Regulation of mitogen-stimulated human T-cell proliferation, interleukin-2 production, and interleukin-2 receptor expression by protein kinase C inhibitor, H-7

Recently published reports suggest that the activation of protein kinase C (PKC) plays an important role in the activation pathway of many cell types. In this study, we examined the role of PKC in human T-cell proliferation, IL-2 production, and IL-2R expression, when cultured with the mitogen PHA, the PKC inhibitor H-7, and H-7 control HA1004. H-7 inhibited the PHA-stimulated [3H]thymidine uptake, IL-2 production, and IL-2R expression in a dose-related manner. Further, we found H-7 inhibited T-cell proliferation, IL-2 production, IL-2 mRNA from PHA plus PMA-stimulated cultures. We also found that H-7 inhibited the early-stage activation of PHA-stimulated cells. The presence of exogenous purified human IL-2 or rIL-4 partly reversed the immunosuppression caused by H-7. In contrast, HA1004 had no effect on cell proliferation, IL-2 production, or IL-2R expression. Our results demonstrate that PKC activation is one major pathway through which T-cells become activated.     

Reversal of immune dysfunction in osteopetrotic rats by interferon-gamma: augmentation of macrophage Ia expression and lymphocyte interleukin-2 production and proliferation

Lymphocytes from osteopetrotic (op) rats, compared to their normal (n) littermates, exhibit defective immune functions associated with their inability to resorb bone. Among these immune defects are the failure of their spleen cells to proliferate normally to mitogens and to generate IL-2. Addition of exogenous IL-2 failed to reverse the suppressed proliferation in the op spleen cells, indicating that additional defects were involved in the suppression. Phenotypic analysis of cellular constituents of op and n spleens revealed that the percentages of T cells, macrophages, and IL-2 receptor positive cells were not different. Furthermore, there was no difference in CD4 (W3/25) and CD8 (OX8) cells. However, the Ia+ (OX3) cells in the op spleen represented less than 50% of those found in the n spleen, but the op had higher levels of transferrin receptor (OX26). On the basis of the ability of interferon-gamma (IFN-gamma) to increase Ia expression, this cytokine was added to op spleen cells (10-50 U/ml) and found to increase the number of Ia+ cells to the level found in n spleen cells. Moreover, pretreatment of op spleen cells with IFN-gamma restored their ability to proliferate to mitogens and their responsiveness to IL-2. Not only did IFN-gamma reverse the defective response to IL-2, but it also augmented the defective IL-2 production by op spleen cells. Taken together, these findings demonstrate that IFN-gamma can reverse many of the impaired immune functions characteristic of op spleen cells in vitro. Furthermore, these data suggest that IFN-gamma may provide an important avenue of treatment in these animals that may contribute to restoration of normal bone resorption.     

Notch signaling augments T cell responsiveness by enhancing CD25 expression

Notch receptors signal through a highly conserved pathway to influence cell fate decisions. Notch1 is required for T lineage commitment; however, a role for Notch signaling has not been clearly defined for the peripheral T cell response. Notch gene expression is induced, and Notch1 is activated in primary CD4(+) T cells following specific peptide-Ag stimulation. Notch activity contributes to the peripheral T cell response, as inhibition of endogenous Notch activation decreases the proliferation of activated T cells in a manner associated with the diminished production of IL-2 and the expression of the high affinity IL-2R (CD25). Conversely, forced expression of a constitutively active Notch1 in primary T cells results in increased surface expression of CD25, and renders these cells more sensitive to both cognate Ag and IL-2, as measured by cell division. These data suggest an important role for Notch signaling during CD4(+) T cell responses, which operates through augmenting a positive feedback loop involving IL-2 and its high affinity receptor.     

Regulation of lymphocyte proliferation and death by FLIP

Lymphocyte homeostasis is a balance between lymphocyte proliferation and lymphocyte death. Tight control of apoptosis is essential for immune function, because its altered regulation can result in cancer and autoimmunity. Signals from members of the tumour-necrosis-factor receptor (TNF-R) family, such as Fas and TNF-R1, activate the caspase cascade and result in lymphocyte death by apoptosis. Anti-apoptotic proteins, such as FLIP (also known as FLICE/caspase-8 inhibitory protein) have recently been identified. FLIP expression is tightly regulated in T cells and might be involved in the control of both T-cell activation and death. Abnormal expression of FLIP might have a role not only in autoimmune diseases, but also in tumour development and cardiovascular disorders.     

