Pro-inflammatory Cytokines IFN-g Research Articles

Targeting BRD4 to Counteract Cytokine-Induced Expression of PD-L1 and PD-L2 on Vascular, Osteoblastic and Stromal Niche Cells

The stem cell niche plays a crucial role in promoting the expansion of leukemic stem cells (LSC) and in mediating resistance of LSC against various anti-neoplastic drugs in different types of leukemias. Recent data suggest that targeting niche cells may be an effective approach to overcome niche-mediated drug resistance of LSC. However, niche cells themselves are considered to be often resistant against therapeutic intervention. This may especially be relevant in those myeloid neoplasms where niche cell expansion is a pathognomonic feature and the resulting neo-angiogenesis, bone marrow fibrosis and/or sclerosis are important prognostic variables. We and others have recently shown that several of the anti-neoplastic drugs used to treat myeloid leukemias can also counteract niche cell expansion. So far, however, little is known about mechanisms underlying resistance of niche cells against anti-neoplastic drugs. Recent studies have shown that certain immune checkpoint antigens are overexpressed in cancer cells and/or their microenvironment and that these checkpoint antigens contribute to drug resistance. Other studies have shown that checkpoint molecules, including PD-L1 and CD47, are displayed by LSC in acute myeloid leukemia, chronic myeloid leukemia and other myeloid neoplasms. We asked whether niche-related cells also display these checkpoint molecules. Furthermore, we screened for cytokines that promote expression of checkpoint antigens on niche cells and asked for drugs that counteract growth and cytokine-induced checkpoint expression in these cells. The following niche cells were examined: primary human umbilical vein endothelial cells (HUVEC), the human endothelial cell line HMEC-1, the human osteoblast-like cell line CAL-72, primary osteoblasts, and the human stromal cell line HS5. Expression of the checkpoint antigens PD-L1, PD-L2, PD1, CD28, CD47, CD80, CD83, CD86 and TIM-3 was determined by flow cytometry. All niche cells were found to constitutively express PD-L1, PD-L2, and CD47. The other checkpoint antigens were expressed at low or undetectable levels in these cells. Expression of PD-L1, PD-L2 and CD47 in niche cells was confirmed by quantitative PCR and by immunocytochemistry. Of all cytokines tested (n=12), interferon-alpha (IFN-A; effective range 50-500 ng/ml) and interferon-gamma (IFN-G; 10-500 ng/ml) were found to promote surface expression of PD-L1 and PD-L2 in HMEC-1, CAL72, and HS5 cells in our immunocytochemical and flow cytometry experiments. Moreover, we found that tumor necrosis factor-alpha (TNF-A; 100 ng/ml) enhances IFN-G-induced expression of PD-L1 and PD-L2 in HMEC-1, CAL-72 and HS5 cells. The BRD4-targeting drug JQ1 and the BRD4 degraders dBET1 and dBET6 suppressed cytokine-induced upregulation of surface expression of PD-L1 and PD-L2 in HMEC-1, HUVEC, CAL-72 and HS5 cells, suggesting a role for BRD4 and MYC in checkpoint regulation. In all cell lines examined, dBET6 was found to be a more effective drug compared to JQ1 and dBET1 (IFN-G-induced expression of PD-L1 in: HMEC-1: IC 50 for JQ1 0.5 µM vs dBET1 1 µM vs dBET6 0.05 µM; CAL-72: IC 50 for JQ1 >0.5 µM vs dBET1 1 µM vs dBET6 <0.05 µM). Together, we show that endothelial cells, osteoblasts and stromal cells display PD-L1, PD-L2 and CD47 in a constitutive manner. Our data also show that the pro-inflammatory cytokines IFN-G and IFN-A augment expression of PD-L1 and PD-L2 in these cells, and that TNF-A can further enhance IFN-A/IFN-G-induced expression of PD-L1 and PD-L2 in niche cells. In addition, we show that the BRD4-targeting drugs JQ1, dBET1 and dBET6 suppress cytokine-induced PD-L1 and PD-L2 expression on niche cells. Whether checkpoint expression in niche cells is associated with drug resistance in myeloid neoplasms and whether drug-induced suppression of checkpoint expression in niche cells can overcome resistance remains unknown.

