Lymphocytotoxins in leprosy and in asymptomatic hepatitis B virus infection.

Serum lymphocytotoxic antibodies (LCAs) were detected in 67% of Papua New Guinean lepromatous leprosy patients who were persistent carriers of hepatitis B surface antigen (HBsAg). Lymphocytotoxins were not associated with asymptomatic HBsAg in either healthy controls or tuberculoid leprosy patients. It was apparent that, although HBsAg itself is a poor indicator of in vitro lymphocytotoxicity, when the antigen occurred in a host with impaired immune response, lymphocytotoxicity, when the antigen occurred in a host with impaired immune response, lymphocytotoxicity was enhanced. In contrast to this finding, lepromatous leprosy patients without HBsAg had significantly depressed LCA production in comparison with tuberculoid patients and controls. The interaction between leprosy and hepatitis B virus was highly significant (P = 0.001) in an analysis of variance of cytotoxicity scores. It is proposed that the previously reported equivocal results regarding autoantibodies in leprosy patients may be explained by this unusual interaction between lepromatous leprosy and hepatitis B virus infection.     

Brain reactivity of lymphocytotoxic antibodies in systemic lupus erythematosus with and without cerebral involvement.

A prospective clinical study of cold-reactive lymphocytotoxic antibodies in systemic lupus erythematosus has been completed. A highly significant association between serum lymphocytotoxicity and the development of cerebral manifestations was observed. While lymphocytotoxic antibodies from patients with cerebral lupus were absorbed by homogenates of human brain, those from patients who at no time had evidence of cerebral disease failed to cross-react with brain. It is suggested that subpopulations of lymphocytotoxic antibodies differ in their brain reactivity, and that one population may be causally related to the development of some of the features of cerebral lupus.     

Production in vitro of antibodies directed against alloantigen-specific recognition sites on T cells and on lymphocytotoxic HLA antibodies.

We have examined the mechanism of immunological unresponsiveness in a recipient (P.S.) with a long-term functioning renal allograft. P.S., whose HLA type is A1, A30; B14, B18; DR1, w8; DRw52; DQw1 and in whose serum we had earlier demonstrated the presence of antiidiotypic antibodies, received a kidney from a cadaver donor of HLA type A1, A10, B8 in March, 1970. Peripheral blood B lymphocytes from the patient were transformed with Epstein-Barr virus (EBV), and by the cluster-picking technique a B cell line was propagated with continuous production of antibodies. Antiidiotypic antibodies with two distinct biological functions were demonstrable; one specifically inhibiting the lymphocytotoxic activity of anti-HLA-B8, B5, and DR3 reference typing sera, and the other specifically inhibiting proliferative responses in MLC of the recipient's lymphocytes and of third party cells sharing B14, DR1, DQw1 with the patient against stimulator cells carrying B8, DR3 antigens. Immunodepletion experiments demonstrated that the inhibitory activity was associated with the IgM fraction. Absorption experiments suggested that different antibodies may be responsible for the inhibition of lymphocytotoxic activity of anti-HLA sera and of the proliferative responses in MLC. Antiidiotypic antibodies have been postulated to be important in maintaining allograft tolerance in vivo, thereby enhancing renal allograft survival. The availability of such antibodies in large quantities, produced in vitro, could provide antisera for the immunochemical characterization of specific idiotypic receptors on immunoglobulins and T lymphocytes.     

Complexes of soluble HLA antigens and anti-HLA autoantibodies in human sera: Possible role in maintenance of self-tolerance

