Immunologically mediated cytotoxicity against human eye muscle and thyroid cells in euthyroid and thyrotoxic Graves' ophthalmopathy

We have studied the role of eye muscle and thyroid autoimmunity in patients with Graves' hyperthyroidism with or without ophthalmopathy in an area of relatively low iodine intake. Antibody dependent cell mediated cytotoxicity (ADCC) and complement mediated antibody dependent cytotoxicity (CMAC) against thyroid and eye muscle cells, and levels of antibodies against TSH receptor antigen and the thyroid microsomal antigen (thyroid peroxidase) were determined in three groups of patients: (1) thyrotoxic with exophthalmos (TX, n = 28), (2) thyrotoxic without ophthalmopathy (GR, n = 10), and (3) euthyroid ophthalmopathy (EU, n = 12). The thyroid glandular mass of the EU group was significantly less (P less than 0.01) compared with TX or GR. Mean (+/- SD) TSH receptor antibody (TRAb) level was 27 +/- 14% in EU which was significantly lower compared with TX (52.4 +/- 20%) and GR (59 +/- 18%). The prevalence of microsomal antibodies were similar and not significantly different in the three groups. On the other hand the prevalence of positive ADCC and CMAC tests was significantly greater, and at higher levels, in EU (ADCC THY CELLS 10.9 +/- 8.9% SL, ADCC Eye muscle = 25.9 +/- 20% SL, CMAC = 70.2 +/- 43% SL) and TX (ADCC THY CELLS = 9.3 +/- 9.2% SL, ADCC Eye muscle = 20.1 +/- 19% SL, CMAC = 62.4 +/- 30% SL) compared to GR (ADCC THY CELLS = 4.4 +/- 9.5% SL, ADCC Eye muscle = 7.7 +/- 6.7% SL, CMAC = 24.7 +/- 23% SL).(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity in cellular and humoral immune responses against Toxoplasma gondii antigen in humans

Protection against Toxoplasma gondii in infected patients is mainly attributed to cellular immunity. We here attempt to improve the characterization of the proteins that induce cellular immunity in naturally infected patients. Cellular immunity was evaluated by flow cytometry after 7 days of blood culture from 31 chronically T. gondii infected and 8 noninfected pregnant women, in the presence of soluble T. gondii antigen (ST-Ag) or fractionated proteins from ST-Ag, separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Blood cultures from infected patients with ST-Ag induced 39.5 +/- 12.7% of activated (CD25+) CD4+ T cells using flow cytometry. This contrasts with the absence of activated CD4+ T cells after either culture with PBS or in blood cultures from noninfected women. The protein fraction between 21 and 41.9 kD induced the highest response (14.7 +/- 10.0%). Blood samples from 20 infected and 5 uninfected women were cultured in presence of 12 protein subfractions of 2-208 kD. The highest frequencies of response among infected patients were seen with fractions (Fr) 26-31.9 kD (C.I. 85-100%) and Fr 32-36.9 kD (C.I. 77-100%). Although we note a good concordance between cellular and humoral response, Western blot analysis of ST-Ag does not completely predict the panel of proteins recognized by cellular immunity. Two-dimensional separation of the ST-Ag revealed more than 200 protein spots in these fractions. However, only two proteins in the 20-40 kD range induced a significant humoral response. Further studies are necessary to determine which proteins in the Fr 26-31.9 kD and 32-36.9 kD are superior immunogens for cellular responses.     

Cellular and humoral immune responses against cancer: implications for cancer vaccines.

The key issue in tumor immunology is to identify antigens as target structures for a cancer-selective immunological attack in the tumor-bearing host, resulting in tumor rejection. There is a growing detailed understanding of structural and regulatory gene alterations giving rise to candidate rejection antigens and peptides in tumor cells. As well as reviewing the development of new adjuvant and recombinant vector systems, new approaches are suggested for the construction of cancer vaccines.     

