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Children and adults with Philadelphia chromosome–like B cell acute lymphoblastic leukemia (Ph-like B-ALL) experience high relapse rates despite best-available conventional chemotherapy. Ph-like ALL is driven by genetic alterations that activate constitutive cytokine receptor and kinase signaling, and early-phase trials are investigating the potential of the addition of tyrosine kinase inhibitors (TKIs) to chemotherapy to improve clinical outcomes. However, preclinical studies have shown that JAK or PI3K pathway inhibition is insufficient to eradicate the most common cytokine receptor–like factor 2–rearranged (CRLF2-rearranged) Ph-like ALL subset. We thus sought to define additional essential signaling pathways required in Ph-like leukemogenesis for improved therapeutic targeting. Herein, we describe an adaptive signaling plasticity of CRLF2-rearranged Ph-like ALL following selective TKI pressure, which occurs in the absence of genetic mutations. Interestingly, we observed that Ph-like ALL cells have activated SRC, ERK, and PI3K signaling consistent with activated B cell receptor (BCR) signaling, although they do not express cell surface μ-heavy chain (μHC). Combinatorial targeting of JAK/STAT, PI3K, and “BCR-like” signaling with multiple TKIs and/or dexamethasone prevented this signaling plasticity and induced complete cell death, demonstrating a more optimal and clinically pragmatic therapeutic strategy for CRLF2-rearranged Ph-like ALL.

// Nathan H. Roy 1 , Mahinbanu Mammadli 2 , Janis K. Burkhardt 1 and Mobin Karimi 2 1 Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia Research Institute and Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA 2 Department of Microbiology and Immunology, SUNY Upstate Medical University, Syracuse, NY 13210, USA Correspondence to: Mobin Karimi, email: karimim@upstate.edu Keywords: graft-versus-host disease; CrkL; T cell; migration; inflammation Received: January 03, 2020 Accepted: February 17, 2020 Published: April 28, 2020 ABSTRACT The success of cancer therapies based on allogeneic hematopoietic stem cell transplant relies on the ability to separate graft-versus-host disease (GvHD) from graft-versus-tumor (GVT) responses. Controlling donor T cell migration into peripheral tissues is a viable option to limit unwanted tissue damage, but a lack of specific targets limits progress on this front. Here, we show that the adaptor protein CrkL, but not the closely related family members CrkI or CrkII, is a crucial regulator of T cell migration. In vitro , CrkL-deficient T cells fail to polymerize actin in response to the integrin ligand ICAM-1, resulting in defective migration. Using a mouse model of GvHD/GVT, we found that while CrkL-deficient T cells can efficiently eliminate hematopoietic tumors they are unable to migrate into inflamed organs, such as the liver and small intestine, and thus do not cause GvHD. These results suggest a specific role for CrkL in trafficking to peripheral organs but not the lymphatic system. In line with this, we found that although CrkL-deficient T cells could clear hematopoietic tumors, they failed to clear the same tumor growing subcutaneously, highlighting the role of CrkL in controlling T cell migration into peripheral tissues. Our results define a unique role for CrkL in controlling T cell migration, and suggest that CrkL function could be therapeutically targeted to enhance the efficacy of immunotherapies involving allogeneic donor cells.

Summary The parasite Cryptosporidium invades and replicates in intestinal epithelial cells and is a leading cause of diarrheal disease and early childhood mortality. The molecular mechanisms that underlie infection and pathogenesis are largely unknown. Here, we delineate the events of host cell invasion and uncover a mechanism unique to Cryptosporidium. We developed a screen to identify parasite effectors, finding the injection of multiple parasite proteins into the host from the rhoptry organelle. These factors are targeted to diverse locations within the host cell and its interface with the parasite. One identified effector, rhoptry protein 1 (ROP1), accumulates in the terminal web of enterocytes through direct interaction with the host protein LIM domain only 7 (LMO7) an organizer of epithelial cell polarity and cell-cell adhesion. Genetic ablation of LMO7 or ROP1 in mice or parasites, respectively, impacts parasite burden in vivo in opposite ways. Taken together, these data provide molecular insight into how Cryptosporidium manipulates its intestinal host niche.

Robertson et al. show that unlike T cells responding to conventional chemokines, T cells responding to the lipid chemoattractant S1P utilize a bleb-based mode of motility. This response involves myosin-induced increases in intracellular pressure and is directed by the cortical cytoskeletal proteins ezrin and moesin., Ezrin, radixin, and moesin (ERM) family proteins regulate cytoskeletal responses by tethering the plasma membrane to the underlying actin cortex. Mutations in ERM proteins lead to severe combined immunodeficiency, but the function of these proteins in T cells remains poorly defined. Using mice in which T cells lack all ERM proteins, we demonstrate a selective role for these proteins in facilitating S1P-dependent egress from lymphoid organs. ERM-deficient T cells display defective S1P-induced migration in vitro, despite normal responses to standard protein chemokines. Analysis of these defects revealed that S1P promotes a fundamentally different mode of migration than chemokines, characterized by intracellular pressurization and bleb-based motility. ERM proteins facilitate this process, controlling directional migration by limiting blebbing to the leading edge. We propose that the distinct modes of motility induced by S1P and chemokines are specialized to allow T cell migration across lymphatic barriers and through tissue stroma, respectively.

The parasite Cryptosporidium invades and replicates in intestinal epithelial cells and is a leading cause of diarrheal disease and early childhood mortality. The molecular mechanisms that underlie infection and pathogenesis are largely unknown. Here we delineate the events of host cell invasion and uncover a mechanism unique to Cryptosporidium. We develop a screen to identify parasite effectors and show injection of multiple parasite proteins into the host from the rhoptry organelle. These factors are targeted to diverse locations within the host cell and its interface with the parasite. One of them, ROP1 accumulates in the enterocyte’s terminal web through direct interaction with the host protein LMO7, an organizer of epithelial cell polarity and cell-cell adhesion. Genetic ablation of LMO7 and ROP1 in mice and parasites respectively, show that both impact parasite burden in vivo. Taken together, these studies provide molecular insight into how Cryptosporidium manipulates its intestinal host niche.

T cell entry into inflamed tissue requires firm adhesion, cell spreading, and migration along and through the endothelial wall. These events require the T cell integrins LFA-1 and VLA-4 and their endothelial ligands ICAM-1 and VCAM-1, respectively. T cells migrate against the direction of shear flow on ICAM-1 and with the direction of shear flow on VCAM-1, suggesting that these two ligands trigger distinct cellular responses. However, the contribution of specific signaling events downstream of LFA-1 and VLA-4 has not been explored. Using primary mouse T cells, we found that engagement of LFA-1, but not VLA-4, induces cell shape changes associated with rapid 2D migration. Moreover, LFA-1 ligation results in activation of the phosphoinositide 3-kinase (PI3K) and ERK pathways, and phosphorylation of multiple kinases and adaptor proteins, whereas VLA-4 ligation triggers only a subset of these signaling events. Importantly, T cells lacking Crk adaptor proteins, key LFA-1 signaling intermediates, or the ubiquitin ligase cCbl (also known as CBL), failed to migrate against the direction of shear flow on ICAM-1. These studies identify novel signaling differences downstream of LFA-1 and VLA-4 that drive T cell migratory behavior. This article has an associated First Person interview with the first author of the paper.

