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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 in Recent Literature | Clinical Immunology Highlights | Basic Immunology & Novel Therapies | ImmunphenotypingPDF VersionPrevious Issues

Highlights from Recent Literature

Signaling via the EGF Receptor Increases Immunologic Visibility

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

NK cells and cytolytic T cells can recognize and destroy stressed, damaged and malignant cells via the interaction of the lymphocyte activating receptor NKG2D with NKG2D ligands expressed on stressed or damaged cells. The expression of NKG2D ligands therefore enhances “immunologic visibility” of these cells. The authors studied the mechanisms by which NKG2D ligands are up regulated on human epithelial cells.

• The authors stressed human epithelial cells by exposing them to ultraviolet radiation, osmotic shock, oxidative stress and treatment with growth factors.

• They observed that up-regulation of NKG2D ligands after these treatments were mediated by activation of epidermal growth factor receptor (EGFR) signaling.

• The authors found that a family of proteins, termed AUF1 proteins, normally destabilizes NKG2D ligand mRNAs by targeting a conserved AU-rich element present in the NKG2D ligand mRNAs of humans. Interestingly, NKG2D ligand mRNAs in mice did not have these elements.

• Triggering of the EGFR led to intracellular relocalization of these AUF1 genes, blocking their destabilization of NKG2D ligand mRNAs and allowing NKG2D ligand expression on the surface of cells.

• EGFR signaling is commonly hyper activated in human epithelial carcinomas, and NKG2D ligand expression by primary human carcinomas correlated with EGFR expression.

• Clinically available EGFR inhibitors reduced NKG2D ligand expression and might be expected to decrease immunologic visibility of these tumors.

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


Nanoring Adjuvants Show Potent Vaccine Adjuvant Activity

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.
• The activity of D-ODN is dependent on their ability to form complex multimeric structures, complicating attempts to manufacture them for use in clinical trials.

• The authors used the HIV derived cationic peptide Tat(47-57) to nucleate the formation of aggregates from usually non-multimerizing K-ODNs. They found that these structures formed stable, nuclease resistant nanorings.

• These nanorings, when added to human pDC in culture, were targeted to the early endosomes and induced to robust secretion of IFNα.

• In a mouse vaccination model, these ODN nanorings increased Th1 responses after immunization with a vaccine composed of inactivated foot and mouth virus.

• In a mouse tumor immunotherapy model, a therapeutic tumor vaccine containing ODN nanorings induced superior antitumor immunity against EG.7 thymoma tumors.

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.

Reviewed by Rachael A. Clark, MD, PhD, Brigham and Women's Hospital


The 'Big Dig' - Leveraging Electronic Media Records and Genomic Data

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

Commentary:
E Bowton, et al. Biobanks and Electronic Medical Records: Enabling Cost Effective Research. Science Translational Medicine. 6, 234cm3 (2014). PMID: 24786321

As electronic medical records (EMR) become more common, there is an increasing body of research that delves into their details to better understand human health. These two studies use very different techniques to extract biomedical knowledge from EMR. The first focuses on the theory that clinical parameters that are not directly connected with disease (‘traits’) and diseases can be connected by identifying shared single nucleotide polymorphisms, thus identifying new risk factors for those diseases. The second presents a linked biorespository and EMR within the Vanderbilt medical system as an exemplar of building systems to facilitate both cost and time efficient research.

SUMMARY

  • Li et al. briefly review the extensive genome wide association study (GWAS) literature that has mapped single nucleotide polymorphisms linked to traits (i.e. clinical demographics, personal behaviors and disease susceptibility). Given that some SNPs can be linked to multiple conditions and traits, the question of whether those traits and diseases may share a common mechanism has been raised.

    • This study seeks to link non-disease traits and diseases by identifying pairs with shared genetic variation. This could then allow assessment or measurement of traits which could serve as predictive risk factors for disease initiation or progression.
    • VARIMED, a database of disease-SNP associations, is used to discover the disease-trait pairs. They began by identifying 801 genes linked to 69 diseases and 796 genes linked to 85 non-disease traits (requiring at least 3 significant associations per disease or trait).

      • Interestingly, the three diseases with the most associated genes were rheumatoid arthritis, membranous nephropathy and myocardial infarction.

    • Next, they found 120 disease and non-disease trait pairs that shared genetic variants (when correlated with the literature, 94 were published previously, and were either risk factors, diagnostic tests or consequences of progression).

    • These disease-trait pairs were used to build a network, and disease-trait pairs that shared genetic variation with other pairs were grouped together, leading to 8 disease modules (diseases linked to one or a few traits via shared genetic variation) and 7 trait modules.

      • Some of these disease modules were already known, including autoimmunity linking to antibody titers due to association with MHC I and II.

    • For the novel disease-trait pairs (26 out of 120), they examined the EMR data from three medical centers across the US and validated the 5 that could be purely electronically assessed. For example, they had identified MCV and acute lymphoblastic leukemia as both being linked to variants in IZKF1 and found in EMR data that patients with an abnormal MCV were more likely to be diagnosed within 12 months with ALL than patients with normal MCV. In addition, they validated a link between abnormal serum magnesium and an increased likelihood of gastric cancer, prostate specific antigen (PSA) and lung cancer, abnormal alkaline phosphatase and venothromoboembolism and abnormal platelet counts and alcohol dependence syndrome.

