Immunomodulating activity of p-hydroxyphenyllactic and ascorbic acids

p-Hydroxyphenyl lactic acid (PHA) in a concentration of 5 . 10(-5) M produced a significant inhibition of cell proliferation in response to alloantigens in a one-way mixed lymphocyte culture (MLC) in colonic cancer patients and in blast transformation in response to suboptimal doses of Con A. Multiple administration of ascorbic acid in an optimal concentration to the culture increased the proliferative response of lymphocytes to alloantigens and Con A. PHA and ascorbic acid did not exhibit any immunomodulating action during the use of healthy donors' lymphocytes or lymphocytes from colonic cancer patients, transformed with optimal mitogen doses. PHA did not affect the production of cytotoxic T lymphocytes in the MLC of the spleens of allogeneic mice but inhibited lymphocyte proliferation in response to alloantigens in the MLC of the spleens obtained from B6 and vitamin A deficient animals.     

Mesenchymal stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by different mechanisms

Human mesenchymal stem cells (MSCs) have immuno-modulatory properties. They inhibit T-cell proliferation to mitogens and alloantigens in vitro and prolong skin graft survival in vivo. We found that MSCs inhibited the proliferation of peripheral blood lymphocytes (PBLs) to phorbol myristate acetate (PMA), suggesting that MSCs exert an inhibitory effect downstream of the receptor level. We analyzed cytokine profiles of PBLs co-cultured with MSCs. MSCs increased interleukin (IL)-2 and soluble IL-2 receptor in mixed lymphocyte cultures (MLCs), while IL-2 and IL-2R decreased in phytohemagglutinin (PHA)-stimulated PBL cultures. MSCs inhibited IL-2 induced proliferation, without absorbing IL-2. IL-10 levels increased in MLCs co-cultured with 10% MSCs, while the levels were not affected in PHA cultures. In MLCs inhibited by MSCs, antibodies against IL-10 further suppressed proliferation but had no effect in PHA cultures. Addition of indomethacin, an inhibitor of prostaglandin-synthesis, restored part of the inhibition by MSCs in PHA cultures. However, indomethacin did not affect MSC-induced inhibition in MLCs. To conclude, our data indicate that MSC-induced suppression is a complex mechanism affecting IL-2 and IL-10 signaling and may function differently, depending on T-cell stimuli. Prostaglandins are important in the inhibition by MSCs when the T cells were activated by PHA, but not alloantigens.     

Functional properties of the 50 kd protein associated with the E-receptor on human T lymphocytes: suppression of IL 2 production by anti-p50 monoclonal antibodies

Monoclonal antibody 9.6 is specific for a 50 kd T cell surface protein (p50) associated with the sheep erythrocyte (E)-receptor on human T lymphocytes. This antibody interferes with many T cell functions. We have examined the effect of antibody 9.6 on lymphocyte proliferation and interleukin 2 (IL 2) production triggered by mitogens, soluble antigens, and alloantigens to elucidate the mechanism(s) of its immunosuppressive action. At concentrations as low as 50 ng/ml, 9.6 suppressed lymphocyte proliferation and the elaboration of IL 2 by T cells stimulated by PHA, alloantigens, or low concentrations of the phorbol ester TPA (less than or equal to ng/ml). Furthermore, in cultures stimulated by a combination of PHA plus TPA, 9.6 did not inhibit the acquisition of IL 2 receptors but inhibited proliferation and IL 2 production. Immunoaffinity-purified IL 2 completely restored lymphocyte proliferation in cultures inhibited by 9.6. Studies of kinetics of inhibition by 9.6 showed that this antibody inhibited lymphocyte proliferation induced by PHA, alloantigen, and PPD even when added at 24, 48, and 72 hr, respectively, after the initiation of these cultures, suggesting that 9.6 does not block lectin binding or antigen recognition by T cells and that it can inhibit lymphocyte proliferation even after cells have undergone one or more rounds of cell division. A dose-response analysis of lymphocyte proliferation induced by PHA or by TPA demonstrated that the degree of inhibition by 9.6 decreased with increasing concentrations of these mitogens. Antibody 9.6 did not inhibit lymphocyte response induced by optimal concentrations of PHA (50 to 100 micrograms/ml; PHA-M) but inhibited proliferation of maximally induced lymphocytes by using a synergistic combination of low concentrations of PHA (5 micrograms/ml, PHA-M) plus TPA (1 ng/ml). Taken together, these findings indicate that 1) 9.6 inhibits lymphocyte proliferation by affecting IL 2 production, 2) 9.6 does not inhibit the acquisition of 9.6 receptors induced by a synergistic combination of PHA plus TPA, and 3) p50 molecules may be involved in multiple pathways of T cell activation.     

