TGF-beta Production Research Articles

Preliminary results of atezolizumab and pirfenidone in non-small cell lung cancer.

e15197 Background: Checkpoint inhibitors (CPI) targeting the PD1/PD-L1 axis improved outcomes for stage IV non-small cell lung cancer (NSCLC). Poor response to PD-L1 antibody has been linked to increased cancer-associated fibroblasts (CAF), which interact with cytotoxic T cells (CTLs) similarly to regulatory T cells (Tregs). CAFs, through cytokine production, impair antitumor immunity by affecting CTL function (TGF beta) and inhibiting CTL recruitment into tumors, suggesting that CAFs could be a therapeutic target in CPI-resistant NSCLC. Pirfenidone (P) exhibits anti-fibrotic effects by blocking fibroblast differentiation into CAFs and suppressing TGF beta production and associated signaling pathways/collagens. Atezolizumab (A) targets PD-L1, inhibiting its interaction with PD-1 and B7-1 (CD80) receptors on T cells. We initiated a phase I/II trial to test the combination of AP in recurrent NSCLC after CPI progression. The phase I study aims to determine the maximum tolerated dose (MTD) of AP and assess its safety and tolerability. The phase II study will evaluate its efficacy. The Exploratory Objectives are to measure circulating levels of TGF beta and investigate the expression of CAF-related proteins. Methods: phase I will enroll 3 patients using P at 801 mg PO TID and A at 1200mg IV every 3 weeks. If there is ≤ 1 Dose Limiting Toxicity (DLT), the study will proceed to phase II. If there are ≥2 DLTs, P will be reduced to 534 mg TID. If there is ≤ 1 DLT at this dose, we will proceed to phase II. If there are ≥2 DLTs, then the study will be terminated. Phase II will enroll 16 patients to assess efficacy. The main inclusion criteria are patients with recurrent NSCLC without actionable mutation, after progression with first-line therapy CPI with or without platinum-based doublet chemotherapy, no more than 2 lines of therapy, measurable disease, ECOG 0-2, and adequate organ function. Results: The study enrolled a total of 5 patients (4 males, 1 female), median age of 63.4; 5 were white; 3 patients did not complete the required 21 days of therapy for evaluation (1 non-compliance, 1 disease progression,1 unrelated Urinary Tract Infection); 2 patients were evaluable for toxicity, without DLT; 1 exhibited a partial response to cutaneous metastatic disease, with overall stable disease, 1 had disease progression. Adverse events (AE) in more than 5% of patients included blood disorders, cardiac, gastrointestinal, administration site, metabolic, musculoskeletal, psychiatric, respiratory, and skin disorders. Serious AEs were noted in 2 patients: one experienced gastrointestinal bowel perforation due to disease progression resulting in death, and the other had a respiratory disorder. Conclusions: The combination of AP did not produce unexpected side effects or reach the dose-limiting toxicity threshold. After the phase I portion is completed, the study will proceed to phase II expansion to enroll patients with recurrent NSCLC. Clinical trial information: NCT04467723 .

Regulatory T and B cells in pediatric Henoch–Schönlein purpura: friends or foes?

Background and objectivesHenoch–Schönlein purpura (HSP) is the most common immunoglobulin A-mediated systemic vasculitis in childhood. We studied immune dysregulation in HSP by analyzing regulatory T (Treg), T helper 3 (Th3), and regulatory B cell (Breg) subpopulations that might intervene in immune activation, IgA production, and HSP clinical manifestations.MethodsThis prospective study included 3 groups of children: 30 HSP on acute phase, 30 HSP on remission, and 40 healthy controls (HCs) matched on age. Treg, Breg, and Th3 were analyzed by flow cytometry. Serum immunoglobulin and cytokine levels were quantified by ELISA and Luminex.ResultsTreg frequencies were higher in acute HSP than in remitting HSP and HCs (6.53% [4.24; 9.21] vs. 4.33% [3.6; 5.66], p = 0.002, and vs. 4.45% [3.01; 6.6], p = 0.003, respectively). Activated Th3 cells (FoxP3 + Th3 cells) tend to be more abundant in HSP than in HCs (78.43% [50.62; 80.84] vs. 43.30% [40.20; 49.32], p = 0.135). Serum IgA, IL-17, and latency-associated peptide (a marker of the anti-inflammatory cytokine TGF-beta production) were significantly and inflammatory cytokines TNF-alpha, IL-1-beta, and IL-6 were non-significantly higher in HSP than HCs. Bregs were identical between the groups, but, in patients with renal impairment, Breg percentage was lower compared to those without. Treg removal in PBMC culture resulted in an increase in IgA production in HSP proving a negative regulatory role of Tregs on IgA production.ConclusionsIn pediatric HSP, immune activation persists in spite of an increase in Th3 and Tregs. Th3 could be involved in IgA hyperproduction, inefficiently downregulated by Tregs. Lack of Bregs appears linked to renal impairment.

