Induction of phenotypic differentiation, interleukin 2 production, and PHA responsiveness of "immature" human thymocytes by interleukin 1 and phorbol ester

Small human thymocytes (ST) representing 70% of the thymocytes were isolated according to size by centrifugal elutriation. Although these ST contained approximately 30% PNA-cells, they failed to respond to lectins, indicating the existence of a PNA-ST subset that can be considered to belong to the "immature" thymocyte population. The ST were induced to proliferate if, in addition to PHA, IL 1-containing supernatants of highly purified monocyte cultures or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were present. The incubation of the ST for 90 hr with TPA or IL 1 in the absence of PHA resulted in a strong reduction in the percentage of cells reacting with the immature thymocyte markers TdT and PNA. In addition, the OKT6+ cells were partially reduced after incubation with IL 1. Concomitantly, an increase in the percentage of cells reacting with the mature T cell markers OKT1 and OKT3 was observed, whereas HLA antigens became strongly expressed on all ST. Although IL 1 or TPA were unable to induce proliferation of the ST, these substances induced IL 2 production by these cells. These shifts to cells with more "mature" phenotypes that are able to produce IL 2 were not observed if the ST were incubated with PHA or culture medium only. The responder capacity of the ST to PHA plus TPA was not significantly affected by the depletion of the more "mature" OKT3+ and OKT1+ cells. In addition, in this situation OKT1+, OKT3+, OKT6- cells were found to be generated from OKT1-, OKT3-, OKT6+ cells. Therefore, it could be excluded that the proliferative responses were due to a selective expansion of a preexisting mature T cell population. Our results indicate that TPA mimics IL 1 in the induction of differentiation of the ST to a stage in which subpopulations of these cells are able to produce IL 2 and to respond to PHA. Because only the proliferating ST were found to react with a monoclonal antibody, which is thought to be directed at the IL 2 receptor (anti-Tac), our data suggest that PHA is required for the induction of expression of receptors for IL 2 in those ST subpopulations that are able to proliferate in the presence of IL 2 generated in situ.     

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.     

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.     

The recognition specificity of a murine helper T cell for hemagglutinin of influenza virus A/PR/8/34

Human large granular lymphocytes (LGL), which are known to be responsible for natural killer (NK) cell activity, also produced a variety of lymphokines including interleukin 2 (IL 2), colony stimulating factor (CSF), and interferon (IFN) in response to phytohemagglutinin (PHA) or concanavalin A (Con A). Human peripheral blood LGL, which were purified by removal of monocytes adhering to plastic flasks and nylon columns, followed by separation on a discontinuous Percoll gradient, and additional treatment with anti-OKT3 and Leu-M1 plus complement, were more potent producers of these lymphokines than unseparated mononuclear cells (MNC), nylon column-eluted cells, or purified T lymphocytes. Moreover, IL 2 production by LGL could be further distinguished in that it was not enhanced by the addition of macrophages or macrophage-derived factor, i.e., IL 1, whereas addition of macrophages did potentiate IL 2 production by T lymphocytes. Further analysis of cells in the LGL population using various monoclonal antibodies revealed that removal of cells with OKT11 or AF-10, a monoclonal antibody against human HLA-DR antigen, decreased IL 2 production, whereas removal of OKT8+, OKM1+, Leu-M1+, or Leu-7+ cells led to enhanced IL 2 production. The LGL population is therefore heterogeneous and includes at least three functionally and phenotypically distinct subsets. An atypical T cell subset (OKT3-, Leu-1-, OKT11+) rather than the myeloid subset of LGL (Leu-M1+ or OKMI+) was the source of LGL-derived IL 2, whereas the latter subset and/or another subset of OKT8+ cells appear to regulate this IL 2 production. In addition to performing NK activity, LGL on a per cell basis seem to be more effective than T lymphocytes in producing lymphokines, namely, IL2, CSF, and IFN.     

