Regulation of an in vitro anti-DNA antibody response by thymocytes and T cells from NZB/W and normal mice

The spontaneous in vitro anti-DNA antibody response generated by preautoimmune and many normal mouse spleen cells was suppressed by the addition of syngeneic thymocytes or splenic T cells. Suppressive activity was found in normal mice (DBA/2J) and to an equivalent degree in the autoimmune (New Zealand Black X New Zealand White)F1 (B/W) strain. The suppressor cells were cortisone-resistant, radiosensitive and carried Lyt 1 and Lyt 2 markers. Nonspecific suppression was not involved since the primary and primed in vitro anti-sheep erythrocyte (anti-SRBC) responses were unaffected. Both spontaneous and lipopolysaccharide-stimulated anti-DNA antibody responses could be suppressed. There was no difference in the suppressive activity of cells from young or old, normal or autoimmune mice. These T cells may therefore play a role in preventing the anti-DNA antibody response in normal and young B/W mice, but evidently fail to influence the development of in vivo anti-DNA autoimmune responses in the old B/W mice.     

Production of high affinity autoantibodies in autoimmune New Zealand Black/New Zealand white F1 mice targeted with an anti-DNA heavy chain

Lupus-prone, anti-DNA, heavy (H) chain "knock-in" mice were obtained by backcrossing C57BL/6 mice, targeted with a rearranged H chain from a VH11(S107)-encoded anti-DNA hybridoma (D42), onto the autoimmune genetic background of New Zealand Black/New Zealand White (NZB/NZW) F1 mice. The targeted female mice developed typical lupus serologic manifestations, with the appearance of transgenic IgM anti-DNA autoantibodies at a young age (2-3 mo) and high affinity, somatically mutated IgM and IgG anti-DNA Abs at a later age (6-7 mo). However, they did not develop clinical, lupus-associated glomerulonephritis and survived to at least 18 mo of age. L chain analysis of transgenic anti-DNA Abs derived from diseased NZB/NZW mouse hybridomas showed a very restricted repertoire of Vkappa utilization, different from that of nonautoimmune (C57BL/6 x BALB/c)F1 transgenic anti-DNA Abs. Strikingly, a single L chain was repetitively selected by most anti-DNA, transgenic NZB/NZW B cells to pair with the targeted H chain. This L chain had the same Vkappa-Jkappa rearrangement as that expressed by the original anti-DNA D42 hybridoma. These findings indicate that the kinetics of the autoimmune serologic manifestations are similar in wild-type and transgenic lupus-prone NZB/NZW F1 mice and suggest that the breakdown of immunologic tolerance in these mice is associated with the preferential expansion and activation of B cell clones expressing high affinity anti-DNA H/L receptor combinations.     

Aberrant genetic control of invariant TCR-bearing NKT cell function in New Zealand mouse strains: possible involvement in systemic lupus erythematosus pathogenesis

Both suppressive and promoting roles of NKT cells have been reported in the pathogenesis of systemic lupus erythematosus (SLE). Herein, we found that although New Zealand mice have normal frequencies of NKT cells, their in vitro potential to produce IL-4 and IFN-gamma in response to alpha-galactosylceramide was remarkably impaired in New Zealand Black (NZB) mice prone to mild SLE, while production was highly up-regulated in nonautoimmune New Zealand White (NZW) mice and at intermediate levels in (NZB x NZW)F(1) mice, which are prone to severe SLE. Because this aberration is evident in young mice before disease onset, genetic mechanisms are thought to be involved. Genome-wide quantitative trait locus analysis and association studies revealed that a locus linked to D11Mit14 on chromosome 11 may be involved in the difference in cytokine-producing potential between NZB and NZW NKT cells. Additionally, (NZB x NZW)F(1) x NZB backcross progeny with the NZW genotype for D11Mit14 showed significantly increased frequencies of age-associated SLE phenotypes, such as high serum levels of IgG, IgG anti-DNA Abs, and lupus nephritis. In coculture studies, alpha-galactosylceramide-stimulated NKT cells from NZW and (NZB x NZW)F(1) mice, but not from NZB mice, showed significantly enhanced Ig synthesis by B cells. These findings suggest that the D11Mit14-linked NZW locus may contribute to the development of SLE in (NZB x NZW)F(1) mice through a mechanism that up-regulates NKT cell function. Thus, this NZW allele may be a candidate of the NZW modifiers that act to promote (NZB x NZW)F(1) disease.     

