Analysis of a Genetic Polymorphism in the Costimulatory Molecule TNFSF4 with Hematopoietic Stem Cell Transplant Outcomes.

Despite stringent procedures to secure the best HLA matching between donors and recipients, life-threatening complications continue to occur after hematopoietic stem cell transplantation (HSCT). Studying single nucleotide polymorphism (SNP) in genes encoding costimulatory molecules could help identify patients at risk for post-HSCT complications. In a stepwise approach we selected SNPs in key costimulatory molecules including CD274, CD40, CD154, CD28, and TNFSF4 and systematically analyzed their association with post-HSCT outcomes. Our discovery cohort analysis of 1157 HLA-A, -B, -C, -DRB1, and -DQB1 matched cases found that patients with donors homozygous for the C variant of rs10912564 in TNFSF4 (48%) had better disease-free survival (P = .029) and overall survival (P = .009) with less treatment-related mortality (P = .006). Our data demonstrate the TNFSF4C variant had a higher affinity for the nuclear transcription factor Myb and increased percentage of TNFSF4-positive B cells after stimulation compared with CT or TT genotypes. However, these associations were not validated in a more recent cohort, potentially because of changes in standard of practice or absence of a true association. Given the discovery cohort, functional data, and importance of TNFSF4 in infection clearance, TNFSF4C may associate with outcomes and warrants future studies.

Costimulation-dependent expression of microRNA-214 increases the ability of T cells to proliferate by targeting Pten.

T cell activation requires signaling through the TCR and costimulatory molecules, such as CD28. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally and are also known to be involved in lymphocyte development and function. In this paper, we set out to examine potential roles of miRNAs in T cell activation, using genome-wide expression profiling to identify miRNAs differentially regulated following T cell activation. One of the miRNAs upregulated after T cell activation, miR-214, was predicted to be capable of targeting Pten based on bioinformatics and reports suggesting that it targets Pten in ovarian tumor cells. Upregulation of miR-214 in T cells inversely correlated with levels of phosphatase and tensin homolog deleted on chromosome 10. In vivo, transcripts containing the 3' untranslated region of Pten, including the miR-214 target sequence, were negatively regulated after T cell activation, and forced expression of miR-214 in T cells led to increased proliferation after stimulation. Blocking CD28 signaling in vivo prevented miR-214 upregulation in alloreactive T cells. Stimulation of T cells through the TCR alone was not sufficient to result in upregulation of miR-214. Thus, costimulation-dependent upregulation of miR-214 promotes T cell activation by targeting the negative regulator Pten. Thus, the requirement for T cell costimulation is, in part, related to its ability to regulate expression of miRNAs that control T cell activation.

Reducing number of laboratories performing complement dependent cytotoxicity crossmatching: Reasons and conclusions.

Complement dependent cytotoxicity crossmatch (CDC-XM) has been the original standard crossmatch test, whereas, flow cytometry crossmatch (FCXM) is an enhanced and highly sensitive crossmatch assay performed to detect donor specific anti-HLA antibodies (DSA). We analyzed American Society for Histocompatibility and Immunogenetics (ASHI) proficiency testing data (2011-2020) and examined the number of laboratories performing CDC-XM vs. FCXM, the overall efficiency of laboratories in reporting ≥80% consensus CDC-XM vs. FCXM result, and reasons for non-consensus results in the two assays. Of 600 crossmatches in each crossmatch category, the percentage of laboratories reporting T cell CDC-XMs reduced from 40% in 2011 to 13% in 2020, T cell anti-human globulin (AHG) CDC-XM reduced from 56% in 2011 to 21% in 2020, and B cell CDC-XM reduced from 51% in 2011 to 20% in 2020. The percentage of laboratories performing T cell and B cell FCXM remained at approximately 80% throughout. CDC-XM performed on par with FCXM in providing a consensus negative result using negative DSA serum, but under-performed in comparison to FCXM in providing a consensus positive result using positive DSA serum. In addition, only minority of CDC-XMs was reported positive in presence of complement fixing DSA. This study shows that non-consensus CDC-XM was always in presence of HLA IgG DSA and that laboratories may be struggling to interpret the low sensitive CDC-XM results, where highly sensitive solid phase multi-antigen or single antigen assay shows the presence of HLA IgG DSA in serum.

