bluuebunny/crossref_metadata_2025 huggingface dataset:
Column-free isolation of untouched mouse plasmacytoid dendritic cells (P4149)
Authors: Tabatabaei-Zavareh Nooshin , Moor Sameet , Thomas Terry , Fairhurst Maureen
Published: January 2023

Plasmacytoid dendritic cells (pDC) are a DC subset distinct from conventional dendritic cells (cDC) in that they are able to produce large amounts of type I interferon (IFN) after challenge with pathogens. Viruses activate pDC mainly through Toll-like receptors (TLR). Unlike cDC, pDC are poor antigen-presenting cells. In line with the functional differences, pDC express lower levels of CD11c and MHCII as compared to cDC. Moreover, a number of markers expressed by pDC are absent on cDC including: PDCA-1, Siglec-H, B220 and Ly-6C. pDC are found in both lymphoid and non-lymphoid organs. Here, we describe a negative selection method to isolate untouched pDC from mouse spleen. This method uses an immunomagnetic, column-free cell separation technology (EasySepTM). Briefly, single cell suspensions of splenocytes are labeled with biotinylated antibodies against non-pDC. Bi-specific antibody complexes against biotin and dextran are used to cross-link non-pDC with dextran-coated magnetic particles. The unwanted cells are then removed using an EasySepTM magnet. The procedure can be automated using RoboSepTM. Starting with 0.4±0.2% CD11c+PDCA-1+ pDC in spleen, purities of 79±9% (n=21) are achieved. The isolated pDC are functional and produce IFN-α in response to TLR9 ligand, ODN-1585 as measured in ELISA. pDC play an important role in anti-viral immunity and autoimmunity, therefore the isolation of untouched pDC is critical in studies examining their role in these immune responses.
Costimulation through GITR increases follicular helper T cell formation and leads to control of a chronic viral infection (P1021)
Authors: Pascutti Maria Fernanda , Brandao Claudia , van Olffen Ronald , Poplonski Tomasz , van Lier Rene , Nolte Martijn
Published: January 2023

We have previously shown that enhanced costimulation through GITR by overexpression of GITRL increases the formation of both effector and regulatory CD4+ T cells. Here we explored whether it could also affect humoral immunity and T cell help to B cells. Although development of mature B cells was not affected in GITRL transgenic (tg) mice, we found that the number of follicular helper T cells (CXCR5+ PD1+ CD4+ T cells, Tfh) was significantly increased, including the absolute number of Tfh-B cell conjugates, as revealed by ImageStream analysis. Tfh from GITRL tg mice had normal expression levels of ICOS, SLAM and CD44 and slightly lower levels of CD62L and CCR7 compared to wild-type (WT) littermates. Interestingly, Tfh from GITRL tg mice produced more IFN-γ and IL-10, which was accompanied by a biased antibody repertoire (decreased IgG3 and increased IgA, IgG2a and IgG2b). Since Tfh have been implicated in the late control of viral replication, we infected WT and GITRL tg mice with LCMV Clone 13. Surprisingly, at day 30 after infection, we could not detect viral genome in spleen and liver from GITRL tg mice, while WT mice were still infected. Also, PD-1 expression was strongly decreased on virus-specific CD8+ T cells, which correlated with faster viral clearance. All in all, these results indicate that GITRL signaling enhances the control of chronic viral infections, by boosting and modulating Tfh cell responses.
Galectin-9 controls the activity of 4-1BB (CD137) (P1009)
Authors: Madireddi Shravan , Eun So-Young , Mehta Amit , Nemcovicova Ivana , Lee Seung-Woo , Niki Toshiro , Hirashima Mitsuomi , Zajonc Dirk , Croft Michael
Published: January 2023

