J.M. Rojo

CD4 Dependence of Activation Threshold and TCR Signalling in Mouse T Lymphocytes

The effect of CD4 expression on the activation threshold of mouse T lymphocytes has been analysed. To do this, the authors studied the response to antigen and other T cell receptor (TCR) ligands in a series of CD4− mutants obtained from the SR.D10 clone. This non‐tumour clone spontaneously arose from the Th2 clone D10.G4.1, and characteristically shows a low threshold for antigen activation as well as reactivity to syngeneic antigen presenting cells (APC). Although SR.D10 CD4− mutant cells can be stimulated by antigen, they need higher antigen concentration or more APC than SR.D10 or CD4 transfectants to yield optimal antigen responses. Furthermore, CD4− clones are not activated by syngeneic APC or by clonotypic antibodies. These effects do not correlate with changes in the expression of cell surface molecules implicated in antigen recognition, like TCR/CD3, CD2, LFA‐1, or CD45, or with lower p56lck or p59fyn activity in the mutant cells. Since inhibition experiments using anti‐CD4 antibodies have previously shown that activation of the CD4+ T cell clone D10.G4.1 by antigen or alloantigens is largely dependent on CD4, our results indicate that activation by antigen‐plus self MHC may become CD4‐independent if the activation threshold is lowered enough, e.g. in cells like SR.D10. Expression of CD4 further lowers the activation threshold of the cells, allowing the detection of low‐affinity TCR reactivities like those directed at self MHC. Moreover, by using anti‐TCR/CD3 antibodies, the authors have confirmed the importance of CD4‐associated tyrosine kinase activity in early TCR/CD3 signalling in this Th2 cell line, as (1) upon TCR/CD3 ligation, tyrosine phosphorylation is detected only in those CD3 chains co‐precipitating with CD4; and (2) CD4 expression is needed for efficient early tyrosine phosphorylation and detectable p56lck‐TCR co‐precipitation.

Enhancement of lymphocyte proliferation, interleukin-2 production and NK activity by inmunoferon (AM-3), a fungal immunomodulator: Variations in normal and immunosuppressed mice

The in vivo effect of Inmunoferon (AM-3), a glycophosphopeptide of fungal origin, has been studied on T and B lymphocyte mitogenesis, Interleukin 2 (IL-2) synthesis and natural killer (NK) activity. Inmunoferon (30 mg/kg/day) was administered to several groups of mice 2, 3 or 7 days/week for 2 weeks, and its effect assessed on day 15 of treatment. Every treatment assayed enhanced IL-2 and NK activity in the spleen. The greater effect was produced by daily administration of the immunomodulator. No enhancement was found in mitogen-induced proliferation of T and B lymphocytes. Similar treatments with Inmunoferon enhanced NK activity in the spleens of mice treated with 250 mg/kg cyclophosphamide. In addition, mitogenic responses of T lymphocytes, but not IL-2 production, were also increased in immunosuppressed mice after treatment with the immunomodulator.

Polyerga, a biological response modifier enhancing T-lymphocyte-dependent responses

Cancer patients are often treated with biological response modifiers to enhance immunological functions. However, little is known about the actual mechanism of action of many of these substances. Therefore, we were interested in the effect of i.p. treatment with porcine low-molecular-weight spleen peptides, which are used during supportive cancer therapy, on lymphoid cell populations and function in mice. After treatment with 0.5 μg peptides/kg body weight for 14 consecutive days, lymphokine secretion and the generation of cytotoxic T-cells were significantly enhanced as compared with controls. However, there was no effect on the number of cells or the percentage of cells expressing functional surface markers in secondary lymphoid organs.

Sodium diethyldithiocarbamate restores T lymphocyte proliferation, interleukin-2 production and NK activity in cyclophosphamide-immunosuppressed animals

The in vivo effect of sodium diethyldithiocarbamate (DTC) on IL-2 production, mitogen-induced proliferation and natural killer (NK) activity of lymphocytes from normal as well as cyclophosphamide (CY)-treated mice has been investigated. DTC was given in a single subcutaneous injection (25 mg/kg) to normal or cyclophosphamide-treated mice (250 mg/kg i.p. simultaneously to DTC). An enhancement of T lymphocyte proliferation in CY-treated animals and an increase of IL-2 production in both normal and immunosuppressed mice was observed. When DTC (10 mg/kg) was administered daily for two weeks an increase in concanavalin A-induced mitogenesis, IL-2 production and NK activity in CY-treated animals was observed. These immune parameters were reduced 12 days after CY treatment by a factor of 2 to 3 times, while DTC treatment restored these responses to normal levels. LPS-induced mitogenesis was not significantly enhanced. The effect of DTC could be partially mediated by changes in IL-2 activity. According to these results some functional parameters of the immune system of the suppressed host can be partially or completely restored by means of an appropriate immunomodulator treatment. DTC could be of interest in the treatment of diseases where immune functions are impaired or in combined treatments with immunosuppressants.

In vivo effect of isoprinosine on interleukin-2 production, lymphocyte mitogenesis and NK activity in normal and cyclophosphamide immunosuppressed mice

The in vivo effect of isoprinosine on IL-2 production, mitogen-induced proliferation and NK activity of lymphocytes from normal as well as cyclophosphamide (CY) treated mice has been investigated. Isoprinosine was given in a single dose (50 mg/kg or 5 mg/kg) to normal or CY treated mice (250 mg/kg i.p. simultaneously to isoprinosine). An enhancement of T lymphocyte proliferation and IL-2 production was observed in both cases. There was no correlation between the small effect observed in T lymphocyte proliferation and the enhancement of IL-2 levels found in the supernatants of Con A activated spleen cells, specially in normal mice. Administration of isoprinosine every day (50 mg/kg) augmented Con A induced mitogenesis, IL-2 production and NK activity in animals treated with cyclophosphamide, but not in normal mice. Isoprinosine could be of interest in a combined treatment with immunosuppressants for the restoration of certain immune functions. The effect of isoprinosine on immune responses may be mediated in part by changes in IL-2 activity.

