Biological Activities of Murine Low-Affinity Fc Receptors for IgG

Murine low-affinity Fc receptors for IgG (FcγRIIbl, FcγRIIb2, and FcγRIII) bind the same IgG subclasses and are not distinguished by available anti-FcγRII/III mAbs (2.4G2). They trigger various biological activities, among which are the internalization of soluble and particulate immune complexes, cell activation, and its regulation. To determine the biological properties of the three murine receptors, each was expressed by stable transfection of corresponding cDNAs in two model cells: the murine lymphoma B cell IIA1.6 and the rat basophilic leukemia cell RBL-2H3. Biological activities of recombinant receptors were triggered with soluble immune complexes or 2.4G2 IgG in IIA1.6 cells, which express no FcγR, and with 2.4G2 Fab or F(ab′)₂, cross-linked with mouse anti-rat F(ab′)₂ in RBL, which express rat FcγR. Conditions for studying cell activation and endocytosis in both cell models are described, as are conditions for studying phagocytosis in RBL cells and antigen presentation or regulation of cell activation in IIA1.6 cells. Internalization of immune complexes was triggered by FcγRIIb2 and FcγRIII, but not by FcγRIIb1. Intracytoplasmic sequences required for phagocytosis and endocytosis could be distinguished in FcγRIIb2, but not in FcγRIII. Cell activation was restricted to FcγRIII. FcγRIII-mediated endocytosis, phagocytosis, and cell activation involved the consensus tyrosine-containing activation motif found in the intracytoplasmic domain of the γ subunit. Regulation of cell activation was induced by both FcγRII isoforms and depended on the same sequence as endocytosis. As a consequence, a single motif can determine more than one biological response of the cell, and a given response may be triggered by several motifs, borne by different FcγR.     

The Fc receptor for IgG expressed in the villus endothelium of human placenta is Fc gamma RIIb2

To evaluate the potential role of human placental endothelial cells in the transport of IgG from maternal to fetal circulation, we studied Fc gamma receptor (Fc gamma R) expression by immunohistology and immunoblotting. Several pan-Fc gamma RII Abs that label the placental endothelium displayed a distribution pattern that correlated well with transport functions, being intense in the terminal villus and nil in the cord. In contrast, the MHC class 1-like IgG transporter, FcRn, and the classical Fc gamma RIIa were not expressed in transport-related endothelium of the placenta. Our inference, that Fc gamma RIIb was the likely receptor, we confirmed by analyzing purified placental villi, enriched in endothelium, by immunoblotting with a new Ab specific for the cytoplasmic tail of Fc gamma RIIb. These experiments showed that the Fc gamma RII expressed in villus endothelium was the b2 isoform whose cytoplasmic tail is known to include a phosphotyrosyl-based motif that inhibits a variety of immune responses. We suggest that this receptor is perfectly positioned to transport IgG although as well it may scavenge immune complexes.     

Antibody production in mice deficient for complement receptors 1 and 2 can be induced by IgG/Ag and IgE/Ag, but not IgM/Ag complexes

Deficiencies in C factors C2, C3, or C4 as well as lack of C receptors 1 and 2 (CR1/2) lead to impaired Ab production. Classical pathway activation plays a major role, as mice deficient in factor B, a key factor in the alternative pathway, have normal Ab production. Abs in complex with their specific Ag are known to feedback regulate the Ab response, and enhanced responses are initiated by IgM, IgE, and IgG. IgM acts via the C system, whereas IgE and IgG can operate independently of C via Fc receptors. Here we have investigated whether these isotypes are able to enhance Ab responses in mice lacking CR1/2. SRBC-specific IgM, administered with SRBC, does not enhance Ab responses in these animals. In contrast, 2,4, 6-trinitrophenyl-specific IgE and IgG2a, administered with BSA-2,4, 6-trinitrophenyl, induce potent Ab responses in CR1/2-deficient mice. Additionally, BSA administered with CFA or alum induced strong Ab responses in the absence of CR1/2. These results indicate that CR1/2 is needed to promote IgM-mediated induction of primary Ab responses. The data also show that the need for CR1/2 can be circumvented by Abs typical of a secondary immune response forming complexes with Ag or by conventional adjuvants, presumably mimicking physiological inflammatory reactions.     

