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Peripheral blood involvement by cutaneous T-cell lymphoma is typically assessed by flow cytometry and plays a critical role in diagnosis, classification, and prognosis. Simplified strategies to detect tumor cells (Sezary cells) fail to exclude reactive subsets, whereas tumor-specific abnormalities are subtle and inconsistently present. We implemented a flow cytometric strategy to detect clonal Sezary cells based on the monotypic expression of one of two mutually exclusive TCR constant β chains, TRBC1 and TRBC2. Analysis of CD4+T-cell subsets and TCR variable β classes from healthy donors showed polytypic TRBC1 staining. Clonal Sezary cells were identified by TRBC1 staining in 56 of 111 (50%) samples from patients with cutaneous T-cell lymphoma, accounting for 7–18,155 cells/μl and including 13 cases (23%) lacking tumor-specific immunophenotypic abnormalities. CD4+T-cell subsets from 86 patients without T-cell lymphoma showed polytypic TRBC1 staining, except for five patients (6%) with minute T-cell clones of uncertain significance accounting for 53–136 cells/μl. Assessment of TRBC1 expression within a comprehensive single-tube flow cytometry assay effectively overcomes interpretative uncertainties in the identification of Sezary cells without the need for a separate T-cell clonality assay.

Subluxation of the radial head is a common injury in young children. It occurs after longitudinal traction on the extended arm with the forearm in pronation. Diagnosis is made from history and clinical presentation. Closed reduction achieved by supination and flexion or by hyperpronating the affected arm can usually restore normal anatomy satisfactorily. Rarely, it requires open reduction. There are very few cases in the literature that have required open reduction and no article shows intraoperative photographs. We present two cases of delayed diagnosis owing to an atypical clinical history that required open reduction.

Confluences are locations of complex hydrodynamic conditions within river systems. The effects on hydrodynamics and mixing of temperature‐induced density differences between incoming flows are investigated at a small‐size, concordant bed confluence. To evaluate density effects, results of eddy‐resolving simulations for a densimetric Froude number Fr= 4.9 (weak‐density‐effects cases) and Fr= 1.6 (strong‐density‐effects cases) are compared to results of simulations in which the densities of the incoming flows do not differ (no‐density‐effects cases). Flow patterns predicted for both weak‐ and strong‐density‐effects cases show that secondary flow develops with increasing distance from the confluence apex. The pattern of secondary flow is characterized by denser fluid on one side of the confluence moving near the bed toward the side of the downstream channel corresponding to the less dense fluid and the less dense fluid moving near the free surface in the opposite direction. This pattern of fluid motion is similar to a spatially evolving lock‐exchange cross flow. In the strong‐density‐effects simulations, a cross‐stream cell of secondary flow develops at the density interface between the flows, similar to interfacial billows generated in classical lock‐exchange flows. Density effects increase global mixing with respect to corresponding no‐density‐effects cases regardless of whether the high‐momentum stream contains the higher‐density fluid or the lower‐density fluid. When density effects are weak, the lock‐exchange mechanism either reinforces the pattern of mixing associated with secondary flow induced by inertial forces, particularly helical motion, or opposes this pattern of mixing, depending on which tributary contains the denser fluid. When density effects are strong, flow from the upstream channel with the denser fluid moves under the flow from the upstream channel with the less dense fluid. Weak and strong density effects lead to the development of secondary flow with increasing distance from the confluence apexDensity difference between the incoming fluids affects the size of the stagnation zone and the type and coherence of MI billowsFor high densimetric Froude numbers a new type of cross‐stream cell of secondary flow are generated

Background: Small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) is a lymphoproliferative disorder of small mature B-cells, most commonly presenting with peripheral blood and bone marrow involvement. Tumoral lesions are variably encountered, resulting from either lymph node involvement or, less commonly, infiltration into virtually any extramedullary site. The prognostic assessment of patients with SLL/CLL relies strongly on the identification of recurrent chromosomal abnormalities, which are routinely tested by fluorescence in situhybridization (FISH) on peripheral blood or bone marrow specimens. However, the incidence of such recurrent chromosomal abnormalities on tumoral lesions has only been studied on small series, and its concordance with peripheral blood and/or bone marrow testing remains unknown. We hereby report a large series of chromosomal abnormalities detected on tumoral SLL/CLL lesions, based on a validated FISH panel for formalin-fixed/paraffin-embedded (FFPE) biopsy sections.

Horna: MorphoSys AG: Research Funding. Nowakowski:Selvita: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Bayer: Consultancy, Research Funding; Curis: Research Funding; F. Hoffmann-La Roche Ltd: Research Funding; Genentech, Inc.: Research Funding; MorphoSys: Consultancy, Research Funding; NanoString: Research Funding. Endell:MorphoSys AG: Employment, Patents & Royalties. Boxhammer:MorphoSys AG: Employment, Patents & Royalties.

Horna: MorphoSys AG: Research Funding.

