bluuebunny/crossref_metadata_2025 huggingface dataset:
Host and microbe adaptation underlying true fungal commensalism
Authors: Han Geongoo , Yunker Rebecca , Hasan Mohammad , Leblanc Brian , Pacia Jessica , Luong Hien , Beura Lalit , Vaishnava Shipra
Published: August 2023

Fungi are found ubiquitously in the mammalian gut however the role of commensal fungi in the host is poorly understood. Laboratory mice are used to model host-fungi interactions however due to ultra-clean housing they do not harbor fungi. The human commensal fungus Candida albicans is widely studied to dissect host-commensal fungi interactions however prior dysbiosis of commensal bacteria is required to colonize laboratory mice. Insights from such forced host-fungal interactions are unsuitable for extrapolating mechanisms underlying fungal commensalism and its breakdown. To discover the natural fungal commensal of mice, we used pet-store “dirty” mice. We found that pet-store mice have a high fungal burden in their feces and their gut is predominantly colonized by single species of fungus, Kazachstania pintolopesii. Furthermore, we found that K. pintolopesii isolated from pet-store mice, stably colonized laboratory mice to high levels in the presence of an intact immune system and commensal bacteria. Laboratory mice colonized with K. pintolopesii exhibited increased neutrophil frequency in blood but did not show any pathological symptoms. Surprisingly, even a systemic K. pintolopesii challenge did not show any pathology in laboratory mice indicating unique adaptations of this fungus to the murine host. Remarkably, we found that laboratory mice stably colonized with K. pintolopesii are significantly protected from systemic C. albicans challenge suggesting that commensal fungi promote systemic antifungal responses in their host. In summary, our work established a novel unmanipulated murine model of fungal commensalism and provide unique insight into cellular and molecular mechanisms of mammalian host and fungal mutualism.

This research was supported by grants from NIH (R01 DK113265), Searle Scholars program, and AAI Careers in Immunology Fellowship.
Innate lymphoid cells and interferons limit neurologic complications of Brucellosis
Authors: Moley Charles R , Chambers Catherine , Dadelahi Alexis , Ponzilacqua-Silva Bárbara , Abushahba Mostafa , Lacey Carolyn , Skyberg Jerod A
Published: August 2023

Brucellosis is a globally significant zoonotic disease. Human brucellosis patients develop flu-like symptoms and focal complications including arthritis and neurobrucellosis. The purpose of our study was to uncover the role of innate lymphoid cells (ILCs) in the pathogenesis of focal brucellosis. After footpad infection, we found NK cells and ILC1 cells both limit joint colonization by Brucella. Mice lacking NK cells, and in particular mice lacking all ILCs also developed marked arthritis after footpad infection. Following pulmonary infection, mice lacking adaptive immune cells and ILCs developed arthritis, neurologic complications, and meningitis. Adaptive immune cells and ILCs both limited colonization of the brain by Brucella following pulmonary infection. Transcriptional analysis of Brucella-infected brains revealed marked upregulation of genes associated with inflammation and interferon responses, as well as downregulation of genes associated with neurologic function. Type II interferon deficiency resulted in colonization of the brain by Brucella, but mice lacking both type I and type II interferon signaling more rapidly developed clinical signs of neurobrucellosis, exhibited hippocampal neuronal loss, and had higher levels of Brucella in their brains than mice lacking type II interferon signaling alone. Collectively, our findings indicate ILCs and interferons play an important role in prevention of focal complications during Brucella infection, and that mice with deficiencies in ILCs or interferons can be used to study pathogenesis of neurobrucellosis.

Supported by grants from NIH (T32 OD011126-4, R21 AI153074-02)
Neonatal helminth infection and chronic type 2 inflammation promotes immune suppression through FcγRIIb
Authors: Alexander Bridget , Miller Mindy M , Reinhardt R Lee
Published: August 2023

Allergic diseases are rising in industrialized countries. This contrasts with rural communities where decreased allergic incidence correlates with increased prevalence of intestinal helminth infections. While helminths and their byproducts can ameliorate allergic inflammation in adult mice with established disease, it remains unknown if early-life exposure can prevent or delay allergic disease onset. We utilized a neonatal infection model with the gastrointestinal helminth Heligmosomoides polygyrus and found that neonatal H. polygyrus colonization results in chronic infection and type 2 inflammation. This study investigates how chronic and systemic type 2 hallmarks IL-4 and IgG1 may be involved in the long-term suppressive mechanisms induced by neonatal helminth colonization. We hypothesize that IL-4-induced upregulation of the sole inhibitory Fc receptor, FcγRIIb, allows for sustained suppression of T cell mediated inflammation by increasing the activation threshold of antigen presenting cells. Increasing this threshold prevents allergen-specific T cells from becoming optimally activated and instead promotes T cell tolerance. In addition to IL-4 increasing expression of FcγRIIb on macrophages, it promotes the production of its ligand IgG1, completing the suppressive circuit. This model predicts that elimination of FcγRIIb and/or IL-4 will result in greater T cell activation and allergic inflammation. Our in vivo and in vitro data show that FcγRIIb is necessary for reducing allergic airway inflammation and T cell activation after allergen challenge. Taken together, these data support a model in which chronic helminth infection naturally mimics the suppressive effects of intravenous immunoglobulin therapy.

