Double-positive Research Articles

Characterizing unconventional T cells in the tumor microenvironment of metastatic osteosarcoma

Abstract Osteosarcoma is the most common bone cancer and typically affects patients in the second decade of life. Current treatment methods have not proven effective for treating reoccurring or metastatic osteosarcoma (mOS) given the 5-year survival rate of 15–30%. Previous work has shown anti-PD-L1 combined with anti-CTLA-4 improves survival of mOS in mice from 0% in no treatment mice to 60% in treated mice. To further improve survival, we analyzed tumor infiltrating lymphocytes (TILs) in mice over time and discovered CD4hiCD8hiPD-1hi T cells consistently appeared in the tumor microenvironment (TME) after approximately two months. To determine whether these CD4hiCD8hi, or double positive (DP) cells, were candidates for immune therapy, TILs from mice with long-term cancer were stained for CD3 to confirm T cell lineage, CD44 to verify activation, and Qa-2, which is first expressed during the stage IV single positive phase of T cell maturation and is therefore a marker to distinguish mature from immature T cells. The DP TILs were found to be activated, mature T cells that are distinct from DP immature thymocytes. The DP TILs were further compared to splenocytes and were determined to be concentrated in the TME rather than resulting from a systemic change in the immune system. Finally, the presence of DP T cells in the lungs was found to be unique to long-term lung metastases when lung tissues from age-matched healthy mice were obtained and processed alongside experimental lung metastases. Overall, mature and activated DP T cells concentrate in the TME of long-term lung metastases of mOS.

Molecular Signatures of IL-10/IFN-γ Co-Producing CD4 T Cells During Acute Dengue Virus Infection

Abstract Dengue virus (DENV) is an emerging pathogen and a serious public health issue. It is estimated that ~390 million people are infected yearly. DENV infection is associated with a range of clinical manifestations, from asymptomatic to more severe presentations including dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). There is currently no specific therapy available for the treatment of dengue diseases other than supportive care. Thus, a better understanding of the host’s immune response against DENV, especially during the acute phase of infection, is of great public health interests. Although previous studies suggest that increased IL-10 level in the serum and plasma is associated with severe dengue disease, whether dengue-specific CD4 T cells produce IL-10 remains unknown. Here we show evidence of DENV-specific IL-10-producing CD4 T cells during acute DENV infection. Surprisingly, an IL-10+IFN-γ+ double positive (DP) CD4 T cell population is prominent in acute hospitalized DENV cases but disappear at the convalescent stage. These cells are not conventional regulatory T (Treg) cells as they lack Foxp3 expression. In contrast, RNA-sequencing and high-dimensional CyTOF analyses reveal that these cells partially exhibit previously identified signatures associated with cytotoxic CD4 T cells and type 1 regulatory T (Tr1) cells. Notably, we identified several novel signatures of DP cells including IL-19, IL-21, and IL-22, suggesting that these cells may have complex cytokine activities and functions. Overall, this study reveals unique phenotypes of DENV-specific IL-10/IFN-γ co-producing CD4 cells and provides insights into antigen-specific T cell responses during acute DENV infection.

Characterization of adiponectin‐expressing cells in thymus

ObjectiveAdiponectin, a protein considered as the most abundant adipokine, was found to be expressed in the thymus of wild type mice. The present study characterized the identity and function of adiponectin‐expressing cells in thymus.MethodsThymus was collected from mice with (WT) or without (AKO) adiponectin for isolation and enrichment of thymic nurse cell complexes (TNCs). Different populations of lymphocytes were analyzed by flow cytometry and confocal microscopy. Protein expressions were evaluated by immunofluorescence staining and/or Western blotting.ResultsAdiponectin‐expressing cells were present in TNCs, the specific epithelial cell complexes which engulf thymocytes and provide microenvironment for T lymphocyte development. Compared with age‐matched WT mice, the number and size of TNCs in thymus of AKO mice were significantly reduced. The percentage composition of CD3+ double positive (DP) lymphocytes was significantly decreased, whereas the total amount of double negative (DN) thymocytes significantly increased in TNCs of AKO mice. Adiponectin‐expressing cells were derived from a subpopulation of early T progenitors, which facilitated the formation and function of TNCs. Treatment with adiponectin in vivo or ex vivo could promote the formation and functions of TNCs.ConclusionsAdiponectin‐expressing cells play important roles in the positive and negative selection process of TNCs, thus regulating T‐cell maturation and development in thymus. The results have important implications on the anti‐inflammatory, anti‐diabetic and anti‐tumorigenic properties of adiponectin.Support or Funding InformationThis work was supported in part by grants from Seeding Funds for basic research of the University of Hong Kong, Research Council Grants (17153016) and Hong Kong Health and Medical Research Fund (04151796), Collaborative Research Fund of Hong Kong (C7055‐14G) and the National Basic Research Program of China (973 Program 2015CB553603).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A critical regulator of Bcl2 revealed by systematic transcript discovery of lncRNAs associated with T-cell differentiation

Normal T-cell differentiation requires a complex regulatory network which supports a series of maturation steps, including lineage commitment, T-cell receptor (TCR) gene rearrangement, and thymic positive and negative selection. However, the underlying molecular mechanisms are difficult to assess due to limited T-cell models. Here we explore the use of the pro-T-cell line P5424 to study early T-cell differentiation. Stimulation of P5424 cells by the calcium ionophore ionomycin together with PMA resulted in gene regulation of T-cell differentiation and activation markers, partially mimicking the CD4-CD8- double negative (DN) to double positive (DP) transition and some aspects of subsequent T-cell maturation and activation. Global analysis of gene expression, along with kinetic experiments, revealed a significant association between the dynamic expression of coding genes and neighbor lncRNAs including many newly-discovered transcripts, thus suggesting potential co-regulation. CRISPR/Cas9-mediated genetic deletion of Robnr, an inducible lncRNA located downstream of the anti-apoptotic gene Bcl2, demonstrated a critical role of the Robnr locus in the induction of Bcl2. Thus, the pro-T-cell line P5424 is a powerful model system to characterize regulatory networks involved in early T-cell differentiation and maturation.