An inherited disorder makes WAVEs The WAVE regulatory complex (WRC) is a multiunit complex that regulates actin cytoskeleton formation. Although other actin-regulatory proteins modulate human immune responses, the precise role for the WRC has not yet been established. Cook et al. studied five patients from four unrelated families who harbor missense variants of the gene encoding the WRC component HEM1. These patients presented with recurrent infections and poor antibody responses, along with enhanced allergic and autoimmune disorders. HEM1 was found to be required for the regulation of cortical actin and granule release in T cells and also interacted with a key metabolic signaling complex contributing to the disease phenotype. By linking these interactions to immune function, this work suggests potential targets for future immunotherapies. Science , this issue p. 202

T cell entry into inflamed tissue requires firm adhesion, cell spreading, and migration along and through the endothelial wall. These events require the T cell integrins LFA-1 and VLA-4 and their endothelial ligands ICAM-1 and VCAM-1, respectively. T cells migrate against the direction of shear flow on ICAM-1 and with the direction of shear flow on VCAM-1, suggesting that these two ligands trigger distinct cellular responses. However, the contribution of specific signaling events downstream of LFA-1 and VLA-4 has not been explored. Using primary mouse T cells, we found that engagement of LFA-1, but not VLA-4, induces cell shape changes associated with rapid 2D migration. Moreover, LFA-1 ligation results in activation of the PI3K and ERK pathways, and phosphorylation of multiple kinases and adaptor proteins, while VLA-4 ligation triggers only a subset of these signaling events. Importantly, T cells lacking Crk adaptor proteins, key LFA-1 signaling intermediates, or the ubiquitin ligase cCbl, failed to migrate against the direction of shear flow on ICAM-1. These studies identify novel signaling differences downstream of LFA-1 and VLA-4 that drive T cell migratory behavior.Summary StatementInflammatory responses require leukocyte migration along the vascular wall. We show that signaling from β2, but not β1, integrins induces cytoskeletal changes needed for upstream migration under shear flow.

Dendritic cell (DC) migration is required for efficient presentation of antigen to T cells and the initiation of an adaptive immune response. In spite of its importance, many aspects of DC migration have not been characterized. DCs encounter a variety of environments with different stiffness and geometry, but the effect of these parameters on DC migration has not yet been determined. We addressed this question by comparing DC motility on standard migration surfaces (polydimethylsiloxane (PDMS)-coated coverslips) and micropost array detectors (mPADs). These two surfaces differ in both stiffness and geometry. We found that DC migration was affected by substrate type, with significant increases in speed and significant decreases in persistence time on mPADs made of PDMS as compared to spin-coated PDMS coverslips. To determine whether the geometry or compliance of the post arrays was responsible for these changes in DC migration, we quantified DC motility on mPADs of identical geometry but different stiffness. Migration was indistinguishable on these mPADs, suggesting that DCs are responsive to geometry of ligand presentation and not stiffness. Further, by micropatterning ligands on flat PDMS surfaces in similar geometries to the mPAD arrays, we determined that DCs respond to the geometry of printed ligand. Finally, we used a variety of small molecule inhibitors to identify pathways involved in geometry sensing. We saw a significant role for myosin contractility and α(5)β(1) integrin engagement. We also noted significant reorganization of the actin cytoskeleton into dynamic actin rings when DCs were motile on posts. From these experiments, we conclude that DCs are insensitive to substrate compliance in the range tested but respond to changes in geometry via a mechanism that involves integrin function, myosin contractility, and remodeling of the actin cytoskeleton. As a possible explanation, we postulate a consistent role for filopodial extension and contraction as the driver of DC motility.

T cell activation requires force production at the immunological synapse. Blumenthal and Burkhardt describe how distinct actin structures contribute to this process, addressing canonical immunological models from a mechanobiology perspective., Activation of naive T cells by antigen-presenting cells (APCs) is an essential step in mounting an adaptive immune response. It is known that antigen recognition and T cell receptor (TCR) signaling depend on forces applied by the T cell actin cytoskeleton, but until recently, the underlying mechanisms have been poorly defined. Here, we review recent advances in the field, which show that specific actin-dependent structures contribute to the process in distinct ways. In essence, T cell priming involves a tug-of-war between the cytoskeletons of the T cell and the APC, where the actin cytoskeleton serves as a mechanical intermediate that integrates force-dependent signals. We consider each of the relevant actin-rich T cell structures separately and address how they work together at the topologically and temporally complex cell–cell interface. In addition, we address how this mechanobiology can be incorporated into canonical immunological models to improve how these models explain T cell sensitivity and antigenic specificity.

Immunodeficiency often coincides with immune hyperresponsiveness such as autoimmunity, lymphoproliferation, or atopy, but the molecular basis of this paradox is typically unknown. We describe four families with immunodeficiency coupled with atopy, lymphoproliferation, cytokine overproduction, hemophagocytic lymphohistocytosis, and autoimmunity. We discovered loss-of-function variants in the gene NCKAP1L, encoding the hematopoietic-specific Hem1 protein. Three mutations cause Hem1 protein and WAVE regulatory complex (WRC) loss, thereby disrupting actin polymerization, synapse formation, and immune cell migration. Another mutant, M371V encodes a stable Hem1 protein but abrogates binding of the Arf1 GTPase and identifies Arf1 as a critical Hem1 regulator. All mutations reduce the cortical actin barrier to cytokine release explaining immune hyperresponsiveness. Finally, Hem1 loss blocked mTORC2-dependent AKT phosphorylation, T cell proliferation, and effector cytokine production during T cell activation. Thus, our data show that Hem1 independently governs two key regulatory complexes, the WRC and mTORC2, and how Hem1 loss causes a combined immunodeficiency and immune hyperresponsiveness disease.One sentence summaryHem1 loss of function mutations cause a congenital immunodysregulatory disease and reveal its role regulating WAVE2 and mTORC2 signaling.

T cell activation by dendritic cells (DCs) involves forces exerted by the T cell actin cytoskeleton, which are opposed by the cortical cytoskeleton of the interacting APC. During an immune response, DCs undergo a maturation process that optimizes their ability to efficiently prime naïve T cells. Using atomic force microscopy, we find that during maturation, DC cortical stiffness increases via process that involves actin polymerization. Using stimulatory hydrogels and DCs expressing mutant cytoskeletal proteins, we find that increasing stiffness lowers the agonist dose needed for T cell activation. CD4+ T cells exhibit much more profound stiffness-dependency than CD8+ T cells. Finally, stiffness responses are most robust when T cells are stimulated with pMHC rather than anti-CD3ε, consistent with a mechanosensing mechanism involving receptor deformation. Taken together, our data reveal that maturation-associated cytoskeletal changes alter the biophysical properties of DCs, providing mechanical cues that costimulate T cell activation.

