ABSTRACT. Next generation sequencing is a powerful technology to monitor the reconstitution of the immune system after allogeneic hematopoietic stem cell transplantation (HSCT), especially to characterize in depth the diversity of the T-cell receptor (TCR) repertoire. Immune reconstitution is a critical factor for patient’s prognosis and long term survival. Previously, using high throughput sequencing of the TCRβ CDR3 region (ImmunoSEQ), we could demonstrate in a cohort of 116 patients that the repertoire is mainly reconstituted de novo with limited overlap of clonotypes between donors and full chimeric recipients at one year post-HSCT. The repertoire was often skewed with a predominance of oligoclonal profiles at one year. Age alongside Cytomegalovirus (CMV) serological status and infection/reactivation were the most significant clinical parameters driving the shift of diversity. In this project, we reviewed the TCR repertoire of the 116 patients with in silico analysis of the specificity of the sequenced clonotypes using a public database enriched by more than 20,000 CMV-specific clonotypes. Then we analysed 25 patients with a follow up of 6 years, in order to investigate whether and how their TCR repertoire had evolved since our prior analysis. Interestingly, while the overlap of clonotypes remained very low in recipients at 6 years in comparison to their donor’s profile, a larger overlap was retrieved when comparing sequences at one year and at 6 years post-HSCT. This was especially evident for D+/R+ transplants in case of CMV reactivation/infection. The clonality of the repertoire didn’t change or even slightly increased over time in D-/R-, D+/R- and D-/R+ transplants, yet with each group exhibiting contrasted levels of diversity at the different time points. Conversely, in D+/R+ transplants, which represented the group exhibiting the most drastic shift of clonality at one year, the repertoire showed an opposite trend with recovery of more diverse profiles after 6 years.

ABSTRACT. Knowing the donation probability of a potential stem cell donor allows for flexible communication with registered donors or adapted donor center practice, e.g. more efficient donor retention processes. We have examined the dependency of the donation probabilities on donor age, gender, CMV status (known vs. unknown) and HLA genotype frequency. We clustered the genotypes according to their frequency in 10 groups, each group containing a similar number of donors. All donors from DKMS Germany with high resolution typing for HLA-A, -B, -C, -DRB1 and -DQB1 (n=3.85 Mio) where included. We considered 14,348 donations from 2017 to 2019. The distribution of donors’ parameters from donations was compared to the overall distribution of these parameters. For the most common HLA genotypes, young male donors with known CMV status are strongly preferred. The probability of donation for a 18-20-year old male donor with known CMV status is 12.5 times higher than for a female donor of same genotype frequency, age and known CMV status. For female donors of same age and genotype frequency but unknown CMV status this ratio raises to a factor of almost 70. For the rarest HLA genotypes, the donation probabilities do not depend strongly on the other parameters. In this case the probability of donation for a 18-20-year old male donor and known CMV status is equal to that for a female donor of same age, genotype frequency and known CMV status. Compared to a 55-60-year old female donor with unknown CMV status, a 18-20-year old male donor with known CMV status has only a 1.5-fold higher donation probability. Correspondingly, about 7.5% of all collections from donors with the rarest HLA genotypes are from female donors of age >45 without known CMV status. In common genotypes, this group does not contribute to the donations at all. Although older donors have a significantly smaller probability to be selected for donation, they may advance to stem cell donation if they have a rare HLA genotype.

ABSTRACT. CD147 (BSG, EMMPRIN) is a multifunctional immunoglobulin involved in pathogenesis of many diseases. It was shown to facilitate various viral infections, such as measles virus and SARS-CoV-2, and was reported to have a role in cytomegalovirus (CMV) infection. While mostly working as a membrane-bound protein, it can be released by cells in a soluble form that can be detected in serum. Our recent studies showed that CD147 polymorphism and serum level may be associated with risk and survival in acute myeloid leukaemia (AML) patients. This prompted us to investigate CD147 serum levels and genotypes in AML patients with CMV infection after allogeneic stem cell transplantation (SCT). CD147 was measured using Quantikine ELISA Human EMMPRIN/CD147 Immunoassay in serum of 16 AML patients undergoing SCT, of whom 9 developed CMV infection, and 25 controls. Genotyping of CD147 variants was performed on 170 DNA samples, including 135 healthy individuals and 35 patients (18 with CMV infection) using Taqman assays. Serum CD147 levels were higher in patients with CMV infection than in those without detectable CMV infection (p=0.026) and controls (p<0.001). CD147 allele rs4919859 C, a marker of risk and worse survival in AML, tended to be more common in patients with CMV infection than those without infection (p=0.094). Furthermore, we built a logistic regression model incorporating the type of conditioning, sex of transplant donor and recipient, type of donor (related/unrelated), donor and recipient serologic CMV status, and presence/absence of the rs4919859 C allele. The analysis confirmed allele rs4919859 C (p=0.044) and recipient CMV status (p=0.035) as independent markers of CMV infection. Our results suggest that higher serum CD147 levels and presence of CD147 the rs4919859 C allele may be markers of CMV infection in AML patients undergoing SCT. This work was supported by National Science Centre (Poland) projects No. 2018/29/N/NZ5/02022 and 2018/31/B/NZ2/03065.

ABSTRACT. Patient/Donor HLA mismatch is a critical variable affecting the outcomes of hematopoietic stem cell transplantation (HSCT). We hypothesize that mismatches in alleles that differ by AA that are not directly involved in peptide binding may not be immunogenic and could be classified as permissible mismatches. Similarly, mismatches in which the peptide binding repertoire of patient alleles is included in that of the patient may not be immunogenic in the GvH vector. We included 4417 patients reported to the CIBMTR, aged 0-60, receiving their first 8/8 or 7/8 UD HSCT for hematological malignancies between 2003 and 2011. HSCT were grouped in two discovery cohorts: putative permissible (P) (n=95) and non-permissible (N) (n=1034), and an HLA-matched cohort (M) (n=3288). We compared clinical outcomes [OS, DFS, NRM, grade III-IV acute (a)GvHD] of patients receiving P or N HSCT versus M HSCT, and of patients receiving P versus N HSCT. For the multiple regression analyses, we adjusted for variables with an ASD >= 0.2 between groups in a Cox proportional hazards model. Using a log rank test, no significant differences in OS, DFS, NRM and aGvHD were observed between M and P cohorts. Conversely, when compared to the M cohort, the N cohort had dismal OS, DFS, NRM, and higher aGvHD (p-value < 0.0001). We also observed a significant difference in OS between P and N cohorts (p-value = 0.004). In N cohort - based on Cox model – the risk of death was 51% higher [HR = 1.51 (1.15-1.99)]. The lower OS in the N versus M and P cohorts is sustained by an increased incidence of aGvHD (27.8% vs 18%, p-value<0.0001; 27.8% vs 22.1%, p-value<0.05, respectively). In conclusion, we developed a new classification of HLA-allele mismatches considering differences in peptide binding profiles; it will most likely change clinical practice for donor eligibility, prioritization and selection. Based on this dataset, at least 8.4% of 7/8 UDs was functionally equivalent to 8/8 UDs.

