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Supplementary Table from P2RX7 Enhances Tumor Control by CD8+ T Cells in Adoptive Cell Therapy

Expression of the purinergic receptor P2RX7 by CD8+ T cells promotes the generation of memory populations following acute infections. However, data suggest that P2RX7 may limit the efficacy of antitumor responses. Herein, we show that P2RX7 is beneficial for optimal melanoma control in a mouse CD8+ T-cell adoptive transfer model. Tumor-specific P2rx7–/– CD8+ T cells exhibited impaired mitochondrial maintenance and function but did not display signs of overt exhaustion early in the antitumor response. However, as the tumor burden increased, the relative frequency of P2RX7-deficient CD8+ T cells declined within the tumor; this correlated with reduced proliferation, increased apoptosis, and mitochondrial dysfunction. Extending these studies, we found that the transient in vitro stimulation of P2RX7 using the ATP analogue BzATP led to enhanced B16 melanoma control by CD8+ T cells. These findings are in keeping with the concept that extracellular ATP (eATP) sensing by P2RX7 on CD8+ T cells is required for their ability to efficiently eliminate tumors by promoting mitochondrial fitness and underscore the potential for P2RX7 stimulation as a novel therapeutic treatment to enhance tumor immunotherapy.

Supplementary Figure from P2RX7 Enhances Tumor Control by CD8+ T Cells in Adoptive Cell Therapy

Optimal ex vivo expansion protocols of tumor-specific T cells followed by adoptive cell therapy must yield T cells able to home to tumors and effectively kill them. Our previous study demonstrated ex vivo activation in the presence of IL-12–induced optimal CD8+ T cell expansion and melanoma regression; however, adverse side effects, including autoimmunity, can occur. This may be due to transfer of high-avidity self-specific T cells. In this study, we compared mouse low- and high-avidity T cells targeting the tumor Ag tyrosinase-related protein 2 (TRP2). Not surprisingly, high-avidity T cells provide superior tumor control, yet low-avidity T cells can promote tumor regression. The addition of IL-12 during in vitro expansion boosts low-avidity T cell responsiveness, tumor regression, and prevents T cell exhaustion. In this study, we demonstrate that IL-12–primed T cells are resistant to PD-1/PD-L1–mediated suppression and retain effector function. Importantly, IL-12 preconditioning prevented exhaustion as LAG-3, PD-1, and TOX were decreased while simultaneously increasing KLRG1. Using intravital imaging, we also determined that high-avidity T cells have sustained contacts with intratumoral dendritic cells and tumor targets compared with low-avidity T cells. However, with Ag overexpression, this defect is overcome, and low-avidity T cells control tumor growth. Taken together, these data illustrate that low-avidity T cells can be therapeutically beneficial if cocultured with IL-12 cytokine during in vitro expansion and highly effective in vivo if Ag is not limiting. Clinically, low-avidity T cells provide a safer alternative to high-avidity, TCR-engineered T cells, as IL-12–primed, low-avidity T cells cause less autoimmune vitiligo.

SUMMARY The immune system adapts to constitutive antigens to preserve self-tolerance, which is a major barrier for anti-tumor immunity. Antigen-specific reversal of tolerance constitutes a major goal to spur therapeutic applications. Here, we show that robust, iterative, systemic stimulation targeting tissue-specific antigens in the context of acute infections reverses established CD8+ T cell tolerance to self, including in T cells that survive negative selection. This strategy results in large numbers of circulating and resident memory self-specific CD8+ T cells that are widely distributed and can be co-opted to control established malignancies bearing self-antigen without concomitant autoimmunity. Targeted expansion of both self- and tumor neoantigen-specific T cells acts synergistically to boost anti-tumor immunity and elicits protection against aggressive melanoma. Our findings demonstrate that T cell tolerance can be re-adapted to responsiveness through robust antigenic exposure, generating self-specific CD8+ T cells that can be used for cancer treatment., Graphical Abstract, In Brief Nelson et al. show that immune tolerance to self is not a fixed state and can be overcome with robust, iterative stimulation in the context of infection. Autoreactive CD8+ T cells expanded with this method can be co-opted to target tumors bearing shared self-antigen without associated autoimmunity.