IgG2a-mediated enhancement of antibody and T cell responses and its relation to inhibitory and activating Fc gamma receptors

A number of studies in experimental animal models point to an important role of Fc gamma Rs in autoimmunity and allergy. In this study, we investigate how the production of IgG, an early step in the chain of events leading to inflammation, is regulated by activating and inhibitory Fc gamma Rs. IgG Abs are known to feedback-enhance Ab responses to soluble Ags, and this effect requires activating Fc gamma Rs. To test proliferation of Th cells, mice were adoptively transferred with CD4(+) T cells expressing a transgenic OVA-specific TCR before immunization with IgG2a anti-2,4,6-trinitrophenyl (TNP) plus OVA-TNP or with OVA-TNP alone. IgG2a induced a significant increase in OVA-specific T cell numbers, which preceded the OVA-specific Ab response and was dependent on the Fc gamma chain. The role of the inhibitory Fc gamma RIIB in Ab responses was studied in mice lacking this receptor. Although IgG2a enhanced primary Ab responses, development of germinal centers, and immunological memory in wild-type mice, enhancement was markedly stronger in Fc gamma RIIB(-/-) mice. The presented data are compatible with the hypothesis that the mechanism behind IgG2a-mediated up-regulation of Ab responses involves increased Ag presentation to CD4(+) T cells by Fc gamma R(+) APCs. Our observations also illustrate the intricate immunoregulatory role of IgG Abs. On the one hand, they enhance Ab responses via activating Fc gamma Rs, and on the other hand, they set an upper limit for the same Ab response via Fc gamma RIIB.     

Characterization and crystallization of soluble human Fc gamma receptor II (CD32) isoforms produced in insect cells.

Fc gamma RII (CD32), the receptor for the Fc part of IgG, is responsible for the clearance of immunocomplexes by macrophages and plays a role in the regulation of antibody production by B cells. To investigate the process of immunocomplex binding in terms of stoichiometry and stability of the Fc gamma RII:IgG complex, we produced both Fc gamma RII isoforms (Fc gamma RIIa and Fc gamma RIIb) as soluble proteins in insect cells. The expressed proteins could be purified in high yields and were biologically active as judged by their ability to bind IgG. Thus, the minor glycosylation performed by the insect cells is not crucial for the binding of the usually highly glycosylated Fc gamma RII to IgG. The dissociation constant of the sFc gamma RIIa:IgG-hFc complex was determined by fluorescence titration (KD = 2.5 x 10(-)7 M). Complementary sFc gamma RIIa antagonizes immunocomplex binding to B cells. Here sFc gamma RIIa showed a comparable dissociation constant (KD = 1.7 x 10(-)7 M) which was almost 10-fold lower than the constant for Fc gamma RIIb. The stoichiometry of the FcRIIa:IgG-hFc complex was determined by equilibrium gel filtration and shows that IgG is able to bind alternatively one or two Fc gamma RII molecules in a noncooperative manner. Furthermore, in an ELISA-based assay the isotype specificity of various anti-Fc gamma RII monoclonal antibodies was measured as well as their ability to interfere with the IgG recognition through its receptors. To further investigate the molecular basis of the Fc gamma RII-ligand interaction, we crystallized Fc gamma RIIb. Trigonal crystals diffracted to 3 A and the structure solution is in progress.     

Cutting edge: biasing immune responses by directing antigen to macrophage Fc gamma receptors

An immune response can deviate toward either a Th1- or Th2-like response. In this work we examine the contribution that activated macrophages and IgG Abs make toward this deviation. The use of activated macrophages as APCs resulted in a strong polarized T cell response that was predominated by IFN-gamma. However, when Ag was targeted to FcgammaRs on these macrophages, the T cell response was reversed and biased toward a Th2-like response. This Th2-like phenotype was stable and was retained when the T cells were subsequently restimulated under nonbiasing conditions. The T cell biasing and its reversal via FcgammaR was also observed in vivo. Mice vaccinated with IgG-opsonized OVA made high levels of IgG Ab of the IgG1 isotype. These studies demonstrate that the ligation of FcgammaR on activated macrophages can reverse the Th1 biasing that occurs as a result of innate immune responses to microbial products.     

