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The cellular source of positive signals that reinvigorate T cells within the tumor microenvironment (TME) for the therapeutic efficacy of programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) blockade has not been clearly defined. We now show that Batf3-lineage dendritic cells (DCs) are essential in this process. Flow cytometric analysis, gene-targeted mice, and blocking antibody studies revealed that 4-1BBL is a major positive co-stimulatory signal provided by these DCs within the TME that translates to CD8 + T cell functional reinvigoration and tumor regression. Immunofluorescence and spatial transcriptomics on human tumor samples revealed clustering of Batf3 + DCs and CD8 + T cells, which correlates with anti-PD-1 efficacy. In addition, proximity to Batf3 + DCs within the TME is associated with CD8 + T cell transcriptional states linked to anti-PD-1 response. Our results demonstrate that Batf3 + DCs within the TME are critical for PD-1/PD-L1 blockade efficacy and indicate a major role for the 4-1BB/4-1BB ligand (4-1BBL) axis during this process., Competing Interests: Declaration of interests T.F.G. has served on scientific advisory boards for Pyxis Oncology, Jounce Therapeutics, Allogene, MAIA, Samyang, Portal Innovations, Fog Pharma, Adaptimmune, Catalym, Bicara, and Merck; is a scientific co-founder and shareholder of Jounce Therapeutics and Pyxis Oncology; and has received research support from Bristol-Myers Squibb, Merck, Pyxis, FogPharma, and Bayer. K.H. is currently working at Merck, and D.E.C.K. is currently working at Slalom., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract: Myocardial fibrosis, a common complication of myocardial infarction (MI), is characterized by excessive collagen deposition and can result in impaired cardiac function. The specific role of CD137 in the development of post-MI myocardial fibrosis remains unclear. Thus, this study aimed to elucidate the effects of CD137 signaling using CD137 knockout mice and in vitro experiments. CD137 expression levels progressively increased in the heart after MI, particularly in myofibroblast, which play a key role in fibrosis. Remarkably, CD137 knockout mice exhibited improved cardiac function and reduced fibrosis compared with wild-type mice at day 28 post-MI. The use of Masson's trichrome and picrosirius red staining demonstrated a reduction in the infarct area and collagen volume fraction in CD137 knockout mice. Furthermore, the expression of alpha-smooth muscle actin and collagen I, key markers of fibrosis, was decreased in heart tissues lacking CD137. In vitro experiments supported these findings because CD137 depletion attenuated cardiac fibroblast differentiation, and migration, and collagen I synthesis. In addition, the administration of CD137L recombinant protein further promoted alpha-smooth muscle actin expression and collagen I synthesis, suggesting a profibrotic effect. Notably, the application of an inhibitor targeting the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway attenuated the profibrotic effects of CD137L. To conclude, this study provides evidence that CD137 plays a significant role in promoting myocardial fibrosis after MI. Inhibition of CD137 signaling pathways may hold therapeutic potential for mitigating pathological cardiac remodeling and improving post-MI cardiac function., Competing Interests: The authors report no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Anxiety manifestations and cognitive dysfunction are common sequelae in patients with sepsis-associated encephalopathy (SAE). Microglia-mediated inflammatory signaling is involved in anxiety, depression, and cognitive dysfunction during acute infection with bacterial lipopolysaccharide (LPS). However, the molecular mechanisms underlying microglia activation and behavioral and cognitive deficits in sepsis have not been in fully elucidated. Based on previous research, we speculated that the CD137 receptor/ligand system modulates microglia function during sepsis to mediate classical neurological SAE symptoms. A murine model of SAE was established by injecting male C57BL/6 mice with LPS, and cultured mouse BV2 microglia were used for in vitro assays. RT-qPCR, immunofluorescence staining, flow cytometry, and ELISA were used to assess microglial activation and the expression of CD137L and inflammation-related cytokines in the mouse hippocampus and in cultured BV2 cells. In addition, behavioral tests were conducted in assess cognitive performance and behavioral distress. Immunofluorescence and RT-qPCR analyses showed that hippocampal expression of CD137L was upregulated in activated microglia following LPS treatment. Pre-treatment with the CD137L neutralizing antibody TKS-1 significantly reduced CD137L levels, attenuated the expression of M1 polarization markers in microglia, and inhibited the production of TNF-α, IL-1β, and IL-6 in both LPS-treated mice and BV2 cells. Conversely, stimulation of CD137L signaling by recombinant CD137-Fc fusion protein activated the synthesis and release of pro-inflammatory cytokines in cultures BV2 microglia. Importantly, open field, elevated plus maze, and Y-maze spontaneous alternation test results indicated that TKS-1 administration alleviated anxiety-like behavior and spatial memory decline in mice with LPS-induced SAE. These findings suggest that CD137L upregulation in activated microglia critically contributes to neuroinflammation, anxiety-like behavior, and cognitive dysfunction in the mouse model of LPS-induced sepsis. Therefore, therapeutic modulation of the CD137L/CD137 signaling pathway may represent an effective way to minimize brain damage and prevent cognitive and emotional deficits associated with SAE., (© 2023. The Author(s).)

Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of hematological malignancies but has yet to achieve similar success in solid tumors due to a lack of persistence and function in the tumor microenvironment. We previously reported the augmentation of CAR T cell therapy in an engineered solid tumor model through the secretion of anti-PD-1 single-chain fragment variable region (scFv), as shown by enhanced CAR T cell antitumor efficacy, expansion, and vitality. We have since improved the platform to create a superior cellular product-CAR T cells secreting single-chain trimeric 4-1BB ligand fused to anti-PD-1 scFv (αPD1-41BBL). 4-1BB signaling promotes cytotoxic T lymphocyte proliferation and survival but targeting 4-1BB with agonist antibodies in the clinic has been hindered by low antitumor activity and high toxicity. CAR T cells using 4-1BB endodomain for costimulatory signals have demonstrated milder antitumor response and longer persistence compared to CAR T cells costimulated by CD28 endodomain. We have, for the first time, engineered CD28-costimulated CAR T cells to secrete a fusion protein containing the soluble trimeric 4-1BB ligand. In vitro and in vivo , CAR19.αPD1-41BBL T cells exhibited reduced inhibitory receptor upregulation, enhanced persistence and proliferation, and a less differentiated memory status compared to CAR T cells without additional 4-1BB:4-1BBL costimulation. Accordingly, CAR19.αPD1-41BBL T cell-treated mice displayed significantly improved tumor growth control and overall survival. Spurred on by our preclinical success targeting CD19 as a model antigen, we produced mesothelin-targeting CAR T cells and confirmed the enhanced solid tumor efficacy of αPD1-41BBL-secreting CAR T cells.

Many microRNAs (miRNAs) are selectively expressed in mammalian immune cells and have been linked to immune responses in host defense and autoimmune disease. In macrophages, miRNAs regulate cell metabolism by repressing the expression of genes such as transcription factors, enzymes, and metabolism-related molecules, as well as the expression of genes that impact inflammatory responses and phenotype determination. Previous studies showed that miR-22 plays a role in a variety of biological processes, such as cancer cell growth, cell survival, and cell expansion. In CD4 +  T cells of inflammatory bowel disease patients, miR-22 is upregulated and regulates inflammasome-mediated responses. However, it has not yet been determined how miR-22 contributes to the activation of innate immune cells. In this study, we identified a mechanism of toll-like receptors- (TLR-) dependent miR-22 induction that regulates the downstream signaling pathway linking inflammatory responses and macrophage polarization. MiR-22 is induced via TLR-signaling, which regulates the induction of Slc2a1 (glucose transporter 1 and Glut1) and Tnfsf9 (tumor necrosis factor 9, 4-1BB ligand, and 4-1BBL) mRNAs that contribute to sustained inflammatory responses and the polarization of macrophages. Our observations support further efforts to explore a potential therapeutic strategy using miR-22 for the modulation of excessive macrophage activation for the treatment of inflammatory diseases., Competing Interests: The author declares that there is no conflicts of interest., (Copyright © 2023 Young Jun Kang.)

Since NKG2D ligands (NKG2DLs) are primarily overexpressed on multiple types of solid tumors but absent on most normal tissues, NKG2DLs could be optimal antigens for CAR-T cells. To date, there have been two types of NKG2DL CARs: (i) the extracellular domain of NKG2D fused to the CD8a transmembrane domain, signaling domains of 4-1BB and CD3ζ (NKBz) and (ii) full-length NKG2D fused to the CD3ζ signaling domain (chNKz). Although NKBz- and chNKz-engineered T cells both showed antitumor activities, a comparison of their functions has not been reported. In addition, use of the 4-1BB signaling domain into the CAR construct could prolong the persistence and resistance to antitumor activities of CAR-T cells, we designed a new NKG2DL CAR, full-length NKG2D fused to the signaling domains of 4-1BB and CD3ζ (chNKBz). Among the two types of NKG2DL CAR-T cells reported in previous studies, we found that chNKz T cells had stronger antitumor ability than NKBz T cells in vitro, but their antitumor activity in vivo is similar. The chNKBz T cells showed antitumor activity superior to that of chNKz T cells and NKBz T cells in vitro and in vivo, providing a new option for the immunotherapy of NKG2DL-positive tumor patients., (© 2023 the Australian and New Zealand Society for Immunology, Inc.)

