bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of the JAK2/STAT3 Signaling Pathway by the Lupus Susceptibility Gene Pbx1
Authors: Roach Tracoyia A , Choi Seung-Chul , Abboud Georges , Cornaby Caleb , Morel Laurence
Published: January 2023

Systemic Lupus Erythematosus (SLE) is an autoimmune disease in which poorly characterized genetic factors lead to the production of autoreactive or inflammatory T cells. Pre-B cell leukemia homeobox 1 (Pbx1) is a transcription factor whose dominant negative splice isoform (Pbx1-D) is overexpressed in CD4 T cells of lupus patients and lupus-prone mice as compared to the normal isoform (Pbx1-B). Pbx1-D overexpression impairs Treg cell development and function while favoring the production of follicular helper T cells. Based on previous studies showing that Pbx1 promotes cell proliferation via JAK2/STAT3 signaling pathway in a renal cell carcinoma, we hypothesized that Pbx1-D decreases T cell proliferation and viability by attenuating the JAK2/STAT3 signaling pathway. Pbx1-B or Pbx1-D expression plasmids increased pSTAT3 protein levels but there was no difference between the two isoforms in 293T cells. U3A STAT3 reporter cells transfected with Pbx1-B plasmid have greater luciferase expression, and hence pSTAT3 transcriptional activity in comparison to cells transfected with Pbx1-D or control plasmids. We also observed that Pbx1-B increases the expression of Jak2 and Stat3 message as compared to Pbx1-D. These results suggest a mechanism by which Pbx1 contributes to impaired T cells in lupus pathogenesis is via the JAK2/STAT3 pathway. Current studies are conducted to dissect the mechanism by which Pbx1 regulates this pathway in primary T cells.

Supported by a grant from the NIH (RO1AI04505021) to LM
Elucidating roles of the Rag1 N-terminus and RAG DSBs in shaping the cellular response to TCRa recombination
Authors: Miot Charline , Arya Rahul , Burn Thomas , Behrens Edward M. , Bassing Craig
Published: January 2023

The RAG1/RAG2 (RAG) endonuclease generates lymphocyte antigen receptor gene diversity via V(D)J recombination. The large numbers of V, D, and J segments and inherent imprecision in repair of RAG DNA double strand breaks (DSBs) together establish a vast diversity of antigen receptor specificities, including self-reactive receptors. Mechanisms have evolved to negatively select self-reactive cells and inhibit autoimmunity. In humans, deletion of the RAG1 N-terminus causes Omenn Syndrome, a fatal immunodeficiency with ab T cell-based autoimmunity. We discovered impaired negative selection in a spontaneous homozygous Rag1 mutant mouse with loss of the Rag1 N-terminus. This Rag1 region, which is not required for V(D)J recombination, has intrinsic ubiquitin ligase activity and interacts with another ubiquitin ligase and a kinase. We hypothesize the RAG1 N-terminus signals from RAG DSBs induced during TCRa recombination to shape the thymocyte proteome and enhance negative selection. To test our hypothesis, we have made mice whose thymocytes lack RAG DSBs or harbor RAG DSBs induced by wild-type Rag1 or mutant Rag1 lacking the N-terminus or intrinsic ubiquitin ligase activity. Our preliminary data suggest that RAG DSBs and distinct RAG1 N-terminus activities up-regulate expression of Zap70, an intracellular protein that transmits TCR signals, and CD80, a transmembrane protein that co-stimulates T cells engaging antigen. These data are consistent with a model wherein the RAG1 N-terminus facilitates negative selection through stimulating TCR signaling in thymocytes that bind self-antigens presented by thymocytes, dendritic cells, or thymic epithelial cells.
CD49a mediates cutaneous resident memory CD8+ T cell persistence and response
Authors: Bromley Shannon K , Akbaba Hasan , Mani Vinidhra , Mora-Buch Rut , Chasse Alexandra Y , Luster Andrew D
Published: January 2023

Resident memory CD8+ T cells (TRM) persist at sites of previous infection where they provide rapid local protection against pathogen challenge. CD8+ TRM expressing the a1 chain (CD49a) of integrin VLA-1 have been identified within sites of resolved skin infection as well as in vitiligo lesions. This study examines the signals that regulate CD8+ T cell CD49a expression and its requirement for cutaneous TRM formation and response. We demonstrate that CD49a was expressed early following T cell activation in vivo, and that TGF-b as well as IL-12 induced CD49a expression by CD8+ T cells in vitro. Despite this rapid expression, CD49a was not required for the generation of a primary CD8+ T cell response to cutaneous herpes simplex virus (HSV) infection. Rather, CD49a mediated epidermal CD8+ TRM interaction with the extracellular matrix, supported CD8+ TRM persistence within the skin, and increased the frequency of CD8+ TRM that secrete IFN-g following local antigen challenge. Thus, our results suggest CD49a promotes optimal cutaneous CD8+ TRM-mediated immunity.
Naïve T cells surpass pre-existing memory to dominate post-vaccine responses in humans
Authors: Su Laura F. , Pan Yi-Kan , Aiamkitsumrit Benjamas , Wang Yifeng , Marc Adam , Lavery criswell , Rappazzo Garrett C. , Hensley Scott , Birnbaum Michael
Published: January 2023

Background

There is a critical unmet need for vaccines against emerging diseases and common infections, such as HIV, malaria, and tuberculosis. Defining the mechanisms by which memory T cell responses are generated in humans is essential for developing efficacious vaccines. We have previously described that human adults possess pre-existing memory phenotype CD4+ T cells specific for viral antigens to which they have never been expose. The goal of our study is to test how memory precursors respond to antigen challenge and to identify other key aspects of the human precursor repertoire that are required for a robust vaccine response.

