Induction Of Peripheral Tolerance Research Articles

Tumor-derived immuno-modulators induce overlapping pro-tolerogenic gene expression signatures in human dendritic cells

Immature dendritic cells (iDCs) and tolerogenic DCs are essential for the induction and maintenance of peripheral tolerance. Tumors produce immuno-modulatory factors which imprint a pro-tolerogenic, maturation-resistant state in DCs. Here we asked for common markers of differentially tolerized human monocyte-derived DC populations. For this, PBMC-derived monocytes were differentiated to DCs in the presence of established immuno-modulators as released by tumors (IL-6, IL-10, TGF-β, glucocorticoid [GC], prostaglandin E2 [PGE2]). Most unstimulated pro-tolerogenic DC populations commonly over-expressed some tolerance-associated markers (ILT-4, IL-10, HO-1) as compared with iDCs. These markers may contribute to imprint a pro-tolerogenic state in DCs. Furthermore, some tolerance markers were overexpressed in an immuno-modulator specific manner in DCs differentiated in the presence of TGF-β (overexpressed tolerance markers: B7-H3, CD103, TGF-β1), IL-10 (B7-DC, ILT-3) and PGE2 (IDO). Upon stimulation, matured control DCs (mDCs) down-regulated most pro-tolerogenic markers monitored, while some were upregulated (IkBα, IDO, B7-H1, B7-DC). In contrast, the different groups of tolerized DCs largely retained expression of pro-tolerogenic markers after stimulation. In contrast to mDCs, most groups of tolerized DCs showed impaired upregulation of CD80, and all groups retained IL-10 cytokine production after stimulation. All tolerized DC populations commonly exerted an attenuated allogenic T cell stimulatory capacity as compared with mDCs.

NKT cells in the liver are important for periferal tolerance induction

Abstract NKT cells are mainly located in the liver in mice. The role of liver inherent NKT cells are still unclear. Method In this study, we employed mouse orthotopic liver transplantation and heterotopic heart transplantation models to critical examine the role of liver NKT cells in liver and peripheral tolerance induction. Results NKT cells were increased in the tolerated liver grafts which expressed higher levels of CD95L and PD-L1, while the CD4+CD25+Foxp3+ regulatory T cells (Treg) were markedly increased in the both liver and spleen at day 7 post transplantation. The heart allograft survival were prolonged significantly in the recipient which accepted donor spleen cells by portal vein (p.v.) injection in contract to the recipient which received the donor spleen cells by tail vein (i.v.) injection. In vitro immunological assay revealed that the number of NKT cells in the liver was increased and Treg were increased in both liver and spleen from the p.v. treated mice significantly. The IL-2 expression was decreased, IL-4 and IL-10 were increased. Further, significantly increased IL-4, IL-10, and IFN-γ production was detected from liver NPCs under aGalCer stimulation. Conclusion NKT cells are important to liver transplant tolerance induction, appear to play a key role in down regulation of peripheral immune responses and facilitate CD4+CD25+Foxp3+ Treg induction.

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.

IL‐12 and IL‐4 activate a CD39‐dependent intrinsic peripheral tolerance mechanism in CD8+ T cells

Immune responses to protein antigens involve CD4+ and CD8+ T cells, which follow distinct programs of differentiation. Naïve CD8 T cells rapidly develop cytotoxic T‐cell (CTL) activity after T‐cell receptor stimulation, and we have previously shown that this is accompanied by suppressive activity in the presence of specific cytokines, i.e. IL‐12 and IL‐4. Cytokine‐induced CD8+ regulatory T (Treg) cells are one of several Treg‐cell phenotypes and are Foxp3− IL‐10+ with contact‐dependent suppressive capacity. Here, we show they also express high level CD39, an ecto‐nucleotidase that degrades extracellular ATP, and this contributes to their suppressive activity. CD39 expression was found to be upregulated on CD8+ T cells during peripheral tolerance induction in vivo, accompanied by release of IL‐12 and IL‐10. CD39 was also upregulated during respiratory tolerance induction to inhaled allergen and on tumor‐infiltrating CD8+ T cells. Production of IL‐10 and expression of CD39 by CD8+ T cells was independently regulated, being respectively blocked by extracellular ATP and enhanced by an A2A adenosine receptor agonist. Our results suggest that any CTL can develop suppressive activity when exposed to specific cytokines in the absence of alarmins. Thus negative feedback controls CTL expansion under regulation from both nucleotide and cytokine environment within tissues.