4-1BB is an inducible stimulatory molecule in the TNFR superfamily expressed on activated T cells, dendritic cells, NK cells, and other lymphoid cell types. Although known to bind the TNF family molecule 4-1BBL, we searched for novel binding partners that might modulate or control the activity of 4-1BB. We found that 4-1BB can bind to Galectin-9, a member of the beta-galactoside binding lectins containing homologous carbohydrate recognition domains (CRDs). Surface plasmon resonance analysis for mouse and human molecules demonstrated that the equilibrium binding constant (KD) of this interaction was very strong at ~11 nM and ~85 nM respectively. Functional studies in vitro showed that the stimulatory activity of 4-1BB was dependent on Galectin-9 in that Galectin-9-/- NK cells, CD4 T cells, or CD8 T cells, were largely unresponsive when 4-1BB was ligated but had normal activity when other stimulatory receptors were engaged. Similarly, 4-1BB activity on dendritic cells was impaired when Galectin-9 was absent. Additionally, in EAE and Asthma models, where an agonist of 4-1BB can suppress disease via induction of CD8β+CD11c+ regulatory cells, we found that this protective effect of anti-4-1BB was lost in Galectin-9-/- mice. Thus, our data demonstrate a novel function of Galectin-9 in facilitating signaling through 4-1BB, and suggest that Galectin-9 may allow 4-1BB to oligomerize and/or cluster in a manner that allows efficient engagement of intracellular signaling pathways.
Cell type specific role of B7 for Ag-specific T and B cell activation and differentiation in T-dependent antibody response in vivo (P1017)
Authors: Watanabe Masashi , Hodes Richard
Published: January 2023

B7.1/B7.2 molecules are essential for eliciting immune responses through CD28-mediated T cell activation, as well as for CTLA-4-mediated negative regulation of T cell responses. Although many cell types (DC, MΦ, B and T) express B7 in vivo, the role of B7 on each cell type is not well defined. Utilizing cell type-specific B7cKO mice, cell type specific roles of B7 for T-dependent GC and Ab responses were analyzed. Upon NP-OVA/Alum immunization, NP-specific IgG1 production was profoundly defective in mice lacking B7 expression on DC (DC-B7KO) but was intact in mice lacking B7 on B cells (B-B7KO). Consistent with this, NP-binding GC B cells and Ag-specific CD4+ T cell expansion were not observed in DC-B7KO. Surprisingly, however, GC formation and total numbers of GC B and TFH were undiminished in DC-B7KO; whereas B-B7KO showed reduction of total number of GC B and TFH despite normal number of NP-binding GC B. In addition, NP-specific secondary Ab was absent in DC-B7KO but intact in B-B7KO. These results clearly demonstrate that B7 on DC but not B cells is critical for Ag-specific CD4+ T cell expansion and IgG1 production as well as for secondary Ab responses. In contrast, a distinct pathway dependent upon B7 on B cells but not DC supports apparently Ag-nonspecific GC response. Further studies are ongoing to evaluate cell type-specific roles of B7 for affinity maturation, memory T/B formation, and potentially nonspecific or autoreactive components during T-dependent Ab response.
Discovering the determinants of diversity in the human NK cell repertoire by mass cytometry (P3018)
Authors: Horowitz Amir , Strauss-Albee Dara , Nemat-Gorgani Neda , Dogan Ozge , Dekker Cornelia , Mackey Sally , Swan Gary , Davis Mark , Norman Paul , Guethlein Lisbeth , Parham Peter , Blish Catherine
Published: January 2023

