T-cell Receptor Signal Transduction Research Articles

A dynamic biomimetic model of the membrane-bound CD4-CD3-TCR complex during pMHC disengagement

The coordinated (dis)engagement of the membrane-bound T cell receptor (TCR)-CD3-CD4 complex from the peptide-major histocompatibility complex (pMHC) is fundamental to TCR signal transduction and T cell effector function. As such, an atomic-scale understanding would not only enhance our basic understanding of the adaptive immune response but would also accelerate the rational design of TCRs for immunotherapy. In this study, we explore the impact of the CD4 coreceptor on the TCR-pMHC (dis)engagement by constructing a molecular-level biomimetic model of the CD3-TCR-pMHC and CD4-CD3-TCR-pMHC complexes within a lipid bilayer. After allowing the system complexes to equilibrate (engage), we use steered molecular dynamics to dissociate (disengage) the pMHC. We find that 1) the CD4 confines the pMHC closer to the T cell by 1.8 nm at equilibrium; 2) CD4 confinement shifts the TCR along the MHC binding groove engaging a different set of amino acids and enhancing the TCR-pMHC bond lifetime; 3) the CD4 translocates under load increasing the interaction strength between the CD4-pMHC, CD4-TCR, and CD4-CD3; and 4) upon dissociation, the CD3-TCR complex undergoes structural oscillation and increased energetic fluctuation between the CD3-TCR and CD3-lipids. These atomic-level simulations provide mechanistic insight on how the CD4 coreceptor impacts TCR-pMHC (dis)engagement. More specifically, our results provide further support (enhanced bond lifetime) for a force-dependent kinetic proofreading model and identify an alternate set of amino acids in the TCR that dominate the TCR-pMHC interaction and could thus impact the design of TCRs for immunotherapy.

Functional heterogeneity and adaptation of naive T cells in response to tonic TCR signals.

Mature CD4+ and CD8+ T cells constitutively experience weak T cell receptor (TCR) stimulation in response to self-antigens, termed tonic (or basal) signaling. How tonic TCR signal strength impacts T cell responses to foreign antigens is an active area of investigation. Such studies rely on surrogate markers of tonic signal strength, including CD5, Ly6C, and transgenic reporters of Nr4a genes. Recent research indicates that strong tonic TCR signal strength influences basal T cell metabolism, effector differentiation, and TCR signal transduction. T cells that experience the strongest tonic TCR signaling exhibit features of T cell activation and negative regulation. These data suggest a model whereby adaptation to tonic signaling has lasting effects that alter T cell activation and differentiation.

Phosphorylation of CrkL S114 induced by common gamma chain cytokines and T-cell receptor signal transduction

T-cell activation and cellular expansion by common gamma chain cytokines such as Interleukin-2 is necessary for adaptive immunity. However, when unregulated these same pathways promote pathologies ranging from autoimmune disorders to cancer. While the functional role of Interleukin-2 and downstream effector molecules is relatively clear, the repertoire of phosphoregulatory proteins downstream of this pathway is incomplete. To identify phosphoproteins downstream of common gamma chain receptor, YT cells were radiolabeled with [32P]-orthophosphate and stimulated with Interleukin-2. Subsequently, tyrosine phosphorylated proteins were immunopurified and subjected to tandem mass spectrometry—leading to the identification of CrkL. Phosphoamino acid analysis revealed concurrent serine phosphorylation of CrkL and was later identified as S114 by mass spectrometry analysis. S114 was inducible through stimulation with Interleukin-2 or T-cell receptor stimulation. Polyclonal antibodies were generated against CrkL phospho-S114, and used to show its inducibility by multiple stimuli. These findings confirm CrkL as an Interleukin-2 responsive protein that becomes phosphorylated at S114 by a kinase/s downstream of PI3K and MEK/ERK signaling.

