ABSTRACT
Shp1, encoded by the gene Ptpn6, is a protein tyrosine phosphatase that transduces inhibitory signals downstream of immunoreceptors in many immune cell types. Blocking Shp1 activity represents an exciting potential immunotherapeutic strategy for the treatment of cancer, as Shp1 inhibition would be predicted to unleash both innate and adaptive immunity against tumor cells. Antibodies blocking the interaction between CD47 on tumor cells and SIRPα on macrophages enhance macrophage phagocytosis, show efficacy in preclinical tumor models, and are being evaluated in the clinic. Here we found that Shp1 bound to phosphorylated peptide sequences derived from SIRPα and transduced the anti-phagocytic signal, as Shp1 loss in mouse bone marrow-derived macrophages increased phagocytosis of tumor cells in vitro. We also generated a novel mouse model to evaluate the impact of global, inducible Ptpn6 deletion on anti-tumor immunity. We found that inducible Shp1 loss drove an inflammatory disease in mice that was phenotypically similar to that seen when Ptpn6 is knocked out from birth. This indicates that acute perturbation of Shp1 in vivo could drive hyperactivation of immune cells, which could be therapeutically beneficial, though at the risk of potential toxicity. In this model, we found that Shp1 loss led to robust anti-tumor immunity against two immune-rich syngeneic tumor models that are moderately inflamed though not responsive to checkpoint inhibitors, MC38 and E0771. Shp1 loss did not promote anti-tumor activity in the non-inflamed B16F10 model. The observed activity in MC38 and E0771 tumors was likely due to effects of both innate and adaptive immune cells. Following Shp1 deletion, we observed increases in intratumoral myeloid cells in both models, which was more striking in E0771 tumors. E0771 tumors also contained an increased ratio of effector to regulatory T cells following Shp1 loss. This was not observed for MC38 tumors, though we did find increased levels of IFNγ, a cytokine produced by effector T cells, in these tumors. Overall, our preclinical data suggested that targeting Shp1 may be an attractive therapeutic strategy for boosting the immune response to cancer via a mechanism involving both innate and adaptive leukocytes.
Subject(s)
Adenocarcinoma/enzymology , Breast Neoplasms/enzymology , Colonic Neoplasms/enzymology , Melanoma, Experimental/enzymology , Protein Tyrosine Phosphatase, Non-Receptor Type 6/deficiency , Skin Neoplasms/enzymology , Tumor-Associated Macrophages/enzymology , Adaptive Immunity , Adenocarcinoma/immunology , Adenocarcinoma/pathology , Adenocarcinoma/therapy , Animals , Antigens, Differentiation/metabolism , Breast Neoplasms/immunology , Breast Neoplasms/pathology , Breast Neoplasms/therapy , Colonic Neoplasms/immunology , Colonic Neoplasms/pathology , Colonic Neoplasms/therapy , Female , Humans , Immunity, Innate , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Melanoma, Experimental/immunology , Melanoma, Experimental/pathology , Melanoma, Experimental/therapy , Mice, Inbred C57BL , Mice, Knockout , Phagocytosis , Protein Tyrosine Phosphatase, Non-Receptor Type 6/genetics , Receptors, Immunologic/metabolism , Signal Transduction , Skin Neoplasms/immunology , Skin Neoplasms/pathology , Skin Neoplasms/therapy , THP-1 Cells , Tumor Burden , Tumor Microenvironment , Tumor-Associated Macrophages/immunologyABSTRACT
The protein tyrosine phosphatase SHP2 binds to phosphorylated signaling motifs on regulatory immunoreceptors including PD-1, but its functional role in tumor immunity is unclear. Using preclinical models, we show that RMC-4550, an allosteric inhibitor of SHP2, induces antitumor immunity, with effects equivalent to or greater than those resulting from checkpoint blockade. In the tumor microenvironment, inhibition of SHP2 modulated T-cell infiltrates similar to checkpoint blockade. In addition, RMC-4550 drove direct, selective depletion of protumorigenic M2 macrophages via attenuation of CSF1 receptor signaling and increased M1 macrophages via a mechanism independent of CD8+ T cells or IFNγ. These dramatic shifts in polarized macrophage populations in favor of antitumor immunity were not seen with checkpoint blockade. Consistent with a pleiotropic mechanism of action, RMC-4550 in combination with either checkpoint or CSF1R blockade caused additive antitumor activity with complete tumor regressions in some mice; tumors intrinsically sensitive to SHP2 inhibition or checkpoint blockade were particularly susceptible. Our preclinical findings demonstrate that SHP2 thus plays a multifaceted role in inducing immune suppression in the tumor microenvironment, through both targeted inhibition of RAS pathway-dependent tumor growth and liberation of antitumor immune responses. Furthermore, these data suggest that inhibition of SHP2 is a promising investigational therapeutic approach. SIGNIFICANCE: Inhibition of SHP2 causes direct and selective depletion of protumorigenic M2 macrophages and promotes antitumor immunity, highlighting an investigational therapeutic approach for some RAS pathway-driven cancers.
