bluuebunny/crossref_metadata_2025 huggingface dataset:
Tumor RIPK1 signaling mediates intrinsic and extrinsic mechanisms of resistance to immune checkpoint blockade therapy
Authors: Cucolo Lisa , Qiu Jingya , Klapholz Max , Shi Junwei , Gilliland Gary , Jordan Martha , Minn Andy
Published: January 2023

Immune checkpoint blockade (ICB) therapy can result in impressive clinical responses of multiple cancer types, however resistance is common. Mechanisms of resistance include both tumor intrinsic evasion of immune mediated killing and tumor extrinsic features in the immune-microenvironment, such as poor infiltration of tumor reactive T cells and accumulation of immunosuppressive cells. By examining transcriptomic features associated with response or resistance to ICB, we found the expression of Receptor Interacting Protein Kinase 1 (RIPK1), a critical regulator of inflammation and cell death, is significantly higher in ICB resistant murine tumor cells. We hypothesized that RIPK1 expression in the tumor cells may influence both intrinsic and extrinsic mechanisms regulating response to ICB. To test this hypothesis, we genetically deleted RIPK1 in murine tumor cell lines and found improved overall survival upon treatment compared to tumors expressing RIPK1. Mechanistically, deletion of RIPK1 in the tumor cells results in favorable changes in the tumor microenvironment, including an increased frequency of effector T cells, a decreased frequency of immunosuppressive myeloid cells, and changes in inflammatory cytokine secretion that can impact these changes in immune infiltrate. Further, deletion of the apoptosis effector Caspase 8 reversed RIPK1 KO tumor sensitization to ICB, suggesting that tumor sensitization to cell death also contributes to improved response to ICB upon RIPK1 deletion. In conclusion, RIPK1 is an important regulator at the intersection of tumor intrinsic and extrinsic pathways governing response to ICB, and therefore is a potential target for combinatorial therapies to improve patient response to ICB.
Topoisomerase-I inhibitors induce the expression of clinically relevant immunomodulators in colon cancer cells
Authors: Samuel Temesgen , Shaddox Sage H , Bass Danielle , Bedi Deepa , Datta Pran
Published: January 2023

Immune checkpoint inhibitors have dramatically improved the treatment of certain cancers, but not of most colon cancer types. The reasons are unknown. Camptothecin (CPT), Topotecan (TOP) and Irinotecan (IRI) are chemotherapy drugs that non-specifically inhibit topoisomerase-I, causing side effects. Modulation of immune targets may synergize with topoisomerase inhibitors and reduce the drug dose and side effects. The aim of this study was to examine NF-κB pathway activation and the expression of immunomodulatory proteins after treatment. Colon cancer cells were grown in vitro and treated with either CPT, TOP or IRI. The expression of selected proteins identified through RNA analysis was examined by immunoblotting, ELISA or flow cytometry. We show that topoisomerase inhibitors activate NF-κB pathway with no correlation between peak activation and the concentrations that cause S-phase arrest. On the other hand, the drugs upregulated the immune checkpoint protein Programmed Death Ligand 1 (PD-L1) in colorectal cancer cells in a dose-dependent manner. Osteopontin (OPN) expression was also upregulated in cells grown and treated in medium with regular FBS, while those in charcoal-stripped FBS did not reveal a notable difference in OPN expression. Toll-like Receptor 9 (TLR-9) expression did not markedly vary among CPT treatments under both culture conditions. Topo-I protein expression was downregulated by the treatments, indicating drug-target engagement. Our results hint that PD-L1 and OPN may be potential molecular targets for combination therapy of colon cancer with topoisomerase-I inhibitors. The pros or cons of NF-κB activation by colon cancer cells after chemotherapy remains unknown, and is the subject of further studies.
Neuropilin-1, an immune checkpoint for controlling tumor-specific CD8 T-cell functions whose blockade improves anti-PD-1 immunotherapy
Authors: Mami-Chouaib Fathia
Published: January 2023

Neuropilin-1 (Nrp-1) has been defined as a marker of murine CD4+Foxp3+ regulatory T (Treg) cells and a subset of human CD4+ Treg cells. It is also expressed by a subpopulation of CD8+ T cells infiltrating some human solid tumors. However, little is known about its contribution to regulation of tumor-specific CD8 T-cell functions. We show that Nrp-1 defines a subset of CD8+ T cells displaying PD-1high (PD-1hi) status and infiltrating human non-small cell lung cancer (NSCLC). Using a human cytotoxic T lymphocyte (CTL) clone model, we show that the interaction of Nrp-1 with its ligand semaphorin-3A (Sema-3A), secreted by autologous NSCLC cells, inhibits both CTL migration and tumor-specific lytic function. In vivo experiments revealed that this Nrp-1+PD-1hi CD8+ tumor-infiltrating T lymphocyte (TIL) subset is also found in engrafted B16F10 mouse melanoma, and is enriched with tumor-specific T cells exhibiting an exhausted state, with co-expression of Tim-3, LAG-3 and CTLA-4 inhibitory receptors. Functionally, anti-Nrp-1 neutralizing antibodies increase the migratory capacity of Nrp+PD-1hi CD8+ TIL toward autologous cancer cells and enhance their specific cytotoxic activity ex vivo. Remarkably, in vivo immunotherapeutic blockade of Nrp-1 results in control of tumor growth, with a parallel increase in tumor infiltration by CD8+ TIL and enhanced proliferative and cytotoxic potential. This blockade is cumulative with anti-PD-1 treatment in improving tumor regression. Thus, Nrp-1 appears to be a novel immune checkpoint target on CD8+ tumor-specific T cells for combined antibody-based cancer immunotherapies. More recent advances on Nrp-1 expression regulation and functions in CD4+ and CD8+ T cells will be presented.
Thrombin contributes to cancer immune evasion via proteolysis of platelet-bound GARP for LTGF-β activation
Authors: Metelli Alessandra , Li Zihai
Published: January 2023

