bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of IL-4-induced insulin receptor substrate-2 activation in allergic inflammation (P6294)
Authors: Gowda Nagaraj , Fang Jessie , Heller Nicola
Published: January 2023

Allergic asthma is a chronic airway disease driven by Th2 cytokines, IL-4, -5 and -13. IL-4 receptors activate IRS-2/STAT6 signaling to induce differentiation of M2 macrophages, cells associated with allergic lung inflammation in mice and humans. IL-4, but not IL-13, strongly induced tyrosine phosphorylation (pY) of IRS-2 and M2 gene expression. Understanding regulation of IRS-2 activation is essential for designing novel therapeutics. We hypothesized that both induction of SOCS proteins and serine phosphorylation (pS) of IRS-2 downregulates IRS-2 signaling. To test this, we stimulated both human monocytes (U937 cells) and mouse primary macrophages with IL-4 and observed robust pY-IRS2 that peaked at 60 min and returned to baseline by 150 min. Kinetics of induction of mRNA for SOCS1, SOCS3 and CIS by IL-4 matched the decrease in pY-IRS-2. Using a siRNA knockdown approach, we found that SOCS1 is responsible for downregulating pY-IRS2 but not CIS and SOCS3. SOCS proteins bind to ubiquitin ligase complexes and target proteins for proteasomal degradation. Proteasome inhibitor-treated, IL-4-stimulated U937 cells showed extended pY-IRS2 signaling. In U937 cells, IL-4-induced pS-IRS-2 had an opposite pattern to pY-IRS-2 and when serine phosphatases were inhibited, pY-IRS-2 dramatically decreased. Using serine kinase inhibitors, we found that inhibition of mTORC1 prolonged pY-IRS-2 signaling in BMM and U937 cells, thus uncovering novel, targetable regulators of allergic inflammation.
Engineering DNA nanoparticles as immunomodulatory reagents (P1076)
Authors: Li Lingqian , Huang Lei , Lemos Henrique , Chandler Phillip , Pacholczyk Gabriela , Baban Babak , Mellor Andrew
Published: January 2023

Cytoplasmic DNA sensing triggers anti-microbial immunity by inducing interferon type I (IFNαβ) via STING-TBK1 activation but sustained STING activation incites autoimmunity. Systemic DNA nanoparticle (DNP) treatment triggered rapid and potent tolerogenic responses in mice via selective activation of the STING-IFNαβ pathway in myeloid dendritic cells (DCs) to induce indoleamine 2, 3 dioxygenase (IDO) activity. DNPs associated rapidly with splenic marginal zone (MZ) macrophages (MΦs) and some DCs, and IFNαβ and IL-1β were induced soon after DNP injection. IFNβ1 was primarily induced in myeloid (CD11b+) DCs and this response was STING dependent, while IL-1β was induced primarily in granulocytes and was STING independent. IFNαβ-mediated IDO induction caused DCs and regulatory T cells (Tregs) to acquire potent tolerogenic phenotypes. Physiologic depletion of (CD11c+) DCs and STING ablation abolished IDO induction and tolerogenic responses to DNPs, but MZ (CD169+) MΦ depletion did not. Moreover, systemic treatment with the cyclic dinucleotide (c-diGMP) to activate STING directly induced IDO and suppressed antigen-specific Th1 responses. Thus myeloid DCs sense circulating cargo DNA and c-diGMP to induce selective IFNαβ release that promotes dominant tolerogenic responses via IDO. Targeting this distinctive tolerogenic pathway may be an effective strategy to suppress hyper-immune syndromes, including autoimmune diseases incited by excessive IFNαβ release.
Trp53 inactivation in lymphoid-like stroma enhances their survival and immunoregulatory function (P1092)
Authors: Guo Gang , Marrero Luis , Del Valle Luis , Ochoa Augusto , Cui Yan
Published: January 2023

The tumor suppressor p53, a crucial gene in controlling cell cycle and apoptosis, is inactivated via somatic mutations in inflamed tissues, such as synovial fibroblasts of rheumatoid arthritis and cancer infiltrating fibroblasts. Recent studies by our laboratory and others suggest that p53 inactivation in immune cells promotes a pro-inflammatory host microenvironment - elevated serum inflammatory cytokines/chemokines, and enhanced Th17 cells, and augmented differentiation of myeloid cells, including myeloid derived suppressors (MDSCs). Recently, compelling evidence suggests that stroma of the secondary lymphoid organs (SLO) also plays a vital role in immune regulation. We, thus, examined the potential immunomodulatory function of p53null stroma for host immune microenvironment. While the frequency of CD45- stroma in the SP and BM of p53null mice did not differ from that of WT mice, p53null, but not WT, stroma was readily expanded in culture, as well as in the spleen of B16F1tumor bearing p53nullmice. The stromal cells were CD106hiCD54+GP38+Sca-1lo/- fibroblastic reticular cell (FRC)-like that expressed high levels of proinflammatory cytokine/chemokine and immunosuppressive mediators. They enhanced the survival and differentiation of MDSCs and likely regulate the function of other immune cells. Together, our results suggest that p53null stroma is highly immunosuppressive, which modulates host immune-microenvironment via cytokine/chemokine and stroma-immune cell interaction.
Langerhans cell and CD103+ dermal dendritic cell promote Tfh in the steady-state (P1094)
Authors: Igyarto Botond , Jarrett Elizabeth , Zurawski Sandra , Zurawski Gerard , Kaplan Daniel
Published: January 2023

