bluuebunny/crossref_metadata_2025 huggingface dataset:
Generation of large numbers of functional NK cells without feeders or serum
Authors: Le Fevre Tim , Ang Elaine , Wognum Albertus W , Szilvassy Stephen J , Eaves Allen C , Louis Sharon A , Tabatabaei-Zavareh Nooshin
Published: January 2023

Natural killer (NK) cells are innate lymphocytes that modulate immune responses by secreting proinflammatory cytokines, and provide antitumor and antivirus activity. The ability to generate therapeutically relevant numbers of functional NK cells is critical for the development of advanced cellular immunotherapies. We have developed a streamlined culture workflow that enables expansion of functional NK cells without using either feeder cells or serum. Fresh human NK cells were isolated from peripheral blood using EasySep™, then cultured in ImmunoCult™ NK Cell Expansion Medium in plates coated with ImmunoCult™ NK Cell Expansion Coating Material. NK cells were fed, harvested, and replated on days 7 and 10 – 11 prior to phenotyping and functional assessment on day 14. The average frequency of CD56+ CD3− NK cells was 88% (range 75 – 96%, n=34), 74% (range 49 – 92%) of which expressed CD16, with an average expansion of 88 fold (range 3 – 454). Expanded NK cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, or co-cultured with K562 cells. As detected by intracellular flow cytometry, the average frequency of interferon-γ+ among NK cells was 68% (range 41 – 90%, n=7) and 43% (range 12 – 55%) when stimulated by PMA/ionomycin or K562 cells, respectively. Similarly, the frequency of degranulated NK cells (CD107a+) was 81% (range 35 – 97%, n=7) and 64% (range 35 – 97%), respectively. The ability of expanded NK cells to kill target K562 cells was visualized by activated caspase in co-cultures with labeled K562 cells using the Incucyte® imaging system. At an effector:target ratio of 1:1, an average of 50% of K562 cells were killed (range 45 – 56, n=3). These results show large numbers of functional NK cells can be generated.
Characterization of a novel mouse model for deletion of a floxed gene in alternatively activated macrophages
Authors: Vo Thao T , Patial Sonika , Saini Yogesh
Published: January 2023

V-maf musculoaponeurotic fibrosarcoma oncogene family, protein B (MafB) promoter has been identified as macrophage-specific promoter, but it has never been characterized for pulmonary macrophages. Therefore, we hypothesized that MafB-driven Cre Recombinase expression will allow recombination in alveolar macrophages that contain floxed alleles. MafB-Cre+/− \Rosa-mTOMFlox/Flox/mEGFP (MafB-EGFP) mice was generated by crossbreeding MafB-Cre+/+ and Rosa-mTOMFlox/Flox/mEGFP parents. To determine the extent of macrophage-specific gene recombination, we performed bronchoalveolar lavage, fluorescent microscopy, and flow cytometry on BALF and lung digest of 42 days old MafB-EGFP mice. In addition, we assess the beneficial effect of in vitro and in vivo macrophage activation on recombination efficiency. Flow cytometry analyses on macrophages from MafB-EGFP offspring revealed that: 1) only 30–40% of the alveolar macrophages are amenable to the Cre-Lox mediated recombination, 2) majority of bone marrow-derived macrophages are amenable to the Cre-Lox mediated recombination, 3) Classical activation status did not affect the Cre-Lox recombination efficiency in freshly cultured alveolar macrophages. However, the alternative activation increased the recombination efficiency to ~80–90% in cultured alveolar macrophages. Intratracheal IL-33 treatment of MafB-EGFP mice also increased Cre-Lox mediated recombination of alveolar macrophages to ~80%. Our data suggest that while MafB-EGFP mice do not allow Cre-Lox recombination in the homeostatic lung and inflamed lungs with classically activated macrophages, these mice allow significant recombination in alternatively activated macrophages.

Supported by NIEHS (R01, ES030125)
RenMice™ HiTS platform enables identification of novel therapeutic antibodies
Authors: Frame Jenna , Zhang Xiaoqian , Jin James , Soto Rebecca , Zhang Shujin , Li Xin , Zhang Jing , Shen Yuelei
Published: January 2023

Despite an increase in approved cancer-targeting antibody drugs over the last decade, the process of identifying novel therapeutic antibodies is routinely hampered by limitations in the discovery process. Such barriers include immune tolerance of highly homologous genes, antibody sequence humanization, clone selection and models for drug efficacy/safety evaluation. To overcome these challenges and increase the diversity of antibody paratopes and sequences that recognize functional epitopes, we developed the RenMice™ HiTS (Hyperimmune Target Specific) Platform, which consists of chromosome engineered mice with fully human immunoglobulin variable domains replacing the mouse loci, each with a specific target gene knocked out. Immunization of target-specific RenMice™ generates a sizeable diversity of antibodies, including those that recognize conserved regions between the antigen and the endogenous proteins of the immunized species. The platform is ideal for challenging targets, such as proteins with high homology across species, or multi-pass transmembrane proteins, such as GPCRs/ion channels, and can be used to generate antibodies that cross-react with human, monkey, dog, and mouse targets using a hybrid immunization strategy with both human and mouse/dog antigen. Generation of these species cross-reactive antibodies can be used for high-throughput in vivo efficacy screening in wild-type mice, and the preliminary response and toxicity can be assessed in dogs. Altogether, the RenMice™ HiTS platform facilitates the generation of antibodies that recognize novel epitopes and challenging targets while simultaneously allowing for a streamlined and successful preclinical phase based on in vivo efficacy and safety.
Effects of sample storage using tumor and tissue preservation reagent on genomic data quality
Authors: Shi Xiaoshan , Tran Aimee , Widmann Stephanie , Tyznik Aaron J
Published: January 2023

