Establishment Of Immune Tolerance Research Articles

Immune tolerance and the prevention of autoimmune diseases essentially depend on thymic tissue homeostasis.

The intricate balance of immune reactions towards invading pathogens and immune tolerance towards self is pivotal in preventing autoimmune diseases, with the thymus playing a central role in establishing and maintaining this equilibrium. The induction of central immune tolerance in the thymus involves the elimination of self-reactive T cells, a mechanism essential for averting autoimmunity. Disruption of the thymic T cell selection mechanisms can lead to the development of autoimmune diseases. In the dynamic microenvironment of the thymus, T cell migration and interactions with thymic stromal cells are critical for the selection processes that ensure self-tolerance. Thymic epithelial cells are particularly significant in this context, presenting self-antigens and inducing the negative selection of autoreactive T cells. Further, the synergistic roles of thymic fibroblasts, B cells, and dendritic cells in antigen presentation, selection and the development of regulatory T cells are pivotal in maintaining immune responses tightly regulated. This review article collates these insights, offering a comprehensive examination of the multifaceted role of thymic tissue homeostasis in the establishment of immune tolerance and its implications in the prevention of autoimmune diseases. Additionally, the developmental pathways of the thymus are explored, highlighting how genetic aberrations can disrupt thymic architecture and function, leading to autoimmune conditions. The impact of infections on immune tolerance is another critical area, with pathogens potentially triggering autoimmunity by altering thymic homeostasis. Overall, this review underscores the integral role of thymic tissue homeostasis in the prevention of autoimmune diseases, discussing insights into potential therapeutic strategies and examining putative avenues for future research on developing thymic-based therapies in treating and preventing autoimmune conditions.

A novel missense mutation in the AIRE gene underlying autoimmune polyglandular syndrome type 1.

The immune regulator gene AIRE plays an essential role in the establishment of immune tolerance and the prevention of autoimmunity. This transcription factor plays a critical role in promoting self-tolerance in the thymus by regulating the expression of a large number of self-antigens that share the common feature of being tissue-restricted in their expression pattern in the periphery. Dysfunction of AIRE in humans causes a rare disease, autoimmune polyglandular syndrome type 1 (APS1), characterized by an autoimmune response against peripheral tissues, particularly endocrine tissues. Although a few dominant mutations have been described, the inactivation of AIRE is usually caused by recessive mutations. Recent data suggests that alterations in AIRE function contribute not only to APS1 but also to more common forms of autoimmune disease. Here, we present a previously unreported missense mutation (NM_000383.2:c.260T > C) in exon 2 of the AIRE gene, predicted to cause the substitution (p.(Leu87Pro)) in the CARD domain of the AIRE protein. When inherited in conjunction with another dysfunctional AIRE allele, this mutation was associated with immune dysregulation in a pediatric patient. The presence of hypergammaglobulinemia, malabsorption syndrome, ectodermal dysplasia, mucocutaneous candidiasis, vitiligo, and hypothyroidism as well as the presence of multiple autoantibodies allowed us to confirm an APS1 diagnosis.

Role of Kupffer cells in tolerance induction after liver transplantation.

Currently, liver transplantation has reached a level of maturity where it is considered an effective treatment for end-stage liver disease and can significantly prolong the survival time of patients. However, acute and chronic rejection remain major obstacles to its efficacy. Although long-term use of immunosuppressants can prevent rejection, it is associated with serious side effects and significant economic burden for patients. Therefore, the investigation of induced immune tolerance holds crucial theoretical significance and socio-economic value. In fact, the establishment of immune tolerance in liver transplantation is intricately linked to the unique innate immune system of the liver. Kupffer cells, as a crucial component of this system, play a pivotal role in maintaining the delicate balance between inflammatory response and immune tolerance following liver transplantation. The important roles of different functions of Kupffer cells, such as phagocytosis, cell polarization, antigen presentation and cell membrane proteins, in the establishment of immune tolerance after transplantation is comprehensively summarized in this paper. Providing theoretical basis for further study and clinical application of Kupffer cells in liver transplantation.

Membrane-bound IL-2 improves the expansion, survival, and phenotype of CAR Tregs and confers resistance to calcineurin inhibitors.

