Selenium promotes T-cell response to TCR-stimulation and ConA, but not PHA in primary porcine splenocytes

There is controversy in the literature over whether the selenium (Se) influences cellular immune responses, and the mechanisms possibly underlying these effects are unclear. In this study, the effects of Se on T-cell proliferation and IL-2 production were studied in primary porcine splenocytes. Splenocytes were treated with different mitogens in the presence of 0.5-4 μmol/L sodium selenite. Se significantly promoted T-cell receptor (TCR) or concanavalin A (ConA)-induced T-cell proliferation and IL-2 production but failed to regulate T-cell response to phytohemagglutinin (PHA). In addition, Se significantly increased the levels of cytosolic glutathione peroxidase (GPx1) and thioredoxin reductase 1 (TR1) mRNA, the activity of GPx1 and the concentration of reduced glutathione (GSH) in the unstimulated, or activated splenocytes. These results indicated that Se improved the redox status in all splenocytes, including unstimulated, TCR, ConA and PHA -stimulated, but only TCR and ConA-induced T-cell activation was affected by the redox status. N-acetylcysteine (NAC), a pharmacological antioxidant, increased T-cell proliferation and IL-2 production by TCR and ConA stimulated splenocytes but had no effect on the response to PHA in primary porcine splenocytes confirming that PHA-induced T-cell activation is insensitive to the redox status. We conclude that Se promotes GPx1 and TR1 expression and increases antioxidative capacity in porcine splenocytes, which enhances TCR or ConA -induced T-cell activation but not PHA-induced T-cell activation. The different susceptibilities to Se between the TCR, ConA and PHA -induced T-cell activation may help to explain the controversy in the literature over whether or not Se boosts immune responses.     

B7-H4 Treatment of T Cells Inhibits ERK, JNK, p38, and AKT Activation

B7-H4 is a newly identified B7 homolog that plays an important role in maintaining T-cell homeostasis by inhibiting T-cell proliferation and lymphokine-secretion. In this study, we investigated the signal transduction pathways inhibited by B7-H4 engagement in mouse T cells. We found that treatment of CD3(+) T cells with a B7-H4.Ig fusion protein inhibits anti-CD3 elicited T-cell receptor (TCR)/CD28 signaling events, including phosphorylation of the MAP kinases, ERK, p38, and JNK. B7-H4.Ig treatment also inhibited the phosphorylation of AKT kinase and impaired its kinase activity as assessed by the phosphorylation of its endogenous substrate GSK-3. Expression of IL-2 is also reduced by B7-H4. In contrast, the phosphorylation state of the TCR proximal tyrosine kinases ZAP70 and lymphocyte-specific protein tyrosine kinase (LCK) are not affected by B7-H4 ligation. These results indicate that B7-H4 inhibits T-cell proliferation and IL-2 production through interfering with activation of ERK, JNK, and AKT, but not of ZAP70 or LCK.     

Discovery of 2-amino-heteroaryl-benzothiazole-6-anilides as potent p56(lck) inhibitors

A series of structurally novel benzothiazole based small molecule inhibitors of p56(lck) was prepared to elucidate their structure-activity relationships (SAR), selectivity and cell activity in the T-cell proliferation assay. BMS-350751 (2) and BMS-358233 (3) are identified as potent Lck inhibitors with excellent cellular activities against T-cell proliferation.     

The non-genomic effects on Na+/H+-exchange 1 by progesterone and 20alpha-hydroxyprogesterone in human T cells

Progesterone is an endogenous immunomodulator and can suppress T-cell activation during pregnancy. We have previously shown that the non-genomic effects of progesterone, especially acidification, are exerted via plasma membrane sites and suppress cellular genomic responses to mitogens. This study aimed to show that acidification is due to a non-genomic inhibition of Na(+)/H(+)-exchange 1 (NHE1) by progesterone and correlate this with immunosuppressive phytohemagglutinin (PHA)-induced T-cell proliferation. The presence of amiloride-sensitive NHE 1 was identified in T cells. The activity of NHE1 was inhibited by progesterone but not by 20alpha-hydroxyprogesterone (20alpha-OHP). Furthermore, 20alpha-OHP was able to compete with progesterone and release the inhibitory effect on the NHE1. The inhibition of NHE1 activity by progesterone-BSA demonstrated non-genomic action via plasma membrane sites. Finally, co-stimulation with PHA and progesterone or amiloride, (5-(N, N-dimethyl)-amiloride, DMA), inhibited PHA-induced T-cell proliferation, but this inhibition did not occur with 20alpha-OHP and PHA co-stimulation. However, when DMA was applied 72 h after PHA stimulation, it was able to suppress PHA-induced T-cell proliferation. This is the first study to show that progesterone causes a rapid non-genomic inhibition of plasma membrane NHE1 activity in T cells within minutes which is released by 20alpha-OHP. The inhibition of NHE1 leads to immunosuppressive T-cell proliferation and suggests that progesterone might exert a major rapid non-genomic suppressive effect on NHE1 activity at the maternal-fetal interface in vivo and that 20alpha-OHP may possibly be able to quickly release the suppression when T cells circulated away from the interface.     

