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.     

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.     

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.     

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.     

Wheat germ agglutinin decreases expression of the T8 antigen on human peripheral mononuclear cells

We examined dynamics of expression of the human T-suppressor specific antigen, T8, following interaction of peripheral lymphocytes with wheat germ agglutinin (WGA). Cells were incubated at 37 degrees C with or without WGA (15 micrograms/ml) for 18 hrs, washed sequentially with N-acetylglucosamine (to remove bound WGA) and plain medium, then analyzed by flow cytometry for binding of lectins and monoclonal antibodies OKT8(T-suppressor specific) and OKT3 (pan-T specific). WGA pretreatment induced an overall 65% reduction in WGA binding and concomitant 30% reduction in percentage of T8+ cells. Furthermore, residual T8+ cells showed 50% reduction in T8 expression. Taking into account reductions in both percentages of T8+ cells and also antigen densities, WGA reduced T8 expression by greater than 60% overall. By contrast, binding of OKT3 and the lectins, concanavalin A (con A) and Ricinus communis agglutinin (RCA-I), was unaffected by WGA. The decreased T8 expression could not be explained by residual cell bound WGA and was fully reversible within 48 hours of removal of cells from WGA-containing medium. Therefore, WGA caused downregulation of T8 antigen expression. The effect of WGA was time- and concentration-dependent. Downregulation did not occur at 4 degrees C nor in the presence of azide, thereby demonstrating a requirement for cellular metabolism. The data suggest that WGA may bind to the T8 antigen, and they provide the possibility that similar downregulation of T8 by WGA may underlie certain of the in vitro immunoregulatory effects of this lectin.     

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.     

OKT3 induces suppressor cells for mixed lymphocyte and PHA mitogenic responses in human peripheral lymphocytes

Human peripheral lymphocytes pretreated with the Orthoclone monoclonal anti- T cell antibody OKT3 for 48 h markedly suppressed the proliferative response of autologous lymphocytes in one-way MLC and the mitogenic response to PHA. The ability to induce suppression is specific to OKT3 since other monoclonal antibodies to human T cells (OKT1, OKT4 and OKT8) did not elicit similar responses. OKT3 is mitogenic but further proliferation of OKT3 pretreated lymphocytes was not required for the suppression of autologous lymphocytes since mitomycin-C treated cells were fully effective. Kinetic studies indicated that pretreatment of lymphocytes with OKT3 for 24 h was sufficient to induce marked inhibition of the mitogenic response of autologous lymphocytes to PHA whereas suppression in MLC was not observed until lymphocytes were pretreated for 48 h.These studies support the previous observations that OKT3 may be reaching with an important molecule on the T cell surface and that interaction of OKT3 with this molecule induces profound functional changes.     

OKT3治疗移植排斥所致T细胞抗原调变

目的：研究了OKT3所致的体内T细胞抗原调变作用。方法：采用流式细胞术免疫荧光三色分析法,动态监测了19例肾移植术后应用OKT3治疗的患者的T细胞及其亚群的表达率和CD3抗原荧光强度的变化。结果：当注射OKT3后,CD3^ 、CD3^ CD4^ 、CD3^ CD8^ 细胞百分率明显下降甚至消失,相对地出现CD3^-CD4^ 和CD3^-CD8^ 细胞;同时,CD3^ 细胞的荧光强度也较注射前下降了86％;CD3^ CD4^ /CD3^ CD8^ 比值以及CD3^-CD4^ /CD3^-CD8^ 比值波动很大,甚至表现为明显的倒置。当停用OKT3后,CD3^ 、CD3^CD4^ 和CD3^ CD8^ 细胞逐渐回升,而CD3^-CD4^ 和CD3^-CD8^ 细胞开始下降直至消失;同时CD3^ 细胞的荧光强度逐渐恢复正常;CD3^ CD4^ ／CD3^ CD8^ 比值也很快恢复正常,但CD3^-CD4^ 和CD3^-CD8^ 比值倒置更明显,直至为0。结论：OKT3所致的T细胞抗原调变会出现CD3^-CD4^＋和CD3^-CD8^ 细胞,提示使用CD4／CD8比值监测移植排斥反应有一定的局限性。     

