ABSTRACT
BACKGROUND: Cutaneous viral infections and immune suppression are risk factors for some forms of nonmelanoma skin cancer; however, their interrelationship is poorly understood. OBJECTIVES: To examine cross-sectional associations between cutaneous viral infections and circulating forkhead-box P3 (FOXP3)-expressing T-regulatory (Treg) cells, suppressive cells that dampen effective antitumour immunity. MATERIALS AND METHODS: Blood, eyebrow hair (EBH) and skin swab (SSW) samples were collected from 352 patients 60 years and older undergoing skin screening, without prevalent skin cancer, while participating in an ongoing prospective cohort study of cutaneous viral infections and skin cancer. DNA corresponding to 98 cutaneous human papillomavirus (HPV) types and five human polyomaviruses (HPyV) was assessed in EBH and SSW. Distinct classes of circulating Treg-cell subpopulations were defined by flow cytometry including cutaneous lymphocyte antigen (CLA) and CCR4high Treg cells, both previously associated with cutaneous diseases. Age- and sex-adjusted associations between circulating T-cell populations and infection were estimated using logistic regression. RESULTS: Total Treg-cell proportion in peripheral blood was not associated with ß HPV or HPyV infection. However, the proportion of circulating CLA+ Treg cells was inversely associated with γ HPV EBH infection [odds ratio (OR) 0·54, 95% confidence interval (CI) 0·35-0·84]. Interestingly, circulating Treg cells expressing markers indicative of antigen activation (CD27- CD45RA- FOXP3+ CD4+ ) were also inversely associated with γ HPV infection in SSW (OR 0·55, 95% CI 0·30-0·99) and EBH (OR 0·56, 95% CI 0·36-0·86). CONCLUSIONS: Inverse associations between circulating Treg cells and γ HPV infection suggest that localized viral infection may promote immunosuppressive cell migration into skin.
Subject(s)
Gammapapillomavirus/isolation & purification , Immune Tolerance , Papillomavirus Infections/immunology , Skin Diseases, Viral/immunology , T-Lymphocytes, Regulatory/immunology , Aged , Carcinogenesis/immunology , Cross-Sectional Studies , DNA, Viral/isolation & purification , Eyebrows/immunology , Eyebrows/virology , Female , Gammapapillomavirus/genetics , Gammapapillomavirus/immunology , Humans , Male , Middle Aged , Papillomavirus Infections/blood , Papillomavirus Infections/virology , Polyomavirus/genetics , Polyomavirus/immunology , Polyomavirus/isolation & purification , Polyomavirus Infections/blood , Polyomavirus Infections/immunology , Polyomavirus Infections/virology , Prospective Studies , Skin/immunology , Skin/virology , Skin Diseases, Viral/blood , Skin Diseases, Viral/virology , Skin Neoplasms/immunology , Tumor Virus Infections/blood , Tumor Virus Infections/immunology , Tumor Virus Infections/virologyABSTRACT
Lenalidomide is the first karyotype-selective therapeutic approved for the treatment of myelodysplastic syndromes (MDS) owing to high rates of erythroid and cytogenetic response in patients with chromosome 5q deletion [del(5q)]. Although haploinsufficiency for the RPS14 gene and others encoded within the common deleted region (CDR) have been implicated in the pathogenesis of the del(5q) phenotype, the molecular basis of the karyotype specificity of lenalidomide remains unexplained. We focused our analysis on possible haplodeficient enzymatic targets encoded within the CDR that play key roles in cell-cycle regulation. We show that the dual specificity phosphatases, Cdc25C and PP2Acalpha, which are coregulators of the G(2)-M checkpoint, are inhibited by lenalidomide. Gene expression was lower in MDS and acute myeloid leukemia (AML) specimens with del(5q) compared with those with alternate karyotypes. Lenalidomide inhibited phosphatase activity either directly (Cdc25C) or indirectly (PP2A) with corresponding retention of inhibitory phospho-tyrosine residues. Treatment of del(5q) AML cells with lenalidomide induced G(2) arrest and apoptosis, whereas there was no effect in nondel(5q) AML cells. Small interfering RNA (shRNA) suppression of Cdc25C and PP2Acalpha gene expression recapitulated del(5q) susceptibility to lenalidomide with induction of G(2) arrest and apoptosis in both U937 and primary nondel(5q) MDS cells. These data establish a role for allelic haplodeficiency of the lenalidomide inhibitable Cdc25C and PP2Acalpha phosphatases in the selective drug sensitivity of del(5q) MDS.
