T-cell Receptor Signaling Pathway Research Articles

Abstract 546: Systemic Transcriptional Alterations of Innate-Adaptive Immune Signaling Pathways in Atherosclerosis, Ischemic Stroke and Myocardial Infarction

Objective: The immune system regulates inflammation associated with chronic cardiovascular disease. The primary aim of this study was to compare gene expression profiles of three innate and adaptive immune signaling pathways --the toll-like receptor (TLR), T-cell receptor (TCR), and B-cell receptor (BCR) signaling pathways in patients with subclinical atherosclerosis, acute ischemic stroke (IS), and myocardial infarction (MI). Methods: Peripheral blood gene expression profiles from human atherosclerotic diseases were downloaded from Gene Expression Omnibus (GEO): Atherosclerosis (GSE20129: 48 patients, 71 controls), IS (GSE16561: 39 patients - samples within 24 hours from symptom onset, 24 controls), and MI (GSE29111, 18 MI patients (7 and 30 days) samples after experiencing MI). Genes in the TLR (106 genes), TCR (104 genes), and BCR (72 genes) pathways were retrieved from the NCBI BioSystems database. Gene set enrichment analysis was used to evaluate the statistical significance of gene enrichment in each signaling pathway for each study. Weighted Gene Co-expression Network Analysis was used to identify gene modules and evaluate network connectivity across the disease states. Results: All three immune pathways significantly enriched differentially expressed genes in each disease state (P<0.01) - TLR (38% in atherosclerosis, 47% in IS, 30% in MI); TCR (51% in atherosclerosis, 54% in IS, and 53% in MI); BCR (57% in atherosclerosis; 60% in IS; 43% in MI). Transcriptional changes in the pathways are highly concordant across the diseases. The correlation of expression changes between any two diseases ranges from 0.53 to 0.76 (mean±SD: 0.66±0.09, P<0.00001). Although TLR genes were all up-regulated in each condition, network analysis revealed a striking down-regulation of gene modules in the TCR and BCR pathways. Of significance, the TCR-CD3 complex and CD79A/B were significantly down-regulated in patients of all three disease states. Conclusion: We found a strong overlap in expression between innate and adaptive immune pathways in three atherosclerotic related diseases. The down regulation of the TCR-CD3 complex suggests immune suppression is a common occurrence in either chronic or acute inflammation associated atherosclerotic disease.

CRACR2A encodes a novel Ca2-binding Rab GTPase important for T cell receptor signalling (IRM10P.748)

Abstract Ca2+ signalling is important for T cell stimulation and its impairment leads to immune deficiency. In T cells, Ca2+ entry is mediated by CRAC (Ca2+-release activated Ca2+) channels that consist of the pore component, Orai1 and the endoplasmic reticulum Ca2+ sensor, STIM1. We recently identified CRACR2A (CRAC channel regulator 2A) as an interacting partner of Orai1 and STIM1. Here we investigate the role of a novel isoform of CRACR2A that is predominantly expressed in immune cells. CRACR2A contains multiple functional domains including Ca2+-binding motifs and a C-terminal Rab GTPase domain. Different from small Rab GTPases, CRACR2A utilized a two-step activation mechanism of GTP binding and T cell receptor (TCR) stimulation to mediate its translocation into the immunological synapse. Defect in GTP binding or hydrolysis activity results in loss and gain of function respectively. CRACR2A played a predominant role in Ca2+ and mitogen-activated protein kinase (MAPK) signalling pathways. Furthermore, CRACR2A-deficient T cells showed impairment in Th1 differentiation. In support of these data, mice with T cell-specific deletion of CRACR2A showed a strong resistance to experimental autoimmune encephalomyelitis. Previously, small GTPase has been shown to play an important role in TCR signaling. Our study identifies the unexpected usage of a large Rab GTPase as a molecular switch to regulate TCR signalling pathways.

The A2 gene of alcelaphine herpesvirus-1 is a transcriptional regulator affecting cytotoxicity in virus-infected T cells but is not required for malignant catarrhal fever induction in rabbits

