bluuebunny/crossref_metadata_2025 huggingface dataset:
Quick and Easy Isolation of Immune Cells From Large-Volume Samples
Authors: Posarac Vesna , MacDonald G Neil , Buck Chris A , Toombs Eric , Gellner Savannah D , de Jong Susan , Williamson Mark E , Egeler Oliver , Dalton Bob , Kokaji Andy I , Eaves Allen C , Louis Sharon A , Antignano Frann
Published: January 2023

Large-scale cell isolation is commonly performed in labs and core facilities for cell banking and drug discovery, and as a critical step in cell therapy manufacturing. Current methods can be a significant bottleneck in a lab’s workflow, often requiring a full day for sample processing and cell isolation. To address this need, we have developed two methods: manual column-free isolation using the Easy 250 EasySep™ magnet, and automated cell isolation in a closed system using RoboSep™-C.

The Easy 250 magnet allows users to isolate cells from a full leukopak of up to 20 billion cells in under 30 minutes by simply pipetting out their target cells. Negative selection protocols have been optimized to achieve 95.3% T cell, 97.2% CD4+ T cell, 91.9% CD8+ T cell, 99.4% B cell, 96.5% NK cell, and 91.5% monocyte purities. Positive selection protocols obtain 95.4% CD3+ cell, 94.4% CD4+ T cell, 93.9% CD8+ T cell, and 96.2% CD14+ cell purities.

RoboSep™-C automates this cell isolation procedure, along with the cell washing steps for sample preparation, in as little as 50 minutes. The instrument features a scale tower, pump and clamp modules to direct fluid through defined paths, and a magnet for separation of labelled cells. The system uses a sterile single-use tubing set that incorporates a cartridge for cell washing and concentration, and a magnet chamber for cell separation. Starting with fresh leukopaks, we obtained 95.9% T cell, 95.8% CD4+ T cell, and 89.6% CD8+ T cell purities following negative selection, and 92.0% CD4+ T cell and 89.5% CD8+ T cell purities following positive selection.

These approaches offer efficient and user-friendly cell isolation that allow researchers to scale up their operations, and can be easily integrated upstream of existing workflows.
Extending the capabilities of a high-parameter immunophenotyping assay with cytoplasmic staining applications for mass cytometry
Authors: Rogers Clare E , Yao Huihui , Cohen Michael , Loh Christina
Published: January 2023

Maxpar® Direct™ Immune Profiling Assay™ (Cat. No. 201325) is a 30-marker panel for suspension mass cytometry. This panel provides an unprecedented sample-to-answer solution for detecting and analyzing 30 surface markers in a single experiment. The 18 open mass channels in the Maxpar Direct Assay facilitate panel expansion and enable flexibility for higher multiplexity and applications. Among the potential complementary applications, intracellular cytokine staining (ICS) is of particular interest as it may be used to assess infiltrating immune cell phenotypes in the tumor microenvironment. However, for the purpose of assessing cell viability in this workflow, the effectiveness of the Cell-ID™ Intercalator-Rh (103Rh, Cat. No. 201103) included in the Maxpar Direct Assay is in question, as cell permeabilization during ICS can potentially damage the DNA-intercalator bond. In this study, we investigated the compatibility of 103Rh with intracellular staining. We stained either human peripheral blood mononuclear cell or whole blood samples with the Maxpar Direct Assay followed by intracellular staining for the detection of expressed cytokines. We demonstrate that 103Rh provides equivalent functionality as a cell viability indicator during intracellular staining for cytoplasmic proteins compared to the benchmark Cell-ID Cisplatin-194Pt (Cat. No. 201194). This work was designed to support use of the Maxpar Direct Immune Profiling Assay in combination with additional intracellular markers. Overall, these findings expand the applicability of Cell-ID Intercalator-Rh (103Rh) to processes that involve cytoplasmic staining.

For Research Use Only. Not for use in diagnostic procedures.
Tregs: The Sertoli cell’s secret weapon in transplant survival
Authors: Hibler Taylor , Kaur Gurvinder , Dufour Jannette
Published: January 2023

Advanced germ cells develop after central tolerance is established and express non-self antigen but are not destroyed by the immune system. They owe their survival in part to the physical and immunological barrier created by Sertoli cells (SC). The protective nature of SC has sparked interest in the field of transplant immunology as a way to prevent transplant rejection. Indeed, SC prolong the survival of various co-transplanted tissues. However, exactly how SC achieve this protection remains largely unexplored, as does their effectiveness in female recipients. Data from transplantation studies indicates that T regulatory cells (Tregs) are critical in graft survival. This study aims to determine if SC survive at comparable rates in males and females, and if SC survival is dependent on Tregs. Therefore, we transplanted SC from B6 mice into allogeneic BALB/c male and female immunosuppressive drugs. At day 20, all SC grafts survived in male recipients and at least 67% survived in female recipients. In both males and females, Tregs were detected in high numbers in surviving grafts. To further explore their role in SC graft survival, Tregs were depleted in male graft recipients. Analysis of SC grafts at day 20 showed that 57% of SC grafts survived and saw a return of Tregs, as detected through flow cytometry. Tregs remained depleted in the rejected grafts. Together, this data indicates that Tregs must be present for SC grafts to survive and that SC themselves may induce Tregs. To conclude, SC survive allotransplantation in males and females, possibly through the activation of Tregs.