P08 Successful treatment of paediatric morphoea with tofacitinib

Abstract Morphoea is a chronic autoimmune fibrosing condition causing inflammation, skin sclerosis and underlying soft tissue. The Janus kinase (JAK)-STAT pathway is considered to be the downstream mediator in the process of promoting fibrosis by transforming growth factor-β, interleukin-6 and other factors and plays a signal transduction role in the process of fibrosis. JAK inhibitors have been shown in preclinical studies to inhibit the fibrotic pathway in systemic sclerosis (Celada LJ, Kropski JA, Herazo-Maya JD et al. PD-1 up-regulation on CD4(+) T cells promotes pulmonary fibrosis through STAT3-mediated IL-17A and TGF-beta1 production. Sci Transl Med 2018; 10). Here, we report the successful use of tofacitinib in two paediatric patients. Case 1 was a 6-year-old girl with a 4-year history of morphoea; case 2 was a 13-year-old boy, also with a 4-year history of morphoea. In case 1, we initiated tofacitinib therapy 2.5 mg twice daily, reduced the dose after 2 months and stopped using it after 8 months. For case 2, the dose was 5 mg twice daily for 5 months, considering the patient’s height and weight. We have seen a significant improvement in the appearance and pathology of the lesions. The Localized Scleroderma Cutaneous Assessment Tool (LoSCAT) was used to assess the disease activity and damage, and the post-treatment scores for both cases were significantly lower than before treatment. Skin biopsy in both cases after tofacitinib therapy revealed that collagen bundles were decreased in size and number, with decreased lymphocytic infiltration in the peripheral tissue. Immunofluorescence staining for case 1 showed that activated α-smooth muscle actin and myofibroblasts were significantly decreased after treatment of tofacitinib, mostly in the dermal papillary layer. The two patients remain under follow-up; neither of them has so far relapsed. To our knowledge, these are the first two paediatric cases of morphoea treated with tofacitinib to be reported in the literature. Disease progression was found to have been inhibited clinically and pathologically. The traditional treatment of morphoea with methotrexate, cyclophosphamide or mycophenolate mofetil is not invariably effective in our experience, especially in antifibrosis (Zhao M, Wu J, Wu H et al. Clinical treatment options in scleroderma: recommendations and comprehensive review. Clin Rev Allergy Immunol 2022; 62:273–91). Our results provide the rationale for large prospective cohort studies of tofacitinib in morphoea and other fibrotic disorders.

Role of TGF-beta1 and TNF-alpha1 produced by neoplastic cells in the pathogenesis of fibrosis in patients with hematologic neoplasms.

We conducted this study with the objective of elucidating the mechanism of development of fibrosis in hematologic neoplasms and develop treatments for these patients. Among the suggested mechanisms of development of fibrosis is cases of hematologic neoplasms is the production of TGF-beta1 (transforming growth factor-beta-1) and TNF-alpha1 (tumor necrotizing factor-alpha-1) by the tumor cells, both of which are fibrosis-stimulating cytokines that act on fibroblasts to promote fibrosis. However, there are few reports based on human clinical pathology studies. We conducted an immunohistochemical study on paraffin-embedded formalin-fixed specimens obtained from 104 patients with various pathologic conditions (acute leukemia, malignant lymphoma, inflammation, cancer, etc.). The association of tissue fibrosis with positive immunohistochemistry for TGF- beta1 and/or TNF-alpha1, TGF-beta1 was found to be strongly associated with tissue fibrosis, and in cases with positive immunohistochemistry for TGF-beta1, the odds ratio for fibrosis was 12.8, which was significantly high. Combined positivity for TGF-beta1 and TNF-alpha1 was also associated with a significant odds ratio for fibrosis of 3.4, suggesting that TGF-beta1 expression is an important prerequisite. TGF-beta1 has been suggested as playing a relatively important role in tissue fibrosis. Future clinical application of these cytokines for both diagnosis and treatment is expected.

P1526: AUTOIMMUNE HEMOLYTIC ANEMIAS: RELATIONSHIP BETWEEN SINGLE NUCLEOTIDE POLYMORPHISMS OF CYTOKINE GENES AND CLINICAL/HEMATOLOGICAL PARAMETERS

Background: Autoimmune hemolytic anemia (AIHA) is highly heterogeneous and involves several pathogenic mechanisms including autoantibody production, complement activation, and cytokine dysregulation. Hb levels at baseline and the degree of bone marrow compensation are the main predictors of outcome. The contribution of high, intermediate, and low producer genetic profile cytokine genes may give further insights into the pathogenesis of the disease. Aims: To evaluate the frequency of cytokine single nucleotide polymorphisms (SNPs) in warm (wAIHA) and cold agglutinin disease (CAD) and their relationship with clinical and hematological parameters, including the degree of anemia, treatment refractoriness and number of therapies lines. Methods: We investigated SNPs of five cytokine genes [tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, transforming growth factor (TGF)-beta, interleukin (IL)-6 and IL-10] in 72 Italian warm AIHA patients, using the Cytokine Tray (One Lambda, Canoga Park, CA) according to the manufacturer’s recommendations. Allele and genotype frequency results were compared to those described in the Italian population by Uboldi de Capei et al. (2003) and Poli et al. (2002). Results: One hundred and twenty AIHA patients have been evaluated: median age 63 years (range 17-90), classified as wAIHA (n=75) and CAD (n=45). Demographic and hematological characteristics of patients are described in Figure 1A. Regarding cytokine SNP frequencies (Figure 1B), we observed that the TNF-alpha-308 high producers genotypes (G/G and G/A) were significantly more frequent in AIHA patients (37% vs 16%, p=0.0003 or 37% vs 23%, p=0.01) and in wAIHA (43% vs 16%, p<0.0001 or 43% vs 23%, p=0.0014) compared to controls. No differences were observed in CAD patients versus controls. Likewise, no significant differences between AIHA (including wAIHA and CAD subtypes) and controls were noted regarding other cytokine SNPs. Concerning the clinical severity at presentation, the frequency of TGF-beta (82% vs 58%, p=0.01) and IL-10 (17% vs 5%, p=0.03) high producers was higher in patients with Hb ≤7.5 g/dL (median of all AIHA) versus those with less severe presentation. Similarly, the frequency of high/intermediate IFN-gamma producers was higher within the group with Hb levels ≤7.5 g/dL at onset (75% vs 58%, p=0.03). No characteristic patterns were observed regarding IL-6 and TNF-alpha. Finally, the patterns of TNF-alpha and TGF-beta production were differently distributed considering the number of therapy lines: the first both in all AIHA and in CAD patients (p=0.004 and p=0.01, respectively), the second only in CAD patients (p=0.03). No differences in genotype frequencies of cytokine SNPs were observed regarding the number of relapses. Image:Summary/Conclusion: These preliminary data show that AIHA patients display a more pro-inflammatory cytokine signature at molecular level (TNF-alpha high producer genotype) as compared to the Italian control population. Moreover, cytokine SNPs were also associated with clinical severity at onset, and requirement of multiple treatments.