A Direct Comparison of the In Vivo Efficacy and in Vitro Signaling of Chimeric Antigen Receptors and Endogenous T Cell Receptors

The adoptive transfer of chimeric antigen receptor (CAR) expressing T cells targeting the CD19 antigen induces remission in 70-90% of pediatric patients with relapsed and/or refractory acute lymphoblastic leukemia (ALL). Despite initial enthusiasm, emerging data indicate the durability of remission is limited in a substantial number of patients, therefore, addressing relapse will be critical for increasing the clinical efficacy of CAR T cell therapy. CARs are synthetic receptors designed to redirect the antigen specificity of T cells while recapitulating the T cell activating properties of the endogenous T cell receptor (TCR). Clinical and pre-clinical observations have demonstrated that CAR T cells have inferior sensitivity to low levels of antigen and have diminished persistence in vivo compared to T cells responding to TCR activation. These observations suggest the CAR does not fully replicate the signaling of a TCR which subsequently leads to suboptimal T cell function which may underlie the inability of CAR T cell therapy to induce durable remissions in a significant number of patients.In these studies, we directly compared the in vivo and in vitro consequences of CAR versus TCR activation, as well as the signal transduction of each of these receptors. To accomplish this, we created a novel, syngeneic model system in which a 2nd generation CAR is introduced into OTI TCR transgenic T cells to generate a population of T cells (CAR-OTI cells) in which each cell recognizes the CD19 antigen via its CAR as well as the ovalbumin (OVA) antigen via its TCR, thus providing a model in which the function and signaling of each receptor could be studied within a uniform population of T cells. To stimulate either the CAR or the TCR, we generated antigen-expressing cell lines from the pre-B ALL cell line, E2aPbx, that expressed either the cognate antigen for the CAR (CD19), the TCR (OVA) or both. These cell lines allowed us to stimulate either receptor in a model which preserved the properties of the cell-to-cell interactions expected in vivo . We validated the activity of each receptor in this model, by demonstrating similar in vitro degranulation, proliferation and target cell lysis in CAR-OTI cells upon co-incubation with CAR-antigen or TCR-antigen positive E2aPbx cells. Despite the similarities seen in vitro, CAR-OTI cells adoptively transferred into mice with established E2aPbx leukemia demonstrated enhanced expansion at 4 (p<0.001) and 6 days (p<0.01) post-infusion in TCR-activated CAR-OTI cells, relative to CAR-activated CAR-OTI cells. Likewise, clearance of leukemia from the bone marrow (p<0.001) and survival (p<0.01) were each superior in mice in which CAR-OTI cells were activated through the TCR, relative to the CAR. These results demonstrate the significant difference in in vivo behavior and efficacy of CAR- versus TCR-mediated T cell activation, and suggest these differences may result from differences in the signals generated by each receptor.Using our model of CAR-OTI cells and E2aPbx cells expressing either the CAR- or TCR-antigen, we interrogated proteins in the canonical TCR signal transduction cascade after CAR or TCR activation. We demonstrated that CAR activation resulted in increased and prolonged phosphorylation of Zap70, a kinase central to canonical TCR signal transduction. Despite the increased activation of Zap70, the CAR signal demonstrated diminished activation of the MAPK family members, Erk (p<0.001) and p38 (p<0.05), which correlated with the diminished in vivo efficacy seen in this model. Furthermore, this model was used to define the global phosphoproteome of CAR- and TCR-activated CAR-OTI cells by a Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) methodology in conjunction with mass spectrometry (MS). Our SILAC-MS analysis demonstrated multiple differences in the signal transduction cascade of CARs relative to TCRs, including proteins of the signalosome as well as the NFAT and NF-kB pathways. These data demonstrate that a CAR generates an “altered” signal, relative to that of a TCR, correlating with diminished in vivo expansion and efficacy in the treatment of established leukemia. These findings underscore the need to better understand the biology of chimeric antigen receptors as a means of rationally altering their design to improve their clinical effectiveness in mediating durable remissions in patients with relapsed/refractory ALL. DisclosuresNo relevant conflicts of interest to declare.

PLCγ1 promotes phase separation of T cell signaling components.

