TGF-β Neutralizing Research Articles

Abstract 321: MUC1 regulates TGFβ function in pancreatic cancer

Abstract In 2016, Pancreatic Cancer (PC) has moved to 3rd leading cause of cancer-related deaths in the USA with 94% dying within 5 years of diagnosis. 90% of PC is Pancreatic Ductal Adenocarcinoma (PDA), of which 80% of PDA overexpress tumor associated Mucin-1 (tMUC1), a membrane bound glycoprotein that is hypoglycosylated. Overexpression of tMUC1 is associated with metastasis and poor prognosis. However the mechanism remains obscure. Transforming growth factor-β (TGF-β) plays a role in poor prognosis. TGF-β is a cytokine with dual functionality. Within normal cells and early carcinogenesis, TGF-β functions as a tumor suppressor and induces apoptosis. This effect is mediated by activation of the canonical Smad pathway via engagement of TGF-β Receptor 1 (TGF-βRI). However during later stages of cancer, TGF-β becomes a tumor promoter and stimulates epithelial to mesenchymal transition, migration, and invasion thus enhancing metastasis. This TGF-β effect is mediated by the noncanonical Erk1/2 pathway. We have recently shown correlation between tMUC1 and TGF-β within an exogenous tMUC1 model of PDA. Therefore, it is hypothesized that the mechanism between the two newly connected pathways exists in PDA tumors with high endogenous tMUC1 levels. We propose that the tyrosine kinases present in the cytoplasmic tail of tMUC1 are intermediary between the two pathways, thus leading to enhanced metastasis. First the secretion levels of TGF-β1 were assessed in a panel of 13 PDA cell lines with variable expression levels of tMUC1 by ELISA. The effects of TGF-β1 to induce apoptosis versus invasiveness on a variety of tMUC1 high and tMUC1 low PDA cell lines were determined. The effects of tMUC1 knockdown were evaluated in tMUC1 high cell lines via siRNA. We studied the role of tyrosine kinases and other protein complexes in mediating the interactions between tMUC1 and TGF-β via proteomics analysis. Finally, we evaluated the anti-tumor efficacy of neutralizing TGF-β1 in vivo in high and low tMUC1-expressing PDA tumors. In tMUC1 high PDA cells, TGF-β1 is secreted less when compared to tMUC1 low PDA cells, allowing for the more resistant cells to diminish TGF-β mediated effects endogenously. It is shown that with TGF-β1 treatment, tMUC1 high cells are more invasive, resist apoptosis, and activate Erk pathway, while tMUC1 low cells activate SMAD pathway. However, tMUC1 knockdown via siRNA does not affect TGF-β receptor levels. When studying the tyrosine kinases, c-Src becomes more phosphorylated in the presence of TGF-β1 in tMUC1 high cells, while in tMUC1 low cells c-Src becomes more deactivated in the presence of TGF-β1. Finally, within the mouse model, tMUC1-high PDA tumors respond to the neutralization of TGF-β1 by decreasing tumor size, while having no statistical effect on tMUC1-low PDA tumors. tMUC1 expression is influential in TGF-β function in an endogenous model. Neutralizing TGF-β in tMUC1 high expressing tumors can be considered as a possible treatment. This has high clinical significance for patients with PDA. Citation Format: Priyanka Grover, Monica D. Nye, Mahboubeh Yazdanifar, Mohammad Ahmad, Ru Zhou, Lopamudra Das Roy, Kajal Grover, Shu-ta Wu, Sritama Nath, Pinku Mukherjee. MUC1 regulates TGFβ function in pancreatic cancer [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 321. doi:10.1158/1538-7445.AM2017-321

The Effect of TGF-β on Treg Cells in Adverse Pregnancy Outcome upon Toxoplasma gondii Infection.

Toxoplasma gondii (T. gondii) is a ubiquitous intracellular protozoan parasite that causes adverse pregnancy outcomes. Its infection downregulates the Treg cell population and TGF-β level, which may contribute to adverse pregnancy outcomes. TGF-β plays a critical role in Treg cell development, but whether TGF-β treatment can affect the number and function of Treg cells and hence alleviate adverse pregnancy outcomes caused by T. gondii infection remains elusive. In this study, T. gondii-infected pregnant mice were treated with TGF-β or TGF-β-neutralizing antibody. The pregnancy outcomes were observed on gestational day 14. The numbers of Treg cells and pSmad3, programmed death 1 (PD-1), and Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) expression of Treg cells were analyzed by flow cytometry. Histological changes were assessed using HE staining, while IL-10 and TNF-α levels were measured using ELISA. The results indicated that TGF-β treatment improved the T. gondii-induced adverse pregnancy outcomes, with alleviation of hemorrhage, restoration of uterine spiral arteries of the placenta, and increased Treg cell numbers; meanwhile, TGF-β neutralization resulted in more serious adverse pregnancy outcomes, with serious hemorrhage, more dilated uterine spiral arteries, and decreased Treg cell numbers. pSmad3 expression in CD4+ cells and CTLA-4 and PD-1 levels on Treg cells were upregulated by TGF-β treatment, but downregulated by TGF-β neutralization. The ratio of IL-10/TNF-α also increased after TGF-β treatment, but decreased after TGF-β neutralization. Our data indicate that TGF-β treatment could improve adverse pregnancy outcomes caused by T. gondii infection by upregulating Treg cell differentiation and function via the TGF-β/Smad3 signaling pathway, but not the proliferation of Treg cells.

