June 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 Vantrourout, P., et al, Immunological Visibility: Posttranscriptional Regulation of Human NKG2D Ligands by the EGF Receptor Pathway. Science Translational Medicine. 6, 231ra49 (2014). PMID: 24718859
• The authors stressed human epithelial cells by exposing them to ultraviolet radiation, osmotic shock, oxidative stress and treatment with growth factors.
Interaction of immune cell NKG2D receptor with NKG2D ligands on cells is a major way that immune cells detect and destroy damaged, stressed and malignant cells. The authors found that expression of NKG2D ligands on the surface of human cells was directly linked to EGFR signaling. This represents an advance in our understanding of how NKG2D ligand expression is regulated. However it is also a cautionary tale because it could be expected that the EGFR inhibitors in current use for the treatment of cancer may have the unanticipated effect of reducing the immunologic visibility of tumors.
Reviewed by Rachael A. Clark, MD, PhD, Brigham and Women's Hospital
Gungor, B., et al. CpG ODN Nanorings Induce IFNa from Plasmacytoid Dendritic Cells and Demonstrate Potent Vaccine Adjuvant Activity. Science Translational Medicine. 6, 235ra61 (2014). PMID: 24807558
CgG oligonucleotides (ODN) are synthetic short single-stranded DNA molecules that have potent activities to activate the immune system. Different structures of ODN have different immunologic effects. D-type ODN (D-ODN) stimulate interferon-α (IFNα) release from plasmacytoid dendritic cells (pDC) and can be potent vaccine adjuvants. In this manuscript, the authors describe the design and activity of a novel ring form of ODN.
D-ODN induce IFNα production from pDC and can markedly enhance the effectiveness of vaccines against both tumors and infectious organisms. However, these molecules need to be aggregated into complex structures before they have the desired effect. The authors have shown that they can artificially aggregate K-ODN, which usually act alone, into stable higher ordered structures that have immunologic effects comparable to D-ODN. These results are significant because it provides a way to manufacture stable adjuvants that have the immunologic activity of D-ODN. This advance helps to pave the way for the use of these novel molecules in clinical trials of vaccination against cancer and infectious diseases.
A review of Li, L., et al. Disease Risk Factors Identified Through Shared Genetic Architecture and Electronic Medical Records. Science Translational Medicine 6, 234ra57 (2014). PMID: 24786325
In Bowton et al. in an era with increasing coverage by EMR, decreasing costs for genomic studies and stiff competition for research funding, the efficiencies of cost and time that are created by well-structured and maintained linked EMR-biorepository are very exciting. Encouraging further development of and innovation in these systems nationally and internationally, thus building the capacity for studying rare patients and rare side effects across many centers and quantifying the effects of incremental interventions on common disease may yield fundamental changes in our understanding of human health and disease.
In Li et al. a model is presented for utilizing our increasingly deep and broad understanding of genetic variation in disease and non-disease traits to identify shared underlying mechanisms that can be exploited both for direct clinical applications (diagnosis and prognosis) but also the clarification of our definitions of disease and our understanding of disease mechanisms (hopefully facilitating novel therapies or the use of older therapeutics in novel scenarios based on insights about shared genetic variation that yield deeper pathophysiological understanding). This is a technique that will require additional testing and exploration, but is very exciting in its potential to change how we work with and use our ‘big’ translational datasets.
A review of Duffy, D., et al. Functional Analysis via Standardized Whole-Blood Stimulation Systems Defines the Boundaries of a Healthy Immune Response to Complex Stimuli. Immunity. 40, 2014; 436-450. PMID: 24656047
The immune system is a complex network that includes cells and soluble factors to recognize and assess antigens and determine an appropriate set of responses. While many groups are undertaking studies of human immunology, there has been a focus on defining the immune system in settings of pathology, from infection to inflammation and cancer. However, while these studies are fascinating, the reality of the human immune response is that there is great potential variability among individuals in their baseline states and their responses to stimuli. Therefore, there is a need for reproducibility and the definition of ‘normal’ ranges. This study attempts both, by carefully defining assays that can be conducted on whole blood (to minimize the effects of storing, freezing and transferring cells) and defining preliminary ‘normal’ ranges for these assays.
