Whole blood stimulation with Toll-like receptor (TLR)-7/8 and TLR-9 agonists induces interleukin-12p40 expression in plasmacytoid dendritic cells in rhesus macaques but not in humans.

Macaques provide important animal models in biomedical research into infectious and chronic inflammatory disease. Therefore, a proper understanding of the similarities and differences in immune function between macaques and humans is needed for adequate interpretation of the data and translation to the human situation. Dendritic cells are important as key regulators of innate and adaptive immune responses. Using a new whole blood assay we investigated functional characteristics of blood plasmacytoid dendritic cells (pDC), myeloid dendritic cells (mDC) and monocytes in rhesus macaques by studying induction of activation markers and cytokine expression upon Toll-like receptor (TLR) stimulation. In a head-to-head comparison we observed that rhesus macaque venous blood contained relatively lower numbers of pDC than human venous blood, while mDC and monocytes were present at similar percentages. In contrast to humans, pDC in rhesus macaques expressed the interleukin (IL)-12p40 subunit in response to TLR-7/8 as well as TLR-9 stimulation. Expression of IL-12p40 was confirmed by using different monoclonal antibodies and by reverse transcription-polymerase chain reaction (RT-PCR). Both in humans and rhesus macaques, TLR-4 stimulation induced IL-12p40 expression in mDC and monocytes, but not in pDC. The data show that, in contrast to humans, pDC in macaques are able to express IL-12p40, which could have consequences for evaluation of human vaccine candidates and viral infection.

Peptide arrays for the determination of humoral responses induced by active immunization with a monoclonal antibody against EpCAM.

Epithelial cell adhesion molecule (EpCAM) is an attractive target for monoclonal antibody serotherapy because it is over-expressed in approximately 70% of epithelial cancers and their metastatic lesions. IGN101, the immunogenic formulation of the murine monoclonal anti-EpCAM antibody Mab17-1A, has been shown to evoke a strong humoral immune response in both monkey studies and early clinical trials. Notably, there was a reduction in the number of circulating EpCAM-positive tumor cells in the peripheral blood of treated cancer patients. In contrast to earlier publications by other groups, we could not detect an anti-EpCAM immune response upon treatment with Mab17-1A using a conventional but optimized anti-EpCAM ELISA. Therefore, in a novel experimental setup, sera of healthy immunized monkeys, normal human donors and cancer patients immunized with IGN101 were tested for reactivity against a series of overlapping synthetic peptides encompassing the entire sequence of EpCAM prepared by SPOT synthesis on cellular supports. Using this method, sera from normal donors reacted with different peptides compared to sera from healthy monkeys. However, the peptides were clustered in the same regions of EpCAM. Cancer patients generally had a lower reactivity to EpCAM peptides and immunization with IGN101 induced reactivity against a different set of peptides. Antibodies cross-reacting with both the IgG2a framework and with the Mab17-1A idiotype were identified. In summary, our data indicate that some EpCAM peptides may be recognized in a species-specific manner. At least seven EpCAM-derived peptides could be of diagnostic interest (QCQCTSVGAQ, ERVRTYWIII, ALQKEITTRY, TYWIIIELKH, IADVAYYFEK, AYYFEKDVKG, GQTLIYYVDE), while four out of these seven peptides may also possess therapeutic relevance (TYWIIIELKH, ALQKEITTRY, IADVAYYFEK, AYYFEKDVKG).

Qualification and application of a surface plasmon resonance-based assay for monitoring potential HAHA responses induced after passive administration of a humanized anti Lewis-Y antibody.