Subject(s)
Antineoplastic Agents/pharmacology , Chromosome Deletion , Chromosomes, Human, Pair 5 , Myelodysplastic Syndromes/drug therapy , Protein Phosphatase 2/antagonists & inhibitors , Thalidomide/analogs & derivatives , cdc25 Phosphatases/antagonists & inhibitors , Apoptosis/drug effects , G2 Phase/drug effects , Humans , Lenalidomide , Myelodysplastic Syndromes/genetics , Protein Phosphatase 2/genetics , Thalidomide/pharmacology , U937 Cells , cdc25 Phosphatases/geneticsABSTRACT
Large granular lymphocyte (LGL) leukemia is commonly associated with poor hematopoiesis. The first case of pulmonary artery hypertension (PAH) was observed in a 57-year-old woman with natural killer (NK)-LGL leukemia and transfusion-dependent anemia. Using a genetic approach, we demonstrated that killing of pulmonary endothelial cells by patient NK cells was mediated by dysregulated balance in activating and inhibitory NK-receptor signaling. Elevated pulmonary artery pressure and erythroid differentiation improved after disrupting the NK-receptor signaling pathway with 4 courses of a farnesyltransferase inhibitor, tipifarnib. Coincidental association between PAH and LGL leukemia suggest a causal relationship between the expanded lymphocyte population and these clinical manifestations. This trial is registered at www.ClinicalTrials.gov as NCI 6823.
Subject(s)
Antineoplastic Agents/therapeutic use , Farnesyltranstransferase/antagonists & inhibitors , Leukemia, Large Granular Lymphocytic/drug therapy , Receptors, Natural Killer Cell/genetics , Cells, Cultured , Enzyme Inhibitors/therapeutic use , Female , Humans , Hypertension, Pulmonary/drug therapy , Hypertension, Pulmonary/etiology , K562 Cells , Leukemia, Large Granular Lymphocytic/complications , Leukemia, Large Granular Lymphocytic/genetics , Middle Aged , Quinolones/therapeutic use , Signal Transduction/genetics , Treatment OutcomeABSTRACT
We report on the clinico-biological characteristics of 20 cases of gammadelta T cell large granular lymphocyte (LGL) leukemia. All the data were compared to that of 196 cases with alphabeta T cell subtype, which represents the majority of T cell LGL leukemias. Clinical findings were quite similar in the two groups regarding age, sex ratio, recurrent infections, and association with auto-immune diseases especially rheumatoid arthritis. Gammadelta LGL predominantly expressed a CD3+/CD4-/CD8+/CD16+/CD57+ phenotype, in 50% of cases. Clinical outcome was favorable for these patients with overall survival of 85% at 3 years. Fifty percent of gammadelta patients required treatment and the response to therapy was estimated at 55%. gammadelta and alphabeta T cell LGL leukemia harbor a very similar clinico-biological behavior and represent part of an antigen-driven T cell lymphoproliferation.
Subject(s)
Leukemia, T-Cell/diagnosis , Receptors, Antigen, T-Cell, gamma-delta , Adult , Aged , Aged, 80 and over , Autoimmune Diseases/complications , Clone Cells , Female , Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor , Humans , Immunophenotyping , Leukemia, T-Cell/immunology , Leukopenia/diagnosis , Male , Middle Aged , Receptors, Antigen, T-Cell, alpha-beta , Splenomegaly/diagnosisABSTRACT
Acquired amegakaryocytic thrombocytopenia and pure red cell aplasia rarely occur concurrently. We report a case in which these disorders were associated with an occult large granular lymphocyte leukemia. The peripheral blood cytopenias improved after glucocorticoids and intravenous immunoglobulin were administered, and response was maintained with cyclosporine. Large granular lymphocyte leukemia should be suspected in the setting of unexplained bone marrow failure.