Cytokinetic basis for the impaired activation of lymphocytes from patients with primary intracranial tumors

Patients with malignant brain tumors have a variety of immunologic abnormalities, including the impaired responsiveness of peripheral blood lymphocytes (PBL) to mitogens and alloantigens. We further investigated this impairment of lymphocyte reactivity by employing the techniques of limiting dilution analysis and cytokinetic analysis. PBL preparations from patients have approximately six times fewer phytohemagglutinin (PHA)-responsive cells than PBL from normal subjects. Similar results were obtained with purified T cell preparations. Cytokinetic analysis of PHA-induced [3H]thymidine incorporation employing colchicine blocking of mitosis demonstrated that the number of first generation cells entering the S-phase of mitosis for each 24-hr period was less for PBL from patients than for PBL from normal individuals. First generation responding cells from patients and normal subjects entered DNA synthesis at the same time (48 to 72 hr). Cytokinetic analysis over a period of 168 hr demonstrated that whereas PBL from normal individuals demonstrated second generation responding cells, PBL from the majority of patients did not, thus indicating a defect in their ability to undergo clonal expansion. Measurement of interleukin 2 (IL 2) activity in culture fluids from PHA-activated PBL from normal subjects and patients revealed significantly lower IL 2 levels in culture fluids from PBL from patients. The addition of various concentrations of lectin-free IL 2 to PBL from patients stimulated with PHA did not restore responsiveness to normal values. There was no difference between the levels of interleukin 1 (IL 1) produced by lipopolysaccharide-activated monocytes from normal subjects and patients. Overall, these results suggest that an intrinsic defect exists in T cells obtained from brain tumor patients that renders them unable to enter into normal mitogen-induced blastogenesis.     

OT-CLL: a human T cell chronic lymphocytic leukemia that produces IL 2 in high titer

This report identifies and describes a human T cell chronic lymphocytic leukemia, OT-CLL, which can be triggered by selected mitogens (either PHA or Con A) to produce IL 2 in high titer. Optimal IL 2 production requires culturing OT-CLL cells at 2 to 5 X 10(6)/ml for 24 hr in the presence of 1 to 2% PHA-M. Under these conditions, the titer of IL 2 generated is greater than 20-fold that obtained from conventional sources, e.g., from mitogen-activated tonsillar lymphocytes. Two lines of experimental evidence suggest that the tumor cell product(s) is IL 2. First, in functional assays, suprenatants derived from cultures of PHA-activated OT-CLL cells trigger the proliferation and long-term growth of IL 2-dependent human TCL cells. Second, a partial biochemical purification of the active moiety(ies) derived from OT-CLL demonstrates marked similarity to conventional human IL 2. Thus, the biologically active material(s) precipitates in 50 to 70% saturated (NH4)2 SO4 solutions; elutes from DEAE-Sepharose in the presence of 0.04-0.08 M NaCl; and has an apparent m.w. of approximately 14,000, as determined by Sephadex G-100 gel filtration. In addition, analysis of OT-CLL cells by indirect immunofluorescence, utilizing a panel of monoclonal antibodies, confirms not only that these tumor cells are of T cell lineage but that they display surface antigens that define the normal human peripheral T cell subset subserving helper or inducer function: OKT3+, OKT4+ , OKT8-.     

The interleukin 2 secretion defect in vitro in systemic lupus erythematosus is reversible in rested cultured T cells

Interleukin 2 (IL 2) secretion in response to mitogenic stimulation in vitro is strongly reduced in circulating T lymphocytes from patients with SLE. It is still not clear how this abnormality relates to the B cell hyperactivity in the disease. Some investigators proposed that an intrinsic T helper cell defect could lead to suppressor cell dysfunction and autoimmunity. Others have found that in fact increased suppressor cell activity can cause IL 2 hyposecretion. In the present study we report that the IL 2 secretion in response to PHA plus PMA by T cells from patients with SLE, which initially was decreased by a factor of 10 as compared with the IL 2 secretion in blood donor T cells, was restored when the T cells were rested for 2 to 3 days in culture before stimulation. IL 2 hyposecretion in SLE T cells and the kinetics of normalization in culture were not changed by the addition of normal adherent cells during the stimulation with PHA/PMA, occurred in the absence of significant cell death or proliferation or change of the T4:T8 cell ratio during the resting culture, were not due to a maturation of immature T6-positive cells (less than 1.5% T6 cells in SLE T cells), and also occurred in T8-depleted T4 cells alone. Furthermore, a normalization of IL 2 secretion took place in the presence of either SLE serum or normal serum, and the addition of fresh autologous T cells to 3-day-cultured SLE T cells did not cause suppression of IL 2 secretion. These data show that some rapidly reversible defect occurs in circulating T helper cells in SLE. That this could reflect an exhaustion of T helper cells that have been activated in vivo is discussed.     