Background: Chronic myelomonocytic leukemia (CMML), a myeloid neoplasm with overlapping features of myelodysplastic syndromes and myeloproliferative neoplasms (MPN), is characterized by peripheral blood (PB) monocytosis and a risk for leukemic transformation (LT). Gene mutations commonly seen in CMML include, TET2 (~60%), SRSF2 (~50%)and ASXL1 (~40%). MPN associated-driver mutations such as JAK2V617F (~10%), MPL (<1%)and CALR (<1%)are uncommon, and in fact, the 2016 World Health Organization (WHO) guidelines state that the presence of these mutations tends to support a diagnosis of MPN with monocytosis. We carried out this study to assess the i)clinical correlates and ii)survival outcomes of MPN associated-driver mutations in CMML.

Introduction: Outcomes of multidisciplinary and molecular tumor boards have been reported. Lymphoma specific tumor board outcomes have not been reported in the molecular era. In addition, the utility of multi-site, interactive tumor boards using videoconference technology are not widely reported. We prospectively followed the outcomes of a multidisciplinary multi-site lymphoma tumor board in the molecular era and during a change in the revision of the World Health Organization Classification in first published in 2016.

Determination of metanephrines from blood plasma plays an important role in the diagnosis of chromaffin cell tumors pheochromocytoma (PHEO) and paraganglioma (PGL). It is highly preferable to use this method for the diagnosis as it is more sensitive than the other methods.

Nonhepatosplenic/noncutaneous γδ peripheral T-cell lymphoma (NHNCγδ PTCL) represents a miscellaneous group of unrelated T-cell lymphomas of which only isolated cases have been reported. We describe two cases of transformation from T-lymphoblastic leukemia/lymphoma to NHNCγδ PTCL. Transformation into more aggressive disease is a rare event in T-cell lineage-derived hematologic malignancies compared to B-cell neoplasms. Nevertheless, both of our cases involved relapse as PTCL manifested with skin involvement and an overt shift from blastic morphology to large granular leukemia-like mature T cells. Among other notable molecular characteristics, expression of immature markers such as TdT was lost in both cases. Based on cytogenetics, phenotype, and morphology, both patients represent a novel phenomenon of clonal transformation from T-ALL to PTCL which has rarely been reported in the literature. Such transformation may carry important diagnostic and biological implications.

Inhibiting Aurora kinase A and JAK2 signal transduction pathways permits the differentiation of potent regulatory T cells while neutralizing alloreactive T cells and preventing graft-versus-host disease without impairing antitumor responses.

Background

Phoneticians traditionally describe American English /&edh;/ as a voiced, nonsibilant, interdental fricative. However, in flowing speech, /&edh;/ often sounds stoplike. This study investigates the acoustic attributes of stoplike, as well as continuant, variants of /&edh;/ in “the” occurring in various phonological conditions in text‐reading and conversation modes. The results of spectrographic analysis allow for the definition of five degrees of constriction and two types of constriction release, burst and smooth formant transition into the following segment. The degree of constriction is a coarticulatory effect of the preceding phoneme. Smooth formant transitions tend to occur with less constricted degrees of constriction; a burst may occur with any degree of constriction and is prevalent across phonological conditions. Articulatory interpretations that explain these and other variations of /&edh;/ are forwarded. The results also show intermodal differences in /&edh;/ in that there is a higher occurrence of some coarticulatory effects of the preceding phoneme in the conversation mode than in the text‐reading mode.

Purpose. The goal was to describe our experience in the surgical management and treatment of four patients with congenital upper eyelid colobomas. Methods. A descriptive, observational, retrospective study was performed including patients with congenital eyelid colobomas referred to Asociación para Evitar la Ceguera en México I.A.P. “Dr. Luis Sánchez Bulnes” between 2004 and 2014 and assessed by the Oculoplastics and Orbit Service. Results. The four cases required surgical treatment of the eyelid defects before one year of age and their evolution was monitored from the time of referral to the present day. One of the patients needed a second surgical procedure to repair the eyelid defect and correct the strabismus. Conclusions. Eyelid colobomas are a potential threat to vision at an early age, which requires close monitoring of the visual development of patients.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder (LPD) derived from mature B-cells with heterogeneous outcomes. High-grade lymphoma can arise from CLL in 3-10% of cases, a process known as Richter's transformation (RT). The majority of RT results in high grade B-cell lymphomas, though rarely transformation to Hodgkin lymphoma (HL) has been reported. Epstein Barr virus (EBV) viremia can arise in patients with CLL secondary to immunosuppression and can lead to an EBV driven LPD. In patients with CLL, this has been seen with fludarabine based treatments but has been reported with alemtuzumab. HL is also been associated with EBV virus in tumor cells but cases of HL and EBV viremia have not been reported. We report the first two cases in the English literature of CLL with EBV viremia and HL.

