bluuebunny/crossref_metadata_2025 huggingface dataset:
Virtual graduate immunology and immunotherapy education during COVID-19
Authors: Sterner Rosalie , Hedin Karen E
Published: February 2023

The objective was to ascertain the impact of an online, virtual course in immunology and chimeric antigen receptor (CAR)-T cell therapy on trainee’s knowledge and interest in the field. The course occurred May 4–8, 2020. Students taking the course for graduate school credit received a pre-course and post-course survey. The survey included questions covering background, knowledge/confidence in skills, interests, and perceived ability to learn using an online platform. Students worked in teams to develop a research grant specific aims page. A total of 27 students took the course for graduate level credit and were surveyed, and all 27 (100%) returned the surveys. Students came from variable backgrounds and were at different stages of graduate training. After completion of the course, there was a statistically significant increase in self-perceived knowledge of immunotherapy (mean score of 3.7 +/− 1.0 standard deviation post-course vs 2.8 +/− 1.3 pre-course, on a 5 point Likert scale, P=0.0075), knowledge of the translational process (3.8 +/− 0.9 post-course vs 3.2 +/− 1.3 pre-course, P=0.0082), knowledge of the cancer treatment/immunotherapy patient experience (3.5 +/− 1.2 post-course vs 2.4 +/− 1.3 pre-course, P=0.0003), and confidence in immunotherapy grant writing skills (3.7 +/− 0.8 post-course vs 2.7 +/− 1.2 pre-course, P<0.0001). This course can serve as a model to help encourage training in translational research and enhance knowledge and interest in immunology, immunotherapy, CAR-T cell therapy, and regenerative sciences via an online platform in this era of COVID-19 and potentially beyond.
Old is Gold! Old School is Back to a New School of Learning.
Authors: Fazal Nadeem , Brehm Danielle , Jimenez Julianne
Published: February 2023

COVID-19 has challenged the way students' learn and gain knowledge. The purpose of this research project was to investigate how students adapted to remote learning due to COVID-19. We selected a group of undergraduate Biomedical Science students at the University of Birmingham, England with the intent of understanding their new approaches to learning and whether remote teaching discriminated against the less fortunate. Our google form survey received 40 responses. 27 students said they forgot how to learn, with only 15 obtaining new ways of learning. This demonstrated how the adjustment to online teaching detracted from the time and energy students could put into learning. 75% of students did not enjoy university as much as they did pre COVID-19, and 3 students liked no aspects of online teaching. 22 students learnt worse online, while 10 felt no difference. Choosing when to watch lectures improved some students time management, self motivation and discipline. However, lack of externally-imposed routine brought new challenges as students had no time pressure, making it easier to fall behind. Cancelled laboratory work meant students missed out on vital skills. Our data showed that student satisfaction decreased, traditional learning methods were lost, and new ways of learning were hard to attain when transitioning to online teaching. With the prospect of learning returning to what the student’s consider normal, students fear this will accentuate how they have forgotten to learn in the traditional way, posing another disruption to their studies as they readjust to a post COVID world, whenever it comes. We anticipate that lessons learnt and forgotten in a pandemic-induced remote environment will remain a challenge to current students at large.
Instructor attitudes towards digital assessment in undergraduate immunology courses.
Authors: Pearson Todd F , Brayton Kate A , Twitchell Elizabeth A , Bolster Ruth E
Published: February 2023

To be better able to meet the instructional needs of undergraduate immunology instructors, we sought to understand the attitudes of individuals teaching this course. We conducted a survey, inviting 230 randomly selected instructors to participate, of which 38 completed the survey. Our results highlight that while very few instructors (2/38, 5.26%) currently use digital assessment in their immunology course, a strong majority (26/38, 72.22%) indicated that they would use it if it were available with their text. A similar number of instructors (23/38, 63.89%) expressed interest in adaptive quizzing functionality in a digital assessment platform. Additionally, we explored instructor attitudes about the goal of digital assessment. Despite a wide range of responses, over half of the survey respondents identified terminology/vocabulary (19/38, 52.78%) and reading questions that test student understanding of basic concepts (19/38, 52.78%) as important goals of a digital assessment platform. These results highlight that most instructors see digital assessment, especially in a formative setting, as an important instructional modality that can address student learning challenges.
Cell Health Analysis on Guava Muse Analyzer – A Capstone Study
Authors: Chen Thurston , Ghanimifard Bita , Francia Marielle Joy , Gillis Katherine , Salow Lisa , Dickey David , Husarik Aarti , Madiraju Charitha
Published: February 2023

Guava® Muse® Cell Analyzer is a fluorescence detection instrument that performs flow cytometry analysis in an easy-to-use and simple format. The Muse® Cell Analyzer is loaded with immunological and cell health assays optimized using validated kits from the manufacturer. A student-driven research project that is part of a Research & Scholarship (R&S) course series (previously Capstone) incorporated Guava® Muse® Cell Analyzer-based experimental protocols undertaken in collaboration with Luminex Corp. The specific goal is to perform initial characterization of a myeloma cell line for its inherent ability to proliferate and survive. Three proprietary kits designed for cellular assays were helpful in assessing myeloma cell health on Guava®Muse® Cell Analyzer. Students enrolled into the research project worked under the mentorship of a research faculty, reviewed the scientific literature, charted schematics prior to experimentation with the Muse® CellAnalyzer-based protocols for cell death and cell survival, performed quantitative analysis and evaluation of the data. Training on the Muse® Cell Analyzer instrument during R&S course series helped students understand the miniaturized version of the flow cytometry-based technology, evaluate the three Guava®Muse®Cell Analyzer-based immunological methods for cell health assessment, and integrate the knowledge captured during didactic coursework with experimental evaluation of an immune cell type.
COVID-19 affected “face to face” interaction with the professors. Lessons learned from traditional learning.
Authors: Olk Sandra R , Stempek Ryan , Renteria Sahira , Fazal Nadeem
Published: February 2023

