bluuebunny/crossref_metadata_2025 huggingface dataset:
CD28 regulates T cell metabolic reprogramming through ARS2-dependent effects on alternative splicing
Authors: Holling G. Aaron , Sharda Anand , Honikel Mackenzie M. , Qiao Guanxi , Repasky Elizabeth A , Lee Kelvin P , Olejniczak Scott H
Published: January 2023

An adaptive immune response requires activation of naïve T cells via T cell receptor (TCR) ligation and engagement of costimulatory molecules such as CD28, which enables cell growth, rapid proliferation, and effector differentiation. To accomplish such bioenergetically demanding processes, T cells must dynamically regulate their metabolism upon activation. After initial TCR triggering of aerobic glycolysis, CD28 co-stimulation maintains uptake and catabolism of glucose required for CD8+ T cells to attain full effector status. Despite evidence that CD28 signaling through the PI3-kinase pathway induces glucose uptake via increased GLUT1 surface expression, knockin T cells expressing PI3-kinase signaling-deficient CD28 are functional in vivo and displayed only transient defects in glycolysis. In contrast, knockin T cells expressing Grb2/Vav signaling-deficient CD28 lacked in vivo function and displayed sustained defects in glycolysis. Here we identify a novel link between CD28-Grb2/Vav signaling and T cell glycolytic reprogramming through the RNA binding protein ARS2, a protein central to co-transcriptional processing of nascent RNA polymerase II transcripts. Data show that ARS2 is a critical downstream mediator of CD28-Grb2/Vav signaling that supports alternative splicing (AS) of the rate-limiting glycolytic enzyme pyruvate kinase M from the M1 to M2 isoform. Mechanistically, ARS2 binds known Pkm splicing factors and promotes their interaction with Pkm mRNA. Consistent with this, ARS2 knockout T cells displayed reduced Pkm2 AS, defective glycolytic reprogramming and lacked the ability to control in vivo tumor growth, suggesting a novel mechanism by which co-stimulation drives T cell metabolic reprogramming.
The Inhibition of Phosphatidylinositol-3-Kinase in activated T-cells by Hibiscone C derivatives
Authors: Rawlings Jason S , Irsik Jeremy , Wheibe Elias , Johar Shamael , Besley Caroline , Goess Brian
Published: January 2023

The phosphatidylinositol-3-kinase (PI3K) pathway is of significant interest due to its ability to regulate cell proliferation, growth, and migration in numerous contexts including in T cells. Furthermore, gain-of-function mutations in PI3K can lead to an upregulation of the pathway, resulting in tumorigenesis, autoimmunity, and leukemia. Specific furanosesquiterpenoids, such as wortmannin, have been known to inhibit the PI3K pathway in T cells. However, wortmannin has unfavorable characteristics as a chemotherapeutic or immunomodulatory drug due to its insolubility in neutral buffers, instability, and high toxicity in vivo. Hibiscone C, another furanosesquiterpenoid, lacks many of the functional groups as wortmannin, but contains the same diacyl furan ring that is critical for interacting with the ATP binding pocket of PI3K. Via analysis of phosphorylation of the downstream effector molecule Akt in activated T cells, we demonstrate that Hibiscone C also can irreversibly inhibit PI3K activity, however not as effectively as wortmannin. These results led us to test other derivatives of hibiscone C, to see if modifications to the molecule’s structure would affect potency of the molecule.
Characterization of the bidirectional activity of the human TET2 gene promoters
Authors: Li Hongchuan , Lou Hong , Dean Michael , Anderson Stephen
Published: January 2023

Tet methylcytosine dioxygenase 2 (TET2) is a tumor suppressor gene that is inactivated in a wide range of hematological cancers. TET2 enzymatic activity converts 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC), an essential step in DNA demethylation. Human TET2 is highly expressed in pluripotent cells and down-regulated in differentiated cells. However, transcriptional regulation of the human TET2 gene underlying functional and biological implications has not been investigated in detail. We previously reported three promoters, designated as Pro1, Pro2, and Pro3, generate three alternative first exons, and their presence in TET2 mRNAs varies with cell type and developmental stage. In the present study, we found that the human TET2 promoters has in transcriptional activity in either orientation. The functional identification of these promoters was performed and the results showed distinct bidirectional expression by a broad range of tissues. The antisense activity from Pro2 is significantly higher than Pro1 or Pro3 promoters. The distinct properties and bidirectional nature of the human TET2 promoter elements may play an important role in human cancer development and differentiation and should provide new insights into understanding pathogenesis and development of new therapeutic approaches.
The differential pathological outcome between Chlamydia infection and vaccination is mediated by microRNA dysregulation
Authors: Eko Francis , Howard Simone , Benyeogor Ifeyinwa , Richardson Shakyra S , Lundy Stephanie , Adebayo Olayinka Omoyitola , Igietseme Joseph U. , Omosun Yusuf
Published: January 2023