Directing antigen to dendritic cells via antibody/antigen conjugates that target antigen-uptake receptors is a highly efficient vaccination technique and also allows for in vivo examination of the effect of antigen presentation by particular DC subsets. Classic experiments determined that antigen presentation in the absence of DC activation led to deletional tolerance. To study the effects of antigen presentation isolated to LC or CD103+ dDC in the steady-state, we performed a series of experiments in which either 1) transgenic mice that express human Langerin only in LC were immunized with anti-human Langerin/antigen complexes or 2) mice lacking LC were immunized with anti-mouse Langerin/antigen complexes. In both cases the DC remained unactivated and antigen was exclusively found on either LC or CD103+ dDC, respectively. Both DC subsets, were able to promote proliferation of MHC-II tetramer binding endogenous CD4 T cells that peaked at day 7 and slowly declined over time without evidence of deletion. Both subsets induced strong expansion of a population of antigen-specific CXCR5+, PD-1+ CD4 T cells that expressed IFNγ, IL-21 and IL-4 which is most consistent with follicular helper T cells (Tfh). In addition, immunization led to the formation of germinal centers in regional LN and was accompanied with a robust humoral response. Thus, presentation in the steady-state by LC and CD103+ dDC promotes Tfh and humoral responses but not deletional tolerance.
Myeloid-derived suppressor cells ameliorate the pathogenesis of autoimmune arthritis (P1058)
Authors: Crook Kristen , Weeks Michael , Rampersad Rishi , Baldi Robert , Liu Peng
Published: January 2023

Myeloid-derived suppressor cells (MDSCs) are subtypes of monocytes and granulocytes that suppress T cell activity in diseases like cancer and multiple sclerosis. Our previous studies suggested a subset of monocytes may act as MDSCs in autoimmune arthritis. Using the collagen-induced arthritis (CIA) model we identified an increase in the Ly6Chigh subset of monocytes in the periphery of WT mice with CIA compared to naïve mice. This subset was absent from the periphery of CCR2-deficient mice and these mice developed more severe CIA disease. Ly6Chigh monocytes isolated from immunized mice inhibited collagen-activated CD4+T cell proliferation as well as reduced the overall T cell numbers. In the presence of Ly6Chigh monocytes we observed high levels of nitric oxide in the supernatants and an increase in Annexin V positive CD4+T cells. To examine the biological role of these monocytes, we performed a cell-based treatment assay. Compared to control mice, in vivo transfer of Ly6Chigh monocytes to collagen-immunized mice resulted in delayed onset of disease, reduced joint swelling, decreased IL-6, IL-1β, and IFNγ expression in the joints and decreased IL-6 and IL-17 levels in the serum. In summary, Ly6Chigh monocytes function as MDSCs to inhibit CD4+T cell proliferation and survival. In vivo transfer of Ly6Chigh monocytes ameliorated autoimmune arthritis in the CIA model suggesting these cells could potentially be used in cell-based therapies for treatment of rheumatoid arthritis.
Cytokine profiles in patients with Staphylococcus aureus skin infections (P3136)
Authors: Chen Luqiu , Bartman Caroline , Chong Anita , Daum Robert , Alegre Maria-Luisa
Published: January 2023

Methicillin-resistant Staphylococcus aureus (MRSA) causes skin infections in epidemic proportions among otherwise healthy individuals. Previous evidence shows that Th17 cells may be crucial to prevent localized S. aureus infections, as they occur in patients deficient in STAT3 or mice lacking IL-17A and F. In this study, we hypothesized that patients presenting to the emergency room (ER) with skin infections but no known genetic deficiency may have a reduction in Th17 differentiation, either globally or specifically to S. aureus antigens because of inhibitory factors produced by MRSA strains. Sera from 72 patients and 143 controls were collected and cytokines were measured by MultiPlex beads assay. It showed elevated cytokines in patients with skin abscesses on day 0. PBMCs from the same groups were stimulated with MRSA lysates, or anti-CD3, and supernatants were collected at 24h for detection of cytokines by ELISA. The production of IFNγ and IL-17 in response to anti-CD3 was similar in patients and controls, but MRSA stimulation triggered greater secretion of IL-6 and IFNγ in select groups of patients than in controls. These data suggest that our skin infected patients do not have a global defect in Th17 differentiation but may have a specific increase in IFNγ production in response to S. aureus antigens. Future experiments in mice will investigate whether this response may be detrimental to prevent S. aureus infections.
Environmental perturbation and behavioral fatigue in healthy and virus-infected mice (P3112)
Authors: Trammell Rita , Toth Linda
Published: January 2023

Introduction: High antibody titers to Epstein-Barr virus occur in some patients with chronic fatigue syndrome. We hypothesized that inflammatory immune responses to reactivation of latent virus may trigger or exacerbate fatigue particularly under stressful conditions. To test this hypothesis, we exposed mice latently infected with murine gammaherpesvirus (MuGHV) and uninfected mice to environmental stressors or basal non-stressful conditions and measured the impact of these challenges on indices of behavioral fatigue and markers of inflammation. Results: Exposure of mice to environmental stressors during the normal somnolent phase was associated with behavioral signs consistent with fatigue during the subsequent active phase. These signs of behavioral fatigue develop despite an intervening rest period and were only marginally affected by infection status. Under basal non-stressful conditions, there was increased inflammation in lung of infected versus uninfected mice with no significant differences observed in serum. In contrast, exposure to stress for 6 h was associated with inflammatory responses in both serum and lung regardless of infection status. Conclusion: Our study has shown that exposure of mice to environmental stressors is associated with signs of behavioral fatigue and systemic inflammation in a manner that is largely independent of latent viral infection with MuGHV.
Chemically induced intestinal inflammation was suppressed in progeny of Trichinella spiralis infected mice. (P3118)
Authors: Cho Min Kyoung , Park Mi Kyung , Kang Shin Ae , Yu Hak Sun
Published: January 2023

