Current Concepts and Advances in Graft-Versus-Host Disease Immunology.

Worldwide, each year over 30,000 patients undergo an allogeneic hema-topoietic stem cell transplantation with the intent to cure high-risk hematologic malignancy, immunodeficiency, metabolic disease, or a life-threatening bone marrow failure syndrome. Despite substantial advances in donor selection and conditioning regimens and greater availability of allograft sources, transplant recipients still endure the morbidity and mortality of graft-versus-host disease (GVHD). Herein, we identify key aspects of acute and chronic GVHD pathophysiology, including host/donor cell effectors, gut dysbiosis, immune system and cytokine imbalance, and the interface between inflammation and tissue fibrosis. In particular, we also summarize the translational application of this heightened understanding of immune dysregulation in the design of novel therapies to prevent and treat GVHD.

Medication effects on the gut microbiome in allo-HCT.

Decreased gut microbiome diversity has been associated with negative outcome in allogeneic hematopoietic stem cell transfer (HCT). A study published in this issue of Cell identifies associations between non-antibiotic drug administration, microbiome state transitions, and response to HCT, highlighting the potential impact of such drugs on microbiome and HCT outcome.

HIV-1 remission and possible cure in a woman after haplo-cord blood transplant.

Previously, two men were cured of HIV-1 through CCR5Δ32 homozygous (CCR5Δ32/Δ32) allogeneic adult stem cell transplant. We report the first remission and possible HIV-1 cure in a mixed-race woman who received a CCR5Δ32/Δ32 haplo-cord transplant (cord blood cells combined with haploidentical stem cells from an adult) to treat acute myeloid leukemia (AML). Peripheral blood chimerism was 100% CCR5Δ32/Δ32 cord blood by week 14 post-transplant and persisted through 4.8 years of follow-up. Immune reconstitution was associated with (1) loss of detectable replication-competent HIV-1 reservoirs, (2) loss of HIV-1-specific immune responses, (3) in vitro resistance to X4 and R5 laboratory variants, including pre-transplant autologous latent reservoir isolates, and (4) 18 months of HIV-1 control with aviremia, off antiretroviral therapy, starting at 37 months post-transplant. CCR5Δ32/Δ32 haplo-cord transplant achieved remission and a possible HIV-1 cure for a person of diverse ancestry, living with HIV-1, who required a stem cell transplant for acute leukemia.

High-resolution analyses of associations between medications, microbiome, and mortality in cancer patients.

Discerning the effect of pharmacological exposures on intestinal bacterial communities in cancer patients is challenging. Here, we deconvoluted the relationship between drug exposures and changes in microbial composition by developing and applying a new computational method, PARADIGM (parameters associated with dynamics of gut microbiota), to a large set of longitudinal fecal microbiome profiles with detailed medication-administration records from patients undergoing allogeneic hematopoietic cell transplantation. We observed that several non-antibiotic drugs, including laxatives, antiemetics, and opioids, are associated with increased Enterococcus relative abundance and decreased alpha diversity. Shotgun metagenomic sequencing further demonstrated subspecies competition, leading to increased dominant-strain genetic convergence during allo-HCT that is significantly associated with antibiotic exposures. We integrated drug-microbiome associations to predict clinical outcomes in two validation cohorts on the basis of drug exposures alone, suggesting that this approach can generate biologically and clinically relevant insights into how pharmacological exposures can perturb or preserve microbiota composition. The application of a computational method called PARADIGM to a large dataset of cancer patients' longitudinal fecal specimens and detailed daily medication records reveals associations between drug exposures and the intestinal microbiota that recapitulate in vitro findings and are also predictive of clinical outcomes.

Mobilization-based chemotherapy-free engraftment of gene-edited human hematopoietic stem cells.