Methods

We are using yellow fever virus (YFV) vaccination as a model to examine the link between precursor T cell composition and cellular response to cognate antigen stimulation. We screened study participants for HLA allele of interest and the absence of prior exposure to YFV by serology. We then use peptide-MHC tetramers to identify YFV-specific T cells before vaccination and at multiple time points after YFV vaccination.

Results

Direct ex vivo analyses of YFV-specific CD4+ T cells in the blood revealed three key observations. First, precursor T cells vary in size, but a larger precursor frequency does not predict better post-vaccination response. Second, naïve precursors outcompete memory precursors to generate a larger effector population. Third, the magnitude of effector cell frequency predicts clonotypic selection into the stable memory pool.

Conclusions

Durable memory response to primary immune challenge is dependent on successful preservation of naïve T cell repertoire. Age-associated erosion of naïve T cell pool may contribute to the decreased effectiveness of vaccines in older individuals.
Conditional ablation of H-2Kb and H-2Db on discrete APC subsets modulate outcomes in experimental cerebral malaria
Authors: Fain Cori E. , Rana Sabhya , Huggins Matthew A , Malo Courtney , Sieck Gary C. , Johnson Aaron J
Published: January 2023

Cerebral malaria (CM) is a severe complication arising from Plasmodium falciparum infection which results in high mortality. Lack of mechanistic insight into this pathology has resulted in ineffective therapeutic strategies. Using the Plasmodium berghei ANKA model of experimental cerebral malaria (ECM) our lab recapitulated hallmarks of human CM using small animal imaging, including blood-brain barrier (BBB) permeability, severe edema and microhemorrhage visible via T1 and T2-weighted MRI. Previously it had been demonstrated that infiltration of CD8 T cells with cytotoxic capacity is required to induce BBB permeability and ECM. The observation that not only infiltration, but cytotoxic killing is required provoked us to analyze differential MHC class I-restricted priming among discrete antigen presenting cell (APC) types. Using new MHC class I conditional knockout mice generated by our team, we addressed CD8 T cell priming and capacity to disrupt the BBB in ECM. Our findings revealed that mice lacking the H-2Kb MHC class I molecule expression in CD11c+ cells, but not LysM+ cells, were protected from the development of lethal ECM. Subsequently, conditional ablation of the H-2Db on these same subsets yielded similar results. Therefore, we demonstrate that both Db and Kb class I molecule expression by CD11c+ cells enable CD8 T cell priming that results in BBB permeability during ECM. In contrast, CD8 T cells primed by H-2Kb or H-2Db expressing LysM+ APCs are not capable of inducing BBB disruption. These results imply discrete APC subsets prime functionally distinct CD8 T cell responses in the CNS and modulate the outcome of cerebral malaria. This work begins to bridge the mechanistic gap and will impact future therapeutic strategies.
Deeper comparison of the human and mouse microbiota reveals the utility of mouse models
Authors: Beresford-Jones Benjamin Samuel , Kumar Nitin , Stares Mark , Vervier Kevin , Lawley Trevor , Pedicord Virginia Ahn
Published: January 2023

Mouse models are the backbone of many immunological studies and understanding their relevance to human disease is critical when interpreting results and moving research into the clinic. The intestinal microbiota performs essential roles in host physiology including immune development and metabolic encoding. However, it is currently uncertain to what degree the microbiota of mice is taxonomically and functionally comparable to humans. In addition, the microbiota has been identified as a leading cause of irreproducibility in mouse models but current mouse commensal genome databases are inadequate to define the microbes responsible. Better characterization of the mouse microbiota is therefore necessary to understand the relevance of mouse studies to human medicine, as well as produce cleaner, more reproducible science.

We performed high-throughput culturing and whole-genome sequencing of commensals from the faeces of specific pathogen-free mice. These genomes represent hundreds of novel species with which we define the taxonomic and functional relatedness of the mouse and human microbiota. We highlight functional pathways that are common and unique to each host species, thus describing areas of study where mouse models may be used validly. We also demonstrate the requirement for mouse commensal-specific databases for analysis of mouse samples.

Our work brings a new level of understanding to the mouse microbiota. By supplying an extensive selection of mouse commensal genomes, we facilitate comparison of the microbiota of mice, not only providing a means to understand issues of irreproducibility in mouse research but also delivering new opportunities to correlate and validate specific commensals with phenotypes of interest.
Data driven identification of divergent host immune subgroups in tuberculosis
Authors: Coarfa Cristian , Rajapakshe Kimal , Gandhi Tanmay , Grimm Sandra L , Heyckendorf Jan , Lange Christoph , van Crevel Reinout , Netea Mihai , Cirillo Jeffrey , Kaufmann Stefan H.E. , Mandalakas Anna Maria , DiNardo Andrew
Published: January 2023

BACKGROUND

Immunity is a critical factor of clinical outcomes after Mycobacterial tuberculosis (Mtb) infection, however, the lack of identification of immune correlates of protection has prevented development of host directed therapeutics. TB studies have identified incongruous immune responses that can lead to a similar TB disease phenotype. Therefore, instead of envisioning susceptibility to TB to follow a unique path, we sough evidence by which divergent host immunity could result in TB.

