Juan Dubrot

Immune tolerance. Group 3 innate lymphoid cells mediate intestinal selection of commensal bacteria-specific CD4⁺ T cells.

Innate lymphoid cells keep gut T cells in check Trillions of bacteria inhabit our guts. So do many types of immune cells, including T cells, which might be expected to attack these bacteria. How, then, do our bodies manage to keep the peace? Working in mice, Hepworth et al. report one such mechanism. A population of immune cells, called innate lymphoid cells, directly killed CD4+ T cells that react to commensal gut microbes. Some of the specifics of this process parallel how the immune system keeps developing self-reactive T cells in check in the thymus. Furthermore, this peacekeeping process may be disrupted in children with inflammatory bowel disease. Science, this issue p. 1031 Innate lymphoid cells delete commensal bacteria–specific CD4+ T cells from the intestine in mice. Inflammatory CD4+ T cell responses to self or commensal bacteria underlie the pathogenesis of autoimmunity and inflammatory bowel disease (IBD), respectively. Although selection of self-specific T cells in the thymus limits responses to mammalian tissue antigens, the mechanisms that control selection of commensal bacteria–specific T cells remain poorly understood. Here, we demonstrate that group 3 innate lymphoid cell (ILC3)–intrinsic expression of major histocompatibility complex class II (MHCII) is regulated similarly to thymic epithelial cells and that MHCII+ ILC3s directly induce cell death of activated commensal bacteria–specific T cells. Further, MHCII on colonic ILC3s was reduced in pediatric IBD patients. Collectively, these results define a selection pathway for commensal bacteria–specific CD4+ T cells in the intestine and suggest that this process is dysregulated in human IBD.

Influence of bevacizumab, sunitinib and sorafenib as single agents or in combination on the inhibitory effects of VEGF on human dendritic cell differentiation from monocytes

Vascular endothelial growth factor (VEGF) inhibits differentiation and maturation of dendritic cells (DC), suggesting a potential immunosuppressive role for this proangiogenic factor. Bevacizumab, sorafenib and sunitinib target VEGF-mediated angiogenesis and are active against several types of cancer, but their effects on the immune system are poorly understood. In this study, VEGF and supernatants of renal carcinoma cell lines cultured under hypoxia were found to alter the differentiation of human monocytes to DC. Resulting DC showed impaired activity, as assessed by the alloreactive mixed T-lymphocyte reaction. Bevacizumab and sorafenib, but not sunitinib, reversed the inhibitory effects of VEGF, but not of those mediated by tumour supernatants. Dendritic cells matured under the influence of VEGF expressed less human leukocyte antigen-DR (HLA-DR) and CD86, and this effect was restored by bevacizumab and sorafenib. Finally, tumour-cell supernatants decreased interleukin-12 (IL-12) production by mature DC, and such inhibition was not restored by any of the tested drugs, delivered either as single agents or in combination. The deleterious effects of tumour-cell supernatants were mainly mediated by thermostable molecules distinct from VEGF. These results indicate that inhibition of the differentiation of monocytes to DC is a multifactorial effect, and that they support the development of combinations of angiogenesis inhibitors with immunological modulators.

Agonist Anti-CD137 mAb Act on Tumor Endothelial Cells to Enhance Recruitment of Activated T Lymphocytes

Agonist monoclonal antibodies (mAb) to the immune costimulatory molecule CD137, also known as 4-1BB, are presently in clinical trials for cancer treatment on the basis of their costimulatory effects on primed T cells and perhaps other cells of the immune system. Here we provide evidence that CD137 is selectively expressed on the surface of tumor endothelial cells. Hypoxia upregulated CD137 on murine endothelial cells. Treatment of tumor-bearing immunocompromised Rag(-/-) mice with agonist CD137 mAb did not elicit any measurable antiangiogenic effects. In contrast, agonist mAb stimulated tumor endothelial cells, increasing cell surface expression of the adhesion molecules intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin. When adoptively transferred into mice, activated T lymphocytes derived from CD137-deficient animals entered more avidly into tumor tissue after treatment with agonist mAb. This effect could be neutralized with anti-ICAM-1 and anti-VCAM-1 blocking antibodies. Thus, stimulation of CD137 not only enhanced T-cell activation but also augmented their trafficking into malignant tissue, through direct actions on the blood vessels that irrigate the tumor. Our findings identify an additional mechanism of action that can explain the immunotherapeutic effects of agonist CD137 antibodies.