In this study, a diverse set of robust assays for whole blood stimulation and assessment of human blood is developed and presented. This group of 25 patients is analyzed and shows clear variability in responses to these standard assays, including identifying subsets of patients who do not respond to the anti-CD3/28 assay and a separate subset of patients who lack of the ability to secrete IL-1α in response to the set of assays. The ease of use for this syringe-based system is attractive, especially for a POC scenario, though it does sacrifice the ability to assess individual cell populations (i.e. stimulation of isolated bulk T cells or subpopulations like Th-17 cells). In order to move forward and harness the power of large data sets in human immunology, there is a need for standardized, robust and easy to use assays that we can use as a community and thus facilitate cross-study comparison of data. This study proposes a novel method, with interesting preliminary data, and it will be fascinating to see how it is applied to larger ‘healthy’ populations and disease cohorts, and paired with flow cytometry, genomics, metabolomics, microbiome analysis and clinical demographics and outcomes, going forward.
A review of Muraro, P.A., et al. T Cell Repertoire Following Autologous Stem Cell Transplantation for Multiple Sclerosis. Journal of Clinical Investigation. 2014; 124(3): 1168-1172. PMID: 24531550
Autologous hematopoietic stem cell transplant has been performed in the context of clinical trials for patients with severe, refractory autoimmune diseases as a means of recalibrating the immune system to produce a new, non-autoimmune repertoire. Insights into the characteristics of the post-transplant repertoire have been limited. Prior work has shown that new thymic output (even in adults) and expansion of residual cells not eliminated during conditioning both contribute to the regenerated T cell compartment. These studies relied upon analysis of T cell receptor excision circles (TRECs) or CDR3 spectra typing for TCR clonal analysis. However, these methods do not enable robust analysis of the regenerating TCR repertoire. In this report, Muraro, Robins, Turka, and colleagues leverage high-throughput deep TCRβ sequencing to evaluate the T cell repertoire before and at two time points after autologous transplant in 24 patients with poor-prognosis multiple sclerosis (MS) treated in the context of a phase II trial. For each patient, 1 million CD4 and CD8 T cells were sorted and sequenced before and one year post transplant. Due to lower numbers of circulating T cells, ‘only’ 200,000 sorted CD4 and CD8 T cells from each patient were sequenced two months post-transplant. Remarkably, from the 24 patients on study, productive TCRβ sustained from almost 100 million T cells. This work provides several important insights.
These results support the notion that reestablishing repertoire diversity is critical for recalibrating the immune system and reestablishing tolerance. While the authors caution that larger patients’ numbers are needed, the observation that early repertoire diversity correlates with outcome supports a need for further studies to validate whether this may be a useful biomarker. It will be important to evaluate whether these findings extend to transplant for other autoimmune diseases.
Reviewed by Michelle L. Hermiston, MD, PhD, University of California, San Francisco
A review of Tjon A.S.W., van Gent R., Jaadar H., van Hagen P.M., Mancham, S., et al. Intravenous Immunoglobulin Treatment in Humans Suppresses Dendritic Cell Function via Stimulation of IL-4 and IL-13 Production. Journal of Immunology. 2014. PMID: 24808368
The authors conclude that IVIg stimulates an IL-33-Th2 axis in humans, similar to observations in mice. However, in contrast to mouse models, IVIg appears to mediate a sustained decrease in the activating receptor FcγRIIa and a transient decrease in FcγRIIb. While the mechanism is quite different, the net effect is similar to that seen in mice (which lack FcγRIIa) with an overall net increase in the level of inhibitory receptor FcγRIIb relative to the activating receptor. An important and interesting next step will be to determine whether these processes are deficient in patients that fail to respond to IVIg.
