Spatial Distribution of Non-Immune Cells Expressing Glycoprotein A Repetitions Predominant in Human and Murine Metastatic Lymph Nodes.

Several types of cancer spread through the lymphatic system via the sentinel lymph nodes (LNs). Such LN-draining primary tumors, modified by tumor factors, lead to the formation of a metastatic niche associated with an increased number of Foxp3+ regulatory T cells (Tregs). These cells are expected to contribute to the elaboration of an immune-suppressive environment. Activated Tregs express glycoprotein A repetitions predominant (GARP), which binds and presents latent transforming growth factor beta 1 (TGF-ß1) at their surface. GARP is also expressed by other non-immune cell types poorly described in LNs. Here, we mapped GARP expression in non-immune cells in human and mouse metastatic LNs. The mining of available (human and murine) scRNA-Seq datasets revealed GARP expression by blood (BEC)/lymphatic (LEC) endothelial, fibroblastic, and perivascular cells. Consistently, through immunostaining and in situ RNA hybridization approaches, GARP was detected in and around blood and lymphatic vessels, in (αSMA+) fibroblasts, and in perivascular cells associated with an abundant matrix. Strikingly, GARP was detected in LECs forming the subcapsular sinus and high endothelial venules (HEVs), two vascular structures localized at the interface between LNs and the afferent lymphatic and blood vessels. Altogether, we here provide the first distribution maps for GARP in human and murine LNs.

Diagnosing borderline personality disorder: Reports and recommendations from people with lived experience.

Borderline personality disorder (BPD) is a severe mental health condition marked by impairments in self and interpersonal functioning. Stigma from health staff may often result in a reluctance to diagnose, impacting recovery trajectories. Qualitative interviews were conducted with participants (N = 15; M Age = 36.4 years, SD = 7.5; 93.3% female) with lived experience of BPD exploring topics of illness onset, insight, experience of diagnosis and treatment. Qualitative responses were analysed within a co-design framework with a member of the research team who identifies as having a lived experience of BPD. On average, participant symptoms emerged at 12.1 years of age (SD = 6.6 years, range 1.5-27), but diagnoses of BPD were delayed until 30.2 years (SD = 7.8 years, range 18-44) resulting in a 'diagnosis gap' of 18.1 years (SD = 9.6 years, range 3-30). Participant explanations for BPD emergence varied from biological, psychological and social factors. Benefits of diagnosis (e.g., fostering insight, aiding treatment planning and reducing isolation) were contrasted with challenges (e.g., stigma and treatment unavailability). Delay in diagnosis was common, and no participants reported receiving a diagnosis of BPD during their adolescence yet 85% felt they would have benefited from a diagnosis in adolescence. Only a quarter (27%) felt highly supported in the diagnostic process. An ideal four-step diagnosis procedure was outlined based on recommendations from participants with a lived experience; this involved the following: (1) explain the process, (2) assess thoroughly, (3) explore how the features are active in everyday life and (4) link diagnosis to evidence-based treatment planning.

p16Ink4a, a marker of cellular senescence, is associated with renal disease in the B6.NZMSle1/Sle2/Sle3 mouse model of lupus.

OBJECTIVES: Despite treatment, one-third of patients with lupus nephritis (LN) show a decline in renal function. Prognostic markers of poor outcome as well as novel therapeutic targets are therefore highly sought. We showed that p16INK4a, a marker of cellular senescence, is observed in baseline kidney biopsies from patients with LN, and is associated with renal disease. Here, we set out to assess for whether these findings are recapitulated in the B6.NZMSle1/Sle2/Sle3 (B6.Sle1.2.3) mouse model of spontaneous lupus. METHODS: We evaluated the occurrence and time of onset of p16Ink4a staining by immunohistochemistry on kidney sections, and tested for its association with multiple renal and systemic disease parameters, fibrosis and CD8+ T cell infiltration, in two cohorts of B6.Sle1.2.3 mice. RESULTS: The presence of p16Ink4a-positive cells in kidney was significantly associated with increased urine albumin/creatinine ratio, histopathological scores, CD8+ T cell infiltration and fibrosis, in both B6.Sle1.2.3 cohorts. In contrast, p16Ink4a staining was not associated with systemic disease parameters. A time course showed that systemic disease parameters as well as glomerular IgG deposits appeared in B6.Sle1.2.3 mice by 4 months of age; the appearance of p16Ink4a-positive cells occurred later, by 8 months of age, overlapping with renal disease. CONCLUSION: We report, for the first time, the presence of p16Ink4a-positive cells, a marker of cellular senescence, in the B6.Sle1.2.3 kidney, and their association with renal disease severity. This provides a preclinical model in which to test for the role of cellular senescence in the pathogenesis of LN, as a potential kidney-intrinsic disease mechanism.