Previous studies found that in contrast to immunization with an IL-10 knockout dendritic cell (DC)-based cellular or Vibrio cholerae ghost (VCG)-based subunit vaccine, immunization with live chlamydial elementary bodies does not protect against upper genital tract pathology following Chlamydia genital infection. The molecular mechanism regulating live Chlamydia- and vaccine-induced immunity is currently incompletely understood. To elucidate the mechanisms underlying this intriguing outcome, we examined the characteristic miRNA expression profiles in the upper genital tract (UGT) tissues of mice after immunization with live, subunit and DC-based Chlamydia vaccines using the NanoString nCounter Mouse miRNA assay. There were notable differences in miRNA expression profiles after immunization with the different vaccine formulations, with the DC vaccine-immunized group showing the highest number of uniquely differentially expressed (DE) miRNAs. Only 2 miRNAs, miR-146a and miR-2140 were commonly expressed in the genital tracts of the 3 immunization groups. Pathway analysis showed that the DE miRNAs regulate a number of pathways involved in disease and biological functions and targeted genes regulating immune response and inflammation. A number of central focus molecules associated with the miRNAs, including E2F Transcription Factor 1 (E2F1), Peroxisome Proliferator Activated Receptor Alpha (PPARA), and Interferon gamma (IFNG) were identified. Taken together, the results suggest that miRNA dysregulation plays an important role in the pathological outcome associated with chlamydial infection versus vaccination.
Novel Mechanism of Regulating IL-17 Signaling by 14-3-3zeta
Authors: Chakravarti Ritu , Veerman Brent , McGowan Jenna , Saul-McBeth Jessica , Conti Heather , Chattopadhyay Saurabh
Published: January 2023

Interleukin (IL)-17A is an important cytokine associated with autoimmune diseases and host defense. The mechanism of its action depends upon its ability to induce pro-inflammatory cytokines, e.g., IL-6, IL-8 as well as anti-fungal genes, e.g., b-defensin, CCL-20, CXCL-1. Most non-hematopoietic cells express IL-17 receptors and respond to IL-17A. Several regulators of each of the two independent arms of IL-17A signaling- gene induction and mRNA stabilization of inflammatory genes, are known; however, there is limited knowledge of how two arms influence each other. We previously reported the increased presence of 14-3-3 proteins in the inflamed aortic tissue, an autoimmune disease with increased IL-17A levels. This study led us to investigate the role of 14-3-3zeta(z) in the IL-17A signaling. By genetic manipulations in human and mouse cells, we observed that 14-3-3z is required for the IL-17A-induced IL-6 production by fibroblast and epithelial cells. Contrary to IL-6, 14-3-3z suppressed the CXCL-1 production in response to IL-17A. In the absence of 14-3-3z, IL-17A induction of anti-fungal genes (b-defensin) was completely suppressed. The mechanism of 14-3-3z action depends upon its interaction with Traf/Act1/IL-17R signaling. Our results show that 14-3-3z is an essential component of IL-17A signaling and its interaction with receptor complex provides a new model of regulating the output in IL-17A effector cells. To the best of our knowledge, this is the first report of the 14-3-3z role in regulating IL-17A induced outputs.
IL-21 from CD4 T cells drives differentiation of brain-resident CD8 T cells during polyomavirus CNS infection
Authors: Ren Heather , Ren Mingqiang , Kolawole Elizabeth , Jin Ge , Netherby-Winslow Colleen , Shwetank  , Evavold Brian , Lukacher Aron
Published: January 2023

CD4 T cells help CD8 T cells differentiate into competent effector and memory cells. We recently showed that CD4 T cells are essential for CD8 T cell differentiation into brain-resident memory (bTRM) during polyomavirus (PyV) infection. Here we identify IL-21 as the CD4 T cell help. Using MHC-II tetramers for two epitopes in mouse PyV, we show brain PyV-specific CD4 T cells express PD-1 and CXCR5. IL21-VFP reporter mice revealed CD4 T cells as the only cellular source of IL-21 in the brain. 2D micropipette adhesion assays, measuring TCR affinity, showed brain IL-21-producing CD4s have higher affinity TCRs than IL-21− cells. RNAseq analysis of IL-21+ vs. IL-21− brain CD4s corroborates that IL-21 production is positively associated with TCR affinity and that IL-21 producers are positively enriched for TFH signature genes. We further found that IL-21 receptor (IL21R) is required for CD8 TRM differentiation. Brain CD8 T cells upregulated IL21R at 15 days post-infection, coincident with expression of the TRM marker CD103. Few CD8 T cells in brains of IL-21R−/− mice expressed CD103, a finding confirmed using donor anti-PyV CD8 T cells lacking IL-21R in infected wild type (WT) recipients. Unlike IL21R-sufficient CD8s, IL21R−/− CD8 T cells are not maintained in the brain upon systemic CD8 antibody depletion. IL21R−/− CD8s also have a diminished response to a rechallenge in the brain than what we observed with WT CD8 T cells. RNAseq analysis revealed IL21R−/− CD8 T cells were negatively enriched for TRM core signature genes and oxidative metabolism genes compared to IL-21R-sufficient CD8 T cells. Thus, we conclude that IL-21 produced by high-affinity, PyV-specific CD4 T cells in the brain is required for differentiation of CD8 bTRM during PyV CNS infection.
PD-1 Signals are Critical for Homeostatic Maintenance of Memory CD8 T Cells
Authors: Sarkar Surojit , Yuzefpolskiy Yevgeniy , Vegaraju Adithya , Xiao Hanxi , Baumann Florian , Jatav Shashank , Church Candice , Earnest-bernhart Kayser , Prlic Martin , Jha Abhishek , Nghiem Paul , Riddell Stanley , Sedensky Margaret , Morgan Philip , Kalia Vandana
Published: January 2023