Supported by the National Institutes of Health NIAID training grant (Training Program in Immunology; T32-AI07405)
CD8+ lymphocytes contribute to immunologic bottlenecks in Mycobacterium tuberculosis infection
Authors: Simonson Andrew W , Chao Michael , Hopkins Forrest , Kelly Ryan , Chase Mike , Vickers Andrew , Chishti Harris , Borish Jake , Maiello Pauline , Lin Philana , Fortune Sarah , Flynn JoAnne L
Published: August 2023

Significance

Meta-analysis of human data suggests that M. tuberculosis (Mtb) infection confers protection against subsequent exposure. Our lab demonstrated this in a controlled experimental system using non-human primates. As the best model for natural immunity against TB, it is crucial to explore the mechanisms behind concomitant immunity. We hypothesized that CD8+ T cells would play a key role in preventing disease following secondary challenge.

Methods

Chinese cynomolgus macaques were challenged intrabronchially with a genetically barcoded library (LibP) of Mtb, allowing an adaptive response to develop. After clearing the primary infection with anti-TB drugs, CD8+ cells were depleted in half of the cohort. This was followed by a secondary Mtb challenge with a unique barcoded library (LibS). PET-CT scans enabled tracking of lesions that developed after secondary exposure. All granulomas and lymph nodes were isolated at necropsy and sequenced to determine the bacterial source (LibP vs. LibS). Spectral flow cytometry was used to analyze lymphocytes in individual tissues isolated at necropsy, examining the cytokines and other effector molecules produced by that may drive protection in the presence and absence of CD8+ cells.

Results

Our data support that CD8 depleted animals lose the ability to contain Mtb after reinfection. CD8+ cells do not appear to prevent establishment of infection independently. However, the bottleneck restricting dissemination, especially to lymph nodes, is loosened in the absence of this population. Thus, CD8+ lymphocytes are critical to immunological protection against Mtb and should be considered as a key component for vaccine and host directed therapy development.

Supported by NIH IMPAc-TB (HI-IMPACT) and T32 (5T32AI089443-13)
CD4+ T cells show a cytolytic phenotype functions in human visceral leishmaniasis
Authors: Singh Om Prakash , Chauhan Shashi Bhushan , Singh Siddharth Sankar , Engwerda Christian , Kumar Rajiv , Sundar Shyam
Published: August 2023

Control of Visceral leishmaniasis (VL), a parasitic disease caused by L. donovani, requires robust CD4+ T cells response to control the parasite replication by IFN-γ production and activation of macrophages. Our recent study on transcriptional signature of CD4+ T cell isolated from peripheral blood of active VL patients showed enhanced expression of genes related to cytotoxicity. In present study, we investigated expression of these cytotoxic molecules including granzyme B (GZMB), Granulysin, Perforin and NKG7 on different CD4+ T cell subsets to establish the role of GZMB in regulating CD4+ T cells functional capacity. PBMCs cells were isolated from active and post treated VL patients and endemic controls, and flow cytometry was performed. LAMP assay (CD107b expression) was performed to study the degranulation capacity of CD4+ T cells. Whole blood assay followed by ELISA was performed to measure antigen specific cytokine / GZMB production by CD4+ T cells. We found that activated and degranulating CD4+ T cells (CD38+CD107+) had higher expression of GZMB, Granulysin, perforin and NKG-7. Similarly, GZMB level in plasma and antigen stimulated whole blood culture supernatant were significantly elevated in active VL patient compared to post treatment and EC. There was no change is IFN-γ secretion when whole blood culture was stimulated with soluble leishmania antigen in presence of Con-A but a significant decline in IL-6 production was observed. In summary, VL CD4+ T cells show a cytolytic phenotype and further investigations are required to understand their differentiation and function, particularly for promoting anti-parasitic immunity for host protection and effective intervention or therapy.

Extramural Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health (TMRC grant number U19AI074321)
Metabolism and ontogeny of alveolar macrophages contribute to peripheral trained immunity and confer protection against Mycobacterium tuberculosis
Authors: Andrews James Tucker , Mercado Miguel , Huey Fischer , Pierce Katie , Ahmed-Braimah Yasir , Li Lin-Xi , Huang Lu
Published: August 2023

Lung alveolar macrophages (AMs) reside in the alveolar space and function as key sentinels to promote lung immune resilience. Notably, as the major cell target of early Mycobacterium tuberculosis (Mtb) infection, AMs provide a permissive cellular niche for rapid Mtb growth. However, mechanisms which regulate the permissiveness in AMs during Mtb infection remain elusive. Here, we aimed to identify intrinsic features of AMs that could be exploited to promote protective trained immunity against Mtb. Employing an intranasal β-glucan exposure model, we observed that AMs in mice treated with β-glucan acquire sustained and enhanced activation as well as metabolic reprogramming. These phenotypes are attributed to the development of monocyte-derived AMs (MoAMs) in CCR2- and innate-derived IFN-γ dependent manners. Intriguingly, MoAMs rather than pre-existing AMs exhibit canonical features of trained immunity, including a metabolic shift towards glycolysis, enhanced cytokine production and phagocytic activity. Chromatin accessibility analysis further revealed that ontogeny of AMs dictates their epigenetic landscape. Lastly, β-glucan treatment significantly reduces Mtb burden in the lung, associated with enhanced AM functions and increased productions of key proinflammatory cytokines from MoAMs. These results suggest that while tissue signals restrict the plasticity of AMs and limit their capability of being reprogrammed for vaccination and therapeutic purposes, ontogeny and metabolism are two essential intrinsic features regulating functions of AMs against Mtb infection. Furthermore, our study also highlights the importance of macrophage ontogeny as a novel determining factor of peripheral trained immunity.

