Inhibition Of CD4 Research Articles

Tofacitinib Represses the Janus Kinase-Signal Transducer and Activators of Transcription Signalling Pathway in Keratinocytes.

Tofacitinib is a Janus kinase (JAK) inhibitor, which has shown efficacy in treating psoriasis. The mode of action of tofacitinib is not completely understood but it has been thought to be mediated by the inhibition of CD4+ T-cell activation. Here, we investigated whether the molecular targets of tofacitinib are expressed in keratinocytes, and whether tofacitinib can modulate the activity of the JAK/Signal Transducer and Activators of Transcription (STAT)-pathway in keratinocytes. Transcriptomic profiling of human keratinocytes treated with IL-22 in combination with tofacitinib revealed that tofacitinib could prevent the majority of IL-22-mediated gene expression changes. Pathway analysis of tofacitinib-regulated genes in keratinocytes revealed enrichment of genes involved in the JAK/STAT signalling pathway. Quantitative real-time-PCR confirmed the upregulation of S100A7 and downregulation of EGR1 expression by IL-22, which was prevented by tofacitinib pre-treatment. These results indicate a direct effect of tofacinitib on keratinocytes, which can have relevance for systemic as well as for topical treatment of psoriasis with tofacitinib.

Third-party regulatory T cells prevent murine acute graft-versus-host disease.

Background/AimsAdoptive therapy with regulatory T (Treg) cells to prevent graft-versus-host disease (GVHD) would benefit from a strategy to improve homing to the sites of inflammation following hematopoietic stem cell transplantation (HSCT). Although donor-derived Treg cells have mainly been used in these models, third-party-derived Treg cells are a promising alternative for cell-based immunotherapy, as they can be screened for pathogens and cell activity, and banked for GVHD prevention. In this study, we explored major histocompatibility complex (MHC) disparities between Treg cells and conventional T cells in HSCT to evaluate the impact of these different cell populations on the prevention of acute GVHD, as well as survival after allogeneic transplantation.MethodsTo induce acute GVHD, lethally irradiated BALB/c (H-2d) mice were transplanted with 5 × 105 T cell-depleted bone marrow cells and 5 × 105 CD4+CD25– splenic T cells from C57BL/6 (H-2b) mice. Recipients were injected with 5 × 105 cultured donor-, host-, or third-party-derived CD4+CD25+CD62L+ Treg cells (bone marrow transplantation + day 1).ResultsSystemic infusion of three groups of Treg cell improved clinicopathological manifestations and survival in an acute GVHD model. Although donor-derived Treg cells were immunologically the most effective, the third-party-derived Treg cell therapy group displayed equal regulation of expansion of CD4+CD25+- Foxp3+ Treg cells and suppressive CD4+IL-17+ T-helper (Th17) cells in ex vivo assays compared with the donor- and host-derived groups.ConclusionsOur findings demonstrate that the use of third-party Treg cells is a viable alternative to donor-derived Treg cellular therapy in clinical settings, in which human leukocyte antigen-matched donors are not always readily available.

Delayed and short course of rapamycin prevents organ rejection after allogeneic liver transplantation in rats.

To test whether a delayed and short course of rapamycin would induce immunosuppressive effects following allogeneic orthotopic liver transplantation (OLT) in rats. Allogeneic OLTs were performed using Dark Agouti livers transplanted into Lewis recipients, and syngeneic OLTs were performed using the Lewis rat strain. Rapamycin (1 mg/kg per day) was administered by gavage from day 4 to day 11 post-transplantation. Lymphocyte cellular compartments were analyzed by flow cytometry in draining lymph nodes, non-draining lymph nodes and the spleen at days 11 and 42 in rapamycin-treated rats, untreated control rats and syngeneic grafted rats. Skin grafts from Dark agouti or from F344 RT were performed at day 30 on liver grafted rats treated with rapamycin. An 8-d course of rapamycin treatment initiated 4 d following transplantation resulted in the survival of grafted rats for more than 100 d. In contrast, untreated rats died of liver failure within 13 to 21 d. The analysis of the cellular compartment revealed an increase in two cellular subpopulations, specifically myeloid-derived suppressor cells (MDSCs) and CD8+CD45RClow T cells, without major modifications in the regulatory T cell (Treg) compartment in treated rats in the early stages after grafting. We evaluated the ability of treated rats to reject third-party allogeneic skin grafts to confirm their immune competence. In contrast, when skin was collected from rats syngeneic to the grafted liver, it was not rejected. Our results demonstrate that short and delayed rapamycin treatment allows for tolerance in allogeneic OLT. The results also allowed for the identification of the mechanisms of tolerance induced by rapamycin by identifying MDSCs and CD8+CD45RClow T cells as associated with the state of tolerance.

