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.     

Peripheral T Cell Lymphomas: An Immunological Study of Seven Unusual Cases

A multiparameter study of malignant lymph node cells and peripheral blood lymphocytes of seven patients with peripheral T cell lymphoma is presented. The results of monoclonal marker studies showed three cases of helper-suppressor T cell lymphoma (OKT4+, OKT8+), one case of suppressor T cell lymphoma (OKT8+), and three cases of helper T cell lymphoma (OKT4+). Immunophenotypic heterogeneity of neoplastic T cells with expression of pan-T antigens, OKT3+, and OKT11+ (erythrocyte rosetting+) was observed in most patients. Six of the seven cases tested showed Ia and DR antigens. No relationship was detected between patterns of reactivity with T cell reagents and histological types. When tested, the in-vitro malignant T cells of five patients proliferated in response to concanavalin A (Con A), but had poor response to phytohaemagglutinin. The interleukin 2 receptors showed maximum expression on Con A-activated T cells of five patients, and phytohaemagglutinin-activated T cells of one patient. The neoplastic T cells (OKT4+, OKT8+) of one patient studied had suppressor activity for IgG and IgA, and helper activity for IgM synthesis on pokeweed mitogen-induced normal B cell differentiations.     

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.     

Two color flow cytometry of lymphocytes from patients with adult T-cell leukemia

Lymphocytes from eight patients with adult T-cell leukemia were analyzed by two color flow cytometry. Monoclonal antibodies (Leu 3 a, Leu 8, Leu 2 a and Leu 15) labelled with fluorescein isothiocyanate or phycoerythrin were used. The purpose was to identify the subsets of the lymphocytes as helper, suppressor/inducer, suppressor or cytotoxic by the surface marker of the cells. All eight patients had antibodies for ATLA. Proviral DNA in the lymphocytes was found in six patients. Summarising the results, OKT4-positive ATL cells were all of the helper T-cell subset, not the inducer subset. OKT8-positive ATL cells were also positive for OKT4 and were all of the cytotoxic T cell subset, not the suppressor subset. In two patients, some ATL cells had both OKT4 and OKT8 on the same cells, especially in the lymph nodes. In our study, ATL cells from eight cases of ATL had all of the helper T subset. These results suggest that the target cells of the human T cell leukemia/lymphoma virus type will be helper T cells.     

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.     

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.     

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.     

Sézary's syndrome: a case with blood T-lymphocytes of helper phenotype, elevated IgE levels and circulating immune complexes

A patient with Sézary's syndrome is described. Surface marker analysis of her peripheral blood lymphocytes exhibit a phenotype characteristic of mature helper T cells (OKT3+, OKT4+, OKT8-, OKT6-). Serological studies revealed a polyclonal hyperimmunoglobulinemia with large amounts of IgE and IgA and circulating immune complexes that activate the complementary system via alternative pathway. The patient's cells showed a helper activity on normal B cell differentiation after a 7-day co-culture with pokeweed mitogen. The relevance of this helper phenotype and the function on in vivo polyclonal B cell activation is discussed.     

Monoclonal antibody analysis of T-lymphocyte subsets in young and aged adults

Eleven monoclonal antibodies identifying surface antigens present on human T lymphocytes were utilized to investigate the effects of advanced age on peripheral blood lymphocyte subsets. Both the proportion and number of lymphocytes recognized by five antibodies reactive with 'pan' T cell antigens (OKT3, OKT11, Leu4, T101 and Lyt3) decreased with age. The percentage of helper/inducer (OKT4+, Leu3a+) cells remained constant; however the proportion of Leu2a+, suppressor/cytotoxic cells declined. There was no change with age in the percent representation of OKT9+ or OKT10+ cells, nor in the ratio of helper/inducer to suppressor/cytotoxic cells (OKT4+/OKT8+ or Leu3a+/Leu2a+). Absolute numbers of helper/inducer (OKT4+, Leu3a+), suppressor/cytotoxic (OKT8+, Leu2a+), OKT9+ and OKT10+ cells were lower in elderly individuals as the result of lymphocytes constituting a lower percentage of the peripheral blood white cell population in this age group. While only small differences existed between the lymphocyte populations of young and aged men; aged women, compared to young women, had more dramatic shifts in their T cell populations. Comparison of antibodies putatively identifying similar (the same) functional groups of T cells demonstrated excellent correlation between the percentage of cells enumerated with the antibodies OKT3+: Leu4+ (r = .951), OKT4+: Leu3a+ (r = .914), OKT8+: Leu2a+ (r = .896), and in the ratio of helper/inducer to cytotoxic/suppressor OKT4+/OKT8+: Leu3a+/Leu2a+ (r = .926) cells.     