Supported by grants from American Lung Association (CA-828143) and NIH (P20GM103625)
Plasmablast Ig repertoire dynamics through repeat Plasmodium falciparum challenges
Authors: Ferrer Patricia , Frank Andrew , Berry Andrea A , Lyke Kirsten E , Williamson Kim C
Published: August 2023

The development of natural immunity against malaria requires multiple exposures and even after protection develops against clinical disease individuals continue to be susceptible to infections. To study the dynamics of the immune response to repetitive P. falciparum (PF) infected mosquito exposures, we used controlled human malaria challenges with the same strain of PF. The homologous strain was used to eliminate differences in immune responses due to strain polymorphisms and focus on the maturation of the human immune response to repeat exposures. Consistent with the gradual immune response observed in the field, after 4 sequential exposures over the course of 2 years, symptoms decreased and the time to parasite detection on a blood smear increased significantly, but all the volunteers continued to be susceptible to infections. Anti-PF antibody levels increased over time and were able to inhibit liver cell invasion in an in vitro assay. The Ig repertoire of plasmablasts collected 7 days after peak parasitemia demonstrated large clonal expansions in one or two sequential infections that decreased after subsequent exposures. This pattern suggests a marked difference in the lineage of plasmablast and memory cells populations. Naïve B cells stimulated by PF to proliferate and differentiate into plasmablasts may fail to differentiate into memory cells that can produce both plasmablasts and memory cells following subsequent exposures. This differentiation pattern would lead to new sets of expanded clones at each PF exposure, instead of further maturation of the same antibodies generated during the first response. Such a response could enhance the diversity of the Ig pool, but would delay the generation of a strong, durable memory response

U0110852
Intestinal Epithelial Cell Intrinsic Zinc Homeostasis is Critical for Host-Microbiome Symbiosis
Authors: Yunker Rebecca , Han Geon Goo , Luong Hien , Vaishnava Shipra
Published: August 2023

A robust intestinal barrier is essential for maintaining host-microbiome symbiosis. Defects in the intestinal barrier are associated with a broad range of inflammatory and metabolic diseases. Pharmacological doses of Zinc (Zn) are widely used to improve intestinal barrier function; however, the precise cellular role of Zn in regulating the intestinal barrier during health and disease is not known. A better understanding of how Zn regulates the gut barrier and intestinal epithelial cells (IEC) could enable the development of targeted Zn-based therapeutic approaches. To understand the impact of Zn homeostasis we developed a mouse model with the transcription factor MTF-1 specifically deleted in IECs (MTFΔIEC). MTF-1 controls the expression of many different zinc importers and exporters. We find that MTFΔIEC mice had reduced expression of Zn homeostasis machinery, demonstrated a leaky barrier, and showed heightened neutrophil activation in the periphery. Additionally, MTFΔIEC mice had a higher bacterial load at the mucosa and were more susceptible to infection by Crohn’s disease associated adherent invasive E.coli. MTFΔIEC mice showed a striking loss of Paneth cells (PCs) from the small intestinal crypts. PCs are specialized antimicrobial-secreting cells with a unique requirement for Zn, but the exact role of Zn in PCs is not known. Based on our findings we conclude that Zn homeostasis is needed for regulating host-microbiome interaction via modulation of intestinal barrier permeability and maintenance of Paneth cells. Our work demonstrating the impact of zinc homeostasis on host-microbe interactions lays the groundwork for understanding zinc in the context of inflammatory bowel disease and dysbiosis in the mucosal microbiome.

This work was supported by the NIH [R01DK113265] to Shipra Vaishnava
Role of Bacteroides acidifaciens in gut inflammation during colitis
Authors: Staley Shanieka Caroline , Busbee Philip Brandon , Nagarkatti Mitzi , Nagarkatti Prakash S
Published: August 2023

Colitis is an inflammatory bowel disease characterized by chronic inflammation of the colon with significant disease-associated alterations in the gut microbiome, a term known as microbial dysbiosis. IBDs, are becoming more prevalent in industrialized areas, impacting over 1.6 million Americans. Our lab has previously shown that in mouse models of colitis, Bacteroides acidifaciens (BA) significantly increases in abundance during disease. In fact, disease severity directly correlates with the increase of abundance of this microbe. BA are obligately anaerobic, non-sporing, non-motile, gram-negative rods. This bacterium can degrade protective mucus in the colon and has properties linked to inflammation, such as enhancing immunoglobulin A (IgA) responses and producing short-chain fatty acid (SCFA) acetic acid. This study investigates the effects of the overgrowth of BA in mice with colitis as well as germ-free (GF) mice to access the ability of this microbe to induce or enhance colitis symptoms. Colitis was induced by administering the 3% dextran sulfate sodium (DSS) model. Results showed that compared to disease controls, conventional C57BL/6 mice inoculated with a high dose of BA via oral gavage had more severe colitis symptoms. These mice lost over 20% of their initial body weight, their colons were shorter, and their colons displayed ulcers, redness, and mucosal crystallization, suggesting BA significantly exacerbated the symptoms of DSS-induced colitis. Introducing BA into GF C57BL/6 mice resulted in a colitis-like phenotype with significant reduction in weight-loss and colon lengths. From this data, we deduced that BA may have the potential to induce colitis if overgrowth of this microbe reaches pathogenic levels.