Inhibition of mitogen-stimulated T lymphocyte proliferation by calcitonin gene-related peptide

The effect of calcitonin gene-related peptide (CGRP) on mouse lymphocyte proliferation stimulated by mitogens was studied. CGRP (10(-10)-10(-7) M) dose-dependently inhibited the proliferative response of mouse lymph node cells and spleen cells stimulated by T cell mitogens concanavalin A (Con A) and phytohemagglutinin (PHA), whereas a B cell mitogen lipopolysaccharide (LPS) did not inhibit this response. The maximal inhibition by this peptide was 50% to 80% at 10(-8) and 10(-7) M. The addition of 10(-8) and 10(-7) M CGRP to lymph node cell cultures 24 hr after stimulation with Con A or PHA also had a significant inhibitory effect on the proliferative response. Furthermore, in the same concentration range (10(-10)-10(-7) M) CGRP increased intracellular cyclic AMP concentration in nylon wool nonadherent cells, but not in nylon wool adherent cells. CGRP had no significant effect on intracellular cyclic GMP concentration. In addition, specific binding of CGRP was observed in mouse spleen cells. Our present study suggests that CGRP inhibits the proliferative response of T lymphocytes to the mitogens by interacting with cell receptors coupled with adenylate cyclase. CGRP may be implicated in the regulation of T cell function.     

Induction of suppressor cells in autologous mixed lymphocyte culture (AMLC) in humans

T cells stimulated for 6-7 days in autologous mixed lymphocyte culture (AMLC) showed suppressive effects when added to fresh mixed cultures where autologous lymphocytes (A) were stimulated by Mitomycin C-treated allogeneic lymphocytes (Xm), in a ratio of A:Xm:AMLC-activated cells of 1:1:0.5. Both cytotoxic and proliferative activities in second cultures, as assayed after 6 days of incubation, were significantly inhibited (percentage suppression of cytotoxic activity observed in 17 experiments was 75.3 +/- 22.4; percentage suppression of proliferation was 60.6 +/- 18.2). Suppressor cells (SC) generated in AMLC were Mitomycin C sensitive and nonspecific in their action; not only A/Xm but also X/Am and X/Ym cultures were suppressed to the same extent. AMLC-Activated cells showed a considerable degree of proliferation in response to alloantigens but failed to express any cytotoxic activity against autologous or allogeneic phytohemagglutinin blasts. Thus, the inhibitory effect observed in this system is not due to cytotoxic elimination of responding or stimulating cells in the second culture but rather reflects a true regulatory (suppressive) mechanism.     

Effect of novel non-peptidic delta opioid receptor antagonists on human T and B cell activation

The effects of the antagonist naltrindole (NTI) on cells of the immune system have been largely studied although the mechanisms of action are still unclear. The aim of this study is to evaluate, in vitro, the immunomodulatory activity of four new delta-selective opioid compounds structurally related to naltrindole. The effects at different concentrations of these opioid antagonists on proliferative response were studied on normal human peripheral blood mononuclear cells (PBMC) stimulated with different stimuli: mitogens, the antigen PPD, the anti-CD3 monoclonal antibodies (mAb), the superantigen Staphylococcus aureus Cowan strain 1 (SAC) and alloantigens in the mixed lymphocyte cultures (MLR). The immunomodulatory capacity of these compounds was evaluated by determining the interleukin-2 (IL-2) release in mitogen activated PBMC. The present study shows that all the new delta opioid antagonists at 10(-5) M concentration are immunosuppressive. The inhibitory action is also evident at lower concentrations when anti-CD3 mAb and SAC were used as stimulators. In addition, the production of IL-2 was inhibited by the opioid treatment, but this might not be the only mechanism of action.     

