High resolution HLA genotyping with third generation sequencing technology-A multicentre study.

Molecular HLA typing techniques are currently undergoing a rapid evolution. While real-time PCR is established as the standard method in tissue typing laboratories regarding allocation of solid organs, next generation sequencing (NGS) for high-resolution HLA typing is becoming indispensable but is not yet suitable for deceased donors. By contrast, high-resolution typing is essential for stem cell transplantation and is increasingly required for questions relating to various disease associations. In this multicentre clinical study, the TGS technique using nanopore sequencing is investigated applying NanoTYPE™ kit and NanoTYPER™ software (Omixon Biocomputing Ltd., Budapest, Hungary) regarding the concordance of the results with NGS and its practicability in diagnostic laboratories. The results of 381 samples show a concordance of 99.58% for 11 HLA loci, HLA-A, -B, -C, -DRB1, -DRB3, -DRB4, -DRB5, -DQA1, -DQB1, -DPA1 and -DPB1. The quality control (QC) data shows a very high quality of the sequencing performed in each laboratory, 34,926 (97.15%) QC values were returned as 'passed', 862 (2.4%) as 'inspect' and 162 (0.45%) as 'failed'. We show that an 'inspect' or 'failed' QC warning does not automatically lead to incorrect HLA typing. The advantages of nanopore sequencing are speed, flexibility, reusability of the flow cells and easy implementation in the laboratory. There are challenges, such as exon coverage and the handling of large amounts of data. Finally, nanopore sequencing presents potential for applications in basic research within the field of epigenetics and genomics and holds significance for clinical concerns.

NK and T cell repertoire is established early after allogeneic HSCT and is profoundly imprinted by CMV reactivation.

Besides genetic influences, non-genetic factors such as graft-versus-host disease (GvHD) and viral infections have been shown as important shapers of the immune reconstitution and diversification processes after hematopoietic stem cell transplantation (HSCT). However, the differential susceptibility to immune modulation by non-genetic factors is not fully understood. We determined to follow the reconstitution of the T cell receptor (TCR) repertoire through immune-sequencing, of natural killer (NK) cells using a 35-marker spectral flow cytometry panel, and in relation to clinical events. Longitudinal investigation was performed on samples derived from 54 HSCT recipients during the first-year post-HSCT. We confirmed a significant contraction in TCR repertoire diversity with a remarkable stability over time. CMV reactivation had the ability to significantly change TCR repertoire clonality and composition, with a long-lasting imprint. Our data further revealed skewing of NK cell reconstitution in CMV reactivated recipients, with an increased frequency of KIR2DL2L3S2+ adaptive, cytolytic and functional CD107a+ NK cells concomitant with a reduced pool of NKG2A+ NK cells. We provided support that CMV might act as one of the more important driver of peripheral homeostatic proliferation of circulating specific T and NK cells, which can be viewed as a compensatory mechanism to establish a new peripheral repertoire.

HLA variants and TCR diversity against SARS-CoV-2 in the pre-COVID-19 era.

HLA antigen presentation and T-cell mediated immunity are critical to control acute viral infection such as COVID-19 caused by SARS-CoV-2. Recent data suggest that both the depth of peptide presentation and the breadth of the T-cell repertoire are associated with disease outcome. It has also been shown that unexposed subjects can develop strong T-cell responses against SARS-CoV-2 due to heterologous immunity. In this study, we explored the anti-SARS-CoV-2 T-cell repertoire by analyzing previously published T-cell receptor (TCR) CDR3ß immunosequencing data in a cohort of 116 healthy donors and in the context of immune reconstitution after allogeneic hematopoietic stem cell transplantation in 116 recipients collected during the pre-COVID-19 era. For this, 143,310 publicly available SARS-CoV-2 specific T-cell sequences were investigated among the 3.5 million clonotypes in the cohort. We also performed HLA class I peptide binding predictions using the reference proteome of the virus and high resolution genotyping data in these patients. We could demonstrate that individuals are fully equipped at the genetic level to recognize SARS-CoV-2. This is evidenced by the 5% median cumulative frequency of clonotypes having their sequence matched to a SARS-CoV-2 specific T-cell. In addition, any combination of HLA class I variants in this cohort is associated with a broad capacity of presenting hundreds of SARS-CoV-2 derived peptides. These results could be explained by heterologous immunity and random somatic TCR recombination. We speculate that these observations could explain the efficacy of the specific immune response against SARS-CoV-2 in individuals without risk factors of immunodeficiency and infected prior to vaccination.

