bluuebunny/crossref_metadata_2025 huggingface dataset:
Preloading of CD4+CD25+ Tregs to donor islets in vitro prolongs islet allograft survival
Authors: Dai Zhenhua , Zhang Shuzi
Published: January 2023

Despite extensive studies on regulatory T cells (Treg) in transplantation (Tx), Treg transfer alone remains ineffective in wild-type (WT) animal recipients, because only a small fraction of systemically administered Tregs can reach an allograft. The objective of this study is to optimize Treg suppression of islet allograft rejection via preloading Tregs to donor islets before Tx. We developed a unique system in which donor islets were activated and incubated with CD4+CD25+ Treg cells, resulting in intraislet recruitment of Tregs before Tx. Donor islets were stimulated by cytokines (IL-1β+ IL-6 + IFNβ) for 24 h and incubated with CD4+CD25+FoxP3+ Tregs for 48 h so that Tregs infiltrated the islets. Islets then were rested in the media for another 24 h before Tx. We found that “Treg-preloaded” islet allografts survived much longer than control islets with long-term islet allograft survival in the hepatic parenchyma in 43% of WT recipient mice (n=7) without any immunosuppressive drug. Importantly, Treg-preloaded islet allografts also survived significantly longer when transplanted in the hepatic parenchyma than in kidney capsule (MST = 62 vs. 39 days, n=7–8, P < 0.05) or the portal vein (MST = 62 vs. 41 days, n=7, P < 0.05). As a control, cytokine-stimulated islets with the similar culture without Treg cells were rejected at the same pace as fresh control islets (14 vs. 16 days, n=7–8, P>0.05). Moreover, BrdU staining demonstrated that graft-infiltrating Thy1.1- T cells of recipients proliferated at a slower pace in islets preloaded with Tregs than in control islets (BrdU+: 21% vs. 43%) one week after Tx. In conclusion, transplanting Treg-preloaded islets in the hepatic parenchyma is a novel approach to prolonging islet allograft survival.
IL-17C ameliorates murine acute graft-versus-host disease by promoting regulatory T cell differentiation and maintaining intestinal epithelial barrier functions
Authors: Gong Huanle , Ma Shoubao , Dong Chen , Wu Depei , Liu Haiyan
Published: January 2023

Among IL-17 family cytokines, functions of IL-17A, IL-17F, and IL-17E (also named IL-25) are well documented in host defense against infections and inflammatory diseases. Recent studies have demonstrated that IL-17C binds to a unique heterodimeric receptor complex composed of IL-17RA and IL-17RE subunits. IL-17C was reported in the regulation of DSS-induced colitis, experimental autoimmune encephalomyelitis and other inflammatory immune disorders. However, the function of IL-17C in acute graft-versus-host-disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains unknown.

In this study, IL-17C deficiency mice as donors showed a significantly accelerated aGVHD-related mortality compared with wild type donors in a fully MHC-mismatched bone marrow transplantation model (C57BL/6 to BALB/c). We found that the productions of IFN-γ by T cells were significantly increased in spleen, liver, lung as well as intestinal epithelia lymphocytes with IL-17C deficient donors. Furthermore, a reduction of FoxP3+ regulatory T cell population was observed with IL-17C−/− donors. In vitro studies showed that the percentage of Treg cells were decreased in IL-17C−/− splenocytes in a Treg polarizing condition compared with WT splenocytes. Moreover, the expression of tight junction protein occludin was decreased with IL-17C−/− donors when compared with WT controls.

In summary, IL-17C protects against aGVHD by possibly promoting Treg differentiation and maintaining intestinal barrier functions. Further studies are required to explore detailed mechanisms of its action and its potential therapeutic applications for clinical prevention and treatment of aGVHD.
Immunochemistry of the Proteins of Dog Hair
Authors: Wodehouse R. P.
Published: January 2023

Summary and Conclusions

From dog hair there can be dissolved out with weak alkali a small amount of protein. Part of this, the “alkali metaprotein,” can be precipitated by neutralization while the rest remains dissolved but can be recovered from solution by dialysing out the salt and evaporating to dryness. This we have called “dog hair peptone.”.

These two proteins show themselves, by their skin reactions with asthmatics, to be immunologically distinct and also distinct from a peptone obtained from pure keratin, which is itself inactive, by hydrolysis, and from the serum proteins of the same species.
On the Non-Influence of Injections of Pure Proteins upon the Proportions of Globulin and Albumin in Blood Serum
Authors: Schmidt Esther Skolfield , Schmidt Carl L. A.
Published: January 2023

Leucocyte counts and determinations of the serum proteins of rabbits receiving doses of antigenic, non-antigenic, toxic and non-toxic proteins have been made for extended periods. This was likewise done with rabbits injected with benzol to produce a leucopenia. The figures show that while the quantity of serum proteins in normal rabbits is fairly constant the protein ratio varies considerably in different animals and to a somewhat lesser extent in individual rabbits. The injection of proteins in doses used was not followed by any decided change in the protein quotient. A decrease of serum proteins follows the injection of benzol, though no change occurs in the protein quotient. A rise of globulins is not necessary for immune body production. Immunity in rabbits may be produced with well regulated dosage of antigen without giving rise to an increase of globulins. Our results are essentially in accord with recent work on the non-dependence of immunity on the serum proteins, as ordinarily determined.
The Effect of Alexin in Virus-Antivirus Mixtures
Authors: Mueller J. Howard
Published: January 2023

In most experiments with filterable viruses designed to show neutralizing or viricidal action by specific immune sera, the possibility of alexin exerting an effect during a preliminary incubation seems largely to have been neglected. It has been assumed that, because such mixtures may be tested only in the animal body for survival or destruction of the virus, and since alexin is there available, that it would be superfluous to add it as a separate component of the mixtures before injection. Although at first view this appears reasonable, certain observations may well cast some doubt on its validity.