Subject(s)
Breast Neoplasms/immunology , Immunosuppressive Agents/pharmacology , Macrophages/immunology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Tumor Microenvironment/immunology , Allosteric Regulation , Animals , Apoptosis , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Breast Neoplasms/prevention & control , Cell Proliferation , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Signal Transduction , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Oncogenic alterations in the RAS/RAF/MEK/ERK pathway drive the growth of a wide spectrum of cancers. While BRAF and MEK inhibitors are efficacious against BRAFV600E-driven cancers, effective targeted therapies are lacking for most cancers driven by other pathway alterations, including non-V600E oncogenic BRAF, RAS GTPase-activating protein (GAP) NF1 (neurofibromin 1) loss and oncogenic KRAS. Here, we show that targeting the SHP2 phosphatase (encoded by PTPN11) with RMC-4550, a small-molecule allosteric inhibitor, is effective in human cancer models bearing RAS-GTP-dependent oncogenic BRAF (for example, class 3 BRAF mutants), NF1 loss or nucleotide-cycling oncogenic RAS (for example, KRASG12C). SHP2 inhibitor treatment decreases oncogenic RAS/RAF/MEK/ERK signalling and cancer growth by disrupting SOS1-mediated RAS-GTP loading. Our findings illuminate a critical function for SHP2 in promoting oncogenic RAS/MAPK pathway activation in cancers with RAS-GTP-dependent oncogenic BRAF, NF1 loss and nucleotide-cycling oncogenic KRAS. SHP2 inhibition is a promising molecular therapeutic strategy for patients with cancers bearing these oncogenic drivers.
Subject(s)
Biomarkers, Tumor/genetics , Guanosine Triphosphate/metabolism , Mutation , Neoplasms/enzymology , Neoplasms/genetics , Neurofibromin 1/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Enzyme Inhibitors/pharmacology , Extracellular Signal-Regulated MAP Kinases/metabolism , Genetic Predisposition to Disease , HEK293 Cells , Humans , Mice, Inbred BALB C , Mice, Nude , Mitogen-Activated Protein Kinase Kinases/metabolism , Neoplasms/drug therapy , Neoplasms/pathology , Phenotype , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , SOS1 Protein/metabolism , Signal Transduction , Tumor Burden/drug effects , Xenograft Model Antitumor Assays , raf Kinases/metabolismABSTRACT
BACKGROUND: Proton-pump inhibitors (PPIs) have been demonstrated to reduce rates of gastrointestinal events in patients requiring dual antiplatelet therapy (DAPT). Data are limited regarding the efficacy and safety of PPIs in high-risk cardiovascular subsets after acute coronary syndrome or percutaneous coronary intervention. METHODS: All patients enrolled in COGENT (Clopidogrel and the Optimization of Gastrointestinal Events Trial) were initiated on DAPT (with aspirin and clopidogrel) for various indications within the prior 21 days. These post hoc analyses of the COGENT trial evaluated the efficacy and safety of omeprazole compared with placebo in subsets of patients requiring DAPT for the 2 most frequent indications: 1) patients undergoing percutaneous coronary intervention (for any indication) within 14 days of randomization (n = 2676; 71.2%); and 2) patients presenting with acute coronary syndrome managed with or without percutaneous coronary intervention (n = 1573; 41.8%). Unadjusted Cox proportional hazards models were used to estimate effect sizes through final follow-up. RESULTS: Median follow-up duration was 110 days (interquartile range 55-167). In percutaneous coronary intervention-treated patients, omeprazole significantly reduced rates of composite gastrointestinal events at 180 days (1.2% vs 2.7%; hazard ratio [HR] 0.43; 95% confidence interval [CI], 0.22-0.85; P = .02) without increasing composite cardiovascular events (5.4% vs 6.3%; HR 1.00; 95% CI, 0.67-1.50; P = 1.00). Similarly, omeprazole lowered risk of the primary gastrointestinal endpoint at 180 days in patients presenting with acute coronary syndrome (1.1% vs 2.7%; HR 0.37; 95% CI, 0.13-1.01; P = .05) without a significant excess in cardiovascular events (5.6% vs 4.5%; HR 1.40; 95% CI, 0.77-2.53; P = .27). CONCLUSIONS: PPI therapy attenuates gastrointestinal bleeding risk without significant excess in major cardiovascular events in high-risk cardiovascular subsets, regardless of indication for DAPT. Future studies will be needed to clarify optimal gastroprotective strategies for higher-intensity and longer durations of DAPT.