OKT4 epitope deficiency in significant proportions of the black population. A cause for underestimation of helper/suppressor lymphocyte ratios

Monoclonal antibodies such as OKT4, OKT4A, Coulter T4, and Leu 3a are commonly used to define subsets of human peripheral blood lymphocytes having helper activity in vitro. Analysis of peripheral blood lymphocyte populations is a useful aid in the diagnosis of immunodeficiency syndromes. Peripheral blood lymphocytes of certain black and oriental patients do not mark with the conventional OKT4 antibody but do mark with OKT4A and Leu 3a antibodies. We determined helper subset populations of peripheral blood lymphocytes as delineated by OKT4, OKT4A, and Coulter T4 antibodies in 103 unselected patients from two tertiary care hospitals. Twenty percent of black patients without clinical immunodeficiency demonstrated marked reduced numbers of cells marking with OKT4 antibody, but normal numbers of cells marking with OKT4A and Coulter T4 antibodies. No white patients demonstrated this trait to the degree seen in black patients. Use of OKT4 antibody to define helper cell percentages in black patients may lead to underestimation of these percentages in certain hospital populations.     

Peripheral blood CD4+CD8+ lymphocytes in cynomolgus monkeys are of resting memory T lineage

In this study, we analyzed peripheral blood CD4+CD8+ double-positive (DP) lymphocytes in adult cynomolgus monkeys (Macaca fascicularis). Forty of 55 monkeys had > 5% of the peripheral blood DP subpopulation (9.3 +/- 5.9%; mean +/- SD) in peripheral blood lymphocytes (PBL) in contrast to a low percentage of peripheral blood DP cells in humans and mice. In a cross-sectional study, the peripheral blood DP cells were found to increase in proportion with age. To clarify whether peripheral blood DP lymphocytes were immature precursors released from thymus without prior differentiation, the expressions of CD8 chains and CD1b on peripheral blood DP lymphocytes were compared with those on thymocytes. The peripheral blood DP lymphocytes were CD8 alpha + beta- and CD1b-, while thymic DP lymphocytes were CD8 alpha + beta + and CD1b +, suggesting that the peripheral blood DP cells are extrathymic T lymphocytes. Furthermore, the peripheral blood DP lymphocytes exhibited a resting memory T cell phenotype with CD2hiCD3+CD28-CD29hiCD49dhiCD69- CD80lo. Taken together, adult cynomolgus monkeys possess a unique peripheral blood DP T cell subpopulation which expresses a resting memory T cell phenotype. In addition, similar phenotypic properties of DP lymphocytes were distributed in the spleen and lymph nodes, although the proportion was less in the spleen and much less in lymph nodes than in PBL.      

A clinicopathological review of 12 autopsied cases of adult T-cell leukemia

Twelve autopsied cases with adult T-cell leukemia (ATL) were reviewed clinicopathologically. The prognosis of three cases who had suffered from severe cutaneous lesions was much better than that of the other nine cases with no or negligible cutaneous lesions. The surface marker of leukemic cells from six cases was ordinary inducer/helper phenotype (OKT4+ and 8-), but in one case leukemic cells showed OKT4+ and 8+. In another case, a significant amount of leukemic cell infiltration was found in the thymic cortex. Calcium content in the bone of ATL cases was lower than that of the patients without ATL (control group), and six cases with ATL (50%) were complicated by severe hypercalcemia. Neither adenoma nor hyperplasia of the parathyroid glands was found in any case. In most severely hypercalcemic patients, bone trabeculae were actively absorbed by numerous osteoclasts and partly replaced by fibrous tissues. In two normocalcemic patients, skeletal calcium content was also markedly reduced by osteoporosis, but the activation of osteoclasts was inconspicuous. It was speculated that the manner of bone resorption in ATL cases was diverse and there were some clinicopathological subtypes in ATL from the viewpoints of cutaneous lesions, hypercalcemia, and bone lesions.     

