Antibody OKT3 Research Articles

Langerhans cells in oral epithelium of chronically inflamed human gingivae

This study describes the histopathological features and the distribution of oral epithelial Langerhans cells in 19 gingival biopsies originating from an adult Tanzanian population characterized by very poor oral hygiene and severe gingival inflammation. Light-microscopically, all biopsies contained often large inflammatory connective tissue infiltrates, 6 of which predominantly contained plasma cells while the rest were dominated by lymphocytes. Seven specimens contained peculiar accumulations of round lymphoid and dendritic cells in the lower cell layers of the oral epithelium. These phenomena have not previously been demonstrated in human gingiva and deserve further attention in studies on the pathogenesis of periodontal diseases. Immuno-histochemical staining with OKT6, OKT4 and OKT8 antibodies showed markedly increased numbers of OKT6-positive cells in 7 specimens and clusters of OKT4- and OKT8-positive cells in the oral epithelium of 4 specimens. High numbers of OKT6-positive cells were not related to the presence of intra-epithelial, non-keratinocyte infiltrates or large connective tissue infiltrates. The variable numbers of oral epithelial Langerhans cells may therefore result from different bacterial antigens elucidating different responses or, alternatively, reflect different responses to similar plaque antigens penetrating the surface of the oral epithelium.

Interleukin 2 secretion by lymphocytes of patients with squamous cell cancer of the oropharynx or larynx

The various strengthening influences of interleukin-2 (IL-2) on the immunological system is of intrinsic importance to tumour defense. IL-2 concentration can be measured by the determination of the IL-2 dependent CTLL cell line in a micro-Elisa-assay. The IL-2 secretion is dose dependent after stimulation with the mitogens phytohaemagglutinin A (PHA), phorbolester (PMA) or the monoclonal T cell antibody OKT3. Maximal IL-2 release is achieved by concomitant activation with phytohaemagglutinin A (PHA) and phorbolester (PMA). As controls healthy donors with low and high alcohol and cigarette consumption were used. In patients with cancer of the oropharynx, a significant reduction in the IL-2 secretion was observed when compared with the larynx cancer group or with healthy donors. This might suppose an immunological influence of the squamous cell carcinoma which grows in the oropharynx in close proximity to lymphatic cells and vessels which are numerously present in the tonsillar ring of Waldeyer.

Treatment with recombinant IFN-gamma decreases cell surface CD4 levels on peripheral blood monocytes and on myelomonocyte cell lines.

The human cell surface protein CD4 is not only an important accessory molecule in the activation of MHC class-II-restricted T cells, but has also been implicated to be a receptor for the human immunodeficiency virus HIV-I on lymphoid and monocytic cells. We have found that a 24-h treatment of the promonocytic leukemia cell line U937 with rIFN-gamma decreases the expression of the CD4 Ag by 50% as measured by cytofluorographic analysis. The decrease in CD4 expression was dependent on the concentration of rIFN-gamma, with maximal effects occurring at 20 to 200 U/ml. The decrease appeared to be due to actual loss of the CD4 molecule from the cell surface rather than masking of a particular epitope, inasmuch as similar results were obtained with the OKT4 and OKT4A antibodies. The effect of rIFN-gamma to decrease CD4 expression was not due to a general loss of cell surface Ag, because the binding of OKM1 and anti-HLe-1 increased after rIFN-gamma treatment. Treatment of rIFN-gamma also decreased cell surface CD4 expression on the promyelocytic leukemia cell line HL-60, and on the monocytic cell line THP-1, although the extent of the decrease was less than on U937 cells. Freshly isolated normal peripheral blood monocytes treated for 48 h with rIFN-gamma bound much less OKT4 or OKT4A antibody than cells incubated in the absence of rIFN-gamma. Moreover, treatment with rIFN-gamma reduced the percentage of peripheral blood monocytes that were positive for the CD4 Ag. In contrast with the decrease in CD4 levels on rIFN-gamma-treated monocytes, treatment with rIFN-gamma had no effect on CD4 levels on peripheral blood T lymphocytes or T cell lines.

Immunohistochemical detection of tissue-infiltrating lymphocytes in bladder tumors.