RESULTS

Inspired by multiomics identification of cancer subtypes improving clinical care, we implemented unbiased, RUV clustering from 12 publicly available datasets consisting of data from 717 TB patients and 527 controls, including 12,468 common genes, and identified 4 distinct immune responses among TB patients. The two largest sub-groups displayed divergent metabolic, epigenetic and immune pathways. Cluster A, consisting of 333 TB patients was characterized by increased glycolysis; up-regulation of epigenetic-modifying genes and increased IFN-γ and TNF signaling. In contrast, Cluster B, consisting of 313 TB patients enriched for gene expression signatures similar to patients with sepsis and immune exhaustion and was characterized by up-regulated NFAT and hormone metabolism and decreased glycolysis, IFN-γ and TNF signaling. In silico evaluation suggests that drugs beneficial for TB patients in Cluster A are detrimental in Cluster B type disease, and vice versa. Multiplex ELISA using a validation cohort of TB patients confirmed divergent immune sub-types in TB.

CONCLUSION

Host immunity to TB is heterogenous and applying precision medicine may enabled sub-stratification guided host directed therapies for management of TB.
The role of the ER stress response during C. rodentium infection
Authors: Sweet Lydia , Duerkop Sharon , Keestra-Gounder Marijke
Published: January 2023

C. rodentium is a mucosal pathogen of mice that shares several pathogenic mechanisms with enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC), which are two clinically important human gastrointestinal pathogens. The C. rodentium model of infection is used to study host pathogen interactions in the gastrointestinal tract and to study ulcerative colitis (UC). Mutations in unfolded protein response (UPR) pathways have been linked to a higher risk of UC development. The ER is an important organelle for protein synthesis and protein folding. Dysregulation of ER stress results in the activation of the UPR to restore homeostasis. Chop is activated downstream of the PERK arm of the UPR and is a transcription factor for multiple targets including cell death and inflammatory pathways. We hypothesized that Chop activation increases cell death and inflammation during C. rodentium infection which then promotes infection and tissue damage. We found that when a mouse epithelial cell line was treated with an ER stress inducing drug or C. rodentium there was an upregulation of Il6 and Il23 but when we inhibited Chop expression the upregulation was lost. We next infected Chop knockout mice with C. rodentium and these mice showed less colon pathology and colon colonization at Day 10. These data suggest that Chop has a role in the ability of C. rodentium to trigger inflammation and in tissue pathology.
Sytemic and Cellular Metabolic Phenotype of Infection and Immune Response to Listeria monocytogenes
Authors: Schwartz Elizabeth Hiltbold , Johnson Robert , Olatunde Adesola , Woodie Lauren , Greene Michael
Published: January 2023

It is generally accepted that immune response and pathogen clearance are metabolically demanding. Additionally, shifts in cellular metabolism are known to occur during immune responses. However, we lack a grasp of how the numerous components of protective immunity contribute to the metabolic demand of illness. Previous studies using model antigens or purified PAMPs have been unable to represent the dynamics of live pathogen infection and immune response. Therefore, we sought to measure the systemic and cellular metabolism of infection and immune response to a well-characterized bacterial pathogen, Listeria monocytogenes. Mice infected with Listeria were monitored over the course of 12 days in multi-parameter metabolic cages. The metabolic phenotype data collected included body weight, activity, sleep, respiratory exchange rate, and energy expenditure. In a parallel study, mice infected with Listeria were utilized to measure bacterial burden, Listeria-specific T cell response, and cellular metabolism of the immune system. We found the early stages of infection (day 1–5), corresponding to the kinetics of the innate immune response, had the greatest metabolic impact on the organism as indicated by weight loss, reduced activity, increased sleep, and decreased energy expenditure. At later timepoints, corresponding to the timing of the adaptive response, there was little evidence of metabolic impact over uninfected controls. We also observed changes in myeloid cell metabolism over the course of the infection. Overall, our results indicate that the innate immune response is more metabolically demanding, compared to the adaptive immune response, linking “sickness behavior” to the timing of the innate immune response.
The Function of Phosphatidylinositol 3-Kinase delta (

PI3Kδ

) Enzyme in Protective Immunity to Trypanosoma congolense Infection in Mice: The Role of Regulatory B cells
Authors: Olayinka-Adefemi Folayemi , Onyilagha Chukwunonso , Jayachandran Nipun , Hou Sen , Jia Ping , Uzonna Jude E , Marshall Aaron J
Published: January 2023

The delta isoform of PI 3-kinase (PI3Kδ) has important functions in B cell activation. Trypanosoma parasites utilize several mechanisms to exploit and evade host B cell and antibody responses critical for immunity.

We sought to determine the impact of PI3Kδ in immunity to Trypanosoma congolense infection. We found that PI3KδD910A mutant mice show a surprisingly enhanced control of parasitemia in early infection (7–9 days) when compared to wild-type (WT) C57BL/6 mice. Drug treatment with Idelalisib to acutely inhibit PI3Kδ during infection in WT mice led to improved early control of parasitemia, consistent with results from genetically deficient mice. Both mutant mice and Idelalisib-treated mice showed a delay in polyclonal B cell activation and CD80/86 expression.