Supported by NIH grant (5P20GM103641)
Nonencapsulated Streptococcus pneumoniae avoid innate immunity clearance through reactive oxygen species resistance
Authors: McDaniel Larry S , Thompson Courtney D , Holcomb Shelby , Keller Lance E
Published: August 2023

Neutrophils provide an important mechanism of clearance for Streptococcus pneumoniae. Therefore, evasion of the innate immune response is essential for pneumococci to cause disease. The pneumococcal capsule is protective against phagocytosis but nonencapsulated Streptococcus pneumoniae (NESp) lack this capsule. Despite the lack of capsule NESp effectively colonize and cause invasive and noninvasive disease. All invasive isolates of NESp express the oligopeptide transporters AliC and AliD, which are required for virulence in multiple animal models. AliC and AliD alter downstream gene expression, and it is hypothesized that one of these regulated genes is responsible for increased immune evasion of NESp. A mutant library of AliC/AliD regulated genes was created and tested for virulence in a Galleria mellonella model of infection. Genes of interest from the in vivo screen were tested for the ability to resist phagocytic killing by neutrophil-like cells. Resistance to killing through reactive oxygen species (ROS) was examined. Of the seven AliC/AliD regulated genes tested, LytFN1 and MgtC mutants were found to have significantly reduced virulence compared to wildtype. A 40% decrease in resistance to phagocytic killing was observed in both mutants, as well as significantly decreased survival during exposure to ROS from growth in 2.5 mM hydrogen peroxide. Expressing AliD in a pneumococcal background that does not natively have the oligopeptide transporter significantly increased virulence and ROS resistance. Regulation of gene expression by AliC and AliD promote pneumococcal survival and virulence by increasing resistance to ROS mediated clearance and partially complements the lack of the opsonophagocytosis resistant capsule.

Institutional funds
A gut commensal protist protects against virus-mediated loss of oral tolerance
Authors: Sanchez Luzmariel Medina , Zeng Yanlin , Siller Magdalena , Brigleb Pamela H , Sangani Kishan A , Dermody Terence S , Jabri Bana , Verdu Elena F , Meisel Marlies , Hinterleitner Reinhard
Published: August 2023

Loss of oral tolerance (LOT) to gluten, characterized by a T helper 1 (Th1) gluten-specific immune response, is a hallmark of celiac disease (CeD) and can be triggered by enteric viral infections. We hypothesized that certain gut microbes have the capacity to protect against virus-mediated LOT. By using our previously defined reovirus-mediated LOT CeD model, we discovered that the gut colonizing protist Tritrichomonas promotes oral tolerance and protects against reovirus-mediated LOT by suppressing the reovirus-induced proinflammatory program of dietary-antigen-presenting CD103+ dendritic cells. Importantly, Tritrichomonas-mediated protection against T1L-induced LOT is not attributable to differences in antiviral host responses and is independent of the microbiota. Mechanistically, we show that Tritrichomonas colonization restrains reovirus-induced inflammatory responses in dendritic cells and thus limit their ability to promote Th1 immune responses. Finally, our studies using human stool samples support a role for Tritrichomonas sp. colonization in protecting against development of CeD.

Supported by grants from NIH (T32 AI089443)
Type I and II interferons induce granulopoiesis and correlate with organ damage during infection-induced shock
Authors: Bahr Amber , Howard Jennifer , MacNamara Kate C
Published: August 2023

The mechanisms that drive pathology in infection-induced shock are not well understood. The tick-borne pathogen Ixodes ovatus erhlichia (IOE) induces severe inflammation, vascular damage, liver injury, and death. Mice that lack the receptor for type I IFN, or receptors for both type I and II IFNs, improves survival by 50–70 and 90%, respectively. IOE infection induced a significant increase in circulating granulocytes that depended on both type I and II IFNs. We also observed an IFN-dependent increase in eosinophils and basophils in the bone marrow in IOE-infected C57BL/6TA (WT). Although IOE infection induced an increase in bone marrow IL-5 and GM-CSF levels independent of IFN signaling, bone marrow concentrations of IL-3 and IL-13, both known to promote eosinophil differentiation and survival, increased in an IFNAR-dependent manner. Furthermore, we demonstrate an infection- and IFN-dependent increase of CCL3 and CCL5, two chemokines that promote eosinophil activation and migration. While bone marrow and blood eosinophils and basophils were increased, we observed a specific depletion of eosinophils in WT liver at day 7 post-IOE infection, correlating with increased cell death and severe liver pathology. In contrast, basophil frequencies were increased in IOE-infected WT mice. Additionally, Ifnar-deficient and Ifnar-Ifngr-deficient mice exhibited normal eosinophil and basophil frequencies and reduced liver inflammation, similar to uninfected mice. Together, our data reveal an IFN-driven cytokine and chemokine response that correlates with rampant granulopoiesis and mortality and provide evidence that IFN-induced eosinophil activation may contribute to vascular leak and liver damage in infection-induced shock.

