bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel mechanism of action of Anti-malarial drugs in the inhibition of Type I interferon production
Authors: An Jie , Woodward Joshua J , Minie Mark , Peng Yufeng , Sasaki Tomikazu , Elkon Keith B
Published: January 2023

Anti-malarial drugs (AMD) such as Hydroxychloroquine (HCQ) and Quinacrine (QC) are effective in the treatment of skin rash and arthritis in systemic lupus erythematosus (SLE). However which mechanism(s) are responsible for their beneficial action is uncertain. Type I interferon, (IFN-I) is strongly implicated in the pathogenesis of SLE and ‘interferonopathies’ such as Aicardi-Goutieres Syndrome (AGS). A new DNA activated IFN-I pathway, cyclic GMP-AMP (cGAMP) synthase (cGAS), was recently discovered and linked to AGS and mouse models of Lupus.

In silico screening of drug libraries identified several antimalarial drugs (AMD) which could potentially inhibit cGAS activity by interacting with cGAS/DNA dimer complex. Electrophoretic Mobility Shift Assay revealed that AMD disrupted the double stranded DNA-cGAS complex in a dose dependent manner. These AMD also inhibited IFN-I expression in THP1 cells transfected with dsDNA and in 293T cells transfected with cGAS/STING plasmids validating that cGAS is a target of AMD. We synthesized several new AMD. One of these compounds, X6, had excellent water solubility and cell penetration. X6 localized to the cytosol and had a lower toxicity profile compared to QC. Biochemical and cellular assay revealed that X6 was a more potent inhibitor of IFN-I production than HCQ.

Our studies identify new DNA sensor cGAS as a target of AMD activity, which provide a novel mechanism of action of these AMD. We have synthesized new AMD like drugs that are also able to inhibit cGAS as well as Toll pathways. These drugs could be beneficial for the treatment of AGS and/or Lupus.
Interferon-γ signaling inhibition by a suppressor of cytokine signaling-1 (SOCS-1) mimetic peptide ameliorates lupus pathology in a spontaneous mouse model.
Authors: Collins Teresa D. , Lam Brandon K. , Mohamed Zaynab Sidi , Mishra Shiwangi , Larkin Joseph
Published: January 2023

Cytokine-mediated dysregulation of the Jak/STAT pathway is closely associated with systemic lupus erythematosus (SLE) onset and progression. In the MRLlpr/lprmouse model of spontaneous SLE, aberrant IFN-γ signaling is critical for onset and progression. Notably, Suppressor of Cytokine Signaling-1 (SOCS-1) is known to regulate proinflammatory cytokine production. We have previously shown that a peptide mimicking SOCS1 signaling (SOCS1-KIR) inhibited IFN-γ signaling and auto-inflammatory disease progression. SOCS1-KIR mimics the ability of native SOCS1 to inactivate signal cascade propagation by Jak2 and Tyk2. In this study we test the hypothesis that SOCS1-KIR administration will inhibit lupus pathology in MRLlpr/lpr mice. A 3x/week administration of SOCS1-KIR mediated a statistically significant reduction in lupus associated lymphadenopathy. Mechanistically, SOCS1-KIR inhibited the generation of IFN-γ+ CD8+ and IFN-γ+ CD4+ memory T lymphocytes in vivo and in vitro. Additionally, the mechanistic specificity of SOCS1-KIR was delineated through in vitro culturing experiments with a Jak/STAT agonist, which restored the generation of IFN-γ+ memory T lymphocytes. These data show that administration of a SOCS1-KIR peptide has the ability to limit lupus-associated pathology, in part through regulation of IFN-γ signaling. In addition, these data suggest that strategies targeting regulation of excessive Jak/STAT signaling such as, the use of SOCS1-KIR, should be considered as a therapeutic, due to its ability to dampen excessive cytokine help often observed in disease onset and progression.
Therapeutic blockade of immune complex-mediated glomerulonephritis by highly selective inhibition of Bruton’s tyrosine kinase.
Authors: Chalmers Samantha A. , Doerner Jessica L , Bosanac Todd , Khalil Sara , Smith Dustin , Harcken Christian , Dimock Janice , Der Evan B , Herlitz Leal , Webb Deborah , Seccareccia Elise , Feng Di , Fine Jay S , Ramanujam Meera , Klein Elliott , Putterman Chaim
Published: January 2023

Lupus nephritis (LN) is a potentially dangerous end organ pathology that affects upwards of 60% of SLE patients. Bruton’s tyrosine kinase (BTK) is important for B cell development, Fc receptor signaling, and macrophage polarization. In this study, we investigated the effects of a novel, highly selective and potent BTK inhibitor, BI-BTK-1, in an inducible model of LN in which mice receive nephrotoxic serum (NTS) containing anti-glomerular antibodies. Mice were treated once daily with vehicle alone or BI-BTK-1 (0.3–10 mg/kg, n=16/group), either prophylactically or therapeutically.

When compared with control treated mice, NTS-challenged mice treated prophylactically with BI-BTK-1 exhibited significantly attenuated disease which was dose dependent, as measured by proteinuria, serum creatinine, and serum BUN. Histological assessment confirmed marked renal protection in the BI-BTK-1 treatment groups. BI-BTK-1 treatment resulted in decreased recruitment of inflammatory monocytes from the splenic reservoir, and a decrease in infiltrating IBA-1+ cells, as well as C3 deposition, within the kidney. RT-PCR on whole kidney RNA and serum profiling indicated that BTK inhibition significantly decreased levels of LN-relevant inflammatory cytokines and chemokines. Renal RNA expression profiling by RNA-seq revealed that BI-BTK-1 dramatically modulated pathways related to inflammation and glomerular injury. Importantly, when administered therapeutically, BI-BTK-1 reversed established proteinuria and improved renal histopathology.