Metalloproteases regulate T-cell proliferation and effector function via LAG-3

Tight control of T-cell proliferation and effector function is essential to ensure an effective but appropriate immune response. Here, we reveal that this is controlled by the metalloprotease-mediated cleavage of LAG-3, a negative regulatory protein expressed by all activated T cells. We show that LAG-3 cleavage is mediated by two transmembrane metalloproteases, ADAM10 and ADAM17, with the activity of both modulated by two distinct T-cell receptor (TCR) signaling-dependent mechanisms. ADAM10 mediates constitutive LAG-3 cleavage but increases approximately 12-fold following T-cell activation, whereas LAG-3 shedding by ADAM17 is induced by TCR signaling in a PKCtheta-dependent manner. LAG-3 must be cleaved from the cell surface to allow for normal T-cell activation as noncleavable LAG-3 mutants prevented proliferation and cytokine production. Lastly, ADAM10 knockdown reduced wild-type but not LAG-3(-/-) T-cell proliferation. These data demonstrate that LAG-3 must be cleaved to allow efficient T-cell proliferation and cytokine production and establish a novel paradigm in which T-cell expansion and function are regulated by metalloprotease cleavage with LAG-3 as its sole molecular target.     

Interleukin 1 secretion is not required for human macrophage support of T-cell proliferation

Interleukin 1 (IL-1) is a soluble factor secreted by stimulated monocytes (Mo) and animal macrophages (Mx). We have previously demonstrated that human Mo cultured in vitro for 1-6 days transform to Mx, and retain their ability to support concanavalin A (Con A)-driven T-cell proliferation. We have also shown that, paradoxically, these Mx do not secrete IL-1, when stimulated by endotoxin (LPS). In this study we examined two alternative hypotheses: T cells plus mitogen induce Mx IL-1 production, and human Mx deliver a second signal to T cells via a non-IL-1 mechanism. IL-1 was assayed in a mouse CD-1 thymocyte system without concanavalin A. Mo/Mx were cultured with T cells at low (2 X 10(4)/200 microliters) or high (1 X 10(5)/200 microliters) concentrations for 2 or 4 days, in the presence of Con A. Six hours prior to quantitation of proliferation, 50 microliters of supernatant was removed and assayed for IL-1. As expected both Mo and Mx enhanced T-cell proliferation eight- to tenfold. Mo secreted large amounts of IL-1; there was no demonstrable IL-1 activity present in supernatants from cultures containing either T cells and Mx, or Mx alone. Similar results were obtained by preincubating the cells (Mo, Mx, and T cells) with Con A for 12 hr and removing Con A prior to a 36-hr coculture. We examined the possibility that a small amount of IL-1 may be able to support Con A-stimulated T-cell proliferation and yet may not induce thymocyte proliferation. The highest dilutions of Mo supernatant (1:125) which supported T-cell proliferation also caused a fivefold increase in thymocyte proliferation. Supernatants from Mx failed to stimulate thymocyte proliferation or support Con A-driven T-cell proliferation. However, Mo and Mx lysates contain Il-1 activity. We conclude that human Mx support Con A-induced T-cell proliferation in the absence of IL-1 secretion. Mx may support T-cell proliferation by cell-bound IL-1 or by a non-IL-1 mechanism.     