Alcelaphine herpesvirus-1 (AlHV-1) causes malignant catarrhal fever (MCF). The A2 gene of AlHV-1 is a member of the bZIP transcription factor family. We wished to determine whether A2 is a virulence gene or not and whether it is involved in pathogenesis by interference with host transcription pathways. An A2 gene knockout (A2ΔAlHV-1) virus, revertant (A2revAlHV-1) virus, and wild-type virus (wtAlHV-1) were used to infect three groups of rabbits. A2ΔAlHV-1-infected rabbits succumbed to MCF, albeit with a delayed onset compared to the control groups, so A2 is not a critical virulence factor. Differential gene transcription analysis by RNAseq and qRT-PCR validation of a selection of these was performed in infected large granular lymphocyte (LGL) T cells obtained in culture from the MCF-affected animals. A2 was involved in the transcriptional regulation of immunological, cell cycle and apoptosis pathways. In particular, there was a bias towards γδ T cell receptor (TCR) expression and downregulation of αβ TCR. TCR signalling, apoptosis, cell cycle, IFN-γ and NFAT pathways were affected. Of particular interest was partial inhibition of the cytotoxicity-associated pathways involving perforin and the granzymes A and B in the A2ΔAlHV-1-infected LGLs compared to controls. In functional assays, A2ΔAlHV-1-infected LGLs were significantly less cytotoxic than wtAlHV-1- and A2revAlHV-1-infected LGLs using rabbit corneal epithelial cells (SIRC) as targets. This implies that A2 is involved in a pathway enhancing the expression of LGL cytotoxicity. This is important as virus-infected T cell cytotoxicity in vivo has been suggested as a potential mechanism of disease induction in MCF.

Lead poisoning influences TCR-related gene expression patterns in peripheral blood T-lymphocytes of exposed workers

Previous studies have shown that occupational lead (Pb) exposure might influence human T-lymphocyte function, including such as changes in T-cell receptor (TCR) Vβ and Vγ repertoire and in expression of the TCRζ gene. Thus, the study here further investigated expression of TCRζ-related factors and the FcɛRIγ gene (whose product has a functional role complementary to the TCRζ chain) and the Elf-1 gene whose product is involved in regulation of TCR expression. Quantitative real-time RT-PCR was used to measure expression of TCRζ, FcɛRIγ, and Elf-1 genes in peripheral blood mononuclear cells (PBMC) isolated from 17 Pb-exposed workers. Samples were collected before and after the workers had undergone chelation therapy regimens. Twenty-three healthy individuals served as controls. The results showed that TCRζ, FcɛRIγ, and Elf-1 gene expression in Pb-exposed workers before chelation therapy was significantly lower than in PBMC from healthy individuals. After chelation therapy, expression of TCRζ appeared to trend toward normal levels; in comparison, lower expressions of FcɛRIγ and Elf-1 persisted. In conclusion, the previously-documented impairment of T-lymphocyte functions and T- lymphocyte-mediated immune responses seen previously in response to occupational Pb exposure might be attributable, in part, to effects on TCR signaling pathways - including those related to TCRζ and FcɛRIγ - and to any down-regulation of membrane TCRζ expression/activity that might be associated with Pb-induced effects on Elf-1 expression.

Alternative Capture of Noncoding RNAs or Protein-Coding Genes by Herpesviruses to Alter Host T Cell Function

In marmoset Tcells transformed by Herpesvirus saimiri (HVS), a viral U-rich noncoding (nc) RNA, HSUR 1, specifically mediates degradation of host microRNA-27 (miR-27). High-throughput sequencing of RNA after crosslinking immunoprecipitation (HITS-CLIP) identified mRNAs targeted by miR-27 as enriched in the Tcell receptor (TCR) signaling pathway, including GRB2. Accordingly, transfection of miR-27 into human Tcells attenuates TCR-induced activation of mitogen-activated protein kinases (MAPKs) and induction of CD69. MiR-27 also robustly regulates SEMA7A and IFN-γ, key modulators and effectors of Tcell function. Knockdown or ectopic expression of HSUR 1 alters levels of these proteins in virally transformed cells. Two other T-lymphotropic γ-herpesviruses, AlHV-1 and OvHV-2, do not produce a noncoding RNA to downregulate miR-27 but instead encode homologs of miR-27 target genes. Thus, oncogenic γ-herpesviruses have evolved diverse strategies to converge on common targets in host Tcells.

Lymphoid-Specific Tyrosine Phosphatase (Lyp): A Potential Drug Target For Treatment of Autoimmune Diseases

Lymphoid-tyrosine phosphatase (Lyp), encoded by the PTPN22 gene, is a member of the protein tyrosine phosphatase family enzymes. Human genetics studies have shown that a single-nucleotide polymorphism in PTPN22 is often mutated in patients suffering from autoimmune diseases such as type 1 diabetes, rheumatoid arthritis, and systemic lupus erythematosis. Because of its critical role in the regulation of T-cell Receptor (TCR) signaling pathways, Lyp recently emerged as a candidate target for therapy of autoimmune diseases. Herein, we review the structure and splice isoforms of Lyp, the biochemistry of the disease-predisposing allele, discuss the function of the phosphatase in TCR signaling and the association with human autoimmune diseases. Especially, we summarized recent progress in the development of Lyp inhibitors, intending to provide a basis for the Lyp-based treatment of autoimmunity. Moreover, the emphasis and direction for future study of Lyp in autoimmune diseases were prospected.

