Background: Allogeneic hematopoietic progenitor cell transplantation (HCT) can cure patients with hematologic malignancies such as acute myeloid leukemia (AML) by inducing durable graft versus leukemia (GvL) immune responses. However, this procedure is often complicated by graft versus host disease and opportunistic infections. The development of less toxic immunotherapies such as chimeric antigen receptor (CAR) T or bispecific T cell engager therapies is hindered by the lack of unique tumor-specific antigens. In patients with AML, who were cured after allogeneic HCT, we demonstrated the presence of antibody producing B cells that target the U5 snRNP200 complex (Gillissen et. al. Blood 2018). In non-malignant cells, the U5 snRNP200 complex is a nuclear protein that is part of the spliceosome. We found that in 30-50% of AML patients, U5 snRNP200 is expressed on the surface of AML blasts but not on normal hematopoietic stem cells. This finding was recently confirmed in an independent cohort (Knorr et. al. Nat Cancer 2023). Here, we investigated the durability of the U5 snRNP200 antibody response in the same patients, who are now over 10 years after allogeneic HCT. Methods: Characteristics of the 2 patients, who remain in complete remission 12 years after allogeneic HCT for myelomonocytic AML, were described previously (Gillissen et.al. Blood 2018, pts 58 and 59). Memory IgG B-cells (CD19+, CD27+, IgM-, IgA-) were sorted from peripheral blood cells, immortalized by overexpression of BCL6 and Bcl-xL, seeded 10-20 cells/well, and cultured for 2-3 weeks. B-cell supernatants containing secreted antibodies were screened for the presence of U5 snRNP200 antibodies by snRNP200 protein based fluorescent ELISA and assessed for their capacity to bind AML cell lines THP-1, Molm13, SH2, and U937. Monoclonal antibodies were retrieved after single cell culture, sequenced and heavy and light chain domains cloned. To study cross-reactivity to commensal bacteria, U5 snRNP200 antibodies were incubated with bacteria derived from frozen stool of healthy donors and the patients. Results: Blood samples were collected from patients cured of AML in whom U5 snRNP200 antibody producing B cells were identified 10 years ago, 2 years after allogeneic HCT. These patients have remained AML-free, for over 12 years after transplantation. Screening of their current B cell repertoire identified 6 B cell clones producing U5 snRNP200 antibodies, as tested by U5 snRNP200 ELISA. The specificity of these antibodies to AML cells was confirmed by binding to the AML cell lines THP-1, Molm13, SH2, and U937 via FACS. Sequencing of these antibodies demonstrated involvement of different IGHV families and a higher number of somatic hypermutations in their CDR3s compared to the U5 snRNP200 antibodies identified 10 years ago. U5 snRNP200 antibodies have not been detected in healthy individuals or in patients receiving allogeneic HCT for hematologic malignancies other than AML. The sustained presence of U5 snRNP200 specific B-cell immunity in these patients for years, presumably long after AML clearance, suggests cross-reactivity with another factor. This, combined with findings from checkpoint inhibitor and CAR T cell studies indicating that tumor immunity is co-dependent on microbiome related factors prompted us to study cross-reactivity of U5 snRNP200 antibodies to commensal bacteria. Five out of 6 U5 snRNP200 antibodies bind commensal bacteria, while isotype controls and RSV or SARS-CoV-2 specific antibodies did not, as measured by FACS. We are currently identifying the specific bacterial strains to which these antibodies are cross-reacting. Conclusions:U5 snRNP200 is a tumor specific antigen expressed on the surface of AML cells. Allogeneic HCT recipients who successfully cleared AML generated U5 snRNP200-specific (donor) B-cells early after transplantation, and these B cell responses persisted for more than a decade. Cross-reactivity with commensal bacteria may contribute to this long-lasting immune response. Identification of AML-specific antibodies from allogeneic HCT recipients who successfully cleared AML helps discover novel targets for AML treatment. Additionally, U5 snRNP200 antibodies can be developed into therapeutic formats, such as CAR-T cells and bispecific antibodies. Disclosures HS, TB and MDH hold a patent covering human U5 snRNP antibodies (PCT/NL2014/050873).