Therapeutic activity of GARP:TGF-ß1 blockade in murine primary myelofibrosis.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by the clonal expansion of myeloid cells, notably megakaryocytes (MKs), and an aberrant cytokine production leading to bone marrow (BM) fibrosis and insufficiency. Current treatment options are limited. TGF-ß1, a profibrotic and immunosuppressive cytokine, is involved in PMF pathogenesis. While all cell types secrete inactive, latent TGF-ß1, only a few activate the cytokine via cell type-specific mechanisms. The cellular source of the active TGF-ß1 implicated in PMF is not known. Transmembrane protein GARP binds and activates latent TGF-ß1 on the surface of regulatory T lymphocytes (Tregs) and MKs or platelets. Here, we found an increased expression of GARP in the BM and spleen of mice with PMF and tested the therapeutic potential of a monoclonal antibody (mAb) that blocks TGF-ß1 activation by GARP-expressing cells. GARP:TGF-ß1 blockade reduced not only fibrosis but also the clonal expansion of transformed cells. Using mice carrying a genetic deletion of Garp in either Tregs or MKs, we found that the therapeutic effects of GARP:TGF-ß1 blockade in PMF imply targeting GARP on Tregs. These therapeutic effects, accompanied by increased IFN-γ signals in the spleen, were lost upon CD8 T-cell depletion. Our results suggest that the selective blockade of TGF-ß1 activation by GARP-expressing Tregs increases a CD8 T-cell-mediated immune reaction that limits transformed cell expansion, providing a novel approach that could be tested to treat patients with myeloproliferative neoplasms.

BRAFV600E Expression in Thyrocytes Causes Recruitment of Immunosuppressive STABILIN-1 Macrophages.

Papillary thyroid carcinoma (PTC) is the most frequent histological subtype of thyroid cancers (TC), and BRAFV600E genetic alteration is found in 60% of this endocrine cancer. This oncogene is associated with poor prognosis, resistance to radioiodine therapy, and tumor progression. Histological follow-up by anatomo-pathologists revealed that two-thirds of surgically-removed thyroids do not present malignant lesions. Thus, continued fundamental research into the molecular mechanisms of TC downstream of BRAFV600E remains central to better understanding the clinical behavior of these tumors. To study PTC, we used a mouse model in which expression of BRAFV600E was specifically switched on in thyrocytes by doxycycline administration. Upon daily intraperitoneal doxycycline injection, thyroid tissue rapidly acquired histological features mimicking human PTC. Transcriptomic analysis revealed major changes in immune signaling pathways upon BRAFV600E induction. Multiplex immunofluorescence confirmed the abundant recruitment of macrophages, among which a population of LYVE-1+/CD206+/STABILIN-1+ was dramatically increased. By genetically inactivating the gene coding for the scavenger receptor STABILIN-1, we showed an increase of CD8+ T cells in this in situ BRAFV600E-dependent TC. Lastly, we demonstrated the presence of CD206+/STABILIN-1+ macrophages in human thyroid pathologies. Altogether, we revealed the recruitment of immunosuppressive STABILIN-1 macrophages in a PTC mouse model and the interest to further study this macrophage subpopulation in human thyroid tissues.

Blocking GARP-mediated activation of TGF-ß1 did not alter innate or adaptive immune responses to bacterial infection or protein immunization in mice.

