Cell-based gene therapies offer unprecedented promise for medicine. One day, we will be able to cure degenerative diseases that consist of the loss of specialized cell functions, as well as decelerate and perhaps even revert the deterioration our bodies experience during aging. However, several hurdles must be surpassed in order to establish cell-based therapies, a major one being rejection by the recipient's immune system. This challenge is particularly prominent for autoimmune disorders, such as type 1 diabetes and multiple sclerosis, where the immune system destroys self-tissues. In my Ph.D. thesis work at Harvard University, I focused on understanding immune tolerance using pregnancy as a model. I was also the first to show successful genome editing in primary human T cells and hematopoietic stem cells using CRISPR/Cas9 and was part of a team creating universally compatible human pluripotent stem cells. As an NIH NIDDK HIRN Emerging Leader in Type 1 Diabetes at the University of California San Francisco, I worked on next-generation regulatory T cell therapies for type 1 diabetes and organ transplant rejection. Currently, I am an Assistant Professor of Microbiology and Immunology and, by courtesy, of Regenerative Medicine and Cell Biology at the Medical University of South Carolina and the Hollings Cancer Center. My goal is to design and develop engineered immune cell therapies for autoimmune disease, cancer, and aging. I envision that we will be able to use our capacity to engineer the human genome with high precision and efficiency to manipulate our immune system, enabling us to treat several devastating illnesses now labeled as incurable. I am highly committed to make that vision become a reality.
Reinterpretation of cuisines from around the world. Locally sourced. Only ingredients you can pronounce.
Poulet au riz sauvage, carottes arc-en-ciel et brocoli
Oeufs ensoleillés avec bacon, carottes et riz au fromage
Oeufs pochés au chou-fleur, brocoli, tomates et camembert
Poulet aux pâtes, tomates roma et avocat
Saumon avec riz brun, carottes, brocoli et tomates
Saucisses aux pâtes, carottes et chou frisé
Poulet aux spaghettis, œufs durs et bok choy
Mandrin de boeuf avec macaroni, carottes, brocoli et tomates
Seminars and talk shows
One of the most powerful tools at a scientist's disposal: the seminar!
Reprogramming human regulatory T cell specificity with a novel anti-HLA-A2 chimeric antigen receptor
COVID-19: an Immunologist's Perspective
October 7, 2020
HOW TO SOLVE THE AGING PROBLEM with Leonardo Ferreira, Harvard PhD & UCSF Stem Cells Researcher
July 2, 2020
Terapias celulares para el rechazo de transplantes
June 16, 2020
Modificación genética de células humanas, terapia para el futuro
Genetic Modification of Human Cells: Designing Medicine for the 21st Century
Dr. Leonardo Ferreira - Immunodietica
Designing Next-Generation Regulatory T Cell Therapies with Single-Cell Proteomics
Ciência aos Domingos (Radio University of Coimbra): Immunodietica
Bioquímicos pelo Mundo: Leonardo Ferreira
Cognitum S2 E1: Immunodietica
Jornal 2 ep. 195 - Leonardo Ferreira
Designing cells as living drugs for autoimmune disease and organ transplant rejection
Newton News: Immunodietica
Cognitum S1 E4: regulatory T cells (Tregs) and type 1 diabetes (T1D)
Engineering designer cell therapies for immune diseases
Cognitum S1 E3: CRISPR and HIV
A Ciência nos Estados Unidos também se faz em Português
Leonardo Ferreira at the American Association of Immunologists (AAI) StoryBooth 2018
The future is here: cell therapies for complex diseases
Reescrevendo o nosso DNA com CRISPR/Cas9
How to cure type 1 diabetes using immunology
Designing the Next Generation of Chimeric Antigen Receptors for Regulatory T Cell Therapy
HLA-G and maternal-fetal tolerance
Transcriptional control of maternal-fetal immune tolerance
Long-range chromatin interactions control trophoblast-restricted HLA-G expression during pregnancy
Caminhos da Investigação: Leonardo Ferreira
The emerging role of regulatory cell-based therapy in autoimmune disease
December 14, 2022
Autoimmune disease, caused by unwanted immune responses to self-antigens, affects millions of people each year and poses a great social and economic burden to individuals and communities. In the course of autoimmune disorders, including rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes mellitus, and multiple sclerosis, disturbances in the balance between the immune response against harmful agents and tolerance towards self-antigens lead to an immune response against self-tissues. In recent years, various regulatory immune cells have been identified. Disruptions in the quality, quantity, and function of these cells have been implicated in autoimmune disease development. Therefore, targeting or engineering these cells is a promising therapeutic for different autoimmune diseases. Regulatory T cells, regulatory B cells, regulatory dendritic cells, myeloid suppressor cells, and some subsets of innate lymphoid cells are arising as important players among this class of cells. Here, we review the roles of each suppressive cell type in the immune system during homeostasis and in the development of autoimmunity. Moreover, we discuss the current and future therapeutic potential of each one of these cell types for autoimmune diseases.