Background aimsThe success of allogeneic hematopoietic cell transplantation (HCT) as therapy for hematologic conditions is negatively impacted by the occurrence of graft-versus-host disease (GVHD). Tissue damage, caused, for example, by chemotherapy and radiotherapy, is a key factor in GVHD pathogenesis. Innate lymphoid cells (ILCs) are important mediators of tissue repair and homeostasis. The presence of ILCs before, and enhanced ILC reconstitution after, allogeneic HCT is associated with a reduced risk to develop mucositis and GVHD. However, ILC reconstitution after allogeneic HCT is slow and often incomplete. A way to replenish the pool of ILC relies on the differentiation of hematopoietic progenitor cells (HPCs) into ILC.MethodsWe developed an ex vivo stromal cell–containing culture system to study the capacity of HPCs to differentiate into all mature helper ILC subsets.ResultsILC development depended on the source of HPCs. ILCs developed at high frequencies from umbilical cord blood– and fetal liver–derived HPC and at low frequencies when HPCs were obtained from allogeneic or autologous adult HCT grafts or healthy adult bone marrow. Although all helper ILC subsets could be generated from adult HPC sources, development of tissue protective ILC2 and NKp44+ ILC3 was notoriously difficult.ConclusionsOur data suggest that slow ILC recovery after allogeneic HCT may be related to an intrinsic incapability of adult HPC to develop into ILC.

Allogeneic hematopoietic cell transplantation (HCT) is often complicated by graft versus host disease (GvHD), an alloreactive immune response triggered by tissue damage. Interleukin (IL)-22 producing type 3 innate lymphoid cells (ILC3) protect epithelial tissues against chemo(radio)therapy-induced damage, suppress alloreactive T cells and mitigate acute GvHD symptoms after allogeneic HCT. Relatively high numbers of ILC before and after allogeneic HCT has been associated with significantly reduced tissue damage and less acute GvHD. While most transplantation conditioning and GvHD prophylaxis regimens are aimed at eliminating host and alloreactive donor lymphocytes, the effect of these regimens on ILC remain elusive. Here, we studied the effect of conditioning chemotherapy and immunosuppressive agents on the survival, proliferation, activation and function of human ILC3 in vitro . Tonsil-derived ILC3 were activated and incubated with agents commonly used to prevent and treat GvHD. While fludarabine, rapamycin, mycophenolic acid and prednisolone suppressed ILC3 to a similar degree as T cells, the effect of other agents, including cyclosporine A, methotrexate, imatinib, ibrutinib and ruxolitinib, was milder on ILC3 than on T cells. ILC3 are less sensitive to immunosuppressants potentially because of their expression of functionally active ATP Binding Cassette Subfamily B Member 1 (ABCB1) drug exporter proteins. This suggests less intracellular accumulation of immunosuppressive agents, which renders ILC3 resistant to these compounds. The present findings may help to develop strategies to simultaneously maintain the tissue protective properties of ILC3 and at the same time suppress alloreactive lymphocytes, which is important in the prevention and treatment of acute GvHD.

Disruption of the intestinal bacterial microbiota is frequently observed in the context of allogeneic hematopoietic cell transplantation (HCT) and is particularly pronounced in patients who develop graft-versus-host disease (GVHD). Donor fecal microbiota transplantation (FMT) restores gut microbial diversity and reduces GVHD in HCT recipients. The composition of the intestinal fungal community in patients with GVHD, and whether fungal taxa are transferred during FMT are currently unknown. We performed a secondary analysis of our clinical trial of FMT in patients with steroid-refractory GVHD with a focus on the mycobiota. We characterized the fecal mycobiota of 17 patients and healthy FMT donors using internal transcribed spacer amplicon sequencing. The donor who provided the majority of FMT material in our study represents an n-of-one study of the intestinal flora over time. In this donor, mycobiota composition fluctuated over time while the bacterial microbiota remained stable over 16 months. Fungal DNA was detected more frequently in baseline stool samples from patients with steroid-refractory GVHD than in patients with steroid-dependent GVHD. We could detect fungal taxa in the majority of samples but did not see evidence of mycobiota transfer from donor to recipient. Our study demonstrates the feasibility of profiling the mycobiota alongside the more traditional bacterial microbiota, establishes the methodology, and provides a first insight into the mycobiota composition of patients with GVHD.