Our results highlight the important role for BTK in the pathogenesis of immune complex-mediated nephritis, and BTK inhibition as a promising therapeutic target for LN.
Tumor antigen specific Treg from the bone marrow migrate towards increased S1P and CCL2 gradients established in the blood of breast cancer patients
Authors: Rathinasamy Anchana , Böhm Hans-Henning , Ge Yingzi , Dettling Steffen , Umansky Ludmila , Domschke Christoph , Herold-Mende Christel , Schütz Florian , Beckhove Philipp
Published: January 2023

High regulatory T cell (Treg) infiltration in breast tumors is associated with reduced survival. However, the source of tumor infiltrating Treg and signals underlying their migration from lymphoid organs to the tumor tissue remain elusive. We here demonstrate that pronounced Treg infiltration in human breast tumors correlates with a selective reduction of tumor antigen specifc Treg from the bone marrow. Furthermore using MHC-II tumor peptide tetramers we show that tumor antigen specific bone marrow Treg selectively express the egress receptor Sphingosine-1-phosphate receptor 1 (S1P1) and CCR2. S1P1 and CCR2 were upregulated in Treg upon TCR stimulation and triggered selective Treg but not Tcon cell migration in response to S1P and CCL2 gradients between bone marrow and blood which we found to be significantly increased in breast cancer patients. Taken together, our data suggest that tumor antigen specific Treg population in the bone marrow are mobilized into the blood after TCR stimulation through elevated S1P and CCL2 gradients in breast cancer patients.
Stellate cells as mediators of immune suppressive features in pancreatic cancer
Authors: McQuinn Christopher William , Mace Thomas A , Farren Matthew R , Komar Hannah , Kitano Mio , Yu Lianbo , Bloomston Mark , Zhong Xiaoling , Zimmers Teresa , Bekaii-Saab Tanios , Lesinski Gregory Brian
Published: January 2023

One hallmark of pancreatic ductal adenocarcinoma (PDAC) is the dense stroma that includes activated myofibroblast-like cells termed ‘pancreatic stellate cells’ (PSC). We hypothesized that antitumor immunity is restrained by PSC, and that key pathways can be identified to augment efficacy of immunotherapy. To gain insight into the most relevant soluble factors and pathways, primary PSC (n=10) were isolated from PDAC patients. A normal human pancreatic fibroblast (HPF) cell line was used as a control. RNA obtained from PSC or HPF underwent nanostring analysis using the nCounter PanCancer Immune Profiling panel. Cytokine and chemokine expression in supernatants was measured via bioplex analysis or ELISA. Unsupervised clustering analysis revealed the greatest fold change in IL-6 transcript between PSC and HPF, versus other cytokines in the panel. Abundant IL-6 expression was validated in supernatants by bioplex analysis, and was accompanied by other cytokines, most notably, MCP-1, VEGF and SDF-1. Supernatants from human PSC also expanded suppressive myeloid cells from bulk, human peripheral blood mononuclear cells. Analysis of a separate cohort of human pancreatic tumors (n=12) via immunohistochemistry further revealed abundant IL-6 protein in stromal regions of tumors. This prominent stromal localization of IL-6, and STAT3 phosphorylation STAT3 was also evident in pancreata from mice with mutant Kras-driven, PDAC. These data point to stromal IL-6 as a target in PDAC, and complement our data confirming plasma IL‑6 as a poor prognostic factor in a large cohort (n=73) of treatment naïve PDAC patients. Ongoing studies are exploring the role of IL-6 in PSC and the effect of targeting this cytokine in combination with immunotherapy.
CD8α+ Dendritic Cells Induce Leukemia-Specific T cell Tolerance
Authors: Kline Douglas E , Chen Xiufen , Fosco Dominick , Kline Justin
Published: January 2023

Antigen presenting cells (APCs) are critical for the acquisition of tumor-derived antigens and the orchestration of anti-tumor T cell responses. Batf3-dependent CD8α+ and CD103+ dendritic cells (DCs) have been implicated as the critical APCs that initiate and maintain spontaneous CD8+ T cell priming against solid tumors. In contrast, little is known about the APCs that regulate immunity against malignancies of hematopoietic origin. Using a murine model of acute myeloid leukemia (AML) that induces T cell tolerance, we demonstrate that leukemia-derived antigens are exclusively acquired and cross-presented by CD8α+ DCs. In the steady state, CD8α+ DCs rapidly induce leukemia-specific T cell tolerance, which can be prevented upon the activation of these DCs. Together, our data reveal that the same DC lineage can imprint disparate T cell fates in mice with solid verses hematopoietic malignancies. In the context of solid tumors, Batf3-dependent DCs stimulate productive effector responses; however, in AML-bearing mice, CD8α+ DCs actively promote T cell tolerance.
B cell subset biology in a tumor microenvironment model
Authors: Riggs James Edward , Goldman Naomi , Valiuskyte Kornelija , DePierri Kelley , Somerville John
Published: January 2023

