Receptor Alpha Chain Research Articles

Heterodimeric IL15 Treatment Enhances Tumor Infiltration, Persistence, and Effector Functions of Adoptively Transferred Tumor-specific T Cells in the Absence of Lymphodepletion.

Purpose: Adoptive cell transfer (ACT) is a promising immunotherapeutic approach for cancer. Host lymphodepletion is associated with favorable ACT therapy outcomes, but it may cause detrimental effects in humans. We tested the hypothesis that IL15 administration enhances ACT in the absence of lymphodepletion. We previously showed that bioactive IL15 in vivo comprises a stable complex of the IL15 chain with the IL15 receptor alpha chain (IL15Rα), termed heterodimeric IL15 (hetIL15).Experimental Design: We evaluated the effects of the combination regimen ACT + hetIL15 in the absence of lymphodepletion by transferring melanoma-specific Pmel-1 T cells into B16 melanoma-bearing mice.Results: hetIL15 treatment delayed tumor growth by promoting infiltration and persistence of both adoptively transferred Pmel-1 cells and endogenous CD8+ T cells into the tumor. In contrast, persistence of Pmel-1 cells was severely reduced following irradiation in comparison with mice treated with hetIL15. Importantly, we found that hetIL15 treatment led to the preferential enrichment of Pmel-1 cells in B16 tumor sites in an antigen-dependent manner. Upon hetIL15 administration, tumor-infiltrating Pmel-1 cells showed a "nonexhausted" effector phenotype, characterized by increased IFNγ secretion, proliferation, and cytotoxic potential and low level of PD-1. hetIL15 treatment also resulted in an improved ratio of Pmel-1 to Treg in the tumor.Conclusions: hetIL15 administration improves the outcome of ACT in lymphoreplete hosts, a finding with significant implications for improving cell-based cancer immunotherapy strategies. Clin Cancer Res; 23(11); 2817-30. ©2016 AACR.

Expression of CD25 on leukemic stem cells in BCR-ABL1+ CML: Potential diagnostic value and functional implications.

Chronic myeloid leukemia (CML) is a stem cell-derived leukemia in which neoplastic cells exhibit the Philadelphia chromosome and the related oncoprotein BCR-ABL1. The disease is characterized by an accumulation of myeloid precursor cells in the peripheral blood and bone marrow (BM). A small fraction of neoplastic cells in the CML clone supposedly exhibits self-renewal and thus long-term disease-propagating ability. However, so far, little is known about the phenotype, function, and target expression profiles of these leukemic stem cells (LSCs). Recent data suggest that CML LSCs aberrantly express the interleukin-2 receptor alpha chain CD25. Whereas normal CD34+/CD38- BM stem cells display only low amounts of CD25 or lack CD25 altogether, CD34+/CD38- LSCs express CD25 strongly in more than 90% of all patients with untreated CML. As a result, CD25 can be used to identify and quantify CML LSCs. In addition, it has been shown that CD25 serves as a negative growth regulator of CML LSCs. Here, we review the value of CD25 as a novel marker and potential drug target in CML LSCs.

Immunosuppressive Effects of Bryoria sp. (Lichen-Forming Fungus) Extracts via Inhibition of CD8+ T-Cell Proliferation and IL-2 Production in CD4+ T Cells.

Lichen-forming fungi are known to have various biological activities, such as antioxidant, antimicrobial, antitumor, antiviral, anti-inflammation, and anti proliferative effects. However, the immunosuppressive effects of Bryoria sp. extract (BSE) have not previously been investigated. In this study, the inhibitory activity of BSE on the proliferation of CD8+ T cells and the mixed lymphocytes reaction (MLR) was evaluated in vitro. BSE was non-toxic in spleen cells and suppressed the growth of splenocytes induced by anti-CD3. The suppressed cell population in spleen cells consisted of CD8+ T cells and their proliferation was inhibited by the treatment with BSE. This extract significantly suppressed the IL-2 associated with T cell growth and IFN-γ as the CD8+ T cell marker. Furthermore, BSE reduced the expression of the IL-2 receptor alpha chain (IL-2Rα) on CD8+ T cells and CD86 on dendritic cells by acting as antigen-presenting cells. Finally, the MLR produced by the co-culture of C57BL/6 and MMC-treated BALB/c was suppressed by BSE. IL-2, IFN-γ, and CD69 on CD8+ T cells in MLR condition were inhibited by BSE. These results indicate that BSE inhibits the MLR via the suppression of IL-2Rα expression in CD8+ T cells. BSE has the potential to be developed as an anti-immunosuppression agent for organ transplants.

