Vicki Seyfert-Margolis

Identification of a B cell signature associated with renal transplant tolerance in humans

Establishing long-term allograft acceptance without the requirement for continuous immunosuppression, a condition known as allograft tolerance, is a highly desirable therapeutic goal in solid organ transplantation. Determining which recipients would benefit from withdrawal or minimization of immunosuppression would be greatly facilitated by biomarkers predictive of tolerance. In this study, we identified the largest reported cohort to our knowledge of tolerant renal transplant recipients, as defined by stable graft function and receiving no immunosuppression for more than 1 year, and compared their gene expression profiles and peripheral blood lymphocyte subsets with those of subjects with stable graft function who are receiving immunosuppressive drugs as well as healthy controls. In addition to being associated with clinical and phenotypic parameters, renal allograft tolerance was strongly associated with a B cell signature using several assays. Tolerant subjects showed increased expression of multiple B cell differentiation genes, and a set of just 3 of these genes distinguished tolerant from nontolerant recipients in a unique test set of samples. This B cell signature was associated with upregulation of CD20 mRNA in urine sediment cells and elevated numbers of peripheral blood naive and transitional B cells in tolerant participants compared with those receiving immunosuppression. These results point to a critical role for B cells in regulating alloimmunity and provide a candidate set of genes for wider-scale screening of renal transplant recipients.

Development of a cross-platform biomarker signature to detect renal transplant tolerance in humans.

Identifying transplant recipients in whom immunological tolerance is established or is developing would allow an individually tailored approach to their posttransplantation management. In this study, we aimed to develop reliable and reproducible in vitro assays capable of detecting tolerance in renal transplant recipients. Several biomarkers and bioassays were screened on a training set that included 11 operationally tolerant renal transplant recipients, recipient groups following different immunosuppressive regimes, recipients undergoing chronic rejection, and healthy controls. Highly predictive assays were repeated on an independent test set that included 24 tolerant renal transplant recipients. Tolerant patients displayed an expansion of peripheral blood B and NK lymphocytes, fewer activated CD4+ T cells, a lack of donor-specific antibodies, donor-specific hyporesponsiveness of CD4+ T cells, and a high ratio of forkhead box P3 to alpha-1,2-mannosidase gene expression. Microarray analysis further revealed in tolerant recipients a bias toward differential expression of B cell-related genes and their associated molecular pathways. By combining these indices of tolerance as a cross-platform biomarker signature, we were able to identify tolerant recipients in both the training set and the test set. This study provides an immunological profile of the tolerant state that, with further validation, should inform and shape drug-weaning protocols in renal transplant recipients.

Prerequisites for cytokine measurements in clinical trials with multiplex immunoassays

BackgroundGrowing knowledge about cellular interactions in the immune system, including the central role of cytokine networks, has lead to new treatments using monoclonal antibodies that block specific components of the immune system. Systemic cytokine concentrations can serve as surrogate outcome parameters of these interventions to study inflammatory pathways operative in patients in vivo. This is now possible due to novel technologies such as multiplex immunoassays (MIA) that allows detection of multiple cytokines in a single sample. However, apparently trivial underappreciated processes, (sample handling and storage, interference of endogenous plasma proteins) can greatly impact the reliability and reproducibility of cytokine detection.Therefore we set out to investigate several processes that might impact cytokine profiles such as blood collecting tubes, duration of storage, and number of freeze thawing cycles.ResultsSince under physiological conditions cytokine concentrations normally are low or undetectable we spiked cytokines in the various plasma and serum samples. Overall recoveries ranged between 80-120%. Long time storage showed cytokines are stable for a period up to 2 years of storage at -80°C. After 4 years several cytokines (IL-1α, IL-1β, IL-10, IL-15 and CXCL8) degraded up to 75% or less of baseline values. Furthermore we show that only 2 out of 15 cytokines remained stable after several freeze-thawing cycles. We also demonstrate implementation of an internal control for multiplex cytokine immunoassays.ConclusionAll together we show parameters which are essential for measurement of cytokines in the context of clinical trials.

