The classical complement pathway in transplantation: unanticipated protective effects of C1q and role in inductive antibody therapy.

Though complement (C) deposition within the transplant is associated with allograft rejection, the pathways employed have not been established. In addition, evidence suggests that C-mediated cytolysis may be necessary for the tolerance-inducing activities of mAb therapies. Hence, we assessed the role of the classical C pathway in acute allograft rejection and its requirement for experimental mAb therapies. C1q-deficient (C1q-/-) recipients rejected allografts at a faster rate than wild-type (WT) recipients. This rejection was associated with exacerbated graft pathology but not with enhanced T-cell responses in C1q-/- recipients. However, the humoral response to donor alloantigens was accelerated in C1q-/- mice, as an early IgG response and IgG deposition within the graft were observed. Furthermore, deposition of C3d, but not C4d was observed in grafts isolated from C1q-/- recipients. To assess the role of the classical C pathway in inductive mAb therapies, C1q-/- recipients were treated with anti-CD4 or anti-CD40L mAb. The protective effects of anti-CD4 mAb were reduced in C1q-/- recipients, however, this effect did not correlate with ineffective depletion of CD4+ cells. In contrast, the protective effects of anti-CD40L mAb were less compromised in C1q-/- recipients. Hence, this study reveals unanticipated roles for C1q in the rejection process.

Transforming growth factor beta-induced connective tissue growth factor and chronic allograft rejection.

Late loss of allograft function is primarily attributed to chronic rejection (CR). There are no effective treatments for CR and the underlying cause of the disease is unknown. This study compared events that occurred within cardiac allografts placed in mice that received either anti-CD4 therapy and develop CR or anti-CD40L therapy and do not develop CR. Both TGFbeta and connective tissue growth factor (CTGF), which is induced by TGFbeta, were expressed in grafts with CR but were not expressed in grafts without CR. TGFbeta transfection of allografts in anti-CD40L-treated recipients resulted in CTGF expression and CR. However, TGFbeta transfection of syngeneic grafts did not result in CTGF expression or CR. These data indicate that TGFbeta alone is insufficient to induce CR and that CTGF is required. Further, antigenic stimulation is required for TGFbeta induction of CTGF. Thus, CTGF may serve as a therapeutic target for CR.

Cutting edge: dichotomy of homing receptor dependence by mucosal effector B cells: alpha(E) versus L-selectin.

The common mucosal immune system may be compartmentalized because lymphocyte homing to the upper respiratory tract appears to be mediated by L-selectin interactions rather than alpha(4)beta(7) interactions, as is the case for gut-associated lymphoreticular tissue. To assess the role of L-selectin in effector B cell immunity, L-selectin-deficient mice were intranasally immunized with cholera toxin (CT), and mucosal immune responses were compared with C57BL/6 mice. The absence of L-selectin correlated with a reduction in CT-specific secretory-IgA responses in nasal passages and reproductive tract, but not intestinal lamina propria. Cell sorting experiments showed that an L-selectin-dependent subset was responsible for CT-specific responses in nasal passages and reproductive tract, whereas an alpha(E)beta(7)(+) B cell subset was responsible for L-selectin-independent intestinal immunity. This study provides evidence for compartmentalization of the common mucosal immune system into "intestinal" vs "nonintestinal" effector sites.

M cell-targeted DNA vaccination.

DNA immunization, although attractive, is poor for inducing mucosal immunity, thus limiting its protective value against most infectious agents. To surmount this shortcoming, we devised a method for mucosal transgene vaccination by using an M cell ligand to direct the DNA vaccine to mucosal inductive tissues and the respiratory epithelium. This ligand, reovirus protein final sigma1, when conjugated to polylysine (PL), can bind the apical surface of M cells from nasal-associated lymphoid tissues. Intranasal immunizations with protein final sigma1-PL-DNA complexes produced antigen-specific serum IgG and prolonged mucosal IgA, as well as enhanced cell-mediated immunity, made evident by elevated pulmonary cytotoxic T lymphocyte responses. Therefore, targeted transgene vaccination represents an approach for enabling DNA vaccination of the mucosa.

Gene transfer facilitated by a cellular targeting molecule, reovirus protein sigma1.

To facilitate eventual genetic vaccination of mucosal tissues, a receptor-mediated gene transfer system was devised using the reovirus adhesin, protein sigma1. Highly efficient uptake and internalization of protein sigma1 polylysine (PL) DNA complexes could be demonstrated by fluorescent microscopy. Successful cellular transfection of rodent and human cell lines was obtained with the recombinant protein sigma1 as a PL-DNA complex, and could be shown to be receptor-specific. Transfection efficiency was dependent upon the ratio of DNA complexed to protein sigma1-PL and chloroquine treatment improved transfection efficiency dramatically. To test its ability to bind a mucosal inductive tissue, recombinant protein sigma1 was specifically bound to the nasal-associated lymphoid tissues (NALT). Thus, recombinant protein sigma1-PL-DNA conjugates can efficiently bind and transfect cells that express the receptor for protein sigma1. Gene Therapy (2000) 7, 61-69.

Nasal-associated lymphoid tissue: phenotypic and functional evidence for the primary role of peripheral node addressin in naive lymphocyte adhesion to high endothelial venules in a mucosal site.

Nasal-associated lymphoid tissue (NALT), a mucosal inductive site for the upper respiratory tract, is important for the development of mucosal immunity locally and distally to intranasally introduced Ag. To more fully understand the induction of nasal mucosal immunity, we investigated the addressins that allow for lymphocyte trafficking to this tissue. To investigate the addressins responsible for naive lymphocyte binding, immunofluorescent and immunoperoxidase staining of frozen NALT sections were performed using anti-mucosal addressin cell adhesion molecule-1 (MAdCAM-1), anti-peripheral node addressin (PNAd), and anti-VCAM-1 mAbs. All NALT high endothelial venules (HEV) expressed PNAd, either associated with MAdCAM-1 or alone, whereas NALT follicular dendritic cells expressed both MAdCAM-1 and VCAM-1. These expression profiles were distinct from those of the gut mucosal inductive site, Peyer's patches (PP). The functionality of NALT HEV was determined using a Stamper-Woodruff ex vivo assay. The anti-L-selectin MEL-14 mAb blocked >90% of naive lymphocyte binding to NALT HEV, whereas the anti-MAdCAM-1 mAb, which blocks almost all naive lymphocyte binding to PP, minimally blocked binding to NALT HEV. NALT lymphocytes exhibited a unique L-selectin expression profile, differing from both PP and peripheral lymph nodes. Finally, NALT HEV were found in increased amounts in the B cell zones, unlike PP HEV. These results suggest that NALT is distinct from the intestinal PP, that initial naive lymphocyte binding to NALT HEV involves predominantly L-selectin and PNAd rather than alpha4beta7-MAdCAM-1 interactions, and that MAdCAM-1 and VCAM-1 expressed by NALT follicular dendritic cells may play an important role in lymphocyte recruitment and retention.