Memory CD8(+) T cells colocalize with IL-7(+) stromal cells in bone marrow and rest in terms of proliferation and transcription.

It is believed that memory CD8(+) T cells are maintained in secondary lymphoid tissues, peripheral tissues, and BM by homeostatic proliferation. Their survival has been shown to be dependent on IL-7, but it is unclear where they acquire it. Here we show that in murine BM, memory CD8(+) T cells individually colocalize with IL-7(+) reticular stromal cells. The T cells are resting in terms of global transcription and do not express markers of activation, for example, 4-1BB (CD137), IL-2, or IFN-γ, despite the expression of CD69 on about 30% of the cells. Ninety-five percent of the memory CD8(+) T cells in BM are in G0 phase of cell cycle and do not express Ki-67. Less than 1% is in S/M/G2 of cell cycle, according to propidium iodide staining. While previous publications have estimated the extent of proliferation of CD8(+) memory T cells on the basis of BrdU incorporation, we show here that BrdU itself induces proliferation of CD8(+) memory T cells. Taken together, the present results suggest that CD8(+) memory T cells are maintained as resting cells in the BM in dedicated niches with their survival conditional on IL-7 receptor signaling.

Tumour necrosis factor receptor superfamily member 9 (TNFRSF9) is up-regulated in reactive astrocytes in human gliomas.

AIMS: The prognosis of patients with malignant gliomas is still dismal despite maximum treatment. Novel therapeutic alternatives targeting tumorigenic pathways are, therefore, demanded. In murine glioma models, targeting of tumour necrosis factor receptor superfamily (TNFRSF) 9 led to complete tumour eradication. Thus, TNFRSF9 might also constitute a promising target in human diffuse gliomas. As there is a lack of data, we aimed to define the expression pattern and cellular source of TNFRSF9 in human gliomas. METHODS: We investigated TNFRSF9 expression in normal human central nervous system (CNS) tissue and glioma specimens using immunohistochemistry, immunofluorescence and Western blotting techniques. RESULTS: Our results show that TNFRSF9 is considerably up-regulated in human gliomas when compared with normal brain tissue. In addition, our data provides evidence for an immune cell-independent de novo expression pattern of TNFRSF9 in mainly non-neoplastic reactive astrocytes and excludes classic immunological cell types, namely lymphocytes and microglia as the source of TNFRSF9. Moreover, TNFRSF9 is predominantly expressed in a perivascular and peritumoural distribution with significantly higher expression in IDH-1 mutant gliomas. CONCLUSIONS: Our findings provide a novel, TNFRSF9-positive, reactive astrocytic phenotype and challenge the therapeutic suitability of TNFRSF9 as a promising target for human gliomas.

Human T cells engineered to express a programmed death 1/28 costimulatory retargeting molecule display enhanced antitumor activity.

Adoptive transfer of T cells genetically modified to express cancer-specific receptors can mediate impressive tumor regression in terminally ill patients. However, T cell function and persistence over time could be hampered by the activation of inhibitory costimulatory pathways, such as programmed death 1 (PD1)/programmed death ligand 1, leading to T cell exhaustion and providing tumor cells with an escape mechanism from immunosurveillance. In addition, the lack of positive costimulation at the tumor site can further dampen T cell response. Thus, as T cell genetic engineering has become clinically relevant, we aimed at enhancing T cell antitumor activity by genetically diverting T cell-negative costimulatory signals into positive ones using chimeric costimulatory retargeting molecules and which are composed of the PD1 extracellular domain fused to the signaling domains of positive costimulatory molecules such as CD28 and 4-1BB. After characterizing the optimal PD1 chimera, we designed and optimized a tripartite retroviral vector that enables the simultaneous expression of this chimeric molecule in conjunction with a cancer-specific TCR. Human T cells, transduced to express a PD1/28 chimeric molecule, exhibited enhanced cytokine secretion and upregulation of activation markers upon coculture with tumor cells. These engineered cells also proliferated better compared with control cells. Finally, we tested the function of these cells in two xenograft models of human melanoma tumors and show that PD1/28-engineered human T cells demonstrated superior antitumor function. Overall, we propose that engineering T cells with a costimulatory retargeting molecule can enhance their function, which bears important implications for the improvement of T cell immunotherapy.