Correction of the abnormal ratio of OKT4+ to OKT8+ peripheral blood lymphocytes in patients suffering from juvenile arthritis (JCA) by the extract from calf thymuses

Children suffering from JCA showed an elevated percentage of OKT4+ T lymphocytes and an increased ratio of OKT4+ to OKT8+ lymphocytes. Several children showing a significant aggravation of the disease, were selected for a 3-week treatment with TFX. In 7 out of 10 patients a pronounced clinical and laboratory improvement was registered, accompanied by a normalization of the OKT4+ to OKT8+ lymphocyte ratio.     

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.     

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.     

Administration of prednisolone in vivo affects the ratio of OKT4/OKT8 and the LDH-isoenzyme pattern of human T lymphocytes

Oral administration of prednisolone (in single doses of 10, 30, or 60 mg) to healthy volunteers was found to affect the T lymphocytes in the blood with regard to binding of monoclonal antibodies and lactate dehydrogenase isoenzyme pattern. The findings indicate that these effects are dependent on the dose of the drug and the time after the administration of the drug. Prednisolone induces a T lymphocytopenia in the peripheral blood that affects OKT4-positive lymphocytes more than OKT8-positive lymphocytes, resulting in a slight decrease in the ratio OKT4/OKT8. Moreover, the lactate dehydrogenase isoenzyme pattern changes, resulting in a decrease of the H/M ratio of this enzyme. The proliferative responses of peripheral blood lymphocytes are not affected after a single dose of 10 mg. However, after administration of either 30 or 60 mg of prednisolone, the proliferative responses are decreased to a different extent, depending on the stimulus used. In vitro experiments are presented showing that any effect of prednisolone on nonstimulated lymphocytes is reversible. Based on the observed changes in OKT pattern and lactate dehydrogenase isoenzyme profile of the T lymphocytes induced by administration of prednisolone, it is concluded that the drug induces a temporary depletion from the peripheral blood, preferentially of high-reactive T lymphocytes. As a consequence, the peripheral blood compartment is enriched for T lymphocytes with a low H/M ratio of lactate dehydrogenase isoenzymes, known to be less reactive to proliferative stimuli.     

Dog erythrocyte rosette-forming lymphocyte: blockage by OKT11 monoclonal antibody

Human peripheral blood mononuclear cells (PBM) were separated into sheep erythrocyte rosette-forming (Es+) and non Es+ cells by the Ficoll-Hypaque gradient sedimentation method. Thirty-eight percent of the Es+ cells formed rosettes with dog erythrocytes and were designated as Es+Ed+ cells. The remaining Es+ cells were designated as Es+Ed- cells. Only a few non Es+ cells formed rosettes with dog erythrocytes. Among Es+Ed+ cells, T4 antigen-positive cells were observed approximately 1.7 times as often as T8 antigen-positive cells, when measured by staining with OKT4 or OKT8 monoclonal antibody. Among Es+Ed- cells, however, T4 and T8 antigen-positive cells were observed in almost equal proportion. Preincubation of PBM with OKT11 monoclonal antibody, but not with OKT4 monoclonal antibody, inhibited the rosette formation with dog as well as sheep erythrocytes. These results indicated that Es+Ed+ cells were a subpopulation of T-cells in which a majority of the cells were T4 antigen-positive, and that the binding sites of dog erythrocytes on human T-cells was closely linked with that of sheep erythrocytes.     

B cell activation by pokeweed mitogen in cultures of normal peripheral blood lymphocytes depleted of T regulator subsets by treatment with OKT4 and OKT8 monoclonal antibodies.

OKT4+ (T helper/inducer) and OKT8+ (T cytotoxic/suppressor) subsets were depleted from peripheral blood lymphocytes (PBL) by complement-mediated lysis and residual cells examined for responsiveness to pokeweed mitogen (PWM) using a protein A haemolytic plaque assay for immunoglobulin secreting B cells. It was shown that: (1) three cycles of cell killing were required to totally abolish T helper function; (2) OKT4- PBL did not respond to PWM, but in a co-culture system, an equal number of unfractionated normal PBL could entirely reconstitute responsiveness of the residual B cells; (3) OKT8- PBL gave enhanced numbers of PWM-induced plaque forming cells (PFC); (4) addition of 4 micrograms/ml concanavalin A (con A) to PWM stimulated OKT8- PBL failed to suppress PFC generation, but suppression was induced by 12.5 and 25 micrograms/ml con A and (5) kinetics of PWM-induced PFC development were similar in the presence or absence of OKT8+ cells.     

