bluuebunny/crossref_metadata_2025 huggingface dataset:
CHOP depletion does not prevent gut inflammation in experimental spondyloarthritis
Authors: Navid Fatemeh , Gill Tejpal , Fones Lilah , Brooks Stephen , Colbert Robert A
Published: January 2023

HLA-B27, which predisposes to spondyloarthritis (SpA), can misfold, generate endoplasmic reticulum stress, and activate the unfolded protein response (UPR). The UPR may contribute to SpA pathogenesis by promoting pro-inflammatory cytokine production. CHOP, which is upregulated by the PERK arm of the UPR, is required for excess IL-23 production during UPR-TLR co-activation. Here, we examined whether CHOP deficiency in HLA-B27-transgenic (B27+) rats affects pro-inflammatory cytokine production and gut inflammation.

CHOP-deficient (CHOP-) Lewis rats were generated using CRISPR/CAS9 editing and crossed with B27+ Lewis animals to create B27+CHOP− rats. CHOP expression was not detectable by western blot in CHOP− cells when treated with thapsigargin, to induce UPR and CHOP expression. In contrast to CHOP+ macrophages, CHOP-cells did not express Il23a upon UPR-TLR co-activation. Histological scoring of colon samples from 6-month-old animals showed a trend to increased inflammation in B27+CHOP− compared to B27+CHOP+ rats (8.05±1.11 vs 6.87±1.40; p=0.07). B27-CHOP− rats had no gut inflammation similar to B27-CHOP+ WT rats (scores ≤ 2). RNASeq analysis of colon tissue showed significantly increased expression of 73 genes in B27+CHOP− vs B27+CHOP+ rats, including pro-inflammatory genes like Il1a, Tnf, and Il4r. TOPPGENE analysis revealed that the differentially expressed genes are involved in processes like inflammation, IL-6 production and cytokine secretion. Our data demonstrate that CHOP depletion does not prevent SpA development, but rather it may exacerbate gut inflammation. This suggests that CHOP may play a protective role in HLA-B27 associated disease. This axis warrants further investigation as a therapeutic avenue.
Chimeric antigen receptor signaling confers antitumor activity to human regulatory T cells
Authors: Ferreira Leonardo , Muller Yannick D. , Kaul Anupurna M. , Shaikh Hashim , Guerrero-Moreno Ryan , Yao Lilian E. , Goodman Daniel B. , Bluestone Jeffrey A , Tang Qizhi
Published: January 2023

Regulatory T cells (Tregs) are a subset of T cells dedicated to suppressing immune responses to ensure immune self tolerance. Treg therapy offers the opportunity to treat transplant rejection and autoimmunity without the toxicity associated with immunosuppressive regimens. Conferring antigen specificity to Tregs using a chimeric antigen receptor (CAR) dramatically expands what targets can be pursued using Treg therapies. However, the consequences of CAR-mediated signaling for human Treg biology remain poorly understood. To address this, we generated anti-CD19 CAR Tregs with a tandem CD28-TCRζ intracellular domain. Upon in vitro co-incubation with CD19-expressing cells, CAR Tregs upregulated activation markers, proliferated, secreted IL-10, and suppressed T cell proliferation, while maintaining high FOXP3 expression and a demethylated TSDR. In humanized mice harboring CD19+ tumor cells, CAR Tregs suppressed CAR T cell proliferation. Strikingly, CAR Tregs also suppressed tumor growth, even in the absence of CAR T cells. This phenomenon was observed across three tumor cell types (B-cell leukemia, myeloid leukemia, and epithelial carcinoma) and two routes of delivery (intravenous and subcutaneous). Real-time monitoring of tumor cell survival and proliferation in vitro confirmed that CAR Tregs suppress tumor cell growth. Interestingly, single-cell cytokine analysis revealed that CAR-mediated activation of Tregs leads to higher production of IFN-γ, TNF-α, perforin, and granzyme B than anti-CD3/28 stimulation. CRISPR/Cas9-mediated ablation of granzyme B in CAR Tregs diminished their antitumor activity in vivo. These results demonstrate that CAR Treg cytotoxicity is an important concern for safe clinical translation.
Novel mRNA-based Therapeutics to Modulate T and B Cell Signaling and Function
Authors: Kenney Laurie Lea , Fu Ying , Stein Paul , Huang Eric
Published: January 2023

Autoimmunity is considered a T cell driven disease and in many cases B cells contribute to the underlying pathology. Many of the current therapeutics used to treat autoimmunity and organ transplantation are broadly immunosuppressive and can result in increased susceptibility to infection and cancer. We have designed several mRNA-encoded chimeric polypeptides containing an inhibitory motif and a location motif that are expected to disrupt signaling downstream of the T and B cell receptor (T/BCR) and blunt activation. We found that lipid nanoparticle (LNP)-encapsulated mRNA encoding TCR disruptors (Immune LNP-TCRi) containing either a constitutively active CSK (CA.Csk), ITIM, or SHIP1 inhibitory motif reduced in vitro proliferation and cytokine production in primary T cells. In an in vivo xeno-GVHD model Immune LNP-TCRi significantly delayed T cell-mediated mortality. BCR disruptors were engineered by creating mRNA encoding chimeric proteins containing components of the BCR complex (CD79a, CD79b and CD19) and either the CD22 intracellular ITIM or SHIP1 inhibitory motif. Encapsulated in Immune LNP, these BCR disruptors downregulated B cell activation as measured by phosphorylation of Syk, suppressed BCR-induced IgM/G, IL6 and IL10 secretion, as well as reduced B cell in vitro proliferation. In NSG mice engrafted with healthy human PBMC, Immune LNP-BCRi also suppressed humoral immunity against tetanus toxoid and reduced serum IgM and IgG levels. Unlike corticosteroids and anti-CD20 Ab, co-engagement of chimeric T/BCR disruptors did not significant decrease the number of lymphocytes. Thus, amplification of T/BCR disruptor pathway may represent a novel therapeutic approach for autoimmune diseases.
Combining tolerogenic agents with dexamethasone synergistically inhibits innate inflammatory responses
Authors: Genito Christopher J , Stiepel Rebeca T , Batty Cole J , Bachelder Eric M , Ainslie Kristy M
Published: January 2023