OKT8 antibody inhibits OKT3-induced IL 2 production and proliferation in OKT8+ cells

We studied IL 2 production and proliferation induced by OKT3 mitogenic monoclonal antibody in the OKT8+ T cell subset. OKT3 antibody induced IL 2 production and proliferation in OKT8+ cells in a typical time-dependent manner: maximal IL 2 levels were found in 24 hr culture supernatants; maximal proliferation was found on day 3. OKT3 antibody was mitogenic over a wide range of concentrations (0.125 to 500 ng/ml). The presence of OKT8 antibody (greater than or equal to 100 ng/ml) in these cultures resulted in almost complete inhibition of IL 2 production and proliferation. Kinetic studies demonstrate that OKT8 antibody suppresses both IL 2 production and response to exogenous IL 2 in OKT8+ cells when added within the first 2 hr of culture. After 14 to 20 hr of culture, addition of OKT8 only blocks IL 2 production but not the IL 2 response of activated OKT8+ cells. The specificity of inhibition by OKT8 antibody of OKT3 mitogenicity on OKT8+ cells was confirmed by the failure of Leu-I and OKT4 antibody to produce the same effect and by the lack of inhibition by OKT8 antibody of OKT3-induced IL 2 production and proliferation in OKT4+ cells.     

Human bone marrow allograft recipients: production of, and responsiveness to, interleukin 2

The production and utilization of interleukin 2 (IL 2) by peripheral blood mononuclear cells (PBMC) from 14 bone marrow allograft recipients was examined. PBMC from all patients were impaired in their ability to produce IL 2 when stimulated by phytohemagglutinin (PHA). On the average, the IL 2 activity produced by patients' PBMC was 8% of that from normal donors' PBMC. To examine the basis for this impaired T cell function in marrow recipients, the ability of a resting T lymphocyte population isolated from PBMC to respond to PHA and exogenously supplied IL 2 was analyzed. The proliferative response of resting T cells to PHA and IL 2, although low at early times post-transplant, reached near normal levels by 8 mo. Only two of 11 patients had normal numbers of precursor T cells that could respond. For all other patients the average number of precursor T cells was 10-fold lower than the average determined for normal donors. The impaired production of IL 2 by patients' PBMC may be due to this low precursor frequency. For some patients the rate and/or extent of clonal expansion of activated T cells appears to be greater than that of normal donors. The data suggest that the therapeutic application of IL 2 to such patients is unlikely to be successful in overcoming defects of T cell function before 8 mo post-marrow transplant.     

