Restoring immune tolerance by engineered regulatory T cell (Treg) therapy is a promising strategy to treat patients suffering from autoimmune and inflammatory diseases. However, in many of these conditions, relevant disease-driving antigens are unknown. Therefore, suitable target (auto-)antigens for antigen-specific Treg cell therapy are rarely available. We present a novel artificial immune biosensor for Treg cells that circumvents this limitation by targeting the immune costimulatory protein CD40-ligand (CD40L), transiently expressed by activated T cells. The artificial immune receptor (AIR) comprises a CD40-derived extracellular binding domain, an intracellular costimulatory signaling domain, and a T cell receptor signaling domain of the CD3-ζ-chain. After interaction with its membrane-bound ligand, this synthetic receptor triggers a TCR-like activation program in Treg cells including induction of Treg effector molecules and cell proliferation. In a mouse model of graft-versus-host disease, transfer of CD40-AIR Treg cells significantly improved survival and demonstrated immune control of the alloantigen-reactive T cell compartment. Expression and signaling of the corresponding human CD40-AIR illustrate the potential for translating this concept. Engineering Treg cells with a CD40L-targeting sensor that detects activated T cells presents a promising therapeutic approach for a broad range of T cell-mediated inflammatory diseases.

Abstract Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is curative for hematological malignancies but limited by graft-versus-host disease (GvHD), in which donor T cells damage host tissues. Current prophylaxis broadly suppresses donor immunity, often compromising the beneficial graft-versus-leukemia (GvL) response, highlighting the need for strategies that uncouple GvHD from GvL. The cGAS–STING pathway can be strongly activated during conditioning-induced tissue damage and represents a potential therapeutic target. However, its context-dependent roles in inflammation and homeostasis have constrained its clinical translation. Here, we show that “timed” administration of the covalent STING inhibitor H151 before conditioning reduced GvHD-associated mortality without impairing GvL in murine allo-BMT models. Donor T cell activation and effector function were preserved, indicating that the protective effect operates at the level of GvHD target tissues rather than through systemic immunosuppression. Timed STING inhibition protected the intestinal epithelium by limiting apoptosis, preserving intestinal stem cell function, and sustaining metabolic fitness during conditioning-induced injury, independently of type I interferon (IFN-I) signaling. In allo-HSCT patients, low intestinal STING expression is associated with reduced transplant-related mortality. Together, these findings identify timed STING inhibition as a tissue-protective prophylactic strategy that could be incorporated into existing conditioning regimens to enhance efficacy while minimizing toxicity.

ABSTRACT:Chronic graft-versus-host disease (cGVHD) remains a major hurdle to the success of hematopoietic stem cell transplantation, directly affecting patient morbidity and mortality. Impaired regulatory T-cell (Treg) recovery in patients with cGVHD has led to clinical studies aiming to increase peripheral Treg numbers. We conducted phase 1 dose-escalation clinical trials testing the feasibility and safety of using freshly isolated donor-derived Treg infusions in steroid-refractory/dependent cGVHD. The phase 1 was extended to a preliminary phase 2 trial, resulting in a total of 33 treated patients. We report that Treg purification from donor leukapheresis using CliniMACS were feasible and that Treg infusions were safe. Importantly, Treg infusions resulted in improved symptoms, particularly at higher Treg doses. Global responses were observed in 71% of patients, and 52% of patients had at least a 2-point improvement in the cGVHD severity scale. Furthermore, improvement in cGVHD symptoms resulted in reductions in corticosteroids, ruxolitinib, and mycophenolate in 58%, 83%, and 33% of patients, respectively, whereas calcineurin inhibitors were discontinued in 75% of patients. Exploratory analyses revealed the detection of infused Treg clonotypes up to 12 months after infusion and suggest increased Treg numbers in circulation. We observed increases in serum levels of interleukin-7 and interferon gamma and decreases in soluble CD13 and suppression of tumorigenicity 2 over time, which were not statistically significant after adjustment for multiple comparisons. Although these studies were not powered to assess efficacy, they suggest potential therapeutic benefits of donor-derived Treg in cGVHD treatment and highlight the need for larger phase 2 clinical trials. The trials were registered at www.clinicaltrials.gov as #NCT02385019 and #NCT03683498.

