September 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 Xiang-ni Wu., et al. Defective PTEN regulation contributes to B cell hyperresponsiveness in systemic lupus erythematosus. Sci Transl Med 6, 246ra99 (2014). PMID: 25101889
PTEN is a key negative regulatory of B cell activation in healthy individuals. The authors report that PTEN levels are decreased in B cells from patients with SLE and suggest that decreased PTEN levels may contribute to B cell activation and autoantibody production in SLE. The authors found inappropriately high expression of the microRNA miR-7 in SLE B cells, leading to decreased protein levels of PTEN. This work is significant because it identifies decreased PTEN signaling may contribute to chronic B cell activation in SLE and suggests that agents that decrease miR-7 levels could be effective new therapeutics for SLE.
Reviewed by Rachael A. Clark, MD, PhD, Brigham and Women's Hospital
A review of Saito N., et al. An annexin A1–FPR1 interaction contributes to necroptosis of keratinocytes in severe cutaneous adverse drug reactions. Sci Transl Med 6, 245ra95 (2014).
Toxic epidermal necrolysis (TEN) and Stevens-Johnson syndrome (SJS) are severe, potentially fatal drug reactions that involve widespread keratinocyte death and sloughing of the skin. Because of widespread loss of skin, TEN patients must be managed in burn units and this drug reaction is uniformly fatal when advanced facilities are not available. In this study, the authors find that immune cell production of annexin A1 induces keratinocyte death in these patients and that inhibition of annexin A1-keratinocyte signaling may be an effective therapy for TEN and SJS.
SJS/TEN are severe, life threatening drug reactions with no specific therapy. The authors found that keratinocyte cell death in these patients occurs via necroptosis- a type of signaling induced cell death that under normal conditions is thought to be a defense against viral infection. The authors identified that annexin A1 was produced by drug-reactive T cells after exposure to the causative drug and it was this factor that mediated keratinocyte death. These studies are important because they identify annexin-A1 signaling as a causative agent in the life threatening loss of skin viability and integrity in SJS/TEN patients. These results suggest that annexin-A1 neutralizing or inhibitors of necroptosis may be effective new therapies for patients with SJS and TEN.
Reviewed by Rachael A. Clark, MD, PhD, Brigham and Women's Hospital
A review of Seumois G., et al. Epigenomic analysis of primary human T cells reveals enhancers associated with Th2 memory cell differentiation and asthma susceptibility. Nature Immunology. (2014) PMID:24997565
Over the past few decades we have seen global increases in asthma in both adults and children. Asthma is a chronic inflammatory illness with significant morbidity and mortality, which is associated for many patients with atopy. However, though it is the most common chronic condition in childhood and has been the subject of deep investigation, the fundamental mechanisms remain unclear. CD4 T cells that secrete type 2 cytokines (Th2 cells) have been implicated as a key cell subset in the pathogenesis of asthma. In this study Seumois et al compare the genome wide histone modification profiles of a single epigenomic mark (H3K4me2) using ChIP-seq in naïve, T helper 1 (Th1) and T helper 2 (Th2) patients with asthma versus healthy controls. This single mark was chosen as an assay that could be used to identify enhancers that are either active or poised to become active in rare cell subsets from human samples with inherently limited volumes of blood available for research. The investigators isolated peripheral blood CD4+ T cells from 12 healthy controls and 12 asthmatic patients and then isolated naïve and memory cells, and divided the memory cells into CCR4 expressing (and denoted these as Th2, given that they were enriched for Th2 cells) and CCR4 negative (and denoted these as Th1, given that they were depleted of Th2 cells and enriched for Th1 cells). Within these three cell subsets in each patient group, ChIP-seq was used to determine the DNA regions associated with H3K4me2. The assay was extensively optimized and microscaled (to be reproducible down to 10,000 cells). The major findings were:
This epigenome-wide study of 24 patients, including 12 healthy controls, 6 mild asthmatics (never treated with corticosteroids) and 6 moderate asthmatics (treated with inhaled corticosteroids) presents evidence that this microscaled assay of a single epigenomic mark could be used to identify novel pathways (and potentially therapies) in human disease. As with any initial study demonstrating a novel technique, there are caveats. For example, the use of CCR4 as the differentiating factor for Th2 vs. Th1 cells provides enrichment, rather than pure populations of Th2 and Th1 cells. In addition, asthma is a very inclusive term and we are only beginning to understand the sub-phenotypes within that diagnosis (which have different ages of onset, key cell type and response to therapy) and this study included mild and moderate patients. It is possible that severe adult patients (or moderate pediatric patients) would have different results. However, this is a very powerful technique for deepening our understanding of the genomics underlying human disease, and it will likely be applied broadly in the next few years.
