Background.Cytomegalovirus (CMV) antiviral drugs can cause side effects and promote the emergence of drug-resistance after transplantation. T cell-mediated immunity to CMV (CMI) can be used to limit and optimize the duration of antiviral therapy. However, interferon-gamma release assays show varying rates of CMI in CMV-seronegative recipients (R-), who are at the highest risk of CMV infection with CMV-seropositive donors (D+). Here, we characterize CMI with CMV-specific T cells that express CD154 in R- liver transplant and intestine transplant recipients.Methods.Pretransplant blood leukocyte samples from 42 R- recipients were stimulated with a 15-mer overlapping peptide mixture representing the pp65 CMV antigen. CMI was measured by flow cytometry with CMV-specific CD154+ T-cell frequencies.Results.Recipients, median age (range) 3.3 (0.4-36.5 y), included 39 liver transplant and 3 intestine transplant. CMV serostatus was D+/R- in 21, and D-/R- in 21. Protective CMI levels of >1.7% previously established in 142 liver or intestine recipients were observed in 25 of 42 total R- recipients (60%), including 11 of 21 D+/R- (52%) and 14 of 21 D-/R- (67%) participants. CMV DNAemia was observed in the first 60 d after transplantation in 10 of 42 (24%) R- recipients, including 8 of 17 with CMI <1.7% and 2 of 25 with CMI >1.7%. Adjusted for D+/R-, CMI<1.7% was independently associated with a higher risk of DNAemia (hazard ratio 6.04 [1.2-29.4], P = 0.026).Conclusions.CMV-specific T cells that express CD154 demonstrate protective levels of CMI in half of all seronegative recipients and can be used to optimize antiviral prophylaxis across all risk categories.

Acute T-cell-mediated transplant rejection, the earliest contributor to immunological graft failure responds variably to T-cell suppression. Early rejection can co-exist with donor-specific antibodies which also shorten graft survival, without affecting graft histology visibly. Here, during early liver transplant rejection, blood leukocytes manifest primed T-cells with heat shock protein (HSP) signaling and transcriptional programs for memory cell expansion. Corresponding biopsies demonstrate upregulated immune synapse and proteasomal genes in T-cell-infiltrated portal and central regions of the liver lobule. With donor-specific antibodies, the intervening intralobular region demonstrates a germinal-center-like allograft response with upregulated CD40, chemokine, T-follicular and complement signaling, which intensifies along the direction of sinusoidal blood flow from portal to central regions. Proteasomal and HSP90 inhibitors suppress donor-specific alloresponses of T- and B-cells in blood samples from patients with early rejection. The molecular injury spectrum of early rejection is complex, spatially differentiated and reveals novel early immunosuppressive strategies to extend graft survival. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND:Belatacept-treated kidney transplant recipients (KT) experience a lower incidence of de novo donor-specific anti-HLA antibody (DSA) formation despite their higher risk of acute cellular rejection. In vitro studies show that concentrations associated with half-maximal suppression (EC50) of target cells correlate with therapeutic trough concentrations (C0). PURPOSE:To determine whether belatacept inhibits B-cell alloantigen presentation and B-cell alloresponse at known therapeutic C0. METHODS:Peripheral blood leukocytes (PBL) from healthy adults were cultured with HLA-mismatched PBL or fluorochrome-labeled alloantigenic lysate and increasing belatacept concentrations. EC50 was calculated with best-fit four-parameter log-logistic function under a Poisson assumption, as described. RESULTS:After overnight allostimulation, frequencies of alloreactive CD154 + B-cells and their subsets decreased with increasing belatacept concentrations (n = 10). Median (range) EC50s were 1.3 (0.01-37) µg/ml for unfractionated B-cells, 2 (0.03-50) µg/ml for naïve, 3.6 (0.2-92) µg/ml for unswitched memory, 6.2 (0.05-32) µg.ml for transitional, and 7.8 (0.01-101) µg/ml for plasmablasts. Median EC50s were highest at 19 and 31 µg/ml, respectively for CD27- and CD27 + isotype-switched memory B-cells. No effect was seen on B-cell presentation of alloantigen. CONCLUSIONS:At lower C0 levels of < 10 µg/ml, belatacept suppresses alloresponses of B-cells and most B-cell subsets, thereby explaining lower levels of DSA in kidney transplant patients.