Regulatory T cell homeostasis: Requisite signals and implications for clinical development of biologics
December 5, 2022
Novel biologics are currently being tested in clinical trials for the treatment of autoimmune diseases and the prevention of transplant allograft rejection. Their premise is to deliver highly efficient immunosuppression while minimizing side-effects, as they specifically target inflammatory mediators involved in the dysregulation of the immune system. However, the pleiotropism of soluble mediators and cell-to-cell interactions with potential to exert both proinflammatory and regulatory influences on the outcome of the immune response can lead to unpredictable results. Predicting responses to biologic drugs requires mechanistic understanding of the cell type-specific effect of immune mediators. Elucidation of the central role of regulatory T cells (Treg), a small subset of T cells dedicated to immune homeostasis, in preventing the development of auto- and allo-immunity has provided a deeper understanding of the signaling pathways that govern immune tolerance. This review focuses on the requisite signals that promote Treg homeostasis and discusses the anticipated outcomes of biologics targeting these signals. Our goal is to inform and facilitate the design of cell-specific biologics that thwart T effector cells (Teff) while promoting Treg function for the treatment of autoimmune diseases and the prevention of transplant rejection.
The dichotomous roles of TNFa signaling in CD4+ T cells
November 16, 2022
TNFa blocking agents were the first-in-class biologic drugs used for the treatment of autoimmune disease. Paradoxically, however, exacerbation of autoimmunity was observed in some patients. TNFa is a pleiotropic cytokine that has both proinflammatory and regulatory effects on CD4+ T cells and can influence the adaptive immune response against autoantigens. Here, we critically appraise the literature and discuss the intricacies of TNFa signaling that may explain the controversial findings of previous studies. The pleiotropism of TNFa is based in part on the existence of two biologically active forms of TNFa, soluble and membrane-bound, with different affinities for two distinct TNF receptors, TNFR1 and TNFR2, leading to activation of diverse downstream molecular pathways involved in cell fate decisions and immune function. Distinct membrane expression patterns of TNF receptors by CD4+ T cell subsets and their preferential binding of distinct forms of TNFα produced by a diverse pool of cellular sources during different stages of an immune response are important determinants of the differential outcomes of TNFa-TNF receptor signaling. Targeted manipulation of TNFa-TNF receptor signaling on select CD4+ T cell subsets may offer specific therapeutic interventions to dampen inflammation while fortifying immune regulation for the treatment of autoimmune diseases.
Modeling human T1D-associated autoimmune processes
December 10, 2021
Type 1 diabetes (T1D) is an autoimmune disease characterized by impaired immune tolerance to β-cell antigens and progressive destruction of insulin-producing β-cells. Animal models have provided valuable insights for understanding the etiology and pathogenesis of this disease, but they fall short of reflecting the extensive heterogeneity of the disease in humans, which is contributed by various combinations of risk gene alleles and unique environmental factors. Collectively, these factors have been used to define subgroups of patients, termed endotypes, with distinct predominating disease characteristics. Here, we review the gaps filled by these models in understanding the intricate involvement and regulation of the immune system in human T1D pathogenesis. We describe the various models developed so far and the scientific questions that have been addressed using them. Finally, we discuss the limitations of these models, primarily ascribed to hosting a human immune system (HIS) in a xenogeneic recipient, and what remains to be done to improve their physiological relevance. To understand the role of genetic and environmental factors or evaluate immune-modifying therapies in humans, it is critical to develop and apply models in which human cells can be manipulated and their functions studied under conditions that recapitulate as closely as possible the physiological conditions of the human body. While microphysiological systems and living tissue slices provide some of these conditions, HIS mice enable more extensive analyses using in vivo systems.
IL-6 and TNFα Drive Extensive Proliferation of Human Tregs Without Compromising Their Lineage Stability or Function
December 23, 2021
Treg therapies are being tested in clinical trials in transplantation and autoimmune diseases, however, the impact of inflammation on Tregs remains controversial. We challenged human Tregs ex-vivo with pro-inflammatory cytokines IL-6 and TNFα and observed greatly enhanced proliferation stimulated by anti-CD3 and anti-CD28 (aCD3/28) beads or CD28 superagonist (CD28SA). The cytokine-exposed Tregs maintained high expression of FOXP3 and HELIOS, demethylated FOXP3 enhancer, and low IFNγ, IL-4, and IL-17 secretion. Blocking TNF receptor using etanercept or deletion of TNF receptor 2 using CRISPR/Cas9 blunted Treg proliferation and attenuated FOXP3 and HELIOS expression. These results prompted us to consider using CD28SA together with IL-6 and TNFα without aCD3/28 beads (beadless) as an alternative protocol for therapeutic Treg manufacturing. Metabolomics profiling revealed more active glycolysis and oxidative phosphorylation, increased energy production, and higher antioxidant potential during beadless Treg expansion. Finally, beadless expanded Tregs maintained suppressive functions in vitro and in vivo. These results demonstrate that human Tregs positively respond to proinflammatory cytokines with enhanced proliferation without compromising their lineage identity or function. This property can be harnessed for therapeutic Treg manufacturing.
CAR T-Cell Therapy: Is CD28-CAR Heterodimerization Its Achilles’ Heel?
November 17, 2021
Chimeric antigen receptor (CAR) T-cell therapy has dramatically expanded the success rate of cancer immunotherapy, especially in CD19-expressing blood cancers. Yet, it has also given rise to new complications, notably cytokine release syndrome, neurotoxicity, and, sometimes, fatal cerebral edema. The exact mechanisms of such toxicities across different CD19 CAR T-cell products, however, remain hotly debated. It was recently demonstrated that CARs containing a CD28 transmembrane domain (TMD) can heterodimerize with the endogenous CD28 receptor. Here, we hypothesize that, upon on-target activation, this heterodimerization is responsible for the increased sensitivity of CD19 CAR to CD19low brain mural cells, resulting in increased risk of developing severe neurotoxicity. This hypothesis may only be confirmed with a clinical trial comparing two CD19-CD28-TMD CARs differing only by targeted amino-acid mutations in the CD28 transmembrane domain.
