Normal T-cells Research Articles

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The role of T-cells in the pathogenesis of chronic lymphocytic leukemia has recently gained much attention due to the importance of the constant interaction between neoplastic B-cells with microenvironment substratum and T-cells. It is believed that these interactions modulate the clinical course of the disease, mainly through the regulation of the expansion, differentiation, and survival of chronic lymphocytic leukemia B-cells. Importantly, this crosstalk may also change the number, function, and memory phenotype of normal T-cells, thereby altering the amplitude and/or efficiency of adaptive immunity in chronic lymphocytic leukemia patients. The present study presents an overview on important aspects of this immunological crosstalk, particularly on the abnormalities of chronic lymphocytic leukemia B-cells and the alterations in normal T-cells, with focus on the CD4 memory T-cell compartment that could offer survival signals to chronic lymphocytic leukemia B-cell clone(s) and contribute to the establishment and progression of the disease. The authors believe that understanding the biological consequences of the interaction between normal T- and neoplastic B-cells in chronic lymphocytic leukemia may allow for improvements in the prognostic information and therapeutic approaches for this disease.© 2014 Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular. All rights reserved.

In-Vivo Activation Of STAT3 In Angioimmunoblastic T Cell Lymphoma, PTCL Not Otherwise Specified, and ALK Negative Anaplastic Large Cell Lymphoma: Implications For Therapy

T cell lymphoma (TCL) is a heterogeneous group of malignant lymphomas and is subdivided in three major subgroups, (i) cutaneous (mycosis fungoides (MF), sezary syndrome (SS) and primary CTCL), (ii) nodal (peripheral T-cell lymphoma, PTCL NOS, anaplastic large cell lymphoma (ALCL), angioimmunoblastic (AITL)) (iii) and extranodal. Because of this broad morphological spectrum and immunophenotypic variation the pathogenesis of most of the TCL is poorly understood. Constitutive STAT3 signaling provides growth-promoting signals in many forms of malignancy; however in-vivo activation of phosphorylated STAT3 and its clinical correlation in subtypes of TCLs has not been extensively studied. In this study we examined constitutive STAT3 activation in various subtypes of TCL tumors and performed pre-clinical testing to determine whether STAT3 inhibitors will be effective.First, we assessed the STAT3 activation (pSTAT3Tyr705) by immunohistochemistry (IHC) on a tissue microarray (TMA) from 169 untreated TCL patient samples. Using a cutoff of ≥30% pSTAT3 nuclear staining, 38% (64/169) of TCL tumors were pSTAT3+. Interestingly, pSTAT3 was differentially expressed among the various TCL subgroups. 27% (16/59) PTCL NOS, 29% (11/38) AITL, 93% (14/15) ALK+ ALCL, 57% (13/28) ALK- ALCL, 50% (6/12) extranodal NK/T cell tumors and 17% (1/6) primary cutaneous showed pSTAT3 positivity. Using either a 30% or 80% cut-off, pSTAT3 positivity was associated with inferior event free survival for AITL, PTCL NOS and ALK neg ALCLs TCLs. Due to small sample size within each group statistical significance (p=0.03) was met only for ALK neg ALCL (80% cutoff). Further analysis in the human TCL cell lines and normal control T-cells also revealed differential expression of pSTATs. ALCL cell lines, Karpas 299 and SR786 were pSTAT3 positive but pSTAT5 negative. Both the CTCL (SS) cell lines HuT78 and SeAx were pSTAT3 negative, however MyLa a cell line of MF origin was strongly pSTAT3+ and pSTAT5+. The normal control CD4 T-cells was pSTAT3- and pSTAT5+. These data clearly suggest that pSTAT3 is differentially expressed among the TCL tumors and is a potential therapeutic target. Potential mechanisms for STAT3 activation could be aberrant secretion of cytokines/chemokines produced by the tumor itself or the tumor microenvironment. We compared the cytokine profile of the culture supernatant from pSTAT3 positive SUDHL1 and pSTAT3 negative HuT78 TCL cell lines using a 30-plex ELISA. The most significantly different cytokines among these 2 cell lines were interleukin -10 (IL-10) and soluble interleukin-2 receptor alpha (sIL-2Ra). The SUDHL1 was found to secrete a very high level (1700 pg/ml) of IL-10, in contrast to only 167 pg/mL in the HuT78 supernatant. Moreover, SUDHL1 secretes 3.5 times more sIL-2Ra (6600 pg/ml) than the pSTAT3 negative cell line HuT78 (1800 pg/ml). In-vitro exposure to IL-10 but not sIL-2Ra was able to further upregulate pSTAT3 in SUDHL1 and Karpas 299. Overall, these data suggest that autocrine secretion of IL-10 but not sIL-2Ra is important for STAT3 activation in ALCL TCLs. Next we compared the cytokine profile of 2 pSTAT3 positive cells from different origin; SUDHL1 (ALCL) and MyLa (MF). In contrast to SUDHL1 (which produce high level of IL-10), MyLa was producing very little IL-10 (18pg/ml). The other most significantly different cytokines between these cells was RANTES with MyLa producing a very high level (28897 pg/ml) and SUDHL1 a very low level (8pg/ml). We also noticed differential level of IL-13 between these cells; MyLa producing 113pg/ml while SUDHL1 only 13 pg/ml. SAR302 (Sanofi, Cambridge, MA), a selective JAK2 inhibitor currently in clinical trial for myeloproliferative neoplasms was able to inhibit autocrine secretion of IL-10 but not sIL-2Ra in a dose-dependent manner followed by STAT3 dephosphorylation and decreased lymphoma cell survival.In summary, our data provide first evidence of in-vivo STAT3 activation in AITL, PTCL NOS, ALK negative ALCL TCLs using TMA from patient samples. Comprehensive cytokine analysis determined IL-10 as an autocrine factor driving STAT3 activation in ALCLTCLs, while RANTES and IL-13 might be important drivers for STAT3 activation in MF TCLs. Finally our data provide a mechanistic basis of selecting and targeting TCL tumor cells with high IL-10 levels and/or constitutive STAT3 activity with potent and novel JAK2 inhibitors such as SAR302 or other STAT3 inhibitors. Disclosures:No relevant conflicts of interest to declare.

