bluuebunny/crossref_metadata_2025 huggingface dataset:
The effects of αPD-1 and αPD-L1 therapy on macrophage remodeling within the solid tumor microenvironment
Authors: Kwok Tina , Da Silva Ildefonso Alves , Dong Haidong , Lancaster Jessica
Published: August 2023

PD-1- and PD-L1-targeted immune checkpoint blockade therapy has shown great clinical potential. Unfortunately, this therapy has been ineffective in a majority of patients bearing solid tumors due to the highly immunosuppressive nature of the tumor microenvironment. As macrophages compose the most abundant myeloid cell subset within the tumor, current macrophage-targeted treatments aim to deplete macrophages within the tumor to reduce immune suppression. However, our goal is to determine whether αPD-1 and αPD-L1 therapy can harness macrophage potential by shifting tumor associated macrophages (TAMs) from a suppressive M2 to a pro-inflammatory M1 phenotype. Our preliminary data has shown that αPD-1/αPD-L1 treatment of mice bearing B16 murine melanoma leads to controlled tumor growth and this was accompanied by a higher M1:M2 ratio and CD8 T-cell abundance. Therefore, we hypothesized that αPD-1/αPD-L1 therapy may drive TAMs towards a pro-inflammatory phenotype and to become better activators of CD8 T-cells. By analyzing antigen presentation machinery on TAMs, we show that in the presence of tumor cells, αPD-L1 blockade upregulates expression of MHC1 and MHC2 on TAMs. Unexpectedly, direct αPD-L1 blockade on TAMs leads to reduced CD80 surface due to mechanisms still to be elucidated. Further, we analyzed co-cultures of αPD-L-treated TAMs loaded with ovalbumin and naïve OT1 cells, which led to greater proliferation of naïve CD8 T-cells compared to their isotype control counterparts. Our observations thus far support the ability of αPD-1/αPD-L1 therapy to induce an M1-phenotype shift among macrophages and also αPD-L1 blockade alone can drive TAMs to become better CD8 T-cell activators.

Mayo Clinic David and Margaret Grohne Cancer Immunotherapy and Mayo Clinic Immune Monitoring Core Pilot Awards
Hijacking Suppression: Anti-CD40 Converts Regulatory T Cells Into Type I Effectors
Authors: Maltez Vivien Ileana , Arora Charu , Sor Rina , Lian Qiaoshi , Vonderheide Robert H , Germain Ronald N , Byrne Kateyln T
Published: August 2023

Background

Pancreatic ductal adenocarcinoma (PDA) is an aggressive and heterogeneous cancer that is often refractory to current treatments. We found that a combination of anti-PD1, anti-CTLA-4, and anti-CD40 results in tumor regression. Thus, 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.

Results and Conclusions

In the TME of responsive tumors, our therapy severely depleted regulatory T cells. We found this Treg loss to be ADCC–independent, but anti-CD40–dependent, a surprising result given that anti-CTLA-4 was thought to mediate Treg depletion. Residual Tregs were restricted to the tumor periphery by 48 hours post anti-CD40 administration. We used tamoxifen-inducible Foxp3 lineage tracing mice to label all Foxp3–expressing cells prior to therapy initiation. Strikingly, we found that many of the lineage marked Tregs no longer expressed Foxp3. These ExTregs now had high levels of Tbet and IFNg, and had evidence of cognate antigen recognition, as assessed via imaging of NFAT1 nuclear translocation in situ. Single and combination treatments in the absence of anti-CD40 failed to induce this ExTreg phenomenon. Blockade of MHC-II, IL-12, or IFNg or tumor implantation into Batf3 KO, IL12p40 KO, or IFNg KO mice also ablated this phenomenon. Combined, these data reveal a unique mechanism by which the clinically relevant agonistic anti-CD40 amplifies the anti-tumor immune compartment through the conversion of immunosuppressive Tregs to an effector population within the tumor microenvironment.

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.
NSCLC patient-derived xenografts perpetuating tumor-associated, tumor-reactive T cells that respond to immunotherapy but do not cause GVHD in host mice
Authors: Radhakrishnan Vijay , Venkateshaiah Sathisha Upparahalli , Mahadevappa Chandrashekara Puthanapura , Okpasuo Onyekachi Juliet , Cheatham Christa , Nagaraj Suvilesh Kanve , Manjunath Yariswamy , Li Guangfu , Kaifi Jussuf T , Schrum Adam G , Gil Diana , Avella Diego M
Published: August 2023

Non-small cell lung cancer (NSCLC) is the leading cause of malignancy-related deaths worldwide. We wished to overcome a current major limitation of PDX modeling, by providing autologous human T cells suitable for immunotherapy responses, but notably, without graft versus host disease (GVHD). We have developed PDXs from NSCLC by implanting surgically resected patient tumors subcutaneously in NOD scid gamma (NSG) mice. We used a metastatic PDX model to test potential therapeutic efficacy of a novel T cell molecule, anti-human-CD3ɛ monovalent Fab fragment (Mono-OKT3-Fab) and compared tumor growth results with anti-human PD-L1 therapy. Overall engraftment rate of unique, primary patient tumors was 52%. Over multiple passages in vivo, PDX tumors retained expression of primary tumor-matched NSCLC-diagnostic pathological markers. Moreover, primary-tumor-associated T cells were present and passaged in mice in vivo, without producing any signs of GVHD. Immunotherapy showed significant response from a PDL-1+ tumor, consistent with functional tumor-specific T cell passaging in our system. In contrast, control experiments using patient-matched human PBMCs showed vigorous GVHD in the presence or absence of co-transplanted PDX tumor; this demonstrated that non-tumor-associated lymphocytes from these patients were capable of xenoreaction, even though tumor-associated T cells were not. We observed that PDX-NSG mice treated with either modality showed significantly reduced tumor burden and increased survival over control-Ig-treated mice. Overall, our study shows that PDXs implanted in NSG mice from surgically resected-NSCLC retain T cells sufficient for preclinical evaluation of novel T cell-targeting immunotherapies.