ABSTRACT. Permissive HLA-DPB1 mismatches defined by the T-cell epitope (TCE) model are an established criterion for selection of unrelated donors in hematopoietic cell transplantation (alloHCT). One of the TCE groups, TCE group 3 (TCE3) houses a large number of alleles with different structural and functional characteristics. We hypothesized that HLA-DPB1 mismatches involving alleles encoding structurally distant allotypes within TCE3 could have differential impacts on clinical outcomes. Multidimensional scaling based on 28 polymorphic positions (amino acids 8-215) among 51 alleles in a cohort of 5,140 10/10-matched patient-donor pairs reported to the Center for International Blood and Marrow Transplant Research (CIBMTR) identified a subgroup of four frequent and structurally related TCE3 alleles (i.e. DPB1*02:01, 04:01, 04:02, 23:01) forming a separate cluster. In addition, principal component analysis identified the HLA-DP84-87 DEAV/GGPM motif as a major factor driving structural variability among TCE3 alleles. “Core” alleles display similar bound-peptide motifs and elicited lower in vitro alloreactivity from permissive responders compared to “non-core” alleles (mean CD137% 18.5 vs 37.2; p<0.001). Compared to allele-matched pairs (N=785), the risks of aGVHD II-IV increased progressively for “core” TCE3-permissive (N=930; HR 1.12 [0.98-1.28]; p=0.1012), “non-core” TCE3-permissive (N=1,286; HR 1.24 [1.06-1.46]; p=0.0082), and non-permissive mismatches (N=2,023; HR 1.32 [1.16-1.50]; p<.0001). Similar albeit less significant results were obtained with the DEAV/GGPM model stratifying patients into DEAV/GGPM-matched (N=1209) and mismatched (N=1007) pairs. “Core” TCE3-permissive transplants also showed lower TRM risks than “non-core” TCE3-permissive (HR 0.82 [0.70-0.96]; p=0.0118) and non-permissive transplants (HR 0.78 [0.68-0.88]; p=0.0002). Our results suggest that “core” TCE3 donors are main drivers of HLA-DPB1 permissiveness and its favorable outcomes after alloHCT.

ABSTRACT. Following hematopoietic stem cell transplantation (alloHSCT), T cells require up to one year for the CD4 and two years for the CD8 to achieve normal counts and full function. The delayed immune reconstitution contributes to various transplant complications. Acquiring further knowledge about the reconstitution pattern of T cells is crucial to improving transplant outcomes. This study aims to evaluate the reconstitution of sorted CD4- and CD8-T cell subsets of 26 HLA-matched donor/recipient pairs before and one year after transplantation. First, we characterized phenotypically the donor/patient pair's naive and memory subpopulations by flow cytometry. Further, the T-cell receptor (TCR) repertoire of donor-recipient pairs was evaluated by next-generation sequencing of the TCR beta complementarity-determining region 3. While naïve (CD45RO-/CCR7+/CD95-) donor's T cells represent 50% of the total T cells, memory T cells are dominant in post-HSCT samples (~80%), with a marked increase of effector memory (CD45RO-/CCR7-/CD95+) phenotype in the CD8 compartment. The TCR repertoire was primarily oligoclonal at one-year post-HSCT in both populations, with CD8-T cells being less diverse. The TCR overlap is minimal between donors and recipients at one year, suggesting de novo reconstitution. Interestingly, 53% of the CD4 and 34% of CD8 clones shared before and after transplantation increased in frequency. Among the expanded clones, the most abundant are CMV-specific and mainly found in the CD8 fraction. Longitudinal analysis is ongoing to track and identify the clones that expand accordingly to the immune recovery and clinical events during the first year after transplantation. This information could be helpful to predict transplantation outcomes.

ABSTRACT. A common genetic variant within the TCRA-TCRD locus (rs2204985) has been recently identified to correlate with thymic function and T cell repertoire diversity. T cell mediated pathways are known to play a significant role in immunological processes affecting HSCT outcome. Aim of this study was to investigate the potential impact of donor rs2204985 genotype on patient’s outcome after unrelated HSCT. To this end, we retrospectively analyzed 2,016 adult patients who received their first unrelated graft for a hematologic malignancy between 2000 and 2013. Genotyping of rs2204985 was performed by NGS. Overall survival (OS), disease free survival (DFS), relapse (RI), non-relapse mortality (NRM), acute GvHD (aGvHD) and chronic GvHD (cGvHD) were evaluated; p<0.05 was considered significant and donor rs2204985 GG/AG genotype was set as reference vs the AA genotype. Genotyping of rs2204985 showed codominant prevalence of the two alleles (A and G) and the frequencies found were in line with those previously reported for Caucasian populations. Although no significant effect was detected in the complete cohort with respect to donor rs2204985 genotype, subanalysis on account of HLA incompatibility revealed an association between donor AA genotype and significantly inferior OS (HR: 1.48, p=0.003) and DFS (HR: 1.50, p=0.001) as compared to the AG/GG genotypes in patients transplanted with single HLA mismatched grafts (n=624). This effect was driven by a combined higher risk of RI (HR: 1.38, p=0.035) and NRM (HR: 1.38, p=0.042). No association was found between donor rs2204985 genotype and risk of acute or chronic GvHD. This is the first study to date exploring the role of this thymus-relevant polymorphism in an allogeneic HSCT context. Our findings suggest that donor rs2204985 AA genotype in combination with single HLA mismatches may adversely affect HSCT outcome and should therefore be avoided. Further studies are warranted before final conclusions are drawn.