Cancer-associated thrombocytosis and high concentration of circulating TGFβ1 are hallmarks of progressive tumor disease in cancer patients. Using genetic and pharmacological approaches, we show a direct link between thrombin catalytic activity and maturation of LTGFβ bound to platelet GARP. We found that GARP protein has two conserved thrombin binding sites through which thrombin cleaves platelet-bound GARP to release active TGFβ from GARP/LTGFβ complex. Crystal structure models docked thrombin onto the concave surface of GARP supporting the notion that thrombin is able to bind and cleave GARP/LTGFβ complex. We observed that inhibition of thrombin obliterates TGFβ maturation in platelet releasate and in serum of WT mice, which translates in better tumor control alone and in combination with checkpoint blockade therapy, in two tumor preclinical models. Of note, the reduction of serum active TGFβ and the anti-tumor properties of thrombin were not observed in absence of platelet GARP. In the tumor microenvironment we found that blocking thrombin reduced active TGFβ signaling and increased the infiltrating CD8+ T cells as well as the innate NK cells and neutrophils. Also, we demonstrated that thrombin cleavage releases soluble GARP/LTGFβ complex and that high concentration of GARP in plasma, alone or in complex with LTGFβ, is a biomarker of poor clinical outcome in prostate cancer patients. We conclude that thrombin-GARP interaction and consequent GARP cleavage induce the activation of TGFβ derived from platelets. Considering that platelets are the major TGFβ reservoir, these observations demonstrated that blocking GARP cleavage opens a new therapeutic window to overcome TGFβ-mediated resistance to immune check point inhibitors.
Human endogenous retroviruses form a reservoir of T cell targets in hematological cancers with low mutational burden
Authors: Saini Sunil Kumar , Ørskov Andreas , Bjerregaard Anne-Mette , Unnikrishnan Ashwin , Holmberg-Thydén Staffan , Borch Annie , Anande Govardhan , Bentzen Amalie , Marquard Andrea , Tamhane Tripti , Gang Anne , Defva Inge , Szallasi Zoltan , Eklund Aron , Pimanda John , Grønbæk Kirsten , Hadrup Sine
Published: January 2023

Human endogenous retroviruses (HERVs) form a substantial part of the human genome. However, most elements remain transcriptionally silent. Malignant transformation and epigenetic therapies may induce transcription of HERV elements. Such HERV elements could potentially trigger adaptive immune responses and provide targets for immunotherapy applications. Here, we evaluate the potential for T cell recognition of HERV elements in myeloid malignancies. We studied 66 HERV genes reported to retain transcriptional activity and generated a library of 1,169 potential antigenic HERV-derived peptides predicted for presentation by 4 HLA class I molecules. Through the use of DNA barcode-labeled MHC-I multimers, we identified CD8+ T cell populations recognizing 29 HERV-derived peptides, represented by 18 different HERVs. T cell recognition of HERVs was detected in half of the 34 patients analyzed and was significantly enhanced in patients compared to healthy donors. Patients with HERV-derived T cell reactivity demonstrated improved viral-antigen recognition following treatment, indicating that HERV-derived T cell recognition may contribute to the therapy-induced clinical improvement and immune reconstitution. In line with the T cell data, transcriptomic analyses revealed a significantly enhanced transcription of the HERVs in patients compared to healthy donors. DNA-demethylating therapy did not result in additional transcriptional enhancement of the HERVs evaluated for T cell recognition. Our study demonstrates substantial T cell recognition of HERVs in myeloid malignancies. These HERVs could serve as a reservoir of antigens potentially exploited as immunotherapeutic targets in malignancies with low mutational burden.
Identifying host-intrinsic resistance mechanisms to therapeutic anti-tumor antibody
Authors: Glassbrook James E , McCarthy Claire , Hackett Justin , Muñiz Maria , Dyson Greg , McCasland Jennifer , Gibson Heather
Published: January 2023

Monoclonal antibody (mAb) therapies have greatly improved outcomes for many cancer subtypes, many of which target tumor-associated antigens (TAA). Trastuzumab targets the TAA human epidermal growth factor 2 (HER2), inducing antibody dependent cellular cytotoxicity and phagocytosis (ADCC/ADCP) as well as interfering with receptor signaling. Despite its reputation as the gold standard treatment for HER2-overexpressing cancers, response rates remain low in metastatic breast cancer (≤30% as a monotherapy, ≤50% with chemotherapy). Multiple studies suggest induction of endogenous anti-tumor immunity is a determinant of therapeutic success. Utilizing the genetically heterogeneous Diversity Outbred (DO) mouse model, we seek to identify host-intrinsic mechanisms of resistance to TAA-mediated immunotherapy. We find a divergent response to αNeu (HER2 homolog) mAb clone 7.16.4 in (DOxBALB/c)F1 mice bearing established BALB/c-syngeneic TUBO mammary carcinoma. We find host genetic background regulates anti-tumor immune activity. ~25% showed complete response (CR) to therapy, ~60% a partial response (PR), and ~15% failed to respond (NR). CR mice were protected from a contralateral rechallenge, suggesting adaptive anti-tumor immunity was promoted by mAb therapy. Peripheral anti-tumor IgG, measured by flow cytometry, and IFNγ producing T cells, measured via ELISpot, also trend toward CR>PR>NR. Genetic linkage analysis using R/qtl2 identified loci associated with robust response to mAb therapy. Genes within these loci may represent potential routes of ingress toward addressing innate and acquired resistance during anti-TAA mAb therapy, particularly as regulators of immunogenicity of ADCC and ADCP. NIH R37 CA220482.
Irreversible electroporation can alter immune cell composition and enhance antigen presentation potential in pancreatic cancer
Authors: Brock Rebecca Michaela , Beitel-White Natalie , Hendricks Alissa Danielle , Zeher Allison , Manuchehrabadi Navid , Davalos Rafael V , Allen Irving C
Published: January 2023

