Functional analysis of human T cell subsets defined by monoclonal antibodies. V. Suppressor cells within the activated OKT4+ population belong to a distinct subset

In the present report, we characterize a monoclonal antibody directed at a surface differentiation antigen on human T cells. The monoclonal antibody, OKT17, recognizes a cell surface antigen present on the majority of resting normal peripheral T cells. In contrast, OKT17 is unreactive with normal B cells, B cell lines, T cell lines, or SIg+ CLL. Interestingly, after activation, the antigen recognized by OKT17 is lost from a subset of OKT4+ cells. We took advantage of this finding to explore further the functional heterogeneity within activated OKT4+ cells. Evidence was obtained that the PWM-activated OKT4+ subset remaining after depletion of OKT17-reactive T cells (OKT4+ 17-) contains radiosensitive helperr cells but is devoid of suppressor cells. In contrast, the activated OKT4+ 17+ population contains potent radiosensitive suppressor cells as well as radioresistant helpe cells. Taken together, these studies suggest that the OKT17 monoclonal antibody can differentiate two functionally mature, activated OKT4+ human T cells: OKT4+ OKT17+ radiosensitive suppressor cells and OKT4+ 17- radiosensitive helper cells.     

Functional analysis of human T cell subsets defined by monoclonal antibodies. III. Regulation of helper factor production by T cell subsets

In the present report we extended our previous studies demonstrating that obligatory T-T interactions are important in regulating human immune responses in vitro. Functionally distinct human T cell subsets were isolated by complement-mediated lysis using the monoclonal antibodies OKT4 and OKT8. Evidence was obtained that during allogeneic interactions, OKT4+, but not OKT8+, responder T cells are required to generate helper factor(s) capable of polyclonally activating human B cells independent of additional T cell help. Importantly, the alloantigen-induced helper factor(s) production and/or release was found to be suppressed by addition of graded numbers of radiosensitive OKT8+ cells. On the other hand, no evidence was obtained that supernatant derived from alloactivated OKT8+ cells could counterbalance the helper activity generated in the presence of supernatant from alloactivated OKT4+ cells. Furthermore, OKT8+ cells, known to suppress PWM-driven B cell differentiation in the presence of OKT4+ cells, do not suppress B cell differentiation induced by preformed helper factor even in the presence of OKT4+ cells. These data further underscore the importance of functional T-T interactions in immunoregulation in vitro and support the idea that the target of suppression of B cell differentiation, induced either by alloantigen-triggered helper factor or PWM, are OKT4+ cells and not B cells themselves.     

A defect in the suppressor circuits among OKT4+ cell populations in patients with systemic lupus erythematosus occurs independently of a defect in the OKT8+ suppressor T cell function

The autologous mixed lymphocyte reaction (MLR) is thought to be part of a regulatory role of T cells on B cell function. OKT4+, but not OKT8+, cells can proliferate in response to autologous non-T cells. Moreover, the OKT4+ cell population activated early in the course of autologous MLR functioned as inducer cells for the differentiation of B cells, whereas later in the response, the activated OKT4+ cells were particularly enriched in suppressor cells. A part of the autologous MLR appears to be an important pathway for the activation of feedback suppression mechanisms among cells contained within the OKT4+ populations. Patients with systemic lupus erythematosus (SLE) were studied with regard to the following OKT4+ cell functions in vitro after activation in the autologous MLR: a) proliferative response, and b) helper and suppressor activities for differentiation of B cells. A marked reduction in the proliferative response of OKT4+ cells was observed in SLE patients. SLE OKT4+ cells activated in the autologous MLR could function as helper cells but could not exert any suppressor activity. This OKT4+ cell abnormality was present regardless of the disease activity, and occurred in the absence of autoantibodies including anti-T cell antibodies. Instead, SLE anti-T cell antibodies could preferentially eliminate cells bearing the OKT8+ phenotype characteristic of suppressor cells in populations of normal T cells. These results suggest that the defect in the suppressor circuits among OKT4+ cell populations is intrinsic to SLE lymphocytes and that the OKT8+ suppressor T cell defect is caused by antibodies produced by the B cells of SLE patients.     