Supported by CH Foundation, NIAID
Dysregulated regulatory T cell responses target fibroblasts and lead to chronic rejection after heart transplantation
Authors: Dwyer Gaelen K , Fan Lu , Zhang Xue , Li Tengfang , Mathews Lisa , Saba Johnny , Colon Emmanuel , Calderon Michael , Helfrich Katherine , Ross Mark , Watkins Simon , Turnquist Heth Roderick
Published: January 2023

Suppressive and reparative regulatory T cell (Treg) functions are described. However, in transplantation (Tx), recipient immune responses to persistent MHC differences will sustain injury and impact repair. The development of immunosuppressant-resistant graft vasculopathy and fibrosis, or chronic rejection (CR) remains a leading cause of death post-heart Tx (HTx) and may reflect failed tissue repair. We tested the hypothesis that Treg repair responses become dysregulated due to sustained alloreactive immune responses. Our studies utilized IL-33 as a model injury signal, or damage-associated molecular pattern (DAMP) and B6 (H-2b) CD45.1+ mice received heterotopic HTx from MHCII-mismatched Bm12 (H2-I-Abm12) donors that were IL-33-deficient or competent. On day (d)14, scRNAseq and CITE-seq were used to compare CD45.1+ cells from HTx digests (n=4). IL-33 modulated >800 genes in HTx-infiltrating Treg, including the growth factor amphiregulin (Areg; 2.25x; P=0.0241). Areg is implicated in tissue repair, yet when B6 Foxp3YFP-Cre xAregfl/fl and Foxp3YFP-Cre mice receiving Bm12 HTx were assessed at d100 (n=6–10), the deletion of Areg in Treg protected against CR. Using IncuCyte Live-Cell imaging, it was clear that Areg from Treg stimulated fibroblast proliferation and migration. Tregs stimulated with IL-2 alone or with IL-33 were assessed at d14 or d21 for CpG methylation of Treg-associated genes. These data revealed that sustained exposure to IL-33 increased methylation of Foxp3, suggesting that sustained DAMP stimulation makes Treg unstable. In total, our studies identify how a sustained and dysregulated repair response involving interactions between fibroblasts and recipient Treg contributes to the progression of HTx CR.

Supported by grants from NIH (F30AI147437, T32 CA082084) (GKD), NIH (R01HL122489, R01AR073527) (HRT)
Engineering T cells to prevent graft-versus-host disease and leukemia relapse following allogeneic stem cell transplantation
Authors: Mo Feiyan , Watanabe Norihiro , Burkhardt Phillip M , Heslop Helen E , Brenner Malcolm K , Mamonkin Maksim
Published: January 2023

Acute graft-versus-host disease (aGvHD) and leukemia relapse remain major causes of mortality after allogeneic hematopoietic stem cell transplantation (alloHSCT). Prophylaxis and treatment of aGvHD rely on generalized immunosuppression, increasing the risk of tumor relapse and opportunistic infections and emphasizing the need for more targeted therapies.

Alloreactive donor CD4+ T cells play a central role in aGvHD pathogenesis, we thus hypothesized that elimination of activated CD4+ T cells with engineered T cells would mitigate aGvHD while preserving protective CD8+ T cell immunity post alloHSCT. We developed an alloimmune defense receptor (ADR) targeting OX40, a surface marker predominantly upregulated on activated CD4+ T cells. OX40 ADR-expressing T cells eliminated activated CD4+ T cells during coculture but spared the majority of activated CD8+ T cells, including virus-specific T cells, and had no discernible activity against resting lymphocytes. A single infusion of ADR T cells fully protected mice from fatal xenogeneic aGvHD induced by intravenous injection of human PBMC, minimizing signs of aGvHD and maximizing survival. To enable simultaneous activity of engineered T cells against aGvHD and leukemia relapse, we further armed ADR T cells with a CD19-directed chimeric antigen receptor (CAR). In a mouse model of residual leukemia post alloHSCT, administration of T cells co-expressing OX40 ADR and CD19 CAR mediated dual protection against tumor relapse and aGvHD. These results support the feasibility of a bi-functional CAR.ADR T cell product to improve outcomes post alloHSCT and reduce transplant-related mortality.

Supported by grant from Leukemia & Lymphoma Society.
Enhancing the antitumor response of CD8+ T cells by 4-1BB (CD137) co-stimulation with distinct inactivation of the type 2 adenosine receptors.
Authors: Ahn Jihae , Chen Siqi , Fan Jie , Zhang Bin
Published: January 2023

Activating the immune co-stimulatory receptor 4-1BB (CD137) with agonist antibody binding and crosslinking-inducing agents that elicit 4-1BB intracellular signaling potentiates the anti-tumor responses of CD8+ T cells. However, the underlying in-depth mechanisms remain to be defined. The type 2 adenosine receptors (primarily the high affinity receptor, A2AR) predominantly expressed on CD8+ T cells inhibit T cell activation and expansion by blocking TCR signaling in a cAMP-dependent manner. Here, we show that inactivation of the low affinity receptor A2BR rather than A2AR by continuous treatment of antagonists and/or genetic deletion induces superior survival advantage of effector CD8+ T cells with agonistic 4-1BB co-stimulation especially upon chronic TCR stimulation and/or long-term antigen exposure. Mechanistically, A2BR inactivation helps sustain the increased energy and biosynthetic requirements through the accumulation of intracellular glutathione (GSH) in response to agonistic 4-1BB co-stimulation. Importantly, A2BR inactivation in combination with agonistic 4-1BB co-stimulation displays a greater ability to modulate mitochondrial fitness for anti-tumor CD8+ T cell expansion while minimize T cell exhaustion. Thus, the A2BR pathway plays an unexpected role in metabolic reprogramming of GSH upon agonistic 4-1BB co-stimulation that allows the fine-tuning of the anti-tumor responses of CD8+ T cells.