DNMT3a Mutations Define a Pre-Leukemic Stem Cell Reservoir In Human Acute Myeloid Leukemia

Leukemia in humans arises from the multistep accumulation of mutations. However, the identity of the cell of origin, the nature of the first genetic lesion and the order of subsequent mutations remain poorly understood, as most cases of de novo acute myeloid leukemia (AML) are diagnosed without prior observation of a pre-leukemic clonal expansion. As part of studies to examine intra-tumoral genetic heterogeneity in AML, we carried out deep targeted sequencing (read depth 250×) of 101 commonly mutated leukemia genes on samples from 12 patients at diagnosis. Normal T-cells from each sample were expanded in vitro to provide a non-leukemic hematopoietic tissue for comparison. In 3 of 4 patients, we unexpectedly identified DNMT3a mutation not only in AML cells but also in T-cells at a low allele frequency (1-20%). Other genetic alterations such as NPM1 mutation (mutNPM1) were found only in AML cells and not in T-cells, ruling out contaminating AML cells as the source of the DNMT3a signal in cultured T-cells. To investigate the prevalence of T cell involvement, an additional 71 samples from AML patients at diagnosis were screened by Sanger sequencing for DNMT3a mutations. 17 of 71 AML samples (24%) carried R882 codon mutations (mutDNMT3a), and 15 of 17 (88%) also carried mutNPM1. Mutant allele frequency in freshly isolated T-cells was measured by droplet digital PCR (ddPCR). mutDNMT3a with no evidence of mutNPM1 was detected in T-cells of 12 of 17 patients (70.5%), suggesting that DNMT3a mutation occurs before NPM1 mutation in an ancestral stem/progenitor cell that gives rise to both T-cells and the dominant AML clone present at diagnosis. To directly determine whether phenotypic stem/progenitor cells that carried the mutDNMT3a allele could be identified within the non-leukemic hematopoietic compartment of AML blood and bone marrow samples, we undertook genetic analysis of highly-resolved phenotypically-defined normal stem, progenitor and mature lymphoid cell fractions from 10 patient samples. mutDNMT3a without detectable mutNPM1was present in stem cells and all downstream progenitors, with mean allele frequency among multipotent progenitor (MPP), multilymphoid progenitor (MLP) and common myeloid progenitor (CMP) of 31.7%. mutDNMT3a and mutNPM1 were found together only in granulocyte monocyte progenitor (GMP) and CD33+ blasts. Importantly, even for patients in whom mutDNMT3a was not detected in mature lymphoid populations, mutDNMT3a without mutNPM1 was found in MPP, MLP, CMP, providing strong evidence that mutDNMT3a precedes mutNPM1 during leukemogenesis. Analysis of diagnostic and remission samples revealed similar or higher proportion of cells with mutDNMT3a alone at remission compared to diagnosis. Xenotransplantation of cells from the diagnostic samples of 2 patients with mutDNMT3a and mutNPM1generated predominantly non-leukemic multilineage grafts (18 of 19 mice) with predominance of cells bearing mutDNMT3a without mutNPM1 (mean allele frequency 57%), confirming that mutDNMT3a was present in HSC. Kinetic analysis at 8 and 16 weeks revealed increasing mutDNMT3a allele frequency in multilineage xenografts over time, suggesting that mutDNMT3a confers a competitive growth advantage over non-mutated HSC. Collectively, our results are consistent with the clonal expansion in AML patients of mutDNMT3a HSC that survive chemotherapy. These cells may therefore represent a reservoir for further genomic progression leading to relapse. Our findings now offer the possibility of therapeutic intervention during remission to eliminate these surviving pre-leukemic clones and prevent relapse in a large proportion of AML patients carrying mutDNMT3a. As well, our work provides a framework for the identification of other early events in leukemogenesis and examination of how these changes disrupt normal HSC function and lead to leukemia. Disclosures:Wang:Trillium Therapeutics/Stem Cell Therapeutics: Research Funding.

PKC Inhibitor AEB071 Demonstrates Pre-Clinical Activity In Chronic Lymphocytic Leukemia

Targeting B-cell receptor (BCR) downstream pathways is of therapeutic importance in eradicating chronic lymphocytic leukemia (CLL) cells. Members of the protein kinase C (PKC) family play an important role in B-cell activation. PKC-β has recently been shown to be over-expressed in CLL and essential to CLL development in the TCL1 mouse model. Mice deficient in PKC-β exhibit a survival defect in response to BCR stimulation, correlating with an inability to induce the NF-κB-dependent anti-apoptotic proteins as Bcl-xL and A1. Moreover, PKC-β-dependent activation of NF-κB in stromal cells is pivotal for the survival of B-CLL cells in vivo; wherein PKC-β inhibition was shown to prevent microenvironment protection of CLL. Additionally, PKC-β lies downstream of PLC-γ2 where activating mutations have been noted in BTK (ibrutinib) resistant patients, which conveys a potential mechanism to target resistance related to mutations in this target protein. Therefore exploration of a PCK-β inhibitor in CLL is highly justified and innovative. Sotrastaurin (AEB071) is an orally administered potent inhibitor of classical and novel PKC isotypes; with strong and specific activity on PKC-α, PKC-β and PKC-θ and lesser activity on PKC-δ, PKC-ε, and PKC-η. Pre-clinically, AEB071 has demonstrated in vivo pre-clinical activity in activated B-cell diffuse large B-cell lymphoma (DLBCL) models and is currently being tested for efficacy in CD79b mutated DLBCL. Since PKC-β is indispensable for BCR-induced NF-κB activation and B-cell survival, herein we evaluate the impact of AEB071 on CLL cell survival as a promising therapeutic to target this pathway.Our preliminary work demonstrated that AEB071 was markedly cytotoxic to CLL cells in a dose-dependent (≤6.25uM, p<0.001) and time-dependent manner (p=0.011) as measured by MTS analysis. In a whole blood assay, AEB071 exhibits a retained selective cytotoxicity against tumor cells with a modest reduction in B-CLL cells whereas no effect on T-cells or natural killer cells was detected in CLL patient samples. Notably, upon treatment of blood from healthy subjects, AEB071 showed no toxic effects on normal B-cells, T-cells and natural killer cells. AEB071 inhibits CPG-induced survival of CLL cells in vitro (p<0.01), and effectively blocks the protection induced by soluble factors such as CD40L, IL-4, and TNF (p<0.01), which are known to reduce the spontaneous apoptosis associated with CLL cells. Similar effects were observed with stromal cell contact; wherein AEB071 showed enhanced cytotoxic potency on CLL cells under co-culture conditions with stromal cells compared to CLL alone (p<0.05). Additionally, AEB071 attenuated anti-IgM-induced survival of CLL cells with a modest induction of apoptosis (p<0.001). Furthermore, treatment of PMA- or BCR-activated CLL cells with AEB071 could effectively abrogate downstream survival pathways including ERK1/2, p38MAPK, AKT, GSK3β, and NF-κB as revealed by immunoblot analysis. Collectively, this data indicate that therapeutic strategies to inhibit PKC-β have the potential to disrupt signaling from the microenvironment that lead to in vivo CLL cell survival and potentially drug resistance. Current studies are ongoing to evaluate the in vivo tolerability and therapeutic efficacy of AEB071 in the Eμ-TCL1 transgenic mouse model of CLL.In conclusion, PKC-β represents an innovative target for CLL and therefore, future efforts targeting PKC with the PKC inhibitor AEB071 as monotherapy in clinical trials of relapsed and refractory CLL patients may be warranted. Disclosures:No relevant conflicts of interest to declare.

