Antigen-driven induction of polyreactive IgM during intracellular bacterial infection

Polyreactivity is well known as a property of natural IgM produced by B-1 cells. We demonstrate that polyreactive IgM is also generated during infection of mice with Ehrlichia muris, a tick-borne intracellular bacterial pathogen. The polyreactive IgM bound self and foreign Ags, including single-stranded and double-stranded DNA, insulin, thyroglobulin, LPS, influenza virus, and Borrelia burgdorferi. Production of polyreactive IgM during infection was Ag driven, not due to polyclonal B cell activation, as the majority of polyreactive IgM recognized ehrlichial Ag(s), including an immunodominant outer membrane protein. Monoclonal polyreactive IgM derived from T cell-independent spleen plasmablasts, which was germline-encoded, also bound cytoplasmic and nuclear Ags in HEp-2 cells. Polyreactive IgM protected immunocompromised mice against lethal bacterial challenge infection. Serum from human ehrlichiosis patients also contained polyreactive and self-reactive IgM. We propose that polyreactivity increases IgM efficacy during infection but may also exacerbate or mollify the response to foreign and self Ags.     

Distortion of the self-reactive IgG antibody repertoire in multiple sclerosis as a new diagnostic tool

To date, none of the myelin-associated Ag targets definitively discriminates between the immune response observed in multiple sclerosis (MS) patients and healthy subjects. However, it has been shown recently that analysis of global immune Ab profiles such as natural autoantibody reactivities can help to distinguish between normal individuals and patients suffering from various immune diseases. The aim of our study was to compare the global IgG immune response against brain self-Ags in sera from 82 MS patients and 27 healthy subjects. The analysis of the immune profiles was performed by Western blotting, and data were subjected to linear discriminant analysis. Particular patterns of IgG reactivity were found in healthy subjects, Sj?gren patients, and MS patients. Moreover, this approach separated the three clinical forms of MS with a high concordance rate with the clinical data (kappa value, 77.8%). Our study suggests, for the first time, that serum IgG Ab repertoires are able to distinguish MS patients. In addition, our data suggest that patterns of IgG reactivity could model the pathological processes underlying the various forms of MS. Further characterization of such discriminant Ags could provide useful information regarding their potent role in pathogenesis or regulatory processes in MS.     

The rearranged V(H) domain of a physiologically selected anti-single-stranded DNA antibody as a precursor for formation of IgM and IgG antibodies to diverse antigens

It has been proposed that autoreactivity of modest affinity contributes to positive selection of a preimmunization B cell repertoire, whereas high-affinity autoreactivity leads to negative selection. This hypothesis predicts that a B cell producing a physiologically selected unmutated ssDNA-binding Ab should be a precursor of cells that respond to diverse exogenous Ags. To test this prediction, we prepared transgenic mice bearing the rearranged V(H) domain of an IgM Ab from a nonautoimmune mouse immunized with a DNA-protein complex, poly(dC)-methylated BSA. The Ab, dC1, binds both poly(dC) and ssDNA. It is encoded by V(H) and V(L) gene segments with no mutations, suggesting that the producing cell may have been selected before and activated during immunization. The dC1V(H) transgene was targeted to the IgH locus. In heterozygous mice, on a nonautoimmune C57BL/6 background, the transgene allotype was expressed on B cell surfaces and in serum Ig, but about one-third of B cells expressed the endogenous allele instead. Total serum Ig concentrations were normal and included both transgene- and endogenous gene-coded IgM and IgG. The transgene V(H) D(H)J(H) was expressed in splenic IgM cDNA with few or no mutations, and in IgG cDNA with multiple mutations. The transgene allotype was also expressed in Abs formed on immunization with thyroglobulin, pneumococcal polysaccharide, and ssDNA-methylated BSA. Consistent with the hypothesis, cells with a rearranged autoreactive V(H) domain selected for reactivity with a form of ssDNA did serve as precursors for cells producing IgM and IgG Abs to diverse Ags.     

