Analysis of the T-cell repertoire and transcriptome identifies mechanisms of regulatory T-cell suppression of GVHD.

CD4+FOXP3+ regulatory T cells (Tregs) have demonstrated efficacy in the prevention and treatment of graft-versus-host disease (GVHD). Preclinical and clinical studies indicate that Tregs are able to protect from GVHD without interfering with the graft-versus-tumor (GVT) effect of hematopoietic cell transplantation (HCT), although the underlying molecular mechanisms are largely unknown. To elucidate Treg suppressive function during in vivo suppression of acute GVHD, we performed paired T-cell receptor (TCRα and ΤCRß genes) repertoire sequencing and RNA sequencing analysis on conventional T cells (Tcons) and Tregs before and after transplantation in a major histocompatibility complex -mismatched mouse model of HCT. We show that both Tregs and Tcons underwent clonal restriction, and Tregs did not interfere with the activation of alloreactive Tcon clones and the breadth of their TCR repertoire but markedly suppressed their expansion. Transcriptomic analysis revealed that Tregs predominantly affected the transcriptome of CD4 Tcons and, to a lesser extent, that of CD8 Tcons, thus modulating the transcription of genes encoding pro- and anti-inflammatory molecules as well as enzymes involved in metabolic processes, inducing a switch from glycolysis to oxidative phosphorylation. Finally, Tregs did not interfere with the induction of gene sets involved in the GVT effect. Our results shed light onto the mechanisms of acute GVHD suppression by Tregs and will support the clinical translation of this immunoregulatory approach.

Regulation of HLA class I expression by non-coding gene variations.

The Human Leukocyte Antigen (HLA) is a critical genetic system for different outcomes after solid organ and hematopoietic cell transplantation. Its polymorphism is usually determined by molecular technologies at the DNA level. A potential role of HLA allelic expression remains under investigation in the context of the allogenic immune response between donors and recipients. In this study, we quantified the allelic expression of all three HLA class I loci (HLA-A, B and C) by RNA sequencing and conducted an analysis of expression quantitative traits loci (eQTL) to investigate whether HLA expression regulation could be associated with non-coding gene variations. HLA-B alleles exhibited the highest expression levels followed by HLA-C and HLA-A alleles. The max fold expression variation was observed for HLA-C alleles. The expression of HLA class I loci of distinct individuals demonstrated a coordinated and paired expression of both alleles of the same locus. Expression of conserved HLA-A~B~C haplotypes differed in distinct PBMC's suggesting an individual regulated expression of both HLA class I alleles and haplotypes. Cytokines TNFα /IFNß, which induced a very similar upregulation of HLA class I RNA and cell surface expression across alleles did not modify the individually coordinated expression at the three HLA class I loci. By identifying cis eQTLs for the HLA class I genes, we show that the non-coding eQTLs explain 29%, 13%, and 31% of the respective HLA-A, B, C expression variance in unstimulated cells, and 9%, 23%, and 50% of the variance in cytokine-stimulated cells. The eQTLs have significantly higher effect sizes in stimulated cells compared to unstimulated cells for HLA-B and HLA-C genes expression. Our data also suggest that the identified eQTLs are independent from the coding variation which defines HLA alleles and thus may be influential on intra-allele expression variability although they might not represent the causal eQTLs.

Detection of circulating highly expanded T-cell clones in at-risk individuals for rheumatoid arthritis before the clinical onset of the disease.

OBJECTIVES: To quantitatively profile the T-cell repertoire in the peripheral blood of individuals genetically at risk for RA, namely first-degree relatives of RA patients (RA-FDR) at different phases of disease development. METHODS: Next-generation sequencing of the TCR CDR3ß repertoire was performed on genomic DNA isolated from whole blood samples of RA-FDR selected at three different pre-clinical stages and of matched RA patients (n = 20/group). T-cell clones were identified by their unique sequence and their degree of expansion (frequency) within each sample was characterized. Clones with a frequency over 0.5% were considered highly expanded clones (HEC). RESULTS: The absolute number of HEC was significantly higher in established RA patients (mean 4.65) and tended to be higher in symptomatic RA-FDR (mean 3.4) compared with asymptomatic RA-FDR (mean 1.55, P =0.003 and P =0.07, respectively). Asymptomatic individuals with high levels of ACPA did not differ from asymptomatic RA-FDR in terms of absolute number and frequency of clones. The number of HEC tended to be slightly higher at the time of RA onset (P =0.055). Neither clones shared by several patients, nor clones previously associated with RA, were preferentially present within or between the different groups. Finally, a longitudinal analysis did not allow to uncover a kinetic expansion of RA-specific clones closely correlated with disease development. CONCLUSIONS: HEC were detected in the peripheral blood before the clinical onset of RA, in particular in the later pre-clinical phase of RA development, and their presence increased over time.