Performance characteristics of chimerism testing by next generation sequencing.

Chimerism testing provides informative clinical data regarding the status of a biological sample mixture. For years, this testing was achieved by measuring the peaks of informative short tandem repeat (STR) loci using capillary electrophoresis (CE). With the advent of next generation sequencing (NGS) technology, the quantification of the percentage of donor/recipient mixtures is more easily done using sequence reads in large batches of samples run on a single flow cell. In this study, we present data on using a FORENSIC NGS chimerism platform to accurately measure the percentage of donor/recipient mixtures. We were able to detect chimerism to a limit threshold of 1% using both STR and single nucleotide polymorphism (SNP) informative loci. Importantly, a significant correlation was observed between NGS and CE chimerism methods when compared at donor detection ranges from 1% to 10%. Furthermore, 100% accuracy was achieved through proficiency testing over six surveys. Its usefulness was expanded beyond this to help identify suitable donors for solid organ transplant patients using ancestry SNP profiles. In summary, the NGS method provides a sensitive and reliable alternative to traditional CE for chimerism testing of clinical samples.

Induction of transplantation tolerance to fully mismatched cardiac allografts by T cell mediated delivery of alloantigen.

Induction of transplantation tolerance has the potential to allow for allograft acceptance without the need for life-long immunosuppression. Here we describe a novel approach that uses delivery of alloantigen by mature T cells to induce tolerance to fully allogeneic cardiac grafts. Adoptive transfer of mature alloantigen-expressing T cells into myeloablatively conditioned mice results in long-term acceptance of fully allogeneic heart transplants without evidence of chronic rejection. Since myeloablative conditioning is clinically undesirable we further demonstrated that adoptive transfer of mature alloantigen-expressing T cells alone into mice receiving non-myeloablative conditioning resulted in long-term acceptance of fully allogeneic heart allografts with minimal evidence of chronic rejection. Mechanistically, tolerance induction involved both deletion of donor-reactive host T cells and the development of regulatory T cells. Thus, delivery of alloantigen by mature T cells induces tolerance to fully allogeneic organ allografts in non-myeloablatively conditioned recipients, representing a novel approach for tolerance induction in transplantation.

Genetic Polymorphism in Cytokines and Costimulatory Molecules in Stem Cell and Solid Organ Transplantation.

There is growing evidence supporting the genetic variability outside of HLA system that is contributing to the variation in transplant outcomes. Determining novel predictors could help to identify patients at risk and tailor their immunosuppressive regimens. This article discusses the various single nucleotide polymorphisms in costimulatory molecules and cytokines that have been evaluated for their effect on transplantation. An overview of how gene polymorphism studies are conducted and factors to consider in the experimental design to ensure meaningful data can be concluded are discussed.

MHC class I and integrin ligation induce ERK activation via an mTORC2-dependent pathway.

The aim of this study was to characterize the interaction between mTOR and ERK in primary endothelial cells (EC) following MHC class I and integrin ligation. Ligation of MHC class I molecules or integrins on the surface of EC leads to phosphorylation of ERK at Thr202/Tyr204. We utilized small interfering RNA (siRNA) blockade of mTOR and proteins involved in mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) to define a relationship between mTOR and ERK following MHC class I signaling. We found mTORC2 was responsible for MHC class I and integrin induced phosphorylation of ERK at Thr202/Tyr204. We corroborated these results demonstrating that long-term exposure to rapamycin also inhibited ERK pathway activation in response to MHC class I signaling. Our results demonstrate, for the first time, that engagement of either MHC class I or integrin on the surface of EC leads to ERK activation through an mTORC2-dependent pathway.

Human Leukocyte Antigen Epitope Matching in Solid Organ Transplantation.

HLA epitope matching provides a better approach to stratify patients at risk of developing antibody-mediated rejection compared with counting HLA mismatches. However, several immunologic parameters are not incorporated into these algorithms used to assess HLA epitopes, raising questions about the predictive value of these programs. Therefore, it is imperative to obtain more 3D structural data of antibody-antigen binding to "train" these computer algorithms. Also, mechanistic studies should be performed to prove these theoretic "epitopes." Most important, more information is needed to ensure these predictive computer algorithms are equitable and safe to use in clinical diagnostics before wide-scale implementation.