Supported by NCI CCSG P30 CA060553 R01CA258857
Spontaneous class-switched antibody responses at endometrial cancer tumor bed drives superior patients’ outcome
Authors: Mandal Gunjan , Biswas Subir , Anadon Carmen M , Yu Xiaoqing , Gatenbee Chandler D , Prabhakaran Sandhya , Payne Kyle K , Chaurio Ricardo A , Martin Alexandra , Innamarato Patrick , Moran Carlos , Powers John J , Harro Carly M , Mine Jessica A , Sprenger Kimberly B , Rigolizzo Kristen E , Wang Xuefeng , Curiel Tyler J , Rodriguez Paulo C , Anderson Alexander R A , Saglam Ozlen , Conejo-Garcia Jose R
Published: January 2023

The role of humoral responses in endometrial cancer remains insufficiently investigated. Using a cohort of 107 patients with different histological subtypes of endometrial carcinoma, we report that concomitant accumulation of T, B and plasma cells at tumor beds predicts better survival. However, only B cell markers predict survival specifically in high-grade endometrioid type and serous tumors. Accordingly, immune protection is associated with class-switched IgA and, to a lesser extent, IgG. Notably, expression of polymeric immunoglobulin receptor (pIgR) by tumor cells and its occupancy by IgA are superior predictors of outcome, and correlate with defects in methyl mismatch repair. Mechanistically, pIgR-dependent, antigen-independent IgA occupancy drives inflammatory pathways associated with IFN and TNF signaling in tumor cells, along with apoptotic and ER stress pathways, while thwarting DNA repair mechanisms. Therefore, coordinated humoral and cellular immune responses, characterized by IgA:pIgR interactions in tumor cells, determine the progression of human endometrial cancer, and therefore the potential for effective immunotherapies.

Supported by grants from NIH (R01CA157664, R01CA124515, R01CA178687 and R01CA211913), and from Cancer Center Support Grant (CCSG) CA076292
Inhibition of DNMTs and RNA editing increases immunogenic transposable element RNA to reduce tumor burden and prolong survival in a murine ovarian cancer model
Authors: Gomez Stephanie , Cox Olivia L , Rentia Uzma , Balick Veronica , Hadley Melissa , Grundy Erin E , Kanholm Tomas , McDonald James I , Kobyra Julie , Palmer Erica , Saunthararajah Yogen , Chiappinelli Katherine B
Published: January 2023

Novel therapies are urgently needed for ovarian cancer (OC), the fifth deadliest cancer in women. Preclinical data shows great promise for the treatment of OC with epigenetic therapy. The inhibition of DNMT activates transcription of double-stranded (ds)RNA, including transposable elements. These dsRNAs activate sensors in the cytoplasm and trigger the induction of type I interferon signaling. Adenosine deaminase 1 (ADAR1) is induced by interferon signaling and edits mammalian dsRNA with an A-to-I nucleotide change. These edited dsRNAs cannot be sensed by dsRNA sensors, and thus ADAR1 inhibits the type I interferon response. We show that transposable elements including Alus, LINEs, and ERVs, are edited by ADAR1 in human OC cell lines after DNMTi treatment.

We hypothesized that increased ADAR1 editing reduces the DNMTi immune response and treated Adar1 knockdown cells with DNMT1 in vitro and in vivo. Combining ADAR1 knockdown with DNMTi significantly increased pro-inflammatory cytokine production compared to either perturbation alone. We treated an immunocompetent mouse model of OC with DNMTi after implanting them with Adar1 knockdown cells or control cells (ID8 Trp53−/−) and found that DNMTi treatment and Adar1 loss reduces tumor burden and extends survival in this model, and the combination of both is the most effective. Combining Adar1 loss and DNMTi transforms the immune microenvironment, including increased recruitment of natural killer cells and M1 macrophage polarization. This survival benefit is a result of type I interferon signaling and CD8+ T cells. Thus, epigenetically inducing transposable element transcription, followed by inhibition of RNA editing, is a novel therapeutic strategy to reverse immune evasion in OC.

Research was supported by the National Cancer Institute R00CA204592 and R37CA251270 (to KBC), the DOD Ovarian Cancer Research Program (W81XWH2010273 to KBC), and by the Marlene and Michael Berman Endowed Fund for Ovarian Cancer Research. JIM was supported by an NCI NRSA Institutional Research Training Program grant (T32 CA 247756). SG was supported by a NRSA Predoctoral Fellowship (NIH/NCI 1F31CA254315-01). The authors would like to acknowledge the Institute for Biomedical Sciences at the George Washington University for graduate student support and training (SG, EEG, TK, VB).
Effects of tumor-derived extracellular vesicles on T cell fate and function
Authors: Ma Feiya , Wang Yuqi , Peng Guangyong
Published: January 2023

Immunosuppressive microenvironments created by malignant tumors represent a major obstacle for effective anti-tumor immunity. There are multiple immunosuppressive mechanisms operative in the tumor microenvironment (TME) that interfere with T cell function. Extracellular vesicle (EV) in tumor microenvironment is an important way employed by tumor cells to control T cell fate and function. However, the mechanisms responsible for EV-mediated T cell suppression and dysfunction are still largely unknown. Here, we purified EVs from human melanoma A375 and breast cancer MCF7 cell lines and then explored EV impacts on T cell function and fate in anti-tumor immunity. We found that tumor derived EVs can suppress T cell proliferation and induce T cell senescence, which is a novel and fundamentally important phenomenon that contributes to tumor-induced immunosuppression in the TME. Mechanistically, tumor derived EVs promoted DNA damage responses and cell cycle arrest of T cell that induces T cell senescence. Blockage of DNA damage signaling in T cells via the specific inhibitor KU55933 can prevent T cell senescence and recover T cell impaired effector functions. Importantly, we further performed in vivo studies to investigate that tumor-derived EVs can induce T cell suppression and senescence, which can also be reversed by specific signaling inhibitors or/and targeting signaling molecules via the CRISPR knockdown strategy. These studies facilitate a better understanding of the novel suppressive mechanism utilized by the TME and provide insights relevant to the development of effective strategies to reverse immune suppression for enhanced anti-tumor immunity.