Metformin down‐regulates TGF beta signal transduction and production of PAR2 N‐terminus cleaving protease activity in an NR4a1 dependent manner in a PC3 prostate cancer cell line

OBJECTIVEOur aim is to understand the mechanisms of metformin’s antitumor actionsHYPOTHESISMetformin is known to have antitumour effects in cancers like head &amp; neck cancer, that may involve its regulation of TGF‐β signalling, but the metformin receptor mechanisms for this action are not known. We recently discovered that the action of metformin to preserve hypoglycaemia‐impaired endothelial cell function regulated by proteinase‐activated receptor‐2 (PAR2) requires its interaction with the orphan nuclear receptor, NR4A1 (PubMed ID 34452975). PAR2 also plays a role in regulating prostate cancer PC3 cell function whereby autocrine signaling occurs in PC3 cells which secrete a PAR2‐cleaving/activating matrix metalloproteinase, MMP2. We hypothesized that metformin can act in PC3 prostate cancer‐derived cells via NR4A1 to change TGF‐β signalling and to alter the production of PAR‐cleaving proteinases.METHODSThe ability of TGF‐β to upregulate connective tissue growth factor (CTGF) mRNA in PC3 cells was measured for metformin treated cells (&lt;/=100 μM) that were transfected with NR4A1, C‐terminally tagged with red‐fluorescent protein (RFP): either wild‐type (WT) or mutant (PPGG) murine NR4A1 that does not interact with metformin. TGF‐β upregulated CTGF mRNA in PC3 cells was monitored after metformin treatment for 20 hours. In similarly transfected WT and mutant PPGG NR4A1‐expressing PC3 cells, treated or not with metformin (&lt;/=100 μM), the cleavage status of dually tagged transfected N‐terminal RFP/C‐terminal GFP‐PAR2 was monitored: (confocal imaging: yellow = intact receptor; green = cleaved receptor). Concurrently, the abundance of MMP activity of PC3 cell supernatants was measured by a fluorogenic MMP substrate, Mca‐RPKPVE‐Nval‐WRK(Dnp)‐NH2 (ES002).RESULTSThe expected TGF‐β upregulated CTGF mRNA in PC3 cells was prevented by metformin treatment, but only for cells transfected with the WT and not the mutant PPGG NR4A1. Metformin acted at concentrations (&lt;/=100 μM) far lower than those that affect mitochondrial complex I (&gt; mM). We also found that in WT, but not PPGG mutant‐transfected PC3 cells, metformin (&lt;/=100 μM) down‐regulated MMP PAR2‐cleaving proteinase activity.CONCLUSIONSOur results show that low concentrations of metformin (&lt;/=100 μM), in the presence of wild‐type but not PPGG mutant NR4A1 which does not enable metformin interactions at the NR4A1 alternative ligand binding site, can both block TGF‐ β signaling and reduce cell‐secreted MMP activity that in turn can drive tissue invasion and metastasis via PAR2 activation. Thus, NR4A1 may play a role in the antitumour actions of metformin.

Combination of atezolizumab and pirfenidone in second-line and beyond NSCLC: A phase I/II study.

TPS2678 Background: Checkpoint inhibitors (CPI) targeting the PD1/PD-L1 axis significantly improved patient outcomes in stage IV non-small cell lung cancer (NSCLC). However, these patients will eventually develop resistance and progression. There is a need to identify novel treatment options. Poor response to PD-L1 antibody was correlated with increase in cancer-associated fibroblasts (CAF), which is known to interact with cytotoxic T cells (CTLs) by suppressing their function in a manner similar to regulatory T cells (Tregs). Production of cytokines by CAFs leads to impaired antitumor immunity by impairing CTL function (TGF beta) and prevent recruitment/mobilization of CTLs into tumors. These effects suggesting that CAF can be a therapeutic target in lung cancer resistant to checkpoint inhibitors. Pirfenidone (P) is approved to treat pulmonary fibrosis with anti-fibrotic effect by blocking the differentiation of fibroblasts into CAFs and suppress the production of TGF beta and TGF beta-induced signaling pathways/collagens. Atezolizumab is a humanized immunoglobulin (Ig) G1 monoclonal antibody that targets PD-L1 and inhibits the interaction between PD-L1 and its receptors, PD-1 and B7-1 (CD80), both of which function as inhibitory receptors expressed on T cells. We proposed a phase I/II trial to test the combination of atezolizumab (A) with P in patients with recurrent non-small cell lung cancer (NSCLC) after progression with CPI. The primary objective of phase I is to determine the maximum tolerated (MTD) dose of P in combination with A and assess the safety and tolerability of this combination. The secondary objective is to determine the efficacy of AP in all NSCLC participants treated in this study. Exploratory objectives include the measurement of circulating levels of TGF beta and research in expression of CAF related proteins. Methods: The initial phase I will enroll 3 patients using P at 801 mg po TID. A will be at 1200mg iv every 3 weeks. If there is ≤ 1 DLT, the study will proceed to phase II If there are 2- 3 DLT, P will be reduced to 534 mg TID. If there is ≤ 1 DLT, then this dose will proceed to phase II. If there is 2-3 DLT, then the study will be terminated. The phase II will enroll 16 patients to assess efficacy. Main inclusion criteria are patients with recurrent NSCLC after progression with first-line therapy CPI with or without chemotherapy, measurable disease, ECOG 0-2, and adequate organ function. Clinical trial information: NCT04467723.

