T-cell Receptor-mediated Activation Research Articles

The role of bystander activation among CD8αα T cells in controlling Mycobacterium tuberculosisinfection

Abstract Classical cytotoxic T cells have a CD8 coreceptor with an αβ heterodimer and recognize peptide antigens via major histocompatibility (MHC) molecules. CD8 T cells with an αα homodimer structure are nonclassical and can recognize non-MHC molecules independently of antigen. CD8αα T cells have a higher propensity to undergo T cell receptor (TCR) independent bystander activation via pro-inflammatory cytokines. Bystander activation of CD8 T cells has shown a protective effect during Mycobacterium tuberculosis (M.tb) infection, but the distinct roles of CD8αα and CD8αβ T cells have not been elucidated. To begin assessing this, we compared the levels of CD8αα and CD8αβ T cell proliferation in response to TCR dependent stimulation using anti-CD3/CD28 beads. We observed that CD8αα T cells had lower levels of proliferation compared to CD8αβ T cells after three days of stimulation. After seven days, a population of CD8αα T cells did not proliferate while nearly all CD8αβ T cells showed high proliferative capacity. We next stimulated healthy peripheral blood mononuclear cells with IL-12, IL-15, and IL-18 to induce bystander activation and found 52.8% of CD8αα T cells secreted granzyme B compared to 25.1% of CD8αβ T cells. Interestingly, CD8αα T cells also had higher levels of CD69 expression than CD8αβ T cells (62.6% and 38.2%, respectively). Finally, we present plans for defining the role of bystander activated CD8αα and CD8αβ T cells during M.tb infection using tonsil organoids. These data reveal that CD8αα T cells have a distinct bimodal response to TCR mediated activation as well as an increased cytotoxic and activated phenotype after bystander activation. We further establish a foundation for future bystander activation studies in M.tb infection. Supported by grants from the NIH (R01 AI146072)

Atherosclerotic plaque T cells produce cytokines but do not exhibit signs of T-cell receptor-mediated activation

Abstract T-cell activation in the atherosclerotic plaque has been proposed to be a key event promoting lesion inflammation and destabilization. Although T cells and antigen-presenting cells are present in the plaque, it is unclear whether primary activation or secondary re-activation of T cells occurs in the lesion. We sought to quantify and characterize activated plaque T-cells in experimental atherosclerosis utilizing transgenic reporter mice of TCR-signalling (Nur77-GFP) or cytokine production (IFN-γ-YFP). As has been previously described, splenic Nur77hi T cells displayed markers consistent with a recently activated phenotype (PD-1+CD44+). Importantly, we observed very low levels of Nur77hi CD4+ or CD8+ T cells in the atherosclerotic aorta of Nur77-GFP-Apoe−/− mice compared to other tissues. In line with these findings, adoptive transfer of splenic Nur77-GFP CD4+ T cells to lymphodeficient atherosclerotic Rag1−/−Apoe−/− mice revealed markedly lower levels of Nur77hi T cells in plaque compared to lymphoid organs 10 weeks post-transfer, suggesting limited TCR-mediated activation in plaques. Conversely, analysis of atherosclerotic plaques from Apoe−/−IFN-γ-YFP reporter mice demonstrated robust IFN-γ production by CD4+ and CD8+ T cells. Altogether these results indicate that levels of cytokine release are above that of TCR-mediated T-cell activation, suggesting that antigen-driven activation of T cells in the plaque is not key for mediating T-cell driven atherosclerosis.

Cytotoxic T-Lymphocyte-Associated Protein 4 Haploinsufficiency-Associated Inflammation Can Occur Independently of T-Cell Hyperproliferation.