T cell-independent regulation of IgE antibody production induced by surface-linked liposomal antigen

Control of IgE Ab production is important for the prevention of IgE-related diseases. However, in contrast to the existing information on the induction of IgE production, little is known about the regulation of the production of this isotype, with the exception of the well-documented mechanism involving T cell subsets and their cytokine products. In this study, we demonstrate an alternative approach to interfere with the production of IgE, independent of the activity of T cells, which was discovered during the course of an investigation intended to clarify the mechanism of IgE-selective unresponsiveness induced by surface-coupled liposomal Ags. Immunization of mice with OVA-liposome conjugates induced IgE-selective unresponsiveness without apparent Th1 polarization. Neither IL-12, IL-10, nor CD8(+) T cells participated in the regulation. Furthermore, CD4(+) T cells of mice immunized with OVA-liposome were capable of inducing Ag-specific IgE synthesis in athymic nude mice immunized with alum-adsorbed OVA. In contrast, immunization of the recipient mice with OVA-liposome did not induce anti-OVA IgE production, even when CD4(+) T cells of mice immunized with alum-adsorbed OVA were transferred. In the secondary immune response, OVA-liposome enhanced anti-OVA IgG Ab production, but it did not enhance ongoing IgE production, suggesting that the IgE-selective unresponsiveness induced by the liposomal Ag involved direct effects on IgE, but not IgG switching in vivo. These results suggest the existence of an alternative mechanism not involving T cells in the regulation of IgE synthesis.     

Interaction with FcγRIIB is critical for the agonistic activity of anti-CD40 monoclonal antibody

A high activatory/inhibitory FcγR binding ratio is critical for the activity of mAb such as rituximab and alemtuzumab that attack cancer cells directly and eliminate them by recruiting immune effectors. Optimal FcγR binding profiles of other anti-cancer mAb, such as immunostimulatory mAb that stimulate or block immune receptors, are less clear. In this study, we analyzed the importance of isotype and FcγR interactions in controlling the agonistic activity of the anti-mouse CD40 mAb 3/23. Mouse IgG1 (m1) and IgG2a (m2a) variants of the parental 3/23 (rat IgG2a) were engineered and used to promote humoral and cellular responses against OVA. The mouse IgG1 3/23 was highly agonistic and outperformed the parental Ab when promoting Ab (10-100-fold) and T cell (OTI and OTII) responses (2- to >10-fold). In contrast, m2a was almost completely inactive. Studies in FcγR knockout mice demonstrated a critical role for the inhibitory FcγRIIB in 3/23 activity, whereas activatory FcγR (FcγRI, -III, and -IV) was dispensable. In vitro experiments established that the stimulatory effect of FcγRIIB was mediated through Ab cross-linking delivered in trans between neighboring cells and did not require intracellular signaling. Intriguingly, activatory FcγR provided effective cross-linking of 3/23 m2a in vitro, suggesting the critical role of FcγRIIB in vivo reflects its cellular distribution and bioavailability as much as its affinity for a particular Ab isotype. In conclusion, we demonstrate an essential cross-linking role for the inhibitory FcγRIIB in anti-CD40 immunostimulatory activity and suggest that isotype will be an important issue when optimizing reagents for clinical use.     

Murine recombinant Fc gamma RIII, but not Fc gamma RII, trigger serotonin release in rat basophilic leukemia cells.

