MAGI-2 scaffold protein is critical for kidney barrier function.

MAGUK Inverted 2 (MAGI-2) is a PTEN-interacting scaffold protein implicated in cancer on the basis of rare, recurrent genomic translocations and deletions in various tumors. In the renal glomerulus, MAGI-2 is exclusively expressed in podocytes, specialized cells forming part of the glomerular filter, where it interacts with the slit diaphragm protein nephrin. To further explore MAGI-2 function, we generated Magi-2-KO mice through homologous recombination by targeting an exon common to all three alternative splice variants. Magi-2 null mice presented with progressive proteinuria as early as 2 wk postnatally, which coincided with loss of nephrin expression in the glomeruli. Magi-2-null kidneys revealed diffuse podocyte foot process effacement and focal podocyte hypertrophy by 3 wk of age, as well as progressive podocyte loss. By 5.5 wk, coinciding with a near-complete loss of podocytes, Magi-2-null mice developed diffuse glomerular extracapillary epithelial cell proliferations, and died of renal failure by 3 mo of age. As confirmed by immunohistochemical analysis, the proliferative cell populations in glomerular lesions were exclusively composed of activated parietal epithelial cells (PECs). Our results reveal that MAGI-2 is required for the integrity of the kidney filter and podocyte survival. Moreover, we demonstrate that PECs can be activated to form glomerular lesions resembling a noninflammatory glomerulopathy with extensive extracapillary proliferation, sometimes resembling crescents, following rapid and severe podocyte loss.

High plasma leptin levels confer increased risk of atherosclerosis in women with systemic lupus erythematosus, and are associated with inflammatory oxidised lipids.

BACKGROUND: Patients with systemic lupus erythematosus (SLE) are at increased risk of atherosclerosis, even after accounting for traditional risk factors. High levels of leptin and low levels of adiponectin are associated with both atherosclerosis and immunomodulatory functions in the general population. OBJECTIVE: To examine the association between these adipokines and subclinical atherosclerosis in SLE, and also with other known inflammatory biomarkers of atherosclerosis. METHODS: Carotid ultrasonography was performed in 250 women with SLE and 122 controls. Plasma leptin and adiponectin levels were measured. Lipoprotein a (Lp(a)), oxidised phospholipids on apoB100 (OxPL/apoB100), paraoxonase, apoA-1 and inflammatory high-density lipoprotein (HDL) function were also assessed. RESULTS: Leptin levels were significantly higher in patients with SLE than in controls (23.7±28.0 vs 13.3±12.9 ng/ml, p<0.001). Leptin was also higher in the 43 patients with SLE with plaque than without plaque (36.4±32.3 vs 20.9±26.4 ng/ml, p=0.002). After multivariate analysis, the only significant factors associated with plaque in SLE were leptin levels in the highest quartile (≥29.5 ng/ml) (OR=2.8, p=0.03), proinflammatory HDL (piHDL) (OR=12.8, p<0.001), age (OR=1.1, p<0.001), tobacco use (OR=7.7, p=0.03) and hypertension (OR=3.0, p=0.01). Adiponectin levels were not significantly associated with plaque in our cohort. A significant correlation between leptin and piHDL function (p<0.001), Lp(a) (p=0.01) and OxPL/apoB100 (p=0.02) was also present. CONCLUSIONS: High leptin levels greatly increase the risk of subclinical atherosclerosis in SLE, and are also associated with an increase in inflammatory biomarkers of atherosclerosis such as piHDL, Lp(a) and OxPL/apoB100. High leptin levels may help to identify patients with SLE at risk of atherosclerosis.

Cardiovascular disease in systemic lupus erythematosus.

There is a well-known increased risk for cardiovascular disease that contributes to morbidity and mortality in systemic lupus erythematosus (SLE). Major adverse cardiovascular events and subclinical atherosclerosis are both increased in this patient population. While traditional cardiac risk factors do contribute to the increased risk that is seen, lupus disease-related factors, medications, and genetic factors also impact the overall risk. SLE-specific inflammation, including oxidized lipids, cytokines, and altered immune cell subtypes all are likely to play a role in the pathogenesis of atherosclerotic plaques. Research is ongoing to identify biomarkers that can help clinicians to predict which SLE patients are at the greatest risk for cardiovascular disease (CVD). While SLE-specific treatment regimens for the prevention of cardiovascular events have not been identified, current strategies include minimization of traditional cardiac risk factors and lowering of overall lupus disease activity.