A sensitive, surface plasmon resonance (SPR)-based assay monitoring potential human-anti-human antibody (HAHA) reactions against the monoclonal antibody (mAb) IGN311 is presented. The latter is a fully humanized Lewis-Y carbohydrate specific mAb that is currently tested in a passive immune therapy approach in a clinical phase I trial. For the SPR experiments a BIACORE 3000 analyzer was used. The ligand IGN311 was covalently coupled to the carboxy-methylated dextran matrix of a CM5 research grade chip (BIACORE). In the course of a fully nested experimental design, a four parameter logistic equation was identified as appropriate calibration model ranging from 0.3 microg/mL (lower limit of quantitation, LLOQ) to 200 microg/mL (upper limit of quantitation, ULOQ) using an anti-idiotypic mAb ('HAHA mimic') as calibrator. The bias ranged from -2.4% to 5.5% and the intermediate precision expressed as 95% CI revealed values from 5.6% to 8.3%. Specificity was evaluated using six human serum matrices from healthy donors spiked with calibrator at the limit of quantitation (LOQ) with >80% of values being recovered with less than 25% relative error. The qualified assay was applied to monitor potentially induced HAHA reactivity in 11 patients from a clinical phase I trial with passively administered IGN311. Of the 11 patients, one high HAHA responder and several low responders were identified. Protein-G depletion experiments with human serum samples revealed that the observed response is predominantly caused by IgG binding to the ligand. The characteristics of these HAHA responses were all of the so-called 'Type I' which is defined by a peak response around day 15 that decreases from this point steadily suggesting that some kind of tolerance is established. Therefore, this type of HAHA response is regarded as non critical for the patient's safety.

IgE-positive Langerhans cells and Th2 allergen-specific T cells in atopic dermatitis.

In atopic dermatitis (AD) IgE-positive Langerhans cells (LC) may be present in the epidermis. These LC are able to capture allergens by means of their specific IgE, inducing an allergen-specific T-cell response in autologous peripheral blood T cells. Epicutaneous patch testing (EPT) may induce an eczematous reaction when IgE is present on the LC. Thus, both in vivo and in vitro, it appears that IgE may be crucial for induction of allergen-specific T-cell responses. Indeed, the cloning of infiltrating T cells from a positive 12-h EPT produced allergen-specific T cells, wheras no in vivo activated bystander T cells have yet been cloned. Moreover, greater than 85% of the T cells cloned were of Th2 phenotype after anti-CD3 and phorbol myristate acetate stimulation, whereas all clones were Th2 after allergen-specific stimulation, and they were able to induce IgE production in normal B cells. This completes the circle of events, because IgE produced by peripheral B cells may bind to LC and facilitate new allergen-specific reactions in the skin.

MDP(Lysyl)GDP, a nontoxic muramyl dipeptide derivative, inhibits cytokine production by activated macrophages and protects mice from phorbol ester- and oxazolone-induced inflammation.

High levels of pro-inflammatory cytokines and nitric oxide are proposed to orchestrate pathophysiologic mechanism(s) associated with various inflammatory dermatoses. This study examines whether a water soluble 3-O-[N-acetylmuramyl-L-lysyl-D-iso]-2-di-on-glycine [MDP(Lysyl)GDP], a nontoxic and nonpyrogenic derivative of muramyl dipeptide (MDP), can inhibit the in vitro production of inflammatory mediators by lipopolysaccharide- or interferon-gamma-activated macrophages, and whether such an inhibitory effect can translate into in vivo protection of mice from irritant and allergic contact dermatitis. Thioglycollate-elicited peritoneal macrophages cultured in medium alone or in medium supplemented with MDP(Lysyl)GDP (1-100 microg per ml) expressed neither mRNA transcripts for inducible nitric oxide synthase, interleukin-1beta, and tumor necrosis factor-alpha, nor cytokine proteins and nitric oxide activity. Incubation of the cells with either lipopolysaccharide or interferon-gamma for 6 h resulted in a significant induction of inducible nitric oxide synthase, interleukin-1beta, and tumor necrosis factor-alpha mRNA, and the accumulation of high levels of monokines and nitrites in cultures by 24 h. Co-incubation of the macrophages with lipopolysaccharide or interferon-gamma and MDP(Lysyl)GDP (1-100 microg per ml) resulted in a concentration-dependent suppression of the steady-state mRNA transcripts for inducible nitric oxide synthase, tumor necrosis factor-alpha, and interleukin-1beta, induced by lipopolysaccharide, but not by interferon-gamma. In mouse models of phorbol ester- and oxazolone-induced ear inflammation, topical application of MDP(Lysyl)GDP significantly suppressed ear swelling in a dose-dependent manner. Likewise, oral treatment with MDP(Lysyl)GDP at days -3, -2, and -1 before elicitation with oxazolone also significantly inhibited ear inflammation. Taken together, our findings suggest that MDP(Lysyl)GDP has the potential to be a therapeutic application in the treatment of inflammatory conditions in which overproduction of pro-inflammatory mediators are implicated to play a pathogenic role.

