bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from An Anticancer Drug Cocktail of Three Kinase Inhibitors Improved Response to a Dendritic Cell–Based Cancer Vaccine
Authors: Guo Jitao , Muse Elena , Christians Allison J. , Swanson Steven J. , Davila Eduardo
Published: April 2023

Abstract
Monocyte-derived dendritic cell (moDC)–based cancer therapies intended to elicit antitumor T-cell responses have limited efficacy in most clinical trials. However, potent and sustained antitumor activity in a limited number of patients highlights the therapeutic potential of moDCs. In vitro culture conditions used to generate moDCs can be inconsistent, and moDCs generated in vitro are less effective than natural DCs. On the basis of our study highlighting the ability for certain kinase inhibitors to enhance tumor antigenicity, we therefore screened kinase inhibitors for their ability to improve DC immunogenicity. We identified AKT inhibitor MK2206, DNA-PK inhibitor NU7441, and MEK inhibitor trametinib as the compounds most effective at modulating moDC immunogenicity. The combination of these drugs, referred to as MKNUTRA, enhanced moDC activity over treatment with individual drugs while exhibiting minimal toxicity. An evaluation of 335 activation and T-cell–suppressive surface proteins on moDCs revealed that MKNUTRA treatment more effectively matured cells and reduced the expression of tolerogenic proteins as compared with control moDCs. MKNUTRA treatment imparted to ICT107, a glioblastoma (GBM) DC-based vaccine that has completed phase II trials, an increased ability to stimulate patient-derived autologous CD8+ T cells against the brain tumor antigens IL13Rα2(345-354) and TRP2(180-188). In vivo, treating ICT107 with MKNUTRA, prior to injection into mice with an established GBM tumor, reduced tumor growth kinetics. This response was associated with an increased frequency of tumor-reactive lymphocytes within tumors and in peripheral tissues. These studies broaden the application of targeted anticancer drugs and highlight their ability to increase moDC immunogenicity.
Data from Efficient Tumor Clearance and Diversified Immunity through Neoepitope Vaccines and Combinatorial Immunotherapy
Authors: Lee Karin L. , Benz Stephen C. , Hicks Kristin C. , Nguyen Andrew , Gameiro Sofia R. , Palena Claudia , Sanborn John Z. , Su Zhen , Ordentlich Peter , Rohlin Lars , Lee John H. , Rabizadeh Shahrooz , Soon-Shiong Patrick , Niazi Kayvan , Schlom Jeffrey , Hamilton Duane H.
Published: April 2023

Abstract
Progressive tumor growth is associated with deficits in the immunity generated against tumor antigens. Vaccines targeting tumor neoepitopes have the potential to address qualitative defects; however, additional mechanisms of immune failure may underlie tumor progression. In such cases, patients would benefit from additional immune-oncology agents targeting potential mechanisms of immune failure. This study explores the identification of neoepitopes in the MC38 colon carcinoma model by comparison of tumor to normal DNA and tumor RNA sequencing technology, as well as neoepitope delivery by both peptide- and adenovirus-based vaccination strategies. To improve antitumor efficacies, we combined the vaccine with a group of rationally selected immune-oncology agents. We utilized an IL15 superagonist to enhance the development of antigen-specific immunity initiated by the neoepitope vaccine, PD-L1 blockade to reduce tumor immunosuppression, and a tumor-targeted IL12 molecule to facilitate T-cell function within the tumor microenvironment. Analysis of tumor-infiltrating leukocytes demonstrated this multifaceted treatment regimen was required to promote the influx of CD8+ T cells and enhance the expression of transcripts relating to T-cell activation/effector function. Tumor-targeted IL12 resulted in a marked increase in clonality of T-cell repertoire infiltrating the tumor, which when sculpted with the addition of either a peptide or adenoviral neoepitope vaccine promoted efficient tumor clearance. In addition, the neoepitope vaccine induced the spread of immunity to neoepitopes expressed by the tumor but not contained within the vaccine. These results demonstrate the importance of combining neoepitope-targeting vaccines with a multifaceted treatment regimen to generate effective antitumor immunity.
Data from Siglec-9 Regulates an Effector Memory CD8+ T-cell Subset That Congregates in the Melanoma Tumor Microenvironment
Authors: Haas Quentin , Boligan Kayluz Frias , Jandus Camilla , Schneider Christoph , Simillion Cedric , Stanczak Michal A. , Haubitz Monika , Seyed Jafari Seyed Morteza , Zippelius Alfred , Baerlocher Gabriela M. , Läubli Heinz , Hunger Robert E. , Romero Pedro , Simon Hans-Uwe , von Gunten Stephan
Published: April 2023

Abstract
Emerging evidence suggests an immunosuppressive role of altered tumor glycosylation due to downregulation of innate immune responses via immunoregulatory Siglecs. In contrast, human T cells, a major anticancer effector cell, only rarely express Siglecs. However, here, we report that the majority of intratumoral, but not peripheral blood, cytotoxic CD8+ T cells expressed Siglec-9 in melanoma. We identified Siglec-9+ CD8+ T cells as a subset of effector memory cells with high functional capacity and signatures of clonal expansion. This cytotoxic T-cell subset was functionally inhibited in the presence of Siglec-9 ligands or by Siglec-9 engagement by specific antibodies. TCR signaling pathways and key effector functions (cytotoxicity, cytokine production) of CD8+ T cells were suppressed by Siglec-9 engagement, which was associated with the phosphorylation of the inhibitory protein tyrosine phosphatase SHP-1, but not SHP-2. Expression of cognate Siglec-9 ligands was observed on the majority of tumor cells in primary and metastatic melanoma specimens. Targeting the tumor-restricted, glycosylation-dependent Siglec-9 axis may unleash this intratumoral T-cell subset, while confining T-cell activation to the tumor microenvironment.
Data from Blockade of β-Adrenergic Receptors Improves CD8+ T-cell Priming and Cancer Vaccine Efficacy
Authors: Daher Clara , Vimeux Lene , Stoeva Ralitsa , Peranzoni Elisa , Bismuth Georges , Wieduwild Elisabeth , Lucas Bruno , Donnadieu Emmanuel , Bercovici Nadège , Trautmann Alain , Feuillet Vincent
Published: April 2023