Hepatosplenic T-cell lymphoma (HSTL) is a rare form of lymphoma, comprising less than 1% of the cases. However, HSTL extracts a highly disproportionate toll on patients with a median age of diagnosis of 35 years and an expected median survival of less than two years. The vast majority of HSTL patients eventually succumb to their disease. The genetic basis of the disease is largely unknown. Although abnormalities of chromosome 7, including isochromosome 7q occur commonly in the disease, the role of specific genes and genetic mutations to the disease remains essentially unknown.In this study, we sought to define the genetic features of HSTL through the whole genome sequencing and exome sequencing of 32 HSTL tumors and germline DNA (where available) from the same patients. Exome enrichment of DNA was carried out using the Agilent solution-based system of exon capture, which uses RNA baits to target all protein coding genes as well as ∼700 human microRNAs. Both whole genome and exome sequencing were performed using the Illumina platform.We identified 28 candidate cancer genes that were recurrently mutated in HSTL. Commonly implicated biological processes comprising these genes included signal transduction (e.g. PIK3CD, KRAS) and chromatin modification (e.g. TET1, SETD2 and MLL3), accounting for 16% and 23% of the total genetic events, respectively. Nearly all of these genes have been implicated in HSTL for the first time and provide new insights into the pathogenesis of the disease and potential targets for therapy. Whole genome sequencing confirmed isochromosome 7q as the most common recurrent chromosomal abnormality in HSTL and additional structural genetic alterations in chromosome 7.Our study provides the most comprehensive genetic portrait of HSTL to date, and is a significant step in defining the genetic causes of this disease.No relevant conflicts of interest to declare.

Chronic myelomonocytic leukemia (CMML) is a myelodysplastic and myeloproliferative neoplasm (MDS/MPN) characterized by cytopenias, persistent monocytosis, morphologic dysplasia and a tendency to transform to acute myeloid leukemia (AML). Similar to MDS, multiple genetic, molecular, microenvironmental and immunologic mechanisms of pathogenesis have been linked to CMML. Autoimmunity results from inflammation and tissue damage caused by inappropriate T and B cell self-recognition. The prevalence of autoimmunity in the U.S. population (not age adjusted) was estimated to be 3.2% prior to 1996 and is now 5 –8% according to 2004 data with a female prevalence (M: F= 1:3) (http://www.niaid.nih.gov/topics/autoimmune/documents/adccfinal.pdf). The prevalence of autoimmunity is estimated to be higher (10–17%) in MDS and MDS derived AML compared to the general population (Giannouli S et al.2012, Emadi A abstract 2012 ASCO).Recent investigations have shown a close link between autoimmune disorders and cancer. A recent study indicated that a history of autoimmununity might predict a better clinical response to DNA methyltransferase inhibitors (DNMTI) in MDS [Emadi A abstract 2012 ASCO]. The current study was conducted to identify the prevalence of autoimmune diseases in patients with CMML and compare it with the general population and to patients with a non-MDS-related MPN chronic myelogenous leukemia (CML).A retrospective analysis was performed of a series of 123 patients with CMML at Moffitt Cancer Center diagnosed between January 1999 and June 2011. A consecutive collection of 116 patients who were diagnosed with CML between January 2007 and June 2011 was included for comparison. Patients with a past medical history of autoimmunue disorders e.g. idiopathic thrombocytopenic purpura (ITP), Sjogren's syndrome, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), chronic autoimmune hemolytic anemia, inflammatory bowel diseases (IBD, ulcerative colitis or Crohn disease), psoriasis etc. were retrieved and clinical findings were correlated to published prevalence of these conditions in the general population.Among 123 CMML patients (average age of 68.6, ranging from 30 to 90 y/o, M: F = 2.61), 20 (16.26%, 8 Female, 12 Male) had a history of autoimmune related disorders: RA(3, 2.4%), MS (2, 1.6% ), Sjogren's syndrome (1, 0.8%), IBD(1, 0.8%), autoimmune anemia (1, 0.8%), psoriasis (4, 3.25%), polymyalgia rheumatica (1, 0.8%), ITP(6, 4.9%), hyperthyroidism (2, 1.6%); and 3 patients have more than 1 autoimmune disorder. In addition, 9 of 123 cases (7.3%) had a history of the other malignancies (7 with solid tumors and 2 with hematopoietic neoplasms other than MDS/CMML/AML, but no CML). In the CML group (average age of 55.5, ranging from 21 to 84 y/o, M: F = 1.2), history of autoimmunity was found in 6 of 116 patients (5.2%), which is similar to reports of these conditions in the general population. The autoimmune phenomenon included 2 RA, 2 polymyalgia rheumatica, 1 IBD, and 1 psoriasis. There was a statistically significant difference in the rate of autoimmune diseases between CMML and CML (p=0.0066, X2test). Of note, 5 CMML patients were considered secondary CMML; however, none of the 5 patients in the study had documented autoimmununity in his/her past medical history.The presence of autoimmune disorders is significantly higher in CMML compared to historical data on the general population and Moffitt Cancer Center patients with CML. Identifying autoimmune process in CMML may relevance to development of novel therapeutic strategies. A larger study to explore the detailed relationship between a history of autoimmune diseases and CMML is needed to define the molecular pathogenesis.No relevant conflicts of interest to declare.

Abstract 5085

Martin: Millennium Pharmaceuticals, Inc.: Speakers Bureau.

