Background: While the human fetal immune system defaults to a program of tolerance, there is concurrent need for protective immunity to meet the antigenic challenges encountered after birth. Activation of T cells in utero is associated with the fetal inflammatory response with broad implications for the health of the fetus and of the pregnancy. However, the characteristics of the fetal effector T cells that contribute to this process are largely unknown. Methods: We analyzed primary human fetal lymphoid and mucosal tissues and performed phenotypic, functional, and transcriptional analysis to identify T cells with pro-inflammatory potential. The frequency and function of fetal-specific effector T cells was assessed in the cord blood of infants with localized and systemic inflammatory pathologies and compared to healthy term controls. Results: We identified a transcriptionally distinct population of CD4+ T cells characterized by expression of the transcription factor Promyelocytic Leukemia Zinc Finger (PLZF). PLZF+ CD4+ T cells were specifically enriched in the fetal intestine, possessed an effector memory phenotype, and rapidly produced pro-inflammatory cytokines. Engagement of the C-type lectin CD161 on these cells inhibited TCR-dependent production of IFNγ in a fetal-specific manner. IFNγ-producing PLZF+ CD4+ T cells were enriched in the cord blood of infants with gastroschisis, a natural model of chronic inflammation originating from the intestine, as well as in preterm birth, suggesting these cells contribute to fetal systemic immune activation. Conclusion: Our work reveals a fetal-specific program of protective immunity whose dysregulation is associated with fetal and neonatal inflammatory pathologies.
Joanna Halkias, Elze Rackaityte, Sara L. Hillman, Dvir Aran, Ventura F. Mendoza, Lucy R. Marshall, Tippi C. MacKenzie, Trevor D. Burt
BACKGROUND. African American (AA) patients have higher cancer mortality rates and shorter survival times compared to European American (EA) patients. Despite a significant focus on socioeconomic factors, recent findings strongly argue the existence of biological factors driving this disparity. Most of these factors have been described in a cancer-type specific context rather than a pan-cancer setting. METHODS. A novel in silico approach based on Gene Set Enrichment Analysis (GSEA) coupled to Transcription Factor enrichment was carried out to identify common biological drivers of pan-cancer racial disparity using The Cancer Genome Atlas (TCGA) dataset. Mitochondrial content in patient tissues was examined using a multi-cancer tissue microarray approach (TMA). RESULTS. Mitochondrial oxidative phosphorylation was uniquely enriched in AA tumors compared to EA tumors across various cancer types. AA tumors also showed strong enrichment for the ERR1-PGC1α-mediated transcriptional program, which has been implicated in mitochondrial biogenesis. TMA analysis revealed that AA cancers harbor significantly more mitochondria compared to their EA counterparts. CONCLUSIONS. These findings highlight changes in mitochondria as a common distinguishing feature between AA and EA tumors in a pan-cancer setting, and provide the rationale for the repurposing of mitochondrial inhibitors to treat AA cancers.
Danthasinghe Waduge Badrajee Piyarathna, Akhila Balasubramanian, James M. Arnold, Stacy M. Lloyd, Balasubramanyam Karanam, Patricia Castro, Michael M. Ittmann, Nagireddy Putluri, Nora Navone, Jeffrey A. Jones, Wendong Yu, Vlad C. Sandulache, Andrew G. Sikora, George Michailidis, Arun Sreekumar
Background: Chimeric antigen receptor (CAR) T cells are a promising therapy for hematologic malignancies. B-cell maturation antigen (BCMA) is a rational target in multiple myeloma (MM). Methods: We conducted a phase I study of autologous T cells lentivirally-transduced with a fully-human, BCMA-specific CAR containing CD3ζ and 4-1BB signaling domains (CART-BCMA), in subjects with relapsed/refractory MM. Twenty-five subjects were treated in 3 cohorts: 1) 1-5 x 108 CART-BCMA cells alone; 2) Cyclophosphamide (Cy) 1.5 g/m2 + 1-5 x 107 CART-BCMA cells; and 3) Cy 1.5 g/m2 + 1-5 x 108 CART-BCMA cells. No pre-specified BCMA expression level was required. Results: CART-BCMA cells were manufactured and expanded in all subjects. Toxicities included cytokine release syndrome and neurotoxicity, which were grade 3-4 in 8 (32%) and 3 (12%) subjects, respectively, and reversible. One subject died at day 24 from candidemia and progressive myeloma, following treatment for severe CRS and encephalopathy. Responses (based on treated subjects) were seen in 4/9 (44%) in cohort 1, 1/5 (20%) in cohort 2, and 7/11 (64%) in cohort 3, including 5 partial, 5 very good partial, and 2 complete responses, 3 of which were ongoing at 11, 14, and 32 months. Decreased BCMA expression on residual MM cells was noted in responders; expression increased at progression in most. Responses and CART-BCMA expansion were associated with CD4:CD8 T cell ratio and frequency of CD45RO-CD27+CD8+ T cells in the pre-manufacturing leukapheresis product. Conclusion: CART-BCMA infusions with or without lymphodepleting chemotherapy are clinically active in heavily-pretreated MM patients. Trial Registration: NCT02546167. Funding: University of Pennsylvania-Novartis Alliance and NIH.