Aujeszky's disease in the guinea pig: Cellular and humoral responses following immunization

Summary The fatal disease caused by virulent ADV in guinea pigs was found to be identical to that seen in sheep and cattle. Intramuscular (i.m.) injection of an avirulent strain of ADV (Bartha) yielded better immunity to challenge after 3 weeks than did intranasal (i.n.) immunization, and this was reflected in differences in histopathological changes in the brain.Serum antibodies active in antibody-dependent cell-mediated cytotoxicity (ADCC) were titrated using polymorphonuclear leukocytes as effector cells. ADCC correlated fairly well with virus neutralization and was a far more sensitive technique. There was good, but not complete, correlation between ADCC and protection. Lymphocyte responsiveness to virus antigensin vitro was assessed by³H-thymidine uptake and lymphokine tests. Lymphocyte stimulation and mitogenic factor responses were low grade but blood lymphocyte stimulation was more pronounced in the better-protected animals. Macrophage migration inhibition correlated neither with serum ADCC nor with protection, being equally demonstrable in the two immunized groups.This work was carried out while this establishment under its former title Microbiological Research Establishment was under the management of the Ministry of Defence.     

Antibody-dependent cell-mediated cytotoxicity against human eye muscle cells and orbital fibroblasts in Graves' ophthalmopathy--roles of class II MHC antigen expression and gamma-interferon action of effector and target cells

We have studied the significance of antibody dependent cell-mediated cytotoxicity (ADCC) against human orbital fibroblasts (OF) and eye muscle (EM) cells in the pathogenesis of the orbital autoimmune reactions of Graves' ophthalmopathy (GO). Possible roles of Class II MHC antigen expression on the surface of orbital target cells and of gamma-interferon (gamma-IFN) modulation of ADCC were also studied. Both OF and EM expressed HLA-DR antigen when stimulated with gamma-IFN and phytohemagglutinin, but not spontaneously, and not by thyroid stimulating hormone or alpha-IFN. Intrathyroidal T cells from a patient with GO induced greater DR expression on both OF and EM cells than equal numbers of her peripheral blood T cells. gamma-IFN treated EM and OF were more susceptible to lysis in ADCC assays than untreated targets. gamma-IFN also enhanced lysis in ADCC assays by an effect on the killer cell population. On the other hand treatment with alpha-IFN, which is a potent inducer of Class I antigen expression, did not affect the susceptibility of target cells to lysis in ADCC. When sera from patients with GO were tested in ADCC, tests were positive (% specific lysis greater than mean + 2 s.d. for normals) in 10 of 20 patients with EM cells, but in only two of 25 with OF. The degree of killing of EM cells was significantly positively correlated to that of abdominal skeletal muscle cells and, to a lesser degree, normal thyroid cells, but not OF. In sera showing killing of EM cells and OF, ADCC activity against EM cells was absorbed by preincubation with EM and orbital connective tissue membranes but not thyroglobulin and, conversely, lysis of THY cells was absorbed by preincubation of positive sera on monolayers of THY and EM cells and OF, but not vascular endothelial (VE) cells. Finally, killing of 51Cr labelled EM cells was inhibited by addition of unlabelled ('cold') thyroid cells, EM cells and OF, but not VE cells. Our findings suggest that ADCC is likely to be an important mechanism for the eye muscle cell damage of GO, but not for the associated orbital connective tissue inflammation. Since ADCC is not MHC-restricted the enhanced lysis of HLA-DR positive target cells presumably reflects other effects of gamma-IFN treatment on both the killer cell population and the target cells.     