NIH, National Cancer Institute, IOTN(U01 CA244314)

NIH, NIAID, R01 AI097187

Mizzou Start-up funds
Understanding the cellular and molecular mechanism of anti-CTLA4 mediated tumor rejection
Authors: Liu Na , Kazmi Rida , Jin Wenwen , Gauthier Thierry , Patino Liliana C , Jin Wenwen
Published: August 2023

Although the clinical success of checkpoint inhibitors treatment is significant to prolong the patient survival, there is a substantial gap in our understanding of their mechanism in patients due to several technical and ethical limitations. Recent work suggests that the therapeutic effect of anti-CTLA-4 antibodies may not only block CTLA-4 interaction with its ligands, but also selectively deplete intratumoral Tregs (the highest expresser of CTLA-4) through ADCC. This releases the immunosuppressive state of the tumor microenvironment, thereby, promoting the expansion of effector CD4 and CD8, and increasing the secretion of IFN-g and TNF to reject the tumors. We observed a rapid Treg reduction characterized with fragile phenotype as early as 24 hours after anti-CTLA4 treatment. Using FOXP3-DTR mice, we confirmed that Treg depletion is the major contributor against tumor progression as anti-CTLA4 effect was abrogated in the DT-treated mice, and Treg function was maintained in the FcR KO mice. This suggested that FcR engagement is responsible for the Treg weakness. We hypothesized that there might be additional signal being transmitted when anti-CTLA4 is engaging with FcR which is capable of inducing Treg reduction. We made this hypothesis when we found the important receptor CD25 expressed on the Treg was significantly decreased after engagement. This receptor can also sense surrounding IL2 to maintain Treg cell survival, function, and proliferation. Interestingly, the CD25 expression was not inhibited when lacking FcR to induce CTLA4 engagement. Hence, there is still a need to study the underlying mechanism to see why Treg cells express lower CD25 after CTLA4 engagement.
Development of effective immunotherapy for triple negative breast cancer by WEE1 kinase inhibition
Authors: Choi Mi Ran , Xie Ping , Tang Hui , Fan Jie , Ahn Ji Hae , Orpano Rachel , Zhang Bin
Published: August 2023

Treatment options for triple negative breast cancer (TNBC) are limited because they are usually insensitive to most available hormonal or targeted therapeutic agents owing to their triple negative nature. Exciting clinical results using immune checkpoint inhibitors (ICI) directed against PD-1/PD-L1 have been reported. However, only a subset of patients (~20%) respond to ICI. Therefore, identifying rational combinations is needed to enhance the efficacy of immunotherapy in TNBC. We demonstrated there that inhibition of WEE1, a G2/M cell cycle checkpoint regulator induced by DNA damage, increased expression levels of MHC-I, and pro-inflammatory chemokines (CXCL9 and CXCL10) together with PD-L1 in human and mouse TNBC cells. Consequently, WEE1 inhibition enhanced cytotoxic T-lymphocyte (CTL)-mediated killing of tumor cells, and CTL infiltration into the tumor, making tumors susceptible to anti-PD-1 mAb immune checkpoint blockade in mouse TNBC models in a CD8+ T cell–dependent manner. Mechanistically, WEE1 inhibition triggered the cellular damage stress and interferon response through the double-stranded DNA/RNA (dsDNA/RNA) by activating the TLR9/IRF7 pathway. Furthermore, combining WEE1 inhibition and TLR9 agonist CpG DNA resulted in tumor regression, facilitating an effective antitumor T cell response. Our findings have rapid translational implications for the exploration of WEE1 inhibitors alongside agents that stimulate CD8+ T cell activity, including ICI and TLR9 agonists, which are both clinically available to treat TNBC patients.

We acknowledge the funding support from NCI R01CA222963 and DoD W81XWH-19-1-0106.
A novel tumor-bearing humanized mouse model for evaluating the efficacy and predictive safety of bispecific T cell engager
Authors: Yang Guoxiang , Yang Hongyuan , Ordones Evie , Yao Li-Chin , Yang Jiwon , Keck James G , Cheng Mingshan
Published: August 2023

