Georgina H. Cornish

Phosphatidylinositol-3-OH kinase and nutrient-sensing mTOR pathways control T lymphocyte trafficking

Phosphatidylinositol-3-OH kinase (PI(3)K) and the nutrient sensor mTOR are evolutionarily conserved regulators of cell metabolism. Here we show that PI(3)K and mTOR determined the repertoire of adhesion and chemokine receptors expressed by T lymphocytes. The key lymph node–homing receptors CD62L (L-selectin) and CCR7 were highly expressed on naive T lymphocytes but were downregulated after immune activation. CD62L downregulation occurred through ectodomain proteolysis and suppression of gene transcription. The p110δ subunit of PI(3)K controlled CD62L proteolysis through mitogen-activated protein kinases, whereas control of CD62L transcription by p110δ was mediated by mTOR through regulation of the transcription factor KLF2. PI(3)K-mTOR nutrient-sensing pathways also determined expression of the chemokine receptor CCR7 and regulated lymphocyte trafficking in vivo. Hence, lymphocytes use PI(3)K and mTOR to match metabolism and trafficking.

Ig gene-like molecule CD31 plays a nonredundant role in the regulation of T-cell immunity and tolerance

CD31 is an Ig-like molecule expressed by leukocytes and endothelial cells with an established role in the regulation of leukocyte trafficking. Despite genetic deletion of CD31 being associated with exacerbation of T cell-mediated autoimmunity, the contribution of this molecule to T-cell responses is largely unknown. Here we report that tumor and allograft rejection are significantly enhanced in CD31-deficient mice, which are also resistant to tolerance induction. We propose that these effects are dependent on an as yet unrecognized role for CD31-mediated homophilic interactions between T cells and antigen-presenting cells (APCs) during priming. We show that loss of CD31 interactions leads to enhanced primary clonal expansion, increased killing capacity, and diminished regulatory functions by T cells. Immunomodulation by CD31 signals correlates with a partial inhibition of proximal T-cell receptor (TCR) signaling, specifically Zap-70 phosphorylation. However, CD31-deficient mice do not develop autoimmunity due to increased T-cell death following activation, and we show that CD31 triggering induces Erk-mediated prosurvival activity in T cells either in conjunction with TCR signaling or autonomously. We conclude that CD31 functions as a nonredundant comodulator of T-cell responses, which specializes in sizing the ensuing immune response by setting the threshold for T-cell activation and tolerance, while preventing memory T-cell death.

NF-κB signaling mediates homeostatic maturation of new T cells

Significance Interleukin (IL)-7 is critical for the maintenance of the peripheral T-cell compartment of the adaptive immune system. Our study identifies a role for the Nuclear Factor κ-B (NF-κB) signalling pathway in the control of IL-7 receptor expression by T cells. Following thymic selection, new T cells specifically up-regulate IL-7R even as they leave the thymus, and we reveal that this expression is strictly NF-κB dependent. NF-κB signaling was only required transiently, however, and once fully mature, naive T cells did not require NF-κB signaling to maintain IL-7R expression. Therefore, we reveal a developmental role for NF-κB signaling for the normal maturation and function of new T cells. Interleukin (IL)-7 is critical for the maintenance of the peripheral T-cell compartment of the adaptive immune system. IL-7 receptor α ( IL-7Rα) expression is subject to developmental regulation and new T cells induce expression as they leave the thymus, which is essential for their long-term survival. It is not understood how this expression is regulated. Here, we identify a role for the Nuclear Factor κ-B (NF-κB) signaling pathway in controlling expression of IL-7Rα in new T cells. Perturbations to NF-κB signaling, either by deletion of Inhibitor of Kappa-B Kinase-2 (IKK2) or by inhibiting Rel dimer activity, prevented normal IL-7Rα expression in new T cells. Defective IL-7Rα expression resulted in impaired survival and homeostatic cell division responses by T cells that could be attributed to their failure to express IL-7Rα normally. Surprisingly, NF-κB signaling was only required transiently in new T cells to allow their normal expression of IL-7Rα, because IKK2 deletion in mature T cells had no effect on IL-7Rα expression or their normal homeostatic responsiveness. Therefore, we identify a developmental function for NF-κB signaling in the homeostatic maturation of new T cells, by regulating IL-7Rα expression.