Suppressors of signaling protein 3 inhibits glucocorticoid‐induced M2 polarization of monocytic cells by attenuation of ROS/p38 and GILZ‐mediated anti‐inflammatory cytokine production

Macrophages respond to diverse environmental cues to acquire different phenotypes, ranging from pro-inflammatory (M1) to anti-inflammatory (M2) types. Suppressors of signaling protein (SOCS) have emerged as critical regulators of inflammation participating in distinct phases of macrophage activation and differentiation. We have recently reported that both SOC1 and SOCS3 attenuated LPS/TLR4-induced inflammatory signaling via down-regulation of ROS levels and inflammasome activation associated with M1 differentiation. We have subsequently observed that SOCS3 also suppresses M2 polarization of THP1 monocytic cells induced by glucocorticoid (GC) and investigated the signaling mechanisms regulated by SOCS3.Treatment of PMA-differentiated THP1 cells with dexamethasone (Dex) induced M2 polarization by reducing TNF-alpha, IL-1 beta and IL-6 and by promoting IL-10 and TGF-beta production with the increased expression of Arg1 and CD163.SOCS3 over-expression reduced, while SOCS3 ablation increased IL-10 and TGF-beta in ROS and p38-dependent manners.A potential mediator of Dex-induced M2 response, glucocorticoid-induced leucine zipper (GILZ) was down-regulated by SOCS3 and up-regulated by shSOCS3. GILZ ablation blocked ROS generation and p38 activation leading to the inhibition of Dex-induced anti-inflammatory cytokines and the expression of M2 markers. Together the data suggest that SOCS3 target ROS/p38-dependent GILZ expression to suppress Dex-induced M2 polarization of THP1 cells. The critical role and detailed regulation mechanism of GILZ as a target of SOCS3 action during GC-induced M2 polarization is under investigation.

Abstract 15530: Single-cell Analysis in Aortic Aneurysmal Tissue From Patients With Marfan Syndrome Reveals Increased Tgf-beta Production but Downregulation of Downstream Canonical Tgf-beta Signaling Pathways

Background: Marfan syndrome (MFS) is caused by mutations in the gene for fibrillin-1 ( FBN1 ); however, the mechanisms by which these mutations cause aortic aneurysms are poorly understood. Although it was hypothesized previously that dysregulation of the complex TGF-β signaling pathway leads to aortic aneurysm formation, FBN1 mutations appear to have a paradoxical effect on TGF-β signaling in MFS. In this study, we evaluated cell-specific TGF-β expression in non-immune cells in MFS aortic tissue. Methods: We performed single-cell RNA sequencing of ascending aortic aneurysm tissues from MFS patients (n=3) undergoing aneurysm repair and age-matched, non-aneurysmal control tissue from cardiac transplant donors and recipients (n=4). Non-immune cells were separated out from the data and analyzed using the Seurat package in R. Differentially expressed genes were identified using edgeR. Results: Conserved gene expression was used to identify populations of smooth muscle cells (SMCs; n=6), fibroblasts (n=3), and endothelial cells (ECs; n=3). We found that TGFB1 was significantly upregulated in quiescent fibroblasts (identified by increased expression of DCN , LUM , and complement factors) with log2FC of 1.30 and FDR 8.25x10 -8 , as well as in activated fibroblasts (identified by increased expression of genes involved in blood vessel repair and healing including ACTA2 , NOTCH3 , THBS2 , and PDGFRB ) with log2FC of 1.25 and FDR 6.15x10 -22 . Despite this increase in TGFB1 , expression of TGF-β receptor genes (predominately TGFBR2 ) as well as downstream SMAD genes was downregulated significantly in the SMC, fibroblast, and endothelial cell clusters. Finally, genes involved in the non-canonical TGF-β pathway, including ERK , JNK, and p38, were not differentially expressed in non-immune cells in MFS compared with control tissues. Conclusion: Increased expression of TGFB1 in non-immune cells in MFS was driven by two clusters of fibroblasts. Despite this, our data do not support associated upregulation of other genes in the canonical or non-canonical TGF-β pathways and in fact support downregulation of canonical TGF-β signaling in non-immune cells of aneurysmal tissues from MFS patients with advanced aortic disease.

