Modulation of IFN-mediated Ly-6E antigen induction by cAMP in a T cell lymphoma: opposite effects on the responses to IFN-gamma and IFN-alpha/beta.

This study was initiated to examine the role of cyclic nucleotides in the regulation of the expression of the Ly-6E cell surface Ag by IFN. As a model system, we used the YAC T cell lymphoma where this Ag is constitutively absent but is highly inducible by both IFN-gamma and IFN-alpha/beta. Treatment with cAMP or cGMP analogs did not cause Ly-6E expression in the absence of IFN. However, treatment with cAMP analogs, but not with cGMP analogs, markedly altered Ly-6E expression triggered by IFN, both at the mRNA and at the cell surface protein levels. Interestingly, these effects depended on whether Ly-6E induction was mediated by IFN-gamma or IFN-alpha/beta. Thus, the response to IFN-gamma was enhanced up to tenfold, whereas the response to IFN-alpha/beta was suppressed by 90-95%. Similar differential modulations of Ly-6E induction were also exerted by forskolin and cholera toxin, which are known to elevate intracellular cAMP concentration through distinct mechanisms. A YAC cell variant (C10) was isolated, where cAMP analogs or cAMP inducers could not modify Ly-6E induction. Although resistant to the biological effect of cAMP, the C10 mutant exhibited normal IFN-mediated Ly-6E responses. Moreover, in this mutant, Ly-6E induction was still affected by the PKC activator PMA, as in wild-type YAC cells. Altogether, our data demonstrate that cAMP (and cGMP) is not directly involved as second messenger in Ly-6E induction mediated by IFNs. However, a rise of cAMP modulates in an opposite fashion the Ly-6E-inducing actions of IFN-gamma and IFN-alpha/beta, which suggests that the two types of IFN utilize separate biochemical pathways to regulate Ly-6E expression.     

Role of protein kinase C in IFN-mediated Ly-6E antigen induction.

The YAC T cell lymphoma normally does not express Ly-6E mRNA or Ly-6E surface molecules but can be induced to do so on incubation with either IFN-gamma or IFN-alpha/beta. This system afforded a model to assess the possible role of protein kinase C (PKC) in IFN-mediated Ly-6E induction. First, we used various pharmacologic agents known to interfere with the function of PKC or other kinases. The PKC inhibitors H-7 and phloretin were found to block Ly-6E induction by IFN-gamma or IFN-alpha/beta both at the mRNA and protein levels. In contrast, inhibitors of cyclic nucleotide-dependent kinases (HA1004), of myosin L chain kinase (ML-9, A-3) or of calmodulin (R24157, W-7) failed to suppress this induction. Next, we investigated the effects of the PKC activators PMA and mezerein (MEZ) on Ly-6E expression. Although neither PMA nor MEZ by themselves could induce Ly-6E in YAC cells, both agents enhanced by up to fivefold the induction of Ly-6 mRNA and Ly-6E surface expression triggered by IFN-gamma. However, the induction of Ly-6E expression caused by IFN-alpha/beta was only marginally increased by cotreatment of YAC cells with PMA or MEZ. Altogether, these observations demonstrate that PKC or a related kinase is involved in the transduction mechanisms that lead to Ly-6E induction. However, activation of PKC is not sufficient for this induction and requires other unidentified signal(s) provided by IFN. Our data also indicate that IFN-gamma and IFN-alpha/beta induce Ly-6E through overlapping but distinct intracellular pathways with different sensitivities to PKC activators.     