Transmembrane protein GARP binds latent TGF-ß1 to form GARP:(latent)TGF-ß1 complexes on the surface of several cell types including Tregs, B-cells, and platelets. Upon stimulation, these cells release active TGF-ß1. Blocking TGF-ß1 activation by Tregs with anti-GARP:TGF-ß1 mAbs overcomes resistance to PD1/PD-L1 blockade and induces immune-mediated regressions of murine tumors, indicating that Treg-derived TGF-ß1 inhibits anti-tumor immunity. TGF-ß1 exerts a vast array of effects on immune responses. For example, it favors differentiation of TH17 cells and B-cell switch to IgA production, two important processes for mucosal immunity. Here, we sought to determine whether treatment with anti-GARP:TGF-ß1 mAbs would perturb immune responses to intestinal bacterial infection. We observed no aggravation of intestinal disease, no systemic dissemination, and no alteration of innate or adaptative immune responses upon oral gavage of C. rodentium in highly susceptible Il22r-/- mice treated with anti-GARP:TGF-ß1 mAbs. To examine the effects of GARP:TGF-ß1 blockade on Ig production, we compared B cell- and TH cell- responses to OVA or CTB protein immunization in mice carrying deletions of Garp in Tregs, B cells, or platelets. No alteration of adaptive immune responses to protein immunization was observed in the absence of GARP on any of these cells. Altogether, we show that antibody-mediated blockade of GARP:TGF-ß1 or genetic deletion of Garp in Tregs, B cells or platelets, do not alter innate or adaptive immune responses to intestinal bacterial infection or protein immunization in mice. Anti-GARP:TGF-ß1 mAbs, currently tested for cancer immunotherapy, may thus restore anti-tumor immunity without severely impairing other immune defenses. PRéCIS: Immunotherapy with GARP:TGF-ß1 mAbs may restore anti-tumor immunity without impairing immune or inflammatory responses required to maintain homeostasis or host defense against infection, notably at mucosal barriers.

Combined Blockade of GARP:TGF-ß1 and PD-1 Increases Infiltration of T Cells and Density of Pericyte-Covered GARP+ Blood Vessels in Mouse MC38 Tumors.

When combined with anti-PD-1, monoclonal antibodies (mAbs) against GARP:TGF-ß1 complexes induced more frequent immune-mediated rejections of CT26 and MC38 murine tumors than anti-PD-1 alone. In both types of tumors, the activity of anti-GARP:TGF-ß1 mAbs resulted from blocking active TGF-ß1 production and immunosuppression by GARP-expressing regulatory T cells. In CT26 tumors, combined GARP:TGF-ß1/PD-1 blockade did not augment the infiltration of T cells, but did increase the effector functions of already present anti-tumor T cells. Here we show that, in contrast, in MC38, combined GARP:TGF-ß1/PD-1 blockade increased infiltration of T cells, as a result of increased extravasation of T cells from blood vessels. Unexpectedly, combined GARP:TGF-ß1/PD-1 blockade also increased the density of GARP+ blood vessels covered by pericytes in MC38, but not in CT26 tumors. This appears to occur because anti-GARP:TGF-ß1, by blocking TGF-ß1 signals, favors the proliferation of and expression of adhesion molecules such as E-selectin by blood endothelial cells. The resulting densification of intratumoral blood vasculature probably contributes to increased T cell infiltration and to the therapeutic efficacy of GARP:TGF-ß1/PD-1 blockade in MC38. We conclude from these distinct observations in MC38 and CT26, that the combined blockades of GARP:TGF-ß1 and PD-1 can exert anti-tumor activity via multiple mechanisms, including the densification and normalization of intratumoral blood vasculature, the increase of T cell infiltration into the tumor and the increase of the effector functions of intratumoral tumor-specific T cells. This may prove important for the selection of cancer patients who could benefit from combined GARP:TGF-ß1/PD-1 blockade in the clinics.

Functional Th1-oriented T follicular helper cells that infiltrate human breast cancer promote effective adaptive immunity.