Local immunocompetence in patients with bladder tumors was evaluated immunohistochemically using monoclonal antibodies. As well as investigating the subsets of peripheral blood lymphocytes by flow cytometry, the subpopulations of tissue-infiltrating lymphocytes were investigated in frozen specimens from bladder cancers (n = 25) and from noncancerous tissues (n = 4). Six serial sections were stained with Leu4, Leu7, Leu10, LeuM3, OKT4, and OKT8 antibodies, respectively. Marked lymphocytic infiltration into bladder tumors was a favorable prognostic sign. T cell infiltration was marked in the noninvasive group and was prominent around tumor cells, whereas B cells were rare in the stroma. T cells predominated over B cells in 19 of 25 bladder tumors. In patients with low-stage tumors, OKT8 cells were more prominent than OKT4 cells. NK cells accumulated around the cancer cells and infiltrated within cancer nests; NK cell infiltration was scanty in high-stage cases. There was a slight scattering of macrophages in 13 cases. In contrast to the findings in subsets of tissue-infiltrating lymphocytes, subsets of peripheral blood lymphocytes did not correlate with stage of disease. These findings suggest the presence of local immunosurveillance against bladder tumors. To elucidate host immune response against tumor, it may be important to characterize tissue-infiltrating lymphocytes.

Immunological studies of the pathogenesis of multiple sclerosis Cell-mediated cytotoxicity by peripheral blood lymphocytes against basic protein of myelin, encephalitogenic peptide, cerebrosides and gangliosides

Cell-mediated cytotoxic reactions against myelin basic protein (MBP), encephalitogenic peptide, cerebrosides and gangliosides were studied in 165 MS cases. The cytotoxicity, which is detectable only in the autologous system, is mediated by T lymphocytes bearing antigens reactive with antibody OKT8. The cytotoxicity is highest during active MS regardless of whether it is relapsing or chronic progressive. During inactive MS cytotoxicity is significantly less and remains virtually unchanged at a low level over long periods. The degree of cytotoxicity is thus, predominantly, dependent on the activity stage of the disease. Further, there is a correlation with the severity of the clinical deficiencies. The pathogenetic significance of cytotoxic reactions can be recognized by the close correlation between the stage and course of MS, which can be demonstrated both statistically and individually.

Proliferation Antigens in Cutaneous Melanocytic Tumors – an Immunohistochemical Study Comparing the Transferrin Receptor and the Ki 67 Antigen

The cellular reactivities with the monoclonal antibodies OKT9 and Ki 67 have been demonstrated to be closely related to proliferation in various malignant neoplasms. In this study a total of 25 melanocytic skin tumors was examined immunohistochemically with both antibodies and the results were evaluated semiquantitatively for OKT9 and quantitatively for Ki 67 by stereological methods. All cases of primary and metastatic malignant melanoma expressed a strong stainability for OKT9, whereas benign melanocytic nevi were almost completely negative. Our results with the monoclonal antibody Ki 67 revealed highly significant differences in the numerical density of Ki-67-positive cells between metastatic malignant melanoma (number of positive cells: 47.0 +/- 9.2 X 10(3)/mm3), primary malignant melanoma (6.3 +/- 1.9 X 10(3)/mm3) and benign melanocytic nevi (2.2 +/- 0.7 X 10(3)/mm3). Correlation analysis between mean percentage of OKT9-positive cells and numerical density of Ki-67-positive cells revealed a significant correlation of both parameters (r = 0.58; p less than or equal to 0.05), indicating a positive relationship of OKT9 and Ki 67 expression. Especially in primary malignant melanoma, however, the amount of OKT9-positive cells considerably exceeds that of Ki-67-positive cells. The monoclonal antibodies OKT9 and Ki 67 reflect 'proliferative activity' in melanocytic skin tumors, as both are expressed in significantly higher amounts in primary and metastatic malignant melanomas. The combined application of these antibodies in cutaneous melanocytic lesions might be of diagnostic and prognostic value.

Isoelectric focusing of cross-linked monoclonal antibody heterodimers, homodimers and derivatized monoclonal antibodies.