Novel Predictor Of Lenalidomide Response In Non-del5q MDS Reveals Linkage To Molecular Mechanism: First Characterization Of T-Cell Function In Cereblon Homozygous Deficient Mice

BackgroundDurable responses to lenalidomide (LEN, an immunomodulatory drug, IMiDTM) occur in both non-del5q and del5q Myelodysplastic Syndrome (MDS). In non-del5q MDS and multiple myeloma, modulation of the bone marrow microenvironment by an unknown mechanism has been theorized. IMiDs initiate numerous effects on the immune system, some of which may induce an anti-leukemic and anti-inflammatory response. The connection between the drug's immune modulating activity and its hematopoietic stimulatory activity in MDS is currently untested. We and others have shown that LEN expands T-cells and increases IL-2 (p<0.001) by substituting for CD28 co-stimulation. In support of this, we found that LEN activates a CD28-specfic transcription factor pCREB in the absence of extracellular ligation of the CD28 receptor suggesting that it is acting to suppress a pharmacologic target that is involved specifically in costimulatory signaling. Normally, several negative regulators provide immunotolerance to T-cells to limit self-reactivity and protect against autoimmune disease. Anti-cancer immunosurveillance is often circumvented through acquired resistance to costimulation. MethodsThe critical role of immune modulation in LEN-induced hematologic response was examined by testing the performance of a predictive biomarker in patients stratified by del5q status in a training cohort and in a blinded validation cohort. Molecular analyses were performed on sorted populations of cells and knock-down of genes in normal human T-cells. Finally, we demonstrate the relevance of cereblon, a molecular target of LEN, in the negative regulation of costimulatory CD28 signaling using a homozygous germline knockout mouse. ResultsComparison of sorted CD28+CD8+ and CD28-CD8+ human T-cells illustrates the importance of the expression of this receptor in LEN-induced T-cell activation. CD28- T-cells were found to be resistant to LEN. Accumulation of CD28- cells is a form of accelerated immunosenescence that is often associated with cancer and has been reported in MDS. To determine if CD28- T-cell accumulation is associated with LEN hematopoietic outcome in patients, we performed T-cell profiling before treatment in a training cohort (n=21) and a blinded validation cohort (n=35). Of the T-cell markers studied, a higher percentage of CD28- T-cells was associated with LEN hematopoietic failure in the training cohort (p=0.001). Using a R-Part-defined cutoff of CD28+ vs CD28- T-cells, the biomarker sensitivity was found to be 86%, specificity 82%, positive predictive value (PPV) 67%, and negative PV (NPV) 93% in non-del5q MDS patients, but did not improve response discrimination in del5q MDS. The association between CD28 and LEN response led us to hypothesize that the molecular target of LEN is involved in signaling by the CD28 receptor. A landmark study identified cereblon (CRBL), a RING-domain E3-Ubiquitin Ligase (UbL) to be the primary target of thalidomide and LEN. To examine the effect of CRBL on CD28 signaling, germ-line crbl knock-out mice (crbl-/-) were examined compared to wild-type (WT) C57BL6 mice. We found that crbl deficiency augments T-cell proliferation (Fig. 1A) and contextual activation of IL2 in the absence of CD28 co-stimulation similar to LEN. Examination of the bone marrow, thymus, and peripheral blood shows that crbl is a negative regulator of lymphopoiesis in the spleen and thymocyte with increased peripheral blood lymphocyte counts (Fig. 1B) and increased double negative (DN)-1 (P=0.01), DN3 (p=0.03), and reduced DN4 (p=0.005) thymocytes in crbl-/- compared to WT mice. [Display omitted] ConclusionsOur results indicate that immune reconstitution mediated by T-cell CD28 signaling is important for hematologic response in non-del5q MDS, but not del5q MDS, and is consistent with different molecular mechanisms of action in these two groups of patients. LEN may improve T-cell function by directly suppressing CRBL a novel E3-UbL regulator involved in lymphopoiesis, thymocyte development and CD28 co-stimulation. Our data suggests that CRBL acts similarly to the E3 UbL proteins Cbl-b, GRAIL, and ITCH that mechanistically induce immunotolerance. In response to LEN, T-cell functional activation may sustain anti-leukemic immunosurveillance or act to remove suppressive immature cell populations from the bone marrow microenvironment allowing hematologic improvement in non-del5q MDS. Disclosures:Epling-Burnette:Celgene: Research Funding. McDaniel:Celgene: Employment. List:Celgene: Membership on an entity's Board of Directors or advisory committees.

Absence Of The E3 Ubiquitin Ligase SKP2 Attenuates Notch-Induced T-Cell Leukemogenesis

Acute Lymphoblastic Leukemia (ALL) is the most common malignancy in childhood, accounting for almost 30% of pediatric cancers. In pediatric T-cell acute lymphoblastic leukemia (T-ALL), around 30% of the children still undergo disease relapse, which is associated with poor prognosis. Hence, novel therapeutic strategies for the treatment of T-ALL are necessary, since conventional therapies still fail to cure a significant number of patients. Notch signaling contributes to the regulation of normal T-cells homeostasis, whereas oncogenic, activated form of Notch is present in the majority of the T-ALL cases.Previous studies in our laboratory demonstrated that Notch1 signaling induces transcriptional activation of SKP2, the F-box protein of the SCF E3-ubiquitin ligase complex. SKP2 targets the major cell cycle inhibitors, the CKIs p21Cip1, p27Kip1, p57Kip2 and p130, for proteasome-mediated degradation. Increased Skp2 expression accelerates cell cycle progression in hematopoietic cells, and its overexpression is frequently found in cancers, in particular lymphomas and leukemias, in which is associated with poor prognosis.We found that Skp2 expression is dynamically regulated during T-cell differentiation coinciding with the Notch expression pattern. Moreover, primary thymocytes cultured in vitro responded to Notch stimulation by the Delta1 ligand increasing their Skp2 expression and their cell cycle activity. As anticipated, we found that Skp2 expression is increased in T-ALL patient samples. Our hypothesis is that Notch activation promotes T-cell leukemogenesis by altering the cell cycle control through upregulation of SKP2. To better define the role of SKP2 in T-ALL, we analyzed the Skp2 expression in a murine model of Notch-induced T-cell leukemia. Mice transplanted with stem/progenitor cells transduced with the constitutive active form of Notch (ICN), developed T-ALL and died by week 12 after transplant. Analysis of T-ALL cells revealed a 5 fold upregulation of Skp2 expression compared to controls. Next, we tested whether SKP2 was required for T-ALL development. To this end, we evaluated leukemia development in mice transplanted with SKP2 deficient cells overexpressing ICN. Stem/progenitor cells derived from Skp2+/+, Skp2+/- or Skp2-/- mice were transduced with ICN and then transplanted into WT recipients. Loss of SKP2 significantly delayed the development of T-cell leukemia in transplanted mice and increased their survival by 60% at 12 weeks.Taken together, these results demonstrate a previously unrecognized role for SKP2 in the initiation and progression of T-ALL and its potential role as a therapeutic target. Disclosures:No relevant conflicts of interest to declare.

