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Allogeneic hematopoietic cell transplantation (alloHCT) provides a potential curative treatment for haematological malignancies. The therapeutic Graft-versus-Leukaemia (GvL) effect is induced by donor T cells attacking patient hematopoietic (malignant) cells. However, if healthy non-hematopoietic tissues are targeted, Graft-versus-Disease (GvHD) may develop. After HLA-matched alloHCT, GvL and GvHD are induced by donor T cells recognizing polymorphic peptides presented by HLA on patient cells, so-called minor histocompatibility antigens (MiHAs). The balance between GvL and GvHD depends on the tissue distribution of MiHAs and T-cell frequencies targeting these MiHAs. T cells against broadly expressed MiHAs induce GvL and GvHD, whereas those targeting MiHAs with hematopoietic-restricted expression induce GvL without GvHD. Recently, the MiHA repertoire identified in natural immune responses after alloHCT was expanded to 159 total HLA-I-restricted MiHAs, including 14 hematopoietic-restricted MiHAs. This review explores their potential relevance to predict, monitor, and manipulate GvL and GvHD for improving clinical outcome after HLA-matched alloHCT., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Septo-optic dysplasia (SOD) syndrome is a rare congenital disorder characterized by a classic triad of optic nerve/chiasm hypoplasia, agenesis of septum pellucidum and corpus callosum, and hypoplasia of the hypothalamic-pituitary axis.Herein, we report the clinical case of 2-year-old boy presenting with psychomotor delay, nystagmus, congenital hypothyroidism, and a clinically relevant growth delay. The neuroradiological examination showed partial segmental agenesis of the corpus callosum, agenesis of the septum pellucidum, optic nerve hypoplasia, and a small pituitary gland with a small median pituitary stalk. A whole-exome sequencing analysis detected a novel heterozygous de novo variant c.1069_1070delAG in SON , predicted as likely pathogenic.To date, SON pathogenic variants have been described as responsible for Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome, a multisystemic neurodevelopmental disorder mainly characterized by intellectual disability, facial dysmorphisms, visual abnormalities, brain malformations, feeding difficulties, and growth delay. The herein described case is the first recognized clinic-radiological occurrence of SOD syndrome with hypothalamic-pituitary dysfunction in a patient carrying a SON gene variant, considered responsible of ZTTK syndrome, suggesting a possible relationship between SOD and SON gene alterations, never described so far, making the search for SON gene mutations advisable in patients with SOD., Competing Interests: None declared., (Thieme. All rights reserved.)

Diphthamide is a modified histidine residue unique for eukaryotic translation elongation factor 2 (eEF2), a key ribosomal protein. Loss of this evolutionarily conserved modification causes developmental defects through unknown mechanisms. In a patient with compound heterozygous mutations in Diphthamide Biosynthesis 1 (DPH1) and impaired eEF2 diphthamide modification, we observe multiple defects in neural crest (NC)-derived tissues. Knockin mice harboring the patient's mutations and Xenopus embryos with Dph1 depleted also display NC defects, which can be attributed to reduced proliferation in the neuroepithelium. DPH1 depletion facilitates dissociation of eEF2 from ribosomes and association with p53 to promote transcription of the cell cycle inhibitor p21, resulting in inhibited proliferation. Knockout of one p21 allele rescues the NC phenotypes in the knockin mice carrying the patient's mutations. These findings uncover an unexpected role for eEF2 as a transcriptional coactivator for p53 to induce p21 expression and NC defects, which is regulated by diphthamide modification., (© 2024. The Author(s).)

Insulin is packaged into secretory granules that depart the Golgi and undergo a maturation process that involves changes in the protein and lipid composition of the granules. Here, we show that insulin secretory granules form physical contacts with the endoplasmic reticulum and that the lipid exchange protein oxysterol-binding protein (OSBP) is recruited to these sites in a Ca 2+ -dependent manner. OSBP binding to insulin granules is positively regulated by phosphatidylinositol-4 (PI4)-kinases and negatively regulated by the PI4 phosphate (PI(4)P) phosphatase Sac2. Loss of Sac2 results in excess accumulation of cholesterol on insulin granules that is normalized when OSBP expression is reduced, and both acute inhibition and small interfering RNA (siRNA)-mediated knockdown of OSBP suppress glucose-stimulated insulin secretion without affecting insulin production or intracellular Ca 2+ signaling. In conclusion, we show that lipid exchange at endoplasmic reticulum (ER)-granule contact sites is involved in the exocytic process and propose that these contacts act as reaction centers with multimodal functions during insulin granule maturation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Minor histocompatibility antigens (mHAg) composed of peptides presented by HLA molecules can cause immune responses involved in graft-versus-host disease (GVHD) and graft-versus-leukemia effects after allogeneic hematopoietic cell transplantation (HCT). The current study was designed to identify individual graft-versus-host genomic mismatches associated with altered risks of acute or chronic GVHD or relapse after HCT between HLA-genotypically identical siblings. Our results demonstrate that in allogeneic HCT between a pair of HLA-identical siblings, a mHAg manifests as a set of peptides originating from annotated proteins and non-annotated open reading frames, which i) are encoded by a group of highly associated recipient genomic mismatches, ii) bind to HLA allotypes in the recipient, and iii) evoke a donor immune response. Attribution of the immune response and consequent clinical outcomes to individual peptide components within this set will likely differ from patient to patient according to their HLA types., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Martin, Levine, Storer, Zheng, Jain, Heavner, Norris, Geraghty, Spellman, Sather, Wu and Hansen.)