Located contiguously on the long arm of the second chromosome are gene paralogs encoding the immunoglobulin-family co-activation receptors CD28 and cytotoxic T-lymphocyte-associated protein 4 (CTLA4). CD28 and CTLA4 share the same B7 ligands yet each provides opposing proliferative signals to T cells. Herein, we describe for the first time two unrelated subjects with coexisting CD28 and CTLA4 haploinsufficiency due to heterozygous microdeletions of chromosome 2q. Although their clinical phenotype, multi-organ inflammatory disease, is superficially similar to that of CTLA4 haploinsufficient autoimmune lymphoproliferative syndrome type V (ALPS5) patients, we demonstrate our subjects’ underlying immunopathology to be distinct. Unlike ALPS5 T cells which hyperproliferate to T-cell receptor-mediated activation and infiltrate organs, T cells from our subjects are hypoproliferative and do not. Instead of T cell infiltrates, biopsies of affected subject tissues demonstrated infiltrates of lineage negative lymphoid cells. This histologic feature correlated with significant increases in circulating type 3 innate lymphoid cells (ILC3s) and ILC3 cytokines, interleukin 22, and interleukin-17A. CTLA4-Ig monotherapy, which we trialed in one subject, was remarkably effective in controlling inflammatory diseases, normalizing ILC3 frequencies, and reducing ILC3 cytokine concentrations.

Single-cell analysis identifies cellular markers of the HIV permissive cell.

Cellular permissiveness to HIV infection is highly heterogeneous across individuals. Heterogeneity is also found across CD4+ T cells from the same individual, where only a fraction of cells gets infected. To explore the basis of permissiveness, we performed single-cell RNA-seq analysis of non-infected CD4+ T cells from high and low permissive individuals. Transcriptional heterogeneity translated in a continuum of cell states, driven by T-cell receptor-mediated cell activation and was strongly linked to permissiveness. Proteins expressed at the cell surface and displaying the highest correlation with T cell activation were tested as biomarkers of cellular permissiveness to HIV. FACS sorting using antibodies against several biomarkers of permissiveness led to an increase of HIV cellular infection rates. Top candidate biomarkers included CD25, a canonical activation marker. The combination of CD25 high expression with other candidate biomarkers led to the identification of CD298, CD63 and CD317 as the best biomarkers for permissiveness. CD25highCD298highCD63highCD317high cell population showed an enrichment of HIV-infection of up to 28 fold as compared to the unsorted cell population. The purified hyper-permissive cell subpopulation was characterized by a downregulation of interferon-induced genes and several known restriction factors. Single-cell RNA-seq analysis coupled with functional characterization of cell biomarkers provides signatures of the “HIV-permissive cell”.

Small-molecule inhibitors of PD-1/PD-L1 immune checkpoint alleviate the PD-L1-induced exhaustion of T-cells.

Antibodies targeting the PD-1/PD-L1 immune checkpoint achieved spectacular success in anticancer therapy in the recent years. In contrast, no small molecules with cellular activity have been reported so far. Here we provide evidence that small molecules are capable of alleviating the PD-1/PD-L1 immune checkpoint-mediated exhaustion of Jurkat T-lymphocytes. The two optimized small-molecule inhibitors of the PD-1/PD-L1 interaction, BMS-1001 and BMS-1166, developed by Bristol-Myers Squibb, bind to human PD-L1 and block its interaction with PD-1, when tested on isolated proteins. The compounds present low toxicity towards tested cell lines and block the interaction of soluble PD-L1 with the cell surface-expressed PD-1. As a result, BMS-1001 and BMS-1166 alleviate the inhibitory effect of the soluble PD-L1 on the T-cell receptor-mediated activation of T-lymphocytes. Moreover, the compounds were effective in attenuating the inhibitory effect of the cell surface-associated PD-L1. We also determined the X-ray structures of the complexes of BMS-1001 and BMS-1166 with PD-L1, which revealed features that may be responsible for increased potency of the compounds compared to their predecessors. Further development may lead to the design of an anticancer therapy based on the orally delivered immune checkpoint inhibition.

Immunosuppression after sepsis: systemic inflammation and sepsis induce a loss of naïve T-cells but no enduring cell-autonomous defects in T-cell function.