Tu1747 - Reduced Neutrophil Granulocyte-Macrophage Colony Stimulating Factor Signaling is Associated with Granulocyte-Macrophage Colony Stimulating Factor Receptor Alpha Chain Gene Mutations and Stricturing Disease Complications in Pediatric Crohn Disease

Tu1747 - Reduced Neutrophil Granulocyte-Macrophage Colony Stimulating Factor Signaling is Associated with Granulocyte-Macrophage Colony Stimulating Factor Receptor Alpha Chain Gene Mutations and Stricturing Disease Complications in Pediatric Crohn Disease

Human Epistatic Interaction Controls IL7R Splicing and Increases Multiple Sclerosis Risk

Multiple sclerosis (MS) is an autoimmune disorder where Tcells attack neurons in the central nervous system (CNS) leading to demyelination and neurological deficits. A driver of increased MS risk is the soluble form of the interleukin-7 receptor alpha chain gene (sIL7R) produced by alternative splicing of IL7R exon 6. Here, we identified the RNA helicase DDX39B as a potent activator of this exon and consequently a repressor of sIL7R, and we found strong genetic association of DDX39B with MS risk. Indeed, we showed that a genetic variant in the 5' UTR of DDX39B reduces translation of DDX39B mRNAs and increases MS risk. Importantly, this DDX39B variant showed strong genetic and functional epistasis with allelic variants in IL7R exon 6. This study establishes the occurrence of biological epistasis in humans and provides mechanistic insight into the regulation of IL7R exon 6 splicing and its impact on MS risk.

Adenovirus Serotype 5 Vaccination Results in Suboptimal CD4 T Helper 1 Responses in Mice.

Adenovirus serotype 5 (Ad5) is one of the most widely used viral vectors and is known to generate potent T cell responses. While many previous studies have characterized Ad5-induced CD8 T cell responses, there is a relative lack of detailed studies that have analyzed CD4 T cells elicited by Ad5 vaccination. Here, we immunized mice with Ad5 vectors encoding lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) and examined GP-specific CD4 T cell responses elicited by Ad5 vectors and compared them to those induced by an acute LCMV infection. In contrast to LCMV infection, where balanced CD4 T helper 1 (Th1) and T follicular helper (Tfh) responses were induced, Ad5 immunization resulted in a significantly reduced frequency of Th1 cells. CD4 T cells elicited by Ad5 vectors expressed decreased levels of Th1 markers, such as Tim3, SLAM, T-bet, and Ly6C, had smaller amounts of cytotoxic molecules like granzyme B, and produced less interferon gamma than CD4 T cells induced by LCMV infection. This defective CD4 Th1 response appeared to be intrinsic for Ad5 vectors and not a reflection of comparing a nonreplicating vector to a live viral infection, since immunization with a DNA vector expressing LCMV-GP generated efficient CD4 Th1 responses. Analysis at early time points (day 3 or 4) after immunization with Ad5 vectors revealed a defect in the expression of CD25 (interleukin-2 [IL-2] receptor alpha chain) on Ad5-elicited CD4 T cells, and administration of exogenous IL-2 following Ad5 immunization partially restored CD4 Th1 responses. These results suggest that impairment of Th1 commitment after Ad5 immunization could be due to reduced IL-2-mediated signaling.IMPORTANCE During viral infection, generating balanced responses of Th1 and Tfh cells is important to induce effective cell-mediated responses and provide optimal help for antibody responses. In this study, to investigate vaccine-induced CD4 T cell responses, we characterized CD4 T cells after immunization with Ad5 vectors expressing LCMV-GP in mice. Ad5 vectors led to altered effector differentiation of LCMV GP-specific CD4 T cells compared to that during LCMV infection. CD4 T cells following Ad5 immunization exhibited impaired Th1 lineage commitment, generating significantly decreased Th1 responses than those induced by LCMV infection. Our results suggest that suboptimal IL-2 signaling possibly plays a role in reduced Th1 development following Ad5 immunization.

Selenoprotein K modulate intracellular free Ca2+ by regulating expression of calcium homoeostasis endoplasmic reticulum protein

Selenoprotein K (SelK) is an 11-kDa selenoprotein, which may be involved in the regulation of oxidative stress, endoplasmic reticulum (ER) stress and immune response. To explore the function of SelK in the process of immune response, several short-hairpin RNAs (shRNA) were designed for the construction of recombinant plasmids to down-regulate the expression of SelK gene in vitro. These shRNAs specifically and efficiently interfered with the expression of SelK at both mRNA and protein levels. The expression of calcium homoeostasis endoplasmic reticulum protein (CHERP) and the intracellular free Ca2+ concentration were significantly down-regulated in anti-CD3 stimulated SelK-knockdown cells. The expression of Interleukin 2 receptor alpha chain (IL-2Rα) and the secretion of Interleukin 4 (IL-4), which play a significant role in the process of T cell activation and proliferation, were also reduced in SelK-knockdown cells. Selenomethionine (Se-Met) at an optimum concentration of 5 μM could up-regulate SelK expression and reverse the change of the expression of CHERP and the intracellular free calcium caused by SelK-knockdown. These results hereby imply SelK may regulate the release of Ca2+ by CHERP and play an important role in the proliferation and differentiation of T cell by TCR stimulation.