The MHC plays an essential role in regulating the process of self-nonself discrimination. T cells recognize nonself antigens only in the context of self-MHC gene products. It is not clear, however, whether B cells are also endowed with immune receptors for self-MHC antigens. We have explored the existence of antibodies against self-MHC antigens (HLA) in the human by analyzing the specificity of anti-HLA antibodies developed by a population of 727 dialysis patients who had been monitored monthly over a period of 3-78 months. Anti-HLA autoantibodies were identified by serum screening in 119 patients. Twenty-five of these 119 patients had not been exposed to alloantigens before, indicating that the production of anti-HLA autoantibodies is not necessarily stimulated by allogeneic HLA antigens. Cross-matching of sera with autologous lymphocytes, confirmed the autoreactive nature of these anti-HLA antibodies which were of IgM or of IgG isotype in approximately equal numbers of patients. The fine specificities of anti-HLA autoantibodies was ascertained in studies which showed that antibodies can be adsorbed, and the eluted, from the membranes of target cells carrying then relevant HLA antigen. Since the antibodies characterized in our studies may be functionally active in vivo we examined the possibility that their level is controlled by anti-idiotypic antibodies or by soluble HLA antigens. We found that the titer of anti-HLA autoantibodies increased significantly if soluble HLA antigens were depleted from the serum. Our data suggest that circulating HLA antigens form immune complexes with anti-HLA autoantibodies and contribute to the maintenance of self-tolerance by inhibiting antibody binding to membrane HLA antigens. The finding that the immune repertoire includes B cells with receptors for self-MHC opens new perspectives for the study of network perturbations in autoimmune diseases.     

Specific removal of anti-red cell blood group activity from anti-HLA lymphocytotoxic sera and purification of anti-A and anti-B antibodies with lymphocytotoxic activity

Lymphocytotoxic antibodies to the red cell blood groups A and B were purified from human sera by elution from synthetic immunoadsorbents (Synsorbs from Chembiomed). The lymphocytotoxic activity of the eluted antibodies was better preserved when elution was carried out at pH 11, whereas it was less stable when elution was performed at pH3. In addition, complete removal of anti-red cell blood group activity from an anti-HLA serum was easily obtained by adsorption on the corresponding Synsorb without loss of anti-HLA activity.     

Specific and sensitive detection of purified HLA molecules in an ELISA using mouse, rabbit and human anti-HLA antibodies

An ELISA detecting anti-HLA antibodies of rabbit, mouse or human origin was developed using plates coated with HLA molecules purified on affinity columns. The sensitivity of the assay was optimal when coating was performed in PBS, pH 7.8 at 4 degrees C for 6-16 h and using a serum incubation period of 16 h at 4 degrees C. The optimum protein concentration for coating was estimated to be 1 micrograms/ml. With monoclonal anti-HLA sera, antipeptide antibodies from mice or rabbit and human alloantisera, this method appeared to be highly sensitive, very specific and reproducible.     

A rapid solid-phase rosette-inhibition assay for the detection of Fc receptor (FcRII)-blocking alloantibodies [corrected

A procedure for the detection of FcR-blocking alloantibodies is described which uses human B lymphocytes immobilised on plastic by poly-L-lysine. Antibodies which inhibited rosette formation between B lymphocytes or Daudi cells and ox erythrocytes coated with rabbit antibodies (EA) were detected in 10 out of 10 sera containing anti-HLA A2 antibodies and 3 out of 3 sera containing anti-HLA class II antibodies. Inhibition of rosette formation (EAI activity) was mediated by protein G-separated IgG. Analysis of rosette formation using these 13 sera and lymphocytes from 39 donors revealed that the degree of inhibition was bimodal; most sera were either clearly inhibitory or non-inhibitory in the assay. However, there was no correlation between inhibition of rosette formation (EAI activity) and lymphocytotoxicity. Four pairs of sera showed similar patterns of reactivity (r greater than 0.6, p less than 0.01; 2 x 2 chi-square test), and cells from 2 donors showed antithetical reactions with 12 of 13 sera (r = -0.86, p less than 0.001). These data suggest that the solid-phase rosette inhibition assay is a rapid and reproducible means of detecting antibodies reactive with non-HLA class I or class II antigens on human B cells.     