Expression of the purinergic receptor P2RX7 by CD8+ T cells promotes the generation of memory populations following acute infections. However, data suggest that P2RX7 may limit the efficacy of antitumor responses. Herein, we show that P2RX7 is beneficial for optimal melanoma control in a mouse CD8+ T-cell adoptive transfer model. Tumor-specific P2rx7–/– CD8+ T cells exhibited impaired mitochondrial maintenance and function but did not display signs of overt exhaustion early in the antitumor response. However, as the tumor burden increased, the relative frequency of P2RX7-deficient CD8+ T cells declined within the tumor; this correlated with reduced proliferation, increased apoptosis, and mitochondrial dysfunction. Extending these studies, we found that the transient in vitro stimulation of P2RX7 using the ATP analogue BzATP led to enhanced B16 melanoma control by CD8+ T cells. These findings are in keeping with the concept that extracellular ATP (eATP) sensing by P2RX7 on CD8+ T cells is required for their ability to efficiently eliminate tumors by promoting mitochondrial fitness and underscore the potential for P2RX7 stimulation as a novel therapeutic treatment to enhance tumor immunotherapy.

Hypertension often occurs in concurrence with obesity and diabetes mellitus, commonly referred to as metabolic syndrome. Renal denervation (RDNx) lowers arterial pressure (AP) and improves glucose metabolism in drug-resistant hypertensive patients with high body mass index. In addition, RDNx has been shown to reduce renal inflammation in the mouse model of angiotensin II hypertension. The present study tested the hypothesis that RDNx reduces AP and renal inflammation and improves glucose metabolism in obesity-induced hypertension. Eight-week-old C57BL/6J mice were fed either a low-fat diet (10 kcal%) or a high-fat diet (45 kcal%) for 10 weeks. Body weight, food intake, fasting blood glucose, and glucose metabolism (glucose tolerance test) were measured. In a parallel study, radiotelemeters were implanted in mice for AP measurement. High fat–fed C57BL/6J mice exhibited an inflammatory and metabolic syndrome phenotype, including increased fat mass, increased AP, and hyperglycemia compared with low-fat diet mice. RDNx, but not Sham surgery, normalized AP in high-fat diet mice (115.8±1.5 mm Hg in sham versus 96.6±6.7 mm Hg in RDNx). RDNx had no significant effect on AP in low-fat diet mice. Also, RDNx had no significant effect on glucose metabolism or renal inflammation as measured by the number of CD8, CD4, and T helper cells or levels of inflammatory cytokines in the kidneys. These results indicate that although renal nerves play a role in obesity-induced hypertension, they do not contribute to impaired glucose metabolism or renal inflammation in this model.

During acute infections, naive Ag-specific CD8 T cells are activated and differentiate into effector T cells, most of which undergo contraction after pathogen clearance. A small population of CD8 T cells persists as memory to protect against future infections. We investigated the role of adhesion- and degranulation-promoting adapter protein (ADAP) in promoting CD8 T cell responses to a systemic infection. Naive Ag-specific CD8 T cells lacking ADAP exhibited a modest expansion defect early after Listeria monocytogenes or vesicular stomatitis virus infection but comparable cytolytic function at the peak of response. However, reduced numbers of ADAP-deficient CD8 T cells were present in the spleen after the peak of the response. ADAP deficiency resulted in a greater frequency of CD127+ CD8 memory precursors in secondary lymphoid organs during the contraction phase. Reduced numbers of ADAP-deficient killer cell lectin-like receptor G1− CD8 resident memory T (TRM) cell precursors were present in a variety of nonlymphoid tissues at the peak of the immune response, and consequently the total numbers of ADAP-deficient TRM cells were reduced at memory time points. TRM cells that did form in the absence of ADAP were defective in effector molecule expression. ADAP-deficient TRM cells exhibited impaired effector function after Ag rechallenge, correlating with defects in their ability to form T cell–APC conjugates. However, ADAP-deficient TRM cells responded to TGF-β signals and recruited circulating memory CD8 T cells. Thus, ADAP regulates CD8 T cell differentiation events following acute pathogen challenge that are critical for the formation and selected functions of TRM cells in nonlymphoid tissues.

The inflammatory response regulates congestive heart failure (CHF) development. T cell activation plays an important role in tissue inflammation. We postulate that CD28 or B7 deficiency inhibits T cell activation and attenuates CHF development by reducing systemic, cardiac, and pulmonary inflammation. We demonstrated that chronic pressure overload–induced end-stage CHF in mice is characterized by profound accumulation of activated effector T cells (CD3 + CD44 high cells) in the lungs and a mild but significant increase of these cells in the heart. In knockout mice lacking either CD28 or B7, there was a dramatic reduction in the accumulation of activated effector T cells in both hearts and lungs of mice under control conditions and after transverse aortic constriction. CD28 or B7 knockout significantly attenuated transverse aortic constriction–induced CHF development, as indicated by less increase of heart and lung weight and less reduction of left ventricle contractility. CD28 or B7 knockout also significantly reduced transverse aortic constriction–induced CD45 + leukocyte, T cell, and macrophage infiltration in hearts and lungs, lowered proinflammatory cytokine expression (such as tumor necrosis factor-α and interleukin-1β) in lungs. Furthermore, CD28/B7 blockade by CTLA4-Ig treatment (250 μg/mouse every 3 days) attenuated transverse aortic constriction–induced T cell activation, left ventricle hypertrophy, and left ventricle dysfunction. Our data indicate that CD28/B7 deficiency inhibits activated effector T cell accumulation, reduces myocardial and pulmonary inflammation, and attenuates the development of CHF. Our findings suggest that strategies targeting T cell activation may be useful in treating CHF.