Pancreatic cancer has a devastating survival rate of only 9%. A major treatment limitation is the immunosuppressive nature of pancreatic tumors created by high amounts of tumor-associate stromal cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Tregs). These form a barrier that inhibits anti-tumor immunity and has yet to be overcome by current immunotherapy options. Irreversible electroporation (IRE), an ablative technology that utilizes short electrical pulses to disrupt tumor cell membranes, shows evidence of inducing proinflammatory cell death in pancreatic tumors, but its effect on innate and adaptive immune responses, vital for long-term survival, has not been investigated. We hypothesize that IRE treatment for pancreatic cancer can reduce pro-tumor immune cells, recruit anti-tumor immune cells such as cytotoxic T-cells, and increase tumor-specific antigen presentation potential. We utilized in vivo immunocompetent Pan02 murine pancreatic cancer models to assess progression-free survival and tumor immune cell populations via flow cytometry, as well as an ex vivo antigen presentation model with murine bone marrow-derived dendritic cells and splenic T-cells to determine the ability of IRE to generate viable antigens. Our findings show a temporal increase in anti-tumor immune cell populations, reduction in tumor-associated immune cell populations, and strong antigen presentation potential. However, we also observed an increase in Pdl-1, normally down-regulated in pancreatic cancer, in response to increased immune signaling. This may make IRE a candidate for cotherapy options such as anti-Pdl-1 antibodies or other immunotherapies that could increase patient survival.
IRF1 regulates apoptotic caspases to thwart the development of colorectal cancer
Authors: Karki Rajendra , Sharma Bhesh , Kanneganti Thirumala-Devi
Published: January 2023

Interferon regulatory factor 1 (IRF1) is a transcription factor that is induced in response to interferons and inflammatory cytokines. We have previously identified the role of IRF1 in mediating inflammasome activation. These inflammasomes are associated with providing protective responses against colorectal tumorigenesis. The relevance of IRF1 to the immunobiology of cancer is still emerging, and the crucial role of IRF1 in colorectal cancer (CRC) remains unknown. The multiple inflammatory mediators produced during colorectal tumorigenesis have the ability to mediate cellular death; this may represent alternative forms of apoptosis that cannot readily be recapitulated in vitro by using classical apoptosis inducers. The precise regulatory mechanisms by which IRF1 governs the activation of cell death in response to inflammatory milieu remains elusive. Given that IRF1 is a transcription factor, it may induce the expression of upstream cell death mediators that are dependent on cytokines and that subsequently activate the executioner caspases. Here, we found that Irf1−/− mice were hyper-susceptible to CRC. IRF1 played roles in both myeloid and epithelial compartments to confer protection against CRC. Interestingly, IRF1 did not regulate inflammatory cytokines production and inflammasome activation in colons. However, IRF1 promoted cell death mediated by the inflammatory cytokines. The decreased cell death in the colons of Irf1−/− mice was associated with impaired activation of apoptotic caspases. Mechanistically, we demonstrated that IRF1 deficiency attenuated the induction of Fas and activation of caspase-8 that lead to reduced effector caspase-3 and caspase-7 activation in the colons.
Elucidating the immune response in metastatic ovarian cancer using single cell transcriptomics
Authors: Olalekan Susan , Xie Bingqing , Back Rebecca , Eckart Heather , Basu Anindita
Published: January 2023

Ovarian cancer is a highly metastatic and the most lethal malignancy of the female reproductive tract. In high grade serous ovarian carcinoma, the presence of tumor infiltrating lymphocytes suggests that this disease can be treated using an immunotherapeutic approach. However, determining candidates who will benefit from immunotherapies such as immune checkpoint inhibitor remains a crucial hurdle. Since immune checkpoint inhibitors work by reactivating an already existing anti-tumor response, a knowledge of the immune cell types within the tumor is required to predict the efficacy of this therapy. The objective of this study was to determine the cellular composition of metastatic ovarian cancer samples using single cell RNA sequencing. Hierarchical clustering of 6 patient samples revealed 2 main groups: a high T cell (2 samples) and 2) a high monocyte signature group (4 samples) suggesting differences in immune response. To assess the immune response in these patient samples, we performed an unsupervised clustering of the T cell population in the two groups. The T cell population clustered into 4 and 3 subpopulations in the high T cell and high monocyte group respectively. A granulysin expressing population was unique to the High T cell group. Interestingly, although both groups had tissue-resident memory CD8+T (CD8+Trm) cells, only the CD8+Trm cells in the high T cell group expressed TOX, a recently described transcription factor. TOX is expressed by exhausted CD8+ T cells that are reactivated in response to PD-L1 immunotherapy. In conclusion, our data reveals a subset of patients that may benefit from novel therapy.
Leveraging the power of high parameter cell sorting and single-cell multi-omics to profile intratumoral immune cells in a model of B cell lymphoma
Authors: Shi Xiaoshan , Baracho Gisele Vanessa , Lin Wai , Sedy John , Lomas Chip , Widmann Stephanie , Tyznik Aaron J
Published: January 2023