Hematopoietic stem/progenitor cell gene therapy (HSPC-GT) is proving successful to treat several genetic diseases. HSPCs are mobilized, harvested, genetically corrected ex vivo, and infused, after the administration of toxic myeloablative conditioning to deplete the bone marrow (BM) for the modified cells. We show that mobilizers create an opportunity for seamless engraftment of exogenous cells, which effectively outcompete those mobilized, to repopulate the depleted BM. The competitive advantage results from the rescue during ex vivo culture of a detrimental impact of mobilization on HSPCs and can be further enhanced by the transient overexpression of engraftment effectors exploiting optimized mRNA-based delivery. We show the therapeutic efficacy in a mouse model of hyper IgM syndrome and further developed it in human hematochimeric mice, showing its applicability and versatility when coupled with gene transfer and editing strategies. Overall, our findings provide a potentially valuable strategy paving the way to broader and safer use of HSPC-GT.

Mucus-degrading Bacteroides link carbapenems to aggravated graft-versus-host disease.

The intestinal microbiota is an important modulator of graft-versus-host disease (GVHD), which often complicates allogeneic hematopoietic stem cell transplantation (allo-HSCT). Broad-spectrum antibiotics such as carbapenems increase the risk for intestinal GVHD, but mechanisms are not well understood. In this study, we found that treatment with meropenem, a commonly used carbapenem, aggravates colonic GVHD in mice via the expansion of Bacteroides thetaiotaomicron (BT). BT has a broad ability to degrade dietary polysaccharides and host mucin glycans. BT in meropenem-treated allogeneic mice demonstrated upregulated expression of enzymes involved in the degradation of mucin glycans. These mice also had thinning of the colonic mucus layer and decreased levels of xylose in colonic luminal contents. Interestingly, oral xylose supplementation significantly prevented thinning of the colonic mucus layer in meropenem-treated mice. Specific nutritional supplementation strategies, including xylose supplementation, may combat antibiotic-mediated microbiome injury to reduce the risk for intestinal GVHD in allo-HSCT patients.

Human hemato-lymphoid system mice: current use and future potential for medicine.

To directly study complex human hemato-lymphoid system physiology and respective system-associated diseases in vivo, human-to-mouse xenotransplantation models for human blood and blood-forming cells and organs have been developed over the past three decades. We here review the fundamental requirements and the remarkable progress made over the past few years in improving these systems, the current major achievements reached by use of these models, and the future challenges to more closely model and study human health and disease and to achieve predictive preclinical testing of both prevention measures and potential new therapies.

Mesenchymal stem cell: keystone of the hematopoietic stem cell niche and a stepping-stone for regenerative medicine.

Mesenchymal stem cells (MSCs) are self-renewing precursor cells that can differentiate into bone, fat, cartilage, and stromal cells of the bone marrow. Recent studies suggest that MSCs themselves are critical for forming a niche that maintains hematopoietic stem cells (HSCs). The ease by which human MSC-like and stromal progenitor cells can be isolated from the bone marrow and other tissues has led to the rapid development of clinical investigations exploring their anti-inflammatory properties, tissue preservation capabilities, and regenerative potential. However, the identity of genuine MSCs and their specific contributions to these various beneficial effects have remained enigmatic. In this article, we examine the definition of MSCs and discuss the importance of rigorously characterizing their stem cell activity. We review their role and that of other putative niche constituents in the regulation of bone marrow HSCs. Additionally, how MSCs and their stromal progeny alter immune function is discussed, as well as potential therapeutic implications.

A latent subset of human hematopoietic stem cells resists regenerative stress to preserve stemness.

Continuous supply of immune cells throughout life relies on the delicate balance in the hematopoietic stem cell (HSC) pool between long-term maintenance and meeting the demands of both normal blood production and unexpected stress conditions. Here we identified distinct subsets of human long-term (LT)-HSCs that responded differently to regeneration-mediated stress: an immune checkpoint ligand CD112lo subset that exhibited a transient engraftment restraint (termed latency) before contributing to hematopoietic reconstitution and a primed CD112hi subset that responded rapidly. This functional heterogeneity and CD112 expression are regulated by INKA1 through direct interaction with PAK4 and SIRT1, inducing epigenetic changes and defining an alternative state of LT-HSC quiescence that serves to preserve self-renewal and regenerative capacity upon regeneration-mediated stress. Collectively, our data uncovered the molecular intricacies underlying HSC heterogeneity and self-renewal regulation and point to latency as an orchestrated physiological response that balances blood cell demands with preserving a stem cell reservoir.

