bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multiplex microscopy reveals unique spatiotemporal effects of cancer immunotherapies
Authors: Maltez Vivien Ileana , Byrne Katelyn T , Germain Ronald N 
Published: January 2023

Abstract
Pancreatic ductal adenocarcinoma (PDA) is an aggressive and heterogeneous cancer that is often refractory to current treatments. To address this issue, we employ a mouse PDA-derived tumor clone library where individual clones elicit a spectrum of unique immune cell infiltration profiles in the tumor microenvironment (TME). We found that a combination of anti-PD1, anti-CTLA-4, and anti-CD40 results in tumor regression only in tumor clones with a high number of infiltrating T cells. The aim of our study is to gain mechanistic understanding into this therapeutic combination. To that end, we leverage quantitative high multiplex microscopy, enabling us to decipher the complexities of cellular behaviors, interactions, and phenotypes in an intact TME. We found that our therapy distinctively alters the TME of both responsive and non-responsive tumors. Unique to non-responsive tumors, we found discrete myeloid clusters scattered through the TME that increase in frequency and size post therapy. Additionally, we witnessed prominent cellular movement within myeloid clusters using 2-photon intravital imaging. In contrast, in the TME of responsive tumors our therapy severely depleted, reprogrammed, and restricted regulatory T cells to the tumor periphery within 48 hours post anti-CD40 administration. We found this to be ADCC–independent, but anti-CD40–dependent, with the mechanistic factors driving the fate of these cells under detailed investigation. Combined, these data reveal mechanistic insight into anti-CD40 combination therapies and provide a platform for investigating the factors driving the formation and maintenance of an immunosuppressive TME.
This work was supported by (1) Intramural Research Program of NIAID, NIH, (2) Postdoctoral Research Associate Training (PRAT) Fellowship of NIGMS, NIH, (3) Parker Institute for Cancer Immunotherapy, and (4) Bench to Bedside Award.
Identification of immunogenic peptide neoantigens expressed in sarcomas and their therapeutic potential
Authors: Sedlacek Abigail L , Osei-Hwedieh David , Agyekum-Yamoah Archibald , Mena Luis , Iyer Swati , Weiss Kurt , Binder Robert J 
Published: January 2023

Abstract
T cells recognize tumor neoantigens (TNag) in the context of MHC and eliminate cells which express these antigens, leaving those lacking immunodominant epitopes. Central to investigating tumor immunoediting is the ability to define immunogenic TNag. Differential aggretope index (DAI) is a measure of the difference in MHC binding affinity between mutated and unmutated peptides and has emerged as a highly efficient way of identifying immunogenic TNag. Our previous studies have demonstrated that in mice, TNag with a DAI &gt;8 are considered immunogenic and elicit T cells that lead to the tumor elimination. Thus, tumors expressing TNag of DAI &gt;8 are only found in immunodeficient mice. Given these observations, we investigated the mutational burden and immune phenotype of bone proximal tumors in 122 patients. Tumors included are of bone, surrounding tissue and distal metastases, and were analyzed by flow cytometry, whole exome and TCR-Vβ sequencing. We hypothesized that tumors with high DAI TNag would be immunoedited in patients with robust T cell responses as measured by T cell clonality. Similar to observations in mice, this correlation was only observed in TNag with DAI &gt;8. In contrast, there was no correlation between DAI and frequency of CD8 TILs indicating that the repertoire of TILs is a better indicator of immune reactivity than their frequency. Among tumor types analyzed, osteosarcomas were the least immunoedited, suggesting greatest potential for immunotherapy. We confirmed this potential in a mouse model of osteosarcoma, where we show that therapeutic HSP-mediated responses, augmented by PD-1 blockade, conferred the best survival. These studies provide compelling rationale for novel immunotherapies in patients with osteosarcoma.
Supported by NIH R01 CA233803 and an HCC UPMC Developmental Award
RNA-protein Co-detection using spatial analysis to profile tumor-infiltrated immune cells
Authors: Dikshit Anushka , Basak Sayantani , Doolittle Emerald , Bunting Michaeline 
Published: January 2023