The role of interleukin-12-PRMT5 axis in generating suppressive Th1-like induced regulatory T cells

Abstract After activation, naïve CD4 T cells can differentiate into T helper cell subsets (Th1, Th2, Th17, and Follicular helper T cells) or into regulatory T cells (Tregs), depending on the type of antigen-presenting cells (APC) and cytokine milieu encountered during differentiation. Recent studies suggest that CD4+Foxp3+ Tregs can produce the pro-inflammatory cytokine IFNg when stimulated in a Th1 cytokine environment, such as in the presence of IL-12, and stimulation under these conditions makes iTregs sensors for inflammatory cytokines. The stability and suppressive functions of the iTregs depend on multiple factors. The goal of this study is to explore an epigenetic enzyme protein arginine methyltransferase 5 (PRMT5) and to understand the significance of this protein in the development of Th1-like iTregs. PRMT5 can either represses or activate transcription of target genes via symmetric di-methylation of arginine residues on histones. However, the role of PRMT5 in the differentiation of Th1-like iTregs has not been thoroughly investigated. We demonstrate that differentiating iTregs in a Th1 cytokine environment generated a specific population of Th1-like iTregs with increased suppressive function. In Th1-like iTregs, we observed symmetric arginine di-methylation on histone (H3R2me2s) mediated by PRMT5, while this epigenetic marker is absent in iTregs. We also demonstrate that si-RNA mediated knockdown of PRMT5 reduced the suppressive capacity of Th1-like iTregs. Therefore, we hypothesize that the IL12-PRMT5 axis is crucial for developing suppressive Th1-like iTregs. Goals of further research are to generate stable immunosuppressive iTregs for therapeutic use.

Abstract 4159: The COX-2/PGE2 pathway as a mediator of resistance to anti-PD-1 therapy

Abstract Background: We previously found upregulation of the cyclooxygenase-2/prostaglandin E2 (COX-2/PGE2) pathway in the tumor microenvironment (TME) of cancer types that respond poorly to anti-PD-1 therapy, and showed that TME-resident cytokines such as IL-1B induce COX-2 expression in human tumor and myeloid cells. To investigate a potential role for COX-2/PGE2 in anti-PD-1 resistance, we studied PGE2-mediated effects on human myeloid and T cells and their dependence on prostaglandin (EP) receptor signaling. Methods: Monocytes (Monos) and T cells were enriched from normal donor peripheral blood by negative selection. PGE2 effects were tested on Monos activated with IFN-g (PD-L1 expression; FACS) or LPS (cytokine secretion; ELISA). PGE2 effects on monocytic dendritic cells (DCs) generated with GM-CSF+IL-4 and matured with CD40L were assessed by phenotyping for costimulatory molecules (CD80, CD86) and subpopulation markers (CD14, CD16, CD83). T cells activated with anti-CD3/CD28 +/- PGE2 for 2-6 days were assessed for expression of CD25, CD69, CD107a, CD137, OX40, GITR, LAG-3, PD-1, PD-1, PD-L1 and TIM3 (FACS); proliferation markers (Ki67, CFSE; FACS); cytokine secretion (IFN-g, IL-2, IL-10, TGF-b; ELISA); and metabolic function (Seahorse assay). In some experiments, cells were pre-incubated with inhibitors of EP2 (PF-04418948), EP4 (ONO-AE3-208), or the dual inhibitor TPST-1495 (Tempest Therapeutics). Results: Myeloid cell functions were suppressed by PGE2, which reduced IFNg-induced PD-L1 expression and modulated LPS-induced cytokine secretion. Furthermore, PGE2 inhibited DC maturation, evidenced by persistent CD14 and CD16 expression and a decreased CD83+ subset. In CD4 and CD8 T cells activated with anti-CD3/CD28, PGE2 reduced expression of CD25 and CD69 (early activation markers), CD107a (cytolytic potential), Ki67 (cell proliferation), co-stimulatory molecules (CD137, OX40, GITR) and checkpoint molecules (LAG-3, PD-1, PD-L1, TIM3) in a dose-dependent manner. PGE2 also reduced the CFSE-low (proliferating) population in activated CD4, CD8 and Treg cells. Secretion of proinflammatory cytokines IFN-g and IL-2, and inhibitory cytokines IL-10 and TGF-b from activated T cells was also suppressed by PGE2. These effects were specifically reversed by chemical inhibitors of EP2 and/or EP4. PGE2 exposure at the time of T cell activation was associated with depressed mitochondrial oxidation and cellular glycolysis, suggesting global effects on T cell metabolism. Conclusion: Understanding and preventing anti-PD-1 resistance is a critical goal in oncology. Our results suggest that the COX-2/PGE2 pathway exerts immunosuppressive effects on myeloid and T cells via the EP2 and EP4 receptors. COX-2 and PD-L1 are distinct immune checkpoints, providing a rationale to test anti-PD-1 in conjunction with COX-2 pathway inhibitors such as IL-1B antagonists and highly specific EP2 and EP4 inhibitors in patients with cancer. Citation Format: Shuming Chen, Seoho Lee, Tracee L. McMiller, Isaac Morales, Preethi Sankaran, Brian Francica, Robert D. Leone, Tom W. Dubensky, Suzanne L. Topalian. The COX-2/PGE2 pathway as a mediator of resistance to anti-PD-1 therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4159.