Focal thermal therapy (Heat), cryosurgery (Cryo) and irreversible electroporation (IRE) are increasingly used to treat cancer. However, local recurrence and systemic spread are persistent negative outcomes. Nevertheless, emerging work with immunotherapies (i.e., checkpoint blockade or dendritic cell (DC) vaccination) in concert with focal therapies may improve outcomes. To understand the role of focal therapy in priming the immune system for immunotherapy, an in vitro model of T cell response after exposure to B16 melanoma cell lysates after lethal exposures was designed. Exposure included: Heat (50 °C, 30 min), Cryo (−80 °C, 30 min) and IRE (1250 V/cm, 99 pulses, 50 µs pulses with 1 Hz intervals). After viability assessment (CCK-8 assay), cell lysates were collected and assessed for protein release (BCA assay), protein denaturation (FTIR-spectroscopy), TRP-2 antigen release (western blot), and T cell activation (antigen-specific CD8 T cell proliferation). Results showed IRE released the most protein and antigen (TRP-2), followed by Cryo and Heat. In contrast, Cryo released the most native (not denatured) protein, compared to IRE and Heat. Finally, IRE dramatically outperformed both Cryo and Heat in T cell activation while Cryo modestly outperformed Heat. This study demonstrates that despite all focal therapies ability to destroy cells, the ‘quantity’ (i.e., amount) and ‘quality’ (i.e., molecular state) of tumor protein (including antigen) released can dramatically change the ensuing priming of the immune system. This suggests protein-based metrics whereby focal therapies can be designed to prime the immune system in concert with immunotherapies to eventually achieve improved and durable cancer treatment in vivo.

The maintenance of T cell repertoire diversity involves the entry of newly developed T cells, as well as the maintenance of memory T cells generated from previous infections. This balance depends on competition for a limited amount of homeostatic cytokines and interaction with self-peptide MHC class I. In the absence of prior infection, memory-like or memory phenotype (MP) CD8 T cells can arise from homeostatic cytokine exposure during neonatal lymphopenia. Aside from downstream cytokine signaling, little is known about the regulation of the conversion of naive CD8 T cells to MP CD8 T cells during acute lymphopenia. We have identified a novel negative regulatory role for adhesion and degranulation–promoting adapter protein (ADAP) in CD8 T cell function. We show that in the absence of ADAP, naive CD8 T cells exhibit a diminished response to stimulatory Ag, but an enhanced response to weak agonist-altered peptide ligands. ADAP-deficient mice exhibit more MP CD8 T cells that occur following thymic emigration and are largely T cell intrinsic. Naive ADAP-deficient CD8 T cells are hyperresponsive to lymphopenia in vivo and exhibit enhanced activation of STAT5 and homeostatic Ag-independent proliferation in response to IL-15. Our results indicate that ADAP dampens naive CD8 T cell responses to lymphopenia and IL-15, and they demonstrate a novel Ag-independent function for ADAP in the suppression of MP CD8 T cell generation.

CD8+ T cell immunosurveillance dynamics influence the outcome of intracellular infections and cancer. Here we used two-photon intravital microscopy to visualize the responses of CD8+ resident memory T cells (TRM cells) within the reproductive tracts of live female mice. We found that mucosal TRM cells were highly motile, but paused and underwent in situ division after local antigen challenge. TRM cell reactivation triggered the recruitment of recirculating memory T cells that underwent antigen-independent TRM cell differentiation in situ. However, the proliferation of pre-existing TRM cells dominated the local mucosal recall response and contributed most substantially to the boosted secondary TRM cell population. We observed similar results in skin. Thus, TRM cells can autonomously regulate the expansion of local immunosurveillance independently of central memory or proliferation in lymphoid tissue. Masopust and colleagues show that mucosal tissue-resident memory T cells proliferate in situ in response to local antigen and dominate the local recall response.

