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Graft-derived cell-free DNA (gdcfDNA) quantification is a promising, minimally invasive tool for detecting acute T cell-mediated rejection (ATCMR) following liver transplantation (LT). We investigated the utility of measuring hepatocyte-specific methylation in cfDNA (HS-cfDNA) to quantify gdcfDNA, examining its accuracy in detecting ATCMR in a prospective, cross-sectional study. Blood was collected from LT recipients immediately prior to graft biopsy for suspected rejection. HS-cfDNA was quantified using droplet-digital polymerase chain reaction. Prebiopsy liver function tests (LFTs) and HS-cfDNA levels were correlated with biopsy results and the primary outcome of treated biopsy-proven acute rejection (tBPAR). A total of 51 patients were recruited; 37 had evidence of rejection on biopsy and 20 required treatment. As much as 11 patients needed inpatient treatment for rejection. HS-cfDNA significantly outperformed LFTs in identifying patients with tBPAR, particularly those needing inpatient treatment (area under the curve, 73.0%; 95% confidence interval, 55.4%-90.6%; P = 0.01). At a threshold of33.5% of the total cfDNA fraction, HS-cfDNA had a specificity of 97%, correctly excluding tBPAR in 30/31 patients. Quantifying graft-specific methylation in cfDNA has a major advantage over previous gdcfDNA techniques: it does not require genotyping/sequencing, lending it greater feasibility for translation into transplantation care. Low levels of HS-cfDNA were a strong negative predictor for tBPAR (negative predictive value, 86%) and may have a future role in triaging patients prior to invasive graft biopsies.

Recent studies have established that memory B cells, largely thought to be circulatory in the blood, can take up long-term residency in inflamed tissues, analogous to widely described tissue-resident T cells. The dynamics of recruitment and retention of memory B cells to tissues and their immunological purpose remains unclear. Here, we characterized tissue-resident memory B cells (B RM ) that are stably maintained in the lungs of mice after pulmonary influenza infection. Influenza - specific B RM were localized within inducible bronchus-associated lymphoid tissues (iBALTs) and displayed transcriptional signatures distinct from classical memory B cells in the blood or spleen while showing partial overlap with memory B cells in lung-draining lymph nodes. We identified lung-resident markers, including elevated expression of CXCR3, CCR6, and CD69, on hemagglutinin (HA)– and nucleoprotein (NP)–specific lung B RM . We found that CCR6 facilitates increased recruitment and/or retention of B RM in lungs and differentiation into antibody-secreting cells upon recall. Although expression of CXCR3 and CCR6 was comparable in total and influenza-specific memory B cells isolated across tissues of human donors, CD69 expression was higher in memory B cells from lung and draining lymph nodes of human organ donors relative to splenic and PBMC-derived populations, indicating that mechanisms underpinning B RM localization may be evolutionarily conserved. Last, we demonstrate that human memory B cells in lungs are transcriptionally distinct to populations in lung-draining lymph nodes or PBMCs. These data suggest that B RM may constitute a discrete component of B cell immunity, positioned at the lung mucosa for rapid humoral response against respiratory viral infections.

We aimed to facilitate the donation of tissue samples for research by establishing a centralized system integrated in the organ donation program for collection, storage, and distribution of samples (the Australian Donation and Transplantation Biobank [ADTB]).Feasibility of a research biobank integrated within the deceased organ and tissue donation program was assessed. DonateLife Victoria sought consent for ADTB donation after consent was received for organ donation for transplantation from the donor's senior available next of kin. ADTB samples were collected during donation surgery and distributed fresh to researchers or stored for future research. The main outcome measures were ADTB donation rates, ADTB sample collection, ADTB sample use, and to identify ethical considerations.Over 2 y, samples were collected for the ADTB from 69 donors (28% of 249 donors). Samples were obtained from the spleen (n = 59, 86%), colon (n = 57, 83%), ileum (n = 56, 82%), duodenum (n = 55, 80%), blood (n = 55, 80%), bone marrow (n = 55, 80%), skin (n = 54, 78%), mesenteric lymph nodes (n = 56, 81%), liver (n = 21, 30%), lung (n = 29, 42%), and lung-draining lymph node (n = 29, 42%). Heart (n = 20), breast (n = 1), and lower urinary tract (n = 1) samples were obtained in the second year. Five hundred fifty-six samples were used in 19 ethics-approved research projects spanning the fields of immunology, microbiology, oncology, anatomy, physiology, and surgery.The integration of routine deceased donation and transplantation activities with a coordinated system for retrieval and allocation of donor samples for use in a range of research projects is feasible and valuable.

Supplemental Digital Content is available in the text., Background. Assessment of donor-specific cell-free DNA (dscfDNA) in the recipient is emerging as a noninvasive biomarker of organ rejection after transplantation. We previously developed a digital polymerase chain reaction (PCR)-based approach that readily measures dscfDNA within clinically relevant turnaround times. Using this approach, we characterized the dynamics and evaluated the clinical utility of dscfDNA after liver transplantation (LT). Methods. Deletion/insertion polymorphisms were used to distinguish donor-specific DNA from recipient-specific DNA. Posttransplant dscfDNA was measured in the plasma of the recipients. In the longitudinal cohort, dscfDNA was serially measured at days 3, 7, 14, 28, and 42 in 20 recipients. In the cross-sectional cohort, dscfDNA was measured in 4 clinically stable recipients (>1-y posttransplant) and 16 recipients (>1-mo posttransplant) who were undergoing liver biopsies. Results. Recipients who underwent LT without complications demonstrated an exponential decline in dscfDNA. Median levels at days 3, 7, 14, 28, and 42 were 1936, 1015, 247, 90, and 66 copies/mL, respectively. dscfDNA was higher in recipients with treated biopsy-proven acute rejection (tBPAR) when compared to those without. The area under the receiver operator characteristic curve of dscfDNA was higher than that of routine liver function tests for tBPAR (dscfDNA: 98.8% with 95% confidence interval, 95.8%-100%; alanine aminotransferase: 85.7%; alkaline phosphatase: 66.4%; gamma-glutamyl transferase: 80.1%; and bilirubin: 35.4%). Conclusions. dscfDNA as measured by probe-free droplet digital PCR methodology was reflective of organ health after LT. Our findings demonstrate the potential utility of dscfDNA as a diagnostic tool of tBPAR.