Excessive activation of innate immune cells drives many diverse inflammatory human diseases, such as septic shock and autoimmunity. Many modern therapies employ immunosuppressants and other tolerogenic agents (TAs) to treat inflammation through inhibiting inflammatory cytokines and other effector molecules secreted from innate immune cells. TAs carry side effects due to general immunosuppression and off-target effects. One alternative is TA combination therapy, which could reduce side-effects through dose sparing and may improve therapeutic efficacy. To this end, multiple commonly used TAs were screened for synergistic inhibition of inflammation. Screened TAs included immunosuppressants and steroids used in the clinical treatment of allergy, arthritis, and autoimmune disease, as well as candidate drugs in preclinical testing. Anti-inflammatory potential was determined by inhibition of reactive oxygen species from activated macrophages in culture. Synergistic inhibition was calculated mathematically for TAs combined in various ratios. At least 4 synergistic hits were obtained from the screen, with minimum combination indices ranging from 0.5 (good synergy) to 0.04 (extreme synergy). All other tested TAs showed additive improvement in combination. Each synergistic combination included dexamethasone (DXM). Using DXM in combination with an otherwise ineffective TA may have reduced serum levels of inflammatory cytokines in a mouse model of septic shock. We conclude that using DXM in combination with other TAs may improve the inhibition of macrophage inflammation in multiple clinical applications.
Antigen-specific B cell depletion with desmoglein 3 chimeric autoantibody receptor T cells (DSG3-CAART) for targeted therapy of mucosal pemphigus vulgaris
Authors: Lundgren Daniel Kjell , Lee Jinmin , Mao Xuming , Manfredo-Vieira Silvio , Wang Baomei , Nunez-Cruz Selene , Williams Erik , Fraietta Joseph , Milone Michael , Payne Aimee
Published: January 2023

Chimeric autoantibody receptors (CAARs) direct T cells to kill autoantigen-specific B cells. We previously established proof-of-concept for the CAAR approach using gene-engineered T cells expressing CAARs comprising DSG3, the autoantigen in the autoimmune blistering disease mucosal pemphigus vulgaris, fused to CD137-CD3ζ signaling domains. Here, we present results of preclinical studies that supported the DSG3-CAART investigational new drug application.

DSG3-CAART demonstrated activity related to dose in a passive transfer polyclonal anti-DSG3 hybridoma mouse model of mucosal pemphigus vulgaris, resulting in reversal of the rising serum anti-DSG3 antibody titers, target cell burden, and IgG deposition in epithelial tissues, as well as increased DSG3-CAART engraftment relative to dose. We further evaluated DSG3-CAART efficacy in a novel exploratory active immune mouse model with physiologic levels of circulating anti-DSG3 IgG, which demonstrated decreased antibody responses against pathogenic DSG3 epitopes and autoantibody binding to epithelial tissues, leading to clinical and histologic resolution of blisters. High-throughput screening of a membrane protein array with soluble DSG3 CAAR did not identify verifiable off-target toxicities.

These preclinical studies have enabled a first-in-human clinical trial of DSG3-CAART in mucosal pemphigus vulgaris and provide a foundation that may inform the preclinical development of future CAAR T cell therapies for antigen-specific B cell depletion in other autoantibody-mediated diseases.
JMJD3-deficient impair CD4 T cell trafficking in autoimmune disease models and adoptive immunotherapy models by targeting cytoskeleton genes
Authors: Fu Chuntang , Xing Changsheng , Yin Bingnan , Chu Junjun , Duan Tianhao , Liu Xin , Wang Rongfu
Published: January 2023

Histone H3K27 demethylase, JMJD3 plays a critical role in gene expression and T-cell differentiation. However, the role and mechanisms of JMJD3 in T cell trafficking remain poorly understood. Here we show that JMJD3 deficiency in CD4+ T cells resulted in T cell trafficking deficiency autoimmune disease models and adoptive immunotherapy models. We identified PDLIM4 and S1P1 as significantly down-regulated target genes in JMJD3-deficient CD4+ T cells by gene profiling and ChIP-seq analyses. The further study showed that PDLIM4 regulate actin cytoskeleton, thus serving as a key regulator of T cell trafficking. These results demonstrate that manipulating JMJD3 expression can benefit autoimmune disease treatment and generate better T cell grafts for adoptive immunotherapy.
Use of CART cells to selectively target autoantigen-specific T cells for the treatment of autoimmune diabetes
Authors: Yu Hua , Bettini Maria , Ellis Gavin , Riley James L , Collins Jesse , Preston-Hurlburt Paula , Korah Maria , Mallone Roberto , Deng Songyan , Wang Xiaoli , Fremont Daved H. , Spiegel David , Cresswell Peter , Herold Kevan C
Published: January 2023

Previous clinical trials using biologics-based broad-spectrum T cell- and B cell-depleting molecules for the treatment of autoimmune diabetes have shown promising, yet mixed, results. Their varied extent of success may be due to their non-specific action and failure to permanently and completely remove the pathogenic subpopulations. As CD8+ T cells, the most dominant cell type in human insulitis, are thought to be the primary mediator of β-cells damage, we thus designed a strategy by adapting chimeric antigen receptor engineered T (CART) technology to directly target these pathogenic T cells.