Recently, there are many reports that worm infections during pregnancy have important immunological effects on the progeny. In previous study, we confirmed that the Trichinella spiralis infection could inhibit chemically induced intestinal inflammation. In this study, we investigated if the immune regulation in T. spiralis infected mice could be persisted to their progeny. We obtained the progeny from T. spiralis infected female mice or no infected mice and induced intestinal inflammation by dextran sulfate sodium (DSS). As the results, loss of body weight and increased disease activity index (DAI) value were caused by DSS, but these responses were significantly suppressed in the progeny of T. spiralis infected mice than those of the progeny of uninfected mice. Moreover, IL-1β, TNF-α, IL-6 and IL-17 cytokines level was decreased in the progeny of T. spiralis infected mice. Additionally, this down-regulation of intestinal inflammation was observed in the breast milk-fed progeny from T. spiralis infected mice, but is not generated from infected mice. In the progeny fed breast milk of T. spiralis infected mice, DAI value, degree of weight loss and the level of inflammatory cytokines were significantly lower than those in the progeny fed breast milk of no infected mice. In order to find T. spiralis molecules that could be transferred to the progeny, we performed western blot. In the result, we confirmed three T. spiralis molecules in the breast milk of T. spiralis infected mice.
Bystander CD8 T cells produce granzyme B and promote exacerbated disease during Leishmania major infection (P3079)
Authors: Crosby Erika , Wherry E. , Scott Phillip
Published: January 2023

CD8 T cells are recruited to inflamed tissue independent of their antigen specificity, but whether CD8 T cells with irrelevant specificities contribute to the immune response is unclear. We found that Lymphocytic choriomeningitis virus (LCMV)-specific CD8 T cells from a previous LCMV infection were recruited in large numbers to leishmanial lesions in mice infected with L. major. Importantly, LCMV immune mice developed larger leishmanial lesions than naïve mice, associated with increased CD8 T cell, neutrophil and monocyte infiltration into the lesions. The recruited CD8 T cells produced granzyme B and increased levels of circulating granzyme B were detected in the serum of LCMV immune mice that had been challenged with L. major, suggesting that granzyme B may be promoting the increased inflammation. This prompted us to determine what cytokines might stimulate the production of granzyme B from previously activated CD8 T cells in the absence of T cell receptor signaling. We found that IL-15 induced granzyme B, but not IFN-γ, expression. In contrast, IL-12 + IL-18 induced IFN-γ but little granzyme B expression. Thus, our findings demonstrate that bystander CD8 T cells can influence the outcome of subsequent infections, and that the cytokine milieu may dictate how the bystander memory CD8 T cells respond within inflamed sites.
Lung infiltrates containing myeloid-derived suppressor cells impair protective immunity in pneumonic tularemia caused by Francisella tularemia (P3082)
Authors: Periasamy Sivakumar , Sellati Timothy , Harton Jonathan
Published: January 2023

Inhalation of low (~10) numbers of Francisella tularensis, a biothreat category ‘A’ agent, causes acute pneumonia and death; two key features of pneumonic tularemia emphasizing deficiency in the host response to pathogen. We previously reported that early establishment of an immunosuppressive milieu in the lungs favored unfettered and exponential growth of F. tularensis in mice, suggesting a role for immunosuppressive cells in the pathogenesis of tularemia. A comprehensive multicolor flow cytometer analysis for immune cells in the lungs and spleen was performed to address this issue. F. tularensis-infected mice had a high frequency of infiltrating myeloid (CD11b+) cells, but no change in the frequencies of B220+, CD4+, or CD8+ lymphoid cells in lungs and spleen. A majority of the infiltrating myeloid cells were CD11b+CD11c-Ly6G+Ly6C+, a phenotype consistent with immature or myeloid-derived suppressor cells (MDSC). The frequencies of neutrophils (Gr-1+F4/80-) and macrophages (Gr-1-F4/80+) were also increased. Consistent with myeloid cell recruitment, levels of mouse KC, MCP-1 and IL-17A were increased in the lungs. Despite a rapid accumulation of myeloid cells in the lungs, these cells fail to control bacterial replication. Our results suggest that a significant portion of the CD11b+CD11c-Ly6G+Ly6C+ infiltrating cells are likely MDSC, which impair protective immune responses and cause necrotizing inflammation in the lungs and death of F. tularensis-infected mice.
Liver induced immunosuppression by Salmonella-specific CD4 T cells during persistent infection (P3097)
Authors: Kurtz Jonathan , McLachlan James
Published: January 2023