The influence of T cell depletion on recovery of T cell proliferation to herpesviruses and Candida after allogeneic bone marrow transplantation.

To test the influence of T cell depletion of the marrow in allogeneic bone marrow transplantation on functional T cell recovery, in vitro lymphocyte proliferation tests (LPTs) to microbial antigens were regularly performed in 23 recipients of normal BM and in 25 patients receiving BM with a fixed low number of T cells (1 x 10(5) T cells/kg body weight; recipients of T-depleted BM). The long-term recovery of positive LPT to at least 1 of the 4 tested microbial antigens--Candida, herpes simplex virus (HSV), varicella-zoster virus (VZV), and cytomegalovirus--was nearly similar in both groups: 16/23 versus 18/25. Recovery of LPT to Candida and HSV in the first 3 months appeared to be greatly influenced by prophylactic measures; only 2/23 recipients of normal BM, receiving amphotericin B, showed a positive LPT to Candida versus 13/25 recipients of T-depleted BM (P less than 0.01). In contrast, only 1/23 seropositive recipients of T-depleted BM, receiving acyclovir, showed a positive LPT to HSV versus 9/22 recipients of normal BM (P less than 0.05). A positive LPT to CMV in the first 3 months was found in 9/9 seropositive recipients of normal BM, versus in 5/11 seropositive recipients of T-depleted BM (P less than 0.05). Five of the 6 patients with a negative LPT died of CMV-interstitial pneumonia versus 1/14 with positive LPT (P less than 0.01). We conclude that in CMV-seropositive recipients of allogeneic BM, T cell depletion of the graft affects the early recovery of T cell proliferation to CMV, which is associated with a higher risk of fatal CMV-interstitial pneumonia.

The 'monocytic' cell line I937 displays typical B cell characteristics.

The monocytic cell line I937 was derived from U937 by sorting for cells with high expression of MHC class II molecules. Further analysis of these class II molecules revealed the presence of the HLA-DR3 subtype suggesting that the cell line was a potential candidate for testing antigen presentation to T cells restricted by HLA-DR3. We found that the T cell clones CFTS4:2.80 and CFTS4:2.6 with the required restriction element responded to the house dust mite antigen DPT presented by I937 but not U937, whereas CFTS4:3.1, which is not HLA-DR restricted, did not respond to either cell line. Subsequent analysis of surface markers on I937, however, indicated that the cell line is of B cell origin. In contrast to the parental cell line U937, I937 was tested negative for CD4, CD31 and CD64 but expressed CD19, CD21 and CD40. Although neither surface nor cytoplasmic Ig molecules were detected in either I937 or U937, Southern blot analysis revealed IgH gene rearrangement in I937. In addition, a fragment specific for Epstein-Barr virus nuclear antigen (EBNA2) was amplified in I937 by PCR technique. Therefore, we conclude that I937 is an EBV-transformed B cell line, presumably derived from the same donor and not as reported originally as a subline of U937, which expresses high MHC class II levels.

IgE production after antigen-specific and cognate activation of HLA-DPw4-restricted T-cell clones, by 78% of randomly selected B-cell donors.

The frequency of expression of the MHC class II antigen, HLA-DPw4, in the caucasoid population is approximately 78%, and is unmatched by phenotypic frequencies of other HLA class II molecules. Here we describe three human Der-P1-specific T-cell clones (TCC), restricted by the HLA-DPw4-variant HLA-DPB1*0401, of which two TCC also responded to antigen, presented on HLA-DPB1*0402. Thus, randomly selected caucasoid donors present a 78% chance for a correct match with these HLA-DPw4-restricted TCC. This allows comparative in vitro antigen presentation studies with various antigen presenting cells (APC) from different (healthy or diseased) donors without the variable influence of responding T cells. It was subsequently demonstrated that the TCC can be used to study antigen-induced IgE production in randomly selected primary B cells. Cognate HLA-DPw4-restricted antigen presentation caused enhanced immunoglobulin production of IgE, IgG1, IgA and IgM, of which only IgE induction was reversed by addition of anti-IL-4 antibodies.

