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.

Allogeneic CD19-targeted CAR-T therapy in patients with severe myositis and systemic sclerosis.

Allogeneic chimeric antigen receptor (CAR)-T cells hold great promise for expanding the accessibility of CAR-T therapy, whereas the risks of allograft rejection have hampered its application. Here, we genetically engineered healthy-donor-derived, CD19-targeting CAR-T cells using CRISPR-Cas9 to address the issue of immune rejection and treated one patient with refractory immune-mediated necrotizing myopathy and two patients with diffuse cutaneous systemic sclerosis with these cells. This study was registered at ClinicalTrials.gov (NCT05859997). The infused cells persisted for over 3 months, achieving complete B cell depletion within 2 weeks of treatment. During the 6-month follow-up, we observed deep remission without cytokine release syndrome or other serious adverse events in all three patients, primarily shown by the significant improvement in the clinical response index scores for the two diseases, respectively, and supported by the observations of reversal of inflammation and fibrosis. Our results demonstrate the high safety and promising immune modulatory effect of the off-the-shelf CAR-T cells in treating severe refractory autoimmune diseases.

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.

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.

An enterococcal phage-derived enzyme suppresses graft-versus-host disease.

Changes in the gut microbiome have pivotal roles in the pathogenesis of acute graft-versus-host disease (aGVHD) after allogenic haematopoietic cell transplantation (allo-HCT)1-6. However, effective methods for safely resolving gut dysbiosis have not yet been established. An expansion of the pathogen Enterococcus faecalis in the intestine, associated with dysbiosis, has been shown to be a risk factor for aGVHD7-10. Here we analyse the intestinal microbiome of patients with allo-HCT, and find that E. faecalis escapes elimination and proliferates in the intestine by forming biofilms, rather than by acquiring drug-resistance genes. We isolated cytolysin-positive highly pathogenic E. faecalis from faecal samples and identified an anti-E. faecalis enzyme derived from E. faecalis-specific bacteriophages by analysing bacterial whole-genome sequencing data. The antibacterial enzyme had lytic activity against the biofilm of E. faecalis in vitro and in vivo. Furthermore, in aGVHD-induced gnotobiotic mice that were colonized with E. faecalis or with patient faecal samples characterized by the domination of Enterococcus, levels of intestinal cytolysin-positive E. faecalis were decreased and survival was significantly increased in the group that was treated with the E. faecalis-specific enzyme, compared with controls. Thus, administration of a phage-derived antibacterial enzyme that is specific to biofilm-forming pathogenic E. faecalis-which is difficult to eliminate with existing antibiotics-might provide an approach to protect against aGVHD.

Human γδ T cells are quickly reconstituted after stem-cell transplantation and show adaptive clonal expansion in response to viral infection.

To investigate how the human γδ T cell pool is shaped during ontogeny and how it is regenerated after transplantation of hematopoietic stem cells (HSCs), we applied an RNA-based next-generation sequencing approach to monitor the dynamics of the repertoires of γδ T cell antigen receptors (TCRs) before and after transplantation in a prospective cohort study. We found that repertoires of rearranged genes encoding γδ TCRs (TRG and TRD) in the peripheral blood of healthy adults were stable over time. Although a large fraction of human TRG repertoires consisted of public sequences, the TRD repertoires were private. In patients undergoing HSC transplantation, γδ T cells were quickly reconstituted; however, they had profoundly altered TCR repertoires. Notably, the clonal proliferation of individual virus-reactive γδ TCR sequences in patients with reactivation of cytomegalovirus revealed strong evidence for adaptive anti-viral γδ T cell immune responses.

Gut microbiome-derived metabolites modulate intestinal epithelial cell damage and mitigate graft-versus-host disease.