Naive CD4 T-cell activation identifies MS patients having rapid transition to progressive MS

Objective:Our objective was to determine whether altered naive CD4 T-cell biology contributes to development of disease progression in secondary progressive multiple sclerosis (SPMS).Methods:We compared the naive CD4 T-cell gene expression profiles of 19 patients with SPMS and 14 healthy controls (HCs) using a whole-genome microarray approach. We analyzed surface protein expression of critical genes by flow cytometry after T-cell receptor (TCR) stimulation of naive CD4 T cells isolated from HCs and patients with SPMS.Results:Hierarchical clustering segregated patients with SPMS into 2 subgroups: SP-1, which had a short duration of relapsing-remitting multiple sclerosis (MS), and SP-2, which had a long duration of relapsing-remitting MS. SP-1 patients upregulated numerous immune genes, including genes within TCR and toll-like receptor (TLR) signaling pathways. SP-2 patients showed immune gene downregulation in comparison with HCs. We identified an SP-1–specific transcriptional signature of 3 genes (TLR4, TLR2, and chemokine receptor 1), and these genes had higher surface protein expression in SP-1 than in SP-2. After TCR stimulation for 48 hours, only SP-1 showed a progressive linear increase in TLR2 and TLR4 protein expression.Conclusions:Differences in naive CD4 T-cell biology, notably of TCR and TLR signaling pathways, identified patients with MS with more rapid conversion to secondary progression, a critical determinant of long-term disability in MS.

A genome-wide association study combining pathway analysis for typical sporadic amyotrophic lateral sclerosis in Chinese Han populations

Sporadic amyotrophic lateral sclerosis (sALS) is a severe neurodegenerative disease that causes progressive motor neuron death. Although the etiology of sALS remains unknown, genetic variants are thought to predispose individuals to the disease. Several recent genome-wide association studies have identified a number of loci that increase sALS susceptibility, but these only explain a small proportion of the disease. To extend the current genetic evidence and to identify novel candidates of sALS, we performed a pooling genome-wide association study by 859,311 autosomal single-nucleotide polymorphisms of IlluminaHumanOmniZhongHua-8 combining pathway analysis in 250 typical sALS cases precluding age, clinical course, and phenotype interference and 250 control subjects from Chinese Han populations (CHP). The results revealed that 8 novel loci of 1p34.3, 3p21.1, 3p22.2, 10p15.2, 22q12.1, 3q13.11, 11q25, 12q24.33, and 5 previously reported loci of CNTN4 (kgp11325216), ATXN1 (kgp8327591), C9orf72 (kgp6016770), ITPR2 (kgp3041552), and SOD1 (kgp10760302) were associated with sALS from CHP. Furthermore, the pathway analysis based on the Gene Set Analysis Toolkit V2 showed that 10 top pathways were strongly associated with sALS from CHP, and among them, the 7 most potentially candidate pathways were phosphatidylinositol signaling system, Wnt signaling pathway, axon guidance, MAPK signaling pathway, neurotrophin signaling pathway, arachidonic acid metabolism, and T-cell receptor signaling pathway, a total of 39 significantly associate genes in 7 candidate pathways was suggested to involve in the pathogenesis of sALS from CHP. In conclusion, our results revealed several new loci and pathways related to sALS from CHP and extend the association evidence for partial loci, genes, and pathways, which were previously identified in other populations. Thus, our data provided new clues for exploring the pathogenesis of sALS.

Development and maturation of the immune system in preterm neonates: results from a whole genome expression study.

To expand the knowledge about the consecutive expression of genes involved in the immune system development in preterm neonates and to verify if the environment changes the gene expression after birth we conducted a prospective study that included three cohorts: (A) extremely (gestational age (GA): 23–26 weeks; n = 41), (B) very (GA: 27–29 weeks; n = 39), and (C) moderately preterm infants (GA: 30–32 weeks; n = 33). Blood samples were drawn from the study participants on the 5th and 28th day of life (DOL). The mRNA samples were evaluated for gene expression with the use of GeneChip Human Gene 1.0ST microarrays. Differential expression analysis revealed small subsets of genes that presented positive or negative monotone trends in both the 5th (138 genes) and 28th DOL (308 genes) in the three subgroups of patients. Based on pathway enrichment analysis, we found that most of the pathways that revealed a positive monotone trend were involved in host immunity. The most significantly GA dependent pathways were T-cell receptor signaling pathway and intestinal immune network for IgA production. Overall 4431 genes were differentially expressed between the 5th and 28th DOL. Despite differences in gestational age, patients with the same postconceptional age have a very similar expression of genes.

