Isotype selection for antibody-based cancer therapy.

The clinical application of monoclonal antibodies (mAbs) has revolutionized the field of cancer therapy, as it has enabled the successful treatment of previously untreatable types of cancer. Different mechanisms play a role in the anti-tumour effect of mAbs. These include blocking of tumour-specific growth factor receptors or of immune modulatory molecules as well as complement and cell-mediated tumour cell lysis. Thus, for many mAbs, Fc-mediated effector functions critically contribute to the efficacy of treatment. As immunoglobulin (Ig) isotypes differ in their ability to bind to Fc receptors on immune cells as well as in their ability to activate complement, they differ in the immune responses they activate. Therefore, the choice of antibody isotype for therapeutic mAbs is dictated by its intended mechanism of action. Considering that clinical efficacy of many mAbs is currently achieved only in subsets of patients, optimal isotype selection and Fc optimization during antibody development may represent an important step towards improved patient outcome. Here, we discuss the current knowledge of the therapeutic effector functions of different isotypes and Fc-engineering strategies to improve mAbs application.

IgG is elevated in obese white adipose tissue but does not induce glucose intolerance via Fcγ-receptor or complement.

BACKGROUND/OBJECTIVES: In obesity, B cells accumulate in white adipose tissue (WAT) and produce IgG, which may contribute to the development of glucose intolerance. IgG signals by binding to Fcγ receptors (FcγR) and by activating the complement system. The aim of our study was to investigate whether activation of FcγR and/or complement C3 mediates the development of high-fat diet-induced glucose intolerance. METHODS: We studied mice lacking all four FcγRs (FcγRI/II/III/IV-/-), only the inhibitory FcγRIIb (FcγRIIb-/-), only the central component of the complement system C3 (C3-/-), and mice lacking both FcγRs and C3 (FcγRI/II/III/IV/C3-/-). All mouse models and wild-type controls were fed a high-fat diet (HFD) for 15 weeks to induce obesity. Glucose metabolism was assessed and adipose tissue was characterized for inflammation and adipocyte functionality. RESULTS: In obese WAT of wild-type mice, B cells (+142%, P<0.01) and IgG (+128% P<0.01) were increased compared to lean WAT. Macrophages of FcγRI/II/III/IV-/-mice released lower levels of cytokines compared to wild-type mice upon IgG stimulation. Only C3-/- mice showed reduced HFD-induced weight gain as compared to controls (-18%, P<0.01). Surprisingly, FcγRI/II/III/IV-/- mice had deteriorated glucose tolerance (AUC +125%, P<0.001) despite reduced leukocyte number (-30%, P<0.05) in gonadal WAT (gWAT), whereas glucose tolerance and leukocytes within gWAT in the other models were unaffected compared to controls. Although IgG in gWAT was increased (+44 to +174%, P<0.05) in all mouse models lacking FcγRIIb, only FcγRI/II/III/IV/C3-/- mice exhibited appreciable alterations in immune cells in gWAT, for example, increased macrophages (+36%, P<0.001). CONCLUSIONS: Lack of FcγRs reduces the activity of macrophages upon IgG stimulation, but neither FcγR nor C3 deficiency protects against HFD-induced glucose intolerance or reduces adipose tissue inflammation. This indicates that if obesity-induced IgG contributes to the development of glucose intolerance, this is not mediated by FcγR or complement activation.

Enhanced uptake of blood coagulation factor VIII containing immune complexes by antigen presenting cells.

