Patricia L. Mottram

Size-Dependent Immunogenicity: Therapeutic and Protective Properties of Nano-Vaccines against Tumors

Infection can protect against subsequent disease by induction of both humoral and cellular immunity, but inert protein-based vaccines are not as effective. In this study, we present a new vaccine design, with Ag covalently conjugated to solid core nano-beads of narrowly defined size (0.04–0.05 μm) that localize to dendritic cells (DEC205+ CD40+, CD86+) in draining lymph nodes, inducing high levels of IFN-γ production (CD8 T cells: precursor frequencies 1/5000 to 1/1000) and high Ab titers in mice. Conjugation of Ag to these nano-beads induced responses that were significantly higher (2- to 10-fold) than those elicited by other bead sizes, and higher than a range of currently used adjuvants (alum, QuilA, monophosphoryl lipid A). Responses were comparable to CFA/IFA immunization for Abs and ex vivo peptide-pulsed dendritic cell immunization for CD8 T cells. A single dose of Ag-conjugated beads protected mice from tumors in two different model challenges and caused rapid clearance of established tumors in mice. Thus, a range of Ags conjugated to nano-beads was effective as immunogens in both therapeutic and prophylactic scenarios.

The dendritic cell subtype-restricted C-type lectin Clec9A is a target for vaccine enhancement.

A novel dendritic cell (DC)-restricted molecule, Clec9A, was identified by gene expression profiling of mouse DC subtypes. Based on sequence similarity, a human ortholog was identified. Clec9A encodes a type II membrane protein with a single extracellular C-type lectin domain. Both the mouse Clec9A and human CLEC9A were cloned and expressed, and monoclonal antibodies (mAbs) against each were generated. Surface staining revealed that Clec9A was selective for mouse DCs and was restricted to the CD8(+) conventional DC and plasmacytoid DC subtypes. A subset of human blood DCs also expressed CLEC9A. A single injection of mice with a mAb against Clec9A, which targets antigens (Ags) to the DCs, produced a striking enhancement of antibody responses in the absence of added adjuvants or danger signals, even in mice lacking Toll-like receptor signaling pathways. Such targeting also enhanced CD4 and CD8 T-cell responses. Thus, Clec9A serves as a new marker to distinguish subtypes of both mouse and human DCs. Furthermore, targeting Ags to DCs with antibodies to Clec9A is a promising strategy to enhance the efficiency of vaccines, even in the absence of adjuvants.

FcγRI-Deficient Mice Show Multiple Alterations to Inflammatory and Immune Responses

The inactivation of the mouse high-affinity IgG Fc receptor FcgammaRI resulted in a wide range of defects in antibody Fc-dependent functions. These studies showed the primary importance of FcgammaRI in endocytosis of monomeric IgG, kinetics, and extent of phagocytosis of immune complexes, in macrophage-based ADCC, and in immune complex-dependent antigen presentation to primed T cells. In the absence of FcgammaRI, antibody responses were elevated, implying the removal of a control point by the deletion of FcgammaRI. In addition, FcR-gamma chain-deficient mice were found to express partially functional FcgammaRI. Thus, FcgammaRI is an early participant in Fc-dependent cell activation and in the development of immune responses.

Increased expression of IL-4 and IL-10 and decreased expression of IL-2 and interferon-gamma in long-surviving mouse heart allografts after brief CD4-monoclonal antibody therapy.

In a mouse model for vascularized heart transplantation, CBA recipients of BALB/c hearts were treated with 0.25 mg of anti-CD4 (GK1.5) given intraperitoneally on the day of grafting and on days 1, 2, and 3 thereafter. This reduced splenic CD4+ cells to < 1% and all grafts survived > 100 days, compared with 8-10 days in untreated recipients. Despite recovery of the CD4+ cells after day 21, mice did not reject donor-type skin grafts at > 30 days, but rapidly rejected third-party skin, showing alloantigen-specific tolerance. The surviving heart grafts had significant mononuclear cell infiltration at time points from 7 to 100 days after transplantation. In the normal rejection process, where extensive myocyte necrosis was seen at 7 days, graft-infiltrating T cells produced IL-2 and IFN-gamma. These cells responded in vitro to IL-2 and displayed donor-specific CTL activity. In contrast, cells from CD4-mAb-treated hearts did not show significant growth in IL-2 or kill donor cells in CTL assays. In these nonrejecting hearts, immunohistology showed a diffuse infiltrate of T cells and macrophages by day 3. The allograft infiltrate increased rapidly thereafter in both rejecting and nonrejecting grafts, peaking at day 6-7 in rejecting grafts, when CD4+, CD8+, and IL-2R+ cells were present, with expression of IL-2, IFN-gamma, and IL-4, but only trace levels of IL-10. From 14 to 100 days, nonrejecting allografts showed a characteristic cytokine profile of dense IL-4 and IL-10 expression on intragraft leukocytes and endothelial cells, with low levels of IL-2 and IFN-gamma. This cytokine profile, characteristic of Th2 responses, was seen in all nonrejecting grafts and was not present in rejecting grafts. Allograft tolerance can studied by examination of the functions and cytokine profile of the cells within the graft, and tolerance develops in the presence of a Th2 response within the graft.