Subject(s)
Leukemia, Large Granular Lymphocytic/complications , Purpura, Thrombocytopenic/complications , Red-Cell Aplasia, Pure/complications , Adult , Antigens, CD/analysis , Female , Humans , Leukemia, Large Granular Lymphocytic/diagnosis , Leukemia, Large Granular Lymphocytic/drug therapy , Leukemia, Large Granular Lymphocytic/immunology , Receptors, Immunologic/analysis , Signaling Lymphocytic Activation Molecule FamilyABSTRACT
Selected patients with Myelodysplastic Syndromes (MDS) are responsive to immunosuppressive therapy, suggesting that hematopoietic suppressive T cells have a pathogenic role in ineffective hematopoiesis. We assessed T-cell receptor (TCR) clonality through combined flow cytometry and molecular analysis of the complementarity determining region (CDR)-3 of the T-cell receptor-Vbeta gene. We identified clonal T cells in 50% of MDS patients (n=52) compared to 5% of age-matched normal controls (n=20). The presence of T-cell clones was not associated with features linked previously to immunosuppression response, including WHO diagnostic category, karyotype, marrow cellularity, IPSS category, sex or age Subject(s)
Myelodysplastic Syndromes/genetics
, Myelodysplastic Syndromes/immunology
, T-Lymphocytes, Regulatory/immunology
, T-Lymphocytes/immunology
, Antigens, CD/blood
, Antigens, CD/genetics
, Female
, Humans
, Karyotyping
, Male
, Middle Aged
, Polymerase Chain Reaction/methods
, Receptors, Antigen, T-Cell/genetics
ABSTRACT
The interplay between tumor heterogeneity and microenvironmental factors is a critical mechanism for clonal selection in leukemia. Evidence of unique clonal capacities to engraft within patient-derived xenograft (PDX) models suggests that intrapatient genetic architecture may be defined by functional differences at the clonal level. However, methods to detect functional differences assigned to genetically defined clones remain limited. Here, we describe a scalable method to directly measure the functional properties of clones within the same leukemia patient by coupling intracellular flow cytometry and next-generation sequencing (NGS). We provide proof of concept utilizing primary chronic myelmonocytic leukemia (CMML) samples and granulocyte-macrophage colony stimulating factor (GM-CSF) to elucidate the interaction between tumor heterogeneity and microenvironmental factors. Mixtures of human leukemia cell lines, with known response to GM-CSF, were used to validate the accuracy of our methodology. Using this approach, we confirm that our method is capable of discriminating GM-CSF sensitive cell lines, identifies somatic variants in primary leukemia samples, and resolves functional clonal architecture in an illustrative patient. Taken together, our data describes a novel method to determine intrapatient functional clonal heterogeneity and provides proof-of-concept for future investigation aimed at elucidating the clinical relevance of functional clonal differences.
Subject(s)
Flow Cytometry/methods , High-Throughput Nucleotide Sequencing/methods , Leukemia/genetics , Leukemia/pathology , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Humans , K562 Cells , Tumor Cells, CulturedABSTRACT
Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are associated with disease-initiating stem cells that are not eliminated by conventional therapies. Transcriptomic analysis of stem and progenitor populations in MDS and AML demonstrated overexpression of STAT3 that was validated in an independent cohort. STAT3 overexpression was predictive of a shorter survival and worse clinical features in a large MDS cohort. High STAT3 expression signature in MDS CD34+ cells was similar to known preleukemic gene signatures. Functionally, STAT3 inhibition by a clinical, antisense oligonucleotide, AZD9150, led to reduced viability and increased apoptosis in leukemic cell lines. AZD9150 was rapidly incorporated by primary MDS/AML stem and progenitor cells and led to increased hematopoietic differentiation. STAT3 knockdown also impaired leukemic growth in vivo and led to decreased expression of MCL1 and other oncogenic genes in malignant cells. These studies demonstrate that STAT3 is an adverse prognostic factor in MDS/AML and provide a preclinical rationale for studies using AZD9150 in these diseases.
Subject(s)
Leukemia, Myeloid, Acute , Myelodysplastic Syndromes , Neoplasm Proteins , Neoplastic Stem Cells , Oligonucleotides/pharmacology , STAT3 Transcription Factor , Animals , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Female , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Male , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Myelodysplastic Syndromes/drug therapy , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/metabolism , Myelodysplastic Syndromes/pathology , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , STAT3 Transcription Factor/antagonists & inhibitors , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Large granular lymphocyte (LGL) leukemia is characterized by the expansion of antigen-activated cytotoxic T lymphocytes. These leukemic cells are resistant to Fas-mediated apoptosis despite expressing high levels of Fas. We found that leukemic LGL from 19 patients displayed high levels of activated STAT3. Treatment of leukemic LGL with the JAK-selective tyrosine kinase inhibitor AG-490 induced apoptosis with a corresponding decrease in STAT-DNA binding activity. Moreover, using an antisense oligonucleotide approach to diminish STAT3 expression, we found that Fas sensitivity was restored in leukemic LGL. AG-490-induced apoptosis in leukemic LGL was independent of Bcl-xL or Bcl-2 expression. However, we found that the Bcl-2-family protein Mcl-1 was significantly reduced by AG-490 treatment. Activated STAT3 was shown to bind an SIE-related element in the murine mcl-1 promoter. Using a luciferase reporter assay, we demonstrated that v-src overexpression in NIH3T3 induced STAT3-dependent transcriptional activity from the mcl-1 promoter and increased endogenous Mcl-1 protein levels. We conclude that STAT3 activation contributed to accumulation of the leukemic LGL clones. These findings suggest that investigation should focus on novel strategies targeting STAT3 in the treatment of LGL leukemia.
