Early Stages Of T-cell Activation Research Articles

T-cell activation decreases miRNA-15a/16 levels to promote MEK1–ERK1/2–Elk1 signaling and proliferative capacity

While miRs have been extensively studied in the context of malignancy and tumor progression, their functions in regulating T-cell activation are less clear. In initial studies, we found reduced levels of miR-15a/16 at 3 to 18 h post–T-cell receptor (TCR) stimulation, suggesting a role for decreased levels of this miR pair in shaping T-cell activation. To further explore this, we developed an inducible miR15a/16 transgenic mouse model to determine how elevating miR-15a/16 levels during early stages of activation would affect T-cell proliferation and to identify TCR signaling pathways regulated by this miR pair. Doxycycline (DOX)-induced expression of miR-15a/16 from 0 to 18 h post-TCR stimulation decreased ex vivo T-cell proliferation as well as in vivo antigen-specific T-cell proliferation. We also combined bioinformatics and proteomics approaches to identify the mitogen-activated protein kinase kinase 1 (MEK1) (Map2k1) as a target of miR-15a/16. MEK1 targeting by miR-15a/16 was confirmed using miR mimics that decreased Map2k1 mRNA containing the 3′-UTR target nucleotide sequence (UGCUGCUA) but did not decrease Map2k1 containing a mutated control sequence (AAAAAAAA). Phosphorylation of downstream signaling molecules, extracellular signal–regulated protein kinase 1/2 (ERK1/2) and Elk1, was also decreased by DOX-induced miR-15a/16 expression. In addition to MEK1, ERK1 was subsequently found to be targeted by miR-15a/16, with DOX-induced miR-15a/16 reducing total ERK1 levels in T cells. These findings show that TCR stimulation reduces miR-15a/16 levels at early stages of T-cell activation to facilitate increased MEK1 and ERK1, which promotes the sustained MEK1–ERK1/2–Elk1 signaling required for optimal proliferation.

Anti-inflammatory Effects of First-line Anti-arthritic Drugs on T-cell Activation.

The in vitro effects of commonly used first-line anti-arthritic drugs on early stages of T-cell activation were examined. The 2B4.11 murine T cell hybridoma cell line recognizing pigeon cytochrome c (PCC) as the antigen was co-cultured with the histocompatible antigen presenting B cell hybridoma line LK35.2, PCC, and anti-arthritic drugs, including methotrexate, hydroxychloroquine, salazopyrine, cyclosporin, and leflunomide. After 16 hours of incubation, the supernatant was removed, and cytokines were assayed. Anti-arthritic drugs inhibited the production of pro-inflammatory cytokines IL-2, IL-6, IFN-γ, GM-CSF, and TNF-α (Th1 cytokines) to a varying extent. Surprisingly, leflunomide, salazopyrine, prednisone and indomethacin as well as blocking Th1 cytokines, stimulated the production of the anti-inflammatory cytokine IL-10, a Th2 cytokine. Anti-arthritic medications can inhibit the production of pro-inflammatory cytokines and in some cases, incite a Th2 response that could potentially inhibit the progression of the immune response.

Ipilimumab-induced thrombotic thrombocytopenic purpura (TTP)

BackgroundCTLA-4 (Cytotoxic T-lymphocyte-associated protein 4) was the first immune checkpoint receptor clinically targeted for use in cancer treatment. It is expressed exclusively on T-cells where its primary role is to regulate the amplitude of the early stages of T-cell activation.1 Ipilimumab, a CTLA-4 blocking antibody, has been widely used for the treatment of patients with high risk and metastatic melanoma. Given its mechanism of action and consequent immune activation, the side effect profile of this drug greatly differs from that of standard cytotoxic chemotherapy. Adverse events are from the most part immune-mediated, ranging from the more common, such as rash and fatigue, to the less common, such as immune endocrinopathy and colitis.Case presentationWe describe a case of immune-mediated thrombotic thrombocytopenic purpura (TTP) in a 68 year-old woman with high risk, stage III melanoma occurring after 3 cycles of adjuvant treatment with ipilimumab as part of a clinical trial.ConclusionThe range of immune-mediated adverse events during treatment with ipilimumab is wide and varied and clinicians should have a high degree of suspicion when managing these patients.