Abstract
β-Adrenergic receptor (β-AR) signaling exerts protumoral effects by acting directly on tumor cells and angiogenesis. In addition, β-AR expression on immune cells affects their ability to mount antitumor immune responses. However, how β-AR signaling impinges antitumor immune responses is still unclear. Using a mouse model of vaccine-based immunotherapy, we showed that propranolol, a nonselective β-blocker, strongly improved the efficacy of an antitumor STxBE7 vaccine by enhancing the frequency of CD8+ T lymphocytes infiltrating the tumor (TIL). However, propranolol had no effect on the reactivity of CD8+ TILs, a result further strengthened by ex vivo experiments showing that these cells were insensitive to adrenaline- or noradrenaline-induced AR signaling. In contrast, naïve CD8+ T-cell activation was strongly inhibited by β-AR signaling, and the beneficial effect of propranolol mainly occurred during CD8+ T-cell priming in the tumor-draining lymph node. We also demonstrated that the differential sensitivity of naïve CD8+ T cells and CD8+ TILs to β-AR signaling was linked to a strong downregulation of β2-AR expression related to their activation status, since in vitro–activated CD8+ T cells behaved similarly to CD8+ TILs. These results revealed that β-AR signaling suppresses the initial priming phase of antitumor CD8+ T-cell responses, providing a rationale to use clinically available β-blockers in patients to improve cancer immunotherapies.
Data from Differential Effects of Depleting versus Programming Tumor-Associated Macrophages on Engineered T Cells in Pancreatic Ductal Adenocarcinoma
Authors: Stromnes Ingunn M. , Burrack Adam L. , Hulbert Ayaka , Bonson Patrick , Black Cheryl , Brockenbrough J. Scott , Raynor Jackson F. , Spartz Ellen J. , Pierce Robert H. , Greenberg Philip D. , Hingorani Sunil R.
Published: April 2023

Abstract
Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy resistant to therapies, including immune-checkpoint blockade. We investigated two distinct strategies to modulate tumor-associated macrophages (TAM) to enhance cellular therapy targeting mesothelin in an autochthonous PDA mouse model. Administration of an antibody to colony-stimulating factor (anti-Csf1R) depleted Ly6Clow protumorigenic TAMs and significantly enhanced endogenous T-cell intratumoral accumulation. Despite increasing the number of endogenous T cells at the tumor site, as previously reported, TAM depletion had only minimal impact on intratumoral accumulation and persistence of T cells engineered to express a murine mesothelin-specific T-cell receptor (TCR). TAM depletion interfered with the antitumor activity of the infused T cells in PDA, evidenced by reduced tumor cell apoptosis. In contrast, TAM programming with agonistic anti-CD40 increased both Ly6Chigh TAMs and the intratumoral accumulation and longevity of TCR-engineered T cells. Anti-CD40 significantly increased the frequency and number of proliferating and granzyme B+ engineered T cells, and increased tumor cell apoptosis. However, anti-CD40 failed to rescue intratumoral engineered T-cell IFNγ production. Thus, although functional modulation, rather than TAM depletion, enhanced the longevity of engineered T cells and increased tumor cell apoptosis, ultimately, anti-CD40 modulation was insufficient to rescue key effector defects in tumor-reactive T cells. This study highlights critical distinctions between how endogenous T cells that evolve in vivo, and engineered T cells with previously acquired effector activity, respond to modifications of the tumor microenvironment.
Data from A High-Avidity T-cell Receptor Redirects Natural Killer T-cell Specificity and Outcompetes the Endogenous Invariant T-cell Receptor
Authors: Landoni Elisa , Smith Christof C. , Fucá Giovanni , Chen Yuhui , Sun Chuang , Vincent Benjamin G. , Metelitsa Leonid S. , Dotti Gianpietro , Savoldo Barbara
Published: April 2023

Abstract
T-cell receptor (TCR) gene transfer redirects T cells to target intracellular antigens. However, the potential autoreactivity generated by TCR mispairing and occurrence of graft-versus-host disease in the allogenic setting due to the retention of native TCRs remain major concerns. Natural killer T cells (NKT) have shown promise as a platform for adoptive T-cell therapy in cancer patients. Here, we showed their utility for TCR gene transfer. We successfully engineered and expanded NKTs expressing a functional TCR (TCR NKTs), showing HLA-restricted antitumor activity in xenogeneic mouse models in the absence of graft-versus-mouse reactions. We found that TCR NKTs downregulated the invariant TCR (iTCR), leading to iTCR+TCR+ and iTCR−TCR+ populations. In-depth analyses of these subsets revealed that in iTCR−TCR+ NKTs, the iTCR, although expressed at the mRNA and protein levels, was retained in the cytoplasm. This effect resulted from a competition for binding to CD3 molecules for cell-surface expression by the transgenic TCR. Overall, our results highlight the feasibility and advantages of using NKTs for TCR expression for adoptive cell immunotherapies. NKT-low intrinsic alloreactivity that associated with the observed iTCR displacement by the engineered TCR represents ideal characteristics for “off-the-shelf” products without further TCR gene editing.
Data from Broad Cytotoxic Targeting of Acute Myeloid Leukemia by Polyclonal Delta One T Cells
Authors: Di Lorenzo Biagio , Simões André E. , Caiado Francisco , Tieppo Paola , Correia Daniel V. , Carvalho Tânia , da Silva Maria Gomes , Déchanet-Merville Julie , Schumacher Ton N. , Prinz Immo , Norell Haakan , Ravens Sarina , Vermijlen David , Silva-Santos Bruno
Published: April 2023

