Humoral immune response to keyhole limpet haemocyanin, the protein carrier in cancer vaccines.

Keyhole limpet haemocyanin (KLH) appears to be a promising protein carrier for tumor antigens in numerous cancer vaccine candidates. The humoral immune response to KLH was characterized at the single-cell level with ELISPOT combined with separations of cell populations according to their expression of homing receptors (HRs). The analysis of HR expressions is expected to reveal the targeting of the immune response in the body. Eight orally primed and four nonprimed volunteers received KLH-vaccine subcutaneously. Circulating KLH-specific plasmablasts were found in all volunteers, 60 KLH-specific plasmablasts/10(6) PBMC in the nonprimed and 136/10(6) in the primed group. The proportion of L-selectin(+) plasmablasts proved high and integrin α(4)ß(7) (+) low. KLH serving as protein carrier in several vaccines, the homing profile of KLH-specific response may be applicable to the cancer antigen parts in the same vaccines. The present data reflect a systemic homing profile, which appears advantageous for the targeting of immune response to cancer vaccines.

T-cell regulation of murine IgA synthesis.

In studies reported here, the polyclonal activator lipopolysaccharide was used to stimulate the synthesis and secretion of IgM, IgA, and IgG in cultures of mouse lymphoid cells. The total immunoglobulin of each class which resulted was measured by specific double-antibody radioimmunoassays. The effect of Con A-activated T cells from various tissues on such immunoglobulin synthesis was then assessed. Variations in regulatory T-cell activity among the various lymphoid tissues for IgA but not for IgM or IgG was observed. In particular, Peyer's patches T cells were found to contain a high level of IgA T-cell helper activity compared to that of spleen or peripheral lymph node. The independent variation of T-cell regulatory activity for IgA as compared to that for IgM and IgG among the different tissues is most consistent with there being a separate subset of T cells specifically regulating IgA. The significance of these findings for the understanding of the secretory immune system is discussed.

CD4+ T cells reactive to enteric bacterial antigens in spontaneously colitic C3H/HeJBir mice: increased T helper cell type 1 response and ability to transfer disease.

C3H/HeJBir mice are a new substrain that spontaneously develop colitis early in life. This study was done to determine the T cell reactivity of C3H/HeJBir mice to candidate antigens that might be involved in their disease. C3H/HeJBir CD4+ T cells were strongly reactive to antigens of the enteric bacterial flora, but not to epithelial or food antigens. The stimulatory material in the enteric bacteria was trypsin sensitive and restricted by class II major histocompatibility complex molecules, but did not have the properties of a superantigen. The precursor frequency of interleuken (IL)-2-producing, bacterial-reactive CD4+ T cells in colitic mice was 1 out of 2,000 compared to 1 out of 20,000-25,000 in noncolitic control mice. These T cells produced predominately IL-2 and interferon gamma, consistent with a T helper type 1 cell response and were present at 3-4 wk, the age of onset of the colitis. Adoptive transfer of bacterial-antigen-activated CD4+ T cells from colitic C3H/HeJBir but not from control C3H/HeJ mice into C3H/HeSnJ scid/scid recipients induced colitis. These data represent a direct demonstration that T cells reactive with conventional antigens of the enteric bacterial flora can mediate chronic inflammatory bowel disease.

Helper T cell subsets for immunoglobulin A responses: oral immunization with tetanus toxoid and cholera toxin as adjuvant selectively induces Th2 cells in mucosa associated tissues.