BACKGROUND AIMS:Regulatory T-cell (Treg) infusions are an emerging therapy for the prevention or treatment of autoimmune, alloimmune and inflammatory disorders. Tregs that are modified to express a chimeric antigen receptor (CAR) are an additional promising method to expand the potential of Treg therapies. However, the development of CAR Treg is hampered by limitations in pre-clinical murine models, including poor murine Treg expansion and the need to use transgenic mouse strains to maintain pure Treg cultures. To address these issues and facilitate improved pre-clinical testing of CAR Treg products, we developed a novel protocol using wild-type (WT) mice. METHODS:Splenic CD4+CD8-CD25highCD62L+ murine T cells were flow cytometry sort-purified from WT mice to achieve >99% Foxp3 positivity. Tregs were activated by anti-CD3/CD28 beads and supported by exogenous human interleukin-2 for a total of 7 days. Retroviral transduction was performed on day 2 or day 3 of the culture. Tregs were analyzed by flow cytometry and used in in vitro and in vivo assays at the end of the 7-day culture. RESULTS:Robust WT Treg expansion (>30 fold) was seen without a loss of Foxp3 expression and while maintaining high CD25 and CD62L levels. Expanded Tregs were highly suppressive in vitro and protective in vivo in an acute graft-versus-host disease model. Retroviral transduction on day 3 resulted in high transduction efficiency (>90%) without compromising overall expansion potential. When transduced with a CD19-CAR, CAR Tregs eliminated CD19+ malignant cells and maintained suppressive activity. CONCLUSIONS:We developed a novel WT murine Treg culture protocol that produces highly functional CAR Treg with robust expansion, Foxp3 purity and retroviral transduction efficiency. This protocol may facilitate broader application of murine CAR Treg pre-clinical studies and may result in an expansion of future CAR Treg products for patients.

Paradoxically, immune cell infiltration correlates with worse prognosis in renal cell carcinoma (RCC) patients, with tumor-associated myeloid cells playing a key role in tumor progression. However, little is known about factors driving their polarization. Here, we investigated the link between RCC-related glycolysis, hypoxia-inducible factor (HIF)1α-associated myeloid inflammation, and patient prognosis. TCGA data revealed a strong correlation between the expression of monocarboxylate transporter 4 (MCT4), the myeloid marker CD14 and patient survival in ccRCC patients. scRNAseq data confirmed high MCT4 expression in both tumor and myeloid cells, suggesting lactate transport. In vitro analyses proved lactate uptake by CD14+ monocytes, which stabilized HIF1α and induced an MDSC-like, HLA-DR low phenotype. In line, the HIF1α-stabilizing drug Roxadustat increased the number of CD14+ HLA-DR low cells. Lactate uptake also increased protein lactylation. To further investigate the interplay between RCC tumor and myeloid cells, we established a 3D spheroid co-culture model and analyzed the effects of MCT inhibitors. This 3D model reflected tumor-myeloid cell interactions, as spheroid-infiltrating myeloid cells exhibited spontaneous IL-6 secretion comparable to patient-derived RCC cultures. Inhibition of lactate secretion reduced lactate and IL-6 secretion while increasing CD14+ HLA-DR+ cells. These findings were validated in patient-derived RCC cultures treated with anti-glycolytic drugs. Our data dissect the intratumoral network of RCC and show that tumor-derived lactate promotes a pro-tumorigenic myeloid phenotype with low MHC-II but high immune-checkpoint, LOX-1 and S100A8/9 expression. Blocking MCT disrupts this interplay, offering a promising strategy to re-educate tumor-associated myeloid cells and enhance tumor immune surveillance.

