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OBJECTIVE: The major human Fc receptor, FcgammaRIIa, is the most widespread activating FcR. Our aim was to determine the role of FcgammaRIIa in a transgenic mouse model of immune complex-mediated autoimmunity and to characterize the development of spontaneous autoimmune disease. METHODS: Arthritis was induced in normal and FcgammaRIIa-transgenic mice by immunization with type II collagen (CII) or by transfer of arthritogenic anti-CII antibodies. Also, mice that spontaneously developed autoimmune disease were assessed by clinical scoring of affected limbs, histology and serology, and measurement of autoantibody titers and cytokine production. RESULTS: FcgammaRIIa-transgenic mice developed collagen-induced arthritis (CIA) more rapidly than did archetypal CIA-sensitive DBA/1 (H-2q) mice, while nontransgenic C57BL/6 (H-2b) mice did not develop CIA when similarly immunized. Passive transfer of a single dose of anti-CII antibody induced a more rapid, severe arthritis in FcgammaRIIa-transgenic mice than in nontransgenic animals. In addition, most immune complex-induced production of tumor necrosis factor alpha by activated macrophages occurred via FcgammaRIIa, not the endogenous mouse FcR. A spontaneous, multisystem autoimmune disease developed in aging (>20 weeks) transgenic mice (n = 25), with a 32% incidence of arthritis, and by 45 weeks, all mice had developed glomerulonephritis and pneumonitis, and most had antihistone antibodies. Elevated IgG2a levels were seen in mice with CIA and in those with spontaneous disease. CONCLUSION: The presence of enhanced passive and induced autoimmunity, as well as the emergence of spontaneous autoimmune disease at 20-45 weeks of age, suggest that FcgammaRIIa is a very important factor in the pathogenesis of autoimmune inflammation and a possible target for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Most adjuvants require danger signals to promote immune responses against vaccine antigens. Our previous studies have characterised a powerful nano-particulate antigen delivery system, which by itself does not induce inflammation, and which further appears to induce substantial immune responses in mice and sheep without the requirement for added stimulators of toll like receptors or other pathogen recognition receptors. In the present study we dissect the nature of the early induction phase of the immune response stimulated by such a vaccine comprising 40 nm polystyrene nano-particles conjugated to the antigen. We analyse the kinetics of export from an individual draining lymph node from the sheep, of antibodies and cytokines as well as antigen responsive CD4 and CD8 T cells. Our results indicate that simple inert nano-bead based antigen delivery into the draining area of the lymph node is highly efficient at priming combined humoral and T cell antigen specific immunity without the need for added 'danger signals'. Furthermore this nano-bead adjuvant is a potent agent capable of promoting cross-priming for CD8 T cell induction in sheep. Interestingly, using nano-beads, similarly to what has been observed with natural pathogen based lymph node stimulation, a phase of CD4 T cell priming and export preceded CD8 T cell induction, suggesting the engagement of natural priming processes and kinetics., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The major human Fc receptor, huFcgammaRIIa, is implicated in the development of autoimmune arthritis in humans but until recently has not been studied in mouse models. We evaluated potential roles of FcgammaRIIa by using transgenic mice expressing the receptor. We examined two models of induced autoimmune arthritis pristane-induced arthritis (PIA) and collagen-induced arthritis (CIA) as well as the anti-collagen-II antibody-induced arthritis (CAIA) model. In the induced arthritis models PIA and CIA, the transgenic mice developed a more severe arthritis than the other arthritis-prone SJL or DBA1 mice. Interestingly, anti-collagen-II antibodies were elevated in PIA in the susceptible mice. In the CIA model, the highly susceptible transgenic mouse had IgG subclass levels equivalent to the unaffected and disease resistant C57BL/6 mouse strain implying that the FcgammaRIIa lowers the threshold of IgG dependent leukocyte activation. This is consistent with the greatly enhanced sensitivity of the FcgammaRIIa transgenic mice to CAIA which clearly indicates a role for the receptor at least at the inflammatory effector cell level. Other roles for huFcgammaRIIa or other gene products in the development of autoimmunity cannot be ruled out however, especially as the mice exhibited elevated Th1 or Th17 CD4 T cells in the draining lymph nodes., (Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.)

The interaction of immune complexes with the human Fc receptor, FcgammaRIIa, 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 FcgammaRIIa 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 FcgammaRIIa, 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 FcgammaRIIa 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 FcgammaRIIa receptor antagonists demonstrated their potential as anti-inflammatory agents for autoimmune diseases involving immune complexes.