A37 DEFECTS IN INTESTINAL MUCUS DISRUPTS THE REPERTOIRE OF DEVELOPING T CELLS IN THE THYMUS

Severe, chronic inflammation of the intestinal tract is a defining hallmark of inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn’s disease. IBD is postulated to result from a complex combination of environmental and genetic factors that lead to excessive bacterial stimulation of the mucosal immune system. An overlying layer of mucus functions to segregate contents of the intestinal lumen from the intestinal epithelium and the underlying immune system. Importantly, decreased mucus production and impaired mucus function have been associated with UC pathogenesis. MUC2 is the primary intestinal mucin and acts as a structural scaffold for mucus formation - forming a physical barrier to shield the intestinal wall. However, recent work suggests that mucus may also function to maintain immunological tolerance towards luminal antigens by promoting the regulatory activity of intestinal antigen-presenting cells. Currently, it is unclear what role mucus plays in influencing the T cell repertoire and modulating T cell immunity both at local and distal sites. We hypothesize that mucus is essential for maintaining T cell tolerance towards intestinal luminal antigens and preventing disease caused by constitutive T cell activation. To investigate whether peripheral T cells become highly activated in young, mildly colitic Muc2-/- mice following oral antigen gavage. To test our hypothesis, young (6–10 week old) wild type and MUC2-deficient (Muc2-/-) mice were gavaged with the model antigen ovalbumin (OVA) and the elicited OVA-specific T cell responses monitored at various time points post-treatment. We found that oral administration of OVA resulted in the antigen rapidly escaping the gut and disseminating throughout the blood and lymphoid tissues of Muc2-/- but not wild type mice. Given the systemic spread of antigen in the Muc2-/- mice, we next sought to investigate whether the selection of developing T cells in the thymus of Muc2-/- mice was impacted by dietary antigens. Remarkably, we found that gavage of Muc2-/- mice led to presentation of OVA by thymic dendritic cells and the deletion of OVA-specific thymocytes whereas T cell development in wild type mice appeared unaffected. Based on our results, we concluded that oral antigens are rapidly disseminated throughout Muc2-/- mice. The heightened concentration of oral antigens induced rapid depletion of oral antigen-specific double positive (DP) thymocytes, and altered the T cell repertoire. Future studies will explore whether IBD patients suffering from impaired gut mucus structure/function also suffer defects in their T cell repertoires. CIHR

Vasculature-associated fat macrophages readily adapt to inflammatory and metabolic challenges.

Tissue-resident macrophages are the most abundant immune cell population in healthy adipose tissue. Adipose tissue macrophages (ATMs) change during metabolic stress and are thought to contribute to metabolic syndrome. Here, we studied ATM subpopulations in steady state and in response to nutritional and infectious challenges. We found that tissue-resident macrophages from healthy epididymal white adipose tissue (eWAT) tightly associate with blood vessels, displaying very high endocytic capacity. We refer to these cells as vasculature-associated ATMs (VAMs). Chronic high-fat diet (HFD) results in the accumulation of a monocyte-derived CD11c+CD64+ double-positive (DP) macrophage eWAT population with a predominant anti-inflammatory/detoxifying gene profile, but reduced endocytic function. In contrast, fasting rapidly and reversibly leads to VAM depletion, while acute inflammatory stress induced by pathogens transiently depletes VAMs and simultaneously boosts DP macrophage accumulation. Our results indicate that ATM populations dynamically adapt to metabolic stress and inflammation, suggesting an important role for these cells in maintaining tissue homeostasis.

Association of anti-nuclesome and anti C1q antibodies with lupus nephritis in an Egyptian cohort of patients with systemic lupus erythematosus

IntroductionAnti-nucleosome and anti-C1q antibodies demonstrated an association with the development of glomerulonephritis in systemic lupus erythematosus (SLE). Some investigators have proposed that monitoring anti-C1q and anti-nucleosome antibodies might be valuable for making predictions about lupus nephritis (LN) and assessment of disease activity as a non-invasive biological marker of renal disease.ObjectivesThe current study was proposed to investigate the presence of anti-C1q and anti-nucleosome antibodies in the sera of Egyptian patients with SLE and their association with LN.MethodsEighty patients with SLE were included. Patients were classified into, a LN group including 40 cases with active LN (based on the results of renal biopsy and renal SLEDAI≥4) and a non renal SLE group including 40 patients (with no clinical or laboratory evidence of renal involvement that were attributed in the past or present to SLE). They were subjected to full medical history taking, clinical examination, routine laboratory investigations, measurement of antinuclear antibody (ANA), anti-ds DNA, anti-C1q & anti-nucleosome antibodies.ResultsAnti-C1q antibody showed a statistically significant association with the presence of vasculitis and nephritis while anti-nucleosome antibody didn’t show a significant association with the presence of any clinical features. Double positivity of anti-nucleosome and anti-C1q antibodies showed a statistically significant association with the presence of vasculitis and photosensitivity, high ECLAM score, elevated ESR, low serum albumin and low C3 levels.ConclusionSerum anti-C1q antibody has a significant association with LN while double positive antibodies have a significant association with vasculitis and low C3 levels in Egyptian patients with SLE.