Supported by the grants from the Melanoma Research Alliance and National Institutes of Health.
Spatial whole transcriptome profiling of the tumor microenvironment in FFPE prostate carcinoma using the Visium platform
Authors: Reyes Daniel , Giangarra Valeria , Williams Stephen R , Turkekul Mesruh , Mielinis Paulius , Chell James , Taylor Sarah E B
Published: January 2023

Despite years of prostate cancer research, balancing the risks of treatment complications and tumor progression remains a challenge. The inability to dissect tumor microenvironments (TME) and immune compartments contributes to a knowledge gap. Spatially resolved molecular profiling allows understanding these complexities for potential personalized treatments.

We used 10× Genomics Visium Spatial Gene Expression for FFPE tissue for spatial whole transcriptome analysis of two prostate cancer sample TMEs. The tissues were processed over~5,000 molecularly barcoded, spatially encoded capture probes in spots for spatially resolved transcriptional readout.

We resolved whole transcriptome tumorigenic profiles in sections of normal, stage III adenocarcinoma, and stage IV acinar cell carcinoma FFPE human prostate tissues. Computational clustering identified spatial gene expression patterns that aligned with pathologist annotations. The data revealed spatial disorganization of basal and luminal cells in tumor samples. T lymphocytes were dispersed throughout the whole tissue in the adenocarcinoma, while plasma B cells were in the peritumoral region impacting patient prognosis. Computational methods inferred copy number variation, identifying aneuploidy regions and specific loci driving the genomic profile of the cancerous regions.

We demonstrated that spatial whole transcriptome analysis resolves FFPE prostate samples. Our data rapidly confirms known cell type patterns and tumor region-specific gene expression while enhancing understanding of the TME for drug target or biomarker discovery for tailored therapies and patient stratification.
Dissecting anti-tumor immunity in glioblastoma
Authors: Chen Dan , Varanasi Siva Karthik
Published: January 2023

Glioblastoma multiforme (GBM) is the deadliest form of brain cancer, for which there is currently no effective treatment. Despite success of immunotherapies such as checkpoint inhibitors (CPI) in treating some cancers, less than 8% of GBM patients responded to anti-PD-1 therapy. Thus, we need to understand what mechanisms underlie tumor resistance to immunotherapies. In our GBM mouse model, we have found that anti-CTLA-4 treatment greatly improved survival and suppressed tumor growth, but surprisingly, its efficacy was lost in mice that were depleted of CD4+ but not CD8+ T cells, suggesting that CD4+  T cells are critical for protection against GBM. This matches clinical data showing decreased populations of CD4+ T cells in patients with adverse clinical outcomes such as high-grade GBM. Our preliminary results from flow cytometry and single-cell RNA sequencing (sc-RNAseq) also suggested enhanced cytokine production (IFNγ, TNFα, IL-2) in CD4+ T cells after anti-CTLA-4 treatment. Similarly, we found that microglia, the local sentinels of the central nervous system, also play a significant role in tumor control, as indicated by faster GBM growth and reduced number of tumor-infiltrating CD4+ T cells when mice are depleted of microglia by a CSF1R inhibitor (PLX3397). Additionally, microglia in GBM may serve as potential antigen-presenting cells, as shown by their high MHC class II (MHC-II) expression. Therefore, we postulate that microglia are necessary to sustain anti-tumor immunity by CD4+ T cells. Importantly, through a more detailed exploration of how CD4+ T cells and microglia interact to impact tumor control, our study will provide new insights for developing therapeutic strategies against “difficult-to-treat” cancers such as GBM.
Target the activin receptor 1c on CD4+ T cells to achieve anti-tumor therapeutic effects.
Authors: Zheng Ying , Lebid Andriana , Fu Juan , Patel Chirag , Wang Xiaoxu , Pardoll Drew
Published: January 2023

Activin signaling molecules and their receptors have not been substantially studied in the context of the immune system and cancer. We show here that tumor-bearing mice had elevated Activins levels, which correlated directly with tumor burden. Likewise, cancer patients had elevated plasma Activins compared to healthy controls. Importantly, our in vitro studies suggested that Activins promoted differentiation of naive wild type CD4+ cells into induced FoxP3-expressing Tregs. Database and qRT-PCR analysis of sorted major immune cell subsets in mice revealed that Activin Receptor 1C (ActR1c) was uniquely expressed on Tregs. ActR2b, as paired to ActR1c, was found to be highly upregulated during iTreg differentiation. In humans, these receptors were expressed on CD4+ Foxp3− cells. Thus, while Activins may have many cellular targets, signaling through these receptors on CD4+ cells likely promotes Treg-mediated immunosuppression. In vivo B16 melanoma and MC38 colon tumor studies have demonstrated that mice lacking ActR1c were more resistant to cancer progression compared to wild type mice. This phenotype correlated with reduced expression of the FoxP3 transcription factor in CD4+ cells. The anti-tumor effect was T-cell dependent, as T cell-deficient mice reconstituted with naïve ActR1c knock out CD4+ cells had delayed tumor progression. In vitro, naïve CD4+ cells lacking ActR1c had a defect in their ability to gain FoxP3 expression when Activin A was present and when TGF-β was limited. Thus, blocking Activins signaling through this receptor is a promising and disease-specific strategy for preventing the accumulation of immunosuppressive iTregs in cancer.