The effect of alterations in intestinal microbiota on microbial metabolites and on disease processes such as graft-versus-host disease (GVHD) is not known. Here we carried out an unbiased analysis to identify previously unidentified alterations in gastrointestinal microbiota-derived short-chain fatty acids (SCFAs) after allogeneic bone marrow transplant (allo-BMT). Alterations in the amount of only one SCFA, butyrate, were observed only in the intestinal tissue. The reduced butyrate in CD326(+) intestinal epithelial cells (IECs) after allo-BMT resulted in decreased histone acetylation, which was restored after local administration of exogenous butyrate. Butyrate restoration improved IEC junctional integrity, decreased apoptosis and mitigated GVHD. Furthermore, alteration of the indigenous microbiota with 17 rationally selected strains of high butyrate-producing Clostridia also decreased GVHD. These data demonstrate a heretofore unrecognized role of microbial metabolites and suggest that local and specific alteration of microbial metabolites has direct salutary effects on GVHD target tissues and can mitigate disease severity.

T Cell Recruitment to the Intestinal Stem Cell Compartment Drives Immune-Mediated Intestinal Damage after Allogeneic Transplantation.

The key sites within the gastrointestinal (GI) tract where T cells mediate effector responses and the impact of these responses on intestinal stem cells (ISCs) remain unclear. Using experimental bone marrow transplantation to model immune-mediated GI damage and 3D imaging to analyze T cell localization, we found that the ISC compartment is the primary intestinal site targeted by T cells after transplantation. Recruitment to the crypt base region resulted in direct T cell engagement with the stem cell compartment and loss of crypt base columnar ISCs, which expressed both MHC classes I and II. Vasculature expressing the adhesion molecule MAdCAM-1 clustered near the crypt base, preferentially regulating crypt compartment invasion and ISC reduction without affecting T cell migration to villi. These findings indicate that allogeneic T cells rapidly access the stem cell niche after transplantation, and this targeted recruitment to the stem cell compartment results in ISC loss during immune-mediated GI damage.

Sequential CD7 CAR T-Cell Therapy and Allogeneic HSCT without GVHD Prophylaxis.

BACKGROUND: Patients with relapsed or refractory hematologic cancers have a poor prognosis. Chimeric antigen receptor (CAR) T-cell therapy as a bridge to allogeneic hematopoietic stem-cell transplantation (HSCT) has the potential for long-term tumor elimination. However, pre-HSCT myeloablation and graft-versus-host disease (GVHD) prophylaxis agents have toxic effects and could eradicate residual CAR T cells and compromise antitumor effects. Whether the integration of CAR T-cell therapy and allogeneic HSCT can preserve CAR T-cell function and improve tumor control is unclear. METHODS: We tested a novel "all-in-one" strategy consisting of sequential CD7 CAR T-cell therapy and haploidentical HSCT in 10 patients with relapsed or refractory CD7-positive leukemia or lymphoma. After CAR T-cell therapy led to complete remission with incomplete hematologic recovery, patients received haploidentical HSCT without pharmacologic myeloablation or GVHD prophylaxis drugs. Toxic effects and efficacy were closely monitored. RESULTS: After CAR T-cell therapy, all 10 patients had complete remission with incomplete hematologic recovery and grade 4 pancytopenia. After haploidentical HSCT, 1 patient died on day 13 of septic shock and encephalitis, 8 patients had full donor chimerism, and 1 patient had autologous hematopoiesis. Three patients had grade 2 HSCT-associated acute GVHD. The median follow-up was 15.1 months (range, 3.1 to 24.0) after CAR T-cell therapy. Six patients remained in minimal residual disease-negative complete remission, 2 had a relapse of CD7-negative leukemia, and 1 died of septic shock at 3.7 months. The estimated 1-year overall survival was 68% (95% confidence interval [CI], 43 to 100), and the estimated 1-year disease-free survival was 54% (95% CI, 29 to 100). CONCLUSIONS: Our findings suggest that sequential CD7 CAR T-cell therapy and haploidentical HSCT is safe and effective, with remission and serious but reversible adverse events. This strategy offers a feasible approach for patients with CD7-positive tumors who are ineligible for conventional allogeneic HSCT. (Funded by the National Natural Science Foundation of China and the Key Project of Science and Technology Department of Zhejiang Province; ClinicalTrials.gov numbers, NCT04599556 and NCT04538599.).

High-Dose ERT, Rituximab, and Early HSCT in an Infant with Wolman's Disease.