In-depth understanding of the intrinsic properties and complexity of the tumor microenvironment is crucial to develop both diagnostic and therapeutic strategies for cancer treatment. The tumor microenvironment is composed of various cell types, all of which interact and collectively determine cancer progression and response to therapy. Additionally, tumor cells have a strong ability to adapt to environmental stimuli and can evade treatment modalities. As a result of those features, it is essential to understand the dynamic gene expression profile to decipher tumor heterogeneity and develop efficient and safe therapeutic strategies to fight cancer. Challenges are faced by downstream genomic assay design where tumor specimens cannot be processed immediately in the clinical site. In this case, samples need to be preserved properly to maintain good cell quality and transcriptomic profile. In this work, we propose a storage solution named BD® Tumor and Tissue Preservation Reagent (TTPR). We evaluated the RNA quality of sample storage in this solution up to three days. Expression of cellular stress-related genes were assessed over the storage time course. Importantly, samples stored in TTPR proved compatible with single cell capture workflow using BD RhapsodyTM system and the single-cell gene expression is comparable to that of cells from fresh sample. In conclusion, TTPR is a useful and convenient solution for short-term sample storage prior to downstream genomic assays including single-cell sequencing. For Research Use Only. Not for use in diagnostic or therapeutic procedures.

BD, the BD Logo and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2021 BD. All rights reserved. MSA Approved 1121

Supported by NA
Development of a cell free virus free beads-based SARS-Cov-2 virus neutralization assay
Authors: Hou Xiaohong , Sims Emily , Pan Wenjing , Brown Brittany , Steele Miranda , Song Stephanie , Han Jian
Published: January 2023

Development of a simple, fast, and safe method to detect neutralizing antibody levels to a pathogen is urgent to help understand the immune status of convalescent patients and the immune responses after vaccine. Here we report the development of a cell-free and virus-free beads-based virus neutralization assay. We focused on the detection of neutralization antibody (nAb) in blood circulation to SARS-Cov-2 spike protein receptor binding domain (RBD). This assay uses histidine tagged recombinant RBD protein to replace living virus and uses streptavidin coated polystyrene beads as a carrier of human angiotensin-converting enzyme 2 (hACE2). This bead serves as a surrogate for target cells. Neutralization was detected by the decrease or absence of fluorescence signal associated with RBD. The assay was compared to a cell-based neutralization assay and ELISA, and these results are reported. The bead-based assay is simple, fast, safe and importantly, can truly reflect the immune status of individuals. The result of the assay can help assess the response of vaccine recipients and assist in selecting serum from convalescent patients for therapeutic purposes.
Development of a multiplex microbead-based immunoassay for quantitative analysis of mouse immune checkpoint proteins
Authors: Lie Wen-Rong , Kornmeier Christine , Williams Sasha , Pfeifer Justin , Banerjee Munmun , Hoberg James
Published: January 2023

Immune checkpoint protein research plays a significant role in improving antitumor immune response as well as achieving a deeper understanding of the immune system and the disease states of inflammation, sepsis, and autoimmunity. Various mouse models have contributed to the clinical successes of immune checkpoint inhibitor immunotherapies and the understanding of checkpoint molecule-related inflammatory processes. Here we report the assay verifications and applications of a newly developed Luminex bead-based mouse immune-oncology checkpoint protein multiplex immunoassay for the quantitative profiling of 28 mouse immune checkpoint molecules and immune regulators (PD-1, PD-L1, PD-L2, CTLA-4/CD152, TIM-3, LAG-3, B7-H2/ICOSL, B7-H3/CD276, BTLA, GITR, HVEM, CD27, CD40, CD80/B7-1, CD226/DNAM-1, 4-1BBL/CD137L/TNFSF9, CD137/4-1BB, 5’-NT/CD73, BCA-1/CXCL-13, CD25/IL-2Rα, Granzyme B, E-cadherin, Galectin-1, Galectin-3, IFNγ, IL-10, TLR-2, and TNFα). We present quantitative protein biomarker profiling of mouse serum/plasma samples and conditioned media and lysates from mouse tumor cell lines, including LLC, B16-F10, MCA205, ID8, MB49, AT3, CT-2A, and YUMM1.7, as well as DC2.4 (dendritic cell line), C2C12 (myoblast cell line), and bEnd.3 (endothelial cell line). Our results demonstrate that this mouse immune checkpoint protein multiplex immunoassay is a powerful research tool for the quantitation of key immune regulator proteins and has potential application in biomarker discovery and translational research.
Linking TCR to its cognate antigens with high-throughput and high-dimensional Smart Immune Receptor Sequencing in single cells
Authors: Guo Yuwan , Malone Michael J , Jiang Ning
Published: January 2023

With advances in next generation sequencing, researchers are able to enjoy the ease of sequencing tens of thousands of single cells in one experiment. However, the large cell loss associated poses great challenges for immune receptor profiling from rare clinical samples. To address these challenges, we previously developed Smart Immune Receptor Sequencing (SmartIR-Seq) to simultaneously profile T cell receptor (TCR) sequences and whole transcriptome in single cells more efficiently. However, to better understand T cell functions, it is essential to develop a method to link TCR sequence information to its cognate antigens in the same assay. Here, we describe a high-dimensional SmartIR-Seq technology, SmartIR-SeqHD, which allows us to multiplex cognate antigen specificities, paired TCR sequences, and whole transcriptome in single cells.