Microbiota dictate T cell clonal selection to augment graft-versus-host disease after stem cell transplantation.

Allogeneic T cell expansion is the primary determinant of graft-versus-host disease (GVHD), and current dogma dictates that this is driven by histocompatibility antigen disparities between donor and recipient. This paradigm represents a closed genetic system within which donor T cells interact with peptide-major histocompatibility complexes (MHCs), though clonal interrogation remains challenging due to the sparseness of the T cell repertoire. We developed a Bayesian model using donor and recipient T cell receptor (TCR) frequencies in murine stem cell transplant systems to define limited common expansion of T cell clones across genetically identical donor-recipient pairs. A subset of donor CD4+ T cell clonotypes differentially expanded in identical recipients and were microbiota dependent. Microbiota-specific T cells augmented GVHD lethality and could target microbial antigens presented by gastrointestinal epithelium during an alloreactive response. The microbiota serves as a source of cognate antigens that contribute to clonotypic T cell expansion and the induction of GVHD independent of donor-recipient genetics.

Integrated Single-Cell Analysis Maps the Continuous Regulatory Landscape of Human Hematopoietic Differentiation.

Human hematopoiesis involves cellular differentiation of multipotent cells into progressively more lineage-restricted states. While the chromatin accessibility landscape of this process has been explored in defined populations, single-cell regulatory variation has been hidden by ensemble averaging. We collected single-cell chromatin accessibility profiles across 10 populations of immunophenotypically defined human hematopoietic cell types and constructed a chromatin accessibility landscape of human hematopoiesis to characterize differentiation trajectories. We find variation consistent with lineage bias toward different developmental branches in multipotent cell types. We observe heterogeneity within common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) and develop a strategy to partition GMPs along their differentiation trajectory. Furthermore, we integrated single-cell RNA sequencing (scRNA-seq) data to associate transcription factors to chromatin accessibility changes and regulatory elements to target genes through correlations of expression and regulatory element accessibility. Overall, this work provides a framework for integrative exploration of complex regulatory dynamics in a primary human tissue at single-cell resolution.

Rapid Mobilization Reveals a Highly Engraftable Hematopoietic Stem Cell.

Hematopoietic stem cell transplantation is a potential curative therapy for malignant and nonmalignant diseases. Improving the efficiency of stem cell collection and the quality of the cells acquired can broaden the donor pool and improve patient outcomes. We developed a rapid stem cell mobilization regimen utilizing a unique CXCR2 agonist, GROß, and the CXCR4 antagonist AMD3100. A single injection of both agents resulted in stem cell mobilization peaking within 15 min that was equivalent in magnitude to a standard multi-day regimen of granulocyte colony-stimulating factor (G-CSF). Mechanistic studies determined that rapid mobilization results from synergistic signaling on neutrophils, resulting in enhanced MMP-9 release, and unexpectedly revealed genetic polymorphisms in MMP-9 that alter activity. This mobilization regimen results in preferential trafficking of stem cells that demonstrate a higher engraftment efficiency than those mobilized by G-CSF. Our studies suggest a potential new strategy for the rapid collection of an improved hematopoietic graft.

EBF1-deficient bone marrow stroma elicits persistent changes in HSC potential.

Crosstalk between mesenchymal stromal cells (MSCs) and hematopoietic stem cells (HSCs) is essential for hematopoietic homeostasis and lineage output. Here, we investigate how transcriptional changes in bone marrow (BM) MSCs result in long-lasting effects on HSCs. Single-cell analysis of Cxcl12-abundant reticular (CAR) cells and PDGFRα+Sca1+ (PαS) cells revealed an extensive cellular heterogeneity but uniform expression of the transcription factor gene Ebf1. Conditional deletion of Ebf1 in these MSCs altered their cellular composition, chromatin structure and gene expression profiles, including the reduced expression of adhesion-related genes. Functionally, the stromal-specific Ebf1 inactivation results in impaired adhesion of HSCs, leading to reduced quiescence and diminished myeloid output. Most notably, HSCs residing in the Ebf1-deficient niche underwent changes in their cellular composition and chromatin structure that persist in serial transplantations. Thus, genetic alterations in the BM niche lead to long-term functional changes of HSCs.

