TGF-β Neutralizing Research Articles

TGF-β neutralization attenuates tumor residency of activated T cells to enhance systemic immunity in mice

A tissue resident-like phenotype in tumor infiltrating Tcells can limit systemic anti-tumor immunity. Enhanced systemic anti-tumor immunity is observed in head and neck cancer patients after neoadjuvant PD-L1 immune checkpoint blockade (ICB) and transforming growth factor β (TGF-β) neutralization. Using Tcell receptor (TCR) sequencing and functional immunity assays in a syngeneic model of oral cancer, we dissect the relative contribution of these treatments to enhanced systemic immunity. The addition of TGF-β neutralization to ICB resulted in the egress of expanded and exhausted CD8+ tumor infiltrating lymphocytes (TILs) into circulation and greater systemic anti-tumor immunity. This enhanced egress associated with reduced expression of Itgae (CD103) and its upstream regulator Znf683. Circulating CD8+ Tcells expressed higher Cxcr3 after treatment, an observation also made in samples from patients treated with dual TGF-β neutralization and ICB. These findings provide the scientific rationale for the use of PD-L1 ICB and TGF-β neutralization in newly diagnosed patients with carcinomas prior to definitive treatment of locoregional disease.

Abstract 3615: Targeting high-risk neuroblastoma by TGFβ imprinted natural killer cells

Abstract Background: Neuroblastoma (NB) is a common malignant extracranial solid tumor in children, responsible for 11% of cancer deaths in pediatric patients under 15 (Smith et al, Cancer, 2014). High-risk neuroblastoma is a metastatic disease in children aged 18 months or older with L2, M, or MS disease with amplification of the MYCN oncogene (Chuang Pediatr Blood Cancer 2021). Recent analyses by COG show that HR Neuroblastoma still has a 5-year event free survival of 51% despite an extensive treatment regimen (Steven et al, ASCO, 2022). To address improved tumor targeting and overcome antigen escape, chimeric antigen receptor (CAR) technology to Natural Killer (NK) cell therapy has been adapted (Chu/Cairo, et al, Frontiers in Oncology, 2019). TGFβ is a potent mechanism of the NK cell anti-tumor resistance and is present at elevated levels in the NB tumor microenvironment (TME) (Foltz, Cancers, 2018). Studies demonstrated that blockade of TGF-β signaling in NK cells caused the accumulation of NK cells that produce IFN-γ and neutralization of TGF-β prevented NKG2D downregulation and also restored NK cell anti-tumor reactivity (Nayyar/Chu/Cairo, et al Frontierls in Oncology, 2018). ROR1, a transmembrane protein is highly expressed on the majority of NB (Rebagay/Yan Front Oncol, 2012). Our group has successfully expanded functional and active peripheral blood NK cells (exPBNK) with irradiated feeder cells and electroporated CAR mRNA to exPBNK (Chu/Cairo, et al Cancer Immunol Res, 2015). Objective: To determine in vitro cytotoxicity against neuroblastoma cells by ex vivo expanded TGFβ imprinting (TGFβi) NK cells. Methods: TGFβi NK cells are generated by culturing expanded NK cells with irradiated K562-41BBL-mbIL21 in 50 IU/mL IL-2 plus 10 ng/mL TGFβ every 2-3 days for two weeks. NK and TGFβi NK cells specific in-vitro cytotoxicity were compared via luciferase reporter assay against ROR1+ NB cell lines as we previously described (Chu/Cairo, et al, JITC, 2022). Cytokine/chemokine levels secreted by NK and TGFβi NK cells were compared using ELISA assays (Chu/Cairo, et al, JITC, 2022). Results: We have successfully expanded TGFβi NK cells. These TGFβi NK cells showed significantly enhanced in vitro cytotoxicity against NB cells compared to NK cells under TGFβ environment (20+2.4% vs 0%; p<0.001) at E:T=3:1 and consistently, TGFβ-imprinting NK secreted significantly high level of TNF-alpha (393.7pg/ml+16.77pg/ml) against NB cells (p<0.001) compared to NK cells (79.7pg/ml+14.7pg/ml) under TGFβ environment. Conclusion: Our data indicate TGFβi NK cells may be a promising novel therapy to overcome the immunosuppression of TGFβ in the tumor microenvironment for pediatric NB tumors. We will further engineer these TGFβi NK cells to express anti-ROR1 CAR to enhance the targeting specificity against NB tumors and investigate the anti-tumor effects of anti-ROR1 CAR TGFβi NK cells against NB tumors in-vitro and in-vivo (Funded by U54 CA232561). Citation Format: Jessica A. Ayala-Cuesta, Yaya Chu, Keira Foley, Meijuan Tian, Kayleigh Klose, Alyssa S. Mendelowitz, Timothy P. Cripe, Dean A. Lee, Mitchell S. Cairo. Targeting high-risk neuroblastoma by TGFβ imprinted natural killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3615.

Identification of a novel, MSC-induced macrophage subtype via single-cell sequencing: implications for intervertebral disc degeneration therapy.