Adam D. Cohen, Alfred L. Garfall, Edward A. Stadtmauer, J. Joseph Melenhorst, Simon F. Lacey, Eric Lancaster, Dan T. Vogl, Brendan M. Weiss, Karen Dengel, Annemarie Nelson, Gabriela Plesa, Fang Chen, Megan M. Davis, Wei-Ting Hwang, Regina M. Young, Jennifer L. Brogdon, Randi Isaacs, Iulian Pruteanu-Malinici, Don L. Siegel, Bruce L. Levine, Carl H. June, Michael C. Milone
BACKGROUND. Chimeric antigen receptor (CAR) T cells can induce remission in highly refractory leukemia and lymphoma subjects, yet the parameters for achieving sustained relapse-free survival are not fully delineated. METHODS. We analyzed 43 pediatric and young adult subjects participating in a Phase I trial of defined composition CD19CAR T cells (NCT02028455). CAR T cell phenotype, function and expansion, as well as starting material T cell repertoire, were analyzed in relation to therapeutic outcome (defined as achieving complete remission within 63 days) and duration of leukemia free survival and B cell aplasia. RESULTS. These analyses reveal that initial therapeutic failures (n = 5) were associated with attenuated CAR T cell expansion and/or rapid attrition of functional CAR effector cells following adoptive transfer. The CAR T products were similar in phenotype and function when compared to products resulting in sustained remissions. However, the initial apheresed peripheral blood T cells could be distinguished by an increased frequency of LAG-3+/TNF-αlow CD8 T cells and, following adoptive transfer, the rapid expression of exhaustion markers. For the 38 subjects who achieved an initial sustained MRD-neg remission, remission durability correlated with therapeutic products having increased frequencies of TNF-α-secreting CAR CD8+ T cells, and was dependent on a sufficiently high CD19+ antigen load at time of infusion to trigger CAR T cell proliferation. CONCLUSION. These parameters have the potential to prospectively identify patients at risk for therapeutic failure and support the development of approaches to boost CAR T cell activation and proliferation in patients with low levels of CD19 antigen. TRIAL REGISTRATION. ClinicalTrials.gov NCT02028455. FUNDING. Partial funding for this study was provided by Stand Up to Cancer & St. Baldrick’s Pediatric Dream Team Translational Research Grant (SU2C-AACR-DT1113), RO1 CA136551-05, Alex Lemonade Stand Phase I/II Infrastructure Grant, Conquer Cancer Foundation Career Development Award, Washington State Life Sciences Discovery Fund, Ben Towne Foundation, William Lawrence & Blanche Hughes Foundation, and Juno Therapeutics, Inc., a Celgene Company.