Type 3 innate lymphoid cells (ILC3) are important in tissue homeostasis. In the gut, ILC3 repair damaged epithelium and suppress inflammation. In allogeneic hematopoietic cell transplantation (HCT), ILC3 protect against graft-versus-host disease (GvHD), most likely by restoring tissue damage and preventing inflammation. We hypothesize that supplementing HCT grafts with interleukin-22 (IL-22)-producing ILC3 may prevent acute GvHD. We therefore explored ex vivo generation of human IL-22-producing ILC3 from hematopoietic stem and progenitor cells (HSPC) obtained from adult, neonatal and fetal sources. We established a stroma-free system culturing human cord blood-derived CD34+ HSPC with successive cytokine mixes for 5 weeks. We analyzed the presence of phenotypically defined ILC, their viability, proliferation and IL-22 production (after stimulation) by flow cytometry and enzyme-linked immunosorbent assay (ELISA). We found that the addition of recombinant human IL-15 and the enhancer of zeste homolog 1/2 inhibitor UNC1999 promoted ILC3 generation. Similar results were demonstrated when UNC1999 was added to CD34+ HSPC derived from healthy adult granulocyte colony-stimulating factor mobilized peripheral blood and bone marrow, but not fetal liver. UNC1999 did not negatively impact IL-22 production in any of the HSPC sources. Finally, we observed that autologous HSPC mobilized from the blood of adults with hematological malignancies also developed into ILC3, albeit with a significantly lower capacity. Together, we developed a stroma-free protocol to generate large quantities of IL-22-producing ILC3 from healthy adult human HSPC that can be applied for adoptive transfer to prevent GvHD after allogeneic HCT.

Regeneration of functional naïve T lymphocytes following the onset of human immunodeficiency virus (HIV) infection remains a crucial issue for people living with HIV (PLWH), even when adhering to antiretroviral therapy (ART). Thus far, reports on the impact of HIV-1 infection on the entry of thymic precursors and the egress of functional naïve T lymphocytes to and from the thymus are limited. We examined the impact of HIV-1 on Sphingosine-1-phosphate (S1P) signaling, which governs the egress of functional naïve thymocytes from the thymus to the periphery. Using in vitro experiments with primary human thymocytes and in vivo and ex vivo studies with humanized mice, we show that HIV-1 infection results in upregulation of the expression of S1P receptor 1 (S1PR1) in the human thymus. Intriguingly, this upregulation occurs during intrathymic infection (direct infection of the human thymic implant) as well as systemic infection in humanized mice. Moreover, considering the dysregulation of pro- and anti-inflammatory cytokines in infected thymi, the increased expression of S1PR1 in response to in vitro exposure to Interferon-Beta (IFN-β) and Tumor Necrosis Factor-Alpha (TNF-α) indicates that cytokine dysregulation following HIV infection may contribute to upregulation of S1PR1. Finally, an increased presence of CD3hiCD69− (fully mature) as well as CD3hiCD69+ (less mature) T cells in the spleen during HIV infection in humanized mice, combined with earlier expression of S1PR1 during thymocyte development, suggests that upregulation of S1PR1 may translate to increased or accelerated egress from the thymus. The egress of thymocytes that are not functionally mature from the thymus to peripheral blood and lymphoid organs may have implications for the immune function of PLWH.

Many patients with hematological malignancies, such as acute myeloid leukemia, receive an allogeneic hematopoietic cell transplantation (HCT) to cure their underlying condition. Allogeneic HCT recipients are exposed to various elements during the pre-, peri- and post-transplant period that can disrupt intestinal microbiota, including chemo- and radiotherapy, antibiotics, and dietary changes. The dysbiotic post-HCT microbiome is characterized by low fecal microbial diversity, loss of anaerobic commensals, and intestinal domination, particularly by Enterococcus species, and is associated with poor transplant outcomes. Graft-versus-host disease (GvHD) is a frequent complication of allogeneic HCT caused by immunologic disparity between donor and host cells and results in tissue damage and inflammation. Microbiota injury is particularly pronounced in allogeneic HCT recipients who go on to develop GvHD. At present, manipulation of the microbiome for example, via dietary interventions, antibiotic stewardship, prebiotics, probiotics, or fecal microbiota transplantation, is widely being explored to prevent or treat gastrointestinal GvHD. This review discusses current insights into the role of the microbiome in GvHD pathogenesis and summarizes interventions to prevent and treat microbiota injury.

Innate lymphoid cells (ILC) are important barrier tissue immune regulators. They play a pivotal role in early non-specific protection against infiltrating pathogens, regulation of epithelial integrity, suppression of pro-inflammatory immune responses and shaping the intestinal microbiota. GATA2 haploinsufficiency causes an immune disorder that is characterized by bone marrow failure and (near) absence of monocytes, dendritic cells, B cells and natural killer (NK) cells. T cells develop normally, albeit at lower numbers. Here, we describe the absence of ILCs and their progenitors in blood and bone marrow of two patients with GATA2 haploinsufficiency and show that all subsets of ILCs appear after allogeneic hematopoietic stem cell transplantation, irrespective of the preparative conditioning regimen. Our data indicate that GATA2 is involved in the development of hematopoietic precursor cells (HPC) towards the ILC lineage.