The newly generated CAR construct maintains original transmembrane and intracellular components, while the extracellular scFv antigen-binding domain was replaced with HLA-A2/β2-microglobulin (B2M) complex that is linked with either diabetes-associated immunodominant peptide zinc transporter 8(ZnT8)186–194 or negative control peptide HIV Gag77–85. We have shown that HLA-A2/B2M complexes were correctly folded and presenting right peptide epitopes on CART cells. The CAR signaling was also sustained, as the expression levels of CD25 and CD69 were significantly elevated only on CAR-transduced Jurkat cells presenting ZnT8186–194 peptide co-cultured with a T-cell line that expresses TCRs recognizing the same peptide. To determine the killing ability of CART cells, we further engineered primary human T cells to express our CAR construct and demonstrated that CART cells can selectively deplete human antigen-specific CD8+ T cells in vitro. Therefore, our results have provided proof-of-principle for the development of a novel immunotherapy for disease treatment.
Identification of T cells specific for DNAJB1-PRKACA fusion neoantigens in fibrolamellar carcinoma
Authors: Kirk Allison M , Crawford Jeremy Chase , Zamora Anthony E , Allen Kaity , Zhang Xiaoyu , Strome Scott E , Thomas Paul G
Published: January 2023

Fibrolamellar carcinoma (FLC) is a rare liver tumor with poor outcomes and for which novel therapies are urgently needed. A potential therapeutic vulnerability in FLC is a gene fusion between DNAJB1 and PRKACA that is expressed in all FLC tumors and has been shown to drive FLC tumorigenesis. Genetic mutations that alter amino acid sequence, including gene fusions, can be presented as neoantigens by MHC class I molecules on tumor cells and thereby elicit anti-tumor CD8 T cell responses. We hypothesize that DNAJB1-PRKACA fusion neoantigens can elicit T cell responses in FLC patients and serve as targets for T cell-based immunotherapies for FLC. To test this hypothesis, T cells isolated and expanded from an FLC patient tumor were stimulated with DNAJB1-PRKACA fusion peptides, which reproducibly induced cytokine production measured by intracellular cytokine staining. Single cell gene expression and T cell receptor (TCR) repertoire analysis of patient tumors using the 10X Genomics platform identified clonally expanded TCR clonotypes that were associated with expression of cytokines and activation markers in response to fusion peptide stimulation. These TCRs were cloned and expressed in Jurkat 76 cells to test their fusion specificity and their ability to mediate in vitro functional responses to fusion neoantigens. When stimulated with their cognate fusion neoepitope, TCR-expressing cells produced cytokines, expressed the early activation marker CD69, and downregulated surface expression of TCR and CD3. These studies have therefore identified T cell responses against the DNAJB1-PRKACA fusion for the first time, as well as identified DNAJB1-PRKACA-specific TCR clonotypes that could be used to develop novel immunotherapies for FLC.
Treatment of CEA-positive solid tumors with anti-CEA chimeric antigen receptor T-cells in CEA transgenic mice
Authors: Cha Seung , Yazaki Paul J , Brown Christine , Shively John
Published: January 2023

Chimeric antigen receptors (CARs) combine specificity of antibody to target antigen on cancer cells with the potent cytotoxic activity of T-cells without the need for MHC recognition. Chimeric antigen receptor (CAR) T-cells that were designed to express the single chain variable fragment binding domain of human anti-CEA antibody BW431/26 on mouse splenocytes were able to reduce CEA-expressing pancreatic adenocarcinoma tumor in CEA-transgenic (CEAtg) mice without significant off-target effects (Chmielewski et al., 2012).

In this study, an anti-CEA CAR construct derived from murine anti-CEA antibody (T84.66) expressed on human T-cells targeted CEA+ colon carcinoma cells (LS174T/CEA and MC38CEA) with high specificity in vitro. When co-incubated with CEA-positive or CEA-negative mouse cancer cells, anti-CEA CAR expressed on mouse T-cells specifically targeted CEA-positive cancer cells in vitro. Compared to mock T-cells control, anti-CEA CAR T-cells injected intravenously delayed subcutaneous MC38CEA tumor growth in CEAtg mice. Lymphodepletion via cyclophosphamide injected before anti-CEA T-cells therapy further delayed subcutaneous MC38CEA tumor growth in CEAtg mice. To improve the therapeutic potential of anti-CEA CAR T-cells, IL2 was conjugated to a humanized anti-CEA antibody to form an immunocytokine (ICK). The ICK was injected interperitoneally after anti-CEA CAR T-cells into lymphodepleted CEAtg mice. A single injection of the ICK delayed subcutaneous MC38CEA tumor growth even further. Four injections of ICK after anti-CEA CAR T-cells eradicated subcutaneous MC38CEA tumors. These data show the therapeutic potential of anti-CEA CAR T-cells to target CEA-positive tumors.
Immunotherapy for solid tumors: Chimeric antigen receptor (CAR) T-cells and the tumor microenvironment
Authors: Nwanze Chiadika , Kratzmeier Sabina , Kaplan Rosandra
Published: January 2023