Regulatory T cells (Tregs) play an important role in the maintenance of immune homeostasis and the establishment of immune tolerance. Since Tregs do not secrete endogenous IL-2, they are especially dependent on external IL-2. IL-2 deficiency leads to lower Treg numbers, instability of the Treg phenotype and loss of immune regulation. After organ transplantation, patients are treated with calcineurin inhibitors (CNIs), which further limits available IL-2. Application of low-dose IL-2 expands Tregs but also activates NK and CD8+ T cells. It was recently shown that graft-specific Tregs recognizing mismatched MHC I molecules via a chimeric antigen receptor were far more potent than polyclonal Tregs in the regulation of immune responses after solid organ transplantation in a humanized mouse model. Therefore, our aim was to enhance the function and stability of transferred CAR-Tregs via expression of membrane-associated IL-2 (mbIL-2). mbIL-2 promoted higher survival, phenotypic stability, and function among CAR-Tregs than observed in clinical trials. The cells were also more stable under inflammatory conditions. In a preclinical humanized mouse model, we demonstrated that mbIL-2 CAR Tregs survive better in the Treg niche than control CAR Tregs and are even resistant to CNI therapy without affecting other Tregs, thus acting mainly in cis. The functional and phenotypic improvements observed after membrane-attached IL-2 expression in CAR-Tregs will be important step for enhancing CAR-Treg therapies currently being tested in clinical trials for use after kidney and liver transplantation as well as in autoimmune diseases.

Decidual CXCR4+ CD56bright NK cells as a novel NK subset in maternal-foetal immune tolerance to alleviate early pregnancy failure.

Natural killer (NK) cells preferentially accumulate at maternal–foetal interface and are believed to play vital immune‐modulatory roles during early pregnancy and related immunological dysfunction may result in pregnant failure such as recurrent miscarriage (RM). However, the mechanisms underlying the establishment of maternal–foetal immunotolerance are complex but clarifying the roles of decidual NK (dNK) cells offers the potential to design immunotherapeutic strategies to assist RM patients. In this report, we analysed RNA sequencing on peripheral NK (pNK) and decidual NK cells during early pregnancy; we identified an immunomodulatory dNK subset CXCR4+CD56brightdNK and investigated its origin and phenotypic and functional characteristics. CXCR4+CD56brightdNK displayed a less activated and cytotoxic phenotype but an enhanced immunomodulatory potential relative to the CXCR4 negative subset. CXCR4+CD56brightdNK promote Th2 shift in an IL‐4‐dependent manner and can be recruited from peripheral blood and reprogramed by trophoblasts, as an active participant in the establishment of immune‐tolerance during early pregnancy. Diminished CXCR4+ dNK cells and their impaired ability to induce Th2 differentiation were found in RM patients and mouse models of spontaneous abortion. Moreover, adoptive transfer of CXCR4+ dNK cells to NK‐deficient (Nfil3–/–) mice showed great therapeutic potential of CXCR4+ dNK via recovering the Th2/Th1 bias and reducing embryo resorption rates. The identification of this new dNK cell subset may lay the foundation for understanding NK cell mechanisms in early pregnancy and provide potential prognostic factors for the diagnosis and therapy of RM.

Harnessing Treg Homeostasis to Optimize Posttransplant Immunity: Current Concepts and Future Perspectives.

CD4+CD25+Foxp3+ regulatory T cells (Tregs) are functionally distinct subsets of mature T cells with broad suppressive activity and have been shown to play an important role in the establishment of immune tolerance after allogeneic hematopoietic stem cell transplantation (HSCT). Tregs exhibit an activated phenotype from the stage of emigration from the thymus and maintain continuous proliferation in the periphery. The distinctive feature in homeostasis enables Tregs to respond sensitively to small environmental changes and exert necessary and sufficient immune suppression; however, on the other hand, it also predisposes Tregs to be susceptible to apoptosis in the inflammatory condition post-transplant. Our studies have attempted to define the intrinsic and extrinsic factors affecting Treg homeostasis from the acute to chronic phases after allogeneic HSCT. We have found that altered cytokine environment in the prolonged post-HSCT lymphopenia or peri-transplant use of immune checkpoint inhibitors could hamper Treg reconstitution, leading to refractory graft-versus-host disease. Using murine models and clinical trials, we have also demonstrated that proper intervention with low-dose interleukin-2 or post-transplant cyclophosphamide could restore Treg homeostasis and further amplify the suppressive function after HSCT. The purpose of this review is to reconsider the distinctive characteristics of post-transplant Treg homeostasis and discuss how to harness Treg homeostasis to optimize posttransplant immunity for developing a safe and efficient therapeutic strategy.

Characterization of an Expanded IL-10-Producing-Suppressive T Cell Population Associated with Immune Tolerance.