Precision engineering of an anti-HLA-A2 chimeric antigen receptor in regulatory T cells for transplant immune tolerance
September 20, 2021
Infusion of regulatory T cells (Tregs) engineered with a chimeric antigen receptor (CAR) targeting donor-derived human leukocyte antigen (HLA) is a promising strategy to promote transplant tolerance. Here, we describe an anti-HLA-A2 CAR (A2-CAR) generated by grafting the complementarity-determining regions (CDRs) of a human monoclonal anti-HLA-A2 antibody into the framework regions of the Herceptin 4D5 single-chain variable fragment and fusing it with a CD28-ζ signaling domain. The CDR-grafted A2-CAR maintained the specificity of the original antibody. We then generated HLA-A2 mono-specific human CAR Tregs either by deleting the endogenous T-cell receptor (TCR) via CRISPR/Cas9 and introducing the A2-CAR using lentiviral transduction or by directly integrating the CAR construct into the TCR alpha constant locus using homology-directed repair. These A2-CAR+TCRdeficient human Tregs maintained both Treg phenotype and function in vitro. Moreover, they selectively accumulated in HLA-A2-expressing islets transplanted from either HLA-A2 transgenic mice or deceased human donors. A2-CAR+TCRdeficient Tregs did not impair the function of these HLA-A2+ islets, whereas similarly engineered A2-CAR+TCRdeficientCD4+ conventional T cells rejected the islets in less than 2 weeks. A2-CAR+TCRdeficient Tregs delayed graft-versus-host disease only in the presence of HLA-A2, expressed either by co-transferred peripheral blood mononuclear cells or by the recipient mice. Altogether, we demonstrate that genome-engineered mono-antigen-specific A2-CAR Tregs localize to HLA-A2-expressing grafts and exhibit antigen-dependent in vivo suppression, independent of TCR expression. These approaches may be applied towards developing precision Treg cell therapies for transplant tolerance.
The CD28-Transmembrane Domain Mediates Chimeric Antigen Receptor Heterodimerization With CD28
March 23, 2021
Anti-CD19 chimeric antigen receptor (CD19-CAR)-engineered T cells are approved therapeutics for malignancies. The impact of the hinge domain (HD) and the transmembrane domain (TMD) between the extracellular antigen-targeting CARs and the intracellular signaling modalities of CARs has not been systemically studied. In this study, a series of 19-CARs differing only by their HD (CD8, CD28, or IgG4) and TMD (CD8 or CD28) was generated. CARs containing a CD28-TMD, but not a CD8-TMD, formed heterodimers with the endogenous CD28 in human T cells, as shown by co-immunoprecipitation and CAR-dependent proliferation of anti-CD28 stimulation. This dimerization was dependent on polar amino acids in the CD28-TMD and was more efficient with CARs containing CD28 or CD8 HD than IgG4-HD. The CD28-CAR heterodimers did not respond to CD80 and CD86 stimulation but had a significantly reduced CD28 cell-surface expression. These data unveiled a fundamental difference between CD28-TMD and CD8-TMD and indicated that CD28-TMD can modulate CAR T-cell activities by engaging endogenous partners.
Immunodietica: interrogating the role of diet in autoimmune disease
August 18, 2020
Diet is an environmental factor in autoimmune disorders, where the immune system erroneously destroys one’s own tissues. Yet, interactions between diet and autoimmunity remain largely unexplored, particularly the impact of immunogenetics, one’s human leukocyte antigen (HLA) allele make-up, in this interplay. Here, we interrogated animals and plants for the presence of epitopes implicated in human autoimmune diseases. We mapped autoimmune epitope distribution across organisms and determined their tissue expression pattern. Interestingly, diet-derived epitopes implicated in a disease were more likely to bind to HLA alleles associated with that disease than to protective alleles, with visible differences between organisms with similar autoimmune epitope content. We then analyzed an individual’s HLA haplotype, generating a personalized heatmap of potential dietary autoimmune triggers. Our work uncovered differences in autoimmunogenic potential across food sources and revealed differential binding of diet-derived epitopes to autoimmune disease-associated HLA alleles, shedding light on the impact of diet on autoimmunity.
Plasma-based COVID-19 treatments in low- and middle-income nations pose a high risk of an HIV epidemic
July 6, 2020
Convalescent plasma therapy holds promise as a transient treatment for COVID-19. Yet, blood products are important sources of HIV infection in low- and middle-income nations. Great care must be taken to prevent plasma therapy from fueling HIV epidemics in the developing world.
Intermediary metabolism: An intricate network at the crossroads of cell fate and function
June 26, 2020
Intermediary metabolism is traditionally viewed as the large, highly integrated network of reactions that provides cells with metabolic energy, reducing power and biosynthetic intermediates. The elucidation of its major pathways and molecular mechanisms of energy transduction occupied some of the brightest scientific minds for almost two centuries. When these goals were achieved, a sense that intermediary metabolism was mostly a solved problem pervaded the broader biochemical community, and the field lost its vitality. However, intermediary metabolism has recently been re-energized by several paradigm-shifting discoveries that challenged its perception as a self-contained system and re-positioned it at the crossroads of all aspects of cell function, from cell growth, proliferation and death to epigenetics and immunity. Emphasis is now increasingly placed on the involvement of metabolic dysfunction in human disease. In this review, we will navigate from the dawn of intermediary metabolism research to present day work on this ever-expanding field.