Mucosal-Associated Invariant T (MAIT) Cells Are Functionally Impaired in Pediatric Patients Following HCT and Their Recovery Is Associated with the Onset of GvHD

Delayed quantitative and qualitative immune recovery, in particular of the T-cell lineage, is associated with an increased risk of infections, disease relapse and adverse clinical outcomes in patients receiving allogeneic hematopoietic cell transplantation (allo-HCT). This delay is particularly relevant for recipients of T cell-depleted grafts who do not benefit from the adoptive transfer of mature donor T cells and experienced profound and prolonged immunodeficiency which lasts until the T-cell pool is newly generated from donor hematopoietic precursor cells. A better understanding of the reconstitution dynamics of all the components of the immune system after allo-HCT is crucial to optimize the control of infections, leukemia relapse and graft-versus-host disease (GvHD). We performed a longitudinal observational study to explore the kinetics of T-cell recovery in patient peripheral blood by combining multiparameter flow-cytometry and functional ex vivo assays. Ninety-three children with hematological malignancies who received transplantation after myeloablative conditioning regimen were included in the study: 59 patients received T-cell receptor (TCR) alpha-beta/CD19-depleted HLA-haploidentical HCT (alpha/beta-haploHCT), while 34 unmanipulated matched unrelated donor (MUD) HCT (32 MUD recipients received BM and 2 received PBSC). Among the different T-cell populations assessed, we found that mucosal-associated invariant T (MAIT) cells displayed profound alterations in frequency and function across all time points analyzed. MAIT cells are a class of unconventional αβ T cells that recognize microbial proteins presented by non-polymorphic MHC class I related-molecule (MR1) and possess innate-like capacity. While total TCRαβ T cells gradually started to recover approximately from 3 months reaching normal levels between one to two years post transplantation (Figure 1), MAIT cells defined as CD3+ TCRαβ+ MR1-5-OP-RU-tetramer+ showed long-term impaired reconstitution, and their numbers did not reach normal levels up to two-years post-HCT (Figure 1). Compared to MUD HCT, MAIT cell recovery after alpha/beta-haploHCT was significantly delayed, in agreement with the fact that TCRab reconstitution in this clinical setting is primarily driven by de novo T cell formation. Previous studies demonstrated that MAIT cells are characterized by the binding to MR1-Ag tetramers and simultaneous expression of the C-type lectin-like receptor CD161, a marker associated with memory phenotype and trafficking of immune cells. Interestingly, across all time points of the study, we observed that most MAIT cells following were CD161-. We also found increased expression of the activation marker CD38 and the senescence marker CD57, and downregulation of CD28 in MAIT cells following allo-HCT, suggesting a dysfunctional or hyperactivated status. In agreement with these data, ex vivo stimulation of PBMCs at day +360 post allo-HCT with Escherichia Coli or anti-CD3/anti-CD28 showed that MAIT cells had suboptimal upregulation of the activation markers CD25 and CD69, and reduced production of pro-inflammatory cytokines IFNg, TNFa and IL2, when compared to healthy control samples. Previous studies suggested that a low MAIT cell number was associated with an increased risk of acute GvHD, In the MUD cohort of our study, we did not observe significant correlations between the kinetics of MAIT cell recovery and the occurrence of GvHD. On the other hand, we found a significantly higher frequency of CD161+ MAIT cells at day +30 in alpha/beta-haploHCT patients who developed acute GvHD (grade 1 or greater). Trends toward higher frequency of CD161+ MAIT cells in patients developing acute GvHD were also observed at day +60 and +90, although not statistically significant. Our study shows that MAIT cell recovery is delayed in children for up to two years following allo-HCT. MAIT cells exhibited altered functional status as demonstrated by the expression of activation/senescence makers as well as the suboptimal response to TCR stimulation. Interestingly, we found that a large proportion of MAIT cells are CD161- after transplantation, which can be the result of recruitment of CD161+ cells to peripheral tissues or down regulation of CD161 after cell activation. Finally, our data suggested that the evaluation of CD161 expression on MAIT cells after alpha/beta-haploHCT may identify patients at risk of acute GvHD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Glycoside oleandrin downregulates toll-like receptor pathway genes and associated miRNAs in human melanoma cells