Olivia C. Finney, Hannah M. Brakke, Stephanie Rawlings-Rhea, Roxana Hicks, Danielle Doolittle, Marisa Lopez, Robert B. Futrell, Rimas J. Orentas, Daniel Li, Rebecca A. Gardner, Michael C. Jensen
Background: Systems vaccinology allows cutting-edge analysis of innate biomarkers of vaccine efficacy. We have been exploring novel strategies to shape the adaptive immune response, by targeting innate immune cells through novel immunization routes. Methods: This randomized phase I/II clinical study (n=60 healthy subjects aged 18-45 years old) used transcriptomic analysis to discover early biomarkers of immune response quality after transcutaneous (t.c.), intradermal (i.d.), and intramuscular (i.m.) administration of a trivalent influenza vaccine (TIV season 2012-2013) (1:1:1 ratio). Safety and immunogenicity (hemagglutinin inhibition (HI), microneutralization (MN) antibodies and CD4, CD8 effector T cells) were measured at baseline Day (D)0 and at D21. Blood transcriptome was analyzed at D0 and D1. Results: TIV-specific CD8+GranzymeB+(GRZ) T cells appeared in more individuals immunized by the t.c. and i.d. routes, while immunization by the i.d. and i.m. routes prompted high levels of HI antibody titers and MN against A/H1N1 and A/H3N2 influenza viral strains. The early innate gene signature anticipated immunological outcome by discriminating two clusters of individuals with either distinct humoral or CD8 cytotoxic responses. Several pathways explained this dichotomy confirmed by nine genes and serum level of CXCL10 were correlated with either TIV-specific cytotoxic CD8+GRZ+ T-cell or antibody responses. A logistic regression analysis demonstrated that these nine genes and serum levels of CXCL10 (D1/D0) best foreseen TIV-specific CD8+GRZ+ T-cell and antibody responses at D21. Conclusion: This study provides new insight into the impact of immunization routes and innate signature in the quality of adaptive immune responses.
Eléna Gonçalves, Olivia Bonduelle, Angèle Soria, Pierre Loulergue, Alexandra Rousseau, Marine Cachanado, Henri Bonnabau, Rodolphe Thiebaut, Nicolas Tchitchek, Sylvie Behillil, Sylvie van der Werf, Annika Vogt, Tabassome Simon, Odile Launay, Behazine Combadière
BACKGROUND. Recent genomic and bioinformatic technological advances have made it possible to dissect the immune response to personalized neoantigens encoded by tumor-specific mutations. However, timely and efficient identification of neoantigens is still one of the major obstacles to using personalized neoantigen-based cancer immunotherapy. METHODS. Two different pipelines of neoantigens identification were established in this study: (1) Clinical grade targeted sequencing was performed in patients with refractory solid tumor, and mutant peptides with high variant allele frequency and predicted high HLA-binding affinity were de novo synthesized. (2) An inventory-shared neoantigen peptide library of common solid tumors was constructed, and patients' hotspot mutations were matched to the neoantigen peptide library. The candidate neoepitopes were identified by recalling memory T-cell responses in vitro. Subsequently, neoantigen-loaded dendritic cell vaccines and neoantigen-reactive T cells were generated for personalized immunotherapy in six patients. RESULTS. Immunogenic neo-epitopes were recognized by autologous T cells in 3 of 4 patients who utilized the de novo synthesis mode and in 6 of 13 patients who performed shared neoantigen peptide library, respectively. A metastatic thymoma patient achieved a complete and durable response beyond 29 months after treatment. Immune-related partial response was observed in another patient with metastatic pancreatic cancer. The remaining four patients achieved the prolonged stabilization of disease with a median PFS of 8.6 months. CONCLUSIONS. The current study provided feasible pipelines for neoantigen identification. Implementing these strategies to individually tailor neoantigens could facilitate the neoantigen-based translational immunotherapy research. TRIAL REGSITRATION. ChiCTR.org ChiCTR-OIC-16010092, ChiCTR-OIC-17011275, ChiCTR-OIC-17011913; ClinicalTrials.gov NCT03171220. FUNDING. This work was funded by grants from the National Key Research and Development Program of China (Grant No. 2017YFC1308900), the National Major Projects for “Major New Drugs Innovation and Development” (Grant No.2018ZX09301048-003), the National Natural Science Foundation of China (Grant No. 81672367, 81572329, 81572601), and the Key Research and Development Program of Jiangsu Province (No. BE2017607).