A Panel of Biomarkers Associates With Increased Risk for Cardiovascular Events in Women With Systemic Lupus Erythematosus.

OBJECTIVE: The increase in cardiovascular events (CVEs) in systemic lupus erythematosus (SLE) is not fully explained by traditional risk factors. We previously identified four biomarkers (proinflammatory high-density lipoprotein, leptin, soluble TNF-like weak inducer of apoptosis (sTWEAK), and homocysteine) that we combined with age and diabetes to create the predictors of risk for elevated flares, damage progression, and increased cardiovascular diseasein patients with SLE (PREDICTS) risk profile. PREDICTS more accurately identified patients with SLE at risk for progression of subclinical atherosclerosis than any individual variable. We examined whether PREDICTS can also identify patients with SLE at risk for future CVEs. METHODS: A total of 342 patients with SLE and 155 matched control subjects participated in this longitudinal prospective study. A high PREDICTS score was defined as three or more predictors or diabetes + one or more predictor. The biomarkers were measured at baseline using published methods. All major adverse CVEs (MACEs) were confirmed by medical record review. RESULTS: During 116 months of follow-up, 5% of patients with SLE died, 12% had a cerebrovascular event, and 5% had a cardiac event. Overall, 20% of patients with lupus experienced any new MACE compared with 5% of control subjects (P < 0.0001). More patients with SLE with a new MACE had high PREDICTS score at baseline (77%) versus patients with no new events (34%) (P < 0.0001). High baseline PREDICTS score also associated with cerebrovascular (P < 0.0001) and cardiac events (P < 0.0001) in SLE. Using Cox regression, a baseline high PREDICTS score associated with a 3.7-fold increased hazard ratio (HR) for a new MACE (P < 0.0001) in SLE. Hypertension (HR = 2.1; P = 0.006) was also a risk. CONCLUSION: A high PREDICTS score and hypertension confer increased risk for new MACEs in patients with SLE.

Dysfunctional, pro-inflammatory HDL directly upregulates monocyte PDGFRß, chemotaxis and TNFα production.

Accelerated atherosclerosis is a major co-morbid condition in autoimmune diseases. Monocytes are the main immune cell involved in atherosclerosis initiation. We hypothesized that dysfunctional, pro-inflammatory HDL (piHDL), which occurs in approximately half of SLE patients, might directly influence monocyte gene expression and function. SLE subjects were stratified into three groups: 1) carotid artery plaque+piHDL+,2) plaque-piHDL+,and 3) plaque-piHDL- (n=18/group). PDGFRß was upregulated in primary monocytes from plaque+piHDL+patients and in THP-1 cells acutely treated in vitro with piHDL compared to normal HDL. THP-1 chemotaxis was enhanced after treatment with piHDL versus normal HDL. Abnormal migration was restored to normal levels by treatment with imatinib or an apoJ mimetic peptide. Increased piHDL-mediated TNFα protein levels were reduced with both inhibitors. Dysfunctional piHDL directly influences expression of a small number of transcripts and proteins, and piHDL inhibition through reducing piHDL oxidation or blocking PDGFRß kinase activity restored normal monocyte chemotaxis.

Sequential ABL kinase inhibitor therapy selects for compound drug-resistant BCR-ABL mutations with altered oncogenic potency.

Molecularly targeted kinase inhibitor cancer therapies are currently administered sequentially rather than simultaneously. We addressed the potential long-term impact of this strategy in patients with chronic myelogenous leukemia (CML), which is driven by the fusion oncogene BCR-ABL. Analysis of BCR-ABL genotypes in CML patients who relapsed after sequential treatment with the ABL inhibitors imatinib and dasatinib revealed evolving resistant BCR-ABL kinase domain mutations in all cases. Twelve patients relapsed with the pan-resistant T315I mutation, whereas 6 patients developed novel BCR-ABL mutations predicted to retain sensitivity to imatinib based on in vitro studies. Three of these patients were retreated with imatinib (or the chemically related compound nilotinib) and responded; however, selection for compound mutants (2 or 3 BCR-ABL mutations in the same molecule) can substantially limit the potential effectiveness of retreating patients with inhibitors that have previously failed. Furthermore, drug-resistant mutations, when compounded, can increase oncogenic potency relative to the component mutants in transformation assays. The Aurora kinase inhibitor VX-680, currently under clinical evaluation based on its activity against the T315I mutation, is also effective against the other commonly detected dasatinib-resistant mutation in our analysis, V299L. Our findings demonstrate the potential hazards of sequential kinase inhibitor therapy and suggest a role for a combination of ABL kinase inhibitors, perhaps including VX-680, to prevent the outgrowth of cells harboring drug-resistant BCR-ABL mutations.

