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Predicted Indirectly ReCognizable Human Leukocyte Antigen (HLA) Epitopes (PIRCHE) are known to be a significant risk factor for the development of donor HLA-specific antibodies after organ transplantation. Most previous studies on PIRCHE limited their analyses on the presentation of the HLA-DRB1 locus, although HLA-DRB3/4/5, -DQ, and -DP are also known for presenting allopeptides to CD4+ T cells. In this study, we analyzed the impact of predicted allopeptides presented by these additional loci on the incidence of HLA-specific antibodies after an immunization event. We considered pregnancy as a model system of an HLA immunization and observed child-specific HLA antibody (CSA) development of 231 mothers during pregnancy by samples being taken at delivery. Our data confirm that PIRCHE presented by HLA-DRB1 along with HLA-DRB3/4/5, -DQ, and -DP are significant predictors for the development of CSA. Although there was limited peptidome overlap observed within the mothers' presenting HLA proteins, combining multiple presenting loci in a single predictor improved the model only marginally. Prediction performance of PIRCHE further improved when normalizing scores by the respective presenters' binding promiscuity. Immunogenicity analysis of specific allopeptides could not identify significant drivers of an immune response in this small cohort, suggesting confirmatory studies., Competing Interests: MN works for PIRCHE AG, which develops and operates the PIRCHE web service. The UMC Utrecht has filed a patent application on the prediction of an alloimmune response against mismatched HLA. ES is listed as an inventor on this patent. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Niemann, Matern, Spierings, Schaub and Hönger.)

The functional Fc gamma receptor (FcγR) IIIA polymorphism FCGR3A -V/F158 was earlier suggested to determine the potential of donor-specific HLA antibodies to trigger microcirculation inflammation, a key lesion of antibody-mediated renal allograft rejection. Associations with long-term transplant outcomes, however, have not been evaluated to date. To clarify the impact of FCGR3A -V/F158 polymorphism on kidney transplant survival, we genotyped a cohort of 1,940 recipient/donor pairs. Analyzing 10-year death-censored allograft survival, we found no significant differences in relation to FCGR3A -V/F158. There was also no independent survival effect in a multivariable Cox model. Similarly, functional polymorphisms in two other activating FcγR, FCGR2A -H/R131 (FcγRIIA) and FCGR3B -NA1/NA2 (FcγRIIIB), were not associated with outcome. There were also no significant survival differences among patient subgroups at increased risk of rejection-related injury, such as pre-sensitized recipients (> 0% panel reactivity; n = 438) or recipients treated for rejection within the first year after transplantation (n = 229). Our study results suggest that the earlier shown association of FcγR polymorphism with microcirculation inflammation may not be strong enough to exert a meaningful effect on graft survival., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wahrmann, Döhler, Arnold, Scherer, Mayer, Haindl, Haslacher, Böhmig and Süsal.)

The outcome of organ transplantation is largely dictated by selection of a well-matched donor, which results in less chance of graft rejection. An allogeneic immune response is the main immunological barrier for successful organ transplantation. Donor and recipient human leukocyte antigen (HLA) mismatching diminishes outcomes after solid organ transplantation. The current evaluation of HLA incompatibility does not provide information on the immunogenicity of individual HLA mismatches and impact of non-HLA-related alloantigens, especially in vivo . Here we demonstrate a new method for analysis of alloimmune responsiveness between donor and recipient in vivo by introducing a humanized mouse model. Using molecular, cellular, and genomic analyses, we demonstrated that a recipient's personalized humanized mouse provided the most sensitive assessment of allogeneic responsiveness to potential donors. In our study, HLA typing provided a better recipient-donor match for one donor among two related donors. In contrast, assessment of an allogeneic response by mixed lymphocyte reaction (MLR) was indistinguishable between these donors. We determined that, in the recipient's humanized mouse model, the donor selected by HLA typing induced the strongest allogeneic response with markedly increased allograft rejection markers, including activated cytotoxic Granzyme B-expressing CD8 + T cells. Moreover, the same donor induced stronger upregulation of genes involved in the allograft rejection pathway as determined by transcriptome analysis of isolated human CD45 + cells. Thus, the humanized mouse model determined the lowest degree of recipient-donor alloimmune response, allowing for better selection of donor and minimized immunological risk of allograft rejection in organ transplantation. In addition, this approach could be used to evaluate the level of alloresponse in allogeneic cell-based therapies that include cell products derived from pluripotent embryonic stem cells or adult stem cells, both undifferentiated and differentiated, all of which will produce allogeneic immune responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ajith, Mulloy, Musa, Bravo-Egana, Horuzsko, Gani and Horuzsko.)

