Although quantitative PCR (qPCR) has been explored for chimerism monitoring after allogeneic stem cell transplantation (SCT), evidence regarding its clinical utility compared with standard short tandem repeat (STR) is still limited. We retrospectively studied commercial qPCR and STR chimerism with respective positivity thresholds of .1% and 1% in 359 peripheral blood (PB) and 95 bone marrow (BM) samples from 30 adult patients after first HLA-matched SCT for myeloid malignancies or acute lymphatic leukemia. Concordance between the 2 methods was 79.5%, with all discordant samples positive in qPCR but negative in STR. Of the latter, sporadic qPCR positivity without clinical correlates was seen mostly in BM samples early post-transplant. In 7 of 21 patients with available follow-up samples in the first months after transplantation, qPCR but not STR revealed low levels (<1%) of sustained host chimerism in PB, reflecting delayed engraftment or persistent mixed chimerism (PMC). These conditions were associated with donor-recipient cytomegalovirus (CMV) serostatus and early CMV reactivation but not with immunosuppressive regimens or clinical outcome. qPCR predicted all 8/8 relapses with samples in the 6 months before onset by sustained positivity in both PB and BM compared with 1/8 relapses predicted by STR mainly in BM. The response kinetics to donor lymphocyte infusions for the treatment of PMC or relapse was shown by qPCR but not STR to be protracted over several months in 3 patients. Our results demonstrate the superior clinical utility of qPCR compared with STR for monitoring subtle changes of host chimerism associated with different clinical conditions, making a case for its use in the clinical follow-up of transplant patients.

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Comprehensive knockout of HLA class II (HLA-II) b-chain genes is complicated by their high polymorphism. In this study, we developed CRISPR/Cas9 genome editing to simultaneously target HLA-DRB,-DQB1, and-DPB1 through a single guide RNA recognizing a conserved region in exon 2. Abrogation of HLA-II surface expression was achieved in five different HLA-typed, human EBV-transformed B lymphoblastoid cell lines (BLCLs). Next-generation sequencing-based detection confirmed specific genomic insertion/deletion mutations with 99.5% penetrance in sorted cells for all three loci. No alterations were observed in HLA-I genes, the HLA-II peptide editor HLA-DMB, or its antagonist HLA-DOB, showing high on-target specificity. Transfection of full-length HLA-DPB1 mRNA into knockout BLCLs fully restored HLA-DP surface expression and recognition by alloreactive human CD4 T cells. The possibility to generate single HLA-II-expressing BLCLs by one-shot genome editing opens unprecedented opportunities for mechanistically dissecting the interaction of individual HLA variants with the immune system.

T cell alloreactivity is mediated by a self-human leukocyte antigen (HLA)-restricted T cell receptor (TCR) repertoire able to recognize both structurally similar and dissimilar allogeneic HLA molecules (i.e., differing by a single or several amino acids in their peptide-binding groove). We hypothesized that thymic selection on self-HLA molecules could have an indirect impact on the size and diversity of the alloreactive response. To test this possibility, we used TCR Vβ immunophenotyping and immunosequencing technology in a model of alloreactivity between self-HLA selected T cells and allogeneic HLA-DPB1 (DPB1) differing from self-DPB1*04:02 by a single (DPB1*02:01) or several (DPB1*09:01) amino acids in the peptide-binding groove. CD4+ T cells from three different self-DPB1*04:01,*04:02 individuals were stimulated with HeLa cells stably transduced with the relevant peptide processing machinery, co-stimulatory molecules, and HLA-DP. Flow cytometric quantification of the DPB1-specific T cell response measured as upregulation of the activation marker CD137 revealed significantly lower levels of alloreactivity against DPB1*02:01 compared with DPB1*09:01 (mean CD4+CD137+ frequency 35.2 ± 9.9 vs. 61.5 ± 7.7%, respectively, p < 0.0001). These quantitative differences were, however, not reflected by differences in the breadth of the alloreactive response at the Vβ level, with both alloantigens eliciting specific responses from all TCR-Vβ specificities tested by flow cytometry, albeit with higher levels of reactivity from most Vβ specificities against DPB1*09:01. In line with these observations, TCRB-CDR3 immunosequencing showed no significant differences in mean clonality of sorted CD137+CD4+ cells alloreactive against DPB1*02:01 or DPB1*09:01 [0.39 (0.36–0.45) and 0.39 (0.30–0.46), respectively], or in the cumulative frequencies of the 10 most frequent responding clones (55–67 and 58–62%, respectively). Most of the clones alloreactive against DPB1*02:01 (68.3%) or DPB1*09:01 (75.3%) were characterized by low-abundance (i.e., they were not appreciable among the pre-culture T cells). Interestingly, however, their cumulative frequency was lower against DPB1*02:01 compared with DPB1*09:01 (mean cumulative frequency 35.3 vs. 50.6%, respectively). Our data show that, despite lower levels of alloreactivity, a similar clonal diversity can be elicited by structurally similar compared with structurally dissimilar HLA-DPB1 alloantigens and demonstrate the power of TCRB immunosequencing in unraveling subtle qualitative changes not appreciable by conventional methods.