Sepsis describes the life-threatening systemic inflammatory response (SIRS) of an organism to an infection and is the leading cause of mortality on intensive care units (ICU) worldwide. An acute episode of sepsis is characterized by the extensive release of cytokines and other mediators resulting in a dysregulated immune response leading to organ damage and/or death. This initial pro-inflammatory burst often transits into a state of immune suppression characterised by loss of immune cells and T-cell dysfunction at later disease stages in sepsis survivors. However, despite these appreciations, the precise nature of the evoked defect in T-cell immunity in post-acute phases of SIRS remains unknown. Here we present an in-depth functional analysis of T-cell function in post-acute SIRS/sepsis. We document that T-cell function is not compromised on a per cell basis in experimental rodent models of infection-free SIRS (LPS or CpG) or septic peritonitis. Transgenic antigen-specific T-cells feature an unaltered cytokine response if challenged in vivo and ex vivo with cognate antigens. Isolated CD4+/CD8+ T-cells from post-acute septic animals do not exhibit defects in T-cell receptor-mediated activation at the the level of receptor-proximal signalling, activation marker upregulation or expansion. However, SIRS/sepsis induced transient lymphopenia and gave rise to an environment of immune attenuation at post acute disease stages. Thus, systemic inflammation has an acute impact on T-cell numbers and adaptive immunity, but does not cause major cell-autonomous enduring functional defects in T-cells.

SDF1α-induced interaction of the adapter proteins Nck and HS1 facilitates actin polymerization and migration in Tcells.

Noncatalytic region of tyrosine kinase (Nck) is an adapter protein that comprises one SH2 (Src homology) domain and three SH3 domains. Nck links receptors and receptor-associated tyrosine kinases or adapter proteins to proteins that regulate the actin cytoskeleton. Whereas the SH2 domain binds to phosphorylated receptors or associated phosphoproteins, individual interactions of the SH3 domains with proline-based recognition motifs result in the formation of larger protein complexes. In Tcells, changes in cell polarity and morphology during T-cell activation and effector function require the T-cell receptor-mediated recruitment and activation of actin-regulatory proteins to initiate cytoskeletal reorganization at the immunological synapse. We previously identified the adapter protein HS1 as a putative Nck-interacting protein. We now demonstrate that the SH2 domain of Nck specifically interacts with HS1 upon phosphorylation of its tyrosine residue 378. We report that in human Tcells, ligation of the chemokine receptor CXCR4 by stromal cell-derived factor 1α (SDF1α) induces a rapid and transient phosphorylation of tyrosine 378 of HS1 resulting in an increased association with Nck. Consequently, siRNA-mediated downregulation of HS1 and/or Nck impairs SDF1α-induced actin polymerization and T-cell migration.

Cytotoxic response of human regulatory T cells upon T-cell receptor-mediated activation: a matter of purity.

Regulatory T cells (Tregs) are major players in immune homeostasis, and defects in their number and/or function are associated with a broad range of immunological disorders including autoimmunity, graft-versus-host disease (GvHD), transplant rejection, chronic infections or malignant diseases.1, 2 One way of how Tregs actively damp an immune response might be direct induction of apoptosis in activated antigen-presenting cells as well as in conventional effector T cells (Teffs) by secretion of lytic granzyme- and perforin-containing granula. Despite the substantial progress made in our understanding of Treg biology, the cytotoxic potential of Tregs is still under debate. As Tregs are under intense investigation as cellular therapeutics for treatment of GvHD and graft rejection, and might also interfere with novel antitumor strategies,3 this question is not a purely academic one but also implies major consequences for the prospective development of clinical treatment strategies involving Tregs. To shed some light on this controversial issue, we decided to analyze this question using peripheral human Tregs, as these cells are one major source for potential therapeutic applications.1, 2 As Tregs are a very rare population in human peripheral blood, it seems currently technically too challenging to isolate sufficient Treg numbers with defined antigen-specificity in a way as it can be done for—for example, virus- or tumor-specific Teffs. Therefore, in this study recombinant bispecific antibodies (bsAb) were applied for antigen-specific redirection of polyclonal human T cells as surrogate for T-cell receptor (TCR)-mediated stimulation. BsAb comprise binding sites for two different target antigens (for example, CD3 and a cell surface antigen) and can directly cross-link T cells and antigen-expressing cells. This results in a major histocompatibility complex (MHC)- and TCR-independent activation and triggering of effector mechanisms of the cross-linked immune cell.4 T-cell-engaging bsAb have been proven to be highly effective in inducing the lytic capacity of both CD4+ and CD8+ Teffs in vitro, in vivo and even in clinical trials and closely resemble signal cascades induced upon binding of a TCR to its target peptide/MHC complex.4, 5, 6

