Nonhematopoietic Tissues Research Articles

Novel coenzyme Q6 genetic variant increases susceptibility to pneumococcal disease.

Acute lower respiratory tract infection (ALRI) remains a major worldwide cause of childhood mortality, compelling innovation in prevention and treatment. Children in Papua New Guinea (PNG) experience profound morbidity from ALRI caused by Streptococcus pneumoniae. As a result of evolutionary divergence, the human PNG population exhibits profound genetic variation and diversity. To address unmet health needs of children in PNG, we tested whether genetic variants increased ALRI morbidity. Whole-exome sequencing of a pilot child cohort identified homozygosity for a novel single-nucleotide variant (SNV) in coenzyme Q6 (COQ6) in cases with ALRI. COQ6 encodes a mitochondrial enzyme essential for biosynthesis of ubiquinone, an electron acceptor in the electron transport chain. A significant association of SNV homozygosity with ALRI was replicated in an independent ALRI cohort (P = 0.036). Mice homozygous for homologous mouse variant Coq6 exhibited increased mortality after pneumococcal lung infection, confirming causality. Bone marrow chimeric mice further revealed that expression of variant Coq6 in recipient (that is, nonhematopoietic) tissues conferred increased mortality. Variant Coq6 maintained ubiquinone biosynthesis, while accelerating metabolic remodeling after pneumococcal challenge. Identification of this COQ6 variant provides a genetic basis for increased pneumonia susceptibility in PNG and establishes a previously unrecognized role for the enzyme COQ6 in regulating inflammatory-mediated metabolic remodeling.

Chemokine receptors are required for effector T-cell trafficking to GVHD tissues but not to bone marrow

AbstractIn allogeneic hematopoietic stem cell transplantation (allo-SCT), alloreactive donor T cells mediate the graft-versus-leukemia effect but also attack nonhematopoietic tissues, causing graft-versus-host disease (GVHD). Reducing alloreactive T-cell trafficking to GVHD target tissues while allowing their access to bone marrow (BM) and spleen, major sites of malignant hematopoiesis, is a rational strategy for reducing the GVHD risk when using alloreactive T cells as a therapeutic. Here, we show that effector T-cell (Teff) entry into BM and spleen in unmanipulated mice and in mice that received transplantation without alloreactive T cells is augmented by pertussis toxin (PTX)-sensitive chemokine receptor signaling. However, unexpectedly, in the presence of a GVH response, chemokines no longer draw T cells into BM and spleen but remain critical for their recruitment to GVHD target tissues. Consistent with this, PTX-treated Teff cells were as efficacious as untreated T cells in killing leukemia cells in BM and spleen in mice with a concurrent GVHD response. These results suggest a strategy to improve the safety of alloreactive T-cell therapeutics in treating leukemias in the context of an allo-SCT.

Vasoactive Intestinal Polypeptide in the Enteric Neural Plexus Promotes Microbiome Diversity and Resistance to GvHD

Abstract Graft-versus-host disease (GvHD) in allo-transplant recipients is characterized by donor T cell- mediated damage to recipient organs and changes in the gut microbiome. Vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, directs T cell responses towards a Th2 phenotype while inhibiting proinflammatory cytokine production. VIP-producing enteric neurons contribute to the mucosal barrier and maintain gut microbiome homeostasis. The impact of VIP production by host cells on GvHD remains unclear. We examined the effect of endogenous VIP synthesis in modulating GvHD in multiple murine allo-BMT models. VIP knock-out mice were exquisitely sensitive to GvHD, experiencing activation and expansion of Th1- and Th17-polarized T cells and early death. Transplanting radiation chimeric mice in which the VIP gene was knocked out in either hematopoietic or non-hematopoietic cells demonstrated that VIP production by non-hematopoietic tissues limited the GvHD activity of donor T cells. Immunofluorescence staining demonstrated VIP expression in enteric neurons in allo-BMT recipients, consistent with local effects of VIP on T cells and the gut microbiome. VIP- KO mice had lower gut pH, higher frequencies of Bacillota species, and less microbiome diversity post-transplantation. These data support the crucial role of VIP in maintaining the diversity of the gut microbiome with beneficial commensal species and in modulating the alloreactivity of donor T cells after allo-BMT.

Anti-CD45 PBD-based antibody-drug conjugates are effective targeted conditioning agents for gene therapy and stem cell transplant

Stem cell gene therapy and hematopoietic stem cell transplantation (SCT) require conditioning to ablate the recipient's hematopoietic stem cells (HSCs) and create a niche for gene-corrected/donor HSCs. Conventional conditioning agents are non-specific, leading to off-target toxicities and resulting in significant morbidity and mortality. We developed tissue-specific anti-human CD45 antibody-drug conjugates (ADCs), using rat IgG2b anti-human CD45 antibody clones YTH24.5 and YTH54.12, conjugated to cytotoxic pyrrolobenzodiazepine (PBD) dimer payloads with cleavable (SG3249) or non-cleavable (SG3376) linkers. Invitro, these ADCs internalized to lysosomes for drug release, resulting in potent and specific killing of human CD45+ cells. In humanized NSG mice, the ADCs completely ablated human HSCs without toxicity to non-hematopoietic tissues, enabling successful engraftment of gene-modified autologous and allogeneic human HSCs. The ADCs also delayed leukemia onset and improved survival in CD45+ tumor models. These data provide proof of concept that conditioning with anti-human CD45-PBD ADCs allows engraftment of donor/gene-corrected HSCs with minimal toxicity to non-hematopoietic tissues. Our anti-CD45-PBDs or similar agents could potentially shift the paradigm in transplantation medicine that intensive chemo/radiotherapy is required for HSC engraftment after gene therapy and allogeneic SCT. Targeted conditioning both improve the safety and minimize late effects of these procedures, which would greatly increase their applicability.