Bispecific T cell engager (BiTE) is one of the most promising therapeutic approaches for treatment of various malignancies. Humanized mice engrafted with human peripheral blood mononuclear cells (PBMCs) have currently become the preclinical platform for evaluating the effectiveness of various cancer immunotherapies including BiTE. However, the wide application of this humanized animal model is limited by a strong xenoreaction of human T cells against the mouse tissues. In this study, we used humanized murine major histocompatibility class I and class II double knock-out NSG mice (referred to DKO) to evaluate the antitumor effect of BiTE. The DKO mice were implanted with human pre-B acute lymphoblastic leukemia cell line (Nalm6). Five days later, the mice were reconstituted with PBMCs. Following CD19xCD3 BiTE treatment whole-body tumor burden was quantified by a in vivo imaging system. Our data demonstrated that the mice developed consistent clinical signs of leukemia-related body weight loss and hind-limb paralysis within 4 weeks. With serial dose of CD19xCD3 BiTE treatment, the tumor cell growing was significantly suppressed. A lower incidence of hind-limb paralysis with prolonged survival were observed in BiTE-treated mice compared with controls. In addition, after treatment with CD19xCD3 BiTE, the humanized mice exhibited elevated serum levels of human IFN-γ, TNF-a, IL-6, IL-10, IL-2, and IL-4. The cytokine production was dose-dependent and associated with the increase of CD19xCD3 BiTE effectiveness. This tumor-bearing humanized model provides a promising preclinical platform for evaluating the efficacy, toxic cytokine release, and dosing requirements of any T cell-based immunotherapies for cancer treatment.
A case for teaching basic immunology through scientific journalism-lesson taught by a Coronavirus went viral!
Authors: Fazal Nadeem , Alsiyaghy Jassam , Eshetie Kirubel , Wilson Kiara D , Newaz Mohammad , Faridi Hafeez , Tauseef Mohammad , Graefen Bahar , Aliyeva Leyla , Najafova Nurana
Published: August 2023

Sudden switch to online mode of learning in the March of 2020 for a Basic Immunology course lead to a cascade of experimenting teaching strategies. We at College of Pharmacy Chicago State University experimented such innovative teaching techniques for two consecutive spring semesters. One such teaching method was to deliver course content material through use of mainstream media (MSM) via scientific journalism. Basic immunology course content was divided into media-inspired topics of public concern and the students were made to research and review selected topics before we discussed those in virtual classrooms. The most common topics included immunology terms (antigen, antibody, immune reaction, innate and adaptive immunity, immunology of infection, ARDS, non-pharmacological interventions (NPI) like lockdown, masking and herd immunity, development of vaccines and therapeutics of infectious diseases. The students researched those topics in daily news outlets, i.e., TV, Radio, Newspaper, Social media (YouTube, Facebook, Twitter, Instagram), and presented to the class the underlying and foundational immunology concepts making the breaking news. Subjective data was collected through student opinion surveys and objective data was analyzed through students’ performance, assessments, grades, and Course and Faculty evaluations. The study found a positive correlation between the use of scientific journalism as a teaching tool and students’ engagement, comprehension, retention and academic outcomes. The publications authored by Scientific Journalists presented the complex and complicated immunology material in a very simple and understandable way for the public, which complemented immunology students’ learning as well.
A Perfect Fit: 3D-Printed Kit to Teach Students Principles of Antigen-Antibody Recognition and Herd Immunity
Authors: Ohashi Sarah N , Hughes Lindsey D , Connolly Kelli , Wang Jennifer , Velazquez Sofia , Tabachnikova Sasha , Kavathas Paula
Published: August 2023

The immunological principles underlying vaccines are a critical part of understanding human health and disease, but are typically not included in general K-12 education curriculum. The COVID-19 pandemic has underscored both the power of science and the importance of science literacy for our country. Though multifactorial, vaccine hesitancy is associated with patients’ lack of information surrounding vaccine biology. Recognizing this gap, we aimed to develop innovative learning activities that would distill the complex concepts of adaptive immunity and herd immunity to make them accessible to middle and high school students.

Herein, we present one such learning activity intended to help students visualize the structural features of an antibody and the specificity of antibody-antigen interactions. Each student is provided with a base 3D-printed antibody, along with three sets of exchangeable variable domains and three model pathogens. Students explore the ability of their antibody combinations to recognize different pathogens, learning the structure and specificity of the antibody and the role they play in fighting pathogens. The set of kits for a classroom are distinct, with kits sharing some, but not all, variable regions. This feature allows for modeling of herd immunity and how different infections spread through a community depending on vaccination rates. This tangible model therefore can convey to students how vaccines elicit protection against pathogens at both an individual and population level. Inclusion of this learning activity during STEM outreach events or integration into classroom curriculum provides an opportunity for early educational intervention that would promote vaccine literacy in our communities.
The Use and Creation of Analogies as a Teaching Tool for Understanding Immunology
Authors: Bernard Aimee Pugh , Spencer Brady L
Published: August 2023

Utilizing existing knowledge of the familiar and known can build associations toward learning, understanding, retaining, and communicating complex concepts. The use of analogies while teaching complex concepts allows the unknown to become familiar and act as an aid toward deeper understanding. Analogies were used as a teaching tool during an immunology course for dental, medical, physician assistant, and graduate students to explain complex concepts. Analogies were used to communicate and enhance the learning and retention of a wide variety of complex concepts and included, but was not limited to, those related to B cell development and activation and the modifications (i.e. somatic hypermutation, affinity maturation, isotype switching) within the germinal center reaction. Analogies were also used to provide students with a teaching tool that they could imitate to create analogies to enhance their understanding and communication of complex concepts. The impact of learning complex concepts through the use of analogies resulted in self-reported increased confidence in understanding and retention of the material. Students reported that learning from and working with analogies not only enhanced their learning of the content but also provided them with a solid foundation from which to build and augment complex concepts with increased detail and added complexity toward a deeper and more nuanced understanding of complex concepts. The incorporation of analogies into the learning process augments scientific communication skills and acts as a model for effective communication while speaking to future patients about medical conditions and/or associated treatments and the general public about scientific discoveries and advancements.

