TGFß1 neutralization displays therapeutic efficacy through both an immunomodulatory and a non-immune tumor-intrinsic mechanism.

BACKGROUND: Transforming growth factor-ß (TGFß) is emerging as a promising target for cancer therapy, given its ability to promote progression of advanced tumors and to suppress anti-tumor immune responses. However, TGFß also plays multiple roles in normal tissues, particularly during organogenesis, raising toxicity concerns about TGFß blockade. Dose-limiting cardiovascular toxicity was observed, possibly due to the blockade of all three TGFß isoforms. The dominant isoform in tumors is TGFß1, while TGFß2 and TGFß3 seem to be more involved in cardiovascular development. Recent data indicated that selective targeting of TGFß1 promoted the efficacy of checkpoint inhibitor anti-PD1 in transplanted preclinical tumor models, without cardiovascular toxicity. METHODS: To further explore the therapeutic potential of isoform-specific TGFß blockade, we developed neutralizing mAbs targeting mature TGFß1 or TGFß3, and tested them, in parallel with anti-panTGFß mAb 1D11, in two preclinical models: the transplanted colon cancer model CT26, and the autochthonous melanoma model TiRP. RESULTS: We observed that the blockade of TGFß1, but not that of TGFß3, increased the efficacy of a prophylactic cellular vaccine against colon cancer CT26. This effect was similar to pan-TGFß blockade, and was associated with increased infiltration of activated CD8 T cells in the tumor, and reduced levels of regulatory T cells and myeloid-derived suppressor cells. In contrast, in the autochthonous TiRP melanoma model, we observed therapeutic efficacy of the TGFß1-specific mAb as a single agent, while the TGFß3 mAb was inactive. In this model, the anti-tumor effect of TGFß1 blockade was tumor intrinsic rather than immune mediated, as it was also observed in T-cell depleted mice. Mechanistically, TGFß1 blockade increased mouse survival by delaying the phenotype switch, akin to epithelial-to-mesenchymal transition (EMT), which transforms initially pigmented tumors into highly aggressive unpigmented tumors. CONCLUSIONS: Our results confirm TGFß1 as the relevant isoform to target for cancer therapy, not only in combination with checkpoint inhibitors, but also with other immunotherapies such as cancer vaccines. Moreover, TGFß1 blockade can also act as a monotherapy, through a tumor-intrinsic effect blocking the EMT-like transition. Because human melanomas that resist therapy often express a gene signature that links TGFß1 with EMT-related genes, these results support the clinical development of TGFß1-specific mAbs in melanoma.

Soluble HLA-G and TGF-ß in couples attending assisted reproduction - A possible role of TGF-ß isoforms in semen?

Soluble isoforms of the non-classical Human Leukocyte Antigen (HLA)-G as well as Transforming Growth Factor (TGF)-ß is expressed in seminal plasma possibly influencing the pregnancy potential. We wanted to examine the association of seminal plasma sHLA-G, TGF-ß1, TGF-ß2 and TGFß3 with pregnancy success in a cohort of 127 couples and 4 single women attending fertility treatment with the use of assisted reproduction technologies (ART). Soluble HLA-G, TGF-ß1, TGF-ß2 and TGF-ß3 in seminal plasma did not fluctuate significantly over time. We did not find any impact of seminal plasma sHLA-G, TGF-ß1, TGF-ß2 and TGF-ß3 on time-to-pregnancy measured as number of treatment cycles. There was a significant association between concentrations of seminal plasma sHLA-G and HLA-G variations in the 3'untranslated region (3'UTR) of the HLA-G gene, supporting and extending previous findings. Furthermore, by comparing seminal plasma concentrations of sHLA-G, TGF-ß1, TGF-ß2 and TGF-ß3 in male subjects with reduced semen quality, male subjects with normal semen quality, and sperm donors, we found that TGF-ß2 was significantly lower, and TGF-ß3 was significantly higher, in seminal plasma from sperm donors. These findings suggest that TGF-ß isoforms may influence semen quality and fertility.