The Pathogenic Transforming Growth Factor-β Overdrive Hypothesis in Aortic Aneurysms and Dissections: A Mirage?

For >10 years ago, an unexpected role for the transforming growth factor-β cytokine pathway has been put forward in driving thoracic aortic aneurysms and dissections. Here, we reassess the evidence for a detrimental transforming growth factor-β overdrive in thoracic aortic aneurysms and dissections. In our view, most of the available mechanistic data argue against this theory. Syndromic thoracic aortic aneurysms and dissections (TAADs) develop in patients with connective tissue disorders because of genetic mutations that affect structural components of the extracellular matrix and the cell contractile machinery. Early pathogenic hypotheses attributed the aortopathy to structural failure of the aortic tissue. Over 14 years ago, Neptune et al,1 Habashi et al,2 and Lindsay and Dietz3 proposed a novel hypothesis to explain how fibrillin-1 ( FBN1 ) mutations in Marfan syndrome (MFS) lead to pulmonary emphysema and aortic aneurysm and pointed to increased transforming growth factor-β (TGFβ) activation as the culprit mechanism. This constituted a major paradigm shift, and a new hope emerged that the life-threatening manifestations of MFS might be prevented by a simple medical treatment, losartan, shown to prevent the disease in mice through its TGFβ-antagonizing properties.2 In 2010, we serendipitously discovered that TGFβ neutralization in mice treated with AngII (angiotensin II) unexpectedly induced fatal aortic dissections.4 Despite differences in the mouse models, the critical vasculoprotective role of TGFβ in our experiments highly contrasted with the reported pathogenic role of TGFβ in MFS and Loeys–Dietz syndrome (LDS), leading us to question the validity of the previous assumptions. Moreover, recent clinical testing of the concept in MFS patients failed to show any benefit of losartan over placebo or β-blockade.5 Thus, the time has come for a reassessment of the scientific evidence that supports a causal role for increased TGFβ signaling in TAADs. ### Marfan Syndrome The paradigm stipulates that FBN1 …

Low proportion of follicular regulatory T cell in renal transplant patients with chronic antibody-mediated rejection

Follicular regulatory T (Tfr) cell can effectively regulate humoral immunity, but its function and mechanism in antibody-mediated rejection (AMR) after organ transplantation remains unclear. Here we detected follicular helper T (Tfh) cell subsets in 88 renal transplant patients with chronic renal allograft dysfunction (40 with AMR and 48 without AMR). The ratio of Tfr cells in renal graft tissues and peripheral blood of AMR patients significantly decreased, while the ratio of IL-21-producing Tfh cells (Tfh2 and Tfh17) significantly increased, compared to non-AMR patients. When tested in functional assays, Tfr cells from both AMR and non-AMR patients exerted equivalent inhibitory function. Tfr cell transplantation or CTLA-4 virus transfection could significantly inhibit IL-21 secretion from Tfh cells of these patients, further suppress the proliferation and differentiation of B cells. CTLA-4 blocking, IL-10 and TGF-β neutralization could partially weaken such inhibitory effect of Tfr cells. Besides, our study found that sirolimus reduced the ratio of Tfr cells, while cyclosporine and tacrolimus had no significant effect on Tfr cells. In a word, renal transplant patients with AMR have low proportion of Tfr cells but these cell exerted normal function.

TGFβ superfamily members regulate radiation-induced anti-tumor immunity.

Abstract Transforming Growth Factor-beta (TGFβ) and activin A (actA) are TGFβ superfamily members with overlapping functions in many processes including regulation of inflammation and immunity. We have recently found that actA release by breast cancer cells is enhanced by radiotherapy (RT). Interestingly, prolonged exposure to TGFβ inhibitors also resulted in actA upregulation, consistent with a previously described compensatory mechanism. Here we hypothesized that actA and TGFβ regulate RT-induced anti-tumor immunity. 4T1 mouse breast cancer cell derivatives with inducible actA knockdown (KD) (4T1shActA) or non-silencing control (4T1shNS) were injected s.c. in mice (day 0). ActA KD was induced by doxycycline at day 8. TGFβ neutralizing antibody (1D11) was given i.p. every other day starting on day 12. 6Gy RT was delivered to the tumor on days 13, 14, 15, 16 and 17. TGFβ blockade improved RT-mediated tumor control, an effect mediated by T cells. However, tumor recurred. Notably, ActA KD or 1D11 increased intratumoral Tregs (Control: 11%; 1D11: 26%, shActA: 21%) and enhanced Tregs infiltration induced by RT (RT: 15%; RT+1D11: 27%; RT+shActA: 30%). When both TGFβ and actA were blocked, Tregs significantly decreased in both untreated (1D11+shActA: 13%) and RT-treated tumors (RT+1D11+shActA: 8%). Tumor-specific IFNγ production by CD8+ T cells was higher in RT+1D11+shActA compared to RT+1D11 (*p) and RT+shActA (**p). ActA KD in mice treated with RT+1D11 reduced tumor recurrence and improved survival (RT+1D11 vs RT+1D11+shActA **p; RT+shActA vs RT+1D11+shActA ***p). Data indicate that both TGFβ and actA impair RT-induced anti-tumor immune responses. Concomitant inhibition of actA and TGFβ is required for optimal in situ vaccination by RT.