Supported by grants from NIH R35GM131842
Exploring the Role of TVM Cells in Age-Related CD8+ T Cell Dysfunction through ATAC-seq and RNA-seq Analysis
Authors: Thiele Daniel , Nguyen Angela , Hussain Tabinda , Barugahare Adele , Bennett Taylah , Li Jasmine , Russ Brendan , Quinn Kylie , Turner Stephen J , La Gruta Nicole L
Published: August 2023

Virtual memory T (TVM) cells are a unique subset of CD8+ T cells that have both classical memory and innate-like immune function. Unlike other CD8+ T cell subsets, TVM cells are highly dependent on cytokines for their homeostasis and are selectively depleted in mice lacking IL-15. In aged mice and humans, TVM cells acquire T cell receptor-associated defects, which may contribute to the decline in overall CD8+ T cell function seen with aging. TVM cells make up a large proportion of the naive CD8+ T cell pool in aged individuals, making them an ideal target for age-specific therapeutic interventions. In this study, we performed ATAC-seq and RNA-seq analysis on young and aged mouse CD8+ T cell subsets and identified several key signatures associated with age-related dysfunction specifically in TVM cells. These signatures reveal a bifurcation in the aging process whereby memory-phenotype CD8+ T cells are affected differently by the aging environment. The aged-specific TVM signature is distinct from that of conventional memory CD8+ T cells and results in the upregulation of immunomodulatory molecules such as Fcgr2b, which encodes the low-affinity inhibitory Fcγ receptor. Expression of FcγRIIB on CD8+ T cells has been shown to play a role in suppressing anti-tumor immunity and mitigating graft-versus-host disease. We show that a subset of aged TVM cells upregulate FcγRIIB independently of antigen exposure and are prone to apoptosis in vitro. Blockade of FcγRIIB has been shown to augment CD8+ T cell responses in vivo, making it a potential therapeutic target for restoring function in aged TVM cells. These findings provide insight into the mechanisms of age-related immune dysfunction and potential strategies for targeting TVM cells in the elderly.

Supported by grants from NHMRC/ARC
BMI-1 repression of the CD8+ T cell effector program is essential for memory formation
Authors: Turner Stephen John , Udupa Vibha , Bruer Thomas , Sng Xavier , O’Hara Jessie , Barugahare Adele , Bennett Taylah , Zhang Zhongming , Degli-Esposti Mariapia , Russ Brendan
Published: August 2023

NaïveCD8+ T cell activation results in a program of proliferation and differentiation that results in large scale changes in phenotype and function that is critical for pathogen control and establishment of immunological memory. While these changes are underpinned by structural and biochemical changes in genome regulatory elements, the mechanisms that mediate these changes are not fully elucidated. Polycomb Repressive complexes (PRC) are chromatin remodeling complexes implicated in silencing genes encoding crucial developmental regulators. We demonstrated that upregulation of PRC1 components occurs rapidly upon naïve CD8+ T cell activation and was associated with maintenance of ubiquitination at key effector transcriptional regulators. Importantly, in vitro activation of PRC1-deficient CD8+ T cells resulted in greater proliferation and more rapid acquisition of effector function compared to wildtype cells. Use of two different viral infection models demonstrated that T cell specific PRC1 deficiency resulted in a greater effector expansion and effector differentiation with establishment of dysfunctional memory T cell populations. Strikingly, even after acute infection, the resulting memory T cells exhibited phenotypic and molecular markers of T cell exhaustion and were unable to be recalled. Our data suggest that PRC1 regulation early after CD8+ T cell activation is essential for restraining early effector T cell commitment and is a key step in establishing immunological CD8+ T cell memory.

Supported by grants from National Health and Medical Research Council of Australia (Ideas Grant 2020562).
TCF-1 dictates lung tissue resident helper T cell and memory B cell development during flu
Authors: McGee Michael C , Magazine Nicholas , Zhang Tianyi , Babawale Pius , Huang Weishan
Published: August 2023

Influenza (flu) infections are a significant cause of annual human morbidity and mortality. The efficacy of seasonal flu vaccines depends on the accuracy of the prediction of the circulating strains for the upcoming season. To develop flu vaccines that are broadly effective and more sustainable, induction of a cross-reactive memory immunity is desirable. Flu infection induces cross-reactive tissue resident memory T (Trm) and B (Brm) cells which rapidly respond to reinfection. Tissue resident helper T (Trh) cells are a population of T follicular helper (Tfh)-like CD4+ Trm cells that promote the development of Brm cell responses following flu infection. The transcription factor TCF-1, encoded by Tcf7, is critical in the development of both memory T cells and Tfh cells following viral infections. However, the role of TCF-1 in Trh cell development has not been investigated. Utilizing a Tcf7fl/flCD4Cre conditional knockout mouse model in conjunction with intravascular labeling and influenza A peptide MHC Class II tetramers, we found that TCF-1 is required for the differentiation of flu-specific Trh cells, as well as optimal Brm development following flu infections. Most significantly, upon heterosubtypic reinfection, the Trh population in the Tcf7fl/fl control mice expanded while it became undetectable in the Tcf7fl/flCD4Cre mice, coinciding with a severely impaired lung resident germinal center B cell and CD138+ plasma cell response. Together, our data indicates that TCF-1 is critical in the development of lung tissue resident helper cells and tissue resident memory B cell response following flu infections. This work has implications for the development of universal flu vaccines.
Towards molecular mechanisms for pathogenic Th17 cell differentiation in ocular autoimmunity
Authors: Bass Victor , Mattapallil Mary J , Xu Biying , Caspi Rachel , Shih Han-Yu
Published: August 2023

CD4+ T helper (Th) cells play critical roles in coordinating proper immune responses by differentiating into effector subtypes specialized against different types of pathogens, while dysregulated Th cells can drive autoimmune disease by triggering inappropriate immune responses. Pathogenic Th17 (pTh17) cells, which combine behaviors of classic Th1 and Th17 effector cells by producing both interferon-gamma (IFNg) and interleukin-17A (IL17A), have been implicated in autoimmune diseases. Our goal is to map regulatory networks and identify novel regulators underlying pTh17 differentiation and activation using the murine model experimental autoimmune uveitis (EAU), in which ocular autoimmunity is driven by pTh17 cells.