CD8+ T cell-mediated suppression of autoimmunity in a murine lupus model of peptide-induced immune tolerance depends on Foxp3 expression

Systemic lupus erythematosus is an autoimmune disease caused by autoantibodies, including IgG anti-DNA. New Zealand Black/New Zealand White F(1) female mice, a model of spontaneous polygenic systemic lupus erythematosus, tolerized with an artificial peptide (pConsensus) based on anti-DNA IgG sequences containing MHC class I and class II T cell determinants, develop regulatory CD4+CD25+ T cells and CD8+ inhibitory T cells (CD8+ Ti), both of which suppress autoantibody production. CD8+ Ti inhibit primarily via secretion of TGF-beta. In the present study, we show that the inhibitory function of CD8+ T cells from tolerized mice is sustained for up to 8 wk and at all times depends on expression of Foxp3. Both CD28-positive and CD28-negative CD8+ T cells contain inhibitory cells, but the expression of mRNA for Foxp3 and for TGF-beta is higher and lasts longer in the CD28- subset. In vitro addition of TGF-beta (in the presence of IL-2) induces Foxp3 expression in a dose-response manner. Gene inhibition or blockade with small interfering RNA of Foxp3 abrogates the ability of the CD8+ Ti to inhibit anti-DNA production and the proliferation of CD4+ Th cells. Moreover, a significant correlation between expression of Foxp3 and ability of CD8+ Ti to secrete TGF-beta is observed. Therefore, CD8+ Ti in this system of tolerance are similar to CD4+CD25+ regulatory T cells in their dependence on expression of Foxp3, and there may be a bidirectional Foxp3/TGF-beta autocrine loop that determines the ability of the CD8+ T cells to control autoimmunity.     

Autoimmunity after induction of neonatal tolerance to alloantigens: role of B cell chimerism and F1 donor B cell activation

BALB/c mice rendered tolerant by the neonatal injection of semiallogeneic (C57BL/6 X BALB/c)F1 spleen cells develop features of autoimmune disease. The possible mechanisms involved in autoantibody production, particularly anti-DNA antibodies, were investigated. In the first 5 wk, there was polyclonal B cell activation, as indicated by marked hypergammaglobulinemia, with a predominance of IgG1 and an increased production of antihapten antibodies. IgG1 anti-SSDNA and anti-DSDNA antibodies were detected with similar kinetics, but at higher titers than the anti-hapten antibodies. Also, there was a correlation between the effective induction of tolerance, as evaluated by the measurement of alloantigen-specific cytolytic T lymphocyte precursors, the persistence of B cell chimerism, and the production of anti-DNA antibodies. Anti-DNA antibodies were observed only in mice exhibiting a persistence of immunoglobulins bearing the donor's allotype. To determine the origin of anti-DNA antibodies, experiments were conducted whereby newborn BALB/c (Igh-1a) mice were injected with F1 cells from mice resulting from a crossing between Igh congenic BALB/c mice bearing the IgCHb allotype and conventional C57BL/6 mice (Igh-1b). All anti-DNA and anti-hapten antibodies exhibited the Igb allotype and thus were produced by the F1 donor B cells. The initial phase of tolerance induction was apparently associated with an allogeneic helper effect, because DNP-KLH-primed F1 donor cells transferred to newborn BALB/c could be stimulated after challenge with DNP-BGG. The triggering of persisting auto-reactive F1 donor B cells may reflect an activation by "incompletely" tolerant semiallogeneic T cells.     

Autoimmune alterations induced by the New Zealand Black Lbw2 locus in BWF1 mice

The New Zealand Black (NZB) Lbw2 locus (lupus NZB x New Zealand White (NZW) 2 locus) was previously linked to mortality and glomerulonephritis, but not to IgG autoantibodies, suggesting that it played a role in a later disease stage. To define its contribution, (NZB x NZW)F1 hybrids (BWF1) containing two, one, or no copies of this locus were generated. Lack of the NZB Lbw2 indeed reduced mortality and glomerulonephritis, but not serum levels of total and anti-DNA IgG Abs. There were, however, significant reductions in the B cell response to LPS, total and anti-DNA IgM and IgG Ab-forming cells, IgM Ab levels, and glomerular Ig deposits. Furthermore, although serum IgG autoantibody levels correlated poorly with kidney IgG deposits, the number of spontaneous IgG Ab-forming cells had a significant correlation. Genome-wide mapping of IgM anti-chromatin levels identified only Lbw2, and analysis of subinterval congenics tentatively reduced Lbw2 to approximately 5 Mb. Because no known genes associated with B cell activation and lupus are in this interval, Lbw2 probably represents a novel B cell activation gene. These findings establish the importance of Lbw2 in the BWF1 hybrid and indicate that Lbw2, by enhancing B cell hyperactivity, promotes the early polyclonal activation of B cells and subsequent production of autoantibodies.     

