bluuebunny/crossref_metadata_2025_split huggingface dataset:
B Cell Leukemia Elicits a Suppressive Tr1-like CD4-T cell Response that can be circumvented with PDL1-blockade
Authors: Venkatesh Hrishi , Tracy Sean , Qiu Yinjie , Knutson Todd , Farrar Michael 
Published: November 2024

Abstract
Although inhibitory receptor (IR)hi CD4-T cells are associated with relapse in B cell leukemia (B-ALL), how they facilitate relapse is not understood. Using a murine B-ALL model, we show that leukemia induced IRhi CD4-T cells express genes associated with suppressive FOXP3- Tr1 CD4-T cells. These CD4-T cells do not have epigenetic silencing of effector cytokines, suggesting a functional state distinct from exhaustion. While anti-PDL1 or nilotinib (targeted therapy) alone had a minimal impact on survival, concurrent treatment with anti-PDL1 and nilotinib had a significant CD4-T cell-dependent synergistic effect. In contrast, IL10R blockade and nilotinib had a minimal impact on survival. IL10R blockade did improved the effector function of leukemia-specific CD4-T cells, but also impairing their long-term maintenance. Treatment with nilotinib and anti-PDL1 promoted the expansion of leukemia-specific CD4-T cells with lower IL10 expression and higher expression of helper molecules (CD40L, CCL4). The expansion of these CD4-T cells correlated with an increase in GzmB+ CD8-T cells, and resulted in a memory response that protected mice from a leukemia re-challenge. Nilotinib and IL10R blockade improve CD4-T cell function while also impairing their persistence, resulting in sub-optimal leukemia control. In contrast, nilotinib and anti-PDL1 induce CD4-T cells with a lower level of IL10 and improved helper function-this results in a potent and durable anti-leukemia T cell response.
NAC1 deficient regulatory T cell support tumor growth by CD36 mediated metabolic adaptation in tumor microenvironment.
Authors: Kumar Anil 
Published: November 2024

Abstract
In regulatory T cells, NAC1 enhances the acetylation of FoxP3 and protect from proteasomal degradation. Elevated levels of FoxP3+ Tregs within the tumor microenvironment (TME) showed a positive correlation with poor prognosis in various cancer patients. To address this dichotomy, we studied the role of NAC1 deletion on tumor growth. Here we report that NAC1 deficiency support tumor growth by enhanced infiltration of NAC1 deficient Tregs cells. This study confirmed that NAC1 deficient intra-tumoral Tregs are metabolically more able to adapt to acidic and nutrient deficient tumor microenvironment through enhanced FoxP3 expression mediated increased lipid metabolism and mitochondrial biogenesis. These results revealed that the NAC1-FoxP3-CD36–PGC1a signal sustains survival and functional fitness in intratumoral Treg cells by modulating mitochondrial fitness, lipid metabolism and oxidative phosphorylation driven protection from lipotoxicity and metabolizing lactate in the TME. These results highlight the unexplored NAC1-FoxP3-CD36–PGC1a modulated metabolic adaptation, which allows intratumoral Treg cells to utilize lactate and fatty acid in tumors. In adoptive Tregs transfer experiment we confirmed that Treg specific NAC1 deficiency is sufficient and a key factor to support tumor initiation and growth. This study that targeting metabolic adaptation in intratumoral Treg cells would be a hopeful strategy for reprogramming the TME.
Comparative Analysis of PD-1, PD-L1 and CTLA-4 Expression in Infiltrating Leukocytes in Tumor Tissue and Peripheral Blood in Patients with Renal Cell Carcinoma.
Authors: Gonzalez Garza Raquel , Salinas Carmona Mario , Gutierrez Gonzalez Adrian , Flores Gutierrez Juan , Castro Corona Maria , Mejia Torres Manuel 
Published: November 2024

Abstract
Clear cell renal cell carcinoma (ccRCC) is the most prevalent subtype of kidney cancer. Despite the availability of treatment options, the overall prognosis for ccRCC remains poor, mainly for the acquired resistance to conventional therapies. Current evidence indicates that the immune system is indirectly involved in tumor progression, where T cells play a critical role in antitumor immunity. In addition, the functional state of infiltrating leukocytes predicts response to therapy in most types of cancer. In the present study, we describe the nature of infiltrating leukocytes in tumor tissue and the exhaustion-associated markers PD-1, PD-L1 and CTLA-4 in blood and renal tissue obtained from patients diagnosed with ccRCC. Our results indicated that within the tumor microenvironment, the main source of inhibitory molecule PD-L1 are the infiltrating monocytes whereas the most expressed inhibitory marker in lymphoid cells are PD-1 in CD8+ cells, but CTLA-4 in CD4+ cells. More importantly, the phenotype remains consistent across central and peripheral tissues, suggesting that exhaustion-associated markers could be measured in blood, which is a less invasive alternative to conventional histopathologic evaluation.
Digital PCR as a novel way to assess chimerism of humanized immune system mice
Authors: Smith Nicholas , Hansen Sarah , Livingston Robert , Smith Steve , Roesch Paula , MacBride Megan 
Published: November 2024