Inhibitory signaling in dysfunctional CD8 T cells through the PD-1 axis is well established in chronic viral infections and cancers. PD-1 is also transiently induced to high levels during priming of acute infections and immunizations, yet its impact on the development of long-lived antigen-independent T cell memory remains unclear. Here we show that in addition to its expected role in restraining clonal expansion, PD-1 expression on antigen-specific CD8 T cells during priming and activation is critically required for the development of a durable CD8 T cell memory pool after antigen clearance. Loss of T cell-specific PD-1 signaling led to increased contraction and a striking defect in antigen-independent renewal of memory CD8 T cells in response to homeostatic cytokine signals, thus resulting in attrition and near ablation of the memory pool over time. Notably, in the setting of PD-1 checkpoint blockade immunotherapy of chronic viral infection, while the exhausted CTLs expectedly regained function, the pre-existing pool of resting functional memory cells established in response to a previously administered vaccine underwent attrition. Metabolically, PD-1 signals were necessary for regulating the critical balance of anabolic glycolysis and fatty acid oxidation programs through mTOR to meet the bioenergetics needs of quiescent memory. These studies define PD-1 as a key metabolic regulator of protective T cell immunity, and have potential clinical implications for pre-existing T cell memory to prior infections and vaccinations during PD-1 checkpoint-blockade immunotherapy in cancer.
Long-term skin resident memory T cells proliferate clonally and contribute to inflammation in human graft-versus-host disease
Authors: Strobl Johanna M , Pandey Ram V , Krausgruber Thomas , Bayer Nadine , Kleißl Lisa , Vieyra-Garcia Pablo , Wolf Peter , Stingl Georg , Bock Christoph , Stary Georg
Published: January 2023

The skin contains a population of tissue-resident memory T cells (Trm) that is thought to contribute to local tissue homeostasis and protection against environmental injuries. While much information about the regulation, survival program and pathophysiological roles of Trm has been obtained from murine studies, less is known about the biology of human cutaneous Trm. Here we demonstrate that in patients undergoing allogeneic hematopoietic stem cell transplantation host-derived CD69+ αβ memory T cell clones in the epidermis and dermis remain stable and functionally competent over one decade. Transcriptional profiling revealed that long-term persisting T cells in the skin are characterized by high expression of tissue retention genes and stem cell markers and, in addition to known Trm markers, may be defined by galectin-3 and RUNX3. Furthermore, skin lesions from patients developing graft-versus-host disease showed an unprecedented large number of locally proliferating host-derived Trm, suggesting a hitherto unrecognized contribution of these cells to the pathogenesis of this condition. Together, our studies highlight the intimate relationship between the local human skin environment and long-term persisting T cell clones which differs from murine skin. Our results further point towards a new understanding of local tissue destruction through both host- and donor-derived Trm following allogeneic hematopoietic stem cell transplantation.
T cell response during Group B Streptococcus vaginal colonization and ascending infection
Authors: Spencer Brady , Alhajjar Norhan , Doran Kelly
Published: January 2023

Pre-term birth, miscarriage, and other adverse pregnancy outcomes remain the primary cause of neonatal death and are associated with bacterial ascending infections within the female reproductive tract. Group B Streptococcus (GBS), a vaginal tract colonizer, is a leading contributor to adverse pregnancy outcomes and neonatal invasive disease. We have developed a mouse model of GBS vaginal colonization and ascending infection and have observed increased IL-17 production in mice that cleared GBS colonization compared to those still colonized. Thus, we hypothesize that IL-17 and IL-17-producing cells may be important for host defense; yet, the immune dynamics during GBS vaginal persistence and ascending infection/adverse pregnancy outcomes remain unknown.

T cells are a major source of IL-17 in the mucosa. To assess the role of adaptive immunity during GBS colonization, we colonized RAG1+/+ and RAG1−/− mice with GBS and observed increased persistence of GBS in the vaginal tract of the RAG1−/− mice. To determine the cellular source of IL-17 within the reproductive tract of GBS-colonized mice, we isolated immune cells from vaginal, cervical, and uterine tissues at the time of GBS clearance and performed flow cytometry. TCRɣδ+ cells comprised the highest percentage of RORɣT+ immune cells in the vagina, cervix, and uterus. We further observed an increased percentage of TCRɣδ+ cells in the uterus of GBS-colonized mice compared to naïve mice at day 9 post-inoculation, at which point half the mice had cleared. We hypothesize that IL-17-producing TCRɣδ+ cells contribute to GBS clearance in the murine reproductive tract. Future studies will assess GBS vaginal persistence and ascending infection in IL-17- and TCRɣδ-deficient mouse lines.
Wnt5A Signaling Restrains Leishmania donovani infection in a mouse model
Authors: MAITY SHREYASI , CHAKRABORTY ARIJIT , SEN MALINI
Published: January 2023

Objective

In view of the emergence of drug resistant strains of L. donovani and persistence of Kala-azar, it is important to understand how host derived factors promote immune resistance to L. donovani infection. Accordingly, considering the role of Wnt5A in maintenance of immune homeostasis, our objective is to evaluate the potential of host Wnt5A signaling in restraining L. donovani infection independent of drug sensitivity or resistance, utilizing a mouse model.

Methods

Effect of Wnt5A depletion on L. donovani infection was deciphered using Wnt5A heterozygous mice with reference to wild type cohorts, where mice were infected with L. donovani promastigotes through iv route. Effect of augmented Wnt5A signaling on L. donovani infection was investigated by iv injection of recombinant Wnt5A protein (rWnt5A) prior to infection. Infection load in liver and spleen was enumerated by Leishman Donovan Unit (LDU) and evaluated by histology. Plasma cytokine levels were checked by ELISA.

Results

Wnt5A heterozygous mice display substantially more disorganized splenic germinal centers and liver granulomas and about 50–60% more LDU upon L. donovani infection as compared to their wild type partners. rWnt5A treatment causes 40–80% reduction in infection load, which correlates with decrease or abolition of splenic germinal center disorganization and liver granulomas, and increase in pro-inflammatory cytokine to anti-inflammatory cytokine ratio.

