In Vitro Effect of Inosiplex on T Lymphocytes. I Influence on T Cells with Receptors for IgG (T γ)

Abstract

Inosiplex, a complex of inosine and 2-hydroxypropyldimethyl ammonium-4-(acetylamino) benzoate, 1:3 molar ratio, originally developed for antiviral use, is now under wider investigation because of its immunopotentiating properties.

This compound can have some actions on T cells at various stages of differentiation, thus promoting an enhancement of their blastogenic responses to varied mitogenic agents (PHA, Con A, PWM, MLC, tetanus toxoid, and viral antigens).

Our studies demonstrate that under the influence of inosiplex human peripheral blood T lymphocytes bearing Fc IgG receptors have an augmented receptor avidity for SRBC which result in an increased E active rosette formation, and that T cells preincubated with the drug at the appropriate concentrations express more Fc IgG receptors.

Even though Tγ cells exert “in vitro” immunoregulatory properties, the increase in percentage of Tγ lymphocytes do not correlate with a potentiation of the Con A-induced suppressor activity of T cells.

Moreover, the lymphocytes treated with the substance in the absence of Con A exert helper functions, increasing the mitogenic responses of the second culture PHA - treated lymphocytes.

These data appear to suggest a pro-proliferative inosiplex-induced effect which could mask a concomitant suppressor cell induction.     

Mechanism of T lymphocyte activation by OKT3 antibodies. A general model for T cell induction

The OKT3 monoclonal antibody has unique reactivity for human T cells and induces a mitogenic response equal to that of concanavalin A (Con A). However, significant differences were found in activation requirements for the two T cell mitogens. Although both OKT3 and Con A required living accessory cells for stimulation of T cells, the OKT3 antibody was optimally mitogenic at a molar concentration 10(3) times less than Con A. Under such conditions, the two mitogens showed similar kinetics as well as magnitude or responses. Furthermore normal IgG blocked the OKT3-induced proliferation selectively. The inhibition is most likely caused by the Fc portion of IgG competing for Fc receptors on accessory cells. The different requirements for activation are discussed in relation to the T cell receptor and its role in T cell activation.     

Characterization of lymphocytes which inhibit proliferation of human T cells exposed to lymphocyte-derived mitogenic factors.

Culture supernatants of PHA-activated human lymphocytes (active SUPs) contain factors which are strongly mitogenic for fractionated peripheral T cells. This stimulation is suppressed by certain non-T lymphocytes. It is shown that these suppressor cells can inhibit an ongoing response of T cells to active SUP and that this inhibition is reversible. Using various rosette sedimentation techniques for fractionating subpopulations of lymphocytes it is concluded that the suppressor cells lack membrane-associated receptors for C3 but possess receptors for the Fc part of IgG. This subset of lymphocytes may be an important regulator of lymphocyte proliferation during immune responses.     

T cell chronic lymphocytic leukaemia with suppressor phenotype

The peripheral blood cells from a patient with T cell chronic lymphocytic leukaemia were examined for surface marker and functional characteristics. Eighty-91% of the peripheral blood cells formed SRBC rosettes and 22-49% possessed Fc receptors; 73% of the peripheral blood cells were reactive with the OKT8 antiserum and 61% expressed DR antigens. Response to PHA stimulation was markedly reduced, whereas allogeneic responsiveness in mixed leucocyte culture was intact. The ability of Con A-stimulated peripheral blood cells to generate suppressor activity in a mixed leucocyte reaction was deficient, whereas suppression of in vitro immunoglobulin synthesis was greater than normal. The leukaemic peripheral blood cell population expressed a T suppressor phenotype. Functional studies suggest that these cells were derived from the subset of T lymphocytes with regulatory activity for immunoglobulin synthesis as opposed to mitogenic responsiveness.     

The Effect of Levamisole on Peripheral T-Lymphocytes of Patients with Malignant Lymphomas

The effect of various concentrations (0.015-10 μg/ml) of Levamisole (LMS) on the peripheral lymphocytes of patients with malignant lymphomas (ML) and normal donors was investigated in vitro. The parameters studied include : E rosettes forming cells (total T lymphocytes), active E rosettes (early T lymphocytes) and DNA synthesis induced by pitogens PHA and Con A.