Essentials Anti-factor (F) VIII antibody formation is a major complication in the treatment of hemophilia A. We investigated uptake of FVIII and FVIII immune complex by bone marrow derived dendritic cells. Immune complex formation increased uptake of FVIII 3-4 fold in a Fcγ receptor dependent manner. FVIII immune complex binding to Fcγ receptors may modulate immune tolerance induction. SUMMARY: Background A major complication in the treatment of hemophilia A is the development of inhibitory antibodies targeting coagulation factor VIII (FVIII). Eradication of these inhibitors can be established by immune tolerance induction (ITI), which consists of daily administration of high dosages of FVIII. FVIII immune complexes (FVIII-IC) could be formed following FVIII infusion in patients with pre-existing anti-FVIII antibodies. Objectives Here we studied endocytosis of FVIII-IC by bone marrow-derived dendritic cells (BMDCs). Methods BMDCs were pulsed with FVIII/FVIII-IC and uptake was assessed by flow cytometry and confocal imaging. Results BMDCs were able to efficiently internalize FVIII-IC in a dose-dependent manner, 3-4-fold more efficiently when compared with equimolar concentrations of non-complexed FVIII. Uptake of FVIII-IC, but not FVIII alone, could be inhibited with anti-Fcγ receptor (FcγR) antibody 2.4G2, indicating functional involvement of FcγR. No internalization of FVIII-IC was observed in BMDCs lacking FcγRI, FcγRIIb, FcγRIII and FcγRIV. Genetic ablation of FcγRIIb, FcγRIII or FcγRIV individually did not affect the ability of anti-FVIII IgG to promote the uptake of FVIII. BMDCs lacking FcγRI showed lower FVIII-IC uptake levels when compared with other single FcγR null BMDCs. Expression of the inhibitory FcγRIIb alone was sufficient to internalize FVIII-IC more efficiently than FVIII. Conclusions FcγR are critical in the internalization of FVIII-IC by BMDCs and multiple FcγR can contribute independently to this process. Our findings provide a basis for future studies to address whether the outcome of ITI is dependent on the interplay between FVIII-IC and inhibitory and activating FcγR.

FcgammaRI (CD64) contributes substantially to severity of arthritis, hypersensitivity responses, and protection from bacterial infection.

The high-affinity receptor for IgG, FcgammaRI, shares its capacity to bind IgG2a immune complexes (IgG2a-IC) with the low-affinity receptor FcgammaRIII and complement factors, hampering the definition of its biological role. Moreover, in vivo, FcgammaRI is occupied by monomeric IgG2a, reducing its accessibility to newly formed IgG2a-IC. By using a variety of FcgammaR(-/-) mice, we demonstrate that in the absence of FcgammaRI, the IgG2a-IC-induced cellular processes of phagocytosis, cytokine release, cellular cytotoxicity, and antigen presentation are impaired. FcgammaRI(-/-) mice showed impaired hypersensitivity responses, strongly reduced cartilage destruction in an arthritis model, and impaired protection from a bacterial infection. We conclude that FcgammaRI contributes substantially to a variety of IgG2a-IC-dependent immune functions and immunopathological responses.

Complement facilitates early prion pathogenesis.

New-variant Creutzfeldt-Jakob disease and scrapie are typically initiated by extracerebral exposure to the causative agent, and exhibit early prion replication in lymphoid organs. In mouse scrapie, depletion of B-lymphocytes prevents neuropathogenesis after intraperitoneal inoculation, probably due to impaired lymphotoxin-dependent maturation of follicular dendritic cells (FDCs), which are a major extracerebral prion reservoir. FDCs trap immune complexes with Fc-gamma receptors and C3d/C4b-opsonized antigens with CD21/CD35 complement receptors. We examined whether these mechanisms participate in peripheral prion pathogenesis. Depletion of circulating immunoglobulins or of individual Fc-gamma receptors had no effect on scrapie pathogenesis if B-cell maturation was unaffected. However, mice deficient in C3, C1q, Bf/C2, combinations thereof or complement receptors were partially or fully protected against spongiform encephalopathy upon intraperitoneal exposure to limiting amounts of prions. Splenic accumulation of prion infectivity and PrPSc was delayed, indicating that activation of specific complement components is involved in the initial trapping of prions in lymphoreticular organs early after infection.

Markedly different pathogenicity of four immunoglobulin G isotype-switch variants of an antierythrocyte autoantibody is based on their capacity to interact in vivo with the low-affinity Fcgamma receptor III.

