Anti-TNF-alpha antibody (infliximab) therapy supports the recovery of eNOS and VEGFR2 protein expression in endothelial cells.

The aim of this study is to investigate the effect of sera obtained from patients of Crohn's disease treated by anti-TNF-alpha antibody (Infliximab) on the expression of endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor receptor-2 (VEGFR2) protein in human umbilical vein endothelial cells (HUVEC) cultured in vitro. HUVEC was cultured in the presence of sera derived from patients before and after treatment, or from healthy individuals. Effects of sera on the expression of eNOS and VEGFR2 were monitored by determination of mRNA and protein levels using real time quantitative PCR and Western blot analysis, respectively. The serum of Crohn's patients contained elevated levels of TNF-alpha (34±1.80 pg/mL), which resulted in a decrease in the protein level of eNOS in HUVEC with a simultaneous induction of VEGFR2. Infliximab treatment normalized the expression level of these proteins by decreasing TNF-alpha level, particularly in those cases when clinical healing was also recorded, and it also conferred restitution of the level of angiogenic cytokines. Results suggest that altered angiogenesis possibly contributes to the initiation and perpetuation of inflammatory processes in inflammatory bowel disease (IBD). Endothelial dysfunction, a selective feature of Crohn's disease is beneficially affected by intravascular TNF-alpha neutralization.

Modeling MHC class II molecules and their bound peptides as expressed at the cell surface.

A detailed insight to the structure of a given major histocompatibility complex (MHC)-peptide complex can strongly support and also improve the analysis of the peptide binding capabilities of the MHC molecule and the characterization of the developing T cell response. The number of MHC class II-peptide crystal structures is limited, therefore constructing and analyzing computer models can serve as efficient complementary tools when someone deals with experimentally determined binding and/or functional data. Commercial programs are available for modeling protein and protein-protein complexes, in general. However, more accurate results can be obtained if the parameters are directly optimized to a given complex, especially in the case of special proteins as MHC class II, an integral membrane protein, whose functional parts behave like regular globular proteins. Here, we present the optimization of an approach used for modeling MHC class II molecules complexed with various peptides fitting into the binding groove and several ways to analyze them with the help of experimental data.

Serum amyloid P component inhibits influenza A virus infections: in vitro and in vivo studies.

Serum amyloid P component (SAP) binds in vitro Ca(2+)-dependently to several ligands including oligosaccharides with terminal mannose and galactose. We have earlier reported that SAP binds to human influenza A virus strains, inhibiting hemagglutinin (HA) activity and virus infectivity in vitro. These studies were extended to comprise five mouse-adapted influenza A strains, two swine influenza A strains, a mink influenza A virus, a ferret influenza A reassortant virus, a influenza B virus and a parainfluenza 3 virus. The HA activity of all these viruses was inhibited by SAP. Western blotting showed that SAP bound to HA trimers, monomers and HA1 and HA2 subunits of influenza A virus. Binding studies indicated that galactose, mannose and fucose moieties contributed to the SAP reacting site(s). Intranasal administration of human SAP to mice induced no demonstrable toxic reactions, and circulating antibodies against SAP were not detected. Preincubation of virus (A/Japan/57) with SAP prevented primary infection of mice and development of antiviral antibodies. After a single intranasal administration of SAP (40 microg) 1 h before primary infection with virus (2LD(50)), nine out of 10 mice survived on day 10 and these mice approached normal body weight, whereas control mice (one out of five surviving on day 10) died. The data provide evidence of the potential of intranasally administered SAP for prophylactic treatment of influenza A virus infections in humans.

Anti-cholesterol antibodies (ACHA) in patients with different atherosclerotic vascular diseases and healthy individuals. Characterization of human ACHA.