Down Regulation of T Cell Receptor Expression in COPD Pulmonary CD8 Cells

CD8 cells may contribute towards an autoimmune process in COPD. Down regulation of T cell receptor (TCR) signalling molecules occurs in autoimmune diseases with consequent T cell dysfunction. We hypothesise that TCR signalling is abnormal in COPD pulmonary CD8 cells. Micro-array gene expression analysis of blood and pulmonary COPD CD8 samples was performed and compared to pulmonary CD8 cells from smoker controls (S). We focused on the TCR signalling pathway, with validation of key findings using polymerase chain reaction and immunofluorescence. TCR signalling molecules in COPD pulmonary CD8 cells were down regulated compared to blood CD8 cells (CD247: fold change (FC) −2.43, Q = 0.001; LCK: FC −2.25, Q = 0.01). Micro-array analysis revealed no significant differences between COPD and S pulmonary CD8 cells. However, PCR revealed significantly lower gene expression levels of CD247 (FC −1.79, p = 0.04) and LCK (FC −1.77, p = 0.01) in COPD compared to S pulmonary CD8 cells. CD247 down regulation in COPD CD8 cells was confirmed by immunofluorescent staining of bronchoalveolar lavage cells: Significantly fewer COPD CD8 cells co-expressed CD247 compared to healthy non-smoker CD8 cells (mean 88.9 vs 75.2%, p<0.05) There is down regulation of TCR signalling molecules in COPD pulmonary CD8 cells. This may cause T cell dysfunction.

Quantitative profiling of tyrosine phosphorylation revealed changes in the activity of the T cell receptor signaling pathway upon cisplatin-induced apoptosis

In order to better understand the cellular responses to the chemotherapeutic drug cisplatin and the mechanisms leading to apoptosis and potential side effects, we performed a SILAC-based quantitative phosphotyrosine analysis of Jurkat T cells exposed to cisplatin. Signaling molecules in the T cell receptor (TCR) pathway were enriched among proteins displaying reduced phosphorylation levels. The results were verified by immunoblotting and/or phospho-flow cytometry for a selected set of proteins, including the tyrosine kinases Lck and Zap70, and downstream targets Itk, Plcγ1 and Erk. In contrast to the effects on the T cell signaling pathways, the dually phosphorylated form of p38α MAPK was increased in treated cells, and activation of this signaling pathway was verified by immunoblot analysis of phosphorylation levels of p38α MAPK and the downstream targets Atf2 and MAPKAPK2. Activation of the p38α MAPK signaling pathway has been suggested to be one of the main mechanisms by which cisplatin induces apoptosis. Our results indicate that cisplatin may reduce the activity of proteins involved in the TCR signaling pathway, which has an important role in regulating proliferation of T cells, and may contribute to explain previous observations where cisplatin has been reported to inhibit proliferation of T cells. In this study, a quantitative phosphotyrosine analysis was performed to identify changes of the phosphoproteome during exposure of Jurkat T cells by cisplatin. The results of the phosphoproteome analysis were complemented with immunoblotting and temporal phospho-flow analysis. An initial activation of the p38α MAPK signaling pathway was detected at early time points of cisplatin treatment, a response previously suggested to be part of the mechanism by which cisplatin induces apoptosis. Furthermore, reduced phosphorylation levels of proteins involved in signaling downstream of the TCR during apoptosis were found by the phosphotyrosine proteome analysis. Our study can support to elucidate the mechanism behind the previously observed immunosuppressive effect of cisplatin.

CD25 and CD69 induction by α4β1 outside-in signalling requires TCR early signalling complex proteins

Distinct signalling pathways producing diverse cellular outcomes can utilize similar subsets of proteins. For example, proteins from the TCR (T-cell receptor) ESC (early signalling complex) are also involved in interferon-α receptor signalling. Defining the mechanism for how these proteins function within a given pathway is important in understanding the integration and communication of signalling networks with one another. We investigated the contributions of the TCR ESC proteins Lck (lymphocyte-specific kinase), ZAP-70 (ζ-chain-associated protein of 70 kDa), Vav1, SLP-76 [SH2 (Src homology 2)-domain-containing leukocyte protein of 76 kDa] and LAT (linker for activation of T-cells) to integrin outside-in signalling in human T-cells. Lck, ZAP-70, SLP-76, Vav1 and LAT were activated by α4β1 outside-in signalling, but in a manner different from TCR signalling. TCR stimulation recruits ESC proteins to activate the mitogen-activated protein kinase ERK (extracellular-signal-regulated kinase). α4β1 outside-in-mediated ERK activation did not require TCR ESC proteins. However, α4β1 outside-in signalling induced CD25 and co-stimulated CD69 and this was dependent on TCR ESC proteins. TCR and α4β1 outside-in signalling are integrated through the common use of TCR ESC proteins; however, these proteins display functionally distinct roles in these pathways. These novel insights into the cross-talk between integrin outside-in and TCR signalling pathways are highly relevant to the development of therapeutic strategies to overcome disease associated with T-cell deregulation.