Reviewed by Melissa Ruck, MD, PhD, and Michelle Hermiston, MD, PhD, University of California, San Francisco
Oral Tolerance Can be Established via Gap Junction Transfer of Fed Antigens from CX3CR1+ Macrophages to CD103+ Dendritic Cells
A review of Mazzini et al. Oral Tolerance Can be Established via Gap Junction Transfer of Fed Antigens from CX3CR1+ Macrophages to CD103+ Dendritic Cells. Immunity. 40, 2014; 246-261. PMID: 24462723
The authors used fluorescent Ovalbumin as soluble oral antigen and a mouse model deficient in CX3CR1 to demonstrate that indeed CX3CR1+ macrophages are responsible for antigen uptake in proximal intestinal region, and that the lack of CX3CR1 compromises lumenal antigen uptake and the downstream tolerance induction by CD103+ DCs. These initial observations lead to the hypothesis that CX3CR1+ macrophages, in some way, are able to transfer the processed soluble antigen to CD103+ DCs. Gap junction hemichannel proteins-connexins emerged as putative key players in this transfer process. Thus to further test their hypothesis, the authors generated and validated a mouse model with CD11c+ APCs defective in Connexin 43. Going further, in their lynchpin set of experiments, the authors saw that Connexin 43 indeed enables unidirectional transfer of soluble antigens captured and processed by CX3CR1+ macrophages to CD103+ DCs. Contrary to WT mice; this transfer process was thus blocked in mice harboring CD11c+ APCs lacking Connexin 43 hampering the downstream oral tolerance induction.
Whether these results based on Ovalbumin as a model soluble antigen would hold true for various other food antigens/allergens needs to be tested. Also, the mechanism behind the directionality of the demonstrated transfer is yet to be elucidated. Nevertheless, the current findings by Mazinni et. al., provide crucial leads in advancing our understanding of mechanism of oral tolerance induction.
Reviewed by Kari Nadeau, MD, PhD, Stanford School of Medicine
|Highlights From Clinical Immunology, the Official Journal of FOCIS|
A review of Szymula, A. et al. T Cell Epitope Mimicry between Sjögren's Syndrome Antigen A (SSA)/Ro60 and Oral, Gut, Skin and Vaginal Bacteria. Clinical Immunology 125, 1-9, 21670014. PMID: 24576620
The findings from this study support the link between commensal flora and autoimmunity. The finding that multiple T cell epitopes of Ro60 are mimicked in various commensal bacteria suggests that the immune system can be readily exposed to proteins that can stimulate autoreactive T cell responses. The authors argue that the bacteria that produce the peptides they identified in this study might play a role in initiating and sustaining pathogenic autoimmune anti-Ro60 immune responses in Sjögren's syndrome and SLE.
Reviewed by Andrew H. Lichtman, MD, PhD, Brigham and Women’s Hospital
A review of Lu, et al. Identifying Functional Anti-Staphylococcus Aureus Antibodies by Sequencing Antibody Repertoires of Patient Plasmablasts. Clinical Immunology 152, 77-89, 2014 PMID: 24589749
This study used sophisticated individual plasmablast Ig gene cloning and sequencing approaches identify and interrogate specificity and function of antibodies produced at the height of acute infection with a pathogen. Technical limitations did not permit the identity of antibodies based on shared CDR structures which would likely have similar antigen binding properties. Nonetheless, the authors showed that S. aureus specific antibodies could be characterized by DNA sequencing of blood plasmablasts. They propose that such an approach applied to more patents should identify the important antigens targeted by the anti-bacterial antibody response. The applications of this approach could include the development of diagnostic and therapeutic antibodies, design methods for monitoring humoral immune responses over time, and identifying epitopes for vaccine development.