Murine Fc gamma RII and Fc gamma RIII have highly homologous extracellular domains, but unrelated transmembrane and intracytoplasmic (IC) domains. Murine Fc gamma RIIb1 and b2 are two isoforms of single-chain receptors which differ only by 47 aa in their IC domain. Murine Fc gamma RIII are composed of an IgG-binding alpha-chain, the intracellular portion of which is unrelated to that of Fc gamma RII, and of a homodimeric gamma-chain which also associates with Fc epsilon RI. Murine mast cells express Fc gamma RII, Fc gamma RIII, and Fc epsilon RI. They can be induced to degranulate by murine IgG immune complexes or by F(ab')2 fragments of the rat anti-murine Fc gamma RII/III mAb 2.4G2, complexed to mouse anti-rat (MAR) F(ab')2. In order to determine which murine Fc gamma R can activate mast cells, cDNA encoding murine Fc gamma RIIb1, Fc gamma RIIb2 or Fc gamma RIII alpha were stably transfected into RBL-2H3 cells. Murine Fc gamma RIII but not Fc gamma RIIb1 or Fc gamma RIIb2 induced serotonin release when aggregated by (2.4G2-MAR) F(ab')2 complexes. The respective roles of the IC domains of murine Fc gamma RIII subunits in signal transduction were investigated by stably transfecting cDNA encoding IC-deleted or chimeric murine Fc gamma R into RBL-2H3 cells. The substitution of the IC domain of murine Fc gamma RII for that of murine Fc gamma RIII gamma, but not that of murine Fc gamma RIII alpha, conferred the ability to trigger serotonin release. The deletion of IC sequences of the alpha subunit did not alter the ability of murine Fc gamma RIII to trigger serotonin release. It follows that 1) murine Fc gamma RIII, but not Fc gamma RII, can induce RBL cells to release serotonin, 2) the aggregation of the IC domain of the murine Fc gamma RIII gamma subunit is sufficient, but 3) the IC domain of the murine Fc gamma RIII alpha subunit is neither sufficient nor necessary for triggering serotonin release.     

Activation of human peripheral IgM+ B cells is transiently inhibited by BCR-independent aggregation of Fc gammaRIIB

Immune complexes can trigger a SHIP-1-independent proapoptotic signal in mouse class-switched IgG(+) B cells and plasma cells by binding to Fc gammaRIIB, in the absence of concomitant coaggregation with BCR, hence regulating plasma cell survival and participating in the selection of B cells producing high affinity Abs during secondary Ab responses. By contrast, we demonstrate in the present study that the unique aggregation of Fc gammaRIIB on human peripheral IgM(+) B cells does not induce apoptosis but transiently inhibits B cell proliferation and calcium influx triggered by BCR cross-linking. Using human peripheral B cells and IIA1.6 lymphoma B cells expressing wild-type human Fc gammaRIIB (IIA1.6-Fc gammaRIIB), we also show that the unique aggregation of human Fc gammaRIIB induces ITIM phosphorylation. This aggregation provokes the recruitment of phosphorylated SHIP-1 by Fc gammaRIIB and inhibits the constitutive phosphorylation of Akt in human IIA1.6-Fc gammaRIIB cells. This inhibitory signaling pathway is abrogated in IIA1.6 cells expressing ITIM-mutated Fc gammaRIIB (Fc gammaRIIB(Y292G)), suggesting that ITIM phosphorylation is necessary for Fc gammaRIIB-induced B cell blockade. Overall, we demonstrate that the unique aggregation of Fc gammaRIIB on human peripheral IgM(+) B cells is sufficient to transiently down-regulate their activation without inducing apoptosis. Our results suggest that Fc gammaRIIB could negatively regulate IgM(+) B cells before class-switch occurrence and that its unique engagement by immune complexes represents a reversible checkpoint for peripheral IgM(+) B cells.     

