Notch signaling drives intestinal graft-versus-host disease in mice and nonhuman primates.

Notch signaling promotes T cell pathogenicity and graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) in mice, with a dominant role for the Delta-like Notch ligand DLL4. To assess whether Notch's effects are evolutionarily conserved and to identify the mechanisms of Notch signaling inhibition, we studied antibody-mediated DLL4 blockade in a nonhuman primate (NHP) model similar to human allo-HCT. Short-term DLL4 blockade improved posttransplant survival with durable protection from gastrointestinal GVHD in particular. Unlike prior immunosuppressive strategies tested in the NHP GVHD model, anti-DLL4 interfered with a T cell transcriptional program associated with intestinal infiltration. In cross-species investigations, Notch inhibition decreased surface abundance of the gut-homing integrin α4ß7 in conventional T cells while preserving α4ß7 in regulatory T cells, with findings suggesting increased ß1 competition for α4 binding in conventional T cells. Secondary lymphoid organ fibroblastic reticular cells emerged as the critical cellular source of Delta-like Notch ligands for Notch-mediated up-regulation of α4ß7 integrin in T cells after allo-HCT. Together, DLL4-Notch blockade decreased effector T cell infiltration into the gut, with increased regulatory to conventional T cell ratios early after allo-HCT. Our results identify a conserved, biologically unique, and targetable role of DLL4-Notch signaling in intestinal GVHD.

Irreversible electroporation augments checkpoint immunotherapy in prostate cancer and promotes tumor antigen-specific tissue-resident memory CD8+ T cells.

Memory CD8+ T cells populate non-lymphoid tissues (NLTs) following pathogen infection, but little is known about the establishment of endogenous tumor-specific tissue-resident memory T cells (TRM) during cancer immunotherapy. Using a transplantable mouse model of prostate carcinoma, here we report that tumor challenge leads to expansion of naïve neoantigen-specific CD8+ T cells and formation of a small population of non-recirculating TRM in several NLTs. Primary tumor destruction by irreversible electroporation (IRE), followed by anti-CTLA-4 immune checkpoint inhibitor (ICI), promotes robust expansion of tumor-specific CD8+ T cells in blood, tumor, and NLTs. Parabiosis studies confirm that TRM establishment following dual therapy is associated with tumor remission in a subset of cases and protection from subsequent tumor challenge. Addition of anti-PD-1 following dual IRE + anti-CTLA-4 treatment blocks tumor growth in non-responsive cases. This work indicates that focal tumor destruction using IRE combined with ICI is a potent in situ tumor vaccination strategy that generates protective tumor-specific TRM.

Evolutionarily conserved resistance to phagocytosis observed in melanoma cells is insensitive to upregulation of pro-phagocytic signals and to CD47 blockade.

Therapeutic activation of macrophage phagocytosis has the ability to restrain tumour growth through phagocytic clearance of tumour cells and activation of the adaptive immune response. Our objective for this study was to evaluate the effects of modulating pro- and anti-phagocytic pathways in malignant melanoma. In order to identify evolutionarily conserved mechanisms of resistance that may be important for melanoma cell survival, we utilized a multi-species approach and examined the phagocytosis of human, mouse, and dog melanoma cells. We observed that melanoma cells from all three species displayed unexpected resistance to phagocytosis that could not be fully mitigated by blockade of the 'don't eat me' signal CD47 or by chemotherapeutic enhancement of known 'eat me' signals. Additionally, CD47 blockade failed to promote anti-melanoma immune responses or tumour regression in vivo. This melanoma resistance to phagocytosis was not mediated by soluble factors, and it was unaffected by siRNA-mediated knockdown of 47 prospective 'don't eat me' signals or by CRISPR-Cas-mediated CD47 knockout. Unexpectedly, CD47 knockout also did not enhance phagocytosis of lymphoma cells, but it eliminated the pro-phagocytic effect of CD47 blockade, suggesting that the pro-phagocytic effects of CD47 blockade are due in part to Fc receptor engagement. From this study, we conclude that melanoma cells possess an evolutionarily conserved resistance to macrophage phagocytosis. Further investigation will be needed to overcome the mechanisms that mediate melanoma cell resistance to innate immunity.