The pathogenesis and progression of B-cell lymphomas is a dynamic process involving reciprocal interactions between malignant cells and surrounding components in the tumor microenvironment. Neoplastic B cells migrate to lymphoid sites and prompt the immune cell infiltration. The infiltrated cytotoxic lymphocytes potentially contribute to tumor cell killing and good prognosis. However, most immune cell types in the tumor niche often become unresponsive or end up promoting tumor growth. In this study we present a sorting strategy using the BD FACSymphony™ S6 Cell Sorter that enables concurrent isolation of six cell populations from a tumor niche in a murine B-cell lymphoma model. Single-cell sequencing was used downstream to unravel the respective roles of each cell subset for the tumor’s progression and/or evasion. The six cell subsets were isolated from mice that had been inoculated with an OVA-expressing B-cell lymphoma cell line and subsequently with either wild-type or BTLA-deficient OVA-specific CD8+ T cells. The cell sorting aided more robust single-cell profiling of rare cell subsets within the sorted populations. In summary, we demonstrated a high-throughput approach that enabled a comprehensive characterization of critical cell populations in tumor responses. Since these cell populations constitute potential targets for the treatment of B-cell lymphomas, this platform, notably through the analysis of protein expression, offers an opportunity for identification of new therapeutic targets.

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Identifying regulators of reversible MHC I loss in Merkel cell carcinoma through genome-scale screens
Authors: Lee Patrick C , Le Phuong , Korthauer Keegan , Cheng Jingwei , Doench John , DeCaprio James A. , Keskin Derin B. , Wu Catherine J.
Published: January 2023

Loss of surface MHC I is an important mechanism by which cancer cells evade immune surveillance, and it can correlate with worse prognosis and resistance to immunotherapy. MHC I surface expression can be affected by genomic or transcriptional alterations to HLA-A/B/C, β2M, or the class I antigen processing machinery. In the case of transcriptional loss, we hypothesized that certain cancers might employ specific epigenetic programs to enforce downregulation of the MHC I pathway. Thus, we sought to identify novel MHC I regulators in MHC I-low cancers. In particular, we focused on Merkel cell carcinoma (MCC), a rare but aggressive neuroendocrine skin cancer that is caused by the Merkel cell polyomavirus in 80% of cases. Notably, MHC I downregulation is prevalent in MCC. We first generated and characterized a series of patient-derived MCC cell lines, in which MHC I surface expression was low but inducible with IFN-γ. We then conducted genome-scale CRISPR-KO and open reading frame (ORF) gain-of-function screens in one of our virus-positive MCC lines, using FACS to select for perturbations that upregulated surface MHC I. One of the top hits from these screens was PRC1.1, a non-canonical Polycomb repressive complex. Polycomb complexes are known to mediate gene silencing through chromatin modification and play an important role in development and cancer. These studies suggest a possible role for PRC1.1 in suppressing MHC I in MCC.
Genome-wide CRISPR screen reveals cancer cell resistance to NK cells induced by NKderived IFN-γ
Authors: Zhuang Xiaoxuan , Long Eric O
Published: January 2023

The anti-leukemia activity of NK cells is important for preventing relapse during HSCT in leukemia patients. However, the factors that determine susceptibility of leukemia cells in the context of NK-mediated killing are not well established. Here we performed a genome wide CRIPSR screen in the human CML cell line K562 to identify genes that regulate vulnerability of leukemia cells to killing by primary human NK cells. Distribution of gRNAs in K562 cells that survived co-incubation with NK cells showed that loss of NCR3LG1 (encodes B7H6), the ligand of the activating receptor NKp30, predominantly protected K562 cells from killing. It indicates that B7H6 is a major ligand on K562 cells. In contrast, loss of genes that regulate pathways for antigen-presentation (B2M, HLA-I, PSMB5, TAP1, TAPBP) and IFN-γ signaling (IFNGRs, JAKs, STATs) increased the vulnerability of K562 cells. MHC-I on K562 cells was upregulated after co-culture with NK. Addition of IFN-γ neutralizing antibody during the co-culture increased the susceptibility of K562 cells to NK-mediated lysis. The results suggest that cancer cells respond to NK-derived IFN-γ and lead to NK-resistance and immune evasion. Moreover, Analysis of RNA-seq data from The Cancer Genome Atlas (TCGA) showed that low IFNGR2 expression in cancer tissues associated with improved overall survival in AML patients, indicating that the IFNGR-JAK-STAT pathway can be a contributor to worse prognosis of cancer patients, potentially related to IFN-γ–induced NK-resistance, despite the fact that IFN-γ signaling promotes T cell mediated immune surveillance. Our results suggest that targeting IFN-γ responses might be a promising approach to enhance NK cell anti-cancer efficacy.
Profiling the immune infiltrate in tumor samples at single cell resolution
Authors: Cheung Denise , Adams Bruce A. , McDonnell Wyatt J. , Jaffe David B. , Puleo Alaina R. , Sukovich David J , Reyes Daniel J , Royall Ariel J , Chi Javelin C. , Srinavas Niranjan J , Krishnan Sreenath J , Carli Natasha J , Montesclaros Luz J , Lau Julia J , Stubbington Michael J.T. , Taylor Sarah E.B.
Published: January 2023