The high myeloid to lymphoid ratio of cultured peritoneal cavity (PerC) leukocytes can serve as an in vitro model to study the tumor microenvironment (TME). C57BL/6J PerC T cell responses to TCR ligation (anti-CD3) and mitogen (ConA) are suppressed in these cultures. Likewise, the PerC B cell response to BCR (F[ab’2] anti-IgM) and TLR4 (TLR4L/LPS) ligation are suppressed. T cell suppression can be liberated by neutralizing IFNγ or by blocking iNOS with 1-methyl arginine. The BCR response is recovered by blocking prostaglandin production with indomethacin; the LPS response by neutralizing IL10. To dissect putative receptor-ligand signals in this model, we investigated expression of the inhibitory cell surface marker PDL1/B7H1/CD274 on PerC cells. TCR ligation increased PDL1 expression on macrophages (Mfs), B1, and B2 cells and was reduced by blocking IFNγ. TLR4 ligation increased PDL1 expression on Mfs and both B cell subsets and was reduced by blocking IFNAR1. Interestingly, BCR ligation increased B7H1 and Class II expression on Mfs and B2 cells, but not B-1 cells. Increased B7H1 expression could be reduced by neutralizing IL6, IFNβ, IFNγ, or IFNAR1. TCR ligation led to B1, but not B2, cell division and proliferating B cells increased IL10 production. These findings illustrate that TCR, and surprisingly BCR, ligation can foster immune suppression in macrophage-dense cultures and suggest that regulatory B cells must be considered when designing immunomodulatory cancer therapies.
PRMT5 is a novel cofactor of Foxp3 and can be a target for tumor immunotherapy
Authors: Nagai Yasuhiro , Ji Mei , Xiao Yan , Zhang Hongtao , Li Bin , Ohtani Takuya , Greene Mark I
Published: January 2023

Regulatory T cells (Tregs) are a subpopulation of T cells specialized for suppressing host immune systems, whose dysfunction can lead to severe autoimmunity and inflammatory disease in humans and animals. In a tumor bearing host, an enhanced activity of Tregs may limit host immunity against tumor antigens. Our laboratory has studied the Foxp3 transcriptional complex at the molecular and atomic levels. We identified a protein arginine methyl transferase 5 (PRMT5) as a binding partner of Foxp3. Mice whose expression of PRMT5 was deleted in Tregs developed severe autoimmunity, scurfy like phenotypes and died at ~3 weeks of age. A limited number of Tregs were found in the spleen, but there were normal numbers of Tregs in the peripheral lymph nodes. Tregs that lacked PRMT5 displayed limited suppressive function when compared to wild type cells. Two types of PRMT5 inhibitors, EPZ004777 and EPZ015666, were studied to determine if inhibition of PRMT5 reduces human Treg functions. While EPZ004777 could inhibit human Treg functions in vitro, EPZ015666 had limited ability to limit these Treg activities. EPZ004777 could also inhibit the methylation of Foxp3. Injection of EPZ004777 to p185/erbB2/neu tumor bearing mice enhanced the effect of anti-erbB2/neu monoclonal antibody therapy. Furthermore, treatment with EPZ004777 reduced the numbers of tumor infiltrating Tregs, indicating that EPZ004777 functions by inhibiting Treg functions and/or migration of Tregs into p185 expressing tumors. These studies support the concept that controlling PRMT5 activity is a promising strategy in a p185 targeted monoclonal antibody therapy in which host immunity against tumors is augmented in a Foxp3 dependent manner.
Growth inhibitory effects of Withaferin A on MDS-L cells – A human 5q Myelodysplastic Syndrome (MDS) cell line
Authors: Oben Karine Zinkeng , Gachuki Beth , Akunuru Shailaja , Jinpeng Liu , Brandon Jason , St Clair Daret , Wang Chi , Geiger Hartmut , Gupta Ramesh , Bondada Subbarao
Published: January 2023

Myelodysplastic syndrome (MDS) affects bone marrow production of white blood cells and erythrocytes. At least 40% of MDS patients who go into remission after treatment eventually progress to acute myelogenous leukemia (AML) which is very difficult to treat. Hence, there is a compelling need for new and better therapies. Withaferin A (WFA), a withanolide isolated from Winter cherry (Withania somnifera), has been shown to have potent anti-cancer properties in several animal models of cancer. We found that WFA inhibited the growth of MDS-L cells, a human cell line derived from a 5q MDS patient. Treatment with WFA induced apoptosis of MDS-L cells, indicated by the cleavage of caspase-3 as early as 6hrs after treatment and a dose dependent increase of annexin positive cells. Cell cycle analysis revealed that WFA caused MDS-L cells to arrest at S and G2/M and accordingly there was a reduction in cyclin A/CDK2 and cyclin B/CDK1 respectively. WFA also inhibited the activity of the heat shock protein (Hsp 90) chaperone as shown by the degradation of its client proteins such as Akt, an important pro-survival kinase and increased expression of Hsp70 which partially compensates for decrease in Hsp90 function. Interestingly, the mechanism by which WFA acts on MDS-L cells is independent of its well-recognized action on Nf-κB. Microarray analysis of gene expression in MDS-L cells showed a dramatic increase in several genes that may have a role in Nf-κB independent growth inhibitory effects of WFA. Studies with a xenograft model of MDS-L cells in NSGS mice suggest WFA might also be effective in vivo. The potent anti-proliferative and anti-survival effects of WFA on MDS-L cells, suggest that it could be a novel therapeutic agent for the treatment of 5q MDS.
Aberrant tumor derived LPA production suppresses CD8+ T cells though the LPA5R to promote tumor growth.
Authors: Mathew Divij , Strauch Pamela , Berry Karin Zemski , Schroeder Kristin Shotts , Murphy Robert , Torres Raul M
Published: January 2023

As a bioactive signaling lipid, lysophosphatidic acid (LPA) is in the same phospholipid family as sphingosine-1-phosphate (S1P) yet its impact on immune cells remains unclear, despite that all immune cells express specific LPA receptors. Generated systemically through the extracellular enzyme Autotaxin, LPA exerts its biological influence through 6 known G protein couple receptors (GPCRs), LPAR1-6. Aberrant LPA production has been linked to multiple “hallmarks” of tumorigenesis, including enhancing tumor promoting inflammation, metastasis, and angiogenesis. We propose that up regulation of tumor-derived LPA can also dampen adaptive immune responses, thereby creating an immune suppressive tumor microenvironment. CD8+ T cells are known to be able to eliminate nascent tumors, yet over time are often rendered functionally silent due to tumor-derived signals that act on inhibitory receptors such as CTLA4 and PD1. Work from our lab has found that pathological levels of LPA can also dampen TCR signaling suggesting an additional mechanism by which cancers avoid elimination. Strikingly, ablation of LPAR5 on CD8+ T cells significantly reduced tumor burden compared to wild type CD8+ T cells, suggesting that LPAR5 is a novel inhibitory lipid receptor. Given the recent success of checkpoint blockade therapy for the treatment of cancers, we are currently characterizing this novel inhibitory interaction between CD8+ T cells and LPA generated in the tumor microenvironment.
A multikinase inhibitor and DNA-PK inhibitor combination reduces tumor growth and alters the tumor microenvironment of B16 melanoma
Authors: Tsai Alexander , Davila Eduardo
Published: January 2023