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.     

Regeneration of natural antibody repertoire after massive ablation of lymphoid system: robust selection mechanisms preserve antigen binding specificities

Natural Abs (NAbs) are Igs present in the serum and body fluids of healthy vertebrate animals, without any previous intentional immunization. NAbs often exhibit autoreactivity but also play an essential role in immunity, being a first line of defense against infectious microorganisms. We have previously analyzed the natural serum IgM Ab repertoire of normal mice, characterizing their reactivity with hundreds/thousands of self Ags; a significant similarity among different individuals was observed, and it was found that many reactivities of NAbs stably kept during adulthood were established early in life, implicating that period as a critical time window in the physiology of NAb repertoire selection. In the work reported here, experiments were conducted to address the role of normal lymphocyte ontogeny to the formation and stability of adult NAb repertoire. The massive destruction of the lymphoid system was promoted in adult mice with gamma-irradiation, and regeneration of hemopoietic tissues was granted by bone marrow or fetal liver inoculum. NAb repertoire regeneration was followed for 60 days after gamma-irradiation in bone marrow or fetal liver chimeric animals. The analysis of serum IgM reactivity with hundreds/thousands of self Ags showed that the NAb repertoire regenerated most of its original format after massive destruction of lymphoid compartments, characterizing autoreactive repertoire selection as a robust biological process. The data also show that regeneration of the NAb repertoire occurred similarly in fetal liver and bone marrow chimeras, although the latter animals poorly reconstituted their CD5(+) B1 cell compartment, suggesting that B1 cells are not essential for natural Ab regeneration.     

Antibody repertoire development in fetal and neonatal piglets. VIII. Colonization is required for newborn piglets to make serum antibodies to T-dependent and type 2 T-independent antigens

Cesarean-derived piglets were reared for 5 wk under germfree conditions or monoassociated with a benign Escherichia coli (G58-1) or a enterohemorrhagic strain (933D) derived from O157:H7, and immunized i.p. with the T-dependent (TD) Ags fluorescein-labeled (FL) keyhole limpet hemocyanin or trinitrophenylated (TNP) keyhole limpet hemocyanin and the type 2 T-independent Ags TNP-Ficoll or FL-Ficoll. Only colonized piglets showed an increase in serum IgG, IgA, and IgM and had serum Abs to FL, TNP, and colonizing bacteria. While serum Abs to FL or TNP appeared following colonization alone, secondary responses were restricted to piglets immunized using TD carriers. While animals colonized with 933D had significantly higher total serum IgG and IgM levels and specific IgG Abs than those colonized with G58-1, no differences were seen in serum IgA levels, B cell diversification in the ileal Peyer's patches, and specific activity (ELISA activity per micrograms of Ig) of pre-boost serum IgG and IgM anti-TNP and anti-FL Abs. Serum IgA Abs to TNP, FL, or bacteria were not detected. Ag-driven responses, as measured by an increase in specific Ab activity, were only observed in secondary responses to TD Ags and to colonizing, pathogenic E. coli. We propose that germline-encoded, isotype-switched B cells in newborn piglets differentiate to Ab-secreting cells 1) after stimulation by bacteria-activated APCs or 2) through direct stimulation by bacterial products. We further propose that Ag-driven systemic responses require both bacterial colonization and TD Ags translocated to the peritoneum.     

Human memory B lymphocyte subsets fulfill distinct roles in the anti-polysaccharide and anti-protein immune response

There is controversy on the role of IgM memory and switched memory B lymphocytes in the Ab response to T cell-independent and T cell-dependent Ags. We transplanted SCID/SCID mice with human B lymphocyte subsets and immunized them with heat-inactivated Streptococcus pneumoniae or with a pneumococcal vaccine. Inactivated S. pneumoniae and soluble pneumococcal capsular polysaccharides elicited an IgM anti-polysaccharide and anti-protein Ab response from IgM memory B lymphocytes and an IgG anti-polysaccharide and anti-protein response from switched memory B lymphocytes. In addition to the IgM Ab response, IgM memory B cells elicited an IgG anti-polysaccharide and anti-protein Ab response after immunization with inactivated S. pneumoniae or soluble pneumococcal capsular polysaccharides. In conclusion, our findings provide evidence for a versatile role of IgM memory B cells in T-independent and T-dependent immune responses.     