Antigen-specific B cell responses to mucosally delivered proteins are dependent upon CD4-positive T helper (Th) cells, and the frequency of Th1 and Th2 cell responses after oral immunization may determine the level and isotype of mucosal antibody responses. We have used a protein-based vaccine, tetanus toxoid (TT), together with the mucosal adjuvant cholera toxin (CT), for oral immunization of mice to study the nature of antigen-specific Th cell subsets induced in Peyer's patches (PP) of the gastrointestinal (GI) tract and in the spleen (SP) during peak antibody responses. Mice orally immunized with TT and CT responded with antigen-specific secretory immunoglobulin A (S-IgA) antibodies in the GI tract, and with both IgG and IgA antibody responses in serum. PP and SP CD4+ T cells from mice orally immunized with TT plus CT were cultured with antigen-coated latex microspheres for induction of proliferative responses and for enumeration of cytokine producing CD4+ T cells. Interestingly, both PP and SP CD4+ T cell cultures showed increased numbers of IL-4- and IL-5 (Th2-type)-producing, spot-forming cells (SFCs) after 21 d of immunization, while essentially no interferon-gamma (IFN-gamma) or IL-2 (Th1-type) SFCs were noted. Cytokine-specific Northern blots and RT-PCR also revealed that significant IL-4 and IL-5 mRNA levels, but not IFN-gamma or IL-2 mRNA, were present in CD4+ T cells isolated from antigen-stimulated cultures. However, systemic immunization with TT and CT induced antigen-specific IgG and IgM but not IgA antibodies in serum. Further, both IL-2 and IFN-gamma-producing Th1-type cells as well as IL-4- and IL-5-secreting Th2-type cells were generated in SP. Our results show that oral immunization with TT and the mucosal adjuvant CT selectively induced antigen-specific Th2-type responses which may represent the major helper cell phenotype involved in mucosal IgA responses in the GI tract.

Linkage of Crohn's disease-related serological phenotypes: NFKB1 haplotypes are associated with anti-CBir1 and ASCA, and show reduced NF-kappaB activation.

BACKGROUND AND AIMS: Genetics studies of the serum expression of antibodies to microbial antigens may yield important clues to the pathogenesis of Crohn's disease. Our aim was to conduct a linkage study using expression of anti-CBir1, anti-I2, anti-OmpC and ASCA as quantitative traits. METHODS: Expression of antibodies to microbial antigens was measured by enzyme-linked immunosorbant assay (ELISA) and a standard approximately 10 cM whole genome microsatellite study was conducted. Single nucleotide polymorphism genotyping was performed using either Illumina or TaqMan MGB technology. Nuclear factor Kappa B (NF-kappaB) activation in cells from Epstein-Barr virus (EBV)-transformed cell lines was assessed using an electrophoretic mobility shift assay and protein was measured using ELISA and western blotting. RESULTS: Evidence for linkage to anti-CBir1 expression was detected on human chromosome 4 (logarithm of odds (LOD) 1.82 at 91 cM). We therefore directly proceeded to test the association of haplotypes in NFKB1, a candidate gene. One haplotype, H1, was associated with anti-CBir1 (p = 0.003) and another, H3, was associated with ASCA (p = 0.023). Using cell lines from Crohn's disease patients with either H1 or H3, NF-kappaB activation and NF-kappaB p105 and p50 production were significantly lower for patients with H1 compared to patients with H3. CONCLUSIONS: These results suggest that NFKB1 haplotypes induce dysregulation of innate immune responses by altering NF-kappaB expression. The results also show the use of EBV-transformed lymphoblastoid cell lines to conduct phenotypic studies of genetic variation.

Expanded TCRß CDR3 clonotypes distinguish Crohn's disease and ulcerative colitis patients.