Life expectancy of patients with severe transfusion-dependent beta-thalassemia (TDT) remains below that of the general population. Allogenic hematopoietic stem cell transplantation (HSCT) is the standard curative treatment. Due to the paucity of matched donor (MD) availability, haploidentical HSCT (haplo-HSCT) is a reasonable alternative. Twenty patients with TDT (median age 10 years; range 2–23) received either a matched sibling donor (MSD; n = 7) or a haplo-HSCT (n = 13) in a single center (Regensburg, Germany) between 2016 and 2022, including two patients referred for a haplo-HSCT as rescue failing prior MD- and haplo-HSCT, respectively. The conditioning regimen consisted of anti-thymocyte globulin (ATG; Grafalon®), treosulfan, thiotepa, and fludarabine (FTT). Immunosuppression consisted of a calcineurin inhibitor and mycophenolate mofetil (MMF). At a median follow-up of 37 months (range 6–90), overall survival (OS) was 100% with a disease-free survival (DFS) of 100% in MSD and 92% in haplo-HSCT, respectively. Two patients in haplo-HSCT experienced graft failure, one achieving DFS after a second haplo-HSCT. No acute graft-versus-host disease (aGvHD) ≥ °III or severe chronic GvHD (cGvHD) were observed. No sinusoidal obstruction syndrome (SOS) was observed in this high-risk population. Treosulfan-based T-cell depleted haplo-HSCT can achieve comparable OS and DFS even in young adult TDT patients with no SOS/VOD.

Background: Chronic graft-versus-host disease (cGvHD) remains a major cause of late morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Aberrant germinal center (GC) activity, driven by dysregulated T-B cell interactions, contributes to disease pathogenesis. PD-1+ CXCR5+ follicular helper T (TFH) cells orchestrate GC responses by promoting memory B cell and plasmablast formation, and elevated circulating (c) TFH frequencies have been linked to cGvHD severity. Here, we report the identification of a cytotoxic TFH subset, previously published by us and referred to as killer TFH or TFK cells, co-expressing canonical TFH markers with high levels of the cytotoxic effector molecules NKG-7 and granzyme B (GRZB). This dual helper–killer phenotype suggests a unique capacity to both disrupt GC homeostasis and mediate direct tissue injury on MHC-II-upregulating epithelia. Integrating these cells into the immunopathogenic framework of cGvHD offers a novel perspective on disease initiation and progression. Methods: In a prospective trial, peripheral blood from 80 allo-HSCT patients was collected on median day +113 (range +91 to +190). In 24 patients, additional samples were obtained at cGvHD onset (median day +187, range +98 to +410) and 4 and 8 weeks later (9 de novo, 15 quiescent/progressive cases). CD4+ T cell subsets, particularly cTFH cells, were analyzed by flow cytometry. Results: At a median of day +113 post allo-HSCT, patients without current or future GvHD exhibited significantly higher frequencies of PD-1+ CXCR5+ cTFH cells (median 7.98%, range 16.92%) compared to those who later developed cGvHD (median 3.81%, range 21.36%, p<0.002). In the latter group, cTFH frequencies were already reduced at day +113 and remained low following disease onset.Subtype analysis revealed that cTFH1 (CXCR3+CCR6-) and cTFH1/17 (CXCR3+CCR6+) frequencies at day +113 were markedly elevated in patients who never developed GvHD (cTFH1: median 4.38%, range 13.25%; cTFH1/17: median 1.18%, range 4.2%) compared to those with subsequent disease (cTFH1: median 2.53%, range 9.96%, p<0.03; cTFH1/17: median 0.31%, range 2.02%, p<0.0002). cTFH2 (CXCR3-CCR6-) and cTFH17 (CXCR3-CCR6+) frequencies declined from day +113 to disease onset (week 0) in untreated cGvHD patients.A prominent difference was also observed for cytotoxic NKG-7+ (anti-TIA-1) GRZB+ CD4+ T cells: Patients not developing any GvHD later on displayed significantly higher levels of NKG-7+ and NKG-7+ GRZB+ T cells at day +113 (NKG-7+: 24.56%, range 64.85%; NKG-7+ GRZB+: median 6.03%, range 23.00%, p<0.03) than patients who later developed cGvHD (NKG-7+: median 12.07%, range 51.98%; NKG-7+ GRZB+: median 0.99%, range 25.95%). A trend mirrored in the TFH compartment, where killer cTFH cells (NKG-7+ GRZB+) were most abundant in patients without developing any form of GvHD.Receiver operating characteristic (ROC) analysis assessed on day +113 identified several populations with strong prognostic potential subsequent for cGvHD: cTFH cells (AUC = 0.77; p = 0.0019), NKG-7+ GRZB+ CD4+ cells (AUC = 0.73; p = 0.0081), cTFH1 cells (AUC = 0.73; p = 0.0072), and cTFH1/17 cells (AUC = 0.84; p<0.0001). Conclusion: Our findings reveal that early post-transplant preservation of cTFH subsets, particularly cTFH1, cTFH1/17, and killer cTFH cells, can be found in patients without future or present cGvHD, whereas their decline in peripheral blood precedes disease onset. The identification of a killer TFH phenotype with high GRZB and NKG-7 expression adds a novel layer to the immunopathogenesis of cGvHD. Furthermore, the predictive value of the cytotoxic NKG-7+ GRZB+ CD4+T cells indicates a potential benefit for risk stratification and will be correlated further with clinical parameters.