Abstract 624: Ptpn1 deficiency collaborates with a NUP98::HOXD13 fusion gene to generate B cell precursor acute lymphoblastic leukemia

Abstract Background: Acute lymphoid leukemia is characterized by inherited or acquired mutations that effect critical differentiation and proliferation pathways. We have shown that Mcm2 deficient mice developed T cell acute lymphoblastic leukemia, due to copy number variations, most commonly interstitial deletions throughout the genome. When crossed with mice that expressed a NUP98::HOXD13 (NHD13) fusion gene, Mcm2−/−NHD13 mice developed B cell precursor ALL (BCP-ALL). A majority of these BCP-ALL had acquired homozygous deletions of Ptpn1, a protein tyrosine phosphatase, leading to the hypothesis that Ptpn1 deficiency combined with NHD13 expression leads to BCP ALL. Objective: To investigate the role of Ptpn1 deletion in BCP-ALL development using mouse models. Methods: Mice expressing an NHD13 fusion gene were crossed with Ptpn1 knockout mice, generating 6 possible genotypes. Mice were followed for 18 months. Mice with signs of leukemia were characterized by clinical evaluation, CBC, flow cytometry, and IHC. Primary BCP-ALL and derived BCP-ALL cell lines were also evaluated with RNA-seq and molecular pathway analysis. Results: NHD13+Ptpn1-/- mice developed BCP-ALL with 65% penetrance, characterized by hyperleukocytosis, anemia, thrombocytopenia, and invasion of non-hematopoietic tissues. Similar to human BCP-ALL, NHD13+Ptpn1-/- BCP-ALL had clonal IGH as well as clonal Tcrd gene rearrangements. Flow cytometry revealed CD19 and/or B220 expression. NHD13+Ptpn1+/- mice with BCP-ALL frequently lost the wild-type (WT) Ptpn1 allele in leukemic cells, reinforcing the hypothesis that Ptpn1 can function as a classic tumor suppressor gene in this context. Whole exome sequencing revealed acquired mutations in B-cell differentiation genes (Pax5 or Bcor) and activating mutations in tyrosine kinase genes (Jak1/3 and Flt3). Transcription signature analysis showed significant upregulation of Hoxa/b gene clusters, RNase12 and LncRNAs subsets. Conclusion: This study demonstrates that Ptpn1 loss combined with expression of NHD13 fusion gene leads to highly penetrant BCP-ALL in mice, suggesting a role for Ptpn1 in preventing malignant transformation. These findings present a collaborative model for BCP-ALL in which the NHD13 transgene leads to increased stem cell self-renewal, somatic Bcor or Pax5 mutations block normal B cell differentiation, and somatic signaling mutations (Jak1/3,Flt3) lead to hyperproliferation, which is potentiated by Ptpn1 deficiency. Citation Format: Nupur Nigam, Toshihiro Matsukawa, Ryan Bertoli, Michel L. Tremblay, Mianmian Yin, Peter D. Aplan. Ptpn1 deficiency collaborates with a NUP98::HOXD13 fusion gene to generate B cell precursor acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 624.

Genetic ablation of Immt induces a lethal disruption of the MICOS complex.

The mitochondrial contact site and cristae organizing system (MICOS) is important for crista junction formation and for maintaining inner mitochondrial membrane architecture. A key component of the MICOS complex is MIC60, which has been well studied in yeast and cell culture models. However, only one recent study has demonstrated the embryonic lethality of losing Immt (the gene encoding MIC60) expression. Tamoxifen-inducible ROSA-CreERT2-mediated deletion of Immt in adult mice disrupted the MICOS complex, increased mitochondria size, altered cristae morphology, and was lethal within 12 d. Pathologically, these mice displayed defective intestinal muscle function (paralytic ileus) culminating in dehydration. We also identified bone marrow (BM) hypocellularity in Immt-deleted mice, although BM transplants from wild-type mice did not improve survival. Altogether, this inducible mouse model demonstrates the importance of MIC60 in vivo, in both hematopoietic and non-hematopoietic tissues, and provides a valuable resource for future mechanistic investigations into the MICOS complex.

