Cell-Free DNA Maps Tissue Injury and Correlates with Disease Severity in Lung Transplant Candidates.

Rationale: Plasma cell-free DNA levels correlate with disease severity in many conditions. Pretransplant cell-free DNA may risk stratify lung transplant candidates for post-transplant complications. Objectives: To evaluate if pretransplant cell-free DNA levels and tissue sources identify patients at high risk of primary graft dysfunction and other pre- and post-transplant outcomes. Methods: This multicenter, prospective cohort study recruited 186 lung transplant candidates. Pretransplant plasma samples were collected to measure cell-free DNA. Bisulfite sequencing was performed to identify the tissue sources of cell-free DNA. Multivariable regression models determined the association between cell-free DNA levels and the primary outcome of primary graft dysfunction and other transplant outcomes, including Lung Allocation Score, chronic lung allograft dysfunction, and death. Measurements and Main Results: Transplant candidates had twofold greater cell-free DNA levels than healthy control patients (median [interquartile range], 23.7 ng/ml [15.1-35.6] vs. 12.9 ng/ml [9.9-18.4]; P < 0.0001), primarily originating from inflammatory innate immune cells. Cell-free DNA levels and tissue sources differed by native lung disease category and correlated with the Lung Allocation Score (P < 0.001). High pretransplant cell-free DNA increased the risk of primary graft dysfunction (odds ratio, 1.60; 95% confidence interval [CI], 1.09-2.46; P = 0.0220), and death (hazard ratio, 1.43; 95% CI, 1.07-1.92; P = 0.0171) but not chronic lung allograft dysfunction (hazard ratio, 1.37; 95% CI, 0.97-1.94; P = 0.0767). Conclusions: Lung transplant candidates demonstrate a heightened degree of tissue injury with elevated cell-free DNA, primarily originating from innate immune cells. Pretransplant plasma cell-free DNA levels predict post-transplant complications.

Emergency myelopoiesis distinguishes multisystem inflammatory syndrome in children from pediatric severe COVID-19.

BACKGROUND: Multisystem inflammatory syndrome in children (MIS-C) is a hyperinflammatory condition caused by recent SARS-CoV-2 infection, but the underlying immunological mechanisms driving this distinct syndrome are unknown. METHODS: We utilized high dimensional flow cytometry, cell-free (cf) DNA, and cytokine and chemokine profiling to identify mechanisms of critical illness distinguishing MIS-C from severe acute COVID-19 (SAC). RESULTS: Compared to SAC, MIS-C patients demonstrated profound innate immune cell death and features of emergency myelopoiesis (EM), an understudied phenomenon observed in severe inflammation. EM signatures were characterized by fewer mature myeloid cells in the periphery and decreased expression of HLA-DR and CD86 on antigen presenting cells. IL-27, a cytokine known to drive hematopoietic stem cells towards EM, was increased in MIS-C, and correlated with immature cell signatures in MIS-C. Upon recovery, EM signatures decreased, and IL-27 plasma levels returned to normal levels. Despite profound lymphopenia, we report a lack of cfDNA released by adaptive immune cells and increased CCR7 expression on T cells indicative of egress out of peripheral blood. CONCLUSIONS: Immune cell signatures of EM combined with elevated innate immune cell-derived cfDNA levels distinguish MIS-C from SAC in children and provide mechanistic insight into dysregulated immunity contributing towards MIS-C, offering potential diagnostic and therapeutic targets.

Drebrin 1 in dendritic cells regulates phagocytosis and cell surface receptor expression through recycling for efficient antigen presentation.

Phagocytosis, macropinocytosis and antigen presentation by dendritic cells (DC) requires reorganization of the actin cytoskeleton. Drebrin (Dbn1) is an actin binding and stabilizing protein with roles in endocytosis, formation of dendrite spines in neurons and coordinating cell-cell synapses in immune cells. However, its role in DC phagocytosis and antigen presentation is unknown. These studies now report that silencing of Dbn1 in DC resulted in restrained cell surface display of receptors, most notably MHC class I and II and co-stimulatory molecules. This, as expected, resulted in impaired antigen-specific T-cell activation and proliferation. Studies additionally revealed that knockdown of Dbn1 in DC impaired macropinocytosis and phagocytosis. However, there was a concomitant increase in fluid-phase uptake, suggesting that Dbn1 is responsible for the differential control of macropinocytosis versus micropinocytosis activities. Taken together, these findings now reveal that Dbn1 plays a major role in coordinating the actin cytoskeletal activities responsible for antigen presentation in DC.