4-1BB (CD137) is an activation-induced costimulatory receptor that regulates immune responses of activated CD8 T and natural killer cells, by enhancing proliferation, survival, cytolytic activity, and IFNγ production. The ability to induce potent antitumor activity by stimulating 4-1BB on tumor-specific cytotoxic T cells makes 4-1BB an attractive target for designing novel immuno-oncology therapeutics. To minimize systemic immune toxicities and enhance activity at the tumor site, we have developed a novel bispecific antibody that stimulates 4-1BB function when co-engaged with the tumor-associated antigen 5T4. ALG.APV-527 was built on the basis of the ADAPTIR bispecific platform with optimized binding domains to 4-1BB and 5T4 originating from the ALLIGATOR-GOLD human single-chain variable fragment library. The epitope of ALG.APV-527 was determined to be located at domain 1 and 2 on 4-1BB using X-ray crystallography. As shown in reporter and primary cell assays in vitro, ALG.APV-527 triggers dose-dependent 4-1BB activity mediated only by 5T4 crosslinking. In vivo, ALG.APV-527 demonstrates robust antitumor responses, by inhibiting growth of established tumors expressing human 5T4 followed by a long-lasting memory immune response. ALG.APV-527 has an antibody-like half-life in cynomolgus macaques and was well tolerated at 50.5 mg/kg. ALG.APV-527 is uniquely designed for 5T4-conditional 4-1BB-mediated antitumor activity with potential to minimize systemic immune activation and hepatotoxicity while providing efficacious tumor-specific responses in a range of 5T4-expressing tumor indications as shown by robust activity in preclinical in vitro and in vivo models. On the basis of the combined preclinical dataset, ALG.APV-527 has potential as a promising anticancer therapeutic for the treatment of 5T4-expressing tumors., (©2022 The Authors; Published by the American Association for Cancer Research.)

Aberrant expression of the proto-oncogene BCL6 is a driver of tumorigenesis in diffuse large B cell lymphoma (DLBCL). Mice overexpressing BCL6 from the B cell-specific immunoglobulin heavy chain μ intron promoter (Iμ-Bcl6 Tg/+ ) develop B cell lymphomas with features typical of human DLBCL. While the development of B cell lymphoma in these mice is tightly controlled by T cells, the mechanisms of this immune surveillance are poorly understood. Here we show that CD4 T cells contribute to the control of lymphoproliferative disease in lymphoma-prone Iμ-Bcl6 Tg/+ mice. We reveal that this CD4 T cell immuno-surveillance requires signaling by the co-stimulatory molecule CD137 ligand (CD137L; also known as 4-1BBL), which may promote the transition of pre-malignant B cells with an activated phenotype into the germinal center stage via reverse signaling, preventing their hazardous accumulation. Thus, CD137L-mediated CD4 T cell immuno-surveillance adds another layer of protection against B cell malignancy to that provided by CD8 T cell cytotoxicity., (© 2022 The Authors. Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

The complex structure of the tumor microenvironment leads to the poor efficacy of tumor immunotherapy. The therapeutic adjuvant designed to enhance the effect of T cells by acting on the costimulatory molecule tumor necrosis factor superfamily member 9 (TNFSF9) has achieved good results. However, because some tumors are characterized by reduced T-cell infiltration, adjuvants acting on T cells alone may have limitations. On the other hand, the blockade of TNFSF9 reverse signalling can have an antitumor effect by reshaping the tumor microenvironment. Therefore, this paper mainly discusses the current status and potential of TNFSF9 bidirectional signalling in antitumor immunotherapy to provide new ideas for tumor immunotherapy., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Recombinant-modified vaccinia virus Ankara (rMVA) is known to elicit potent antitumor immune responses in preclinical models due to its inherent ability to activate the innate immune system and the activation of adaptive responses mediated by the expression of tumor antigens and costimulus-providing molecules, such as CD40L and CD137L. Here, we evaluated different rMVA vectors in preclinical peritoneal carcinomatosis models (ID8.OVA- Vegf /GFP and MC38). We compared rMVA vectors expressing a tumor antigen (OVA or gp70) either alone or co-expressed with CD40L or/and CD137L. In tumor-free mice, the vector coding for the triple combination was only slightly superior, whereas, in tumor-bearing animals, we observed a synergistic induction of T lymphocytes specific against vector-encoded and non-encoded tumor-associated antigens. The enhanced activation of the immune response was associated with improved survival in mice with peritoneal carcinomatosis treated with a rMVA vector encoding both CD40L and CD137L. Thus, the triple transgene combination in vaccinia viral vectors represents a promising strategy for the treatment of peritoneal carcinomatosis., (© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Senile osteoporosis is a chronic skeletal disease, leading to increased bone brittleness and risk of fragile fractures. With the acceleration of population aging, osteoporosis has gradually become one of the most serious and prevalent problems worldwide. Bone formation is highly dependent on the proper osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in the bone marrow microenvironment, which is generated by the functional relationship among different cell types, including osteoblasts, adipogenic cells, and bone marrow stromal cells in the bone marrow. It is still not clear how osteoporosis is caused by its molecular mechanism. With aging, bone marrow is able to restrain osteogenesis. Discovering the underlying signals that oppose BMSC osteogenic differentiation from the bone marrow microenvironment and identifying the unusual changes in BMSCs with aging is important to elucidate possible mechanisms of senile osteoporosis. We used 3 gene expression profiles (GSE35956, GSE35957, and GSE35959) associated with osteoporosis. And a protein-protein interaction (PPI) network was also built to identify the promising gene CD137. After that, we performed in vivo experiments to verify its function and mechanism. In this experiment, we found that significant bone loss was observed in aged (18-month-old) mice compared with young (6-month-old) mice. The adipose tissue in bone marrow cavity from aged mice reached above 10 times more than young mice. Combining bioinformatics analysis and vivo experiments, we inferred that CD137 might be involved in the p53 and canonical Wnt/β-catenin signaling pathways and thereby influenced bone mass through regulation of marrow adipogenesis. Importantly, osteoporosis can be rescued by blocking CD137 signaling in vivo . Our research will contribute to our understanding not only of the pathogenesis of age-related bone loss but also to the identification of new targets for treating senile osteoporosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Han, Wang, Zhou, Zhang and Wan.)

4-1BB is a T cell costimulatory receptor and a member of the tumor necrosis factor receptor superfamily. Here, we show that Galectin-3 (Gal-3) decreases the cellular response to its ligand (4-1BBL). Gal-3 binds to both soluble 4-1BB (s4-1BB) and membrane-bound 4-1BB (mem4-1BB), without blocking co-binding of 4-1BBL. In plasma, we detected complexes composed of 4-1BB and Gal-3 larger than 100 nm in size; these complexes were reduced in synovial fluid from rheumatoid arthritis. Both activated 4-1BB + T cells and 4-1BB-transfected HEK293 cells depleted these complexes from plasma, followed by increased expression of 4-1BB and Gal-3 on the cell surface. The increase was accompanied by a 4-fold decrease in TNFα production by the 4-1BB high Gal-3 + T cells, after exposure to 4-1BB/Gal-3 complexes. In RA patients, complexes containing 4-1BB/Gal-3 were dramatically reduced in both plasma and SF compared with healthy plasma. These results support that Gal-3 binds to 4-1BB without blocking the co-binding of 4-1BBL. Instead, Gal-3 leads to formation of large soluble 4-1BB/Gal-3 complexes that attach to mem4-1BB on the cell surfaces, resulting in suppression of 4-1BBL's bioactivity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Nielsen, Juul-Madsen, Stegmayr, Gao, Mehta, Greisen, Kragstrup, Hvid, Vorup-Jensen, Cummings, Leffler and Deleuran.)