Expanding the repertoire reveals recurrent, cryptic, and hematopoietic HLA class I minor histocompatibility antigens

AbstractAllogeneic stem cell transplantation (alloSCT) is a curative treatment for hematological malignancies. After HLA-matched alloSCT, antitumor immunity is caused by donor T cells recognizing polymorphic peptides, designated minor histocompatibility antigens (MiHAs), that are presented by HLA on malignant patient cells. However, T cells often target MiHAs on healthy nonhematopoietic tissues of patients, thereby inducing side effects known as graft-versus-host disease. Here, we aimed to identify the dominant repertoire of HLA-I-restricted MiHAs to enable strategies to predict, monitor or modulate immune responses after alloSCT. To systematically identify novel MiHAs by genome-wide association screening, T-cell clones were isolated from 39 transplanted patients and tested for reactivity against 191 Epstein-Barr virus transformed B cell lines of the 1000 Genomes Project. By discovering 81 new MiHAs, we more than doubled the antigen repertoire to 159 MiHAs and demonstrated that, despite many genetic differences between patients and donors, often the same MiHAs are targeted in multiple patients. Furthermore, we showed that one quarter of the antigens are cryptic, that is translated from unconventional open reading frames, for example long noncoding RNAs, showing that these antigen types are relevant targets in natural immune responses. Finally, using single cell RNA-seq data, we analyzed tissue expression of MiHA-encoding genes to explore their potential role in clinical outcome, and characterized 11 new hematopoietic-restricted MiHAs as potential targets for immunotherapy. In conclusion, we expanded the repertoire of HLA-I-restricted MiHAs and identified recurrent, cryptic and hematopoietic-restricted antigens, which are fundamental to predict, follow or manipulate immune responses to improve clinical outcome after alloSCT.

Autophagy differentially regulates tissue tolerance of distinct target organs in graft-versus-host disease models.

Tissue-intrinsic mechanisms that regulate severity of systemic pathogenic immune-mediated diseases, such as acute graft-versus-host disease (GVHD), remain poorly understood. Following allogeneic hematopoietic stem cell transplantation, autophagy, a cellular stress protective response, is induced in host nonhematopoietic cells. To systematically address the role of autophagy in various host nonhematopoietic tissues, both specific classical target organs of acute GVHD (intestines, liver, and skin) and organs conventionally not known to be targets of GVHD (kidneys and heart), we generated mice with organ-specific knockout of autophagy related 5 (ATG5) to specifically and exclusively inhibit autophagy in the specific organs. When compared with wild-type recipients, animals that lacked ATG5 in the gastrointestinal tract or liver showed significantly greater tissue injury and mortality, while autophagy deficiency in the skin, kidneys, or heart did not affect mortality. Treatment with the systemic autophagy inducer sirolimus only partially mitigated GVHD mortality in intestine-specific autophagy-deficient hosts. Deficiency of autophagy increased MHC class I on the target intestinal epithelial cells, resulting in greater susceptibility to damage by alloreactive T cells. Thus, autophagy is a critical cell-intrinsic protective response that promotes tissue tolerance and regulates GVHD severity.

A3 INVESTIGATING MACROPHAGE-TISSUE CELL COMMUNICATION AXES IN THE GASTROINTESTINAL TRACT

Abstract Background The gastrointestinal tract boasts the highest concentration of macrophages (MPs) in the body, acting as vital immune sentinels that oversee tissue balance. Three distinct subsets of MPs, namely CCR2+, Tim4+, and CCR2,Tim4 double-negative (DN) MPs, populate distinct gut niches. Their functional specialization suggests unique interactions within their non-hematopoietic tissue microenvironments. Despite their crucial roles, the intricacies of these interactions remain elusive. One recently discovered interaction involves a communication axis between MPs and neurons within the muscularis layer of the gut. Here, neurons secrete Colony Stimulating Factor 1 (CSF1), a survival factor for MPs. These MPs reciprocate by producing Bone Morphogenetic Protein 2 (BMP2), assisting neurons in regulating gut motility. Aims Based on the aforementioned interaction, we hypothesize that BMP2, derived from tissue-resident MPs, might influence functions of other tissue niches, subsequently prompting survival of MP via CSF1 production. Our aims are twofold: (1) Identify the role of BMP2 in gut macrophages; (2) Identify BMP2-responsive CSF1-producing tissue cells within the gut. Methods To discern the roles of MP subsets, we developed a conditional knockout mouse model, targeting Bmp2 expression in MPs. We validated successful Bmp2 deletion(BMP2 KO) through quantitative polymerase chain reaction (qPCR) and an in-situ hybridization immunofluorescence assay (RNAscope). Subsequently, single cell RNA (scRNAseq) was performed, combining samples from both control and KO small intestines. This was done to further confirm BMP2 KO and to identify BMP2-responsive CSF1-producing cells. Flow cytometry was then used to characterize the intestinal MPs in control and KO mice. Results Tim4+ MPs emerged as the primary source of BMP2. Comprehensive analysis using qPCR, RNAscope, and scRNAseq validated the complete KO of BMP2 across all macrophage subsets. The loss of MP-produced BMP2 led to a disruption in MP development. Notably, a marked reduction in DN and Tim4+ MPs was observed following deletion of Bmp2 in MPs, emphasizing the critical role of BMP2-driven CSF1 in sustaining these subsets. CSF1 producing cells co-expressing the cognate BMP receptor in the gut, were predominantly non-hematopoietic tissue cells. Among these, distinct fibroblast populations emerged as a potent but not the sole source of CSF1. Conclusions Our work underscores the involvement of multiple tissue cell populations, revealing an intricate network of interactions that extends beyond fibroblasts. This study unveils the multifaceted nature of tissue-MP interactions and their critical roles in maintaining gut homeostasis. Disturbances of these pathways may pave the way for disease by altering tissue homeostasis. Funding Agencies NSERC

CD45-Directed CAR-T Cells with CD45 Knockout Efficiently Kill Myeloid Leukemia and Lymphoma Cells In Vitro Even after Extended Culture.