The T cell receptor (TCR) pathway receives, processes, and amplifies the signal from pathogenic antigens to the activation of T cells. Although major components in this pathway have been identified, the knowledge on how individual components cooperate to effectively transduce signals remains limited. Phase separation emerges as a biophysical principle in organizing signaling molecules into liquid-like condensates. Here, we report that phospholipase Cγ1 (PLCγ1) promotes phase separation of LAT, a key adaptor protein in the TCR pathway. PLCγ1 directly cross-links LAT through its two SH2 domains. PLCγ1 also protects LAT from dephosphorylation by the phosphatase CD45 and promotes LAT-dependent ERK activation and SLP76 phosphorylation. Intriguingly, a nonmonotonic effect of PLCγ1 on LAT clustering was discovered. Computer simulations, based on patchy particles, revealed how the cluster size is regulated by protein compositions. Together, these results define a critical function of PLCγ1 in promoting phase separation of the LAT complex and TCR signal transduction.

A Role for Human Renal Tubular Epithelial Cells in Direct Allo-Recognition by CD4+ T-Cells and the Effect of Ischemia-Reperfusion.

Direct allorecognition is the earliest and most potent immune response against a kidney allograft. Currently, it is thought that passenger donor professional antigen-presenting cells (APCs) are responsible. Further, many studies support that graft ischemia-reperfusion injury increases the probability of acute rejection. We evaluated the possible role of primary human proximal renal tubular epithelial cells (RPTECs) in direct allorecognition by CD4+ T-cells and the effect of anoxia-reoxygenation. In cell culture, we detected that RPTECs express all the required molecules for CD4+ T-cell activation (HLA-DR, CD80, and ICAM-1). Anoxia-reoxygenation decreased HLA-DR and CD80 but increased ICAM-1. Following this, RPTECs were co-cultured with alloreactive CD4+ T-cells. In T-cells, zeta chain phosphorylation and c-Myc increased, indicating activation of T-cell receptor and co-stimulation signal transduction pathways, respectively. T-cell proliferation assessed with bromodeoxyuridine assay and with the marker Ki-67 increased. Previous culture of RPTECs under anoxia raised all the above parameters in T-cells. FOXP3 remained unaffected in all cases, signifying that proliferating T-cells were not differentiated towards a regulatory phenotype. Our results support that direct allorecognition may be mediated by RPTECs even in the absence of donor-derived professional APCs. Also, ischemia-reperfusion injury of the graft may enhance the above capacity of RPTECs, increasing the possibility of acute rejection.

T cell receptor–dependent S-acylation of ZAP-70 controls activation of T cells

ZAP-70 is a tyrosine kinase essential for T cell immune responses. Upon engagement of the T cell receptor (TCR), ZAP-70 is recruited to the specialized plasma membrane domains, becomes activated, and is released to phosphorylate its laterally segregated targets. A shift in ZAP-70 distribution at the plasma membrane is recognized as a critical step in TCR signal transduction and amplification. However, the molecular mechanism supporting stimulation-dependent plasma membrane compartmentalization of ZAP-70 remains poorly understood. In this study, we identified previously uncharacterized lipidation (S-acylation) of ZAP-70 using Acyl-Biotin Exchange assay, a technique that selectively captures S-acylated proteins. We found that this posttranslational modification of ZAP-70 is dispensable for its enzymatic activity. However, the lipidation-deficient mutant of ZAP-70 failed to propagate the TCR pathway suggesting that S-acylation is essential for ZAP-70 interaction with its protein substrates. The kinetics of ZAP-70 S-acylation were consistent with TCR signaling events indicating that agonist-induced S-acylation is a part of the signaling mechanism controlling T cell activation and function. Taken together, our results suggest that TCR-induced S-acylation of ZAP-70 can serve as a critical regulator of T cell-mediated immunity.

Packed red blood cells inhibit T-cell activation via ROS-dependent signaling pathways

Numerous observations indicate that red blood cells (RBCs) affect T-cell activation and proliferation. We have studied effects of packed RBCs (PRBCs) on T-cell receptor (TCR) signaling and the molecular mechanisms whereby (P)RBCs modulate T-cell activation. In line with previous reports, PRBCs attenuated the expression of T-cell activation markers CD25 and CD69 upon costimulation via CD3/CD28. In addition, T-cell proliferation and cytokine expression were markedly reduced when T-cells were stimulated in the presence of PRBCs. Inhibitory activity of PRBCs required direct cell–cell contact and intact PRBCs. The production of activation-induced cellular reactive oxygen species, which act as second messengers in T-cells, was completely abrogated to levels of unstimulated T-cells in the presence of PRBCs. Phosphorylation of the TCR-related zeta chain and thus proximal TCR signal transduction was unaffected by PRBCs, ruling out mechanisms based on secreted factors and steric interaction restrictions. In large part, downstream signaling events requiring reactive oxygen species for full functionality were affected, as confirmed by an untargeted MS-based phosphoproteomics approach. PRBCs inhibited T-cell activation more efficiently than treatment with 1 mM of the antioxidant N-acetyl cysteine. Taken together, our data imply that inflammation-related radical reactions are modulated by PRBCs. These immunomodulating effects may be responsible for clinical observations associated with transfusion of PRBCs.

