Effect of cyclosporin A on the early activation of human T helper lymphocytes: inhibition of RNA-synthesis and modification of the expression of activation antigens.

The ability of cyclosporin A (CS-A) to inhibit induced lymphocyte activation and to modify expression of membrane receptors was assessed on human T helper cells. Flow cytometric cell cycle analyses of acridine orange-stained cells showed that CS-A (0.5 micrograms/ml) inhibits the G0-G1 activation process of a substantial proportion of PHA- and Con A-stimulated lymphocytes. The expression of Tac, OKT9 and 4F2 antigens (previously shown to be expressed or increased on activated cells) was investigated by immunofluorescence. Fewer cells expressed the Tac and OKT9 antigens after activation in presence of CS-A, but the percentage of 4F2-positive cells remained unchanged. Analyses of receptor densities measured by fluorescence intensity revealed for all three investigated antigens a decreased receptor density on positive cells in presence of CS-A. Thus, CS-A not only inhibited cell activation (G0-G1 transition) and the expression of Tac, OKT9 and 4F2 antigens, but it also diminished the number of Tac, OKT9 and 4F2 antigens per cell. Assessing specifically the activation of OKT4 (helper) and OKT8 (cytotoxic) cells after 24 h, either by double-fluorescence or by cell fractionation with anti-OKT4 or anti-OKT8 antibodies plus complement, showed that preferentially OKT4 cell activation as well as expression of Tac and OKT9 antigens on those cells was inhibited in the presence of CS-A.

Effect of monoclonal antibodies (MoAb) to class I and class II HLA antigens on lectin- and MoAb OKT3-induced lymphocyte proliferation.

We have examined the effect of several monoclonal antibodies (MoAb) to monomorphic determinants of class II HLA antigens, and MoAb to monomorphic determinants of class I HLA antigens and to beta-2-microglobulin (beta 2-mu) on lectin- and MoAb OKT3-induced proliferation of human peripheral blood mononuclear cells (PBMNC) and cultured T cells (CTC). Some, but not all, anti-class II HLA MoAb inhibited the proliferative response of PBMNC to MoAb OKT3 and pokeweed mitogen (PWM). The degree of inhibitory effect varied considerably. This effect was not limited to anti-class II HLA MoAb since anti-class I HLA MoAb and anti-beta 2-mu MoAb also inhibited MoAb OKT3- or PWM-induced proliferative responses. In contrast, the response of PBMNC to phytohemagglutinin (PHA) and concanavalin A (Con A) was not blocked by any anti-class II HLA MoAb. However, some anti-class II HLA MoAb also inhibited the proliferative response of CTC plus allogeneic peripheral blood adherent accessory cells (AC) to PHA or Con A as well as to MoAb OKT3 or PWM. This may be attributable to the substantially greater class II HLA antigen expression by CTC than by fresh lymphocytes. Pretreatment of either CTC or AC with anti-class II HLA MoAb inhibited OKT3-induced proliferation. In contrast, pretreatment of CTC, but not AC, with anti-class I HLA MoAb inhibited the proliferative response of CTC to OKT3. Pretreatment of CTC with anti-class I HLA MoAb inhibited PHA-, Con A and PWM-induced proliferation, to a greater degree than the anti-class II HLA MoAb. It appears as if lymphocyte activation by different mitogens exhibits variable requirements for the presence of cells expressing major histocompatibility determinants. Binding of Ab to membrane markers may interfere with lymphocyte-AC cooperation, perhaps by inhibiting binding of mitogens to their receptors or by interfering with lymphocyte and AC function. We also have examined the role of class II HLA antigens on CTC by depleting class II HLA-positive cells. As expected, elimination of class II HLA-positive AC with anti-class II HLA MoAb plus complement caused a decrease in proliferation of CTC in response to all the mitogens tested. In contrast, elimination of class II HLA-positive CTC was shown to clearly increase proliferation of CTC, perhaps because this may deplete class II HLA-positive suppressor cells.