Profiling the complex interactions of immune infiltrate with tumor cells in a tumor microenvironment is critical for advancing our understanding of tumor biology for developing personalized cancer therapies. Using a droplet-based single cell RNA sequencing (scRNA-seq) platform, we profiled the transcriptome and immune repertoire of gastric, kidney, and lung cancer cells that are primarily immune cells from three different donors. scRNA-seq analysis also identified similar fractions of CD45+ immune cells, CD4+ and CD8+ T cells, CD19+ B cells, and myeloid cells compared to flow. Targeted scRNA-seq was also used to identify paired, full length B-cell (BCR) and T-cell (TCR) receptors. In the gastric adenocarcinoma tumor cells, gene expression analysis identified a large population of B cells, but with no clonal expansion. T cells expressing CD8A constituted about 10% of cells with the top clonotype being 1% of all clones. The clear cell RCC sample had a modest fraction of infiltrating T cells with the top clonotype representing 7.4% of all clones, and no B cell infiltrate. The NSCLC cells were mainly T and B cells but limited clonal expansion was observed; the top TCR clonotype was present on 2.2% of all T cells, no expansion was seen in any of the B cell clonotypes. These findings highlight the value of profiling of tumor immune cells holistically and not relying on the presence of B or T cells alone to understand the immune dynamics of the tumor microenvironment. The presence of tumor-infiltrating lymphocytes is associated with favorable clinical outcomes in some cancers, but understanding their cellular subtype and clonality by high resolution profiling is key in the development of immune-based cancer treatments
Single-cell transcriptomic profile reveals macrophage heterogeneity in medulloblastoma and their distinct responses to different treatment modalities
Authors: Haldar Malay , Dang Mai T
Published: January 2023

Tumor-associated macrophages (TAMs) are an important component of the tumor microenvironment (TME). Pro-inflammatory macrophages can suppress while anti-inflammatory macrophages can promote tumor growth. Despite their abundance in many tumors, the origins and diversity of TAMs are not well understood, especially in brain tumors. Our long-term goal is to uncover ways to modulate TAMs for the treatment of pediatric brain tumors.

Medulloblastoma (MB) is one of the most common pediatric brain malignancy. MB is heterogeneous with four molecular subgroups: WNT, sonic hedgehog (SHH), group 3, and group 4. Treatment includes surgical resection, cytotoxic chemotherapy, and radiation. Radiation is effective, but severe neurological side-effects limit its use to non-infants (> 3 years). Therefore, treatment choices are even more limited in infants. SHH-MB dominates this age group and is characterized by constitutive activation of the SHH signaling pathway. Ptch1 is a negative regulator of SHH signaling that is often mutated in SHH-MB. In mice, germline deletion of Ptch1 and loss of Tp53 leads generates MB with 100% penetrance.

Using single-cell RNA sequencing (ScRNA-Seq) in the genetically engineered mouse model (Ptch+/−:p53−/−) of SHH-MB, we describe macrophage heterogeneity in medulloblastoma, demonstrate differential recruitment and function of macrophages under distinct modalities of tumor therapy (molecular-targeted vs. radiation), and use these insights to uncover potential strategies for immunomodulation as adjunctive therapy.
Thousands of new antigens are recognized in mice via endogenous antibodies after being cured of a B78 melanoma via immunotherapy
Authors: Hoefges Anna , Erbe-Gurel Amy K , McIlwain Sean J , Melby Andrew S , Xu Angie , Mathers Nicholas , Rakhmilevich Alexander L , Hank Jacquelyn A , Baniel Claire , Pinapati Richard , Garcia Brad , Patel Jigar , Morris Zachary S , Ong Irene M , Sondel Paul M
Published: January 2023

Antibodies can play an important role in innate and adaptive immune responses against cancer. Using a high-density peptide array, we assessed potential protein-targets for antibodies activated in mice cured of their melanoma through a combined immunotherapy regimen. Our goal was to determine the linear peptide sequences recognized by anti-tumor antibodies produced in mice cured of their melanoma with immunotherapy.

Mice with GD2-expressing syngeneic B78 melanoma were treated with a combination immunotherapy capable of inducing an “in situ vaccine” effect (ISV), enabling mice to be cured of their tumors with long-term immune memory. Naïve and immune sera were collected from these mice. Using flow cytometry, immune sera showed strong antibody-binding against B16 (parental cell line of B78 without GD2 expression). These sera were then used on a Nimble Therapeutics’ peptide-array (either whole proteome or a curated list of ~650 proteins) to determine specific antibody-binding sites, and data were analyzed using a probabilistic model. Using the “curated list” peptide array, proteins were selected if the protein was bound in immune sera but not bound in the sera from naïve or non-responding tumor-bearing mice. When focusing on the whole mouse proteome data, thousands of peptides were targeted by 2 or more mice and exhibited strong antibody binding only by immune sera.

We are continuing to refine our analytical methods and are further investigating all identified proteins. These peptides may be new targets for antibody-based or cellular therapies and some of the tumor-specific endogenous antibodies that we have identified may be used as biomarkers to predict response to our ISV regimen and potentially other immunotherapy treatments.
Multi-dimensional analysis of tumor-specific and bystander CD8+ tumor infiltrating lymphocytes with TetTCR-SeqHD
Authors: Schonnesen Alexandra A , Parker Patrik , Ma Ke-Yue , McKinney Jennifer , Flemming Declan , Fox Matthew , Eckhardt Gail , Jiang Ning
Published: January 2023

Objective

Antigen specific CD8+ T cells were isolated from primary tumor tissue and analyzed for on the single cell level to determine tumor-specific signatures for further study.