Intervertebral disc (IVD) degeneration is a common pathological condition associated with low back pain. Recent evidence suggests that mesenchymal signaling cells (MSCs) promote IVD regeneration, but underlying mechanisms remain poorly defined. One postulated mechanism is via modulation of macrophage phenotypes. In this manuscript, we tested the hypothesis that MSCs produce trophic factors that alter macrophage subsets. To this end, we collected conditioned medium from human, bone marrow-derived STRO3+ MSCs. We then cultured human bone marrow-derived macrophages in MSC conditioned medium (CM) and performed single cell RNA-sequencing. Comparative analyses between macrophages cultured in hypoxic and normoxic MSC CM showed large overlap between macrophage subsets; however, we identified a unique hypoxic MSC CM-induced macrophage cluster. To determine if factors from MSC CM simulated effects of the anti-inflammatory cytokine IL-4, we integrated the data from macrophages cultured in hypoxic MSC CM with and without IL-4 addition. Integration of these data sets showed considerable overlap, demonstrating that hypoxic MSC CM simulates the effects of IL-4. Interestingly, macrophages cultured in normoxic MSC CM in the absence of IL-4 did not significantly contribute to the unique cluster within our comparison analyses and showed differential TGF-β signaling; thus, normoxic conditions did not approximate IL-4. In addition, TGF-β neutralization partially limited the effects of MSC CM. In conclusion, our study identified a unique macrophage subset induced by MSCs within hypoxic conditions and supports that MSCs alter macrophage phenotypes through TGF-β-dependent mechanisms.

Hepatocyte FoxO1 Deficiency Protects From Liver Fibrosis via Reducing Inflammation and TGF-β1-mediated HSC Activation

The O-class of the forkhead transcription factor FoxO1 is a crucial factor mediating insulin→PI3K→Akt signaling and governs diverse cellular processes. However, the role of hepatocyte FoxO1 in liver fibrosis has not been well-established. In his study, we investigated the role of hepatocyte FoxO1 in liver fibrosis and uncovered the underlying mechanisms. Liver fibrosis was established by carbon tetrachloride (CCL4) administration and compared between liver-specific deletion of FoxO1 deletion (F1KO) and control (CNTR) mice. Using genetic and bioinformatic strategies invitro and invivo, the role of hepatic FoxO1 in liver fibrosis and associated mechanisms was established. Increased FoxO1 expression and FoxO1 signalingactivation were observed in CCL4-induced fibrosis. Hepatic FoxO1 deletion largely attenuated CCL4-induced liver injury and fibrosis compared with CNTR mice. F1KOmice showed ameliorated CCL4-induced hepatic inflammation and decreased TGF-β1 mRNA and protein levels compared with those of CNTR mice. In primary hepatocytes, FoxO1 deficiency reduced TGF-β1 expression andsecretion. Conditioned medium (CM) collected from wild-type hepatocytes treated with CCL4 activated humanHSC cell line (LX-2); such effect was attenuated byFoxO1 deletion in primary hepatocytes or neutralization of TGF-β1 in the CM using TGF-β1 antibody. Hepatic FoxO1overexpression in CNTR mice promoted CCL4-induced HSC activation; such effect was blocked in L-TGF-β1KO mice. Hepatic FoxO1 mediates CCL4-inducled liver fibrosis via upregulating hepatocyte TGF-β1 expression, stimulating hepatic inflammation and TGF-β1-mediated HSC activation. Hepatic FoxO1 may be a therapeutic target for prevention and treatment of liver fibrosis.

Latency-associated peptide (LAP)+CD4+ regulatory T cells prevent atherosclerosis by modulating macrophage polarization

RationaleA persistent autoimmune and inflammatory response plays a critical role in the progression of atherosclerosis. The transcription factor forkhead box P3 (Foxp3)+CD4+ regulatory T cells (Foxp3+ Tregs) attenuate atherosclerosis. Latency-associated peptide (LAP)+CD4+ T cells are a new class of Tregs whose role in atherosclerosis is unknown. ObjectiveTo investigate the function of CD4+LAP+ Tregs in inhibiting inflammation and preventing atherosclerosis. Methods and resultsDepletion of CD4+LAP+ Tregs results in aggravated inflammation and atherosclerotic lesions. Mechanistically, CD4+LAP+ Treg depletion was associated with decreased M2-like macrophages and increased Th1 and Th17 cells, characterized by increased unstable plaque promotion and decreased expression of inflammation-resolving factors in both arteries and immune organs. In contrast, adoptive transfer of CD4+LAP+ Tregs to ApoE−/− mice or CD4−/−ApoE−/− mice led to decreased atherosclerotic lesions. Compared with control animals, adoptive transfer of CD4+LAP+ Tregs induced M2-like macrophage differentiation within the atherosclerotic lesion and spleen, associated with increased collagen and α-SMA in plaques and decreased expression of MMP-2 and MMP-9. Mechanistic studies reveal that isolated CD4+LAP+ Tregs exhibit a tolerance phenotype, with increased expression of inhibitory cytokines and coinhibitory molecules. After coculture with CD4+LAP+ Tregs, monocytes/macrophages display typical features of M2 macrophages, including upregulated expression of CD206 and Arg-1 and decreased production of MCP-1, IL-6, IL-1β and TNF-α, which was almost abrogated by transwell and partially TGF-β1 neutralization. RNA-seq analysis showed different gene expression profiles between CD4+LAP+ Tregs and LAP−CD4+ T cells and between CD4+LAP+ Tregs of ApoE−/− mice and CD4+LAP+ Tregs of C57BL/6 mice, of which Fancd2 and IL4i1 may contribute to the powerful inhibitory properties of CD4+LAP+ Tregs. Furthermore, the number and the suppressive properties of CD4+LAP+ Tregs were impaired by oxLDL. ConclusionsOur data indicate that the remaining CD4+LAP+ Tregs play a protective role in atherosclerosis by modulating monocyte/macrophage differentiation and regulatory factors, which may partly explain the protective effect of T cells tolerance in atherosclerosis. Moreover, adoptive transfer of CD4+LAP+ Tregs constitutes a novel approach to treat atherosclerosis.