Impact of NID dielectric–magnetic medium on reflection and transmission characteristics of a Lorentz–Drude slab

Impact of non-integer dimensional (NID) dielectric–magnetic medium on the behavior of power transmitted and reflected from a planar NID Lorentz–Drude slab is analyzed for uniform electromagnetic plane wave excitation. The dispersive slab is terminated by NID dielectric–magnetic medium and dispersion in the slab has been indulge through the Lorentz–Drude model. It is assumed that medium inside the Lorentz–Drude slab and medium backing the slab are NID in direction perpendicular to surface of the slab but their dimensions are described by different NID parameters. According to the characteristics of the model, while the operating frequency increases the NID Lorentz–Drude slab operates as epsilon negative (ENG), double negative (DNG), mu negative (MNG), double positive (DPS) metamaterial respectively. Effect of NID parameter of the terminating medium, dielectric magnetic properties of the terminating medium and impact of their combination on the powers is noted one by one. It is noticed that parameters describing the NID dielectric–magnetic medium significantly change the behavior of powers. These parameters can be effectively used to control the power according to the requirements.

Cutting Edge: HDAC3 Protects Double-Positive Thymocytes from P2X7 Receptor-Induced Cell Death.

Intricate life-versus-death decisions are programmed during T cell development, and the regulatory mechanisms that coordinate their activation and repression are still under investigation. In this study, HDAC3-deficient double-positive (DP) thymocytes exhibit a severe decrease in numbers. The thymic cortex is rich in ATP, which is released by macrophages that clear apoptotic DP thymocytes that fail to undergo positive selection. We demonstrate that HDAC3 is required to repress expression of the purinergic receptor P2X7 to prevent DP cell death. HDAC3-deficient DP thymocytes upregulate the P2X7 receptor, increasing sensitivity to ATP-induced cell death. P2rx7/HDAC3-double knockout mice show a partial rescue in DP cell number. HDAC3 directly binds to the P2rx7 enhancer, which is hyperacetylated in the absence of HDAC3. In addition, RORγt binds to the P2rx7 enhancer and promotes P2X7 receptor expression in the absence of HDAC3. Therefore, HDAC3 is a critical regulator of DP thymocyte survival and is required to suppress P2X7 receptor expression.

Thymic expression of IL-4 and IL-15 after systemic inflammatory or infectious Th1 disease processes induce the acquisition of "innate" characteristics during CD8+ T cell development.

Innate CD8+ T cells express a memory-like phenotype and demonstrate a strong cytotoxic capacity that is critical during the early phase of the host response to certain bacterial and viral infections. These cells arise in the thymus and depend on IL-4 and IL-15 for their development. Even though innate CD8+ T cells exist in the thymus of WT mice in low numbers, they are highly enriched in KO mice that lack certain kinases, leading to an increase in IL-4 production by thymic NKT cells. Our work describes that in C57BL/6 WT mice undergoing a Th1 biased infectious disease, the thymus experiences an enrichment of single positive CD8 (SP8) thymocytes that share all the established phenotypical and functional characteristics of innate CD8+ T cells. Moreover, through in vivo experiments, we demonstrate a significant increase in survival and a lower parasitemia in mice adoptively transferred with SP8 thymocytes from OT I—T. cruzi-infected mice, demonstrating that innate CD8+ thymocytes are able to protect against a lethal T. cruzi infection in an Ag-independent manner. Interestingly, we obtained similar results when using thymocytes from systemic IL-12 + IL-18-treated mice. This data indicates that cytokines triggered during the acute stage of a Th1 infectious process induce thymic production of IL-4 along with IL-15 expression resulting in an adequate niche for development of innate CD8+ T cells as early as the double positive (DP) stage. Our data demonstrate that the thymus can sense systemic inflammatory situations and alter its conventional CD8 developmental pathway when a rapid innate immune response is required to control different types of pathogens.

Effect of Taurine on Thymus Differentiation of Dex-Induced Immunosuppressive Mice.

Taurine (2-aminoethanesulfonic acid) has positive effects on the formation of immune systems. In this study, we evaluated the effects of taurine on the development of T lymphocyte subpopulations in thymus of immunosuppresive mice. The immunosuppressed mice model was established by intraperitoneal injection of dexamethasone (Dex) for 7days. Mice (male, Kunming strain) were randomly divided into three groups, the normal control group (Cont.), the Dex-induced immunosuppressive model group (Dex + PBS), and the taurine intervention group (Dex + TAU). Taurine was administered at a dose of 200mg/kg for 30days or until euthanasia. Total cell numbers in the thymi of mice were evaluated by cell count, and the flow cytometry was used to determine the proportion of different cell subsets. Our results showed that the size and weight of thymi of Dex + PBS group were significantly smaller than those of Cont. group, and taurine administration efficiently increased the thymus index. Taurine also significantly increased the number of CD4- CD8- double negative (DN), CD4+ CD8+ double positive (DP), CD4+ single positive (CD4+) and CD8+ SP (CD8+) cells compared with the Dex + PBS group, but did not affect the CD4+/CD8+ cell ratio in thymus of Dex-induced immunoseppressive mice. Our results suggested that taurine has a positive effect on thymus differentiation in Dex-induced immunosuppressive mice.