Mechanism of action of combined short-term CTLA4Ig and anti-CD40 ligand in murine systemic lupus erythematosus

Short-term combination therapy with the costimulatory antagonists CTLA4Ig and anti-CD40 ligand induces prolonged suppression of disease onset in New Zealand Black/New Zealand White F(1) systemic lupus erythematosus-prone mice. To determine the mechanism for this effect, 20- to 22-wk-old New Zealand Black/New Zealand White F(1) mice were treated with six doses each of CTLA4Ig and anti-CD40 ligand Ab over 2 wk. Combination-treated mice, but not mice treated with either agent alone, had prolonged survival and the production of pathogenic IgG anti-dsDNA Ab was suppressed. Twenty weeks after completion of treatment the frequency of activated B cells producing anti-dsDNA Ab was decreased, and the abnormal transition of T cells from the naive to the memory compartment was blocked. Combination treatment partially suppressed class switching and decreased the frequency of somatic mutations in the V(H)BW-16 gene, which is expressed by pathogenic anti-DNA Abs. Treated mice were still able to respond to the hapten oxazolone when it was given 8 wk after treatment initiation, and they mounted a somatically mutated IgG anti-oxazolone response that was noncross-reactive with dsDNA. Fifty to 60% of previously treated mice, but only 14% of previously untreated mice, responded within 2-3 wk to a second course of therapy given at the onset of fixed proteinuria and remained well for a further 3-4 mo. Although this treatment had no immediate effect on serum anti-dsDNA Abs or on the abnormal T cell activation observed in sick mice, 25% of treated mice lived for >18 mo compared with 5% of untreated controls. These results suggest that the effect of costimulatory blockade in remission induction must be mediated by a different mechanism than is demonstrated in the disease prevention studies.     

Calreticulin as a potential diagnostic biomarker for lung cancer

Calreticulin (CRT) is an endoplasmic reticulum luminal Ca(2+)-binding chaperone protein. By immunizing mice with recombinant fragment (rCRT/39-272), six clones of monoclonal antibodies (mAbs) were generated and characterized. Based on these mAbs, a microplate chemiluminescent enzyme immunoassay (CLEIA) system with a measured limit of detection of 0.09?ng/ml was developed. Using this CLEIA system, it was found that soluble CRT (sCRT) level in serum samples from 58 lung cancer patients was significantly higher than that from 40 healthy individuals (only 9 were detectable, P?相似文献   

The effects of pepsin on the natural progression of autoimmunity in glomerulonephritis in the NZB/W F1 mouse

The effects of pepsin on autoimmune glomerulonephritis of New Zealand Black and White F1 hybrid (NZB/W F1) mice were investigated. Intravenous pepsin significantly improved survival rate and suppressed progressive increase in urinary protein and histopathological changes in kidney. Increased serum levels of immune complexes, anti-DNA antibody and natural thymocytotoxic autoantibody were decreased and abnormalities in lymphocyte subsets were ameliorated by pepsin. Pepsin suppressed autoantibody production and enhanced antibody production against xenogeneic substance in these mice. The fact that pepsin ameliorates abnormalities in immune function may contribute to the preventive effects of pepsin against pathogenesis and progression of immune complex nephritis.     

Reactivity of anti-proliferating cell nuclear antigen (PCNA) murine monoclonal antibodies and human autoantibodies to the PCNA multiprotein complexes involved in cell proliferation

Proliferating cell nuclear Ag (PCNA) occurs as a component of multiprotein complexes during cell proliferation. We found the complexes to react with murine anti-PCNA mAbs, but not with anti-PCNA Abs in lupus sera. The complexes were purified from rabbit thymus extract by affinity chromatography using anti-PCNA mAbs (TOB7, TO17, and TO30) and analyzed by ELISA, immunoprecipitation, immunoblotting, and HPLC gel filtration. That PCNA was complexed with other proteins was demonstrated by its copurification with a group of proteins excluded by an HPLC G3000 SW column. Although immunoblot analysis showed the mAbs to react exclusively with the 34-kDa PCNA polypeptide, they nonetheless immunoprecipitated the same group of proteins, confirming the interaction of the isolated PCNA with other proteins. Anti-PCNA sera, including AK, which reacts with biologically functional sites on PCNA, did not react with complexed PCNA, but did react with it once it was dissociated from the complexes. PCNA complexes in turn reacted with murine anti-DNA mAbs, as well as with Abs against p21, replication protein A, DNA helicase II, cyclin-dependent kinases 4 and 5, and topoisomerase I. These findings suggest that the PCNA complexes purified using anti-PCNA mAbs comprise the "protein machinery" for DNA replication and cell cycle regulation. They also suggest that anti-PCNA mAbs are useful tools with which to characterize the protein-protein interactions within PCNA complexes, as well as the autoimmune responses to proteins interacting with PCNA, which may shed light on the mechanisms of autoantibody production in lupus patients.     