A new Natural Killer cell therapy to target relapse Acute Lymphoblastic Leukemia

Abstract Acute Lymphoblastic Leukemia (ALL) is the most common childhood cancer. Drug resistance and relapse are two major problems in ALL. Pre-B ALL cells express B-cell activating factor receptor (BAFF-R) on their surface, which is not present on normal pre-B cells. This makes it an attractive therapeutic target for ALL. We used a BAFF-R antibody optimized for antibody dependent cell cytotoxicity (ADCC) to enhance NK cell mediated killing of drug resistant and relapsed ALL cells, which are difficult to treat by conventional chemotherapy. We found that anti-BAFF-R antibody enhanced NK cell mediated killing of drug resistant ALL cells in vitro. Early treatment with anti-BAFF-R antibody and NK cells significantly reduced disease burden in xenograft ALL models and increased overall mice survival. However, in advanced disease, the efficacy of NK cells to mediate ADCC is reduced. ALL cells are known to produce TGF beta, a cytokine that causes NK cell dysfunction. We found that ALL cells produce TGF-β and co-culturing ALL cells with NK cells lead to a decrease in CD16 expression on NK cells, which is reversible by addition of EW-7197, a potent TGF-beta receptor I inhibitor. We found that EW-7197 treatment improved anti-BAFF-R antibody mediated ADCC of drug resistant ALL cells, in vivo, even if treatment is started late. Our data validate this novel BAFF-R antibody as a very promising tool for pre-B ALL therapy.

Mycobacterium tuberculosis Heat-Shock Protein 16.3 Induces Macrophage M2 Polarization Through CCRL2/CX3CR1

Mycobacterium tuberculosis, the pathogen of tuberculosis (TB), can survive in host macrophages and induce macrophages to M2 phenotype might result in latent MTB infection. During the latent phase, the expression of MTB heat-shock protein 16.3 (Hsp16.3) is markedly increased among most of bacterial proteins, but the role of Hsp16.3 in macrophage M2 polarization is not clear. In this work, we found that macrophages incubated with 100 ng/ml MTB Hsp16.3 increased the production of Arg-1, IL-10, TGF-beta, and CD206. These results showed that MTB Hsp16.3 may induce macrophage M2 phenotype. And the interaction of Hsp16.3 with macrophages was found to depend on chemokine receptors CCRL2 and CX3CR1. Additionally, we used overexpression and silencing techniques to further verify the effect of CCRL2 and CX3CR1 on MTB Hsp16.3-induced M2 polarization macrophages. Furthermore, we explored the downstream signaling molecules of CCRL2 and CX3CR1 and we found MTB Hsp16.3 altered the signal transduction of the AKT/ERK/p38-MAPK. Taken together, this study provides evidence that MTB Hsp16.3 promotes macrophages to M2 phenotype and explores its underlying mechanism.

Abstract 3186: A novel combination therapy for treating advanced drug resistant acute lymphoblastic leukemia

Abstract Purpose: Drug resistance and relapse are two major problems in Acute Lymphoblastic Leukemia (ALL) as these patients do not respond to chemotherapy. We observed BAFF-R expression on drug resistant and relapse B-ALL patient cells. We wanted to explore the in vivo therapeutic efficacy of a BAFF-R antibody, optimized for antibody dependent cellular cytotoxicity (ADCC), against drug resistant ALL cells. Experimental design: ADCC assay was performed using primary human natural killer (NK) cells isolated from normal donors or ALL patients. NK cell mediated cytotoxicity was measured using Calcein-AM assay. Immunocompromised NSG mice were used for in vivo xenograft model of ALL. Results: We found that anti-BAFF-R antibody enhanced NK cell mediated killing of drug resistant ALL cells in vitro. Early treatment with anti-BAFF-R antibody and NK cells significantly reduced disease burden in xenograft ALL models and increased overall mice survival. However, in advanced disease, the efficacy of NK cells to mediate ADCC is reduced. ALL cells are known to produce TGF beta, that causes NK cell dysfunction. We found that ALL cells produce TGF-β and co-culturing ALL cells with NK cells lead to a decrease in CD16 expression on NK cells, which is reversible by addition of EW-7197, a potent TGF-beta receptor I inhibitor. We found that EW-7197 treatment improved anti-BAFF-R antibody mediated ADCC of drug resistant ALL cells, in vivo, even if treatment is started late. Conclusion: Combination treatment using BAFF-R antibody, NK cells and EW-7197 is a potential strategy to treat advanced drug resistant ALL patients with BAFF-R expressing blasts. Citation Format: Yorleny M. Vicioso, Rose Beck, Herman Gram, Abhishek Asthana, keman Zhang, Derek Wong, John Letterio, Reshmi Parameswaran. A novel combination therapy for treating advanced drug resistant acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3186.

The subpopulation of CD105 negative mesenchymal stem cells show strong immunomodulation capacity compared to CD105 positive mesenchymal stem cells

Introduction: Human mesenchymal stem cells (MSCs) are the most popular stem cells applied in disease treatment. MSCs can be isolated and in vitro expanded from various sources such as bone marrow, peripheral blood, umbilical cord blood, umbilical cord tissue, and adipose tissue. According to Dominici et al. (2006), MSCs should express CD105, an essential marker used to confirm MSCs. However, some recent studies have show that MSCs contained a subpopulation that is negative for CD105. This study aimed to compare the immune modulation capacity of 2 populations of CD105 positive (CD105+) and negative (CD105-) MSCs derived from 2 sources: human adipose tissue (AT) and human umbilical cord (UC).&#x0D; Methods: MSCs were isolated from human adipose tissues (adipose tissue-derived mesenchymal stem cells – AT-MSCs) and human umbilical cord (umbilical cord-derived mesenchymal stem cells – UC-MSCs) according to previously published protocols. The two populations of CD105- and CD105+ MSCs were sorted based on the expression of CD105 from AT-MSCs and UC-MSCs. Four populations of CD105 (AT-MSCs, CD105+ AT-MSCs, CD105- UC-MSCs, and CD105+ UC-MSCs) were used to compare the phenotype as well as in vitro differentiation potential; then they were used to evaluate the immune modulation capacity by allogeneic T cell suppression and cytokine release.&#x0D; Results: The results showed that CD105- MSCs from AT and UC exhibited an immune modulation capacity that was much stronger than CD105+ MSCs from the same source of AT and UC. The strong immunomodulation of CD105- MSCs may relate to autocrine production of TGF-beta 1 by MSCs.&#x0D; Conclusion: The results suggested that CD105- MSCs are promising MSCs for application in regenerative medicine, especially for the treatment of diseases related to inflammation.&#x0D;