Growth Factor-like Gene Regulation Is Separable from Survival and Maturation in Antibody-Secreting Cells.

Recurrent mutational activation of the MAP kinase pathway in plasma cell myeloma implicates growth factor-like signaling responses in the biology of Ab-secreting cells (ASCs). Physiological ASCs survive in niche microenvironments, but how niche signals are propagated and integrated is poorly understood. In this study, we dissect such a response in human ASCs using an in vitro model. Applying time course expression data and parsimonious gene correlation network analysis (PGCNA), a new approach established by our group, we map expression changes that occur during the maturation of proliferating plasmablast to quiescent plasma cell under survival conditions including the potential niche signal TGF-ß3. This analysis demonstrates a convergent pattern of differentiation, linking unfolded protein response/endoplasmic reticulum stress to secretory optimization, coordinated with cell cycle exit. TGF-ß3 supports ASC survival while having a limited effect on gene expression including upregulation of CXCR4. This is associated with a significant shift in response to SDF1 in ASCs with amplified ERK1/2 activation, growth factor-like immediate early gene regulation and EGR1 protein expression. Similarly, ASCs responding to survival conditions initially induce partially overlapping sets of immediate early genes without sustaining the response. Thus, in human ASCs growth factor-like gene regulation is transiently imposed by niche signals but is not sustained during subsequent survival and maturation.

Dental Follicle Stem Cells Ameliorate Lipopolysaccharide-Induced Inflammation by Secreting TGF-ß3 and TSP-1 to Elicit Macrophage M2 Polarization.

BACKGROUND/AIMS: Increasing evidence has demonstrated the novel roles of mesenchymal stem cells (MSCs) in immunotherapy. However, difficulty in acquiring these cells and possible ethical issues limited their application. Recently, we have isolated a unique MSC population from dental follicles with potent stem cell-like properties. This study focused on the effects of dental follicle stem cells (DFSCs) on macrophage activation and polarization to determine their role in immunomodulation and to test if DFSCs are a promising cell source for MSC-based immunotherapy. METHODS: Rat acute lung injury (ALI) models induced by Lipopolysaccharide (LPS) were applied to test the immune-modulatory effects of DFSC/DFSC-CM in vivo. The pulmonary permeability was determined by the dry / wet weight ratios of the left upper lung lobe. The lung histopathological damage was graded on a 0 to 4+ scale. And the inflammatory cytokines in bronchoalveolar lavage fluid (BALF) were tested by ELISA. Then we established LPS-induced inflamed macrophage models in vitro. Inflammatory cytokine production and polarization marker expression were measured by RT-qPCR, ELISA, western blot and flow cytometric analysis in macrophages following DFSC-CM treatment. The paracrine factors in DFSC-CM were revealed by a RayBiotech Protein Array. Thereafter, neutralization studies were performed to confirm the potential immune regulators in DFSC-CM. RESULTS: The DFSC/DFSC-CM not only attenuated histopathological damage and pulmonary permeability, but also downregulated pro-inflammatory cytokines MCP-1, IL-1, IL-6 and TNF-α, and upregulated anti-inflammatory cytokine IL-10 in BALF. Immunofluorescence staining revealed the increased expression of macrophage M2 marker Arg-1. Further in vitro study revealed that macrophages switched to an anti-inflammatory M2 phenotype when co-cultured with DFSC-CM, characterized by suppressed production of pro-inflammatory cytokines MCP-1, IL-1, IL-6, TNF-α and M1-polarizing markers iNOS and CD86; and increased expression of the anti-inflammatory cytokine IL-10 and the M2-polarizing markers Arg-1 and CD163. A RayBiotech Protein Array revealed 42 differentially expressed (> 2-fold) paracrine factors in DFSC-CM compared with the serum-free Ham's F-12K medium, among which TGF-ß3 and Thrombospondin-1 (TSP-1) were upregulated by 18- and 105-fold, respectively. Neutralization studies confirmed the immunoregulatory roles of TGF-ß3 and TSP-1 in macrophage activation and polarization. CONCLUSION: These results indicated that DFSCs can reprogram macrophages into the anti-inflammatory M2 phenotype, the paracrine factors TGF-ß3 and TSP-1 may be one of the underlying mechanisms. This study supports the hypothesis that DFSCs are promising for MSC-based immunotherapy.