Many university programs have implemented/utilized online teaching platforms for various reasons over the last decade. This has allowed for both students and professors to track progress in the course, as well as providing a method to submit assignments over the traditional paper submission. These are just a few of the reasons that online platforms have gained popularity, though many have been caught off-guard by the sudden full transition from traditional/hybrid learning to online learning. Through the use of surveys, we have aimed to study the effects, both positive and negative, that this transition to online learning has had on university students. Our group has pooled our questions into a larger set of survey questions that have been broken into smaller surveys, so that we can encourage a response rate that allows us to maximize our results without having students lose interest in answering all of our questions. In this current research, we have focused on the major loss in the current pandemic induced learning. Our preliminary data suggests that one of the major things that students feel is that they miss face to face interactions with their professors. Since we looked at a cohort of professional pharmacy school students where experiential education is one of the major aspects of training future pharmacists. The students believe that the practical aspect of learning needs supervision, oversight, mentoring, and physical presence of teachers. Most of the students feel that no matter how realistic the internet world of learning seems, it can not compensate the training and practice required in a professional school of pharmacy. We conclude that nothing can compensate or simulate the loss of profound student-teacher interaction.
The integration of Immunology based Pharmacological elements for Health Sciences education
Authors: Sika-Paotonu Dianne
Published: February 2023

Health Sciences education supports learning across multiple discipline areas. Appropriate integration of different subject aspects for students when using particular conditions as in-class teaching examples is important. This work highlights the use of integrated teaching approaches within Health Sciences education to support learning of Immunology based Pharmacology elements relevant to the drug design context for the prevention of Acute Rheumatic Fever (ARF) and Rheumatic Heart Disease (RHD). ARF is the body’s autoimmune reaction to an untreated Group A Streptococcal (GAS) infection of the throat or skin. If a severe, or repeated ARF episodes occur that are left untreated, permanent cardiac damage can occur known as RHD. In the absence of a GAS vaccines, the most effective management plan for ARF/RHD involves painful monthly Benzathine Penicillin G injections for at least 10 years or more. These BPG injections prevent recurrent GAS infections that may trigger ARF and lead to RHD. Adherence to BPG injection regimens remain problematic, and a new Penicillin for ARF/RHD prevention that is less painful and longer lasting, is urgently needed. Local and international research efforts are well underway to support the generation of a new Penicillin for this purpose. When integrated teaching approaches that included online interactive websites were used to support learning of Immunology based Pharmacology elements relevant to the drug design context for ARF/RHD prevention, students demonstrated better in-class engagement, participation and concept understanding, expressing approval and improved confidence with the teaching content.
A novel subset of follicular helper-like MAIT cells has capacity for B cell help and antibody production in the mucosa.
Authors: Jensen Owen , Trivedi Shubhanshi , Meier Jeremy D , Fairfax Keke , Hale Scott , Leung Daniel T
Published: February 2023

Mucosal-associated invariant T (MAIT) cells are innate-like T lymphocytes that respond to conserved microbial metabolites presented on the evolutionarily conserved MHC class I-related protein, MR1. MAIT cells are enriched in the mucosa and aid in protection against bacterial pathogens via release of inflammatory cytokines and cytotoxic molecules. Recent evidence suggests MAIT cells are capable of providing B cell help in vitro and in vivo. In this study, we used primary human samples and mouse models to uncover the role of MAIT cells in B cell help at the mucosa. Here we describe a novel population of CXCR5+ T follicular helper (Tfh)-like MAIT cells (MAITfh) that are enriched in human mucosal lymphoid tissues. Using in vitro culture assays and fluorescence imaging, we found that MAITfh cells preferentially locate within germinal centers in human tonsils and express the canonical Tfh transcription factor, B-cell lymphoma 6 (BCL-6), co-stimulatory markers, inducible T cell costimulator (ICOS), and programmed death receptor 1 (PD-1), and cytokines, interleukin (IL)-21, IL-10, and BAFF. Furthermore, we investigate the sufficiency of MAIT cells to aid in antibody mediated immunity in vivo using a mouse model of MAIT adoptive transfer followed by mucosal challenge with live Vibrio cholerae. We show that adoptive transfer of MAIT cells into αβ T cell deficient mice promotes B cell differentiation and increased serum V. cholerae-specific IgA and bactericidal responses. Our data demonstrate the capacity of MAIT cells to participate in adaptive immune responses at the mucosa, and suggest MAIT cells may be potential targets to improve mucosal vaccines.
Origin of human uterine natural killer cells
Authors: Porrett Paige , Gonzalez Michael , Garifallou James , Smiler Jacqueline , Mafra Fernanda , Silva Renata Pellegrino Da , Johnston Sarah Addie , Vieyra Mark , Testa Giuliano , Johannesson Liza , O‘Neill Kathleen
Published: February 2023