The anti-lymphoma activities of anti-CD137 monoclonal antibodies are enhanced in FcγRIII(-/-) mice.

Agonistic monoclonal antibodies (mAbs) directed against the co-signaling molecule CD137 (4-1BB) elicit potent anti-tumor immunity in mice. This anti-tumor immunity has traditionally been thought to result from the ability of the Fab portion of anti-CD137 to function as an analog for CD137L. Although binding of CD137 by anti-CD137 mAbs has the potential to cross-link the Fc fragments, enabling Fc engagement of low to moderate affinity Fc gamma receptors (FcγR), the relative import of such Fc-FcγR interactions in mediating anti-CD137 associated anti-tumor immunity is unknown. We studied the ability of a rat anti-mouse CD137 mAb (2A) to mediate the anti-tumor response against the EL4E7 lymphoma in WT and FcγR(-/-) strains. 2A-treated FcRγ(-/-) mice had improved anti-tumor immunity against EL4E7, which could be completely recapitulated in FcγRIII(-/-) animals. These improved anti-tumor responses were associated with increased splenic CD8ß T cell and dendritic cell (DC) populations. Furthermore, there was an increase in the number of DCs expressing high levels of the CD40, CD80, and CD86 molecules that are associated with more effective antigen presentation. Our results demonstrate an unexpected inhibitory role for FcγRIII in the anti-tumor function of anti-CD137 and underscore the need to consider antibody isotype when engineering therapeutic mAbs.

CD4 T-cell memory responses to viral infections of humans show pronounced immunodominance independent of duration or viral persistence.

Little is known concerning immunodominance within the CD4 T-cell response to viral infections and its persistence into long-term memory. We tested CD4 T-cell reactivity against each viral protein in persons immunized with vaccinia virus (VV), either recently or more than 40 years ago, as a model self-limited viral infection. Similar tests were done with persons with herpes simplex virus 1 (HSV-1) infection as a model chronic infection. We used an indirect method capable of counting the CD4 T cells in blood reactive with each individual viral protein. Each person had a clear CD4 T-cell dominance hierarchy. The top four open reading frames accounted for about 40% of CD4 virus-specific T cells. Early and long-term memory CD4 T-cell responses to vaccinia virus were mathematically indistinguishable for antigen breadth and immunodominance. Despite the chronic intermittent presence of HSV-1 antigen, the CD4 T-cell dominance and diversity patterns for HSV-1 were identical to those observed for vaccinia virus. The immunodominant CD4 T-cell antigens included both long proteins abundantly present in virions and shorter, nonstructural proteins. Limited epitope level and direct ex vivo data were also consistent with pronounced CD4 T-cell immunodominance. We conclude that human memory CD4 T-cell responses show a pattern of pronounced immunodominance for both chronic and self-limited viral infections and that this pattern can persist over several decades in the absence of antigen.

Up-regulation of alternate co-stimulatory molecules on proinflammatory CD28null T cells in bronchiolitis obliterans syndrome.

Bronchiolitis obliterans syndrome (BOS) is associated with lack of immunosuppression of T cell proinflammatory cytokines and increased T cell granzyme B. Repeated antigen-driven proliferation down-regulates T cell CD28. We hypothesized that down-regulation of CD28 and up-regulation of alternate co-stimulatory molecules (CD134, CD137, CD152 and CD154) on T cells may be associated with BOS. Co-stimulatory molecules, granzyme B, perforin and intracellular cytokines were measured by flow cytometry on T cells from stable lung transplant patients (n = 38), patients with BOS (n = 20) and healthy controls (n = 10). There was a significant increase in the percentage of CD4/28(null) and CD8/28(null) T cells producing granzyme B, interferon (IFN)-γ and tumour necrosis factor (TNF)-α in BOS compared with stable patients. Down-regulation of CD28 was associated with steroid resistance and up-regulation of CD134, CD137, CD152 and CD154 on CD4(+) T cells and CD137 and CD152 on CD8(+) T cells. There was a significant correlation between increased CD28(null) /CD137 T cells producing IFN-γ, TNF-α with BOS grade (r = 0·861, P < 0·001 for CD28(null) /CD137 IFN-γ/CD8) and time post-transplant (r = 0·698, P < 0·001 for CD28(null) /CD137 IFN-γ/CD8). BOS is associated with down-regulation of CD28 and up-regulation of alternate co-stimulatory molecules on steroid-resistant peripheral blood proinflammatory CD4(+) and CD8(+) T cells. Therapeutic targeting of alternate co-stimulatory molecules on peripheral blood CD28(null) T cells and monitoring response using these assays may help in the management of patients with BOS.