IkappaB kinase beta inhibition induces cell death in Imatinib-resistant and T315I Dasatinib-resistant BCR-ABL+ cells.

Chronic myelogenous leukemia is a malignant disease of the hematopoietic stem cell compartment, which is characterized by expression of the BCR-ABL fusion protein. Expression of BCR-ABL allows myeloid cells to grow in the absence of the growth factors interleukin-3 and granulocyte-macrophage colony-stimulating factor. The tyrosine kinase activity of BCR-ABL constitutively activates signaling pathways associated with Ras and its downstream effectors and with the Jak/STAT pathway. Additionally, we reported previously that BCR-ABL activates the transcription factor nuclear factor-kappaB (NF-kappaB) in a manner dependent on Ras and that inhibition of NF-kappaB by expression of a modified form of IkappaBalpha blocked BCR-ABL-driven tumor growth in a xenograft model. Here, we show that a highly specific inhibitor of IkappaB kinase beta, a key upstream regulator of the NF-kappaB pathway, induces growth suppression and death in cells expressing wild-type, Imatinib-resistant, or the T315I Imatinib/Dasatinib-resistant forms of BCR-ABL. Cell cycle variables were not affected by this compound. These data indicate that blockage of BCR-ABL-induced NF-kappaB activation via IkappaB kinase beta inhibition represents a potential new approach for treatment of Imatinib- or Dasatinib-resistant forms of chronic myelogenous leukemia.

Induction of immune tolerance by activation of CD8+ T suppressor/regulatory cells in lupus-prone mice.

Multiple CD8(+) suppressive T cell (Ts) subtypes are now recognized as essential regulators of the immune system that prevent autoimmunity through secretion of multiple cytokines and the subsequent inhibition of effector lymphocyte function. CD8(+) Ts are an exciting area of study because of the possible therapeutic implications of inducing suppressive cells that are able to subdue or anergize autoimmune manifestations. Current research in systemic lupus erythematosus (SLE), a disease in which most effective therapies are widely immunosuppressive, is often focused on novel and highly targeted ways in which to treat this multiorgan disease. CD8(+) Ts have been impaired in human and murine SLE. Our group and others have utilized tolerogenic peptides to induce and study CD8(+) Ts to understand their function, as well as investigate a possible new SLE therapy. This review will discuss the similarities and differences in CD8(+) Ts subsets, the concept of tolerance as a therapy, and the current understanding of CD8(+) Ts in mouse SLE models.

The direct recruitment of BLNK to immunoglobulin alpha couples the B-cell antigen receptor to distal signaling pathways.

Following B-cell antigen receptor (BCR) ligation, the cytoplasmic domains of immunoglobulin alpha (Ig alpha) and Ig beta recruit Syk to initiate signaling cascades. The coupling of Syk to several distal substrates requires linker protein BLNK. However, the mechanism by which BLNK is recruited to the BCR is unknown. Using chimeric receptors with wild-type and mutant Ig alpha cytoplasmic tails we show that the non-immunoreceptor tyrosine-based activation motif (ITAM) tyrosines, Y176 and Y204, are required to activate BLNK-dependent pathways. Subsequent analysis demonstrated that BLNK bound directly to phospho-Y204 and that fusing BLNK to mutated Ig alpha reconstituted downstream signaling events. Moreover, ligation of the endogenous BCR induced Y204 phosphorylation and BLNK recruitment. These data demonstrate that the non-ITAM tyrosines of Ig alpha couple Syk activation to BLNK-dependent pathways.

Laquinimod delays and suppresses nephritis in lupus-prone mice and affects both myeloid and lymphoid immune cells.