It has been shown, for example, by Andrewes (1), Bedson (2) and Todd (3) that inactive mixtures of certain viruses with their specific antisera are readily dissociated by physical means (adsorption, dilution or filtration) in such a way that an active virus is again obtained. Moreover, Pentimalli (4) and Duran-Reynals and Murphy (5) have found that the virus of the Rous chicken tumor is readily adsorbed onto or into susceptible cells.
Hypersensitiveness to Diphtheria Bacterial Products
Authors: Sugg John Y. , Richardson Lurline V. , Neill James M.
Published: January 2023

This paper reports further experiments on guinea pigs actively sensitized against diphtheria toxoid filtrate. Five points are made. First, that the anaphylactic reaction of the actively sensitized guinea pigs is inhibited by incubation of the test dose of antigen (toxin or toxoid filtrate) in mixture with antitoxic horse globulin prior to its intravenous injection. Second, that actively sensitized guinea pigs are made refractory by intravenous injection of the antitoxic horse globulin or serum shortly before the test injection of the toxin or toxoid filtrate antigen. Third, that guinea pigs anaphylactically reactive to toxoid filtrate are made refractory by subcutaneous injections of toxoid filtrate ten and seven days before the time of intravenous test, but not by similar injections of diphtheria bacterial vaccine. Fourth, that the anaphylactic reaction is inhibited if filtrate absorbed with antitoxic rabbit serum is used as the intravenous antigen, but not if filtrate similarly absorbed with antibacterial rabbit serum is used. Fifth, that the capacity of toxoid filtrate either to invoke antitoxic immunity or to actively sensitize, is greatly reduced, and under certain quantitative conditions entirely lost, if filtrate absorbed with antitoxic rabbit serum is used as immunizing or sensitizing material, while the same filtrate similarly treated with antibacterial rabbit serum retains the capacity both to invoke antitoxin production and to sensitize against the toxoid filtrate.
Neuritin Is Required for T Regulatory Cell-Mediated Suppression of the Long-Lived Plasma Cell Niche
Authors: Cooper James J. M. , Newman Stephanie , Egan Stephanie , Utley Adam , Lee Kelvin P. , Barbi Joseph J.
Published: January 2023

T Regulatory cells (Tregs) are critical mediators of the immune system both during active immune responses and in the maintenance of immune homeostasis. Neuritin, a highly conserved gpi-anchored protein, has been shown to regulate Treg maintenance and function in vivo. Recent data demonstrates a critical role for Tregs in the survival of bone marrow (BM) resident plasma cells (BMPCs), and that CTLA-4 expressed on Tregs limits the BMPC niche. However, the molecular mechanisms by which Tregs in the bone marrow both promote BMPC survival and limit the niche space are unknown. Here we demonstrate that Treg intrinsic neuritin levels correlate with high expression of CD44, and that activation increases the population of CD44 expressing cells in neuritin-high Tregs. This suggests that neuritin positively regulates the induction of effector Tregs. However, there is not a significant difference in the percentage of Tregs in the BM of wild type (WT) vs neuritin knock-out (KO) mice. Interestingly, in neuritin KO BM, there is a significant increase in the % of B220-ve CD138+ve BMPCs, implying that neuritin negatively regulates the BMPC pool. Previously published data from our collaborators show that CD28 is required for BMPC survival. This occurs via interaction with BM resident CD80/86 expressing dendritic cells (DCs). CTLA-4, possibly from Tregs, can also bind CD80/86 on DCs. This may sequester the survival signals to BMPCs from CD80/86. This leads to a model wherein neuritin-mediated regulation of bone marrow Treg effector functionality negatively regulates the BMPC survival niche. With the ability to target CTLA-4 with novel immunotherapeutic strategies, this understanding may allow us to enhance vaccine design and augment humoral immunity.
Tumour-elicited neutrophils engage mitochondrial metabolism to circumvent nutrient limitations and maintain immune inhibition
Authors: Rice Christopher M. , Davies Luke C. , Subleski Jeffery J. , Maio Nunziata , Andrews Caroline , Cotto Marieli Gonzalez , Lee Jung-min , Annunziata Christina M. , Rouault Tracey K. , Durum Scott K. , McVicar Daniel W.
Published: January 2023

Neutrophils are a vital component of the immune system, however in cancer they often play major roles in tumour progression, partly by generating reactive oxygen species (ROS) which disrupts lymphocyte function. Metabolically, neutrophils are often overlooked as purely glycolytic, however we show that immature subsets, defined by c-Kit expression, possess the capacity for oxidative mitochondrial metabolism. In limited glucose, oxidative neutrophils use mitochondrial fatty acid oxidation to fuel NADPH-oxidase (NOX) dependent ROS production. In 4T1 tumour bearing mice, mitochondrial fitness is enhanced in circulating and splenic neutrophils and is associated with the expression of c-Kit. Concordantly, tumour-elicited oxidative neutrophils are able to maintain ROS production and suppression of T-cell when glucose utilization is restricted. In accordance with these findings, peripheral blood neutrophils from cancer patient are more immature, with increased mitochondrial content and higher subsequent oxidative phosphorylation. Together our data suggest that tumours elicit oxidative neutrophil populations which are adapted to maintain immune-suppressive activity in the glucose-restricted tumour microenvironment.
The role of ERK2 in bone marrow-derived dendritic cells development and function
Authors: Jeffreys Sean Alec , Forsthuber Thomas , Negron Austin Joseph
Published: January 2023

