ALBERT

Description: Altered DNA methylation is a well-known feature of acute myeloid leukemia (AML) genomes, but the mechanisms underlying these changes and their relevance for AML pathogenesis are unclear. We previously showed that DNMT3A is the predominant de novo methyltransferase expressed in AML cells, and that the DNMT3AR882H mutation in AML creates a dominant negative protein that reduces in vitro DNA methylation activity by ~80%. Since DNMT3A provides themajority of the methylation activity in AML cells, we hypothesized that AML samples with and without DNMT3AR882H could reveal novel insights about the role of this enzyme in AML initiation and progression. We performed whole-genome bisulfite sequencing (WGBS) of 38 primary human AML samples and 17 normal human hematopoietic cell samples, as well as a remission sample from a patient with a persistent DNMT3AR882H mutation, and blood samples from a non-leukemic patient with a constitutional DNMT3AR882H mutation. We first identified 3,848 differentially methylated regions ('DMRs') between DNMT3AR882H and DNMT3AWT AMLs, virtually all of which were hypomethylated in the DNMT3AR882H AMLs. Further, 28% (1,087/3,848) of these DMRs were also hypomethylated when compared to CD34 cells, implying that these regions are truly hypomethylated in the AML cells with the R882H mutation. In contrast, 72% (2,759/3,848) of the DMRs were unmethylated in bothDNMT3AR882H AMLs and CD34 cells, but were hypermethylated in the DNMT3AWT AML samples. These loci were associated with CpG dense regions, suggesting that they represent abnormal CpG island hypermethylation that occurs only in AML samples with wild-type DNMT3A. Analysis of 21 additional primary AML samples with wild-type DNMT3A identified 4,912 hypermethylated regions compared to CD34 cells, of which 4,544 (92%) were significantly less methylated in DNMT3AR882H AMLs, implying that functional DNMT3A mediates abnormal CpG island hypermethylation in AML. WGBS analysis of two non-leukemic hematopoietic samples with DNMT3AR882H mutations was also performed to understand the direct effects of DNMT3AR882H in non-transformed myeloid cells. These samples included peripheral blood (PB) neutrophils and monocytes from a newly identified 9-year old patient with an overgrowth syndrome and developmental delay (Tatton-Brown et. al., Nature Genetics 2014), who was found to have a heterozygous DNMT3AR882H mutation in all skin and peripheral blood cells. His CBC was normal, and he had no evidence of clonal hematopoiesis by exome sequencing. We identified 2,051 DMRs in his PB myeloid cells, all of which were hypomethylated compared to control PB myeloid cells from his healthy 13-year old brother (and also normal CD34 cells), demonstrating that DNMT3AR882H directly causes focal methylation loss. We also performed WGBS on cells expanded from single stem/progenitor cells from an AML patient with a persistent DNMT3AR882H mutation during remission. Expanded cells with DNMT3AR882H were hypomethylated relative to wild-type DNMT3A cells expanded from the same sample. The majority of the hypomethylated regions were also present in the patient's AML cells, implying that DNMT3AR882H-associated hypomethylation in pre-leukemic cells is maintained during AML progression. These findings demonstrate that DNMT3AR882H-associated hypomethylation precedes leukemia development, and may therefore represent an important initiating phenotype for AML. Our data also suggest that the abnormal hypermethylation of CpG islands in AML cells is DNMT3A-dependent, and must occur during disease progression. This hypermethylation is absent in AMLs with DNMT3AR882H, revealing that it is not required for leukemia progression. We therefore propose a model where DNMT3A-dependent DNA methylation in AML cells acts as a 'brake' that prevents abnormal self-renewal; the abnormal CpG island hypermethylation in DNMT3AWT AMLs may be an adaptive response that is ultimately overcome during leukemia progression. The absence of this 'braking' activity in AMLs with DNMT3AR882H may contribute directly to leukemia initiation. The restoration of DNMT3A activity in AML cells with the DNMT3AR882H mutation is therefore a therapeutic goal. Disclosures Spencer: Cofactor Genomics: Consultancy.

Description: Key Points C/EBPα is needed for transition from stem/progenitor cells to common dendritic cell progenitors. C/EBPα is dispensable in later stages of dendritic cell maturation.

Description: Key Points Anti–third-party Tcm kill malignant B cells in a T-cell receptor–independent mechanism while sparing naive B cells.