Inhibition of macrophage-induced antigen-specific T-cell proliferation by interferon-gamma

The effects of IFN-gamma on macrophage (M phi)-mediated antigen-specific T-cell proliferation was investigated. A well-defined assay system using purified resident populations of antigen-pulsed peritoneal M phi and immune T cells was used to measure M phi-induced antigen-specific T-cell proliferation. Antibody affinity purified or recombinant IFN-gamma inhibited M phi-induced T-cell proliferation when KLH-pulsed M phi from mice given IFN-gamma prior to KLH were cultured with KLH immune T cells from normal mice. Monoclonal rat anti-IFN-gamma antibody neutralized the inhibitory effect of IFN-gamma. This inhibition of T-cell proliferation occurred despite the fact that these M phi appeared to be activated by IFN-gamma treatment as measured by increased tumoricidal activity. The mechanism for the inhibition was unrelated to class II (Ia) expression, IL-1 secretion, and prostaglandin secretion. These results demonstrate the complex and sensitive role IFN-gamma has in regulating the immune response.     

Phorbol myristate acetate-induced proliferation of an IL-2-dependent T-cell line: action of PMA is independent of IL-2 and cannot be mimicked by diacylglycerols

Phorbol myristate acetate (PMA) at concentrations of 2 X 10(-10) to 2 X 10(-7) M was able to sustain proliferation of the interleukin 2 (IL-2)-dependent murine T-cell line CTLL-K. This line, which died within 24 hr without exogenously added IL-2, survived for at least 96 hr and completed two to three cycles of replication in the presence of an optimal dose of PMA. PMA did not increase proliferation induced by saturating amounts of IL-2, but it mimicked IL-2 activity when no IL-2 or only suboptimal doses of IL-2 were present. This effect was completely independent of any residual IL-2 activity and was not mediated by the endogenous production of IL-2. The finding that stimulation of CTLL-K cells with 1-oleoyl-2-acetyl-glycerol or 1,2-dioctanoyl-glycerol rather than with PMA was not sufficient to induce any proliferation suggests that diacylglycerols and phorbol esters have qualitatively different effects on protein kinase C activity. Falsely positive results could occur when IL-2-dependent T-cell lines were used as indicator cells for IL-2, since evidence is presented that PMA-responsive cells could emerge spontaneously from T-cell clones that originally were not responsive or only weakly influenced by PMA.     

Alpha and lambda interferon together mediate suppression of CD4 T cells induced by respiratory syncytial virus

The mechanism by which respiratory syncytial virus (RSV) suppresses T-cell proliferation to itself and other antigens is poorly understood. We used monocyte-derived dendritic cells (MDDC) and CD4 T cells and measured [(3)H]thymidine incorporation to determine the factors responsible for RSV-induced T-cell suppression. These two cell types were sufficient for RSV-induced suppression of T-cell proliferation in response to cytomegalovirus or Staphylococcus enterotoxin B. Suppressive activity was transferable with supernatants from RSV-infected MDDC and was not due to transfer of live virus or RSV F (fusion) protein. Supernatants from RSV-infected MDDC, but not MDDC exposed to UV-killed RSV or mock conditions, contained alpha interferon (IFN-alpha; median, 43 pg/ml) and IFN-lambda (approximately 1 to 20 ng/ml). Neutralization of IFN-alpha with monoclonal antibody (MAb) against one of its receptor chains, IFNAR2, or of IFN-lambda with MAb against either of its receptor chains, IFN-lambdaR1 (interleukin 28R [IL-28R]) or IL-10R2, had a modest effect. In contrast, blocking the two receptors together markedly reduced or completely blocked the RSV-induced suppression of CD4 T-cell proliferation. Defining the mechanism of RSV-induced suppression may guide vaccine design and provide insight into previously uncharacterized human T-cell responses and activities of interferons.     

Cytokines correlate with age in healthy volunteers, dialysis patients and kidney-transplant patients