Abstract
Interrogating complex tumor microenvironment requires a multi-omics approach. Detecting target immune cell markers using immunohistochemistry/Immunofluorescence (IHC/IF) and visualizing cytokine expression with in situ hybridization (ISH) can provide comprehensive information about the activation states of immune cells. Here, we demonstrate a newly developed integrated ISH and IHC/IF workflow compatible with manual and automated platforms with improved RNA-protein co-detection.
We demonstrate the use of our RNA-Protein Co-detection assay in combination with the automated and manual RNAscope Multiplex Fluorescent assay as well as the automated RNAscope Chromogenic Duplex assay to detect T cell markers, macrophage markers and checkpoint markers in the tumor microenvironment by using a microarray with different tumor samples.
We identified CD4+ helper T cells, CD8+ cytotoxic T cells and determined their activation states by visualizing IFNG, GZMB and IL-2 expression. We were also able to identify macrophages detected by CD68 protein expression and differentiate the M1 and M2 subtypes by using M2-specific marker, CD163. We could also delineate tumor-stroma border in the samples by using the Pan-CK probe which distinctly marks the tumor cells and visualize expression of immunoregulatory receptors PD-L1 and CTLA4 in the tumor.
This assay is enables for multiplexing by combining with our fluorescent assays or enables target detection while retaining morphological context when combined with our chromogenic assays. Overall, the new RNAscope-ISH-IHC/IF co-detection workflow and reagents enable simultaneous visualization of RNA and protein targets by enhancing the compatibility of antibodies with minimal optimization.
Thymic stromal lymphopoietin (TSLP) receptor signaling controls a tumorigenic Treg to promote colorectal cancer
Authors: Obata-Ninomiya Kazushige , Ziegler Steven F 
Published: January 2023

Abstract
Regulatory T cell (Treg) is a hallmark of progression of cancer. However, the subset of Tregs and the factor regulating Tregs in colorectal cancers (CRCs) have not been defined. We identified a novel subset of Tregs expressing TSLP receptor (TSLPR) and ST2 was predominantly associated with CRC in human and mouse. Mef2c, a transcription factor, exclusively expressed on this Treg controlled the expression of CTLA-4 on this Treg under TSLPR signaling. To study the role of TSLP signaling on this Treg, we examined mice lacking TSLPR on Tregs (TSLPRΔTreg) in AOM/DSS-induced CRC model. The results showed that the number and the expression of CTLA-4 on this Treg were lower in TSLPRΔTreg mice and the size and number of tumors were reduced compared to control mice, concomitant with increase of Th1 cells. In addition, in human CRCs, the number of TSLPR+ ST2+ Tregs is negatively correlated with the number of Th1 cells. These findings suggested that TSLPR+ ST2+ Tregs play a pivotal role in progression of CRC through suppression of anti-tumor immunity in CRCs in human and mouse. Furthermore, to address the cure of CRC, we administrated mice with neutralization antibody for TSLP after development of colorectal tumors. Blockage of TSLP signaling reduced size and number of tumors via reduction of TSLPR+ ST2+ Tregs, indicating that neutralization of TSLP inhibits progression of CRCs. Taken together, and TSLP signaling on tumorigenic TSLPR+ ST2+ Treg can be good therapeutic targets for CRC.
Cloning ospA of Borrelia Burgderferi for Plant Expression
Authors: Browne Quinton Ambrose , Puthoff David P , Taylor Rebekah 
Published: January 2023

Abstract
Borrelia burgdorferi (Lyme disease) is a vector-borne disease that is both very common in the Mid-Atlantic region and which causes long term negative health effects. It is primarily spread to humans by deer ticks, and because one of the main ways that it is passed between ticks is by previously infected mouse blood, our goal is to design an oral vaccine which decreases infection rates in mice. Our goal is to do this by inserting outer surface protein A (ospA) of B. burgdorferi into a plant vector: pCambia 1201. Agrobacterium tumefaciens was used as a means of introducing the vector (bearing B. burgdorferi ospA) into Arabidopsis thaliana. The pCambia vector was introduced into A. tumefaciens competent cells after being purified from E. coli colonies which had undergone a similar transformation process, as E. coli is more suitable for cloning. The successfully electroporated A. tumefaciens was then plated and colonies were picked and grown as liquid cultures. Those liquid cultures transformed with both the ospA bearing pCambia and a control pCambia were used for floral dip of Arabidopsis thaliana (both ospA bearing and pCambia only control plants). These dipped plants’ seeds have been tested for successful transformation, and one plant has been found to contain the sequence for ospA. Continued floral dips will be undertaken to increase the number of positive plants, and to develop a proper control for testing efficacy as a vaccine. Any additional seeds of positive plants will be planted and crossed to ensure use of only plants which express pCambia in later testing, and to ensure that the process has been successful.
Supported by Frostburg State University Department of Biology and FSU Foundation
A recombinant Chlamydia muridarum MOMP encapsulated PLGA 85/15 nanovaccine induced antibodies elicited cross-recognition of serovar-D elementary bodies
Authors: Sahu Rajnish , Verma Richa , Sharma Homa Nath , Tadjuidje Vanella , Joshi Sameer , Forte Othreniel A , Singh Shree R , Dennis Vida A 
Published: January 2023