Design of Zeroth-order resonance antenna array with a pair of DPS and ENG materials

This paper proposes one-dimensional antenna arrays of the four-element and the eight-element using composite materials. Firstly, the single element is designed to resonate at Zeroth-order using a pair of Double positive (DPS) and Epsilon negative (ENG) materials meta-structured transmission line (MTL). Secondly, three of 1:2 T-Junction power dividers and seven of 1:2 T-Junction power dividers based on micro-strip technology are designed for feeding the four-element and the eight-element array antennas, respectively. Finally, the proposed arrays are optimized using FEM-based simulation to operate at the frequency of 8,5 GHz. The simulated results show that both antenna arrays have Zeroth-order resonance (ZOR) property, in which the four-element array has a bandwidth spreading from 8.39 to 8.61 GHz and a maximum gain of 8.82 dB while the other one of the eight-element array is 8.39 – 8.60 GHz and 12.2 dB, respectively. The proposed array antennas can be used for wireless applications or mobile communications.

Plasma membrane sphingomyelin modulates thymocyte development by inhibiting TCR-induced apoptosis.

Sphingomyelin (SM) in combination with cholesterol forms specialized membrane lipid microdomains in which specific receptors and signaling molecules are localized or recruited to mediate intracellular signaling. SM-microdomain levels in mouse thymus were low in the early CD4+CD8+ double-positive (DP) stage prior to thymic selection and increased >10-fold during late selection. T-cell receptor (TCR) signal strength is a key factor determining whether DP thymocytes undergo positive or negative selection. We examined the role of SM-microdomains in thymocyte development and related TCR signaling, using SM synthase 1 (SMS1)-deficient (SMS1-/-) mice which display low SM expression in all thymocyte populations. SMS1 deficiency caused reduced cell numbers after late DP stages in TCR transgenic models. TCR-dependent apoptosis induced by anti-CD3 treatment was enhanced in SMS1-/- DP thymocytes both in vivo and in vitro. SMS1-/- DP thymocytes, relative to controls, showed increased phosphorylation of TCR-proximal kinase ZAP-70 and increased expression of Bim and Nur77 proteins involved in negative selection following TCR stimulation. Addition of SM to cultured normal DP thymocytes led to greatly increased surface expression of SM-microdomains, with associated reduction of TCR signaling and TCR-induced apoptosis. Our findings indicate that SM-microdomains are increased in late DP stages, function as negative regulators of TCR signaling and modulate the efficiency of TCR-proximal signaling to promote thymic selection events leading to subsequent developmental stages.

Post-Transplant Cyclophosphamide Ameliorates Lethal Thymic GVHD By Restoring Regulatory and Effector T Cell Homeostasis in Recipients with Prior PD-1 Blockade Therapy

Allogeneic HSCT has a curative potential for patients with hematological malignancies. However, graft-versus-host disease (GVHD) remains to be a significant cause of morbidity and mortality after HSCT. Regulatory T cells (Tregs) are critical mediator for immune tolerance after HSCT and we recently reported that PD-1 plays an essential role for Treg survival (Asano et al, Blood 2017). Clinical studies suggested that PD-1 blockade prior to HSCT could be a risk of increasing severe GVHD. However, the mechanisms about GVHD induced by PD-1 blockade have largely unclear and there remains a paucity of data on appropriate GVHD prophylaxis for patients who undergo HSCT after PD-1 blockade. To address these issues, we investigated the impact of PD-1 expression on donor T cells on immune reconstitution with murine BMT models. First, lethally irradiated B6D2F1 mice were transplanted with 10 million of C57BL/6-background PD-1+/+ or PD-1-/- spleen cells with 5 million of bone marrow cells from normal C57BL/6, and GVHD scores and overall survival was monitored. Recipients receiving PD-1-/- graft developed severe GVHD resulting in a significant shorter survival than recipients receiving PD-1-/- graft (P<0.0001). We analyzed lymphocytes in spleen and thymus on day3, 7, and 14. We found that CD8 T cells in PD-1-/- group showed markedly higher Ki67 expression and CFSE-dilution until day3. Interestingly, PD-1-/- Tregs increased aggressively at day3 but it could not maintain until day14, while PD-1-/- CD8 T cells and conventional CD4 T cells (CD4 Tcons) continued to increase until day+14, resulting in the significant higher CD8/Treg ratio in PD-1-/- group (P<0.05, vs PD-1+/+ group). PD-1-/- Tregs showed significantly higher expression of Annexin V on day+7 and thymus CD4- and CD8- double-positive (DP) cells were in the extremely low levels in PD-1-/- group on day+14 (P<0.05, vs PD-1+/+ group). Thymic analysis showed that donor PD-1-/- graft-derived CD8 T cells infiltrated thymus in PD-1-/- group, suggesting reconstruction of thymic function was critically disturbed by severe GVHD. These data suggest that loss of PD-1 signaling resulted in unbalanced reconstitution of donor-derived T cell subsets as a consequence of continuous CTL expansion and increased Treg apoptosis. Next, to evaluate the impact of post-transplant cyclophosphamide (PTCy) on the abnormal reconstitution after PD-1 blockade, we administered 50mg/kg of Cy or control vehicle on day3. PTCy efficiently ameliorated GVHD in PD-1-/- group and extended overall survival by safely regulating the proliferation and apoptosis of T cell subsets. Of note, after PTCy, Tregs regained the ability of continuous proliferation in the first 2 weeks, resulting in well-balanced reconstitution of donor-derived T cell subsets. Thymic DP cells on day 14 was markedly increased in PD-1-/- group with PTCy intervention as compared to without PTCy, suggesting PTCy could rescue thymus from PD-1 blockade-related severe GVHD. Finally, to evaluate GVL activity, we performed BMT with co-infusion of P815L tumor cells on day0 and we confirmed that PTCy treatment for PD-1-/- recipients reduced the severity of GVHD with maintaining sufficient GVL effect. In summary, our data suggested three insights about the impact of PD-1 signaling on immune reconstitution. First, PD-1 inhibition influenced graft-derived T cells very differently within T cell subsets. PD-1-/- Tregs increased transiently but it was counterbalanced by accelerated apoptosis, while PD-1-/- CD4+Tcons and CD8 T cells continued the drastic expansion. Second, we found that PD-1-/- donor T cells developed severe GVHD in thymus. Few reports have concentrated on the impact of donor graft PD-1 expression to thymus after BMT and acute GVHD in thymus could lead late central immune disturbance. Third, PTCy successfully ameliorated GVHD induced by PD-1-/- donor T cells preserving GVL effect. Cell proliferation study implied that PD-1-/- graft-derived CD8 T cells might be more susceptible for PTCy because of the high-rate proliferation. In conclusion, PD-1-/- graft cause lethal thymic GVHD and PTCy successfully ameliorated it. The influence of PD-1 inhibition was different within T cell subtypes. PTCy might be appropriate GVHD prophylaxis strategy for patients who had prior usage of PD-1 blockade. DisclosuresNo relevant conflicts of interest to declare.