Oroxylin A inhibits the generation of Tregs in non-small cell lung cancer.

Oroxylin A (OA), a naturally occurring monoflavonoid isolated from Scutellariae radix, has previously been reported to inhibit the proliferation of several cancer cell lines. CD4+CD25+Foxp3+ regulatory T cells (Tregs) play an important role in maintenance of immunologic self-tolerance. Tregs also increase in cancer and take part in suppressing antitumor immune responses. Here, we explored how OA affected the Tregs in lung cancer environment and the involved underlying mechanism. It is found that OA reversed the generation of Tregs induced by H460 lung cancer cells co-culture. Furthermore, in vivo, OA reduced tumor formation rate and attenuated Foxp3 expression in tumor-infiltrating lymphocytes. We also found that transforming growth factor-β1 (TGF-β1) neutralizing antibody reversed the enhancement of Treg number and expression of p-Smad3ˎ p-p38ˎp-JNKˎp-ERK1/2 in the co-culture model. Moreover, OA reduced the secretion of TGF-β1 and down-regulated the activation of NF-κB signaling in H460 cells. OA also inhibited Treg activity by a direct inhibition of the T cells' response to TGF-β1. In conclusion, our study demonstrated that OA inhibits the generation of Tregs in lung cancer environment by inhibiting the T cells' response to TGF-β1 and decreasing the secretion of TGF-β1 in lung cancer cells via NF-κB signaling.

Binding and biologic characterization of recombinant human serum albumin-eTGFBR2 fusion protein expressed in CHO cells

ABSTRACT Transforming growth factor-β1 (TGF-β1) signaling is involved in cell metabolism, growth, differentiation, carcinoma invasion and fibrosis development, which suggests TGF-β1 can be treated as a therapeutic target extensively. Because TGF-β1 receptor type α(TGFBR2) is the directed and essential mediator for TGF-β1 signals, the extracellular domain of TGFBR2 (eTGFBR2), binding partner for TGF-β1, has been produced in a series of expression systems to inhibit TGF-β1 signaling. However, eTGFBR2 is unstable with a short half-life predominantly because of enzymatic degradation and kidney clearance. In this study, a fusion protein consisting of human eTGFBR2 fused at the C-terminal of human serum albumin (HSA) was stably and highly expressed in Chinese Hamster Ovary (CHO) cells. The high and stable expression sub-clones with Ig kappa signal peptide were selected by Western blot analysis and used for suspension culture. After fed-batch culture over 8 d, the expression level of HSA-eTGFBR2 reached 180 mg/L. The fusion protein was then purified from culture medium using a 2-step chromatographic procedure that resulted in 39% recovery rate. The TGF-β1 binding assay revealed that HSA-eTGFBR2 could bind to TGF-β1 with the affinity constant (KD of 1.42 × 10−8 M) as determined by the ForteBio Octet System. In addition, our data suggested that HSA-eTGFBR2 exhibited a TGF-β1 neutralizing activity and maintained a long-term activity more than eTGFBR2. It concluded that the overexpressing CHO cell line supplied sufficient recombinant human HSA-eTGFBR2 for further research and other applications.

TGFβ1 Suppresses Neuronal IGF1 via a Let-7F-Dependent Mechanism During Azoxymethane-Induced Hepatic Encephalopathy in Mice

Background and Aim: Hepatic encephalopathy (HE) is a neurological complication that arises after a loss of liver function. HE is associated with increased cerebral edema, neuroinflammation and the onset of cognitive decline. We have previously shown that there is increased expression and secretion of transforming growth factor β1 (TGFβ1) from hepatocytes during the azoxymethane (AOM) model of acute liver failure and HE. Increased circulating TGFβ1 was shown to promote blood-brain barrier permeability and also led to progressive neurological complications via increased activation of its receptor TGFβR2. Insulin-like growth factor 1 (IGF1) is a neuroprotective peptide that can be suppressed by TGFβ1 signaling in other organs. IGF1 expression is regulated by a number of microRNAs, including Let-7f, which is positively regulated by TGFβ1. Therefore, we hypothesize that circulating hepatic-derived TGFβ1 suppresses neural IGF1 via a mechanism involving Let-7f during HE, which subsequently exacerbates neurological decline. Methods: In order to determine the exact contribution of TGFβ1 liver-brain axis signaling in this model, male C57Bl/6 (WT) mice, hepatocyte-specific TGFβ1 knockout mice (floxed TGFβ1 × albumin-Cre) or neuron-specific TGFβR2 knockout mice (floxed TGFβR2 × Thy1-Cre) were injected with the hepatotoxin azoxymethane (AOM). In parallel, WT mice were treated with an anti-TGFβ neutralizing antibody 1 h prior to AOM, or were infused with rIGF1 into the lateral ventricle for 3 days prior to AOM injection. Cognitive impairment was monitored and livers, serum and whole brains were collected at coma. IGF1, TGFβ1, Let-7f and proinflammatory cytokine expression were assessed by immunoblotting, immunohistochemistry and/or qPCR. Microglia were stained by IBA1 and cortex field staining and cell morphology were assessed. In vitro, mouse neurons were transfected with a specific Let-7f inhibitor prior to treatment with rTGFβ1 and the expression of Let-7f, IGF1 and CCL2 were measured. Results: Mice injected with AOM had increased Let-7f and suppressed cortical IGF1 expression. Strategies to inhibit TGFβ1 signaling in the brain (ie hepatocyte-specific TGFβ1 knockout, neuron-specific TGFβR2 knockout or pretreatment with TGFβ neutralizing antibody) attenuated the (i) induction of Let-7f expression, (ii) suppression of cortical IGF1, (iii) neuroinflammation and (iv) neurological decline when compared to WT mice treated with AOM alone. In vitro, treatment of neurons with rTGFβ1 increased the expression of Let-7f and reduced the expression of IGF1 in a Let-7f-dependent manner, while also inducing CCL2 expression. Lastly, rIGF1 infusion in the brain delayed AOM-induced neurological decline and reduced neuroinflammation. Conclusion: Elevated TGFβ1 signaling during HE leads to increased cortical Let-7f expression, decreased IGF1 expression and worse outcomes in AOM-treated mice. These deleterious effects can be reversed by inhibiting neuronal TGFβ1 signaling or by increasing IGF1 concentrations in the brain, indicating that elevated TGFβ1 and suppressed IGF1 contribute to the progression of HE. The authors have none to declare.