Analysis of cytokine levels in the blood and peritoneal cavity showed higher IFNg and lower IL-10 levels in the drug-treated group at early time points, indicating a more pro-inflammatory environment. B1 cells were identified as the major IL-10 producing cells in the peritoneal cavity in early infection, and B1 cell production of IL-10 was significantly impaired by PI3Kδ-inhibitor treatment. We further observed increased Nitric oxide production in drug-treated mice, which correlated with increased parasite killing in early infection. Despite the improved early parasite control, there was a 100% mortality in PI3KδD910A mutants and 25% mortality in Idelalisib treated mice, presumably due to compromised generation of parasite-specific antibodies required to clear the first wave of blood infection.

Our findings suggest that PI3Kδ inhibition impacts both regulatory B cell functions, affecting the early innate response, and generation of critical protective antibodies.
Myeloid and lymphoid cell immune exhaustion profiles during murine visceral leishmaniasis
Authors: Valadares Diogo Garcia , Scorza Breanna , Davis Richard , Petersen Christine , Wilson Mary E
Published: January 2023

Leishmaniasis is a chronic disease of reticuloendothelial organs that is usually controlled by TH1-type CD4 T cells. Anti-microbicidal responses are ineffective during disease progression, and it is reported that T cells in humans and dogs with visceral leishmaniasis (VL) express markers of cell exhaustion. We hypothesized that myeloid cells provide counter-receptors for inhibitory receptors on T cells at the local sites of Leishmania infantum infection, inhibiting T cell effector functions. We examined inhibitory receptors PD1, LAG3, CTLA4 and TIM3 on lymphoid cells, and ligands PDL1, MHCII, CD80 and Galectin 9 on myeloid cells using flow cytometry, throughout 10 weeks of infection. In livers of infected mice, an increased population of neutrophils with DC features (CD11c+MHCII+) expressing PD-L1 and IL-10 was also observed. In all infected samples an increased DC subset with reduced expression of MHCII, CD80 and CCR7 and higher expression of Galectin 9, PD-L1 and IL-10, was also detected suggesting a pro-exhaustion profile of those cells in infected mice. Furthermore, CD4 and CD8 T cells in infected mice expressed higher levels of the exhaustion markers LAG3, CTLA4 and PD-1 than uninfected mice, with greater proportions of exhausted CD8 than CD4 T cells in livers and spleens. Majority of Ag-experienced CD8 T PD-1+ cells in liver and spleens produced IL-10, whereas most Ag-experienced CD4 T PD-1+ cells produced IFN-γ at the time points studied, suggesting that CD8 T cells assume an exhausted phenotype earlier than CD4 T cells. Data also suggest that both DC and neutrophil subsets could represent a pro-exhaustion myeloid population that may influence the unresponsiveness of lymphocytes in infected tissues during visceral leishmaniasis.
The herbal medicine ‘Indigo naturalis’ altered gut microbiota to accumulate IL-22+ ILC3s
Authors: Yoshimatsu Yusuke , Teratani Toshiaki , Sujino Tomohisa , Mikami Yohei , Ichikawa Masataka , Hagihara Yuya , Takada Yoshiaki , Tanemoto Shun , Umeda Satoko , Nomura Ena , Ono Keiko , Sugimoto Shinya , Nanki Kosaku , Naganuma Makoto , Kanai Takanori
Published: January 2023

Ulcerative colitis (UC) is a chronic inflammation of the large intestine led by the dysregulation of adaptive and innate immune responses. Recently, specific microbiota has been reported to instruct the immune cells and immune response. We have recently proved the clinical efficacy of “indigo naturalis (IN)”, a Chinese herbal medicine, on patients with UC by the multicenter randomized-controlled trial (INDIGO study, Gastroenterology 2018).

4%IN-fed mice ameliorated DSS-colitis (IN-DSS mice) compared to the normal diet-fed mice (ctrl-DSS mice). The number of IL-22+ ILC3s in the colonic lamina propria in IN-DSS mice increased more than the one in ctrl-DSS mice. We also confirmed that IN-fed diet mice significantly altered gut microbiota composition compared to the normal diet-fed mice.

We next inoculated feces obtained from IN-fed mice or normal diet-fed mice to antibiotic treated mice (IN-FMT mice, ctrl-FMT mice respectively). Interestingly, IN-FMT mice significantly restored the pathology of DSS-induced colitis than ctrl-FMT mice with increased number of IL-22+ ILC3s. We showed IN induced specific microbiota which accumulated IL-22+ ILC3s in gut.
Toll-like Receptor 2 is crucial for commensal-specific immune responses on the ocular surface
Authors: Zhu Wenjie , Xu Xiaoyan , St. Leger Anthony J , Almaghrabi Fatimah , Horai Reiko , Caspi Rachel R
Published: January 2023

Conjunctiva of the eye is a mucosal immune tissue and harbors a variety of immune cells. It is exposed to diverse microorganisms that come in contact with the ocular surface. Previously, we demonstrated that colonization of the ocular mucosa with the commensal bacterium, Corynebacterium mastitidis (C. mast), results in increased resistance of the ocular surface to infectious fungal and bacterial pathogens, and that this effect is mediated by IL-17A produced by γδ T cells that respond to C. mast. In this study, we show that Toll-like receptor 2 (TLR2) is important for activation and proliferation of γδ T cells and for their IL-17A production in response to C. mast.