Live-Cell FRET Imaging Reveals a Role of ERK Activity Dynamics in Thymocyte Motility

Thymocyte motility is key to orchestrating migration between different thymic microenvironments at the appropriate developmental stage. Several lines of evidence suggest that extracellular signal-regulated kinase (ERK) signaling pathways plays critical roles in T cell development. Nevertheless, the dynamics of ERK activity and its role in regulating cell motility remain largely unknown due to technical difficulties. Therefore, there is an increasing demand for genetic reporter systems that provide the information of thymocyte motility and ERK signaling under physiological condition in real time.To meet the demand, we developed transgenic mice expressing fluorescence resonance energy transfer (FRET) based biosensor for ERK incorporated into the ROSA26 locus conditionally. EKAREV is a genetically encoded intramolecular FRET biosensor for monitoring ERK activity in living cells. We introduced cDNAs of EKAREV into the ROSA26 locus to generate mouse lines named R26-EKAREV-NLSfl/+. In these mouse lines, EKAREV will be expressed in the nucleus after Cre-mediated excision of the loxP-flanked tdKeima-coding sequence. To study ERK activity dynamics in T cells, R26-EKAREV-NLSfl/+ were crossed with Lck-Cre mice to generate R26-EKAREV-NLSlck/+. R26-EKAREV-NLSlck/+ showed uniform and high-level expression of EKAREV in lymphocytes and allowed us to examine the ERK activity of T cells in vivo.After initial characterization of R26-EKAREV-NLSlck/+, we attempted to unravel the potential crosstalk between ERK activity dynamics and cell motility within the thymic microenvironment. Long-term in vivo imaging of thymus was difficult due to the anatomical location abutting the heart. To circumvent this problem, thymocytes obtained from R26-EKAREV-NLSlck/+ were overlaid on C57BL/6 (WT) thymic slices and observed under two-photon excitation microscopy. During the course of experiments, we noticed a negative correlation between ERK activity and migration speed of thymocytes. To confirm this observation, thymocytes were cultured in the presence or absence of an mitogen-activated protein kinase/ERK kinase (MEK) inhibitor prior to transfer onto thymic slices. Treatment of thymocytes with MEK inhibitor increased averaged migration speed in both double-positive (DP) and single-positive (SP) subsets. Moreover, time-lapse imaging of each subsets of thymocytes revealed that the deviation of ERK activity from the average of individual cells regulated cell motility in CD4-SP, while the absolute value of ERK activity regulated cell motility in DP and CD8-SP. Collectively, these results suggest that CD4-SP is unique in that the ERK activity dynamics negatively regulate cell motility.Which signal regulates ERK activity and cell motility of CD4-SP in the medulla? Interaction between T cell receptor (TCR) and major histocompatibility complex (MHC) is known to regulate ERK activity and cell motility of T cells, but their direct relationship remains unknown in tissues. To examine this possibility, CD4-SP thymocytes were overlaid onto WT or MHC class II knockout (KO) thymic slices. When overlaid onto KO thymic slices, the average migration speed was significantly increased, indicating that CD4-SP cells arrest upon TCR-MHC II interaction. Moreover, the variance of ERK activity, but not the average of it, in CD4-SP cells on KO thymic slices was decreased, suggesting that TCR-MHC II interaction contribute the variance of ERK activity in CD4-SP.Our findings unravel that the deviation of ERK activity induced by TCR-MHC interactions negatively regulate cell motility of CD4-SP in the medulla. The live-cell FRET imaging of ERK activity will open a new window to understand the dynamic nature and the diverse functions of ERK signaling in T cell biology. DisclosuresTakaori-Kondo:Janssen Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Celgene: Honoraria, Research Funding.