CD40-CD40L cross-talk drives fascin expression in dendritic cells for efficient antigen presentation to CD4+ T cells.

Fascin is an actin-bundling protein that, among immune cells, is restricted to expression in dendritic cells (DCs). Previous reports have suggested that fascin plays an important role in governing DC antigen presentation to CD4+ T cells. However, no report has clearly linked the receptor-ligand engagement that can direct downstream regulation of fascin expression. In this study, bone marrow-derived DCs from wild-type versus CD40-knockout C57BL/6 mice were used to elucidate the mechanisms of fascin expression and activity upon CD40-CD40 ligand (CD40L) engagement. These investigations now show that CD40 engagement governs fascin expression in DCs to promote CD4+ T-cell cytokine production. Absence of CD40 signaling resulted in diminished fascin expression in DCs and was associated with impaired CD4+ T-cell responses. Furthermore, the study found that loss of CD40-CD40L engagement resulted in reduced DC-T-cell contacts. Rescue by ectopic fascin expression in CD40-deficient DCs was able to re-establish sustained contacts with T cells and restore cytokine production. Taken together, these results show that cross-talk through CD40-CD40L signaling drives elevated fascin expression in DCs to support acquisition of full T-cell responses.

Racial Differences in Donor-Derived Cell-Free DNA and Mitochondrial DNA After Heart Transplantation, on Behalf of the GRAfT Investigators.

BACKGROUND: Black heart transplant patients are at higher risk of acute rejection (AR) and death than White patients. We hypothesized that this risk may be associated with higher levels of donor-derived cell-free DNA (dd-cfDNA) and cell-free mitochondrial DNA. METHODS: The Genomic Research Alliance for Transplantation is a multicenter, prospective, longitudinal cohort study. Sequencing was used to quantitate dd-cfDNA and polymerase chain reaction to quantitate cell-free mitochondrial DNA in plasma. AR was defined as ≥2R cellular rejection or ≥1 antibody-mediated rejection. The primary composite outcome was AR, graft dysfunction (left ventricular ejection fraction <50% and decrease by ≥10%), or death. RESULTS: We included 148 patients (65 Black patients and 83 White patients), median age was 56 years and 30% female sex. The incidence of AR was higher in Black patients compared with White patients (43% versus 19%; P=0.002). Antibody-mediated rejection occurred predominantly in Black patients with a prevalence of 20% versus 2% (P<0.001). After transplant, Black patients had higher levels of dd-cfDNA, 0.09% (interquartile range, 0.001-0.30) compared with White patients, 0.05% (interquartile range, 0.001-0.23; P=0.003). Beyond 6 months, Black patients showed a persistent rise in dd-cfDNA with higher levels compared with White patients. Cell-free mitochondrial DNA was higher in Black patients (185 788 copies/mL; interquartile range, 101 252-422 133) compared with White patients (133 841 copies/mL; interquartile range, 75 346-337 990; P<0.001). The primary composite outcome occurred in 43% and 55% of Black patients at 1 and 2 years, compared with 23% and 27% in White patients, P<0.001. In a multivariable model, Black patient race (hazard ratio, 2.61 [95% CI, 1.35-5.04]; P=0.004) and %dd-cfDNA (hazard ratio, 1.15 [95% CI, 1.03-1.28]; P=0.010) were associated with the primary composite outcome. CONCLUSIONS: Elevated dd-cfDNA and cell-free mitochondrial DNA after heart transplant may mechanistically be implicated in the higher incidence of AR and worse clinical outcomes in Black transplant recipients. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02423070.

Cell-free chromatin immunoprecipitation to detect molecular pathways in heart transplantation.