Abstract
Acute myeloid leukemia (AML) remains a clinical challenge due to frequent chemotherapy resistance and deadly relapses. We are exploring the immunotherapeutic potential of peripheral blood Vδ1+ T cells, which associate with improved long-term survival of stem-cell transplant recipients but have not yet been applied as adoptive cell therapy. Using our clinical-grade protocol for expansion and differentiation of “Delta One T” (DOT) cells, we found DOT cells to be highly cytotoxic against AML primary samples and cell lines, including cells selected for resistance to standard chemotherapy. Unlike chemotherapy, DOT-cell targeting did not select for outgrowth of specific AML lineages, suggesting a broad recognition domain, an outcome that was consistent with the polyclonality of the DOT-cell T-cell receptor (TCR) repertoire. However, AML reactivity was only slightly impaired upon Vδ1+ TCR antibody blockade, whereas it was strongly dependent on expression of the NKp30 ligand, B7-H6. In contrast, DOT cells did not show reactivity against normal leukocytes, including CD33+ or CD123+ myeloid cells. Adoptive transfer of DOT cells in vivo reduced AML load in the blood and target organs of multiple human AML xenograft models and significantly prolonged host survival without detectable toxicity, thus providing proof-of-concept for DOT-cell application in AML treatment.
Data from Radiotherapy and Cisplatin Increase Immunotherapy Efficacy by Enabling Local and Systemic Intratumoral T-cell Activity
Authors: Kroon Paula , Frijlink Elselien , Iglesias-Guimarais Victoria , Volkov Andriy , van Buuren Marit M. , Schumacher Ton N. , Verheij Marcel , Borst Jannie , Verbrugge Inge
Published: April 2023

Abstract
To increase cancer immunotherapy success, PD-1 blockade must be combined with rationally selected treatments. Here, we examined, in a poorly immunogenic mouse breast cancer model, the potential of antibody-based immunomodulation and conventional anticancer treatments to collaborate with anti–PD-1 treatment. One requirement to improve anti-PD-1–mediated tumor control was to promote tumor-specific cytotoxic T-cell (CTL) priming, which was achieved by stimulating the CD137 costimulatory receptor. A second requirement was to overrule PD-1–unrelated mechanisms of CTL suppression in the tumor microenvironment (TME). This was achieved by radiotherapy and cisplatin treatment. In the context of CD137/PD-1–targeting immunotherapy, radiotherapy allowed for tumor elimination by altering the TME, rather than intrinsic CTL functionality. Combining this radioimmunotherapy regimen with low-dose cisplatin improved CTL-dependent regression of a contralateral tumor outside the radiation field. Thus, systemic tumor control may be achieved by combining immunotherapy protocols that promote T-cell priming with (chemo)radiation protocols that permit CTL activity in both the irradiated tumor and (occult) metastases.
Data from Activation of CD8+ T Cells Contributes to Antitumor Effects of CDK4/6 Inhibitors plus MEK Inhibitors
Authors: Teh Jessica L.F. , Erkes Dan A. , Cheng Phil F. , Tiago Manoela , Wilski Nicole A. , Field Conroy O. , Chervoneva Inna , Levesque Mitch P. , Xu Xiaowei , Dummer Reinhard , Aplin Andrew E.
Published: April 2023

Abstract
Concurrent MEK and CDK4/6 inhibition shows promise in clinical trials for patients with advanced-stage mutant BRAF/NRAS solid tumors. The effects of CDK4/6 inhibitor (CDK4/6i) in combination with BRAF/MEK-targeting agents on the tumor immune microenvironment are unclear, especially in melanoma, for which immune checkpoint inhibitors are effective in approximately 50% of patients. Here, we show that patients progressing on CDK4/6i/MEK pathway inhibitor combinations exhibit T-cell exclusion. We found that MEK and CDK4/6 targeting was more effective at delaying regrowth of mutant BRAF melanoma in immunocompetent versus immune-deficient mice. Although MEK inhibitor (MEKi) treatment increased tumor immunogenicity and intratumoral recruitment of CD8+ T cells, the main effect of CDK4/6i alone and in combination with MEKi was increased expression of CD137L, a T-cell costimulatory molecule on immune cells. Depletion of CD8+ T cells or blockade of the CD137 ligand–receptor interaction reduced time to regrowth of melanomas in the context of treatment with CDK4/6i plus MEKi treatment in vivo. Together, our data outline an antitumor immune-based mechanism and show the efficacy of targeting both the MEK pathway and CDK4/6.
Data from Mammary Tumor Cells with High Metastatic Potential Are Hypersensitive to Macrophage-Derived HGF
Authors: Kitamura Takanori , Kato Yu , Brownlie Demi , Soong Daniel Y.H. , Sugano Gaël , Kippen Nicolle , Li Jiufeng , Doughty-Shenton Dahlia , Carragher Neil , Pollard Jeffrey W.
Published: April 2023

Abstract
Metastasis-associated macrophages (MAM) promote persistent growth of breast cancer cells at the metastatic site and are, thus, an attractive therapeutic target to treat breast cancer metastasis, a leading cause of cancer-related death in women. However, the precise mechanisms behind MAM-mediated metastatic tumor outgrowth have not been fully elucidated. Using mouse models of metastatic breast cancer, we showed that MAMs uniquely expressed hepatocyte growth factor (HGF) in metastatic tumors. We also demonstrated that a selected population of cancer cells with high metastatic potential (cancer cells that can establish metastatic tumors in mice with higher number and incidence than parental cells) had higher expression of HGF receptor, MNNG HOS transforming gene (MET), and were more responsive to HGF released from macrophages compared with the parental cells. Blockade of MET signaling in cancer cells suppressed metastatic tumor expansion, in part, through activation of natural killer cells. Results from this study suggest an approach to prevent life-threatening metastatic tumor formation using blockade of MAM-induced MET signal activation in metastatic cancer cells.
Data from Cross-dressing of CD8α+ Dendritic Cells with Antigens from Live Mouse Tumor Cells Is a Major Mechanism of Cross-priming
Authors: Das Mohapatra Alok , Tirrell Isaac , Bénéchet Alexandre P. , Pattnayak Shashmita , Khanna Kamal M. , Srivastava Pramod K.
Published: April 2023