Combination of Flow Cytometry and Functional Imaging for Monitoring of Residual Disease in Myeloma

IntroductionThe iliac crest is the usual sampling site for minimal residual disease (MRD) monitoring in Multiple Myeloma (MM). However, the disease distribution in the bone marrow (BM) is often heterogeneous. Functional imaging can be used to complement MRD detection at a single site, thereby accounting for asymmetrically distributed disease. Diffusion weighted MRI with background suppression (DWIBS) is a novel functional imaging method that can detect disease in a higher proportion of newly diagnosed MM (NDMM) patients than 18F-fluorodeoxyglucose positron emission tomography (PET), as it is independent of the tumor metabolism. Yet, its performance for monitoring of residual disease has not been described. The aims of this study were 1) to compare DWIBS to PET for the detection of residual disease in patients achieving complete remission (CR), and 2) to test whether DWIBS and PET could complement MRD flow cytometry with a sensitivity of 1x10-5. To address these aims, we investigated 168 NDMM and 33 relapsed patients for whom DWIBS, PET, and MRD were available at the onset of CR during first-line and salvage therapy, respectively.MethodsAll patients signed written consent in accordance with the Declaration of Helsinki. Residual focal lesions (FLs) were defined as well delineated focal intensities above the surrounding BM background. For DWIBS FLs were considered if restriction could be confirmed on ADC maps. 8-color MRD flow cytometry with a limit of detection of 1x10-5 was available for 83 NDMM and all 33 salvage therapy patients. The Kaplan-Meier method was used for survival analyses. PFS time was measured from onset of CR to relapse or death from any cause or censored at the date of last contact. Paired-end whole exome sequencing of CD138-enriched MM cells was performed on an Illumina HiSeq 2500. Mutations were called from BWA aligned sequencing reads using MuTect. Subclonal reconstruction was done using SciClone.ResultsCompared to PET, DWIBS detected more CR patients with residual FLs (21% vs. 6%), and the concordance between PET and DWIBS was low. Only 6 of the DWIBS-positive patients also presented with FLs in PET. Yet, 5 patients had PET+/DWIBS- FLs, suggesting that the two techniques are complementary. Both, DWIBS+ and PET+ FLs negatively impacted PFS (p<0.05).For 83 patients MRD data were available. Combining MRD and imaging, residual disease was detectable in 53 patients (64%). The best outcome was seen for 30 double negative (MRD-/Imaging-) patients (3 events with a median follow-up of 3.6 years), the worst outcome was seen for 10 double positive (MRD+/Imaging+) patients (median PFS: 2.1 years). Only 4 of 86 patients were MRD-/Imaging+, indicating that residual FLs are rare in MRD-negative NDMM patients at a sensitivity of 1x10-5.A heterogeneous disease distribution is a common feature of late-stage patients. To test if this increased heterogeneity confounded MRD, we investigated a set of 33 heavily pretreated patients who achieved CR during salvage therapy. Combining MRD and imaging data, we detected residual disease in 25 patients (76%). Of note, the proportion of patients, who were MRD-negative but had residual FLs on functional imaging was significantly higher compared to NDMM (8/16 vs 4/34 patients, p=0.01). At the same time, 10 patients (30%) were MRD+ but Imaging-, supporting the idea that a combined MRD/Imaging approach can improve detection of residual disease and should be used in late-stage patients.To obtain insights in the underlying biology, we performed longitudinal multi-region sequencing of a subset of these CR patients. Our findings support the concept of persistence and progression of multiple spatially separated clones in the BM irrespective of being in an MRD-negative CR. Thereby, focal residual disease could be shown to contribute to relapse.ConclusionDWIBS is a promising tool for detection of residual disease and complements PET. The combination of MRD diagnostics and functional imaging improves prediction of outcome, with double-negativity and double positivity defining groups with excellent and dismal PFS, respectively. Prospective trials using this information to tailor therapy are warranted. From a biological perspective, this study highlights the confounding effects of spatial heterogeneity and limited dissemination of clones within the BM on MRD diagnostics. This may especially be true for patients achieving deep responses during salvage therapies. DisclosuresRoy Choudhury:University of Arkansas for Medical Sciences: Employment, Research Funding. Epstein:University of Arkansas for Medical Sciences: Employment. Barlogie:International Workshop on Waldenström's Macroglobulinemia: Other: travel stipend; Millenium: Consultancy, Research Funding; Multiple Myeloma Research Foundation: Other: travel stipend; ComtecMed- World Congress on Controversies in Hematology: Other: travel stipend; Myeloma Health, LLC: Patents & Royalties: : Co-inventor of patents and patent applications related to use of GEP in cancer medicine licensed to Myeloma Health, LLC; European School of Haematology- International Conference on Multiple Myeloma: Other: travel stipend; Celgene: Consultancy, Research Funding; Dana Farber Cancer Institute: Other: travel stipend. Davies:Takeda: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Morgan:Celgene: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Research Funding.