Anti-tumor monoclonal antibodies were cross-linked to the anti-CD3 T-cell antibody OKT3 by the use of the heterobifunctional cross-linker succinimidyl-3-(2-pyridyldithio)propionate. Derivatized monoclonal antibodies, heterodimers, and homodimers were resolved by analytical isoelectric focusing in polyacrylamide gels containing 1% Triton X-100. Isoelectric points of the derivatized antibodies were lower than native antibodies, consistent with lysine derivatization. Antibodies derivatized with 2-iminothiolane were equivalent or slightly higher in pI compared with native antibodies. Heterodimers focused in microheterogeneous bands between the pI extremes of the parent derivatized antibodies. The isoelectric points of homodimers were lower than those of parental antibodies and could be distinguished from heterodimers. Reduced and alkylated heterodimers were resolved into their constituent antibodies by isoelectric focusing.

Spontaneous Thyroglobulin Autoantibody Production by Peripheral Blood Lymphocytes is Regulated by T Cells and is Augmented by Soluble T Cell Factors

In this study we report the spontaneous production of autoimmune anti-thyroglobulin antibodies (Tg Ab) by peripheral blood lymphocytes from patients with autoimmune thyroid disease and the use of these cells to study the regulation of this production in vitro. Using a mitogen free microculture system we have found a significant correlation between an individuals' serum titre and the amount of Tg Ab produced in vitro. At low B:T cell ratios Tg Ab production decreased. Specific depletion of suppressor T cells and helper T cells was accomplished using the monoclonal antibodies OKT 8 and OKT 4 respectively. Suppressor cell depletions resulted in increases in Tg Ab production while depletion of helper cells had no consistent effect. The addition of pokeweed mitogen had no stimulatory effect on spontaneous thyroglobulin antibody production. In contrast, when T cell conditioned media was added to the cells, Tg Ab production increased substantially in all patient cultures tested but not in normal control cultures. The results of this study are consistent with the existence of functional thyroid-antigen specific suppressor cells in the peripheral blood which are actively involved in the regulation of autoantibody producing B cells. The results obtained with the cultures maintained in conditioned media show that autoimmune lymphocytes are extremely sensitive to one or more stimulatory factors present in the media and suggests an important role for the production of soluble lymphocyte factors and the expression of receptors for these factors in the aetiology of autoimmune thyroid disease.

Antigen-specific electrophoretic cell separation for immunological investigations.

Preincubation of human blood lymphocytes with cell surface antigen specific antibodies under non-capping conditions reduces the electrophoretic mobility of the corresponding lymphocyte subpopulation. Antigen-positive and antigen-negative cells can be separated by free flow electrophoresis with high yield, purity and viability. The use of fluorescence-labelled second antibodies augments the induced decrease in net surface charge density, and allows rapid detection of antigen-positive cells in the fractions of electrophoresis. Carrier-free cell electrophoresis of human peripheral blood lymphocytes after reaction with anti-IgM-antibody or the monoclonal antibodies OKT4 or OKT8, and sandwich staining with tetrarhodamine isothiocyanate-labelled anti-IgG resulted in the large-scale separation of high pure human B and T lymphocyte subpopulations. Their functional integrity was shown in assays of lymphocyte transformation and of antigen-specific induction and regulation of antibody synthesis in vitro. These separate lymphocyte subpopulations are useful tools for immunological investigations. While, for instance, the effects of drugs on human lymphocytes are obscured by coincident changes in cell composition of the peripheral blood tested that do not by themselves reflect whole body immunocompetence, the cell separation and in vitro assays at a defined cell number and cell composition allow the recording of quantitative changes in the function of different cell subpopulations. We studied the influence of the anesthetic thiopental on separated human lymphocyte subsets. In both polyclonal lectin stimulation and in vitro antibody production, thiopental exhibited a noncytotoxic suppression of lymphocyte functions. B-Cells, T-helper and T-suppressor cells were equally affected and showed the same dose response.(ABSTRACT TRUNCATED AT 250 WORDS)

Flow cytometric studies of the binding of monoclonal antibodies OKT3, OKT4 and OKT8

The binding of monoclonal antibodies (OKT3, OKT4 and OKT8) to human T cells was investigated by flow cytometry. A flow cytometer was calibrated with standard fluorescence microspheres, which permitted quantitation of the number of bound antibody molecules. Considerable care was taken to perform the flow cytometric assay at a constant temperature and the effect of temperature on the binding reaction was examined. The binding of OKT3, OKT4 and OKT8 exhibited saturation kinetics. The maximum binding varied with temperature. Kinetic analysis according to the Hill equation revealed that the value of the Hill coefficient for OKT3 changed from 1.8 to 1.0 when the temperature was raised from 12°C to 36°C, whereas the corresponding values for OKT4 and OKT8 did not vary with temperature. Thermodynamic functions obtained from the Van't Hoff plot showed that the binding of OKT3 was exothermic whereas the binding of OKT4 and OKT8 were endothermic.