CAR-T cell therapy has shown impressive results and is now part of standard-of-care treatment of B-lineage malignancies, whereas the treatment of myeloid diseases has been limited by the lack of suitable targets. CD45 is expressed on almost all types of blood cells including myeloid leukemia cells, but not on non-hematopoietic tissue, making it a potential target for CAR-directed therapy. Because of its high expression on T and NK cells, fratricide is expected to hinder CD45CAR-mediated therapy. Due to its important roles in effector cell activation, signal transduction and cytotoxicity, CD45 knockout aimed at preventing fratricide in T and NK cells has been expected to lead to considerable functional impairment. CD45 knockout was established on T and NK cell lines using CRISPR/Cas9-RNPs and electroporation, and the successful protocol was transferred to primary T cells. A combined protocol was developed enabling CD45 knockout and retroviral transduction with a third-generation CAR targeting CD45 or CD19. The functionality of CD45ko effector cells, CD45ko/CD45CAR-T and CD45ko/CD19CAR-T cells was studied using proliferation as well as short- and long-term cytotoxicity assays. As expected, the introduction of a CD45-CAR into T cells resulted in potent fratricide that can be avoided by CD45 knockout. Unexpectedly, the latter had no negative impact on T- and NK-cell proliferation in vitro. Moreover, CD45ko/CD45CAR-T cells showed potent cytotoxicity against CD45-expressing AML and lymphoma cell lines in short-term and long-term co-culture assays. A pronounced cytotoxicity of CD45ko/CD45CAR-T cells was maintained even after four weeks of culture. In a further setup, we confirmed the conserved functionality of CD45ko cells using a CD19-CAR. Again, the proliferation and cytotoxicity of CD45ko/CD19CAR-T cells showed no differences from those of their CD45-positive counterparts in vitro. We report the efficient production of highly and durably active CD45ko/CAR-T cells. CD45 knockout did not impair the functionality of CAR-T cells in vitro, irrespective of the target antigen. If their activity can be confirmed in vivo, CD45ko/CD45CAR-T cells might, for example, be useful as part of conditioning regimens prior to stem cell transplantation.

Inborn errors of immunity with kidney and urinary tract disorders: a review.

Human inborn errors of immunity (IEIs), previously referred to as primary immunodeficiency disorders (PIDs), are a heterogeneous spectrum of inherited abnormalities of the immune system with different organ involvement. The number of identified IEIs is rapidly increasing, highlighting the non-negligible role of an interdisciplinary approach in clinical diagnosis. Kidney disorders are one of the important comorbidities in some of the affected patients and play a significant role in the diagnosis and course of disease. According to recent studies, 22 types of human IEI with renal manifestations have been identified so far, including immunodeficiency with congenital thrombocytopenia, thymic defects with additional congenital anomalies, complement deficiencies, type 1 interferonopathies, immunity related to non-hematopoietic tissues, congenital neutropenia's, common variable immunodeficiency disorder (CVID) phenotype and immuno-osseous dysplasia. Based on this classification, we herein review IEIs with renal features and explain the genetic defect, inheritance, and type of renal manifestations.

ERCC6L2 Disease and Shwachman-Diamond Syndrome Exhibit Alterations in Metabolomes in Patient Fibroblasts