GPR146 Deficiency Protects against Hypercholesterolemia and Atherosclerosis
November 27, 2019
Although human genetic studies have implicated many susceptible genes associated with plasma lipid levels, their physiological and molecular functions are not fully characterized. Here we demonstrate that orphan G protein-coupled receptor 146 (GPR146) promotes activity of hepatic sterol regulatory element binding protein 2 (SREBP2) through activation of the extracellular signal-regulated kinase (ERK) signaling pathway, thereby regulating hepatic very low-density lipoprotein (VLDL) secretion, and subsequently circulating low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG) levels. Remarkably, GPR146 deficiency reduces plasma cholesterol levels substantially in both wild-type and LDL receptor (LDLR)-deficient mice. Finally, aortic atherosclerotic lesions are reduced by 90% and 70%, respectively, in male and female LDLR-deficient mice upon GPR146 depletion. Taken together, these findings outline a regulatory role for the GPR146/ERK axis in systemic cholesterol metabolism and suggest that GPR146 inhibition could be an effective strategy to reduce plasma cholesterol levels and atherosclerosis.
HSP90: A Key Player in Metal-Induced Carcinogenesis?
November 5, 2019
Three extensively used metals, cadmium, chromium and nickel, are established human carcinogens. The elucidation of the molecular and cellular mechanisms underlying the carcinogenicity of these metals has centered mostly on the signalling pathways that regulate cellular growth, differentiation and death. Unfortunately, our understanding of the involvement of these pathways in metal-induced carcinogenesis is still very incomplete. More recently, research has extended to include the impact of these metals on mechanisms not traditionally associated with cancer, but that are now increasingly viewed as playing a critical role in carcinogenesis. Among them is the stress response, a highly conserved mechanism employed by all cells for protection against protein damage. Indeed, all three metals induce proteotoxic stress, which warrants following this line of research. The present chapter will critically review published studies on the impact of carcinogenic metals on the expression of the heat shock protein 90 family (HSP90), one of the protein families that mediate the stress response. HSP90 has been consistently found to be overexpressed in many types of cancer and, significantly, HSP90 overexpression has been correlated with increased tumor growth, metastatic potential and resistance to chemotherapy.
Universal donor stem cells and related methods
October 10, 2019
Disclosed herein are universal donor stem cells and related methods of their use and production. The universal donor stem cells disclosed herein are useful for overcoming the immune rejection in cell-based transplantation therapies. In certain embodiments, the universal donor stem cells disclosed herein do not express one or more MHC-I and MHC-II human leukocyte antigens. Similarly, in certain embodiments, the universal donor stem cells disclosed herein do not express one or more human leukocyte antigens (e.g., HLA-A, HLA-B and/or HLA-C) corresponding to MHC-I and MHC-II human leukocyte antigens, thereby rendering such cells hypoimmunogenic.
Impact of Carcinogenic Chromium on the Cellular Response to Proteotoxic Stress
October 3, 2019
Worldwide, several million workers are employed in the various chromium (Cr) industries. These workers may suffer from a variety of adverse health effects produced by dusts, mists and fumes containing Cr in the hexavalent oxidation state, Cr(VI). Of major importance, occupational exposure to Cr(VI) compounds has been firmly associated with the development of lung cancer. Counterintuitively, Cr(VI) is mostly unreactive towards most biomolecules, including nucleic acids. However, its intracellular reduction produces several species that react extensively with biomolecules. The diversity and chemical versatility of these species add great complexity to the study of the molecular mechanisms underlying Cr(VI) toxicity and carcinogenicity. As a consequence, these mechanisms are still poorly understood, in spite of intensive research efforts. Here, we discuss the impact of Cr(VI) on the stress response—an intricate cellular system against proteotoxic stress which is increasingly viewed as playing a critical role in carcinogenesis. This discussion is preceded by information regarding applications, chemical properties and adverse health effects of Cr(VI). A summary of our current understanding of cancer initiation, promotion and progression is also provided, followed by a brief description of the stress response and its links to cancer and by an overview of potential molecular mechanisms of Cr(VI) carcinogenicity.
regulatory T cell therapy
Sepptember 20, 2019
Regulatory T cells (Treg cells) are a small subset of immune cells that are dedicated to curbing excessive immune activation and maintaining immune homeostasis. Accordingly, deficiencies in Treg cell development or function result in uncontrolled immune responses and tissue destruction and can lead to inflammatory disorders such as graft-versus-host disease, transplant rejection and autoimmune diseases. As Treg cells deploy more than a dozen molecular mechanisms to suppress immune responses, they have potential as multifaceted adaptable smart therapeutics for treating inflammatory disorders. Indeed, early-phase clinical trials of Treg cell therapy have shown feasibility, tolerability and potential efficacy in these disease settings. In the meantime, progress in the development of chimeric antigen receptors and in genome editing (including the application of CRISPR–Cas9) over the past two decades has facilitated the genetic optimization of primary T cell therapy for cancer. These technologies are now being used to enhance the specificity and functionality of Treg cells. In this Review, we describe the key advances and prospects in designing and implementing Treg cell-based therapy in autoimmunity and transplantation.