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.     

Modulation of peripheral blood T-lymphocytes in kidney transplant recipients treated with low dose OKT3 therapy

The immunosuppressive effect of OKT3 depends upon both T cell depletion and antigenic modulation of CD3 complex. To establish the effect of low doses of OKT3 on peripheral T lymphocytes, we analyzed 47 kidney transplant recipients receiving OKT3 for the first time. OKT3 was used as rescue therapy in 39 patients and as part of induction protocols in 8. The mean age of patients was 39+/-10 years, 30 were females and 9 were re-transplants. Half of them (51.1%) received kidney from cadaver donors. Among those receiving OKT3 as rescue therapy, 82% recovered graft function, including patients with severe BANFF-graded rejections. After the first dose of OKT3, it a pronounced T cell depletion was observed followed by an increase in CD4 and CD8 expression in CD3 negative T cells, supporting the idea that T cell modulation was present. In conclusion, low dose OKT3 was effective in treating severe allograft rejection by inducing a sustained TCR/CD3 down modulation without long-lasting T cell depletion.     

OKT4+ T cell abnormality in patients with active systemic lupus erythematosus: HLA-DR antigen expressions

The objective of this study was to define immunologic T cell abnormality characteristic of active systemic lupus erythematosus (SLE). Eight of nine patients who had severe clinical and laboratory manifestations of active SLE had a characteristically marked increase in OKT4+ and a decrease in OKT8+ T cells. Using OKIa1 and OKDR monoclonal antibody, we found that, in circulating blood of all patients with active SLE, an increased percentage of Ia+ and DR+ T cells is present compared to inactive SLE. Five of these active SLE patients had Tac+ antigens, an interleukin 2 receptor on OKT4+ and OKT8+ T cell subsets in resting blood. The present study demonstrates that Ia+ and DR+ antigens are selectively expressed on the majority of OKT4+ T cell subsets of all patients with active SLE, whereas Ia+ and DR+ antigens are expressed almost equally on both OKT4+ and OKT8+ T cell subsets in inactive SLE. The elevated percentage of Ia+, DR+, OKT4+ T cells in active SLE was accompanied by a highly depressed proliferative response to T cell mitogens, phytohemagglutinin and concanavalin A. However, OKT8+ T cell subsets in active SLE possessed a normal proliferative response to these T cell mitogens. We conclude that this abnormality of activated OKT4+ T cells bearing HLA-DR antigens may play a role in the development of active SLE.     

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.     

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.     

Familial OKT4 epitope deficiency: studies on antigen density and lymphocyte function

The family of a case of hereditary deficiency of OKT4 epitope on helper T cells was investigated. A 30-year-old woman was found to have hardly any OKT4+ T cells (0.7%, normal 24-51%), but normal OKT3, OKT8, OKT11, OKIa1, Leu1, Leu2a, Leu3a, Leu7, and B1 positive cells. The response to mitogens (PHA, PWM, and Con A) and helper-or suppressor-T-cell functions of her peripheral lymphocytes were normal. She had normal helper-T-cell populations detected with OKT4A (39.1%) and Leu3a (39.5%) monoclonal antibodies. One of her sisters had the same defect, but other members of the family had normal lymphocyte subsets. Lymphocyte functions were also found to be normal in all family members examined. The peak position of the fluorescence intensity of OKT4+ cells was about half that of normal controls in five members of this family. Considering that these were carriers of OKT4 epitope deficiency, the OKT4 epitope abnormality was inherited as an autosomal codominant trait in this family. Similar OKT4 epitope deficiency with normal Leu3a+ and OKT4A+ cells was found in 38 of 8866 (0.43%) other subjects in Japan by the examination of routine blood samples.