Defining the lymphoid stress surveillance response in human skin

BackgroundThe skin contains many tissue-resident immune cells increasingly acknowledged as having key roles in maintaining tissue integrity and excluding infection. In lymphoid stress surveillance, lymphocytes are activated rapidly and in synchrony by stress ligands expressed by tissue cells after cellular stress and engage activating receptors such as NKG2D. Although much of this process has been worked out in model systems, whether this capability exists in human skin is not entirely clear. Therefore, we have characterised the human skin-resident lymphocyte compartment, and measured functional responsiveness of cells to see whether certain T cells have the capacity to respond rapidly to tissue stress. MethodsWe used normal adult human skin discarded after cutaneous surgery. Protocols were established to isolate and characterise lymphocytes by tissue disaggregation or three-dimensional explant culture. Skin-resident lymphocytes were characterised by multicolour flow cytometry, including cell-surface receptor expression and cytokine production after in-vitro stimulation. FindingsAfter ex-vivo isolation, distinct skin-resident lymphocyte subsets were identified, including the unconventional lymphocytes, γδ T cells and natural killer (NK) cells. Subsets were consistent across individuals; expressed as a mean (SD) percentage of leucocytes isolated, there were 46·1% CD4 T cells (15·2), 20·2% CD8 T cells (14·8), 1·6% γδ T cells (1·0), and 6·2% NK cells (5·0) (n=7–10). Explant culture greatly increased lymphocyte yield, enabling detailed functional characterisation. In addition, culture conditions greatly amplified the γδ T-cell compartment (1·6–4·0%, p=0·01, Student's t test). Skin-resident lymphocytes displayed skin-homing surface markers and a memory phenotype. Lymphocytes differed in expression of co-stimulatory receptors, with CD8 T cells, γδ T cells, and NK cells expressing the activating receptor NKG2D. Upon in-vitro activation, skin-resident lymphocytes readily produced cytokines and displayed distinct cytokine-producing profiles, with the response dependent on the strength of T-cell receptor-mediated activation. Skin-derived NKG2D+ lymphocytes had clear potential to produce both tumour necrosis factor α and interferon γ. InterpretationSkin-resident lymphocytes could be isolated from healthy human skin. Consistent with published data, cultured skin-resident αβ T cells maintained a tissue-resident memory (TRM) phenotype. In addition, that skin γδ T cell and NK cells also had a TRM-type phenotype was a novel finding. Distinct NKG2D+ lymphocytes were identified, which showed variable expression of additional activatory or inhibitory co-receptors, and were functionally competent with distinct cytokine-producing subsets. This system provides a platform to further characterise human skin-resident lymphocytes and their potential to be directly activated by surrounding tissue dysregulation. FundingUK Medical Research Council.

Allosteric Noncompetitive Small Molecule Selective Inhibitors of CD45 Tyrosine Phosphatase Suppress T-Cell Receptor Signals and Inflammation In Vivo

CD45 is a receptor-like member of the protein tyrosine phosphatase (PTP) family. We screened in silico for small molecules binding at a predicted allosteric pocket unique to the CD45 intracellular domain, and validated inhibitors by in vitro phosphatase assays. Compound 211 exhibited a CD45 IC50 value of 200 nM and had >100-fold selectivity over six related PTPs. The relevance of the allosteric pocket was verified through site-directed mutagenesis. Compound 211 has a noncompetitive mechanism of action, and it is extremely effective at preventing dephosphorylation of substrate Lck phosphotyrosine (pY)-505 versus preventing dephosphorylation of Lck pY-393. In cultured primary T cells, compound 211 prevents T-cell receptor-mediated activation of Lck, Zap-70, and mitogen-activated protein kinase, and interleukin-2 production. In a delayed-type hypersensitivity reaction in vivo, compound 211 abolished inflammation. This work demonstrates a novel approach to develop effective allosteric inhibitors that can be expanded to target the corresponding allosteric domains of other receptor PTPs.

