Littérature scientifique sur le sujet « INTERFERON-GAMMA ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION GVHD »

Background: Recent studies highlight immunoregulatory functions of type I interferons (IFN-I) during the pathogenesis of graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We demonstrated that selective activation of IFN-I pathways including RIG-I/MAVS and cGAS/STING prior to allo-HSCT conditioning therapy can ameliorate the course of GVHD. However, direct effects of IFN-Is on immune cells remain ill characterised. Methods: We applied selective RIG-I agonists (3pRNA) to stimulate IFN-I production in murine models of conditioning therapy with total body irradiation (TBI) and GVHD. Results: Using IFNAR1-deficient donor T and hematopoietic donor cells, we found that endogenous and RIG-I-induced IFN-Is do not reduce GVHD by acting on these respective cell types. However, 3pRNA applied before conditioning therapy reduced the ability of CD11c+ recipient cells to stimulate proliferation and interferon gamma expression of allogeneic T cells. Consistently, RIG-I activation before TBI reduced the proliferation of transplanted T-cells after allo-HSCT. The reduced allogenicity of CD11c+ recipient cells was dependent on IFN-I signalling. Notably, this immunosuppressive function of DCs was restricted to a scenario of genotoxic tissue damage as neither RIG-I activation and IFN-I induction in naive (non-irradiated) mice altered allogeneic T cell activation. Conclusion: Our findings uncover a hitherto unknown IFN-I- and context dependent immunosuppressive function of dendritic cells. This needs to be considered in the development of IFN-I based therapeutic approaches to modulate donor T cell activation after allo-HSCT. Disclosures No relevant conflicts of interest to declare.

Abstract Stimulator of interferon genes (STING) plays an important role in eliciting innate immune responses by sensing tumor and microbial DNA in anti-tumor and anti-infection responses, respectively. How the STING signal affects allogeneic response is not clear. To address this question, we utilized murine models of allogeneic hematopoietic stem cell transplantation (allo-HCT). By transferring donor bone marrow (BM) and T cells into allogeneic recipients, we found that significantly more severe graft-versus-host disease (GVHD) was induced in STING−/− recipients as compared to WT controls. By generating BM-chimeric mice in which STING was deficient in hematopoietic or non-hematopoietic antigen-presenting cells (APCs), we confirmed that STING on hematopoietic cells was primarily responsible for constraining host APC function. We further demonstrated that STING on host CD11c+ APCs played a predominant role in the regulation of allogenic T-cell responses. Mechanistically, we found that host CD11c+IAb+ cells deficient for STING could survive better and be activated more strongly after allo-HCT. As a consequence, STING-deficient APCs augmented donor T-cell expansion, chemokine receptor expression and migration into intestinal tissues, resulting accelerated/exacerbated GVHD after allo-HCT. Using pharmacologic approaches, we further demonstrated that systemic administration of STING agonist (c-diGMP) on recipient mice before irradiation significantly reduced GVHD mortality. In conclusion, we reveal a novel role of STING in APC activity that dictates T-cell allogenic responses, and validate STING as a potential therapeutic target for controlling GVHD after allo-HCT.

Abstract Stimulator of interferon genes (STING) is an innate immune sensor of cytoplasmic dsDNA originating from microorganisms and host cells. STING plays an important role in the regulation of murine graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and may be similarly activated during other transplantation modalities. In this review, we discuss STING in allo-HSCT and its prospective involvement in autologous HSCT (auto-HSCT) and solid organ transplantation (SOT), highlighting its unique role in nonhematopoietic, hematopoietic, and malignant cell types.