Subject(s)
CD8-Positive T-Lymphocytes/physiology , DNA-Binding Proteins/antagonists & inhibitors , Leukemia/physiopathology , Neoplasm Proteins/biosynthesis , Proto-Oncogene Proteins c-bcl-2 , Trans-Activators/antagonists & inhibitors , Apoptosis/drug effects , Blotting, Western , CD8-Positive T-Lymphocytes/drug effects , Cell Line , DNA-Binding Proteins/metabolism , Dimerization , Enzyme Inhibitors/pharmacology , Gene Expression Regulation , Humans , Myeloid Cell Leukemia Sequence 1 Protein , Neoplasm Proteins/genetics , Oncogene Protein pp60(v-src) , Phosphorylation , STAT3 Transcription Factor , Signal Transduction , Trans-Activators/metabolism , Tumor Cells, Cultured , Tyrphostins/pharmacologyABSTRACT
T cells expressing human leukemia virus (HTLV) type 1, the etiological agent of adult T-cell leukemia, are remarkably resistant to conventional chemotherapy, and the need for drugs that effectively kill these cells is apparent. Here we show that roscovitine, an inhibitor of cyclin-dependent kinases (CDKs), induces the apoptosis of the HTLV-1-transformed T-cell line MT-2. Roscovitine prevented the tyrosine phosphorylation and consequent activation of the transcription factor signal transducer and activator of transcription (STAT) 5 when presented to MT-2 cells in the presence or absence of a caspase-3 inhibitor, and ectopic expression of a dominant-negative form of STAT5 in MT-2 cells induced apoptosis. Roscovitine and dominant-negative STAT5 also reduced the expression of the antiapoptotic protein XIAP, and STAT5 was associated with the XIAP promoter in vivo. Antibody to platelet-derived growth factor (PDGF) alpha receptors coprecipitated STAT5 from extracts of untreated but not roscovitine-treated cells. The tyrosine phosphatase inhibitor sodium orthovanadate ablated the inhibitory effects of roscovitine on STAT5/PDGF alpha receptor interaction, STAT5 activity, and cell survival. We suggest that roscovitine reduces the abundance of tyrosine-phosphorylated PDGF alpha receptors; as a result, STAT5 does not become active, and STAT5 gene products required for cell survival are not expressed.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , DNA-Binding Proteins/antagonists & inhibitors , Human T-lymphotropic virus 1/physiology , Milk Proteins , Purines/pharmacology , T-Lymphocytes/drug effects , Trans-Activators/antagonists & inhibitors , Apoptosis/physiology , Cell Line, Transformed , DNA-Binding Proteins/metabolism , Humans , Promoter Regions, Genetic , Protein Biosynthesis , Proteins/antagonists & inhibitors , Proteins/genetics , Receptor, Platelet-Derived Growth Factor alpha/antagonists & inhibitors , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Roscovitine , STAT5 Transcription Factor , T-Lymphocytes/cytology , T-Lymphocytes/virology , Trans-Activators/metabolism , X-Linked Inhibitor of Apoptosis ProteinABSTRACT
Aberrant T cell phenotype is one of the characteristics of myelodysplastic syndromes (MDS). In this study, we detected an increased concentration of IL-15 in the plasma of MDS patients (n = 20) compared with that in the plasma of healthy controls (n = 20). In MDS patients, reduced naïve CD4+ and CD8+ T cells [16.11 ± 6.56 vs. 24.11 ± 7.18 for CD4+ T cells (p < 0.001) and 13.15 ± 5.67 vs. 23.51 ± 6.25 for CD8+ T cells (p < 0.001)] were observed. The reduced naïve and increased effector memory T cells were significantly correlated with IL-15 plasma level. Then, the effect of IL-15 and IL-7 was tested in vitro. Peripheral blood mononuclear cells from MDS were treated for 15 days with IL-15. This treatment significantly decreased naïve CD4+ (p < 0.001) and CD8+ (p < 0.001) T cells and correspondingly increased terminal memory CD4+ and CD8+ T cells (p < 0.001). Treatment with IL-7 increased naïve CD4+ (p < 0.05) and CD8+ (p < 0.001) T cells. Our results indicated that exposure to high levels of IL-15 may be involved in the T cell phenotype conversion observed in MDS. IL-7 may be one of the promising therapeutic candidates for recovering the effector immune compartment in MDS patients.