Notch1 Gene Mutations Target KRAS G12D-expressing CD8+ Cells and Contribute to Their Leukemogenic Transformation

Acute T-cell lymphoblastic leukemia/lymphoma (T-ALL) is an aggressive hematopoietic malignancy affecting both children and adults. Previous studies of T-ALL mouse models induced by different genetic mutations have provided highly diverse results on the issues of T-cell leukemia/lymphoma-initiating cells (T-LICs) and potential mechanisms contributing to T-LIC transformation. Here, we show that oncogenic Kras (Kras G12D) expressed from its endogenous locus is a potent inducer of T-ALL even in a less sensitized BALB/c background. Notch1 mutations, including exon 34 mutations and recently characterized type 1 and 2 deletions, are detected in 100% of Kras G12D-induced T-ALL tumors. Although these mutations are not detected at the pre-leukemia stage, incremental up-regulation of NOTCH1 surface expression is observed at the pre-leukemia and leukemia stages. As secondary genetic hits in the Kras G12D model, Notch1 mutations target CD8(+) T-cells but not hematopoietic stem cells to further promote T-ALL progression. Pre-leukemia T-cells without detectable Notch1 mutations do not induce T-ALL in secondary recipient mice compared with T-ALL tumor cells with Notch1 mutations. We found huge variations in T-LIC frequency and immunophenotypes of cells enriched for T-LICs. Unlike Pten deficiency-induced T-ALL, oncogenic Kras-initiated T-ALL is not associated with up-regulation of the Wnt/β-catenin pathway. Our results suggest that up-regulation of NOTCH1 signaling, through either overexpression of surface NOTCH1 or acquired gain-of-function mutations, is involved in both T-ALL initiation and progression. Notch1 mutations and Kras G12D contribute cooperatively to leukemogenic transformation of normal T-cells.

Eosinophils affect functions of in vitro-activated human CD3-CD4+ T cells

BackgroundThe recent development of eosinophil-targeting agents has raised enthusiasm for management of patients with hypereosinophilic syndromes. Roughly half of anti-IL-5-treated patients with corticosteroid-responsive lymphocytic (L-HES) and idiopathic disease variants can be tapered off corticosteroids. Potential consequences of corticosteroid-withdrawal on clonal expansion of pre-malignant CD3-CD4+ T-cells associated with L-HES are a subject of concern. Indeed, corticosteroid treatment inhibits T-cell activation and may lower blood CD3-CD4+ cell counts. On the other hand, previous studies have shown that eosinophils support CD4 T-cell activation, suggesting that targeted eosinophil depletion may negatively regulate these cells.ObjectivesEffects of eosinophils on CD4 T-cell activation in vitro were investigated as an indirect means of exploring whether treatment-induced eosinophil depletion may affect pathogenic T-cells driving L-HES.MethodsHelper (CD4) T-cells and CD3-CD4+ cells from healthy controls and L-HES patients, respectively, were cultured in vitro in presence of anti-CD3/CD28 or dendritic cells. Effects of eosinophils on T-cell proliferation and cytokine production were investigated.ResultsEosinophils enhanced CD3-driven proliferation of CD4 T-cells from healthy subjects in vitro, while inhibiting TCR-independent proliferation and IL-5 production by CD3-CD4+ T-cells.ConclusionsWhile this study confirms previous work showing that eosinophils support activation of normal helper T-cells, our in vitro findings with CD3-CD4+ T-cells suggest that eosinophil-depletion may favor activation and expansion of this pathogenic lymphocyte subset. With the ongoing development of eosinophil-targeted therapy for various eosinophilic conditions, the indirect consequences of treatment on the underlying immune mechanisms of disease should be investigated in detail in the setting of translational research programs.

Increased expression of miR-221 is associated with shorter overall survival in T-cell acute lymphoid leukemia

BackgroundCD56 expression has been associated with a poor prognosis in lymphoid neoplasms, including T-cell acute lymphoblastic leukemia (T-ALL). MicroRNAs (miRNAs) play an important role in lymphoid differentiation, and aberrant miRNA expression has been associated with treatment outcome in lymphoid malignancies. Here, we evaluated miRNA expression profiles in normal thymocytes, mature T-cells, and T-ALL samples with and without CD56 expression and correlated microRNA expression with treatment outcome.MethodsThe gene expression profile of 164 miRNAs were compared for T-ALL/CD56+ (n=12) and T-ALL/CD56- (n=36) patients by Real-Time Quantitative PCR. Based on this analysis, we decided to evaluate miR-221 and miR-374 expression in individual leukemic and normal samples.ResultsmiR-221 and miR-374 were expressed at significantly higher levels in T-ALL/CD56+ than in T-ALL/CD56- cells and in leukemic blasts compared with normal thymocytes and peripheral blood (PB) T-cells. Age at diagnosis (15 or less vs grater than 15 years; HR: 2.19, 95% CI: 0.98-4.85; P=0.05), miR-221 expression level (median value as cut off in leukemic samples; HR: 3.17, 95% CI: 1.45-6.92; P=0.004), and the expression of CD56 (CD56-vs CD56+; HR: 2.99, 95% CI: 1.37-6.51; P=0.006) were predictive factors for shorter overall survival; whereas, only CD56 expression (HR: 2.73, 95% CI: 1.03-7.18; P=0.041) was associated with a shorter disease-free survival rate.ConclusionsmiR-221 is highly expressed in T-ALL and its expression level may be associated with a poorer prognosis.

Diagnostic Value of CD200 Expression for the Differentiation Between Chronic Lymphocytic Leukemia (CLL), CLL with Increased Prolymphocytes (CLL/PL) and Mantle Cell Lymphoma.