Graft-versus-host disease (GVHD) and infection are major obstacles to successful allogeneic hematopoietic stem cell transplantation (HSCT). Intestinal goblet cells form the mucus layers, which spatially segregate gut microbiota from host tissues. Although it is well known that goblet cell loss is one of the histologic features of GVHD, effects of their loss in pathophysiology of GVHD remain to be elucidated. In mouse models of allogeneic HSCT, goblet cells in the colon were significantly reduced, resulting in disruption of the inner mucus layer of the colon and increased bacterial translocation into colonic mucosa. Pretransplant administration of interleukin-25 (IL-25), a growth factor for goblet cells, protected goblet cells against GVHD, prevented bacterial translocation, reduced plasma concentrations of interferon-γ (IFN-γ) and IL-6, and ameliorated GVHD. The protective role of IL-25 was dependent on Lypd8, an antimicrobial molecule produced by enterocytes in the colon that suppresses motility of flagellated bacteria. In clinical colon biopsies, low numbers of goblet cells were significantly associated with severe intestinal GVHD, increased transplant-related mortality, and poor survival after HSCT. Goblet cell loss is associated with poor transplant outcome, and administration of IL-25 represents an adjunct therapeutic strategy for GVHD by protecting goblet cells.

Abstract Bone marrow transplantation is an accepted therapy for hematologic malignancies, aplastic anemia, metabolic disorders, and solid tumors. However, graft-versus-host disease (GVHD) and failure of engraftment have limited the widespread application of this technology to nonmalignant disease states. The use of purified bone marrow stem cells has been suggested as an approach to promote engraftment yet avoid GVHD. Although bone marrow stem cells, purified by cell sorting, engraft and repopulate lethally irradiated genetically identical recipients, they do not engraft in major histocompatibility complex (MHC)-disparate allogeneic recipients. We report for the first time the characterization of a novel cell population of donor bone marrow origin, separate from the hematopoietic stem cell, that facilitates engraftment of purified allogeneic bone marrow stem cells in an MHC- specific fashion without causing GVHD. Although 1,000 purified stem cells (c-kit+/Sca-1+/lineage-) reliably repopulate syngeneic mouse recipients, 10 times that number do not engraft in MHC-disparate allogeneic recipients. The addition of as few as 30,000 facilitating cells (CD8+/CD45R+/TCR-) is sufficient to permit engraftment of purified stem cells in MHC-disparate recipients. The cell surface phenotype of this purified cellular population differs significantly from other characterized lineages of lymphoid or myeloid origin. Based on multiparameter rare-events cell sorting, the facilitating fraction is CD8+, CD3+, CD45R+, Thy 1+, class IIdim/intermediate but alpha beta- TCR- and gamma delta-TCR-. This cellular population comprises approximately 0.4% of the total bone marrow and is separate from the hematopoietic stem cell. The coadministration of purified facilitating cells plus stem cells to optimize engraftment yet avoid GVHD may expand the potential application of bone marrow transplantation to disease states in which the morbidity and mortality associated with conventional BMT cannot be justified.

Bone marrow transplantation is an accepted therapy for hematologic malignancies, aplastic anemia, metabolic disorders, and solid tumors. However, graft-versus-host disease (GVHD) and failure of engraftment have limited the widespread application of this technology to nonmalignant disease states. The use of purified bone marrow stem cells has been suggested as an approach to promote engraftment yet avoid GVHD. Although bone marrow stem cells, purified by cell sorting, engraft and repopulate lethally irradiated genetically identical recipients, they do not engraft in major histocompatibility complex (MHC)-disparate allogeneic recipients. We report for the first time the characterization of a novel cell population of donor bone marrow origin, separate from the hematopoietic stem cell, that facilitates engraftment of purified allogeneic bone marrow stem cells in an MHC- specific fashion without causing GVHD. Although 1,000 purified stem cells (c-kit+/Sca-1+/lineage-) reliably repopulate syngeneic mouse recipients, 10 times that number do not engraft in MHC-disparate allogeneic recipients. The addition of as few as 30,000 facilitating cells (CD8+/CD45R+/TCR-) is sufficient to permit engraftment of purified stem cells in MHC-disparate recipients. The cell surface phenotype of this purified cellular population differs significantly from other characterized lineages of lymphoid or myeloid origin. Based on multiparameter rare-events cell sorting, the facilitating fraction is CD8+, CD3+, CD45R+, Thy 1+, class IIdim/intermediate but alpha beta- TCR- and gamma delta-TCR-. This cellular population comprises approximately 0.4% of the total bone marrow and is separate from the hematopoietic stem cell. The coadministration of purified facilitating cells plus stem cells to optimize engraftment yet avoid GVHD may expand the potential application of bone marrow transplantation to disease states in which the morbidity and mortality associated with conventional BMT cannot be justified.