Subject(s)
Interleukin-15/immunology , Interleukin-7/immunology , Myelodysplastic Syndromes/immunology , T-Lymphocytes/immunology , Adult , Aged , Aged, 80 and over , Case-Control Studies , Female , Humans , Immunologic Memory/drug effects , Immunologic Memory/immunology , Interleukin-15/pharmacology , Interleukin-7/blood , Interleukin-7/pharmacology , Male , Middle Aged , Myelodysplastic Syndromes/blood , Phenotype , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/immunology , T-Lymphocytes/drug effectsABSTRACT
Chronic myelogenous leukemia (CML) is a myeloproliferative disease characterized by the BCR-ABL genetic translocation and constitutive activation of the Abl tyrosine kinase. Among members of the Signal Transducers and Activators of Transcription (STAT) family of transcription factors, Stat5 is activated by the Bcr-Abl kinase and is implicated in the pathogenesis of CML. We recently identified PD180970 as a new and highly potent inhibitor of Bcr-Abl kinase. In this study, we show that blocking Bcr-Abl kinase activity using PD180970 in the human K562 CML cell line resulted in inhibition of Stat5 DNA-binding activity with an IC(50) of 5 nM. Furthermore, abrogation of Abl kinase-mediated Stat5 activation suppressed cell proliferation and induced apoptosis in K562 cells, but not in the Bcr-Abl-negative myeloid cell lines, HEL 92.1.7 and HL-60. Dominant-negative Stat5 protein expressed from a vaccinia virus vector also induced apoptosis of K562 cells, consistent with earlier studies that demonstrated an essential role of Stat5 signaling in growth and survival of CML cells. RNA and protein analyses revealed several candidate target genes of Stat5, including Bcl-x, Mcl-1, c-Myc and cyclin D2, which were down-regulated after treatment with PD180970. In addition, PD180970 inhibited Stat5 DNA-binding activity in cultured primary leukemic cells derived from CML patients. To detect activated Stat5 in CML patient specimens, we developed an immunocytochemical assay that can be used as a molecular end-point assay to monitor inhibition of Bcr-Abl signaling. Moreover, PD180970 blocked Stat5 signaling and induced apoptosis of STI-571 (Gleevec, Imatinib)-resistant Bcr-Abl-positive cells. Together, these results suggest that the mechanism of action of PD180970 involves inhibition of Bcr-Abl-mediated Stat5 signaling and provide further evidence that compounds in this structural class may represent potential therapeutic agents for CML.
Subject(s)
Cell Division/drug effects , DNA-Binding Proteins/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Milk Proteins , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyridones/pharmacology , Pyrimidines/pharmacology , Trans-Activators/antagonists & inhibitors , Apoptosis/drug effects , Apoptosis/genetics , Base Sequence , DNA Primers , DNA-Binding Proteins/metabolism , Fusion Proteins, bcr-abl , G1 Phase , Humans , Immunohistochemistry , STAT5 Transcription Factor , Trans-Activators/metabolism , Tumor Cells, CulturedABSTRACT
Liver injury is a clinical prognostic indicator in acute pancreatitis (AP). We have demonstrated that Kupffer cell-derived FasL mediates liver injury during AP and sought to determine its role in AP-induced hepatocyte apoptosis. AP was induced in National Institutes of Health (NIH) Swiss mice, C57/C57, and Fas-/-, FasL-/- mice by a choline-deficient ethionine-supplement diet. Liver Fas, FasL, p38-mitogen activated phosphokinase (p38-MAPK), poly-ADP ribose polymerase (PARP), and cytochrome C were measured by immunoblotting. Apoptosis was assessed by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) and DNA fragmentation (ELISA). AP upregulated liver FasL (4280 +/- 580 vs. 733 +/- 336), Fas (2866 +/- 595 vs. 649 +/- 111), cytochrome C (6980 +/- 237 vs. 903 +/- 156), and PARP (6393 +/- 591 vs. 466 +/- 261) as well as increased TUNEL staining (40 +/- 2 vs. 14 +/- 1) and DNA fragmentation (all P < 0.03 vs. control). In FasL-/- and Fas-/- mice, AP-induced upregulation of p38-MAPK, PARP, and cytochrome C was significantly attenuated (all P < 0.01 compared to C57/C57 control). In addition, AP-induced DNA fragmentation was reduced 60% in Fas-/- and FasL-/- mice (P < 0.01 vs. C57/C57). AP induces apoptosis by transcriptional activation of Fas/FasL. AP-induced apoptosis was significantly reduced in Fas and FasL knockout mice along with downregulation of p38-MAPK, PARP, and cytochrome C, thereby suggesting a central role for Fas/FasL in hepatocyte apoptosis. The manipulation of interactions between Kupffer cell-derived FasL and hepatocytes may have important therapeutic implications.
