bluuebunny/crossref_metadata_2025 huggingface dataset:
Teaching Immunology in an Integrated System-Based Undergraduate Medical Curriculum using Team-Based Learning (TBL) in the Virtual Classroom: A Learning Experience
Authors: Benoit Lora , Qureshi Mahboob
Published: February 2023

Teaching immunology in the absence of a traditional lecture format presents certain challenges that require special consideration. In part because immunology is often identified as one of the most difficult pre-clinical biomedical courses, requiring a comprehensive understanding of cellular and molecular biology, genetics, anatomy, and infectious diseases and because it is presented in the context of an exclusive vocabulary. To help address the challenge of teaching immunology in the context of the SARS-CoV-2 Pandemic, we developed a four-week, 1st year medical school immunology course using a mostly active learning type modality that could be effectively delivered virtually. In particular, the course content was delivered using a combination of ZOOM, HoloLens, InteDashboard, Brightspace, and the Panopto™ Video platform. Most of the content was delivered using the Team-Based Learning (TBL) pedagogy which incorporated clinically oriented exercises that inspired and engaged students in achieving a mastery of immunology while honing their critical thinking skills; thus, efficiently preparing them for future clinical practices and licensure exams while avoiding an overwhelming perception of the course. Despite the inherent limitations of virtual delivery, the outcome of the course was an unexpected 100% pass rate. A description of the content, course design, formative /summative assessment methods as well as category performance on exams is provided. The expressed perception of the students is reported as are opportunities for improving the educational experience.
Disadvantaged students at minority serving institutions became disproportionally affected by the current COVID-19 pandemic.
Authors: Renteria Sahira , Stempek Ryan , Olk Sandra R , Fazal Nadeem
Published: February 2023

Learning has been a complex human skill. Humans have gone through a gradual learning experience spanning hundreds of years. Never in the history mankind have been tested with learning a tsunami of information in a very short period. Last couple of decades have stressed out not only the neurobiology of learning but tested the maximum limits of human capacities. If not all, some of the population group like students have burnt out. To add further stress to depleting limits of human capacities the current pandemic totally exhausted even their reserves. We at College of Pharmacy Chicago State University tested and other stressor being minority serving university and having a major population of disadvantage students. We did a survey of under graduate students and asked them this question to identify only one and most important challenge they faced in this time of pandemic induced remote learning. Not to our surprise we found that a statistically significant number of students declared that the cost of paying for technology to survive in this remote learning was the major factor that impacted their learning and lead to a major challenge for them. Most of the students suffered financial stress with loss of jobs, declining college loans, lack of motivation for a successful career. More specifically these disadvantage students could not keep up with the rising cost of wifi, laptop, cell phones and opting for additional online learning tools. To give an example, one student told us that she would go to near by Starbucks to get online assignments done in a timely manner. In the current lock down even the public libraries are closed and students struggle. Our study suggests that the universities set aside a special budget to financially help these disadvantage students.
Pan-academic in Pandemic! Remote learning goes viral, mostly out of control.
Authors: Fazal Nadeem , Alakbarova Gulgaz
Published: February 2023

A pandemic of viral origin has caused a pan-academic in the educational world as well. The younger student population though felt safe owing to their robust immune response but took a big hit in their emergency academic response to COVID-19 times. A pandemic needed a pan-academic response. The students were obviously not prepared and equipped for such an abrupt behavioral adaptation, with no second choice offered. Now it has evolved into global research in progress and Educators are experimenting on no-so-willing students. We studied a robust group of 138 medical students across 3 medical schools in Azerbaijan and gathered enormous amounts of data set points. Some of the major takes from our surveys proved the dichotomy and split among the medical students have in terms of coping with the unprecedented circumstances. The challenges included using combination of distance-learning platforms, i.e., synchronous lessons (similar to zoom sessions), pre-recorded lectures, powerpoint presentations, question-banks, etc. A significant difference was found between time spent on online platforms before COVID-19 (10.86%), as compared to (46.37%) during lock-down, while average students were online >15 hours per week. The commonly perceived barriers to using online teaching included, poor internet connection (72.5%), cost of technology, and distractions while studying at home (56.5%). For one group of students remote learning seemed cheaper and flexible than traditional class (saved rent, food, traffic woes). Most of the students’ settled for hybrid learning, thus giving equal weight to in-class vs remote learning experiences. We conclude that there seems to be no one size fits all-approach, we suggest a blended, hybrid, multi thronged system.
Adaptation of a CURE course aimed at characterization of host-pathogen interactions for on-line learning
Authors: Snyder Michelle , Snyder Greg A
Published: February 2023

Course-based undergraduate research experiences (CUREs) aim to engage a large, diverse group of students in authentic research, promote student research skills, content knowledge, scientific literacy, critical thinking and analysis skills, support student self-efficacy, and at the same time provide a means to advance faculty research productivity. Addressing each of these varied goals of a CURE course is a challenge, and became even more challenging when classes were forced on-line for the COVID-19 pandemic. In Fall of 2020, we adapted our CURE course, which seeks to engage students at Towson University with research ongoing at the University of Maryland, School of Medicine, for virtual learning. To move the course on-line, we focused on activities that could easily be adapted to a virtual environment, such as those focusing on bioinformatics analysis, research design, data analysis, scientific writing and on-line lab simulations. Students were tasked with becoming ‘project managers’—designing hypotheses and protocols and choosing experimental variables and controls, while instructors and an undergraduate learning assistant carried out the experiments, videotaping so students could become familiar with the techniques. While the drawbacks of the virtual CURE course were many, including the lack of student involvement in hands-on bench work, benefits of the virtual CURE included more time and attention devoted to critical skills such as research design, data analysis and scientific writing. Future designs of CURE courses may benefit from increased attention to these skills, even upon return to in-person learning.
Active learning strategies incorporating Immunology based teaching examples for Health Sciences Education
Authors: Sika-Paotonu Dianne
Published: February 2023