Background: Mucosal-associated invariant T (MAIT) cells are unconventional T cells which recognize microbial metabolites presented by the major histocompatibility complex class I-related molecule MR1. Although MAIT cells have been shown to reside in human and murine skin, their contribution to atopic dermatitis (AD), an inflammatory skin disease associated with barrier dysfunction and microbial translocation, has not yet been determined., Methods: Genetic deletion of MR1 and topical treatment with inhibitory MR1 ligands, which result in the absence and functional inhibition of MAIT cells, respectively, were used to investigate the role of MR1-dependent immune surveillance in a MC903-driven murine model of AD., Results: The absence or inhibition of MR1 arrested AD disease progression through the blockade of both eosinophil activation and recruitment of IL-4- and IL-13-producing cells. In addition, the therapeutic efficacy of phototherapy against MC903-driven AD could be increased with prior application of folate, which photodegrades into the inhibitory MR1 ligand 6-formylpterin., Conclusion: We identified MAIT cells as sentinels and mediators of cutaneous type 2 immunity. Their pathogenic activity can be inhibited by topical application or endogenous generation, via phototherapy, of inhibitory MR1 ligands., (© 2021 European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Patients undergoing allogeneic stem cell transplantation as treatment for hematological diseases face the risk of Graft-versus-Host Disease as well as relapse. Graft-versus-Host Disease and the favorable Graft-versus-Leukemia effect are mediated by donor T cells recognizing polymorphic peptides, which are presented on the cell surface by HLA molecules and result from single nucleotide polymorphism alleles that are disparate between patient and donor. Identification of polymorphic HLA-binding peptides, designated minor histocompatibility antigens, has been a laborious procedure, and the number and scope for broad clinical use of these antigens therefore remain limited. Here, we present an optimized whole genome association approach for discovery of HLA class I minor histocompatibility antigens. T cell clones isolated from patients who responded to donor lymphocyte infusions after HLA-matched allogeneic stem cell transplantation were tested against a panel of 191 EBV-transformed B cells, which have been sequenced by the 1000 Genomes Project and selected for expression of seven common HLA class I alleles (HLA-A ∗ 01:01, A ∗ 02:01, A ∗ 03:01, B ∗ 07:02, B ∗ 08:01, C ∗ 07:01, and C ∗ 07:02). By including all polymorphisms with minor allele frequencies above 0.01, we demonstrated that the new approach allows direct discovery of minor histocompatibility antigens as exemplified by seven new antigens in eight different HLA class I alleles including one antigen in HLA-A ∗ 24:02 and HLA-A ∗ 23:01, for which the method has not been originally designed. Our new whole genome association strategy is expected to rapidly augment the repertoire of HLA class I-restricted minor histocompatibility antigens that will become available for donor selection and clinical use to predict, follow or manipulate Graft-versus-Leukemia effect and Graft-versus-Host Disease after allogeneic stem cell transplantation., (Copyright © 2020 Fuchs, Honders, van der Meijden, Adriaans, van der Lee, Pont, Monajemi, Kielbasa, ’t Hoen, van Bergen, Falkenburg and Griffioen.)

Minor histocompatibility antigens are the main targets of donor-derived T-cells after allogeneic stem cell transplantation. Identification of these antigens and understanding their biology are a key requisite for more insight into how graft vs. leukemia effect and graft vs. host disease could be separated. We here identified four new HLA class II-restricted minor histocompatibility antigens using whole genome association scanning. For one of the new antigens, i.e., LB-PIP4K2A-1S, we measured strong T-cell recognition of the donor variant PIP4K2A-1N when pulsed as exogenous peptide, while the endogenously expressed variant in donor EBV-B cells was not recognized. We showed that lack of T-cell recognition was caused by intracellular cleavage by a protease named asparagine endopeptidase (AEP). Furthermore, microarray gene expression analysis showed that PIP4K2A and AEP are both ubiquitously expressed in a wide variety of healthy tissues, but that expression levels of AEP were lower in primary acute myeloid leukemia (AML). In line with that, we confirmed low activity of AEP in AML cells and demonstrated that HLA-DRB1 * 03:01 positive primary AML expressing LB-PIP4K2A-1S or its donor variant PIP4K2A-1N were both recognized by specific T-cells. In conclusion, LB-PIP4K2A-1S not only represents a novel minor histocompatibility antigen but also provides evidence that donor T-cells after allogeneic stem cell transplantation can target the autologous allelic variant as leukemia-associated antigen. Furthermore, it demonstrates that endopeptidases can play a role in cell type-specific intracellular processing and presentation of HLA class II-restricted antigens, which may be explored in future immunotherapy of AML., (Copyright © 2020 Kremer, Bausenwein, Lurvink, Kremer, Rutten, van Bergen, Kretschmann, van der Meijden, Honders, Mazzeo, Watts, Mackensen, Falkenburg and Griffioen.)

A subset of MHC-associated self-peptides presented by the recipient's cells and immunologically foreign to the donor can induce an allogeneic immune response after hematopoietic stem cell transplantation (HSCT). These immunogenic peptides originate from the genomic polymorphisms and are known as minor histocompatibility antigens (MiHA). MiHA mismatches trigger the post-transplant immune response, which could manifest in both the deleterious "graft-vs.-host" disease and the beneficial "graft-vs.-leukemia" effect. Importantly, some MiHAs are considered to be promising targets for posttransplant T-cell immunotherapy of hematopoietic malignancies. This creates a demand for a robust and fast approach to genotyping MiHA-encoding polymorphisms. We report a multiplex real-time PCR method for the genotyping of 20 polymorphisms that are encoding HLA-A * 02:01-restricted MiHAs. This method uses allele-specific primers and gene-specific hydrolysis probes. In 1 h it allows for the detection of MiHA mismatches in a donor-recipient pair without the need for electrophoresis, sequencing, or other time-consuming techniques. We validated the method with Sanger and NGS sequencing and demonstrated good performance over a wide range of DNA concentrations. We propose our protocol as a fast and accurate method of identifying mismatched MiHAs. The information on the MiHA mismatches is useful for studying the allogeneic immune response following HSCT and for selecting the targets for post-transplant T-cell therapy.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative therapy for hematopoietic malignancies. The graft-derived donor lymphocytes are capable of eliminating the residual recipient malignant cells in the course of allogeneic immune response, thus decreasing the chances of a relapse of the disease. Foreign peptides of the recipient presented by the MHC molecules are able to elicit the immune response immunologically. These polymorphic peptides are known as minor histocompatibility antigens (MiHAs). MiHAs occur due to the nonsynonymous single nucleotide polymorphisms in human genome. Transfusion of T cells specific to MiHAs presented predominantly in the cells of hematopoietic origin will allow the targeted elimination of residual malignant clones avoiding undesirable damage to healthy tissues. To induce the immune response, the donor must be homozygous by the MiHA allele and the recipient must either be homozygous or heterozygous by the alternative MiHA allele. The therapeutic mismatch occurs in 25% of cases under the optimal frequency of allelic variants. Minor antigen ACC-1Y originates from polymorphism in the BCL-2A1 gene; its immunogenic mismatch occurrence approaches the theoretical maximum. In addition, BCL2A1 is overexpressed in cells of various lymphomas. ACC-1Y is presented on allele HLA-A*24:02, which is relatively frequent in the Russian population. Combination of these factors makes the minor antigen ACC-1Y a promising target for immunotherapy. Transfusion of donor CD8^(+) lymphocytes modified with transgenic MiHA-specific TCR is one of the promising methods of posttransplant leukemia therapy and relapse prophylaxis. We obtained a sequence of high-affinity ACC-1Y-specific TCR after the antigen-specific expansion of T cells derived from a healthy ACC-IY^(-/-) donor. We cloned this sequence into the lentiviral vector and obtained the assembled viral particles. Further, we transduced the CD8^(+) lymphocyte culture and demonstrated its antigen-specific cytotoxic activity. It is suggested that CD8^(+) lymphocytes modified by the described method could be potentially transferred to recipients as a therapy against relapse after allo-HSCT.