Abstract
Chlamydia trachomatis (Ct), caused by various serovars, remains the leading sexually transmitted bacterial infection worldwide. An efficient delivery system is critical to developing whole or subunit vaccines against Ct, and vaccine developmental efforts are rapidly targeting biodegradable nanoparticles based vaccines with encapsulated antigens. We have developed a nanovaccine employing the recombinant major outer membrane protein (rMOMP) of C. muridarum (Cm) encapsulated in poly (lactic acid-co-glycolic acid) (PLGA) 85/15 (PLGA-rMOMP). We previously compared the humoral responses of priming routes; subcutaneous (SC) or intramuscular (IM-p) followed by two sc-boosts to evaluate rMOMP specific serum antibodies [Th1 (IgG2a, IgG2b), Th2 (IgG1)] produced by PLGA-rMOMP immunization of female BALB/c mice. Here we explored the ability of systemic antibodies produced in immunized mice to recognize UV-inactivated whole elementary bodies (EB) of Cm and one of the prevalent human serovar D (Ct-D) using antigen-specific ELISA. Serum from phosphate-buffered saline (PBS) or PBS encapsulated (PLGA-PBS) groups of mice served as a negative control to validate the EB recognition by immunized sera. We observed higher EB specific total IgG (4-fold) and isotypes IgG2b (16-fold), IgG1 (2-fold) antibodies recognizing Ct-D compared to Cm for the SC and IM-p immunization groups. Evaluation of the Th1 (IgG2b)/Th2 (IgG1) antibody titer ratio revealed a higher Th1 antibody recognition of Cm (4-fold) and Ct-D (16-fold), for the SC and IM-p immunization routes. Our data show that mice immunized with PLGA-rMOMP produced Th1 systemic antibodies against EB, but more importantly, there was robust serological cross recognition of the human serovar D.
This research was supported by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Number R21AI111159, NIH-NIGMS-RISE (1R25GM106995-01) and the National Science Foundation (NSF)-CREST (HRD-1241701) and NSF-HBCU-RISE (HRD-1646729) grants.
The use of outer membrane vesicles as novel, mucosal adjuvants against intracellular bactiera
Authors: Harrell Jaikin , Morici Lisa A , McLachlan James B 
Published: January 2023

Abstract
Many pathogens first enter the body via mucosal surfaces where they can then invade and disseminate systemically to cause disease. Despite this, most vaccines are given parenterally and are unable to induce mucosal immunity. Immunizing directly at the mucosa could solve this problem, however delivering vaccines at these surfaces often doesn’t invoke robust immunity. One way to alleviate this is to use adjuvants that can evoke an immune response. Most adjuvants, like aluminum salts, are unable to induce mucosal immunity and so novel adjuvants must be employed. Outer membrane vesicles (OMVs) from Burkholderia pseudomallei are potent immune mediators and have been shown to have adjuvant capabilities. The goal of this study is to highlight the role of OMVs as a novel adjuvant that can be used in the next generation of mucosal vaccines. To test this, we created an OMV-adjuvanted inactivated whole-cell vaccine against two intracellular pathogens – Salmonella Typhimurium and Francisella holarctica LVS that could be delivered mucosally. An oral vaccine against S. Typhimurium adjuvanted with OMVs showed protection against lethal challenge in addition to evoking antigen specific CD4 T cells, B cells, and anti-Salmonella antibodies. These antibodies induced greater bacterial killing in macrophages. We are currently exploring an OMV-adjuvanted oropharyngeally delivered vaccine against F. holarctica LVS. Immunity against Francisella requires both CD4 and CD8 T cells and we are determining how an OMV-adjuvanted vaccine will influence these immune cell populations. This study represents a novel approach to mucosal vaccines using OMVs as adjuvants.
Supported by NIH U01 AI124289 NIH BAA HHSN72201800045C
Mechanisms of impaired muscle repair following influenza infection in aged mice.
Authors: Cadar Andreia , Keilich Spencer R , Martin Dominique E , Torrance Blake L , Haynes Laura , Bartley Jenna M 
Published: January 2023