Dose-Dependent Effect of Anti-CTLA-4 on Survival in Sepsis

Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is one of the critical inhibitory regulators of early stages of T-cell activation and proliferation, which opposes the actions of CD28-mediated costimulation. Anti-CTLA-4 therapy has been effective clinically in enhancing immunity and improving survival in patients with metastatic cancer. Sepsis is a lethal condition that shares many of the same mechanisms of immune suppression with cancer. Given the similarities in immune defects in cancer and sepsis, we examined the ability of anti-CTLA-4 antibody to block apoptosis, reverse the immunosuppression of sepsis, and improve survival in the cecal ligation and puncture model. Mice underwent sham or cecal ligation and puncture, and spleens harvested at various time points after surgery. Expression of CTLA-4 on CD4, CD8, and regulatory T cells was quantitated. Anti-CTLA-4 was administrated 6 and 24 h after surgery. Spleens were harvested at 48 h after surgery, and apoptosis and cytokine production determined. Seven-day survival studies were also conducted. Expression of CTLA-4 on CD4, CD8, and regulatory T cells increased during sepsis. Anti-CTLA-4 therapy decreased sepsis-induced apoptosis but had little effect on proinflammatory or anti-inflammatory cytokines. There was a dose-dependent effect of anti-CTLA-4 on survival. At high dose, anti-CTLA-4 worsened survival, but at lower doses, survival was significantly improved. Survival in sepsis depends on the proper balance between the proinflammatory and anti-inflammatory/immunologic systems. Anti-CTLA-4-based immunotherapy offers promise in the treatment of sepsis, but care must be used in the timing and dose of administration of the drug to prevent adverse effects.

Functional analysis of a tripartite stability element within the CD40 ligand 3′ untranslated region

We previously identified a cis-acting element within the 3' untranslated region of CD40 ligand messenger RNA (mRNA) that is composed of three complex binding sites and acts to increase mRNA stability in both in vitro and in vivo systems. We now demonstrate the functional consequences of the three binding sites with respect to increasing both luciferase activity and mRNA stability in a heterologous transcript expressed in a T-cell line. The internal region B was shown to be a bona fide stability element because its presence increased luciferase activity fourfold over the unmodified transcript and its removal from the XbaI-HaeIII region resulted in rapid degradation of the transcript. Region A contained both a binding site for a polypyrimidine-tract-binding protein (PTB)-mediated complex (Complex I) and an upstream, adjacent sequence that was a negative regulator of mRNA stability. Region C bound Complex II, which contained both PTB and heterogeneous nuclear ribonucleoproteinL (hnRNPL), and was less effective as a stability element on its own compared to region B. Our findings demonstrate differential levels of activity for the three binding sites relative to the turnover of CD40 ligand mRNA, suggesting that the lack of binding of Complex I/II during the early stages of T-cell activation contributes to the rapid degradation of the CD40 ligand mRNA transcript.

Depletion of LAG-3 Positive Cells in Cardiac Allograft Reveals Their Role in Rejection and Tolerance

