bluuebunny/crossref_metadata_2025 huggingface dataset:
PD-1 mediated regulation of CD8+ T cells in the murine submandibular gland
Authors: Borys Samantha , Zemmour David , ImmGen T , Brossay Laurent
Published: August 2023

Immune checkpoints, including programmed cell death protein 1 (PD-1), restrain the immune response, maintain self-tolerance, and prevent excessive collateral damage. Immune checkpoint blockade has revolutionized cancer immunotherapy by unleashing exhausted immune cells to kill tumor cells, but can lead to immune related adverse events (IrAEs). Patients receiving PD-1 blockade can develop IrAEs including vitiligo, colitis, endocrinopathies, and sicca syndrome, an autoimmune disease characterized by the accumulation of immune cells in the salivary and lacrimal glands, leading to deterioration and dysfunction. Because PD-1 is highly expressed on CD8+ T cells in the naive submandibular gland (SMG), we hypothesized that loss of PD-1 is likely to disrupt the previously reported hyporesponsive microenvironment of the SMG, leaving patients particularly susceptible to IrAEs affecting this gland. Our data indicates that genetic loss of PD-1 results in increased CD8+ T cell number and frequency in the SMG of naïve C57BL/6 animals. To characterize this expanding population, we recently collaborated with the ImmGen T consortium to perform single-cell RNA-seq on SMG T cells of wildtype and PD-1 knockout mice. Preliminary analysis indicates that the expanding CD8+ T cells in the salivary gland of PD-1 deficient animals have a unique phenotype. These cells exhibit exhaustion characteristics including expression of transcription factors Nr4a2 and Tox, and inhibitory receptors LAG-3 and TIGIT. Altogether, our data provide insights for IrAE prevention and treatments in patients receiving PD-1 blockade.

This work was supported by NIH research Grants AI46709 (L.B.), AI-072073 (ImmGen T), and a Brown University Presidential Award (S.B.).
PI3Kδ: a key driver of effector differentiation under conditions of T cell exhaustion
Authors: Pichler Andrea C , Cannons Jennifer L , Golec Dominic P , Reilley Julie , McGavern Dorian B , Schwartzberg Pamela L
Published: August 2023

In chronic infection and cancer, prolonged antigen exposure drives a state of T cell hyporesponsiveness, called exhaustion. A small subset of TCF1+ progenitor exhausted stem-like T cells (pTex) cells, is required to maintain exhausted T (Tex) cells and respond to checkpoint blockade. Understanding mechanisms regulating these populations may help identify approaches to counter exhaustion. Phosphatidylinositol 3-kinase-d (PI3Kd) is an important component of T cell signaling. We have found that TCF-1 is repressed in CD8+ T cells from patients with Activated PI3K Delta Syndrome and in a mouse model (Pik3cd E1020K/+ mice) in acute viral infections. How activated-PI3Kδaffects T cell exhaustion is unknown. Using chronic LCMV clone 13 infection, we found that Pik3cdE1020K/+ mice rapidly die, yet surviving mice recover faster than WT. RNA sequencing revealed decreased expression of Tcf7, encoding TCF-1, in antigen-specific CD8+ T cells, along with loss of TCF-1+Tim3− CD8+pTex cells in Pik3cdE1020K/+ mice upon cl13 infection. Tex cells that were maintained during chronic infection in Pik3cdE1020K/+ mice displayed an effector-like phenotype, with expression of CX3CR1+ and KLRG1+, and increased effector functions, suggesting that these cells might induce early immunopathology yet increase viral clearance. P14 cell transfers, in conjunction with CRISPR-mediated gene disruption, revealed that loss of FoxO1 leads to loss of TCF-1+ cells, whereas the increase of effector cells is at least partially cell-extrinsic. Our findings suggest that PI3Kδ is a critical rheostat that balances effector vs exhaustion differentiation, providing insight for therapeutic strategies for exhaustion.
Pinpointing molecular regulators of CD8 T cell differentiation through multimodal single-cell profiling
Authors: Green William D , Gomez Amber , Mullins Genevieve Nicole , Green Jarred , Baldwin Albert , Milner Justin
Published: August 2023

CD8 T lymphocytes control infection and malignancy through differentiation into specialized cell states with discrete functional attributes. While CD8 T cell fate commitment is a transcriptionally and epigenetically controlled process, the transcription factors and chromatin modifiers governing CD8 T cell differentiation remain ill-defined. Here, we leveraged mouse models of viral infection and cancer to simultaneous profile the chromatin and transcriptomic landscapes of virus-specific and tumor-specific CD8 T cells at single-cell resolution. Through in-depth comparison of antigen-specific CD8 T cells across distinct disease settings, we define uncharacterized CD8 T cell states and clarify roles for molecular regulators of T cell heterogeneity. Comparison of uniquely accessible regions of chromatin along with predicted transcription factor activity uncover regulatory networks that both guide and enforce CD8 T cell fate commitment into novel and established states spanning T cell memory, residency, and exhaustion. Further, we have functionally defined novel roles for several transcriptional regulators in controlling CD8 T differentiation during infection and cancer, including undescribed roles for the transcription factor Runx3. Together, our findings clarify CD8 T cell heterogeneity and differentiation trajectories across acute infection, chronic infection, and cancer. We show our findings can be leveraged to enhance promising immunotherapy approaches.