In SmartIR-SeqHD, we in vitro transcribed and translated peptide encoding DNA oligos to generate a library of peptides, which were then loaded onto MHCs using a previously developed UV exchange method. By crosslinking streptavidin molecule with a universal oligo, we annealed a DNA oligo which serves as the DNA barcode for that antigen-specificity. Next, tetramer-stained cells were single-cell sorted into plates and lysed, enabling DNA barcodes to be amplified together with mRNAs, including TCR transcripts. We added cell barcodes during PCR amplification and included molecular identifier on the DNA barcodes. The addition of one more “dimension” to SmartIR-Seq using DNA-barcoded pMHC tetramers allows screening for large panels of antigens on primary T cells affordably and quickly, thus further improving the efficiencies and flexibility in the analysis of CD8+ T cells from rare clinical samples.

Supported by grants from NIH R33 IMAT
Enriching for tumor-reactive CD8 TIL (AGX148) leads to effective tumor clearance in a patient-derived xenograph model
Authors: Thalhofer Colin J , Ballinger Elaine A , Montler Ryan D , Morris Nicholas P , Rios Joshua F , Moudgil Tarsem , Weinberg Andrew D
Published: January 2023

Adoptive Cell Therapy (ACT) with Tumor Infiltrating Lymphocytes (TIL) can induce durable clinical responses in a subset of patients with melanoma; however, the efficacy of standard bulk TIL therapy can be limited. We hypothesize that the therapeutic efficacy of TIL ACT is dependent on the frequency and functionality of tumor-reactive T cells when infused back into patients. Our approach enhances the activity of the ACT product by enriching for the tumor-reactive CD8 TIL prior to ex vivo expansion by sorting on cells that co-express CD103 and CD39. The CD103/CD39 double positive (DP) CD8 TIL can be found in most human solid tumors but at varying frequencies in any given patient. Surgically resected human tumor samples were provided through a collaboration with the EACRI. Tumor samples were flow sorted and expanded ex vivo and patient-matched autologous tumor lines were generated. Tumors were then cocultured with the expanded autologous ACT product (AGX148) in vitro or used in xenograph mouse experiments. The AGX148 TIL products are effective at recognizing and killing autologous tumor cells, in vitro. Upon adoptive transfer into patient-derived tumor-bearing huIL-2 NOG mice, the transferred human T cells increased in frequency, migrated to the tumor, became activated in the tumor and recognized/destroyed established tumors. Interestingly there was little to no in vivo activity of CD8 TIL that did not express these two markers. We have generated a potent therapeutic ACT TIL product (AGX148) through the selective isolation and expansion of tumor-reactive T cells. This TIL product effectively induces durable curative responses in hosts harboring autologous tumors providing rationale for clinical investigation.
Targeting the high-affinity IL-2R with high-dose mIL-2/CD25 induces effective antitumor responses
Authors: Laporte Kathryn M , Hernandez Rosmely , Malek Thomas R
Published: January 2023

Much interest remains to harness the T cell activating properties of IL-2 to promote antitumor immunity. Current efforts largely utilize approaches directed toward the intermediate-affinity IL-2R to broadly stimulate memory-phenotypic CD8+ T and NK cells while minimizing off-target Tregs. However, this approach does not effectively engage tumor-reactive CD4+ and CD8+ T effector cells that express the high-affinity IL-2R. Recently, we developed a fusion protein where mouse IL-2 was linked to CD25 (mIL-2/CD25 or FP) with selectivity toward the high-affinity IL-2R. At a low dose, the FP is Treg-selective to limit autoimmunity, but at a high dose with a tumor vaccine it expands tumor-reactive T cells to develop antitumor immunity. Here, we assessed the efficacy of high-dose (HD) FP as a cancer monotherapy or with checkpoint blockade. HD FP supports antitumor responses in mice bearing CT26, MC38, or B16.F10 tumors. Although HD FP led to increases in Tregs in immune tissues, Tregs were at a lower frequency in the tumor microenvironment (TME), leading to a high CD8+ T cell:Treg ratio. Further, adding anti-PD-1 to HD FP treatment was more effective in controlling tumors in CT26-bearing mice. Surprisingly, at the doses tested, HD FP supported more robust antitumor responses in each model than IL-2/S4B6 anti-IL-2 complexes (IL2/IC), directed to the intermediate-affinity IL-2R. In the CT26 TME, the number of tumor antigen-specific CD8+ T cells trended upward with HD FP over IL2/IC. These studies indicate that targeting the high-affinity IL-2R on activated tumor-specific T cells with HD FP alone or with PD-1 blockade supports antitumor responses and suggests that this strategy may be more effective than targeting the intermediate-affinity IL-2R.