ABSTRACT. It was shown by Petersdorf et al. (Blood 2013) that SNPs are accumulated in the proximity of mismatched HLA in MHC haplotypes. This favors the coding for proteins with altered amino acid sequences in these regions. Moreover, strong linkage disequilibria across the MHC region cause dissimilar antigenic peptides to be associated with mismatched HLA loci. Graft versus host disease (GvHD) may be a measure of the permissiveness of HLA mismatch. Our goal was to determine whether the incidence and/or clinical course of GvHD after HSC transplantation from a mismatched donor is related to the phase of HLA mismatch within MHC diplotype. Methods. High-resolution HLA typing (SSO, SSP, SBT). Population-based genotype phasing of donors and patients (PHASE 2.0), clinical assessment of 664 patients after HSCT. We found that the TRM, occurrence of aGvHD, and incidence of high-grade aGvHD were significantly dependent on both, the number of mismatched HLA molecules (p=0.023, p=0.0016, and p=0.017, respectively) and the number of mismatched extended MHC haplotypes (p=0.054, p=0.00022, and p=0.049, respectively). The occurrence of cGvHD and incidence of extended cGvHD were highly dependent on the number of mismatched extended MHC haplotypes with switched phases (p=0.00012 and p=0.000024, respectively). The trend for the incidence of high-grade cGvHD was highly significant when numbers of mismatched HLA molecules and mismatched extended MHC haplotypes with switched phases increased from 0 to 2 (p=2.3e10-8, HR=1.09, 95%CI 1.05-1.12). Multivariate analysis showed an independent predictive value of the number of mismatched extended MHC haplotypes for the risk of chronic GvHD and overall survival of patients after HSC transplantation from an unrelated donor. We conclude that the permissiveness of the HLA mismatch may be dependent on the phase localization of the mismatched HLA gene in extended MHC haplotypes.

ABSTRACT. Anti-tumor immune-surveillance by T cells is dependent on the ability of Human Leukocyte Antigens (HLA) to present immunogenic tumor associated (TAA) or neomutated antigens. HLA class II peptide processing is crucial for determining which antigenic peptides will be displayed to CD4+ T cells, and is regulated by the endosomal peptide editor HLA-DM. Here, we investigated the influence of HLA-DM mediated peptide editing in the presentation of TAA by the HLA class II molecule DP in the monocytic leukemia line THP-1. Leukemia cells expressing single HLA-DP molecules were generated by CRISPR/Cas9 mediated knock-out of HLA class II beta chains, followed by replacement with two model HLA-DP beta chain allotypes (DP402 and DP10) with or without HLA-DM. HLA-DP immunopeptidome characterization was performed by mass spectrometry of eluted peptides after immuneaffinity chromatography, allowing us to identify a total of 5.148 peptides (1.972/1.775 for DP402 and 1.850/1.127 for DP10 with/without HLA-DM). Of these, 647 and 405 or 733 and 442 were significantly up- and down-regulated by the presence of HLA-DM in DP402 or DP10, respectively. A search of the TANTIGEN 2.0 database (http://projects.met-hilab.org/tadb/) revealed that 273/5.148 (5.3%) of peptides were annotated as TAA, 161 of these in DP402 and 123 in DP10. Significant regulation by HLA-DM was found for 48 (29.8%) of these peptides for DP402 (21 up- and 27 down-regulated), and for 96 (78%) for DP10 (42 up- and 54 down-regulated), for a total of 144/273 (52.7%) TAA peptides regulated by HLA-DM. Our data demonstrate that HLA-DM is an important modulator of TAA antigen presentation by HLA-DP in THP-1 leukemia cells, suggesting a rationale for previous observations that HLA-DM loss of function mutations can be involved in immune escape of hematologic tumors. Moreover, TAA peptides up-regulated in the absence of HLA-DM might be exploited for cancer immunotherapy after iatrogenic targeting of HLA-DM in leukemia.

ABSTRACT. Chimpanzees (Pan troglodytes) and humans share an evolutionary ancestor that lived approximately 5 million years ago, as is reflected by a high sequence similarity of 98.7% at the nonrepetitive DNA level. Particular Mhc class I and II lineages are old entities and predate speciation. Evident orthologs of the HLA-A gene are only traced back in great ape species. In humans, two different Mhc-A lineages (A2 and A3), each consisting of three different families, are recognized. Previously, it was suggested that all chimpanzee Mhc-A alleles cluster into the human A3 lineage. However, to more precisely elucidate such an evolutionary relationship, a large panel of intron sequences might provide the definitive answer. In the present study we have characterized a substantial number of Patr-A alleles at the full-length genomic DNA level, and compared its introns 1 to 8 sequences with the extensive number of full-length genomic HLA-A data available at the IPD-database as well as with data from other chimpanzee/bonobo cohorts. This analysis highlighted that in particular introns 3, 4, 5 and 6 have strong genetic signatures illustrating the close evolutionary relationship of the known Patr-A alleles with the HLA-A3 lineage.

ABSTRACT. MICA is a ligand for the NKG2D receptor and regulates the activity of NK and T cells. The MICA gene is located inside the MHC complex in close proximity and linkage disequilibrium to HLA-B. Since 2019, we have genotyped over two million potential stem cell donors for MICA. Thereby, we discovered a frequent MICA duplication and adapted our analysis pipeline to detect certain copy number variations in MIC genes. Until today, over 23,000 samples (1%) show evidence of three MICA alleles. Linkage analysis revealed that 91% of these samples share one distinct haplotype: HLA-C*02:02:02G~HLA-B*27:02:01G~MICA*007+MICA*008~MICB*003/005/006/010. Out of all HLA-B*27:02 carriers, 67% harbor the MICA duplication. This observation might be significant because HLA-B*27:02 is one of the alleles with high susceptibility to arthritis. Analysis of cohorts from Germany, Poland and UK reveals that the frequency of the MICA duplication correlates to population frequencies of HLA-B*27:02, which are higher in Eastern European countries. Interestingly, MICA duplications were also identified in several samples from Chile (0.4%) or India (0.1%) without the presence of HLA-B*27:02. This observation suggests the occurrence of several independent MICA duplication events, which might indicate a selective advantage.