In pre-sensitizing events, immunological memory is mainly created via indirect allorecognition where CD4 + T cells recognize foreign peptides in the context of self-HLA class II (pHLA) presented on antigen-presenting cells. This recognition makes it possible for naive CD4 + T-helper cells to differentiate into memory cells, resulting in the creation of further antibody memory. These responses contribute to effective secretion of donor-specific anti-HLA antibodies (DSA) after second encounters with the same peptide. Preformed donor-reactive CD4 + memory T cells may induce early immune responses after transplantation; however, the tools to evaluate them are limited. This study evaluated shared T cell epitopes (TEs) between the pre-sensitizing and donor HLA using an in silico assay, an alternative to estimate donor-reactive CD4 + memory T cells before transplantation. In 578 living donor kidney transplants without preformed DSA, 69 patients had anti-HLA antibodies before transplantation. Of them, 40 had shared TEs and were estimated to have donor-reactive CD4 + memory T cells. De novo DSA formation in the early phase was significantly higher in the shared TE-positive group than in the anti-HLA antibody- and shared TE-negative groups (p=0.001 and p=0.02, respectively). In conclusion, evaluation of shared TEs for estimating preformed donor-reactive CD4 + memory T cells may help predict the risk of early de novo DSA formation after kidney transplantation., Competing Interests: MN is an employee of PIRCHE AG. The UMC Utrecht has filed a patent application on the prediction of an alloimmune response against mismatched HLA. ES is listed as inventor on this patent. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tomosugi, Iwasaki, Sakamoto, Niemann, Spierings, Nahara, Futamura, Okada, Hiramitsu, Takeda, Goto, Narumi, Watarai and Kobayashi.)

Prognostic, diagnostic or predictive biomarkers are urgently needed for assessment of chronic graft-versus-host disease (cGvHD), a major risk for patients undergoing allogeneic hematopoietic stem cell transplantation. The main goal of this review generated within the COST Action EUROGRAFT "Integrated European Network on Chronic Graft Versus Host Disease" was to identify potential novel biomarkers for cGvHD besides the widely accepted molecular and cellular biomarkers. Thus, the focus was on cellular biomarkers, alloantibodies, glycomics, endothelial derived particles, extracellular vesicles, microbiome, epigenetic and neurologic changes in cGvHD patients. Both host-reactive antibodies in general, and particularly alloantibodies have been associated with cGvHD and require further consideration. Glycans attached to IgG modulate its activity and represent a promising predictive and/or stratification biomarker for cGVHD. Furthermore, epigenetic changes such as microRNAs and DNA methylation represent potential biomarkers for monitoring cGvHD patients and novel targets for developing new treatment approaches. Finally, the microbiome likely affects the pathophysiology of cGvHD; bacterial strains as well as microbial metabolites could display potential biomarkers for dysbiosis and risk for the development of cGvHD. In summary, although there are no validated biomarkers currently available for clinical use to better inform on the diagnosis, prognosis or prediction of outcome for cGvHD, many novel sources of potential markers have shown promise and warrant further investigation using well characterized, multi-center patient cohorts., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer BB declared a shared affiliation, with no collaboration, with one of the authors, FP, to the handling editor at the time of review., (Copyright © 2020 Crossland, Perutelli, Bogunia-Kubik, Mooney, Milutin Gašperov, Pučić-Baković, Greinix, Weber, Holler, Pulanić, Wolff, Dickinson, Inngjerdingen and Grce.)