Cellular signaling domain engineering in chimeric antigen receptor-modified regulatory T cells
September 19, 2019
Chimeric antigen receptor (CAR)-expressing T regulatory cells (Tregs) include intracellular co-stimulatory or inhibitory domains based on the biology, functions and activities of Tregs. The co-stimulatory or inhibitory domains modulate the Treg response, thereby, activating or suppressing an effector T cell (Teff) immune response to specific antigens.
developing nations: Can scientists be effective non-state diplomats?
September 3, 2019
Foreign education efforts by institutions of developed nations often meet resistance abroad, when the need for national sovereignty stands in opposition to collaborative opportunities with outside states. Strict isolationist policies can not only impact diplomatic ties, but also prevent the transfer of technologies, knowledge, and innovation that ultimately drive economic strength. Developing nations are particularly vulnerable to stagnation—both economic and scientific—when divorced from the march of global progress. We believe there is a balance to be struck whereby advances can be shared to mutual benefit on all sides while respecting local contexts, and science education may serve as an effective vehicle for such cross-border connection.
Scientists as non-state
actors of public diplomacy
August 19, 2019
Establishing trust between distant nations requires a level of openness that fosters mutual understandings at individual and population scales. This is especially true in countries separated by degrees of economic development, where feelings of distrust felt by a less developed nation may cast perceptions of imperialistic intents upon the activities of more industrialized nations. Furthermore, between countries that do not enjoy advanced diplomatic relationships, there may appear to be no clear path to realizing cross-border connections in a top-down manner. We present here the argument that, under conditions of suboptimal diplomatic ties between developed and developing governments, there is an opportunity for non-state actors to fill the void and foster nationwide trust. The vehicle, in our case? Science education.
Effective participatory science education in a diverse Latin American population
June 25, 2019
Particular challenges exist for science education in the developing world, where limited resources require curricula designed to balance state-of-the-art knowledge with practical and political considerations in region-specific contexts. Project-based biology teaching is especially difficult to execute due to high infrastructural costs and limited teacher training. Here, we report the results of implementing short, challenging, and low-cost biology courses to high school and college students in Bolivia, designed and taught in collaboration between scientists from developed nations and local science instructors. We find our approach to be effective at transmitting advanced topics in disease modeling, microscopy, genome engineering, neuroscience, microbiology, and regenerative biology. We find that student learning through this approach was not significantly affected by their background, education level, socioeconomic status, or initial interest in the course. Moreover, participants reported a heightened interest in pursuing scientific careers after course completion. These results demonstrate efficacy of participatory learning in a developing nation, and suggest that similar techniques could drive scientific engagement in other developing economies.
Immunodietica: A data-driven approach to investigate interactions between diet and autoimmune disorders
May 19, 2019
Autoimmunity is on the rise around the globe. Diet has been proposed as a risk factor for autoimmunity and shown to modulate the severity of several autoimmune disorders. Yet, the interaction between diet and autoimmunity in humans remains largely unstudied. Here, we systematically interrogated commonly consumed animals and plants for peptide epitopes previously implicated in human autoimmune disease. A total of fourteen species investigated could be divided into three broad categories regarding their content in human autoimmune epitopes, which we represented using a new metric, the Gershteyn-Ferreira index (GF index). Strikingly, pig contains a disproportionately high number of unique autoimmune epitopes compared to all other species analyzed. This work uncovers a potential new link between pork consumption and autoimmunity in humans and lays the foundation for future studies on the impact of diet on the pathogenesis and progression of autoimmune disorders.
Generation of hypoimmunogenic human pluripotent stem cells
May 21, 2019
To enable cell therapy on a broader scale, the development of universal donor stem cell products that can be administered to multiple patients in need has been proposed, yet a strategy controlling both adaptive and innate immune rejection has not been reported. In this study, we employed multiplex genome editing to selectively ablate the highly polymorphic HLA class Ia and class II molecules and introduced the immunoregulatory factors PD-L1, HLA-G, and CD47 to control T cell- and NK cell-mediated immune responses and macrophage engulfment in vitro and in vivo. Our strategy demonstrates the power of cell engineering and informs future studies aiming to generate “off-the-shelf” universal cell products that may make cell therapy available to a larger pool of patients.
Cells lacking B2M surface expression and methods for allogeneic administration of such cells
May 24, 2018
Disclosed herein are cells and populations of cells comprising a genome in which the B2M gene has been edited to eliminate surface expression of MHC Class I protein in the cells or population of cells, and methods for allogeneic administration of such cells to reduce the likelihood that the cells will trigger a host immune response when the cells are administered to a subject in need of such cells.
Hexavalent chromium, a lung carcinogen, confers resistance to thermal stress and interferes with heat shock protein expression in human bronchial epithelial cells
March 16, 2018
Exposure to hexavalent chromium [Cr(VI)], a lung carcinogen, triggers several types of cellular stresses, namely oxidative, genotoxic and proteotoxic stresses. Given the evolutionary character of carcinogenesis, it is tempting to speculate that cells that survive the stresses produced by this carcinogen become more resistant to subsequent stresses, namely those encountered during neoplastic transformation. To test this hypothesis, we determined whether pre-incubation with Cr(VI) increased the resistance of human bronchial epithelial cells (BEAS-2B cells) to the antiproliferative action of acute thermal shock, used here as a model for stress. In line with the proposed hypothesis, it was observed that, at mildly cytotoxic concentrations, Cr(VI) attenuated the antiproliferative effects of both cold and heat shock. Mechanistically, Cr(VI) interfered with the expression of two components of the stress response pathway: heat shock proteins Hsp72 and Hsp90α. Specifically, Cr(VI) significantly depleted the mRNA levels of the former and the protein levels of the latter. Significantly, these two proteins are members of heat shock protein (Hsp) families (Hsp70 and Hsp90, respectively) that have been implicated in carcinogenesis. Thus, our results confirm and extend previous studies showing the capacity of Cr(VI) to interfere with the expression of stress response components.