Memory CD8+ T cells populate non-lymphoid tissues (NLT) and can protect against malignancies, but little is known about the establishment of tumor-specific tissue resident memory CD8+ T cells (TRM) by therapeutically initiated immune responses. We used a transplantable mouse model of prostate carcinoma and MHCI tetramers to track and quantify tumor-specific CD8+ T cells. Tumor challenge led to systemic expansion of endogenous, naïve tumor antigen-specific CD8+ T cells and established a small population of TRM (CD44hi, CD62Llo, CD69+, CD103+) in several NLTs. Previously, we demonstrated that debulking primary tumors using irreversible electroporation (IRE) induced expansion of tumor-specific T cells. We hypothesized that IRE would cooperate with checkpoint blockade to increase antigen-specific TRM and promote tumor clearance in vivo. Combination treatment with IRE followed by α-CTLA4 promoted a robust 50-fold expansion of proliferating (Ki67+) tumor-specific T cells in blood, a 10-fold increase of TRM in NLT, and was associated with complete remission of residual tumor. Conversely, monotherapy allowed tumor outgrowth and failed to boost T cell numbers, resulting in small populations of PD-1hi tumor-specific CD8+ T cells. Similarly, anti-PD-1 alone or following IRE was also ineffective, suggesting that combined IRE/α-CTLA4 therapy targets newly primed T cells. IRE also enhanced antigen presentation by XCR1+ dendritic cells, promoting T cell priming in regional lymph nodes. These results indicate that combining α-CTLA4 checkpoint immunotherapy with focal tumor ablation, such as IRE, capable of releasing high quantities and qualities of endogenous tumor antigens may serve as a potent in situ tumor vaccination strategy.

The Ag-specific interactions between T cells and dendritic cells progress through dynamic contact stages in vivo consisting of early long-term stable contacts and later confined, yet motile, short-lived contacts. The signaling pathways that control in vivo interaction dynamics between T cells and dendritic cells during priming remain undefined. Adhesion and degranulation promoting adapter protein (ADAP) is a multifunctional adapter that regulates “inside-out” signaling from the TCR to integrins. Using two-photon microscopy, we demonstrate that, in the absence of ADAP, CD4 T cells make fewer early-stage stable contacts with Ag-laden dendritic cells, and the interactions are characterized by brief repetitive contacts. Furthermore, ADAP-deficient T cells show reduced contacts at the late motile contact phase and display less confinement around dendritic cells. The altered T cell interaction dynamics in the absence of ADAP are associated with defective early proliferation and attenuated TCR signaling in vivo. Regulation of multistage contact behaviors and optimal T cell signaling involves the interaction of ADAP with the adapter src kinase–associated phosphoprotein of 55 kDa (SKAP55). Thus, integrin activation by the ADAP-SKAP55–signaling module controls the stability and duration of T cell–dendritic cell contacts during the progressive phases necessary for optimal T cell activation.

Targeted deletion of Actb demonstrates that the β-actin gene, in contrast to the γ-actin gene, is an essential gene uniquely required for cell growth and migration. Cell motility and growth defects in β-actin–knockout primary cells are due to a specific role for β-actin in regulating gene expression through control of the cellular G-actin pool., Ubiquitously expressed β-actin and γ-actin isoforms play critical roles in most cellular processes; however, their unique contributions are not well understood. We generated whole-body β-actin–knockout (Actb−/−) mice and demonstrated that β-actin is required for early embryonic development. Lethality of Actb−/− embryos correlated with severe growth impairment and migration defects in β-actin–knockout primary mouse embryonic fibroblasts (MEFs) that were not observed in γ-actin–null MEFs. Migration defects were associated with reduced membrane protrusion dynamics and increased focal adhesions. We also identified migration defects upon conditional ablation of β-actin in highly motile T cells. Of great interest, ablation of β-actin altered the ratio of globular actin (G-actin) to filamentous actin in MEFs, with corresponding changes in expression of genes that regulate the cell cycle and motility. These data support an essential role for β-actin in regulating cell migration and gene expression through control of the cellular G-actin pool.

NF-kappaB activation following engagement of the antigen-specific T cell receptor involves protein kinase C-theta-dependent assembly of the CARMA1-BCL10-MALT1 (CBM) signalosome, which coordinates downstream activation of IkappaB kinase (IKK). We previously identified a novel role for the adhesion- and degranulation-promoting adapter protein (ADAP) in regulating the assembly of the CBM complex via an interaction of ADAP with CARMA1. In this study, we identify a novel site in ADAP that is critical for association with the TAK1 kinase. ADAP is critical for recruitment of TAK1 and the CBM complex, but not IKK, to protein kinase C-theta. ADAP is not required for TAK1 activation. Although both the TAK1 and the CARMA1 binding sites in ADAP are essential for IkappaB alpha phosphorylation and degradation and NF-kappaB nuclear translocation, only the TAK1 binding site in ADAP is necessary for IKK phosphorylation. In contrast, only the CARMA1 binding site in ADAP is required for ubiquitination of IKKgamma. Thus, distinct sites within ADAP control two key activation responses that are required for NF-kappaB activation in T cells.