Activation-induced CD137 is a fast assay for identification and multi-parameter flow cytometric analysis of alloreactive T cells.

Detection and isolation of viable alloreactive T cells at the single-cell level requires a cell surface marker induced specifically upon T cell receptor activation. In this study, a member of the tumour necrosis factor receptor (TNFR)-family, CD137 (4-1BB) was investigated for its potential to identify the total pool of circulating alloreactive T cells. Optimal conditions for sensitive and specific detection of allogeneic-induced CD137 expression on circulating T cells were established. Thereafter, CD137(+) alloreactive T cells were phenotypically and functionally characterized by multi-parameter flow cytometry. Alloantigen-induced CD137 expression identified both alloreactive CD8(+) T cells (mean ± standard error of the mean: 0·21 ± 0·07%) and alloreactive CD4(+) T cells (0·21 ± 0·05%). CD137(+) alloreactive T cells were detected in different T cell subsets, including naive T cells, but were found preferentially in CD28(+) T cells and not in the terminally differentiated T cell subset. Upon allogeneic (re-)stimulation, the cytokine-producing as well as proliferative capacity of T cells resided mainly within the CD137-expressing fraction. About 10% of the CD137(+) alloreactive T cells produced any combination of interferon (IFN)-γ, interleukin (IL)-2 and TNF-α. Polyfunctional alloreactive T cells, defined by multiple cytokine expression, were observed infrequently. In conclusion, activation-induced CD137 expression is a fast assay allowing for detection and functional analysis of the total alloreactive T cell compartment at the single-cell level by multi-parameter flow cytometry.

CD137 on inflamed lymphatic endothelial cells enhances CCL21-guided migration of dendritic cells.

CD137/TNFR9/41BB was originally described as a surface molecule present on activated T and NK cells. However, its expression is broader among leukocytes, and it is also detected on hypoxic endothelial cells and inflamed blood vessels, as well as in atherosclerotic lesions. Here, we demonstrate that lymphatic endothelial cells (LECs) up-regulate CD137 expression from undetectable baseline levels on stimulation with TNF-α, LPS, and IL-1ß. CD137 cross-linking with an agonistic mAb results in NF-κB nuclear translocation, followed by up-regulation of VCAM and a 3-fold increase in the production of the chemokine CCL21. Accordingly, there is a 50% increase in CCR7-dependent migration toward conditioned medium from activated LECs on CD137 cross-linking with the agonistic mAb or the natural ligand (CD137L). Such an enhancement of cell migration is also observed with monocyte-derived dendritic cells transmigrating across CD137-activated LEC monolayers. Using explanted human dermal tissue, we found that inflamed skin contains abundant CD137(+) lymphatic vessels and that ex vivo incubation of explanted human dermis with TNF-α induces CD137 expression in lymphatic capillaries. More interestingly, treatment with CD137 agonistic antibody induces CCL21 expression and DC accumulation close to lymphatic vessels. Collectively, our results demonstrate that the inflammatory function of lymphatic vessels can be regulated by CD137.

CD137-mediated pathogenesis from chronic hepatitis to hepatocellular carcinoma in hepatitis B virus-transgenic mice.

Chronic hepatitis B virus (HBV) infection is characterized by sustained liver inflammation with an influx of lymphocytes, which contributes to the development of cirrhosis and hepatocellular carcinoma. The mechanisms underlying this immune-mediated hepatic pathogenesis remain ill defined. We report in this article that repetitive infusion of anti-CD137 agonist mAb in HBV-transgenic mice closely mimics this process by sequentially inducing hepatitis, fibrosis, cirrhosis, and, ultimately, liver cancer. CD137 mAb initially triggers hepatic inflammatory infiltration due to activation of nonspecific CD8(+) T cells with memory phenotype. CD8(+) T cell-derived IFN-γ plays a central role in the progression of chronic liver diseases by actively recruiting hepatic macrophages to produce fibrosis-promoting cytokines and chemokines, including TNF-α, IL-6, and MCP-1. Importantly, the natural ligand of CD137 was upregulated significantly in circulating CD14(+) monocytes in patients with chronic hepatitis B infection and closely correlated with development of liver cirrhosis. Thus, sustained CD137 stimulation may be a contributing factor for liver immunopathology in chronic HBV infection. Our studies reveal a common molecular pathway that is used to defend against viral infection but also causes chronic hepatic diseases.