Vaccines that facilitate antigen entry into dendritic cells

Although vaccines have been highly successful in preventing and treating many infectious diseases (including smallpox, polio and diphtheria) diseases prevalent in the developing world such as malaria and HIV, that suppress the host immune system, require new, multiple strategies that will be defined by our growing understanding of specific immune activation. The definition of adjuvants, previously thought of as any substance that enhanced the immunogenicity of antigen, could now include soluble mediators and antigenic carriers that interact with surface molecules present on DC (e.g. LPS, Flt3L, heat shock protein) particulate antigens which are taken up by mechanisms available to APC but not other cell types (e.g. immunostimulatory complexes, latex, polystyrene particles) and viral/bacterial vectors that infect antigen presenting cells (e.g. vaccinia, lentivirus, adenovirus). These approaches, summarized herein, have shown potential in vaccinating against disease in animal models, and in some cases in humans. Of these, particle‐antigen conjugates provide rapid formulation of the vaccine, easy storage and wide application, with both carrier and adjuvant functions that activate DC. Combined vaccines of the future could use adjuvants such as virus‐like particles and particles targeted towards a predominant cellular type or immune response, with target cell activation enhanced by growth factors or maturation signals prior to, or during immunization. Collectively, these new additions to adjuvant technology provide opportunities for more specific immune regulation than previously available.

Anti-gal Antibody-mediated Allograft Rejection In α1,3-galactosyltransferase Gene Knockout Mice: A Model of Delayed Xenograft Rejection

Background. The key role of anti-galactosea1,3-galactose (anti-aGal) xenoantibodies in initiating hyperacute xenograft rejection has been clearly demonstrated using a variety of in vitro and in vivo approaches. However, the role of anti-aGal antibodies in mediating post-hyperacute rejection mechanisms, such as antibody-dependent cellular cytoxicity, remains to be determined, primarily because of the lack of a small animal model with which to study this phenomena. Methods. Hearts from wild-type mice were transplanted heterotopically into a1,3-galactosyltransferase knockout (Gal KO) mice, which like humans develop antibodies to the disaccharide galactosea1,3-galactose (Gal). At the time of rejection, hearts were examined histologically to determine the mechanism of rejection. Results. Hearts from wild-type mice transplanted into high-titer anti-aGal recipients were rejected in 8 ‐13 days. Histological examination demonstrated a cellular infiltrate consisting of macrophages (80 ‐90%), natural killer cells (5‐10%), and T cells (1‐5%). In contrast, wild-type hearts transplanted into low anti-Gal titer recipients demonstrated prolonged (>90 day) survival. However, a significant proportion (30 ‐ 40%) of these underwent a minor rejection episode between 10 and 13 days, but then recovered (“accommodated”). Conclusions. The results of this study suggest that the Gal KO mouse is a useful small animal vascularized allograft model, in which the role of anti-aGal antibody in graft rejection can be studied in isolation from other rejection mechanisms. The titer of antiaGal antibody was found to be the critical determinant of rejection. The histopathological features of rejection in this model are very similar to other models of delayed xenograft rejection, in both the timing and composition of the cellular infiltrate. The Gal KO mouse therefore provides a new rodent model, which will aid in the identification of the distinct components involved in the pathogenesis of delayed xenograft rejection.