We aimed to determine whether the TCR repertoires of Crohn's disease (CD) patients contain highly prevalent disease-specific T-cell clonotypes reflective of the characteristic and highly shared aberrant serum antibody reactivity to gut commensal flagellin antigens. The CD4 TCRß CDR3 sequence repertoires from active CD (n = 20) and ulcerative colitis (UC) (n = 10) patients were significantly more diverse, and individual sequences over-represented, compared to healthy controls (HC) (n = 97). While a very small number of expanded public CDR3 sequences are highly shared between active CD and UC, the majority of significantly expanded TCRß CDR3 clonotypes are private to CD and UC patients with equivalent prevalence among IBD patients. Further defining TCR clonotypes by Vß-CDR3 linkage showed significant differences in the TCR repertoires between UC and CD. Flagellin antigen exposure induced expansion of several TCRß CDR3 sequences in CD4 cells from a flagellin-seropositive subject including sequences highly shared by or relatively private to CD (and UC) patients. These data suggest that flagellin-reactivity contributes to the expansion of a small number of CD4 clonotypes but does not support flagellin antigens as predominantly driving CD4 cell proliferation in CD. Disease-specific expanded TCRß CDR3 clonotypes characterize CD and UC and the shared exposure to the gamut of gut microbial antigens.

Deficiency of the autologous mixed lymphocyte reaction in patients with primary biliary cirrhosis.

In this study we show that patients with primary biliary cirrhosis (PCB) have a marked deficiency in the ability to generate an autologous mixed lymphocyte reaction (AMLR) but have a normal ability to generate an allogeneic mixed lymphocyte reaction (MLR). This deficiency is not due to differences in the time-course of the proliferative response or to an altered response to variable numbers of stimulator cells. The deficiency was consistently found irrespective of the methods used to isolate autologous stimulator cells. Both responder cells and stimulator cells obtained from patients with PBC were similar to normal cells in their ability to generate an MLR in allogeneic normal human serum. In addition, serum from patients with PBC inhibited the ability of normal lymphocytes to generate both the AMLR and MLR to a similar degree, suggesting that the defect of the AMLR in PBC is not due to a serum factor. It has been shown that the responder cell population in the AMLR contains a subpopulation of cells that mediate suppression. Therefore, it is possible that the deficiency of the AMLR may be related to previously described abnormalities of suppressor function in patients with PBC.

A randomized, double-blind, placebo-controlled trial of CDP571, a humanized monoclonal antibody to tumour necrosis factor-alpha, in patients with corticosteroid-dependent Crohn's disease.

AIM: To evaluate CDP571, a humanized monoclonal antibody to tumour necrosis factor-alpha, for the treatment of corticosteroid-dependent Crohn's disease. METHODS: Patients with corticosteroid-dependent Crohn's disease (use of prednisolone 15-40 mg/day or budesonide 9 mg/day for at least 8 weeks, a previous failed attempt to discontinue corticosteroids within 8 weeks, and Crohn's Disease Activity Index score 150 points or less) were enrolled in a 16-week, randomized, double-blind, placebo-controlled trial. The patients received intravenous CDP571 (20 mg/kg at week 0 and 10 mg/kg at week 8) or placebo. Corticosteroid therapy was decreased following a predefined schedule. The primary efficacy end-point was the percentage of patients with corticosteroid-sparing [i.e. no disease flare (Crohn's Disease Activity Index score > or =220 points) and no longer requiring corticosteroid therapy] at week 10. The major secondary efficacy end-point was corticosteroid-sparing at week 16. RESULTS: Seventy-one patients received treatment. Corticosteroid-sparing was achieved by 19 of 39 (48.7%) CDP571 patients and 13 of 42 (40.6%) placebo patients (P = 0.452) at week 10, and by 18 of 39 (46.2%) CDP571 patients and seven of 32 (21.9%) placebo patients (P = 0.032) at week 16. CDP571 therapy was well-tolerated and the incidence of serious adverse events was similar to placebo. CONCLUSIONS: The CDP571 was effective for corticosteroid-sparing at week 16 but not week 10, and was well-tolerated in patients with corticosteroid-dependent Crohn's disease.

Cholera toxin and its subunits as potential oral adjuvants.