Characterization of cell lines enhancing IL-2 production by human phytohemagglutinin-stimulated lymphocytes.

Certain cell lines were found to significantly enhance IL-2 production by phytohemagglutinin-stimulated T lymphocytes, and the mechanisms involved in mediating such an enhancement have been studied. All B-lymphoblastoid cell lines (B-CL) tested had an enhancing capability, even lines which were immature, surface immunoglobulin negative, or negative for EBV-associated antigens or Fc receptors. Furthermore, a B-CL lacking HLA-A, B, and C, antigens as well as a HLA-DR-deficient mutant line enhanced IL-2 production. Autologous B-CL as well as allogeneic lines were able to augment IL-2 production. Cell lines from patients with T-cell acute lymphoblastic leukemia did not stimulate, while more mature, DR-positive T-cell lines did. Although all HLA-DR positive cell lines, regardless of their derivation, provided enhancement, several lines of evidence, including blocking experiments with anti-DR antibodies, indicated that the reaction was not HLA-DR mediated. The enhancing determinant(s) appeared to be cell associated since CL supernatants were ineffective, and it may serve as an important additional signal to the preactivated IL-2-producer T-cell.

Lymphokine regulation of human lymphocyte proliferation: formation of resting G0 cells by removal of interleukin 2 in cultures of proliferating T lymphocytes.

The question of whether lymphocytes which have once been activated and have completed one or several cell cycle(s) can return to the G0 phase and stay ready for a new activation (G0-G1 transition), rather than simply die, was investigated. To do so interleukin 2 (IL-2) was removed from cultures of continuously proliferating human T lymphocytes and the formation of resting (G0) cells was measured. Kinetic analyses in freshly prepared peripheral blood lymphocytes (PBL) revealed that the onset of detectable RNA synthesis and the appearance of structures binding the anti-Tac antibody occurred simultaneously. This allowed the expansion of the definition of G0 T lymphocytes as cells having a low RNA (and DNA) content, and no Tac antigen. When cultured human T cells proliferating continuously by means of IL-2 were characterized in terms of their distribution in the cell cycle, 7 days after the initial PHA stimulation, it could be demonstrated that very few cells were in the G0 phase, supporting the concept of direct S/G2/M-G1 transition. However, when IL-2 was removed from the cultures, the [3H]thymidine incorporation per 10(4) cells and correspondingly the number of cells in the S/G2/M and G1 phases were reduced drastically and during the following 72-hr period, the number of G0 cells increased markedly. Restimulation of such in vitro formed G0 cells, under conditions permitting observation of their shift from the G0 to G0 phase, demonstrated that most cells could respond normally. Based on these observations, it was concluded that IL-2 not only ensures T-lymphocyte survival and proliferation, but IL-2 starvation induces many continuously proliferating T lymphocytes to stop cycling and to return to the G0 phase of the cell cycle where they remain functional.

Modulation of glucocorticoid receptors by mitogenic stimuli, glucocorticoids and retinoids in normal human cultured T cells.

Human T lymphocytes can be maintained in cell culture by the addition of conditioned medium (CM) containing a T cell growth factor (TCGF). This system provides an opportunity to study the presence and modulation of glucocorticoid receptors (GR) by various factors in these cells. GR were present in T cells grown from each of 22 normal individuals; the binding capacity (mean = 3851 +/- 2880 sites/cell) and affinity (Kd = 7.4 X 10(-9)) were similar in rested cultured T cells (CTC) to those reported in peripheral blood T lymphocytes. Treatment of rested CTC with stimuli such as phytohemagglutinin (PHA), CM, or 12-O-tetradecanoylphorbol-13-acetate (TPA) results in a mean increase in GR binding capacity of 3.1-, 3.2- or 2.4-fold respectively without modification of binding affinity. Using an exchange assay to measure occupied and unoccupied GR, we examined the effects of cortisol on its own receptor. Treatment of rested CTC with 10(-7) M cortisol for 24 h decreased GR by more than 50%. Cortisol treatment also blocked the induction of GR by PHA and CM. Since retinoids have been shown to modulate the immune response and to alter the effects of PHA and phorbol esters on lymphocytes, we examined their effects on GR. Retinol decreased GR activity in CTC but only at concentrations which inhibit cell growth. It is concluded that GR activity in human T cells can be modulated by several important factors involved in lymphocyte function.