Next Generation Genomic Analyses in T-ALL Patients Identify Recurrent and Novel Genomic Abnormalities

Background and Objective 15% of pediatric and 40% of adult T-cell Acute Lymphoblastic Leukemia (T-ALL) patients fail conventional therapy highlighting the need for novel therapeutic strategies based on genomic alterations of individuals. We interrogated genomic alterations in Australian T-ALL patients for patterns of mutation and druggable targets. A subset of samples were used to establish patient-derived xenograft (PDX) models to evaluate novel therapies. Methods T-ALL patients' samples underwent next generation genomic analyses (128 total samples including diagnosis, refractory and relapse timepoints from 118 patients of all age groups). mRNA sequencing (mRNAseq) identified gene fusions and structural variants and assessed gene expression (n=101 patients). Fusions were called when identified by 2/3 predictors (FusionCatcher, SOAPfuse, JAFFA). Variant calling utilized GATK HaplotypeCaller and underwent several filtering steps to eliminate possible germline alterations and common SNPs. DNA copy number variations (CNVs) were detected via Multiplex Ligation-dependent Probe Amplification (MLPA: P202, P335, P383; n=64 patients). Establishment of PDX models from patient material (bone marrow or peripheral blood) is ongoing. Recapitulation of human disease was confirmed by mRNAseq in a subset of xenografts. Results Genomic fusion genes were identified in 46/101 samples (46%) by mRNAseq; the most common fusion identified was STIL-TAL1 (n=6). Increased expression of LCK and/or LAT (encoded proteins are involved in T-cell receptor (TCR) signal transduction) was observed in 100% of patients with the STIL-TAL1 gene fusion indicating TCR signaling pathways may be perturbed in this sub-group. Other common gene fusions were MLLT10-DDX3X (n=5) and KMT2A-MLLT4/AFDN (n=4). We also observed the previously reported fusions SET-NUP214, KMT2A-MLLT1, PICALM-MLLT10, NUP214-ABL1, several fusions involving TCR subunits as well as novel fusions involving KMT2A, NOTCH1, LMO1, ZEB2. Numerous nonsynonymous mutations were identified in 81/101 patients (80%) with mRNAseq data available (Figure 1). Broadly, the mutated genes encode proteins in the following categories: oncoproteins (NRAS, KRAS); tumour suppressors (TP53, CHEK2, BRCA1, BRCA2, PTEN), epigenetic regulators (EZH2, SETD5, DNMT3A); regulators of NOTCH signalling (NOTCH1, NOTCH2, FBXW7); transcription factors and regulators (IKZF1, KMT2A, EP400, SMARCD1, RUNX1, AFF1, AFF3); kinase and cytokine signal regulators (ATM, JAK2, JAK3, TYK2, FLT3, PTPN11). INDELS in clinically relevant genes were identified in 37/101 patients (37%) including alterations to: NOTCH1, PHF6, PTEN, STAT1, IL7R, CDKN2A, LYL1, WT1, JAK3, LEF1. The most common copy number alterations identified in our patient cohort were CDKN2A/B deletions (30/64 patients, 47%), PHF6 duplication (20/64 patients, 31%) and MLLT3 deletion (13/64 patients, 20%). In patients with CDKN2A/B deletions and additional CNVs, PHF6 duplication (n=6) and MYB duplication (n=4) were mutually exclusive. However, one patient without CDKN2A/B deletions harbored both MYB and PHF6 duplications. MLLT3 deletion always co-occurred with CDKN2A/B deletions (13/13 patients with MLLT3 deletion), but was never observed with PTEN deletion (0/7 patients with PTEN deletion). Patients with either CDKN2A/B deletions or PHF6 duplications frequently harbored NOTCH1 abnormalities: 13/32 patients (41%) and 8/21 patients (38%), respectively. PDX primagrafts investigated the engraftment latency and peripheral organ infiltration. Primagrafts established from patients harboring NUP214-SET1 or NUP214-ABL1 fusions engrafted at a slower rate (82 d and 91 d, respectively) than primagrafts from patients harboring a STIL-TAL1 fusion (45 d) or CDKN2A/B deletions (31 d and 48 d). Conclusions In our T-ALL cohort we demonstrate that the majority of cases harbor rearrangements, structural variations and duplication/deletion of genes associated with malignant transformation. We identified several co-occurring lesions as well as mutually exclusive genomic abnormalities. The top 20 mutated genes in our patient cohort differ to those reported for a pediatric cohort (Roberts et al 2019 Blood 134:649), indicating an association between patient age and genomic alteration. Secondary PDX models investigating novel targeted treatment strategies are ongoing. Disclosures Hughes: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. White:Bristol-Myers Squibb: Honoraria, Research Funding; Amgen: Honoraria.