Patients with solid tumors continue to experience decreased survival rates, with metastasis being the leading cause of mortality in these patients. This has prompted the development of novel immunotherapies, with cellular immunotherapy giving rise to the development of Chimeric Antigen Receptor (CAR) T-cells, an approach combining specificity for tumor antigens and cytotoxicity independent of major histocompatibility complex molecule co-stimulation. This modality has proven efficacious in Acute Lymphoblastic Leukemia and B-cell Lymphoma. However, progress has remained limited in solid tumors despite targeting of various antigens, one of such being disialoganglioside (GD2) which has proven a viable target in neuroblastoma. This study investigates the efficacy of GD2 as an antigen target for CAR T-cell therapy in osteosarcoma. Surface flow cytometric analysis revealed the presence of GD2 on human 143B and Hu09H3 osteosarcoma cell lines. Of note, the tumor microenvironment offers an additional potential therapeutic target, as we observed increases in myeloid-derived suppressor cells alongside decreases in CD4+ T cells in primary tumor and metastatic tissues. In summary, we report the utility of GD2 as a target for cellular immunotherapy in osteosarcoma and the role of the tumor microenvironment in anti-tumor therapeutic modalities, a therapeutic combination that could be applicable to optimizing potent therapeutic modalities for solid tumors.
THERAPEUTIC EFFECTS OF TRAIL-SECRETING MESENCHYMAL STEM CELLS WITH FOCUSED ULTRASOUND-INDUCED TEMPORARILY OPENING OF BLOOD-BRAIN BARRIERS IN THE BRAIN TUMOR
Authors: Park Sang In , Jung Ho Yong , Lee Eui-Jin , Maeng Lee-So
Published: January 2023

Mesenchymal stem cell (MSC)-based gene therapy is a promising tool for the treatment of various neurological disease including brain tumor. The tumor necrosis factor-related apoptotic ligand (TRAIL) is believed to have promise as a cancer the therapy. For stem cell therapy in the brain tumor, therapy efficiency of stem cells by non-invasive method is extremely limited due to blood brain barrier (BBB). Noninvasive pulsed focused ultrasound (FUS) can temporarily opening the BBB of specific areas in the brain. Herein, we investigated the tumor-targeted BBB temporarily opening by FUS and therapeutic efficiency of NIR fluorescence-labeled MSCs-TRAIL by intravenous injection in brain tumor. In addition, we observed the inflammatory changes of microglia cells by FUS using PET-CT and histological analysis. As a results, In vivo survival experiments showed that FUS treated-MSC-TRAIL group has greater therapeutic efficacy than FUS untreated-groups. The number of MSCs accumulated specifically site in the tumor region. MSC migration toward FUS treated-tumor site greater compared to the FUS untreated-tumors. Furthermore, the changes of microglia activity by FUS was no difference between the other groups. Collectively, these results suggest that therapeutic efficacy of MSC-TRAIL increases by temporarily opening the BBB using the FUS, which may be a more useful strategy for cancer therapy.
Lactate is critical for cytokines production of T cell after TCR activation
Authors: Zhang Yaguang , Wen Jing , Sun Bing
Published: January 2023

Glycolysis is considered to be a key regulator of T cell maturation and function. After TCR activation, T cell accelerates the metabolic process of glycolysis, which produces a large amount of lactic acid. Because the dissociation of lactic acid in solution is effectively complete and there is a strong systemic acid-base equilibrium in our body, lactate exists in the main form of lactic acid ion after TCR activation. This neutral environment with a high concentration of lactic acid ion gives us a hint that T cells may recycle this free lactic acid ion for energy recovery or other vital processes. In this study, we found that lactate as the end-product of glycolysis is critical for T cell cytokines production, especially in T cell-dependent tumor immunology. In in vitro T cell activation, the addition of excess sodium lactate promoted more cytokines production (such as IL-2/TNF) than sodium chloride or sodium pyruvate, whose difference depended on TCR/CD3 activation, but not CD28. Further work showed that those “up-regulated” cytokines were owing to post-transcription, which was regulated by enhanced glycolysis after TCR activation with sodium lactate. This enhanced glycolysis attenuated GAPDH binding ability to cytokines mRNA, which permitted more cytokine protein translation. In vivo study showed that sodium lactate treatment could inhibit tumor growth in the MC38 tumor model with more cytokines production from T cells compared to sodium chloride treatment group. Taken together, our finding indicates a new regulatory mechanism of glycolysis in T cell function.
Suppressive effects of fucoidan-agaricus mix (CUA-fucoidan) on angiogenesis and tumor growth in B16 melanoma-bearing mice
Authors: Miyazaki Yoshiyuki , Bak Juneha , Nakano Hayato , Takeuchi Shugo , Takeuchi Hideaki , Tachikawa Daisuke
Published: January 2023

Fucoidan is a series of natural sulfated polysaccharides contained in brown sea algae. In previous studies, we proved that fucoidans derived from Cladosiphon okamuranus and Undaria pinnatifida effectively augmented anti-tumor immunity in combination with an Agaricus blazei mycelium extract. In this study, we evaluated dietary effects of the mixture consist of the two high-molecular weights fucoidan and the agaricus extract (named CUA-fucoidan) on tumor angiogenesis. For that purpose, C57BL/6 mice were subcutaneously injected basement membrane matrix (Geltrex) enclosing 5×104 mitomycin C-treated B16 melanoma cells or 500 ng/mL vascular endothelial growth factor (VEGF), and fed 2% CUA-fucoidan containing diet for 7 days before and after the matrix inoculation. The accumulation of hemoglobin and CD31-positive cells in the B16-enclosed Geltrex was lower in the CUA-fucoidan-fed mice than in the control mice, whereas the hemoglobin contents in the VEGF-enclosed Geltrex were not affected by the CUA-fucoidan feeding. These results suggested that CUA-fucoidan was effective in suppressing tumor angiogenesis, but did not inhibit the physiological action of VEGF. Rather, it was guessed that CUA-fucoidan suppressed VEGF production because the expression of VEGF mRNA was reduced in the matrix resected from CUA-fucoidan-fed mice. In this context, the infiltration of CD206-positive macrophages, which known to produce VEGF, were declined by the CUA-fucoidan feeding. Furthermore, CUA-fucoidan decreased accumulation of Treg in the B16-included Geltrex, and delayed B16 tumor growth. In conclusion, CUA-fucoidan was expected to be effective in inhibition of tumor angiogenesis by improving immunosuppressive tumor microenvironments.
Development of a Humanized 3D Tumor Spheroid Models and Discovery of Effective Immunotherapy Strategies Against Resistant Microsatellite Stable Colorectal Cancers
Authors: Torres Adorno Angie M , Fong Julie , Grein Jeff , Blumenschein Wendy , Cheng Danye , Porth Katherine , Yang Peng , Yearley Jennifer , Beebe Amy , Joyce-Shaikh Barbara
Published: January 2023