Uterine NK cells (uNK) are a distinct immune population which influence pregnancy outcome. While murine studies have suggested that tissue-resident uNKs predominate in virgin endometrium, the origin of uNKs in humans is unknown. To define subsets of uterine NKs in humans and determine their developmental origin, we performed single-cell RNA-sequencing on CD56+ uNKs sorted from endometrial biopsies of 4 healthy controls (HCs) (17,146 cells) and 5 uterus transplant (UTx) recipients (30,351 cells). SNP polymorphisms and HLA alleles were used to identify cells of donor (tissue-resident) or recipient (peripheral) origin in UTx recipients. Developmental trajectories were built with Monocle v.3.0. Ten major clusters were identified in HCs, with 48% of endometrial NK cells (eNKs) occupying 3 clusters (eNK1–3) that shared gene signatures with known human decidual NK cell subsets (dNK1–3). Pseudotime analysis revealed that the eNK1–3 clusters arose from proliferating subsets and were less mature than cytotoxic uNKs expressing ZEB2, TBX21, and FCGR3A (CD16). uNKs from UTx recipients were similarly distributed across the clusters, with a trend towards fewer cells in the FCGR3A-expressing mature subset. Notably, recipient-derived uNKs dominated all uNK subsets within four months post-transplant but became less frequent in UTx recipients >1 year after transplant (95% vs. 72%; early vs. late). The proportion of donor-derived to recipient-derived cells remained constant across subsets in all UTx recipients. In contrast to mouse models, our study suggests that human uNKs derive primarily from blood-borne peripheral immigrants and that environmental factors influence uNK differentiation more than genotype or developmental origin.
Spatial Map of Human B Cell and Plasma Cell Compartments
Authors: Liu Ailing , Rothrauff Abigail Elizabeth , Borghesi Lisa A
Published: February 2023

Naive B cells differentiate into antibody secreting plasma cells (PC) and memory B cells (MBC) that together dictate the quality and persistence of the humoral immune response. However, a comprehensive analysis of human B lineage population structure that integrates surface phenotype and intracellular functional status is lacking. One reason for this knowledge gap is the difficulty of obtaining diverse tissues from normal individuals. Here we have performed a comprehensive analysis of human lymphoid tissue from relatively inaccessible sites including SPL, BM and LN to visualize B cell organization across diverse tissues from normal donors. We exploited a novel algorithmic pipeline for unbiased analysis. Further, we are aligning findings across mouse and man. Here we report the identification of select markers that are involved in a shared BM precursor/PC program including CD28, CLEC12A, CCR2, CD7 and CD79b by LEGENDScreening. We have validated these markers by RNA-seq analysis of murine splenic PC and conventional flow cytometry analysis of human gut PC. We hypothesize that long-lived PCs returning to BM extinguish the B cell program and re-engage with the ancestral long-lived program. Overall, our findings constitute the basis for a geographic understanding of human B lineage population structure that will inform studies on the human humoral immune response and the mechanisms of PC longevity.
Easy isolation of F4/80 positive macrophages from mouse tissues
Authors: Antignano Frann , Poon Grace , Liang Alice , Eaves Allen , Louis Sharon , Kokaji Andy
Published: February 2023

Macrophages are important and also have critical roles in the development and homeostasis of tissues and organs. Our understanding of macrophages is constantly evolving and expanding, as they are actively studied in many areas of research, including infectious diseases, wound healing, and tumor immunology. Adding to the complexity of macrophage research, subsets of macrophages can be identified throughout the body with diverse phenotypes and functions. Furthermore, macrophage frequency can be highly variable across different tissues, presenting a challenge to obtain highly pure macrophages. To address these challenges, we have developed a simple method to isolate macrophages by targeting F4/80, a well-established murine macrophage marker. Starting with a single-cell suspension from mouse peritoneal cavity, lung, or spleen, F4/80-positive cells were labeled with an antibody complex that links F4/80-positive cells to magnetic particles, then separated using an EasySep™ magnet. Using this method, F4/80-positive cells were enriched from 37.3 ± 9.3% to 94.4 ± 2.9% (mean ± SD; n = 12) from peritoneal lavage fluids, 26.5 ± 2.7% to 94.3 ± 2.8 % (n = 9) from lungs, and 8.0 ± 2.4% to 88.8 ± 3.4% (n = 18) from spleens. Protocols have been optimized to accommodate a range of sample sizes from the various tissue sources. EasySep™-isolated F4/80-positive macrophages are functional, as demonstrated by their ability to uptake FITC-dextran and secrete inflammatory mediators upon activation. Overall, the EasySep™ Mouse F4/80 Positive Selection Kit enables simple and easy isolation of F4/80-positive macrophages in under 25 minutes, streamlining the experimental workflows for researchers studying macrophage biology.
Androgens regulate sex differences in lung-resident ILC2 functional responses during influenza virus infection
Authors: Kadel Sapana , Turner Sean , Karlik Anna , Miller Reegan , Ainsua-Enrich Erola , Kovats Susan
Published: February 2023

