Mechanism and function of Vav1 localisation in TCR signalling

The antigen-specific binding of T cells to antigen presenting cells results in recruitment of signalling proteins to microclusters at the cell-cell interface known as the immunological synapse (IS). The Vav1 guanine nucleotide exchange factor plays a critical role in T cell antigen receptor (TCR) signalling, leading to the activation of multiple pathways. We now show that it is recruited to microclusters and to the IS in primary CD4+ and CD8+ T cells. Furthermore, we show that this recruitment depends on the SH2 and C-terminal SH3 (SH3B) domains of Vav1, and on phosphotyrosines 112 and 128 of the SLP76 adaptor protein. Biophysical measurements show that Vav1 binds directly to these residues on SLP76 and that efficient binding depends on the SH2 and SH3B domains of Vav1. Finally, we show that the same two domains are critical for the phosphorylation of Vav1 and its signalling function in TCR-induced calcium flux. We propose that Vav1 is recruited to the IS by binding to SLP76 and that this interaction is critical for the transduction of signals leading to calcium flux

Acute Inhibition of Selected Membrane-Proximal Mouse T Cell Receptor Signaling by Mitochondrial Antagonists

T cells absorb nanometric membrane vesicles, prepared from plasma membrane of antigen presenting cells, via dual receptor/ligand interactions of T cell receptor (TCR) with cognate peptide/major histocompatibility complex (MHC) plus lymphocyte function-associated antigen 1 (LFA-1) with intercellular adhesion molecule 1. TCR-mediated signaling for LFA-1 activation is also required for the vesicle absorption. Exploiting those findings, we had established a high throughput screening (HTS) platform and screened a library for isolation of small molecules inhibiting the vesicle absorption. Follow-up studies confirmed that treatments (1 hour) with various mitochondrial antagonists, including a class of anti-diabetic drugs (i.e., Metformin and Phenformin), resulted in ubiquitous inhibition of the vesicle absorption without compromising viability of T cells. Further studies revealed that the mitochondrial drug treatments caused impairment of specific membrane-proximal TCR signaling event(s). Thus, activation of Akt and PLC-γ1 and entry of extracellular Ca2+ following TCR stimulation were attenuated while polymerization of monomeric actins upon TCR triggering progressed normally after the treatments. Dynamic F-actin rearrangement concurring with the vesicle absorption was also found to be impaired by the drug treatments, implying that the inhibition by the drug treatments of downstream signaling events (and the vesicle absorption) could result from lack of directional relocation of signaling and cell surface molecules. We also assessed the potential application of mitochondrial antagonists as immune modulators by probing effects of the long-term drug treatments (24 hours) on viability of resting primary T cells and cell cycle progression of antigen-stimulated T cells. This study unveils a novel regulatory mechanism for T cell immunity in response to environmental factors having effects on mitochondrial function

The Cell Cycle Time of CD8+ T Cells Responding In Vivo Is Controlled by the Type of Antigenic Stimulus

A hallmark of cells comprising the mammalian adaptive immune system is the requirement for these rare naïve T (and B) lymphocytes directed to a specific microorganism to undergo proliferative expansion upon first encounter with this antigen. In the case of naïve CD8+ T cells the ability of these rare quiescent lymphocytes to rapidly activate and expand into effector T cells in numbers sufficient to control viral and certain bacterial infections can be essential for survival. In this report we examined the activation, cell cycle time and initial proliferative response of naïve murine CD8+ T cells responding in vivo to Influenza and Vaccinia virus infection or vaccination with viral antigens. Remarkably, we observed that CD8+ T cells could divide and proliferate with an initial cell division time of as short as 2 hours. The initial cell cycle time of responding CD8+ T cells is not fixed but is controlled by the antigenic stimulus provided by the APC in vivo. Initial cell cycle time influences the rate of T cell expansion and the numbers of effector T cells subsequently accumulating at the site of infection. The T cell cycle time varies with duration of the G1 phase of the cell cycle. The duration of G1 is inversely correlated with the phosphorylation state of the retinoblastoma (Rb) protein in the responding T cells. The implication of these findings for the development of adaptive immune responses and the regulation of cell cycle in higher eukaryotic cells is discussed

Systems model of T cell receptor proximal signaling reveals emergent ultrasensitivity