Background. Enhanced B-cell presentation of donor alloantigen relative to presentation of HLA-mismatched reference alloantigen is associated with acute cellular rejection (ACR), when expressed as a ratio called the antigen presenting index (API) in an exploratory cohort of liver and intestine transplant (LT and IT) recipients. Methods. To test clinical performance, we measured the API using the previously described 6-h assay in 84 LT and 54 IT recipients with median age 3.3 y (0.05–23.96). Recipients experiencing ACR within 60 d after testing were termed rejectors. Results. We first confirmed that B-cell uptake and presentation of alloantigen induced and thus reflected the alloresponse of T-helper cells, which were incubated without and with cytochalasin and primaquine to inhibit antigen uptake and presentation, respectively. Transplant recipients included 76 males and 62 females. Rejectors were tested at median 3.6 d before diagnosis. The API was higher among rejectors compared with nonrejectors (2.2 ± 0.2 versus 0.6 ± 0.04, P value = 1.7E-09). In logistic regression and receiver-operating-characteristic analysis, API ≥1.1 achieved sensitivity, specificity, and positive and negative predictive values for predicting ACR in 99 training set samples. Corresponding metrics ranged from 80% to 88% in 32 independent posttransplant samples, and 73% to 100% in 20 independent pretransplant samples. In time-to-event analysis, API ≥1.1 predicted higher incidence of late donor-specific anti-HLA antibodies after API measurements in LT recipients (P = 0.011) and graft loss in IT recipients (P = 0.008), compared with recipients with API <1.1, respectively. Conclusions. Enhanced donor antigen presentation by circulating B cells predicts rejection after liver or intestine transplantation as well as higher incidence of DSA and graft loss late after transplantation

Assessment of T-cell immunity to the COVID-19 coronavirus requires reliable assays and is of great interest, given the uncertain longevity of the antibody response. Some recent reports have used immunodominant spike (S) antigenic peptides and anti-CD28 co-stimulation in varying combinations to assess T-cell immunity to SARS-CoV-2. These assays may cause T-cell hyperstimulation and could overestimate antiviral immunity in chronically immunosuppressed transplant recipients, who are predisposed to infections and vaccination failures. Here, we evaluate CD154-expressing T-cells induced by unselected S antigenic peptides in 204 subjects-103 COVID-19 patients and 101 healthy unexposed subjects. Subjects included 72 transplanted and 130 non-transplanted subjects. S-reactive CD154+T-cells co-express and can thus substitute for IFNγ (n=3). Assay reproducibility in a variety of conditions was acceptable with coefficient of variation of 2-10.6%. S-reactive CD154+T-cell frequencies were a) higher in 42 healthy unexposed transplant recipients who were sampled pre-pandemic, compared with 59 healthy non-transplanted subjects (p=0.02), b) lower in Tr COVID-19 patients compared with healthy transplant patients (p<0.0001), c) lower in Tr patients with severe COVID-19 (p<0.0001), or COVID-19 requiring hospitalization (p<0.05), compared with healthy Tr recipients. S-reactive T-cells were not significantly different between the various COVID-19 disease categories in NT recipients. Among transplant recipients with COVID-19, cytomegalovirus co-infection occurred in 34%; further, CMV-specific T-cells (p<0.001) and incidence of anti-receptor-binding-domain IgG (p=0.011) were lower compared with non-transplanted COVID-19 patients. Healthy unexposed transplant recipients exhibit pre-existing T-cell immunity to SARS-CoV-2. COVID-19 infection leads to impaired T-cell and antibody responses to SARS-CoV-2 and increased risk of CMV co-infection in transplant recipients.