Delineation of suppressor and helper activity within the OKT4-defined T lymphocyte subset in human newborns

Lymphocytes taken from the cord blood of newborns have active suppressor activity. Using in vitro PWM-stimulated cocultures, unfractionated T cells from newborns potently suppressed the expected immunoglobulin G (IgG) synthesis of their mothers' peripheral blood lymphocytes (PBL). Using positive and negative selection techniques, we characterized the active suppressor cell as expressing the OKT4+T8- phenotype. This cord blood lymphocyte subset suppressed maternal IgG synthesis after depletion of maternal suppressor cells, implicating the ability of newborn T cells to suppress directly rather than by inducing adult suppressor activity. Sublethal amounts (1500 rad) of gamma-irradiation fully abrogated the suppressor activity of cord blood T lymphocytes. Radioresistant cord T cells provided T cell help. Irradiation of cord OKT4+ and OKT8+ populations and their subsequent culture with maternal B cells determined that helper activity was a radioresistant subpopulation of the OKT4+ subset. These results indicate significant differences in the functional properties of T cell subsets from adults and newborns. Population studies determined that cord blood lymphocytes had a greater proportion of OKT4+ cells and lower proportion of OKT8+ cells than PBL from unrelated adults. The mothers tested had similar proportions of OKT4+ cells as their babies, and these levels are significantly higher than those of unrelated adults.     

Identification of the T cell subset that produces human gamma interferon

Positive and negative selection procedures combined with cytofluorographic analysis and lysis with monoclonal antibodies were utilized to identify the T lymphocyte subset that produces human gamma interferon (gamma-IFN) (formerly referred to as "immune" or "type II" interferon) in response to mitogen stimulation. Lymphocytes were separated on the basis of their Fc receptors for IgG or IgM, their nonreactivity with IgM or IgG antibodies, and their reactivity with the monoclonal antibodies OKT4, OKT8, OKT11a, and OKM1. Isolated T cell subsets were incubated with the gamma-IFN inducer, phytohemagglutinin. Three days after induction, the cell supernatants were harvested and assayed for interferon. The T cell subset that produces gamma-IFN was identified as E rosette positive with the phenotype: T gamma, T non-micro, OKM1+, OKT4-, OKT8- and OKT11a+. gamma-IFN production by cells was resistant to doses of x-irradiation that abrogate mitogen-induced T suppressor function but was highly sensitive to low doses of 4-hydroperoxycyclophosphamide. These data demonstrate that gamma-IFN is produced by the T gamma, OKM1+ lymphocyte subset, but these cells may also require the presence of accessory monocytes for elaboration of gamma-IFN. The anti-proliferative activity of gamma-IFN may be responsible for the previously described suppressor function of this subset, and gamma-IFN production by T gamma cells may distinguish this subset from the suppressor/cytotoxic functions of the OKT8+ subset or the mitogen-induced OKT4+ suppressor.     

A monoclonal antibody reactive with the human cytotoxic/suppressor T cell subset previously defined by a heteroantiserum termed TH2

A hybridoma-secreting monoclonal antibody was produced from the spleen cells of a mouse immunized with human thymocytes. This hybridoma antibody, termed OKT5, was reactive by indirect immunofluorescence with 80% of human thymocytes but only 20% of peripheral blood T cells. Moreover, OKT5 was unreactive with normal B cells, null cells, and macrophages at any dilution tested. A similar pattern of reactivity was seen with an equine antiserum to human thymocytes termed anti-TH2. Fluorescence-activated cell sorting demonstrated that the OKT5 antibody reactivity on peripheral T cells was restricted to the majority of the previously defined TH2+ subpopulation. In functional studies, the OKT5+ subset, like the TH2+ subset, proliferated well to the mitogen Con A and to alloantigens, and contained cytotoxic effector cells after sensitization in MLC, and suppressor effector cells after activation with Con A. In addition, like the TH2+ T cell, the OKT+ T cell was virtually unresponsive to soluble antigen. Thus, the OKT5 monoclonal antibody is reactive with the cytotoxic/suppressor T cell subset. OKT5 should provide an important probe to assess the status of suppressor cells in human disease.     