Dendritic cells (DCs) regulate antigen-specific T cell responses by providing stimulatory or inhibitory signals through surface receptors and cytokines, the expression of which relies on the mitogen-activated protein kinase (MAPK) signaling pathway. In lymphocytes, the MAPK pathway is involved in differentiation, proliferation, and survival, using extracellular signal regulated kinases 1 and 2 (ERK1/2) as primary effector molecules. Importantly, ERK1 and ERK2 are not functionally redundant, despite 85% sequence homology. The role of ERK1 in DCs has been explored, however that of ERK2 remains unknown due to the lack of Erk2 Δ transgenic models. Our lab has developed a novel mouse model in which Cre-mediated deletion of Erk2 is accompanied by the expression of an eYFP reporter gene, allowing us to identify and isolate viable Erk2 Δ cells using flow cytometry. Preliminary data has shown that DCs cultured from bone marrow progenitor cells (BMDCs) exhibit altered expression of several surface receptors associated with both antigen presentation and T cell stimulation when compared to wild type BMDCs. We therefore hypothesize that ERK2 deficiency will significantly affect the ability of DCs to prime naïve T cells. We are currently investigating the functional competency of these BMDCs regarding migration, cytokine secretion, and antigen presentation. Our results will help to elucidate the role ERK2 plays in regulating DC function, potentially offering a new approach to treating T cell-driven diseases.
T-cell intrinsic RIP2 regulates pathogenic Th17 differentiation in chronic lung and vascular inflammation
Authors: Porritt Rebecca A. , Shimada Kenichi , Crother Timothy R. , Rivas Magali Noval , Chen Shuang , Arditi Moshe
Published: January 2023

RIP2 plays a role in sensing intracellular pathogens as an adaptor molecule for NOD1 and NOD2 intracellular receptors. While these pathways are present in many cells types, the role of RIP2 in T-cells has been much less studied. Th17 cells are a subset of CD4+ T-helper cells that produce IL17A. While Th17 cells and IL17A play an important protective role at mucosal surfaces under homeostatic conditions, and are important for immunity against certain pathogens, aberrant Th17 responses can lead to severe and chronic inflammatory diseases. Using a Chlamydia pneumoniae lung infection model, we found that Rip2 deficiency led to chronic and severe IL17A mediated inflammation. This was a T-cell intrinsic effect as naive Rip2−/− CD4+ T-cells transferred into Rag1− /− mice resulted in increased Th17 cell formation in the lungs of C. pneumoniae infected mice. This was further confirmed in an atherosclerosis model, in which naive Rip2−/− CD4+ T-cells transferred into Rag1−/− mice caused exacerbated disease, which was IL17A dependent. Intriguingly, our in vitro studies found that while RIP2 deficiency resulted in reduced Rorc expression and reduced homeostatic Th17 differentiation, loss of RIP2 also led to significantly enhanced differentiation of pathogenic Th17 cells, which was RORa dependent, but NOD1 and NOD2 independent. Conversely, overexpression of Rip2 resulted in suppression of pathogenic Th17 differentiation, which was independent of RIP2 kinase activity. Taken together, we have discovered a novel T-cell intrinsic mechanism for RIP2 in regulating the balance between pathogenic and homeostatic Th17 cells. This has broad-reaching implications for many chronic inflammatory diseases where IL17A plays a major role.
A critical role of NLRP3 inflammasome in boosting antigen-specific immunity
Authors: Qian Youcun
Published: January 2023

While NLPR3 inflammasome is activated by pathogen signals and danger signals like ATP and MSU, the physiological triggers of NLRP3 inflammasome activation still remain to be explored. We identified perforin as a new physiological NLRP3 inflammasome activator. We found that NLRP3 inflammasome was activated during co-cultures of antigen-specific CD8+T cells (also called cytotoxic T lymphocytes, CTL) and antigen presenting cells (APCs) in both OT-1 CTL as well as allo-antigen CTL systems. We further found that antigen presentation was required for the NLRP3 inflammasome activation in the co-culture system in vitro and in mice in vivo. The NLRP3 inflammasome activation required the direct contact of CTLs and APCs. We demonstrated that perforin in CTLs activated NLRP3 inflammasome in APCs. Through utilizing Nlrp3, Caspase-1, IL1r or perforin-deficient mice, we demonstrated that perforin-activated NLRP3 inflammasome was critical for antigen-specific CTL-mediated anti-tumor immunity and GVHD (graft-versus-host disease) pathogenesis. However, the antigen-specific CTL-mediated NLRP3 inflammasome activation was not important for host defense against intracellular bacterial pathogen and virus infections. Thus, our study reveals a positive feedback loop of adaptive immunity to promote innate immunity to amplify antigen-specific adaptive immune responses in situations of antitumor immunity and GVHD pathogenesis where innate immunity is not strongly activated.
The impact of target stiffness on NK cell activation
Authors: Friedman Daniel , Hale Alexander , White Michael , Davis Daniel M.
Published: January 2023