Phenotypic changes induced by interferon in resting T cells: major enhancement of Ly-6 antigen expression

The murine Ly-6 locus controls multiple cell surface antigenic specificities with distinct cellular and tissue distributions. Although the functions of Ly-6 antigens are unknown, several of these antigens represent interesting markers of T cell differentiation and activation. In this work we used a panel of monoclonal antibodies (MAb) in conjunction with flow cytofluorometry (FCF) analysis to investigate the effect of interferon (IFN) on the surface representation of T cell-associated Ly-6 antigens. It was found that in vitro treatment of purified T cells from both C57Bl/6 (Ly-6.2) and BALB/c (Ly-6.1) mice with 10 to 10(4) U IFN-alpha/beta/ml results in a dose-dependent enhancement of Ly-6 antigen expression. This effect was already detectable after 12 to 18 hr and culminated after 48 hr of incubation. Both frequencies and brightness of Ly-6 bearing cells were increased. The most dramatic shifts were observed for the Ly-6A, D, and E antigens, which were augmented by eightfold to 20-fold upon exposure to 10(4) U IFN alpha/beta/ml. Expression of Ly-6C antigens was enhanced by fourfold to sixfold under the same conditions. Immunochemical analyses and use of metabolic inhibitors additionally demonstrated that such IFN-alpha/beta-induced phenotypic alterations of T cells reflect augmented de novo biosynthesis of Ly-6 molecules. Comparison of purified IFN-alpha and IFN-beta revealed that both are equally active in influencing Ly-6. IFN-gamma also enhanced Ly-6 expression but less efficiently than IFN-alpha/beta. Additional experiments were carried out to determine the selectivity of IFN-alpha/beta action on T cell phenotype. These studies demonstrated that IFN-induced Ly-6 enhancement occurs without emergence of interleukin 2 or transferrin receptors. Expression of H-2 and beta 2m antigens, previously known to be sensitive to IFN, was increased but to a much lesser extent than Ly-6. Most other cell surface antigens examined were minimally affected by IFN-alpha/beta with the exception of Ly-11 and Ly-23. Augmentation of these latter markers was lower than for Ly-6 antigens, however. Therefore the Ly-6 locus appears to be preferentially activated by IFN-alpha/beta in resting T cells. Additional exploration of this phenomenon should provide insight into both the biological significance of Ly-6 antigens and the mechanism(s) by which IFN affect T cell functions.     

Divergent effects of cyclosporine A on interferon- and mitogen-induced Ly-6A antigen suggest autocrine regulation of Ly-6A expression in activated T cells

The murine Ly-6A cell surface antigen is normally present on a minor subset of mature T cells. This marker has been shown to become highly expressed on mitogen-activated T cells. We found that expression of Ly-6A is also markedly increased in resting T cells by incubation with IFN-alpha/beta or IFN-gamma. Here, we compared the effect of the immunosuppressant cyclosporine A (CsA) on Ly-6A induction by IFN and concanavalin A (Con A). The augmentation of Ly-6A expression produced by treatment of T cells with IFN-alpha/beta or IFN-gamma was found not to be affected by CsA concentrations up to 2 micrograms/ml. In contrast, at doses as low as 50 ng/ml, CsA prevented the enhancement of Ly-6A expression in Con A-treated T-cell cultures. Culture supernatant transfer experiments were performed to further explore this effect of CsA. It was found that supernatants from Con A-activated T cells enhanced Ly-6A expression in resting T cells. This activity could be neutralized with an anti-IFN-gamma monoclonal antibody. Supernatants from T cells treated with Con A in presence of CsA lacked Ly-6A-enhancing activity. Taken together, these data suggest that the inhibition by CsA of Ly-6A induction in Con A-treated T cells reflects the known inhibitory effects of the drug on IFN-gamma secretion. This may imply the existence in T cells of an autocrine circuit involving IFN-gamma and regulating Ly-6A expression.     