BMP6 Increases TGF-Beta1 Production by Up-Regulating Furin Expression in Human Granulosa-Lutein Cells

Background: Bone morphogenetic protein 6 (BMP6) and transforming growth factor--β1 (TGF--β1) are key intraovarian regulators that play essential roles in regulating mammalian follicular function and promoting oocyte maturation. Dysregulated BMP6 or TGF-β1 signaling is associated with polycystic ovary syndrome. Furin is a proprotein convertase that promotes the activation of diverse functional proteins by cleaving protein precursors. Methods: Primary and immortalized (SVOG) human granulosa-lutein (hGL) cells were used as study models. RT-qPCR, western blot analyses, and enzyme immunoassay were used to examine the effects of recombinant human BMP6 on the expression of furin and production of TGF-β1. Dual inhibition approaches (kinase receptor inhibitors and small interfering RNAs targeted knockdown) were used to investigate the underlying molecular mechanisms by which BMP6 regulated the expression of furin. Findings: BMP6 significantly up-regulated the expression of furin and increased the production of TGF-β1. Additionally, both activin receptor-like (ALK)2 and ALK3 were involved in BMP6-induced up-regulation of furin. Furthermore, knockdown of furin abolished BMP6-induced increases in TGF--β1 production, and knockdown of endogenous SMAD4 reversed the increase in furin expression induced by BMP6. Interpretation: The ALK2/3-mediated canonical SMAD signaling pathway is required for the stimulatory effect of BMP6 on furin expression, which in turn increases the production of TGF-β1 in hGL cells. Our findings shed insights into the molecular interactions and mechanisms of two intrafollicular growth factors in hGL cells, which could be potentially applied as therapeutic targets for patients with polycystic ovary syndrome. Funding Statement: Canadian Institutes of Health Research Foundation Scheme Grant #143317. Declaration of Interests: The authors have no conflict of interests to declare. Ethics Approval Statement: All participants signed a written informed consent. The study was approved by Research Ethics Board from the University of British Columbia.

Decreased cellular and humoral immunity in patients with breast cancer.

4 Background: A growing body of evidence over the past few years suggests that the presence of immune elements within the tumor or in the tumor stroma has prognostic and predictive value in breast cancer. Immunotherapy is successfully used in many types of cancer, and modulation of immune system became standard part of the cancer patients treatment Responses in breast cancer have been more recently reported. However it has yet to be determined whether predictable biomarkers of response can be identified. Therefore in recent years, research focused on a precise description of the status and function of the immune system.The purpose of the study was to monitor immune responses in patients with breast cancer, particularly the examination of cellular (CD4, CD8, B cells) as well as humoral immunity (IgG, IgG1, IgG2, IgG3, IgG4. It appears that a factor contributing to the immunosupression may be a transforming factor-beta (TGF-beta).It is highly immunosuppressive factor that inhibits the natural and specific immunity against tumors. Methods: 50 patients included in the research project were implemented routine cancer treatment. Basic parameters (histological type and grade, the degree of expression of ER and PR, HER2, and the proliferative marker) were established. Patients were evaluated by a cancer clinical immunologist to exclude immune disorders, allergic or autoimmune origin. Anti-tumor cellular immunity (CD4, CD8, CD19) was measured by flow citometry, humoral immunity (IgG, IgG1, IgG2, IgG3, IgG4) was measured and TGF beta and VEGF production was monitored by ELISA. Results: In breast cancer patients mainly depression in cellular immunity was found. Immunglobuline plasma level was decreased as well (mainly IgG4 subtype). TGF beta as well as VEGF plasma level were increased. Most patients have shown clinical symptoms of immunodeficiency (frequent infections of respiratory or urinary tract, herpetic infections).Those patient could benefit from immunomodulation. Conclusions: The state of anticancer immunity could contribute to the selection of targeted immune therapy in breast cancer patients and to help to find optimal combination of immunotherapy. This project was supported by governmental grant AZV CR 15-28188A.

Sitagliptin inhibit human lymphocytes proliferation and Th1/Th17 differentiation in vitro