Antigen focusing by specific monomeric immunoglobulin E bound to CD23 on Epstein-Barr virus-transformed B cells.

Monomeric IgE bound to the low-affinity receptor for IgE (FcERII-CD23) on EBV-transformed human B cells selectively enhances binding of antigen and therefore presentation to specific T-cell clones. To demonstrate the role of monomeric IgE in antigen focusing, we have made use of a system consisting of human T-cell clones specific for Der-P1 (major allergen of the Dermathophagoides pteronyssinus), Der-P1 coupled to NIP (Der-P1-NIP), and the commercially available chimeric (human-murine) monoclonal IgE antibodies with specificity for the hapten NIP. We have found that monomeric IgE binds to CD23 and remains detectable on the surface of the B cells for a period of at least 16 hours at 37 degrees C. Pulsing of these IgE-anti-NIP (1 microgram/ml) treated B cells for 1 hour at 37 degrees C with low amounts (10 ng/ml) of Der-P1-NIP antigen allows the B cells to stimulate Der-P1-specific T cells. Even with IgE concentrations as low as 20 ng/ml, which were not detectable by immunofluorescence, we were able to induce a significant T-cell response. Furthermore, ongoing specific T-cell-B-cell interactions were not inhibited by the presence of high concentrations of nonspecific IgE molecules (incubated with up to 25 micrograms/ml) on the surface of the B cells. Our data confirm the hypothesis that IgE, bound by either CD23 or the high-affinity receptor for IgE, potentiates the immune response. Therefore, IgE may be seen as the fourth general mechanism for antigen capture by (nonspecific) antigen-presenting cells.

Induction of IgE and IgG1 in human B cell cultures with staphylococcal superantigens: role of helper T cell interaction, resistance to interferon-gamma.

Non-antigen-specific activation of human B lymphocytes for IgE production in vitro requires the presence of interleukin 4 and non-cognate physical interaction with T cells. The latter can be replaced by antibodies directed against the B cells' CD40 structure. Antigen-specific induction of immunoglobulin responses, including IgE, is difficult in human lymphocyte cultures. Thus, we developed a model system which might resemble physiological B lymphocyte stimulation by antigen. Co-cultures of purified tonsillar B cells from normal donors with non-HLA matched T helper clones obtained from the skin of atopic dermatitis patients produced significant levels of IgE and IgG1 after stimulation with appropriate types of staphylococcal exotoxins, provided that IL-4 was also induced in the T cells. Such responses were further enhanced by addition of low doses of anti-CD40 antibodies. Concentrations of anti-CD40, optimal for stimulation of B cells in the absence of T helper lymphocytes, were less effective in this regard and even inhibitory in some experiments. Most powerful immunoglobulin induction was observed when the cultures were spiked with low amounts of IL-4 and anti-CD40 which did not elicit substantial immunoglobulin production in the absence of the staphylococcal exotoxins. Induction of IL-2 in T/B cell cultures by superantigens without production of appreciable quantities of IL-4 provoked considerable IgG1 titer but no IgE. High amounts of interferon-gamma generated by the T cells in vitro in the presence of superantigens did not appear to interfere with immunoglobulin induction. Addition of recombinant interferon at the beginning of the culture period at doses which effectively suppressed IL-4 plus anti-CD40 induced immunoglobulin responses did not inhibit T helper and superantigen dependent B cell activation. Superantigen mediated B cell stimulation for immunoglobulin production was dependent on cell-cell contact. The experimental results presented suggest that this cellular interaction did not necessarily involve T-B cell bridging by superantigens.

Cytomegalovirus seronegative platelets and leukocyte-poor red blood cells from random donors can prevent primary cytomegalovirus infection after bone marrow transplantation.

The effect of a deliberate transfusion policy for prevention of primary cytomegalovirus (CMV) infection, consisting of leukocyte-poor red blood cells from random donors and platelets from CMV-negative donors, was studied in 29 CMV-negative negative recipients of an allogeneic (from CMV-negative donors) or autologous bone marrow transplant. All transplant recipients remained CMV-negative with this approach. Such a policy depends on the availability of CMV-negative platelet donors. Siblings from CMV-negative marrow transplantation candidates appeared to be more often CMV-negative than siblings from CMV-positive transplantation candidates (77% versus 34%, p less than 0.001). Selection of CMV-negative blood bank donors for transfusion of blood products is also easy to perform. As a consequence, CMV-negative marrow transplant recipients have a good chance of receiving CMV-negative marrow transplants and blood products and primary CMV infection can thus be prevented by this transfusion policy.