Despite the current success of immunotherapies, only microsatellite instable (MSI) Colorectal Cancer (CRC) typically respond to T cell-driven immunotherapies while microsatellite stable (MSS) CRC displays poor response. Our goal is to identify resistance mechanisms in MSS-CRC to uncover novel therapeutic targets. Current syngeneic CRC models are largely immunogenic and lack complex structure. Therefore, we developed novel 3D humanized CRC mouse models which form complex human-like tumors which are typically absent in syngeneic systems. We investigated molecular profiles of human MSI-versus MSS-CRC tumor xenografts following Keytruda immunotherapy and identified an immunosuppressive profile in MSS models that was not observed in MSI-CRC. We evaluated the therapeutic potential of blocking inflammation in combination with other immuno-modulatory therapies in both cold and hot CRC models and identified a significant regression of tumor burden in MSS-CRC mice. Our studies provide a novel CRC in vivo model and a unique opportunity to better define how immune cells interact in human MSS- and MSI-CRC microenvironments, to enable clinical therapeutic implementation.
Immunological responses through miRNA signatures in GBM plasma extracellular vesicles from patients receiving experimental immunotherapy
Authors: Garcia Luz Milbeth Cumba , Dehankar Mrunal K , Nair Asha A , Dietz Allan B , Parney Ian F
Published: January 2023

Glioblastoma (GBM) patients have a median survival of 15 months despite aggressive treatment. Treatment-related pseudo-progression confounds outcome assessment by MRI, particularly in patients receiving immunotherapy. Extracellular vesicles (EVs) contain tumor-specific miRNA that could serve as a liquid biopsy to distinguish true progression from treatment-related pseudo-progression. Small plasma EVs were isolated by serial density gradient ultracentrifugation from 20 newly-diagnosed GBM patients enrolled in a clinical trial of allogeneic tumor lysate-pulsed autologous dendritic cell (DC) vaccination. Short non-coding RNA sequencing was performed for each patient at three time points (TP): pre-vaccine (TP1), post-initial vaccine (TP2), and at end of treatment (TP3). Ingenuity Pathway Analysis from 31 differentially expressed miRNAs across time points (p-value <0.05, |logFC|>1) revealed that glioma-related signaling pathways previously seen in small EVs from glioma patients compared to normal donors were replicated when comparing patients at the beginning of the treatment (TP1-vs-TP2) and between early to late time points (TP1-vs-TP3). In addition, pathways corresponding to immunological responses typically seen in GBM patients undergoing treatment, such as IL-10, IL-6, and Toll-like Receptor (TLR) signaling, were exclusively found after the initial vaccine towards the end of treatment (TP2-vs-TP3). In conclusion, signaling pathways derived from differentially expressed miRNAs across time points suggest that as patients progress through treatment, consistent differences in plasma small EVs miRNA expression profile and immunological responses could be utilized as predictors of treatment response.
Reprograming of Tumor-Associated Macrophages in Human BrafV600EMutant Melanoma
Authors: Santiesteban Gretel Maria Torres , Bhandari Rajan , Yang Heetaek , Spezza Paul , Shabaneh Tamer Basel , Liby Karen , Turk Mary Jo , Pioli Patricia A
Published: January 2023

Melanoma tumors are highly immunogenic, making them an attractive target for immunotherapy. However, many patients do not mount robust clinical responses to targeted therapies, which is attributable, at least in part, to suppression of immune responses by tumor-associated macrophages (TAMs) in the tumor microenvironment (TME). Using a human in vitro culture system, we have shown that the synthetic triterpenoid CDDO-Me enhances immune activation by reprogramming macrophages from immuno-suppressive to immuno-stimulatory. CDDO-Me significantly reduced CCL2, VEGF and IL-6 secretion by macrophages and inhibited surface expression of CD163, a marker associated with poor clinical outcomes. Furthermore, CDDO-Me mediated both contact-dependent and independent effects on macrophage activation in tri-cultures of macrophages with activated autologous T cells and BRAFV600E mutant melanoma cells. Signaling pathway activation was interrogated to identify the potential mechanism by which CDDO-Me attenuates the pro-tumorigenic macrophage activation profile. Our results demonstrated that CDDO-Me inhibited STAT3 phosphorylation, which is a known regulator of TAM activation. Given these results, we hypothesize that, in addition to mediating direct anti-tumorigenic effects, CDDO-Me may also enhance the efficacy of additional immunotherapies, including BRAF inhibition. Collectively, our studies suggest that the redirection of immuno-suppressive myeloid cell activation may provide both a direct means of inhibiting melanoma growth and may enhance the efficacy of additional targeted immunotherapies through relief of immune suppression in the TME.
ST8Sia6 Overexpression Accelerates Tumorigenesis in a Siglec E Dependent Manner Through Reshaping the Tumor Immune Microenvironment
Authors: Friedman David J , Shapiro Michael J , Rajcula Matthew , McCue Shaylene , Khazaie Khashayarsha , Dong Haidong , Shapiro Virginia Smith
Published: January 2023