What role microenvironments play in dictating immune outcomes during infection is largely unknown. Our lab uniquely combines a murine model of persistent S. typhimurium infection with recombineered Salmonella strains and MHC-II tetramers to visualize endogenous Salmonella-specific CD4 T cell responses during infection in various organs. We hypothesize that an immunological stalemate between bacterial persistence and the host immune response is determined by anatomical location, which dictates T cell function and infection outcome. We show Salmonella-specific CD4 T cells adoptively transferred from lymphoid organs protect mice from subsequent challenge, while CD4 T cells transferred from infected livers increase susceptibility. Furthermore, lymphoid Salmonella-specific Th1-cells produce higher levels of interferon-γ, while FoxP3- liver CD4 T cells produce larger amounts of interleukin-10, with a decreased proliferative capacity. Additionally, we show lymphoid Th1 cells are potent activators of iNOS and reduce macrophage bacterial loads, while liver-derived Tr1-like cells fail to control bacterial replication, through attenuated iNOS activation. Whether these immunosuppressive Tr1-like cells are induced in the liver, or phenotypically switch upon entering the organ, remains under investigation. These results demonstrate that during persistent Salmonella infection different immunological responses occur at different anatomical sites, which may dictate the outcome of infection.
CD4+CD25+ T regulatory cells dampen inflammatory responses in murine mycoplasma pneumonia and are associated with promotion of IL-17 AND IFN-γ production, but not IL-10 or TGF-β. (P3055)
Authors: Simecka Jerry , Odeh Adam
Published: January 2023

Mycoplasmas cause respiratory diseases characterized by persistent infection and chronic airway inflammation. Mycoplasma lung disease is immunopathologic, with Th cells determining disease severity and resistance to infection. Th2 cell responses promote immunopathology; Th1 cells confer resistance to infection. However, little is known about the role of Treg cells in mycoplasma respiratory diseases. We hypothesized Treg cells control the severity of the inflammatory lesions and may also promote persistence of infection, as found in other diseases. To examine this, mice, given anti-CD25 antibody to deplete Treg cells, had increased disease severity due to Mycoplasma pulmonis infection. There was however no affect on mycoplasma numbers recovered from the lungs. Treg cells promote the secretion of IFN-γ and IL-17 mycoplasma-specific CD4+ T cell responses in vitro. We were unable to detect significant changes in IL-10 and TGF-β production. In addition, Treg cell depletion increased Th2 (IL-13) responses. Populations of Treg cells from lymph nodes had increased expression of IFN-γ or IL-17 after infection. Thus, Treg cells play an important role in controlling damaging immune responses in mycoplasma respiratory infection but do not influence the level of mycoplasma infection. It appears that Treg cells dampen mycoplama inflammatory disease through a novel mechanism mediated production of IFN-γ and IL-17.
The Eph/Ephrin molecules are modulated in organs of parasitized red blood cells sequestration and may contribute to disease pathogenesis in rodent malaria. (P3059)
Authors: Mimche Patrice , Brady Lauren , Cox Daniel , Lamb Tracey
Published: January 2023

Eph receptors and their Ephrin ligands are the largest family of receptor tyrosine kinases. Beyond their role in cell trafficking/adhesion and cancer, nothing is known about their activation during malaria infection. We explored the regulation of Eph/Ephrin in malaria using the rodent malaria model P. berghei ANKA, a parasite that causes experimental cerebral malaria in C57BL/6 mice. Infection with P. berghei ANKA leads to modulation of transcription for some members of the Eph/Ephrin in C57BL/6 mouse brain and spleen. This may be driven by inflammatory cytokines upregulated in these organs in response to sequestration of iRBCs because TNFR2-/- mice which are known to be resistant from death by ECM did not display any upregulation of Eph/Ephrin. To further examine their modulation, subsets of antigen presenting cells and T cells were FACS sorted from splenocytes at different time points post infection. Transcription was found to be modulated between days 2 and 3, particularly for EphB2 on CD11b+ macrophage/neutrophil subset. To support the modulation of transcription in the brain during ECM, mouse primary brain microvascular endothelial cells were isolated from naïve mice and stimulated with inflammatory cytokines and lysates of iRBCs. We observed an increase in the transcription of EphB4 and EphB6 in response to inflammatory cytokine signaling and an increase in the protein expression of EphB4 in response to iRBC lysates. The implications of these results will be discussed.
Acute inflammatory asthma protects influenza infected mice from bacterial superinfection (P3065)
Authors: Samarasinghe Amali , LeMessurier Kim , Iverson Amy , Woolard Stacie , McCullers Jonathan
Published: January 2023

The Centers of Disease Control identified asthmatics to suffer more severe morbidity from influenza during the 2009 pandemic. However, our data indicate that pre-existing allergic inflammation during viral infection is protective against influenza associated disease in mice. Since bacterial super-infections during influenza increase morbidity and mortality, we hypothesized that acute inflammatory asthma promotes bacterial colonization of the lungs after influenza infection thereby leading to a more severe outcome. We created a triple variant model system by infecting Aspergillus fumigatus sensitized and challenged mice with 1000 TCID50/ml of A/CA/4/2009 virus a week after the last fungal challenge. Mice were co-infected with 600 cfu of Streptococcus pneumoniae strain A66.1 a week later and monitored for six days. Mice with allergic asthma lost less weight after co-infection, had less airway resistance after methacholine challenge, and had at least a 50% reduction in mortality compared to non-asthmatic controls. Fewer inflammatory cells were recruited into the airways of allergic mice after co-infection. Allergic asthma delayed S. pneumoniae colonization in the airways and lungs but not blood compared to co-infection alone. Mice in the asthma and co-infection model had less normal flora outgrowth in the airways, lungs, and blood compared to those in the co-infection model. Allergic asthma protects against, or delays, bacterial super-infection after influenza challenge.
Contribution of differentially expressed micro-RNA to functional differences in human blood monocyte subpopulations (P1194)
Authors: Dang Minh , Wong Wing Cheong , Ong Siew Min , Chen Jinmiao , Poidinger Michael , Zolezzi Francesca , Wong Siew Cheng
Published: January 2023