Lymphocyte-activated gene-3 (LAG-3, CD223) is upregulated during the early stages of T-cell activation and could be the target of cytotoxic antibodies for induction therapy in transplantation. Fully vascularized heterotopic allogeneic heart transplantation was performed in rats across a full major histocompatibility complex-mismatch barrier (LEW.1W into LEW.1A). Recipients received two injections (day 0 and 3) of cytotoxic antibodies directed to the extra-loop of LAG-3 immunoglobulin (Ig)-like N-terminal domain or control antibodies. LAG-3 mRNA transcripts accumulated in cardiac allografts undergoing rejection, but not in peripheral lymphoid organs. Administration of anti-LAG-3 antibodies on the day of transplantation did not modify alloreactivity of T lymphocytes from the spleen and did not change the alloantibody response. However, it inhibited graft infiltration by effector mononuclear cells, reduced intragraft levels of interferon-gamma mRNA and prolonged allograft survival from 6 days in controls to a median of 27 days. Anti-LAG-3 antibodies were also active in prolonging survival when administered in a delayed manner, after rejection onset. LAG-3 being also expressed by activated regulatory T (Treg) cells, we tested the effect of anti-LAG-3 antibodies on graft acceptance after donor blood transfusions, a Treg-dependent tolerance induction model. We found that tolerance induction was prevented by anti-LAG-3 antibodies. Targeting LAG-3-positive cells with cytotoxic antibodies is immunosuppressive in transplantation by depleting effectors T cells and therefore may represent a treatment for rejection episodes focused only on pathogenic cells. However, it might not be compatible with tolerance-induction strategies.

Monoclonal Antibody Specific for TIRC7 Induces Donor-specific Anergy and Prevents Rejection of Cardiac Allografts in Mice

T cell immune response c-DNA (TIRC7) is up-regulated during the early stages of T-cell activation in response to alloantigens. In this study, we analyzed the effects of newly developed monoclonal antibodies (mAb) against TIRC7 in acute cardiac allograft rejection. Fully vascularized heterotopic allogeneic heart transplantation was performed in mice across a full-mismatch barrier (C57Bl/10 into CBA). Recipients received seven injections (day 0–7) of a novel anti-TIRC7 mAb or remained untreated. Graft survival, histology and ex vivo lymphocyte functions were tested. Targeting of TIRC7 with an anti-TIRC7 mAb diminishes lymphocyte infiltration into grafts resulting in delay of morphological graft damage and prolongation of allograft survival. The lymphocytes from anti-TIRC7 mAb-treated animals exhibit hypo-responsiveness without evidence of lymphocyte depletion against the donor allo-antigens. Proliferation and expression of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) were down-regulated while interleukin-4 (IL-4) and IL-10 expression were spared. Moreover, anti-TIRC7 mAb enhanced up-regulation of CTLA-4 expression but suppressed up-regulation of CD25 on stimulated lymphocytes in vitro and in vivo. Ligation of TIRC7 has important effects on the regulation of co-stimulatory signaling pathways associated with suppressing of T-cell activation. Targeting of TIRC7 may therefore provide a novel therapeutic approach for modulating T cell immune responses during organ transplantation.

Allogeneic bone marrow inhibits T-cell activation and clonal expansion in vitro.

Donor bone marrow infusion has long been used to enhance graft survival or induce tolerance in T cell depleted solid organ allograft recipients. However, the mechanisms through which bone marrow cells affect tolerance remain obscure. We studied the affect of allogeneic bone marrow cells on the activation of allospecific T cells in vitro. Carboxyfluorescein-diacetate succinimidyl ester-labeled CBA/Ca strain CD8+ splenocytes, bearing T-cell receptor alpha and beta transgenes from the BM3.3 T-cell clone specific for the major histocompatibility complex class I antigen Kb, were placed in culture with irradiated C57BL/6J stimulator cells in the presence of increasing numbers of C57BL/6J or Balb/cJ bone marrow cells for 1 to 3 days. Responder cells were individually analyzed for proliferative history, expression of activation-associated antigens, and intracellular cytokine production. Allogeneic bone marrow cells exert a dose-dependent inhibitory effect on proliferation of allospecific CD8+ T cells in mixed lymphocyte culture. However, the inhibited T-cell subpopulations show physiologic changes associate with the early stages of T-cell activation, including expression of CD69 and early decrease of surface T-cell expression. Unlike cells not co-cultured with bone marrow, these cells fail to reexpress the T-cell receptor (TCR) by 72 hr of culture. The observed inhibitory effect is also associated with a decrease in the proportion of CD8+ cells expressing interleukin-2 and interferon-gamma. Collectively, these results suggest that peripheral allospecific T cells undergo the initial stages of activation on exposure to antigen in the presence of bone marrow cells, but the cell cycle is arrested and TCR reexpression is inhibited. We speculate that bone marrow cells effect this inhibition through a receptor-ligand interaction that modulates the transmembrane signal pathway for the TCR.