Previous studies compared uptake by dendritic cells (DC) of 20, 40, 100, 200, 500, 1000, and 2000 nm beads in vivo. When beads were used as antigen carriers, bead size influenced antibody responses and induction of IFN-gamma-producing CD4 and CD8 T cells. Beads of 40-50 nm were taken up preferentially by DC and induced particularly strong immunity. Herein, we examine immunity induced by minute differences in nanobead size, specifically within a narrow viral-sized range (20, 40, 49, 67, 93, 101, and 123 nm), to see if bead carrier size influenced the induction of type 1 or type 2 cells as demonstrated by the production of IFN-gamma or IL-4. In vivo uptake by DC was assessed for selected sizes in this range. Responses to whole ovalbumin (OVA) or the OVA-derived CD8 T cell peptide epitope (SIINFEKL) were tested. After one immunization with beads-OVA, IFN-gamma responses to both OVA and SIINFEKL were significantly better with 40 and 49 nm beads than other sizes, while, in contrast, IL-4 responses to OVA were higher after immunization with OVA conjugated to larger beads (93, 101, and 123 nm). Thus IFN-gamma induction from CD8 T cells was limited to 40-49 nm beads, while CD4 T cell activation and IL-4 were induced by 93-123 nm beads-OVA. After two immunizations, there were comparable high levels of IFN-gamma produced with 40 and 49 beads and IL-4 reactivity was still higher for larger beads (93, 101, 123 nm). Production of IgG1 was seen across the full range of bead sizes, increasing after two immunizations. Since protection against respiratory syncytial virus (RSV) depends on strong IFN responses, while IL-4 responses are reported to cause asthma-like symptoms, immunization with RSV antigens on the 49 nm carrier beads could provide the basis for a suitable vaccine. When the 49 nm beads were conjugated to RSV proteins G88 (surface) or M2.1 (internal capsid), one immunization with G88 induced high levels of IFN-gamma and low levels of IL-4. IL-4 increased with two immunizations. Beads-M2.1 induced only moderate levels of IFN-gamma and low titer antibody after two immunizations. Mice vaccinated once with G88-conjugated 49 nm beads and challenged intranasally with RSV strain A2 subtype showed reduced viral titers and recovered from weight loss more rapidly than mice immunized with M2.1-conjugated 49 nm beads or naive control mice. These results show that precise selection of nanobead size for vaccination can influence the type 1/type 2 cytokine balance after one immunization, and this will be useful in the development of effective vaccines against common human pathogens such as RSV.

The FcR are a crucial link in the immune response between humoral and cellular immunity and cell-based effector systems, mediating a wide variety of physiological and biochemical responses. The FcR for IgG (FcgammaR) and in particular the most widely expressed of these, FcgammaRII, are important in regulating adaptive immunity. Disruption of their function is a key factor in the development of autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), which are characterized by chronic, multi-organ inflammation. Studies of the FcgammaRII include structure/function relationships, investigation of the associations between FcR polymorphisms and human disease and animal studies using knockout or transgenic mouse models. These investigations showed that the various forms of FcgammaRII interact with immune complexes to either initiate or inhibit inflammation. In conjunction with environmental antigens and genotype, the FcgammaRII activating and inhibitory receptors determine the nature and magnitude of response to antigens. In this review, the structure and function of the FcgammaRIIs and their role in immune complex-mediated auto-immunity are discussed.

Nano- and microparticles have long been used for the delivery of drugs and are currently being evaluated as vaccine delivery systems. Particulates can elicit potent immune responses, either by direct immuno-stimulation of antigen presenting cells (APC) or/and by delivering antigen to specific cellular compartments and promoting antigen uptake by appropriate stimulatory cell types. Herein, we describe a detailed method for the preparation of a novel nanoparticle-based antigen delivery system which induces strong cellular and humoral immune responses in mice and sheep. This simple system is based on the use of 40 nanometer (nm) inert solid carrier beads to which antigen is covalently coupled before injection. Covalent conjugation of antigen to the nanobeads, assessment of conjugation efficiency, characterisation and measurement of in vivo immunogenicity by cytokine ELISPOT (to measure antigen-specific T-cell responses) and ELISA (to measure antibody titers), are described. Emphasis is placed on providing trouble-shooting advice to enable the reproducible production of soluble nano-size formulations that do not suffer from common problems such as aggregation, as well as understanding the causes and thus avoiding a range of prevalent technical problems that occur when using immune response detection assays, such as the cytokine ELISPOT assay and ELISA.

The use of particulate carriers holds great promise for the development of effective and affordable recombinant vaccines. Rational development requires a detailed understanding of particle up-take and processing mechanisms to target cellular pathways capable of stimulating the required immune responses safely. These mechanisms are in turn based on how the host has evolved to recognize and process pathogens. Pathogens, as well as particulate vaccines, come in a wide range of sizes and biochemical compositions. Some of these also provide 'danger signals' so that antigen 'senting cells (APC), usually dendritic cells (DC), acquire specific stimulatory activity. Herein, we provide an overview of the types of particles currently under investigation for the formulation of vaccines, discuss cellular uptake mechanisms (endocytosis, macropinocytosis, phagocytosis, clathrin-dependent and/or caveloae-mediated) for pathogens and particles of different sizes, as well as antigen possessing and presentation by APC in general, and DC in particular. Since particle size and composition can influence the immune response, inducing humoral and/or cellular immunity, activating CD8 T cells and/or CD4 T cells of T helper 1 and/or T helper 2 type, particle characteristics have a major impact on vaccine efficacy. Recently developed methods for the formulation of particulate vaccines are presented in this issue of Methods, showcasing a range of "cutting edge" particulate vaccines that employ particles ranging from nano to micro-sized. This special issue of Methods further addresses practical issues of production, affordability, reproducibility and stability of formulation, and also includes a discussion of the economic and regulatory challenges encountered in developing vaccines for veterinary use and for common Third World infectious diseases.