Description: Key Points Low doses of donor iNKT infusion prevent and reverse murine cGVHD. iNKT efficacy in treating established cGVHD is dependent on donor Treg expansion.

Description: Abstract 2436 Poster Board II-413 Sensitive in vivo imaging methods have advanced the fields of stem cell transplantation, graft-versus–host disease (GVHD) and graft-versus-tumor responses (GVT). Near-infrared (NIF) fluorescent proteins (FP) appear advantageous for deeper tissue penetration due to minimized absorbance by hemoglobin, water and lipids. Therefore we tested whether a recently published NIF FP (FP635, “Katushka”) could serve as a single reporter for whole body and single cell imaging. To compare signal intensities of eGFP and FP635 we generated fluorescent MOSEC cell lines (mouse ovarian cancer), titrated them in vitro and subcutaneously (s.c.) in vivo in Balb/c nu/nu mice. MOSEC FP635 showed twice the signal intensities compared to MOSEC eGFP in vitro by spectral fluorescence imaging (FLI). In vivo the eGFP signal was attenuated 〉60% in contrast to only 20% for FP635 from subcutaneous sites. However, FP635 signals from deep tissue layers were quenched. To address whether reduced signal attenuation of FP635 may allow sensitive visualization of immune processes by FLI and multiphoton-laser-scanning-microscopy (MPM) we generated transgenic mice in the genetic C57Bl/6 (B6) background, expressing FP635 under the ubiquitin promoter. Transgenic founders were selected upon signal intensities of leukocyte populations measured by flow cytometry in the PerCP channel. Combination of FP635 with colors other than red were possible for multiparameter flow cytometry. Next, eGFP, DsRed and FP635 splenocytes from transgenic donors were titrated as described above. In vitro signal intensities of FP635 splenocytes were 〉5 times lower compared to the other two FPs. FP635 signal absorption in vivo was low (30%) which is consistent with MOSEC titration results. In vivo DsRed detection was most sensitive and signals were similarly attenuated as FP635 in contrast to eGFP (60%). Subsequently, we aimed to visualize FP635 in a model of GVHD, where alloreactive T cells undergo massive expansion. Balb/c nu/nu mice were lethally irradiated and transplanted with 5×106 B6.WT bone marrow cells plus either 2×107 B6.DsRed+Luciferase+ or 2×107 B6.FP635 splenocytes. Sensitivity for DsRed cell detection was superior over FP635 cells. FP635 signal was only weakly detectable in lymph nodes (LN) by ex vivo FLI, where DsRed signals were detectable at earlier timepoints and LNs were even visualized by in vivo FLI. DsRed+ Luciferase+ double transgenic splenocytes allowed direct comparison of bioluminescence imaging (BLI) to FLI. Timely in vivo visualization of immune cells in deep tissues was feasible only by BLI. After whole body imaging the suitability of FP635 for MPM was checked by co-injecting eGFP B cells and either DsRed or FP635 T cells intravenously into RAG-/- mice. As FP635 is a NIF FP we expected to achieve deeper tissue penetration in hemoglobin rich organs, such as the spleen, in single cell microscopy. After 6 weeks of adoptive cell transfer we imaged spleens by MPM. Tissue penetration depths of DsRed or FP635 T cells were compared to eGFP B cells. No advantage in penetration depth of FP635 over DsRed was measured. Photobleaching is an important factor for microscopy, especially if cells are to be tracked over long time. FP635 transfected 293T cells bleached faster (t1/2=108 sec) than 293T cells transfected with eGFP (t1/2〉900 sec) or DsRed (t1/2=411 sec). These experiments indicate that very high expression levels of FP635 need to be achieved for imaging. The signal attenuation of FP635 is low which may increase the sensitivity but in our hands DsRed showed comparable characteristics. Yet, the fast photobleaching of FP635 compared to the broadly established FPs DsRed and eGFP may be disadvantageous for long term microscopic tracking of cells. Our data indicate that BLI is by far superior over FLI in sensitivity and tissue penetration for whole body imaging of immune cells. However, FLI of red or near-infrared clonally selectable tumor cell lines may provide a welcome color addition to study immune cell-tumor interactions in combined models of BLI and FLI. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3286 Background. Lymphocytes infiltrate most human tissues, comprising a critical component of the adaptive immune response. Infiltrating lymphocyte populations have previously been associated with outcomes for a variety of clinical issues, including cancer, alloimmunity, and autoimmunity. Quantitative detection of lymphocyte infiltration has potential as a clinical biomarker of disease activity. Furthermore, locally infiltrating disease-associated clones can be monitored longitudinally in peripheral blood, enabling less invasive follow-up. Deep sequencing of lymphocyte variable regions is an ideal candidate for clinical characterization of tissue-infiltrating lymphocytes. Tissue biopsies produce low genomic DNA yields generally and also contain only a small percentage of cells that have undergone variable region recombination. In this study, we first performed two analytical validity experiments to determine the sensitivity of T cell receptor (TCR) repertoire sequencing, and then made measurements of infiltrating lymphocytes in non-small cell lung carcinomas (NSCLCs), gastrointestinal (GI) biopsies, and cerebrospinal fluid (CSF). Methods. The first analytical validity experiment was designed to determine whether we could accurately characterize rare T cells in a background of cells with unrearranged TCRs. We therefore spiked genomic DNA from three unique T cell clones into background unrearranged human genomic DNA derived from immortal lung cancer carcinoma cells. The dilution series was designed such that the high end of the range surveyed the equivalent of 40,000 T cells, and the low end of the range surveyed the equivalent of 1,200 T cells. We then performed commercial TCR repertoire sequencing (GigaMune Rep-Seq™). The second analytical validity experiment was designed to determine reproducibility and accuracy across a dilution series. Here, we spiked two TCR clones into blood genomic DNA from a normal human individual. These DNA mixes were created at clonal cell to normal blood genomic DNA ratios of 1:1000, 1:100, 4:100, and 1:10. Then, we subjected these DNA mixtures to commercial TCR repertoire sequencing in triplicate. Finally, as proof-of-principle, we used deep TCR sequencing to characterize tissue-infiltrating lymphocytes and peripheral blood from patients with NSCLC, multiple sclerosis (MS), and graft-versus-host disease (GVHD). Results. In the two analytical validity experiments, we detected all expected clones, at quantities as low as 50 copies per reaction. In the first experiment, as a metric of detection noise, we calculated a normalized count of primer-dimer reads (“background”) across the dilution series. The background for the lowest spike-in mixture was 96% lower than a no-template negative control. In the second analytical validity experiment, the average variation coefficient across replicate measurements was 10%; regression analysis of observed versus expected TCR counts had an r2of 0.93. In our proof-of-principle experiments, we were able to detect oligoclonal TCR in CSF biopsies from two MS subjects. Each CSF contained a single clone at 〉39% of the total TCR repertoire. Matched peripheral blood from one of these subjects contained the CSF-infiltrating clone at 8% of the total TCR repertoire. We were also able to reproducibly detect TCR clones in GI biopsies from two GVHD subjects. For each of the GI biopsies, top GI-infiltrating clones represented 〉10% of the total peripheral blood TCR repertoire +30 days after GVHD diagnosis. Finally, we were able to detect tumor-infiltrating TCR clones in NSCLCs and matched peripheral blood from two subjects. In one of the NSCLC subjects, three of the top five tumor-infiltrating clones were among the four most common TCR clones in matched peripheral blood. Conclusions. TCR repertoire sequencing can be used to quantitatively, sensitively, and accurately detect tissue-infiltrating T cell clones. We have shown that tissue-infiltrating T cell clones proliferate significantly in the peripheral blood, across disease conditions as divergent as MS, GVHD, and NSCLC. In the future, we will conduct analytical and clinical validity studies that will eventually be used as evidence to justify use of deep TCR sequencing as a clinical diagnostic in autoimmunity, alloimmunity, and oncology. Disclosures: Johnson: GigaGen Inc.: Employment, Equity Ownership. Löhr:GigaGen Inc.: Employment, Equity Ownership. Howell:GigaGen Inc.: Employment, Equity Ownership. Hsu:GigaGen Inc.: Employment, Equity Ownership. Meyer:GigaGen Inc.: Consultancy, Equity Ownership.