GARP inhibits allergic airway inflammation in a humanized mouse model.

Regulatory T cells (Treg) represent a promising target for novel treatment strategies in patients with inflammatory/allergic diseases. A soluble derivate of the Treg surface molecule glycoprotein A repetitions predominant (sGARP) has strong anti-inflammatory and regulatory effects on human cells in vitro as well as in vivo through de novo induction of peripheral Treg. The aim of this study was to investigate the immunomodulatory function of sGARP and its possible role as a new therapeutic option in allergic diseases using a humanized mouse model. To analyze the therapeutic effects of sGARP, adult NOD/Scidγc(-/-) (NSG) mice received peripheral blood mononuclear cells (PBMC) derived from allergic patients with sensitization against birch allergen. Subsequently, allergic inflammation was induced in the presence of Treg alone or in combination with sGARP. In comparison with mice that received Treg alone, additional treatment with sGARP reduced airway hyperresponsiveness (AHR), influx of neutrophils and macrophages into the bronchoalveolar lavage (BAL), and human CD45(+) cells in the lungs. Furthermore, the numbers of mucus-producing goblet cells and inflammatory cell infiltrates were reduced. To elucidate whether the mechanism of action of sGARP involves the TGF-β receptor pathway, mice additionally received anti-TGF-β receptor II (TGF-βRII) antibodies. Blocking the signaling of TGF-β through TGF-βRII abrogated the anti-inflammatory effects of sGARP, confirming its essential role in inhibiting the allergic inflammation. Induction of peripheral tolerance via sGARP is a promising potential approach to treat allergic airway diseases.

Classical dendritic cells are required for dietary antigen-mediated induction of peripheral T(reg) cells and tolerance.

Oral tolerance prevents pathological inflammatory responses towards innocuous foreign antigens via peripheral regulatory T cells (pTreg cells). However, whether a particular subset of antigen-presenting cells (APCs) is required during dietary antigen exposure to instruct naïve CD4+ T cells to differentiate into pTreg cells has not been defined. Using myeloid lineage-specific APC depletion in mice, we found that monocyte-derived APCs are dispensable, while classical dendritic cells (cDCs) are critical for pTreg cell induction and oral tolerance. CD11b− cDCs from the gut-draining lymph nodes efficiently induced pTreg cells, and conversely, loss of IRF8-dependent CD11b− cDCs impaired their polarization, although oral tolerance remained intact. These data reveal the hierarchy of cDC subsets in pTreg cell induction and their redundancy during oral tolerance development.

Loss of Programmed Death Ligand-1 Expression on Donor T Cells Lessens Acute Graft-Versus-Host Disease Lethality