ABSTRACT. HLA frequencies show widespread variation across human populations. Demographic factors as well as selection are thought to have shaped HLA variation across continents. In this study, we performed a worldwide comparison of HLA class I and class II diversity applying multidimensional scaling techniques to 50 HLA class I as well as 13 HLA-DRB1 allele group frequencies in 204 (1,020,810 chromosomes) and 198 (349,334 chromosomes) not recently admixed populations from all continents, respectively. Our results show a strong effect of geography on the distribution of HLA class I allele groups, with principal coordinates analysis closely resembling the geographical location of populations, in particular in the “Old World”. Conversely, class II frequencies stratify populations along a continuum of differentiation less clearly correlated to actual geographic location. Dual clustering analysis revealed finer intra-continental sub-clusters (e.g. Northern and Western Europe vs South East Europe, North Africa and Southwest Asia), and their characteristic HLA allele group patterns. Ancient (Austronesian expansion) and more recent (Romani people in Europe) migrations, as well as extreme differentiation (Taiwan aborigines; Native Americans), and interregional gene flow (Sámi, Egyptians) are also reflected by the results. Barrier analysis comparing DST and geographic distance identified genetic discontinuities caused by natural barriers or human behavior explaining inter and intra-continental HLA borders, which appear more pronounced for class I. Overall, a progressive reduction in HLA diversity (median number of class I allele groups detected 38-40 vs 16.5-20, p<0.0001) from African to Oceanian and Native American populations is noted. This large comparison of global HLA frequencies shows striking similarity of class I frequencies to geography, and a more complex development for class II, with implications for both human evolutionary studies and biomedical research.

ABSTRACT. Herpesviruses are ubiquitous, genetically diverse DNA viruses, with long-term presence in humans associated with infrequent but significant pathology. Human Leukocyte Antigen (HLA) class I presents intracellularly derived peptide fragments from infected tissue cells to cytotoxic lymphocytes, thereby directing immunity to diverse pathogens, including viruses. Allotypes of highly polymorphic HLA class I are distinguished by their peptide binding repertoires. Because this HLA class I variation is a major determinant of herpesvirus disease, we examined if sequence diversity of virus proteins reflects evasion of HLA presentation. Using population genomic data from Epstein-Barr virus (EBV), Human cytomegalovirus (HCMV), and Varicella-Zoster virus (VZV), we tested whether diversity differed between the regions of herpesvirus proteins that can be recognized, or not, by HLA class I. Herpesviruses exhibit lytic and latent infection stages, with the latter better enabling tissue-specific immune evasion. Whereas HLA-binding peptides of lytic proteins are conserved, we found that EBV and HCMV proteins expressed during latency have increased peptide sequence diversity. Similarly, latent, but not lytic, herpesvirus proteins have greater population structure in HLA-binding than non-binding peptides. Finally, we found patterns consistent with EBV adaption to the local HLA environment, with less efficient recognition of EBV isolates by high frequency HLA class I allotypes. Here, the frequency of CD8+ T cell epitopes inversely correlated with the frequency of HLA class I recognition. Previous analyses have shown pathogen-mediated natural selection maintains exceptional polymorphism in HLA residues that determine peptide recognition. Here we show HLA class I peptide recognition impacts diversity of globally widespread pathogens.

ABSTRACT. Inadequately representing substantial sequence, structure and worldwide diversity of this medically important genomic region, the current human reference has two complete and six partial sequences for the 5Mbp MHC. We have expanded the reference to complete ten MHC region haplotypes, including representatives from the four major HLA class II structures -DR1, -3, -4 and -5. To ensure accurate assembly and annotation, we used targeted short and long-read sequencing, and IHWG cell lines established as homozygous through the MHC. We show the mapping efficiency of genome sequence reads is increased for individuals having genotypes corresponding to the newly-completed haplotypes. To enhance utilization of the expanded reference set, we are developing a population reference graph (PRG) that captures both the nature and frequency of variation through the entire genomic region. To maximize worldwide genomic diversity represented in the PRG, we have sequenced an additional 100 IHWG cells and 100 individuals we also identified as MHC homozygous. In collaboration with multiple immunogenetics researchers around the globe we are collecting a further 300 individuals known to be fully or partially homozygous through the MHC. These haplotypes are chosen to represent worldwide genomic diversity. The haplotype sequences and newly developed bioinformatic tools are publicly available. The completed PRG will allow alignment, phasing, and annotation of the MHC for sample cohorts, enabling studies of human disease and evolution at a level of detail not previously possible.

ABSTRACT. A fine characterization of the HLA molecular profiles of populations from distinct origins and ethnical backgrounds is essential for both clinical issues in histocompatibility and transplantation and fundamental research in population and evolutionary genetics. Recent developments of high throughput sequencing technologies (including NGS) have allowed generating high resolution data at most HLA loci for thousands of individuals at rather accessible costs, leading many laboratories to type or retype large population samples with the aim to fill gaps in the landscape of HLA-typed populations, thereby improving significantly the quality of the molecular data available. Under the umbrella of the EFI Population Genetics Working Group and the Population Genetics, Anthropology and Evolution (PGAE) Component of the 18th International HLA & Immunogenetics Workshop, as many as 30 laboratories were involved in a collective effort to improve the HLA molecular map of a large geographic region encompassing Europe, North Africa, West Asia and South Asia and also including populations of European and mixed descent from North, Central and South America. In addition to 15 populations represented by very large samples (in total more than 270,000 individuals) typed at the 2nd-field level of resolution, the final dataset includes 20 populations sequenced by NGS and reported at the 4th-field level for up to 11 HLA loci. Statistical and graphical analyses of this newly generated dataset, combined with previously available worldwide data, reveal a much more precise topography of the HLA molecular landscape across populations and hence also improve the reference datasets used in histocompatibility and transplantation. The observed HLA molecular diversity is further contrasted to that of equivalent MHC-Patr loci analysed in several chimpanzee cohorts (with implementation of new sequence data), enabling novel interpretations of the genetic history and evolution of these crucial immune genes.

ABSTRACT. Alternative splicing (AS) is a form of post transcriptional modification that is observed in approximately 95% of human multi-exon type of genes. Different mechanisms of AS exist, such as exon skipping and intron retention. The introduction of next generation sequencing techniques enabled the large scale discovery of alternative spliced transcripts. In the present study we characterized the Mhc class I and II transcriptome of a population of cynomolgus macaques (Macaca fascicularis) using a PacBio sequencing platform. Especially the Mhc-DQ and -DP repertoire revealed the presence of alternative spliced transcripts that may encode for different isoforms. A more detailed inventory showed that, in particular Mafa-DQB is subject to alternative splicing. The skipping of exon 4, that codes for the transmembrane region, was observed for several Mafa-DQB1 alleles, but most prevalent for the DQB1*06 lineage, which predates the speciation of humans and Old World monkeys. The generation of such an isoform is also predicted on the basis of certain HLA-DQB transcripts. The conserved character of this specific splice event between humans and cynomolgus macaques suggests a biological relevance. For instance, single-chain murine MHC-II and soluble HLA molecules are known to be involved in apoptosis and modulation of the immune response.