Checkpoint blockade has improved patient outcome through reinvigorating T cells against cancer. Despite this success, many patients don’t respond, leaving them with few options. Tumors utilize many immunosuppressive mechanisms, including altering the function of innate immune cells. Cancer cells can promote differentiation of macrophages into tolerogenic “M2-like” macrophages, which support tumor growth. Macrophage differentiation by cancer cells is influenced by many receptors, including Siglec-E. Siglec-E binds to α2,8-linked disialic acids, generated by the enzyme ST8Sia6, on O-linked glycoproteins, and functions to inhibit activation of innate immune cells. Cancer patients with high expression of ST8Sia6 exhibit reduced survival. Therefore, ST8Sia6 overexpressing tumors may engage Siglec-E, leading to immune suppression and accelerating tumor growth. Mice injected with ST8Sia6 overexpressing MC38 and B16-F10 tumors had increased tumor burden, reduced survival and an increase M2 macrophage polarization. The acceleration in tumor growth was abrogated when injected into Siglec-E knockout mice. Thus, the primary function of ST8Sia6 in tumors is an extrinsic alteration of the immune response, rather than an intrinsic effect on cell growth. Similarly, in a spontaneous colon cancer model, ST8Sia6 overexpression in a murine colon cancer model accelerated the development of tumors, decreasing survival to 2.5 months instead of 6 months. The overexpression of ST8Sia6 on tumors leads to increased Arg-1 expression by CD11b+ CD11c+Ly6C−Ly6G− cells, which suppresses the immune response. Therefore, ST8Sia6 expression in tumors inhibits the immune response to promote tumor growth in a Siglec-E dependent manner.
Isolation of human CD45+ leukocytes from tissues and human tumor xenografts in humanized mice
Authors: Antignano Frann , Posarac Vesna , Ewen Catherine L , Jovanovic Vida K , Liang Alice , Woodside Steven M , Kokaji Andy I , Eaves Allen C , Thomas Terry E
Published: January 2023

The study of immune cell function in non-lymphoid tissue and tumors promises to elucidate novel strategies to treat immune disorders, infectious diseases, and cancer. To address the challenge of isolating leukocytes from complex and variable tissues and tumors, we have developed a new protocol to isolate particle-free, human CD45+ leukocytes. Using the EasySep™ Release Human CD45 Positive Selection Kit, leukocytes are labeled with antibody complexes linked to magnetic particles and separated using an EasySep™ magnet. The magnetic particles are then removed from the desired cells by resuspension in EasySep™ Release Buffer and a final magnetic separation.

To assess performance, NRG-3GS mice were first engrafted with human CD34+ cells followed by xenotransplant with human breast (MDA-MB-231) or ovarian (SKOV3) cancer cell lines. In humanized mouse lungs, bone marrow and spleen, the starting and isolated human CD45+ frequency ranges were 6.0 – 57.2% and 90.9 – 99.4%, respectively (n = 3). Starting with human tumor xenografts, tumor infiltrating leukocytes were enriched from a starting range of 0.4 – 18.0% to 76.6 – 92.7% (n = 4). The final immune cell frequencies are representative of the starting population, and further separation of immune subsets can be achieved with additional downstream isolation.

Humanized mouse models of clinical disease are instrumental in furthering our understanding of complex mechanisms of disease progression and resolution. This new kit for the isolation of human immune cells from tissues and tumors will facilitate further examination of the roles of immunity in disease and the evaluation of immune-based treatment strategies.
Bone microenvironment-suppressed T cells increase osteoclast formation and breast cancer bone metastases
Authors: Arellano Danna L , Corral-Avila Juan A , Drescher Florian , Verdugo Andrea , Jimenez Samanta , Olvera-Rodriguez Felipe , Juarez Patricia , Fournier Pierrick
Published: January 2023

Bone metastases is a frequent (>75%) complication of advanced breast cancer (BCa). BCa cells in bone are supported by cytokines released during the osteoclastic resorption, and remain untreatable. T cells during immunotherapy could turn against BCa cells in bone but could also increase osteoclasts and bone metastases.

Comparing the development of bone metastases from 4T1 BCa cells between Balb/C and SCID or T cell-depleted mice, the absence of T cells decreased bone metastases (−72% and −38%, respectively), while orthotopic tumors were increased. Histology confirmed that the osteoclast number was decreased at the tumor-bone interface of mice lacking T cells. Ex vivo addition of non-activated T cells increased osteoclast formation, and flow cytometry confirmed that >85% of T cells in bone metastases were not expressing activation markers.

In sharp contrast, activated T cells were potent inhibitors of osteoclast formation, which could be beneficial for the treatment of patients. Although activated T cells expressed the anti-osteoclastic Ifng and Il4, their effect was not reversed by neutralizing antibodies, or due to a bystander effect by cytotoxic T cells. Thus, we sought to activate T cells of bone metastases. However, it was not possible to activate T cells in bone marrow cultures ex vivo. This could be due to an increase of MDSCs in 4T1 bone metastases, including M-MDSCs that were PD-L1+ (>85%) and could suppress PD-1+ T cells (>70%) in bone.