In animal experiments the protective role of anti-cholesterol antibodies (ACHA) in the development of atherosclerosis has been demonstrated. Despite the fact that ACHA are present in the serum of healthy humans, no data on the occurrence of these antibodies in human diseases are available. We determined serum concentrations of IgG type ACHA by an enzyme immunosorbent assay in 600 patients with atherosclerotic vascular diseases (86 patients with peripheral occlusive atherosclerosis, 146 patients with cerebrovascular diseases, 341 patients with severe coronary heart disease (CHD) who received aorto-coronary by-pass, 27 patients with myocardial infarction who did not undergo by-pass operation), in 57 patient controls (complaints of CHD, without coronarographic alterations) and in 218 healthy individuals. ACHA were present in the sera of all persons tested. No serum cofactor is needed for the binding of human ACHA to solid phase cholesterol, binding can be inhibited dose-dependently by LDL and even more strongly with LDL/VLDL preparations purified from human serum. ACHA levels were found to be considerably lower in patients with peripheral occlusive atherosclerosis and cerebrovascular diseases compared with the levels in healthy individuals. By contrast, the ACHA levels of patients with CHD were considerably higher. No differences in the IgG subclass distribution and binding efficiency of ACHA in the sera of CHD patients and controls were found. Thus, our present findings indicate that both low and high ACHA production may be associated with different atherosclerotic vascular diseases.

Function-related regulation of the stability of MHC proteins.

Proteins must be stable to accomplish their biological function and to avoid enzymatic degradation. Constitutive proteolysis, however, is the main source of free amino acids used for de novo protein synthesis. In this paper the delicate balance of protein stability and degradability is discussed in the context of function of major histocompatibility complex (MHC) encoded protein. Classical MHC proteins are single-use peptide transporters that carry proteolytic degradation products to the cell surface for presenting them to T cells. These proteins fulfill their function as long as they bind their dissociable ligand, the peptide. Ligand-free MHC molecules on the cell surface are practically useless for their primary biological function, but may acquire novel activity or become an important source of amino acids when they lose their compact stable structure, which resists proteolytic attacks. We show in this paper that one or more of the stabilization centers responsible for the stability of MHC-peptide complexes is composed of residues of both the protein and the peptide, therefore missing in the ligand-free protein. This arrangement of stabilization centers provides a simple means of regulation; it makes the useful form of the protein stable, whereas the useless form of the same protein is unstable and therefore degradable.

Effect of chain length on the conformation and T cell recognition of synthetic hemagglutinin fragments.

Circular dichroism and Fourier-transform infrared spectroscopies were used to compare the conformational mobility of 13-mer peptides covering the 317-329 region of the envelope protein hemagglutinin of human influenza A virus subtypes H1, H2 and H3 with that of their truncated deca- and nonapeptide analogs. These peptides were demonstrated to bind to the murine I-Ed major histocompatibility complex encoded class II and human HLA-B*2705 class I molecules. Despite the amino acid substitutions in the three 13-mer subtype sequences, no significant differences in the conformational properties could be shown. Deletion of the N-terminal three residues resulted in a shift to an increased alpha-helical conformer population in the 317-329 H1 peptide and the breakage of the 3(10) or weakly H-bonded (nascent) alpha-helix in the H2 and H3 peptides. The conformational change observed upon deletion did not influence the efficiency of I-Ed peptide interaction, however, the C-terminal Arg had a beneficial effect both on MHC class II and class I binding without causing any remarkable change in solution conformation.

Mapping of a protective helper T cell epitope of human influenza A virus hemagglutinin.

The synthetic peptide comprising the 317-341 region of human influenza A virus (H1N1 subtype) hemagglutinin elicits peptide-specific antibody and helper T cell responses and confers protection against lethal virus infection. Molecular mapping of the 317-329 region, which encompasses the epitope recognized by peptide-specific T cells, revealed that the minimal size required for T cell activation was the 317-326 segment. The most likely peptide alignment, which placed 320Leu to pocket 1 of the I-E(d) peptide binding groove, was predicted by molecular mechanics calculations performed with the parental and with the Ala-substituted analogs. In line with the prediction data, the results of the peptide binding assay, where the relative binding efficiency to I-E(d) molecules expressed on the surface of antigen-presenting cells was monitored, identified the 320-326 core sequence interacting with the major histocompatibility class II peptide binding groove. Functional analysis of Ala-substituted variants by functional assays and by calculating the surface-accessible areas of the single peptidic amino acids in the I-E(d)-peptide complexes demonstrated that 324Pro is a primary contact residue for the T cell receptor. Our results show that this type of analysis offers a suitable tool for molecular mapping of helper T cell epitopes and thus provides valuable data for subunit vaccine design.

