ALBERT

Description: Compelling evidences indicate a key role for regulatory T cells (Tregs) on the host response to cancer and recent studies indicated that the generation of effective WT1-specific cytotoxic T cells can be largely affected by the presence of Tregs. This is the first study to describe human Tregs generated specifically against the WT1 antigen which is overexpressed in several human leukemias and provide the mechanism by which these anti-WT1 Tregs inhibit the immune response in leukemia patients. We have generated T cell lines and clones that specifically recognized a WT1-84 peptide in an HLA DRB1*0402/TCR-Vb8-restricted manner. Importantly, they recognized HLADRB1* 04-matched fresh leukemic cells expressing the WT1 antigen. These clones exerted a Th2 cytokine profile, had a CD4+CD25+Foxp3+GITR+CD127− Tregs phenotype, and significantly inhibited the proliferative activity of allogeneic T cells independently of cell-contact. Priming of allo-reactive T cells in the presence of Tregs strongly inhibited the expansion of NK; NK-T and CD8+ T cells, had an inhibitory effect on NK/NK-T cytotoxic activity but not on CD8+ T cells. Furthermore, priming of T cells with the WT1- 126 HLA-A0201-restricted peptide in the presence of Tregs strongly inhibited the induction of anti-WT1-126 CD8+ CTL responses as evidenced by both very low cytotoxic activity and IFN-g production. Moreover, these Tregs clones specifically produced Granzyme-B and selectively induced apoptosis in WT1-84 pulsed-autologous APCs but not in apoptoticresistant DR4-matched leukemic cells. Importantly, we have also detected anti-WT1-84 IL-5+/Granzyme-B+/Foxp3+ CD4+ Tregs in 5 out of 8 HLA-DR4+ AML patients. These findings suggest a critical role for anti-WT1 Tregs in the inhibition of T cell responses against leukemia. This study may have important implications for the clinical manipulation of Tregs such as immuno-targeting of TCR-Vb-8 with mAbs to eliminate anti-WT1 Tregs in leukemia patients in order to enhance GVL before vaccination with the WT1 antigen. On the other hand, leukemia patients with GVHD should be clinically-tried for vaccination with the current WT1-84 peptide or adoptively-treated with ex-vivo anti-WT1 Treg cells to specifically enhance their frequency, which is known to be very low in these patients.

Description: Background Myelodysplastic syndrome (MDS) is a pre-leukemia disease affecting the erythroid, myeloid and megakaryocytic bone marrow production. MDS patients are classified according to the WHO classification of myeloid neoplasms. During the past 15 years management of MDS patients has been stratified according to the International Prognostic Scoring System (IPSS) risk score. Recently a revised version of IPSS has been introduced (IPSS-R). One quarter of LR-MDS in this new IPSS-R were reclassified as having a higher risk and a substantial subset of high risk-MDS (HR-MDS) were reclassified as lower risk. In LR-MDS a differentiation block is observed in the erythroid lineage. The diagnosis and follow up of cytopenias in particular anemia must be the main objective (1). The soluble transferrin receptor (sTfR) directly reflects the erythropoietic activity in individuals without iron defiency and may appreciate ineffective dysplastic erythropoiesis in LR-MDS. In LR-MDS there is also an inverse relationship between EPO level and the degree of anemia but a wide range of EPO levels is found in patients with similar Hb concentrations. Thus the highest EPO levels are found in patients with erythroid hypoplasia in bone marrow. Aims The combination of several biomarkers: Hb, ferritin, EPO and sTfR may be useful in LR-MDS for diagnosis and follow up. Methods A total of 192 patients with LR-MDS were investigated. Median age of the 192 patients was 71 years (21-92) with 56% males, median survival: 54 months, median follow up: 102 months. The stratification according to the WHO criteria and IPSS risk score was realized. Bone marrows were studied and cytogenetic assessment was realized in the same time. Serum concentrations of ferritin, EPO and sTfR has been analyzed by immuno-assays. Hb level was determined on Beckman Coulter apparatus. The follow up of Hb, ferritin, EPO and sTfR was realized every 2 months in patients with supportive care only until the first specific treatment. A multivariate logistic regression analysis to ascertain the correlations between disease progression and studied biological parameters was realized. Results The logistic regression analysis of our results is significant to define a biological evolutive profile of LR-MDS patients with these biomarkers. The combination of these routine parameters may represent a functional erythropoietic follow up in LR-MDS patients (table 1). This biological tool is an easy method to observe the red cell lineage of LR-MDS patients. This combination informs about the progressive ineffective and dysplatic erythropoiesis in LR-MDS patients. The measurement of ferritin which is a correlated parameter in LR-MDS shows the level of iron overload. A normal or high level without inflammation condition excludes an iron deficiency. The EPO level can give a predictive information about the future efficacy of ESA (endogenous EPO 〈 500 U/l). Conclusion With our results and a correlative logistic regression analysis, we can propose a biological scoring system to appreciate the evolutive anemia of LR-MDS progression in patients. In LR-MDS the management of patients may be based on personalized medicine according a risk assessment with IPSS-R, cytogenetics, mutations and HLA typing (2). But an additional biological and functional predictive scoring system informs about the important independent role of dysplasias particularly anemia in LR-MDS patients before to choose a suitable therapy: transfusions, iron chelation, ESA, TGF-ï¢ pathway inhibitors, G-CSF, immun suppressive treatment, lenalidomide, azacytidine, allogeneic HSCT Table 1. Hb ± EPO ±  sTfRDysplastic erythropoiesis without anemia Hb ±  EPO  sTfRStabilized dysplastic erythropoiesis Hb  EPO  sTfRUnstabilized dysplactic erythropoiesis Hb  EPO  sTfRIneffective dysplastic erythropoiesis EPO 〈 500 U/l : ESA may be efficient〉 500 U/l : ESA will be inefficientFerritin level : iron overload References Giagounidis A Management of low-risk myelodysplastic syndromes Hematology Education, 2015, 9 (1), 219-225 Platzbecker U et al Personalized medicine in myelodysplastic syndromes: wishful thinking or already clinical reality? Hematologica, 2015, 100 (5), 568-571 Disclosures No relevant conflicts of interest to declare.