ABSTRACT. Eliminating the need for IS medications after kidney transplantation (KTx) remains a hoped-for goal. Reports of successful tolerance induction protocols are based on transient or persistent chimerism that develops after the infusion of donor hematopoietic progenitor cells. We aimed to determine whether IS-free tolerance could be induced in fully HLA-matched kidney and hematopoietic cell transplant patients. In this phase 2 study, 6 adult patients who received KTx from 10/10 HLA allele matched siblings, were given a conditioning regimen comprising of total lymphoid irradiation and rATG in the 10 days post-KTx. Donor hematopoietic stem cells were collected before kidney transplantation and cryopreserved. Highly enriched donor CD34+ stem cells and a defined dose of T cells were infused IV on day 11 - from the same donor. Median age was 36 (range 27-45) years. Etiology of kidney failure was IGA nephropathy, diabetes, malignant hypertension, Alport syndrome, reflux and idiopathic ESRD. Patients received MMF for 1 month and tacrolimus for at least 6 months. IS was gradually tapered off starting from 6 months post KTx until complete withdrawal as long as donor chimerism persisted for at least 6 months and there was no evidence of graft-versus-host (GvHD) or graft rejection. The preparatory regimen was well tolerated by all patients. Median time from KTx to last follow-up is 15 (range 3-42) months. All recipients achieved mixed donor chimerism that was maintained until last follow-up. No recipients experienced clinical kidney rejection or GvHD. 3 patients discontinued IS 9, 8 and 11 months after KTx and a fourth patient is expected to discontinue IS within a month. Time from IS discontinuation to last follow-up is 33, 13 and 4 months, respectively. Combined kidney and hematopoietic cell transplantation for tolerance induction between HLA matched sibling donor-recipient pairs is safe and applicable. We intend to expand our program to haploidentical donor-recipient pairs.

ABSTRACT. T follicular helper (Tfh) cells play an important role during the development of antibody-mediated rejection (ABMR) following organ transplantation by stimulation of antibody-secreting B-cell proliferation and differentiation. Circulating T follicular helper (cTfh) cells, which are considered to represent a memory Tfh cell population, have previously been shown to promote B-cell differentiation and antibody secretion. In the current study, we aimed to unravel the functional phenotype of these cTfh cells. To do so, CD3+CD4+CXCR5+PD1+ cTfh cells and CD3+CD4+CXCR5- T cells were isolated via FACS sorting of PBMCs of a renal allograft recipient who experienced ABMR. To analyze the RNA expression patterns of these T cells, single-cell RNA sequencing (scRNAseq) was performed. In addition, single-cell TCR sequencing was performed to link the scRNAseq data to TCR clonotypes. Compared to CD3+CD4+CXCR5- T cells, cTfh cells expressed high levels of several genes including FOS and JUN, transcriptional factors that play a vital role in effector T-cell differentiation, proliferation, and function, and JUNB, which has previously been shown to be highly expressed by cTfh cells. Clustering of the cTfh cells showed four clusters, three of which clustered together and one separate cluster. Compared to the rest of the cells and to the CXCR5- T cells, this separate cluster was observed to be more clonal. In addition, a high expression of cytotoxic genes such as GZMK, GZMA, and NKG7 was observed, which might suggest cytotoxic capacity. In conclusion, our preliminary data provide the first deep insight into the functional phenotype of cTfh cells during antibody-mediated kidney allograft rejection.

ABSTRACT. Transplant vasculopathy (TV) is a frequent and deleterious complication after organ transplantation. TV results from a process of intimal hyperplasia developing inside the intra-graft arterial tree, due to an abnormal proliferation of vascular smooth muscle cells (VSMC), leading to neointima formation, loss of organ perfusion and function. Anti-HLA class I antibodies (Ab) are able to induce VSMC proliferation in vitro. When injected to immunodeficient SCID/beige mice, transplanted with portions of human mesenteric arteries, these Ab trigger the development of a human-like TV inside the grafts. The detection of anti-HLA Ab inside the neointima suggests their ability to cross the endothelial layer. Our study aimed to characterized the mechanisms involved in the permeability of the vascular endothelium to anti-HLA Ab. Mouse and Fc-humanized Anti-HLA class I (clone W6/32) monoclonal Ab, were tested in an in vitro model of endothelial layer, mimicked by seeding human microvascular endothelial cells (HMEC-1) on Transwell inserts. The amount of Ab crossing the endothelial layer from the apical to the basal compartment was quantified by immunoblotting and the underlying molecular mechanisms were studied thanks to the use of various chemical and biological agents blocking the main ways of transcytosis. Our results revealed that clathrin inhibitors efficiently block Ab transport through the endothelium. We concluded that anti-HLA Ab are internalized by two main processes: micropinocytosis and clathrin-dependent endocytosis. These results open potential new therapeutic avenues for further mechanistic investigations (involvement of Fc receptors…) to prevent TV by limiting anti-HLA Ab infiltration inside the vascular wall and promising perspectives for chronic allograft rejection treatment.

ABSTRACT. Multiple studies have shown that HLA mismatch analysis at the eplet level is superior in predicting the development of de novo donor specific antibodies than the HLA antigen level. Antibody verified eplets are particularly informative, since they have been shown to directly interact with HLA antibodies, confirming their immunogenicity. While eplets can be antibody verified by using monoclonal antibodies (mAbs), determining the crucial amino acids for antibody reactivity is limited by the number and nature of alleles available in single antigen bead (SAB) panels. Multiple eplets or amino acids, unlikely to cover the same paratope, are often included as candidates. To resolve this uncertainty, site-directed mutagenesis was performed to identify residues vital to the eplet or functional epitope. The human mAb VDK1D12 from a producer carrying A*03:01, A*31:01 immunized by A*01:01, showed high MFI values for A*01:01, A*01:02 and A*36:01 beads in Luminex SAB assay. Three residues (K44, V150 and V158) on these reactive alleles were deduced to be (part of) the potential functional epitope using HLA-EMMA and HLA Fusion MM. Single amino acid mutations (K44R, K44A, V150A or V158A) on the wildtype A*01:01 allele were made and transiently expressed. These mutants did not significantly alter the expression level compared to wildtype as detected by a pan HLA CI mAb in flow cytometry, indicating that each mutation did not perturb the overall protein structure. In contrast, VDK1D12 binding was lost with K44R and K44A mutants, but not V150A and V158A mutants. The same results were recapitulated on stable cell pools post antibiotic selection. In this proof of concept study, we were able to reliably identify binding patterns using flow cytometry on cells expressing HLA with defined amino acid mutations. With this experimental setup, we plan to further our understanding of HLA immunogenicity and contribute to the antibody-verification of HLA eplets.