Melanoma accounts for the majority of skin cancer-related deaths. Nerium oleander is a plant known to be toxic and consumed due to the cardiac glycosides it contains. Oleandrin is a cardiac glycoside obtained from of N. oleander. Beside capable of inhibiting proliferation and metastasis of cancer cells, cardiac glycoside derivative compounds cause cardiovascular side effects. Because of cardiovascular toxicity of clinically used cardiac glycosides, it is necessary to investigate cardiac glycoside derivative compounds capable of inhibiting proliferation and metastasis of cancer cells.It is known that oleandrin has anticarcinogenic effects in other cancers. Previous studies have shown that toll-like receptors (TLRs) and their related microRNAs (miRNAs) are associated with cancer. Therefore, aim was to investigate the effect of oleandrin on genes and miRNAs associated with TLRs in A375 melanoma cells in this study. The effects of oleandrin on cell viability, cytokines, apoptosis were evaluated using XTT, ELISA and TUNEL analyses, respectively. The effect of oleandrin on expression of TLR genes and 5 associated miRNAs in A375 cells has been determined by qRT-PCR. In addition, the levels of MyD88, TLR2 and TLR4 proteins were analyzed by western blot method.ELISA indicated that oleandrin treatment (47 nM at 48 h) reduced the level of proinflammatory cytokine IFNG. TUNEL analysis showed that apoptosis rate was significantly increased in the oleandrin dose group. According to qRT-PCR results, there was a significant decrease in IRAK1, IRAK4, MyD88, TLR2-TLR7 and TRAF3 expressions in the oleandrin treated group compared to the control (untreated cell). Also, a significant decrease in TLR4 protein expression has been observed. In addition, oleandrin significantly downregulated the levels of hsa-miRNA-146a-5p and hsa-miRNA-21-5p.In conclusion, it has been observed that oleandrin has an effect on TLR pathway-related genes and miRNAs in melanoma cells. We show that TLRs pathways and hsa-miR-146a-5p and hsa-miR-21-5p can participate in the oleandrin molecular mechanism of action.

111 Changes in proinflammatory cytokines and chemokines in beef steers chronically exposed to endophyte-infected tall fescue seed

Abstract Endophyte-infected tall fescue consumption adversely effects health and immune response. The aim of this study was to evaluate the impact of protein supplementation on cytokine response in steers consuming ergovaline, found in endophyte-infected tall fescue. Thirty-two beef steers were fed a total mixed ration (TMR) and randomly assigned to receive one of four treatments for 56 days: endophyte-free seed (0 ug/kg ergovaline) with either 18% (EF-18; n = 8) or 14% dietary protein (EF-14; n = 8) and endophyte-infected seed (500 ug/ kg ergovaline) with 18% (EI-18; n = 8) or 14% dietary protein (EI-14; n = 8). Blood samples were collected for cytokine analysis on d 28 and d 42 of the feeding period representing when steers vaccinated and boostered for bovine viral diarrhea (BVD) types 1 and 2 and infectious bovine rhinotracheitis (IBR). Cytokine concentrations were evaluated using Quantibody Bovine Cytokine Arrays. Data were analyzed using PROC MIXED of SAS with repeated measures. Statistical significance was determined at P0.05). Interestingly, concentrations of cytokines, IL13 (912±148 vs. 410.9±135 pg/mL) and IL21 (1636.0±295 vs. 629±305 pg/mL), which are involved in chronic inflammatory process were greater (P < 0.05) in EI steer compared to EF steers on d 28 and d 42. Concentrations of chemokine CCL4 and proinflammatory cytokines IFNG and IL-1alpha tended to be greater in EI steers compared to EF steers (P < 0.10). Overall, these data demonstrate that consumption of ergovaline increased proinflammatory cytokines and induced a chronic immune response. Developing a better understanding of overall immunity in cattle exposed to endophyte-infected tall fescue is fundamental to improve cattle health within the beef industry.