Pre-transplant donor-specific HLA antibodies and risk for poor first-year renal transplant outcomes: results from the Swiss Transplant Cohort Study.

The aim of this study was to analyze first year renal outcomes in a nationwide prospective multicenter cohort comprising 2215 renal transplants, with a special emphasis on the presence of pre-transplant donor-specific HLA antibodies (DSA). All transplants had a complete virtual crossmatch and DSA were detected in 19% (411/2215). The investigated composite endpoint was a poor first-year outcome defined as (i) allograft failure or (ii) death or (iii) poor allograft function (eGFR ≤25 ml/min/1.73 m2 ) at one year. Two hundred and twenty-one (221/2215; 10%) transplants showed a poor first-year outcome. Rejection (24/70; 34%) was the most common reason for graft failure. First-year patient's death was rare (48/2215; 2%). There were no statistically significant differences between DSA-positive and DSA-negative transplants regarding composite and each individual endpoint, as well as reasons for graft failure and death. DSA-positive transplants experienced more frequently rejection episodes, mainly antibody-mediated rejection (both P < 0.0001). The combination of DSA and any first year rejection was associated with the overall poorest death-censored allograft survival (P < 0.0001). In conclusion, presence of pre-transplant DSA per se does not affect first year outcomes. However, DSA-positive transplants experiencing first year rejection are a high-risk population for poor allograft survival and may benefit from intense clinical surveillance.

Analysis of biological models to predict clinical outcomes based on HLA-DPB1 disparities in unrelated transplantation.

HLA compatibility is a key factor for survival after unrelated hematopoietic stem cell transplantation (HSCT). HLA-A, -B, -C, -DRB1, and -DQB1 are usually matched between donor and recipient. By contrast, HLA-DPB1 mismatches are frequent, although it is feasible to optimize donor selection and DPB1 matching with prospective typing. Because classical DPB1 allele mismatches are often unavoidable, however, several biological models have been developed to predict the optimal DPB1 mismatch combination for less graft-versus-host disease (GVHD) and better overall survival. In 909 recipient/donor pairs, we analyzed the role of 3 biological models: T-cell epitopes (TCEs) based on the immunogenicity of DPB1, cell surface expression of DPB1 molecules based on a single-nucleotide polymorphism located in the 3' untranslated region, and the Predicted Indirectly ReCognizable HLA Epitopes (PIRCHE) model based on the presentation of allogeneic peptides derived from mismatched HLA, compared with the classical allele mismatch. Matching for both DPB1 alleles remains the best option to prevent acute GVHD. In the situation of one DPB1 allele mismatch, the donor associated with the lowest acute GVHD risks is mismatched for an allele with a low expression profile in the recipient, followed by a permissive TCE3/4 mismatch and/or the absence of PIRCHE II potential against the recipient. In the context of 2 DPB1 mismatches, the same considerations apply for a permissive TCE3/4 mismatch and no PIRCHE II. By combining the biological models, the most favorable DPB1 constellation can be defined. This approach will help optimize donor selection and improve post-HSCT complications and patient prognosis.

Peripheral Donor-specific Antibodies Are Associated With Histology and Cellular Subtypes in Protocol Liver Biopsies of Pediatric Recipients.