Following TCR stimulation, T cells utilize the hematopoietic specific adhesion and degranulation-promoting adapter protein (ADAP) to control both integrin adhesive function and NF-κB transcription factor activation. We have investigated the molecular basis by which ADAP controls these events in primary murine ADAP−/− T cells. Naive DO11.10/ADAP−/− T cells show impaired adhesion to OVAp (OVA aa 323–339)-bearing APCs that is restored following reconstitution with wild-type ADAP. Mutational analysis demonstrates that the central proline-rich domain and the C-terminal domain of ADAP are required for rescue of T:APC conjugate formation. The ADAP proline-rich domain is sufficient to bind and stabilize the expression of SKAP55 (Src kinase-associated phosphoprotein of 55 kDa), which is otherwise absent from ADAP−/− T cells. Interestingly, forced expression of SKAP55 in the absence of ADAP is insufficient to drive T:APC conjugate formation, demonstrating that both ADAP and SKAP55 are required for optimal LFA-1 function. Additionally, the ADAP proline-rich domain is required for optimal Ag-induced activation of CD69, CD25, and Bcl-xL, but is not required for assembly of the CARMA1/Bcl10/Malt1 (caspase-recruitment domain (CARD) membrane-associated guanylate kinase (MAGUK) protein 1/B-cell CLL-lymphoma 10/mucosa-associated lymphoid tissue lymphoma translocation protein 1) signaling complex and subsequent TCR-dependent NF-κB activity. Our results indicate that ADAP is used downstream of TCR engagement to delineate two distinct molecular programs in which the ADAP/SKAP55 module is required for control of T:APC conjugate formation and functions independently of ADAP/CARMA1-mediated NF-κB activation.

The hemopoietic specific adapter protein ADAP (adhesion and degranulation-promoting adapter protein) positively regulates TCR-dependent, integrin-mediated adhesion and participates in signaling pathways downstream of the TCR that result in T cell activation. The specific role of ADAP in regulating Ag-dependent T cell interactions with APCs and T cell activation following Ag stimulation is not known. We used ADAP−/− DO11.10 T cells to demonstrate that ADAP promotes T cell conjugation to Ag-laden APCs. Complementary in vitro and in vivo approaches reveal that ADAP controls optimal T cell proliferation, cytokine production, and expression of the prosurvival protein Bcl-xL in response to limiting Ag doses. Furthermore, ADAP is critical for clonal expansion in vivo independent of Ag concentration under conditions of low clonal abundance. These results suggest that ADAP regulates T cell activation by promoting Ag-dependent T cell-APC interactions, resulting in enhanced T cell sensitivity to Ag, and by participating in prosurvival signaling pathways initiated by Ag stimulation.

Summary: Integrin adhesion receptors are critical for antigen recognition by T cells and for regulated recirculation and trafficking into and through various tissues in the body. T-cell receptor (TCR) signaling induces rapid increases in integrin function that facilitate T-cell activation by promoting stable contact with antigen-presenting cells and extracellular proteins in the environment. In this review, we outline the molecular mechanisms by which the TCR signals to integrins and present a model that highlights four key events: (i) initiation of proximal TCR signals nucleated by the linker for activated T cells (LAT) adapter protein and involving Itk, phospholipase C-γ1, Vav1, and Src homology 2 domain-containing leukocyte-specific phosphoprotein of 76 kDa; (ii) transmission of integrin activation signals from the LAT signalosome to integrins by protein kinase (PK) C and the adapter protein, adhesion and degranulation-promoting adapter protein; (iii) assembly of integrin-associated signaling complexes that include PKD, the guanosine triphosphatase Rap1 and its effectors, and talin; and (iv) reorganization of the actin cytoskeleton by WAVE2 and other actin-remodeling proteins. These events coordinate changes in integrin conformation and clustering that result in enhanced integrin functional activity following TCR stimulation.

The adapter protein ADAP regulates T lymphocyte adhesion and activation. We present evidence for a previously unrecognized function for ADAP in regulating T cell receptor (TCR)-mediated activation of the transcription factor NF-kappaB. Stimulation of ADAP-deficient mouse T cells with antibodies to CD3 and CD28 resulted in impaired nuclear translocation of NF-kappaB, a reduced DNA binding, and delayed degradation and decreased phosphorylation of IkappaB (inhibitor of NF-kappaB). TCR-stimulated assembly of the CARMA1-BCL-10-MALT1 complex was substantially impaired in the absence of ADAP. We further identified a region of ADAP that is required for association with the CARMA1 adapter and NF-kappaB activation but is not required for ADAP-dependent regulation of adhesion. These findings provide new insights into ADAP function and the mechanism by which CARMA1 regulates NF-kappaB activation in T cells.