Recent breakthroughs in targeted therapies and immunotherapies for melanoma have prolonged survival in certain patients. Yet, many patients are not candidates for targeted therapies and response rates to immunotherapies remain low. Thus, there is an unmet need to develop novel melanoma therapies that generate: the high response rates that are characteristic of targeted therapies, the durable responses often observed with immunotherapies, and can be used in a majority of melanoma patients. Therefore, a high-throughput approach evaluating nearly 2,000 compounds was taken to identify FDA-approved therapies that alter the expression of immunologically relevant molecules commonly found on human melanomas. Eight compounds were identified based on their ability to reduce melanoma proliferation without negatively impacting T cell proliferation and function. Studies examining the expression of immunosuppressive molecules on melanoma, melanoma proliferation, and T cell-melanoma co-cultures were used to select a multikinase inhibitor and DNA-PK inhibitor for further investigation. The compounds sensitized melanoma to T cell cytotoxicity which was associated with increased MHC class I and antigen expression. In mice with established B16 tumors, the therapeutic combination significantly reduced tumor growth while prolonging survival. Preliminary data suggests that the drug combination impacts the tumor microenvironment and alters various immune cells present in the tumor. Studies using alternative models of melanomas and additional therapeutic combinations are ongoing. These studies represent a proof of concept demonstrating the ability to repurpose drugs to immunosensitize tumors to T cell-based therapies.
Epigenetic silencing of TH1-type chemokines shapes tumour immunity and immunotherapy
Authors: Kryczek Ilona , Peng Dongjun , Nagarsheth Nisha B , Zhao Lili , Wei Shuang , Zhao Ende , Vatan Linda , Szeliga Wojciech , Liu Rebecca , Kotarski Jan , Tarkowski Rafal , Wang Weimin , Zou Weiping
Published: January 2023

Epigenetic silencing including histone modifications and DNA methylation is an important tumorigenic mechanism. However, its role in cancer immunopathology and immunotherapy is poorly understood. Using human ovarian cancers as our model, here we show that enhancer of zeste homologue 2 (EZH2)-mediated histone H3 lysine 27 trimethylation (H3K27me3) and DNA methyltransferase 1 (DNMT1)-mediated DNA methylation repress the tumour production of T helper 1 (TH1)-type chemokines CXCL9 and CXCL10, and subsequently determine effector T-cell trafficking to the tumour microenvironment. Treatment with epigenetic modulators removes the repression and increases effector T-cell tumour infiltration, slows down tumour progression, and improves the therapeutic efficacy of programmed death-ligand 1 (PD-L1; also known as B7-H1) checkpoint blockade and adoptive T-cell transfusion in tumour-bearing mice. Moreover, tumour EZH2 and DNMT1 are negatively associated with tumour-infiltrating CD8(+) T cells and patient outcome. Thus, epigenetic silencing of TH1-type chemokines is a novel immune-evasion mechanism of tumours. Selective epigenetic reprogramming alters the T-cell landscape in cancer and may enhance the clinical efficacy of cancer therapy
NF-κB acts as a molecular link between tumor cells and Th1/Tc1 T cells in the tumor microenvironment to exert radiation-mediated tumor suppression.
Authors: Redd Priscilla S , Bardhan Kankana , Chen May R , Paschall Amy V , Lu Chunwan , Bollag Roni J , Kong Fengchong , Jin JianYue , Kong Feng-Ming , Waller Jennifer L , Pollock Raphael E , Liu Kebin
Published: January 2023

Radiation is a local treatment for many types of solid cancers. About two thirds of cancer patients require radiation during the course of their disease treatment. Radiation modulates both tumor cells and immune cells in the tumor microenvironment to exert anti-tumor activity, but the molecular connection between tumor cells and immune cells that mediates radiation-exerted tumor suppression activity is largely unknown. We report here that radiation induces rapid activation of the p65/p50 and p50/p50 NF-κB complexes in human soft tissue sarcoma (STS) cells. Radiation-activated p65/p50 and p50/p50 bind to the TNFα promoter to activate its transcription in STS cells. Radiation-induced TNFα then induces tumor cell death in an autocrine manner. Smac mimetic BV6 induces cIAP1 and cIAP2 degradation to increase tumor cell sensitivity to radiation-induced cell death in vitro and to enhance radiation-mediated suppression of STS xenografts in vivo. Inhibition of caspases, RIP1, or RIP3 blocks radiation/TNFα-induced cell death, whereas inhibition of RIP1 blocks TNFα-induced caspase activation, suggesting that caspases and RIP1 act sequentially to mediate the non-compensatory cell death pathways. We determined in a syngeneic sarcoma mouse model that radiation up-regulates the NF-κB target genes IRF3, IFNβ, and the T cell chemokines CCL2 and CCL5 in the tumor microenvironment, which is associated with activation and increased infiltration of Th1/Tc1 T cells in the tumor microenvironment. Consequently, combined BV6 and radiation completely suppressed tumor growth in vivo. Radiation-induced NF-κB functions as a molecular link between tumor cells and immune cells in the tumor microenvironment for radiation mediated tumor suppression.
Following the fate of genetically modified T cells in melanoma patients
Authors: Moore Tamson V , Scurti Gina , Regan Courtney , Hutchens Kelli , Godellas Constatine , Clark Ann Lau , Moxley Kelly , Dropulic Boro , Embree Heather , Shirai Keisuke , Garrett-Mayer Elizabeth , Li Mingli , Eby Jonathan , Clark Joseph I. , Le Poole Caroline , Nishimura Michael I.
Published: January 2023

