bluuebunny/crossref_metadata_2025 huggingface dataset:
PSGL-1-directed regulation of calcium signaling modulates PD-1 expression following T cell activation
Authors: Otero Dennis C , Ma Jiadai , Hope Jennifer L , Bradley Linda M
Published: January 2023

We previously identified PSGL-1 (P-selectin glycoprotein-1) as a T cell-intrinsic, cell surface checkpoint inhibitor during chronic viral infection. In this model, we found that cross-linking PSGL-1 with antibody reduced signaling through the T cell receptor (TCR) in exhausted T cells and further upregulated PD-1 expression. However, the mechanism responsible for PD-1 regulation by PSGL-1 remained to be described. Here we show that, in naïve T cells, PSGL-1 ligation results in enhanced PD-1 expression following activation through the T cell receptor. Furthermore, naïve T cells lacking PSGL-1 fail to upregulate PD-1 to the same extent as wild type T cells following activation. Strikingly we have discovered that T cells lacking PSGL-1 showed reduced calcium signaling. In T cells, the calcium/calcineurin/NFAT pathway is important for induction of many genes including PD-1. Consequently, PSGL-1 deficient T cells also exhibit reduced nuclear translocation of NFAT explaining the reduced PD-1 expression. Conversely, we found that ligating PSGL-1 enhanced NFAT nuclear translocation with TCR signaling thus contributing to upregulation of PD-1. Therefore, by regulating calcium signaling through the T cell receptor, PSGL-1 modulates the extent of PD-1 expression early in activation and subsequently influencing the extent of T cell exhaustion. Further studies aim to elucidate how PSGL-1 regulates calcium signaling through the T cell receptor.
Insights from an in vitro derived T cell receptor
Authors: Alonso Jesus A , Singh Nishant K , Kranz David M , Evavold Brian D , Baker Brian M
Published: January 2023

Heterodimeric alpha-beta T cell receptors (TCRs) play a key role in cell-mediated immune responses of adaptive immunity. T cells continuously probe peptides presented by major histocompatibility complex (MHC) found on the cell surface. T cell activation initiates through the binding of antigenic peptides on MHCs. Yeast display technology has proven an effective way to gain insight into the underpinnings of peptide-MHC specificity enabling the switching of TCR affinity and specificity to unrelated antigens through directed evolution. Interestingly, in vitro directed evolution also generates mutants that lose peptide specificity. Here, we provide further detail of one of these cross-reactive TCRs, S3–4, which binds tightly but with a highly unusual geometry. Despite binding with an affinity that is characteristic of a strong agonist, S3–4’s odd binding geometry does not support T cell signaling. Further investigation with functional experiments and 2D kinetics shows that lack of signaling by S3–4 is ultimately attributable to the TCR’s inability to reach ligand. Although S3–4 is an example from a constrained system generated outside the bounds of usual immune function, our results show how unusual binding can influence T cell function and further demonstrate how divergences between 3D and 2D binding parameters can emerge.
Structure-guided design of the TIL1383I T cell receptor
Authors: Alonso Jesus A , Singh Nishant K , Devancaze Lauren , Devlin Jason , Baker Brian M
Published: January 2023

T cell therapy is an exciting form of cancer immunotherapy in which T cells are engineered to target specific tumor antigens. We employed a structure-guided design approach with the goal of engineering a safer and more effective variant of the TIL1383I T cell receptor (TCR) currently under study in clinical trials for malignant melanoma. Using our unpublished structure of TIL1383I we are in process of designing a panel of TCR variants with the goal of identifying candidates that improve “focus” towards the tyrosinase antigen presented on the MHC class I molecule HLA-A2. Structural analysis of TIL1383I revealed key residues, particularly beta-chain residues E97, G101, L102, responsible for engaging the tyrosinase peptide bound to HLA-A2. The crystal structure of TIL1383I in complex with tyrosinase-HLA-A2 also highlighted its uncharacteristic binding geometry and we therefore hypothesize that this binding orientation is associated with the observed CD8 co-receptor independence of TIL1383I. Indeed, functional analysis with TIL1383I-transduced CD8-positive and CD8-negative T cells, transduced T cells expressing a truncated CD8 lacking the intracellular LCK signaling domain, and tyrosinase peptide variants presented by HLA-A2 mutants outline this co-receptor independence. Combined with our interrogation of tyrosinase peptide cross-reactivity via a peptide positional scanning library approach, structure-guided design resulted in the identification of TIL1383I variants with improved binding affinities to the tyrosinase peptide as well as an understanding of structural characteristics that may contribute to TIL1383I’s co-receptor independence.
LAG3 mediates its inhibitory function by associating with the TCR:CD3 complex and inducing co-receptor:p56lck dissociation
Authors: Workman Creg , Guy Cliff , Mitrea Diana , Chou Po-Chien , Temirov Jamshid , Vignali Kate , Liu Xueyan , Zhang Hui , Kriwacki Richard , Bruchez Marcel , Watkins Simon , Vignali Dario
Published: January 2023

Lymphocyte activation gene 3 (LAG3) is an inhibitory receptor that is highly expressed on exhausted T cells in the tumor microenvironment. Despite over ten LAG3-targeting immunotherapeutics in clinical trials, we still do not know how LAG3 mediates its inhibitory activity. We show that LAG3 can function in the absence of MHC class II ligation and that in the absence of LAG3 there is a 2–3 fold increase in T cell receptor signaling resulting in increased proliferation. We show that both murine and human LAG3 molecules track to the immunological synapse (IS) by close association with the T cell receptor (TCR):CD3 complex. A phylogenetically conserved, acidic tandem glutamic acid–proline repeat in the LAG3 cytoplasmic tail can lower local pH at the IS and chelate Zn2+, limiting T cell activating signals by causing the dissociation of CD4:p56lck, which interact via a di-cystine:Zn2+ complex. This in turn causes a loss of co-receptor:TCR signaling. This novel inhibitory pH-mediated, Zn2+-displacement mechanism highlights LAG3 as a “signal disruptor” that can function in an MHC class II independent manner, providing critical insight into the mechanism of action of LAG3-targeting immunotherapies.
Therapeutic manipulation of memory T cell homeostasis in vivo
Authors: Wolf Gideon S , Singh Nevil J
Published: January 2023