Glucocorticoids Paradoxically Induce Intrinsic Steroid Resistance through a STAT5-Mediated Survival Mechanism in T-Cell Acute Lymphoblastic Leukemia

▪Upfront resistance to glucocorticoids (GCs) confers a poor prognosis for children with T-cell acute lymphoblastic leukemia (T-ALL). Using primary diagnostic samples from the Children's Oncology Group trial AALL1231, we previously demonstrated that one-third of patient T-ALL samples are intrinsically resistant to GCs when cultured in the presence of interleukin-7 (IL7), a cytokine that is abundant in the T-ALL microenvironment. Furthermore, we demonstrated that inhibiting JAK/STAT signaling downstream of the IL7 receptor (IL7R) with the JAK1/2 inhibitor ruxolitinib (RUX) overcomes GC resistance in these samples. The objective of the present study was to determine the mechanism of IL7-induced GC resistance in T-ALL and to identify novel therapeutic targets to enhance GC sensitivity. We utilized CCRF-CEM cells, a human T-ALL cell line, as a model system in conjunction with primary patient samples. Exposing CCRF-CEM cells to IL7 induced phosphorylation of STAT5, the predominant downstream effector of IL7R signaling. When cultured in the presence of IL7 and the GC dexamethasone (DEX), CCRF-CEM cells recapitulated the IL7-induced GC resistance phenotype observed in patient samples. This resistance could be overcome with RUX, and Bliss index analysis demonstrated a synergistic relationship between DEX and RUX in the presence of IL7. Furthermore, CRISPR/Cas9 mediated knockout of STAT5 (STAT5 KO) was sufficient to overcome resistance, implicating STAT5 as the critical mediator of IL7-induced GC resistance. DEX exposure potently induced upregulation of IL7R expression in CCRF-CEM cells. Using a luciferase reporter construct containing a series of STAT5 response elements, we demonstrated that in the presence of IL7, DEX-induced upregulation of IL7R expression is associated with increased downstream signal transduction, leading to a significant increase in STAT5 transcriptional output. We then performed RNA-seq to further assess the functional consequences of this enhanced STAT5-mediated transcription. Gene set enrichment analysis (GSEA) revealed that STAT5 target genes were significantly upregulated in cells exposed to DEX and IL7 relative to IL7 alone (normalized enrichment score -2.27; p < 0.001; FDR < 0.001), suggesting that DEX exposure augments activation of the STAT5 transcriptional program. One critical component of this program that was induced by the combination of DEX and IL7 was the anti-apoptotic family member BCL2, which was not induced by DEX alone. Further analysis of its protein expression in CCRF-CEM cells confirmed this paradoxical upregulation of BCL2 specifically by the combination of DEX and IL7. Furthermore, BCL2 was not upregulated by DEX and IL7 in STAT5 KO cells, consistent with this being a STAT5-mediated effect. IL7-induced GC resistance could be overcome with shRNA-mediated knockdown of BCL2 and with pharmacologic inhibition of BCL2 by venetoclax. Similar to the effect observed with RUX, Bliss index analysis demonstrated synergy between DEX and venetoclax in the presence of IL7. Consistent with our observations in CCRF-CEM cells, an analysis of primary diagnostic T-ALL samples revealed DEX-induced upregulation of IL7R expression in samples with IL7-induced GC resistance, which was associated with increased BCL2 expression in the presence of DEX and IL7. Finally, we performed a similar analysis in healthy murine thymocytes, and found that CD4/CD8 double negative (DN) and CD4 or CD8 single positive (SP) thymocytes, but not double positive (DP) thymocytes, exhibited profound IL7-induced GC resistance that was associated with DEX-induced upregulation of IL7R expression and increased BCL2 expression in the presence of DEX and IL7. These data are consistent with the known role of IL7 specifically at the DN and SP stages of development, and suggests that IL7-induced GC resistance is a physiologic mechanism of GC resistance in normal thymocyte development that is retained during leukemogenesis in a subset of T-ALL samples. Taken together, these data demonstrate that GCs paradoxically induce their own resistance in a subset of T-ALLs and in normal developing T-cells by augmenting a STAT5-mediated pro-survival program that results in upregulation of BCL2. Furthermore, we demonstrate that inhibition of JAK/STAT signaling or of BCL2 may have considerable therapeutic benefit to enhance GC sensitivity in T-ALL patients with IL7-induced GC resistance. DisclosuresTeachey:La Roche: Consultancy; Amgen: Consultancy.

Suppression of GATA3 Binding Drives Selective Abrogation of NOTCH1-MYC Enhancer Activity By Nucleosome Invasion in Thymocyte Development and Leukemia