  • Bowton et al. present the Vanderbilt Electronic Systems for Pharmacogenomic Assessment (VESPA), which includes almost 180,000 DNA samples, with more than 11,000 genotyped subjects, and was assessed in this study for 28 clearly defined phenotypes.

    • They highlight the importance of disease agnostic patient inclusion and the inclusion of stakeholders from multiple specialties in the design of the pipeline and project.

    • They were able to develop both fully and semi-automated phenotype analysis algorithms, which used ICD-9 codes, medication lists and laboratory results for classification, in addition to natural language processing and other advanced techniques in some cases, with special attention to the temporal relationships between symptoms and drug administration (given the focus on pharmacogenomics).

    • This study is able to assess the potential cost savings (though, as they point out, the costs of establishing some of the components of VESPA, as well as the EMR itself, are not included as they had already been completed), which are substantial when compared to a traditional prospective, question/disease focused sample collection study. For example, they calculate that their cost per genotyped subject of $393, versus $1419 in a more standard study (calculated based on medians for NIH funded studies with similar goals).

    • They discuss the challenges of uniting data from different genotyping platforms and the inherent loss to follow-up when repositories are de-identified (so that EMR data is only available up to the point of sampling and inclusion in the biorepository).

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. 

Both studies challenge us to imagine how to synergize the massive datasets that we are collecting clinically and in our labs on a daily basis, and how to bring those datasets to bear on human health.

Reviewed by Sarah Henrickson, MD, PhD, Children’s Hospital of Philadelphia


What Does it Mean to be Healthy?

 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.

SUMMARY

  • The study begins describing how the 27 assays were established (from a starting point of 54 potential assays). All of the assays were designed to be conducted using a commercially available system that allows blood to be drawn directly into a syringe-based apparatus that is pre-loaded with culture media with the stimuli of interest. Using a whole blood (rather than PBMC based) system precludes the need for manipulation of the samples (with the potential for contamination during transfers and cell loss and response alteration with cryopreservation), but also prevents the isolation of individual cell subsets. All assays are evaluated using Luminex assays to assess protein levels of 29 chemokines, cytokines and growth factors.

  • The selected assays include whole microbes (e.g. heat killed E. coli), purified TLR and other microbe associated molecular pattern (MAMP) agonists (e.g. LPS), cytokines (e.g. IFN-α) and T cell agonists (e.g. anti-CD3, anti-CD28) among others. They describe their dose selection process, with the goal of minimizing the variation secondary to technical artifacts.

  • Next, they describe their patient sample collection procedures, which are done as part of the Milleu Interieur project. This study includes samples from 25 healthy donors of European background, with gender balance.

  • First, they examine the entire data set, including the levels of all 29 assessed proteins in response to all 27 stimuli using PCA analysis of all 25 donors. The first three PC vectors account for more than half of the total variance, and include PC1 (based on chemokines and cytokines), PC2 (based on induction of IL4, 5, 2 and GM-CSF) and PC3 (based on induction of CXCL10 (IP-10) and IL-12p70.

  • When they focus on two TCR stimuli (anti-CD3 and anti-CD28), they interestingly identify 6/25 of their donors who respond to SEB, but not to anti-CD3/28 (though they bind anti-CD3 appropriately).

  • When microbes, MAMPs and cytokine stimulations were assessed (using hierarchical clustering and PCA analysis) they were able to separate each category into sub-categories, with differential induction of proteins (both in terms of which proteins were induced and the degree to which they were induced). For example, E. coli and C. albicans each yielded unique signatures.

  • In order to derive a preliminary understanding of the range of ‘normal’ immune responses to their assays, they constructed radar plots to allow simultaneous visualization of the range of responses among their patients. Interestingly, they found that 2/25 patients did not secrete IL-1α in response to any of the stimulation conditions (while producing other cytokines in ranges like the rest of the cohort), and argue that having developed robust, quantifiable assays allows investigators to identify outlier values within a normal population (and get a more accurate definition of ‘normal’ itself).

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.

Reviewed by Sarah Henrickson, MD, PhD, Children’s Hospital of Philadelphia 


Reestablishing the T Cell Repertoire Post Stem Cell Transplant

 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.

  • The clones were grouped based on their V-J usage and ranked according to their clonotypic frequency, which was calculated as a fraction of the whole library for each sample. Pre-transplant, CD4+ T cells showed greater diversity relative to CD8+ T cells.

  • Interestingly, transplant led to further restriction in diversity for both CD4 and CD8 T cells. However, the dominant CD4 T cell clones present prior to transplant were generally undetectable following reconstitution with most patients developing a predominantly new CD4+ repertoire.

  • One potential explanation for this finding is that the alterations in the repertoire are due is natural repertoire ‘drift’ over time in patients. However, analysis of the T cell repertoire taken 1 year apart in healthy donors suggested that the T cell repertoire in adults is normally quite stable. The stability of the repertoire in non-transplanted MS patients was not addressed.

  • In contrast, the reconstituted CD8+ repertoire continued to retain a high proportion of pre-transplant CD8+ cells, suggesting that the CD8 repertoire was created primarily by clonal expansion of pre-transplant cells.

  • Importantly, the diversity of the T cell repertoire at two months post-transplant correlated with treatment outcome. Patients who responded had a trend to a more diverse repertoire of both CD4+ and CD8+ T cells relative to non-responders.