Clinical studies have shown that renal denervation (RDNX) decreases arterial pressure (AP) and improves glucose metabolism in drug resistant obese hypertensive patients. In the present study, we used a murine model of obesity-induced hypertension (HTN) to test the hypothesis that RDNX lowers AP and improves glucose metabolism via an interaction of renal nerves with inflammatory mediators in the kidney. 8-week old C57Bl/6J mice were fed either a low fat diet (LFD; 10 KCal% from fat) or a high fat diet (HFD; 45 KCal% from fat) for 10 weeks. Two parallel protocols were conducted. In a metabolic protocol, body weight, food intake, body-composition and glucose metabolism were measured. In a cardiovascular protocol, radiotelemeters were implanted for measurement of AP. C57Bl6J mice on a HFD exhibited an inflammatory and metabolic syndrome phenotype including increase splenic and renal total T cells, increased fat mass, high blood pressure, hyperglycemia and glucose intolerance as compared to LFD mice. RDNX but not Sham surgery after 12 weeks of HFD normalized AP (116 ± 4 in sham vs. 97± 6 mmHg in RDX HFD mice). RDX had no effect on AP in LFD diet mice. RDX in obese hypertensive mice had no effect on renal T cells or cytokines. Finally, RDX had no effect on glucose metabolism in HFD mice as determined by the glucose tolerance test at 2 weeks after RDX. We conclude that the antihypertensive effect of RDX in obesity-induced hypertension is not associated with improvement in glucose metabolism or renal inflammation in the mouse.

B82L mouse fibroblasts respond to fibronectin or vitronectin via a syndecan-1-mediated activation of the alphavbeta5 integrin. Cells attached to syndecan-1-specific antibody display only filopodial extension. However, the syndecan-anchored cells extend lamellipodia when the antibody-substratum is supplemented with serum, or low concentrations of adsorbed vitronectin or fibronectin, that are not sufficient to activate the integrin when plated alone. Integrin activation is blocked by treatment with (Arg-Gly-Asp)-containing peptides and function-blocking antibodies that target alphav integrins, as well as by siRNA-mediated silencing of beta5 integrin expression. In addition, alphavbeta5-mediated cell attachment and spreading on high concentrations of vitronectin is blocked by competition with recombinant syndecan-1 ectodomain core protein and by downregulation of mouse syndecan-1 expression by mouse-specific siRNA. Taking advantage of the species-specificity of the siRNA, rescue experiments in which human syndecan-1 constructs are expressed trace the activation site to the syndecan-1 ectodomain. Moreover, both full-length mouse and human syndecan-1 co-immunoprecipitate with the beta5 integrin subunit, but fail to do so if the syndecan is displaced by competition with soluble, recombinant syndecan-1 ectodomain. These results suggest that the ectodomain of the syndecan-1 core protein contains an active site that assembles into a complex with the alphavbeta5 integrin and regulates alphavbeta5 integrin activity.

Syndecan-1 was overexpressed in T47D, MCF-7, or Hs578t human breast carcinoma cell lines, mimicking overexpression observed in carcinomas in vivo. Overexpression of syndecan-1, or its ectodomain alone fused to a glycosylphosphatidylinositol anchor (GPI-mS1ED), promotes cell rounding in 2D culture. Deletions within the syndecan-1 ectodomain (S1ED) implicate an active site within the core protein between the glycosaminoglycan attachment region and the transmembrane domain. Polyclonal antibodies directed against the ectodomain, or treatment with the tyrosine kinase inhibitor genistein, block activity and revert GPI-mS1ED overexpressing cells to a normal morphology. Extracellular matrix (ECM)-dependent signaling appears to be targeted, as GPI-mS1ED cells attach and spread similarly to control cells in response to E-cadherin engagement, but fail to spread on integrin-dependent ligands. However, integrin-dependent cell attachment, and integrin activation and subsequent FAK phosphorylation are unaffected, suggesting that the syndecan regulates the integration of signaling following matrix adhesion. In 3D culture, where syndecan-1 may have a more critical role in cell behavior, the disrupted signaling leads to poorly cohesive, invasive colonies. Thus, altered matrix-dependent signaling due to increased levels of cell surface syndecan-1 may lead to epithelial cell invasion during early stages of tumorigenesis.