Reevaluation of Pluripotent Cytokine TGF-ß3 in Immunity.

Transforming growth factor (TGF)-ßs are pluripotent cytokines with stimulatory and inhibitory properties for multiple types of immune cells. Analyses of genetic knockouts of each isoform of TGF-ß have revealed differing expression patterns and distinct roles for the three mammalian isoforms of TGF-ß. Considerable effort has been focused on understanding the molecular mechanisms of TGF-ß1-mediated immune regulation, given its pivotal role in prohibiting systemic autoimmune disease. In recent years, functional similarities and differences between the TGF-ß isoforms have delineated their distinct roles in the development of immunopathology and immune tolerance, with increased recent attention being focused on TGF-ß3. In addition to the characteristic properties of each TGF-ß isoform, recent progress has identified determinants of context-dependent functionality, including various cellular targets, cytokine concentrations, tissue microenvironments, and cytokine synergy, which combine to shape the physiological and pathophysiological roles of the TGF-ßs in immunity. Controlling TGF-ß production and signaling is being tested as a novel therapeutic strategy in multiple clinical trials for several human diseases. This review highlights advances in the understanding of the cellular sources, activation processes, contextual determinants, and immunological roles of TGF-ß3 with comparisons to other TGF-ß isoforms.

The effects of TGF-ßs on immune responses.

Transforming growth factor (TGF)-ß family is a cytokine family with various biological processes and forms a highly homologous group of three mammalian isoforms, TGF-ß1, TGF-ß2, and TGF-ß3. Most of the attention on TGF-ß family in immunology has been mainly focused on TGF-ß1 in that TGF-ß1 induces anti-inflammatory regulatory T cells (Treg), and inflammatory T helper 17 (Th17) cells in combination with interleukin-6. Although little attention has been focused on the immunological roles of TGF-ß2 and TGF-ß3, the function of TGF-ß3 for maintaining immunological homeostasis has recently been identified such as the induction of Th17 cells and direct regulatory effects on humoral immunity. TGF-ß1 and TGF-ß3 shares similar anti-inflammatory or pro-inflammatory functions, but exhibits significantly different effects on fibrosis and chondrogenesis. For the clinical application of TGF-ßs, the mechanisms by which each TGF-ß isoform regulates immunity has to be elucidated. In this review, we provide an overview of the effects, cellular targets, and therapeutic potential of TGF-ßs on immune responses and autoimmune diseases.

Role of TGF-ß3 in the regulation of immune responses.

Transforming growth factor-betas (TGF-ßs) are multifunctional cytokines that have been implicated in the regulation of a broad range of biological processes, including cell proliferation, cell survival, and cell differentiation. The three isoforms identified in mammals, TGF-ß1, TGF-ß2, and TGF-ß3, have high sequence homology, bind to the same receptors, and show similar biological functions in many in vitro studies. However, analysis of the in vivo functions of the three isoforms and mice deficient for each cytokine reveals striking differences, illustrating their unique biological importance and functional non-redundancy. Although increasing evidence suggests that TGF-ß1 and, to a lesser extent, TGF-ß2 play an integral role in maintaining immune tolerance, the immunological role of TGF-ß3 has not been carefully evaluated. Recent studies have focused on the multifunctional role of TGF-ß3. In this review, we provide an overview of the role of TGF-ß3 in immunity, with comparison to TGF-ß1 and -ß2.

Lyn mitigates mouse airway remodeling by downregulating the TGF-ß3 isoform in house dust mite models.