Supported by grants from NIH (R01AI137046)
The role of lymphocyte subsets in preventing tuberculosis following intravenous vaccination with BCG
Authors: Simonson Andrew W , Bucsan Allison N , Zeppa Joseph J , Winchell Caylin G , Myers Amy J , Sutton Matthew , Ameel Cassaundra L , Darrah Patricia A , Roederer Mario , Seder Robert A , Flynn JoAnne L
Published: January 2023

Background

We previously demonstrated in non-human primates (NHP) that intravenous (IV) administration of BCG induces substantial T cell responses and often sterilizing immunity against Mycobacterium tuberculosis (Mtb) infection. Here, we investigated the role of T cell populations in this unmatched response in the context of necessary immune mechanisms for protection against tuberculosis (TB).

Methods

Beginning 20 weeks after vaccination and through the end of the study, rhesus macaques were infused with antibodies to deplete CD4, CD8α, or CD8β expressing T cells, or with control IgG/saline. NHPs inoculated with ~15 CFU Mtb Erdman via bronchoscope were monitored for 8 weeks. Granulomas, areas of disease, lung tissue and thoracic lymph nodes (LNs) were harvested at necropsy using FDG PET CT as a map to identify inflamed lesions. Immune responses were analyzed by spectral flow cytometry and homogenates were plated for colony forming unit (CFU) quantification.

Results

Successful depletion of expected cell types was seen in blood, airways, LNs and lung tissue. Depletion of CD4 T cells ablated IV BCG mediated protection in all NHPs. CD8α and CD8β depleted NHPs retained varying levels of protection, leading to mixed outcomes by bacterial burden. Preliminary results indicate the loss of protection following CD8α (both innate and adaptive cells) and CD8β (mainly adaptive cells) depletion is similar, suggesting a key role for conventional CD8 T cells in vaccination.

Conclusions

Our results provide critical insight regarding roles played by CD4 and CD8 T cell populations in terms of defining desired responses for vaccination against progressive TB. Functionality of these cells in immunized NHPs will clarify the purposes of T cell subsets.

Supported by grants from Bill and Melinda Gates Foundation and NIH NIAID (75N93019C00071)
Adjuvanted multivalent vaccine targeting clinically relevant Pf bacteriophage creates cross-reactive antibodies
Authors: Román-Cruz Valery Cristina , Miller Shannon , Schoener Roman , Lukasiewicz Chase , Secor Patrick , Burkhart David , Evans Jay T
Published: January 2023

Pseudomonas aeruginosa (Pa) is a bacterial pathogen associated with chronic diabetic wounds as well as potentially deadly lung infections in cystic fibrosis patients. Pa is listed by the WHO as “Priority 1” in the Priority Pathogens list and results in hundreds of deaths each year. Pa diversity presents a challenge in developing a vaccine or therapeutic capable of resolving infections caused by different Pa clinical isolates. Filamentous Pf phages (a virus) infect the majority of Pa strains. Additionally, the presence Pf phage is a key virulence factor contributing to Pa infections. Pf is a filamentous-lysogenic phage that is primarily comprised of repeats of coat protein (CoaB). A consensus peptide derived from the coat protein was identified and used as the antigen for our phage vaccine targeted vaccine. This conjugate vaccine targeting Pf phage induced protection against Pa wound challenges in mice. Additionally, our team has implemented the use of a novel TLR4 agonist to further enhance antibody response against Pf coat protein intact Pf phage virions. Based on their amino acid sequence homology, clinical isolates of Pa and associated Pf phage were divided into two major clades. We prepared CRM-197 conjugates using the consensus peptides from both clades. Vaccination with these multivalent conjugates in combination with a novel synthetic TLR4 agonist (adjuvant) boosts antibody response against the peptides and promotes antibody cross-recognition between peptides. Creating a safe and effective vaccine that can mount a robust immune response against both clades of Pf phage is an important advancement towards the development and clinical testing of phage based vaccine targeting deadly Pa infections.

Supported by R01 AI138981-01
Differential sex-specific immune responses following Prime-and-Trap vaccination alters protection against malaria in mice
Authors: Duncombe Caroline J , Watson Felicia N , Kalata Anya C , Shears Melanie J , Murphy Sean C
Published: January 2023

Generating liver resident-memory CD8+ T cells (TRM cells) is critical for effective liver-stage malaria vaccine. The role of biological sex in liver-stage vaccine protection is understudied. Here we report sex-specific immune responses and protection outcomes for the two-step heterologous ‘Prime-and-Trap’ liver-stage malaria vaccine designed to induce liver TRM cells. The Prime-and-Trap strategy combines sporozoite antigen DNA priming with a single intravenous dose of liver-homing radiation-attenuated sporozoites (RAS) to direct and “trap” activated and expanding T cells in the liver. This strategy induces robust liver CD8+ TRM cell responses and confers sterile protection in the P. yoelii (Py) rodent malaria model in female mice. However, when tested in male mice, it induces weaker liver CD8+ TRM cell responses and little to no protection from sporozoite challenge. We examined sex-divergent immune responses in the spleen and liver following each step of the Prime-and-Trap regimen. We found that sexually dimorphic activation of certain immune pathways drives attenuated downstream retention of TRM cells in the liver, leading male mice to generate a sub-optimal immune response that ultimately fails to protect from Py sporozoite challenge. Our findings emphasize the importance of incorporating sex as a variable for evaluation in the design of robust liver-stage malaria vaccines.