Human blood monocytes can be broadly divided into two subpopulations, CD14+CD16- (CD16-) and CD14+/loCD16+ (CD16+). Although numerous phenotypic and functional differences between the two subsets have already been described, much remains to be learned about the roles contributed by each subset during homeostasis or disease conditions. In this study, we characterize these two monocyte subsets using microRNAs (miRNAs) and messenger RNA (mRNA) array approach. We identified 66 miRNAs to be differentially expressed (DE) between CD16+ and CD16- monocytes. By integrating mRNA expression data with miRNA expression data via negative correlation, we were able to demonstrate that several of these DE miRNAs regulate mRNAs that play important roles in inflammatory response, cell death, cellular development, cellular movement and cell-to-cell signaling and interaction. We validated experimentally the association of miR-432 (expressed higher in CD16+ monocytes) with apoptosis and this might explain why CD16+ monocytes are more susceptible to spontaneous apoptosis seen in our previous study. The association of miR-19a with cell motility was also validated in our experiments and this might explain why CD16+ monocytes (with higher expression of miR-19a) move slower in comparison with CD16- monocytes. Together, our data showed that differentially expressed miRNA contribute to the functional differences in human blood monocyte subpopulations.
TRAF3 restrains regulatory T cell development by modulating IL-2 signaling (P1219)
Authors: Yi Zuoan , Lin Waiwai , Stunz Laura , Bishop Gail
Published: January 2023

The signaling protein TNF receptor associated factor 3 (TRAF3) has multiple diverse effects on signaling transduction. We recently produced a new strain of T cell specific TRAF3 deficient mice (CD4CreTRAF3flox/flox (T-T3-/-)), and found that the frequency and number of Foxp3+ regulatory T cells (Treg) are increased 2-3 times compared with litter mate control mice (LMC), though total CD4+ and CD8+ T cell numbers do not change. These Treg are Helios+ and the increase is cell intrinsic. Interestingly, thymic Treg emerge as early as day 1 after birth in T-T3-/- mice, compared to day 3 after birth in LMC. Notably, the percentage of Treg precursor cells is unchanged. However, following IL-2 stimulation, TRAF3-deficient Treg precursor cells convert into Foxp3+ Treg more efficiently than those of LMC, though expression levels of IL-2 receptor components are comparable. We further found that phosphorylation of STAT5 is remarkably enhanced in TRAF3-/- Treg precursors in response to IL-2 stimulation. We also noticed that T-T3-/- conventional CD4 T cells exhibit robustly enhanced p-STAT5, but not p-ERK and p-AKT, when stimulated with IL-2. Additionally, TRAF3 deficiency affects the plasma membrane localization of T Cell Protein Tyrosine Phosphatase (TCPTP), an enzyme that plays a negative role in IL-2 signaling. Taken together, our data show for the first time that TRAF3 is an important regulator of IL-2 signaling and affects Treg development.
Twist1 impairs inflammatory cytokine production in T helper 17 cells (P1227)
Authors: Pham Duy , Walline Crystal , Blum Janice , Kaplan Mark
Published: January 2023

Subsets of T helper cells are specialized for the production of specific cytokines, and this specificity is controlled by the function of transcription factors in each subset. Twist1, a basic helix-loop-helix (bHLH) transcription factor, is induced by STAT3-activating cytokines and inhibited by TGF-β in Th17 cells. Ectopic Twist1 expression in Th17 cells reduced IL-17A and IL-17F production while Twist1-deficient Th17 cells produced more IL-17A, IL-17F, and other inflammatory cytokines than wild type cells. In a myelin oligodendrocyte glycoprotein (MOGp35-55)-induced EAE disease model, Twist1-deficient mice manifest earlier onset of paralysis compared to wild type mice. Mechanistically, Twist1 controls cytokine production in Th17 cells by directly repressing Il6ra expression, limiting activation of the IL-6-STAT3 signaling pathway. Thus, Twist1 inhibits inflammatory cytokine production in Th17 cells, suggesting a central role for Twist1 in the regulatory network controlling autoimmune disease.
Regulated cytoskeletal control of ICAM-1 mobility on the dendritic cell plasma membrane enhances T cell activation (P1205)
Authors: Comrie William , Boyle Sarah , Li Shuixing , Burkhardt Janis
Published: January 2023

T cell activation involves actin-dependent assembly and centripetal movement of signaling microclusters at the Immune Synapse (IS). Model stimulatory surfaces that limit microcluster dynamics enhance TCR signaling, but the possibility that antigen presenting cells can modulate T cell signaling by regulating ligand distribution is largely unexplored. Dendritic cells (DCs) form a multi-focal IS rather than the concentric pattern seen with freely mobile ligands, suggesting that DCs can control receptor-ligand localization. We have measured the lateral mobility of T cell ligands on the DC membrane, and find that ICAM-1, the key ligand for the integrin LFA-1, is selectively constrained via interactions with the actin cytoskeleton. ICAM-1 mobility decreases further upon LPS-induced DC maturation, concomitant with upregulation moesin and α-actinin 1, two actin-binding proteins that bind the cytoplasmic tail of ICAM-1. ICAM-1 co-localizes with moesin at punctate sites on the DC surface. Mutation of the ICAM-1 cytoplasmic tail or suppression of moesin and/or α-actinin liberates ICAM-1 lateral mobility. Importantly, liberating ICAM-1 from cytoskeletal constraints decreases the ability of DCs to induce the proliferation of naïve CD4 T cells. Taken together, our data show that the DC actin cytoskeleton actively controls ligand mobility and thus enhances T cell activation, and raise the possibility that DCs modulate T cell signaling by actively regulating molecular dynamics at the IS.
FoxP3+ Tregs cells sensitivity to low doses of IL-2 can be rapidly and easily monitored by p-STAT-5 detection in ex-vivo whole blood samples. (P1211)
Authors: Malergue Fabrice , Mallet Valérie , Scifo Caroline , Van Agthoven Andreas , Miloud Tewfik , Montero-Julian Felix A
Published: January 2023