Base editing is an exciting new genome engineering technology. C-to-T mutations in genomic DNA have been achieved using ribonucleoprotein complexes comprised of rat APOBEC1 single-stranded DNA deaminase, Cas9 nickase (Cas9n), uracil DNA glycosylase inhibitor (UGI), and guide (g)RNA. Here, we report the first real-time reporter system for quantification of APOBEC-mediated base editing activity in living mammalian cells. The reporter expresses eGFP constitutively as a marker for transfection or transduction, and editing restores functionality of an upstream mCherry cassette through the simultaneous processing of two gRNA binding regions that each contain an APOBEC-preferred 5'TCA target site. Using this system as both an episomal and a chromosomal editing reporter, we show that human APOBEC3A-Cas9n-UGI and APOBEC3B-Cas9n-UGI base editing complexes are more efficient than the original rat APOBEC1-Cas9n-UGI construct. We also demonstrate coincident enrichment of editing events at a heterologous chromosomal locus in reporter-edited, mCherry-positive cells. The mCherry reporter also quantifies the double-stranded DNA cleavage activity of Cas9, and may therefore be adaptable for use with many different CRISPR systems. The combination of a rapid, fluorescence-based editing reporter system and more efficient, structurally defined DNA editing enzymes broadens the versatility of the rapidly expanding toolbox of genome editing and engineering technologies.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is routinely used to treat hematopoietic malignancies. The eradication of residual tumor cells during engraftment is mediated by donor cytotoxic T lymphocytes reactive to alloantigens. In a HLA-matched transplantation context, alloantigens are encoded by various polymorphic genes situated outside the HLA locus, also called minor histocompatibility antigens (MiHAs). Recently, MiHAs have been recognized as promising targets for post-transplantation T-cell immunotherapy as they have several appealing advantages over tumor-associated antigens (TAAs) and neoantigens, i.e., they are more abundant than TAAs, which potentially facilitates multiple targeting; and unlike neoantigens, they are encoded by germline polymorphisms, some of which are common and thus, suitable for off-the-shelf therapy. The genetic sources of MiHAs are nonsynonymous polymorphisms that cause differences between the recipient and donor proteomes and subsequently, the immunopeptidomes. Systematic description of the alloantigen landscape in HLA-matched transplantation is still lacking as previous studies focused only on a few immunogenic and common MiHAs. Here, we perform a thorough in silico analysis of the public genomic data to classify genetic polymorphisms that lead to MiHA formation and estimate the number of potentially available MiHA mismatches. Our findings suggest that a donor/recipient pair is expected to have at least several dozen mismatched strong MHC-binding SNP-associated peptides per HLA allele (116 ± 26 and 65 ± 15 for non-related pairs and siblings respectively in European populations as predicted by two independent algorithms). Over 70% of them are encoded by relatively frequent polymorphisms (minor allele frequency > 0.1) and thus, may be targetable by off-the-shelf therapeutics. We showed that the most appealing targets (probability of mismatch over 20%) reside in the asymmetric allele frequency region, which spans from 0.15 to 0.47 and corresponds to an order of several hundred (213 ± 47) possible targets per HLA allele that can be considered for immunogenicity validation. Overall, these findings demonstrate the significant potential of MiHAs as targets for T-cell immunotherapy and emphasize the need for the systematic discovery of novel MiHAs.

Minor histocompatibility antigen (miHA) mismatches have been related to graft-versus-host disease (GVHD) after allogeneic stem cell transplantation, but this association remains controversial due to the lack of consistency in the results obtained by different groups. The CTLA-4 genotype of the donor has been reported to be relevant in the appearance of acute GVHD. We explored the effect of the donor's CTLA-4 genotype in the incidence of acute GVHD associated with HA-1, HA-8, or H-Y miHA mismatches in a large cohort of 1295 patients receiving an allogeneic transplant from an HLA-identical sibling donor. The incidence of acute GVHD was higher if the donor and recipient were mismatched for HA-1, HA-8, or H-Y, but only when the donor had the CTLA-4 rs231775 AA genotype (hazard ratio [HR], 2.18; 95% confidence interval [CI], 1.27 to 3.75; P = .005; HR, 2.11, 95% CI, 1.06 to 4.18; P = .033; and HR, 1.50; 95% CI, 1.05 to 2.15; P = .025, respectively). In contrast, this increased risk of developing acute GVHD was not found when the donor presented the CTLA-4 rs231775 AG or GG genotypes. We conclude that the immune response to specific miHA mismatches is modulated by the CTLA-4 genotype of the donor., (Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Donor lymphocyte infusions (DLI) can induce durable remissions in multiple myeloma (MM) patients, but this occurs rather infrequently. As the graft-versus-tumor (GvT) effect of DLI depends on the presence of host-dendritic cells (DCs), we tested in a phase I/II trial whether the efficacy of DLI could be improved by simultaneous vaccination with host-DCs. We also analyzed the possibility of further improving the GvT effect by loading the DCs with peptides of mismatched hematopoietic cell-specific minor histocompatibility antigens (mHags). Fifteen MM patients not responding to a first DLI were included. Eleven patients could be treated with a second equivalent dose DLI combined with DC vaccinations, generated from host monocytes (moDC). For four patients, the DC products did not meet the quality criteria. In four of the treated patients the DCs were loaded with host mHag peptides. Toxicity was limited and no acute GvHD occurred. Most patients developed objective anti-host T-cell responses and in one patient a distinct mHag-specific T-cell response accompanied a temporary clinical response. These findings confirm that DLI combined with host-DC vaccination, either unloaded or loaded with mHag peptides, is feasible, safe and capable of inducing host-specific T-cell responses. The limited clinical effects may be improved by developing more immunogenic DC products or by combining this therapy with immune potentiating modalities like checkpoint inhibitors.

T cells specific for hematopoietic system restricted minor Histocompatibility (H) antigens target normal and malignant hematopoietic cells. Thus, cellular immune responses against the latter miHAS eradicate the recipient's hematopoiesis including residual leukemic cells after HLA-matched minor H antigen-mismatched stem-cell transplantation (SCT). However, there are controversial reports on the role of HA-1 in the development of graft-versus-host-disease (GVHD) as well. Here, we address the behavior of HA-1-specific cytotoxic T cells (CTLs) in an ex vivo in situ skin explant model wherein HA-1-expressing dendritic cells (DCs) were added as antigen-presenting cells (APCs). Infiltration and activation of HA-1 CTLs occurred only in those cases where both HLA-A2 and HA-1 were expressed, either by the skin or by the DCs, or by the combination of HLA-A2(+) skin and HA-1(+) DCs. These results point toward the role of recipient's HA-1(+) DCs in the chimeric patient suffering from GVHD after HA-1-mismatched SCT. Although in our model the infiltrated and activated CTLs did not cause skin tissue destruction, our results provide a first step in understanding the reported association of HA-1 mismatching with clinical GVHD., (2011. Published by Elsevier Inc.)