Two phenotypically distinct suppressor T cell subpopulations inhibit the induction of B cell differentiation by phytohemagglutinin

Human B lymphocytes can be induced to differentiate into antibody-secreting plasma cells by Leu-3+ T lymphocytes stimulated with pokeweed mitogen (PWM), a polyclonal T cell activator. In contrast, other polyclonal T cell mitogens, such as phytohemagglutinin (PHA), also activate Leu-3+ T cells but are relatively ineffective inducers of B cell differentiation. We have performed a series of experiments to investigate the mechanism underlying this apparent paradox. When human B cells were cultured with unfractionated T cells and PWM or PHA, only PWM was able to induce plasma cell formation and immunoglobulin (Ig) secretion. However, when the T cells were treated with mitomycin C (MMC) before culture, both PWM and PHA were able to induce significant B cell differentiation. These data indicated that both mitogens were able to activate the helper T cells required for B lymphocyte differentiation and suggested that MMC-sensitive suppressor T cells were responsible for inhibiting the induction of antibody-secreting cells by MMC-untreated T cells stimulated with PHA. Phenotypic analysis of the T cells capable of suppressing PHA-induced B cell differentiation revealed that small numbers of either Leu-2+ or Leu-3+ T cells could profoundly suppress the B cell differentiation induced by PHA. In contrast, significant suppression of PWM-stimulated B cell differentiation was observed only with relatively large numbers of Leu-2+ T cells. These data confirm previous reports that OKT4+/Leu-3+ T cells can suppress human B cell differentiation and indicate that the difference in B cell differentiation induced by PWM and PHA with MMC-untreated T cells is largely a reflection of the relative potency of these mitogens to activate these phenotypically distinct suppressor T cell subpopulations.     

Lymphocyte subpopulations in the neonate: identification of an immature subset of OKT8-positive, OKT3-negative cells

T cell subpopulations of lymphocytes from cord blood (CBL) of 24 newborns and from peripheral blood (a-PBL) of 24 healthy adult volunteers were assessed in T cell-enriched, T cell depleted and unseparated lymphocyte fractions by using OKT3, 4, 6, and 8 monoclonal antibodies. The results show that T cell-enriched CBL include adult numbers of OKT3+, OKT4+, OKT6+ and OKT8+ lymphocytes whereas the T cell-depleted fraction consists of a high percentage of OKT8+, OKT3-, non-E rosette-forming cells bearing a PNA receptor. The presence of the PNA receptor and the lack of the OKT3+ antigen strongly support the hypothesis that the subset of OKT8+ cells in cord blood includes immature T lymphocytes that may represent an intermediate stage between thymocytes and mature peripheral T cells.     

Effector activity of OKT4+ and OKT8+ T-cell subsets in lectin-dependent cell-mediated cytotoxicity against adherent HEp-2 cells

The role of OKT4+ and OKT8+ T-cell subsets was studied in lectin-dependent cell-mediated cytotoxicity (LDCC) against adherent HEp-2 human epipharynx carcinoma target cells. LDCC was evaluated by detachment from the monolayer of [3H]thymidine prelabeled HEp-2 cells in a 24-hr assay with a concanavalin A (Con A) dose of 25 microgram/ml at effector:target cell ratios of 5:1, 25:1, and 50:1. Under these conditions but without Con A considerable natural cell-mediated cytotoxicity (NCMC) was not elicited; however, the cytotoxicity was significantly augmented in the presence of Con A (=LDCC) by sheep erythrocyte rosette-forming T lymphocytes and by both OKT4+ and OKT8+ T-cell fractions. LDCC activity by isolated OKT8+ T cells was superior to that by OKT4+ T cells and unfractionated T lymphocytes. By contrast, addition of either OKT4+ or OKT8+ T cells together with unfractionated T lymphocytes, or OKT4+ and OKT8+ T cells mixed at ratios of 1:1, 1:2, and 2:1, to target cells did not result in major differences in comparison of LDCC activities by these mixed effector cell populations with each other or with that by unfractionated T lymphocytes. Parallel studies were carried out to determine the effect of OKT4+ and OKT8+ T-cell subsets on the Con A-induced proliferation of peripheral blood mononuclear cells (PBMC). While OKT8+ T cells inhibited the mitogenic response to Con A, OKT4+ T lymphocytes had no major effect. A higher responsiveness of the OKT8+ to OKT4+ T-cell subset in LDCC to HEp-2 targets and in Con A-induced lymphocyte proliferation is suggested.     