Conclusion

Our results indicate that (i) Wnt5A signaling protects the host from L. donovani infection independent of drug sensitivity or resistance and (ii) rWnt5A has potential as a prophylactic agent for prevention of L. donovani infection and incidence of Kala-azar.
VCAM1/VLA4 interaction mediates Ly6Clow monocyte recruitment to the brain in a TNFR signaling dependent manner during fungal infection
Authors: Shi Meiqing , Sun Donlei , Strickland Ashley , Chen Yanli , Liu Gongguan
Published: January 2023

Monocytes exist in two major populations, termed Ly6Chi and Ly6Clow monocytes. Compared to Ly6Chi monocytes, less is known about Ly6Clow monocyte recruitment and mechanisms involved in the recruitment of this subset. Furthermore, the role of Ly6Clow monocytes during infections is largely unknown. Here, using intravital microscopy, we demonstrate that Ly6Clow monocytes are predominantly recruited to the brain vasculature following infection with Cryptococcus neoformans, a fungal pathogen causing meningoencephalitis. The recruitment depends primarily on the interaction of VCAM1 expressed on the brain endothelium with VLA4 expressed on Ly6Clow monocytes. Furthermore, TNFR signaling is essential for the recruitment through enhancing VLA4 expression on Ly6Clow monocytes. Interestingly, the recruited Ly6Clow monocytes internalized C. neoformans and carried the organism while crawling on and adhering to the luminal wall of brain vasculature and migrating to the brain parenchyma. Our study reveals a substantial recruitment of Ly6Clow monocytes to the brain and highlights important properties of this subset during infection.
Adaptive γδ T cell function is subverted by Yersinia pseudotuberculosis outer proteins
Authors: Chu Timothy H , Khairallah Camille F , Cho Rhea , Zhang Yue , Lin Xun , Bliska James B , Sheridan Brian S
Published: January 2023

γδ T cells are an unconventional subset of multifunctional T cells enriched in mucosal barrier tissues. After foodborne Listeria monocytes (Lm) infection, an activated CD44hiCD27loVγ4 subset of γδ T cells formed in the mesenteric lymph nodes (MLN) that shared numerous characteristics with traditional T cells including anamnestic responses. While attempting to uncover the specificity of Lm-elicited memory cells utilizing heterologous challenge infections we observed that Lm-elicited γδ T cell function was induced with heat-killed Yersinia pseudotuberculosis (Yptb) but not live Yptb suggesting that Yptb employs immune evasion strategies to limit anti-pathogen adaptive γδ T cell functions. Live Yptb inhibited CD44hiCD27loVγ4 T cell IL-17A and IFNγ production integral for anti-pathogen immunity. Tracking Yptb translocation through a FRET-based reporter assay revealed preferential translocation of Yptb outer proteins (Yop) into adaptive γδ T cells over conventional T cells and an abrogated cytokine response compared to their non-translocated counterparts within the same environment. Using a series of Yptb mutants deficient in translocated effector proteins, we determined that YopH inhibited IL-17A production while YopJ inhibited IFNγ production. Inhibition of Stat4 phosphorylation in γδ T cells was observed in a translocation, YopJ, and IL-12/IL-23p40 dependent manner, suggesting that YopJ inhibits IFNγ production through Stat4 phosphorylation. Collectively, these data demonstrate direct inhibitory mechanisms of γδ T cell function mediated by the Yptb effectors YopH and YopJ and identify an unknown pathogenesis pathway utilized by Yptb to subvert immune function.
Metabolic ‘de-programming’ of neutrophils protects against fatal bloodstream fungal infections during kidney disease
Authors: Biswas Partha Sarathi , Jawale Chetan V , Ramani Kritika , Coleman Bianca , Oberoi Rohan S , Kupul Saran , Prokopienko Alexander J , Desai Jigar V , Delgoffe Greg M. , Lionakis Michail S , Nolin Thomas D , Gaffen Sarah L
Published: January 2023

Disseminated candidiasis (DC) is the third most common cause of mortality in hospital acquired infections. Disseminated candidiasis caused by the fungus Candida albicans is a major clinical problem in individuals with kidney disease and accompanying uremia. DC fatality is twice as common in patients with uremia as those without renal impairments. Many antifungal drugs are nephrotoxic, making treatment of these patients challenging. The underlying basis for this impaired capacity to control infections in uremic individuals is poorly understood. Here we show that uremic mice show an increased susceptibility to DC. Uremia inhibits Glucose transporter 1 (Glut1)-mediated uptake of glucose in neutrophils by causing aberrant activation of Glycogen synthase kinase 3 beta (GSK3beta), resulting in reduced ROS generation and hence impaired killing of C. albicans in both mice and human cells. Consequently, pharmacological inhibition of GSK3beta ‘de-programs’ neutrophil function and restores glucose uptake, ROS production and candidacidal activity of neutrophils in uremic mice. These findings reveal a central mechanism of neutrophil dysfunction during uremia and suggest a potentially translatable therapeutic avenue for treatment of DC, with broader implications for other fatal systemic infections.
ESX5 associated PE proteins of Mycobacterium tuberculosis drives Th1 type polarization: an immunomodulatory paradigm
Authors: Ehtram Aquib , Shariq Muhammed , Ali Sabeeha , Ehtesham Nasreen Z. , Hasnain Seyed E.
Published: January 2023