Ethanol-exposed lung fibroblasts cause airway epithelial barrier dysfunction.

Chronic alcohol ingestion predisposes to lung injury and disrepair during sepsis. Our previous studies outlined roles for transforming growth factor-beta 1 (TGFβ1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in epithelial barrier homeostasis and how alcohol perturbs their expression and signaling. Here we hypothesize that ethanol-exposed lung fibroblasts (LF) are a source of dysregulated TGFβ1 and GM-CSF and thereby alter airway epithelial barrier function. Human or rat LF were cultured ± ethanol for 2 weeks and then co-cultured with human or rat airway epithelial cells (AEC) seeded on Transwell permeable supports. In selected groups, a TGFβ1 receptor type 1 (TGFβR1) inhibitor (SB431542) or a TGFβ1 neutralizing antibody was applied. Transepithelial electrical resistance (TER) was measured prior to co-culture and on day 5 of co-culture. AEC were then analyzed for the expression of selected tight junction and mesenchymal proteins, and transwell membranes were analyzed by immunofluorescence microscopy for ZO-1 expression and localization. TGFβ1 and GM-CSF levels in conditioned media from the co-cultures were quantified by ELISA. AEC co-cultured with ethanol-exposed LF (ELF) showed a significant reduction in TER and corresponding decreases in ZO-1 expression, whereas collagen type 1A1 and α-smooth muscle actin protein expression were increased. In parallel, in conditioned media from the ELF + AEC co-cultures, activated TGFβ1 levels increased and GM-CSF levels decreased. Notably, all the effects of ELF on the AEC were prevented by blocking TGFβ1 activity. Prior ethanol exposure to LF induces barrier dysfunction in naive AEC in a paracrine fashion through activation of TGFβ1 signaling and suppression of GM-CSF. These experimental findings provide a potential mechanism by which chronic alcohol ingestion impairs airway epithelial integrity and renders individuals susceptible to lung injury.

Circulating macrophages as the mechanistic link between mosaic loss of Y-chromosome and cardiac disease

BackgroundGenetics evidences have long linked mosaic loss of Y-chromosome (mLOY) in peripheral leukocytes with a wide range of male age-associated diseases. However, a lack of cellular and molecular mechanistic explanations for this link has limited further investigation into the relationship between mLOY and male age-related disease. Excitingly, Sano et al. have provided the first piece of evidence directly linking mLOY to cardiac fibrosis through mLOY enriched profibrotic transforming growth factor β1 (TGF-β1) regulons in hematopoietic macrophages along with suppressed interleukin-1β (IL-1β) proinflammatory regulons. The results of this novel finding can be extrapolated to other disease related to mLOY, such as cancer, cardiac disease, and age-related macular degeneration.ResultsSano et al. used a CRISPR-Cas9 gRNAs gene editing induced Y-chromosome ablation mouse model to assess results of a UK biobank prospective analysis implicating the Y-chromosome in male age-related disease. Using this in vivo model, Sano et al. showed that hematopoietic mLOY accelerated cardiac fibrosis and heart failure in male mice through profibrotic pathways. This process was linked to monocyte-macrophage differentiation during hematopoietic development. Mice confirmed to have mLOY in leukocytes, by loss of Y-chromosome genes Kdm5d, Uty, Eif2s3y, and Ddx3y, at similar percentages to the human population were shown to have accelerated rates of interstitial and perivascular fibrosis and abnormal echocardiograms. These mice also recovered poorly from the transverse aortic constriction (TAC) model of heart failure and developed left ventricular dysfunction at higher rates. This was attributed to aberrant proliferation of cardiac MEF-SK4 + fibroblasts promoted by mLOY macrophages enriched in profibrotic regulons and lacking in proinflammatory regulons. These pro-fibrotic macrophages localized to heart and eventually resulted in cardiac fibrosis via enhanced TGF-β1 and suppressed IL-1β signaling. Furthermore, treatment of mLOY mice with TGFβ1 neutralizing antibody was able to improve their cardiac function. This study by Sano et al. was able to provide a causative link between the known association between mLOY and male cardiac disease morbidity and mortality for the first time, and thereby provide a new target for improving human health.ConclusionsUsing a CRISPR-Cas9 induced Y-chromosome ablation mouse model, Sano et al. has proven mosaic loss of Y-chromosome in peripheral myeloid cells to have a causative effect on male mobility and mortality due to male age-related cardiac disease. They traced the mechanism of this effect to hyper-expression of the profibrotic TGF-β1 and reduced pro-inflammatory IL-1β signaling, attenuation of which could provide another potential strategy in improving outcomes against age-related diseases in men.