Abstract
Live cells are the most abundant sources of antigen in a tumor-bearing host. Here, we used live tumor cells as source of antigens to investigate the mechanism underlying their immunogenicity in murine tumor models. The live tumor cells were significantly more immunogenic than irradiated or apoptotic tumor cells. We examined the interaction of live and apoptotic tumor cells with major subsets of antigen-presenting cells, i.e., CD8α+ dendritic cells (DC), CD8α− DCs, plasmacytoid DCs, and CD169+ macrophages at skin draining lymph nodes. The CD8α+ DCs captured cell-associated antigens from both live and apoptotic tumor cells, whereas CD169+ macrophages picked up cell-associated antigens mostly from apoptotic tumor cells. Trogocytosis and cross-dressing of membrane-associated antigenic material from live tumor cells to CD8α+ DCs was the primary mechanism for cross-priming of tumor antigens upon immunization with live cells. Phagocytosis of apoptotic tumor cells was the primary mechanism for cross-priming of tumor antigens upon immunization with apoptotic or irradiated cells. These findings clarify the mechanism of cross-priming of cancer antigens by DCs, allowing for a greater understanding of antitumor immune responses.
Data from STING Signaling in Melanoma Cells Shapes Antigenicity and Can Promote Antitumor T-cell Activity
Authors: Falahat Rana , Perez-Villarroel Patricio , Mailloux Adam W. , Zhu Genyuan , Pilon-Thomas Shari , Barber Glen N. , Mulé James J.
Published: April 2023

Abstract
STING (stimulator of IFN genes) signaling is an innate immune pathway for induction of a spontaneous antitumor T-cell response against certain immunogenic tumors. Although antigen-presenting cells are known to be involved in this process, insight into the participation of tumor cell–intrinsic STING signaling remains weak. In this study, we find diversity in the regulation of STING signaling across a panel of human melanoma cell lines. We show that intact activation of STING signaling in a subset of human melanoma cell lines enhances both their antigenicity and susceptibility to lysis by human melanoma tumor-infiltrating lymphocytes (TIL) through the augmentation of MHC class I expression. Conversely, defects in the STING signaling pathway protect melanoma cells from increased immune recognition by TILs and limit their sensitivity to TIL lysis. Based on these findings, we propose that defects in tumor cell–intrinsic STING signaling can mediate not only tumor immune evasion but also resistance to TIL-based immunotherapies.
Data from GM-CSF Promotes Antitumor Immunity by Inducing Th9 Cell Responses
Authors: Kim Il-Kyu , Koh Choong-Hyun , Jeon Insu , Shin Kwang-Soo , Kang Tae-Seung , Bae Eun-Ah , Seo Hyungseok , Ko Hyun-Ja , Kim Byung-Seok , Chung Yeonseok , Kang Chang-Yuil
Published: April 2023

Abstract
GM-CSF as an adjuvant has been shown to promote antitumor immunity in mice and humans; however, the underlying mechanism of GM-CSF–induced antitumor immunity remains incompletely understood. In this study, we demonstrate that GM-CSF potentiates the efficacy of cancer vaccines through IL9-producing Th (Th9) cells. GM-CSF selectively enhanced Th9 cell differentiation by regulating the COX2–PGE2 pathway while inhibiting the differentiation of induced regulatory T (iTreg) cells in vitro and in vivo. GM-CSF–activated monocyte-derived dendritic cells converted tumor-specific naïve Th cells into Th9 cells, and delayed tumor growth by inducing antitumor CTLs in an IL9-dependent manner. Our findings reveal a mechanism for the adjuvanticity of GM-CSF and provide a rationale for the use of GM-CSF in cancer vaccines.
Data from Immunosuppressive Mediators Impair Proinflammatory Innate Lymphoid Cell Function in Human Malignant Melanoma
Authors: Ercolano Giuseppe , Garcia-Garijo Andrea , Salomé Bérengère , Gomez-Cadena Alejandra , Vanoni Giulia , Mastelic-Gavillet Beatris , Ianaro Angela , Speiser Daniel E. , Romero Pedro , Trabanelli Sara , Jandus Camilla
Published: April 2023

Abstract
Innate lymphoid cells (ILC) are a family of immune cells that are emerging as potent orchestrators of immune responses. In cancer, ILCs display both pro- and antitumorigenic functions depending on the nature of the tumor and the involved ILC subset. Little is known about the ILC–tumor cross-talk in human melanoma. Here, we showed that ILC1s were enriched but functionally impaired in cytokine secretion in both peripheral blood mononuclear cells and tumor-infiltrated lymph nodes of melanoma patients. These findings were confirmed in vivo in murine cutaneous melanoma. Multiple immunosuppressive mechanisms are described in the melanoma microenvironment. Among others, adenosine and kynurenines were shown to suppress antitumor immune responses. By exposing ILCs to adenosine and kynurenines, we observed a similar shift toward the ILC1 subset distribution and impairment in proinflammatory cytokine production to that of patient samples studied ex vivo. Thus, we hypothesized that the immunosuppressive microenvironment of malignant melanoma might shape ILC subpopulations. Hence, we provide a rationale for the use of drugs targeting adenosine and kynurenine pathways in melanoma patients.
Data from CD39 Identifies the CD4+ Tumor-Specific T-cell Population in Human Cancer
Authors: Kortekaas Kim E. , Santegoets Saskia J. , Sturm Gregor , Ehsan Ilina , van Egmond Sylvia L. , Finotello Francesca , Trajanoski Zlatko , Welters Marij J.P. , van Poelgeest Mariette I.E. , van der Burg Sjoerd H.
Published: April 2023