Antiviral B cell memory in the absence of mature follicular dendritic cell networks and classical germinal centers in TNFR1-/- mice

TNFR1-/- mice have been shown to lack networks of mature follicular dendritic cells (FDCs) and they do not form germinal centers. With nonreplicating Ags, IgG titers were inefficiently induced and not maintained. In this study, the neutralizing Ab response and the establishment of B cell memory in TNFR1-/- mice after infection with vesicular stomatitis virus (VSV) were analyzed histologically and functionally. Immunization with VSV-derived protein Ags without adjuvant induced only IgM but no IgG Abs in TNFR1-/- mice, whereas VSV glycoprotein emulsified in CFA or IFA induced IgM and IgG responses that were short-lived and of moderate titer. However, infection with live VSV induced excellent neutralizing IgM and IgG responses in TNFR1-/- mice, and adoptively transferable B cell memory was generated and persisted for more than 300 days. In contrast, IgG levels and Ab-forming cells in the bone marrow declined within 300 days by 90-95% compared with controls. These findings suggest that 1) increased Ag dose and time of Ag availability can substitute for FDC-stored Ab-complexed Ag in the induction of efficient IgG responses in TNFR1-/- mice devoid of classical germinal centers; 2) the induction and maintenance of adoptively transferable B cell memory can occur in the absence of Ag bound to mature FDCs; and 3) the long-term maintenance of elevated IgG titers is largely dependent on FDC-associated persisting Ag. However, about 5-10% of the Ab production remained in the absence of detectable persisting Ag in TNFR1-/- mice, probably either due to immature FDCs being partially functional and/or due to long-lived plasma cells.     

Mice deficient in complement receptors 1 and 2 lack a tissue injury-inducing subset of the natural antibody repertoire

Intestinal ischemia-reperfusion (IR) injury is initiated when natural Abs recognize neoantigens that are revealed on ischemic cells. Cr2(-/-) mice, deficient in complement receptors (CR)1 and CR2, demonstrate defects in T-dependent B-2 B cell responses to foreign Ags and have also been suggested to manifest abnormalities of the B-1 subset of B lymphocytes. To determine whether these CRs might play a role in the generation of the natural Abs that initiate intestinal IR injury, we performed experiments in Cr2(-/-) and control Cr2(+/+) mice. We found that Cr2(-/-) mice did not demonstrate severe intestinal injury that was readily observed in control Cr2(+/+) mice following IR, despite having identical serum levels of IgM and IgG. Pretreatment of Cr2(-/-) mice before the ischemic phase with IgM and IgG purified from the serum of wild-type C57BL/6 mice reconstituted all key features of IR injury, demonstrating that the defect involves the failure to develop this subset of natural Abs. Pretreatment with IgM and IgG individually demonstrates that each contributes to unique features of IR injury. In sum, CR2/CR1 play an unanticipated but critical role in the development of a subset of the natural Ab repertoire that has particular importance in the pathogenesis of IR injury.     

Bead Arrays for Antibody and Complement Profiling Reveal Joint Contribution of Antibody Isotypes to C3 Deposition