Dipeptidyl peptidase-4 (DPP-4) inhibitors are a new class of anti-diabetic agents that are widely used in clinical practice to improve glycemic control in patients with type 2 diabetes. DPP-4 is also known as lymphocyte cell surface protein, CD26, and plays an important role in T-cell immunity. Recent studies suggest that DPP-4 inhibitors improve beta-cell function and attenuate autoimmunity in type 1 diabetic mouse models. To investigate the direct effect of DPP4 in immune response, human peripheral blood mononuclear cells (PBMC) from healthy volunteers were obtained by Ficoll gradient and cultivated in the absence (control) or presence of phytohemagglutinin (PHA), or stimulated with PHA and treated with sitagliptin. The immune modulation mechanisms analyzed were: cell proliferation, by MTT assay; cytokine quantification by ELISA or cytometric bead array (CBA), Th1/Th2/Th17 phenotyping by flow cytometric analysis and CD26 gene expression by real time PCR. The results showed that sitagliptin treatment inhibited the proliferation of PBMC-PHA stimulated cells in a dose dependent manner and decreased CD26 expression by these cells, suggesting that sitagliptin may interfere in CD26 expression, dimerization and cell signaling. Sitagliptin treatment not only inhibited IL-10 (p<0.05) and IFN-gamma (p=0.07) cytokines, but also completely abolish IL-6 expression by PBMCs (p<0.001). On the other hand, IL-4 were secreted in culture supernatants from sitagliptin treated cells. A statistically significant increase (p<0.05) in the ratio of TGF-beta/proliferation index after sitagliptin treatment (2627.97±1351.65), when comparing to untreated cells (646.28±376.94), was also demonstrated, indicating higher TGF-beta1 production by viable cells in cultures. Sitagliptin treatment induced a significantly (p<0.05) decrease in IL-17 and IFN-gamma intracellular expression compared with PHA alone. Also, the percentage of T CD4+IL-17+, T CD4+IFNgamma+ and T CD4+IL-4+ cells were significantly reduced (p<0.05) by sitagliptin. Our data demonstrated an immunosuppressive effect of sitagliptin on Th1, Th17 and Th2 lymphocytes differentiation that leads to the generation of regulatory TGF-beta1 secreting cells with low CD26 gene expression that may influence the state of pancreatic beta-cells and controlling DM1 patients.

Inhibition of Wound TGF Beta-1 by Celecoxib: A Possible Therapeutic Route for Scar Free Wound

Background: Wound healing is a highly ordered dynamic process associated with inflammation at early stages and with permanent scarring at late stages. Scars could be disfiguring and could advance to be hypertrophic or keloid scars, this would have a strong physical and psychological impact on the patients afterward. The role of inflammatory mediators which could be pro- or anti-inflammatory, pro-or antifibrotic was the focus of wound healing research for decades and the balance between them is the key factor determining the outcome of healing.Aims: In this study, we investigate the correlation and the interrelation between the pro-inflammatory Cyclooxygenase-2 (COX-2) and the pro fibrotic (TGF-Beta-1) in an in vivo model of incisional dermal wound healing and the effect of a selective COX-2 inhibiton the progression of repair and scar formation.Materials and methods: Adult male Sprague-Dawley rats received four full thickness dermal wounds. a selective COX-2 inhibitor was applied to the wounds immediately postwounding twice daily for two days. Wounds and scars were then harvested and at different time points and processed for COX-2 and TGF Beta-1 immunostaining and for collagen staining. Immunoreactivity was semi quantified using Image J.Results: We have shown upregulation of COX-2, co-upregulation and colocalization of TGF-Beta-1 and COX-2 two days postwounding during the inflammatory phase. Celecoxib application significantly inhibited both COX- 2 (P<0.01) and TGF Beta-1 (P<0.001). It improved wound healing microscopically and macroscopically, through reducing inflammatory cell infiltrate, granulation tissues formation and early closure of the incision. Additionally, there was marked improvement in the postwounding scarring. There was a significantly (P<0.01) correlation between COX-2 and TGF Beta-1 (Pearson Correlation=0.94).Conclusion: The overall effect of COX-2 inhibitor was shortening of the inflammatory phase of wound healing with subsequent minimization of the associated tissue destruction and consequently improvement of the scar quality. COX-2 inhibitors regulate inflammatory phase of the wound. They could regulate collagen deposition byregulating the produc-tion of the pro fibrotic TGF Beta-1 production, through autocrine/paracrine effect. Therefore, early application of COX-2 inhibitors to wounds immediately after injury/surgery could enhance the repair and improve the quality of scar.

Identification of Bone Marrow-Derived and Circulating Fibrocytes in Gata1low Mice with Myelofibrosis

Introduction: It has recently been shown that the population of CD45+ Col1+ CXCR4+ (characteristic fibrocyte markers) cells increases in the bone marrow after bleomycin-induced lung injury. But little is known about their existence in primary or secondary myelofibrosis. Questions also remain about the exact origin of fibrocytes and how their development/differentiation is altered in response to tissue damage. Fibrocytes are believed to be a bone marrow-derived cell type, differentiated from monocytes, with essential roles in both normal tissue repair and multiple fibrotic diseases. Fibrocytes are unique in that they express both stromal and hematopoietic markers. Fibrocytes have been isolated and cultured from murine and human blood PMBCs, however, this method yields a low number of cells per animal. The murine splenic monocyte reservoir has also been shown to be able to give rise to fibrocyte cultures. Methods: Long bones (humeri and femurs) were obtained from adult C57BL/6J mice ± 14 Gy and Gata-1low C57Bl/6J mice bred with CD1 mice. Bones were aseptically flushed with sterile PBS. Collected bone marrow was passed through a 22 gauge needle to further dissociate the cells. Cells were centrifuged at 300 x g for 10 min at room temperature. Cells were washed and red blood cells were lysed. After a wash cells were resuspended in supplemented Fibrolife media (10 mM, 2 x non-essential amino acids, 2 mM sodium pyruvate, 4 mM glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, 2× ITS-3, and 50 μM 2-mercaptoethanol) and plated at 1.25x106 cells/mL. Cells differentiated for 7-10 days before analysis. Immunocytochemical staining and qPCR analysis was performed using standard methods. Results: Using this new method to isolate fibrocytes from total murine bone marrow, we found that Fibrocytes differentiated as early as 5 days in serum-free media. In addition to exhibiting the characteristic spindle shape morphology, cultured fibrocytes also expressed both hematopoietic (CD45) and stromal markers (collagen I). Importantly, these cells were negative for the fibroblast specific marker fibronectin, confirming that these cells were not fibroblasts. Cultured fibrocytes also appeared to be terminally differentiated, as they could not be passaged. Bone marrow-derived fibrocytes were found to upregulate collagen I in response to TGFβ treatment, mirroring the response reported in blood-derived fibrocytes in lung fibrosis. We further identified fibrocytes from the peripheral blood of heterozygous Gata-1low mice, which displayed myelofibrosis. Irradiation of shielded mice with 14 Gy resulted in myelofibrosis by 60 days (as opposed to ~12 months in Gata-1low mice). Furthermore, there was associated increased TGFbeta production. This correlated with a marked increase in fibrocytes. Conclusions: Our new culturing method will further facilitate the study of fibrocyte differentiation and response to both pro- and anti-inflammatory signals. We are currently utilizing flow cytometry to characterize fibrocyte kinetics and to quantify these cells in mice and patients with myelofibrosis. Our data also suggest that fibrocytes may play a contributing role to myelofibrosis in part through TGFb pathways. We have also developed an radiation-induced model of myelofibrosis with characteristic TGFbeta and circulating fibrocytes. Because myelofibrosis occurs in a much shorter time period, this model may be useful to more rapidly evaluate novel anti-fibrotic therapies.This work was supported by a Translational Award from the Leukemia and Lymphoma Society. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of PLGA containing anti-CTLA4 inhibited endometriosis progression by regulating CD4 + CD25 + Treg cells in peritoneal fluid of mouse endometriosis model