AAI Intersect Fellowship Program for Computational Scientists and Immunologists (2022-2023)
Investigating memory CD8 T cell-mediated immunopathology
Authors: Wall Sarah M , Jarjour Nicholas N , Jameson Stephen C
Published: August 2023

Bystander memory CD8 T cells can respond to other pathogens independently of their T cell receptors (TCRs). Current dogma is that these memory CD8 T cells can improve control of unrelated acute infections via TCR independent, innate-like functions. Contrary to this, we observed that mice with memory CD8 T cells with the transgenic P14 TCR that is specific for lymphocytic choriomeningitis virus (LCMV), or P14 memory chimera mice, succumb to a typically sublethal bacterial infection with Listeria monocytogenes (Lm). However, mice with a reduced number of transferred P14 T cells are less susceptible to Lm, suggesting that a high frequency of P14 CD8 T cells leads to increased lethality of Lm infection. At day 3 post Lm infection, LCMV-experienced mice have significantly higher bacterial burdens in the spleen and liver compared to controls. Preliminary data also suggests that certain cytokines may be involved in this process. We are investigating possible mechanisms of bystander memory CD8 T cell-mediated immunopathology. This work highlights that bystander CD8 memory T cells are not unequivocally protective but can also enhance susceptibility to non-cognate infections.

Supported by NIH grant, R01 AI38903.
Viral infection in the brain induces priming and maintenance of a novel antigen specific CD8 T cell population within the bone marrow that dysregulates the stem cell niche
Authors: Wolf Delaney , Tritz Zachariah P , Reesman Rachael A , Rangel Frances A , Bamkole Michael A , Pfaller Christian K , Maynes Mark A , Hansen Michael J , Jin Fang , Fain Cori E , Johnson Aaron J , Ayasoufi Katayoun
Published: August 2023

Bone marrow is a primary immune organ responsible for stem cell maintenance and hematopoiesis. While the bone marrow niche is mainly studied in the context of hematopoiesis, aging, and mechanisms underlying stem cell and plasma cell survival, changes of the niche during brain viral infections and primary T cell responses is unknown. Intracranial infection with the neurotropic Theiler’s Murine Encephalomyelitis Virus (TMEV) results in an acute infection in the C57BL/6 mouse which is cleared 30 days post infection due to generation of an antigen specific CD8 T cell response raised against the immunodominant epitope VP2121–130. We determined that 7–10% of CD8 T cells present in the sternal and femoral bone marrows and 15–20% in the cranial bone marrow were antigen specific 7 days post intracranial TMEV infection. Over 70% of these CD8 T cells were located outside of the vasculature as determined by IV labeling. Importantly, continuous treatment with FTY720, which sequesters T cells outside of the blood, did not eliminate antigen specific CD8 T cells in any bone marrow compartment. CD8 T cells in the bone marrow established durable memory following viral clearance and were reactivated upon cognate antigen reencounter. Both acute TMEV infection and antigen specific reactivation caused an increase in lineage−, Sca-1+, ckit+ (LSK) cells in the bone marrow. This LSK dysregulation was abrogated upon CD8 T cell depletion. We conclude that brain viral infections induce in situ effector and memory T cell responses within the bone marrow compartment. Both acute and memory recall CD8 T cell responses cause stem cell dysregulation. Our data hence paves the way for crucial studies of bone marrow resident antigen specific CD8 T cells in health and disease.

Supported by grants from NIH (K99NS117799-01A1 (KA), R01NS103212 (AJJ), and RF1NS122174(AJJ)
Harnessing the microbiome to regulate systemic innate immunity
Authors: Mihori Saki , Nichols Frank , Provatas Anthony , Blesso Christopher N , Clark Robert B
Published: August 2023

The mechanisms used by the gastrointestinal microbiome to regulate systemic immunity remain unclear. We have identified a microbiome Bacteroidota-derived lipopeptide—L654—that is a TLR2 ligand, accesses the systemic circulation, and thus potentially links the microbiome and systemic innate immunity. We previously documented that Multiple Sclerosis patients have significantly lower serum L654 levels than healthy controls, leading us to postulate that: 1) L654 regulates systemic innate immunity by entering the systemic circulation and mediating TLR2 interactions that maintain “normal” levels of innate immune signaling feedback inhibitors; and 2) inadequate systemic L654 levels result in poorly regulated, enhanced innate immune responses. In prior proof-of-concept studies, we reported that increasing systemic L654 levels by administering exogenous L654 intravenously significantly diminishes systemic innate immune responses and attenuates murine autoimmunity. In the present study, our goal was to document the converse effect, i.e., that decreasing microbiome L654 production enhances systemic innate immune responses. Using wild-type C57BL/6 mice, we report that decreasing microbiome Bacteroidota using specific non-absorbable oral antibiotics reduces fecal and serum L654 levels and significantly enhances innate immune responses. Moreover, the intravenous administration of L654 to antibiotic-treated mice prevents this innate immune enhancement. Overall, our results suggest that L654 is: 1) used by the microbiome to regulate systemic innate immunity; and 2) a manipulatable target capable of enhancing or decreasing innate immunity in the context of infectious, malignant, or autoimmune diseases.

UConn Health Center Academic Advancement Fund
Mucus sialylation determines intestinal host-commensal homeostasis
Authors: Kim Girak , Yao Yikun , Chen Zuojia , Wu Chuan , Lenardo Michael J
Published: August 2023

Intestinal mucus forms the first line of defense against bacterial invasion while providing nutrition to support microbial symbiosis. How the host controls mucus barrier integrity and commensalism is unclear. We show that terminal sialylation of glycans on intestinal mucus by ST6GALNAC1 (ST6), the dominant sialyltransferase specifically expressed in goblet cells and induced by microbial pathogen-associated molecular patterns, is essential for mucus integrity and protecting against excessive bacterial proteolytic degradation. Glycoproteomic profiling and biochemical analysis of ST6 mutations identified in patients show that decreased sialylation causes defective mucus proteins and congenital inflammatory bowel disease (IBD). Mice harboring a patient ST6 mutation have compromised mucus barriers, dysbiosis, and susceptibility to intestinal inflammation. Based on our understanding of the ST6 regulatory network, we show that treatment with sialylated mucin or a Foxo3 inhibitor can ameliorate IBD.