Regulation of B lymphocyte responses to IL-4 and IFN-gamma by activation through Ly-6A/E molecules

The Ly-6 family of cell surface molecules has previously been shown to participate in T cell activation. We show that Ly-6A/E proteins also modulated the response of normal B lymphocytes in three separate in vitro assays. First, unfractionated or small resting B cells proliferated when cultured with IFN-gamma, IL-4, and an anti-Ly-6A/E mAb. Second, this anti-Ly-6A/E mAb restored B cell proliferation responses that were inhibited when coculturing the B cells in IFN-gamma, IL-4, and anti-IgM. Third, anti-Ly-6A/E specifically up-regulated the cell surface expression of its own Ag, and this response was dependent upon co-stimulation with IFN-gamma. Mixing of T and B cells in culture suggested that T cells did not contribute substantially to the B cell proliferative response. Moreover, up-regulation of Ly-6A/E was observed for one B cell lymphoma, WEHI-231. Therefore, it appeared that modulation of B cell function by anti-Ly-6A/E was due to a direct effect of the mAb binding to the B cells. Taken together, these data suggest Ly-6A/E proteins are functional on B cells and may play a regulatory role in B cell activation.     

Down-regulation of IL-2 production by activation of T cells through Ly-6A/E

Ly-6A/E molecules are expressed on the surface of T cells and have been shown to function in activation by the capacity of anti-Ly-6A/E mAb to induce T cell hybridomas or normal T cells to produce IL-2. Recent evidence suggests that activation through Ly-6A/E may be linked to the TCR signaling pathway. To further investigate the relationship between Ly-6- and TCR-induced T cell activation, we have examined whether an anti-Ly-6A/E mAb (D7) modulates TCR signaling in vitro. We now report that mAb D7 specifically inhibited IL-2 production by T cells also activated through TCR. Such inhibition was noted for normal T cells stimulated by soluble anti-CD3 or alloantigen and for T hybridomas stimulated by soluble anti-CD3. The ability of D7 to inhibit IL-2 production by T hybridomas was dependent on the nature of the TCR activating signal because IL-2 production was not inhibited when T hybridomas were stimulated with Ag or immobilized anti-CD3. Inhibition of IL-2 production by D7 apparently required cross-linking of the mAb because D7 F(ab')2 fragments were not effective for inhibition of IL-2 production. Similar to its ability to enhance anti-Ly-6A/E-induced activation of T and B cells, IFN-gamma enhanced the D7-induced inhibition of IL-2 production by alloantigen-activated normal T cells. These data further support the notion that Ly-6 and TCR signaling pathways are interrelated.     

CD73 and Ly-6A/E distinguish in vivo primed but uncommitted mouse CD4 T cells from type 1 or type 2 effector cells

Primed CD4 T cells may develop into effector T cells such as Th1 and Th2, or remain uncommitted as Th primed precursor (Thpp) cells that can subsequently differentiate into Th1 and Th2 cells. Although mouse Thpp-like cells have also been identified among spleen and particularly lymph node cells, further characterization of these cells has been difficult without a defining cell surface marker. Using Affymetrix GeneChips followed by FACS analysis, we found that in vitro-derived Thpp cells expressed CD73 but not Ly-6A/E, whereas Th1 and Th2 cells showed the reciprocal pattern. CD73+ Ly6A/E- memory CD4 T cells were identified in normal C57BL/6 mice, and the proportion of these cells was highest in lymph nodes, lower in spleens, and lowest in the lungs. These cells produced IL-2 and MIP-1alpha, but much less IL-4 and IFN-gamma than CD73- Ly6A/E+ cells. Similar results were obtained with additional Ly-6.2 mouse strains, but not Ly-6.1 strains. Restimulation of Thpp-like CD73+ Ly-6A/E- cells in Th1- or Th2-polarizing conditions induced differentiation into populations producing mainly IFN-gamma or mainly IL-4, respectively. In contrast, the effector-like CD73- Ly-6A/E+ population was more committed, and continued to produce both IL-4 and IFN-gamma in both conditions. CD73 and Ly-6A/E expression therefore identify a population of Thpp-like cells in C57BL/6 mice and at least some other Ly-6.2 mice.     