LMS improved significantly lymphocyte response both in patients with ML and normal donors when the cells were stimulated by Con A. In both groups no significant effect was observed on the response to PHA nor on the percentage of E-rosettes, whereas the mean number of active E rosettes was significantly increased on all concentrations of the drug.

While in the normal subjects a positive statistical correlation between active E-rosettes and Con A response was observed, in patients with ML an inverse correlation was found. This latter correlation was partially reversed by LMS.     

Unusual phenotype and function of an expanded subpopulation of T cells in patients with haemopoietic disorders.

We have studied two patients, one with red cell aplasia and the other with neutropenia. Both showed lymphocytosis. In both cases, 90-100% of E rosetting cells were T cells as defined by the monoclonal antibodies UCHT1 and OKT3. The majority of these cells also carried the OKT8 suppressor/cytotoxic marker and were HLA-DR- and Fc gamma R-positive. In spite of the similarity of this phenotype to that reported for suppressor cells, these cells failed to suppress pokeweed mitogen-induced polyclonal Ig synthesis. Cells from both patients also failed to respond significantly to Con A and PHA. They were, however, unable to suppress the Con A responses of normal donors although cells from one patient were able to suppress completely a normal PHA response. These results demonstrate the existence of a genuine subset of T cells with Fc gamma receptors but suggest that not all such cells have typical suppressor function.     

Characterization and Mitogenic Activity of Haemophilus Influenzae Type B Capsular Polysaccharide

Studies were conducted on the characterization of Haemophilus influenzae type b polysaccharide (HITB-PS) and its mitogenic activity upon peripheral lymphocytes. This capsular polysaccharide was found to contain hexosamines and hexoses in addition to the main components of ribose and ribitol phosphate. The molecular weight of HITB-PS was determined as 585,000. The affinity constant of HITB-PS to unfraction-ated lymphocytes was 3.13 × 10³ M^-1 with 1.11 × 10⁴ binding sites per cell.

HITB-PS was found to be mitogenic for both numan T and B lymphocytes. At optimum doses, a three to five fold increase in ³H-thymidine incorporation into T and B cells was observed. Higher than optimum doses resulted in suppression of this mitogenicity. The effect of concanavalin A (Con A) mitogenicity was detected in T and B cells treated with effective as well as suppressive doses of HITB-PS; the mitozenic activities of Con A and HITB-PS were found to be independent of each other.     

Human T-cell subset changes during culture

When human peripheral blood lymphocytes (PBL) are cultured with either concanavalin A (Con A)-treated or control autologous T lymphocytes, the mitogenic responses of the PBL co-cultured with Con A-treated cells are much lower. We have investigated the cell surface receptor changes during culture of T cells with and without mitogen in an attempt to explain this differential regulatory phenomenon. We present data here which show that human T cells cultured in complete medium alone gain helper cells with time. Con A-treated T cells are known to lose helper cells during culture. Erythrocyte rosette-purified T cells were cultured with or without Con A for 84 h and the numbers of cells with receptors for the Fc regions of either IgM (T mu) or IgG (T gamma) were enumerated daily. T mu cells have been associated with helper activity while T gamma cells have predominantly suppressor activity. Treatment with 10 micrograms/ml of Con A decreased T mu by approximately 50%. Untreated cells, however, showed significant increases in T mu (44 +/- 30.5% in twelve individuals). The great variance in T mu increases is due to the fact that individuals having higher initial T mu values showed smaller increases. These changes probably represent the gain or loss of receptors because total cell numbers did not change. There was no significant change in the number of T gamma cells in either control or Con A-treated cultures during the same 84 h period. In co-culture experiments in which the responses of fresh autologous PBL were determined, 60-h control T-cell cultures enhanced the mitogen responses of the fresh cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences in Mitogenic Responses of Murine T Cells to Two Distinct Phytohemagglutinin (Pha) Subcomponents