BACKGROUND: The cellular infiltrate in protocol liver biopsies (PB) following pediatric liver transplantation remains mostly uncharacterized, yet there is increasing concern about the role of inflammation and fibrosis in long-term liver allografts. We aimed to define cell types in PB and to analyze their relationship with donor-specific antibodies (DSA) and histological phenotype. METHODS: PB were performed at least 1 year after transplantation. We identified 4 phenotypes: normal, fibrosis, inflammation, inflammation with fibrosis. Cell types were counted after immunostaining for CD3, CD4, CD8, CD68, CD20, MUM1, and FoxP3. RESULTS: Forty-four patients underwent 1 PB between 2000 and 2015. Eleven percent (5/44) of PB displayed normal histology, 13.6% (6/44) fibrosis, 34.1% (15/44) inflammation, and 40.9% (18/44) inflammation and fibrosis. The main cell types in the portal tracts and lobules were CD3+ and CD68+ cells. Frequency of de novo DSA was 63% (27/44). The presence of CD8+ cells in the lobules was associated with fibrosis. Inflammation and fibrosis in PB were associated with the presence of circulating de novo DSA, number of de novo DSA, and C1q binding activity when compared to other phenotypes. CONCLUSIONS: T cells (CD3+) and macrophages (CD68+) were the most prevalent cell-types in PB. In the presence of inflammation, portal tracts were enriched in CD3+, CD20+ but displayed fewer CD68+. This coincided with the presence and number of de novo DSA. How these cellular and humoral actors interact is unclear, but peripheral DSA may be a marker of immune cellular activity in the seemingly quiescent allograft.

Characterization of the novel HLA-DQB1*03:01:46 variant allele in a French hematopoietic stem cell donor.

HLA-DQB1*03:01:46 differs from HLA-DQB1*03:01:01:01 by one nucleotide substitution at codon 142.3 in exon 3.

Correction: High-resolution HLA phased haplotype frequencies to predict the success of unrelated donor searches and clinical outcome following hematopoietic stem cell transplantation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genetic T-cell receptor diversity at 1 year following allogeneic hematopoietic stem cell transplantation.

After allogeneic hematopoietic stem cell transplantation (HSCT), immune reconstitution leads to the development of a new T-cell repertoire. Immune reconstitution could be influenced by events such as conditioning, infections, and graft versus host disease (GVHD). Factors influencing the TCR diversity are of great interest to fine-tune the strategy for donor selection and to optimize standard of care. In this work, immunosequencing of the TCR CDR3ß region was carried out in a large cohort of 116 full chimeric recipients at 1 year post-HSCT and their respective donors prior to transplantation. The repertoire overlap before and after HSCT was minimal, supporting de novo reconstitution as a primary pathway at any age. Among the parameters investigated, increased patient and/or donor age as well as positive CMV serologic status reinforced by CMV infection/reactivation were the ones significantly associated with a reduced diversity at 1 year post-HSCT. CMV-specific T-cell clones were shown to influence the clonality of the repertoire alongside the expansion of limited numbers of non-CMV T-cell populations. Interestingly, at the exception of CMV infection/reactivation, TCR diversity was not predictive of GVHD, relapse, death, or infections post-HSCT.

Preformed and de novo DSA are associated with T-cell-mediated rejection in pediatric liver transplant recipients requiring clinically indicated liver biopsy.

Despite growing interest about the impact of donor-specific HLA antibodies (DSA) in LT limited data are available for pediatric recipients. Our aim was to perform a retrospective single-center chart review of children (0-16 years) having undergone LT between January 1, 2005 and December 31, 2017, to characterize DSA, to identify factors associated with the development of de novo DSA, and to analyze potential associations with the diagnosis of TCMR. Information on patient- and donor-characteristics and LB reports were analyzed retrospectively. Serum obtained before LT and at LB was analyzed for presence of recipient HLA antibody using Luminex® technology. MFI > 1000 was considered positive. In 63 pediatric LT recipients with a median follow-up of 72 months, the overall prevalence of de novo DSA was 60.3%. Most were directed against class II antigens (33/38, 86.8%). Preformed DSA were present in 30% of patients. Twenty-eight (28/63) patients (44.4%) presented at least one episode of TCMR, mostly (12/28, 43%) moderate (Banff 6-7). De novo DSA were significantly more frequent in patients with TCMR than in patients without (75% vs 48.6%, P = .03), and patients with preformed and de novo DSA had a significantly higher rate of TCMR than patients without any DSA (66.7% vs 20%, P = .02). Neither preformed DSA nor de novo DSA were associated with frequency or severity of TCMR. Recipients with lower weight at LT developed de novo DSA more frequently (P = .04). De novo DSA were highly prevalent in pediatric LT recipients. Although associated with the development of TCMR, they did not appear to impact the frequency or severity of TCMR or graft survival. Instead, de novo DSA may suggest a state of insufficient IS.