NLRP2 is a suppressor of NF-kB signaling and HLA-C expression in human trophoblasts
March 7, 2017
During pregnancy, fetal extravillous trophoblasts (EVT) play a key role in the regulation of maternal T cell and NK cell responses. EVT display a unique combination of human leukocyte antigens (HLA); EVT do not express HLA-A and HLA-B, but do express HLA-C, HLA-E, and HLA-G. The mechanisms establishing this unique HLA expression pattern have not been fully elucidated. The major histocompatibility complex (MHC) class I and class II transcriptional activators NLRC5 and CIITA are expressed neither by EVT nor by the EVT model cell line JEG3, which has an MHC expression pattern identical to that of EVT. Therefore, other MHC regulators must be present to control HLA-C, HLA-E, and HLA-G expression in these cells. CIITA and NLRC5 are both members of the nucleotide-binding domain, leucine-rich repeat (NLR) family of proteins. Another member of this family, NLRP2, is highly expressed by EVT and JEG3, but not in maternal decidual stromal cells. In this study, transcription activator-like effector nuclease technology was used to delete NLRP2 in JEG3. Furthermore, lentiviral delivery of shRNA was used to knockdown NLRP2 in JEG3 and primary EVT. Upon NLRP2 deletion, Tumor Necrosis Factor-α (TNFα)-induced phosphorylation of NF-KB p65 increased in JEG3 and EVT, and more surprisingly a significant increase in constitutive HLA-C expression was observed in JEG3. These data suggest a broader role for NLR family members in the regulation of MHC expression during inflammation, thus forming a bridge between innate and adaptive immune responses. As suppressor of proinflammatory responses, NLRP2 may contribute to preventing unwanted antifetal responses.
HLA-G: at the interface of maternal-fetal tolerance
March 6, 2017
During pregnancy, semiallogeneic fetal extravillous trophoblasts (EVT) invade the uterine mucosa without being rejected by the maternal immune system. Several mechanisms were initially proposed by Peter Medawar half a century ago to explain this apparent violation of the laws of transplantation. Then, three decades ago, an unusual human leukocyte antigen (HLA) molecule was identified: HLA-G. Uniquely expressed in EVT, HLA-G has since become the center of the present understanding of fetus-induced immune tolerance. Despite slow progress in the field, the last few years have seen an explosion in our knowledge of HLA-G biology. Here, we critically review new insights into the mechanisms controlling the expression and function of HLA-G at the maternal–fetal interface, and discuss their relevance for fetal tolerance.
Generating antigen-specific regulatory T cells in the fast lane
January 19, 2017
Human regulatory T cells engineered to express a chimeric antigen receptor targeting HLA‐A2 efficiently suppress alloimmune responses in humanized mouse models of transplantation.
Modified T cells and methods of making and using the same
December 1, 2016
Disclosed herein are modified primary human T cells and populations thereof comprising a genome in which the CTLA4, PD1, TCRA, TCRB, and/or B2M genes have been edited to generate an off-the-shelf universal CAR T cell from allogeneic healthy donors that can be administered to any patient while reducing or eliminating the risk of immune rejection or graft versus host disease, and which are not prone to T cell inhibition, and methods for allogeneic administration of such cells to reduce the likelihood that the cells will trigger a host immune response when the cells are administered to a subject in need of such cells.
A distant trophoblast-specific enhancer controls HLA-G expression at the maternal-fetal interface
April 13, 2016
Successful pregnancy poses an immunological paradox, as the mother’s immune system does not reject a fetus, even though it is a partially foreign tissue. Fetal extravillous trophoblasts (EVTs) deeply invade the uterus and interact with maternal immune cells without facing rejection. The nonclassical major histocompatibility complex (MHC) molecule HLA-G is essential for immune tolerance induction in pregnancy, yet the mechanism by which EVTs uniquely express HLA-G remains unknown. Using high-throughput cis-regulatory element dissection and genome editing tools, we discovered a remote enhancer essential for HLA-G expression in human EVTs, describing the basis for its selective expression at the maternal–fetal interface. These findings provide insight into immune tolerance induction during pregnancy and may yield new therapeutic targets for pregnancy-related disorders.