SL-401 Mediates Potent Cytotoxicity Against CD123+ AML and MDS with Excess Blasts and Demonstrates Therapeutic Benefit in PDX Model

Acute myeloid leukemia (AML) is a heterogeneous myeloid neoplasm with complex pathophysiology. The diversities of karyotype, genetic mutations and epigenetic aberrations in AML result in difficult risk stratification and treatment selection. Despite the application of standard chemotherapies and immunotherapies, many AML patients relapse at least in part due to the failure to eradicate AML leukemic stem cells (LSC). New therapies to target these cells would be immensely valuable. CD33 is a widely expressed myeloid marker present on the majority of AML cells, and CD33-targeted immunotherapies have shown promising results. However, the majority of LSC lack CD33 expression and are not eliminated with such agents. CD123, the alpha chain of the interleukin-3 receptor (IL-3R) heterodimer, is expressed on the majority of LSC and in many AML tumor cells. Several efforts to target CD123 to eradicate LSC have emerged. To date, however, each of these agents exhibited shortcomings that limited their development.SL-401 (from Stemline Therapeutics, NY) is a recombinant fusion protein consisting of human IL-3 and truncated diphtheria toxin. IL-3 dictates the specificity for CD123expressing cells, and the catalytic unit of diphtheria toxin upon internalization inhibits the translational machinery to initiate cell death. SL-401 induced potent, dose-dependent (10, 100, 1000 ng/ml) cytotoxicity in AML patient cells ex vivo, as observed by annexin V/propidium iodide staining. (N=16, 48hr trend p<0.0001). In addition, a rare CD33-/CD123+ AML cell population also responded to SL-401, suggesting that SL-401 may be useful for treating CD33- AML subsets that express CD123. Notably, FLT3-ITD high risk AML cells were strongly positive for CD123 expression and sensitive to SL-401. Moreover, SL-401 treatment (1µg/ml, 2µg/ml) for 120hours specifically depleted CD123+ blasts in samples from patients with myelodysplastic syndrome (MDS) with refractory anemia with excess blasts (RAEB) while preserving lymphoid cells (N=5 MDS-RAEB, p=0.0183), as documented by CD45 /CD34 /CD33 staining. No significant correlation was observed between the cytotoxic activity of SL-401 and the levels of CD123 cell surface expression. Interestingly, AML cells expressing as few as 10,000 CD123 molecules were sensitive to SL-401 mediated cytotoxicity.Bone marrow-derived mesenchymal stromal cells (MSC) from AML patients exhibit overlapping as well as distinct cytogenetic abnormalities versus AML blasts and provide a conducive cytokine and growth milieu for leukemia cells and LSCs. Therefore, it is critical to assess potential AML therapies in the context of the bone marrow microenvironment with autologous MSCs. Assessment of SL-401 in an autologous MSC co-culture system developed from the bone marrow of AML patients abrogated the growth of AML cells. (N=4 AML; protective effect of MSC p=0.0107). Presence of MSC had no significant inhibitory effect on SL-401 (p=0.7297).To demonstrate the therapeutic effect of SL-401 in vivo, we used an AML patient-derived xenograft (PDX) model. CD123+ AML cells were used to engraft NRG-SG3 (NRGS) mice, which are immunocompromised and express stem cell factor (SCF), granulocyte monocyte-colony stimulating factor (GM-CSF) and IL-3 transgenes. Treatment of patient AML xenografted mice with SL-401 (50µg/kg, i.p, 15 doses at 3 doses per week) significantly increased survival rates compared to the vehicle control (N=4/group, p= 0.0069). The presence of AML tumor in the animals was confirmed in spleen, bone marrow and blood using a multi-color flow cytometric panel (CD45 /CD33 /CD34 /CD123 /CD19 /CD3 /HLA-DR /mCD45 /viability stain).Together, these data indicate that SL-401 is active against AML/MDS cells and LSCs. Importantly, the ability of SL-401 to deliver its toxin payload selectively to cancer cells and LSCs could limit side effects by preventing exposure to other cells. Thus, SL-401 has the potential to become an effective agent for CD123+ AML and other myeloid neoplasms and is currently being evaluated in a Phase 2 trial of AML patients in remission with minimal residual disease, a setting associated with the persistence of CD123+ LSCs (clinical trial information: NCT02270463). DisclosuresBrooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Lozanski:Boehringer Ingelheim: Research Funding; Beckman Coulter: Research Funding; Genentech: Research Funding; Stemline Therapeutics Inc.: Research Funding.