Abstract
Humanized immune system (HIS) mice contain human and murine immune cells. Analysis of chimerism in HIS mice is typically accomplished via flow cytometry, but this requires collecting a 75 µl blood sample and access to a flow cytometer. Repeated bleeding can negatively affect the health of HIS mice, limiting serial analysis. Digital PCR offers a novel method for chimerism analysis, which uses much smaller blood volumes and can be performed on clotted or frozen samples, sample types unsuitable for flow cytometry. A digital PCR assay directly comparing the presence of three human genes and one murine gene was developed. This assay was validated in the huNOG-EXL HIS mouse engrafted with CD34+ hematopoetic stem cells from 3 unique donors via blind comparison with chimerism data from flow cytometry. At 10 weeks post engraftment, the digital PCR assay showed excellent correlation for all three human genes against chimerism as measured by flow cytometry of the peripheral blood. This assay represents a new, user-friendly, rapid tool for the analysis of HIS mice and may permit serial chimerism measurement using 10 μL whole blood. This assay may be useful to investigate other mouse-human chimera models, including mice harboring functional human hepatocytes, tumors, and other cells.
Development of a T-cell activation panel using image cytometry in rheumatoid arthritis patient samples
Authors: Chan Leo , Stuckey Tyanna , Pierce Mackenzie , Musgrove Blessing , Hernandez Veronica , Lin Bo , Rapson Sean , McDonald James , Reading Julian 
Published: November 2024

Abstract
Rheumatoid arthritis (RA) is an autoimmune disease that results in cartilage damage and bone loss due to chronic inflammation of joints. Treatments for RA include disease-modifying antirheumatic drugs and nonsteroidal anti-inflammatory drugs, as well as the inhibition of activated T-cells. Consequently, there is a need to screen RA patient samples rapidly and effectively to monitor treatment efficacy, disease progression, immune response, and predictive markers. In this work, we developed and demonstrated a multiplex detection method to monitor T-cell activation in a panel consisting of CD3, CD69, CD25, and CD279 (PD-1) surface markers using the Cellaca™ PLX Image Cytometer (Revvity). First, a dosage titration of PHA-P was performed on PBMCs to stimulate T-cell activation. Once stimulated, CD3 positive T-cells were evaluated for CD69, CD25, and the checkpoint molecule CD279. After 24 hours, the percent surface marker positive T-cells and the level of marker expression (median fluorescent intensity values) were used to determine optimal PHA-P dosage. Results showed concentration dependent increases in the expression levels with a calculated EC50 value for PHA-P ranging from 20.16-30.78 µg/mL with a 95% CV based on the CD279 titration curve. The proposed image cytometric surface marker detection method may prove to be an efficient tool to rapidly screen RA patient samples for T-cell activation in the setting of clinical disease treatment or progression.
Functional single-cell screening of chimeric antigen receptor (CAR)-T cells based on multiple cytokines using Nanovial Technology
Authors: Kuo Wei-Ying , Iyer Neha , Liang Jesse , Bosler Lucienne , Wu Alex , Padgett Sean , Di Carlo Dino , de Rutte Joseph 
Published: November 2024

