Uremia is accompanied by profound disturbance of the immune response comprising both impaired immune defence and enhanced inflammation. We have shown that this complex picture is explained by profound perturbation of the human transcriptome mediated through the effects of cMyc, SP1 and other transcription factors which markedly down-regulate both innate and adaptive components. The resulting changes in expression of cytokine, chemokine and other genes gradually recover after transplantation though the timing, extent and diversity of recovery vary widely between subjects and may be related to ethnicity, genetic polymorphisms, age, co-morbid disease and prior antigenic exposure. The individual recovery in immune competence is unmapped, however, and is critically important since rapid recovery may be an important risk for AMR, while persistent dysfunction may be a risk factor for viral infection.
The inability to precisely quantitate these immunological changes has been a major impediment to precision care in transplantation, but is gradually yielding to modern molecular methods. We have previously mapped acute cellular rejection (ACR) at the cellular, genomic and proteomic level; we have developed predictive tools, bioinformatics processes for quality assessment, shown the profound distortion of gene expression in organ failure, the rapid activation of inflammatory genes post-surgery, the recovery of chemokine and interleukin expression in the first 3 months post-transplant, the transcriptomic and proteomic signatures of cytoskeletal organization, signal activation and miRNA expression patterns characteristic of ACR in kidney and heart, and have provided computational biomarker pipelines for development of multi-marker bio-signatures.
We have demonstrated the transient appearance of class I and class II DSA after kidney transplantation, the variable relationship of these measures to early graft rejection and the strong relationship to chronic AMR later in the course. And we have developed rigorous quality standards and national programs to detect and avoid pre-formed donor specific IgG antibodies (DSA) that would trigger AMR post-transplant, and finally reported early measures of graft cellular injury by detection of cell-free DNA. But we lack two sets of precise and critical tools to guide patient management: (a) robust measures of global immune competence to quantitate immune suppression and minimize the risk of infection and malignancy, and (b) reliable measures of the specific response to donor antigens to enable intervention before progression to AMR and irreversible graft injury. We will extend our prior studies as outlined below to produce simple, reproducible and quantitative measures for these purposes thatcan be adopted quickly and simply by all transplant programs.
To Monitor Immune Recovery and Competence
Despite the large number of kidney transplant patients under care, we have virtually no knowledge of the functional recovery of the immune response after transplantation. Our aim is to develop rapid and reliable tests for monitoring immune recovery and measuring immune competence following transplantation. We will employ sophisticated measures, including multi-parametric phenotyping, genotyping, cellular activation and assessment of regulatory T cell subsets in peripheral blood mononuclear cells derived from transplant recipients.
(1) Phenotype: We will map the phenotypic changes first by mass cytometry (CyTOF, UBC, Rossi lab), using panels of 38 antibodies identifying key activation where signals that have already reported. The process will then be transferred for clinical application using multi-parameter 5-laser flow cytometry to map and visualize high-dimensional single cell data of the most informative changes.
(2) Genomics: We will characterize longitudinal immune quiescence or activity, including reduced expression of T-cell signaling molecules and IgG framework genes using Affyetrix Clarion D Genome arrays. Candidate polymorphisms and copy number variations in key immune response genes will be explored using the 700K Affymetrix SNP arrays.
(3) Activation We will assess antigen-nonspecific cellular activation via TCR-dependent (CD3+/CD28+ beads; Agilent) and TCR-independent (PMA-ionomycin) pathways. The cellular response will be measured via activation of the glycolytic pathway using the Seahorse XFp analyzer, which permits rapid recording within 120 minutes. Subjects with informative responses will be further tested using antibodies targeting phosphorylation and other post-translational modifications to precisely quantitate individual signaling steps and to explore activation of immediate early genes.
(4) Regulation : We will use a novel multi-parametric clonal T-cell strategy for in-depth analysis of T-cell subsets and Treg cells in peripheral blood mononuclear cells from patients with or without AMR. Treg cells represent a major checkpoint mechanism for transplant acceptance. Reliable markers for Treg- mediated immune regulation would aid in their detection in transplant recipients and provide data regarding graft acceptance. Furthermore, the presence (or absence) of these cells in transplant patients undergoing various therapeutic regimens would also be of prognostic value.
How are we going to positively impact transplant patients?