Although a number of adjuvants are currently approved for use in veterinary species, only alum has been widely used in humans. While it induces strong antibody responses, cell mediated responses are often low and inflammatory reactions at the site of injection are common. We investigated the immunological properties of a novel nano-bead adjuvant in a sheep large-animal model. In contrast to alum, antigen covalently coupled to nano-beads induced substantial cell mediated responses along with moderate humoral responses. No adverse reactions were seen at the site of immunisation in the sheep. Thus, nano-bead adjuvants in veterinary species may be useful for the induction of immunity to viral pathogens, where a cell mediated response is required. These findings also highlight the potential usefulness of nano-bead vaccines for intracellular pathogens in humans.

The immune system is a tightly regulated network that is able to maintain a balance of immune homeostasis under normal physiological conditions. Normally, when challenged with foreign antigen, specific appropriate responses are initiated that are aimed at restoring homeostasis. However under particular circumstances, this balance is not maintained and immune responses either under or over react. Cancer is an example of a situation where the immune response can be inefficient or unresponsive, resulting in uncontrolled growth of the cancer cells. Conversely, when the immune response over-reacts, this can result in conditions such as autoimmunity or pathology following infection. Many drug therapies have been developed that aim to alleviate or prevent such immune disorders and restore immune homeostasis. This review highlights recent advances in immunotherapies, with an emphasis on specific examples in the treatment of cancer, autoimmune disease (multiple sclerosis) and viral infection (respiratory syncytial virus).

Objective: The major human Fc receptor, FcgammaRIIa, is the most widespread activating FcR. Our aim was to determine the role of FcgammaRIIa in a transgenic mouse model of immune complex-mediated autoimmunity and to characterize the development of spontaneous autoimmune disease., Methods: Arthritis was induced in normal and FcgammaRIIa-transgenic mice by immunization with type II collagen (CII) or by transfer of arthritogenic anti-CII antibodies. Also, mice that spontaneously developed autoimmune disease were assessed by clinical scoring of affected limbs, histology and serology, and measurement of autoantibody titers and cytokine production., Results: FcgammaRIIa-transgenic mice developed collagen-induced arthritis (CIA) more rapidly than did archetypal CIA-sensitive DBA/1 (H-2q) mice, while nontransgenic C57BL/6 (H-2b) mice did not develop CIA when similarly immunized. Passive transfer of a single dose of anti-CII antibody induced a more rapid, severe arthritis in FcgammaRIIa-transgenic mice than in nontransgenic animals. In addition, most immune complex-induced production of tumor necrosis factor alpha by activated macrophages occurred via FcgammaRIIa, not the endogenous mouse FcR. A spontaneous, multisystem autoimmune disease developed in aging (>20 weeks) transgenic mice (n = 25), with a 32% incidence of arthritis, and by 45 weeks, all mice had developed glomerulonephritis and pneumonitis, and most had antihistone antibodies. Elevated IgG2a levels were seen in mice with CIA and in those with spontaneous disease., Conclusion: The presence of enhanced passive and induced autoimmunity, as well as the emergence of spontaneous autoimmune disease at 20-45 weeks of age, suggest that FcgammaRIIa is a very important factor in the pathogenesis of autoimmune inflammation and a possible target for therapeutic intervention.

Targeting antigen to dendritic cells (DC) in vivo might be an effective method of modulating immune responses. Given the functional specializations among DC subsets, we investigated how targeting different receptors on different DC subsets may influence antibody (Ab) production. We show here that targeting FIRE (F4/80-like receptor) or CIRE (C-type lectin receptor), two molecules expressed on the surface of immature CD8- DC in the mouse, increases Ab production 100-1000-fold over a non-targeted control. This response was equivalent to that achieved with CpG adjuvant. In contrast, targeting CD205, which is primarily expressed on CD8+ DC, did not elicit an Ab response unless an adjuvant was added. Strong Ab responses in FcRgamma-/- mice, and with the use of F(ab')2 fragments, confirmed that FIRE and CIRE targeting was due to specific rather than FcR or complement binding. Our findings may reflect differences in the ability of CD8+ and CD8- DC subsets to stimulate immune responses in vivo. Although the consensus view is that Ag presentation on DC in their steady state leads to tolerance, the Ab enhancement from FIRE and CIRE targeting in the apparent absence of any "danger" or inflammatory signal would suggest that targeting certain DC molecules can supplant the need for external adjuvants for eliciting immune responses.

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 microm) that localize to dendritic cells (DEC205(+) CD40(+), CD86(+)) in draining lymph nodes, inducing high levels of IFN-gamma 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.

Historically, treatment of complex autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus has aimed to relieve symptoms, and in severe cases, use broad-spectrum immunosuppressive treatments in attempts to induce permanent remission. Recent research into the causes of chronic autoimmune inflammatory activation have not only explored the mechanism of action of known therapies, but also provided a number of new targets for therapy, by identifying the cells, cytokines and signalling pathways activated during autoimmune antibody mediated processes. This review briefly outlines progress in the understanding of the autoimmune nature of rheumatoid diseases and the expansion of treatment options, from broad to specific immunotherapies for these closely related diseases.