The augmentation of surface Ly-6A/E molecules in activated T cells is mediated by endogenous interferon-gamma

The murine Ly-6A.2 and Ly-6E.1 antigens, which can transduce triggering signals in T cells, have been shown to become highly expressed after mitogenic stimulation. It has recently been found that enhanced expression of Ly-6A/E antigens is also induced by interferon-gamma (IFN-gamma) in resting T cells. Here, the possibility is investigated that Ly-6A/E induction on activated T cells may be due to the IFN-gamma known to be secreted by these cells. A potent neutralizing anti-IFN-gamma monoclonal antibody (mAb) (H-22.10) was used. This mAb was found to abrogate the augmentation of Ly-6A/E antigens produced in resting T cells by supernatants from T cells stimulated with concanavalin A. When added directly into cultures of T cells stimulated with concanavalin A or by the combination of ionomycin with the protein kinase C activator phorbol myristate acetate (PMA), the H-22.10 mAb inhibited Ly-6A/E enhancement without affecting the blastogenesis or the emergence of interleukin 2 receptors and transferrin receptors. Such a selective effect of the anti-IFN-gamma mAb indicated that IFN-gamma is involved in the up-regulation of Ly-6A/E antigens during T cell activation. In determining whether other activation signals, in addition to IFN-gamma receptor occupancy, may contribute to Ly-6A/E enhancement, it was found that suboptimal stimulation of BALB/c T cells provided by a 3-hr pulse with ionomycin plus PMA or by culture with PMA alone potentiated by about twofold the increase of Ly-6E.1 induced by exogenous IFN-gamma. Therefore, Ly-6A/E augmentation in activated T cells reflects primarily an action of endogenous IFN-gamma that is amplified (in BALB/c mice) by a protein kinase C-dependent step.     

Tumor necrosis factor-alpha production by astrocytes. Induction by lipopolysaccharide, IFN-gamma, and IL-1 beta

Astrocytes have the capacity to secrete or respond to a variety of cytokines including IL-1, IL-6, IL-3, and TNF-alpha. In this study, we have examined the capacity of astrocytes to secrete TNF-alpha in response to a variety of biologic stimuli, particularly cytokines such as IL-1 and IFN-gamma, which are known to be present in the central nervous system during neurologic diseases associated with inflammation. Rat astrocytes do not constitutively produce TNF-alpha, but have the ability to secrete TNF-alpha in response to LPS, and can be primed by IFN-gamma to respond to a suboptimal dose of LPS. IFN-gamma and IL-1 beta alone do not induce TNF-alpha production, however, the combined treatment of IFN-gamma and IL-1 beta results in a striking synergistic effect on astrocyte TNF-alpha production. Astrocyte TNF-alpha protein production induced by a combined treatment of either IFN-gamma/LPS or IFN-gamma/IL-1 beta occurs in a dose- and time-dependent manner, and appears to require a "priming signal" initiated by IFN-gamma, which then renders the astrocyte responsive to either a suboptimal dose of LPS or IL-1 beta. Astrocyte TNF-alpha production by IFN-gamma/LPS stimulation can be inhibited by the addition of anti-rat IFN-gamma antibody, whereas IFN-gamma/IL-1-induced TNF-alpha production is inhibited by antibody to either IFN-gamma or IL-1 beta. Polyclonal antisera reactive with mouse macrophage-derived TNF-alpha neutralized the cytotoxicity of IFN-gamma/LPS and IFN-gamma/IL-1 beta-induced astrocyte TNF-alpha, demonstrating similarities between these two sources of TNF-alpha. We propose that astrocyte-produced TNF-alpha may have a pivotal role in augmenting intracerebral immune responses and inflammatory demyelination due to its diverse functional effects on glial cells such as oligodendrocytes and astrocytes themselves.     

Role of Ly-6A/E and T cell receptor-zeta for IL-2 production. Phosphatidylinositol-anchored Ly-6A/E antagonizes T cell receptor-mediated IL-2 production by a zeta-independent pathway.