T-cell functions are currently used as biomarkers for the pharmacodynamic monitoring of immunosuppressive drugs or as disease biomarkers of inflammation/sepsis and organ rejection. In order to evaluate co-factors potentially influencing the expression of the immunological biomarkers, we explored T-cell proliferation, T-cell activation (CD25 and CD71 expressions) and intra-lymphocyte cytokine production (interleukin (IL)-2 and tumor necrosis factor (TNF)-α) in healthy volunteers, dialysis patients and stable kidney-transplant patients treated with standard immunosuppressive therapy, i.e. tacrolimus, mycophenolic acid with or without steroids. Age was positively correlated with TNF-α expression in all three patient populations, and with IL-2 expression in healthy volunteers and kidney-transplant patients. Further age was correlated with inhibition of lymphocyte proliferation in healthy volunteers and with the T-cell activation marker CD25 in kidney-transplant patients. In healthy volunteers lymphocyte proliferation was higher in woman as compared to men. Other biomarkers of T-cell function were independent of the gender. In the kidney-transplant patient group a significantly lower expression of all biomarkers of T-cell functions compared to healthy volunteers and dialysis patients. In dialysis patients we found significant increased IL-2 expression compared to healthy volunteers, while the other T-cell functions were not significantly different. Further time on dialysis had no effect on the level of biomarker expression. In conclusion we found decreased T-cell functions in kidney-transplant patients compared to healthy volunteers and dialysis patients, increased IL-2 expression in dialysis patients compared to healthy volunteers and in all three populations we found a correlation of age and intra-T-lymphocyte TNF-α expression.     

Arginase-dependent suppression by CpG-ODN plus IFA-induced splenic myeloid CD11b(+)Gr1(+) cells

The ability of synthetic oligodeoxynucleotides containing unmethylated cytosine guanine motifs (CpG-ODN) to induce both stimulatory and counter-regulatory responses offers novel opportunities for using these molecules as immunomodulatory agents in different therapeutic strategies. Here, we investigated the potential of CpG-ODN to activate the arginase (ARG) enzyme in vivo and focused on the consequences of this activation in T-cell proliferation. Challenging mice subcutaneously with CpG-ODN emulsified in incomplete Freund's adjuvant (IFA) induced ARG and reduced T-cell proliferation associated with CD3ζ chain downregulation. Interestingly, impaired T-cell expansion correlated with elevated levels of CD11b(+)Gr1(+) myeloid cells localized near T-cell areas in the spleen. In addition, purified CD11b(+) cells obtained from the spleen of CpG-ODN+IFA-treated mice exhibited increased ARG activity and ARG I expression along with an augmented [(3)H]-L-arginine uptake. CD11b(+) myeloid cells significantly suppressed T-cell proliferation and CD3ζ chain expression induced by a polyclonal stimulus. Furthermore, these effects could be recovered by the addition of excess L-arginine or by treatment of CD11b(+) cells with a specific ARG inhibitor. This study provides a novel evidence that CpG-ODN+IFA are able to induce splenic CD11b(+) cells with ARG activity, with this population being responsible for the impaired T-cell proliferation observed after the treatment with CpG-ODN+IFA. These results underscore a key role of CpG-ODN on ARG activity in vivo and add support to the growing body of evidence in favor of a counter-regulatory role for CpG-ODN in an immune response.     

Different roles of the CD2 and LFA-1 T-cell co-receptors for regulating cytotoxic, proliferative, and cytokine responses of human V gamma 9/V delta 2 T cells

BACKGROUND: Human V gamma 9/V delta 2 T lymphocytes recognize nonpeptidic antigens in a manner distinct from the classical antigen recognition by alpha beta T cells. The apparent lack of major histocompatibility (MHC) restriction and antigen processing allows very fast responses against pathogenic insults. To address the potential functional requirement for accessory molecules, we investigated the roles of the CD2 and lymphocyte function-associated antigen (LFA)-1 T-cell co-receptors in antigen-induced activities of human V gamma 9/V delta 2 T-cell clones. MATERIALS AND METHODS: Human peripheral blood V gamma 9/V delta 2 T lymphocytes were cloned and their cytotoxicity against Daudi lymphoma was measured by a standard 51Cr-release assay. The responses of V gamma 9/V delta 2 T lymphocytes to nonpeptidic antigens were assessed using DNA synthesis and cytokine ELISA assays. Monoclonal antibodies specific for various molecules with potential T-cell accessory functions were utilized in blocking assays. RESULTS: All of our V gamma 9/V delta 2 T-cell clones displayed the Th1 phenotype. The anti-LFA-1 antibody strongly inhibited the cytotoxicity of V gamma 9/V delta 2 T cells against Daudi B-cell lymphoma; whereas, it had no influence on the antigen-induced cytokine release or proliferation. In contrast, antibodies against CD2 and LFA-3 had no effect on the lytic activity of V gamma 9/V delta 2 T cells, but strongly inhibited the cytokine release and proliferation. However, the CD2-LFA-3 interaction was not an absolute requirement for the cytokine release and the DNA synthetic activity of antigen-stimulated V gamma 9/V delta 2 T cells, since the inhibitory effect could be reversed by addition of exogenous interleukin 2 (IL-2). CONCLUSIONS: These novel observations indicate that the signals generated by different accessory molecules and IL-2 can contribute in an integrated fashion to the regulation of V gamma 9/V delta 2 T cells. These interactions may be important for the effectiveness of V gamma 9/V delta 2 T-cell responses.     