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


New Insight into IVIg Therapy in Humans: IL-4 and IL-13 Suppresses DC Maturation in vivo

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

Intravenous immunoglobulin (IVIg) therapy has been efficacious in treating various autoimmune and immunodeficient syndromes. Despite its success, its mechanism of action is incompletely understood. Research using murine models suggests that IVIg treatment induces a cascade of IL-33-T helper 2 (Th2) cytokines in the spleen leading to upregulation of the inhibitory receptor FCγRIIb on macrophages and dendritic cells (DCs) so that they are no longer activated by immune complexes. Other studies suggest that IVIg may also modulate regulatory T cell activity. However, splenic architecture in humans is quite distinct from the mouse and whether these processes function similarly in humans is less well understood. Moreover, splenectomized patients with idiopathic thrombocytopenic purpura (ITP) often still respond to IVIg, suggesting the spleen may not be essential for IVIg action. To address these knowledge gaps, the authors study patients with autoimmune disease or immunodeficiency receiving high dose (HD; >6g/kg) IVIg therapy and immunodeficient patients receiving low dose IVIg therapy (LD; <6g/kg). All patients responded to the therapy. There are several interesting findings from this work.

  • Similar to murine models, plasma levels of IL-33 increased significantly for at least 7 days after both low and high dose IVIg. This correlated with increases in plasma levels of the TH-2 cytokines IL-4 and IL-13. Only transient increases in Th1 cytokine IFN-γ and IL-10 were observed.

  • Surprisingly, unlike the murine models, IVIg was not associated with upregulation of the inhibitory receptor FcγRIIb. Rather, HD, but not LD, IVIg was associated with a sustained decrease in expression of the activating receptor FcγRIIa and IFN-γR2 and a transient decrease in the inhibitory receptor FcγRIIb on circulating myeloid dendritic cells.

  • Unlike murine models where IVIg has been proposed to cause Th2 cytokine-mediated upregulation of FcγRIIb on macrophages, exposure of human monocyte-derived macrophages did impact FcγRIIa or FcγRIIb expression.

  • In vitro exposure of myeloid dendritic cells from healthy human donors to recombinant IL-4 or IL-13 recapitulated the decrease in FcγRIIa and IFN-γR2 expression and prevented activation by immune complex. Incubation in vitro with IL-33 had no effect.

  • Using cells procured from organ donors, the authors demonstrate that human lymph node cells but not splenocytes were a potential source of IL-33 production. Macrophages, but not dendritic cells, derived from circulating monocytes also produced IL-33 in response to IVIg exposure.

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

Establishment of oral tolerance to food antigens is a complex process involving various immune and non-immune cells that reside at the intestinal tissue. Among these, CX3CR1+ macrophages exhibit remarkable efficiency in sending protrusions to the intestinal lumen to capture antigens. On the other hand, CD103+ dendritic cells, though relatively inefficient in lumenal antigen capture, play a crucial role in establishing tolerance by migrating to the draining mesenteric lymph nodes (MLNs) on antigen acquisition and inducing regulatory T cells via the release of retinoic acid and TGF-beta. Recent findings by Mazzini and coworkers reveal an interesting mechanism of division of labor between these two APC subsets of distinct functional specialties by which the gap between lumenal antigen capture and tolerance induction at MLNs is bridged.

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

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Highlights From Clinical Immunology, the Official Journal of FOCIS

Commensal Bacteria, Antigen Mimicry and Autoimmunity

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

Antibodies specific for the ribonucleoprotein Ro60 (also called Sjögren's syndrome Antigen A (SSA)) are commonly found in patients with Sjögren's syndrome and SLE, and their presence is associated with pathological and clinical severity of these disorder. The authors had previously shown that Ro60 antibody responses required a loss of T cell tolerance to Ro60, and they propose that the autoantibody responses in SLE and Sjögren's s depend on T cell responses to these antigens. Furthermore, they hypothesized that molecular mimicry with commensal bacterial may underlie these T cell dependent autoantibody responses. To test his hypothesis, the authors first generated HLA-DR3 transgenic mice, which lack mouse DR, because of the strong association of DR3 with SLE and Sjögren's syndrome. They immunized these mice with human Ro60, restimulated draining lymph node cells with Ro60, and then generated and cloned Ro60-reactive T cell hybridomas. They then performed Ro60 epitope mapping studies to determine exactly which Ro60-derived peptides the hybridomas were responding to and they followed up with BLAST searches for homologous commensal microbial peptides. The major findings were:

  • HLA-DR3 restricted T cell hybridomas were generated that recognized 3 epitope regions of Ro60: Ro60221–250, Ro60241–260 and Ro60361–390. The first two were identified in a previous study from this group, but Ro60221–250 was a newly defined T cell epitope.

  • A hybdridoma reactive to Ro60361–390 was tested for cross reactive with 22 homologous bacterial peptides, and 11 tested positive.

  • The bacterial peptides that cross reacted with Ro60361–390 were derived from the von Willebrand factor (vWFA) domain superfamily of proteins, and the strongest reacting peptide came from a vWFA family protein from the oral cavity commensal Gram negative bacteria C. ochracea.

  • Antigen presenting cells (APCs) pulsed with whole recombinant C. ochracea vWFA protein or APCs exposed to E. coli expressing the protein were able to activate the Ro60-specific hybridomas. Attempts to activate the hybridomas with intact C. ochracea were not successful, presumably to do suppressive factors made by the bacterium.