Allogeneic Tcrα/β Deficient CAR T-Cells Targeting CD123 Prolong Overall Survival of AML Patient-Derived Xenografts

Acute myeloid leukemia (AML) is a disease with a high incidence of relapse and mortality. Relapse is attributed to the inability of current chemotherapeutic agents to eliminate leukemia stem cells (LSCs). Thus, to improve leukemia therapy, it is critical to identify agents that effectively target LSCs, e.g. via unique cell surface antigens. A target of major interest is CD123, the transmembrane alpha chain of the interleukin-3 receptor, expressed on blasts, leukemic progenitor and LSCs in the majority of AML patients. We have developed an allogeneic chimeric antigen receptor (CAR) T-cell platform using T-cells from third-party healthy donors to generate engineered T-cells targeting CD123 (UCART123). UCART123 cells no longer express a TCR, having undergone a disruption of the TCRα gene using TALEN¨ gene editing technology followed by elimination of TCRα/β-positive cells, thus minimizing the potential for engineered T-cells to cause graft versus host disease (GvHD). We tested the activity of UCART123 cells in vitro using primary AML samples, normal bone marrow (nBM) and cord blood (CB) cells. Additionally, we established patient derived xenograft (PDX), nBM- or CB- humanized xenografts (HuX) and a competitive nBM/AML xenograft model to evaluate the in vivo potential of UCART123 cells to preferentially eliminate AML over normal BM cells.In vitro studies reveled that UCART123 cells eliminate AML cells and had minimum effect on normal cells at effector:target ratios as low as 0.5:1. Next, we evaluated the in vivo activity of UCART123 against PDX (AML37, TP53 mutant relapsed AML and AML20, FLT3-ITD+ and TP53 mutant AML) and normal-HuX mice (n=3). At 3 weeks post T-cell injection we found that UCART123 treatment eliminated the leukemic cells when using 10M or 3M UCART123 cells per mouse and no significant difference between PBS or TCR-deficient T-cells (TCRkoT; 10M/mouse). Toxicity to normal cells was dose dependent, doses of 2.5M UCART123 cells did not significantly affect hematopoietic cells. T-cells were detected in the BM at day 14 after treatment, without evidence of GvHD.Since we found complete elimination of human AML cells in the BM of the PDX precluding serial transplantation to evaluate LSC activity, we initiated two new sets of PDX-AML mice [AML2 (NPM1+FLT3-ITD+) and AML37 (TP53 mutant)] to evaluate long-term survival, and time to relapse. Animals were treated with PBS, UCART123 (2.5M or 1M), TCRkoT (2.5M), or Ara-C (60mg/kg 5 days). Animal weight and peripheral blood (PB) was monitored. Cytokines changes were evaluated at day 2. We found that the cytokine release and the kinetics of AML targeting by UCART123 were dose dependent. We found a significant overall survival (OS) benefit with UCART123 in both PDX tested. For example, all PDX-AML2 mice treated with UCART123 are alive to date (day 167; updates will be presented). All other cohorts were lost (PBS day124, TCRkoT day126, AraC day144) (Figure 1A).Finally, to determine selectivity of UCART123 cells for AML cells over nBM cells, we generated a competitive model bearing both nBM and AML (NPM1+FLT3-ITD+). With PB monitoring, treatment with 1M UCAR123 cells resulted in selective elimination of AML cells. Untreated and TCRkoT treated mice showed a rapid progression of AML, while treated mice showed normal hematopoiesis (Figure 1B). NPM1 transcripts were also monitored in the mice and confirmed molecular remission in mice.Taken together, our data show that UCART123, an "off-the-shelf" allogeneic engineered CAR-T product targeting CD123 potently eliminates AML cells in vivo, prevents relapse, and improves OS in PDX mice. Also, UCART123 cells preferentially targets AML cells in a competitive BM/AML model. A phase I trial of UCART123 in AML is under development. [Display omitted] DisclosuresGuzman:Cellectis: Research Funding. Sugita:Cellectis: Research Funding. Galetto:Cellectis SA: Employment. Gouble:Cellectis: Employment. Smith:Cellectis SA: Employment. Roboz:Agios, Amgen, Amphivena, Astex, AstraZeneca, Boehringer Ingelheim, Celator, Celgene, Genoptix, Janssen, Juno, MEI Pharma, MedImmune, Novartis, Onconova, Pfizer, Roche/Genentech, Sunesis, Teva: Consultancy; Cellectis: Research Funding.