NKG2D is a potent activating immunoreceptor expressed on nearly all cytotoxic lymphocytes promoting their cytotoxicity against self-cells expressing NKG2D ligands (NKG2DLs). NKG2DLs are MHC class I-like glycoproteins that usually are not expressed on "healthy" cells. Rather, their surface expression is induced by various forms of cellular stress, viral infection, or malignant transformation. Hence, cell surface NKG2DLs mark "dangerous" cells for elimination by cytotoxic lymphocytes and therefore can be considered as "kill-me" signals. In addition, NKG2DLs are up-regulated on activated leukocytes, which facilitates containment of immune responses. While the NKG2D receptor is conserved among mammals, NKG2DL genes have rapidly diversified during mammalian speciation, likely due to strong selective pressures exerted by species-specific pathogens. Consequently, NKG2DL genes are not conserved in man and mice, although their NKG2D-binding domains maintained structural homology. Human NKG2DLs comprise two members of the MIC (MICA/MICB) and six members of the ULBP family of glycoproteins (ULBP1-6) with MICA representing the best-studied human NKG2DLs by far. Many of these studies implicate a role of MICA in various malignant, infectious, or autoimmune diseases. However, conclusions from these studies often were limited in default of supporting in vivo experiments. Here, we report a MICA transgenic (MICAgen) mouse model that replicates central features of human MICA expression and function and, therefore, constitutes a novel tool to critically assess and extend conclusions from previous in vitro studies on MICA. Similarly to humans, MICA transcripts are broadly present in organs of MICAgen mice, while MICA glycoproteins are barely detectable. Upon activation, hematopoietic cells up-regulate and proteolytically shed surface MICA. Shed soluble MICA (sMICA) is also present in plasma of MICAgen mice but affects neither surface NKG2D expression of circulating NK cells nor their functional recognition of MICA-expressing tumor cells. Accordingly, MICAgen mice also show a delayed growth of MICA-expressing B16F10 tumors, not accompanied by an emergence of MICA-specific antibodies. Such immunotolerance for the xenoantigen MICA ideally suits MICAgen mice for anti-MICA-based immunotherapies. Altogether, MICAgen mice represent a valuable model to study regulation, function, disease relevance, and therapeutic targeting of MICA in vivo ., (Copyright © 2020 Kim, Born, Bléry and Steinle.)

Objective: To evaluate the diagnostic performance of donor-derived plasma cell-free DNA (cfDNA) in discriminating antibody-mediated rejection (ABMR) or de novo donor-specific antibodies (DSA) without histological lesions in kidney allograft recipients., Methods: In this prospective single center observational study, we enrolled kidney allograft recipients between November, 2016 and September, 2017 at the First Affiliated Hospital of Sun Yat-sen University. Kidney allograft recipients with ABMR, de novo DSA but no histological lesions or negative DSA, and stable renal function were included. The plasma cfDNA fraction was measured using a targeted, single nucleotide polymorphism (SNP)-based assay. Pathological diagnosis was made according to the 2015 Banff Kidney Rejection Classification. The area under the ROC curve (AUC-ROC) was determined using the bootstrapping method to estimate median and 95% confidence interval (95% CI). The sensitivity, specificity and Youden index, positive predictive value (PPV), and negative predictive value (NPV) were calculated for specific cfDNA fractions., Results: Totally 37 consecutive patients received kidney allografts, including 18 recipients in the ABMR group and 19 recipients in the stable allograft group (7 DSA-positive and 12 DSA-negative). All patients in the ABMR group were DSA positive and 7 patients in the stable group were DSA positive but had no pathologically proven ABMR. The median donor-derived plasma cfDNA fraction was 2.4% (Q1 1.52% -Q3 3.70%) in the ABMR group, and was significantly higher than that of the stable group (0.65%, Q1 0.57% -Q3 0.97%; P < 0.001), but comparable with that of the DSA-positive patients in the stable allograft group ( P = 0.074). The AUC-ROC of cfDNA was 0.90 (95% CI, 0.79-0.98). When a cfDNA threshold of 1% was chosen, it had a sensitivity of 88.9% and a specificity of 73.7%. The PPV was 76.2% and the NPV was 87.5%., Conclusion: Donor-derived plasma cfDNA fraction increased in kidney allograft recipients with ABMR. Detection of donor-derived plasma cfDNA fraction may contribute to the discrimination between ABMR and stable renal allograft function and may aid early recognition of earlier stage antibody-mediated injury., (Copyright © 2020 Zhang, Zheng, Li, Fu, Li, Su, Zeng, Liu, Wang, Huang, Xu, Ye, Liu and Wang.)