While CD19-directed chimeric antigen receptor (CAR) T cells can induce remission in patients with B cell acute lymphoblastic leukemia (ALL), a large subset relapse with CD19- disease. Like CD19, CD22 is broadly expressed by B-lineage cells and thus serves as an alternative immunotherapy target in ALL. Here we present the composite outcomes of two pilot clinical trials ( NCT02588456 and NCT02650414 ) of T cells bearing a 4-1BB-based, CD22-targeting CAR in patients with relapsed or refractory ALL. The primary end point of these studies was to assess safety, and the secondary end point was antileukemic efficacy. We observed unexpectedly low response rates, prompting us to perform detailed interrogation of the responsible CAR biology. We found that shortening of the amino acid linker connecting the variable heavy and light chains of the CAR antigen-binding domain drove receptor homodimerization and antigen-independent signaling. In contrast to CD28-based CARs, autonomously signaling 4-1BB-based CARs demonstrated enhanced immune synapse formation, activation of pro-inflammatory genes and superior effector function. We validated this association between autonomous signaling and enhanced function in several CAR constructs and, on the basis of these observations, designed a new short-linker CD22 single-chain variable fragment for clinical evaluation. Our findings both suggest that tonic 4-1BB-based signaling is beneficial to CAR function and demonstrate the utility of bedside-to-bench-to-bedside translation in the design and implementation of CAR T cell therapies.

Smallpox and monkeypox pose severe threats to human health. Other orthopoxviruses are comparably virulent in their natural hosts, including ectromelia, the cause of mousepox. Disease severity is linked to an array of immunomodulatory proteins including the B22 family, which has homologs in all pathogenic orthopoxviruses but not attenuated vaccine strains. We demonstrate that the ectromelia B22 member, C15, is necessary and sufficient for selective inhibition of CD4+ but not CD8+ T cell activation by immunogenic peptide and superantigen. Inhibition is achieved not by down-regulation of surface MHC- II or co-stimulatory protein surface expression but rather by interference with antigen presentation. The appreciable outcome is interference with CD4+ T cell synapse formation as determined by imaging studies and lipid raft disruption. Consequently, CD4+ T cell activating stimulus shifts to uninfected antigen-presenting cells that have received antigen from infected cells. This work provides insight into the immunomodulatory strategies of orthopoxviruses by elucidating a mechanism for specific targeting of CD4+ T cell activation, reflecting the importance of this cell type in control of the virus., Author summary Orthopoxviruses pose considerable threats to their hosts by producing a battery of proteins that disable the immune system at many levels through mechanisms that remain poorly understood. An essential part of most immune responses is the activation of CD4+ T cells by antigen-presenting cells through formation of a supramolecular structure termed the immunological synapse. We show here that the C15 protein of ectromelia, the cause of mousepox, inhibits CD4+ T cell activation through a novel immunoevasion mechanism that results in disruption of synapse formation. As many poxviruses encode C15 homologs, these studies could provide insights into the virulence of other family members including monkeypox and smallpox, both of great concern to human populations.

T cell entry into inflamed tissue involves firm adhesion, spreading, and migration of the T cells across endothelial barriers. These events depend on “outside-in” signals through which engaged integrins direct cytoskeletal reorganization. We investigated the molecular events that mediate this process and found that T cells from mice lacking expression of the adaptor protein Crk exhibited defects in phenotypes induced by the integrin lymphocyte function–associated antigen 1 (LFA-1), namely, actin polymerization, leading edge formation, and two-dimensional cell migration. Crk protein was an essential mediator of LFA-1 signaling–induced phosphorylation of the E3 ubiquitin ligase c-Cbl and its subsequent interaction with the phosphatidylinositol 3-kinase (PI3K) subunit p85, thus promoting PI3K activity and cytoskeletal remodeling. In addition, we found that Crk proteins were required for T cells to respond to changes in substrate stiffness, as measured by alterations in cell spreading and differential phosphorylation of the force-sensitive protein CasL. These findings identify Crk proteins as key intermediates coupling LFA-1 signals to actin remodeling and provide mechanistic insights into how T cells sense and respond to substrate stiffness.

Improved diagnostic and treatment methods are needed for chronic graft-versus-host disease (cGVHD), the leading cause of late nonrelapse mortality (NRM) in long-term survivors of allogenic hematopoietic cell transplantation. Validated biomarkers that facilitate disease diagnosis and classification generally are lacking in cGVHD. Here, we conducted whole serum proteomics analysis of a well-established murine multiorgan system cGVHD model. We discovered 4 upregulated proteins during cGVHD that are targetable by genetic ablation or blocking antibodies, including the RAS and JUN kinase activator, CRKL, and CXCL7, CCL8, and CCL9 chemokines. Donor T cells lacking CRK/CRKL prevented the generation of cGVHD, germinal center reactions, and macrophage infiltration seen with wild-type T cells. Whereas antibody blockade of CCL8 or CXCL7 was ineffective in treating cGVHD, CCL9 blockade reversed cGVHD clinical manifestations, histopathological changes, and immunopathological hallmarks. Mechanistically, elevated CCL9 expression was present predominantly in vascular smooth muscle cells and uniquely seen in cGVHD mice. Plasma concentrations of CCL15, the human homolog of mouse CCL9, were elevated in a previously published cohort of 211 cGVHD patients compared with controls and associated with NRM. In a cohort of 792 patients, CCL15 measured at day +100 could not predict cGVHD occurring within the next 3 months with clinically relevant sensitivity/specificity. Our findings demonstrate for the first time the utility of preclinical proteomics screening to identify potential new targets for cGVHD and specifically CCL15 as a diagnosis marker for cGVHD. These data warrant prospective biomarker validation studies.

The actin-binding proteins moesin and α-actinin-1 limit the lateral mobility and clustering of ICAM-1 in dendritic cells and thereby promote antigen-dependent conjugate formation and T cell priming., Integrity of the dendritic cell (DC) actin cytoskeleton is essential for T cell priming, but the underlying mechanisms are poorly understood. We show that the DC F-actin network regulates the lateral mobility of intracellular cell adhesion molecule 1 (ICAM-1), but not MHCII. ICAM-1 mobility and clustering are regulated by maturation-induced changes in the expression and activation of moesin and α-actinin-1, which associate with actin filaments and the ICAM-1 cytoplasmic domain. Constrained ICAM-1 mobility is important for DC function, as DCs expressing a high-mobility ICAM-1 mutant lacking the cytoplasmic domain exhibit diminished antigen-dependent conjugate formation and T cell priming. These defects are associated with inefficient induction of leukocyte functional antigen 1 (LFA-1) affinity maturation, which is consistent with a model in which constrained ICAM-1 mobility opposes forces on LFA-1 exerted by the T cell cytoskeleton, whereas ICAM-1 clustering enhances valency and further promotes ligand-dependent LFA-1 activation. Our results reveal an important new mechanism through which the DC cytoskeleton regulates receptor activation at the immunological synapse.