Immunohistochemical Expression of CD123 Is Associated with an Inferior Clinical Outcome in Acute Myeloid Leukemia

Background:The aberrant expression of the interleukin-3 receptor (IL3RA or CD123) alpha chain is frequently observed in a subset of leukemic disorders, including acute myeloid leukemia (AML), particularly in leukemia stem cells. Using flow cytometric (FCM) analysis, studies have shown that increased CD123 expression is associated with a poor prognosis of AML. Although FCM is a sensitive technique, the analysis may be limited in a clinical setting because live leukemia cells are required. Immunohistochemistry (IHC) is a more useful alternative compared with FCM because it can be tested long after specimens are collected; however, it is less sensitive. Here we evaluated the impact of blast CD123 expression via IHC analysis for the prognosis of AML.Patients and methods:This study was performed as a retrospective analysis of 70 patients who were diagnosed with de novo AML (M0-M5, n = 48) and AML with myelodysplasia-related changes (MRC) (n = 22) at our hospital from February 2008 to September 2015. The median age at diagnosis was 64.5 years (range: 21-93 years). The median follow-up period was 498 days (range: 2-3052 days). Morphological findings were obtained using HE stains of 3-µm sections. Formalin-fixed, paraffin-embedded specimens were used for immunohistochemical analysis. We analyzed the relationships between the patients’ clinical outcome and CD123, p53, CD34, CD71, CD56, and c-kit expressions. [(CD123, CD34, CD71, CD56, and c-kit immunostained slides were defined as positive if >10% of the blast cells had a moderate to strong membranous staining. p53 was evaluated as positive when more than 5% of the cells were positively stained. For each case, the following data were obtained: patient age (>60 years or <60 years), sex, karyotype (good, intermediate, or poor), SCT (undergone or not), 1st induction (failure or complete remission (CR), and overall survival (OS)].Results:Of the 70 cases, percentages of the positive immunohistochemical study dates were as follows: CD123: 25.7%; p53: 30%; CD34: 52.8%; CD71: 46.3%; CD56: 25.7%; and c-kit: 77.1%. There were 48 cases with de novo AML: CD123: 29.1%; p53: 75%; CD34: 52%; CD71: 37.5%; CD56: 27%; and c-kit: 79.1%. Moreover, there were 22 cases with MRC: CD123: 18.1%; p53: 31.8%; CD34: 54.5%; CD71: 66.6%; CD56: 22.7%; and c-kit: 72.7%. CD71 is highly expressed in MRC than de novo AML (P = 0.036). Among all patients, the CR rate following first induction therapy was 62.3%. Age (≥60 years), high p53 expression, disease (MRC), and poor karyotype were associated with induction failure (P = 0.011, P = 0.002, P <0.001, and P = 0.008, respectively). Among patients with de novo AML, only elevated CD123 expression was associated with the failure to obtain a CR following the first remission induction chemotherapy (P = 0.044). In contrast, among patients with MRC, CD123 expression was not associated with any clinical data or the CR rate. The 2-year OS rates were 45.9%. Age (≥60), high p53 expression, MRC, poor karyotype were significantly associated with poor OS (P = 0.003, P = 0.036, P <0.001, and P = 0.002, respectively). In Multivariate analysis, age (≥60 years), MRC, poor karyotype are significantly associated with poor OS (P = 0.028, P = 0.001, and P = 0.014, respectively). Among de novo AML patients, CD123 expression, p53 expression, and induction failure were independently associated with poor OS (P = 0.036, 0.003 and P < 0.001, respectively), and increased p53 expression and induction failure was associated with poor OS in the multivariate analysis (P = 0.001 and P = 0.002). Among patients with MRC, only induction failure was associated with the OS (P = 0.026).Conclusion:This is the first study to demonstrate that CD123 expression using IHC is associated with poor a CR rate and OS in de novo AML patients; however, this association was not observed in MRC patients. Our results suggest that CD123 expression may predict the refractory to induction therapy and poor OS in de novo AML. Moreover, these results support previous reports using FCM. Therefore, CD123 expression may become one of the important factors used to characterize leukemia blasts and predict the prognosis of AML. In addition, novel therapy with antibodies targeting CD123 is currently under development. Therefore, we suggest that an analysis of CD123 expression using IHC is a clinically important assessment for de novo AML patients at the time of diagnosis. DisclosuresNo relevant conflicts of interest to declare.