Abstract
Older adults are susceptible to infectious diseases including Influenza (flu), due to widely accepted declines in immune response with age. Flu infection in older adults commonly results in hospitalization and catastrophic disability, leading to loss of independence. Previous findings demonstrate that flu infection induces severe and prolonged muscle injury in aged mice. We aimed to explore the effects of flu infection on skeletal muscle gene expression and immune cell populations to elucidate mechanisms of flu-induced disability in older adults. Young and aged C57BL/6 male mice were infected with a sublethal dose of PR8 (H1N1) influenza. Gastrocnemius muscles were harvested for RNA sequencing, confocal microscopy, and flow cytometry. Our findings showed differential gene expression changes between young and aged mice, including downregulation of muscle regeneration and organization genes and upregulation proinflammatory cytokine and migratory cell pathways in aged muscle. Pathway analysis revealed significant increases in T cell activation pathways in the aged muscle. Confocal microcopy confirmed increased leukocytes (CD45+) and T cells (CD3+) intramuscularly following flu infection. Flow cytometric analyses confirmed that aged mice have increased infiltrating CD8 T cells during infection compared to young mice, while young muscle have increased infiltrating regulatory T cells (Tregs) compared to their aged counterparts. Further analysis suggested that differences in IL-33 levels between young and aged mice influence these infiltrating populations. Future research is necessary to determine if IL-33 supplementation may prevent or ameliorate flu-induced disability in older adults.
Supported by NIH/NIA R01 AG051647 (Co-I) NIH/NIA R01 AR075346 (Co-I) NIH/NIA R33 AG061456 (Co-I) NIH/NIA R21 AG071292 (Co-I) American Federation for Aging Research Reboot (PI)
Identification and Characteristics of HLA-restricted HMPV-specific CD8+ T cells
Authors: Lan Jie , Parks Olivia , Zhang Yu , Walters Sara , Sojati Jorna , Williams John V 
Published: January 2023

Abstract
Human metapneumovirus (HMPV) is a leading cause of respiratory tract infection in pediatric, elderly, and immunocompromised populations. All individuals have been exposed to HMPV by the age of 5, but humoral immunity does not fully protect against reinfection of adults. Despite the clinical burden, there are currently no FDA-approved vaccines or therapeutics for HMPV. T cell responses are important for protection and viral clearance. The aim of this study was to better understand the human T cell response to HMPV and guide vaccine development. To accomplish these goals, we sought to identify HLA class I-restricted viral epitopes. In this study, 5 transgenic mouse strains that express human HLA-A*01:03, HLA-A*24:01, HLA-B*35:01, HLA-B*15:02, or HLA-Cw*07:01 were used to map HMPV-specific epitopes and characterize HMPV-specific CD8+ T cells. We used ELISpot screening of overlapping peptides and predictopes to discover several epitopes and generate MHC-I tetramers for each genetic background. We found that human CD8+ T cells of subjects expressing the same HLA types could recognize viral epitopes by ELISpot and tetramer staining. Our results suggest that the transgenic mouse is a useful model to identify HLA-restricted viral-specific epitopes and novel targets for vaccination against HMPV.
Supported by grants from NIH (R01 AI085062)
Induction of blood brain barrier disruption through reactivation of virus antigen specific brain resident memory T cells
Authors: Ayasoufi Katayoun , Namen Shelby L , Wolf Delaney , Tritz Zachariah Peter , Goddery Emma , Pfaller Christian K , Gulbicki Lauren , Fain Cori E , Jin Fang , Hansen Michael , Johnson Aaron J 
Published: January 2023