Alteration of human blood cell transcriptome in uremia

BackgroundEnd-stage renal failure is associated with profound changes in physiology and health, but the molecular causation of these pleomorphic effects termed “uremia” is poorly understood. The genomic changes of uremia were explored in a whole genome microarray case-control comparison of 95 subjects with end-stage renal failure (n = 75) or healthy controls (n = 20).MethodsRNA was separated from blood drawn in PAXgene tubes and gene expression analyzed using Affymetrix Human Genome U133 Plus 2.0 arrays. Quality control and normalization was performed, and statistical significance determined with multiple test corrections (qFDR). Biological interpretation was aided by knowledge mining using NIH DAVID, MetaCore and PubGeneResultsOver 9,000 genes were differentially expressed in uremic subjects compared to normal controls (fold change: -5.3 to +6.8), and more than 65% were lower in uremia. Changes appeared to be regulated through key gene networks involving cMYC, SP1, P53, AP1, NFkB, HNF4 alpha, HIF1A, c-Jun, STAT1, STAT3 and CREB1. Gene set enrichment analysis showed that mRNA processing and transport, protein transport, chaperone functions, the unfolded protein response and genes involved in tumor genesis were prominently lower in uremia, while insulin-like growth factor activity, neuroactive receptor interaction, the complement system, lipoprotein metabolism and lipid transport were higher in uremia. Pathways involving cytoskeletal remodeling, the clathrin-coated endosomal pathway, T-cell receptor signaling and CD28 pathways, and many immune and biological mechanisms were significantly down-regulated, while the ubiquitin pathway and certain others were up-regulated.ConclusionsEnd-stage renal failure is associated with profound changes in human gene expression which appears to be mediated through key transcription factors. Dialysis and primary kidney disease had minor effects on gene regulation, but uremia was the dominant influence in the changes observed. This data provides important insight into the changes in cellular biology and function, opportunities for biomarkers of disease progression and therapy, and potential targets for intervention in uremia.

New insights into the catalytic mechanism of histidine phosphatases revealed by a functionally essential arginine residue within the active site of the Sts phosphatases

Sts (suppressor of T-cell receptor signalling)-1 and Sts-2 are HPs (histidine phosphatases) that negatively regulate TCR (T-cell receptor) signalling pathways, including those involved in cytokine production. HPs play key roles in such varied biological processes as metabolism, development and intracellular signalling. They differ considerably in their primary sequence and substrate specificity, but possess a catalytic core formed by an invariant quartet of active-site residues. Two histidine and two arginine residues cluster together within the HP active site and are thought to participate in a two-step dephosphorylation reaction. To date there has been little insight into any additional residues that might play an important functional role. In the present study, we identify and characterize an additional residue within the Sts phosphatases (Sts-1 Arg383 or Sts-2 Arg369) that is critical for catalytic activity and intracellular function. Mutation of Sts-1 Arg383 to an alanine residue compromises the enzyme's activity and renders Sts-1 unable to suppress TCR-induced cytokine induction. Of the multiple amino acids substituted for Arg383, only lysine partially rescues the catalytic activity of Sts-1. Although Sts-1 Arg383 is conserved in all Sts homologues, it is only conserved in one of the two sub-branches of HPs. The results of the present study highlight an essential role for Sts-1 phosphatase activity in regulating T-cell activation and add a new dimension of complexity to our understanding of HP catalytic activity.

Regulation of T cell function by the ubiquitin-specific protease USP9X via modulating the Carma1-Bcl10-Malt1 complex

The ubiquitin conjugation system plays an important role in immune regulation; however, the ubiquitin-specific proteases (USPs) that carry out deubiquitination of cellular substrates are poorly understood. Here we show that in vivo knockdown of the deubiquitinating enzyme USP9X attenuates T-cell proliferation. In addition, naïve CD4(+) T cells from USP9X knockdown chimeric mice display decreased cytokine production and T helper cell differentiation in vitro, which we confirmed in vivo by performing adoptive transfer of transgenic T cells and subsequent immunization. USP9X silencing in both a human T-cell line and mouse primary T cells reduced T-cell receptor (TCR) signaling-induced NF-κB activation. Mechanistically, USP9X interacts with Bcl10 of the Carma1-Bcl10-Malt1 (CBM) complex and removes the TCR-induced ubiquitin chain from Bcl10, which facilitates the association of Carma1 with Bcl0-Malt1. These results demonstrate that USP9X is a crucial positive regulator of the TCR signaling pathway and is required for T-cell function through the modulation of CBM complex formation.