  • In addition, several other microbial peptides derived from other commensal were identified in the BLAST search, and many proved to activate Ro60-speifcic hybridomas. Some of these peptides were derived from vWFA proteins, but not all. The proteins were products of microbes in oral flora (Prevotella disiens, Capnocytophaga sputigena and Capnocytophaga ochracea), gut flora (Bacterioides finegoldii, Bacteroides intestinalis, Bacteroides fragilis and Alistipes finegoldii), and skin (Corynebacteriium amycolatum and A. johnsonii) and vagina (Prevotella disiens).

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


Individual Plasmablast Ig Sequencing in Acute S. Aureus Infection

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

Staphylococcus aureus (S. aureus) infection, including infections with antibiotic resistant strains (e.g. MERSA), is a serious clinical problem of increasing frequency. Although it is clear that both humoral and T cell immunodeficiency cause increased risk for such infections, the mechanisms of immune defense against S. aureus are incompletely understood. The authors of this paper sought to better characterize the antibody responses to S. aureus by cloning genes encoding bacteria specific antibodies from circulating plasmablasts of acutely infected bacteremia patients. They determined the specificities of the antibodies, and determined the mechanisms they may engage for anti-microbial defense. This study relied on FACS sorting and cloning individual plasmablasts, and DNA barcoding-enabled deep sequencing of the paired heavy- and light-chain Ig genes in the cloned cells. The major results and findings were:

  • Circulating plasmablasts (CD19+/int/CD20/CD27++/CD38++) accounted for on average 3.2% of blood B cells in patients with S. aureus bacteremia, a 10 fold increase over normal controls.

  • Single cell sequencing of IgG heavy and light chain genes in the circulating plasmablasts of bacteremic patients revealed clonal “families” with shared IGHV and IGHJ alleles, and recombinant antibodies from these families were enriched for S. aureus binding Igs.

  • S. aureus proteome microarrays were used to identify the antigens the S. aureus-specific recombinant Igs bound to. The antigens included various virulence factors and surface antigens.

  • In vitro assays showed that some of the recombinant S. auresu binding antibodies promoted opsonization for phagocytosis, and one of the antibodies prevented S. aureus colony growth.

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

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Developments in Basic Immunology and Novel Therapies

Immune Responses in Atherosclerosis: An Opportunity for New 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.

The molecules that simulate innate and adaptive immune responses in the setting of atherosclerosis are, in large part, generated in the unique pathological microenvironment of the intima of muscularized arteries at particular predilection sites. At these locations, several factors including turbulent blood flow, toxins, and advanced glycation end-products work in concert to enhance the permeability of the endothelium for LDL, which promotes LDL binding to and retention by extracellular matrix beneath the endothelial basement membrane. Elevated LDL cholesterol levels will result in more LDL deposition and retention. Once retained, LDL is oxidatively modified, generating a variety of oxidation specific epitopes of the various components of LDL, including phospholipids, cholesterol, fatty acids, and the ApopB100 protein. Oxidation specific epitopes (OSEs) generated by LDL-oxidation are recognized by innate immune pattern recognition receptors, including scavenger receptors and TLRs. Remarkably, a large fraction of natural IgM antibodies produced in mice and humans specifically bind LDL-derived OSEs, such as phosphorylcholine (PC). In the typical fashion of innate immune driven inflammatory responses, inflammatory monocytes (Ly6Ghi-mouse; CD14hi -human) are recruited to the intima because oxidized-LDL (Ox-LDL) stimulates EC adhesion molecule expression (e.g. VCAM-1) and chemokine expression (e.g. MCP1). Once within the intima, the monocytes differentiate into phagocytic and pro-inflammatory (M1-like) macrophages, which engulf retained Ox-LDL, and they become cholesterol-laden foam cells. Cytosolic cholesterol crystals that form in early foam-cells can activate the inflammasome, generating IL-β, which promotes the local inflammatory milieu. In response to growth factors and chemokine’s elaborated by activated macrophages, medial smooth muscle cells migrate into the expanding intima, and change their phenotype form contractile to synthetic cells, producing extracellular matrix proteins, including collagen. Eventually, cholesterol accumulation in macrophages leads to their death, and the generation of a necrotic core of pro-inflammatory and pro-thrombotic material. Over time, a classic chronic inflammatory site develops in the arterial wall, with fibrosis, dystrophic calcification, activated lymphocytes and macrophages, and tissue remodeling. The contributions of various components of the innate immune system to lesion development have been interrogated in mouse models (mainly LDL-receptor [LDLR-R] or Apolipoprotein E [ApoE] deficient mice). In these mouse models, hypercholesterolemia is readily achievable and lesions develop in the aorta and its proximal branches with most of the major features of human lesions. Antibody-blockade or genetic deficiency of innate immune cytokines and chemokines (e.g. IL-1, MCP1), or pattern recognition receptors (e.g. TLRs) reduce disease burden in atherosclerosis-prone mice, while targeting IL-10 or TGFβ enhance disease.