Methods

Primary tumor, metastatic lesions, adjacent healthy tissue and peripheral blood were from collected from patients at Dell Seton Medical Center at the University of Texas. Matched tumor and healthy samples underwent exome and RNA sequencing to allow prediction of tumor-specific epitopes. We then synthesized DNA-barcoded tetramers for all predicted tumor associated-epitopes and an HLA-matched library of exogenous epitopes. Tetramer stained CD8+ T cells were then sorted and analyzed by TetTCR-SeqHD, a technology that we recently developed to link TCR antigen specificity, TCR sequence, gene expression, and phenotype of single T cells.

Results

We analyzed ~2900 tumor infiltrating lymphocytes (TILs) and T cells from adjacent healthy tissue and observed enrichment for cytotoxic effector and tissue resident memory cells within the tumor versus the healthy tissues. We also note an enrichment of bystander cells, specifically those reactive to Epstein Barr Virus epitopes, within the cytotoxic cluster. Finally, we analyze the distribution and differential expression between tumor-specific lymphocytes and bystander T cells, noting reduced expression of CD57 and CD49d in the tumor-specific TILs, as previously described.

Conclusions

We validate the use of TetTCR-SeqHD for gaining insight into phenotypic and gene expression differences between bystander and tumor-specific TILs. We identified markers for future exploration and demonstrate key differences between shared clonal populations within healthy and cancerous tissues.
USP22 stabilizes FoxP3 to maintain Regulatory T cell function through a dual mechanism resulting in tumor immune evasion
Authors: Montauti Elena , Zhang Yusi , Cortez Jessica , Marson Alexander , Fang Deyu
Published: January 2023

Aggressive cancers are less sensitive to standard cancer treatments available, reducing overall survival rates and increasing relapse percentage in cancer patients. As a result, there is an increasing need for alternative therapeutics that will prolong the survival of patients. Within the last decade, methods to attenuate tumor immune evasion have become a centerpiece of tumor therapies. However, a major hurdle in tumor immunotherapy is the immunosuppression mediated by Regulatory T (Treg) cells, which can promote tumor progression through the suppression of tumor immune evasion. Therefore, the ability to modulate Treg function in the context of cancer could lead to more effective therapies. Here, we identify the ubiquitin-specific peptidase 22 (USP22), a member of the deubiquitination module of the SAGA chromatin modifying complex, as a specific regulator of Foxp3, the lineage-specifying transcription factor of Tregs. Treg-specific ablation of Usp22 in mice reduced Foxp3 at both the transcriptional and post-translational level, creating defects in their suppressive function and leading to spontaneous autoimmunity. Importantly, USP22 ablation protected against tumor growth in multiple cancer models. Collectively, our findings reveal a previously underappreciated physiological function of USP22 in maintaining Treg stability and identify USP22 as a potential target for cancer immunotherapy.
Human influenza reactivity to conserved influenza hemagglutinin and its relation to age and repeat vaccination
Authors: Moritzky Savannah A , Sant Andrea J
Published: January 2023

The first exposures to influenza have been shown to bias the immune system towards a certain strain which is then boosted by subsequent infection and vaccination throughout life. The impact of repeated stimulation of hemagglutinin (HA) epitopes of the CD4 T cell repertoire is unknown. We are interested in the CD4 T cells specific to conserved regions of HA as the conserved stalk region is a target for new vaccine designs and antibodies to HA are a correlate of protection from infection. We are also interested in how CD4 T cell phenotypes after repeated antigen exposure from conserved HA epitopes could change the CD4 T cell repertoire. In order to probe these questions, we analyzed PBMCs collected from healthy human donors using cytokine ELISpots and flow cytometry. We created overlapping peptide pools containing conserved or unique regions of HA, to compare differences in cytokine secretion, abundance, and phenotypes of CD4 T cells elicited to different regions of HA. Our hypothesis is that CD4 T cells specific to repeatedly stimulated epitopes will have a higher frequency and different phenotypes compared to unique epitopes. We have found that there is a robust circulating CD4 T cell response to influenza antigens. The reactivity to conserved regions of HA varies between individuals as well as between strains of influenza in healthy human donors and in some donors, there are CD4 T cells that are biased towards conserved regions of HA. Those CD4 T cells to conserved regions of the stalk could be recalled after vaccination with stalk-based vaccine strategies. We plan to further examine the phenotypes of those CD4 T cells and to determine if decade of birth and vaccination history will impact the abundance of CD4 T cells to conserved HA regions.
Immune tolerance limits effective immunity to epitopes targeted by universal influenza vaccines
Authors: McGargill Maureen Ann , Pillai Meenu R , Labombarde Jocelyn , Lin Chun-Yang , Crawford Jeremy Chase , Chang Ti-Cheng , Keating Rachael , Lewis Carlessia , Guthmiller Jenna , Li Quan-Zhen , Wilson Patrick C , Thomas Paul Glyndwr
Published: January 2023