Suppression of cytotoxic T-lymphocyte activation by pyruvate and L-alanine

The activation of cytotoxic T lymphocytes (CTL) in allogeneic mixed-lymphocyte cultures was found to be strongly inhibited if 1-3 X 10(-2) M L-alanine or the structurally and biochemically related substance pyruvate was present in the period from 7 to 19 or from 19 to 120 hr. The cytotoxic response was not inhibited when L-alanine or pyruvate was present during the first 7 hr of the culture period. L-Alanine produced also little or no suppression, if added on Day 3 of the culture. L-Lactate or D-alanine at similar concentrations was not suppressive during the entire culture period. The suppression by pyruvate and L-alanine was strongly reduced by 1 X 10(-4) M adenosine. Adenosine in combination with an interleukin-2 (IL-2)-containing EL-4-cell supernatant was even more effective. Pyruvate and alanine (1-3 X 10(-2) M) also inhibited the DNA synthesis in mixed-lymphocyte cultures on Day 5 by about 50%, but both substances had practically no effect on DNA synthesis in cultures that had been supplemented with an IL-2-containing EL-4 supernatant. They had also no effect on the IL-2-dependent proliferation of several T-cell clones or of concanavalin A-activated thymocytes. These relatively selective regulatory effects of pyruvate and L-alanine may be useful for the analysis of the biochemical pathways during lymphocyte activation and/or for a selective manipulation of the immune response.     

Lysozyme-induced inhibition of the lymphocyte response to mitogenic lectins

Both human lysozyme (HL) and hen egg white lysozyme (HEWL) inhibited the proliferative response of peripheral blood lymphocytes to T cell mitogens such as the lectins phytohemagglutinin and concanavalin A. This inhibition was observed both when HL or HEWL was added to the lymphocyte cultures in combination with phytohemagglutinin or concanavalin A and when lymphocytes were pretreated with either lysozyme and extensively washed prior to culture with mitogens. Under both conditions, the effects were strictly dose dependent; the lysozyme concentrations yielding maximal inhibitory effect were 5 micrograms/ml for HL and 1 microgram/ml for HEWL, while both lower and higher concentrations were less effective. Specific antilysozyme rabbit sera completely prevented the inhibitory effects of both HL and HEWL on the proliferative response of lymphocytes to phytohemagglutin or concanavalin A. Chitotriose (a lysozyme inhibitor) caused a strong reduction in the inhibitory effects of the two lysozymes on the lymphocyte response to either lectin. HL and HEWL also were found to markedly inhibit the polyclonal B cell proliferation and differentiation induced by pokeweed mitogen and T cells. A less marked inhibition was also obtained when T cells, but not B cells, were pretreated with HL or HEWL. Again, as in the experiments with T cell mitogens, the effects were dose dependent and 5 micrograms/ml HL and 1 microgram/ml HEWL proved to be the most effective concentrations. The possible mechanisms by which lysozyme inhibits the lymphocyte response to mitogenic lectins are considered and discussed. The enzymatic activity seemed to perform an essential function, as shown by the loss of effect when the heat- or trypsin-inactivated lysozymes were used and by the fact that only the enzymatically active compound, among certain semisynthetic derivatives of HEWL, inhibited the lymphocyte response to the mitogens. However, the cationic properties of the lysozyme molecule appeared to be essential too, since enzymes with a similar specificity of action showed effects similar to those observed with HL or HEWL only when they carried a strong positive charge. It is suggested that lysozyme, which is naturally secreted by monocytes and macrophages, might interact with lymphocyte surface receptor sites and participate in the complex mononuclear phagocyte-lymphocyte interactions and in the modulation of lymphocyte activation.     

Blockade of physiologically secreted IFN-gamma inhibits human T lymphocyte and natural killer cell activation

The role of physiologically secreted human IFN-gamma in T lymphocyte and NK cell activation has been probed with a panel of mouse mAb directed against various epitopes of the human IFN-gamma molecule, or human IFN-gamma R. Addition to the culture medium of those mAb that neutralize the antiviral activity of IFN-gamma or interact with its receptor inhibited proliferative and cytotoxic responses elicited in PBL by HLA alloantigens, anti-CD3 mAb, and IL-2, but not the proliferative response to PHA. The IFN-gamma blockade also inhibited IFN-gamma, IL-2, and TNF-alpha release during MLC. Kinetic experiments showed that reduction of proliferative and cytotoxic responses to HLA alloantigens is maximal when IFN-gamma is blocked within the first 48 h. Exogenous rIFN-gamma restored the proliferative response only when added at the beginning. Moreover, when IFN-gamma was blocked, T lymphocytes recovered from 6-day MLC displayed a profound decrease in their expression of p55 and p75 chains of the IL-2R, as well as in the number of high-affinity IL-2 binding sites. These findings strongly suggest that IFN-gamma is required in the early phases of induction of the oligo- and polyclonal proliferative and cytotoxic responses of lymphocytes.     