Reviewed by Sarah Henrickson, MD, PhD, Children’s Hospital of Philadelphia
A review of Pai, S.Y., et al. Transplantation outcomes in severe combined immunodeficiency, 2000-2009. NEJM. (2014) 371: 434-436.
In severe combined immunodeficiency, infants have reduced T cell counts and impaired T and B cell immune responses, and the many etiologies of the condition are fatal in infancy if left untreated. Allogeneic hematopoetic stem cell transplantation can be curative, and there are many possible types of transplants (from bone marrow, T cell depleted bone marrow, peripheral blood derived stem cells and umbilical cord blood) and donors (siblings, parents and unrelated donors). With so many variables, including age at transplant and whether or not to condition (and what type of conditioning), a study surveying the current landscape of practice and outcomes was undertaken to identify important clinical factors.
The records from 240 infants with SCID who were transplanted at 25 centers who participate in the Primary Immune Deficiency Treatment Consortium (PIDTC) 1from 2000-2009 were collated to study the factors associated with better outcomes.
The improved outcomes of infants less than 3.5 months of age (and those without prior infection) emphasize the importance of SCID newborn screening, improving our ability to identify and transplant infants prior to significant infections. These programs continue to spread and this data further supports the importance of these programs. This study permits an assessment of many key questions in SCID transplantation and provides a rich dataset for consideration
Reviewed by Sarah Henrickson, MD, PhD, Children’s Hospital of Philadelphia
A review of Deau MC., et al. A human immunodeficiency caused by mutations in the PIK3R1 gene. J Clin Invest. 2014 Sep 2;124(9):3923-8. PMID:25133428
Phosphoinositide 3-kinase (PI3K) is a heterodimeric enzyme composed of a catalytic p110 subunit (P110α, P110β, or P110δ) bound to a regulatory subunit (P85α/P55α, P85β, or P55γ). PI3K coordinates signals emanating from a wide variety of receptors and modulates an array of cellular functions including proliferation, differentiation, survival, and metabolism in multiple cell types. While the P110α and β subunits are ubiquitously expressed, the P110δ subunit is restricted to lymphocytes. Deletion of P110δ in mice results in impaired B cell development and loss of T and B cell activation and function. It was thus notable when two recent studies identified activating mutations in the P110δ subunit of PI3K in several unrelated patients with primary immunodeficiency (PID). The syndrome was named activated P110δ syndrome (APDS). New work by Deau et al. suggests that hyperactivation of the PI3K signaling axis may be a more common theme in PID syndromes. There are several key findings in this study:
These findings phenocopy those in APDS syndrome leading the authors to suggest naming PID syndromes due to p85α splice mutations activated PI3Kδ syndrome 2 (APDS2) since uncontrolled activation of the p110 subunit is responsible for many of the observed phenotypes. These data are especially intriguing given that mutations that decrease PI3K activity can also cause a PID phenotype. Together, these data suggest that careful titration of PI3K activity is critical for normal immune cell function and have important implications for drugs targeting this pathway. The data also suggest that this pathway should be interrogated in patients with abnormal immune cell function or recurrent infections. One could envision treating APDS1 and 2 patients with PI3K pathway inhibitors. However, given that aberrant PI3K activity has been implicated in lymphomagenesis, further studies modeling the phenotypic consequences of these mutations in murine models would be of value.