Subject(s)
Acute Coronary Syndrome/drug therapy , Aspirin/therapeutic use , Gastrointestinal Hemorrhage/prevention & control , Omeprazole/therapeutic use , Percutaneous Coronary Intervention/methods , Platelet Aggregation Inhibitors/therapeutic use , Proton Pump Inhibitors/therapeutic use , Aspirin/administration & dosage , Aspirin/adverse effects , Clopidogrel , Double-Blind Method , Drug Therapy, Combination , Humans , Omeprazole/adverse effects , Percutaneous Coronary Intervention/adverse effects , Percutaneous Coronary Intervention/statistics & numerical data , Platelet Aggregation Inhibitors/administration & dosage , Platelet Aggregation Inhibitors/adverse effects , Proton Pump Inhibitors/adverse effects , Ticlopidine/administration & dosage , Ticlopidine/adverse effects , Ticlopidine/analogs & derivatives , Ticlopidine/therapeutic use , Treatment OutcomeABSTRACT
BACKGROUND: The COGENT (Clopidogrel and the Optimization of Gastrointestinal Events Trial) showed that proton-pump inhibitors (PPIs) safely reduced rates of gastrointestinal (GI) events in patients requiring dual antiplatelet therapy (DAPT). However, utilization of appropriate prophylactic PPI therapy remains suboptimal, especially with low-dose aspirin. OBJECTIVES: The authors investigated the safety and efficacy of PPI therapy in patients receiving DAPT in low- and high-dose aspirin subsets. METHODS: Randomized patients with available aspirin dosing information in COGENT (N = 3,752) were divided into "low-dose" (≤ 100 mg) and "high-dose" (>100 mg) aspirin groups. The primary GI and cardiovascular endpoints were composite upper GI events and major adverse cardiac events, respectively. All events were adjudicated by independent, blinded gastroenterologists and cardiologists. RESULTS: Median duration of follow-up was 110 days. Low-dose aspirin users (n = 2,480; 66.1%) were more likely to be older, female, and have higher rates of peripheral artery disease, prior stroke, and hypertension, whereas high-dose aspirin users (n = 1,272; 33.9%) had higher rates of hyperlipidemia, smoking, a history of percutaneous coronary intervention, and were more than twice as likely to be enrolled from sites within the United States (80.4% vs. 39.8%). High-dose aspirin was associated with similar 180-day Kaplan-Meier estimates of adjudicated composite GI events (1.7% vs. 2.1%; adjusted hazard ratio: 0.88; 95% confidence interval: 0.46 to 1.66) and major adverse cardiac events (4.8% vs. 5.5%; adjusted hazard ratio: 0.73; 95% confidence interval: 0.48 to 1.11) compared with low-dose aspirin. Randomization to PPI therapy reduced 180-day Kaplan-Meier estimates of the primary GI endpoint in low-dose (1.2% vs. 3.1%) and high-dose aspirin subsets (0.9% vs. 2.6%; p for interaction = 0.80), and did not adversely affect the primary cardiovascular endpoint in either group. CONCLUSIONS: Gastroprotection with PPI therapy should be utilized in appropriately selected patients with coronary artery disease requiring DAPT, even if the patients are on low-dose aspirin. (Clopidogrel and the Optimization of Gastrointestinal Events Trial [COGENT]; NCT00557921).
Subject(s)
Aspirin/administration & dosage , Omeprazole/therapeutic use , Platelet Aggregation Inhibitors/administration & dosage , Proton Pump Inhibitors/therapeutic use , Ticlopidine/analogs & derivatives , Aged , Aspirin/adverse effects , Clopidogrel , Dose-Response Relationship, Drug , Drug Therapy, Combination , Dyspepsia/chemically induced , Dyspepsia/prevention & control , Female , Gastrointestinal Hemorrhage/chemically induced , Gastrointestinal Hemorrhage/prevention & control , Humans , Intestinal Obstruction/chemically induced , Intestinal Obstruction/prevention & control , Intestinal Perforation/chemically induced , Intestinal Perforation/prevention & control , Male , Middle Aged , Myocardial Infarction/epidemiology , Myocardial Revascularization/statistics & numerical data , Pain/chemically induced , Pain/prevention & control , Peptic Ulcer/chemically induced , Peptic Ulcer/prevention & control , Platelet Aggregation Inhibitors/adverse effects , Prospective Studies , Stroke/epidemiology , Ticlopidine/administration & dosage , Ticlopidine/adverse effectsABSTRACT