After a primary immune response, a cohort of antigen-specific memory T cells are retained in vivo conferring long term protection against that specific pathogen. Over a lifetime, the accumulation of a memory T cell repertoire with specificities to multiple pathogens, collectively keeps the host safe against prevalent environmental threats. Other than booster vaccinations which narrowly increase the frequency of a single antigen-specific cohort, there are no viable strategies available to globally alter the total memory T cell population in vivo. Since memory T cells are thought be maintained by trophic signals from cytokines or self-MHC- both of which are available from endogenous antigen-presenting cells (APC)- we hypothesized that enhancing APC numbers in vivo can be a viable strategy to amplify memory T cells. Towards this end we treated mice with FMS-like tyrosine kinase 3 ligand (Flt3l) or specific self-peptides known to increase the peripheral survival of cohorts of CD4 T cells. After an acute expansion of dendritic cells (DCs) following Flt3l treatment, we observed an increase in effector memory CD4s and CD8s, but not in naïve T cells. Over the short term, this increase was independent of alterations in the thymus. On cessation of Flt3l treatment, the increase in DCs was not sustained, and the population of memory T cells also returned to steady state, suggesting restoration of competitive homeostasis. In contrast to this global effect, administering an endogenous self-peptide used by a small subset of peripheral T cells did not significantly alter the overall number or distribution of the memory repertoire. Taken together, these data suggest new options to manipulate memory T cell populations in clinically relevant situations.
Naive brain harbors resident memory T cells that respond to neurological insults prior to infiltration of antigen specific T cells
Authors: Ayasoufi Katayoun , Namen Shelby L , Goddery Emma , Tritz Zachariah Peter , Yokanovich Lila T , Fain Cori , Jin Fang , Hansen Michael , Johnson Aaron J
Published: January 2023

Brain resident memory T cells (TRM) have recently been phenotypically characterized. However, the function of these cells in the brain, and their changes during neurological insults, is not well understood. Using the described phenotype of brain TRMs (TCRB+, CD69+, CD4 or CD8+, CD103−, CD44+), we evaluated cellular responses of this population in the naïve brain and upon various neurological insults. We found that numbers of brain TRMs increased as a function of age. Additionally, TRM cells in the naïve brain produce TNFα constitutively, setting them apart from TRMs in other organs. Parabiosis studies revealed that circulating pools of memory T cells contributes to maintenance of TRM cells in the naïve brain. During neurological injury, we observed an increase in numbers of TRMs as early as 24 hours following physical insult induced by intracranial injection of PBS. During brain infection with Theiler’s Murine Encephalomyelitis virus, brain TRMs increased in number prior to detection of any virus-specific CD8 T cells. This implies TRMs respond to CNS viral infections prior to generation of virus antigen specific responses. This response is due to proliferation of TRM cells in the brain, as treatment with FTY720 did not inhibit TRM proliferation 24 hours post infection. In short, naïve brains harbor populations of TNFα producing TRM cells. And TRMs rapidly respond to neurological injuries through proliferation in an antigen independent manner. Understanding brain TRMs is crucial in investigating their role in neurodegenerative disorders or targeting this potent population of brain resident T cells in cancers of the CNS.
Characterization of macrophages in the liver of total body irradiated mice
Authors: Okamoto Norio , Asai Akira , Matsui Masahiro , Yasuoka Hidetaka , Nishikawa Tomohiro , Yokohama Keisuke , Nakamura Ken , Ohama Hideko , Tsuchimoto Yusuke , Fukunishi Shinya , Tsuda Yasuhiro , Higuchi Kazuhide
Published: January 2023

Two hepatic macrophage (MΦ) populations have been described in the inflamed liver: F4/80+CD11b+ cells and F4/80+CD68+ cells. In normal conditions, hepatic F4/80+CD11b+ cells are involved in host antibacterial resistance. However, these cells in total body irradiated mice (TBI mice) are unfunctional for the elimination of bacteria translocated from intestinal tracts. In this study, the properties of hepatic MΦ populations in TBI mice were characterized. Normal mice and mice 7 days after exposure to 4 Gy of X-rays were infected i.v. with 107 CFU/mouse of Enterococcus faecalis (E. faecalis). Three day after infection, F4/80+CD11b+ cells and F4/80+CD68+ cells isolated from the liver of these mice were cultured for 24 hrs, and culture fluids were assayed for IL-12, IL-10 and CCL1 by ELISA. NSG mice were inoculated i.v. with these MΦ preparations (2 × 105 cells/mouse), then infected i.v. with 5 × 104 CFU/mouse of E. faecalis. In the results, all TBI mice died within 2 weeks, while all normal mice survived. IL-12 was produced by F4/80+CD11b+ cells from normal mice, but not by both MΦ preparations from TBI mice. IL-10 was produced by F4/80+CD68+ cells from TBI mice (529.9 ± 272.6 pg/ml) and normal mice (445.8 ± 147.7 pg/ml). Large amounts of IL-10 (912.1 ± 103.7 pg/ml) was produced by F4/80+CD11b+ cells from TBI mice, and these cells produced CCL1 (327.5 ± 9.1 pg/ml), while other MΦ preparations did not. NSG mice inoculated with F4/80+CD11b+ cells from normal mice were resistant to infection; however, NSG mice inoculated with F4/80+CD11b+ cells from TBI mice did not. These results indicate hepatic F4/80+CD11b+ cells are polarized to M2bMΦ in response to total body irradiation, and these MΦ are involved in the decreased host antibacterial resistance in TBI mice.
Circulating memory is sufficient for protective immunity to infection with Chlamydia muridarum
Authors: Labuda Jasmine C , McSorley Stephen J
Published: January 2023

