Induction Of T-cell Tolerance Research Articles

G-CSF Induces MAIT Cells to Exert Anti-Intestinal Gvhd Effects Dependent on CXCR6 Mediated Chemotaxis

Background: Graft-versus-host disease (GVHD) is a major complication leading to transplantation failure and affecting overall survival after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Granulocyte colony-stimulating factor (G-CSF) can induce T-cell tolerance and altering graft cell components to enhance GVHD prevention in HSCT. MAIT cells are a group of innate immune T cells. It has unique biological properties, including mucosal tissue enrichment, activation of microbial riboflavin derivatives and tissue repair properties, which determine MAIT cells play an important role in mucosal immunity. Previous studies found that a high MAIT cell counts in G-CSF-induced grafts (G-grafts) is associated with a low incidence of gut acute GVHD (aGVHD). However, the effect of G-CSF mobilization on the phenotype, function and its regulatory mechanism in GVHD of MAIT cells has not been elucidated. Methods: MAIT cells in PB samples from four healthy donors before and after G-CSF treatment were isolated for Single-cell RNA sequencing (Figure A). Flow cytometry, In vitro experiment and NOD-SCID-IL-2Rg−/− mice (NSG mice) were performed to observe the phenotypic, functional changes and the dynamic distribution of G-CSF induced donor-derived MAIT cells in recipients. Results: A total of 80709 MAIT cells were divided into 14 clusters. Based on key type-associated marker genes and gene sets, MAIT0 (clusters 5 and 9), MAIT1 (cluster 7), MAIT2 (clusters 4 , 11), Cycling MAIT (clusters 12, 13), Activation (clusters 0, 8), ISG (cluster 10) and MAIT17 (clusters 1,2,3,6) cells were identified (Figure B,C). The subset frequencies and expression signatures of immunosuppression (MAIT2 and Cycling cells from cluster 12) and tissue repair (Activation and MAIT17) were both increased at both transcriptome (Figure D) and protein levels, suggesting that G-CSF enhanced the immune tolerance and tissue repair of MAIT cells. In vitro further confirmed that G-CSF-induced MAIT cells showed a stronger inhibition for T cell proliferation (Figure E). Besides, overexpressed genes related to immune regulation in G-CSF induced MAIT cells were strongly enriched in cluster 12 with high CSF3R (G-CSF receptor) expression (Figure F). The cluster 12 also showed the enrichment of IL-17 and TNF signaling pathway favoring tissue repair function, while downregulation of Graft-versus-host disease pathway upon G-CSF mobilization, indicating that G-CSF-CSF3R interaction is the potential mechanism by which G-CSF mediates the immune regulation of MAIT cells. Based on whether recipients develop gut aGVHD or not after allo-HSCT, the matched donor grafts after G-CSF mobilization were divided into two groups termed as post-G-CSF-no gut aGVHD (n=3) and post-G-CSF-gut aGVHD group (n=1), respectively. MAIT cells in post-G-CSF-no gut aGVHD group overexpressed CSF3R and its related target genes, immunosuppression and tissue repair signatures compared with post-G-CSF-gut aGVHD. Besides, MAIT cells from G-PB of recipients without gut aGVHD had a higher CD114 expression (encoded by CSF3R) compared with gut aGVHD (Figure G). These results suggest that the enhanced immunosuppression and tissue repair function of MAIT cells induced by G-CSF may exert anti-gut aGVHD effects. The NSG mice confirmed MAIT cells in G-PB to tissue chemotaxis especially gut tissues in the recipient. A higher frequency of MAIT cells was detected in gut tissues in the low GVHD score group (GVHD Score＜3) compared with the high GVHD score group on post-transplant day 14 (Figure H), suggesting recruited MAIT cells in gut tissues favor anti-gut aGVHD effect. Moreover, CXCR6 was the most differentially expressed chemotactic receptor gene between post-G-CSF-no gut aGVHD and post-G-CSF-gut aGVHD group (Figure I). At the protein level, the expression of CXCR6 were significantly upregulated upon G-CSF administration (Figure I), while that of CCR6 and CXCR6 was non-significant. These results suggest that CXCR6 is necessary for G-CSF induced MAIT cell recruitment to the gut tissue. Conclusions G-CSF-CSF3R interactions enhance immunosuppression and tissue repair functions of MAIT cells, while CXCR6 recruits these functional MAIT cells from PB to gut tissues to exert anti-gut aGVHD effect. Hence, CSF3R and CXCR6 expression in G-CSF mobilized MAIT cells is crucial to exert anti-gut aGVHD effect, and has the potential to become predictive markers of gut aGVHD.