Cellular basis of in vitro anti-DNA antibody production: evidence for T cell dependence of IgG-class anti-DNA antibody synthesis in the (NZB X NZW)F1 hybrid

An in vitro system was designed to measure anti-DNA antibody synthesis, and the cellular basis of this autoantibody production in NZB X NZW (B/W)F1 (B/W F1) mice was analyzed. The spleen cells from old B/W F1 mice contained a number of B cells that spontaneously produced anti-DNA antibodies of both IgM and IgG classes in the absence of stimulants, thereby demonstrating that these B cells had been activated in vivo. These activated B cells could be removed by Sephadex G-10 column (G-10) filtration. Such G-10-passed, homogeneously small B cells were activated by the stimulant lipopolysaccharide (LPS) and produced both IgM and IgG class anti-DNA antibodies. The G-10-passed cells contained both B and T cells, and the cytotoxic treatment of the cells with monoclonal antibodies to T cells, anti-Thy-1 and anti-L3T4, abolished the LPS-induced IgG class, but not IgM class, anti-DNA antibody syntheses. Thus, the LPS-induced production of IgG class anti-DNA antibodies in B/W F1 mice is regulated by T cells. Reconstitution experiments revealed the requirement of T-B cell contact but not of the proliferative response of T cells. Moreover, there was no apparent adherent cell requirement. Such IgG class anti-DNA antibodies were produced only by spleen cells from old B/W F1 mice, but not from young B/W F1, NZB, NZW, and C57BL/6 mice. Like IgM class anti-DNA antibodies, LPS-induced synthesis of polyclonal IgM was T cell-independent. Only a slight reduction in the polyclonal IgG synthesis was observed after the G-10-passed cells had been treated with anti-Thy-1 antibody plus complement. This study should facilitate investigation of cell to cell interactions in the formation of autoantibodies and their correlations to immunologic abnormalities in autoimmune disease.     

A peptide DNA surrogate accelerates autoimmune manifestations and nephritis in lupus-prone mice

Lupus-associated anti-DNA Abs display features of Ag selection, yet the triggering Ag in the disease is unknown. We previously demonstrated that the peptide DWEYSVWLSN is bound by a pathogenic anti-DNA Ab, and that immunization of nonautoimmune mice with this peptide induces autoantibodies and renal Ig deposition. To elucidate differences in the induced B cell responses in mice genetically predisposed to autoimmunity, young (NZB x NZW)F(1) mice were immunized with this peptide DNA mimetope. DWEYSVWLSN-immunized mice had significantly increased IgG anti-dsDNA, anti-laminin, and anti-cardiolipin Ab titers compared with controls. In addition, glomerular histopathology in the form of endocapillary disease and crescent formation was markedly more severe in DWEYSVWLSN-immunized mice. Analysis of mAbs from DWEYSVWLSN-immunized mice revealed that anti-peptide Abs were often cross-reactive with DNA. Genetic elements used in the Ab response in immunized mice were homologous to those used in the spontaneous anti-DNA response in (NZB x NZW)F(1) mice, as well as in other, experimentally induced anti-DNA Abs. Our results indicate that peptide immunization can induce a molecular genetic response common to a variety of stimuli that break tolerance to mammalian dsDNA. Based on the similarity between spontaneously arising anti-DNA Abs and several types of induced anti-DNA Abs, we suggest that there may be more than a single Ag that can trigger systemic lupus erythematosus.     