NK cells respond to infected and transformed cells using germline-encoded NK receptors (NKRs). Collectively, these NKRs combine to form distinct repertoires that tune the NK cell response. To provide a framework for understanding how perturbations in expression of NKRs influence disease pathogenesis, we defined the phenotypic heterogeneity of NK cells in 22 healthy individuals (ages 21-62; M=10, F=12), including 5 sets of monozygotic twins. Using mass cytometry, surface expression of 38 markers specific for NKRs and lineage markers to identify B cells, T cells and myeloid cells were examined. KIR and HLA class I genotypes were determined by Luminex, and KIR gene content was further assessed using pyrosequencing. NK cell populations, as defined by inhibitory NKRs, were highly concordant between twins (R2 = 0.90) in comparison to unrelated individuals (R2 = 0.66), indicating that the inhibitory repertoire is highly genetically determined. However, when analyzing all twenty-five NKRs, including activating receptors, the concordance in twins (R2=0.34) and in unrelated individuals (R2=0.11) was greatly decreased, indicating significant environmental influence in determining the overall NK cell repertoire. Overall, these analyses reveal tremendous diversity within the NK repertoire that is both genetically and environmentally determined, and offer the ability to examine the functional capacity of NK cells with far greater resolution.
Loss of CD19 expression alters the CDR-H3 content of marginal zone and follicular B cells, changing the composition of the immunoglobulin antigen receptor repertoire (P3022)
Authors: Khass Mohamed , Freeberg Larry , Zhuang Ynigxin , Carter Robert , Justement Louis , Schroeder Harry
Published: January 2023

Expression of CD19 on B cells modulates B cell receptor signaling threshold. Reduced CD19 expression has been observed in some human patients with common variable immune deficiency (CVID), a disease which is characterized by low production of mature B cells with a paradoxical increase in auto-reactivity. Development of the B cell repertoire in the absence of CD19 expression has not been well characterized. We previously showed that the B cell repertoire is controlled by the sequence characteristics of the complementary determining region heavy chain (CDR-H3). We sought to examine the CDR-H3 repertoire in mice lacking CD19 expression, using our novel bioinformatics system, to test whether its absence can affect the control of B cell repertoire content, yielding altered development and auto reactive antibody production. We analyzed the CDR-H3 based on its length, reading frame usage and average hydrophobicity of its amino acid contents. Follicular B cells lacking CD19 expression were found to express an increase in the use of both highly charged and highly hydrophobic CDR-H3 sequences, sequences that are normally excluded from the follicles. Conversely, when compared to C57BL/6 controls, hydrophobic CDR-H3s were suppressed among marginal zone B cells. These findings indicate a loss of normal repertoire control in the absence of CD19, which may help explain why CD19 deficient patients have altered responses to self and non-self antigens.
The role of endogenous galectin-9 in T cells (P3038)
Authors: Wang Li-Chieh , Li Chi-Shan , Chen Huan-Yuan , Chiang Bor-Luen , Liu Fu-Tong
Published: January 2023

BACKGROUND: Galectins are members of a β-galactoside-binding animal lectin family. Recombinant galectin-9 is a negative regulator of Th1 cells through the Tim3 inhibitory signaling pathway, but the function of endogenous galectin-9 in the immune response is not well studied. Here we examined the role of endogenous galectin-9 in T cells by a mouse model of contact hypersensitivity (CHS), mediated by T cells. RESULTS: CHS response was less severe in galectin-9-deficient (Gal-9 -/-) mice than in Gal-9 +/+ mice by checking the ear thickness change. CD4+ T cells from Gal-9 -/- mice secreted less IFN-γ, IL-17, IL-4 and IL-13 than those from Gal-9 +/+ mice when stimulated by anti-CD3/anti-CD28 mAbs in vitro. CD4+ T cells from Gal-9 -/- mice sensitized with dinitrofluorobenzene topically secreted less IL-17, but not other three cytokines, than those from comparably sensitized Gal-9 +/+ mice. The adoptive transfer with sensitized CD4+ T cells from Gal-9 -/- mice evoked less severe CHS response than those from Gal-9 +/+ mice. Further, CD4+ T cells from Gal-9 -/- mice contained a lower amount of phosphorylated IκBα than those from Gal-9 +/+ mice, when activated by anti-CD3/anti-CD28. CONCLUSIONS: Galectin-9-deficient mice exhibit less CHS response, which might be related to lower IκBα phosphorylation and lower cytokine production in CD4+ T cells. Additional studies of the function of endogenous Gal-9 may provide a new insight on treatment of allergic contact dermatitis.
Critical role for inflammasome-independent IL-1β production in osteomyelitis (P6211)
Authors: Lukens John , Kanneganti Thirumala-Devi
Published: January 2023