Background: Allogeneic hematopoietic cell transplantation (HCT) can be devastating when graft-versus-host disease (GvHD) develops. GvHD is characterized by mucosal inflammation due to breaching of epithelial barriers. Innate lymphoid cells (ILCs) are immune modulatory cells that are important in the maintenance of epithelial barriers, via their production of interleukin (IL)-22 and their T cell suppressive properties. After chemo-and radiotherapy, ILCs are depleted, and recovery after remission-induction therapy and after allogeneic HCT is slow and incomplete in a significant number of patients, which is associated with an increased risk to develop acute GvHD. Objective: To investigate whether the presence of mature ILCs within G-CSF-mobilized HCT grafts is correlated with the development of acute GvHD after allogeneic HCT. Study Design: We analyzed ILCs in a cohort of 36 patients who received allogeneic HCT for a hematologic malignancy, by flow-cytometric immune-phenotyping of prospectively collected, cryopreserved peripheral blood mononuclear cells (PBMCs) and donor-derived HCT grafts collected for the same patients. Biased analysis, with ILCs defined as CD3(-)lineage(-)CD45(+)CD127(+)CD161(+) lymphocytes, was performed using FlowJo version 10 software. Unbiased analysis was done using FlowSOM, which uses a self-organizing map (SOM) with a minimal spanning tree (MST) to define and visualize different clusters present in the samples. Results: Remission-induction therapy significantly depleted ILCs from the blood, and patients who had a relatively low percentage of ILCs before allogeneic HCT were significantly more prone to develop acute GvHD, confirming previous findings in a separate cohort. Allogeneic HCT grafts, which were all obtained from the blood of G-CSF-mobilized healthy donors, contained ILCs at a frequency very similar to the peripheral blood of healthy individuals. The ILC subset composition was also comparable to that of the blood of healthy individuals, with the exception of NKp44(+) ILC3s, which were significantly more abundant in HCT grafts. The relative ILC content of the graft tended to correlate with ILC reconstitution after allogeneic HCT, suggesting that peripheral expansion of transplanted mature ILCs may contribute to early ILC reconstitution after allogeneic HCT. Patients who received a relatively ILC-poor HCT graft had a significantly increased risk to develop acute GvHD, compared with patients who received relatively ILC-rich allogeneic HCT grafts. Unbiased phenotypic analysis with the FlowSOM algorithm confirmed that allogeneic HCT grafts of patients who developed acute GvHD contained a lower frequency of ILCs that clustered in NKp44(+) ILC3 signature groups. Conclusion: The presence of ILCs in allogeneic HCT grafts is associated with a reduced risk to develop acute GvHD. These data suggest that enhancement of ILC reconstitution of ILC3s in particular, for example via adoptive transfer of ILCs, may prevent acute GvHD and has the potential to improve outcome of allogeneic HCT recipients. (C) 2021 International Society for Cell & Gene Therapy. Published by Elsevier Inc.

CD4+CD25+FOXP3+ regulatory T (Treg) cells control immunological tolerance. Treg cells are generated in the thymus (tTreg) or in the periphery. Their superior lineage fidelity makes tTregs the preferred cell type for adoptive cell therapy (ACT). How human tTreg cells develop is incompletely understood. By combining single-cell transcriptomics and flow cytometry, we in this study delineated three major Treg developmental stages in the human thymus. At the first stage, which we propose to name pre-Treg I, cells still express lineage-inappropriate genes and exhibit signs of TCR signaling, presumably reflecting recognition of self-antigen. The subsequent pre-Treg II stage is marked by the sharp appearance of transcription factor FOXO1 and features induction of KLF2 and CCR7, in apparent preparation for thymic exit. The pre-Treg II stage can further be refined based on the sequential acquisition of surface markers CD31 and GPA33. The expression of CD45RA, finally, completes the phenotype also found on mature recent thymic emigrant Treg cells. Remarkably, the thymus contains a substantial fraction of recirculating mature effector Treg cells, distinguishable by expression of inflammatory chemokine receptors and absence of CCR7. The developmental origin of these cells is unclear and warrants caution when using thymic tissue as a source of stable cells for ACT. We show that cells in the major developmental stages can be distinguished using the surface markers CD1a, CD27, CCR7, and CD39, allowing for their viable isolation. These insights help identify fully mature tTreg cells for ACT and can serve as a basis for further mechanistic studies into tTreg development.

