bluuebunny/crossref_metadata_2025 huggingface dataset:
In vitro generated human microglia derived from blood precursors (111.14)
Authors: Etemad Samar , Ruitenberg Marc , Filgueira Luis
Published: January 2023

Microglia as the resident innate immune cells of the brain share common characteristics with monocyte-macrophage lineage such as expression of surface markers and chemokine/cytokine receptors. To date, there is no standardized simple model available to investigate the biology of human microglia. The aim of this study is to establish a new in vitro microglia model using blood-derived precursor cells. For that purpose, human peripheral blood monocytes were cultured in serum free medium in the presence of M-CSF, GM-CSF, NGF and CCL2 to generate monocyte-derived microglia (M-MG).M-MG were clearly different in morphology, phenotype and function from freshly isolated monocyte-derived dendritic cells (M-DC).M-MG acquired a ramified morphology with primary and secondary processes .M-MG displayed a comparable phenotype to the human microglia cell line HMC3, expressing very low levels of CD45, CD14 and HLA-DR, CD11b and CD11c; and a distinct pattern of chemokine receptors (CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR3, CX3CR1).In comparison with M-DC, M-MG displayed lower T-lymphocyte stimulatory capacity as well as lower phagocytosis activity.The described protocol for the generation of human monocyte-derived microglia is feasible, well standardized and reliable, as it uses well defined culture medium and recombinant cytokines, but no serum or conditioned medium. This protocol will certainly be very helpful for future studies investigating the biology and pathology of human microglia.
Development of a novel method for in vitro analysis of γc-cytokine effects on CD8 T cells positive selection (111.15)
Authors: Rafei Moutih , Rouette Alexandre , Ruiz Vanegas Juan , Perreault Claude
Published: January 2023

Interleukin (IL)7 is the only γc-cytokine known to support double-positive thymocytes differentiation to the single-positive (SP) stage. To determine if additional γc-cytokines can support T-cell development, we studied their differential effects on positive selection in vitro using a monolayer of OP9 bone marrow-derived stromal cells. We chose the OTI transgenic TCR as our working model due to the previous identification of positively selecting peptides in FTOC assays. In addition to rIL7, rIL4 and rIL13 (but not rIL2, rIL9, rIL15 or rIL21) were able to induce SP CD8 T-cell development. Interestingly, rIL4 induces the de novo expression of EOMES and triggers the development of 2 distinct SP CD8 T cell populations, which we termed CD8int and CD8hi. Flow-cytometry analysis showed that ex vivo generated CD8int, but not CD8hi, expressed high levels of CD69, PD-L1 and CD44 whereas rIL7 or rIL13 treatments did not up regulate these markers. Furthermore, rIL4 has a unique ability to enhance expression of FOXO1, KLF2 and S1PR1. Flow-cytometry analysis of signaling events demonstrated that every γc-cytokine leads to differential activation of AKT, ERK, STAT3, STAT5 and STAT6. Taken together, our findings show that CD8 T-cell positive selection can be studied with an in vitro model that is more convenient than FTOC assays and can be customized to a high-throughput format. Notably, we validate a novel role for rIL4 and rIL13 in supporting positive selection of thymocytes.
A Pre-BCR/NFκB Pathways Suppresses STAT5-dependent Pre-B Cell Leukemia (111.18)
Authors: Katerndahl Casey , Willette Mark , Heltemes-Harris Lynn , Ramsey Laura , Koeuth Thearith , Baechler Emily , Farrar Michael
Published: January 2023

Mice expressing a constitutively active form of STAT5b (called Stat5b-CA mice) develop pre-B leukemia with low frequency (~1-2%). Microarray analysis of these leukemias shows decreased expression of genes involved in pre-BCR signaling. Stat5b-CA mice crossed to mice with defects in a coherent signaling pathway downstream of the pre-BCR involving BLNK, BTK, or PKCβ developed pre-B leukemia in > 75% of progeny. These leukemias exhibited reduced expression of a subset of NFκB target genes, which was important for transformation as Stat5b-CA x Nfkb1-/- mice also developed pre-B leukemia. Our results point to an unexpected role for NFκB1 as a tumor suppressor in pre-B cells. We found that STAT5 cooperates with defects in this pre-BCR-dependent pathway to repress expression of the potential tumor suppressor genes Foxo1, Foxo3a, Ikzf1, and Ikzf3. Our findings demonstrate that STAT5 activation antagonizes a pre-BCR-dependent pathway that limits pre-B cell proliferation, promote pre-B cell differentiation, and inhibit pre-B cell transformation.
Specialized role of thymic transendothelial dendritic cells in antigen acquisition. (111.23)
Authors: Vollmann Elisabeth , von Andrian Ulrich
Published: January 2023

A functional immune system has to protect the body from pathogens while being tolerant to self-antigens (sAg). To achieve this goal, developing thymocytes are subjected to tolerance mechanisms. Central tolerance takes place within the thymus and removes the bulk of auto-reactive thymocytes by either clonal deletion (negative selection) or redirection into a regulatory T cell lineage (Treg). Dendritic cells (DCs) are key players in this process. Thymic DCs can be distinguished and subdivided into distinct subsets according to their surface markers as well as by their origin. Thymus resident DCs differentiate locally from a progenitor cell, while homed DCs enter the thymus fully differentiated from the blood. Here, we report a functional sub-specialization between thymus resident and homed periphery-derived DCs. While the latter can deliver peripheral sAg to the thymus as their cargo, some resident DCs are strategically positioned in the thymus to extend processes across the endothelial barrier of capillaries and are able to acquire, process and present circulating Ag.
Enumeration and characterization of self-reactive T cells in polyclonal populations (111.25)
Authors: Stritesky Gretta , Hogquist Kristin
Published: January 2023