Despite their essential role in host protection, immunoglobulins are also involved in autoimmune processes where antibodies recognize the host's 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 FcgammaRIIa 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.

The mouse Fc gamma RI 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 Fc gamma RI of BALB/c and the majority of in-bred mouse strains. Three different mAb were obtained that detected Fc gamma RI encoded by the more common Fcgr1(a) and Fcgr1(b) alleles, and although they identified different epitopes, none inhibited the binding of IgG to Fc gamma RI. When bound to Fc gamma RI, these mAb induced calcium mobilization upon cross-linking. Several novel observations were made of the cellular distribution of Fc gamma RI. Resting and IFN-gamma-induced macrophages expressed Fc gamma RI as well as mast cell lines. Both bone marrow-derived and freshly isolated dendritic cells from spleen and lymph nodes expressed Fc gamma RI. A class of DC, uniquely found in s.c. lymph nodes, expressed the highest level of Fc gamma RI and also high levels of MHC class II, DEC205, CD40, and CD86, with a low level of CD8 alpha, corresponding to the phenotype for Langerhans-derived DC, which are highly active in Ag processing. Thus, in addition to any role in effector functions, Fc gamma RI 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.

Diabetes in NOD mice is an autoimmune disease similar to Type I diabetes in humans. Prior to hypoglycemia, changes in the islet infiltrate led to autoreactive T cell activation and destruction of the insulin-producing beta cells. If T cell activation can be inhibited before beta cell destruction is complete, islet cell rescue and regeneration can occur. Female NOD mice > 100 days old with blood glucose levels > 20 mM/l for less than 7 days were selected as 'recent onset' mice. Untreated, all of these animals would die of diabetes in < 40 days. Mice treated with anti-CD4 (GK1.5) achieved 14.3% permanent remission, while those treated with anti-CD8 (53.6.7) showed 33.3% permanent remission. Mice treated with anti-CD3 (145-2C1) also achieved 33.3% permanent remission, but 14% of these died of first dose syndrome. In mice treated with a low dose of anti-CD3 (10 microg KT3), which did not induce first dose syndrome, 50% remained in remission for > 100 days. This dose of mAb reduced insulitis but did not deplete splenic CD3 cells. When mice in remission were challenged with a vascularized pancreas isograft at 50 days, 9/22 remained normal and 13/22 had recurrent disease in both transplanted and native pancreas. Of the long-surviving isografts 7/9 were in KT3 treated recipients. Histology showed activated T cell infiltration in the native and transplanted pancreases of mice with transient remission. Benign insulitis with macrophages, B cells, CD4 > CD8 T cells and low levels of IL-2R, IL-2, IFN-gamma and IL-4 was seen in islets from the native pancreas and in long surviving pancreas isografts in mice that remained in remission. Thus, using low dose KT3, it was possible to halt the development of diabetes in 50% of animals treated soon after diagnosis, despite significant islet cell destruction at this stage. Of the KT3 treated mice in permanent remission, 70% had re-established tolerance to autoantigen and did not destroy vascularized pancreas isografts.

Previously, we demonstrated that combination CTLA4-Fc and anti-CD40L mAb treatment results in tolerance to concordant, cellular islet xenografts. The aim of this study was to determine its effectiveness in a model of fetal pig pancreas (FPP) xenotransplantation. Survival of FPP fragment grafts were compared to the survival of rat islet or cardiac xenografts following short term CTLA4-Fc and anti-CD40L mAb treatment. Rat islet and FPP fragment grafts survived long-term. However, rat cardiac grafts were rejected by 52-91 days. Both rat islet and FPP grafts showed similar histology with intact islet structures and adjacent 'nests' of lymphocytes. Concordant vascularised rat hearts showed extensive polymorphonuclear infiltrate, concentric vasculitis and a perivascular infiltrate predominantly of CD8+ T cells. This suggests that this therapy is effective for prolonging islet xenografts and demonstrates that the cellular mechanism of rejection for vascularised and non-vascularised xenografts are different.