Chlamydia trachomatis is the most prevalent bacterial sexually transmitted infection worldwide and yet no vaccine currently exists to protect the public from this reproductive tract pathogen. Recent reports suggest that CD4 tissue resident memory cells (TRM) are essential for vaccine-induced protection against Chlamydia trachomatis in a mouse infection model. Additionally, CD4 TRM in the female reproductive tract (FRT) have been shown to co-localize with APCs in memory lymphocyte clusters (MLCs) to enhance secondary adaptive immunity to reproductive tract infections. Here, we examine a requirement for CD4 TRM and MLCs in protective immunity against infection with Chlamydia muridarum (Cm). Using parabiosis of naïve and Chlamydia-immune mice, we demonstrate that although CD4 TRM form in the FRT after primary Cm infection, they are not required for protection. Furthermore, histological analysis of FRT tissue from parabionts revealed that circulating memory is sufficient for protection against intravaginal challenge with Cm in the absence of CD4 TRM or MLCs in the reproductive tract. Moreover, this protective circulating memory can be induced through intravaginal or intranasal challenge with Chlamydia, indicating that a strong mucosal immunization can generate circulating protection in a vaccine setting. Ongoing work using adoptive transfers and antibody depletions of CD4 T cells and B cells will reveal which memory lymphocytes are responsible for the observed circulating protection.
GM-CSF+ Tc17 cells mediate vaccine-immunity, but not immunopathology, against lethal fungal pneumonia under CD4+ T-cell lymphopenia
Authors: Mudalagiriyappa Srinivasu , Vieson Miranda , Nanjappa Som G
Published: January 2023

Although opportunistic fungal infections are rising at alarming rates in immunocompromised individuals, there is no licensed vaccine available nor the clear understanding of the protective host responses. In a mouse model of fungal vaccine-immunity in CD4+ T-cell lymphopenia, we have previously shown that vaccine-induced IL-17A producing CD8+ T (Tc17) cells are necessary for controlling lethal fungal pneumonia. However, we found a significant number of Tc17 cells that co-expressed GM-CSF (>50%), but their role has been implicated as ‘pathogenic’ in autoimmune disorders. In this study, using state-of-the-art approaches, we found that GM-CSF +Tc17 cells are essential for vaccine-immunity against lethal fungal pneumonia without precipitating an adverse immunopathology. Induction of GM-CSF+Tc17 cells, following vaccination, required signaling from cytokines, IL-1 and IL-23, that promoted differentiation and proliferation of effectors. However, memory homeostasis of GM-CSF+Tc17 cells was mostly independent of IL-23. Following the lethal challenge by the pulmonary route, IL-23 was required for the GM-CSF+Tc17 cells responses, but not for their re-differentiation, and was necessary to mitigate the immunopathology of the lungs. Collectively, GM-CSF+Tc17 cells are required for vaccine-mediated immunity against lethal fungal pneumonia.
Regions of mouse chromosomes 2 and 11 in SJL mice contain critical alleles for resistance to Cryptococcus neoformans
Authors: Davis Michael J , Rodriguez-Gil Jorge , Hoke Liz , Pinheiro Giovana , Chang Yun , Khillan Jaspal , Paven William , Kwon-Chung June
Published: January 2023

Cryptococcus neoformans (Cn) remains a leading cause of deadly mycosis. While previous studies have elucidated some of the host immune processes, little is known about the fundamental host factors that determine the outcome of Cn infection. Unfortunately, all commonly used inbred mice (including the common C57BL/6) are highly susceptible to virulent Cn strains, whereas humans are resistant to Cn unless immunocompromised. Thus, we sought a murine Cn infection system that more closely models humans. Testing the susceptibility of about 20 inbred strains of mice, we found that the SJL strain is, by far, the most Cn resistant inbred mouse strain in contrast to the closely related FVB strain being completely susceptible. While the early stages of Cn infection seem similar, SJL mice eventually cleared Cn and resolved pulmonary pathology, with little CNS dissemination. In contrast, susceptible mice showed significant and increasing fungal loads in both lungs and brains with associated pathology and death. F1 mice from FVB × SJL crosses were highly resistant to Cn suggesting that the resistant phenotype is dominant. Twenty percent of N2 mice (FVB × SJL F1 mice backcrossed to FVB) survived infection suggesting the involvement of multiple loci. Gene mapping uncovered two areas of genetic linkage to Cn resistance. These areas of interest were in portions of chromosomes 2 and 11. Genomic analysis found several candidate genes which are currently under evaluation. Mapping these cryptococcal resistance gene alleles may illuminate pathways of natural resistance which could be utilized for improved therapeutic options or to identify human populations at risk for cryptococcosis, making SJL mice an important tool for understanding cryptococcosis.
Dietary Antigens Drive Protective Innate-like CD4 T Cell Immunity at the Mucosal Barrier of the Small Intestine
Authors: Chen Angeline , Thiault Nicolas , Verstichel Greet , Iwaya Hitoshi , Cheroutre Hilde
Published: January 2023

CD4 T helper (Th) cells reprogram to cytotoxic T lymphocytes (CTLs) in the intestinal epithelium during steady state. The unique localization of mucosal CD4 CTLs, in the absence of a pathogenic threat, suggests local non-pathogen antigens and organ-specific properties direct the CTL conversion.