Characterizing Zap70 signaling mutants in primary T cells using CRISPR/Cas9

Abstract T cell activation is controlled by a complex series of signaling events that originate from the T cell receptor (TCR) at the plasma membrane. These signaling events are both spatially and temporally regulated to ensure that T cells activate only in a correct and specific context. A key aspect of T cell activation is the ability of the TCR to recognize peptides with specific affinities while simultaneously discounting a large proportion of MHC-TCR interactions with lower affinities. Our lab has identified Zap70 interaction dynamics with phosphorylated motifs in the TCR as a key mediator of T cell activation thresholds. However, studying these signaling events within primary T cells remains challenging because manipulating the signaling threshold through germline mutations will cause abnormal T cell development and selection in the thymus. To investigate the role of Zap70 in regulating the activation threshold in mature, peripheral T cells, we have developed a CRISPR/Cas9 expression system in primary murine T cells. Using a retroviral construct, we can simultaneously knock down endogenous Zap70 and replace its expression with ectopic Zap70 mutants with differing TCR interaction dynamics and signal transduction. Ectopic Zap70 is expressed up to 41 days after infection and 33 days after adoptive transfer to Rag1−/− mice. In addition, the primary cells are sensitive to antigenic stimulation comparable to uninfected primary T cells. Using this system, we can both dissect the mechanisms by which Zap70-TCR dynamics regulate signal transduction and determine whether manipulating Zap70-TCR dynamics can affect T cell activity under various pathological conditions.

Molecular Mechanisms for the Stochastic Condensation of LAT Assemblies in T Cells

T cells discriminate between self and agonist by requiring long dwelling binding events between peptide-MHC (pMHC) and the T Cell Receptor (TCR). A consequence of successful signal transduction is the formation of a multi-molecular assembly of downstream molecules that is crosslinked by the phosphorylated scaffold protein Linker for Activation of T cells (LAT). While it is known that after ligand binding Zap70 kinase recruits to the TCR and begins phosphorylating LAT, the early molecular events precipitating LAT condensation in relation to a single ligated TCR is unknown. Using supported lipid bilayer technology and TIRF microscopy, we directly measure the intracellular responses to single pMHC-TCR binding events in living primary T cells with high spatiotemporal, multi-color resolution. This provides the ability to accurately determine the order of events in TCR signal transduction in relation to a single TCR, as opposed to bulk stimulation methods. We observe that after a pMHC binds to a TCR that LAT condensation occurs after an extended delay. While this timing is likely set, in part, by a competition of kinase/phosphatase pressure and LAT diffusion, the phosphorylation of LAT has a similarly extended delay. This suggests that diffusive phospho-LAT requires an additional event to trigger aggregation. These data demonstrate novel roles for key signaling proteins and, in contrast to ensemble methods, are the first to visualize the decisive steps leading to LAT condensation and completion of kinetic proofreading.