Cholera toxin has been shown to have adjuvant effects in multiple different systems. The dose, timing and genetic background of the recipient all seem to be important variables. The role of the two subunits in both the immunogenicity and the adjuvanticity of this molecule remain unclear. The mechanisms of the adjuvant effect likely involves effects on regulatory T cells; there is evidence that the adjuvant effect is due at least in part to inhibition of suppressor T cells. When KLH is used as a model antigen, the adjuvanticity of cholera toxin appears to be related to its immunogenicity in that both properties occur mainly in mouse strains that are high responders to cholera toxin. The genetic engineering of chimeric neoantigens consisting of cholera toxin subunits coupled to antigens of interest has been shown to be technically possible and is an attractive future approach for the generation of effective oral vaccines.

Liver failure with steatonecrosis after jejunoileal bypass: recovery with parenteral nutriton and reanastomosis.

Two women, aged 41 and 51 years, developed jaundice, encephalopathy, and hypoprothrombinemia during rapid weight loss four and 12 months after jejunoileal bypass for refractory obesity. Both were treated for liver failure and received a prolonged course of nutrition parenterally and orally. Serial liver biopsy specimens demonstrated extensive alcoholic-like hepatitis and cirrhosis that improved with nutritional repletion and reanastomosis. Postoperative biopsy specimens later demonstrated minimal portal fibrosis in one patient and inactive mild cirrhosis in the other. Although previous reports indicate that patients usually die when they develop liver failure of this severity after jejunoileal bypass, prolonged intensive nutritional repletion was associated with sufficient clinical and histologic improvement in these two patients so that intestinal reanastomosis could be performed safely.

Interleukin (IL)-21 promotes intestinal IgA response to microbiota.

Commensal microbiota-specific T helper type 17 (Th17) cells are enriched in the intestines, which can convert into T follicular helper (Tfh) in Peyer's patches, and are crucial for production of intestinal immunoglobulin A (IgA) against microbiota; however, the role of Th17 and Tfh cytokines in regulating the mucosal IgA response to enteric microbiota is still not completely known. In this study, we found that intestinal IgA was impaired in mice deficient in interleukin (IL)-17 or IL-21 signaling. IL-21, but not IL-17, is able to augment B-cell differentiation to IgA(+) cells as mediated by transforming growth factor ß1 (TGFß1) and accelerate IgA class switch recombination (CSR). IL-21 and retinoic acid (RA) induce IgA(+) B-cell development and IgA production and drives autocrine TGFß1 production to initiate IgA CSR. Repletion of T-cell-deficient TCRßxδ(-/-) mice with Th17 cells specific for commensal bacterial antigen increased the levels of IgA(+) B cells and IgA production in the intestine, which was blocked by neutralizing IL-21. Thus IL-21 functions to strongly augment IgA production under intestinal environment. Furthermore, IL-21 promotes intestinal B-cell homing through α4ß7 expression, alone or with TGFß and RA. Together, IL-21 from microbiota-specific Th17 and/or Tfh cells contributes to robust intestinal IgA levels by enhancing IgA(+) CSR, IgA production and B-cell trafficking into the intestine.

New perspectives in mucosal immunity with emphasis on vaccine development.

In this review, we have purposely focused on five areas that are currently receiving extensive research attention and will be of major importance for development of mucosal and systemic immunity to oral vaccines. These five areas include the following: (1) helper T-cell (Th) subsets and cytokines for mucosal IgA responses; (2) Th1- and Th2-type subsets in regulation of mucosal IgA responses; (3) antigen uptake and presentation in the mucosal immune system; (4) the importance of memory in mucosal immunity to vaccines; and (5) the determination of whether oral immunization alone induces immunity in all mucosal effector tissues. It is now established that the mucosal immune system can be divided into discrete mucosal inductive sites where vaccines/antigens are encountered and taken up, processed, and presented to B and T cells, and separate areas where immune cells actually function (mucosal effector tissues). Further, through the help provided by Th cells and cytokines, the B cells respond to antigen and undergo expansion including memory cell formation. Following the migration of memory B cells to mucosal effector tissues, the cells rapidly develop into IgA plasma cells, and the prevalence of the latter cell type represents a major characteristic of mucosal effector tissues. It also appears that antigen-specific Th cells and perhaps even CD8+ cytotoxic T lymphocytes can make this circular journey (along with memory/activated B cells) from inductive to mucosal effector sites, and this is termed the common mucosal immune system (CMIS). The major implications of the CMIS for development of vaccines would include each of the five components that are discussed.