TPA, tumor promoter-induced suppression of immunoglobulin secretion in human blood lymphocytes

12-O-Tetradecanoylphorbol-13-acetate (TPA) modulates DNA synthesis and differentiation of normal and malignant human lymphoid cells. Using the reverse plaque forming assay and radioimmunoassay, we showed that nontoxic concentrations of TPA (5 to 10 ng/ml) inhibited Ig secretion of peripheral blood lymphocytes. This inhibition was dependent on T lymphocytes and not monocytes; TPA treatment of the B cell-enriched fraction slightly enhanced Ig secretion. Suppression was evident when the proportion of TPA-pretreated T lymphocytes exceeded 50%. TPA-induced suppressor cells were present in both OKT8+ (suppressor/cytotoxic) and OKT4+ ("helper/inducer") subpopulations. The suppression was diminished but not abolished by the irradiation of T lymphocytes. In addition, TPA treatment modulated the expression of OKT4 antigen, whereas the expression of OKT8, 9.6 (sheep erythrocyte receptors) and surface Ig remained unchanged. Modulation of OKT4 was energy dependent and was not blocked by a maximal saturation of TPA receptors at 4 degrees C. We postulate that TPA-induced suppression of Ig secretion is T cell dependent and is likely to be associated with proliferation and activation of OKT8+ and OKT4+ lymphocytes and the induction of OKT4+ suppressor cells.     

Cellular origin and interactions involved in gamma-interferon production induced by OKt3 monoclonal antibody

OKT3 monoclonal antibody, a human T cell mitogen, induced interferon production by cultured mononuclear cells at 10(-11) M concentrations. Interferon was secreted only under conditions wherein OKT3 was mitogenic, and production was correlated with cell proliferation. Thus, like mitogenesis, interferon secretion reached a peak 3 days after OKT3 stimulation, was inhibited by a factor(s) in human serum, and required 1000 times higher concentrations of Fab and F(ab')2 fragments of OKT3 for induction. The interferon was most likely of "gamma" (immune) type, because pH 2 and 56 degrees C treatments denatured it, whereas anti-alpha or -beta interferon antibodies did not. Mononuclear cells were fractionated into subpopulations that contained OKT4+ cells (helper/inducer T cells), OKT8+ cells (cytotoxic/suppressor T cells), and OKM1+ cells (monocytes) by combining sheep red blood cell rosetting and complement-mediated lysis using monoclonal antibodies against specific cell types. Both OKT4+ and OKT8+ cells proliferated upon OKT3 stimulation with the absolute requirement of OKM1+ cells. However, OKT4+ cells plus OKM1+ cells were necessary for the secretion of interferon. Studies with selective pretreatments with mitomycin C suggested that gamma-interferon was secreted by the OKT4+ cells and that the OKM1+ population subserved an accessory function.     

Human autologous rosette-forming cells. I. Expression of cell surface antigens in relation to age and lymphoid organ distribution

Autologous rosette-forming cells (auto-RFC) were characterized with monoclonal antibodies to various cell surface antigens using a technique combining immunofluorescence and rosette formation. In peripheral blood, auto-RFC were T cells (Leu 1+/OKT3+) the majority being derived from the helper/inducer subset (Leu 3a+/OKT4+). A small proportion of the circulating auto-RFC were Leu 2a+/OKT8+ and virtually none of them bore T10, T6, and DR antigens or peanut agglutinin (PNA) receptors. In the elderly, the percentages of Leu 3a+ auto-RFC increased significantly along with the augmentation of the Leu 3a+ circulating pool. After Con A stimulation of peripheral blood lymphocytes the autorosette population was expanded and therefore their phenotype was again that of helper cells. In the thymus, high levels of autorosettes are found (30 to 50%). Simple or double labeling of the rosetting cells with various monoclonal antibodies permitted the confirmation of the existence of distinct thymocyte subpopulations and moreover to identify the location of the auto-RFC in the intrathymic differentiation scheme. Nearly 70% of the rosetting cells were derived from common thymocytes, those cells defined by the coexpression of T10, T6, T4, and T8 antigens whether or not they were also stained by OKT3 antibodies. The remaining auto-RFC were found with similar frequency among the T4+ and T8+ mature thymocytes. In the spleen low percentages of auto-RFC were found and the majority resided in the Leu 3a+/OKT4+ population, similarly to peripheral blood autorosettes. Taken together, these data suggest that the expression of autologous erythrocyte receptors is acquired in the thymus and is gradually lost during T-cell maturation.     