Two distinct mitogenic subcomponents of phytohemagglutinin (PHA) — leucoagglutinin (LA) and “purified” PHA — apparently stimulated different subpopulations of murine T cells. In the DBA/2J strain, the mitogenic responses of splenic lymphocytes to LA reached maximal levels after 24 to 36 hr exposure and almost completely disappeared by 48 hr, whereas maximal responses to PHA were maintained after 48 hr incubation. The levels of LA-responding T cells were highest in DBA/2J spleens at 5 weeks of age but markedly declined by 9 weeks of age, whereas thymic levels of LA-responding T cells reached a maximum at 9 weeks of age and remained maximal past 15 weeks of age. PHA-responding cells, in contrast, reached maximal levels in both the spleen and thymus of DBA/2J mice at 9 weeks of age. In C57BL/6J mice, splenic and thymic lymphocytes responded similarly to both components, except that the response of splenic lymphocytes to PHA reached a maximum after shorter incubation time and declined sooner than their response to LA. The mitogenic responses of C57BL/6J thymocytes to both components were already at their peak by 5 weeks of age and almost totally disappeared by 9 week, whereas the responses of splenic lymphocytes were maximal at 9-15 weeks of age. The responses of DBA/2J splenocytes to LA was significantly augmented by PHA, but LA markedly suppressed the proliferative responses to PHA.     

Neuraminidase treatment of human T lymphocytes: effect on Fc receptor phenotype and function

Purified peripheral blood T cells or T mu cells from normal healthy donors were treated in vitro with neuraminidase and examined for the expression of IgM Fc and IgG Fc receptors. Increasing concentrations of neuraminidase selectively removed IgM Fc receptors, whereas the number of T cells expressing IgG Fc receptors was significantly increased. Following neuraminidase treatment, IgM Fc receptors could be regenerated by reincubation of T cells at 37 degrees C. The regeneration of IgM Fc receptors could be blocked by treatment with cycloheximide. Neuraminidase treatment of purified T mu cells resulted in the expression of IgG Fc receptors on a subpopulation of T mu lymphocytes. A small percentage of the neuraminidase-treated T cells expressed receptors for both IgG and IgM. Treatment of T cells with neuraminidase did not effect T cell-mediated spontaneous cytotoxicity (SLMC) or antibody-dependent cellular cytotoxicity (ADCC). Our results indicate that T cell Fc receptor phenotypes can be modulated in vitro without significantly altering their functional capacity.     

Theoylline Induced Non Specific Suppressor Activity in Human Peripheral Blood Lymphocytes

It is wellknown that theophylline yields phenotypic changes on suppressor cells. In the present study we investigated the possibility that theophylline could directly induce a suppressor activity on a lymphocyte subpopulation. We observed that a short preincubation (120 min at 37°C) with theophylline (1mM) activates human peripheral blood lymphocytes to suppress mitogenic response of autologous cells. This activity was not evident on a T cell subpopulation depleted of theophylline-sensitive (T-sens) lymphocytes.

Theophylline mediated suppressor activity is only present in the Concanavalin A stimulated cultures, thus suggesting a synergism between Concanavalin A and theophylline in the expression of non specific suppression. Moreover we observed that after a 24 hrs preincubation of lymphocytes in complete culture medium there was a complete loss of theophylline-induced suppression. Such a preincubation time also produced a decrease in the theophylline-mediated enhancement of intracellular 3',5' cyclic adenosine monophosphate levels and the impairment of E-rosette formation, suggesting that theophylline acts mainly on a “short-lived” suppressor lymphocyte subset.     

Suppression of B lymphocyte differentiation by newborn T lymphocytes with an Fc receptor for IgM

The IgM (Fc)-binding subpopulation of human newborn T lymphocytes (T_M) suppressed the pokeweed mitogen induced differentiation of adult B lymphocytes. Positively isolated IgG (Fc)-binding T lymphocytes (T_G) also suppressed, but lymphocyte preparations negatively enriched for T_G by the depletion of T_M cells were not suppressive. The Fc receptor specificity of the human newborn suppressor appears to resemble that of the human Con A suppressor.     