The immune system plays an important role in the pathophysiology of many acute and chronic bone disorders, but the specific inflammatory networks that regulate individual bone disorders remain to be elucidated. Here, we characterized the osteoimmunological underpinnings of osteolytic bone disease in Pstpip2cmo mice. These mice carry a homozygous L98P missense mutation in the Pombe Cdc15 homology (PCH) family phosphatase PSTPIP2 that is responsible for the development of a persistent autoinflammatory disease resembling chronic recurrent multifocal osteomyelitis (CRMO) in humans. We found that improper regulation of IL-1β production resulted in secondary induction of inflammatory cytokines, inflammatory cell infiltration in the bone and unremitting bone inflammation. Aberrant Il1β expression precedes the development of osteolytic damage in young Pstpip2cmo mice, and genetic deletion of Il1β, but not Il1α, rescued osteolytic bone disease in mutant mice. Intriguingly, caspase-1 activation in the inflammasome complex was dispensable for Pstpip2cmo-mediated bone disease. Thus, our findings establish a critical role for inflammasome-independent production of IL-1β in osteolytic bone disease.
IL-10-mediated modulation of cochlear inflammation (P6228)
Authors: Moon Sung , Woo Jeong-Im , Oh Sejo , Lee Yoojin , Ahn Okjin , Lim David
Published: January 2023

Inflammatory response is commonly involved in the pathogenesis of various cochlear diseases such as acoustic trauma and cisplatin ototoxicity. Previously, we have demonstrated that the spiral ligament fibrocytes play a pivotal role in cochlear inflammation through secretion of chemokines such as CCL2 and CXCL2 in response to pro-inflammatory signals. In this study, we aim to determine molecular mechanism involved in negative modulation of cochlear inflammation. We found that IL-10 inhibits SLF’s CCL2 up-regulation and migration of THP-1 cells in response to SLF-derived molecules. The SLFs appeared to up-regulate HMOX1 expression via NRF-2 activation. The inhibitory effect of IL-10 was mimicked by CoPP and CORM-3 but was blocked by silencing of IL-10RA and HMOX1. IL-10 was found to inhibit binding of p65 NFκB to the enhancer region of the CCL2 gene. IL-10 deficiency appeared to enhance cochlear inflammation secondary to nontypeable H. influenzae-induced otitis media. Furthermore, we demonstrated that the cochlear macrophages serve as a local source of IL-10, which appeared to be primarily distributed in the stria vascularis. Our results suggest that cochlear macrophage-derived IL-10 modulates cochlear inflammation by inhibition of SLF’s CCL2 up-regulation through HMOX1-mediated CO production. This study is anticipated providing us with a novel strategy for the management of inflammation-associated cochlear diseases.
Antigen-specific CD4+ T cell derived IL-10 is uniquely pro-inflammatory in the context of acute influenza (P6234)
Authors: Dutta Avijit , Chen Tse-Ching , Lin Chun-Yen , Lin Yung-Chang , Chang Chia-Shiang , He Yueh-Chia , Huang Ching-Tai
Published: January 2023

IL-10 is regarded as an anti-inflammatory cytokine which prevents damage from immunopathology with exacerbated immune responses. However, severe lung inflammation in fatal human influenza is accompanied with increased levels of IL-10 as well as other pro-inflammatory cytokines. This information raises questions regarding the role IL-10 plays in inflammation associated with acute influenza. We found that IL-10 was produced by CD4+ T cells in synchronization with pro-inflammatory cytokines during an acute influenza virus infection in mice. IL-10 supplementation during the acute phase increased inflammation leading to death, while IL-10 knockout mice displayed decreased lung inflammation and survival benefits. In the absence of IL-10, influenza-specific CD4+ T cells divided less and produced less pro-inflammatory cytokines despite unaffected Th1 polarity, no increase of regulatory T cell populations, and no alteration in the kinetics of viral eradication. Influenza-specific CD4+ T cell derived IL-10 was more important than IL-10 from other sources for the detrimental effect. In this context of acute influenza in mice, IL-10 also impeded the release of mature TGF-β with decreased neuraminidase-mediated desialylation of host immune cells. The abundance of neuraminidase may contribute to the pro-inflammatory role of IL-10 we demonstrated in this context of acute influenza virus infection.
Catechin modulates inflammation in TNBS induced colitis. (P6238)
Authors: Hajj Hussein Inaya , Zeenny Marie-Noel , Shams Ali , Eid Assaad , Jurjus Abdo
Published: January 2023