We previously demonstrated that tumor-infiltrating lymphocytes (TIL) in human breast cancer sometimes form organized tertiary lymphoid structures (TLS) characterized by CXCL13-producing T follicular helper (Tfh) cells. The present study found that CD4+ Tfh TIL, CD8+ TIL, and TIL-B, colocalizing in TLS, all express the CXCL13 receptor CXCR5. An ex vivo functional assay determined that only activated, functional Th1-oriented Tfh TIL (PD-1hiICOSint phenotype) provide help for immunoglobulin and IFN-γ production. A functional Tfh TIL presence signals an active TLS, characterized by humoral (immunoglobulins, Ki-67+ TIL-B in active germinal centers) and cytotoxic (GZMB+CD8+ and GZMB+CD68+ TIL plus Th1 gene expression) immune responses. Analysis of active versus inactive TLS in untreated patients revealed that the former are associated with positive clinical outcomes. TLS also contain functional T follicular regulatory (Tfr) TIL, which are characterized by a CD25+CXCR5+GARP+FOXP3+ phenotype and a demethylated FOXP3 gene. Functional Tfr inhibited functional Tfh activities via a glycoprotein A repetitions predominant (GARP)-associated TGF-ß-dependent mechanism. The activity of tumor-associated TLS was dictated by the relative balance between functional Tfh TIL and functional Tfr TIL. These data provide mechanistic insight into TLS processes orchestrated by functional Th1-oriented Tfh TIL, including TIL-B and CD8+ TIL activation and immunological memory generation. Tfh TIL, regulated by functional Tfr TIL, are an expected key target of PD-1/PD-L1 blockade.

Targeting immunosuppression by TGF-ß1 for cancer immunotherapy.

The TGF-ß1 cytokine is a key mediator of many biological processes. Complex regulatory mechanisms are in place that allow one single molecule to exert so many distinct indispensable activities. The complexity of TGF-ß1 biology is further illustrated by the opposing dual roles it plays during cancer progression. Risks of toxicities combined with lack of convincing therapeutical efficacy explain at least in part why therapies targeting TGF-ß1 have lagged behind in past decades. However, recent successes of immunostimulatory antibodies for the immunotherapy of cancer and findings that TGF-ß1 activity associates with resistance to immunotherapeutic drugs have revived the field. In this review, we discuss the biology of TGF-ß1 with a special focus on its roles in regulating immune responses in the context of cancer. We describe the various therapeutic approaches available to inhibit TGF-ß signalling, and more recent findings that allow selective targeting of specific sources of TGF-ß activity, which may prove relevant to increase the efficacy and reduce the toxicity of cancer immunotherapy.

TGFß drives NK cell metabolic dysfunction in human metastatic breast cancer.

BACKGROUND: Natural killer (NK) cells provide important immune protection from cancer and are a key requirement for particular immunotherapies. There is accumulating evidence that NK cells become dysfunctional during cancer. Overcoming NK cell exhaustion would be an important step to allow them to function optimally in a range of NK cell therapies, including those that depend on autologos circulating NK cells. We have previously demonstrated that NK cells undergo a normal metabolic reprogramming in response to cytokine activation and that this is required for optimal function. The objective of this work was to investigate if cellular metabolism of circulating NK cells is dysregulated in patients with metastatic breast cancer and if so, to gain insights into potential mechanisms underpinning this. Such discoveries would provide important insights into how to unleash the full activity of NK cells for maximum immunotherapy output. METHODS: Single-cell analysis, metabolic flux and confocal analysis of NK cells from patients with metastatic breast cancer and healthy controls RESULTS: In addition to reduced interferon-γ production and cytotoxicity, peripheral blood NK cells from patients had clear metabolic deficits including reduced glycolysis and oxidative phosphorylation. There were also distinct morphologically alterations in the mitochondria with increased mitochondrial fragmentation observed. Transforminggrowth factor-ß (TGFß) was identified as a key driver of this phenotype as blocking its activity reversed many metabolic and functional readouts. Expression of glycoprotein-A repetitions predominant (GARP) and latency associated peptide (LAP), which are involved with a novel TGFß processing pathway, was increased on NK cells from some patients. Blocking the GARP-TGFß axis recapitulated the effects of TGFß neutralization, highlighting GARP as a novel NK cell immunotherapy target for the first time. CONCLUSIONS: TGFß contributes to metabolic dysfunction of circulating NK cells in patients with metastatic breast cancer. Blocking TGFß and/or GARP can restore NK cell metabolism and function and is an important target for improving NK cell-based immunotherapies.