In an open clinical trial for Stage IV melanoma patients, we are testing a promising new therapy in which the patient’s own T cells are genetically engineered with a T cell receptor (TIL 1383I TCR) targeting the melanoma antigen tyrosinase. Included in the TIL 1383I TCR vector is a nonfunctional CD34 marker that can be readily tracked by flow cytometry. These transgenic T cells are infused into the patient with the goals of killing tumor cells and promoting anti-tumor inflammatory responses.

We find that the transgenic T cells persist at low levels for at least twenty months in vivo. Many of the transgenic T cells are activated in the patient, indicating that they are responding to antigen. A subset of activated cells express inhibitory receptors, including PD-1 and CTLA-4. Upon further analysis of patient samples, we found that the percent of transgene-expressing T cells can be increased up to three-fold with either cytokine stimulation and/or CD3/CD28-bead stimulation.

When cultured in vitro, transduced T cells lose transgene expression over time, and culturing in the absence of cytokines dramatically increases the rate of transgene expression loss. As seen ex vivo, restimulation of in vitro cultured T cells restores transgene expression. Cells undergoing division express substantially more transgene than resting cells. These data suggest that mitogenic factors causing cell division may promote transgene expression. We next plan to test mitogenic (high dose IL-2, IL-15) and anti-inhibition (PD-1 and CTLA-4 blockade) therapies with T cell transfer utilizing murine mouse models and potentially amend our protocol in the future to reduce the effects of exhaustion and transgene loss on transgenic anti-tumor T cells.
Toll-like receptor agonist therapy augments the antitumor activity of adoptively transferred CD8+ T cells without host preconditioning
Authors: Paulos Chrystal M , Bowers Jacob S , Bailey Stefanie R , Diven Marshall A , Fugle Caroline W , Kaiser Andrew D , Wrzesinski Claudia , Liu Bei , Restifo Nicholas P , Nelson Michelle
Published: January 2023

Lymphodepletion enhances adoptive T cell transfer (ACT) therapy by activating the innate immune system via microbes released from the radiation-injured gut. Microbial LPS is a key mediator of lymphodepletion enhancement, but our ability to use these TLR agonists to bolster the potency of T cell-based cancer therapies remains elusive. Herein, we used LPS as a tool to address how and when to use TLR agonists to improve cancer immunotherapy. We utilized the pmel-1 melanoma mouse model. B16F10-bearing mice were lymphodepleted with 5Gy total body irradiation (TBI) and given a tripartite ACT therapy (consisting of transferred pmel-1 CD8+ T cells, vaccination with fowlpox encoding gp100, and IL-2) along with TLR4 agonist LPS. The timing of LPS administration and the requirement of individual components of the tripartite therapy were evaluated. We discovered that while exogenous administration of LPS was able to enhance CD8+ T cells’ tumor destruction, LPS treatment alone did not replace individual components of the tripartite regimen. Interestingly, administering LPS one day before ACT compromised tumor regression. Conversely, administering LPS after ACT potentiated the antitumor effectiveness of the regimen, thereby supporting the expansion of transferred CD8+ T cells over host Treg cells. Non-toxic TLR agonists MPL and CpG also improved ACT therapy. Finally, TBI preconditioning was no longer needed to regress tumors in mice depleted of host CD4+ T cells, given a tripartite ACT regimen and then treated with a TLR agonist. Collectively, our results identify how and when to administer TLR agonists to augment ACT in the absence of host preconditioning. These findings have implications for the design of next generation T cell therapies.
First generation CAR-T therapies can be rendered therapeutically effective when engineered into a novel human CD26high CD4+ T cell
Authors: Nelson Michelle , Bailey Stefanie R , Huff Logan , Bowers Jacob , Paulos Chrystal M
Published: January 2023

Despite a few very exciting results from adoptive cell transfer (ACT) therapy-based clinical trials, most studies have infused less than ideal cell populations into patients. Human CD4+ T cells differentiate into multiple effector subsets, but their distinct roles in anti-tumor immunity remain elusive. Interestingly, we found that CD26high CD4+ T cells were more polyfunctional than the other CD4+ T cells subsets- with increased production of IL-17, IFN-γ, IL-22 and TNF-α. Additionally, CD26high T cells had increased expression of anti-apoptotic molecules c-flip, bcl2, AKT and CEBPD. Moreover, CD26high T cells had a more pathogenic phenotype compared to Th17 cells. To test the therapeutic potential, cells were genetically redirected to recognize human mesothelioma using a 1st generation chimeric antigen receptor (CAR, scFv from monoclonal antibodies that are linked to the CD3z intracellular signaling domain of the TCR complex). To date, effective antitumor responses were only seen in 2nd generation CARs with additional costimulatory molecules. Surprisingly, 1st generation CAR-engineered CD26high T cells possessed a superior ability to kill large human tumors when infused into NSG mice compared to Th1, Th2 or Th17 cells. Furthermore, CD26high T cells strongly bolster infused CD8+ T cells, as seen by an increase in both cell number and functionality. Contrary to Th17 cells, CD26high T cells do not require CD8+ T cells to aid in tumor killing, as the antitumor response was similar with or without CD8+ T cell infusion. These results suggest that the generation of multifunctional, long-lived human CD26high T cells could be instrumental to the design of novel, effective T cell-based therapies, targeted to any number of malignancies.
Increased CD4+ T cell proliferation upon treatment correlates with improved overall survival of patients with metastatic melanoma upon anti-CTLA-4 therapy
Authors: Miller William L , Contrad Valerie , Gonzalez Iliana , Meeuwsen Tanisha , Howells-Ferreira Ana Ana , Poplonski Tomasz , Curti Brendan , Urba Walter , Bahjat Keith , Koguchi Yoshinobu
Published: January 2023