The PD-1/PD-L1 pathway plays an important role in regulation of alloimmune responses and in induction and maintenance of peripheral tolerance. Because GVHD is driven by donor T cells and PD-L1 expression can be markedly elevated on T cells during activation, we investigated the functional significance of PD-L1 expressed by donor T cells in regulating murine models of acute GVHD.PD-L1 expression was up-regulated on donor CD4 and CD8 T cells during GVHD. We considered the possibility that PD-L1 expression on activated donor T cells might inhibit GVHD by down regulating donor anti-host T cell responses, consistent with PD-L1 co-inhibitory activity when expressed on host parenchymal cells during GVHD. Surprisingly, T cell mediated GVHD lethality was markedly reduced in recipients of PD-L1-/- compared to WT donor T cells in both B6 to BALB/c model of GVHD(P<0.0001; Fig 1A) and in B6 to B10.BR model (P =0.0047; Fig 1B), suggesting that PD-L1 expression on donor T cells is involved in interactions that enhance T cell mediated effector function. Survival data confirmed that PD-L1-/- Teffs and not Tregs were responsible for reduced lethality in recipients of PD-L1-/- donor T cells (Fig 1C). During GVHD, PD-L1-/- donor CD4 and CD8 T cells had reduced expression of gut homing receptors (Fig 1D), and recipients of PD-L1-/- donor T cells had reduced T cell infiltration into lymphoid organs and gut, retained intestinal epithelial integrity, and had lower inflammatory cytokine production. PD-L1-/- donor CD4 and CD8 T cells had increased expression of multiple inhibitory receptors (Fig 1E, 1F), reduced T cell proliferation, and increased T cell apoptosis by transcriptional profiling and cell surface marker expression. Four pathways, including proteasome activity showed decreased expression in PD-L1-/- donor T cells. In vitro T cell activation in the presence of single (PD-L1:B7-1) vs. dual (PD-L1:B7-1 and PD-L1:PD-1) pathway blocking anti-PD-L1 mAb confirmed that T-T interaction between PD-1 and PD-L1 is important for proliferation and survival, whereas sensitive in vitro assays with supported lipid bilayers found no evidence for a functionally relevant cis interaction of PD-L1 and PD-1 on T cells. We found a significant increase in glucose transporter (GLUT1) expression in proliferating WT vs. PD-L1-/- donor CD4 and CD8 T cells, along with increased glycolysis, OXPHOS, glutamine consumption and glutamate production. We also observed increased fatty acid (FA) uptake and FA oxidation, and enhanced pharmacologic inhibition of FA oxidation in WT donor T cells, suggesting that PD-1:PD-L1 interactions are important for FA metabolism, which may further support T cell survival. Studies using stable isotope carbon tracers highlighted the divergent roles for glutamine and glucose in energy generation and biosynthetic pathways. Given the importance of acetyl-CoA as a high energy thioester intermediate in the TCA cycle and a lipogenic precursor for T cells undergoing expansion, significantly enhanced production of acetyl-CoA from glucose by WT donor T cells support the notion that PD-L1 on T cells promotes clonal expansion of alloreactive T cells. In summary, these data are the first to show that PD-L1 expression on donor T cells can provide positive signals for T cell survival, activation, and metabolism. Greater understanding of the function of PD-L1 expression by activated donor T cells will provide new insight into the regulation of GVHD and suggest strategies to selectively inhibit PD-L1 on donor T cells that may be clinically useful to prevent GVHD. [Display omitted] DisclosuresAoyama:CHUGAI PHAMACEUTICAL CO.,LTD: Honoraria; Mochida Pharmaceutical Co.,Ltd: Honoraria; Kyowa Hakko Kirin Company,Limited: Honoraria. Milone:Novartis: Patents & Royalties, Research Funding. Miller:Coronado: Speakers Bureau; BioSciences: Speakers Bureau; Celegene: Speakers Bureau. Sharpe:Costim Pharmaceuticals: Patents & Royalties.

T Cell Motility as Modulator of Interactions with Dendritic Cells.

It is well established that the balance of costimulatory and inhibitory signals during interactions with dendritic cells (DCs) determines T cell transition from a naïve to an activated or tolerant/anergic status. Although many of these molecular interactions are well reproduced in reductionist in vitro assays, the highly dynamic motility of naïve T cells in lymphoid tissue acts as an additional lever to fine-tune their activation threshold. T cell detachment from DCs providing suboptimal stimulation allows them to search for DCs with higher levels of stimulatory signals, while storing a transient memory of short encounters. In turn, adhesion of weakly reactive T cells to DCs presenting peptides presented on major histocompatibility complex with low affinity is prevented by lipid mediators. Finally, controlled recruitment of CD8+ T cells to cognate DC–CD4+ T cell clusters shapes memory T cell formation and the quality of the immune response. Dynamic physiological lymphocyte motility therefore constitutes a mechanism to mitigate low avidity T cell activation and to improve the search for “optimal” DCs, while contributing to peripheral tolerance induction in the absence of inflammation.