A repetitive sequence of Epstein-Barr virus nuclear antigen 6 comprises overlapping T cell epitopes which induce HLA-DR-restricted CD4(+) T lymphocytes.

Most human adults carry the Epstein-Barr virus (EBV) and develop immunological memory against the structural and the virus-encoded cellular proteins. The EBV nuclear antigen 6 (EBNA6) elicits cytotoxic T cell responses and it also maintains a persistent antibody response. The majority of sera from EBV-seropositive individuals reacts with a synthetic peptide, p63, comprising 21 amino acids of a repetitive region of EBNA6. CD4(+) T lymphocytes, with specificity for p63, could be recalled from the T cell repertoire of EBV carriers that expressed certain HLA-DR allotypes which were identified as good binders of p63 by an in vitro flow cytometric assay. Analysis of the HLA-DR/p63 interaction by molecular mechanics calculations indicated the presence of multiple overlapping epitopes which were predicted to bind in a HLA-DRB1 allo- and subtype-specific manner. Specific activation of p63-selected long-term CD4(+) T cell cultures resulted in a proliferative response, in the production of IL-2 and in the secretion of high levels of tumor necrosis factor as measured by bioassays. Proliferation and cytokine production of p63-specific T cells could be induced by p63-loaded HLA-DR-matched antigen-presenting cells and by B cells co-expressing relevant HLA-DR molecules and EBNA6. Our results show that peptides of an EBNA6 repeat region induce CD4(+) T cells which can react with EBNA6-carrying cells in many individuals. We suggest that these T(h) cells may be important in conditioning dendritic cells for initiation potent virus-specific immune responses, provide help for EBV-specific B cells, drive IgG isotype switch and support the sustained effector function of memory cytotoxic T lymphocytes.

Soluble interleukin-6 receptor (sIL-6R) makes IL-6R negative T cell line respond to IL-6; it inhibits TNF production.

The receptor for interleukin-6 (IL-6) consists of two subunits: a ligand specific IL-6Ralpha and gp130 that is responsible for signal-transduction. A soluble form of the ligand specific chain was described that when complexed to IL-6 is capable of binding to the membrane-bound gp130 subunit and thus can elicit signal-transduction. This soluble receptor can act on cells that express only the gp130 but not the ligand-specific subunit of the IL-6R. This phenomenon, called trans-signaling, introduced a novel aspect of cytokine action. In this study we examined the response of Jurkat cells, that are known not to express IL-6Ralpha, to IL-6, the soluble IL-6 receptor (sIL-6R) and a covalent complex of IL-6 and sIL-6R termed Hyper-IL-6. We studied the expression of tumour necrosis factor (TNF) and interferon-gamma (IFN-gamma). The complex of IL-6+sIL-6R and Hyper-IL-6 inhibited significantly the production of TNF in a gp130-dependent manner, whereas no differences in IFN-gamma expression were found. IL-6 and sIL-6R alone were not effective. Because we did not detect major differences in the TNF mRNA levels upon treatments, we conclude that the inhibition of TNF production should occur at the post-transcriptional level. These results provide another example of trans-signaling and underline the physiological importance of sIL-6R, and in the case of Hyper-IL-6 its possible therapeutic application can also be considered.

Synthesis and immunological studies of alpha-conotoxin chimera containing an immunodominant epitope from the 268-284 region of HSV gD protein.