A universal influenza vaccine could save millions of lives in the event of a deadly pandemic. It is not clear why antibodies specific for conserved regions of influenza viruses are so rare. One possibility is that these antibodies have a higher potential to cross-react to self-proteins, and therefore B cells that generate these antibodies are deleted through tolerance mechanisms. Therefore, we examined whether cross-reactive influenza antibodies had a higher potential to be autoreactive than antibodies specific for one subtype of influenza. We previously demonstrated that H3N2-vaccinated mice treated with a low dose of rapamycin had more cross-reactive influenza antibodies and were better protected against subsequent lethal infections of multiple subtypes. Thus, we utilized rapamycin to increase the frequency of influenza cross-reactive antibodies, and tested whether these antibodies were more reactive to self-proteins than strain-specific antibodies. We found that mice with increased levels of cross-reactive influenza antibodies also had more IgM antibodies specific for self-antigens. Importantly, the increase in autoreactive IgM antibodies correlated with increased susceptibility to disease in mouse models of multiple sclerosis, lupus, and Guillian-Barre Syndrome. Together, our results suggest that influenza cross-reactive antibodies have the potential to be autoreactive. These data have important implications for developing universal influenza vaccines designed to generate durable influenza cross-reactive antibodies.
Regression of murine breast tumors after hetIL-15 monotherapy correlates with a novel population of intratumoral dendritic cells, in addition to increased infiltration of T, NK and cDC1 cells
Authors: Pavlakis George N , Karaliota Sevasti , Stellas Dimitri , Bergamaschi Cristina , Stravokefalou Vasiliki , Nagy Bethany , Felber Barbara K.
Published: January 2023

Background

We studied mechanisms of therapeutic efficacy of the heterodimeric IL-15 (hetIL-15) of human, macaque or mouse in several mouse tumor models, including the murine EO771 orthotopic breast cancer model in syngeneic C57BL/6 mice.

Methods

The effects of hetIL-15 on immune cells were compared in tumors, lymph nodes and spleen by flow cytometry, immunohistochemistry (IHC), gene expression profiling (Nanostring) and metabolic profiling of tumor infiltrating T cells.

Results

hetIL-15 administration resulted in tumor regression in 40% of the treated animals and resistance to tumor rechallenge, inducing long term immunological memory. Activated NK and CD8+ T cells were increased in hetIL-15 treated tumors. hetIL-15 administration resulted in increased intratumoral infiltration of conventional Dendritic cells (cDC1) and also induced a new population of intratumoral DC, (F4/80high DCs), which were correlated negatively with the tumor size. Phenotypic profiling of F4/80high DCs identified expression of several cDC1 specific markers, including CD103, IRF8 and XCR1. tSNE analysis clustered F4/80high DCs close to cDC1s and macrophages.

Conclusion

Complete eradication of EO771 tumors by hetIL-15 treatment correlates with different tumor infiltrating immune cell types. We propose that the anti-cancer activity of hetIL-15 in primary tumors is orchestrated by the interplay of CD8+T cells, cDC1 and a novel subset of antigen presenting cells with a distinct phenotypic profile. Effective hetIL-15 treatment leads to development of long term anti-tumor memory. hetIL-15 supported a favorable metabolic profile of intratumoral effector lymphocytes, important for their function.
Glycolipid-loaded nanoparticles harness iNKT cells for tumor immunotherapy
Authors: Shute Travis J , Dudley Elizabeth , Lai Andrew , Salas Briana , Angel Daniel , Nash Kelly , Leadbetter Elizabeth
Published: January 2023

Invariant natural killer T cells (iNKT) have a well-documented role in anti-tumor immunity through their release of proinflammatory cytokines and cytotoxic compounds. As iNKT cells can have direct and indirect killing effects on tumor cells, we propose a novel strategy for activating iNKT cells, via a PLGA nanoparticle delivery platform, to promote anti-tumor immune responses. Poly-lactic-co-glycolic acid (PLGA) nanoparticles can be reproducibly loaded with an iNKT cell glycolipid agonist, alpha-galactosylceramide (αGalCer), and a tumor associated antigen, ovalbumin (OVA). These dual-loaded PLGA nanoparticles rapidly activate iNKT cells in vivo to produce IFNgamma. Furthermore, in an in vivo model of melanoma, using B16F10-OVA cells, both prophylactic and therapeutic administration of nanoparticles containing αGalCer and OVA led to decreased tumor cell growth and increased survival. Ongoing studies are extending these concepts to PLGA nanoparticles loaded with αGalCer plus an immunogenic peptide from the naturally expressed melanocyte protein glycoprotein 100, gp100 25-33. This novel delivery system provides a platform with tremendous potential to harness iNKT cells for cancer immunotherapy purposes and as part of combinational therapies with other approaches such as checkpoint inhibitors.
De novo DNA methylation programs regulate CAR T-cell exhaustion
Authors: Zebley Caitlin C , Petersen Christopher T. , Prinzing Brooke , Bell Matthew , Fan Yiping , Crawford Jeremy Chase , Houke Haley , Haydar Dalia , Yi Zhongzhen , Nguyen Phuong , DeRenzo Christopher , Lazzarotto Cicera , Tsai Shengdar , Miller Shondra , Langfitt Deanna , Gottschalk Stephen , Krenciute Giedre , Youngblood Benjamin
Published: January 2023