Supported by Bristol Myers Squibb grant support
Targeting immunosuppression for enhanced focused ultrasound efficacy in triple negative breast cancer.
Authors: Petricca Lydia E , Witter Alexandra R , Sheybani Natasha D , Thim Eric A , Bullock Timothy N , Price Richard J
Published: January 2023

Immunologic rejection of triple-negative breast cancer (TNBC) is rare. Focused Ultrasound (FUS) promotes anti-tumor immunity by inducing tissue destruction and inflammation. We hypothesized that thermally-ablative FUS (tFUS) would increase the immune response to TNBC. However, TNBC is commonly accompanied by the expansion of immunosuppressive myeloid cells. By implanting a metastasizing TNBC cell line, 4T1, into BALB/c mice, we showed that a combinatorial therapy of tFUS and Gemcitabine (GEM), a myeloablative chemotherapy, was able to control primary tumor growth and reduce mortality. This response is dependent on T cell-mediated immunity. While this treatment remarkably resulted in some cures, the effects were not durable in every case; suggesting immunosuppression may be re-established. We hypothesize GEM is synergizing with FUS to immunologically control tumor growth by either depleting myeloid derived suppressor cells (MDSCs) or inducing tumor cell death. To discriminate between these possibilities, we will use a Ly6G-specific 1A8 antibody to deplete the MDSC population. In parallel, we are interrogating the ability of GEM to induce immunogenic cell death in this model. Current studies are focused on understanding alterations and the role of CD4 and CD8 T cells in tumor control after FUS+GEM therapy. Finally, RNAseq analysis has been performed on treated samples to develop hypotheses to explain the effectiveness of dual therapy. Notably, similar therapeutic approaches are now ongoing as a clinical trial at our institution. We expect our studies to reveal the mechanistic basis in which FUS and GEM are eliciting an immunological response resulting in primary tumor growth control and an overall survival advantage.

Supported by grants from NIH (R01 164985 101 GB10846 41017)
A novel linked TCR: MyD88 receptor improves anti-tumor responses while maintaining antigen specificity
Authors: Magno Joey C , Guo Jitao , Davila Eduardo
Published: January 2023

The treatment of malignant tumors utilizing engineered T cell receptor (TCR) therapies in the clinic have demonstrated encouraging therapeutic potential in a subset of patients. Limitations to T cell-based therapies include down-regulation of tumor-antigen presentation by the tumor cells, poor TCR activation, and the immunosuppressive effects of the tumor microenvironment (TME). Attempts to ameliorate these issues by utilizing T cells engineered with tumor-reactive TCRs with increased affinity showed enhanced responses. However, affinity-modified TCRs are more promiscuous and can lead to off-target toxicity. We and others have demonstrated that activating MyD88 signaling (a key adapter protein in toll-like receptor signaling) in T cells enhances effector function and expansion while simultaneously reducing their propensity to enter an exhausted state. In these studies, we designed novel receptors in which the death and intermediate domains of MyD88 are fused to the TCR. T cells engineered to express this novel TCR:MyD88 receptor showed improved responses in a tumor-antigen dependent manner, as demonstrated by increased proliferation and expression of effector molecules, without reducing the specificity of the TCR. In mouse models, TCR:MyD88 T cells conferred improved anti-tumor responses against tumors expressing suboptimal levels of target antigen which was associated with increased T cell infiltration, reduced T cell exhaustion, and an altered TME. These results demonstrate the ability of TCR:MyD88 to improve current therapies without sacrificing the specificity of the TCR which we believe can be utilized to improve the outcome of patients suffering from advanced cancer while reducing toxicities.

Supported by -US Department of Veteran Affairs -Leukemia Lymphoma Society -University of Colorado -Shoots Medical Center -NCI (R01CA207913)
Targeting Autophagy with Carbon-monoxide Reprograms Anti-Tumor T Cells with Robust Immunometabolic Phenotype
Authors: Chakraborty Paramita , Parikh Rasesh Y , Choi Seungho , Mehrotra Meenal , Maldonado Eduardo N , Wang Hongjun , Diehl J Alan , Gangaraju Vamsi K , Mehrotra Shikhar
Published: January 2023

Mitochondria and Endoplasmic reticulum (ER) share structural and functional networks and activates well-orchestrated signaling processes to shape a cell’s fate and function. While persistent ER stress (ERS) response leads to mitochondrial collapse, moderate ERS condition promotes mitochondrial function. Strategies to boost anti-tumor T cell function by targeting ER-Mitochondria cross talk have not been exploited yet. Thus, we used carbon monoxide (CO), a short-lived gaseous molecule, to test if engaging moderate ERS conditions can improve T cells mitochondrial function and anti-tumor function. Using melanoma antigen specific T cells, we identify that CO induced transient activation of ERS sensor ‘protein kinase R-like endoplasmic reticulum kinase (PERK)’ dramatically increase anti-tumor T cell function. Furthermore, CO-induced PERK activation temporarily halts protein translation and induces protective autophagy (that includes mitophagy). To get further insight, we used LC3-GFP to differentiate between the cells that prepare themselves to undergo active autophagy (LC3-GFPpos) and those that fail to enter into the process (LC3-GFPneg). We found that LC3-GFPpos T cells show robust anti-tumor potential whereas, LC3-GFPneg cells show T regulatory-like phenotype, harbor dysfunctional mitochondria, and accumulate abnormal metabolite content. These anomalous ratios of metabolites render the cells with a hypermethylated state and distinct epigenetic profile limiting their anti-tumor activity. Overall, the study highlights that ERS activated autophagy pathways modify mitochondrial function and epigenetically reprogram the T cells towards a superior anti-tumor phenotype to achieve robust tumor control.
IFNγ from IL-12 virotherapy stably controls tumors independent of T cell cytotoxicity and IFNγ sensing by tumor cells
Authors: Walsh Michael , Ali Lestat , Stump Courtney , Lenehan Patrick , Dougan Michael , Knipe David , Dougan Stephanie
Published: January 2023