Lymphokine regulation of activated (G1) lymphocytes. II. Glucocorticoid and anti-Tac-induced inhibition of human T lymphocyte proliferation.

The regulation of the first cell cycle of human, activated (G1) PBL was analyzed by flow cytometry and [3H]thymidine incorporation. Endogenous IL 2 production was blocked in situ by pharmacologic concentration of DEX (100 to 1000 nM), resulting in an 80 to 90% reduction of thymidine uptake. Although T lymphocyte activation (G0-G1a transition) by PHA was unaltered, cells remained in the G1a phase of the cell cycle due to insufficient RNA synthesis for proliferation. The addition of IL 2-containing supernatants reversed this inhibitory effect of DEX by allowing the cells to synthesize more RNA (G1a-G1b transition). Such cells could enter the S phase and proliferate. Similar studies were performed on cells treated with a monoclonal antibody (anti-Tac) against the IL 2 receptor. In these studies, IL 2-induced RNA synthesis, and subsequent proliferation of DEX-treated and PHA-stimulated cells was inhibited by anti-Tac. Anti-Tac did not, however, inhibit the effect of endogenous IL 2 (PHA-stimulated PBL without DEX treatment), although it did bind equally well to such cells. Thus, IL 2 directly or indirectly regulates human T cell proliferation at the level of RNA synthesis. Furthermore, anti-Tac can inhibit the mitogenic signal given by endogenous IL 2, but not by in situ produced IL 2, an observation of importance to further investigations of the mechanisms by which IL 2 interacts with specific receptors to elicit proliferation.

Antitumor reactivity in vitro and in vivo of lymphocytes from normal donors and cancer patients propagated in culture with T cell growth factor (TCGF).

Peripheral blood lymphocytes (PBL) from 38 normal donors and from 27 cancer patients were propagated in bulk cultures for 3-6 weeks using T cell growth factor (TCGF). In addition, cultures derived from lymphocyte preparations enriched for or depleted of natural killer (NK) cells and several clones of cultured cells were studied. The following main observations were made: (a) PBL of both patients and healthy donors could be expanded to large numbers (up to 2500-fold); (b) CLC derived from unfractionated PBL exhibited intermediate levels of cytotoxic activity against autologous and allogeneic fresh lung tumor cells and strong cytotoxicity toward several cultured adherent tumor cells; (c) whereas cultures originated from populations enriched for NK cells were highly cytotoxic against both adherent tumor target cells and against an NK-sensitive leukemic cell line (K562), cultures derived from populations depleted of NK cells were preferentially cytotoxic to adherent target cells; (d) clones of CLC were also strongly cytotoxic, but 2 out of 3 clones tested showed a narrower spectrum of target cytotoxicity than that of uncloned CLC; (e) CLC, when mixed with two carcinoma cell lines, were able to inhibit tumor growth in nude mice.

Different requirements for Ia-bearing accessory cells in mitogen versus antigen induction of human B-cell responses.