To track pTh17 differentiation, we bred a double reporter mouse with IL17A-cre/tdTomato-flox tracer and IFNg-YFP. We sorted both Tomato+YFP+ and Tomato+YFP- CD4+ T cells from eyes and spleens after EAU induction for single-cell RNA-seq. Clustering of single-cell transcriptomes revealed distinct transcriptomes between eyes and spleen. In the eye, we identified clusters similar to pTh17 and non-pathogenic (np)Th17 populations defined in a myelin autoimmunity model. The pTh17 cluster contained both double positive and Tomato single positive cells, suggesting a heterogeneous pathogenic population. In contrast, the npTh17 cluster only contained single positives. Differential gene expression analysis showed that pathogenic and non-pathogenic clusters utilize distinct sets of transcription factors. We are further characterizing epigenetic differences between pTh17 and npTh17 to better understand how distinct regulatory networks shape pTh17 differentiation.

Supported by the NEI Intramural Research Program fund (ZIAEY000569-01)
Divergent roles for STAT4 in shaping effector differentiation of ILC1 and NK cells during gut inflammation
Authors: Sciume Giuseppe , Mazej Julija , Laffranchi Mattia , Candelotti Arianna Maria , Gadina Massimo , Santoni Angela , Scarno Gianluca
Published: August 2023

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1) require the transcription factor STAT4 to elicit rapid effector responses during infections. Herein, by combining genetic and transcriptomic approaches, we revealed that STAT4 played an unexpected divergent role in regulating effector differentiation of murine ILC1 and NK cells during intestinal inflammation. Stat4 deletion in Ncr1-expressing cells led to an increased generation of cytotoxic ILC1 in the inflamed large intestine. By contrast, Stat4-deficient NK cells showed impaired terminal differentiation, characterized by lower levels of IRF-8 and KLRG1. STAT4 expression in NCR+ innate lymphocytes restrained gut inflammation and type 2 adaptive T cells in the large intestine. Collectively our data shed light on distinctive mechanisms of transcriptional regulation driven by STAT4 in NK cells and ILC1 required for protection during intestinal inflammation.

Italian Association for Cancer Research (AIRC) [MFAG-21311], by Institut Pasteur (France), Transversal Research Program program.
Myeloid cell-cycle-related-kinase drives tumor immunosuppression in hepatocellular carcinoma
Authors: Zhou Jingying
Published: August 2023

Myeloid-derived suppressor cell (MDSC) comprises a heterogeneous population of immature myeloid cells featured by potent immunosuppression. We have uncovered a significant role of hepatoma-intrinsic cyclin-dependent-kinase 20 (CDK20), or cell-cycle-related-kinase (CCRK) in promoting tumor progression and immunotherapy resistance in hepatocellular carcinoma (HCC). As emerging evidence has highlighted the importance of immune cell intrinsic CDKs, we further explored the potentials of CCRK expressions and functions in different immune cells from HCC-bearing mice and patients. Of note, we uncovered specific CCRK overexpression in MDSCs but not lymphocytes, which functioned in regulating MDSC proliferation, immunosuppression, and differentiation. Intratumoral injection of bone-marrow-derived MDSCs with Ccrk knockdown resulted in impaired tumorigenicity, accompanied with increased CD8+T cell responses. Mechanistically, myeloid CCRK upregulation was functional associated with IL-6-mediated MDSC expansion and immunosuppression. Inhibition of myeloid CCRK in turn suppressed STAT3 to revert E4-binding protein 4-dependent transcriptional alterations. Using a transgenic mouse bearing Ccrk-indel mutation (Ccrkindel/indel), we found that impaired Ccrk signal reprogramed the pro-tumoral MDSCs into an antitumoral phenotype, resulting in suppression of tumor growth in our established HCC orthotopic mouse models. As we also showed CCRK overexpression in HCC patient-derived MDSCs, the findings of our study not only unravel mechanistic insights in MDSC maintenance and immunosuppression, but also offer a novel therapeutic kinase-target in HCC therapies.

The project is supported by RGC/GRF14108219.

Supported by grants from general research fund (GRF)/research grants council (RGC) of Hong Kong, 14108219.
Induction of STING/IL-29 signaling in human lung adenocarcinoma/tumor-associated neutrophil cocultures
Authors: Monterroza Lenore , Selvaraj Periasamy , Tirouvanziam Rabindra M
Published: August 2023