Abstract 2660 Background:Chronic lymphocytic leukemia (CLL) is diagnosed by the immunophenotye as published by Matutes et al. with strong expression of CD5 and CD23, weak expression of sCD22, CD79b and immunoglobulins and negativity for FMC7. CLL is readily differentiated from mantle cell lymphoma (MCL) which shares CD5 positivity but features strong expression of sCD22, CD79b and FMC7. CLL with increased prolymphocytes (CLL/PL), which cytomorphologically features 5–55% prolymphocytes, displays an immunophenotype in between CLL and MCL. The cytogenetic finding of a t(11;14) separates MCL from CLL/PL. The analysis of CD200 expression has been suggested to improve differentiation between CLL and MCL based on a limited number of cases and has not yet been assessed in CLL/PL. Aims:To assess the diagnostic usefulness of CD200 expression in the flow cytometric assessment of CLL, CLL/PL and MCL. Methods:We studied 100 patients with CLL (n=59), CLL/PL (n=27) and MCL (n=14) for expression of CD200. t(11;14) was confirmed in all cases with MCL and excluded in all cases with CLL/PL. 37 were female, 63 male, median age was 71.2 yrs (range 39.3–87.1), sample material was peripheral blood (n=68) or bone marrow (n=32). CD200 was analyzed using the antibody clone MRC OX-104 conjugated to phycoerythrin (BD Biosciences, Franklin Lakes, NJ). Cells were rated positive if they expressed CD200 stronger than the cut-off set by an isotype control. Cases were rated positive if ≥20% of the pathologic population as identified based on coexpression of CD19 and CD5 expressed CD200. Results:Mean±SD % values for CD200 positive cells were 94.9±14.0% in CLL, 78.7±27.0% in CLL/PL and 6.6±13.3% in MCL. Thus, besides significant differences between MCL with low CD200 expression and the two other entities with high CD200 expression (p<0.001 for both), there was also a significant difference between CLL and CLL/PL (p=0.006) with higher values for CLL. The same was true for the analysis of mean fluorescence intensities (MFIs): CLL cases expressed CD200 stronger (mean±SD 5.8±2.9) as compared to CLL/PL cases (3.9±2.8, p=0.007) while MCL cases had clearly the lowest MFI (0.3±0.3, p<0.001 for comparisons to both CLL and CLL/PL). In order to use a more reliable parameter than MFI of the malignant cells per se, we included normal T-cells as controls and calculated the MFI ratio “malignant cells:normal T-cells”. This again resulted in the highest values for patients with CLL (mean±SD 14.8±9.0) although the difference to patients with CLL/PL (12.3±10.4) was not significant. Patients with MCL also for this parameter had clearly lower values amounting to 1.2±0.9 (p<0.001 for comparisons to both CLL and CLL/PL).We than tested whether a cut-off for the CD200 expression parameters would be discriminative of the three entities. Regarding positivity for CD200 expression based on a cut-off of 20% malignant cells we found the following rates of positivity for CD200: 58/59 (98.3%) in patients with CLL, 26/27 (96.3%) in patients with CLL/PL and 2/14 (14.3%) in patients with MCL. Thus, although there were overall significant differences in the CD200 expression the application of a 20% cut-off was not capable of completely differentiating CLL and CLL/PL on the one hand from MCL on the other hand. Importantly, the “misclassified” cases mostly were not near to the cut-off (11.5% in CLL, 2.8% in CLL/PL, 47.0% and 22.5% in MCL). To improve classification we applied a cut-off of 0.48 for the MFI of CD200 expression in malignant cells. All 59 cases with CLL and all cases with CLL/PL had MFIs >0.48, however, only 12/14 cases with MCL had MFIs <0.48. Thus, although this analysis reveals highly significant differences between the three entities (p<0.001) there remain two misclassified MCL cases. While in one of these 2 cases the encountered MFI of 0.84 was only slightly above the cut-off and low-level bone marrow infiltration of 1% may have contributed to imprecision of MFI determination this cannot be considered for the second case (MFI 1.26, bone marrow infiltration 8%). The use of a cut-off for the MFI ratio “malignant cells:normal T-cells” yielded no improvement in classification. Conclusions:Assessment of CD200 expression may be applied in the differential diagnosis of CLL, CLL/PL and MCL to predict MCL with high probability based on a low-level expression of CD200, however, the sensitivity of this approach is limited by the infrequent MCL cases with higher expression of CD200. Disclosures:Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Schabath:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Mesenchymal Stromal Cells Support Follicular Lymphoma-Associated Follicular Helper T-Cells, in Part, Through an IL-6 and IL-21-Dependent Mechanism.

Abstract 2707The biology of follicular lymphoma (FL) is largely dictated by the immune-effector and stromal cells that comprise its tumor microenvironment. Recent studies have demonstrated that various infiltrating T-cell populations can be predictive of clinical outcome, with two such populations, follicular helper T-cells (TFH) and regulatory T-cells (Treg) likely playing critical roles in the biology of this disease. TFH are normally responsible for the activation and maintenance of germinal center B-cells while Tregs suppress effector T-cell priming and function as well as suppress normal B-cell proliferation and differentiation. There is also data to support the notion that both of these cells have direct and/or indirect effects on FL B-cells. These cells and other immune-effector cells within the FL microenvironment have dynamic interactions with mesenchymal stromal cells (MSC), multipotent cells residing in most adult tissues, which can be recruited by FL B-cells into the tumor microenvironment where they inhibit anti-lymphoma T-cell responses and FL B-cell apoptosis. We hypothesized that such MSC play a role in modulating TFH viability, similar to what has been previously reported for Treg, and herein we show such a novel role for MSC, that being the selective support of FL-derived TFH populations.To determine the effect of MSC on the viability of discrete T-cell populations infiltrating FL lymph nodes (FLN), un-separated single cell suspensions (SCS) from FLN (n=13) were cultured for 48 hours alone or with normal tonsillar-derived MSC. The fold change in the proportion of CD4 T-cells that are TFH (CXCR5+PD-1+Bcl-6+), Tregs (CD25+FoxP3+), TH1 (T-bet+), TH2 (GATA-3+) or TH17 (ROR-gamma-t+) cells in SCS incubated with MSC was compared to that of SCS incubated without MSC. A 2.3-fold increase in the percentage of TFH (p=0.006) cells and a 2-fold increase in the percentage of Tregs (p=0.007) were observed in the SCS cultured with MSC compared to those cultured without MSC. The effect of MSC in maintaining TFH and Tregs was selective, as in contrast, the proportion of T-cells expressing the canonical TH1, TH2 and TH17 transcription factors was not increased after culture with MSC. B-cells provide survival signals to TFH and stromal cells provide survival signals to B-cells, therefore we next determined whether the stromal support of TFH was mediated via B-cells. Both purified FL T-cells and B-cell-depleted FL SCS cultured on MSC showed a similar increase in TFH (and Treg) populations as seen with un-separated FL-B SCS, indicating that MSC support of TFH and Tregs was independent of B-cells. MSC only partially supported TFH and Treg populations in transwell experiments suggesting a role for both cell-cell contact and soluble factors. In this regard, IL-6 and IL-21 are known to be secreted by MSC and to signal through gp130 receptors on TFH to induce the transcription of Bcl-6, which is required for TFH differentiation. Blocking IL-6 and IL-21 decreased MSC support of TFH by 42% (p=0.021), while reducing MSC support of Tregs by only 17% (p=0.018) suggesting that IL-6 and IL-21 mediate, in part, the protective effect that MSC have on TFH but that other cytokines are likely to play a role in supporting Tregs. Finally, we have shown that FLN-derived MSC support both FL T-cells and normal lymph node T-cells to a similar extent as the tonsillar MSC used in these experiments. This provides support for the use of tonsillar MSC in these experiments as we were able to generate and expand these more consistently than MSC from FLN, which allowed us to use a consistent MSC product for all experiments.These findings therefore demonstrate a new role for MSC in FL, that being to support FL TFH in addition to Treg populations in the tumor microenvironment. MSCs have been shown to support FL B-cell viability and suppress anti-lymphoma T-cell responses. This finding that MSCs support TFH cells, a population that may provide survival signals to FL B-cells, further supports the potential of targeting MSC as a novel therapeutic strategy for patients with FL. Disclosures:No relevant conflicts of interest to declare.