No relevant conflicts of interest to declare.

In previous studies we have shown that Signal Transducer and Activator of Transcription 3 (STAT3) negatively regulates inflammatory responses in myeloid cells and plays a central role in determining immune activation versus immune tolerance of antigen-specific CD4+ T-cells. Indeed, in Stat3 knock out mice (LysMcre/Stat3flox/−) in which macrophages and neutrophils are devoid of Stat3, we found that in response to a tolerogenic stimulus (high dose peptide-induced tolerance or tumor-induced tolerance models) adoptively transferred antigen-specific CD4+ T-cells are not tolerized but instead are effectively primed as determined by their production of IL-2 and IFN-gamma in response to cognate antigen. Such an observation led us to investigate which cell population(s) is required for the priming effect observed in Stat3 KO mice. First, we used anti-Gr-1 antibody to deplete neutrophils in wild type BALB/c mice as well as Stat3 KO mice. Briefly, half the mice in each group were treated with 0.5mg of the antibody given i.p. every 3 days from day-3 until day +15. On day zero, all the mice were adoptively transferred with 2.5 x 106 naïve transgenic CD4+ T-cells specific for a MHC class II restricted epitope of hemaglutinin (HA). On day +2, animals received high dose of HA peptide (275 mcg) given i.v. Mice were sacrificed on day +15 and clonotypic T-cells were re-isolated from their spleens to assess their functional status following their in vivo exposure to this tolerogenic stimuli. A striking difference was observed in T-cells isolated from Stat3 KO mice with an intact neutrophil compartment (non-depleted) versus T-cells from anti-Gr-1 treated LysMcreStat3flox/− mice. Unlike T-cells from the former group in which priming was the functional outcome, clonotypic T-cells from LysMcreStat3flox/− mice depleted of neutrophils, were found to be anergic. Therefore, the T-cell priming effect observed in LysMcreStat3flox/− mice requires an intact neutrophil compartment given that in the absence of this population, tolerance not priming was the functional T-cell outcome. To gain insight into the potential mechanism(s) by which neutrophils devoid of Stat3 influence T-cell responses, we next analyzed the phenotypic and functional properties of neutrophils isolated from Stat3 KO mice and wild type controls. First, the lack of expression of MHC class II molecules by neutrophils from WT and KO mice made unlikely the possibility that neutrophils devoid of Stat3 could directly present antigen to CD4+ T-cells. However, when neutrophils from Stat3−/− conditional mice were added to macrophages monolayers in vitro, the antigen-presenting capabilities of macrophages was significantly enhanced as determined by the increased production of IL-2 and IFN-gamma by antigen-specific T-cells encountering cognate antigen in these APCs. Furthermore, macrophages cultured in vitro with neutrophils from Stat3−/− conditional mice were able to restore the responsiveness of tolerant CD4+ T-cells. This effect that was not observed when tolerized T-cells encountered cognate antigen in macrophages incubated with neutrophils from wild type mice. Trans-well experiments demonstrated that the regulatory effect of neutrophils upon APCs function required cell-cell contact. Taken together, we have unveiled a previously unrecognized role of neutrophils in determining the functional outcome of antigen-specific T-cell responses, effect that is dependent upon the interaction of neutrophils with antigen-presenting cells.

Signal transducer and activator of transcription 3 (Stat3) is a key mediator of several cytokines and growth factors signaling pathways. On myeloid cells, activation of Stat3 to its phosphorylated form (pStat3) has been shown to negatively regulate inflammatory responses. Recently, we have unambiguously demonstrated that Stat3 signaling in APCs also play a central role in the decision leading to immune activation versus immune tolerance of antigen-specific T-cells1. In spite of these advances, there is however a paucity of therapeutic strategies targeting this signaling pathway in immune cells. Using a high throughput cytoblot screening for phospho-Stat3 inhibition, we have recently identified a family of natural compounds known as Cucurbitacins that effectively disrupt Stat3 signaling at different levels2. Three compounds have been identified, Cucurbitacin A (CuA) that inhibits phospho-JAK-2, Cucurbitacin I (CuI) a dual inhibitor of p-JAK2 and p-Stat3 and Cucurbitacin Q (CuQ) a selective inhibitor of p-Stat3. In vitro treatment of peritoneal elicited macrophages (PEM) and bone marrow-derived dendritic cells (DCs) with increasing concentrations of CuA or CuI resulted in inhibition of p-Stat3 and enhanced antigen presentation to naive CD4+ T cells specific for a MHC class II restricted epitope of influenza hemagglutinin (HA). Indeed, these clonotypic T cells displayed increased antigen-specific proliferation and IL-2 production as compared to clonotypic T cells encountering cognate antigen on untreated APCs. Furthermore, unlike untreated PEM or DCs, which are unable to trigger IFN-gamma production by CD4+ T-cells, Cucurbitacin-treated APCs efficiently trigger the production of this cytokine by naïve CD4+ T-cells in response to cognate antigen. Given the above results, we explored next whether inhibition of Stat3 signaling in B-cell lymphomas by Cucurbitacins might increase the intrinsic antigen-presenting capabilities of these malignant B-cells. Reminiscent of our findings with bone marrow derived APCs, Cucurbitacin-treated A20 lymphoma cells also display enhanced antigen-presenting cell function leading to increased proliferation, IL-2 and IFN-gamma by naive antigen-specific CD4+ T-cells. More importantly, tolerant CD4+ T-cells (isolated from lymphoma bearing mice) exposed to Cucurbitacin-treated A20 B-cells regained their ability to proliferate and produce significant amounts of IL-2 and IFN-gamma in response to cognate antigen stimulation. Taken together, the ability of Cucurbitacins to inhibit p-Stat3 in normal APCs as well as in malignant B-cells make these natural compounds a promising agents to overcome the remarkable barrier that tolerance to tumor antigens has imposed to cancer immunotherapeutic strategies.

