March 17, 2014
Editor: Andrew H. Lichtman, MD, PhD, Brigham & Women's Hospital
Editorial Board: Abul K. Abbas, MD, University of California, San Francisco | Carla J. Greenbaum, MD, Benaroya Research Institute | Andrew H. Lichtman, MD, PhD, Brigham & Women's Hospital
|Highlights from Recent Literature|
A review of De la Herrán-Arita, et al., CD4+ T Cell Autoimmunity to Hypocretin/Orexin and Cross-Reactivity to a 2009 H1N1 Influenza A Epitope in Narcolepsy. Science Translational Medicine 5, 216ra176 (2013).
• The authors identified two immune epitopes of the hypocretin neuropeptide that bound to DQ0602. These two epitopes of hypocretin activated a population of CD4+ T cells in patients with narcolepsy but not in DQ0602-positive healthy controls.
Narcolepsy, a lifelong disease with devastating impact on quality of life has long been associated with a particular HLA molecule but an immune etiology has been elusive. The authors have demonstrated that T cells responding to the H1N1 influenza A strain pH1N1 and possibly to a single hemagglutinin epitope of this virus, likely cross-react with two immune epitopes for the critical neuropeptide hypocretin. These results suggest that cross-reactivity induced by antiviral immunity may be responsible for the destruction of hypocretin producing posterior hypothalamus neurons and therefore for some cases of narcolepsy developing after 2009. This elegant study suggests a role for molecular mimicry in the development of narcolepsy and sets the stage for further evaluation of immune epitopes in this neurologic disease.
Reviewed by Rachael A. Clark, MD, PhD, Brigham and Women's Hospital
A review of Robertson, et al., A Zebrafhish Compound Screen Reveals Modulation of Neutrophil Reverse Migration as an Anti-flammatory Mechanism. Science Translational Medicine 5, 225ra29 (2014). PMID: 24554340
• The authors developed an in vivo screen for molecules that helped resolve inflammation in using transgenic zebrafish. In this model, sterile tissue injury was induced and a panel of known compounds was screened for their ability to accelerate resolution of inflammation.
Resolution of inflammation is a process that is poorly understood and novel therapeutics that enhance inflammatory resolution are needed for the treatment of autoimmune and inflammatory disorders. The authors identified a novel enhancer of immune resolution using a new in vivo zebrafish assay. The identified compound, tanshinone IIA, promoted neutrophil death in both humans and zebrafish and had the interesting effect of inducing increased random migration of neutrophils, thereby dispersing them away from the site of inflammation. These studies are significant because they identify a novel therapeutic that can now be further tested in humans and they demonstrate the utility of using a zebrafish screen to identify novel agents that enhance inflammatory resolution.
Reviewed by Rachael A. Clark, MD, PhD, Brigham and Women's Hospital
A review of Schlapach, C., et al. Human Th9 Cells are Skin-Tropic and have Autocrine and Paracrine Proinflammatory Capacity. Science Translational Medicine 6, 219ra8 (2014). PMID: 24431112
The immune system contains a panoply of cells and soluble factors to recognize and assess antigens and then determine an appropriate set of responses. Within the adaptive cell-mediated immune response, T cells have been subdivided into numerous subsets, with increasing appreciation of the existence of many intermediate states and functions in previously defined subsets, as well as entirely new subsets. Th9 cells are one of the newer subsets of CD4+ T cells, and they have led to extensive study in mouse models as well as studies on differentiating them from naïve or memory T cell populations or in a few human diseases. In this study, Schlapbach et al provide a detailed analysis of Th9 cells in healthy human donor PBMCs, including their frequency, their tissue tropism and their response to common pathogens and polyclonal stimulation. This is taken deeper, with investigations into Th9 cells from tissues and in samples from patients with varied skin disorders, and provides the most comprehensive picture yet of this cell subset.