This study assessed the effect of catechin in modulating the inflammation of colitis. Thirty rats were randomized into three groups: Group I: chemically induced with colitis and received no treatment, Group II: chemically induced with colitis and received daily injection of EGCG, Group III: control group provided with water only. In groups I and II, colitis was induced by TNBS solution. Rats were sacrificed on days 3, 10 and 17 and biopsies of distal colons were assessed by histology and molecular techniques for ROS and changes in cytokines like IL-6 and IL1-β. Colitis induced by TNBS and non-treated with EGCG group, showed high inflammatory scores resulting in colonic mucosa and submucosa damage, necrosis, absence of goblet cells, infiltration of inflammatory cells, crypt abscesses and granulomas. Infiltration of inflammatory cells and tissue damage peaked at day 17 after the induction. At day 3 and day 10, the treated rats with EGCG showed amelioration in the severity of inflammation, but at day 17, the state of the colonic crypts and goblet cells was close to normal. The number of mast cells in the colon of treated rats decreased by more than 30% at day 17 compared to the non- treated rats. In addition, the expression of ROS decreased significantly. IL-1B and IL-6 expressions decrease with time by 50% in the treated group. Our data confirmed the anti-inflammatory effect of EGCG in TNBS-induced colitis and provided a clue to a potential therapeutic agent.
Differential regulation of type 2 innate lymphoid cells by IL-25 and IL-33 (P6254)
Authors: Chen Chien-Chang , Iijima Koji , Kobayashi Takao , Kita Hirohito
Published: January 2023

Type 2 Innate Lymphoid Cells (ILC2s) are a novel innate cell populations that plays important roles in Th2-type immune response. They are lineage-negative, CD44+ and require IL-7 signal during their development. ILC2s produce type 2 cytokines in response to IL-33 in vitro. Animal studies demonstrated the production of type 2 cytokines by ILC2s after intranasal administration of IL-33. In addition to IL-33, IL-25 is also able to stimulate ILC2s to secret type 2 cytokines. However, detail mechanism to explain how ILC2s response to IL-33 and IL-25 has been unknown. The goal of this project is to examine the roles of IL-33 and IL-25 in proliferation and activation of ILC2s. We cultured lung cells with IL-33+IL-7 or IL-25+IL-7 and examined the production of IL-5 and IL-13. Lung ILC2s produced a large quantity of IL-13, but little IL-5 and highly expressed ICOS and ST2 in response to IL-25+IL-7 in vitro. When incubated with IL-33, the majority of ILC2s secreted both IL-5 and IL-13 and expressed intermediate level of ICOS and ST2. When IL-25 was administered intranasally to IL-13-GFP mice, a large proportion of ILC2s highly expressed IL-13-GFP. When IL-33 was administered, ILC2s showed modest expression of IL-13-GFP. These findings suggest that IL-33 and IL-25 are involved in overlapping but maybe different immunological and pathological process.
CXCL17 is a novel macrophage recruitment factor (P6215)
Authors: Burkhardt Amanda , Maravillas-Montero Jose Luis , Carnevale Christina , Selman Moises , Zlotnik Albert
Published: January 2023