Prior epigenetic priming of cytokine genes in naive T cells is required for their subsequent activation by inducible enhancers

The inducible lL-3 and GM-CSF genes can only be efficiently expressed in T cells once they have been through a previous cycle of activation and undergone a process termed blast cell transformation [1]. An initial stimulus is required to bring naive T cells out of the resting state and into the cell cycle. This process is triggered by T cell receptor (TCR) stimulation, takes about 24 hours, and is associated with extensive nuclear remodeling. Once primed by a cycle of activation, T blast cells can maintain their ability to express lL-3 and GM-CSF for many cell divisions without the continual need for additional stimuli. In contrast, the lL-3 and GM-CSF genes cannot be induced in naive T cells that have never received a TCR stimulus. We show that this pattern of regulation of the IL-3/GM-CSF locus is controlled at two distinct levels: (1) During blast cell transformation, the IL-3/GM-CSF locus acquires an extensive array of DNase I Hypersensitive Sites (DHSs) which are then maintained indefinitely for many cell cycles [1,2]. These primed DHSs are marked by me2K4 histone H3, and they also persist in non-dividing memory T cells in the peripheral blood [1]. These DHSs are absent in the thymus, spleen T cells, and naive T cells in the blood. These DHSs do not function as classical enhancers, and we propose that they serve to maintain an active chromatin structure in previously activated T cells. (2) The expression of the lL-3 and GM-CSF genes is in each case dependent on the activation of inducible upstream enhancers. The lL-3 and GM-CSF enhancers appear as inducible DHSs T blast cells within 20 min of stimulation, and only acquire the me2K4H3 modification after stimulation. These enhancers are dependant on the TCR inducible factors NFAT and AP-1. However, although AP-1 and NFAT family mRNAs are efficiently expressed in both naive T cells and in T blast cells, the enhancers only respond to induction of these factors in T blast cells. The inducible DHSs remain completely undetectable in naive T cells even after 4 hours of stimulation with direct activators of TCR signaling pathways (PMA and Calcium ionophore). We propose that T blast cells and memory T cells have developed a strategy of maintaining a discrete class of DHS as epigenetic memory modules that support an accessible chromatin environment and thereby prime inducible genes for efficient re-activation when memory cells re-encounter Ag stimuli. In the absence of the priming elements, the lL-3/GM-CSF locus appears to remain inaccessible to transcription factors that are otherwise very efficient at recruiting the chromatin remodelers that create DHSs within the inducible enhancers.

Histone deacetylase 1 and 2 are essential for normal T-cell development and genomic stability in mice

AbstractHistone deacetylase 1 and 2 (HDAC1/2) regulate chromatin structure as the catalytic core of the Sin3A, NuRD and CoREST co-repressor complexes. To better understand the key pathways regulated by HDAC1/2 in the adaptive immune system and inform their exploitation as drug targets, we have generated mice with a T-cell specific deletion. Loss of either HDAC1 or HDAC2 alone has little effect, while dual inactivation results in a 5-fold reduction in thymocyte cellularity, accompanied by developmental arrest at the double-negative to double-positive transition. Transcriptome analysis revealed 892 mis-regulated genes in Hdac1/2 knock-out thymocytes, including down-regulation of LAT, Themis and Itk, key components of the T-cell receptor (TCR) signaling pathway. Down-regulation of these genes suggests a model in which HDAC1/2 deficiency results in defective propagation of TCR signaling, thus blocking development. Furthermore, mice with reduced HDAC1/2 activity (Hdac1 deleted and a single Hdac2 allele) develop a lethal pathology by 3-months of age, caused by neoplastic transformation of immature T cells in the thymus. Tumor cells become aneuploid, express increased levels of c-Myc and show elevated levels of the DNA damage marker, γH2AX. These data demonstrate a crucial role for HDAC1/2 in T-cell development and the maintenance of genomic stability.

Gene Expression Profile of Peripheral Blood Lymphocytes from Renal Cell Carcinoma Patients Treated with IL-2, Interferon-α and Dendritic Cell Vaccine

Lymphocytes are a key component of the immune system and their differentiation and function are directly influenced by cancer. We examined peripheral blood lymphocyte (PBL) gene expression as a biomarker of illness and treatment effect using the Affymetrix Human Gene ST1 platform in patients with metastatic renal cell carcinoma (mRCC) who received combined treatment with IL-2, interferon-?-2a and dendritic cell vaccine. We examined gene expression, cytokine levels in patient serum and lymphocyte subsets as determined by flow cytometry (FCM). Pre-treatment PBLs from patients with mRCC exhibit a gene expression profile and serum cytokine profile consistent with inflammation and proliferation not found in healthy donors (HD). PBL gene expression from patients with mRCC showed increased mRNA of genes involved with T-cell and TREG-cell activation pathways, which was also reflected in lymphocyte subset distribution. Overall, PBL gene expression post-treatment (POST) was not significantly different than pre-treatment (PRE). Nevertheless, treatment related changes in gene expression (post-treatment minus pre-treatment) revealed an increased expression of T-cell and B-cell receptor signaling pathways in responding (R) patients compared to non-responding (NR) patients. In addition, we observed down-regulation of TREG-cell pathways post-treatment in R vs. NR patients. While exploratory in nature, this study supports the hypothesis that enhanced inflammatory cytotoxic pathways coupled with blunting of the regulatory pathways is necessary for effective anti-cancer activity associated with immune therapy. This type of analysis can potentially identify additional immune therapeutic targets in patients with mRCC.