Description: Castleman disease is a rare lymphoproliferative disorder known to represent at least four distinct clinico-pathologic sub-types. Large advancements in our clinical and histopathologic description of these diverse diseases have been made, resulting in subtyping based on degree of nodal involvement (unicentric versus multicentric) and according to viral infection (human herpes virus 8 - HHV8 and Human Immunodeficiency Virus - HIV). However, we continue to lack a foundational understanding of the biologic mechanisms driving at least a subset of patients with these diseases. In recent years, significant molecular and genetic abnormalities that may be associated with this disease have been described; yet we are still unclear as to a unified vision of what loci and genes are involved in at least a subset of cases of Castleman disease. In our study we performed a meta-analysis of all cases (unicentric and multicentric) of Castleman disease described in literature to date, and correlate cytogenetic, molecular and genetic abnormalities with disease subtypes, phenotypes and clinical outcomes. We further performed targeted DNA deep sequencing and RNA-sequencing on a cohort of Castleman Disease cases. Our results demonstrate that genetic abnormalities in the MAPK-pathways, PI3K-pathway and Interleukin signaling are frequent in unicentric Castleman disease, whereas abnormalities in DNA methylation are seen more commonly in idiopathic multicentric Castleman disease, along with abnormalities in MAPK-pathways. We conclude that UCD and iMCD may share similar pathways of MAPK pathogenesis but can also be distinguished by cytokine and other DNA modification pathways that differ. Disclosures Ohgami: Agilent technologies: Other: received support/funding.

Description: Abstract 1900 GVHD is a major complication of bone marrow transplantation (BMT) and results from donor T cells becoming activated and reacting to host antigens. Recently, lymphocyte activation gene-3 (LAG-3) has emerged as an important molecule, negatively regulating T cell activation and has been proposed to play an important role in CD4+CD25+Foxp3+ regulatory T cell (Treg) function. We investigated the functional in vivo role of LAG-3 in Treg and conventional T cells in murine GVHD with the hypothesis that LAG-3 engagement diminishes alloreactive T cell responses after BMT. Using murine models of acute GVHD in which allogeneic bone marrow cells are transplanted into lethally irradiated hosts, we and others have shown previously that donor Treg are able to suppress GVHD induced by donor allogeneic conventional T cells (Tcon). The role of LAG-3 in Treg function was evaluated both in vitro and in vivo by directly comparing Treg isolated from LAG-3−/− donor mice to Treg isolated from wild type donors (WT Treg). In vitro, in a mixed lymphocyte reaction assay, LAG-3−/− Treg efficiently suppressed the proliferation of alloreactive T cells in a manner similar to WT Treg. In vivo, a bioluminescent imaging assay (BLI) was utilized that allows for quantitative assessment of Tcon proliferation in addition to traditional metrics of GVHD severity including weight loss, survival and GVHD score. Both LAG-3−/− Treg and WT Treg were equally potent at suppressing Tcon proliferation as illustrated by BLI of luc+ T cells and demonstrated a significant increase in median survival time (MST) as compared to mice receiving Tcon only (35 days for Tcon vs. 58 and 68 days for WT and LAG-3−/− Treg, respectively, P=0.03), but there was no significant difference in MST between the groups receiving WT and LAG-3−/− Treg. Interestingly, when LAG-3−/− Tcon were used to induce GVHD in the absence of Treg, GVHD lethality was accelerated. Thus, all mice receiving LAG-3−/− Tcon showed decreased survival and significantly lower body weights than mice receiving WT Tcon (P=0.017). GVHD scores of LAG-3−/− Tcon recipients were also significantly higher than WT Tcon recipients at Day 20 post BMT (6.0 vs. 2.2, P=