Introduction Metabolomics is an emerging tool to study metabolic alterations in cancers. It has also the potential to identify clinically relevant biomarkers for diagnosis, risk appraisal, and targeted drug development. In hematology, metabolic reprogramming is essential for normal regulation of hematopoiesis. Association of discrete metabolic alterations with explicit somatic mutations have also been reported in hematological malignancies. We have previously observed glutaminase (GLS) overexpression and improved oxygen consumption rate with excess glutamine in low glucose conditions in patient-derived fibroblasts in ERCC6L2 disease (unpublished). This prompted us to study metabolomic alterations in ERCC6L2 disease and Shwachman-Diamond syndrome (SDS) induced strictly by the germline ERCC6L2 and SBDS mutations. Both inherited bone marrow failure (BMF) syndromes carry considerable tendency of somatic TP53 mutagenesis predisposing patients to a high risk myeloid malignancy. Our aim here was to identify potential drug targets by targeted metabolomics. Methods We examined patient-derived skin fibroblasts (ERCC6L2 disease, n=4; SDS, n=4) and healthy controls (n=4) and performed targeted metabolomics with U-13C-D-glucose and U-13C-L-glutamine labeling. Fibroblasts were seeded on six-well plates and on the following day exposed to 13C-glucose (25mM) or 13C-glutamine (2mM) label containing medium for 30 or 120 minutes. Next, metabolites were extracted and quantified with liquid chromatography mass spectrometer. The results were normalized to total metabolite intensity per sample. Results In 13C-glucose labeled samples, we detected statistically significantly lower intensity of pyruvate M+3 in both ERCC6L2 and SDS fibroblasts compared to control at 30 minutes, which persisted at 120 minutes in SDS cells. In the TCA cycle, malate M+3 labeling indicates pyruvate carboxylase (PC) activity and M+2 labeling pyruvate dehydrogenase activity (PDH) (Figure 1). We found a significantly higher portion of malate M+3 than M+2 in SDS cells at all time points, suggesting lower PDH and higher PC activity in SDS cells. Additionally, we detected higher levels of alanine M+3 in SDS samples. As pyruvate is a source for alanine synthesis, we suspect an alteration in pyruvate metabolism; potentially to overcome challenges in pyruvate oxidation due to either decrease in the respiratory chain function or PDH activity. In 13C-glutamine labeled samples, we detected statistically significantly higher intensity of glutamine M+5 and M+4 in ERCC6L2 samples at 30 minutes compared to control and SDS cells. Glutamine is first converted to glutamate by glutaminase (GLS), after which it enters the TCA cycle as ketoglutarate (KG) (Figure 1). In addition to glutamine, we detected an increase, although not statistically significant, in glutamate M+5 and M+4, as well as KG M+5, in ERCC6L2 cells at 30 minutes. A similar trend was observed in the subsequent TCA cycle intermediates succinic acid M+4, fumarate M+4, and malate M+4. At 120 minutes, there was no difference in metabolite intensities between the groups, suggesting a potentially faster glutamine uptake by ERCC6L2 cells. Conclusions Our analysis using non-hematopoietic tissue provides understanding of metabolic weak links not confounded by somatic events occurring early in the disease course in ERCC6L2 and SDS patients. Our findings imply increased glutamine utilization in ERCC6L2 cells and altered pyruvate metabolism in SDS cells. Therefore, these metabolic pathways are attractive drug targets (summarized in Figure 1). Further studies are needed to determine their potential in supporting the compromised hematopoiesis to reduce the stress for somatic mutagenesis.

Randomized Trial of Prophylactic Donor Lymphocyte Infusion with Positively Selected CD4+ T Cells after HLA-Identical Allogeneic Stem Cell Transplantation

Introduction Allogeneic stem cell transplantation (alloSCT) can cure patients with hematological malignancies by T-cell mediated alloreactivity against patient hematopoiesis, the graft-versus-leukemia (GvL) effect. GvL can coincide with graft-versus-host disease (GvHD) when donor T cells target non-hematopoietic tissues. Increase of donor chimerism in bone marrow (BM) or peripheral blood (PB) after alloSCT indicates GvL response. After T-cell depleted alloSCT, donor lymphocyte infusion (DLI) can restore GvL. Dose and timing of DLI influence the degree of alloreactivity. Low dose of unmodified DLI as low as 0.3x10 6 T cells/kg 3 months after alloSCT appeared not to cause GvHD. (Zouwen et al. Ann Hematol, 2023) We hypothesized that infusion of a higher dose of purified CD4+ donor T cells would not cause GvHD, while inducing GvL by selective targeting of the MHC class II positive hematopoietic cells. Method A randomized controlled phase 2 trial was conducted in patients with hematological malignancies after HLA-identical sibling alloSCT with alemtuzumab added to the graft. Patients without GvHD or relapse were randomized at 2.5 months for prophylactic DLI with 1.0x10 6/kg CD4+ T cells (CD4+ DLI) 3 months after alloSCT or conventional care without DLI (CC). Both groups were eligible for prophylactic unmodified DLI at 6 months. Cumulative incidence (CI) of GvHD requiring immunosuppressive treatment 3 months after randomization was the primary endpoint. Changes in the trajectory of chimerism in BM and subsets in PB (CD4+ T cells, CD8+ T cells, NK cells, B cells and monocytes) were compared by means of mixed effects beta regression as secondary endpoints. Results 66 patients were included (table 1). CI of GvHD did not differ between groups (CD4+DLI: 6.1% vs CC: 9.1%, Gray's test p>0.9). Relapse was comparable (CD4+DLI: 15% vs CC: 12%), although heterogeneity of diseases prevented a formal comparison. In the CD4+ DLI group, BM chimerism was full donor (FD, >95% donor) at baseline in 23/33 patients, while 10/33 had mixed chimerism (MC, ≤95%). In the CC group, 23/33 were FD and 10/33 MC. Some patients with MC at baseline converted to FD in both groups (CD4+ DLI: 1/10 vs CC: 1/10). Changes in BM chimerism did not differ between CD4+ DLI and CC (p = 0.9). Chimerism of B cells, NK cells and monocytes in PB resembled bone marrow chimerism with most patients being FD at baseline. Changes in chimerism did not differ between CD4+ DLI and CC (p = 1.0, 0.5 and 0.9 for B cells, NK cells and monocytes respectively). In the CD4+ DLI group, 25/30 evaluable patients were MC in the CD4+ T-cell subset in PB at baseline. 3 month after randomization 7/25 had converted to FD, 17/25 remained MC and 1/25 was unevaluable. Of the 17 patient who remained MC, CD4+ donor chimerism increased (absolute increase >5%) in 13, decreased (>5%) in 2 and was stable in 2. In the CC group, 26/32 were MC in CD4+ T cells at baseline, of which 6 converted to FD, 18 remained MC and 2 were unevaluable. Of the 18 patients remaining MC, CD4+ chimerism increased in 5, decreased in 9 and remained stable in 4. The CD4+ donor chimerism increased significantly after CD4+ DLI compared to CC (p = 0.01) (Figure 1). In the CD4+ DLI group, 23/29 evaluable patients were MC in the CD8 subset at baseline. 7/23 converted to FD, 15/23 remained MC and 1 was unevaluable. Of these 15 MC patients at 3 months, 9 had increasing, 5 stable and 1 decreasing donor chimerism after 3 months. In CC, 22/31 were MC at baseline. 4/22 converted to FD at 3 months and 17/22 remained in MC. Of the 17 MC patients, 6 had increasing, 3 decreasing and 8 stable chimerism. The CD8+ donor chimerism increased significantly in the CD4+ DLI group compared to CC (p = 0.03). Conclusion CD4+ selected DLI at a dose of 1x10 6 CD4+/kg 3 months after HLA identical T-cell depleted alloSCT did not cause GvHD. Chimerism in BM leukocytes, B cells, NK cells or monocytes did not indicate GvL mediated by CD4+ DLI, possibly because most patients were already FD at baseline. CD4+ and CD8+ T-cell donor chimerism increased significantly after CD4+ DLI, suggesting a GVL-effect - earlier demonstrated in vitro for 4 selected patients. (Van Balen et al. Front Immunol, 2018) However, most patients did not convert to FD in the T-cell compartment, suggesting the GvL-effect of CD4+ DLI was limited. We considered this GvL-effect insufficient to prevent relapse and stress the importance of CD8+ donor T cells as main drivers of alloreactivity after DLI following HLA-identical alloSCT.