Wolman's disease, a severe form of lysosomal acid lipase deficiency, leads to pathologic lipid accumulation in the liver and gut that, without treatment, is fatal in infancy. Although continued enzyme-replacement therapy (ERT) in combination with dietary fat restriction prolongs life, its therapeutic effect may wane over time. Allogeneic hematopoietic stem-cell transplantation (HSCT) offers a more definitive solution but carries a high risk of death. Here we describe an infant with Wolman's disease who received high-dose ERT, together with dietary fat restriction and rituximab-based B-cell depletion, as a bridge to early HSCT. At 32 months, the infant was independent of ERT and disease-free, with 100% donor chimerism in the peripheral blood.

Lipocalin-2: a novel potential therapy for GVHD.

Czech et al. used mouse models of allogeneic hematopoietic stem cell transplantation (allo-HCT) to investigate the role of lipocalin-2 (LCN2) as a newfound regulator of intestinal graft-versus-host disease (GVHD). Administration of recombinant LCN2 protein after disease onset prevented GVHD progression, suggesting that it may play a role in reversing tissue damage that has already begun.

Transfer of Cell-Surface Antigens by Scavenger Receptor CD36 Promotes Thymic Regulatory T Cell Receptor Repertoire Development and Allo-tolerance.

The development of T cell tolerance in the thymus requires the presentation of host proteins by multiple antigen-presenting cell (APC) types. However, the importance of transferring host antigens from transcription factor AIRE-dependent medullary thymic epithelial cells (mTECs) to bone marrow (BM) APCs is unknown. We report that antigen was primarily transferred from mTECs to CD8α+ dendritic cells (DCs) and showed that CD36, a scavenger receptor selectively expressed on CD8α+ DCs, mediated the transfer of cell-surface, but not cytoplasmic, antigens. The absence of CD8α+ DCs or CD36 altered thymic T cell selection, as evidenced by TCR repertoire analysis and the loss of allo-tolerance in murine allogeneic BM transplantation (allo-BMT) studies. Decreases in these DCs and CD36 expression in peripheral blood of human allo-BMT patients correlated with graft-versus-host disease. Our findings suggest that CD36 facilitates transfer of mTEC-derived cell-surface antigen on CD8α+ DCs to promote tolerance to host antigens during homeostasis and allo-BMT.

Chronic graft-versus-host disease: unresolved complication or ancient history?

ABSTRACT: Chronic graft-versus-host disease (cGVHD) is associated with morbidity, mortality, impaired quality of life, prolonged immunosuppressive therapy, and infection risk after allogeneic hematopoietic cell transplantation (HCT). Major strides have occurred in the understanding of cGVHD biology; National Institutes of Health Consensus meetings have refined rigorous approaches to diagnosis, staging, and response criteria; major interventional trials have established standard benchmarks for treatment outcome; and 3 agents to date have been US Food and Drug Administration approved for treating corticosteroid-refractory cGVHD. Promising results from several recent trials have led some, but not others, to conclude that the risk of developing cGVHD is sufficiently low to be considered a major post-HCT complication of the past. We propose that it is time to critically examine the results of contemporary graft-versus-host disease (GVHD) prophylaxis regimens and discuss the state of the science and associated controversies in the spectrum of conclusions reached as to the risk of cGVHD. With these data, the current cGVHD incidence can be most precisely determined, and the present and future burden of cGVHD-affected patients can be accurately modeled. Through review of existing evidence, we highlight unresolved needs and opportunities to refine best GVHD prophylaxis or preemptive therapy approaches and optimize established cGVHD therapy, and make the argument that support of preclinical and clinical research is critical in improving patient outcomes.

Management of adult patients with CMML undergoing allo-HCT: recommendations from the EBMT PH&G Committee.