Introduction: Phospho-epitopes are difficult to detect by flow cytometry, currently requiring dedicated techniques and buffers, a total work time of 2 to 4 hours, 37°C and ice incubations, and 4 to 8 wash steps. The reference methods also uses methanol which is toxic for the user and harmful for the other cell markers, what compromises some multi-parametric studies. The FoxP3 marker is incompatible with “harsh” P-epitopes detection methods, rendering even more challenging the studies of Tregs functions. Methods: Here, we used a new commercially available kit; named PerFix EXPOSE (Phospho Epitopes Exposure Kit; for Research Use Only) based on multiple innovations in the permeabilization, staining, and wash steps. It is devoid of methanol and supports staining of all common P-epitopes with one single procedure of about 1 hour. Since many extra-cellular markers can be combined together with the anti-phospho markers, we evaluated also various FoxP3 clones and conjugates, and tried some procedure optimisations. The best conditions were then used to analyse the ex-vivo functionality of FoxP3+ cells: The IL-2 signalling pathway especially. Results: PerFix EXPOSE allows for the simultaneous detection of some antigens that are otherwise incompatible, such as FOXP3 and p-STATs. An IL-2 dose-response curve could be easily generated directly from fresh whole blood that confirmed on normal donors a 10 to 100-fold difference in sensitivity to IL-2 between Tregs and other T cells.
Enhanced susceptibility of RIPK3 deficient mice to influenza A virus infection (P6196)
Authors: Sharma Shalini , Sturm Oliver , Green Douglas , Thomas Paul
Published: January 2023

Viral infection can activate cytokine expression and trigger cell death. Programmed necrosis, like apoptosis, eliminates pathogen-infected cells as a component of host defense while also regulating the function and survival of professional immune cells, including T cells. Receptor-interacting protein kinase (RIPK3) has been shown to mediate programmed necrosis induced by certain viral infections. Deficiency of RIPK3 results in compromised elimination of pathogen-infected cells and increased viral burdens. We present evidence for this effect in mice acutely infected with influenza A virus (IAV). RIPK3-deficient (RKO) mice have significantly increased body weight loss after both mild IAV x31 infection and severe IAV PR8 infections, with decreased survival in the latter case and significantly increased viral loads in the lungs compared to littermate controls. However these effects were primarily observed after day 7 post infection, indicating that regulation of death in the virally-targeted epithelial cell was not the cause of the observed pathogenic phenotype. Indeed, ex-vivo tetramer and ICCS assays showed significantly diminished virus-specific CD8 T cell responses with a reduced frequency of bifunctional IFNγ+ and TNFα+ cells and a lower MFI of IFNγ production by RKO CD8 T cells suggesting that the increased viral loads and weight loss in RKO animals may be a result of reduced T cell activity. Current studies are addressing the role of RIPK3 in regulating T cell function.
Natural Killer cells in defense against parainfluenza virus (P6178)
Authors: Hendricks Deborah , Kohlmeier Jacob , Lanier Lewis
Published: January 2023

Respiratory viral infections, including parainfluenza virus (PIV), are a significant cause of illness and death worldwide, especially among young children and the elderly. Understanding the immune response to these viruses is important for reducing pathology and developing vaccines. As a first line of defense, Natural Killer (NK) cells provide critical host protection against viruses. Their presence in the lung, a key site of pathogen entry, suggests an important role for these cells in respiratory viral infection. Sendai virus (SeV), a natural mouse pathogen, is closely related to human PIV, and causes respiratory infection similar to that seen in humans. No studies have directly assessed NK cells in host protection during SeV infection. Our research shows that NK cells are recruited into the lung airways by day 2 post infection (p.i.), and continue to accumulate in the lungs. NK-depleted mice have higher viral titers in the lungs on days 3 and 5 p.i. NK cells upregulate the activating receptor NKG2D during infection, and in the absence of NK cells, a higher percentage of CD8 T cells expresses NKG2D. NKG2D antibody blockade results in higher viral titers on day 4 p.i. Our data show that NK cells play a protective role and are an important cell population during PIV infection. These studies will deepen our understanding of NK cell biology, and may also help in the design of new methods to treat, and possibly prevent, these diseases.
Critical role of PD-L2 in limiting pulmonary immunopathology during RSV infection (P6182)
Authors: McDermott Daniel , Varga Steven
Published: January 2023

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract-associated hospitalizations in children under the age of 2. Acute RSV infection induces both Th1- and Th2-associated responses, with elevated airway hyperactivity being highly correlated with increased Th2-associated responses. However, the mechanism for induction of Th1 vs. Th2-associated responses following RSV infection is currently unknown. The inhibitory ligands PD-L1 and PD-L2 serve a critical role in the induction and regulation of Th1 and Th2-associated responses, respectively, during acute infections as well as asthma. Following acute RSV infection, lung epithelial cells upregulate PD-L1 within 24 hours post-infection (p.i.) with peak expression occurring at day 6 p.i.. Furthermore, both lung and mediastinal lymph node (MedLN) CD11c+ cDCs and pDCs upregulate PD-L1 expression early following RSV infection and retain increased PD-L1 expression throughout the course of infection. In contrast, PD-L2 expression is primarily restricted to CD11c+ cDCs. PD-L2 expression peaks at day 6 p.i. on both lung and MedLN CD11c+ cDCs, correlating with increased airway resistance following acute RSV infection. PD-L2-blockade early during RSV infection results in increased airway hyperactivity, whereas PD-L1-blockade exhibited no significant effect. These data suggest that PD-L2 plays a critical role in the regulation of RSV-induced immunopathology.
TCR avidity for thymic and peripheral self peptide-MHC sets and sustains intrinsic CD4+ T cell sensitivity (P1170)
Authors: Persaud Stephen , Weber K. , Allen Paul
Published: January 2023