A large number of CD4+ T-cells from either aged mice or humans display surface markers associated with an activated/memory phenotype. In spite of these changes however, these T-cells have a markedly decreased ability to proliferate and produce IL-2 in response to antigen stimulation in vitro. The cellular and molecular mechanisms involved in this age-related unresponsiveness of the CD4+ T-cell compartment remain poorly understood. Utilizing a well-established experimental system in which transgenic CD4+ T cells specific for a MHC class II restricted epitope of influenza hemagglutinin (HA) are adoptively transferred into non-transgenic recipients, we have previously elucidated important mechanisms involved in the induction and maintenance of CD4+ T-cell tolerance. Our studies were however limited to the analysis of T-cell function in lymphoma bearing young mice (4 to 10 weeks old). Here, we assessed the influence of the aged microenvironment in determining the phenotype and function of antigen-specific T-cells. CD4+ T-cells from young TCR transgenic mice (2 months old) were adoptively transferred into either old (20–24 months) or young (2 months old) non-transgenic mice. Two weeks later, clonotypic and non-clonotypic CD4+ T-cells were isolated from the spleens of these animals and their phenotype and function were determined in vitro. Reminiscent of the age-related changes observed within the normal CD4+ T-cell repertoire, young transgenic T-cells transferred into aged hosts have acquired an activated/memory phenotype but displayed a significant impairment in antigen-specific proliferation and IL-2 production in response to cognate antigen in vitro. These changes were not due to homeostatic proliferation of the transferred T-cells into the relatively lymphopenic aged host. To determine whether the changes observed in “aged” T-cells were reversible or not, we adoptively transfer old T-cells back into young hosts or into control old mice. While old transgenic T-cells transferred into an old environment remained fully unresponsive, the adoptive transfer of the same old T-cells into a young host restored their ability to proliferate and produce IL-2. Surprisingly, these “old” T-cells were able to produce significantly higher levels of IFN-gamma indicative of their memory/effector phenotype. Furthermore, young animals adoptively transferred with “aged” antigen-specific T-cells were now capable of rejecting A20 B-cell lymphomas expressing HA as a model tumor antigen (A20HA). Taking together, factor(s) present in the aged microenvironment are responsible for limiting the effector function of CD4+ T-cells that seem otherwise well equipped to become fully activated if the proper environment is provided (young microenvironment). The potential role of soluble suppressive factors as well as regulatory T-cells (Tregs) in the unresponsiveness observed in the T-cell compartment of aged hosts will be discussed.

Professional antigen-presenting cells (APCs) play an important role in the initiation of antigen-specific T-cell responses. The demonstration that these cells are also required for the induction of T-cell tolerance, placed APCs at the crossroads of immune activation versus immune tolerance. Recent studies have demonstrated that the inflammatory status of the APC at the time of antigen presentation is the central determinant of T-cell priming versus T-cell tolerance. As such, therapeutic induction of inflammatory APCs might override immune tolerance and enhance the efficacy of immunotherapeutic strategies targeting hematologic tumors. Lenalidomide (CC5013) is a thalidomide analogue with immunomodulatory properties. Phase I and Phase II clinical trials in patients with myelodysplastic syndrome (MDS) have shown high frequency of erythropoietic responses, particularly in patients with 5q31 deletion associated with emergence of polyclonal lymphoid infiltrate in responding patient bone marrows. This observation raised the question as to whether immunological mechanism(s) may mediate, at least in part, the beneficial effect of CC5013 in patients with MDS. To gain further insight into the effects of Lenalidomide on APC’s function and regulation of antigen-specific CD4+ T-cell responses, we treated peritoneal elicited macrophages (PEM) and bone marrow-derived dendritic cells (DCs) with escalating concentration of Lenalidomide in vitro. Enhanced expression of both B7.1 and B7.2 co-stimulatory molecules was observed in Lenalidomide-treated APCs relative to untreated APCs. No difference in the expression of MHC class II molecules or CD40 was detected. Assessment of cytokine production by ELISA showed that Lenalidomide-treated APCs produce higher levels of TNF-a, IL-6 and IL-10 in response to LPS stimulation as compared to untreated APCs. Next, we evaluated the ability of Lenalidomide-treated APCs to present cognate antigen to naïve and tolerant CD4+ T-cells specific for a MHC class II restricted epitope of influenza hemagglutinin (HA). We found that treatment of either PEM or DC with low doses of Lenalidomide (range: 1.5–12.5 uM) significantly enhanced their antigen-presenting capabilities leading to effective priming of naïve CD4+ T-cells confirmed by their increased production of IL-2 and IFN-gamma in response to cognate antigen. Taken together, our results shows that by inducing inflammatory APCs, Lenalidomide directs the outcome of antigen-specific T-cell responses. Furthermore, they have broadened the scope of this drug as a promising adjuvant in cancer immunotherapy.