Chronic airway remodeling is a serious consequence of asthma, which is caused by complex but largely unknown mechanisms. Despite versatile functions, the role of Lyn in chronic airway remodeling remains undefined. Using Lyn(-/-) mice, we show that continual exposure (for 8 wk) of house dust mite extracts induced a severe phenotype of chronic airway remodeling, including exacerbated mucus production, collagen deposition, dysregulated cytokine secretion, and elevated inflammation. Strikingly, a significant increase in TGF-ß3 rather than TGF-ß1 was observed in Lyn(-/-) mouse lungs compared with lungs in wild-type mice. Furthermore, TGF-ß3 neutralizing Abs not only inhibited the expression of STAT6 and Smad2/3 but also decreased phosphorylation of Smad2 and NF-κB in Lyn(-/-) mouse lungs. In addition, both recombinant and adenoviral TGF-ß3 significantly promoted epithelial-to-mesenchymal transition and intensified collagen I production and MUC5AC expression. Further examination of chronic asthma patients showed that a decreased Lyn correlated with the severity of airway inflammation and mucus hypersecretion. Finally, Lyn may critically regulate airway remodeling by directly interacting with TGF-ß3. Collectively, these findings revealed that Lyn regulates TGF-ß3 isoform and modulates the development of airway remodeling, which may have therapeutic implications for severe chronic asthma.

Transforming growth factor Beta 3 is required for excisional wound repair in vivo.

Wound healing is a complex process that relies on proper levels of cytokines and growth factors to successfully repair the tissue. Of particular interest are the members of the transforming growth factor family. There are three TGF-ß isoforms-TGF- ß 1, 2, and 3, each isoform showing a unique expression pattern, suggesting that they each play a distinct function during development and repair. Previous studies reported an exclusive role for TGF-ß 3 in orofacial development and a potent anti-scarring effect. However, the role of TGF- ß 3 in excisional wound healing and keratinocyte migration remains poorly understood. We tested the effect of TGF-ß 3 levels on excisional cutaneous wounds in the adult mouse by directly injecting recombinant TGF-ß 3 or neutralizing antibody against TGF-ß 3 (NAB) in the wounds. Our results demonstrate that TGF-ß 3 does not promote epithelialization. However, TGF-ß 3 is necessary for wound closure as wounds injected with neutralizing antibody against TGF-ß 3 showed increased epidermal volume and proliferation in conjunction with a delay in keratinocyte migration. Wild type keratinocytes treated with NAB and Tgfb3-deficient keratinocytes closed an in vitro scratch wound with no delay, suggesting that our in vivo observations likely result from a paracrine effect.

Induction and molecular signature of pathogenic TH17 cells.

Interleukin 17 (IL-17)-producing helper T cells (T(H)17 cells) are often present at the sites of tissue inflammation in autoimmune diseases, which has led to the conclusion that T(H)17 cells are main drivers of autoimmune tissue injury. However, not all T(H)17 cells are pathogenic; in fact, T(H)17 cells generated with transforming growth factor-ß1 (TGF-ß1) and IL-6 produce IL-17 but do not readily induce autoimmune disease without further exposure to IL-23. Here we found that the production of TGF-ß3 by developing T(H)17 cells was dependent on IL-23, which together with IL-6 induced very pathogenic T(H)17 cells. Moreover, TGF-ß3-induced T(H)17 cells were functionally and molecularly distinct from TGF-ß1-induced T(H)17 cells and had a molecular signature that defined pathogenic effector T(H)17 cells in autoimmune disease.

TRIM28 prevents autoinflammatory T cell development in vivo.