Comprehensive immunoproteogenomic analyses of malignant pleural mesothelioma.

We generated a comprehensive atlas of the immunologic cellular networks within human malignant pleural mesothelioma (MPM) using mass cytometry. Data-driven analyses of these high-resolution single-cell data identified 2 distinct immunologic subtypes of MPM with vastly different cellular composition, activation states, and immunologic function; mass spectrometry demonstrated differential abundance of MHC-I and -II neopeptides directly identified between these subtypes. The clinical relevance of this immunologic subtyping was investigated with a discriminatory molecular signature derived through comparison of the proteomes and transcriptomes of these 2 immunologic MPM subtypes. This molecular signature, representative of a favorable intratumoral cell network, was independently associated with improved survival in MPM and predicted response to immune checkpoint inhibitors in patients with MPM and melanoma. These data additionally suggest a potentially novel mechanism of response to checkpoint blockade: requirement for high measured abundance of neopeptides in the presence of high expression of MHC proteins specific for these neopeptides.

Author Correction: Glomerulocapillary miRNA response to HLA-class I antibody in vitro and in vivo.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Glomerulocapillary miRNA response to HLA-class I antibody in vitro and in vivo.

Changes in miRNA expression glomerular of capillaries during antibody-mediated rejection (ABMR) are poorly understood and could contribute to the deleterious inflammation and fibrosis of ABMR via suppression of target genes. A better understanding could lead to novel diagnostic tools and reveal novel therapeutic targets. We explored deregulated miRNAs in an glomeruloendothelial in vitro model of ABMR due to class I human leukocyte antigen (HLA) with and without complement activation. We studied a set of 16 promising candidate miRNAs in microdissected glomeruli a confirmation set of 20 human transplant biopsies (DSA+) compared to 10 matched controls without evidence for ABMR. Twelve out of these 16 glomerulocapillary miRNAs could successfully be confirmed as dysregulated in vivo with 10 upregulated (let-7c-5p, miR-28-3p, miR-30d-5p, miR-99b-5p, miR-125a-5p, miR-195-5p, miR-374b-3p, miR-484, miR-501-3p, miR-520e) and 2 downregulated (miR29b-3p, miR-885-5p) in DSA+ vs. CONTROLS: A random forest analysis based on glomerular miRNAs identified 18/20 DSA+ and 8/10 controls correctly. This glomerulocapillary miRNA signature associated with HLA class I-DSA could improve our understanding of ABMR and be useful for diagnostic or therapeutic purposes.

Anti-HLA antibodies can induce endothelial cell survival or proliferation depending on their concentration.

Patients exhibiting a humoral immune response to the transplanted organ are at increased risk of antibody-mediated rejection and development of transplant vasculopathy. Historically, antibodies were thought to elicit transplant rejection through complement mediated damage of the endothelium of the graft. More recently, studies from our laboratory and others have shown that antibody ligation of class I molecules on the surface of endothelial cells transduces signals resulting in functional changes including expression of cell survival proteins and cell proliferation. The intracellular events initiated by antibody ligation are dependent upon the degree of molecular aggregation and influenced by the concentration of the antibody and level of human leukocyte antigen (HLA) expression. Herein we describe our recent findings on the effect of molecular aggregation on the class I signaling pathway in human endothelial cells.

Pediatric Liver Transplantation Across the ABO Blood Group Barrier: Is It an Obstacle in the Modern Era?