Carbimazole and the autoimmune response in Graves' disease

Microsomal antibodies and antibodies directed toward the receptor for thyroid-stimulating hormone (TSH) decreased in parallel while patients with Graves' disease were taking carbimazole, whereas no significant changes were observed during treatment with placebo or propranolol. The changes in autoantibody levels during carbimazole treatment were independent of changes in serum thyroxine and could have been due to a direct effect of the drug on autoantibody synthesis. Evidence for this suggestion was provided when low doses of methimazole (the active metabolite of carbimazole) were found to inhibit thyroid-autoantibody production in cultured lymphocytes. Since thyroid lymphocytes are probably a major site of thyroid-antibody synthesis in Graves' disease and methimazole is concentrated in the thyroid during treatment, a local action of the drug on antibody production seems likely. This possibility could be important in the use of carbimazole to control hyperthyroidism.     

Intrastructural help in diversification of humoral autoimmune responses

IL-4 plays a key role in the contact sensitivity skin reaction. This has several implications. First, the view that contact sensitivity (CS) is only mediated by cells with a Th1 profile of cytokine secretion needs modification, in the light of the essential role of IL-4 at the effector stage. Second, the concept of a single cell involved in the systemic transfer of CS is no longer tenable, as it is known that both αβ and γδ cells are required. Studies with the cell lines (which contain both αβ and a few γδ cells) suggest that this double requirement may involve the action of IL-4 on γδ cells, which bear receptors for IL-4. Finally, the view that T cell lines only transfer CS when injected locally, but not when injected intravenously (systemic transfer), is correct but incomplete, as T cell lines actually give systemic transfer of CS, providing the cell line or the recipient is treated with IL-4.     

Cellular and humoral immune responses and protection against schistosomes induced by a radiation-attenuated vaccine in chimpanzees

The radiation-attenuated Schistosoma mansoni vaccine is highly effective in rodents and primates but has never been tested in humans, primarily for safety reasons. To strengthen its status as a paradigm for a human recombinant antigen vaccine, we have undertaken a small-scale vaccination and challenge experiment in chimpanzees (Pan troglodytes). Immunological, clinical, and parasitological parameters were measured in three animals after multiple vaccinations, together with three controls, during the acute and chronic stages of challenge infection up to chemotherapeutic cure. Vaccination induced a strong in vitro proliferative response and early gamma interferon production, but type 2 cytokines were dominant by the time of challenge. The controls showed little response to challenge infection before the acute stage of the disease, initiated by egg deposition. In contrast, the responses of vaccinated animals were muted throughout the challenge period. Vaccination also induced parasite-specific immunoglobulin M (IgM) and IgG, which reached high levels at the time of challenge, while in control animals levels did not rise markedly before egg deposition. The protective effects of vaccination were manifested as an amelioration of acute disease and overall morbidity, revealed by differences in gamma-glutamyl transferase level, leukocytosis, eosinophilia, and hematocrit. Moreover, vaccinated chimpanzees had a 46% lower level of circulating cathodic antigen and a 38% reduction in fecal egg output, compared to controls, during the chronic phase of infection.     

Cellular and humoral response to Kunin antigen (CA) in ulcerative colitis and Crohn's disease

In 18 patients with ulcerative colitis and in 8 with Crohn's disease two tests were performed simultaneously: a) the leukocyte migration inhibition test using Kunin antigen, and b) titration of serum antibodies against this antigen. Leukocyte migration was studied by the agarose plate technique. At the concentration of 25 microgram/ml, Kunin antigen inhibited migration in five cases of ulcerative colitis and in one with Crohn's disease. This phenomenon was not observed in any of 33 control subjects. All patients in whom leukocyte migration was inhibited were in the active phase of the disease. Titers of antibodies against Kunin antigen were determined by the passive hemagglutination test in an expended group of patients comprising 61 with Crohn's disease. The antibody titer, expressed as the geometric mean of hemagglutinin titers, was nearly three times as high in patients as in 324 healthy controls. The titers were not correlated either with clinical activity of both diseases or with the results of the leukocyte migration inhibition test. The significance of these findings is discussed in the light of other data indicating that Kunin antigen plays a role in the pathogenesis of inflammatory bowel disease.     