SARS-CoV-2 S and N protein peptides drive invasion abilities of colon cancer cells through TGF-β1 regulation

The COVID-19 pandemic led to the delay of colorectal cancer (CRC) diagnosis, which causes CRC to be treated at more advanced, often metastatic stages. Unfortunately, there is no effective treatment for metastatic CRC stages, which are considered the leading cause of patients' death. The mortality induced by SARS-CoV-2 is significantly higher in cancer patients than in patients with other diseases. Interestingly, COVID-19 patients often develop fibrosis which depends on epithelial-mesenchymal transition (EMT) – the process also involved in cancer progression. The study aimed to verify whether SARS-CoV-2 induces EMT and consequently increases the invasion potential of colon cancer cells.CRC cells were stimulated with SARS-CoV-2 S and N protein peptides and epithelial and mesenchymal markers were analysed with Western blotting to detect the occurrence of the EMT. The migration, invasion assays and MMP-7 secretion were employed to evaluate the potential of SARS-CoV-2 to stimulate the cells invasion in vitro. ELISA assay, TGF-β1 neutralizing antibodies, TGF-βR silencing and inhibitors were used to investigate the role of the TGF-β1 signalling pathways in the SARS-CoV-2-dependent CRC stimulation.The SARS-CoV-2 induced EMT, which increased the invasion ability of CRC cells. Moreover, the SARS-CoV-2 proteins drive colon cancer cell invasion through TGF-β1. Additionally, secreted TGF-β1 induced a bystander effect in colon cancer cells. However, blocking TGF-β1/Smad- and -non-Smad-dependent pathways suppressed the SARS-CoV-2-induced invasiveness of CRC. In conclusion, we revealed that SARS-CoV-2 stimulates the invasion abilities of CRC by regulating TGF-β1-induced EMT. Our results provide a theoretical basis for using anti-TGF-β1 therapy to reduce the risk of CRC metastasis during SARS-CoV-2 infection.

Neutralization of TGF-β in combination with IL-6 ameliorates septic arthritis by altering RANKL/OPG interaction in murine lymphocytes

Septic arthritis is an inflammatory joint disease caused by S. aureus. Hematogenous entry of the bacteria to the joint space secretespro-inflammatory cytokines TGF-β and IL-6, which alter the Th17/Treg switch. Inhibition of TGF-β and IL-6 to modulate Th17/Treghomeostasis and RANKL/OPG interaction are not done so far in septic arthritis. Role of lymphocyte-derived TGF-β, IL-10, IL-21 alongwith OPN, OPG, cellular H2O2, SOD and catalase activities, and the expressions of RANKL and MMP2 were studied in total lymphocytesof blood, spleen and synovial tissues of Swiss albino mice treated with antibody against TGF-β along with IL-6 Ab after induction ofseptic arthritis. Simultaneous neutralization of TGF-β along with IL-6 is effective in shifting the Th17 cell into Treg cell of the arthriticmice and modulates RANKL and MMP2 expression that leads to the down-regulation of osteoclastic activity and reduces the production of OPN. Additionally, such treatment reduces oxidative stress via enhancing the activities SOD and catalase enzymes in lymphocytes. So, simultaneous neutralization of TGF-β and IL-6 reduces S. aureus infection-associated inflammatory joint damage by increasing Treg numbers and decreasing the number of pro-inflammatory Th17 cells.

Overexpression of SMAD7 improves the function of EGFR-targeted human CAR-T cells against non-small-cell lung cancer.

Recent advancements in immunotherapy led to the development of Chimeric antigen receptor (CAR) T-cell therapy. CAR-T cell therapy in non-small cell lung cancer (NSCLC) is hindered by overexpression of transforming growth factor (TGFβ) in the cancer cells that have a negative regulatory role on T-cells activity. This study characterized CAR-T with overexpression of mothers against decapentaplegic homologue 7 (SMAD), a negative regulator of TGFβ downstream signalling. We have generated three types of CAR-T: epidermal growth factor receptor (EGFR)-CAR-T, EGFR-dominant-negative TGFbeta receptor 2 (DNR)-CAR-T, and EGFR-SMAD7-CAR-T by transducing human T-cells with the lentivirus constructs. We characterized the proliferation, expression of proinflammatory cytokines, activation profile, and lysis capacity in co-cultures with A549 lung carcinoma cells with and without TGFβ neutralizing antibodies. We also tested the therapeutic potential of EGFR-SMAD7-CAR-T in the A549 cells tumour-bearing mice model. Both EGFR-DNR-CAR-T and EGFR-SMAD7-CAR-T demonstrated a higher proliferation rate and lysis capacity to A549 than traditional EGFR-CAR-T. Neutralization of TGFβ by the antibodies resulted in increased performance of EGFR-CAR-T. In vivo, both EGFR-DNR-CAR-T and EGFR-SMAD7-CAR-T resulted in complete tumour resorption by day 20, whereas conventional CAR-T only has a partial effect. We demonstrated the high efficacy and resistance to negative TGFβ regulation of EGFR-SMAD7-CAR-T comparable with EGFR-DNR-CAR-T and without the systemic effect of TGFβ inhibition.