CD4+ CD52lo T-cell expression contributes to the development of systemic lupus erythematosus

The cell-surface glycoprotein CD52 is widely expressed in lymphocytes. CD4+CD52hi T cells are functioning suppressor CD4+T cells. We investigated the role of the immune regulation of CD4+CD52 T cells in systemic lupus erythematosus (SLE). CD4+CD52lo T cells were increased in SLE patients, in positive correlation with SLEDAI, anti-ds-DNA antibody, and IgG concentration. Circulating follicular helper-like T cells (Tfh-like cells) were also increased in SLE, in positive correlation with CD4+CD52lo T cells. Chemokine receptor 8 (CCR8) expression in CD4+CD52lo T cells was increased. In vitro experiments using CD4 T cells of SLE patients showed that thymus and activation-regulated chemokine (TARC), a ligand of CCR8, contributed to the development of CD4+CD52hi T cells into CD4+CD52lo T cells. Our findings suggest that CD4+CD52lo T-cell upregulation is involved in the production of pathogens by autoantibodies, and TARC may contribute to the development of SLE through an aberrant induction of CD4+CD52lo T cells.

Graphene quantum dots suppress proinflammatory T cell responses via autophagy-dependent induction of tolerogenic dendritic cells.

Graphene quantum dots (GQD) are atom-thick nanodimensional carbon sheets with excellent physico-chemical and biological properties, making them attractive for application in theranostics. However, their immunoregulatory properties are insufficiently investigated, especially in human primary immune cells. We found that non-toxic doses of GQD inhibit the production of proinflammatory and T helper (Th)1 cytokines, and augment the production of anti-inflammatory and Th2 cytokines by human peripheral blood mononuclear cells. While unable to affect T cells directly, GQD impaired the differentiation and functions of monocyte-derived dendritic cells (DC), lowering their capacity to stimulate T cell proliferation, development of Th1 and Th17cells, and T-cell mediated cytotoxicity. Additionally, GQD-treated DC potentiated Th2 polarization, and induced suppressive CD4+CD25highFoxp3+ regulatory T cells. After internalization in a dynamin-independent, cholesterol-dependent manner, GQD lowered the production of reactive oxygen species and nuclear translocation of NF-κB in DC. The activity of mammalian target of rapamycin (mTOR) was reduced by GQD, which correlated with the increase in transcription of autophagy genes and autophagic flux in DC. Genetic suppression of autophagy impaired the pro-tolerogenic effects of GQD on DC. Our results suggest that GQD-triggered autophagy promotes tolerogenic functions in monocyte-derived DC, which could be beneficial in inflammatory T-cell mediated pathologies, but also harmful in GQD-based anti-cancer therapy.

Obese donor mice splenocytes aggravated the pathogenesis of acute graft-versus-host disease via regulating differentiation of Tregs and CD4+ T cell induced-type I inflammation.