The thymic processes of positive and negative selection are crucial for the formation of a functional T cell repertoire. TCR transgenic models have provided crucial insight into these processes, but it has not been possible to quantitate positive and negative selection in normal diverse polyclonal populations. We developed a Nur77GFP transgenic reporter mouse that specifically expresses GFP in T cells in response to TCR stimulation. Using Nur77GFP mice crossed to mice deficient in the pro-apoptotic molecule Bim, we found that two times more DP thymocytes are negatively selected by high affinity interactions than are positively selected by low affinity interactions. We also identified the fraction of SP thymocytes undergoing further deletion in the medulla, after positive selection. Cells that are rescued from negative selection have increased Nur77GFP compared to foxP3+ T regulatory cells, suggesting that T cells undergoing negative selection have TCRs with higher affinity for self compared to developing T regulatory cells. Finally, our results identify a population of self-reactive CD25-CD44+ CD4 T cells in mesenteric LNs, which have a phenotype consistent with clonal anergy. Future studies using this model will allow us to identify and quantitate selection defects in various mutant or autoimmune prone animals.
Infection-induced dysregulation of the hematopoietic stem cell niche (111.36)
Authors: McCabe Amanda , Zhang Lu , Jones Maura , MacNamara Katherine
Published: January 2023

Hematopoietic stem cells (HSCs) are critical in replenishing all cells of the blood. At homeostasis, HSCs reside in close proximity to bone-lining osteoblasts within the endosteal niche. Osteoblasts produce CXCL12, a chemokine that supports HSC retention in the bone marrow (BM). BM-resident macrophages have recently been shown to retain HSCs in the endosteum through regulation of osteoblast function. How BM-resident macrophages contribute to HSC function during infection has not been addressed. Ehrlichiosis, a tick-born disease, results in anemia and thrombocytopenia, stemming, in part, from alterations in hematopoiesis. Using Ehrlichia muris infection of C57BL/6 mice we observed an IFNγ-dependent increase in F480+Ly6G+CD169+ BM-resident macrophages. These macrophages exhibited increased expression of MHCII and CD80 during infection indicating an activated phenotype. Infection also resulted in reduced expression of osteoblast-specific genes within cells of the endosteum, including osteocalcin and Cxcl12. By histology, we observed a diminished osteoblast lining. Furthermore, HSCs were mobilized into the bloodstream, indicating a dysregulation of the endosteal HSC niche. Our current hypothesis is that during ehrlichial infection, expansion and activation of resident macrophages results in altered osteoblast function and subsequent HSC mobilization from the BM. Future experiments will address mechanisms of macrophage-mediated alterations to the HSC niche during infection.
Development of CD4+ PLZF+ T Cells in the Absence of Itk (111.41)
Authors: Prince Amanda , Evans John , Berg Leslie
Published: January 2023

Innate-like lymphocytes develop in the thymus and share characteristics of the innate and adaptive immune systems. These lymphocytes undergo antigen receptor rearrangement and are able to exert their effector function immediately upon ex vivo stimulation. Our lab is interested in how TCR signaling affects the development of conventional versus innate-like lymphocytes. Itk is a nonreceptor protein tyrosine kinase that signals downstream of the TCR. Previously, our lab has shown that in the absence of Itk, CD8+ Eomesodermin+ T cells develop that have a diverse TCR repertoire and produce IFNγ immediately upon ex vivo stimulation. Recently, several labs have demonstrated that the development of these CD8+ Eomesodermin+ T cells is dependent upon CD4+ PLZF+ thymocytes producing IL-4. We have determined that there are two CD4+ PLZF+ populations consisting of αβ and γδ T cells in Itk-/- thymi. In the absence of γδ T cells, Itk-/- CD4+ PLZF+ αβ T cells retain the ability to induce Eomesodermin expression in CD8+ T cells. Furthermore, Itk-/- CD4+ PLZF+ αβ T cells do not bind CD1d tetramer and develop independently of IL-15, class II MHC, and β2m. We have also found that Itk-/- CD4+ PLZF+ αβ T cells are found in the spleen and mesenteric lymph node in the periphery and have a restricted TCR repertoire. Thus, in the absence of Itk, a non-canonical innate-like population of CD4+ PLZF+ αβ T cells develop that influences the development of conventional T cells.
Inhibition of T cell Protein Tyrosine Phosphatase (TC-PTP) Enhances IL-18-dependent Hematopoietic Progenitor Expansion (111.57)
Authors: Bourdeau Annie , Trop Sebastien , Doody Karen , Dumont Daniel , Tremblay Michel
Published: January 2023

The clinical application of hematopoietic progenitor cell-based therapies for the treatment of hematological diseases is hindered by current protocols, which are cumbersome and have limited efficacy to expand the progenitor cell pool. We report that inhibition of T cell protein tyrosine phosphatase (TC-PTP), an enzyme involved in the regulation of cytokine signaling, through gene knockout results in a 9-fold expansion of functional hematopoietic progenitors in murine bone marrow (BM). This effect could be reproduced using a short (48 h) treatment with a pharmacological inhibitor of TC-PTP in murine BM, as well as in human BM, peripheral blood and cord blood. We also demonstrate that BM stem cells treated ex-vivo with a TC-PTP inhibitor can differentiate into functional progeny in vivo. We establish that the mechanism whereby inhibition of TC-PTP mediates its effects involves the IL-18 signaling pathway, leading to increased production of IL-12 and IFN-γ by progenitor cells. Together, our results reveal a previously unrecognized role for IL-18 in regulating progenitor cell expansion, and provide a novel and simple method to rapidly expand progenitor cells from a variety of sources using a pharmacological compound.
Notch Induces T Cell Receptor γδ Thymocytes to Differentiate along a Bipotent CD4 CD8 Double Positive Pathway (111.6)
Authors: Vandekerckhove Bart , Van Coppernolle Stefanie , Verstichel Greet , Snauwaert Sylvia , Langerak Anton , Kerre Tessa
Published: January 2023