1538-P: Brain TGFß Regulates Glucose Tolerance in Male Mice with Metabolic Syndrome

Hypothalamic inflammation contributes to insulin resistance and obesity. Transforming growth factor beta (TGFβ) is among the inflammatory mediators inducing hypothalamic inflammation. In this study, we investigated the role of brain TGFβ on metabolic function. We hypothesized that TGFβ in the brain promotes insulin resistance and reduces glucose tolerance in diabetic and obese mice. C57BL/J male mice were fed either hypercaloric diet (HCD, 52% fat) or regular diet (RD) from weaning. At 3 months age, intraperitoneal glucose tolerance test (GTT), insulin tolerance test (ITT) and weight confirmed the acquisition of metabolic syndrome. Hypothalamic protein expression was assessed for TGFβ receptor 2, phosphorylated SMAD3, SMAD6 and SMAD7, as downstream signal transducer proteins for TGFβ. Compared to RD male mice (n=6), mice on HCD (n=6) showed glucose intolerance (AUC; 60587 ±2460 vs. 30802±1923, p<0.0001), insulin intolerance (AUC; Mean±SEM; 19025836.0 vs.11436±1087, p=0.02) and weight gain (33.67±2.028 vs. 26.89±0.4231 g). HCD mice also exhibited increased protein levels of TGFβR2 (normalized to total protein, Fold Change (FC)±SEM to RD group; 3.54±0.3, p<0.0001), pSMAD3 (FC 2±0.5, p=0.04) while inhibitors for TGFβ signaling such as SMAD6 and SMAD7, were downregulated (FC 0.7±0.1, p=0.03 and 0.8±0.04, p=0.03, respectively). To understand the role of brain TGFβ on metabolic regulation, 3 months HCD mice were subjected to intracerebroventricular (ICV) infusion with either TGFβ neutralizing antibody (nAb, n=4) or IgG control antibody (n=4) using subcutaneous osmotic pumps connected to ICV cannula for 2 weeks. GTT was measured after 2-week infusion. Treatment with TGFβ nAb resulted in lower fasting blood glucose compared to before treatment (166.1 ±6.878 vs. 229.9 ±3.923 mg/dL) and at 120 min time point (229.9 ±28.69 vs. 382.2 ±24.33 mg/dL). In conclusion, brain TGFβ plays a key role in regulating glucose metabolism probably through inflammation-induced insulin resistance. Disclosure M.Elgazzaz: None. K.Miao: None. A.R.Nuzzo: None. L.Restivo: None. K.Moles: None. E.D.Lazartigues: None. Funding American Diabetes Association (1-19-IBS-291 to E.D.L.)

Immunoregulatory Roles of TGF-β and IL-4 during Mouse-adapted SARS-CoV-2 Infection

Abstract Severe COVID-19 is due to a dysregulated immune response; however, the mechanisms preventing recovery of airway function are unclear. Cytokines regulate the quality of inflammation, and drive both anti-viral and disease tolerance mechanisms. There was a mostly monocytic infiltration in the lungs with alveolar thickening and consolidation 3 days post-infection with mouse-adapted SARS-CoV-2 (CMA3p20) at the peak of weight loss. Based on the cytokine profiles observed in human and animal studies, we targeted IL-4 and TGF-β as potentially regulatory cytokines, and quantified changes using flow cytometry. Neutralizing TGF-β, which regulates inflammatory cytokine production, significantly increased cellular infiltration of the lung. Neutralization of TGF-β particularly affected inflammatory monocyte (Ly6C+) recruitment, with a slight decrease in alveolar macrophages (CD11c-int). Neutralizing IL-4 prolonged weight loss to 4 dpi but visibly improved resolution of alveolar thickening on 7 dpi, suggesting that IL-4 is protecting from prolonged peak pathology, but delaying resolution. Neutralizing IL-4 also increased the fraction of polymorphonuclear cells and Tregs, as well as improving the ratio of type II to type I alveolar epithelial cells in the lungs compared to the isotype group. To begin to understand mechanisms connecting IL-4 to epithelial repair, we are studying the quality of the monocyte response and found alternatively activated macrophages with Ym1 staining in activated macrophages in the alveoli of infected mice in an IL-4 dependent manner. In order to improve therapeutics for severe COVID-19, it will be important to determine the cellular mechanisms linking regulatory cytokines to epithelial repair. R01AI135061 (RS, LPC), R01AI166668 (RR, MGO), R01AI131634 (MCS, HGF)

Phenotypic plasticity and reduced tissue retention of exhausted tumor-infiltrating Tcells following neoadjuvant immunotherapy in head and neck cancer.

Neoadjuvant immunotherapies (NITs) have led to clinical benefits in several cancers. Characterization of the molecular mechanisms underlying responses to NIT may lead to improved treatment strategies. Here we show that exhausted, tumor-infiltrating CD8+ T (Tex) cells display local and systemic responses to concurrent neoadjuvant TGF-β and PD-L1 blockade. NIT induces a significant and selective increase in circulating Tex cells associated with reduced intratumoral expression of the tissue-retention marker CD103. TGF-β-driven CD103 expression on CD8+ Tcells is reversed following TGF-β neutralization invitro, implicating TGF-β in Tcell tissue retention and impaired systemic immunity. Transcriptional changes implicate Tcell receptor signaling and glutamine metabolism as important determinants of enhanced or reduced Tex treatment response, respectively. Our analysis illustrates physiological and metabolic changes underlying Tcell responses to NIT, highlighting the interplay between immunosuppression, tissue retention, and systemic anti-tumor immunity and suggest antagonism of Tcell tissue retention as a promising neoadjuvant treatment strategy.

Prenatally Educated Friendly NK Cells regulate Allo-specific T cell Responses through expansion of Foxp3+CD4+Treg Cells and suppression of Type 1 and 17 CD8+T cell responses.