Clinically relevant Immunology based teaching examples for postgraduate Health Sciences education offer valuable opportunities to support and reinforce understanding and learning of basic principles. The appropriate reference to significant health conditions as teaching examples that are of immediate relevance to specific population groups requires care. Active learning strategies that incorporate Immunology based teaching examples and references to Māori and Pacific population groups living in Aotearoa/New Zealand and the Pacific Region, are much needed to support Health Sciences education. The autoimmune condition known as Acute Rheumatic Fever (ARF) resulting from untreated Group A Streptococcal (GAS) infection of the throat and skin, and subsequent cardiac damage known as Rheumatic Heart Disease (RHD) that occurs if repeated or severe untreated ARF episodes happen, disproportionately affect Māori and Pacific population groups living in Aotearoa/New Zealand and the Pacific Region. These conditions were incorporated into active teaching approaches to support postgraduate Health Sciences students, with appropriate cultural context. Students participating in postgraduate classes that utilised active learning strategies with appropriate incorporation and reference to the ARF/RHD setting, demonstrated high levels of engagement and in-class participation, and as evidenced by formal and informal student feedback, fostered in-depth understanding of the Immunology principles being taught. This highlights the importance of appropriate reference and context for active learning strategies that incorporate Immunology based teaching examples when relating to specific population groups.
Mesenteric lymph node priming licenses intestinal CD103+ CD8 tissue-resident memory T cell development
Authors: Qiu Zhijuan , Khairallah Camille , Chu Timothy H. , Imperato Jessica N. , Romanov Galina , Puddington Lynn , Sheridan Brian S.
Published: February 2023

CD8 tissue-resident memory T (TRM) cells provide front-line protective immunity at barrier tissues; however, mechanisms regulating their development are not completely understood. Priming dictates the migration of effector CD8 T cells to the tissue, while factors in the tissue induce in situ TRM cell differentiation. Whether priming also regulates in situ differentiation of CD8 TRM cells has not been addressed. Here, we demonstrate that T cell priming in the mesenteric lymph nodes (MLN) was the principal determinant of CD103+ CD8 TRM cell differentiation in the intestine. In contrast, CD8 T cells primed in the spleen were inefficient at differentiating into intestinal CD103+ TRM cells. Moreover, both CD103− and CD103+ naïve T cells were highly efficient in differentiating into CD103+ TRM cells in the intestine after priming in the MLN, suggesting preconditioning of naïve T cells by TGF-b during homeostasis had little impact on intestinal CD103+ TRM cell differentiation. We further demonstrate that MLN priming initiated CD103+ CD8 TRM cell signature gene expression and licensed rapid CD103+ CD8 TRM precursor cell differentiation in response to factors in the tissue by providing retinoic acid signaling to CD8 T cells. Thus, MLN priming is specialized to license intestinal CD103+ CD8 TRM cell development.
Accumulation of Tissue Resident NK cells in Female Genital Tract during follicular phase of HIV seronegative women
Authors: Govindaraj Sakthivel , Babu Hemalatha , Endress Daniel , Rahman Sadia J , Ibegbu Chris , Kozlowski Debra Susan , Doraivelu Kamini , Herring Gina Bailey , Mehta Christina , Smith Alicia K , Haddad Lisa , Velu Vijayakumar
Published: February 2023

Female sex hormones are known to regulate the immune functions of the female genital tract (FGT). While the majority of immune cells found in the FGT are T cells and Natural Killer cells (NK cells), very few studies have focused on NK cells in the FGT in contrast to numerous studies focusing on T-cells. Here, we characterized the distribution, phenotype and function of NK cells in FGT of HIV seronegative women using different mucosal sampling methods; cervicovaginal lavage (CVL), endocervical cytobrush (CB), and cervicovaginal biopsy and compared them to blood. We isolated cells from blood and FGT and performed multi-color flow cytometry to identify cellular phenotypes. First, we looked at the distribution of CD56brightand CD56dimpopulations of NK cells across the samples and found that CD56brightNK cells were lower in the FGT tissues compared to blood. Between the CD56brightand CD56dimpopulations, the CD56dimpopulation was significantly reduced in the follicular phase compared to the luteal phase in tissues. The NK cells within the FGT samples express higher levels of tissue resident markers CD69 and CD103. The co-expression of CD69+CD103+ markers seem to be significantly higher in follicular phase compared to luteal phase in tissues. These cells have high proliferative capacity with high levels of HLADR expression, suggesting that these cells are highly functional and activated. The gut tissue homing marker alpha4beta7 expression significantly reduced in the follicular phase, however other homing markers CCR5 and CCR7 expression were elevated in the follicular phase. Altogether, these data demonstrate that FGT is enriched with tissue resident NK cells with high activation and proliferation profile with distinct homing potential.
NKG2A educates uterine NK cells to optimise pregnancy outcomes in humans and mice
Authors: Shreeve N , Traherne JA , Sovio U , Hawkes D , Depierreux D , Huhn O , Jayaraman J , Horowitz A , Ghadially H , Perry JRB , Moffett A , Sled JG , Sharkey AM , Colucci F
Published: February 2023

The conserved CD94/NKG2A receptor binds HLA-E in humans and Qa-1 in mice. Besides inhibiting natural killer (NK) cell activation, NKG2A drives NK-cell education, a process influenced by HLA-B alleles that promotes NK cell function. In human populations some individuals are genetically programmed to favour NKG2A education and have more robust NK cell function. NKG2A is expressed by nearly all human and roughly half mouse uterine NK cells (uNK), but the importance of NK-cell education in physiology is unknown.