Abstract The clinical goal of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is to minimize GVHD while maintaining GvL. Here, we show that interferon γ receptor-deficient (IFNγR−/−) allogeneic Tconv, which possess normal alloreactivity and cytotoxicity, induce significantly less GVHD than wild-type (WT) Tconv. This effect is mediated by altered trafficking of IFNγR−/− Tconv to GVHD target organs, especially the gastrointestinal (GI) tract. We show that the chemokine receptor CXCR3 is induced via IFNγR-mediated signaling and partially contributes to the trafficking of WT Tconv to GVHD target organs. Indeed, CXCR3−/− Tconv recapitulate the reduced GVHD potential of IFNγR−/− Tconv in a minor-mismatched GVHD model. Most importantly, IFNγR−/− (and CXCR3−/−) Tconv mediate a robust and beneficial GvL effect. In addition, we show that IFNγR−/− regulatory T cells (Tregs) are fully suppressive in vitro although defective in suppressor function in vivo and that WT Tregs suppress GVHD in vivo only when allogeneic Tconv produce interferon γ (IFNγ), suggesting that the IFNγR signaling pathway is the major mechanism for both Tregs and Tconv to migrate to GVHD target organs. Finally, pharmacologic inhibition of IFNγR signaling with inhibitors of JAK1/JAK2, which are mediators of IFNγR signaling, results in the decreased expression of CXCR3 and reduced GVHD and improved survival after allo-HSCT and this effect is mediated by altered trafficking of Tconv to GVHD target organs.

Abstract Background: Graft-versus-host disease (GVHD) is a dreaded complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Such inflammatory diseases are fostered by damage to the intestinal barrier after transplantation. Consequently, the integrity and regeneration of the intestinal barrier is a key factor in the prevention of GVHD. On one side the main driver for regeneration of damaged gut epithelium are intestinal stem cells (ISC), but on the other side these cells are themselves primary targets of donor-derived T cells. One known mechanism of T cell mediated damage to the stem cell compartment is through IFN-γ dependent ISC toxicity. Yet, little is known about how T cells are contributing to the regeneration of damaged tissue after allo-HSCT and GVHD. Methods: To address this, we used preclinical models for allo-HSCT and GVHD including transplantation of recipient mice with escalating doses of Wildtype or IFN-γ-deficient allogeneic T cells and in the presence or absence of the JAK-1/2 inhibitor ruxolitinib. Intestinal regeneration was assessed by RNA-seq, flow cytometry and a newly established ex vivo organoid recovery assay. GVHD outcome was assessed by clinical scoring, histology and survival. Additionally, we established an allogeneic co-culture system of murine or human intestinal organoids with CD4+ conventional T cells or T regs -/+ Ruxolitinib. Effects on organoid growth and cell death were assessed by size measurements and manual counting after passaging. Results: We here demonstrate that recipient mice with increasingly dense intestinal infiltration by allogeneic T cells not only developed more severe GVHD (Fig. 1A), but also showed augmented recovery potential early after allo-HSCT (Fig. 1B). This was associated with intestinal gene signatures related to epithelial regeneration and protection from GVHD. Utilizing ex vivo cultures of intestinal organoids generated from murine allo-HSCT recipients, we found that development of GVHD but also regenerative capacity of ISCs were dependent on interferon (IFN)-γ-producing T cells in the intestine (Fig. 2A-B). Mice with fulminant GVHD and enhanced organoid recovery showed accumulation of intestinal regulatory T cells (Tregs) (Fig. 2C). Ex vivo, T regs nurtured growth of intestinal organoids in an IFN-γ dependent manner (Fig. 2D-E). This effect was diminished in intestinal organoids lacking IFNγR signaling, but was independent of T reg intrinsic IFNγR signaling (Fig. 2E-F). Intriguingly, treatment of murine allo-HSCT recipients with the JAK-1/2 inhibitor ruxolitinib enhanced epithelial organoid regeneration and numbers of intestinal Tregs (Fig. 3A-B). Similarily, growth of human intestinal organoids co-cultured with allogeneic T cells could be augmented by ruxolitinib treatment (Fig. 3C). We thus propose that the level and differentiation of infiltrating intestinal T cells determines both ISC damage and epithelial regeneration during immune-mediated tissue injury, leading to a sensitive equilibrium that can be modulated by therapeutic intervention. We also provide evidence that ruxolitinib improves ISC regeneration via IFNγ-producing Treg cells. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The feasibility and toxicity of allogeneic stem cell transplantation after nonmyeloablative conditioning including thiotepa, fludarabine, and cyclophosphamide have been investigated in 6 patients with breast cancer and 7 patients with renal cell cancer. The program included the use of escalating doses of donor lymphocyte infusions (DLI) and/or interferon alpha (IFNα) for patients showing no tumor response and no graft-versus-host disease (GVHD). Patients were at high risk of transplant-related mortality (TRM) because of age, advanced stage, and previous treatments. We observed a partial remission in 4 renal cancer and in 2 breast cancer patients (one at the molecular level in the bone marrow), occurring after cyclosporine withdrawal or after DLI and/or IFNα. All the responses were accompanied by the occurrence of acute GVHD. We conclude that reduced-intensity allogeneic stem cell transplantation is a feasible procedure in renal and breast cancer, and that the exploitation of graft-versus-tumor effect after DLI is a promising finding.