Ly-6A/E molecules were originally implicated in regulation of T cell activation because anti-Ly-6A/E mAb induce IL-2 production. More recently we have shown that anti-Ly-6A/E also inhibits IL-2 production induced by anti-CD3. In the present study we used mutant and transfected cell lines that varied in expression of Ly-6A/E or TCR-zeta to test whether the positive and negative modulations of IL-2 production by anti-Ly-6A/E occur by distinct mechanisms. Anti-Ly-6A/E inhibited anti-CD3-induced IL-2 production for Ly-6E.1-transfected EL4J cells, but did not affect IL-2 production of the parental Ly-6A/E-negative EL4J cells. These results indicate that TCR-mediated IL-2 production can occur in the absence of Ly-6A/E expression and establish that anti-Ly-6A/E-induced inhibition of IL-2 production was the result of antibody binding to Ly-6A/E. As expected, MA5.8 (zeta-negative) or CT108 (zeta-truncated) variants of the 2B4.11 T cell hybridoma did not produce IL-2 when stimulated with anti-Thy-1 or anti-Ly-6A/E mAb. In contrast, anti-Ly-6A/E inhibited anti-CD3-induced IL-2 production by MA5.8 and CT108. Furthermore, anti-Ly-6A/E-induced IL-2 production was restored for zeta-transfected MA5.8. Thus, although induction of IL-2 by anti-Ly-6A/E depends on zeta expression, inhibition of IL-2 by anti-Ly-6A/E occurs by a zeta-independent mechanism. Interestingly, anti-Ly-6A/E, but not anti-Thy-1, inhibited anti-CD3-induced IL-2 production by MA5.8 and Ly-6E.1-transfected EL4J. Therefore, inhibition of IL-2 production by anti-Ly-6A/E was not a general property of a mAb binding to a phosphatidylinositol-linked molecule, as has been suggested for induction of IL-2 production. Taken together these data suggest that the molecular mechanisms of induction and inhibition of IL-2 production by anti-Ly-6A/E are separable and expression of TCR-zeta is one variable that distinguishes these two pathways.     

Cytokine secretion by C3H-lpr and -gld T cells. Hypersecretion of IFN-gamma and tumor necrosis factor-alpha by stimulated CD4+ T cells.

Mice homozygous for lpr and gld develop profound lymphadenopathy characterized by the expansion of two unusual T cell subsets, a predominant Ly-5(B220)+ CD4- CD8- double negative (DN) population and a minor CD4 dull+ Ly-5(B220)+ population. The mechanisms promoting lymphoproliferation are unknown, but one possibility is a abnormality in the production of cytokines that regulate T cell growth. In the present report, unfractionated LN cells and sorted T cell subsets from C3H-lpr, -gld, and -+/+ mice were compared for spontaneous and induced secretion of a spectrum of lymphokines. In addition, CD4+, CD4 dull+ Ly-5(B220)+, and DN T cells were examined for expression of CD3 epsilon, TCR-alpha/beta heterodimers, Ly-6C, and CD44 and for proliferative responses to immobilized anti-TCR mAb and cofactors. These studies revealed that sorted DN T cells did not secrete IL-3, IL-4, IL-5, IL-6, GM-CSF, TNF-alpha, or IFN-gamma spontaneously or after TCR-alpha/beta cross-linking. In contrast, stimulated unfractionated lpr and gld LN cells proliferated strongly and secreted high levels of IFN-gamma and TNF-alpha and low levels of IL-3, IL-4, and IL-6. Despite a 5- to 10-fold deficit in the frequency of CD4+ and CD8+ T cells, cytokine secretion by lpr and gld LN generally exceeded that of +/+ LN. Comparisons of cytokine secretion by stimulated CD4+ T cells revealed that +/+, lpr, and gld CD4+ Ly-5(B220)- T cells proliferated strongly, but only lpr and gld cells produced significant levels of IFN-gamma. The lpr and gld CD4+ T cells also produced higher levels of TNF-alpha and IL-2 than +/+ cells. In contrast to normal CD4+ T cells, lpr and gld CD4+ Ly-5(B220)+ T cells proliferated weakly and did not secrete TNF-alpha, IL-2, or, in most experiments, IFN-gamma after stimulation. Phenotypic studies of T cell subsets revealed that unstimulated lpr and gld CD4+ Ly-5(B220)- T cells express significantly higher levels of CD44 than +/+ CD4+ T cells. In addition, CD4 dull+ Ly-5(B220)+ cells closely resembled DN T cells in size and expression of TCR-alpha/beta, CD3epsilon, CD44, and Ly-6C. Since elevated CD44 expression is generally associated with T cell activation and only previously activated normal CD4+ T cells produce high levels of IFN-gamma in vitro, our data suggest that lpr and gld CD4+ Ly-5(B220)- T cells contain a higher than normal proportion of primed or memory T cells and thus may be polyclonally activated in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ly-6I, a new member of the murine Ly-6 superfamily with a distinct pattern of expression