A lavage technique allowing repeated measurement of IgA antibody in mouse intestinal secretions.

Mouse intestinal secretions can be readily obtained without harm to the mice by administering a lavage solution to them intragastrically followed by pilocarpine intraperitoneally. These secretions are rich in proteases but this enzyme activity can be blocked by addition of a mixture of inhibitors. Both total and specific IgA antibody could be measured in these secretions using ELISA techniques. The total IgA recovered was found to vary considerably, even in the same group of mice sampled on multiple occasions. Specific IgA anti-cholera toxin antibody was easily demonstrable in the intestinal secretions of mice fed cholera toxin but not of mice fed an irrelevant antigen. Expression of the specific IgA antibody per unit of total IgA recovered is desirable in order to correct for the variable recovery of IgA.

A method of obtaining, processing, and analyzing human intestinal secretions for antibody content.

Human intestinal secretions can be readily obtained using a commercially available intestinal lavage solution. Although such secretions contained abundant protease activity, significant loss of immunoglobulins was prevented by the addition of a mixture of protease inhibitors. The total content of IgA, IgM, and IgG antibody in secretions was measured using sandwich ELISA. In the secretions of ten normal volunteers IgA was most abundant (197 micrograms/ml +/- 103 SD) followed by IgM (12.5 micrograms/ml +/- 6.8 SD) and IgG (0.24 micrograms/ml +/- 0.04 SD). The IgA in secretions was predominantly secretory IgA as shown by sucrose density centrifugation. The effect of intestinal secretions on the sensitivity of the antigen-specific ELISA was tested by adding murine myeloma IgA anti-TNP added to samples of human secretions. IgA anti-TNP activity could be detected as low as 1 ng/ml, and there was no evidence of interference with the ELISA by other constituents in the secretions. Using these methods an antigen-specific secretory IgA anti-cholera toxin B subunit response in the secretions of volunteers given an oral B subunit vaccine was readily demonstrated.

The basis of current and future therapy for inflammatory bowel disease.

The majority of patients with IBD can be managed with currently available therapy, but the currently available agents do not seem to alter the natural history of the disease. An explosion of information about the immune system and the mediators of inflammation has now generated a host of new strategies that will likely benefit patients with IBD as well as patients with other chronic inflammatory and autoimmune disorders. Molecules of the immune system are being harnessed to treat disorders of the immune system. Some of these new concepts and therapies have been discussed here. Some are already in clinical trials, others have yet to be tried. As more specific immune modulators are tested in patients, it is likely that some will be effective in Crohn's disease but not ulcerative colitis and vice versa, due to, and revealing, the differences in the pathogenesis of these two diseases. The hope is that the application of these new modalities will alter the natural history of these diseases to the benefit of our patients.

The C3H/HeJBir mouse model: a high susceptibility phenotype for colitis.

C3H/HeJBir is a substrain of C3H/HeJ mice that was generated by selective breeding for the phenotype of spontaneous colitis. These mice show increased B cell and T cell reactivity to antigens of the enteric bacterial flora. CD4+ T cells from this strain cause colitis, when activated by enteric bacterial antigens and transferred to histocompatible severe combined immunodeficient recipients. The expression of the disease phenotype of spontaneous colitis is greatly influenced by housing conditions and probably requires an immunostimulatory enteric flora. This strain seems to carry multiple susceptibility genes for colitis as does the parental C3H/HeJ strain; the genes involved are being mapped. This strain represents a high susceptibility phenotype for colitis that is providing insight into the interactions among immune, environmental and genetic factors that can result in IBD.

The effect of immunosuppressive agents on monocyte generation and cytokine expression.