Membrane-Associated Proteinase 3 On Granulocytes and Myeloid Leukemia Mediates Reversible Inhibition of T Cell Proliferation Associated with Altered ZAP70 and ERK Signaling

Abstract 1031Proteinase 3 (P3), a serine protease constitutively expressed in primary granules and on the membrane of some resting granulocytes, is the target of T cell-mediated autoimmunity in Wegener's granulomatosis (WG) and of anti-leukemia immunity mediated by PR1 (VLQELNVTV)-specific cytotoxic T lymphocytes (PR1-CTL). We have previously shown anti-CD3/CD28 induced proliferation of healthy donor T-cells to be significantly inhibited by peripheral blood polymorphonuclear neutrophils (PMNs) expressing membrane P3 (mP3) at a ratio of 3 PMNs to 1 PBMC. Our results indicate that mP3+ PMNs begin to exert inhibitory effects on T cell proliferation at a ratio of 2.2 mP3+ PMNs to 1 PBMC, a ratio greater than that seen in normal homeostatic conditions in peripheral blood. The inhibition was predominantly enzyme-independent and dose-dependent. Notably soluble P3 exerted similar effects on T cells as was seen with mP3. Additionally soluble P3 induced a G0 cell cycle arrest. Of significance, soluble P3 in acute myeloid leukemia (AML) patient serum can be up to 5-fold higher than that seen in healthy control serum.To confirm mP3 specificity, we FAC-sorted PMNs based on the mP3 co-expressed CD177 molecule to obtain highly purified (>98%) mP3+ and mP3− PMNs. Compared to activated PBMC alone, activated PBMC co-cultured at a ratio of 1:3 with mP3+ PMNs showed 58% and 57% inhibition of CD8+ and CD4+ T cells, respectively (CD8+ and CD4+: p< 0.003). PBMC co-cultured at that same ratio with mP3− PMNs showed less inhibition - only 29% and 26% inhibition of CD8+ and CD4+ T cells, respectively (CD8+: p<0.05; CD4+: p=ns). Inhibition of T cell proliferation by both mP3+PMNs and soluble P3 was blocked by anti-P3 mAbs but not by isotype-matched mAb. Furthermore, P3-mediated inhibition of T-cell proliferation is reversible since removal of PMNs or soluble P3 restored the proliferative capacity of the T cells.Because P3 is over-expressed in AML and chronic myeloid leukemia (CML), we hypothesized that mP3+ leukemia may suppress T-cell proliferation. Healthy donor T cell proliferation was studied with CFSE after stimulation with anti-CD3/CD28 mAbs in the presence or absence of mP3+ AML for five days. AML mediated a dose-dependent inhibition of T-cell proliferation contingent upon the level of mP3 expression (p<0.0001). Co-incubation of PBMC with AML displaying 91% mP3 positivity, reduced proliferation of CD8+ T cells to 29.6%, compared to 94.7% in the PBMC culture alone. This inhibition could be completely abrogated by addition of anti-P3 mAb, restoring the proliferation of CD8+ T cells to a level comparable to that seen in control. In contrast, no inhibition of CD8+ T cell proliferation was observed in co-cultures of T cells with AML in which only 6% of the AML cells expressed mP3. Thus, there is an inverse correlation between percent proliferation of T cells and the amount of mP3 on AML (R=0.4539, p<0.0001). In addition, AML expressing 91% mP3+ induced apoptosis of > 70% of the T cells at a ratio of 10 AML: 1 PBMC as assessed by uptake of aqua dye. The association between the amount of mP3 on AML and percent of apoptosis was significant (R=0.7852, p<0.0001), and apoptosis induced by mP3+AML appeared to be specific since T cells did not undergo apoptosis when anti-P3 mAb was added. The same correlation was not seen after PMNs from healthy donors (% mP3+: 62%±21.7, n=14) were co-incubated with PBMC. The percentage of cells undergoing apoptosis was less than 10%, regardless of the extent of PMN mP3 positivity. Of note, mP3 expression is significantly higher in bone marrow myeloid derived suppressor cells (MDSC) from leukemia patients compared to MDSC from healthy donors (79.4±5.23% (n=7), 22.4±11.55% (n=3), respectively; p= 0.0007). Because mP3 inhibited proliferation of T cells stimulated via the T cell receptor (TCR), i.e. anti-CD3/CD28, we compared effects of mP3 on ZAP70 and ERK signaling by phosphoflow cytometry. ZAP70 phosphorylation in CD8+ and CD4+ T cells was reduced by 78% and 80%, respectively, within 5 minutes of co-incubation with mP3+ PMNs compared with T cells stimulated with anti-CD3/CD28 mAbs alone, while ERK1/2 phosphorylation was completely blocked within 10 minutes, suggesting that P3-mediated inhibition of T cell proliferation involves downstream TCR signaling pathways. Taken together, these data support an important new function of membrane-bound P3 on PMN and leukemia in controlling adaptive T cell immunity. Disclosures:No relevant conflicts of interest to declare.

