanized antibody, hD1V1G2/G4 (hD1), directed against the C-type lectin DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) to explore its capacity to serve as a target receptor for vaccination purposes. hD1 was cross-linked to a model antigen, keyhole limpet hemocyanin (KLH). We observed that the chimeric antibody-protein complex (hD1-KLH) bound specifically to DC-SIGN and was rapidly internalized and translocated to the lysosomal compartment. To determine the targeting efficiency of hD1-KLH, monocyte-derived DCs and peripheral blood lymphocytes (PBLs) were obtained from patients who had previously been vaccinated with KLH-pulsed DCs. Autologous DCs pulsed with hD1-KLH induced proliferation of patient PBLs at a 100-fold lower concentration than KLH-pulsed DCs. In addition, hD1-KLH-targeted DCs induced proliferation of naive T cells recognizing KLH epitopes in the context of major histocompatibility complex (MHC) classes I and II. We conclude that antibody-mediated targeting of antigen to DCs via DC-SIGN effectively induces antigen-specific naive as well as recall T-cell responses. This identifies DC-SIGN as a promising target molecule for DC-based vaccination strategies.

Proinflammatory cytokine expression in the endolymphatic sac during inner ear inflammation.

The inner ear is capable of rapidly mounting an immune response that can ultimately lead to cochlear degeneration and permanent hearing loss. The role of the endolymphatic sac in this immune process is not clear. In order to investigate the cytokine expression of cells within the endolymphatic sac, a secondary inner ear immune response to keyhole limpet hemocyanin (KLH) was created in mice. The animals were sacrificed 3-48 h and 7 days following initiation of the immune response. The cochleas and endolymphatic sacs were assayed by immunocytochemistry for IL-1beta, TNFalpha, and IL-6. Three hours after KLH challenge of the scala tympani, the perisaccular tissue of the endolymphatic sac contained more inflammatory cells than the scala tympani or endolymphatic sac lumen. Only a few of these cells, however, expressed the proinflammatory cytokines IL-1beta and TNFalpha between 3 and 12 h after KLH injection. On the other hand, TNFalpha, which plays an important role in the cochlear secondary immune response, was expressed in cells in the endolymphatic sac lumen. The maximum percentage of cells expressing TNFalpha was seen later than in the scala tympani. Animals treated with systemic injection of the TNF blocker, etanercept, showed a reduction in the number of cells in the endolymphatic sac lumen. It is concluded that the cells in the endolymphatic sac lumen contribute to the amplification of the adaptive immune response by expressing TNFalpha, while the infiltration of cells into the perisaccular connective tissue is part of the nonspecific, innate, cochlear immune response.

Connection between the inner ear and the lymphatic system.

OBJECTIVE: The aim of this study was to identify the lymphatic drainage of the inner ear in guinea pigs. STUDY DESIGN: Prospective study. METHODS: The prospective study was performed in guinea pigs by injection of keyhole limpet hemocyanin (KLH) into either the right-side scala tympani or the middle ear cavity. The left side was not injected and served as a control. Fifteen minutes after injection, the animals were killed by intracardiac perfusion with paraformaldehyde and tissue specimens (right and left temporal bones, cervical lymph nodes, and the spleen) were collected. The presence of KLH in each specimen was determined by immunohistochemical assay of frozen sections using polyclonal mouse anti-KLH antibodies. RESULTS: After injection into the middle ear, labeled cells were identified in the parotid, superficial ventral, mandibular, and deep cranial cervical lymph nodes. However, after inner ear injections KLH was present in only the parotid and superficial ventral cervical nodes. The spleen contained KLH-positive cells following injection into either the middle or inner ear, but not all animals contained labeled spleen cells. CONCLUSIONS: The inner ear has a connection to the lymphatic drainage system. Because fewer lymph nodes contained labeled cells after inner ear injection than after middle ear injection, it is concluded that the inner ear does not simply drain to the middle ear and subsequently to the lymph nodes but seems likely to have its own connections.

Alveolar macrophages, surfactant lipids, and surfactant protein B regulate the induction of immune responses via the airways.

The influences of alveolar macrophages (AM) and pulmonary surfactant on the induction of immune responses via the airways were assessed. Mice were depleted of their AM by intratracheal instillation of multilamellar vesicles containing dichloromethylene-diphosphonate followed by intratracheal instillation of a T cell--dependent antigen, trinitrophenyl--keyhole limpet hemocyanin, in vesicles of various compositions. The primary immune response was determined in the spleen of these animals using an ELI-Spot assay. The secondary immune responses in the sera of the mice were assessed using enzyme-linked immunosorbent assays. An immune response was detected in animals depleted of their AM and intratracheally instilled with antigen in small unilamellar vesicles consisting of either phosphatidylcholine cholesterol or surfactant lipids. Incorporation of surfactant protein (SP)-B in the antigen vesicles enhanced the immune response, whereas SP-A or SP-C in the antigen vesicle did not have an effect. Strikingly, intratracheal instillation of SP-B containing antigen vesicles can induce an immunoglobulin M immune response in mice without depletion of AM. These results indicate that SP-B containing vesicles can enhance the induction of immune responses via the airways and further illustrate the important roles of both AM and pulmonary surfactant in the pulmonary immune system.