Description: Invariant natural killer T (iNKT) cells are an interesting subpopulation of T cells that can potently inhibit graft-versus-host-disease (GVHD) through the production of Interleukin 4 (IL-4), while also carrying anti-tumor potential (Leveson-Gower et al. Blood. 2011;117:3220-9; Schneidawind et al. Blood. 2014;124:3320-8). Murine iNKT cells differentiate during thymic development into three distinct sublineages, named according to the classification of conventional T cells: Th1-like iNKT (iNKT1) cells, Th2-like iNKT (iNKT2) cells, and Th-17 like iNKT (iNKT17) cells (Brennan et al. Nat Rev Immunol. 2013; 13:101-1). In this study we investigated the immune regulatory and anti-tumor potential of iNKT1, iNKT2 and iNKT17 cell subsets. Thymic iNKT1 cells, iNKT2 cells and iNKT17 cells were isolated from 8-10 week-old FVB/NJ mice by flow cytometry based on PBS-57-CD1d-Tetramer and a combination of cell surface molecules (iNKT1: ICOS- PD1- CD27+ CD24-; iNKT2: ICOS+ PD1+ CD27+ CD4+ CD24-; iNKT17: ICOS+ PD1+ CD4- CD27- CD24-) and purity was confirmed by intra-nuclear staining for the transcription factors PLZF and RORγT. RNA sequencing analysis determined that iNKT1 cells were the main subset expressing proinflammatory and cytotoxic genes, such as Interferon gamma (IFN-γ), Fas Ligand (FasL), Perforin, and Granzyme B (Gzmb), whereas IL-4 was expressed by iNKT2 cells and, at a lesser extent, by iNKT17 cells. To assess the immuno-regulatory potential of the three iNKT sublineages, we employed a murine major histocompatibility complex (MHC)-mismatched bone marrow transplantation model. BALB/c (H-2Kd) recipients were lethally irradiated with 8.8 Gy; on the same day, 4 x 106 TCD-BM cells and 1 x 106 conventional CD4 and CD8 T cells (Tcon) from FVB/NJ (H-2kq) mice were injected intravenously. Additionally, 5 x 104 purified iNKT1, iNKT2 or iNKT17 cell subsets from FVB donors were injected. A significant survival benefit was observed when iNKT2 (p=0.017) and iNKT17 (p=0.033) cells were adoptively transferred compared to mice that only received TCD-BM and Tcon, whereas there was no survival benefit in the group that received iNKT1 cells. In addition, body weight was improved in mice that received iNKT2 (day +41: p=0.009, day +49: p=0.005 and day +59: p= 0.005) or iNKT17 (day +59: p= 0.006) compared to mice that received iNKT1 cells. Clinical GVHD scores were also improved in mice that received iNKT2 (day +41: p= 0.012, day +51: p= 0.005) or iNKT17 (day +28: p=0.05, day +51: p=0.007) compared to mice that received iNKT1 cells. Interestingly, we found that even 1 x104 iNKT2 (p= 0.008) and iNKT17 (p= 0.04) significantly suppressed GVHD. As iNKT1, iNKT2 and iNKT17 have a very different gene expression profile, we tested the ability of the sublineages to kill a B-cell lymphoma cell line transduced to express high levels of CD1d (A20-CD1d) in vitro and found that iNKT1 cells killed A20-CD1d cells significantly better than iNKT2 (p=0.006) or iNKT17 (p=0.0001) cells. These findings are in line with the sequencing data mentioned above, showing that iNKT1 cells express a more inflammatory phenotype. In summary, we demonstrate here that only iNKT2 and iNKT17 cells protect from GVHD, whereas iNKT1 cells have cytotoxic function. To our knowledge, this is the first study to show functional differences between the iNKT sublineages, suggesting that iNKT1, iNKT2 and iNKT17 cells have diverse functions. Therefore, these data provide new biological insights, which will be useful for developing iNKT cell-based cell therapy. Disclosures Chang: Spring Discovery: Membership on an entity's Board of Directors or advisory committees; Epinomics and Accent Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Description: Recent work has established that heterozygous germline GATA2 mutations predispose carriers to familial myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML), “MonoMAC” syndrome, and DCML deficiency. Here, we describe a previously unreported MDS family carrying a missense GATA2 mutation (p.Thr354Met), one patient with MDS/AML carrying a frameshift GATA2 mutation (p.Leu332Thrfs*53), another with MDS harboring a GATA2 splice site mutation, and 3 patients exhibiting MDS or MDS/AML who have large deletions encompassing the GATA2 locus. Intriguingly, 2 MDS/AML or “MonoMAC” syndrome patients with GATA2 deletions and one with a frameshift mutation also have primary lymphedema. Primary lymphedema occurs as a result of aberrations in the development and/or function of lymphatic vessels, spurring us to investigate whether GATA2 plays a role in the lymphatic vasculature. We demonstrate here that GATA2 protein is present at high levels in lymphatic vessel valves and that GATA2 controls the expression of genes important for programming lymphatic valve development. Our data expand the phenotypes associated with germline GATA2 mutations to include predisposition to primary lymphedema and suggest that complete haploinsufficiency or loss of function of GATA2, rather than missense mutations, is the key predisposing factor for lymphedema onset. Moreover, we reveal a crucial role for GATA2 in lymphatic vascular development.