Blockade of cytotoxic T-lymphocyte antigen 4 (CTLA-4) augments anti-tumor immunity and has shown success in treating patients with metastatic melanoma. Mechanisms of anti-CTLA-4 efficacy have been proposed in animal models, yet the mechanisms in humans remain to be elucidated. Increased frequency of ICOS+ CD4+ T cells has been shown to be a marker of drug bioactivity, but discovery of prognostic indicators and baseline indicators of which patients are most likely to benefit from CTLA-4 blockade remains a priority.

PBMCs from 29 metastatic melanoma patients in a multicenter compassionate use trial of anti-CTLA-4 antibody were evaluated using flow cytometry. At baseline and 12 weeks following initiation of treatment, CTLA-4, HLA-DR, ICOS, PD-L1, PD-1, and Ki-67 frequencies were assessed on CD4+ and CD8+ T cells, as well as CD3+CD4+ regulatory T cells.

Independent of survival, anti-CTLA-4 treatment significantly increased the frequency of ICOS+ CD4+ T cells, corroborating previous findings. Interestingly, this increase was also observed in ICOS+ Tregs, but not in CD8+ T cells. Increased proliferative Ki67+ CD4+ T cells and Ki67+ Tregs upon CTLA-4 blockade were both associated with prolonged overall survival. Additionally, when the frequency of Ki67+ CD4+ T cells post-treatment was compared to the baseline frequency, patients with a ratio greater than 1.5 had greater overall survival. Increased post-treatment absolute lymphocyte count—a known prognostic biomarker—also correlated with clinical outcome. Our results suggest that anti-CTLA-4 therapy may be mediated by proliferation of CD4+ T cells, and we identify this proliferation as a novel prognostic biomarker in metastatic melanoma patients treated with anti-CTLA-4 antibody.
Hybrid Th1/17 cells possess unique immuno-metabolic phenotype and surpiror anti-tumor activity
Authors: Chatterjee Shilpak , Daenthanasanmak Anusara , Meek Megan , Toth Kyle , Chakraborty Paramita , Paulos Chrystal M , Yu Xuezhong , Mehrotra Shikhar
Published: January 2023

Immunotherapy of cancer is a promising approach for successful eradication of large and established tumor. Accumulating evidence indicates that CD4+ T helper (Th) cells including Th1 and Th17 subsets are also effective in tumor immunotherapy. While the anti-tumor potential of Th17 cells primarily depends upon IFN-γ secretion and persistence, a long-term tumor control has still remained elusive. In current study, we established an optimal protocol to program CD4 T cells ex vivo into a combinational phenotype of Th1 and Th17 subset (named hybrid Th1/17 cells), which express high levels of IFN-γ, CD107a, T-bet, Granzyme B, IL23R, IL22, Bcl6, and Tcf7. A detailed microaray analysis followed by Ingenutity Pathway Analysis for hybrid Th1/17 cells further established its unique molecular signature. Importantly, a comprehensive analysis for about 400 metabolites also established that hybrid Th1/17 cells have unique metaboliomic signature. Furthermore, using two types of TCR transgenic models and established B16 tumor model either administrated subcutaneously or intravenously, we demonstrated that the hybrid Th1/17 cells exhibit a superior activity in controlling tumor as compared to Th1 or Th17 alone. We observed that the unique immuno-metabolic phenotype rendered to hybrid Th1/17 cells corrected with their increased ability to survive, overcome tumor mediated immunosuppression, resulting the superior effectiveness to control tumor in long-term.
Serial monoclonal antibody administration demonstrates therapeutic effects via induction of apoptosis in HPV related cervical cancer
Authors: Bhadauria Preeti , Hockenberry Kristi , Jiang Zewei , Christensen Neil , Dadachova Ekaterina , Phaëton Rebecca
Published: January 2023

Introduction

All cervical cancer is caused by the persistent expression of human papillomavirus (HPV) E6 and E7 oncoproteins that interfere with p53 and Rb pathways, respectively. Virally specific treatments form an excellent basis for highly specific, low toxicity therapies.

Methods

Serial doses of anti-HPV 16/18 E6 C1P5 (Abcam) were compared against Cisplatin in the treatment of human cervical cancer derived tumors (CaSki). C1P5 and Cisplatin were administered on days 1, 4, and 7 after CaSki tumor size reached 3–5mm on the flank of athymic nude mice. Measurements were performed every 3 days during the 40 day observation period. After which the tumors were procured, paraffin embedded, and stained with H&E. SiHa cells which compare to HPV copies in patient explants (previously published) were used in mechanistic studies. Dual staining flow cytometry with PI and Annexin was performed to evaluate cell death and MTT assay was used to confirm findings.

Results

Tumor growth was inhibited in all treatment groups compared to untreated controls (p <0.05). No statistically significant differences between C1P5 treatment groups were noted when compared to cisplatin. However, on H&E we noted increased necrosis proportional to the number of doses: 1 dose 30%, 2 doses 60%, and 3 doses 85%. Dual-staining of Annexin and PI on flow cytometry demonstrated increased apoptosis as a result of C1P5 administration suggesting this as the underlying mechanism of increased necrosis. These results were corroborated by MTT assay (p<0.05).