Supported by grants from NIH (R01 AI141857).
Role of p16 expressing cells in formation and function of T cell memory with age
Authors: Torrance Blake , Lorenzo Erica C , Panier Hunter A , Cadar Andreia N , Martin Dominique E , Bartley Jenna M , Haynes Laura
Published: January 2023

Aging results in the accumulation of senescent cells that have been shown to cause dysfunction in many contexts but the effect on the function of T cell immunity is still unclear. Here, we aimed to probe the effects of clearing senescent cells on T cell responses to influenza infection. We utilized a powerful p16 trimodality reporter mouse model (p16-3MR) that includes cassettes encoding luciferase, RFP, as well as herpesvirus thymidine kinase (HSV-TK) all under the control of p16 promoter. p16 is commonly upregulated in senescent cells so this model allows us to detect senescent cells via luciferase activity, RFP expression, and selectively delete those cells by treating with ganciclovir (GCV). In aged p16-3MR mice following flu infection, we observed a transient induction of RFP+ p16 expressing cells at two weeks post infection. We hypothesized that p16 expressing cells are important for resolution of inflammation and fostering the effector to memory transition. Aged p16-3MR mice lacking p16 expressing cells following GCV treatment developed fewer flu-specific tissue resident memory CD8 T cells (Trm, CD103+ CD69+) in the lung compared to control treated aged counterparts. Interestingly, deletion of p16 expressing cells also increased the proportion of flu-specific Trm cells in the draining lymph node 30 days post infection. As a result, these mice were less effective in controlling viral burden following a secondary challenge. Conversely, during primary infection, deletion of p16 expressing cells induced a more youthful response in aged mice. This suggests that targeting senescent cells is a double-edged sword that may potentiate primary responses to infection while severely limiting the ability to form durable and protective memory.

Supported by grants from NIH (R21 AG060707, R21 AG071292)
T cell-intrinsic IL-17RA signaling in the spleen supports the establishment of chronic gammaherpesvirus infection.
Authors: Jondle Christopher Ned , Tarakanova Vera of
Published: January 2023

Human gammaherpesviruses, such as Epstein-Barr virus (EBV), are human specific gammaherpesviruses that establish lifelong infection in >95% of adults and are associated with multiple cancers, including B cell lymphomas. These viruses have a natural tropism for B cells and usurp B cell differentiation to drive a robust polyclonal germinal center response, needed to establish a latent reservoir in memory B cells. The viral and host factors that influence the gammaherpesvirus-driven germinal center response are not clearly defined.

Murine gammaherpesvirus 68 (MHV68) is a natural rodent pathogen that is genetically and biologically related to human gammaherpesviruses and provides a tractable animal model to study viral and host factors that impact chronic infection. Utilizing MHV68, we discovered that IL-17RA signaling is proviral and supports the establishment of chronic gammaherpesvirus infection. Specifically, loss of IL-17RA signaling during MHV68 infection significantly attenuated viral latency, reactivation, and the germinal center response.

Importantly, the mechanism by which IL-17A signaling promotes MHV68-driven germinal center response and latency remained unclear. To gain mechanistic insight, we generated a mouse model of T cell specific IL-17RA deficiency. Excitingly, loss of IL-17RA specifically in T cells during MHV68 infection attenuated viral latency, reactivation, and the gammaherpesvirus-driven germinal center response in the spleen, similar to what was observed in the global IL-17RA−/− model. As T cells are not infected by MHV68, our finding unveils a novel crosstalk between T cell intrinsic IL-17A signaling and gammaherpesvirus infection of B cells, including virus-driven B cell differentiation.

Supported by American Cancer Society Postdoctoral Award (134165-PF-19-176-01-MPC, C.N.J.)
Influenza A virus-specific memory T cell persistence and functional recall are retained during pregnancy
Authors: Malavet Valeria Flores , Satchmei Ali , Prokop Emily , Strutt Tara M
Published: January 2023

Influenza A virus (IAV)-specific memory T cells induced by infection or through vaccination with live attenuated cold adapted influenza virus (LAIV) vaccine strains are well known to be able to confer protection against secondary homotypic as well as heterotypic IAV challenge. Whether IAV-specific memory T cell responses induced by LAIV vaccination can persist and retain their capacity to protect the maternal-fetal unit when generated prior to the altered physiological state of pregnancy, during which systemic levels of several hormones with putative immunomodulatory functions increase, is largely unknown. We show here that LAIV A/Alaska/6/77 CR-29 (H3N2)-vaccination prior to pregnancy induces IAV-specific memory CD4 and CD8 T cells that do indeed persist and functionally respond during recall in a manner similar to memory cells present in non-pregnant mice. This recall response can fully protect pregnant dams as well as their offspring from the adverse outcomes of lethal heterotypic infection with A/Puerto Rico/8/1934 (PR8-H1N1) virus. Using an adoptive transfer model and congenic HNT TcR transgenic CD4 T cells specific for PR8, we verify the observations observed with endogenous memory T cells that the recovery, surface marker phenotype, transcription factor profile, and TNF, IFN-g, and IL-2 multi-cytokine producing capacity of memory CD4 T cells responding to infection are largely unaltered by the pregnancy environment. These observations thus support that LAIV-vaccination prior to pregnancy results in the generation of IAV-specific memory T cells that have the potential to universally protect the maternal-fetal unit from the adverse outcomes of seasonal as well as pandemic strains of IAV.