Reviewed by Michelle L. Hermiston, MD, PhD, University of California, San Francisco
A review of Xing L., et al. Alopecia areata is driven by cytotoxic T lymphocytes and is reversed by JAK inhibition. Nat. Med. 2014 Sep;20(9):1043-1049. PMID: 2529481
Alopecia areata (AA) is a T-cell-mediated autoimmune disease characterized by hair loss. While not life threatening, the psychological impact for those with severe disease can be significant. Current therapy for AA most commonly involves broad-acting steroids and is met frequently with limited success. The study by Xing, et al. provides new hope for these patients. Building upon a previous genome-wide association study showing that AA is associated with polymorphisms of the NKG2D receptor and additional work demonstrating infiltration of CD8+NKG2D+ T cells in the peribulbar region of human AA hair follicles, the authors explored the role of these cells in disease pathogenesis. They exploit the C3H/HeJ mouse model that develops spontaneous alopecia and recapitulates many histopathologic features of human AA. There are several interesting findings from this work.
Based on these findings, the authors propose a feed-forward model for AA where CD8+NKG2D+ T cells produce IFNγ that then induced production of IL-15 by follicular epithelial cells. This IL-15 further promotes survival and expansion of the infiltrating CD8+NKG2D+ T cells and loss of immune privilege in the hair follicle. The authors postulate that JAK inhibitors break this cycle by interfering with cytokine-mediated signals. Further clinical trials that address frequency of response and long-term safety issues in AA patients are clearly warranted.
Reviewed by Michelle Hermiston, MD, PhD, University of California San Francisco
A review of Cooper, Z.A., et al. Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade. Cancer Immunol Res, 2014; 2(7): 643-654. PMID: 24903021
Therapies targeting the oncogenic BRAF V600E mutation have yielded impressive but short-lived responses, with half of cases progressing after only 6 months. The authors have shown previously that treatment with BRAF inhibitors (BRAFi) leads to an upregulation in known melanoma antigens, resulting in T cell infiltration. The tumors seem primed for an immune response, however high levels of the inhibitory ligand PD-L1 in the microenvironment, along with other immunosupressive signals stall this response. Combining the two approaches of BRAF inhibition and immune checkpoint blockade is a promising strategy and, indeed clinical trials are ongoing. In this study, Cooper et al. show that this combination has strong potential for clinical success using a novel mouse model.
In this study, the authors demonstrate that BRAFi treatment leads to enhanced CD8+ Teff cell infiltration into tumors, but immune regulatory signals, such as the PD1-PD-L1 axis, oppose the infiltrating T cells and suppress the antitumor response. The possibility of combining the two powerful therapies to achieve a durable tumor response has inspired several clinical trials for which data is not yet available . Cooper et al. provide further evidence that these approaches are on the right track, while also creating a model system for elucidating mechanisms of synergy and resistance, and also determining optimal dosing schedules.
Reviewed by Alexander Hopkins and Eric Lutz, PhD, Johns Hopkins University
A review of Hong Qin, et al., Generation of a New Therapeutic Peptide That Depletes Myeloid-Derived Suppressor Cells in Tumor-Bearing Mice. Nature Medicine. 2014; 20(6): 676-81. PMID:24859530
Induction of a potent antitumor response requires circumventing the immunosuppressive environment of the tumor, including myeloid-derived suppressor cells (MDSCs). However, there are few defined surface markers for MDSCs, which limits studies of MDSCs within the tumor microenvironment. Using a peptide phage display platform, Qin et al. identify two peptides that specifically bind murine MDSCs and, when fused with the Fc region of mouse IgG2b to form a peptibody, can deplete MDSCs and delay tumor growth in mice. Immunoprecipitation experiments indicate that the proteins S100A9 and S100A8 may be the targets of these two peptibodies.
MDSCs are potent inhibitors of T cell function with few identified surface markers. The peptides identified by the competitive phage display platform identify two potential molecular markers of both monocytic and granulocytic MDSCs, S100A9 and S100A8, with limited or no cross-reactivity with proinflammatory DCs and lymphocytic cell populations. Furthermore, treatment with MDSC-specific peptibodies resulted in the depletion of both granulocytic and monocytic MDSCs; and delayed tumor growth with no visible off-target effects when compared to control tumor-bearing mice. These data suggest that this platform may be useful for identifying therapeutic targets and cell surface markers for specific cell subsets, including human MDSCs.
