bluuebunny/crossref_metadata_2025_split huggingface dataset:
MARCKS mediates the AXL/PD-L1 signaling axis and contributes to an immunosuppressive environment in lung cancer
Authors: Yoo Estelle Heerim , Li Ji-Min , Hsu Ssu-Wei , Chen Ching-Hsien 
Published: January 2023

Abstract
The advent of immunotherapy with immune checkpoint blockade (ICB) has led to significant advances in lung cancer management; unfortunately, many issues remain to be resolved, especially regarding a low objective response rate. Through integrated large-scale omics data and biomarkers from published immune ICB trials, we identified the PIP2-binding protein, myristoylated alanine-rich C-kinase substrate (MARCKS), and the AXL receptor as top-ranked therapeutic targets in mediating immunotherapy resistance. Both immunofluorescence and immunoprecipitation data demonstrated that MARCKS forms a molecular complex with AXL in aggressive lung cancer cells. In a screening of 200 patients using immunohistochemistry staining, we observed a positive correlation between AXL phosphorylation and MARCKS expression in advanced lung cancer. MARCKS knockdown abated the activity of AXL and its downstream pathways including PI3K/AKT and STAT3/Src signaling. Data from our cytokine arrays showed upregulation of anti-tumoral cytokines including GM-CSF and CXCL10 in MARCKS-knockout cells. We further discovered that MARCKS acts in accordance with PD-L1 to modulate T cell activity and killing. Upon co-culture with MARCKS-expressing lung cancer cells, cytotoxic T cells expressed higher PD-1 levels on cell surfaces, whereas T cell killing activity was potentiated in the MARCKS-knockout group. In mouse xenograft models, mice bearing MARCKS-deficient tumors displayed increased sensitivity to PD-L1 treatment. Our data suggest an oncogenic role of the AXL-MARCKS axis in modulating the tumor immune microenvironment. Therapeutic targeting of MARCKS may warm up an immunologically “cold” microenvironment, sensitizing tumors to immunotherapy.
This work was supported by grants funded by the Department of Defense Kidney Cancer Research Program (KCRP) W81XWH1910831 (log# KC180170) and the California UCOP Tobacco-Related Disease Research Program (TRDRP) T29IR0704.
In situ analysis of the metastatic immune milieu in breast cancer brain metastasis at the single-cell level.
Authors: Longworth Aaron J , Evans Katrina , Lawson Devon 
Published: January 2023

Abstract
Despite advances in early detection and treatment, breast cancer remains the second-leading cause of death among women. Among these cases, incidence of breast cancer brain metastasis (BCBM) carries a poor prognosis with a mean survival of 6 months following diagnosis, demonstrating a drastic unmet need for effective treatments. As the most abundant resident immune cell in the brain, microglia are poised to be effective first-responders to combat tumor infiltration, yet their response to metastases has not been fully characterized. Prevailing literature suggests that microglia promote metastatic outgrowth, however these works fail to adequately discriminate microglia from other brain resident and infiltrating macrophages. Employing a mouse model harboring a deletion in a super-enhancer of macrophage colony-stimulating factor 1 receptor which results in a near-complete loss of microglia, we find more tumor outgrowth compared to animals with microglia. We subsequently compared the metastatic immune milieu in the microglia replete and deficient mice using single-cell RNA sequencing and Akoya’s CODEX (CO-Detection by indEXing) high-parameter immunofluorescence multiplexing platform. We demonstrate that microglia undergo a pro-inflammatory shift in response to BCBM and infiltrating lymphocytes fail to express the inducible costimulatory molecule, ICOS, in the absence of microglia. To understand how microglia promote productive T cell responses to BCBM, we used CODEX to interrogate 20+ different biomarkers in BCBM with or without microglia. Through spatial phenotyping of immune cells from mice with and without microglia we uncover the importance of the microglia/lymphocyte crosstalk in controlling BCBM outgrowth.
Supported by NIH Grant (R01 CA237376-02)
Spatial profiling of immune cell markers in FFPE tumor tissues using the RNAscope&[trade] HiPlex v2 in situ hybridization assay
Authors: Dikshit Anushka , Basak Sayantani , Chang Ching-Wei , Collins Kim , Bunting Michaeline 
Published: January 2023