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 (相似文献   

Modulation of mouse complement receptors 1 and 2 suppresses antibody responses in vivo

A mAb, 7G6, that binds to mouse CR1 and CR2 and down-modulates their expression on splenic B cells in vivo, was used to determine whether a decrease in CR1 and CR2 expression affects antibody responses to different T-dependent and T-independent Ag. Injection of mice with the mAb 7G6 prior to immunization with FITC haptenated Salmonella typhimurium (SH5771), Salmonella montevideo (SH5770), SRBC, or Ficoll dramatically decreased subsequent antibody responses to FITC. Although both IgM and IgG primary antibody responses were affected similarly, the antibody levels were most inhibited during early phases of the response. In contrast, down-modulation of the CR did not affect memory antibody responses, because injection of mice with 7G6 before a second immunization with FITC-SH5771 had no effect on subsequent anti-FITC antibody production. Moreover, polyclonal in vivo activation of the mouse immune system by anti-mouse IgD antibodies was not affected by previous administration of 7G6, because anti-IgD-induced increases in Ia expression and serum IgG1 levels were not affected. Taken together, these observations suggest that CR1 and CR2 may play an important role in enhancing primary antibody responses to many T-dependent and T-independent Ag and may contribute to a host's response to naturally occurring antigens such as bacteria.     

Antigen-induced Ca2+ signaling and desensitization in B cells

Cross-linking of B cell surface Ig (sIg) by anti-Ig results in transmembrane signaling. However, the capacity of a thymus-dependent (TD) Ag to mediate B cell signal transduction has been less well documented. Therefore, we examined Ag-induced intracellular free calcium concentration [( Ca2+]) in B cells by using TD Ag that would be expected to either cross-link or not cross-link sIgM and/or induce the coupling of sIgM to FcR. Stimulation of mouse TA3 hybridoma B cell transfectants that express the SP6 anti-TNP specific sIgM with either TNP-OVA or anti-IgM antibodies resulted in a maximal fourfold increase in [Ca2+]i. The net increase in [Ca2+]i in response to TNP-OVA was dependent upon both the Ag dose and the TNP:OVA molar ratio. Because occupancy of several cell-surface receptor types leads to a loss of response to subsequent stimulation by ligand (homologous desensitization), we examined the ability of Ag to induce homologous desensitization of sIgM in these B cells. TNP1-OVA at all concentrations tested (up to 500 micrograms/ml) did not lead to any change in [Ca2+]i or desensitization. Cross-linking of TNP1-OVA (10 micrograms/ml) with F(ab')2 of anti-OVA antibody induced both a rise in [Ca2+]i and homologous desensitization of sIg, suggesting that cross-linking of sIgM by Ag is sufficient to induce both these processes. TNP6-OVA at a concentration of 10 micrograms/ml induced changes in [Ca2+]i and partially desensitized TNP-specific B cells to stimulation by anti-IgM. Interestingly, a high dose (180 micrograms/ml) of TNP6-OVA stimulated minimal changes in [Ca2+]i yet did not lead to desensitization. However, cross-linking of TNP6-OVA at this high dose with F(ab')2 of rabbit anti-OVA elevated [Ca2+]i and elicited partial desensitization. Complete desensitization of sIgM by Ag was achieved when intact (Fc-containing) anti-OVA antibody was used, suggesting that the FcR can play a role in desensitization. Ag- and antibody-mediated desensitization was not caused by steric hindrance of sIg. Thus, we have observed two forms of Ag-induced desensitization of sIgM, both of which involve sIg cross-linking and one of which is mediated by the physiologic coupling of sIg to FcR.     

Fc receptors on cultured myeloma and hybridoma cells

The specificity of the Fc gamma receptors on the X63.Ag8.653 nonproducing myeloma cell line has been examined for binding to IgG1-, IgG2a-, and IgG2b-containing antigen-antibody complexes. Complexes containing each of these subclasses bind, and the binding of each is inhibited by the others. Trypsin treatment did not inhibit the binding of any of these subclasses. Furthermore, the monoclonal anti-Fc receptor antibody 2.4G2 inhibits the binding of all three subclasses. These results, together with those of other investigators, suggest that there is a single FcR for IgG1, IgG2a, and IgG2b on mouse B cells which differs in its specificity from the macrophage Fc gamma R. This is confirmed by the fact that a mutant IgG2b myeloma protein which binds to the macrophage Fc gamma 1/gamma 2b receptor does not bind to the Fc gamma R on X63.Ag8.653.     