Fc gamma receptors (FcγRs) play a major role in the regulation of humoral immune responses. Single-nucleotide polymorphisms (SNPs) of FCGR2A and FCGR3A can impact the expression level, IgG affinity and function of the CD32 and CD16 FcγRs in response to their engagement by the Fc fragment of IgG. The CD16 isoform encoded by FCGR3A [158V/V] controls the intensity of antibody-dependent cytotoxic alloimmune responses of natural killer cells (NK) and has been identified as a susceptibility marker predisposing patients to cardiac allograft vasculopathy after heart transplant. This study aimed to investigate whether FCGR2A and FCGR3A polymorphisms can also be associated with the clinical outcome of lung transplant recipients (LTRs). The SNPs of FCGR2A ([131R/H], rs1801274) and FCGR3A ([158V/F], rs396991) were identified in 158 LTRs and 184 Controls (CTL). The corresponding distribution of genotypic and allelic combinations was analyzed for potential links with the development of circulating donor-specific anti-HLA alloantibodies (DSA) detected at months 1 and 3 after lung transplant (LTx), the occurrence of acute rejection (AR) and chronic lung allograft dysfunction (CLAD), and the overall survival of LTRs. The FCGR3A [158V/V] genotype was identified as an independent susceptibility factor associated with higher rates of AR during the first trimester after LTx (HR 4.8, p < 0.0001, 95% CI 2.37-9.61), but it could not be associated with the level of CD16- mediated NK cell activation in response to the LTR's DSA, whatever the MFI intensity and C1q binding profiles of the DSA evaluated. The FCGR2A [131R/R] genotype was associated with lower CLAD-free survival of LTRs, independently of the presence of DSA at 3 months (HR 1.8, p = 0.024, 95% CI 1.08-3.03). Our data indicate that FCGR SNPs differentially affect the clinical outcome of LTRs and may be of use to stratify patients at higher risk of experiencing graft rejection. Furthermore, these data suggest that in the LTx setting, specific mechanisms of humoral alloreactivity, which cannot be solely explained by the complement and CD16-mediated pathogenic effects of DSA, may be involved in the development of acute and chronic lung allograft rejection.