Trends and developments in the pharmacological treatment of psoriasis.

In the history of psoriasis pharmacotherapy , the milestone developments have largely occurred by chance. Serendipitous observations in single patients have led to the discovery of such important treatments as methotrexate (1), deltanoids (active form of vitamin D and its analogues) (2), cyclosporin (3) and, most recently, tumour necrosis factor alpha (TNF-a) inhibitors (4). While these observations have been of therapeutic importance, they have also had a major impact on current views on the pathogenesis of this skin disease. From the initial model, where the hyperproliferation of epidermal keratinocytes was considered to be a central event, the current understanding places the immune system at the hub of the pathogenic series of events. Vigorous research and constantly increasing insight into the mechanisms of psoriasis have led to the identi® cation of a number of potential targets for therapeutic intervention. For the ® rst time, the rational, mechanism-based development of new anti-psoriatic therapeutic has become a reality. The purpose of this article is to present a systematic review of emerging drug therapies for psoriasis that, although in the early stage of development today, may enter the clinical practice of tomorrow. The current, established treatments are not mentioned; readers are referred to recently published excellent reviews on this subject (5 ± 8). The compounds included in this review have been selected from a screening of the Medline records; where references are not cited the information has been obtained from IDdb (Investigationa l Drugs Database, Current Drugs Ltd., http://www.iddb3.com/) and PharmaProjects (PJP Publications Ltd.# 2002, http://www.pharmaprojects.co.uk). IMMUNOSUPPRESSIVE AND ANTIINFLAMMATORY DRUGS The autoimmune cutaneous reaction is believed to play a causative role in the development of skin lesions in psoriasis (9). Most of the compounds under current development for psoriasis belong to different classes of immunosuppressives and anti-in ammatory drugs. The central cell in the current pathogenic model is the memory (CD45RO ) T-lymphocyte mediating the type 1 immune response. A type 1 immune response is mediated by TH1 and TC1 lymphocytes secreting a speci® c cytokine pro® le (IFN-g, TNF-a, IL-12). The recently reviewed immunopathogenesis of psoriasis (9, 10) will be mentioned only to the extent necessary for an understanding of the mechanism of action of the drugs included here. The memory T-lymphocytes secrete three major cytokines, IL-2, TNF-a and IFN-g. IL-2 acts at the early stages of T-cell activation and clone expansion, while TNF-a and IFN-g have a twofold role: to drive and stabilize the type I immune reaction (11) and by direct action on keratinocytes to stimulate their growth leading to epidermal hyperproliferation (12). The latter aspect is believed to be an aberrant regenerative response of the epidermal stem cells (12, 13).

Leflunomide induces apoptosis of thymocytes and T-cell hybridoma: differences in sensitivity and signaling pathways