Using three different Fcgamma receptor (FcgammaR)-deficient mouse strains, we examined the induction of autoimmune hemolytic anemia by each of the four immunoglobulin (Ig)G isotype-switch variants of a 4C8 IgM antierythrocyte autoantibody and its relation to the contributions of the two FcgammaR, FcgammaRI, and FcgammaRIII, operative in the phagocytosis of opsonized particles. We found that the four IgG isotypes of this antibody displayed striking differences in pathogenicity, which were related to their respective capacity to interact in vivo with the two phagocytic FcgammaRs, defined as follows: IgG2a > IgG2b > IgG3/IgG1 for FcgammaRI, and IgG2a > IgG1 > IgG2b > IgG3 for FcgammaRIII. Accordingly, the IgG2a autoantibody exhibited the highest pathogenicity, approximately 20-100-fold more potent than its IgG1 and IgG2b variants, respectively, while the IgG3 variant, which displays little interaction with these FcgammaRs, was not pathogenic at all. An unexpected critical role of the low-affinity FcgammaRIII was revealed by the use of two different IgG2a anti-red blood cell autoantibodies, which displayed a striking preferential utilization of FcgammaRIII, compared with the high-affinity FcgammaRI. This demonstration of the respective roles in vivo of four different IgG isotypes, and of two phagocytic FcgammaRs, in autoimmune hemolytic anemia highlights the major importance of the regulation of IgG isotype responses in autoantibody-mediated pathology and humoral immunity.

A codominant role of Fc gamma RI/III and C5aR in the reverse Arthus reaction.

Recent attempts to specify the relative contribution of FcR and complement in various experimental systems of immune complex disease have led to opposing conclusions. As concluded in IgG FcRgamma-/- mice, manifestation of disease is almost exclusively determined by FcgammaR on effector cells, arguing for a minor role of complement. In contrast, data obtained with C5aR-/- mice suggested that, dependent on the tissue site, complement is more important than FcgammaR. In this paper, we demonstrate that, in response to IgG immune complex formation, FcgammaRI/III- and C5aR-mediated pathways are both necessary and only together are they sufficient to trigger the full expression of inflammation in skin and lung. Moreover, both effector systems are not entirely independent, suggesting an interaction between FcgammaR and C5aR. Therefore, FcgammaR-mediated responses can be integrated through C5aR activation, which may explain why these two receptor pathways have previously been considered to dominate each other.

Mast cells induce autoantibody-mediated vasculitis syndrome through tumor necrosis factor production upon triggering Fcgamma receptors.

The generation of autoantibodies and deposition of immune complexes (ICs) in tissue play a primary role in autoimmune diseases. However, the IC-triggered response consists of complex mechanisms that make it difficult to identify the pathogenesis and develop specific therapy. We clarified here a sequential mechanism for the induction of hypersensitivity angiitis by analyzing the responsible Fc receptor (FcR), effector cells, and mediators in an animal model using FcR-deficient mice. In this model, rheumatoid factor-mediated skin vasculitis was induced in wild-type mice, whereas FcRgamma-deficient mice did not develop the vasculitis. Adoptive transfer of various FcR(+) cells into FcRgamma-deficient mice showed that mast cells but not macrophages derived from wild-type mice triggered skin vasculitis. Mast cells derived from either FcgammaRIII-deficient or tumor necrosis factor (TNF)-deficient mice did not possess the inducibility of skin vasculitis. These results indicate that triggering of vascular inflammation was induced by mast cells through IC binding on FcgammaRIII. TNF produced by such activated mast cells was mainly responsible for the pathogenesis of autoantibody-mediated vasculitis. These findings illustrate the clinical significance of mast cells, Fcgamma receptors, and TNF in IC-induced vasculitis syndrome.

Amyloid fibril formation is progressive and correlates with beta-cell secretion in transgenic mouse isolated islets.