A new member of the mouse Ly-6SF, designated Ly-6I, has been isolated as a gene homologous to a segment of the Ly-6C gene. A single allelic difference in the mature protein sequence was identified, which is similar to other Ly-6SF members. Ly-6I mRNA has been detected in a wide range of tissues and cell lines, and a rabbit polyclonal Ab has been used to determine that Ly-6I protein is present at a low constitutive level on cell lines from several different lineages. In contrast to Ly-6C and Ly-6A/E, the Ly-6I gene is only weakly responsive to IFNs. Expression in vivo is most abundant on bone marrow populations and is coexpressed with Ly-6C on granulocytes and macrophages. However, Ly-6I is also expressed on immature B cell populations that do not express Ly-6C. Expression on mature B cells in spleen is uniformly low. Similarly, Ly-6I is expressed on TCRlow/int, but not TCRhigh, thymocytes. Ly-6I is re-expressed on Ly-6Chigh T cells in the periphery. Thus, Ly-6I may be a useful marker to define maturation stages of both T and B lymphocytes as well as subsets of monocytes and granulocytes.     

Ly-6 superfamily members Ly-6A/E,Ly-6C,and Ly-6I recognize two potential ligands expressed by B lymphocytes

Most hemopoietic cells express one or more members of the Ly-6 supergene family of small glycosylphosphatidylinositol-linked proteins. Although levels of Ly-6 proteins vary with stages of differentiation and activation, their function largely remains unknown. To ascertain whether ligands for Ly-6 proteins exist, chimeric proteins were constructed in which Ly-6E, Ly-6C, and Ly-6I were fused to the murine IgM heavy chain. These chimeras specifically stained both developing and mature B lymphocytes, as assessed by flow cytometry. Analysis of variants of the CH27 B cell lymphoma revealed that Ly-6A/E and Ly-6I recognized different molecules. CH27 cells with low levels of Ly-6A/E ligand activity also lost expression of CD22, and cells transfected with CD22 gained the ability to bind the Ly-6A/E chimera and, to a lesser extent, the Ly-6C and Ly-6I chimeric proteins. As many mature B cells coexpress Ly-6A/E and CD22, the function of Ly-6 molecules may be to associate with other membrane proteins, possibly concentrating these ligands in lipid rafts, rather than acting directly as cell:cell adhesion molecules.     

Direct stimulation of cytokines (IL-1 beta, TNF-alpha, IL-6, IL-2, IFN-gamma and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation.