BACKGROUND: Gastrointestinal complications are an important problem of antithrombotic therapy. Proton-pump inhibitors (PPIs) are believed to decrease the risk of such complications, though no randomized trial has proved this in patients receiving dual antiplatelet therapy. Recently, concerns have been raised about the potential for PPIs to blunt the efficacy of clopidogrel. METHODS: We randomly assigned patients with an indication for dual antiplatelet therapy to receive clopidogrel in combination with either omeprazole or placebo, in addition to aspirin. The primary gastrointestinal end point was a composite of overt or occult bleeding, symptomatic gastroduodenal ulcers or erosions, obstruction, or perforation. The primary cardiovascular end point was a composite of death from cardiovascular causes, nonfatal myocardial infarction, revascularization, or stroke. The trial was terminated prematurely when the sponsor lost financing. RESULTS: We planned to enroll about 5000 patients; a total of 3873 were randomly assigned and 3761 were included in analyses. In all, 51 patients had a gastrointestinal event; the event rate was 1.1% with omeprazole and 2.9% with placebo at 180 days (hazard ratio with omeprazole, 0.34, 95% confidence interval [CI], 0.18 to 0.63; P<0.001). The rate of overt upper gastrointestinal bleeding was also reduced with omeprazole as compared with placebo (hazard ratio, 0.13; 95% CI, 0.03 to 0.56; P = 0.001). A total of 109 patients had a cardiovascular event, with event rates of 4.9% with omeprazole and 5.7% with placebo (hazard ratio with omeprazole, 0.99; 95% CI, 0.68 to 1.44; P = 0.96); high-risk subgroups did not show significant heterogeneity. The two groups did not differ significantly in the rate of serious adverse events, though the risk of diarrhea was increased with omeprazole. CONCLUSIONS: Among patients receiving aspirin and clopidogrel, prophylactic use of a PPI reduced the rate of upper gastrointestinal bleeding. There was no apparent cardiovascular interaction between clopidogrel and omeprazole, but our results do not rule out a clinically meaningful difference in cardiovascular events due to use of a PPI. (Funded by Cogentus Pharmaceuticals; ClinicalTrials.gov number, NCT00557921.).
Subject(s)
Coronary Artery Disease/drug therapy , Gastrointestinal Hemorrhage/prevention & control , Omeprazole/therapeutic use , Platelet Aggregation Inhibitors/therapeutic use , Proton Pump Inhibitors/therapeutic use , Ticlopidine/analogs & derivatives , Aged , Aspirin/adverse effects , Aspirin/therapeutic use , Cardiovascular Diseases/epidemiology , Clopidogrel , Drug Interactions , Drug Therapy, Combination , Female , Gastrointestinal Hemorrhage/chemically induced , Humans , Kaplan-Meier Estimate , Male , Middle Aged , Omeprazole/adverse effects , Platelet Aggregation Inhibitors/adverse effects , Proton Pump Inhibitors/adverse effects , Ticlopidine/adverse effects , Ticlopidine/therapeutic useABSTRACT
BACKGROUND: Cells derived from native rodents have limits at distinct steps of HIV replication. Rat primary CD4 T-cells, but not macrophages, display a profound transcriptional deficit that is ameliorated by transient trans-complementation with the human Tat-interacting protein Cyclin T1 (hCycT1). RESULTS: Here, we generated transgenic rats that selectively express hCycT1 in CD4 T-cells and macrophages. hCycT1 expression in rat T-cells boosted early HIV gene expression to levels approaching those in infected primary human T-cells. hCycT1 expression was necessary, but not sufficient, to enhance HIV transcription in T-cells from individual transgenic animals, indicating that endogenous cellular factors are critical co-regulators of HIV gene expression in rats. T-cells from hCD4/hCCR5/hCycT1-transgenic rats did not support productive infection of prototypic wild-type R5 HIV-1 strains ex vivo, suggesting one or more significant limitation in the late phase of the replication cycle in this primary rodent cell type. Remarkably, we identify a replication-competent HIV-1 GFP reporter strain (R7/3 YU-2 Env) that displays characteristics of a spreading, primarily cell-to-cell-mediated infection in primary T-cells from hCD4/hCCR5-transgenic rats. Moreover, the replication of this recombinant HIV-1 strain was significantly enhanced by hCycT1 transgenesis. The viral determinants of this so far unique replicative ability are currently unknown. CONCLUSION: Thus, hCycT1 expression is beneficial to de novo HIV infection in a transgenic rat model, but additional genetic manipulations of the host or virus are required to achieve full permissivity.