Abstract
The accumulation of tumor-specific CD4+ and CD8+ effector T cells is key to an effective antitumor response. Locally, CD4+ T cells promote the recruitment and effector function of tumor-specific CD8+ T cells and activate innate killer cells in the tumor. Here, we show that tumor-specific CD4+ T cells were predominantly present in the CD39+ subset of tumor-infiltrating lymphocytes (TIL). The CD39+ CD4+ and CD8+ TILs were detected in three different tumor types, and displayed an activated (PD-1+, HLA-DR+) effector memory phenotype. CD4+CD39+ single-cell RNA-sequenced TILs shared similar well-known activation, tissue residency, and effector cell–associated genes with CD8+CD39+CD103+ TILs. Finally, analysis of directly ex vivo cell-sorted and in vitro expanded pure populations of CD39-positive and negative CD4+ and CD8+ TILs revealed that tumor-specific antigen reactivity was almost exclusively detected among CD39+ cells. Immunotherapy of cancer is based on the activation of tumor-reactive CD4+ and CD8+ T cells. We show that the expression of CD39 can be used to identify, isolate, and expand tumor-reactive T-cell populations in cancers.
Data from Multiple Immune-Suppressive Mechanisms in Fibrolamellar Carcinoma
Authors: Kim Amy K. , Gani Faiz , Layman Andrew J. , Besharati Sepideh , Zhu Qingfeng , Succaria Farah , Engle Elizabeth L. , Bhaijee Feriyl , Goggins Maria B. , Llosa Nicolas J. , Pawlik Timothy M. , Yarchoan Mark , Jaffee Elizabeth M. , Simons Howard C. , Taube Janis M. , Anders Robert A.
Published: April 2023

Abstract
Fibrolamellar carcinoma (FLC) is a rare type of liver cancer that affects adolescents and young adults. The most effective treatment for FLC is surgical resection, but no standardized systemic therapy exists for patients with recurrent or unresectable FLC. As a first step to understand the immune microenvironment of FLC, we investigated targetable immune-checkpoint pathways, PD-1, PD-L1, B7-H3, IDO-1, and LAG3, in relation to CD8+ cytotoxic T-lymphocyte density. Thirty-two FLC tumor specimens were analyzed using IHC staining for PD-L1, CD8, PD-1, IDO, LAG3, and B7-H3. Sixty-three percent of FLC cases demonstrated membranous PD-L1 expression on tumor cells, and almost 70% of cases demonstrated PD-L1+ tumor-infiltrating lymphocytes and tumor-associated macrophages (TIL/TAM). Myeloid-derived cells appeared to be a major component of PD-L1+ tumor-infiltrating immune cells. Forty percent of the cases showed B7-H3 expression in the tumor zone, with 91% cases showing B7-H3 expression in TILs and TAMs. IDO and PD-1 expression was highest in the tumor interface zone. B7-H3 or IDO expression on tumor cells significantly correlated with higher CD8+ T-cell density. In conclusion, a high proportion of FLC cases showed robust expression of PD-1, PD-L1, B7-H3, and IDO in an adaptive immune-resistance pattern. Our findings provide further basis for targeting these different immune-checkpoint axes in FLC.
Data from Tumor Vessel Normalization, Immunostimulatory Reprogramming, and Improved Survival in Glioblastoma with Combined Inhibition of PD-1, Angiopoietin-2, and VEGF
Authors: Di Tacchio Mariangela , Macas Jadranka , Weissenberger Jakob , Sommer Kathleen , Bähr Oliver , Steinbach Joachim P. , Senft Christian , Seifert Volker , Glas Martin , Herrlinger Ulrich , Krex Dietmar , Meinhardt Matthias , Weyerbrock Astrid , Timmer Marco , Goldbrunner Roland , Deckert Martina , Scheel Andreas H. , Büttner Reinhard , Grauer Oliver M. , Schittenhelm Jens , Tabatabai Ghazaleh , Harter Patrick N. , Günther Stefan , Devraj Kavi , Plate Karl H. , Reiss Yvonne
Published: April 2023

Abstract
Glioblastoma (GBM) is a non-T-cell–inflamed cancer characterized by an immunosuppressive microenvironment that impedes dendritic cell maturation and T-cell cytotoxicity. Proangiogenic cytokines such as VEGF and angiopoietin-2 (Ang-2) have high expression in glioblastoma in a cell-specific manner and not only drive tumor angiogenesis and vascular permeability but also negatively regulate T-lymphocyte and innate immune cell responses. Consequently, the alleviation of immunosuppression might be a prerequisite for successful immune checkpoint therapy in GBM. We here combined antiangiogenic and immune checkpoint therapy and demonstrated improved therapeutic efficacy in syngeneic, orthotopic GBM models. We observed that blockade of VEGF, Ang-2, and programmed cell death protein-1 (PD-1) significantly extended survival compared with vascular targeting alone. In the GBM microenvironment, triple therapy increased the numbers of CTLs, which inversely correlated with myeloid-derived suppressor cells and regulatory T cells. Transcriptome analysis of GBM microvessels indicated a global vascular normalization that was highest after triple therapy. Our results propose a rationale to overcome tumor immunosuppression and the current limitations of VEGF monotherapy by integrating the synergistic effects of VEGF/Ang-2 and PD-1 blockade to reinforce antitumor immunity through a normalized vasculature.
Data from Mediators of Inflammation-Driven Expansion, Trafficking, and Function of Tumor-Infiltrating MDSCs
Authors: Tannenbaum Charles S. , Rayman Patricia A. , Pavicic Paul G. , Kim Jin Sub , Wei Wei , Polefko Alexandra , Wallace Wesley , Rini Brian I. , Morris-Stiff Gareth , Allende Daniela S. , Hamilton Thomas , Finke James H. , Diaz-Montero C. Marcela
Published: April 2023