Effect of TGF-β1 neutralizing antibody on intestinal fibrosis in a mouse model of chronic colitis induced with trinitrobenzene sulfonic acid

Effect of TGF-β1 neutralizing antibody on intestinal fibrosis in a mouse model of chronic colitis induced with trinitrobenzene sulfonic acid

A novel highly potent trivalent TGF-β receptor trap inhibits early-stage tumorigenesis and tumor cell invasion in murine Pten-deficient prostate glands.

The effects of transforming growth factor beta (TGF-β) signaling on prostate tumorigenesis has been shown to be strongly dependent on the stage of development, with TGF-β functioning as a tumor suppressor in early stages of disease and as a promoter in later stages. To study in further detail the paradoxical tumor-suppressive and tumor-promoting roles of the TGF-β pathway, we investigated the effect of systemic treatment with a TGF-β inhibitor on early stages of prostate tumorigenesis. To ensure effective inhibition, we developed and employed a novel trivalent TGF-β receptor trap, RER, comprised of domains derived from the TGF-β type II and type III receptors. This trap was shown to completely block TβRII binding, to antagonize TGF-β1 and TGF-β3 signaling in cultured epithelial cells at low picomolar concentrations, and it showed equal or better anti-TGF-β activities than a pan TGF-β neutralizing antibody and a TGF-β receptor I kinase inhibitor in various prostate cancer cell lines. Systemic administration of RER inhibited prostate tumor cell proliferation as indicated by reduced Ki67 positive cells and invasion potential of tumor cells in high grade prostatic intraepithelial neoplasia (PIN) lesions in the prostate glands of Pten conditional null mice. These results provide evidence that TGF-β acts as a promoter rather than a suppressor in the relatively early stages of this spontaneous prostate tumorigenesis model. Thus, inhibition of TGF-β signaling in early stages of prostate cancer may be a novel therapeutic strategy to inhibit the progression as well as the metastatic potential in patients with prostate cancer.

Predominance of weakly cytotoxic, T-betLowEomesNeg CD8+ T-cells in human gastrointestinal mucosa: implications for HIV infection

The gastrointestinal mucosa is an important site of HIV acquisition, viral replication, and pathogenesis. Immune cells in mucosal tissues frequently differ in phenotype and function from their non-mucosal counterparts. Although perforin-mediated cytotoxicity as measured in blood is a recognized correlate of HIV immune control, its role in gastrointestinal tissues is unknown. We sought to elucidate the cytotoxic features of rectal mucosal CD8+ T-cells in HIV infected and uninfected subjects. Perforin expression and lytic capacity were significantly reduced in rectal CD8+ T-cells compared with their blood counterparts, regardless of HIV clinical status; granzyme B (GrzB) was reduced to a lesser extent. Mucosal perforin and GrzB expression were higher in participants not on antiretroviral therapy compared with those on therapy and controls. Reduction in perforin and GrzB was not explained by differences in memory/effector subsets. Expression of T-bet and Eomesodermin was significantly lower in gut CD8+ T-cells compared with blood, and in vitro neutralization of TGF-β partially restored perforin expression in gut CD8+ T-cells. These findings suggest that rectal CD8+ T-cells are primarily non-cytotoxic, and phenotypically shaped by the tissue microenvironment. Further elucidation of rectal immune responses to HIV will inform the development of vaccines and immunotherapies targeted to mucosal tissues.