Subject(s)
CD4-Positive T-Lymphocytes/virology , Cyclins/metabolism , HIV-1/physiology , Transcription, Genetic , Virus Replication , Animals , Cells, Cultured , Cyclin T , HIV-1/growth & development , Humans , Macrophages/virology , Rats , Rats, TransgenicABSTRACT
Loss of interleukin (IL)-7 or the IL-7 receptor alpha (IL-7Ralpha, CD127) results in severe immunodeficiencies in mice and humans. To more precisely identify signals governing IL-7 function in vivo, we have disrupted the IL-7Ralpha Y449XXM motif in mice by knock-in mutagenesis (IL-7Ralpha(449F)). Thymic precursors were reduced in number in IL-7Ralpha(449F) mice, but in marked contrast to IL-7Ralpha(-/-) knockout mice, thymocytes and peripheral T cells developed normally. Strikingly, Listeria infection revealed that CD4 and CD8 T cells had different requirements for IL-7Ralpha signals. CD4 T cells failed to mount a primary response, but despite normal CD8 primary responses, maintenance of CD8 memory was impaired in IL-7Ralpha(449F) mice. Furthermore, we show that Bcl-2 is IL-7Ralpha Y449 independent and insufficient for IL-7-mediated maintenance of CD8 memory.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Immunologic Memory/genetics , Receptors, Interleukin-7/genetics , Animals , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/pathology , Cell Survival/genetics , Cell Survival/immunology , Cells, Cultured , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Mutant Strains , Mutagenesis, Site-Directed , Signal Transduction/geneticsABSTRACT
Granulocyte colony-stimulating factor (G-CSF) is essential for the host response to bacterial infection by controlling neutrophil production in the bone marrow. The G-CSF receptor (G-CSFR) activates the Jak/STAT pathway, although little is understood about how these signals regulate basal and stress-induced granulopoiesis. We examined STAT3 function in granulocytes using a bone marrow conditional knockout mouse model. Our results show that STAT3 has a crucial role in emergency granulopoiesis and mature neutrophil function. STAT3-deficient mice have an aberrant response to G-CSF in vivo, characterized by failure to accumulate immature granulocytes and an increased ratio of mature to immature neutrophils in the bone marrow, peripheral blood, and spleen. Acute neutrophil mobilization is impaired in STAT3-deficient mice as judged by their failure to up-regulate circulating neutrophils following short-term G-CSF exposure. STAT3 also controls neutrophil chemotactic responses to natural ligands for CXCR2 and regulates the magnitude of chemoattractant-induced actin polymerization. These functions of STAT3 are independent of its principal target gene Socs3, which encodes a crucial feedback inhibitor of cytokine signaling. Our results demonstrate the existence of distinct STAT3 target pathways in neutrophils required for granulopoiesis and innate immunity.
Subject(s)
Chemotaxis/physiology , Immunity, Innate/physiology , Myelopoiesis/physiology , Neutrophils/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction/physiology , Animals , Bacterial Infections/genetics , Bacterial Infections/metabolism , Bone Marrow/metabolism , Cell Differentiation/physiology , Granulocyte Colony-Stimulating Factor/metabolism , Mice , Mice, Knockout , Receptors, Granulocyte Colony-Stimulating Factor/metabolism , STAT3 Transcription Factor/deficiency , Suppressor of Cytokine Signaling 3 Protein , Suppressor of Cytokine Signaling Proteins/metabolismABSTRACT
To enter human cells, HIV-1 usually uses CD4 and 1 of 2 coreceptors: CCR5 and CXCR4. Interestingly, even though CCR5 is expressed on far fewer T cells than is CXCR4, many patients in early- and late-stage HIV disease maintain high levels of CCR5-tropic (R5) viruses. We hypothesized that such high R5 viral loads may be sustained because, relative to CXCR4-tropic (X4) HIV-1 infection, R5 HIV-1 infection of permissive CD4(+)CCR5(+)CXCR4(+) T cells results in the production of significantly more infectious virus particles per target cell. To investigate this possibility, we compared the levels of virus production per target cell after isogenic R5 and X4 HIV-1 infection of 2 in vitro primary human lymphocyte culture systems: T-cell receptor-stimulated blood-derived CD4(+) T cells and tonsil histoculture (which requires no exogenous stimulation for ex vivo infection). We provide evidence that R5 HIV-1 does indeed compensate for a small target cell population by producing, on average, 5 to 10 times more infectious virus per CCR5(+) target cell than X4 HIV-1. This replicative advantage may contribute to the predominance of R5 HIV-1 in vivo.
Subject(s)
CD4-Positive T-Lymphocytes/virology , HIV-1/physiology , CD4-Positive T-Lymphocytes/metabolism , Cells, Cultured , HIV-1/pathogenicity , Humans , Receptors, CCR5/metabolism , Receptors, CXCR4/metabolism , Virulence , Virus ReplicationABSTRACT
The requirement for receptor components and the signalling effector, signal transducer and activator of transcription (STAT) 5A/5B, was assessed genetically in a lymphoma development model induced by interleukin-7 (IL-7). This growth factor for T- and B-cell progenitors and mature lymphocytes activates survival and proliferative pathways including Bcl-2, phosphatidylinositol-3 kinase and STAT5. Overexpression of IL-7 in vivo causes early mortality from lymphoma development. Mice overexpressing IL-7 that were heterozygous for the IL-7Ralpha subunit showed improved survival compared to wild-type mice. In addition, STAT5A/5B+/- compound heterozygous mice with one targeted allele each of STAT5A and STAT5B showed striking amelioration of IL-7-induced mortality and disease development. STAT5A/5B+/- compound heterozygous mice were otherwise normal in stem cell and lymphocyte development and cellularity. Lower STAT5 protein levels accompanied the reduction in STAT5A/5B copy number, which suggests that STAT5 haploinsufficiency is a modifier of IL-7 signal strength.