ELISA ANTI-HLA ANTIBODY SCREENING IDENTIFIES NON-COMPLEMENT-FIXING ANTIBODIES RESPONSIBLE FOR ACUTE GRAFT REJECTION. A CASE REPORT

We report on a kidney transplant recipient experiencing an unexpected early acute vascular graft rejection. Retrospective analysis of patient serum samples, utilizing a new ELISA HLA screening technique, revealed that the rejection crisis and the subsequent graft loss were due to a pretransplant donor-specific pre-sensitization caused by a non-complement-fixing antibody of IgG2 class. The case illustrates the clinical significance of non-complement-fixing anti-HLA antibodies. In addition it is shown that ELISA methods are suitable for detecting potentially harmful donor pre-sensitization in waiting-list patients not detectable by standard lymphocytotoxicity techniques. Hence ELISA could be an alternative to flow cytometry for this purpose. It is concluded that screening and cross-matching techniques which detect non-complement-fixing anti-HLA antibodies could improve graft outcome, and should form part of the immunological monitoring of kidney transplant waiting-list patients.     

Detection of donor-specific-antibodies by solid phase assay and its relevance to complement-dependent-lymphocytotoxicity cross-matching in kidney transplantation

Presensitization against a broad array of HLA is associated with prolonged waiting times and inferior kidney allogaft survival. Although the use of solid phase assay (SPA) for the detection and characterization of anti-HLA antibodies provides greater sensitivity than complement-dependent lymphocytotoxicity (CDC) assay, it often detects donor specific antibodies (DSA) which turn out to be clinically irrelevant. Our data reinforce the concept that these two types of assays should be used in parallel for pre-and post-transplantation monitoring of anti-HLA antibodies in recipients of solid organ allografts.     

Effects of anti-lymphocytic globulin in human subjects.

Some of the immunological effects of anti-lymphocytic globulin (ALG) were examined in patients taking part in a clinical study of equine ALG in ulcerative colitis. ALG administration led to reduction in the percentage of peripheral blood lymphocytes forming spontaneous rosettes with sheep erythrocytes (E rosettes). Evidence was obtained that this reduction was the result of genuine depletion of circulating T (thymus-dependent) lymphocytes, rather than blockade of E-rosette formation by ALG on lymphocyte surfaces. Using an immunofluorescence technique, horse Ig was demonstrated on lymphocytes from patients receiving ALG. By combining this technique with E-rosette formation, horse Ig was detected on patients' E-rosette-positive and -negative lymphocytes. Horse Ig levels in patients' sera were measured by haemagglutination inhibition, and the sera were also examined for anti-lymphocytic antibodies. Although lymphocyte-binding horse Ig was detectable in teh patients' sera, lymphocytotoxic antibody could not be demonstrated. It can be concluded that lymphocytotoxic antibody was rapidly removed from the serum of patients receiving ALG, by binding to circulating lymphocytes.     

Study of procainamide hapten-specific antibodies in rabbits and humans

Procainamide (PA) is the drug most commonly associated with the induction of autoantibodies and drug-related lupus (DRL). While the majority of these patients express autoantibodies, antibodies to the parent drug and metabolites, PA-hydroxylamine (PAHA) or nitroso-PA (NOPA), have not been reported in humans.Hapten-carrier conjugates were prepared using human hemoglobin (HgB) or autologous rabbit erythrocytes with PAHA or NOPA. PA was conjugated to rabbit serum albumin (RSA) or egg albumin (OVA) via diazotization and condensation methods. Rabbits were immunized with hapten conjugates in Freund's adjuvant. These hapten-carrier compounds (5 – 10 μg/ml) were used as test antigens for antibodies in sera from the rabbits and 40 patients on chronic PA treatment, 10 SLE patients, 33 elderly and 20 young normal controls by ELISA. Type I and II collagens were also used as test antigens for human sera.Sera from rabbits immunized with the PA compounds had elevated IgG antibody values to PA, PAHA and NOPA, but no autoantibodies. Absorption of the rabbit sera with the PA compounds reduced the antibody levels; ssDNA and histones failed to inhibit the total binding values. Mean binding to PA - OVA was 0.95 ± 0.41 for PA patients and 1.37 ± 0.26 standard error of means (S.E.M.) in the SLE patients compared to 0.37 ± 0.14 S.E.M. in the normal sera (P⩽0.05); similar binding values to PAHA - HgB and NOPA - HgB were also observed. Sixty-eight percent of the PA patients had antibodies to type II collagen. Elevated binding values to PA compounds were inhibited by absorption of human sera with ssDNA or total histones; absorption with PA or PAHA had no significant effect. These findings suggest that sera from PA patients containing high titers of autoantibodies cross-react in vitro with unrelated antigens.     