Selective inhibition of TGF-ß1 produced by GARP-expressing Tregs overcomes resistance to PD-1/PD-L1 blockade in cancer.

TGF-ß1, ß2 and ß3 bind a common receptor to exert vastly diverse effects in cancer, supporting either tumor progression by favoring metastases and inhibiting anti-tumor immunity, or tumor suppression by inhibiting malignant cell proliferation. Global TGF-ß inhibition thus bears the risk of undesired tumor-promoting effects. We show that selective blockade of TGF-ß1 production by Tregs with antibodies against GARP:TGF-ß1 complexes induces regressions of mouse tumors otherwise resistant to anti-PD-1 immunotherapy. Effects of combined GARP:TGF-ß1/PD-1 blockade are immune-mediated, do not require FcγR-dependent functions and increase effector functions of anti-tumor CD8+ T cells without augmenting immune cell infiltration or depleting Tregs within tumors. We find GARP-expressing Tregs and evidence that they produce TGF-ß1 in one third of human melanoma metastases. Our results suggest that anti-GARP:TGF-ß1 mAbs, by selectively blocking a single TGF-ß isoform emanating from a restricted cellular source exerting tumor-promoting activity, may overcome resistance to PD-1/PD-L1 blockade in patients with cancer.

The importance of naturally attenuated SARS-CoV-2in the fight against COVID-19.

The current SARS-CoV-2 pandemic is wreaking havoc throughout the world and has rapidly become a global health emergency. A central question concerning COVID-19 is why some individuals become sick and others not. Many have pointed already at variation in risk factors between individuals. However, the variable outcome of SARS-CoV-2 infections may, at least in part, be due also to differences between the viral subspecies with which individuals are infected. A more pertinent question is how we are to overcome the current pandemic. A vaccine against SARS-CoV-2 would offer significant relief, although vaccine developers have warned that design, testing and production of vaccines may take a year if not longer. Vaccines are based on a handful of different designs (i), but the earliest vaccines were based on the live, attenuated virus. As has been the case for other viruses during earlier pandemics, SARS-CoV-2 will mutate and may naturally attenuate over time (ii). What makes the current pandemic unique is that, thanks to state-of-the-art nucleic acid sequencing technologies, we can follow in detail how SARS-CoV-2 evolves while it spreads. We argue that knowledge of naturally emerging attenuated SARS-CoV-2 variants across the globe should be of key interest in our fight against the pandemic.

Combination of immune checkpoint blockade with DNA cancer vaccine induces potent antitumor immunity against P815 mastocytoma.

DNA vaccination against cancer has become a promising strategy for inducing a specific and long-lasting antitumor immunity. However, DNA vaccines fail to generate potent immune responses when used as a single therapy. To enhance their activity into the tumor, a DNA vaccine against murine P815 mastocytoma was combined with antibodies directed against the immune checkpoints CTLA4 and PD1. The combination of these two strategies delayed tumor growth and enhanced specific antitumor immune cell infiltration in comparison to the corresponding single therapies. The combination also promoted IFNg, IL12 and granzyme B production in the tumor microenvironment and decreased the formation of liver metastasis in a very early phase of tumor development, enabling 90% survival. These results underline the complementarity of DNA vaccination and immune checkpoint blockers in inducing a potent immune response, by exploiting the generation of antigen-specific T cells by the vaccine and the ability of immune checkpoint blockers to enhance T cell activity and infiltration in the tumor. These findings suggest how and why a rational combination therapy can overcome the limits of DNA vaccination but could also allow responses to immune checkpoint blockers in a larger proportion of subjects.

Structural basis of latent TGF-ß1 presentation and activation by GARP on human regulatory T cells.