Type II NKT cells directed immune regulatory mechanism(s) control spontaneous lupus nephritis in mice

Abstract NKT cells recognize lipid antigens presented by CD1d molecules and can be categorized as iNKT cells and type II NKT cells. Type II NKT cells recognize sulfatide or lysophosphatidylcholine (LPC), and their activation results in cross-regulation of iNKT cells, tolerization of DC and control of inflammatory diseases. iNKT cells have been shown to play a pathogenic role in (NZB x NZW)F1 or BWF1 mice, a prototype for human SLE. Interestingly, anti-glycosphingolipid antibody responses have been found in lupus patients. Since sulfatide is enriched in kidney glomeruli, we have examined the role of type II NKT cells in the spontaneous development of lupus in BWF1 mice. We found that both iNKT (αGalCer/CD1d-tetramer+) and type II (sulfatide/CD1d-tetramer+) NKT cells accumulate in kidney tissues with progression of disease in BWF1 mice. Notably, iNKT cells are also activated in SLE patients and secrete the pro-inflammatory cytokine IFNg. Sulfatide-mediated activation of type II NKT cells leads to a significant inhibition of nephritis in BWF1 mice. Importantly, a clinically relevant structural analog of LPC, Miltefosine, also binds to CD1d, activates type II NKT cells and upon oral administration induces inhibition of proteinuria, anti-DNA Abs as well as infiltration of CD4+/CD8+T cells and B cells into kidney of BWF1 mice. Interestingly, the phenotype and frequency of type I interferon-secreting pDC is also altered in kidney of mice treated with Miltefosine. These studies suggest a key role for a type II NKT cell-based immune regulatory mechanism in the control of lupus and, since the CD1d-dependent pathway is highly conserved from mice to human, they have important implications for repurposing a drug for immunotherapeutic for lupus.

Protection efficacy of Argentinian isolates of Mycobacterium avium subsp. paratuberculosis with different genotypes and virulence in a murine model

Paratuberculosis is a chronic disease caused by Mycobacterium avium subsp. paratuberculosis (Map). The disease causes economic losses and, therefore, it is imperative to follow proper control strategies, which should include an effective vaccine. Several strategies have assessed the virulence and immune response of Map strains that could be used as a vaccine. This study evaluates the degree of virulence, immune response, and protection of Argentinian strains of Map with different genotype in a murine model. Four local isolates (Cattle type) with different genotypes (analyzed by MIRU-VNTR and SSRs) were selected and evaluated in a virulence assay in BALB/c mice. This assay allowed us to differentiate virulent and low-virulence Map strains. The less virulent strains (1543/481 and A162) failed to induce a significant production of the proinflammatory cytokine IFNg, whereas the virulent strain 6611 established infection along with a proinflammatory immune response. On the other hand, the virulent strain 1347/498 was efficient in establishing a persistent infection, but failed to promote an important Th1 response compared with 6611 at the evaluated time. We selected the low-virulence strain 1543/498 as a live vaccine and the virulent strain 6611 as a live and inactivated vaccine in a protection assay in mice. Strain 1543/481 failed to protect the animals from challenge, whereas strain 6611, in its live and inactivated form, significantly reduced the CFUs count in the infected mice, although they had different immunological response profiles. The inactivated virulent strain 6611 is a potential vaccine candidate against paratuberculosis to be tested in cattle.

Seminal plasma pro-inflammatory cytokines interferon-γ (IFNG) and C-X-C motif chemokine ligand 8 (CXCL8) fluctuate over time within men.

Do seminal plasma pro-inflammatory cytokines interferon-γ (IFNG) and C-X-C motif chemokine ligand 8 (CXCL8) vary within individual men over time? IFNG exhibits substantial variation that is independent of duration of abstinence but correlates with lipopolysaccharide (LPS) content, while CXCL8 varies moderately in association with duration of abstinence. Pro-inflammatory cytokines IFNG and CXCL8 in seminal fluid can adversely impact male and female fertility. Other cytokines as well as sperm parameters fluctuate considerably within individuals over time, but whether IFNG and CXCL8 vary similarly, and the determinants of variance, are unknown. Between two and seven semen samples were collected from 14 proven fertile donors at 6-10 week intervals over the course of ~12 months, to assess variation over time in cytokines and LPS, and to investigate relationships with sperm parameters and possible regulatory factors. The concentrations and total amounts per ejaculate of IFNG and CXCL8 were determined using commercial ELISA. Sperm parameters were assessed according to World Health Organization (WHO) IV standards and LPS was measured by limulus amebocyte lysate (LAL) assay. Mixed model analysis was utilized to determine the relative contribution of between- and within-individual factors in explaining variance. Relationships between cytokines, LPS and sperm parameters, as well as effect of age and duration of abstinence, were investigated by correlation analysis. Within-individual variability contributed to total variance particularly for both IFNG, CXCL8 and LPS, and was a stronger determinant than between-individual variability for IFNG and LPS. Normal sperm motility correlated inversely with CXCL8, and sperm concentration correlated inversely with LPS. Duration of abstinence was a determinant of total CXCL8, but not IFNG or LPS. Associations between LPS, IFNG and CXCL8 suggest IFNG and perhaps CXCL8 are influenced by microbial populations. A limited number of donors from a single clinic were investigated. Clinical information on complete microbiology, BMI, nutrition, smoking and other lifestyle factors was unavailable. Further studies are required to determine whether the findings can be generalized to larger populations and different ethnicities. These data reveal substantial variation over time in pro-inflammatory seminal fluid cytokines and imply existence of microbial or other environmental regulatory factors. This study was supported by grants from the National Health and Medical Research Council of Australia. The authors have no competing interests to disclose.