Our study investigated poly(lactic-co-glycolic acid) (PLGA) as protein delivery vehicles encapsulate CTLA-4-antibody (anti-CTLA-4) which is essential for CD4+CD25+Treg cells suppressive function exposing superior potential for inhibiting endometriosis progress in mouse model than single anti-CTLA-4. Anti-CTLA-4 loaded PLGA combined to ligands CTLA-4 in surface of CD4+CD25+Treg cells which distributed in peritoneal fluid of mouse endometriosis model. The particle size, zeta potential of the anti-CTLA-4 loaded nanoparticles was detected by dynamic light scattering. Morphology of nanoparticles was evaluated by transmission electron microscopy (TEM). Confocal laser scanning microscopy (CLSM) indicated distribution of anti-CTLA-4 with PLGA or without in peritoneal fluid. Cumulative anti-CTLA-4 release from nanoparticles was evaluated by Micro BCA assay. The percentage of CD4+CD25+Treg cells in peritoneal fluid was demonstrated by flow cytometer. In vitro experiment we co-culture ectopic endometrial cells (EEC) with isolated CD4+CD25+Treg cells in peritoneal fluid (PF), proliferation and invasion of ectopic endometrial cells (EEC) was measured by BrdU ELISA assay and Matrigel invasion assay. In comparison with anti-CTLA-4 without nanoparticles, the bioconjugates PLGA/anti-CTLA-4 were tolerated in peritoneal fluid with a controlled release of anti-CTLA-4 in 3, 7, 14days. Moreover, PLGA/anti-CTLA-4 had superior protective regulation ability to reduce level of CD4+CD25+Treg cells in peritoneal fluid. Most strikingly, in vitro experiment, PLGA/anti-CTLA-4 exhibited better ability in inhibiting proliferation and invasion of ectopic endometrial cells in co-culture system compared with anti-CTLA-4. Progressively, PLGA/anti-CTLA-4 had better suppressive activity to inhibited IL-10 and TGF-beta secreted by CD4+CD25+Treg cells which indicating that PLGA/anti-CTLA-4 suppressed cells proliferation and invasion through reduced IL-10 and TGF-beta production. Thus, PLGA/anti-CTLA-4 may be a potential strategy for endometriosis therapy.

637. Targeting of Myeloid Leukemia by IL-10-Engineered Human CD4+ Tr1 Cells

T regulatory type 1 (Tr1) cells, characterized by the co-expression of CD49b and LAG-3 and the ability to secrete high amounts of IL-10, control immune responses by IL-10 and TGF-beta production and by killing of myeloid cells via a Granzyme B-dependent mechanism. Tr1 cells are induced in vitro in the presence of recombinant human IL-10 or tolerogenic dendritic cells secreting high amounts of IL-10 (DC-10). Proof-of-principle clinical trials suggest that Tr1 cells can modulate Graft-versus Host Disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, their ability to mediate anti-leukemic activity or their effects of Graft versus Leukemia is largely unknown. We previously showed that enforced IL-10 expression converts human CD4+ T cells into Tr1-like (CD4IL-10) cells that suppress effector T cells in vitro and prevent xenogeneic-GvHD in humanized models. We now demonstrate that these CD4IL-10 cells selectively kill myeloid cell lines and myeloid blasts in vitro in HLA-class I-dependent but antigen-independent manner. Moreover, cytotoxic activity of CD4IL-10 cells is Granzyme B-dependent, is specific for CD13+ cells, and requires CD54 and CD112 expression on target cell lines or primary leukemic blast. Adoptive transfer of CD4IL-10 cells in humanized models mediates direct anti-leukemic activity, and does not compromise the anti-leukemic effect of allogeneic T cells while inhibits xeno-GvHD. These findings provide a strong rationale for designing personalized immunotherapy approaches using CD4IL-10 cells after allo-HSCT to cure myeloid malignancies.