High-resolution HLA phased haplotype frequencies to predict the success of unrelated donor searches and clinical outcome following hematopoietic stem cell transplantation.

HLA matching is a critical factor for successful allogeneic hematopoietic stem cell transplantation. For unrelated donor searches, matching is usually based on high-resolution typing at five HLA loci, looking for a 10/10 match. Some studies have proposed that further matching at the haplotype level could be beneficial for clinical outcome. In this study, we determined the phased haplotypes of 291 patients using family members and segregation analysis. The sum of ranks of the haplotypes carried by patients was used as a surrogate predictor of a successful unrelated donor search. The putative impact of haplotypes was then analyzed in a cohort of 211 recipients transplanted with 10/10 matched unrelated donors. A logistic regression analysis showed a highly significant effect of the haplotypes in the outcome of a search, but we did not find any significant effect on overall survival, graft versus host disease or relapse/progression following HSCT. This study provides useful data for the optimization of unrelated bone marrow donor searches, but does not confirm previous reports that matching at the haplotype level has a clinical impact following HSCT. Due to the extreme polymorphism of HLA genes, further studies are warranted to better understand the many factors at play.

Clinical and histological evolution after de novo donor-specific anti-human leukocyte antigen antibodies: a single centre retrospective study.

BACKGROUND: Donor-specific anti-human leukocyte antigen (HLA) antibodies (DSA) can be preformed or de novo (dn). Strategies to manage preformed DSA are well described, but data on the management and outcomes of dnDSA are lacking. METHODS: We performed a retrospective analysis of data from a single centre of the management and outcomes of 22 patients in whom a dnDSA was identified with contemporary and follow up biopsies. RESULTS: Evolution from baseline to follow up revealed a statistically significant loss of kidney function (estimated glomerular filtration rate: 45.9 ± 16.7 versus 37.4 ± 13.8 ml/min/1.73 m2; p = 0.005) and increase in the proportion of patients with transplant glomerulopathy (percentage with cg lesion ≥1: 27.2% vs. 45.4%; p = 0.04). Nine patients were not treated at the time of dnDSA identification, and 13 patients received various drug combinations (e.g., corticosteroids, plasmapheresis, thymoglobulins and/or rituximab). No significant pathological changes were observed for the various treatment combinations. CONCLUSION: Our retrospective analysis of a small sample suggests that dnDSA should be considered a risk factor for the loss of kidney function independent of the baseline biopsy, and multidisciplinary evaluations of the transplant patient are a necessary requirement. Further confirmation in a multicentre prospective trial is required.

Expression of killer cell immunoglobulin-like receptors (KIRs) by natural killer cells during acute CMV infection after kidney transplantation.

Human cytomegalovirus (CMV) infection may be a serious complication related to immunosuppression after solid organ transplantation. Due to their cytotoxicity, T-cells and natural killer (NK) cells target and clear the virus from CMV-infected cells. Although immunosuppressive drugs suppress T-cell proliferation and activation, they do not affect NK cells that are crucial for controlling the infection. The regulation of NK cells depends on a wide range of activating and inhibitory receptors such as the family of killer-cell immunoglobulin-like receptors (KIRs). Several human genetic studies have demonstrated the association of KIR genes with the clearance of infections. Since the respective activities of the different KIR proteins expressed by NK cells during CMV infection have not been extensively studied, we analyzed the expression of KIRs in a cohort of 22 CMV-IgG(+) renal transplant patients at the time of CMV reactivation, after antiviral therapy and 6 months later. Our data revealed a marked expression of KIR3DL1 during the acute phase of the reactivation. We set up an in vitro model in which NK cells, derived either from healthy donors or from transplanted patients, target allogeneic fibroblasts, CMV-infected or uninfected. Our results demonstrate a significant correlation between the lysis of CMV-infected fibroblasts and the expression of KIR3DL1. Blocking experiments with antibodies to MHC-I, to NKG2D and to NKG2C confirmed the importance of KIR3DL1. Consequently, our results suggest that KIR proteins and especially KIR3DL1 could play an important role during CMV-infection or CMV reactivation in immunosuppressed patients.