It is well documented that in wildtype mice TCRγδ+ cells differentiate along a double negative (DN) pathway whereas TCRαβ+ cells differentiate along the double positive (DP) pathway, suggesting that the TCR itself induces lineage differentiation. Under experimental conditions and in genetically modified mice, however, evidence was presented suggesting that rather than TCR itself, TCR signal strength and notch determine lineage choice. In the human thymus, a “chimeric” DP TCRγδ+ cell population is present and constitutes a sizeable fraction of the γδ population. We asked the question whether these cells belong to the αβ or γδ lineage or whether these cells are bipotent. We found that TCRγδ DP cells are bipotent cells since strong TCR signals induces differentiation to TCRγδ cells, whereas Notch activation induces TCRαβ lineage differentiation. We furthermore could show that Notch signaling diverts TCRγδ DN cells to the DP pathway and induces strong proliferation. In line with these findings, TCRγδ+ acute lymphoblastic leukemias (ALL) with activating Notch1 mutations follow the DP differentiation pathway, whereas the DN ALL cells are devoid of these activating Notch1 mutations. We were able to confirm that also in vivo TCRγδ DP have rearranged TCRβ chains, actively rearrange the TCRα locus and delete the TCRδ locus (αβ lineage). Using TCRα rearrangements as a lineage marker, we could show that a subpopulation of mature TCRγδ cells is derived from DP cells.
Chitinase 3-like 1 enhances bacterial adhesion to colonic epithelial cells through the bacterial chitin-binding motif (112.9)
Authors: Mizoguchi Emiko , Lee In-Ah , Kamba Alan , Santhanam Radhika
Published: January 2023

Chitinase 3-like-1 (CHI3L1) is an inducible molecule in colonic epithelial cells (CECs), and the chitin-binding motif (CBM) in CHI3L1 regulates host-microbial interactions. To examine the existence of CBMs in E. coli, we used ChtBD3 as a search in the SMART database. We found that 11 genomes of E. coli strains contain a gene encodes for ChiA, which has multiple CBMs. There are 5 different amino acids (Q362K, E370K, V378A, V388E & E548V) in CBMs, and the E.coli are grouped into pathogenic- and non-pathogenic strains by substitution. To examine the role of CBMs in E. coli, we generated a deletion mutant (ΔchiA) of the Adherent Invasive E. coli (AIEC) LF82, and performed in vitro assays. LF82 ΔchiA has 50% less adhesion to CECs as compared to LF82. This phenotype is fully recovered when the mutant carries out pHSG575 with LF82 ΔchiA, but not withLF82 chiA plus 5 mutations or E. coli K12 chiA. To test the in vivo influence, B6 mice were treated 2% DSS to enhance the CHI3L1 on CECs and were orally inoculated with LF82 (WT or ΔchiA) for 15 days. In contrast to LF82, mice infected with LF82 ΔchiA showed less body weight loss, a lower clinical score, less stool bacteria and less cellular infiltration in the colon. A significantly increased number of E. coli adhered/invaded the colon in LF82-infected mice, as compared to LF82 ΔchiA-infected or control mice. In conclusion, CBMs of AIEC play a critical role in their adhesion to CHI3L1-expressing CECs during colitis.
HCV genotype 1a representative sequence elicits broad CD8+ T cell responses (113.10)
Authors: Burke Kelly , Munshaw Supriya , Osburn William , Levine Jordana , Liu Lin , Ray Stuart , Cox Andrea
Published: January 2023

Vaccines designed to prevent or to treat hepatitis C viral infection must achieve maximum cross-reactivity against widely divergent circulating strains. Computer-generated sequences minimize genetic distance between circulating strains, and can be generated as a consensus sequence (most common amino acid at each position) or a representative sequence (derived with Bayesian and maximal likelihood phylogenetic tools). Broad recognition of such sequences in HCV has not been demonstrated. Consensus and representative sequences were generated from 390 full-length HCV genotype 1a polypeptide sequences. Sequence mutations in known epitopes were identified by longitudinal sequencing of HCV-infected subjects. Peptides encoding consensus, representative, and variant epitope sequences were tested for the capacity to expand CD8 T cells from HCV-infected subjects and to elicit cross-reactive responses by IFNg ELISpot. The representative and consensus sequences were identical for 9/11 epitopes examined. T cell lines generated against representative sequence peptides were generally cross-reactive to consensus sequence and natural sequence variants. Furthermore, representative sequence peptides generated more robust T cell responses than did natural sequence variant peptides. The broadest recognition of highly diverse circulating HCV strains was achieved using CD8+ T cells expanded with representative sequence HCV. These data support the use of representative sequence in HCV vaccine design.
Transcription factor expression profiling to characterize differentiation of antigen-specific CD8 T cells induced by a live attenuated viral vaccine. (113.18)
Authors: Billingsley James , Rajakumar Premeela , Salisch Nadine , Kuzmichev Yury , Hong Henoch , Connole Michelle , Reeves R. , Johnson R.
Published: January 2023