Preclinical Evaluation of Allogeneic Anti-Bcma Chimeric Antigen Receptor T Cells with Safety Switch Domains and Lymphodepletion Resistance for the Treatment of Multiple Myeloma

Multiple myeloma (MM) is a hematological disease of plasma B cells that remains incurable despite the availability of numerous therapies. The plasma cell-specific expression of the TNF superfamily receptor BCMA may allow targeting of normal and malignant plasma cells. Genetically engineered chimeric antigen-receptor T cells (CAR T) have shown tremendous promise in the treatment of several hematological diseases, including MM. However, conventional autologous CAR T therapies use patient-derived T cells and the logistics of on-demand CAR T manufacture limits their availability to a broad patient pool.Here we describe the preclinical evaluation of an allogeneic CAR T therapy targeting BCMA that has the potential for a readily available, off-the-shelf therapy for MM and other malignancies expressing BCMA. Human T cells were transduced with recombinant lentiviral vectors encoding three BCMA CAR candidates designed with fully human anti-BCMA scFvs, CD8a transmembrane domains and the intracellular signaling domains of 4-1BB and CD3zeta. All CAR T efficiently killed BCMA-expressing multiple myeloma cell lines (KMS12BM, MM1.S, Molp-8 and OPM-2), but not BCMA-negative REH cells in vitro and in vivo. Whereas 2 of the 3 candidates exhibited target-independent cytokine production, accelerated T cell differentiation and reduced target cell-induced expansion in vitro, the third candidate did not exhibit this scFv-induced autoactivation and was chosen as the lead molecule.Due to the allogeneic nature of this T cell therapy, the possibility of graft-versus-host (GvH) reactions can be a safety concern. We applied Cellectis’ know-how and TALEN® technology for the gene inactivation of the T cell receptor (TCR) alpha chain to significantly reduce the probability for TCR-mediated GvH reactions and found that TCR knockout did not affect CAR T activity in vitro or in vivo. Furthermore, we incorporated intra-CAR rituximab-recognition domains into the CAR molecule to enable depletion of CAR T cells from patients when necessary. We found that this modified CAR retained anti-BCMA CAR T activity and enabled CAR T depletion by rituximab.Another aspect of allogeneic CAR T therapies is the rejection of the CAR T by host-versus-graft (HvG) reactions. Lymphodepletion prior to CAR T infusion enhances CAR T efficacy in autologous CAR T trials and may also prevent anti-CAR HvG reactions in allogeneic therapy settings. Engineering lymphodepletion resistance into CAR T cells could therefore enable sustained lymphodepletion for enhanced allogeneic CAR T persistence and efficacy. CD52 is expressed on all lymphocytes and administration of the anti-CD52 antibody alemtuzumab for prolonged lymphodepletion is an approved treatment for multiple sclerosis. TALEN®-mediated knockout of CD52 protected BCMA CAR T from alemtuzumab-induced cytotoxicity and did not alter BCMA CAR T anti-tumor activity.Taken together these results support allogeneic BCMA CAR T as an off-the-shelf adoptive immunotherapy for the treatment of multiple myeloma and other BCMA-positive malignancies. DisclosuresBoldajipour:Pfizer: Employment. Galetto:Cellectis SA: Employment. Sommer:Pfizer Inc.: Employment. Pertel:Pfizer Inc.: Employment. Valton:Cellectis Inc.: Employment. Park:Pfizer Inc.: Employment. Gariboldi:Cellectis SA: Employment. Chen:Alexo Therapeutics: Employment. Geng:Kodiak Sciences: Employment. Dong:Pfizer Inc.: Employment. Boucher:Pfizer Inc.: Employment. Van Blarcom:Pfizer Inc.: Employment. Chaparro-Riggers:Pfizer Inc.: Employment. Rajpal:Pfizer Inc.: Employment. Smith:Cellectis SA: Employment. Kuo:Pfizer Inc.: Employment. Sasu:Pfizer Inc.: Employment.

Influence of Genetic Modifiers on Levels of Invariant Natural Killer T-Cells in Patients with Sickle Cell Anemia