ABSTRACT. The degree of HLA class II eplet mismatch has been previously demonstrated to determine whether kidney transplant recipients (KTRs) tolerate lower tacrolimus through levels without formation of de novo donor-specific antibodies (dnDSA). Recently, the clinical phase I Triton study demonstrated that mesenchymal stromal cell (MSC) therapy is a safe method to facilitate tacrolimus withdrawal in KTRs. Here, we analyzed dnDSA development in MSC-treated KTRs and determined the association with HLA amino acid mismatch (AA MM) loads. Patients and donor were HLA typed on 11 loci by next-generation sequencing and AA MM loads were calculated by using HLA-EMMA. Sera were tested by luminex single antigen bead assays for DSA assignment. In total, 4 out of 27 patients (14.8%) in the control group and 11 out of 29 patients (37.9%) in the MSC group developed dnDSA (defined as MFI >1000), of which the majority was directed against HLA-DQ. The dnDSA in the MSC group often had higher mean fluorescent intensity and persisted longer. Mean HLA-DQ AA MM loads in the two groups were not significantly different. HLA-DQ AA MM load was divided in 4 quartiles (Q1: <7, Q2: 7-13, Q3: 14-35, Q4: >35). In contrast to historical data showing a dose-dependent effect of HLA-DQ AA MM load on dnDSA development, the incidence of dnDSA in the MSC group in Q1, Q2, Q3 and Q4 was 0%, 43%, 43% and 56% respectively. This illustrates that already at lower HLA AA MM loads, these patients are at risk for DSA formation after tacrolimus withdrawal, despite MSC treatment. Importantly, regardless of increased dnDSA formation in MSC-treated patients, the incidence of rejection or graft loss was not increased and kidney function was stable, raising the question on the pathogenicity of these antibodies. Further research is warranted to explore HLA AA MM load as a biomarker to guide personalized immunosuppression in transplantation.

ABSTRACT. Evidence for HLA specific antibodies in peripheral blood usually leads to the exclusion of donors carrying the corresponding HLA antigens. Conversely, the absence of donor-specific HLA antibodies leads to the acceptance of donors, even in the presence of repeated HLA mismatches. Despite close monitoring of recipients, humoral organ rejections occur. As a secondary immune response might remain unseen the need to provide a robust method for immune monitoring before SOT is obvious. Recent studies have demonstrated that the existence of circulating antibodies secreted by plasma cells located in the bone marrow cannot be translated into reliable information on the memory B cell compartment. Re-exposure to a repeated HLA mismatch results in activation of memory B cells, which eventually differentiate into long-lived antibody-secreting plasma cells. We generated an artificial secondary immune response by re-exposure a recipient’s B cells to repeated HLA mismatches in vitro. A sophisticated ELISPOT assay has been developed using recombinant HLA molecules as targets for secreted IgGs from activated memory B cells. A specific subset of peripheral CD19+/27+/138+/38+ B cells has been exposed to selected recombinant HLA-I or -II subtypes. Autologous recombinant HLA antigens served as negative controls. This artificial secondary immune response allowed unequivocal detection of memory B cells against a specific HLA antigen. The ELISPOT allowed quantitative and qualitative detection of HLA-specific memory B cells. In living kidney Tx, this method has been applied to adjust donor selection to prevent humoral immune response. This detection of a secondary immune response has the potential to be used in routine diagnostics, as it provides robust and incontrovertible data on the individual immunization status.

ABSTRACT. BACKGROUND Acute rejection is a risk factor for the later allograft dysfunction and long-term graft loss after kidney transplantation. Previous studies have shown that the genomic mismatch level between donor and recipient is associated with the complications of transplantation. In the present study, we determined the association of genome-level matching with acute rejection and survival in single-center kidney transplantation study cohort. METHODS Altogether 1025 pairs of deceased donors and first kidney transplant recipients transplanted in 2007–2017 were genotyped. The associations between the sums of whole genome missense variant mismatches and missense mismatches in transmembrane, secretory, and kidney-related proteins, with acute rejection were estimated using Cox proportional hazards model. Additionally, mismatches in 40 common deletion-tagging variants were analyzed using Cox model. RESULTS The increasing mismatch sum in kidney-related proteins associated with acute rejection with an unadjusted HR of 1.15 (95% CI, 1.01–1.30; P=0.029). The sums of other mismatches were not associated with acute rejection. In deletion analysis, we found that a mismatch in rs7542235, genotype GG tagging a homozygous deletion at the complement Factor H-related (CFHR) proteins locus, predisposed to acute rejection with adjusted HR of 2.97 (95% CI, 1.46–6.04; P=0.003). CONCLUSIONS The present study suggests that mismatches in gene deletions and kidney-related proteins are novel histocompatibility factors in kidney transplantation. The relative importance of different gene deletions varies between populations, as we found evidence for the CFH-related gene deletion but could not replicate the previously reported LIMS1 deletion.

ABSTRACT. COVID-19 vaccination seems to be the best strategy for controlling the pandemic. However the possibility of vaccination triggering the formation of anti-HLA antibodies (anti-HLA Abs), especially de novo donor-specific Ab(dnDSA) that could affect graft function, is still under consideration. The aim of our study was to estimate the increase of sensitization after anti-SARS-CoV-2 vaccination in kidney transplant recipients. Before, one month after the 2nd vaccine dose (BNT162b2) against SARS-CoV-2 in 66 patients and one month after the 3rd dose in 41 of them, anti-HLA Abs were estimated by Luminex, IgG antibodies against Receptor Binding Domain(RBD) of SARS-CoV-2 and neutralizing antibodies (Nab) by Chemilluminescence Immunoassay. The 39.4% (26 patients) had anti-HLA before vaccination with 10±8 anti-HLA specificities. Only one patient presented dnDSA after the 2nd dose, and all patients were positive in 9±6 anti-HLA specificities. Of the 41 patients tested after the 3rd dose, two more patients became positive and one negative regarding anti-HLA Abs. Concerning DSAs, 13.6% (9/66 patients-7 vs. class II) were positive before vaccination. No patient had dnDSAs while one with low DSA titer before vaccination became negative. In anti-HLA positive patients, MFImax was not changed after the 2nd dose (6458 vs 6930 p=0.88). No change was observed in MFImax (8067 vs 7069 p=0.83) or in MFIsum (53851 vs 46361 p=0.44) in those tested after the 3rd dose. In patients with DSAs the corresponding MFIs did not change after the 2nd dose (19618 vs 18615 p=0.86). Anti-HLA Abs before vaccination were not associated with a higher chance of developing IgG RBD antibodies after the 2nd (p=0.36) or 3rd vaccine dose (p=0.45) nor with a higher chance of developing Nab (p=0.7 and 0.83 respectively). Vaccination against SARS-CoV-2 seems to be safe regarding the development of anti-HLA Abs in transplanted patients as neither their MFI nor their HLA specificities increased after vaccination.