Abstract
The tumor microenvironment (TME) is highly complex, comprised of tumor cells, immune cells, stromal cells, and extracellular matrix. This study demonstrates a highly sensitive and specific multiplexed technique, the RNAscope HiPlex v2 in situ hybridization (ISH) assay for spatial and transcriptomic profiling of target genes to assess immune regulation in FFPE tumor tissues.
The RNAscope HiPlex v2 assay iteratively multiplexes up to 12 targets in frozen and FFPE tissues. The novel FFPE reagent effectively reduces background autofluorescence, improving the signal to noise ratio. Here we investigate spatial expression of 12 immuno-oncology target genes, including tumor markers, immune checkpoint markers, immunosuppression markers, immune cell markers and secreted chemokines The targets were simultaneously registered using HiPlex image registration software v2 that enables background subtraction.
We visualized T cell infiltration and activation within tumors using CD3, CD8 and IFNG expression. Subsets of pro- and anti-inflammatory macrophages were detected by CD68 and CD163 expression Hypoxia markers HIF1A and VEGF indicated immunosuppressive state of tumor cells. Preliminary analysis and quantification of the HIF1A expression using HALO® image analysis showed higher copy numbers in the lung tumor as compared to the other tumors. We additionally showed the differential expression of immune checkpoint markers PDCD1, and CD274 within the TME.
Using a highly sensitive multiplexed RNAscope HiPlex v2 ISH assay, 12 key targets were spatially resolved in four different tumor types. The assay can provide valuable understanding of the biological crosstalk among various cell types in complex and heterogeneous tissues.
Peripheral CD4 T cell resistance to type I interferon defines outcome of PD1 blockade therapy in human cancer
Authors: Boukhaled Giselle M , Gadalla Ramy , Elsaesser Heidi J , Sacher Adrian , Butler Marcus O , Brooks David G 
Published: January 2023

Abstract
Type I interferons (IFN-Is) are paradoxically associated with both the success and failure of immune checkpoint blockade (ICB), with increased inflammation in the tumor associated with better response. To understand how IFN-Is modulate the immune response to ICB, we developed a mass cytometry approach to quantify pro- and anti-inflammatory features of IFN-I responsiveness at the single cell level. Using high dimensional analysis we show that the inflammatory responses that are beneficial in the peripheral blood are the opposite of what is considered beneficial in the tumor, thus identifying a functional disconnect between the peripheral and tumor immune compartments. We demonstrate that an initial resistance to IFN-I by CD4 T effector (Teff) cells in the peripheral blood is strongly associated to long-term benefit of anti-PD1 therapy in patients with melanoma, head and neck and lung cancers. By contrast, a strong IFN-I response correlates with progression and low survival probability. Upregulation of PDL1 by IFN-I did not relate to response, however patients with myeloid cells that initially reacted to IFN-I with higher IDO1 induction survived longer after anti-PD1. Single-cell RNA-sequencing stratified based on response to IFN-I identified transcriptional programs associated with therapy response, providing mechanistic insight into the peripheral cell states prior to therapy conducive to anti-PD1 success. Thus, contrary to a benefit of an initially inflamed tumor environment, an initially restrained CD4 Teff inflammatory response to IFN-I averts therapy failure and enables tumor control. This IFN-I response potential is a promising new biomarker for prediction of which patients will benefit most from anti-PD1 therapy.
Supported by Canadian Institutes of Health Research (CIHR) Foundation Grant FDN148386, the Canadian Cancer Society (CCSRI) Innovation Award No. 706230, the National Institutes of Health (NIH) grant AI085043, The Terry Fox New Frontiers Grant the Scotiabank Research Chair to D.G.B, the Princess Margaret Hold’em for Life Cancer Research Fellowship (G. M. B).
TCR-engineered neoantigen-specific CD4+ T cells mediate immunotherapy of a class II-negative murine squamous cell carcinoma
Authors: Brightman Spencer E , Becker Angelica , Thota Rukman , Naradikian Martin S , Griswold Ryan , Dolina Joseph S , Schoenberger Stephen P 
Published: January 2023

