Monoclonal antibodies OKT 11 and OKT 11A have pan-T reactivity and block sheep erythrocyte “receptors”

Monoclonal antibodies OKT11 (γ1) and OKT11A (γ2) are described and appear to have similar binding specificities. They bind, in immunofluorescence, with >95% of infant thymocytes, staining both cortical and medullary cells, 65-80% of blood lymphocytes and selectively stain the T cell-dependent paracortical areas of tonsil. A small proportion (9-12%) of bone marrow lymphocytes stain, but this population excludes the terminal transferase-positive cells. Both the γ1 and γ2 antibodies stain the surface membrane Ig-negative lymphocytes in blood and tonsil and are able to block sheep E rosette formation (to normal or leukemic T cells). In contrast, other monoclonal anti-T reagents tested (OKT1, OKT3, OKT4, OKT6, OKT8, OKT9, OKT10) did not block E rosette formation. E rosette formation and OKT11 binding are coincident on T-ALL cell lines and both are trypsin-sensitive. In a series of 145 leukemias and 26 leukemic cell lines investigated, only leukemias with a T cell phenotype including E rosette positivity were reactive with OKT11 and OKT11A. OKT11A binds to a polypeptide of approximately 50000 molecular weight on thymic lymphocytes. This structure may carry the recognition site for sheep erythrocytes. These antibodies provide additional useful markers for T cell analysis and are of potential therapeutic value.     

A subset of OKT4+ peripheral T cells can generate colonies containing mixed progeny with OKT4+ helper and OKT8+ suppressor cells

The membrane phenotype of human T cell colony progenitors and that of their clonal progeny was studied for expression of the T4 and T8 determinants. Using clonal culture conditions, the colonies were grown in semi-solid agar medium from peripheral blood cells. Clonality was assessed using the glucose-6-phosphate-dehydrogenase isoenzyme marker. Combination of this marker with the culture of sorted cell fractions allowed us to ascribe the colony progenitors to a subset of OKT4+ lymphocytes. The progeny consisted of the mixture of single OKT4+, single OKT8+ and double OKT4+8+ cells, as determined by double staining. Double staining was performed on mass-harvested colony cells and on individual colonies expanded in liquid culture with fresh interleukin 2. Expression of the OKT8 positivity on colony cells deriving from OKT4+ progenitors required an interaction with radioresistant OKT8+ cells that were co-cultured with these progenitors. Furthermore, the functional capacities of the cell progeny were assayed on the pokeweed mitogen-driven immunoglobulin production by B cells. It was found that OKT4+ colony cells were helper whereas OKT8+ colony cells were suppressor cells. It is concluded that a subset of OKT4+ peripheral blood T lymphocytes can generate colonies containing both helper OKT4+ cells and suppressor OKT8+ cells.     

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 degrees C to 36 degrees 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.     

Immunophenotypic characterization of acute leukemia by immunocytology

The potential for specific immunophenotypic characterization of the acute leukemias has been enhanced greatly by the development of monoclonal antibodies. Currently, this immunologic data is obtained most commonly by flow cytometric analysis or cellular cytotoxicity assays. The former is an expensive technic that lacks morphologic evaluation unless cell sorting is performed. The latter precludes morphologic assessment by the nature of the assay. The authors have developed an immunostaining procedure utilizing cytospin preparations and immunoperoxidase methods that are relatively inexpensive and allow simultaneous assessment of the immunologic markers and cellular morphology. Although a comparison of flow cytometry and immunocytology revealed quantitative differences for individual cell surface markers, the "qualitative" immunologic phenotype of the leukemic population was virtually identical by the two technics.     

Adult T-cell leukemia/lymphoma with OKT4 lack of OKT8 in peripheral blood and with OKT4 and OKT8 in lymph node. Study of two color flow cytometry

Adult T-cell leukemia (ATL) cells usually express the helper/inducer associated antigen OKT 4 with lack of OKT8. However, there are a few case reports indicating that there are atypical cell phenotypes in ATL including OKT4+/OKT8+. The analysis of surface phenotype of peripheral lymphocytes and abnormal cells in lymph node was done with monoclonal antibodies. ATL cells of peripheral blood and small lymphocytes of lymph node had the usual phenotype, OKT4+/OKT8-, but neoplastic cells of large lymphocytes of lymph node had the unusual phenotype OKT4+/OKT8+. The neoplastic cells were immunophenotyped by two-color flow cytometry analysis. OKT4+/OKT8- cells had a helper cell phenotype. OKT4+/OKT8+ cells had a helper cell and a cytotoxic cell. These findings suggest that in this case ATL cells arise from common thymocyte and one of them mature in peripheral blood other remain in lymph node.     