Adaptive immunity in atherosclerosis includes both T cell and antibody responses. T lymphocytes are recruited into developing intimal lesions of atherosclerosis as early in the process as the fatty-streak stage, when a foam cells have already formed, but little arterial wall remodeling has occurred. T cells continue to be present throughout the decades-long life history of the lesions. These T cells are mostly αβ TCR+ CD4+ cells that produce IFNγ. IL-17 or IFNγ/IL-17 producing T cells are also reported to be present in human and mouse lesions. CD8+ T cells are consistently present but less frequent than CD4+ T cells, but their numbers increase when certain immunoregulatory mechanisms are blocked. The specificity of arterial lesional T cells has not been established clearly, as is the case for the spectrum of human autoimmune disease in which T cells contribute, however T cells reactive with 0x-LDL and apoB100 peptides have been detected. Furthermore, IgGs specific for 0x-LDL that show the hallmarks of T cell help (class switched, high affinity) are readily detectable and significantly higher in patients with atherosclerotic disease than healthy controls. T cells and IgGs specific for HSP60 are also markers of atherosclerotic disease in mice, rabbits and humans. Interestingly, intimal dendritic cells (DCs) are present specifically in atherosclerosis prone areas of the arterial tree, such as branch points and the lesser curvature of the aortic arch, even before any lesion development is delectable, and monocyte-derived DC increase in numbers as inflammatory events ensue. These lesional DCs and the monocyte-derived macrophages express class II MHC. The antigen-presenting capabilities of these lesional DCs in mice have been demonstrated two-photon intravital microscopic methods. Antibody or genetic targeting of molecules required for lymphocyte development (e.g. Rag1/Rag2, CD4), naïve T cell activation (e.g. B7-1/2), and TH1 cell differentiation or function (e.g. IFNγ, T-bet, IL-12, IL-18, CD40 ligand), all reduce atherosclerotic disease and reduce lesional inflammatory phenotypes in LDL-R or ApoE KO mice. Treatment or genetic manipulations that reduce Treg numbers or functions enhance lesion development (e.g. anti-CD25), while methods to increase Treg (e.g. adoptive transfer of Treg or IL-2/anti-IL-2 treatment) reduce lesion development. Targeting T cell inhibitory molecules (e.g. PD-1 or PD-L1) enhance lesion development and both CD4+ and CD8+ responses in atherosclerotic-prone mice. Manipulation of B cells and antibody responses have yielded complex results, reflecting the possible protective role of some anti-0xLDL antibody responses (e.g. natural antibodies that may prevent foam cell formation) and pro-atherogenic roles of other B cells subsets, perhaps though their effects on T cells.

A significant majority of clinical events in coronary artery atherosclerotic disease reflect acute structural changes to advanced lesions (plaque rupture, hemorrhage), resulting the exposure of circulating blood to the highly thrombogenic material in the necrotic core. Good mouse models of acute athero-thrombosis are not available, but clinical-pathological correlations of human lesions taken at autopsy indicate that the “unstable” plaques that are associated luminal thrombosis and acute myocardial infarction often have thin fibrous plaques with focal rupture overlying the lesional necrotic core. Importantly, compared to stable plaques, in the unstable plaques there is evidence of more inflammation, including increase numbers of macrophages and T cells, matrix metalloproteinase expression, and T cell-macrophage interactions. Studies of peripheral blood and of coronary artery blood sampled during percutaneous coronary intervention also indicate that patients with acute coronary syndrome/acute myocardial infarction have a pro-inflammatory state (more activated T cells, less Treg, more inflammatory cytokines) compared to patients with stable angina (which correlates with thick fibrous caps and less plaque inflammation).

The evidence reviewed above that innate and adaptive immune responses promote atherosclerotic lesion development and decrease plaque stability has stimulated efforts to develop immunotherapies for atherosclerosis. Statin therapy reduces LDL cholesterol, and has a clear and significant impact in reducing risk for clinical atherosclerotic disease. Statin therapy is associated with a reduction in a variety of systemic inflammatory responses to hypercholesterolemia, a reduction in plaque inflammation, and stabilization of plaques. Although much of the anti-inflammatory effects of statins is likely to be related to reducing the central driver of lesion inflammation, namely LDL-cholesterol, statins have in addition, cholesterol-independent anti-inflammatory effects on endothelial cells, macrophages, and T cells. Nonetheless, even with ubiquitous statin-therapy, approximately 30% of cardiovascular disease patients do not have undesirable blood levels of LDL-cholesterol nor other traditional risk factors, suggesting that targeting inflammation may be a reasonable strategy to impact disease in this population. Clinical trials ( e.g. JUPITER) that showed the effectiveness statin therapy in reducing cardiovascular risk in patients with elevated hsCRP but not elevated cholesterol, supports this approach. The first drug trials that target inflammation in order to reduce myocardial infarction or stroke are underway, including CANTOS, which targets IL-1β with a monoclonal antibody, and CIRT, which uses low dose methotrexate, in a similar manner as has been used in RA patients. Importantly, patients at high risk for clinical atherosclerotic disease because of various autoimmune diseases are now being treated with cytokine inhibitors (TNF, IL-6, IL-17, IL-12/-17, BAFF), T cell costimulatory inhibitors (CTLA4-Ig), and B cell depleting antibodies (anti-CD20). Many of the targets of these drugs have been shown to be pro-atherogenic in mouse models. The influence on these treatments in reducing cardiovascular events in patients, and the impact of the treatments on arterial lesion development, stability, and inflammation, requires careful study in order for us to better refine safe interventions that have an impact on atherosclerosis.

References
1. Libby P, Lichtman AH and Hansson GK. Immune Effector Mechanisms Implicated in Atherosclerosis: From Mice to Humans. Immunity 2013; 38:1092-1104.

2. Witztum J. and Lichtman AH. The Influence of Innate and Adaptive Immune Responses on Atherosclerosis. Ann Rev Path: Mech Dis. 2014; 9:73–102.