An increase in the number of glucocorticoid-induced tumor necrosis factor receptor-family related gene/protein (GITR)+CD25- (or fork-head box protein 3: Foxp3-) CD4+ T cells, after treating a mouse model of arthritis with fingolimod (FTY720), and a pathogenic antigen may play a key role in the establishment of immune tolerance. In this study, we characterized a specific expanded T cell subset in this population. Mice with glucose-6-phosphate isomerase peptide (GPI325-339)-induced arthritis were treated with FTY720 (1 mg/kg, per os) and GPI325-339 (10 µg/mouse, intravenously) for five days, starting from the onset of symptoms. The expanded GITR+CD25- (or Foxp3-) CD4+ T cell population and its cytokine production were examined using flow cytometry. Furthermore, time-dependent changes in T-bet and/or early growth response gene 2 (Egr-2) expression in this T cell subset were examined. The density of T cell immunoreceptors with immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibition motif domains (TIGIT)+CD39+ cell subset in the GITR+Foxp3-CD4+ T cell population was significantly increased only in the combined treatment group, compared to that in the untreated and single-treatment groups. In the TIGIT+CD39+GITR+Foxp3-CD4+ T cell population, T-bet+Egr-2+/T-bet+Egr-2- cell ratio increased in the latter stage of the treatment. Furthermore, this T cell subset, which corresponded to a T helper 1 (Th1) response, produced high levels of both interleukin (IL)-10 and interferon (IFN)-γ. In conclusion, expanded TIGIT+CD39+GITR+Foxp3-CD4+ T cells shifted from an effector Th1 to IL-10-producing-suppressor T cell phenotype, which may promote an immune-tolerant state.

Leukemia Relapse after Allogeneic Hematopoietic Stem Cell Transplantation: From Recapitulation/Acquisition of Leukemogenic Hits to Immune Escape Due to Somatic Class I/ II HLA Mutations

Curative potential of allogeneic hematopoietic cell transplantation (aHCT) in myeloid malignancies is principally related to the graft versus leukemia (GvL) effect exerted by donor-derived immune effectors on leukemic cells. However, different pathways may drive post-transplant relapse, including perturbation/attenuation of T cell-mediated GvL responses. For example, decreased expression of HLA alleles has been described in post-aHCT relapse.1 This could be due to down-regulation of HLA or 6p HLA locus deletion. However, the occurrence of chromosome 6p uniparental disomy (UPD) along with del6p (first described by our group in the context of aplastic anemia)2 suggests that HLA loss of heterozygosity (LOH) and inherent loss of one allele (presenting immunodominant peptides) may be a significant contributor to leukemic relapse either directly, causing decreased HLA expression or as additive effect to HLA down-regulation. In relapses after haploidentical or mismatched aHCT, LOH of mismatched allele has been described.3 Here, we hypothesize that not only copy neutral LOH or haploinsufficient HLA expression but also defective function due to HLA mutations may lead to immune escape from GvL. Such a mechanism would involve the loss of an allele responsible for the presentation of an immunodominant antigenic peptide. Moreover, unlike relapse due to the acquisition of additional myeloid mutations, HLA mutant relapse would also acquire resistance to donor lymphocyte infusion (DLI) therapy. In order to dissect the immunogenomic mechanisms leading to leukemic immune-evasion, we performed a comprehensive genetic analysis of specimens sequentially collected from a cohort of patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) relapsed after aHCT. Specifically, we applied a deep-targeted NGS panel to study HLA region along with 173 genes known to have a role in leukemogenesis and cancer ontogeny. So far, 57 paired/serial biospecimens from 25 transplanted patients have been analyzed (25 samples at AML/MDS diagnosis, 25 at the moment of relapse after aHCT and 7 samples at relapse after chemotherapy) Overall, we found the acquisition of 8 disruptive HLA somatic mutations in 6 patients at post-transplant relapse (24%), 4 in class I and 4 in class II loci. None of those events were found in samples at diagnosis or at post-chemotherapy relapse, suggesting the possibility of an "immune-escape relapse". Those somatic hits accounted for 4 intronic indels, 1 frameshift insertion, one splicing site and 2 point mutations in 3′ and 5′ untranslated regions (UTRs). Median variant allele frequency (VAF) was 17% (range 2-58%). Of note is that all HLA mutant patients received a matched aHCT (4 from related and 2 from a 10/10 matched unrelated donors) suggesting that this mechanism is independent from the deletion and the loss of an immune privileged mismatched allele. Interestingly, median time to relapse was 514 (range 119-935) days for HLA-mutated patients vs 126 (62-543) for HLA wild type cases (p=.00042), consistent with the hypothesis that the establishment of immune-tolerance, and the presence of a GvL effect (less likely in early relapses) are required for the selection of those mutations. When somatic genotype of these patients prior and after transplant was studied, we found that post-aHCT relapses were associated in most cases with a new genomic configuration with either loss of previous events or acquisition of new subclonal mutations in myeloid or cancer related genes (in 17/25 cases). However no difference was seen in terms of number and patterns of new events among the HLA mutated and HLA wild type patients. Results shown here represent an important proof-of-concept for the role played by somatic mutations in HLA genes in the setting of post-aHCT AML/MDS relapses. These events, in analogy to the deletion or copy neutral LOH, may promote immune escape relapse resistant to immunologic manipulations and may coexist or be an alternative pathway to the progression/relapse characterized by acquisition of myeloid subclonal driver mutations. In that context, HLA mutations would be considered facilitator lesions. Disclosures Hsi: CytomX: Consultancy, Honoraria; Eli Lilly: Research Funding; Abbvie: Research Funding; Miltenyi: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria. Hamilton:Syndax Pharmaceuticals: Consultancy, Honoraria. Carraway:Abbvie: Other: Independent Advisory Committe (IRC); BMS: Consultancy, Other: Research support, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Jazz: Consultancy, Speakers Bureau; Stemline: Consultancy, Speakers Bureau; Takeda: Other: Independent Advisory Committe (IRC); ASTEX: Other: Independent Advisory Committe (IRC). Majhail:Incyte: Honoraria; Mallinckrodt: Honoraria; Nkarta Therapeutics: Honoraria; Anthem, Inc.: Consultancy. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