Similar to myeloid antigen presenting cells, intestinal epithelial cells (IECs) constitutively express MHC class II (MHCII), indicating crosstalk with nearby CD4 T cells. IECs constantly absorb and process luminal digested proteins and present these dietary antigens to neighboring CD4 T cells on their basolateral side. We have generated solid evidence with murine conditional knock-out and bone marrow chimera models that MHCII+ IECs and the dietary epitopes they present are instrumental in the development of small intestinal CD4 CTLs in a homeostatic, non-inflammatory environment.

These dietary antigen-specific CD4 CTLs accumulate over time as innate-like protective cells that fortify the epithelium with a pre-existing protective arm. As fully differentiated and functional resident T cells, CD4 CTLs are aptly designed to rapidly respond, independent of pathogen specificity, and contain infections. Using Salmonella as an enteric pathogen model, the data show that dietary antigen-specific CD4 CTLs have the capacity to rapidly and effectively kill infected epithelial cells, thereby preserving the integrity of the barrier and blocking systemic spreading of the pathogen. The double-pronged ability for CD4 CTLs to protect against invaders while also mitigating immunopathology, by differentiating inflammatory CD4 T cells into excellently trained CD4 CTLs, reveal previously unknown mechanisms of mucosal lymphocyte biology.
Indole metabolites are altered in maternal high fat diet exposed offspring and can modulate type 3 innate lymphoid cell function
Authors: Mirpuri Julie , Jaleel Sana , Niu Xinying
Published: January 2023

Background

We have shown that maternal high fat diet exposure (mHFD) results in a microbiota-dependent expansion of IL-17 producing type 3 innate lymphoid cells (ILC3) in offspring. We hypothesized that the unique mHFD microbiome in offspring would result in an altered metabolite profile that could potentiate Ahr signaling and ILC3 function.

Methods

Murine offspring exposed to 60% mHFD were examined at 2-weeks old. Stool was examined by high-throughput, non-targeted mass spectroscopy-based metabolic analysis. Microbial colonization was examined by next generation sequencing. 2-week-old small intestine (SI) from regular diet (RD) offspring was exposed to selected indole metabolites for 24 hours and supernatant examined by ELISA. For in vivo experiments, bacteria was gavaged to RD offspring beginning at 5 days old. Flow cytometry of SI lamina propria cells was used to examine ILC3.

Results

mHFD offspring had a differential pattern of colonization and metabolite composition. mHFD offspring had a consistent expansion of Lactobacillus murinus. In vivo exposure to L. murinus resulted in a 30% increase in IL-17 producing ILC3 not seen with exposure to L. rhamnosus. Metabolomics of stool from mHFD offspring showed a decrease in indole-3-acetate and 3-methyldiozyindole (p<0.00001). SI stimulated with indole metabolites showed a 50% decrease in production of IL-17 (p<0.05) and IL-22 (p<0.05).

Conclusion

L. murinus is specifically expanded in mHFD offspring and can increase IL-17 producing ILC3. L. murinus may expand ILC3 by differential metabolism of indole metabolites. Metabolism of indole metabolites by L. murinus and their role on Ahr signaling need to be further examined as a mechanism of modulating ILC3 response in offspring.
Ingested microplastics pose a potentially serious risk to the gastrointestinal microenvironment
Authors: Castillo Eliseo F
Published: January 2023

BACKGROUND

Plastic pollution has increased exponentially over the past 70 years. Slow breakdown of plastic materials primarily leads to smaller and smaller microplastic particles (MP), but not actual degradation of the material. We now face extensive MP contamination of our food and water sources, raising ecological and public health concerns. MP ingestion by humans is now an inevitable consequence of global plastic pollution, and we are only now becoming aware of this burgeoning crisis. Given this crisis, there is also an important gap in knowledge regarding how MP affect the major direct organ of contact, the gastrointestinal tract. We provide compelling evidence that MP affect both intestinal epithelium and macrophages.

DESIGN

We utilized human colonic organoids and colonic cell lines to examine the effects of MP on intestinal permeability and wound healing. Additionally, we examined the effects of microplastics on macrophage function and metabolism. Lastly, we examined the in vivo response to microplastics in animal models.

RESULTS

Regarding the intestinal barrier, we show MP impede wound healing and affects intestinal permeability. In macrophages, phagocytosis of MP leads to the upregulation of costimulatory molecules and induces metabolic shift towards glycolysis. In vivo MP ingestion affects the weight and microbiota composition of mice.

CONCLUSION

In summary, our findings indicate MP ingestion may pose a serious human health hazard by disrupting both intestinal epithelial barrier and macrophage function subsequently causing intestinal permeability, dysbiosis, and an inflammatory state that could lead to intestinal and systemic inflammation.
Viable bacterial colonization is limited in the human intestine in utero
Authors: Rackaityte Elze , Halkias Joanna , Fukui Elle M , Mendoza Ventura F , Hayzelden Clive , Crawford Emily D , Fujimura Kei E , Burt Trevor D , Lynch Susan V
Published: January 2023