Reviewed by Heather Kinkead and Eric Lutz, PhD, John Hopkins University
|Highlights From Clinical Immunology, the Official Journal of FOCIS|
A review of Liu, et al. Toll-like receptor signaling directly increases functional IL-17RA expression in neuroglial cells. Clinical Immunology. 154:127–140. 2014. PMID:25076485
This paper helps clarify the progression of inflammatory events that occur during the development of EAE. An early event is innate signaling in microglial cells, via TLRs, to induce IL-17RA expression, allowing these cells to be receptive to IL-17 secreted by antigen-specific Th17 cells. The microglial cells then respond to the IL-17 by producing a variety of chemokines that promote further recruitment of inflammatory cells. IL-17 may also promote down-regulation of IL-17RA in astrocytes as a negative feedback mechanism.
Reviewed by Andrew H. Lichtman, MD, PhD, Brigham and Women’s Hospital
A review of Dai C., et al. Interferon alpha on NZM2328.Lc1R27:Enhancing autoimmunity and immune complex-mediated glomerulonephritis without end stage renal failure. Clinical Immunology. 154, 66–71. 2014. PMID: 24981059
A significant role for type I interferons, especially interferon α, in the pathogenesis of SLE has been inferred from gene expression profiles of blood leukocytes of SLE patients. Furthermore, some patients treated with IFNα show ANA, anti-dsDNA antibody responses and some develop SLE. However, longitudinal studies of IFNα levels do not appear to reflect disease activity in SLE patients, and trials of anti-IFNα antibody treatment have not been successful. The authors of this paper previously described the NZM2328 and NZM2328.Lc4 mouse models of lupus nephritis (LN), which they used to show that LN and end stage renal disease (ESRD) are under separate genetic control from autoantibody production. Those previous studies indicated that ANA and anti-dsDNA antibodies in their mice are not necessary for LN/ESRD. The authors also previously described a congenic derivative of NZM2328 line, called R27, which spontaneously develop immune complex-mediated glomerulonephritis (GN) but, unlike the parent strain, do not progress to chronic GN/ESR. In the current paper, the authors use the NZM2328 and R27 mouse lines, together with IFNα treatment via an adenovirus vector, to explore the genetic influence on IFNα induced SLE and LN. The major findings are:
The implications of this study are that IFNα can induce autoimmunity in mice, as previously described, but end organ damage is not directly mediated by IFNα and is regulated by distinct genetic factors. The authors suggest that the IFN signature in SLE patients and the lack of correlation of IFNα levels with clinical activity may reflect the importance of IFNα in initiating autoimmunity, but secondarily induced cytokine/inflammatory responses and susceptibility to end organ damage depend on independent genetic factors, as in their mice studies.
Reviewed by Andrew H. Lichtman, MD, PhD, Brigham and Women’s Hospital
|Human Immunophenotyping Update|
Holden T. Maecker, PhD, Stanford University
Building on the concept of the gene ontology, efforts have been made to construct a cell ontology (https://code.google.com/p/cell-ontology), which has been updated for hematopoietic cell types (2). This is a most noble effort, because the current state of cellular immunology lacks standard definitions for the different types of cells that it studies. Ideally, a cell ontology would provide minimal essential definitions of immune cell types based on their expressed markers and/or functions. Imagine the help that this would provide the field, if every time we referred to, for example, a regulatory T cell, one could be sure that it was a cell defined by a discreet set of commonly agreed-upon markers and/or functions.
Unfortunately, cells are much more diverse than genes. With high-dimensional techniques such as mass cytometry, hundreds of different phenotypes of CD8+ T cells can be defined by combinatorial expression of cytokines, for example (3). Similar diversity has been demonstrated in the NK cell compartment using combinations of stimulatory and inhibitory receptors (4). One could postulate that the number of discreet cell types is limited only by the number of markers being analyzed (and of course, by the total number of cells in the organism).