CD33-Directed Chimeric Antigen Receptor (CAR) T Cells for the Treatment of Acute Myeloid Leukemia (AML)

Therapies for acute myeloid leukemia (AML) have not improved patient long-term survival for decades. Novel treatment options, such as chimeric antigen receptor (CAR) T cells, are needed for patients with this disease. However, AML cells lack ideal targeting antigens that are safe to target with CAR T cells. CD33, a commonly targeted antigen, is expressed in about 85-90% of AML cases but is also present on normal myeloid progenitors and myelocytes. Our aim was to engineer and validate CD33-directed CAR T cells, with the intention to open a phase I clinical trial in patients with relapsed AML. Given the potential toxicity associated with targeting CD33 in patients, an elimination gene was included in the construct design to allow CAR T cell clearance after disease eradication.We constructed a CAR specific for CD33 by utilizing the variable heavy and light chains of the humanized M-195 antibody (HuM-195), CD28 and zeta signaling domains, and the IL-12 gene. To facilitate CAR T cell elimination if necessary, a truncated epidermal growth factor receptor (EGFRt) gene was also inserted into the retroviral vector to create the EGFRt/HuM195-28z/IL-12 CAR construct. Retroviral transduction with this tri-cistronic construct resulted in high transduction efficiency of human T cells, as determined by detection of EGFRt with fluorescently-labeled cetuximab or CAR with fluorescently-labeled CD33 molecule by flow cytometry. T cells transduced with the EGFRt/HuM195-28z/IL-12 CAR secreted functional IL-12, as assessed by culturing CAR cell supernatant with peripheral blood mononuclear cells and detecting IFN-g produced in response to IL-12. When stimulated through the CAR by co-culturing with the CD33+ AML cell line Molm-13, EGFRt/HuM195-28z/IL-12 CAR T cells proliferated and produced significant levels of the pro-inflammatory cytokines IFN-g and IL-2. In addition, EGFRt/HuM195-28z/IL-12 CAR T cells mediated significantly cytotoxicity against Molm-13 at a range of effector-to-target ratios in standard 51Cr-release assays, as compared to control CAR T cells.The in vivo anti-tumor efficacy of EGFRt/HuM195-28z/IL-12 CAR T cells was tested in two preclinical mouse models of AML. First, SCID/Beige mice were xenografted with Molm-13 cells. Mice subsequently treated with EGFRt/HuM195-28z/IL-12 CAR T cells exhibited significant long-term survival, as compared to untreated or control CAR T cell-treated mice (p = <0.0001). In addition, NSG mice were engrafted with patient-derived CD33+ AML (PDX) cells. PDX mice treated with EGFRt/HuM195-28z/IL-12 CAR T cells exhibited reduction of peripheral CD33+ disease, as compared to untreated or control CAR T cell-treated mice. These studies demonstrate the capacity of the EGFRt/HuM195-28z/IL-12 CAR to redirect the specific anti-tumor function of T cells to CD33+ AML tumor cells. Ongoing studies aim to validate elimination of CAR T cells via EGFRt in vitro in antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays, as well as in mice are currently underway. These data support the utilization of CD33-specific CAR T cells in the clinic for patients with relapsed AML as a means to decrease disease burden prior to consolidative therapies such as allogeneic transplantation. DisclosuresBrentjens:Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

CD19(+) CD20(-) CD27(hi) IL-s10-producing B cells are overrepresented in R-CHOP-treated DLBCL patients in complete remission.