Whole-Exome Sequencing Links CARD11 Inactivation with SCID

AbstractAbstract 258Primary immunodeficiencies represent model diseases for the mechanistic understanding of the human innate and the adaptive immune response and are per se clinically highly relevant, because in SCID patients infections by opportunistic pathogens are typically life-threatening early in life. We identified an infant of consanguineous parents suffering from a novel form of SCID, who presented with a life-threatening Pneumocystis jirovecii pneumonia. This entity was characterized by agammaglobulinemia and profoundly deficient T-cell function despite quantitatively normal T- and B-lymphocytes. Lymphocyte proliferation was strongly inhibited after stimulation of PBMCs with T-cell mitogens such as PHA, Con A, or anti-CD3 monoclonal antibody. The expression of several T-cell response associated cytokines upon stimulation with PMA/ionomycin was dramatically reduced in comparison to normal controls. By contrast, proliferation induced by the classical B-cell mitogen PWM was almost comparable to healthy controls. Immunophenotyping revealed a predominantly naïve phenotype (CD45RA+ CCR7+) in CD4+ and CD8+ T-lymphocytes, whereas central memory T-lymphocytes (CD45RA− CCR7+) were nearly absent. B-lymphocytes from peripheral blood were mainly naïve B-cells (CD27−) with a uniformly immature transitional B-lymphocyte phenotype (CD24++, CD38++). Patient B-lymphocytes retained the ability to proliferate and differentiate in response to BCR-independent stimuli, while their response to BCR activation was defective. Our findings thus revealed a combined defect of TCR-mediated T-lymphocyte functions and BCR-mediated B-lymphocyte functions but did not enable us to link the immunological phenotype with one of the known molecularly defined categories of SCID. Diagnostic whole-exome sequencing and systematic variant categorization revealed a single pathogenic homozygous nonsense mutation of the caspase recruitment domain 11 (CARD11) gene. CARD11 is a scaffold protein that is known to be required for the assembly and activation of the NF-kB complex. In reconstitution assays we demonstrated that the patient derived truncated CARD11 protein is defective in antigen receptor signaling and NF-kB activation. Several lines of evidence substantiate the involvement of the identified CARD11 mutation in the new form of SCID that we report here. First, PCR and Sanger re-sequencing validated the truncating CARD11 mutation to be homozygous in the patient and heterozygous in the parents, in agreement with the recessive transmission of the mutation through the healthy consanguineous parents. Second, CARD11 is a scaffold protein required for TCR- and BCR-induced NF-kB activation as well as lymphocyte activation and proliferation, which is specifically expressed in hematopoietic cells, consistent with a causative role of CARD11 mutations in the context of an immune disorder. Third, the GUK domain of CARD11, which is missing in the mutated form of CARD11 due to truncation, was previously reported to be necessary for NF-kB activation by PMA/ionomycin treatment, further supporting the presumed damaging nature of the homozygous CARD11 mutation observed in the female patient reported here. Finally, the immunological findings in this patient are compatible with the phenotype of a previously described Card11 −/− k.o. mouse, which shows a selective defect in NF-κB activation leading to diminished antigen receptor or PKC mediated proliferation and defective cytokine production in T-cells and B-cells. Thus, we have identified an inactivating CARD11 mutation linking defective NF-kB signaling with a novel cause of autosomal recessive SCID, which must be considered in the diagnostic assessment of patients with suspected SCID but with quantitatively normal T-cells. Disclosures:No relevant conflicts of interest to declare.

A Pilot Study to Assess Inflammatory Biomarker Changes When Raltegravir is Added to a Virologically Suppressive Haart Regimen in HIV-1-Infected Patients with Limited Immunological Responses

Despite successful suppression of HIV-1 with HAART, some patients do not have robust immunological recovery. Chronic inflammation from persistent immune activation could contribute to this poor response, resulting in HIV-1 disease progression and the development of some non-HIV-1 comorbidities. We conducted a pilot study of 30 HIV-1-infected patients with undetectable viral loads and poor CD4(+) T-cell responses on long-term stable HAART to assess whether the addition of raltegravir would have an effect on biomarkers of chronic inflammation. A total of 26 patients were followed for 1 year on the intensified regimen. In addition to T-cell responses, we evaluated changes in activated CD4(+) and CD8(+) T-cells, several pro-inflammatory cytokines and chemokines and memory cell responses to HIV-1-associated peptides. Although there was no improvement in CD4(+) T-cell counts, the percentage change in CD4(+)%, CD4(+)/CD8(+) ratios and RANTES (regulated on activation normal T-cells expressed and secreted) increased significantly while the percentage change in CD8(+) T-cell counts and CD8(+)%, activated CD4(+) T-cells and several pro-inflammatory chemokines and cytokines decreased significantly. The percentage change in HIV-1-specific nef, pol set 1, gag and env memory T-cells also declined. The addition of raltegravir to a virologically suppressive HAART regimen in patients with poor immunological responses resulted in the reduction of several pro-inflammatory biomarkers; increases were seen in RANTES levels and CD4(+)/CD8(+) T-cell ratios. The clinical relevance of these observations is beyond the scope of this study.

Aberrant expression of the transcriptional factor Twist1 promotes invasiveness in ALK-positive anaplastic large cell lymphoma

The transcriptional factor Twist1 has been shown to play a key role in regulating epithelial mesenchymal transition, invasiveness and migratory properties in solid tumors. We found that Twist1 is aberrantly expressed in ALK-positive anaplastic large cell lymphoma (ALK+ALCL), a type of T-cell lymphoid malignancy. Using RT-PCR and Western blots, Twist1 was detectable in all 3 ALK+ALCL cell lines examined but absent in normal T-cells. By immunohistochemistry, Twist1 was detectable in all 10 cases of ALK+ALCL examined; benign lymphoid tissues were consistently negative. Twist1 expression in ALK+ALCL cells can be attributed to the NPM-ALK/STAT3 signaling axis, the key oncogenic driving force in this tumor type. Twist1 is biologically important in ALK+ALCL cells, as Twist1 knockdown resulted in a significant decrease in their invasiveness in an in-vitro assay. Further investigation revealed that this increase in invasiveness is linked to the activation of AKT and down-regulation of p66Shc, two signaling proteins known to be involved in NPM-ALK-mediated oncogenesis. Lastly, knockdown of Twist1 sensitizes ALK+ALCL cells to the growth inhibitory effect of PF-2341066 (Crizotinib®), an ALK inhibitor being used in clinical trials. In conclusion, Twist1 expression, owing to the abnormal NPM-ALK/STAT3 signaling, contributes to its invasiveness and decreased sensitivity to PF-2341066 in ALK+ALCL.

Gene Expression Profiling Identifies the JAK/STAT and NFκB Pathways to Be Important in Peripheral T-Cell Lymphomas and Natural-Killer T-Cell Lymphomas