Abstract
CAR-T therapies, while potent against some cancers, face challenges including toxicities and tumor relapse, particularly in solid tumors with hostile TMEs and sub-optimal CAR designs. A major obstacle is the lack of efficient functional screening methods for CAR-T cells, crucial for T-cell-based immunotherapy progress.
Our study introduces a novel functional screening approach using hydrogel microparticles (Nanovials) to analyze millions of individual CAR-T cells based on antigen specificity and functional cytokine secretion. We modified Nanovials with Mesothelin (MSLN) antigen, enriching and binding specific CAR-T cells. Additionally, Nanovials were adapted with antibodies to detect key cytokines (IFN-gamma, GranzymeB) from activated CAR-T cells, identified through fluorescent markers. This setup allows for analyzing and sorting CAR-T cells with unique functional traits using standard flow cytometers.
Using the Nanovial assay we distinguished MSLN CAR-T variants by cytokine profiles, revealing minor CAR engineering differences and enabling early functional assessment. This technique identified distinct CAR-T cell subpopulations within patient samples, surpassing traditional bulk analysis methods like ELISA and cell killing assays.
In summary, our Nanovial-based method offers a scalable, efficient solution for CAR-T cell functional screening, enhancing CAR design and improving therapeutic manufacturing quality control.
Role of Tregs in maintaining alloreactive Tconv hypofunction in transplantation tolerance
Authors: Cassano Alexandra , Mora Cartin Ricardo , Wang Peter , Wang Ying , McIntosh Christine , Andrade Michael , Chong Anita , Alegre Maria-Luisa 
Published: November 2024

Abstract
Whether Tregs are necessary to maintain Tconv dysfunction long-term after tolerance has been achieved remains to be established. Our preliminary data indicate that complete, transient depletion of Tregs using diphtheria toxin (DT) in Foxp3-DTR mice at the maintenance phase of tolerance precipitated the rejection of previously tolerized cardiac allografts and resulted in de novo production of graft-specific antibodies. These Tconvs showed a trend toward renewed cytokine production after Treg depletion. RNA-sequencing of graft-specific Tconvs after Treg depletion in tolerant mice demonstrated transcriptional distinction from Tconvs in tolerant mice that retain Tregs. Conversely, graft-reactive Tconvs from tolerant mice infected with Listeria monocytogenes or receiving an injection of plasmids encoding IFNb and IL-6 to break tolerance, were transcriptionally similar to Tconvs from unmanipulated tolerant mice. GSEA revealed that B cell mediated immune response related pathways were enriched in differentially expressed genes between graft-specific Tconvs from tolerant and Treg-depleted mice. Tconvs from Treg-depleted mice expressed genes associated with Tfh cell differentiation compared to both tolerant and naïve graft-reactive Tconvs. This indicates a unique state of potential reinvigoration and implicates Tregs in actively maintaining dysfunction of graft-specific Tconvs, and suggests that graft-reactive Tconvs may become Tfh cells when reinvigorated by Treg depletion.
Comparing the immunogenicity of different SARS-CoV-2 spike vaccine platforms in mice
Authors: Lew Min Han , Sanchez Sarah , Penaloza-Macmaster Pablo 
Published: November 2024

Abstract
Comparing the immunogenicity of different SARS-CoV-2 spike vaccine platforms in mice
Min Han Lew1Y, Sarah Sanchez1Y, Pablo Penaloza-MacMaster1
1Department of Microbiology and Immunology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611.
YEqual contribution.
Multiple vaccine platforms have been deployed during the COVID-19 pandemic, and it is still unclear how these differ in terms of their immunogenicity and efficacy. Here, we compare the immunogenicity of different vaccine platforms in C57BL/6 mice, including Ad5-, mRNA- and protein-based regimens expressing the SARS-CoV-2 spike antigen. After vaccination with different vaccine vectors, immune responses were measured using enzyme-linked immunosorbent assays (ELISA) and tetramer binding assays to measure antibody responses and CD8 T cell responses to the SARS-CoV-2 spike protein, respectively. Immunization with Ad5 vectored vaccines elicited the highest CD8 T cell and antibody response relative to other vaccine platforms, but only when administered into Ad5 seronegative mice. However, when administered into Ad5 seropositive mice, mRNA vaccines elicited the highest immune response. These data suggest that the relative efficacy of adenovirus- or mRNA-based vaccines depends on whether the host has pre-existing immunity to the adenovirus vector itself.
Design and testing of mosaic enterovirus vaccine to prevent hand, foot and mouth disease
Authors: Greening Gage , McLennan Rebecca , Geanes Eric , Khanal Santosh , Rangaraj Suryabalan , Bradley Todd 
Published: November 2024