Hergen Spits' retirement symposium was held in the Zuiderkerk, Amsterdam, the Netherlands, on 18 February 2019. As the moderator of this symposium, and co-author of this article, Bianca Blom, made a telling analogy between the time-magnitude relationship of Hergen Spits' research career and the adaptive immune response. While Hergen had made enormous impact in his first wave of immune discoveries during 1980–2006, his second wave was even stronger. Indeed, Hergen was using his remarkable memory, generated during his groundbreaking studies of T- and NK cells, to make the paradigm-shifting discovery of several subsets of innate lymphoid cells (ILCs) in humans. By using his wide knowledge and capacity for disruptive and truly innovative thinking, Hergen has cross-fertilized different fields of immunology to make an impressing number of groundbreaking discoveries. That makes Hergen a legend. Hergen graduated (with honors) in medical sciences at the University of Amsterdam in 1983. During these years, Hergen Spits and his supervisor Jan De Vries (Figure 1A–C) had developed the techniques and tools needed to make fundamental discoveries in human T-cell biology. Hergen discovered the principle of CD3-mediated redirected killing in humans,1 which is now generally used for bispecific T-cell engager antibodies in cancer therapy and he was the first to demonstrate that non-specific transient adhesion precedes TCR—antigen interaction. The 80 s represented a new era in immunology, with the cloning of the first cytokines and the discovery of Th1 and Th2 cell subsets. In 1985, Hergen left for research in industry. First at Schering Plough, France, where he played a key role in dissecting the immune system in severe combined immunodeficiency (SCID) patients transplanted with fully mismatched hematopoietic stem cells. These pioneering studies in human chimeras paved the way to the discovery of IL-10 producing type 1 regulatory T cells. Later on at DNAX research institute, Palo Alto, USA (Figure 1D), he was a driving force in the early studies of IL-4 and IL-10,2 and their role in T-cell biology. Hergen's contributions continued in the 90 s, when he returned to Amsterdam and the National Cancer Institute (NKI) and later to the Academic Medical Center (AMC). Here, Hergen described the transcriptional (predominantly inhibitor of DNA binding (Id-) and E-proteins) and molecular programs governing development of T-, NK-, and dendritic cells (DC) from lymphoid precursors in human liver and thymus. In parallel, Hergen founded the biotech company Impact with Ton Schumacher and Donald Kalff, in 2004 named AIMM Therapeutics, utilizing a B-cell immortalization technique which would eventually lead up to the MedImmune-acquired Nirsevimab, currently in phase 3 clinical trials for respiratory syncytial virus (RSV) infections in infants. Hergen was also a major player in a global effort, supported by the Bill and Melinda Gates Foundation, aimed at developing humanized mouse models for studies of immunity, for example, in vaccine responses. Today such models are greatly aiding our understanding of human immunity. In 2006, Hergen was recruited to Genentech, San Francisco, USA. This was right at the beginning of an era where discovery of the IL-23/IL-17-axis laid the foundation for the description of a new branch of T-cell immunity, initially characterized in mice. It did not take long until Hergen had identified the human Th22 subset.3 In addition, Hergen quickly realized that the IL-22 production that he observed in the T-cell system had an equivalent among innate lymphocytes resembling NK cells. Hergen returned to the AMC in 2009. This was an exceptionally exiting time where Hergen, through his life-long contribution in the fields of T- and NK cell development and differentiation, could capitalize on his unique know-how for the discovery of the innate equivalents of T cells, ILCs, in humans (Figure 2A). This work was greatly aided by the pioneering work on lymphoid-tissue inducer (LTi) cells in mice by Reina Mebius and others, eventually leading up to Hergen's and Tom Cupedo's characterization of human fetal LTi cells.4 Such LTi-like cells, later termed group 3 ILC, could also be found in human mucosal tissues after birth where they provided an innate source of IL-23-induced IL-22. In parallel, Hergen's description of human ILC2,5 which produce type 2 cytokines, including IL-5 and IL-13 in response to alarmins, such as IL-25 and IL-33, was guided by three landmark papers on the mouse equivalents. Importantly, Hergen's translational discovery filled a knowledge gap for understanding non–T-cell driven (non-allergic) type 2 inflammation in, for example, asthma. Today, multiple drugs targeting the ILC2 pathway show life-changing efficacy for patients with asthma. Similarly, Hergen's discovery of ILC1, provided a source of IFN-γ in the intestine of Crohn's disease patients, where ILC1 were proven to accumulate. Not only did Hergen discover subsets of ILCs, he also described how they develop from naïve precursors, similar to naïve T cells. His most recent contribution to the ILC field was the description of the fantastically plastic nature of ILCs. Hergen and his team (Figure 1E) described the factors that keep lineage stability of ILC2 and ILC3 but also that these cells, if exposed to a specific microenvironment, can acquire features associated with other ILC lineages (Figure 2B). Being very interested in sports, Hergen knows how to make an exit as a true super star. It must be an exceptional feeling to retire when you are at the top of your game. But in the end… did he in fact retire? No, of course not. Hergen continues to supervise students and mentor the next generation of excellent scientists at the AMC, Amsterdam, and beyond. Hergen has a never-ending intellectual curiosity, an unbeatable scientific integrity, and an exceptional capability for motivating others. Working with Hergen has been one of the most intense and rewarding experiences of the co-authors lives, shaping our scientific trajectory. But even more important, Hergen has contributed to improving the lives of patients suffering from immune-mediated diseases. We thank him for his contribution and inspiration to generations that will follow. Dr Mjösberg declares that Hergen Spits is a collaborator and mentor. Dr Blom and Prof Roncarolo have nothing to declare. The authors would like to thank Dr. Whitney Weigel for the illustrations presented in Figure 2.