ABSTRACT. HLA molecules are key restrictive elements to present intracellular antigens for an effective T-cell response against SARS-CoV-2. HLA alleles vary with respect to their potential to present immunogenic viral epitopes and may therefore determine disease severity. Therefore, we set out to investigate the impact of individual HLA genotypes on the severity of SARS-CoV-2 infections. In August 2020 and July 2021 we performed cross-sectional studies among stem cell donors registered with DKMS in Germany. Volunteers registered for stem cell donation represent a comparable healthy subset of the working age population. Available genetic information was linked to self-reported COVID-19 outcome data. Multivariable regression models were fitted to determine the risk of contracting SARS-CoV-2, severe respiratory tract infection and respiratory hospitalization. More than 200,000 registered donors provided informed consent and participated in the study. Their age ranged from 18 to 61 years. Altogether 16,121 participants donors reported a history of COVID-19. Asymptomatic courses were reported by 1,428 participants, mild/moderate symptoms by 10,353 participants, severe respiratory infections by 3,913 not requiring hospitalization and respiratory hospitalizations by 427 patients. Notably, we did not observe a heterozygote advantage. The risk for severe infections was not statistically different among individuals with or without homozygosity at HLA-A, -B, -C, -DRB1, -DQB1 and –DPB1. Of 84 HLA-A, -B, -C, -DRB1, -DQB1 and –DPB1 alleles which were prevalent in more than 400 participants only the presence of HLA-B*39:01 had significant impact on the risk for respiratory hospitalization (OR 2.23, p=0.01) at a significance level of 1%. These findings suggest that the HLA genotype is no major factor determining COVID-19 severity. It is therefore possible that the relatively large viral genome of 29.8 kb encodes for abundant epitopes to mount T-cell responses not limited by the HLA genotype.

ABSTRACT. HLA haplotypes were found to be associated with increased risk for viral infections or disease severity in various diseases, including SARS. Several genetic variants are associated with COVID-19 severity. Studies have proposed associations, based on a very small sample and a large number of tested HLA alleles, but no clear association between HLA and COVID-19 incidence or severity has been reported. We conducted a large-scale HLA analysis of Israeli individuals who tested positive for SARS-CoV-2 infection by PCR. Overall, 72,912 individuals with known HLA haplotypes were included in the study, of whom 6413 (8.8%) were found to have SARS-CoV-2 by PCR. A total of 20,937 subjects were of Ashkenazi origin (at least 2/4 grandparents). One hundred eighty-one patients (2.8% of the infected) were hospitalized due to the disease. None of the 66 most common HLA loci (within the five HLA subgroups: A, B, C, DQB1, DRB1) was found to be associated with SARS-CoV-2 infection or hospitalization in the general Israeli population. Similarly, no association was detected in the Ashkenazi Jewish subset. Moreover, no association was found between heterozygosity in any of the HLA loci and either infection or hospitalization. We conclude that HLA haplotypes are not a major risk/protecting factor among the Israeli population for SARS-CoV-2 infection or severity. Our results suggest that if any HLA association exists with the disease it is very weak, and of limited effect on the pandemic.

ABSTRACT. COVID-19 is a highly heterogeneous disease, and its more severe clinical manifestations have been linked to aberrant host immune responses. To investigate the role of HLA polymorphism in COVID-19 clinical heterogeneity, we collected biological samples and clinical data from 3'393 Italian patients with proven SARS-CoV-2 infection, performed NGS-based typing of 12 HLA loci, compared allele and haplotype frequencies in our cohort with those collected by the Italian Bone Marrow Donor Registry, and investigated associations between immunogenetic and clinical variables. HLA types, heterozygosity, and evolutionary distance, as well as HLA class I antigen load, did not show significant association with COVID-19 and its severity. Interestingly, we documented a previously unrecognized anti-correlation between disease severity and the in silico predicted load of viral antigens presented by the patient HLA class II. This effect appeared to be mainly driven by genes of the HLA-DP locus and remained significant in a multi-variable logistic-regression model (p-value<0.05). Concordantly, HLA-DP allele frequencies were unbalanced between patients with mild vs severe infection (p-value<0.01). Our large-scale analysis evidenced for the first time an association between HLA class II antigen load and disease severity, in agreement with experimental evidence of downregulation of these genes in SARS-CoV-2 infected cells.

ABSTRACT. The polymorphism of the HLA system has been extensively studied in COVID-19 infection, however to our knowledge there are no data about the role of HLA on vaccine response. We report here the HLA-A, -B, -C and DRB1 allelic frequencies of n=111 individuals after BNT162b2 mRNA vaccine, selected on the basis of lower antibody levels (<5% percentile) early after the 2nd dose among a total of n=2,569 vaccinees (healthcare workers) of the ASST Grande Ospedale Metropolitano Niguarda in Milano, Italy, and compare them with the frequencies of a reference population obtained from healthy hematopoietic stem cell donors of the Italian Bone Marrow Donor Registry (Genova, Italy). We found that statistically significant differences in the frequencies of the alleles HLA-A*03:01, A*33:03, B*58:01 and at least one haplotype (HLA-A*24:02~C*07:01~B*18:01~DRB1*11:04) are associated with a weaker antibody response after vaccination, together with the age of vaccinees. In conclusion, our results might suggest a role played by some HLA alleles or haplotypes in antibody production after the BNT162b2 mRNA vaccine, giving insights into the tracking of potentially susceptible individuals across populations. Further studies and clinico-biological follow-up are needed to better define our exploratory findings and dissect the role of HLA polymorphism on response to anti-COVID-19 vaccines.