A large number of CD4+T-cells from either aged mice or humans display surface markers associated with an activated/memory phenotype. In spite of these changes however, these T-cells have a markedly decreased ability to proliferate and produce IL-2 in response to antigen stimulation in vitro. The cellular and molecular mechanisms involved in this age-related unresponsiveness of the CD4+T-cell compartment remain poorly understood. Utilizing a well-established experimental system in which transgenic CD4+T cells specific for a MHC class II restricted epitope of influenza hemagglutinin (HA) are adoptively transferred into non-transgenic recipients, we have previously elucidated important mechanisms involved in the induction and maintenance of CD4+T-cell tolerance. Our studies were however limited to the analysis of T-cell function in lymphoma bearing young mice (4 to 10 weeks old). Here, we assessed the influence of the aged microenvironment in determining the phenotype and function of antigen-specific T-cells. CD4+T-cells from young TCR transgenic mice (2 months old) were adoptively transferred into either old (20–24 months) or young (2 months old) non-transgenic mice. Two weeks later, clonotypic and non-clonotypic CD4+T-cells were isolated from the spleens of these animals and their phenotype and function were determined in vitro.Reminiscent of the age-related changes observed within the normal CD4+T-cell repertoire, young transgenic T-cells transferred into aged hosts have acquired an activated/memory phenotype but displayed a significant impairment in antigen-specific proliferation and IL-2 production in response to cognate antigen in vitro. These changes were not due to homeostatic proliferation of the transferred T-cells into the relatively lymphopenic aged host. To determine whether the changes observed in “aged” T-cells were reversible or not, we adoptively transfer old T-cells back into young hosts or into control old mice. While old transgenic T-cells transferred into an old environment remained fully unresponsive, the adoptive transfer of the same old T-cells into a young host restored their ability to proliferate and produce IL-2. Surprisingly, these “old” T-cells were able to produce significantly higher levels of IFN-gamma indicative of their memory/effector phenotype. Furthermore, young animals adoptively transferred with “aged” antigen-specific T-cells were now capable of rejecting A20 B-cell lymphomas expressing HA as a model tumor antigen (A20HA). Taking together, factor(s) present in the aged microenvironment are responsible for limiting the effector function of CD4+T-cells that seem otherwise well equipped to become fully activated if the proper environment is provided (young microenvironment). The potential role of soluble suppressive factors as well as regulatory T-cells (Tregs) in the unresponsiveness observed in the T-cell compartment of aged hosts will be discussed.

Professional antigen-presenting cells (APCs) play an important role in the initiation of antigen-specific T-cell responses. The demonstration that these cells are also required for the induction of T-cell tolerance, placed APCs at the crossroads of immune activation versus immune tolerance. Recent studies have demonstrated that the inflammatory status of the APC at the time of antigen presentation is the central determinant of T-cell priming versus T-cell tolerance. As such, therapeutic induction of inflammatory APCs might override immune tolerance and enhance the efficacy of immunotherapeutic strategies targeting hematologic tumors.