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive malignancy of CD4+ T-cells associated with HTLV-1 infection. In this study, we used the model of immunodeficient NSG mice reconstituted with a functional human immune system (HIS) to investigate early events in HTLV-1 pathogenesis. Upon infection, human T-cells rapidly increased in the blood and lymphoid tissues, particularly CD4+CD25+ T-cells. Proliferation of CD4+ T-cells in the spleen and mesenteric lymph nodes (MLN) correlated with HTLV-1 proviral load and CD25 expression. In addition, splenomegaly, a common feature of ATLL in humans, was also observed. CD4+ and CD8+ T-cells predominantly displayed an effector memory phenotype (CD45RA−CCR7−) and expressed CXCR3 and CCR5 chemokine receptors, suggesting the polarization into a Th1 phenotype. Activated CD8+ T-cells expressed granzyme B and perforin; however, the interferon-γ response by these cells was limited, possibly due to elevated PD-1 expression and increased frequency of CD4+FoxP3+ regulatory T-cells in MLN. Thus, HTLV-1-infected HIS-NSG mice reproduced several characteristics of infection in humans, and it may be helpful to investigate ATLL-related events and to perform preclinical studies. Moreover, aspects of chronic infection were already present at early stages in this experimental model. Collectively, we suggest that HTLV-1 infection modulates host immune responses to favor viral persistence.

Key Points Thymic-derived Tregs are the most attractive cell type for adoptive cell therapy. Tregs that express the surface marker GPA33 can stably be expanded with high purity. Developing Tregs become GPA33+ around thymic exit, but TGF-β–induced Tregs are GPA33−. Visual Abstract FOXP3-expressing regulatory T (Treg) cells safeguard immunological tolerance. Treg cells can be generated during thymic development (called thymic Treg [tTreg] cells) or derived from mature conventional CD4+ T cells that underwent TGF-β–mediated conversion in the periphery (called peripheral Treg [pTreg] cells). Murine studies have shown that tTreg cells exhibit strong lineage fidelity, whereas pTreg cells can revert into conventional CD4+ T cells. Their stronger lineage commitment makes tTreg cells the safest cells to use in adoptive cell therapy, increasingly used to treat autoimmune and inflammatory disorders. Markers to distinguish human tTreg cells from pTreg cells have, however, not been found. Based on combined proteomic and transcriptomic approaches, we report that the Ig superfamily protein GPA33 is expressed on a subset of human Treg cells. GPA33 is acquired late during tTreg cell development but is not expressed on TGF-β–induced Treg cells. GPA33 identifies Treg cells in human blood that lack the ability to produce effector cytokines (IL-2, IFN-γ, IL-17), regardless of differentiation stage. GPA33high Treg cells universally express the transcription factor Helios that preferentially marks tTreg cells and can robustly and stably be expanded in vitro even without rapamycin. Expanded GPA33high Treg cells are suppressive, unable to produce proinflammatory cytokines, and exhibit the epigenetic modifications of the FOXP3 gene enhancer CNS2, necessary for indelible expression of this critical transcription factor. Our findings thus suggest that GPA33 identifies human tTreg cells and provide a strategy to isolate such cells for safer and more efficacious adoptive cell therapy.