The accessory cell requirements for the induction of proliferative and specific antibody responses of human lymphocytes stimulated with either antigen or mitogen were examined. An Ia-negative human myeloid tumor cell line, K562, could substitute for monocytes in the proliferation of monocyte-depleted lymphocytes in response to pokeweed mitogen (PWM) stimulation. K562 cells could also act as accessory cells in the PWM-induced anti-keyhole limpet hemocyanin (KLH) antibody synthesis of cells from a KLH-immunized donor. In contrast, only monocytes and not K562 cells could function as accessory cells in antigen-induced lymphocyte proliferation as well as in antigen-induced, antigen-specific antibody production. However, K562 cells, like monocytes, were able to positively and negatively regulate polyclonal immunoglobulin responses. Thus, Ia-bearing accessory cells can function in antigen-induced proliferation and antibody responses while non-Ia-bearing cells can function in mitogen-induced, but not antigen-induced responses. These studies indicate a dichotomy in the nature of required accessory cells in antigen-induced versus mitogen-induced human lymphocyte responses and strongly suggest an obligatory role of Ia or an Ia-related molecule on accessory cells in antigen-induced responses of human lymphocytes.

Limiting dilution analysis of the frequency of human T cells and large granular lymphocytes proliferating in response to interleukin 2. II. Regulatory role of interferon on proliferative and cytotoxic precursors.

Limiting dilution analysis has shown that large granular lymphocytes (LGL) and small T cells undergo proliferation in response to lectin-free IL 2. The latter is critically dependent on prior stimulation with lectin. Depending on conditions, alpha or beta interferons (IFN) were found to have either of two opposing effects on the frequency of proliferating cells. Pretreatment of responder cells with IFN resulted in dose-dependent augmentation of proliferative progenitors such that at 500 IU/ml, a fivefold increase of progenitor frequency was apparent. Under such conditions, approximately 5% of LGL could be expanded, and at least a proportion of the cultured cells killed the NK-sensitive K562 cell line. Similar results were apparent with T cell responders: whatever the initial frequency of proliferation (dependent on the PHA dose), augmentation was obtained with IFN pretreatment, although no killing of K562 was induced. In contrast, when IFN was present throughout the 7-day assay period, proliferative frequencies were reduced. This inhibitory effect was mediated through the presence of irradiated T cells in the feeder populations. With purified monocytes as feeder cells, little reduction was seen, whereas addition of T cells resulted in a 19-fold inhibition. Separation of OKT4+ and OKT8+ subsets demonstrated that both were essential for optimum induction of suppression. These data indicate that IFN has a powerful immunodulatory role on both NK and T cell proliferation, and that it may function both by induction of IL 2 receptors and activation of suppressive T cells. The interaction between different soluble mediators provides a complex immunoregulatory circuit in vitro.

Limiting dilution analysis of the frequency of human T cells and large granular lymphocytes proliferating in response to interleukin 2. I. The effect of lectin on the proliferative frequency and cytotoxic activity of cultured lymphoid cells.

Human peripheral blood LGL that mediated NK and small T cells were isolated in high purity (98% by morphology) by density sedimentation on discontinuous Percoll gradients. The proliferative frequency of these subpopulations in the presence of lectin-free conditioned media containing IL 2 was determined by limiting dilution analysis. LGL showed a 20-fold greater frequency of proliferative cell precursors than small T cells (1/200 and 1/4970, respectively). The NK-like nature of cells expanded from LGL preparations in IL 2 was confirmed by parallel testing of the cytotoxicity against K562. Whereas T cell microcultures showed no lytic activity against K562 (cytotoxic precursor frequency less than 1/10,000), LGL cultures showed frequencies of cytotoxic precursors (1/170) comparable to those of proliferative precursors. Neither responder cell type gave rise to detectable lytic activity against NK-insusceptible mouse lymphoma RL male 1 or alloblasts. LGL proliferation was only minimally affected by the presence of PHA at the onset of culture (rise to 1/74 with 2 micrograms/ml PHA). By contrast, small T cells showed a dose-dependent increase of proliferative frequency, to reach 1/11 with 2 micrograms/ml PHA, provided accessory cells in the form of PBMC, monocytes, or LGL but not T cells were present. The cytotoxic activity of LGL and small T cells expanded in IL 2 was confirmed in bulk cultures. LGL-CLC showed high lytic activity against NK-susceptible cell lines and a majority of freshly isolated allogeneic human tumor targets. T cell-CLC showed little activity against cell line targets (K562, Raji, L1210, RL male 1) but were lytic for some fresh tumor cells. These data establish optimal conditions for the growth of human LGL in IL 2-dependent culture and suggest that a major contributor to lysis of allogeneic human tumors by CLC is likely to be NK cells. The data indicate that large numbers of activated T cells cannot be detected in vivo and that in vitro induction of IL 2 receptors by lectin/antigen is necessary for the establishment of antigen-reactive T cell lines. In contrast, a proportion of LGL appear to be spontaneously activated and susceptible to IL 2-dependent growth. Thus, in the absence of stimulation, culture of unfractionated lymphoid cells in the presence of IL 2 is likely to select for the growth of LGL with NK activity.