Acute graft-versus-host disease (aGVHD) remains one of the most severe complications in organ and bone marrow transplantation, leading to much morbidity and mortality. Obesity has been associated with increased risk of development of various inflammatory diseases. Here, we investigated the in vitro and in vivo effects of obese donor splenocytes on the development of acute graft-versus-host disease (aGVHD). In this study, mixed lymphocyte reactions (MLR) in vitro showed that obese donor mouse CD4+ T cell promoted the production of interleukin-2 (IL-2), interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Meanwhile, the inducible Tregs population decreased greatly in obese donor mouse CD4+ T cells’ induction group, compared with normal group. Then in the murine aGVHD model, we found that obese donor splenocytes dramatically increased the severity of aGVHD through down-regulating immune tolerance while enhancing systemic and local immunity. Moreover, we showed that aGVHD induced by obese donors resulted in massive expansion of donor CD3+ T cells, release of Th1-related cytokines, interleukin-17 (IL-17) and chemokines, significant increase of Th17 cells and inhibition of CD4+CD25+Foxp3+ regulatory T cells (Tregs) and impaired suppressive ability of donor Tregs. Expression of sphingosine-1-phosphate receptor 1 (S1PR1), phosphorylated Akt, mammalian target of rapamycin (mTOR) and Raptor increased, while the phosphorylation level of SMAD3 was decreased in the skin, intestine, lung and liver from obese donor splenocytes-treated aGVHD mice. Furthermore, at mRNA and protein levels, we defined several molecules that may account for the enhanced ability of obese donor splenocytes to migrate into target organs, such as IL-2, IL-17, IFN-γ, TNF-α, CXCR3, CXCL9, CXCL10, CXCL11 and CCL3. Therefore, these results imply that obese donor cells may be related to the risk of aGVHD and helping obese donor individuals lose weight represent a compulsory clinical strategy before implementing transplantation to control aGVHD of recipients.

Anti-CD25 monoclonal antibody enhances the protective efficacy of Schistosoma japonicum GST vaccine via inhibition of CD4+CD25+Foxp3+ regulatory T cells.

The effect of anti-CD25 monoclonal antibody (anti-CD25 mAb) on the protection efficacy of Schistosoma japonicum 26kDa GST (glutathione-S-transferase) vaccine was evaluated. Mice were immunized with GST before infection with S. japonicum cercariae and then injected with anti-CD25 mAb. The worm reduction rate was promoted from 24.18% in mice with GST immunization to 47.09% in mice with GST plus anti-CD25 mAb. Compared with the control group, the percentages of splenic CD4+CD25+Foxp3+ regulatory T cells (Tregs) were significantly lower after administration of anti-CD25 mAb; meanwhile, elevated levels of IFN-γ and IL-2 were secreted by splenocytes. These results indicate that the poor protective efficacy of the GST vaccine against S. japonicum results from the presence of CD4+CD25+Foxp3+ Tregs, while anti-CD25 mAb can partially block CD4+CD25+Foxp3+ Tregs and thus enhance the protective efficacy of the GST vaccine.

Characterization and Expansion of Autologous GMP-ready Regulatory T Cells for TREG-based Cell Therapy in Patients with Ulcerative Colitis.

A local imbalance between regulatory (Treg) and effector T cells is believed to play a major role in gut-specific inflammation, including ulcerative colitis (UC). Restoration of this balance through an adoptive Treg transfer is an attractive new treatment approach in patients who are refractory to current standard therapies. It was our goal to develop a Good Manufacturing Practices (GMP)-conform protocol for expansion of UC Treg cells as a rational backbone for future studies on Treg therapy in UC. CD25 blood T cells derived from patients with UC were ex vivo expanded in the presence of IL-2, rapamycin, and anti-CD3/anti-CD28 expander beads using a GMP-conform protocol. Cells were subsequently assessed for stability and function. Patient-derived ex vivo rapamycin-expanded GMP-ready CD25 cells were polyclonal, hypomethylated at intron 1 of the FoxP3 locus, and suppressive in carboxyfluorescein succinimidyl ester-dilution assays against autologous peripheral blood-derived and allogeneic colon-derived responder cells. Function was mediated by soluble factors, including toxic granules. In addition to CD4 T cells, suppressive hypermethylated CD8 T-cell subsets were also induced during the expansion process. Patient-derived rapamycin-expanded CD25 cells are stable and functional, and as such, ready to serve in a phase I dose-escalation safety study in UC.

T cell-intrinsic TLR2 stimulation promotes IL-10 expression and suppressive activity by CD45RbHi T cells.