Alterations in the Mitogen Responsiveness of Lymphocytes from Mice Bearing Moloney Sarcoma Virus Induced Tumors

The effects of Moloney Sarcoma Virus (MSV) induced tumor growth dynamics on the blastogenic responsiveness of lymphocytes from BALB/c mice were investigated. Lymphocytes from spleen, thymus and lymph node pools were tested for responsiveness to phytohemagglutinin (PHA) and concanavalin A (Con A). Results showed a significant decrease in PHA-induced blastogenesis of all lymphocytes tested at the time of maximal tumor volume, with a return to normal responsiveness as the tumor regressed. In contrast, a differential dose dependent Con A response occurred in spleen and thymus lymphocytes. A decreased ³H-thymidine uptake occurred at optimal Con A dose, correlating with the PHA decrease. However, at a lower Con A dose an increased response was observed, beginning shortly before the PHA depression and continuing until regression of tumor began. This phenomena was not observed in lymph node lymphocytes.

Based upon these observations, we suggest that the cell or cells responsible for the transient suppression of PHA responsiveness may be Con A responsive T lymphocytes.     

Alpha-Naphthyl Acetate Esterase Activity in Human Lymphocytes: Distribution in Lymphocyte Subpopulations and in Mitogen-activated Cells

The cytochemical demonstration of nonspecific alpha-naphthyl acetate esterase (ANAE) activity in human peripheral blood mononuclear cells was studied. Different staining patterns were found, allowing differentiation of mononuclear cells into macrophages (strong granular cytoplasmic activity), B lymphocytes (negative reaction), Tγ lymphocytes, i.e. bearing IgG Fc receptors (granular scattered reaction), and T non-γ lymphocytes, i.e. devoid of IgG Fc receptors (single cytoplasmic ANAE spot). During the early phases of phytohaemagglutinin (PHA)- and concanavalin A (Con A)-induced activation, the reactivity of most lymphocytes became granular and scattered, similar to that found in T_γ cells. Blast cells generating in successive phases, appeared devoid of detectable enzymatic activity. The hypothesis is put forth that T cells showing granular, scattered reactivity represent a population of activated cells and that the redistribution of enzymatic activity could represent a preliminary step leading to secretion (lymphokine-like?) of enzyme from cytoplasm in the course of cell activation.     

Heterogeneity of concanavalin A-induced suppressor T cells in man defined with monoclonal antibodies.

Human peripheral blood T lymphocytes were enriched for OKT4+ or OKT8+ subpopulations using complement mediated lysis with OKT8 or OKT4 monoclonal antibodies. These subpopulations and unfractionated T cells were separately stimulated with concanavalin A (Con A) for a period of 48 hr and were then examined for their suppressive influence on proliferative response of autologous T cells to phytohaemagglutinin (PHA) or allogeneic non-T cells. Con A-activated unfractionated T cells, OKT4+ and OKT8+ T cell subsets markedly suppressed both these responses. Both OKT4+ and OKT8+ T cell subsets when enriched following Con A-activation of unfractionated T cells also caused significant suppression of proliferative responses of autologous T cells to PHA and allogeneic non-T cells in mixed lymphocyte cultures. The suppressive influence of Con A-activated T subsets was abolished by irradiation (2,000 rad) of activated cells. These studies indicate that Con A-induced suppressor T cells are heterogeneous. Precursors of Con A-induced suppressor T cells appear to reside in both OKT4+ and OKT8+ T cell populations.     

The effect of the plant lectins phytohaemagglutinin and concanavalin A on human T cell populations bearing receptors for IgG and IgM.

The effect of long-term culture with the plant lectins phytohaemagglutinin (PHA) and concanavalin A (con A) on the percentages of human T cells bearing Fc receptors for IgG (TG) and IgM (TM) was investigated. Con A produced an early increase in the percentage of TG cells as compared to control cells cultured without mitogen. TM cells decreased. PHA suppressed the percentages of both TG and TM. These changes were not due to loss of cell viability nor to loss of cell surface receptors in general since up to 98% of the cells continued to form sheep erythrocyte rosettes in the virtual absence of IgM Fc receptors.     