Calcineurin Inhibitors Inhibit Tolerance Induction By Suppressing Terminal Differentiation of Donor Exhausted T Cells after Allogeneic SCT

[Introduction] Calcineurin inhibitors (CNIs) reduce acute GVHD by inhibiting NFAT signaling in donor T cells but fail to prevent chronic GVHD (cGVHD). We have previously shown that donor alloreactive T cells differentiated into exhausted T cells with severely impaired alloreactivity after mouse allogeneic hematopoietic stem cell transplantation (allo-SCT) (Asakura S: JCI 2010). Given the critical role of NFAT signaling in T-cell exhaustion, we explored the role of CNIs in donor T-cell exhaustion and tolerance induction after allo-SCT using mouse SCT models with host alloantigen (allo-Ag)-specific 2C TCR-transgenic (2C) T cells and single cell RNA sequencing (scRNAseq) of donor T cells. [Methods] B6D2F1 recipients (H-2b/d) were lethally irradiated and transplanted with 1 x 106 2C T cells (CD8.1+) plus 1 x 106 wild type T cells (WT, CD45.1+) of B6 background (H-2b) together with T-cell-depleted bone marrow cells (TCD-BM) from WT B6 (CD45.2+) on day 0. In some experiments, recipients were p.o. injected with 25 mg/kg cyclosporin (CSP) daily on days 0-14. [Results] Flowcytometric analysis (FCM) showed 2C CD8+ T cells differentiated into terminally exhausted T cells (Tex) expressing multiple co-inhibitory receptors such as PD-1 and TIGIT on day 14 after allo-SCT, that led to unresponsiveness of donor T cells to PD-1 blockade. Next, we performed scRNAseq of donor T cells harvested from CSP- or vehicle-treated recipients on day 14 after allo-SCT. UMAP analysis identified 4 clusters of CD4+ T cells (C10-C13) and 9 clusters of CD8+ T cells (C1-C9), and all of them expressed Tox and Pdcd1, indicating that all donor T cells entered differentiation pathway to exhaustion. CSP remarkably increased C10 (CD4+) and C1 (CD8+) characterized by lower expression of Tox and Pdcd1 and higher expression of Ly6c2 compared to other clusters (Fig 1). FCM confirmed CSP significantly expanded PD-1+TIGIT-, and TOXlow cells in 2C and WT CD8+ and WT CD4+ donor T cells on day 14, and these cells persisted at least 2 wks after cessation of CSP. After adoptively transferred into irradiated secondary B6D2F1 recipients, Ly6C+ T cells purified from CSP-treated recipients were differentiated into Ly6C- Tex, while Ly6C- T cells remained to be Ly6C-, indicating that CSP-induced Ly6C+ donor T cells are precursors of Tex (pTex) like cells. We named these cells as pTex-CNI, based on lower TCF-1 expression compared to TCF-1high canonical pTex. We found that, among various agents known to suppress GVHD or Tex differentiation, only CSP and ibrutinib induced ToxlowPD-1+TIGIT-Ly6C+ pTex-CNI, suggesting that NFAT inhibition is critical for pTex-CNI induction. Importantly, only Ly6C+ donor T cells, but not Ly6C- cells, induced cGVHD after adoptive transfer, suggesting that pTex-CNI play a critical role in pathophysiology of cGVHD (Fig 2). pTex-CNI demonstrated marked proliferative response and differentiation toward Tex upon PD-1 blockade as canonical pTex did in tumor and viral infection models in the previous reports (Sade-Feldman M: Cell 2018, Hudson WH: Immunity 2019). Administration of PD-L1 mAbs from day 14 enhanced OXPHOS of donor T cells and eradicated host-type leukemia cells only in CSP-treated recipients. Post-transplant cyclophosphamide (PTCy) is widely used in clinical allo-SCT, and CNIs are started either before SCT or after PTCy typically on day 5. We found that pTex-CNI were induced only when CSP was added to PTCy from day -1, but not from day 5, suggesting that NFAT inhibition early after allo-SCT is critical for induction of pTex-CNI. Finally, we confirmed that T cells from the patients who underwent HLA-matched PBSCT with tacrolimus (TAC)-based GVHD prophylaxis started from day -1 expressed significantly lower levels of PD-1, TIGIT, and TOX on day 28 compared to those from patients underwent haploidentical PBSCT with PTCy-based GVHD prophylaxis combined with TAC started from day 5. [Conclusion] Our study for the first time demonstrated that CNI inhibits terminal differentiation of donor exhausted T cells after allo-SCT. In mouse models, pTex-CNI contributed to development of cGVHD and PD-1 blockade-induced anti-tumor response after allo-SCT. The timing of CNI initiation in PTCy-based allo-SCT could determine the persistence of donor T cell alloreactivity after allo-SCT, that could impact on the strategies of cessation of immunosuppressants and treatment of relapsed diseases. These should be tested in the future clinical studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification of the Immunological Changes Appearing in the CSF During the Early Immunosenescence Process Occurring in Multiple Sclerosis.