Although some work has been done to understand the equilibrium phase of cancer immunoediting, it is still common for immunotherapies to generate stable disease or for patients to relapse after being seemingly tumor-free. We have repurposed the non-lytic herpes simplex virus (HSV)-1 d106S vaccine vector for local delivery of IL-12. Our replication-defective d106S virus releases a large burst of IL-12 locally within the tumor environment, synergizing with a type I IFN response induced by the virus. We have shown that d106S-IL12 induces regression and long-term stable immune equilibrium in murine B16 melanoma. Intriguingly, individual depletion of CD8+ T cells, CD4+ T cells, or NK cells does not affect stable tumor control. However, triple depletion of these cell types leads to tumor outgrowth suggesting these cells have redundant functions. Indeed, IFNγ, likely secreted by these lymphocytes, is essential for controlling tumors in response to d106S-IL12 treatment. The inability of tumors to respond to IFNγ and present antigen to CD8+ T cells through MHC-I often renders tumors resistant to checkpoint blockade and other immunotherapies reliant on direct T cell cytotoxicity. Surprisingly, B16 tumors defective in IFNγ-sensing or MHC-I presentation are still stably maintained in response to IL-12 virotherapy. This suggests that while IFNγ secretion by lymphocytes is necessary, IFNγ and the lymphocytes themselves are not required to act directly on cancer cells to stably control tumor growth. Single-cell RNA-sequencing of immune cells infiltrating the tumor indicates that IFNγ is primarily acting on myeloid cells. Thus, myeloid cells activated by IFNγ may be a method to control growth of checkpoint blockade-resistant tumors.

Supported by grants from NIH (P01-AI098681) and Melanoma Research Alliance
Molecular regulation of CD8 T cell activity and heterogeneity in pancreatic cancer
Authors: Green William D , Mullins Genevieve Nicole , Green Jarred Michael , Baldwin Albert , Milner J Justin
Published: January 2023

T cell dysfunction or ‘exhaustion’ is a hallmark of solid malignancies. Despite the critical importance of CD8 T lymphocytes in controlling tumor growth and immunotherapy efficacy, the mechanisms governing T cell exhaustion remain unclear. Here, we focused on resolving the heterogeneity and molecular programming of exhausted CD8 T cell states in the context of pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a discouraging 5-year survival rate of approximately 8%. PDAC tumors commonly present as immunologically ‘cold’, devoid of protective T cell populations, and are typically resistant to promising immunotherapies such as immune checkpoint blockade or adoptive cell therapy. Therefore, understanding the unique microenvironmental and CD8 T cell-intrinsic signals governing T cell exhaustion and accumulation in PDAC tumors may reveal crucial insight for engineering CD8 T cells for optimal immunotherapy efficacy. Here, we developed preclinical PDAC models for profiling tumor-specific murine CD8 T cell responses. Through application of spectral flow cytometry and multimodal single-cell sequencing approaches, we have uncovered impressively diverse antigen-specific CD8 T cell states in PDAC tumors. Select CD8 T cell populations identified within the PDAC tumor microenvironment included canonical populations of terminally-exhausted and progenitor-exhausted CD8 T cells as well as unexpected and uncharacterized CD8 T cell populations. Accordingly, we also have identified critical transcription factors controlling key features of CD8 T cell heterogeneity and persistence in PDAC tumors. These findings may ultimately provide insight for tailoring CD8 T cell fate and function in PDAC.

Supported by 2022 AAI Intersect Fellowship for Computational Scientists and Immunologists
Tumors bearing defective transcription elongation are immune hot but resistant to immune checkpoint inhibitors
Authors: Modur Vishnu , Guo Fukun
Published: January 2023

Tumors infiltrated with abundant T lymphocytes are generally thought to respond to immune checkpoint inhibitors (ICI). However, a significant proportion of immune hot tumors are resistant to ICI. The immune nature of such tumors is poorly understood. We recently reported a tumor type that is defective in transcription elongation (TEdef) and irresponsive to ICI in several clinical cohorts. Here, we show that TEdef tumors are unexpectedly infiltrated with cytotoxic T cells (CTLs) in patients and in animal models. Mechanistically, T cell infiltration is facilitated by cytoplasmic DNA/double strand RNA-nucleic acid sensing-TBK1-ccl5/cxcl/9/10 signaling cascade. However, single-cell RNA sequencing combined with flow cytometry reveals that CTLs in TEdef tumors are exhausted. Furthermore, immune suppressive regulatory T cells are enriched in TEdef tumors. ICI treatment of TEdef tumors in mice and human patients fails to further induce T cell infiltration. This study reveals a mechanism underlying inert inflammation and ICI resistance of TEdef tumors. We suggest that TEdef, alongside the surrogate markers of tumor mutational burden and CTLs, should be assessed in immunotherapy-candidate patients.

Supported by 5R01CA234038-03
Suppression of local IFN-I by commensal microbiota-derived butyrate impairs antitumor effects of ionizing radiation
Authors: Yang Kaiting , Fu Yang-Xin , Weichselbaum Ralph R
Published: January 2023

The antitumor effects of ionizing radiation (IR) are mediated in part through activation of innate and adaptive immunity. Here, we report that gut microbiota influences tumor control following IR. Vancomycin decreased the abundance of butyrate-producing gut bacteria, accompanied with reduction of butyric acid in circulation and tumor tissues, and enhanced antitumor responses to IR. Oral administration of Lachnospiraceae, a family of vancomycin-sensitive bacteria, associated with increased systemic and intratumoral butyric acid levels and impaired the efficacy of IR in germ-free (GF) mice. Local butyrate inhibited STING-activated type I IFN expression in dendritic cells (DCs) through blockade of TBK 1 and IRF3 phosphorylation, which abrogated IR-induced tumor-specific cytotoxic T cell immune responses without directly protecting tumor cells from radiation. Our findings demonstrate that the selective targeting of butyrate-producing microbiota may provide a novel therapeutic option to enhance tumor radiation sensitivity.