Genome editing for human gene therapy
December 14, 2014
The rapid advancement of genome-editing techniques holds much promise for the field of human gene therapy. From bacteria to model organisms and human cells, genome editing tools such as zinc-finger nucleases (ZNFs), TALENs, and CRISPR/Cas9 have been successfully used to manipulate the respective genomes with unprecedented precision. With regard to human gene therapy, it is of great interest to test the feasibility of genome editing in primary human hematopoietic cells that could potentially be used to treat a variety of human genetic disorders such as hemoglobinopathies, primary immunodeficiencies, and cancer. In this chapter, we explore the use of the CRISPR/Cas9 system for the efficient ablation of genes in two clinically relevant primary human cell types, CD4 + T cells and CD34 + hematopoietic stem and progenitor cells. By using two guide RNAs directed at a single locus, we achieve highly efficient and predictable deletions that ablate gene function. The use of a Cas9-2A-GFP fusion protein allows FACS-based enrichment of the transfected cells. The ease of designing, constructing, and testing guide RNAs makes this dual guide strategy an attractive approach for the efficient deletion of clinically relevant genes in primary human hematopoietic stem and effector cells and enables the use of CRISPR/Cas9 for gene therapy.
Efficient Ablation of Genes in Human Hematopoietic Stem and Effector Cells using CRISPR/Cas9
November 6, 2014
Genome editing via CRISPR/Cas9 has rapidly become the tool of choice by virtue of its efficacy and ease of use. However, CRISPR/Cas9 mediated genome editing in clinically relevant human somatic cells remains untested. Here, we report CRISPR/Cas9 targeting of two clinically relevant genes, B2M and CCR5, in primary human CD4+ T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). Use of single RNA guides led to highly efficient mutagenesis in HSPCs but not in T cells. A dual guide approach improved gene deletion efficacy in both cell types. HSPCs that had undergone genome editing with CRISPR/Cas9 retained multi-lineage potential. We examined predicted on- and off-target mutations via target capture sequencing in HSPCs and observed low levels of off-target mutagenesis at only one site. These results demonstrate that CRISPR/Cas9 can efficiently ablate genes in HSPCs with minimal off-target mutagenesis, which could have broad applicability for hematopoietic cell-based therapy.
Impact of hexavalent chromium on mammalian cell bioenergetics: phenotypic changes, molecular basis and potential relevance to chromate-induced lung cancer
March 25, 2014
Occupational exposure to hexavalent chromium [Cr(VI)] has been firmly associated with the development of several pathologies, notably lung cancer. According to the current paradigm, the evolution of normal cells to a neoplastic state is accompanied by extensive metabolic reprogramming, namely at the level of energy-transducing processes. Thus, a complete understanding of the molecular basis of Cr(VI)-induced lung cancer must encompass the elucidation of the impact of Cr(VI) on metabolism. Research in this area is still in its infancy. Nonetheless, Cr(VI)-induced metabolic phenotypes are beginning to emerge. Specifically, it is now well documented that Cr(VI) exposure inhibits respiration and negatively affects the cellular energy status. Furthermore, preliminary results suggest that it also upregulates glucose uptake and lactic acid fermentation. From a mechanistic point of view, there is evidence that Cr(VI) exposure can interfere with energy transducing pathways at different levels, namely gene expression, intracellular protein levels and/or protein function. Loss of thiol redox control likely plays a key role in these processes. The transcriptional networks that control energy transduction can likewise be affected. Data also suggest that Cr(VI) exposure might compromise energy transducing processes through changes in the intracellular pools of their substrates. This article reviews, for the first time, the information available on Cr(VI) impact on mammalian cell bioenergetics. It aims to provide a framework for the understanding of the role played by bioenergetics in Cr(VI)-induced carcinogenesis and is also intended as a guide for future research efforts in this area.
Gammadelta T cells: innately adaptive immune cells?
April 25, 2013
T cells employ a cell surface heterodimeric molecule, the T cell receptor (TCR), to recognize specific antigens (Ags) presented by major histocompatibility complex (MHC) molecules and carry out adaptive immune responses. Most T cells possess a TCR with an α and a β chain. However, a TCR constituted by a γ and a δ chain has been described, defining a novel subset of T cells. γδ TCRs specific for a wide variety of ligands, including bacterial phosphoantigens, nonclassical MHC-I molecules and unprocessed proteins, have been found, greatly expanding the horizons of T cell immune recognition. This review aims to provide background in γδ T cell history and function in mouse and man, as well as to provide a critical view of some of the latest developments on this still enigmatic class of immune cells.
How induced pluripotent stem cells are redefining personalized medicine
May 10, 2013
Since the generation of the first induced pluripotent stem (iPS) cells, the stem cell field has grown at an unparalleled pace. Today, these cells have become the major tools in the advancement of personalized medicine. Here we review the experiments that lead to their discovery as well as the latest developments in iPS cell biology. By emphasizing the current applications and limitations of induced pluripotency, we discuss how iPS cells are shaping innovation in personalized therapies. In addition, we analyze the major landmarks in direct lineage reprogramming, a potentially faster alternative to the use of iPS cells in therapy. Finally, we present the current progress in disease modeling and future directions of the treatment of genetic disorders.
Changing cell identity to create true personalized medicine
November 19, 2012
The 2012 Nobel Prize in Physiology or Medicine was awarded to Sir John Gurdon and Shinya Yamanaka for their breakthroughs in cellular reprogramming. These discoveries not only changed our view of the process of cell fate determination, but also hold the potential to revolutionize medicine. By providing a brief historical context and a succinct summary of the current methodologies, we present the major advances in basic research, as well as their potential applications to the clinic. This review aims to provide a concise overview of the current state of the field and its implications for therapy.