AIMS/HYPOTHESIS: Amyloid fibrils are formed in islets isolated from transgenic mice expressing the gene for human islet amyloid polypeptide (IAPP) by an unknown mechanism. This model of islet amyloidosis in Type II (non-insulin-dependent) diabetes mellitus has been used to investigate the temporal and glucose dependency of fibril formation. METHODS: To determine the time course and nature of amyloid-like accumulations and the role of glucose, transgenic mouse islets were cultured for 2-12 days in medium containing glucose (4.2 mmol/l, 11.1 mmol/l or 16.7 mmol/l) or 3.3 mmol/l glucose plus non-glucose secretagogues, 10 mmol/l leucine, 10 mmol/l leucine + 0.1 mmol/l tolbutamide, 10 mmol/l alpha-ketoisocaproic acid + 10 mmol/l glutamine. The extent of fibril formation was determined by quantitative immuno-electron microscopy. Insulin and islet amyloid polypeptide secretion into the media was measured by radioimmunoassay. RESULTS: Extracellular amyloid fibrils immunoreactive for islet amyloid polypeptide were visible initially after 6 days of culture in 11.1 mmol/l glucose and formed 2.3 +/- 0.8 % of the islet area after 12 days; small accumulations of intracellular fibrils and amorphous extracellular islet amyloid polypeptide-immunoreactive material were present at 6-12 days. Beta-cell secretion was increased significantly by 16.7 mmol/l glucose and by alpha-ketoisocaproic acid + glutamine. The proportion of fibrillar amyloid (amyloid area/islet area%) correlated with the amount of insulin (r = 0.55, p < 0.05) and IAPP (r = 0.5, p < 0.05) in the culture media. Evidence of cellular damage was present in less than 10 % cells and correlated with the degree of fibril deposition (r = 0.8, p < 0.0001). CONCLUSION/INTERPRETATION: These data suggest that islet amyloid polypeptide amyloid is formed primarily at extracellular sites in isolated transgenic mouse islets and progressive fibril formation correlates with beta-cell secretion. [Diabetologia (1999) 42: 1219-1227]

IgG-mediated enhancement of antibody responses is low in Fc receptor gamma chain-deficient mice and increased in Fc gamma RII-deficient mice.

Immunization with IgG/Ag or IgE/Ag complexes leads to a higher production of specific Abs than immunization with Ag alone. The enhancing effect of IgE is exclusively dependent upon the low-affinity receptor for IgE, Fc epsilon RII, whereas the mechanism behind IgG-mediated enhancement is unknown. We have investigated whether receptors for the Fc part of IgG are required for responses to IgG/Ag. Mice lacking the gamma subunit of Fc receptors (FcRs) (FcR gamma-/-), Fc gamma RII (Fc gamma RII-/-), or Fc gamma RIII (Fc gamma RIII-/-) were immunized with BSA-2,4,6-trinitrophenyl (TNP) alone or BSA-TNP complexed to monoclonal TNP-specific IgG1, IgG2a, or IgG2b. As expected, all subclasses enhanced the Ab-response to BSA in wild-type mice. Enhancement was in the same order of magnitude in Fc gamma RIII-/- mice (

Human immunoglobulin A receptor (FcalphaRI, CD89) function in transgenic mice requires both FcR gamma chain and CR3 (CD11b/CD18).

Even though more immunoglobulin A (IgA) is produced in humans than all other isotypes combined, relatively little is known about receptors that bind the Fc part of IgA. The myeloid IgA receptor, FcalphaRI (CD89), triggers various effector functions in vitro, but its in vivo role remains unclear. Here, a transgenic mouse model is described in which FcalphaRI is expressed under its own regulatory sequences. Receptor expression and regulation by cytokines was comparable to the human situation and hFcalphaRI can trigger phagocytosis and lysis of tumor cells. To analyze the contribution of the FcR gamma chain or the beta2 integrin CR3 (CD11b/CD18) in FcalphaRI biological function, FcalphaRI transgenic mice were crossed with either FcR gamma chain -/- or CR3 -/- mice. In contrast to in vitro data, FcR gamma chain was essential for surface expression of hFcalphaRI in vivo. Functional studies in hFcalphaRI/ gamma-/-mice were, therefore, limited. In vitro studies showed FcR gamma chain to be necessary for phagocytosis. Neither hFcalphaRI expression nor phagocytosis, triggered via hFcalphaRI, were influenced by CR3. Remarkably, the capacity to lyse tumor targets was ablated in hFcalphaRI transgenic/ CR3-/- mice, although binding of neutrophils to tumor cells was intact. This shows a previously unrecognized importance of CR3 for hFcalphaRI-mediated antibody-dependent cellular cytotoxicity (ADCC).