We have synthesized and characterized new chimeric peptides by inserting an epitope of the glycoprotein D (gD) of herpes simplex virus (HSV) serotype 1 as 'guest' sequence in the 'host' structure of alpha-conotoxin GI, a 13-residue peptide (ECCNPACGRHYSC) isolated from the venom of Conus geographus. The 276-284 region of HSV gD-1 selected for these studies is highly hydrophilic and adopts a beta-turn. The alpha-conotoxin GI also contains a beta-turn in the 8-12 region, stabilized by two disulfide bridges at positions 2-7 and 3-13. Thus, the tetramer sequence of alpha-conotoxin, 8Arg-His-Tyr-Ser12 has been replaced by Asp-Pro-Val-Gly (DPVG), identified previously as the epitope core. The syntheses were performed by Fmoc strategy on Rink resin and DTNB or air oxidation were applied for the formation of the first 3-13 disulfide bond in the presence of guanidinium hydrochloride. For the formation of the second disulfide Cys2-Cys7 three different oxidation procedures [iodine in 95% acetic acid, air oxidation in dimethyl sulfoxide/1 M HCl or Tl(tfa)3 in trifluoroacetic acid (TFE)] were compared. The high-performance liquid chromatography purified peptides were characterized by electrospray mass spectrometry and amino acid analysis. The bicyclic HSV-alpha-[Tyr1]-conotoxin chimeric peptide and native alpha-conotoxin GI showed similar circular dichroism spectra in phosphate-buffered saline (PBS) and in a PBS-TFE 1:1 (v/v) mixture, which might suggest that these compounds also share similar secondary structures. In immunologic studies the characteristics of the primary and of the memory immunoglobulin (Ig) M- and IgG-type antibody responses showed that the bicyclic HSV-alpha-[Tyr1]-conotoxin chimera is capable to induce strong antibody responses in C57/Bl/6 mice but was poorly immunogenic in CBA and BALB/c mice. Data obtained with the C57/Bl/6 serum indicate that the polyclonal antibodies recognize the DPVG motif presented in the bicyclic HSV-alpha-[Tyr1]-conotoxin and some reactivity was also found with the monocyclic but not with the linear form of the chimera. Results with two IgM type monoclonal antibodies from a bicyclic HSV-alpha-[Tyr1]-conotoxin immunized C57/Bl/6 mouse also point to the specific interaction with the DPVG sequence. Taken together these studies suggest, that the relative intensity of DPVG-specific responses was found to be dependent on the mouse strain and on the conformation of the chimeric molecules. We found that the IgM monoclonal antibodies are able to recognize the linear DPVG sequence, while the majority of IgG antibodies is directed to the same motif in a conformation stabilized by double cyclization.

Carrier design: new generation of polycationic branched polypeptides containing OH groups with prolonged blood survival and diminished in vitro cytotoxicity.

For the construction of macromolecule-drug conjugates, it is important to provide rational basis to the selection of proper carrier. With respect to the importance of the side-chain structure and charge of the branched polypeptides in biological properties, we have prepared a new class of branched polypeptides with single or multiple hydroxyl groups and studied their solution conformation, in vitro cytotoxicity, biodistribution, and immunoreactivity. For comparative studies, polypeptides were designed to contain serine at various positions of the side chains, varying also the number. Ser was attached to the end of oligo(DL-Ala) side chains grafted to polylysine resulting polypeptides with the general formula poly[Lys(Ser(i)-DL-Ala(m))], (SAK). Ser was also coupled directly to the polylysine backbone poly[Lys(Ser(i))] (S(i)K) and then elongated by polymerization of N-carboxy-DL-Ala anhydride resulting poly[Lys(DL-Ala(m)-Ser(i))] (ASK). An additional polymer was also prepared, but instead of the oligo(DL-Ala) branches, oligo(DL-Ser) side chains were introduced (poly[Lys(DL-Ser(m))], SK). The presence of hydroxyl groups resulted in compounds with improved of water solubility. CD spectra of polypeptides showed significant differences correlating with the position and numbers of Ser residues in the side chains. Under physiological conditions, polycationic polypeptides assumed ordered secondary structure (S(i)K and LSK) or partially unordered conformation (SK, SAK, and ASK). Data of selected polymers demonstrate that these polycationic compounds are essentially nontoxic in vitro on normal rat liver or mouse spleen cells and have no cytostatic effect on mouse colorectal carcinoma C26 cells. The blood clearance and biodistribution of these derivatives were greatly dependent on the position and number of Ser residues in the branches and possess a rather extended blood survival in mice. Polypeptides were taken up predominantly by the liver and kidney (S(i)K, LSK, and ASK) or kidney and lung (SK and SAK). The best survival in the blood was found with SAK, representing the first polycationic branched polypeptide, which show extended blood clearance. The relative position of Ser residue had also a marked influence on the immunogenicity of polypeptides. The characteristics of the antibody response to polypeptide containing Ser at the end of the branches (SAK) or adjacent to the polylysine backbone (ASK) was also dependent on the genetic background of the mouse strains. We also found that these compounds have no effect on to the SRBC-specific humoral immune response, indicating the lack of nonspecific immunostimulatory potential. In conclusion, these studies suggest that synthetic branched polypeptides with Ser can be considered as candidates for constructing suitable conjugates for drug/epitope delivery. It is not only due to the presence of hydroxyl group to be used for oxime chemistry but also to their beneficial biological features.