Transforming growth factor-ß1 (TGF-ß1) is one of very few cytokines produced in a latent form, requiring activation to exert any of its vastly diverse effects on development, immunity, and cancer. Regulatory T cells (Tregs) suppress immune cells within close proximity by activating latent TGF-ß1 presented by GARP (glycoprotein A repetitions predominant) to integrin αVß8 on their surface. We solved the crystal structure of GARP:latent TGF-ß1 bound to an antibody that stabilizes the complex and blocks release of active TGF-ß1. This finding reveals how GARP exploits an unusual medley of interactions, including fold complementation by the amino terminus of TGF-ß1, to chaperone and orient the cytokine for binding and activation by αVß8. Thus, this work further elucidates the mechanism of antibody-mediated blockade of TGF-ß1 activation and immunosuppression by Tregs.

Activation of latent transforming growth factor-ß1, a conserved function for pregnancy-specific beta 1-glycoproteins.

STUDY QUESTION: Do all 10 human pregnancy-specific beta 1-glycoproteins (PSGs) and murine PSG23 activate latent transforming growth factor-ß1 (TGF-ß1)? SUMMARY ANSWER: All human PSGs and murine PSG23 activated latent TGF-ß1. WHAT IS KNOWN ALREADY: Two of the 10 members of the PSG1 family, PSG1 and PSG9, were previously shown to activate the soluble small latent complex of TGF-ß1, a cytokine with potent immune suppressive functions. STUDY DESIGN, SIZE, DURATION: Recombinant PSGs were generated and tested for their ability to activate the small latent complex of TGF-ß1 in a cell-free ELISA-based assay and in a bioassay. In addition, we tested the ability of PSG1 and PSG4 to activate latent TGF-ß bound to the extracellular matrix (ECM) or on the membranes of the Jurkat human T-cell line. PARTICIPANTS/MATERIALS, SETTING, METHODS: Recombinant PSGs were generated by transient transfection and purified with a His-Trap column followed by gel filtration chromatography. The purified PSGs were compared to vehicle (PBS) used as control for their ability to activate the small latent complex of TGF-ß1. The concentration of active TGF-ß was measured in an ELISA using the TGF-ß receptor II as capture and a bioassay using transformed mink epithelial cells that express luciferase in response to active TGF-ß. The specificity of the signal was confirmed using a TGF-ß receptor inhibitor. We also measured the binding kinetics of some human PSGs for the latent-associated peptide (LAP) of TGF-ß using surface plasmon resonance and determined whether PSG1 and PSG4 could activate the large latent complex of TGF-ß1 bound to the ECM and latent TGF-ß1 bound to the cell membrane. All experiments were performed in triplicate wells and repeated three times. MAIN RESULTS AND THE ROLE OF CHANCE: All human PSGs activated the small latent complex of TGF-ß1 (P < 0.05 vs. control) and showed similar affinities (KD) for LAP. Despite the lack of sequence conservation with its human counterparts, the ability to activate latent TGF-ß1 was shared by a member of the murine PSG family. We found that PSG1 and PSG4 activated the latent TGF-ß stored in the ECM (P < 0.01) but did not activate latent TGF-ß1 bound to glycoprotein A repetitions predominant (GARP) on the surface of Jurkat T cells. LIMITATIONS, REASONS FOR CAUTION: The affinity of the interaction of LAP and PSGs was calculated using recombinant proteins, which may differ from the native proteins in their post-translational modifications. We also utilized a truncated form of murine PSG23 rather than the full-length protein. For the studies testing the ability of PSGs to activate membrane-bound TGF-ß1, we utilized the T-cell line Jurkat and Jurkat cells expressing GARP rather than primary T regulatory cells. All the studies were performed in vitro. WIDER IMPLICATIONS OF THE FINDINGS: Here, we show that all human PSGs activate TGF-ß1 and that this function is conserved in at least one member of the rodent PSG family. In vivo PSGs could potentially increase the availability of active TGF-ß1 from the soluble and matrix-bound latent forms of the cytokine contributing to the establishment of a tolerogenic environment during pregnancy. LARGE-SCALE DATA: None. STUDY FUNDING/COMPETING INTEREST(S): The research was supported by a grant from the Collaborative Health Initiative Research Program (CHIRP). No conflicts of interests are declared by the authors.