To compare the effectiveness of transgenic and systemic monoclonal antibody therapy for pancreas transplantation, vascularised segmental pancreas allografts from wild-type or transgenic pancreatic tissue that secreted monoclonal anti-CD4 were placed in CBA recipients in which diabetes had been induced chemically by streptozotocin (STZ, non-autoimmune diabetes). In untreated CBA recipients, wild-type BALB/c or C57BL/6 bml pancreas transplants were rejected in a mean survival time (MST) of 27 and 30 days, respectively. BALB/c and C57BL/6 graft survival improved when recipients were given a short course of T cell depleting monoclonal anti-CD4 antibody, (GK 1.5, 2 mg total on days -1, 0, 1, 2 with grafting on day 0) with MST +/- S.D. of 71 +/- 29 and 44 +/- 36 days, respectively. Thus, transient depletion of CD4 was effective in delaying pancreas allograft rejection in these strain combinations. The use of C57BL/6 bml mice transgenic for a rat anti-CD4 antibody (GK5 mice) as pancreas donors provided allografts that secreted sufficient anti-CD4 antibody to cause CD4 T cell depletion in the recipients (CD4 cells decreased from 30 to < 5% of small lymphocytes). This degree of depletion was not sustained and the CD4 recovery inversely correlated with graft survival. Mice with > 20% CD4 cells in the splenic lymphocyte population 4 weeks post-transplant rejected their grafts (3 of 10 mice). However, in 7 of 10 mice CD4 cells remained low (< 15%) and allografts survived for > 80 days. The GK5 allografts survived significantly longer than those from non-transgenic bml controls (MST 83 +/- 32 days, compared with 30 days, P < 0.0005). This survival time was similar to that of BALB/c allografts in CBA recipients treated with a high dose of anti-CD4 antibody. Thus, transgenic secretion of anti-CD4 antibody by the pancreas allograft was very effective in prolonging its survival.

Background: Investigations of the role of CD4 T lymphocytes in allograft rejection and tolerance have relied on the use of mouse models with a deficiency in CD4 cells. However, in mice treated with depleting monoclonal antibody (mAb) and in MHC class II knockout (KO) mice, there are residual populations of CD4 cells. CD4 KO mice had increased CD4- CD8-TCRalphabeta+ helper T cells, and both strains of KO mice could reject skin allografts at the normal rate. In this study, transgenic mice with no peripheral CD4 cells were the recipients of skin and heart allografts. Results were compared with allograft survival in CD4 and MHC class II KO mice., Methods: GK5 (C57BL/6 bml mice transgenic for a chimeric anti-CD4 antibody) had no peripheral CD4 cells. These mice, and CD4 and class II KO mice, received BALB/c or CBA skin or cardiac allografts. Some GK5 mice were treated with anti-CD8 mAb to investigate the role of CD8 cells in rejection. CD4 and CD8 cells were assessed by FACS and immunohistochemistry., Results: BALB/c skin on GK5 mice had a mean survival time +/- SD of 24+/-6 days, compared with 9+/-2 days in wild-type mice. Anti-CD8 mAb prolonged this to 66+/-7 days. BALB/c skin survived 10+/-2 days on class II KO and 14+/-2 days on CD4 KO, both significantly less than the survival seen on GK5 recipients (P<0.001). BALB/c hearts survived >100 days in GK5 recipients and in wild-type recipients treated with anti-CD4 mAb at the time of grafting, in contrast to a mean survival time of 10+/-2 days in untreated wild-type mice. Immunohistochemistry revealed that long-term surviving heart allografts from the GK5 recipients had CD8 but no CD4 cellular infiltrate. These hearts showed evidence of transplant vasculopathy., Conclusions: The GK5 mice, with a complete absence of peripheral CD4 cells, provide the cleanest available model for investigating the role of CD4 lymphocytes in allograft rejection. Prolonged skin allograft survival in these mice compared with CD4 and MHC class II KO recipients was clearly the result of improved CD4 depletion. Nevertheless, skin allograft rejection, heart allograft infiltration, and vascular disease, mediated by CD8 cells, developed in the absence of peripheral CD4 T cells.

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., (Copyright 1999 Wiley-Liss, Inc.)

Background: To evaluate cardiac allografts from recipients that had achieved peripheral tolerance after transient CD4+ T cell depletion, we analyzed cellular infiltrate, cytokine expression, and vascular thickening. Long-surviving cardiac allografts from tolerant recipients were compared with acutely rejecting allografts and isografts., Methods and Results: In CBA mice treated with anti-CD4 (GK1.5, 0.5 mg intraperitoneally on days 1-28), BALB/c cardiac allografts survived >100 days. These recipients were tested for tolerance at >70 days, by challenge with donor and third-party (C57BL/6) skin grafts. BALB/c skin grafts survived >30 days, although C57BL/6 skin was rejected in <12 days, reflecting alloantigen-specific peripheral tolerance. When vascular thickening in graft arteries was assessed and computerized measurements performed, heart allografts from tolerant recipients showed significantly increased percentage of luminal occlusion compared with isografts (47% compared with 1.2%). Semiquantitative reverse transcriptase-polymerase chain reaction was used to assess normalized intragraft mRNA transcripts for cytokines and T cell markers, with immunoperoxidase staining of frozen sections to confirmed the presence of protein. Compared with rejecting grafts, well-preserved hearts from tolerant mice had lower levels of macrophage and T cell infiltration and decreased transcription of interferon-gamma, interleukin (IL)-2, IL-10, and inducible nitric oxide synthase. IL-4 expression was similar in both groups., Conclusions: The degree of tolerance achieved allowed specific acceptance of donor skin grafts, preserved primary graft function, and reduced inflammatory activation. Tolerance did not, however, completely prevent macrophage and T cell infiltration of the graft or the development of vascular lesions typical of chronic rejection.