Patients with multiple sclerosis (MS) suffer with age an early immunosenescence process, which influence the treatment response and increase the risk of infections. We explored whether lipid-specific oligoclonal IgM bands (LS-OCMB) associated with highly inflammatory MS modify the immunological profile induced by age in MS. This cross-sectional study included 263 MS patients who were classified according to the presence (M+, n=72) and absence (M-, n=191) of LS-OCMB. CSF cellular subsets and molecules implicated in immunosenescence were explored. In M- patients, aging induced remarkable decreases in absolute CSF counts of CD4+ and CD8+ T lymphocytes, including Th1 and Th17 cells, and of B cells, including those secreting TNF-alpha. It also increased serum anti-CMV IgG antibody titers (indicative of immunosenescence) and CSF CHI3L1 levels (related to astrocyte activation). In contrast, M+ patients showed an age-associated increase of TIM-3 (a biomarker of T cell exhaustion) and increased values of CHI3L1, independently of age. Finally, in both groups, age induced an increase in CSF levels of PD-L1 (an inductor of T cell tolerance) and activin A (part of the senescence-associated secretome and related to inflammaging). These changes were independent of the disease duration. Finally, this resulted in augmented disability. In summary, all MS patients experience with age a modest induction of T-cell tolerance and an activation of the innate immunity, resulting in increased disability. Additionally, M- patients show clear decreases in CSF lymphocyte numbers, which could increase the risk of infections. Thus, age and immunological status are important for tailoring effective therapies in MS.

Dissection of T-cell tolerance induction by dendritic cell surface glycoproteins

Abstract Tolerogenic dendritic cells (DC) induce T-cell clonal deletion, anergy, generation and activation of regulatory T cells. In vitro production of TolDC has been accomplished, however the full molecular circuitry is not known. We aim to decipher the chemical signals and physical characteristics leading to T-cell tolerance in an artificial system. We have performed surface proteomic analysis of human peripheral blood cDC2 subset without and with in vitro stimulation with polyIC in vitro. The surface proteomics is based on biotinylation of sialic acids and the signal strength was 55-fold higher in the resting compared to the activated condition, presumably due to decreased sialation of glycoproteins in the activated cDC2. We found that label free quantification (LFQ), corrected for differences in sialation, agreed well with quantitative surface expression by flow cytometry. We combined the two datasets and ranked the top 120 glycoproteins with extracellular domains (ECD) > 60 amino acids by LFQ with approximately one third invariant, one third up-regulated/induced and one one-third down-regulated/lost on activation. The 120 ECD were expressed in HEK293 cells with C-terminal 10His tags and purified by Ni2+ affinity chromatography. Supported lipids bilayers (SLB) representing the tolerogenic and activating DC are being designed with different combinations of the 120 ECDs. We will adjust sialation to match resting and activated states. The SLB compositions will be tested for immunological synapse formation (fluorescent self-pMHC/CD80, ICAM-1 and CD58 for the cSMAC, pSMAC and dSMAC) and tolerance/activation spectrum with identification of relevant molecular combinations by machine learning.

Recombinant allergens for immunotherapy: state of the art.

Purpose of reviewMore than 30 years ago, the first molecular structures of allergens were elucidated and defined recombinant allergens became available. We review the state of the art regarding molecular AIT with the goal to understand why progress in this field has been slow, although there is huge potential for treatment and allergen-specific prevention.Recent findingsOn the basis of allergen structures, several AIT strategies have been developed and were advanced into clinical evaluation. In clinical AIT trials, promising results were obtained with recombinant and synthetic allergen derivatives inducing allergen-specific IgG antibodies, which interfered with allergen recognition by IgE whereas clinical efficacy could not yet be demonstrated for approaches targeting only allergen-specific T-cell responses. Available data suggest that molecular AIT strategies have many advantages over allergen extract-based AIT.SummaryClinical studies indicate that recombinant allergen-based AIT vaccines, which are superior to existing allergen extract-based AIT can be developed for respiratory, food and venom allergy. Allergen-specific preventive strategies based on recombinant allergen-based vaccine approaches and induction of T-cell tolerance are on the horizon and hold promise that allergy can be prevented. However, progress is limited by lack of resources needed for clinical studies, which are necessary for the development of these innovative strategies.