Mycobacterium tuberculosis (M.tb) harbors various effector molecules that affect human immune system. There exists a varied difference between the slow growing pathogenic mycobacteria compared to the fast growing non-pathogenic strains. One marked difference is the presence of ESX5, a type VII secretion system that is found to be most recently evolved and has been acquired by the pathogenic M.tb. ESX5 is involved in the secretion of an enigmatic PE/PPE family of proteins that are exclusive to the genus mycobacteriacea. PE/PPE are the effector virulent molecules that play vivid role in modulating host immune system. ESX5 locus has 2 pe genes and 3 ppe genes. Of the translated products, two PE proteins are paralogs, but contrastingly found to have different PPE binding partners as these proteins function as heterodimers, homodimers and individual proteins. THP1 macrophages (differentiated human monocytes) when treated with different micro-concentrations of PE 1X and PE 1Y induce differential Th1 type cytokine responses. PE 1X and PE 1Y induced stronger TNF-α response at 24hrs of protein exposure that increased markedly at 48hrs. IL-12 p70 was found to decrease from 24hrs to 48hrs during PE 1X treatment whereas PE 1Y exposure led to the secretion of IL-12 p70 which was constantly maintained from 24hrs to 48hrs of exposure time. PE 1X specifically mediated TLR2 dependent activation of macrophages that was responsible for the secretion of pro-inflammatory cytokines. On the contrary PE 1Y activated macrophages through TLR4 dependent cascade. These results therefore, conclude that PE 1X and PE 1Y contribute profoundly towards Th1 polarization of the immune response.
Immune responses to Dengue infected microvascular endothelial cells
Authors: Sekaran Shamala Devi , Soe Hui Jen , Raju Chandramathi Samudi , Manikam Rishya , Vellasamy Shalini
Published: January 2023

Severe dengue is associated with manifestations such as bleeding, fluid accumulation and organ impairment. This study aimed to investigate the role of dengue non-structural 1 (NS1) protein and host factors contributing to severe dengue. Electrical cell-substrate impedance sensing system was used to investigate the changes in barrier function of microvascular endothelial cells treated NS1 protein and serum samples from dengue patients. Cytokines and metabolites profiles were also assessed. Our study revealed that dengue NS1 protein was able to induce the loss of barrier function of the microvascular endothelium in a dose dependent manner. However, the level of NS1 did not correlate with the extent of vascular leakage observed in the microvascular endothelium treated with serum samples from patients with dengue virus infection. This implies presence of other host factors that might overshadow the direct effect of NS1 in inducing vascular leakage during dengue virus infection. Most of the cytokines that were highly expressed in dengue patients including CCL2, CCL5, CCL20 and CXCL1 are involved in leukocyte infiltration where the rearrangement of junctional complex proteins such as ICAM-1, which was detected significantly higher in patients with SD, may lead to the disruption of inter-endothelial junctions. Altered lipid metabolism was detected in in patients with DWWS and SD. This might be associated with phospholipid metabolism, which may affect the membrane permeability of cells including the microvascular endothelium. The identification of these altered metabolites could facilitate dengue diagnosis or be used as a potential target for new therapeutic options.
Modeling a human STAT4 mutation that predisposes to disseminated Coccidioidomycosis in mice
Authors: Powell Daniel A , Shubitz Lisa F , Butkiewicz Christine D , Moale Hilary , Trinh Hien T , Doetschman Thomas , Hsu Amy P , Holland Steven M , Galgiani John N , Frelinger Jeffrey A
Published: January 2023

Coccidioidomycosis is a common fungal respiratory disease caused by the fungus Coccidioides spp. fungi in Arizona and the central valley of California. Disseminated Coccidioidomycosis (DCM) occurs in 8% of identified cases, causing significant morbidity and mortality. DCM occurred in 3 generations of a family in Tucson, Arizona who all shared a heterozygous missense mutation (p.E626G) in STAT4. Transfection of STAT4 E626G in WT human cells showed dampened responses to STAT4-dependent cytokine gene induction, suggesting the mutation is a dominant negative.

To understand the function of E626G mutation, we generated the homologous mutation in Stat4 in B6 mice. Mice carrying E626G were grossly normal with normal numbers of innate and adaptive immune cells in primary and secondary lymphoid organs. To examine the effect of E626G on susceptibility to Coccidioides infection, we infected mice with C. posadasii strain 1038 intranasally. After infection E626G heterozygous mice all died by 50 days post infection (dpi), median time to death 45.5 days; range 30–50 dpi. 87% of WT B6 controls were alive when the experiment ended at a predetermined 70 dpi, resulting in a median time to death of >70 days. Mediastinal lymph nodes in E626G heterozygous mice had reduced numbers of B cells and activated (CD62Llow) T cells compared to B6 controls on 14 dpi. Cells from infected lungs from E626G heterozygotes produced less IFN-gamma after infection on 28, 35 and 42 dpi. Interferon stimulated cytokines (IP-10, MIG, MIP-2) were also significantly reduced compared to WT controls. Ongoing work includes examining the cells requiring STAT4 signaling and IFN-gamma production as well as the mechanism of reduced accumulation of immune cells in the draining lymph nodes.
Clostridia protect from food allergy by promoting intestinal barrier integrity and modulating retinoic acid metabolism
Authors: Kemter Andrea Maria , Wang Shan , Nagler Cathryn
Published: January 2023