Treatment of diffuse large B cell lymphoma (DLBCL) with rituximab, an anti-CD20 monoclonal antibody, has resulted in significantly improved patient responses with longer event-free intervals and higher overall survival rates. However, since rituximab depletes all CD20-expressing cells, including noncancerous B cells, the effects of rituximab on the normal immunity of DLBCL patients under remission need to be examined. Here, we observed that DLBCL patients under remission contained significantly lower frequencies of total B cells, with a significantly overrepresented interleukin (IL)-10-producing B cell (B10) population in the peripheral blood. Further examination confirmed that a large fraction of B10 cells was CD20(-) CD27(hi) plasmablasts, possibly explaining the persistence of B10 cells after R-CHOP treatment. We also observed that the percentage of B10 cells in DLBCL patients in remission gradually reduced during the first year of achieving complete remission, primarily due to the replenishment of non-B10 B cells. Despite this, the percentage of B10 cells in DLBCL patients after 1year of achieving complete remission was still higher than that in controls. CD4(+) and CD8(+) T cells cocultured with B10-enriched B cells secreted significantly lower levels of proinflammatory cytokines IFN-g and TNF-a, compared to those incubated with B10-depleted B cells. Together, our data observed a long-lasting overrepresentation of B10 cells in DLBCL patients under remission. Whether this change could impact on the overall anti-tumor immunity during remission requires further studies.

Inhibition of β‐catenin transactivation by IFNγ is regulated by Akt1 and 14.3.3z (650.1)

The proinflammatory cytokine IFNg influences intestinal epithelial cell (IEC) homeostasis in a biphasic manner by acutely stimulating proliferation that is followed by sustained inhibition of proliferation despite continued mucosal injury. B‐catenin activation has been classically associated with increased IEC proliferation. However, we have observed that IFNg inhibits IEC proliferation despite sustained activation of Akt/B‐catenin signaling. Here we show that inhibition of Akt/B‐catenin‐mediated cell proliferation by IFNg requires the formation of a protein complex containing phosphorylated B‐catenin 552 (pB‐cat552) and 14.3.3Z. We identify Akt1 as a bimodal switch that promotes or inhibits B‐catenin transcriptional activity in response to IFNg stimulation. We demonstrate that IFNg initially induces B‐catenin transactivation through Akt‐dependent c‐terminal phosphorylation of B‐catenin to promote its association with 14.3.3Z. Augmented B‐catenin transactivation leads to increased Akt1 protein and active Akt1 accumulates in the nucleus where it phosphorylates 14.3.3Z to induce relocalization of 14.3.3Z/B‐catenin from the nucleus thereby inhibiting B‐catenin transactivation and IEC proliferation. These results outline an unexpected dual function of Akt1 that suppresses IEC proliferation during intestinal inflammation.Grant Funding Source: Supported by NIH (CP and AN) and Conacyt (PN) grants.

A single non-synonymous polymorphism of TLR2 is sufficient to recapitulate the variability of Experimental Multiple Sclerosis

A single non-synonymous polymorphism of TLR2 is sufficient to recapitulate the variability of Experimental Multiple Sclerosis

Novel Foxo1-dependent transcriptional programs control Treg cell function

Regulatory T (T(reg)) cells, characterized by expression of the transcription factor forkhead box P3 (Foxp3), maintain immune homeostasis by suppressing self-destructive immune responses. Foxp3 operates as a late-acting differentiation factor controlling T(reg) cell homeostasis and function, whereas the early T(reg)-cell-lineage commitment is regulated by the Akt kinase and the forkhead box O (Foxo) family of transcription factors. However, whether Foxo proteins act beyond the T(reg)-cell-commitment stage to control T(reg) cell homeostasis and function remains largely unexplored. Here we show that Foxo1 is a pivotal regulator of T(reg )cell function. T(reg) cells express high amounts of Foxo1 and display reduced T-cell-receptor-induced Akt activation, Foxo1 phosphorylation and Foxo1 nuclear exclusion. Mice with T(reg)-cell-specific deletion of Foxo1 develop a fatal inflammatory disorder similar in severity to that seen in Foxp3-deficient mice, but without the loss of T(reg) cells. Genome-wide analysis of Foxo1 binding sites reveals ~300 Foxo1-bound target genes, including the pro-inflammatory cytokine Ifng, that do not seem to be directly regulated by Foxp3. These findings show that the evolutionarily ancient Akt-Foxo1 signalling module controls a novel genetic program indispensable for T(reg) cell function.