Full T cell activation requires coordination of signals from multiple receptor-ligand pairs that interact in parallel at a specialized cell-cell contact site termed the immunological synapse. Signaling at the immunological synapse is intimately associated with actin dynamics; TCR engagement induces centripetal flow of T cell actin network, which in turn enhances the function of ligand-bound integrins by promoting conformational change. Here, we have investigated the effects of integrin engagement on actin flow, and on associated signaling events downstream of the TCR. We show that integrin engagement significantly decelerates centripetal flow of the actin network. In primary CD4+ T cells, engagement of either LFA-1 or VLA-4 by their respective ligands ICAM-1 and VCAM-1 slows actin flow. Slowing of actin flow is greatest when T cells interact with low mobility integrin ligands, supporting a predominately drag-based mechanism. Using integrin ligands presented on patterned surfaces, we demonstrate that the effects of localized integrin engagement are distributed across the actin network, and that focal adhesion proteins such as talin, vinculin, and paxillin are recruited to sites of integrin engagement. Further analysis shows that talin and vinculin are interdependent upon one another for recruitment, and that ongoing actin flow is required. Suppression of vinculin or talin partially relieves integrin-dependent slowing of actin flow, indicating that these proteins serve as molecular clutches that couple engaged integrins to the dynamic actin network. Finally, we found that integrin-dependent slowing of actin flow is associated with reduction in tyrosine phosphorylation downstream of the TCR, and that this modulation of TCR signaling depends on expression of talin and vinculin. More generally, we found that integrin-dependent effects on actin retrograde flow were strongly correlated with effects on TCR signaling. Taken together, these studies support a model in which ligand-bound integrins engage the actin cytoskeletal network via talin and vinculin, and tune TCR signaling events by modulating actin dynamics at the immunological synapse.

Dendritic cell migration to the T-cell-rich areas of the lymph node is essential for their ability to initiate the adaptive immune response. While it has been shown that the actin cytoskeleton is required for normal DC migration, the role of many of the individual cytoskeletal molecules is poorly understood. In this study, we investigated the contribution of the Arp2/3 complex binding protein, haematopoietic lineage cell-specific protein 1 (HS1), to DC migration and force generation. We quantified the random migration of HS1−/− DCs on 2D micro-contact printed surfaces and found that in the absence of HS1, DCs have greatly reduced motility and speed. This same reduction in motility was recapitulated when adding Arp2/3 complex inhibitor to WT DCs or using DCs deficient in WASP, an activator of Arp2/3 complex-dependent actin polymerization. We further investigated the importance of HS1 by measuring the traction forces of HS1−/− DCs on micropost array detectors (mPADs). In HS1 deficient DCs, there was a significant reduction in force generation (3.96 ± 0.40 nN per cell) compared to WT DCs (13.76 ± 0.84 nN per cell). Interestingly, the forces generated in DCs lacking WASP were only slightly reduced compared to WT DCs. Taken together, these findings show that HS1 and Arp2/3 complex-mediated actin polymerization are essential for the most efficient DC random migration and force generation.

T cell signaling is inextricably linked to actin cytoskeletal dynamics at the immunological synapse (IS). This process can be imaged in living T cells expressing GFP actin or fluorescent F-actin binding proteins. Because of its planar nature, the IS provides a unique opportunity to image events as they happen, monitoring changes in actin retrograde flow in T cells interacting with different stimulatory surfaces or after pharmacological treatments. Here, we described the imaging methods and analytical procedures used to measure actin velocity across the IS in T cells spreading on planar stimulatory surfaces.

We recently conducted a clinical trial of CD22-directed chimeric antigen receptor (CAR) T cells in children and adults with relapsed or refractory B-cell acute lymphoblastic leukemia (ALL). While we did observe some transient responses, overall outcomes were inferior to another recent trial of CD22 CAR T cells in ALL performed at the NCI (Fry, T.J. et al. Nat Med, 2018). Intriguingly, these trials used a CAR that employed the same antigen-binding and intracellular signaling domains, and differed only in the length of linker connecting the variable regions of the single chain variable fragment (scFv). Based on these clinical observations, we sought to identify how the scFv linker impacts CAR biology and regulates CAR-driven T cell activity. The University of Pennsylvania's CD22 CAR contained a long 20 amino acid scFv linker ("CAR22-L") while the NCI's CAR had a 5 amino acid linker ("CAR22-S"). We began by investigating the impact of linker length on CAR biochemistry. Both CAR22-L and CAR22-S had similar antigen-binding affinities (KD of 1.67nM and 6.05nM, respectively). Chromatography revealed that CAR22-L remained monomeric in solution while CAR22-S formed homodimers. To explore how dimerization influenced surface-membrane biology, we developed GFP-tagged versions of each CAR and performed confocal microscopy on CAR+ T cells. CAR22-L exhibited homogenous surface membrane expression, while CAR22-S appeared to self-aggregate and cluster (Fig. 1a). We investigated the impact of this clustering on receptor signaling and found that CAR22-S demonstrated high levels of signaling molecule activation (i.e. Akt, p70-S6 and STAT3) in the absence of antigen engagement. This is consistent with previous reports establishing that CAR clustering can lead to tonic signaling (Long, A.H. et al. Nat Med, 2015). Importantly, this tonic signaling did not lead to autonomous T cell proliferation. We proceeded to evaluate how clustering and tonic signaling impacted CAR function upon antigen engagement. Microscopic evaluation of CAR T cells combined with CD22+ Nalm6 cells revealed greater actin and microtubule organizing complex polarization (P = 0.02 and 0.01, respectively) in CAR22-S cells, consistent with superior immune synapse formation. This was accompanied by increased phosphorylation of PI3K, MAPK and calcium signaling proteins (Fig. 1b) after CAR engagement. RNA sequencing revealed significantly greater activation of immune response gene programs in CAR22-S cells as compared to CAR22-L after overnight exposure to Nalm6. We next investigated the impact that this enhanced receptor-driven activity had on CAR T cell anti-tumor function. CAR T cells were combined with Nalm6 in vitro and residual Nalm6 was serially quantified, revealing that CAR22-S mediated greater tumor control than CAR22-L, particularly at later time periods (P < 0.001). This was associated with greater secretion of IFNg, IL-2 and TNFa (all P < 0.001). Finally, we compared anti-tumor efficacy in xenograft models of systemic Nalm6. NOD/SCID/cg-/- mice were engrafted with Nalm6 and received 1x106 CAR T cells 7 days later. CAR22-S demonstrated greater in vivo expansion (P < 0.0001) and enhanced control of systemic disease (Fig. 1c,P = 0.017), resulting in prolongation of animal survival (Fig. 1d,P = 0.013). Based on these observations, we have designed a novel, affinity-enhanced CD22 CAR and confirmed that shorter linker length improves anti-tumor activity of this CAR. T cells expressing this CAR are currently undergoing evaluation in a phase I clinical trial (ClinicalTrials.org Identifiers NCT03620058 and NCT02650414). Thus far, 4 children and 2 adults have been infused with manageable toxicity. Early outcomes are promising, with 67% achieving complete remission at day 28, compared to 50% in our original CART22 trials. In summary, by investigating the potential mechanisms for an apparent discrepancy in outcomes between two different clinical trials, we demonstrate that reducing the length of the scFv linker results in significant changes to CAR biochemistry that directly lead to antigen-independent receptor activity. In contrast to previously published data demonstrating that tonic signaling of CD28-costimulated CARs is detrimental to T cell function (Long, A.H. et al. Nat Med, 2015), we found that tonic signaling of 4-1BB-costimulated CARs may be beneficial, possibly by priming T cells for rapid response to antigen. Disclosures Singh: University of Pennsylvania: Patents & Royalties. Frey:Novartis: Research Funding. Engels:Novartis: Employment. Zhao:Novartis: Employment. Peng:Novartis: Employment. Granda:Novartis: Employment. Ramones:Novartis: Employment. Lacey:Novartis: Research Funding; Novartis: Patents & Royalties: Patents related to CAR T cell biomarkers; Tmunity: Research Funding. Young:novartis: Research Funding. Brogdon:Novartis: Employment. Grupp:Roche: Consultancy; GSK: Consultancy; Novartis: Consultancy, Research Funding; Humanigen: Consultancy; CBMG: Consultancy; Novartis: Research Funding; Kite: Research Funding; Servier: Research Funding; Jazz: Other: study steering committees or scientific advisory boards; Adaptimmune: Other: study steering committees or scientific advisory boards; Cure Genetics: Consultancy. June:Novartis: Research Funding; Tmunity: Other: scientific founder, for which he has founders stock but no income, Patents & Royalties. Maude:Novartis: Consultancy; Kite: Consultancy. Gill:Novartis: Research Funding; Tmunity Therapeutics: Research Funding; Carisma Therapeutics: Research Funding; Amphivena: Consultancy; Aro: Consultancy; Intellia: Consultancy; Sensei Bio: Consultancy; Carisma Therapeutics: Equity Ownership. Ruella:AbClon: Membership on an entity's Board of Directors or advisory committees; Nanostring: Consultancy, Speakers Bureau; Novartis: Patents & Royalties: CART for cancer.