Subject(s)
DNA-Binding Proteins/analysis , DNA-Binding Proteins/genetics , Interleukin-7/physiology , Lymphoma/genetics , Milk Proteins/analysis , Milk Proteins/genetics , Trans-Activators/analysis , Trans-Activators/genetics , Animals , Cell Proliferation , Disease Models, Animal , Gene Dosage , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Haplotypes , Humans , Interleukin-7/genetics , Lymphoma/immunology , Lymphoma/pathology , Mice , Receptors, Interleukin-7/physiology , STAT5 Transcription Factor , Tumor Suppressor ProteinsABSTRACT
Infection of macrophages has been implicated as a critical event in the transmission and persistence of human immunodeficiency virus type 1 (HIV-1). Here, we explore whether primary X4 HIV-1 isolates can productively infect tissue macrophages that have terminally differentiated in vivo. Using immunohistochemistry, HIV-1 RNA in situ hybridization, and confocal immunofluorescence microscopy, we demonstrate that macrophages residing in human tonsil blocks can be productively infected ex vivo by primary X4 HIV-1 isolates. This challenges the model in which macrophage tropism is a key determinant of the selective transmission of R5 HIV-1 strains. Infection of tissue macrophages by X4 HIV-1 may be highly relevant in vivo and contribute to key events in HIV-1 pathogenesis.
Subject(s)
HIV-1/growth & development , Macrophages/virology , Antigens, CD/analysis , Cells, Cultured , HIV-1/classification , HIV-1/isolation & purification , Humans , Macrophages/immunology , Microscopy, Confocal , Monocytes/virologyABSTRACT
OBJECTIVE: STAT5A/5B-deficient mice are recognized to manifest defects in multiple cell types and tissues. In particular, the hematopoietic defects in these mice are widespread, affecting multiple lineages and multiple stages of development. Previous studies indicate that deficiencies intrinsic to hematopoietic cells contribute substantially to the observed defects. However, in light of the broad physiologic effects of STAT5 in the context of the organism outside the blood system, we wished to investigate the possibility of STAT5-dependent environmental influence of nonhematopoietic origin on hematopoietic development in these mice. MATERIALS AND METHODS: We transplanted wild-type bone marrow into STAT5A/5B-deficient mice to determine the effects of loss of STAT5 in nonhematopoietic tissue on hematopoietic development. RESULTS: We observed that transplantation of wild-type marrow completely corrects hematopoietic defects in STAT5A/5B-deficient recipient mice, including peripheral blood counts, bone marrow cellularity, and reductions in specific progenitor subsets. CONCLUSION: These results indicate that the important role of STAT5 in hematolymphoid development are mediated directly through effects on hematopoietic cells and not indirectly.
Subject(s)
Bone Marrow Transplantation , DNA-Binding Proteins/deficiency , Milk Proteins , Trans-Activators/deficiency , Animals , Blood Cells/cytology , Bone Marrow Cells/cytology , DNA-Binding Proteins/genetics , Hematopoiesis/genetics , Hematopoietic Stem Cells/cytology , Hematopoietic System/growth & development , Lymphatic System/growth & development , Mice , Mice, Knockout , STAT5 Transcription Factor , Trans-Activators/geneticsABSTRACT
OBJECTIVE: Cytokines bind high-affinity receptors expressed on hematopoietic cells to initiate signaling cascades that regulate differentiation, proliferation, and survival. Previous studies have established a role for STAT5 in transducing survival signals for hematopoietic progenitor cells in response to cytokines. MATERIALS AND METHODS: To determine if constitutive expression of a member of the bcl-2 family of anti-apoptotic proteins could compensate for the loss of STAT5, we utilized combinatorial genetics to generate STAT5A/5B-deficient mice expressing a bcl-2 transgene. RESULTS: Although bcl-2 expression restored peripheral blood counts to normal in STAT5A/5B(-/-) mice, we noted a striking failure of this transgene to correct defects in hematopoietic stem and progenitor cells. CONCLUSION: These data imply important effects of STAT5 in modulating hematopoietic cells in addition to promoting survival per se.