Naked Antibody-Based Conditioning Targeting CD47 and CD117 Plus Janus Kinase Inhibition Enables Toxin-Free, Radiation-Free, and Chemotherapy-Free Fully Mismatched Allogeneic Hematopoietic Stem Cell Transplantation

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for numerous malignant and non-malignant hematologic diseases. To enable engraftment of donor hematopoietic stem cells (HSCs), recipients undergo conditioning with chemotherapy and irradiation to make marrow space for incoming donor HSCs and suppress host immune responses that may lead to graft rejection. These cytotoxic conditioning regimens carry risks of severe toxicities which prevents many patients from accessing the lifesaving benefit of allo-HSCT. Previously, we developed fully ablative CD45- and cKit-targeted antibody-drug conjugates (ADC) which, when combined with Janus kinase 1/2 (JAK1/2) inhibitors, enabled fully mismatched allo-HSCT with full donor chimerism and minimal toxicity while providing durable antileukemia activity (Persaud et al (2022), ASH Abstract). However, even ADCs carry potential risks of acute and chronic toxicities that may preclude their common use as HSCT conditioning agents. Moreover, unlike malignant diseases, allo-HSCT for non-malignant diseases does not require the conditioning regimen to provide antitumor benefit and partial donor chimerism may be sufficient for cure, strongly favoring the use of less intense conditioning strategies with the fewest possible toxicities. We hypothesized that naked antibody-based conditioning strategies can achieve the therapeutic benefits of allo-HSCT for non-malignant hematologic diseases, obviating exposure to toxic ADC payloads. METHODS: We combined unconjugated antibodies targeting CD47 and cKit with JAK1/2 inhibition to perform minor histocompatibility antigen (miHA)-mismatched (129/Sv→B6-Ly5.1) and fully mismatched (BALB/c→B6) murine allo-HSCT. In some full mismatch HSCT experiments, recipients included B6-gp91phox -/- mice, which serve as a model of chronic granulomatous disease (CGD) due to their deficiency in a catalytic subunit of NADPH oxidase. Mice received 100 µg anti-CD47 (clone mIAP410; BioXcell) 8 days before HSCT (d-8) as a priming dose to reduce the severity of CD47 blockade-induced anemia. Next, mice received 500 µg anti-cKit (clone ACK2; BioLegend) on d-6 and daily 500 µg doses of anti-CD47 from d-6 through d-2. For JAK1/2 inhibition, mice received oral ruxolitinib (Incyte; 2 mg drug/g chow) starting d-3 or d-1 and continuing until d+28. On d0, recipients were transplanted with 20 x 10 6 whole bone marrow cells from 129/Sv (miHA) or BALB/c (fully mismatched) mice. Peripheral blood donor chimerism was followed monthly by flow cytometry for CD45.2 + (miHA) or H-2D d+ (fully mismatched) cells. Oxidative burst among granulocytes in B6-gp91phox -/- recipients was assessed by dihydrorhodamine 123 (DHR123) staining after phorbol 12-myristate 13-acetate (PMA) stimulation. RESULTS: CD47 and cKit antibodies plus ruxolitinib enabled multilineage engraftment in both miHA and fully mismatched allo-HSCT, including myeloid lineage donor chimerism of 95-99% (Figure 1A). As expected, CD47 blockade induced significant but transient morbidity from anemia during the peritransplant period; notably, a shorter duration of ruxolitinib treatment before HSCT (starting d-1 instead of d-3) improved the tolerability of the regimen without impacting engraftment success. Allo-HSCT of B6-gp91phox -/- mice led to near complete replacement of host granulocytes in peripheral blood with donor granulocytes, whose DHR123 positivity upon PMA stimulation indicated an intact oxidative burst capacity (Figure 1B). CONCLUSIONS: Combination of antibodies targeting CD47 and cKit with JAK1/2 inhibition provides a novel conditioning strategy for allo-HSCT in mouse models, achieving high-level, stable multilineage engraftment without radiation, chemotherapy, or exposure to toxic ADC payloads. The near complete donor chimerism among granulocytes made it particularly well suited to correcting phagocyte oxidase function in the gp91phox -/- model of CGD. Future studies seek to 1) mitigate the peritransplant anemia caused by CD47 blockade using Fc-silenced anti-CD47 reagents, 2) determine the proportions of donor- versus recipient-derived myeloid cells in non-hematopoietic tissues of allo-HSCT recipients, and 3) use murine infection models to evaluate the impact of allo-HSCT on host defense in transplanted B6-gp91phox -/- recipients.