FLT3-Kinase Inhibitors Quizartinib and Midostaurin Do Not Impair T-Cell Reactivity and Activation

AbstractAbstract 1045In patients with FLT3-ITD mutated AML, FLT3-inhibitors have been used successfully as a ‘bridging therapy' before allogeneic transplantation. Inhibitors of other kinases (such as imatinib for BCR-ABL positive CML) have previously been used successfully after allogeneic transplantation – even before discontinuation of immunosuppressive medication. However, it is known that some BCR-ABL inhibitors such as dasatinib exert strong inhibitory effects on primary T-cells through inhibition of Src-kinases relevant for T-cell receptor signaling. Even imatinib and nilotinib - although not affecting Src kinase activity – showed decreased T-cell activation and reactivity to some extent. Thus, the influence of FLT3-kinase inhibitors on T-cell function may be critical in the context of allogeneic bone marrow transplantation for FLT3-ITD-positive AML. Besides inhibition of FLT3-kinase, midostaurin (PKC412) exerts activity against PDGFR, VEGFR or c-KIT. In contrast, second generation inhibitors such as quizartinib (AC220) act in a far more FLT3-specific manner.Therefore, we aimed to investigate the effects of both clinically relevant FLT3-inhibitors on T-cell receptor signaling in comparison to the well characterized and potent BCR-ABL inhibitor dasatinib. Investigating primary T-cells derived from healthy donors, we applied a dose range of 10–50 nM dasatinib, 5–50nM midostaurin and 10–50 nM quizartinib. These dose ranges have been previously described to be achievable as trough levels during inhibitor therapy in early clinical trials. Upon incubation with dasatinib (10nM and 50nM), we found overall reduction in global tyrosine phosphorylation as detected by Western-blotting using the 4G10 antibody. In contrast, treatment with midostaurin left the activation of T-cell receptor signaling pathways unaffected. Comparable to DMSO control, overall phosphorylation was induced almost immediately after stimulation. Western-blotting of LCK and Plcg1 showed similar time dependent activation compared to total phosphorylation. Likewise, quizartinib did not reduce overall tyrosine phosphorylation level and left activation of downstream kinases (ZAP70, MAPK, LCK, Plcg1) largely unaffected.As activation of primary T-cells is a critical step in immune responses against viral and tumor antigens we aimed to investigate the influence of FLT3-kinase inhibitors quizartinib and midostaurin on activation of CD8+ T-cells. T-cells from healthy donors were stimulated using either PHA 0.5% or CD3/CD28 beads to ensure a more T-cell receptor specific stimulation. Using CD3/CD28 stimulation, CD69 expression was almost abrogated following dasatinib treatment. Applying clinically relevant doses of midostaurin or quizartinib to isolated T-cells did not influence CD69 expression. Expression levels upon PHA or CD3/CD28 stimulation were comparable to DMSO-control - even in the presence of 50nM midostaurin or quizartinib. Proliferation of T-cells upon CD3/CD28 stimulation was impaired by dasatinib treatment, while midostaurin and quizartinib left T-cell proliferation largely unaffected – as determined by CSFE staining. In order to investigate the T cell allo-reactivity, mixed lymphocyte culture was performed, where human pan-T-cells are co-cultured with allogeneic antigen presenting cells. T-cell proliferation – as measured by 3H-thymidine incorporation – was significantly impaired by dasatanib but neither midostaurin nor quizartinib treatment. Investigation of leukemia- and virus-antigen-specific T-cell responses are currently under way to gain deeper insight regarding this clinically relevant scenario.Overall, we found FLT3-kinase inhibitors midostaurin and quizartinib to leave T-cell activation, proliferation and function unaffected in-vitro. This information may be useful for the design of up-coming clinical trials testing the safety and efficacy of FLT3-kinase inhibitors in combination with allogeneic stem-cell transplantation. Disclosures:Lipka:Novartis Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Heidel:Novartis Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.