Introduction. Genomic loss of an HLA haplotype encoding incompatible alleles ("HLA loss") has been described in previous single-center studies as a mechanism by which leukemic cells evade the graft-versus-leukemia effect mediated by alloreactive donor T cells and outgrow into a clinically evident relapse. HLA loss accounts for up to 30% of relapses after HLA-haploidentical transplants (Crucitti, Leukemia 2015), but the actual frequency and clinical relevance of this phenomenon in unrelated donor HSCTs, including cord blood transplants, are largely unknown. Here we present the first global collaborative study to investigate the incidence of HLA loss across different transplant settings. Methods. Twenty transplant centers from Europe (n=16), North America (n=3) and Asia (n=1) joined to form the HLALOSS consortium. To date, we collected a total of 619 cases of hematologic relapse from adult patients with acute myeloid leukemia (78.5%), acute lymphoblastic leukemia (13.9%), myelodysplastic syndromes (4%) or myeloproliferative neoplasms (1.1%) after allogeneic HSCT from HLA-haploidentical relatives (31.7%), HLA-mismatched unrelated donors (MMUD, 21.3%), 10/10-matched unrelated donors (MUD, 37.2%), or unrelated cord blood units (UCB, 9.8%). Where available, the donor and patient germlines and the patient pre-transplant disease were collected in parallel. Until today, 476 cases were analyzed using conventional HLA typing of sorted leukemic blasts, the recently developed HLA-KMR assay (Ahci and Toffalori, Blood, 2017) or a novel Next-Generation Sequencing (NGS) method. The latter was developed adapting the HLA typing strategy in use at the DKMS (Lange, BMC Genomics 2013) to the study of chimeric samples, and allowing to cover all possible HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 alleles and to analyze at least 48 different cases in a single run. Results. Out of the 476 relapses analyzed to date, 396 (83.2%) were informative for the study of HLA loss. Of these, 155 occurred after haploidentical HSCT, 101 after MMUD HSCT, 93 after 10/10-matched, HLA-DPB1 mismatched MUD, and 47 after UCB HSCTs. Three-hundred-two (76.2%) of cases were analyzed using the NGS platform. This method resulted particularly robust, reliable and sensitive in analyzing large sample series: the mean coverage across the 6 sequenced loci was over 8500x, up to 0.5% of the HLA allele of interest could be detected in artificial chimerism curves, and relapse samples tested in parallel via the sequencing platform and HLA-KMR (n=10) showed remarkable concordance between the two methods (R2=0.86, p<0.0001). In total, we detected 51 HLA loss post-transplantation relapses out of the 396 cases analyzed (12.8%). Of these, 35 occurred after haploidentical HSCT (22.6% of relapses in this setting), 12 after MMUD HSCT (11.9%), 4 after 10/10 MUD HSCT (4.3%) and, notably, none after UCB HSCT. Conclusions. The present data, obtained from the largest collaborative study on the immunobiology of relapse to date, confirm the clinical relevance of HLA loss as a major mechanism of immune evasion and post-transplantation relapse after allogeneic HSCT, with an incidence which is proportional to the number of donor-recipient HLA mismatches. The only exception is represented by UCB HSCT which, despite being often performed across multiple major HLA incompatibilities, does not appear to be associated with this relapse modality. This finding might reflect the fact that in UCB HSCT, multiple HLA mismatches are often not encoded in cis on the same chromosome, thereby reducing the selective advantage for leukemic cells that undergo an HLA haplotype loss. This phenomenon might in turn contribute to the lower incidence of relapse reported for UCB HSCT compared to other stem cell sources. Disclosures Vago: Moderna TX: Research Funding; GENDX: Research Funding. Stoelzel:Neovii: Speakers Bureau. Gojo:Novartis: Membership on an entity's Board of Directors or advisory committees; Merck inc: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees. Busca:Novartis: Speakers Bureau; Jazz Pharmaceuticals: Honoraria; Pfizer Pharmaceuticals: Honoraria, Speakers Bureau; Merk: Honoraria, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Luznik:WIndMIL Therapeutics: Equity Ownership, Patents & Royalties. Kobbe:Amgen: Honoraria, Research Funding; Celgene: Honoraria, Other: Travel Support, Research Funding; Roche: Honoraria, Research Funding. Kroeger:Novartis: Honoraria, Research Funding; Sanofi: Honoraria; Riemser: Honoraria, Research Funding; Neovii: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; JAZZ: Honoraria. Finke:Neovii: Consultancy, Honoraria, Other: travel grants, Research Funding; Medac: Consultancy, Honoraria, Other: travel grants, Research Funding; Riemser: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Other: travel grants, Research Funding. Mohty:Takeda: Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria; Servier: Consultancy; MaaT Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; Molmed: Consultancy; Jazz Pharmaceuticals: Honoraria, Research Funding, Speakers Bureau; Bristol Myers: Consultancy, Research Funding; Janssen: Honoraria, Research Funding, Speakers Bureau. Beelen:Medac: Consultancy, Other: Travel Support. Fleischhauer:GENDX: Research Funding.

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