NA
Simulated lab module to teach vaccine biology: From disease transmission to vaccine efficacy, all in a matter of three lab sessions
Authors: Kucknoor Ashwini S
Published: August 2023

Since the COVID-19 pandemic, it has become very important to teach undergraduate students at all levels, about disease transmission and vaccine biology. Just using lectures and reading assignments on these topics often will not be very effective for the student learning. In resource limited undergraduate teaching environment, it is often not possible to use advanced immunology lab techniques. To combat this problem, a lab-module was incorporated into the undergraduate immunology course. In this module, students will follow the process of vaccine development from the identification of a novel disease through the creation and clinical testing of a vaccine This hands-on module was run over the course of three-week lab period, including important concepts of disease transmission, disease diagnosis, mRNA vaccine development, vaccine efficacy and herd immunity. All the exercises included paper-simulations and simulated samples using sodium hydroxide pellets. Overall concepts of innate and adaptive immunity, and detailed concepts like antigenic epitopes, primary and secondary immune responses, and booster doses are emphasized. Students also learn to design mRNA vaccines using gene sequences of Coronavirus spike protein. They also learn principles of ELISA and phases of clinical trial of a novel vaccine. A survey was conducted at the end of the module to evaluate the student learning and student responses to this module. 90% of the students in class were very pleased with this module and stated that this module helped them consolidate their understanding of vaccines. This module can also be modified to be used in a molecular biology or upper-level microbiology courses, with minimum resources.

None
Meta-Lecture: The educational experience of reversed roles
Authors: Ghittoni Raffaella S
Published: August 2023

According to Bloom’s taxonomy classification create, meant as the production of new and original work, represents the highest and most advanced level of cognitive domain in the learning process. Through creative expression students are encouraged to demonstrate their knowledge by combining elements together and by building something coherent, tangible or conceptual.

Meta-Lecture is intended as a non-traditional method to assess mastery of course concepts incorporating short lectures prepared by students, individually, in pair or in a small group, on selected topic, with emphasis on application of course content. This pedagogical approach in teaching is suitable for undergraduate introductory courses as well as for more advanced courses. It represents a student engagement method in both face-to-face and hybrid environment, that aims to improve student commitment and class participation.

Through this alternative method, by simulating a professional environment such as public speaking, poster and seminar scientific presentation, students benefit from the experience of reversed role and from instructor’s and audience feedback on the individual evaluation.

This report provides introduction to the Meta-Lecture assignment, guidance on the feedback analysis and discussion on the learning process.
Trick or Treat-Use of ChatGPT in Education
Authors: Fazal Nadeem , Qasmieh Samah , Graefen Bahar
Published: August 2023

ChatGPT, a free and easy-to-use AI search tool has put Educators scrambling to preserve learning. An education technology company says ChatGPT can be thought of as a “virtual best friend”. ChatGPT launched last November as part of a larger set of technologies developed by the San Francisco-based company OpenAI is part of a new generation of AI systems that can have a discussion and create written work. Millions of people have played with the tool over the past month. Many school systems in the U.S. are still deciding how to set policies on the use of AI programs and how they can be used. Its launch came with little guidance for how to use it. But there is no stopping a student from using ChatGPT from home or on a personal device. We planned to work with both educators and students to learn how people are experimenting with ChatGPT in the real world. Academics are raising tough questions about the future of AI in education. We concluded ChatGPT became so popular quickly across the globe because it is one of the first AI technologies of its kind to be available to the public in a way the public can understand it. We surveyed a global educator and student population about ChatGPT. We report that out of 100 respondents, 85% know about it, 80% are keen to use in education, 80% are still trying to sign up but report high internet traffic to hinder their registration. Finally, younger generation are more willing to experiment this tool, when it becomes available. Nevertheless, it was a talk of first day of spring semester. An OpenAI spokesperson said the company is already working with educators to address their concerns about ChatGPT. We conclude that using such a tool doesn’t build critical-thinking and problem-solving skills which are essential for academic and lifelong success.

Fulbright Association and CTRE Chicago State University, Chicago, IL
Enhancing immunology based health sciences education for addressing health inequities - in the context of COVID-19
Authors: Sika-Paotonu Dianne
Published: August 2023

Māori and Pacific peoples in Aotearoa/New Zealand and the Pacific Region, remain disproportionately and inequitably impacted and affected by a number of adverse health conditions. The global COVID-19 pandemic exacerbated pre-existing inequities in health for different groups, including Māori and Pacific peoples. The consideration of addressing health inequities for Pacific peoples in the context of immunologically relevant health conditions, was explored in the online learning setting for the health sciences students. This work employed use of a Pacific method and means of communication and dialogue, known as Talanoa, that was adapted for the online learning setting as a result of the COVID-19 pandemic. The Talanoa approach was utilised as a tool for students to try and gain better understanding of how to engage, communicate and tackle health inequities appropriately and effectively for Pacific peoples. Undergraduate health sciences students received instruction into the Talanoa approach for communication within the Pacific setting. Online opportunities for presentations and discussion pertaining to developing appropriate solutions to address health inequities for Pacific peoples were facilitated. Students were able to adopt and apply elements of Talanoa in their presentations, discussions and interactions in the online setting. This work highlights the importance of the inclusion of Talanoa, as a means for enhancing online immunology based health sciences education for addressing health inequities appropriately in the context of COVID-19.