Lung-resident group 2 innate lymphoid cells (ILC2s) produce type 2 cytokines and facilitate tissue repair in response to alarmins such as IL-33 released during respiratory virus infection, but they also may be functionally suppressed by type 1 cytokines. Lung ILC2s express notably high levels of intracellular androgen receptor (AR) compared to ILC1s, T and B cells. To test the hypothesis that females and males show differential ILC2 responses in influenza virus (IAV) infection, we analyzed the numbers and functional responses of lung ILCs in IAV infected mice. On days 3–10 post-infection, lungs of female mice contained greater numbers of ILC2s and ILC1s compared to males. However, the female ILC2s were preferentially suppressed at the peak of infection, with a dampened type 2 program manifest as attenuated proliferation, decreased IL-5 and amphiregulin production, reduced expression of GATA-3 and IL-33R and increased surface IFNGR. IFNG levels in the lung were comparable between sexes at day 7 post-infection, suggesting intrinsic differences in ILC2 responses to interferons. Indeed, naïve female ILC2s showed higher expression of IFNGR and higher phospho-STAT1 levels following stimulation by IFNG. Early life orchiectomy revealed that endogenous androgens decreased ILC2 numbers but protected males from suppression of ILC2s in IAV infection. Furthermore, single cell RNA-sequencing experiments revealed that female ILC2s were enriched in a “suppressed ILC2” cluster while male ILC2s were enriched in a “proliferating ILC2” cluster on day 7 post-infection. Taken together, our data show that AR activity preserves canonical ILC2 function in males during IAV infection and may help to explain known gender differences in immunity to IAV.
Histotripsy ablation stimulates the innate immune system and modulates anti-tumor immunity in pancreatic cancer
Authors: Hendricks Alissa Danielle , Sereno Jaqueline , Gannon Jessica , Zeher Allison , Brock Rebecca M. , Betiel-White Natalie , Simon Alexander , Davalos Rafael V. , Coutermarsh-Ott Sheryl L , Vlaisavljevich Eli , Allen Irving C
Published: February 2023

With an overall 5-year survival rate of less than 9%, pancreatic cancer is a substantial cause of cancer related death in the United States. This high mortality has been associated with the immunosuppressive tumor microenvironment (TME) and the shortage of effective late-stage therapeutic options. Histotripsy is a non-thermal, non-ionizing, non-invasive cancer ablation modality that rapidly lyses targeted cells. To investigate how histotripsy can change the pancreatic TME, a Pan02 cell culture model of pancreatic adenocarcinoma was employed to determine the neoantigen release following histotripsy treatment and other standard ablation modalities. Histotripsy was found to release potential neoantigens at a level similar to other non-thermal ablations and superior to thermal ablation, which paralleled the increased innate immune system activation in vivo. For in vivo studies, subcutaneous Pan02 tumors in mice were treated with histotripsy. To monitor changes in the TME after treatment, rtPCR and flow cytometry were used and compared to untreated animals. Progression-free and overall survival were significantly improved with histotripsy treatment. Within 24 hours of treatment there was increased activation of the innate immune system and by 14 days there were decreased tumor-associated immune cell populations. This work illustrates the feasibility of using histotripsy to treat pancreatic cancer and shift the TME as well as providing mechanistic understanding into the activation of the innate immune system post-treatment. Future studies are needed to establish the mechanism of immunomodulation of the TME and the extent of potential systemic effects.
Effects of glucose concentration in the tumor microenvironment on Non Small Lung Cancer (NSCLC) Progression
Authors: Gähler Alexander , Hohenberger Katja , Tausche Patrick , Chiriac Mircea T. , Rauh Manfred , Trufa Denis I , Geppert Carol-Immanuel , Rieker Ralf J , Sirbu Horia , Finotto Susetta
Published: February 2023

Lung cancer is one of the most common cancer types in the western world, with a high lethality and increasing incidence. One major factor, supporting tumor growth is a high sugar diet. Thus, reducing the sugar intake by different diets might be a way to improve current cancer treatment. These metabolic changes are accompanied by regulation of systemic levels of Insulin and insulin like growth factors. As oppose to normal cells, tumor cells metabolism mainly depends on glycolysis. Thus, reducing diet carbohydrate intake might be a way to improve current immunotherapy by inducing immunological changes caused by the extracellular glucose concentration in tumor microenvironment.

Therefore, we cultured the human lung adenocarcinoma (ADC) cell line A549 with different extracellular glucose concentrations to analyze effects by flow cytometry and qPCR. Moreover, lung tissue samples from patients with non small cell lung cancer (NSCLC), were analyzed by qPCR. In a murine model of lung ADC, tumor cells were injected intravenously and mice were fed with different diets. Lung cells were isolated and analyzed by flow cytometry.

Here we show that, IGF1R expression is upregulated in A549 cells line, depending on increasing extracellular glucose concentration. Moreover, in studies on human cohorts of patients with NSCLC, we found a direct correlation between BMI and IGF1R expression and its downstream signaling. Finally, in a murine model of disease, diet glucose restriction decreased significantly tumor load associated with increased lung infiltrating CD8+ T-cells and tissue-resident memory T-cells.