Agar human T cell colony growth promoted by a B + null cell-derived lymphokine distinct from IL 2

Human T cell agar colonies can be grown under PHA stimulation from either mature T cells or their E rosette-negative (E-), OKT3- peripheral blood and bone marrow precursors. Colonies comprise a majority of mature E+, OKT3+ cells and a minor (5 to 10%) population of immature E-, T3-, T8-, T4-, DR+, T10+, RFB1+ cells, which upon replating in subculture, can generate secondary colonies of OKT3+, E+, OKT4+, OKT8+ cells. Secondary colony formation can serve as a test for growth requirement of colony precursors, because it depends on the presence of both PHA and a colony-promoting activity (CPA) recovered in PHA-stimulated B + null or T + adherent cell supernatants. CPA production by B + null cells was not affected by their treatment with OKT3 or D66 (T11-like) monoclonal antibodies (MAB) + complement but was abolished by an anti-HLA-DR MAB + complement. However, B cells sorted by panning with the same anti-HLA-DR MAB did not release CPA, demonstrating the requirement of both B cells and null cells for CPA production. Neither IL 2 nor IL 1 could account for B + null cell-derived CPA.     

Immune deficiency in the X-linked lymphoproliferative syndrome. II. Immunoregulatory T cell defects

Surface phenotypic markers and the function of lymphocytes in patients affected with the X-linked lymphoproliferative syndrome (XLP) were studied. This syndrome is characterized by a defective response to infection with Epstein Barr virus (EBV). Normal numbers of B and T cells were detected with anti-Ig and monoclonal OKT3 antisera, respectively. T cell subset values, however, were persistently altered: cells reacting with OKT8 were significantly elevated in five of nine patients, accompanied by a slight decrease in the percentage of OKT4-positive cells, leading to abnormally low OKT4 to OKT8 ratios. One patient had a high OKT4 to OKT8 ratio due to low number of OKT8-positive cells. Lymphocytes from patients showed normal proliferation after stimulation with T and B cell mitogens. In contrast, Ig synthesis by lymphocytes after stimulation with B cell mitogens was markedly deficient: low or undetectable levels of one or all classes of Ig were detected, whereas cell lines established from EBV-infected B lymphocytes from patients produced normal quantities of Ig. These studies imply immune regulatory impairments in the patient with XLP.     

In vitro maturation of B cells in chronic lymphocytic leukemia. I. Synergistic action of phorbol ester and interleukin 2 in the induction of Tac antigen expression and interleukin 2 responsiveness in leukemic B cells

Recent evidence indicates that interleukin 2 (IL 2), formerly thought to serve as growth factor exclusively for activated T cells, is directly involved in human B cell differentiation. We have investigated the role of IL 2 and IL 2 receptors (as defined by monoclonal anti-Tac antibody) in the phorbol ester-induced in vitro maturation of leukemic B cells from patients with chronic lymphocytic leukemia (CLL). Peripheral blood lymphocytes from B cells from CLL patients with high (greater than 10(5)/microliters) white blood cell counts were depleted of residual T lymphocytes and low-density cells (primarily macrophages) by consecutive steps of E rosetting, complement-mediated lysis of OKT3+ and OKT4+ cells, and Percoll density gradient centrifugation. No OKT3+ T cells were detectable in these cell populations before or after culture. When incubated for 3 days with phorbol ester plus recombinant human IL 2 (rIL 2), 12 to 57% of highly purified B cells from four of five tested patients expressed Tac antigen. Both phorbol ester and rIL 2 were required for maximal Tac antigen expression. Functional studies revealed that phorbol ester-activated (but not resting) CLL B cells responded to rIL 2 with [3H]thymidine incorporation and with enhanced secretion of IgM. Tac+ B cells were isolated in two cases on a fluorescence-activated cell sorter. In one patient, stimulation of Tac+ B cells with rIL 2 resulted in enhanced [3H]thymidine incorporation but no change in IgM secretion, as compared with Tac- B cells; in the second patient, stimulation of Tac+ B cells with rIL 2 did not result in [3H]thymidine uptake, but did result in significant IgM secretion. These findings indicate that certain leukemic B lymphocytes can be induced to express IL 2 receptors and respond to IL 2. The use of resting clonal B cell populations arrested at distinct stages of differentiation may help to better define the stage(s) at which IL 2 acts directly on B cells to induce proliferation and/or terminal differentiation.     