Base specificity and idiotypy of anti-DNA autoantibodies reactive with synthetic nucleic acids

Synthetic nucleic acid reactivities and the distribution of idiotypes associated with poly(dA) and poly(dT) specificities were evaluated among both monoclonal and polyclonal anti-DNA antibodies from autoimmune New Zealand mice. Ten monoclonal anti-DNA antibodies (IgG2a or IgG2b), derived from NZB/NZW mice and reactive with natural DNA (duplex and/or heat-denatured), were found to collectively exhibit a diverse binding pattern with six deoxyribohomopolymers. Several monoclonal antibodies displayed reactivity with poly(dT) comparable to that with natural DNA. Serologic studies indicated that polyclonal anti-DNA autoantibodies from NZW/NZW mice and both parental strains also cross-reacted with various homopolymers and bound preferentially with those containing pyrimidines, particularly poly(dT), relative to purines. Detailed binding analyses with two poly(dT)-reactive monoclonal antibodies demonstrated that stable DNA/anti-DNA complexes were formed with synthetic oligomers containing six to 10 nucleotides; binding to such antigens was relatively insensitive to ionic strength and inversely dependent on temperature. Both antibodies exhibited preferential binding (greater than or equal to 10-fold) with poly(dT) relative to poly(dU), suggesting the importance of the C5-methyl group and/or helical conformation in pyrimidine base recognition. Idiotypes on poly(dA)-specific and poly(dT)-specific monoclonal antibodies were found to be reciprocally distinct, localized at or near active site residues, and expressed at low levels (less than 10 to 130 ng/ml) in anti-DNA sera from all three New Zealand strains. These findings suggest that: nucleotide base determinants are significantly involved in DNA/anti-DNA interactions; poly(dT) represents a major cross-reactive synthetic antigen; and idiotype expression among lupus autoantibodies which recognize such determinants may be diverse.     

Lysyl tRNA synthetase is required for the translocation of calreticulin to the cell surface in immunogenic death

In response to immunogenic cell death inducers, calreticulin (CRT) translocates from its orthotopic localization in the lumen of the endoplasmic reticulum (ER) to the surface of the plasma membrane where it serves as an engulfment signal for antigen-presenting cells.¹ Here, we report that yet another ER protein, the lysyl-tRNA synthetase (KARS), was exposed on the surface of stressed cells, on which KARS co-localized with CRT in lipid rafts. Depletion of KARS with small interfering RNAs suppressed CRT exposure induced by anthracyclines or UVC light. In contrast to CRT, KARS was also found in the supernatant of stressed cells. Recombinant KARS protein was unable to influence the binding of recombinant CRT to the cell surface. Moreover, recombinant KARS protein was unable to stimulate macrophages in vitro. These results underscore the contribution of KARS to the emission of (one of) the principal signal(s) of immunogenic cell death, CRT exposure.     

Calreticulin exposure dictates the immunogenicity of cancer cell death

Anthracyclin-treated tumor cells are particularly effective in eliciting an anticancer immune response, whereas other DNA-damaging agents such as etoposide and mitomycin C do not induce immunogenic cell death. Here we show that anthracyclins induce the rapid, preapoptotic translocation of calreticulin (CRT) to the cell surface. Blockade or knockdown of CRT suppressed the phagocytosis of anthracyclin-treated tumor cells by dendritic cells and abolished their immunogenicity in mice. The anthracyclin-induced CRT translocation was mimicked by inhibition of the protein phosphatase 1/GADD34 complex. Administration of recombinant CRT or inhibitors of protein phosphatase 1/GADD34 restored the immunogenicity of cell death elicited by etoposide and mitomycin C, and enhanced their antitumor effects in vivo. These data identify CRT as a key feature determining anticancer immune responses and delineate a possible strategy for immunogenic chemotherapy.     

7, 8-diacetoxy-4-methylcoumarin induced cell death in human tumor cells is influenced by calreticulin

Calreticulin (CRT), an endoplasmic reticulum resident protein demonstrates transacetylase activity in presence of 7, 8 diacetoxy-4-methyl coumarin (DAMC) in vitro. To investigate the possible role of CRT and DAMC mediated protein acetylation in cells, we investigated the effects of DAMC in tumor cells with different levels of CRT. DAMC was more toxic (clonogenicity, metabolic viability and proliferation) to human glioma cells (BMG-1) expressing low endogenous CRT level as compared to head and neck carcinoma cells (KB) with a high CRT level. The cytotoxicity was accompanied by loss of mitochondrial membrane potential in both the cells, which correlated with corresponding changes in the levels of pro-apoptotic (Bax) and anti-apoptotic (NFkB) regulators. Manipulation of CRT protein level in KB cells by application of small RNA interference enhanced the sensitivity by four folds while over expression of CRT in BMG-1 cells reduced their sensitivity to DAMC by ∼20% strongly suggesting the influence of CRT on DAMC induced cytotoxicity. The partial rescue of CROE cells from DAMC induced toxicity was accompanied by changes in NFkB levels and over all protein acetylation status, besides increase in the NADPH-cytochrome c reductase activity related to its well known antioxidant property. Since CRT is over-expressed in cancer cells, which are generally resistant to radio- and chemotherapy; targeting CRT transacetylase system, may be an attractive approach for increasing the efficacy of anticancer therapies.     