Abstract Hematopoietic stem cell transplantation (HSCT) is associated with significant posttransplantation gonadotoxicity. This deficit has been mainly attributed to pretransplantation conditioning, but lower sperm counts in humans also appear to be associated with graft-versus-host disease (GVHD) following allogeneic HSCT. However, the mechanisms leading to diminished spermatocyte levels during GVHD remain unknown. Here we demonstrate that injury to intratesticular cells occurs in unconditioned F1 mice following the infiltration of donor alloreactive T cells during an acute graft-versus-host reaction (GVHR). Using computer-aided quantitative microscopic morphometry we demonstrate that the nadir of Leydig cell volume density coincides with the peak of intratesticular infiltration by donor T cells. Injury to Leydig cells correlates with an intratesticular inflammatory response characterized by interferon-γ and tumor necrosis factor-α production. These results demonstrate impairment of testosterone-producing Leydig cells during a local alloresponse, thus representing a mechanism that contributes to gonadal insufficiency following allogeneic HSCT.

Introduzione La GVHD è la principale complicanza del trapianto allogenico di CSE ed è associata ad considerevole morbidità e mortalità. Una diagnosi precoce può migliorare l'outcome del trapianto. L'effetto delle citochine Th1 come l'IFN-γ è cruciale nella patogenesi del GVHD. Scopo della tesi e metodi La nostra ipotesi è che il monitoraggio dell'INF-Y dopo stimolo con PHA può aiutare il clinico nella gestione della GVHD. Un recente test ELISA (QuantiFERON®-CMV) è in grado di misurare la produzione di IFN-γ in whole blood in seguito a stimolo aspecfico. Lo scopo dello studio è quello di validare il metodo come biomarker di GVHD. Methods: QuantiFERON®‑CMV è un test diagnostico in vitro che usa un cocktail di antigeni di cytomegalovirus (CMV) per stimolare le cellule di sangue intero a produrre INF-Y. I campioni di plasma vengono stimolati da mitogeno (PHA) come controllo positivo. Dosaggio di IFN-γ con ELISA identifica le risposte. Per associare la GVHD con la produzione di INF-Y indotta da PHA abbiamo considerato 3 valori differenti di cutoff: 1) 0,5 IU/mL definito dal produttore, 2) 9 IU/mL definito sperimentalmente come la mediana dei campionamenti dei dati preliminari, 3) 5 IU/mL definito dalla mediana dei campionamenti dello studio prospettico. La diagnosi di GVHD era classificata in accordo con i criteri NCI. Trentasei pazienti sono stati arruolati e monitorati prospetticamente dopo trapianto ai seguenti timepoints: ogni 2-3 settimane fino a +180gg, successivamente mensilmente. Gli eventi(GVHD acuta e cronica, recidiva, morte) venivano registrati e i dati associati alla produzione di INF-Y indotta da PHA; in caso di GVHD era raccomandata la biopsia. Un modello di Cox a 2 step è stato utilizzato per l'analisi statistica per definire i fattori di rischio per lo sviluppo di GVHD. Results INF-Y indotto da PHA aumenta successivamente nei primi 3 mesi dal trapianto ed è associato indipendentemente allo sviluppo di GVHDall'analisi multivariata the multivariate analysis (p= 0.021 al primo mese, 0.015 al secondo, 0.027 al terzo) assieme al sesso (femmina), età, fonte di staminali, profilassi della GvHD e infezioni. La sensibilità del test era del 97% al cutoff 1 (0.5 IU/mL). La riduzione dell'INF-Y indotto da PHA è stata rilevata in tutti i pazienti trattati con immunosoppressione per GVHD eccetto in uno che ha sviluppato in una GVHD cronica steroide resistente. Conclusioni L'INF-Y indotto da PHA è un marker affidabile di GVHD e può aiutare il clinico nella gestione del trattamento. Ulteriori studi sono necessari per definire meglio i setting di pazienti che possono beneficiare di questo test in modo ottimale.