OBJECTIVE: Lupus nephritis depends on autoantibody deposition and activation of multiple immune cell types that promote kidney inflammation, including lymphocytes and monocyte/macrophages. Laquinimod, currently in clinical trials for multiple sclerosis and lupus nephritis, reduces infiltration of inflammatory cells into the spinal cord in experimental autoimmune encephalomyelitis. Activated monocyte/macrophages infiltrate the kidneys during nephritis in systemic lupus erythematosus (SLE). We undertook this study to determine whether using laquinimod to reduce monocyte/macrophage-driven tissue damage as well as to alter lymphocytes in SLE nephritis could have greater therapeutic benefit than current treatments that primarily affect lymphocytes, such as mycophenolate mofetil (MMF). METHODS: To test laquinimod efficacy, we used the (NZB × NZW)F1 mouse model of SLE, in which disease manifests as nephritis. Preventive and therapeutic studies were performed to determine whether laquinimod could prevent or delay nephritis, as measured by proteinuria, serum creatinine, survival, and renal pathology. Spleen and kidney leukocyte populations and suppression assays were analyzed by flow cytometry. RESULTS: Laquinimod prevented or delayed lupus manifestations at levels equal to or better than MMF. Laquinimod treatment was associated with reduced numbers of monocyte/macrophages, dendritic cells, and lymphocytes, as well as with induction of myeloid-derived suppressor cells in spleens and kidneys. Laquinimod suppressed macrophage-secreted tumor necrosis factor α and induced production of interleukin-10 (IL-10). In addition, laquinimod suppressed interferon-γ and IL-17 production by lymphocytes and down-regulated expression of activation/costimulatory markers on antigen-presenting cells. CONCLUSION: The effects of laquinimod on myeloid and lymphoid cells may contribute to improvements in (NZB × NZW)F1 mouse survival, proteinuria, and glomerulonephritis. Future development of laquinimod as a therapeutic agent for lupus nephritis is promising.

A panel of biomarkers is associated with increased risk of the presence and progression of atherosclerosis in women with systemic lupus erythematosus.

OBJECTIVE: An increased frequency of atherosclerosis (ATH) in systemic lupus erythematosus (SLE) is well-documented but not fully explained by the presence of traditional cardiac risk factors. Several nontraditional biomarkers, including proinflammatory high-density lipoprotein (piHDL) and leptin, have been individually associated with subclinical ATH in SLE. The aim of this study was to examine whether these and other biomarkers can be combined into a risk profile, the Predictors of Risk for Elevated Flares, Damage Progression, and Increased Cardiovascular Disease in Patients with SLE (PREDICTS), that could be used to better predict future progression of ATH. METHODS: In total, 210 patients with SLE and 100 age-matched healthy control subjects (all women) participated in this prospective cohort study. The longitudinal presence of carotid plaque and intima-media thickness (IMT) were measured at baseline and followup (mean ± SD 29.6 ± 9.7 months). RESULTS: At followup, carotid plaque was present in 29% of SLE patients. Factors significantly associated with plaque, determined using Salford Predictive Modeling and multivariate analysis, included age ≥48 years (odds ratio [OR] 4.1, P = 0.002), high piHDL function (OR 9.1, P < 0.001), leptin levels ≥34 ng/dl (OR 7.3, P = 0.001), plasma soluble TWEAK levels ≥373 pg/ml (OR 28.8, P = 0.004), and history of diabetes (OR 61.8, P < 0.001). Homocysteine levels ≥12 µmoles/liter were also a predictor. However, no single variable demonstrated an ideal combination of good negative predictive values (NPVs), positive predictive values (PPVs), sensitivity, and specificity. A high-risk PREDICTS profile was defined as ≥3 positive biomarkers or ≥1 positive biomarker plus a history of diabetes; for high-risk SLE patients, the PPV was 64%, NPV was 94%, sensitivity was 89%, and specificity was 79%. In multivariate analysis, SLE patients with the high-risk profile had 28-fold increased odds for the longitudinal presence of plaque (P < 0.001) and increased progression of IMT (P < 0.001). CONCLUSION: A high-risk PREDICTS score confers 28-fold increased odds of the presence of any current, progressive, or acquired carotid plaque, both in patients with SLE and in control subjects, and is significantly associated with higher rates of IMT progression.

Accelerated atherosclerosis in patients with SLE--mechanisms and management.