Tumor antigen-specific T-cell tolerance imposes a significant barrier to the development of effective therapeutic cancer vaccines. Bone marrow-derived antigen presenting cells (APCs) are critical in the induction of this unresponsive state. Recently, we have identified STAT3 signaling in APCs as an important regulatory pathway that determines the functional outcome of antigen-specific CD4+ T-cells in response to cognate antigen. Indeed, while disruption of this signaling pathway in APCs led to effective T cell priming, enhanced STAT3 activity resulted in the induction of T cell unresponsiveness1. Given the above results, we explored in this study whether disruption of STAT3 signaling in APCs may preserve the responsiveness of antigen-specific CD4+ T-cells during the growth of a tumor that induces antigen specific T-cell tolerance. First, mice with a genetic disruption of Stat3 in macrophages, neutrophils and a sub-population of myeloid DCs (LysMcre/Stat3flox/− mice) or control C57BL/6 mice were given subcutaneously 1x106 B16 melanoma tumor cells engineered to express Ovalbumin as a model tumor antigen (B16-OVA). Four days later, naive CD4+ T-cells (1x106) specific for a MHC class II-restricted epitope of Ovalbumin (OT-II cells) were adoptively transferred intravenously into tumor bearing mice as well as into tumor-free controls. Two weeks later animals were sacrificed and antigen-specific CD4+ T-cell responses to in vitro re-stimulation with OVA-peptide were evaluated. As expected, antigen-specific T cells re-isolated from tumor-bearing C57BL/6 mice were fully tolerant (lack of HA-specific proliferation and cytokine production). In sharp contrast, anti-OVA CD4+ T-cells isolated from tumor bearing LysMcre/Stat3flox/− mice remained fully functional as determined by their capacity to proliferate and produce IL-2 and IFN-gamma in response to cognate OVA-peptide. The demonstration that tumor-induced antigen-specific CD4+ T-cell tolerance occurs in mice with an intact STAT3 signaling in APCs, but not in mice with genetic disruption of this signaling pathway, led us next to evaluate the efficacy of pharmacologic inhibitors of STAT3 in preventing and or overcoming tumor-induced T-cell tolerance. In vivo treatment of tumor bearing mice with Tyrphostin AG490 (0.5 mg/ i.p. /twice a day x 5 days), a well-known inhibitor of STAT3 signaling, also resulted in preservation of the responsiveness of tumor-antigen specific CD4+ T-cells. Furthermore, in vitro treatment of APCs with this compound led to effective priming of naive antigen-specific T cells and breaking of antigen-specific T-cell anergy. More recently, we have evaluated the efficacy of a novel STAT3 inhibitor, compound 295558, which efficiently inhibits the DNA-binding activity of STAT3. Treatment of DCs or macrophages with this specific inhibitor led to the generation of inflammatory APCs capable of restoring the responsiveness of tolerized CD4+ T-cells isolated from tumor bearing mice. Taken together, our findings establish a critical role for STAT3 signaling in the induction of tolerance to tumor antigens in vivo. Inhibition of this signaling pathway in APCs provides a novel molecular target to overcome the remarkable barrier that tolerance to tumor antigens imposes to cancer vaccination strategies.

The immune system is very effective in fighting infections but seems not to be as efficient in recognizing and destroying cancer cells. In the cancer setting, tumor antigen uptake and presentation by APCs to antigen-specific T-cells often occurs in the absence of inflammation resulting therefore in tolerance induction. It is plausible therefore that by converting APCs from a non-inflammatory to an inflammatory phenotype through ligation of TLRs we may well overcome immune tolerance and tip the balance towards productive tumor antigen-specific T-cell responses. Among all the TLR-ligands identified to date, flagellin is the only one with a strictly proteinic nature, characteristic that make it a suitable candidate for cloning and transfection into tumor cells to generate novel tumor cell based vaccines. In this study, we first evaluated whether treatment with purified flagellin could prevent tolerance induction in vivo. Naïve CD4+ T-cells (2.5x106) specific for a MHC class II-restricted epitope of influenza hemagglutinin (HA) were adoptively transferred intravenously into BALB/c mice, 24 hours after mice were given either a tolerogenic dose of HA-peptide (200 mg), or a combination of this high dose of peptide together with Flagellin (10 mg I.V.). Two weeks later animals were sacrificed and antigen-specific CD4+ T-cell responses towards the cognate antigen evaluated in vitro. As expected clonotypic T-cells isolated from animals treated with high dose peptide were fully tolerant, in sharp contrast with those isolated from flagellin treated animals that displayed normal responses in terms of cytokine production and proliferation. Surprisingly, this preservation of T-cell function following in vivo treatment with flagellin was not observed when animals were treated with high dose HA-peptide in the presence of the TLR4 ligand, LPS. To better understand the mechanism(s) by which flagellin, and not LPS, preserved the responsiveness of antigen-specific CD4+ T-cells to cognate antigen presented by APCs, we assessed the phenotypic characteristics and the cytokine profile of macrophages and DCs treated in vitro with these TLRs ligands. Although LPS-treated APCs produce higher levels of IL-12, relative to flagellin-treated APCs, the production of this pro-inflammatory cytokine was accompanied by a parallel induction of the anti-inflammatory cytokine, IL-10. Interestingly, flagellin-treated APCs produced IL-12 but were unable to produce IL-10. This effect was dependent on ligation of TLR5, since it was not observed when RAW264.7 cells -which lack TLR5- were treated with flagellin. In vivo studies further confirmed our observations since IL-10 was not detected in the serum of animals treated with flagellin, but it was present in significant amounts in LPS-treated animals. This inhibitory effect of flagellin on IL-10 production was seen even when APCs were stimulated in vitro with strong inducers of IL-10. Given the above properties of flagellin, we generated two novel approaches to be used in the formulation of tumor cell based vaccines: 1) A bystander cell line transfected with the fliC gene from Salmonella typhimurim for flagellin expression (B78H1-Flagellin) and 2) Microspheres beads coated with flagellin. Both vaccination strategies are being currently studied in the in vivo and in vitro settings