In this study, Th9 cells are identified in healthy human skin and PBMCs and the paper demonstrates that the secretion of IL-9 is from skin tropic CD4+ memory cells in PBMCs or skin resident memory CD4+ T cells and can be stimulated using tissue relevant pathogens or polyclonal stimulation techniques. They demonstrated that the production of IL-9 is co-incident with TNFa and granzyme B and comes from a CD3+ population (i.e. does not seem to be exclusively from ILCs in humans). In addition, the early and transient secretion of IL-9 by Th9 cells was shown to enhance the secretion by other core helper T cell subsets of their own signature cytokines (i.e. IL-17 from Th17 cells). Finally, psoriatic lesions from human patients showed an increase in Th9 cells versus healthy skin. Much work remains to be done to link these cells to the pathogenesis of disease, and starting with inflammatory skin disorders seems like a perfect first step. The ways in which this cell type are tied into the complex network of inhibitory and activating factors that drive helper T cell differentiation and function will also likely fuel efforts for years to come. This study provides many new avenues for investigation and a clear set of questions to pursue next.
Reviewed by Sarah Henrickson, MD, PhD, Children’s Hospital of Philadelphia
A review of Newel, E.W. and Davis, M.M., et al. Beyond Model Antigens: High Dimensional Methods for the Analysis of Antigen-Specific T Cells. Nature Biotechnology. 32, 147-149, (2014). PMID: 24441473
The complexity of the human immune system has dramatic variability among individuals based on genetic diversity as well as unique exposures to pathogens, commensal organisms and myriad environmental factors. Recent reviews in Nature Immunology and Nature Biotechnology (Feb. 2014) highlight the many challenges and tools available and under development for analysis of the increasingly deep and massive data sets that are being generated in this field.
Newell et al focuses on the challenges and strategies in deriving a complete understanding of the antigen specific T cell response to immunological challenge. Kidd et al focuses on massively multidimensional datasets and the best strategies for getting our hands on the full implications of these datasets, making use of the best algorithms and packages that are currently available.
Both reviews provide overviews for the generalist and the specialist of both the available tools, the most recent articles developing and exploiting these tools and the challenges that remain. Among these challenges remains the need for resources to allow systematic collection of appropriate samples (both in healthy donors from pediatric, young adult and elderly populations across races and ethnicities as well as in human disease) as well the development of analysis packages that can be used by non-computational biology researchers with deep domain knowledge in the samples under study.
Reviewed by Sarah Henrickson, MD, PhD, Children’s Hospital of Philadelphia
A review of Joseph, C.G., et al. Association of the Autoimmune Disease Scleroderma with an Immunologyic Response to Cancer. Science. 2014; 343(6167): 152-157. PMID: 24310608
• The authors first compared the antitumor efficacy of systemic versus local administration of CTLA-4 blocking antibody against subcutaneously implanted murine colon carcinoma MC-38 tumors expressing Ovalbumin (MC-38-OVA). They demonstrate that a single local injection of 50 μg of antibody in Montanide ISA-51 was just as effective as treatment with two systemic doses of 200 μg of antibody. They also showed that localized treatment with CTLA-4 blocking antibodies induced antitumor responses in mice bearing MC-38 tumors that do not express OVA, and mice bearing more aggressive OVA-expressing EG7 thymoma tumors.
• Low dose treatment with 50 μg of antibody was shown to only be effective when delivered in the tumor-draining area, and not when given systemically, or in the contralateral flank of tumor-bearing mice.
• Similar to systemic treatment, localized treatment with CTLA-4 blocking antibody was shown to enhance systemic tumor-specific T cell responses, and as a result, was capable of controlling distant tumors.
• By depleting CD4+ and CD8+ T cells in treated mice, the authors demonstrated that CD8+ T cells, and not CD4+ T cells, were responsible for the antitumor effect in their model, indicating that CTLA-4 blocking antibodies can operate directly on CD8+ T cells.
• Analysis of serum from treated mice showed that antibody levels were more than 1,000-fold lower in mice treated with local doses of antibody compared to mice treated with systemic doses. Levels of the liver enzymes ALT and AST were also lower in locally-treated mice suggesting that local slow-release administration of the antibody decreases the induction of adverse effects.