Background: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype occurring in 15-40% of older children, adolescents, and adults with B-ALL and is associated with high relapse rates and poor survival. Approximately 50% of Ph-like ALL cases have CRLF2 (cytokine receptor-like factor 2 rearrangements (CRLF2-R), often with concomitant JAK2 point mutations. We and others have shown that targeted inhibition of JAK/STAT and PI3K/AKT signaling in CRLF2-rearranged Ph-like ALL only partially inhibits leukemia proliferation. Our preliminary data indicates that modified B cell receptor signaling results in ERK activation and Ph-like ALL cell growth that appears independent of CRLF2-activated signaling. This study aimed to define the extent to which BCR signaling mediates drug resistance in Ph-like ALL after cytokine receptor signaling inhibition and to develop novel combinatorial treatment strategies. Methods and Results: We first validated high basal levels of JAK/STAT and PI3K pathway phosphoproteins via immunoblotting of cell lysates from >20 primary childhood and adult Ph-like ALL samples and patient-derived xenograft (PDX) models. Despite lack of B-cell receptor (BCR) μ heavy chain on Ph-like ALL cells by flow cytometry analysis, we detected high expression levels of the BCR-associated proteins CD79A, CD79B, and BLNK. We further identified constitutive phosphorylation of downstream BCR-associated signaling molecules, including SRC family kinases (SFKs), BTK, and ERK. Given these new observations, we then sought to delineate more precisely alternative pathways in Ph-like ALL (beyond JAK/STAT and PI3K) that may contribute to adaptive signaling regulation and confer resistance to kinase inhibitor monotherapy. We hypothesized that JAK inhibitor (JAKi) resistance is mediated by compensatory activation of PI3K/AKT/mTOR through 'BCR-like' signaling via CD79/CD19/SRC/BTK/ERK. To test this hypothesis, we treated CRLF2-R Ph-like ALL cells in vitro with selective inhibitors of JAK1/2 (ruxolitinib) or PI3Kd (INCB050465) and assessed effects upon signaling via immunoblotting. We observed i) effective dephosphorylation of STAT5 and moderate dephosphorylation of AKT with full reactivation within 72h following JAKi or PI3Kdi monotherapy, respectively, ii) combined JAKi and PI3Kdi prevented PI3K pathway, reactivation, and iii) JAKi and PI3Kdi had no effects upon ERK signaling. These results suggested that oncogene-independent activation of BCR and PI3K pathway signaling occurs in Ph-like ALL. We then performed immunofluorescence analysis and detected distinct clustering and homodimerization of CD79B at the cell membrane of Ph-like ALL cells, as well as physical proximity of CD79B and CD19, suggesting that CD19 could be phosphorylated by SRC/SFKs independently of an activated BCR. Interestingly however, CRISPR-mediated deletion of CD19 or CD79B resulted in hyperactivation of ERK signaling. Finally, we tested if triple inhibition of JAK/STAT, PI3K, and SRC/SFKs signaling is sufficient to eradicate Ph-like ALL. Strikingly, combined JAKi (ruxolitinib), PI3Kdi (INCB050465), and SRCi (dasatinib) silenced STAT5, PI3K, and ERK signaling and induced near-complete cytotoxicity in vitro and in vivo in Ph-like ALL cell lines and patient-derived xenograft (PDX) models. While triple inhibitor treatment appeared well-tolerated in mice, we also assessed dual kinase inhibition of JAK2 and PI3K with dexamethasone (a known repressor of BCR signaling) as a more clinically relevant combinatorial strategy. We observed similarly robust inhibition of signaling pathways and leukemia eradication in vitro and in vivo in our Ph-like ALL cell lines and PDX models. Conclusions: Children and adults with Ph-like ALL have a >60% risk of relapse despite maximally intensive chemotherapy, demonstrating need for alternative therapeutic approaches. While earlier studies by our group and others have reported partial anti-leukemia activity of JAKi and PI3Ki monotherapies, our new data provide compelling evidence that three essential cooperating signaling pathways are required for Ph-like leukemogenesis. We postulate that triple JAK, PI3K, and SRC inhibition or dual kinase inhibition with chemotherapy can overcome innate compensatory mechanisms of signaling resistance and may in fact be necessary to eradicate Ph-like ALL in patients. Disclosures Perl: Daiichi Sankyo: Consultancy, Honoraria, Other, Research Funding; Arog: Consultancy, Other: Non-financial support included travel costs for advisory board meetings.; AbbVie: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; Actinium Pharmaceuticals: Consultancy, Honoraria, Other: Clinical Advisory Board member, Research Funding; Agios: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Non-financial support included travel costs for advisory board meetings.; Jazz: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; NewLink Genetics: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; Astellas: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings as well as a medical writing company that assisted with manuscript preparation/submission and slide deck assembly for academic meeting presentations of trial data., Research Funding; Takeda: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; Bayer: Research Funding; BioMed Valley Discoveries: Research Funding; FujiFilm: Research Funding; Novartis: Honoraria, Other: Advisory board, Non-financial support included travel costs for advisory board meetings as well as a medical writing company that assisted with manuscript preparation/submission and slide deck assembly for academic meeting presentations of the data., Research Funding. Carroll:Astellas Pharmaceuticals: Research Funding; Incyte: Research Funding; Janssen Pharmaceuticals: Consultancy. Tasian:Gilead Sciences: Research Funding; Incyte Corportation: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees.