LEFLUNOMIDE is a novel antiphlogistic and immunomodulating agent that has been shown to be efficacious in preventing and healing autoimmune disorders and reactions leading to organ transplantation rejection. However, the mechanisms of its action are not sufficiently elucidated. It has been postulated that the immunomodulatory effect of the drug is probably a consequence of reversible inhibition of T-cell proliferation by blocking the early stage of T-cell activation and preventing the expression of interleukin 2 receptor (IL-2R). The effect is partly dependent on inhibition of certain protein tyrosine kinases (PTK). In this work we show evidence that A77 1726, an active metabolite of leflunomide, induces apoptosis of rat thymocytes and rat thymocyte hybridoma in vitro. Experiments were done on thymocytes of AO rats (9 weeks old) and BWRT8 hybridoma cells. The following chemicals were obtained from ICN (Costa Mesa, Calif, USA); RPMI 1640 medium; fetal calf serum (FCS); Z-PheAla-CH2F, an IL-1b converting enzyme (ICE)-like inhibitor; Z-Val-Ala-Asp(Ome)-CH2F, a caspase inhibitor; and dexamethasone. Anti-rat abT-cell receptor (TCR) monoclonal antibody (mAb) was purchased from Serotec (Oxford, UK). Merocyanine (MC) 540, propidium iodide (PI), phorbol myristate acetate (PMA), genistein, and herbimycin A were obtained from Sigma (Munich, Germany). Cyclosporin A was obtained from Novartis (Basel, Switzerland), whereas A77 1726 was obtained from Hoechst, AG (Wiesbaden, Germany). Apoptosis induced by A77 1726, genistein, herbimycin A, dexamethasone, or immobilized R73 mAb was measured after cultivation of the cells for 20 hours (RPMI 1 10% FCS) using morphological criteria, MC 540, and PI staining. The percentage of specific apoptosis was determined as previously described. We found that A77 1726 induced apoptosis of both thymocytes and BWRT8 hybridoma cells, as judged by staining with MC 540 (Fig 1A), morphological analysis of nuclei (chromatin condensation, pyknosis, and kariorexis) (Fig 1B), and staining with PI (Fig 1C). All the methods gave comparable results. As shown in Fig 1A and 1B, A77 1726 at concentrations between 10 mmol/L and 100 mmol/L induced apoptosis of thymocytes in a dose-dependent manner. Approximately 10 times lower concentrations of the metabolite were necessary for apoptosis induction in BWRT8 cells. This is the first report showing direct proapoptotic effect of leflunomide on thymocytes and T-cell hybridoma. Peripheral T cells are more resistant, but the compound significantly modulates activation-induced death of these cells (Colic et al, (manuscript in preparation). It is well known that glucocorticoids and TCR crosslinking promote apoptosis of thymocytes. A similar phenomenon was published for BWRT8 cells. Therefore, in the next experiments we studied the interference of A77 1726 with dexamethasone and an mAb to rat abTCR (R73) immobilized to plastic in inducing apoptosis. As shown in Fig 1, the leflunomide metabolite increased apoptosis both of thymocytes and BWRT8 cells triggered by these agents. The finding may be relevant for selection processes in the thymus because the glucocorticoid-mediated apoptosis is supposed to be a model of “death by neglect,” whereas TCR cross-linking mimics the processes of negative selection. When we studied the effect of PMA, a PKC stimulator, and CsA, an inhibitor of calcineurin, the agents known to modulate apoptosis of thymocytes, it was found that PMA potentiated apoptosis of thymocytes but significantly inhibited apoptosis of BWRT8 cells. In contrast, CsA slightly inhibited the hybridoma cell death, but did not modulate apoptosis of thymocytes, either alone or in combination with A77 1726 (Fig 1C). PMA has been shown to either promote or inhibit apoptosis of T cells. The effect probably depends on whether this compound activates and/or subsequently depletes PKC and also on the type of cells or their activation state. In our experiments, a shortterm preincubation of the cells with PMA probably reflects activation of PKC, and the differences between the two cell types might be related to the differences in their activation state and intracellular signaling pathways. This is in agreement with the requirements for calcineurin, as shown using CsA. CsA has been found to prevent the activation-induced cell death (AICD) by inhibiting the Fas-L expression. However, it is not efficacious in modulating apoptosis of

Effect of aging on T lymphocyte activation

Studies of the early stages of T-cell activation reveal that T cells from aged mice show multiple abnormalities within the first few minutes after stimulation, including decline in the activation of the Raf-1/MEK/ERK kinases and in JNK protein kinase. Zap-70 kinase associated with the CD3 ζ chain shows a 2-fold increase with age in resting CD4 T cells, despite a three-fold decline with age in the levels of tyrosine phosphorylation of CD3 ζ; nonetheless, there is no effect of aging on Zap-70 kinase function in activated T cells as measured by in vitro kinase methods. Age-related impairment of the translocation of PKC θ from cytoplasm to the site of T-cell interaction with antigen-presenting cells may underlie downstream defects in the activation cascade.