Spontaneously diabetic nonobese diabetic (NOD/Lt) mice were treated with anti-T-cell monoclonal antibodies (mAbs) at the time of grafting with vascularized segmental pancreas isografts. Recipients were either untreated or given anti-CD4 and/or anti-CD8 mAbs (0.5 mg/20-g mouse on each of 4 consecutive days), which reduced target cell levels to <5% of normal. Graft function was monitored by measuring blood glucose (BG) levels. Transplants were removed for histological examination when BG returned to >20 mmol/l for two consecutive readings. Isografts from 3- to 4-week-old prediabetic mice placed in untreated diabetic NOD mice ceased functioning in 9-13 days with a mean survival time (MST) +/- SD of 10 +/- 2. Treatment with anti-CD4 prolonged survival significantly (MST = 61 +/- 35 days, P < 0.05 compared with untreated control mice). Anti-CD8 treatment was less effective, but it still significantly improved graft survival (MST = 24 +/- 9 days, P < 0.05 compared with untreated control mice). Anti-CD8 plus anti-CD4 treatment was highly effective in inhibiting autoimmune destruction of the grafts (MST = 97 +/- 8 days). This clearly demonstrates that transient inactivation of most T-cells with anti-CD4 plus anti-CD8 mAbs effectively controls autoimmune disease in the isograft, despite recovery of CD4 and CD8 T-cells to normal levels. Although insulitis developed in the long-term grafts, insulitis scores did not increase between 33 and 100 days, and none of the mice progressed to IDDM in 100 days. Histology showed a predominantly peri-islet T-cell and macrophage infiltrate with ductal expression of the cytokines interleukin (IL)-4, IL-2, and interferon-gamma. There was little infiltrate or expression of cytokines within the islets. Thus, mAb treatment at the time of grafting allowed isograft survival and prevented progression from insulitis to beta-cell destruction.

To study the role of IL-4 in tolerance induction and transplant arteriosclerosis, BALB/c hearts were transplanted into C57BL/6J wild-type or IL-4 knockout (IL-4(-/-)) recipients. A 30-day course of anti-CD4/8 mAb was used to induce long term graft survival. Primary graft survival was 50% (5 of 10) in IL-4(-/-) recipients comparable to 63% (5 of 8) in wild-type recipients. Mice with allografts surviving >80 days were tested for tolerance by challenge with a second donor or third party (CBA) heart. Secondary donor-strain heart grafts survived >30 days, but showed histologic evidence of ongoing alloimmune response. Third party hearts rejected rapidly. Although immunostaining and 32P RT-PCR assays showed no differences in the mononuclear cell infiltration and T cell activation between IL-4(-/-) and wild-type tolerant recipients, some monokines (IL-12, TNF-alpha, and allograft inflammatory factor-1) were up-regulated in grafts from IL-4(-/-) recipients. Computer-assisted analysis of elastin-stained vessels revealed that the severity of vascular thickening (percentage of luminal occlusion, mean +/- SD, n = 329) was similar in grafts from IL-4(-/-) (63.7 +/- 16.9%) and wild-type (69.5 +/- 17.6%) recipients. Thus, IL-4 deficiency did not alter primary or secondary graft survival, infiltration, or vascular thickening. The selective alterations in monokine expression suggests that alternative pathways are activated and may compensate in IL-4(-/-) mice.

To study the role of cytokines in long-term cardiac allografts we have used recipient mice with targeted gene deletions (-/-) in IFN-gamma, IL-4, or IL-10. In wild-type and IL-4 -/- recipients immunosuppressed with a 30-d course of anti-CD4 and anti-CD8, graft survival was > 87 d. This time was significantly reduced in IFN-gamma -/- (62 +/- 19 d, P < 0.05) and IL-10 -/- recipients (55 +/- 4 d, P < 0.0001). Histology showed mononuclear cell infiltration, patchy necrosis, fibrosis, and vascular thickening in all groups. Intragraft transcript levels measured by 32P-reverse transcriptase PCR showed different inflammatory patterns. IFN-gamma -/- recipients had higher IL-2 transcripts and selective alteration in macrophage activation that may have contributed to decreased graft survival. Decreased graft survival in IL-10 -/- recipients was associated with increases in iNOS and IFN-gamma-driven responses. Finally, in grafts from IL-4 -/- recipients, there were increases in CD3 transcripts concurrent with TNF-alpha levels. This increase suggests that IL-4 may regulate T cell infiltration through TNF-alpha-mediated inflammatory cell recruitment. Concurrent evaluation of these three isolated cytokine deletions has shown that the recipient environment caused distinct graft modifications.

