Ulrike Lorenz

SHP‐1 and SHP‐2 in T cells: two phosphatases functioning at many levels

Summary:  Tyrosine phosphorylation and dephosphorylation of proteins play a critical role for many T‐cell functions. The opposing actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) determine the level of tyrosine phosphorylation at any time. It is well accepted that PTKs are essential during T‐cell signaling; however, the role and importance of PTPs are much less known and appreciated. Both transmembrane and cytoplasmic tyrosine phosphatases have been identified in T cells and shown to regulate T‐cell responses. This review focuses on the roles of the two cytoplasmic PTPs, the Src‐homology 2 domain (SH2)‐containing SHP‐1 and SHP‐2, in T‐cell signaling, development, differentiation, and function.

Deficiency of the Src Homology Region 2 Domain-Containing Phosphatase 1 (SHP-1) Causes Enrichment of CD4+CD25+ Regulatory T Cells

A subpopulation of T cells, named regulatory T cells (Treg cells), has been shown to play a key role in tolerance and the prevention of autoimmunity. It is not known how changes in TCR signal strength during thymic T cell development affect the generation of a Treg population. In this study, we took two different strategies to modulate the TCR signal strength: an intrinsic approach, where signaling was enhanced by the loss of a negative regulator, and an extrinsic approach, where signaling strength was altered through variations in the concentrations of the selecting peptide. The tyrosine phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP-1) is a known negative regulator of TCR-mediated signaling. motheaten mice, lacking expression of SHP-1, showed a 2- to 3-fold increase in the percentage of CD4+CD25+ Treg cells within the CD4+ T cells. Similarly, the percentage of Treg cells was heightened in fetal thymic organ cultures (FTOCs) derived from motheaten mice compared with wild-type FTOCs, thus establishing the thymic origin of these Treg cells. Using FTOCs derived from DO11.10 TCR transgenic mice, we demonstrated that exposure to increasing concentrations of the cognate OVA peptide favored the appearance of Treg cells. Our data suggest that the development of CD4+CD25+ Treg cells is intrinsically different from non-Treg cells and that Treg cells are selectively enriched under conditions of enhanced negative selection. Our data also reveal a key role for the SHP-1-mediated regulation of TCR signal strength in influencing the ratio of Treg vs non-Treg cells.

Regulatory T Cells in Central Nervous System Injury: A Double-Edged Sword

Previous research investigating the roles of T effector (Teff) and T regulatory (Treg) cells after injury to the CNS has yielded contradictory conclusions, with both protective and destructive functions being ascribed to each of these T cell subpopulations. In this work, we study this dichotomy by examining how regulation of the immune system affects the response to CNS trauma. We show that, in response to CNS injury, Teff and Treg subsets in the CNS-draining deep cervical lymph nodes are activated, and surgical resection of these lymph nodes results in impaired neuronal survival. Depletion of Treg, not surprisingly, induces a robust Teff response in the draining lymph nodes and is associated with impaired neuronal survival. Interestingly, however, injection of exogenous Treg cells, which limits the spontaneous beneficial immune response after CNS injury, also impairs neuronal survival. We found that no Treg accumulate at the site of CNS injury, and that changes in Treg numbers do not alter the amount of infiltration by other immune cells into the site of injury. The phenotype of macrophages at the site, however, is affected: both addition and removal of Treg negatively impact the numbers of macrophages with alternatively activated (tissue-building) phenotype. Our data demonstrate that neuronal survival after CNS injury is impaired when Treg cells are either removed or added. With this exacerbation of neurodegeneration seen with both addition and depletion of Treg, we recommend exercising extreme caution when considering the therapeutic targeting of Treg cells after CNS injury, and possibly in chronic neurodegenerative conditions.

Cutting Edge: Dependence of TCR Antagonism on Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase Activity

The mechanism by which antagonist peptides inhibit T cell responses is unknown. Mice deficient in Src homology 2 domain-containing protein tyrosine phosphatase (SHP-1) have revealed its importance in the negative regulation of lymphocyte signaling. We investigated a possible role for SHP-1 in T cell antagonism and demonstrate, for the first time, a substantial increase in SHP-1 activity during antagonism of CD4+ T cells. Furthermore, the removal of functional SHP-1 prevents antagonism in these cells. Our data demonstrate that T cell antagonism occurs via a negative intracellular signal that is mediated by SHP-1.