Supported by the Intramural Research Program of the NIAID, NIH, United States and by National Multiple Sclerosis Society Career Transition Award, United States (TA 3059-A-2 to C.W.).
Bi-allelic mutation of CARD11 resulting in fatal immune dysregulation with dramatic B cell expansion
Authors: Markle Janet G , Bhattad Sagar , Gil Deb , Ruchinskas Allison , Snow Andrew L
Published: August 2023

Caspase recruitment domain family member 11 (CARD11) functions as a scaffold to bridge antigen receptors with downstream immune signaling pathways that enable antigen-dependent lymphocyte activation. Given its pleiotropic role in regulating adaptive immunity, different genetic variants in CARD11 can result in diverse phenotypic consequences in humans. Here we describe a patient harboring a novel bi-allelic CARD11 mutation that results in potentially fatal immune dysregulation with massive B cell expansion. He presented with splenomegaly, amenia, leukocytosis and thrombocytopenia within the first year of life. A sibling showed similar symptoms before passing away in infancy. Due to parental consanguinity, an autosomal recessive inborn error of immunity was suspected; whole exome sequencing revealed a homozygous missense mutation in the coiled-coil (CC) domain of CARD11 (p.Arg331Pro, R331P). Functional testing revealed the R331P mutation caused a mild gain-of-function effect relative to wild-type CARD11 in driving constitutive NF-kB activation, suggesting that R331P may disrupt interactions needed to maintain CARD11 autoinhibition in resting lymphocytes. Dramatic B cell lymphocytosis noted in the patient was not present in his heterozygous parents, indicating that 2 mutant alleles are required for this phenotype. In contrast, the patient’s T cells responded poorly to TCR stimulation with severely reduced proliferation. Collectively, these results indicate that the novel R331P CARD11 mutation manifests as the first known recessive form of B cell Expansion with NF-kB and T cell Anergy (BENTA disease), and illustrate how rare monogenic inborn errors of immunity provide a unique discovery platform for basic immunology.
Differential responses to R848 and IgG/IgM in EBV-transformed B cells from individuals with Down syndrome compared to disomic individuals
Authors: James Stephanie , Rosastri Eustacia
Published: August 2023

Down syndrome (Trisomy 21; T21) results from an extra copy of chromosome 21 and causes a myriad of abnormalities, including immune dysfunction. Recent studies of PBMCs in those with T21 have shown reduced numbers of naive B cells, and increases in differentiated B cells, perhaps contributing to increased autoreactive and inflammatory conditions. The receptor for IFNα is located on chromosome 21, and has been shown to be expressed and functional on cells with T21. IFNα has been suggested to play a role in B cell responses, but little is known about the role of IFNα and B cell interaction in those with T21. Herein, we sought to further evaluate B cells from individuals with T21 following in vitro stimulation, with the hypothesis that cells with T21 would have greater B cell activation and cytokine production due to the extra IFNα receptor, compared to their disomic counterparts. EBV-transformed B cells from individuals with and without T21 were cultured and stimulated with R848/IL2, IgG/IgM cocktail or media as a control. In our unstimulated controls our results were in alignment with previous studies that suggest B cells from those with T21 are more terminally differentiated; we also observed significantly increased expression of the activation marker CD86 on these cells compared to disomic individuals. However, when stimulated with either R848 or IgG/IgM we did not observe a significant difference in the ability of B cells to express activation marker CD86. No significant differences were observed among cytokine production. Thus, the extra IFNα receptor may not help in B cell responses to infections and instead drive B cells into a more consistent inflammatory state.
Development and application of a high-content biology-based assay to quantify effects of modulators of NETosis in primary human neutrophils
Authors: Loomans C J M , Lin Xin-Xuan , Sciarrillo Rocco , Scandiuzzi Lisa , Stallen Jan , Stavenuiter Fabian , Vlaming Marijn
Published: August 2023

Neutrophils represent the first line of defense against pathogens by using multiple mechanisms, including phagocytosis, degranulation, and the release of neutrophil extracellular traps (NETs). NET formation can lead to a unique form of programmed cell death in neutrophils, called NETosis. In addition to its microbicidal function, compelling evidence has linked NETosis to the pathogenesis of diseases including autoimmune diseases, atherosclerosis and thrombosis.

To facilitate drug discovery programs for NETosis modulators, a high content biology-based method was developed and applied to blood derived primary human neutrophils. In summary, NETosis was triggered by PMA or BSA-Immune Complexes and the loss of membrane integrity together with spreading of NET formation beyond cell membranes as well as nuclear decondensation were captured in a live-cell setting on a CellVoyager (CV8000) platform. Furthermore, after addition of NETosis modulators (DPI and R406) to the triggered-neutrophils, a dose-dependent NETosis reduction was observed with optimal assay windows between 2.5 and 4 hours after trigger addition. Based on their distinct changes in nuclear morphology and loss of membrane integrity, this method was also able to distinguish various mechanisms of neutrophil cell death including NETosis and apoptosis. This procedure was successfully applied in medium-throughput screenings (96-well plate format), assessing dose-dependent inhibitory effects of selected large molecules, small molecules, and lipids on NETosis processes.

In conclusion

We developed a high content biology-based approach that can be employed to advance drug discovery for therapeutic areas associated with NETosis.