Adverse influence of recipient lymphoid resistance to in vitro immunosuppression on the outcome of kidney transplants.

The study investigated whether preoperative in vitro sensitivity of lymphocytes from potential renal transplant recipients could identify patients at increased risk of acute rejection following transplantation and immunosuppression with cyclosporine, azathioprine, and prednisolone. Mixed lymphocyte culture responses were measured preoperatively in the presence of methylprednisolone, CsA, and antithymocyte globulin, and without immunosuppressive agents in 50 transfused recipients of primary cadaver renal transplants. Patients were classified as sensitive if all three immunosuppressive agents produced more than 50% inhibition of their MLC responses, and as resistant if one or more agents failed to produce 50% inhibition. All patients received postoperatively a standardized triple immunosuppressive regimen. Acute rejection was confirmed histologically and treated with Pred with or without ATG or monoclonal antibody OKT3. A total of 29 patients (58%) were sensitive and 21 (42%) were resistant; 4 patients were resistant to 3 agents, 5 were resistant to MP and ATG, 6 were resistant to MP and CsA, and 6 were resistant to MP alone. Sensitive and resistant groups did not differ in age, sex, transfusion history, HLA A, B and DR mismatches or duration of follow-up. The resistant group had a higher rate of graft loss from acute rejection (chi 2 = 6.0, d.f. = 1, P less than 0.02), more episodes of acute rejection (chi 2 = 8.7, d.f. = 3, P less than 0.05), and a higher proportion of patients in whom reflux nephropathy was the cause of renal failure (chi 2 = 18.3, d.f. = 1, P less than 0.001). The resistant group also had a higher proportion of highly sensitized patients and higher serum creatinine concentrations than the sensitive group, although the differences did not reach statistical significance. The study indicates that patients at high risk of acute rejection of renal allografts can be identified by a pretransplant in vitro assay, a finding that could influence recipient selection and immunosuppression.

Relationship between internalization and cytotoxicity of ricin A-chain immunotoxins.

Immunotoxins (ITs) appear to vary considerably in their killing efficiency towards antigen positive cells. In order to unravel the mechanisms underlying these differences, the parameters responsible for these variations were studied. The efficacy of the monoclonal antibodies (MoAbs) WT32 (CD3), OKT4 (CD4), T101 (CD5), WT1 (CD7) and WT82 (CD8) conjugated to ricin A-chain was expressed by the extent of protein synthesis inhibition of four leukaemic T cell lines (CEM, GH1, Jurkat and HPB-ALL). Large differences in cytotoxicity were observed. Efficient inhibition of protein synthesis was seen with anti-CD3 IT, anti-CD5 IT and anti-CD7 IT. In these cases the cytotoxicity was related to the antigen density on the target cell membrane. Anti-CD4 IT inhibited poorly and anti-CD8 IT was ineffective, even on cell lines with a high expression of the corresponding antigen. When antigen density and cytotoxicity were plotted for all CD antigens, no correlation could be found. Subsequently, internalization was studied with 125Iodine-labelled antibodies. Anti-CD7 showed the fastest internalization rate, followed by anti-CD3 and anti-CD5. Anti-CD4 and anti-CD8 antibodies were respectively moderately and hardly internalized. When the absolute amount of internalized MoAb was calculated, a highly significant correlation with cytotoxicity was found. We conclude that the degree of antigen expression is not so important as the absolute amount of antibody internalized in predicting the efficacy of ITs.