ABSTRACT:Chronic graft-versus-host disease (cGVHD) is characterized by dysregulation of the adaptive immune system, including an aberrant B-cell homeostasis after allogeneic hematopoietic stem cell transplantation (allo-SCT). It is uncertain, however, whether this B-cell dysregulation is a result of manifest cGVHD or develops as a sign of aberrant B lymphopoiesis after allo-SCT before cGVHD becomes apparent. To gain insight into the development of B-cell dysregulation before the onset of cGVHD, we analyzed B-cell subpopulations by multiparameter flow cytometry on days 90, 180, and 356 after allo-SCT in a prospective study design. After completion of follow-up, patients were assigned retrospectively to 3 groups according to onset of GVHD: (1) no GVHD (n = 17); (2) acute GVHD (aGVHD) without subsequent cGVHD (n = 32); and (3) cGVHD (n = 59). Although CD21lowCD11c+ B cells were increased in all groups, the frequency of CD20-CD38hi plasmablasts was significantly elevated already 90 days after allo-SCT in patients who subsequently developed cGVHD, compared to patients without GVHD or with aGVHD only (median of CD19+ cells, 5.9% vs 2.2% vs 2.2%; P = .0016 and .0304, respectively). Detailed molecular analysis of expanded plasmablasts revealed a dominance of the immunoglobulin A isotype, with molecular evidence for recent generation in mucosal sites and markers for intestinal homing. A large fraction of the clonally expanded plasmablasts produced antibodies that bound to subgroups of commensals known to produce short-chain fatty acids. In summary, our data suggest that dysregulated intestinal antibody responses against commensals contribute to the pathophysiology of cGVHD.

Intestinal stem cells (ISCs) promote tissue repair after genotoxic or immune-mediated injury. However, ISCs are particularly sensitive to various stressors and primary targets of overwhelming immune responses, such as interferon γ (IFNγ)-mediated killing. In mouse models of radiation therapy-induced gut damage and in biopsies from patients who underwent allogeneic hematopoietic stem cell transplantation, we observed IFNγ expression by intestinal Treg cells. Treg cells leverage combined IFNγ and interleukin 10 (IL-10) stimulation of ISCs to nurture the growth of intestinal organoids through the activation of the mTORC1 and Myc pathways. Similarly, Treg cells or the combined addition of recombinant IFNγ and IL-10 promoted the regeneration of organoids after irradiation, and both cytokines were essential for ensuring epithelial regeneration following acute intestinal tissue injury in vivo. The exposure of organoids to growth factor-free culture conditions revealed distinct EGF-like properties of IFNγ and Wnt-like properties of IL-10. While IFNγ rapidly induced epithelial proliferation, it depleted the pool of ISCs in vitro. Only the combination of IFNγ and IL-10 led to epithelial proliferation and organoid growth while simultaneously ensuring ISC maintenance over time. Our results reveal a context-dependent role of inflammatory signaling in ISCs, through which Treg cells promote epithelial repair following therapy-induced injury.