NA
GIMAP5 regulates a lymphocyte longevity assurance pathway
Authors: Gabrielski Justin Q , Park Ann Y , Lenardo Michael J
Published: August 2023

Elucidating the molecular mechanisms of immunodeficiency diseases is a powerful approach to discovering new immunoregulatory pathways in humans. One such previously unknown pathway is that of GIMAP5, an evolutionarily conserved GTPase expressed in T and NK lymphocytes, also known as the gene mutated in the Biobreeding diabetic rat and Sphinx lymphopenic mice. To better understand this disease association, we identified causative GIMAP5 mutations in 17 individuals of 9 kindreds with lymphopenia, liver disease, splenomegaly, thrombocytopenia, lymphadenopathy, lung infections, and lymphoma. As the mechanism of action, we now report that GIMAP5 controls the pathological accumulation of long chain ceramides. The ceramide synthase inhibitor, fumonisin B1, rescues GIMAP5-deficient T cells from ceramide overaccumulation and cell deterioration. Concordant with these findings, induction of ceramide accumulation and cell death by exogenous C6-ceramides in human T cell blasts was reduced by fumonisin B1 and GIMAP5 overexpression. Our work reveals the underlying long-chain ceramide mechanics of GIMAP5, opening new opportunities for further metabolomic studies of immunodeficiency diseases and targets for the development of therapeutics. This research was supported by NIAID by the Intramural Research Program of the NIH.
ITK inhibition improves the response to immune checkpoint blockade in solid tumors
Authors: Li Ling , Zhao Manzhi , Kiernan Caoimhe H , Castro Eiro Melisa D , Dammeijer Floris , van Meurs Marjan , Brouwers-Haspels Inge , Wilmsen Merel , Grashof Dwin G B , van de Werken Harmen J G , Hendriks Rudi W , Aerts Joachim G , Mueller Yvonne M , Katsikis Peter D
Published: August 2023

Cytotoxic CD8+ T cell (CTL) exhaustion is driven by persistent T cell receptor (TCR) stimulation in chronic infections and tumors. The exhausted state of T cells is characterized by impaired effector function and proliferative capacity, upregulation of multiple inhibitory receptors and altered transcriptional profiles. The phenotypic and epigenetic changes of these cells hinder effective antitumor immunity and immunotherapy. Therefore, reinvigorating exhausted T cells may provide significant clinical benefits to patients with cancer. Our approach is to reduce chronic TCR stimulation by inhibiting IL2-inducible T-cell kinase (ITK), a pivotal component of TCR signaling. In our study, we found that intermittent ITK inhibitor treatment significantly improved ICB response in three immune checkpoint blockade (ICB) -resistant solid tumors (melanoma, mesothelioma or pancreatic cancer) in vivo. In vitro, we demonstrated that specific ITK inhibitor and ibrutinib, an inhibitor of Bruton’s tyrosine kinase (BTK) and ITK, directly reversed the exhaustion-related phenotypes in exhausted CTLs. The changes included enhanced cytokine production, decreased inhibitory receptor expression and changes in transcription factors. Subsequently, RNA-seq data revealed that exhaustion-related transcriptional profile of in vitro CTLs is reduced by ibrutinib treatment. Ibrutinib mediated reversal of CTL exhaustion was BTK independent. To conclude, our study demonstrates that ITK inhibition can be used to directly ameliorate CTLs exhaustion and overcomes immune checkpoint blockade resistance in solid tumors.

This work was supported by the department of Immunology, KWF Grant 128371 (PDK), in part from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 779295 (PDK), and by the China Scholarship Council for funding PhD fellowships (No. 201506160120 for MZ and 201906210055 for LL).
A new PD-L1 antibody H1A causes PD-L1 degradation and sensitizes cancer cells for cytotoxic therapy
Authors: Tu Xinyi , Mutter Robert , Lou Zhenkun , Dong Haidong
Published: August 2023

PD-L1/PD-1 receptor pathway represents an important way of cancer cells to escape immune surveillance, yet only small portions of patients can benefit from PD-1/PD-L1 inhibitors. This is at least partially due to incomplete understanding of PD-1/PD-L1 biology in cancer. Although the interaction of extracellular domains between PD-L1 and PD-1 on cancer cells and immune cells is well studied, the function of PD-L1 inside cells, however, has not been well established. In our previous data, we found that intracellular PD-L1 acts as an RNA binding protein and interacts with NBS1 and BRCA1 mRNAs. Furthermore, intracellular PD-L1 protects targeted RNAs from degradation by RNA exosome, therefore increasing cellular resistance to DNA damage. Since Clinical approved PD-1/PD-L1 inhibitors have little effect on intracellular PD-L1, we sought to find new drugs to target intracellular PD-L1. Here we developed a new PD-L1 antibody, H1A, which has a different effect on PD-L1. Instead of blocking of PD-1/PD-L1 interaction, we found that this antibody disrupts the interaction of PD-L1 and CMTM6, which leads to degradation of PD-L1 by the lysosome. Therefore, H1A can unleash host antitumor activity and enhance the effects of DNA damaging therapy in cancer cells at the same time. Indeed, applying H1A in vivo significantly sensitized tumors to radiation and chemotherapy in a PDX models. Moreover, in a humanized immune competent TNBC PDX model, H1A monotherapy exhibited better therapeutic efficacy compared with clinically available PD-L1 inhibitor, suggesting that H1A activates anti-tumor immunity through PD-L1 degradation. Therefore, H1A may be a potential new drug to target both intracellular and extracellular PD-L1 to improve cancer therapy.
Neutrophils Extracellular Traps (NETs) Inhibition Improves PD-1 Blockade Immunotherapy in Triple-Negative Breast Cancer
Authors: Zhang Hongji , Fa Pengyan , Tsung Allan
Published: August 2023

