Karen Keslar

Urinary-Cell mRNA Profile and Acute Cellular Rejection in Kidney Allografts

BACKGROUND The standard test for the diagnosis of acute rejection in kidney transplants is the renal biopsy. Noninvasive tests would be preferable. METHODS We prospectively collected 4300 urine specimens from 485 kidney-graft recipients from day 3 through month 12 after transplantation. Messenger RNA (mRNA) levels were measured in urinary cells and correlated with allograft-rejection status with the use of logistic regression. RESULTS A three-gene signature of 18S ribosomal (rRNA)-normalized measures of CD3ε mRNA and interferon-inducible protein 10 (IP-10) mRNA, and 18S rRNA discriminated between biopsy specimens showing acute cellular rejection and those not showing rejection (area under the curve [AUC], 0.85; 95% confidence interval [CI], 0.78 to 0.91; P<0.001 by receiver-operating-characteristic curve analysis). The cross-validation estimate of the AUC was 0.83 by bootstrap resampling, and the Hosmer-Lemeshow test indicated good fit (P=0.77). In an external-validation data set, the AUC was 0.74 (95% CI, 0.61 to 0.86; P<0.001) and did not differ significantly from the AUC in our primary data set (P=0.13). The signature distinguished acute cellular rejection from acute antibody-mediated rejection and borderline rejection (AUC, 0.78; 95% CI, 0.68 to 0.89; P<0.001). It also distinguished patients who received anti-interleukin-2 receptor antibodies from those who received T-cell-depleting antibodies (P<0.001) and was diagnostic of acute cellular rejection in both groups. Urinary tract infection did not affect the signature (P=0.69). The average trajectory of the signature in repeated urine samples remained below the diagnostic threshold for acute cellular rejection in the group of patients with no rejection, but in the group with rejection, there was a sharp rise during the weeks before the biopsy showing rejection (P<0.001). CONCLUSIONS A molecular signature of CD3ε mRNA, IP-10 mRNA, and 18S rRNA levels in urinary cells appears to be diagnostic and prognostic of acute cellular rejection in kidney allografts. (Funded by the National Institutes of Health and others.).

CXC Chemokine Ligand (CXCL) 9 and CXCL10 Are Antagonistic Costimulation Molecules during the Priming of Alloreactive T Cell Effectors

Donor Ag-reactive CD4 and CD8 T cell production of IFN-γ is a principal effector mechanism promoting tissue injury during allograft rejection. The CXCR3-binding chemokines CXCL9 and CXCL10 recruit donor-reactive T cells to the allograft, but their role during the priming of donor-reactive T cells to effector function is unknown. Using a murine model of MHC-mismatched cardiac transplantation, we investigated the influence of CXCL9 and CXCL10 during donor-reactive T cell priming. In allograft recipient spleens, CXCL9 and CXCL10 were expressed as early as 24 h posttransplant and increased with similar kinetics, concurrently with CXCR3 expression on T cells. CXCL9, but not CXCL10, expression required NK cell production of IFN-γ. The absence of CXCL9 in donor allografts, recipients, or both significantly decreased the frequency of donor-reactive CD8 T cells producing IFN-γ and increased the frequency of donor-reactive CD8 T cells producing IL-17A. In contrast, the absence of CXCL10 increased the frequency of IFN-γ–producing CD8 T cells in a CXCL9-dependent manner. These data provide novel evidence that donor-reactive CD8 T cells use the CXCR3 chemokine axis as a costimulation pathway during priming to allografts where CXCL9 promotes the development of IFN-γ–producing CD8 T cells, and CXCL10 antagonizes this skewing.

Complement gene expression in human cardiac allograft biopsies as a correlate of histologic grade of injury.

Complement activation contributes to antibody-mediated allograft rejection, but increasing evidence also implicates complement proteins produced locally within the graft, in part by infiltrating mononuclear cells, as important mediators of tissue injury. To test this concept in transplant recipients, we evaluated complement, complement regulator, and T cell/proinflammatory marker gene expression by quantitative real-time polymerase chain reaction in 71 archived heart transplant biopsies and correlated the results with the histologic grade of rejection. Significantly more transcripts encoding alternative pathway components factor B, C3 and properdin, and C3a receptor and C5a receptor were detected in grade 3 versus grade 0 or 1 biopsies. The grade 3 rejections also contained significantly higher amounts of CD3, interferon &ggr;, perforin, and granzyme B genes. In addition to providing supportive evidence for a pathogenic role of graft-derived complement in human heart transplant injury, these correlations suggest that molecular profiling of complement gene expression could be useful in the diagnosis of human allograft rejection.