A diverse number of cell functions and behaviour are regulated by the mechanical properties of surrounding tissue. In addition, mechanical forces have been shown to impact synapse formation and signalling by T and B cells. Here, we set out to establish whether or not target cell tension impacts NK cell responses. To address this, human primary NK cells were placed on polyacrylamide substrates of varying rigidity coated with activating antibodies against NKp30 and LFA-1. NK cell activation measured by IFNγ secretion and degranulation were reduced on softer substrates. To better model the 3D interaction between NK cells and target cells, we synthesised sodium alginate cell-sized microbeads crosslinked with calcium chloride, where the inherent stiffness of the beads was controlled to be low (2-15kPa), medium (25-50kPa), or high stiffness (75-400kPa). Beads were coated with increasing concentrations of antibody to create surrogate targets with specific ligands and controlled stiffness. NK cells conjugated to stiff beads coated with mAb against NKp30, showed greater actin clustering, as well as perforin and MTOC polarisation towards the bead, to a far greater extent than those conjugated to softer beads. Thus, gel microbeads can be used to characterise the mechanosensitivity of immune synapses in 3D, and establish a role for target stiffness in NK cell activation.
Regulation of TCR signaling by CD2AP in CD4 T cells modulates antibody responses to chronic LCMV infection
Authors: Raju Saravanan , Shaw Andrey Shin-Yee , Egawa Takeshi
Published: January 2023

CD4 T cell-mediated help to CD8 T cells and B cells is a critical arm of the adaptive immune system required for control of pathogens. CD4 T cells express cytokines and co-stimulatory molecules that support a sustained CD8 T cell response and also enhance generation of protective antibody by germinal center B cells. However, the molecular components that regulate CD4 T cell functions in response to viral infection or vaccine is incompletely understood. Here we demonstrate that inactivation of the signaling adaptor CD2AP promotes CD4 T cell differentiation towards the follicular helper lineage, leading to enhanced control of viral infection by augmented germinal center response in chronic LCMV infection. The enhanced follicular helper differentiation is associated with extended duration of TCR signaling and enhanced cytokine production of CD2AP-deficient CD4 T cells specifically under TH1 conditions, while neither prolonged TCR signaling nor enhanced follicular helper differentiation was observed under conditions that induce other helper effector subsets. These results suggest that tuning of TCR signaling by targeting CD2AP improved protective antibody responses in viral infection.
Identification of a T cell immunomodulatory domain in histidyl-tRNA synthetase
Authors: Mertsching Elisabeth C. , Ampudia Jeanette , Adams Ryan , Rosengren Sanna , Nangle Leslie , Mendlein John , Cubitt Andrea , Ramsdell Fred , Ogilvie Kathy , King David
Published: January 2023

Histidyl-tRNA synthetase (HARS) is the autoantigen target of Jo-1 antibodies, which occur in the major form of anti-synthetase syndrome. These patients are characterized by an autoimmune myositis and interstitial lung disease. Circulating extracellular HARS is detected in healthy individuals, but is reduced or undetectable in Jo-1-positive individuals. Administration of ATYR1940, a recombinant form of HARS, ameliorates lung fibrosis and reduces T cell cytokine production in the bleomycin-induced lung injury model. Similar effects were observed with the N-terminal domain of HARS (the iMod domain) conjugated to IgG Fc, suggesting that this domain confers the immunomodulatory activity of HARS.

To confirm primary immune effects of ATYR1940 and ATYR1923 (iMod.Fc), human T cells were isolated from PBMC from healthy individuals and stimulated with anti-CD3/anti-CD28. Proteins containing the HARS iMod domain reduced in vitro activation of human CD4+ and CD8+ T cells, as evidenced by reduced secretion of IL-2, IFNγ, TNFα, IL-17, IL-13, and granzyme B, as well as decreased upregulation of activation markers such as CD69 and CD40L. ATYR1940 and ATYR1923 also inhibited cytokine release after ex vivo stimulation of human memory T cells in a NSG mouse xenogeneic GVHD model. T cell inhibition by ATYR1940 was dependent on its iMod domain, as demonstrated using an iMod-specific blocking monoclonal antibody. The ATYR1940-induced T cell gene signature reflected a general inhibitory effect on activation as well as on cell cycle protein expression. These results suggest that circulating levels of HARS may act to control the threshold stimulatory signal required to activate T cells. We propose circulating HARS as a soluble immune set-point modulator.
GRK2 transactivation of CXCR4 is required for TCR-mediated TCR-CXCR4 complex formation
Authors: Dinkel Brittney A. , Kremer Kimberly N. , Rollins Meagan R. , Medlyn Michael J. , Hedin Karen E.
Published: January 2023

T cell cytokine production was recently shown to depend not only on the T cell antigen receptor (TCR), but also on the chemokine receptor CXCR4. Our lab recently published that stimulation of the TCR results in the TCR associating with and transactivating CXCR4, which, in turn, activates a PREX-1/Rac-1 pathway that stabilizes cytokine mRNA. This pathway therefore normally significantly increases IL-2, IL-4, and IL-10 production by activated T cells. Here, we further characterize the molecular mechanisms responsible for the first step of this pathway: TCR-mediated TCR-CXCR4 complex formation. First, we used FRET and proximity ligation assay (PLA) to define the required structural motifs of CXCR4, showing that this pathway requires the CXCR4 cytoplasmic tail domain and phosphorylation of CXCR4-S339. Second, we showed that TCR-induced TCR-CXCR4 complex formation requires TCR-induced activity of Src family and ZAP-70 tyrosine kinases, as well as one or more Ser/Thre kinases. Third, we found that the G protein-coupled receptor kinase-2 (GRK2) is required both for TCR-induced phosphorylation of CXCR4-S339 and for the formation of TCR-CXCR4 complexes. Finally, we found that GRK2 is required for robust IL-2, IL-4, and IL-10 cytokine secretion by normal human T cells. Together these results identify a novel role for TCR-induced GRK-2 in inducing the TCR-CXCR4 complex formation that subsequently signals to increase TCR-mediated cytokine production. Targeting GRK2 or other mechanisms required for TCR-induced TCR-CXCR4 formation may therefore be therapeutically useful for limiting production of T cell cytokines in humans, for example, during graft versus host disease or immunotherapy.
Improve Your Publications Using Research Resource Identifiers
Authors: Vita Randi J. , Bandrowski A. E. , Grethe J. S. , Martone M. E.
Published: January 2023