TRIM28 is a component of heterochromatin complexes whose function in the immune system is unknown. By studying mice with conditional T cell-specific deletion of TRIM28 (CKO mice), we found that TRIM28 was phosphorylated after stimulation via the T cell antigen receptor (TCR) and was involved in the global regulation of CD4(+) T cells. The CKO mice had a spontaneous autoimmune phenotype that was due in part to early lymphopenia associated with a defect in the production of interleukin 2 (IL-2) as well as incomplete cell-cycle progression of their T cells. In addition, CKO T cells showed derepression of the cytokine TGF-ß3, which resulted in an altered cytokine balance; this caused the accumulation of autoreactive cells of the T(H)17 subset of helper T cells and of Foxp3(+) T cells. Notably, CKO Foxp3(+) T cells were unable to prevent the autoimmune phenotype in vivo. Our results show critical roles for TRIM28 in both T cell activation and T cell tolerance.

TGF ß 3 immunoassay standardization: comparison of NIBSC reference preparation code 98/608 with avotermin lot 205-0505-005.

Juvista™ drug product contains human recombinant active transforming growth factor beta 3 (TGFß3; avotermin). Juvista is being developed for the prevention and reduction of human scarring. Phase II and III clinical and development batches of Juvista were assayed for content by an immunoenzymometric assay (IEMA) using a National Institute for Biological Standards and Control (NIBSC) TGFß3 reference material (98/608) and avotermin standard (Lot 205-0505-005). Paired Juvista TGFß3 data were compared directly, pooled, and processed using the statistical analysis described by Bland and Altman. A direct comparison of the two standards was also made. The Bland-Altman result was 1.958, the best estimate of the relationship between Lot 205-0505-005 and reference material 98/608. By IEMA, reference material 98/608 has approximately 50% of the immunoreactivity of Lot 205-0505-005. During clinical development, no change in Juvista TGFß3 dosage was made, but the standard used for Juvista TGFß3 assay was changed from 98/608 to 205-0505-005. The stated amount of Juvista TGFß3 in phase III trials was approximately one-half of that in phase II trials. This article highlights the importance of early adoption of an appropriate and representative standard to achieve accurate quantification of protein drug during clinical development.

Characterization of OP9 as authentic mesenchymal stem cell line.

Mesenchymal stem cells (MSCs) are multipotent stem cells capable of differentiating into various cell types, including osteocytes, chondrocytes, adipocytes, myocytes, and tenocytes. However, the difficulty or failure in expanding the mouse MSCs in vitro greatly hampered important research in animal models. The OP9, a stromal cell line from mouse bone marrow, has hematopoietic supportive capacity. Here, we report that the OP9 has the immunophenotype (CD45(-), CD11b(-), FLK-1(-), CD31(-), CD34(-), CD44(+), CD29(+), Sca-1(+), CD86(-), and MHCII(-)) identical to canonical mouse MSCs. The expression of CD140a(+), CD140b(+), alpha-SMA(+) and Calponin(+) suggested the perivascular origin of OP9. Functionally, the OP9 had strong clonogenic ability and could be induced into osteocytes, chondrocytes and adipocytes. The lymphocyte transformation test (LTT) and mixed leukocyte reaction (MLR) showed that the OP9 could suppress T lymphocyte proliferation stimulated by nonspecific mitogens (PHA) or allogeneic lymphocytes (BALB/c T cells). Finally, the migration of OP9 could be efficiently induced by bFGF, IGF-1, IL-3, PDGF-BB, TGF-beta1 and TGF-beta3. In conclusion, the OP9 were bona fide MSCs, and such homogenous cell line will be helpful to delineate biological features of MSCs at the stem cell level.

Key roles for transforming growth factor beta in melanocyte stem cell maintenance.