An approach to induction of tolerance to pig cardiac xenografts in neonates.

There is a continuing need for donor hearts for infants with complex congenital heart defects. The transplantation of hearts from neonatal pigs would be an alternative to human organs, particularly if donor-specific immunological tolerance could be achieved. The great majority of infant humans do not make natural (preformed) antibodies against triple-knockout (TKO) pigs (that do not express any of the three known pig antigens against which humans have natural anti-pig antibodies). The transplantation of a heart from a TKO pig into an infant would therefore minimize any risk of early antibody-mediated rejection, and, with adequate immunosuppressive therapy, prolonged graft survival may well be achieved. Total host thymectomy (commonly carried out at the time of orthotopic heart transplantation in this age group)±residual T-cell depletion and donor-specific pig thymus tissue transplantation might induce T-cell tolerance and allow immunosuppressive therapy to be discontinued (if there is in vitro evidence of T-cell and B-cell nonresponsiveness to donor-specific pig cells). Even if tolerance were not achieved, with continuing immunosuppressive therapy, the graft would likely "bridge" the patient until a suitable allograft became available or be associated with prolonged xenograft function.

Amelioration of progressive autoimmune encephalomyelitis by epigenetic regulation involves selective repression of mature neutrophils during the preclinical phase

We have recently demonstrated that treatment of NOD mice with the epigenetic drug Trichostatin A (TSA) ameliorated myelin peptide induced progressive experimental autoimmune encephalomyelitis (P-EAE). Protection was accompanied by induction of antigen-specific T-cell tolerance in the periphery and reduced influx of T cells into the spinal cord. In this investigation, we examined whether the epigenetic drug could impact the innate immune system as well. Whereas the mature (MHC class II+) CD11b+Ly-6G+ neutrophils expanded substantially in the peripheral lymphoid compartment during the preclinical phase, the MHC class II+, CD11b+Ly-6C+ mature monocytes increased modestly throughout the disease course. Amelioration of the clinical disease by TSA treatment was accompanied by diminished abundance of CD11b+Ly-6Gdim activated neutrophils in secondary lymphoid organs and their influx into the spinal cord without affecting monocytes. Interestingly, the co-inhibitory ligand CD274+ (PD-L1+) but not CD275+ (ICOS-L+), CD39+ or CD11c+ dendritic cells were decreased in the peripheral lymphoid compartment of drug treated mice. Thus, in addition to myelin-specific T cell tolerance induction observed previously, selective repression of mature neutrophils and PD-L1+ cells is critically involved in the epigenetic regulation of P-EAE.

Thymic epithelial cells require p53 to support their long-term function in thymopoiesis in mice

AbstractThymic epithelial cells (TECs) provide crucial microenvironments for T-cell development and tolerance induction. As the regular function of the thymus declines with age, it is of fundamental and clinical relevance to decipher new determinants that control TEC homeostasis in vivo. Beyond its recognized tumor suppressive function, p53 controls several immunoregulatory pathways. To study the cell-autonomous role of p53 in thymic epithelium functioning, we developed and analyzed mice with conditional inactivation of Trp53 in TECs (p53cKO). We report that loss of p53 primarily disrupts the integrity of medullary TEC (mTEC) niche, a defect that spreads to the adult cortical TEC compartment. Mechanistically, we found that p53 controls specific and broad programs of mTEC differentiation. Apart from restraining the expression and responsiveness of the receptor activator of NF-κB (RANK), which is central for mTEC differentiation, deficiency of p53 in TECs altered multiple functional modules of the mTEC transcriptome, including tissue-restricted antigen expression. As a result, p53cKO mice presented premature defects in mTEC-dependent regulatory T-cell differentiation and thymocyte maturation, which progressed to a failure in regular and regenerative thymopoiesis and peripheral T-cell homeostasis in the adulthood. Lastly, peripheral signs of altered immunological tolerance unfold in mutant mice and in immunodeficient mice that received p53cKO-derived thymocytes. Our findings position p53 as a novel molecular determinant of thymic epithelium function throughout life.

Antigen presenting cell-targeted proinsulin expression converts insulin-specific CD8+ T-cell priming to tolerance in autoimmune-prone NOD mice.