Oncolytic viruses (OVs) can have utility for direct killing of cancer cells, but may also serve to activate the immune system against cancer cells. While viruses alone can serve as immune stimulators, there is great interest in arming OVs with genes encoding immune stimulatory proteins to amplify their effects. In this work, we have tested the efficacy of conditionally-replicating adenoviruses (CRAds) with and without selected immunostimulatory payloads, murine CD40L (mCD40L) or 4-1BBL (m4-1BBL), in an immune competent mouse model of melanoma. When CRAd657-m4-1BBL and CRAd657-mCD40L were injected into B16-hCAR murine melanoma tumors, both single vectors delayed tumor growth and prolong survival compared to empty CRAd657. However, combined injection of both CRAd-m4-1BBL and CRAd-mCD40L mediated significantly better control of tumor growth. All of the payloads increased immune cell infiltration into tumors and notably reduced expression of PD-1 exhaustion marker on T cells. Tumor volumes were negatively associated with total infiltrating T cell population. We found that the payloads increased immune cell infiltration into tumors with some specificities: recruitment of CD8 + T cells was higher with m4-1BBL expression, while mCD40L expression induced more CD4 + T cell infiltration. Importantly, the combination of CRAd657-m4-1BBL and CRAd657-mCD40L induced the highest immune cells/T cell infiltration and the highest anti-TRP-2 tumor-associated antigen T cell responses than empty or single gene vector. This combination also caused depigmentation in areas adjacent to the tumor sites in more animals. These data indicate that driving two axes of the immune system with combined immune stimulatory payloads can lead to improved anticancer immune responses and better tumor control in an immune competent model of cancer.

Leveraging the T cell immunity against tumors represents a revolutionary type of cancer therapy. 4-1BB is a well-characterized costimulatory immune receptor existing on activated T cells and mediating their proliferation and cytotoxicity under infectious diseases and cancers. Despite the accumulating interest in implementing 4-1BB as a therapeutic target for immune-related disorders, less is known about the pattern of its intracellular behaviors and regulations. It has been previously demonstrated that 4-1BB is heavily modified by N -glycosylation; however, the biological importance of this modification lacks detailed elucidation. Through biochemical, biophysical, and cell-biological approaches, we systematically evaluated the impact of N -glycosylation on the ligand interaction, stability, and localization of 4-1BB. We hereby highlighted that N -glycan functions by preventing the oligomerization of 4-1BB, thus permitting its membrane transportation and fast turn-over. Without N -glycosylation, 4-1BB could be aberrantly accumulated intracellularly and fail to be sufficiently inserted in the membrane. The N -glycosylation-guided intracellular processing of 4-1BB serves as the potential mechanism explicitly modulating the "on" and "off" of 4-1BB through the control of protein abundance. Our study will further solidify the understanding of the biological properties of 4-1BB and facilitate the clinical practice against this promising therapeutic target.

CD160 is a member of the immunoglobulin superfamily with a pattern of expression mainly restricted to cytotoxic cells. To assess the functional relevance of the HVEM/CD160 signaling pathway in allogeneic cytotoxic responses, exon 2 of the CD160 gene was targeted by CRISPR/Cas9 to generate CD160 deficient mice. Next, we evaluated the impact of CD160 deficiency in the course of an alloreactive response. To that aim, parental donor WT (wild-type) or CD160 KO (knock-out) T cells were adoptively transferred into non-irradiated semiallogeneic F1 recipients, in which donor alloreactive CD160 KO CD4 T cells and CD8 T cells clonally expanded less vigorously than in WT T cell counterparts. This differential proliferative response rate at the early phase of T cell expansion influenced the course of CD8 T cell differentiation and the composition of the effector T cell pool that led to a significant decreased of the memory precursor effector cells (MPECs) / short-lived effector cells (SLECs) ratio in CD160 KO CD8 T cells compared to WT CD8 T cells. Despite these differences in T cell proliferation and differentiation, allogeneic MHC class I mismatched (bm1) skin allograft survival in CD160 KO recipients was comparable to that of WT recipients. However, the administration of CTLA-4.Ig showed an enhanced survival trend of bm1 skin allografts in CD160 KO with respect to WT recipients. Finally, CD160 deficient NK cells were as proficient as CD160 WT NK cells in rejecting allogeneic cellular allografts or MHC class I deficient tumor cells. CD160 may represent a CD28 alternative costimulatory molecule for the modulation of allogeneic CD8 T cell responses either in combination with costimulation blockade or by direct targeting of alloreactive CD8 T cells that upregulate CD160 expression in response to alloantigen stimulation., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Renal lymphangiogenesis is a new field of international nephrology in recent years and plays an important role in the progression of chronic renal disease. CD137 was originally described as a surface molecule present on activated T and NK cells and detected on hypoxic endothelial cells and inflamed blood vessels, but its function on lymphatic endothelial cells remains unclear. We investigated the relationships among CD137, lymphangiogenesis and macrophages, which are involved in interstitial fibrosis. Similar to other chronic inflammatory diseases, we found lymphangiogenesis and expression of CD137 in the renal tissue of patients with IgA nephropathy. CD137-positive lymphatic vessels were involved in the development process of IgA nephropathy and positively correlated with serum creatinine, serum urea nitrogen, serum uric acid, and urinary 24 h total protein. The expression of these indicators was negatively correlated with eGFR, plasma albumin, and HB. In mouse models of UUO, we verified that CD137 expression was significantly elevated during lymphangiogenesis and that its ligand CD137L was released by macrophages after VEGF-C stimulation in the kidney. In vitro, recombinant CD137L significantly enhanced LEC proliferation, migration and tube formation, and these effects were inhibited by CD137 siRNA. Mechanistically, the CD137L interaction with CD137 induced the transition from LC3-I to LC3-II and the expression of Atg5, Atg7, Atg12 and p62 proteins by activating the PI3K/AKT/mTOR pathway to promote autophagy. Knockdown of Atg5 and Atg7 blocked CD137L-induced autophagy. Thus, we propose that CD137L secretion by macrophages interacts with CD137 on lymphatic endothelial cells to prompt lymphangiogenesis in the kidney, which further drives fibrogenic responses. Our findings suggest that inhibition of the CD137-CD137L pathway is a novel therapeutic approach for obstructive nephropathy., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The effect of tumor necrosis factor superfamily member 9 (TNFSF9) on the metastasis of pancreatic cancer (PC) and the underlying mechanism remain unclear. We studied the expression of TNFSF9 in pancreatic cancer and its relationship with immune cells. We further explored the effect of TNFSF9 on pancreatic cancer metastasis by inducing macrophage polarization, and evaluated the expression of Src/FAK/p-Akt/IL-1β signals in macrophages after knocking down TNFSF9. The data shows that TNFSF9 expression is elevated in pancreatic cancer and is related to the poor prognosis of patients with pancreatic cancer. In addition, TNFSF9 may induce the M2 polarization of macrophages through Src/FAK/p-Akt/IL-1β signals, thereby promoting the migration of pancreatic cancer cells. In conclusion, our data reveals that TNFSF9 may become a predictive biomarker of pancreatic cancer and provides a new intervention target for the immunotherapy of pancreatic cancer., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Early metastasis of pancreatic cancer (PC) leads to high mortality, and the underlying mechanism of metastasis remains unclear. Tumor necrosis factor superfamily member 9 (TNFSF9) is associated with poor prognosis in PC. Here, we investigated the effect of TNFSF9 on PC proliferation and apoptosis, and focused on the effect of TNFSF9 on PC metastasis and its potential mechanism. We found that TNFSF9 promotes PC metastasis in vivo and in vitro , and may be partially dependent on the Wnt/Snail signaling pathway. In addition, TNFSF9 also regulates the release of cytokines IL-10 and transforming growth factor-β (TGF-β) in pancreatic cancer cells through Wnt signaling to induce the M2 polarization of macrophages and promote the migration of PC cells. Overall, our study found that TNFSF9 may directly promote PC metastasis or indirectly promote PC metastasis through macrophage M2 polarization. Our study provides a new costimulatory target for the treatment of PC.

Recombinant agonists that activate co-stimulatory and cytokine receptors have shown limited clinical anticancer utility, potentially due to narrow therapeutic windows, the need for coordinated activation of co-stimulatory and cytokine pathways and the failure of agonistic antibodies to recapitulate signaling by endogenous ligands. RTX-240 is a genetically engineered red blood cell expressing 4-1BBL and IL-15/IL-15Rα fusion (IL-15TP). RTX-240 is designed to potently and simultaneously stimulate the 4-1BB and IL-15 pathways, thereby activating and expanding T cells and NK cells, while potentially offering an improved safety profile through restricted biodistribution. We assessed the ability of RTX-240 to expand and activate T cells and NK cells and evaluated the in vivo efficacy, pharmacodynamics and tolerability using murine models. Treatment of PBMCs with RTX-240 induced T cell and NK cell activation and proliferation. In vivo studies using mRBC-240, a mouse surrogate for RTX-240, revealed biodistribution predominantly to the red pulp of the spleen, leading to CD8 + T cell and NK cell expansion. mRBC-240 was efficacious in a B16-F10 melanoma model and led to increased NK cell infiltration into the lungs. mRBC-240 significantly inhibited CT26 tumor growth, in association with an increase in tumor-infiltrating proliferating and cytotoxic CD8 + T cells. mRBC-240 was tolerated and showed no evidence of hepatic injury at the highest feasible dose, compared with a 4-1BB agonistic antibody. RTX-240 promotes T cell and NK cell activity in preclinical models and shows efficacy and an improved safety profile. Based on these data, RTX-240 is now being evaluated in a clinical trial., (© 2021. The Author(s).)