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.     

The effect of NPT 15392, 9-(erythro-2-hydroxy-3-nonyl)-6-hydroxypurine, on the phytohemagglutinin of OKT3+, OKT4+, OKT8+, and OKM1+ cell-depleted and undepleted peripheral blood mononuclear cells

NPT 15392, 9-(erythro-2-hydroxy-3-nonyl)-6-hydroxypurine, has been reported to influence immunological responses involving different cell types. Herein data are obtained by studying the influence of NPT 15392 on the phytohemagglutinin (PHA)-induced proliferative responses of unfractionated peripheral blood lymphocytes (PBLs) and of cell suspensions, which have been depleted of the cell subsets recognized by the monoclonal antibodies OKT3, OKT4, OKT8, and OKM1, in an attempt to identify which cell types respond to NPT 15392 in the PHA-driven blast transformation assay. The proliferative responses of unfractionated peripheral blood lymphocytes are potentiated when the drug is employed at the concentration of 0.1 microgram/ml and inhibited when NPT 15392 is added to the cell suspensions at concentrations over 5 micrograms/ml. The data reported here suggest that this phenomenon is a composite effect, made up of a combination of the counteracting effects caused by the OKT4+ cells on the one hand, and the OKT8+ and OKM1+ cells on the other.     

Absence of the OKT4 epitope on blood T cells and thymus cells in a patient with thymoma, hypogammaglobulinemia, and red blood cell aplasia

Human helper/inducer T-lymphocytes that express the T4 antigen are important in the regulation of B and T cell functions. Several epitopes of the T4 molecule have now been recognized; however, the precise role of these molecules in the function of helper/inducer T cells is unclear. We studied a patient with thymoma, hypogammaglobulinemia, and red blood cell aplasia whose blood lymphocytes and thymus cells did not express the epitope recognized by OKT4 monoclonal antibody but did display the T4 epitopes recognized by OKT4A and Leu3A monoclonal antibodies. The absence of the OKT4 epitope on the patient's thymus cells suggested that the abnormality occurred during early T cell differentiation. The patient had intact delayed hypersensitivity to 4/4 antigens, and his blood lymphocytes proliferated normally to phytohemagglutinin, concanavalin A, pokeweed mitogen, tetanus toxoid, and allogeneic cells. The patient's T cells demonstrated augmented suppressor activity that was localized to the OKT8+ population rather than to the unusual T4 subset. Irradiation abrogated suppressor activity and rendered his T cells capable of providing help for polyclonal B cell differentiation. The data emphasize the limitations of OKT4 as the sole reagent for characterizing the subset of human helper/inducer cells and demonstrate that the expression of the T4 epitope recognized by OKT4 monoclonal antibody is not required for certain helper/inducer T cell functions in vitro and in vivo.     

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.     

A monoclonal antibody and functional study of malignant T cells of a patient with suppressor-T-cell lymphoma

We describe the immunologic characteristics of malignant T cells of a T-cell lymphoma patient. The neoplastic cells in lymph node expressed OKT3+, OKT11-, E-rosetting-, OKT4-, OKT8+, OKIa1+, OKDR+ suppressor-T-cell phenotype. Functionally, these malignant T cells did not respond to phytohemagglutinin, but produced a good response to concanavalin A. A defect in expression of interleukin 2 receptors was evident in phytohemagglutinin-activated T cells. In vitro immunoglobulin production experiments demonstrated that patient's malignant T cells possess helper function on normal B cells to produce IgM, and suppressor function to produce IgG.     