BACKGROUND: The initial experience with ABO incompatible (ABOi) orthotopic liver transplantations (OLTs) was dismal. In the current study, we investigated whether ABOi pediatric OLTs could achieve acceptable patient outcomes. The option for ABOi transplantation is vital because critically ill children have limited access to donor liver allografts. STUDY DESIGN: Kaplan-Meier and multivariate Cox analysis was performed on data collected from 13,179 pediatric OLT recipients in the United Network for Organ Sharing database, including 540 ABOi recipients. We also analyzed 18 pediatric recipients of ABOi OLTs at Texas Children's Hospital. Recipients were divided into 2 groups: transplanted between 1987 to 2002 (remote era) and 2002 to 2013 (modern era). RESULTS: Analysis revealed 4 main points. First, there was a significant (p < 0.01) improvement in ABOi OLT survival in the modern era. Second, threshold analysis revealed superior outcomes (p < 0.01) for OLT recipients younger than 2 years of age. Third, survival outcomes for ABOi and ABO-identical OLTs were the same for recipients younger than 2 years: ABOi was 91.8% (1 year) and 88.4% (5 year), and ABO identical was 91.5% (1 year) and 86.7% (5 year) (p = 0.94). Lastly, we found identical OLT results when analyzing our own institutional experience. To date, there has been a 92.9% survival rate in the modern era compared with 75% in the remote era. All recipients younger than 2 years (n = 9) are still alive, compared with 78% of those older than 2 years. CONCLUSIONS: This analysis revealed a significant improvement in the survival of ABOi liver transplant recipients in the modern era. Importantly, ABOi liver transplantation can be performed in recipients younger than 2 years of age with equivalent outcomes compared with ABO-identical recipients.

Anti-HLA class I antibodies activate endothelial cells and promote chronic rejection.

Transplant recipients exhibiting a humoral immune response to the allograft demonstrate lower graft survival and increased risk for the development of chronic rejection and transplant arteriosclerosis. Our studies suggest that anti-HLA class I antibodies (Ab) play an important role in controlling endothelial cell (EC) function by binding to class I molecules on the surface of the EC and transducing intracellular signals. Anti-HLA Ab exhibit two primary effector functions: stimulation of cell proliferation and up-regulation of cell survival genes. Importantly, the intracellular events initiated by class I ligation appear to be influenced by the concentration of the Ab. High-titered anti-HLA Ab stimulate cell proliferation whereas low-titered Ab activate the PI3K/Akt pathway and promote expression of cell survival proteins including Bcl-2 and Bcl-xL. Anti-HLA class I Ab may contribute to the process of chronic allograft rejection by promoting EC survival and proliferation.

Anti-HLA class I antibody-mediated activation of the PI3K/Akt signaling pathway and induction of Bcl-2 and Bcl-xL expression in endothelial cells.

Anti-human leukocyte antigen (HLA) antibodies (Ab) have long been implicated in the process of acute and chronic allograft rejection, yet their mechanism(s) of action is not well understood. The aim of this study was to determine whether ligation of HLA class I molecules by anti-HLA Ab on the surface of human endothelial cells (EC) activates the PI3 Kinase (PI3K)/Akt signaling pathway and downstream target proteins of the cell death apparatus. We report that Ab ligation of major histocompatibility complex (MHC) class I molecules on the surface of EC triggers phosphorylation of Akt, PI3K, and recruitment of PI3K and Akt into a signaling unit with focal adhesion kinase. Signaling through class I also stimulated phosphorylation of Bad and upregulated expression of Bcl-2 and Bcl-xL. Pretreatment of EC with the PI3K inhibitor wortmannin blocked class I-mediated expression of Bcl-2, but not Bcl-xL, suggesting a role for the PI3K/Akt signaling pathway in regulation of class I-induced Bcl-2 expression. The intracellular events initiated by class I ligation were influenced by the concentration of the anti-HLA Ab with the lowest tested concentrations of Ab stimulating the highest level of Akt phosphorylation, Bcl-xL and Bcl-2 expression. Consistent with the in vitro experiments, analysis of biopsy samples from heart transplant recipients with evidence of Ab-mediated rejection exhibited increased Bcl-2 expression on the vascular endothelium. These results suggest that exposure of the graft endothelium to low concentrations of anti-HLA Ab may promote cell survival by transducing signals resulting in upregulation of cell survival genes.

HLA class I antibody-mediated endothelial cell proliferation via the mTOR pathway.