Rapid-onset cardiovascular disease (CVD) is a major concern for many patients with systemic lupus erythematosus (SLE). Cardiovascular events occur more frequently and with earlier onset in patients with SLE compared with healthy individuals. Traditional risk factors, such as altered lipid levels, aging and smoking, do not fully explain this increased risk of CVD, strongly suggesting that autoimmunity contributes to accelerated atherosclerosis. Altered immune system function is recognized as the primary contributor to both the initiation and progression of atherosclerosis. Multiple manifestations of autoimmunity, including changes in cytokine levels and innate immune responses, autoantibodies, adipokines, dysfunctional lipids, and oxidative stress, could heighten atherosclerotic risk. In addition, multiple SLE therapeutics seem to affect the development and progression of atherosclerosis both positively and negatively. SLE-specific cardiovascular risk factors are beginning to be discovered by several groups, and development of a comprehensive, clinically feasible biomarker panel could be invaluable for identification and treatment of patients at risk of developing accelerated atherosclerosis. Here, we discuss the epidemiology of CVD in SLE and the implications of immune system dysfunction on the development and progression, monitoring and treatment of atherosclerosis in individuals with this disease.

Systemic lupus erythematosus and cardiovascular disease: prediction and potential for therapeutic intervention.

Patients with systemic lupus erythematosus have a significantly increased risk of cardiovascular events due to atherosclerosis. Traditional cardiac risk factors cannot fully explain this increased risk. Recent evidence strongly suggests that atherosclerotic plaque is largely driven by inflammation and an active immunological response, in contrast to the long-held belief that plaque is a passive accumulation of lipids in the arterial wall. Current approaches to the prevention of atherosclerosis in systemic lupus erythematosus involve targeting modifiable cardiac risk factors. Future preventive strategies may include therapies that counteract the immunologic responses that lead to plaque formation.

CD8+ Tregs in lupus, autoimmunity, and beyond.

While CD4(+)CD25(high) regulatory T cells (Tregs) have garnered much attention for their role in the maintenance of immune homeostasis, recent findings have shown that subsets of CD8(+) T cells (CD8(+) Tregs) display immunoregulatory functions as well. Both CD4(+) Tregs and CD8(+) Tregs appear impaired in number and/or function in several autoimmune diseases and in experimental animal models of autoimmunity, suggesting the possibility of immunotherapeutic targeting of these cells for improved management of autoimmune conditions. Our group has developed a strategy to induce CD8(+) Tregs in autoimmune mice through the use of a tolerogenic self-peptide, and new information has been gained on the phenotype, function and role of induced CD8(+) Tregs in autoimmunity. Here we present an overview of the role and mechanisms of action of CD8(+) Tregs in autoimmunity, with a special focus on lupus. We also discuss the potential role of CD8(+) Tregs in other diseases, including chronic infection and cancer.

Low physical activity is associated with proinflammatory high-density lipoprotein and increased subclinical atherosclerosis in women with systemic lupus erythematosus.

OBJECTIVE: To investigate the association between physical activity, functional activity of high-density lipoprotein (HDL), and subclinical cardiovascular disease in patients with systemic lupus erythematosus (SLE). METHODS: A total of 242 SLE patients (all women) participated in this cross-sectional study from February 2004 to February 2008. Carotid plaque and intima-media thickness (IMT), antioxidant function of HDL, and traditional cardiac risk factors were measured. Physical activity was assessed from self-reports by calculating the metabolic equivalents (METS) per week and by the physical function domain of the Medical Outcomes Study Short Form 36 (SF-36). Data were analyzed using bivariate and multivariate regression analyses. RESULTS: Number of METS per week spent performing strenuous exercise was negatively correlated with IMT (r = -0.4, P = 0.002) and number of plaques (r = -0.30, P = 0.0001). Physical function as assessed by the SF-36 was also negatively correlated with IMT (r = -0.14, P = 0.03) and number of plaques (r = -0.14, P = 0.04). In multivariate analyses, number of strenuous exercise METS was significantly associated with IMT (t = -2.2, P = 0.028) and number of plaques (t = -2.5, P = 0.014) when controlling for markers of SLE disease activity and damage, but not after controlling for traditional cardiac risk factors. Low physical activity, defined as <225 total METS per week, was associated with the presence of proinflammatory HDL (P = 0.03). CONCLUSION: Low physical activity is associated with increased subclinical atherosclerosis and proinflammatory HDL in patients with SLE. Increased strenuous exercise may reduce the risk of atherosclerosis in SLE.

Altered lipoprotein metabolism in chronic inflammatory states: proinflammatory high-density lipoprotein and accelerated atherosclerosis in systemic lupus erythematosus and rheumatoid arthritis.