Wellington School of Medicine & Health Sciences, University of Otago.
The appropriate application and utilisation of a Pacific model of health and wellbeing for immunology based health sciences education
Authors: Sika-Paotonu Dianne , Taramai Aliesha , Teumohenga Roneima , Tagi Selafi
Published: August 2023

The clinical relevance of immunology in the education setting can be demonstrated by drawing upon examples of specific health conditions affecting populations, but these must be contextualised appropriately. Cancer is a major cause of morbidity and mortality worldwide, and remains a significant health issue that continues to disproportionately impact and affect Pacific peoples in Aotearoa/New Zealand and the Pacific Region. Pacific models of health, clearly and succinctly illustrate key elements important for the health and well being of Pacific communities. Utilisation of these models of health are helpful tools when seeking to address health inequities. This work explored the utilisation of the Fonofale Pacific model of health, to support better understanding of cancer related issues of relevance to Pacific peoples, with immunologically based principles and concepts applicable to the health sciences. This model was also applied as a framework to support better understanding of how to address health inequities appropriately and effectively for Pacific peoples. Undergraduate health sciences students demonstrated understanding and ability in applying and utilising the Fonofale model when developing more appropriate responses for addressing health inequities for Pacific peoples. Students also demonstrated active engagement and participation in the associated activities, with strong interest expressed by some in further work through subsequent research efforts. This work highlighted the importance of the appropriate utilisation and application of a Pacific model of health and wellbeing for immunology based health sciences education and when seeking to reduce health inequities for vulnerable communities.

Wellington School of Medicine & Health Sciences, University of Otago
PI3Kdelta coordinates transcriptional, epigenetic and metabolic changes to promote effector CD8 T cells differentiation
Authors: Cannons Jennifer Leigh , Pichler Andrea C , Villarino Alejandro , Kapnick Senta , Shih Han-Yu , Uzel Gulbu , Su Helen , McGuire Peter , O’Shea John J , McGavern Dorian B , Schwartzberg Pamela
Published: August 2023

Activated PI3K-delta syndrome (APDS) is a primary immunodeficiency caused by heterozygous activating mutations of PIK3CD, resulting in dysregulated immunity, recurrent infections and lymphoproliferation, yet underlying mechanisms behind these phenotypes remain unclear. Using patient samples and Pik3cdE1020K/+ mouse model, we evaluated CD8 cell function both in vitro and in response to infectious agents, assessing cellular phenotypes, metabolism, gene expression and chromatin organization. Pik3cdE1020K/+ CD8 cells exhibited accelerated differentiation to short-lived effectors, associated with increased mTORC1 and c-Myc pathways, as well as altered metabolic, transcriptional and epigenetic circuits characterized by a pronounced IL-2/STAT5 signature associated with heightened IL-2 responses that prevented IL-15 differentiation to memory-like cells. Moreover, Pik3cdE1020K/+ CD8 cells failed to sustain expression of proteins critical for maintenance of long-lived memory cells, including TCF1, and mounted inadequate central memory responses in vivo with enhanced generation of long-lived effector populations. In response to chronic LCMV Clone 13 infection, Pik3cdE1020K/+ mice rapidly die, yet the surviving mice recover more rapidly than WT counterparts. Pik3cdE1020K/+ mice fail to sustain TCF1+ Ly108+ stem-like CD8 cells, with a simultaneous reduction in exhausted cells. However, Pik3cdE1020K/+ mice also have an expansion of CX3CR1+ effector cells associated with increased viral clearance and increased immunopathology. Our data position PI3Kd as a central hub integrating multiple signaling nodes that promote an accelerated effector T cell program during both acute and chronic infections.

This work was supported by the Intramural Research Program of NIAID and NHGRI, NIH.
Patients who develop insulin resistance 4-months post-COVID-19 exhibited higher NETosis and miR-21-5p expression than COVID-19 patients without metabolic alterations
Authors: Cabrera-Garcia Camilo Daniel , Quiroga Romina Andrea , Novoa Sergio Andrés Sanhueza , Cáceres Bárbara Evelyn Antilef , Grez Camila Paz Muñoz , Fraga Marco , Llerena Faryd , Vidal Mabel , Henriquez Mario , Labarca Gonzalo , Garcia-Hidalgo Maria C , Barbé Ferran , De Gonzalo-Calvo David , Verdugo Ricardo , Nova-Lamperti Estefania , Fernandez Liliana Lampert
Published: August 2023

Post COVID-19 has been associated with alterations in the glucidic metabolism and the presence of metabolic-related microRNAs (miRNAs). Since neutrophils response depends on glycolysis, it is unclear whether the formation of neutrophil extracellular traps (NETosis) is also affected in post COVID-19 patients that developed insulin resistance (IR) post infection. In addition, miR-21-5p has been described as a direct gene regulator of the innate immune response against COVID-19 and the presence of IR. In this study, we evaluated NETosis at 4-months post-COVID-19 and its association with the development of IR and the expression of circulating miR-21-5p. Sixty post-COVID-19 patients were recruited from Víctor-Rios-Ruiz Hospital, and post COVID-19 was evaluated 4-months after acute phase. Serum samples were used to determine biochemical parameters. NETosis was measured in patients at basal condition and in response to TLR agonists using flow cytometry. miR-21-5p expression was measured with RT-qPCR. From our cohort, 19 patients were excluded because of they had IR or type 2 diabetes mellitus before COVID-19, 25 patients developed new IR and 16 remained without glucidic alteration. Our analysis showed that COVID-19 patients with IR-Post-COVID-19 had a significant increment of NETosis in basal condition, reducing the capacity to respond efficiently to TRL3/7/8 agonists. We also found a significant increment of miR-21-5p expression in IR-Post-COVID-19 patients and a significant and positive correlation between miR-21-5p and vital NETosis. These results demonstrated that patients who developed IR post-COVID-19 exhibited higher basal NETosis and higher miR-21-5p expression in comparison with patients without alterations.