Allogeneic hematopoietic stem cell transplants (HSCT) are used to treat many malignancies, but the prevalence of graft-versus-host disease (GvHD) limits their overall success. Manipulating T-cell trafficking has emerged as an effective countermeasure, yet downstream integrin signaling pathways have yet to be targeted. We previously found that T-cells lacking the adapter proteins Crk and CrkL exhibit a robust antitumor response while causing little GvHD. We now find that T-cells from mice lacking Crk adapter proteins exhibit defects in LFA-1/ICAM-1 induced actin polymerization, leading edge formation, 2D migration, and integrin-mediated mechanosensing. Under shear flow, Crk/CrkL deficient T-cells fail to migrate upstream on ICAM-1 but migrate normally on VCAM-1, suggesting these integrin ligands relay different outside-in signals. Analysis of LFA-1 signaling reveals that Crk protein expression is required for phosphorylation of c-Cbl and its subsequent interaction with the PI3K subunit p85. Through this mechanism, Crk proteins promote PI3K activity and cytoskeletal remodeling downstream of LFA-1 engagement. Interestingly, this signaling pathway was largely specific to the LFA-1/ICAM-1 interaction, as WT-cells plated on VCAM-1 (which binds and signals through VLA-4) failed to induce high levels of phospho-Cbl, showed diminished PI3K activity, and did not migrate with a defined actin-rich leading edge. Finally, we show that knocking out CrkL alone is sufficient to alleviate GvHD, pointing toward unique roles of the Crk isoforms in T-cell biology. Together, these studies identify key signaling differences downstream of β2 vs β1 integrins that drive T-cell migratory behavior, and identify CrkL as an important factor during GvHD. Importantly, these data provide insight into integrin signaling that could be used to manipulate T-cell trafficking. Citation Format: Nathan H. Roy, Joanna L. MacKay, Alexander Buffone Jr., Krista Newell, Tanner F. Robertson, Sangya Agarwal, Mobin Karimi, Daniel A Hammer, Janis K. Burkhardt. LFA-1 signals via Crk adapter proteins to induce actin-dependent T-cell migration and mechanosensing [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B183.

The immunological synapse formed between a cytotoxic T lymphocyte (CTL) and an infected or transformed target cell is a physically active structure capable of exerting mechanical force. Here, we investigated whether synaptic forces promote the destruction of target cells. CTLs kill by secreting toxic proteases and the pore forming protein perforin into the synapse. Biophysical experiments revealed a striking correlation between the magnitude of force exertion across the synapse and the speed of perforin pore formation on the target cell, implying that force potentiates cytotoxicity by enhancing perforin activity. Consistent with this interpretation, we found that increasing target cell tension augmented pore formation by perforin and killing by CTLs. Our data also indicate that CTLs coordinate perforin release and force exertion in space and time. These results reveal an unappreciated physical dimension to lymphocyte function and demonstrate that cells use mechanical forces to control the activity of outgoing chemical signals.

It is well known that F-actin dynamics drive micron-scale cell shape changes required for migration and immunological synapse formation. In addition, recent evidence points to a more intimate role for the actin cytoskeleton in promoting T cell activation. Mechanotransduction, the conversion of mechanical input into intracellular biochemical changes, is thought to play a critical role in several aspects of immunoreceptor triggering and downstream signal transduction. Multiple molecules associated with signaling events at the immunological synapse have been shown to respond to physical force, including the TCR, costimulatory molecules, adhesion molecules, and several downstream adapters. In at least some cases, it is clear that the relevant forces are exerted by dynamics of the T cell actomyosin cytoskeleton. Interestingly, there is evidence that the cytoskeleton of the antigen presenting cell also plays an active role in T cell activation, by countering the molecular forces exerted by the T cell at the immunological synapse. Since actin polymerization is itself driven by TCR and costimulatory signaling pathways, a complex relationship exists between actin dynamics and receptor activation. This review will focus on recent advances in our understanding of the mechano-sensitive aspects of T cell activation, paying specific attention to how F-actin directed forces applied from both sides of the immunological synapse fit into current models of receptor triggering and activation.

Actomyosin dynamics and T cell receptor signaling are tightly coupled to ensure proper dynamics and function of signaling microclusters within the immunological synapse., Activation of T cells by antigen-presenting cells involves assembly of signaling molecules into dynamic microclusters (MCs) within a specialized membrane domain termed the immunological synapse (IS). Actin and myosin IIA localize to the IS, and depletion of F-actin abrogates MC movement and T cell activation. However, the mechanisms that coordinate actomyosin dynamics and T cell receptor signaling are poorly understood. Using pharmacological inhibitors that perturb individual aspects of actomyosin dynamics without disassembling the network, we demonstrate that F-actin polymerization is the primary driver of actin retrograde flow, whereas myosin IIA promotes long-term integrity of the IS. Disruption of F-actin retrograde flow, but not myosin IIA contraction, arrested MC centralization and inhibited sustained Ca2+ signaling at the level of endoplasmic reticulum store release. Furthermore, perturbation of retrograde flow inhibited PLCγ1 phosphorylation within MCs but left Zap70 activity intact. These studies highlight the importance of ongoing actin polymerization as a central driver of actomyosin retrograde flow, MC centralization, and sustained Ca2+ signaling.

Clonal selection of a T cell for use in the immune response appears to necessitate proliferative expansion and terminal effector differentiation of some cellular progeny, while reserving other progeny as less-differentiated memory cells. It has been suggested that asymmetric cell division may promote initial cell diversification. Stem cell-like models of adaptive immunity might predict that subsequent encounters with a pathogen would evoke reiterative, self-renewing, asymmetric division by memory T cells. In this study, we show that murine memory CD8+ T cells can divide asymmetrically in response to secondary encounter with pathogen. Critical regulators of signaling and transcription are partitioned to one side of the mitotic spindle in rechallenged memory T cells, and two phenotypically distinct populations of daughter cells are evident from the earliest divisions. Memory T cells may thus use asymmetric cell division to generate cellular heterogeneity when faced with pathogen rechallenge.