Abstract
The expansion of neoantigen (NeoAg)-specific T cells often accompanies clinical responses to immunotherapies such as immune checkpoint blockade (ICB), adoptive cellular therapy (ACT), and personalized cancer vaccines (PCV), highlighting their importance for antitumor immunity. While the functions of NeoAg-specific CD8+ T cells have been characterized, the role of NeoAg-specific CD4+ T cells is less well understood. To study the antitumor mechanisms of NeoAg-specific CD4+ T cells, we sorted single CD4+ T cells specific for a mutated clathrin heavy chain epitope (Cltc H129Q) expressed by the murine squamous cell carcinoma VII (SCC VII) tumor. T cell receptor (TCR) sequencing analysis revealed the presence of four distinct TCR clonotypes and expression of these TCRs in primary cells was sufficient to confer preferential recognition of Cltc H129Q as compared to the corresponding wildtype Cltc epitope. Despite differences in TCR avidity, both low and high avidity CD4+ T cells were capable of proliferating to similar degrees in response to tumor antigen in vivo and enhancing primary tumor immunity in a CD8+ T cell- and CD40L-dependent manner. ACT with Cltc H129Q specific CD4+ T cells also synergized with cyclophosphamide treatment to reduce disease burden in mice with established tumors. Overall, these findings illuminate the mechanistic role of NeoAg-specific CD4+ T cells in tumor immunity, provide insights into the impact of TCR avidity on the helper functions of CD4+ T cells, and highlight the therapeutic potential of ACT with TCR-engineered CD4+ T cells.
Assessing Immunogenicity of Vaccines and Adjuvant Combinations for Q Fever
Authors: Jan Sharon , Gregory Anthony , Felgner Jiin , Davies Jenny , Nakajima Rie , Jasinskas Algis , Davies David Huw , Felgner Philip L 
Published: January 2023

Abstract
There is an unmet need for developing a safer, effective, single-dose vaccine for Coxiella burnetii conferring prolonged resistance, due to its high infectivity and biothreat potential. C. burnetii causes Q fever, which acutely causes fever-like symptoms but may manifest chronically and escalate into pneumonia and endocarditis. Prior sensitization has been documented to exacerbate the current vaccine’s reactogenicity and there have been considerable efforts in developing alternatives. Our lab has been studying the effects of combinations of adjuvants and their synergistic effects in eliciting antibody and T cell responses. Here we describe an effective adjuvant combination using synthetic TLR4 agonist monophosphoryl lipid A (MPLA), TLR9 agonist CpG ODN 1018, and Addavax, a squalene oil-in-water emulsion. In vitro assays were performed to assess the immunogenicity of these adjuvants in the context of NFκB activation and inducible nitric oxide synthase (iNOS) production.
Model antigens from C. burnetii were selected based on previous protein microarray data and formulated with varying adjuvant combinations. These formulations were administered to C57BL/6 mice and assessed for immunogenicity. Sera collected at regular intervals were screened against protein microarrays to monitor the dynamics and isotypes of the antibody response. Here we observed robust, durable and antigen-specific T cell and antibody responses. Our top vaccine formulations were administered to Hartley guinea pigs in a challenge study. Initial data demonstrate specific IgG production against immunizing antigens. Downstream analysis for this data set is currently underway.
Supported by DTRA contract HDTRA1-16-C-0009 DTRA grants HDTRA1-18-1-0035 and HDTRA1-18-1-0036
The novel adjuvant, ADA-1, restores age-associated defects in the adaptive immune response to Clostridioides difficile infection and vaccination in an aging mouse model
Authors: Bell Matthew , Bernui Mariana , Shah Neev , Connors Jennifer R , Kutzler Michele A 
Published: January 2023