Introduction GVHD is associated with a high morbidity and mortality in alloSCT patients. An early diagnosis of GVHD could reduce this adverse impact on the outcome of alloSCT. The effect of Th1 cytokine IFN-γ is crucial in the pathogenesis of GVHD and, as expected, higher protein levels are reported in the serum of patients with active chronic GVHD. Aims and Methods Our hypotesis is that the monitoring of IFN-γ basal levels as well as IFN-γ induced by mitogen stimulation in the blood samples of patients after alloSCT could help the management and the prediction of GVHD. A recent ELISA based test (QuantiFERON®-CMV) could measure specific (anti-CMV) and aspecific production of IFN-γ in whole blood. The aim of this study is to assess the reliability of the positive control of the QuantiFERON®-CMV kit as new marker for GVHD early diagnosis during immune system reconstitution after bone marrow transplantation. Methods: QuantiFERON®‑CMV is an in vitro diagnostic test that use an antigenic human cytomegalovirus proteins (CMV) peptide cocktail to stimulate cells from whole blood and is used after alloSCT to monitor the occurrence of CMV infection. The mitogen-stimulated (PHA) plasma sample is used as a positive control for each specimen tested. Detection of interferon-γ (IFN-γ) by ELISA is used to identify responses. In order to assess the association between PHA stimulation IFN-γ production and GVHD, we decided to consider 3 different positivity of the test: 1) 0,5 IU/mL as defined by manufacturer, 2) 9 IU/mL as experimentally defined by the median of the observations in our preliminary data set. 3) 5 IU/mL as defined by the median of the values of the prospective study. GVHD extension was defined by Seattle criteria and/or the number of involved sites. A prospective study has been designed in order to confirm these data. Thirty-six patients were prospectively monitored after transplant with QuantiFERON®‑CMV according the following timepoints: every 2-3 weeks until +180d since transplantation, every month until +365d. Lymphocyte subpopulations +28, +56, +84, every month thereafter. Events (acute GVHD, chronic GVHD, relapse, death are registered in the database and matched with the production of PHA induced INF-y; when GVHD was suspected a biopsy of the target organ was recommended. A two step cox regression model for time dependent variables was implemented in order to define risk factors for cGvHD. Results PHA-induced INF-Y increase in the first three months after alloHSCT was independently associated with cGvHD at the multivariate analysis (p= 0.021, 0.015, 0.027 respectively) together with recipient sex (female), age, stem cell source, GvHD prophylaxis and infections. The sensitivity of the test was 97% at the cutoff 1 (0.5 IU/mL). The reduction of PHA-induced INF-Y was detected in all patients treated for a cGvHD but one who did not respond to immune-suppression. Conclusions PHA-induced INF-Y is a reliable biomarker of cGvHD and could help the physician in the management of cGvHD treatment. Further studies are needed in order to better define subsets of patients who could benefit from this test.