Management of immunosuppression after solid organ transplantation in context of chimeric antigen receptor T cell therapy (CART) is challenging. Although required to prevent graft rejection, systemic immunosuppression can interfere with biological functions of apheresis products and adoptively transferred T cells. We treated a 33-year-old kidney transplant recipient who developed relapsed/refractory posttransplant lymphoproliferative disorder with CD19-directed CART as a fourth-line therapy. Immunosuppression was discontinued before leukapheresis for CART and not reinitiated ever since. Although the posttransplant lymphoproliferative disorder remained in complete remission, we did not observe any signs of graft rejection (clinically and by determination of donor-derived cell-free DNA) until last follow-up at 23 months after CART. Phenotyping of peripheral blood immune cell subsets showed stable recovery of T and B cell compartments with dominant naïve differentiation. Immune responses against foreign antigens were shown by T cell cytokine production after stimulation with virus-derived peptide pools and absence of torque teno virus DNA. The lack of human leukocyte antigen antibodies and absence of T cell proliferation in a mixed leukocyte reaction with peripheral blood cells of the kidney donor confirmed tolerance against donor antigens. We envision CD19-directed CART as a therapeutic option to prevent organ rejection without conventional long-term immunosuppression in selected patients.

Background: Graft-versus-host disease (GVHD) is a severe complication of allogeneic stem-cell transplantation (allo-HSCT). Previously, we and others showed that activation of type I interferon (IFN-I) inducing pathways, such as cGAS/STING, can promote intestinal barrier integrity and limit GVHD. In contrast, there is limited knowledge regarding whether inhibiting the STING pathway can help prevent GVHD and about the optimal timing for such inhibition. Methods: The covalent STING inhibitor H-151 was evaluated at various time points in preclinical GvHD and GvL mouse models. For mechanistic studies, murine intestinal damage models were used and outcomes assessed using an organoid recovery assay in addition to established read-outs (histology, immune infiltrate). In vitro organoid assays were used to assess the impact of STING inhibition on epithelial regeneration after genotoxic stress from irradiation or chemotherapy. These assays were expanded to include co-cultures with allogeneic T cells to assess immune-modulatory effects. Human immune cells were treated with H151 in vitro and subsequently analyzed using flow cytometry and single-cell RNA sequencing (scRNA-seq). Ongoing scRNA-seq of epithelial and immune cells after transplantation in mice seeks to further unravel the underlying mechanisms and will be shared. Results: In preclinical models of GvHD, administering H151 before irradiation, but not at later time points, significantly decreased GvHD severity and enhanced survival. Recipients treated with H151 prior to conditioning showed a decreased loss of intestinal stem cells and reduced epithelial apoptosis after conditioning. Consequently, we observed enhanced intestinal regeneration, as demonstrated by improved organoid formation ex vivo. In vitro, H-151 treatment improved the resistance of organoids to genotoxic stress induced by irradiation or chemotherapy. Co-culture assays indicated that transient STING inhibition did not affect resistance to T cell-mediated damage, though prolonged inhibition impaired organoid growth, highlighting STING's role in intestinal homeostasis and regeneration. In immune cell assays, H151 minimally affected T cell activation but reduced APC activation, suggesting that STING inhibition modulates inflammatory signals without compromising T cell function. Data from GvL models showed that transient STING inhibition did not impair the GvL effect. Ongoing scRNA-seq experiments aim to clarify the cellular mechanisms driving these outcomes, focusing on epithelial cells, T cells, and APCs following allo-BMT. Conclusions: Our findings show that transient modulation of the STING pathway before conditioning protects the intestinal epithelium and reduces GvHD without affecting the GvL effect. While STING activity is harmful during genotoxic damage, it is crucial for maintaining intestinal homeostasis and regeneration later. This study underscores the importance of timing in STING modulation and its potential as a therapeutic strategy to improve allo-HSCT outcomes.