Introduction

Despite significant advances using immune checkpoint blockade therapy for patients with advanced cancer, 40–85% of patients fail to respond to therapy. Increasing evidence suggests that the tumor immune microenvironment plays a critical role in rendering the immune checkpoint blockade ineffective. As such, targeting the tumor immune microenvironment is an attractive strategy for the treatment of solid tumors and an important approach to improve the efficacy of checkpoint inhibitors. Our study explores the combination therapy of DNase I and PD-1 antibody to prevent tumor growth. We demonstrate that this combination treatment for triple-negative breast (TNBC) cancer shows superior efficacy in comparison to a single-agent treatment, both in vivo and in vitro.

Methods

For 4T1 tumor models, 0.1-ml sample containing 104 tumors was injected into the mammary fat pad of female BALB/c mice. Tumor diameters were measured using calipers. The tumor volume was calculated. Anti-PD-1 or IgG2a isotype Ab (10mg/kg, every 3 days) and DNase I (5 mg/kg, daily) were administered intraperitoneally starting from day 5 after tumor cell inoculation.

Results

DNase I plays a pivotal role in the degradation of NETs, significantly dampening the resistance to anti-PD-1 blockade in a mouse TNBC model by attenuating tumor growth. Remarkably, DNase I decreases tumor-associated neutrophils and the formation of NETs in vivo. Mechanistically, the inhibition of NETs with DNase I results in the reversal of anti-PD-1 blockade resistance through increasing CD8+ T cell infiltration and cytotoxicity. These findings signify a novel approach to targeting the tumor microenvironment using DNase I alone or in combination with immune checkpoint inhibitors.

R01CA214865-06 (Renewal) Surgery triggered immune response and liver metastases. NIH/NCI 1R01 CA214865-01. Surgery triggered immune response and liver metastases.
Pharmacologic tumor PDL1 depletion as a novel approach to overcome treatment resistance
Authors: Murray Clare , Bai Haiyan , Ontiveros Carlos Oscar , Blinkiewicz Paige , Galvan Eva , Deng Yilun , Souto-Padron Alvaro , Curiel Tyler J
Published: August 2023

Tumor surface PDL1 canonically engages immune cell PD1 to inhibit anti-tumor immunity. However, PDL1 also mediates tumor-intrinsic pathologic signals, including promoting the DNA damage response (DDR) and suppressing immunogenic STING. Genetic tumor PDL1 depletion (CRISPR/Cas9, shRNA) induces novel treatment vulnerabilities that are not replicated by αPDL1 antibodies, but genetic tumor PDL1 depletion is not clinically feasible. We hypothesized that pharmacologic tumor PDL1 depletion could replicate genetic PDL1 depletion effects as a highly translational strategy to create novel treatment vulnerabilities. We conducted a high-throughput drug screen that identified 15 tumor PDL1 depleting drugs (PDDs) that potently reduce tumor PDL1 and are FDA-approved. Cefepime (β-lactam antibiotic) is a PDD that induced PDL1-dependent Chk2 DDR protein depletion (immunoblots), Chk1 inhibitor sensitivity in vitro (MTT, p<0.0001) and in vivo in wild-type and NSG mice (p=0.03), and immunogenic STING signals (immunoblots, RT-qPCR). The PDD telmisartan (angiotensin II receptor blocker) induced PARP inhibitor sensitivity in vitro (p<0.0001), impaired homologous recombination DDR (DR-GFP reporter, immunoblots), and induced STING signals and immunogenic cell death (flow cytometry). The PDD chlorambucil (alkylating agent) selectively depleted tumor PDL1 in vivo and sensitized αPDL1-resistant tumors to αPDL1 in a tumor PDL1 and NK cell-dependent manner. Thus, PDDs deplete tumor PDL1 to inhibit its pathologic cell-intrinsic signals, induce novel treatment vulnerabilities, improve tumor immunogenicity, and are rapidly translatable.

Supported by grants from NIH (Graduate Research in Immunology Program training grant T32 AI138944, TL1 TR002647, T32GM113896, CA204965, CA054515) and the Clayton Foundation (no grant number).
DRIVE: Diversity, Representation, and Inclusion for Value in Education – interprofessional faculty development for a bias-free curriculum at UMass Chan Medical School
Authors: Gerstein Rachel , Carter Yasmin , Haley Heather-Lyn , MacGinnis Christine , Peterson Kenneth , Hogan Susan , Whitehouse Amanda , Taweh Omar , Igiesuoboro Oghomwen , Herbert Carly , Fischer Melissa
Published: August 2023

Objective

The DRIVE Initiative engages interprofessional faculty and learners at UMass Chan Medical School to build tools and support for educators to identify and mitigate bias in their educational materials and teaching.

Methods

Key DRIVE resources include a core skills workshop, taught by representatives from the Graduate, Nursing and Medical schools. To encourage active application of these skills, there is a printable curriculum appraisal tool and an independent learning module. The DRIVE team also offers individual consultation for faculty as well as discussion across our academic community through weekly DRIVE-in cafés.

Results

In 2021, 16 core skills workshops were held with 84 faculty attending. Monday DRIVE-in cafés were started; the associated interactive library guide which links to curated resources and the anonymous reporting tool was accessed 922 times.