TCR interactions with pMHC are crucial to T cell development and function. It is not known how the strength of the positively selecting signal impacts peripheral T cell function. To pursue this issue, we utilized the LLO56 and LLO118 TCR transgenic mice, each of which bears CD4+ T cells that recognize the same epitope from Listeriolysin O. During in vivo responses to L. monocytogenes, LLO118 T cells expand better upon primary challenge, whereas LLO56 prevails upon rechallenge. Surprisingly, naive LLO56 T cells showed stronger IL-2 responses than LLO118 to nonspecific in vitro stimulation, associated with greater ERK activation and higher basal phospho-TCRζ levels. The greater basal TCRζ phosphorylation, along with higher CD5 expression, suggested that LLO56 has a higher avidity for self-pMHC, possibly underlying its higher sensitivity. Indeed, the respective sensitivities of LLO56 and LLO118 cells emerged as they matured into CD4SP thymocytes after positive selection, during which LLO56 thymocytes received a stronger signal from selecting self-pMHC. Peripheral self-pMHC was required to actively maintain mature LLO56 and LLO118 T cell sensitivity, as transfer of these cells into MHC II-/- mice rendered them similarly unresponsive to stimulation. In summary, our data demonstrate unanticipated roles for TCR:self-pMHC avidity in establishing T cell sensitivity centrally, sustaining it in the periphery, and affecting CD4+ T cell responses to pathogens.
DLG1 alternative splice variants: molecular conduits specifying proximal TCR signals to distinct CTL functional responses (P1174)
Authors: Silva Oscar , Crocetti Jillian , Humphries Lisa , Miceli M.
Published: January 2023

CD8+ cytotoxic T lymphocytes (CTLs) are a critical component of the adaptive immune system because they mediate contact-dependent cytolysis and produce proinflammatory cytokines. Although both of these CTL activities require T cell receptor (TCR) activation these functions are not always coordinately invoked, suggesting that signaling pathways downstream of the TCR can be selectively activated to guide specific functional outcomes. Here we investigated the role of Discs large homolog 1 (DLG1) alternative splice variants, DLG1 i1A-i1B and DLG1 i1B, in coupling proximal TCR signals to select downstream pathways to specify cytotoxicity and proinflammatory cytokine transcription in CD8+ T cells. We found that DLG1 i1A-i1B, but not DLG1 i1B selectively directs TCR-induced p38/NFAT-dependent proinflammatory cytokine production by binding Lck, promoting alternative p38 activation and downstream NFAT mediated upregultion of IFNγ and TNFα gene expression. Conversely, DLG1 i1A-i1B mediated alternative p38 activation had no effect on actin polymerization, granzyme B gene expression, granzyme B release, or degranulation. Rather, either DLG1 variant expressed in CD8+ T cells promoted antigen-induced degranulation, suggesting that both DLG1 variants have p38-independent activity guiding granule-dependent cytotoxicity. Thus CD8+ T cells contain two distinct DLG1 scaffolds that act as molecular conduits for proximal TCR signals to allow differential CTL functional responses.
mTOR complex signaling inhibits expression of a specific set of microRNAs during T cell activation that regulate G1 cell cycle progression (P1180)
Authors: Podshivalova Katie , Wang Eileen , Salomon Daniel
Published: January 2023

microRNAs are a class of small non-coding RNAs that post-transcriptionally regulate gene expression. T cell activation results in dramatic changes in the expression of individual microRNAs. Mammalian target of rapamycin (mTOR) is activated promptly after TCR stimulation via the PI3K signaling branch and promotes T cell exit from the quiescent resting state into a highly proliferative activated state. Bringing these two important regulatory pathways together, we show that mTOR signals the down-regulation of several specific microRNAs after activation of T cells. Inhibition of mTOR in human T cells by rapamycin or PP242 results in up-regulation of a unique set of microRNAs. Conversely, the same miRNAs are dramatically reduced with T cell activation. Regulation of these miRNAs by mTOR appears to be T cell specific, as their expression does not change in B cells, fibroblasts or epithelial cells treated with mTOR inhibitors. Mechanistically, we show that mTOR-dependent microRNAs negatively regulate proliferation and induce G1 cell cycle arrest by targeting multiple genes, acting either additively or synergistically with mTOR inhibitors. Our data demonstrate that several microRNAs help maintain the quiescent state of resting T cells, are down-regulated with activation due to negative regulation by mTOR and because these microRNAs contribute to the induction of a cell cycle arrest, they may have potential roles as tumor suppressor genes.
Nck proteins are recruited directly to the BCR and regulate PI3K signaling thereby shaping B cell immune responses (P1154)
Authors: Castello Angelo , Engelke Michael , Tucholski Johannes , Oellerich Thomas , Tafuri Anna , Pawson Tony , Batista Facundo
Published: January 2023