Although patients homozygous for the sickle cell disease (SCD) mutation have an identical genotype, the severity of the disease can be extremely variable. The hemoglobin (Hb) S mutation has been associated with five different beta S-globin gene cluster haplotypes (βS-haplotypes) that show different clinical expression. Because genetic modifiers can modulate treatment response, we hypothesized that βS-haplotypes can affect levels of invariant natural killer T (iNKT) cells, which are known to play a key role in the pathogenesis of SCD, and are being considered as a potential target to treat acute crises in SCD patients. Herein we evaluated the impact of βS-haplotypes and HbF concentration on iNKT cell and dendritic cell (DC) subsets in a well-defined group of patients with SCD in a steady state. Sickle cell anemia patients were selected based on the history of patients, clinical examination and hematological findings. The βS-haplotypes were identified by polymerase chain reaction-restriction fragment length polymorphism analysis for seven restriction sites. The iNKT cell subsets were characterized by the positive-staining of Vα24Jα18 T cell receptor alpha chain, along with CD3, CD4 and CD8 surface markers and the intra-cellular cytokine production of interferon-gamma (Th1-like), interleukin-4 (Th2-like) and interleukin-17 (Th17-like) cells using flow cytometry and cell culture. The myeloid DC (mDC) and plasmacytoid DC (pDC) cells were identified by the expression of HLA-DR, CD123, CD11c, Lin and CD1d, a non-classical molecule that induces the activation of iNKT cells by flow cytometry. Comparisons among βS-haplotypes were performed using ANOVA and unpaired t test, while the Spearman’s correlation was used to assess associations. Among the 125 sickle cell anemia patients studied, Benin haplotype (40%) was the most common followed by Bantu (21%), Arab/Indian (18%), Atypical (12%) and Benin/Bantu or Benin/Arab-Indian or Bantu/Arab-Indian (9%). The majority of subjects with Benin/Benin βS-haplotype had a severe to moderate clinical profile similar to Bantu/Bantu or Arab/Indian βS-haplotype groups (P=0.23). A trend toward increased levels of CD3iNKT, CD4iNKT and CD8iNKT cell subsets was observed in the subjects with the Benin/Benin βS-haplotype, when compared to other βS-haplotype groups (P=0.06). Interestingly, subjects with the Benin/Benin βS-haplotype exhibited slightly higher levels of Th1-like cells, but not Th2-like and Th17-like cells, when compared to subjects with the Bantu/Bantu or Arab/Indian βS-haplotype groups (P=0.047). Comparisons between the levels of mDC and pDC cell subsets, as well as the expression of CD1d on these DC cells, showed no statistically significant differences among the βS-haplotype groups (P=0.42). Similarly, levels of iNKT cell subsets, Th1-like, Th2-like, and Th17-like cells as well as DC subsets were similar among patients who received or not hydroxyurea therapy, independently of βS-haplotype groups (P= 0.09). Likewise, coexistence of high HbF and βS-haplotypes did not show any significant association with iNKT cell subsets. Collectively, our findings suggest that neither the βS-haplotypes or HbF levels nor the hydroxyurea therapy or clinical severity appeared to be associated with iNKT cell or DC cell subsets in patients with SCD. The effects of other genetic modifiers such as alpha/beta-thalassemia and G6PD deficiency on iNKT cell subsets need to be evaluated in future studies. DisclosuresNo relevant conflicts of interest to declare.

T cell receptor repertoires after adoptive transfer of expanded allogeneic regulatory T cells

Regulatory T cell (Treg ) therapy has been exploited in autoimmune disease, solid organ transplantation and in efforts to prevent or treat graft-versus-host disease (GVHD). However, our knowledge on the in-vivo persistence of transfused Treg is limited. Whether Treg transfusion leads to notable changes in the overall Treg repertoire or whether longevity of Treg in the periphery is restricted to certain clones is unknown. Here we use T cell receptor alpha chain sequencing (TCR-α-NGS) to monitor changes in the repertoire of Treg upon polyclonal expansion and after subsequent adoptive transfer. We applied TCR-α-NGS to samples from two patients with chronic GVHD who received comparable doses of stem cell donor derived expanded Treg . We found that in-vitro polyclonal expansion led to notable repertoire changes in vitro and that Treg cell therapy altered the peripheral Treg repertoire considerably towards that of the infused cell product, to different degrees, in each patient. Clonal changes in the peripheral blood were transient and correlated well with the clinical parameters. We suggest that T cell clonotype analyses using TCR sequencing should be considered as a means to monitor longevity and fate of adoptively transferred T cells.

Design, synthesis and evaluation of anti-CD123 antibody drug conjugates

Leukemia stem cells (LSCs) account for the development of drug resistance and increased recurrence rate in acute myeloid leukemia (AML) patients. Targeted drug delivery to leukemia stem cells remains a major challenge in AML chemotherapy. Overexpressed interleukin-3 receptor alpha chain, CD123, on the surface of leukemia stem cells was reported to be a potential target in AML treatment. Here, we designed and developed an antibody drug conjugate (CD123-CPT) by integrating anti-CD123 antibody with a chemotherapeutic agent, Camptothecin (CPT), via a disulfide linker. The linker is biodegradable in the presence of Glutathione (GSH, an endogenous component in cells), which leads to release of CPT. Anti-CD123 antibody conjugates showed significant higher cellular uptake in CD123-overexpressed tumor cells. More importantly, CD123-CPT demonstrated potent inhibitory effects on CD123-overexpressed tumor cells. Consequently, these results provide a promising targeted chemotherapeutical strategy for AML treatment.