Abstract 2658 Background:Peripheral T-cell lymphomas (PTCL) carry a poorer prognosis compared to their B-cell counterparts and the molecular pathogenesis of PTCL is still largely unknown. The aims of this study are to characterize the molecular signatures and identify signaling pathways involved in the different subsets of PTCL and NKTCL.Materials and Methods: RNA was extracted from tumors of 60 patients with newly diagnosed PTCL and NKTCL: 21 with angioimmunoblastic T-cell lymphoma (AITL), 12 anaplastic large-cell lymphoma (ALCL), 15 peripheral T-cell lymphoma not-otherwise-specified (PTCL-NOS) and 12 with natural-killer T-cell lymphoma (NKTCL). Comparisons were made using published gene expression data files of normal T and NK T-cells. Gene expression profiling was performed using the Affymetrix HG-U133 Plus 2.0 GeneChip platform. Results:The Affymetrix expression profiling distinguishes the 48 PTCL samples from normal T-cell controls (p<0.005). Similarly, the molecular signature of 12 NKTCL significantly differs from normal NK cells. The JAK/STAT pathway is significantly upregulated in AITL and a subset of PTCL-NOS compared to normal T cells and ALCL (p<0.005). [Figure 1] Pathways related to NFκB significantly distinguish PTCL from normal T-cells and it also distinguishes NKTCL from normal NK T-cells. Unsupervised hierarchical clustering by gene expression profiles is able to separate the subsets of PTCL (AILT, ALCL, NKTCL) in concordance with their immmunohistochemical diagnosis. PTCL-NOS however, are dispersed across the clusters reflecting its molecular heterogeneity. [Figure 2] Discussion:Gene expression profiling identifies distinct molecular pathways in PTCL. In particular, the JAK-STAT pathway is upregulated in AITL and the NFκB pathway is dysregulated in PTCL and NKCL. Our results suggest that targeting the key kinases in these pathways may be effective in the treatment for this subset of lymphomas with poor prognosis. [Display omitted] [Display omitted] Disclosures:Tan:Janssen: Honoraria, Research Funding; Celgene: Honoraria; Novartis: Research Funding.

Regression of Adult T-Cell Leukemia with Anti-CD25 Recombinant Immunotoxin LMB-2 Preceded by Chemotherapy

Abstract 2575 Background:Adult T-cell leukemia (ATL) is a highly aggressive CD25+ HTLV-1-associated malignancy with median survivals of 4 and 9 months for leukemic and lymphomatous forms, respectively. There is no standard therapy capable of altering median survival by more than a few months. The anti-CD25 recombinant immunotoxin LMB-2 as a single agent was previously reported in phase I testing to have activity, but limited by rapid tumor growth between cycles and immunogenicity. Human CD25+ xenografts in mice contained soluble CD25 (sCD25) levels >100-fold higher than in serum, which were rapidly depleted by chemotherapy. Treatment with fludarabine was previously reported to be associated with lower immunogenicity to murine antibodies (HAMA), and the fludarabine-cyclophosphamide (FC) combination was reported associated with reductions in normal T- and B-cells. Methods:To prevent the formation of neutralizing antibodies and enhance anti-tumor activity, leukemic or lymphomatous ATL patients received FC chemotherapy with fludarabine 25 mg/m2 and cyclophosphamide 250 mg/m2 on days 1, 2, and 3, and 2 weeks later began cycles every 3 weeks in which FC was administered on days 1, 2 and 3, followed by LMB-2 30–40 ug/Kg on days 3, 5 and 7. Formation of neutralizing antibodies and clinical response were determined with each cycle. Results:Interim results with 8 evaluable patients receiving 28 cycles (2–5 each) include an ORR of 50% with 2 CRs and 2 PRs. In 6 patients with circulating ATL cells detectable in the blood, reductions of ATL cells varied from 90.8 to 100% compared to baseline. CRs were associated with complete resolution of chest wall and pelvic masses, circulating ATL cells, and bone marrow involvement as assessed by immunohistochemistry and flow cytometry. One of 2 PRs is ongoing for nearly 1 year after complete resolution of skin involvement and partial decrease in circulating ATL cells. One of 2 CRs recurred after 6 months only in a sanctuary site (testis). The toxicity of LMB-2 was not increased by FC and 40 ug/Kg of LMB-2 days 3, 5 and 7 was not associated with dose-limiting toxicity (DLT). While the original doses of FC used were also without DLT, escalation to 30 and 300 mg/m2/day x3 resulted in severe thrombocytopenia in 1 of 2 patients. The most common toxicities, based on percent of cycles observed, were neutropenia (69%), lymphopenia (52%), leukopenia and nausea (48%), and anemia, thrombocytopenia and fever (41%). With FC, normal T-cells were reduced 0–95% (median 70%), but normal B-cells were reduced 91–100% (median 96%). Two (25%) patients made high levels of neutralizing antibodies after 3–4 cycles of LMB-2. Conclusions:LMB-2 is effective in ATL when preceded by FC, and can achieve major responses including CR. Additional patients will be needed to determine if FC can delay immunogenicity significantly, and allow enough cycles to result in long-term remission from this disease (Supported in part by NCI, intramural research program, NIH). Disclosures:Kreitman:NIH: Patents & Royalties. Off Label Use: Use of investigational agent and FC chemotherapy for treatment of ATL. Pastan:NIH: Patents & Royalties.

Proof of Y-Loss As Clonal Abnormality in MDS by Comparative Analysis of CD34+ and CD3+ Peripheral Blood Cells,

Abstract 3800 Introduction:In an ongoing diagnostic study we are currently following chromosomal anomalies in immunomagnetically enriched CD34+ peripheral blood cells in patients with suspected or proven myelodysplastic syndromes (MDS) at short intervals using fluorescence in situ hybridization (FISH) analysis every two to three months over three years. A loss of the Y chromosome was detected in 4% of these patients, as a single anomaly in 2%. Since it is controversially discussed whether loss of the Y chromosome is an age-related or a clonal event in patients with MDS, we aimed to examine whether a Y-loss is clonal or an age-related event in our patients. Methods:For patients with known Y-loss, we used peripheral blood not only to immunomagnetically enrich clonal CD34+ cells, but also CD3+ T-cells not belonging to the MDS clone. Subsequently, we performed FISH analysis to compare the clone sizes of cells with Y-loss in CD34+ and CD3+ cells. As our laboratory threshold for the FISH probe in CD34+ peripheral blood cells is 5%, we included 18 patients with clone sizes exceeding this threshold in CD34+ cells. The median age of the patients was 76 years (range 62–89). To establish a laboratory threshold for the FISH probe in CD3+ peripheral blood cells, we analyzed T-cells of 25 healthy men with a median age of 27 years (range 19–35). Furthermore, we just initialized an investigation of the laboratory threshold for the FISH probe in CD3+ peripheral blood cells of elder men by measuring the frequency of loss of the Y chromosome in T-cells of this control cohort not suffering from hematopoietic diseases. Until now we could recruit 15 men with a median age of 75 years (range 66–84) for this purpose, further will follow soon. Results:In patients with suspected or proven MDS, the number of cells with -Y was significantly increased in CD34+ cells compared to CD3+ cells (p<0.0001). The median clone size was 64% (range 12–97) in CD34+ cells and 5% (range 1–14) in CD3+ T-cells. The clone size in CD34+ cells was at least four times higher than in CD3+ cells in all patients. We could not detect further chromosomal abnormalities in 16 patients. Chromosomal banding analysis revealed that cells with -Y and cells with -Y and +8 occurred in parallel in two patients. In men below the age of 35 Y-loss could not be detected. The median clone size of 0.5% (range 0–2) resulted in a laboratory threshold of 2%. Interim analysis of men over the age of 65 resulted in a median clone size of 2.5% (range 1–14) and a laboratory threshold of 13%. So far the FISH-signal corresponding to the Y chromosome was significantly more frequent missing in T-cells of elder than in T-cells of younger men (p=0.005). Conclusion:Regarding the absence of Y-loss in CD3+ peripheral blood cells of young healthy men compared to up to 14% -Y in CD3+ peripheral blood cells of elder men, the low proportion of -Y in CD3+ cells of our patients suggests an age related Y-loss in normal T-cells. As the number of CD34+ peripheral blood cells with -Y exceeds the number of CD3+ peripheral blood cells with -Y in all patients, we assume that Y-loss is clonal to some extent in all of them. We established a reliable method to test if loss of the Y chromosome is disease- or age-related in individual MDS patients. It can be used to determine a clonal disease in patients with suspected MDS and Y-loss as sole abnormality. Disclosures:No relevant conflicts of interest to declare.