While Toll-like receptors (TLRs) represent one of the best characterized innate immune pathways, evidence suggests that TLRs are not restricted to innate leukocytes and some epithelial cells, but are also expressed in T cells. Specifically, published evidence focusing on FoxP3+ regulatory T cells demonstrate that they express functional TLR2, which is already known among the TLR family for its association with immune suppression; however, little is known about the relationship between T cell-intrinsic TLR2 binding and cytokine production, T cell differentiation, or T cell receptor (TCR) stimulation. Here, we demonstrate that TCR and TLR2 co-stimulation provides a T cell-intrinsic signal which generates a dramatic, synergistic cytokine response dominated by IL-10. Importantly, the response was not seen in either CD4+CD25+ or CD4+FoxP3+ Tregs, yet resulted in the expansion of a suppressive CD4+CD25+CD62L-CD44+CD45Rbhi effector/memory T cell subset not typically associated with immune inhibition. This study reveals the striking ability of a prototypical innate immune receptor to trigger a potent and suppressive IL-10 response in effector/memory T cells, supporting the notion that TLR2 is a co-regulatory receptor on T cells.

Immunotherapy with Listeria reduces metastatic breast cancer in young and old mice through different mechanisms

ABSTRACTCancer immunotherapy is one of the most promising and benign therapies against metastatic cancer. However, most cancer patients are old and elderly react less efficient to cancer vaccines than young adults, due to T cell unresponsiveness. Here we present data of cancer vaccination in young and old mice with metastatic breast cancer (4T1 model). We tested adaptive and innate immune responses to foreign antigens (Listeria-derived) and self-antigens (tumor-associated antigens (TAA)) and their contribution to elimination of metastases at young and old age. Three different protocols were tested with Listeria: a semi- and exclusive-therapeutic protocol both one-week apart, and an exclusive therapeutic protocol frequently administered. Adaptive and innate immune responses were measured by ELISPOT in correlation with efficacy in the 4T1 model. We found that Listeria induced immunogenic tumor cell death, resulting in CD8 T cell responses to multiple TAA expressed by the 4T1 tumors. Only exclusive therapeutic frequent immunizations were able to overcome immune suppression and to activate TAA- and Listeria-specific CD8 T cells, in correlation with a strong reduction in metastases at both ages. However, MHC class Ia antibodies showed inhibition of CD8 T cell responses to TAA at young but not at old age, and CD8 T cell depletions in vivo demonstrated that the T cells contributed to reduction in metastases at young age only. These results indicate that CD8 T cells activated by Listeria has an antitumor effect at young but not at old age, and that metastases at old age have been eliminated through different mechanism(s).

Abstract 2780: High nuclear FTH1 protein expression predicts early disease recurrence for BRCA1/2 mutation carriers

Abstract FTH1 is a 21kD subunit of the ferritin complex, which is involved in intracellular iron storage. In a previous study we found that FTH1 proteins can be located either in the cytoplasm or nucleus and, depending on its cellular localization, is associated with good or poor prognosis in triple negative breast cancer (TNBC). Furthermore, cytoplasmic localization of cFTH1 was associated with an increased anti-tumor (CD8+) T-cell / tumor-promoting (CD4+) T-cell ratio suggesting an immune modulatory role for FTH1. In the current study we have assessed the prognostic value of FTH1 expression for BRCA1/2 mutation carriers. FTH1 proteins were visualized by immunohistochemistry on a tissue microarray containing primary breast cancer specimens from 222 BRCA1/2 mutation carriers that were diagnosed between 1982 and 2008. Primary breast cancers from BRCA1 carriers were predominantly of the TNBC subtype, whereas primary breast cancers from BRCA2 mutation carriers were mainly ER-positive. The association between FTH1 expression and clinical outcome was evaluated by a Cox proportional hazards regression model using left truncation to adjust for study inclusion bias. The median follow-up time of the study was 9.2 years. High nuclear FTH1 (nFTH1), but not cytoplasmic FTH1 (cFTH1), was associated with decreased metastasis-free and disease-free survival as compared with low nFTH1 (multivariable HR=3.54, 95% CI=1.45-8.66, P=0.0056 adjusted for tumor size and nodal status; multivariable HR=2.71, 95% CI=1.49-4.92, P=0.0011 adjusted for tumor size). Neither nFTH1 nor cFTH1 were associated with breast cancer-specific or overall survival. This suggests that high nFTH1 expression in primary breast cancer predicts earlier disease recurrence for BRCA1/2 mutation carriers. Since we had observed that CD4+:CD8+ T-cell ratios were increased among TNBCs with high nFTH1, we hypothesize that nFTH1 promotes recruitment of immune suppressive CD4+/FOXP3+ regulatory T-cells in TNBC and BRCA1/2-associated tumors, suppressing CD4+/FOXP3- and CD8+ T-cell anti-tumor immunity. Citation Format: Antoinette Hollestelle, Mieke Timmermans, Renée Broeren-Foekens, Anita Trapman - Jansen, Carolien H. van Deurzen, Arzu Umar, John W. Martens, Maartje Hooning. High nuclear FTH1 protein expression predicts early disease recurrence for BRCA1/2 mutation carriers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2780. doi:10.1158/1538-7445.AM2017-2780