Checkpoint inhibitors and T-cell therapies have highlighted the critical role of T cells in anti-cancer immunity. However, limitations associated with these treatments drive the need for alternative approaches. Here, we engineer red blood cells into artificial antigen-presenting cells (aAPCs) presenting a peptide bound to the major histocompatibility complex I, the costimulatory ligand 4-1BBL, and interleukin (IL)-12. This leads to robust, antigen-specific T-cell expansion, memory formation, additional immune activation, tumor control, and antigen spreading in tumor models in vivo. The presence of 4-1BBL and IL-12 induces minimal toxicities due to restriction to the vasculature and spleen. The allogeneic aAPC, RTX-321, comprised of human leukocyte antigen-A*02:01 presenting the human papilloma virus (HPV) peptide HPV16 E7 11-19 , 4-1BBL, and IL-12 on the surface, activates HPV-specific T cells and promotes effector function in vitro. Thus, RTX-321 is a potential 'off-the-shelf' in vivo cellular immunotherapy for treating HPV + cancers, including cervical and head/neck cancers.

While CD19-directed chimeric antigen receptor (CAR) T cells can induce remission in patients with B cell acute lymphoblastic leukemia (ALL), a large subset relapse with CD19 - disease. Like CD19, CD22 is broadly expressed by B-lineage cells and thus serves as an alternative immunotherapy target in ALL. Here we present the composite outcomes of two pilot clinical trials ( NCT02588456 and NCT02650414 ) of T cells bearing a 4-1BB-based, CD22-targeting CAR in patients with relapsed or refractory ALL. The primary end point of these studies was to assess safety, and the secondary end point was antileukemic efficacy. We observed unexpectedly low response rates, prompting us to perform detailed interrogation of the responsible CAR biology. We found that shortening of the amino acid linker connecting the variable heavy and light chains of the CAR antigen-binding domain drove receptor homodimerization and antigen-independent signaling. In contrast to CD28-based CARs, autonomously signaling 4-1BB-based CARs demonstrated enhanced immune synapse formation, activation of pro-inflammatory genes and superior effector function. We validated this association between autonomous signaling and enhanced function in several CAR constructs and, on the basis of these observations, designed a new short-linker CD22 single-chain variable fragment for clinical evaluation. Our findings both suggest that tonic 4-1BB-based signaling is beneficial to CAR function and demonstrate the utility of bedside-to-bench-to-bedside translation in the design and implementation of CAR T cell therapies.

Background: Adoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells has shown remarkable efficacy for the treatment of hematologic malignancies. The primary mechanism of action of these infused T cells is the direct killing of tumor cells expressing the cognate antigen. However, understanding why only some T cells are capable of killing, and identifying mechanisms that can improve killing has remained elusive., Methods: To identify molecular and cellular mechanisms that can improve T-cell killing, we utilized integrated high-throughput single-cell functional profiling by microscopy, followed by robotic retrieval and transcriptional profiling., Results: With the aid of mathematical modeling we demonstrate that non-killer CAR T cells comprise a heterogeneous population that arise from failure in each of the discrete steps leading to the killing. Differential transcriptional single-cell profiling of killers and non-killers identified CD137 as an inducible costimulatory molecule upregulated on killer T cells. Our single-cell profiling results directly demonstrate that inducible CD137 is feature of killer (and serial killer) T cells and this marks a different subset compared with the CD107a pos (degranulating) subset of CAR T cells. Ligation of the induced CD137 with CD137 ligand (CD137L) leads to younger CD19 CAR T cells with sustained killing and lower exhaustion. We genetically modified CAR T cells to co-express CD137L, in trans, and this lead to a profound improvement in anti-tumor efficacy in leukemia and refractory ovarian cancer models in mice., Conclusions: Broadly, our results illustrate that while non-killer T cells are reflective of population heterogeneity, integrated single-cell profiling can enable identification of mechanisms that can enhance the function/proliferation of killer T cells leading to direct anti-tumor benefit., Competing Interests: Competing interests: LJNC is the CEO of Ziopharm. LJNC and NV are founders of CellChorus., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: TNFSF9 gene has been found to play an anti-tumor role and regulate the function of immune cells. However, the prognostic role of TNFSF9 in pancreatic cancer and its relationship with immune cell infiltration have not been studied., Methods: We used Oncomine, UALCAN, and GEPIA databases to analyze the expression of TNFSF9 in pancreatic cancer. We used Kaplan-Meier plotters, GEPIA, and UALCAN to evaluate the effect of TNFSF9 on clinical prognosis. We further used TIMER to study the correlation between TNFSF9 and cancer immune infiltrate cells. In addition, we used GEPIA to analyze the correlation between TNFSF9 expression and gene markers of immune infiltrate cells., Results: TNFSF9 mRNA expression level was remarkably increased in pancreatic cancer than that in normal tissues (both P < 0.05). In addition, high TNFSF9 expression was significantly related to poor overall survival (OS) and relapse-free survival (RFS) in pancreatic cancer (OS HR = 2.02, P = 0.0012; RFS HR = 2.63, P = 0.022). Moreover, high TNFSF9 expression in pancreatic cancer patients was associated with worse OS in stage 1 to 2 but not stage 3 and stage 4. Specifically, TNFSF9 expression and CD8 + T cell infiltration of pancreatic cancer was negatively correlated. TNFSF9 expression showed strong correlations with M1 macrophages in pancreatic cancer., Conclusions: Our results suggest that TNFSF9 is associated with prognosis and CD8 + T cell infiltration levels in patients with pancreatic cancer. Further, TNFSF9 expression potentially contributes to the modulation of M1 polarization of macrophages. These findings indicate that TNFSF9 can be serves as a prognostic biomarker in determining the prognosis of pancreatic cancer and is associated with different types of phenotypes of immune cell infiltration.

We have established an immune cell therapy with immortalized induced pluripotent stem-cell-derived myeloid lines (iPS-ML). The benefits of using iPS-ML are the infinite proliferative capacity and ease of genetic modification. In this study, we introduced 4-1BBL gene to iPS-ML (iPS-ML-41BBL). The analysis of the cell-surface molecules showed that the expression of CD86 was upregulated in iPS-ML-41BBL more than that in control iPS-ML. Cytokine array analysis was performed using supernatants of the spleen cells that were cocultured with iPS-ML or iPS-ML-41BBL. Multiple cytokines that are beneficial to cancer immunotherapy were upregulated. Peritoneal injections of iPS-ML-41BBL inhibited tumor growth of peritoneally disseminated mouse melanoma and prolonged survival of mice compared to that of iPS-ML. Furthermore, the numbers of antigen-specific CD8 + T cells were significantly increased in the spleen and tumor tissues treated with epitope peptide-pulsed iPS-ML-41BBL compared to those treated with control iPS-ML. The number of CXCR6-positive T cells were increased in the tumor tissues after treatment with iPS-ML-41BBL compared to that with control iPS-ML. These results suggest that iPS-ML-41BBL could activate antigen-specific T cells and promote their infiltration into the tumor tissues. Thus, iPS-ML-41BBL may be a candidate for future immune cell therapy aiming to change immunological "cold tumor" to "hot tumor".

CD137 is a costimulatory molecule expressed on activated T cells. CD137 ligand (CD137L) is expressed by antigen presenting cells (APC), which use the CD137-CD137L system to enhance immune responses. It was, therefore, surprising to discover CD137 expression on regulatory T cells (Treg). The function of CD137 in Treg are controversial. While some studies report that CD137 signalling converts Treg to effector T cells (Teff), other studies find that CD137-expressing Treg display a stronger inhibitory activity than CD137 - Treg. Here, we describe that CD137 on Treg binds to CD137L on APC, upon which one of the two molecules is transferred via trogocytosis to the other cell, where CD137-CD137L forms a complex that is internalized and deprives APC of the immune-stimulatory CD137L. Truncated forms of CD137 that lack the cytoplasmic domain of CD137 are also able to downregulate CD137L, demonstrating that CD137 signalling is not required. Comparable data have been obtained with human and murine cells, indicating that this mechanism is evolutionarily conserved. These data describe trogocytosis of CD137 and CD137L as a new mechanism employed by Treg to control immune responses by downregulating the immunostimulatory CD137L on APC.