Lymph node stromal cells acquire peptide–MHCII complexes from dendritic cells and induce antigen-specific CD4+ T cell tolerance

LNSCs present peptide–MHCII complexes acquired from DCs to CD4+ T cells and induce T cell dysfunction by preventing their proliferation and survival.

Multi-layered action mechanisms of CD137 (4-1BB)-targeted immunotherapies

CD137 (also known as 4-1BB) is a surface co-stimulatory glycoprotein originally described as present on activated T lymphocytes. Artificial stimulation of this molecule with monoclonal antibodies or other agonist moieties therapeutically augments the cellular immune response against tumors, regardless of the absence of CD137 on tumor cells. These pharmacological agents, when administered systemically, surpass the immune effects of the membrane-bound natural ligand (CD137 or 4-1BB ligand), the activity of which is confined to cell-to-cell interactions. Greater affinity and broader distribution of the CD137 pharmacological agonists cause much more intense receptor crosslinking and stronger intracellular signals than the natural ligand. Target engagement on a variety of immune cell types such as T, natural killer and dendritic cells and on tumor vessels could switch on multiple mechanisms of action. As an agonist, anti-CD137 monoclonal antibody has entered Phase II clinical trials; elucidation of the mechanisms behind the antitumor efficacy requires further research in mice and patients to understand and rationally combine these new treatments.

Therapeutic antitumor efficacy of anti-CD137 agonistic monoclonal antibody in mouse models of myeloma.

Purpose: Eradication of post-treatment residual myeloma cells is needed to prevent relapses, and immunostimulatory monoclonal antibodies (mAb) such as anti-CD137, CTLA-4, CD40, etc., which enhance the immune response against malignancies, represent a means of achieving this purpose. This study explores anti-CD137 mAbs for multiple myeloma treatment in preclinical models of the disease because they safely augment tumor immunity and are in clinical trials for other cancers. Experimental Design: The antitumor effect of anti-CD137 mAb on mouse plasmacytomas derived from HOPC and NS0 cell lines was studied and compared with that of anti-CTLA-4, anti-CD40, and anti-ICAM-2 mAbs. The antitumor effect of anti-CD137 mAb was also examined in a mouse syngeneic disseminated myeloma (5TGM1) model, which more closely resembles human multiple myeloma. Depletions of specific cell populations and gene-targeted mice were used to unravel the requirements for tumor rejection. Results: Agonistic mAb against CD137 and blocking anti-CTLA-4 mAb showed activity against i.p. HOPC tumors, resulting in extended survival of mice that also became immune to rechallenge. Anti-CD137 mAbs induced complete eradications of established s.c. NS0-derived tumors that were dependent on IFN-γ, natural killer cells, and CD8+ T lymphocytes. Natural killer cells accumulated in tumor draining lymph nodes and showed increased IFN-γ production. Antitumor efficacy of anti-CD137 mAb was preserved in CD28-deficient mice despite the fact that CD28 signaling increases the expression of CD137 on CD8+ T cells. Importantly, anti-CD137 mAb treatment significantly decreased systemic tumor burden in the disseminated 5TGM1 model. Conclusions: The immune-mediated antitumor activity of anti-CD137 mAb in mouse models holds promise for myeloma treatment in humans.

Treatment with anti-CD137 mAbs causes intense accumulations of liver T cells without selective antitumor immunotherapeutic effects in this organ

Background/aimsCancer therapy with agonist anti-CD137 mAbs has been shown to induce immune-mediated tumor rejections in mice, and equivalent agents of this kind are currently being tested in cancer patients. Previous reports indicated that CD137 stimulation induced polyclonal infiltrates of T lymphocytes in the liver. This study characterizes the liver infiltrates and the target dependency of the phenomena and addresses the question of whether tumors nested in the liver are a more favorable target for CD137-based immunotherapy.MethodsLiver infiltrates were studied with conventional histology and multiple color flow cytometry of total liver leukocytes. CD137−/− mice, mice with a single rearrangement of the TCR (OT-1 mice) and Rag−/− mice were used to clarify molecular requirements. Mice implanted with MC38 colon carcinomas either subcutaneously or inside the liver were used for comparative studies under treatment with agonist anti-CD137 mAbs.ResultsCD137 treatment caused mononuclear inflammation in the portal spaces of the liver, which gave rise to moderate increases in transaminases without signs of cholestasis. Marked increases in the numbers of CD8+ T cells were observed, including CD8+ T lymphocytes co-expressing CD11c. Infiltrates were absent in CD137−/− mice and mitigated in mice harboring a single transgenic TCR on their CD8 T cells. Despite the tumor-independent accumulation of T cells in the liver, immunotherapeutic effects were not more prominent against tumors located in this organ.ConclusionsTarget-dependent effects of CD137 stimulation lead to liver infiltration with T cells, but lymphocyte enrichment in this organ does not privilege this site for immunotherapeutic effects against transplanted tumors.