Graft-versus-host disease is locally maintained in target tissues by resident progenitor-like T cells.

In allogeneic hematopoietic stem cell transplantation, donor αß T cells attack recipient tissues, causing graft-versus-host disease (GVHD), a major cause of morbidity and mortality. A central question has been how GVHD is sustained despite T cell exhaustion from chronic antigen stimulation. The current model for GVHD holds that disease is maintained through the continued recruitment of alloreactive effectors from blood into affected tissues. Here, we show, using multiple approaches including parabiosis of mice with GVHD, that GVHD is instead primarily maintained locally within diseased tissues. By tracking 1,203 alloreactive T cell clones, we fitted a mathematical model predicting that within each tissue a small number of progenitor T cells maintain a larger effector pool. Consistent with this, we identified a tissue-resident TCF-1+ subpopulation that preferentially engrafted, expanded, and differentiated into effectors upon adoptive transfer. These results suggest that therapies targeting affected tissues and progenitor T cells within them would be effective.

Intestinal microbiota controls graft-versus-host disease independent of donor-host genetic disparity.

Acute graft-versus-host disease (aGVHD) remains a major limitation of allogeneic stem cell transplantation (SCT), and severe intestinal manifestation is the major cause of early mortality. Intestinal microbiota control MHC class II (MHC-II) expression by ileal intestinal epithelial cells (IECs) that promote GVHD. Here, we demonstrated that genetically identical mice of differing vendor origins had markedly different intestinal microbiota and ileal MHC-II expression, resulting in discordant GVHD severity. We utilized cohousing and antibiotic treatment to characterize the bacterial taxa positively and negatively associated with MHC-II expression. A large proportion of bacterial MHC-II inducers were vancomycin sensitive, and peri-transplant oral vancomycin administration attenuated CD4+ T cell-mediated GVHD. We identified a similar relationship between pre-transplant microbes, HLA class II expression, and both GVHD and mortality in a large clinical SCT cohort. These data highlight therapeutically tractable mechanisms by which pre-transplant microbial taxa contribute to GVHD independently of genetic disparity.

Allogeneic immunity clears latent virus following allogeneic stem cell transplantation in SIV-infected ART-suppressed macaques.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) from donors lacking C-C chemokine receptor 5 (CCR5Δ32/Δ32) can cure HIV, yet mechanisms remain speculative. To define how alloHSCT mediates HIV cure, we performed MHC-matched alloHSCT in SIV+, anti-retroviral therapy (ART)-suppressed Mauritian cynomolgus macaques (MCMs) and demonstrated that allogeneic immunity was the major driver of reservoir clearance, occurring first in peripheral blood, then peripheral lymph nodes, and finally in mesenteric lymph nodes draining the gastrointestinal tract. While allogeneic immunity could extirpate the latent viral reservoir and did so in two alloHSCT-recipient MCMs that remained aviremic >2.5 years after stopping ART, in other cases, it was insufficient without protection of engrafting cells afforded by CCR5-deficiency, as CCR5-tropic virus spread to donor CD4+ T cells despite full ART suppression. These data demonstrate the individual contributions of allogeneic immunity and CCR5 deficiency to HIV cure and support defining targets of alloimmunity for curative strategies independent of HSCT.

Ambient oxygen levels regulate intestinal dysbiosis and GVHD severity after allogeneic stem cell transplantation.