Evaluation of three different laboratory methods to detect preformed human leukocyte antigen antibodies in a South African kidney transplant population

BackgroundAnti-human leukocyte antigen antibodies (anti-HLA) play a crucial role in graft. Detection of anti-HLA, both pre- and post-transplant is a crucial investigation in clinical organ transplantation.ObjectivesThree methodologies for the detection of lymphocytotoxic antibodies were compared to establish which of these is best suited to optimise pre-transplant donor-recipient matching.MethodsSerum samples from 15 renal transplant patients were tested for the presence of anti-HLA by i) cytotoxic-dependent cross-match (CDCXM), ii) flow cytometric cross-match (FCXM) and iii) Luminex-based donor specific antibody cross-match (DSAXM) method, Confirmatory tests for the presence of preformed HLA antibodies were tested using Luminex methodology.ResultsTwo (13%) of the 15 patients had positive HLA Class I antibodies (Ab) using all 3 methods. An additional 2 HLA Class I Ab were identified with FCXM/CDCXM. DSAXM identified 1 HLA Class I positive, not indicated by CDCXM/FCXM.High HLA Class II positivity (40%), identified by CDCXM, while DSAXM and FCXM identified two and one patients, respectively. CDCXM produced 4 false-positive results confirmed by lymphocyte single antigen (LSA) assay.ConclusionsThe DSAXM method appears to add value in pre-transplantation screening to identify pre-sensitised patients that may not reject the donor graft due to the absence of donor-specific antibodies.     

Transplantation in miniature swine: analysis of graft-infiltrating lymphocytes provides evidence for local suppression

Previous studies from this laboratory have demonstrated that swine tolerant of class I disparate renal allografts show peripheral antidonor cellular reactivity which can be augmented by skin grafting. To assess the possibility of local suppression, cell-mediated lymphocytotoxicity of graft-infiltrating lymphocytes was compared to that of peripheral blood lymphocytes from three tolerant and four acutely rejecting recipients of class I--disparate renal allografts. Mixed lymphocyte cultures using peripheral blood lymphocytes or graft-infiltrating lymphocytes and an equal number of irradiated peripheral blood lymphocyte stimulators were incubated for 6 days and tested in a 6-hr 51Cr release assay. Graft-infiltrating lymphocytes from rejecting animals had potent antidonor cell-mediated lymphocytotoxic activity with or without in vitro stimulation. Anti-third-party reactivity was seen with appropriate stimulation, suggesting heterogeneity of graft-infiltrating lymphocyte cultures. Peripheral blood lymphocytes from rejectors generated donor-specific cell-mediated lymphocytotoxicity. Graft-infiltrating lymphocytes from tolerant animals generated no antidonor cell-mediated lymphocytotoxicity with or without in vitro stimulation, but generated an anti-third-party response. Peripheral blood lymphocytes from tolerant animals displayed both antidonor and anti-third-party reactivity with appropriate in vitro stimulation. These data support the hypothesis that local suppression may contribute significantly to maintenance of tolerance to class I disparate renal allografts in miniature swine.     

Study of the antibody-dependent lymphocytotoxicity test as a function of the nature of the antibodies]

It is well known that IgM antibodies are inactive for the LDA test. Moreover, this work showed 7 S IgM to be just as inactive as 19 S IgM. With HLA antibodies, usually IgG, this work showed that the LDA test was not inhibited by a temperature of + 15 instead of + 37 degrees C. With cold lymphocytotoxic antibodies 19 S and 7 S IgM the LDA test was negative even at + 15 degrees C (optimum temperature for these complement dependant lymphocytotoxic antibodies) with or without prolonged incubation. Under these same conditions, a negative result was obtained using auto-lymphocytes as a target. Finally, our results confirmed that IgM antibodies with anti-HLA activity could not act as mediators even for targets with corresponding HLA activity.     