Abstract
The contribution of antiviral resident memory T cells (TRM) to blood-brain barrier disruption has not been defined despite the implication of this lymphocyte in clinical encephalitis. We therefore evaluated the capacity of TRM cells to induce CNS vascular permeability in mice which resolved Theiler’s murine encephalomyelitis (TMEV) infection. Following TMEV infection of the brain, persistent populations of virus antigen specific resident memory T cells are induced within the brain. This population of resident memory T cells can be further reactivated at 60 dpi through administration of the cognate immunodominant virus peptide antigen, VP2, inducing blood brain barrier disruption in the process. We determined that brain resident memory T cells expand following reactivation with administered VP2 peptide. This reactivation is associated with infiltration of T cells, and inflammatory myeloid cells into the brain. Importantly, using depletion and T cell sequestrating strategies with low dose anti CD8 antibodies and FTY720, we determined that TRM induced blood-brain barrier disruption is independent of peripheral T cells. We conclude that reactivation of brain TRM cells during peptide administration occurs without dependency on peripheral responses, highlighting the importance of this cell type in inducing neuroinflammation and underlying neuropathology.
Exploring the role of c-Myc in defining the fate of exhausted CD8 T cells
Authors: Toumi Ryma , Deo Rucha , Bhise Shruti , Sarkar Surojit , Kalia Vandana 
Published: January 2023

Abstract
c-Myc is a major transcription factor expressed upon activation of CD8 T cells that drives their clonal expansion and metabolism. The role of c-Myc in regulating the outcome of exhausted CD8 T cells during chronic infection and cancer remains poorly defined. In this study, we used a unique model of c-Myc conditional ablation following antigen priming of CD8 T cells to investigate whether c-Myc exerts a role in CD8 T cell exhaustion in chronic antigenic settings. Our studies show that targeted deficiency of c-Myc in antigen-specific CD8 T cells resulted in decreased cell numbers but promoted the generation of stem-cell-like TCF-1+ less exhausted CD8 T cells. These cells are key therapeutic target for overcoming CD8+ T cells exhaustion by providing the proliferative burst after PD1 checkpoint blockade immunotherapy (CBI). We, therefore combined anti-PD-L1 therapy and c-Myc inhibition to determine whether c-Myc inhibition might synergize to improve the therapeutic outcome. Our data using c-Myc inhibitor alone revealed an increased frequency of TCF-1+ CD8 T cells. However, the number of antigen specific CD8 T cells and the production of effector cytokines (IFN-γ and TNF-α) remained identical to untreated group. Notably, the combination of c-Myc inhibitor and PD-1 CBI significantly promoted the proliferation of antigen-specific CD8 T cells and the production of IFN-γ and TNF-α. Overall, findings support a role for c-Myc in defining the fate of exhausted CD8 T cells, and illustrate a potential pathway for the optimization of check point blockade immunotherapy of chronic infections and cancer.
Supported by NIH R21AI154363 NIH R01AI32819
Persistence of SARS-CoV-2 in long COVID-19 patients
Authors: Goh Denise , Lim Jeffrey Chun Tatt , Fernandez Sonia Bilbao , Lee Justina Nadia , Joseph Craig Ryan , Guerrero Sílvia , Neo Zhen Wei , Lau Mai Chan , Yeong Joe Poh Sheng 
Published: January 2023

Abstract
The World Health Organization has defined long COVID-19 (LC) as a condition where patients exhibit persistent symptoms over time after its acute phase, which cannot be explained by alternative diagnosis. Since we have previously reported residual viral antigens in tissues of convalescent patients, we now aim to assess the presence of such antigens in post-convalescent tissues. Here, we established the presence of residual virus within the appendix and breast tissue of 2 patients who exhibited LC symptoms, 163 to 462 days upon positive diagnosis, using immunohistological techniques. We observed positive staining for viral nucleocapsid protein (NP) in the appendix, and tumour-adjacent region of the breast, but not within the tumour. Multiplex immunohistochemistry further detected colocalization of viral NP with macrophage marker CD68, in support of immune cells being direct targets. Notably, with RNA scope, both positive-sense and negative-sense (replicative intermediate) viral RNA were detected. As a single-stranded virus, SARS-CoV-2, have to produce a replicative intermediate as a template to synthesize new genomic RNAs. Thus, the detection of negative-sense viral RNA suggests ongoing viral replication. While viral RNA and antigen from gastrointestinal and stool samples of convalescent patients has been extensively reported, we believe this is the first study to detect viable virus. Furthermore, our positive finding in the breast tissue also corroborated with recent reports that immunocompromised patients had also experienced LC symptoms and persistent viral replication. Overall, our findings, along with emerging LC studies, question the possibility of the gastrointestinal tract functioning as a reservoir.
Supported by A*STAR Career Development Award (C21112056)