Abstract
Clostridioides difficile is the leading cause of healthcare-associated infection in the United States. The elderly (≥65) have the greatest risk of C. difficile infection (CDI), severe morbidity, and mortality due to CDI. Though antibody responses against C. difficile Toxin A/B (TcdA/B) following primary infection or vaccination are protective against symptomatic disease, the elderly have impaired humoral responses. Using an aging mouse model, we show that there is significantly greater morbidity and mortality in aged mice during primary CDI compared to young adult mice, as seen in elderly CDI patients. Additionally, during both primary CDI and vaccination against TcdA/B, aged mice have significantly lower anti-TcdA/B antibody titers. This impaired humoral response is the result of poor Tfh and B cell responses following infection and vaccination as aged mice have a significantly lower frequency of Tfh cells and number of anti-TcdA/B B cells compared to young adult mice. While these age-associated defects in the humoral response allow for increased vulnerability to CDI, we show that these defects can be restored with the addition of the novel adjuvant ADA-1 during vaccination of aged mice with TcdA/B-encoding DNA. With ADA-1, the anti-TcdA/B antibody response in aged mice is restored to levels seen in young adult mice. ADA-1 also boosts the frequency of Tfh cells in aged mice following vaccination, suggesting that ADA-1 can restore the development of the aged germinal center reaction following vaccination. As such, this work highlights both the need for the development of a C. difficile vaccine with the elderly in mind and presents ADA-1 as a solution to overcoming age-associated defects in the vaccine response of the elderly.
Supported by Department of Defense Grant: W81XWH0910382 Pennsylvania Department of Health CURE Grant
Examining effector functions of lung CD8+ tissue resident memory T cells in humans.
Authors: Mattingly Cameron , Elliott Jennifer L , Thomas Jeronay K , Lobby Jenna L , Michalets Sarah E , Kohlmeier Jacob E 
Published: January 2023

Abstract
Due to their position in the lung tissue, CD8+ tissue resident memory T cells (TRM) act as sentinels of the respiratory tract that rapidly respond to, and mediate protection against, respiratory viruses. In mice, TRM have been shown to mediate protection at barrier sites by producing cytokines, chemokines, and performing cell lysis. In the lungs specifically, our lab has shown that influenza-specific CD8+ TRM rapidly produce IFNγ, but airway TRM are poorly cytolytic in mice. In humans, less is known about the effector functions of virus-specific CD8+ TRM in the lungs, and thus this study seeks to fill that gap in knowledge. Using cells from healthy human lungs, we first identified and quantified the frequency of antigen-specific cells in our lung donors by performing intracellular cytokine staining (IFNγ+) and activation induced marker assays (CD137+ CD25+). Then, by performing a series of in vitro peptide stimulation and cytokine neutralization experiments, we investigated which cytokines are produced by lung CD8+ TRM, and how those cytokines impact local innate and epithelial cells. Initial results show that, when stimulated with their cognate antigen, lung CD8+ TRM produce cytokines that directly activate innate immune cells. Results of this study suggests that human CD8+ TRM in the lungs act to rapidly reprogram local immune cells, and these data will ultimately help us understand how CD8+ TRM fit into the overall immune response to respiratory viruses.
Supported by grants from the NIH/NHLBI (R35 HL150803) and by NIH/NIAD through the Centers of Excellence for Influenza Research and Response (75N93019R00028).
MicroRNA-29a attenuates exhaustion and promotes memory-like CD8 T cells
Authors: Leng Xuebing , Buchness Lance , Ristin Svetlana , Villarino Alejandro , Stelekati Erietta 
Published: January 2023