The development of antigen arrays has provided researchers with great tools to identify reactivities against self or foreign antigens from body fluids. Yet, these approaches mostly do not address antibody isotypes and their effector functions even though these are key points for a more detailed understanding of disease processes. Here, we present a bead array-based assay for a multiplexed determination of antigen-specific antibody levels in parallel with their properties for complement activation. We measured the deposition of C3 fragments from serum samples to reflect the degree of complement activation via all three complement activation pathways. We utilized the assay on a bead array containing native and citrullinated peptide antigens to investigate the levels of IgG, IgM and IgA autoantibodies along with their complement activating properties in serum samples of 41 rheumatoid arthritis patients and 40 controls. Our analysis revealed significantly higher IgG reactivity against the citrullinated fibrinogen β and filaggrin peptides as well as an IgA reactivity that was exclusive for citrullinated fibrinogen β peptide and C3 deposition in rheumatoid arthritis patients. In addition, we characterized the humoral immune response against the viral EBNA-1 antigen to demonstrate the applicability of this assay beyond autoimmune conditions. We observed that particular buffer compositions were demanded for separate measurement of antibody reactivity and complement activation, as detection of antigen-antibody complexes appeared to be masked due to C3 deposition. We also found that rheumatoid factors of IgM isotype altered C3 deposition and introduced false-positive reactivities against EBNA-1 antigen. In conclusion, the presented bead-based assay setup can be utilized to profile antibody reactivities and immune-complex induced complement activation in a high-throughput manner and could facilitate the understanding and diagnosis of several diseases where complement activation plays role in the pathomechanism.     

Memory B cells and pneumococcal antibody after splenectomy

Splenectomized patients are susceptible to bloodstream infections with encapsulated bacteria, potentially due to loss of blood filtering but also defective production of anticarbohydrate Ab. Recent studies propose that a lack of Ab is related to reduced numbers of IgM(+) CD27(+) memory B cells found after splenectomy. To test this, we analyzed CD27(+) memory B cell subsets, IgG, and IgM pneumococcal Ab responses in 26 vaccinated splenectomized subjects in comparison to memory B cell subsets and Ab responses in healthy controls. As shown previously, the splenectomized autoimmune subjects had fewer total, isotype switched, and IgM(+) CD27(+) memory B cells as compared with controls, but there was no difference in memory B cells subsets between controls and splenectomized subjects with spherocytosis. There was no difference between the geometric mean IgG Ab response between normal controls and splenectomized subjects (p = 0.51; p = 0.81). Control subjects produced more IgM Ab than splenectomized autoimmune subjects (p = 0.01) but the same levels as subjects with spherocytosis (p = 0.15.) There was no correlation between memory B cell subsets and IgG or IgM Ab responses for controls or splenectomized subjects. These data suggest that splenectomy alone may not be the sole reason for loss of memory B cells and reduced IgM antipneumococcal Ab. Because subjects with autoimmunity had splenectomy at a significantly older age than participants with spherocytosis, these data suggest that an age-related loss of extra splenic sites necessary for the maintenance or function of memory B cells may lead to impaired immunity in these subjects.     

HIV-1 envelope triggers polyclonal Ig class switch recombination through a CD40-independent mechanism involving BAFF and C-type lectin receptors

Switching from IgM to IgG and IgA is essential for antiviral immunity and requires engagement of CD40 on B cells by CD40L on CD4(+) T cells. HIV-1 is thought to impair CD40-dependent production of protective IgG and IgA by inducing progressive loss of CD4(+) T cells. Paradoxically, this humoral immunodeficiency is associated with B cell hyperactivation and increased production of nonprotective IgG and IgA that are either nonspecific or specific for HIV-1 envelope glycoproteins, including gp120. Nonspecific and gp120-specific IgG and IgA are sensitive to antiretroviral therapy and remain sustained in infected individuals with very few CD4(+) T cells. One interpretation is that some HIV-1 Ags elicit IgG and IgA class switch DNA recombination (CSR) in a CD40-independent fashion. We show that a subset of B cells binds gp120 through mannose C-type lectin receptors (MCLRs). In the presence of gp120, MCLR-expressing B cells up-regulate the CSR-inducing enzyme, activation-induced cytidine deaminase, and undergo CSR from IgM to IgG and IgA. CSR is further enhanced by IL-4 or IL-10, whereas Ab secretion requires a B cell-activating factor of the TNF family. This CD40L-related molecule is produced by monocytes upon CD4, CCR5, and CXCR4 engagement by gp120 and cooperates with IL-4 and IL-10 to up-regulate MCLRs on B cells. Thus, gp120 may elicit polyclonal IgG and IgA responses by linking the innate and adaptive immune systems through the B cell-activating factor of the TNF family. Chronic activation of B cells through this CD40-independent pathway could impair protective T cell-dependent Ab responses by inducing immune exhaustion.     

