The relative radioresistance of interleukin-2 production by human peripheral blood lymphocytes: consequences for the development of a new limiting dilution assay for the enumeration of helper T lymphocyte precursor frequencies.

We describe a limiting dilution assay for the enumeration of alloreactive helper T lymphocyte precursor frequencies in human peripheral blood. The proliferation rate of the murine indicator cell line, cytotoxic T lymphoblastic line 2 (CTLL-2) induced by interleukin-2 (IL-2) culture supernatants was determined by staining with the fluorescent DNA dye propidium-iodide. Lymphocytes from healthy individuals as well as from patients with end stage kidney disease and no previous allosensitization exhibited a relative radioresistance of their IL-2 production up to gamma irradiation doses of 40-60 Gy. This differs from previous findings in the literature, showing a total inhibition of the IL-2 production in unsensitized individuals using a gamma irradiation dose of 20 Gy. The consequences of this relative radioresistance are that for a reliable stimulator cell inactivation in assays for the enumeration of helper T lymphocyte precursors gamma irradiation doses of at least 50 (-60) Gy are needed. Increasing the gamma irradiation dose for the inactivation of the stimulator cells can result in a decrease of the antigen presenting capacity of these cells.

Characterization of two human monoclonal antibodies reactive with HLA-B12 and HLA-B60, respectively, raised by in vitro secondary immunization of peripheral blood lymphocytes.

We have developed an in vitro immunization system for the production of B-cell lines that secrete HLA-specific human mAbs. For this purpose, peripheral blood lymphocytes of parous women were stimulated with pools of allogeneic lymphocytes. Preferential outgrowth of B-lymphocytes was effected by inclusion of rIL-2 and a B-cell specific nucleoside analogue. Stimulated B cells were immortalized by EBV transformation, and specific antibody-producing transformants were fused to heteromyeloma or mouse myeloma cell lines, yielding stable hybridomas. This approach has led to the successful development of two human heterohybridomas producing HLA-specific mAbs reactive by complement-mediated cytotoxicity. The specificities of these human mAbs, reactive with HLA-B12(44 + 45) and HLA-B60, respectively, are fully concordant with those of HLA-typing sera.

The grasping response in rats: interaction between gamma-type endorphins and haloperidol.

gamma-Type endorphins mimic neuroleptics in inducing a grasping response in rats. It was studied whether the haloperidol-induced grasping response was altered after blockade of gamma-type endorphin activity in the rat brain. To achieve this blockade rats were injected i.c.v. with gamma-endorphin antiserum or with a monoclonal anti-idiotype desenkephalin-gamma-endorphin antibody, which may bio-inactivate the gamma-type endorphins or block the putative receptors for gamma-type endorphins, respectively. The results showed that both treatments attenuated the haloperidol-induced grasping response, particularly 3 h after haloperidol treatment. The influence of these antibodies appeared to be specific, since other sera were without effect. Thus there may be an interaction between the endogenous gamma-type endorphin activity and the haloperidol-induced grasping response.

The influence of cytomegalovirus carrier status on lymphocyte subsets and natural immunity.

The influence of the cytomegalovirus (CMV) carrier status on peripheral lymphocyte subsets was studied in 70 healthy individuals. IgG-class antibodies against CMV late antigen were used as markers for the presence of CMV in those individuals. The 39 CMV-seropositive individuals had significantly higher numbers of CD3+ (P = 0.009), CD8+ (P = 0.005) and HNK1+ (P = 0.002) cells than the 31 CMV-seronegative individuals. Two-colour immunofluorescence studies revealed that the HNK1+ cells coexpressing CD4 or high density CD8 were particularly increased in the number under the influence of CMV, but not the HNK1+ cells coexpressing CD16. Since HNK1 and CD16 are markers associated with natural killer (NK) activity and antibody-dependent cellular cytotoxicity (ADCC), we investigated the influence of the CMV carrier status on those functions. The NK and ADCC functions of peripheral blood mononuclear cells (PBMC), HNK1+ and HNK1- cells were correlated with the percentages of CD16+ cells among those cells. Although CMV-seropositive individuals had significantly less CD16+ cells among their HNK1+ cells than CMV-seronegative individuals (mean and s.d.: 39 and 19%, versus 58 and 11%, P = 0.003), the NK and ADCC functions of the HNK1+ cells were similar in both groups. Also, the CMV carrier status did not influence significantly those functions of PBMC and HNK1- cells. We conclude that the CMV carrier status, i.e. CMV-seropositivity, is associated with a significant increase in the numbers of HNK1+ lymphocytes coexpressing T cell markers. That situation may reflect the continuing interaction between CMV and the immune system of its host.

Automated reading of Propidium Iodide lymphocytotoxicity tests for HLA-DR, MB, MT typing.