Description: We present a deep near-infrared (NIR; J , H , and K s bands) photometric catalogue of sources from the Parkes H i Zone of Avoidance (HIZOA) survey, which forms the basis for an investigation of the matter distribution in the Zone of Avoidance. Observations were conducted between 2006 and 2013 using the Infrared Survey Facility (IRSF), a 1.4-m telescope situated at the South African Astronomical Observatory site in Sutherland. The images cover all 1108 HIZOA detections and yield 915 galaxies. An additional 105 bright 2MASS galaxies in the southern ZOA were imaged with the IRSF, resulting in 129 galaxies. The average K s -band seeing and sky background for the survey are 1.38 arcsec and 20.1 mag, respectively. The detection rate as a function of stellar density and dust extinction is found to depend mainly on the H i mass of the H i detected galaxies, which in principal correlates with the NIR brightness of the spiral galaxies. The measured isophotal magnitudes are of sufficient accuracy (errors ~0.02 mag) to be used in a Tully–Fisher analysis. In the final NIR catalogue, 285 galaxies have both IRSF and 2MASS photometry (180 HIZOA plus 105 bright 2MASX galaxies). The K s -band isophotal magnitudes presented in this paper agree, within the uncertainties, with those reported in the 2MASX catalogue. Another 30 galaxies, from the HIZOA northern extension, are also covered by UKIDSS Galactic Plane Survey (GPS) images, which are one magnitude deeper than our IRSF images. A modified version of our photometry pipeline was used to derive the photometric parameters of these UKIDSS galaxies. Good agreement was found between the respective K s -band isophotal magnitudes. These comparisons confirm the robustness of the isophotal parameters and demonstrate that the IRSF images do not suffer from foreground contamination, after star removal, nor underestimate the isophotal fluxes of ZoA galaxies.

Description: Dust extinction and stellar confusion by the Milky Way reduce the efficiency of detecting galaxies at low Galactic latitudes, creating the so-called Zone of Avoidance (ZoA). This stands as a stumbling block in charting the distribution of galaxies and cosmic flow fields, and therewith our understanding of the local dynamics in the Universe (cosmic microwave background dipole, convergence radius of bulk flows). For instance, ZoA galaxies are generally excluded from the whole-sky Tully–Fisher (TF) surveys (| b | ≤ 5°) even if catalogued. We show here that by fine-tuning the near-infrared (NIR) TF relation, there is no reason not to extend peculiar velocity surveys deeper into the ZoA. Accurate axial ratios ( b / a ) are crucial to both the TF sample selection and the resulting TF distances. We simulate the effect of dust extinction on the geometrical properties of galaxies. As expected, galaxies appear rounder with increasing obscuration level, even affecting existing TF samples. We derive correction models and demonstrate that we can reliably reproduce the intrinsic axial ratio from the observed value up to extinction level of about A J ~= 3 mag ( A V  ~ 11 mag); we also recover a fair fraction of galaxies that otherwise would fall out of an uncorrected inclination limited galaxy sample. We present a re-calibration of the 2MTF (The Two Micron All Sky Survey Tully–Fisher Survey) relation in the NIR J , H , and K s bands for isophotal rather than total magnitudes, using their same calibration sample. Both TF relations exhibit similar scatter at high Galactic latitudes. However, the isophotal TF relation results in a significant improvement in the scatter for galaxies in the ZoA, and low surface brightness galaxies in general, because isophotal apertures are more robust in the face of significant stellar confusion.