Multicenter evaluation of a standardized protocol for noninvasive gene expression profiling

Gene expression profiling of transplant recipient blood and urine can potentially be used to monitor graft function, but the multitude of protocols in use make sharing data and comparing results from different laboratories difficult. The goal of this study was to evaluate the performance of current methods of RNA isolation, reverse transcription and quantitative polymerase chain reaction (qPCR) and to test whether multiple centers using a standardized protocol can obtain the same results. Samples, reagents and detailed instructions were distributed to six participating sites that performed RNA isolation, reverse transcription and qPCR for 18S, PRF, GZB, IL8, CXCL9 and CXCL10 as instructed. All data were analyzed at a single site. All sites demonstrated proficiency in RNA isolation and qPCR analysis. Gene expression measurements for all targets and samples had correlations >0.938. The coefficient of variation of fold‐changes between pairs of samples was less than 40%. All sites were able to accurately quantify a control sample of known concentration within a factor of 1.5. Collectively, we have formulated and validated detailed methods for measuring gene expression in blood and urine that can yield consistent results in multiple laboratories.

IL‐15 Induces Alloreactive CD28− Memory CD8 T Cell Proliferation and CTLA4‐Ig Resistant Memory CD8 T Cell Activation

The presence of CD28− memory CD8 T cells in the peripheral blood of renal transplant patients is a risk factor for graft rejection and resistance to CTLA‐4Ig induction therapy. In vitro analyses have indicated poor alloantigen‐induced CD28− memory CD8 T cell proliferation, raising questions about mechanisms mediating their clonal expansion in kidney grafts to mediate injury. Candidate proliferative cytokines were tested for synergy with alloantigen in stimulating CD28− memory CD8 T cell proliferation. Addition of IL‐15, but not IL‐2 or IL‐7, to co‐cultures of CD28− or CD28+ memory CD8 T cells and allogeneic B cells rescued proliferation of the CD28− and enhanced CD28+ memory T cell proliferation. Proliferating CD28− memory CD8 T cells produced high amounts of interferon gamma and tumor necrosis factor alpha and expressed higher levels of the cytolytic marker CD107a than CD28+ memory CD8 T cells. CTLA‐4Ig inhibited alloantigen‐induced proliferation of CD28+ memory CD8 T cell proliferation but had no effect on alloantigen plus IL‐15‐induced proliferation of either CD28− or CD28+ memory CD8 T cells. These results indicate the ability of IL‐15, a cytokine produced by renal epithelial during inflammation, to provoke CD28− memory CD8 T cell proliferation and to confer memory CD8 T cell resistance to CTLA‐4Ig‐mediated costimulation blockade.

γδ T Cells Coexpressing Gut Homing α4β7 and αE Integrins Define a Novel Subset Promoting Intestinal Inflammation

γδ T lymphocytes, dominant T cell subsets in the intestine, mediate both regulatory and pathogenic roles, yet the mechanisms underlying such opposing effects remain unclear. In this study, we identified a unique γδ T cell subset that coexpresses high levels of gut-homing integrins, CD103 and α4β7. They were exclusively found in the mesenteric lymph node after T cell–mediated colitis induction, and their appearance preceded the inflammation. Adoptive transfer of the CD103+α4β7high subsets enhanced Th1/Th17 T cell generation and accumulation in the intestine, and the disease severity. The level of generation correlated with the disease severity. Moreover, these cells were also found to be elevated in a spontaneous mouse model of ileitis. Based on the procolitogenic function, we referred to this subset as “inflammatory” γδ T cells. Targeting inflammatory γδ T cells may open a novel strategy to treat inflammatory diseases where γδ T cells play a pathogenic role including inflammatory bowel disease.

Acute and Chronic Rejection: Compartmentalization and Kinetics of Counterbalancing Signals in Cardiac Transplants

Heart disease is the major cause of mortality in the United States and other parts of the world. Heart transplantation is the treatment of choice for patients with end stage heart failure. However, transplanted organs fail due to either acute or chronic rejection. This acute and chronic rejection impacts distinct compartments of cardiac allografts. Acute rejection is characterized by infiltration of mononuclear cells whereas chronic rejection is characterized by progressive narrowing of coronary arteries. In a minor histoincompatibility mismatch mouse model we found hearts transplanted from male to female C57BL/6 mice undergo an acute rejection with diffuse interstitial infiltrates at 2 weeks that resolve by 6 weeks when about half of the large arteries develop CAV. These processes are dependent on T cells because no infiltrate developed in T cell deficient mice. Markers of M1 macrophages were upregulated in the interstitium acutely and then decreased as markers of M2 macrophages increased chronically. Interstitial and arterial infiltrates were microdissected and expression of an array of 86 genes was screened by real time PCR. Programmed cell death protein 1 (PD1), a negative costimulator, and its ligand PDL1 were highly upregulated in the interstitium during the resolution of acute rejection. Flow cytometry analysis of graft infiltrating cells confirmed an enrichment of macrophages expressing PDL1. Treatment with a blocking antibody to PDL1 in the acute phase increased interstitial T cell infiltrates. In the arterial compartment, Toll Like

Myeloid-derived suppressor cells increase and inhibit donor-reactive T cell responses to graft intestinal epithelium in intestinal transplant patients