Reproducibility was assessed in the scientific literature and the field of immunology did not fare well, as described in Vasilevsky et al 2013. Antibodies and constructs used in immunological publications could not be identified in more than 50% of the cases studied. Similarly, in publications describing the recognition of immune epitopes by the adaptive immune system, crucial details regarding the host, the source protein of the epitope, and the sequence of the epitope often could not be identified (Vita et al 2016). Newly trained scientists can remedy this situation through consistent use of Research Resource Identifiers (RRIDs) in their scientific publications. RRIDs are persistent and unique authentication tags for key biological resources such as antibodies, transgenic organisms, cell lines and software tools. The use of RRIDs promotes research resource identification, discovery, and reuse and is now required in NIH grants (NOT--OD--16--011). By including RRIDs, you can make your scientific publications more reproducible, findable and more widely read, as well as supporting NIH’s new guidelines for Rigor and Transparency in biomedical publications. Many well respected journals are starting to require RRIDs, such as Cell and Immunity, making this an increasingly common practice. SciCrunch (scicrunch.org ) is a data sharing and display platform that offers a central location for obtaining RRIDs. Here we describe Scicrunch as a free and easy to use resource that exists to make the inclusion of RRIDs in publications as simple as possible.
The use of concept maps in a non-majors virology class.
Authors: Minges Wols Heather A.
Published: January 2023

Columbia College Chicago is a leader in the arts and media. While the Science & Mathematics Department houses a major and 3 minors (including one in biology), the majority of the students matriculating through our courses are fulfilling general education requirements. One popular course is The Biology of AIDS: Life of a Virus. After teaching this course for nearly a decade, the HIV replication cycle continues to be a tough concept for students to grasp. This semester I utilized the free concept mapping software, Cmap, to help students tie together concepts, and prepare better for quizzes and exams, with the ultimate goal to reinforce the steps of the replication cycle. Because I found students were unfamiliar with, and thus initially uncomfortable with, the concept mapping approach, I introduced the methodology during week 2, starting with simplistic maps which required less input from the students, who worked in groups to complete. As the students became more comfortable with the practice, I provided fewer completed “concepts” and less verbal help. The students began to expect (and want) the concept maps, requesting them even when I didn’t have some prepared! When that happened, I “flipped” the concept and had the students create their own maps in groups to aid in their comprehension of the material. In the end, it was difficult to assess whether students performed “better” on quizzes and exams because of the concept mapping (no control group, varying levels of preparation, small “n,” etc.), but students did appreciate another approach to studying the material. In addition, the use of concept maps helped me better organize my lecture slides and connect them to student assessment.
Just-In-Time Active Learning Instructional Pedagogy for PharmD Integrated Immunology Course
Authors: Madiraju Charitha , Tellez-Corrales Eglis , Hua Henry , Brown Deborah M.
Published: January 2023

Just-In-Time Teaching (JiTT) is an active learning pedagogy that has been successfully adopted by various disciplines. Our previous studies showed that JiTT presents value to a pharmacy curriculum. The current study implemented JiTT as an active-learning tool for an Integrated Immunology course offered during the first year of a PharmD program. At the end of the course, 38 students provided feedback on a survey that asked for their degree of agreement to various statements about JiTT usage in the course: 18 items were phrased positively (e.g., “Overall, JiTT questions are beneficial to me”), and 4 items were phrased negatively (e.g., “JiTT made the course more difficult”). These statements were rated on a scale ranging from 1 (strongly disagree) to 5 (strongly agree). The aggregate raw score for the 4 negatively-phrased items (M = 2.20, SD = 0.80) suggests that, overall, students typically disagreed with negatively-phrased statements about JiTT. After the negatively-phrased items were reverse-coded, all items were aggregated into a single measure (Cronbach’s alpha = .95) such that higher scores indicate positive overall perceptions of JiTT. This overall mean score for the survey indicates that students strongly perceive JiTT as beneficial (M = 4.00, SD = 0.84). Our results suggest that students in a pharmacy program perceive JiTT to be beneficial and valuable.
The Mammalian Target of Rapamycin (mTOR) / Peroxisome Proliferator-Activated Receptor γ (PPARγ) axis drives immune dysfunction and outcome after burn injury.
Authors: Maile Robert , Stepp Wesley H. , Eitas Timothy , Cairns Bruce A.
Published: January 2023