Abstract
CD8 T cells are crucial for immunity against pathogens and tumor cells. However, persistent antigens during chronic infections and cancers drive the differentiation of exhausted T cells (TEX). TEX do not generate optimal immune responses to persistent antigens. The epigenetic profile of TEX is altered and cannot be reversed with current immunotherapy, such as PD-L1 blockade, providing a challenge for the efficacy of such therapies. Despite considerable work interrogating the transcriptional regulation of TEX, the post-transcriptional control of TEX remains poorly understood. Here, we interrogated the role of microRNAs (miRs) in CD8 T cells responding to acutely resolved or chronic viral infection and identified miR-29a as a key regulator of TEX. Enforced expression of miR-29a improved CD8 T cell responses, attenuated exhaustion, and fostered a memory-like CD8 T cell differentiation state during chronic infection. Importantly, miR-29a combined with anti-PD-L1 therapy increased CD8 T cell accumulation, promoted a progenitor-like TEX subset and enhanced a memory-like phenotype long-term. These data indicate that miR-29a can instruct CD8 T cell differentiation and promote memory-like responses. Therefore, we suggest miR-29a as a novel immunotherapeutic to regulate TEX differentiation and promote functional CD8 T cell responses long-term.
Genetic analysis of CNS autoimmunity using the diversity of the Collaborative Cross reveals unique phenotypes and mechanisms
Authors: Holt Emily A , Lahue Karolyn G , Mahoney J Matthew , Teuscher Cory , Krementsov Dimitry N 
Published: January 2023

Abstract
Multiple Sclerosis (MS) is a complex disease with remarkable heterogeneity in disease course and progression, the genetic basis of which remains obscure. Here we leveraged the Collaborative Cross (CC) - a highly genetically diverse mouse strain panel - and myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55) induced experimental autoimmune encephalomyelitis (EAE), to model genetics of MS disease course. 33 CC strains were selected based on compatible MHC haplotypes (H2b and H2g7), which captured a wide spectrum of distinct EAE phenotypes, compared with typical chronic EAE in C57BL/6 mice. CC028 mice exhibited severe and rapidly progressing disease. In contrast, several strains, including CC011 and CC040, were highly resistant to EAE. Sex differences in EAE course were observed in 4 strains, including CC042. Remitting-relapsing EAE was observed in 4 strains, including CC002. In addition to classical EAE clinical signs (ascending paralysis), we identified two strains, CC004 and CC083 that exhibited high incidence of axial rotary (AR)-EAE, including profound ataxia, head tilt, and axial rotation. Preliminary quantitative trait locus (QTL) analysis revealed a distinct linkage pattern in each sex for classical EAE severity, with several emerging peaks on chromosome (Chr) 2, 8, 12, 18, and X in females, Chr10 in males, and Chr4 in both sexes. QTL analysis of AR-EAE severity revealed a narrow interval on Chr18 (39.2–41.0Mb) passing suggestive linkage significance at the 90% confidence threshold. Experiments are ongoing to determine the immunopathologic basis of distinct genetically controlled EAE phenotypes in CC strains of interest.
Supported by grants from NIH (R21 AI145306-01) and the National MS Society (RG-1901-33309)
Common gamma chain-dependent cytokines drive antigen-independent proliferation of circulating and resident memory CD8+ T cells
Authors: Jarjour Nicholas N , Wanhainen Kelsey M , Peng Changwei M , da Silva Henrique Borges , Martinez Ryan J , Dalzell Talia S , Huggins Matthew A , Urban Joseph F , Hamilton Hart Sara E , Jameson Stephen C 
Published: January 2023