Regulation of human T lymphocyte mitogenesis by antibodies to CD3

The inhibitory and mitogenic effects of anti-CD3 antibodies (anti-CD3) were examined in cultures of human peripheral blood T cells. Resting T cells required the presence of accessory cells (AC) or phorbol myristate acetate (PMA) to be stimulated by soluble anti-CD3 (OKT3 and 64.1). Anti-CD3 was unable to induce activation of AC-depleted T cells as determined by IL 2 receptor expression, IL 2 production, cell cycle analysis, or detectable DNA synthesis. Although T cell responses to PHA also required AC, far fewer were necessary to generate responses. Anti-CD3 inhibited PHA-stimulated T cell IL 2 production, IL 2 receptor expression and proliferation in partially AC-depleted cultures. Moreover, anti-CD3 was able to inhibit PHA responses when added to culture as late as 24 to 42 hr after the initiation of a 96-hr incubation. Increasing concentrations of PHA reduced the inhibitory effect of anti-CD3 on PHA-stimulated T cell proliferation, whereas IL 2 production remained suppressed. Anti-CD3 linked to Sepharose beads effectively inhibited PHA-stimulated T cell DNA synthesis, indicating that internalization of the CD3 molecule was not required for inhibition of PHA responses. Although inhibition of IL 2 production was a major effect of anti-CD3 in PHA-stimulated cultures, it was not the only apparent inhibitory effect because the addition of exogenous IL 2 could not prevent inhibition completely. Intact AC but not IL 1 also reduced anti-CD3-mediated inhibition of PHA responsiveness, whereas the addition of both IL 2 and AC largely prevented inhibition. Thus, anti-CD3 in the absence of adequate AC signals exerted a number of distinct inhibitory effects on mitogen-induced T cell activation. These results suggest that the CD3 molecular complex may play a role in regulating T cell responsiveness after engagement of the T cell receptor by a number of mechanisms, some of which involve inhibition of IL 2 production.     

Studies on human blood lymphocytes with iC3b (type 3) complement receptors. I. Granular, Fc-IgG receptor positive and negative subsets in healthy subjects and patients with systemic lupus erythematosus

By using the OKM1 monoclonal antibody and the fluorescence-activated cell sorter to identify lymphocytes bearing iC3b (type 3) complement receptors, two principal populations of OKM1+ lymphocytes have been identified in human peripheral blood. One subset exhibited azurophilic granules and Fc receptors for IgG stained by Leu-11. The other population did not display FcR, but was enriched in cells reacting with OKT3 and OKT8 (low intensity). In healthy subjects, approximately 60% of CR3+ lymphocytes were granular FcR-bearing cells and only 18% co-expressed OKT3 determinants. In patients with systemic lupus erythematosus (SLE), CR3+ lymphocytes were predominantly FcR negative cells and 71% lacked granules. Only 33% reacted with Leu-11, but 50% co-expressed OKT3, 44% reacted with OKT8+, and 15% were OKT4+. We tested the hypothesis that agranular OKT3+ Leu-11- lymphocytes, such as those found in SLE patients, contained the precursors of natural killer (NK) cells. Leu-11+ cells were removed from normal lymphocytes by complement lysis, and the remaining cells were treated with recombinant IFN-alpha, IFN-gamma, or IL 2. These procedures were ineffective in generating typical NK effector cells. Our studies do not support the hypothesis that CR3+ Leu-11- lymphocytes are the precursors of granular Leu-11+ NK cells.     

Mechanisms of human T cell response to mitogens: IL 2 induces IL 2 receptor expression and proliferation but not IL 2 synthesis in PHA-stimulated T cells

Human peripheral blood T cells were purified by a four-step procedure which included depletion of plastic-adherent cells, rosetting with sheep red blood cells, nylon wool passage, and treatment with mouse monoclonal antibodies to human Ia antigens plus complement. The purified T cells completely failed to proliferate to phytohemagglutinin (PHA). Bacterially derived recombinant human interleukin 2 (IL 2) reconstituted the proliferative response of resting T cells to PHA. The optimal concentration of IL 2 required was 100 to 200 U/ml. IL 2 alone caused no T cell proliferation. Both PHA and IL 2 needed to be present together for the proliferation of T cells to occur. Incubation of T cells with either PHA or IL 2 alone for up to 18 hr, followed by washing, then by the addition of the reciprocal reagent, resulted in no T cell proliferation. Expression of IL 2 receptors and of Ia antigens, as assessed by indirect immunofluorescent staining, revealed that both PHA and IL 2 needed to be present for Tac and Ia antigen expression by T cells. T cells incubated with PHA and IL 2 for 18 to 42 hr acquired responsiveness to IL 2. These T cells remained absolutely dependent on IL 2 for proliferation to occur. In contrast to T cells stimulated with PHA in the presence of monocytes, T cells stimulated with PHA and IL 2 released no detectable IL 2. The failure of IL 2 secretion was not caused by down-regulation of IL 2 production by IL 2 itself, because the addition of IL 2 to cultures of T cells stimulated with PHA in the presence of monocytes did not interfere with IL 2 production. These results indicate that IL 2 is a sufficient signal to induce the expression of its receptor in PHA-stimulated T cells and subsequent proliferation but is not sufficient to cause endogenous IL 2 release.     