Several studies in HLA-matched sibling hematopoietic stem cell transplantation (HSCT) have reported an association between mismatches in minor histocompatibility antigens (mHAg) and outcomes. We assessed whether single and multiple minor mHAg mismatches are associated with outcomes in 730 unrelated donor, HLA-A, B, C, DRB1, and DQB1 allele-matched hematopoietic stem cell transplants (HSCT) facilitated by the National Marrow Donor Program (NMDP) between 1996 and 2003. Patients had acute and chronic leukemia or myelodysplastic syndrome (MDS), received myeloablative conditioning regimens and calcineurin inhibitor-based graft-versus-host-disease (GVHD) prophylaxis, and most received bone marrow (BM; 85%). Donor and recipient DNA samples were genotyped for mHAg including: HA-1, HA-2, HA-3, HA-8, HB-1 and CD31(125/563). Primary outcomes included grades III-IV acute GVHD (aGVHD) and survival; secondary outcomes included chronic GVHD (cGVHD), engraftment, and relapse. Single disparities at HA-1, HA-2, HA-3, HA-8, and HB-1 were not significantly associated with any of the outcomes analyzed. In HLA-A2-positive individuals, single CD31(563) or multiple mHAg mismatches in the HVG vector were associated with lower risk of grades III-IV aGVHD. Based on these data, we conclude that mHAg incompatibility at HA-1, HA-2, HA-3, HA-8, HB-1, and CD31 has no detectable effect on the outcome of HLA matched unrelated donor HSCT.

Graft-versus-host disease (GVHD) is a potentially fatal complication after allogeneic bone marrow transplantation. However, few data exist thus far on the molecular signals governing leukocyte trafficking during the disease. We therefore investigated the sequential pattern of distinct adhesion, costimulatory, and apoptosis-related molecules in GVHD organs (ileum, colon, skin, and liver) after transplantation across minor histocompatibility barriers (B10.D2 --> BALB/c, both H-2d). To distinguish changes induced by the conditioning regimen from effects achieved by allogeneic cell transfer, syngeneic transplant recipients (BALB/c --> BALB/c) and irradiated nontransplanted mice were added as controls. Irradiation upregulated the expression of vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-l, and B7-2 in ileum, as well as VCAM-1 and B7-2 in colon, on day 3 in all animals. Whereas in syngeneic mice these effects were reversed from day 9 on, allogeneic recipients showed further upregulation of VCAM-1, ICAM-1, B7-1, and B7-2 in these organs on day 22, when GVHD became clinically evident. Infiltration of CD4+ and CD8+ donor T cells was noted on day 9 in skin and liver and on day 22 in ileum and colon. Surprisingly, the expression of several other adhesion molecules, such as ICAM-2, platelet-endothelial cell adhesion molecule 1, E-selectin, and mucosal addressin cell adhesion molecule 1, did not change. Proapoptotic and antiapoptotic markers were balanced in GVHD organs with the exception of spleen, in which a preferential expression of the proapoptotic Bax could be noted. Our results indicate that irradiation-induced upregulation of VCAM-1, ICAM-1, and B7-2 provides early costimulatory signals to incoming donor T cells in the intestine, followed by a cascade of proinflammatory signals in other organs once the alloresponse is established.

CXB-2/By (CXB-2) recombinant inbred mice express a subset of the minor histocompatibility antigen (miHA) repertoire expressed by C.B10-H2(b)/LiMcdJ (BALB.B) mice. On lethal irradiation and the transplantation of H2(b)-matched C57BL/6 (B6) T cell-depleted bone marrow cells, along with naive unfractionated T cells, both strains succumb to acute graft-versus-host disease (GVHD). Although alloreactive B6 CD4(+) T cells are a necessary source of T-cell help for the B6 CD8(+) component of the GVHD response in both recipient strains, they are capable of mediating severe GVHD by themselves only in BALB.B mice. Previous CD4(+) T-cell receptor repertoire analysis demonstrated overlapping oligoclonal Vbeta use between the CD4(+) B6 anti-BALB.B and B6 anti-CXB-2 responses, with indications of additional BALB.B unique T-cell responses (Vbeta2 and Vbeta11). We report here that the more severe B6 anti-BALB.B response is not due to a quantitative difference in the responding cells, because the frequency of alloreactive donor CD4(+) T cells over time was equivalent in the spleens of BALB.B versus CXB-2 recipients. The responses were also similar in the number of infiltrating B6 CD4(+) T cells in the lingual epithelium of the 2 recipients. In contrast, a significantly greater degree of infiltration and injury of BALB.B intestinal epithelium correlated with the increased level of clinical GVHD severity. Of most significance, despite the involvement of at least 11 Vbeta-associated CD4(+) T-cell families in the overall B6 anti-BALB.B response, the development of severe GVHD correlated with the presence of Vbeta2- and Vbeta11-positive donor T cells. Transplantation of donor CD4(+) T cells from Vbeta-associated families that were shared between the B6 anti-BALB.B and anti-CXB-2 responses resulted in minimal GVHD potential. These data suggest that severe GVHD across miHA barriers depends on the involvement of a restricted number of potent T-cell specificities and implies that there are only a limited number of corresponding responsible miHAs.

The graft-versus-leukemia (GVL) effect of HLA-identical allogeneic stem cell transplantation is mainly mediated by alloreactive T-cells directed at the minor histocompatibility antigens (H ags) expressed on the leukemic cells of the recipient. Minor H ags are major histocompatibility complex-bound polymorphic peptides that are derived from intracellular proteins and that can show ubiquitous or hematopoietic system-restricted expression. Whereas ubiquitous minor H ags are involved both in the GVL effect and in graft-versus-host disease (GVHD), hematopoietic system-specific minor H ags expressed on leukemic cells are considered important targets for leukemia-specific cellular immunotherapy with a low risk of GVHD. This review will summarize the current knowledge of the immunobiology of minor H ags and discuss the advantages and drawbacks of cellular immunotherapy strategies that aim to separate the GVL effect from GVHD by targeting donor T-cells to hematopoietic system-specific minor H ags.

Donor lymphocyte infusions (DLI) can induce a graft-versus-leukaemia (GvL) reaction in patients with relapsed disease. However, the mechanisms involved in remission induction are not completely known. A patient with chemotherapy-refractory relapse 1 year after human leucocyte antigen (HLA)-identical, unrelated stem cell transplantation (SCT) for bcr/abl-positive common acute lymphoblastic leukaemia (ALL) received a DLI from the original donor, and achieved complete cytogenetic and molecular remission concomitantly with extensive graft-versus-host disease (GvHD). Seven CD8+, donor-derived, alloreactive T-cell clones were generated by stimulating post-DLI remission cells with the patient's pretransplant mature dendritic cells. The minor histocompatibility antigen (mHag) recognized by these T-cell clones was identified as HA-1, a mHag associated with acute GvHD after SCT. Our finding provides evidence of HA-1-associated GvL effects after DLI that paralleled the eradication of full-blown, chemotherapy-refractory ALL relapse after allogeneic SCT.