Cellular and humoral immune responses to a human pancreatic cancer antigen, coactosin-like protein, originally defined by the SEREX method

Among a number of human tumor antigens identified using the serological analysis of recombinant cDNA expression libraries (SEREX), only MAGE-1, tyrosinase, and NY-ESO-1 have been reported to be immunogenic tumor antigens that have the potential to elicit both humoral and cellular immunity. In this study, we determined whether our SEREX-defined pancreatic cancer antigens could be recognized by CTL, and report that one SEREX-defined antigen, coactosin-like protein (CLP), encoded cellular epitopes recognized by HLA-A2-restricted and tumor-reactive CTL. Three CLP peptides at positions 15-24, 57-65, and 10-113 possessed the ability to induce HLA-A2-restricted and tumor-reactive CTL from the PBMC of cancer patients. Subsequently, humoral responses to these peptides were investigated. IgG antibodies specific to the CLP 15-24, 57-65, and 104-113 peptides were detected in sera from 12, 0, and 12 of 12 cancer patients tested, and were also found in 5, 0, and 0 of 9 healthy donors, respectively. IgE antibodies specific to these peptides were also detected in sera from certain cancer patients and healthy donors. Since peptide-specific IgE was detected, type-I allergy to these peptides was tested. Unexpectedly the CLP 57-65 peptide, to which IgE was found in only 2 healthy donors, but not the other two peptides, was found to elicit an immediate-type hypersensitivity in all 10 healthy volunteers tested. These results indicate that identical antigenic peptides can be recognized by both cellular and humoral immune systems to a tumor-associated antigen. The CLP 15-24 and 104-113 peptides might be appropriate vaccine candidates for peptide-based immunotherapy of HLA-A2(+) cancer patients.     

Cellular and humoral immune responses in Campylobacter pylori-associated chronic gastritis

Gastric cellular and humoral immune response investigated by immunoperoxidase staining of 53 antral biopsies showed significant differences in Campylobacter pylori-associated gastritis as compared with non-bacterial chronic gastritis and normal controls. IgA, secretory component, and complement C3 coated bacilli were seen in all cases of active chronic gastritis. C3 was always associated with coating by IgA, IgM, or both, which were rarely seen in gastritis without polymorphonuclear neutrophil infiltration. Intraepithelial mononuclear cellular infiltration was seen in 18 of 26 cases of C. pylori-associated chronic gastritis. The intraepithelial mononuclear cells stained positively for T cells and histiocytes.     

Major histocompatibility complex (MHC) factors predisposing to and protecting against Graves' eye disease

We investigated the influence of gene mapping within the major histocompatibility complex on the susceptibility to Graves' eye disease. We studied 133 randomly selected patients with Graves' disease, many of whom had eye disease. HLA B8 and DR3 carried the greatest risk for disease but the difference between the two patient groups was not statistically significant. An earlier finding that Hungarian patients with a subset of B8, (B8 + DR7 +) had a greatly enhanced risk for eye disease was confirmed in Newfoundland patients. HLA B8 and DR7 are probably carried on different homologous chromosomes and their interaction enhances eye disease. HLA-DR4 was negatively correlated with eye disease. In particular, a subset of DR4 (B35 + DR4 +) appears to protect against eye disease. We have also derived the haplotypes of 22 probands, half of whom had eye disease. The haplotype data emphasized the high frequency of HLA A1 B8 DR3 C4A*QO and C4B*1 in both patient groups, 15% of the haplotypes in the group with eye disease and 25% in that without eye disease. Forty-one percent of haplotypes in the eye disease group and 32% in the no eye disease group were either C4A*QO or C4B*QO. In one proband with eye disease B8 and DR7 were carried on separate chromosomes. The phenotype DR4, C4A*3 C4B*1 was found in 3/20 haplotypes of patients without eye disease but in 0/20 of patients with eye disease. This finding is in keeping with the increased frequency of the DR4 C4A*3 C4B*1 in the patient group with no eye disease when 94 patients were phenotyped.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular and humoral immune responses against autoreactive T cells in multiple sclerosis patients after T cell vaccination.