Binding of antigen to the B cell antigen receptor (BCR) triggers the activation of different kinases and the initiation of many signaling pathways required for mounting B-dependent immune responses. Scaffolding proteins, recruited downstream of the BCR, act as signal integrators through the recruitment of multiple signaling effectors. Nck is a family of scaffolding proteins best known for linking phosphotyrosine signals to cytoskeleton regulation but until now uncharacterized in the context of B lymphocytes. In our study, Nck was found to be directly recruited to the Igα tail of the BCR and to be important to regulate immune-receptor signaling. Through the generation of a novel Nck1 and Nck2 B cell conditional KO mouse model, we found these adaptors to be essential for mounting T-independent immune response and differentiate into extrafollicular plasma cells during T-dependent responses. The phenotype reported was shown to be due to defective PI3K signaling and Foxo1 downregulation in response to antigen challenge. Our work therefore provides the first compelling evidence for a critical role of Nck in BCR signaling, which is independent of its cytoskeleton regulation yet essential to mount B cell responses.
Micron-scale exclusion of CD45 enhances activation of TCR zeta associated protein of 70 kDa tyrosine kinase. (P1192)
Authors: Lee Joung-Hyun , Dustin Michael , Kam Lance
Published: January 2023

CD45, a transmembrane protein tyrosine phosphatase, is critical for activation of T cells. CD45 is known to modulate T cell signaling by dephosphorylating the inhibitory Tyr 505 of Lck, but at the same time it has been argued that exclusion of CD45 from the vicinity of the TCR is necessary for activation of T cells. While many data support the notion that CD45 exclusion is necessary for triggering of TCR signaling, there is no evidence that CD45 exclusion is sufficient. In this work, we designed substrates to test if local exclusion of CD45 can trigger TCR without aid of TCR ligation. To test this hypothesis, mouse CD4 T cells were seeded onto surfaces micropatterned with an antibody targeting the extracellular domain of CD45. These surfaces were created by first defining 1- or 2-µm diameter features of PLL-g-PEG onto glass coverslips, followed by adsorption of anti-CD45 to the remaining regions. These substrates depleted CD45 from the PLL-g-PEG patterned regions and enhanced production of pZap70 over levels observed on uniformly coated anti-CD45 surfaces as determined by total internal reflection fluorescence. Surprisingly, the areas of higher pZap70 were not localized in the specific regions of CD45 exclusion, but were concentrated in the center of the interface, approximating early stages of signaling in a T cell immunological synapse. These results demonstrate that local control over CD45 distribution can alter T cell signaling, independent of TCR-MHC recognition.
STIM2 drives calcium oscillations through store-operated calcium entry caused by mild store depletion (P1160)
Authors: Thiel Markus , Penner Reinhold , Lis Annette
Published: January 2023

The main source of calcium in T cells is through a mechanism known as store-operated calcium entry (SOCE). STIM1 and STIM2 serve as ER calcium sensors that, upon store depletion, activate calcium release-activated calcium (CRAC) channels (Orai1-3,CRACM1-3) in the plasma membrane. In the absence of sustained calcium influx through CRAC channels, numerous lymphocyte functions are severely compromised, as demonstrated in patients suffering from SCID. Agonist-induced calcium oscillations in many cell types are triggered by calcium release from intracellular stores and driven by SOCE. However, their relative roles in agonist-mediated calcium oscillations remain ambiguous. We here report that while both STIM1 and STIM2 contribute to store-refilling during calcium oscillations in T cells, RBL and HEK cells, they do so dependent on the level of store depletion. Molecular silencing of STIM2 by siRNA or inhibition by G418 selectively suppresses SOCE and agonist-mediated calcium oscillations at low levels of store depletion, without interfering with STIM1-mediated signals induced by full store depletion. Thus, STIM2 is preferentially activated by low-level physiological agonist concentrations that cause mild reductions in ER calcium levels. We conclude that with increasing agonist concentrations, SOCE is mediated initially by endogenous STIM2 and incrementally by STIM1, enabling differential modulation of calcium entry over a range of stimulus intensities and levels of store depletion.
Sustained Treg expansion by repeated in vivo injection of anti-TNFRSF25 mAb (P4142)
Authors: Zhang Hong , Pahuja Anil , Lundsten Kelly , Li Sky , Lin Xiao , Ransom John , Yang Xi Feng
Published: January 2023

FOXP3+ Tregs play key roles in maintenance of immune tolerance and autoimmune reactions. TNFRSF25 (also known as DR3) is one of the more recently discovered TNFRSF member, which is expressed by mouse Tregs, naïve CD4, CD8, NKT cells and TLR4-induced DC and macrophages. A significant Treg in vivo expansion by TNFRSF25 stimulation has been reported. Here, we show that by single dose i.p. injection of an agonistic mAb, 4C12, Treg expansion starts to be observed on day 3, reached a peak (up to 30-40% of total CD4+ T-cells) on day 5-6, and then returned to baseline (5-7%) by day 10. Upon repeated 3-day interval injections, high level (about 20%) of expanded Tregs can be sustained more than 10 days. As reported by other authors, followed by one dose 4C12 injection, the absolute number of CD8+T cells, B cells, dendritic cells and macrophages was not affected. In the in vitro study, 4C12 antibody did not significantly induce Treg expansion in CD3/CD28/IL-2 culture system. Moreover, we characterized the TNFRSF25 expanded Tregs in vivo, we found that the expression level of CD304, CD152, Helio, GARP, LAP (TGF-b1), FR4, GITR was significantly increased in comparison to the control. These data suggest that multiple injections of 4C12 can long term sustain expanded Tregs at certain level, the biological significance of Treg need to be further studied.