Different profiles of alloantibody responses are observed in the clinic, with those that persist, often despite targeted treatment, associated with poorer long-term transplant outcomes. Although such responses would suggest an underlying germinal center (GC) response, the relationship to cellular events within the allospecific B cell population is unclear. Here we examine the contribution of germinal center (GC) humoral alloimmunity to chronic antibody mediated rejection (AMR). A murine model of chronic AMR was developed in which T cell deficient ( Tcrbd -/- ) C57BL/6 recipients were challenged with MHC-mismatched BALB/c heart allografts and T cell help provided by reconstituting with 10 3 "TCR75" CD4 T cells that recognize self-restricted allopeptide derived from the H-2K d MHC class I alloantigen. Reconstituted recipients developed Ig-switched anti-K d alloantibody responses that were slow to develop, but long-lived, with confocal immunofluorescence and flow cytometric characterization of responding H-2K d -allospecific B cells confirming persistent splenic GC activity. This was associated with T follicular helper (T FH ) cell differentiation of the transferred TCR75 CD4 T cells. Heart grafts developed progressive allograft vasculopathy, and were rejected chronically (MST 50 days), with explanted allografts displaying features of humoral vascular rejection. Critically, late alloantibody responses were abolished, and heart grafts survived indefinitely, in recipients reconstituted with Sh2d1a -/- TCR75 CD4 T cells that were genetically incapable of providing T FH cell function. The GC response was associated with affinity maturation of the anti-K d alloantibody response, and its contribution to progression of allograft vasculopathy related principally to secretion of alloantibody, rather than to enhanced alloreactive T cell priming, because grafts survived long-term when B cells could present alloantigen, but not secrete alloantibody. Similarly, sera sampled at late time points from chronically-rejecting recipients induced more vigorous donor endothelial responses in vitro than sera sampled earlier after transplantation. In summary, our results suggest that chronic AMR and progression of allograft vasculopathy is dependent upon allospecific GC activity, with critical help provided by T FH cells. Clinical strategies that target the T FH cell subset may hold therapeutic potential. This work is composed of two parts, of which this is Part II. Please read also Part I: Alsughayyir et al., 2019.

Humoral alloimmunity is now recognized as a major determinant of transplant outcome. MHC glycoprotein is considered a typical T-dependent antigen, but the nature of the T cell alloresponse that underpins alloantibody generation remains poorly understood. Here, we examine how the relative frequencies of alloantigen-specific B cells and helper CD4 T cells influence the humoral alloimmune response and how this relates to antibody-mediated rejection (AMR). An MHC-mismatched murine model of cardiac AMR was developed, in which T cell help for alloantibody responses in T cell deficient ( Tcrbd -/- ) C57BL/6 recipients against donor H-2K d MHC class I alloantigen was provided by adoptively transferred "TCR75" CD4 T cells that recognize processed H-2K d allopeptide via the indirect-pathway. Transfer of large numbers (5 × 10 5 ) of TCR75 CD4 T cells was associated with rapid development of robust class-switched anti-H-2K d humoral alloimmunity and BALB/c heart grafts were rejected promptly (MST 9 days). Grafts were not rejected in T and B cell deficient Rag2 -/- recipients that were reconstituted with TCR75 CD4 T cells or in control (non-reconstituted) Tcrbd -/- recipients, suggesting that the transferred TCR75 CD4 T cells were mediating graft rejection principally by providing help for effector alloantibody responses. In support, acutely rejecting BALB/c heart grafts exhibited hallmark features of acute AMR, with widespread complement C4d deposition, whereas cellular rejection was not evident. In addition, passive transfer of immune serum from rejecting mice to Rag2 -/- recipients resulted in eventual BALB/c heart allograft rejection (MST 20 days). Despite being long-lived, the alloantibody responses observed at rejection of the BALB/c heart grafts were predominantly generated by extrafollicular foci: splenic germinal center (GC) activity had not yet developed; IgG secreting cells were confined to the splenic red pulp and bridging channels; and, most convincingly, rapid graft rejection still occurred when recipients were reconstituted with similar numbers of Sh2d1a -/- TCR75 CD4 T cells that are genetically incapable of providing T follicular helper cell function for generating GC alloimmunity. Similarly, alloantibody responses generated in Tcrbd -/- recipients reconstituted with smaller number of wild-type TCR75 CD4 T cells (10 3 ), although long-lasting, did not have a discernible extrafollicular component, and grafts were rejected much more slowly (MST 50 days). By modeling antibody responses to Hen Egg Lysozyme protein, we confirm that a high ratio of antigen-specific helper T cells to B cells favors development of the extrafollicular response, whereas GC activity is favored by a relatively high ratio of B cells. In summary, a relative abundance of helper CD4 T cells favors development of strong extrafollicular alloantibody responses that mediate acute humoral rejection, without requirement for GC activity. This work is composed of two parts, of which this is Part I. Please read also Part II: Chhabra et al., 2019.