Release of arachidonic acid by stimulation of opsonic receptors in human monocytes: the FcgammaR and the complement receptor 3 pathways

The role of the opsonic receptors FcgammaR and CR3 on the release of arachidonic acid (AA) by human monocytes was studied using IgG-ovalbumin (OVA) equivalence immune complexes (IC), anti-OVA IgG bound to OVA-coupled latex beads, and C3bi-bound IC. Release of AA was produced by IC and latex-OVA beads bound to IgG, whereas binding of C3bi to IC inhibited the ability of IC to release AA. In contrast, coating of zymosan particles with C3bi enhanced AA release as compared with that produced by non-coated particles. Masking of C3bi on C3bi-bound IC by incubation with anti-C3 IgG resulted in the recovery of their ability to release AA, thereby suggesting that binding of C3b by IC reduces their flogogenic effects, whereas opsonization of microbial walls by complement may enhance their proinflammatory potential. The binding/uptake of opsonized zymosan particles was inhibited by anti-CR3 Ab and C3bi-bound IC, but not by beta-glucan, mannan, and anti-Toll-like receptor 2 Ab. These findings show that cooperative engagement of CR3 on both the lectin-like site involved in beta-glucan binding and the I-domain involved in C3bi binding, as it can be observed in the innate immune response, produces AA release, whereas the unique interaction of C3bi-bound IC with the I-domain of CR3, as it may occur in the adaptive immune response, diverts the IC lattice from a productive interaction with FcgammaR linked to AA release.     

Inhibition of tumor rejection by gammadelta T cells and IL-10

Although many tumors express tumor-specific antigens, most fail to stimulate effective immune responses. Tumors generally lack co-stimulatory molecules, which can lead to tolerance of tumor-specific T cells and progressive tumor growth. Here, we demonstrate that the ovalbumin (OVA) transfected EL4 tumor, E.G7-OVA, grows progressively in syngeneic mice even though the tumor can be rejected if the mice are immunized with OVA in adjuvant. E.G7-OVA grew more rapidly in RAG-1 deficient than sufficient mice suggesting that normal mice make an abortive immune response to this tumor. Depletion of gammadelta T cells or IL-10 augmented the ability of B6 mice to reject E.G7-OVA. Spleen cells from normal, but not IL-10 knockout, mice reconstituted rapid tumor growth in gammadelta T cell-deficient mice. Thus, gammadelta T cells play an important role in preventing immune elimination of this tumor by a mechanism that directly or indirectly involves IL-10.     

Differential modulation of stimulatory and inhibitory Fc gamma receptors on human monocytes by Th1 and Th2 cytokines

Immune complex-mediated inflammatory responses are initiated by Fc gamma R on phagocytes. We report in this study that an inhibitory receptor, Fc gamma RIIb2, is expressed on circulating human monocytes, and when co-cross-linked with stimulatory Fc gamma R it down-regulates effector function. Fc gamma RIIb2 expression is increased by IL-4 and decreased by IFN-gamma, in contrast to the activating receptor, Fc gamma RIIa, which is increased by IFN-gamma and decreased by IL-4. Thus, Th1 and Th2 cytokines differentially regulate the opposing Fc gamma R systems, altering the balance of activating and inhibiting Fc gamma R. The detection and cytokine modulation of Fc gamma RIIb2 in human myeloid cells provide evidence of a negative regulator of immune complex-mediated responses in human phagocytes and offer a new approach to limit Ab-triggered inflammation in autoimmune disease.     