FcgammaRIII (CD16)-deficient mice show IgG isotype-dependent protection to experimental autoimmune hemolytic anemia.

In autoimmune hemolytic anemia (AIHA), there is accumulating evidence for an involvement of FcgammaR expressed by phagocytic effector cells, but demonstration of a causal relationship between individual FcgammaRs and IgG isotypes for disease development is lacking. Although the relevance of IgG isotypes to human AIHA is limited, we could show a clear IgG isotype dependency in murine AIHA using pathogenic IgG1 (105-2H) and IgG2a (34-3C) autoreactive anti-red blood cell antibodies in mice defective for FcgammaRIII, and comparing the clinical outcome to those in wild-type mice. FcgammaRIII-deficient mice were completely resistent to the pathogenic effects of 105-2H monoclonal antibody, as shown by a lack of IgG1-mediated erythrophagocytosis in vitro and in vivo. In addition, the IgG2a response by 34-3C induced a less severe but persistent AIHA in FcgammaRIII knock-out mice, as documented by a decrease in hematocrit. Blocking studies indicated that the residual anemic phenotype induced by 34-3C in the absence of FcgammaRIII reflects an activation of FcgammaRI that is normally coexpressed with FcgammaRIII on macrophages. Together these results show that the pathogenesis of AIHA through IgG1-dependent erythrophagocytosis is exclusively mediated by FcgammaRIII and further suggest that FcgammaRI, in addition to FcgammaRIII, contributes to this autoimmune disease when other IgG isotypes such as IgG2a are involved.

Murine IgG1 complexes trigger immune effector functions predominantly via Fc gamma RIII (CD16).

Previously, we have demonstrated that phagocytosis of IgG1-coated particles by macrophages in vitro is impaired by deletion of Fc gamma RIII in mice, suggesting that IgG1 may interact preferentially with Fc gamma RIII. In the present study, the biologic relevance of this observation was addressed by triggering various effector functions of the immune system in Fc gamma RIII(-/-) mice, using panels of mAbs of different IgG subclasses. Both binding and phagocytosis of IgG1-coated sheep or human erythrocytes by Fc gamma RIII(-/-) macrophages in vitro were strongly impaired, indicating that the impaired ingestion of complexed IgG1 by Fc gamma RIII(-/-) macrophages is due to a defect in binding. An in vivo consequence of the defective phagocytosis was observed by resistance of Fc gamma RIII-deficient mice to experimental autoimmune hemolytic anemia, as shown by a lack of IgG1-mediated erythrophagocytosis in vivo by liver macrophages. Furthermore, trapping of soluble IgG1-containing immune complexes by follicular dendritic cells in mesenteric lymph nodes from Fc gamma RIII(-/-) mice was abolished. Whole blood from Fc gamma RIII(-/-) mice was unable to induce lysis of tumor cells in the presence of IgG1 antitumor Abs. Finally, IgG1 mAbs proved unable to mount a passive cutaneous anaphylaxis in Fc gamma RIII(-/-) mice. Together, these results demonstrate that IgG1 complexes, either in particulate or in soluble form, trigger in vitro and in vivo immune effector functions in mice predominantly via Fc gamma RIII.

Targeted overexpression of the neurite growth-associated protein B-50/GAP-43 in cerebellar Purkinje cells induces sprouting after axotomy but not axon regeneration into growth-permissive transplants.