T cell subpopulations in CLL: methods of T cell enrichment artificially alter proportions of OKT4 and OKT8 positive cells

There is growing speculation about the meaning of reported imbalances in subpopulations of T lymphocytes in patients with chronic lymphocytic leukaemia (CLL). This study compared two techniques for producing T cell enriched subpopulations from both patients with CLL and normal individuals and the effects of these techniques on relative proportions of OKT4 and OKT8 positive cells. A sheep red cell rosetting technique resulted in significantly larger OKT8 and smaller OKT4 positive populations than did nylon wool column elution. Similar results were obtained in normal individuals. The nylon wool column elution technique produced less distortion of unfractionated OKT4/OKT8 ratios in normals than did the rosetting technique. Studies of T lymphocyte subpopulations should be interpreted with great caution as the methods used to study them can influence the results.     

Identification and isolation of OKT4+ suppressor cells with the monoclonal antibody WR16.

The monoclonal antibody WR16 was secreted by a hybridoma produced by fusing splenocytes from a BALB/c mouse immunized with human T-cell chronic lymphocytic leukaemia cells and the murine myeloma cell line NS-O. WR16 reacts specifically with human lymphocytes and binds to 48% of OKT4+ T lymphocytes and the majority of B lymphocytes. Human OKT4+ tonsil lymphocytes were subfractionated into WR16-/OKT4+ and WR16+/OKT4+ subpopulations by the panning technique. The capacity of these cells to help or suppress pokeweed mitogen-induced immunoglobulin (Ig) secretion by autologous B lymphocytes was monitored after 9 days of coculture. Cells of phenotype WR16-/OKT4+ enhanced Ig secretion in excess of that found with non-fractionated OKT4+ lymphocytes, whilst WR16+/OKT4+ lymphocytes suppressed Ig secretion when added to a mixture of B lymphocytes and non-fractionated OKT4+ cells. The WR16-/OKT4+ subpopulation was further fractionated by use of the MoAb Leu 8. Forty-two percent of WR16-/OKT4+ lymphocytes bound Leu 8, and cells of the phenotype WR16-/OKT4+/Leu 8+ were found to induce B-lymphocyte Ig secretion, whilst WR16-/OKT4+/Leu 8- lymphocytes were less active in this system. These data confirm the heterogeneity of the human T helper/inducer subset and indicate the existence of a population of OKT4+ lymphocytes that can suppress Ig secretion in the absence of OKT8+ lymphocytes.     

Activation of immune regulatory circuits among OKT4+ cells by autologous mixed lymphocyte reactions

We examined the nature of an autologous mixed lymphocyte reaction (AMLR) using T cell subsets defined by monoclonal antibodies. Cells capable of proliferating in the AMLR were demonstrated to reside in an OKT4+, but not an OKT8+, cell subset. With regard to the role of the T cell subsets recoverable from AMLR in the immune regulation, OKT4+ cells isolated from cells that had been activated for 3 days in AMLR did help pokeweed mitogen (PWM) stimulated immunoglobulin synthesis by autologous B cells. However, the OKT4+ cells activated for 6 days in AMLR exerted strong suppressor activity for PWM-induced immunoglobulin synthesis. Irradiation with 1,500 rad on activated OKT4+ cells in AMLR for 6 days not only eliminated the suppressor function but allowed for re-emergence of helper function. Cells exerting suppressor activity alone were recovered from OKT8+ cells stimulated with or without autologous non-T cells. These data suggest that OKT4+ cells activated in AMLR contain two functionally different subsets; one as helper cells and the other as suppressor cells. In addition, the emergence of OKT4+ suppressor function follows activation of the OKT4+ helper population, suggesting that a part of AMLR reflects a mechanism of 'feedback suppression' among OKT4+ cells.     

Radiosensitivity of isolated subsets of human lymphocytes (E+, OKT4+, OKT8+): protective role of monocytes and monokines.