In this review, the authors discuss the formation and structure of high-density lipoproteins (HDLs) and how those particles are altered in inflammatory or stress states to lose their capacity for reverse cholesterol transport and for antioxidant activity. In addition, abnormal HDLs can become proinflammatory (piHDLs) and actually contribute to oxidative damage. The assay by which piHDLs are identified involves studying the ability of test HDLs to prevent oxidation of low-density lipoproteins. Finally, the authors discuss the potential role of piHDLs (found in some 45% of patients with systemic lupus erythematosus and 20% of patients with rheumatoid arthritis) in the accelerated atherosclerosis associated with some chronic rheumatic diseases.

Phosphorylation of the ATP-binding loop directs oncogenicity of drug-resistant BCR-ABL mutants.

The success of targeting kinases in cancer with small molecule inhibitors has been tempered by the emergence of drug-resistant kinase domain mutations. In patients with chronic myeloid leukemia treated with ABL inhibitors, BCR-ABL kinase domain mutations are the principal mechanism of relapse. Certain mutations are occasionally detected before treatment, suggesting increased fitness relative to wild-type p210 BCR-ABL. We evaluated the oncogenicity of eight kinase inhibitor-resistant BCR-ABL mutants and found a spectrum of potencies greater or less than p210. Although most fitness alterations correlate with changes in kinase activity, this is not the case with the T315I BCR-ABL mutation that confers clinical resistance to all currently approved ABL kinase inhibitors. Through global phosphoproteome analysis, we identified a unique phosphosubstrate signature associated with each drug-resistant allele, including a shift in phosphorylation of two tyrosines (Tyr253 and Tyr257) in the ATP binding loop (P-loop) of BCR-ABL when Thr315 is Ile or Ala. Mutational analysis of these tyrosines in the context of Thr315 mutations demonstrates that the identity of the gatekeeper residue impacts oncogenicity by altered P-loop phosphorylation. Therefore, mutations that confer clinical resistance to kinase inhibitors can substantially alter kinase function and confer novel biological properties that may impact disease progression.

Comparative analysis of two clinically active BCR-ABL kinase inhibitors reveals the role of conformation-specific binding in resistance.

Structural studies suggest that most point mutations in the BCR-ABL kinase domain cause resistance to the ABL kinase inhibitor imatinib by impairing the flexibility of the kinase domain, restricting its ability to adopt the inactive conformation required for optimal imatinib binding, rather than by directly interfering with drug contact residues. BMS-354825, currently in clinical development for imatinib-resistant chronic myelogenous leukemia, is a dual SRC/ABL kinase inhibitor that binds ABL in both the active and inactive conformation. To examine the potential role of conformational binding properties in drug resistance, we mapped the mutations in BCR-ABL capable of conferring resistance to BMS-354825. Through saturation mutagenesis, we identified 10 such BCR-ABL mutations, 8 of which occurred at drug contact residues. Some mutants were unique to BMS-354825, whereas others also conferred imatinib resistance. Remarkably, the identity of the amino acid substitution at either of two contact residues differentially affects sensitivity to imatinib or BMS-354825. The combination of imatinib plus BMS-354825 greatly reduced the recovery of drug-resistant clones. Our findings provide further rationale for considering kinase conformation in the design of kinase inhibitors against cancer targets.

Receptor-facilitated antigen presentation requires the recruitment of B cell linker protein to Igalpha.

Ags that cross-link the B cell Ag receptor are preferentially and rapidly delivered to the MHC class II-enriched compartment for processing into peptides and subsequent loading onto MHC class II. Proper sorting of Ag/receptor complexes requires the recruitment of Syk to the phosphorylated immunoreceptor tyrosine-based activation motif tyrosines of the B cell Ag receptor constituent Igalpha. We postulated that the Igalpha nonimmunoreceptor tyrosine-based activation motif tyrosines, Y(176) and Y(204), contributed to receptor trafficking. Igalpha(YDeltaF(176,204))/Igbeta receptors were targeted to late endosomes, but were excluded from the vesicle lumen and could not facilitate the presentation of Ag to T cells. Subsequent analysis demonstrated that phosphorylation of Y(176)/Y(204) recruited the B cell linker protein, Vav, and Grb2. Reconstitution of Igalpha(YDeltaF(176,204))/Igbeta with the B cell linker protein rescued both receptor-facilitated Ag presentation and entry into the MHC class II-enriched compartment. Thus, aggregation accelerates receptor trafficking by recruiting two separate signaling modules required for transit through sequential checkpoints.