We previously reported that intestinal bacteria of the Clostridia class modulate epithelial barrier function through induction of IL-22 and hypothesized that this was crucial for their allergy protective effect. We now confirm that hypothesis and further describe the mechanisms underlying this protection. Clostridia colonization of weanling germ free mice induced colonic IL-22 production and expression of the antimicrobial peptides Reg3b and Reg3g within three days. At the same time, the numbers of CD11b+, CD103+ and CD11b+CD103+ dendritic cells (DC) in the colonic lamina propria were reduced, while the number of macrophages remained unaltered. Seven days post colonization, the numbers of those same DC subsets were increased in the colon draining mesenteric lymph node (cMLN) and we detected an increased ability to produce retinoic acid (RA) in CD11c+ cells in the cMLN. We also observed increased expression of Reg3b, Reg3g and the retinol dehydrogenase Rdh7 in the ileum and the RA-responsive gene Isx in colon epithelial cells. At 14 days, Clostridia colonization increased percentages of RORγt+ Foxp3+ regulatory T cells (Tregs), which are known to efficiently suppress type 2 immune responses. Finally, preliminary data showed that the TLR5 ligand flagellin replicates Clostridia-induced production of IL-22 and intestinal barrier protection but not modulation of RA metabolism. With the rapid induction of IL-22, which supports intestinal barrier integrity, and the slower modulation of retinoic acid metabolism and Treg induction we describe two mechanisms by which Clostridia promote tolerance to food antigens. Detailed knowledge of these mechanisms will enable us to develop new strategies to combat the dramatic rise of food allergies.
Microbial enzyme stereoselectively converts EPA into 17(S),18(R)-epoxyeicosatetraenoic acid useful for the amelioration of contact hypersensitivity
Authors: Saika Azusa , Nagatake Takahiro , Kishino Shigenobu , Park Si-Bum , Honda Tetsuya , Matsumoto Naomi , Shimojou Michiko , Morimoto Sakiko , Tiwari Prabha , Node Eri , Hirata So-ichiro , Hosomi Koji , Kabashima Kenji , Ogawa Jun , Kunisawa Jun
Published: January 2023

Dietary intake of ω3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid are beneficial for health control because ω3 fatty acids are metabolized to pro-resolution and anti-inflammatory lipid metabolites. We previously identified 17,18-epoxyeicosatetraenoic acid (17,18-EpETE) as a new lipid metabolite endogenously generated from EPA that exhibits potent anti-allergic and anti-inflammatory properties. However, the chemically synthesized 17,18-EpETE is enantiomeric mixture concerning to its epoxy group, i.e., 17(S),18(R)-EpETE and 17(R),18(S)-EpETE, and the stereostructural differences in anti-inflammatory effect of 17,18-EpETE has not been clarified. Because it is necessary to identify which isomer shows physiological function to develop it as a medicine, we evaluated stereospecific effects of 17(S),18(R)-EpETE and 17(R),18(S)-EpETE in amelioration of skin contact hypersensitivity. As a result, we found that anti-inflammatory activity was detected in 17(S),18(R)-EpETE, but not 17(R),18(S)-EpETE. In addition, we found that cytochrome P450 BM-3 derived from Bacillus megaterium stereoselectively converts EPA into 17(S),18(R)-EpETE, which effectively inhibited the development of contact hypersensitivity by inhibiting neutrophil migration in a G protein-coupled receptor 40-dependent manner. These results demonstrated that immune cells (e.g., neutrophils) distinguish biological active lipid metabolites stereoselectively for the control of their activity, which is a new strategy for the development of efficient immunotherapy.
Regulation of thymic T-cell development by intestinal microbiota
Authors: Majumdar Shubhabrata , Ruiz Daniel Zegarra , Wu Wan-Jung , Orozco Stephanie , Bettini Maria , Diehl Gretchen , Bettini Matthew L.
Published: January 2023

Host commensal microbiota plays an important role in the development and functioning of a healthy immune system. Early life interference with commensal colonization is associated with immune disorders like inflammatory bowel disease (IBD), asthma, allergic rhinitis and atopic dermatitis. One important aspect of microbiota mediated immune regulation is its impact on T-cell differentiation and function. Although most studies show that microbial regulation of T-cells is limited to the periphery, some studies have suggested that the microbiota might impact antigen specific T-cell development in the thymus. However, there has been insufficient data. Interestingly, we are able to detect bacterial signatures by 16s sequencing within mouse and human thymus. Additionally, we observed development of antigen specific thymocytes in SFB colonized 7B8 TCR transgenic mice and in wildtype mice through SFB tetramer staining. We therefore, hypothesize that antigen-presenting cells (APCs) from the gut sample local microbial antigens and migrate to the thymus to present them to the developing thymocytes and influence selection. To test this hypothesis, we tracked the migration of intestinal APCs to the thymus using mice that express the photoconvertible Kikume protein. We performed targeted photo-conversion of the cecum through live surgery and discovered photoconverted APCs in the thymus 48 hours later. These observations open up the possibility that intestinal APCs migrate to the thymus early in life to shape the microbiota specific TCR repertoire.
Innate CD8αα cells orchestrate intraepithelial lymphocytes balance and protect against colitis
Authors: Nazmi Ali , Hoek Kristen L. , Tansie Stephen , Piazuelo M. Blanca , Olivares-Villagómez Danyvid
Published: January 2023

Innate CD8αα+ cells (iCD8α) are TCRneg intraepithelial lymphocytes (IEL) that possess immune functions such as phagocytosis, bacteria killing, and antigen processing and presentation. Recently our group and others reported that iCD8α cells promote IEL survival via osteopontin. Herein, we investigate whether iCD8α cells sustain intestinal homeostasis through maintaining IEL balance.

We compared the number of IEL in the colon of WT and iCD8α-deficient mice (E8i−/−). E8i is an enhancer required for CD8α homodimer expression in IEL. Despite the dramatic reduction of CD8αα expression on TCRαβ+ and TCRgd+ IEL in E8i−/− mice, the total cell numbers were comparable to WT mice. E8i−/− mice had higher number of TCRβ+CD4+, and lower number of TCRβ+CD4+CD8α+ and TCRβ+CD8αα+ IEL. There were no differences in TCRβ+CD8αβ+ and TCRneg IEL.