Recent advances in immunosuppressive regimens have decreased acute cellular rejection (ACR) rates and improved intestinal and multivisceral transplant (ITx) recipient survival. We investigated the role of myeloid‐derived suppressor cells (MDSCs) in ITx. We identified MDSCs as CD33+CD11b+ lineage(CD3/CD56/CD19)−HLA‐DR−/low cells with 3 subsets, CD14−CD15− (e‐MDSCs), CD14+CD15− (M‐MDSCs), and CD14−CD15+ (PMN‐MDSCs), in peripheral blood mononuclear cells (PBMCs) and mononuclear cells in the grafted intestinal mucosa. Total MDSC numbers increased in PBMCs after ITx; among MDSC subsets, M‐MDSC numbers were maintained at a high level after 2 months post ITx. The MDSC numbers decreased in ITx recipients with ACR. MDSC numbers were positively correlated with serum interleukin (IL)‐6 levels and the glucocorticoid administration index. IL‐6 and methylprednisolone enhanced the differentiation of bone marrow cells to MDSCs in vitro. M‐MDSCs and e‐MDSCs expressed CCR1, ‐2, and ‐3; e‐MDSCs and PMN‐MDSCs expressed CXCR2; and intestinal grafts expressed the corresponding chemokine ligands after ITx. Of note, the percentage of MDSCs among intestinal mucosal CD45+ cells increased after ITx. A novel in vitro assay demonstrated that MDSCs suppressed donor‐reactive T cell–mediated destruction of donor intestinal epithelial organoids. Taken together, our results suggest that MDSCs accumulate in the recipient PBMCs and the grafted intestinal mucosa in ITx, and may regulate ACR.

CD4+ T lymphocytes produce adiponectin in response to transplants

Adiponectin is a pleiotropic cytokine with diverse immunomodulatory effects on macrophages and lymphocytes. In the current paradigm, lymphocytes and macrophages respond to adiponectin that is produced by adipocytes and other parenchymal cells. Using a model of chronic arterial inflammation in cardiac transplants, we found that T cells derived from the recipient migrate to the heart and produce adiponectin locally. The evidence that T cells produce significant amounts of adiponectin is based on 3 experimental approaches. First, CD4+ T cells isolated from the blood and spleen after cardiac transplantation express mRNA for adiponectin. Second, reconstitution of T cell-deficient recipients with transgenic CD4+ T cells that express receptors for donor antigens results in arterial infiltrates containing T cells and increased mRNA expression for adiponectin in cardiac transplants. Third, CD4+ T cells isolated from the allograft secrete adiponectin in vitro. Taken together, these data indicate that adiponectin-competent cells originating in the recipient migrate into the transplant. Establishing T cells as a source of adiponectin provides a new dimension, to our knowledge, to the modulatory effects of adiponectin on immune responses.

Inflammatory cytokines in the papillary tips and urine of nephrolithiasis patients

INTRODUCTION Intrarenal inflammation has been implicated in the pathogenesis of nephrolithiasis, with prior work showing increased urine levels of IL-6, IL-8, and CCL-2 in stone patients. However, no studies have assessed for inflammation in the renal papillae. We sought to characterize novel papillary tip and urinary biomarkers in stone patients. MATERIALS AND METHODS Ninety-two patients with nephrolithiasis undergoing percutaneous nephrolithotomy were enrolled. Papillary tip biopsies, kidney urine, and bladder urine were collected, as well as voided urine from eight healthy volunteers. Quantitative polymerase chain reaction was performed to measure inflammatory gene expression. RESULTS Initial 84-gene polymerase chain reaction array revealed significant elevation of several cytokines in stone patients vs controls (fold change 2.3-694). Twenty-four genes were selected for final analysis. In 41 pairs of urine samples, levels of CCL5, CD40, FasL, RIPK2, SELE, TLR3, and IL-15 were significantly elevated in kidney vs bladder urine (p0.0001-0.04). In 23 triplets of samples, expression of these cytokines plus CCL2, CCL7, CCR2, CSF1, CXCL9, and CXCL10, was significantly greater in papillary tips vs urine samples (p0.001-0.05). Cytokine elevation was independent of maximum postoperative heart rate, respiratory rate, temperature, leukocyte count, urinary tract infection in the past year, presence or absence of antibiotics at the time of surgery, and stone composition (all p > 0.05). CONCLUSION Expression of CCL-2, CCL-5, CCL-7, CCR-2, CD40, CSF1, CXCL-9, CXCL-10, Fas-L, RIPK2, SELE, and TLR-3 is markedly elevated in the papillary tips, kidney urine, and bladder urine of nephrolithiasis patients. Cytokine elevation was independent of signs of systemic inflammation. These findings further support the role of inflammation in nephrolithiasis and imply that the inflammatory process likely begins at the renal papillae. These may represent novel biomarkers of stone disease, which may be useful in basic nephrolithiasis research, disease diagnosis, and prognosis.