Fc gamma receptor IIB on follicular dendritic cells regulates the B cell recall response

Generation of the B cell recall response appears to involve interaction of Ag, in the form of an immune complex (IC) trapped on follicular dendritic cells (FDCs), with germinal center (GC) B cells. Thus, the expression of receptors on FDC and B cells that interact with ICs could be critical to the induction of an optimal recall response. FDCs in GCs, but not in primary follicles, express high levels of the IgG Fc receptor Fc gamma RIIB. This regulated expression of Fc gamma RIIB on FDC and its relation to recall Ab responses were examined both in vitro and in vivo. Trapping of IC in spleen and lymph nodes of Fc gamma RII-/- mice was significantly reduced compared with that in wild-type controls. Addition of ICs to cultures of Ag-specific T and B cells elicited pronounced Ab responses only in the presence of FDCs. However, FDCs derived from Fc gamma RIIB-/- mice supported only low level Ab production in this situation. Similarly, when Fc gamma RIIB-/- mice were transplanted with wild-type Ag-specific T and B cells and challenged with specific Ag, the recall responses were significantly depressed compared with those of controls with wild-type FDC. These results substantiate the hypothesis that FcgammaRIIB expression on FDCs in GCs is important for FDCs to retain ICs and to mediate the conversion of ICs to a highly immunogenic form and for the generation of strong recall responses.     

Interleukin-2/liposomes potentiate immune responses to a soluble protein cancer vaccine in mice

A critical element in improving the potency of cancer vaccines, especially pure protein or peptide antigens, is to develop procedures that can strongly but safely increase their ability to induce immune responses. Here, we describe that encapsulation of a pure protein antigen and interleukin-2 (IL-2) together into liposomes significantly improves immune responses and tumor protection. Groups of C57Bl/6 mice were immunized weekly ×4 with –0.1 mg of ovalbumin (OVA) injected subcutaneously in PBS or encapsulated in liposomes with or without human recombinant IL-2. Control groups included mice immunized to irradiated E.G7-OVA cells (that express ovalbumin), or to PBS. Sera were collected and pooled by immunization group at baseline and at weeks 2 and 4 to measure antibody responses to OVA by ELISA. Splenocytes obtained at week 4 were tested for anti-OVA cellular responses by ELISPOT. Mice were then challenged to a lethal dose of E.G7-OVA cells to measure tumor-protective immunity. IL-2 liposomes caused no detectable toxicity. Antibody, CD8⁺ T cell, and tumor-protective immune responses were markedly enhanced in mice immunized to OVA + IL-2 in liposomes compared to mice immunized to OVA, either alone or encapsulated into liposomes without IL-2. These results indicate that IL-2 liposomes enhance antibody, cellular, and tumor-protective immune responses to immunization with a soluble protein. This may provide a simple, safe, and effective way to enhance the immunogenicity of vaccines that consist of pure protein antigens. Supported by grant CA096804 (DJ)     

Characterization of the antibody response against NeuGcGM3 ganglioside elicited in non-small cell lung cancer patients immunized with an anti-idiotype antibody

1E10 mAb is an anti-Id murine mAb (Ab2 mAb) specific for an Ab1 mAb that reacts with NeuGc-containing gangliosides, sulfatides, and Ags expressed in some human tumors. In preclinical studies, this Ab2 Ab was able to mimic NeuGc-containing gangliosides only in animals lacking expression of these Ags in normal tissues. In this study, we report on the immune responses elicited in 20 non-small cell lung cancer patients treated with 1 mg of aluminum hydroxide-precipitated 1E10 mAb. In the hyperimmune sera from 16 of 20 patients, a strong specific Ab response of both IgM and IgG isotypes against NeuGcGM3 ganglioside was observed. Patient immune sera were able to induce complement-independent cell death of NeuGcGM3-expressing X63 murine myeloma target cells. Significant immunoreactivity to NeuGcGM3 was still detected after the complete abrogation of the reactivity against 1E10 mAb by the adsorption of patient sera with this Ab. We hypothesize that Id(-)Ag(+) Abs could reflect the activation of an autologous idiotypic cascade into the patients. Both Id(+)Ag(+) and Id(-)Ag(+) fractions were separated by affinity chromatography and characterized. Although IgG isotype Abs were found in both fractions, IgM isotype Abs were found only in the Id(-)Ag(+) fraction. Both Id(+)Ag(+) and Id(-)Ag(+) Abs were able to specifically recognize and induce cell death in NeuGcGM3-expressing X63 myeloma target cells. Patients that developed IgG and/or IgM Abs against NeuGcGM3 showed longer median survival times.