Production of interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), interferon gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) after stimulation by lipopolysaccharide (LPS) and phytohemagglutinin (PHA) was studied in 1/10 diluted whole blood (WB) culture and in peripheral blood mononuclear cell (PBMC) culture. Cytokines IL-1 beta, TNF-alpha and IL-6 are preferentially stimulated by LPS whereas IL-2, IFN-gamma and GM-CSF are stimulated by PHA. Combination of 5 micrograms/ml PHA and 25 micrograms/ml LPS gave the most reliable production of the six cytokines studied. IL-1 beta, TNF-alpha and IL-6 represent a homogeneous group of early-produced cytokines positively correlated among themselves and with the number of monocytes in the culture (LeuM3). Furthermore, IL-1 beta was negatively correlated with the number of T8 lymphocytes. IL-2, IFN-gamma and GM-CSF represent a group of late-produced cytokines. Kinetics and production levels of IL-6 and GM-CSF are similar in WB and PBMC cultures. In contrast, production levels of TNF-alpha and IFN-gamma are higher in WB than in PBMC whereas production levels of IL-6 and IL-2 are lower in WB than in PBMC. Individual variation in responses to PHA + LPS was always higher in PBMC cultures than in WB cultures. The capacity of cytokine production in relation to the number of mononuclear cells is higher in WB, or in PBMC having the same mononuclear cell concentration as WB, than in conventional cultures of concentrated PBMC (10(6)/ml). Because it mimics the natural environment, diluted WB culture may be the most appropriate milieu in which to study cytokine production in vitro.     

Stimulation of lymphocyte migration by endotoxin, tumor necrosis factor, and interferon

Since several studies have demonstrated that lipopolysaccharide (LPS), tumor necrosis factor (TNF), and interleukin-1 (IL-1) enhanced lymphocyte binding to endothelial cells in vitro, we examined the effects of these agents on lymphocyte migration in vivo. Small peritoneal exudate lymphocytes (sPEL), which perferentially migrate into inflammatory sites, were radiolabeled with 111In and injected iv into rats. The id injection of LPS was a strong stimulus for the migration of these cells into the skin. TNF alpha was also a good stimulator of lymphocyte migration, while TNF beta and IL-1 alpha were weak or nearly inactive. Kinetic analysis demonstrated that migration to TNF was rapid, with a peak at 6 hr, followed by a steady decline, while migration to LPS was sustained for 24 hr. TNF alpha, TNF beta, and IL-1 alpha, when combined with interferon-gamma (IFN-gamma) or IFN-alpha/beta produced striking synergistic increases in lymphocyte migration. Combinations of the TNFs and IL-1 had less than additive effects, as did combinations of the IFNs. Qualitatively similar migration responses were found when spleen T cells instead of sPEL were studied.     

Treatment of resting T lymphocytes with interferon-alpha/beta augments their proliferative response to activation signals delivered through their surface Ly-6 antigen

Although the exact significance of Ly-6 antigens is unknown, recent evidence suggests they may provide an important alternative pathway for murine T-cell activation. Thus, Shevach et al. (1986, Fed. Proc. 45, 1131) discovered that cross-linking of Ly-6 antigens on the cell surface acts in concert with phorbol myristate acetate to trigger mitogenesis in T cells. Previously, we reported that surface expression of Ly-6 antigens on T cells is markedly increased following exposure to interferon-alpha/beta (IFN-alpha/beta). The purpose of the present work was to determine the effect of IFN-induced Ly-6 enhancement on Ly-6-mediated T-cell stimulation. Purified T cells were incubated in vitro for 1-27 hr with various doses (10-10(4) units/ml) of IFN-alpha/beta. This was found to result in various degrees of augmentation of the proliferative responses of these T cells to stimulation through their Ly-6 antigen. Surprisingly, while maximal enhancement of Ly-6 expression occurred only after the longest pulses with the highest IFN concentrations, treatment with as little as 100 units IFN/ml for 12 hr was sufficient to induce a dramatic (25-fold) and nearly maximal enhancement of proliferation. This high sensitivity to IFN-alpha/beta of the Ly-6 pathway of T-cell activation led us to speculate that this pathway may play a role in the immunomodulatory activities of IFN-alpha/beta.