INKT cells with high PLZF expression are recruited into the lung via CCL21-CCR7 signaling to facilitate the development of asthma tolerance in mice.

Establishment of immune tolerance is crucial to protect humans against asthma. Promyelocytic leukemia zinc finger (PLZF) is an emerging suppressor of inflammatory responses. CCL21-CCR7 signaling mediates tolerance development. However, whether PLZF and CCL21-CCR7 are required for the development of asthma tolerance is unknown. Here, we found that Zbtb16 (coding PLZF) and Ccl21 were upregulated in OVA-induced asthma tolerance (OT) lungs by RNA-seq. PLZF physically interacted with GATA3 and its expression was higher in GATA3+ Th2cells and ILC2s in OT lungs. Zbtb16-knockdown in lymphocytes promoted the differentiation of CD3e+ CD4+ Tcells, particularly those producing IL-4 and IL-5. Moreover, iNKTcells with high expression of PLZF were recruited into the lungs via draining lymph nodes during tolerance. Blockade of CCL21-CCR7 signaling in OT mice decreased the PLZF+ cell population, abolished CCR7-induced PLZF+ iNKT recruitment to the lungs, enhanced Th2responses and exacerbated lung pathology. In OT mice, respiratory syncytial virus (RSV) infection impeded PLZF+ cell and CCR7+ PLZF+ iNKTcellrecruitment to the lungs and increased airway resistance. Collectively, these results indicate that PLZF could interact with GATA3 and restrain differentiation of IL-4- and IL-5-producing Tcells, iNKTcells with high PLZF expression are recruited to the lungs via CCL21-CCR7 signaling to facilitate the development of asthma tolerance.

Tethering Innate Surface Receptors on Dendritic Cells: A New Avenue for Immune Tolerance Induction?

Dendritic cells (DCs) play a key role in immunity and are highly potent at presenting antigens and orienting the immune response. Depending on the environmental signals, DCs could turn the immune response toward immunity or immune tolerance. Several subsets of DCs have been described, with each expressing various surface receptors and all participating in DC-associated immune functions according to their specific skills. DC subsets could also contribute to the vicious circle of inflammation in immune diseases and establishment of immune tolerance in cancer. They appear to be appropriate targets in the control of inflammatory diseases or regulation of autoimmune responses. For all these reasons, in situ DC targeting with therapeutic antibodies seems to be a suitable way of modulating the entire immune system. At present, the field of antibody-based therapies has mainly been developed in oncology, but it is undergoing remarkable expansion thanks to a wide variety of antibody formats and their related functions. Moreover, current knowledge of DC biology may open new avenues for targeting and modulating the different DC subsets. Based on an update of pathogen recognition receptor expression profiles in human DC subsets, this review evaluates the possibility of inducing tolerant DCs using antibody-based therapeutic agents.

Characterization of dendritic cell (DC)-10 in recurrent miscarriage and recurrent implantation failure.