Leukocyte-associated immunoglobulin-like receptor 1 inhibits T-cell signaling by decreasing protein phosphorylation in the T-cell signaling pathway

Multiple observations implicate T-cell dysregulation as a central event in the pathogenesis of rheumatoid arthritis. Here, we investigated mechanisms for suppressing T-cell activation via the inhibitory receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1). To determine how LAIR-1 affects T-cell receptor (TCR) signaling, we compared 1) T cells from LAIR-1-sufficient and -deficient mice, 2) Jurkat cells expressing either LAIR-1 mutants or C-terminal Src kinase (CSK) mutants, and 3) T cells from mice that contain a CSK transgene susceptible to chemical inhibition. Our results indicated that LAIR-1 engagement by collagen or by complement C1q (C1Q, which contains a collagen-like domain) inhibits TCR signaling by decreasing the phosphorylation of key components in the canonical T-cell signaling pathway, including LCK proto-oncogene SRC family tyrosine kinase (LCK), LYN proto-oncogene SRC family tyrosine kinase (LYN), ζ chain of T-cell receptor-associated protein kinase 70 (ZAP-70), and three mitogen-activated protein kinases (extracellular signal-regulated kinase, c-Jun N-terminal kinase 1/2, and p38). The intracellular region of LAIR-1 contains two immunoreceptor tyrosine-based inhibition motifs that are both phosphorylated by LAIR-1 activation, and immunoprecipitation experiments revealed that Tyr-251 in LAIR-1 binds CSK. Using CRISPR/Cas9-mediated genome editing, we demonstrate that CSK is essential for the LAIR-1-induced inhibition of the human TCR signal transduction. T cells from mice that expressed a PP1 analog-sensitive form of CSK (CskAS) corroborated these findings, and we also found that Tyr-251 is critical for LAIR-1's inhibitory function. We propose that LAIR-1 activation may be a strategy for controlling inflammation and may offer a potential therapeutic approach for managing autoimmune diseases.

Urate crystals trigger B-cell receptor signal transduction and induce B-cell proliferation.

Background Urate in its crystal form is a known danger-associated molecular pattern, which after its internalization activates cells of the innate immune system. However, by inducing lipid raft sequestration and clustering of membrane-bound proteins with immunoreceptor tyrosine-based activation motifs, urate crystals can also activate cells of the innate immune system without previous internalization. Also, urate crystals trigger T-cell receptor signal transduction and induce T-cell proliferation. In this study, we evaluated whether urate crystals can also initiate B-cell receptor (BCR) signal transduction and promote B-cell proliferation. Methods B cells were isolated from the blood of 10 individuals and cultured with or without urate at a concentration of 10 mg/dL, at which crystallization occurs. Phosphorylated Igα (CD79A) and c-Myc were assessed by Western blotting and B-cell proliferation with BrdU assay. Results Urate increased the level of phosphorylated Igα, a component of the BCR complex. Phosphorylation of Igα is the very proximal event in BCR signal transduction. Also, urate increased the expression of c-Myc, an essential transcription factor for BCR-induced B-cell proliferation. Finally, urate induces B-cell proliferation. Conclusions Urate crystals trigger BCR signal transduction and induce B-cell proliferation. The clinical significance of urate-induced B-cell activation remains to be elucidated.

Mg2+ regulation of kinase signaling and immune function.

Mg2+ is required at micromolar concentrations as a cofactor for ATP, enzymatic reactions, and other biological processes. We show that decreased extracellular Mg2+ reduced intracellular Mg2+ levels and impaired the Ca2+ flux, activation marker up-regulation, and proliferation after T cell receptor (TCR) stimulation. Reduced Mg2+ specifically impairs TCR signal transduction by IL-2-inducible T cell kinase (ITK) due to a requirement for a regulatory Mg2+ in the catalytic pocket of ITK. We also show that altered catalytic efficiency by millimolar changes in free basal Mg2+ is an unrecognized but conserved feature of other serine/threonine and tyrosine kinases, suggesting a Mg2+ regulatory paradigm of kinase function. Finally, a reduced serum Mg2+ concentration in mice causes an impaired CD8+ T cell response to influenza A virus infection, reduces T cell activation, and exacerbates morbidity. Thus, Mg2+ directly regulates the active site of specific kinases during T cell responses, and maintaining a high serum Mg2+ concentration is important for antiviral immunity in otherwise healthy animals.