A novel vaccine against bovine viral diarrhea (BVD) induced pathogenic antibody production in 5-10% of BVD-vaccinated cows. Transfer of these antibodies via colostrum caused Bovine neonatal pancytopenia (BNP) in calves, with a lethality rate of 90%. The exact immunological mechanisms behind the onset of BNP are not fully understood to date. To gain further insight into these mechanisms, we analyzed the immune proteome from alloreactive antibody producers (BNP cows) and non-responders. After in vitro stimulation of peripheral blood derived lymphocytes (PBL), we detected distinctly deviant expression levels of several master regulators of immune responses in BNP cells, pointing to a changed immune phenotype with severe dysregulation of immune response in BNP cows. Interestingly, we also found this response pattern in 22% of non-BVD-vaccinated cows, indicating a genetic predisposition of this immune deviant (ID) phenotype in cattle. We additionally analyzed the functional correlation of the ID phenotype with 10 health parameters and 6 diseases in a retrospective study over 38 months. The significantly increased prevalence of mastitis among ID cows emphasizes the clinical relevance of this deviant immune response and its potential impact on the ability to fight infections.

Heterologous immunity of virus-specific T cells poses a potential barrier to transplantation tolerance. Cross-reactivity to HLA-A and -B molecules has broadly been described, whereas responses to allo-HLA-C have remained ill defined. In contrast to the transplant setting, HLA-C is the only polymorphic HLA molecule expressed by extravillous trophoblasts at the maternal-fetal interface during pregnancy. Uncontrolled placental viral infections, accompanied by a pro-inflammatory milieu, can alter the activation status and stability of effector T cells. Potential cross-reactivity of maternal decidual virus-specific T cells to fetal allo-HLA-C may thereby have detrimental consequences for the success of pregnancy. To explore the presence of cross-reactivity to HLA-C and the other non-classical HLA antigens expressed by trophoblasts, HLA-A and -B-restricted CD8+ T cells specific for Epstein-Barr virus, Cytomegalovirus, Varicella-Zoster virus, and Influenza virus were tested against target cells expressing HLA-C, -E, and -G molecules. An HLA-B * 08:01-restricted EBV-specific T cell clone displayed cross-reactivity against HLA-C * 01:02. Furthermore, cross-reactivity of HLA-C-restricted virus-specific CD8+ T cells was observed for HCMV HLA-C * 06:02/TRA CD8+ T cell lines and clones against HLA-C * 03:02. Collectively, these results demonstrate that cross-reactivity against HLA-C can occur and thereby may affect pregnancy outcome.

Acute graft- vs. -host disease (GVHD) is an important cause of morbidity and death after allogeneic hematopoietic cell transplantation (HCT). We identify a new approach to prevent GVHD that impairs monocyte-derived dendritic cell (moDC) alloactivation of T cells, yet preserves graft- vs.-leukemia (GVL). Exceeding endoplasmic reticulum (ER) capacity results in a spliced form of X-box binding protein-1 (XBP-1s). XBP-1s mediates ER stress and inflammatory responses. We demonstrate that siRNA targeting XBP-1 in moDCs abrogates their stimulation of allogeneic T cells. B-I09, an inositol-requiring enzyme-1α (IRE1α) inhibitor that prevents XBP-1 splicing, reduces human moDC migration, allo-stimulatory potency, and curtails moDC IL-1β, TGFβ, and p40 cytokines, suppressing Th1 and Th17 cell priming. B-I09-treated moDCs reduce responder T cell activation via calcium flux without interfering with regulatory T cell (Treg) function or GVL effects by cytotoxic T lymphocytes (CTL) and NK cells. In a human T cell mediated xenogeneic GVHD model, B-I09 inhibition of XBP-1s reduced target-organ damage and pathogenic Th1 and Th17 cells without impacting donor Tregs or anti-tumor CTL. DC XBP-1s inhibition provides an innovative strategy to prevent GVHD and retain GVL.