During pregnancy, the maternal immune system must tolerate the persistence of semi-allogeneic fetus in the maternal tissue. Inadequate recognition of fetal antigens may lead to pregnancy complications, such as recurrent miscarriage (RM) and recurrent implantation failure (RIF). Dendritic cells (DCs) are key regulators of protective immune responses and the development and maintenance of tolerance. Regarding that DCs are important in the establishment of immune tolerance in human pregnancy, it would be important to study the microenvironment in which DCs reside or are activated may affect their functions toward tolerance rather than active immune response. IL-10 plays a critical role in the maintenance of normal pregnancy, and the increased production of IL-10 is associated with successful pregnancy. In this study, we provide an in-depth comparison of the phenotype and cytokine production by DC-10 and other DC subsets, such as iDC and mDC. CD14+ monocyte-derived DCs were differentiated in the presence of IL-10 (DC-10) in vitro from ten normal fertile controls, six RM women and seven RIF women, and characterized for relevant markers. DC-10 was characterized by relatively low expression of costimulatory molecule CD86, as well as MHC class II molecule HLA-DR, high expression of tolerance molecules HLA-G, ILT2, ILT4 and immunosuppressive cytokine IL-10, but produced little or no proinflammatory cytokines, such as TNF-α, IL-6 and IL-12p70. Our study provides a better understanding of the phenotypical properties of DC-10, which may participate in the complex orchestration that leads to maternal immune tolerance and homeostatic environment in human pregnancy.

Isolation of Highly Viable Thymic Epithelial Cells for Use in In Vitro and In Vivo Experiments.

Thymic epithelial cells (TECs) play multiple essential roles in T-cell development and the establishment of immune tolerance, but their isolation can be challenging, and their low viability upon isolation complicates downstream experiments. A method that allows TECs to be isolated easily and to survive afterward will be useful for elucidating key questions in TEC biology. Here, we demonstrate a simple method to isolate highly viable TECs. Primary TECs isolated using papain together with collagenase IV and DNase I survive and proliferate in vitro. Moreover, these primary TECs functionally engraft after intrathymic transplantation into recipient mice. Thus, the methods described herein will be useful for elucidating the roles of TECs and TEC subsets in T-cell development and immune tolerance.

Low-dose interleukin-2 as a modulator of Treg homeostasis after HSCT: current understanding and future perspectives.

CD4+CD25+Foxp3+ Treg is a functionally distinct subset of mature T cells with broad suppressive activity and has been shown to play an important role in the establishment of immune tolerance after HSCT. Altered cytokine environment in post-HSCT lymphopenia with a relative functional deficiency of IL-2 could hamper the reconstitution of Treg, leading to refractory GVHD. Based on the theory of low-dose IL-2 in which Treg can be selectively stimulated through the high-affinity IL-2 receptor, clinical studies have been conducted and demonstrated that low-dose IL-2 administration can restore Treg homeostasis and promote the expansion of this subset on the polymorphic processes of Treg reconstitution after HSCT. The new therapeutic indication of IL-2 for immune tolerance has launched in the field of HSCT and is spreading to the other fields including the treatment for autoimmune diseases. To further extend the indication of low-dose IL-2 to more patients with various immunological problems, the optimization of the timing and dosing of IL-2 intervention and the concomitant immune suppressive therapy according to each patient-based assessment are to be desired in the near future. Further prospective studies may facilitate the development of novel therapeutic algorithms for the effective and safe induction of immune tolerance after HSCT.

Glioblastoma progression is assisted by induction of immunosuppressive function of pericytes through interaction with tumor cells

The establishment of immune tolerance during Glioblastoma Multiforme (GBM) progression, is characterized by high levels expression of anti-inflammatory cytokines, which suppress the function of tumor assocciated myeloid cells, and the activation and expansion of tumor antigen specific T cells. However, the mechanisms underlying the failed anti-tumor immune response around the blood vessels during GBM, are poorly understood. The consequences of possible interactions between cancer cells and the perivascular compartment might affect the tumor growth. In this work we show for the first time that GBM cells induce immunomodulatory changes in pericytes in a cell interaction-dependent manner, acquiring an immunosuppresive function that possibly assists the evasion of the anti-tumor immune response and consequently participates in tumor growth promotion. Expression of high levels of anti-inflammatory cytokines was detected in vitro and in vivo in brain pericytes that interacted with GBM cells (GBC-PC). Furthermore, reduction of surface expression of co-stimulatory molecules and major histocompatibility complex molecules in GBC-PC correlated with a failure of antigen presentation to T cells and the acquisition of the ability to supress T cell responses. In vivo, orthotopic xenotransplant of human glioblastoma in an immunocompetent mouse model showed significant GBM cell proliferation and tumor growth after the establishment of interspecific immunotolerance that followed GMB interaction with pericytes.

SATB1 Plays a Critical Role in Establishment of Immune Tolerance.