Regulation of lymphocyte responses by human gangliosides. I. Characteristics of inhibitory effects and the induction of impaired activation

Gangliosides obtained from normal human brain were found to inhibit the in vitro activation of human lymphocytes by nonspecific mitogens and allogeneic cells at concentrations between 3 to 50 microgram/1.5 to 1.7 X 10(5) lymphocytes/0.2 ml culture. Ganglioside inhibition did not represent cytotoxic effects or altered lectin binding and was independent of the mitogen concentration. In addition to concentration, the degree of inhibition was dependent on the mode of presentation to lymphocytes, since gangliosides incorporated within liposomal membranes displayed a synergistic inhibitory effect greater than predicted from the cultures receiving either gangliosides or liposomes alone. In binding experiments, radiolabeled ganglioside GM1 became associated with human lymphocytes within 10 min. However, approximately 72 hr pre-exposure of human lymphocytes to gangliosides was required to induce impaired lymphocyte responses to mitogens and allogeneic cells. Thus, concentrations of human gangliosides equivalent to the levels occurring in the sera of patients with certain malignancies are capable of actively inhibiting lymphocyte stimulation in addition to inducing impaired lymphocyte responses.     

Effect of cyclosporin A on human lymphocyte responses in vitro. III. CsA inhibits the production of T lymphocyte growth factors in secondary mixed lymphocyte responses but does not inhibit the response of primed lymphocytes to TCGF

The mechanism whereby Cyclosporin A (CsA) inhibits secondary mixed lymphocyte responses was assessed. CsA added to secondary MLR cultures inhibited proliferation and induction of cytolytic lymphocyte activity. This inhibition was found to be associated with the inhibition of T lymphocyte stimulating growth factor(s) (TCGF) production in the supernatants of secondary MLR cultures. As little as 1.0 micrograms/ml of CsA added to secondary MLR cultures resulted in no measurable TCGF activity. In contrast, moderate doses of CsA (1.0, 2.5 micrograms/ml), which completely inhibited the secondary MLR response to alloantigen, did not inhibit the proliferative and CML response of alloantigen-primed lymphocytes to these stimulating growth factors. Even at high doses of CsA (20 micrograms/ml), substantial levels of proliferation (50% of control response) and CML induction (60% of control response) were observed when the primed cells were exposed to secondary MLR supernatants containing TCGF activity. It was concluded that inhibition of secondary mixed lymphocyte responses by CsA may be due in part to the inhibition of TCGF production rather than the inhibition of the effect of TCGF on mature cytotoxic T lymphocytes.     

In vitro glucocorticoid inhibition of steroid-treated residual circulatory human lymphocytes

Various doses of glucocorticoids given in vivo caused a similar degree of maximal lymphopenia. The sensitivities of mitogen-induced proliferation to the suppressive effects of glucocorticoid added in vitro were studied in residual lymphocytes obtained after steroid injection. Methylprednisolone (MP) administered intravenously depleted circulatory lymphocytes and reduced markedly the proliferative responses of residual cells to mitogens (phytohemagglutinin and concanavalin A) 4 to 8 hr after the injection. The addition of MP in vitro to the residual cells further inhibited the cell proliferation. The degrees of proliferation inhibition induced by in vitro MP were compared in cells obtained at various intervals after MP injection. At each specific mitogen concentration, lymphocytes obtained at various intervals were inhibited to a similar degree by MP in the cultures. There was no evidence that cells obtained at the period of maximal lymphopenia, 4 to 8 hr after MP injection, were more resistant to the inhibition of glucocorticoid added in vitro. Hence, the residual lymphocytes were not “steroid-resistant” in the sense of proliferative responses to T-cell mitogens. These results indicate the mechanism of lymphocyte sequestration is unrelated to the suppressive effects of glucocorticoid on cell proliferation.     