Targeting of influenza epitopes to murine CR1/CR2 using single-chain antibodies.

Single-chain variable fragment (scFv) antibodies are genetically engineered molecules comprising the variable regions responsible for specific binding. scFv that recognize certain surface molecules on professional antigen presenting cells could therefore be suitable for targeting Ag to these cells. We have produced an scFv that recognizes murine complement receptors 1 and 2 (CR1/CR2) and genetically fused it with different numbers of influenza hemagglutinin peptides which contain both B and T cell epitopes. The CR1/CR2 specific hybridoma 7G6 was used for RT-PCR to obtain the variable regions, which were then combined to create an scFv fragment. The influenza hemagglutinin intersubunit peptide HA317-41 (IP) was engineered to the N terminus of the scFv in one, two or three copies. The so obtained IP(1-3)7G6scFv still bound the complement receptors; the peptides in the construct were recognized by the peptide specific monoclonal IP2-11-1 on Western blots and ELISAs. The CR1/CR2 positive B lymphomas A20 and 2PK3 presented the peptide to an I-Ed restricted IP specific T cell hybridoma more efficiently when incubated with the IP(1)7G6 constructs as compared to the free peptide. The results suggest that scFv could work as targeting devices in subunit vaccines.

Interleukin-7: physiological roles and mechanisms of action.

Interleukin-7 (IL-7), a product of stromal cells, provides critical signals to lymphoid cells at early stages in their development. Two types of cellular responses to IL-7 have been identified in lymphoid progenitors: (1) a trophic effect and (2) an effect supporting V(D)J recombination. The IL-7 receptor is comprised of two chains, IL-7R alpha and gamma(c). Following receptor crosslinking, rapid activation of several classes of kinases occurs, including members of the Janus and Src families and PI3-kinase. A number of transcription factors are subsequently activated including STATs, c-myc, NFAT and AP-1. However, it remains to be determined which, if any, previously identified pathway leads to the trophic or V(D)J endpoints. The trophic response to IL-7 involves protecting lymphoid progenitors from a death process that resembles apoptosis. This protection is partly mediated by IL-7 induction of Bcl-2, however other IL-7-induced events are probably also involved in the trophic response. The V(D)J response to IL-7 is partly mediated through increased production of Rag proteins (which cleave the target locus) and partly by increasing the accessibility of a target locus to cleavage through chromatin remodeling.

Horizontal transfer of DNA by the uptake of apoptotic bodies.

In this study we have raised the question of whether DNA can be transferred from one cell to another by phagocytosis of apoptotic bodies. We have used integrated copies of the Epstein-Barr virus (EBV) as a marker to follow the fate and expression pattern of apoptotic DNA in the phagocytotic host. Apoptosis was induced in EBV-carrying cell lines by irradiation before cultivation with either human fibroblasts, macrophages, or bovine aortic endothelial cells. Analysis of the expression pattern of EBV-encoded genes was performed by immunofluorescent staining as well as in situ hybridization. Cocultivation of apoptotic bodies from lymphoid cell lines containing integrated but not episomal copies of EBV resulted in expression of the EBV-encoded genes EBER and EBNA1 in the recipient cells at a high frequency. Fluorescence in situ hybridization analysis showed uptake of human chromatin as well as integrated EBV-DNA into the nuclei of bovine aortic endothelial cells. These data show that DNA may be rescued and reused from apoptotic bodies by somatic cells. In addition, our findings suggest that apoptotic bodies derived from EBV-carrying B lymphocytes may serve as the source of viral transfer to cells that lack receptors for the EBV virus in vivo.