T cell stimulation and expansion using anti-CD3/CD28 beads

Following appropriate stimulation, T lymphocytes will proliferate extensively in vitro. Traditionally, mitogenic lectins such as phytohemagglutinin (PHA) and concanavalin A (Con A) have been used for polyclonal T cell stimulation. A more physiologically relevant approach uses beads coated with anti-CD3 and anti-CD28 to stimulate T cells in a manner that partially mimics stimulation by antigen-presenting cells. This protocol describes the steps involved in T cell stimulation and their subsequent in vitro expansion using anti-CD3/CD28 beads.     

A suppressor cell in the response of rabbit T cells to Con A.

The response to Concanavalin A is regulated by a helper cell, described elsewhere (4), and by a suppressor cell, described in this paper. This suppressor cell is an adherent T cell with Fc receptors. Evidence for properties of the suppressor cell was obtained by two types of experiments: 1) regulatory cells gave more help if Fc-bearing subpopulations were removed from them, i.e. the suppressor cell could not be removed with Degalan beads, coated with anti-allotype antibody, but not with beads, coated with F(ab')2 fragments of the antibody; 2) help for T cells, in their response to Con A, was augmented when T cells were eliminated from the regulating adherent cell preparation. Thus, the response of spleen T cells to Con A is regulated by two adherent cells, a B helper cell and a T suppressor cell. We have previously shown that the response to phytohemagglutinin (PHA) is regulated by an adherent helper cell (4). We have found no evidence, in the present study for an adherent suppressor cell which participates in the T cell response to PHA.     

Immunosuppressive effect of a trichothecene mycotoxin, Fusarenon-X in mice.

The in vitro treatment with Fusarenon-X, a mycotoxin produced by Fusarium nivale Fn 2B, depressed the mitogenic responses of mouse lymphocytes to the T-cell mitogens, phytohaemagglutinin (PHA) and concanavalin A (Con A), but to a lesser extent to B-cell mitogen, a bacterial lypopolysaccharide (LPS). The in vitro treatment of mice with Fusarenon-X also decreased the responsiveness of splenic lymphocytes to the T-cell mitogen, Con A. Administration of Fusarenon-X to BALB/c mice before immunization significantly reduced anti-DNP IgE and IgG1 responses, while the treatment of mice after immunization was less effective. The number of spleen cells of the treated mice was increased with a reduction of T lymphocytes and an increase of granulocytic elements. In vitro antibody responses of spleen cells from Fusarenon-X treated mice by pokeweed mitogen (PWM) or LPS were also suppressed. Reconstruction experiments using sIg positive and negative spleen cells from normal and Fusarenon-X treated mice showed that this inhibitory effect resided in the sIg negative cell population.     

The mitogenic lectin from Phaseolus vulgaris does not recognize the T3 antigen of human T lymphocytes

Human peripheral blood T lymphocytes are stimulated to grow and divide by lectins such as concanavalin A (Con A) and Phaseolus vulgaris phytohemagglutinin (PHA), as well as a few anti-T cell monoclonal antibodies. The latter antibodies recognize the T3 antigen. It has been suggested previously that PHA and Con A mediate T cell growth by interacting with T3. However, as reported in this study, affinity chromatography on immobilized lectins, and immunoprecipitation by lectin plus anti-lectin antibodies showed that T3 binds Con A but not PHA. Fab fragments of a monoclonal antibody against T3 (namely Leu-4) inhibited T lymphocyte proliferation induced by T3 antibodies and Con A, but not by PHA. Nevertheless, co-capping experiments performed with fluorescein-labeled lectins and rhodamine-labeled T3 antibodies showed that T3 co-caps with Con A and PHA receptors, although the co-capping with PHA was incomplete. Since the T cell receptor for antigen (Ti) has been shown to co-cap with T3 on the cell surface, we reasoned that PHA induced capping of the T3 antigen by interacting with Ti. A disulfide-linked heterodimer comprising subunits of about 49 000 and 41 000 mol. wt. that resembled the Ti molecule was detected in PHA-anti-PHA immunoprecipitates of various surface- and biosynthetically-labeled T cells, by two-dimensional (nonreduced vs. reduced) sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. The results suggest that PHA triggers T lymphocytes by interacting with the carbohydrate moieties of Ti and imply that T lymphocytes can be stimulated by mitogens via at least two different cell surface molecules (Ti and T3).