The extracellular matrix regulates the effect of decorin and transforming growth factor beta-2 (TGF-β2) on myoblast migration

Muscular injuries that destroy the basal lamina result in poor functional recovery of skeletal muscle. This is due, in part, to the deposition of structural fibrotic proteins such as fibronectin and collagen by fibroblasts and other cells. Transforming growth factor-β (TGF-β) promotes fibrosis, whereas the proteoglycan decorin is known to act as an anti-fibrotic agent, in part via the binding and neutralization of TGF-β. We have previously established that decorin can alter the migratory response of skeletal muscle myoblasts to the extracellular matrix (ECM) factor collagen, but not fibronectin. We have also shown that TGF-β reduces myoblast migration. In the current study we demonstrate that decorin can dramatically alter the inhibitory role of TGF-β on human myoblast migration and go on to shown that the extracellular matrix can significantly modify this effect. Decorin and TGF-β2 in combination were observed to significantly increase the rate of human myoblast migration, despite the inhibitory effect of TGF-β2 on its own. Furthermore, in the presence of fibronectin, TGF-β2 and decorin no longer acted synergistically to promote migration; while in the presence of collagen I, TGF-β2 failed to inhibit migration. These studies show, for the first time, that decorin can alter the bioactivity of TGF-β2 on human myoblast migration and emphasize the crucial regulatory role of the extracellular matrix in determining the response of skeletal muscle myoblasts to migratory cues.

Role of TGF-β and IL-6 in dendritic cells, Treg and Th17 mediated immune response during experimental cerebral malaria

The role of cytokines in Plasmodium infection have been extensively investigated, but pro and anti inflammatory cytokines mediated imbalance during malaria immune-pathogenesis is still unrevealed. Malaria is associated with the circulating levels of Interleukin-6 (IL-6) and transforming growth factor β (TGF-β), but association between these two cytokines in immune response remains largely obscured. Using mouse model, we proposed that IL-6 and TGF-β are involved in immune regulation of dendritic cells (DC), regulatory T cells (Treg), T-helper cells (Th17) during P. berghei ANKA (PbA) infection. Association between the cytokines and the severity of malaria was established with anti-TGF-β treatment resulting in increased parasitemia and increased immunopathology, whereas; anti-IL-6 treatment delays immunopathology during PbA infection. Further, splenocytes revealed differential alteration of myeloid DC (mDC), plasmocytoid DC (pDC), Treg, Th17 cells following TGF-β and IL-6 neutralization. Interestingly anti-TGF-β reduces CD11c+CD8+ DC expression, whereas anti-IL-6 administration causes a profound increase during PbA infection in Swiss mice. We observed down regulation of TGF-β, IL-10, NFAT, Foxp3, STAT-5 SMAD-3 and upregulation of IL-6, IL-23, IL-17 and STAT-3 in splenocytes during PbA infection. The STAT activity probably plays differential role in induction of Th17 and Treg cells. Interestingly we found increase in STAT-3 and decrease in STAT-5 expression during PbA infection. This pattern of STAT indicates that possibly TGF-β and IL-6 play a crucial role in differentiation of DCs subsets and Treg/Th17 imbalance during experimental cerebral malaria (ECM).

Platelet-Derived Ectosomes Reduce NK Cell Function.

Platelet (PLT) transfusions are potentially life saving for individuals with low PLT numbers; however, previous work revealed that PLT transfusions are associated with increased infection risk. During storage, PLT intended for transfusion continuously shed ectosomes (Ecto) from their surface, which express immunomodulatory molecules like phosphatidylserine or TGF-β1. Recently, PLT-Ecto were shown to reduce proinflammatory cytokine release by macrophages and to favor the differentiation of naive T cells toward regulatory T cells. Whether PLT-Ecto modify NK cells remains unclear. We exposed purified NK cells and full PBMCs from healthy donors to PLT-Ecto. We found a reduced expression of several activating surface receptors (NKG2D, NKp30, and DNAM-1) and decreased NK cell function, as measured by CD107a expression and IFN-γ production. Pretreatment of PLT-Ecto with anti-TGF-β1 neutralizing Ab restored surface receptor expression and NK cell function. We further observed a TGF-β1-mediated upregulation of miR-183, which, in turn, reduced DAP12, an important protein for stabilization and downstream signaling of several activating NK cell receptors. Again, these effects could antagonized, in part, when PLT-Ecto were preincubated with anti-TGF-β1 Ab. Erythrocyte Ecto did not affect NK cells. Polymorphonuclear cell Ecto expressed MHC class I and inhibited NK cell function. In addition, they induced the secretion of TGF-β1 by NK cells, which participated in an auto/paracrine manner in the suppressive activity of polymorphonuclear cell-derived Ecto. In sum, our study showed that PLT-Ecto could inhibit NK cell effector function in a TGF-β1-dependent manner, suggesting that recipients of PLT transfusions may experience reduced NK cell function.

Abstract 4987: Role of the PD-1/PDL-1 pathway in resistance of patients with metastatic breast cancer to treatment with radiotherapy and TGFβ neutralization