Abstract 2639: Development of small molecule selective inhibitors of GCN2 as an immunotherapy aimed at preventing immune escape of tumor cells

Abstract GCN2 is a protein kinase capable of sensing amino acid (AA) shortage. It gets activated by binding of unloaded tRNAs and subsequently phosphorylates translation initiation factor 2 alpha (eIF2α) eventually leading to induction of ATF4-mediated integrated stress response (ISR). Elevated catabolism of one of the essential amino acids - tryptophan (Trp), driven by overexpression of critical enzymes in Trp metabolism - IDO and TDO, leads to immunosuppressive microenvironment in many types of cancer. GCN2 is a key effector signaling component for IDO/TDO and is considered as a metabolic checkpoint of highly Trp-dependent T-cells. GCN2 activation through accumulation of unloaded tRNAs leads to inhibition of CD8+ effector T-cells and increase in generation and activation of regulatory T-cells. According to these findings, selective inhibition of GCN2 may be an effective strategy for targeting Trp-dependent immunosurveillance of tumor cells. In this study, we report the results for a series of novel small molecule GCN2 kinase inhibitors that have been developed at Selvita and, to our best knowledge, are the most potent GCN2 inhibitors reported so far. Newly synthesized compounds exert low nanomolar potency for GCN2, good selectivity as well as ADME profiles. Profound assessment of the compounds potency and efficacy using a selection of in vitro activity/binding assays will be presented. Citation Format: Michał Gałęzowski, Kamil Sitarz, Eliza Majewska, Stefan Chmielewski, Kinga Michalik, Magdalena Masiejczyk, Agnieszka Adamus, Arkadiusz Białas, Joanna Fogt, Marcin Bień, Mateusz Świrski, Maciej Mikulski, Julian Zachmann, Krzysztof Brzózka. Development of small molecule selective inhibitors of GCN2 as an immunotherapy aimed at preventing immune escape of tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2639. doi:10.1158/1538-7445.AM2017-2639

Abstract 4600: Potent and selective C-C chemokine receptor (CCR4) antagonists potentiate anti-tumor immune responses by inhibiting regulatory T cells (Treg)