Here we show that NKG2A was required for uNK cell-education in dams. Genetic ablation of NKG2A caused sub-optimal vascular responses in pregnancy, increased rate of smaller fetuses, which grew asymmetrically with abnormal brain development, and changes in placental gene expression consistent with stress. These are features of the human syndrome pre-eclampsia. In a genome-wide association study of 7,219 cases and 155,660 control pregnancies, we found that the maternal HLA-B allele that does not favour NKG2A education, was associated with a 7% greater relative risk of pre-eclampsia (P=0.005, OR= 1.07).

These results establish the relevance of NK cell education in physiology and show that the maternal HLA-B –> HLA-E –> NKG2A pathway contributes to healthy pregnancy and may influence offspring health.
Zom-biecoming: Single-Cell RNA-seq reveals senescence-like features of alveolar macrophages during aging
Authors: Wu Yue , Zhu Bibo , Zhang Ruixuan , Wang Zheng , Goplen Nick P. , Gao Xiaochen , Li Ying , Cadani Amber , Braciale Thomas J , Sun Jie
Published: February 2023

Alveolar Macrophages (AMs) are unique innate immune cells that reside in the alveolar space and accommodate the ever-changing need of the lungs against internal and external challenges. During homeostasis, AMs maintained themselves through self-renewal without the need for the input of adult hematopoietic stem cells. Currently, little is known about how aging influences AM dynamics, heterogeneity and transcriptional profiles. Here, we identified the transcription factor, Cbfb, has indispensable role in AM self-renew ability. In the bone marrow chimera mice with genetic knock-out of Cbfb specifically in the myeloid compartment, AMs displayed reduced proliferation and an immature phenotype. Moreover, with the analysis of the combined scRNA-seq data of the FACS sorted AMs (CD11+ Siglec F+) in young(~8w) and aged(~2y) mice, we discovered that despite for the similar transcriptome in the proliferating cells, the AMs from the aged mice had reduced embryotic-stem cell (ESC)-like features. The AMs from the aged host also had reduced DNA repair ability, which could contribute to their impaired capacity to pass through the cell cycle checkpoints and elevation of senescence markers. In accordance with the analysis, we observed reduced number of AMs in the aged mice, which had abrogated self-renew ability and were more sensitive to the reduction of GM-CSF. Increased b-galactosidase staining was also observed in the AMs from the aged mice. Taken together, we concluded that AMs in the aged host harbor a senescence-like phenotype. Further investigation is warranted for the mechanism and pathophysiological consequences of the accumulation of AMs with senescence-like phenotype in the aged host.
Large scale analysis of smoking-induced changes in the tumor immune microenvironment
Authors: Alisoltanidehkordi Arghavan , Qiu Xinru , Jaroszewski Lukasz , Sedova Mayya , Iyer Mallika , Li Zhanwen , Godzik Adam
Published: February 2023

Here, we expanded the recent knowledge on the immune landscape of cancer by performing a comparative analysis of tobacco smoking-induced changes in the populations of major immune cell types between smokers and never-smokers (people who have never smoked). To investigate the immune response of smokers and never-smokers, the relative abundance of immune cell types was calculated by both CIBERSORT and xCell based on TCGA RNA-seq data. We further validated the findings using publicly available bulk RNA-seq and single-cell RNA-seq data from cohorts of cancer patients other than TCGA cohort. We showed statistically significant smoking-induced changes in immune cell populations in all studied cancers. The increase in plasma cell populations and the changes in the ratio of activated to resting immune cell types were the most consistent features distinguishing smokers and never-smokers across different cancers, with both being correlated with survival outcomes. We found that the tobacco-induced changes in populations of immune cells were not as marked in cancers of tissues that are not directly exposed to smoke as in lung cancers and head and neck cancer (LUAD, LUSC, and HNSC). However, the significant differences in survival in several cancer types suggest that even a small shift in the frequency of immune cell types might result in adverse outcomes. We also showed that smoking-induced changes in the patterns of immune cell populations and their correlations to survival outcomes are stronger in female smokers than in males. In general, the findings of this study suggest that certain immune cell types could potentially be used as common prognostic markers and therapeutic targets for active smoker patients.
The contradictory functions of NLRX1 in breast cancer allow for suppression or promotion of tumor growth and metastasis in a cell-specific manner.
Authors: Nagai Margaret A , Hendricks-Wenger Alissa , Fletcher Endia , Khan Imran , Morrison Holly , Allen Irving C
Published: February 2023