The value of immunocytochemical staining of lymph node aspirates in diagnostic cytology

This study investigates the applicability of immunocytochemical techniques towards improving the cytological diagnosis of lymph node disorders. Cytocentrifuge preparations of fine needle aspirates were examined using an indirect immunoperoxidase method and the alkaline phosphatase-antialkaline phosphatase method. 36 reactive lymph nodes were evaluated. 23 patients showed T cell predominance as assessed by the presence of CD3+ cells. 13 patients showed an excess of CD24+ cells over CD3+ cells. In all patients the B cell population showed no light chain class restriction. 48 aspirates from patients with B-non-Hodgkin's lymphoma of low malignancy were investigated. The majority of the cells in each of these cases expressed CD24 and 47 cases were monoclonal with respect to their light chain determinants. In 37/48 cases the antibody OKT9 reacted with less than 15% of cells. 18 aspirates were obtained from patients with B-non-Hodgkin's lymphoma of high malignancy. 17 patients showed light chain class restriction and a high percentage of CD24+ (greater than 80%) and OKT9+ (greater than 40%) cells. 8 malignancies were considered to be of T-cell origin. A low percentage (less than 15%) of Ig+CD24+ cells with a high number of CD3+ and/or CD4+ cells suggests a T-cell lymphoma. The majority of neoplastic cells in peripheral T-cell lymphomas (excluding mycosis fungoides) expressed OKT9 and CD3 expression was found to be variable. In 15 cases of Hodgkin's disease, there were 11 correct cytologic diagnoses. A significant number of BerH2 (CD30)+ cells having the morphology of Sternberg-Reed cells supports this diagnosis. 25 aspirates were obtained from patients with metastatic malignant tumours. Marker studies in eight cases helped in distinguishing metastatic malignant tumours from malignant lymphomas. Our findings indicate that the immunocytochemical technique is applicable to cytological material and that the simplicity of the procedure merits application to routine diagnostic cytology.

Two distinct subsets of patients with systemic lupus erythematosus

This study was undertaken to examine the levels and function of peripheral blood immunoregulatory T cell subpopulations in systemic lupus erythematosus (SLE). T cell subpopulations can be distinguished by the T cell differentiation antigens CD4 (recognized by the monoclonal antibodies OKT4 or Leu3) and CD8 (recognized by the monoclonal antibodies OKT8 or Leu2). All SLE patients tested had normal percentages of CD8 cells in their peripheral blood. The SLE patients, however, fell into two groups based on their CD4 cell numbers. Fifty-five percent of the SLE patients had normal levels of CD4 cells (Group A) and therefore normal CD4/CD8 cell ratios, whereas 45% of the SLE patient population had markedly depressed CD4 cell levels (Group B) and significantly low CD4/CD8 cell ratios. T cells from normal donors and SLE patients were further examined for their ability to stimulate allogeneic normal B/Mφ cells cells to secrete IgM in the presence of pokeweed mitogen (PWM). Utilizing this assay system two forms of immunosuppression were observed: (1) that mediated by high concentrations of purified CD4 cells and (2) that mediated by CD8 cells. High concentrations of purified CD4 cells, added to a constant number of allogeneic normal B/Mφ cells, suppressed PWM-stimulated IgM synthesis. Group B SLE patients, with significantly low CD4 cell numbers, had defective CD4 cell-mediated suppression which was concentration dependent. This result was confirmed in a study using identical twins discordant for SLE. In this case CD4 cells from the SLE twin did not induce immunosuppression at a high concentration of CD4 cells whereas similar concentrations of CD4 cells from the normal twin resulted in suppression. SLE patients (Group A) with normal levels of CD4 cells had normally immunosuppressive CD4 cells. Suppression mediated by CD8 cells was demonstrated by the fact that removal of CD8 cells resulted in enhanced IgM synthesis induced by the remaining CD4 cells. Although all the SLE patients in this study had normal peripheral blood levels of CD8 cells, SLE Group A patients had defective CD8 cell suppression whereas CD8 function appeared to be normal in Group B patients. These results suggest that in SLE patients with depressed CD4 cell numbers (Group B) there is a corresponding defect in CD4 cell function. We demonstrate that in SLE Group B patients, defective suppression is due to a subset of T cells that bear the CD4 antigen. The SLE patient population (Group A) with normal CD4/CD8 ratios and normally functioning CD4 cells, however, appear to have normal CD4 cell-mediated suppression but defective CD8 suppressor cell function.

Human cytotoxic T cells specific for autologous melanoma cells: successful generation from lymph node cells in seven consecutive cases.

