Secondary but not primary T cell responses are enhanced in CTLA-4-deficient CD8+ T cells

Negative as well as positive co-stimulation appears to play an important role in controlling T cell activation. CTLA-4 has been proposed to negatively regulate T cell responses. CTLA-4-deficient mice develop a lymphoproliferative disorder, initiated by the activation and expansion of CD4+ T cells. To assess the function of CTLA-4 on CD8+ T cells, CTLA-4(-/-) animals were crossed to an MHC class I-restricted 2C TCR transgenic mouse line. We demonstrate that although the primary T cell responses were similar, the CTLA-4-deficient 2C TCR+ CD8+ T cells displayed a greater proliferative response upon secondary stimulation than the 2C TCR+ CD8+ T cells from CTLA-4 wild-type mice. These results suggest that CTLA-4 regulates antigen-specific memory CD8+ T cell responses.     

Processing and release of IL-16 from CD4+ but not CD8+ T cells is activation dependent

IL-16 is synthesized as a precursor molecule of 68 kDa (pro-IL-16) that is processed by caspase-3, a member of the IL-1 converting enzyme (ICE) family. This cleavage results in a 13-kDa carboxy terminal peptide, which constitutes the bioactive secreted form of IL-16. We have previously reported constitutive IL-16 mRNA expression and pro-IL-16 protein in CD4+ and CD8+ T cells. Although bioactive IL-16 protein is present in unstimulated CD8+ T cells, there is no bioactive IL-16 present in CD4+ T cells. Along these lines, unstimulated CD8+ T cells contain active caspase-3. In the current studies we investigated the regulation of IL-16 protein and mRNA expression in CD4+ T cells and determined the kinetics of secretion following stimulation of the TCR. CD4+ T cells release IL-16 protein following antigenic stimulation, and this release is accelerated in time by costimulation via CD28. However, CD3/CD28 costimulation did not alter IL-16 mRNA appearance or stability in either CD4+ or CD8+ T cells. The secretion of bioactive IL-16 from CD4+ T cells correlated with the appearance of cleavage of pro-caspase-3 into its 20-kDa active form. Thus, resting CD8+ T cells contain active caspase-3 that is capable of cleaving pro-IL-16, whereas CD4+ T cells require activation for the appearance of active caspase-3. The mechanism of release or secretion of bioactive IL-16 is currently unknown, but does not correlate with cellular apoptosis.     

IL-2, but not IL-4 and other cytokines, induces phosphorylation of a 98-kDa protein associated with SHP-2, phosphatidylinositol 3'-kinase, and Grb2

Binding of IL-2 to its receptor activates several biochemical pathways, including JAK-STAT, Ras-mitogen-activated protein kinase, and phosphatidylinositol 3'-kinase (PI 3'-kinase) pathways. Recently, it has been shown that the SH2-containing phosphatase, SHP-2, becomes phosphorylated in response to IL-2 stimulation, associates with PI3'-kinase and Grb2, and can exert a positive regulatory role in IL-2 signaling. We now report the identification of a prominent 98-kDa protein (p98) found to be phosphorylated in response to IL-2 stimulation and coprecipitated with SHP-2, the p85 subunit of PI 3'-kinase and Grb2. Interestingly, whereas IL-4 is known to activate PI 3'-kinase, we did not observe any p98 phosphorylation in response to IL-4 stimulation. p98 can form a multipartite complex with all these proteins as immunodepleting with anti-p85 antiserum substantially reduced the amount of p98 immunoprecipitated by SHP-2 and Grb2; the converse was also true. Furthermore, phosphorylation of p98 did not occur in cells lacking JAK3, suggesting that it may be a JAK substrate. Finally, deglycosylation of p98 did not alter its migration, suggesting p98 is not a member of the recently described SHP substrate/signal-regulatory proteins family of transmembrane glycoproteins. Thus p98 is a prominent IL-2-dependent substrate that associates with multiple proteins involved in IL-2 signaling and may play an important role in coupling the different signal transduction pathways activated by IL-2.     