Breast cancer is a deadly malignancy that is responsible for 15% of all cancer-related deaths in women. Examining the innate immune system in breast cancer has revealed the importance of pattern recognition receptors, such as NLRX1, in disease progression. NLRX1 has been implicated as a tumor suppressor because it suppresses inflammation by many mechanisms, including by inhibiting the NF-κB pathway. Here, we investigated the potential of NLRX1 as a tumor suppressor in breast cancer when it is expressed either by healthy host cells or cancer cells. To determine the role of NLRX1 in healthy host cells, we used the 4T1 breast cancer model in WT and Nlrx1−/− (KO) mice. KO mice exhibited increased lung metastasis and tumor size compared to WT mice. Gene expression data revealed increased expression of proliferation, survival, growth, invasion, and metastatic genes in tumors from KO mice compared to WT mice. Thus, NLRX1 in healthy host cells is protective against breast cancer tumor growth and metastasis. Next, we investigated the role of NLRX1 in breast cancer cells. 4T1 cells were transduced to overexpress NLRX1 and injected into WT (WTOE) and KO(KOOE) mice. Transduction controls were also generated and injected into WT (WTOE-CTL) and KO (KOOE-CTL) mice. KOOE mice developed larger tumors than KOOE-CTL mice. KOOE mice also developed more lung metastasis compared to WTOE, WTOE-CTL, and KOOE-CTL mice. This suggests NLRX1 in breast cancer cells contributes to tumor growth and metastasis, which directly contradicts the function of NLRX1 in healthy host cells and demonstrates a cell-specific function of NLRX1 in breast cancer. These cell-specific differences are critical foundations for the development of NLR-based breast cancer therapies.
Lung tumor immune evasion mechanism in Non-small cell lung cancer (NSCLC) targets pSTAT5 in CD4+CD25+FoxP3− T effector cells
Authors: Tausche Patrick , Trufa Denis I , Geppert Carol-Immanuel , Rieker Ralf J , Sirbu Horia , Finotto Susetta
Published: February 2023

The mechanisms of lung cancer evading the immune system are still not fully understood. As regulatory T cells (Tregs) exhibit a pro-tumoral function they are in focus of immunotherapies. Peripheral Treg differentiation is driven by TCR and its co-stimulation. A further crucial signal displays the binding of IL-2 to the high affinity receptor CD25 with downstream involvement of Signal Transducer and Activator of Transcription 5 (STAT5). STAT5 activation as well as TGF-beta stimulation in the periphery leads to expression of the Treg key factor FoxP3.

In this study, we wanted to investigate the role of STAT5 and its activated status via phosphorylation (pSTAT5) within lung cancer. We thus isolated peripheral blood mononuclear cells (PBMC) from NSCLC patients and healthy donors. PBMCs were cultured under different stimulation conditions for 4 days and analyzed by FACS.

Here we found CD4+ T cells with high induction of pSTAT5A and CD25 upon aCD3 and aCD28 antibody treatment. Moreover, CD4+CD25+FoxP3+ Tregs were detected under T cell activating conditions, and were found to be increased by TGFβ in smokers and NSCLC patients. The Treg population was further inducible by adding low doses of IL-2 while CD4+CD25+FoxP3− effector T cells decreased in the presence of TGFβ. Our findings point to an immunosuppressive microenvironment induced by the tumor with increased TGFβ and reduced IL-2 forcing effector T cells into a Treg state. These preliminary data suggest that current immunotherapy by aCD3/aCD28 antibody treatment could be improved by an additional blockade of TGFβ and induction of IL-2 levels. This treatment would result in an increase of CD4+CD25+pSTAT5+FoxP3− effector cells and a reduction of CD4+CD25+FoxP3+STAT5+ Tregs.
PID1 promotes HCC development through regulating TME and inhibiting inflammatory factors induced apoptosis
Authors: Yang Jian , Xie Mengyuan , Xiang Ming
Published: February 2023

Objective:

PID1 (Phosphotyrosine interaction domain containing 1), an obesity related gene, was reported to induce insulin resistance and play a vital role in lipid metabolism in liver. However, its influence on HCC (hepatocelluar cancer) have not been full clarified. Here, we aims to investigate the mechanism of PID1 in favoring HCC development.

Method:

Hydrodynamic injection was applied to overexpress PID1 specifically in liver. Adaptive immunity was evaluated through flow cytometry. Lipids metabolism was determined by metabonomics. Western blotting was utilized to determine the effects of PID1 on PI3K/AKT and RAS/RAF/ERK pathway.

Result:

Overexpression of PID1 specifically in liver induced HCC development within 16 weeks. The frequency of CD4+ and CD8+ T cells decreased whereas the ratio of Gr1+CD11b+ cells increased in mice bearing PID1 plasmid hydrodynamic injection. Moreover, PID1 induced liver metabolism disorder, resulted in upregulation of TG, TC, NEFA, LDL-C as well as lactate levels, thereby involved in regulating TME (tumor microenvironment) and depressed adaptive immunity. Importantly, PID1 inhibited PI3K/AKT pathway, thus restrained phosphorylation of c-RAF on ser259 site induced by p-AKT and promoted phosphorylation of c-RAF on ser338 site, leading to activation of RAS/RAF/ERK pathway. Activated c-RAF targeted to mitochondria to decrease ROS level, which alleviated toxic effect of inflammatory factors and favored HCC cells survival.