Abstract
Myeloid-derived suppressor cells (MDSC) are induced by and accumulate within many histologically distinct solid tumors, where they promote disease by secreting angiogenic and immunosuppressive molecules. Although IL1β can drive the generation, accumulation, and functional capacity of MDSCs, the specific IL1β-induced inflammatory mediators contributing to these activities remain incompletely defined. Here, we identified IL1β-induced molecules that expand, mobilize, and modulate the accumulation and angiogenic and immunosuppressive potencies of polymorphonuclear (PMN)-MDSCs. Unlike parental CT26 tumors, which recruited primarily monocytic (M)-MDSCs by constitutively expressing GM-CSF– and CCR2-directed chemokines, IL1β-transfected CT26 produced higher G-CSF, multiple CXC chemokines, and vascular adhesion molecules required for mediating infiltration of PMN-MDSCs with increased angiogenic and immunosuppressive properties. Conversely, CT26 tumors transfected with IL1β-inducible molecules could mobilize PMN-MDSCs, but because they lacked the ability to upregulate IL1β-inducible CXCR2-directed chemokines or vascular adhesion molecules, additional PMN-MDSCs could not infiltrate tumors. IL1β-expressing CT26 increased angiogenic and immunosuppressive factors of tumor-infiltrating MDSCs, as did CT26 tumors individually transfected with G-CSF, Bv8, CXCL1, or CXCL5, demonstrating that mediators downstream of IL1β could also modulate MDSC functional activity. Translational relevance was indicated by the finding that the same growth factors, cytokines, chemokines, and adhesion molecules responsible for the mobilization and recruitment of PMN-MDSCs into inflammatory CT26 murine tumors were also coordinately upregulated with increasing IL1β expression in human renal cell carcinoma tumors. These studies demonstrated that IL1β stimulated the components of a multifaceted inflammatory program that produces, mobilizes, chemoattracts, activates, and mediates the infiltration of PMN-MDSCs into inflammatory tumors to promote tumor progression.
Data from Excessive Costimulation Leads to Dysfunction of Adoptively Transferred T Cells
Authors: Wijewarnasuriya Dinali , Bebernitz Christina , Lopez Andrea V. , Rafiq Sarwish , Brentjens Renier J.
Published: April 2023

Abstract
Although clinical responses with CD19-targeting chimeric antigen receptor (CAR) T-cell treatment have been observed in patients with certain hematologic malignancies, high rates of disease relapse highlight the necessity to understand and improve mechanisms of CAR T-cell failure. Because T-cell dysfunction is thought to contribute to CAR T-cell treatment failure, understanding what mechanisms drive T cells into this dysfunctional state may aid optimal design of efficacious CAR T cells. Dysfunctional CAR T cells have been characterized as having upregulated inhibitory receptors and decreased cytolytic capabilities. Previous studies have identified a role for sustained CAR CD3ζ signaling in CAR T-cell dysfunction. Here, we demonstrate a mechanism that drives dysfunction in CAR T cells through excessive costimulation. Fully activated CD19-targeted CAR T cells were rendered dysfunctional upon stimulation with both endogenous CD28 stimulation and CAR-mediated CD28 costimulation. Costimulation-driven dysfunction of CAR T cells was demonstrated in a syngeneic immunocompetent mouse model, in which CAR T cells were activated with signals 1 (CD3ζ), 2 (CD28), and 3 (IL12). Thus, we show that CAR T-cell dysfunction can be driven through excessive CD28 and 4-1BB costimulation.

See related article by Drakes et al., p. 743
Data from ASC Modulates CTL Cytotoxicity and Transplant Outcome Independent of the Inflammasome
Authors: Cheong Melody , Gartlan Kate H. , Lee Jason S. , Tey Siok-Keen , Zhang Ping , Kuns Rachel D. , Andoniou Christopher E. , Martins Jose Paulo , Chang Karshing , Sutton Vivien R. , Kelly Greg , Varelias Antiopi , Vuckovic Slavica , Markey Kate A. , Boyle Glen M. , Smyth Mark J. , Engwerda Christian R. , MacDonald Kelli P.A. , Trapani Joseph A. , Degli-Esposti Mariapia A. , Koyama Motoko , Hill Geoffrey R.
Published: April 2023

Abstract
The adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) is known to facilitate caspase-1 activation, which is essential for innate host immunity via the formation of the inflammasome complex, a multiprotein structure responsible for processing IL1β and IL18 into their active moieties. Here, we demonstrated that ASC-deficient CD8+ T cells failed to induce severe graft-versus-host disease (GVHD) and had impaired capacity for graft rejection and graft-versus-leukemia (GVL) activity. These effects were inflammasome independent because GVHD lethality was not altered in recipients of caspase-1/11–deficient T cells. We also demonstrated that ASC deficiency resulted in a decrease in cytolytic function, with a reduction in granzyme B secretion and CD107a expression by CD8+ T cells. Altogether, our findings highlight that ASC represents an attractive therapeutic target for improving outcomes of clinical transplantation.
Data from CD86+ Antigen-Presenting B Cells Are Increased in Cancer, Localize in Tertiary Lymphoid Structures, and Induce Specific T-cell Responses
Authors: Wennhold Kerstin , Thelen Martin , Lehmann Jonas , Schran Simon , Preugszat Ella , Garcia-Marquez Maria , Lechner Axel , Shimabukuro-Vornhagen Alexander , Ercanoglu Meryem S. , Klein Florian , Thangarajah Fabinshy , Eidt Sebastian , Löser Heike , Bruns Christiane , Quaas Alexander , von Bergwelt-Baildon Michael , Schlößer Hans A.
Published: April 2023