Regulation of cell growth by IL-2: role of STAT5 in protection from apoptosis but not in cell cycle progression

Cytokines play an essential role in the regulation of lymphocyte survival and growth. We have analyzed the pathways activated by IE-2 that lead to protection from apoptosis and cell proliferation. IL-2 can act as a long-term growth factor in 32D cells expressing the wild-type human (hu)IL-2R beta. By contrast, cells expressing a truncated form of the huIL-2R beta, which is able to induce Bcl-2 and c-myc expression but not STAT5 activation, were not protected from apoptosis by IL-2; consequently, they could not be grown long term in the presence of IL-2. However, IL-2 promoted cell cycle progression in cells bearing the truncated huIL-2R beta with percentages of viable cells in the G0/G1, S, and G2/M phases similar to cells expressing the wild-type huIL-2R beta. Transplantation of a region from the erythropoietin receptor, which contains a docking site for STAT5 (Y343) to the truncated huIL-2R beta, restored the ability of IL-2 to signal both activation of STAT5 and protection from apoptosis. By contrast, transplantation of a region from the huIL-4R alpha containing STAT6 docking sites did not confer protection from apoptosis. These results indicate that the IL-2-induced cell cycle progression can be clearly distinguished from protection from apoptosis and that STAT5 participates in the regulation of apoptosis.     

Thrombopoietin induces association of Crkl with STAT5 but not STAT3 in human platelets

Crkl, a 39-kD SH2, SH3 domain-containing adapter protein, is constitutively tyrosine phosphorylated in hematopoietic cells from chronic myelogenous leukemia (CML) patients. We recently reported that thrombopoietin induces tyrosine phosphorylation of Crkl in normal platelets. In this study, we demonstrate that thrombopoietin induces association of Crkl with a tyrosine phosphorylated 95- to 100-kD protein in platelets and in UT7/TPO cells, a thrombopoietin-dependent megakaryocytic cell line. With specific antibodies against STAT5, we demonstrate that the 95- to 100-kD protein in Crkl immunoprecipitates is STAT5. This coimmunoprecipitation was specific in that Crkl immunoprecipitates do not contain STAT3, although STAT3 becomes tyrosine phosphorylated in thrombopoietin-stimulated platelets. The coimmunoprecipitaion of Crkl with STAT5 was inhibited by the immunizing peptide for Crkl antisera or phenyl phosphate (20 mmol/L). After denaturing of Crkl immunoprecipitates, Crkl was still immunoprecipitated by Crkl antisera. However, coimmunoprecipitation of STAT5 was not observed. Coincident with STAT5 tyrosine phosphorylation, thrombopoietin induces activation of STAT5 DNA-binding activity as demonstrated by electrophoretic mobility shift assays (EMSA). Using a beta-casein promoter STAT5 binding site as a probe, we have also demonstrated that Crkl antisera supershift the STAT5-DNA complex, suggesting that Crkl is a component of the complex in the nucleus. Furthermore, interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin also induce Crkl-STAT5 complex formation in responding cells in a stimulation-dependent manner. In vitro, glutathione S-transferase (GST)-Crkl bound to STAT5 inducibly through its SH2 domain. These results indicate that thrombopoietin, IL-3, GM-CSF, and erythropoietin commonly induce association of STAT5 and Crkl and that the complex translocates to the nucleus and binds to DNA. Interestingly, such association between STAT5 and Crkl was not observed in cytokine-stimulated murine cells, suggesting an intriguing possibility that components of the human STAT5-DNA complex may be different from those of the murine counterpart.     

Evidence for a critical role for IL-2 in CD40-mediated activation of naive B cells by primary CD4 T cells

The interaction of CD40 on B cells with the CD40 ligand (CD40L) on preactivated CD4 T cells is critical for the initiation of T-dependent Ab responses. It is believed that signals via CD40 synergize with cytokines (e.g., IL-4 and IL-5) to drive B cell activation. However, primary T cells preactivated via CD3 alone cannot induce B cell proliferation; we have shown previously that costimulation of T cells via CD3 and CD28 stabilizes the expression of the CD40L, which we propose contributes to their capacity to act as competent helper-effector cells. Here we show that an additional, critical reason why CD3-stimulated CD40L-bearing T cells are incompetent helper cells is because they secrete insufficient IL-2. In contrast, CD28/CD3-activated T cells induce B cells to become IL-2 responsive via a combination of CD40L and IL-2-mediated signals, and these two stimuli subsequently drive B cell proliferation and IgM secretion. We therefore propose that T cells must first encounter Ag in conjunction with CD80/86 on APCs. This leads to the stable expression of CD40L and maximal secretion of IL-2, which together render primary T cells competent to activate B cells in an IL-2-dependent fashion.     