Mucosal immunity develops in the human fetal intestine by 11–14 weeks gestation, yet whether viable microbes exist in utero and interact with intestinal immunity is unknown. Structures consistent with coccoid bacterial morphology, embedded in fetal meconium were evident before mid-gestation by high-resolution scanning electron microscopy (n=4). Molecular methods indicated extremely low bacterial burden and simple profiles in fetal meconium (n=40 of 50) compared to controls (n=87). A subset of Micrococcaceae-dominated (n=9) meconium associated with proportions of lamina propria PLZF+ CD161+ CD4+ T cells and divergent intestinal epithelial transcriptomes. Fetal Micrococcus luteus was isolated only in the presence of a monocyte feeder cell line. This strain grew on placental hormones, remained viable within fetal antigen presenting cells, exhibited species-specific immunomodulatory capacity and genomic features indicating fetal adaptation. Thus, viable bacteria are highly limited and inconsistently detectable in human fetal meconium at mid-gestation.
HY209 alleviates inflammation in colitis mice through NLRP3 inflammasome inhibition
Authors: ZOU YUNYUN , Seong Seung-Yong
Published: January 2023

The anti-inflammatory effects of HY209 have been identified in sepsis. However, the anti-inflammatory potential of HY209 in colitis has not yet been investigated. In here, we evaluated the therapy efficacy of HY209 in an acute colitis mice model which induced by using dextran sulfate sodium (DSS). Compare to vehicle mice, HY209 treated mice showed significantly less body weight loss, lower disease activity index, longer colon length, as well as a lower histology disease scores. Meanwhile, the expression of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6) were reduced in colon tissue after HY209 treatment. Furthermore, HY209 treated mice significantly reduced inflammatory macrophage, but increased anti-inflammatory macrophage in lamina propria. In vitro, HY209 showed a strong inhibition to LPS and ATP/BzATP activated NLRP3 inflammasome formation in the bone marrow derived macrophages (BMDMs). In summary, our findings demonstrated that HY209 alleviates inflammation in colitis mice through NLRP3 inflammasome inhibition, discovery the potential of HY209 as a therapeutic option for colitis.
Intestinal commensal bacteria promote gut barrier repair through non-canonical inflammasome pathway activation
Authors: Wu Wan-Jung , Kim Myunghoo , Tsai Ming-Ting , Morales Fatima Beatriz Saldana , Ruiz Daniel Fernando Zegarra , Hill Andrea , Song Hyo Won , Norwood Kendra , Diehl Gretchen
Published: January 2023

The intestinal epithelial barrier critically separates exogenous microbes from our internal tissues. Signals from commensal microbes are known to support intestinal barrier integrity and repair. However, the role of specific microbes in maintaining the intestinal barrier is not well-defined. We hypothesize that signals from distinct commensal microbes play different roles in regulating the barrier. We find that mice develop severe colitis with decreased ability to repair the intestinal barrier after antibiotic depletion of intestinal microbes. We further isolated a mouse commensal E. coli that protects from increased colitis after antibiotic treatment. This protection depends on the presence of intestinal antigen presenting cells (APCs) that express the chemokine receptor CX3CR1. We screened a panel of human mucosal-associated E. coli and identified a subset that also protect from colitis in a CX3CR1+ APC dependent manner. All protective E. coli induce IL-1β secretion by activation of the non-canonical inflammasome pathway in CX3CR1+ APCs. In vivo protection is lost if IL-1β signaling is blocked with anti-IL-1β antibody. This E. coli induced IL-1β production activates type 3 innate lymphoid cells (ILC3) to produce IL-22 which drives epithelial proliferation and barrier repair. Collectively, we identified a novel mechanism by which a subset of mucosal-associated intestinal E. coli activate IL-1β production by APCs to protect the intestinal barrier from damage.
CD8αα intestinal intraepithelial lymphocytes derived from two thymic precursors seed the intestine in early life
Authors: Ruscher Roland , Lee Seungeun Thera , Barrero Oscar Salgado , Breed Elise , Isakson Sara , Jameson Stephen , Hogquist Kristin Ann
Published: January 2023

TCRαβ+CD4−CD8αα+ intestinal intraepithelial lymphocytes (CD8αα IEL) descend from thymic precursors. To better define this IEL precursor (IELp) population, we analyzed their maturation, localization, and emigration. Using rigorous lineage exclusion criteria, we defined two precursors among DN TCRβ+ thymocytes: a nascent PD-1+ population and a T-bet+ population that accumulates with age. Both gave rise to intestinal CD8αα IEL upon adoptive transfer. In adult mice, PD-1+ cells contained more self-reactive clones, localized to the cortex, and were dominant in S1PR1-dependent thymic egress. Gut homing α4β7 was already expressed by these IELp at a thymic stage. To understand the kinetics of CD8αα IEL seeding the intestine, we performed “time-stamp” experiments: We crossed Cd4CreERT2 with Rosa26TdT (stop-floxed tdTomato) mice. In these mice, tamoxifen or its metabolite 4-OHT permanently labels every CD4 expressing cell. As TCRαβ T cells (including CD8αα IEL) go through a CD4+CD8+ stage during thymic development, a single dose of tamoxifen or 4-OHT will label thymic IEL precursors permanently, so that they can be tracked when seeding the gut. Our results indicate that these cells enter the intestine during a narrow time window in early life and that this influx is almost completely shut down by the age of 3 weeks. These data provide an important foundation for understanding the biology of this abundant population of barrier surface T cells.
Potentiating lung-localized immune responses to conserved influenza virus antigen through a novel vaccine platform
Authors: Nelson Sean Alexander , Fischer Nicholas O , Rasley Amy , Sant Andrea J
Published: January 2023