Subject(s)
Apoptosis , Hepatocytes/metabolism , Membrane Glycoproteins/metabolism , Pancreatitis/physiopathology , Receptors, Tumor Necrosis Factor/metabolism , Acute Disease , Animals , Blotting, Western , Cytochromes c/metabolism , Enzyme-Linked Immunosorbent Assay , Fas Ligand Protein , Female , In Situ Nick-End Labeling , Kupffer Cells/metabolism , Mice , Mice, Inbred C57BL , Pancreatitis/metabolism , Up-Regulation , fas Receptor , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
In this study we provide evidence that a human breast carcinoma cell line, MDA-MB-231 (MDA), can be made immunogenic following B7 transfection and that full T cell activation is obtained through cooperation of T-B lymphocytes via CD40-CD40L interactions. Tumor cells transfected with either B7 gene (MDAB7), neomycin-resistant gene only (MDAneo), or untransfected (MDA) were used in an allogeneic mixed lymphocyte tumor culture (MLTC) to investigate their ability to stimulate T cell proliferation and generate cytotoxic T lymphocytes (CTL). MDAB7 induced moderate T cell proliferation while MDAneo or MDA did not. Substantial T cell proliferation and de novo generation of cytolytic T cells was obtained only in response to MDAB7 when B cells were present during the MLTC. CD8+-purified T + B cells proliferated to a greater extent than whole T cell populations + B or CD4+ + B in response to MDAB7. Addition of alpha-B7-1 or alpha-CD40 in the MLTC inhibited T cell proliferation by 65 and 40%, respectively, whereas T cell proliferation and generation of CTL was completely abrogated when MLTC was performed in the presence of both antibodies. These data suggest that the engagement of CD40L on T cells with CD40 on B cells provides a costimulatory signal which, in synergism with TCR-dependent MDAB7-T cell recognition (signal 1) and B7/CD28 interactions (signal 2), leads to full T cell activation.
Subject(s)
B7-1 Antigen/pharmacology , Breast Neoplasms/immunology , CD40 Antigens/pharmacology , Lymphocyte Activation , Membrane Glycoproteins/pharmacology , T-Lymphocytes/immunology , B-Lymphocytes/immunology , B7-1 Antigen/immunology , B7-1 Antigen/metabolism , Breast Neoplasms/metabolism , CD40 Antigens/immunology , CD40 Antigens/metabolism , CD40 Ligand , CD8-Positive T-Lymphocytes/immunology , Humans , Ligands , Lymphocyte Cooperation , Lymphocyte Culture Test, Mixed , Membrane Glycoproteins/immunology , Membrane Glycoproteins/metabolism , Transfection/immunology , Tumor Cells, CulturedABSTRACT
P53 is a key regulator of many cellular processes and is negatively regulated by the human homolog of murine double minute-2 (MDM2) E3 ubiquitin ligase. Single nucleotide polymorphisms (SNPs) of either gene alone, and in combination, are linked to cancer susceptibility, disease progression, and therapy response. We analyzed the interaction of TP53 R72P and MDM2 SNP309 SNPs in relationship to outcome in patients with myelodysplastic syndromes (MDS). Sanger sequencing was performed on DNA isolated from 208 MDS cases. Utilizing a novel functional SNP scoring system ranging from +2 to -2 based on predicted p53 activity, we found statistically significant differences in overall survival (OS) (p = 0.02) and progression-free survival (PFS) (p = 0.02) in non-del(5q) MDS patients with low functional scores. In univariate analysis, only IPSS and the functional SNP score predicted OS and PFS in non-del(5q) patients. In multivariate analysis, the functional SNP score was independent of IPSS for OS and PFS. These data underscore the importance of TP53 R72P and MDM2 SNP309 SNPs in MDS, and provide a novel scoring system independent of IPSS that is predictive for disease outcome.