Burn patients suffer from mixed antagonist response syndrome (MARS) with dysregulation of immune responses. Acutely, a hyper-inflammatory period can result in multiple organ dysfunction (MOD). Patients surviving MOD often exhibit a subsequent immune suppression resulting in bacterial susceptibility, with Pseudomonas pneumonia a common cause of mortality. Induction of the Mechanistic/Mammalian Target of Rapamycin (mTOR) pathway positively regulates inflammatory responses and induces Peroxisome Proliferator-Activated Receptor γ (PPARγ) which in turn represses immune responses. We hypothesized that the mTOR/PPARγ axis regulates immune function after burn injury. Using a mouse model, we inhibited mTOR activity with systemic administration of rapamycin daily, starting 7d before a 20% Total Body Surface Area (TBSA) contact burn or sham. P. aeruginosa was inoculated 3 or 14 days after burn. mTOR pathway inhibition reduced hyper-activation of innate immune cells and inhibited bacterial susceptibility after burn. In peripheral blood mononuclear cells (PBMC) collected from burn patients early following burn injury, heightened mTOR signaling (higher levels of phospho-S6) was observed in moderate/severe burn patients relative to controls. Quantitative global gene expression analysis revealed that expression of mTOR-induced immune-inhibitory molecules PPARγ and SOCS3 were also significantly elevated in severely burned patients. Regression analysis revealed PPARγ expression significantly correlated with patient %TBSA and poor outcome. These data suggest a role for the mTOR/PPARγ axis in the immune pathology following burn injury and highlights therapeutic targets not previously considered for burn patients.
Pore-forming toxins impair pro-inflammatory macrophage responses
Authors: Thapa Roshan , Bhattacharjee Pushpak , Keyel Peter A.
Published: January 2023

Necrotizing soft tissue infections (NSTIs) are lethal microbial infections. One virulence factor common to most bacteria responsible for NSTIs such as Streptococcus pyogenes is a pore-forming toxin (PFT). PFTs promote immune evasion through unknown mechanisms and lyse cells by forming pores. One mechanism by which cells resist these PFTs is intrinsic membrane repair which is sequestering and shedding toxin pores on microvesicles. It is possible that intrinsic membrane repair is hijacked by bacteria to promote immune evasion. We hypothesized that PFTs promote immune evasion by inducing immune receptor shedding during intrinsic membrane repair. We tested this hypothesis by challenging murine bone marrow derived macrophages with sublytic toxin doses prior to LPS or IFNγ challenge. We measured surface receptor expression and intracellular TNFα by flow cytometry in live cells. We found that toxins from one family of PFTs, cholesterol-dependent cytolysins (CDCs), suppressed LPS or IFNγ-stimulated TNFα production and CD69 and CD86 upregulation in a cell-intrinsic manner independently of cell death. CDCs further induced partial shedding of TLR4 and IFNγR, which might contribute to macrophage dysfunction. In contrast, we found that a toxin from a second PFT family, aerolysin, prevented TNFα production by inducing cell death in macrophages at equivalent hemolytic doses. Overall, these results suggest that different PFT families use different strategies to impair macrophages. CDCs may hijack intrinsic membrane repair to impair TNFα production, while aerolysin kills macrophages at similar doses. These findings might explain the immune suppression and lethality observed during NSTIs.
Clostridium difficile TcdB toxin differentially regulates pyrin via differences in activity of RhoA between proliferating and non-proliferating mammalian cells
Authors: Habyarimana Fabien , Napolitano Nina , McAndrew Christian C. , Mitra Srabani , Sarkar Anasuya , Gavrilin Mikhail A. , Wewers Mark
Published: January 2023

Background

Pyrin is a member of the TRIM family of proteins. Pyrin senses RhoA posttranslational modifications that inactivate RhoA, resulting in the formation of a pyrin inflammasome. Clostridium difficile TcdB toxin inactivates RhoA by glycation of threonine 37. Previous studies have shown that active RhoA predominates in proliferating cells. Thus, TcdB may differentially regulate the pyrin inflammasome in proliferating versus non-proliferating mammalian cells.

Methods

We used a RhoA activation assay (Rhotekin) to test this hypothesis in proliferating monocytic cells (THP1), PMA-differentiated THP1 cells and non-proliferating human monocytes. Pyrin function was determined by its interaction with 14-3-3ɛ and IL-1β release.

Results

We noted that TcdB did not inactivate RhoA in proliferating THP1 cells. However, the toxin drastically reduced the level of active RhoA in non-proliferating human monocytes and PMA-differentiated THP1 cells. TcdB synergized with LPS to induce IL1β release from TcdB- and LPS-stimulated THP1 and PMA-differentiated cells. In contrast, TcdB alone was sufficient to generate a robust IL1β release from TcdB-stimulated human monocytes. TcdB disrupted pyrin’s interaction with 14-3-3ɛ in human monocytes and differentiated THP1 cells, suggesting a decrease in the level of phosphorylated pyrin.

Conclusion

Our data indicate that TcdB is capable of turning off active RhoA in non-proliferating cells but not in proliferating cells. Hence, cellular proliferation may impair pyrin inflammasome assembly and activation.
Themis integrates signalling from self pMHC and cytokines in mature CD8+ T cells
Authors: Gascoigne Nicholas R. J. , Zhao Xiang , Mehta Monika , Gautam Namrata , Brzostek Joanna
Published: January 2023

Optimal T cell responses require integration of T cell receptor (TCR) and cytokine signals. We have identified a new role of Themis, a recently discovered LAT signalosome component, in regulation of CD8 T cell responses to self peptide-MHC (pMHC) and cytokines. Themis plays a critical role in modulation of TCR signal strength during thymic development through interaction with the phosphatase Shp1, and we have now demonstrated that Themis affects the phosphatase activity of Shp1 in thymocytes. Germline deletion of Themis leads to severe perturbation in numbers and phenotype of peripheral T cells, but this is a result of the abnormal thymic development. We have now developed a conditional knockout mouse model to investigate the role of Themis in peripheral T cells. Post-selection deletion of Themis reduces the number of CD8+ T cells in the periphery. Unlike thymocytes, Themis-deficient lymphocytes show largely unimpaired functional responses to TCR stimulation in vitro and in vivo. However, Themis deficiency results in reduced Akt phosphorylation after TCR stimulation. Unexpectedly, Themis-deficient CD8+ T cells have reduced homeostatic proliferation in vivo and reduced proliferative responses to self pMHC and pro-inflammatory cytokines in vitro. This reduced responsiveness to self pMHC and cytokines is accompanied by decreased expression of molecules critical for metabolism, such as c-Myc, and nutrient transporters. Our work suggests a novel role for Themis in regulation of signaling from self pMHC and cytokines in mature CD8+ T cells.
Neutrophils and M1 macrophages contribute to vascular injury and lung Pathogenesis during Orientia tsutsugamushi infection
Authors: Trent Brandon J. , Xing Yan , Wei Yang , Liang Yuejin , Shelite Thomas , Bouyer Donald H. , Soong Lynn
Published: January 2023