Description: High-accuracy H i profiles and linewidths are presented for inclined (( b / a ) o 〈 0.5) spiral galaxies in the southern Zone of Avoidance (ZOA). These galaxies define a sample for use in the determinations of peculiar velocities using the near-infrared Tully–Fisher (TF) relation. The sample is based on the 394 H i -selected galaxies from the Parkes H i Zone of Avoidance survey (HIZOA). Follow-up narrow-band Parkes H i observations were obtained in 2010 and 2015 for 290 galaxies, while for the further 104 galaxies, sufficiently high signal-to-noise (S/N) spectra were available from the original HIZOA data. All 394 spectra are reduced and parametrized in the same systematic way. Five different types of linewidth measurements were derived, and a Bayesian mixture model was used to derive conversion equations between these five widths. Of the selected and measure galaxies, 342 have adequate signal to noise (S/N ≥ 5) for use in TF distance estimation. The average value of the S/N ratio of the sample is 14.7. We present the H i parameters for these galaxies. The sample will allow a more accurate determination of the flow field in the southern ZOA which bisects dynamically important large-scale structures such as Puppis, the Great Attractor, and the Local Void.

Description: An obstacle with continued clinical development of CAR T cells is the limited understanding of their biology and mechanisms of anti-tumor immunity. We and others have shown that CARs with a CD28 co-stimulatory domain drive high levels of T cell activation that also lead to exhaustion and shortened persistence. The CD28 domain includes 3 intracellular subdomains (YMNM, PRRP, and PYAP) that regulate signaling pathways post TCR-stimulation, but it is unknown how they modulate activation and/or exhaustion of CAR T cells. A detailed understanding of the mechanism of CD28-dependent exhaustion in CAR T cells will allow the design of a CAR less prone to exhaustion and reduce relapse rates. This led us to hypothesize that by incorporating null mutations of CD28 subdomains (Fig 1A) we could optimize CAR T cell signaling and reduce exhaustion. In vitro, we found mutated CAR T cells with only a functional PYAP (mut06) subdomain secrete significantly less IFNγ, IL6, and TNFα after 24hr stimulation compared to non-mutated CD28 CAR T cells, but greater than the 1st generation m19z CAR. Also, cytotoxicity was enhanced compared to non-mutated CARs (Fig 1B). Using a pre-clinical immunocompetent mouse tumor model, we found the mut06 CAR T cell treated mice had a significant survival advantage compared to non-mutated CD28 CAR T cells (Fig 1C). To examine exhaustion, we ex vivo stimulated CAR T cells with target cells expressing CD19 and PDL1 and found mut06 CAR T cells had increased IFNγ (42%), TNFα (62%) and IL2 (73%) secretion compared to exhausted non-mutated CD28 CAR T cells. This suggests that mut06 CAR T cells are more resistant to exhaustion. To find a mechanistic explanation for this observation we examined CAR T cell signaling. After 24hr stimulation with CD19 target cells mut06 CAR T cells had a significant reduction in pAkt compared to m1928z CAR T cells, which is a critical signaling mediator in the NFAT and NR4A1 transcription factor pathways. Additionally, mut06 had decreased p-NFAT compared to m1928z when examined by western blot. To determine how optimized CAR signaling affected T cell exhaustion we looked at 22 genes that are upregulated when NFAT is constitutively active and overlap with genes identified as important for T cell exhaustion. We found that most of the exhaustion related genes were upregulated in m1928z CAR T cells while they were decreased in m19hBBz. The mut06 CAR T cell gene expression pattern was more similar to m19hBBz with exhaustion related genes downregulated compared to m1928z (Fig 1D). To examine differences in the accessibility of exhaustion related genes we performed ATAC-seq and found NFAT (Nfatc1) and NR4A2 (Nr4a2) had lower chromatin accessibility profiles in mut06 compared to m1928z (Fig 1E). We also found that exhaustion related genes Havcr2 (TIM3), Pdcd1 (PD1), and Lag3 (LAG3) all had greatly reduced chromatin accessibility in mut06 CAR T cells compared m1928z. Overall, these genomic studies support our findings that mut06 optimizes CAR T cell signaling by lowering transcription factors that regulate exhaustion. Figure 1 Disclosures Li: ImmuneBro Therapeutics: Other: sole shareholder . Davila:Atara: Research Funding; Celgene: Research Funding; GlaxoSmithKline: Consultancy; Novartis: Research Funding; Anixa: Consultancy; Bellicum: Consultancy; Adaptive: Consultancy; Precision Biosciences: Consultancy.