SUMMARY: : The purpose of this study was to determine the effect of immunosuppressive drugs on the production of inflammatory cytokines and on the generation of macrophages from bone marrow precursors. Lipopolysaccharide-stimulated peripheral blood mononuclear cells (PBMCs) were cultured in the presence of therapeutic concentrations of 6-mercaptopurine (6-MP), methotrexate (MTX), or prednisolone. The effect of these drugs on the expression of interleukin (IL)-lß, IL-8, and tumor necrosis factor-α (TNF-α) was studied using bioassays and Northern analysis. The generation of monocytes/macrophages from precursors in the bone marrow was examined by culturing murine bone marrow cells with macrophage colony-stimulating factor (M-CSF) in the presence or absence of these agents. 6-MP and MTX did not inhibit the production or messenger RNA (mRNA) expression of the macrophage-derived cytokines, although prednisolone did. Both 6-MP and MTX substantially decreased the generation of macrophages from bone marrow precursors. We conclude that the beneficial effects of these immunosuppressive agents might be due to the suppression of the generation of monocytes in the bone marrow precursors, secondarily diminishing the production of cytokines, eicosanoids, and the production of free radicals.

Heritable susceptibility for colitis in mice induced by IL-10 deficiency.

Severity of inflammatory bowel disease in IL-10 gene-targeted mice is in part determined by genetic background. In the current study, a targeted IL-10 gene was transferred into the C3H/HeJBir substrain, known to exhibit high T-cell and B-cell responses to enteric flora, and to be highly sensitive to colitigenic stress. IL-10-deficient C3H/HeJBir mice developed early onset colitis in contrast to IL-10-deficient C57BL/6J congenic mice. Histopathologic analysis of disease in C3H/HeJBir.Il10-/- and C57BL/6J.Il10-/- mice showed significant differences at all ages studied. Hybrids of these congenic strains (F1.Il10-/-) were produced to study the mode of inheritance as well as subphenotypes that correlated with histopathology. Lesions in F1 mice were intermediate between parental strains. C3H-contributed subphenotypes that correlated best with histopathology were peripheral blood granulocyte percentage, serum amyloid A concentration, spleen weight/body weight ratio, and mesenteric lymph node weight/ body weight ratio. Neither enhanced humoral immunity (secretory IgA, anti-Escherichia coli cellular membrane Ig) characteristic of C3H/HeJBir, nor T-cell percentages in peripheral blood correlated as well. This study represents a necessary step in elucidating murine genetic modifiers controlling colitis sensitivity.

The effects of immunosuppressive agents on cytokines.

Controlled clinical trials have shown that several immunosuppressive drugs are efficacious in patients with inflammatory bowel disease. These drugs each have multiple effects on the immune system and the exact mechanism of their beneficial effect in inflammatory bowel disease is unknown. None of these agents directly inhibits the 'inflammatory cytokines' such as IL-1 beta, IL-6, IL-8, or TNF-alpha. However, they can all inhibit generation of leukocyte precursors in the bone marrow, that is, the cells that produce such cytokines. The effects of current agents on T-cell subsets and their associated cytokines remains unclear. Based on insights from novel mouse models of chronic colitis, the goal of 'immunotherapy' going into the next decade should be either to inhibit specifically the effector T cells mediating disease or to enhance the regulatory T cells that inhibit such effector cells or, ideally, to do both. Therapy meeting this goal, which will need to based on a more thorough understanding of the immunopathogenesis of inflammatory bowel disease, should allow a more specific or 'surgical' approach to immunotherapy and simultaneously reduce its risks and adverse effects.

Early upregulation of T cell IL-10 production plays an important role in oral tolerance induction.

Early in oral tolerance induction, IL-10-producing CD4(+) T cells were increased, and adoptive transfer of IL-10-deficient CD4(+) T cells failed induction of oral tolerance, suggesting a key role of IL-10 production in such a process.