Abstract
The role of B cells in antitumor immunity and their impact on emerging immunotherapies is increasingly gaining attention. B-cell effector functions include not only secretion of antibodies, but also presentation of antigens to T cells. A physiologic B-cell subset with immunostimulatory properties was described in humans, defined by a high expression of CD86 and downregulation of CD21. We used multicolor flow cytometry and IHC to elucidate abundance and spatial distribution of these antigen-presenting B cells (BAPC) in blood (peripheral blood mononuclear cells, PBMC) and tumor samples of 237 patients with cancer. Antigen-specific T-cell responses to cancer testis antigens were determined using tetramer staining and sorted BAPCs in FluoroSpot assays for selected patients. We found that BAPCs were increased in the tumor microenvironment of 9 of 10 analyzed cancer types with site-specific variation. BAPCs were not increased in renal cell carcinoma, whereas we found a systemic increase with elevated fractions in tumor-infiltrating lymphocytes (TIL) and PBMCs of patients with colorectal cancer and gastroesophageal adenocarcinoma. BAPCs were localized in lymphoid follicles of tertiary lymphoid structures (TLS) and were enriched in tumors with increased numbers of TLSs. BAPCs isolated from tumor-draining lymph nodes of patients with cancer showed increased percentages of tumor antigen–specific B cells and induced responses of autologous T cells in vitro. Our results highlight the relevance of BAPCs as professional antigen-presenting cells in tumor immunity and provide a mechanistic rationale for the observed correlation of B-cell abundance and response to immune checkpoint inhibition.
Data from Tumor Fusion Burden as a Hallmark of Immune Infiltration in Prostate Cancer
Authors: Wagle Marie-Claire , Castillo Joseph , Srinivasan Shrividhya , Holcomb Thomas , Yuen Kobe C. , Kadel Edward E. , Mariathasan Sanjeev , Halligan Daniel L. , Carr Adrian R. , Bylesjo Max , McAdam Paul R. , Lynagh Sarah , Marien Koen M. , Kockx Mark , Waumans Yannick , Huang Shih-Min A. , Lackner Mark R. , Mounir Zineb
Published: April 2023

Abstract
Prostate cancer is the second leading cause of cancer-related death in men. Despite having a relatively lower tumor mutational burden than most tumor types, multiple gene fusions such as TMPRSS2:ERG have been characterized and linked to more aggressive disease. Individual tumor samples have been found to contain multiple fusions, and it remains unknown whether these fusions increase tumor immunogenicity. Here, we investigated the role of fusion burden on the prevalence and expression of key molecular and immune effectors in prostate cancer tissue specimens that represented the different stages of disease progression and androgen sensitivity, including hormone-sensitive and castration-resistant prostate cancer. We found that tumor fusion burden was inversely correlated with tumor mutational burden and not associated with disease stage. High fusion burden correlated with high immune infiltration, PD-L1 expression on immune cells, and immune signatures, representing activation of T cells and M1 macrophages. High fusion burden inversely correlated with immune-suppressive signatures. Our findings suggest that high tumor fusion burden may be a more appropriate biomarker than tumor mutational burden in prostate cancer, as it more closely associates with immunogenicity, and suggests that tumors with high fusion burden could be potential candidates for immunotherapeutic agents.
Data from In Human Visualization of Ibrutinib-Induced CLL Compartment Shift
Authors: Mayerhoefer Marius E. , Haug Alexander , Jäger Ulrich , Pichler Verena , Pfaff Sarah , Wester Hans-Jürgen , Hacker Marcus , Kazianka Lukas , Staber Philipp B.
Published: April 2023

Abstract
Bruton tyrosine kinase inhibitor ibrutinib is effective in treating chronic lymphocytic leukemia (CLL). However, after ibrutinib treatment initiation, patients frequently experience an increase of CLL blood cell count. This phenomenon in clinical practice is thought to reflect a “compartment shift” of CLL cells from lymph nodes to the peripheral blood, but the actual shifting has not yet been demonstrated. Using [68Ga]Pentixafor-PET/MRI for in vivo CXCR4 visualization, we here provide images of topical changes of CLL cells upon ibrutinib treatment. Within the first month of ibrutinib treatment, mean standardized [68Ga]Pentixafor uptake decreased in the bone marrow and lymph nodes, whereas [68Ga]Pentixafor uptake increased in the spleen. Leukocytosis rose, as did numbers of CXCR4high (tissue-resident) CLL cells. Volumes of lymph nodes and spleen decreased. Upon longer ibrutinib treatment, leukocytosis decreased, followed by a decrease of [68Ga]Pentixafor uptake in the spleen. These results support the preexisting clinical hypothesis of a “compartment shift” of CLL cells from the lymph nodes to the peripheral blood, but also refine the mechanistic model by describing early clearing of the bone marrow and redistribution of CLL cells to the orthotopic splenic cavernous system in response to ibrutinib treatment.
Data from T Cells Expressing Receptor Recombination/Revision Machinery Are Detected in the Tumor Microenvironment and Expanded in Genomically Over-unstable Models
Authors: Morello Gaia , Cancila Valeria , La Rosa Massimo , Germano Giovanni , Lecis Daniele , Amodio Vito , Zanardi Federica , Iannelli Fabio , Greco Daniele , La Paglia Laura , Fiannaca Antonino , Urso Alfonso M. , Graziano Giulia , Ferrari Francesco , Pupa Serenella M. , Sangaletti Sabina , Chiodoni Claudia , Pruneri Giancarlo , Bardelli Alberto , Colombo Mario P. , Tripodo Claudio
Published: April 2023