Subject(s)
DNA-Binding Proteins/deficiency , Hematopoietic System/drug effects , Lymphatic System/drug effects , Milk Proteins , Proto-Oncogene Proteins c-bcl-2/pharmacology , Trans-Activators/deficiency , Animals , Blood Cells/cytology , Blood Cells/drug effects , Cell Survival/drug effects , DNA-Binding Proteins/genetics , Hematopoiesis/drug effects , Hematopoietic Stem Cells/cytology , Hematopoietic System/growth & development , Lymphatic System/growth & development , Mice , Mice, Knockout , Mice, Transgenic , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/therapeutic use , STAT5 Transcription Factor , Trans-Activators/genetics , Transgenes , Treatment FailureABSTRACT
STAT5 has previously been reported to be dispensable for the maintenance of tolerance in vivo. However, in examining hemopoiesis in mice lacking both isoforms of STAT5, STAT5A, and STAT5B, we noted that a subset of these mice demonstrated dramatic alterations in several bone marrow progenitor populations concomitant with lymphocytic infiltration of the bone marrow. In addition, cellular infiltration affecting the colon, liver, and kidney was observed in these mice. Survival analysis revealed that STAT5A/5B(-/-) mice exhibited early death. The increased mortality and the pathology affecting multiple organs observed in these mice were abrogated on the recombination-activating gene 1(-/-) background. In light of the similarities between STAT5A/5B-deficient mice and mice unable to signal through the IL-2R, we hypothesized that the tolerizing role of STAT5A/5B was triggered via activation of the IL-2R. In agreement with this, we found that IL-2Rbeta chain-deficient mice exhibited similar hemopoietic abnormalities. Because IL-2 signaling is thought to contribute to tolerance through maintenance of a CD4(+)CD25(+) regulatory T cell population, we examined these cells and observed a numerical reduction in STAT5A/5B(-/-) mice along with a higher rate of apoptosis. These data provide strong evidence for a requirement for STAT5 in the maintenance of tolerance in vivo.
Subject(s)
Autoimmune Diseases/genetics , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Immune Tolerance/genetics , Milk Proteins , Trans-Activators/deficiency , Trans-Activators/genetics , Adoptive Transfer , Animals , Apoptosis/genetics , Apoptosis/immunology , Autoimmune Diseases/mortality , Autoimmune Diseases/pathology , Autoimmune Diseases/prevention & control , Bone Marrow Cells/immunology , Bone Marrow Cells/pathology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/pathology , CD4-Positive T-Lymphocytes/transplantation , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Cell Movement/genetics , Cell Movement/immunology , Colon/immunology , Colon/pathology , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/pathology , Homeodomain Proteins/genetics , Immunologic Deficiency Syndromes/genetics , Immunologic Deficiency Syndromes/immunology , Immunologic Deficiency Syndromes/pathology , Immunologic Memory/genetics , Interleukin-2 Receptor beta Subunit , Kidney/immunology , Kidney/pathology , Liver/immunology , Liver/pathology , Lymphoid Tissue/immunology , Lymphoid Tissue/pathology , Lymphopenia/genetics , Lymphopenia/immunology , Lymphopenia/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Receptors, Interleukin/deficiency , Receptors, Interleukin/genetics , Receptors, Interleukin-2/biosynthesis , STAT5 Transcription Factor , Survival Rate , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolismABSTRACT
Retroviruses must gain access to the host cell nucleus for subsequent replication and viral propagation. Human immunodeficiency virus type 1 (HIV-1) and other primate lentiviruses are distinguished from the gammaretroviruses by their ability to infect nondividing cells such as macrophages, an important viral reservoir in vivo. Rather than requiring nuclear membrane breakdown during cell division, the HIV-1 preintegration complex (PIC) enters the nucleus by traversing the central aqueous channel of the limiting nuclear pore complex. The HIV-1 PIC contains three nucleophilic proteins, matrix, integrase, and Vpr, all of which have been implicated in nuclear targeting. The mechanism by which Vpr can display such nucleophilic properties and yet also be available for incorporation into virions assembling at the plasma membrane is unresolved. We recently characterized Vpr as a nucleocytoplasmic shuttling protein that contains two novel nuclear import signals and an exportin-1-dependent nuclear export signal (NES). We now demonstrate that mutation of this NES impairs the incorporation of Vpr into newly formed virions. Furthermore, we find that the Vpr NES is required for efficient HIV replication in tissue macrophages present in human spleens and tonsils. These findings underscore how the nucleocytoplasmic shuttling of Vpr not only contributes to nuclear import of the HIV-1 PIC but also enables Vpr to be present in the cytoplasm for incorporation into virions, leading to enhancement of viral spread within nondividing tissue macrophages.