Background Human cancers are extraordinarily heterogeneous in terms of tumor antigen expression, immune infiltration and composition. A common feature, however, is the host's inability to mount potent immune responses that prevent tumor growth effectively. Often, naturally primed CD8 + T cells against solid tumors lack adequate stimulation and efficient tumor tissue penetration due to an immune hostile tumor microenvironment. Methods To address these shortcomings, we cloned tumor-associated antigens (TAA) and the immune-stimulatory ligand 4-1BBL into the genome of modified vaccinia Ankara (MVA) for intratumoral virotherapy. Results Local treatment with MVA-TAA-4-1BBL resulted in control of established tumors. Intratumoral injection of MVA localized mainly to the tumor with minimal leakage to the tumor-draining lymph node. In situ infection by MVA-TAA-4-1BBL triggered profound changes in the tumor microenvironment, including the induction of multiple proinflammatory molecules and immunogenic cell death. These changes led to the reactivation and expansion of antigen-experienced, tumor-specific cytotoxic CD8 + T cells that were essential for the therapeutic antitumor effect. Strikingly, we report the induction of a systemic antitumor immune response including tumor antigen spread by local MVA-TAA-4-1BBL treatment which controlled tumor growth at distant, untreated lesions and protected against local and systemic tumor rechallenge. In all cases, 4-1BBL adjuvanted MVA was superior to MVA. Conclusion Intratumoral 4-1BBL-armed MVA immunotherapy induced a profound reactivation and expansion of potent tumor-specific CD8 + T cells as well as favorable proinflammatory changes in the tumor microenvironment, leading to elimination of tumors and protective immunological memory., Competing Interests: Competing interests: MH, RG, FG, GF, BB, SW, PC, MS, HL, HH and JM-E are or have been employees of Bavarian Nordic. IM reports advisory roles with Roche-Genentech, Bristol-Myers Squibb, CYTOMX, Incyte, MedImmune, Tusk, F-Star, Genmab, Molecular Partners, Alligator, Bioncotech, MSD, Merck Serono, Boehringer Ingelheim, Astra Zeneca, Numab, Catalym, Bayer, and PharmaMar, and research funding from Roche, BMS, Alligator, and Bioncotech. PB reports advisory roles with Ferring, Tusk and Moderna, research funding from Sanofi, and Bavarian Nordic and speaker honoraria from Ferring, BMS, MSD, Novartis, Boehringer Ingelheim and AstraZeneca., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The tumor microenvironment (TME) contains noncancerous cells such as various types of immune cells and fibroblasts. Cancer cells direct these stromal cells to create a microenvironment favorable for tumor growth and intercellular interactions have a critical role in this process. In established tumors, interactions between CD137 and its ligand (CD137L) contribute to tumor immune evasion and tumor growth. Therefore, it is important to identify cells expressing CD137 and CD137L within tumors. In this chapter, we will introduce a simple, powerful method of analyzing CD137- and CD137L-expressing tumor cells using Fluorescence-activated cell sorting.

T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have produced impressive outcomes for the treatment of B cell malignancies, but different products vary in kinetics, persistence, and toxicity profiles based on the co-stimulatory domains included in the CAR. In this study, we performed transcriptional profiling of bulk CAR T cell populations and single cells to characterize the transcriptional states of human T cells transduced with CD3ζ, 4-1BB-CD3ζ (BBζ), or CD28-CD3ζ (28ζ) co-stimulatory domains at rest and after activation by triggering their CAR or their endogenous T cell receptor (TCR). We identified a transcriptional signature common across CARs with the CD3ζ signaling domain, as well as a distinct program associated with the 4-1BB co-stimulatory domain at rest and after activation. CAR T cells bearing BBζ had increased expression of human leukocyte antigen (HLA) class II genes, ENPP2, and interleukin (IL)-21 axis genes, and decreased PD1 compared to 28ζ CAR T cells. Similar to previous studies, we also found BBζ CAR CD8 T cells to be enriched in a central memory cell phenotype and fatty acid metabolism genes. Our data uncovered transcriptional signatures related to costimulatory domains and demonstrated that signaling domains included in CARs uniquely shape the transcriptional programs of T cells., (Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

The activation of 41BB costimulatory signals by agonistic Abs enhances the expansion and function of tumor-infiltrating lymphocytes (TILs) for treating cancer patients with adoptive cell therapy. However, the impact of 41BB agonism is not limited to enhancing the activity of T cells, and the mechanism by which additional activation of this costimulatory axis in tumor-associated myeloid cells is poorly understood. In this study, we describe that the intratumoral administration of 41BB agonistic Abs led to increases in CD8 T cell infiltration followed by tumor regression in murine models. We found that granulocytes and monocytes rapidly replaced macrophages and dendritic cells in tumors following administration of anti-41BB Abs. Overall, myeloid cells from anti-41BB-treated tumors had an improved capacity to stimulate T cells in comparison with control-treated tumors. In human coculture systems, we demonstrated that the agonism of the 41BB-41BBL axis enhanced costimulatory signals and effector functions among APC and autologous TILs. Overall, these findings suggest that the effect of 41BB agonistic Abs are supported by additional costimulatory signals from tumor-associated myeloid cells,v leading to enhanced TIL expansion and function., (Copyright © 2020 by The American Association of Immunologists, Inc.)

The potent costimulatory effect of CD137 has been implicated in several murine autoimmune disease models. CD137 costimulates and polarizes antigen-specific T cells toward a potent Th1/Tc1 response, and is essential for the development of experimental autoimmune encephalomyelitis (EAE), a murine model of Multiple Sclerosis (MS). This study aimed to investigate a role of CD137 in MS. Immunohistochemical and immunofluorescence staining of MS brain tissues was used to identify expression of CD137. CD137 + cells were identified in MS brain samples, with active lesions having the highest frequency of CD137 + cells. CD137 expression was found on several leukocyte subsets, including T cells, B cells and endothelial cells. In particular, CD137 + B cells were found in meningeal infiltrates. In vitro experiments showed that CD137 engagement on activated B cells increased early TNF and persistent IL-6 secretion with increased cell proliferation. These CD137 + B cells could interact with CD137L-expressing cells, secrete pro-inflammatory cytokines and accumulate in the meningeal infiltrate. This study demonstrates CD137 expression by activated B cells, enhancement of the inflammatory activity of B cells upon CD137 engagement, and provides evidence for a pathogenic role of CD137 + B cells in MS., (Copyright © 2020 Wong, Prasad, Gan, Chua and Schwarz.)

We constructed the CS1-targeted second- and third-generation CAR-T cells with genetic engineered 4-1BB or/and ICOS as a costimulatory signaling molecule by use of lentiviral platform. The CS1-targeted second-generation CAR-T cells with ICOS or 4-1BB had similar anti-neoplastic activity. When effector/target ratio was 1:1, the CAR-T cells with ICOS showed better killing effect on IM9-lucgfp cells than those with 4-1BB. However, The CS1-targeted third-generation CAR-T cells exihibited lower cytolytic capacity against IM9-lucgfp cells than the CS1-targeted second-generation CAR-T cells when the ratio of effector/target was 1:1, 2:1 or 5:1. When the ratio of effector/target was 10:1, the killing efficacy of both the second- and third-generation CAR-T cells against IM9-lucgfp cells was more than 85%, significantly higher than that of the control T cells. Taken together, both the CS1-targeted second- and third-generation CAR-T cells with ICOS or/and 4-1BB could efficiently kill CS1-positive multiple myeloma cells, but the CS1-targeted second-generation CAR-T cells had more potent killing effect on CS1-positive multiple myeloma cells than the CS1-targeted third-generation CAR-T cells.

Background: Adoptive NK cell infusion is a promising immunotherapy for acute myeloid leukemia (AML) patients. The aim of this study was to test the activity of clinical-grade membrane-bound IL-21/4-1BBL-expanded NK cell products against AML in vivo., Methods: Fresh peripheral blood mononuclear cells (PBMCs) were incubated with equal numbers of irradiated membrane-bound IL-21/4-1BBL-expressing K562 cells for 2-3 weeks to induce clinical-grade NK cell expansion., Results: Expansion for 2 and 3 weeks produced ∼4 and 8 × 10 9 NK cells from 2 × 10 7 PBMCs. The production of CD107a and TNF-α in NK cell products in response to AML cell lines and primary blasts was higher than that observed in resting NK cells. The 2-week expanded NK cell products were xenografted into immunodeficient mice with leukemia and were persistently found in the BM, spleen, liver, lung, and peripheral blood for at least 13 days; furthermore, these expanded products reduced the AML burden in vivo. Compared with matched AML patients with persistent or relapsed minimal residual disease (MRD + ) who underwent regular consolidation therapy, MRD + patients who underwent NK treatment had better overall survival and showed no major adverse events., Conclusions: Clinical-grade mbIL-21/4-1BBL-expanded NK cells exhibited antileukemic activity against AML in vitro and in vivo., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