The apoptotic and proliferative fate of cytokine-induced killer cells after redirection to tumor cells with bispecific Ab

BACKGROUND: Cytokine-induced killer (CIK) cells are ex vivo expanded T cells with co-expression of CD3 and CD56 and NK activity. They have recently been evaluated in a phase I/II clinical trial against malignant lymphoma. Bispecific Ab (bsAb) redirect CIK cells to tumor targets, thus enhancing their cytotoxicity. While bsAb may improve T-cell mediated anti-tumor activity, little is known about the fate of effector cells upon redirection to tumor targets using a bsAb. METHODS: Using ex vivo-activated CIK cells, Her2/neu expressing breast and ovarian cell lines and a F(ab')2 Her2/neu x CD3 bsAb, we investigated the anti-tumor activity and the proliferative and apoptotic outcome of CIK cells. RESULTS: When redirected to tumor targets with bsAb, there was a significant increase in anti-tumor activity as well as an increase in both CIK cell proliferation and apoptosis. The addition of agonistic Ab against CD28 did not significantly increase proliferation or apoptosis of CIK cells redirected to CD80- and CD86- tumor targets. To attempt to reduce T-cell apoptosis, we incubated CIK cells in the presence of the pan-caspase inhibitor z-VAD-fmk, which led to a partial reduction in T-cell apoptosis without increasing cellular cytotoxicity. DISCUSSION: bsAb are effective in redirecting activated T cells to tumor targets and such redirection leads to both T-cell proliferation and apoptosis that are not altered by co-stimulation through CD28. Effector cell apoptosis can be reduced by using a caspase inhibitor but this does not increase CIK cell cytotoxicity.     

Dose-dependent changes in influenza virus-infected dendritic cells result in increased allogeneic T-cell proliferation at low, but not high, doses of virus

During the acute phase of infection with influenza A virus, the degree of lymphopenia correlates with severity of disease. Factors that contribute to T-cell activation during influenza virus infection may contribute to this observation. Since the immune response is initiated when dendritic cells (DC) interact with T cells, we have established an in vitro system to examine the effects of influenza virus infection on DC function. Our results show that allogeneic T-cell proliferation was dependent on the dose of A/PR/8/34 used to infect DC, with enhanced responses at low, but not high, multiplicities of infection. The lack of enhancement at high virus doses was not primarily due to the increased rate of DC apoptosis, but required viral replication and neuraminidase (NA) activity. Clusters that formed between DC or between DC and T cells were also dependent on the viral dose. This change in cellular interaction may oppose T-cell proliferation in response to DC infected with high doses of PR8, since the increased contact between DC resulted in the exclusion of T cells. The enhanced alloreactive T-cell response was restored by neutralization of transforming growth factor beta1 (TGF-beta1). It is likely that NA present on viral particles released from DC infected with high doses of PR8 activates TGF-beta1. Future studies will determine the mechanism by which TGF-beta1 modifies the in vitro T-cell response and address the contribution of this cytokine to the lymphopenia observed in severe disease.     

The HIV-1 accessory gene vpr can inhibit antigen-specific immune function

The 14-kDa HIV-1 accessory gene vpr has been reported to have effects on host cell biology. These activities include inhibition of cell proliferation, inhibition of NF-kappaB activation, inhibition of CD4 T-cell proliferation, and induction of apoptosis in tissue culture. This collection of activities could, in theory, impact host cell immune responses. We tested the activity of recombinant Vpr protein to inhibit T-cell activation in vitro. Here, we present data illustrating that the Vpr protein can significantly suppress T-cell activation-related cytokine elaboration and proliferation. In vivo, we observed that covaccination with plasmids expressing the vpr gene product profoundly reduces antigen-specific CD8-mediated cytotoxic T lymphocyte (CTL) activity. This supports that vpr might compromise T-cell immunity in vivo during infection. To study this aspect of Vpr biology, we developed an Adenoviral Vpr expression vector for delivery of Vpr to immune cells and to study Vpr function in the absence of other lentiviral gene products. This vector delivers a functional Vpr protein to immune cells including antigen-presenting cells (APCs). We observe that the Adeno-Vpr vector suppresses human CD4 T-cell proliferation driven by immune activation in vitro. Further study of the biology of Vpr will likely have importance for a clearer understanding of host pathogenesis as well as have important implications for HIV vaccine development.     