Neutrophil, monocyte, natural killer- and B-cell number and function are rapidly restored after bone marrow transplant (BMT), whereas T-cell reconstitution is often quite delayed. Our hypothesis was that V beta T-cell receptor (TCR) repertoire diversity among recipients of allogeneic BMT is influenced by the expression of major and minor HLA antigens in the host. The study population comprised unmanipulated and CD34(+)-selected allogeneic bone marrow grafts, autologous peripheral blood stem cell transplants and recipients of volunteer unrelated donor (VUD) bone marrow transplants. Using flow cytometry, the relative frequencies of 18 V beta TCR families were determined and ranked for each time point studied. Comparisons and correlations were made between paired blood samples obtained within a single patient over time, and between donors and their recipients. The pattern of the V beta TCR repertoire from allogeneic recipients and their HLA-matched donors was very similar, with a correlation coefficient (CC) of 0.59. This similarity was not as marked in VUD pairs (CC = 0.32). By 3 months after transplant, the pattern of the V beta TCR repertoire in recipients of HLA-matched sibling transplants was more similar to the pattern seen in pretransplant recipients than to the donor pattern (CC = 0.40 vs. 0.31). Our data suggest that both major and minor HLA antigens influence V beta TCR repertoire diversity and reconstitution after BMT.

We recently demonstrated that spleen cells primed against dominant BALB.B antigens can inhibit the cytotoxic T lymphocyte (CTL) response against subdominant antigens in vitro. In this study, we show that this interference is dependent on CD8+, but not CD4+, T cells directed against dominant antigens. Similar to immunodominance in vivo, T cell interference in vitro required presentation of dominant and subdominant antigens by the same antigen-presenting cell. In vivo priming with cells expressing dominant and subdominant antigens did not induce long-lasting unresponsiveness against the latter. These results support a model in which immunodominance is mediated by T cell competition. In line with this, we found that the immunodominance effects in the CTL response against these minor histocompatibility antigens could be broken by immunization with live bone marrow-derived dendritic cells.

Minor histocompatibility Ags (HA) play prominent roles in stimulating allograft rejection and are recognized by CTLs that mediate this process. However, there is no information on the diversity of TCRs that are specific for single minor histocompatibility Ag peptides and expressed by CTLs in vivo. We have used the technique of spectratyping to study the diversity of Vbeta usage and beta complementarity-determining region 3 (CDR3) length of TCRs expressed by CTL-infiltrating skin allografts expressing the immunogenic H4 peptide during the process of rejection. Spectratyping revealed overall reduction in diversity of both Vbeta usage and CDR3 length, with sequential application of primary, second-set, and third-set H4-incompatible grafts. This dissection of the array of beta-chains expressed by graft-infiltrating CTLs allowed the direct sequencing of individual beta-chain PCR products. Beta CDR3s were characterized by a net negative charge, as we have observed previously with CDR3s expressed by H4-specific CTL clones selected in vitro. Identical and closely related CDR3 amino acid sequences could be identified that were shared by TCRs that 1) utilized different Vbeta genes, 2) derived from different mice, or 3) derived from different sequential sets of allografts on individual mice. Furthermore, a number of CDR3 sequences expressed by graft-infiltrating CTLs were identical or closely related to sequences we have identified previously in in vitro selected CTL clones that were specific for the H4 peptide.

The existence of transplantation antigens, in addition to those encoded by genes in the MHC, has been known for over half a century. The molecular identification of these additional minor histocompatibility (H) antigens lagged behind that of their MHC counterparts, largely because minor H antigens are recognised by T cells and not by antibodies. In the past year, however, new minor H antigens have been identified at both the genetic and protein level and include Uty, a second novel gene encoding a male-specific epitope in mice, a novel autosomal gene encoding each of the H-13 alleles of mice, and a second male-specific epitope encoded by the SMCY gene.

Cytokines are believed to cause a number of inflammatory diseases. We have investigated the role of 3 inflammatory cytokines, IL-1, IL-2, and TNF alpha, during graft-versus-host disease (GVHD), a paradigm disease of cytokine dysregulation in vivo. Measuring cytokine mRNA transcripts with a quantitative polymerase chain reaction technique, we demonstrate that IL-1 transcript levels are increased several hundred-fold in GVHD target organs, whereas TNF alpha transcripts increase only 4- to 6-fold. Kinetic studies during the first month after transplant unexpectedly show that GVHD never induces IL-2 transcripts in the skin and only induces IL-2 transcripts in the spleen during the first week, whereas levels of IL-1 transcripts continue to increase throughout the entire 4 weeks. Administration of an IL-1 receptor antagonist after the termination of the IL-2 response and after the establishment of GVHD significantly increases long-term survival, confirming the central role of IL-1 as an effector molecule of GVHD and suggesting new therapeutic strategies for this disorder.

Skin tissue of a healthy chimera 7 years after HLA identical bone marrow transplantation was found to express a minor histocompatibility (mH) antigen against which cytotoxic T lymphocytes (CTLs) had been discovered at a time of acute graft-versus-host disease (aGVHD). We were prompted to investigate the apparent tolerance to this persistent mH antigen and used limiting dilution analysis to monitor in vitro anti-host CTL responses in time after bone marrow transplantation. A high anti-host CTL precursor frequency was found during acute GVHD, declining in time until beyond detection level in the healthy chimera 7 years after transplantation. In this case report (i) CTL precursor frequencies are used for the first time to monitor in vitro tolerance induction to persistent host mH antigens after HLA identical BMT in man; and (ii) it is shown that LDA may be a potential tool for quantification and specificity analysis of CTL responses to mH antigens.

This article summarizes work that focuses on alloantibodies that bind to endothelial cells and attempts to provide an evaluation of the apparent role that antigens distinct from classic HLA alleles, and not expressed in lymphocytes, may play in kidney transplant rejection. The most recent experience with the donor-skin crossmatch in prediction of early kidney allograft rejection also is described.

The RMA-S mutant T cell line is defective in H-2b restricted antigen presentation and has markedly reduced surface expression of Kb and Db. We examined RMA-S for the expression of the medial class I histocompatibility antigens Qa1b and Mta. While RMA-S targets varied in their susceptibility to lysis by cytotoxic T lymphocytes (CTL) specific for Qa1b, Mta levels were detectable but consistently low compared to the parent RMA cell line. Addition of synthetic ND1 alpha 1-26 or ND1 alpha 1-17 peptides that mimic MTF alpha (the ligand of Mta) increased killing of RMA-S by anti-Mta alpha CTL to levels comparable to or better than RMA, with 300 nM peptide being fully effective. None of the MTF peptides increased the killing of RMA-S by anti-H-2b or anti-Qa1b CTL, even at the highest (1 microM) peptide concentrations. RMA-S cells treated with 100 microM of either the ND1 alpha 4-26 or ND1 alpha 1-26 peptides showed a small increase in the fluorescent staining for beta 2-microglobulin but not for H-2Kb or H-2Db. These results show that Mta and Qa1b, although affected, are not obliterated by the defect in RMA-S cells; that the association of MTF peptides with HMT is exclusive; and that MTF enters the endoplasmic reticulum in the same fashion as other endogenous peptides.