Myelin basic protein (MBP)-reactive T cells may play an important role in the autoimmune pathogenesis of multiple sclerosis (MS). MBP-reactive T cells can be specifically targeted by T cell vaccination, a procedure whereby MS patients are immunized with attenuated autologous MBP reactive T cells. T cell vaccination induces immune responses to the vaccine cells together with a depletion of MBP reactive T cells. Forty-nine MS patients were treated with T cell vaccination in an extended phase I trial to study the safety, immune responses and clinical effects of T cell vaccination. In the present paper the immune responses towards the vaccine cells were characterized. Substantial long-term in vitro proliferative responses were observed in all treated patients. Some patients, immunized with different clones, displayed distinct proliferative reactivity against the various vaccine clones, suggesting unequal immunogenic properties of these clones. Reactive TCRalphabeta(+), CD8(+)and CD4(+)T cells, and to a lesser extent, gammadelta T cells and NK cells were observed to in vitro stimulation with the vaccine cells. A small fraction only of CD8(+)T cells expressed cytolytic and inhibitory anti-clonotypic reactivity against the vaccine cells. Stimulation with the vaccine clones predominantly induced expression of pro-inflammatory cytokines in these mixed cultures, although one vaccine clone consistently induced production of IL-4. CD4(+)T cells are the major cytokine-producing cells in these anti-vaccine lines. We could not detect upregulated antibody responses to the vaccine cells in most patients, although a temporary antibody response was observed in one patient. In conclusion, immunization with attenuated autoreactive T cells induces a complex cellular response specifically targeted at the vaccine cells, but no antibody responses. These data provide further insights into the mechanisms of T cell vaccination and improve our understanding of the complex regulatory networks of autoreactive T cells.     

Cellular immune responses to salivary antigens in autoimmune liver disease with sicca syndrome.

Twenty-six patients with primary biliary cirrhosis (PBC) and twenty-two with active chronic hepatitis (ACH) were examined for evidence of the sicca syndrome (keratoconjunctivitis sicca, xerostomia). Measurements of tear flow and total saliva flow showed that at least one sicca feature was present in twenty (77%) of the patients with PBC and ten (45%) of those with ACH. Examination of cellular immune responses to a protein fraction of normal human saliva using the leucocyte migration test showed sensitization to the saliva protein in twenty-three of the thirty cases with sicca syndrome but in only two of the eighteen in whom sicca features were not detected. Antisera raised in guinea-pigs against the saliva protein gave specific immunofluorescent staining of bile duct epithelial cells in sections of normal human liver. These findings suggest that damage to structures in the liver may lead to sensitization to various self-antigens which cross-react with other tissues in which a similar disease process may be consequently be initiated.     

Cellular and humoral immune responses to recombinant 65-kD antigen of Mycobacterium leprae in leprosy patients and healthy controls.