Abstract
Memory CD8+ T cells are found throughout the body and can locally control reinfection at barrier sites. Vaccines often fail to elicit durable tissue-resident memory (TRM) CD8+ T cells, likely contributing to unsuccessful efforts to vaccinate against several important pathogens. TRMs locally self-renew by poorly understood mechanisms; a better understanding of this process could help to enhance TRM immunity. We propose that proliferating TRMs flexibly use available members of the common gamma chain-(γC)-dependent cytokine family to persist and as bystanders during other immune responses. We have found that γC-dependent cytokines can mediate cell-intrinsic signaling to drive proliferation of both circulating and resident memory CD8+ T cells, in contrast to naïve T cells. Using adoptive transfer as well as tetramer staining of endogenous populations, we have confirmed that cytokine-stimulated proliferation of memory T cells of known antigen specificities elicited by different pathogens can occur without cognate antigen. Via bulk and single-cell RNA-Seq of resting and in vivo cytokine-stimulated memory CD8+ T cells, we are elucidating the downstream signature of cytokine-driven TRM proliferation. We have also found that memory CD8+ T cells proliferate comparably in response to exogenous cytokines or unrelated pathogens, consistent with pathogen-elicited γC-dependent cytokine signals driving memory CD8+ T cell proliferation. These data support a conserved ability of TRM and circulating memory T cells to use infection-elicited γC-dependent cytokines for enhanced proliferation, likely to promote maintenance, and that treatment with γC-dependent cytokines can drive rapid proliferation without a need to identify antigens.
N. N. J. Damon Runyon Cancer Research Foundation Fellow DRG-2427-21 K. M. W. NCI F30 H. B. d. S. CRI/Paul Shiverick Fellow; NIAID K99/R00 AI139381 S. C. J. NIAID R01 AI038903
Transient Receptor Potential Ankyrin 1 (TRPA1) mediates IL-1β-Induced Thermoregulation
Authors: Silverman Harold A , Tynan Aisling , Chang Eric H , Li Jian Hua , Thompson Dane A , Tracey Kevin J , Chavan Sangeeta S 
Published: January 2023

Abstract
TRPA1 is a non-specific cation channel, that has been previously implicated in temperature regulation and inflammation. Administration of TRPA1 agonists induce significant changes in body temperature. Similarly, IL-1β, a cytokine mediator of inflammation and injury, induces a significant change in body temperature. Here we reasoned that TRPA1 plays a role in IL-1β mediated temperature regulation. To monitor body temperature in conscious and unrestrained mice, telemetry probes were implanted into the peritoneal cavity. Using chemogenetic technology, we generated a novel TRPA1-GqDREADD strain, in which TRPA1+ cells are activated by the administration of clozapine-N-oxide (CNO). Administration of CNO (ip) induces a significant change in body temperature (vehicle vs. CNO; 674.3 ± 29.3 AUC vs. 1646 ± 172 AUC; Welch’s T-test; p&lt;.001). To determine if TRPA1 is responsible for IL-1β induced thermoregulation, wild type mice and TRPA1 KO mice were injected with IL-1β (5.0μg/kg, ip). As expected, wildtype mice had a significant change in body temperature. However, this response was significantly impaired in TRPA1 KO mice (wild type vs. TRPA1 KO; 1045 ± 35.29 AUC vs. 577.9 ± 26.13 AUC; Welch’s T-test; p&lt;0.0001). To investigate whether selective TRPA1 expression in neurons is required for IL-1β-induced thermoregulation, we generated Syn-Cre/TRPA1fl/fl mice selectively devoid of TRPA1 expression in neurons. Selective ablation of TRPA1-expression in neurons significantly attenuates IL-1β-induced changes in body temperature (wild type vs. Syn-Cre/TRPA1fl/fl; 1082 ± 61.49 AUC vs. 775.8 ± 66.89 AUC Welch’s T-test; p&lt;0.01). Together, these studies indicate that IL-1β-induced thermoregulation is mediated via neuronal TRPA1.
Supported by grant from NIH to KJT and SSC