Current influenza vaccines are primarily delivered at peripheral sites and provide immunity characterized by neutralizing antibody responses. However, this strategy often fails to provide broadly-protective immune capacity. Numerous studies have implicated lung localized T cells as key mediators of broadly protective immunity to influenza infection. The goal of our current research is to expand virus-specific CD4 T cells via the respiratory route in order to understand how this mode of priming poises the host for future protective immunity to infection. We have explored a novel nanolipoprotein (NLP) vaccine conjugated to influenza nucleocapsid protein (NP) to boost lung localized immunity. ELISA assays revealed that NLP immunization boosted anti-NP antibody titers, which have been shown to contribute to heterosubtypic immunity. Importantly, immunization with NLP elicited NP-specific CD4 and CD8 T cell responses in the lung, draining lymph node, and spleen. Intravascular labeling and flow cytometry were performed to assess the localization and phenotype of CD4 T cells in the lung. Analyses revealed that intranasal immunization with NLP elicited an increased abundance of lung parenchyma-localized CD4 T cells expressing surface markers associated with tissue residency and cytolytic potential. Using a novel pMHC class II tetramer, we observed that the majority of antigen-specific CD4 T cells were localized to the lung tissue and can rapidly produce antiviral cytokines upon restimulation. These studies will improve our understanding of vaccine strategies deigned to elicit effector T cells at mucousal sites and demonstrate the potential of tissue resident cells to mediate broadly protective immune responses to influenza.
The C-X-C motif chemokine 17 plays a minimal role during lung infection with hypervirulent Mycobacterium tuberculosis
Authors: Choreno-Parra Jose Alberto , Dunlap Micah , Swanson Rosemary , Jiménez-Álvarez Luis A. , Zúñiga Joaquín , Khader Shabaana A
Published: January 2023

The C-X-C motif chemokine 17 (CXCL17) is a novel mucosal chemotactic factor for myeloid cells that displays bactericidal activity against respiratory pathogens in vitro. Although CXCL17 is constitutively expressed along the respiratory tract, its function during lung infections has not been tested in vivo. We previously showed that the CXCL17 gene is overexpressed in innate lymphoid cells (ILCs) isolated from the lung of patients with active pulmonary tuberculosis (TB). Thus, in the current study we further explored the role of CXCL17 in the defense against Mycobacterium tuberculosis (Mtb). In a mouse model of aerosol infection with the prototype W-Beijing lineage Mtb strain, hypervirulent HN878, we found that CXCL17 is actively produced at the lung during the acute and chronic stages of disease, with levels peaking at 30 days post-infection. Using in vitro infection assays, we observed that lung epithelial cells, bone marrow-derived macrophages (BMDMs), and bone marrow-derived dendritic cells (BMDCs) can become cellular sources of CXCL17 in response to Mtb. In humans, the serum levels of CXCL17 are significatively higher among individuals with latent TB infection (LTBI) than in active TB patients, suggesting a protective role. However, low serum levels of CXCL17 do not correlate with increased lung damage among active TB subjects. Furthermore, CXCL17−/− mice do not exhibit increased susceptibility to Mtb HN878 infection as compared to wild type controls. Finally, administration of exogenous recombinant CXCL17 into the airways of Mtb-infected animals does not impact their lung bacterial burden despite increased recruitment of myeloid cells to the pulmonary tissue. Our data suggest that CXCL17 plays a redundant role during TB.
Altered expression of Surfactant and CD74 in the alveolar epithelial cells during Influenza infection disease
Authors: Periolo Natalia , Ibañez Lorena Itati , Peralta Liliana Martinez
Published: January 2023

Background

Influenza A virus (IAV) causes substantial morbidity and mortality in the human population. In the lower lung, the primary targets for IAV replication are type II alveolar epithelial cells (AECII), which are increasingly recognized for their immunological potential. Surfactant components are synthesized primarily by AECII, which play an important role in host defense against infection. On the other side, AECII express high levels of CD74 and contributes to repair the damaged alveolus during infection.

Aim

To analyze the expression of surfactant (proSP-C), CD74 and activation markets (HLA-DR, CD80, CD86) expressed by AECII cells from infected mice and A549 cell line.

Methods

IAV used include the mouse adapted influenza strains. Pandemic H1N1 (pH1N1) and A/Victoria/3/75 (H3N2) were used to infect BALB/c mice. AECII cells were obtained from mice lungs, and cell suspensions obtained from enzymatically digested murine lungs were labeled for surface antigens (CD45/CD31/CD74/proSP-C) followed by FACS analysis. The A549 cell line was used as a human in vitro model and to compare the results.

Results

Analysis of AECII phenotype showed a significant decrease of proSP-C+CD31−CD45−and CD74+CD31−CD45− expression in infected mice comparing to controls, and also in the group infected with H1N1 vs H3N2 (MFI p <0.01; p<0.05, respectively). Expression of HLA-DR was very low for AECII and A549 cell line in infection condition. However, CD80 and CD86 they did not show changes in their expression.

Conclusions

Our results indicate that morphological changes in AECII phenotype suggest very active participation of alveolar epithelial cells in inflammatory viral processes in the lung, and higher effects of H1N1 comparing to H3N2 strain.
Expansion of natural killer cells with optimal killing activity for cancer immunotherapy
Authors: Flynn Kevin , Goetz Christine A. , Van Etten Jamie , Hammerbeck Christopher , Peng Li , Lee Neil , Liang Joyce , O-Reilly Daniel , Herr Greg , Tong Thuy , Finkel Dave , Kubik Mandy , Bonnevier Jody L
Published: January 2023