Discussion

While the workshop is required for core faculty in the School of Medicine and highly encouraged for all others, we have noted gaps in uptake particularly in basic sciences (Graduate School) and are increasing efforts in that area. Challenges have included demonstrating relevance to faculty across disciplines e.g., loose connection to basic science teaching. To increase adoption, materials and programs have been continuously updated and improved based on feedback, and workshops tailored to specific audiences. Recruiting compensated faculty from multiple schools has been vital to our success.

Conclusion

Biased curricula harm our learning environments and those who teach and learn within them, and lead to the perpetuation of disparities: the interprofessional approach of DRIVE has demonstrated success in mitigating this.
Assessment of factors that impact the quality and penetrance of immunology into the curriculum at the secondary education level in Alabama
Authors: Bruns Heather A , Evans Paulette , LaChenaye Jenna , Justement Louis B
Published: August 2023

Immunology as a discipline has significant relevance to health and disease and recent advances have harnessed the immune system to better fight against infectious diseases, as well as to address a wide range of illnesses that include allergy/asthma, autoimmunity, and the broad spectrum of inflammatory disorders. Therefore, immunology is a discipline that warrants increased attention not only at the graduate and professional school level, but at the undergraduate level. In spite of this, an analysis of approved undergraduate majors has shown that there are only a handful of programs at the undergraduate level that have a primary focus on understanding the immune system. Subsequent studies to understand the factors that influence the decision of individuals to pursue an undergraduate degree in immunology revealed that the primary means of exposure is through formal educational experiences and that the timing of the experience is critical. Studies demonstrated that exposure to immunology at a later stage, as an undergraduate versus in high school correlated with the decision to pursue immunology. It was hypothesized that if and when immunology is introduced at the high school level, either the students themselves are not prepared to master the material, or the educational experience is suboptimal. To further assess these possibilities, secondary educators were surveyed to determine the extent to which immunological concepts are integrated into the biology curriculum and the comfort level of the educators with immunological concepts. The study supports the need for programs to expand educator familiarity with immunology and to foster the development of learning activities to reinforce student learning and interest in immunology.

Department support only
Results from a CURE course project reveal novel insights into the structure of bacterial Toll/interleukin-1 receptor (TIR) domain proteins
Authors: Snyder Michelle , Vallejo-Schmidt Tamara E , Palm Cheyenne , Obiorah Trinity E , Koudjra Rachida E , Snyder Greg A
Published: August 2023

Toll-like receptors (TLRs) are critical for innate immune signaling, and various bacteria express Toll/interleukin-1 receptor (TIR) domains to evade this signaling. Bacterial TIRs exhibit NADase activity, although it is unclear what role NADase activity plays in evasion of innate responses. To better characterize the NADase activity of the TIR from Acenitobacter baumanii (AbTIR), we used site directed mutagenesis to identify amino acids predicted to be important for NADase activity. Studies were carried out in a Course-Based Undergraduate Research Experience (CURE) involving 16–20 undergraduate research students per year. Students used Chimera to analyze the AbTIR structure and hypothesize amino acids they predicted would play a role in enzymatic activity. They then performed site-directed mutagenesis to create plasmids for expression of mutant AbTIR. Mutant TIRs were expressed, purified and tested for NADase activity. So far, our mutagenesis results suggest a potential conformational change in the NAD binding site upon NAD binding. Identifying amino acids critical for enzymatic activity may inform drug development for diseases associated with dysregulated TIR activity. In addition, the application of the CURE class to carry out these studies has provided project-based research training experiences for approximately 80 undergraduate students, about half of whom are members of groups traditionally underrepresented in science. Several students, including some who had limited research experience before completing the course, are currently pursuing graduate studies and research-based careers, highlighting the value of CURE-based projects for facilitating the inclusion of students from diverse groups in research.

TVS and TO were supported by the NIH Bridges to Baccalaureate Grant R25GM058264; CP was supported by the NIH Bridges to the Doctorate Grant R25GM119970, and the CURE course was supported by an HHMI Inclusive Excellence Grant
Teaching Methods and Instructional Efficacy of Virtual Course Presentation in a Pre-Clerkship Medical Immunology Module
Authors: Robinson Isabelle L , Ryan Madison R , Edwards Penny S , Chosed Renee J , Grier Jennifer T
Published: August 2023

In March of 2020, education across the world transitioned to virtual delivery and medical education was not exempt. This study explored the efficacy of virtual medical education in a pre-clerkship immunology course during the COVID-19 pandemic at the University of South Carolina School of Medicine Greenville. Course delivery methods and educational resources provided to students during either 2019 or 2020 were evaluated to generate a picture of the learning environment by year. Deidentified summative exam performance data from 204 students was analyzed across 61 matched questions that did not differ between the 2019 and 2020 exams. Statistical significance was determined by unpaired t-test using GraphPad Prism. As expected, in 2020, the content delivery shifted towards remote and asynchronous sessions. The total number of integrated clinical cases decreased in 2020 compared to 2019, although a greater percentage of the clinical cases directly mapped to the USMLE Content Outline in 2020. Formative multi-attempt online quizzes replaced in-person practice questions and the overall number of available review questions increased from 2019 to 2020. There was no significant difference in student performance between the two years on matched exam questions or on exam performance as a whole. While the switch to virtual learning offered fewer synchronous sessions for student-faculty interactions, modifications to course materials appears to have counteracted any potential learning loss. This suggests that virtual medical immunology education with adequate resources can be as effective as in-person delivery, which has implications for undergraduate and medical immunology instruction and for the entirety of medical education.
Reverse Mentoring in STEM: Changing Perspectives to Foster Dialogue
Authors: Banda France-Elvie , Seger Yvette , Brute Maribet
Published: August 2023