Cell-free DNA reveals distinct pathology of multisystem inflammatory syndrome in children (MIS-C).

Multisystem inflammatory syndrome in children (MIS-C) is a rare but life-threatening hyperinflammatory condition induced by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes pediatric COVID-19 (pCOVID-19). The relationship of the systemic tissue injury to the pathophysiology of MIS-C is poorly defined. We leveraged the high sensitivity of epigenomic analyses of plasma cell-free DNA (cfDNA) and plasma cytokine measurements to identify the spectrum of tissue injury and glean mechanistic insights. Compared to pediatric healthy controls (pHC) and pCOVID-19, MIS-C patients had higher levels of cfDNA primarily derived from innate immune cells, megakaryocyte-erythroid precursor cells, and non-hematopoietic tissues such as hepatocytes, cardiac myocytes, and kidney cells. Non-hematopoietic tissue cfDNA levels demonstrated significant inter-individual variability, consistent with the heterogenous clinical presentation of MIS-C. In contrast, adaptive immune cell-derived cfDNA levels were comparable in MIS-C and pCOVID-19 patients. Indeed, the innate immune cells cfDNA in MIS-C correlated with levels of innate immune inflammatory cytokines and non-hematopoietic tissue-derived cfDNA, suggesting a primarily innate immunity-mediated response to account for multi-system pathology. These data provide insight into the pathogenesis of MIS-C and support the value of cfDNA as a sensitive biomarker to map tissue injury in MIS-C and likely other multi-organ inflammatory conditions.

Comparison of the IHC Markers CD138 and CD43 in Oral Leukoplakia: An Original Research.

In the clinical practice, one of the most common suspicious lesions that may be potentially malignant is oral leukoplakia. Globally, the rate at which it turns malignant varies. This study examines the levels of markers CD138 and 43 in oral leukoplakia. Twenty archival blocks of confirmed epithelial dysplasia were taken from the Department of Oral Pathology. These were processed for the identification of markers CD138 and 43 through Immuno Histo Chemistry (IHC). The blocks were divided equally for both the markers. There was a noticeable difference in staining intensity between dysplastic tissue and nondysplastic epithelium. However, CD138 expression was low or weak in dysplastic epithelium. CD43 expression was negative in all nonhematopoietic tissues. Genes that are cancer associated have been found to have incredibly different impacts in numerous tissues during the multistep process of oral carcinogenesis. In tissues undergoing dysplastic changes, CD138 expression was shown to be decreased, which could point out the malignant changes initiated in the epithelium of the oral tissues.

Clinical features and treatment of bone marrow metastasis.

Bone marrow metastasis (BMM) refers to the metastasis of malignant tumours originating from nonhematopoietic tissues to the bone marrow. The nonhematopoietic malignant tumour cells metastasize to the bone marrow via heterogeneous dissemination or direct invasion to form metastases and the bone marrow is infiltrated by tumour cells, resulting in the destruction of its structure and the development of haematopoietic disorders. In the present study, the clinical characteristics, prognosis and treatment of BMMs were investigated. The main clinical manifestations were moderate anaemia and thrombocytopenia. Out of 52 cases, a total of 18 patients were not treated and the remaining patients underwent chemotherapy, radiotherapy, surgery or autologous stem cell transplantation in the Affiliated Tumour Hospital of Tianjin Medical University from September 2010 to October 2021. The primary tumours of bone marrow metastatic cancer were usually neuroblastoma and tumours originating from the breast and stomach. When bone metastases occur, patients are not necessarily accompanied by BMMs. In the present study, bone metastases occurred mainly in patients with breast and prostate cancers. The median overall survival of patients treated with antitumor therapy was significantly higher than that of untreated patients (11.5 vs. 3.3 months P<0.01). For patients with BMM, it is of great importance to actively evaluate the patient's condition and select the appropriate treatment plan for improving their prognosis.