Unique Immunological Behaviour of CD8+ (Suppressor T Cell) Leukaemia Cells

A patient with chronic lymphocytic leukaemia and elevated serum IgM antibody is described. A multiparameter surface marker analysis of his peripheral blood lymphocytes demonstrated a unique phenotype of OKT3-, OKT11+, E-rosetting+, OKT4-, OKT8+, OKIa1+, OKCLL+, OKDR+, Tac+, characterizing the disease as suppressor (CD8+) T cell chronic lymphocytic leukaemia. Because of the uncommon phenotype and the abnormal serum immunoglobulin pattern, in vitro functional assays were performed that showed decreased mitogenic response to the phytohaemagglutinin, but increased response to concanavalin A. However, these leukaemic T cells demonstrated phytohaemagglutinin-specific suppressor cell activity on normal blood lymphocytes. In vitro functional studies indicated that a defect in the patient's T cells may cause IgM hypergammaglobulinaemia. The regulatory function of the patient's T cells on immunoglobulin synthesis by normal B cells was found to be mediated by a soluble factor secreted from neoplastic T cells.     

Identification of lymphocyte subsets in peripheral blood and thyroid gland in Graves' disease by alpha-naphthyl-acetate-esterase reaction

T lymphocyte subsets were prospectively examined in the peripheral blood and thyroid aspirates of 10 patients with hyperthyroid Graves' disease before and after treatment with methimazole and attainment of euthyroidism. T lymphocyte subsets were identified with monoclonal antibodies and pattern of alpha-naphthyl-acetate esterase (ANAE) staining pattern in the case of peripheral blood and ANAE staining pattern with thyroid aspirate smears. Before treatment, OKT8+ lymphocytes were significantly decreased (18.4% +/- 4.8) (S. D.) in the patients compared to control (28.8 +/- 6.7%, p less than 0.05), the OKT4/OKT8 ratio was increased (2.92 vs 2.11). Percent OKT8+ lymphocytes were not different from the controls when the ten patients had been rendered euthyroid. ANAE mononuclear cells with a diffuse pattern (presumed suppressor cells) were 4.2% +/- 1.8 before treatment and 8.3 +/- 2.4 (p less than 0.05) after treatment and 11.5% +/- 2.2 in controls. ANAE mononuclear cells with diffuse pattern represented 4.2% +/- 1.8 of the mononuclear cells infiltrating the thyroid gland of untreated patients and rose to 8.3% +/- 2.4 after the patients had become euthyroid. ANAE negative cells (B cells and some T cells) were increased in the thyroid of untreated patients. It is concluded that mononuclear cells with presumed suppressor T cell phenotype are decreased in the blood and thyroid glands of patients with active Graves' disease and that this defect is corrected when euthyroidism has been established.     

Use of orthoclone monoclonal antibodies in the study of selected dermatologic conditions

Monoclonal antibodies recognizing human T cell differentiation antigens were used to study lymphocyte populations in three cutaneous diseases.Neoplastic lymphocytes from patients with varying phases of cutaneous T cell lymphoma (mycosis fungoides, Sezary syndrome and related presentations) were reactive with OKT1 and OKT3 (pan T cell reagents) and OKT4 (an antibody defining the functional “helper” T cell subset). The malignant cells lacked membrane antigens reactive with OKT5 and OKT8 (markers of “suppressor” T cells). The presence of an OKT1⁺, OKT3⁺, OKT4⁺, OKT5⁻, OKT8⁻ phenotype on the neoplastic T lymphocytes of cutaneous T cell lymphoma (CTCL) supports the clinical impression that all phases of CTCL represent a single disease entity.A patient with pemphigus vulgaris, a disease of autoreactive, antiepidermal antibodies was shown to consistently have a marked expansion of the peripheral blood OKT4 reactive T lymphocyte population. These findings suggest that autoantibodies in pemphigus vulgaris may occur in the context of a profound OKT4/OKT5 immunoregulatory imbalance.Peripheral blood lymphocytes from patients with extensive psoriasis vulgaris had a normal profile of reactivity with the OKT antibodies. In addition, OKT6 (marker of intrathymic T cells) has been shown to react with Ia⁺ dendritic cells in the epidermis suggesting that this antibody may recognize Langerhans' cells.