Superresolution imaging of the cytoplasmic phosphatase PTPN22 links integrin-mediated T cell adhesion with autoimmunity

The tyrosine phosphatase PTPN22 redistributes from clusters to the leading edge in migrating T cells to inhibit integrin-mediated adhesion. Release the phosphatase! T cells need to move through the circulation, attach to endothelial cells, transmigrate into tissues, and stably interact with target cells. The phosphatase PTPN22 targets phosphorylated tyrosines in Src and Syk family kinases, many of which are phosphorylated and activated in migrating T cells in response to the binding of the integrin LFA-1 to its ligand ICAM-1. Burn et al. used superresolution microscopy to show that PTPN22 formed clusters in nonmigrating T cells, which were dispersed in T cells that migrated on surfaces coated with ICAM-1. Freed from these complexes, PTPN22 interacted with its targets near the front of the migrating T cell, which inhibited LFA-1 signaling. In contrast, clusters containing the PTPN22 R620W mutant, a variant that is associated with autoimmune diseases, failed to disaggregate in migrating T cells, and thus, LFA-1 clustering and signaling were not inhibited. Together, these data suggest how a mutation associated with autoimmunity dysregulates T cell adhesion and migration. Integrins are heterodimeric transmembrane proteins that play a fundamental role in the migration of leukocytes to sites of infection or injury. We found that protein tyrosine phosphatase nonreceptor type 22 (PTPN22) inhibits signaling by the integrin lymphocyte function-associated antigen–1 (LFA-1) in effector T cells. PTPN22 colocalized with its substrates at the leading edge of cells migrating on surfaces coated with the LFA-1 ligand intercellular adhesion molecule–1 (ICAM-1). Knockout or knockdown of PTPN22 or expression of the autoimmune disease–associated PTPN22-R620W variant resulted in the enhanced phosphorylation of signaling molecules downstream of integrins. Superresolution imaging revealed that PTPN22-R620 (wild-type PTPN22) was present as large clusters in unstimulated T cells and that these disaggregated upon stimulation of LFA-1, enabling increased association of PTPN22 with its binding partners at the leading edge. The failure of PTPN22-R620W molecules to be retained at the leading edge led to increased LFA-1 clustering and integrin-mediated cell adhesion. Our data define a previously uncharacterized mechanism for fine-tuning integrin signaling in T cells, as well as a paradigm of autoimmunity in humans in which disease susceptibility is underpinned by inherited phosphatase mutations that perturb integrin function.

Primed T cell responses to chemokines are regulated by the immunoglobulin-like molecule CD31.

CD31, an immunoglobulin-like molecule expressed by leukocytes and endothelial cells, is thought to contribute to the physiological regulation T cell homeostasis due to the presence of two immunotyrosine-based inhibitory motifs in its cytoplasmic tail. Indeed, loss of CD31 expression leads to uncontrolled T cell-mediated inflammation in a variety of experimental models of disease and certain CD31 polymorphisms correlate with increased disease severity in human graft-versus-host disease and atherosclerosis. The molecular mechanisms underlying CD31-mediated regulation of T cell responses have not yet been clarified. We here show that CD31-mediated signals attenuate T cell chemokinesis both in vitro and in vivo. This effect selectively affects activated/memory T lymphocytes, in which CD31 is clustered on the cell membrane where it segregates to the leading edge. We provide evidence that this molecular segregation, which does not occur in naïve T lymphocytes, might lead to cis-CD31 engagement on the same membrane and subsequent interference with the chemokine-induced PI3K/Akt signalling pathway. We propose that CD31-mediated modulation of memory T cell chemokinesis is a key mechanism by which this molecule contributes to the homeostatic regulation of effector T cell immunity.

Evidence of STAT5-dependent and -independent routes to CD8 memory formation and a preferential role for IL-7 over IL-15 in STAT5 activation

Interleukin (IL)‐7 and IL‐15 have non‐redundant roles in promoting development of memory CD8+ T cells. STAT5 is activated by receptors of both cytokines and has also been implicated as a requirement for generation of memory. To determine whether STAT5 activity was required for IL‐7 and IL‐15‐mediated generation of memory, we expressed either wild type (WT) or constitutively active (CA) forms of STAT5a in normal effector cells and then observed their ability to form memory in cytokine replete or deficient hosts. Receptor‐independent CA‐STAT5a significantly enhanced memory formation in the absence of either cytokine but did not mediate complete rescue. Interestingly, WT‐STAT5a expression enhanced memory formation in a strictly IL‐7‐dependent manner, suggesting that IL‐7 is a more potent activator of STAT5 than IL‐15 in vivo. These data suggest that the non‐redundant requirement for IL‐7 and IL‐15 is mediated through differential activation of both STAT5‐dependent and STAT5‐independent pathways.