Late antibody-mediated rejection by de novo donor HLA-DP-specific antibody after renal transplantation: a case report.

The role of donor HLA-DP-specific antibodies after renal transplantation is controversial, and only preformed HLA-DP-specific antibodies have been shown to mediate rejection. Here we present a case of late humoral rejection mediated by de novo donor HLA-DP-specific antibodies in a non-sensitized recipient. This unique case demonstrates the pathogenic role of de novo anti-DP antibodies and suggests that HLA-DP matching might be relevant for renal transplantation.

The clinical relevance of pre-formed anti-HLA and anti-MICA antibodies after cord blood transplantation in children.

Preformed anti-HLA antibodies (AHA) are known to be associated with delayed engraftment and reduced overall survival after adult hematopoietic stem cell transplantation. However, limited data is available in pediatric patients. In this study, we explored the role of AHA on clinical outcomes in 70 pediatric patients who received a single unit of HLA mismatch cord blood for hematologic malignancies, immunodeficiencies or metabolic diseases. The presence of AHA was detected in 44% (31/70) of the patients. Preformed class I AHA was associated with an increased occurrence of grade 1-4 acute graft-versus host disease (p<0.05). The presence of anti- major-histocompatibility-complex class I-related chain A antigens (MICA) antibodies was significantly associated with a reduced platelet recovery after transplantation (p<0.05). AHA of class II with the strength of antibody titer measured as the mean fluorescence intensity above 2000 was associated with reduced event-free survival (p<0.05). A reduction of high titer of AHA and anti-MICA antibodies might have to be considered before cord blood transplantation in pediatric patients for better outcomes.

Effects of an interleukin-15 antagonist on systemic and skeletal alterations in mice with DSS-induced colitis.

Inflammatory bowel diseases are commonly complicated by weight and bone loss. We hypothesized that IL-15, a pro-inflammatory cytokine expressed in colitis and an osteoclastogenic factor, could play a central role in systemic and skeletal complications of inflammatory bowel diseases. We evaluated the effects of an IL-15 antagonist, CRB-15, in mice with chronic colitis induced by oral 2% dextran sulfate sodium for 1 week, followed by another 1% for 2 weeks. During the last 2 weeks, mice were treated daily with CRB-15 or an IgG2a control antibody. Intestinal inflammation, disease severity, and bone parameters were evaluated at days 14 and 21. CRB-15 improved survival, early weight loss, and colitis clinical score, although colon damage and inflammation were prevented in only half the survivors. CRB-15 also delayed loss of femur bone mineral density and trabecular microarchitecture. Bone loss was characterized by decreased bone formation, but increased bone marrow osteoclast progenitors and osteoclast numbers on bone surfaces. CRB-15 prevented the suppression of osteoblastic markers of bone formation, and reduced osteoclast progenitors at day 14, but not later. However, by day 21, CRB-15 decreased tumor necrosis factor α and increased IL-10 expression in bone, paralleling a reduction of osteoclasts. These results delineate the role of IL-15 on the systemic and skeletal manifestations of chronic colitis and provide a proof-of-concept for future therapeutic developments.

Contact with stimulated T cells up-regulates expression of peptidylarginine deiminase 2 and 4 by human monocytes.