Analysis of sirtuin 1 expression reveals a molecular explanation of IL-2–mediated reversal of T-cell tolerance

The type III histone deacetylase sirtuin 1 (Sirt1) is a suppressor of both innate and adoptive immune responses. We have recently found that Sirt1 expression is highly induced in anergic T cells. However, the transcriptional program to regulate Sirt1 expression in T cells remains uncharacterized. Here we report that the early responsive genes 2 and 3, which can be up-regulated by T-cell receptor-mediated activation of nuclear factor of activated T-cell transcription factors and are involved in peripheral T-cell tolerance, bind to the sirt1 promoter to transcript sirt1 mRNA. In addition, the forkhead transcription factor, FoxO3a, interacts with early responsive genes 2/3 on the sirt1 promoter to synergistically regulate Sirt1 expression. Interestingly, IL-2, a cytokine that can reverse T-cell anergy, suppresses sirt1 transcription by sequestering FoxO3a to the cytoplasm through activating the PI3K-AKT pathway. Expression of the constitutively active form of FoxO3a blocks IL-2-mediated reversal of T-cell tolerance by retaining sirt1 expression. Our findings here provide a molecular explanation of IL-2-mediated reversion of T-cell anergy.

Modulatory effect of insulin on T cell receptor mediated calcium signaling is blunted in long lasting type 1 diabetes mellitus

AbstractInsulin significantly influences Ca2+ signals evoked by various stimulants. In type 1 recent onset diabetes mellitus the proliferative response of T cells is significantly decreased. The number of clinical trials exploring the role of anti-CD3 monoclonal antibodies (mAb) as a therapeutic agent in recent onset diabetes mellitus type 1 is increasing last years. Therefore, a better understanding of the interplay between T cell receptor (TCR) dependent Ca2+ increase, and insulin is of vital clinical significance. The aim of the study was to assess the effect of insulin on TCR evoked Ca2+ responses in T lymphocytes obtained from healthy volunteers and patients suffering from long lasting diabetes mellitus type 1. Analysis was performed with use of the flow cytometer. We demonstrated that T cells ability to mobilize Ca2+ was significantly reduced in long lasting diabetes mellitus type 1. Ca2+ decrease achieved by the long term incubation with anti-CD3 mAb in T cells from healthy volunteers was restored by insulin. Strong interrelationship between baseline Ca2+ level and plateau phase response to TCR stimulation was observed in the cytoplasm of cells pre-incubated with insulin from both healthy subjects and diabetic patients (r=0.95, p<0.0001 and r=0.94, p<0.0001, respectively). We postulate the existence of the interplay between TCR mediated activation and insulin. The TCR-insulin interplay is blunted in long lasting diabetes mellitus type 1. These observations may have an important implication for future therapeutic options in diabetes.