This work was supported by a grant from the Ludwig Foundation to R.R. Weichselbaum, and National Institutes of Health/National Cancer Institute Provocative Questions grants R21 CA227528 and R21 CA231273-01 (R.R. Weichselbaum).
Metabolic reprogramming of antitumor CD8+ T cells by modulation of GSH-Gpx4 axis
Authors: Chen Siqi , Fan Jie , Xie Ping , Ahn Jihae , Fernandez Michelle , Chandel Navdeep , Zhang Bin
Published: January 2023

The CD8+ T cells within tumor microenvironment are the most essential component in tumor immunity. Activation of CD8+ T cells is coupled to various metabolic reprogramming. Glutathione (GSH) as the key role in oxidative metabolismto buffer increased reactive oxygen species (ROS) in activated T cells, is important for T cells effector functions in the context of inflammation. The function and mechanism by GSH metabolism in regulating anti-tumor immunity of CD8+ T cells remain unknown. Here, we show that GSH is dependent on glutathione peroxidase 4 (Gpx4) to maintain CD8+ T cell activity, and A2AR signaling pathway interacts with the GSH-Gpx4 axis to reprogram the metabolism and survival of functional CD8+ T cells. Interestingly, A2AR signaling blockade increases effector function of antitumor CD8+ T cells, but facilitates the assumption of intracellular GSH, leading to rapid ferroptosis. Notably, combination treatment with a potent ferroptosis inhibitor liproxstatin-1 (Lip-1) and A2AR antagonists elicits a synergistic antitumor efficacy and enhanced mitochondrial functionality of antitumor CD8+ T cells in multiple mouse tumor models. Finally, we generate a gene expression signature for GSH metabolism in tumor-infiltrating CD8+ T cells positively correlating with favorable clinical outcomes. Our work demonstrate a critical role of GSH metabolism in modulating antitumor CD8+ T cell survival and functionality, pointing to new strategies of targeting these cells for cancer immunotherapy.
CD8 T cells licensed with immune checkpoint blockade kill murine tumors lacking MHC-I
Authors: Lerner Emily C , Tomaszewski William , D’Anniballe Vincent , Perera Jonathan , Cui Xiuyu , Wilkinson Daniel S , Waibl-Polania Jessica , Gunn Michael , Fecci Peter E , Woroniecka Karolina
Published: January 2023

MHC-I downregulation is a well described mechanism of tumor immune escape. Thus, targeting tumors with low or no MHC-I expression remains a significant challenge for T cell-based immunotherapies, including immune checkpoint blockade (ICB).

We previously demonstrated that the combination of PD-1 blockade and 4-1BB agonism has marked efficacy against intracranial murine CT2A glioma in a CD8 T cell-dependent manner. Surprisingly, this combination therapy remained effective in a β2 microglobulin knockout CT2A line overexpressing TRP2 (CT2A-TRP2-β2mKO). These tumors lack MHC-I but retain the Trp2 rejection antigen. Remarkably, the persisting efficacy remained dependent on CD8 T cells, but independent of NK cells, CD4 T cells, and B cells. Furthermore, the efficacy was demonstrated to be antigen-specific in vivo, as adoptively transferred Trp2-specific T cells prolonged survival of mice bearing CT2A-TRP2-β2mKO tumors, but not those bearing CT2A-β2mKO, which lacked the TRP2 antigen.

To analyze the mechanism driving antigen-dependent killing of tumor cells lacking MHC-I, we performed a series of in vitro cytotoxicity experiments using Trp2-specific T cells in the presence or absence of Trp2 antigen-loaded bone marrow-derived macrophages (BMDM). These studies demonstrated that in the absence of BMDM, Trp2-specific T cells efficiently killed MHC-I-expressing CT2A-Trp2, but failed to kill MHC-I deficient CT2A-Trp2-B2mKO tumors. However, killing was restored in the presence of Trp2 loaded BMDMs; the latter of which possessed little to no cytotoxic effect alone. These findings suggest a novel role for myeloid populations in mediating the influence of ICB on T cell function and challenge the traditional model of T cell tumor killing.
Adoptive transfer of Trac-targeted T cell receptor engineered T cells with defective Tgfbr2 signaling promotes pancreatic cancer eradication
Authors: Rollins Meagan R , Burrack Adam L , Miller Ebony , Stromnes Ingunn
Published: January 2023

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy characterized by a suppressive tumor microenvironment (TME) with elevated Tgfb. Adoptive transfer of T cell receptor (TCR) engineered T cells specific to mesothelin (Msln) effectively targets autochthonous PDA, but the TME promotes engineered T cell dysfunction and limits efficacy. To test our hypothesis that Tgfb is a key mediator of engineered T cell dysfunction in PDA, we first created a Msln-specific TCR Trac knock-in mouse model to generate a uniform source of Msln406-414:H-2Db-specific T cells. Using CRISPR/Cas9, we next knocked out Tgfbr2 in Msln-specific T cells. Both Tgfbr2WT and Tgfbr2KO engineered T cells preferentially accumulate in orthotopic KPC tumors, comprising over 70% of the total CD8+ intratumoral T cells. In combination with vaccination, engineered T cells cause a 10-fold reduction in tumor weight at day 12 post tumor implantation. Loss of Tgfbr2 in effector T cells increases Klrg1, IFNg, TNFa and decreases exhaustion markers PD1, Lag3 and Tox in engineered T cells. Further supporting our data, Tgfbr2 cKO mice receiving orthotopic KPC2a PDA implantation had reduced tumor growth compared to Tgfbr2 WT littermates. Analysis of the endogenous tumor-specific T cells using a tetramer support the role of Tgfb in promoting T cell exhaustion and tumor growth in PDA. Ongoing 2-photon studies on the extent Tgfβ impacts engineered T cell migration, interactions with dendritic cells and stromal modifications will be discussed. Overall, disruption of Tgfβ is a key mediator of both engineered and endogenous tumor-antigen-specific T cell fate by promoting potent Klrg1+ T cells at the expense of Tox+ exhausted T cells and leading to durable tumor control in PDA.