IL-4 induces functional cell-surface expression of CXCR4 on human T cells

Here we report that IL-4 specifically enhances cell surface expression of CXCR4 on resting peripheral and cord blood T cells. Whereas polarized Th2 clones express variable levels of CXCR4, expression of this receptor is undetectable on polarized Th1 clones but can be induced on the latter cells as well, following short-term culture in the presence of IL-4. The IL-4-induced CXCR4 is functional since interaction with its ligand, stromal-derived factor (SDF)-1, activates the p42 MAP-kinase ERK-2. In addition, although CXCR4 expression is down-regulated following stimulation of T cells and T cell clones via CD28 or CD3 and CD2 cell surface molecules, respectively, it is re-induced by IL-4. These data indicate an important role for IL-4 in rendering CD4+ T cells susceptible to infection with HIV via CXCR4, as well as in promoting SDF-1-induced migration of these cells.     

Cobalt-chromium-molybdenum but not titanium-6aluminium-4vanadium alloy discs inhibit human T cell activation in vitro

The Authors studied the morphological, biochemical, physico-chemical and biological characteristics of Vibrio anguillarum cultured on different growth conditions, characterized by low osmolarity and high temperature (37 degrees). One culture was subcultured for several days in tryptone soya agar with 0.5% Nacl at 37 degrees C incubation until the cell morphology was stabilized. The low osmolarity, through an osmotic shock, induced remarkable morphological modifications in the strain, evidenced by optic and electron microscopic studied; in addition SDS-PAGE analysis of saline extracts from the culture at 37 degrees C showed a specific new protein band of about 66KDa. This band was correlated with remarkable differences in outer membrane protein composition (OMPs) evidenced by Ag/Ah cross-reactions with rabbit hyperimmune sera against the modified and the reference V. anguillarum strains. Finally, the modified strain proved to be non pathogenic for trout and sea bass.     

IL-7 promotes the survival and maturation but not differentiation of human post-thymic CD4+ T cells

This study examines the influence of IL-7 on post-thymic CD4+ T cells using cord blood as a model system. Survival of naive cord blood T cells in the presence of IL-7 alone was significantly prolonged by up-regulating bcl-2, thereby preventing apoptosis while maintaining maximal cell viability. Cultures without IL-7 showed high rates of apoptosis resulting in 50% cell death by day 5 of culture. Upon phorbol 12-myristate 13-acetate + ionomycin stimulation, accumulation of cytoplasmic IL-2 was similar to that observed in freshly isolated cells, but no IL-4- or IFN-gamma-positive cells were detected. IL-7 maintained the naive T cells in a quiescent state expressing the CD45RA antigen. A significant finding was the loss of CD38 antigen expression on the naive cord blood T cells to levels similar to that observed on adult naive T cells. In contrast to the reduced proliferative response of fresh cord blood T cells to anti-CD2 + CD28 stimulation, the proliferative response of IL-7-treated cells was similar to that of adult naive T cells. This study shows that as well as maintaining the naive T cell pool by enhancing cell survival and up-regulating bcl-2 expression, IL-7 also functions as a maturation factor for post-thymic naive T cells.     

High lytic activity against human leukemia cells after activation of allogeneic NK cells by IL-12 and IL-2