A unique T cell subset, Th40, are pathogenic and diagnostic in mulitple sclerosis (101.30)

Abstract Auto-aggressive T cells have been implicated in the pathogenesis and maintenance of MS, including mouse models and human disease. We described a T cell subset, Th40 cells, which proved highly pathogenic and auto-aggressive in autoimmune diabetes. Using flow cytometry, we compared Th40 levels in 90 MS subjects of all subtypes, 238 T1DM and 208 control patients. A subset of MS patients were followed during and after pregnancy as well. RRMS subjects have significantly increased percentages of Th40 cells in PBL compared to non-autoimmune controls. MS subjects (75) had Th40 cell levels equal to or greater than autoimmune T1D (238) subjects. The majority of Th40 cells from MS subjects' are T helpers producing pro-inflammatory cytokines IFNg, TNFa and IL-17. Th40 cells produce high levels of IL-6, an acute phase response cytokine that shunts regulatory T cells into Th17 pathway. During RRMS Th40 cell expansions occur regardless of age or gender and are responsive to known MS associated self antigens including MOG, MBP and PLP. A clonal expansion of TRAV 8.1.3+ cells within the Th40 population was evident. During pregnancy Th40 cell levels dropped dramatically corresponding with remission returning to pathogenic levels postpartum. In the mouse EAE model of MS Th40 cells are highly expanded. Th40 cells could prove diagnostic in RRMS and prove diagnostic to predict relapse. Th40 cells are pathogenic in MS and identification of a clonal expansion in MS further suggests autoimmunity.

Novel population of CD39-expressing T cells enriched in the inflamed site (89.21)

Abstract CD39-mediated hydrolysis of proinflammatory ATP is a newly described mechanism of Treg suppression. We describe the presence and function of a novel population of CD39+ Foxp3- CD4+ T cells enriched in the human inflammatory site. 36 patients with juvenile idiopathic arthritis (JIA) and 25 healthy controls were included in this study with full ethical approval (LREC). Mononuclear cells purified from peripheral blood (PBMC) and synovial fluid (SFMC) were analysed by FACS, functional suppression and ATP assays. CD39+ Foxp3- CD4 T cells were significantly enriched (p&amp;lt;0.001) in the inflamed joint, compared to PBMC from patients or healthy controls (18.6%, 2.9%, 4.2%, respectively). Unlike CD39+Foxp3+ Treg, this novel population produced the proinflammatory cytokines IFNg and IL-17 and also IL-2. CD39+Foxp3- cells expressed lower CCR4 and higher levels of CD127 than classical Treg. Upon cell sorting, CD39+Foxp3- cells had comparable ATP hydrolysis activity to Treg, but crucially, failed to suppress cell proliferation in vitro. Thus, the expression of Foxp3 with CD39 may be dissociated in the inflamed site and CD39 expression on CD4 T cells may not result in regulatory status. This will be important in understanding the complex effects of extracellular ATP in human inflammation. This study was supported by SPARKS UK and the Big Lottery Fund. KN is supported by an ARC Fellowship (Ref. 17998).

Repeat catheterization in ventricular septal defect and pulmonary hypertension.

NKT cells recognize lipid antigens presented by CD1d molecules and can be categorized as iNKT cells and type II NKT cells. Type II NKT cells recognize sulfatide or lysophosphatidylcholine (LPC), and their activation results in cross-regulation of iNKT cells, tolerization of DC and control of inflammatory diseases. iNKT cells have been shown to play a pathogenic role in (NZB x NZW)F1 or BWF1 mice, a prototype for human SLE. Interestingly, anti-glycosphingolipid antibody responses have been found in lupus patients. Since sulfatide is enriched in kidney glomeruli, we have examined the role of type II NKT cells in the spontaneous development of lupus in BWF1 mice. We found that both iNKT (αGalCer/CD1d-tetramer⁺) and type II (sulfatide/CD1d-tetramer⁺) NKT cells accumulate in kidney tissues with progression of disease in BWF1 mice. Notably, iNKT cells are also activated in SLE patients and secrete the pro-inflammatory cytokine IFNg. Sulfatide-mediated activation of type II NKT cells leads to a significant inhibition of nephritis in BWF1 mice. Importantly, a clinically relevant structural analog of LPC, Miltefosine, also binds to CD1d, activates type II NKT cells and upon oral administration induces inhibition of proteinuria, anti-DNA Abs as well as infiltration of CD4⁺/CD8⁺T cells and B cells into kidney of BWF1 mice. Interestingly, the phenotype and frequency of type I interferon-secreting pDC is also altered in kidney of mice treated with Miltefosine. These studies suggest a key role for a type II NKT cell-based immune regulatory mechanism in the control of lupus and, since the CD1d-dependent pathway is highly conserved from mice to human, they have important implications for repurposing a drug for immunotherapeutic for lupus.