Tumor necrosis factor receptor subfamily 9 (Tnfrsf9) gene is expressed in distinct cell populations in mouse uterus and conceptus during implantation period of pregnancy.

Tumor necrosis factor receptor subfamily 9 (TNFRSF9) plays a potentially important general role in immune function. Tnfrsf9 gene expression has previously been characterized in late pregnant mouse uterus and placenta. However, little is known about its expression in the uterus during the implantation phase of early pregnancy. We have assessed the levels and localization of Tnfrsf9 expression in the mouse uterus and conceptus during implantation. Relative Tnfrsf9 mRNA levels were significantly higher in implantation than in non-implantation site tissue on days 6.5-8.5 of pregnancy. This increase did not depend on the presence of the conceptus, as mRNA levels were not significantly different between pregnant implantation sites and artificially induced deciduomas. Localization by in situ hybridization revealed a subpopulation of endothelial and uterine natural killer cells expressing Tnfrsf9 in the endometrium during implantation. In the developing conceptus, primary trophoblast giant and ectoplacental cells expressed Tnfrsf9 on days 6.5-8.5, followed by expression in the trophoblast giant cell layers surrounding the conceptus on day 9.5 of pregnancy. Two main splice forms of Tnfrsf9 mRNA exist and encode proteins with distinct biological functions; both mRNA splice forms were present in uterine and conceptus tissues as determined by reverse transcription with the polymerase chain reaction. Thus, both membrane and soluble forms of Tnfrsf9 are expressed in specific cell types of the uterus and conceptus during the progression of implantation in mice and possibly have an important function in this process.

Endogenous 4-1BB ligand plays a critical role in protection from influenza-induced disease.

A critical issue during severe respiratory infection is whether it is the virus or the host response that does the most damage. In this study, we show that endogenous 4-1BBL plays a critical role in protecting mice from severe effects of influenza disease. During mild respiratory influenza infection in which virus is rapidly cleared, the inducible costimulatory receptor 4-1BB is only transiently induced on lung T cells and 4-1BB ligand (4-1BBL) is completely dispensable for the initial CD8 T cell response and mouse survival. In contrast, during more severe respiratory influenza infection with prolonged viral load, 4-1BB expression on lung CD8 T cells is sustained, and 4-1BBL-deficient mice show decreased CD8 T cell accumulation in the lungs, decreased viral clearance, impaired lung function, and increased mortality. Transfer of an optimal number of naive Ag-specific T cells before infection protects wild-type but not 4-1BBL-deficient mice from an otherwise lethal dose of influenza virus. Transfer of T cells lacking the proapoptotic molecule Bim extends the lifespan of 4-1BBL-deficient mice by one to three days, suggesting that at least part of the role of 4-1BB/4-1BBL is to prolong effector cell survival long enough to clear virus. Intranasal delivery of 4-1BBL by recombinant adenovirus marginally improves survival of 4-1BBL-deficient mice at low dose, but exacerbates disease at high dose. These findings suggest a rationale for the evolutionary accumulation of inducible costimulatory molecules, thereby allowing the immune system to sustain the expression of molecules such as 4-1BB to a level commensurate with severity of infection.

4-1BB enhances proliferation of beryllium-specific T cells in the lung of subjects with chronic beryllium disease.