Conclusion

Virally specific therapy is promising for the treatment of women with cervical cancer. To extend the paucity of options, further mechanistic understanding must be developed to derive maximal therapeutic benefit.
Tumor specific epitopes on Podocalyxin as a target for ADC and CAR T cell therapy
Authors: von Rossum Anna , Samudio Ismael , Bergqvist Peter , Cruz Eric , Wee Kathlee , Hughes Michael , Canals Diana , Babcook John , Roskelly Calvin , McNagny Kelly , Bond Chris
Published: January 2023

Podocalyxin (Podxl) is a heavily glycosylated sialomucin type 1 membrane protein that has been shown to be involved in cell adhesion, migration, and polarity. Although Podxl is found on a variety of normal cell types, it is aberrantly expressed in acute myeloid leukemia (AML) blasts, and its presence in breast, colorectal and glial malignancies is correlated with poor clinical outcome. Using the human triple negative breast cancer line MDA-MB-231, we have demonstrated a functional role for Podxl in tumor growth and metastasis in xenografted mice, and have recently developed high affinity antibodies to a tumor-specific post-translational modification of Podxl. Preliminary IHC data suggests that this post-translational modification on Podxl may found in glioblastoma, melanoma, and female reproductive tract tumors, but not on matched normal tissue. Staining intensity in melanoma samples correlates with stage of disease and is strongest in tissues collected from metastatic lesions. Preliminary experiments indicate that expression of this glyco-epitope is increased in myeloid leukemia cells lines by co-culture with bone marrow stromal cells or hypoxia, suggesting that the tumor microenvironment may contribute to the observed reactivity. Our lead antibody, Podo447, binds this tumor-associated glycoform of Podxl with high affinity and induces cytotoxicity in various tumor cell lines as an antibody drug conjugate (ADC), and when expressed as a human IgG1 Fc chimera stimulates antibody dependent cellular cytotoxicity. With its exquisite specificity for malignant tissues, and the number of potential indications, Podo447 is an ideal targeting arm for an antibody drug conjugate or CAR-T enabled therapeutic.
Modeling anti-CD20 chimeric antigen receptor therapy in canine B cell lymphoma patients
Authors: Panjwani M. Kazim , Gnanandarajah Josephine S. , MaloneyHuss Martha A. , Mason Nicola J.
Published: January 2023

Chimeric antigen receptor (CAR) therapy has demonstrated great promise in treating human leukemias, but preclinical murine models are limited in their ability to predict safety and efficacy in humans. Given the rapid and on-going advances in CAR T cell technology in the laboratory, it now becomes necessary to identify and develop an outbred, large animal spontaneous cancer model in which the safety of novel targets and therapeutic effectiveness of re-directed T cells can be evaluated and optimized.

Canines naturally develop spontaneous B cell lymphoma, and we have previously shown the feasibility of evaluating CAR T cell therapy in dogs using CD20-targeting RNA CAR T cells. To parallel human CAR T cell approaches, we have now optimized primary canine T cell transduction with lentivirus. Once peak T cell activation and artificial antigen presenting cell elimination timeframes were established, transduction yielded up to 26% CAR+ canine T cells. Canine T cells transduced with a second generation CD20-targeting CAR (CD20-28-ζ) demonstrated CAR-mediated, antigen-specific proliferation and efficient cytolysis of a CD20+ canine B cell lymphoma line in vitro.

Three relapsed canine B cell lymphoma patients were treated with autologous CD20-28-ζ CAR T cells. Despite the administration of low numbers of CAR T cells, transient anti-tumor effects were observed in the first 2 patients. Following administration of ~700,000 CAR T cells/kg intravenously to the third patient, CAR T cells were found to persist and expand in the periphery and malignant nodes and disease progression was halted. This work establishes the feasibility of using the dogs with spontaneous cancer as pre-clinical models for advancing human CAR T cell therapy.
Small-molecule probe targeting viral PPxY L-domain inhibits filovirus and arenavirus egress.
Authors: Prugar Laura I , Dye John M , Loughran Marie , Wrobel Jay E , Reitz Allen B , Freedman Bruce D , Harty Ronald N , Herbert Andrew S
Published: January 2023

Viral matrix proteins containing a highly conserved PPxY Late (L) domain motif are critical for efficient viral budding of several emerging RNA viruses. The L domain motif functions by recruiting host cell proteins necessary for separation of virus particles from the cell surface during the budding process. The conserved nature of the PPxY motif and its importance for viral egress make it an attractive drug target with potential broad spectrum antiviral activity. Here we describe a PPxY inhibitor capable of reducing viral budding of four highly lethal, category A RNA viruses: Ebola, Marburg, Lassa, and Junín viruses.
Genetic background influences the humoral responses to vaccines
Authors: Nouri-Shirazi Mahyar A , Abu-Nuwar Emily , Nourishirazi Erika
Published: January 2023

Background

The assessment of TLR agonists as promising candidate adjuvants for the induction of effective Th1 immunity continues to rely on the use of inbred mouse. However, the genetic variation among commonly used inbred mice may influence the efficacy of candidate adjuvants and bias the study conclusion. Indeed, we have found significant differences in the recruitment, phenotypes and cytokines production of DCs and NK cells and the generation of effector T cell in lymphoid tissues of two genetically non-identical mouse strains immunized with a protein based vaccine formulated with TLR agonists.

Objective

In this study, we further evaluated the nature and amplitude of humoral immune responses of B6 and BALB/c strains immunized with protein antigen ovalbumin plus Alum, TLR3, TLR4, TLR7/8, or TLR9 agonists.

Results

Our data suggest that compared to the Alum, all other adjuvants induced more effectively the recruitment of B cells and production of OVA-specific antibodies in both strains. We also observed significant differences between two strains. BALB/c strain recruited less B cells but produced significantly higher amounts of IgG2a in response to any of the TLR agonists. In addition, both strains produced similar amounts of IgG1 and IgG2b in response to TLR agonist except for MPLA which induced significantly more of IgG2b production in B6 strain.