Humoral anti-KLH responses in cancer patients treated with dendritic cell-based immunotherapy are dictated by different vaccination parameters

Purpose

Keyhole limpet hemocyanin (KLH) attracts biomedical interest because of its remarkable immunostimulatory properties. Currently, KLH is used as vaccine adjuvant, carrier protein for haptens and as local treatment for bladder cancer. Since a quantitative human anti-KLH assay is lacking, it has not been possible to monitor the dynamics of KLH-specific antibody (Ab) responses after in vivo KLH exposure. We designed a quantitative assay to measure KLH-specific Abs in humans and retrospectively studied the relation between vaccination parameters and the vaccine-induced anti-KLH Ab responses.

Experimental design

Anti-KLH Abs were purified from pooled serum of melanoma patients who have responded to KLH as a vaccine adjuvant. Standard isotype-specific calibration curves were generated to measure KLH-specific Ab responses in individual serum samples using ELISA.

Results

KLH-specific IgM, IgA, IgG and all IgG-subclasses were accurately measured at concentrations as low as 20?μg/ml. The intra- and inter-assay coefficients of variation of this ELISA were below 6.7 and 9.9?%, respectively. Analyses of 128 patients demonstrated that mature DC induced higher levels of KLH-specific IgG compared to immature DC, prior infusion with anti-CD25 abolished IgG and IgM production and patients with locoregional disease developed more robust IgG responses than advanced metastatic melanoma patients.

Conclusions

We present the first quantitative assay to measure KLH-specific Abs in human serum, which now enables monitoring both the dynamics and absolute concentrations of humoral immune responses in individuals exposed to KLH. This assay may provide a valuable biomarker for the immunogenicity and clinical effectiveness of KLH-containing vaccines and therapies.     

Thymus-dependent antiidiotype and anti-antiidiotype responses to a dinitrophenyl-specific monoclonal antibody

BALB/c mice were inoculated i.p. with graded doses of a DNP-specific, IgM mAb (designated 57.1). Injection with unmodified 57.1 in the absence of adjuvants resulted in the generation of an anti-Id response (Ab2) and an anti-anti-Id response (Ab3). The generation of serum anti-Id antibodies was found to be thymus dependent. Nude mice immunized with 57.1 were unable to produce a serum Ab2 response above nonimmunized controls whereas euthymic mice receiving identical doses of 57.1 produced strong Ab2 responses. To examine the specificity of serum anti-Id, sera from mice receiving 57.1 were screened against a panel of mAb representing at least five distinct VH gene families. Serum titers were significantly higher against 57.1 than against any of the other antibodies in the panel. Three of the antibodies in this panel bind FD5-1, a monoclonal anti-Id (Ab2) that also binds 57.1. However, sera from mice receiving 57.1 bound 57.1 only. Thus, the serum Ab2 response appears to be highly specific for idiotopes on 57.1. The predominant isotype of these anti-Id antibodies was IgG1. The number of isotypes detected increased in a dose dependent manner with all IgG subclasses having anti-Id specificity in sera from animals receiving the higher doses of 57.1. Further analysis of the serum demonstrated that approximately 8% of the Ab2 response was paratope-specific (inhibitable by the monovalent hapten DNP-lysine). The same sera were analyzed for the presence of Ab3 by binding to the monoclonal anti-Id antibody FD5-1. Lower serum titers of Ab3 were generated in comparison to serum titers of Ab2. Analysis of the binding specificity of the Ab3 response revealed that DNP-BSA was able to partially inhibit the binding of serum IgM and IgG Ab3 to FD5-1. A subset of the Ab3 response. Ab1' that is specific for DNP was observed in a direct binding assay where detectable amounts of DNP binding IgM, IgG1, and IgG3 isotypes were present. We have thus described a complete circuit (Ab1----Ab2----Ab3) of antibodies within the Id network by immunizing animals with an unmodified mAb in the absence of Ag or adjuvants.     