Regulatory T cells control toxicity in a humanized model of IL-2 therapy.

While patient selection and clinical management have reduced high-dose IL-2 (HDIL2) immunotherapy toxicities, the immune mechanisms that underlie HDIL2-induced morbidity remain unclear. Here we show that dose-dependent morbidity and mortality of IL-2 immunotherapy can be modeled in human immune system (HIS) mice. Depletion of human T cell subsets during the HDIL2 treatment reduces toxicity, pointing to the central function of T cells. Preferential expansion of effector T cells secondary to defective suppressive capacity of regulatory T (Treg) cells after HDIL2 therapy further underscores the importance of Treg in the maintenance of immune tolerance. IL-2 toxicity is induced by selective depletion or inhibition of Treg after LDIL2 therapy, and is ameliorated in HDIL2-treated HIS mice receiving the PIM-1 kinase inhibitor, Kaempferol. Modeling IL-2 pathophysiology in HIS mice offers a means to understand the functions of effector and regulatory T cells in immune-mediated toxicities associated with cancer immunotherapy.

Blocking immunosuppression by human Tregs in vivo with antibodies targeting integrin αVß8.

Human regulatory T cells (Tregs) suppress other T cells by converting the latent, inactive form of TGF-ß1 into active TGF-ß1. In Tregs, TGF-ß1 activation requires GARP, a transmembrane protein that binds and presents latent TGF-ß1 on the surface of Tregs stimulated through their T cell receptor. However, GARP is not sufficient because transduction of GARP in non-Treg T cells does not induce active TGF-ß1 production. RGD-binding integrins were shown to activate TGF-ß1 in several non-T cell types. Here we show that αVß8 dimers are present on stimulated human Tregs but not in other T cells, and that antibodies against αV or ß8 subunits block TGF-ß1 activation in vitro. We also show that αV and ß8 interact with GARP/latent TGF-ß1 complexes in human Tregs. Finally, a blocking antibody against ß8 inhibited immunosuppression by human Tregs in a model of xenogeneic graft-vs.-host disease induced by the transfer of human T cells in immunodeficient mice. These results show that TGF-ß1 activation on the surface of human Tregs implies an interaction between the integrin αVß8 and GARP/latent TGF-ß1 complexes. Immunosuppression by human Tregs can be inhibited by antibodies against GARP or against the integrin ß8 subunit. Such antibodies may prove beneficial against cancer or chronic infections.

Role of GARP in the activation of latent TGF-ß1.

TGF-ß1, 2 and 3 cytokines are involved in many cellular processes including cell proliferation, differentiation, migration and survival. Whereas TGF-ß2 and 3 play important roles in embryonic development, TGF-ß1 is mostly implicated in controlling immune responses after birth. The production of TGF-ß1 is a tightly regulated process, occurring mostly at a post-translational level. Virtually all cells produce the latent, inactive form of TGF-ß1. In latent TGF-ß1, the mature TGF-ß1 dimer is non-covalently associated to the Latency Associated Peptide, or LAP, which prevents binding to the TGF-ß1 receptor. Activation of the cytokine implies release of mature TGF-ß1 from LAP. Only a few cell types activate latent TGF-ß1, via mechanisms that are cell type specific. Proteins such as integrins, proteases and thrombospondin-1 activate TGF-ß1 in epithelial cells, fibroblasts and dendritic cells. More recently, the protein GARP was shown to be involved in TGF-ß1 activation by regulatory T cells (Treg), a subset of CD4+ T lymphocytes specialized in suppression of immune responses. GARP is a transmembrane protein that binds latent-TGF-ß1 and tethers it on the Treg surface. The role of GARP was studied mostly in Tregs, and this was recently reviewed in L. Sun, H. Jin and H. Li, Oncotarget, 2016, 7, 42826-42836. However, GARP is also expressed in non-immune cells. This review focuses on the roles of GARP in latent TGF-ß1 activation by immune and non-immune cells.