Although systemic treatment with CTLA-4 blocking antibodies can induce significant antitumor effects in some patients, and has been FDA approved for the treatment of advanced melanoma, this treatment can also cause severe autoimmune and inflammatory side effects. This study provides a simple, yet novel delivery system for distributing CTLA-4 blocking antibody in the tumor-draining area. In preclinical models, use of this delivery approach resulted in antitumor responses that were comparable to systemic antibody treatment, but were associated with 1,000-fold lower serum levels of antibody. These data indicate that use of this delivery approach may make it possible to achieve the same antitumor effects in patients without inducing unwanted side effects. This strategy could also be useful for other immunomodulatory agents being developed for cancer treatment.
Reviewed by Elizabeth Jaffe, MD, John Hopkins Institute for Clinical and Translational Research and Eric Lutz, PhD, John Hopkins University, Sidney Kimmel Cancer Center
A review of Beatty, G.L., et al. Mesothelin-Specific Chimeric Antigen Receptor mRNA-Engineered T Cells Induce Antitumor Activity in Solid Malignancies. Cancer immunol. Res. 2014; 2(2): 112-120. PMID: 24579088
• The authors established a platform for engineering transient CAR-expressing T cells using in vitro transcribed mRNA.
A review of Xue, et al. Transcriptome-Based Network Analysis Reveals a Spectrum Model of Human Macrophage Activation. Immunity. 40, 2014; 274-288. PMID: 249590056
• Thorough analysis of this data set through powerful analytical tools such as coregulation analysis (CRA), self-organizing-map (SOM) clustering, and correlation coefficient matrices (CCM) revealed a more extensive ‘spectrum’ model of macrophage activation that indeed goes beyond the existing M1 versus M2 model. The spectrum model was endorsed by in phenotypic and functional characterization studies.
Reviewed by Kari Nadeau, MD, PhD, Stanford School of Medicine
A review of Lee, et al. Bitter and Sweet Taste Receptors Regulate Human Upper Respiratory Innate Immunity. Journal of Clinical Investigation. 2014; 124(2): 1393-1405. PMID: 24531552
• Using surgically removed human explants, the author set-up an air-liquid interface (ALI) to mimic the polarized upper airway epithelium. Administration of the bitter compound denatonium benzoate to apical nonciliated human sinonasal chemosensory cells in ALI cultures induces a calcium response that propagates to adjacent cells via gap junctions. Apical co-expression and activation of sweet receptors T1R2/3 by glucose and sugar substitutes overrides denatonium-induced calcium responses, thereby regulating T2R calcium-dependent signaling pathways.
In summary, the authors postulate that in a healthy individual normal glucose levels repress bitter receptor expression of α-microbial peptides. Upon upper airway infections, microbial glucose consumption and shedding of bitter-derived microbial compounds relieve inhibition of T2R-dependent α-microbial peptide secretion.
A review of Passeerini, L., et al. CD4+ T Cells from IPEX Patients Convert into Functional and Stable Regulatory T Cells by FOXP3 Gene Transfer. Science Translational Medicine 5, 215ra174 (2013). PMID: 24337481
Immune dysregulation, polyendocrinopathy, enteropahty, X-linked (IPEX) syndrome is a primary immunodeficiency disorder that is characterized by loss of function of thymus-derived CD4+CD25+ regulatory T cells (Treg). Hematopoietic stem cell transplantation (HSCT) is the only curative available for IPEX syndrome patients. Data from animal models and recent successful gene therapy trials in graft-versus-host-disease patients undergoing allogeneic HSCT show promise for the use of Treg cells as therapeutic agents in diseases. In this study, the authors explore the possibility to restore immune regulatory function in FOXP3 mutated T cells by wild-type FOXP3 gene transfer thereby converting these conventional T cells in fully functional Treg like cells with suppressor function.