3. Ridker PM. Moving beyond JUPITER: will inhibiting inflammation reduce vascular event rates? Curr Atheroscler Rep. 2013 15:29

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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
J. Philip McCoy, Jr. 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.

References
1. Maecker, H. T., J. P. McCoy and R. Nussenblatt (2012). "Standardizing Immunophenotyping for the Human Immunology Project." Nat Rev Immunol 12(3): 191-200.

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.


Figure 1.
The myriad of B cell subsets that can be defined by high dimensional flow cytometry.
The area highlighted in blue shows representative subsets that can be identified by the FOCIS HIPC panel. The unshaded area shows subsets identified by a larger 15-color panel. Even more subsets could be identified when using more markers.

update


The area in blue shows a gating strategy to identify specific B cell populations. The area outside of the blue shows the array of markers that could be examined on each of these populations. Please note that not all of these markers may be expressed on each population, and in some instances could be used to verify the lack of expression of a marker on various subsets.  Not all of the potentially identifiable populations listed here have been biologically described.

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

Clinical Trials:
Wallace, D., et al. Efficacy and Safety of Epratuzumab in Patients with Moderate/Severe Active Systemic Lupus Erythematosus: Results from EMBLEM, a Phase IIb, Randomized, Double-blind, Placebo-controlled, Multicentre Study. Ann Rheum Dis. 2014 Jan; 73(1): 183-190.
PMID: PMC3888603

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.

Study design:

  • Phase 2b randomized, double-blind, placebo controlled multicenter trial

  • 227 patients with moderate/severe active SLE randomly assigned to one of six treatment groups: placebo or one of five different cumulative doses (cd) of epratuzumab from 200 mg to 3600 mg. Infusions were given weekly at weeks 0, 1, 2, and 3.

  • Efficacy evaluations at weeks 4, 8, and 12.

  • The primary efficacy endpoint was the response rate at 12 weeks according to a composite endpoint, BICLA (BILAG-based Combined Lupus Assessment,) and no treatment failure.

Results:

  • No statistically significant differences between placebo and treatment arms at week 12 were found, though the responder rate in the placebo arm was lower than in the epratuzumab arms.

  • Exploratory analysis demonstrated clinical improvements by week 8 only in patients receiving a total dose of 2400 mg epratuzumab.

  • Rates of AEs and serious AEs were similar across all groups.

Why The Trial is of Interest to the Broader FOCIS Community:

There is an unmet clinical need for better SLE treatments. Clinical trials in SLE are difficult to conduct due to marked disease heterogeneity and the difficulty of predicting disease prognosis for individuals. A better understanding of the pathogenesis of SLE has led to the development of therapies targeting B cells, but results have been mixed and have raised many important questions about how to study SLE generally and how to target B-cells in particular.

This is the third randomized trial with Epratuzumab which targets CD22, expressed mostly on mature B cells. Results are inconclusive. As noted, though exploratory analysis suggested clinical effect in one dose group, this study failed to meet its primary endpoint. The two previous trials with this drug suggested efficacy with reduced disease activity scores and corticosteroid use (5), but were terminated early due to interruption of drug supply.

Similarly, despite early exploratory studies hinting at efficacy (6-8), two phase III clinical trials using rituximab (an anti-CD 20 monoclonal Ab) also did not meet primary endpoints(1,2).

These studies are in contrast with two phase III trials of belimumab (an anti-BLys mAb which blocks B cell maturation and antibody production) which demonstrated clinical efficacy (3, 4). Belimumab is now FDA approved for treatment of SLE.

A key question is whether these different clinical trial outcomes are due to differences in clinical trial design and implementation or whether these results are informative about the pathophysiology of disease since each agent targets B cells at different stages of development and function.

While all clinical trials involve multiple decisions that can markedly impact study results, clinical trial design in SLE is particularly difficult. Key issues include (a) entry criteria; or how to select trial subjects from the highly clinically heterogeneous SLE population, (b) endpoint measures; different trials have used different disease activity scoring systems, all of which are somewhat subjective, (c) powering a study to detect important effects; in addition to disease heterogeneity, subjects often continue their usual SLE treatments, including corticosteroids, making it difficult to see a treatment effect. As suggested in this study, it is also likely that dose and dosing interval are important, yet enrolling sufficient subjects with SLE to test multiple dosing variations may not be feasible.

Collectively, the primary clinical outcomes alone in trials of B cell depletion in SLE are unlikely to provide answers to the etiopathology of disease and serve to emphasize the importance of carefully considered mechanistic based ancillary studies to provide hints for future therapies.

References:
1. Furie R, Looney RJ, Rovin B, et al Efficacy and safety of rituximab in subjects with active proliferative lupus nephritis (LN): results from the randomized, double blind phase III LUNAR study Arthritis Rheum 2009;60:S429

2. Merrill JT, Neuwelt CM, Wallace DJ, et al Efficacy and safety of rituximab in moderately to severely active systemic lupus erythematosus: the randomized, double-blind, phase II/III systemic lupus erythematosus evaluation of rituximab trial. Arthritis Rheum 2010;62:222-33

3. Navarra SV, Guzman RM, Gallacher AE, et al Efficacy and safety of belimumab in patients with active systemic lupus erythematosus: a randomized, placebo-controlled, phase 3 trial Lancet 2011;377:721-31

4. Furie R, Petri M, Zamani O, et al A phase III, randomized, placebo-controlled study of belimumab, a monoclonal antibody that inhibits B lymphocyte stimulator, in pateints with systemic lupus erythematosus, Arthritis Rheum 2011;63:3918-30