Supported by grants from NIH (R21HD093948), the University of Central Florida Foundation and the Florida Education Fund McKnight Doctoral Dissertation Fellowship.
Phenotypical changes in peripheral NK cells and their impact on the clinical outcomes of patients with severe pandemic influenza A(H1N1)
Authors: Ramírez-Noyola Jazmín Ariadna , Fernández-López Luis Alejandro , Pacheco-Hernández Lynette Miroslava , Ignacio-Cortés Sergio , Gómez-García Itzel Alejandra , Garcia-Padrón Beatriz , Ramírez-Martínez Gustavo , Zúñiga Joaquín , Choreño-Parra José Alberto
Published: January 2023

NK cells are crucial antiviral effectors. However, their role against emerging respiratory pathogens has not been extensively addressed in humans. Here, we characterized the phenotype of peripheral NK cells in patients with severe pandemic influenza A(H1N1) and age- and sex-matched healthy controls (HC) using an extended panel of flow cytometry antibodies against markers of activation, cytotoxicity, cytokine production, immune memory, and surface receptors. Enrolled patients were grouped according to their clinical outcome (survival vs. death). The study included 42 influenza patients (73% men) with a median age of 48 years, from which eight (26%) died during the follow-up period. All patients were admitted to the intensive care unit and required mechanical ventilation. Individuals with influenza differentiated from HC by reduced numbers of total, CD56bright, CD56dimCD16+, CD27+ memory-like, NKp46+, and perforin+ NK cell subpopulations, but increased CD69+ NK cells. Notably, lower percentages of CD27+ memory-like NK cells correlated with increased alkaline phosphatase levels, a readout of lung damage in patients with acute respiratory distress syndrome. Strikingly, a higher expression of the senescence marker CD57 in CD27+ memory-like NK cells and expansion of double-positive CD27+CD57+ NK cells was characteristic of deceased influenza patients, but not survivors. Finally, despite being reduced in the overall influenza group with respect to HC, increased percentages of IFN-γ+ NK cells distinguished death vs. survival outcomes. Collectively, our results demonstrate that alterations in the phenotype of NK cells influence with the progression and outcomes of patients with severe pandemic influenza A(H1N1) infection.

Supported by grants from CONACyT (FORDECyT/10SE/2020/05/14-06, FORDECyT/10SE/2020/05/14-07) and SECTEI CDMX (SECTEI/050/2020) to JZ. JAR-N received a scholarship from CONACyT (CVU-1097402) for achieving her M.S.
Development of uveitis in a mouse model of spontaneous autoimmunity correlates with frequency of autoantigen-specific regulatory T cells
Authors: Yin Mianmian , Hiam Kamir J , Proekt Irina , Chan Jackie , Hu Yongmei , Lowell Clifford A , Gould Douglas B , Spitzer Matthew , Anderson Mark S , DeFranco Anthony L
Published: January 2023

B6 AireGW/+ Lyn−/− mice are a model of spontaneously autoimmunity in which 50% of mice develop uveitis by 9 weeks of age, and the remaining mice do not develop autoimmunity. Uveitis results from CD4+ T cells responding to the retina protein interphotoreceptor retinoid-binding protein (IRBP). To define cellular mechanisms that determine whether mice developed autoimmunity or not, we did single-cell RNA sequencing (scRNA-seq) of eye-draining lymph node (LN) CD4+ T cells that recognize the P2 epitope of IRBP (amino acid 271–290). Uniform manifold approximation and projection (UMAP) analysis showed that these CD4+ T cells included distinct subsets that were Ly6c1hi, Pdcd1hi and Treg, with the Treg population being overrepresented in the mice without disease. Pseudotime analysis indicated that the Ly6c1hi T cell subset represented an earlier stage in activation, whereas the Pdcd1hi subset represented a later stage. The two types of effector CD4+ T cells of mice with and without disease largely clustered together relative to their gene expression. Similar analysis of P2-specific CD4+ T cells from retinas with inflammation indicated a progression from proliferative (Mki67hi) cells to a Lag3hi subset to a Lag3hi/Pdcd1hi subset. In mice without uveitis, depletion of Treg by treatment of AireGW/+ Lyn−/− Foxp3DTR+/Y mice with diphtheria toxin resulted in rapid expansion of P2–specific CD4+ T cells in the draining LN, development of inflammation in the retina, and immune activation in other locations leading to lethality. Ongoing experiments are further testing the hypothesis that the fraction of Treg within the IRBP-specific CD4+ T cells determines whether or not these genetically susceptible mice develop eye-specific autoimmunity.

Supported by AI138479
Interstitial lung disease patients exhibit augmented germinal center responses in lung lymph nodes and increased serum reactivities to novel autoantigens
Authors: Yoon Young me , Velez Tania , Upadhyay Vaibhav , Vazquez Sara , Lee Cathryn T , Blaine Kelly , Decker Donna , Guzy Robert , Adegunsoye Ayodeji , Strek Mary , Noth Imre , Anderson Mark S , DeRisi Joseph , Shum Anthony , Sperling Anne I
Published: January 2023