Orally-Induced Intestinal CD4+ CD25+ FoxP3+ Treg Controlled Undesired Responses towards Oral Antigens and Effectively Dampened Food Allergic Reactions

The induction of peripheral tolerance may constitute a disease-modifying treatment for allergic patients. We studied how oral immunotherapy (OIT) with milk proteins controlled allergy in sensitized mice (cholera toxin plus milk proteins) upon exposure to the allergen. Symptoms were alleviated, skin test was negativized, serum specific IgE and IgG1 were abrogated, a substantial reduction in the secretion of IL-5 and IL-13 by antigen-stimulated spleen cells was observed, while IL-13 gene expression in jejunum was down-regulated, and IL-10 and TGF-β were increased. In addition, we observed an induction of CD4+CD25+FoxP3+ cells and IL-10- and TGF-β-producing regulatory T cells in the lamina propria. Finally, transfer experiments confirmed the central role of these cells in tolerance induction. We demonstrated that the oral administration of milk proteins pre- or post-sensitization controlled the Th2-immune response through the elicitation of mucosal IL-10- and TGF-β-producing Tregs that inhibited hypersensitivity symptoms and the allergic response.

Extra-thymically induced T regulatory cell subsets: the optimal target for antigen-specific immunotherapy.

Antigen-specific immunotherapy aims to selectively restore tolerance to innocuous antigens in cases of autoimmune or allergic disease, without the need for general immune suppression. Although the principle of antigen-specific immunotherapy was discovered more than a century ago, its clinical application to date is limited, particularly in the control of autoimmunity. This has resulted mainly from a lack of in-depth understanding of the underlying mechanism. More recently, the differentiation of extra-thymically induced T regulatory (Treg) cell subsets has been shown to be instrumental in peripheral tolerance induction. Two main types of inducible Treg cells, interleukin-10-secreting or Foxp3(+) , have now been described, each with distinct characteristics and methods of therapeutic induction. It is crucial, therefore, to identify the suitability of either subset in the control of specific immune disorders. This review explores their natural function, the known mechanisms of therapeutic differentiation of either subset as well as their in vivo functionality and discusses new developments that may aid their use in antigen-specific immunotherapy, with a focus on autoimmune disease.

The atypical chemokine receptor ACKR4 facilitates dendritic cell migration during inflammation by scavenging CCL19 (CCR3P.205)

Abstract The migration of dendritic cells from tissues to draining lymph nodes is a critical step in the induction of peripheral tolerance and the initiation of adaptive immune responses. This is dependent on CCR7 expression by dendritic cells. In response to the chemokine CCL21, CCR7 directs dendritic cells into lymphatic vessels in the tissue, and permits their transit from the subcapsular sinus into the lymph node parenchyma. The CCR7 ligands CCL19 and CCL21 also bind ACKR4, an atypical chemokine receptor expressed by keratinocytes in the skin and lymphatic endothelial cells lining the subcapsular sinus. In mice, ACKR4 controls interfollicular CCL21 gradients, and enhances dendritic cell entry into the lymph node parenchyma from the subcapsular sinus. Here we report that Ackr4 deficiency disrupts CCR7-dependent dendritic cell arrival at skin-draining lymph nodes during cutaneous inflammation. We show that this, at least in part, is due to the defective departure of dendritic cells from inflamed skin, and is accompanied by dysregulation of bioavailable CCL19 and CCL21 in the skin. Strikingly, genetic deletion of CCL19 completely rescues the defective inflammation-driven trafficking of dendritic cells caused by Ackr4 deficiency. Thus, by regulating CCL19, ACKR4 helps maintain CCR7-dependent dendritic cell departure from inflamed tissues.