A hemagglutinin-based multipeptide construct elicits enhanced protective immune response in mice against influenza A virus infection.

Multipeptide constructs, comprising adjacent sequences of the 317-341 intersubunit region of immature influenza A hemagglutinin (H1N1), were designed and the functional properties of these branched peptides were compared to that of the corresponding linear peptides. In vivo studies revealed that the immunogenicity of the peptides was dependent on the presence of the hydrophobic fusion peptide (comprised in FP3), encompassing the N-terminal 1-13 sequence of the HA2 subunit. Antibody and T cell recognition, however, was directed against the 317-329 HA1 sequence, comprised in the P4 peptide. Multiple copies of P4, covalently linked by branched lysine residues, significantly enhanced the efficiency of antibody binding and the capacity of peptides to elicit B- and T-cell responses. A fraction of peptide induced antibodies reacted with immature or with proteolitically cleaved hemagglutinin (HA) molecules pretreated at low pH. Immunization with a multipeptide construct, (P4)4-FP3, not only resulted in elevated antibody and T cell responses but conferred enhanced protection against lethal A/PR/8/34 (H1N1) infection as compared to its subunit peptides. The beneficial functional properties of this artificial peptide antigen may be acquired by multiple properties including: (i) stabilized peptide conformation which promotes strong, polyvalent binding to both antibodies and MHC class II molecules; (ii) appropriate P4 conformation for antibody recognition stabilized by the covalently coupled fusion peptide, resulting in the production of virus cross reactive antibodies which inhibit the fusion activity of the virus; (iii) activation of peptide specific B cells which potentiate antigen presentation and peptide specific T cell responses; and (iv) generation of helper T cells which secrete lymphokines active in the resolution of infection.

Characterizing immunodominant and protective influenza hemagglutinin epitopes by functional activity and relative binding to major histocompatibility complex class II sites.

In the present study the analysis of functional activity and major histocompatibility complex (MHC) binding of two adjacent MHC class II-restricted epitopes, located in the C-terminal 306-329 region of human influenza A virus hemagglutinin 1 subunit (HA1) conserved with subtype sequences and not affected by antigenic drift, was undertaken to explore the hierarchy of local immunodominance. The functional activity of two T cell hybridomas of the memory/effector Th1 phenotype in combination with in vivo immunization studies provided a good tool for investigating the functional characteristics of the T cell response. The in vitro binding assays performed with a series of overlapping, N-terminal biotinylated peptides covering the 306-341 sequence enabled us to compare the relative binding efficiency of peptides, comprising two distinct epitopes of this region, to I-Ed expressed on living antigen-presenting cells. Our studies revealed that (i) immunization of BALB/c mice with the 306-329 H1 or H2 peptides resulted in the activation and proliferation of T cells recognizing both the 306-318 and the 317-329 epitopes, while the 306-329 H3 peptide elicits predominantly 306-318-specific T cells, (ii) the 317-329 HA1 epitope of the H1 and H2 but not the H3 sequence is recognized by T cells and is available for recognition not only in the 317-329 peptide but also in the extended 306-329 or 306-341 peptides, (iii) the 306-318 and the 317-329 hemagglutinin peptides encompassing the H1, H2 but not the H3 sequence bind with an apparently similar affinity to and therefore compete for I-Ed binding sites, and (iv) the 317-341, the 317-329 peptides and their truncated analogs show subtype-dependent differences in MHC binding and those with lower binding capacity represent the H3 subtype sequences. These results demonstrate that differences in the binding capacity of peptides comprising two non-overlapping epitopes located in the C-terminal 306-329 region of HA1 of all three subtype-specific sequences to MHC class II provide a rationale for the local and also for the previously observed in vivo immunodominance of the 306-318 region over the 317-329 epitope in the H3 but not in the H1 or H2 sequences. In good correlation with the results of the binding and functional inhibition assays, these data demonstrate that in the H1 and H2 subtypes both regions are available for T cell recognition, they compete for the same restriction element with an apparently similar binding efficiency and, therefore, function as co-dominant epitopes. Due to the stabilizing effect of the fusion peptide, peptides comprising the 306-341 or 317-341 H1 sequences are highly immunogenic and elicit a protective immune response which involves the production of antibodies and interleukin-2 and tumor necrosis factor producing effector Th1 cells both directed against the 317-329 region. Based on the similarity of the I-Ed and HLA-DR1 peptide binding grooves and motifs, these results suggest that amino acid substitutions inserted to the H3 subtype sequence during viral evolution can modify the relative MHC binding capacity and invert the local hierarchy of immunodominance of two closely situated epitopes that are able to bind to the same MHC class II molecule.