HIV-1 tat protein recruits CIS to the cytoplasmic tail of CD127 to induce receptor ubiquitination and proteasomal degradation

HIV-1 Tat protein down regulates expression of the IL-7 receptor alpha-chain (CD127) from the surface of CD8 T cells resulting in impaired T cell proliferation and cytolytic capacity. We have previously shown that soluble Tat protein is taken up by CD8 T cells and interacts with the cytoplasmic tail of CD127 to induce receptor degradation. The N-terminal domain of Tat interacts with CD127 while the basic domain directs CD127 to the proteasome. We have also shown that upon IL-7 binding to its receptor, CD127 is phosphorylated resulting in CIS-mediated proteasomal degradation. Here, we show that Tat mimics this process by recruiting CIS to CD127 in the absence of IL-7 and receptor phosphorylation, leading to CD127 ubiquitination and degradation. Tat therefore acts as an adapter to induce cellular responses under conditions where they may not otherwise occur. Thusly, Tat reduces IL-7 signaling and impairs CD8 T cell survival and function.

Regulation of the IL-7 receptor alpha chain gene in primary human CD8 T-cells

Regulation of the IL-7 receptor alpha chain gene in primary human CD8 T-cells

Transcriptional regulation of the IL-7Rα gene by dexamethasone and IL-7 in primary human CD8 T cells.

Interleukin-7 is essential for the development and maintenance of T cells, and the expression of the IL-7 receptor is tightly regulated at every stage of the T cell's lifespan. In mature CD8 T cells, IL-7 plays important roles in cell survival, peripheral homeostasis, and cytolytic function. The IL-7 receptor alpha-chain (CD127) is expressed at high levels on naïve and memory cells, but it is rapidly downregulated upon IL-7 stimulation. In this study, we illustrate the dynamicity of the CD127 promoter and show that it possesses positive as well as negative regulatory sites involved in upregulating and downregulating CD127 expression, respectively. We cloned the CD127 gene promoter and identified key cis-regulatory elements required for CD127 expression in mature resting primary CD8 T cells. The core promoter necessary for efficient basal transcription is contained within the first 262bp upstream of the TATA box. Additional positive regulatory elements are located between -1200 and -2406bp, conferring a further 2- to 4-fold enhancement in gene expression. While transcription of the CD127 gene is increased directly through a glucocorticoid response element located between -2255 and -2269bp upstream of the TATA box, we identified a suppressive region that lies upstream of 1760bp from the TATA box, which is likely involved in the IL-7-mediated suppression of CD127 transcription. Finally, we illustrated IL-7 does not bias alternative splicing of CD127 transcripts in primary human CD8 T cells.

An asthma-associated IL4R variant exacerbates airway inflammation by promoting conversion of regulatory T cells to TH17-like cells.

Mechanisms by which regulatory T (Treg) cells fail to control inflammation in asthma remain poorly understood. We show that a severe asthma-associated polymorphism in the interleukin-4 receptor alpha chain (IL4RA R576) promotes conversion of induced Treg (iTreg) cells towards a T helper 17 (TH17) cell fate. This skewing is mediated by the recruitment by IL-4Rα-R576 of the growth factor receptor-bound protein 2 (GRB2) adaptor protein, which drives IL-17 expression by activating a pathway involving extracellular signal-regulated kinase, IL-6 and STAT3. Treg cell-specific deletion of Il6ra or Rorc, but not Il4 or Il13, prevented exacerbated airway inflammation in Il4raR576 mice. Furthermore, treatment of Il4raR576 mice with a neutralizing anti-IL-6 antibody prevented iTreg cell reprogramming into TH17-like cells and protected against severe airway inflammation. These findings identify a novel mechanism for the development of mixed TH2-TH17 cell inflammation in genetically prone individuals, and point to interventions that stabilize iTreg cells as potentially effective therapeutic strategies.

Single-cell TCRseq: paired recovery of entire T-cell alpha and beta chain transcripts in T-cell receptors from single-cell RNAseq.

Accurate characterization of the repertoire of the T-cell receptor (TCR) alpha and beta chains is critical to understanding adaptive immunity. Such characterization has many applications across such fields as vaccine development and response, clone-tracking in cancer, and immunotherapy. Here we present a new methodology called single-cell TCRseq (scTCRseq) for the identification and assembly of full-length rearranged V(D)J T-cell receptor sequences from paired-end single-cell RNA sequencing reads. The method allows accurate identification of the V(D)J rearrangements for each individual T-cell and has the novel ability to recover paired alpha and beta segments. Source code is available at https://github.com/ElementoLab/scTCRseq.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-016-0335-7) contains supplementary material, which is available to authorized users.

Histone deacetylase 3 regulates the inflammatory gene expression programme of rheumatoid arthritis fibroblast-like synoviocytes

ObjectivesNon-selective histone deacetylase (HDAC) inhibitors (HDACi) have demonstrated anti-inflammatory properties in both in vitro and in vivo models of rheumatoid arthritis (RA). Here, we investigated the potential contribution of specific...