In contrast to naive T cells, reactivation of memory cells is less dependent on CD28-mediated costimulation. We have shown that circulating beryllium-specific CD4(+) T cells from chronic beryllium disease patients remain CD28-dependent, while those present in the lung no longer require CD28 for T cell activation. In the present study, we analyzed whether other costimulatory molecules are essential for beryllium-induced T cell function in the lung. Enhanced proliferation of a beryllium-responsive, HLA-DP2-restricted T cell line was seen after the induction of 4-1BB ligand expression on the surface of HLA-DP2-expressing fibroblasts. Following beryllium exposure, CD4(+) T cells from blood and bronchoalveolar lavage of chronic beryllium disease patients up-regulate 4-1BB expression, and the majority of beryllium-responsive, IFN-gamma-producing CD4(+) T cells in blood coexpress CD28 and 4-1BB. Conversely, a significant fraction of IFN-gamma-producing bronchoalveolar lavage (BAL) T cells express 4-1BB in the absence of CD28. In contrast to blood, inhibition of the 4-1BB ligand-4-1BB interaction partially blocked beryllium-induced proliferation of BAL CD4(+) T cells, and a lack of 4-1BB expression on BAL T cells was associated with increased beryllium-induced cell death. Taken together, these findings suggest an important role of 4-1BB in the costimulation of beryllium-responsive CD4(+) T cells in the target organ.

Decreased 4-1BB expression on HIV-specific CD4+ T cells is associated with sustained viral replication and reduced IL-2 production.

CD4+ T cell dysfunction in subjects with chronic HIV infection is in part due to an imbalance of costimulatory and coinhibitory receptors. We report that virus-specific CD4+ T cells expressing 4-1BB (CD137) or OX40 (CD134) produced more IL-2 than cells lacking these costimulatory receptors (P<0.05) and that 4-1BB was expressed at a lower level on HIV- than CMV-specific IFN-gamma and IL-2 producing CD4+ T cells (P<0.0001 and P<0.01, respectively). Suppression of viral replication with antiretroviral therapy was associated with increased 4-1BB expression on HIV- and CMV-specific IL-2 producing CD4+ T cells (P<0.05 and P<0.01, respectively) and the percentage of IL-2 producing HIV-specific CD4+ T cells that expressed 4-1BB was inversely correlated with HIV plasma viral load (r=-0.75, P=0.007). These findings indicate that the loss of 4-1BB on HIV-specific CD4+ T cells is associated with viral replication and that it may contribute to reduced IL-2 production observed during chronic infection.

Characterisation of soluble murine CD137 and its association with systemic lupus.

CD137 is a member of the tumor necrosis factor receptor family, and is involved in the regulation of activation, proliferation, differentiation and apoptosis of T cells, B cells, monocytes, dendritic cells, natural killer cells and granulocytes. Here report that soluble forms of murine CD137 (sCD137) are generated by differential splicing and are released by activated T cells. Levels of sCD137 correlate with cell activation and the extent of cell death but not with cellular proliferation. While CD8+ T cells express significantly more cell surface CD137 than CD4+ T cells, both T cell subsets express similar levels of sCD137, resulting a twofold increased ratio of soluble to cell surface CD137 for CD4+ T cells. sCD137 exists as a trimer and a higher order multimer, can bind to CD137 ligand, and inhibits secretion of IL-10 and IL-12. sCD137 is present in sera of mice with autoimmune disease but is undetectable in sera of healthy mice.

Analysis of CD137 and CD137L expression in human primary tumor tissues.

AIM: To assess the expression of CD137 and CD137L in human primary tumor tissues and their potential role in tumor immunity. METHODS: Expression of CD137 and CD137L was assessed by immunohistochemistry in frozen sections of 12 human normal tissues, 15 benign tumors of epithelial or mesenchymal origin (adenoma and leiomyoma), and 36 malignant tumors of epithelial origin (squamous cell carcinoma and adenocarcinoma). The expression of CD137L on 9 human tumor cell lines (3 hepatocarcinoma, 2 lung carcinoma, 2 colon carcinoma, 1 lymphoma, and 1 leukemia) was detected by reverse transcription polymerase chain reaction. To analyze the role of CD137L expressed on tumor cells, we co-cultured tumor cells expressing CD137L with activated T lymphocytes expressing CD137 or with Chinese hamster ovary cells expressing CD137 and then detected by ELISA the levels of cytokines (IL-8, IFN-gamma) secreted by tumor cells or activated T cells. RESULTS: The expression of CD137 and CD137L was observed only in human benign (2/15, 3/15) or malignant tumors (15/36, 21/36), but not in normal tissues (0/12, 0/12). CD137 was expressed on the vessel walls within tumor tissues, whereas CD137L was expressed on tumor cells. The expression of CD137 and CD137L was more common in malignant tumors, especially in moderate or low-differentiated tumors. Furthermore, CD137L expression found on tumor cell lines was functional because the ligation of CD137L on lung squamous carcinoma cells L78 with CD137 on T cells induced IFN-gamma production by T cells, and ligation of CD137L on hepatocarcinoma cells HepG2.2.15 with CD137 triggered tumor cells to produce IL-8. CONCLUSION: CD137 and CD137L are expressed in different human primary tumor tissues, suggesting that they may influence the progression of tumors.