Inhibition of interleukin-6 release and T-cell proliferation by synthetic mirror pseudo cord factor analogues in human peripheral blood mononuclear cells

Abstract The effects of synthetic alkyl ((alkyl 6-deoxy-a- d - gluco -heptopyranosyluronate) 6-deoxy-a- D -gluco-heptopyranoside) uronates, a novel type of mirror pseudo cord factor, on the in vitro modulation of interleukin-6 production and T-cell proliferation in human peripheral blood mononuclear cells, were investigated. Synthetic mirror pseudo cord factors with alkyl chains ranging from C₁₆ to C₁₈ have very weak interleukin-6-inducing capacities and lack mitogenic activities for T-cell proliferation. However, they could inhibit IL-6 release induced by sonicated Bacillus Calmette-Guérin (S-BCG), bacterial endotoxin, and phytohaemagglutinin in a dose-dependent manner. Inhibition was observed not only with mononuclear cells but also with purified monocytes. Furthermore, these synthetic compounds could suppress T-lymphocyte proliferation stimulated by sonicated Mycobacterium tuberculosis H37Rv (S-H37Rv) antigens, S-BCG antigens, as well as by recombinant 65 kDa mycobacterial heat-shock protein. In contrast, these compounds failed to inhibit the phytohaemagglutinin-induced T-cell proliferation. We conclude that the inhibition of cytokine release and T-cell proliferation by synthetic mirror pseudo cord factors was due to direct blocking of the function and/or activity of monocytes or antigen-presenting cells.     

Establishment and Characterization of Japanese Encephalitis Virus-Specific, Human CD4+ T-Cell Clones: Flavivirus Cross-Reactivity, Protein Recognition, and Cytotoxic Activity

We analyzed the CD4⁺ T-lymphocyte responses of two donors who had received Japanese encephalitis virus (JEV) vaccine 6 or 12 months earlier. Bulk culture proliferation assays showed that peripheral blood mononuclear cells (PBMC) responded to JEV antigens (Ag) but also responded at lower levels to West Nile virus (WNV) and dengue virus type 1, 2, and 4 (D1V, D2V, and D4V, respectively) Ag. Five JEV-specific CD4⁺ human T-cell clones and one subclone were established from PBMC of these two donors. Two clones responded to WNV Ag as well as to JEV Ag, whereas the others responded only to JEV Ag. Three of five CD4⁺ T-cell clones had JEV-specific cytotoxic activity and recognized E protein. The HLA restriction of the JEV-specific T-cell clones was examined. Three clones were HLA-DR4 restricted, one was HLA-DQ3 restricted, and the HLA restriction of one clone was not determined. T-cell receptor analysis showed that these clones expressed different T-cell receptors, suggesting that they originated from different T lymphocytes. These results indicate that JEV vaccine induces JEV-specific and flavivirus-cross-reactive CD4⁺ T lymphocytes and that these T lymphocytes recognize E protein. The functions and HLA restriction patterns of these T lymphocytes are, however, heterogeneous.     

Changes in the balance between mitogenic and antimitogenic lipid second messengers during proliferation, cell arrest, and apoptosis in T-lymphocytes.