BackgroundOperational tolerance after retransplantation of the intestine has never been reported. PurposeTo two recently described intestine transplant recipients with operational tolerance, we now add a third. MethodsReview of case record and immunological testing to confirm donor-specific hyporesponsiveness in multiple immune cell compartments. ResultsRe-transplanted with a multivisceral liver- and kidney-inclusive intestine allograft at age 12 years, this recipient self-discontinued immunosuppression 14 years after the retransplant and has been rejection free for 2 years thereafter. As in the two previous reports, immunological testing demonstrated decreased donor-specific inflammatory response of T-cytotoxic memory cells and B-cells, decreased presentation of donor antigen by B-cells and monocytes, absence of donor-specific anti-HLA antibodies, circulating FOXP3 + T-helper cells, and intact cellular and humoral immunity to cytomegalovirus and Epstein-Barr virus. Additionally, our recipient demonstrated enhanced donor-activation-induced apoptosis of alloreactive T-cytotoxic memory cells. ConclusionsDespite variable paths to tolerance which include graft versus host disease in two previous cases, and rejection-related loss of the primary isolated intestinal allograft in our recipient, the three cases with operational tolerance are bound by common themes: a relatively large donor antigenic load transmitted during intestine transplantation, and donor-specific hyporesponsiveness. Cell-based assays suggest enhanced donor-induced apoptosis of recipient T-cells and circulating T-regulatory cells as mechanistic links between antigenic load and donor-specific hyporesponsiveness.

Background & Aims: Biliary atresia (BA) is poorly understood and leads to liver transplantation (LT), with the requirement for and associated risks of lifelong immunosuppression, in most children. We performed a genome-wide association study (GWAS) to determine the genetic basis of BA. Methods: We performed a GWAS in 811 European BA cases treated with LT in US, Canadian and UK centers, and 4,654 genetically matched controls. Whole-genome sequencing of 100 cases evaluated synthetic association with rare variants. Functional studies included whole liver transcriptome analysis of 64 BA cases and perturbations in experimental models. Results: A GWAS of common single nucleotide polymorphisms (SNPs), i.e. allele frequencies 1%, identified intronic SNPs rs6446628 in AFAP1 with genome-wide significance (p = 3.93E-8) and rs34599046 in TUSC3 at sub-threshold genome-wide significance (p = 1.34E-7), both supported by credible peaks of neighboring SNPs. Like other previously reported BA -associated genes, AFAP1 and TUSC3 are ciliogenesis and planar polarity effectors (CPLANE). In gene-set-based GWAS, BA was associated with 6,005 SNPs in 102 CPLANE genes (p = 5.84E-15). Compared with non-CPLANE genes, more CPLANE genes harbored rare variants (allele frequency <1%) that were assigned Human Phenotype Ontology terms related to hep-atobiliary anomalies by predictive algorithms, 87% vs. 40%, p <0.0001. Rare variants were present in multiple genes distinct from those with BA-associated common variants in most BA cases. AFAP1 and TUSC3 knockdown blocked ciliogenesis in mouse tracheal cells. Inhibition of ciliogenesis caused biliary dysgenesis in zebrafish. AFAP1 and TUSC3 were expressed in fetal liver organoids, as well as fetal and BA livers, but not in normal or disease-control livers. Integrative analysis of BA -associated variants and liver transcripts revealed abnormal vasculogenesis and epithelial tube formation, explaining portal vein anomalies that co-exist with BA. Conclusions: BA is associated with polygenic susceptibility in CPLANE genes. Rare variants contribute to polygenic risk in vulnerable pathways via unique genes.