Supported by grants from PROYECTO COVID1005, ACT210085, Fondecyt Regular 1211480, Instituto de Salud Carlos III (COV20/00110, CP20/00041), Programa de donaciones “estar preparados” UNESPA (Madrid, Spain), Fundación Francisco Soria Melguizo (Madrid, Spain).
Immune Regulatory Markers in Clinical Cases of COVID-19
Authors: Madiraju Charitha , Mallarpu Chandra Shekar , Chelluri Srinivasa Ikswaja , Singarapu Maneendra , Krishna Tapaswi , Chelluri Lakshmi Kiran
Published: August 2023

The objective of this research study is to profile anti-viral and pro-inflammatory cytokines, major histocompatibility complex (MHC) class I & II molecules and cell death markers in clinical cases of sepsis. Research study design included three patient cohorts namely, COVID-19 with sepsis (C19wSepsis, N=17), COVID-19 with no sepsis (C19NoSepsis, N=17) and sepsis alone (N=24). Healthy subjects served as controls (N=16). Serum samples derived from patient cohorts and control subjects were analyzed by ELISA for anti-viral and pro-inflammatory cytokines (IFN-gamma and IL-1beta), MHC class I (MHCA) & MHC class II (MHCDRb1) molecules and cell death markers. Data and statistical analyses were performed using GraphPad Prism 7.0. ELISA of serum samples identified a significant increase in serum IFN-gamma, MHCA and MHCDRb1 levels in C19wSepsis cohort compared to C19NoSepsis cohort and control subjects [p < 0.0001]. Although there was a significant increase in IL-1beta levels in serum samples of C19wSepsis compared to control subjects and sepsis alone cohort [p < 0.0005], the levels were comparable between C19wSepsis and C19NoSepsis cohorts [p >= 0.05]. Cell death markers, Caspase-3 and p62/SQSTM1, showed significantly altered levels between the three patient cohorts when compared to controls [p < 0.01]. Representative data from flow cytometry confirm ELISA results. Results from this study identify key immune regulatory markers in clinical cases of COVID-19.
Delayed CD4+ T-cell response in older adults is associated with reduced immunogenicity and reactogenicity after COVID-19 mRNA vaccination
Authors: Jo Norihide , Hidaka Yu , Kikuchi Osamu , Fukahori Masaru , Sawada Takeshi , Aoki Masahiko , Yamamoto Masaki , Nagao Miki , Morita Satoshi , Nakajima Takako E , Muto Manabu , Hamazaki Yoko
Published: August 2023

While the newly developed COVID-19 mRNA vaccines are highly effective, characteristics of the immune response after the vaccination and its association with the adverse effects, especially for an older population, remain elusive. In this study, we recruited a total of 216 SARS-CoV-2-naïve healthy donors of various ages (23–81 years) who received two doses of the BNT162b2 vaccine and assessed age-associated differences in vaccine-induced T-cell responses and adverse effects over 3 months. We observed that older adults (≥65 years) had fewer vaccine-induced Spike-specific CD4+ T cells, including CXCR3+ cTfh and Th1 cells, after the first dose, which correlated with their lower peak IgG levels and fewer systemic adverse effects after the second dose, compared with younger adults. Moreover, Spike-specific Th1 cells in older adults expressed higher levels of PD-1, a negative regulator of T-cell activation, which was associated with low Spike-specific CD8+ T-cell response. These data indicated that delayed induction of CD4+ T-cell response in older adults is linked to impaired humoral and cellular immunity and reduced systemic reactogenicity. Therefore, enhancing CD4+ T-cell response following the first dose is key to improving vaccine efficacy in older adults.

This work was supported by the Japan Agency for Medical Research and Development (grant numbers JP21gm5010005, JP20fk0108454, and JP223fa627009; Y.H.), iPS Cell Research Fund (Y.H.), the COVID-19 Private Fund to the Shinya Yamanaka Laboratory, CiRA, Kyoto University (Y.H.), Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT)/Japan Society for the Promotion of Science (grant number 19K23862; N.J.), Kansai Economic Federation (KANKEIREN; Y.H.), Sumitomo-Mitsui Trust Bank Fund for Covid-19 Research, Kyoto University (Y.H.), and Takeda Science Foundation (Y.H.).
Paradoxically Increased Inflammation in Immunosuppressed Individuals with COVID-19
Authors: Moore-Stanley James Oscar , Knight Sean Blandin , Bakerly Nawar , Kirkham Sara , Williams Thomas , Hussell Tracy
Published: August 2023