Reverse mentoring provides organizations with key insights into the personal and professional needs and preferences of staff members from a variety of backgrounds and experiences. Although pairing a senior-level mentee with a junior-level mentor has been successfully utilized by the corporate world for over a decade to help employees gain appreciation of colleagues’ lived experiences, traditional mentoring remains the norm in academic research settings. Given the well-documented barriers to participation and attrition of individuals from historically excluded groups from the professoriate, FASEB established a reverse mentoring program for scientists and staff of scientific societies to assess the ability of this strategy to foster a greater understanding and appreciation of differences between individuals at different career stages and lived experiences. Through participation in reverse mentoring, pairs learn to develop inclusive spaces and foster a culture of inclusive decision-making in their spheres of influence. This engagement will enable participants to better assess existing procedures and policies that create institutional barriers for historically excluded groups. Our presentation will summarize key findings from our pilot cohort of four reverse mentoring pairs, including applicant statistics, reverse mentoring curriculum, and preliminary participant feedback.
Chronic opioid use is associated with higher flu vaccine induced Ab response in HIV+ and HIV− individuals
Authors: Dang Christine Minh , Iyer Akshay , Nelson C Mindy , Feaster Daniel , Forrest David , Ghanta Priya , Pahwa Rajendra , Jayaweera Dushyantha , Rodriguez Allan , Tookes Hansel , Pallikkuth Suresh , Pahwa Savita
Published: August 2023

Opioid dependence is frequent in people with HIV (PWH), but impact of opioids on immune system of PWH is unknown. This study tested the hypothesis that chronic opioid use exacerbates known impairment of flu vaccine responses in PWH. Chronic opioid users were compared to non-opioid users in HIV+ and HIV− groups termed HIV+OP+ (n=28), HIV-OP+ (n=55), HIV+OP− (n=53), HIV-OP− (controls, n=58). HIV+ individuals were on ART with plasma virus load <200 copies/mL. Flu antibody titers were determined at pre- (T0) and 4 weeks post (T2) seasonal quadrivalent influenza vaccinations (2020–2022) by hemagglutination inhibition (HAI) along with a plasma cytokine score at T0. Vaccine response (VR) was defined as a 4-fold change (FC) in titer from T0 to T2. In Kruskal Wallis analysis, all 4 groups had increases in HAI titer from T0 to T2 (p<0.01 to p<0.0001) for antigens (H1N1, H3N2, B Victoria) and whole vaccine. HIV+ status was associated with decreased responses to all antigens but OP+ status in HIV+ and HIV− had higher response than HIV+OP−, both in T2/T0 FC and T2 titer for one or more vaccine antigens (p<0.05 to p<0.001). In regression analysis controlling for demographics, opioid use was associated with increased response to H1N1 and B Yamagata antigens. In a random forest model, predicted probability of VR was lower for HIV+ status and higher for OP+ status. Cytokine score was highest in HIV+OP+. Results of quantitative Ab response to flu vaccine refute our hypothesis that flu vaccine response would be impaired in PWH who chronically use opioids. Qualitative assessments of Ab and mechanism of opioid intersection with B cell function are ongoing to understand if opioid use overcomes humoral immune deficits in PWH.
Role of IgG glycans in severe COVID-19 inflammatory cytokine responses
Authors: Scherer Erin M , Sherman Jacob D , Karmali Vinit , Kumar Bhoj , Simon Trevor W , Bechnak Sarah , Ciric Caroline R , Gray Natalie , Huerta Christopher , Hussaini Laila , Johnson Brandi , Lee Matthew , Panganiban Bernadine , Panjwani Anusha , Samaha Hady , Wang Dongli , Anderson Evan J , Azadi Parastoo , Collins Matthew H , Rostad Christina A , Rouphael Nadine
Published: August 2023

Severe COVID-19 and multisystem inflammatory syndrome in children (MIS-C) are characterized by hyperinflammation. Severe COVID in adults is associated with development of afucosylated SARS-CoV-2 IgG that induce monocytes to secrete inflammatory cytokines. This study aimed to analyze the relationship between spike IgG Fc glycosylation and inflammatory markers during acute pediatric COVID or MIS-C or following COVID vaccination in adults.

We assessed cross-sectional and longitudinal samples from 195 participants: Adult vaccinees, acute COVID patients, and healthy controls, as well as pediatric acute COVID patients, MIS-C patients, and healthy controls. We developed capillary electrophoresis (CE) methods to analyze bulk and spike IgG Fc glycans and measured ten relevant cytokines/chemokines by multiplexed assay.

Analysis of healthy control bulk IgG by CE found 95.3–99.6% fucosylated, 2.7–6.9% bisected, 55.8–66.8% galactosylated, and 4.6–15.6% sialylated Fc glycans, compared to 99.8%, 6.7%, 45.4%, and 8.2% by mass spectrometry, respectively. IFN-γ, IL-10, CXCL10, and CCL3 were significantly increased in MIS-C compared to pediatric healthy controls; IL-10, CCL2, and TNF were only increased in severe adult COVID compared to healthy controls or less severe COVID. Initial results confirmed significantly more afucosylated spike IgG in adult severe COVID than afucosylated bulk IgG in controls.

Our high-throughput methods for IgG Fc glycan profiling generated comparable results to mass spectrometry. Continued assessment will determine if COVID- or vaccine-induced spike IgG glycoforms correlate with inflammatory markers. Future mechanistic work will examine how modified glycosylation induces inflammatory cytokines.