Cellular and humoral immune responses to recombinant 65-kD antigen of Mycobacterium leprae (rML65) were studied in leprosy patients and healthy contacts from a leprosy-endemic population. Peripheral blood mononuclear cells from a considerable proportion of tuberculoid leprosy patients, healthy contacts and non-contacts showed proliferative response to rML65 in vitro. A strong positive correlation was observed between the responses to rML65 and bacille Calmette-Guérin (BCG) or leprosin A. Addition of recombinant IL-2 (rIL-2) enhanced the proportion of responders to rML65 considerably in all groups of leprosy patients, healthy contacts and non-contacts. Among lepromatous patients this enhancement was more pronounced in the bacterial index (BI)-negative group. These results indicate that the 65-kD antigen of Myco. leprae is a dominant T cell immunogen in our study population. Though lepromatous patients showed poor lymphoproliferative response to rML65, their IgG antibody levels to the same antigen were markedly high. Most of the BI-positive lepromatous patients with elevated anti-rML65 IgG levels did not show T cell reactivity even with the addition of rIL-2. On the other hand, tuberculoid leprosy patients, healthy contacts and non-contacts showed good T cell reactivity but low levels of IgG antibodies to rML65, thus indicating the presence of an inverse relationship between cell-mediated and humoral immune responses to a defined protein antigen of Myco. leprae in humans. A significant proportion of individuals among tuberculoid leprosy patients, healthy contacts and non-contacts showed neither T cell reactivity nor elevated levels of IgG antibody to rML65. However, in most of these subjects, a T cell response to rML65 was demonstrable with the addition of rIL-2. These results are discussed with reference to the immunoregulatory mechanisms occurring during Myco. leprae infection on the basis of differential activation of Th1 and Th2 subsets.     

Cellular and molecular mechanisms of autoimmune disease

Autoimmune disease (AIDx) results from failure to sustain tolerance to self molecules. Dozens of AIDx involving one or multiple organ systems afflict 3% or more of people worldwide (>75% women). Predisposing factors for AIDx include genetic background, hormonal status, pathogens, and xenobiotic exposures. The incidence of AIDx is higher in individuals living in developed nations, including recent immigrants. Patients may have several AIDx simultaneously. Certain AIDx can prevent other AIDx. A history of AIDx raises the risk for developing hematopoietic neoplasia. Some common mechanisms for losing self-tolerance include reduced deletion or enhanced activation of autoreactive CD4(+) T-helper (Th) lymphocytes, defective immunomodulation by CD4(+) regulatory (Treg) and CD8(+) suppressor (Ts) T-lymphocytes, dysregulated signaling (leading to a relative increase in pro-inflammatory cytokines), comparable structure between self-antigens and foreign molecules, or expression of new epitopes on previously hidden or xenobiotic-modified self proteins. Organ-specific AIDx is generally a cell-mediated (Th1 or Th17) process, while multi-organ AIDx also incorporates a robust autoantibody (Th2) component. Cytokine signatures of different AIDx overlap incompletely; for a given AIDx, different patients have divergent cytokine profiles. Newer anti-AIDx agents are based on our increasing knowledge of AIDx pathogenesis and usually attempt to reverse lymphocyte dysfunction, quell pro-inflammatory signaling, or restore self-tolerance.     

Cellular but not humoral immune responses generated by vaccination with dendritic cells protect mice against leukaemia

Dendritic cells (DC) are extremely efficient at generating both prophylactic and therapeutic anti‐tumour immunity. We aimed to analyse the respective roles of humoral and cellular immune responses generated in mice vaccinated with bone marrow (BM)‐derived DC in terms of in vivo anti‐leukaemia effect. We used the murine L1210 B lymphocytic leukaemia genetically modified to express on the cell surface of human CD4 (hCD4) (L1210/hCD4) as a model tumour‐associated antigen (TAA). DC cultures were loaded with either purified soluble hCD4 (shCD4) protein or unfractionated L1210/hCD4 extracts and injected as vaccine into mice. The efficacy of these vaccinations was compared with that of vaccination with shCD4 protein emulsified in Freund’s adjuvant (FA). We evaluated the immune responses generated after these vaccinal protocols and the survival rate of vaccinated mice subsequently challenged with a lethal injection of L1210/hCD4 cells. Our results demonstrated that vaccination with shCD4 protein or tumour extract‐loaded DC mainly generated an hCD4 antigen‐specific cell‐mediated cytotoxic immune response that was associated with a specific protection against leukaemia. In contrast, vaccination with the protein emulsified in FA only generated potent humoral immune responses that were not protective against leukaemia. Altogether, our results indicate that the unique property of loaded DC to trigger an anti‐leukaemia protective effect is mainly associated with cellular immune responses.