Enhancing PET Signal at Target Tissue in Vivo: Dendritic and Multimeric Tris(hydroxypyridinone) Conjugates for Molecular Imaging of αvβ3 Integrin Expression with Gallium-68

Tris(hydroxypyridinone) chelators conjugated to peptides can rapidly complex the positron-emitting isotope gallium-68 (68Ga) under mild conditions, and the resulting radiotracers can delineate peptide receptor expression at sites of diseased tissue in vivo. We have synthesized a dendritic bifunctional chelator containing nine 1,6-dimethyl-3-hydroxypyridin-4-one groups (SCN-HP9) that can coordinate up to three Ga3+ ions. This derivative has been conjugated to a trimeric peptide (RGD3) containing three peptide groups that target the αvβ3 integrin receptor. The resulting dendritic compound, HP9-RGD3, can be radiolabeled in 97% radiochemical yield at a 3-fold higher specific activity than its homologues HP3-RGD and HP3-RGD3 that contain only a single metal binding site. PET scanning and biodistribution studies show that [68Ga(HP9-RGD3)] demonstrates higher receptor-mediated tumor uptake in animals bearing U87MG tumors that overexpress αvβ3 integrin than [68Ga(HP3-RGD)] and [68Ga(HP3-RGD3)]. However, concomitant nontarget organ retention of [68Ga(HP9-RGD3)] results in low tumor to nontarget organ contrast in PET images. On the other hand, the trimeric peptide homologue containing a single tris(hydroxypyridinone) chelator, [68Ga(HP3-RGD3)], clears nontarget organs and exhibits receptor-mediated uptake in mice bearing tumors and in mice with induced rheumatoid arthritis. PET imaging with [68Ga(HP3-RGD3)] enables clear delineation of αvβ3 integrin receptor expression in vivo.

Protein Tyrosine Phosphatase PTPN22 regulates IL‐1β dependent Th17 responses by modulating dectin‐1 signaling in mice

A single nucleotide polymorphism within the PTPN22 gene is a strong genetic risk factor predisposing to the development of multiple autoimmune diseases. PTPN22 regulates Syk and Src family kinases downstream of immuno‐receptors. Fungal β‐glucan receptor dectin‐1 signals via Syk, and dectin‐1 stimulation induces arthritis in mouse models. We investigated whether PTPN22 regulates dectin‐1 dependent immune responses. Bone marrow derived dendritic cells (BMDCs) generated from C57BL/6 wild type (WT) and Ptpn22−/− mutant mice, were pulsed with OVA323‐339 and the dectin‐1 agonist curdlan and co‐cultured in vitro with OT‐II T‐cells or adoptively transferred into OT‐II mice, and T‐cell responses were determined by immunoassay. Dectin‐1 activated Ptpn22−/− BMDCs enhanced T‐cell secretion of IL‐17 in vitro and in vivo in an IL‐1β dependent manner. Immunoblotting revealed that compared to WT, dectin‐1 activated Ptpn22−/− BMDCs displayed enhanced Syk and Erk phosphorylation. Dectin‐1 activation of BMDCs expressing Ptpn22R619W (the mouse orthologue of human PTPN22R620W) also resulted in increased IL‐1β secretion and T‐cell dependent IL‐17 responses, indicating that in the context of dectin‐1 Ptpn22R619W operates as a loss‐of‐function variant. These findings highlight PTPN22 as a novel regulator of dectin‐1 signals, providing a link between genetically conferred perturbations of innate receptor signaling and the risk of autoimmune disease.

Protein clustering and spatial organization in T-cells

T-cell protein microclusters have until recently been investigable only as microscale entities with their composition and structure being discerned by biochemistry or diffraction-limited light microscopy. With the advent of super resolution microscopy comes the ability to interrogate the structure and function of these clusters at the single molecule level by producing highly accurate pointillist maps of single molecule locations at ~20nm resolution. Analysis tools have also been developed to provide rich descriptors of the pointillist data, allowing us to pose questions about the nanoscale organization which governs the local and cell wide responses required of a migratory T-cell.

Protein tyrosine phosphatase PTPN22 is dispensable for dendritic cell antigen processing and promotion of T-cell activation by dendritic cells

The PTPN22R620W single nucleotide polymorphism increases the risk of developing multiple autoimmune diseases including type 1 diabetes, rheumatoid arthritis and lupus. PTPN22 is highly expressed in antigen presenting cells (APCs) where the expression of the murine disease associated variant orthologue (Ptpn22R619W) is reported to dysregulate pattern recognition receptor signalling in dendritic cells (DCs) and promote T-cell proliferation. Because T-cell activation is dependent on DC antigen uptake, degradation and presentation, we analysed the efficiency of these functions in splenic and GM-CSF bone marrow derived DC from wild type (WT), Ptpn22-/- or Ptpn22R619W mutant mice. Results indicated no differential ability of DCs to uptake antigen via macropinocytosis or receptor-mediated endocytosis. Antigen degradation and presentation was also equal as was WT T-cell conjugate formation and subsequent T-cell proliferation. Despite the likely presence of multiple phosphatase-regulated pathways in the antigen uptake, processing and presentation pathways that we investigated, we observed that Ptpn22 and the R619W autoimmune associated variant were dispensable. These important findings indicate that under non-inflammatory conditions there is no requirement for Ptpn22 in DC dependent antigen uptake and T-cell activation. Our findings reveal that perturbations in antigen uptake and processing, a fundamental pathway determining adaptive immune responses, are unlikely to provide a mechanism for the risk associated with the Ptpn22 autoimmune associated polymorphism.