Localization of Src Homology 2 Domain-Containing Phosphatase 1 (SHP-1) to Lipid Rafts in T Lymphocytes: Functional Implications and a Role for the SHP-1 Carboxyl Terminus

The protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1) has previously been shown to be a negative regulator of signaling mediated via the TCR. A growing body of evidence indicates that the regulated localization of proteins within certain membrane subdomains, referred to as lipid rafts, is important for the successful transduction of signaling events downstream of the TCR. However, considerably less is known about the localization of negative regulators during these lipid raft-dependent signaling events. In this study we have investigated the subcellular localization of SHP-1 and its role in regulation of TCR-mediated signaling. Our studies demonstrate that in a murine T cell hybridoma as well as in primary murine thymocytes, a fraction of SHP-1 localizes to the lipid rafts, both basally and after TCR stimulation. Interestingly, although SHP-1 localized in the nonraft fractions is tyrosine phosphorylated, the SHP-1 isolated from the lipid rafts lacks the TCR-induced tyrosine phosphorylation, suggesting physical and/or functional differences between these two subpopulations. We identify a requirement for the C-terminal residues of SHP-1 in optimal localization to the lipid rafts. Although expression of SHP-1 that localizes to lipid rafts potently inhibits TCR-mediated early signaling events and IL-2 production, the expression of lipid raft-excluded SHP-1 mutants fails to elicit any of the inhibitory effects. Taken together these studies reveal a key role for lipid raft localization of SHP-1 in mediating the inhibitory effects on T cell signaling events.

Identification of a Novel Lipid Raft-Targeting Motif in Src Homology 2-Containing Phosphatase 1

The tyrosine phosphatase Src homology 2-containing phosphatase 1 (SHP-1) is a key negative regulator of TCR-mediated signaling. Previous studies have shown that in T cells a fraction of SHP-1 constitutively localizes to membrane microdomains, commonly referred to as lipid rafts. Although this localization of SHP-1 is required for its functional regulation of T cell activation events, how SHP-1 is targeted to the lipid rafts was unclear. In this study, we identify a novel, six-amino acid, lipid raft-targeting motif within the C terminus of SHP-1 based on several biochemical and functional observations. First, mutations of this motif in the context of full-length SHP-1 result in the loss of lipid raft localization of SHP-1. Second, this motif alone restores raft localization when fused to a mutant of SHP-1 (SHP-1 ΔC) that fails to localize to rafts. Third, a peptide encompassing the 6-mer motif directly binds to phospholipids whereas a mutation of this motif abolishes lipid binding. Fourth, whereas full-length SHP-1 potently inhibits TCR-induced tyrosine phosphorylation of specific proteins, expression of a SHP-1-carrying mutation within the 6-mer motif does not. Additionally, although SHP-1 ΔC was functionally inactive, the addition of the 6-mer motif restored its functionality in inhibiting TCR-induced tyrosine phosphorylation. Finally, this 6-mer mediated targeting of SHP-1 lipid rafts was essential for the function of this phosphatase in regulating IL-2 production downstream of TCR. Taken together, these data define a novel 6-mer motif within SHP-1 that is necessary and sufficient for lipid raft localization and for the function of SHP-1 as a negative regulator of TCR signaling.

The Protein Tyrosine Phosphatase SHP-1 Modulates the Suppressive Activity of Regulatory T Cells

The importance of regulatory T cells (Tregs) for immune tolerance is well recognized, yet the signaling molecules influencing their suppressive activity are relatively poorly understood. In this article, through in vivo studies and complementary ex vivo studies, we make several important observations. First, we identify the cytoplasmic tyrosine phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP-1) as an endogenous brake and modifier of the suppressive ability of Tregs; consistent with this notion, loss of SHP-1 expression strongly augments the ability of Tregs to suppress inflammation in a mouse model. Second, specific pharmacological inhibition of SHP-1 enzymatic activity via the cancer drug sodium stibogluconate potently augmented Treg suppressor activity both in vivo and ex vivo. Finally, through a quantitative imaging approach, we directly demonstrate that Tregs prevent the activation of conventional T cells and that SHP-1–deficient Tregs are more efficient suppressors. Collectively, our data reveal SHP-1 as a critical modifier of Treg function and a potential therapeutic target for augmenting Treg-mediated suppression in certain disease states.