Abstract Experimental data and clinical observations indicate that local radiation therapy (RT) converts the irradiated tumor into an in situ vaccine and improves responses to immunotherapy. Preclinical breast cancer models show that TGFb neutralization was required to achieve CD8+ T cell priming specific for multiple tumor antigens, and rejection of the irradiated tumor and non-irradiated metastases (Vanpouille-Box et al., Cancer Res 2015). Tumor response and survival were improved by PD-1 blockade. We explored the role of the PD-1/PDL-1 pathway in resistance to combined RT + TGFβ neutralizing antibody (fresolimumab) in metastatic breast cancer patients (pts). 22 pts were treated in a prospective trial at NYU and UCLA (NCT01401062, supported by DOD MTA BC100481). Pts were randomly assigned to 2 doses of fresolimumab (freso) (1mg/kg and 10 mg/kg, q 3 weeks). Image guided RT was delivered to one metastasis on weeks 2 and 7, 7.5 GyX3. 1 pt achieved objective response, with 28% reduction of tumor burden. The response lasted 11 months, anthracycline-induced acute myelogenous leukemia developed. Pts randomized to the higher freso dose had a hazard ratio of 2.17 (95% CI: 0.753-6.272) for risk of death at 1 year follow up. Currently, at a median follow up of 2 years, 20/22 pts have died. To explore reasons for the limited response, peripheral blood mononuclear cells collected at baseline, 5 and 15 weeks into treatment, were analyzed. Responses to survivin, as a tumor antigen, were assessed using tetramers. Single cell network profiling (SCNP) was used to evaluate expression of 6 immunomodulatory receptors plus immune signaling in T cells collected from 7 healthy donors (HD) and 15 breast cancer pts (6 at 1mg/kg, 9 at 10mg/kg fresolimumab). In vitro activation of p-AKT and p-Erk was quantified following in vitro T cell receptor (TCR) modulation with anti-CD3/αnti-CD28. 6/12 HLA-A2.1+ pts had pre-existing survivin-specific CD8+ T cells, and 3 showed an increase over time. 2 pts who were negative generated modest responses after RT and 10 mg/kg freso. Prior to treatment, PDL-1 expression was increased in monocytes (p = 0.01) and CD4+ T cells (p = 0.037) compared to HD. Elevated PD-1 (p = 0.054) and OX-40 (p = 0.014) expression were identified on CD4+ T cells of pts. Upon TCR modulation, CD4+ and CD8+ T cells from pts showed reduced signaling through p-AKT and, to a lesser extent, p-Erk, compared to HD. Signaling was lower in PD-1+ vs PD-1- CD8+ and CD4+ T cells. Addition of anti-PD-1 pembrolizumab partially restored TCR-mediated signaling through p-AKT and p-Erk in PD-1+ but not PD-1- T cells. Overall, data indicate impaired T cell signaling in PD-1+ T cells of metastatic breast cancer pts, which may explain the inability to respond to RT + TGFβ blockade. In vitro addition of anti-PD-1 improved T cell signaling through p-AKT and p-Erk. Together with the preclinical data, this supports adding anti-PD-1 to the combination of RT +TGFβ blockade. Citation Format: Sandra Demaria, Claire Vanpouille-Box, Rachael E. Hawtin, Christie Fanton, Andy Conroy, Erik Evensen, Neha Dixit, Alan Barber, Dorthe Schaue, William H. McBride, Mary Helen Barcellos-Hoff, Silvia C. Formenti. Role of the PD-1/PDL-1 pathway in resistance of patients with metastatic breast cancer to treatment with radiotherapy and TGFβ neutralization. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4987.

Abstract 3239: Cancer-associated fibroblasts induce immunosuppressive macrophages in head and neck squamous cell carcinoma

Abstract Background Cancer-associated fibroblasts (CAFs) are one of main elements in the tumor microenvironment (TME). Among the innate and adaptive immune cells in the TME, tumor-associated macrophages (TAMs) are particularly abundant and play a protumoral role. However, an interaction between CAFs and TAMs in head and neck squamous cell carcinoma (HNSCC) still remains unclear. In the present study, we investigated whether CAFs skewed macrophage function toward the immune suppressive phenotype. Materials and Methods In vitro assay: CAFs were prepared from surgical tissue of HNSCC patients, and culture supernatants of CAFs were collected. Using this supernatants and peripheral blood mononuclear cells collected from healthy donors, immunological interactions between CAFs and TAMs was investigated. In vivo assay: The immunohistochemistry (IHC) of 73 patients with HNSCC was performed. Anti-α-smooth muscle actin, CD68, and CD163 antibodies were used to identify CAFs and TAMs, respectively. The relationship between immunohistochemical parameters and clinical parameters was evaluated. Results The expression levels of CD68, CD163, CD14, CD80, CD86, and HLA-G were up-regulated in CD14+ cells co-cultured with the supernatants of CAFs (CAF-educated cells) compared with control cells. Moreover, the gene expressions of ARG1, IL6, IL10 and TGFB1 were also increased in CAF-educated cells. The CAF-educated cells suppressed T-cell proliferation more intensively than control cells, and neutralization of TGF-β and IL-10 led to significant restoration of T-cell proliferation. In IHC, the intensity of CAFs correlated with the number of CD68-positive macrophages, especially with CD163-positive macrophages. It also correlated with several clinical parameters such as lymphatic invasion, vascular invasion, nodal status, tumor stages and recurrence rate. Moreover, the high intensity of CAFs correlated with poor PFS and OS. The high intensity of CAFs and M2 macrophage infiltration were independent prognostic factors of PFS and OS in HNSCC. Conclusion The present study suggests that CAFs can induce an immunosuppressive phenotype of macrophages and contribute to establishing the immunosuppressive networks in TME, resulting poor prognosis in HNSCC patients. Citation Format: Hideyuki Takahashi, Koichi Sakakura, Kazuaki Chikamatsu. Cancer-associated fibroblasts induce immunosuppressive macrophages in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3239.