Purpose of reviewTissue injury often occurs as collateral damage after chemotherapy and radiotherapy and is associated with significant comorbidity and mortality. The arsenal of options to prevent tissue injury other than dose reduction is limited, and treatment is mostly aimed at symptom relief and prevention of complications, such as bacterial translocation and malnourishment. Novel approaches directed at prevention and early repair of damaged tissues are highly anticipated.Recent findingsInnate lymphoid cells (ILC) are important in tissue homeostasis and wound healing. Most knowledge of ILC is based on studies in mice, and the contribution of ILC to repair therapy-induced tissue damage in humans is relatively understudied. A picture is nevertheless emerging, suggesting that ILC have several means to maintain tissue homeostasis. Subsets of ILC produce, for example, interleukin (IL)-22 or amphiregulin (AREG) that induce epithelial tissue repair and the release of microbiome modulating proteins. In addition, ILC have immune-regulatory capacities given that adoptive transfer of ILC in a mouse model of graft versus host disease (GvHD) attenuated tissue inflammation.SummaryILC are important in tissue maintenance and damage repair and as such have the potential to be developed as (adoptive) therapy to prevent and repair therapy-induced tissue damage.

Disruption of the intestinal microbiota occurs frequently in allogeneic hematopoietic cell transplantation (allo-HCT) recipients and predisposes them to development of graft-versus-host disease (GvHD). In a prospective, single-center, single-arm study, we investigated the effect of donor fecal microbiota transplantation (FMT) on symptoms of steroid-refractory or steroid-dependent, acute or late-onset acute intestinal GvHD in 15 individuals who had undergone allo-HCT. Study participants received a fecal suspension from an unrelated healthy donor via nasoduodenal infusion. Donor FMT was well tolerated, and infection-related adverse events did not seem to be related to the FMT procedure. In 10 of 15 study participants, a complete clinical response was observed within 1 month after FMT, without additional interventions to alleviate GvHD symptoms. This response was accompanied by an increase in gut microbial α-diversity, a partial engraftment of donor bacterial species, and increased abundance of butyrate-producing bacteria, including Clostridiales and Blautia species. In 6 of the 10 responding donor FMT recipients, immunosuppressant drug therapy was successfully tapered. Durable remission of steroid-refractory or steroid-dependent GvHD after donor FMT was associated with improved survival at 24 weeks after donor FMT. This study highlights the potential of donor FMT as a treatment for steroid-refractory or steroid-dependent GvHD, but larger clinical trials are needed to confirm the safety and efficacy of this procedure.

Background:Disruption of the intestinal microbiome has a negative influence on the outcome of allogeneic hematopoietic stem cell transplantation (HSCT). Decreased gut flora diversity with loss of commensals and pathobiont domination, is associated with an increased risk to develop graft‐versus‐host disease (GvHD). Fecal microbiota transplantation (FMT) has successfully been used to treat Clostridium difficile enteritis but the safety and efficacy of FMT in immunocompromised GvHD patients remains to be elucidated.Aims:1. To assess the safety and effectiveness of FMT as treatment for GvHD patients in a prospective, single‐arm pilot study2. To analyze the microbiome dynamics of GvHD patients receiving an FMTMethods:Fifteen patients with steroid‐refractory or steroid‐dependent intestinal GvHD received a single FMT from an unrelated, healthy donor via nasoduodenal infusion. Follow‐up included collection of blood and fecal samples prior to FMT and at 1, 4, 12 and 24 weeks after FMT as well as clinical follow‐up up to 6months.Results:FMT was well tolerated by all patients, there were no serious adverse events observed that could be attributed to FMT. Response evaluation at 4 weeks after FMT identified 11 participants with a complete response (CR), defined as resolution of all GvHD, without other interventions to alleviate symptoms. In 6 CR patients, the normalization of stool frequency and consistency was sustained throughout the whole period of follow‐up, with successful taper of immunosuppressants. Five other responders initially showed improvement of GvHD after FMT but relapsed upon prednisolone taper (CR/sf; complete responders with secondary failure). A durable response to FMT was associated with a better prognosis (Figure 1). Analysis of peripheral blood immune cell subsets did not reveal significant differences. Alpha diversity of fecal samples, analyzed by 16S ribosomal RNA sequencing, was low in patients pre‐FMT but improved upon response to treatment in CR and CR/sf patients. One week after FMT, the fecal microbial composition of CR patients resembled that of the donor the most, suggesting a better engraftment of fecal donor species in patients with persistent responses.Summary/Conclusion:This pilot study shows the potential of donor FMT to safely restore microbial diversity and improve symptoms of steroid‐refractory or steroid‐dependent GvHD patients. These encouraging data promote further investigation of this therapy in larger cohorts.image