Chronic GVHD (cGVHD) following allogeneic stem cell transplantation (allo-SCT) is marked by disruption of the adaptive immune system, including aberrant B cell homeostasis, and numerous studies suggest that B cells play a role in cGVHD pathogenesis. However, which specific B cell populations contribute to cGVHD development, their pathogenic role in the disease, and how these cells could potentially be targeted remains uncertain. To gain insight into B cell dysregulations before onset of cGVHD, peripheral blood samples of 172 allo-SCT patients were examined at defined time points between 56 days and 365 days after allo-SCT using multiparameter flow cytometry in a prospective study design. Retrospective grouping categorized the patients into three cohorts: no GVHD (n=17), acute GVHD without later cGVHD (n=32), and cGVHD (n=59; de novo n=11; quiescent n=49), which emerged at a median of day 180. Patients who later developed cGVHD revealed a markedly higher frequency of antibody-producing CD21low CD20neg CD38hi plasmablasts at day 90 than those who never experienced GVHD or patients with only acute GVHD (median 5.9% vs 2.2% vs 2.2% of CD19+ cells; p=0.0016 and p=0.0304, respectively) and the elevation persisted until day 365. The expansion of the plasmablast population already occurred prior to the onset of cGVHD and a Cox proportional hazards model with cGVHD diagnosis as an endpoint identified an elevated frequency of plasmablasts higher than 3% of CD19+ cells 90 days after allo-SCT as an independent risk factor for subsequent cGVHD development taking into account multiple clinical transplant parameters as co-variables (hazard ratio, 3.07; 95% CI, 1.05 to 8.99; p value, 0.04). As demonstrated by scRNA sequencing data (n=5), expanded plasmablasts of cGVHD patients revealed clonal expansions, a high frequency of somatic mutations, and a selection of certain VH genes in their antibody repertoire. Most plasmablasts were found class switched to IgG and even more frequently to the mucosa-associated isotype IgA. Many IgA+ plasmablasts additionally expressed surface markers for mucosal homing like CCR9 or α4β7-integrin. Notably, 12 out of 24 IgA+ antibody clones derived from cGVHD plasmablasts recognized specific short-chain fatty acid-producing taxa of intestinal commensals such as Bacteroides, Blautia, Faecalibacterium, or Ruminococcus indicating a connection between peripheral plasmablasts and the microbiome. In a new patient cohort, we investigated whether plasmablast frequencies were elevated at disease onset and how first-line therapy for cGVHD affected B cell subpopulations. Samples from 20 patients (de novo, n=6; quiescent, n=14) were collected at the day of cGVHD onset (median day 183, range 98 to 402 days) and 4 and 8 weeks after disease onset and initiation of steroid therapy using prednisolone. At the day of cGVHD onset a remarkably elevated frequency of plasmablasts compared to patients 180 days after allo-SCT who never experienced GVHD was found by multiparameter flow cytometry (median 7.0% vs 1.9% of CD19+ cells; p=0.009). Steroid therapy led to a reduction of cGVHD severity with a concomitant decrease of plasmablast frequencies already 4 weeks after onset treatment (median 7.0% vs 1.6% of CD19+ cells; p=0.008). This was accompanied by a substantial decline in serum BAFF levels under steroid therapy (median 5.4 ng/ml vs 1.9 ng/ml; p=0.009). Interestingly, the frequency of other B cell subsets such as CD11c+ T-bet+ B cells remained unaltered in the first 8 weeks after treatment (median 3.1% vs 3.0% of CD19+ cells), suggesting a complex dynamic of B cell dysregulation at the onset of therapy. Overall, our new findings confirm the prominent role of plasmablasts as important predictors for the onset of cGVHD.

Changes in the intestinal microbiome and microbiota-derived metabolites predict clinical outcomes after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Here, we report that desaminotyrosine (DAT), a product of bacterial flavonoid metabolism, correlates with improved overall survival and reduced relapse rates in patients receiving allo-HSCT. In preclinical mouse models, treatment with synthetic DAT prevents graft-versus-host disease by protecting the intestinal barrier and promoting intestinal regeneration and contributes to graft-vs.-leukemia responses. DAT´s beneficial effects on intestinal regeneration remain effective despite broad-spectrum antibiotics-induced dysbiosis, also when administered by fecal microbiota transfer with flavonoid-degrading F. plautii. Mechanistically, DAT promotes mTORC1-dependent activation and proliferation of intestinal stem cells, with concomitant engagement of the innate immune receptor STING required to mitigate metabolic stress and maintain an undifferentiated stem cell state independently of type-I interferon responses. Additionally, DAT can skew T cells towards an effector phenotype to modulate graft-versus-leukemia responses. Our data uncover DAT's dual, tissue- and immune-modulating properties and underscore its potential in precision microbiome-based therapies to improve tissue regeneration and minimize immune-mediated side effects.