Acute lung injury (ALI) leading to acute respiratory distress syndrome is a prevailing pathologic manifestation during O. tsutsugamushi-induced severe scrub typhus in humans. In this study, we tested a hypothesis that lung pathology in scrub typhus is due in part to dysregulated activation of inflammatory responses, leading to vascular malfunction and ALI. Following infection with a lethal dose of O. tsutsugamushi in mice, lung tissues had a significant increase in ICAM1, VEGFR2, and angiopoietin-2 (Ang2), as measured by flow cytometry and immunofluorescence. Several unique features were also observed. First, a progressive decrease or loss of endothelial functional markers (Ang1 and Tie2) and CD41+ platelets, even after the peak of bacterial replication (around day 6), implying continued endothelial cell stress and tissue damage. Second, a sustained neutrophil influx and activation as disease progressed, and lung-recruited neutrophils became highly activated in releasing azurophilic granules or myeloperoxidase around day 10 (prior to host death). Finally, lung-derived macrophages were highly polarized to an M1 phenotype (CD80+CD64+CD11b+Ly6G−) at day 6-day 10, with no signs of M2 activation (CD206+CD64+CD11b+Ly6G−). This study reveals specific biomarkers for vascular stress/dysfunction and uncovers a type 1-skewed, but type 2-suppressed, immune responses in the lungs of lethally-infected mice. More importantly, it furthers findings from human patients and cells, implying an immunopathogenic role in ALI development during severe scrub typhus. Understanding of leukocyte effector molecules and endothelial stress pathways triggered by bacterial vs. host factors will help control this neglected tropical disease.
Differential expression of PD-L1 and Th1 response of lymphocytes co-cultured with human gastric organoids infected with Helicobacter pylori strains isolates from different gastric pathologies
Authors: Peniche Alex , Estes Mary K. , Yamaoka Yoshio , Reyes Victor E.
Published: January 2023

Helicobacter pylori (Hp) bacteria successfully establishes chronic infection leading to chronic gastritis, peptic ulcer disease (PUD), and gastric cancer (GC). Since we have previously shown that Hp hijacks expression of checkpoint immunoregulators (i.e., PD-L1), we hypothesized that strains from different gastric pathologies differ in their ability to evade the host response. We used human gastric organoids (hGOs), which recapitulate polarized epithelium, gastric gland and pit cell markers observed in the stomach. We cultured hGOs in transwell inserts which were exposed to Hp on the apical surface and cultured with naïve T cells on the basolateral side of polarized epithelial cells, thereby reproducing the interactions observed in vivo. After 7 days of culture, we recovered all cells and evaluated levels of PD-L1, B7-H3, B7-H4, and CTLA4; and in CD4+ T cells markers of Th1, Th2, Th17 and Treg. Infection of hGOs with Hp strains isolated from cases of gastritis, PUD or GC, all led to increased expression of negative immune checkpoint regulators, predominantly PD-L1, which binds PD-1 on T cells and promotes loss of effector functions, apoptosis, and reduced T cell-target cell contact. Interestingly, PUD strains are the stronger inducers of PD-L1 and GC strains were the lowest inducers of Th1 cells (Tbet+, IFNγ+), which suggest that different strains differ in their orchestration of the host response, which may contribute to the type of pathology elicited. Our data suggest that this co-culture system to evaluate expression of immune checkpoint regulators and CD4+ T cell differentiation in the context of strains comparison may provide insights in differential expression of candidate biomarkers of disease and future therapies.
Intracellular Salmonella enterica infection is controlled in mononuclear phagocytes that attract CXCR3+ Th1 cells in vivo and persists in those that do not
Authors: Goldberg Michael F. , Roeske Elizabeth , Ward Lauren N. , Kotov Dmitri Ivanovich , Dileepan Thamotharampillai , Jenkins Marc K.
Published: January 2023

Salmonella enterica (Se) is a prototypical bacterial pathogen that persists in myeloid cells in secondary lymphoid organs and is controlled by interferon-g-producing CD4+ Th1 cells. How Th1 cells limit the infection without eliminating it is not understood. Using a PhoNdTomato S. enterica strain, we found Se bacteria inside tissue macrophages and recruited monocytes, some of which expressed inducible nitric oxide synthase (iNOS) or CXCL9 and CXCL10 ligands for CXCR3 expressed by Se-specific Th1 cells. The iNOS+ cells were clustered in red pulp regions devoid of T cells and surrounded by CXCL9/CXCL10+ cells and interspersed Th1 cells. Ablation of CD4+ T cells or blockade of CXCR3 lead to preferential loss of bacterial control in the CXCL9/CXCL10+ areas. These results suggest that Se bacteria are controlled inside myeloid cells in regions that attract CXCR3+ Th1 cells and harbored inside myeloid cells in areas that do not.
Polysaccharide capsule allows Group B Streptococcus to avoid killing in a newborn pneumonia model
Authors: Lund Sean J. , Patras Kathryn A. , Kimmey Jacqueline M. , Yamamura Asami , Hernandez Gilberto , Lakhdari Omar , McCoy Alyssa M. , Nizet Victor , Prince Lawrence S.
Published: January 2023