Selecting the right immunosuppressant to ensure rejection-free outcomes poses unique challenges in pediatric liver transplant (LT) recipients. A molecular predictor can comprehensively address these challenges. Currently, there are no well-validated blood-based biomarkers for pediatric LT recipients before or after LT. Here, we discover and validate separate pre- and post-LT transcriptomic signatures of rejection. Using an integrative machine learning approach, we combine transcriptomics data with the reference high-quality human protein interactome to identify network module signatures, which underlie rejection. Unlike gene signatures, our approach is inherently multivariate and more robust to replication and captures the structure of the underlying network, encapsulating additive effects. We also identify, in an individual-specific manner, signatures that can be targeted by current anti-rejection drugs and other drugs that can be repurposed. Our approach can enable personalized adjustment of drug regimens for the dominant targetable pathways before and after LT in children.

BackgroundTranscriptional regulation of liver transplant (LT) rejection may reveal novel predictive and therapeutic targets.PurposeTo test the role of differential DNA methylation in children with biopsy-proven acute cellular rejection (rejectors, R) after LT.MethodsPaired peripheral blood DNA samples were obtained before and after LT from 17 children, including 4R and 13 non-rejector (NR), and assayed with MethylC capture sequencing (MCC-Seq) approach covering 5 million CpGs in immune-cell specific regulatory elements. Differentially methylated CpGs (DMCs) were identified using generalized linear regression models adjusting for sex and age and merged into differentially methylated regions (DMR) comprising 3 or more DMCs.ResultsContrasting R vs NR, we identified 2238 DMCs in post-LT and 2620 DMCs in pre-LT samples, which clustered in 216 and 282 DMRs respectively. DMCs associated with R were enriched in enhancers and depleted in promoters. The proportion of hypomethylated versus hypermethylated DMRs increased from 22% to 48% (p<0.0001) in pre-LT vs. post-LT DMCs, respectively. The highest-ranked biological processes enriched in post-LT DMCs were antigen processing and presentation via MHC class I, MHC class I complex, and peptide binding (p<7.92E-17), respectively. Top-ranked DMRs mapped to genes which mediate B-cell receptor signaling (ADAP1) or regulate several immune cells(ARRB2)(p<3.75E-08). DMRs in MHC class I genes were enriched for SNPs which bind TFs, affect gene expression and splicing, or alter peptide-binding amino acid sequences.ConclusionsDynamic methylation in distal regulatory regions reveals known transplant-relevant MHC-dependent rejection pathways, and identifies novel loci for future mechanistic evaluations in pediatric transplant subcohorts.

We evaluated post-vaccination immunity after COVID-19 vaccination with serial changes in cellular and antibody responses to the spike protein S, its S2 component which is conserved between SARS-CoV-2 and human coronaviruses, and the S1 component, which is specific to SARS-CoV-2 and also contains its receptor binding domain (RBD). In 21 healthy immunocompetent subjects all of whom demonstrated circulating IgG antibodies 4 months after mRNA1273 or BNT162b vaccination, a) the strength of S-IgG was stable while RBD-IgG declined, b) S2-reactive B-cell frequencies increased progressively (p=0.002) c) S1-reactive CD8+T-cells and CD19+B-cells were undetectable after a transient increase, and d) monocytic and polymorphonuclear myeloid-derived suppressor cells (M-MDSC, PMN-MDSC) increased after the first vaccine dose. Compared with 4-month measurements from immunocompetent subjects, single samples from 20 vaccinated immunocompromised (IC) subjects revealed a) circulating S-IgG and RBD-IgG in 13 (65%) and 9 (45%) subjects, respectively, b) no differences in S2-reactive T- and B-cells, c) undetectable S1-reactive T- and B-cells, and d) fewer S-reactive CD8+T-cells and CD19+B-cells (p<0.05). Among 11 IC recipients who failed to make RBD-IgG, frequencies of PMN-MDSC were significantly higher (p<0.0004) compared with IC or immunocompetent subjects with RBD-IgG. COVID-19 vaccination induces stable antibodies to the spike protein and expands circulating B-cells reactive to the conserved spike protein sequence in immunocompetent subjects. MDSC which are known to suppress T- and B-cells, and which increase after vaccination, may limit post-vaccination responses especially among immunocompromised subjects. Antibody and cellular responses to SARS-CoV-2-specific spike antigenic sequences appear to be less durable.