[Evaluation of the new algorithm in ANCA testing]. / Présentation des nouveaux algorithmes de dosage d'ANCA.

Testing for ANCA is useful for the diagnosis and monitoring of disease activity in ANCA associated vasculitis. ANCA testing is also sometimes requested for other indications. The relevance of indirect immunofluorescence in ANCA testing is controversial and has recently been the object of new international recommendations. The new approach certainly improves the performance of this test, particularly in Switzerland where, due to the large number of requests, the pre-test probability of ANCA associated vasculitis is relatively low. However, in this article we discuss the possible flaws of the new algorithm based on two recent cases, and highlight the importance of a good communication with the laboratory in order to improve the interpretation of the results.

La recherche des ANCA est utilisée pour le diagnostic et le suivi de l'activité des vasculites associées aux ANCA, mais aussi parfois pour d'autres indications plus controversées. La place de l'immunofluorescence indirecte dans l'algorithme de dépistage et de suivi des vasculites à ANCA est contestée depuis plusieurs années et a récemment fait l'objet de nouvelles recommandations internationales. Cette nouvelle approche améliore certainement le rendement de ce test, en particulier en Suisse où, en raison du grand nombre d'analyses demandé, la probabilité prétest d'une vasculite à ANCA est relativement faible. Cependant, nous discutons dans cet article les possibles failles de la nouvelle approche en nous basant sur deux cas récents au laboratoire et rendons les prescripteurs attentifs sur l'importance de la communication avec le laboratoire afin d'améliorer l'interprétation des résultats.

Correction to: HLA class I molecular variation and peptide-binding properties suggest a model of joint divergent asymmetric selection.

The above article originally published with an incomplete bibliographic information for Bitarello et al. (2016) and presented correctly in this article.

The HLA-B landscape of Africa: Signatures of pathogen-driven selection and molecular identification of candidate alleles to malaria protection.

Human leukocyte antigen (HLA) genes play a key role in the immune response to infectious diseases, some of which are highly prevalent in specific environments, like malaria in sub-Saharan Africa. Former case-control studies showed that one particular HLA-B allele, B*53, was associated with malaria protection in Gambia, but this hypothesis was not tested so far within a population genetics framework. In this study, our objective was to assess whether pathogen-driven selection associated with malaria contributed to shape the HLA-B genetic landscape of Africa. To that aim, we first typed the HLA-A and -B loci in 484 individuals from 11 populations living in different environments across the Sahel, and we analysed these data together with those available for 29 other populations using several approaches including linear modelling on various genetic, geographic and environmental parameters. In addition to relevant signatures of populations' demography and migrations history in the genetic differentiation patterns of both HLA-A and -B loci, we found that the frequencies of three HLA alleles, B*53, B*78 and A*74, were significantly associated with Plasmodium falciparum malaria prevalence, suggesting their increase through pathogen-driven selection in malaria-endemic environments. The two HLA-B alleles were further identified, by high-throughput sequencing, as B*53:01:01 (in putative linkage disequilibrium with one HLA-C allele, C*04:01:01:01) and B*78:01 in all but one individuals tested, making them appropriate candidates to malaria protection. These results highlight the role of environmental factors in the evolution of the HLA polymorphism and open key perspectives for functional studies focusing on HLA peptide-binding properties.

HLA class I molecular variation and peptide-binding properties suggest a model of joint divergent asymmetric selection.

The main function of HLA class I molecules is to present pathogen-derived peptides to cytotoxic T lymphocytes. This function is assumed to drive the maintenance of an extraordinary amount of polymorphism at each HLA locus, providing an immune advantage to heterozygote individuals capable to present larger repertories of peptides than homozygotes. This seems contradictory, however, with a reduced diversity at individual HLA loci exhibited by some isolated populations. This study shows that the level of functional diversity predicted for the two HLA-A and HLA-B genes considered simultaneously is similar (almost invariant) between 46 human populations, even when a reduced diversity exists at each locus. We thus propose that HLA-A and HLA-B evolved through a model of joint divergent asymmetric selection conferring all populations an equivalent immune potential. The distinct pattern observed for HLA-C is explained by its functional evolution towards killer cell immunoglobulin-like receptor (KIR) activity regulation rather than peptide presentation.

A significant effect of the killer cell immunoglobulin-like receptor ligand human leucocyte antigen-C on fibrosis progression in chronic C hepatitis with or without liver transplantation.