Background:Graft‐versus‐host disease (GvHD) is a common and life threatening complication of allogeneic stem cell transplantations (ASCT), and is thought to be initiated by chemotherapy‐induced tissue damage. Innate lymphoid cells are involved in tissue remodeling and repair and may therefore have a protective role in the development of GvHD. Indeed, in a previous study we observed that relatively high frequencies of activated ILC3 s before and/or after ASCT were associated with a lower risk to develop GvHD. While rapid reconstitution of ILCs thus seems favorable, ILCs in fact recover slowly and do not reach normal levels within 6 months post‐ASCT.Aims:By investigating the composition of stem cell grafts and the ILC‐development potential of graft‐derived multipotent hematopoietic progenitor cells (HPCs), we here aim to gain more insight into how ILCs reconstitute from stem cell grafts.Methods:Peripheral blood‐mobilized stem cell grafts of healthy adult donors were thawed and ILCs were phenotyped by flow cytometry. Lineage− CD34+ CD45RA+ HPCs were FACS‐sorted and cultured for a maximum of 4 weeks on JAG1‐expressing OP9 stromal cells in the presence of IL‐2, IL‐7, stem cell factor and Flt3‐ligand. This method was previously shown to support ILC development.Results:We detected mature ILC1 s, ILC2 s and ILC3 s in all grafts, and the median frequency of total CD127+ ILCs was 0.26% of the lymphocytes. The distribution of the ILC subsets differed substantially between stem cell grafts. HPCs isolated from human fetal liver successfully developed into ILCs within 4 weeks culture, however, HPCs derived from adult stem cell grafts did not have the capacity to develop into ILCs. We are currently investigating whether the age and the source of the isolated HPCs matters by testing the ILC developmental potential of cord‐blood and bone‐marrow derived HPCs. At the same time we are extending our graft and post‐ASCT analyses to be able to correlate our in vitro findings to in vivo ILC reconstitution.Summary/Conclusion:While mature ILCs present in stem cell grafts may be a good source for reconstituting ILCs, the ILC‐developmental potency of (adult) graft‐derived HPCs seems to be poor, possibly reflecting a decline in the potency of stem cells to develop into ILCs with increasing age. These results offer an explanation for the slow reconstitution of ILCs after ASCT.image

Innate lymphoid cells (ILCs) have attracted considerable attention in the past years. As modulators of epithelial barrier immunology and homeostasis they play important roles in (auto)immunity and inflammation. Here we review the role of ILCs in hematologic malignancies, where ILCs act as efficient killer cells and as tissue healers, in the context of chemotherapy, radiotherapy and after allogeneic hematopoietic stem cell transplantation (HSCT).

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is often associated with chemotherapy- and radiotherapy-induced host tissue damage, leading to graft-versus-host disease (GVHD). Innate lymphoid cells (ILC) have an essential role in tissue homeostasis and tissue repair via their production of interleukin (IL)-22, which acts on intestinal stem cells. The tissue healing capacities of ILC via IL-22 in the context of allo-HSCT and GVHD has previously been demonstrated in a mouse model for acute GVHD. We investigated potential other ways of ILC-mediated tissue protection against GVHD. Tissue injury leads to the release of danger-associated molecular patterns (DAMPs). DAMPs interact with purinergic receptors and ectoenzymes on immune cells and induce pleiotropic effects, including activation of proinflammatory antigen-presenting cells and immunosuppressive effects via the generation of adenosine. Here, we report a novel subset of human ILC3 that coexpress the ectoenzymes CD39 and CD73 (ecto+ ILC3). Ecto+ ILC3 express RORγt and were present in the oral-gastrointestinal tract and bone marrow. ILC3 ectoenzyme expression is modulated by the proinflammatory cytokine IL-1β. Extracellular adenosine triphosphate (eATP) stimulated ecto+ ILC3 to produce IL-22 and adenosine. Activated ecto+ ILC3 suppressed autologous T-cell proliferation in coculture experiments via the production of adenosine. In allo-HSCT recipients, intestinal GVHD was associated with reduced proportions of ecto+ ILC3 and decreased levels of adenosine and its metabolite inosine. Taken together, ecto+ ILC3 have immunosuppressive properties, but in patients with GVHD, ecto+ ILC3 are depleted. A lack of ecto+ ILC3 and subsequent reduced capacity to neutralize DAMPs may contribute to the development of GVHD.