In vitro IgE but not IgG production of canine peripheral blood B cells is inhibited by CD40 ligation.

The aim of this study was to investigate in vitro IgE induction in peripheral canine B cells. CD21(+) B cells were purified from the peripheral blood of beagle dogs by positive selection via magnetic separation to a purity of >/=95%. Subsequently, proliferation, and IgG and IgE production of canine B cells were investigated after stimulation with human recombinant Interleukin-4 (hrIL-4) and human recombinant Interleukin-2 (hrIL-2) in the presence or absence of CD40L-CD8 fusion protein (CD40L) of mouse origin. We could demonstrate that canine B cells react on hrIL-2 alone by proliferation and IgG production but not by IgE secretion, whereas activation with hrIL-4 induced proliferation and mainly IgE production. Together, both cytokines synergistically increased B cell proliferation as well as IgG and IgE production. We could also show that mouse CD40L induces proliferation of dog B cells, which is further enhanced by addition of hrIL-4. Unexpectedly, CD40L led to a dramatic decrease in the IL-4 mediated IgE secretion (82% inhibition on an average). In contrast, IgG production was not affected significantly by CD40L. The same effects of CD40L were observed when B cells were stimulated by a combination of IL-2 and IL-4 and this inhibition could not be abrogated by increasing the amounts of IL-4. In summary, activation of canine B cells from peripheral blood by hrIL-4 in the presence or absence of hrIL-2 led to marked IgE production that is strongly and in a dose-dependent manner inhibited by CD40L. Stimulation of IgG production is not influenced by CD40L.

The presence of IgE molecules on epidermal Langerhans cells in atopic dermatitis and their significance for its pathogenesis.

Aeroallergens can induce a delayed patch test reaction, which is specific for patients with atopic dermatitis (AD). Histopathology of this aeroallergen induced skin reaction suggests the involvement of Langerhans cells (LC), T cells and eosinophils. Since LC from AD patients may bear Fc sigma, R-bound IgE molecules, it was hypothesized that aeroallergens after contact with the skin bind to allergen-specific IgE on LC leading to efficient presentation to T cells. In vitro studies show that LC from AD patients can indeed present aeroallergens to T cells, provided LC-bound IgE molecules are present. These results, firstly, explain the reaction mechanism behind the patch test reaction to aeroallergens; secondly, they point to a new allergic reaction mechanism in which a link is provided between type I and type IV allergic reactions according to Gell and Coombs; thirdly, they throw a new light on the role of aeroallergens in the pathogenesis of AD. Aeroallergens may after contact with skin of AD patients not only induce eczematous skin lesions but also be involved in the induction of the synthesis of allergen specific IgE molecules. From a practical point of view, the results of this study show that aeroallergens play a more important role in the pathogenesis of AD than has been accepted so far.

Introducing antigen-binding sites in structural loops of immunoglobulin constant domains: Fc fragments with engineered HER2/neu-binding sites and antibody properties.

Yeast surface display libraries of human IgG1 Fc regions were prepared in which loop sequences at the C-terminal tip of the CH3 domain were randomized. A high percentage of these library members bound to soluble CD64 and Protein A indicating that the randomization step did not grossly interfere with the overall structure of the displayed Fc. Sorting these libraries by FACS for binders against HER2/neu yielded antigen-specific Fc binders (Fcab; Fc antigen binding) of which one was affinity matured, resulting in Fcab clone H10-03-6 which showed >10-fold improvement in antigen-binding activity versus the parental clone. Pre-equilibrium surface plasmon resonance experiments revealed a K(D) value of 69 nM. H10-03-6 did not react with other members of the HER family and specifically interacted with HER2-positive but not with HER2-negative cells. Importantly, Fcab H10-03-6 elicited potent antibody-dependent cellular cytotoxicity in vitro. Finally, the in vivo half-life in mice was similar to wild-type Fc indicating that the amino acid changes in the CH3 domain did not affect the pharmacokinetic behavior of the recombinant Fc. Our data demonstrate that the Fcab scaffold combines all features of normal antibodies in a small 50 kD homodimeric protein: antigen binding, effector functions and long half-life in vivo.