Inhibition of murine suppressor cell function by progesterone

The effects of progesterone on murine suppressor cell function generated in allogeneic MLCs were investigated. BALB/c splenic lymphocytes stimulated in vitro with C3H/He cells significantly suppressed the proliferative response of BALB/c lymphocytes in a secondary MLC. This suppression was highly specific for the sensitizing alloantigens since the suppressor cells had no effect on the proliferative response of BALB/c lymphocytes to third-party alloantigens. In addition, BALB/c lymphocytes stimulated with syngeneic cells were observed to nonspecifically suppress the MLC response to a lesser extent. One to 10 micrograms/ml progesterone added at initiation to suppressor cell generating cultures diminished the ability of both alloantigen specific and nonspecific suppressor cell populations to suppress the proliferative response of homologous lymphocytes to alloantigens. Experiments with pyrilamine, an antihistamine, which blocks cytotoxic T lymphocyte (CTL) generation, suggests that progesterone has a direct inhibitory effect on suppressor cell function independent of its ability to block CTL induction. The effects of progesterone on suppressor cells were not due to shifts in peak response time in MLC or induction of radiosensitive cells in progesterone-treated cultures. Estradiol at doses between 5 and 10 micrograms/ml, and cortisol at dose of 1 microgram/ml, also significantly inhibited suppressor cell function. These results suggest that the steroid hormone milieu within the placenta may effect the activity of allogeneic or nonspecific suppressor cell activity.     

In vitro effects of retinoids on murine thymus-dependent and thymus-independent mitogenesis

The effects of three retinoids: all-trans-retinoic acid (RA), 13-cis-retinoic acid (13-cis-RA), and N-(4-hydroxyphenyl)retinamide (4-HPR) on murine splenic lymphocyte proliferative responses to mitogens were evaluated. The responses to T-cell mitogens, PHA and Con A, and a T-cell-dependent B-cell mitogen, PWM were significantly potentiated by these retinoids. However proliferative responses to a B-cell mitogen, Escherichia coli LPS were unaffected or inhibited. All three retinoids at concentrations ranging from 10(-6) to 10(-15) M significantly potentiated Con A-induced proliferative responses. In response to PWM, 10(-13) M RA, 10(-12) M 13-cis RA, and 10(-11) M 4-HPR were the lowest concentrations producing significant potentiation. Endpoint concentrations of retinoids significantly potentiating responses to PHA were; 10(-9) M RA, 10(-8) M 13-cis RA, and 10(-6) M 4-HPR. These responses were independent of retinol contained in fetal calf serum supplemented medium since responses were reproduced in serum-free medium devoid of retinol. Optimal potentiation by retinoids of responses to these T-cell-dependent mitogens were found at superoptimal concentrations of mitogen suggesting a selective inhibition of T-suppressor cells. Thus, potentiation of T-cell-dependent mitogen responses provides the most sensitive biological assay yet described for detection of retinoid activity and is a reproducible system to explore the cellular and molecular mechanisms of retinoid-mediated immunopotentiation.     

Effects of N-acyl-2-hydroxymethyl aziridines on in vitro proliferative responses of human lymphocytes stimulated by mitogens

Aziridines have been shown to possess marked immunotropic activity. The aim of this work was to study the in vitro effects of different concentrations of three novel aziridines, 2-hydroxy-methyl-1-(N-phtaloylglycyl) aziridine (aziridine 1), 2-hydroxy-methyl-1-(N-phtaloylalanyl) aziridine (aziridine 2) and 2-hydroxy-methyl-1-(N-phtaloylphenylalanyl) aziridine (aziridine 3), on the proliferative responses of human lymphocytes stimulated by mitogens (concanavalin A (Con A) and lipopolysaccharide (LPS)), and interleukin-2 (IL-2), interleukin-6 (IL-6) secretion. The results showed that aziridines 1 and 3 significantly stimulated the resting and Con A or LPS lymphocyte proliferation at concentrations between 1 micromol/l and 1 mmol/l, in a dose-dependent manner, the action of aziridine 3 being the highest. They also increased IL-2 and IL-6 secretion. However, aziridine 2 had no effect on the resting lymphocyte proliferation in the absence of mitogens, at any concentration used, reduced Con A-stimulated T lymphocyte proliferation and LPS- stimulated B lymphocyte proliferation in a dose dependent manner and diminished IL-2 and IL-6 production. None of the three aziridines affected cell viability. In conclusion, the three aziridines used in this study displayed immunomodulatory properties. Aziridines 1 and 3 are potentially immunostimulant while aziridine 2 is immunosuppressive and could be used to provide nonspecific cell-mediated immune responses.     