Reviewed by Andrew H. Lichtman, MD, PhD, Brigham and Women’s Hospital
|Developments in Basic Immunology and Novel Therapies|
Andrew Lichtman, MD, PhD, Brigham Women's Hospital
Atherosclerosis is the pathological process underlying most cases of myocardial infarction, embolic stroke, and peripheral arterial ischemic disease, and is therefore a far greater cause of morbidity and mortality worldwide than all well-defined autoimmune diseases combined. There are two compelling reasons why clinicians and investigators with a primary interest either in immunological diseases or in cardiovascular disease should both be interested in atherosclerosis. First, ample evidence indicates that atherosclerosis is chronic inflammatory process driven by innate and adaptive immune responses to lipoprotein deposition in the arterial intima. In a sense, atherosclerosis is at its core, autoimmune. Second, patients with chronic autoimmune diseases carry significantly elevated risks for atherosclerosis, perhaps best documented for rheumatoid arthritis, psoriasis, and systemic lupus erythematosus, but also true for many other chronic immune inflammatory disorders. These associations suggest that new therapeutic approaches in practice or under development for the treatment of autoimmunity may be of direct benefit for atherosclerosis, even in individuals without RA, psoriasis, SLE or other diseases of immune dysregulation.
|Human Immunophenotyping Update|
Immunophenotyping of B Cells in Human Peripheral Blood, an Endeavor Well Suited for High Dimensional Flow Cytometry
Angélique Biancotto, PhD, National Institute of Health
Unlike the immunophenotyping of human T cells, which most often yields subsets with distinct surface markers and with discrete patterns of staining, B cell immunophenotyping can be much more complex with overlapping patterns of marker expression and subsets defined by the intensity of staining rather than by staining positive of negative for a particular marker. Despite these obstacles, B cell immunophenotyping is considered crucial in many studies, particularly those examining autoimmunity and in measuring responses to vaccine, or viral infections.
Whereas the sub setting of T cells generally begins with a simple bifurcation based on the expression of CD4 or CD8 (or a trifurcation if one considers the double negative T cell), subdivisions of B cells are not universally so easy. Initial sub classification of B cells can be based on their origin (B1 or B2), relative maturational state (naïve, memory, switched memory), anatomic location (follicular, marginal zone). These classification schemes can are overlapping and one can identify populations of B cells based on a combination of these classifications.
There is scant agreement on a single panel of markers to best sub classify B cells, and it is likely that no one set of markers would reach universal consensus. The recent FITMaN conference selected a combination of markers for the FOCIS Human Immunophenotyping Consortium panel which represents a consensus for basic immunophenotyping of major immune cell populations found in peripheral blood, composed of five 8-color stainings (Maecker, McCoy et al. 2012). The B cell staining is consisted of CD19, CD20, CD3, CD24, CD27, CD38, and IgD. In this panel, CD3 is used to exclude T cells and CD19 is used as an inclusive marker for B cells, leaving the 5 additional markers to classify the B cells. CD20 is used to detect mature B cells, while IgD and CD27 can be used to identify naïve B cells (CD27-IgD+) and memory B cells (switched CD27+IgD-, unswitched CD27+IgD+, and DN CD27-IgD-). CD24 and CD38 are added to identify transitional B cells (CD24high CD38+), and CD38 can be used to identify plasmablasts as well as transitional B cells). This panel allows identification and quantification of these 6 subpopulations, but more complex B cell subsets cannot be phenotyped. For this reason, even higher dimensional immunophenotyping of B cells is worth pursuing in many studies (see figure for the myriad of phenotypes that can be identified with the HIPC panel and a larger panel). As an example of high dimensional immunophenotyping of B cells is a 15-color panel that we developed for routine use in studies at the NIH Center for Human Immunology (Biancotto, Fuchs et al. 2011). Two markers were added as gating tools: a viability stain and CD45. The former assures that artifacts arising from nonspecific staining of dead cells are excluded and the latter eases identification of lymphocytes, particularly if staining is performed on B cells isolated from tissues. In addition to CD45 are five of the markers used in the HIPC panel for the B lineage: CD19, CD20, IgD, CD27, CD38.