Secretions of anti-myelin-associated glycoprotein antibodies by B cells from patients with neuropathy and nonmalignant monoclonal gammopathy

Four patients with peripheral neuropathy and nonmalignant monoclonal gammopathy with anti-myelin-associated glycoprotein (MAG) antibodies were studied to determine whether secretion of anti-MAG IgM antibodies by B cells was autonomous, or whether the monoclonal B cells were responsive to T cells. Secretion of anti-MAG IgM by isolated B cells was stimulated by the addition of increasing numbers of pokeweed mitogen (PWM)-activated autologous OKT4+ helper T cells in all four patients. Secretion of anti-MAG IgM by peripheral blood lymphocytes was dependent on the ratio of OKT4+ T helper cells to OKT8+ T suppressor/cytotoxic cells. In three patients with an OKT4+ to OKT8+ T-cell ratio of 2:1, PWM activation stimulated secretion of anti-MAG IgM; in one patient with an OKT4+ to OKT8+ ratio of 1:2, activation by PWM suppressed anti-MAG IgM secretion. These studies suggest that the monoclonal B cells that secrete anti-MAG IgM are responsive to regulatory T cells.     

Accessory cell-T cell interactions involved in anti-CD3-induced T4 and T8 cell proliferation: analysis with monoclonal antibodies

The effect of monoclonal antibodies (Mab) directed at T cell and accessory cell (AC) surface molecules on OKT3-induced T4 and T8 cell proliferation was examined. Mab directed at nonpolymorphic class I (W6/32, MB40.5) and class II (L243) major histocompatibility complex (MHC)-encoded gene products, an epitope common to LFA-1, CR3, and the p150, 95 molecule (60.3), and a heterodimer present on monocytes (M phi) and activated T cells (4F2) inhibited M phi-supported OKT3-induced proliferation of both T4 and T8 cells. Moreover, an Mab directed at the CD4 molecule (66.1) inhibited OKT3-induced T4 but not T8 cell proliferation, whereas an Mab directed at the CD8 molecule (OKT8) inhibited T8 but not T4 cell responses. With the exception of 66.1, each inhibited OKT3-induced T cell proliferation when added as late as 15 hr after the initiation of culture. Inhibition could not be explained by competition for Fc receptors on the AC. A variety of other Mab including OKT11 and those directed at other HLA-DR and DQ determinants were not inhibitory. The inhibitory Mab were found to diminish T4 cell IL 2 production and IL 2 receptor expression. Consequently, IL 2 reversed some but not all of the Mab-mediated inhibition of T cell proliferation. In contrast to the effects noted with M phi-supported responses, 60.3 and 66.1 but neither L243 nor 4F2 inhibited OKT3-induced T4 cell proliferation supported by Ia- or IFN-gamma-treated Ia+ endothelial cells. None of the Mab tested inhibited T cell proliferation induced by the AC-independent stimuli OKT3 and phorbol myristate acetate (PMA) or calcium ionophore and PMA in the presence or absence of added AC. The data therefore suggest that the Mab inhibit OKT3-induced activation of T4 and T8 cells by preventing necessary interactions between AC and T cell surface proteins. Moreover, the results suggest that different arrays of interaction molecules are involved in OKT3-induced T cell proliferation depending on the nature of the AC and the responding T cell subset.     