5. Petri M, Hobbs K, Gordon C et al, Clinically meaningful improvements with epratuzumab (anti CD22 mab targeting b-cells) in patients (pts) with moderate/severe systemic lupus erythematosus (SLE) flares: results from 2 randomized controlled trials, Arthritis Rheum 2008; 57(Suppl): 1088

6. Leandro MJ, Cambridge G, Edwards JC, Ehrenstein MR, Isenberg DA: B-cell depletion in the treatment of patients with systemic lupus erythematosus: a longitudinal analysis of 24 patients, Rheumatology 2005, 44:1542-1545

7. Ng KP, Leandro MJ, Edwards JC, Ehrenstein MR, Cambridge G, Isenberg DA: Repeated B cell depletion in treatment of refractory systemic lupus erythematosus, Ann Rheum Dis 2006, 65:942-945

8. Ng KP, Cambridge G, Leandro MJ, Edwards JC, Ehrenstein M, Isenberg DA: B cell depletion therapy in systemic lupus erythematosus: long-term follow up and predictors of response, Ann Rheum Dis 2007, 66:1259-1262

Reviewed by Sandra Lord, MD, Benaroya Research Center


Atacicept in Multiple Sclerosis (ATAMS): A Randomixed, Placebo-Controlled, Double-Blind, Phase 2 Trial

Clinical Trial:
Kappos, L., et al. Atacicept in Multiple Sclerosis (ATAMS): a Randomized, Placebo-Controlled, Double-Blind, Phase 2 Trial. The Lancet Neurology, 2014; 13; 4: 353-363. PMID: 24613349

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.

Study design:

  • Phase II randomized, double-blind, placebo controlled multicenter trial assessing efficacy and safety in patients with relapsing-remitting multiple sclerosis

  • 255 subjects randomly assigned to receive weekly injections of either placebo or atacicept for 36 weeks.

  • Primary endpoint was the change in mean number of gadolinium-enhancing lesions on T1-weighted MRI per patient per scan between weeks 12 and 36.

Results:

  • Study was terminated early after relapse rates in all atacicept groups more than doubled compared to placebo.

  • Mean numbers of gadolinium-enhancing T1 lesions per scan were similar in all groups

  • Seven patients (6 in atacicept and 1 in placebo) discontinued due to adverse events.

Why The Study is of Interest to the Broader FOCIS community:

Increased expression of BLyS and APRIL is seen in monocytes and T cells of patients with MS (1-3), which makes these cytokines attractive targets. Furthermore, the anti-CD 20 monoclonal antibodies rituximab and ocrelizumab have been shown to suppress clinical relapses and inflammatory lesions on MRI in patients with RRMS (4-7). CD 20 is expressed at all stages of B cell development except the first and last (plasma cells) which suggests that rituximab and ocrelizumab (anti CD 20 antibodies) cause broad suppression of B cell function. Atacicept blocks a later stage of B cell development compared to rituximab and ocrelizumab; notably, B cell progenitors and memory B cells are not depleted by atacicept. There is no clear explanation for the unexpected negative outcome of this study; however, it may be that Atacicept depleted IL-10 producing B cells which would curb regulatory function.

While the study was stopped due to increase in clinical relapse, the planned primary endpoint measurement in this study was a change in mean number of inflammatory lesions seen on MRI. It is of interest that this did not differ between placebo and treatment groups despite the increase in clinical relapse with therapy. A recent meta-analysis showed a close correlation between a treatment-induced remission and reduced MRI lesions (8). The lack of a strong correlation between relapse and MRI findings seen in this study raises the question of whether a spontaneous relapse is different from a treatment-induced relapse. In other words, a treatment-induced relapse could be driven by direct cytokine effects, whereas a spontaneous relapse is related to cellular invasion and damage, which would be more apparent on MRI.

Like the different outcomes seen with various anti B cell therapies in SLE, the transient worsening of clinical disease using Atacicept in MS only highlights how little is understood about disease mechanisms, the potential targets, and most importantly the “downstream” effects of targeted therapy.

References:
1. Thangarajh M, Gomes A, Masterman T, Hillert J, Hjelmstrom P Expression of B-cell-activating factor of the TNF family (BAFF) and its receptors in multiple sclerosis, J Neuroimmunol 2004; 152: 183-90

2. Thangarajh M, Masterman T, Rot U, et al Increased levels of APRIL (a proliferation-inducing ligand) mRNA in multiple sclerosis J Neuroimmunol 2005; 167: 210-14

3. Thangarajh M, KisiswaL, Pirskanen R, Hillert J The expression of BAFF-binding receptors is not altered in multiple sclerosis or myasthenia gravis Scand J Immunol 2007; 65: 461-66

4. Bar-Or A, Calabresi PA, Arnold D, et al Rituximab in relapsing-remitting multiple sclerosis: a 73-week, open label, phase 1 trial, Ann Neurol 2008; 3: 395-400

5. Hawker K, O’Connor P, Freedman MS, et al, Rituximab in patients with primary progressive multiple sclerosis: results of a randomized double-blind placebo-controlled multicenter trial, Ann Neurol 2009; 66: 460-71

6. Kappos L, Li D, Calabresi PA, et al, Ocrelizumab in relapsing-remitting multiple sclerosis: a phase 2, randomized, placebo-controlled, multicenter trial, Lancet 2011; 378:1779-87