Type 1 diabetes (T1D) results from autoimmune destruction of insulin-producing pancreatic β cells. Therapies need to incorporate strategies to overcome the genetic defects that impair induction or maintenance of peripheral T-cell tolerance and contribute to disease development. We tested whether the enforced expression of an islet autoantigen in antigen-presenting cells (APC) counteracted peripheral T-cell tolerance defects in autoimmune-prone NOD mice. We observed that insulin-specific CD8+ T cells transferred to mice in which proinsulin was transgenically expressed in APCs underwent several rounds of division and the majority were deleted. Residual insulin-specific CD8+ T cells were rendered unresponsive and this was associated with TCR downregulation, loss of tetramer binding and expression of a range of co-inhibitory molecules. Notably, accumulation and effector differentiation of insulin-specific CD8+ T cells in pancreatic lymph nodes was prominent in non-transgenic recipients but blocked by transgenic proinsulin expression. This shift from T-cell priming to T-cell tolerance exemplifies the tolerogenic capacity of autoantigen expression by APC and the capacity to overcome genetic tolerance defects.

Cyclic MOG35–55 ameliorates clinical and neuropathological features of experimental autoimmune encephalomyelitis

EAE is induced to susceptible mice using linear peptides of myelin proteins of the central nervous system. Specific peptide motifs within the peptide-binding groove of the MHC peptide-complex determines the affinity of the peptide in each animal and the consequent T-cell receptor recognition and activation of the cell. Altered peptide ligand (APL) vaccination is a novel approach based on an effort to induce T-cell tolerance or alter cytokine profile from pro-inflammatory to anti-inflammatory. In the present study we synthesized the MOG35–55 peptide and altered its 3-dimensional conformation to make it a cyclic one (c-MOG35–55). EAE was induced in C57BL/6 mice and pathology was studied on acute and chronic phase of the disease. Our data indicates that c-MOG35–55 peptide alone induces a mild transient acute phase without chronic axonopathy. Administration of the c-MOG35–55 peptide at a 1:1 ratio during disease induction significantly ameliorates clinical disease and underlying pathology, such as demyelination and axonopathy in the acute and chronic phases. Binding and structural studies revealed milder interactions between the c-MOG35–55 and mouse or human MHC class II alleles (H2-IAb and HLA-DR2). Collectively, we provide data supporting for the first time the concept that the cyclic modification of an established encephalitogenic peptide ameliorates the clinical outcomes and underlying pathological processes of EAE. Such a cyclic modification of linear peptides could provide a novel treatment approach for future, patient-selective, immunomodulative treatments of multiple sclerosis.

Short-course rapamycin treatment enables engraftment of immunogenic gene-engineered bone marrow under low-dose irradiation to permit long-term immunological tolerance

BackgroundApplication of genetically modified hematopoietic stem cells is increasingly mooted as a clinically relevant approach to protein replacement therapy, immune tolerance induction or conditions where both outcomes may be helpful. Hematopoietic stem and progenitor cell (HSPC)-mediated gene therapy often requires highly toxic pretransfer recipient conditioning to provide a ‘niche’ so that transferred HSPCs can engraft effectively and to prevent immune rejection of neoantigen-expressing engineered HSPCs. For widespread clinical application, reducing conditioning toxicity is an important requirement, but reduced conditioning can render neoantigen-expressing bone marrow (BM) and HSC susceptible to immune rejection if immunity is retained.MethodsBM or HSPC-expressing OVA ubiquitously (actin.OVA) or targeted to MHC II+ cells was transferred using low-dose (300 cGy) total body irradiation. Recipients were administered rapamycin, cyclosporine or vehicle for 3 weeks commencing at BM transfer. Engraftment was determined using CD45 congenic donors and recipients. Induction of T-cell tolerance was tested by immunising recipients and analysing in-vivo cytotoxic T-lymphocyte (CTL) activity. The effect of rapamycin on transient effector function during tolerance induction was tested using an established model of tolerance induction where antigen is targeted to dendritic cells.ResultsImmune rejection of neoantigen-expressing BM and HSPCs after low-dose irradiation was prevented by a short course of rapamycin, but not cyclosporine, treatment. Whereas transient T-cell tolerance developed in recipients of OVA-expressing BM administered vehicle, only when engraftment of neoantigen-expressing BM was facilitated with rapamycin treatment did stable, long-lasting T-cell tolerance develop. Rapamycin inhibited transient effector function development during tolerance induction and inhibited development of CTL activity in recipients of OVA-expressing BM.ConclusionsRapamycin acts to suppress acquisition of transient T-cell effector function during peripheral tolerance induction elicited by HSPC-encoded antigen. By facilitating engraftment, short-course rapamycin permits development of long-term stable T-cell tolerance.