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.     

Monoclonal antibody OKT11A inhibits and recombinant interleukin 2 (IL 2) augments expression of IL 2 receptors at a pretranslational level

The expression of receptors for interleukin 2 (IL 2) represents a critical event regulating the growth of normal T lymphocytes. We investigated the effects of the inhibitory monoclonal antibody OKT11A (anti-sheep erythrocyte receptor) and of purified recombinant IL 2 (rIL 2) on the expression of IL 2 receptors by activated T cells at both the protein and the mRNA levels. Adding OKT11A antibody (0.5 microgram/ml) to phytohemagglutinin (PHA)-stimulated cultures of human peripheral blood mononuclear cells (PBMC) markedly suppressed cellular proliferation (assessed by [3H]thymidine incorporation) and IL 2 receptor expression (determined by immunofluorescence assay by using the anti-IL 2-receptor antibody, anti-Tac). Northern blot analysis performed with the use of a cDNA probe specific for the human IL 2 receptor gene demonstrated that OKT11A antibody also decreased the accumulation of IL 2 receptor mRNA induced by PHA in PBMC. Purified rIL 2 (10 U/ml) alone had little effect on the expression of IL 2 receptors in unstimulated PBMC cultures. In combination with PHA or with PHA plus OKT11A, however, rIL 2 augmented both the expression of IL 2 receptor protein on PBMC and the accumulation of IL 2 receptor mRNA in PBMC. Adding anti-Tac antibody to PBMC cultures to block the interaction of IL 2 with its receptor diminished the accumulation of IL 2 receptor mRNA induced by PHA. Taken together, these data demonstrate that OKT11A antibody inhibits and IL 2 augments expression of IL 2 receptors on PHA-stimulated T cells, at least in part, at a pretranslational level.     

Failure of OKT3 monoclonal antibody to induce lymphocyte mitogenesis: a familial defect in monocyte helper function

Monoclonal antibodies to the T3 molecule on human T cells have mitogenic activity. Although anti-T3 antibodies of the IgG1 subclass (e.g., UCHT1) induce mitogenesis in lymphocyte cultures from only 60 to 70% of normal donors, antibodies of Ig2a subclass (e.g., OKT3) invariably have been found to be mitogenic in all subjects tested up to the present. This paper describes a family (a mother, six daughters, and one son) in which five members failed to respond mitogenically to OKT3 although the proportion of OKT3-reactive cells in their peripheral blood was normal. Mitogenic responses to PHA, Con A, and PWM were normal. Five members comprising four OKT3 nonresponders were also unresponsive to UCHT1. Unresponsiveness to OKT3 and unresponsiveness to UCHT1 were not absolutely linked to each other, nor were they linked to an HLA haplotype inherited from the mother. Upon stimulation by OKT3, lymphocyte preparations from OKT3-nonresponders failed to produce interleukin 2 (IL 2) and to display IL 2 receptors. OKT3 unresponsiveness was due to defective monocyte help: thus, responsiveness to OKT3 of T cells from an OKT3-nonresponder was restored by the addition of monocytes from an HLA-identical sister who had a normal response to OKT3. Inversely, T cells from the OKT3 responder had no reactivity to OKT3 when cultured in the presence of monocytes from an HLA-identical, OKT3-nonresponsive sister. Unresponsiveness to OKT3 could not be overcome by the addition of phorbol myristate acetate to the cultures. These data on a familial, non-HLA-linked deficiency of monocytes to exert their auxiliary function provide better insight into the mechanism of anti-T3-induced T cell activation.     