Glutathione modulates activation-dependent proliferation of human peripheral blood lymphocyte populations without regulating their activated function

L-Buthionine-(S,R)-sulfoximine (BSO) specifically depletes GSH synthesis by inactivating gamma-glutamylcysteine synthetase, whereas 2-ME augments intracellular GSH concentration. These reagents were used to examine GSH regulation of the proliferation and function of human PBL in response to IL-2 or OKT-3 mAb directed at the CD3 T cell Ag. 2-ME enhanced both IL-2-induced proliferation of PBL and CD3- large granular lymphocytes (LGL) and OKT-3 mAb-induced proliferation of CD3+ T cells. BSO partially suppressed activation-induced proliferation in CD3- LGL and CD3+ T cells and totally inhibited the positive co-proliferative regulation by 2-ME in these cells. By contrast, neither BSO nor 2-ME appeared to affect the activation-dependent differentiation of cytotoxic lymphocytes. The absence of effect of 2-ME or BSO on activation-induced PBL NK activity and T cell cytotoxic potential was supported by their negligible effect on the induction of two different markers of activated cytotoxic lymphocytes, namely pore-forming protein gene expression and benzoyloxycarbonyl-1-L-lysine thiobenzylester-esterase activity. BSO inhibition of CD3- LGL proliferation accounted for the inhibitory effects of BSO on both IFN-gamma production in IL-2-stimulated PBL cultures and IL-2-induced PBL lymphokine activated killer activity. The modulatory effects of 2-ME and BSO on lymphocyte proliferation regardless of phenotype (LGL vs T cell) or stimulation (IL-2, via CD3, lectin, etc.) and the functional differentiation of cytotoxic lymphocytes independent of proliferation suggests that these cells share a common site of GSH regulation close to or at the level of DNA synthesis.     

Natural regulatory cells in murine bone marrow: inhibition of in vitro proliferative and cytotoxic responses to alloantigens

A lymphocyte-enriched fraction of murine bone marrow (BML), obtained by sucrose density fractionation, contains natural regulatory cells that can profoundly suppress the proliferative and cytotoxic response of syngeneic lymph node cells to irradiated alloantigens in a mixed lymphocyte culture (MLC). A close correlation exists between the inhibition of alloantigen-induced proliferation and the generation of cytotoxic effectors. The suppression of proliferation is dependent on the dose of BML added to the cultures but is not due to cell crowding, since red blood cells, thymocytes, and irradiated splenocytes, all syngeneic to the lymph node responders, do not inhibit proliferation to the degree observed with BML. The addition of BML to cultures does not cause the maximum proliferative response to change from the usual day 5 peak, indicating that there is no change in culture kinetics. The release of nonradioactive thymidine by BML cannot explain the suppression. The target of suppression is maximally affected during the first 24 hr of culture, since adding BML to MLC later than this resulted in negligible inhibition of proliferation. Thus, the natural regulatory cell-mediated suppression reflects inhibition of "early" events in the proliferative response to alloantigens.     

Chemokine monokine induced by IFN-gamma/CXC chemokine ligand 9 stimulates T lymphocyte proliferation and effector cytokine production