Retention and tolerance of autoreactive CD4+ recent thymic emigrants in the liver (HEM2P.249)

Abstract Mechanisms of peripheral tolerance play a critical role in preventing T cells that escape from negative selection in the thymus from initiating autoimmune reactions. To investigate the site of peripheral tolerance induction, we examined migration and activation of recent thymic emigrants (RTEs) in liver, spleen, lymph node and peripheral blood. We show that a fraction of RTE precursors were retained in the liver independent of the secondary lymphoid organs. Compared to RTEs from the lymph nodes, RTEs from the liver proliferated more and many exhibited an activated phenotype with the capability of producing IL-10 upon activation. Liver RTEs also responded poorly to interleukin (IL)-7 and were more prone to apoptosis. Following transfer into RAG-/- recipients, liver RTEs induced more severe inflammation and T cell infiltration in the lung and colon. The extrathymic expression of MHC and Aire is required for the acquisition of tolerogenic phenotype of newly generated thymic emigrants in the liver. These results suggest that the liver is the first checkpoint in the periphery to filter, retain, and enforce tolerance to autoreactive CD4+ thymic emigrants that escape from negative selection.

The contribution of NF-κB signalling to immune regulation and tolerance.

Immune regulation is necessary to control inflammatory responses and to prevent autoimmune diseases. Therefore, mechanisms of central and peripheral tolerance have evolved to ensure that T cells recognize antigens as self- or non-self-antigens. The thymus is crucially important for central tolerance induction to self-antigens via negative selection of T cells. However, if T cells escape negative selection in the thymus and enter the periphery, peripheral mechanisms are active to warrant immune tolerance. Secondary lymphoid organs, as well as tolerogenic dendritic cells and regulatory T cells, play an important role in peripheral tolerance. In chronic inflammatory diseases, tertiary lymphoid organs are sometimes formed that may also be involved in the induction of peripheral tolerance. This review discusses the main processes that are involved in immune regulation and tolerance, and focuses on the contribution of NF-κB signalling to these processes. This narrative review is based on peer-reviewed publications listed on PubMed up to December 2014. The focus of our literature search was on studies investigating the role of (non)canonical NF-κB signalling in central and peripheral mechanisms of tolerance. Only studies published in English language were considered. This review discusses the immune phenotype of mutant mice with defective (non)canonical NF-κB signalling, corroborated with human data, and emphasizes the contribution of the noncanonical NF-κB pathway to immune regulation and tolerance induction. Noncanonical NF-κB signalling has an important immunoregulatory role in the immune system and contributes to both central and peripheral mechanisms of tolerance.

Carbon black nanoparticle exposure during middle and late fetal development induces immune activation in male offspring mice.

Increasing exposure to nanoparticles (NPs) has raised concerns regarding their health and safety profiles in humans and animals, especially in developing organisms, which may display increased sensitivity to NP toxicity. The present study examined the effects of gestational exposure to carbon black NP (CB-NP) on the development of the offspring immune system. Pregnant mice were exposed to CB-NP (95μg/kg body weight) by intranasal instillation on gestational days 9 and 15. The thymus and spleen were collected from their offspring mice on postnatal day (PND) 1, 3 and 5. Thymocyte and splenocyte phenotypes were examined by determining the expression of cell-surface molecules using flow cytometry. Gene expression in the thymus and spleen was examined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Prenatal exposure to CB-NP increased total thymocytes and their immunophenotypes (CD4(-)CD8(-) and CD4(+)CD8(+) cells). It also induced an increase in total lymphocytes, and CD4(-)CD8(-), particularly CD3(-)B220(-)cells, at PND 5 in the spleen of newborn male offspring, reflecting the stimulation of immature splenocytes. Furthermore, mRNA expression of genes related to the induction of peripheral tolerance (i.e. thymic Traf6) was upregulated. These data suggest that respiratory exposure to CB-NP during middle and late gestation may have allergic or inflammatory effects in male offspring, and may provide initial information on the potential developmental immunotoxicity of nanoparticles.