The combination of the Propidium Iodide method and automatic reading and scoring with a microscope fluorimeter is an accurate, reproducible and stable procedure for HLA-DR, MB and MT typing.

Automated reading of HLA-A,B,C typing and screening. The propidium iodide (PI) method.

A routine method has been developed and tested in the laboratory for the automatic reading of HLA typing and screening for antibodies with the microlymphocytotoxicity test. The assay is more sensitive than the NIH technique, is rapid, produces objective results, and can be easily linked up with existing manual procedures. Multipurpose reading machines are now commercially available.

Inhibition of responder cell activity in mixed leukocyte culture reactions. II. Influence of antibody binding and shedding properties within a B7-cross-reactivity group.

An HLA-B7 antiserum showed cross-reactivity with HLA-B8, Bw41, a split of B40 (Bw60) and possibly Bw22 and B27, thus detecting one or more determinants on these antigens similar to an antigenic site on HLA-B7 usually not detected by other B7-antisera. The cross-reactions were demonstrable by adsorption of antibodies on lymphocytes followed by release at 37 degrees C, which enabled the detection of weak and otherwise hardly detectable reactivity. Released antibody molecules were detected in two different assays: (1) Antibody-dependent cellular cytotoxicity (ADCC) with HLA-B7 positive target cells (fluorochromasia micro ADCC). (2) Inhibition of MLC reactions with B7 positive stimulator cells. The B7-antibody, as detected in both assays, was released in decreasing activity from Bw41 greater than B8 greater than Bw60 much greater than B7 greater than (B27 = Bw22) positive cells. The order of sensitivity in which the various antigens were detected in ADCC assays in which the antiserum activity was measured directly on various target cells was different, viz. HLA-B7 greater than Bw60 = B27 greater than Bw41 greater than B8. Bw22 was not detected. Absorption studies demonstrated that HLA-B7 positive cells bound more B7 antibody activity than B8 positive cells. However, antibody molecules bound to B7 positive cells were mainly released as immune complexes, which could be dissociated by treatment with acid. In contrast, B7 antibody molecules bound to B8 positive cells were released as free antibody molecules. This marked difference in shedding properties further explained the previously described B7 specific unresponsiveness in MLC of HLA-B8 (and also Bw41) positive responder cells after sensitization with the B7 antiserum (de Rooij et al. 1980).

On the dynamics of HLA-A2 antigen-antibody complexes on the cell membrane of human peripheral blood lymphocytes. A biochemical and electron microscopical autoradiographic study.

This report deals with some dynamic aspects of membrane bound immune complexes of HLA antigens and antibodies. Radiolabelled HLA antibodies were used to sensitize peripheral blood lymphocytes. Upon in vitro incubation of these cells, two different events were detected: 1) Immune complexes and a limited amount of free antibody were shed into the culture supernate; from the immune complexes specific antibodies could be regenerated. 2) Immune complexes were also internalized by means of endocytosis as visualized by electron microscopical autoradiography. Antibodies were found above dense bodies (lysosomes) and above multivesicular bodies. The results are discussed in relation to the biology of membrane bound HLA molecules.

Inhibition of responder cell activity in mixed leukocyte culture reactions. I. Evidence of cross-reactivity of B7-antibody with HLA-B8.

In a previous report we described a human allo-antiserum which showed MHC restricted dual specific inhibition of mixed leukocyte culture (MLC) reactions (de Rooij et al. 1980). Responder cells were preincubated with the antiserum and washed. Inhibition of MLC reactions occurred if HLA-B8 positive responder cells and HLA-B7 or B40 positive stimulator cells were used. The mechanism of inhibition by this antiserum was investigated and described in this report. The results strongly suggested that the observed inhibition was due to HLA-B7 specific antibody molecules cross-reacting with HLA-B8 antigens. We decided on the following mechanism: HLA-B7 specific antibody molecules bind to HLA-B8 antigens on responder cells at 4 degrees C. Bound antibody molecules area readily released at 37 degrees C and the bind preferentially to HLA-B7 or -B40 positive stimulator cells. Stimulator cells are then lysed by antibody dependent cellular cytotoxicity (ADCC), resulting in impaired MLC reaction. This conclusion was derived from the following observations: 1. HLA-B7 positive cells could be lysed due to antibody molecules bound to B8 positive cells: a. Cells coated with antibody molecules lysed B7 or B40 positive target cells. b. Antibody molecules released from B8 positive cells lysed B7 or B40 positive target cells in ADCC. 2. The active antibody molecules were HLA-B7 specific: a. The inhibiting antibody molecules in the antiserum could be absorbed on and eluted from HLA-B7 positive platelets (de Rooij et al. 1980). b. Antibody molecules bound to and released from HLA-B8 positive cells carried the same B7 specific inhibiting and cytotoxic activity as the original antiserum. 3. The site of recognition on HLA-B8 positive cells is the HLA-B8 antigen: a. F(ab')2 preparations from HLA-B8 and -Bw6 specific antisera inhibited antibody binding to B8 positive cells. b. Antibody binding was not restricted to B, T, FcR+, FcR- or non adherent cell subpopulations. 4. The antibody molecules bind to B8 positive cells with the antigen binding site and not with the Fc part of the molecule: a. Antibody binding could be blocked partially with a F(ab')2 preparation from the original antiserum but not with a Fc preparation. b. The antigen binding site of antibody molecules bound to HLA-B8 positive cells was not freely available, since the MLC inhibiting activity of sensitised B8 positive cells could not be blocked by soluble HLA-B7 antigens. c. The antiserum was cytotoxic for HLA-B8 positive target cells in ADCC. The observed inhibition of MLC by antibody molecules with apparent dual specificity is thus the consequence of a shared or a similar antigenic determinant of HLA-B7 and HLA-B8 antigen molecules.