Human T-cell populations specifically cytotoxic for autologous melanoma cells have been successfully generated from lymph node cells obtained from seven consecutive patients. The lymph node cells were stimulated in vitro with autologous irradiated melanoma cells; stimulation was repeated every 10-15 days at a tumor cell-to-lymphocyte ratio of approximately 1:20. Cytotoxic activity was assessed by a 4-hour 51Cr release assay. Mean lysis of autologous tumor cells was 47% at an effector-to-target cell ratio of 20:1, while mean lyses of the human myeloid leukemia cell line K562, allogeneic melanoma cells, and an osteosarcoma cell were 20%, 13%, and 11%, respectively. There was no lysis of autologous fibroblasts, fresh lymphocytes, or phytohemagglutinin-stimulated blasts. Three grades of specificity developed sequentially. In grade I, lysis of autologous tumor cells exceeded lysis of allogeneic tumor cells but did not exceed lysis of K562 cells. In grade II, lysis of autologous tumor cells exceeded lysis of K562 cells and all allogeneic tumor cells tested. In grade III, potent lysis of autologous tumor cells (greater than 40%) exceeded lysis of K562 cells and of all allogeneic tumor cells tested. All seven lymphocyte populations reached or exceeded grade I. Six reached or exceeded grade II. Two progressed to grade III. The generated cells were T cells, as determined by phenotypic analysis with flow cytometry. CD4+ cells and CD8+ cells accounted for 83%-100% of the cells. CD8+ T cells were separated from CD4+ T cells by panning with OKT8 and OKT4 antibodies. The resulting CD8-enriched and CD4-enriched populations were compared as effectors in cytotoxicity assays. The results suggest that the cell responsible for lysis of autologous tumor cells is CD8+. The methods used in this study have repeatedly resulted in the successful generation of cytotoxic T lymphocytes specifically cytotoxic for autologous melanoma cells; it is suggested that these cells have potential application for adoptive immunotherapy of melanoma.

Human T lymphocyte differentiation antigens as target for immunotoxins or complement-mediated cytotoxicity.

Graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT) is initiated by immunocompetent T cells present in the graft. Selective elimination of distinct T-cell subsets or a sufficient, but not complete T-cell depletion, might abolish severe GVHD without graft rejection and loss of the anti-tumour potential. In this study we analysed the efficacy of different monoclonal antibodies (MoAb) WT32 (CD3), OKT4 (CD4), T101 (CD5), WT1 (CD7), and WT82 (CD8) with respect to their cytotoxicity to T cells either as immunotoxin (IT) or in combination with complement. The cytotoxic potential was assessed by protein synthesis inhibition and clonogenic assays. The ricin A conjugated MoAb exerted only a minor effect on blood or bone marrow T cells, although they were highly inhibitory to T-cell lines. However, in the presence of 20 mM ammonium chloride, IT directed against CD3, CD5, and CD7 were highly cytotoxic. IT directed against CD4 and CD8 were less effective, due to a low internalization. The complement-mediated cytotoxicity was efficient for all antigens used. The natural killer (NK) activity, as measured by cytotoxicity to K562, was hardly depressed by anti-CD3, anti-CD4, anti-CD5, and anti-CD8, but was eliminated by anti-CD7. All procedures used had only a minimal effect on haematopoietic progenitors as measured by CFU-GM and BFU-E assays. We concluded that, although the T-cell population can be eliminated with the combination of anti-CD3, anti-CD5, and anti-CD7 antibodies plus complement, IT with 20 mM NH4Cl appear to kill higher amounts of T cells. Selective elimination of CD4- and CD8-positive cells is effectively obtained by MoAb with complement.

EXPRESSION OF HUMAN CD4 BY TWO HUMAN‐MOUSE INTERLINEAGE HYBRIDS

Two hybrid cell lines expressing human CD4 were prepared by fusing human B-lymphoid cells with the mouse T-lymphoma BW5147. Hybrid TF42 was derived from a human B-lymphoblastoid line and TF53.1 from a human B-ALL. Variants of these hybrids expressing or lacking CD4 were isolated by sorting cells stained with the monoclonal antibody (mAb) OKT4 on a fluorescence-activated cell sorter (FACS). Cytogenetic, isoenzyme and DNA analysis confirmed the presence of human chromosome 12 in the CD4+ hybrids, and revealed that CD4 expression by TF42 was associated with multiple copies of this chromosome. Of seventy mAb recognizing human T-cell antigens screened on the CD4+ and CD4- variants of the two hybrids, only mAb recognizing CD4 and Leu 8 reacted with the CD4+ cells. These hybrids should be useful in the preparation, screening and analysis of anti-CD4 monoclonal antibodies, and in studies of CD4 epitopes recognized by HIV.