Long range enhancers play critical roles in the control of gene expression during development and have emerged as key regulators of lineage commitment and oncogenic programs in hematopoiesis and leukemia. The MYC oncogene is dynamically regulated in the hematopoietic system under the control of a network of clustered distal enhancers, which provide modular regulation of MYC expression during lymphoid and myeloid development. In thymocyte development MYC transcription critically depends on the activity of N-Me, a distinct T-cell specific enhancer controlled by NOTCH1 signaling and located 1.4 Mb telomeric to the MYC transcription start site. Yet, the specific mechanisms governing N-Me enhancer activity and lineage specific control of MYC expression remain rudimentarily understood. Analysis of chromatin looping by 4C and chromatin accessibility by ATACseq revealed an unanticipated high density of chromatin contacts between N-Me and additional regulatory elements in the Myc locus and showed a distinct pattern of N-Me chromatin accessibility -opening as progenitors mature into T cell committed CD4 CD8 double negative (DN) 2b cells and returning to a closed configuration in CD4 CD8 double positive (DP) thymocytes-. To explore potential regulators of N-Me activity we performed Mass Spectrometry proteomic profiling of N-Me binding proteins and ChIPseq analyses identifying numerous factors involved in hematopoietic and lymphoid development (ERG, ETS1, GATA3, RUNX1, TCF3 and TCF12) and transcription factor oncogenes with prominent roles in the pathogenesis of T-ALL (HOXA9, MYB, MYC, LMO1, LMO2, TAL1 and TLX1). Moreover, phylogenetic footprinting analyses across vertebrate species identified two ultraconserved elements matching GATA factor binding motifs (GS1 and GS2). To test the functionality of these elements we introduced targeted mutations in the N-Me sequence at these sites using CRISPR/CAS9 directed mutagenesis. Mice homozygous for combined N-Me GS1 and GS2 mutations (GS1+2mut) revealed a marked defect in thymus cellularity with characteristic accumulation of DN and intermediate single positive (ISP) thymocytes and decreased numbers of more mature populations. Mechanistically, immunohistochemical, flow cytometry and single cell RNaseq analyses revealed decreased Myc protein levels in thymocyte poulations of GS1+2 mutant animals. In this context, we hypothesized that GATA3, a prominent N-Me binding transcription factor in our ChIP and proteomic analyses critically implicated in T-cell commitment, could play a major role in N-Me regulation via interaction with the GS1 and GS2 N-Me GATA sites. Consistent with this hypothesis analysis of Gata3 ChIPs from heterozygous GS1+2 mutant mice recovered only the N-Me wild type sequence, formally demonstrating the strict requirement of these sites for N-Me Gata3 binding. Mechanistically, ATACseq analysis revealed a marked reduction in chromatin accessibility and nucleosome invasion in thymocytes from GS1+2 mutant mice in support of a critical pioneering activity for GATA3 in the control of N-Me activity. Finally, given the important role of NOTCH1 induced MYC upregulation in the pathogenesis of T-ALL, we hypothesized that disruption of N-Me activity via targeted mutation of N-Me GATA sites could effectively impair the development of NOTCH1-driven T-ALL in N-Me GS1+2 mutant mice. To test this possibility we infected hematopoietic progenitors from N-Me wild type and N-Me GS1+2 homozygous mice with retroviruses driving the expression of an oncogenic constitutively active form of NOTCH1 (DE-NOTCH1) and transplanted them into sublethally irradiated recipients. In these experiments, mice transplanted with DE-NOTCH1 infected N-Me wild type cells developed overt T-ALL 6 weeks postransplant with 100% penetrance. In contrast, mice transplanted with DE-NOTCH1-expressing N-Me GS1+2 homozygous cells showed complete protection from NOTCH1 induced T-ALL (P <0.001). In all these results identify GATA3 binding to the N-Me enhancer as a critical driver of nucleosome eviction and enhancer activation strictly required for thymocyte development and NOTCH1-induced T-cell transformation. DisclosuresNo relevant conflicts of interest to declare.

Renal Cell Carcinoma (RCC) Tumors Display Large Expansion of Double Positive (DP) CD4+CD8+ T Cells With Expression of Exhaustion Markers.

Checkpoint inhibitors target the inhibitory receptors expressed by tumor-infiltrating T cells in order to reinvigorate an anti-tumor immune response. Therefore, understanding T cell composition and phenotype in human tumors is crucial. We analyzed by flow cytometry tumor-infiltrating lymphocytes (TILs) from two independent cohorts of patients with different cancer types, including RCC, lung, and colon cancer. In healthy donors, peripheral T cells are usually either CD4+ or CD8+ with a small percentage of CD4+ CD8+ DP cells (<5%). Compared to several other cancer types, including lung, and colorectal cancers, TILs from about a third of RCC patients showed an increased proportion of DP CD4+CD8+ T cells (>5%, reaching 30–50% of T cells in some patients). These DP T cells have an effector memory phenotype and express CD38, 4-1BB, and HLA-DR, suggesting antigen-driven expansion. In fact, TCR sequencing analysis revealed a high degree of clonality in DP T cells. Additionally, there were high levels of PD-1 and TIM-3 expression on DP T cells, which correlated with higher expression of PD-1 and TIM-3 in conventional single positive CD8 T cells from the same patients. These results suggest that DP T cells could be dysfunctional tumor-specific T cells with the potential to be reactivated by checkpoint inhibitors.

Metabolic regulation of T cell development by Sin1-mTORC2 is mediated by pyruvate kinase M2.

Glucose metabolism plays a key role in thymocyte development. The mammalian target of rapamycin complex 2 (mTORC2) is a critical regulator of cell growth and metabolism, but its role in early thymocyte development and metabolism has not been fully studied. We show here that genetic ablation of Sin1, an essential component of mTORC2, in T lineage cells results in severely impaired thymocyte development at the CD4−CD8− double negative (DN) stages but not at the CD4+CD8+ double positive (DP) or later stages. Notably, Sin1-deficient DN thymocytes show markedly reduced proliferation and glycolysis. Importantly, we discover that the M2 isoform of pyruvate kinase (PKM2) is a novel and crucial Sin1 effector in promoting DN thymocyte development and metabolism. At the molecular level, we show that Sin1–mTORC2 controls PKM2 expression through an AKT-dependent PPAR-γ nuclear translocation. Together, our study unravels a novel mTORC2−PPAR-γ−PKM2 pathway in immune-metabolic regulation of early thymocyte development.