Melanocyte stem cells in the bulge area of hair follicles are responsible for hair pigmentation, and defects in them cause hair graying. Here we describe the process of melanocyte stem cell entry into the quiescent state and show that niche-derived transforming growth factor beta (TGF-beta) signaling plays important roles in this process. In vitro, TGF-beta not only induces reversible cell cycle arrest, but also promotes melanocyte immaturity by downregulating MITF, the master transcriptional regulator of melanocyte differentiation, and its downstream melanogenic genes. In vivo, TGF-beta signaling is activated in melanocyte stem cells when they reenter the quiescent noncycling state during the hair cycle and this process requires Bcl2 for cell survival. Furthermore, targeted TGF-beta type II receptor (TGFbRII) deficiency in the melanocyte lineage causes incomplete maintenance of melanocyte stem cell immaturity and results in mild hair graying. These data demonstrate that the TGF-beta signaling pathway is one of the key niche factors that regulate melanocyte stem cell immaturity and quiescence.

Mice that lack activity of alphavbeta6- and alphavbeta8-integrins reproduce the abnormalities of Tgfb1- and Tgfb3-null mice.

The arginine-glycine-aspartate (RGD)-binding integrins alphavbeta6 and alphavbeta8 activate latent TGFbeta1 and TGFbeta3 in vivo, but it is uncertain whether other RGD-binding integrins such as integrins alphavbeta5 and alphavbeta3 activate these TGFbeta isoforms. To define the combined role of alphavbeta6- and alphavbeta8-integrin in TGFbeta activation, we analyzed mice lacking function of both integrins by means of gene deletion and/or pharmacologic inhibition. Most Itgb6-/-;Itgb8-/- embryos die at mid-gestation; those that survive develop cleft palate-as observed in Tgfb3-/- mice. Itgb8-/- mice treated with an anti-alphavbeta6-integrin antibody develop severe autoimmunity and lack Langerhans cells-similar to Tgfb1-null mice. These results support a model in which TGFbeta3-mediated palate fusion and TGFbeta1-mediated suppression of autoimmunity and generation of Langerhans cells require integrins alphavbeta6 and alphavbeta8 but not other RGD-binding integrins as TGFbeta activators.

Prevention of spontaneous abortion in the CBA x DBA/2 mouse model by intravaginal TGF-beta and local recruitment of CD4+8+ FOXP3+ cells.

PROBLEM: Activation of latent transforming growth factor (TGF)-beta in seminal plasma has been suggested by Robertson et al. to promote maternal tolerance to paternal antigens. A possible consequence reported by Tremellen et al. is increased pregnancy rates in women undergoing IVF. A decreased spontaneous abortion rate has also been postulated. Seminal plasma contains many factors besides TGF-beta, and a critical test of the hypothesis was required. The purpose of the present study was to directly test the effect of pure TGF-beta. METHOD OF STUDY: Pharmaceutical grade bioactive TGF-beta3 with a bovine serum albumin (BSA) carrier 0.1-1% in phosphate-buffered saline (PBS) was given into the vaginal tract of CBA/J female mice at the time of mating with DBA/2 males. One microgram Salmonella enteritidis lipopolysaccharide was given intraperitoneally to augment occult losses and spontaneous resorptions assessed on day 13.5 of pregnancy. The effect of TGF-beta3 on recruitment of lymphomyeloid cells to the vaginal wall and vaginal lumen of unmated mice in estrus was assessed using immunohistochemistry and flow cytometry. RESULTS: Two nanogram of intravaginal TGF-beta3 in 0.1% BSA-PBS or 20 ng in 1% BSA-PBS reduced abortion rates. Protection was comparable to that achieved by immunization with BALB/c spleen cells. Fraction V BSA, a binder of TGF-betas, had some activity, and could reduce availability of added TGF-beta3. CD11c dendritic cells, CD3+ T cells, and CD25+ cells were recruited to the vaginal wall by 48 hr after TGF-beta3 treatment, and cellularity of vaginal exudates increased. Foxp3+ cells were present in increased numbers, and appeared to be CD8+ and CD4+ 8+. Semen, but not TGF-beta3, stimulated a physiological polymorphonuclear leukocyte exudate. CONCLUSION: Intravaginal bioactive TGF-beta3 can enhance success of pregnancy in vivo in an established model of abortion. The result could be explained by the independent ability of TGF-beta to promote a regulatory T-cell response.