A rapid technique for isolation of viable tumor cells from solid tumors: use of the tumor cells for induction and measurement of cell-mediated cytotoxic responses.

A rapid and simple technique for the isolation of viable tumor cells from human and mouse solid neoplasms is described. It consists of a 5 to 10-min treatment with trypsin-collagenase-DNase mixture, followed by mechanical disaggregation of the tumor tissue and subsequently by a brief centrifugation on a discontinuous Percoll gradient. With the tumors employed, this procedure usually requires less than 1 hr and results in preparations comprising greater than 80% tumor cells with viability of 80-90%. Cell-mediated cytotoxic response was measured with: (a) unsensitized lymphocytes freshly obtained from tumor-bearing hosts; (b) lymphocytes propagated in culture with T cell growth factor; and (c) lymphocytes stimulated in cocultures with autologous or syngeneic tumor cells. The cytotoxic activity was assessed in a modified [51Cr]-release assay adapted for solid tumor cells, allowing a long incubation period (24 hr) and the use of a low number (200-1000) of highly labeled target cells (2-10 counts/min/cell).

The effect of lentinan on production of interleukin-1 by human monocytes.

Activated human monocytes produce a monokine, interleukin-1 (IL-1) which can amplify the immune response by inducing antigen specific T cells to elaborate T cell growth factor, or interleukin-2, which in turn causes T cells to proliferate. The immune adjuvant lentinan has been shown in this study to augment IL-1 production by human monocytes. Stimulation of IL-1 by lentinan was seen as early as 5 hr, with some effect as late as 60 hr. Optimal effects were seen with very low concentrations, around 0.1 micrograms/ml. Lentinan was also able to stimulate IL-1 production by the leukemic cell line, K-562. The data reported here suggest that the previously reported adjuvant effects of lentinan may in part be mediated via its ability to stimulate IL-1 production.

Cryopreserved human cultured T cells as responder cells for the quantitative measurement of interleukin-2: improvement of the assay.

Large batches of human cultured T cells (CTC) were cryopreserved for later use as responder cells in a proliferation assay for measurement of interleukin (IL)-2 activity. Cryopreservation of CTC could be carried out without considerable loss in viability and cryopreserved and fresh cells showed equally good responses to IL-2. The conditions of IL-2-dependent CTC growth were analyzed, which led to a better evaluation of test results, and had important implications for the calculation of relative IL-2 activity. The repeated use of the same batch of cryopreserved CTC reduced test variability and provided an assay system that allows reliable and reproducible measurement of human IL-2 activity.

Interferon and prostaglandins: effects on human natural and lectin-induced cytotoxicity.