Receptor phosphorylation is thought to be tightly regulated because phosphorylated receptors initiate signaling cascades leading to cellular activation. The T cell antigen receptor (TCR) on the surface of T cells is phosphorylated by the kinase Lck and dephosphorylated by the phosphatase CD45 on multiple immunoreceptor tyrosine-based activation motifs (ITAMs). Intriguingly, Lck sequentially phosphorylates ITAMs and ZAP-70, a cytosolic kinase, binds to phosphorylated ITAMs with differential affinities. The purpose of multiple ITAMs, their sequential phosphorylation, and the differential ZAP-70 affinities are unknown. Here, we use a systems model to show that this signaling architecture produces emergent ultrasensitivity resulting in switch-like responses at the scale of individual TCRs. Importantly, this switch-like response is an emergent property, so that removal of multiple ITAMs, sequential phosphorylation, or differential affinities abolishes the switch. We propose that highly regulated TCR phosphorylation is achieved by an emergent switch-like response and use the systems model to design novel chimeric antigen receptors for therapy

Mitochondrial and Plasma Membrane Pools of Stomatin-Like Protein 2 Coalesce at the Immunological Synapse during T Cell Activation

Stomatin-like protein 2 (SLP-2) is a member of the stomatin – prohibitin – flotillin – HflC/K (SPFH) superfamily. Recent evidence indicates that SLP-2 is involved in the organization of cardiolipin-enriched microdomains in mitochondrial membranes and the regulation of mitochondrial biogenesis and function. In T cells, this role translates into enhanced T cell activation. Although the major pool of SLP-2 is associated with mitochondria, we show here that there is an additional pool of SLP-2 associated with the plasma membrane of T cells. Both plasma membrane-associated and mitochondria-associated pools of SLP-2 coalesce at the immunological synapse (IS) upon T cell activation. SLP-2 is not required for formation of IS nor for the re-localization of mitochondria to the IS because SLP-2-deficient T cells showed normal re-localization of these organelles in response to T cell activation. Interestingly, upon T cell activation, we found the surface pool of SLP-2 mostly excluded from the central supramolecular activation complex, and enriched in the peripheral area of the IS where signalling TCR microclusters are located. Based on these results, we propose that SLP-2 facilitates the compartmentalization not only of mitochondrial membranes but also of the plasma membrane into functional microdomains. In this latter location, SLP-2 may facilitate the optimal assembly of TCR signalosome components. Our data also suggest that there may be a net exchange of membrane material between mitochondria and plasma membrane, explaining the presence of some mitochondrial proteins in the plasma membrane

Equine penile squamous cell carcinoma: expression of biomarker proteins and EcPV2

Equine penile squamous cell carcinoma (EpSCC) is a relatively common cutaneous neoplasm with a poor prognosis. In this study, we aimed to determine the protein expression and colocalisation of FRA1, c-Myc, Cyclin D1, and MMP7 in normal (NT), tumour (T), hyperplastic epidermis and/or squamous papilloma (Hyp/Pap), poorly-differentiated (PDSCC), or well-differentiated (WDSCC) EpSCC using a tissue array approach. Further objectives were to correlate protein expression to (i) levels of inflammation, using a convolutional neural network (ii) equine papillomavirus 2 (EcPV2) infection, detected using PCR amplification. We found an increase in expression of FRA1 in EpSCC compared to NT samples. c-Myc expression was higher in Hyp/Pap and WDSCC but not PDSCC whereas MMP7 was reduced in WDSCC compared with NT. There was a significant increase in the global intersection coefficient (GIC) of FRA1 with MMP7, c-Myc, and Cyclin D1 in EpSCC. Conversely, GIC for MMP7 with c-Myc was reduced in EpSCC tissue. Inflammation was positively associated with EcPV2 infection in both NT and EpSCC but not Hyp/Pap. Changes in protein expression could be correlated with EcPV2 for Cyclin D1 and c-Myc. Our results evaluate novel biomarkers of EpSCC and a putative correlation between the expression of biomarkers, EcPV2 infection and inflammation