Abstract Prenatal alloantigen exposure during embryonic development permanently tolerizes the early gestational fetus and provides a tremendous advantage over transplantation after birth. However, the influence of prenatally educated NK cells on alloantigen-specific T cell immune responses is not well elucidated. In previous experiments, we found that prenatally-educated friendly NK (fNK) cells exert suppressive effects on allo-specific T cell responses in vitro. We hypothesized that prenatally-educated fNK cells exert regulatory effects on allo-specific T cells in vivo. To challenge this hypothesis, we examined the regulatory potential of prenatally educated fNK cells through gain and loss of function experiments in stable prenatal Balb/c --> B6.Thy1.2+ chimeras (engrafter mice). We depleted both (Ly49AFG+) fNK cells and Thy1.2+T cells or Thy1.2+T cells alone in host engrafter mice and then adoptively transferred naïve responder Thy1.1+ T cells along with donor-specific allogeneic or syngeneic target cells into the engrafter hosts. We found that the fNK cell-replete animals exhibited a higher frequency of foxp3+Tregs and a lower frequency of allospecific T-bet+ and RORγt+ CD8 T cells compared to the fNK cell-depleted controls. Furthermore, the neutralization of TGF-β1 decreased the fNK cell-induced expansion of Treg cells in vitro. From these studies, we conclude that prenatally educated fNK cells exert regulatory effects on allo-specific T-cell responses through the TGF-β1 mediated expansion of foxp3+Treg cells. Prenatal antigen experience drives the emergence of antigen-specific suppressors that regulate T cell responses. National Institutes of Health Grant R01HL103745 and Lurie Children’s Hospital Research Foundation (to A.F.S.)

AMPK reduces macrophage endotoxin tolerance through inhibition of TGF-β1 production and its signaling pathway.

Adenosine monophosphate-activated protein kinase (AMPK) is involved in suppression of the development of endotoxin tolerance, which is a driver of the immunosuppression induced by sepsis. However, the mechanism by which AMPK inhibits the development of endotoxin tolerance has not been clearly elucidated. Therefore, the present study was performed to investigate the mechanism by which the AMPK activator, metformin, inhibits the development of endotoxin tolerance. Lipopolysaccharide (LPS) increased the production of transforming growth factor (TGF)-β1 in macrophages, which was inhibited by metformin and resveratrol. Knockdown of AMPKα1 inhibited the suppressive effect of metformin on LPS-induced TGF-β1 production. TGF-β neutralizing antibody and TGF-β type I receptor inhibitor increased the production of TNF-α and IL-6 via LPS restimulation in tolerized macrophages. LPS increased Smad2 phosphorylation, but this was inhibited in cells treated with TGF-β neutralizing antibody or metformin. Smad2 knockdown inhibited the development of endotoxin tolerance, as evidenced by increased TNF-α production in response to LPS restimulation in tolerized macrophages. TGF-β1 expression was increased, and the levels of TNF-α and IL-6 production induced by LPS stimulation were decreased, in splenocytes of cecal ligation and puncture (CLP) model mice compared to sham-operated controls. However, metformin treatment suppressed the production of TGF-β1, and enhanced the production of TNF-α and IL-6 induced by LPS stimulation in splenocytes of CLP mice. These results indicated that AMPK activation inhibits LPS-induced TGF-β1 production and its signaling pathway, thus suppressing the development of endotoxin tolerance in macrophages.

Abstract 155: Platelet-derived Transforming Growth Factor-β1 Ameliorates AAA in a Murine Model

Background: Platelets are rapidly recruited to the forming intraluminal thrombus in abdominal aortic aneurysm (AAA), stabilizing the aortic wall and preventing rupture. Adhesion and activation of platelets releases a mix of inflammatory mediators such as transforming growth factor β (TGFβ), which mediates cellular proliferation, inflammatory signaling, and fibrosis. Importantly, while TGFβ neutralization results in increased rupture and disease progression in mouse models of AAA, the source of protective TGFβ signaling remains controversial. We hypothesized that platelet-derived TGFβ1 attenuates aortic growth in the elastase AAA model. Methods and Results: Ldlr -/- mice with platelet-specific deletion of Tgfβ1 were produced by breeding hemizygous male mice expressing Cre under the control of the Pf4 promoter to female Tgfβ1 floxed mice (Jackson Labs). Mice were subjected to laparotomy and topical elastase application to the abdominal aorta (10 mg/mL porcine pancreatic elastase for 5 minutes) and sacrificed at day 28 to determine AAA progression via aortic diameter. Male TGFβ1-Pf4 Cre+ mice (n=8) had significantly increased abdominal aortic diameters compared to Male TGFβ1-Pf4 Cre- mice (n=7) ( Cre- : 1.70 ± 0.12 mm; Cre+ : 2.66 ± 0.25 mm; P < 0.004). Additionally, 25% of male TGFβ1-Pf4 Cre+ mice died of AAA rupture compared to 0% in the TGFβ1-Pf4 Cre- cohort. To determine if these actions were occurring via TGFβ1 activation of the TGFβ receptor 2 (βr 2 ), Ldlr -/- /βr2 Flox /PF4 Cre+ and Cre- mice were created, similarly to the previously described Tgfβ1 mice. Male βr2-Pf4 Cre+ (n=7) had increased aortic diameter compared to βr2-Pf4 Cre- (n=6) ( Cre- : 1.70± 0.20 mm; Cre+ : 2.45 ± 0.38 mm; P < 0.138). Additionally, female βr2-Pf4 Cre+ (n=9) also had significantly larger aortic diameters compared to βr2-Pf4 Cre- (n = 5) ( Cre- : 1.27± 0.04 mm; Cre+ : 1.90 ± 0.23 mm; P < 0.01). Conclusions: Our results further support TGFβ signaling playing a protective role in AAA. More specifically, platelet-derived TGFβ1 ligand and signaling via platelet TGFβr2 mitigates aortic diameter expansion. Further research into the pertinent TGFβ isoforms and the mechanism of TGFβr2 activation will ideally yield insights into therapeutic targets for an, as yet, intractable disease.