Differentially Expressed Genes Induced by Erythropoietin Receptor Overexpression in Rat Mammary Adenocarcinoma RAMA 37-28 Cells.

The erythropoietin receptor (EPOR) is a transmembrane type I receptor with an essential role in the proliferation and differentiation of erythroid progenitors. Besides its function during erythropoiesis, EPOR is expressed and has protective effect in various non-hematopoietic tissues, including tumors. Currently, the advantageous aspect of EPOR related to different cellular events is still under scientific investigation. Besides its well-known effect on cell proliferation, apoptosis and differentiation, our integrative functional study revealed its possible associations with metabolic processes, transport of small molecules, signal transduction and tumorigenesis. Comparative transcriptome analysis (RNA-seq) identified 233 differentially expressed genes (DEGs) in EPOR overexpressed RAMA 37-28 cells compared to parental RAMA 37 cells, whereas 145 genes were downregulated and 88 upregulated. Of these, for example, GPC4, RAP2C, STK26, ZFP955A, KIT, GAS6, PTPRF and CXCR4 were downregulated and CDH13, NR0B1, OCM2, GPM6B, TM7SF3, PARVB, VEGFD and STAT5A were upregulated. Surprisingly, two ephrin receptors, EPHA4 and EPHB3, and EFNB1 ligand were found to be upregulated as well. Our study is the first demonstrating robust differentially expressed genes evoked by simple EPOR overexpression without the addition of erythropoietin ligand in a manner which remains to be elucidated.

The ESCRT protein CHMP5 controls skeletal muscle homeostasis and coordinates myeloid cell-mediated tissue repair

Abstract Macrophages promote skeletal muscle homeostasis by participating in crosstalk with satellite cells, fibroadipogenic precursors, and myofibers that drives regeneration after injury. Dysregulation of this crosstalk contributes to conditions like myopathy, inefficient wound healing, and sarcopenia. Despite their central role in muscle homeostasis and regeneration, the tissue-intrinsic mechanisms that coordinate the recruitment and activity of these macrophages are poorly understood. In this study, we investigated how skeletal muscle homeostasis is regulated by the ESCRT protein CHMP5. CHMP5 was initially characterized as a member of the ESCRT family of proteins that coordinate membrane scission events in eukaryotic cells. However, recent studies by us and others have revealed non-canonical roles for CHMP5 wherein CHMP5 promotes the stability of client proteins required for cellular differentiation and cell fate decisions in hematopoietic and non-hematopoietic tissues. Using an in vitro model of myogenesis, we found that CHMP5 knockdown impaired the differentiation of C2C12 myoblasts into myotubes. Furthermore, when wild-type BMDMs were cocultured with CHMP5-knockdown C2C12 myoblasts, they failed to polarize into M2-like macrophages. In vivo, mice with muscle-specific CHMP5 heterozygous deletion (CHMP5 hetmice) displayed diminished myeloid cell presence in muscle following cardiotoxin-induced injury. 12 days post-injury, histologic and flow cytometric analyses of muscles from CHMP5 hetmice revealed less regenerative progress than littermate controls. These data suggest a critical function for CHMP5 in promoting muscle homeostasis both in the steady state and in the context of injury and inflammation. Supported in part by intramural funding from the National Cancer Institute, T32 GM007250, T32 NS077888-08, and NIH R01AI143992-03.

Pigment cell progenitor heterogeneity and reiteration of developmental signaling underlie melanocyte regeneration in zebrafish.

Tissue-resident stem and progenitor cells are present in many adult organs, where they are important for organ homeostasis and repair in response to injury. However, the signals that activate these cells and the mechanisms governing how these cells renew or differentiate are highly context-dependent and incompletely understood, particularly in non-hematopoietic tissues. In the skin, melanocyte stem and progenitor cells are responsible for replenishing mature pigmented melanocytes. In mammals, these cells reside in the hair follicle bulge and bulb niches where they are activated during homeostatic hair follicle turnover and following melanocyte destruction, as occurs in vitiligo and other skin hypopigmentation disorders. Recently, we identified melanocyte progenitors in adult zebrafish skin. To elucidate mechanisms governing melanocyte progenitor renewal and differentiation we analyzed individual transcriptomes from thousands of melanocyte lineage cells during the regeneration process. We identified transcriptional signatures for progenitors, deciphered transcriptional changes and intermediate cell states during regeneration, and analyzed cell-cell signaling changes to discover mechanisms governing melanocyte regeneration. We identified KIT signaling via the RAS/MAPK pathway as a regulator of melanocyte progenitor direct differentiation and asymmetric division. Our findings show how activation of different subpopulations of mitfa-positive cells underlies cellular transitions required to properly reconstitute the melanocyte pigmentary system following injury.