We showed previously that transplantation of 10(7) unmanipulated C57BL/6 marrow cells to irradiated LP mice yields healthy (B6-LP) chimeras showing no signs of rejection or graft-versus-host disease (GVHD). The aim of this work was to gain more insight into the mechanism(s) responsible for tolerance to host minor histocompatibility antigens following allogeneic bone marrow transplantation (BMT). (B6-LP) chimeras showed very good immune reconstitution when studied in vitro for proliferative response to mitogens and alloantigens and generation of T cell cytotoxic activity. In co-culture experiments their spleen cells showed no natural suppressor activity. When used as cell donors, their capacity to initiate GVHD in four strains of mice presenting H-2 differences was normal when compared to C57BL/6 donors. However, they provoked no GVHD in the three strains of H-2 compatible mice studied. Re-irradiated (B6-LP) chimeras rapidly died of GVHD following injection of C57BL/6 marrow + spleen cells. (B6-LP.R111) chimera cells appeared tolerant to LP minor antigens presented in the context of H-2r or H-2b. No anamnestic anti-idiotypic suppressor response was noted when stable (B6-LP) chimeras were stimulated with naive C57BL/6 cells. These findings suggest that in BMT chimeras transplanted across minor histocompatibility barriers: (1) both host and donor-derived antigen-presenting cells can present host antigens to donor T cells whose numbers in the marrow inoculum will determine if GVHD or tolerance will ensue, (2) GVHD can be triggered by only a limited number of 'dominant' minor antigens, and (3) we found no evidence for the presence of natural suppressors, veto cells or anti-idiotypic suppressor T cells.

We have previously demonstrated that anti-H-43a CTL response of H-43b responder mice was exclusively restricted by self H-2Kb (Kb) but not by the other nine self MHC class I alleles from independent origins, i.e., Kbml,d,k,s and Db,d,k,q,s. In the present study, we verified that Kf,q,r and Df,r alleles could also not serve as restricting class I elements in the CTL response to H-43a alloantigen. Another notable observation made in the earlier study was the fact that, in H-43 incompatibility of the alternative combination, H-43a mice were incapable of generating CTL activity against H-43b alloantigen. However, by means of employing new in vivo immunization procedures, we discovered that some but not all genetically identical H-43a responder mice could mount anti-H-43b CTL response restricted by self Kb. Again, no anti-H-43b CTL activity could be generated in the context of self Kk, Kj, Db or Dk molecules. Although the number of class I alleles we examined is still limited, these results indicate that antigenic fragments derived from the processed H-43a and H-43b alloantigens possess an indistinguishable epitope (agretope), and that such agretope either interacts only with the privileged Kb molecules or allows to bestow the immunogenic conformation of allodeterminants on the fragments solely in the context of the restricting Kb element.

BACKGROUND: MEDI7247 is a first-in-class antibody-drug conjugate (ADC) consisting of an anti-sodium-dependent alanine-serine-cysteine transporter 2 antibody-conjugated to a pyrrolobenzodiazepine dimer. OBJECTIVE: This first-in-human phase 1 trial evaluated MEDI7247 in patients with hematological malignancies. PATIENTS AND METHODS: Adults with acute myeloid leukemia (AML), multiple myeloma (MM), or diffuse large B-cell lymphoma (DLBCL) relapsed or refractory (R/R) to standard therapies, or for whom no standard therapy exists, were eligible. Primary endpoints were safety and determination of the maximum tolerated dose (MTD). Secondary endpoints included assessments of antitumor activity, pharmacokinetics (PK), and immunogenicity. RESULTS: As of 26 March 2020, 67 patients were treated (AML: n = 27; MM: n = 18; DLBCL: n = 22). The most common MEDI7247-related adverse events (AEs) were thrombocytopenia (41.8%), neutropenia (35.8%), and anemia (28.4%). The most common treatment-related grade 3/4 AEs were thrombocytopenia (38.8%), neutropenia (34.3%), and anemia (22.4%). Anticancer activity (number of responders/total patients evaluated) was observed in 11/67 (16.4%) patients. No correlation was observed between ASCT2 expression and clinical response. Between-patient variability of systemic exposure of MEDI7247 ADC and total antibody were high (AUCinf geometric CV%: 62.3-134.2, and 74.8-126.1, respectively). SG3199 (PBD dimer) plasma concentrations were below the limit of quantification for all patients after Study Day 8. Anti-drug antibody (ADA) prevalence was 7.7%, ADA incidence was 1.9%, and persistent-positive ADA was 5.8%. CONCLUSIONS: Thrombocytopenia and neutropenia limited repeat dosing. Although limited clinical activity was detected, the dose-escalation phase was stopped early without establishing an MTD. The study was registered with ClinicalTrials.gov (NCT03106428).

Human APOBEC3 enzymes are a family of single-stranded (ss)DNA and RNA cytidine deaminases that act as part of the intrinsic immunity against viruses and retroelements. These enzymes deaminate cytosine to form uracil which can functionally inactivate or cause degradation of viral or retroelement genomes. In addition, APOBEC3s have deamination-independent antiviral activity through protein and nucleic acid interactions. If expression levels are misregulated, some APOBEC3 enzymes can access the human genome leading to deamination and mutagenesis, contributing to cancer initiation and evolution. While APOBEC3 enzymes are known to interact with large ribonucleoprotein complexes, the function and RNA dependence are not entirely understood. To further understand their cellular roles, we determined by affinity purification mass spectrometry (AP-MS) the protein interaction network for the human APOBEC3 enzymes and mapped a diverse set of protein-protein and protein-RNA mediated interactions. Our analysis identified novel RNA-mediated interactions between APOBEC3C, APOBEC3H Haplotype I and II, and APOBEC3G with spliceosome proteins, and APOBEC3G and APOBEC3H Haplotype I with proteins involved in tRNA methylation and ncRNA export from the nucleus. In addition, we identified RNA-independent protein-protein interactions with APOBEC3B, APOBEC3D, and APOBEC3F and the prefoldin family of protein-folding chaperones. Interaction between prefoldin 5 (PFD5) and APOBEC3B disrupted the ability of PFD5 to induce degradation of the oncogene cMyc, implicating the APOBEC3B protein interaction network in cancer. Altogether, the results uncover novel functions and interactions of the APOBEC3 family and suggest they may have fundamental roles in cellular RNA biology, their protein-protein interactions are not redundant, and there are protein-protein interactions with tumor suppressors, suggesting a role in cancer biology. Data are available via ProteomeXchange with the identifier PXD044275.

Mutations in the RNA helicase DDX3X, implicated in various cancers and neurodevelopmental disorders, often impair RNA unwinding and translation. However, the mechanisms underlying the impairment and the differential interactions of DDX3X mutants with wild-type (WT) X-linked DDX3X and Y-linked homolog DDX3Y remain elusive. This study reveals that specific DDX3X mutants more frequently found in disease form distinct hollow condensates in cells. Using a combined structural, biochemical, and single-molecule microscopy study, we show that reduced ATPase and RNA release activities contribute to condensate formation and these catalytic deficits result from inhibiting the catalytic cycle at multiple steps. Proteomic investigations further demonstrate that these hollow condensates sequester WT DDX3X/DDX3Y and other proteins crucial for diverse signaling pathways. WT DDX3X enhances the dynamics of heterogeneous mutant/WT hollow condensates more effectively than DDX3Y. These findings offer valuable insights into the catalytic defects of specific DDX3X mutants and their differential interactions with wild-type DDX3X and DDX3Y, potentially explaining sex biases in disease.