Retention and tolerance of autoreactive CD4+ recent thymic emigrants in the liver

Mechanisms of peripheral tolerance play a critical role in preventing T cells that escape from negative selection in the thymus from initiating autoimmune reactions. To investigate the site of peripheral tolerance induction, we examined migration and activation of recent thymic emigrants (RTEs) in liver, spleen, lymph node and peripheral blood. We show that a fraction of RTE precursors were retained in the liver independent of the secondary lymphoid organs. Compared to RTEs from the lymph nodes, RTEs from the liver proliferated more and many exhibited an activated phenotype with the capability of producing IL-10 upon activation. Liver RTEs also responded poorly to interleukin (IL)-7 and were more prone to apoptosis. Following transfer into RAG−/− recipients, liver RTEs induced more severe inflammation and T cell infiltration in the lung and colon. The extrathymic expression of MHC and Aire is required for the acquisition of tolerogenic phenotype of newly generated thymic emigrants in the liver. These results suggest that the liver is the first checkpoint in the periphery to filter, retain, and enforce tolerance to autoreactive CD4+ thymic emigrants that escape from negative selection.

Properties and molecular characteristic of immune regulation of human thymus-derived CD4+HL−G + regulatory T cells in vitro and in vivo

Regulation of effector T cells by regulatory T cells (Treg) is an essential mechanism for induction and maintenance of peripheral immune tolerance. Treg cells represent functionally and phenotypically distinct specialized subpopulations of T lymphocytes and are derived from the thymus or induced in the periphery. In humans, CD4+CD25+FoxP3-expressing T cells represent by far the best-characterized population of thymus-derived Treg (tTreg) cells. Recently, our group has described another subpopulation of CD4+ tTreg cells characterized by the expression of the non-classical major histocompatibility complex class I molecule human leukocyte antigen G (HLA-G), which is involved in immune regulation and parenchymal homeostasis during infectious and autoimmune inflammation.

Contribution of TCR signaling strength to CD8+ T cell peripheral tolerance mechanisms.

Peripheral tolerance mechanisms are in place to prevent T cells from mediating aberrant immune responses directed against self and environmental Ags. Mechanisms involved in the induction of peripheral tolerance include T cell-intrinsic pathways, such as anergy or deletion, or exogenous tolerance mediated by regulatory T cells. We have previously shown that the density of peptide-MHC class I recognized by the TCR determines whether CD8(+) T cells undergo anergy or deletion. Specifically, using a TCR-transgenic CD8(+) T cell model, we demonstrated that persistent peripheral exposure to low- or high-dose peptides in the absence of inflammatory signals resulted in clonal deletion or anergy of the T cell, respectively. In this study, by altering the affinity of the peptide-MHC tolerogen for TCR, we have confirmed that this mechanism is dependent on the level of TCR signaling that the CD8(+) T cell receives. Using altered peptide ligands (APLs) displaying high TCR affinities, we show that increasing the TCR signaling favors anergy induction. Conversely, using APLs displaying a decreased TCR affinity tilted our system in the direction of deletional tolerance. We demonstrate how differential peripheral CD8(+) T cell tolerance mechanisms are controlled by both the potency and density of MHC class I-peptide tolerogen.

Anti-atherogenic peptide Ep1.B derived from apolipoprotein E induces tolerogenic plasmacytoid dendritic cells.

Tolerogenic dendritic cells (DCs) play a critical role in the induction of regulatory T cells (Tregs ), which in turn suppress effector T cell responses. We have previously shown the induction of DCs from human and mouse monocytic cell lines, mouse splenocytes and human peripheral blood monocytes by a novel apolipoprotein E (ApoE)-derived self-peptide termed Ep1.B. We also showed that this C-terminal region 239-252 peptide of ApoE has strong anti-atherogenic activity and reduces neointimal hyperplasia after vascular surgery in rats and wild-type as well as ApoE-deficient mice. In this study, we explored the phenotype of DC subset induced by Ep1.B from monocytic cell lines and from the bone marrow-derived cells. We found Ep1.B treatment induced cells that showed characteristics of plasmacytoid dendritic cells (pDC). We explored in-vitro and in-vivo effects of Ep1.B-induced DCs on antigen-specific T cell responses. Upon in-vivo injection of these cells with antigen, the subsequent ex-vivo antigen-specific proliferation of lymph node cells and splenocytes from recipient mice was greatly reduced. Our results suggest that Ep1.B-induced pDCs promote the generation of Treg cells, and these cells contribute to the induction of peripheral tolerance in adaptive immunity and potentially contribute its anti-atherogenic activity.