Abstract
Enterovirus A (EV-A) has been responsible for recent outbreaks of hand-foot-mouth disease (HFMD); however, there is no vaccine or effective antiviral treatment. The goal of our project is to develop broad vaccines targeting the diverse EV-A serotype viruses that cause HFMD. We used the EV-A viral capsid protein 1 (VP1) as the vaccine antigenic target, because of its genetic diversity across EV-A serotypes, and prior work that showed VP1 has linear neutralizing antibody epitopes. We computationally designed VP1 proteins to create mosaic antigens that optimize B and T cell epitopes among diverse EV-A viral sequences. We identified that a hexavalent mosaic vaccine cocktail had the highest EV-A sequence coverage. We then immunized mice in three groups with recombinant VP1 proteins in adjuvant; Group 1 was given the EV-A Consensus VP1 (Consensus), Group 2 was given the EV-A strain EVA-71 VP1 protein (EVA-71, strain specific) and Group 3 was given our newly designed hexavalent vaccine (EV-A Consensus + 5 mosaic VP1). We measured the antibody binding levels to 9 EV-A VP1 antigens after immunization and found that the EV-A hexavalent vaccine resulted in improved antibody binding breadth during in vivo vaccination. Our results demonstrate the feasibility of using polyvalent VP1 proteins to increase the breath of antibody epitopes among EV-A serotypes. Our approach has the potential to treat and block EV-A infection and serve as a model for responding to other emerging viral diseases.
Swine T cell responses to influenza A virus following live attenuated influenza virus or replicon particle vaccine
Authors: Wymore Brand Meghan , Kunzler Souza Carine , Arruda Bailey , Baker Amy 
Published: November 2024

Abstract
Influenza A virus (IAV) is a significant economic concern in the swine industry. Live attenuated influenza virus vaccines (LAIV) in swine are not available commercially but experimentally shown to induce mucosal cellular immunity. Non-adjuvanted hemagglutinin (HA) replicon particle (HA-RP) vaccines induce robust HA antibody, but little is reported about induction of T cell responses compared to LAIV. This study compared HA-RP and LAIV induction of T cell responses. Pigs were vaccinated with experimental LAIV or HA-RP, challenged with heterologous IAV, and necropsied at 5 or 21 days post infection. Virus titration was performed on nasal swabs and bronchoalveolar lavage fluid (BALF). T cells isolated from blood, lung, lymph node, and BALF were stimulated ex vivo with IAV and evaluated for cytokine production and proliferation. HA-RP-vaccinated pigs had lower macroscopic lesion scores but similar virus titers in nasal swabs and BALF to non-vaccine challenge (NV/CH) pigs. LAIV-vaccinated pigs had similar macroscopic lesion scores compared to NV/CH pigs but minimal virus titers. Both vaccines induced T cell production of IFNγ and TNFα and T cell proliferation after IAV stimulation, though LAIV responded at greater magnitude. In conclusion, both LAIV and HA-RP vaccines induced T cell responses and variable protection from heterologous IAV challenge. Understanding vaccine induction of cellular immunity as a correlate of protection will aid in improved swine IAV vaccine strategies.
Natural intraepithelial lymphocytes are critical intestinal defense against necrotic enteritis in chickens
Authors: Nazmi Ali , Majeed Shuja , Hamad Shaimaa , Shah Bikas , Bielke Lisa 
Published: November 2024

Abstract
In chickens, the subsets of intraepithelial lymphocytes (IEL) and their responses to enteric diseases remain unclear. Therefore, our study aimed to elucidate the roles of IEL populations in necrotic enteritis (NE) in chickens. At 14 days of age, 63 SPF chicks were randomly divided into 3 groups: Control (sham challenge), Eimeria maxima (EM) challenge, and Clostridium Perfringens (CP) challenge. Birds in the EM and CP groups were orally infected with EM at day 14 of age, and while CP birds received additional oral infection with CP bacteria at days 18 and 19 of age. At 20 days of age (1 day post CP infection; dpi), and 27 days of age (7 dpi), 7 birds per group were euthanized, and jejunum was harvested for gross lesion scores, IEL isolation, and gene expression. Birds exhibited subclinical NE based on lesion scores, and CP-infected birds showed lower BWG and shorter colon length. The Most changes in the IEL populations were observed at 1 dpi. The CP group showed substantial increases in the total number of natural IEL subsets, including TCRαβ+CD4-CD8-, TCRαβ+CD8αα+, TCRγδ+, TCRneg and iCD8α IEL. By 7 dpi, only the number of TCRαβ+CD4-CD8- and TCRαβ+CD8αα+ IEL maintain their increase in the CP group. The CP group had significant higher expression of IL-1β and IFN-γ and osteopontin genes at 1 dpi. These findings suggest that natural IEL play a critical role in the host response during subclinical NE, potentially conferring protection against CP infection.
A conserved TRAF binding motif within human 41BB enhances canine CD8+ CAR T cell expansion and cytolysis
Authors: Goodman Emma , Mason Nicola , Payne Aimee 
Published: November 2024