Collaboration of TCR-, CD4- and CD28-mediated signalling in antigen-specific MHC class II-restricted T-cells.

A previously developed experimental system was applied to obtain qualitative and quantitative data on the contribution of TCR-, CD4- and CD28-mediated signalling in the activation of an antigen specific T-cell hybridoma. All the three signal transducing receptors were stimulated by their natural ligands, and intermediate and late responses of an I-Ed restricted, CD4 +, influenza HA specific murine T-hybridoma (IP-12-7) were monitored by measuring the concentration of intracellular calcium [Ca2+]i and secreted IL-2. This type of analysis of T-cell activation revealed: (i) calcium mobilization induced by peptide loaded APC requires rapid conjugate formation; (ii) a direct correlation between the magnitude of the intermediate and the late responses was observed as a consequence of differential TCR ligation modulated by peptide dose or by the presence CD4; (iii) considering the APC/peptide and T/APC ratios, the concentration dependence of the intermediate and late responses was similar in both assays but a substantial difference in the sensitivity of the two methods was observed; (iv) CD4 mediated signalling has a co-stimulatory effect predominantly at suboptimal in vitro conditions; and (v) sustained increase of [Ca2+]i as well as the production of high concentrations of IL-2 is highly dependent on the CD28-B7 interaction. These results demonstrate that distinct peptide doses and the presence or absence of CD4 result in quantitative changes in T-cell responses, while the degree of CD28 mediated signalling has a qualitative affect on the outcome of T-cell activation, revealed by complete or partial inhibition of IL-2 secretion as a result of limited CD28-B7 interaction as well as by alteration in the duration and time kinetics of the calcium response.

Kinetics and isotype profile of rheumatoid factor production during viral infection: organ distribution of antibody secreting cells.

The kinetics and isotype profile of influenza virus-specific IgG antibodies were studied in correlation with the serum titre of IgG-reactive autoantibodies. An increased level of IgG isotype-specific, rheumatoid factor-type autoantibody secretion was observed in the late phase of the virus-specific memory response. These rheumatoid factors were specific for the IgG2a and IgG1 subclasses which dominated the anti-viral antibody response. As revealed by a preparative immunosorbent technique combined with isotype quantitation the majority of IgG2a- or IgG1-bound immunoglobulins isolated from the serum of virus-infected mice belonged to the same subclass as the target antibody. Comparison of the kinetics of appearance and the number of IgM-, IgG- and IgA-type IgG2a-reactive autoantibody secreting cells during the primary and memory anti-viral antibody responses showed isotype switch of IgM rheumatoid factor secreting cells predominantly to IgA. Localization of IgM and IgA antibody secreting cells demonstrated the wide organ distribution of IgM-type rheumatoid factor secreting cells. On the contrary, IgA rheumatoid factor production was observed only in Peyer's patches and at the site of the local virus-specific immune response, i.e. in mediastinal lymph nodes and in the lung. These results demonstrate that B cells specific for self IgG are activated and differentiated in concert with the viruspecific antibody response in similar microenvironments. The predominant involvement of the mediastinal lymph nodes and the spleen in the production of IgG2a-specific IgM-type autoantibodies suggest a regulatory function of this type of autoantibodies in modulating IgG2a production in both systemic and local anti-viral immune responses. The results also suggest a strictly regulated rheumatoid factor production which, however, can be unbalanced by repeated viral infections resulting in the escape of high affinity, isotype-switched autoantibodies.