Ig-reactive CD4+CD25+ T cells from tolerized (New Zealand Black x New Zealand White)F1 mice suppress in vitro production of antibodies to DNA

We have recently shown that tolerogenic administration of an artificial peptide (pConsensus) that is based on sequences within the V(H) regions of several murine anti-dsDNA Ig delays appearance of autoantibodies in female (New Zealand Black (NZB) x New Zealand White (NZW))F(1) (NZB/W F(1)) mice and significantly prolongs their survival. The aim of this study was to characterize the T cell population(s) involved in pConsensus-induced down-regulation of autoimmune responses in tolerized NZB/W F(1) mice. Using MHC class II dimers loaded with tolerogenic peptide, we found that pCons favored expansion of peptide-reactive CD4(+)CD25(+) regulatory T cells (T(R)) that inhibited in vitro production of anti-dsDNA Ab-forming cells. Suppression by T(R) was abrogated by the presence in culture of Ab to glucocorticoid-induced TNFR family member 18 or to TGFbeta latency-associated protein. These findings suggest possible relevance of Ag specificity in the mechanism of T(R)-mediated immune tolerance to Ig-derived peptides in NZB/W F(1) mice.     

The co-translocation of ERp57 and calreticulin determines the immunogenicity of cell death

The exposure of calreticulin (CRT) on the plasma membrane can precede anthracycline-induced apoptosis and is required for cell death to be perceived as immunogenic. Mass spectroscopy, immunofluorescence and immunoprecipitation experiments revealed that CRT co-translocates to the surface with another endoplasmic reticulum-sessile protein, the disulfide isomerase ERp57. The knockout and knockdown of CRT or ERp57 inhibited the anthracycline-induced translocation of ERp57 or CRT, respectively. CRT point mutants that fail to interact with ERp57 were unable to restore ERp57 translocation upon transfection into crt(-/-) cells, underscoring that a direct interaction between CRT and ERp57 is strictly required for their co-translocation to the surface. ERp57(low) tumor cells generated by retroviral introduction of an ERp57-specific shRNA exhibited a normal apoptotic response to anthracyclines in vitro, yet were resistant to anthracycline treatment in vivo. Moreover, ERp57(low) cancer cells (which failed to expose CRT) treated with anthracyclines were unable to elicit an anti-tumor response in conditions in which control cells were highly immunogenic. The failure of ERp57(low) cells to elicit immune responses and to respond to chemotherapy could be overcome by exogenous supply of recombinant CRT protein. These results indicate that tumors that possess an intrinsic defect in the CRT-translocating machinery become resistant to anthracycline chemotherapy due to their incapacity to elicit an anti-cancer immune response.     

Characterization of two monoclonal antibodies, 38F10 and 44D11,against the major envelope fusion protein of Helicoverpa armigera nucleopolyhedrovirus

The envelope fusion protein F of baculoviruses is a class I viral fusion protein which play a significant role during virus entry into insect cells. F is initially synthesized as a precursor(F_0) and then cleaved into a disulfide-linked F_1 and F_2 subunits during the process of protein maturation and secretion. To facilitate further investigation into the structure and function of F protein during virus infection, monoclonal antibodies(mAbs) against the F_2 subunit of Helicoverpa armigera nucleopolyhedrovirus(HearNPV)(Ha F) were generated. Two kinds of mAbs were obtained according to their different recognition epitopes: one kind of mAbs, as represented by 38F10,recognizes amino acid(aa) 85 to 123 of F_2 and the other kind, represented by 44D11, recognizes aa148 to 173 of F_2. Western blot and immunofluorescence assay confirmed that both of the mAbs recognized the F protein expressed in HearNPV infected cells, however, only 44D11 could neutralize HearNPV infection. The results further showed that 44D11 may not interact with a receptor binding epitope, rather it was demonstrated to inhibit syncytium formation in cells expressing the Ha F protein. The results imply that the monoclonal antibody 44D11 recognizes a region within HaF_2 that may be involved in the F-mediated membrane fusion process.