IgG subclass distribution of anti-ADAMTS13 antibodies in patients with acquired thrombotic thrombocytopenic purpura.

BACKGROUND: ADAMTS13-neutralizing IgG autoantibodies are the major cause of acquired thrombotic thrombocytopenic purpura (TTP). OBJECTIVE: To analyze the IgG subclass distribution of anti-ADAMTS13 antibodies and a potential relationship between subclass distribution and disease prognosis. METHODOLOGY: An enzyme-linked immunosorbent assay-based method was used to quantify the relative amounts of IgG subclasses of anti-ADAMTS13 antibodies in acquired TTP plasma. RESULTS: IgG(4) (52/58, 90%) was the most prevalent IgG subclass in patients with acquired TTP, followed by IgG(1) (52%), IgG(2) (50%), and IgG(3) (33%). IgG(4) was found either alone (17/52) or with other IgG subclasses (35/52). IgG(4) was not detected in 10% of the patients. There was an inverse correlation between the frequency and abundance of IgG(4) and IgG(1) antibodies (P < 0.01). Patients with high IgG(4) levels and undetectable IgG(1) are more prone to relapse than patients with low IgG(4) levels and detectable IgG(1). CONCLUSIONS: All IgG subclasses of anti-ADAMTS13 antibodies were detected in patients with acquired TTP, with IgG(4), followed by IgG(1), antibodies dominating the anti-ADAMTS13 immune response. Levels of IgG(4) could be useful for the identification of patients at risk of disease recurrence.

Consequences of IgE/CD23-mediated antigen presentation in allergy.

Allergen-specific IgE antibodies are responsible for the generation of immediate-type hypersensitivity reactions. However, as described here by Geert Mudde, Roy Bheekha and Carla Bruijnzeel-Koomen, IgE may also be involved in the uptake and processing of allergens. Such IgE-mediated antigen presentation may lead to a continuous (over) activation of the immune system due to high titers of IgE and the presence of large numbers of allergen-specific Th2 cells. In addition, it may be a cause for the advance of disease from a 'single allergy' to 'multi-allergy' syndrome.

Maternal antigen stimulation downregulates via mother's milk the specific immune responses in young mice.

BACKGROUND: Different aspects of the vertical transfer of predisposition to allergy from mother to child have been investigated. An issue which is still largely open is the influence of breast-feeding by allergic mothers on the development of the allergic phenotype of the infant. In the current study we employed a murine ovalbumin (OVA) immunization model to investigate possible milk-borne influences of the mother's specific immune status on the primary immune response of the breast-fed pup. METHODS: Naïve and OVA-immunized female mice were mated simultaneously. Immediately after birth litters were exchanged between the immunized and the untreated mothers which allowed the evaluation of maternal influence exerted via milk only. At the age of 3 weeks the pups were injected with a single dose of OVA intraperitoneally and sacrificed 2 weeks later. Serum was obtained for the determination of total and OVA-specific IgE and IgG2a. In addition, lymphocyte proliferation was measured following OVA stimulation of the pups' splenocytes and lymph node cells. During the lactation period milk was collected from the mothers for evaluation of its total and OVA-specific immunoglobulin levels. RESULTS: Breast-feeding of naïve pups by OVA-immunized mothers results in the suppression of the pups' specific IgE response as well as the downregulation of the OVA-induced proliferative response of the pups' lymph node cells and splenocytes. Additionally, splenocytes of naïve pups nursed by immune mothers show a decrease in IL-4 production compared to naïve pups nursed by naïve mothers, whereas the IFN-gamma production is not altered. CONCLUSION: We demonstrated the suppression of the pups' primary humoral and cellular response towards OVA by breast-feeding by mothers exposed to OVA shortly before pregnancy. It appears that such a transfer of the suppressive activity from mother to pups via milk directs the pups' immune response towards a Th1 and away from a Th2 type, thus avoiding the 'allergic' phenotype. Our study suggests that breast-feeding by mothers immune to an antigen may suppress the development of an allergic response to this antigen.