The hematopoietic actin regulatory protein hematopoietic lineage cell-specific protein 1 (HS1) is required for cell spreading and signaling in lymphocytes, but the scope of HS1 function in Ag presentation has not been addressed. We show that dendritic cells (DCs) from HS1−/− mice differentiate normally and display normal LPS-induced upregulation of surface markers and cytokines. Consistent with their normal expression of MHC and costimulatory molecules, HS1−/− DCs present OVA peptide efficiently to CD4+ T cells. However, presentation of OVA protein is defective. Similarly, MHC class I-dependent presentation of VSV8 peptide to CD8+ T cells occurs normally, but cross-presentation of GRP94/VSV8 complexes is defective. Analysis of Ag uptake pathways shows that HS1 is required for receptor-mediated endocytosis, but not for phagocytosis or macropinocytosis. HS1 interacts with dynamin 2, a protein involved in scission of endocytic vesicles. However, HS1−/− DCs showed decreased numbers of endocytic invaginations, whereas dynamin-inhibited cells showed accumulation of these endocytic intermediates. Taken together, these studies show that HS1 promotes an early step in the endocytic pathway that is required for efficient Ag presentation of exogenous Ag by DCs.

Dendritic cells (DCs) are professional APCs that reside in peripheral tissues and survey the body for pathogens. Upon activation by inflammatory signals, DCs undergo a maturation process and migrate to lymphoid organs, where they present pathogen-derived Ags to T cells. DC migration depends on tight regulation of the actin cytoskeleton to permit rapid adaptation to environmental cues. We investigated the role of hematopoietic lineage cell-specific protein 1 (HS1), the hematopoietic homolog of cortactin, in regulating the actin cytoskeleton of murine DCs. HS1 localized to lamellipodial protrusions and podosomes, actin-rich structures associated with adhesion and migration. DCs from HS1−/− mice showed aberrant lamellipodial dynamics. Moreover, although these cells formed recognizable podosomes, their podosome arrays were loosely packed and improperly localized within the cell. HS1 interacts with Wiskott–Aldrich syndrome protein (WASp), another key actin-regulatory protein, through mutual binding to WASp-interacting protein. Comparative analysis of DCs deficient for HS1, WASp or both proteins revealed unique roles for these proteins in regulating podosomes with WASp being essential for podosome formation and with HS1 ensuring efficient array organization. WASp recruitment to podosome cores was independent of HS1, whereas HS1 recruitment required Src homology 3 domain-dependent interactions with the WASp/WASp-interacting protein heterodimer. In migration assays, the phenotypes of HS1- and WASp-deficient DCs were related, but distinct. WASp−/y DCs migrating in a chemokine gradient showed a large decrease in velocity and diminished directional persistence. In contrast, HS1−/− DCs migrated faster than wild-type cells, but directional persistence was significantly reduced. These studies show that HS1 functions in concert with WASp to fine-tune DC cytoarchitecture and direct cell migration.

CD43 interaction with ERM proteins regulates CD43 phosphorylation and T-cell migration. CD43 phosphorylation can also drive CD43 localization in T-cells independently of ERM association., Cell polarization is a key feature of cell motility, driving cell migration to tissues. CD43 is an abundantly expressed molecule on the T-cell surface that shows distinct localization to the migrating T-cell uropod and the distal pole complex (DPC) opposite the immunological synapse via association with the ezrin-radixin-moesin (ERM) family of actin regulatory proteins. CD43 regulates multiple T-cell functions, including T-cell activation, proliferation, apoptosis, and migration. We recently demonstrated that CD43 regulates T-cell trafficking through a phosphorylation site at Ser-76 (S76) within its cytoplasmic tail. Using a phosphorylation-specific antibody, we now find that CD43 phosphorylation at S76 is enhanced by migration signals. We further show that CD43 phosphorylation and normal T-cell trafficking depend on CD43 association with ERM proteins. Interestingly, mutation of S76 to mimic phosphorylation enhances T-cell migration and CD43 movement to the DPC while blocking ERM association, showing that CD43 movement can occur in the absence of ERM binding. We also find that protein kinase Cθ can phosphorylate CD43. These results show that while CD43 binding to ERM proteins is crucial for S76 phosphorylation, CD43 movement and regulation of T-cell migration can occur through an ERM-independent, phosphorylation–dependent mechanism.

Dendritic cells (DCs) play central roles in innate and adaptive immunity. Upon maturation, DCs assemble numerous veil-like membrane protrusions, disassemble podosomes, and travel from the peripheral tissues to lymph nodes to present Ags to T cells. These alterations in morphology and motility are closely linked to the primary function of DCs, Ag presentation. However, it is unclear how and what cytoskeletal proteins control maturation-associated alterations, in particular, the change in cell migration. Fascin1, an actin-bundling protein, is specifically and greatly induced upon maturation, suggesting a unique role for fascin1 in mature DCs. To determine the physiological roles of fascin1, we characterized bone marrow-derived, mature DCs from fascin1 knockout mice. We found that fascin1 is critical for cell migration: fascin1-null DCs exhibit severely decreased membrane protrusive activity. Importantly, fascin1-null DCs have lower chemotactic activity toward CCL19 (a chemokine for mature DCs) in vitro, and in vivo, Langerhans cells show reduced emigration into draining lymph nodes. Morphologically, fascin1-null mature DCs are flatter and fail to disassemble podosomes, a specialized structure for cell-matrix adhesion. Expression of exogenous fascin1 in fascin1-null DCs rescues the defects in membrane protrusive activity, as well as in podosome disassembly. These results indicate that fascin1 positively regulates migration of mature DCs into lymph nodes, most likely by increasing dynamics of membrane protrusions, as well as by disassembling podosomes.

Productive T cell activation requires efficient reorganization of the actin cytoskeleton. We showed previously that the actin-regulatory protein, hematopoietic lineage cell-specific protein 1 (HS1), is required for the stabilization of F-actin and Vav1 at the immunological synapse and for efficient calcium responses. The Tec family kinase IL-2-inducible T cell kinase (Itk) regulates similar aspects of T cell activation, suggesting that these proteins act in the same pathway. Using video microscopy, we show that T cells lacking Itk or HS1 exhibited similar defects in actin responses, extending unstable lamellipodial protrusions upon TCR stimulation. HS1 and Itk could be coimmunoprecipitated from T cell lysates, and GST-pulldown studies showed that Itk’s Src homology 2 domain binds directly to two phosphotyrosines in HS1. In the absence of Itk, or in T cells overexpressing an Itk Src homology 2 domain mutant, HS1 failed to localize to the immunological synapse, indicating that Itk serves to recruit HS1 to sites of TCR engagement. Because Itk is required for phospholipase C (PLC)γ1 phosphorylation and calcium store release, we examined the calcium signaling pathway in HS1−/− T cells in greater detail. In response to TCR engagement, T cells lacking HS1 exhibited diminished calcium store release, but TCR-dependent PLCγ1 phosphorylation was intact, indicating that HS1’s role in calcium signaling is distinct from that of Itk. HS1-deficient T cells exhibited defective cytoskeletal association of PLCγ1 and altered formation of PLCγ1 microclusters. We conclude that HS1 functions as an effector of Itk in the T cell actin-regulatory pathway, and directs the spatial organization of PLCγ1 signaling complexes.