Treatment of patients with new onset Type 1 diabetes with a single course of anti-CD3 mAb teplizumab preserves insulin production for up to 5 years

Anti-CD3 mAbs may prolong beta cell function up to 2 years in patients with new onset Type 1 diabetes (T1DM). A randomized open label trial of anti-CD3 mAb, Teplizumab, in T1DM was stopped after 10 subjects because of increased adverse events than in a previous trial related with higher dosing of drug. Teplizumab caused transient reduction in circulating T cells, but the recovered cells were not new thymic emigrants because T cell receptor excision circles were not increased. There was a trend for reduced loss of C-peptide over 2 years with drug treatment (p=0.1), and insulin use was lower (p<0.001). In 4 drug-treated subjects followed up to 60 months, C-peptide responses were maintained. We conclude that increased doses of Teplizumab are associated with greater adverse events without improved efficacy. The drug may marginate rather than deplete T cells. C-peptide levels may remain detectable up to 5 years after treatment.

Analysis of T-Cell Assays to Measure Autoimmune Responses in Subjects With Type 1 Diabetes Results of a Blinded Controlled Study

Type 1 diabetes is a chronic autoimmune disease mediated by autoreactive T-cells. Several experimental therapies targeting T-cells are in clinical trials. To understand how these therapies affect T-cell responses in vivo, assays that directly measure human T-cell function are needed. In a blinded, multicenter, case-controlled study conducted by the Immune Tolerance Network, we tested responses in an immunoblot and T-cell proliferative assay to distinguish type 1 diabetic patients from healthy control subjects. Peripheral blood cells from 39 healthy control subjects selected for DR4 and 23 subjects with recently diagnosed type 1 diabetes were studied. Autoantibody responses were measured in serum samples. Positive responses in both assays were more common in peripheral blood mononuclear cells from new-onset type 1 diabetic patients compared with control subjects. The proliferative, immunoblot, and autoantibody assays had sensitivities of 58, 91, and 78% with specificities of 94, 83, and 85%, respectively. When cellular assays were combined with autoantibody measurements, the sensitivity of the measurements was 75% with 100% specificity. We conclude that cellular assays performed on peripheral blood have a high degree of accuracy in discriminating responses in subjects with type 1 diabetes from healthy control subjects. They may be useful for assessment of cellular autoimmune responses involved in type 1 diabetes.

Differential gene expression profiles are dependent upon method of peripheral blood collection and RNA isolation

BackgroundRNA isolation and purification steps greatly influence the results of gene expression profiling. There are two commercially available products for whole blood RNA collection, PAXgene™ and Tempus™ blood collection tubes, and each comes with their own RNA purification method. In both systems the blood is immediately lysed when collected into the tube and RNA stabilized using proprietary reagents. Both systems enable minimal blood handling procedures thus minimizing the risk of inducing changes in gene expression through blood handling or processing. Because the RNA purification steps could influence the total RNA pool, we examined the impact of RNA isolation, using the PAXgene™ or Tempus™ method, on gene expression profiles.ResultsUsing microarrays as readout of RNA from stimulated whole blood we found a common set of expressed transcripts in RNA samples from either PAXgene™ or Tempus™. However, we also found several to be uniquely expressed depending on the type of collection tube, suggesting that RNA purification methods impact results of differential gene expression profiling. Specifically, transcripts for several known PHA-inducible genes, including IFNγ, IL13, IL2, IL3, and IL4 were found to be upregulated in stimulated vs. control samples when RNA was isolated using the ABI Tempus™ method, but not using the PAXgene™ method (p < 0.01, FDR corrected). Sequenom Quantiative Gene Expression (QGE) (SanDiego, CA) measures confirmed IL2, IL4 and IFNγ up-regulation in Tempus™ purified RNA from PHA stimulated cells while only IL2 was up-regulated using PAXgene™ purified (p < 0.05).ConclusionHere, we demonstrate that peripheral blood RNA isolation methods can critically impact differential expression results, particularly in the clinical setting where fold-change differences are typically small and there is inherent variability within biological cohorts. A modified method based upon the Tempus™ system was found to provide high yield, good post-hybridization array quality, low variability in expression measures and was shown to produce differential expression results consistent with the predicted immunologic effects of PHA stimulation.