Effects of interleukin-2 therapy on the proliferation and differentiation of CD4/CD25 positive and CD4/CD25 negative cells in HIV+ patients

Interleukin-2 has been widely used in HIV-1+ subjects as an immunoactivating agent. In this study, we investigated cytokine production, Ki67 antigen expression and the modulation of the surface phenotype of the CD4/CD25+ subset as compared to the reciprocal CD4/CD25- subset in IL-2-treated HIV+ patients. Our findings suggest that CD4 T cells are heterogeneous in responding to IL-2, because CD4/CD25+ cells sharply increased their "memory" phenotype, their Ki67 antigen expression and were the main in vivo targets for IL-2-dependent proliferation during therapy, while the percentages of IFN-gamma+ (terminally differentiated) cells remained unchanged at the end of therapy. Conversely, the CD4+/CD25- subpopulation showed an expansion of differentiated cells and a slight increase in the proliferation rate. The use of anti-retroviral therapy alone (HAART) reduced the proliferation and increased the differentiation of both CD4 subsets. Our data suggest that IL-2 has a moderate capacity to activate resting T cells in vivo and is probably unable to boost HIV-1 from latency to the replicative state.     

Polyamine biosynthesis is necessary for interleukin-2-dependent proliferation but not for interleukin-2 production or high-affinity interleukin-2 receptor expression

DL-alpha-Difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase [EC 4.1.1.17] (ODC), inhibited concanavalin A-induced proliferation of splenic mononuclear cells (SMNC). The inhibition was not reversed by interleukin-2 (IL-2) addition. Although DFMO did not affect the production of IL-2 or the expression of high-affinity IL-2 receptor, IL-2-dependent proliferation of SMNC was inhibited by DFMO, and the inhibition was reversed by exogenous putrescine. The inhibition of IL-2-dependent DNA synthesis appeared to be related to the decrease in intracellular polyamines. When the proliferation of SMNC was induced by IL-2, ODC activity was also increased. A similar result was obtained in the proliferation of an IL-2-dependent T cell line, CTLL. The time course of ODC induction was similar to that of IL-2 production by concanavalin A-stimulated SMNC. These results indicate that polyamine biosynthesis is necessary for IL-2-dependent proliferation, but not for IL-2 production or IL-2 receptor expression.     

Regulation of T lymphocyte differentiation and proliferation by thymus hormones, Ca2+ and chalone-T

The regulatory effect on T lymphocyte proliferation of the differentiator thymosin hormone family (thymosin fr. 5, A. L. Goldstein), Ca2+, and purified inhibitory protein fractions prepared from calf thymus was investigated in C57Bl/6 mice. 1.2 mM Ca2+ concentration was the most favourable for murine thymocyte growth in culture. Net protein synthesis was transitorily inhibited by Cu2+ concentrations higher than 2 mM. This inhibition was followed by a marked inhibition of DNA synthesis 2 hrs later. The effect of thymosin fr. 5 was slight, of short duration, and oscillatory in nature; in contrast, chalone-T preparations inhibited thymocyte DNA synthesis permanently up to 12 hrs of cultivation. When spleen cells taken from mice treated with the immunoadjuvant Bacillus Calmette-Guérin (BCG) were exposed to chalone-T in culture, then stimulated with PHA, a reduced proliferative response was measured in chalone-T pretreated cultures compared to controls or spleen cells from normal non-BCG-activated mice. This result had led us to suggest that chalone-T has a dual effect on thymocytes, viz. it inhibits cell cycle progression and induces the phenotypic conversion of suppressor T lymphocytes. The multifactorial concept of T lymphocyte production is discussed.     

Regulation of lymphocyte proliferation and differentiation by macrophages.

In this paper, we consider the role of a macrophage secretory product in promoting thymocyte differentiation, as well as a macrophage-immune T cell interaction that results in augmented secretion of lymphostimulatory factors. When cultured with the thymocyte-differentiating factor (TDF), thymocytes show a physiological increase in H-2D and K, decreased sensitivity to lysis with anti-TL and complement, and acquisition of responsiveness in the mixed lymphocyte culture. Development of the mature phenotype requires 2 to 3 days of culture and, once attained, is stable. The induced antigenic changes do not require cell division. The activity demonstrated by TDF, which is not attributable to interferon and cannot be replaced by 2-mercaptoethanol, is also displayed by normal thymic macrophages themselves. Enhanced secretion of TDF and of a distinct mitogenic protein follows the interaction of macrophages and immune T cells. This interaction is shown to require physical contact of the two cell types and is regulated by products of the I-A region of the major histocompatibility complex.     