Supported by grants from NIH (R01 CA249393, R01 CA255039, T32 AI 007313), Dennis W. Watson Fellowship, American Association for Cancer Research (AACR) Pancreatic Cancer Action Network Career Development Award (17-20-25-STRO), an AACR Pancreatic Cancer Action Network Catalyst Award (19-35-STRO), an American Cancer Society Institutional Research Grant (124166-IRG-58-001-55-IRG65)
Optimizing vaccine performance through improved cross-presentation with a nanoparticle adjuvant
Authors: Hernandez-Franco Juan F , Jan Imran M , Goldsmith Malaycia , HogenEsch Harm
Published: January 2023

Activation of CD8 T cells requires presentation of antigenic peptides by MHC I molecules and costimulatory signals provided by CD80 and CD86 on dendritic cells (DCs). Subunit and purified antigen vaccines often fail to induce CD8 T cell responses because the antigens have limited access to the endogenous MHC I pathway, a phenomenon known as antigen cross-presentation. Vaccine adjuvants are being investigated to promote cross-presentation, but adjuvants in currently licensed vaccines are not effective. The goal of this study was to determine if a combination adjuvant (NanoS100) comprised of cationic plant-derived adjuvant nanoparticles (Nano-11) and the synthetic STING agonist ADU-S100 stimulates cross-presentation in vitro and in vivo after intradermal and intranasal vaccination. Incubation of DCs with OVA adsorbed to NanoS100 significantly enhanced the presentation of antigen to the MHC I-restricted OVA-specific B3Z T cell hybridoma compared with OVA only. The cross-presentation was inhibited by preincubation of DCs with chlorpromazine and chloroquine, suggesting that NanoS100 supports endosomal processing of OVA. NanoS100 also significantly increased the expression of CD80 and CD86 on antigen-presenting cells in vitro. Vaccination of mice with OVA and NanoS100 increased humoral and cellular responses with a significant increase of IFNγ+ CD8 T cells in the spleen and the lungs. Intranasal immunization induced a greater percentage of IFNγ+ CD8 T cells in the lungs compared with intradermal immunization. These findings support the potential utility of NanoS100 as a vaccine adjuvant.
Intranasal administration of a novel synthetic TLR4 agonist suppresses allergen-specific Th2 responses in mice
Authors: Miller Shannon M , Buhl Cassandra , Whitacre Margaret , Ward Janine , Jackson Konner , Khalaf Juhienah K , Bazin Helene G , Evans Jay T
Published: January 2023

Allergen-agnostic immunotherapies to treat seasonal allergic rhinitis are not currently available. Antihistamines are the most common form of seasonal allergic rhinitis treatment – minimizing symptoms instead of addressing underlying immune dysfunction. Allergen specific immunotherapies (AIT) are also available for seasonal allergic rhinitis but require a known allergen and long-term treatment regiments that can be costly and take several years to complete. Here we report a novel synthetic TLR4 agonist that prevented Th2 allergic responses in an OVA-sensitized mouse model. This novel synthetic TLR4 agonist has improved stability compared to other TLR4 agonists used as vaccine adjuvants or immunotherapeutics. After sensitizing Balb/c mice to OVA using IP injections of OVA + alum, intranasal treatment with the novel TLR4 agonist at one, three, or seven days prior to intranasal OVA challenge reduced or ameliorated OVA-specific Th2 allergic responses. Specifically, intranasal TLR4 agonist treatment reduced OVA-induced airway hypersensitivity, eosinophil influx, and Th2 T cell cytokine secretion – hallmarks of Th2 allergic immune responses. Additionally, we have identified a particular formulation of our novel synthetic TLR4 agonist which has shown greater efficacy, stability, and reduced toxicity profile compared to initial formulations. These data suggest this novel synthetic TLR4 agonist can potentially treat the underlying immune dysfunction associated with allergic responses for a durable cure of seasonal allergic rhinitis.

Supported by NIH contract HHSN272201800036C
B cell ontology in a model marsupial
Authors: Sampson Jordan M , Morrissey Kimberly A , Miller Robert D
Published: January 2023

Marsupials, such as kangaroos and opossums, are members of a lineage of mammals that last shared a common ancestor with humans and mice ~160 million years ago. Although they share many of the same components of the adaptive immune system of humans and mice, they also have some significant differences. For example, in addition to conventional αβ and γδ T cells is the presence of a third T cell lineage, the γμ T cell. Like humans and mice, marsupials have homologues of the classical MHC class I and II molecules and many of the non-conventional MHCs such as CD1 and MR1, but in addition, opossums have another MHC class I family called the UT family. Like humans and mice, opossums also have FcRN. However, the development of B cells may also differ significantly. B cell development in the opossum is entirely postnatal. For example, opossums lack the VpreB1 and 2 and λ5 surrogate light chains; however, like birds, they have VpreB3. B cell development also occurs at a slower pace in the opossum and the sites of B cell development in marsupials remain largely unknown. To address these differences and unknowns, we will present our results regarding timing and location of B cell development, and the establishment of immuno-competence in a model marsupial, the laboratory opossum Monodelphis domestica.