Engineering T cell response to cancer antigens by choice of focal therapeutic conditions.

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.

Role of afferent and efferent renal nerves in the development of AngII-salt hypertension in rats.

Hypertension is the leading modifiable risk factor for death worldwide, yet the causes remain unclear and treatment remains suboptimal. Catheter-based renal denervation (RDNX) is a promising new treatment for resistant hypertension, but the mechanisms underlying its antihypertensive effect remain unclear. We recently found that RDNX attenuates deoxycorticosterone acetate-salt hypertension and that this is dependent on ablation of afferent renal nerves and is associated with decreased renal inflammation. To determine if this is common to other models of salt-sensitive hypertension, rats underwent complete RDNX (n = 8), selective ablation of afferent renal nerves (n = 8), or sham denervation (n = 8). Mean arterial pressure (MAP) and heart rate were measure by telemetry and rats were housed in metabolic cages for measurement of sodium and water balance. Rats were then subjected to angiotensin II (AngII)-salt hypertension (10 ng/kg/min, intravenous + 4% NaCl diet) for 2 weeks. At the end of the study, renal T-cell infiltration was quantified by flow cytometry. AngII resulted in an increase in MAP of ~50 mmHg in all three groups with no between group differences, and a transient bradycardia that was blunted by selective ablation of afferent renal nerves. Sodium and water balance were unaffected by AngII-salt treatment and similar between groups. Lastly, AngII infusion was not associated with T-cell infiltration into the kidneys, and T-cell counts were unaffected by the denervation procedures. These results suggest that AngII-salt hypertension in the rat is not associated with renal inflammation and that neither afferent nor efferent renal nerves contribute to this model.

Cytokine-mediated activation of human ex vivo-expanded Vγ9Vδ2 T cells.

Vγ9Vδ2 T cells, the major subset of the human peripheral blood γδ T-cell, respond to microbial infection and stressed cells through the recognition of phosphoantigens. In contrast to the growing knowledge of antigen-mediated activation mechanisms, the antigen-independent and cytokine-mediated activation mechanisms of Vγ9Vδ2 T cells are poorly understood. Here, we show that interleukin (IL) -12 and IL-18 synergize to activate human ex vivo-expanded Vγ9Vδ2 T cells. Vγ9Vδ2 T cells treated with IL-12 and IL-18 enhanced effector functions, including the expression of IFN-γ and granzyme B, and cytotoxicity. These enhanced effector responses following IL-12 and IL-18 treatment were associated with homotypic aggregation, enhanced expression of ICAM-1 and decreased expression of the B- and T-lymphocyte attenuator (BTLA), a co-inhibitory receptor. IL-12 and IL-18 also induced the antigen-independent proliferation of Vγ9Vδ2 T cells. Increased expression of IκBζ, IL-12Rß2 and IL-18Rα following IL-12 and IL-18 stimulation resulted in sustained activation of STAT4 and NF-κB. The enhanced production of IFN-γ and cytotoxic activity are critical for cancer immunotherapy using Vγ9Vδ2 T cells. Thus, the combined treatment of ex vivo-expanded Vγ9Vδ2 T cells with IL-12 and IL-18 may serve as a new strategy for the therapeutic activation of these cells.

Resting Afferent Renal Nerve Discharge and Renal Inflammation: Elucidating the Role of Afferent and Efferent Renal Nerves in Deoxycorticosterone Acetate Salt Hypertension.