Existing monitoring approaches in heart transplantation lack the sensitivity to provide deep molecular assessments to guide management, or require endomyocardial biopsy, an invasive and blind procedure that lacks the precision to reliably obtain biopsy samples from diseased sites. This study examined plasma cell-free DNA chromatin immunoprecipitation sequencing (cfChIP-seq) as a noninvasive proxy to define molecular gene sets and sources of tissue injury in heart transplant patients. In healthy controls and in heart transplant patients, cfChIP-seq reliably detected housekeeping genes. cfChIP-seq identified differential gene signals of relevant immune and nonimmune molecular pathways that were predominantly down-regulated in immunosuppressed heart transplant patients compared with healthy controls. cfChIP-seq also identified cell-free DNA tissue sources. Compared with healthy controls, heart transplant patients demonstrated greater cell-free DNA from tissue types associated with heart transplant complications, including the heart, hematopoietic cells, lungs, liver, and vascular endothelium. cfChIP-seq may therefore be a reliable approach to profile dynamic assessments of molecular pathways and sources of tissue injury in heart transplant patients.

Cell-free Chromatin Immunoprecipitation to detect molecular pathways in Physiological and Disease States.

Patient monitoring is a cornerstone in clinical practice to define disease phenotypes and guide clinical management. Unfortunately, this is often reliant on invasive and/or less sensitive methods that do not provide deep phenotype assessments of disease state to guide treatment. This paper examined plasma cell-free DNA chromatin immunoprecipitation sequencing (cfChIP-seq) to define molecular gene sets in physiological and heart transplant patients taking immunosuppression medications. We show cfChIP-seq reliably detect gene signals that correlate with gene expression. In healthy controls and in heart transplant patients, cfChIP-seq reliably detected housekeeping genes. cfChIP-seq identified differential gene signals of the relevant immune and non-immune molecular pathways that were predominantly downregulated in immunosuppressed heart transplant patients compared to healthy controls. cfChIP-seq also identified tissue sources of cfDNA, detecting greater cell-free DNA from cardiac, hematopoietic, and other non-hematopoietic tissues such as the pulmonary, digestive, and neurological tissues in transplant patients than healthy controls. cfChIP-seq gene signals were reproducible between patient populations and blood collection methods. cfChIP-seq may therefore be a reliable approach to provide dynamic assessments of molecular pathways and tissue injury associated to disease.

Chronic graft-versus-host disease is characterized by high levels and distinctive tissue-of-origin patterns of cell-free DNA.

Chronic graft-versus-host disease (cGvHD) is a devastating complication of hematopoietic stem cell transplantation (HSCT). Effective early detection may improve the outcome of cGvHD. The potential utility of circulating cell-free DNA (cfDNA), a sensitive marker for tissue injury, in HSCT and cGvHD remains to be established. Here, cfDNA of prospectively collected plasma samples from HSCT recipients (including both cGvHD and non-cGvHD) and healthy control (HC) subjects were evaluated. Deconvolution methods utilizing tissue-specific DNA methylation signatures were used to determine cfDNA tissue-of-origin. cfDNA levels were significantly higher in HSCT recipients than HC and significantly higher in cGvHD than non-cGvHD. cGvHD was characterized by a high level of cfDNA from innate immune cells, heart, and liver. Non-hematologic tissue-derived cfDNA was significantly higher in cGvHD than non-cGvHD. cfDNA temporal dynamics and tissue-of-origin composition have distinctive features in patients with cGvHD, supporting further exploration of the utility of cfDNA in the study of cGvHD.