Kinetics of T-cell Receptor Binding by Bivalent HLA-DR·Peptide Complexes That Activate Antigen-specific Human T-cells

Monovalent major histocompatibility complex-peptide complexes dissociate within seconds from the T-cell receptor (TCR), indicating that dimerization/multimerization may be important during early stages of T-cell activation. Soluble bivalent HLA-DR2.myelin basic protein (MBP) peptide complexes were expressed by replacing the F(ab) arms of an IgG2a antibody with HLA-DR2.MBP peptide complexes. The binding of bivalent HLA-DR2.peptide complexes to recombinant TCR was examined by surface plasmon resonance. The bivalent nature greatly enhanced TCR binding and slowed dissociation from the TCR, with a t((1)/(2)) of 2.1 to 4.6 min. Soluble bivalent HLA-DR2.MBP peptide complexes activated antigen-specific T-cells in the absence of antigen presenting cells. In contrast, soluble antibodies to the TCR.CD3 complex were ineffective, indicating that they failed to induce an active TCR dimer. TCR/CD3 antibodies induced T-cell proliferation when bound by antigen presenting cells that expressed Fc receptors. In the presence of dendritic cells, bivalent HLA-DR2. MBP peptide complexes induced T-cell activation at >100-fold lower concentrations than TCR/CD3 antibodies and were also superior to peptide or antigen. These results demonstrate that bivalent HLA-DR. peptide complexes represent effective ligands for activation of the TCR. The data support a role for TCR dimerization in early TCR signaling and kinetic proofreading.

The human homolog of Drosophila cornichon protein is differentially expressed in alloactivated T-cells

To identify novel genes induced in the early stage of T-cell activation, mRNA expression in alloactivated human lymphocytes was examined. Differential display-reverse transcription PCR analysis revealed a 207-bp cDNA fragment which was upregulated 24 h after allostimulation of a human T-cell line. The corresponding complete 1396 bp cDNA, named TGAM77, encodes a predicted 134 amino acid protein which shares 63% homology with the cornichon ( cni) protein of Drosophila melanogaster. Upregulation of TGAM77 mRNA in the early phase of T-cell activation was confirmed by Northern blot and RT-PCR analysis of activated human lymphocytes. TGAM77 mRNA is expressed in a variety of human tissues with various expression levels. In analogy to cni which is involved in an epidermal growth factor-like signaling pathway inducing cellular asymmetry in Drosophila oogenesis, TGAM77 might function in similar signaling establishing vectorial re-localization and concentration of signaling events in T-cell activation.

Inhibition of death receptor signals by cellular FLIP.

The widely expressed protein Fas is a member of the tumour necrosis factor receptor family which can trigger apoptosis. However, Fas surface expression does not necessarily render cells susceptible to Fas ligand-induced death signals, indicating that inhibitors of the apoptosis-signalling pathway must exist. Here we report the characterization of an inhibitor of apoptosis, designated FLIP (for FLICE-inhibitory protein), which is predominantly expressed in muscle and lymphoid tissues. The short form, FLIPs, contains two death effector domains and is structurally related to the viral FLIP inhibitors of apoptosis, whereas the long form, FLIP(L), contains in addition a caspase-like domain in which the active-centre cysteine residue is substituted by a tyrosine residue. FLIPs and FLIP(L) interact with the adaptor protein FADD and the protease FLICE, and potently inhibit apoptosis induced by all known human death receptors. FLIP(L) is expressed during the early stage of T-cell activation, but disappears when T cells become susceptible to Fas ligand-mediated apoptosis. High levels of FLIP(L) protein are also detectable in melanoma cell lines and malignant melanoma tumours. Thus FLIP may be implicated in tissue homeostasis as an important regulator of apoptosis.