Thus, this study supports the need to introduce glucose restrictions in the diet of patients with NSCLC in association with current immunotherapy.
PD-L1-expressing B cells promote murine breast cancer development and mediate response to anti-PD-L1 immune checkpoint inhibitor.
Authors: Cervantes Christian , Khan Mustafa Tamim Alam , Liu Zexuan , Viswanadhapalli Suryavathi , Gupta Harshita , Curiel Tyler , Yan Hui , Vadlamudi Ratna , Xu Zhenming
Published: February 2023

B cells promote tumor development in murine models of breast cancer (BCa), – as we have shown – by producing pleiotropic cytokine IL-27, which upregulates expression of PD-L1 immune checkpoint in BCa cells and tumor-infiltrating B cells (TIL-Bs). Paradoxically, B cell activation has been shown to predict a positive response to immune checkpoint inhibitors (ICIs) in murine BCa tumors and melanoma, sarcoma and kidney cancer in humans. Here, we have addressed the role of B cell-expressed PD-L1 in BCa development and responses to ICIs by generating conditional knockout mice in which Cd274 (encoding PD-L1) was ablated only in activated B cells (AicdacreCd274fl/fl). Tumor development in AicdacreCd274fl/fl mice from engrafted syngeneic ER+ E0071 BCa cells was significantly delayed, albeit not abolished (as occurring in mice lacking B cells), with a marked increase in CD86 B-cell expression, a key agonistic ligand for T cell-expressed CD28 (a co-stimulatory receptor whose function is inhibited by the PD-L1/PD-1 immune checkpoint). The heightened B-cell CD86 expression in AicdacreCd274fl/fl mice was recapitulated in vitro using CD154, a T helper cell stimulus critical for B cell activation and differentiation. Treatment with anti-PD-L1 (aPD-L1) boosted wildtype B cells, stimulated with CD154 in vitro, to express CD86 and differentiate into potentiated plasma cells. Thus, PD-L1+ TIL-Bs use PD-L1 to impose a “brake” on host anti-tumor activity but are directly targeted by aPD-L1 to relieve the brake, which “jump-starts” CD86:CD28 signaling and potentiates the anti-tumor response. Thus, providing a mechanistic explanation for the opposing roles of B cells in murine BCa development and ICI responses.
Neoantigen driven B cell and CD4+ T follicular helper cell collaboration promotes robust anti-tumor CD8+ T cell responses
Authors: Cui Can , Joshi Nikhil S. , Craft Joseph E
Published: February 2023

CD4+ TFH cells provide help to B cells, which is critical for germinal center (GC) formation. Although the presence of these cells is correlated with favorable clinical outcomes in various cancer types, little is known about how TFH and B cells function in anti-tumor immunity. Through the analyses of survival and bulk expression data in The Cancer Genome Atlas (TCGA) and single cell RNA sequencing data, we found that TFH cells correlated with GC B cells and with prolonged survival of lung adenocarcinoma (LUAD) patients. To further investigate if and how TFH-B cell interactions evoke protective anti-tumor responses, we developed the KP-HELLO murine lung adenocarcinoma model, in which tumor cells expressed B-cell- and T-cell-recognized neoantigens (HEL, LCMV GP33–43/FLAG/GP61–80, and codon-Optimized mScarlet). KP-HELLO tumors triggered tumor-specific TFH and GC B cell responses, which were necessary for tumor control, as were effector CD8+ T cell responses. The latter were reduced in the absence of T cell-B cell interactions or the IL-21 receptor. IL-21 was produced primarily by TFH cells, development of which required B cells. Moreover, development of tumor-specific TFH cell-responses was also reliant upon tumors that expressed B-cell-recognized neoantigens, which suggested that tumor-neoantigens themselves can control the fate decisions of tumor-specific CD4+ T cells by facilitating interactions with tumor-specific B cells. Our data highlight the importance of B cell-TFH cell collaborations in driving anti-tumor CD8+ T cell responses via IL-21 and indicated that therapeutics targeting B cell-TFH cell-IL-21 axis may be beneficial in patients with lung and other cancers.
Th17 cells enhance CD8 T cell cytotoxicity via IL-21 production in tumor vaccinated mice.
Authors: Lust Hannah E , Huang Yina H
Published: February 2023

A precise tug-of-war between regulatory T (Treg) cells and IL-17-producing (Th17) CD4 T cells ensures appropriate reactivity to pathogenic challenge. While an imbalance favoring Treg activity leads to suboptimal responses, overactive Th17 activity promotes autoimmunity. Both Th17 and Treg cells represent a significant proportion of the tumor infiltrating lymphocytes (TILs) in melanoma patients. In this disease context, Tregs suppress effector cells via numerous mechanisms. Productive immune responses are believed to result from passive escape from Treg suppression. We report an active role for Th17 cells in dampening Tregs to promote CD8 effector T cell activity. Using a melanoma mouse model, we found that elimination of IL-17-producing cells prevents vaccine-induced anti-melanoma immunity. Using RNA-Seq analysis, we identified a gene expression (GE) signature that implicates the IL-21/IL-21R axis in the ability of Th17 cells to inhibit Tregs and to induce a cytolytic GE signature in CD8 T cells. Similarly compromised vaccine-induced anti-melanoma responses in both IL-21R-deficient and Th17 diminished mice further support a critical role for Th17 cells and IL-21. Ectopic expression of IL-21 rescued compromised anti-tumor responses in Th17 diminished mice, suggesting that Th17-specific production of IL-21 induces effective CD8-mediated anti-tumor immunity. Single-cell RNA-seq analysis elucidated dynamic changes in both the identity and phenotype of diverse TILs populations in response to Th17 cells and IL-21 expression. Together, these data suggest that Th17-specific IL-21 production impedes Treg suppression, resulting in increased CD8 T cell effector function, and changes in TILs populations.
Tumor-infiltrating CD8+ T cells can be resident, but exhaustion markers rather than CD69 correlate with residence and tumor specificity
Authors: Gavil Noah Veis , Weyu Eyob , Scott Milcah C. , Smith Olivia , Wijeyesinghe Sathi , Masopust David
Published: February 2023