Our results suggest that while T cells under the influence of the bone microenvironment promote BCa bone metastases, immunotherapy-activated T cells could suppress bone resorption and eliminate BCa cells in patients with bone metastases.
Protein Kinase D: A new target in the fight against flu?
Authors: Veazey Janelle , Chapman Timothy J , Smyth Timothy R , Hillman Sara E , Eliseeva Sophia I , Georas Steve N
Published: January 2023

Antivirals targeting host proteins may slow development of viral resistance and work against multiple viruses. The serine/threonine kinase protein kinase D (PKD) is one promising target as others have shown inhibiting PKD restricts rhinovirus and hepatitis in cell culture. We show inhibiting PKD restricts Influenza A virus (IAV) in cell culture and in mice.

Methods

The competitive PKD inhibitor, CRT, was given to A549, 16HBE cells and mice, prior to or during stimulation with dsRNA or IAV.

Pulmonary barrier integrity was quantified by assaying total protein leak into the lumen (inside/out leak), and loss of 4kDa FITC-dextran out of the lumen (outside/in leak).

Inflammatory cytokine levels were determined via ELISA/multiplex and mRNA levels via RT-PCR. Transcription factor activity was assayed via luciferase reporter system.

Viral load was determined via RT-PCR of viral M protein. For in vivo IAV experiments, mice were given CRT/vehicle post-infection with lethal dose IAV (PR/8) and morbidity assessed by weight loss.

Results

Conclusion

PKD inhibition promotes barrier integrity, lowers transcription of potentially pathogenic pro-inflammatory cytokines, and restricts viral replication, highlighting the potential of CRT as a novel anti-viral therapeutic.
Immunogenic potency of engineered exosomes for prevention of respiratory syncytial virus
Authors: HONG SUYEON , Ruan Shaobo , Kubota Pamela Gamero , He Mei , McGill Jodi L
Published: January 2023

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease in infants and young children. Bovine RSV is a common cause of respiratory disease in young cattle and is a useful model to study RSV infection. There is currently no vaccine for RSV treatment in humans, and the vaccines available for BRSV have limited efficacy in the field. Exosomes (30–150nm) are cell-derived extracellular vesicles that play critical roles in cellular communication by carrying various molecules. Since exosomes secreted by virus or tumor infected cells can deliver cell-specific antigens and proteins to immune cells, immunogenic exosomes have emerged as a new vaccine platform for prevention of viral infections or therapy for cancer treatment. Here, dendritic-cell derived exosomes were engineered to present peptides from the RSV M, NS and L proteins, purified from PDMS microfluidic systems and were evaluated as an efficacious surface engineered antigenic exosome vaccine against RSV and BRSV in mice. The immunogenicity of the exosomes was confirmed in vitro by measuring their capacity to induce antigen-specific T cell clonal proliferation and IFNγ production. The in vivo immunogenicity and efficacy of the engineered exosomes was investigated by immunizing animals subcutaneously with escalating doses of engineered exosomes and measuring antigen-specific T cell responses in the spleen and lungs, and assessing morbidity and mortality. Collectively, dendritic-cell derived surface engineered antigenic exosomes have the capacity to elicit antigen-specific CD8 T cell activation in vitro and in vivo, and the potential to be developed as a vaccine platform to prevent RSV and other viral infections in humans and animals.
Dissecting human, ferret, and murine antibody and B cell responses to generate a broadly protective synthetic influenza virus hemagglutinin
Authors: Kim Hyunsuh , DeBeauchamp Jennifer , Stigger Evelyn , Ellebedy Ali , Rubrum Adam , El-Shesheny Rabeh , Crumpton Jeri , Kackos Christina , Thomas Paul G , Caniza Miguela , DeVincenzo John , Ahmed Rafi , Webby Richard J
Published: January 2023

A major challenge in developing improved influenza vaccines is the identification of broadly reactive and protective epitopes. While it is generally believed that the specificity of the antibody response to hemagglutinin (HA) is relatively narrow, we and others have identified individuals that mount a cross-reactive neutralizing antibody response after natural infection. Using plasmablasts from one such individual we generated broadly cross-reactive HA-specific monoclonal antibodies (mAbs). Six of these mAbs bound to the globular head of the HA and one to the stalk region; all provided broad protection against multiple subtypes of influenza A viruses including zoonotic strains in passively immunized mice. Using a peptide ELISA approach, we identified the binding regions of each mAb and subsequently immunized mice with these peptides. Immunized mice showed less weight loss and significantly improved survival against challenges with multiple subtypes as compared with control mice. We next used a similar mapping approach with broadly reactive B cells from mice and ferrets. Based on all of the collected data we generated three different composite HA antigens (HF1, HF2 and HF3) that contained a different mix of broadly reactive epitopes in one native HA structure. Mice immunized with the HFs were protected from multiple challenges, including lethal doses of H1, H3, H5, H6, H7 and H9 viruses. We also showed the HFs protected ferrets from H1, H3, and H9 viruses. Overall, by combining data generated from an examination of B cell and antibody responses following infection and/or vaccination in humans, mice, and ferrets we were able to construct a composite HA that was able to induce broad protection against multiple subtypes of influenza virus.
The effect of SMAC mimetics on human T cell activation
Authors: Murray Susan E , Ligman Brittany , Burton Ashley , Kearney Claire
Published: January 2023

Second mitochondria-derived activator of caspase (SMAC) mimetics are small molecule drugs currently in cancer clinical trials. They are thought to act by antagonizing inhibitors of apoptosis (IAPs), which are often overexpressed in malignancies. However, in leukocytes, IAPs inhibit cell activation via antagonizing non-canonical NF-κB. This suggests that, paradoxically, while SMAC mimetics can induce apoptosis in cancer cells, they may enhance activation in T cells. The aim of this project is to determine whether human T cell proliferation and effector function are affected by SMAC mimetics.