Monokine induced by IFN-gamma (MIG; CXC chemokine ligand (CXCL)9) is important in T lymphocyte recruitment in organ transplantation. However, it is not known whether this chemokine, in addition to its chemotactic properties, exerts any effect on T lymphocyte effector functions. For in vivo studies, we used a previously characterized murine model of chronic rejection. The recipient mice were treated with anti-MIG/CXCL9 Ab; graft-infiltrating cells were analyzed for IFN-gamma production. For in vitro studies, exogenous CXCR3 ligands were added to CD4 lymphocytes in MLRs, and the proliferative responses were measured. Separate experiments quantitated the number of IFN-gamma-producing cells in MLRs by ELISPOT. Neutralization of MIG/CXCL9, in the in vivo model, resulted in significant reduction in the percentage of IFN-gamma-producing graft-infiltrating T lymphocytes. In vitro experiments demonstrated that 1) exogenous MIG/CXCL9 stimulated CD4 lymphocyte proliferation in a MHC class II-mismatched MLR, 2) MIG/CXCL9 also increased the number of IFN-gamma-producing CD4 lymphocytes in ELISPOT, 3) neutralization of MIG/CXCL9 in MLR reduced T lymphocyte proliferation, 4) IFN-gamma-inducible protein 10/CXCL10 and IFN-inducible T cell alpha chemoattractant/CXCL11 had similar effects on T lymphocyte proliferation, 5) MIG/CXCL9 stimulated T lymphocyte proliferation in MHC class I- and total MHC-mismatched MLRs, 6) neutralization of CXCR3 reduced MIG/CXCL9-induced T lymphocyte proliferation and the number of IFN-gamma-positive spots on ELISPOT, and 7) the proliferative effects of MIG/CXCL9 were mediated via an IL-2-independent pathway and were controlled by IFN-gamma. This study demonstrates that MIG/CXCL9 stimulates T lymphocyte proliferation and effector cytokine production, in addition to its chemotactic effects. This novel observation expands our current understanding of MIG/CXCL9 biology beyond that of mediating T cell trafficking.     

Effect of cholecystokinin and gastrin on human peripheral blood lymphocyte functions, implication of cyclic AMP and interleukin 2

The effects in vitro of sulphated and desulphated cholecystokinin (CCK)-8, and of gastrin-17 and gastrin-34 were studied at concentrations from 10⁻¹⁴ M to 10⁻⁶ M on several functions of human peripheral blood lymphocytes, i.e.: adherence to substrate, mobility (spontaneous and directed by a chemical gradient or chemotaxis), and spontaneous and phytohaemagglutinin (PHA)-mediated proliferation. All peptides, at concentrations from 10⁻¹⁰ M to 10⁻⁸ M, inhibited significantly the mobility capacity and PHA-induced proliferation, and increased the adherence and spontaneous proliferation. A dose-response relationship was observed, with a maximum response of lymphocyte functions at 10⁻¹⁰ M. These peptides induced a significant increase of intracellular cAMP levels at 30 and 60 sec. Because lymphoproliferation requires production of interleukin 2 (IL-2) by lymphocytes, we also measured the IL-2 production in the presence of the CCK and gastrin peptides, finding that this production was higher than in the respective controls. When peptides were added to samples containing PHA, the IL-2 production was significantly decreased with respect to samples incubated with PHA alone. These results suggest that the CCK and gastrin peptides are negative modulators of lymphocyte mobility (spontaneous mobility and chemotaxis), causing an inhibition of these activities through an increase of intracellular cAMP levels, and of PHA-induced lymphoproliferation, which is mediated by a diminution of the IL-2 production by lymphocytes.     

Modulatory effect of zinc on the proliferative response of murine spleen cells to polyclonal T cell mitogens

To study the effect of zinc on the proliferative response to polyclonal T cell mitogens, spleen cells from C57BL/6 mice were cultured with or without ZnCl2 and stimulated with graded doses of concanavalin A or phytohemagglutinin. Addition of 10(-4) M ZnCl2 inhibited proliferation whereas 10(-5) to 10(-6) M ZnCl2 did not modify the response to suboptimal doses of mitogen but increased DNA synthesis in cultures stimulated with high doses of mitogen (10 or 20 micrograms/ml of concanavalin A and 10 or 25 microliters/ml of phytohemagglutinin) which are supraoptimal for C57BL/6 mice, and inhibited proliferation in cultures of spleen cells from animals of this strain, low responder to T cell mitogens. In contrast, supplementation with ZnCl2 did not enhance the response to mitogen of spleen cells from high responder BALB/c mice. The enhancing effects of ZnCl2 on the proliferative response of C57BL/6 cells were not observed following depletion of adherent cells or in cultures supplemented with 5 X 10(-5) M 2-mercaptoethanol, both conditions capable of abrogating the inhibitory effect of high mitogen doses on the response of C57BL/6 cells.