CD137 (TNFRSF9, 4-1BB) is a potent co-stimulatory molecule of the tumour necrosis factor receptor superfamily (TNFRSF) that is expressed by activated T cells. CD137/CD137 ligand (CD137L) signalling primarily induces a potent cell-mediated immune response, while signalling of cell surface-expressed CD137L into antigen presenting cells enhances their activation, differentiation and migratory capacity. Studies have shown that bidirectional CD137/CD137L signalling plays an important role in the pathogenesis of autoimmune diseases. This review discusses the mechanisms how CD137/CD137L signalling contributes to immune deviation of helper T cell pathways in various murine models, and the potential of developing immunotherapies targeting CD137/CD137L signalling for the treatment of autoimmune diseases., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Aim: CD19 chimeric antigen receptor (CAR) T cells have been approved by the US FDA for treatment of relapsed and refractory (R/R) B-cell malignancies. Patients & methods: This study investigated the safety and efficacy of autologous 4-1BB costimulatory domain-engineered CD19 CAR-T cells in R/R B-cell lymphoma. Results: After CD19 CAR-T-cell infusion, severe cytokine release syndrome occurred in 28.6% (4/14) of the patients. The overall response rate was 77% with complete remission observed in 6/14 patients at 3 months. A higher tumor burden and grade 3-4 of myelosuppression after chemotherapy were associated with severe cytokine-release syndrome. Notably, combining CD19 CAR-T cells and PD-1 blockade, but not CD19 CAR-T cells alone, reduced intracranial tumor burden in a patient with central invasion of lymphoma. Conclusion: CD19 CAR-T cells could effectively induce tumor remission and PD-1 blockade might improve the efficacy in Chinese patients with R/R B-cell lymphoma.

Background: The costimulatory receptor 4-1BB (CD137, TNFRSF9) plays an important role in sustaining effective T cell immune responses and is investigated as target for cancer therapy. Systemic 4-1BB directed therapies elicit toxicity or low efficacy, which significantly hampered advancement of 4-1BB-based immunotherapy. Therefore, targeted delivery of 4-1BB agonist to the tumor side is needed for eliciting antitumor efficacy while avoiding systemic toxicity., Methods: We analyzed the immunostimulatory properties of a fibroblast activation protein (FAP)-targeted 4-1BB agonist (FAP-4-1BBL) by assessing tumor-infiltrating lymphocytes' (TIL) activity from patients with non-small cell lung cancer and epithelial ovarian cancer., Results: Combination treatment with FAP-4-1BBL and T cell receptor stimulation by either anti-CD3 or T cell bispecific antibodies significantly enhanced TIL activation and effector functions, including T cell proliferation, secretion of proinflammatory cytokines and cytotoxicity. Notably, costimulation with FAP-4-1BBL led to de novo secretion of interleukin (IL)-13. This was associated with cytokine-mediated tumor cell apoptosis, which was partially dependent on IL-13 alpha 1/2 receptors and STAT6 phosphorylation., Conclusions: Our study provides mechanistic insights into T cell stimulation induced by FAP-4-1BBL in primary human tumors and supports the investigation of FAP-4-1BBL compound in early clinical trials., Competing Interests: Competing interests: HL, SR and AZ received research funding from Bristol-Myers Squibb. FU, PH and AZ received research funding from Roche Innovation Center Zurich. PU, CK, MB, VK, MA, CC and CF-K are employed by Roche Innovation Center Zurich and declare ownership of stock and patents with Roche. RA was employed by Roche Innovation Center Zurich. CC declares ownerships of patents. AZ received honoraria from Bristol-Myers Squibb, Merck Sharp & Dohme, Hoffmann–La Roche, NBE Therapeutics, Secarna, ACM Pharma and Hookipa. AZ maintains non-commercial research agreements with Hoffmann–La Roche. AZ maintains further non-commercial research agreements with NBE Therapeutics, Secarna, ACM Pharma, Hookipa, and BeyondSpring., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

CD137 modulates type 1 diabetes (T1D) progression in NOD mice. We previously showed that CD137 expression in CD4 T cells inhibits T1D, but its expression in CD8 T cells promotes disease development by intrinsically enhancing the accumulation of β-cell-autoreactive CD8 T cells. CD137 is expressed on a subset of FOXP3 + regulatory CD4 T cells (Tregs), and CD137 + Tregs are the main source of soluble CD137. Soluble CD137 suppresses T cells in vitro by binding to the CD137 ligand (CD137L) upregulated on activated T cells. To further study how the opposing functions of CD137 are regulated, we successfully targeted Tnfsf9 (encoding CD137L) in NOD mice using the CRISPR/Cas9 system (designated NOD .Tnfsf9 -/- ). Relative to wild-type NOD mice, T1D development in the NOD .Tnfsf9 -/- strain was significantly delayed, and mice developed less insulitis and had reduced frequencies of β-cell-autoreactive CD8 T cells. Bone marrow chimera experiments showed that CD137L-deficient hematopoietic cells were able to confer T1D resistance. Adoptive T cell transfer experiments showed that CD137L deficiency on myeloid APCs was associated with T1D suppression. Conversely, lack of CD137L on T cells enhanced their diabetogenic activity. Furthermore, neither CD137 nor CD137L was required for the development and homeostasis of FOXP3 + Tregs. However, CD137 was critical for the in vivo T1D-suppressive activity of FOXP3 + Tregs, suggesting that the interaction between CD137 and CD137L regulates their function. Collectively, our results provide new insights into the complex roles of CD137-CD137L interaction in T1D., (Copyright © 2020 by The American Association of Immunologists, Inc.)

4-1BBL, a member of the TNF superfamily, regulates the sustained production of inflammatory cytokines in macrophages triggered by TLR signaling. In this study, we have investigated the role of 4-1BBL in macrophage metabolism and polarization and in skin inflammation using a model of imiquimod-induced psoriasis in mice. Genetic ablation or blocking of 4-1BBL signaling by Ab or 4-1BB-Fc alleviated the pathology of psoriasis by regulating the expression of inflammatory cytokines associated with macrophage activation and regulated the polarization of macrophages in vitro. We further linked this result with macrophage by finding that 4-1BBL expression during the immediate TLR response was dependent on glycolysis, mitochondrial oxidative phosphorylation, and fatty acid metabolism, whereas the late-phase 4-1BBL-mediated sustained inflammatory response was dependent on glycolysis and fatty acid synthesis. Correlating with this, administration of a fatty acid synthase inhibitor, cerulenin, also alleviated the pathology of psoriasis. We further found that 4-1BBL-mediated psoriasis development is independent of its receptor 4-1BB, as a deficiency of 4-1BB augmented the severity of psoriasis linked to a reduced regulatory T cell population and increased IL-17A expression in γδ T cells. Additionally, coblocking of 4-1BBL signaling and IL-17A activity additively ameliorated psoriasis. Taken together, 4-1BBL signaling regulates macrophage polarization and contributes to imiquimod-induced psoriasis by sustaining inflammation, providing a possible avenue for psoriasis treatment in patients., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Obtaining sufficient numbers of functional natural killer (NK) cells is crucial for the success of NK-cell-based adoptive immunotherapies. While expansion from peripheral blood (PB) is the current method of choice, ex vivo generation of NK cells from hematopoietic stem and progenitor cells (HSCs) may constitute an attractive alternative. Thereby, HSCs mobilized into peripheral blood (PB-CD34 + ) represent a valuable starting material, but the rather poor and donor-dependent differentiation of isolated PB-CD34 + cells into NK cells observed in earlier studies still represents a major hurdle. Here, we report a refined approach based on ex vivo culture of PB-CD34 + cells with optimized cytokine cocktails that reliably generates functionally mature NK cells, as assessed by analyzing NK-cell-associated surface markers and cytotoxicity. To further enhance NK cell expansion, we generated K562 feeder cells co-expressing 4-1BB ligand and membrane-anchored IL-15 and IL-21. Co-culture of PB-derived NK cells and NK cells that were ex-vivo-differentiated from HSCs with these feeder cells dramatically improved NK cell expansion, and fully compensated for donor-to-donor variability observed during only cytokine-based propagation. Our findings suggest mobilized PB-CD34 + cells expanded and differentiated according to this two-step protocol as a promising source for the generation of allogeneic NK cells for adoptive cancer immunotherapy.