We evaluated inflammatory activation and vascular thickening in a heterotopic murine heart transplant model. C57BL/6J recipient mice received anti-CD4 therapy (days 1 to 4 after transplantation) or sustained, combined anti-CD4/CD8 therapy (days 1 to 4, weekly thereafter). Morphometric analysis of grafts (> 95 days) found the mean percentage of vessel occlusion to be 51.7% in allografts treated with anti-CD4, 8.3% in allografts treated with sustained anti-CD4/CD8, and 6.7% in isografts. Mean transcript levels of the adhesion molecules P-selectin, intercellular adhesion molecule 1 (ICAM-1), and leukocyte function-associated antigen 1 (LFA-1) and the cytokines interleukin 4 (IL-4), interferon-gamma (IFN-gamma), inducible nitric oxide synthase (iNOS), allograft inflammatory factor 1 (AIF-1), and monocyte chemoattractant protein 1 (MCP-1) were measured with reverse transcription-polymerase chain reaction [RT-PCR] assays using deoxycytidine triphosphate radiolabeled with phosphorus 32 [32P-dCTP]. The assays were normalized against glyceraldehyde-3-phosphate dehydrogenase [G3PDH] Levels were found to be significantly higher in the anti-CD4 group than in the anti-CD4/CD8 group. A strong correlation was also found between the percentage of luminal occlusion and the expression of these markers of inflammation (r = .92-.99, P < .0001). Sustained therapy involving proximal blockade of CD4 and CD8 interrupts pathways leading to inflammation and vascular thickening. However, long-term heart allografts in mice treated with a short course of anti-CD4 display an ongoing inflammatory cell activation that culminates in arteriosclerosis. This model may help examine the role of targeted immune factors using knockout mice to identify those causally involved in vessel thickening.

Background: In testing new anti-CD3 agents for transplantation tolerance induction, an anti-CD3 monoclonal antibody was used as a carrier for the cytotoxic drug idarubicin (IDA)., Methods: Anti-CD3 (KT3) was covalently coupled with IDA, producing the IDA-KT3 immunoconjugate, which was tested for specificity by fluorometry and for inhibition of proliferation of CD3+ E3 cells ([3H]thymidine uptake). KT3 and IDA-KT3 were used to treat CBA recipients of BALB/c vascularized cardiac allografts. Mice with hearts surviving >100 days were challenged with donor and third-party (C57BL/6) skin., Results: Conjugation to IDA did not reduce binding of KT3 to E3 cells, although the toxicity of IDA was reduced by conjugation. In BALB/c to CBA cardiac allografts (rejected in 12-17 days), both KT3 and IDA-KT3 (0.25-0.5 mg/20 g mouse i.p. at the time of transplantation) induced tolerance. Hearts survived >100 days and skin graft challenge showed indefinite survival of donor grafts but not third-party grafts. KT3 was less toxic, as measured by tumor necrosis factor-a release and blood glucose levels, than equivalent dosages of 145-2C11. At lower dosages (0.1 mg/20 g mouse), KT3-treated animals rejected BALB/c allografts in 15 to 19 days, but IDA-KT3 induced long survival (>100 days) and donor-specific tolerance in 5 of 6 mice., Conclusions: Coupling IDA to anti-CD3 reduced the in vivo toxicity of IDA and improved the immunosuppressive performance of KT3, reducing the side effects seen with other anti-CD3 agents. IDA-KT3 is a new, effective, nontoxic tolerogen in this donor-recipient combination.

We analyzed cytokine expression in recipient spleens and cardiac allografts placed in mice that were unable to synthesize interleukin (IL)-4 due to disruption of the IL-4 gene (IL-4 -/-) and in wild-type (IL-4 +/+) mice. Polyclonal BL-4P and monoclonal 11B11, 1D11, and 24G2 anti-IL-4 antibodies were used to detect cell-surface and cytoplasmic antigens in sections of frozen tissue. All of the antibodies were found to react with non-IL-4 determinants associated with graft-infiltrating cells, and BL-4P, 1D11, and 24G2 bound to cells and connective tissue in the spleens of IL-4 -/- mice. The IL-4-producing cell line, X63Ag8-653 (X63), was used as a positive control for IL-4 staining and to test the ability of recombinant IL-4 to block the binding of antibodies to IL-4.

Human CD46 (membrane cofactor protein) is a type 1 glycoprotein that functions to protect autologous cells from complement-mediated damage by binding C3b and C4b for their factor I-mediated cleavage. We now describe the production and function of recombinant soluble CD46 (rsCD46), which was produced as a truncated form by mutagenesis using the splice overlap extension polymerase chain reaction, by inserting a translational stop codon into the CD46 cDNA at the junction of the transmembrane and extracellular domains. After transfection of an expression construct into 293-EBNA (Epstein-Barr nuclear antigen)-transformed cells, secretion of rsCD46 protein was detected by immunoradiometric assay using monoclonal antibodies. Following a single-step immunoaffinity purification, the protein resolved as a single band of approximately 56,000 MW on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified rsCD46 (51 micrograms/ml) protected Chinese hamster ovary (CHO) cells from lysis initiated by a high titre rabbit anti-CHO antibody and complement from rabbit or human. The protection was specifically mediated by rsCD46 because the monoclonal antibody M177, which blocks interaction between CD46 and C3b/C4b, abrogated the protection. The results demonstrate that rsCD46 is effective as a fluid-phase regulator of complement activation on cell surfaces, even when initiated by the classical complement pathway. The in vivo efficacy of rsCD46 was investigated using a mouse heart to rat xenograft model. Administration of a bolus injection of rsCD46 was effective at delaying hyperacute graft rejection. These data suggest that rsCD46 may have a role as a therapeutic agent.