Abstract 4986: Regulation of radiation-induced in situ tumor vaccination by TGFβ superfamily members

Abstract Transforming Growth Factor-beta (TGFβ) and activin A (actA) are members of the TGFβ superfamily with overlapping as well as distinct functions in many processes including regulation of inflammation and immunity. Similar to TGFβ, actA has been shown to promote the conversion of CD4+CD25- T cells into induced regulatory T cells (iTregs), and to potentiate iTregs generation by TGFβ. Many cancer cells produce actA, and we have recently found that levels of actA released in vitro by breast cancer cells are enhanced by radiotherapy (RT). Interestingly, prolonged exposure to TGFβ inhibitors also resulted in enhanced actA release, consistent with a compensatory mechanism described in development (Carvalho et al., J Cell Sci, 2007). Here we tested the hypothesis that actA plays a role in regulating anti-tumor immunity induced by local tumor RT and TGFβ blockade in vivo (Vanpouille-Box et al., Cancer Res 2015). 4T1 derivatives with conditional actA knockdown (4T1shActA) or non-silencing control (4T1shNS) were engineered using inducible doxycycline (DOX) plasmids and injected s.c. in BALB/c mice (day 0). ActA gene knockdown (KD) was induced by DOX at day 8. TGFβ neutralizing 1D11 or isotype control antibodies were given i.p. every other day starting on day 12. RT was delivered to the primary tumor in 6Gy fractions on five consecutive days starting at day 13. Mice were followed for tumor growth and survival or euthanized at day 22 or 28 for analysis. Consistent with in vitro data, ActA gene expression was upregulated at day 28 in 4T1 tumors of mice after prolonged TGFβ blockade by 1D11. Neither 1D11 nor ActA gene KD by themselves inhibited tumor growth. However, each intervention significantly improved tumor control achieved by RT. ActA KD in mice treated with RT+1D11 prevented tumor recurrence and improved survival (RT+1D11 vs RT+1D11+shActA p = 0.026; RT+shActA vs RT+1D11+shActA p = 0.0008). Interestingly, blockade of TGFβ or actA resulted in increased intratumoral Tregs (Control: 11.6%; 1D11: 26.2%, shActA: 21%) and markedly enhanced the increase in Tregs seen with RT alone (RT: 15.7%; RT+1D11: 27.5%; RT+shActA: 30.3%). In marked contrast, when both TGFβ and actA were inhibited Tregs significantly decreased in both non-irradiated (1D11+shActA: 13.6%) and irradiated tumors (RT+1D11+shActA: 7.9% of Tregs). IFNγ production by CD8+ T cells in response to the tumor-specific AH1 peptide was significantly higher in RT+1D11+shActA treated mice compared to RT+1D11 (p = 0.045) and RT+shActA (p = 0.0147). Additionally, in RT+1D11+shActA mice expression of the activation marker CD69 was markedly increased in intra-tumoral CD8+ PD-1+ T cells (MFI Control: 187; 1D11: 139.5; shActA: 162; 1D11+shActA: 111; RT: 138.5; RT+1D11: 379; RT+shActA: 182; RT+1D11+shActA: 498.5). Overall, data indicate that TGFβ and actA regulate Tregs numbers in tumors and show a complex interaction with RT. Combined blockade of TGFβ and actA during RT may be required to improve in situ vaccination by RT. Citation Format: Claire I. Vanpouille-Box, Silvia C. Formenti, Sandra Demaria. Regulation of radiation-induced in situ tumor vaccination by TGFβ superfamily members. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4986.

Customized Platelet-Rich Plasma for Skeletal Muscle Injuries

Objectives:Skeletal muscle injuries are among the most common sports-related trauma. Current treatment strategies result in formation of fibrous tissue that hinders the healing process before complete recovery. Incomplete recovery impairs muscle function and predisposes to re-injury. Platelet-Rich-Plasma (PRP) contains a multitude of growth factors and is an autologous source of growth factors for various tissue repairs. It is well established that PRP contains beneficial growth factors for muscle repair; however, it also contains high concentrations of deleterious growth factors for optimal muscle healing, such as transforming growth factor-beta 1 (TGF-β1). TGF-β1 leads to increased fibrosis impeding muscle healing. We therefore hypothesized that neutralization of TGF-β1’s action within PRP could improve PRP’s beneficial effect on skeletal muscle repair.Methods:Sixteen week old in-bred Fisher rats were used. Three rats were used for PRP isolation. 10 ml of blood were extracted from abdominal aorta and mixed with citrate phosphate dextrose solution. PRP were isolated by twice centrifugation. 24 rats were randomly assigned to four groups. A small incision was made along the tibialis anterior (TA) muscle; 50 µl cardiotoxin (CTX) (0.15ug/ul) was injected intramuscularly to the TA. One day after CTX injection, the animals were treated with PBS (control), plain PRP (PRP group), customized PRP+Ab-1x, and PRP+Ab-5x. Animals were sacrificed, and TA muscles were dissected on week 1 and 2 for assessment of muscle regeneration, fibrosis, macrophage infiltration, and satellite cell activation.Results:We observed significantly more regenerative myofibers in the PRP and customized PRP groups compared to control (Fig 1A-C). Collagen deposition (fibrosis) was detected in all groups at week 1 and week 2 after injury; while customized PRP group showed significantly decreased collagen deposition at week 1 and week 2 when compare to control and PRP groups (Fig. 1D-F). PRP was also shown to activate satellite cells at day 7 while customized PRP extended satellite cell activation through day 14 (Fig. 1G, H). Macrophage infiltration was significantly decreased in PRP group when compared to control at week 1 after injury; however the macrophage infiltration was increased in customized PRP group when compare with PRP group, specifically, we observed an increased in the percentage of the M2 macrophage subtype in the customized PRP group at week 1 but not week 2(Fig. 1I, J).Conclusion:This study demonstrates that substantial fibrosis occurs in skeletal muscle following CTX injury. PRP treatment significantly increases muscle regeneration by promoting angiogenesis, satellite cell activation, and reducing the infiltration of inflammatory macrophages. However, augmented fibrosis was also observed. Neutralizing TGF-β1 within PRP also significantly promotes muscle regeneration while significantly reducing fibrosis. It has been suggested that M2 macrophages are involved in the suppression, resolution and reversal of fibrosis. Hence, the increase in M2 macrophages in the customized PRP group may have contributed to the observed decrease in fibrosis. Our results demonstrate that neutralization of TGF-β1 within PRP is a promising approach to improve PRP’s beneficial effect on skeletal muscle repair while decreasing deleterious fibrosis.