The sensitivity of human peripheral blood T lymphoid populations to 60Co ionizing radiation was investigated. Dose-response values were determined for populations that are commonly identified by their ability to form spontaneous rosettes with sheep red blood cells (E+ cells), helper T lymphocytes (OKT4+ cells) and suppressor T lymphocytes (OKT8+ cells). OKT4+ and OKT8+ T cell subsets were negatively selected by complement (C)-mediated cytolysis using the C fixing OKT4 and OKT8 monoclonal antibodies (MoAb). The irradiation-induced damage was assessed by the lymphoblast transformation test, using the polyclonal T cell mitogen, phytohaemagglutinin (PHA) and the OKT3 MoAb. (The OKT3 antibodies are mitogenic for T cells only in the presence of monocytes). No significant differences were evident between dose-response values of E+, OKT4+ and OKT8+ lymphoid subpopulations when using PHA as a mitogen. On the other hand, when OKT3 was used to trigger resting irradiated peripheral blood T lymphocytes, e.g. E+ cells, OKT3 stimulated T cells proved to be markedly radioresistant as compared to PHA stimulated cell cultures. This was found to result from the fact that purified T cell cultures were co-cultured with non-irradiated monocytes when OKT3 was employed as a motogen. Similarly co-culturing of irradiated E+, OKT4+ and OKT8+ cells with non-irradiated autologus monocytes partially corrected the irradiation damage, regardless of the mitogen employed. More important, however, was the observation that macrophage derived supernatants containing (interleukin-1) IL-1 could confer a high degree of radioprotection on irradiated E+ cells. It is concluded that monocytes and monocyte products partially protect against irradiation damage.     

Correlation between active E-rosettes and antigens defined on peripheral lymphocytes by OKT 4 and OKT 8 monoclonal antibodies

A relationship has been sought between the classical marker, active E-rosette and the surface antigens of T-lymphocytes, defined by OKT 4 and 8 monoclonal antigens, in the peripheral blood of normal human people. It was found that no significant correlation existed in the case of OKT 4 antigen; conversely the OKT 8-positive cells were significantly enriched after E-rosette-forming cell isolation (about 2.5 times in comparison with the negatively selected cells). In this latter case, some partial correlation could exist between SRBC-high affinity of T-lymphocytes and the subsets defined by OKT 8 antigen.     

Hereditary deficiency of OKT4-positive cells: studies for mode of inheritance and lymphocyte functions.

Two Graves' patients were found to have no OKT4+ cells in their peripheral blood lymphocytes (PBL). However, the reactivity of their lymphocytes with T4(T4A) and anti Leu-3a was normal and no autoantibodies to the OKT4 determinant were found in their sera. Cytofluorographic analysis of PBL from their family members showed three types of immunofluorescence profiles with OKT4. The first type was the complete OKT4+ cell deficiency, the second was the normal percentage of OKT4+ cells with half immunofluorescence intensity and the third was the normal staining pattern with OKT4. Phytohaemagglutinin (PHA) and pokeweed mitogen (PWM) induced blastogenesis; PWM induced IgG synthesis, autologous and allogeneic mixed lymphocyte reaction, and Interleukin-2 (IL-2) production of their PBL were also normal. These results suggest that (i) the expression of determinant to OKT4 is transmitted as autosomal incomplete dominant trait and (ii) OKT4+ cell deficiency is not accompanied by a lack of the inducer/helper subset of T cells.     

OKT4+ and OKT8+ T lymphocytes produce soluble factors that can modulate growth and differentiation of human B cells.

The human T cell subset(s) responsible for the production of soluble factors that can modulate B cell growth and B cell differentiation was investigated in the present study. For this purpose, highly purified OKT4+ and OKT8+ lymphocytes were stimulated with phytohaemagglutinin and phorbol myristate acetate. Subsequently, culture supernatants were analysed for B cell growth factor (BCGF) activity and B cell differentiation factor (BCDF) activity in the following systems: 1) maintenance of a proliferative state of Staphylococcus aureus of strain Cowan I (SAC)-stimulated B cells and 2) induction of plaque-forming cell responses of SAC-stimulated B cells. Mitogenic stimulation led to production of equivalent amounts of either BCGF activity or BCDF activity from both of the OKT4+ and OKT8+ subsets. These findings may provide a basis for further studies of the molecular mechanisms as well as cellular interactions involved in human B cell activation, proliferation and differentiation.     

Molecular identification of human T-lymphocyte antigens defined by the OKT5 AND OKT8 monoclonal antibodies

Three human lymphocyte differentiation antigens, specific of the entire T-cell population, of the helper/inducer T-cell subset, and of the cytotoxic/suppressor T-cell subset have been identified, using mouse monoclonal antibodies obtained from Dr. P. Kung. Various T-cell populations were radio-labelled, the antigens were isolated by immunoprecipitation with the monoclonal antibodies and the resulting immune complexes subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The OKT3 antigen, present on peripheral T-lymphocytes and on functionally mature thymocytes has been identified as an oligomeric protein, composed of 23,000 mol. wt subunits. The OKT4 antigen, specific for the helper/inducer subset, is a single protein of 53,000 mol. wt. The OKT8/OKT5 antigen, defining the cytotoxic/suppressor subpopulation is composed of two subunits of 31,000 and 33,000. From co-capping experiments and biochemical data, the hypothesis is established that OKT5 recognizes a dimer of 140,000 mol. wt and OKT8 recognizes a determinant present on both the monomer 70,000 and the dimer. This hypothesis could explain the OKT5⁻ OKT8⁺ phenotype of some T-cells.     