• They successfully generated CD4 FOXP3 Treg like cells using lentiviral vector system from healthy donors. These cells were stable and showed low levels of cytokine secretion (IL-2, IFN-g, IL-5, and IL-22) even under inflammatory conditions when stimulated with IL-6 and IL-1b.
• Using a humanized murine model of xeno-GVHD (graft-versus host disease) in NSG mice they show in vivo functionality of these CD4FOXP3 Treg cells. They show increased survival and decreased weight loss in NSG mice after transfer of CD4FOXP3 Treg cells. Transfer of these cells resulted in development of substantial chimerism in peripheral blood and these cells did not hamper the engraftment of Teff cells but instead controlled their effector function and proliferation.
• They show that CD4FOXP3 Treg cells generated using naïve CD4 cells are more stable and less proliferative compared to CD4FOXP3 Treg cells generated using antigen experiences memory CD4 T cells indicating that FOXP3 mediated Treg conversion of T cells is more efficient in naïve CD4 T cells compared to memory T cells.
• To next evaluate the possibility of using these cells in a clinical setting, they transduced FOXP3 expression in FOXP3 mutant conventional CD4 T cells from 5 IPEX patients with different FOXP3 mutations. They successfully generated transduced T cells with stable FOXP3 expression that acquired Treg like phenotype- CD25hi, CTLA4hi and CD127low and upregulation of Helios. These transduced FOXP3 cells also showed reduced cytokine secretion similar to that observed in cells generated in healthy donors. These FOXP3 Treg cells also acquired functional properties similar to those of conventional Treg cells- cell suppression of allogenic CD4 effector cells and low self proliferation upon CD3 stimulation. In vivo studies using the lethal GVHD humanized mouse model also demonstrated in vivo functionality of these cells generated from IPEX patients by protecting these animals from lethal GVHD and loss of weight.
Overall these data demonstrate that CD4FOXP3 T cells generated from FOXP3-mutated CD4+ T cells from IPEX patients are fully functional, both in vitro and in vivo. These de novo generated cells are highly stable if generated from naïve T cells and perform regulatory functions even in inflammatory conditions in vitro and in vivo. Taken together these data suggest that gene transfer of FOXP3 can be a potential therapeutic approach for IPEX syndrome patients.
Reviewed by Kari Nadeau, MD, PhD, Stanford School of Medicine
|Highlights From Clinical Immunology, the Official Journal of FOCIS|
A review of Peterson L. et al. SLAP Deficiency Decreases DsDNA Autoantibody Production. Clinical Immunology 150, 201-209, 2014 PMID: 24440645
• dsDNA peptide mimetope immunization induced an anti-dsDNA responses in wild type BALB/c but not SLAP-/- BALB/c mice. There were equivalent amounts of anti-peptide antibodies in both groups.
Reviewed by Andrew H. Lichtman, MD, PhD, Brigham and Women’s Hospital
A review of Ali, M. et al. Cyclization Enhances Function of Linear Anti-arthritic Peptides. Clinical Immunology 150, 121–133, 2014. PMID: 24207019
• In comparison to a control linear peptide, the C1 cyclic peptide was very effective in inhibiting T cell hybridoma IL-2 production in response to SEA plus APCs (an MHC dependent mechanisms), and was less albeit somewhat effective in inhibiting hybridoma IL-2 production in response to anti-CD3, anti-TCRβ, and PMA and ionomycin. The C1 was not toxic to the cells in these assays.
Overall, this study shows that cyclization of a previously characterized linear immunomodulatory peptide greatly enhanced its effectiveness in binding to membranes, blocking T cell activation in vitro, and reducing inflammatory parameters of T cell mediated disease process in rodent models. The authors results suggest that cyclization may be promising approach to enhancing the delivery and therapeutic effectiveness peptide inhibitors of immune mediated diseases.