Abstract
Anti-CD19 chimeric antigen receptor T cells (CART) have transformed cancer therapy by inducing durable remissions of B cell cancers, although incomplete responses are common, highlighting a need for improved therapy. Immune competent dogs develop B cell lymphomas with similar pathophysiology as humans, offering a compelling opportunity for CART preclinical assessment. In a first-in-canine trial, anti-CD20 CART with canine 41BB and CD3ζ (caBBζ) domains exerted selective pressure on B cell lymphomas but did not persist or induce remission. We hypothesize canine CART signaling defects impair cytolytic activity and persistence. To optimize CAR design, we incorporated canine CD28 and CD3ζ (ca28ζ) or human (hu) BBζ and evaluated CART function and immunophenotype, using caBBζ as a reference control. In both an in vitro rechallenge and canine leukemia xenograft model, huBBζ CART elicited greater cytolysis and CD8 T cell outgrowth than caBBζ, and ca28ζ CART did not persist. Mutagenesis studies indicate T235 in the conserved PxQxT TRAF binding motif is required for CD8 outgrowth in huBBζ CART. Dogs and other mammals express A235, but introduction of A235T is not sufficient to confer CD8 outgrowth in caBBζ CART. Ongoing studies are profiling pathways downstream of ca/hu41BB that drive cytolytic activity and CD8 outgrowth. These studies reveal fundamental differences in T cell biology between humans and other mammals that hold promise for enabling clinical efficacy of canine CART.
Ultralong CDR3 cattlebody use biased to switched immunoglobulin isotypes in bovine immune tissues
Authors: Criscitiello Michael , Ott Jeannine , Sheppard Morgan , Whitley Casey , Ebert Nicholas 
Published: November 2024

Abstract
Alongside typical immunoglobulin (Ig), cattle express unique ultralong immunoglobulins (UL) that contain a CDR H3 comprised of a β-ribbon stalk and disulfide-linked knob that reaches over 70 amino acids in length. UL Ig exhibit potential "reach" to recessed pathogen epitopes and can provide broadly neutralizing antibodies to elusive antigens. This study explores the relative amounts of UL Ig in pre-switch isotypes (IgM and IgD) versus post-switch isotypes (IgG, IgA and IgE). Rearranged immunoglobulin heavy chain transcripts from primary and secondary lymphoid tissues of three steer were assessed with primers specific for the variable segments used in ultralong and canonical rearrangements as well as specific subisotype constant regions. Relative expression suggests biased selection for UL Ig in germinal centers and use in mucosal tissues of the ruminant gastrointestinal tract. Despite the explosion of studies exploring the immunogenetic diversification and binding possibilities of the UL Ig knob "picobody", our understanding of the use of the role of these antibodies in the Bovid immune system has been much slower. With a more complete understanding of tissue-specific expression patterns and isotype-specific immune responses using these exceptional paratopes, we'll be better poised to exploit them for improved bovine and human health.
Comparing visio-spatial intelligence in amateur rugby and netball players using hand-eye coordination tests
Authors: Millard Lourens , Breukelman Gerrit J. , Mathe Nonkululeko 
Published: November 2024

Background: Research investigating the differences in visio-spatial skills (VSS) between athletes and non-athletes, as well as variations across sports, presents conflicting findings.Aim: The objective of this study was to determine if there exist significant differences in visio-spatial intelligence (VSI) skills between rugby players and netball players, and whether such disparities are present when comparing both groups to non-athletes.Setting: All participants, including the control group (non-athletes), rugby players and netball players, were recruited from the Zululand region of South Africa’s KwaZulu-Natal province, specifically from all premier league rugby and netball teams in the area.Methods: Participants underwent an optometric assessment, followed by an evaluation of VSS using six established tests: The Hart Near Far Rock, saccadic eye movement, evasion, accumulator, flash memory and ball wall toss tests.Results: The results revealed that rugby players significantly outperformed netball players in speed of recognition, peripheral awareness and hand-eye coordination (P = 0.000). Moreover, both rugby players and netball players performed significantly better than non-athletes in five of the six tests (P = 0.000), with the exception being the visual memory test (P = 0.809).Conclusion: This discrepancy in performance suggests that certain VSS are superior in athletes compared to non-athletes, highlighting potential implications for theories of vision, test selection and the development of sport-specific VSS testing batteries. Furthermore, the use of a hand-eye coordination-specific VSS test battery effectively differentiated between different sports.Contribution: The identified pattern was not consistent across all VSS tests, indicating that further research should explore the training methods employed by both sports, as these factors may contribute to the observed differences.