Live attenuated SIV (SIVΔnef) is the most effective approach to induce protection to pathogenic SIV challenge in macaques. Protective immunity, in part mediated by CD8 T cell responses, correlates with phenotypic maturation of SIV-specific T cells, but remains incompletely understood. Expression profiling of T cell transcription factors (TF) offers a novel approach to characterize antigen-specific T cell differentiation. Highly parallel qRT-PCR was used to characterize the expression of 21 TF in naïve, central, transitional, and effector memory T cell subsets, and in SIV-specific CD8 T cells obtained at times associated with lesser protection (wk 5) and greater protection (wk 20) following SIVΔnef vaccination. Unsupervised clustering organized samples from CD4 and CD8 T cells into groups concordant with cell surface phenotype. TF expression in SIV-specific CD8+ T cells segregated by time, with wk 20 cells exhibiting increased expression of TF associated with both maintenance of quiescence (TCF7, BAZF) and promotion of effector function (Eomes, T-Bet). Different expression profiles were observed in T cells specific for different SIV epitopes, consistent with different epitope escape kinetics. TF expression profiling suggests T cell responses correlated with protection may include characteristics of both memory and effector cells. TF expression profiling can provide data complementary to the analysis of memory cell differentiation based on classical phenotypic markers.
Vaccination of endangered Hawaiian geese (Nene, Branta sandvicensis) against West Nile Virus using a protein-based vaccine (WN-80E) (113.7)
Authors: Jarvi Susan , Hu Darcy , Misajon Kathleen , Coller Beth-Ann , Wong Teri , Lieberman Michael
Published: January 2023

While West Nile Virus (WNV) has not yet been reported in Hawaii, eventual introduction appears unavoidable. Nene are endemic, endangered Hawaiian geese, and are susceptible to WNV. The goal of this study is to demonstrate that a vaccine developed against WNV for humans (WN-80E) is also highly immunogenic in Nene, and does not produce adverse biological effects. Six captive, non-breeding Nene were immunized with two 10 µg doses (four weeks apart) of the WN-80E recombinant protein adjuvanted with Montanide ISA720. Two Nene were similarly injected with mock preparation as controls. Blood samples were collected prior to the first dose, at two weeks and six months after the second dose. Detailed observations were noted for 48 hours post injection, followed by daily observations of health status to monitor for adverse biological effects. WNV-specific antibody titers were determined by an endpoint ELISA. An unpaired t-test demonstrated significantly higher geometric mean titers for immunized vs. control groups at two weeks post dose two (4129 and 100, respectively, p = 0.0096) and at 6 months post dose two (246 and 63, respectively, p = 0.0023). No swelling was reported at any time during the study at the site of injection. No serious adverse biological effects were observed directly due to the immunization. The vaccine containing the WN-80E and Montanide ISA720 adjuvant appears to be safe and effective in Nene, and should be considered for widespread use in Hawaii.
Critical role of the inflammasome during central nervous system Staphylococcus aureus infection (114.1)
Authors: Hanamsagar Richa , Kielian Tammy
Published: January 2023

Staphylococcus aureus (S. aureus) infection in the central nervous system (CNS) can lead to abscess formation typified by widespread inflammation and necrosis. A key cytokine driving inflammation during this process is IL-1β, which is processed into its active form by the inflammasome. Our recent studies demonstrated that the NOD-like receptor 3 (NLRP3) inflammasome and its adapter protein ASC are required for IL-1β secretion by microglia, the resident immune cells of the CNS. To understand the role of NLRP3 inflammasome activation during brain abscess formation, C57BL/6 wild type (WT), ASC knockout (KO), and NLRP3 KO mice received intracerebral infections with 1000 cfu S. aureus. Interestingly, ASC KO mice succumbed to infection within 24 h, yet bacterial burdens were identical to WT animals. Inflammatory mediators including CXCL1, CXCL10, CCL2, and IL-6 were significantly reduced in ASC KO mice, suggesting that ASC loss may impact processes extending beyond IL-1β cleavage. To extend the survival of ASC KO mice, animals were infected with a 1-log lower inoculum, revealing significant reductions in neutrophil and CD4+ T cell infiltrates compared to WT animals. In contrast, NLRP3 loss had minimal impact on brain abscess development. Collectively, these results indicate that ASC is involved in diverse pathways affecting inflammatory mediator expression and immune cell infiltration into the CNS, which may occur independently of its classical inflammasome actions.
HMGB1 facilitates inflammasome-dependent IL-1β release (114.2)
Authors: Keyel Peter , Vernon Philip , Chu Jessica , Lotze Michael , Salter Russell
Published: January 2023

HMGB1 is a potent mediator of late-phase shock, endotoxin toxicity and other inflammatory processes. HMGB1 is released passively from necrotic cells, and also following inflammasome activation in macrophages and dendritic cells. We hypothesized that caspase-1 activated nucleases liberate HMGB1 from the nucleus and allow for subsequent cellular release. To test this hypothesis, we measured inflammasome-mediated HMGB1 release in the presence of various nuclease inhibitors. Inhibitors of DNase γ blocked inflammasome-activated HMGB1 release. Surprisingly, DNase γ inhibitors also blocked IL-1β release, suggesting that nuclear proteins play a role in nonconventional cytokine secretion. To determine if HMGB1 regulates IL-1β release, we tested its intracellular association with IL-1β and the ability of HMGB1-deficient cells to secrete IL-1β. We found that HMGB1 co-immunoprecipitated with IL-1β in cells, indicating an interaction prior to IL-1β release. Furthermore, genetic disruption of HMGB1 in dendritic cells reduced their ability to secrete IL-1β in response to inflammasome activation. Taken together, these data suggest that HMGB1 and other nuclear proteins regulate IL-1β release through an inflammasome dependent process.
PKR is required for NLRP3 inflammasome activation and HMGB1 release (114.7)
Authors: Lu Ben , Nakamura Takahisa , Inouye Karen , Valdes-Ferrer Sergio , Olofsson Peder , Li Jianhua , Wang Haichao , Erlandsson-Harris Helena , Kalb Thomas , Andersson Ulf , Yang Huan , Chavan Sangeeta , Hotamisligil Gökhan , Tracey Kevin
Published: January 2023