Conclusion:

Our results demonstrated that PID1 provoked HCC development through regulating TME to depress adaptive immunity and inhibiting apoptosis induced by inflammatory factors.
Reciprocal interactions between the gut microbiome and mammary tissue microenvironment promote distant regulation of breast tumor metastasis
Authors: Feng Tzu-Yu , Azar Francesca N. , McGinty Mitchell T. , Rutkowski Melanie R.
Published: February 2023

Establishing commensal dysbiosis, defined as an inflammatory gut microbiome with low biodiversity, prior to breast tumor initiation enhances early dissemination of hormone-receptor positive (HR+) mammary tumor cells into the circulation, distal lymph nodes, and lungs. Here, we sought to define mammary tissue mediators of dysbiosis-induced tumor dissemination. Commensal dysbiosis increased mast cell numbers in the pre-malignant mammary tissue, with numbers remaining elevated in the presence of a tumor. During early tumor progression, increased mast cell numbers correlated with increased numbers of activated fibroblasts and collagen I protein levels in mammary tissues. Fibroblast activation and tissue remodeling are associated with enhanced breast tumor metastasis. Although mast cell degranulation is known to trigger fibrosis in other disease models, it is unknown whether mammary tissue mast cells influence dissemination of tumor cells. To investigate the role of mast cells during tumor dissemination, mice were treated with mast cell stabilizers ketotifen or cromolyn. Notably, inhibition of mast cell degranulation significantly reduced fibroblast activation and dissemination of tumor cells into the blood of dysbiotic animals. Collagen levels in mammary tissues from women diagnosed with HR+ breast cancer also correlated with mast cell abundance, suggesting mast cell-mediated fibroblast activation is relevant to human disease. Together, these data demonstrate that mast cells recruited into the pre-malignant mammary tissue in response to commensal dysbiosis orchestrate early dissemination of HR+ breast tumors through activation of tissue fibroblasts.
ST8Sia6 Overexpression Accelerates Tumorigenesis in a Siglec-E Dependent Manner Through Reshaping the Tumor Immune Microenvironment
Authors: Friedman David J , Crotts Sydney B , Shapiro Michael J , Rajcula Matthew , McCue Shaylene , Khazaie Khashayarsha , Dong Haidong , Shapiro Virginia Smith
Published: February 2023

Checkpoint blockade has improved patient outcome through reinvigorating T cells against cancer. Despite this success, many patients don’t respond, leaving them with few options. Tumors utilize many immunosuppressive mechanisms, including altering the function of innate immune cells. Cancer cells can promote differentiation of macrophages into tolerogenic “M2-like” macrophages, which support tumor growth. Macrophage differentiation by cancer cells is influenced by many receptors, including Siglec-E. Siglec-E binds to α2,8-linked disialic acids, generated by the enzyme ST8Sia6, on O-linked glycoproteins, and functions to inhibit activation of innate immune cells. In some tumor types, higher expression of ST8Sia6 correlates with poorer survival. Therefore, ST8Sia6 overexpressing tumors may engage Siglec-E, leading to immune suppression and accelerating tumor growth. Mice injected with ST8Sia6 overexpressing MC38 and B16-F10 tumors had increased tumor burden, reduced survival and increased M2 macrophage polarization, which suppresses the immune response. This acceleration of tumor growth was abrogated when injected into Siglec-E knockout mice. Thus, the primary function of ST8Sia6 in tumors is an extrinsic alteration of the immune response, rather than an intrinsic effect on cell growth. Similarly, in a spontaneous colon cancer model, ST8Sia6 overexpression in a murine colon cancer model accelerated the development of tumors, decreasing survival to 2.5 months instead of 6 months. The overexpression of ST8Sia6 leads to increased Arg-1 expression by colonic tumor macrophages. Therefore, ST8Sia6 expression in tumors inhibits the immune response to promote tumor growth in a Siglec-E dependent manner.
Genetic linkage analysis using Diversity Outbred mice identifies prolactin as a host-intrinsic regulator of immune checkpoint inhibitor response
Authors: Hackett Justin , Glassbrook James , Múniz Maria , Gibson Heather
Published: February 2023

The use of immune checkpoint inhibitors (ICI) targeting CTLA-4 and PD-1 have greatly improved patient survival in metastatic melanoma, but a significant proportion of patients fail to respond to therapy. Incorporating what has been learned in autoimmune conditions, we hypothesize the host genetic background plays a significant role in determining response to immunotherapy. To implement genetic diversity, Diversity Outbred (DO) mice were crossed with C57BL/6 to generate (C57BL/6xDO)F1 mice that reliably develop B16F0 tumors after subcutaneous inoculation. (C57BL/6xDO)F1 mice (n=142) treated with combined anti-PD1/anti-CTLA-4 on days 3, 6, and 10 after inoculation show wide variation in tumor latency up to a maximum of 65 days, with 19 mice never developing tumor. Genetic linkage analysis using the R/qtl2 package shows a suggestive association within the murine prolactin family on chromosome 13. Visualization of QTL effects by DO founder identifies the NZO mouse genotype as a driver of increased tumor latency. Validation was performed using Collaborative Cross (CC) mouse lines that were either the NZO or C57BL/6 genotype within this locus. (C57BL/6xCC)F1 mice containing the NZO genotype have lower tumor burden by day 15 compared to mice with the C57BL/6 genotype, validating the locus as influential on the response to ICI. Combination of systemic prolactin treatment and ICI shows evidence of an interaction effect. Mice treated with both prolactin and ICI show lower tumor burden by day 19 versus single therapy or placebo controls. These results show that the host’s genetic background significantly contributes to response to ICI, and modulation of prolactin expression may augment ICI activity.
Tumor-intrinsic IRF5 reprograms the tumor immune microenvironment to inhibit breast cancer metastasis
Authors: Barnes Betsy J , Li Dan , Somerville Carter , Castro-Piedras Isabel , Pruitt Kevin
Published: February 2023