Background: The success of allogeneic hematopoietic stem cell transplantation (allo-HSCT) hangs on a delicate balance between preventing graft-versus-host disease (GvHD) while simultaneously promoting the elimination of residual malignant cells by an allogeneic immune reaction of donor T cells (graft-versus-leukemia (GvL)). Microbiome diversity and composition have been associated with clinical outcomes after allo-HSCT, including overall survival and incidence of GvHD [1-3]. Still, little is known about whether and how the microbiome modulates GvL. Our group recently established an “Immuno-Modulatory Metabolite Risk Index (IMM-RI)” comprised of microbiota-derived immuno-modulatory metabolites (IMMs) that were predictive of overall survival and relapse in patients receiving allo-HSCT [4]. The effect of IMMs has been partially attributed to type-I interferon (IFN-I) responses, which have been associated with positive or negative outcomes on GvHD, depending on the timing of IFN-I activation. We hypothesized microbiota-derived metabolites engage IFN-I signaling in epithelial and immune cells, poising them to modulate GvHD and GvL. To address the underlying cellular and molecular mechanisms, we screened for IMMs in allo-HSCT patients and tested them as treatment in patient ex vivo and murine in vivo models. Methods Stool samples from patients receiving allo-HSCT were obtained at Rechts der Isar, Munich, and University Hospital Regensburg per IRB-approved study protocols (German Clinical Trials Register (DRKS00034175)). We quantified levels of microbiota-derived metabolites by mass spectrometry. IMMs detected in patients were tested as treatment in murine and human immune cells, intestinal crypt-derived organoids, and organoid/T cell co-culture assays. To test for the requirement for IFN-I signaling, we stimulated WT or IFN-I-signaling-impaired organoids (MAVS-/-, STINGgt/gt, IFNaR-/-) or used small molecule inhibitors and analyzed them regarding growth performance, intestinal stem cell (ISC) numbers, and by scRNAseq. IMMs were used to treat intestinal damage and major mismatch GvHD mouse models. Outcomes were assessed using a novel organoid recovery assay in addition to established read-outs (histology, barrier integrity, immune infiltrate). IMM treatment was also performed in germ-free or antibiotic-induced dysbiosis models to test for the requirement of microbiome signals. Results: We demonstrate that IMMs correlate with improved overall survival and reduced relapse rates in allo-HSCT patients. In preclinical models, IMM treatment showed dose-dependent effects. Low-dose IMM moderately enhanced graft-vs-leukemia responses and prevented graft-vs.-host disease by protecting the intestinal barrier and promoting intestinal regeneration, while high doses promoted T cell activation and risked hyperacute GvHD. IMM treatment remained effective despite broad-spectrum antibiotic treatment, but not in germ-free mice. Mechanistically, IMM promoted mTORC1-dependent activation and proliferation of ISC, with the innate immune receptor STING required to mitigate metabolic stress and maintain an undifferentiated stem cell state. Consequently, the protective effect of IMMs was abrogated in STING-deficient mice. Conclusions: Our findings underscore the potential of IMM's epithelial- and immune-modulating properties, paving the way for the design of microbiome-based therapies that could significantly improve outcomes in allo-HSCT and potentially other cancer immunotherapies. References Peled, J.U., et al., Microbiota as Predictor of Mortality in Allogeneic Hematopoietic-Cell Transplantation. New England Journal of Medicine, 2020. 382(9): p. 822-834. Shono, Y., et al., Increased GVHD-related mortality with broad-spectrum antibiotic use after allogeneic hematopoietic stem cell transplantation in human patients and mice. Science Translational Medicine, 2016. 8(339): p. 339ra71-339ra71. Weber, D., et al., Microbiota Disruption Induced by Early Use of Broad-Spectrum Antibiotics Is an Independent Risk Factor of Outcome after Allogeneic Stem Cell Transplantation. Biology of Blood and Marrow Transplantation, 2017. 23(5): p. 845-852. Thiele Orberg, E., et al., Bacteria and bacteriophage consortia are associated with protective intestinal metabolites in patients receiving stem cell transplantation. Nature Cancer, 2024.