Dysregulated inflammation is central to the morbidity and mortality associated with COVID-19. Treatment with dexamethasone and IL-6 blockade can be life-saving in patients stratified for moderate - severe disease. Patients with suppressed immunity are often excluded from immune blockade therapy due to the assumption that additional suppression of an impaired immune system will be detrimental to viral clearance. We hypothesised that patients with suppressed immune systems would be slower to clear viral infection, leading to increased damage and therefore, paradoxically, a more intense acute phase response to SARS-CoV-2 infection. This was tested by a sub-group analysis of the Coronavirus Immune Response and Clinical Outcomes (CIRCO) cohort, an observational study of acute COVID-19 in Greater Manchester, UK. Patients were included if they were treated with dexamethasone and had a research blood sample retrieved within 48 hours of admission, and excluded if they were treated with IL-6 blockade or antiviral therapy prior to research sampling. Acute phase serum cytokine levels were compared between eight immunosuppressed and 12 immunocompetent patients positive for SARS-CoV-2. In support of our hypothesis, we found that immunosuppressed individuals had higher levels of the inflammatory cytokines IP-10 (p=0.03) and MCP-1 (p=0.01) compared to immunocompetent patients matched for COVID-19 severity. This suggests that immunosuppressed patients may benefit as much as immunocompetent individuals from systemic treatments to dampen inflammation in COVID-19, and current recommendations of excluding these patients from treatment solely on the basis of immune competence may need to be re-evaluated.
Integrated transcriptomic analysis reveals T and B cell immune signatures influencing resolving outcome in MRSA bacteremia
Authors: Parmar Rajesh , Pickering Harry C , Gjertson David W , Chan Liana C , Yeaman Michael R , Reed Elaine F
Published: August 2023

Staphylococcus aureus bacteremia is a potentially lethal infection particularly when involving methicillin-resistant S. aureus (MRSA). Contrary to MRSA bacteremia that resolves on its own (RB), persistent MRSA bacteremia (PB) happens when the infection persists after receiving antibiotic therapy. Host immune responses in PB vs. RB are poorly understood. Our goal was to identify proteomic, epigenetic, and transcriptomic features associated with PB and RB clinical outcomes. We examined the whole-blood transcriptomes of propensity score matched PB (n = 28) and RB (n = 30) patients. Gene expression (GE) modules were analysed by weighted gene co-expression network analysis (WGCNA) and quantified by module eigengene analysis. Enriched pathways and protein-protein interaction networks were identified. The correlation between host DNA methlytransferase-3A (DNMT3A) genotype and serum IL-10 level was examined in the identified GE modules. Upregulation of T and B cell immune response genes early during MRSA infection discriminate RB from PB patients. T and B cell signalling pathways were significantly increased in RB patients and correlated with lower serum IL-10 levels and the DNMT3A heterozygous A/C genotype. These findings indicate that patients with DNMT3A A/A genotypes have dysregulated adaptive immune responses characterized by impaired T and B cell function that increase the risk of PB outcome. These findings suggest that combining the genotype of the host with transcriptome and proteomic profiles might help determine the outcome of MRSA bacteremia. This knowledge may result in a better understanding of S. aureus pathogenesis and development of new anti-infective therapies and vaccines to improve patient outcomes.

These studies were supported by in-part by the following NIH grants U01-AI124319, R01-AI068804 and R33-AI111661(to MRY) and U01-AI124319 and U19AL128913 (to EFR).
The immunodominant SARS-COV-2 CD8+ T cell repertoire is capable of resisting antigenic drift
Authors: Malone Michael J , Guo Yuwan , Su Laura , Jiang Jenny
Published: August 2023

Immunodominance (ID) is a common phenomenon in infection, vaccination, and cancer where a small set of many possible epitopes are targeted by the immune system. Although favorable in many contexts, ID is inherently susceptible to immune escape. Given that a major concern of COVID-19 has been the possibility of emerging variants escaping immune detection, we sought to investigate the extent of ID in T cells and their ability to target SARS-CoV-2 variants after immune challenge. To do so, we probed T cells from vaccinated and infected donors with a library of DNA-barcoded pMHC-tetramers containing epitopes from conserved, wild-type, and variant spike. After single cell sequencing, we recalled each cell’s T cell receptor (TCR) sequence and its cognate specificity, finding a striking ID to few unmutated epitopes. Interestingly, despite low antigenic diversity, many donors had both clonal and diverse repertoires toward the dominant epitopes. We suspected that integration of these diverse repertoires may allow an individual to reap the metabolic benefits of ID, while simultaneously countering drifting antigens during infections and cancers. We thus transduced the ID repertoires of several donors into cell lines. We will next evaluate their tolerance to mutagenesis by probing them with libraries of DNA-barcoded pMHC-tetramers containing all possible single amino acid point mutations of their original cognate ID epitopes. Such analyses will provide deep insight into the antigen binding properties each TCR, as well as how their cooperation at the individual and population levels could resist immune escape. Application of this approach to other infections and cancers could reveal novel avenues for vaccines and cell-based therapies in cancer.