Renal sympathetic denervation (RDNx) has emerged as a novel therapy for hypertension; however, the therapeutic mechanisms remain unclear. Efferent renal sympathetic nerve activity has recently been implicated in trafficking renal inflammatory immune cells and inflammatory chemokine and cytokine release. Several of these inflammatory mediators are known to activate or sensitize afferent nerves. This study aimed to elucidate the roles of efferent and afferent renal nerves in renal inflammation and hypertension in the deoxycorticosterone acetate (DOCA) salt rat model. Uninephrectomized male Sprague-Dawley rats (275-300 g) underwent afferent-selective RDNx (n=10), total RDNx (n=10), or Sham (n=10) and were instrumented for the measurement of mean arterial pressure and heart rate by radiotelemetry. Rats received 100-mg DOCA (SC) and 0.9% saline for 21 days. Resting afferent renal nerve activity in DOCA and vehicle animals was measured after the treatment protocol. Renal tissue inflammation was assessed by renal cytokine content and T-cell infiltration and activation. Resting afferent renal nerve activity, expressed as a percent of peak afferent nerve activity, was substantially increased in DOCA than in vehicle (35.8±4.4 versus 15.3±2.8 %Amax). The DOCA-Sham hypertension (132±12 mm Hg) was attenuated by ≈50% in both total RDNx (111±8 mm Hg) and afferent-selective RDNx (117±5 mm Hg) groups. Renal inflammation induced by DOCA salt was attenuated by total RDNx and unaffected by afferent-selective RDNx. These data suggest that afferent renal nerve activity may mediate the hypertensive response to DOCA salt, but inflammation may be mediated primarily by efferent renal sympathetic nerve activity. Also, resting afferent renal nerve activity is elevated in DOCA salt rats, which may highlight a crucial neural mechanism in the development and maintenance of hypertension.

Renal Denervation Normalizes Arterial Pressure With No Effect on Glucose Metabolism or Renal Inflammation in Obese Hypertensive Mice.

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.

Adhesion- and Degranulation-Promoting Adapter Protein Promotes CD8 T Cell Differentiation and Resident Memory Formation and Function during an Acute Infection.

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-ß 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.

CD28/B7 Deficiency Attenuates Systolic Overload-Induced Congestive Heart Failure, Myocardial and Pulmonary Inflammation, and Activated T Cell Accumulation in the Heart and Lungs.

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-1ß) 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.

Negative Regulation of Memory Phenotype CD8 T Cell Conversion by Adhesion and Degranulation-Promoting Adapter Protein.

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.

ICAM-1-dependent homotypic aggregates regulate CD8 T cell effector function and differentiation during T cell activation.

A hallmark of T cell activation in vitro and in vivo is the clustering of T cells with each other via interaction of the LFA-1 integrin with ICAM-1. The functional significance of these homotypic aggregates in regulating T cell function remains unknown. We used an APC-free in vitro activation system to demonstrate that stimulation of purified naive CD8 T cells results in enhanced expression of ICAM-1 on T cells that is sustained by the inflammatory cytokine IL-12 and associated with robust T cell aggregates. ICAM-1-deficient CD8 T cells proliferate normally but demonstrate a striking failure to aggregate. Interestingly, loss of ICAM-1 expression results in elevated levels of IFN-γ and granzyme B, as well as enhanced cytotoxicity. Similar results were obtained when anti-LFA-1 Ab was used to block the clustering of wild-type T cells. ICAM-1 ligation is not required for IFN-γ regulation, as clustering of ICAM-1-deficient CD8 T cells with wild-type T cells reduces IFN-γ expression. Analysis using a fluorescent reporter that monitors TCR signal strength indicates that T cell clustering limits T cell exposure to Ag during activation. Furthermore, T cell clustering promotes the upregulation of the CTLA-4 inhibitory receptor and the downregulation of eomesodermin, which controls effector molecule expression. Activation of ICAM-1-deficient CD8 T cells in vivo results in an enhanced percentage of KLRG-1(+) T cells indicative of short-lived effectors. These results suggest that T cell clustering represents a mechanism that allows continued proliferation but regulates T cell effector function and differentiation.

Multistage T cell-dendritic cell interactions control optimal CD4 T cell activation through the ADAP-SKAP55-signaling module.

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.

ADAP regulates cell cycle progression of T cells via control of cyclin E and Cdk2 expression through two distinct CARMA1-dependent signaling pathways.

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 ß-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.

ß-Actin specifically controls cell growth, migration, and the G-actin pool.

Ubiquitously expressed ß-actin and γ-actin isoforms play critical roles in most cellular processes; however, their unique contributions are not well understood. We generated whole-body ß-actin-knockout (Actb(-/-)) mice and demonstrated that ß-actin is required for early embryonic development. Lethality of Actb(-/-) embryos correlated with severe growth impairment and migration defects in ß-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 ß-actin in highly motile T cells. Of great interest, ablation of ß-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 ß-actin in regulating cell migration and gene expression through control of the cellular G-actin pool.