T cells from patients successfully treated with OKT3 do not react with the T-cell receptor antibody

The mechanism of OKT3 therapy is complex and may include depletion of circulating CD3 cells, modulation of the CD3 molecule, and/or functional inactivation of T cells. Although the absolute number of circulating CD3 cells in OKT3-treated patients is used to monitor therapy, many laboratories assign CD3 numbers based on reactivity with OKT3. These CD3 numbers could be artificially low since the epitope recognized by OKT3 may already be occupied. Using a monoclonal antibody against a different CD3 epitope, we detected CD3 expression on T lymphocytes from 18/18 OKT3-treated patients. Nonetheless, OKT3 therapy in these patients was clinically successful, suggesting that monitoring patients solely for CD3 is uninformative. Since CD3 is associated with the T-cell receptor (TcR), we also evaluated alpha-TcR-1, a monoclonal antibody which detects a conformational determinant of the CD3/TcR alpha/beta complex, and found that less than 1% of the CD3 cells from OKT3-treated patients reacted. Furthermore, these cells were unresponsive to allogeneic stimulation. However, when patient cells were cultured overnight in the absence of OKT3, both alpha-TcR 1 binding and responsiveness to allogeneic stimulation became detectable. Thus, the monitoring of patients treated with OKT3 can be more informative if lymphocytes are tested for reactivity with alpha-TcR-1 and an alpha-CD3 antibody other than OKT3.     

Specific inhibition of OKT8 binding to peripheral blood mononuclear cells by jacalin

Human T-specific monoclonal antibodies were used to study the interactions between the binding of jacalin to peripheral blood mononuclear cells (PBMC) and the immunoregulatory molecules displayed at the surface of T cells. Jacalin inhibits the binding of OKT8 (anti-CD8) to both fresh PBMC and jacalin-induced T cell blasts. In both cases the binding of anti-CD3 (OKT3) or anti-CD4 (OKT4) was not affected by the lectin. The effect of jacalin on OKT8 binding is abolished by 1-O-alpha-D-methylgalactopyranoside, suggesting its mediation by the lectin saccharide combining sites. Preincubation experiments indicated that the inhibitory effect of jacalin is due to a competition between the lectin and the monoclonal antibody. The effect of the lectin could also be reversed by increasing concentrations of the monoclonal antibody. Taken together this data demonstrates a specific inhibition of OKT8 (anti-CD8) binding by jacalin. This effect is mediated by the binding of the lectin to structures on the cell surface, perhaps the CD8 antigen. The data also points to the discovery of a new mitogen that could be useful for studying the physiological role of CD8 on T cell responses.     

OKT3-activated locomotion of human blood lymphocytes: a phenomenon requiring contact of T cells with Fc receptor-bearing cells.

The OKT3 antibody activates locomotion of human blood T lymphocytes as measured by polarization assays and invasion of collagen gels. The proportion of motile cells increases during a period of 24-48 hr of culture, even following only a brief initial contact with OKT3. The motile cells are the growing population. Locomotion activation is cell-density dependent. Studies with surface-bound mitogens, namely substratum-bound OKT3 and Con A-Sepharose, showed that only lymphocytes in direct contact with the mitogen acquired locomotor capacity. Those separated from it by a cell-impermeable filter were not activated. The response to OKT3 requires the whole antibody molecule. F(ab')2 fragments were inactive. Intact normal human IgG, but not its F(ab')2 fragments, blocked the response. Removal of RFc gamma + cells from the population by rosetting with IgG-Ab-coated sheep red cells prevented the response. These findings suggest that an RFc gamma + population has to be present for T cells to become activated to locomotion by OKT3, and that the OKT3 antibody links the T cell to an FcR+ cell, cell-to-cell contact being essential for activating the locomotor response.     