Then, we tested the role of iCD8α cells during intestinal inflammation in different colitis models. iCD8α-deficient mice had higher susceptibility to Citrobacter rodentium infection as indicated by increased weight loss, disease index, and bacterial load in the colon compared to WT mice. Similar results were obtained using DSS-induced colitis: E8i−/− mice presented increased shortening of colon and pathological scores. Finally, we performed adoptive transfer of naïve CD4 T cells into Rag-2−/− and E8i−/−Rag-2−/− mice. iCD8α-deficient mice rapidly developed chronic colitis, manifested by sever weight loss and bloody diarrhea. These mice also reconstituted with higher number of donor-derived CD4 T cells that mostly developed into pathogenic TCRβ+CD4+ IEL.

Our results indicate that iCD8α cells have a critical role in the maintenance of IEL homeostasis and healthy intestines.
Resident memory CD8 T cells patrol the lung and mediate early protective immunity
Authors: Anthony Scott M , Langlois Ryan A , Varga Steven M , Harty John T
Published: January 2023

In contrast to the long-lived nature of resident-memory CD8 T cells (Trms) residing in the skin or intestine, Trms located in the lung parenchyma wane and their loss coincides with the decline of protective immunity against subsequent heterosubtypic Influenza infections. We utilized intravital 2-photon microscopy of live mice to dissect the relative contribution and examine the real-time spatiotemporal dynamics of secondary CD8 T cell responses containing or lacking lung Trms. Early post-Influenza infection, the lung contained a mixture of phenotypically defined circulating and CD69+/CD103+ resident-memory transgenic P14 cells which exhibited heterogeneous motility patterns, comprised of both highly motile cells and those exhibiting a slow to moderate scanning pattern. The incorporation of IV exclusion during intravital imaging predominantly labeled highly dynamic cells, and largely spared slower CD103+ Trms in the lung parenchyma and surrounding airways. Conversely, the selective loss of this slow to moderate population was observed with either CD103 antibody-mediated depletion of early memory or at 6 months post infection, suggesting that CD103+ CD8 Trms exhibit slow to moderate motility dynamics in the lung at homeostasis. Importantly, upon secondary infection, hosts containing Trms exhibited a higher proportion of CD8 T cells with altered dynamics, more pronounced clustering around infected cells in the lung in an antigen-specific manner and provided superior early heterosubtypic protection compared to hosts containing only circulating memory. Overall, these data indicate that lung Trms exhibit slow to moderate motility patterns, rapidly respond to and mediate early immunity to subsequent infections in the lung.
Ornithine-A urea cycle metabolite enhances autophagy and controls Mycobacterium tuberculosis infection
Authors: Thandi Ramya Sivangala , Radhakrishnan Rajesh kumar , Tripathi Deepak , Paidipally Padmaja , Vankayalapati Ramakrishna
Published: January 2023

Alveolar macrophages (AMs) are the first cells encountered by TB pathogen and serve as the primary defense against Mycobacterium tuberculosis (Mtb) in the lungs. Studies have shown that liver macrophages (Kupffer cells; KCs) can control Mtb growth but, it is not clear how KCs completely eliminate mycobacterial infections. Thus, identification of these mechanisms will facilitate the development of immunomodulatory strategies to boost AM-mediated immunity to Mtb. In the current study, we compared Mtb growth in murine AMs, peritoneal (PMs), liver macrophages (Kupffer cells; KCs) and bone marrow-derived monocytes (BDMs). KCs restricted Mtb growth more efficiently than all other macrophages and monocytes despite equivalent infections. Differences in Mtb growth restriction were not due to differences in cytokine production, expression of Toll-like receptors 2 and 4, M1/M2 paradigm or apoptosis. We provide evidence that the enhanced autophagy efficiently restricts Mtb growth in KCs using flow cytometry, western blot, RT-PCR and confocal microscopy. A metabolomic comparison of Mtb-infected macrophages by liquid chromatography mass spectrometry indicated that ornithine (VIP=1.8) and imidazole (VIP=1.6) were two top-scoring metabolites found in Mtb-infected KCs and that acetylcholine was top-scoring in Mtb-infected AMs. Ornithine and imidazole inhibited Mtb growth in AMs by enhancing AMPK mediated autophagy whereas imidazole directly killed Mtb by reducing cytochrome P450 activity. Intranasal delivery of ornithine or imidazole or together restricted Mtb growth in Mtb-infected mouse lungs. Our study demonstrates that the metabolic differences in Mtb-infected AMs and KCs leads to differences in the restriction of Mtb growth.
Regulation of Respiratory CD8+ T cell Immunity by Suppressive Monocyte Derived Dendritic Cells (moDCs)
Authors: Reagin Katie L , Klonowski Kimberly D
Published: January 2023

Active immune suppression can mediate the balance between protective cellular immunity and harmful immunopathology. This suppression can occur locally, at an infection site, or in regional draining lymph nodes (dLN). Immune regulation is of particular importance in sites such as the lung where aberrant immunopathology can result in loss of tissue function. We have recently identified a novel population of CD11b+CD103+CCR2+monocyte-derived dendritic cells (moDCs) which directly suppress CD8+ T cell proliferation in vitro. Respiratory infection with RNA viruses recruits these moDCs either exclusively to the dLN (after vesicular stomatitis virus infection) or both the dLN and site of viral replication (after influenza infection). Exclusive depletion of moDCs from the dLN using CCR2-DTR bone marrow chimeras results in enhanced respiratory CD8+ lung tissue resident memory cell formation. By contrast, depletion of moDCs from both the dLN and respiratory tract following influenza infection results in enhanced respiratory CD8+ T cell responses coupled with fatal immunopathology. Together, these data suggest that suppressive moDCs govern key aspects of respiratory immunity by regulating the function and development of long-lived CD8+ T cells, thereby balancing immunity and adverse pathology in the context of viral infection. Ongoing experiments seek to uncouple moDC interactions with CD8+ T cells in specific sites in order to better understand, and potentially beneficially modulate, CD8+ T cell immunity within the lung.
Empty-loadable MHC class I tetramers for efficient detection of antigen-specific T cells
Authors: Tamhane Tripti , Saini Sunil Kumar , Anjanappa Raghavendra , Saikia Ankur , Ramskov Sofie , Donia Marco , Swane Inge Marie , Jakobsen Søren Nyboe , Zacharia Martin , Meijers Rob , Springer Sebastian , Hadrup Sine
Published: January 2023