Increased Frequencies of Myelin Oligodendrocyte Glycoprotein/MHC Class II-Binding CD4 Cells in Patients with Multiple Sclerosis

Multiple sclerosis (MS) is an autoimmune disease characterized by infiltration of pathogenic immune cells in the CNS resulting in destruction of the myelin sheath and surrounding axons. We and others have previously measured the frequency of human myelin-reactive T cells in peripheral blood. Using T cell cloning techniques, a modest increase in the frequency of myelin-reactive T cells in patients as compared with control subjects was observed. In this study, we investigated whether myelin oligodendrocyte glycoprotein (MOG)-specific T cells could be detected and their frequency was measured using DRB1*0401/MOG97–109(107E-S) tetramers in MS subjects and healthy controls expressing HLA class II DRB1*0401. We defined the optimal culture conditions for expansion of MOG-reactive T cells upon MOG peptide stimulation of PMBCs. MOG97–109-reactive CD4+ T cells, isolated with DRB1*0401/MOG97–109 tetramers, and after a short-term culture of PMBCs with MOG97–109 peptides, were detected more frequently from patients with MS as compared with healthy controls. T cell clones from single cell cloning of DRB1*0401/MOG97–109(107E-S) tetramer+ cells confirmed that these T cell clones were responsive to both the native and the substituted MOG peptide. These data indicate that autoantigen-specific T cells can be detected and enumerated from the blood of subjects using class II tetramers, and the frequency of MOG97–109-reactive T cells is greater in patients with MS as compared with healthy controls.

Reduced numbers of IL‐7 receptor (CD127) expressing immune cells and IL‐7‐signaling defects in peripheral blood from patients with breast cancer

Interleukin‐7‐receptor‐signaling plays a pivotal role in T‐cell development and maintenance of T‐cell memory. We studied IL‐7Rα (CD127) expression in PBMCs obtained from patients with breast cancer and examined IL‐7 receptor‐mediated downstream effects defined by STAT5 phosphorylation (p‐STAT5). Reduced numbers of IL‐7Rα‐positive cells were identified in CD4+ T‐cells as well as in a CD8+ T‐cell subset defined by CD8α/α homodimer expression in patients with breast cancer. PBMCs obtained from healthy donors (n = 19) and from patients with breast cancer (n = 19) exhibited constitutive p‐STAT5 expression in the range of 0–6.4% in CD4+ T‐cells and 0–4% in CD8+ T‐cells. Stimulation with recombinant human IL‐7 for 15 min increased p‐STAT5 expression up to 36–97% in CD4+T‐cells and to 26–90% in CD8+T‐cells obtained from healthy control donors (n = 19). In contrast, PBMCs obtained from 13/19 patients with breast cancer did not respond to IL‐7 as defined by STAT5 phosphorylation, despite expression of IL‐7Rα on T‐lymphocytes. T‐cells were further characterized for IL‐ 2 and IFN‐γ production induced by PMA/Ionomycin. PBMCs from 9/19 patients with breast cancer showed decreased IL‐2 and IFN‐γ production combined with IL‐7‐signaling defects; PBMCs from 4 patients with breast cancer exhibited deficient IL‐7‐signaling, yet intact cytokine production. Reduced numbers of IL‐7Rα‐positive cells and nonresponsiveness to IL‐7, defined by lack of STAT5 phosphorylation, characterizes the immunological profile in T‐cells from patients with breast cancer.

Validated protocol for FoxP3 reveals increased expression in type 1 diabetes patients

FoxP3 has become a key identifier of regulatory T cells. Investigators have used a variety of antibodies and methods for detecting FoxP3 by flow cytometry. To standardize FoxP3 antibody staining for use in clinical trial samples, we tested various antibodies from different vendors, cell preparation protocols and fix/perm reagents, and cell isolation procedures. Using this optimized staining protocol, we evaluated clinical specimens from patients with multiple sclerosis (MS) or type 1 diabetes.

Power enhancement via multivariate outlier testing with gene expression arrays

Motivation: As the use of microarrays in human studies continues to increase, stringent quality assurance is necessary to ensure accurate experimental interpretation. We present a formal approach for microarray quality assessment that is based on dimension reduction of established measures of signal and noise components of expression followed by parametric multivariate outlier testing. Results: We applied our approach to several data resources. First, as a negative control, we found that the Affymetrix and Illumina contributions to MAQC data were free from outliers at a nominal outlier flagging rate of α=0.01. Second, we created a tunable framework for artificially corrupting intensity data from the Affymetrix Latin Square spike-in experiment to allow investigation of sensitivity and specificity of quality assurance (QA) criteria. Third, we applied the procedure to 507 Affymetrix microarray GeneChips processed with RNA from human peripheral blood samples. We show that exclusion of arrays by this approach substantially increases inferential power, or the ability to detect differential expression, in large clinical studies. Availability: http://bioconductor.org/packages/2.3/bioc/html/arrayMvout.html and http://bioconductor.org/packages/2.3/bioc/html/affyContam.html affyContam (credentials: readonly/readonly) Contact: aasare@immunetolerance.org; stvjc@channing.harvard.edu