Antiviral DNA cytosine deaminases APOBEC3A and APOBEC3B are major sources of mutations in cancer by catalyzing cytosine-to-uracil deamination. APOBEC3A preferentially targets single-stranded DNAs, with a noted affinity for DNA regions that adopt stem-loop secondary structures. However, the detailed substrate preferences of APOBEC3A and APOBEC3B have not been fully established, and the specific influence of the DNA sequence on APOBEC3A and APOBEC3B deaminase activity remains to be investigated. Here, we find that APOBEC3B also selectively targets DNA stem-loop structures, and they are distinct from those subjected to deamination by APOBEC3A. We develop Oligo-seq, an in vitro sequencing-based method to identify specific sequence contexts promoting APOBEC3A and APOBEC3B activity. Through this approach, we demonstrate that APOBEC3A and APOBEC3B deaminase activity is strongly regulated by specific sequences surrounding the targeted cytosine. Moreover, we identify the structural features of APOBEC3B and APOBEC3A responsible for their substrate preferences. Importantly, we determine that APOBEC3B-induced mutations in hairpin-forming sequences within tumor genomes differ from the DNA stem-loop sequences mutated by APOBEC3A. Together, our study provides evidence that APOBEC3A and APOBEC3B can generate distinct mutation landscapes in cancer genomes, driven by their unique substrate selectivity.

In this study, the impact of the apolipoprotein B mRNA-editing catalytic subunit-like (APOBEC) enzyme APOBEC3B (A3B) on epidermal growth factor receptor (EGFR)-driven lung cancer was assessed. A3B expression in EGFR mutant (EGFRmut) non-small-cell lung cancer (NSCLC) mouse models constrained tumorigenesis, while A3B expression in tumors treated with EGFR-targeted cancer therapy was associated with treatment resistance. Analyses of human NSCLC models treated with EGFR-targeted therapy showed upregulation of A3B and revealed therapy-induced activation of nuclear factor kappa B (NF-κB) as an inducer of A3B expression. Significantly reduced viability was observed with A3B deficiency, and A3B was required for the enrichment of APOBEC mutation signatures, in targeted therapy-treated human NSCLC preclinical models. Upregulation of A3B was confirmed in patients with NSCLC treated with EGFR-targeted therapy. This study uncovers the multifaceted roles of A3B in NSCLC and identifies A3B as a potential target for more durable responses to targeted cancer therapy.

The multifaceted chemo-immune resistance is the principal barrier to achieving cure in cancer patients. Identifying a target that is critically involved in chemo-immune-resistance represents an attractive strategy to improve cancer treatment. iRhom1 plays a role in cancer cell proliferation and its expression is negatively correlated with immune cell infiltration. Here we show that iRhom1 decreases chemotherapy sensitivity by regulating the MAPK14-HSP27 axis. In addition, iRhom1 inhibits the cytotoxic T-cell response by reducing the stability of ERAP1 protein and the ERAP1-mediated antigen processing and presentation. To facilitate the therapeutic translation of these findings, we develop a biodegradable nanocarrier that is effective in codelivery of iRhom pre-siRNA (pre-siiRhom) and chemotherapeutic drugs. This nanocarrier is effective in tumor targeting and penetration through both enhanced permeability and retention effect and CD44-mediated transcytosis in tumor endothelial cells as well as tumor cells. Inhibition of iRhom1 further facilitates tumor targeting and uptake through inhibition of CD44 cleavage. Co-delivery of pre-siiRhom and a chemotherapy agent leads to enhanced antitumor efficacy and activated tumor immune microenvironment in multiple cancer models in female mice. Targeting iRhom1 together with chemotherapy could represent a strategy to overcome chemo-immune resistance in cancer treatment.

BackgroundThe upper-airway microbiome is involved in asthma exacerbations despite inhaled corticosteroid (ICS) treatment. Although human genetics regulates microbiome composition, its influence on asthma-related airway bacteria remains unknown.ObjectiveWe sought to identify genes and biological pathways regulating airway-microbiome traits involved in asthma exacerbations and ICS response.MethodsSaliva, nasal, and pharyngeal samples from 257 European patients with asthma were analyzed. The association of 6,296,951 genetic variants with exacerbation-related microbiome traits despite ICS treatment was tested through microbiome genome-wide association studies. Variants with 1 × 10-4 -6 were examined in gene-set enrichment analyses. Significant results were sought for replication in 114 African American and 158 Latino children with and without asthma. ICS-response-associated single nucleotide polymorphisms reported in the literature were evaluated as microbiome quantitative trait loci. Multiple comparisons were adjusted by the false discovery rate.ResultsGenes associated with exacerbation-related airway-microbiome traits were enriched in asthma comorbidities development (ie, reflux esophagitis, obesity, and smoking), and were likely regulated by trichostatin A and the nuclear factor-κB, the glucocorticosteroid receptor, and CCAAT/enhancer-binding protein transcription factors (7.8 × 10-13 ≤ false discovery rate ≤ 0.022). Enrichment in smoking, trichostatin A, nuclear factor-κB, and glucocorticosteroid receptor were replicated in the saliva samples from diverse populations (4.42 × 10-9 ≤ P ≤ .008). The ICS-response-associated single nucleotide polymorphisms rs5995653 (APOBEC3B-APOBEC3C), rs6467778 (TRIM24), and rs5752429 (TPST2) were identified as microbiome quantitative trait loci of Streptococcus, Tannerella, and Campylobacter in the upper airway (0.027 ≤ false discovery rate ≤ 0.050).ConclusionsGenes associated with asthma exacerbation-related microbiome traits might influence asthma comorbidities. We reinforced the therapeutic interest of trichostatin A, nuclear factor-κB, the glucocorticosteroid receptor, and CCAAT/enhancer-binding protein in asthma exacerbations.

Mucosal-associated invariant T (MAIT) cells are unconventional T cells with innate-like antimicrobial responsiveness. MAIT cells are known for MR1 (MHC class I-related protein 1)-restricted recognition of microbial riboflavin metabolites giving them the capacity to respond to a broad range of microbes. However, recent progress has shown that MAIT cells can also respond to several viral infections in humans and in mouse models, ranging from HIV-1 and hepatitis viruses to influenza virus and SARS-CoV-2, in a primarily cognate Ag-independent manner. Depending on the disease context MAIT cells can provide direct or indirect antiviral protection for the host and may help recruit other immune cells, but they may also in some circumstances amplify inflammation and aggravate immunopathology. Furthermore, chronic viral infections are associated with varying degrees of functional and numerical MAIT cell impairment, suggesting secondary consequences for host defense. In this review, we summarize recent progress and highlight outstanding questions regarding the emerging role of MAIT cells in antiviral immunity.