Metastasis of breast cancer and recurrence to incurable disease are leading causes of mortality. Metastasis is driven by extensive cooperation between a tumor and its microenvironment, resulting in the adaptation of molecular mechanisms that evade the immune system and enable pre-metastatic niche (PMN) formation. Little is known of the tumor-intrinsic factors that regulate these mechanisms. Here we show that expression of the transcription factor interferon regulatory factor 5 (IRF5) in breast cancer clinically correlates with tumor immunogenicity, increased T cell and M1 macrophage recruitment and prolonged survival. Conversely, loss of tumor-intrinsic Irf5 establishes a tumor microenvironment and PMN that supports metastasis. Mechanistically, Irf5 alters the composition and secretion of tumor-derived extracellular vesicles (t-dEVs). Upon whole-body pre-conditioning with t-dEVs from Irf5-positive or −negative breast cancer cells, we found increased lung metastatic colonization that replicated findings from orthotopically implanted breast cancer cells. Collectively, our findings uncover an essential role for IRF5 in breast cancer metastasis through its regulation of t-dEV and immune reprogramming.
Hypoxia profoundly alters Natural Killer cell phenotype and function: implications for immunotherapy within the solid tumor microenvironment
Authors: Arvindam Upasana Sunil , Kennedy Philippa , Ettestad Brianna , Phung Shee Kwan , Hinderlie Peter , Lim James , Miller Jeffrey S , Felices Martin
Published: February 2023

For Natural Killer (NK) cell-based immunotherapy to succeed against solid tumors, NK cells need to enter a highly immunosuppressive tumor microenvironment (TME) and retain functionality. The objective of this study is to evaluate how hypoxia exerts an immunosuppressive effect on NK cells. We used the novel AVATAR™ system to model oxygen levels of three key tissues that NK cells inhabit in vivo: the peripheral blood (12% O2), the bone marrow (5% O2) and the TME (1% O2). NK cells incubated at 1% O2 have decreased proliferation and cytotoxicity compared to NK cells incubated at higher oxygen conditions. To assess what contributes to these changes, we conducted a mass cytometry (CyTOF) analysis, real time imaging assays, metabolic assays and gene expression (RNA-seq and ATAC-seq) analysis. We observed oxygen dependent changes in expression of activating receptors, Ki-67, perforin and granzyme. Hypoxia impacts aggregation of perforin and granzyme granules at the immune synapse. Under hypoxic conditions, NK cell metabolism resembles cancer cell metabolism with increased glycolysis, amino acid synthesis and central carbon metabolism. These changes are accompanied by mitochondrial defects. Gene expression analysis revealed that changes in NK cell metabolism and function are mediated at the epigenetic level by histone demethylases. Treatment with histone demethylase inhibitors rescued NK cell cytotoxicity as measured using an IncuCyte machine. These results indicate that NK cells who enter the TME are fundamentally different than those in the bone marrow or blood stream. The insights gained from this study will be leveraged to overcome hypoxia induced immune suppression in the TME to enhance NK cell-based immunotherapy for solid tumors.
Tipifarnib enhances eradication of acute myeloid leukemia by altering CXCL12/CXCR4 signaling in AML and by modifying the bone marrow microenvironment
Authors: Sterner Rosalie , Greif Lauren E , Khan Rehan , Kwok Tina , Peterson Kevin L , Kaufmann Scott H , Kessler Linda , Scholz Catherine , Gualberto Antonio , Hedin Karen E
Published: February 2023

The prognosis of acute myeloid leukemia (AML) remains poor in part due to the leukemic bone marrow microenvironment. Our lab has found that CXCL12, a chemokine abundant within the leukemic bone marrow microenvironment, induces apoptosis of AML cells expressing CXCR4, the receptor for CXCL12. However, this CXCL12/CXCR4-induced apoptosis is inhibited by differentiating osteoblasts, which protect AML cells from apoptosis in the bone marrow. Tipifarnib is a farnesyltransferase inhibitor shown to increase progression-free survival in AML patients that express high levels of CXCL12. Here, we report that tipifarnib inhibits the CXCL12/CXCR4-directed migration of AML cells via an ERK independent pathway. Furthermore, tipifarnib enhances CXCL12/CXCR4-mediated AML cell apoptosis via a mechanism that alters expression of apoptosis-regulating proteins. In addition, tipifarnib disrupts AML protection by osteoblasts, increasing AML cell apoptosis. Tipifarnib inhibits the osteoblast-mediated protection of AML cells via disrupting COL1A1 and TNAP, proteins essential for extracellular matrix production. In conclusion, tipifarnib alters the bone marrow microenvironment which is predicted to enhance eradication of AML via inhibiting CXCL12/CXCR4 directed cellular migration of AML cells, reducing the protective effects of differentiating osteoblasts by disrupting matrix protection proteins, and increasing CXCL12/CXCR4-mediated apoptosis.
Investigating the role of KLRG1 in regulatory T-cells and implications for anti-tumor immunity in Non-small Cell Lung Cancer
Authors: Bag Arup K , Schultz Andrew R , Goala Payal , Karreth Florian A , Adeegbe Dennis O
Published: February 2023