Adhesion and degranulation-promoting adapter protein (ADAP) is a multifunctional scaffold that regulates T cell receptor-mediated activation of integrins via association with the SKAP55 adapter and the NF-κB pathway through interactions with both the CARMA1 adapter and serine/threonine kinase transforming growth factor β-activated kinase 1 (TAK1). ADAP-deficient T cells exhibit impaired proliferation following T cell receptor stimulation, but the contribution of these distinct functions of ADAP to this defect is not known. We demonstrate that loss of ADAP results in a G1-S transition block in cell cycle progression following T cell activation due to impaired accumulation of cyclin-dependent kinase 2 (Cdk2) and cyclin E. The CARMA1-binding site in ADAP is critical for mitogen-activated protein (MAP) kinase kinase 7 (MKK7) phosphorylation and recruitment to the protein kinase C θ (PKCθ) signalosome and subsequent c-Jun kinase (JNK)-mediated Cdk2 induction. Cyclin E expression following T cell receptor stimulation of ADAP-deficient T cells is transient and associated with enhanced cyclin E ubiquitination. Both the CARMA1- and TAK1-binding sites in ADAP are critical for restraining cyclin E ubiquitination and turnover independently of ADAP-dependent JNK activation. T cell receptor-mediated proliferation was most dramatically impaired by the loss of ADAP interactions with CARMA1 or TAK1 rather than SKAP55. Thus, ADAP coordinates distinct CARMA1-dependent control of key cell cycle proteins in T cells.

Adhesion and degranulation promoting adapter protein (ADAP) is a multifunctional hematopoietic adapter protein that regulates TCR-dependent increases in both integrin function and activation of the NF-κB transcription factor. Activation of integrin function requires both ADAP and the ADAP-associated adapter Src kinase-associated phosphoprotein of 55 kDa (SKAP55). In contrast, ADAP-mediated regulation of NF-κB involves distinct binding sites in ADAP that promote the inducible association of ADAP, but not SKAP55, with the CARMA1 adapter and the TAK1 kinase. This suggests that the presence or absence of associated SKAP55 defines functionally distinct pools of ADAP. To test this hypothesis, we developed a novel SKAP–ADAP chimeric fusion protein and demonstrated that physical association of ADAP with SKAP55 is both sufficient and necessary for the rescue of integrin function in ADAP-deficient T cells. Similar to wild-type ADAP, the SKAP–ADAP chimera associated with the LFA-1 integrin after TCR stimulation. Although the SKAP–ADAP chimera contains the CARMA1 and TAK1 binding sequences from ADAP, expression of the chimera does not restore NF-κB signaling in ADAP−/− T cells. A single point mutation in the pleckstrin homology domain of SKAP55 (R131M) blocks the ability of the SKAP–ADAP chimera to restore integrin function and to associate with LFA-1. However, the R131M mutant was now able to restore NF-κB signaling in ADAP-deficient T cells. We conclude that integrin regulation by ADAP involves the recruitment of ADAP to LFA-1 integrin complexes by the pleckstrin homology domain of SKAP55, and this recruitment restricts the ability of ADAP to interact with the NF-κB signalosome and regulate NF-κB activation.

NK cells are key mediators of the innate immune response and anti-tumor surveillance. Adhesion and degranulation-promoting adapter protein (ADAP, formerly known as SLAP-130 or Fyb) is a hematopoietic-specific adapter that is required for efficient TCR signaling and T cell activation. Herein, we examine a potential role for ADAP in NK development and function. ADAP is expressed in primary NK cells and in IL-2 stimulated lymphokine-activated killers. However, ADAP-deficient mice show no defects in NK development. Further, ADAP is dispensable for key NK functions, including cytotoxicity in response to engagement of activating receptors, cytokine production, conjugate formation and tumor suppression in vivo. These results indicate that, unlike events stimulated by TCR engagement, signaling events engaged by immunoreceptor tyrosine-based activation motif-associated and cytokine receptors on NK cells can occur independently of ADAP.

The alpha(v)beta(3) integrin participates in cell morphogenesis, growth factor signaling, and cell survival. Activation of the integrin is central to these processes and is influenced by specific ECM components, which engage both integrins and syndecans. This paper demonstrates that the alpha(v)beta(3) integrin and syndecan-1 (S1) are functionally coupled. The integrin is dependent on the syndecan to become activated and to mediate signals required for MDA-MB-231 and MDA-MB-435 human mammary carcinoma cell spreading on vitronectin or S1-specific antibody. Coupling of the syndecan to alpha(v)beta(3) requires the S1 ectodomain (ED), as ectopic expression of glycosylphosphatidylinositol-linked S1ED enhances alpha(v)beta(3) recognition of vitronectin; and treatments that target this domain, including competition with recombinant S1ED protein or anti-S1ED antibodies, mutation of the S1ED, or down-regulation of S1 expression by small-interfering RNAs, disrupt alpha(v)beta(3)-dependent cell spreading and migration. Thus, S1 is likely to be a critical regulator of many cellular behaviors that depend on activated alpha(v)beta(3) integrins.