7. Naismith RT, Piccio L, Lyons JA, et al Rituximab add-on therapy for breakthrough relapsing multiple sclerosis: a 52-week phase II trial, Neurology 2010; 74: 1860-67

8. Sormani MP, Bruzzi P MRI lesions as a surrogate for relapses in multiple sclerosis: a meta-analysis of randomized trials, Lancet Neurol 2013; 12: 669-76

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

Clinical Trial: 
Feagan B, McDonald J, Panaccione R, et al. Methotrexate in Combination with Infliximab is no More Effective Than Infliximab Alone With Crohn’s Disease. Gastroenterology. 2014; 146(3): 681-688. PMID:24269926

Disease: Crohn’s Disease (CD)

Drug: Methotrexate (MTX,) a purine anti-metabolite, and infliximab, a chimeric monoclonal antibody against TNF-alpha

Study design:

  • 50 week double-blind, placebo-controlled trial comparing MTX and infliximab with infliximab alone

  • 126 patients with CD in the midst of a flare who had started prednisone induction therapy within the previous 6 weeks

  • In both groups, prednisone was tapered, beginning in week 1 and discontinued no later than week 14.

  • Primary outcome measurement was time to treatment failure defined as a lack of prednisone-free remission at week 14 or failure to maintain remission at week 50.

Results:

  • At week 50, treatment failure rate was 30.6% in the combined therapy group and 29.8% in the infliximab group.

  • No differences measured in secondary outcomes (CD Activity Score/CDAI, CRP level, or SF-36 score, which is a quality of life assessment)

  • Higher percent in monotherapy group developed antibodies to infliximab (20%) than in the combined treatment group (4%)

  • Median serum trough infliximab concentration was higher in the combined therapy group than in the monotherapy group, although not statistically significant.

  • Combination therapy was well tolerated.

  • Adverse events similar in both groups

Why This Study Is of Interest to the Broader FOCIS community:

Trials conducted in the 1990s showed that infliximab and MTX each show efficacy for CD when used alone (5, 6). In recent years, comparative efficacy trials have shown that treating inflammatory bowel disease with a combination of an anti-TNF-alpha agent and another purine anti-metabolite, azathioprine, is more effective than either agent alone, and reduces antibodies to anti-TNF agents. (4, 7) Similarly, the efficacy of combined treatment with MTX and infliximab has been well documented in rheumatoid arthritis (RA) and also reduces anti-infliximab antibodies. (1-3)

This study confirmed that treatment with MTX can be effective for prevention of antibodies to infliximab, which is associated with lower trough levels and a loss of responsiveness. However, unlike in RA trials, combination therapy did not correlate with increased efficacy. Thus the dissociation of antibody prevention from efficacy shown in this trial suggests that azathioprine’s augmentation of anti-TNF efficacy is not due to antibody prevention but rather from suppressing CD via a mechanism that is not redundant with TNF blockade and is not targeted by MTX.
This study did not include a prednisone-only treatment arm, and it’s possible that any additional benefit of MTX over infliximab only may have been obscured by the effects of prednisone. Prednisone is an effective induction agent whose effects can be durable when used in combination with other immunosuppressants. It’s possible that the remissions seen in both groups were the result of prednisone induction.

References:
1. Goekoop-Ruiterman, YP, de Vries-Bouwstra JK, Allaart CF, et al, Clinical and radiographic outcomes of four different treatment strategies in patients with early rheumatoid arthritis (the BeSt study): a randomized, controlled trial, Arthritis Rheum 2005; 52: 3381-3390

2. Breedveld FC, Weisman MH, Kavanaugh AF, et al, The PREMIER study: a multicenter, randomized double-blind clinical trial of combination therapy with adalimumab plus methotrexate versus methotrexate alone or adalimumab alone in patients with early, aggressive rheumatoid arthritis who had not had previous methotrexate treatment, Arthritis Rheum 2006;54:26-37

3. Smolen, JS, VanDerHeijde DM, St Clair EW, et al, Predictors of joint damage in patients with early rheumatoid arthritis treated with high-dose methotrexate with or without concomitant infliximab: results from the ASPIRE trial, Arthritis Rheum 2006;54:702-710

4. Baert F, Norman M, Vermeire S, et al, Influence of immunogenicity on the long-term efficacy of infliximab in Crohn’s disease, N Engl J Med 2003;348:601-608

5. Targan SR, Hanauer SB, vanDeveater SJ, A Short Term Study of chimeric monoclonal antibody cA2 to tumor necrosis factor alpha for Crohn’s Disease, N Engl J Med 1997 Oct 9;337(15):1029-35

6. Feagan BG, Rochon J, Fedorak RN, et al, Methotrexate for the Treatment of Crohn’s Disease: The North American Crohn’s Study Group, N Engl J Med 1995 Feb 2;332(5):1292-7

7. Colombel JF, Sandborn WJ, Reinisch W, et al, Infliximab, azathioprine, or combination therapy for Crohn’s Disease, N Engl J Med 2010 April 15; 362(15):1383-95


Reviewed by Sandra Lord, MD, Benaroya Research Center

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Translational Immunology Update is the official publication of the Federation of Clinical Immunology Societies. Scope: Translational Immunology Update aims to provide a knowledge-sharing forum for basic, clinical and translational scientists around all aspects of human immunology including normal and cross-disease physiology, communication between laboratory-based and clinically-based scientists, the lack of negative results that are published and the difficulty for readers to keep up with important literature.

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