Anti-tumor immune response and the prognosis of tumor are the results of competition between stimulatory and inhibitory checkpoints. Except for upregulating inhibitory checkpoints, lowering some immune accelerating molecules to convert an immunostimulatory microenvironment into an immunodormant one through "decelerating the accelerator" might be another effective immune escape pattern. 4-1BBL is a classical transmembrane costimulatory molecule involving in antitumor immune responses. In contrast, we demonstrated that 4-1BBL is predominantly localized in the nuclei of cancer cells in colon cancer specimens and is positively correlated with tumor size, lymph node metastasis, and a lower survival ratio. Furthermore, the nuclear localization of 4-1BBL was also ascertained in vitro. 4-1BBL knockout (KO) arrests the proliferation and impaired the migration and invasion ability of colon cancer cells in vitro and retarded tumor growth in vivo. 4-1BBL KO increased the accumulation of Gsk3β in the nuclei of colon cancer cells and consequently decreased the expression of Wnt pathway target genes and thus alter tumor biological behavior. We hypothesized that unlike membrane-expressed 4-1BBL, which stimulates the 4-1BB signaling of antitumor cytotoxic T cells, the nuclear-localized 4-1BBL could facilitate the malignant behavior of colon cancer cells by circumventing antitumor signaling and driving some key oncotropic signal pathway in the nucleus. Nuclear-localized 4-1BBL might be an indicator of colon cancer malignancy and serve as a promising target of immunotherapy.

Background : The immune-inducing effect of the quorum sensing (QS) molecule autoinducer-2 (AI-2) on macrophages has not been previously comprehensively studied. Methods : We performed proteomic analysis on macrophages cocultured with purified Fusobacterium nucleatum ( F. nucleatum ) AI-2 and performed western blot analysis to verify the differential protein expression. We further used the Gene Expression Profiling Interactive Analysis and Tumor Immune Estimation Resource databases to analyze the expression of differentially expressed proteins in microbial-associated digestive tract tumors. Results : Based on proteomic analysis, we identified 46 upregulated proteins and 11 downregulated proteins. The upregulated proteins were mostly inflammatory factors such as tumor necrosis factor ligand superfamily member 9 (TNFSF9). These proteins have a range of biological functions associated with the regulation of inflammatory responses, apoptosis and tumorigenesis. TNFSF9 is highly expressed in pancreatic adenocarcinoma (PAAD) tissues and is associated with M1 polarization of macrophages. Conclusions : Our data indicated that F. nucleatum AI-2 induced inflammatory responses and activated multiple signaling pathways in macrophages. TNFSF9 is the most significantly differentially expressed protein induced by F. nucleatum AI-2 and is involved in regulating immune cell infiltration in PAAD. Thus, AI-2 may become a new focus for studying the relationship between bacteria and cancer.

Background: Angiogenesis in atherosclerotic plaque is an important factor causing plaque hemorrhage, vulnerability, and rupture, and different phenotypes of macrophages have different effects on angiogenesis. Our previous study has demonstrated CD137-CD137L signaling, a pair of inflammatory costimulatory molecules, can promote angiogenesis in atherosclerotic plaque. Therefore, we aimed to investigate whether this signaling could affect angiogenesis by regulating phenotypic transition of macrophages., Methods: Male mouse primary peritoneal macrophages were extracted by intraperitoneal injection of thioglycollate, and then flow cytometry was used to detect the expression of CD137. Flow cytometry, Western blotting, quantitative real-time PCR, and enzyme-linked immunosorbent assay (ELISA) were used to assess the phenotypic changes of macrophages after different treatment. Mouse brain microvascular endothelial cells (bEnd.3) were cocultured with macrophages, and tube formation was assessed to examine angiogenesis., Results: We found that the number of junctions and branches of bEnd.3 were increased when CD137-CD137L signaling was activated, while such number was further increased when bEnd.3 were cocultured with macrophages. Flow cytometry showed that CD137 was expressed on almost all primary peritoneal macrophages. The expression of CD86 was decreased in the agonist CD137L group and increased in the agonist CD137L + inhibitory anti-CD137 antibody group after adding the CD137 inhibitor. The expression of CD206 in each group exhibited opposite trend compared with CD86. Moreover, the expression of inducible nitric oxide synthase at the mRNA, and protein levels were decreased after stimulating CD137-CD137L signaling, and such downward trend was reversed when CD137-CD137L signaling was inhibited. Furthermore, the expression of arginase-1 was opposite to that of inducible nitric oxide synthase. Enzyme-linked immunosorbent assay indicated that the content of interleukin-12 (IL-12) in the supernatant of macrophages in the agonist CD137L group was lower than that in the control group, and its content in the inhibited group was higher than that in the activated group. The change of interleukin-10 (IL-10) content in macrophage supernatant was opposite to that of IL-12. When AKT serine/threonine kinase 1 (Akt1) inhibitor was used to inhibit the phenotypic transformation of macrophages induced by CD137-CD137L, the number of junctions and branches formed by bEnd.3 was decreased compared with the coculture group., Conclusions: These results indicated that CD137-CD137L signaling could promote angiogenesis by regulating phenotypic transition of macrophages of male mice.

Activated endothelial cells (ECs) play an important role in the development of atherosclerosis (AS) because they form neointima resulting from abnormal vascular smooth muscle cell (VSMC) hyperplasia. TET2 was recently reported as a major regulator of the phenotypic switch of VSMCs, and it also protects ECs from noxious stimuli and represses inflammation in AS. The purpose of the present study is to determine whether activation of CD137 signaling can regulate the function of VSMCs by altering endothelial TET2 expression and to explore the potential mechanisms. VSMCs were isolated from C57BL/6 J mice, and the influence of ECs on VSMCs was analyzed using a co-culture system. Western blotting was used to detect the protein expression levels of TET2 and CD9, CD81, and CD63 as well as VSMC phenotypic markers. RT-PCR was performed to detect the TET2 mRNA levels. EdU proliferation assay and transwell migration assay were employed to assess the functions of VSMCs. EXO-spin kits were used to extract and purify exosomes. Transmission electron microscopy and nanoparticle tracking analysis were applied to characterize exosomes. ECs transduced with the TET2-overexpression lentivirus showed that the activation of endothelial CD137 signaling decreased TET2 expression and the TET2 content in exosomes. EC-derived exosomes were internalized by VSMCs, which inhibited phenotypic switching in vitro and neointimal formation in vivo. However, exosomes derived from CD137-activated ECs exhibited weakened protective effects on VSMCs. In conclusion, activation of endothelial CD137 signaling attenuated the repressive effects of EC-derived exosomes on platelet-derived growth factor (PDGF)-BB-induced phenotypic switching of VSMCs and neointimal formation after carotid injury., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: The expression of PD-L1 and its regulation in tumors remains unclear. The importance of IFN-γ in upregulating the PD-L1 expression in various tumors, and the effects of other essential cytokines in the tumor microenvironment (TME), need to be further elucidated., Methods: Constitutive expression of PD-L1 and CD137L in all 13 lung cancer cell lines were tested by flow cytometry. CD137L mRNA of lung cancer cell lines was detected by RT-PCR. PD-L1 expression rates following stimulation with these cytokines (IFN-γ, TNFα and IL2) were measured. After coculture of cells expressing CD137L (lung cancer cells or 293FT cells transfected with CD137L plasmid) with T cells, the PDL1 expression of lung cancer cells and IFN-γ in supernatant was detected., Results: Our data revealed that adenocarcinoma and squamous cell carcinoma cells had the highest positive expression rate. IFN-γ was the core-inducing factor for enhancing the PD-L1 expression. CD137L was also widely expressed in the lung cancer cell lines at the mRNA level, whereas its expression was generally low at the protein level. However, the low expression of CD137L protein was still enough to induce T cells to produce IFN-γ, which subsequently increased the PD-L1 expression by lung cancer cells. The CD137 signal induces IFN-γ secretion by T cells, which stimulates high-level of PD-L1 expression in cancer cells; this negative immune regulation may represent a mechanism of immune escape regulation., Conclusions: CD137L mRNA was widely expressed in lung cancer cell lines whereas levels of protein expression were generally low. The low level of CD137L protein was still enough to induce T cells to produce IFN-γ that subsequently increased PD-L1 expression. The CD137L-induced negative immune regulation may represent a mechanism of immune escape., (© 2019 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Oncolytic viruses are an excellent platform for developing effective strategies in cancer immunotherapy. Several challenges remain in the use of viro-immunotherapy for cancer, such as the lack of costimulatory signals and negative regulation of immune checkpoints. In this study, we designed a novel adenovirus expressing a soluble fusion protein, programmed cell death protein 1 (PD-1)/CD137L, which contains the extracellular domains of PD-1 and CD137L at each terminus (Ad5-PC). Ad5-PC preserved the costimulatory activity of CD137L and facilitated the persistence of activated CD8 + T cells. Ad5-PC induced strikingly increased antitumor activity in both ascitic and subcutaneous hepatocellular carcinoma (HCC) tumor models, with 70% and 60% long-term cure rates, respectively. The improved antitumor effect of Ad5-PC was attributed to the sustained high-level lymphocyte activation and interferon (IFN)-γ production in the tumor microenvironment, and was essentially dependent on CD8 + T cells rather than natural killer (NK) cells. Moreover, Ad5-huPC-expressing human soluble PD-1/CD137L fusion protein was effective in suppressing tumor growth and improving survival in a humanized mouse model. We confirmed that Ad5-PC induced tumor-specific and systematic protection against tumor rechallenges at both in situ and distant sites. Thus, Ad5-PC harnesses several distinct functions to efficiently overcome several major hurdles of viro-immunotherapy., (Copyright © 2019 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Intracellular pathogens are subject to elimination by a cellular immune response, and were therefore under evolutionary pressure to develop mechanisms that allow them to inhibit especially this arm of immunity. CD137, a T cell costimulatory molecule, and its ligand, CD137 ligand (CD137L), which is expressed on antigen presenting cells (APC), are potent drivers of cellular cytotoxic immune responses. Here, we report that different viruses usurp a negative feedback mechanism for the CD137-CD137L system that weakens cellular immune responses. Latent membrane protein (LMP)-1 and Tax, oncogenes of Epstein-Barr virus (EBV), and human T-cell lymphotropic virus (HTLV)-1, respectively, induce the expression of CD137. CD137 is transferred by trogocytosis to CD137L-expressing APC, and the CD137-CD137L complex is internalized and degraded, resulting in a reduced CD137-mediated T cell costimulation and a weakened cellular immune response which may facilitate the escape of the virus from immune surveillance. These data identify the usurpation of a CD137-based negative feedback mechanism by intracellular pathogens that enables them to reduce T cell costimulation., (© 2019 Wiley Periodicals, Inc.)