Tumor infiltrating lymphocytes (TILs) represent a heterogeneous population of both pathogen-specific bystander and tumor-specific CD8+ T cells. Within select solid tumors, the expression of exhaustion markers PD-1 and CD39 has been used to discriminate between pathogen-specific and tumor-specific cells. Independent studies across numerous types of solid tumors have also shown that CD8+ TILs can exhibit distinct signatures that imply residence, however, the migrational properties of TILs have not been fully elucidated. In this study, we employed parabiosis migration assays in a mouse model of breast cancer and demonstrated that both virus-specific bystander and tumor-specific CD8+ T cells within the tumor microenvironment (TME) can be resident. Canonical markers of resident-memory T cells (TRM), including CD69, failed to discriminate between resident cells and recent migrants. However, the expression of markers associated with T cell dysfunction (PD-1, CD39, Tim-3, Lag-3) identified a population of tumor-specific cells and was tightly correlated with residence within the TME. Thus, TRM exist within tumors, durable intratumoral residence was not well informed by common markers associated with TRM in healthy tissues, and the expression of exhaustion markers correlated with both tumor specificity and durable residence.
Dual function of Bim is switched by ANT2 (SLC25A5) to balance apoptotic and effective signals in CD8+ T cells
Authors: Gicobi Joanina K , Yu Xiatong , Dronca Roxana , Zhu Mojun , Dong Haidong
Published: February 2023

The immune responses to anti-PD-1 therapy in cancer patients are not completely defined due to lack of reliable biomarkers that identifies responsive and functional T cells. Bim, a molecule of the Bcl-2 family, was recently identified as a downstream signaling molecule of PD-1 pathway in CD8+ T cells and has both pro-apoptotic and effective roles in CD8 T cells. Our clinical data shown that high level of Bim in CD8+ T cells (before checkpoint blockade) was predictive of response in metastatic melanoma whereas in colorectal cancer, low level of Bim was associated with treatment response. Using co-immunoprecipitation, high resolution microscopy and FRET we identified ANT2 (Adenine Nucleotide Translocase 2, an inner membrane protein of mitochondria) as a protein interacting with Bim. Accordingly, we found CD8+ T cells with low and high mitochondrial potential demonstrated different ANT2/Bim ratio, and high ANT2/Bim ratio fall in effector T cells with low mitochondrial potential but high cytotoxic activity. Given the pro-survival of ANT2, our results indicate that ANT2 may function to balance the pro-apoptotic and effective role of Bim in mitochondria in order to preserve effector T cells before contraction. Understanding the molecular mechanism of Bim and ANT2 interaction in CD8+ T cells may provide a novel marker to identify resilient CD8+ T cells responsive to anti-PD-1 therapy, and predict clinical response to the therapy.
The role of c-Jun in enhancing TCR-T function in treatment of Hepatocellular Carcinoma
Authors: Hussein Mohamed Ahmed Shawky , He Yukai
Published: February 2023

Hepatocellular carcinoma (HCC) is responsible for most of primary liver cancer cases and is the sixth most common cancer in the world with a general 5 year survival rate of 18% due to lack of effective therapy. Adoptive T-cell therapy (ACT) with T-cell receptor–engineered T-cell (TCR-T) against HCC-associated antigen a-fetoprotein (AFP), can redirect human T cells to recognize and kill HCC tumor cells. However, the clinical efficacy of TCR-T therapy is dependent on the proper expansion, in vivo persistence of T cells that is markedly reduced due to exhaustion-associated dysfunction, and activation induced cell death. To improve persistence of TCR-T cells without modifying TCR affinity, we engineered TCR-T cells to overexpress c-Jun, an AP-1 transcription factor associated with T cell development and activation. In this study, we showed that TCR-T cells overexpressing c-Jun have enhanced expansion capacity in culture, improved cytokine production against HepG2 tumor cells without altering their cytotoxic potential. Interestingly TCR-T cells overexpressing c-Jun showed resistance to activation induced cell death with decreased expression of inhibitory receptors after stimulation with HepG2 tumor cells. Thus, c-Jun overexpression could be a potential therapeutic agent to enhance the anti-tumor efficacy of TCR-T cells against HCC.
CD19 loss by CAR T cell mediated trogoctyosis is regulated by CAR affinity
Authors: Olson Michael , Luetkens Tim , Atanackovic Djordje
Published: February 2023

CD19 CAR T cell therapy has entered the spotlight for the treatment of B cell lymphoma, however, relapse with CD19 negative disease is common in many subtypes of lymphoma. CD19 CAR T cells were recently shown to strip CD19 from the surface of B cell lymphoma cell lines in a process called “trogocytosis,” however, the clinical relevance of this phenomenon remains poorly defined. The purpose of this study was to determine the impact of CD19 trogocytosis on CAR T cell escape in B cell lymphoma and the importance of CAR T cell affinity to trogocytosis mediated tumor escape. We cocultured CD19 CAR T cells with primary CLL, ALL, mantle cell lymphoma and diffuse large B cell lymphoma patient material and identified rapid loss of CD19 from the surface of the malignant cells in all subtypes. CD19 was also observed to be transferred from the lymphoma cell to CD19 CAR T cells, resulting in CAR T cell autoreactivity termed “fratricide.” We then utilized a recently developed low affinity CD19 CAR to assess the importance of CAR affinity in CD19 loss by trogocytosis. Coculture of low affinity CD19 CAR T cells revealed significantly reduced loss of CD19 by trogocytosis from several ALL cell lines and primary samples when compared to high affinity CD19 CAR T cells. Finally, in vivo analysis of CAR affinity revealed increased CAR T cell efficacy and persistence by low affinity CD19 CAR T cells when compared to high affinity CD19 CAR T cells in an established ALL mouse model. Taken together, these data demonstrate that loss of CD19 by CAR T cell mediated trogocytosis occurs in many B cell malignancies and can be ameliorated by modulation of CAR affinity, providing a possible avenue for more effective CAR T cell therapies in the clinic.
Immunotherapeutic effects of RCH-2 on glioblastoma through enhancing NK cellmediated cell lysis
Authors: Lin Yu-Ling , Lin Han-Huei
Published: February 2023