Attenuation by Targeting the B- and T-Cell Attenuator

BTLA is a member of the immunoglobulin family thought to serve as a negative costimulatory molecule on activated T cells. Several recent studies, including the one published in this issue by del Rio and colleagues, have used BTLA-deficient mice and/or anti-BTLA antibodies to investigate the role of BTLA in regulating graft versus host reaction (GvHR) (1–3). Curiously, targeting BTLA either via genetic deletion, or blocking antibody, or agonistic antibody, all resulted in amelioration of GvHR. This suggests that BTLA may have several functions. Indeed, if BTLA’s function were solely inhibitory, agonistic antibody cross-linking should inhibit T cell function, reducing GvHR, whereas genetic deletion or antibody blockade should enhance T cell function, exacerbating GvHR. BTLA is expressed on activated T cells, B cells, dendritic cells, macrophages and some NK cells. Supporting its inhibitory effects, BTLA-deficient T cells display increased proliferation upon mitogenic stimulation and BTLA-KO mice are more susceptible to some autoimmune diseases. BTLA binds the TNFR family member herpes virus entry mediator (HVEM), which can also bind the coinhibitory receptor CD160 and the coactivating ligand LIGHT. The similar phenotype observed in HVEM-deficient and BTLA-deficient mice suggests that BTLA/HVEM is the predominant interaction on T cells (4). To interpret experiments using antibodies that target a surface molecule, it is essential to know whether a given antibody can drive depletion of the targeted cells, block the epitope for the endogenous ligand, or trigger signaling by the targeted molecule. To study the role of BTLA in GvHR, the Murphy group used BTLA-deficient mice as well as the non-depleting and presumably blocking anti-BTLA antibody 6A6 in a hematopoietic stem cell transfer model in which irradiated F1 recipients received parental bone marrow and/or splenocytes (2). Their experiments demonstrated reduced recovery of donor T cells with blocked or genetically absent BTLA, with the protective effect of 6A6 requiring direct engagement of BTLA on donor T cells. Sakoda et al., in a model of GvHR induced by transfer of donor splenocytes into either non-irradiated or lethally irradiated F1 recipients, used their own agonistic, non HVEM-blocking anti-BTLA mAb, BYK-1, and also BTLA-deficient mice (3). Similar to the Murphy model, reduced GvHR observed after transfer of BTLA-KO T cells was associated with diminished recovery of donor T cells whereas BYK-1 did not prevent T cell engraftment. Moreover, these authors demonstrated that reconstitution of BTLA-deficient T cells with a signaling-deficient BTLA molecule restored T cell survival. These 2 studies suggest that T cell-BTLA can serve as a ligand to the receptor HVEM that then delivers a pro-survival signal to T cells. Sakoda also showed that agonistic targeting of BTLA resulted in reduced cytotoxicity and cytokine production against allogenic stimuli (3). In the current study, del Rio and colleagues used their own anti-BTLA antibody, 4G12b, in non-irradiated F1 recipients of parental splenocytes, and confirmed the prevention of GvHR with no reduction in numbers of donor CD4+ and CD8+ T cells (1). Rather, they reported markedly reduced in vivo lysis of host cells and reduced production of cytokines by donor CD8+ T cells in response to alloantigen, suggesting that this agonistic antibody is preventing an alloreactive and cytotoxic pathway independently of any effects on T cell survival. Interestingly, the 6A6 used by the Murphy group and the 4G12b antibody used by del Rio recognize an overlapping epitope on BTLA, but binding of 4G12b to BTLA does not prevent BTLA from engaging HVEM (5), therefore presumably allowing BTLA to drive HVEM-mediated pro-survival effects. Moreover, their distinct Fc portions (hamster IgG for 6A6 versus rat IgG for 4G12b and BYK-1) may trigger different FcR- and complement-mediated signals which may contribute to their distinct consequences on T cell survival in vivo. Together, these 3 studies suggest that targeting BTLA is capable of reducing GvHR by alternative mechanisms depending on whether the endogenous HVEM-BTLA interaction is preserved or not (Figure 1). Figure 1 Schematic model of the dual effects of BTLA engagement Although these studies clarify the dual role of BTLA on T cells, a few issues remain unresolved. For instance, donor B cells express BTLA and may produce alloantibodies in this model. The agonistic anti-BTLA treatment may inhibit B cells either directly or indirectly by reducing T cell help such that reduced production of alloantibodies could contribute to the diminished in vivo cytotoxicity in anti-BTLA-treated animals. In addition, the contribution of interactions by remaining receptor/ligand pairs in the HVEM pathway needs to be considered. This may be challenging given the evidence of bi-directional signaling downstream of the BTLA/HVEM interaction as well as the potential contributions of HVEM binding to CD160 and LIGHT.