The peptide-dependent stability of MHC class I molecules poses a substantial challenge for their use for peptide-MHC multimer-based approaches to comprehensively analyze T cell immunity. We demonstrate here the generation, analysis, and use of empty-loadable (peptide-free) MHC class I tetramers made from disulfide-stabilized MHC molecules.

A disulfide bond links the α1 and α2 helices of MHC class I molecule at the extreme end of the F pocket. It allows in vitro folding of recombinant A2 with a dipeptide and subsequent removal of the dipeptide to yield stable peptide-receptive MHC monomers. Empty-loadable tetramers prepared using disulfide-stabilized MHC monomers can be loaded with peptides within minutes. These tetramers efficiently detects antigen-specific T cells. Furthermore, peptide-MHC tetramers prepared using disulfide-stabilized MHC molecules provide a better staining index for antigen-specific T cell detection compared to multimers prepared with wild-type MHC class I molecules. We demonstrate the value of empty-loadable tetramers that are converted to antigen-specific tetramers by a single-step peptide addition, for the identification of T cells specific to several neo- and cancer-associated antigens in melanoma. Disulfide stabilization has been achieved with several MHC class I allotypes-HLA-A* 02:01, HLA-A*01:01, HLA-A*03:01, HLA-A*11:01, HLA-A*24:02, HLA-B*07:02, and H-2Kb.
Use of TCR-mimic bispecific antibodies against pMHC-II for monitoring antigen processing and redirecting cytolytic effector cells
Authors: Sengupta Srona , Douglass Jacqueline , Wu Fengting , Moskovljevic Milica , Reed Madison , Yu Jeanna , Tabdili Yasmine , Hsiue Emily Han-Chung , Veenhuis Rebecca , Vogelstein Bert , Siliciano Janet , Siliciano Robert , Sadegh-Nasseri Scheherazade
Published: January 2023

HIV-1 infects CD4+ T cells and macrophages and is currently incurable due to a latent reservoir. Targeting infected cells using TCR-mimic antibodies to peptide-MHC (pMHC) would provide a specific, high-affinity platform for immune system activation and monitoring antigen processing. However, antibodies to pMHC are notoriously difficult to isolate. Using a phage display platform, we panned for phage expressing variable fragments against immunodominant HIV pMHC complexes. These Fab fragments were cloned into a bispecific antibody (bsAb) backbone, such that one Fab fragment is specific for an HIV pMHC and the other fragment binds to CD3. These antibodies utilize the sensitivity of T cell signaling as readouts for antigen processing and potentially as therapeutic formats. Using this approach, we have shown that bsAbs specific for the most conserved MHC-II HIV capsid epitope (Gag293) can recognize the processed epitope on human monocyte-derived dendritic cells (moDCs) fed with the whole Gag protein antigen. Recognition is exquisitely epitope-specific. Additionally, human monocyte-derived macrophages infected with HIV were able to present Gag293 using the endogenous MHC-II pathway, as read out by a Gag-specific bsAb. To our knowledge, this is the first description of endogenous class II presentation of HIV in a major cell type that supports viral replication. By developing bsAbs to detect HIV peptide presentation, we can begin to address the kinetics, pathways, and cell types contributing to the priming events in early HIV infection, which will inform improved vaccine therapies. Additionally, given the potential for therapeutic application, these bsAbs may be used to redirect cytolytic effector cells towards infected targets.
A Unique Multiplex Assay to Identify Th1/Th2 Cytokine Profile in MS Patients
Authors: Madiraju Charitha , Karp Jacob Alexander , Oppong Martha , Matson Robert
Published: January 2023

Multiple Sclerosis (MS) is a chronic neuroinflammatory, demyelinating autoimmune disease of the central nervous system (CNS). There are diverse views on the paradigm that disease pathogenesis of MS is due to an imbalance in the proinflammatory T helper 1 (Th1)-type cytokines and anti-inflammatory Th2-type cytokines. Several factors play a crucial role in Th1/Th2 polarization that contributes towards disease activation or progression and this is dependent on MS disease sub-type. The goal of our research study is to investigate a unique and robust multiplex assay for identification of Th1 versus Th2 cytokine expression pattern as applicable to samples derived from MS patients. Towards this end, a sandwich ELISA-based multiplex assay method in a 96-well plate format is designed for establishing Th1/Th2 cytokine profile. Cerebrospinal fluid (CSF) samples derived from MS patients diagnosed with either Relapsing-Remitting MS (RRMS, N = 10) or Secondary progressive MS (SPMS, N = 10) and two unaffected control CSF samples derived from the otherwise healthy human subjects are included in the study. Multiplex ELISA analysis in a 96-well format included TNF, IFN-gamma, IL-4, IL-13 and IL-17E cytokines. Each well is imprinted in a 3*3 format with a capture antibody against a given cytokine of interest. The colorimetric read-outs from multiplex assay are analyzed by ImageJ. Preliminary results identify the multiplex assay to be robust, unique and cost-effective for establishing Th1/Th2 cytokine profiles in two different sub-types of MS. Th1/Th2 cytokine pattern could help predict the course of MS disease progression and remission.