Anti-TGF-β Antibody, 1D11, Ameliorates Glomerular Fibrosis in Mouse Models after the Onset of Proteinuria.

Fibrosis is a final common pathway leading to loss of kidney function, in which the fibrogenic cytokine, transforming growth factor β (TGF-β), plays a central role. While previous studies showed that TGF-β antagonism by various means prevents fibrosis in mouse models, clinical approaches based on these findings remain elusive. 1D11 is a neutralizing antibody to all three isoforms of TGF-β. In both adriamycin (ADR)-induced nephropathy and NEP25 podocyte ablation nephropathy, thrice-weekly intraperitoneal administration of 1D11 from the day of disease induction until the mice were sacrificed (day 14 for ADR and day 28 for NEP25), significantly reduced glomerular COL1A2 mRNA accumulation and histological changes. Consistent with our previous findings, proteinuria remained overt in the mice treated with 1D11, suggesting distinct mechanisms for proteinuria and fibrogenesis. Podocyte numbers determined by WT1 staining were significantly reduced in NEP25-model glomeruli as expected, while WT1-positive cells were preserved in mice receiving 1D11. Even when 1D11 was administered after the onset of proteinuria on day 3, 1D11 preserved WT1-positive cell numbers in glomeruli and significantly reduced glomerular scar score (2.5 ± 0.2 [control IgG] vs. 1.8 ± 0.2 [1D11], P < 0.05) and glomerular COL1A2 mRNA expression (19.3 ± 4.4 [control IgG] vs. 8.4 ± 2.4 [1D11] fold increase over the healthy control, P < 0.05). Transmission electron microscopy revealed loss of podocytes and denuded glomerular basement membrane in NEP25 mice with disease, whereas podocytes remained attached to the basement membrane, though effaced and swollen, in those receiving 1D11 from day 3. Together, these data suggest that TGF-β neutralization by 1D11 prevents glomerular fibrosis even when started after the onset of proteinuria. While overt proteinuria and podocyte effacement persist, 1D11 prevents total podocytes detachment, which might be a key event activating fibrogenic events in glomeruli.

TGF-β-mediated inhibition of IFN-γ production by Mycobacterium tuberculosis-specific T cells in the infected lung

Abstract Tuberculosis (TB) is a major global health problem killing over one million people every year. The emergence of multi and extremely-drug resistant (MDR and XDR) strains of Mycobacterium tuberculosis (Mtb) has made treatment difficult and has spurred interest in developing host-directed immunotherapeutics. A critical roadblock is our failure to understand how Mtb escapes eradication despite an apparently robust adaptive immune response. Interferon-γ (IFN-γ) producing CD4 T cells are critical for immunity, yet their abundance in the blood does not correlate with protection to TB, suggesting that their activity may be blocked locally at the primary site of infection in the lung. A leading candidate for IFN-γ suppression in the lung is transforming growth factor-β (TGF-β), a multifunctional cytokine that is capable of inhibiting IFN-γ. TGF-β is produced in excess at sites of active Mtb infection in both murine and human TB. Neutralization of TGF-β improves T-cell responses of active TB patients in vitro, but the extent of its role during in vivo TB is unknown. Here, we generated mixed bone marrow chimeras using WT mice and mice with a T cell-specific conditional deletion of TGF-β receptor II (dLck-Cre:TGFbRIIfl/fl). Mtb-specific TGF-βRII knockout cells outcompeted their WT counterparts and exhibited increased IFN-γ production during Mtb infection. Furthermore, Mtb-infected dLck-Cre:TGFbRIIfl/fl mice had decreased bacterial loads early during infection. Finally, two pharmacological inhibitors of TGF-β signaling lowered the bacterial loads of lungs from Mtb-infected mice both early and late during infection. Targeting TGF-β may pave the way for new host-directed immunotherapeutics for TB.