Participation of the Cell Polarity Protein PALS1 to T-Cell Receptor-Mediated NF-κB Activation

BackgroundBeside their established function in shaping cell architecture, some cell polarity proteins were proposed to participate to lymphocyte migration, homing, scanning, as well as activation following antigen receptor stimulation. Although PALS1 is a central component of the cell polarity network, its expression and function in lymphocytes remains unknown. Here we investigated whether PALS1 is present in T cells and whether it contributes to T Cell-Receptor (TCR)-mediated activation.Methodology/Principal FindingsBy combining RT-PCR and immunoblot assays, we found that PALS1 is constitutively expressed in human T lymphocytes as well as in Jurkat T cells. siRNA-based knockdown of PALS1 hampered TCR-induced activation and optimal proliferation of lymphocyte. We further provide evidence that PALS1 depletion selectively hindered TCR-driven activation of the transcription factor NF-κB.ConclusionsThe cell polarity protein PALS1 is expressed in T lymphocytes and participates to the optimal activation of NF-κB following TCR stimulation.

Preferential expression of the novel alternative isoform I.3 of hypoxia-inducible factor 1α in activated human T lymphocytes

Hypoxia-inducible factor-1alpha (HIF-1alpha) is critical not only in the regulation of oxygen homeostasis but also in the regulation of innate and adaptive immune systems. We previously reported that T-cell receptor-mediated activation of T cells in mice leads to the expression of an alternative isoform of HIF-1alpha that inhibits activated T cells in a delayed negative feed-back manner. In this paper, we describe a novel mRNA isoform of human HIF-1alpha that is upregulated in peripheral T lymphocytes after T-cell receptor stimulation. This activation- inducible isoform is expressed using the alternative first exon I.3, and it encodes a protein that is 24 amino acids longer than the ubiquitous HIF-1alpha isoform. This mRNA isoform I.3 of HIF-1alpha is expressed in a tissue-specific manner with the highest expression found in peripheral blood leukocytes and the thymus.

Shedded neuronal ICAM-5 suppresses T-cell activation

AbstractIntercellular adhesion molecules (ICAMs) bind to leukocyte β2 integrins, which, among other functions, provide costimulatory signals for T-cell activation. ICAM-5 (telencephalin) is expressed in the somadendritic region of neurons of the mammalian brain. The receptor for ICAM-5 is the integrin LFA-1, a major leukocyte integ-rin expressed in lymphocytes and microglia. In conditions of brain ischemia, epilepsy, and encephalitis, the soluble form of ICAM-5 (sICAM-5) has been detected in physiologic fluids. Here, we report that sICAM-5 attenuates the T-cell receptor-mediated activation of T cells as demonstrated by the decreased expression of the activation markers CD69, CD40L, and CD25 (IL-2R). This effect is most clearly seen in CD45ROLow (naive), and not in CD45ROHigh (memory) T cells, and is most effective early in priming, but not in the presence of strong costimulatory signals. Furthermore, sICAM-5 promotes the mRNA expression of the cytokines TGF-β1 and IFN-γ, but not TNF. The formation of sICAM-5 is promoted by activated T cells through the cleavage of ICAM-5 from neurons. This suggests that ICAM-5 is involved in immune privilege of the brain and acts as an anti-inflammatory agent.

Phosphorylation of Bcl10 Negatively Regulates T-Cell Receptor-Mediated NF-κB Activation

Bcl10 (B-cell lymphoma 10) is an adaptor protein comprised of an N-terminal caspase recruitment domain and a C-terminal serine/threonine-rich domain. Bcl10 plays a critical role in antigen receptor-mediated NF-kappaB activation and lymphocyte development and functions. Our current study has discovered that T-cell activation induced monophosphorylation and biphosphorylation of Bcl10 and has identified S138 within Bcl10 as one of the T-cell receptor-induced phosphorylation sites. Alteration of S138 to an alanine residue impaired T-cell activation-induced ubiquitination and subsequent degradation of Bcl10, ultimately resulting in prolongation of TCR-mediated NF-kappaB activation and enhancement of interleukin-2 production. Taken together, our findings demonstrate that phosphorylation of Bcl10 at S138 down-regulates Bcl10 protein levels and thus negatively regulates T-cell receptor-mediated NF-kappaB activation.