B-Cell–Derived TGF-β1 Inhibits Osteogenesis and Contributes to Bone Loss in Periodontitis

B cells play a vital role in the elimination of periodontal pathogens, the regulation of the immune response, and the induction of tissue destruction. However, the role of B cells in the dysfunction of mesenchymal stem cell (MSC) differentiation to osteoblasts in periodontitis (PD) has been poorly studied. Here we show that the frequency of CD45−CD105+CD73+ MSCs in inflamed periodontal tissues is significantly decreased in patients with PD compared with that of healthy controls. CD19+ B cells dominate the infiltrated immune cells in periodontal tissues of patients with PD. Besides, B-cell depletion therapy reduces the alveolar bone loss in a ligature-induced murine PD model. B cells from PD mice express a high level of TGF-β1 and inhibit osteoblast differentiation by upregulating p-Smad2/3 expression and downregulating Runx2 expression. The inhibitory effect of PD B cells on osteoblast differentiation is reduced by TGF-β1 neutralization or Smad2/3 inhibitor. Importantly, B-cell–specific knockout of TGF-β1 in PD mice significantly increases the number of CD45−CD105+Sca1+ MSCs, ALP-positive osteoblast activity, and alveolar bone volume but decreases TRAP-positive osteoclast activity compared with that from control littermates. Lastly, CD19+CD27+CD38− memory B cells dominate the B-cell infiltrates in periodontal tissues from both patients with PD and patients with PD after initial periodontal therapy. Memory B cells in periodontal tissues of patients with PD express a high level of TGF-β1 and inhibit MSC differentiation to osteoblasts. Thus, TGF-β1 produced by B cells may contribute to alveolar bone loss in periodontitis, in part, by suppressing osteoblast activity.

PKC-ζ mediated reduction of the extracellular vesicles-associated TGF-β1 overcomes radiotherapy resistance in breast cancer

BackgroundRadiotherapy is widely applied in breast cancer treatment, while radiotherapy resistance is inevitable. TGF-β1 has been considered to be an endogenous factor for the development of radiotherapy resistance. As a large portion of TGF-β1 is secreted in an extracellular vesicles-associated form (TGF-β1EV), particularly in radiated tumors. Thus, the understanding of the regulation mechanisms and the immunosuppressive functions of TGF-β1EV will pave a way for overcoming the radiotherapy resistance in cancer treatment.MethodsThe superoxide-Zinc-PKC-ζ-TGF-β1EV pathway in breast cancer cells was identified through sequence alignments of different PKC isoforms, speculation and experimental confirmation. A series of functional and molecular studies were performed by quantitative real-time PCR, western blot and flow cytometry analysis. Mice survival and tumor growth were recorded. Student’s t test or two-way ANOVA with correction was used for comparisons of groups.ResultsThe radiotherapy resulted in an increased expression of the intratumoral TGF-β1 and an enhanced infiltration of the Tregs in the breast cancer tissues. The intratumoral TGF-β1 was found mainly in the extracellular vesicles associated form both in the murine breast cancer model and in the human lung cancer tissues. Furthermore, radiation induced more TGF-β1EV secretion and higher percentage of Tregs by promoting the expression and phosphorylation of protein kinase C zeta (PKC-ζ). Importantly, we found that naringenin rather than 1D11 significantly improved radiotherapy efficacy with less side effects. Distinct from TGF-β1 neutralizing antibody 1D11, the mechanism of naringenin was to downregulate the radiation-activated superoxide-Zinc-PKC-ζ-TGF-β1EV pathway.ConclusionsThe superoxide-zinc-PKC-ζ-TGF-β1EV release pathway was elucidated to induce the accumulation of Tregs, resulting in radiotherapy resistance in the TME. Therefore, targeting PKC-ζ to counteract TGF-β1EV function could represent a novel strategy to overcome radiotherapy resistance in the treatment of breast cancer or other cancers.Trial registration: The using of patient tissues with malignant Non-Small Cell Lung Cancer (NSCLC) was approved by the ethics committees at Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China (NCC2022C-702, from June 8th, 2022).