Regulation of human lymphocyte proliferation by fatty acids

In the present study, the effect of increasing concentrations of palmitic (PA, C16:0), stearic (SA, C18:0), oleic (OA, C18:1, n-9), linoleic (LA, C18:2n-6), docosahexaenoic (DHA, C22:6 n-3) and eicosapentaenoic (EPA, C20:5 n-3) acids on lymphocyte proliferation was investigated. The maximal non-toxic concentrations of these fatty acids for human lymphocytes in vitro were determined. It was also evaluated whether these fatty acids at non-toxic concentrations affect IL-2 induced lymphocyte proliferation and cell cycle progression. OA and LA at 25 microM increased lymphocyte proliferation and at higher concentrations (75 microM and 100 microM) inhibited it. Both fatty acids promoted cell death at 200 microM concentration. PA and SA decreased lymphocyte proliferation at 50 microM and promoted cell death at concentrations of 100 microM and above. EPA and DHA decreased lymphocyte proliferation at 25 and 50 microM being toxic at 50 and 100 microM, respectively. PA, SA, DHA and EPA decreased the stimulatory effect of IL-2 on lymphocyte proliferation, increasing the percentage of cells in G1 phase and decreasing the proportion of cells in S and G2/M phases. OA and LA caused an even greater pronounced effect. The treatment with all fatty acids increased neutral lipid accumulation in the cells but the effect was more pronounced with PA and DHA. In conclusion, PA, SA, DHA and EPA decreased lymphocyte proliferation, whereas OA and LA stimulated it at non-toxic concentrations.     

Lysozyme as a regulator of interleukin-2-activated lymphocyte proliferation

Summary Lysozyme at 1 to 100μg/ml of exposure levels augmented or inhibited proliferative response of human peripheral blood lymphocytes stimulated with interleukin-2 (IL-2). This contradictory effect of lysozyme depended on IL-2 concentration, activating state of lymphocytes, addition time of lysozyme, and serum existence. Lymphocytes increased their IL-2-mediated proliferating ability in response to lysozyme when stimulated with less than suboptimal concentration of IL-2. Lymphocyte activation with anti-CD3 antibody changed the augmented proliferative response into the inhibited response by lysozyme addition whereas elimination of MHC class II molecule-expressing cells augmented the response. Addition of lysozyme within 1 h after IL-2 exposure was most effective in promoting the proliferation whereas additions after 16 to 24 h were ineffective or inhibitory. Addition after longer than 24 h inversely restored the proliferative response. Serum seemed to retard lysozyme action because either sequential serum addition 1 h after exposure of IL-2 and lysozyme to cells or exposure of IL-2 and serum after pretreatment of cells with lysozyme changed the proliferative responsiveness from inhibition into augmentation. Thus lysozyme may regulate lymphocyte proliferation responding to a magnitude of antigenic stimuli and to the progression of cellular events that periodically occur.     

Phytohemagglutinin induced proliferation by aged lymphocytes: reduced expression of high affinity interleukin-2 receptors and interleukin-2 secretion

Human lymphocytes from elderly and young donors were cultured with phytohemagglutinin. Cultures from two groups of aged donors, recruited respectively from our ambulatory clinic and a nursing home, incorporated less tritiated thymidine (3H-TdR) and secreted less interleukin-2 than did young donors. Furthermore, as determined for the first time by a radioligand binding receptor assay, the aged lymphoblasts possessed significantly fewer high affinity IL-2 receptors per cell. Despite a decrease in the number of high affinity receptor cells the dissociation constant (Kd) was comparable for the three groups. It was also shown that the amounts of soluble IL-2 receptors that were released into the supernatants by mitogen stimulated cells did not differ for the aged and young donors. These data suggest that defects in IL-2 production and high affinity IL-2 receptor generation may both be responsible for immune deficiency in the elderly.     

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.     

Interleukin-2 dose,blood monocyte and CD25+ lymphocyte counts as predictors of clinical response to interleukin-2 therapy in patients with renal cell carcinoma

Summary The purpose of this study was to determine immunological parameters in the peripheral blood that correlate with the clinical effect of interleukin-2 (IL-2) in patients with metastatic renal cell cancer. A group of 26 patients with metastatic renal cell cancer underwent IL-2 treatment using a 36-day schedule with continuous intravenous IL-2 infusion (3 × 10⁶ units m^–2 day^–1) administered from days 1 to 5 and days 12 to 16. The white blood cell count and the absolute and relative number of neutrophils, lymphocytes, eosinophils and monocytes were recorded six times in peripheral blood during the treatment. Also the blood counts of T cell and NK cell subsets and cells expressing the T cell activation markers IL-2R and VLA-1 were measured. The lymphokine-activated killer (LAK) cell cytotoxicity was measured either with or without additional in vitro stimulation by IL-2. Multivariate statistical analysis showed that the clinical responses were related to the administered dose of IL-2, to a low number of blood cells expressing IL-2 receptors and to a reduction in the blood monocyte count (P <0.05).     