Reviewed by Andrew H. Lichtman, MD, PhD, Brigham and Women’s Hospital
|Developments in Basic Immunology and Novel Therapies|
Shiv Pillai, MD, PhD, Massachusetts General Hospital, Harvard Medical School
Over the past decade a new disease entity has attracted a lot of interest. Patients presenting with tumescent, often painful, lesions affecting a variety of anatomic sites, initially often thought to have a malignant disease, have had biopsies analyzed that reveal a constellation of features that lead to a diagnosis of an entity now called IgG4-related disease or IgG4-RD. The histological features include storiform fibrosis, obliterative phlebitis, and the presence of a lymphoid and plasmacytic cell infiltrate in which forty percent or more of the IgG expressing plasma cells stain for the IgG4 isotype. Plasma levels of IgG4 may also be elevated.
This concept of IgG4-related disease was established in Japan by Hamano, Kamisawa and others at the beginning of this century (1,2). Virtually any organ in the body may be affected in IgG4-RD. This diagnosis now encompasses patients with salivary gland enlargements once thought to have Mikulicz’s disease, patients who would previously have been described as having Reidel’s thyroiditis, as well as subjects with retroperitoneal fibrosis, autoimmune pancreatitis, hypertrophic pachymeningitis, interstitial lung disease, tubulointerstitial nephritis and a host of other disorders (reviewed in 3, 4).
Why does an inflammatory disease in which fibrosis is a prominent component present with an increase in IgG4 secreting cells? What is the biological role of IgG4? Are disorders that fit the definition of IgG4-RD actually a small subset of a broader group of related fibro-inflammatory conditions? What broader lessons about immunology and disease might be learnt by studying IgG4-RD?
IgG4 is an unusual and relatively poorly understood immunoglobulin isotype. IgG4 antibodies bind poorly to activating Fc receptors and fix complement poorly. By conventional thinking this isotype should be unable to drive inflammation in the context of Fc-dependent effector mechanisms. In addition, when IgG4 is internalized into endosomes by FcRn, it can split down the middle between the two heavy chains and hybrid IgG4 molecules can form in a process called "Fab-arm exchange" (5-7). As a result many IgG4 molecules are functionally monovalent in vivo. IgG4 is therefore not suited for immune complex formation, since bivalency is required for antigen-antibody lattice formation. IgG4 can associate with the inhibitory Fc receptor, FcγRIIb, so it may function primarily as an inhibitory immunoglobulin. However, it has NOT been established beyond any doubt that IgG4 is not inflammatory. IgG4 containing immune complexes have been described in certain renal disorders. It is unclear if these complexes contain both IgG4 and other IgG isotypes and thus fix complement, or if N-Glycans on IgG4 recruits the mannose binding lectin and thus fix complement. The jury is still out on what IgG4 actually does in terms of disease pathogenesis, but it appears likely that IgG4 antibodies do not drive the inflammation and fibrosis seen in patients with IgG4-RD. It is possible that the increase in IgG4 in this disease reflects an attempt to dampen inflammation, albeit an attempt that has not succeeded.
Disease lesions in IgG4-RD share many histological features. CD4+ T cells are abundant at tissue sites of disease and they most likely drive the disease process. Apart from plasmablasts, plasma cells, and activated myofibroblasts, as well as organized tertiary lymphoid organs, essentially follicular structures with germinal centers, are also found in disease lesions. These germinal centers as well as those in draining lymph nodes are presumably major sites of IgG4 class switching. The promoters (so called I regions) adjacent to the IgG4 and IgE switch regions are structurally similar but detailed molecular studies on IgG4 class switching have not been performed. It is likely that T follicular helper cells that secrete IL-4 may participate in the IgG4 class switch, but it is also possible that unique yet to be defined T follicular helper cells are key to this class switching event. IgG4 remains a mysterious isotype and the study of IgG4 related disease may start to provide insights about this immunoglobulin sub-class.