Activation of the HLA-DRA gene in primary human T lymphocytes: novel usage of TATA and the X and Y promoter elements.:

Human T lymphocytes express human leukocyte antigen (HLA)-DR-alpha (DRA) upon mitogenic or antigenic stimulation. DR+ T cells are also found in a number of inflammatory and autoimmune diseases and have a proposed role in these diseases. The molecular mechanism of DR regulation in untransformed blood T lymphocytes was studied here by transient transfection of DRA-chloramphenicol acetyltransferase reporter gene constructs. Several novel features of this regulation were observed. During the early stages of T-cell activation by mitogens or antigens, strong promoter induction was exhibited with the proximal 43 bp of the DRA promoter which contains a TATTA motif. Addition of upstream X and Y DNA elements augmented the response. This contrasts with data from transformed cell lines in which the proximal 43 bp produced no detectable promoter function, and the inclusion of X and Y elements is essential for basal level expression. Mutation of the TATTA motif or substitution with a functional but different TATA element produced errant initiation and greatly reduced gene expression. Interestingly, T lymphocytes from a normal donor were DR+ prior to in vitro stimulation, and again, strong promoter activity was observed with 43 bp of proximal sequence. Unexpectedly, the presence of the X and Y elements correlated with a suppression of class II promoter function and surface antigen expression. This study of nontransformed lymphocytes reveals several novel features of DRA gene regulation and underscores the value and necessity of such studies.

Activation of the HLA-DRA gene in primary human T lymphocytes: novel usage of TATA and the X and Y promoter elements.

Human T lymphocytes express human leukocyte antigen (HLA)-DR-alpha (DRA) upon mitogenic or antigenic stimulation. DR+ T cells are also found in a number of inflammatory and autoimmune diseases and have a proposed role in these diseases. The molecular mechanism of DR regulation in untransformed blood T lymphocytes was studied here by transient transfection of DRA-chloramphenicol acetyltransferase reporter gene constructs. Several novel features of this regulation were observed. During the early stages of T-cell activation by mitogens or antigens, strong promoter induction was exhibited with the proximal 43 bp of the DRA promoter which contains a TATTA motif. Addition of upstream X and Y DNA elements augmented the response. This contrasts with data from transformed cell lines in which the proximal 43 bp produced no detectable promoter function, and the inclusion of X and Y elements is essential for basal level expression. Mutation of the TATTA motif or substitution with a functional but different TATA element produced errant initiation and greatly reduced gene expression. Interestingly, T lymphocytes from a normal donor were DR+ prior to in vitro stimulation, and again, strong promoter activity was observed with 43 bp of proximal sequence. Unexpectedly, the presence of the X and Y elements correlated with a suppression of class II promoter function and surface antigen expression. This study of nontransformed lymphocytes reveals several novel features of DRA gene regulation and underscores the value and necessity of such studies.

Activation of the HLA-DRA gene in primary human T lymphocytes: novel usage of TATA and the X and Y promoter elements.

Human T lymphocytes express human leukocyte antigen (HLA)-DR-alpha (DRA) upon mitogenic or antigenic stimulation. DR+ T cells are also found in a number of inflammatory and autoimmune diseases and have a proposed role in these diseases. The molecular mechanism of DR regulation in untransformed blood T lymphocytes was studied here by transient transfection of DRA-chloramphenicol acetyltransferase reporter gene constructs. Several novel features of this regulation were observed. During the early stages of T-cell activation by mitogens or antigens, strong promoter induction was exhibited with the proximal 43 bp of the DRA promoter which contains a TATTA motif. Addition of upstream X and Y DNA elements augmented the response. This contrasts with data from transformed cell lines in which the proximal 43 bp produced no detectable promoter function, and the inclusion of X and Y elements is essential for basal level expression. Mutation of the TATTA motif or substitution with a functional but different TATA element produced errant initiation and greatly reduced gene expression. Interestingly, T lymphocytes from a normal donor were DR+ prior to in vitro stimulation, and again, strong promoter activity was observed with 43 bp of proximal sequence. Unexpectedly, the presence of the X and Y elements correlated with a suppression of class II promoter function and surface antigen expression. This study of nontransformed lymphocytes reveals several novel features of DRA gene regulation and underscores the value and necessity of such studies.