Conclusion

Thus, our data suggests that genetic background should be taken into consideration when evaluating and comparing the activities of adjuvants for use in new prophylactic and therapeutic vaccines. NIH-NIAID 1R03AI103750-01A1
Type-1 T cell responses and gene signatures driven by a novel virus-like particle (VLP) influenza A (H1N1) vaccine
Authors: Jones Leigh A , Skibinski David A , Xie Lin Wu , Au Bijin , Lee Bernett , Zhu Yuan , Fairhurst Anna-Marie , Lee Audrey , Kaliaperumal Nivashini , Poidinger Michael , Hibberd Martin , Ooi Eng Eong , Hanson Brendon J , Diermayr Veronica , Matter Alex , Bachmann Martin , Saudan Phillipe , Connolly John E
Published: January 2023

Effective influenza vaccination is currently assessed by the induction of antibodies directed against the influenza virus envelope glycoprotein hemagglutinin (HA; HAI test) otherwise known as seroconversion. However, protective antibody responses are sub-optimal in some vaccinated populations, particularly in the elderly. Furthermore, many clinical studies highlight the importance of T cells in driving protection. Therefore, a need exists for vaccine strategies which can engage both the humoral and cell mediated arms of the immune response. Herein, we describe the induction of anti-influenza T cell responses following vaccination with a novel VLP vaccine. Peripheral blood mononuclear cells (PBMCs) stimulated ex vivo with either influenza or vaccine specific antigens led to increased CD4+ and CD8+ T cell proliferation and increased production of cytokines such as IL-17A, IL-17F, IL-5, IL-13, IL-9, IL-10, IL-21 and, most significantly, IFN-γ. A subset of individuals demonstrated a shift from a pre-vaccination IL-5- and IL-13- driven type-2 response to a more protective IFN-γ driven type-1 response following vaccination with VLP. Microarray of whole blood samples collected pre- and post-vaccination with VLP revealed significantly different transcriptional profiles. The top most significantly downregulated gene following VLP vaccination, DEAD Box Helicase 17 (DDX17), was also found to have reduced protein expression in PBMCs by flow cytometry. Finally, weighted gene correlation network analysis (WGCNA) revealed that the post vaccination reduction in protein expression of DDX17, increase in HAI titres and increase in T cell proliferation correlates with particular gene modules.
TGF-β2 decreases surface expression of MHC I and MHC II on equine bone marrow-derived mesenchymal stromal cells.
Authors: Berglund Alix K , Cameron Kristin K , Poole Emma J , Harris Elizabeth A , Schnabel Lauren V
Published: January 2023

Allogeneic bone marrow-derived mesenchymal stromal cells (MSCs) are a promising cell source for treatment of inflammatory diseases, but elicit immune responses in vivo due to recipient recognition of mismatched Major Histocompatibility Complex (MHC) molecules. The purposes of this study were to evaluate the effectiveness of TGF-β2 in decreasing and stabilizing MHC expression on equine bone marrow-derived MSCs before and after IFN-γ stimulation. FACS analysis showed that MSCs treated with 1, 5, or 10 ng/ml of TGF-β2 had significantly reduced MHC I expression. MHC I expression levels on TGF-β2-treated MSCs were also quantified and determined to be similar to fetal fibroblasts. MHC II expression in two positive animals was greatly reduced compared to untreated MSCs. MSCs pre-treated and continuously treated with 1 ng/ml TGF-β2 had significantly lower MHC I expression following stimulation with IFN-γ for 72 hours compared with untreated. Continuously treated MSCs that were baseline positive for MHC II had significantly lower MHC II expression following IFN-γ stimulation, but there was no significant difference in MHC II expression between TGF-β2-treated and untreated MHC II-negative MSCs after IFN-γ stimulation. These results indicate TGF-β2 treatment has significant promise for reducing recipient immune recognition of MHC-mismatched molecules on allogeneic MSCs, but further work is needed to maintain MHC expression stability.
Induction of robust cytotoxic T lymphocytes using adenovirus-vectored multivalent antigen cocktail
Authors: Mwangi Waithaka , Lokhandwala Shehnaz , Waghela Surya
Published: January 2023

Development of subunit vaccines against complex pathogens entails inclusion of multiple targets to improve efficacy and or broaden protection. Importantly, testing of an immunogen in the actual host that require protection can hasten vaccine development and generate knowledge needed to generate other similar vaccines. The African Swine Fever Virus (ASFV) causes a fatal hemorrhagic disease in domestic swine and, at present, no treatment or vaccine is available. Previous studies suggest that induction of ASFV-specific cytotoxic T lymphocytes (CTLs) is critical for protection. We evaluated immunogenicity of adenovirus-vectored ASFV multivalent cocktail formulated in two adjuvants and at two doses in swine. The immunogen induced unprecedented CTL responses against all the antigens in the cocktail. Notably, CTL responses were detected in swine immunized with a low dose (1 × 1010 ifu/each construct) and a high dose (1 × 1011 ifu/each construct). In addition, both adjuvants elicited strong CTL responses. Significant antigen-specific IFN-γ+ responses were detected in PBMCs of vaccinees post-priming and post-boosting. Furthermore, the cocktail induced robust antigen-specific antibody responses against all the antigens and the antibody responses underwent rapid isotype-switching within one week post-priming. The primed antibody responses underwent rapid recall upon boost four months post-priming. Taken together, this study showed for the first time that the adenovirus-vectored ASFV multivalent cocktail safely induced robust cellular and antibody responses in swine following homologous prime-boost immunization. The relevance of the induced immune responses in regards to protection will be evaluated in a challenge study.