Effects of interferons (IFN) and E-series prostaglandins (PGE) were evaluated on subpopulations of human peripheral blood mononuclear lymphocytes (PBL) that mediate natural killer (NK) activity, antibody-dependent cell-mediated cytotoxicity (ADCC), or lectin-induced cellular cytotoxicity (LICC). When PBL were separated into subpopulations depending on the presence or absence of receptors for sheep erythrocytes (E+ or E-, respectively) or for the Fc gamma receptor (Fc gamma R+ or Fc gamma R-, respectively), LICC was mainly associated with the E+Fc gamma R- cells and to a considerably lesser extent with E+Fc gamma R+ cells. In contrast, NK activity and ADCC were associated with the E+ and E- subpopulations that had Fc gamma R. IFN treatment had stronger effects on the Fc gamma R+ subpopulations. Boosting of LICC occurred maximally in the E+Fc gamma R+ subpopulation, and the activity of the E+Fc gamma R- cells increased only modestly. IFN had a greater augmenting effect on the NK activity and ADCC of E+Fc gamma R+ cells than on E-Fc gamma R+ cells. PGE strongly inhibited all three types of cell-mediated cytotoxicity and was effective on IFN-boosted cells as well as on untreated cells.

Immunologic characterization of a helper T-cell lymphoma.

The lymphocytes of a patient with a T-cell non-Hodgkin's lymphoma with peripheral blood involvement and polyclonal hypergammaglobulinemia were characterized in terms of surface markers and immunologic functions. Using the fluorescence-activated cell sorter and employing various monoclonal antibodies against T-cell surface antigens, it was shown that almost all of the patient's peripheral blood lymphocytes were positive for OKT4 and 9.3, antibodies that recognize helper T-cell subset. The circulating lymphoma cells had typical characteristics for T cells; they formed spontaneous rosettes with sheep erythrocytes and stained with the pan-T-cell antibodies 9.6 and 10.2, but did not react with other anti-T-cell monoclonal reagents such as OKT3, UCHT-1, and 3A1. The cells appeared to be mature by the fact that they did not stain with OKT6, and terminal deoxynucleotidyl transferase was undetectable. Functionally, they were able to provide "help" for antibody production, and they could be stimulated to produce moderate amounts of interleukin-2, while unable to proliferate in response to mitogens. Morphologically, some of the lymphocytes showed a deeply cleaved nucleus.

Specific cytotoxicity against autologous tumour and proliferative responses of human lymphocytes grown in interleukin 2.

Peripheral blood lymphocytes of cancer patients were sensitized in vitro to autologous tumour cells in mixed lymphocyte-tumour culture (MLTC). Blast cells were isolated on discontinuous Percoll gradients from MLTC which showed significant stimulation of [3H]-thymidine incorporation. Cultured T cells (CTC) were derived from these blasts by growth in conditioned medium containing interleukin-2 (IL-2) and maintained for up to 51 days by repeated feeding with IL-2 and in some cases by addition of irradiated allogeneic blood mononuclear cells as "fillers". These cultures showed specific cytotoxic reactivity against autologous tumour and in only a few cases was natural killing (NK) of K562 cells apparent. Restimulation of CTC with tumour was measured in primed lymphocyte test (PLT). Increased uptake of [3H]-thymidine was found upon stimulation by autologous tumour and allogeneic tumour of the same site and histology but there was no response to non-related tumours or to a panel of allogeneic lymphocytes. No sensitization to autologous HLA-D/DR could be detected by restimulation or cytotoxicity against monocytes in the majority of cases. These data suggest that, by careful selection of sensitised blasts from MLTC, it is possible to obtain CTC with both helper (proliferative) and cytotoxic T cells and that such CTC have specific reactivity against tumour cells. These cellular reagents will be useful in defining the antigenicity of human neoplasms and possibly in therapy.

Human interleukin 2: production by both TG cells and other T cells.

We have investigated the production of interleukin 2 (IL-2) by human T cells after their stimulation by phytohaemagglutinin (PHA). T cells isolated by rosetting with sheep erythrocytes produced high levels of IL-2. Further separation of rosette-forming cells, according to the expression of Fc receptors for IgG, showed that TG and non-TG cells are equally able to produce IL-2. The release of IL-2 by TG cells did not require DNA synthesis or functional Rc gamma receptors, since positively selected TH cells produced IL-2, even though they lacked lymphoproliferative responses to PHA and antibody-dependent cell-mediated cytotoxicity.