Conformational melding permits a conserved binding geometry in TCR recognition of foreign and self molecular mimics

Molecular mimicry between foreign and self Ags is a mechanism of TCR cross-reactivity and is thought to contribute to the development of autoimmunity. The αβ TCR A6 recognizes the foreign Ag Tax from the human T cell leukemia virus-1 when presented by the class I MHC HLA-A2. In a possible link with the autoimmune disease human T cell leukemia virus-1-associated myelopathy/tropical spastic paraparesis, A6 also recognizes a self peptide from the neuronal protein HuD in the context of HLA-A2. We found in our study that the complexes of the HuD and Tax epitopes with HLA-A2 are close but imperfect structural mimics and that in contrast with other recent structures of TCRs with self Ags, A6 engages the HuD Ag with the same traditional binding mode used to engage Tax. Although peptide and MHC conformational changes are needed for recognition of HuD but not Tax and the difference of a single hydroxyl triggers an altered TCR loop conformation, TCR affinity toward HuD is still within the range believed to result in negative selection. Probing further, we found that the HuD-HLA-A2 complex is only weakly stable. Overall, these findings help clarify how molecular mimicry can drive self/nonself cross-reactivity and illustrate how low peptide-MHC stability can permit the survival of T cells expressing self-reactive TCRs that nonetheless bind with a traditional binding mode.     

Antibody repertoire development in fetal and neonatal piglets. IX. Three pathogen-associated molecular patterns act synergistically to allow germfree piglets to respond to type 2 thymus-independent and thymus-dependent antigens

Newborn piglets maintained germfree (GF) cannot respond to either thymus-dependent (TD) or type 2 thymus-independent Ags (TI-2) unless colonized with bacteria. We show here that pathogen-associated molecular patterns (PAMPs), including muramyl dipeptide (MDP), LPS, and a B-class CpG oligonucleotide (CpG-B), can substitute for gut flora in the induction of neonatal immunoresponsiveness. These PAMPs alone or in combination had little effect on serum IgG and IgA levels, but CpG-B and CpG-B + MDP elevated total IgM levels 3- to 7-fold above that seen in colonized controls after booster immunization. Although only CpG-B could alone stimulate immunoresponsiveness, co-administration of LPS or MDP resulted in a 5-fold increase in the IgG response to both immunogens. Co-administered MDP did not promote secondary IgG responses to either Ag but instead pronounced secondary IgM responses to the epitopes of both immunogens. LPS co-administered with CpG-B may promote class switch recombination or cause differentiation of previously switched cells that become responsive after exposure to CpG-B. Primary and secondary IgG responses equally recognized the epitopes of the TI-2 and TD immunogens, whereas IgM responses favored the TI-2 epitope. Because PAMPs alone can result in Abs to 2,4,6-triitrophenyl and FLU without immunization, it suggests they alone cause differentiation of B cells of the preimmune repertoire. The finding that both bacterial PAMPs and colonization are capable of stimulating Ab responses in both immunized and nonimmunized piglets suggests that PAMPs derived from host flora may play a major role in awakening adaptive immunity in neonates.     