B-50/GAP-43 is a nervous tissue-specific protein, the expression of which is associated with axon growth and regeneration. Its overexpression in transgenic mice produces spontaneous axonal sprouting and enhances induced remodeling in several neuron populations (; ). We examined the capacity of this protein to increase the regenerative potential of injured adult central axons, by inducing targeted B-50/GAP-43 overexpression in Purkinje cells, which normally show poor regenerative capabilities. Thus, transgenic mice were produced in which B-50/GAP-43 overexpression was driven by the Purkinje cell-specific L7 promoter. Uninjured transgenic Purkinje cells displayed normal morphology, indicating that transgene expression does not modify the normal phenotype of these neurons. By contrast, after axotomy numerous transgenic Purkinje cells exhibited profuse sprouting along the axon and at its severed end. Nevertheless, despite these growth phenomena, which never occurred in wild-type mice, the severed transgenic axons were not able to regenerate, either spontaneously or into embryonic neural or Schwann cell grafts placed into the lesion site. Finally, although only a moderate Purkinje cell loss occurred in wild-type cerebella after axotomy, a considerable number of injured transgenic neurons degenerated, but they could be partially rescued by the different transplants placed into the lesion site. Thus, B-50/GAP-43 overexpression substantially modifies Purkinje cell response to axotomy, by inducing growth processes and decreasing their resistance to injury. However, the presence of this protein is not sufficient to enable these neurons to accomplish a full program of axon regeneration.

Impaired IgG-dependent anaphylaxis and Arthus reaction in Fc gamma RIII (CD16) deficient mice.

The family of receptors for IgG (Fc gamma R) plays an essential role in antibody-mediated effector functions of the immune system. However, the specific contribution of each of the Fc gamma R classes to in vivo immune reactions is still unclear. Here, we demonstrate that mice deficient for the ligand-binding alpha chain of Fc gamma RIII lack NK cell-mediated antibody-dependent cytotoxicity and phagocytosis of IgG1-coated particles by macrophages. Strikingly, these mice lack IgG-mediated mast cell degranulation, are resistant to IgG-dependent passive cutaneous anaphylaxis, and exhibit an impaired Arthus reaction. These results indicate a prominent role for Fc gamma RIII in inflammatory and anaphylactic responses, making this receptor a potential target in immunotherapy.

FcR gamma-chain is essential for both surface expression and function of human Fc gamma RI (CD64) in vivo.

Most Ig receptors exist as hetero-oligomeric complexes with separate ligand binding (alpha) and signal transducing (beta, gamma, or zeta) subunits. For Fc gamma RIIIa and Fc epsilon RI, association with the FcR gamma-chain is essential for surface expression. However, the human high affinity IgG receptor, hFc gamma RI, was found to be surface-expressed by itself in transient transfection models. We have now analyzed the integrity of hFc gamma RI expression in more detail in stable transfectants. In vitro we noted that, in the absence of FcR gamma-chain, surface expression of hFc gamma RI rapidly declined to background levels, in both IIA1.6 B cells and NIH3T3 fibroblasts. The effect of FcR gamma-chain on hFc gamma RI surface expression in vivo was evaluated by using two newly generated transgenic mouse lines, selectively expressing hFc gamma RI on myeloid cells. These transgenic mice were crossed with FcR gamma-chain-deficient mice. Analysis of blood monocytes and peritoneal macrophages showed that surface expression of hFc gamma RI was reduced by approximately 80%. The remaining approximately 20% of receptors were still capable of binding IgG-opsonized RBC, suggesting FcR gamma-chain not to be critical for hFc gamma RI ligand-binding capacity. Importantly, however, hFc gamma RI signaling capacity was lost in FcR gamma-chain-deficient cells. No phagocytosis could be observed using either ligand sensitized (EA-IgG2a) or CD64-targeted erythrocytes (using a bispecific antibody) in both hFc gamma RI transgenic lines. This documents the FcR gamma-chain to be indispensable for both surface membrane expression and function of human Fc gamma RI in vivo.

Antigen targeting to myeloid-specific human Fc gamma RI/CD64 triggers enhanced antibody responses in transgenic mice.