Supported by NSF award IOS-2103367
scRNA-Seq profiling of stickleback fish splenocytes: Expansion of myeloid and B cells on immunization
Authors: Attaya Ahmed , Lohman Brian , Steinel Natalie
Published: January 2023

Threespine stickleback is characterized by wide phenotypic variations attributed to repeated evolutions that took place when colonizing new environments. This fish species is hence deemed a powerful model candidate for comparative immunology to gain new insights into the function and evolution of vertebrate immune system, given also fish small size, wide distribution, and easiness of husbandry. Harnessing single-cell RNA sequencing potential to dissect tissue cellular heterogeneity at high resolution, here we employed that technique to profile naïve, and immunized stickleback spleens identifying the different immune cell types present, and reveal the transcriptional changes driven by immunization. Analysis of the compiled splenic data revealed 18 cell clusters, including major leukocyte types (e.g., B cells, macrophages, and neutrophils), and erythrocytes. Surprisingly, none of the clusters could be recognized or presumed as a T cell population based on the genes expressed in each cluster. Fish immunization led to total cell expansion and interestingly to the emergence of distinct myeloid and B cell populations. The myeloid population lacks the expression of MHCII genes, but was found enriched in the expression of GRN2, S100A10, NCCRP, BPIFC, and PRDX1 genes, suggesting that additional cytotoxic roles are possibly played by that population. Despite the B cell population being enriched in the expression of proteolysis associated genes, i.e., TRY and PRSS59, it also, unlike other B cell populations, lacks the expression of MHCII genes. This study creates a comprehensive atlas of threespine stickleback spleen at single-cell resolution and raises new questions about the immune response evolving in such vertebrate species.

Supported by a grants from NIH (Grant number: NIH R01 AI146168)
Prediction of immunodominant CD4+ SARS-CoV-2 epitopes with TCR repertoire sequencing data
Authors: Pogorelyy Mikhail , Minervina Anastasia , Rosati Elisa , Mudd Philip , Ellebedy Ali , Thomas Paul G
Published: January 2023

The T cell receptor (TCR) is a hypervariable molecule defining the specificity of T cells for peptide-MHC complex. Defining exact epitopes is crucial to profile T cell responses in COVID-19 and yet very few MHC-II restricted SARS-CoV-2 epitopes are currently known. Here, we propose a reverse epitope discovery technique, which, instead of using large pools of peptides to identify reactive T cells, utilizes TCR repertoire sequencing data as the means to predict immunodominant epitopes. The core idea of the approach is to combine information from large, publicly available TCR repertoire datasets: TCRbeta repertoires from cohorts of COVID-19 patients and controls, and paired TCR repertoires of single T cells activated by SARS-CoV-2 peptides. Our pipeline allows us to predict the HLA-restriction of public SARS-CoV-2 specific TCR clonotypes, which in turn allows us to predict binding to a specific peptide. We applied this approach to single cell TCR repertoires of CD4+ T cells from COVID-19 patients, and predicted six MHC-II restricted immunodominant epitopes and alpha-beta TCR motifs recognising them and tested our predictions experimentally. We further applied this technique to bulk TCRalpha repertoires from T follicular helper cells from draining lymph nodes of healthy donors after BNT162b2 mRNA vaccination. We found that the DPB1*04 restricted S167–180 epitope is responsible for the largest public CD4+ response, and recognition of this epitope is driven by a semi-invariant TCR alpha chain. This finding led to generation of the DPB1*04 S167–180 tetramer, allowing to track SARS-CoV-2 specific CD4 cells in peripheral blood and lymph node samples with flow cytometry.

This work was partially supported by R01AI136514 grant.

This work was partially supported by R01AI136514 grant.
Spike BATTLE: Simultaneous evaluation of antigen-specific B and T lymphocytes following natural SARS-CoV-2 infection and subsequent mRNA vaccination
Authors: Newell Krista L , Waldran Mitchell J , Thomas Stephen J , Endy Timothy P , Waickman Adam T
Published: January 2023

The development of effective vaccines and therapies against viral diseases requires reliable approaches for assessing durable immunological memory. Although a wealth of information is available from peripheral blood mononuclear cell samples, Conventional methods for quantifying and phenotyping antigen-specific lymphocytes can rapidly deplete irreplaceable specimens. This is due to the fact that antigen-specific T and B cells have historically been analyzed in independent assays each requiring millions of cells. A technique that facilitates the simultaneous detection of antigen-specific T and B cells would allow for more thorough immune profiling with significantly reduced sample requirements. To this end, we developed the B And T cell Tandem Lymphocyte Evaluation (BATTLE) assay, which allows for the simultaneous identification of SARS-CoV-2 Spike reactive T and B cells using an optimized Activation Induced Marker (AIM) T cell assay and a dual-color B cell antigen probes.

Using this assay, we demonstrate that antigen-specific B and T cell subsets can be identified simultaneously using conventional flow cytometry platforms and provide insight into the differential effects of mRNA vaccination on B and T cell populations following natural SARS-CoV-2 infection. While spike-specific B cell and CD8+ T cell responses were higher following mRNA vaccination than natural infection alone, the quantity of CD4+ T cells recognizing spike was enhanced only in subjects with low responses to natural infection. Our findings suggest divergent patterns of B and T cell subset mobilization with repeated antigen encounter and highlight the utility of examining these populations in concert.

Supported by the State of New York.