Chemotherapy-Dependent ATP Release from Leukemia Dying Cells Induces Indoleamine 2,3-Dioxygenase 1 in Dendritic Cells

BACKGROUND: Overall survival of adult acute myeloid leukemia (AML) remains poor due to the lack of novel and effective therapies. The cancer cell death induced by some chemotherapeutic agents, especially anthracyclines, such as daunorubicin (DNR), named immunogenic cell death (ICD), is characterized by intra- and peri-cellular modifications, which favor the induction of anti-tumor T-cell immune response. Among them, the extracellular release of adenosine triphosphate (extracellular ATP, eATP) from dying tumor cells primes dendritic cells (DCs) by activating purinergic P2X7 receptors, thus eliciting the presentation of tumor antigens to T cell. DCs are key regulators of adaptive immunity, promoting or suppressing T-cell responses. One of the suppressive mechanisms involves the expression of indoleamine 2,3-dioxygenase 1 (IDO1), which plays a major role in the induction of T-cell tolerance through the expansion of regulatory T cells (Tregs). The present study aimed at evaluating the involvement of IDO-1 during ATP-driven ICD in AML.METHODS: AML patients were analyzed at diagnosis and after DNR-based chemotherapy. Ex vivo T cells were characterized by FACS and tested for their capacity to produce IFN-γ in response to autologous blasts. Then, CD8+IFN-γ-producing T cells were expanded and further characterized. ATP was used as an ICD representative model. In vitro, murine WEHI-3B and human HL-60 leukemic cell lines and primary blasts were tested for ATP release after DNR treatment. To in vivo investigate DNR-induced ICD, WEHI-3B cells stable transfected with luciferase PmeLUC were inoculated subcutaneously in BALB/c mice to measure ATP release directly from tumor mass. Tumor infiltrating DCs and T cells were characterized by FACS and immunohistochemistry after chemotherapy and plasma levels of cytokines were measured. In vitro DNR-treated AML cells were pulsed into immature DCs, previously generated from healthy donors. DCs maturation and IDO1 expression were examined (by FACS and western blot, respectively) and correlated with the presence of ATP in culture medium. IDO-driven Tregs induction was assessed. Finally, functional immunological tests were performed in vitro to test the ability of Tregs to inhibit leukemia antigen-specific IFN-γ production (FACS analysis) by ICD-activated T cells.RESULTS: After chemotherapy, 15/23 AML patients had an increase in leukemia-specific IFN-γ producing CD4+ and CD8+ T cells. Also an increase of Tregs was observed with a peak at day 21. CD8+ IFN-γ-producing T cells, which resulted in a skewing toward an effector memory phenotype, were activated and cytotoxic against autologous AML blasts but showed features of exhaustion and were defective in perforin production. In vitro and in vivo DNR induced ATP release from AML cells. In vivo the analysis of tumor-infiltrating T cells after treatment has shown an exhausted phenotype of cytotoxic CD8+ cells, increased IFN-γ+ Tregs and decreased TNF-α+ effector T cells. DNR treatment also increased in vivo plasma levels of cytokines IFN-γ, IL-1β, TNF-α, IL-12. Moreover, in DNR-treated mice we observed a significant increase of CD11c+ mature DCs which express IDO1 in tumor infiltrate. In vitro, loading of DNR-treated AML cells into DCs resulted in increased maturation, but also in IDO1 induction. Interestingly, extracellular ATP was directly involved in DCs maturation and IDO1 expression via purinergic receptor P2Y11. ICD-driven DCs were able to expand Tregs in an IDO-dependent manner. Finally, ICD both triggers a leukemia-specific IFN-γ production by CD8+T cells and induces Tregs, via IDO1-expressing DCs, which in turn inhibit leukemia-specific T cell.CONCLUSIONS: Overall, our data indicate that in AML chemotherapy-induced ICD has contrasting, and not fully elucidated, effects on T-cell immune response, resulting in the induction of leukemia-specific CTLs, albeit with defective features, and Tregs. In this scenario, the effects of ATP release from dying leukemia cells on DCs may be pivotal, as indicated by its capacity to concomitantly induce DC maturation and activation as well as tolerogenic function via IDO1. The combination of novel immunological drugs, such as IDO1 and/or checkpoint inhibitors, with conventional chemotherapy may represent an interesting approach to contrast tolerance induction and, then, fully exploit the immunogenic effect of chemotherapy. DisclosuresMartinelli:Genentech: Consultancy; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; MSD: Consultancy; BMS: Speakers Bureau. Cavo:Celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

Mannose receptor induces T-cell tolerance via inhibition of CD45 and up-regulation of CTLA-4.