Activation, proliferation, and differentiation of circulating B cells in autoimmune thyroid disease

Several studies of thyroid autoantibody production in vitro have been reported with the use of pokeweed mitogen, but the conclusions that have resulted regarding the immunoregulation of B cell function in thyroid disease are difficult to interpret due to the relatively nonphysiologic nature of pokeweed mitogen stimulation. We have therefore examined the responses of circulating B cells in Graves' disease and Hashimoto's thyroiditis by using a combination of lymphokines and other stimuli that act at various stages of the B cell cycle. In patients with autoimmune thyroid disease, nonspecific B cell proliferation and differentiation into IgG-secreting cells were both normal. However, a previously unsuspected heterogeneity among patients was found in their ability to produce autoantibodies in vitro. B cells of certain patients produced maximal autoantibody in response to pokeweed mitogen, some in response to Staphylococcus aureus Cowan strain I, and some in response to the lymphokines contained in the supernatants of stimulated T cell cultures. There was no correlation between serum autoantibody levels and those achieved in vitro. Attempts to stimulate antibody production by autoantigen (thyroglobulin) were unsuccessful, even when B cells were cultured with purified autologous OKT4+ T cells to avoid potential suppressor effects in the OKT8+ population. However, OKT4+ T cells enhanced pokeweed mitogen-driven autoantibody production. Our results show that several different functional stages of B cells exist in the circulation of patients with autoimmune thyroid disease, and that circulating B cells from such patients do not manifest a uniform response to B cell stimulators. This is presumably the result of differences in migration of circulating B cells and in their level of activation at the major sites of autoantibody production, such as the thyroid gland itself. In the light of these findings, caution is required in interpreting the results obtained from studies of circulating B cells as a means of elucidating the pathophysiology of autoimmune thyroid disease.     

Effect of lipoxygenase metabolites of arachidonic acid on proliferation of human T cells and T cell subsets

The lipoxygenase products LTB4 and 15 HPETE have been reported to stimulate T suppressor cell function and also to inhibit [3H]thymidine incorporation into mitogen-stimulated T cells. This present report documents that although these compounds do indeed inhibit [3H]thymidine incorporation into unfractionated T cells, they significantly enhance [3H]thymidine incorporation into T cell preparation enriched for cells bearing the cytotoxic suppressor cell phenotype identified by the OKT8 monoclonal antibody. The mitogen response of T cells enriched for OKT4+ helper-inducer cells is inhibited in manner similar to the response of unfractionated T cells. Thus, LTB4 and 15 HPETE stimulate both the function and the proliferation of the cytotoxic-suppressor T cell subset.     

Changes in purine nucleoside phosphorylase activity during thymosin-induced human null cell differentiation

Purine nucleoside phosphorylase (PNP) is a purine salvage pathway enzyme which we have found to be 8-10 times more active (per cell) in human peripheral blood null lymphocytes than in T lymphocytes. To test the hypothesis that null cells are, in part, pre-T lymphocytes we have defined an in vitro system for null cell differentiation into T cells and examined PNP activity during this differentiation process. We found that about 10% of human null cells could be driven to differentiate into T cells using thymosin fraction 5 (TF5) an extract of bovine thymus glands. The response to TF5 was dose related to up to 250 micrograms/ml with a maximum response occurring by 42-46 hr incubation. Exposure to TF5 was necessary for more than 4 hr but no more than 8 hr in order to obtain a maximum response. Both OKT4 and OKT8 positive cells were present in the newly differentiated T cell population but OKT8 positive cells appeared to predominate (OKT4/OKT8 = 0.698 +/- 0.30, mean +/- 1 SD). The differentiation process did not involve DNA synthesis but was inhibited at 4 degrees C. In the newly differentiated T cells PNP activity per cell was 8- to 10-fold lower (36 +/- 23 nm/hr/106 cells) than in null cells (311 +/- 136), and was at a level similar to mature T cells (56 +/- 7). Thus, human peripheral blood null cells can be induced to differentiate into T lymphocytes which can be characterized by both surface markers and biochemical parameters. Future studies will look at the function of TF5-induced T cells and the regulation of PNP activity during the differentiation process.