Chimeric antigen receptor (CAR) T-cell therapy is revolutionizing cancer immunotherapy for patients with B cell malignancies. While clinical trials have demonstrated the curative potential of this approach, a significant and well-established limitation is the heightened contraction and transient persistence that CAR T-cells experience during prolonged antigen exposure. Building upon our prior observation that deleting the de novo DNA methyltransferase 3a (Dnmt3a) prevents T cell exhaustion in the prototypical model of LCMV-induced T cell exhaustion, we assessed the role of DNMT3A programming in the dysfunction of human CAR T-cells. Deletion of DNMT3A in multiple human CAR T-cell systems resulted in a striking preservation of the CAR T-cell’s ability to proliferate and mount an effector response during chronic antigen exposure. Whole genome methylation profiling of DNMT3A KO CAR T-cells established an atlas of epigenetically regulated genes targeted during CAR T-cell dysfunction. Cross-reference of our published murine exhaustion methylation profiles with our newly identified human CAR T-cell methylation atlas revealed conservation of epigenetically regulated exhaustion-associated genes. Using a novel epigenetic-based bioinformatic tool that predicts human T-cell differentiation we further documented the preserved developmental plasticity of the DNMT3A KO CAR T-cells. Lastly, analysis of publicly available RNAseq expression data from CD19-CAR T-cell products prior to infusion into CLL patients demonstrated that DNMT3A programming is significantly coupled to clinical outcome. Collectively our results demonstrate that de novo DNA methylation programming is a key factor limiting T-cell based immunotherapy.
Development of a high affinity and specific CHIKV-E2 monoclonal antibody for Chikungunya diagnosis
Authors: Kim Jaemoo , Yang Jihyun , Kim Young Bong , Lee Hee-Jung , Kim Sehyun , Poo Haryoung
Published: January 2023

Chikungunya fever is a vector-borne viral disease transmitted to humans by Chikungunya virus (CHIKV)-infected mosquitoes. There have been many outbreaks of CHIKV infection in world. Local transmission of chikungunya virus was emerged in the America continents at 2013. The virus poses ongoing risks to global health. To control CHIKV infection, it is important to improve the existing CHIKV diagnostic approaches to allow for the detection of low CHIKV quantity and to correctly discriminate CHIKV infections from those due to other mosquito-transmitted viruses, including Zika virus (ZIKV), Japanese encephalitis virus (JEV), and Dengue virus (DENV). Here, we produced monoclonal antibodies (mAbs) against the CHIKV envelope 2 (CHIKV-E2) protein and compared their affinity and specificity with commercially available mAbs using enzyme-linked immunosorbent assays (ELISA). Two anti-CHIKV-E2 mAbs, 19-1 and 21-1, showed higher binding affinities to CHIKV-E2 protein compared with the commercial mAbs. Especially, the 19-1 mAb had the highest binding affinity to inactivated CHIKV. Moreover, the 19-1 mAb had very little cross-reactivity with other mosquito-borne viruses, such as ZIKV, JEV, and DENV. These results suggest that the newly anti-CHIKV-E2 mAb, 19-1, could be applied for a CHIKV diagnostic approaches.
Transcriptional and clonal characterization of B cell plasmablast diversity following primary and secondary natural DENV infection
Authors: Waickman Adam T , Gromowski Gregory D , Li Tao , Siegfried Hayden , Victor Kaitlin , Kuklis Caitlin , Davidson Edgar , Srikiatkhachorn Anon , Ellison Damon , Rothman Alan , Jarman Richard G , Currier Jeffrey R , Friberg Heather L
Published: January 2023

Antibody-mediated humoral immunity is thought to play a central role in mediating the immunopathogenesis of acute DENV infection, but limited data are available on the molecular-level diversity, specificity, and functionality of the antibody response elicited by primary or secondary DENV infection. In order to close this functional gap in our understanding of DENV-specific humoral immunity, we utilized high-throughput single cell RNA sequencing to investigate B cells circulating in both primary and secondary natural DENV infections. We captured full-length paired immunoglobulin receptor sequence data from 9,027 B cells from a total of 6 subjects, including 2,717 plasmablasts. Unexpectedly, we found a high proportion of the DENV-elicited plasmablasts expressing IgA, principally in individuals with primary DENV infections. These IgA class-switched cells were extensively hypermutated and appeared to be derived from memory B cells, even in individuals with a serologically confirmed primary DENV infection. Utilizing a combination of conventional proteomics and high-throughput shotgun mutagenesis, we determined that DENV-reactive IgA class-switched antibodies represent a significant fraction of DENV-reactive and DENV-neutralizing Igs generated in response to DENV infection, and that they exhibit a comparable epitope specificity to DENV-reactive IgG antibodies. These results identify a heretofore unappreciated role for DENV-reactive IgA in the humoral response to DENV infection, and may offer insight into the differential pathogenesis associated with primary and secondary DENV infections.
Placental myeloid cells protect against Zika virus vertical transmission in a murine model
Authors: Winkler Clayton W , Evans Alyssa B. , Carmody Aaron B. , Peterson Karin E.
Published: January 2023

The ability of Zika virus (ZIKV) to cross the placenta and infect the fetus is a key mechanism by which ZIKV causes microcephaly. How the virus crosses the placenta and the role of the immune response in this process remain unclear. In the current study, we examined how ZIKV infection affected the immune cells within the placenta and developing fetus and whether these cells influenced virus vertical transmission (VTx). We found myeloid cells were elevated in the placenta of pregnant mice infected with ZIKV, primarily at the end of pregnancy, as well as in the fetus several days before birth. These cells, which included maternal monocytes/macrophages, neutrophils and fetal myeloid cells contained high levels of viral RNA suggesting they may be infected and contributing to viral replication and VTx. However, depletion of monocyte/macrophage myeloid cells from the dam during ZIKV infection resulted in increased ZIKV infection in the fetus. Myeloid cells were not depleted in the fetus in this experiment, so the increased virus infection was not the result of an impaired fetal myeloid response. Collectively, these data suggest that monocyte/macrophage myeloid cells in the placenta play a significant role in inhibiting ZIKV VTx to the fetus, possibly through phagocytosis of virus or virus infected cells.