The NLR family pyrin domain-containing 3 (NLRP3) inflammasome, an intracellular oligomeric protein complex, regulates release of caspase-1 activation-dependent cytokines, including IL-1β, IL-18, and high-mobility group box 1 (HMGB1). During the course of studying HMGB1 release mechanisms from macrophages, we unexpectedly observed that double-stranded RNA dependent protein kinase (PKR) is required for NLRP3 inflammasome activation. PKR inactivation by genetic deletion or pharmacological inhibition blocked NLRP3 inflammasome activation in response to double-stranded RNA, ATP, monosodium urate, adjuvant aluminum, and live E coli. Overexpression of PKR significantly enhanced the reconstituted NLRP3 inflammasome-induced caspase-1 activation and IL-1β maturation; whereas blocking PKR significantly inhibited the reconstituted NLRP3 inflammasome activation. PKR physically interacts with NLRP3, and PKR autophosphorylation is required for efficient PKR and NLRP3 interaction. Importantly, PKR autophosphorylation in a cell free system with recombinant NLRP3, ASC and pro-casapse-1 reconstitutes inflammasome activity. Together, these results indicate that PKR is required for inflammasome activation, and that it should be possible to target this molecule to inhibit inflammasome activity during inflammation.
Cytosolic K+ and extracellular ATP as regulators of NLRP3 inflammasome activation and the IL-1β secretion response of macrophages to crystalline stimuli. (114.9)
Authors: Katsnelson Michael , Smith Corey , Dubyak George
Published: January 2023

The crystalline particulate Alum has widely been used as an adjuvant in vaccines. Alum and other crystalline particulates also stimulate NLRP3/caspase-1 inflammasome assembly and secretion of mature IL-1β via poorly understood mechanisms that involve decreases in cytosolic [K+]. This study characterized mechanisms for K+ efflux in LPS-primed murine bone marrow-derived macrophages (BMDM) stimulated with either Imject Alum or the Na+, K+-ATPase inhibitor ouabain. Treatment with Alum for 3 h induced robust secretion of IL-1β that was similar in the absence or presence of antagonists of the P2X7 receptor, an ATP-gated K+ efflux channel known to regulate NLRP3 inflammasome assembly. However, the ATP-scavenging enzyme apyrase markedly reduced this IL-1β secretion, suggesting that NLRP3 activation and IL-1β release induced by Alum crystals is at least partially dependent on a P2X receptor subtype(s) other than P2X7. Current experiments are testing possible involvement of P2X4 receptor channels. Treatment of LPS-primed BMDMs with ouabain for 6 h also induced significant release of IL-1β. We hypothesize that ouabain causes NLRP3 activation and IL-1β secretion by inhibiting the steady-state Na+, K+ATPase activity which counteracts the slow efflux of cytosolic K+ via leak/rectifier-type K+ channels.
CD4+ and CD8+ T cell β antigen receptors have different and predictable V and J gene usage and CDR3 lengths (115.10)
Authors: Robins Harlan , Emerson Ryan , Sherwood Anna , Desmarais Cindy
Published: January 2023

CD8+ and CD4+ T lymphocytes have different and complimentary roles: CD8+ (cytotoxic) T cells directly kill cells presenting non-self epitopes while CD4+ (helper) T cells regulate the immune response to a particular antigen. These T cells recognize antigens by binding to epitope MHC protein complexes, with CD8+ T cells binding class I and CD4+ T cells binding class II MHC protein complexes. While it’s known that the CD8 and CD4 proteins determine which MHC class the T cells bind, it is unknown if the antigen receptor sequences vary between the two populations. The diversity of possible receptors is huge and until recently this diversity precluded the possibility of capturing the antigen receptor repertoire. Adaptive Biotechnologies has developed a novel method that amplifies rearranged T cell receptor β (TCRB) CDR3 sequences and uses high throughput sequencing to sequence millions of TCRB CDR3 chains. To determine if the two populations of T cells have unique TCRB profiles, PBMC from 19 unrelated individuals were sorted 4 ways into CD4+ and CD8+ naïve and memory T cells. V and J gene usage and CDR3 sequence length could distinguish the TCRB CDR3 sequences amplified from CD4+ and CD8+ T cells. These sequence differences can be exploited and used to estimate the proportion of CD4+ to CD8+ T cells in a mixed population of T cells. These data indicate a biological difference between the antigen receptors that bind class I versus class II MHC protein complexes.
Pregnancy alters T-cell development in the mouse thymus (115.11)
Authors: Jaiswal Mukesh , Mallers Timothy , Chaouat Gerard , Gilman-Sachs Alice , Beaman Kenneth
Published: January 2023

The syngeneic and allogeneic mating combinations were used to assess the effect of pregnancy-associated hormones and fetal antigens on different T-cell subsets and their a2V-ATPase (a2V) expression during pregnancy. Our data shows that a2V was significantly (p≤0.05) increased in thymus recovered from normal mating combinations (♀BALB/cX♂BALB/c and ♀CBAX♂BALB/c) when compared with abortion prone mating combinations (♀CBAX♂DBA). The percentage of NK-T cells (CD3+NKp46+ cells) was significantly (p≤0.05) increased in all studied mating combinations on days 12 and 16 of pregnancy when compared to their respective non-pregnant females and pups. The expression of a2V on NK-T cells was also significantly (p≤0.05) increased as their numbers was increased. The numbers of CD3+CD4+CD8- cells were significantly (p≤0.05) increased, while the numbers of CD3-CD4-CD8- and CD3+CD4+CD8+ cells were significantly (p≤0.05) decreased in all the mating combinations when compared to their respective non-pregnant females and pups. This dynamic change in the numbers of T-cells subsets during pregnancy suggests that T-cells development and differentiation is altered in pregnancy. Moreover, a2V may play a crucial role in differentiation of NK-T cells from different T-cells subsets. Future studies to decipher the mechanism of action of a2V in thymic T-cells development and differentiation will lead to valuable knowledge of thymus development as well as its role in maternal fetal allogeneic relationship.
The role of ID3 in iNKT cell development (115.12)
Authors: Verykokakis Mihalis , Kee Barbara
Published: January 2023