Isoprinosine restores in vitro T lymphocyte functions of cyclophosphamide immunosuppressed mice.

The effect and the mechanism of action of isoprinosine has been investigated in several models of in vitro activation of lymphocytes. Isoprinosine added to spleen cell cultures enhanced lymphocyte proliferation induced by concanavalin A or allogeneic stimulation as well as the generation of allospecific cytotoxic T cells. The effect of isoprinosine on T lymphocyte proliferation in vitro was specially marked when mice were treated with cyclophosphamide (75-200 mg/kg) 16-24 h before the onset of cultures. No effect was observed on B cell proliferation to LPS. Addition of inosine or adenosine also enhanced proliferation of cells from both normal and cyclophosphamide treated mice. Isoprinosine and inosine and, more markedly, adenosine, augmented interleukin-2 activity in concanavalin A supernatants of spleen cells from the same animals.

Suppression of Immune Parameters in Animal Models of Morphine Dependence

Implantation of pellets containing 75 mg of morphine induced short term (4 day) morphine dependence and markedly reduced total number of spleen cells of BALB/c mice, without affecting total body or liver weight. Polyclonal responses induced by anti-CD3 antibodies, Concanavalin A or Escherichia coli lipopolysaccharide in the remaining spleen cells of morphine-treated mice were also inhibited. Cytofluorimetric analysis indicated that the proportion of major functional lymphocyte populations (Ig+, CD3+, CD4+ and CD8+ lymphocytes) were not significantly changed in the spleen from morphine-dependent mice. Furthermore, expression levels of surface Ig, CD3, CD4, and CD8, were similar in spleen cells from control or morphine-treated mice. So, morphine dependence in BALB/c mice under these controlled conditions results in a specific defect in lymphoid cell number and function, with no incidence on body weight or particular lymphocyte subsets.

Enhancement and restoration of contact sensitivity reactions by isoprinosine.

Isoprinosine (50 mg/kg i.p.) increased the ear swelling reaction in mice sensitized with trinitro-chlorobenzene (picryl chloride) and challenged with the same agent. Enhancements were observed when the drug was administered either simultaneously to the sensitization or to the challenge with picryl chloride. Similar results were obtained when lymphoid cells from sensitized animals were transferred i.v. to normal animals which were then challenged with picryl chloride. Using the same cell transfer system, we found that treatment of cell donors with Isoprinosine (50 mg/kg/day, i.p.) counteracts the inhibition induced by cyclophosphamide. Isoprinosine (50 mg/kg/day, i.p.) inhibited also the generation of antigen-specific suppressor cells induced by picryl-sulfonic acid as shown by transfer to normal recipients of suppressor cells together with lymphoid cells from sensitized animals. These results indicate that Isoprinosine enhances normal contact sensitivity reactions and can counteract, under certain circumstances, antigen-specific as well as nonspecific immunosuppression.

Modulatory effect of isoprinosine on lymphocyte proliferative response under immunodepressive conditions.

In the present work the effect of Isoprinosine on the mitogenic responses of T and B lymphocytes has been studied. We have found that Isoprinosine can enhance in vitro the response to Concanavalin A. This enhancement was more apparent in cell cultures showing an initially low blastogenic response. In low responses artificially induced by treatments in vivo with cyclophosphamide, our results indicate that Isoprinosine, administered in vivo, does not enhance the response to Con A of treated mice. However, addition of Isoprinosine (75 micrograms/ml) to cultures of spleen cells from mice previously treated with cyclophosphamide enhanced the suppressed response up to normal levels. Neither in vivo nor in vitro Isoprinosine treatments increased the response of lymphocytes to lipopolysaccharide, but usually inhibited the blastogenesis of B cells.

Exogenous additions of prostaglandins variably alter the blastogenic response of B and T lymphocytes from different mice lymphoid organs

Prostaglandins A1 and E2 enhance the 3H-thymidine (TdR) uptake in spleen cell cultures stimulated by two B-lymphocyte mitogens (E.coli lipopolysaccharide (LPS) and dextran sulfate (DXS) at concentrations ranging between 10(-8) and 10(-6) M. The same prostaglandin (PG) concentrations inhibited concanavalin A (Con A) activation of mouse spleen cells depleted or not from a glass-adherent prostaglandin-producing population of suppressor cells. After fractionation of spleen cells by nylon wool adherence, PGs diminished 3H-TdR uptake by nylon nonadherent (T-enriched) cells cultured in the presence of Con A and enhanced the activation of nylon adherent (B-enriched) cells by LPS. The proliferative response of thymic lymphocytes to Con A was drastically inhibited (70-95% by PGE2 10(-8)-10(-6) M. In cultures of purified bone marrow lymphocytes, PGE2 induced a dose-dependent inhibition of either LPS- or DXS-induced activation reaching a 30-40% inhibition at a PGE2 concentration of 10(-6) M. In the spleen, treatment of cells with anti-T sera and complement resulted in abrogation of PG-induced enhancement. Nevertheless, no inhibition of B-cell mitogenesis was observed in the presence of 10(-6) PGE2. From these results, it can be concluded that a different sensitivity of the proliferative response of lymphoid cells to exogenous PGs exists, depending on the subset (T or B) affected and/or the organ used as a source of these cells.