Immune monitoring of anti-thymocyte globulin (ATG) treatment in transplant patients

Over the past few years there has been an increasing array of monoclonal antibodies directed against immunocompetent lymphocytes available for use in the prevention and treatment of rejection in solid organ transplantation. Despite this there has been a continued and expanding use of polyclonal anti-T cell globulins in the management of cardiac, lung and renal transplants in many centres. As with all immunosuppressive drugs over and under-immunosuppression remains a major problem. A number of different methods have been described for monitoring anti-thymocyte globulin (ATG) therapy in transplant patients. These include: monitoring serum IgG levels, the E- rosette assay; flow cytometry and monoclonal antibodies. The measurement of serum IgG levels of the host animal (rabbit or horse) has been shown not to correlate with the presence of acute rejection episodes. The E-rosette assay has been found to be extremely time consuming and unreliable. With flow cytometric techniques, monoclonal antibodies directed against the T lymphocytes have been used to monitor ATG therapy and this has allowed the adjustment of the ATG administered to each individual patient. In patients receiving, renal, cardiac, lung or heart lung transplants we have developed flow cytometric assays to monitor ATG therapy on a daily basis with adjustment of the dosage of ATG administered according to this result. We have progressed from a dual platform assay where the T-cell percent from flow cytometry is combined with the lymphocyte count determined from a Haematology Cell Analyser to a single platform flow cytometric assay using commercially available polyfluorescent beads of two different types.An increasing problem with transplant patients is that ATG once given may induce antibodies against the heterologous rabbit or horse IgG. With time, a number of renal, cardiac and lung transplant patients have lost their grafts and are retransplanted. A similar situation is seen in patients who are given regular ATG therapy for the treatment of aplastic anemia. We have developed a flow cytometric duplex assay for the detection of such antibodies. This has allowed us to screen the sera of transplant patients for the presence of such antibodies before therapy is given and to suggest for that patient the most appropriate ATG to be used. The cellular and humoral assays we have developed are rapid and reproducible and are now used as routine in the clinical management of patients who are receiving or about to receive ATG therapy in transplantation.     

B cell hyperactivity and abnormalities in T cell markers and immunoregulatory function in a patient with nucleoside phosphorylase deficiency.

We describe a 2 year old girl with nucleoside phosphorylase (PNP) deficiency, who had low blood T cell numbers and T lymphocyte blastogenic response to mitogens, hypergammaglobulinaemia, high titres of antibodies to many common antigens, various autoantibodies, a monoclonal IgM-kappa protein, an increased frequency of mature Ig containing blood B cells and a high production of Ig in vitro in unstimulated cultures. E rosetting cells showed faint or no immunofluorescence staining with monoclonal antibodies directed against T cell membrane antigens. In vitro Ig production in response to pokeweed mitogen was defective, and no T cell helper or suppressor activity was observed. It is suggested that the immunoregulatory deficiency might have caused the B cell hyperactivity.     

Autoimmune neutropenia.

Serologic tests for antineutrophil antibodies were used to determine if autoantibodies cause neutropenia. The serums of five patients with idiopathic neutropeniaopsonized normal neutrophils, causing them to be ingested by rabbit macrophages or else to activate glucose oxidation rates of other normal neutrophils by at least twice the rate of controls. Some of the serums inhibited the ability of normal neutrophils to ingest by 62 to 56 per cent. At splenectomy in two of the patients splenic macrophages contained ingested neutrophils, suggesting that the opsonic activity of the serum demonstrated in vitro had pathogenetic importance. In two adults, and possibly in an infant, corticosteroids raised the neutrophil count, although antibody activity remained in the serum of the adults. The findings indicate that autoantibodies are the basis of some cases of idiopathic neutropenia, and that they act by promoting the clearance of neutrophils by mononuclear phagocytes.     

Diagnostic relevance of humoral and cell-mediated immune reactions in patients with acute viral myocarditis.