Identification of Differentially Expressed miRNAs in Peripheral t-Cell Lymphomas

Abstract 773Peripheral T-cell lymphomas (PTCLs) are rare and heterogeneous tumors whose biology is largely unknown. Interestingly, the commonest subtypes (i.e. PTCL not otherwise specified, NOS; angioimmunoblastic T-cell lymphoma, AITL; and anaplastic large cell lymphoma, ALCL) present on one hand few disease-specific molecular features and, on the other hand, several apparently common abnormalities. So far, no data are available regarding miRNA expression in these tumors.In order to identify miRNA deregulated in PTCLs, we performed an extensive miRNA profiling (by studying 379 targets on the TaqMan Array MicroRNA Cards) of 44 PTCLs (including 23 PTCLs/NOS, 12 ALCLs, and 9 AITLs) and 13 sample representative of normal T-cell sub-populations (CD4+ and CD8+, both resting and activated). In addition, for all these cases, gene expression profiles (GEPs) were generated by the Ilumina whole genome DASL-assay. TaqMan Quantitative-PCR (qPCR) was then used for validation.First, we found that PTCLs and normal T-cells could be easily distinguished based on their miRNA profile, by both unsupervised and supervised analysis. Specifically, the latter identified 91 miRNA differentially expressed in PTCLs vs. T-cells with a fold change ≥2 and a pvalue<0.01. Interestingly, the predicted target genes of such miRNA were involved in relevant cellular functions (such as cell proliferation, apoptosis, signal transduction etc.) and were found to be differentially expressed in tumor vs. controls, indicating a functional impact of miRNA deregulation in the molecular phenotype of neoplastic T-cells. Intriguingly, several drugs were estimated in silico to be able to interfere with pathways controlled by the de-regulated miRNA, including flavopiridol, everolimus/ temsirolimus, and forodesine.Secondly, by supervised analysis (ANOVA), we identified miRNA differentially expressed among PTCL/NOS, AITL and ALCL. Indeed, basing on such miRNA it was possible to roughly separate the different entities by hierarchical clustering (HC) (chi-square, p=0.0005). Specifically, we found 7 miRNA differentially expressed in PTCL/NOS vs. AITL, 26 in PTCL/NOS vs. ALK- ALCL, and 16 in ALK- vs. ALK+ ALCL. Selected miRNA were then studied by qPCR in an independent set of PTCLs (including 10 PTCL/NOS, 5 AITL, 5 ALK- ALCL, and 5 ALK+ ALCL) as for validation.Subsequently, in order to assess the impact of miRNA on the GEP, we picked up the miRNA most efficient in differentiating the diverse tumors (miR-431, miR-887, miR-889, and miR-155), and we studied the expression of their predicted target genes. Indeed, we found that miR-431 target genes were differentially expressed in PTCL/NOS vs. AITL (GSEA, q-value=0.45), and basing on their expression, HC could efficiently divide the two diseases (chi-square, p=0.004). Similarly, miR-889 targets were differentially expressed in PTCL/NOS vs. ALK- ALCL (GSEA, q-value=0; chi-square for HC, p=0.01), while miR-155 targets were differentially expressed in ALK+ vs. ALK- ALCL (GSEA, q-value=0; chi-square for HC, p<0.001).In conclusion, we performed for the first time an extensive miRNA profiling of PTCLs and combined it with global GEP. Importantly we identified miRNAs deregulated in PTCLs with possible pathogenetic implications. In addition, we found miRNAs, whose detection might be relevant for the differential diagnosis of PTCL subtypes. Finally, we provided in silico evidences that the cellular pathway controlled by de-regulated miRNA in PTCLs might serve as potential therapeutic targets, thus warranting further functional investigation. Disclosures:No relevant conflicts of interest to declare.

The HIV-1 gp120/V3 modifies the response of uninfected CD4 T cells to antigen presentation: mapping of the specific transcriptional signature

BackgroundThe asymptomatic phase of HIV-1 infection is characterized by a progressive depletion of uninfected peripheral effector/memory CD4+ T cells that subsequently leads to immune dysfunction and AIDS symptoms. We have previously demonstrated that the presence of specific gp120/V3 peptides during antigen presentation can modify the activation of normal T-cells leading to altered immune function. The aim of the present study was to map the specific transcriptional profile invoked by an HIV-1/V3 epitope in uninfected T cells during antigen presentation.MethodsWe exposed primary human peripheral blood monocytes to V3 lipopeptides using a liposome delivery system followed by a superantigen-mediated antigen presentation system. We then evaluated the changes in the T-cell transcriptional profile using oligonucleotide microarrays and performed Ingenuity Pathway Analysis (IPA) and DAVID analysis. The results were validated using realtime PCR, FACS, Western blotting and immunofluorescence.ResultsOur results revealed that the most highly modulated transcripts could almost entirely be categorized as related to the cell cycle or transcriptional regulation. The most statistically significant enriched categories and networks identified by IPA were associated with cell cycle, gene expression, immune response, infection mechanisms, cellular growth, proliferation and antigen presentation. Canonical pathways involved in energy and cell cycle regulation, and in the co-activation of T cells were also enriched.ConclusionsTaken together, these results document a distinct transcriptional profile invoked by the HIV-1/V3 epitope. These data could be invaluable to determine the underlying mechanism by which HIV-1 epitopes interfere with uninfected CD4+ T-cell function causing hyper proliferation and AICD.

Evolving Insights in the Pathogenesis and Therapy of Cutaneous T‐cell lymphoma (Mycosis Fungoides and Sezary Syndrome)

Cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of malignancies derived from skin-homing T cells. The most common forms of CTCL are Mycosis Fungoides (MF) and Sezary Syndrome (SS). Accurate diagnosis remains a challenge due to the heterogeneity of presentation and the lack of highly characteristic immunophenotypical and genetic markers. Over the past decade molecular studies have improved our understanding of the biology of CTCL. The identification of gene expression differences between normal and malignant T-cells has led to promising new diagnostic and prognostic biomarkers that now need validation to be incorporated into clinical practice. These biomarkers may also provide insight into the mechanism of development of CTCL. Additionally, treatment options have expanded with the approval of new agents, such as histone deacetylase inhibitors. A better understanding of the cell biology, immunology and genetics underlying the development and progression of CTCL will allow the design of more rational treatment strategies for these malignancies. This review summarizes the clinical epidemiology, staging and natural history of MF and SS; discusses the immunopathogenesis of MF and the functional role of the malignant T-cells; and reviews the latest advances in MF and SS treatment.