Unique Monoclonal Antibodies Define Expression of FcγRI on Macrophages and Mast Cell Lines and Demonstrate Heterogeneity Among Subcutaneous and Other Dendritic Cells

The mouse FcγRI is one of the most fundamentally important FcRs. It participates in different stages of immunity, being a low affinity receptor for T-independent IgG3 and yet a high affinity receptor for IgG2a, the product of a Th1 immune response. However, analysis of this receptor has been difficult due largely to the failure to generate specific Abs to this FcR. We have made use of the polymorphic differences between BALB/c and NOD/Lt mice to generate mAb specific for the FcγRI of BALB/c and the majority of in-bred mouse strains. Three different mAb were obtained that detected FcγRI encoded by the more common Fcgr1a and Fcgr1b alleles, and although they identified different epitopes, none inhibited the binding of IgG to FcγRI. When bound to FcγRI, these mAb induced calcium mobilization upon cross-linking. Several novel observations were made of the cellular distribution of FcγRI. Resting and IFN-γ-induced macrophages expressed FcγRI as well as mast cell lines. Both bone marrow-derived and freshly isolated dendritic cells from spleen and lymph nodes expressed FcγRI. A class of DC, uniquely found in s.c. lymph nodes, expressed the highest level of FcγRI and also high levels of MHC class II, DEC205, CD40, and CD86, with a low level of CD8α, corresponding to the phenotype for Langerhans-derived DC, which are highly active in Ag processing. Thus, in addition to any role in effector functions, FcγRI on APC may act as a link between innate and adaptive immunities by binding and mediating the uptake of T-independent immune complexes for presentation, thereby assisting in the development of T-dependent immune responses.

The role of FcγRIIa as an inflammatory mediator in rheumatoid arthritis and systemic lupus erythematosus

Despite their essential role in host protection, immunoglobulins are also involved in autoimmune processes where antibodies recognize the hosts own tissue, triggering inflammatory responses that result in extensive tissue damage. A complex interaction of genetic predisposition, together with environment factors, is thought to trigger immune dysfunction. Although recent studies have dissected the essential role of Fc receptors in autoimmune antibody mediated processes, the uniquely human FcγRIIa has not been studied in detail. This Fc receptor is of particular interest, as it is the most abundantly expressed Fc receptor in humans and is implicated in immune complex disease. Investigation of its role has been hampered to date due to lack of suitable animal models. This review examines the evidence for the direct role of this receptor in diseases such as systemic lupus erythematosus and rheumatoid arthritis.

Modified technique for kidney transplantation in mice.

This study describes a new method for joining the donor ureter to the recipient bladder during mouse kidney transplantation. The donor left kidney was harvested using methods previously published, except that bladder tissue was not harvested with the end of the ureter. The recipient left kidney was removed and the donor kidney was attached using end‐to‐side anastomosis. The recipient bladder was pierced with a 21‐gauge needle allowing curved forceps to be inserted through the bladder, to pull through the ureter, and the periuretal tissue was stitched to the exterior wall of the bladder. The donor ureter was allowed to retract inside the bladder. Following a right nephrectomy, grafts were monitored by blood serum creatinine and urea. With a technical success rate of 83%, this technique reduced donor harvest time by 20 minutes and ureter attachment time by 15 minutes making it the best method available for mouse kidney transplantation.

Inhibition of destructive autoimmune arthritis in FcγRIIa transgenic mice by small chemical entities

The interaction of immune complexes with the human Fc receptor, FcγRIIa, initiates the release of inflammatory mediators and is implicated in the pathogenesis of human autoimmune diseases, including rheumatoid arthritis and systemic lupus erythematosus, so this FcR is a potential target for therapy. We have used the three‐dimensional structure of an FcγRIIa dimer to design small molecule inhibitors, modeled on a distinct groove and pocket created by receptor dimerization, adjacent to the ligand‐binding sites. These small chemical entities (SCEs) blocked immune complex‐induced platelet activation and aggregation and tumor necrosis factor secretion from macrophages in a human cell line and transgenic mouse macrophages. The SCE appeared specific for FcγRIIa, as they inhibited only immune complex‐induced responses and had no effect on responses to stimuli unrelated to FcR, for example platelet stimulation with arachidonic acid. In vivo testing of the SCE in FcγRIIa transgenic mice showed that they inhibited the development and stopped the progression of collagen‐induced arthritis (CIA). The SCEs were more potent than methotrexate and anti‐CD3 in sustained suppression of CIA. Thus, in vitro and in vivo activity of these SCE FcγRIIa receptor antagonists demonstrated their potential as anti‐inflammatory agents for autoimmune diseases involving immune complexes.