HC restricted dual specific inhibition of mixed leukocyte culture reactions by human HLA antibody molecules.

A human alloantiserum was found which selectively inhibits responding cells in mixed leukocyte culture reactions. Inhibition was achieved by pre-incubation of responder cells in the antiserum followed by washing. The serum showed dual specificity as an inhibiting agent. First, inhibition was restricted to HLA-B7 or -B40 positive stimulator cells, specificities against which the antiserum also had cytotoxic activity. Second, inhibition was almost exclusively associated with the presence of the phenotype HLA-A1, -B8 on the responder cells The HLA associated specificity for responder cells was unexpected since no alloantibody activity directed to responder alloantigens could be detected by conventional serological-methods. The antiserum donor had not been immunized with HLA-A1, -B8 antigens nor with known crossreactive antigens. Furthermore, the serum donor did not carry HLA-A1, -B8 antigens herself. The inhibiting substance in the antiserum had physicochemical properties of IgG and was specifically reactive with HLA-B7 positive platelets. Pepsin digest preparations were not inhibitory. Fc receptor positive responder cells were required for inhibition. Responder cells, preincubated with the antiserum, suppressed the response of cells not incubated with the antiserum. Three possible explanations of these results are discussed: specific binding of the Fc part of the antibody with Fc receptors of responder cells, specific activation of suppressor cells and cross-reactivity.

The major histocompatibility complex of rhesus monkeys. VIII. Isolation and partial characterization of SD antigens.

The structure and chemical nature of the serologically defined (SD) antigens coded by the major histocompatibility complex (RhLA) of rhesus monkeys was studied. The use of specific anti-SD sera allowed the selective isolation of the corresponding antigens from crude antigen preparations. These were obtained by detergent solubilization after incorporation of 3H, 35S, and 14C amino acids in lymphocytes or mitogen-stimulated lymphoblasts. The results indicate that the SD antigens are of proteinaceous nature and are composed of two polypeptide chains with molecular weights of 44,000 and 12,000, the latter being beta 2-microglobulin. No differences in molecular size and subunit composition were detected between antigens of both segregant series. The results are discussed in relation to similar data available for the analogous human and murine histocompatibility antigens.

HLA-D associated alloantisera react with molecules similar to Ia antigens.

Two human alloantisera specific to bone marrow-derived lymphocytes (B cells) were shown to precipitate polypeptide chains of 29,000 and 34,000 daltons from human lymphoblastoid B cell lines. These molecules are similar to murine Ia antigens and are also precipitated by a rabbit B-cell specific heteroantiserum. Since the alloantisera are thought to recognize determinants coded by HLA-D or closely linked loci, these data support the hypothesis that these B-cell specific alloantigens are the human counterpart of the mouse Ia antigens and may be the products of HLA-D.

The application of radiolabelled HL-A antibodies in the detection of (soluble) transplantation antigens: an alternative for the microcytotoxicity test.

The use of radiolabelled anti HL-A antibody preparations is described. Absorption by platelets, followed by acid elution, fractionation of the eluate by column chromatography, radiolabelling and a second absorption--elution step with platelets yielded specific preparations, irrespective of original cytotoxic titres. The binding of these radiolabelled antibodies to lymphocytes could be inhibited by soluble antigens present in normal plasma or obtained from spleen cells by papain treatment. As a method for the detection of soluble transplantation antigens the use of radiolabelled antibodies appeared as sensitive as the microcytotoxicity test.

Binding of (T,G)-A--L by normal sera.

The synthetic polypeptide poly-L-(Tyr, Glu)-poly-DL-Ala--poly-L-Lys [(T,G)-A--L] was found to be bound not only by mouse immune serum, but also by nonimmune sera from several species. A combination of immunoelectrophoresis and autoradiography showed that albumin, beta-globulins and immunoglobulins in these sera may bind the antigen. This binding pattern is different, however, for the various species investigated.