Active T rosettes, human autologous T rosettes, OKT8 and OKT4 cells, con A-induced suppressive activity, and autoantibodies: clinical correlations

Correlations between various T-cell subsets: OKT4, OKT8, TEh (human autologous T rosettes), and TEa (active T rosettes) cells and the concanavalin A-induced suppressor function assessed on Con A proliferation have been analyzed in 46 patients with various secondary immunological disorders. The different T subsets and the suppressive activity were also compared in patients with and without autoantibodies. Significant positive correlations were found between T-cell markers TEa-OKT8 and TEh-OKT4. Significant inverse correlations were also found between Con A-induced suppressive activity-OKT4/OKT8 ratios, and TEa-OKT4/OKT8 ratios suggesting that the subpopulations identified by the active T rosettes are probably involved in T immunoregulatory mechanisms. There was no association between the Con A-induced suppressive function or the T markers TEa, OKT4, and OKT8, and the presence of autoantibodies. Only subjects with autoantibodies had a lower percentage of T lymphocytes forming autologous rosettes. These observations emphasize the fact that lack of correlation may exist between markers or function and immune status in some patients.     

Regulatory roles of human OKT4/OKT8 subsets in polyclonal immunoglobulin production induced by herpes simplex type 1 virus

T cells, OKT4 cells and OKT8 cells from the peripheral blood of normal individuals seropositive for herpes simplex type 1 virus (HSV) were studied for their capacity to regulate in vitro polyclonal immunoglobulin (Ig) production induced by inactivated HSV. Polyclonal Ig production induced by HSV has been demonstrated to be T-cell dependent. T cells, OKT4 cells and OKT8 cells were co-cultured with autologous non-T cells in the presence of HSV or pokeweed mitogen (PWM) and the number of plaque-forming cells (PFC) was measured with an hemolytic plaque assay after 6 days of culture. The results in the HSV system show that the OKT4 cells provided significantly more helper activity than OKT8 cells (p = 0.002); and the OKT8 cells exhibited more suppressor activity than OKT4 cells for Ig production (p = 0.02). The helper activity of OKT4 cells after HSV stimulation was significantly less than that obtained after pokeweed mitogen stimulation (p = 0.01). The in vitro polyclonal immunoglobulin response to HSV antigen is regulated by the balance of helper/suppressor activity exerted by OKT4 and OKT8 cell subsets.     

Do circulating OKT6-reactive cells belong to Langerhans cell lineage?

A few studies have been made in characterizing the phenotype, ontogeny, ultrastructure, and cytochemistry of circulating cord blood cells. Such studies have been hampered by the difficulty in obtaining pure populations of these cells. We therefore sought to obtain T6-positive cell-enriched populations from human cord blood using panning method. In cord blood, dendritic OKT6-labeled cells were classified in two types: some were similar to lymphocyte-like cells and some were consistent with monocyte-like cells. Both types lacked intracytoplasmic Birbeck granules. Homogeneous labeling of HLA-DR antigens were observed in both types. Heterogeneous labeling of T6 antigen was observed in both types. The monocyte-like cells have developed Birbek granule-like structures after exposure to OKT6 and immuno-adherence. The results suggest several different putative lineages of these OKT6-reactive cells: early precursors of dendritic cells, immature neonatal lymphoid subsets, and immature macrophagic leukocytes.     

Adult T-cell lymphoma/leukemia in a Caribbean patient: cytogenetic, immunologic, and ultrastructural findings

Cytogenetic findings on immunologically and morphologically characterized leukemic cells from a Caribbean patient with adult T-cell lymphoma/leukemia (ATLL) (HTLV+) are reported. Marker studies on peripheral blood lymphoid cells showed a mature postthymic phenotype: TdT-, OKT3+, OKT4+, OKT6-, OKT8-, 3A1-, anti-HLA-DR-. Light and electron microscopic analysis revealed a great cellular pleomorphism with respect to nuclear features. Three main types of leukemic cell were observed: typical multilobed ATLL lymphocytes, Sézary's syndrome (SS) cells, and cells intermediate between those two. Chromosome studies on PHA-stimulated cultures revealed three clones. The predominant clone was hyperdiploid; significant abnormalities were 1q+, 14q+, and 6q- (breakpoint q21), which are known to occur in lymphoid malignancies, together with trisomy 7q and i(17q), which have been reported previously in Japanese ATLL and the small variant of SS, respectively. The 14q+ marker was t(11;14)(q13;q22-24). The incidence of 6q-, trisomy 7q, and i(17q) varied within the main clone, and it is speculated that these chromosome abnormalities might be related to the variation observed in the cell types of this patient. Two minor clones had 6q- (breakpoint q25) and 13q+ markers, respectively. It was not possible to unequivocally establish the relationship between these three clones. The chromosomal, morphologic, and immunologic findings in this case support a close relationship between ATLL in Japan and in the Caribbean basin, as well as between the proliferating cells of ATLL and SS.