Vesicular stomatitis virus glycoprotein displaying retrovirus-like particles induce a type I IFN receptor-dependent switch to neutralizing IgG antibodies

Vesicular stomatitis virus (VSV) infection rapidly induces IFN-alphabeta that confers initial survival, whereas long-term protection is mediated by neutralizing IgG responses. Because coadministration of IFN-alphabeta can enhance Ab responses against soluble Ags, we addressed whether virus-induced IFN-alphabeta also had an impact on the induction of neutralizing Ab responses. To this end, we generated apathogenic retrovirus-like particles (VLP) displaying the VSV gp (VLP-VSV). Reminiscent of live VSV, VLP-VSV induced VSV-neutralizing IgM responses that switched to IgG in a T help-dependent manner. In type I IFN receptor-deficient (IFNAR(-/-)) mice, VLP-VSV injection elicited neutralizing IgM, whereas the IgG switch was absent. The lack of subclass switch was associated with a reduced germinal center reaction. Conditional knockout mice with a lymphocyte-specific IFNAR ablation showed normal Ab responses against VLP-VSV, as well as against live VSV. Thus, IFNAR triggering critically promoted the T help-dependent subclass switch of virus-neutralizing Ab responses against VLP-VSV. Interestingly, in the context of VLP-VSV as well as VSV immunization, IFNAR triggering of B lymphocytes did not play a critical role.     

Quality of recombinant protein determines the amount of autoreactivity detected against the tumor-associated epithelial cell adhesion molecule antigen: low frequency of antibodies against the natural protein

The human epithelial cell adhesion molecule (EpCAM) is expressed on normal epithelial cells and is overexpressed in most carcinomas. EpCAM-targeted immunotherapy has been tried in several clinical studies. High titers of autoantibodies against EpCAM have been reported by different authors. We have generated large amounts of purified protein in S2 Drosophila cells (S2-EpCAM) with a purity of >96%. In contrast, the protein produced in baculovirus-dependent systems (baculo-EpCAM) that has been used in previous studies shows a purity of 79%. (1)H nuclear magnetic resonance spectrum of S2-EpCAM is typical of folded protein, whereas the baculo-EpCAM sample shows a spectrum corresponding to a partially unfolded protein. Using S2-EpCAM, denatured S2-EpCAM, and baculo-EpCAM, we measured EpCAM Abs of different isotypes in the serum of healthy controls and cancer patients. We found Ab titers against EpCAM in a much lower percentage of sera as published previously, and support the hypothesis that Ab reactivity in some published studies might be due to reactivity against denatured protein, to contaminating proteins in the baculovirus preparations, and to reactivity with BSA. Tetanus toxoid-reactive IgG Abs are present in 1000-fold higher titers compared with EpCAM-reactive Abs. Only IgA Abs were found in higher proportions and in higher concentrations than tetanus toxoid-specific Abs. Our study shows that EpCAM only rarely induces autoantibodies against native protein and emphasizes the importance of using extremely purified Ag preparations when evaluating Abs against tumor-associated Ags.     

A panel of candidate tumor antigens in colorectal cancer revealed by the serological selection of a phage displayed cDNA expression library

In the last few years it has been shown that the humoral immune response in cancer patients is a rich source of putative cancer vaccine candidates. To fully explore the complex information present within the Ab repertoire of cancer patients, we have applied a method, serological Ag selection, to molecularly define tumor Ags recognized by the humoral immune response in colorectal cancer (CRC). First, we built a cDNA display library by cloning a cDNA library from CRC cell line HT-29 for expression as a fusion protein with a filamentous phage minor coat protein, pVI. This cDNA display library was then enriched on pooled sera from CRC patients who had undergone active specific immunization with autologous tumor. We identified a panel of 19 clones reactive with the serum pool. Seventeen of 19 (89%) clones showed reactivity with one or more of the eight Ag-reactive sera, conversely six of eight (75%) sera were reactive with at least one of the 19 clones. Sequencing revealed that these 19 clones represented 13 different Ags. A detailed serological analysis of the 13 different Ags showed preferential reactivity to sera of cancer patients for six different Ags. Four of these Ags displayed increased serum reactivity after the active specific immunization procedure. Furthermore, one of the six Ags, a novel Ag homologous to HSPC218, showed restricted expression in normal testis, suggesting that it belongs to the cancer-testis Ag family. Some of the Ags we have identified may be candidates for tumor vaccination, for sero-diagnosis of cancer, as prognostic markers, or as probes for monitoring tumor cell-based vaccination trials.