Thy-1 (CD90) Signaling Preferentially Promotes RORγt Expression and a Th17 Response.

Thy-1 (CD90) is a glycosylphosphatidylinositol-anchored protein (GPI-AP) with signaling properties that is abundant on mouse T cells. Upon antibody-mediated crosslinking, Thy-1 provides a T cell receptor (TcR)-like signal that is sufficient to drive CD4+ T cell proliferation and differentiation into effector cells when costimulatory signals are provided by syngeneic lipopolysaccharide-matured bone marrow-derived dendritic cells. In this study, we investigated the impact of Thy-1 signaling on the production of the T helper (Th) cell subset-associated cytokines, interferon (IFN) γ, interleukin (IL)-4 and IL-17A, as well as the in vitro polarization of highly purified resting CD4+ T cells into Th1, Th2, and Th17 cells. Although CD8+ T cells expressed more Thy-1 than CD4+ T cells, both T cell populations were equally responsive to Thy-1 stimulation. In contrast to TcR stimulation of CD3+ T cells, which favored IFNγ and IL-4 production, Thy-1 signaling favored IL-17 synthesis, indicating a previously unidentified difference between the consequences of Thy-1 and TcR signal transduction. Moreover, Thy-1 signaling preferentially induced the Th17-associated transcription factor RORγt in CD4+ T cells. As with TcR signaling, Thy-1 stimulation of CD4+ T cells under the appropriate polarizing conditions resulted in Th1, Th2 or Th17 cell induction; however, Thy-1 stimulation induced nearly 7- and 2-fold more IL-4 and IL-17A, respectively, but only slightly more IFNγ. The ability to provide a TcR-like signal capable of promoting T helper cell differentiation and cytokine synthesis was not common to all GPI-APs since cross-linking of Ly6A/E with mitogenic mAb did not promote substantial production of IFNγ, IL-4 or IL-17, although there was a substantial proliferative response. The preferential induction of RORγt and Th17 cytokine synthesis as a consequence of Thy-1 signaling suggests a default T helper cell response that may enhance host defense against extracellular pathogens.

T time for ADAR: ADAR1 is required for T cell self-tolerance.

ADAR1, an RNA‐editing enzyme, plays a key role in preventing self‐RNAs from triggering autoinflammatory responses. In this issue of EMBO reports , Nakahama and colleagues uncover a novel role for ADAR1 in T cells [1]. The authors report that in T cells, ADAR1‐mediated suppression of type I interferon‐stimulated gene (ISG) expression is required for thymic T cell self‐tolerance and prevention of colitis. These findings establish a novel function of ADAR1 in T cells and suggest that autoreactive T cells may contribute to disease symptoms in autoinflammatory disorders.

Purification of LAT-Containing Membranes from Resting and Activated T Lymphocytes.

In T lymphocytes, the immune synapse is an active zone of vesicular traffic. Directional transport of vesicular receptors and signaling molecules from or to the immune synapse has been shown to play an important role in T-cell receptor (TCR) signal transduction. However, how vesicular trafficking is regulating the activation of T cells is still a burning question, and the characterization of these intracellular compartments remains the first step to understand this process. We describe herein a protocol, which combines a separation of membranes on flotation gradient with an affinity purification of Strep-tagged fusion transmembrane proteins with Strep-Tactin® resin, allowing the purification of membranes containing the Strep-tagged molecule of interest. By keeping the membranes intact, this protocol leads to the purification of molecules physically associated with the Strep-tagged protein as well as of molecules present in the same membrane compartment: transmembrane proteins, proteins strongly associated with the membranes, and luminal proteins. The example shown herein is the purification of membrane compartment prepared from T lymphocytes expressing LAT fused to a Strep-tag.

SHP1-ing thymic selection.