TLR2−/− mice failed to recruit γδ T cells and neutrophils to the conjunctiva upon C. mast inoculation, and IL-17A-producing γδ T cells were reduced in eye-draining lymph nodes of these mice. In vitro experiments revealed that IL-17A production to C. mast by gd T cells was dependent on APC and that TLR2 expression was needed on both γδ T cells and APCs. Exogenous IL-1 could only partly compensate for the deficiency in IL-17A production by TLR2−/− gdT cells, indicating a need for another TLR2-dependent signal. Flow cytometry and RNA-Seq analyses suggested that dependence on TLR2 for IL-17A production and IL-17A related gene expression in response to C. mast is higher in the Vγ6 than in the Vγ4 subset. Notably, the transcriptomic analysis also implicated dysregulation of mitochondrial pathways in TLR2−/− cells, which was functionally confirmed by the Seahorse assay.

We conclude that TLR2 is required for sensing the commensal C. mast in the conjunctiva and eye-draining lymph nodes, to drive the local IL-17 response and maintain immune homeostasis at the ocular surface.
Mucin degrading bacteria accelerates intestinal stem cells-derived epithelial development
Authors: KIM SEUNGIL , Kim Yeji , Kim Tae-Young , Lee Su-Hyun , Kweon Mi-Na
Published: January 2023

Mucin degrading bacteria are closely located in intestinal epithelium; however, how they associate differentiation and development of intestinal stem cells (ISCs) is not elucidated. To address an exact role of mucin degrading bacteria on the gut homeostasis, mice were fed with Akkermansia muciniphila, which is specialized to mucin degradation, for one month. We found that oral feeding with A. muciniphila accelerated self-renewal of Lgr5+ ISCs and promoted differentiation of lysozyme+ Paneth cells and Muc2+ goblet cells in the small intestine. Co-cultured with Lgr5+ ISCs and Paneth cells from A. muciniphila-fed mice resulted in augmented organoid forming capacity in comparison to those from PBS-fed control mice in a Wnt3-dependant manner. Of note, levels of acetate and propionate were higher in cecum contents of A. muciniphila-fed mice than those of control mice. Organoids treated with cecum contents obtained from A. muciniphila-fed mice exhibited larger size and these effects were diminished by Gpr41/43 antagonists. In addition, oral feeding with A. muciniphila induced change of gut microbiota composition and a positive correlation between several genus including Akkermansia. Most importantly, pre-treatment with A. muciniphila prevented intestinal epithelial cells damage caused by radiation and chemotherapy. These findings suggest that mucin degrading bacteria (i.e., A. muciniphila) play a crucial role in promoting ISCs-mediated epithelial development and contributed to maintain intestinal homeostasis.
Dietary cellulose prevents gut inflammation by modulating lipid metabolism and gut microbiota
Authors: Kim Yeji , Hwang Sung Wook , Kim Seungil , Lee Yong-Soo , Kim Tae-Young , Lee Su-Hyun , Kim Eun Na , Kweon Mi-Na
Published: January 2023

A Western diet comprising high fat, high carbohydrate, and low fiber content has been suggested to contribute to an increased prevalence of colitis. To clarify the effect of dietary cellulose (an insoluble fiber) on gut homeostasis, for 3 months mice were fed a high-cellulose diet (HCD) or a low-cellulose diet (LCD) based on the AIN-93G formulation. Histologic evaluation showed crypt atrophy and goblet cell depletion in the colons of LCD-fed mice. RNA-sequencing analysis showed a higher expression of genes associated with immune system processes, especially those of chemokines and their receptors, in the colon tissues of LCD-fed mice than in those of HCD-fed mice. The HCD was protective against dextran sodium sulfate-induced colitis in mice, while LCD exacerbated gut inflammation; however, the depletion of gut microbiota by antibiotic treatment diminished both beneficial and non-beneficial effects of the HCD and LCD on colitis, respectively. A comparative analysis of the cecal contents of mice fed the HCD or the LCD showed that the LCD did not influence the diversity of gut microbiota, but it resulted in a higher and lower abundance of Oscillibacter and Akkermansia organisms, respectively. Additionally, linoleic acid, nicotinate, and nicotinamide pathways were most affected by cellulose intake, while the levels of short-chain fatty acids were comparable in HCD- and LCD-fed mice. Finally, oral administration of Akkermansia muciniphila to LCD-fed mice elevated crypt length, increased goblet cells, and ameliorated colitis. These results suggest that dietary cellulose plays a beneficial role in maintaining gut homeostasis through the alteration of gut microbiota and metabolites.
High-fat diet causes rapid microbiota-dependent intestinal ILC3 loss and impairs tolerance and immunity
Authors: Boulenouar Selma
Published: January 2023

Innate lymphoid cells (ILCs) are increasingly appreciated to play a critical role in tissue homeostasis, immunity, and tolerance. At steady state, intestinal ILC3 are the main source of IL-22, ensuring epithelial barrier integrity and defense against pathogenic bacteria. In addition, ILC3 support intestinal tolerance to commensal bacteria and dietary antigens. ILC3 can also sense changes in dietary nutrients, such as vitamin A and aryl hydrocarbon receptor ligands that affect their development, function and ultimately intestinal health. Here we show that ILC3 are severely depleted from the small and large intestine of obese mice fed high-fat diet (HFD) but not in leptin or leptin receptor-deficient obese mice. Notably, consumption of HFD for only 24 hours is sufficient to trigger ILC3 cell death. Total loss of ILC3 is reached after one week of HFD without significant weight gain. We found that this short-term consumption of HFD increases host susceptibility to Citrobacter rodentium infection and impairs tolerance to dietary antigens. Unexpectedly, we found that ILC3 were maintained in germ-free mice fed HFD. However, ILC3 were depleted when HFD-fed GF mice were inoculated with either living, heat-killed bacteria or with LPS. Furthermore, HFD-fed TLR4 deficient mice are protected from intestinal ILC3 loss. TNFα blockade is sufficient to prevent HFD-induced ILC3 loss. Collectively, our findings show that short term consumption of HFD, rather than obesity potentiates rapid loss of ILC3 in presence of microbiota that leads to early impairment of gut integrity, tolerance, and protection against pathogens.
Assessment of Antibody Functional Affinity using FluoroSpot
Authors: Kirchenbaum Greg A , Sautto Giuseppe A , Abreu Rodrigo B , Lehmann Paul V , Ross Ted M
Published: January 2023