ABSTRACT. Clinical outcome of immunotherapy depends on tumor cell immunogenicity, including HLA class I and PD-L1 expression, and on tumor microenvironment characteristics. We evaluated HLA-I and PD-L1 co-expression using immunohistology in 77 bladder tumors in correlation with overall and cancer specific patient survival. We defined different tumor phenotypes based on HLA-I/PD-L1 co-expression immunophenotypes: HLA-I/PD-L1 double positive (23 tumors); HLA-I positive/PD-L1 negative (12 tumors); HLA-I negative/PDL1 positive (17 tumors); and HLA-I/PD-L1 double negative (25 tumors). We found that most of the HLA-I negative tumors showed high tumor grade. In fact, all HLA-I negative/PD-L1 positive tumors had high grade. Average survival time of patients with HLA-I positive/PD-L1 negative tumors was higher (161 months) than that in patients with HLA-I negative/PD-L1 positive tumors (54 months). Average overall survival in this last analysis was 152.5 months. Double positive tumors showed a statistically significant higher survival than HLA-I negative/PD-L1 positive tumors. Cancer-specific survival of patients with HLA-I positive/PDL1 negative and double negative tumors was also higher when compared to the tumors with HLA-I negative/PD-L1 positive immune escape phenotype. In summary, we observed that HLA-I negative/PD-L1 positive co-expression pattern in bladder cancer has a significant correlation with high tumor grade and with poor overall and cancer-specific survival. It suggests that loss of HLA-I and upregulation of PD-L1 are essential and common determinants of cancer immune escape in bladder cancer and might cause primary resistance to immune checkpoint inhibitors.

ABSTRACT. Our previous studies showed that HLA-I molecules could restrict MPN development. Whether this principle applies to HLA-II has not been studied so far. Therefore we aimed at the elucidation of the putative protective role of HLA-II alleles for the development of JAK2V617F and CALRmut driven MPNs. We performed NGS typing with Holotype HLA kit (Omixon) of 139 JAK2V617F positive, 46 CALR exon9 mutation positive MPN patients and 1083 healthy controls. Multivariate generalized linear models with age and gender as covariates were fitted for estimation of the association of HLA-II alleles and haplotypes with the presence of JAK2 V617F mutation. Datasets from Gene Expression Omnibus repository were selected to analyze HLA-II pathway gene expression in peripheral blood CD34+ stem cell and granulocytes from MPN patients and healthy controls. To access the global effect of HLA-II genotype on the presence of JAK2 V617F and CALR mutations we calculated Grantham distance as a measure for HLA alleles’ divergence. We did not find any differences between HED per locus and per group. Two alleles: HLA-DPB1*03:01, DQB1*04:02 and 2 haplotypes: DRB1*11:01-DQB1*03:01-DQA1*05:05-DPB1*02:01, DRB1*11:03-DQB1*03:01-DQA1*05:05-DPB1*04:02 were significantly depleted in MPN patients compared to controls. Additionally we observed HLA-II alleles and haplotypes with statistically increased frequencies in CALRmut and JAK2V617F positive patients. Gene expression analysis showed lower HLA-DQA1, HLA-DMA and CD74 expression in JAK2V617F and CALRmut MPN CD34+ cells as compared to normal CD34+ cells. Furthermore JAK2V617F positive CD34+ showed down-regulation of HLA-DMB, HLA-DRB1 and HLA-DRA genes. In conclusion this study provides first immunogenetic evidences that HLA class II may restrict JAK2V617F and CLARmut driven oncogeneis. Specific HLA-II alleles might be a predictive marker for response to immunotherapies up-regulating HLA expression. The work was supported by grant KP-06-H41/2 and Omixon

ABSTRACT. The human leukocyte antigen (HLA) system is the single most important genetic susceptibility factor for many autoimmune diseases and immunological traits. For systematic population-level analysis of HLA-phenotype association landscape we imputed the alleles of classical HLA genes in a discovery cohort of 146,630 and replication cohort of 89,340 Finns of whom SNP genotype data and 3,355 disease phenotypes were available as part of the FinnGen project. In total, 3,649 statistically significant single HLA allele associations in 368 phenotypes were found. Known susceptibility associations clearly dominated the landscape but we discovered also a few previously poorly established HLA associations such as DQA1*01:03 and DQB1*06:03 with mental and behavioural disorders due to cannabinoids (p-value = 10-5; beta = 0.6). As certain HLAs were found to be involved in both autoimmune and infectious diseases, we studied further the independence of their associations and statistical pleiotropy. We found that altogether 11 shared HLA alleles were associated independently with both autoimmune and infectious diseases. The most prominent of these were DQA1*03:01 and DQB1*03:02 both of which associated with three infectious and three autoimmune phenotypes. All the shared HLAs showed risk effects in both disease groups, suggesting that infections can increase the risk for autoimmune diseases. The population-level landscape analysis is an excellent resource for estimating the contribution and genetic models of HLA genes in many different phenotypes and for fine-mapping primary associations.

ABSTRACT. CD8+ memory T cells accumulate in the brain of multiple sclerosis (MS) patients. The effector function of these cells depends on the transcription factors RUNX3, EOMES and T-bet. Interestingly, a single nucleotide polymorphism in RUNX3 (rs6672420) has been identified as one of few coding risk variants for MS. How these factors correspond to brain-homing, cytotoxic and tissue-resident features of CD8+ memory T cells in MS patients remains unknown. In this study, we demonstrate that rs6672420 is associated with increased RUNX3 protein expression in human blood and not brain CD8+ T-cell lines (N = 9). In ex vivo CD8+ memory T cells from MS blood (N = 18), the presence of rs6672420 coincided with a loss in T-bet coexpression. When compared to healthy individuals, the proportion of RUNX3- and EOMES-, but not T-bet-expressing CD8+ memory T cells was reduced in the blood of MS patients. This was reversed after natalizumab therapy, a monoclonal antibody against VLA-4 that blocks migration of immune cells into the brain, implying that RUNX3+EOMES+T-bet- cells have superior brain-homing ability. Indeed, this T-cell subset showed enriched expression of MS-associated tissue-resident markers CCR5, granzyme K, CD20 and CD69, and was selectively increased in paired MS cerebrospinal fluid versus blood samples (N = 7). A similar phenotype was found in EBV(HLA-A2; BMLF1)- but not CMV-specific CD8+ memory T cells in the blood from natalizumab-treated MS patients. In single-cell suspensions from postmortem MS brain tissues (N = 8), RUNX3 and T-bet coexpression was recovered in CD20dim and CD69+ CD8+ T cells, which was accompanied by increased coproduction of granzyme K and B. These results implicate that coexpression of RUNX3 and EOMES, but not T-bet, defines CD8+ memory T cells with a pre-existing tissue-resident phenotype that are prone to enter the central nervous system in MS.