Subject(s)
Myelodysplastic Syndromes/genetics , Polymorphism, Single Nucleotide/genetics , Proto-Oncogene Proteins c-mdm2/genetics , Tumor Suppressor Protein p53/genetics , Adult , Aged , Aged, 80 and over , Disease-Free Survival , Female , Genotype , Humans , Kaplan-Meier Estimate , Male , Middle Aged , Myelodysplastic Syndromes/mortality , Prognosis , Proportional Hazards ModelsABSTRACT
Nonsynonymous TP53 exon 4 single-nucleotide polymorphism (SNP), R72P, is linked to cancer and mutagen susceptibility. R72P associations with specific cancer risk, particularly hematological malignancies, have been conflicting. Myelodysplastic syndrome (MDS) with chromosome 5q deletion is characterized by erythroid hypoplasia arising from lineage-specific p53 accumulation resulting from ribosomal insufficiency. We hypothesized that apoptotically diminished R72P C-allele may influence predisposition to del(5q) MDS. Bone marrow and blood DNA was sequenced from 705 MDS cases (333 del(5q), 372 non-del(5q)) and 157 controls. Genotype distribution did not significantly differ between del(5q) cases (12.6% CC, 38.1% CG, 49.2% GG), non-del(5q) cases (9.7% CC, 44.6% CG, 45.7% GG) and controls (7.6% CC, 37.6% CG, 54.8% GG) (P=0.13). Allele frequency did not differ between non-del(5q) and del(5q) cases (P=0.91) but trended towards increased C-allele frequency comparing non-del(5q) (P=0.08) and del(5q) (P=0.10) cases with controls. Median lenalidomide response duration increased proportionate to C-allele dosage in del(5q) patients (2.2 (CC), 1.3 (CG) and 0.89 years (GG)). Furthermore, C-allele homozygosity in del(5q) was associated with prolonged overall and progression-free survival and non-terminal interstitial deletions that excluded 5q34, whereas G-allele homozygozity was associated with inferior outcome and terminal deletions involving 5q34 (P=0.05). These findings comprise the largest MDS R72P SNP analysis.
Subject(s)
Chromosome Deletion , Myelodysplastic Syndromes/genetics , Tumor Suppressor Protein p53/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Gene Frequency , Humans , Kaplan-Meier Estimate , Male , Middle Aged , Mutation , Myelodysplastic Syndromes/drug therapy , Myelodysplastic Syndromes/pathology , Polymorphism, Single Nucleotide , Treatment OutcomeABSTRACT
The central hypothesis of our laboratory research program in large granular lymphocyte (LGL) leukemia is that leukemic LGL represent antigen-driven cytotoxic T lymphocytes (CTL) with characteristics of dysregulated apoptosis. The clinical features of LGL leukemia highlight the association of autoimmune diseases such as rheumatoid arthritis with the T-cell form of LGL leukemia. We therefore used LGL leukemia as a model disease of dysregulated apoptosis leading to both malignant and autoimmune diseases. Here, we review our understanding of survival signals activated in leukemic LGL in the context of knowledge concerning apoptotic pathways in activated normal lymphocytes.
Subject(s)
Apoptosis/immunology , Leukemia, Lymphoid/immunology , Leukemia, Lymphoid/pathology , Autoimmune Diseases/etiology , Humans , Killer Cells, Natural/pathology , Leukemia, Lymphoid/etiology , Signal Transduction/immunology , T-Lymphocytes, Cytotoxic/pathologyABSTRACT
Large granular lymphocyte (LGL) leukemia is a lymphoproliferative disorder often associated with rheumatoid arthritis. The etiology of LGL leukemia is not known. In order to better understand the pathogenesis of LGL leukemia, we analyzed differential gene expression using microarray technology. We found that approximately 80 genes were up-regulated and 12 genes were down-regulated when compared to normal peripheral blood mononuclear cells (PBMC). In the present study, we were interested in a group of genes involved in cytotoxic function. The up-regulated genes involved in cytotoxic function were serine proteinases (granzymes A, B, H and K) cysteine proteinases [cathepsin C, cathepsin W (lymphopain)], calpain small subunit and caspase-8. In addition, a pore-forming protein perforin, was also up-regulated. Northern blot analysis and RNase protection assays (RPA) confirmed that these genes were over-expressed in the majority of samples from LGL leukemia patients. Of interest, proteolytic inhibitors such as cystatin C, A, alpha-1 antitrypsin and metalloproteinase inhibitors were down-regulated in leukemic LGL when compared to normal peripheral blood mononuclear cells. Importantly, the pattern of gene expression in leukemic LGL resembles that seen in activated cytotoxic T cells (CTL).