Fangjun Chen, Zhengyun Zou, Juan Du, Shu Su, Jie Shao, Fanyan Meng, Ju Yang, Qiuping Xu, Naiqing Ding, Yang Yang, Qin Liu, Qin Wang, Zhichen Sun, Shujuan Zhou, Shiyao Du, Jia Wei, Baorui Liu
BACKGROUND. The human bone marrow (BM) niche contains a population of mesenchymal stromal cells (MSCs) that provide physical support and regulate hematopoietic stem cell (HSC) homeostasis. β-Thalassemia (BT) is a hereditary disorder characterized by altered hemoglobin beta-chain synthesis amenable to allogeneic HSC transplantation and HSC gene therapy. Iron overload (IO) is a common complication in BT patients affecting several organs. However, data on the BM stromal compartment are scarce. METHODS. MSCs were isolated and characterized from BM aspirates of healthy donors (HDs) and BT patients. The state of IO was assessed and correlated with the presence of primitive MSCs in vitro and in vivo. Hematopoietic supportive capacity of MSCs was evaluated by transwell migration assay and 2D coculture of MSCs with human CD34+ HSCs. In vivo, the ability of MSCs to facilitate HSC engraftment was tested in a xenogenic transplant model, whereas the capacity to sustain human hematopoiesis was evaluated in humanized ossicle models. RESULTS. We report that, despite iron chelation, BT BM contains high levels of iron and ferritin, indicative of iron accumulation in the BM niche. We found a pauperization of the most primitive MSC pool caused by increased ROS production in vitro which impaired MSC stemness properties. We confirmed a reduced frequency of primitive MSCs in vivo in BT patients. We also discovered a weakened antioxidative response and diminished expression of BM niche–associated genes in BT-MSCs. This caused a functional impairment in MSC hematopoietic supportive capacity in vitro and in cotransplantation models. In addition, BT-MSCs failed to form a proper BM niche in humanized ossicle models. CONCLUSION. Our results suggest an impairment in the mesenchymal compartment of BT BM niche and highlight the need for novel strategies to target the niche to reduce IO and oxidative stress before transplantation. FUNDING. This work was supported by the SR-TIGET Core grant from Fondazione Telethon and by Ricerca Corrente.
Stefania Crippa, Valeria Rossella, Annamaria Aprile, Laura Silvestri, Silvia Rivis, Samantha Scaramuzza, Stefania Pirroni, Maria Antonietta Avanzini, Luca Basso-Ricci, Raisa Jofra Hernandez, Marco Zecca, Sarah Marktel, Fabio Ciceri, Alessandro Aiuti, Giuliana Ferrari, Maria Ester Bernardo
BACKGROUND. Cryptococcal meningitis (CM) causes an estimated 180,000 deaths annually, predominantly in sub-Saharan Africa, where most patients receive fluconazole (FLC) monotherapy. While relapse after FLC monotherapy with resistant strains is frequently observed, the mechanisms and impact of emergence of FLC resistance in human CM are poorly understood. Heteroresistance (HetR) — a resistant subpopulation within a susceptible strain — is a recently described phenomenon in Cryptococcus neoformans (Cn) and Cryptococcus gattii (Cg), the significance of which has not previously been studied in humans. METHODS. A cohort of 20 patients with HIV-associated CM in Tanzania was prospectively observed during therapy with either FLC monotherapy or in combination with flucytosine (5FC). Total and resistant subpopulations of Cryptococcus spp. were quantified directly from patient cerebrospinal fluid (CSF). Stored isolates underwent whole genome sequencing and phenotypic characterization. RESULTS. Heteroresistance was detectable in Cryptococcus spp. in the CSF of all patients at baseline (i.e., prior to initiation of therapy). During FLC monotherapy, the proportion of resistant colonies in the CSF increased during the first 2 weeks of treatment. In contrast, no resistant subpopulation was detectable in CSF by day 14 in those receiving a combination of FLC and 5FC. Genomic analysis revealed high rates of aneuploidy in heteroresistant colonies as well as in relapse isolates, with chromosome 1 (Chr1) disomy predominating. This is apparently due to the presence on Chr1 of ERG11, which is the FLC drug target, and AFR1, which encodes a drug efflux pump. In vitro efflux levels positively correlated with the level of heteroresistance. CONCLUSION. Our findings demonstrate for what we believe is the first time the presence and emergence of aneuploidy-driven FLC heteroresistance in human CM, association of efflux levels with heteroresistance, and the successful suppression of heteroresistance with 5FC/FLC combination therapy. FUNDING. This work was supported by the Wellcome Trust Strategic Award for Medical Mycology and Fungal Immunology 097377/Z/11/Z and the Daniel Turnberg Travel Fellowship.