We previously showed that non-canonical NF-κB plays an essential intrinsic role in T cell activation in mice. Here, we show that the SMAC mimetic TL32711 (Birinapant) activates non-canonical NF-κB in primary human T cells. Doses of SMAC mimetics that induce 100% apoptosis in the breast cancer cell line, MDA-MB-231, do not affect T cell viability. Birinapant dose-dependently increases IL-2 secretion, but the effects of SMAC mimetics on T cell proliferation and Th1 cytokine production are complex and depend on availability of costimulation and accessory cells.

Endogenous T cells can combat cancer if properly stimulated, a fact underlined by the efficacy of checkpoint blockade. Thus, understanding the effect of SMAC mimetics on T cell responses is a critical consideration in using SMAC mimetics to treat cancer and for designing combination therapies.
Targeting microbial-specific gamma/delta T-cell subset for vaccine development
Authors: Chen Zheng , Shen Ling
Published: January 2023

Tuberculosis (TB), caused by M. tuberculosis (Mtb), remains the top killer among infectious diseases. The current TB vaccine, Bacille Calmette–Guérin (BCG), only protects young children from severe disseminated TB, but not effectively protects against pulmonary TB in adults. Improved TB vaccine or vaccination approach is needed for effective global prevention against TB. Our decade-long mechanistic studies in nonhuman primates (NHP) employed 2 innovative “gain-of-function” manipulations to generate Vγ2Vδ2 T effector cells in vivo for definition of their function and immunity against high-dose TB. These seminal studies established that HMBPP-specific Vγ2Vδ2 T effector cells are fast-acting, multi-functional and protective against high-dose TB in NHP. These novel findings prompted us to perform creative immunization of Vγ2Vδ2 T subset using our innovative vaccine vector Listeria ΔactA prfA*. Respiratory vaccination of macaques with an HMBPP-producing attenuated LM ΔactA prfA*, but not the control, caused prolonged expansion of HMBPP-specific Vγ2Vδ2 T cells in pulmonary and circulating compartments. After pulmonary Mtb challenge, macaques vaccinated with LM ΔactA prfA* exhibited rapid memory-like response of Vγ2Vδ2+ Th1 cells. The selective immunization of Vγ2Vδ2 T cells contained Mtb infection/dissemination and reduced lung TB pathology, enhancing the ability of tissue-resident Vγ2Vδ2 T cells to inhibit Mtb growth in macrophages. Thus, selective immunization of Vγ2Vδ2 T cells elicits fast-acting/durable memory-like responses, providing an approach to creating more effective TB vaccines.
Late-rising CD4 T cells show dramatic control of cytomegalovirus persistence
Authors: Picarda Gaelle Genevieve Alice , Brunel Simon , Ghosh Raima , Gupta Ankan , Greenbaum Jason , Seumois Gregory , Croft Michael , Vijayanand Pandurangan , Benedict Chris A
Published: January 2023

Conventional CD4 T cells typically expand and establish memory pools within the first few weeks of acute viral infection. However, cytomegalovirus (CMV) exhibits an extended persistent replication phase followed by lifelong latency, suggesting the induction, phenotype and/or function of protective memory CD4 T cells may differ. We show that during CMV infection in mice, CD4 T cells recognizing an epitope derived from the viral M09 protein develop long after conventional memory T cells have already formed. These cells are unique compared to their conventional memory counterparts; showing high IFNγ and IL-2 production at times of viral persistence. Ablating these CD4 T cells by mutating the M09 genomic epitope, or inducing them through vaccination, revealed them far superior at resolving persistent replication. RNAseq data reveal that these ‘late-rising’ and conventional CD4 T cells differ dramatically in their transcriptional signatures, and that M09 cells show a uniquely restricted T cell receptor repertoire. We believe these results may help instruct CMV vaccine and cellular immunotherapy approaches in people, and will also provide a blueprint for determining whether similar late-rising CD4 T cells exist in other chronic virus infections.
Loss of anti-inflammatory alveolar macrophages associates with lung inflammation in untreated HIV infection
Authors: Neff Charles Preston , Logue Eric C , Siebert Janet , Görg Carsten , Fiorillo Suzanne , Twigg Homer L , Campbell Thomas B. , Fontenot Andrew P , Palmer Brent E
Published: January 2023

HIV-1 is associated with infectious and noninfectious lung disease, both of which are exacerbated by cigarette smoke. Alveolar macrophages (AMs) are the most prominent immune cell in the alveolar space. These cells play an important role in clearing inhaled pathogens and regulating the inflammatory environment, however, how HIV infection impacts AM function is not well understood, in part due to their autofluorescent properties. The aim of this study was to evaluate the impact of HIV infection on AMs. Utilizing Time of Flight Mass Cytometry (CyTOF) and RNAseq we characterized macrophages from bronchoalveolar lavage (BAL) of HIV-infected and uninfected smokers and nonsmokers. We found that the frequency of alternatively activated AMs, defined by expression of CD163 and CD206, was decreased, while CD163-CCR7+ AM were increased in HIV infection. This phenotype was associated with increased levels of inflammatory cytokines and macrophage activation. While the inflammatory state of macrophages was not impacted by cigarette smoke, other markers such as TLR4 and CXCR4, which are associated with heightened inflammatory response to bacterial products and HIV infection, were altered with smoking. These findings suggest that HIV infection is associated with an increase in pro-inflammatory and depletion of anti-inflammatory AMs, which along with cigarette smoke-induced TLR4 and CXCR4 expression contributes to increased inflammation in the lung. Furthermore, this study demonstrates that CyTOF is a reliable method for immunophenotyping highly autofluorescent cells isolated from the bronchoalveolar lavage of cigarette smokers.