Supported by grants from NIH (U19 AI128949) and the Chan Zuckerberg Initiative Foundation (CZIF)
High Mobility Group Box 1: A Predictive Biomarker of Aberrant Humoral Responses Implicated in Post-Acute Sequelae of SARS-CoV-2 Infection
Authors: Rubas Noelle C , Peres-David Rafael Aniu , Kunihiro Braden P , Phankitnirundorn Krit , McCraken Trevor , Umeda Lesley , Conching Andie , Maunakea Alika K
Published: August 2023

Postacute sequelae of SARS-CoV-2 infection (PASC) is the next public health concern of the pervasive COVID-19 pandemic. Molecular pathophysiology of PASC is undetermined, therefore this study evaluated pathways implicated in humoral responses associated with symptom resolution to identify predictive biomarkers of PASC. Individuals recovered from acute infection were recruited into a 6-week study. Initial health questionnaires and proof of previous SARS-CoV-2 infection were collected prior to study enrollment. Body mass indices were calculated with participant height and weight measurements. Blood samples were collected and isolated for plasma and PBMCs to evaluate a panel of immunological biomarkers and cellular profiling, respectively. A singular stool sample was collected for microbial DNA extraction for metagenomic sequencing. Data was integrated with IPA analysis to establish modulated pathways in PASC. Individuals suffering from symptoms of PASC showed evidence of poor immune responses with diminished rates of symptom resolution. Particularly prevalent in obese individuals, PASC associated with attenuated antibody responses and inability to maintain viral inhibition capacities, partially explained by elevated inflammation and inflammatory cellular landscapes. Individuals with worse humoral responses tended to display characteristics of a compromised gut microbiome, suggesting dysregulation of the immunoepigenetic-microbiome axis. Circulating high mobility group box 1 was shown to be a central mediator of this axis, with elevated levels conducive of poor adaptive immunity. Additionally, mediation analysis revealed baseline levels were predictive of viral inhibition at the time of final follow-up.

This study was supported in part by funding provided by Hawaii Community Foundation under award 20HCF-101573. The comments expressed in this report are the sole responsibility of the authors and do not represent the official view of the Hawaii Community Foundation.
Type I interferons and TGF-β cooperate to induce liver fibrosis during HIV-1 infection under antiretroviral therapy
Authors: Ahodantin James , Nio Kouki , Funaki Masaya , Zhai Xuguang , Wilson Eleanor , Kottilil Shyamasundaran , Cheng Liang , Li Guangming , Su Lishan
Published: August 2023

Liver disease has become a major comorbidity health concern in people living with HIV-1 (PLWH) receiving antiretroviral therapy (ART). To investigate the immune mechanism of liver disease development in ART-treated HIV-infected individuals, we developed humanized mice, NRG mice reconstituted with progenitor cells from human fetal livers, with HIV-1 infection and ART. Animals w/o HIV-1 were treated with TRUVADA (Emtricitabine/ Tenofovir Disoproxil Fumarate) in combination with Raltegravir. Hepatic stellate cells (HepSC) isolated from the liver of healthy donors were used to assess the effect of IFN-I and its potential cooperation with TGF-β in HepSC activation. Finally, identified cellular and molecular mediators of liver disease were analyzed in bloods and liver biopsies from PLWH with ART. We report here that chronic HIV-1 infection with ART induced hepatitis and liver fibrosis in humanized mice, associated with accumulation of M2-like macrophages (M2LMs), elevated TGF-β, and IFN signaling in the liver. Interestingly, IFN-I and TGF-β cooperatively activated HepSCs in vitro. Mechanistically, IFN-I enhanced TGF-β-induced SMAD2/3 activation in HepSCs. Finally, blockade of IFN-I signaling reversed HIV/cART-induced liver diseases in humanized mice. Consistently, we detected elevated markers of liver injury, M2LMs, and of IFN signaling in blood specimens and liver biopsies from PLWH compared with those of healthy individuals. These findings identify the IFN-I/M2LM/HepSC axis in HIV/ART-induced liver diseases and suggest that inhibiting IFN-I signaling or M2LM may provide a novel therapeutic strategy for treating HIV/ART-associated liver diseases in PLWH treated with ART.

This study was supported in part by NIH grants AI127346, AI095097, and DK095962 to Prof. Lishan Su
Immunophenotyping of peripheral blood mononuclear cells in tuberous sclerosis complex
Authors: Balthazard Renaud , Drouin-Engler Rose-Marie , Klement Wendy , Daigneault Audrey , Florès Alina Sreng , Kaufmann Daniel E , Keezer Mark , Larochelle Catherine
Published: August 2023

Tuberous sclerosis complex (TSC) is a multisystem disorder caused by loss-of-function mutations in TSC1/2 proteins. This results in constitutive mTOR activation and predisposes to the development of benign tumours, with renal angiomyolipomas (AMLs) and tuber-related drug-resistant epilepsy (DRE) being major causes of morbidity and mortality in adults. Despite the central role of mTOR in leukocyte biology and the beneficial impact of mTOR inhibitors in TSC, the role of the immune system and inflammation in the development of tumours and DRE in people with TSC remains unknown.

Using peripheral blood mononuclear cells (PBMCs) and serum from people with TSC, healthy controls (HC) and non-TSC DRE, we employed flow cytometry and multiplex assays to interrogate the immune profile associated with chronic loss of mTOR inhibition.

We found that higher levels of the astroglial activation marker GFAP are elevated in TSC and show high intergenotype variability. We also observed differences in circulating immune cell populations, cytokines, chemokines and growth factors related to TSC. We found a higher proportion of B cells in parallel with increased total IgG but a reduction in IL-2 and IL-7 and a lower frequency of T cells. TSC with vs without AML showed differences in CD4:CD8 T cells ratio, monocytes and dendritic cells frequencies. Finally, loss of TSC1/2 affected T cell polarization, leading to an increased proportion of Tc17 CD8+ T cells, especially in subjects with DRE.

Overall, our findings indicate TSC is associated with increased blood biomarkers for inflammation, glial activation, and distribution and profile of circulating PBMCs compared to controls.