ABSTRACT: Chronic myelomonocytic leukemia (CMML) is a heterogeneous disease presenting with either myeloproliferative or myelodysplastic features. Allogeneic hematopoietic cell transplantation (allo-HCT) remains the only potentially curative option, but the inherent toxicity of this procedure makes the decision to proceed to allo-HCT challenging, particularly because patients with CMML are mostly older and comorbid. Therefore, the decision between a nonintensive treatment approach and allo-HCT represents a delicate balance, especially because prospective randomized studies are lacking and retrospective data in the literature are conflicting. International consensus on the selection of patients and the ideal timing of allo-HCT, specifically in CMML, could not be reached in international recommendations published 6 years ago. Since then, new, CMML-specific data have been published. The European Society for Blood and Marrow Transplantation (EBMT) Practice Harmonization and Guidelines (PH&G) Committee assembled a panel of experts in the field to provide the first best practice recommendations on the role of allo-HCT specifically in CMML. Recommendations were based on the results of an international survey, a comprehensive review of the literature, and expert opinions on the subject, after structured discussion and circulation of recommendations. Algorithms for patient selection, timing of allo-HCT during the course of the disease, pretransplant strategies, allo-HCT modality, as well as posttransplant management for patients with CMML were outlined. The keynote message is, that once a patient has been identified as a transplant candidate, upfront transplantation without prior disease-modifying treatment is preferred to maximize chances of reaching allo-HCT whenever possible, irrespective of bone marrow blast counts.

Hope for motherhood: pregnancy after allogeneic hematopoietic cell transplantation (a national multicenter study).

ABSTRACT: Improved long-term survival rates after allogeneic hematopoietic cell transplantation (alloHCT) make family planning for young adult cancer survivors an important topic. However, treatment-related infertility risk poses challenges. To assess pregnancy and birth rates in a contemporary cohort, we conducted a national multicenter study using data from the German Transplant Registry, focusing on adult women aged 18 to 40 years who underwent alloHCT between 2003 and 2018. Of 2654 women who underwent transplantation, 50 women experienced 74 pregnancies, occurring at a median of 4.7 years after transplant. Fifty-seven of these resulted in live births (77%). The annual first birth rate among HCT recipients was 0.45%, which is >6 times lower than in the general population. The probability of a live birth 10 years after HCT was 3.4%. Factors associated with an increased likelihood of pregnancy were younger age at alloHCT, nonmalignant transplant indications, no total body irradiation or a cumulative dose of <8 Gy, and nonmyeloablative/reduced-intensity conditioning. Notably, 72% of pregnancies occurred spontaneously, with assisted reproductive technologies used in the remaining cases. Preterm delivery and low birth weight were more common than in the general population. This study represents the largest data set reporting pregnancies in a cohort of adult female alloHCT recipients. Our findings underscore a meaningful chance of pregnancy in alloHCT recipients. Assisted reproductive technologies techniques are important and funding should be made available. However, the potential for spontaneous pregnancies should not be underestimated, and patients should be informed of the possibility of unexpected pregnancy despite reduced fertility. Further research is warranted to understand the impact of conditioning decisions on fertility preservation.

Tricking the trickster: precision medicine approaches to counteract leukemia immune escape after transplant.

ABSTRACT: Over the last decades, significant improvements in reducing the toxicities of allogeneic hematopoietic cell transplantation (allo-HCT) have widened its use as consolidation or salvage therapy for high-risk hematological malignancies. Nevertheless, relapse of the original malignant disease remains an open issue with unsatisfactory salvage options and limited rationales to select among them. In the last years, several studies have highlighted that relapse is often associated with specific genomic and nongenomic mechanisms of immune escape. In this review we summarize the current knowledge about these modalities of immune evasion, focusing on the mechanisms that leverage antigen presentation and pathologic rewiring of the bone marrow microenvironment. We present examples of how this biologic information can be translated into specific approaches to treat relapse, discuss the status of the clinical trials for patients who relapsed after a transplant, and show how dissecting the complex immunobiology of allo-HCT represents a crucial step toward developing new personalized approaches to improve clinical outcomes.

A phase 2 trial of CD24Fc for prevention of graft-versus-host disease.