Glioblastoma is the most common and dangerous in primary brain tumors. Recent success in cancer immunotherapy is considered a breakthrough in cancer treatment. RCH-2, a phytocompound isolated from Larrea tridentata, is a transcriptional inhibitor of the Sp1 transcription factor. RCH-2 combined with different chemotherapeutic drugs also shows an excellent synergistic effect for cancer therapy in glioblastoma. RCH-2 effectively inhibits tumor growth in vivo, but less RCH-2 accumulated in the tumor area. RCH-2 might trigger an immune response to help tumor suppression. In this study, glioblastoma tumor-bearing mice were treated with RCH-2 daily by oral administration. Their cytokines and anti-tumor antibodies in sera were analyzed. Results showed that RCH-2 induced Th2 cytokines and promoted anti-tumor antibody production that efficiently induced antibody-dependent cell-mediated cytotoxicity (ADCC) to kill tumor cells. Our finding indicated that RCH-2 could induce more anti-tumor antibodies to efficiently target the tumor cell and trigger NK-mediated cell lysis.
Expanded ATLAS™-identified neoantigen-specific peripheral T cells (NPTs) demonstrate considerable neoantigen breadth, polyfunctionality, and effector function.
Authors: Perry James R , Reuter Mercay , DeVault Victoria L , Li Adrienne , Zope Harshal , DeOliveira Daniel B , Khare Pranay D , Jain Manish , Winstead Colleen J , Lam Hubert , Flechtner Jessica Baker
Published: February 2023

Adoptive T cell therapy using ex vivo expanded autologous tumor-infiltrating lymphocytes (TIL) has resulted in notable tumor regression across multiple solid tumor indications. Despite their success, TIL harvest requires invasive surgery and sterile isolation. In addition, extracted cells from an immunosuppressive tumor microenvironment (TME) often have impaired functions, low neoantigen-specificity and require high dose cytokine for expansion. All possible reasons for non-responders or post-treatment relapse. Generating T cell therapies specifically expanded against neoantigen targets and derived from peripheral blood lymphocytes may overcome these limitations.

The ATLAS bioassay identifies patient-specific neoantigen targets of T cells, and Inhibigens™, tumor antigens that are detrimental to protective immune responses. Using PLANET™, a robust and scalable closed manufacturing process, T cells specific for up to 30 ATLAS-identified neoantigens (excluding Inhibigens) are expanded, providing considerable breadth of tumor recognition and the potential to limit tumor escape. Use of peripheral T cells leverages robust cells not suppressed by the TME and eliminates the need for invasive tumor resection. Our data show that NPTs are specific for up to 89% of targeted neoantigens, consist of non-exhausted effector and central memory cells, and express proliferative and tissue homing markers. NPTs are highly polyfunctional, secreting multiple combinations of IFNγ, Granzyme B, TNFα, and MIP1α in response to specific neoantigens. Evaluation of markers for memory-progenitor stem-like features in NPTs are being explored and will be discussed. The TITAN™ clinical trial evaluating GEN-011 NPTs is ongoing (NCT04596033).
Fine-tuning Chimeric Antigen Receptor Signaling by Reducing CD3ζ ITAM Diversity
Authors: Majumdar Shubhabrata , Bettini Matthew L
Published: February 2023

One of the major challenges facing CAR based cancer immunotherapy is the lack of in vivo persistence of functional CAR T-cells due to low memory cell formation and exhaustion. Previous studies have suggested that strong TCR signaling favors the formation of terminally differentiated short-lived effector cells that get exhausted faster while weaker signaling favors formation of less differentiated memory-like cells. It was suggested that reducing CD3 ITAM (immunoreceptor tyrosine-based activation motif) sequence diversity while keeping the number of ITAMs same, attenuates TCR signaling strength to different extents, thus, highlighting the signaling non-redundancy of each individual ITAM sequence. Based on this, we designed 2nd generation murine anti-CD19 CARs with modified CD3ζ domains with the goals of optimizing CAR signaling and studying the non-redundant signaling properties of the individual ITAMs in the context of CARs. The CARs have either three copies of ζ1 ITAMs, ζ2 ITAMs or ζ3 ITAMs instead of the wildtype (WT) ζ1,2 and 3 together. We hypothesize that the non-redundancy of the individual ITAMs and the reduction in ITAM diversity will allow for optimization of CAR T cell signaling quantity, quality and improved T-cell persistence. The single flavor CAR expressing CD8+ T-cells can kill target CD19+ leukemia cells in vitro and are similar to the WT ζ CAR. Interestingly, we observe differences in short-term post-killing live T-cell counts. We also observe different levels of cytokine and perforin production by the activated T cells. When we look at their signaling, we observe differences in Erk, Akt and NFAT signaling. These signaling and functional differences open up the possibility of optimizing CAR persistence and function.
Adjuvanted bacteriophage vaccine targeting Pseudomonas aeruginosa
Authors: Román-Cruz Valery Cristina , Miller Shannon , Buhl Cassandra , Schooner Roman , Walsh Lois , Secor Patrick , Burkhart David , Evans Jay
Published: February 2023