Abstract 6422: Combined TGFβ/PD-L1 blockade enhances systemic antitumor immunity in head and neck cancer

Abstract Head and neck squamous cell carcinoma (HNSCC) unrelated to human papillomavirus (HPV) confers poor prognosis, with half of patients experiencing disease relapse in advanced stages. Response rates to traditional immunotherapies are low and novel approaches such as the bifunctional fusion protein Bintrafusp alfa (BA) that concurrently blocks PD-L1 signaling and neutralizes TGFβ are actively under investigation. Our group showed that when HNSCC patients received neoadjuvant BA therapy, it reversed exhaustion of tumor-infiltrating lymphocytes (TILs) by promoting antigen-mediated signaling. Additionally, we observed that BA promoted recirculation of TILs into peripheral blood possibly through decreased expression of the TGF-β-driven tissue-retention marker CD103. We hypothesize that addition of TGFβ neutralization to PD-L1 blockade can enhance systemic anti-tumor immunity compared to PD-L1 blockade alone. We assessed immune responses to BA in wild-type mice bearing established mouse oral cancer (MOC)-1 tumors that were randomized into the following groups: 1) vehicle; 2) anti-PD-L1 antibody (Avelumab); 3) TGFβ neutralizing construct with a mutated PD-L1 antibody (BA-M); 4) TGFβ neutralizing construct with a functional PD-L1 antibody (BA). Additionally, since our previous data suggests that BA responsiveness depends on CD8 T cell activity, we included two extra groups 5) CD8 depleting antibody alone or 6) in combination with BA. Three days after completing treatment, MOC-1 tumor cells were injected into the contralateral flank and engraftment and progression of secondary challenge tumors was analyzed. Primary tumor progression was significantly inhibited by BA and to a lesser extent avelumab compared to control or BA-M. Engraftment or progression of secondary challenge tumors was significantly inhibited by BA and to a lesser extent avelumab compared to control or BA-M. The growth inhibitory effects of BA were abrogated by CD8 depletion in both the primary and challenge tumors. Flow cytometry analysis of tissue collected on day 7 post-tumor challenge showed that BA-mediated clearance of the tumors was associated with a proportional increase of CD4+ and CD8+ TILs. We also observed that the frequency of CD103 expression among CD4+ TILs was reduced both in the primary and challenge tumors. Although TGFβ neutralization alone fails to induce anti-tumor immunity, the addition of TGFβ neutralization to PD-L1 blockade provides greater control of primary tumor growth and protection from engraftment of a challenge tumor compared to PD-L1 blockade alone. Through adoptive transfer of tumor-specific T cell receptor-engineered T cells, ongoing experiments aim to determine if TGFβ-driven CD103 expression is required for T cell tumor residency and if neutralization of TGFβ in the setting of PD-L1 immune checkpoint blockade allows for recirculation of tumor-specific T cells into the periphery. Citation Format: Magdalena Rainey, Marco Craveiro, Yvette Robbins, Cem Sievers, Clint T. Allen. Combined TGFβ/PD-L1 blockade enhances systemic antitumor immunity in head and neck cancer [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 6422.

Abstract 6677: Preliminary immune correlatives from BCA101 trial show favorable modulation of tumor immune microenvironment

Abstract Background: BCA101 is a bispecific antibody targeting EGFR and TGF-β. TGF-β pathway activation is a hallmark of human immune-excluded tumors, and TGF-β expression is associated with resistance to anti-PD-1 blockade. Neutralization of TGF-β removes an immunosuppressive signal that drives accumulation and polarization of myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) in solid tumors, while EGFR inhibition targets tumor cell-intrinsic oncogenic signaling. Co-targeting of EGFR and TGF-β directly impacts tumor progression while enhancing the immunogenicity of tumors. Methods: Patients with multiple solid tumor types (CRC, pancreatic, HNSCC, SqNSCLC, and others) were treated with escalating doses of either single agent BCA101 or in combination with anti-PD-1 (pembrolizumab) enrolled on NCT04429542 trial. We performed a variety of immune correlatives on pre- and on-treatment tumor biopsies, including Nanostring-based transcriptomic profiling and IHC for immunophenotypic markers, as well as multiparametric flow cytometric profiling of circulating PBMCs. Results: Our preliminary evidence suggests that neutralization of TGF-β positively alters the systemic immune state (PBMCs) and tumor immune phenotype (mRNA, IHC). Circulating HLA-DR+ monocytes were significantly increased in on-treatment PBMC samples relative to screening. Pathway analysis of on-treatment tumor biopsies revealed enhanced costimulatory signaling, cytokine and chemokine signaling, immune infiltration, and interferon signaling. Top differentially regulated genes in on-treatment biopsies included CCL21, CXCL9, CXCL11, and CXCL13, which recruit T and NK cells. HDAC11, which negative regulates type-I interferon signaling, was significantly reduced in on-treatment biopsies. Notably, two patients with EGFR-amplified squamous non-small cell lung cancer, who both progressed on first-line immunotherapy treatment, were treated with BCA101 at 1250 mg and 1500 mg qw and achieved a partial response (ongoing for 10 months at the time of the data cutoff) and a prolonged stable disease for 11 months, respectively. They exhibited increased CD8+ T cell infiltration and a reduction in TAMs following treatment. Conclusions: Increased abundance of circulating HLA-DR+ monocytes following treatment indicated polarization towards a more positive, Th1-like systemic immune state. We observed enhanced immunogenicity of tumors as assessed by a targeted IO transcriptomic analysis. The results of the pathway analysis were supported by IHC on post-treatment biopsies from a subsequent cohort showing enhanced CD8+ T cell infiltration and stable, or reduced expression of TAM marker CD163. These results indicate that neutralization of TGF-β induces a more permissive tumor immune microenvironment. Citation Format: Patrick H. Lizotte, Paul Paik, Liviu Niculescu, Seng-Lai H. Tan, David Bohr, Elham Gharakhani, Ralf Reiners, Rachel Salazar, Avanish Varshney, Shiv Ram Krishn, Pradip Nair, Cloud Paweletz. Preliminary immune correlatives from BCA101 trial show favorable modulation of tumor immune microenvironment. [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 6677.