Control of lymphocyte cell survival and proliferation is critical for both the immune response and for the prevention of autoimmune and infectious diseases. The actions of interleukin-2, the major T-cell regulatory cytokine, are mediated by the complex network of divergent signalling pathways controlled by its high-affinity receptor. Various studies have indicated that the generation of certain lipid second messengers is an important mechanism in the control of proliferation and cell death. We have examined the relationship between diacylglycerol and ceramide and the levels of the lipids phosphatidylcholine and sphingomyelin, their potential precursors, in the human T-cell line Kit 225 cultured in three distinct conditions to favor apoptosis, cell arrest, and proliferation. Our data show that, in proliferating cells, the ratios of diacylglycerol/ceramide and phosphatidylcholine/sphingomyelin are higher than those found in arrested cells and increase with time in culture. These ratios are rapidly reversed in apoptotic cells. Further experiments reveal that de novo synthesis of both diacylglycerol and phosphatidylcholine is greatest in proliferating cells, whereas sphingomyelin synthase activity is increased in cells undergoing apoptosis. In summary, our results demonstrate for the first time that the ratio of mitogenic/antimitogenic lipids changes dramatically during T-cell proliferation and cell death. These results indicate that lipid second messengers and the enzymes that are responsible for their generation may provide targets for novel therapeutic interventions in the clinical management of immunosuppression and autoimmune disease.     

Accessory cell-derived helper signals in human T-cell activation with phytohemagglutinin: induction of interleukin 2-responsiveness by interleukin 6, and production of interleukin 2 by interleukin 1 [corrected

Interleukins (IL-) 1 and 6 have been shown to represent accessory signals for T-cell activation. In the present study, we further examined the effects of both cytokines on accessory cell-depleted human T cells stimulated with phytohemagglutinin (PHA). The addition of IL-6 to the cultures resulted in T-cell proliferation; however, IL-1 was unable to support PHA-induced T-cell growth. The addition of IL-1 consistently induced a low level of IL-2 production and strongly enhanced T-cell proliferation in the presence of IL-6. Thus, the effect of IL-1 on T-cell growth becomes apparent only in the presence of IL-6. Blocking the IL-2-receptor (IL-2R) with the monoclonal antibodies anti-Tac and MikBêta 1 (directed to the alpha and bêta chains of the IL-2R, respectively) had no effect on PHA/IL-6-supported proliferation, but completely eliminated the growth-enhancing effect of IL-1. On the other hand, a neutralizing anti-IL-4-antiserum did not affect PHA/IL-6- or PHA/IL-6/IL-1-induced proliferation. Further experiments showed that IL-6 enhances T-cell responsiveness to IL-2, as evidenced by enhanced IL-2-induced proliferation. However, we could not find an effect of IL-6 on the expression of IL-2R as measured by staining with anti-Tac and with MikBêta 1 or by binding of (125I)-IL-2 to T cells. It can be concluded from these studies that IL-1 and IL-6 have different helper effects on PHA-induced T-cell activation. In the presence of PHA, IL-6 induces limited IL-2/IL-4-independent growth, and more importantly it renders T cells responsive to IL-2. IL-1 provides a signal leading to IL-2 production. The combination of IL-1 and IL-6 represents a synergistic helper signal, leading to an IL-2-dependent pathway of proliferation.     

Properties of Immature Myeloid Progenitors with Nitric-Oxide-Dependent Immunosuppressive Activity Isolated from Bone Marrow of Tumor-Free Mice

Myeloid derived suppressor cells (MDSCs) from tumor-bearing mice are important negative regulators of anti-cancer immune responses, but the role for immature myeloid cells (IMCs) in non-tumor-bearing mice in the regulation of immune responses are poorly described. We studied the immune-suppressive activity of IMCs from the bone marrow (BM) of C57Bl/6 mice and the mechanism(s) by which they inhibit T–cell activation and proliferation. IMCs, isolated from BM by high-speed FACS, inhibited mitogen-induced proliferation of CD4⁺ and CD8⁺ T-cells in vitro. Cell-to-cell contact of T-cells with viable IMCs was required for suppression. Neither neutralizing antibodies to TGFβ1, nor genetic disruption of indolamine 2,3-dioxygenase, abrogated IMC-mediated suppressive activity. In contrast, suppression of T-cell proliferation was absent in cultures containing IMCs from interferon-γ (IFN-γ) receptor KO mice or T-cells from IFN-γ KO mice (on the C57Bl/6 background). The addition of NO inhibitors to co-cultures of T-cells and IMC significantly reduced the suppressive activity of IMCs. IFN-γ signaling between T-cells and IMCs induced paracrine Nitric Oxide (NO) release in culture, and the degree of inhibition of T-cell proliferation was proportional to NO levels. The suppressive activity of IMCs from the bone marrow of tumor-free mice was comparable with MDSCs from BALB/c bearing mice 4T1 mammary tumors. These results indicate that IMCs have a role in regulating T-cell activation and proliferation in the BM microenvironment.