Neil R.H. Stone, Johanna Rhodes, Matthew C. Fisher, Sayoki Mfinanga, Sokoine Kivuyo, Joan Rugemalila, Ella Shtifman Segal, Leor Needleman, Síle F. Molloy, June Kwon-Chung, Thomas S. Harrison, William Hope, Judith Berman, Tihana Bicanic
Background/Purpose: Plasmacytoid dendritic cells (pDC) produce large amounts of type I IFN (IFN-I), cytokines convincingly linked to systemic lupus erythematosus (SLE) pathogenesis. BIIB059 is a humanized mAb that binds BDCA2, a pDC-specific receptor that inhibits the production of IFN-I and other inflammatory mediators when ligated. A first-in-human study was conducted to assess safety, tolerability, pharmacokinetic (PK) and pharmacodynamic (PD) effects of single BIIB059 doses in healthy volunteers (HV) and patients with SLE with active cutaneous disease as well as proof of biological activity and preliminary clinical response in the SLE cohort. Methods: A randomized, double-blind, placebo-controlled, clinical trial was conducted in HV (n=54) and patients with SLE (n=12). All subjects were monitored for adverse events. Serum BIIB059 concentrations, BDCA2 levels on pDCs, and IFN-responsive biomarkers in whole blood and skin biopsies were measured. Skin disease activity was determined using the Cutaneous Lupus Erythematosus Disease Area and Severity Index Activity (CLASI-A).Results: Single doses of BIIB059 were associated with a favorable safety and PK profile. BIIB059 administration led to BDCA2 internalization on pDCs, which correlated with circulating BIIB059 levels. BIIB059 administration in patients with SLE decreased expression of IFN response genes in blood, normalized MxA expression and reduced immune infiltrates in skin lesions, and decreased CLASI-A score. Conclusion: Single doses of BIIB059 were associated with favorable safety and PK/PD profiles, and robust target engagement and biological activity, supporting further development of BIIB059 in SLE. The data suggest that targeting pDCs may be beneficial for patients with SLE, especially those with cutaneous manifestations.
Richard Furie, Victoria P. Werth, Joseph F. Merola, Lauren Stevenson, Taylor L. Reynolds, Himanshu Naik, Wenting Wang, Romy Christmann, Agnes Gardet, Alex Pellerin, Stefan Hamann, Pavan Auluck, Catherine Barbey, Parul Gulati, Dania Rabah, Nathalie Franchimont
Background. Sphingolipids are important components of cellular membranes and functionally associated with fundamental processes such as cell differentiation, neuronal signaling and myelin sheath formation. Defects in the synthesis or degradation of sphingolipids leads to various neurological pathologies, however, the entire spectrum of sphingolipid metabolism disorders remained elusive. Methods. A combined approach of genomics and lipidomics was applied to identify and characterize a human sphingolipid metabolism disorder.Results. By whole-exome sequencing in a patient with a multisystem neurological disorder of both the central and peripheral nervous system, we identified a homozygous p.(Ala280Val) variant in DEGS1, which catalyzes the last step in the ceramide synthesis pathway. The blood sphingolipid profile in the patient showed a significant increase in dihydro sphingolipid species which was further recapitulated in patient-derived fibroblasts, in CRISPR/Cas9-derived DEGS1 knockout cells, and by pharmacological inhibition of DEGS1. The enzymatic activity in patient fibroblasts was reduced by 80% compared to wild type cells which was in line with a reduced expression of mutant DEGS1 protein. Moreover, an atypical and potentially neurotoxic sphingosine isomer was identified in patient plasma and in cells expressing mutant DEGS1. Conclusion. We report DEGS1 dysfunction as cause for a novel sphingolipid disorder with hypomyelination and degeneration of both the central and peripheral nervous system.Trial registration. Not applicable.Funding. RESOLVE: Project number 305707; SNF: Project 31003A_153390/1; Rare Disease Initiative Zurich.
Gergely Karsai, Florian Kraft, Natja Haag, G. Christoph Korenke, Benjamin Hänisch, Alaa Othman, Saranya Suriyanarayanan, Regula Steiner, Cordula Knopp, Michael Mull, Markus Bergmann, J. Michael Schröder, Joachim Weis, Miriam Elbracht, Matthias Begemann, Thorsten Hornemann, Ingo Kurth
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