ABSTRACT: Patients who undergo human leukocyte antigen-matched unrelated donor (MUD) allogeneic hematopoietic stem cell transplantation (HSCT) with myeloablative conditioning for hematologic malignancies often develop acute graft-versus-host disease (GVHD) despite standard calcineurin inhibitor-based prophylaxis in combination with methotrexate. This trial evaluated a novel human CD24 fusion protein (CD24Fc/MK-7110) that selectively targets and mitigates inflammation due to damage-associated molecular patterns underlying acute GVHD while preserving protective immunity after myeloablative conditioning. This phase 2a, multicenter study evaluated the pharmacokinetics, safety, and efficacy of CD24Fc in combination with tacrolimus and methotrexate in preventing acute GVHD in adults undergoing MUD HSCT for hematologic malignancies. A double-blind, placebo-controlled, dose-escalation phase to identify a recommended dose was followed by an open-label expansion phase with matched controls to further evaluate the efficacy and safety of CD24Fc in preventing acute GVHD. A multidose regimen of CD24Fc produced sustained drug exposure with similar safety outcomes when compared with single-dose regimens. Grade 3 to 4 acute GVHD-free survival at day 180 was 96.2% (95% confidence interval [CI], 75.7-99.4) in the CD24Fc expansion cohort (CD24Fc multidose), compared with 73.6% (95% CI, 63.2-81.4) in matched controls (hazard ratio, 0.1 [95% CI, 0.0-0.6]; log-rank test, P = .03). No participants in the CD24Fc escalation or expansion phases experienced dose-limiting toxicities (DLTs). The multidose regimen of CD24Fc was well tolerated with no DLTs and was associated with high rates of severe acute GVHD-free survival after myeloablative MUD HSCT. This trial was registered at ClinicalTrials.gov as #NCT02663622.

How I reduce and treat posttransplant relapse of MDS.

ABSTRACT: Allogeneic hematopoietic stem cell transplantation (HSCT) is the only potentially curative option for patients with high-risk myelodysplastic syndromes (MDS). Advances in conditioning regimens and supportive measures have reduced treatment-related mortality and increased the role of transplantation, leading to more patients undergoing HSCT. However, posttransplant relapse of MDS remains a leading cause of morbidity and mortality for this procedure, necessitating expert management and ongoing results analysis. In this article, we review treatment options and our institutional approaches to managing MDS relapse after HSCT, using illustrative clinical cases that exemplify different clinical manifestations and management of relapse. We address areas of controversy relating to conditioning regimen intensity, chemotherapeutic bridging, and donor selection. In addition, we discuss future directions for advancing the field, including (1) the need for prospective clinical trials separating MDS from acute myeloid leukemia and focusing on posttransplant relapse, as well as (2) the validation of measurable residual disease methodologies to guide timely interventions.

Identification and surveillance of rare relapse-initiating stem cells during complete remission after transplantation.

ABSTRACT: Relapse after complete remission (CR) remains the main cause of mortality after allogeneic stem cell transplantation for hematological malignancies and, therefore, improved biomarkers for early prediction of relapse remains a critical goal toward development and assessment of preemptive relapse treatment. Because the significance of cancer stem cells as a source of relapses remains unclear, we investigated whether mutational screening for persistence of rare cancer stem cells would enhance measurable residual disease (MRD) and early relapse prediction after transplantation. In a retrospective study of patients who relapsed and patients who achieved continuous-CR with myelodysplastic syndromes and related myeloid malignancies, combined flow cytometric cell sorting and mutational screening for persistence of rare relapse-initiating stem cells was performed in the bone marrow at multiple CR time points after transplantation. In 25 CR samples from 15 patients that later relapsed, only 9 samples were MRD-positive in mononuclear cells (MNCs) whereas flowcytometric-sorted hematopoietic stem and progenitor cells (HSPCs) were MRD-positive in all samples, and always with a higher variant allele frequency than in MNCs (mean, 97-fold). MRD-positivity in HSPCs preceded MNCs in multiple sequential samples, in some cases preceding relapse by >2 years. In contrast, in 13 patients in long-term continuous-CR, HSPCs remained MRD-negative. Enhanced MRD sensitivity was also observed in total CD34+ cells, but HSPCs were always more clonally involved (mean, 8-fold). In conclusion, identification of relapse-initiating cancer stem cells and mutational MRD screening for their persistence consistently enhances MRD sensitivity and earlier prediction of relapse after allogeneic stem cell transplantation.