The mannose receptor (MR) is an endocytic receptor involved in serum homeostasis and antigen presentation. Here, we identify the MR as a direct regulator of CD8(+) T-cell activity. We demonstrate that MR expression on dendritic cells (DCs) impaired T-cell cytotoxicity in vitro and in vivo. This regulatory effect of the MR was mediated by a direct interaction with CD45 on the T cell, inhibiting its phosphatase activity, which resulted in up-regulation of cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) and the induction of T-cell tolerance. Inhibition of CD45 prevented expression of B-cell lymphoma 6 (Bcl-6), a transcriptional inhibitor that directly bound the CTLA-4 promoter and regulated its activity. These data demonstrate that endocytic receptors expressed on DCs contribute to the regulation of T-cell functionality.

The Self-Specific Activation Receptor SLAM Family Is Critical for NK Cell Education

NK cell education, a term describing a process for NK cell acquisition of functional competence, is primarily achieved by self-MHC-I-specific inhibitory receptors. In this study, we have demonstrated that SLAM family receptors (SFRs) redundantly expressed on hematopoietic cells function as self-specific activation receptors critical for NK cell education. To overcome gene redundancy, we generated mice simultaneously lacking seven SFRs, revealing that NK-cell-mediatedrejection of semi-allogeneic hematopoietic cells largely depended on the presence of SFRs on target cells. This stimulatory effect was determined by the presence of SFR-coupled adaptors; however, SFR-deficient mice displayed enhanced reactivity to hematopoietic cells. These findings demonstrate that SFRs endow NK cells with an ability to kill hematopoietic cells during the effector phase; however, the sustained engagement of SFRs can desensitize NK cell responses during an education process. Therefore, self-specific activating ligands may be "tolerogens" for NK cells, akin to self-antigens that induce Tcell tolerance.

Donor CD47 controls T cell alloresponses and is required for tolerance induction following hepatocyte allotransplantation.

CD47-deficient hepatocyte transplantation induces rapid innate immune cell activation and subsequent associated graft loss in syngeneic recipients. However, the role of donor CD47 in regulation of T-cell alloresponses is poorly understood. We addressed this question by assessing OVA-specific immune responses in mice following hepatocyte transplantation from CD47-competent or -deficient OVA-transgenic donors. Compared to sham-operated controls, intrasplenic transplantation of CD47-deficient OVA+ hepatocytes significantly accelerated rejection of OVA+ skin grafted 7 days after hepatocyte transplantation. In contrast, mice receiving CD47-competent OVA+ hepatocytes showed prolonged and even indefinite survival of OVA+ skin allografts. T cells from mice receiving CD47-deficient, but not CD47-competent, OVA+ hepatocytes showed significantly enhanced responses to OVA+ stimulators compared to sham-operated controls. In contrast to the production of tolerogenic cytokines (IL-4 and IL-10) in the recipients of CD47-competent hepatocytes, mice receiving CD47-deficient hepatocytes showed elevated production of IFN-γ and IL-1α. Moreover, significant expansion of myeloid-derived suppressor cells was detected in the recipients of CD47-competent hepatocytes, which was required for tolerance induction in these mice. Thus, donor CD47 plays an important role in the control of T-cell alloresponses and tolerance induction following hepatocyte transplantation. Our data also suggest that intrasplenic hepatocyte transplantation may provide a means to induce allograft tolerance.

The role of bone marrow promiscuous gene expression in peripheral tolerance

Abstract For the negative selection of autoreactive T cells, peripheral tissue-restricted antigens (TRAs) are ectopically expressed and presented by medullary thymic epithelial cells (mTECs). This physiological property of mTECs, termed promiscuous gene expression (pGE), is predominantly regulated by the transcription factor, autoimmune regulator (AIRE). In the periphery, pGE was also found in secondary lymphoid organs and can induce peripheral immune tolerance. Bone marrow is an important organ known to prime naïve conventional T cells, as well as being a reservoir of regulatory T cells. However, the occurrence of pGE in the bone marrow is hitherto unknown. We hypothesize that a repertoire of auto-antigens is ectopically expressed and presented in the bone marrow, which maintains the peripheral tolerance of T cells. We investigated the presence of AIRE-expressing cells in bone marrow using a transgenic reporter mouse model in which GFP is expressed under the control of the AIRE promoter. AIRE expression was detected at both the RNA and protein levels. The bone marrow-resident AIRE-expressing cells (BMACs) anatomically locate adjacent to clusters of CD4+ T cells and express high levels of CD45, MHC-II and EpCAM. Their tolerogenic characteristic was conferred by the expression of PD-L1 and lack of costimulatory molecules CD80 and CD86. Gene expression analysis showed the AIRE-dependent TRA genes expressed in BMACs differ from those expressed in mTECs. BMACs were also detected at various frequencies in patients carrying different tumor entities. Our result demonstrates that the AIRE-expressing cells which ectopically express TRAs in the bone marrow could comprise the cellular basis for the induction of peripheral T-cell tolerance.