Thymocyte development and maintenance of peripheral T-cell numbers and functions are critically dependent on T-cell receptor (TCR) signal strength. SHP1 (Src homology region 2 domain-containing phosphatase-1), a tyrosine phosphatase, acts as a negative regulator of TCR signal strength. Moreover, germline SHP1 knockout mice have shown impaired thymic development. However, this has been recently questioned by an analysis of SHP1 conditional knockout mice, which reported normal thymic development of SHP1 deficient thymocytes. Using this SHP1 conditional knockout mice, in this issue of the European Journal of Immunology, Martinez etal. [Eur. J. Immunol. 2016. 46: 2103-2110] show that SHP1 indeed does have a role in the negative regulation of TCR signal strength in positively selected thymocytes, and in the final maturation of single positive thymocytes. They report that thymocyte development in such mice shows loss of mature, post-selection cells. This is due to increased TCR signal transduction in thymocytes immediately post positive-selection, and increased cell death in response to weak TCR ligands. Thus, SHP1-deficiency shows strong similarities to deficiency in the T-cell specific SHP1-associated protein Themis.

Effects of blood transportation on human peripheral mononuclear cell yield, phenotype and function: implications for immune cell biobanking.

Human biospecimen collection, processing and preservation are rapidly emerging subjects providing essential support to clinical as well as basic researchers. Unlike collection of other biospecimens (e.g. DNA and serum), biobanking of viable immune cells, such as peripheral blood mononuclear cells (PBMC) and/or isolated immune cell subsets is still in its infancy. While certain aspects of processing and freezing conditions have been studied in the past years, little is known about the effect of blood transportation on immune cell survival, phenotype and specific functions. However, especially for multicentric and cooperative projects it is vital to precisely know those effects. In this study we investigated the effect of blood shipping and pre-processing delay on immune cell phenotype and function both on cellular and subcellular levels. Peripheral blood was collected from healthy volunteers (n = 9): at a distal location (shipped overnight) and in the central laboratory (processed immediately). PBMC were processed in the central laboratory and analyzed post-cryopreservation. We analyzed yield, major immune subset distribution, proliferative capacity of T cells, cytokine pattern and T-cell receptor signal transduction. Results show that overnight transportation of blood samples does not globally compromise T- cell subsets as they largely retain their phenotype and proliferative capacity. However, NK and B cell frequencies, the production of certain PBMC-derived cytokines and IL-6 mediated cytokine signaling pathway are altered due to transportation. Various control experiments have been carried out to compare issues related to shipping versus pre-processing delay on site. Our results suggest the implementation of appropriate controls when using multicenter logistics for blood transportation aiming at subsequent isolation of viable immune cells, e.g. in multicenter clinical trials or studies analyzing immune cells/subsets. One important conclusion might be that despite changes due to overnight shipment, highly standardized central processing (and analysis) could be superior to multicentric de-central processing with more difficult standardization.

SPA0355 suppresses T-cell responses and reduces airway inflammation in mice

In recent studies, SPA0355, a thiourea analog, has been demonstrated to possess strong anti-inflammatory activity. However, the mechanisms underlying the effects of SPA0355 on immune-mediated diseases have not been fully defined. The present study was designed to investigate the immunological and molecular mechanisms by which SPA0355 modulates cluster of differentiation of (CD4)+ T-cell-mediated immune responses in allergic airway inflammation. In vitro studies have shown that SPA0355 suppresses CD4+ T-cell activation, proliferation, and differentiation via modulation of T-cell receptor (TCR) signal transduction and cytokine-induced Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling. Next, we investigated the efficacy of SPA0355 in ovalbumin (OVA)-induced allergic airway inflammation. Intraperitoneal administration of SPA0355 inhibited inflammatory cell recruitment into the airways as well as the production of Th2 cytokines in bronchoalveolar fluid and suppressed OVA-induced IgE production in serum. Additionally, SPA0355 suppressed mucin production and smooth muscle hypertrophy and prevented the development of airway hyperresponsiveness. Given that allergic airway inflammation is mainly driven by Th2 cell responses, it is highly possible that the defects in CD4+ T-cell activation and Th2 cell differentiation in the draining lymph nodes and suppressed NF-κB activation in the lungs of SPA0355-treated mice illustrate an immunological mechanism of the preventive effect of SPA0355 on the aforementioned asthmatic characteristics. Collectively, our results suggest that SPA0355 directly modulates Th1 and Th2 responses through the suppression of multiple signaling pathways triggered by TCR or cytokine receptor stimulation, and that SPA0355 has protective effects in a murine model of allergic airway inflammation.