Special AT-rich sequence binding protein 1 (SATB1) is a genome organizer that is expressed by T cells. T cell development is severely impaired in SATB1 null mice; however, because SATB1 null mice die by 3 wk of age, the roles of SATB1 in T cell development have not been well clarified. In this study, we generated and analyzed SATB1 conditional knockout (cKO) mice, in which the SATB1 gene was deleted from all hematopoietic cells. T cell numbers were reduced in these mice, mainly because of a deficiency in positive selection at the CD4(+)CD8(+) double-positive stage during T cell development in the thymus. We also found that SATB1 cKO mice developed autoimmune diseases within 16 wk after birth. In SATB1 cKO mice, the numbers of Foxp3(+) regulatory T (Treg) cells were significantly reduced at 2 wk of age compared with wild-type littermates. Although the numbers gradually increased upon aging, Treg cells in SATB1 cKO mice were still less than those in wild-type littermates at adulthood. Suppressive functions of Treg cells, which play a major role in establishment of peripheral tolerance, were also affected in the absence of SATB1. In addition, negative selection during T cell development in the thymus was severely impaired in SATB1 deficient mice. These results suggest that SATB1 plays an essential role in establishment of immune tolerance.

Low-Dose IL-2 Reduces Mitochondrial Priming and Increases Bcl2 Expression in CD4 Memory Regulatory T Cells

CD4+CD25+Foxp3+ regulatory T cells (Treg) play a critical role in establishment of immune tolerance and prevention of graft versus host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). The recovery and maintenance of Treg after HSCT is dependent on homeostatic factors including the generation of naïve Treg from hematopoietic precursor cells, the proliferation and expansion of mature Treg and the survival of Treg in vivo. We previously reported that Treg in healthy donors are more susceptible to mitochondrial apoptotic priming than conventional T cells (Tcon). Mitochondrial priming is increased after hematopoietic stem cell transplantation in all T cell subsets and particularly in patients with chronic GVHD. Interleukin-2 (IL-2) is a critical homeostatic cytokine that is required for Treg development, expansion, activity and survival. We previously demonstrated that daily administration of low-dose IL-2 resulted in selective expansion of Treg in vivo, clinical improvement of chronic GVHD (Koreth et al. NEJM 2011), increased Treg Bcl2 expression and increased Treg resistance to apoptosis in vitro (Matsuoka et al. SciTM, 2013). These observations led us to undertake a more detailed analysis of apoptotic pathways in functionally and phenotypically distinct Treg subsets including naïve Treg, recent thymic emigrants (RTE) Treg and memory Treg.To examine the mechanisms whereby IL-2 affects susceptibility to apoptosis in each Treg subset, we used a functional flow cytometry-based assay (apoptotic profiling) to measure mitochondrial membrane depolarization in response to a panel of pro-apoptotic BH3 peptides (BIM, BID, BAD, NOXA, PUMA, BMF, HRK). This assay allowed us to compare “priming”, which we define as susceptibility to BH3 peptide-induced mitochondrial membrane depolarization, in naïve, RTE and memory subsets. We also examined cytoplasmic expression of Bcl2 as an additional measure of susceptibility to apoptosis in each subset. In resting blood obtained from healthy donors (n=25), memory Treg were more “primed” than either naïve or RTE subsets when exposed to several BH3 peptides (BID, NOXA, PUMA, BMF and the combination of BAD+NOXA) (Figure 1). Memory Treg were also found to have decreased expression of Bcl2 compared to naïve or RTE subsets. Thus, memory Treg in healthy individuals are more susceptible to apoptosis than other Treg subsets through the mitochondrial pathway (Figure 1).To examine the functional effects of IL-2 on T cell homeostasis, Treg and Tcon were purified by cell sorting and cultured for 5 days with different concentrations of IL-2 (0, 10, 100, 1000 U/ml). Low-concentration IL-2 (10 U/ml) lowered apoptotic priming and increased Bcl2 expression in all Treg subsets. Similar effects were observed in all Tcon subsets, but higher concentrations of IL-2 (≥100 U/ml) were required to decrease apoptotic priming and increase Bcl2 expression in Tcon subsets. The peak effect of IL-2 on Treg priming occurred on day 3 but these effects appeared more rapidly in naïve and RTE Treg subsets than in memory Treg. However, the effect of IL-2 on apoptotic priming persisted for a longer period in memory Treg than in naïve and RTE Treg subsets. The peak effect of IL-2 on Bcl2 expression occurred on day 3 in all Treg subsets. To compare the effect of IL-2 on Treg and Tcon subsets, we examined apoptotic priming and Bcl2 expression after 3 day incubation with different IL-2 concentrations (0.001 - 1000 U/ml). Very low-concentration IL-2 (1 U/ml) lowered priming and increased Bcl2 expression in memory Treg, but higher concentrations of IL-2 (≥10 U/ml) were required to increase Bcl2 expression and decrease priming in naïve and RTE Treg. All Tcon subsets showed a similar pattern of response but higher concentrations of IL-2 (≥100 U/ml) were required.Memory Treg are the predominant regulatory population in patients with cGVHD and the high level of priming in this subset contributes to the inability to maintain adequate numbers of CD4 Treg after HSCT. In healthy donors, low concentrations of IL-2 are sufficient to reduce mitochondrial priming and increase Bcl2 expression in memory Treg. IL-2 has similar effects on Tcon but 10-100 fold higher concentrations of IL-2 are required. These concentration-dependent effects of IL-2 likely contribute to the selective expansion and persistence of Treg in patients with cGVHD receiving daily low-dose IL-2 therapy after HSCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Increased mitochondrial apoptotic priming of human regulatory T cells after allogeneic hematopoietic stem cell transplantation.