Invariant NKT cells comprise an unconventional T cell population with innate characteristics. Like the conventional αβ T cell lineage, they develop in the thymus from DP precursors after receiving TCR-dependent positive selection signals, although utilizing different pathways. iNKT cells further mature through a series of differentiation stages and acquire their effector properties intrathymically. The ID3 protein is required to reduce E protein activity for proper selection of DP progenitors towards conventional thymocyte lineages. Here, we investigate the requirement for ID3 in the thymic development of iNKT lineage, in vivo. We show that the frequency of iNKT cells in the Id3-/- thymus is higher than in WT littermates, in a cell-intrinsic manner. Id3-deficient post-selected DP cells express higher levels of the canonical Vα14-Jα18 TCRα chain transcripts. Additionally, the transcription factor PLZF, a molecular determinant of the NKT lineage, is expressed in a subset of post-selected DP cells in the Id3-/- mice. However, further thymic iNKT differentiation is blocked at an immature stage. Taken together these results suggest that ID3 restricts the innate T cell potential of DP precursors thus contributing to the lineage fate decisions at the DP stage. However, ID3 is necessary for the completion of the innate T cell program during iNKT differentiation. Ongoing gene expression analysis will identify potent E protein target genes essential for NKT development
Tcf-1 in early lymphoid development (115.14)
Authors: Germar Kristine , Dose Marei , Konstantinou Tassos , Zhang Jiangwen , Wang Hongfang , Lobry Camille , Arnett Kelly , Blacklow Stephen , Aifantis Iannis , Aster Jon , Gounari Fotini
Published: January 2023

In hematopoiesis, the transcriptional networks governing lineage choice from pluripotent progenitors to mature lymphoid cells remain poorly understood. Although some key factors associated with the specification and commitment of T cells have been described, the functional roles of key regulators in orchestrating these developmental programs remain unclear. Using a loss-of-function model, we established the importance of T-cell factor 1 (Tcf-1), a T-cell specific mediator of Wnt signaling, as a gatekeeper of T-cell fate. We show that Tcf-1 is directly activated by Notch signals. Activation of Notch signaling in uncommitted precursors by the thymic stroma is known to initiate the T-cell differentiation program, and we show that Tcf-1 is one regulator first induced in these precursors. Tcf-1 is required at the earliest phase of T-cell determination for progression beyond the early thymic progenitor (ETP) stage, and this requirement is cell intrinsic and Wnt independent. The global expression profile of Tcf-1 deficient progenitors indicates that basic processes of DNA metabolism are downregulated in its absence, and the blocked T-cell progenitors die by apoptosis. While Tcf-1 is dispensable for the development of bone marrow (BM) progenitors and their migration through the blood, we present evidence for a novel role for Tcf-1 in early BM natural killer (NK) development. Our data thus demonstrate multiple roles for Tcf-1 in the roadmap of lymphoid development.
WNT signaling programs the development of IL-17 producing γδ T cells and Innate Lymphoid Cells (115.17)
Authors: Malhotra Nidhi , Narayan Kavitha , Sylvia Katelyn , Melichar Heather , Kang Joonsoo
Published: January 2023

IL-17/IL-22 producing γδ T cells and other innate lymphoid cell (ILC) subsets safeguards the mucosal epithelia of oral, reproductive, respiratory and intestinal tracts against infection and damages. γδ T cell subsets originate in the thymus and are exported as pre-made effectors, while the other ILC effector subsets differentiate in the gut associated lymphoid tissues. Whether a common gene network program distinct from that operating in adaptive T cells controls IL-17/IL-22 production in all innate lymphoid effectors is unknown. We demonstrate that sry high mobility group (HMG) box transcription factors (TFs), SOX13 and other family members, function in a positive loop to control the intrathymic differentiation of IL-17 producing γδ T cells. In contrast, another HMG TF TCF1, the nuclear effector of canonical WNT signaling, is the primary negative regulator of the network. Furthermore, we show that TCF1 is also required for the functional differentiation of ILCs. Together, the results suggest the existence of a TF network that controls all innate IL-17 producing effectors, independent of tissue origin and T cell antigen receptor expression, distinct from that involved in generating IL-17 producing adaptive T cells.
IFNbeta-1b alters the composition of circulating B cell subsets, and leads to changes in the B cell cytokine secretion profile in patients with relapsing-remitting multiple sclerosis. (116.9)
Authors: Mielcarz Daniel , Bergeron Alan , DeLong John , Smith Kathleen , Heyn Alexandra , Mack Karen , Conrad Mary Ann , Oliver Brant , Kasper Lloyd , Channon Jacqueline
Published: January 2023