Although it is well established that CD4+CD25+Foxp3+ regulatory T cells (Tregs) dampen anti-tumor T cell responses in cancer, their molecular signature and clonal heterogeneity in NSCLC remains poorly understood. In this study, comparative phenotypic assessment of tumor and lymphoid tissue resident Tregs in KRAS/p53 mutant NSCLC mouse model revealed the presence of a distinct tumor infiltrating Treg population which express KLRG1. Compared to their counterparts, tumor associated KLRG1+ Treg showed higher expression of inhibitory receptors and activation markers, suggesting that KLRG1+ Tregs represent an activated subpopulation which may contribute to immunosuppression. Ex-vivo Treg suppression assay revealed that tumor associated KLGR1+ Tregs exhibited higher suppressive activity compared to KLRG1-Tregs. Although T cell development was normal in in-house generated KLRG1 KO mice including thymic and peripheral Treg numbers, KLRG1 KO Tregs showed less suppressive capacity compared to wild type, implying KLRG1 in Tregs functional program. Furthermore, α-KLRG1 antibody treatment in lung tumor bearing KP mice showed prolonged survival, accompanied by reduced proportion of KLRG1+ Tregs in the tumor. Finally, immunogenomic characterization of KLRG1 KO/wt and tumor infiltrating KLRG1+/− Tregs revealed distinct gene expression patterns and associated immune pathways. Collectively, our findings indicate that KLRG1 is not critical for Treg development and maintenance but may play a non-redundant role in Tregs differentiation and function in inflammatory settings. Therefore, targeting KLRG1 to curtail the inhibitory function of the highly suppressive KLRG1+ Treg subset could facilitate antitumor responses in NSCLC.
Perturbed function of natural killer cells by inflammatory cytokines in chronic myeloid leukemia
Authors: Kuznetsova Valeriya , Camacho Virginia , Patel Sweta , Matkins Victoria , Welner Robert S
Published: February 2023

Natural killer (NK) cells’ mature phenotype and counts positively correlate with prolonged treatment-free survival in chronic myeloid leukemia (CML), while dysfunctional NK cells are predictive of relapse in leukemic patients. However, the molecular drivers of NK cell alterations during leukemia remain unclear. Given an established role of inflammatory cytokines in CML progression, we define the leukemic environmental cues impacting NK maturation and function in a BCR-ABL chimeric mouse model of CML where NK cells do not carry the CML oncogene. Reflecting clinical observations, NK cell frequencies and numbers are severely decreased in leukemic mice. Moreover, terminally mature NK subsets are the most affected, implying preferential loss of cells with potent anti-leukemic activity. In addition, this phenotype suggests a shift toward the immature cytokine-producing state of NK development, which could favor the survival of leukemia-initiating cells. We map the transcriptional landscape of NK cells using single-cell RNA-sequencing from multiple tissues. NK cells exposed to CML microenvironment possess a lowered expression of surface activating receptors such as Nkp46 while upregulating inhibitory molecules Tim-3 and TIGIT. BCR-ABL reversion for six weeks partially restored these defects, consistent with findings in patients before and after TKI initiation. Finally, we found that leukemia-associated soluble factors dampen target-specific degranulation of NK cells by applying serum samples from healthy or leukemic mice. These data indicate that the altered cytokine milieu contributes to NK cell dysfunction and represents an optimal target for NK-boosting CML immunotherapies of leukemia.
NK-92 serial killers - characterization of potential limitations to cytotoxic capacity.
Authors: Navarrete-Galvan Lydia T , Guglielmo Michael , Sung Alexander , Smith-Gagen Julie , Lombardi Vincent , Merica Rebecca , Hudig Dorothy
Published: February 2023

The NK cell line NK-92 and transgenic variants are receiving attention as adoptive transfer immunotherapies to treat a range of malignancies. However, since NK-92s are themselves tumors, they require irradiation prior to transfer and are potentially susceptible to attack by patients’ immune systems. In the present study, we investigated NK-92 serial killing against various tumor cells as well as the effects of gamma-irradiation and ligation of the death receptor Fas (CD95) on NK-92 cells and the susceptibility of NK-92s to attack by primary NK cells. To evaluate serial killing, we used a 51Cr-release assay with very low effector to target (E:T) Raji, Daudi or K562 tumor cell ratios at 2, 4, 6, and 8 hour time points. NK-92s that were maintained in high concentrations of recombinant IL-2 were able to kill as many as 14 Raji cells per NK-92 cell in 8 hours at an E:T of 1:32. NK-92 cells retained high cytotoxic activity immediately after irradiation with 10 Gy but lost >50% activity 24 hours after just 7 Gy irradiation. Despite strong expression of CD95, NK-92s maintained viability after 24 hours of Fas-ligation with an anti-CD95 IgM as compared to cells treated with an isotype control antibody. While NK-92s survived 16 hours of exposure to unstimulated primary NK cells, they were killed when exposed to IL-2 activated primary LAK cells. In conclusion, we recommend that adoptive transfer of irradiated NK-92 cells into patients be made as quickly as possible post-irradiation of the NK-92s. Genetic modification of NK-92 cell lines to secrete IL-2 as an autocrine growth factor may need reconsideration because in vivo activation of endogenous LAK cells by IL-2 could reduce the number of NK-92 cells within tumors and thus decrease therapeutic efficacy.
Expression of Tim-3 drives Treg to an effector-like state with enhanced suppressive activity and altered response to tumor
Authors: Banerjee Hridesh , Nieves-Rosado Hector , Murter Benjamin Matthew , Kane Lawrence P
Published: February 2023