In the last few years, great progress has been made in understanding how stromal interacting molecule 1 (STIM1), a protein containing a calcium sensor that is located in the endoplasmic reticulum, and Orai1, a protein that forms a calcium channel in the plasma membrane, interact and give rise to store-operated calcium entry. Pharmacological depletion of calcium stores leads to the formation of clusters containing STIM and Orai that appear to be sites for calcium influx. Similar puncta are also produced in response to physiological stimuli in immune cells. In T cells engaged with antigen-presenting cells, clusters containing STIM and Orai accumulate at the immunological synapse. We recently discovered that in activated T cells, STIM1 and Orai1 also accumulate in cap-like structures opposite the immune synapse at the distal pole of the cell. Both caps and puncta are long-lived stable structures containing STIM1 and Orai1 in close proximity. The function of puncta as sites of calcium influx is clear. We speculate that the caps may provide a secondary site of calcium entry. Alternatively, they may serve as a source of preformed channel complexes that move to new immune synapses as T cells repeatedly engage antigen-presenting cells.

The highly homologous proteins ezrin, radixin, and moesin link proteins to the actin cytoskeleton. The two family members expressed in T cells, ezrin and moesin, are implicated in promoting T cell activation and polarity. To elucidate the contributions of ezrin and moesin, we conducted a systematic analysis of their function during T cell activation. In response to TCR engagement, ezrin and moesin were phosphorylated in parallel at the regulatory threonine, and both proteins ultimately localized to the distal pole complex (DPC). However, ezrin exhibited unique behaviors, including tyrosine phosphorylation and transient localization to the immunological synapse before movement to the DPC. To ask whether these differences reflect unique requirements for ezrin vs moesin in T cell signaling, we generated mice with conditional deletion of ezrin in mature T cells. Ezrin−/− T cells exhibited normal immunological synapse organization based upon localization of protein kinase C-θ, talin, and phospho-ZAP70. DPC localization of CD43 and RhoGDP dissociation inhibitor, as well as the novel DPC protein Src homology region 2 domain-containing phosphatase-1, was also unaffected. However, recruitment of three novel DPC proteins, ezrin binding protein of 50 kDa, Csk binding protein, and the p85 subunit of PI3K was partially perturbed. Biochemical analysis of ezrin−/− T cells or T cells suppressed for moesin using small interfering RNA showed intact early TCR signaling, but diminished levels of IL-2. The defects in IL-2 production were more pronounced in T cells deficient for both ezrin and moesin. These cells also exhibited diminished phospholipase C-γ1 phosphorylation and calcium flux. We conclude that despite their unique movement and phosphorylation patterns, ezrin and moesin function together to promote T cell activation.

Actin dynamics during T-cell activation are controlled by the coordinate action of multiple actin regulatory proteins, functioning downstream of a complex network of kinases and other signaling molecules. The c-Abl nonreceptor tyrosine kinase regulates actin responses in nonhematopoietic cells, but its function in T cells is poorly understood. Using kinase inhibitors, RNAi, and conditional knockout mice, we investigated the role of c-Abl in controlling the T-cell actin response. We find that c-Abl is required for normal actin polymerization and lamellipodial spreading at the immune synapse, and for downstream events leading to efficient interleukin-2 production. c-Abl also plays a key role in signaling chemokine-induced T-cell migration. c-Abl is required for the appropriate function of 2 proteins known to be important for controlling actin responses to T-cell receptor (TCR) engagement, the actin-stabilizing adapter protein HS1, and the Rac1-dependent actin polymerizing protein WAVE2. c-Abl binds to phospho-HS1 via its SH2 domains and is required for full tyrosine phosphorylation of HS1 during T-cell activation. In addition, c-Abl is required for normal localization of WAVE2 to the immune synapse (IS). These studies identify c-Abl as a key player in the signaling cascade, leading to actin reorganization during T-cell activation.

T cell cytoarchitecture differs dramatically depending on whether the cell is circulating within the bloodstream, migrating through tissues, or interacting with antigen-presenting cells. The transition between these states requires important signaling-dependent changes in actin cytoskeletal dynamics. Recently, analysis of actin-regulatory proteins associated with T cell activation has provided new insights into how T cells control actin dynamics in response to external stimuli and how actin facilitates downstream signaling events and effector functions. Among the actin-regulatory proteins that have been identified are nucleation-promoting factors such as WASp, WAVE2, and HS1; severing proteins such as cofilin; motor proteins such as myosin II; and linker proteins such as ezrin and moesin. We review the current literature on how signaling pathways leading from diverse cell surface receptors regulate the coordinated activity of these and other actin-regulatory proteins and how these proteins control T cell function.

Regulatory T cells (Tregs) are a subset of CD4+ T cells that maintain immune tolerance in part by their ability to inhibit the proliferation of conventional CD4+ T cells (Tconvs). The role of the TCR and the downstream signaling pathways required for this suppressive function of Tregs are not fully understood. To yield insight into how TCR-mediated signals influence Treg suppressive function, we assessed the ability of Tregs with altered TCR-mediated signaling capacity to inhibit Tconv proliferation. Mature Tregs deficient in Src homology 2 domain containing leukocyte protein of 76 kDa (SLP-76), an adaptor protein that nucleates the proximal signaling complex downstream of the TCR, were unable to inhibit Tconv proliferation, suggesting that TCR signaling is required for Treg suppressive function. Moreover, Tregs with defective phospholipase C γ (PLCγ) activation due to a Y145F mutation of SLP-76 were also defective in their suppressive function. Conversely, enhancement of diacylglycerol-mediated signaling downstream of PLCγ by genetic ablation of a negative regulator of diacylglycerol kinase ζ increased the suppressive ability of Tregs. Because SLP-76 is also important for integrin activation and signaling, we tested the role of integrin activation in Treg-mediated suppression. Tregs lacking the adaptor proteins adhesion and degranulation promoting adapter protein or CT10 regulator of kinase/CT10 regulator of kinase–like, which are required for TCR-mediated integrin activation, inhibited Tconv proliferation to a similar extent as wild-type Tregs. Together, these data suggest that TCR-mediated PLCγ activation, but not integrin activation, is required for Tregs to inhibit Tconv proliferation.