BACKGROUND & AIMS: The interaction of killer cell immunoglobulin-like receptors with their human leucocyte antigen ligands drives the activation and inhibition of natural killer cells. Natural killer cells could be implicated in the development of liver fibrosis in chronic hepatitis C. METHODS: We analysed 206 non-transplanted and 53 liver transplanted patients, selected according to their Metavir fibrosis stage. Several variables such as the number of activator killer cell immunoglobulin-like receptors or the human leucocyte antigen ligands were considered in multinomial and logistic regression models. Possible confounding variables were also investigated. RESULTS: The killer cell immunoglobulin-like receptors were not significant predictors of the fibrosis stage. Conversely, a significant reduction of the human leucocyte antigen-C1C2 genotype was observed in the most advanced fibrosis stage group (F4) in both cohorts. Furthermore, the progression rate of fibrosis was almost 10 times faster in the subgroup of patients after liver transplantation, and human leucocyte antigen-C1C2 was significantly reduced in this cohort compared with non-transplanted patients. CONCLUSION: This study suggests a possible role of killer cell immunoglobulin-like receptors and their ligands in the development of liver damage. The absence of C1 and C2 ligands heterozygosity could lead to less inhibition of natural killer cells and a quicker progression to a high level of fibrosis in patients infected with hepatitis C virus, especially following liver transplantation.

HLA supertype variation across populations: new insights into the role of natural selection in the evolution of HLA-A and HLA-B polymorphisms.

Supertypes are groups of human leukocyte antigen (HLA) alleles which bind overlapping sets of peptides due to sharing specific residues at the anchor positions-the B and F pockets-of the peptide-binding region (PBR). HLA alleles within the same supertype are expected to be functionally similar, while those from different supertypes are expected to be functionally distinct, presenting different sets of peptides. In this study, we applied the supertype classification to the HLA-A and HLA-B data of 55 worldwide populations in order to investigate the effect of natural selection on supertype rather than allelic variation at these loci. We compared the nucleotide diversity of the B and F pockets with that of the other PBR regions through a resampling procedure and compared the patterns of within-population heterozygosity (He) and between-population differentiation (G ST) observed when using the supertype definition to those estimated when using randomized groups of alleles. At HLA-A, low levels of variation are observed at B and F pockets and randomized He and G ST do not differ from the observed data. By contrast, HLA-B concentrates most of the differences between supertypes, the B pocket showing a particularly high level of variation. Moreover, at HLA-B, the reassignment of alleles into random groups does not reproduce the patterns of population differentiation observed with supertypes. We thus conclude that differently from HLA-A, for which supertype and allelic variation show similar patterns of nucleotide diversity within and between populations, HLA-B has likely evolved through specific adaptations of its B pocket to local pathogens.

A new HLA map of Europe: Regional genetic variation and its implication for peopling history, disease-association studies and tissue transplantation.

OBJECTIVES: HLA genes are highly polymorphic in human populations as a result of diversifying selection related to their immune function. However, HLA geographic variation worldwide suggests that demographic factors also shaped their evolution. We here analyzed in detail HLA genetic variation in Europe in order to identify signatures of migration history and/or natural selection. METHODS: Relationships between HLA diversity and geography were analyzed at 7 loci through several approaches including linear regression on gene diversity and haplotype frequencies. Regional variation was also assessed on HLA multi-locus phenotypes through structure analysis. Deviation from neutrality was tested by resampling. RESULTS: Geographic distance was a strong predictor of HLA variation at 5 loci (A, B, C, DRB1 and DPB1) in Europe, and latitude significantly shaped HLA gene diversity and haplotype frequencies. Whereas the main level of genetic diversity was found within populations, both HLA gene frequencies and phenotypic profiles revealed regional variation, Southeast Europe, Great Britain and Finland being the most distinctive. Effects of natural selection were suggested at the DQ loci. CONCLUSIONS: HLA regional variation was observed in Europe and can be related to population history, locus HLA-A providing by far the strongest signals. This new HLA map of Europe represents an invaluable reference for disease-association studies and tissue transplantation.

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.

Immunogenetics as a tool in anthropological studies.