The expression and clinical correlations of 4-1BB on peripheral CD4+ T cell subsets in patients with coronary artery disease. A cross-sectional pilot study.

BACKGROUND: The expression of 4-1BB on peripheral regulatory T cells (Tregs) and conventional T cells (Tconvs) in coronary artery disease (CAD) patients is unknown. We aimed to investigate the expression and clinical correlations of 4-1BB on peripheral Tregs and Tconvs in CAD patients. METHODS: Flow cytometry analysis was used to analyze 4-1BB expression on peripheral Tregs and Tconvs. We compared the percentages of 4-1BB on Tregs and Tconvs in the control (ctrl) group, the stable ischemic heart disease (SIHD) group, and the acute coronary syndrome (ACS) group. The correlations of 4-1BB expression on Tregs and Tconvs with the Gensini score and CRP were examined in the ACS group. The value of 4-1BB percentage on Tregs for predicting CAD in this cardiovascular risk population was also analyzed. RESULTS: A total of 71 participants were enrolled in this study. In all the groups, the percentages of 4-1BB on Tregs were significantly higher than on Tconvs (all P < .05). After adjusting for sex, age, SBP, HbA1c and LDL, 4-1BB percentages on Tregs and Tconvs were significantly higher in the SIHD and ACS groups compared with the ctrl group (all P < .05). The ratio of 4-1BB percentage on Tregs to 4-1BB percentage on Tconvs was higher in the ACS group compared with the ctrl group (P = .010). In the ACS group, CRP was negatively correlated with the Tregs percentage (in CD4+ T cells) and the Tregs percentage to Tconvs percentage ratio. The Gensini score was positively correlated with the 4-1BB percentage on Tregs in the ACS group. Linear regression analysis showed 4-1BB percentage on Tregs independently predicted the Gensini score. Binary logistic regression showed CRP, HbA1c and 4-1BB percentage on Tregs independently predicted the development of CAD (SIHD+ACS) in the whole population. CONCLUSION: 4-1BB expression on peripheral Tregs and Tconvs was increased in SIHD and ACS patients. 4-1BB percentage on Tregs positively correlated with the severity of coronary artery stenosis in ACS patients. 4-1BB percentage on Tregs independently predicted the severity of coronary artery stenosis in an ACS population and development of CAD in a cardiovascular risk population.

Activation of CD137 Signaling Promotes Angiogenesis in Atherosclerosis via Modulating Endothelial Smad1/5-NFATc1 Pathway.

BACKGROUND: Excessive angiogenesis is a key feature of vulnerable atherosclerotic plaques, and is considered an independent predictor of cardiovascular risk. CD137 signaling has previously been shown to be involved in atherosclerosis. However, the possible role of CD137 signaling in regulating angiogenesis has not been reported. METHODS AND RESULTS: Apolipoprotein E-deficient (ApoE-/-) mice were used as the in vivo model of atherosclerosis. Masson and immunohistochemical analysis of atherosclerotic plaques and Matrigel plug assay were used to evaluate the angiogenesis. Human umbilical vein endothelial cells and mouse brain microvascular endothelial cells were used as in vitro and ex vivo models to study how CD137 signaling affects angiogenesis. Matrigel tube formation assay, mouse aortic ring angiogenesis assay, and migration and proliferation assay were employed to assess angiogenesis. Western blot was used to detect protein expression. We found increased neovessel formation in atherosclerotic plaques of ApoE-/- mice treated with agonist anti-CD137 antibody. Activation of CD137 signaling induced angiogenesis, endothelial proliferation, and endothelial cell migration. CD137 signaling activates the pro-angiogenic Smad1/5 pathway, induces the phosphorylation of Smad1/5 and nuclear translocation of p-Smad1/5, which in turn promotes the expression and translocation of NFATc1. Blocking CD137 signaling with inhibitory anti-CD137 antibody could inhibit this activation and attenuated agonist anti-CD137 antibody-induced angiogenesis. CONCLUSIONS: These findings suggest that CD137 signaling is a new regulator of angiogenesis by modulating the Smad1/5-NFATc1 pathway.