In vivo depletion of DC impairs the anti-tumor effect of agonistic anti-CD137 mAb

Anti‐CD137 mAb are capable of inducing tumor rejection in several syngeneic murine tumor models and are undergoing clinical trials for cancer. The anti‐tumor effect involves co‐stimulation of tumor‐specific CD8+ T cells. Whether antigen cross‐presenting DC are required for the efficacy of anti‐CD137 mAb treatment has never been examined. Here we show that the administration of anti‐CD137 mAb eradicates EG7‐OVA tumors by a strictly CD8β+ T‐cell‐dependent mechanism that correlates with increased CTL activity. Ex vivo analyses to determine the identity of the draining lymph node cell type responsible for tumor antigen cross‐presentation revealed that CD11c+ cells, most likely DC, are the main players in this tumor model. A minute number of tumor cells, revealed by the presence of OVA cDNA, reach tumor‐draining lymph nodes. Direct antigen presentation by tumor cells themselves also participates in anti‐OVA CTL induction. Using CD11c diphtheria toxin receptor‐green fluorescent protein→C57BL/6 BM chimeric mice, which allow for sustained ablation of DC with diphtheria toxin, we confirmed the involvement of DC in tumor antigen cross‐presentation in CTL induction against OVA257–264 epitope and in the antitumor efficacy induced by anti‐CD137 mAb.

Effects of IFN-α as a signal-3 cytokine on human naïve and antigen-experienced CD8(+) T cells.

IFN‐α/β link innate and adaptive immune responses by directly acting on naïve CD8+ T cells. This concept unveiled in mice remains unexplored in humans. To investigate that, human CD8+CD45RO− cells were stimulated with beads coated with anti‐CD3 and anti‐CD28 mAb, mimicking Ag (type‐1) and co‐stimulatory (type‐2) signals, in the presence or absence of IFN‐α and their transcriptional profiles were defined by cDNA‐microarrays. We show that IFN‐α provides a strong third signal directly to human CD8+ T cells resulting in regulation of critical genes for their overall activation. This transcriptional effect was substantiated at the protein level and verified by functional assays. Interestingly, the biological effects derived from this stimulation vary depending on the CD8+ T‐cell population. Thus, whereas IFN‐α increases the proliferative capacity of naïve CD8+ T cells, it inhibits or does not affect the proliferation of Ag‐experienced cells, such as memory and effector CTL, including CMV‐specific lymphocytes. Cytolysis and IFN‐γ‐secretion of all these populations are enhanced by IFN‐α‐derived signals, which are critical in naïve CD8+ T cells for acquisition of effector functions. Our findings in human CD8+ T cells are informative to understand and improve IFN‐α‐based therapies for viral and malignant diseases.

Dendritic cells adhere to and transmigrate across lymphatic endothelium in response to IFN‐α

Migration of DC into lymphatic vessels ferries antigenic cargo and pro‐inflammatory stimuli into the draining LN. Given that tissues under the influence of viral infections produce type I IFN, it is conceivable that these cytokines enhance DC migration in order to facilitate an antiviral immune response. Cultured lymphatic endothelium monolayers pretreated with TNF‐α were used to model this phenomenon under inflammatory conditions. DC differentiated in the presence of either IFN‐α2b or IFN‐α5 showed enhanced adhesion to cultured lymphatic endothelial cells. These pro‐adhesive effects were mediated by DC, not the lymphatic endothelium, and correlated with increased DC transmigration across lymphatic endothelial cell monolayers. Transmigration was guided by chemokines acting on DC, and blocking experiments with mAb indicated a role for LFA‐1. Furthermore, incubation of DC with IFN‐α led to the appearance of active conformation epitopes on the CD11a integrin chains expressed by DC. Differentiation of mouse DC in the presence of IFN‐α also increased DC migration from inflammed footpads toward popliteal LN. Collectively, these results indicate a role for type I IFN in directing DC toward LN under inflammatory conditions.