The genes coding for the main molecules involved in the human immune system--immunoglobulins, human leucocyte antigen (HLA) molecules and killer-cell immunoglobulin-like receptors (KIR)--exhibit a very high level of polymorphism that reveals remarkable frequency variation in human populations. 'Genetic marker' (GM) allotypes located in the constant domains of IgG antibodies have been studied for over 40 years through serological typing, leading to the identification of a variety of GM haplotypes whose frequencies vary sharply from one geographic region to another. An impressive diversity of HLA alleles, which results in amino acid substitutions located in the antigen-binding region of HLA molecules, also varies greatly among populations. The KIR differ between individuals according to both gene content and allelic variation, and also display considerable population diversity. Whereas the molecular evolution of these polymorphisms has most likely been subject to natural selection, principally driven by host-pathogen interactions, their patterns of genetic variation worldwide show significant signals of human geographic expansion, demographic history and cultural diversification. As current developments in population genetic analysis and computer simulation improve our ability to discriminate among different--either stochastic or deterministic--forces acting on the genetic evolution of human populations, the study of these systems shows great promise for investigating both the peopling history of modern humans in the time since their common origin and human adaptation to past environmental (e.g. pathogenic) changes. Therefore, in addition to mitochondrial DNA, Y-chromosome, microsatellites, single nucleotide polymorphisms and other markers, immunogenetic polymorphisms represent essential and complementary tools for anthropological studies.

Characterization of the novel HLA-B*15:514 allele in a French hematopoietic stem cell donor.

The novel HLA-B*15:514 allele was characterized using two next-generation sequencing technologies.

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.

CD8+ T-Cell Repertoire in Human Leukocyte Antigen Class I-Mismatched Alloreactive Immune Response.

In transplantation, direct allorecognition is a complex interplay between T-cell receptors (TCR) and HLA molecules and their bound peptides expressed on antigen-presenting cells. In analogy to HLA mismatched hematopoietic stem cell transplantation (HSCT), the TCR CDR3ß repertoires of alloreactive cytotoxic CD8+ responder T cells, defined by the cell surface expression of CD137 and triggered in vitro by HLA mismatched stimulating cells, were analyzed in different HLA class I mismatched combinations. The same HLA mismatched stimulatory cells induced very different repertoires in distinct but HLA identical responders. Likewise, stimulator cells derived from HLA identical donors activated CD8+ cells expressing very different repertoires in the same mismatched responder. To mimic in vivo inflammation, expression of HLA class l antigens was upregulated in vitro on stimulating cells by the inflammatory cytokines TNFα and IFNß. The repertoires differed whether the same responder cells were stimulated with cells treated or not with both cytokines. In conclusion, the selection and expansion of alloreactive cytotoxic T-cell clonotypes expressing a very diverse repertoire is observed repeatedly despite controlling for HLA disparities and is significantly influenced by the inflammatory status. This makes prediction of alloreactive T-cell repertoires a major challenge in HLA mismatched HSCT.

Characterization of the novel HLA-B*07:398 allele in a French hematopoietic stem cell donor.

HLA-B*07:398 differs from HLA-B*07:02:01:01 by one nucleotide substitution at codon 98.3 in exon 3.

Identification of four novel HLA-A alleles.

Four novel HLA-A alleles were detected using two next-generation sequencing technologies.

Identification of seven novel HLA-C alleles.

Seven novel HLA-C alleles were detected using two next-generation sequencing technologies.

Binding affinities of 438 HLA proteins to complete proteomes of seven pandemic viruses and distributions of strongest and weakest HLA peptide binders in populations worldwide.

We report detailed peptide-binding affinities between 438 HLA Class I and Class II proteins and complete proteomes of seven pandemic human viruses, including coronaviruses, influenza viruses and HIV-1. We contrast these affinities with HLA allele frequencies across hundreds of human populations worldwide. Statistical modelling shows that peptide-binding affinities classified into four distinct categories depend on the HLA locus but that the type of virus is only a weak predictor, except in the case of HIV-1. Among the strong HLA binders (IC50 ≤ 50), we uncovered 16 alleles (the top ones being A*02:02, B*15:03 and DRB1*01:02) binding more than 1% of peptides derived from all viruses, 9 (top ones including HLA-A*68:01, B*15:25, C*03:02 and DRB1*07:01) binding all viruses except HIV-1, and 15 (top ones A*02:01 and C*14:02) only binding coronaviruses. The frequencies of strongest and weakest HLA peptide binders differ significantly among populations from different geographic regions. In particular, Indigenous peoples of America show both higher frequencies of strongest and lower frequencies of weakest HLA binders. As many HLA proteins are found to be strong binders of peptides derived from distinct viral families, and are hence promiscuous (or generalist), we discuss this result in relation to possible signatures of natural selection on HLA promiscuous alleles due to past pathogenic infections. Our findings are highly relevant for both evolutionary genetics and the development of vaccine therapies. However they should not lead to forget that individual resistance and vulnerability to diseases go beyond the sole HLA allelic affinity and depend on multiple, complex and often unknown biological, environmental and other variables.

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.