Neural regeneration: lessons from regenerating and non-regenerating systems
June 21, 2012
One only needs to see a salamander regrowing a lost limb to become fascinated by regeneration. However, the lack of robust axonal regeneration models for which good cellular and molecular tools exist has hampered progress in the field. Nevertheless, the nervous system has been revealed to be an excellent model to investigate regeneration. There are conspicuous differences in neuroregeneration capacity between amphibia and warm-blooded animals, as well as between the central and the peripheral nervous systems in mammals. Exploration of such discrepancies led to significant discoveries on the basic tenets of neuroregeneration in the last two decades, identifying several positive and negative regulators of axonal regeneration. Implications of these findings to the comprehension of mammalian regeneration and to the development of spinal cord injury therapies are also addressed.
Metabolic reprogramming of the tumor
January 9, 2012
Cancer is classically considered as a genetic and, more recently, epigenetic multistep disease. Despite seminal studies in the 1920s by Warburg showing a characteristic metabolic pattern for tumors, cancer bioenergetics has often been relegated to the backwaters of cancer biology. This review aims to provide a historical account on cancer metabolism research, and to try to integrate and systematize the metabolic strategies in which cancer cells engage to overcome selective pressures during their inception and evolution. Implications of this renovated view on some common concepts and in therapeutics are also discussed.
Molecular and cellular mechanisms of hexavalent chromium-induced lung cancer: an updated perspective
March 1, 2012
For over a century, chromium (Cr) has found widespread industrial and commercial use, namely as a pigment, in the production of stainless steel and in chrome plating. The adverse health effects to the skin and respiratory tract of prolonged exposure to Cr have been known or suspected for a long time, but it was much more recently that the toxicity of this element was unequivocally attributed to its hexavalent state. Based on the combined results of extensive epidemiological studies, animal carcinogenicity studies and several types of other relevant data, authoritative regulatory agencies have found sufficient evidence to classify hexavalent chromium [Cr(VI)] compounds as encountered in the chromate production, chromate pigment production and chromium plating industries as carcinogenic to humans. Crucial for the development of novel strategies to prevent, detect and/or treat Cr(VI)-induced cancers is a detailed knowledge of the molecular and cellular mechanisms underlying these pathologies. Unfortunately, in spite of a considerable research effort, crucial facets of these mechanisms remain essentially unknown. This review is intended to provide a concise, integrated and critical perspective of the current state of knowledge concerning multiple aspects of Cr(VI) carcinogenesis. It will present recent theories of Cr(VI)-induced carcinogenesis and will include aspects not traditionally covered in other reviews, such as the possible involvement of the energy metabolism in this process. A brief discussion on the models that have been used in the studies of Cr(VI)-induced carcinogenicity will also be included, due to the impact of this parameter on the relevance of the results obtained.
DNA Damage, Repair and Misrepair in Cancer and in Cancer Therapy
October 26, 2011
The term cancer, which is derived from the Greek word "karkinos", meaning crab, encompasses over 100 distinct diseases that are characterized by an uncontrolled multiplication of abnormal cells. The oldest written description of cancer known to exist dates back to about 1600 BC, but is believed to be based on a much earlier document, from ca. 3000 BC. It is part of the Edwin Smith Papyrus, and describes eight cases of breast tumors or ulcers in Egypt and their treatment by cauterization. Signs of cancer can also be traced back as far as 3000 BC, on the bones of mummies from ancient Egypt and Peru. Humans, though, must have fought against this pathology throughout their existence, for which, according to the above-mentioned papyrus, there was no cure (American Cancer Society, 2010). This opinion was shared by the Greek physician Hippocrates (about 400 BC), known today as the “Father of Medicine”, who believed that it was best to leave cancer alone, as those who received treatment did not survive long. Hippocrates claimed that cancer was due to an excess of black bile, one of the four fluids (or humors) that, according to the humoral theory that he developed, composed the body. Hippocrates was the first to use the words carcinos and carcinoma to describe non-ulcer forming and ulcer-forming tumors, probably to reflect similarities between certain aspects of the tumors’ appearance and that of crabs (Feinberg et al., 2006). In the first century BC, the Roman physician Celsus translated the Greek term into cancer, the Latin word for crab. Later, in the second century AD, Galen, another Roman physician, used the Greek word oncos (meaning swelling) to describe tumors.
Cancer metabolism: The Warburg effect today
September 8, 2010
One of the first studies on the energy metabolism of a tumour was carried out, in 1922, in the laboratory of Otto Warburg. He established that cancer cells exhibited a specific metabolic pattern, characterized by a shift from respiration to fermentation, which has been later named the Warburg effect. Considerable work has been done since then, deepening our understanding of the process, with consequences for diagnosis and therapy. This review presents facts and perspectives on the Warburg effect for the 21st century.
Immunodietica is an emerging field that combines traditional and computational methods to explore and elucidate links between diet and autoimmune disorders. How food affects autoimmunity incidence and severity is essentially mysterious. Many patients find that what they eat affects their symptoms. Numerous books propose various diets for symptom management. Dialing in on specific immune epitopes, HLA (human leukocyte antigen) alleles, and individual microbiomes offers a new way to identify optimal individual diets.
Teaching and science outreach
Your best weapon is to stay informed
Protein synthesis: an epic on the cellular level
Directed in 1971 by Robert Alan Weiss for the Department of Chemistry of Stanford University and imprinted with the "free love" aura of the period, this short film continues to be shown in biology class today. It has since spawn a series of similar funny attempts at vulgarizing protein synthesis. Narrated by Paul Berg, 1980 Nobel prize for Chemistry.
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