In clinical situations, peripheral blood accessible CD3(+)CD4(+)CXCR5(+) T-follicular helper (T-FH) cells may have to serve as a surrogate indicator for dysregulated germinal center responses in tissues. To determine the heterogeneity of T-FH cells in peripheral blood versus tonsils, CD3(+)CD4(+)CD45RA(-)CXCR5(+) cells of both origins were sorted. Transcriptomes, TCR repertoires and cell-surface protein expression were analysed by single-cell RNA sequencing, flow cytometry and immunohistochemistry. Reassuringly, all blood-circulating CD3(+)CD4(+)CXCR5(+) T-cell subpopulations also appear in tonsils, there with some supplementary T-FH characteristics, while peripheral blood-derived T-FH cells display markers of proliferation and migration. Three further subsets of T-FH cells, however, with bona fide T-follicular gene expression patterns, are exclusively found in tonsils. One additional, distinct and oligoclonal CD4(+)CXCR5(+) subpopulation presents pronounced cytotoxic properties. Those 'killer T-FH (T-FK) cells' can be discovered in peripheral blood as well as among tonsillar cells but are located predominantly outside of germinal centers. They appear terminally differentiated and can be distinguished from all other T-FH subsets by expression of NKG7 (TIA-1), granzymes, perforin, CCL5, CCR5, EOMES, CRTAM and CX3CR1. All in all, this study provides data for detailed CD4(+)CXCR5(+) T-cell assessment of clinically available blood samples and extrapolation possibilities to their tonsil counterparts.

The adoptive transfer of regulatory T cells is a promising strategy to prevent graft-versus-host disease after allogeneic bone marrow transplantation. Here, we use a major histocompatibility complex-mismatched mouse model to follow the fate of in vitro expanded donor regulatory T cells upon migration to target organs. Employing comprehensive gene expression and repertoire profiling, we show that they retain their suppressive function and plasticity after transfer. Upon entering non-lymphoid tissues, donor regulatory T cells acquire organ-specific gene expression profiles resembling tissue-resident cells and activate hallmark suppressive and cytotoxic pathways, most evidently in the colon, when co-transplanted with graft-versus-host disease-inducing conventional T cells. Dominant T cell receptor clonotypes overlap between organs and across recipients and their relative abundance correlates with protection efficacy. Thus, this study reveals donor regulatory T cell selection and adaptation mechanisms in target organs and highlights protective features of Treg to guide the development of improved graft-versus-host disease prevention strategies. Graft-versus-Host disease is a major complication after allogeneic bone marrow transplantation and is ameliorated by adoptively transferred donor regulatory T cells. Here, the authors apply transcriptomic and TCR profiling to assess regulatory T cell organ-specific adaptation in murine bone marrow transplantation models.

Vitamin D3 regulates a variety of biological processes irrespective of its well-known importance for calcium metabolism. Epidemiological and animal studies indicate a role in immune regulation, intestinal barrier function and microbiome diversity. Here, we analyzed the impact of different vitamin D3- containing diets on C57BL/6 and BALB/c mice, with a particular focus on gut homeostasis and also investigated effects on immune cells in vitro. Weak regulatory effects were detected on murine T cells. By trend, the active vitamin D3 metabolite 1,25-dihydroxyvitamin D3 suppressed IFN, GM-CSF and IL-10 cytokine secretion in T cells of C57BL/6 but not BALB/c mice, respectively. Using different vitamin D3-fortified diets, we found a tissue–specific enrichment of mainly CD11b+ myeloid cells but not T cells in both mouse strains e.g. in spleen and Peyer’s Patches. Mucin Reg3γ and Batf expression, as well as important proteins for gut homeostasis, were significantly suppressed in the small intestine of C57BL76 but not BALB/c mice fed with a high-vitamin D3 containing diet. Differences between both mouse stains were not completely explained by differences in vitamin D3 receptor expression which was strongly expressed in epithelial cells of both strains. Finally, we analyzed gut microbiome and again an impact of vitamin D3 was detected in C57BL76 but not BALB/c. Our data suggest strain-specific differences in vitamin D3 responsiveness under steady state conditions which may have important implications when choosing a murine disease model to study vitamin D3 effects.