CD137 Stimulation Enhances Cetuximab-Induced Natural Killer: Dendritic Cell Priming of Antitumor T-Cell Immunity in Patients with Head and Neck Cancer.

PURPOSE: Cetuximab, an EGFR-specific antibody (mAb), modestly improves clinical outcome in patients with head and neck cancer (HNC). Cetuximab mediates natural killer (NK) cell:dendritic cell (DC) cross-talk by cross-linking FcγRIIIa, which is important for inducing antitumor cellular immunity. Cetuximab-activated NK cells upregulate the costimulatory receptor CD137 (4-1BB), which, when triggered by agonistic mAb urelumab, might enhance NK-cell functions, to promote T-cell-based immunity. EXPERIMENTAL DESIGN: CD137 expression on tumor-infiltrating lymphocytes was evaluated in a prospective cetuximab neoadjuvant trial, and CD137 stimulation was evaluated in a phase Ib trial, in combining agonistic urelumab with cetuximab. Flow cytometry and cytokine release assays using NK cells and DC were used in vitro, testing the addition of urelumab to cetuximab-activated NK, DC, and cross presentation to T cells. RESULTS: CD137 agonist mAb urelumab enhanced cetuximab-activated NK-cell survival, DC maturation, and tumor antigen cross-presentation. Urelumab boosted DC maturation markers, CD86 and HLA DR, and antigen-processing machinery (APM) components TAP1/2, leading to increased tumor antigen cross-presentation. In neoadjuvant cetuximab-treated patients with HNC, upregulation of CD137 by intratumoral, cetuximab-activated NK cells correlated with FcγRIIIa V/F polymorphism and predicted clinical response. Moreover, immune biomarker modulation was observed in an open label, phase Ib clinical trial, of patients with HNC treated with cetuximab plus urelumab. CONCLUSIONS: These results suggest a beneficial effect of combination immunotherapy using cetuximab and CD137 agonist in HNC. Clin Cancer Res; 23(3); 707-16. ©2016 AACR.

4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors.

Chimeric antigen receptors (CARs) targeting CD19 have mediated dramatic antitumor responses in hematologic malignancies, but tumor regression has rarely occurred using CARs targeting other antigens. It remains unknown whether the impressive effects of CD19 CARs relate to greater susceptibility of hematologic malignancies to CAR therapies, or superior functionality of the CD19 CAR itself. We show that tonic CAR CD3-ζ phosphorylation, triggered by antigen-independent clustering of CAR single-chain variable fragments, can induce early exhaustion of CAR T cells that limits antitumor efficacy. Such activation is present to varying degrees in all CARs studied, except the highly effective CD19 CAR. We further determine that CD28 costimulation augments, whereas 4-1BB costimulation reduces, exhaustion induced by persistent CAR signaling. Our results provide biological explanations for the antitumor effects of CD19 CARs and for the observations that CD19 CAR T cells incorporating the 4-1BB costimulatory domain are more persistent than those incorporating CD28 in clinical trials.

Structural Design of Engineered Costimulation Determines Tumor Rejection Kinetics and Persistence of CAR T Cells.

T cell engineering is a powerful means to rapidly generate anti-tumor T cells. The costimulatory properties of second-generation chimeric antigen receptors (CARs) determine the overall potency of adoptively transferred T cells. Using an in vivo "stress test" to challenge CD19-targeted T cells, we studied the functionality and persistence imparted by seven different CAR structures providing CD28 and/or 4-1BB costimulation. One configuration, which uses two signaling domains (CD28 and CD3ζ) and the 4-1BB ligand, provided the highest therapeutic efficacy, showing balanced tumoricidal function and increased T cell persistence accompanied by an elevated CD8/CD4 ratio and decreased exhaustion. Remarkably, induction of the IRF7/IFNß pathway was required for optimal anti-tumor activity. Thus, 1928z-41BBL T cells possess strikingly potent intrinsic and immunomodulatory qualities.