Objective: To investigate whether CD137-CD137L signaling could affect the secretion of mouse vascular smooth muscle cells (VSMCs) -derived exosomes through autophagy mediated Rab7 pathway. Methods: Primary thoracic aorta VSMCs from C57BL/6J mouse were obtained by tissue block adherence method. VSMCs between the third to fifth passages were used and VSMCs were divided into 4 groups: control group, CD137 agonist group, lentivirus control group, Rab7 lentiviral interference group. VSMCs in CD137 agonist group were treated with recombinant protein of CD137L (10 μg/ml), VSMCs in lentivirus control group were treated with lentiviral followed by recombinant protein of CD137L (10 μg/ml), VSMCs in Rab7 lentiviral interference group were treated with Rab7 lentiviral intervention followed by recombinant protein of CD137L (10 μg/ml). Western blot was used to detect the protein expression of LC3Ⅱ, p62, Rab7, CD9, CD81 and Hsc70. Fluorescence microscopy was used to track the changes of autophagy in cells infected with mRFP-GFP-LC3. Transmission electron microscope was used to observe the morphology and size of VSMCs-derived exosomes. The nanoparticle tracking analysis(NTA) was used to detect the concentration and size of exosomes in each group. Results: (1) The expressions of Rab7, LC3Ⅱ and p62 protein in VSMCs of CD137 activation group were significantly higher than those in control group (all P< 0.05). The expressions of Rab7, LC3Ⅱ and p62 protein in Rab7 lentivirus interference group was lower than in CD137 activation group (all P< 0.05), while the expressions were similar between the lentivirus control group and the CD137 activation group (all P> 0.05). (2) The total number of fluorescent spots and yellow fluorescent spots in the VSMCs of the CD137 activation group were higher than those in the control group (all P< 0.05), and the number of yellow fluorescent spots was higher than that of the red fluorescent spots in the VSMCs of the CD137 activation group ((50.3±0.9) vs. (10.3±1.5)/cell). The total numbers of fluorescent spots and yellow fluorescent spots in VSMCs of Rab7 lentivirus interference group were lower than those of CD137 activation group (both P< 0.05), and the number of red fluorescent spots in VSMCs was higher than that of yellow fluorescent spots ((40.7±4.0) and (10.7±1.2)/cell) in the Rab7 lentiviral interference group. The total numbers of fluorescent spots and yellow fluorescent spots in the VSMCs were similar between the lentivirus control group and the CD137 activation group (all P> 0.05). (3) Under transmission electron microscopy, the size of the VSMCs-derived exosomes was about 30-150 nm. The exosome markers (CD9, CD81) could be detected in vesicles by Western blot. NTA results showed that the concentration of VSMCs-derived exosomes was significantly higher in the CD137-activated group than in the control group ( P< 0.05), which was significantly lower in the Rab7 lentiviral interference group than in the CD137-activation group ( P< 0.05) and was similar between the lentivirus control group and the CD137 activation group ( P> 0.05). The expression of Hsc70 protein in exosomes secreted by CD137 activation group was higher than that in the control group ( P< 0.05). The expression of Hsc70 protein in exosomes was lower in Rab7 lentivirus interference group than in the CD137 activation group ( P< 0.05), which was similar between the lentivirus control group and the CD137 activation group ( P> 0.05). The expression of LC3Ⅱ protein in exosome was higher in CD137 activation group than in control group ( P< 0.05), which was lower in Rab7 lentivirus interference group than in CD137 activation group ( P< 0.05), which was similar between the lentivirus control group and the CD137 activation group ( P> 0.05). Conclusion: The CD137-CD137L signaling may affect the secretion of mouse VSMCs-derived exosomes through modulating the Rab7 pathway mediated autophagy.

The effect of Fusobacterium nucleatum (F. nucleatum) autoinducer-2 (AI-2) on the polarization of macrophages and the underlying mechanism is not known. We investigated the effect of F. nucleatum AI-2 on the migration and polarization of cultured macrophages. We further screened AI-2-interacting proteins in macrophages using a quantitative proteomics strategy, and evaluated the expression of TNFSF9/TRAF1/p-AKT/IL-1β signaling in cultured macrophages and human colorectal cancer (CRC). The data showed that F. nucleatum AI-2 enhanced the mobility and M1 polarization of macrophages, possibly through TNFSF9/TRAF1/p-AKT/IL-1β signaling. Moreover, TNFSF9 and IL-1β expression was significantly increased in human CRCs when compared to normal colon (P < 0.05), and was associated with AI-2 concentration and increased survival. Together, our data suggested that AI-2 induced macrophage M1 polarization by activating the TNFSF9/IL-1β pathway. Thus, AI-2 may serve as a promising novel target for immunotherapy of gut microbiota-related diseases., (Copyright © 2019 Elsevier B.V. All rights reserved.)

To understand and dissect the mechanisms driving human NK cell proliferation, we exploited the methodology used in cell therapy to numerically expand NK cells in the presence of K562-derived artificial APC (aAPCs) and cytokines. For four consecutive weeks, high expression of CD137L by a K562-derived aAPC cell line could sustain NK cell expansion by 3 × 10 5 -fold, whereas low expression of CD137L by the parental K562 cell line only supported the expansion by 2 × 10 3 -fold. The level of expression of CD137L, however, did not modulate the sensitivity of K562 cells to the intrinsic cytotoxicity of NK cells. Similarly, the low NK cell proliferation in the presence of the parental K562 cell line and cytokines was increased by adding agonistic anti-CD137 Abs to levels similar to CD137L-expressing K562-derived aAPCs. Finally, synergy between IL-15 and IL-21 was observed only upon CD137 engagement and the presence of aAPCs. Therefore, we conclude that NK cell proliferation requires cell-to-cell contact, activation of the CD137 axis, and presence of IL-15 (or its membranous form) and IL-21. By analogy with the three-signal model required to activate T cells, we speculate that the cell-to-cell contact represents "signal 1," CD137 represents "signal 2," and cytokines represent "signal 3." The precise nature of signal 1 remains to be defined., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Immunotherapy holds promise for multiple myeloma (MM) patients but little is known about how MM-induced immunosuppression influences response to therapy. Here, we investigated the impact of disease progression on immunotherapy efficacy in the Vk*MYC mouse model. Treatment with agonistic anti-CD137 (4-1BB) mAbs efficiently protected mice when administered early but failed to contain MM growth when delayed more than three weeks after Vk*MYC tumor cell challenge. The quality of CD8+ T cell response to CD137 stimulation was not altered by the presence of MM, but CD8+ T cell numbers were profoundly reduced at the time of treatment. Our data suggest that an insufficient ratio of CD8+ T cells over MM cells (CD8/MM) accounts for the loss of anti-CD137 mAb efficacy. We established serum M-protein levels prior to therapy as a predictive factor of response. Moreover, we developed an in silico model to capture the dynamic interactions between CD8+ T cells and MM cells. Finally, we explored two methods to improve the CD8/MM ratio: anti-CD137 mAb immunotherapy combined with Treg-depletion or administered after chemotherapy treatment with cyclophosphamide or melphalan efficiently reduced MM burden and prolonged survival. Altogether, our data indicate that consolidation treatment with anti-CD137 mAbs might prevent MM relapse.