The nature of the T cells that drive the disease is unclear. Fibrosis can be driven by a range of helper T cell subsets depending on the disease context. Th2 cells drive allergic fibrosis as seen in schistosomiasis and in this context interferon-γ may be inhibitory (8). When one considers the fibrotic nature of lesions in diseases like tuberculosis and Crohn's disease, it is clear that Th1 and Th17 cells may also drive fibrosis in certain contexts. In IgG4-RD the case has sometimes been made that the disease is driven by Th2 cells although a role for Th1 cells has also been suggested (9-11). However recent studies suggest that Th2 cells are only expanded in the circulation of patients with IgG4-RD who have concomitant atopic disease but not in subject with active disease who do not have atopic manifestations (12).
It is unclear whether IgG4-RD is an autoimmune disease and the driving antigens or antigens in this disease remain to be identified. Unlike most autoimmune diseases, IgG4-RD is largely a disease of older males. Studies examining the potential role in disease pathogenesis of genetics, the microbiome and epigenetics remain to be performed.
Is there a role for IgG4 in pathogenesis? Do IgG4 producing B cells collaborate with CD4+ T cells, perhaps by presenting antigen at disease sites? About sixty to seventy percent of subjects respond to steroids, but the most effective therapy is B cell depletion using Rituximab (13). Since B cells help maintain CD4+ effector/memory T cells (reviewed in 14), it is possible that Rituximab, by depleting antigen presenting B cells, indirectly depletes disease-causing rogue CD4+ effector/memory T cells. If IgG4 is not involved in pathogenesis, is it possible that similar mechanisms involving T cells cause disease in patients with IgG4-RD as well as in other fibrotic inflammatory diseases such as systemic sclerosis, lupus nephritis and possible idiopathic pulmonary fibrosis? The advent of immune repertoire analysis by Next Gen Sequencing and the development of many novel tools with which to interrogate the human immune system all suggest that we may soon have answers to a number of these longstanding puzzles.
2. Kamisawa T, Funata N, Hayashi Y, Eishi Y, Koike M, Tsuruta K, Okamoto A, Egawa N, Nakajima H. 2003. A new clinicopathological entity of IgG4-related autoimmune disease. J Gastroenterol 38: 982-4
|Human Immunophenotyping Update|
Holden T. Maecker, PhD, Stanford University
There is a theme among those doing translational immunology that “mice are easy, humans are hard”. One of the more subtle ways that this truism plays out is in the greater need to deal with batch effects in human studies. Partly because of genetic heterogeneity, human studies often involve more subjects than a typical animal study. Also, while animal studies can be manipulated so that all the animals reach a particular endpoint at the same time, human studies often cannot. Thus, in situations requiring fresh samples, one must perform the human assays longitudinally. For both of these reasons (number of subjects and the occasional need for real-time assays), one often faces the prospect of comparing human data across multiple “runs” or batches. Given the variability of many immune monitoring assays, this generally results in some run-to-run variability, or “batch effect”, and the consequent need for normalization of data across batches (1). Here, we will discuss some high-level considerations that should be given when designing studies for downstream normalization, and what normalization options are available for various study designs.
Z-scores. A variation of global normalization is the calculation of a z-score. In this form of normalization, the variance of each measurement is equalized, so that all values in the batch are centered on zero. A value of 1 indicates a sample that is one standard deviation (SD) above the mean; -1 corresponds to one SD below the mean, etc. Z-scores work best with data that is normally distributed, so it may be best used after log transformation (base 10 or base 2), which tends to bring high outliers closer to the mean, and thus force typical biological data to more closely approximate a normal distribution.
Summary. Biological assays have inherent batch effects. Batch design and assay standardization are the most important variables to consider before beginning a study. Batches should be thoughtfully balanced prior to data collection. Once data is collected, one needs to first evaluate the degree of batch variability to determine if there are outlier batches, and/or if normalization is warranted. Second, one needs to choose a normalization strategy, global or control-based. This decision should be driven by the size and balance of the batches, versus the number and reliability of control samples in each batch. See the flow chart summarizing the sequential decisions involved. Careful attention to these considerations should result in improved analysis of data collected over time.
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