The fact that one cell type can give rise to another further complicates the definitions. Are differences that result from short-term activation sufficient to define a cell as a new type? What degree of stability is required to define a discreet cell type? And how do we handle markers that have a continuous distribution of expression, like CD38 in activated T cells? These are just some of the questions that complicate the ability to create a true cell ontology.
This is not to say that cells can’t be grouped into categories based on shared markers and/or functions. For example, most of us would agree that it’s useful to define CD4+ T cells as distinct from CD8+ T cells. And the definition of these groups is relatively easy, requiring only a few markers (CD3, CD4, and CD8). But even this simple case illustrates some difficulties of definitions. For example, CD4+ and CD8+ T cells can have overlapping functions: subsets of both cell types can be cytolytic, and they can produce common cytokines like IFNγ, TNFα, and IL-2. So, an ontology built strictly on functions would look very different from one based only on phenotypic markers.
As we progress to more esoteric cell types, the definitions get harder and harder. Precisely because of this, we have previously used this column to try to create some standardization in the way that important and controversial cell types are defined (e.g., Tregs, dendritic cells, Th17 cells, and B cells; see http://www.focisnet.org). Upcoming issues will continue this effort. Furthermore, the FOCIS Human ImmunoPhenotyping Consortium (HIP-C) has created standard 8-color panels for definition of basic immune cell types (5). Our experience with using these panels in lyophilized format across multiple labs is currently being prepared for publication. Although these panels were chosen by consensus of a large group of experts from academics, industry, and NIH, they don’t necessarily reflect agreement across the entire field of cellular immunology. And differences in how specific cell types are gated (and even how many different cell types to gate) can still be debated.
Another issue of course is the incorporation of new knowledge. At what point do we determine that a particular new marker is “essential” for defining a certain cell type? Is one publication demonstrating its utility enough? In some ways, what the cell ontology attempts to do is to synthesize the whole of the immunology literature, as it relates to cell types, in a single document/database. Such an enormous effort is sure to cause contention, and will require constant curation.
Given all these caveats, some suggestions for the advancement of the cell ontology are perhaps in order. First, it makes sense to concentrate first on the largest and most easily defined cell types. Dissecting the flavors of CD8+ T cells into ever-smaller and more narrowly defined subtypes, for example, is probably not that useful. But standard definitions for naïve, central memory, effector memory, and effector CD8+ T cells could be very helpful.
Second, there needs to be room for multiple options and equivalency in definitions. Building on the above example, the classical definition of a central memory T cell might be given as CD45RA-CCR7+, but under the right conditions, CD62L might be substituted for CCR7 (5). I say “under the right conditions”, because CD62L is labile and can be greatly reduced with cryopreservation or other perturbations (6). Other markers, like CD27 or CD127, might be options as well, but with their own caveats; they are not 100% equivalent to CCR7, but might be suitable for some purposes.
This brings up a final point, namely, the concept of “fit for purpose”. The HIP-C panel, by design, uses only cell-surface markers, for ease of use and standardization. This precludes, for example, the use of FoxP3 for definition of Tregs. Are surface markers truly sufficient to define Tregs? That is debatable, though I would say the loss of resolution is minor. The surface panel is, in effect, fit for the purpose of using a quick and easy staining panel. Another example: Are CD19 and CD20 both required to define mature B cells? Arguably it’s better to have both markers if possible, particularly if the labels used for either of them are dim, and might not provide sufficient resolution on their own. But if one is constrained to designing, say, a 4-color panel, one would probably chose just one of these markers for defining B cells.
All of this is to say that it’s not easy to synthesize the many nuances of immunophenotyping into a single document. Nevertheless, the discussions that take place in attempting this are probably themselves reason enough to pursue the effort. Furthermore, tools that help to apply the knowledge compiled in the cell ontology could also be helpful to the field. In that vein, the Brinkman lab has recently developed a tool, called FlowCL, that can automatically label populations with suggested cell types from the cell ontology (Courtot et al., manuscript submitted).
To summarize, we need to proceed with caution when attempting to summarize the immense diversity and plasticity of immune cells. Just as we don’t want to discount a study that fails to use one specific marker for definition of Tregs, we also don’t want to assume that a minimal definition of such cells is suitable for all applications.
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