Pseudomonas aeruginosa (Pa) is a Gram-negative bacterium which is associated with chronic diabetic wounds as well as potentially deadly lung infections in cystic fibrosis patients. Approximately 51,000 cases of Pa infections occur annually; of these cases, 13% are resistant to antibiotics which results in the deaths of ~400 individuals every year. Diversity between Pa strains presents a challenge in developing a vaccine or therapeutic capable of resolving infections caused by different strains. Pf phage (a filamentous bacteriophage) has been found in Pa from chronic diabetic wound isolates and has been show to increase virulence of Pa. Recent findings by our team indicate that a Pf phage-targeted vaccine or monoclonal antibody provide protection from the establishment of Pa infection in mice. To further improve this innovative new vaccine targeting Pa, adjuvant systems were used to enhance humoral response to the vaccine. A consensus peptide from Pf Phage coat protein was conjugated to the carrier protein CRM197 and combined with novel adjuvants and delivery systems. Selected adjuvants significantly enhanced humoral immunity to the Pf Phage peptide in a dose-dependent manner. Combination adjuvant systems were also used to further enhance antigen-specific immunity to Pf Phage and cell-mediated immunity to the carrier protein. The development of an adjuvanted Pf phage vaccine to protect against Pa infections is a new and innovative strategy with the potential to protect patients at risk for opportunistic bacterial infections.
Prefusion F-based polyanhydride nanovaccine induces robust humoral and cell-mediated immunity resulting in long-lasting protection against respiratory syncytial virus
Authors: Stephens Laura , Ross Kathleen A. , Legge Kevin L. , McLellan Jason S. , Narasimhan Balaji , Varga Steven M.
Published: February 2023

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children, accounting for 7% of deaths in those less than one year of age and leading to more than $300 million in annual medical costs. As natural exposure to the virus generates insufficient immunity, there is a critical need for a safe and efficacious vaccine to prevent RSV-induced disease. We developed an intranasal vaccine, termed RSVNanoVax, composed of polyanhydride nanoparticles encapsulating the RSV prefusion F protein and a CpG1668 oligodeoxynucleotide adjuvant. RSVNanoVax prime-boost vaccination of BALB/c mice induced RSV F-directed IgG serum antibodies specific for both site 0 and site II on the F protein. Systemic antibodies maintained a robust neutralizing capacity out to at least six months post-vaccination. Vaccination with RSVNanoVax also induced F-directed IgA titers within the lungs and nasal passages as well as lung tissue-resident memory CD4 and CD8 T cells, indicating that the vaccine established both a humoral and cell-mediated immune response within the mucosal tissues. Vaccination with RSVNanoVax resulted in protection from RSV-induced disease out to at least six months post-challenge, as measured by weight loss and pulmonary dysfunction. RSVNanoVax vaccination of outbred Swiss Webster mice also elicited protection from RSV-induced disease, and resulted in complete viral clearance as early as day two after RSV challenge. High RSV F-directed antibody titers were also observed in the serum of outbred mice. Based on the robust immune response and the high level of protection observed in both inbred and outbred populations, our prefusion RSV F nanoparticle formulation represents a promising RSV vaccine candidate.
Mucosal delivery of a polyanhydride nanovaccine incorporating CpG and the post-fusion F/ G proteins induces protective immunity against BRSV infection in neonatal calves.
Authors: Maina Teresia , Grego Elizabeth A , Sacco Randy E , Narasimhan Balaji , McGill Jodi L
Published: February 2023

Objective:

Previously, immunization with a single intranasal dose of a BRSV post-fusion F/G nanovaccine resulted in partial protection from a BRSV challenge (McGill et al. 2019, McGill et al. 2018). Knowledge gained from these previous studies was used to optimize the vaccine by incorporating CpG-ODN and to investigate the impact of mucosal vs. systemic vaccination routes. In this study a prime-boost immunization regimen was used.

Method:

36 neonatal, mixed-sex, Holstein calves were divided into 6 groups. Group 1 received a heterologous regime with saline and group 2 a homologous boost of the ‘empty’ nanovaccine CPG. Groups 3 and 4 received a homologous mucosal regime of the nanovaccine ± CpG, while groups 5 and 6 received a heterologous regime of the nanovaccine ± CpG. Vaccine-induced responses were monitored and 6 weeks after boost, all calves were challenged with BRSV. Nasopharyngeal swabs were collected for viral shedding and calves monitored for clinical signs and euthanized on day 7 after infection. Lungs were scored for gross pathology. Blood, nasal secretions and lung tissue samples were analyzed for BRSV specific immune responses.

Results:

Unvaccinated controls and CpG-only nanovaccine controls developed lung pathology consistent with a severe BRSV infection. We observed no evidence of vaccine enhanced disease in any vaccinated group. Calves that received the homologous vaccination of the nanovaccine + CPG had significantly less lung pathology and viral burden in the lungs compared to control calves.

Conclusions:

These results indicate that an intranasal homologous vaccination regimen with the post-fusion F/G + CpG nanovaccine has the capacity to reduce BRSV disease in neonatal calves with preexisting maternal antibodies.