Abstract Naturally suppressive CD4+ Foxp3+ Treg are essential for immune tolerance. Although Treg-mediated suppression of effector cells is important to control inflammation and prevent autoimmune diseases, the presence of Treg in the tumor microenvironment (TME) has been shown to dampen anti-tumor immune responses. Human Treg express CCR4, the receptor for the chemokines CCL17 and CCL22. These chemokines are produced by tumor cells, tumor-associated macrophages and dendritic cells, as well as by effector T cells (Teff). Preclinical and clinical data supports a role for CCR4-mediated recruitment and accumulation of Treg in the TME which can be associated with poor prognosis. Further, recent longitudinal studies in patients receiving IO agents demonstrate an influx of Treg in responding patients which may dampen optimal anti-tumor responses. Therefore, CCR4 is an ideal target to selectively block Treg recruitment into the TME. We have developed structurally unique series of small molecule antagonists of CCR4. These antagonists have cellular potencies in multiple assays (e.g. chemotaxis of primary human Treg in 100% serum) in the low double-digit nM range. Representative compounds are selective against other chemokine receptors, GPCRs and ion channels, including the hERG channel, and lack inhibition of common human CYP450 enzymes. Moreover, compounds have excellent in vitro and in vivo ADME properties, consistent with convenient oral dosing. In preclinical syngeneic tumor models, these CCR4 antagonists block Treg migration and support expansion of activated Teff. In contrast to the non-selective approach of depleting anti-CCR4 antibodies, our compounds reduce Treg in the tumor, but not in peripheral tissues such as blood, spleen or skin. In preclinical efficacy studies, CCR4 antagonists potentiate the anti-tumor effects of various checkpoint inhibitors and immune stimulators such as anti-PD-L1 and anti-CD137 antibodies. We observe enhanced tumor growth inhibition and increased tumor regressions when these agents are combined with CCR4 antagonists, without any gross toxicity. Further characterization of these CCR4 antagonists and their anti-tumor effects will be described. Citation Format: Oezcan Talay, Lisa Marshall, Cesar Meleza, Maureen K. Reilly, Omar Robles, Mikhail Zibisky, Abood Okal, Lisa Seitz, Jenny McKinnell, Scott Jacobson, Erin Riegler, Emily Karbaz, David Chian, Angela Wadsworth, Paul Kassner, David Wustrow, Jordan S. Fridman. Potent and selective C-C chemokine receptor (CCR4) antagonists potentiate anti-tumor immune responses by inhibiting regulatory T cells (Treg) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4600. doi:10.1158/1538-7445.AM2017-4600

TGF-beta/atRA-induced Tregs express a selected set of microRNAs involved in the repression of transcripts related to Th17 differentiation

Regulatory T cells (Tregs) are essential regulators of immune tolerance. atRA and TGF-β can inhibit the polarization of naïve T cells into inflammatory Th17 cells, favoring the generation of stable iTregs, however the regulatory mechanisms involved are not fully understood. In this context, the roles of individual microRNAs in Tregs are largely unexplored. Naïve T cells were immunomagnetically isolated from umbilical cord blood and activated with anti-human CD2/CD3/CD28 beads in the presence of IL-2 alone (CD4Med) or with the addition of TGF-β and atRA (CD4TGF/atRA). As compared to CD4Med, the CD4TGF/atRA condition allowed the generation of highly suppressive CD4+CD25hiCD127−FOXP3hi iTregs. Microarray profiling allowed the identification of a set of microRNAs that are exclusively expressed upon TGF-β/atRA treatment and that are predicted to target a set of transcripts concordantly downregulated. This set of predicted targets were enriched for central components of IL-6/JAK/STAT and AKT-mTOR signaling, whose inhibition is known to play important roles in the generation and function of regulatory lymphocytes. Finally, we show that mimics of exclusively expressed miRs (namely miR-1299 and miR-30a-5p) can reduce the levels of its target transcripts, IL6R and IL6ST (GP130), and increase the percentage of FoxP3+ cells among CD4+CD25+/hi cells.

Hepatic stroma-educated regulatory DCs suppress CD8+ T cell proliferation in mice.

Liver dendritic cells (DCs) display immunosuppressive activities and inhibit the CD4+ T cell response. The present study assessed whether and how liver DCs suppress CD8+ T cells. We found that bone marrow-derived mature DCs incubated with liver stromal cells were characterized by a longer life span, reduced CD11c, IA/IE, CD80, CD86, and CD40 expression, and increased CD11b expression. These unique liver stromal cell-educated mature DCs (LSed-DCs) stimulated CD8+ T cells to express CD25 and CD69, but inhibited their proliferation. CD8+ T cell suppression depended on soluble factors released by LSed-DCs, but not cell-cell contact. Compared with mature DCs, LSed-DCs produced more nitric oxide and IL-10. Addition of a nitric oxide synthase inhibitor, PBIT, but not an IL-10-blocking mAb, reversed LSed-DC inhibition of CD8+ T cell proliferation. We also found that LSed-DCs reduced CD8+ T cell-mediated liver damage in a mouse model of autoimmune hepatitis. These results demonstrate that the liver stroma induces mature DCs to differentiate into regulatory DCs that suppress CD8+ T cell proliferation, and thus contribute to liver tolerance.