Subject(s)
Cystatins/genetics , Endopeptidases/genetics , Gene Expression Regulation, Leukemic , Leukemia, Lymphoid/immunology , T-Lymphocytes, Cytotoxic/immunology , Cathepsin C/genetics , Cathepsin W , Cathepsins/genetics , Cystatin C , Cysteine Endopeptidases/genetics , Down-Regulation , Granzymes , Humans , Leukemia, Lymphoid/enzymology , Leukemia, Lymphoid/genetics , Serine Endopeptidases/geneticsABSTRACT
IL-2, first identified as a T cell growth factor, has been proven to activate many cell types including polymorphonuclear neutrophils (PMN3). However, the mechanisms involved in PMN activation, especially the signaling pathways used by the IL-2R, are currently unknown. Here we demonstrate that IL-2 has the ability to induce protein tyrosine kinases in human PMN, and we provide the first evidence that lyn kinase is activated and physically associated with MAP kinase/ERK1. Co-immunoprecipitation experiments with anti-IL-2Rbeta and Western blotting with anti-p53/56lym revealed that lyn protein was present in IL-2R precipitates and that the association of lyn with IL-2Rbeta was markedly elevated by IL-2 stimulation. Furthermore the activity of lyn kinase, evaluated by an in vitro kinase assay with enolase as a substrate, increased following IL-2 stimulation. Another important finding was that, upon IL-2 activation, MAPK/ERK1 was also phosphorylated in PMN. A direct association between lyn and ERK1 was initially demonstrated by co-immunoprecipitation/Western blotting and then definitively proven by the use of a GST-ERK1 fusion protein. We showed that ERK1 binds lyn only in IL-2 stimulated PMN, but not in unstimulated PMN. These results suggest that IL-2 can promote the association of lyn protein tyrosine kinase with IL-2Rbeta as well as the direct binding of MAPK/ERK1 to lyn. The signaling pathway utilized by human PMN in response to IL-2 may thus involve the association of lyn with IL-2Rbeta and the activation process also triggers the recruitment and activation of a specific ERK.
Subject(s)
Interleukin-2/metabolism , Mitogen-Activated Protein Kinases/metabolism , Neutrophils/metabolism , Receptors, Interleukin-2/metabolism , Signal Transduction/physiology , src-Family Kinases/metabolism , Cells, Cultured , Enzyme Activation , Humans , Interleukin-2/pharmacology , Mitogen-Activated Protein Kinase 3 , Neutrophils/cytology , Neutrophils/drug effects , Phosphorylation , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-hck , Tyrosine/metabolismABSTRACT
Liver injury is an important prognostic indicator during acute pancreatitis. The aim of this study was to determine the role of Fas ligand (FasL) in hepatocyte injury. Liver parenchymal enzymes were measured in cocultures of hepatocytes and Kupffer cells treated with elastase. FasL and FasL mRNA were measured in elastase-treated Kupffer cells. Hepatocytes were treated with FasL and their viability was assessed by monotetrazolium (MTT), apoptosis by flow cytometry, as well as caspase-3 and p38-mitogen-activated protein kinase (MAPK) by immunoblotting. Elastase increased aspartate aminotransferase and lactate dehydrogenase in cocultures of hepatocyte and Kupffer cells (P<0.040). Elastase increased FasL production from Kupffer cells (P=0.02) and upregulated FasL mRNA (FasL/beta-2 microglobulin (BMG): 0.23+/-0.03 vs. 0.11+/-0.003; P=0.04). FasL increased alanine aminotransferase and lactate dehydrogenase (P<0.03) and reduced hepatocyte viability by 45% (P=0.01). FasL increased the number of dually labeled cells with AnnexinV/7AAD (P=0.03) while upregulating cleavage of caspase-3 and the phosphorylation of p38-MAPK. FasL antibody attenuated the FasL-related increase in dually labeled cells (P=0.02), the cleavage of caspase-3, and phosphorylation of p38-MAPK. Pancreatic elastase upregulates FasL within Kupffer cells. FasL induces hepatocyte injury and death and upregulates p38-MAPK and caspase-3 within hepatocytes. The ability to manipulate interactions between Kupffer cells and hepatocytes may have important therapeutic implications.