Studies of B cell immunity often rely upon serologic methodologies that primarily assess antibody specificity. However, functional affinity of the antigen-specific antibody repertoire is a key determinant of protective efficacy. Presently, detailed assessment of single B cell/antibody functional affinity is labor-intensive and low-throughput. Therefore, we sought to evaluate whether B cell FluoroSpot assays would enable distinction between B cells with differential functional affinity since fluorescent intensity is directly proportional to antibody abundance in this assay. To test our prediction, murine B cell hybridomas (P65C6-2, 36–65 and 36–71) secreting monoclonal antibodies (mAb) with differential affinity for the p-azophenylarsonate (Ars) hapten were utilized as model antibody-secreting cells (ASC). Additionally, usage of an anti-idiotypic mAb (17–63) specific for the high-affinity anti-Ars mAb (36–71) confirmed unambiguous segregation of Ars-specific ASC. Moreover, we also evaluated the capacity of a multiplexed FluoroSpot assay to simultaneously distinguish antibody functional affinity among influenza hemagglutinin-reactive murine B cell hybridomas, or in vivo differentiated ASC from immunized mice, secreting various IgG subclasses (IgG1/IgG2a/IgG2b) in parallel. Collectively, our data support the notion that FluoroSpot assays can be used to assess the functional affinity of antigen-specific B cells, and that this approach is well-suited for detailed assessment of humoral immunity.
A new interpretable machine learning approach for single-cell data discovers correlates of clinical outcome in cancer immunotherapy
Authors: Greg Finak , Greene Evan , D’Amico Leonard , Bhardwaj Nina , Church Candice , Morishima Chihiro , Ramchurren Nirasha , Taube Janis , Nghiem Paul , Cheever Martin , Fling Steven , Gottardo Raphael
Published: January 2023

High-dimensional cytometry is routinely used to characterize patient responses to cancer immunotherapy and other treatments, producing a wealth of datasets ripe for exploration but whose biological and technical heterogeneity make them difficult to analyze with current tools. We introduce a new interpretable machine learning method for single-cell mass and flow cytometry studies, FAUST, that robustly performs unbiased cell population discovery and annotation and enables data integration across studies and platforms. FAUST returns biologically interpretable cell phenotypes that can be compared across studies, making it well-suited for the analysis and integration of complex datasets. We use FAUST to perform candidate biomarker discovery and validation by applying it to flow and mass cytometry datasets from melanoma anti-PD-1 trials in order to discover and validate new CD4+ and CD8+ effector-memory T cell correlates of outcome co-expressing PD-1, HLA-DR, and CD28. Existing state-of-the-art computational discovery approaches as well as prior manual analyses did not detect any statistically significant T cell correlates associated with anti-PD-1 treatment these data. We use FAUST to replicate the discovery of published myeloid cell correlates and validate these by identifying them de novo in independent trials. FAUST’s phenotypic annotations are used to perform cross-study integration diverse data sets, allowing hypothesis-driven inference about cell sub-populations through a Phenotypic and Functional Differential Abundance (PFDA) framework. We showcase this approach on data across multiple trials. These results establish FAUST as a powerful new approach for unbiased discovery in single-cell cytometry.
Assessing CD8+ cytotoxic T-cell dysfunction in obesity and its implications in anti-PD-1 blockade therapy
Authors: Baracho Gisele Vanessa , Song Hye-Wong , Taylor Ian , Mortimer Stefanie , Widmann Stephanie , Tyznik Aaron J
Published: January 2023

In chronic conditions, notably cancers, CD8+ cytotoxic T cells gradually lose their effector functions due to constant exposure to antigens and consequent upregulation of inhibitory receptors like PD-1. In this regard, PD-1 blockade therapy was shown to bolster cytotoxic T-cell response and improve disease prognosis in some cases. PD-1 mediated T cell dysfunction is also involved in obesity-induced tumorigenesis. In this study, we used a molecular cytometry approach to better understand the disturbances caused by obesity in immune cells. A detailed single-cell analysis of various cell compartments revealed a unique population of CD8+ cytotoxic T cells in the epididymal fat of mice consuming a high fat diet for 16 weeks. These cells showed high expression of proteins and/or mRNA transcripts for various inhibitory receptors, including PD-1. Based on these results, we created a 28-color panel for high-throughput cell analysis at multiple time points over the 16-week period, using a BD FACSymphony™. The frequency of CD8+PD-1+ T cells in the fat correlated with the numbers of inflammatory cells, suggesting that the dysfunctional phenotype resulted from a growth in inflammation over time. The functional state of the CD8+PD-1+ cells was also examined by challenging these cells with or without PD-1 blockade. Taken together the data delineated a refined application for a broad analysis of immune cells in the fat and provided insights into the effects of obesity-induced T-cell dysfunction in mice.