Abstract
Tumors undergo dynamic immunoediting as part of a process that balances immunologic sensing of emerging neoantigens and evasion from immune responses. Tumor-infiltrating lymphocytes (TIL) comprise heterogeneous subsets of peripheral T cells characterized by diverse functional differentiation states and dependence on T-cell receptor (TCR) specificity gained through recombination events during their development. We hypothesized that within the tumor microenvironment (TME), an antigenic milieu and immunologic interface, tumor-infiltrating peripheral T cells could reexpress key elements of the TCR recombination machinery, namely, Rag1 and Rag2 recombinases and Tdt polymerase, as a potential mechanism involved in the revision of TCR specificity. Using two syngeneic invasive breast cancer transplantable models, 4T1 and TS/A, we observed that Rag1, Rag2, and Dntt in situ mRNA expression characterized rare tumor-infiltrating T cells. In situ expression of the transcripts was increased in coisogenic Mlh1-deficient tumors, characterized by genomic overinstability, and was also modulated by PD-1 immune-checkpoint blockade. Through immunolocalization and mRNA hybridization analyses, we detected the presence of rare TDT+RAG1/2+ cells populating primary tumors and draining lymph nodes in human invasive breast cancer. Analysis of harmonized single-cell RNA-sequencing data sets of human cancers identified a very small fraction of tumor-associated T cells, characterized by the expression of recombination/revision machinery transcripts, which on pseudotemporal ordering corresponded to differentiated effector T cells. We offer thought-provoking evidence of a TIL microniche marked by rare transcripts involved in TCR shaping.
Data from Arginase Therapy Combines Effectively with Immune Checkpoint Blockade or Agonist Anti-OX40 Immunotherapy to Control Tumor Growth
Authors: Badeaux Mark D. , Rolig Annah S. , Agnello Giulia , Enzler Danlee , Kasiewicz Melissa J. , Priddy Leslie , Wiggins Jason F. , Muir Alexander , Sullivan Mark R. , Van Cleef Jessica , Daige Christopher , Vander Heiden Matthew G. , Rajamanickam Venkatesh , Wooldridge James E. , Redmond William L. , Rowlinson Scott W.
Published: April 2023

Abstract
Metabolic dysregulation is a hallmark of cancer. Many tumors exhibit auxotrophy for various amino acids, such as arginine, because they are unable to meet the demand for these amino acids through endogenous production. This vulnerability can be exploited by employing therapeutic strategies that deplete systemic arginine in order to limit the growth and survival of arginine auxotrophic tumors. Pegzilarginase, a human arginase-1 enzyme engineered to have superior stability and enzymatic activity relative to the native human arginase-1 enzyme, depletes systemic arginine by converting it to ornithine and urea. Therapeutic administration of pegzilarginase in the setting of arginine auxotrophic tumors exerts direct antitumor activity by starving the tumor of exogenous arginine. We hypothesized that in addition to this direct effect, pegzilarginase treatment indirectly augments antitumor immunity through increased antigen presentation, thus making pegzilarginase a prime candidate for combination therapy with immuno-oncology (I-O) agents. Tumor-bearing mice (CT26, MC38, and MCA-205) receiving pegzilarginase in combination with anti–PD-L1 or agonist anti-OX40 experienced significantly increased survival relative to animals receiving I-O monotherapy. Combination pegzilarginase/immunotherapy induced robust antitumor immunity characterized by increased intratumoral effector CD8+ T cells and M1 polarization of tumor-associated macrophages. Our data suggest potential mechanisms of synergy between pegzilarginase and I-O agents that include increased intratumoral MHC expression on both antigen-presenting cells and tumor cells, and increased presence of M1-like antitumor macrophages. These data support the clinical evaluation of I-O agents in conjunction with pegzilarginase for the treatment of patients with cancer.
Data from The IKZF1–IRF4/IRF5 Axis Controls Polarization of Myeloma-Associated Macrophages
Authors: Mougiakakos Dimitrios , Bach Christian , Böttcher Martin , Beier Fabian , Röhner Linda , Stoll Andrej , Rehli Michael , Gebhard Claudia , Lischer Christopher , Eberhardt Martin , Vera Julio , Büttner-Herold Maike , Bitterer Katrin , Balzer Heidi , Leffler Magdalena , Jitschin Simon , Hundemer Michael , Awwad Mohamed H.S. , Busch Martin , Stenger Steffen , Völkl Simon , Schütz Christian , Krönke Jan , Mackensen Andreas , Bruns Heiko
Published: April 2023

Abstract
The bone marrow niche has a pivotal role in progression, survival, and drug resistance of multiple myeloma cells. Therefore, it is important to develop means for targeting the multiple myeloma bone marrow microenvironment. Myeloma-associated macrophages (MAM) in the bone marrow niche are M2 like. They provide nurturing signals to multiple myeloma cells and promote immune escape. Reprogramming M2-like macrophages toward a tumoricidal M1 phenotype represents an intriguing therapeutic strategy. This is especially interesting in view of the successful use of mAbs against multiple myeloma cells, as these therapies hold the potential to trigger macrophage-mediated phagocytosis and cytotoxicity. In this study, we observed that MAMs derived from patients treated with the immunomodulatory drug (IMiD) lenalidomide skewed phenotypically and functionally toward an M1 phenotype. Lenalidomide is known to exert its beneficial effects by modulating the CRBN-CRL4 E3 ligase to ubiquitinate and degrade the transcription factor IKAROS family zinc finger 1 (IKZF1). In M2-like MAMs, we observed enhanced IKZF1 levels that vanished through treatment with lenalidomide, yielding MAMs with a bioenergetic profile, T-cell stimulatory properties, and loss of tumor-promoting capabilities that resemble M1 cells. We also provide evidence that IMiDs interfere epigenetically, via degradation of IKZF1, with IFN regulatory factors 4 and 5, which in turn alters the balance of M1/M2 polarization. We validated our observations in vivo using the CrbnI391V mouse model that recapitulates the IMiD-triggered IKZF1 degradation. These data show a role for IKZF1 in macrophage polarization and can provide explanations for the clinical benefits observed when combining IMiDs with therapeutic antibodies.

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