LKB1-mutant lung adenocarcinoma (LUAD), is an intractable form of lung cancer. LKB1-mutant LUAD is poor in T-cells and does not respond to checkpoint inhibitor therapy, and instead is enriched in tumor-infiltrating neutrophils (TINs). STING is a key innate sensor mediating anti-tumor responses that is inhibited in LKB1-mutant LUAD. However, the status of STING signaling and if it can be induced in TINs to antagonize tumors is unknown. Here, we developed a novel human LKB1-mutant LUAD/TIN model to tackle this question. Based on prior work from our lab (Dobosh, STAR Protoc. 2021), we grew the H1944 LKB1-mutant LUAD line at air-liquid interface on Alvetex scaffolds (Reprocell), followed by the transmigration of human blood neutrophils. The effect of the STING agonist MSA-2 was assessed in the model by a 20-plex immune mediator assay(Mesoscale). We successfully manufactured a LKB1-mutant LUAD/TIN model from H1944 cells and human blood neutrophils. MSA-2 treatment resulted in significant increases in the STING-regulated mediators IL-6, IP-10 and MIP-1beta. Most strikingly, MSA-2 treatment induced IL-29 (type III interferon) levels by 27-fold, while neither type I nor type II interferons were induced. The model showcased here mimics LKB1-mutant LUAD/TIN interplay and shows responsiveness to STING agonist treatment through induction of type III (but not type I and II) interferon and other immune mediators. Since IL-29 receptor expression is limited to epithelial and myeloid cells (including neutrophils) and IL-29 is known to mediate immunomodulatory and anti-tumor responses, future work will address if and how the large IL-29 increase observed upon MSA-2 treatment affects LUAD proliferation and TIN activation in this context.
Topology of cell-free DNA from tumour cells influences its immunogenicity in macrophages and dendritic cells in vitro
Authors: Malkin Ethan Z , Brooks Patricia , Lamorte Sara , McGaha Tracy , Bratman Scott V
Published: August 2023

Activation of DNA-sensing pathways can induce an anti-tumour immune response, but the mechanisms by which tumour-derived cell-free DNA (cfDNA) activates pro-inflammatory signaling is not well-understood. In particular, the topological features of cfDNA that contribute to DNA sensor activation in phagocytes are unknown. As such, we investigated how tumour-derived cfDNA topology influences DNA sensor activation in macrophages and dendritic cells. We treated a murine colon adenocarcinoma cell line (MC38) with chemotherapy (topotecan or staurosporine) for varying durations to induce cfDNA release. We then harvested cfDNA, separated the protected (membrane-bound) and accessible (non-membranous) fractions, and quantified nuclear and mitochondrial cfDNA abundance. The cfDNA fractions were then added to murine RAW 264.7 macrophages or bone marrow-derived dendritic cells (BMDCs), and a panel of inflammatory markers was measured by RT-qPCR.

We found that accessible cfDNA from MC38 cells treated with chemotherapy for 48h increased Il6 expression in RAW 264.7, but other inflammatory markers were not elevated. Interestingly, both nuclear and mitochondrial cfDNA abundance directly correlated with Il6 expression. Surprisingly, cfDNA did not induce pro-inflammatory signalling in BMDCs. These results demonstrate that tumour-derived cfDNA immunogenicity depends on its mechanistic origins, abundance, and the immune cell type to which it is exposed. We are currently examining the impact of other cfDNA biophysical properties on macrophage activation, as well as elucidating the DNA sensing pathways responsible for inducing Il6 expression. Our findings will help uncover the role of cfDNA in mediating anti-tumour immunity.

E.Z. Malkin is supported by the Frederick Banting and Charles Best Canada Graduate Scholarship (FRN: FBD-175873) through CIHR. S.V. Bratman is supported by the Gattuso-Slaight Personalized Cancer Medicine Fund at the Princess Margaret Cancer Centre and the Dr. Mariano Elia Chair in Head and Neck Cancer Research at University Health Network and the University of Toronto.
Leukemic Environment Alters Neutrophils Worsening Disease Progression
Authors: Goel Paran , Kuznetsova Valeriya , Franceski Alana , Luca Francesca , Costa Amanda , Crown Brittany , Welner Robert
Published: August 2023

Acute Myeloid Leukemia (AML) is the abnormal differentiation of hematopoietic stem cells in the bone marrow into malignant blasts. The leukemic marrow environment plays an important role during disease progression, by shaping the immune landscape interacting with these blasts. Mounting evidence suggests neutrophils are complicit within tumors by enhancing tumorigenesis and immunosuppression; however, little is known about how these cells contribute to AML even though neutrophils comprise half the marrow cellularity.

Tumor-associated neutrophils are found in an overactivated state, constantly releasing pro-inflammatory cytokines and granular proteins to promote tumorigenesis. Therefore, we investigated the neutrophil phenotype in our AML chimeric mouse model. We observed neutrophils in AML were also in an activated state. More importantly, sorted leukemic-exposed neutrophils were more susceptible to degranulation upon stimulation. Next, our single-cell RNA data showed that neutrophils within the AML environment had higher expression of ELANE, a gene encoding neutrophil elastase (NE). Increased NE levels have been associated with increased tumor burden in many cancers. Although NE levels increase inflammation which drives tumorigenesis, it may also directly increase leukemic blast proliferation. By targeting neutrophils in our AML chimeric model using anti-Ly6G antibody, we found decreased frequency of leukemic blasts in the neutrophil depleted mice. Also, there was a reduced frequency of the Lin-ckit+ CD16/32+ leukemic stem cells. We hypothesize that over-activated neutrophils drive AML progression in the BM by releasing NE, providing a new avenue for disease intervention.
Medullary sinus macrophages limit cancer therapy efficacy by an efferocytosis-induced IL-33/ST2 axis in the tumor-draining lymph node
Authors: Lamorte Sara , Neufeld Luke , Quevedo Rene , Zhang Xin , Ciudad M Teresa , Jin Robbie , Saeed Zaid Kamil , Karlsson Mikael C I , Mak Tak W , Munn David , Brooks David , McGaha Tracy
Published: August 2023