IL-12 is a novel cytokine with interesting features regarding its potential usefulness in peripheral blood stem cell transplantation and leukemia immunotherapy. We used cryopreserved leukemia cells of 18 patients with acute myelogenous (n= 14) or lymphocytic (n= 4) leukemia to investigate the effect of IL-12, alone or in combination with IL-2, on the cytolytic activity of NK cells against human leukemia targets. Effector cells were peripheral blood mononuclear cells from healthy donors which were depleted from CD3+ T cells by immunomagnetic separation. CD3-negative effector cells (mainly CD56+ NK cells) were treated for 24 h with various concentrations of IL-2 (100 U/ml to 1000 U/ml) and IL-12 (1 U/ml to 100 U/ml). Cytotoxicity was measured in a 4 h 51Cr-release assay. Whereas a two-fold enhancement of cytotoxic activity was observed after incubation with optimal doses of IL-2 or IL-12, the combination of both cytokines (500 U/ml IL-2, 100 U/ml IL-12) increased the lytic activity more than six-fold. This effect was accompanied by increased expression of cellular adhesion molecules (CD2, CD18) and CD25 on CD56+ effector cells. Of 18 leukemias investigated, five were completely resistant to lysis by effector cells activated with IL-2 or IL-12 alone. In three of these five cases, however, high cytolytic activity was observed after coincubation with IL-2 and IL-12. In comparison to allogeneic NK cells, autologous cells of three patients in remission demonstrated significantly lower cytotoxic activity. No killing of nonmalignant cells (PHA blasts) by allogeneic NK cells was observed. Our data demonstrate that IL-12 can enhance or even induce MHC-unrestricted cytotoxicity of IL-2-activated allogeneic natural killer cells. Since IL-12 has also been shown to have stem-cell mobilizing capacities, it could be used for the recruitment of both stem cells and antileukemic effector cells in the context of peripheral blood stem cell transplantation.     

Induction of IL-1 beta-converting enzyme-independent apoptosis by IL-2 in human T cell lines

Our previous study demonstrated that IL-2 suppressed growth of human T cell lines, in which the suppression was observed with members among HTLV-I-infected T cell lines independent of IL-2 for growth. In this study, we examined the molecular mechanism of IL-2-induced growth suppression with two HTLV-I-infected T cell lines; TL-OmI expressing endogenously three subunits, i.e. alpha, beta and gamma chains, of the IL-2 receptor, and an MT-1 transfectant expressing the endogenous alpha and gamma chains and exogenous beta chain. Our analysis revealed that IL-2 induced apoptosis in both T cell lines. Experiments with inhibitors for the proteases responsible for apoptosis signals showed that caspase 1 (IL-1 beta-converting enzyme) was not involved in apoptosis induced by IL-2. Other MT-1 sublines introduced with mutant beta chains demonstrated that IL-2-induced apoptosis required signals from both the serine-rich (S) region and acidic (A) region of the IL-2 receptor beta chain, which are essential but not critical for IL-2-mediated cell growth respectively. Collectively, IL-2 functions not only on growth promotion and prevention of apoptosis but also on induction of apoptosis, which may be implicated in physiological regulation of immune reactions by controlling growth and activation of T cells.     

IL-4 inhibits P-glycoprotein in normal and malignant NK cells

Patients presenting with a natural killer (NK) cell leukemia generally have a poor prognosis. NK cell tumors are generally resistant to numerous chemotherapeutic drugs and even combination chemotherapy usually results in only short term remissions. The drug resistance of NK cell leukemias may be at least partially explained by their expression of the multidrug resistant transporter, P-glycoprotein (Pgp). In this study, we demonstrate that the expression and function of Pgp activity on NK cells (leukemic and normal) can be reversed with IL-4.     

Differential regulation of the Janus kinase-STAT pathway and biologic function of IL-13 in primary human NK and T cells: a comparative study with IL-4

We describe new methods for analyzing the apolipoproteins (apo) of the high density lipoproteins (HDL) of several species by two modes of capillary electrophoresis: size separation using a molecular sieving buffer, and capillary zone electrophoresis (CZE) using neutral coated capillaries. By either mode HDL apos were resolved within 25 min. Results for apoA-I and apoA-II mass agreed with those by electroimmunoassay; intra-assay coefficients of variation were 1.8-4.2%. The migration times of human, rat, rabbit, and bovine apoA-I during CZE were proportional to their net charge/Mr ratios. This enabled human and rabbit apoA-I to be quantified simultaneously in transgenic rabbit HDLs. CZE also resolved human apoA-I isoforms, deamidated apoA-I, and pro-apoA-I.     