Immunologic and genomic characterization of high grade serous ovarian cancer (HGSOC) in patients (pts) treated with pembrolizumab (Pembro) in the phase II INSPIRE trial.

5581 Background: Checkpoint inhibitors have shown to be effective in different tumors and are under investigation in HGSOC. Methods: INSPIRE (NCT02644369) is a prospective multi-cohort study investigating tumor genomic and immune landscapes in pts treated with Pembro at 200 mg IV Q3W. Patients underwent tumor biopsy pre, on-treatment and at progression for DNA/RNA sequence, immune-profile, and PD-L1 expression by immunohistochemistry (IHC). Serial blood samples for immunophenotyping were collected. Correlative data are available for 6 pts: 3 with shrinkage in target lesion and 3 with progressive disease (PD). Results: At interim analysis as of January 2017, 18 pts with HGSOC have been enrolled and 16 have platinum-resistant disease, with median 3 prior lines of treatment (range 1-7). Of 14 evaluable pts, best response by RECIST 1.1 was stable disease (SD) in 5 (36%) and PD in 9 (64%). Mean Tumor Proportion Score of PD-L1 by IHC (Qualtek) was 6.4% (range 0-30%). Grade 3/4 adverse events possibly related to Pembro were observed in 4/18 (22%) pts; none was fatal and the most common were fatigue and hyponatremia. Preliminary correlative data showed no significant change in CD4, CD8 and myeloid-derived suppressor cells in peripheral blood after Pembro treatment. Mean PD-1 expression on CD4 and CD8 T cells on baseline tumor tissue (measured as product of PD-1+ cells and the per cell expression of PD-1 [% of mean fluorescence intensity]) was significantly higher in pts with tumor shrinkage compared to pts with PD (CD4: 2658 vs 678, p = .02; CD8: 1999 vs 451, p = .048). Genomic analysis of baseline tumor tissue was available for 3 pts with tumor shrinkage and 2 with PD. Mean mutation burden was higher for pts with tumor shrinkage (2.38 vs 1.0 mutations/Mb covered). The pt with the longest SD in our cohort (6 months) had the highest mutation burden (2.72), including somatic POLE (c.6331-6C > G) and germline BRCA2mutations. Conclusions: In HGSOC, pts with higher PD-1 level on tumor CD4 and CD8 T cells and higher mutation burden at baseline may have a better outcome following treatment with Pembro. POLE mutation is rare in HGSOC but may correlate with checkpoint inhibitor activity. Clinical trial information: NCT02644369.

The Nrf2 activators tBHQ and CDDO-Im have both Nrf2-dependent and – independent effects on human Jurkat T cell activation, as determined by CRISPR-Cas9 gene editing

Abstract Nuclear factor erythroid 2-related factor 2 (Nrf2) is a stress activated transcription factor, activated by such stimuli as reactive oxygen and electrophilic stress. Upon activation Nrf2 accumulates in the nucleus and activates a battery of target genes, many of which are cytoprotective in function. We have previously shown that a common synthetic food additive, tBHQ, is a potent Nrf2 activator and suppresses certain events of human T cell activation, namely, IL-2 secretion, CD25 expression, and transactivation ability of NFkB. The purpose of our current studies was to develop a Nrf2-null human model, in which it can be determined whether the effects of tBHQ upon the early events of T cell activation are Nrf2-dependent. Treatment of both Wild-type and Nrf2-null Jurkat T cells revealed that the suppression of IL-2 and NFkB by tBHQ is largely Nrf2-independent, whereas the suppression of CD25 is partially dependent upon Nrf2. Although interesting, these studies left the question as to the role of Nrf2 in T cell activation largely unanswered. Therefore, we utilized a more potent and selective Nrf2 activator, CDDO-Im. CDDO-Im treatment resulted in a Nrf2-dependent suppression of IL-2 secretion and an inhibition of NFkB activity that was partially Nrf2-dependent. Taken together, our model is the first to show that activation of Nrf2 suppresses early events of human T cell activation (This work is funded by NIH grants ES018885, ES024966 and GM092715).

