Renal transplant recipients require periodic surveillance forimmune-based complications such as rejection and infection. Noninvasive monitoring methods are preferred, particularly for children, for whom invasive testing is problematic. We performed a cross-sectional analysis of adult and pediatric transplant recipients to determine whether a urine-based chemokine assay could non-invasively identify patients with rejection amongst other common clinical diagnoses. Urine was collected from 110 adults and 46 children with defined clinical conditions: healthy volunteers, stable renal transplant recipients, and recipients with clinical or subclinical acute rejection (AR) or BK infection (BKI), calcineurin inhibitor (CNI) toxicity, orinterstitial fibrosis (IFTA). Urine was analyzed using a solid-phase bead-array assay for the interferon gamma-induced chemokines CXCL9 and CXCL10. We found that urine CXCL9 and CXCL10 were markedly elevated in adults and children experiencing either AR or BKI (p=0.0002), but not in stable allograft recipients, or recipients with CNI toxicity or IFTA. The sensitivity and specificity of these chemokine assays exceeded that of serum creatinine. Neither chemokine distinguished between AR and BKI. These data show that urine chemokine monitoring identifies patients with renal allograft inflammation. This assay may be usefulfor non-invasively distinguishing those allograft recipients requiring more intensivesurveillance from those with benign clinical courses.

Kidney transplantation remains limited by toxicities of calcineurin inhibitors (CNIs) and steroids. Belatacept is a less toxic CNI alternative, but existing regimens rely on steroids and have higher rejection rates. Experimentally, donor bone marrow and sirolimus promote belatacept’s efficacy. To investigate a belatacept-based regimen without CNIs or steroids, we transplanted recipients of live donor kidneys using alemtuzumab induction, monthly belatacept and daily sirolimus. Patients were randomized 1:1 to receive unfractionated donor bone marrow. After 1 year, patients were allowed to wean from sirolimus. Patients were followed clinically and with surveillance biopsies. Twenty patients were transplanted, all successfully. Mean creatinine (eGFR) was 1.10±0.07mg/dl (89±3.56ml/min) and 1.13±0.07 mg/dl (and 88±3.48 ml/min) at 12- and 36-months, respectively. Excellent results were achieved irrespective of bone marrow infusion. Ten patients elected oral immunosuppressant weaning, seven of whom were maintained rejection-free on monotherapy belatacept. Those failing to wean were successfully maintained on belatacept-based regimens supplemented by oral immunosuppression. Seven patients declined immunosuppressant weaning and three patients were denied weaning for associated medical conditions; all remained rejection-free. Belatacept and sirolimus effectively prevent kidney allograft rejection without CNIs or steroids when used following alemtuzumab induction. Selected, immunologically low-risk patients can be maintained solely on once monthly intravenous belatacept.

Key Points• CMV reactivation fundamentally resets posttransplant CD8 reconstitution, resulting in massive expansion of CMVspecific CD8 Tem.• CMV reactivation is associated with defects in the underlying TCRb immune repertoire.Although cytomegalovirus (CMV) reactivation has long been implicated in posttransplant immune dysfunction, the molecular mechanisms that drive this phenomenon remain undetermined. To address this, we combined multiparameter flow cytometric analysis and T-cell subpopulation sorting with high-throughput sequencing of the T-cell repertoire, to produce a thorough evaluation of the impact of CMV reactivation on T-cell reconstitution after unrelated-donor hematopoietic stem cell transplant. We observed that CMV reactivation drove a >50-fold specific expansion of Granzyme B high / CD28 low /CD57 high /CD8 1 effector memory T cells (Tem) and resulted in a linked contraction of all naive T cells, including CD31 1 /CD4 1 putative thymic emigrants. T-cell receptor b (TCRb) deep sequencing revealed a striking contraction of CD8 1 Tem diversity due to CMV-specific clonal expansions in reactivating patients. In addition to querying the topography of the expanding CMV-specific T-cell clones, deep sequencing allowed us, for the first time, to exhaustively evaluate the underlying TCR repertoire. Our results reveal new evidence for significant defects in the underlying CD8 Tem TCR repertoire in patients who reactivate CMV, providing the first molecular evidence that, in addition to driving expansion of virus-specific cells, CMV reactivation has a detrimental impact on the integrity and heterogeneity of the rest of the T-cell repertoire. This trial was registered at www.clinicaltrials. gov as #NCT01012492. (Blood. 2015;125(25):3835-3850)

Belatacept is a B7-specific fusion protein used to prevent allograft rejection by blocking T cell costimulation. Generally efficacious, it fails to prevent acute rejection in a sizable minority of patients. In experimental models, memory T cells mediate costimulation blockade-resistant rejection (CoBRR), but this remains undefined in humans. To explore relationships between individuals’ immune cell phenotypes and CoBRR, we studied patients receiving belatacept or conventional calcineurin inhibitor-based immunosuppression. We identified a population of CD57+PD1− CD4 T cells present prior to transplantation that correlated with CoBRR. Contrary to data recognizing CD57 as a marker of senescence on CD8 T cells, we discovered a non-senescent, cytolytic phenotype associated with CD57 on CD4 T cells. Moreover, CD57+ CD4 T cells: expressed high levels of adhesion molecules implicated in experimental CoBRR; were CD28 negative; expressed a transcriptional phenotype broadly defining allograft rejection; and were shown to be present in rejecting human kidney allografts. These data implicate CD57+ CD4 T cells in clinical CoBRR and if prospectively validated, this characteristic could identify patients at higher risk for acute rejection on belatacept-based therapy.