Regulatory T cells (T reg) are mediators effective anti-tumor immunity. We and others previously reported that 40–60% percent of T reg-infiltrating head and neck cancer (HNC) and other tumors highly express Tim-3, compared with about 5% in lymphoid organs. Tumor-infiltrating Tim-3+ T reg also have enhanced suppressive function. Using a novel mouse model with cell type-specific inducible Tim-3 expression, we show here that expression of Tim-3 by T reg is sufficient to drive Treg to a more effector-like phenotype, resulting in enhanced suppressive activity and increased tumor growth. TIM-3 +T reg also seem to be metabolically altered driven by active signaling in ERK and m-TOR pathway. We further see that T reg specific deletion of TIM-3 leads to decrease in T reg frequency in tumor infiltrating lymphocytes and delay in tumor progression. These findings suggest to a possibility of using Tim-3 as a target to alter the tumor progression and clearance highliting a qualitatively different role for TIM-3 in T reg cells. Thus, we propose that Tim-3 regulates anti-tumor immunity at least in part through enhancement of T reg function. To our knowledge, this is the first example in which expression of a single co-stimulatory molecule is sufficient to drive differentiation of T reg and alter immune response in this manner.
Loss of Adar1 increases T cell migration and prolongs survival in ovarian cancer
Authors: Gomez Stephanie , Grundy Erin E , Kanholm Tomas , Hadley Melissa , McDonald James I , Rentia Uzma , Kobyra Julie , Palmer Erica , Saunthararajah Yogen , Chiappinelli Katherine B
Published: February 2023

Novel therapies are urgently needed for ovarian cancer (OC), the fifth deadliest cancer in women. OC is generally characterized by an immunosuppressive tumor microenvironment (TME) and less than 10% of patients respond to immune checkpoint blockade (ICB) therapy, though better OC prognosis is associated with high tumor-infiltrating CD8 T cells. DNA methyltransferase inhibitors (DNMTis) remove methylation and activate transcription of double-stranded (ds)RNA in OC. These dsRNAs are sensed in the cytoplasm and trigger the induction of type I IFN and also the transcription of interferon-stimulated genes (ISGs). Adenosine deaminase 1 (ADAR1) is an ISG that edits mammalian dsRNA with an A-to-I nucleotide change. These edited dsRNAs cannot be sensed by dsRNA sensors, and thus ADAR1 can inhibit this type I IFN response. Our preliminary RNA-seq analysis from human OC cell lines indicates that RNA editing by ADAR1 is increased after DNMTi treatment.

To test the hypothesis that Adar1 loss will amplify the DNMTi-induced IFN response in OC, we implanted mice with syngeneic ID8 murine OC Adar1 knockdown (KD) cells or control cells and treated with DNMTi. We show that DNMTi treatment reduces tumor burden and extends survival in this OC model. Loss of Adar1 significantly prolongs survival in this model, which is further improved with DNMTi treatment. DNMTi treatment of murine OC cells combined with Adar1 KD increases T cell migration in transwell migration assays and also enhances expression of chemokines CCL5, CCL2, and CXCL10. We are currently performing immunophenotyping analyses to interrogate the composition of immune cells in the TME in the mouse model of OC. These studies thus describe a role of Adar1 in the DNMTi-induced immune response in OC.
The Role of CD4+ Tissue Resident Memory Cells in Oncogene Driven Lung Adenocarcinoma
Authors: Melese Etienne Sylvain , Franks Sarah Elizabeth , Cederberg Rachel , Seo Jung Hee , Lam Wan , Bennewith Kevin L , Lockwood William , Abraham Ninan
Published: February 2023

Lung cancer presents a global health risk that is responsible for over 2 million deaths worldwide every year. Tissue resident memory T cells (Trm) are critical for rapid protection to pathogens at entry points in the body, including the lung. Trm are identified as CD69+ CD103+ cells which remain in the tissue (i.e. lung), are longer-lived memory cells, and have antigen specificity. Many studies have shown clinical significance with tumour infiltrating lymphocytes (TIL) markers of CD103+ and CD69+ in solid tumour cancers. Few studies have addressed the direct or indirect mechanism of anti-tumour function of CD4+ Trm cells during tumour progression and how the tumour microenvironment impacts this. One primary role of CD4+ T cells in anti-tumour immunity is to provide aid to CD8+ effector cells. CD4+ Trm are known to have Th1 interferon responses to viral infection and Th2 type responses in allergic response leading to excess fibrosis. Little is known of the diversity of CD4+ Trm and their Type-1 or -2 phenotypes during cancer. In this study, we used a genetically engineered mouse model (GEMM) of lung adenocarcinoma (LAC), with doxycycline-inducible oncogene mutation KRASG12D. We observed a significant increase in PD-1+ CD4+ Trm with a Th2-like phenotype (CD44+ CD62L−CD103+ CD69+ PD-1+GATA3hi) in the KRASG12D tumour bearing lungs. When stimulated ex vivo with PMA/ionomycin, we found CD4+ CD69+ cells produced more IFNγ in the KRASG12DGEMM. To examine clinical relevance, we used the program KMplotter and found signatures of Trm and CD4+ Trm significantly correlated with increased survival in LAC patients. We intend to determine the functional relevance of these Th2-like CD4+ TRM cells in the tumour microenvironment.