Cell death is a fundamental aspect of tissue homeostasis that can expose potentially immunogenic cell components that must be removed to prevent inflammatory autoimmunity. We have shown immune suppression in response to apoptotic cell phagocytosis (a process called efferocytosis) is dependent on tissue-resident macrophages (Mf). Indeed, if we disrupt Mf regulatory responses post-efferocytosis, the same apoptotic cells induce inflammation suggesting Mf responses to dying cells are a key determinant of tolerance. We predict that resident macrophages lining the lymphatic sinus inside the lymph nodes are responsible for tolerance against tumor apoptotic cells. Using our mouse melanoma model, we observed that after either chemotherapy or targeted therapy a subpopulation of macrophages of the TDLN, called medullary sinus Mf (MSM), avidly phagocytosed dying tumor cells and acquired a tolerogenic phenotype. RNA sequencing analysis revealed MSM rapidly and exclusively induced expression of the alarmin IL-33 as opposed to other Mf or dendritic cell populations in the TDLN. Importantly, genetic deletion of Il33 in MSM using a novel MSM-specific deletion model (MSM-IL33ko) generated by our laboratory, or blockade of the IL-33 receptor ST2 with IgG, transformed responses to both chemotherapy or targeted therapy with prolonged, enhanced tumor control and reduced cancer reoccurrence. Functionally, MSM-derived IL-33 triggered accumulation and activation of regulatory T cells in the TDLN which then migrated to the tumor limiting intratumoral CD8+ T cell function. Thus, our data revealed a previously undescribed tumor cell death-induced mechanism limiting anti-cancer immunity and therapy efficacy.

CIHR, NIH
Absence of Toll-like receptor 5 confers survival in mice bearing ovarian tumors treated with anti-PD-L1
Authors: McGinty Mitchell Thomas , Kolli Sree , Putelo Audrey , Feng Tzu-Yu , Rutkowksi Melanie
Published: August 2023

Ovarian cancer accounts for more deaths than any other cancer of the female reproductive system. Patients bearing ovarian tumors infiltrated with high frequencies of T cells associate with a greater survival probability. However, therapies targeting T cells in ovarian cancers are ineffective. We observed in models of late-stage murine ovarian cancer that in absence of Toll-Like Receptor 5 (TLR5) signaling, anti-PD-L1 promotes significant survival and protection against tumor rechallenge. Ovarian tumors from rechallenged TLR5KO mice exhibited significantly higher frequency/number of CD8 T cells expressing functional and activation markers. Thus, in the absence of TLR5 signaling, T cells mount/maintain a stronger response to ovarian tumors after inhibitory receptor blockade. Furthermore, protection against tumor progression in TLR5KO mice corresponds with a significant increase in tumor infiltrating cross presenting and IL-12 producing Dendritic cells (DCs). Additionally, in CD11c Cre x TLR5 fl/fl mice, whose DCs lack TLR5 expression, PD-L1-blockade mediated extended survival in ovarian tumor-bearing mice. Using a mixed bone marrow chimera approach, we demonstrate that direct TLR5 signaling functionally alters DCs to become more suppressive in the tumor microenvironment, impacting T cell differentiation and anti-tumor activity, thereby reducing survival during aPD-L1 therapy. Clinically, roughly 7.5% of the general population harbor a TLR5 SNP that diminishes TLR5 signaling and is associated with increased long-term survival for ovarian cancer patients. Therefore, patients who express the TLR5 SNP may immediately benefit from anti-PDL1 therapy and those without the SNP, TLR5 antagonism.

NIH/NCI 1R01CA253285 UVA Farrow Fellowship
Elucidating the role of heat shock protein receptor CD91 on antigen presenting cells during cancer immunosurveillance
Authors: Nayak Devanshi A , Binder Robert J
Published: August 2023

The premise of immunosurveillance is that the immune system, particularly by T cells, allows detection and elimination of emerging tumor cells. However, the mechanisms that allow efficient T cell priming against nascent tumors despite limited antigen availability remain unclear. We have shown that immunogenic heat shock proteins (HSPs) such as gp96 and their receptor CD91 are one such mechanism. HSPs efficiently chaperone minute levels of peptides from tumors to elicit antigen-specific immunity. Cross presentation of HSP-chaperoned peptides by CD91+ dendritic cells is a key mechanism for priming T cell responses. Diverse DC subsets vary in their capacity to modulate such anti-tumor immune responses. In this project, we aim to understand which CD91+ DC subset(s) is/are important for HSP-mediated cancer immunosurveillance. We generated mouse strains with deletion of CD91 on specific DC subsets. cDC1s are thought to be superior cross-presenters that excel in activation of cytotoxic lymphocytes. CD91 deletion in cDC1 render mice highly susceptible to chemically induced tumors. To better understand the CD91-HSP pathway, we tracked the dispersal of tumor-derived gp96 among myeloid cells using tumor cells that express EGFP-tagged gp96. We show that migratory DC subpopulations, such as CD103+ (cDC1) dendritic cells acquire gp96 from the tumor microenvironment, while a different DC subset (CD11b+ CD11c+) transfers protective antitumor immunity. The hand-off of tumor-derived gp96 between migratory and lymph node resident DCs is currently under investigation. These results advance our understanding of the HSP-CD91 pathway and its role in DC function which can have significant implications for cancer prognosis and therapy.

5R01CA233803-04