Induction of Th2 cell differentiation in the primary immune response: dendritic cells isolated from adherent cell culture treated with IL-10 prime naive CD4+ T cells to secrete IL-4

Rates of hormone replacement therapy (HRT) in women have varied substantially over the last 25 years. Data on the impact of recent recommendations for widespread use to prevent cardiovascular disease and osteoporosis and factors that influence use are needed. We attempted to (1) describe recent trends in HRT use, (2) investigate the relationship between HRT use and prepaid drug benefit, and (3) detail prescribing frequencies by provider specialty. We conducted a cross-sectional analysis of annual HRT pharmacy dispensings from 1986 to 1995 in a large HMO to all female HMO members aged 45 years and older. HRT rates increased among all age categories, although the magnitude of change varied by age. Highest rates of use were found in those 50-59 years old. Although combined estrogen-progestin use increased, 57% of all estrogen users did not receive progestin in 1995. Unopposed estrogen use was largely limited to hysterectomized women. Women of all ages with no prepaid drug benefit as part of their HMO coverage had the lowest HRT rates. Internal medicine, obstetrics/gynecology, and family practice providers prescribed over 90% of HRT, and prescriber specialty varied with user age. HRT use increased in the HMO from 1986 to 1995, especially among younger women. In 1995, about half of women aged 50-64 years received one or more HRT dispensings. As the benefits, risks, and cost effectiveness of HRT depend on the duration of use, additional information on current use duration is needed. Combined estrogen-progestin use increased and appeared appropriate to hysterectomy status. Research is needed to determine if lower HRT use rates among women without a prepaid drug benefit indicate less prophylactic HRT use, particularly among younger women, for whom this lack of coverage was relatively common.     

Evidence for repression of IL-2 gene activation in anergic T cells

The induction of clonal anergy in a T cell inhibits IL-2 secretion because of the development of a proximal signal transduction defect. Fusion of anergic murine T cells to human Jurkat T leukemia cells and formation of heterokaryons failed to result in a complementation of this signaling defect and restoration of murine IL-2 mRNA inducibility. Instead, signal transduction to the human IL-2 gene became disrupted. Heterokaryons formed by the fusion of anergic murine T cells to normal murine T cells also failed to accumulate intracellular IL-2 protein in response to stimulation either with the combination of CD3 and CD28 mAbs or with ionomycin plus a protein kinase C-activating phorbol ester. The results argue against a loss-of-function signaling defect as the sole basis for clonal anergy induction and document the presence of a dominant-acting repressor molecule that inhibits signal transduction to the IL-2 gene within viable anergic T cells.     

SPARC/osteonectin induces matrix metalloproteinase 2 activation in human breast cancer cell lines

Activation of the matrix metalloproteinase 2 (MMP-2) has been shown to play a major role in the proteolysis of extracellular matrix (ECM) associated with tumor invasion. Although the precise mechanism of this activation remains elusive, levels of the membrane type 1-MMP (MT1-MMP) at the cell surface and of the tissue inhibitor of MMP-2 (TIMP-2) appear to be two important determinants. Induction of MMP-2 activation in cells cultivated on collagen type I gels indicated that the ECM is important in the regulation of this process. In this study, we show that SPARC/osteonectin, a small ECM-associated matricellular glycoprotein, can induce MMP-2 activation in two invasive breast cancer cell lines (MDA-MB-231 and BT549) but not in a noninvasive counterpart (MCF-7), which lacks MT1-MMP. Using a set of peptides from different regions of SPARC, we found that peptide 1.1 (corresponding to the NH2-terminal region of the protein) contained the activity that induced MMP-2 activation. Despite the requirement for MT1-MMP, seen in MCF-7 cells transfected with MT1-MMP, the activation of MMP-2 by SPARC peptide 1.1 was not associated with increased steady-state levels of MT1-MMP mRNA or protein in either MT1-MMP-transfected MCF-7 cells or constitutively expressing MDA-MB-231 and BT549 cells. We did, however, detect decreased levels of TIMP-2 protein in the media of cells incubated with peptide 1.1 or recombinant SPARC; thus, the induction of MMP-2 activation by SPARC might be due in part to a diminution of TIMP-2 protein. We conclude that SPARC, and specifically its NH2-terminal domain, regulates the activation of MMP-2 at the cell surface and is therefore likely to contribute to the proteolytic pathways associated with tumor invasion.