Cell-free DNA reveals distinct pathology of multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) is a rare but life-threatening hyperinflammatory condition induced by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes pediatric COVID-19 (pCOVID-19). The relationship of the systemic tissue injury to the pathophysiology of MIS-C is poorly defined. We leveraged the high sensitivity of epigenomics analyses of plasma cell-free DNA (cfDNA) and plasma cytokine measurements to identify the spectrum of tissue injury and glean mechanistic insights. Compared with pediatric healthy controls (pHCs) and patients with pCOVID-19, patients with MIS-C had higher levels of cfDNA primarily derived from innate immune cells, megakaryocyte-erythroid precursor cells, and nonhematopoietic tissues such as hepatocytes, cardiac myocytes, and kidney cells. Nonhematopoietic tissue cfDNA levels demonstrated significant interindividual variability, consistent with the heterogenous clinical presentation of MIS-C. In contrast, adaptive immune cell-derived cfDNA levels were comparable in MIS-C and pCOVID-19 patients. Indeed, the cfDNA of innate immune cells in patients with MIS-C correlated with the levels of innate immune inflammatory cytokines and nonhematopoietic tissue-derived cfDNA, suggesting a primarily innate immunity-mediated response to account for the multisystem pathology. These data provide insight into the pathogenesis of MIS-C and support the value of cfDNA as a sensitive biomarker to map tissue injury in MIS-C and likely other multiorgan inflammatory conditions.

Integrated cell-free DNA and cytokine analysis uncovers distinct tissue injury and immune response patterns in solid organ transplant recipients with COVID-19.

COVID-19 pathogenesis is associated with an exuberant inflammatory response. However, the tissue injury pattern and immune response in solid-organ transplant recipients (SOTRs) taking immunosuppressive therapy have not been well characterized. Here, we perform both cfDNA and cytokine profiling on plasma samples to map tissue damage, including allograft injury and delineate underlying immunopathology. We identified injuries from multiple-tissue types, including hematopoietic cells, vascular endothelium, hepatocyte, adipocyte, pancreas, kidney, heart, and lung in SOTRs with COVID-19 that correlates with disease severity. SOTRs with COVID-19 have higher plasma levels of cytokines such as IFN-λ1, IFN-γ, IL-15, IL-18 IL-1RA, IL-6, MCP-2, and TNF-α as compared to healthy controls, and the levels of GM-CSF, IL-15, IL-6, IL-8, and IL-10 were associated with disease severity in SOTRs. Strikingly, IFN-λ and IP-10 were markedly increased in SOTRs compared to immunocompetent patients with COVID-19. Correlation analyses showed a strong association between monocyte-derived cfDNA and inflammatory cytokines/chemokines in SOTRs with COVID-19. Moreover, compared to other respiratory viral infections, COVID-19 induced pronounced injury in hematopoietic, vascular endothelial and endocrine tissues. Allograft injury, measured as donor-derived cfDNA was elevated in SOTRs with COVID-19, including allografts distant from the primary site of infection. Allograft injury correlated with inflammatory cytokines and cfDNA from immune cells. Furthermore, longitudinal analysis identified a gradual decrease of cfDNA and inflammatory cytokine levels in patients with a favorable outcome. Our findings highlight distinct tissue injury and cytokine features in SOTRs with COVID-19 that correlate with disease severity.

Cell-free DNA maps COVID-19 tissue injury and risk of death and can cause tissue injury.

INTRODUCTIONThe clinical course of coronavirus 2019 (COVID-19) is heterogeneous, ranging from mild to severe multiorgan failure and death. In this study, we analyzed cell-free DNA (cfDNA) as a biomarker of injury to define the sources of tissue injury that contribute to such different trajectories.METHODSWe conducted a multicenter prospective cohort study to enroll patients with COVID-19 and collect plasma samples. Plasma cfDNA was subject to bisulfite sequencing. A library of tissue-specific DNA methylation signatures was used to analyze sequence reads to quantitate cfDNA from different tissue types. We then determined the correlation of tissue-specific cfDNA measures to COVID-19 outcomes. Similar analyses were performed for healthy controls and a comparator group of patients with respiratory syncytial virus and influenza.RESULTSWe found markedly elevated levels and divergent tissue sources of cfDNA in COVID-19 patients compared with patients who had influenza and/or respiratory syncytial virus and with healthy controls. The major sources of cfDNA in COVID-19 were hematopoietic cells, vascular endothelium, hepatocytes, adipocytes, kidney, heart, and lung. cfDNA levels positively correlated with COVID-19 disease severity, C-reactive protein, and D-dimer. cfDNA profile at admission identified patients who subsequently required intensive care or died during hospitalization. Furthermore, the increased cfDNA in COVID-19 patients generated excessive mitochondrial ROS (mtROS) in renal tubular cells in a concentration-dependent manner. This mtROS production was inhibited by a TLR9-specific antagonist.CONCLUSIONcfDNA maps tissue injury that predicts COVID-19 outcomes and may mechanistically propagate COVID-19-induced tissue injury.FUNDINGIntramural Targeted Anti-COVID-19 grant, NIH.