In situ identification of cells in human leprosy granulomas with monoclonal antibodies to interleukin 2 and its receptor

Leprosy is a chronic granulomatous disease with an immunologic spectrum in which lepromatous leprosy patients have defective cell-mediated immune responses, in comparison to tuberculoid leprosy patients. Immunoregulatory aspects of this spectrum were investigated by using monoclonal antibodies to interleukin 2 (IL 2), IL 2 receptors (Tac), and T lymphocyte subpopulations with immunoperoxidase techniques on frozen sections of skin biopsy specimens from 10 tuberculoid and 10 lepromatous patients. A comparison of IL 2+ cells revealed markedly fewer IL 2+ cells in lepromatous specimens (lep. 0.028% +/- 0.02 vs tub. 0.46% +/- 0.28, p less than 0.001). These IL 2+ cells were large, exhibited cytoplasmic staining, and on double immunostaining were Leu-4+, Leu-3a+, Leu-2a-, Tac-, and OKT6-, consistent with the fact they are IL 2 producers. Equivalent numbers of Tac+ cells were observed in both lepromatous and tuberculoid granulomas (lep. 1.5% +/- 0.5 vs tub. 2.1% +/- 0.7, p, NS), suggesting that the responder cells are present in both conditions. The tuberculoid granuloma was highly organized, composed of a central core of mature macrophages, Leu-3a+ and Tac+ cells with a surrounding mantle of Leu-2a+, Leu-3a+, IL 2+, Tac+, and OKT6+ cells. In lepromatous granulomas, Leu-2a+, Leu-3a+, Tac+, and rare IL 2+ cells were randomly admixed with bacilli-laden macrophages. The defective cell-mediated immune responses in lepromatous leprosy appears to be associated with diminished IL 2 production and disorganization of the granuloma.     

Interleukin 2 deficiency in murine Leishmaniasis donovani and its relationship to depressed spleen cell responses to phytohemagglutinin

This study examined the kinetics and mechanisms of depressed spleen cell responses to phytohemagglutinin (PHA) that occur during Leishmania donovani infection of BALB/c mice. In co-culture experiments, neither spleen cells from infected animals nor parasite-infected macrophages suppressed PHA responses of normal spleen cells. In addition, parasite-mediated suppression of PHA-stimulated spleen cell proliferation could not be demonstrated. Mice with 2 wk of infection did manifest an impairment in spleen cell production of interleukin 2 (IL 2) and by 8 wk IL 2 activity in supernatants from these cells was reduced by approximately 95%. This finding was not explained by an alteration in the kinetics of IL 2 production. Furthermore, diminished IL 2 activity in supernatants of PHA-activated spleen cells from infected animals was not caused by suppressive factors in these fluids as shown by their inability to suppress IL 2 stimulation of IL 2-dependent T cells. When spleen cells from mice with 8 wk of infection were cultured with PHA and supplemented with exogenous IL 2, there was an approximately 48% increase in mitogenesis. These data indicate that abnormal PHA-induced spleen cell activation in BALB/c mice with L. donovani infection is associated with impaired production of IL 2. In addition, the observation that supplementation of spleen cells from infected mice with IL 2 resulted in partial reconstitution of the PHA response is consistent with a defect in IL 2 responsiveness.     

Subsets of human large granular lymphocytes (LGL) exhibit accessory cell functions

The present study shows that human large granular lymphocytes (LGL) depleted of OKT3 (T lymphocytes) and Leu-M1-positive (monocytes) cells exhibit accessory cell function for the T lymphoproliferative responses to the soluble stimulants Staphylococcus protein A (SpA) or Streptolysin O (SLO), as well as to surface antigens in the autologous and allogeneic mixed leukocyte reaction (MLR). Fractionation of LGL into subsets according to their reactivity with alpha OKT11, alpha DR, and alpha OKM1 MoAb led to the identification of the subset(s) of LGL with OKT11+, DR+, OKM1+ phenotype as the antigen-presenting cell (APC), whereas the DR-, OKM1- subset(s) of LGL was completely ineffective. Furthermore, virtually all the natural killer (NK) activity of LGL was associated with OKT11+ and OKM1+, DR+ LGL that exerted the observed APC function, suggesting that NK-active cells may also act as effective APC for T lymphocyte activation. These results indicate that human LGL with NK activity may exert other noncytotoxic functions and may play a major role in immunoregulation.     