CD4 regulatory T cells play a critical role in establishment of immune tolerance and prevention of graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. The recovery and maintenance of regulatory T cells is dependent on homeostatic factors including the generation of naïve regulatory T cells from hematopoietic precursor cells, the proliferation and expansion of mature regulatory T cells, and the survival of regulatory T cells in vivo. In this study, quantitation of mitochondrial apoptotic priming was used to compare susceptibility of regulatory T cells, conventional CD4 T cells and CD8 T cells to intrinsic pathway apoptosis in 57 patients after allogeneic hematopoietic stem cell transplantation and 25 healthy donors. In healthy donors, regulatory T cells are more susceptible to mitochondrial priming than conventional T cells. Mitochondrial priming is increased after hematopoietic stem cell transplantation in all T-cell subsets and particularly in patients with chronic graft-versus-host disease. Regulatory T cells express high levels of CD95 and are also more susceptible than conventional T cells to apoptosis through the extrinsic pathway. However, CD95 expression and extrinsic pathway apoptosis is not increased after hematopoietic stem cell transplantation. Decreased expression of BCL2 and increased expression of BIM, a mitochondrial cell death activator protein, in regulatory T cells contributes to increased mitochondrial priming in this T-cell subset but additional factors likely contribute to increased mitochondrial priming following hematopoietic stem cell transplantation.

Peroxisome proliferator-activated receptor-gamma expression in dendritic cells determines immune tolerance versus immune activation (HYP6P.256)

Abstract Dendritic cells (DCs) play a central role in regulating immune tolerance and activation but the molecular axis within DCs that dictates immune outcome is unknown. We investigated the role of Peroxisome proliferator-activated receptor-gamma (PPARγ) in DCs in establishment of immune tolerance in the airways using an experimental model. Using mice with selective deletion of PPARγ in CD11c+ cells (PPARγΔ) to ablate PPARγ expression in DCs, we show that DC-specific PPARγ expression exerts a dual role whose collective goal in response to inhaled antigen is to not only promote de novo Foxp3 expression in T cells but to actively dampen T effector cell development. We demonstrate that PPARγ plays a crucial role in enhancing aldh1a2 expression in lung CD103+ DCs under tolerogenic conditions and when tolerized PPARγΔ mice were antigen-challenged, a complete loss of immune tolerance with increased neutrophil-dominated airway inflammation was observed. Absence of PPARγ promoted the expression of multiple pro-inflammatory cytokines such as IL-6 and IL-23 in DCs, limited Foxp3 induction but augmented IL-17 production in T cells with significant increase in the frequency of dual Foxp3+RORγt+ CD4+ T cells in PPARγΔ mice. In summary, our study identifies PPARγ as a central regulator in DC-mediated programming of the Foxp3/RORγt balance in CD4+ T cells dictating tolerance versus inflammation upon antigen provocation.

Melanoma-derived Wnt5a conditions dendritic cells to promote regulatory T cell differentiation via the upregulation of indoleamine 2,3-dioxygenase: novel pharmacological strategies for augmenting immunotherapy efficacy