The role of lymphokine-activated cell-associated antigen: III. Inhibition of T-cell activation by monoclonal killer-blocking antibody

The addition of monoclonal killer blocking antibodies (KBA MAb) to cultured T cells resulted in significant inhibition of T-cell responses to concanavalin A (Con A), class I antigen and class II antigen, whereas T-cell responses to phytohemagglutinin are insensitive to KBA MAb. The inhibitory effect of KBA MAb is observed only when KBA MAb is added to the culture at an early time. This indicates that the lymphokine-activated cell-associated antigen (LAA) defined by KBA MAb plays an important role in the early stages of T-cell activation. Con A-induced interleukin 2 (IL-2) receptor acquisition and IL-2 production, both of which are required for the early steps of T-cell activation, were greatly inhibited by KBA MAb. However, KBA MAb did not inhibit the action of IL-2, which is required for later stages of T-cell activation.

Regulation of the expression on mouse T lymphocytes of the epitope identified by monoclonal antibody 3A35

Monoclonal antibody 3A35 (MA 3A35) has previously been shown to be an activation marker of macrophages and T lymphocytes. It immunoprecipitated from macrophages a 200-kDa molecule belonging to the T200 family and from T cells a 85-kDa antigen. In the present work, the factors controlling the expression of the epitope identified by MA 3A35 on polyclonal activated T cells and T-cell clones, as well as the ability of 3A35 alone or together with complement to interfere with T-cell functions, were investigated. Corticoresistant thymocytes unreactive with MA 3A35 became fully reactive after 2 days of in vitro stimulation by PMA and IL-2 and the level of reactivity per cell declined to a low level thereafter. In helper and cytolytic T-cell clones, the expression of the epitope defined by MA 3A35 was also maximal soon after antigenic stimulation then declined. In helper-T-cell clones, the epitope remained detectable during the entire culture period, whereas in cytolytic clones its expression was markedly reduced at the end of the culture. The lineage of cytotoxic T lymphocytes (CTL) as studied in a bulk culture of spleen cells primed in vivo against a syngeneic tumor exhibited similar regulation by antigenic stimulation. The CTL precursors were resistant to lysis by MA 3A35 plus complement; after 3 days of culture with the stimulatory antigen, they became highly sensitive but their sensitivity then diminished and mature CTL were completely resistant. MA 3A35 plus complement also killed the activated T cells which responded to macrophage-presented antigens and were thought to be mainly Lyt-1 +. Therefore, the epitope identified by MA 3A35 was expressed predominantly at an early stage of T-cell activation. At a late stage, it persisted almost exclusively on helper and Lyt-1 + cells. In addition, MA 3A35 plus complement lysed NK cells, AK cells, and their precursors present in normal spleen. In the absence of complement, MA 3A35 had no detectable effect on T-cell functions.

Increased density of class I major histocompatibility complex antigens and decreased density of T-cell differentiation antigens in the early stages of T-cell activation

Major histocompatibility complex (MHC) antigens and T-cell differentiation antigens on activated T cells play a central role in T-cell interactions. In the present study, we have analyzed time courses of both quantity and density of the T-cell differentiation antigens, CD3 (T3), CD4 (T4), and CD8 (T8), as well as MHC antigens, on the cell surface of T cells, and made correlated measurements of DNA content with the surface antigen quantity as well with RNA content and cell size following activation of T cells by phytohemagglutinin. We found that the quantity and density of class I MHC antigens increase within 24 hr following activation and then decrease, while the quantity and density of the T-cell differentiation antigens decrease within 24 hr following activation, which suggests that T-cell recognition involving class I MHC gene products occurs at an early stage of T-cell activation. Class II MHC antigens can be detected on more than 40% of T cells as the expression of the T-cell differentiation antigens increase much later in the response. Cell cycle studies demonstrated that the density of class I MHC, CD3, CD4, and CD8 antigens was greater in G 0/G 1 phase cells than G 2 phase cells at all times tested during T-cell activation. Our findings suggest that T cells demonstrate a differential regulation in expression of MHC and T-cell differentiation antigens following activation which may reflect their role in cellular interactions.