In the NOD/Lt recipient mice, disease recurrence in untreated isografts was extremely rapid (median less than 10 days) compared to the rejection of an untreated BALB/c pancreas graft in a CBA mouse (median 26 days). This would be expected since disease recurrence is a secondary response in diabetic mice with lymphocytes primed to respond to the beta-cell autoantigen. The median survival time for the untreated CBA to NOD/Lt pancreas graft falls, as expected, between these two survival times (median 20 days). Although anti-CD4 and/or anti-CD8 were effective in delaying or stopping autoimmune disease recurrence and rejection in the separate models, they were unsuccessful in significantly altering survival times in the combined model, despite using 2-mg doses and dual therapy. Similar doses of anti-CD4 have failed to prevent islet allograft rejection in NOD/Lt mice. Long-term dual treatment may be required to inactivate CD4+ and CD8+ T cells in the NOD/Lt mouse to prevent both autoimmune disease recurrence and rejection. NOD/Lt recipients will require greater immunosuppression to prevent rejection-autoimmune disease recurrence will be easier to prevent. This study shows the value of using NOD/Lt mice, with naturally occurring type 1 diabetes, for assessment of immunosuppressive therapy to prevent failure of pancreas transplants.

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.

Successful pancreas transplantation requires the suppression of both allograft rejection and recurrence of autoimmune disease. In order to study treatments to suppress these two responses, separate models were developed for pancreas allograft rejection and autoimmune disease. In the first model, the diabetic state was induced with streptozotocin in CBA mice prior to the transplantation of pancreas grafts from BALB/c donors. In the absence of autoimmune disease, control mice rejected their grafts in 26 days (median). Antibody treatments (anti-lymphocyte serum and anti-CD4) significantly prolonged allograft survival beyond this time, but not to the extent we have previously reported in the heart graft model. NOD/Lt mice spontaneously developed autoimmune diabetes, and recurrence of disease was seen in isografts at 9.5 days (median). Antibody treatments significantly delayed disease recurrence, with anti-CD4 being the most effective. Heart allografts (CBA donors) in NOD/Lt recipients were rejected within 17 days (median), and the anti-CD4 treatment had a moderate effect in delaying graft survival (median 28 days). Anti-lymphocyte serum did not prolong graft survival. Thus antibody treatment was effective in delaying both rejection and the recurrence of autoimmune disease in segmental pancreas grafts. However, the same doses were not effective in delaying heart rejection in the NOD/Lt model, so it would appear that treatments which inhibit autoimmune disease may not prevent allograft rejection.

To examine the potential use of immunoconjugates of drugs and antibodies as immunosuppressive agents, mice were treated with a short course (4 days) of T-cell-specific anti-Ly-2.1 monoclonal antibody, or MAB conjugated to an anthracycline, idarubicin (IDA). The anti-Ly-2.1 MAB had no significant effect on the survival of BALB/c (Ly-2.2) heart allografts in CBA (Ly-2.1) mice, but was a potent immunosuppressive agent when coupled to IDA, with most grafts surviving for > 100 days following treatment with doses ranging from 10 to 120 micrograms IDA, covalently coupled to 1-8 mg MAB. IDA-MAB treated mice with long-surviving heart grafts showed donor-specific tolerance. They did not reject donor-type skin grafts (these survived for > 50 days), but rejected third-party skin in 10-14 days. Heart allografts in these mice survived for > 100 days. Allografts placed 30 days after treatment were rejected, showing a recovery of peripheral T cell function at this time. Newly derived thymic T cells were, however, not required for this recovery since adult thymectomized, IDA-MAB treated animals also recovered T cell function and rejected heart allografts. FACScan analysis of T cells from mice treated with 80 micrograms IDA-4 mg MAB, which had received a heart allograft, showed 95% T cell depletion in the spleen compared with ungrafted, IDA-MAB treated animals, and untreated controls with or without allografts. Splenic T cell depletion was however not significant in CBA mice immunosuppressed with the lower dose of 10 micrograms IDA-MAB. Thus rapid depletion of splenic T cells was not required for immunosuppression induced by IDA-MAB conjugates. However, the minor subpopulation of Ly-2+, which was activated by alloantigen while carrying IDA-MAB, may be depleted during the T cell response to the allograft, resulting in a state of alloantigen-specific tolerance in mice with long-surviving heart allografts.

A modified technique for the transplantation of primarily vascularized mouse kidneys is described in which the donor was prepared by exposure of the left kidney, dissection of the suprarenal aorta and vena cava and of the ureter and bladder. The recipient was prepared by exposure of the infrarenal aorta and vena cava and isolation of approximately 1 cm of these vessels by proximal and distal 4/0 silk ties. The donor kidney was then perfused and removed to the recipient with the ureter and bladder. The transplant was completed by microsurgical end-to-side anastomosis of the aorta to aorta and vena cava to vena cava and the donor ureter was attached to the recipient bladder via a bladder patch. This technique differed significantly from the previously published technique in using cuffs of donor suprarenal aorta and vena cava rather than dissecting the base of the renal vessels. These changes made the operation easier and reduced ischaemia time from 35-40 to 25-30 min, a substantial improvement which increased both organ and recipient survival.