Breaking Free of Control: How Conventional T Cells Overcome Regulatory T Cell Suppression

Conventional T (Tcon) cells are crucial in shaping the immune response, whether it is protection against a pathogen, a cytotoxic attack on tumor cells, or an unwanted response to self-antigens in the context of autoimmunity. In each of these immune settings, regulatory T cells (Tregs) can potentially exert control over the Tcon cell response, resulting in either suppression or activation of the Tcon cells. Under physiological conditions, Tcon cells are able to transiently overcome Treg-imposed restraints to mount a protective response against an infectious threat, achieving clonal expansion, differentiation, and effector function. However, evidence has accumulated in recent years to suggest that Tcon cell resistance to Treg-mediated suppression centrally contributes to the pathogenesis of autoimmune disease. Tipping the balance too far in the other direction, cancerous tumors utilize Tregs to establish an overly suppressive microenvironment, preventing antitumor Tcon cell responses. Given the wide-ranging clinical importance of the Tcon/Treg interaction, this review aims to provide a better understanding of what determines whether a Tcon cell is susceptible to Treg-mediated suppression and how perturbations to this finely tuned balance play a role in pathological conditions. Here, we focus in detail on the complex array of factors that confer Tcon cells with resistance to Treg suppression, which we have divided into two categories: (1) extracellular factor-mediated signaling and (2) intracellular signaling molecules. Further, we explore the therapeutic implications of manipulating the phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway, which is proposed to be the convergence point of signaling pathways that mediate Tcon resistance to suppression. Finally, we address important unresolved questions on the timing and location of acquisition of resistance, and the stability of the “Treg-resistant” phenotype.

Role of Shc in T-cell development and function.

Shc is a prototype adapter protein that is expressed from the earliest stages of T‐cell development. Shc becomes rapidly tyrosine phosphorylated after T‐cell receptor (TCR) engagement. Expression of dominant negative forms of Shc in T‐cell lines had also suggested a role for this adapter downstream of the TCR. However, until recently, the relative significance of Shc compared to several other adapters in T cells was unclear. Mice lacking Shc expression specifically in the T‐cell lineage together with inducible expression of dominant negative Shc in transgenic mice have revealed an essential and nonredundant role for Shc in thymic T‐cell development. Functional defects in a Jurkat T‐cell line lacking Shc expression also suggest a role for Shc in mature T‐cell functions. While the requirement of Shc in T‐cell signaling is now established, precisely what signaling pathways downstream of Shc make this adapter unique are less clear. Although the Shc‐mediated activation of the extracellular signal regulated kinase (Erk)/mitogen‐activated protein kinase (MAPK) pathway could be one component, Shc likely signals to other pathways in T cells that are not yet discovered. A better molecular understanding of Shc function in the future could provide insights into how multiple adapters coordinate the various outcomes downstream of the TCR.

The tyrosine phosphatase SHP-1 dampens murine Th17 development

Th17 cells represent a subset of CD4+ T helper cells that secrete the proinflammatory cytokine IL-17. Th17 cells have been ascribed both a beneficial role in promoting clearance of pathogenic fungi and bacteria, and a pathogenic role in autoimmune diseases. Here we identify the tyrosine phosphatase SHP-1 as a critical regulator of Th17 development, using 3 complementary approaches. Impaired SHP-1 activity through genetic deletion of SHP-1, transgenic expression of an inducible dominant negative SHP-1, or pharmacologic inhibition of SHP-1 strongly promotes the development of Th17. Ex vivo Th17 skewing assays demonstrate that genetic or pharmacologic disruption of SHP-1 activity in T cells results in a hyper-response to stimulation via IL-6 and IL-21, 2 cytokines that promote Th17 development. Mechanistically, we find that SHP-1 decreases the overall cytokine-induced phosphorylation of STAT3 in primary CD4+ T cells. These data identify SHP-1 as a key modifier of IL-6-and IL-21-driven Th17 development via regulation of STAT3 signaling and suggest SHP-1 as a potential new therapeutic target for manipulating Th17 differentiation in vivo.