Targeting PD-1/PD-L1 in the treatment of metastatic renal cell carcinoma.

Immunostimulatory therapies have been a cornerstone of treatment for metastatic renal cell carcinoma (RCC) since the 1990s. However, the use of traditional immunotherapeutic approaches for RCC, such as high-dose interleukin-2 and interferon-α, has been limited by significant systemic toxicities and the need to deliver these therapies at centers of expertise. Furthermore, in spite of the success of these immunostimulatory therapies for some patients with RCC, it is clear that most patients fail to respond to cytokine therapy. More effective immune therapy for RCC has therefore been necessary. The interaction between programmed death-1 (PD-1, present on T cells), and one of its ligands (PD-L1, present on antigen-presenting cells and tumor cells) constitutes an immune checkpoint through which tumors can induce T-cell tolerance and avoid immune destruction. Monoclonal antibodies that disrupt the PD-1/PD-L1 interaction serve as inhibitors of this immune checkpoint, and have demonstrated favorable activity in RCC as monotherapy and in combination with other active agents. This review summarizes the current landscape of anti-PD-1/PD-L1 therapy for RCC, and highlights challenges for the future development of this promising approach.

Induction of central tolerance by the factor Aire: molecular and epigenetic regulation

The establishment of thymic central tolerance is a critical process to prevent the development of autoimmune diseases. Medullary thymic epithelial cells (mTEC) are essential to this process through the expression of the transcription factor Aire, which controls the transcription of many genes encoding tissue-restricted antigens. Mutations in the Aire gene are responsible for a rare autoimmune disorder called APECED (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy). This review summarizes our current knowledge on the mode of action of Aire at the molecular and epigenetic levels in controlling the expression of tissue-restricted antigens. We also discuss recently described additional roles of this transcription factor in the induction of central T-cell tolerance.

Thymic Crosstalk Coordinates Medulla Organization and T-Cell Tolerance Induction.

The thymus ensures the generation of a functional and highly diverse T-cell repertoire. The thymic medulla, which is mainly composed of medullary thymic epithelial cells (mTECs) and dendritic cells (DCs), provides a specialized microenvironment dedicated to the establishment of T-cell tolerance. mTECs play a privileged role in this pivotal process by their unique capacity to express a broad range of peripheral self-antigens that are presented to developing T cells. Reciprocally, developing T cells control mTEC differentiation and organization. These bidirectional interactions are commonly referred to as thymic crosstalk. This review focuses on the relative contributions of mTEC and DC subsets to the deletion of autoreactive T cells and the generation of natural regulatory T cells. We also summarize current knowledge regarding how hematopoietic cells conversely control the composition and complex three-dimensional organization of the thymic medulla.

DCIR2+ cDC2 DCs and Zbtb32 Restore CD4+ T-Cell Tolerance and Inhibit Diabetes.

During autoimmunity, the normal ability of dendritic cells (DCs) to induce T-cell tolerance is disrupted; therefore, autoimmune disease therapies based on cell types and molecular pathways that elicit tolerance in the steady state may not be effective. To determine which DC subsets induce tolerance in the context of chronic autoimmunity, we used chimeric antibodies specific for DC inhibitory receptor 2 (DCIR2) or DEC-205 to target self-antigen to CD11b(+) (cDC2) DCs and CD8(+) (cDC1) DCs, respectively, in autoimmune-prone nonobese diabetic (NOD) mice. Antigen presentation by DCIR2(+) DCs but not DEC-205(+) DCs elicited tolerogenic CD4(+) T-cell responses in NOD mice. β-Cell antigen delivered to DCIR2(+) DCs delayed diabetes induction and induced increased T-cell apoptosis without interferon-γ (IFN-γ) or sustained expansion of autoreactive CD4(+) T cells. These divergent responses were preceded by differential gene expression in T cells early after in vivo stimulation. Zbtb32 was higher in T cells stimulated with DCIR2(+) DCs, and overexpression of Zbtb32 in T cells inhibited diabetes development, T-cell expansion, and IFN-γ production. Therefore, we have identified DCIR2(+) DCs as capable of inducing antigen-specific tolerance in the face of ongoing autoimmunity and have also identified Zbtb32 as a suppressive transcription factor that controls T cell-mediated autoimmunity.