The severity of T cell-mediated gastrointestinal (GI) diseases such as graft-versus-host disease (GVHD) and inflammatory bowel diseases correlates with a decrease in the diversity of the host gut microbiome composition characterized by loss of obligate anaerobic commensals. The mechanisms underpinning these changes in the microbial structure remain unknown. Here, we show in multiple specific pathogen-free (SPF), gnotobiotic, and germ-free murine models of GI GVHD that the initiation of the intestinal damage by the pathogenic T cells altered ambient oxygen levels in the GI tract and caused dysbiosis. The change in oxygen levels contributed to the severity of intestinal pathology in a host intestinal HIF-1α- and a microbiome-dependent manner. Regulation of intestinal ambient oxygen levels with oral iron chelation mitigated dysbiosis and reduced the severity of the GI GVHD. Thus, targeting ambient intestinal oxygen levels may represent a novel, non-immunosuppressive strategy to mitigate T cell-driven intestinal diseases.

Single-cell analysis reveals the continuum of human lympho-myeloid progenitor cells.

The hierarchy of human hemopoietic progenitor cells that produce lymphoid and granulocytic-monocytic (myeloid) lineages is unclear. Multiple progenitor populations produce lymphoid and myeloid cells, but they remain incompletely characterized. Here we demonstrated that lympho-myeloid progenitor populations in cord blood - lymphoid-primed multi-potential progenitors (LMPPs), granulocyte-macrophage progenitors (GMPs) and multi-lymphoid progenitors (MLPs) - were functionally and transcriptionally distinct and heterogeneous at the clonal level, with progenitors of many different functional potentials present. Although most progenitors had the potential to develop into only one mature cell type ('uni-lineage potential'), bi- and rarer multi-lineage progenitors were present among LMPPs, GMPs and MLPs. Those findings, coupled with single-cell expression analyses, suggest that a continuum of progenitors execute lymphoid and myeloid differentiation, rather than only uni-lineage progenitors' being present downstream of stem cells.

Enhancing Hematopoietic Stem Cell Transplantation Efficacy by Mitigating Oxygen Shock.

Hematopoietic stem cells (HSCs) reside in hypoxic niches within bone marrow and cord blood. Yet, essentially all HSC studies have been performed with cells isolated and processed in non-physiologic ambient air. By collecting and manipulating bone marrow and cord blood in native conditions of hypoxia, we demonstrate that brief exposure to ambient oxygen decreases recovery of long-term repopulating HSCs and increases progenitor cells, a phenomenon we term extraphysiologic oxygen shock/stress (EPHOSS). Thus, true numbers of HSCs in the bone marrow and cord blood are routinely underestimated. We linked ROS production and induction of the mitochondrial permeability transition pore (MPTP) via cyclophilin D and p53 as mechanisms of EPHOSS. The MPTP inhibitor cyclosporin A protects mouse bone marrow and human cord blood HSCs from EPHOSS during collection in air, resulting in increased recovery of transplantable HSCs. Mitigating EPHOSS during cell collection and processing by pharmacological means may be clinically advantageous for transplantation.

Selective haematological cancer eradication with preserved haematopoiesis.

Haematopoietic stem cell (HSC) transplantation (HSCT) is the only curative treatment for a broad range of haematological malignancies, but the standard of care relies on untargeted chemotherapies and limited possibilities to treat malignant cells after HSCT without affecting the transplanted healthy cells1. Antigen-specific cell-depleting therapies hold the promise of much more targeted elimination of diseased cells, as witnessed in the past decade by the revolution of clinical practice for B cell malignancies2. However, target selection is complex and limited to antigens expressed on subsets of haematopoietic cells, resulting in a fragmented therapy landscape with high development costs2-5. Here we demonstrate that an antibody-drug conjugate (ADC) targeting the pan-haematopoietic marker CD45 enables the antigen-specific depletion of the entire haematopoietic system, including HSCs. Pairing this ADC with the transplantation of human HSCs engineered to be shielded from the CD45-targeting ADC enables the selective eradication of leukaemic cells with preserved haematopoiesis. The combination of CD45-targeting ADCs and engineered HSCs creates an almost universal strategy to replace a diseased haematopoietic system, irrespective of disease aetiology or originating cell type. We propose that this approach could have broad implications beyond haematological malignancies.