Expression patterns of syndecan-1, the cell surface heparan sulfate proteoglycan (HSPG) predominant on epithelial cells, were analyzed in tissue samples from 30 infiltrating human breast carcinomas and in 9 human breast carcinoma cell lines. Immunohistochemical staining demonstrates that while a subset of the breast carcinomas lose syndecan-1, this proteoglycan is expressed or overexpressed in a majority of the cases. Interestingly, cells in poor grade tumors contain intracellular syndecan-1, an observation that has not been previously described and was thus further investigated. Examination of cultured breast carcinoma cell lines indicates that they also display the phenotype of the syndecan-1 positive tumors and thereby provide a model system for analysis of intracellular syndecan-1. All cell lines examined express syndecan-1, and poorly differentiated lines such as BT549 cells internalize the proteoglycan from the cell surface where it accumulates as intact HSPG in intracellular vesicles. Colocalization studies using fluorescent markers identify these to be lysosomes. This finding is unexpected, as the accepted mechanism for degradation of syndecan HSPG following endocytosis is fragmentation of the protein core and glycosaminoglycan chains in endosomes, followed by delivery of the fragments to lysosomes. Lysosomal inactivation using ammonium chloride demonstrates that well-differentiated lines such as T47D and MCF-7 cells, which maintain the majority of syndecan-1 on their cell surfaces, also target intact constitutively endocytosed syndecan-1 to lysosomes. Taken together, these results suggest that mammary epithelial cells utilize a previously uncharacterized mechanism for syndecan-1 catabolism. In this pathway the proteoglycan remains intact as it passes through the endosomal system, prior to arriving at its site of intracellular degradation in lysosomes.

A 34-nucleotide A-U sequence located immediately upstream of the editing sites of the Leishmania tarentolaecytochrome b mRNA induces a mitochondrial extract to insert U nucleotides independent of guide RNA. Insertions are localized to positions immediately 5′ and 3′ of the A-U sequence. When placed within an unedited mammalian transcript, the A-U sequence is sufficient to induce U-insertions. The sequence has a high degree of similarity with the templating nucleotides of a cytochrome b guide RNA and with a sequence adjacent to the editing sites in ND7 mRNA, the other characterized kinetoplastid mRNA supporting guide RNA-independent U-insertions. At least one protein specifically interacts with the A-U sequence. The reaction is consistent with a mechanism proposed for guide RNA-directed editing.

Signal transduction pathways regulate T cell activation and differentiation and have a critical influence on the outcome of adaptive immune responses. It has recently been shown that nuclear translocation events within non-adherent tumor cells can be quantified by correlating transnscription factor and nuclear images collected on the ImageStream multispectral imaging flow cytometer. Here we extend the quantitative technique to the study of nuclear localization within primary human or murine T cells in the following systems: translocation of T-bet in CD4 cells derived from murine spleen and lymph node cellstranslocation of NFAT in tetramer positive CD8 cells from human peripheral blood,translocation of NFκB in CD4+CD8+ thymocytes involved in conjugates with APC presenting cognate peptide, andlocalization of phosphorylated STAT4 in human CD4 cells following cytokine stimulation. These data demonstrate image-based nuclear localzation within primary cells with large nuclear to cytoplasmic area ratios using ImageStream technology.

Initiation of antigen-specific adative immune responses requires interaction between a T cell and an antigen presenting cell (APC) that presents a peptide/MHC complex recognized by the T cell receptor. Maintenance of strong contact between the T:APC conjugate and subsequent activation of the T cell involve recruitment towards the conjugate interface (or ‘immune synapse’) of adaptor proteins that regulate cytoskeletal reorganization and T cell gene transcription. Evaluation of molecular recruitment to the immune synapse is traditionally performed using microscopy. However, because manual image acquistion and quantitative image analysis are time consuming processes, analyses of protein recruitment to the immune synapse have remained largely qualitative. Here we describe a method for quantifying events with protein localized to the immune synapse using the ImageStream imaging flow cytometer, which automatically collects large numbers of images per data set and provides quantitative image analysis tools. Lymph node T cells from OVA-specific TCR-transgenic DO11.10 mice were incubated with OVA-pulsed splenocytes to induce T:APC conjugates. Immunofluorescently-stained samples were acquired on the ImageStream and analyzed to 1) specifically distinguish T:APC conjugates from singlets and other doublets using a combination of photometric and morphometric features, and 2) quantify the number of T:APC conjugates with specifc protein recruited to the immune synapse using am intensity-weighted distance metric.