Anti-HLA Abs have been shown to contribute to the process of transplant vasculopathy by binding to HLA class I molecules expressed by the endothelial and smooth muscle cells of the graft and transducing intracellular signals that elicit cell proliferation. The aim of this study was to determine the role of mammalian target of rapamycin (mTOR) in HLA class I-induced endothelial cell proliferation and to explore in depth the relationship between mTOR complexes and their downstream targets following ligation of HLA class I molecules by anti-HLA Abs. We used small interfering RNA technology to abrogate mTOR, rapamycin-insensitive companion of mTOR (rictor), or regulatory associated protein of mTOR (raptor) to study the function of these gene products to activate proteins involved in MHC class I-induced cell proliferation and survival. Knockdown of mTOR inhibited class I-mediated phosphorylation of proteins downstream of mTOR complex 1 and mTOR complex 2. Furthermore, knockdown of mTOR, rictor, or raptor blocked HLA class I-induced endothelial cell proliferation. Long-term pretreatment with the mTOR inhibitor rapamycin significantly blocked both mTOR-raptor and mTOR-rictor complex formation. Interestingly, rapamycin also blocked class I-induced Akt phosphorylation at Ser(473) and Bcl-2 expression. These results support the role of anti-HLA Abs in the process of transplant vasculopathy and suggest that exposure of the graft endothelium to anti-HLA Abs may promote proliferation through the mTOR pathway.

Anti-MHC class I antibody activation of proliferation and survival signaling in murine cardiac allografts.

Anti-MHC class I alloantibodies have been implicated in the process of acute and chronic rejection because these Abs can bind to endothelial cells and transduce signals leading to the activation of cell survival and proliferation pathways. To characterize the role of the MHC class I-signaling pathway in the pathogenesis of Ab-mediated rejection, we developed a mouse vascularized heterotopic cardiac allograft model in which B6.RAG1 KO hosts (H-2K(b)/D(b)) received a fully MHC-incompatible BALB/c (H-2K(d)/D(d)) heart transplant and were passively transfused with anti-donor MHC class I Ab. We demonstrate that cardiac allografts of mice treated with anti-MHC class I Abs show characteristic features of Ab-mediated rejection including microvascular changes accompanied by C4d deposition. Phosphoproteomic analysis of signaling molecules involved in the MHC class I cell proliferation and survival pathways were elevated in anti-class I-treated mice compared with the isotype control-treated group. Pairwise correlations, hierarchical clustering, and multidimensional scaling algorithms were used to dissect the class I-signaling pathway in vivo. Treatment with anti-H-2K(d) Ab was highly correlated with the activation of Akt and p70S6Kinase (S6K). When measuring distance as a marker of interrelatedness, multidimensional scaling analysis revealed a close association between members of the mammalian target of rapamycin pathway including mammalian target of rapamycin, S6K, and S6 ribosomal protein. These results provide the first analysis of the interrelationships between these signaling molecules in vivo that reflects our knowledge of the signaling pathway derived from in vitro experiments.

RNA interference elucidates the role of focal adhesion kinase in HLA class I-mediated focal adhesion complex formation and proliferation in human endothelial cells.

Ligation of class I molecules by anti-HLA Ab stimulates an intracellular signaling cascade resulting in endothelial cell (EC) survival and proliferation, and has been implicated in the process of chronic allograft rejection and transplant-associated vasculopathy. In this study, we used small interfering RNA blockade of focal adhesion kinase (FAK) protein to determine its role in class I-mediated organization of the actin cytoskeleton, cell survival, and cell proliferation in primary cultures of human aortic EC. Knockdown of FAK appreciably inhibited class I-mediated phosphorylation of Src at Tyr(418), p85 PI3K, and Akt at both Thr(308) and Ser(473) sites. FAK knockdown also reduced class I-mediated phosphorylation of paxillin at Try(118) and blocked class I-induced paxillin assembly into focal contacts. FAK small interfering RNA completely abrogated class I-mediated formation of actin stress fibers. Interestingly, FAK knockdown did not modify fibroblast growth factor receptor expression induced by class I ligation. However, FAK knockdown blocked HLA class I-stimulated cell cycle proliferation in the presence and absence of basic fibroblast growth factor. This study shows that FAK plays a critical role in class I-induced cell proliferation, cell survival, and focal adhesion assembly in EC and may promote the development of transplant-associated vasculopathy.