Recent studies provide strong evidence for a role for B cells in the pathology of RRMS. In order to explore the effect of IFNbeta-1b treatment on B cell phenotype and function in RRMS patients, blood was drawn from 10 RRMS patients before treatment and again 2 and 6 months after daily injections of IFNbeta-1b. Cryopreserved PBMCs were thawed and stained with panels of antibodies against B cell surface antigens. At baseline, RRMS patients have increased frequencies of naïve CD19+CD20+ B cells and decreased frequencies of memory B cells when compared with age-matched healthy controls. CpG-stimulated PBMCs from patients treated with IFNbeta-1b show an increase in IL-10 production and a decrease in IL-6 production by naïve, memory and B1 cells (a recently-described subset of autoantibody producing cells that are CD20+CD27+CD43+). These changes in cytokine production are indicative of a change from a pro- to an anti-inflammatory phenotype. In addition, this treatment alters the composition of circulating B cell subsets, leading to an increase after six months in circulating naïve B cells and a decrease in both memory and B1 cells, both cell types of which are potentially pathogenic in RRMS. Although the number of subjects in this study was limited, these findings suggest that alterations in B cell phenotype and function may be a mechanism by which IFNbeta-1b reduces disease activity.
Short chain fatty acids induce IL-1β production: The role of monocarboxylate transporters (MCTs) (117.16)
Authors: Mirmonsef Paria , Makinde Hadijat , Zheng Justin , Spear Greg
Published: January 2023

Short chain fatty acids (SCFAs) are products of anaerobic bacteria at mucosal sites, such as the female genital tract in bacterial vaginosis (BV). IL-1β, a potent proinflammatory cytokine, is found at elevated levels in the vaginal fluids of BV+ women. We recently found that SCFAs induce IL-1β in monocytes. Here, we tested whether SCFAs could control IL-1β production by either: 1) inducing pro IL-1β in the cytoplasm, and/or 2) causing inflammasome activation and release of IL-1β by acting as a “second” danger signal. We found that exposing peripheral blood mononuclear cells (PBMCs) for 24-hours to physiological levels of either acetic or butyric acid resulted in a modest increase in IL-1β levels in vitro. The release of IL-1β was more robust and occurred more rapidly when PBMCs were treated with SCFAs first, followed by exogenous ATP as a “danger” signal. SCFAs themselves were not potent danger signals as they failed to increase the release of LPS-induced pro IL-1β. Since SCFAs can enter cells via MCTs, we blocked these transporters using pharmacological agents and found that MCTs were necessary for SCFA-mediated IL-1β production. These findings show that SCFAs can induce cytoplasmic pro IL-1β production and that their effects are MCT-dependent. Because IL-1β is an important proinflammatory mediator, these results enhance our understanding of how SCFAs may contribute to the inflammatory environment seen in BV.
TLR4 senses proteinase activity to mediate allergic lung disease and macrophage fungistatic response. (117.18)
Authors: Ongeri Valentine , Byrd Tiara , Kheradmand Farrah , Corry David
Published: January 2023

Asthma is characterized by the presence of T-helper type 2 (Th2) cells in the lung. Our lab has shown that active fungal proteinases are requisite for robust Th2 cell generation and allergic airway inflammation that protects against fungal invasion during airway infection. Macrophages (MΦs) interact with inhaled pathogens and control airway infections via toll like receptors (TLRs) which may play a role in host defense against inhaled fungi. We hypothesized that fungal proteinases activate one or more TLRs in MΦs resulting in allergic lung disease and highly efficient fungal killing. Wildtype mice and mice deficient in specific TLRs (TLR4) and their adaptor molecules (MyD88-/- and TRIF-/- mice) were intranasally infected with viable conidia of Aspergillus niger and assessed for the asthma phenotype. Bone marrow cells were also harvested from TLR4-/- and wildtype mice to assess for differentiation of MΦs via RNA expression of canonical markers and cultured with the viable conidia to determine their effect on fungal growth.TLR4-/- mice failed to develop asthma-like disease while MyD88-/- and TRIF-/- mice had normal or exaggerated responses. Fungal proteinase induced differentiation of naive MΦs that profoundly inhibited fungal growth in vitro in a TLR4 dependent manner. Our findings therefore suggest a “Biased Agonism” model of TLR4 signaling in which distinct ligands may activate alternative signaling pathways that give rise to entirely different immune responses.
The generation of M2 monocytes in cultures of human peripheral blood mononuclear cells exposed to 137Cs γ-rays (117.2)
Authors: Nakamura Kiwamu , Okita Yamato , Cornforth Michael , Kobayashi Makiko , Suzuki Fujio
Published: January 2023

Thermally injured patients who are carriers of M2MΦ are greatly susceptible to various opportunistic infections. Since the similarly increased susceptibility to the infection is seen in patients with irradiated injuries, in this study we investigated the effect of 137Cs γ-irradiation on the M2MΦ generation in cultures of human peripheral blood mononuclear cells. Peripheral blood mononuclear cells (PBMC), isolated from healthy volunteers by Ficoll - Hypaque density gradient centrifugation, were exposed to 2 - 8 Gy of 137Cs γ-rays. CD14 positive monocytes were isolated from the irradiated PBMC using magnetic beads, and stimulated with Staphylococcus aureus Cowan strain (0.0075% SAC) for 24 hours. Culture fluids were harvested and analyzed for cytokines by ELISA. In the results, IL-10 was detected and IL-12 was not detected in the culture fluids of monocytes from 137Cs γ-irradiated PBMC, while IL-12 was detected and IL-10 was not detected in the culture fluids of healthy donor monocytes. Also, CCL1 was detected and CXCL13/CCL17 were not detected in the culture fluids of monocytes isolated from 137Cs γ-irradiated PBMC, while all of these chemokines were not produced by healthy donor monocytes. These results suggest that patients with irradiated injuries may be carriers of M2b monocytes.
Pregnane X receptor dependent TLR4 expression regulates innate immune response to intracellular bacteria (117.25)
Authors: Qiu Zhijuan , Schouten Jonathan , Mukherjee Subhajit , Acharya Nandini , Maher Leigh , Salazar Juan , Cervantes Jorge , Mani Sridhar , Khanna Kamal
Published: January 2023