Analysis of early lymphocyte activation events by fluorescence polarization flow cytometry.

After short-term (up to 4 h) stimulation with mitogen or antigen, lymphocytes were incubated with fluorescein diacetate and the polarization of fluorescence from intracellular fluorescein was measured on a specially adapted FACS II. This flow cytofluorimetric method to assay early changes in activated lymphocytes gave a reproducible response to the mitogens phytohaemagglutinin (PHA), concanavalin-A and the monoclonal antibody OKT3, recognized at 1 h by decreased polarization. A response by immune spleen cells to the antigen dinitrophenyl-ovalbumin was revealed at 4 h. The calcium ionophore A23187 induced an increase in polarization after only 10 min. The PHA polarization response was shown to be dependent on PHA binding, PHA dose, T cells, calcium ions and an intact cytoskeleton. The cellular events monitored by the polarization change are presumably altered fluidity of the probe's microenvironment due to conformational change in macromolecules to which the probe has bound or to dissociation of the probe into the aqueous phase. The fluorescein fluorescence polarization assay is a reliable and sensitive monitor of early lymphocyte activation events and, coupled with the use of a flow cytometer, permits study of particular subpopulations of responding cells.

Flow‐cytometric analysis of lymphocyte subsets in relation to virus infections at the onset of Type 1 (insulin‐dependent) diabetes

Alterations in T-lymphocyte subsets have been connected to the autoimmune pathogenesis of Type 1 (insulin-dependent) diabetes. In this study peripheral blood lymphocytes were analysed by flow cytometry using OKT3, OKT4, OKT8, anti-HLA-DR, anti-IL-2 receptor and anti-membrane immunoglobulin antibodies in newly diagnosed Type 1 diabetic children, their healthy siblings and healthy control children. The results were compared to the occurrence of serologically verified recent virus infections, some of which can induce lymphocyte subset alterations and have also been connected with the onset of diabetes. In most diabetic patients the amounts of OKT3, OKT4, OKT8 and membrane-Ig-positive cells were within the normal range. Exceptional helper/inducer and suppressor/cytotoxic T cell profiles were observed in a few patients, most of whom had serologically verified recent Epstein-Barr, rubella, mumps or Coxsackie B virus infection. In addition, increased numbers of activated IL-2 receptor-positive cells were observed in the patient group. These results suggest that significant lymphocyte subset alterations are not characteristic of Type 1 diabetes but can occasionally be induced by recent virus infections in newly diagnosed patients. However, the slight increase in activated lymphocytes could reflect the activation of cellular immune systems to the autoantigens in the pancreas.

Infections with cytomegalovirus and other herpesviruses in 121 liver transplant recipients: transmission by donated organ and the effect of OKT3 antibodies.

One hundred twenty-one adult liver transplant recipients were studied for the incidence, risk factors, and morbidity associated with herpesviruses infections after transplantation. The overall incidence of infection was 59% for cytomegalovirus (CMV), 35% for herpes simplex virus (HSV), 25% for Epstein-Barr virus (EBV), and 7% for varicella-zoster virus (VZV). Primary CMV infection occurred in 46% and reactivation CMV infection in 67% of the susceptible recipients. Symptomatic and disseminated CMV diseases were more common when patients developed primary infection (P less than .01, for both comparisons). The donor organ appeared to be the only important source of CMV infection in seronegative recipients. The use of OKT3 antibodies was associated with disseminated CMV disease in patients with primary infection (P = .04) but not with reactivation infection (P greater than .10). Although most HSV infections were oral or genital reactivations, three cases of HSV hepatitis occurred--one was a primary infection. Symptomatic reactivations of HSV were observed in 53% of HSV-seropositive recipients who received OKT3, versus 31% of seropositive recipients who did not receive OKT3 (P = .05).