Besides their phagocytic effector functions, myeloid cells have an essential role as accessory cells in the induction of optimal humoral immune responses by presenting captured antigens and activating lymphocytes. Antigen presentation by human monocytes was recently found to be enhanced in vitro through the high-affinity Fc receptor for IgG (Fc gamma RI; CD64), which is exclusively present on myeloid cells. To evaluate a comparable role of Fc gamma RI in antigen presentation in vivo, we generated human Fc gamma RI transgenic mice. Under control of its endogenous promoter, human Fc gamma RI was selectively expressed on murine myeloid cells at physiological expression levels. As in humans, expression was properly regulated by the cytokines IFN-gamma, G-CSF, IL-4, and IL-10, and was up-regulated during inflammation. The human receptor expressed by murine macrophages bound monomeric human IgG and mediated particle phagocytosis and IgG complex internalization. To evaluate whether specific targeting of antigens to Fc gamma RI can induce enhanced antibody responses, mice were immunized with an anti-human Fc gamma RI antibody containing antigenic determinants. Transgenic mice produced antigen-specific antibody responses with high IgG1 titers and substantial IgG2a and IgG2b responses. These data demonstrate that human Fc gamma RI on myeloid cells is highly active in mediating enhanced antigen presentation in vivo, and show that anti-Fc gamma RI mAbs are promising vaccine adjuvants.

Functional consequences of the interaction between T-cell antigen receptors and Fc gamma Rs on T cells.

Two series of experiments are presented indicating that Fc gamma II receptors can interfere with the antigen receptor-induced signaling on T cells. It has been previously described that a 13 amino acid motif on the cytoplasmic portion of Fc gamma II beta 1 can abrogate the antigen receptor-initiated signals mediated through consensus motifs present on the cytoplasmic portion of Ig alpha and Ig beta chains. Similar activating motifs are crucial to T-cell receptor (TCR) signaling. A splenic gamma/delta T-cell hybridoma that expressed the Fc gamma RII beta 1 receptor helped to establish that this receptor can also interfere with TCR-induced activation. The cytoplasmic portion of human Fc gamma RIIa has an activation motif similar to the activation motifs present on the TCR. Using a transgenic mouse in which the T cells express the human Fc gamma RIIa transgene, we demonstrated that despite the common activation motif, the TCR and human Fc gamma RIIa-induced signals are different. Additionally, the human Fc gamma RIIa expressing T cells exhibit an enhanced TCR response both in vitro and in vivo.

Intra- and extracellular amyloid fibrils are formed in cultured pancreatic islets of transgenic mice expressing human islet amyloid polypeptide.

Islet amyloid polypeptide (IAPP) is the constituent peptide of amyloid deposits found in the islets of non-insulin-dependent diabetic patients. Formation of islet amyloid is associated with a progressive destruction of insulin-producing beta cells. Factors responsible for the conversion of IAPP into insoluble amyloid fibrils are unknown. Both the amino acid sequence of human IAPP (hIAPP) and hypersecretion of hIAPP have been implicated as factors for amyloid fibril formation in man. We have generated transgenic mice using rat insulin promoter-hIAPP or rat IAPP (rIAPP) gene constructs. No fibrillar islet amyloid was detectable in vivo in these normoglycemic mice, although small amorphous perivascular accumulations of IAPP were observed in hIAPP mice only. To determine the effects of glucose on IAPP secretion and fibrillogenesis, pancreatic islets from transgenic and control mice were examined in vitro. Islet IAPP secretion and content were increased in transgenic islets compared with control islets. IAPP-immunoreactive fibrils were formed at both intra- and extracellular sites in isolated hIAPP islets cultured with glucose at 11.1 and 28 mM for only 7 days. At 28 mM glucose, fibrils were present in deep invaginations of beta cells as observed in non-insulin-dependent diabetic patients. No fibrils were present at low glucose concentrations in hIAPP islets or at any glucose concentration in rIAPP or control islets. Thus, glucose-induced expression and secretion of hIAPP in transgenic mouse islets can lead to formation of amyloid fibrils similar to that found in non-insulin-dependent diabetes mellitus.