To Monitor Donor-Specific Precursor Frequency and Clonal Expansion
Selection, activation and clonal expansion of precursor T cells that recognize mismatched HLA antigens on the transplanted kidney are a primary step in the rejection process. Once activated, these HLA-specific T cells aid in the activation of B cells that produce the anti-graft antibodies. The measurement of precursor cell frequency has been proposed as an indicator of response to donor antigens in both stem cell and solid organ transplantation. However, current established assays require significant quantities for donor cells, are complex and time consuming, and have a relatively low sensitivity. As a result, the assay is of limited value for routine clinical monitoring. Therefore, there is an urgent need for an assay that is rapid, highly specific, and simple to perform. We will employ three important advances in this field:
1) receptor sequencing: We will measure clonal expansion of lymphocytes responding to defined donor/recipient antigenic mismatch by using Next-Generation Sequencing (NGS) technology to precisely monitoring of T-cell and B-cell receptor gene utilization patterns.
2) tetramer technology: We will use MHC multimers to identify and characterize antigen-specific T-cells. Specifically, fluorescently-labeled multimerized MHC-peptide complexes will bind to the TCR, enabling enumeration of these cells by multiparameter flow cytometry. The use of combinatorial colour-coded tetramers enables the simultaneous use of several tetramers to maximize the number of antigen specificities detected, critical in this case where multiple epitope disparities normally exist between donor and recipient.
3) real-time intracellular signaling: We will measure intracellular cell signaling by detecting the activation of the glycolytic pathway using the Seahorse XFp analyzer (Agilent). In addition, we will employ CaFlux assays to measure alterations in intracellular calcium concentration in peripheral blood mononuclear cells (T cells, B cells, NK cells). Alterations in intracellular calcium concentration is a key signal involved in the activation/regulation of more than 75% of genes in mammalian cells. Therefore, measurements of changes in intracellular calcium can identify lymphocyte activation in response to generic activation or specific antigen. The CaFlux assay will enable rapid (seconds), precise and quantitative measurement of both the response to antigenic stimuli and the accurate enumeration of the numbers/proportion of responding cells in transplant recipient blood.
To Monitor Activity of Antibodies to Donor HLA and non-HLA Targets
Current microarray methods can sensitively detect IgG DSA to HLA antigens that are highly predictive of AMR. However, the implication of these DSA in disease progression is unclear; they can appear and then disappear, persist but not result in graft injury, or accompany rapid and devastating AMR. At present, there is no way to distinguish the biological implications of these DSA nor determine whether therapeutic intervention is necessary or effective. Our goal is to clarify the relationship between AMR and the two types of DSA: 1) anti-HLA antibodies and 2) non-HLA antibodies and clinical AMR.
1) Characterization of anti-HLA antibodies: Canadian laboratories currently use flow cytometric crossmatch (FCXM) to detect the presence of DSA. However, these tests have both a high false-positive rate and failed to demonstrate a correlation between the detected DSA and graft loss. Using our large retrospective patient sample set, we will compare FXCM with two alternate commercial assays (OneLambda/Immucor) to first, detect anti-HLA and second, determine whether the specific DSA-detected by the tests correlate with AMR. The assays will be used to screen for anti-HLA antibodies of Class IgM and IgG including its 4 subclasses. In addition, the tests will be used to determine the presence of complement activating antibodies and determine their role in AMR.
2) Characterization of non-HLA antibodies: Several studies have reported that autoantibodies to angiotensin-II type 1 receptor and / or to the LG3 fragment of perlecan may interact with anti-HLA DSA to enhance graft injury and loss in AMR (168). We will use commercial assays (ELISA and Luminex) to determine the presence of non-HLA antibodies in our patient cohort and if present, correlate it with AMR.
To Implement non-invasive Monitoring of Graft Injury
Graft biopsy is invasive, potentially dangerous and unpleasant for the patient, and expensive and time-consuming for the transplant provider. It is therefore performed infrequently, normally only 1 or 2 times in the lifetime of the graft, and usually when deterioration is indicated by a decrease in GFR or increased proteinuria. But biopsy at this point in patients with chronic AMR normally shows that serious deterioration has already occurred, with marked glomerular damage and transplant glomerulopathy, extensive interstitial fibrosis and tubular loss. We will test the precision and accuracy of monitoring the presence of graft-derived cell-free DNA (GcfDNA) in the transplant patient. We will evaluate technologies that detect GcfDNA that have been developed by our commercial partners Chronix (California, US) and Illumina (California, UA). Once validated, these assays will be standardized for use across Canada.