Group B Streptococcus (Streptococcus agalactiae, GBS) causes severe infections in neonates. While not a common adult pathogen, GBS is the leading cause of congenital pneumonia. To determine the molecular mechanisms of neonatal susceptibility, we developed a murine GBS pneumonia model. Neonatal, juvenile, and adult C57BL/6 mice were inoculated intranasally with 2800 CFU/g of GBS (COH1). After 7 d, survival was 100 % in adults and juveniles, with 10 % of neonates dying within 3 d after infection. Histopathological assessments showed severe lung injury scores 24 h after infection, with adult and juvenile mice demonstrating resolution of injury at 3 d post infection and normal histology after 7 d. In contrast, neonatal lungs had persistent lung injury up to 7 d post infection. By FACS, GBS induced rapid neutrophil recruitment into adult lungs and increased CD11b expression on adult lung macrophages. In neonatal lungs, GBS neither increased neutrophil numbers nor altered macrophage marker expression. Confocal imaging showed GBS in adult lungs almost entirely localized to alveolar macrophage phagolysosomes. However in neonatal lungs, GBS was only rarely phagocytosed by macrophages. These data were consistent with GBS killing data, which showed complete GBS killing within 24 h in adult and juvenile mice, but persistent GBS viability within the lung for up to 3 d in neonates. Defective GBS killing in neonates appeared to require the GBS capsule. Neonatal mice were much more efficient in killing the ΔcpsD capsule mutant compared to wild type GBS. Adult mice showed equal killing of ΔcpsD or wild type GBS. Our data suggest that the GBS capsule prevents killing by the newborn lung innate immune system, leading to neonatal disease susceptibility.
Recruited macrophages fail to restrict intracellular growth of Mycobacterium tuberculosis
Authors: Jang Jessica , Ernst Joel D.
Published: January 2023

Mycobacterium tuberculosis (Mtb), a bacterium that predominantly afflicts the lungs, infects over 10 million people per year and causes 1.7 million deaths per year. Immediately following infection, subsets of pulmonary myeloid cells will become infected with Mtb, starting with alveolar macrophages, and eventually spreading to neutrophils, recruited macrophages, monocytes and dendritic cells. These cells differ in their ability to restrict intracellular growth of Mtb. Using reporter Mtb that constitutively express mCherry and express GFP under the tetracycline promoter, we can differentiate between live Mtb (mCherry+ GFP+) and dead Mtb (mCherry+) by flow cytometry. We show that a larger percentage of CD11b+ recruited macrophages and Ly6C+ monocytes in the lungs contain live Mtb and fewer dead Mtb compared to other myeloid populations. This has been validated by sorting infected myeloid cells by flow cytometry and plating colony forming units (CFU). Recruited macrophages averaged 5,000–8,000 live bacilli per 1,000 sorted cells, while alveolar macrophages, neutrophils and dendritic cells harbored 1,000–2,000 live bacteria/1,000 sorted cells. Furthermore, genome-wide transcriptional profiling of these sorted myeloid populations reveal substantial differences in anti-bacterial mechanisms, metabolism and chemokine production between recruited macrophages and other myeloid populations. These include changes in production of interferons, lysosomal genes and tumor necrosis factor superfamily. Altogether, these data reveal that recruited macrophages are unable to restrict the intracellular growth and create a favorable environment for Mtb growth through their deficiency in anti-bacterial cytokines.
T cell IFNγ production is restricted within pulmonary tuberculosis granulomas
Authors: Gern Benjamin Henry , Plumlee Courtney R. , Gerner Michael Y. , Urdahl Kevin B.
Published: January 2023

The direct interaction between CD4 T cells and Mycobacterium tuberculosis (Mtb) infected cells presenting their cognate antigen on MHCII is crucial for optimal bacterial control. Using quantitative imaging (QIM) and a physiologic ultra-low dose (ULD) Mtb infection in mice to visualize this interaction, we have found that while T cell receptor (TCR) signaling in CD4 T cells occurs throughout Mtb-infected lungs, IFNγ production is impaired near infected cells. To examine the antigen-specificity of this observation, we co-transferred Th1-polarized Mtb ESAT-6-specific CD4 cells (C7) and OVA-specific (OTII) CD4 T cells into mice 35 days post ULD Mtb infection. The next day, lungs were harvested for phenotypic and spatial analysis of TCR signaling (using IRF4) and IFNγ production. QIM revealed TCR signaling and IFNγ production in Mtb-specific T cells, but minimal TCR signaling and IFNγ production in control T cells with irrelevant specificity for OVA. Positioning data showed that TCR recognition of Mtb ESAT-6 occurred throughout the lung, including within the center of the granuloma, where most Mtb bacilli are observed. Despite this, only 1–2% of ESAT-6-specific T cells within the granuloma produced IFNγ, whereas 10–15% produced IFNγ outside the granuloma, where Mtb bacilli are sparse. Thus, T cell production of IFNγ is restricted at the site of Mtb infection within the granuloma, despite ongoing TCR signaling at this location. These findings may help explain why IFNγ-producing T cells may be more efficient at preventing extrapulmonary dissemination than in controlling Mtb within the lung. Understanding the mechanisms regulating this local immunosuppression may yield new strategies for vaccine and immunotherapeutic development.