The Pregnane X receptor (PXR) is a member of the nuclear receptor family, which is highly expressed in liver as well as in intestine and regulates the transcription of genes involved in the metabolism, transport and elimination of xenobiotics. Recently, the role of PXR and xenobiotic metabolism in regulating inflammation has become evident. Here we demonstrate that in addition to its role in xenosensing and drug metabolism, PXR signaling is also essential for mediating innate antimicrobial immune responses. Mice lacking PXR were highly susceptible to the intracellular bacteria Listeria Monocytogenes (LM). Bone marrow derived macrophages (BMDMs) from PXR-/- mice were proficient in internalizing bacteria, but incapable of clearing LM. Surprisingly, the over activation of Toll-like receptor 4 (TLR4) signaling in the absence of PXR was the singular determinant of susceptibility in PXR-/- mice since mice that were deficient in both PXR and TLR4 were efficient in clearing LM. Furthermore, administration of TLR4 specific inhibitors rescued the ability of PXR-/- mice and BMDMs to clear bacteria. Our results demonstrate an unexpected role of PXR in controlling innate immunity by negatively regulating TLR4 gene expression. In addition, our study has uncovered a remarkable impact of TLR4 signaling in driving the quality of antimicrobial immune response to gram-positive bacteria.
The defensive role of interferon-γ-producing immature myeloid cells in severe invasive group A Streptococcus infections (118.7)
Authors: Matsumura Takayuki , Ikebe Tadayoshi , Ohnishi Makoto , Watanabe Haruo , Kobayashi Kazuo , Ato Manabu
Published: January 2023

Streptococcus pyogenes (group A Streptococcus; GAS) is one of the most versatile bacteria among human pathogens. Non-invasive GAS infections can cause common diseases, such as pharyngitis and impetigo. Whereas, severe invasive GAS infections can lead to rapid progressive and life-threatening manifestations, including sepsis, necrotizing fasciitis, and streptococcal toxic shock syndrome (STSS). Although GAS-derived superantigens activate T cells to release excessive amounts of cytokines which are considered to participate in the pathogenesis of STSS following severe invasive GAS infections, it remains unclear which factors are protective against such infections and which cell population is the source of them. Here we show that mice infected with severe invasive GAS isolates, but not with non-invasive GAS isolates, exhibit high concentrations of plasma interferon (IFN)-γ during the early stage of infection. IFN-γ is necessary to protect mice, and is produced by a novel population of granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent immature myeloid cells with ring-shaped nuclei (IFN-γ-producing immature myeloid cells; γIMCs). These γIMCs express monocyte and granulocyte markers, and also produce nitric oxide. The adoptive transfer of γIMCs ameliorates infection in wild-type and IFN-γ-deficient mice. Our results indicate that γIMCs have a defensive role during the early stage of severe invasive GAS infections.
Glucocorticoid induced leucine zipper mimic inhibits experimental autoimmune encephalomyelitis (119.7)
Authors: Srinivasan Mythily , Srihari Janardhanam
Published: January 2023

Multiple sclerosis (MS) is a relapsing remitting neurological disease mediated by inflammatory cell infiltration in the brain and spinal cord followed by demyelination. The profound anti-inflammatory activity and the ability promote peripheral deletion of activated lymphocytes make glucocorticoids (GCs) the preferred agents in the management of relapse. The therapeutic efficacy of GCs is largely attributed to the inhibition of nuclear factor-κB (NF-κB), the master regulator of inflammatory responses. Peptides from different subunits of NF-κB complex have been shown to suppress inflammation in experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Here, we report a novel strategy to selectively target activated NF-κB and regulate inflammation. Glucocorticoid induced leucine zipper (GILZ) is a recently described molecule transcriptionally upregulated by GCs. GILZ binds to and inhibits the nuclear translocation of NF-κB thereby suppressing transactivation of inflammatory mediators. This suggests that the anti-inflammatory activity of GCs can be attributed to the inducible upregulation of GILZ. Rationally designed GILZ-mimic protects mice against relapsing remitting EAE. Mechanistically treatment with GILZ-mimic suppresses nuclear translocation of p65, inhibits Th1 specific transcription factor T-bet and upregulates Th2 transcription factor GATA-3. In conclusion, our results suggest that the GILZ: p65 interaction is a potential target for multiple sclerosis.
Effects of low doses of allergen administration and/or probiotics supplementation on cow’s milk protein allergy in a mouse model. (120.10)
Authors: Thang Cin , Boye Joyce , Zhao Xin
Published: January 2023

Cow’s milk allergy (CMA) is one of the most common food allergies among infants and children of developed countries. Accumulating evidence indicates that probiotic supplementation and oral tolerance (OT) induction have food allergy reducing properties. However, there is no report regarding the effects of small doses of allergen and/or multi-strains probiotics (VSL#3) supplementation on CMA. In this study, we examined the effects of OT induction by low doses of allergen and/or VSL#3 supplementation on CMA in a mouse model. Mice were intraperitoneally sensitized and then orally challenged with β-lactoglobulin (BLG), one of the major milk proteins. Allergic responses, including hypersensitivity scores, sera immunoglobulins, fecal IgA and cytokines from spleen lysates, were monitored. Compared to BLG-sensitized control mice, mice supplemented with VSL#3 and/or administered with low doses of allergen showed significantly lowered hypersensitivity scores and reduced BLG-specific serum IgE levels. Allergy reducing effects of VSL#3 supplementation was associated with significantly higher levels of fecal IgA while suppression of both Th-1 and Th-2 responses were observed in OT-induced mice. However, mice that received both low doses of allergen and VSL#3 exhibited allergen-specific protection at the systemic level and the potential to protect non-specific challenges at the mucosal level through increased total intestinal IgA.