Concanavalin A triggers T lymphocytes by directly interacting with their receptors for activation

Anti-HLA-DR antibodies did not inhibit concanavalin A-(Con A) induced T cell proliferation or the generation of suppressor cells capable of inhibiting immunoglobulin synthesis in autologous mononuclear cells after pokeweed mitogen stimulation. Nylon-wool purified T cells (pretreated with anti-HLA-DR antibody and C) exposed to Con A acquired responsiveness to interleukin 2 (IL 2) and were able to absorb this growth factor, whereas nonlectin-treated cells did not respond to IL 2 and could not absorb it. In the presence of interleukin 1 (IL 1), Con A stimulated the synthesis of IL 2 in purified OKT4+ lymphocytes but not OKT8+ cells. However, in the absence of IL 1, neither resting OKT4+ nor Con A-treated OKT4+ cells produced IL 2. Con A by itself did not directly stimulate macrophages to synthesize IL 1, although it could do so in the presence of OKT4+ but not OKT8+ lymphocytes. In addition, Con A induced proliferation of purified T cells provided IL 1 was supplied to the cultures. Cyclosporin A rendered Con A-treated T cells unresponsive to IL 2, made lectin-stimulated OKT4+ lymphocytes unable to respond to IL 1, and inhibited the synthesis of IL 2. Furthermore, this drug abrogated the Con A-stimulated synthesis of IL 1 by acting on OKT4+ lymphocytes and not on macrophages. Finally, cyclosporin-A suppressed the proliferative response and the generation of suppressor T cells induced by Con A. The following are concluded: 1) HLA-DR antigens do not seem to play any role in the triggering of T cells by Con A, and macrophages participate in lectin-induced activation of T cells mainly by providing IL 1. 2) Cyclosporin-A inhibits activation of T cells by interfering with the mechanism by which Con A stimulates T lymphocytes. 3) Con A triggers T lymphocytes by directly interacting with their receptors for activation.     

Preliminary characterization of accessory cells in the nonadherent fraction of human blood mononuclear cells

We investigated why peripheral blood mononuclear cells rigorously depleted of adherent cells by sequential incubation on plastic and nylon wool remained fully responsive to both antigenic and mitogenic signals. Nylon wool nonadherent cells (NWNA) depleted of cells expressing HLA-DR by monoclonal antibody and complement lysis did not respond to tetanus toxoid (TT) or suboptimal concentrations of phytohemagglutinin (PHA). Addition of adherent accessory cells to these NWNA HLA-DR- cells reconstituted the response to stimuli. NWNA, fractionated by discontinuous density gradient centrifugation, contained high density cells which were unresponsive alone to optimal concentrations of both TT and PHA. All the lower density fractions contained cells which were accessory for higher density cell responses to stimuli. The lowest density fraction was approximately 30% monocytes (esterase and peroxidase positive) and less than or equal to 3% B lymphocytes (surface IgG bearing). The other low density fractions contained large granular lymphocytes but rarely monocytes and no B lymphocytes. Depletion of OKT3+, OKM1+, and Leu-11+ cells from lower density cells by monoclonal antibody and complement lysis did not abolish their accessory activity, but depletion of HLA-DR+ cells or gamma irradiation of these cells decreased their accessory activity for PHA and eradicated accessory activity for TT. Thus, the responsiveness of NWNA to soluble antigenic and mitogenic signals is due, in part, to the presence of low density cells which are radiosensitive and phenotypically HLA-DR+ OKT3-OKM1-Leu-11-. Accessory activity in NWNA seems to reside, therefore, in a cell which is not a typical monocyte, natural killer cell, nor B or T lymphocyte.