Previous studies have shown the β-catenin signaling pathway to promote the development of tolerogenic dendritic cells (DCs) that are capable of driving regulatory T cell (Treg) differentiation. Interestingly, tolerogenic DCs have recently been described to play a role in carcinogenesis. However, the molecular mechanisms underlying the establishment of immune tolerance by this DC population are poorly understood and the methods by which developing cancers can co-opt this pathway to subvert immune surveillance are unknown. Using a genetically engineered model, we demonstrate that melanoma-derived Wnt5a ligand is a novel regulator of indoleamine 2,3-dioxygenase-1 (IDO) expression in local myeloid DCs and that Wnt5a induces the durable expression and enzymatic activity of IDO via β-catenin (Figures ​(Figures11,​,2).2). Further, we show that Wnt5a-conditioned DCs promote Treg differentiation in an IDO-dependent manner and that melanoma secretion of Wnt5a both suppresses the generation of anti-tumor immunity and promotes melanoma progression in vivo (Figure ​(Figure3).3). By genetically silencing the PORCN acyl transferase which is necessary for Wnt ligand secretion, we confirm the role of the soluble Wnt ligands in directing DC tolerization both in vitro and in vivo while also establishing a potential pharmacologic target for manipulating this novel pathway (Figure ​(Figure4).4). Indeed, utilizing a small molecule inhibitor of PORCN, we are able to reverse Wnt5a-mediated IDO upregulation by DCs both in vitro and in vivo. These findings were expanded in further melanoma studies where we demonstrated small molecule PORCN inhibition to synergistically suppress melanoma progression while also enhancing anti-melanoma immunity in the setting of combination anti-CTLA-4 therapy (Figure ​(Figure5).5). Additional work in human melanoma confirms the existence of this Wnt5a-mediated paracrine signaling pathway in DCs and reveals by microarray dataset analysis that human melanoma co-expression of Wnt5a and FoxP3 is highly significant. These data prompted us to hypothesize that a Wnt5a-induced gene signature in the melanoma microenvironment may be indicative of immune tolerance. Indeed, we have found that a Wnt5a gene signature identified in purified sentinel lymph node-derived DCs is associated with an inferior clinical prognosis in melanoma patients. This work emphasizes the importance of DC populations in directing tumor immune surveillance and illustrates that the molecular mechanisms involved in DC physiology represent potential targets for pharmacologically enhancing anti-tumor immunity. Figure 1 Wnt3a and Wnt5a upregulate IDO expression and activity in DCs. A. Wnt5a induces durable IDO expression by BMDCs based on Western blot analysis at 24 and 48 hrs. Representative of 3 independent experiments. UT, untreated. XAV939, β-catenin inhibitor. ... Figure 2 Murine melanomas induce paracrine β-catenin signaling activation in tumor and TDLN DCs in vivo. A.Tumor-infiltrating DCs (TIDCs) isolated from Tyr::CreER;BrafCA;Ptenlox/lox primary melanomas exhibit increased expression levels of β-catenin ... Figure 3 Wnt5a conditions DCs to drive regulatory T-cell cifferentiation. A. Schematic of in vitro Treg assay. B. Wnt5a-conditioned DCs stimulate CD4+FoxP3+ Treg differentiation in vitro following co-incubation with total splenic CD4+ T cells at a 1:1, 5:1 or ... Figure 4 Silencing PORCN expression in the B16 melanoma model enhances anti-tumor immunity and suppresses tumor progression in vivo. A. Silencing PORCN expression by B16/F10 cells suppresses paracrine β-catenin activation in a 293T-TCF/LEF1-luciferase ... Figure 5 C59 Inhibition of the Wnt-β-catenin signaling pathway synergistically enhances the efficacy of anti-CTLA-4 antibody immunotherapy in the B16 melanoma model. A. Combination inhibition of Wnt-mediated signaling and anti-CTLA-4 blockade synergistically ...

Double negative Treg cells promote nonmyeloablative bone marrow chimerism by inducing T‐cell clonal deletion and suppressing NK cell function

The establishment of immune tolerance and prevention of chronic rejection remain major goals in clinical transplantation. In bone marrow (BM) transplantation, T cells and NK cells play important roles for graft rejection. In addition, graft-versus-host-disease (GVHD) remains a major obstacle for BM transplantation. In this study, we aimed to establish mixed chimerism in an irradiation-free condition. Our data indicate that adoptive transfer of donor-derived T-cell receptor (TCR) αβ(+) CD3(+) CD4(-) CD8(-) NK1.1(-) (double negative, DN) Treg cells prior to C57BL/6 to BALB/c BM transplantation, in combination with cyclophosphamide, induced a stable-mixed chimerism and acceptance of C57BL/6 skin allografts but rejection of third-party C3H (H-2k) skin grafts. Adoptive transfer of CD4(+) and CD8(+) T cells, but not DN Treg cells, induced GVHD in this regimen. The recipient T-cell alloreactive responsiveness was reduced in the DN Treg cell-treated group and clonal deletions of TCRVβ2, 7, 8.1/2, and 8.3 were observed in both CD4(+) and CD8(+) T cells. Furthermore, DN Treg-cell treatment suppressed NK cell-mediated BM rejection in a perforin-dependent manner. Taken together, our results suggest that adoptive transfer of DN Treg cells can control both adoptive and innate immunities and promote stable-mixed chimerism and donor-specific tolerance in the irradiation-free regimen.