Force Generation upon T Cell Receptor Engagement

T cells are major players of adaptive immune response in mammals. Recognition of an antigenic peptide in association with the major histocompatibility complex at the surface of an antigen presenting cell (APC) is a specific and sensitive process whose mechanism is not fully understood. The potential contribution of mechanical forces in the T cell activation process is increasingly debated, although these forces are scarcely defined and hold only limited experimental evidence. In this work, we have implemented a biomembrane force probe (BFP) setup and a model APC to explore the nature and the characteristics of the mechanical forces potentially generated upon engagement of the T cell receptor (TCR) and/or lymphocyte function-associated antigen-1 (LFA-1). We show that upon contact with a model APC coated with antibodies towards TCR-CD3, after a short latency, the T cell developed a timed sequence of pushing and pulling forces against its target. These processes were defined by their initial constant growth velocity and loading rate (force increase per unit of time). LFA-1 engagement together with TCR-CD3 reduced the growing speed during the pushing phase without triggering the same mechanical behavior when engaged alone. Intracellular Ca2+ concentration ([Ca2+]i) was monitored simultaneously to verify the cell commitment in the activation process. [Ca2+]i increased a few tens of seconds after the beginning of the pushing phase although no strong correlation appeared between the two events. The pushing phase was driven by actin polymerization. Tuning the BFP mechanical properties, we could show that the loading rate during the pulling phase increased with the target stiffness. This indicated that a mechanosensing mechanism is implemented in the early steps of the activation process. We provide here the first quantified description of force generation sequence upon local bidimensional engagement of TCR-CD3 and discuss its potential role in a T cell mechanically-regulated activation process

Encoding optical control in LCK kinase to quantitatively investigate its activity in live cells.

LCK is a tyrosine kinase that is essential for initiating T-cell antigen receptor (TCR) signaling. A complete understanding of LCK function is constrained by a paucity of methods to quantitatively study its function within live cells. To address this limitation, we generated LCK*, in which a key active-site lysine is replaced by a photocaged equivalent, using genetic code expansion. This strategy enabled fine temporal and spatial control over kinase activity, thus allowing us to quantify phosphorylation kinetics in situ using biochemical and imaging approaches. We find that autophosphorylation of the LCK active-site loop is indispensable for its catalytic activity and that LCK can stimulate its own activation by adopting a more open conformation, which can be modulated by point mutations. We then show that CD4 and CD8, T-cell coreceptors, can enhance LCK activity, thereby helping to explain their effect in physiological TCR signaling. Our approach also provides general insights into SRC-family kinase dynamics

HIV Envelope gp120 Activates LFA-1 on CD4 T-Lymphocytes and Increases Cell Susceptibility to LFA-1-Targeting Leukotoxin (LtxA)

The cellular adhesion molecule LFA-1 and its ICAM-1 ligand play an important role in promoting HIV-1 infectivity and transmission. These molecules are present on the envelope of HIV-1 virions and are integral components of the HIV virological synapse. However, cellular activation is required to convert LFA-1 to the active conformation that has high affinity binding for ICAM-1. This study evaluates whether such activation can be induced by HIV itself. The data show that HIV-1 gp120 was sufficient to trigger LFA-1 activation in fully quiescent naïve CD4 T cells in a CD4-dependent manner, and these CD4 T cells became more susceptible to killing by LtxA, a bacterial leukotoxin that preferentially targets leukocytes expressing high levels of the active LFA-1. Moreover, virus p24-expressing CD4 T cells in the peripheral blood of HIV-infected subjects were found to have higher levels of surface LFA-1, and LtxA treatment led to significant reduction of the viral DNA burden. These results demonstrate for the first time the ability of HIV to directly induce LFA-1 activation on CD4 T cells. Although LFA-1 activation may enhance HIV infectivity and transmission, it also renders the cells more susceptible to an LFA-1-targeting bacterial toxin, which may be harnessed as a novel therapeutic strategy to deplete virus reservoir in HIV-infected individuals

Force Measurements of TCR/pMHC Recognition at T Cell Surface

The rupture forces and adhesion frequencies of single recognition complexes between an affinity selected peptide/MHC complex and a TCR at a murine hybridoma surface were measured using Atomic Force Microscopy. When the CD8 coreceptor is absent, the adhesion frequency depends on the nature of the peptide but the rupture force does not. When CD8 is present, no effect of the nature of the peptide is observed. CD8 is proposed to act as a time and distance lock, enabling the shorter TCR molecule to bridge the pMHC and have time to finely read the peptide. Ultimately, such experiments could help the dissection of the sequential steps by which the TCR reads the peptide/MHC complex in order to control T cell activation