Description: Epstein-Barr Virus (EBV)+ lymphomas are an important subgroup of aggressive malignant lymphomas which include lymphomas in the post-transplantation setting, Burkitt’s lymphomas (BL), AIDS-related lymphomas (ARL), and some forms of Hodgkin, T-cell, and natural killer (NK) cell lymphomas. EBV is a member of the herpes virus family, characterized by their ability to support two different life cycles: the productive “lytic” cycle leading to the production and release of new virions and the non-productive “latent” cycle. Most lymphoma cells are infected with latent EBV, and only few viral genes are expressed at low levels. Several groups of broad-acting chemical agents are able to reactivate EBV and induce herpes thymidine kinase (TK) expression in vitro and in vivo. NF-kB has been described to play a critical role in regulating the balance between latency and lytic replication of EBV. Therefore, we hypothesized that the proteasome inhibitor Bortezomib can be used to initiate EBV lytic antigen expression in EBV-related malignancies enabling the antiviral drug Ganciclovir to kill EBV+ lymphoma cells. The human cell line HR-1, derived from a Burkitt’s lymphoma and latently infected with EBV, was cultured in the presence of 50nM bortezomib for 24 hrs. The immediate early lytic phase EBV antigens ZEBRA and RTA were induced and expressed as measured by flow cytometry. The EBV-VCA and EBV-R antigens were not expressed in untreated controls but were induced as demonstrated by western blot analysis, indicating the switch to the lytic life-cycle of EBV. These results were successfully repeated using the EBV+ Akata cell line. Induction of viral thymidine kinase (vTK) was shown by QRT-PCR as well. Histone deacetylase inhibitors are a well known group of broad-acting chemical agents able to reactivate EBV. In combination experiments, we found that Bortezomib plus sodium butyrate or SAHA act at least additive in inducing the EBV lytic life cycle in HR-1 or RAJI cells. Bortezomib sensitizes the EBV+ Akata cell line 2A8-1 to growth inhibitory effects of ganciclovir as shown by MTT assays. The cells were treated with two different non-toxic drug concentrations which were chosen low enough not to induce apoptosis (bortezomib: 1nM and 2nM; ganciclovir:15 and 30μM). Bortezomib induces the lytic EBV life cycle in vivo. In murine xenograft models growing the Akata A.15 line subcutaneously bortezomib induces the immediate-early protein ZEBRA as shown by immunohistochemistry and vTK as shown by QRT-PCR. Experiments to induce lytic phase EBV in murine xenograft models using the Akata cell line and to combine EBV induction with the nucleoside analogue ganciclovir are in progress. Murine studies with EBV-transformed lymphoblastoid cell line (LCL) xenograft models, combination bortezomib plus ganciclovir, and molecular imaging with FHBG specific PET probes are in progress. Reactivating EBV with proteasome inhibitors alone or in combinations with low concentrations of histone deacetylase inhibitors may be a less toxic therapeutic strategy for EBV-associated lymphomas.

Description: Controversy exists as to whether Kaposi's sarcoma–associated herpesvirus (KSHV) is more widespread than originally reported. Recently, Monini et al reported that KSHV is ubiquitous in urogenital and prostate tissues and sperm of healthy Italian adults using nested polymerase chain reaction (PCR). We have examined for the presence of KSHV in 10 normal prostates from Italian men and 10 from men from the United States, as well as 32 prostatic, 30 vulvar, 24 ovarian, 20 cervical, and 30 testicular cancer specimens from patients from the United States. None of the patients had a history of human immunodeficiency virus infection. The samples were tested by nested PCR. The sensitivity of this assay was determined by a dilution study performed by diluting KSHV DNA from the KS-1 cells (a primary effusion lymphoma cell line which is estimated to have 16 copies of KSHV per cell) in DNA from a K562 myeloid cell line. The nested PCR that we used can detect 2.4 copies of KSHV sequences on a background of K562 DNA. All the samples were negative for KSHV sequences. Therefore, we cannot confirm the finding that KSHV sequences are ubiquitous in urogenital and prostate tissues. Furthermore, because our samples were from both the United States and Italy, the discrepancy between results is unlikely to be explained by either ethnic or environmental factors. False-positive results easily occur using nested primer PCR because of contamination. Our data argue that KSHV is not widely disseminated in urogenital tissues from nonimmunosuppressed individuals.