There is now overwhelming evidence that tumor-induced antigen (Ag)-specific T cell tolerance represents a critical problem in tumor immunology. Early studies of CD8 T cell tolerance equated peripheral tolerance with either ignorance or clonal deletion, although more recent evidence has suggested that this may be only partly accurate. While murine modeling outwardly supports the contention that high-affinity tumor-specific CD8 T cell responses are centrally deleted, cognate CD8 T cells displaying an Ag-experienced phenotype can nonetheless be detected in regional draining lymph nodes (dLN) or in non-lymphoid sites where the Ag is present. However, these CD8 T cells are typically deficient in one or more effector functions, including cytokine production, cytotoxicity, or proliferative capacity. To better define the state of Ag-specific CD8 T cell responsiveness in the face of progressive tumor, we adoptively transferred hemagglutinin (HA) Ag-specific Clone 4 (CLN4) CD8 T cells into animals bearing a genetically modified B cell lymphoma expressing HA as a model tumor antigen (A20HA). Analysis of the fate and function of these transferred antigen-specific CD8 T cells revealed that they encountered antigen in vivo, were capable of mounting an initial response to A20HA but this response was not sustained. Indeed, while a prominent CTL activation was observed in the spleen and draining lymph nodes of tumor bearing mice within 14 days of T cell transfer, responses (HA-specific proliferation, IFN-γ production and cytotoxicity) began to wane by day 21 after T cell transfer, and in particular their ability to produce IFN-γ. A similar pattern of transient activation followed by loss of CD8 T cell function has been also observed in an in vivo model of high-dose peptide induced antigen-specific CD8 T cell tolerance. Given our recent demonstration that the disruption of Stat3 signaling in APCs overcomes CD4 T cell tolerance we determined next whether Stat3 deficient APCs may be inherently better at cross-presenting tumor-Ags and elicit therefore a more productive and sustained CD8 T cell response. In an in vitro system in which tumor cells expressing a model tumor antigen (EL4mOVA) were cultured with APCs genetically devoid of Stat3 signaling and anti-OVA CD8 T-cells (OT-I), we found that these T cells displayed an enhanced function relative to antigen-specific CD8 T-cells that encountered antigen on APCs with an intact Stat3 signaling. Currently, we are investigating whether CD8 T-cell tolerance to tumor antigens occurred -or not- in tumor bearing mice with a genetic disruption of Stat3 signaling in APCs. Furthermore, given the emerging role of other members of the STAT family in regulation of APC function, we are exploring whether targeted disruption of Stat1, 4 and 6 can alter the ability of the CD8 T-cell to sustain a protective response or, more importantly to recover function once tolerance is induced.

Signal transducer and activator of transcription 3 (Stat3) is a key mediator of several cytokine and growth factor signaling pathways. On myeloid cells, activation of Stat3 to its phosphorylated form (pStat3) has been shown to negatively regulate inflammatory responses and play a central role in the decision leading to immune activation versus immune tolerance of antigen-specific T-cells1. Little is still known however, about the status of Stat3 signaling in myeloid cells in the steady state and during ongoing immune responses in vivo. To address this question we recently developed flow-cytometric and immuno-histochemistry assays that have allowed us to visualize the in vivodynamics of Stat3 activation in myeloid cells during immune responses leading to divergent outcomes: productive inflammatory response to adjuvant immunization and tumor-induced unresponsiveness or tolerance. In the steady state we found that in peripheral blood only Ly6G+polymorphonuclear cells display a positive nuclear staining for pStat3. Analysis of lymphoid organs revealed that although Stat3 protein was expressed almost ubiquitously on spleen sections of normal mice, only a small number of cells were positive for pStat3. Following immunization with complete Freund adjuvant (CFA) a dramatic increase in the number of cells expressing pStat3 was observed in the peripheral blood and spleen of treated animals. Ly6G+pStat3+were rapidly recruited from the blood to the inflammatory site where they now displayed significantly decreased levels of pStat3. During the growth of a subcutaneous tumor, a similar increase in the number of cells expressing pStat3 was observed in the blood and spleen of tumor-bearing mice. Further analysis by flow cytometry revealed that pStat3 expression was restricted to two sub-populations: a)CD11b+myeloid cells expressing the lineage marker Gr-1 and b)Ly6G−mononuclear cells unable to down-regulate Stat3 activity following their migration from the blood into peripheral tissues. In vivodepletion of Gr-1+cells eliminated most of the pStat3+cells in tumor bearing mice. The immunoregulatory properties of these Gr-1+ cells was highlighted by the demonstration that in their absence, in vivoimmunization with a peptide derived from influenza hemagglutinin (HA) in CFA markedly enhanced the priming of anti-HA specific CD4+T-cells. More importantly, in animals depleted of Gr-1+cells, the outcome in response to a tolerogenic stimuli was T-cell activation rather than tolerance induction. Taken together, although similar changes in the number of cells expressing pStat3 was observed in response to adjuvant immunization and during tumor progression, an important difference might relate to the extent of Stat3 activation in myeloid cells following their migration to the site of stimuli. While down-regulation of Stat3 in myeloid cells at the inflammatory site is an early event during productive inflammatory responses, a sustained Stat3 activation in myeloid cells such as that observed during tumor growth may provide an explanation for the state of immune unresponsiveness associated with malignancies.