Supported by an IDCRC New Investigator Pilot Award to E.M.S. (NIH UM1AI148684), by the Centers for Disease Control and Prevention through a cooperative agreement with the Georgia Emerging Infection Program (grant no. U50CK000485), and by grant from NIH to P.A. (R24GM137782).
Streptococcus pneumoniae coinfection results in increased disease in a murine model of influenza B virus infection
Authors: Lorton Ana T , Beaver Emily , Ray Sean , Page Caroline , Shepard Justin , Tompkins Stephen Mark
Published: August 2023

Secondary Streptococcus pneumoniae (Spn) infection is commonly associated with increased hospitalization and death following seasonal influenza virus infection. The animal models of influenza A virus (IAV) and secondary Spn coinfection have described the increased morbidity and mortality leading to several proposed mechanisms of enhanced disease. Influenza B virus (IBV) causes 25% of seasonal influenza cases and is associated with bacterial coinfection, yet little is known about the immune response to IBV and Spn coinfection. Here, we established a mouse model of IBV and Spn coinfection to study disease in singly and coinfected animals and define mechanism(s) of enhanced disease. Like IAV and Spn coinfection, infection with IBV followed by Spn inoculation resulted in increased morbidity and mortality compared to IBV or Spn infection alone. In IBV primed mice, Spn titers were increased early following bacterial inoculation and Spn rapidly migrated to the lung. Analysis of cytokines in the lung showed increased pro-inflammatory cytokine (TNF-α, IL-1β, and IL-6) responses with secondary Spn infection, which may be associated with enhanced disease. These data suggest that IBV infection initiates innate immune responses that are rapidly enhanced with Spn infection, leading to worsening disease. Ongoing studies include analysis of infiltrating cells during coinfection and histopathologic analysis to complement the innate response data to define mediators of clinical disease.

NIH CIDER Contract #75N93021C00018
Development of a novel serology assay for the detection of IgG antibodies to identify exposures to orthopoxviruses
Authors: Lee William Thomas , Hunt Danielle T , Kulas Karen E , Howard Kelly M , Carson Kyle , Lamson Theresa , Chakravarthi Sandhya , Chave Karen , Bievenue Rachel , Rock Jean M , Styer Linda M , Noone Philip , Hogrefe Wayne , Cai Gianna Y , Bermúdez-González Maria C , Kleiner Giulio , Srivastava Komal , Simon Viviana , Feihel Dennis , McGowan Joseph P , Slifka Mark K , Yates Jennifer L
Published: August 2023

The World Health Organization has declared a Public Health Emergency for an ongoing mpox (formerly monkeypox) outbreak, due to multiple cases of mpox in regions of the world in which infections have not been previously seen. The outbreak is notable for the high degree of human-human transmissions spreading primarily through sexual contacts. Currently, as a prophylactic (and post-exposure) measure, high-risk individuals are being offered immunization with cross-reactive vaccinia virus (VV) vaccines, a strategy previously used for the closely related variola (smallpox) virus. Commercial tests to measure binding antibodies to mpox for diagnostics or surveillance are not (yet) available. We present here a novel IgG detection assay to identify or measure binding antibodies to mpox and VV. This multiplexed, Luminex-based serology assay contains multiple recombinant proteins or peptides derived from VV and mpox. Because the assay contains antigens present on orthopoxviruses including VV, but also contain antigens present on orthopoxviruses except VV, the test allows for serological distinction between infection with virulent orthopoxviruses and attenuated strains of VV. We present data on detection of antibodies elicited by childhood (smallpox) or recent (mpox) vaccination, mpox infection, and breakthrough infection in previously vaccinated individuals. We demonstrate that the baseline antibody profiles of high-risk populations are distinct and must be considered when assessing mpox serology information. Overall, these data support the use of Luminex-based serology as a diagnostic tool for mpox infections and as a surveillance tool to detect the prevalence of mpox infections, even within a vaccinated population.

This work was supported by Cooperative Agreement Number NU50CK000516 funded by the Centers for Disease Control and Prevention. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the Centers for Disease Control and Prevention or the Department of Health and Human Services.
Characterizing the effect of type I interferon signaling within regulatory T cells in HSV-2 infection
Authors: Warrier Lakshmi , Graham Jessica B , Swarts Jessica , Lund Jennifer M
Published: August 2023

Herpes simplex virus type-2 (HSV-2) is a lifelong, recurrent sexually transmitted infection that affects about 13% of the world population between ages 13–49. There is currently no available vaccine for HSV-2 and understanding the nuances of cell-mediated immunity is crucial to developing improved therapeutics and vaccines. HSV-2 infection induces a potent, localized type I interferon (TIIFN) signaling cascade that can stimulate both innate and adaptive antiviral effector responses. Interestingly, regulatory T cells (Tregs), an immunosuppressive CD4+ T cell subset, also respond to TIIFNs. TIIFNs have previously been shown to transiently inhibit Tregs early during a systemic lymphocytic choriomeningitis virus (LCMV) infection to promote the development of antiviral CD4+ and CD8+ T cell responses, and Tregs that lack the interferon alpha receptor (IFNAR) have enhanced suppressive function. The role of TIIFNs in a mucosal infection such as HSV-2 is unclear and studying the tissue-specific effects of TIIFNs warrants further study. To investigate the role of TIIFN signaling within Tregs in the context of HSV-2, we infected IFNARfl/fl x FoxP3Cre mice intravaginally with attenuated or WT HSV-2 and assessed phenotypic Treg markers and viral titers within the vaginal tract by flow cytometry and plaque assays, respectively. Preliminary data indicates that WT, and not attenuated HSV-2 infected mice show enhanced disease progression and increased viral titers. This suggests that TIIFN signaling in Tregs could be aiding in mediating protection early in HSV-2 infection.

NIH R01AI141435