IL-10 producing CD8+ CD122+ PD-1+ regulatory T cells are expanded by dendritic cells silenced for Allograft Inflammatory Factor-1.

Allograft Inflammatory Factor-1 (AIF1) is a cytoplasmic scaffold protein that contains Ca2+ binding EF-hand and PDZ interaction domains important for mediating intracellular signaling complexes in immune cells. The protein plays a dominant role in both macrophage- and dendritic cell (DC)-mediated inflammatory responses. This study now reports that AIF1 expression in DC is important in directing CD8+ T cell effector responses. Silencing AIF1 expression in murine CD11c+ DC suppressed antigen-specific CD8+ T cell activation, marked by reduced CXCR3, IFNγ and Granzyme B expression, and restrained proliferation. These primed CD8+ T cells had impaired cytotoxic killing of target cells in vitro. In turn, studies identified that AIF1 silencing in DC robustly expanded IL-10 producing CD8+ CD122+ PD-1+ regulatory T cells that suppressed neighboring immune effector responses through both IL-10 and PD-1-dependent mechanisms. In vivo studies recapitulated bystander suppression of antigen-responsive CD4+ T cells by the CD8+ Tregs expanded from the AIF1 silenced DC. These studies further demonstrate that AIF1 expression in DC serves as a potent governor of cognate T cell responses and present a novel target for engineering tolerogenic DC-based immunotherapies.

Inhibition of Allograft Inflammatory Factor-1 in Dendritic Cells Restrains CD4+ T Cell Effector Responses and Induces CD25+Foxp3+ T Regulatory Subsets.

Allograft inflammatory factor-1 (AIF1) is a cytoplasmic scaffold protein shown to influence immune responses in macrophages and microglial cells. The protein contains Ca2+ binding EF-hand and PDZ interaction domains important for mediating intracellular signaling complexes. This study now reports that AIF1 is expressed in CD11c+ dendritic cells (DC) and silencing of expression restrains induction of antigen-specific CD4+ T cell effector responses. AIF1 knockdown in murine DC resulted in impaired T cell proliferation and skewed polarization away from T helper type 1 and 17 fates. In turn, there was a parallel expansion of IL-10-producing and CD25+Foxp3+ T regulatory subsets. These studies are the first to demonstrate that AIF1 expression in DC serves as a potent governor of cognate T cell responses and presents a novel target for engineering tolerogenic DC-based immunotherapies.

T follicular helper cells and antibody response to Hepatitis B virus vaccine in HIV-1 infected children receiving ART.

HBV vaccine has 95% efficacy in children to prevent HBV infection and related cancer. We conducted a prospective study in HIV-1 infected children receiving ART (n = 49) and controls (n = 63) to assess humoral and cellular responses to HBV vaccine provided with three doses under an accelerated schedule of 4 weeks apart. At 1 month post-vaccination all children, except 4 HIV-1 infected, displayed protective antibody (ab) titers to HBV vaccine; ab titers were lower in infected children (P < 0.0001). Ab titers decreased (P < 0.0001) in both HIV-1 infected and control children at 6 months. The frequency of circulating Tfh (cTFh) cells was 20.3% for controls and 20.8% for infected children prior to vaccination and remained comparable post-vaccination. Cytokine expression by cTfh cells upon activation with HBV antigen was comparable in the two groups at baseline and 1 month post-vaccination. Higher plasma levels (P < 0.0001) of CXCL13 were found in infected children which correlated with cTfh cell frequency at baseline. In conclusion, a lower ab response to HBV vaccine was measured in HIV-1 infected children. The frequency and activation profile of cTfh cells was comparable in infected children and controls suggesting that cells other than Tfh cells are responsible for impaired ab response to HBV vaccine.