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Comparison of cesium and X-ray irradiation for generation of bone marrow chimeras
Authors: Eng Jason Samuel , Orf Jessica , Perez Kristy , Sawant Deepali , DeVoss Jason J
Published: January 2023

Bone marrow chimeras are mice which have been irradiated with ionizing radiation, thus killing their bone marrow and all bone marrow derived cells, followed by injection of bone marrow from another mouse. Generation of bone marrow chimeras has been a useful tool to interrogate stromal and hematopoietic contributions to immune function. Cesium sourced irradiators have been demonstrated to be effective in generating bone marrow chimeras and have long historic use in academia and industry. However, the increasing cost to maintain a cesium irradiator and greater security measures set in place for using a radioactive source material has led to a need for utilizing an alternative technology – X-ray irradiation. To our knowledge, limited number of publications compare these two modalities.

In our study, we evaluated the X-ray irradiator by analyzing and comparing the reconstitution profile of specific immune cell subsets in the blood and spleens of mice irradiated with a cesium source versus mice irradiated with an X-ray source. Our data suggest that the two modalities provide functionally comparable degrees of chimerism in mice. Thereafter, we transitioned to using the X-ray irradiator and evaluated the effects of irradiation and reconstitution on tumor implantation. Our data shows that there is delayed tumor growth after total body irradiation.

In summary, we have established a protocol for an alternative irradiation modality, confirmed comparable levels of chimerism to traditional generation of bone marrow chimeras, and observed delayed tumor growth following irradiation and reconstitution.
Targeted CRISPR/Cas9-based in vitro and in vivo screening of metabolic pathways in primary T cells
Authors: Sugiura Ayaka , Beier Katherine L. , Rathmell Jeffrey C.
Published: January 2023

Chronic inflammation and immunosuppression are characterized by a dysregulation in the balance between pro-inflammatory effector T (Teff) cells and anti-inflammatory regulatory T (Treg) cells. While many currently available therapies broadly target the immune compartment, selectively targeting the specific T cell subsets that contribute to disease may provide a new avenue for development of improved immunotherapies. We have previously shown that Teff and Treg cells can be distinguished by their reliance on distinct metabolic programs, and that this exposes a way to preferentially target specific T cell subsets. We have developed in vitro and in vivo approaches to CRISPR/Cas9-based genetic screening in primary T cells to interrogate metabolic pathways. In this method, we design small-scale targeted guide RNA libraries, which are transduced into primary T cells. For the in vitro assays, these cells can be cultured under selective pressures, such as with differentiation cytokines, and sorted for populations of interest. For the in vivo assays, the transduced cells are adoptively transferred into host mice of inflammatory or cancer models, such as airway inflammation or melanoma. We have constructed and tested a library targeting the solute carrier (SLC) family of transporters, which import and export essential nutrients required to fuel the metabolic programs. Preliminary results show the glutamine transporter, SLC38a2 (SNAT2), in addition to the previously identified transporter SLC7a5 (LAT1), to be critical for primary T cell proliferative capacity. This approach provides a powerful tool for identifying critical nodes within metabolic pathways of interest in primary T cells that can potentially serve as therapeutic targets.
Detergent wash improves vaccinated lymph node handling ex vivo
Authors: Ball Alexander , Pompano Rebecca R
Published: January 2023

Vaccines target the lymph node (LN) in order to promote an adaptive immune response, but few ex vivo models are available to study this process. We recently developed ex vivo LN slices as a unique tool to study the dynamics of adaptive immunity; however, a pervasive issue occurred between the LN tissue with the embedding biomaterial. Intact lymph nodes from vaccinated animals, which had expanded into the surrounding adipose tissue, consistently failed to adhere to the agarose matrix, preventing downstream analysis. Here we tested the hypothesis that removal of excess lipids with a liquid detergent from the collagen capsule surrounding the LN would improve the interaction with agarose without damaging viability or function. Specifically, we investigated the use of digitonin, to wash LNs before embedding. Harvested LNs were quickly dipped into digitonin solution and washed in saline prior to embedding in agarose and sliced. Fat was visibly removed by this procedure. Slice viability was determined through flow cytometry using Calcein-AM and 7-AAD. Phagocytic capacity across the entire tissue sample was assessed by measuring uptake of fluorescent microparticles through widefield microscopy. Tissue-level responsiveness was tested by incubating the slices with R848 (TLR7 & 8 agonist) for 24 hours and quantified secreted IFNγ by ELISA. None of the above metrics were affected by washing intact LNs with digitonin prior to slicing and we observed significant improvement in adherence to agarose. We anticipate that this novel approach will enable downstream analysis of lymph nodes undergoing an active immune response to vaccination and may improve the slicing other small organs and organ fragments embedded in agarose, such as liver, thymus, and gut.
Resolving the heterogeneity of human circulating innate lymphoid cells via simultaneous, high-dimensional analysis of protein and gene expression
Authors: Tyznik Aaron J , Corselli Mirko , Baracho Gisele V , Lomas Chip , Sa Silin , Widmann Stephanie , Saksena Suraj , Ghanekar Smita
Published: January 2023

Cancer treatment has been revolutionized with the development of immunomodulatory therapies. While these therapies have primarily focused on enhancing T-cell responses, there has been interest in harnessing the potential of other cytotoxic cells such as Natural Killer (NK) cells. Similar to NK cells, innate lymphoid cells (ILCs) may offer another target of these immunotherapy approaches. However, there is a need for better understanding of these recently described cells.