Double-stranded RNA produced during viral replication and transcription activates both protein kinase R (PKR) and ribonuclease L (RNase L), which limits viral gene expression and replication through host shutoff of translation. In this study, we find that APOBEC3B forms a complex with PABPC1 to stimulate PKR and counterbalances the PKR-suppressing activity of ADAR1 in response to infection by many types of viruses. This leads to translational blockage and the formation of stress granules. Furthermore, we show that APOBEC3B localizes to stress granules through the interaction with PABPC1. APOBEC3B facilitates the formation of protein-RNA condensates with stress granule assembly factor (G3BP1) by protecting mRNA associated with stress granules from RNAse L-induced RNA cleavage during viral infection. These results not only reveal that APOBEC3B is a key regulator of different steps of the innate immune response throughout viral infection but also highlight an alternative mechanism by which APOBEC3B can impact virus replication without editing viral genomes.

Rationale: Idiopathic pulmonary arterial hypertension (PAH) is a terminal pulmonary vascular disease characterized by increased pressure, right ventricular failure, and death. PAH exhibits a striking sex bias and is up to four times more prevalent in females. Understanding the molecular basis behind sex differences could help uncover novel therapies. Objectives: We previously discovered that the Y chromosome is protective against hypoxia-induced experimental pulmonary hypertension (PH), which may contribute to sex differences in PAH. Here, we identify the gene responsible for Y-chromosome protection, investigate key downstream autosomal genes, and demonstrate a novel preclinical therapy. Methods: To test the effect of Y-chromosome genes on PH development, we knocked down each Y-chromosome gene expressed in the lung by means of intratracheal instillation of siRNA in gonadectomized male mice exposed to hypoxia and monitored changes in right ventricular and pulmonary artery hemodynamics. We compared the lung transcriptome of Uty knockdown mouse lungs to those of male and female PAH patient lungs to identify common downstream pathogenic chemokines and tested the effects of these chemokines on human pulmonary artery endothelial cells. We further inhibited the activity of these chemokines in two preclinical pulmonary hypertension models to test the therapeutic efficacy. Measurements and Main Results: Knockdown of the Y-chromosome gene Uty resulted in more severe PH measured by increased right ventricular pressure and decreased pulmonary artery acceleration time. RNA sequencing revealed an increase in proinflammatory chemokines Cxcl9 and Cxcl10 as a result of Uty knockdown. We found CXCL9 and CXCL10 significantly upregulated in human PAH lungs, with more robust upregulation in females with PAH. Treatment of human pulmonary artery endothelial cells with CXCL9 and CXCL10 triggered apoptosis. Inhibition of Cxcl9 and Cxcl10 expression in male Uty knockout mice and CXCL9 and CXCL10 activity in female rats significantly reduced PH severity. Conclusions:Uty is protective against PH. Reduction of Uty expression results in increased expression of proinflammatory chemokines Cxcl9 and Cxcl10, which trigger endothelial cell death and PH. Inhibition of CLXC9 and CXLC10 rescues PH development in multiple experimental models.

"Differences of Sexual Development (DSD)," individuals with rearranged Y chromosome breaks in their 46,XY cells are reported with male and female gender phenotypes and differences in germ cell tumour (GCT) risk. This raised the question of whether male or female gender and GCT risk depends on the site of the break and/or rearrangement of the individual´s Y chromosome. In this paper, we report molecular mapping of the breakpoint on the aberrant Y chromosome of 22 DSD individuals with a 45,X/46,XY karyotype reared with a different gender. Their Y chromosome breaks are found at different sites on the long and short Y arms. Our data indicate that gender rearing is, neither dependent on the site of Y breakage, nor on the amount of 45,X0 cells in the individuals' leukocytes. Most prominent are secondary rearrangements of the Y chromosome breaks forming di-centric Y-structures ("dic-Y"). Duplications of the short Y arm and the proximal part of the long Y arm are the results. A putative GCT risk has been analysed with immunohistochemical experiments on some dysgenetic gonadal tissue sections. With specific antibodies for OCT3/4 expression, we marked the pluripotent germ cell fraction being potential tumour precursor cells. With specific antibodies for DDX3Y, TSPY, and UTY we analyzed their putative Gonadoblastoma Y (GBY) tumour susceptibility function in the same specimen. We conclude GBY expression is only diagnostic for GCT development in the aberrant germ cells of these DSD individuals when strong OCT3/4 expression has marked their pluripotency.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that are highly abundant in human blood and tissues. Most MAIT cells have an invariant TCRα-chain that uses T cell receptor α-variable 1-2 (TRAV1-2) joined to TRAJ33/20/12 and recognizes metabolites from bacterial riboflavin synthesis bound to the Ag-presenting molecule MHC class I related (MR1). Our attempts to identify alternative MR1-presented Ags led to the discovery of rare MR1-restricted T cells with non-TRAV1-2 TCRs. Because altered Ag specificity likely alters affinity for the most potent known Ag, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), we performed bulk TCRα- and TCRβ-chain sequencing and single-cell-based paired TCR sequencing on T cells that bound the MR1-5-OP-RU tetramer with differing intensities. Bulk sequencing showed that use of V genes other than TRAV1-2 was enriched among MR1-5-OP-RU tetramerlow cells. Although we initially interpreted these as diverse MR1-restricted TCRs, single-cell TCR sequencing revealed that cells expressing atypical TCRα-chains also coexpressed an invariant MAIT TCRα-chain. Transfection of each non-TRAV1-2 TCRα-chain with the TCRβ-chain from the same cell demonstrated that the non-TRAV1-2 TCR did not bind the MR1-5-OP-RU tetramer. Thus, dual TCRα-chain expression in human T cells and competition for the endogenous β-chain explains the existence of some MR1-5-OP-RU tetramerlow T cells. The discovery of simultaneous expression of canonical and noncanonical TCRs on the same T cell means that claims of roles for non-TRAV1-2 TCR in MR1 response must be validated by TCR transfer-based confirmation of Ag specificity.

Mutations in the RNA helicase, DDX3X, are a leading cause of Intellectual Disability and present as DDX3X syndrome, a neurodevelopmental disorder associated with cortical malformations and autism. Yet, the cellular and molecular mechanisms by which DDX3X controls cortical development are largely unknown. Here, using a mouse model of Ddx3x loss-of-function we demonstrate that DDX3X directs translational and cell cycle control of neural progenitors, which underlies precise corticogenesis. First, we show brain development is sensitive to Ddx3x dosage; complete Ddx3x loss from neural progenitors causes microcephaly in females, whereas hemizygous males and heterozygous females show reduced neurogenesis without marked microcephaly. In addition, Ddx3x loss is sexually dimorphic, as its paralog, Ddx3y, compensates for Ddx3x in the developing male neocortex. Using live imaging of progenitors, we show that DDX3X promotes neuronal generation by regulating both cell cycle duration and neurogenic divisions. Finally, we use ribosome profiling in vivo to discover the repertoire of translated transcripts in neural progenitors, including those which are DDX3X-dependent and essential for neurogenesis. Our study reveals invaluable new insights into the etiology of DDX3X syndrome, implicating dysregulated progenitor cell cycle dynamics and translation as pathogenic mechanisms.