Natural killer (NK) cells play a key role in the elimination of microbial pathogens and cancer cells. NK cells rapidly respond to stressed cells that are infected or bear tumorigenic mutations that reduce “self” markers of MHC class 1 and/or display increased presentation of activating ligands. These signals prompt NK cells to form an immunological synapse with an infected or tumor cell and discharge lytic granules containing cytotoxic proteins such as IFNγ, perforin and granzyme B leading to target cell destruction. Because of their natural ability to recognize and kill cancer cells, a plethora of preclinical studies and clinical trials have shown that NK cell therapies offer a potentially powerful weapon to fight both advanced-stage blood cancers and solid tumors. Given the great potential of NK cell therapies, we developed clinically relevant serum- and xeno-free methods for human NK cell expansion. Using optimized media and cytokines, either with anti-NKp46 coated tissue culture dishes or Cloudz™ CD2/NKp46 microspheres we have greatly expanded highly purified NK cells from human peripheral blood mononuclear cells (PBMCs) that are capable of potent cytolytic activity, as indicated with a novel flow cytometry-based NK cytotoxicity assay. Using this NK expansion methodology, we performed high-throughput screening methods to identify novel agents that enhance NK cell killing activity as assessed by IFNγ and Granzyme B quantikine ELISA assays which were then confirmed with NK cytotoxicity assays. The methods established here provide a novel workflow for generating highly purified NK cells that are applicable to clinical translation. Furthermore, the agents identified in this work provide insight for augmenting NK killing potential.
Use of real-time live-cell analysis with enhanced data analytics to quantify immune cell tumor killing
Authors: Bevan Nicola , Lovell Gillian , Kelsey Lauren , Szybut Clare , McBain Kirsty , Perez-Garrido Sandra , Oliver Miniver , Patel Kalpana , Trezise Del , Dale Tim
Published: January 2023

To develop new therapeutics, researchers are exploring the role of the immune system in defending the body against tumors. Modelling induced malignant cell death in vitro is of paramount importance.

Tumor and immune cell co-cultures were created in 96 well plates and using live-cell analysis, various parameters of tumor killing were quantified in real-time. Red nuclear labeled target cells and various densities of pre-activated PBMCs (α-CD3/IL2, 4 d) were seeded in combination with IncuCyte Annexin V green apoptosis detection reagent. Images were acquired every 2 h for 3 d using IncuCyte. Analysis of the fluorescence images provides measurement of target cell number and apoptosis. Enhancement of the phase contrast image analytics enabled single cell segmentation, permitting determination of effector cell parameters; cell number, shape and, using fluorescently labeled surface marker antibodies, protein expression levels. In addition, studies into spatial interactions of target and effector cells were conducted. To exemplify how these new analytical features can be used to investigate the biology of tumor cell killing, studies of a α-hCD3xCD19 bi-specific T-cell engager antibody induced cytotoxicity were performed.

Further characterization of effects on cell cycle during target cell death and use of more advanced 3D models of immune cell killing were also assessed, demonstrating the flexibility of live-cell analysis as a powerful tool for analyzing immune cell killing.

Advances in data analytics has enabled the multiplexing of target cell quantification alongside the interrogation of effector cell properties in live cells. The added insight gained from these approaches will hopefully lead to improved immuno-therapeutics.
Development of a cells and bead-based, advanced flow cytometry assay for exhausted T cell quantitation
Authors: Carter Mark , Chauhan Sarita , O’Rourke John , Senutovitch Nina
Published: January 2023

T cells can become exhausted due to persistent antigen exposure during chronic infections and cancer. Exhausted T cells exhibit decreased effector functions and express inhibitory receptors (IRs). The exhausted phenotype may be protective in the context of chronic infection, however, it allows tumors to avoid immune surveillance and immune cell mediated killing. Recent therapies block the IRs to reinvigorate exhausted T cells.

A miniaturized, multiplexed flow cytometry assay was developed to measure expression of IRs on T cells while simultaneously measuring viability, proliferation, and cytokine secretion in microtiter plates. Data was acquired on the Intellicyt® iQue platform and analyzed using Forecyt®. Templates were prepared for ease of data analysis, to auto-generate gating, standard curves and sample quantitation and rapid, actionable results.

For proof of concept studies, human PBMCs, purified CD3+, or in vitro exhausted purified CD3+ cells were treated with anti-CD3/CD28 and cells were analyzed at 1, 2, and 3 days post-stimulation. Non-exhausted cells showed a concentration and time dependent increase in IR expression. Persistent, high levels of the IR markers PD-1, LAG-3 and TIM-3 were measured in exhausted cells. Stimulated exhausted T cells secreted stable and moderate levels of IFN-γ (<1650 pg/ml) and TNF (<750 pg/ml) over time whereas non-exhausted cells showed time and concentration dependent increases in IFN-γ (13,319 pg/ml) and TNF (3,711 pg/ml) upon stimulation.

The ability of this assay to examine T cell response allows screening for molecules that reverse T cell exhaustion, screening of modified cells such as CAR-Ts or selection of clones that exhibit low levels of IRs and show robust response to stimulation.
New microfluidic system provides automated alternative to Ficoll® for leukocyte isolation
Authors: Scully Melanie M , Mickool Sarah M , Rivelli Nicholas D , Jonca Aleksander J , Arnal Gustavo , Culligan Laura G , Kou Peng Meng , Smith Kyle C
Published: January 2023

Despite its limitations, Ficoll® has remained the primary method of leukocyte isolation for decades due to a lack of viable alternatives. To address this, MicroMedicine has developed SorterraTM, an automated microfluidic system that enables highly consistent label-free white blood cell separation and concentration.

In a comparative study, Sorterra recovered 25% more viable lymphocytes than Ficoll, 88 ± 7% and 69 ± 13%, respectively (n=48). Sorterra retained <0.01% red blood cells (RBCs) and <0.1% platelets, resulting in a highly pure leukocyte isolate, while Ficoll retained <0.01% RBCs and 8% platelets. Sorterra also demonstrated more consistent performance across operators than Ficoll.