Interstitial lung disease (ILD), a group of disorders characterized by scarring of the lungs, often leads to fatal impairment of respiratory function. While some patients exhibit features of autoimmunity, the role of the immune system within this heterogeneous group of disorders remains a matter of debate. We previously reported the enlargement of lung-draining lymph nodes (LLN) correlates with decreased survival in ILD patients, suggesting that activation of the adaptive immune system may contribute to pathophysiology. To investigate the composition and functional state of cells in the enlarged LLN, we performed flow cytometry analysis of LLN cells from endstage ILD patients undergoing transplantation (n=53) and control organ donors (n=36). We found that T cells from the ILD LLN exhibit effector memory phenotypes and significantly upregulate activation molecules including HLA-DR, CD38, CD154 and CD137, which are indicative of an ongoing immune response. Analyses of CD4 T cells revealed that Foxp3+CD25− regulatory T cells are specifically increased in ILD LLN. Moreover, follicular helper T cells and germinal center (GC) B cells are highly enriched, suggesting that antibody responses are activated in ILD LLN. To test the hypothesis that autoantigens may drive the augmented GC response, we screened circulating antibodies from ILD patients at two medical centers against the entire human peptidome using phage immunoprecipitation sequencing. We defined novel and shared autoantigens and experimentally validated antibody bindings to 5 protein antigens. Collectively, our findings suggest that antigen-specific immune responses may be involved in the pathophysiology and identify novel autoantigens for subsets of ILD.
Lymphotoxin β receptor signaling mediates the formation of high endothelial venule-like vessels in atopic dermatitis-like skin lesions in mice
Authors: Kanameishi Shuto , Ono Sachiko , Keith Yuki Honda , Asahina Ryota , Honda Tetsuya , Kabashima Kenji
Published: January 2023

High endothelial venules (HEVs) are specialized blood vessels contribute to the trafficking of lymphocytes into secondary lymphoid organs by expressing a group of L-selectin ligands called peripheral node addressins (PNAd). In lymph nodes (LNs), lymphotoxin β receptor (LTβR) signaling from dendritic cells (DCs) is essential for the formation of HEVs. On the other hand, PNAd+ HEV-like vessels are observed in the skin under certain inflammatory conditions, such as atopic dermatitis (AD). However, the characteristics and the formation mechanism of HEV-like vessels in the inflammatory skin remains unclear. To address these issues, we first identified PNAd+ HEV-like vessels in the skin using a calcipotriol-induced mouse AD model. HEV-like vessels were located in the postcapillary venules abundant in cellular organelles, which were similar to HEVs in LNs. Blockade of LTβR-signaling by administration of LTβR-Fc fusion protein markedly decreased the formation of HEV-like vessels in the AD-like skin. Analysis using published single-cell RNA-seq data of human AD skin revealed that T cells and DCs highly expressed LTB, a gene encoding LTβ. The expression of LTB was highlighted in T cells by RNAscope in both human AD lesions and mouse AD-like skin. The formation of HEV-like vessels in AD-like skin was normal in Ltbfl/fl CD11cCre mice, but was partially impaired in Ltbfl/fl CD4Cre mice. Collectively, HEV-like vessels in the inflammatory skin show similar characteristics to HEVs in LNs, and LTβR signaling, partly by LTβ from T cells, is responsible for their formation.
Immunomodulatory effects of human myeloid-derived suppressor cells expanded from cord blood on dermatophagoides farinae-induced atopic dermatitis in NC/Nga mice
Authors: Kim Chang-hyun , Sohn Hyun-Jung , Hong Seung-Min , Kim Sueon , Jung Soo-Hyun , Lim Bang-Geul , Kim Eun-ah , Jeong Kwang-Hun , Kim Jongil , Park Jaehee , Kim Stan , Kim Tai-Gyu
Published: January 2023

Myeloid-derived suppressor cells (MDSCs) plays an important role in controlling the immune response against cancer and in suppressing autoimmunity and allergic inflammation. However, the therapeutic effect of MDSCs on experimental mouse model of atopic dermatitis (AD) has not been reported. We investigated the therapeutic efficacy of human myeloid-derived suppressor cells expanded from cord blood (hUCB-MDSCs) in dermatophagoides farinae (Df)-induced AD-like skin lesions in NC/Nga mice and explored the mechanisms involved. The administration of hUCB-MDSCs (1×105 and 1×106 cells per mouse) significantly reduced in clinical severity scores and was associated with the reduction of histopathological changes including inflammatory cellular infiltration, epidermal hyperplasis, and hyperkeratosis. hUCB-MDSCs decreased the serum levels of IgE, Th2-mediated cytokines and chemokines. Particularly, the expression of filaggrin and involucrin in the skin lesions was recovered, whereas the expression of keratin-10 and keratin-14 decreased. These immunomodulatory effects of hUCB-MDSCs in a murine model of AD may provide helpful evidence for the clinical development of cell therapies to treat AD.
Macrophage Clearance of Neutrophil Extracellular Traps (NETs)
Authors: Gronevelt J Paige , Pisano Michael , Rumble Julie
Published: January 2023

Neutrophil Extracellular Traps (NETs) are formed from dying neutrophils expelling their contents to capture pathogens and limit the spread of infection. While they are beneficial during infection, overproduction of NETs or the inability to degrade them can be pathogenic. Different inflammatory disorders and autoimmune diseases have been shown to be associated with an increase in NET production. Therefore, it is important to understand the method in which NETs are removed or broken down.

Cayman Chemical has developed an imaging-based assay to sensitively assay NET uptake by phagocytic cells. Imaging was found to be an ideal method to monitor NET clearance in vitro, in part due to the ability to monitor the uptake of NETs kinetically. Both primary monocyte-derived macrophages and differentiated THP-1 cells, which are frequently used as phagocytic cell models, were shown to take up NETs. Putative modulators of NET uptake were tested in the assay.

We aimed to develop a method in which the process of NET uptake can be monitored and quantified. Modulators of NET uptake can be tested in this assay, allowing for further studies toward treating autoimmune disorders that are driven by aberrant NETs.