Inducing Immunological Tolerance with Synthetic Nanoparticles

Abstract Here we describe the use of synthetic, biodegradable tolerogenic nanoparticles carrying rapamycin, to induce durable and antigen-specific immune tolerance, even in the presence of potent Toll-like receptor (TLR) agonists. Treatment with these synthetic vaccine particles (SVP) and antigen (inside the particles or admixed) results in the inhibition of CD4+ and CD8+ T cell activation, differentiation of T and B regulatory cells, durable B cell tolerance resistant to multiple immunogenic challenges, the inhibition of antigen-specific hypersensitivity reactions, relapsing experimental autoimmune encephalomyelitis (R-EAE) and tolerance to biologic drugs such as the coagulation factor VIII (in hemophilia A animals), adalimumab (Humira in TNFa-transgenic animals), pegylated uricase (in uricase deficient animals), even in animals previously sensitized to antigen. Only encapsulated rapamycin, not the free form, could induce immunological tolerance. SVP therapy represents a potential novel approach for the treatment of treatment of allergies, autoimmune diseases, and prevention of anti-drug antibodies (ADA) against biologic therapies. Tolerogenic nanoparticle therapy for the prevention of ADAs against pegylated uricase in the treatment of hyperuricemia is currently being evaluated in Phase 2 clinical trials. Data from our Phase 1 clinical trial will be presented.

Germinal Center Immune Dynamics in Secondary Lymphoid Organs after Influenza Vaccination in Children

Abstract Introduction The germinal center immune dynamics leading to protective antibody responses after vaccination in humans remains largely unknown. Investigation of such dynamics would provide valuable information regarding the cellular and molecular mechanisms governing the generation of these responses and lead to novel vaccine strategies Materials and methods Paraffin embedded tonsil tissue sections were analyzed by multiplexed confocal microscopy assays and Histo-cytometry. The phenotype of tonsil derived cells was analyzed with polychromatic flow cytometry, flu-specific B cell-responses were detected by ELISPOT to H1N1, H3N2, and B strain and plasmas analyzed with a Luminex multiplex assay. Results 36 children (24 vaccinated with 2015/2016 seasonal influenza vaccine and 12 controls) scheduled for elective tonsillectomy were included. Flow cytometry and Histo-Cytometry analysis showed that vaccination induced the frequency of tonsil CD4 Tfh, especially the PD-1hiCD57lo ones. This profile was associated with a significant reduction of potential tissue suppressor CD4 T cells and positively correlated with all flu-antigen specific B cells. An induction of IL-4R on plasma cells was found after vaccination implying a further mobilization of these cells. Among the circulating cytokines/chemokines tested, CXCL-13 was selectively induced after vaccination, in line with the increased frequency of CD4 Tfh found. Conclusions Considered that children are continuously scheduled for tonsillectomy, tonsils could represent a valuable and approachable secondary lymphoid organ for the investigation of germinal center reactions after vaccination compared to lymph node not easily accessible in this population.

The many ways tissue phagocytes respond to dying cells.

Apoptosis is an important component of normal tissue physiology, and the prompt removal of apoptotic cells is equally essential to avoid the undesirable consequences of their accumulation and disintegration. Professional phagocytes are highly specialized for engulfing apoptotic cells. The recent ability to track cells that have undergone apoptosis in situ has revealed a division of labor among the tissue resident phagocytes that sample them. Macrophages are uniquely programmed to process internalized apoptotic cell-derived fatty acids, cholesterol and nucleotides, as a reflection of their dominant role in clearing the bulk of apoptotic cells. Dendritic cells carry apoptotic cells to lymph nodes where they signal the emergence and expansion of highly suppressive regulatory CD4 T cells. A broad suppression of inflammation is executed through distinct phagocyte-specific mechanisms. A clever induction of negative regulatory nodes is notable in dendritic cells serving to simultaneously shut down multiple pathways of inflammation. Several of the genes and pathways modulated in phagocytes in response to apoptotic cells have been linked to chronic inflammatory and autoimmune diseases such as atherosclerosis, inflammatory bowel disease and systemic lupus erythematosus. Our collective understanding of old and new phagocyte functions after apoptotic cell phagocytosis demonstrates the enormity of ways to mediate immune suppression and enforce tissue homeostasis.