With the addition of IgA, IgG, IgM, one can now obtain information on the isotype class of mature B cells and of the immunoglobulins secreted by plasmablasts. The inclusion in this panel of CD23, CD21, CD86, and CD80 can provide information concerning the state of activation of these cells. The addition of CD10 to this panel can help to identify transitional B cells. Furthermore, the additional of all of these markers may facilitate identification of novel B cell subsets not heretofore described. Thus high polychromatic immunophenotyping can be a highly useful tool for the broad and in depth assessment of the B cell subpopulations.
A recent example of the utility of such detailed B cell phenotyping is in characterizing the immune response to H5N1 vaccine in healthy individuals (Tsang, Schwartzberg et al. 2014). In predictive modeling of antibody responses, day seven levels of CD38+ CD27- IgD+ naïve, CD38+ transitional, and CD38 on both switched and unswitched memory B cell populations, and CD86+IgD+CD27+ memory B cell all positively correlated with antibody endpoint titers. These predictive immune populations could be identified only with high dimensional immunophenotyping. Other high dimensional panels with a variety of markers, such as CXCR3, CD95, and VH-34-encoded idiotype have been used to subset B cells (Wei at al 2011, Kaminsky et al. 2012) and illustrate the power and applicability of such assays.
2. Biancotto, A., J. C. Fuchs, A. Williams, P. K. Dagur and J. P. McCoy, Jr. (2011). "High dimensional flow cytometry for comprehensive leukocyte immunophenotyping (CLIP) in translational research." J Immunol Methods 363(2): 245-261.
3. Tsang, J. S., P. L. Schwartzberg, Y. Kotliarov, A. Biancotto, Z. Xie, R. N. Germain, E. Wang, M. J. Olnes, M. Narayanan, H. Golding, S. Moir, H. B. Dickler, S. Perl, F. Cheung, H. C. Baylor and C. H. I. Consortium (2014). "Global analyses of human immune variation reveal baseline predictors of postvaccination responses." Cell 157(2): 499-513.
4. Wei C, Jung J, and Sanz I (2011). "OMIP-003: Phenotypic Analysis of Human Memory B Cells." Cytometry Part A _ 79A: 894_896.
5. Kaminski DA, Wei C, Yu, et al. (2012) "Advances in human B cell phenotypic profiling." Frontiers Immunol 3 (302) 1-15.
|Selected Recent Clinical Trial Results|
Efficacy and Safety of Epratzuzumab in Patients with Moderate/Severe Active Systemic Lupus Erythematosus: Results from Emblem, a Phase IIb, Randomized, Double-Binded, Placebo-controlled, Mulitcenter Study
Disease: Systemic Lupus Erythematosus (SLE)
Drug: Epratuzumab is a human monoclonal antibody targeted at CD22, a mature B cell transmembrane glycoprotein that affects migration and activation.
Why The Trial is of Interest to the Broader FOCIS Community:
Reviewed by Sandra Lord, MD, Benaroya Research Center
Atacicept in Multiple Sclerosis (ATAMS): A Randomixed, Placebo-Controlled, Double-Blind, Phase 2 Trial
Diseases: Relapsing-remitting Multiple Sclerosis
Drug: Atacicept is a recombinant fusion protein that binds to the cytokines BLyS and APRIL, and therefore suppresses B cell maturation and antibody production.
Why The Study is of Interest to the Broader FOCIS community:
Reviewed by Sandra Lord, MD, Benaroya Research Institute
Methotrexate in Combination with Infliximab is No More Effective than Infliximab Alone in Patients with Crohn's Disease
Disease: Crohn’s Disease (CD)
Drug: Methotrexate (MTX,) a purine anti-metabolite, and infliximab, a chimeric monoclonal antibody against TNF-alpha
Why This Study Is of Interest to the Broader FOCIS community:
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