In this study, we developed a comprehensive approach to further refine the signatures of human circulating ILC subsets. Total ILCs were enriched using the BD FACSAria™ Fusion cell sorter and processed for downstream single-cell multiomic characterization. BD® AbSeq reagents and a targeted BD Rhapsody™ Immune Response Panel enabled simultaneous detection of 42 proteins and 399 genes using the BD Rhapsody™ Single-Cell Analysis System. Differential protein and gene expression analysis in addition to combinatorial expression of CD294 and CD117 confirmed 3 conventional ILC populations as well as the signatures of 3 distinct subsets within ILC1. This discovery approach provided information about relative expression of a small selection of proteins or surface marker-coding genes that enable the discrimination of these ILC subsets. These data were used to design high-parameter flow cytometry panels for high-throughput analysis. ILC subsets differentially distributed across donors were detected and defined using unsupervised computational analysis confirming the result of multiomic analysis.

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A humanized mouse platform supporting in vivo maturation of human antibody responses
Authors: Chupp Daniel P , Acosta Ometeotl , Zan Hong , Casali Paolo
Published: January 2023

Mice are widely used models for research and drug discovery, yet provide limited predictive value due to their divergence from humans in immune elements and functions. This underscores the need for more faithful models of the in vivo human immune response. To address this shortcoming, we have generated a HuMice platform by grafting NSG KitW-41J mice with fresh human cord blood CD34+ stem cells via intracardiac injection. This yielded up to 98% human CD45+ cell reconstitution within all major lymphoid compartments, including GALT. Human leukocyte development and long-term persistence in CD34+ cell-engrafted HuMice revealed robust T and B cell maturation, leading to emergence of IgM, IgG, IgA and IgE forming cells. B cells from HuMice underwent in vitro CSR and plasma cell differentiation as efficiently as B cells isolated from healthy humans. Once conditioned with our immune potentiation treatment, HuMice supported T-dependent and T-independent antibody responses involving formation of germinal center-like structures, class-switching, somatic hypermutation, as well as plasma cell and memory B cell differentiation, this entailed further maturation of the B cell compartment resulting in high B2/B1 cell ratio. HuMice were amenable to hydrocarbon-induced autoimmunity leading to lupus-like disease and were susceptible to infection by and respond to human pathogens such as vaccinia virus and EBV. Thus, we have devised a novel in vivo HuMice platform, which allows for generation and maturation of human antibody responses to microbial agents and lends itself to in vivo modeling of autoimmunity, thereby offering opportunities for identification of therapeutic targets and vaccine development.
Evaluating the WOLF® Cell Sorter impact on neutrophil viability and activation
Authors: Krasny Aly , Morachis Jose
Published: January 2023

Neutrophils have been associated with antimicrobial functions in acute infections and also play critical roles in chronic inflammation. The half-life of circulating neutrophils is estimated at between 13–19 hours and, if activated, they may be cleared even faster. The shear forces and pressure of traditional FACS can be harsh on such sensitive cells and as a result, the use of magnetic beads and centrifugation gradients to isolate neutrophils is sometimes preferred. However, these methods lack the ability to select for viability or multiple markers needed for specific subset gating. NanoCellect’s WOLF Cell Sorter allows for multi-parameter detection and sorting like traditional FACS. However, the WOLF’s microfluidic sorting mechanism uses far lower pressure (<2 psi vs 10–70 psi) that results in gentler sorting and a higher quality of cells post-sort. Here, the ability of this novel instrument to sort neutrophils with minimal cell death or activation was investigated. Live CD16+ neutrophils were sorted from whole blood and evaluated for purity, viability, and expression levels of CD11b, and CD62L. Results demonstrate that the WOLF Cell Sorter can enrich for neutrophils with high purity, viability, and a limited effect on activation, making it an effective method for neutrophil separation. This indicates that this novel microfluidic cell sorting technology can be used to study emerging neutrophil functions, mechanisms of action, and novel cell-based therapeutic approaches.
Analysis of BCR-IgH from genomic DNA with UMI incorporation
Authors: Berngruber Chelsea , Pan Wenjing , Byrne-Steele Miranda , Li Song , Han Jian
Published: January 2023

NGS of T and B cell immune receptors has become routine in immunological studies. While using RNA as a template has its benefits, gDNA is often preferred by those collecting the sample as DNA is often easier to obtain, especially from difficult samples or samples that have been in long-term storage. In addition, since each cell may only have one copy of the successfully rearranged V(D)J, it may reflect the quantity of the repertoire better. In other words, identification of a successfully rearranged V(D)J will not be skewed by expression levels as the relationship should be one cell to one V(D)J rearrangement. Here, we describe the development of a new set of BCR-IgH specific primers to target the gDNA V(D)J region while also incorporating unique molecular indices (UMI) through our proprietary dimer avoided multiplex PCR technology. The inclusion of UMI with gDNA amplification allows each cell to be counted uniquely since the relationship of one V(D)J per cell will be maintained. Thus, both the identity and the frequency of the B-cell can be captured directly, while allowing for the elimination of PCR and sequencing error. The newly developed panel was tested on a BCR-IgH Synthetic Library. The results demonstrate a high degree of correlation with the known BCR-IgH Synthetic Library (R20.956) including coverage of all known V and J gene targets. Currently, gDNA extracted from B-cell enriched PBMC samples are being tested. This method has important implications for monitoring aberrant clonotypes in blood cancers since the frequency of a clonotype can be directly quantified, even under conditions when one clonotype is expanded.