OBJECTIVE: The antigenic targets of the rheumatoid arthritis (RA)-associated autoantibodies to "citrullinated" proteins are generated following citrullination by a peptidylarginine deiminase (PAD). Of the five PAD isotypes, two - PAD2 and PAD4 - are expressed in RA synovial tissue. Within this tissue, the activation of macrophages and fibroblasts mediated by T-cell contact or driven by cytokines plays a prominent part in the pathogenesis. We wanted to determine whether cytokine stimulation and contact with T cells could play a role in PAD expression by peripheral blood monocytes and fibroblastic synoviocytes. METHODS: Human monocytes and T lymphocytes were derived from the blood of healthy donors. HUT-78 cells and T lymphocytes were stimulated with PHA and PMA. Human synovial fibroblasts were isolated after surgical synoviectomy. The expression of PAD was determined by real-time PCR and immunoblot. RESULTS: In monocytes, the PADI2 and PADI4 mRNAs were transiently up-regulated by contact with stimulated HUT-78 and/or T lymphocytes. Positive modulation of the PAD2 and PAD4 proteins were also observed upon contact with stimulated HUT-78 T cells. Stimulation by IL-1ß or IFN-ß did not modify the PADI2 and PADI4 mRNAs, but enhanced PAD4 protein expression. No isotype of PAD was detected at the mRNA or protein level in resting or stimulated synovial fibroblasts. CONCLUSION: Contact between stimulated T cells and monocyte-macrophages or cytokine-activated monocyte-macrophages constitutes a highly likely source of PAD2 and PAD4, which are observed in inflamed synovial tissues. In contrast, it is most unlikely that fibroblastic synoviocytes contribute to PAD expression in rheumatoid synovial membranes.

Natural killer cell receptor--repertoire and functions after induction therapy by polyclonal rabbit anti-thymocyte globulin in unsensitized kidney transplant recipients.

Polyclonal rabbit anti-thymocyte globulin (rATG) is widely used in solid organ transplantation (SOT) as induction therapy or to treat corticosteroid-resistant rejection. In vivo, the effect of rATG on natural killer (NK) cells has not been studied. These cells are of particular relevance after SOT because classical immunosuppressive drugs do not inhibit or even can activate NK cells. A cohort of 20 recipients at low immunological risk, that had been receiving rATG as induction therapy, was analyzed for receptor repertoire, cytotoxicity and capacity of NK cells to secrete IFN-γ before kidney transplantation and at different time points thereafter. NK cells expressed fewer killer-cell immunoglobulin-like receptors (KIR), fewer activating receptors NKG2D, but more inhibitory receptor NKG2A compatible with an immature phenotype in the first 6 months post transplantation. Both cytotoxicity of NK cells and the secretion of IFN-γ were preserved over time after transplantation.

Inhibition of T cell-dependent and RANKL-dependent osteoclastogenic processes associated with high levels of bone mass in interleukin-15 receptor-deficient mice.

OBJECTIVE: T cell production of RANKL, interferon-γ (IFNγ), and other cytokines in inflammatory processes such as rheumatoid arthritis or secondary to conditions such as estrogen deficiency stimulates osteoclast activity, which leads to bone resorption and bone loss. The purpose of this study was to characterize the effects of interleukin-15 (IL-15), a master T cell growth factor whose role in bone remodeling remains unknown. METHODS: We used mice lacking the IL-15 receptor (IL-15Rα(-/-) ) to investigate the effects of IL-15 on osteoclast development, T cell and dendritic cell activation in vitro and in vivo, bone mass, and microarchitecture in intact and ovariectomized (OVX) mice. RESULTS: In wild-type (WT) animals, IL-15 and RANKL provided a costimulatory signal for osteoclast development. Spleens from IL-15Rα(-/-) mice contained few c-Kit+ osteoclast precursors, and the expression of NF-ATc1 and the osteoclastogenic response to RANKL were impaired. In addition, dendritic cell-dependent and T cell-dependent mechanisms of osteoclast activation, including RANKL and IFNγ production, were impaired in IL-15Rα(-/-) mice. In turn, IL-15Rα(-/-) T cells failed to stimulate WT osteoclasts, whereas WT T cells failed to stimulate IL-15Rα(-/-) osteoclasts. Compared with WT mice, both intact and OVX IL-15Rα(-/-) mice had significantly greater bone mineral density and microarchitecture, including a higher trabecular bone volume fraction and cortical thickness. The numbers of osteoclasts on the bone surface as well as markers of bone turnover were significantly decreased in IL-15Rα(-/-) mice. CONCLUSION: In the absence of IL-15 signaling, several converging mechanisms of osteoclastogenesis are inhibited, both directly and indirectly, through T cells, which leads to a high bone mass phenotype. Targeting the IL-15 pathway may represent a novel therapeutic approach to treating primary and secondary osteoporosis.