Subject(s)
Active Transport, Cell Nucleus , Cell Nucleus/metabolism , Gene Products, vpr/metabolism , HIV-1/physiology , Macrophages/virology , Virus Replication , Cell Line , G2 Phase , Humans , Lymphoid Tissue/cytology , Lymphoid Tissue/virology , Protein Transport , vpr Gene Products, Human Immunodeficiency VirusABSTRACT
The destruction of the immune system by progressive loss of CD4 T cells is the hallmark of AIDS. CCR5-dependent (R5) human immunodeficiency virus type 1 (HIV-1) isolates predominate in the early, asymptomatic stages of HIV-1 infection, while CXCR4-dependent (X4) isolates typically emerge at later stages, frequently coinciding with a rapid decline in CD4 T cells. Lymphocyte killing in vivo primarily occurs through apoptosis, but the importance of apoptosis of HIV-1-infected cells relative to apoptosis of uninfected bystander cells is controversial. Here we show that in human lymphoid tissues ex vivo, apoptosis of uninfected bystander CD4 T cells is a major mechanism of lymphocyte depletion caused by X4 HIV-1 strains but is only a minor mechanism of depletion by R5 strains. Further, X4 HIV-1-induced bystander apoptosis requires the interaction of the viral envelope glycoprotein gp120 with the CXCR4 coreceptor on CD4 T cells. These results emphasize the contribution of bystander apoptosis to HIV-1 cytotoxicity and suggest that in association with a coreceptor switch in HIV disease, T-cell killing evolves from an infection-restricted stage to generalized toxicity that involves a high degree of bystander apoptosis.
Subject(s)
Apoptosis , Bystander Effect , CD4-Positive T-Lymphocytes/physiology , Evolution, Molecular , HIV-1/pathogenicity , Receptors, CXCR4/metabolism , CD3 Complex/metabolism , Cells, Cultured , HIV Infections/virology , HIV-1/genetics , HIV-1/physiology , Humans , Palatine Tonsil/immunology , Palatine Tonsil/virology , VirulenceABSTRACT
Kinases of the Jak family (Jak1/2/3 and Tyk2) interact with the membrane proximal domain of different cytokine receptors and play a critical role in the activation of cytokine and growth factor signaling pathways. In this report we demonstrate that both the Box 1 and Box 2 motif collaborate in the association and activation of Jak1 by type I interferons. Mutational analysis of the beta chain of type I interferon receptor (IFNalphaRbetaL/IFNAR2) revealed that Box 1 plays a more significant role in activation than in the association with Jak1. On the contrary, the Box 2 motif contributes more to the association with Jak1 than to kinase activation. Additionally, the study of the Jak1 binding sites on the IL2 receptor beta (IL2Rbeta), IFNgammaRalpha/IFNGR1, and IL10Ralpha/IL10R1 chains suggests that cytokine receptors have two different kinds of interaction with Jak1. One form of interaction involves the Box 1 and the previously described Box 2 motif, which we now designate as Box 2A, characterized by the VEVI and LEVL sequences present in IFNalphaRbetaL/IFNAR2 and IL2Rbeta subunits, respectively. The second form of interaction requires a motif termed Box 2B, which is present in the IFNgammaRalpha/IFNGR1 (SILLPKS) and IL10Ralpha/IL10R1 (SVLLFKK) chains. Interestingly, Box 2B localizes close to the membrane region (8-10 amino acids from the membrane) similar to Box 1, whereas Box 2A is more distal (38-58 amino acids from the membrane).
Subject(s)
Protein-Tyrosine Kinases/metabolism , Receptors, Interferon/metabolism , Amino Acid Sequence , Binding Sites , Humans , Janus Kinase 1 , Mutagenesis , Receptors, Interferon/chemistry , Receptors, Interferon/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
The Sydney Blood Bank Cohort is a group of patients with slowly progressive infection by a human immunodeficiency virus strain containing spontaneous deletions within the nef long terminal repeat region. In 1999, 18 years after the initial infection, one of the members (D36) developed AIDS. In this work, we used an ex vivo human lymphoid cell culture system to analyze two viral isolates obtained from this patient, one prior to the onset of AIDS in 1995 and one after disease progression in 1999. Both D36 isolates were less potent in depleting CD4(+) T cells than a reference dualtropic, nef-bearing viral isolate. However, the 1999 isolate was measurably more cytotoxic to CD4(+) T cells than the 1995 isolate. Interestingly, although both isolates were nearly equally potent in depleting CCR5(+) CD4(+) T cells, the cytotoxic effect of the 1999 isolate toward CCR5(-) CD4(+) T cells was significantly higher. Furthermore, GHOST cell infection assays and blocking experiments with the CXCR4 inhibitor AMD3100 showed that the later D36 1999 isolate could infect both CCR5(+) and CCR5(-) CXCR4(+) cells efficiently, while infection by the 1995 isolate was nearly completely restricted to CCR5(+) cells. Sequence analysis of the V1/V2 and V3 regions of the viral envelope protein gp120 revealed that the more efficient CXCR4 usage of the later isolate might be caused by an additional potential N-glycosylation site in the V1/V2 loop. In conclusion, these data show that an in vivo evolution of the tropism of this nef-deleted strain toward an X4 phenotype was associated with a higher cytopathic potential and progression to AIDS.