The pleckstrin homology domain in the SKAP55 adapter protein defines the ability of the adapter protein ADAP to regulate integrin function and NF-kappaB activation.

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.

ß1 integrin is critical for the maintenance of antigen-specific CD4 T cells in the bone marrow but not long-term immunological memory.

The long-term maintenance of memory CD4 T cells promotes protective immunity against future pathogen reinfection. As a site rich in survival cytokines, the bone marrow is proposed to be a critical niche for the survival of memory CD4 T cells. We demonstrate that endogenous, polyclonal Ag-specific CD4 T cells rapidly enter and are recovered long-term from the bone marrow following i.v. infection with Listeria monocytogenes. ß(1) integrin-deficient CD4 T cells also populate the bone marrow early following an infection, but their numbers in this site rapidly decline. This decline was not caused by increased death of T cells lacking ß(1) integrin but rather by reduced retention in the bone marrow after the primary immune response. The loss of memory CD4 T cells from the bone marrow does not lead to a loss of the predominant source of memory CD4 T cells in the spleen or the ability to mount a memory response. Thus, ß(1) integrin-dependent maintenance of memory CD4 T cells in the bone marrow is not required for long-term CD4 T cell memory.

Control of alpha4beta7 integrin expression and CD4 T cell homing by the beta1 integrin subunit.

The alpha4beta7 integrin promotes homing of T cells to intestinal sites. The alpha4 integrin subunit that pairs with beta7 integrin can also pair with beta1 integrin. In this paper, we show that the preferential pairing of beta1 integrin with alpha4 integrin regulates the expression of alpha4beta7 on T cells. In the absence of beta1 integrin, naive mouse CD4 T cells have increased alpha4beta7 expression, resulting in increased adhesion to mucosal addressin cell adhesion molecule-1 and enhanced homing to Peyer's patches (PP). In a reciprocal manner, overexpression of beta1 integrin causes the loss of alpha4beta7 expression and decreased homing to PP. A similar upregulation of beta1 integrin and suppression of alpha4beta7 expression occurs rapidly after CD4 T cell activation. beta1 integrin thus dominates beta7 integrin for alpha4 integrin pairing, thereby controlling the abundance of unpaired alpha4 integrin. Increasing the abundance of alpha4 integrin relative to beta1 integrin is critical to retinoic acid-mediated expression of alpha4beta7 integrin during T cell activation. In the absence of beta1 integrin, endogenous Ag-specific CD4 T cells uniformly express high levels of alpha4beta7 after Listeria monocytogenes infection. The resulting beta1-deficient early memory T cells have decreased localization to the bone marrow and enhanced localization to PP after infection. Thus, the preferential association of beta1 integrin with alpha4 integrin suppresses alpha4beta7 integrin expression and regulates the localization of memory CD4 T cells.

NF-kappaB activation in T cells requires discrete control of IkappaB kinase alpha/beta (IKKalpha/beta) phosphorylation and IKKgamma ubiquitination by the ADAP adapter protein.

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.

Integrin function in T-cell homing to lymphoid and nonlymphoid sites: getting there and staying there.

The continuous recirculation of naive T cells and their subsequent migration to tissue following activation is crucial for maintaining protective immunity against invading pathogens. The preferential targeting of effector and memory T cells to tissue is instructed during priming and mediated by cell surface expressed adhesion receptors such as integrins. Integrins arc involved in nearly all aspects of T-cell life, including naive T-cell circulation, activation, and finally effector T-cell trafficking and localization. Recent research has revealed that microenvironmental factors present during T-cell priming result in the specific regulation of adhesion/integrin and chemokine receptor expression. Once antigen-experienced T cells enter tissue, further changes in integrin expression may occur that arc critical for T-cell localization, retention, effector function, and survival. This review discusses the function of integrin expression on T cells and the multiple roles integrins play on naive T cells and in directing effector T-cell trafficking to nonlymphoid sites in order to maintain protective adaptive immunity at body barriers.