Streptavidin suppresses T cell activation and inhibits IL-2 production and CD25 expression

Streptavidin is widely used as a detection tool in biology research because of its high affinity and specificity binding to biotin. Biotin-streptavidin system has also been explored for detection of infection and tumor in clinical medicine. Here, we show immunosuppressive property of streptavidin on T cell activation and proliferation. Upon CD3 and CD28 stimulation, CD4(+) T cells produce interleukin 2 (IL-2) and express IL-2 receptor α chain (CD25). Addition of streptavidin in T cell culture suppressed IL-2 synthesis and CD25 expression with no cytotoxicity. The immunosuppressive effect of streptavidin was reversed by excessive biotin. Conjugated to a single chain anti-CD7 variable fragment (scFvCD7), streptavidin was directly delivered to T cells and showed substantially more profound suppressive effect on T cell activation. These results suggest that streptavidin could potentially be used as a novel immunomodulator.     

Differential interleukin-2 and interferon-gamma production by human lymphocyte cultures exceptionally resistant to Epstein-Barr virus immortalization

Epstein-Barr virus (EBV) readily immortalizes human peripheral blood lymphocytes (PBL) in vitro. However, during the past several years, we found that PBL from two exceptional EBV-seropositive healthy adult individuals were refractory to immortalization by EBV. We report here a study aimed at learning about the immunobiological features which differentiate these EBV-resistant (R) PBL from others which are susceptible (S) to EBV immortalization. Results of this investigation indicate that: (a) Following EBV infection, R-PBL produced significantly higher amounts of interferon gamma (IFN-gamma) than S-PBL. There were however no differences in regard to interferon alpha production between these two types (R and S) of EBV-infected cultures. (b) R-PBL had a maximal interleukin-2 (IL-2) production by S-PBL occurred at least 48 hr later, i.e., at Day 7. (c) The percentage of non-B cells expressing the IL-2 receptor was also higher in EBV-infected R-PBL than S-PBL. (d) In contrast, expression of IL-2 receptors after EBV infection was higher on B cells from S-PBL than on B cells from R-PBL. Interestingly, no differences were noted in regard to IL-2 receptor expression between R-PBL and S-PBL treated with mitogens (i.e., phytohemagglutinin and pokeweed mitogen). (e) Finally, using anti-IL-2 and anti-IFN-gamma antibodies in EBV-infected R-PBL cultures, we were able to obtain EBV-induced immortalization of these cultures. Taken together, these results suggest that an early IL-2 synthesis and high IFN-gamma production by EBV-infected PBL play an important role against lymphocyte immortalization by EBV.     

Time-dependent expression of CD25 in phytohemagglutinin- or interleukin-2-stimulated human peripheral blood lymphocytes

The dynamics of the expression of the high-affinity receptor for interleukin-2 (IL-2 receptor) evaluated by the method of flow cytofluorimetry based on changes in the number of cells that express the CD25 marker (CD25+) was studied in human peripheral blood lymphocytes stimulated by various mitogens. It has been shown that, in the resting lymphocyte culture, both phytohemagglutinin (PHA, 10 μg/ml) and 12,13-phorbol dibutyrate (PDBu, 10⁻⁸ M) with ionomycin (IM, 5 × 10⁻⁷ M) induce a long-lasting increase (for 48 h) in the number of CD25+ cells. Interleukin-2 (IL-2) has only been found to be capable of inducing time-dependent CD25 expression in competent (not resting) lymphocytes pretreated with submitogenic doses of PHA (1 μg/ml). A comparison of the dynamics of the number of CD25+ cells and blast transformation has shown that CD25 markers are revealed as early as on small stimulated lymphocytes, while, at the late activation stages, which correspond to the stage of cell growth and transition to DNA synthesis, the overwhelming majority of blasts are CD25+ cells with high-affinity α -receptors for IL-2. The obtained data allow one to suggest that the expression of an α -subunit of IL-2 receptor takes place at the IL-2-dependent stage of T lymphocyte proliferation and may be directly induced by IL-2 via IL-2 receptor.