Sorterra and Ficoll isolates were assessed for B cell and T cell functionality. To assess B cells, the isolates were stimulated with R848 and IL-2 (n=9), and total IgG and IgM secretion were evaluated with ELISpot. Secretion from Sorterra and Ficoll isolates was shown to be equivalent using distribution free resampling analysis. To assess T cell functionality, negatively selected T cells from Sorterra and Ficoll isolates were activated with CD3/28 beads and expanded with IL-2 (n=12). The isolates displayed similar T cell activation (89±4% vs. 88±5% CD25+/CD69+), proliferation, and T cell phenotypes (Tn TCM, TEM, TEFF) at 7 and 10 days post activation.

Compared to Ficoll, Sorterra enables rapid, automated isolation of leukocytes, consistently yielding more lymphocytes with negligible RBCs and platelet contamination while maintaining T cell and B cell function. By minimizing operator variability, Sorterra has the potential to transform cell separation by automating an outdated, variable, and labor-intensive process.
Intravital three-photon microscopy of entire mouse lymph node
Authors: Choe Kibaek , Hontani Yusaku , Wang Tianyu , Ouzounov Dimitre , Lai Kristine , Signh Ankur , Béguelin Wendy , Melnick Ari M. , Xu Chris
Published: January 2023

For the last 20 years, intravital confocal and two-photon microscopy have been powerful tools to explore dynamic immune cell behavior in mouse lymph node. However, they can only image ~100 and ~300 μm depth respectively while a peripheral lymph node of adult mouse has 600 – 1000 μm thickness. Here, we visualized whole thickness of adult mouse popliteal lymph node with intravital three-photon microscopy. Three-photon excitation significantly improved a signal-to-background ratio compared to two-photon excitation even at the same excitation wavelength. The capability to image the full depth enabled to achieve the 3D volume of entire lymph node vasculature in vivo by tiling multiple z-stack images. We observed DsRed-expressing lymphocyte migration in LYVE1+ lymphatic sinus at 600 μm depth of lymph node where conventional two-photon microscopy was normally inaccessible. In addition, we demonstrated the capability multi-color imaging by using Cγ1-Confetti mice of which each germinal center B cells stochastically expresses one of 4 different fluorescent proteins (CFP, GFP, YFP and RFP). Intravital three-photon microscopy has the potential to shed light on unknown immune cell behavior in deeper regions of the lymph node in vivo just as two-photon microscopy did during the last 20 years at the shallower depth.
Development of a Semi-automated Monocyte Monolayer Assay (MMA) to Determine the Clinical Significance of Red Cell Alloantibodies
Authors: Dean Melinda M , Tran Thu V , Ji Yu , Powley Tanya , Gu Yuan Tong , Sauret Emilie , Flower Robert L
Published: January 2023

BACKGROUND

One risk of allogeneic blood transfusion is the development of alloantibodies against red blood cell (RBC) antigens not found on the recipient’s RBC. The monocyte monolayer assay (MMA) is an in vitro model which has been used to determine the clinical significance of alloantibodies and predict post-transfusion RBC survival. We aimed to develop a semi-automated procedure to reduce assay time and facilitate improved patient management.

METHODS

PBMCs isolated using Sepmate tubes (STEMCELL Technologies) were added to Lab-Tek 8-well chamber slides (37°C, 1 hr, CO2). Antigen matched RBC were sensitised with plasma with known anti-RBC antibodies (anti-D n=3, anti-Yta n=4) (37°C, 1 hr, CO2). A modified Wright-Giemsa staining protocol was performed (Aerospray Hematology Pro 2 stainer, ELITechGroup) and the proportion of bound and/or phagocytosed RBC was determined via microscopy (>5% considered clinically significant).

RESULTS

The introduction of PBMC isolation using sepmate tubes and automated cell staining reduced technical requirements, improved assay reproducibility and reduced assay time by >2 hours. For the alloantibodies tested, 67% of the anti-D and none of the anti-Yta were clinically significant.

CONCLUSIONS

We developed a MMA with improved reproducibility and reduced turnaround time. This assay will improve blood transfusion safety and facilitate better patient management by reducing the risk of incompatible blood transfusion.
STING Negatively Regulates Allogeneic T Cell Responses by Constraining Function of Antigen Presenting Cells
Authors: Wu Yongxia , Mealer Corey , Sofi Mohammed , Tian Linlu , Bastian David , Schutt Steven , Choi Hee-Jin , Tang Chih-Hang Anthony , Hu Chih-Chi Andrew , Yu Xue-Zhong
Published: January 2023

Stimulator of interferon genes (STING) plays an important role in eliciting innate immune responses by sensing tumor and microbial DNA in anti-tumor and anti-infection responses, respectively. How the STING signal affects allogeneic response is not clear. To address this question, we utilized murine models of allogeneic hematopoietic stem cell transplantation (allo-HCT). By transferring donor bone marrow (BM) and T cells into allogeneic recipients, we found that significantly more severe graft-versus-host disease (GVHD) was induced in STING−/− recipients as compared to WT controls. By generating BM-chimeric mice in which STING was deficient in hematopoietic or non-hematopoietic antigen-presenting cells (APCs), we confirmed that STING on hematopoietic cells was primarily responsible for constraining host APC function. We further demonstrated that STING on host CD11c+ APCs played a predominant role in the regulation of allogenic T-cell responses. Mechanistically, we found that host CD11c+IAb+ cells deficient for STING could survive better and be activated more strongly after allo-HCT. As a consequence, STING-deficient APCs augmented donor T-cell expansion, chemokine receptor expression and migration into intestinal tissues, resulting accelerated/exacerbated GVHD after allo-HCT. Using pharmacologic approaches, we further demonstrated that systemic administration of STING agonist (c-diGMP) on recipient mice before irradiation significantly reduced GVHD mortality. In conclusion, we reveal a novel role of STING in APC activity that dictates T-cell allogenic responses, and validate STING as a potential therapeutic target for controlling GVHD after allo-HCT.