Regulatory T‐cell function of adult T‐cell leukemia/lymphoma cells

Adult T-cell leukemia/lymphoma (ATLL) patients are highly immunocompromised, but the underlying mechanism responsible for this state remains obscure. Recent studies demonstrated that FOXP3, which is a master control gene of naturally occurring regulatory T (Treg) cells, is expressed in the tumor cells from a subset of patients with ATLL. Since most ATLL cells express both CD4 and CD25, these tumors might originate from CD4(+)CD25(+)FOXP3(+) Treg cells, based on their phenotypic characteristics. However, whether ATLL cells actually function as Treg cells has not yet been clearly demonstrated. Here, we show that ATLL cells from a subset of patients are not only hypo-responsive to T-cell receptor-mediated activation, but also suppress the proliferation of autologous CD4(+) non-ATLL cells. Furthermore, ATLL cells from this subset of patients secrete only small amounts of IFN-gamma, and suppress IFN-gamma production by autologous CD4(+) non-ATLL cells. These are the first data showing that ATLL cells from a subset of patients function as Treg cells in an autologous setting. The present study provides novel insights into understanding the immunopathogenesis of ATLL, i.e., how HTLV-1-infected cells can survive in the face of host immune responses. It also adds to our understanding of ATLL patients' severely immunocompromised state.

The ADAP/SKAP55 Signaling Module Regulates T-Cell Receptor-Mediated Integrin Activation through Plasma Membrane Targeting of Rap1

Adhesion of T cells after stimulation of the T-cell receptor (TCR) is mediated via signaling processes that have collectively been termed inside-out signaling. The molecular basis for inside-out signaling is not yet completely understood. Here, we show that a signaling module comprising the cytosolic adapter proteins ADAP and SKAP55 is involved in TCR-mediated inside-out signaling and, moreover, that the interaction between ADAP and SKAP55 is mandatory for integrin activation. Disruption of the ADAP/SKAP55 module leads to displacement of the small GTPase Rap1 from the plasma membrane without influencing its GTPase activity. These findings suggest that the ADAP/SKAP55 complex serves to recruit activated Rap1 to the plasma membrane. In line with this hypothesis is the finding that membrane targeting of the ADAP/SKAP55 module induces T-cell adhesion in the absence of TCR-mediated stimuli. However, it appears as if the ADAP/SKAP55 module can exert its signaling function outside of the classical raft fraction of the cell membrane.

Direct Inhibition of T-Lymphocyte Activation by Anthrax Toxins In Vivo

The causative agent of anthrax, Bacillus anthracis, produces two toxins that contribute in part to its virulence. Lethal toxin is a metalloprotease that cleaves upstream mitogen-activated protein kinase kinases. Edema toxin is a calmodulin-dependent adenylate cyclase. Previous studies demonstrated that the anthrax toxins are important immunomodulators that promote immune evasion of the bacterium by suppressing activation of macrophages and dendritic cells. Here we showed that injection of sublethal doses of either lethal or edema toxin into mice directly inhibited the subsequent activation of T lymphocytes by T-cell receptor-mediated stimulation. Lymphocytes were isolated from toxin-injected mice after 1 or 4 days and stimulated with antibodies against CD3 and CD28. Treatment with either toxin inhibited the proliferation of T cells. Injection of lethal toxin also potently inhibited cytokine secretion by stimulated T cells. The effects of edema toxin on cytokine secretion were more complex and were dependent on the length of time between the injection of edema toxin and the isolation of lymphocytes. Treatment with lethal toxin blocked multiple kinase signaling pathways important for T-cell receptor-mediated activation of T cells. Phosphorylation of the extracellular signal-regulated kinase and the stress-activated kinase p38 was significantly decreased. In addition, phosphorylation of the serine/threonine kinase AKT and of glycogen synthase kinase 3 was inhibited in T cells from lethal toxin-injected mice. Thus, anthrax toxins directly act on T lymphocytes in a mouse model. These findings are important for future anthrax vaccine development and treatment.