Factors affecting antigen uptake by human intestinal epithelial cell lines.

We assessed the role of size, solubility, and prophagocytic cytokines interferon-gamma (IFN-gamma), and granulocyte-macrophage colony stimulatory factor (GM-CSF) in antigen uptake and kinetics by intestinal epithelial cells using keyhole limpet hemocyanin and ovalbumin. Both fluoresceinated keyhole limpet hemocyanin (3000-7500 kDa) and fluoresceinated ovalbumin (45 kDa) were internalized by human colonic epithelial cell lines, with kinetics similar to those of fluoresceinated tetanus toxoid, and there was decreased uptake of insoluble immune complexes and no enhancement in the uptake of soluble immune complexes. In addition, neither IFN-gamma nor GM-CSF altered the kinetics of uptake nor enhanced antigen internalization by the intestinal epithelial cell lines. These data suggest that regardless of the size of the soluble antigen, the presence of prophagocytic cytokines, or the formation of soluble immune complexes, fluid phase endocytosis of antigen by intestinal epithelial cells appears to be a relatively stable process.

Glutathione and glutathione S-transferase in the rainbow trout olfactory mucosa during retrograde degeneration and regeneration of the olfactory nerve.

In the peripheral olfactory organ, continual olfactory receptor neuron (ORN) turnover exposes neighboring cells to potentially damaging cellular debris such as free radicals. These, in turn, may be inactivated by binding directly onto glutathione (GSH) or by enzymatic conjugation with glutathione S-transferase (GST). In this study, we have investigated GSH and GST during retrograde degeneration and regeneration of the olfactory nerve in rainbow trout. In these fish, prolonged ORN physiological activity and structural integrity following transection of the olfactory nerve may be mediated by GSH and GST. In the olfactory mucosa, early changes following nerve lesion and prior to ORN degeneration included a shift of intense GSH labeling from the dendrites and perikarya of a subpopulation of ORN, and from melanophores, to olfactory nerve fascicles. GSH levels were unchanged, but GST activity decreased by 33% and GST-immunoreactivity (GST-IR) in nerve fascicles diminished slightly. When the process of massive degeneration terminated and ORN were largely absent, GSH levels and GST activity decreased further, GSH labeling was confined to melanophores, and GST-IR was absent. As ORN repopulated the olfactory mucosa, GST-IR was widespread. The combination of increased GST activity (92% of preoperative values) and low GSH levels suggests GSH utilization for GST conjugation reactions. These changes imply that GSH provides protection from cellular debris associated with ORN degeneration. Recovery of GST activity and widespread GST-IR during regeneration indicates modulation of neuroprotective, developmental, and/or physiological processes by GST.

Relative frequencies of secondary B cells activated by cognate vs. other mechanisms.

Three distinct mechanisms for the activation of secondary B cells to antibody-forming cells have been examined in splenic fragment cultures. The clonal response to a protein antigen [cytochrome c (cyt)] was quantified in terms of both the number of B cells activated and the amount of antibody produced by each clone. The vast majority of memory B cells required surface immunoglobulin (sIg) receptor-mediated uptake of antigen followed by cognate interactions with an antigen-specific T helper cell. Nevertheless, a significant number (as many as 12%) could be activated via soluble factor-mediated bystander activation, involving occupation of neither the sIg receptor nor class II major histocompatibility complex (MHC) molecules. This pathway, however, was relatively inefficient in that individual clones secreted less than half as much antibody as clones activated as a result of cognate collaboration with a T helper cell. A similar number of secondary B cells were activated following nonspecific uptake of high concentrations of antigen for which splenic fragment T cells had been primed [i.e., hemocyanin (Hy)], independent of sIg receptor occupancy. Antibody levels were similar to those in cultures where B cells were activated in a cognate manner with sIg receptor occupancy. When Hy-stimulated fragment cultures were supplemented with polymerized cyt, the frequency of activation via this latter pathway increased fourfold, despite the fact that no cyt-primed T cells were present. This observation supports the idea that receptor-mediated uptake of antigen serves not just to focus antigen but also provides an important signal in activating B cells. Bystander B cell activation, however, was not enhanced by providing a sIg cross-linking signal with polymerized cyt. The lower level of antibody production by B cells activated in a bystander fashion and the inability to enhance their frequency of activation with sIg receptor occupancy suggest that there is indeed a fundamental difference between soluble factor-mediated bystander activation and activation via T cell determinant-mediated cognate T helper-B cell interactions, perhaps involving signaling through class II MHC molecules.