Sera of 177 patients with acute myocarditis (10 coxsackie B 3/4, four influenza, four mumps, 15 cytomegalovirus, 144 undefined) were tested by indirect immunofluorescence for autoantibodies against heart and skeletal muscle and vital or air-dried adult cardiocytes. Antibody-dependent cytolysis, lymphocytotoxicity and antibody-dependent cellular lymphocytotoxicity were assessed using viral adult rat cardiocytes as target cells. Muscle-specific anti-sarcolemmal antibodies of the anti-myolemmal type--often associated with non-organ-specific anti-endothelial antibodies--were demonstrated in nine out of 10 patients with coxsackie B, in all patients with influenza and mumps and in 65 out of 144 patients with undefined myocarditis. In contrast, 13 out of 15 patients with cytomegalovirus myocarditis lacked anti-sarcolemmal antibodies but had low titre anti-inter fibrillary antibodies instead. In the presence of complement, anti-myolemmal antibodies induced cytolysis of vital cardiocytes, whereas hepatocytes remained unaffected. Titres of anti-myolemmal antibodies correlated with the degree of cardiocytolysis. The anti-myolemmal immunofluorescent pattern and the cytolytic serum activity could be absorbed with the respective viral antigens suggesting that these antibodies cross-react with moieties of the virus itself and may be both diagnostic and aetiological markers in acute viral myocarditis. Lymphocyte-mediated cytotoxicity against heterologous cardiac target cells could not be observed in our patients with myocarditis of proven viral aetiology. However, lymphocyte-mediated cytotoxicity was demonstrated in 10 ASA-positive and one ASA-negative patient with myocarditis of unknown origin. ASA-positive sera blocked lymphocytotoxicity in three of these patients.     

Autoantibodies to a regulatory T cell subset in human ageing.

Anti-T cell autoantibodies were detected in some aged humans. Non-immunoglobulin-bearing (Ig-) cells were isolated from the peripheral blood of normal human donors by negative selection through Ficoll, using sheep erythrocytes coated with rabbit anti-human Ig. The Ig- cells were then reacted with sera from 83 individuals ranging in age from 60 to 99 years; 36% of the serum samples were noticeably reactive with the Ig- cells (average reactivity 28%). The peripheral blood lymphocytes from some of the aged individuals were also tested for levels of Ig-secreting cells in a reverse haemolytic plaque assay; there was a six- to eight-fold increase in the number of plaque-forming cells (PFC) from those individuals whose sera contained appreciable amounts of anti-T cell antibody, as compared with those whose sera contained little or no anti-T cell antibody. Isolated Ig- cells from these individuals were also examined for the presence of regulatory T cell subsets, using sera from juvenile rheumatoid arthritis (JRA) patients. The Ig- cells from the subjects who had no detectable anti-T cell antibodies in their sera and near normal PFC levels were reactive with the JRA sera, whereas the Ig- cells from individuals with increased numbers of PFC and with serum anti-T cell antibodies were only slightly reactive with the JRA sera. These data suggest that a majority of the regulatory JRA+ subset of T cells had been lost in the latter group. When sera from aged individuals containing anti-T cell autoantibodies were reacted with JRA-, Ig- cells isolated from a normal human donor, little positive reactivity was seen, indicating that the autoantibodies in sera from aged humans and from some JRA patients are directed against similar T cell subsets.     

A screening assay for rapid and quantitative measurement of immunoglobulins and anti-immunoglobulins in unknown sera.

A radioimmunoassay which can measure serum levels of antibody and antigen when both species are immuloglobulins (Igs) is described. The technique is based on competition between insolubilized Ig (bound to Sepharose) and serum Ig for radiolabeled Ig. For measurement of anti-Ig antibodies, labeled Ig antigen is added to a mixture of antibody-Sepharose and unknown or standard antibody solution. For measurement of antigen, labeled, purified antibody is added to a mixture of antigen-Sepharose and unknown or standard antigen solution. Although both assays can detect either Ig antigen or Ig antibody in a given unknown, the antibody assay requires 10-20 times as much antigen as antibody to achieve the same degree of inhibition, and the antigen assay is 100 times more sensitive for antigen than for antibody. By titrating the same unknown in both assays, one can determine whether inhibitory activity is due to antigen or antibody. The routine assay readily detects 1 microgram/ml of antibody or of antigen. A modified assay was also developed which detects levels of antibody as low as 5-10 ng/ml. The technique is simple and allows rapid screening of hundreds of serum samples in a short period of time. The assay can also be modified for measurement of anti-idiotype antibodies.