Biochemical signaling of PD-1 on T cells and its functional implications.

Maintenance of peripheral tolerance is essential for homeostasis of the immune system. While central tolerance mechanisms result in deletion of the majority of self-reactive T cells, T lymphocytes specific for self-antigens also escape this process and circulate in the periphery. To control the development of autoimmunity, multiple mechanisms of peripheral tolerance have evolved, including T cell anergy, deletion, and suppression by regulatory T (Treg) cells. The pathway consisting of the programmed cell death 1 (PD-1) receptor (CD279) and its ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC; CD273) plays a vital role in the induction and maintenance of peripheral tolerance. This pathway also regulates the balance between stimulatory and inhibitory signals needed for effective immunity and maintenance of T cell homeostasis. In contrast to this important beneficial role in maintaining T cell homeostasis, PD-1 mediates potent inhibitory signals that prevent the expansion and function of T effector cells and have detrimental effects on antiviral and antitumor immunity. Despite the compelling studies on the significant functional role of PD-1 in mediating inhibition of activated T cells, little is known about how PD-1 blocks T cell activation. Here, we will provide a brief overview of the signaling events that are regulated by PD-1 triggering, and we will discuss their implications on cell intrinsic and extrinsic mechanisms that determine the fate and function of T effector cells.

Effect of FR104, a Novel Anti-CD28 Fab, in Monkey Cardiac Allograft Transplantation.

Background: Compared to CTLA-4Ig reagents, selective CD28 blockade using antagonist anti-CD28 Abs might favour B7-mediated coinhibitory signals through the receptors of CTLA-4 and/or PDL-1 and facilitate the induction of peripheral allograft tolerance. Here we evaluated the efficacy of a humanized pegylated anti-CD28 Fab Ab fragment, FR104, in cynomolgus monkey heart transplants. Methods: Heterotopic cardiac allograft recipients were untreated or received αCD28 (FR104), αCD154 (hu5C8), or combination. Graft function was monitored by telemetry. Protocol biopsies and explanted grafts were evaluated for ISHLT acute rejection and CAV scores (0, 0%; 1, 1-10%; 2, 11-50%; 3, >50% occlusion). Recipient blood was analyzed at regular intervals for alloantibodies and regulatory T cells (Tregs) frequency. Results: Untreated monkeys rejected their grafts promptly (median graft survival time (MST) 6 days). Treatment with FR104 alone potently prolonged graft survival (50, 167, and 178 days), which was statistically similar to that achieved with hu5C8 (MST 133 days). Combined CD28 and CD154 blockade resulted in an extended graft survival of 184 days. On POD 90-150, hu5C8-treated monkeys exhibited grade 1 to 3 rejection and moderate to severe CAV (score 2.1±0.8), whereas long-term surviving grafts in FR104 (n=2) and FR104+hu5C8 (n=1) groups displayed grade 0 to 1-2 rejection and markedly attenuated CAV (0.1±0.2 with FR104, and 0.3±0.4 with FR104+hu5C8). In all treated groups, the number of CD4+CD25+CD127lo Tregs (cells/μl) was increased (fold change relative to pretreatment baseline: 1.5±0.2 with FR104, 1.4±0.3 with hu5C8, 2.3±0.1 with FR104+hu5C8) on POD 7-21, IgM and IgG donor-reactive alloAbs were generally undetectable until graft failure.Figure: No Caption available.Conclusions: FR104 potently prevented rejection and prolonged cardiac allograft survival in cynomologus monkeys. These promising data demonstrated the high potential of CD28 specific antagonists for clinical transplantation. DISCLOSURE:Vanhove, B.: Stockholder, Tc Land.