Chemokines chemotactic molecules that regulate the development of immune responses, There are 48 members of this human gene superfamily and for most we have a good understanding of their biology. Some, however, remain poorly characterized including CXCL17 which was the last ligand of this superfamily to be described. Recently, we reported that CXCL17 is an important mucosal chemokine that exhibits antimicrobial activity and is elevated in the bronchoalveolar lavage fluid (BALf) of patients with idiopathic pulmonary fibrosis. We have now explored the chemotactic properties of CXCL17. In vitro, CXCL17 induces chemotactic responses in monocytic cell lines and in macrophages. We also analyzed CXCL17 effects in vivo and observed that it recruits F4/80+ macrophages to the peritoneal cavity. We have obtained a Cxcl17 (-/-) mouse and observed that it exhibits decreased numbers of macrophages is several mucosal tissues when compared to wild type mice. We conclude that CXCL17 is an important macrophage chemotactic factor. In addition, this information indicates that CXCL17 is likely involved in macrophage recruitment both under homeostatic conditions and also during inflammatory responses. Importantly, CXCL17 is likely to participate in human disease as we have already observed high levels of this chemokine in IPF. Taken together, our data strongly suggests that CXCL17 may be involved in human inflammatory diseases that affect the mucosa.
Cobalt-chrome micro-particles induce a robust MyD88-independent, Caspase1-dependent type 2 immune response (P1266)
Authors: Mishra Pankaj , Palma Mark , Millman Ariel , Hallab Nadim , Beebe Kathleen , Benevenia Joseph , Kanneganti Thirumala-Devi , Gause William
Published: January 2023

Wear debris particles associated with prosthetic devices may cause inflammation. In our previous study, we demonstrated that micrometer size Titanium particles induce a robust innate and adaptive type 2 in vivo immune response. In this study, we examined the size and mechanisms of particle induced type 2 inflammation, using Cobalt Chrome (COCR). Intra-peritoneal injection of 50 mg COCR (>10micron) still induced a type 2 innate response with marked increases in M2 macrophages, neutrophil and eosinophils independent of MYD88/TRIFF signaling pathways. We observed a marked decrease in the infiltration of neutrophil and M2 macrophages in Caspase-1-/- mice compared to wild type mice suggesting an essential role for inflammasome-mediated pathways. Intra peritoneal injection with smaller size COCR particles (<10micron) showed less infiltration of Th2 innate cells compared to the large size COCR particle (>10micron). We also examined the adjuvant nature of COCR particles by immunizing mice with COCR microparticles + OVA peptide after transfer of CFSE-labeled OVA specific DO11.10 CD4+ T cells. After 7 days marked increases in OVA-specific T cell proliferation and IL-4 production was observed compared to mice immunized with OVA alone. These studies indicate that induction of the type 2 immune response is triggered by solid particles alone similar in size to macroparasites, independently of MYD88 signaling and at least partially dependent on inflammasome activation.
Flagellin induces antibody responses through a Toll-like receptor 5 and inflammasome independent pathway (P1274)
Authors: Lopez-Yglesias Americo , Lai Marvin , Quarles Ellen , Zhao Xiaodan , Strong Roland , Smith Kelly
Published: January 2023

Flagellin is a potent immunogen that is recognized by TLR5 and Naip5/6, activates the innate immune system, and elicits strong T and B cell responses. The adaptor protein MyD88 is critical for both TLR5, as well as IL-1β and IL-18 receptors, major downstream mediators of the Naip5/6. Herein, we define the role of known flagellin receptors and MyD88 in antibody responses generated towards flagellin. To characterize the innate immune components that regulate isotype specific antibody responses, we used several genetically deficient mice: Naip5, caspase-1, MyD88, and TLR5/caspase-1. Using purified flagellin from S. Typhimurium, we tested serum cytokine responses in mice after i.p. injections. We observed that serum levels of IL-12/23p40 were TLR5-dependent, IL-18 levels were Naip5-dependent, and KC and IL-6 levels were dependent on both. We dissected antibody responses produced towards both flagellin and OVA via a prime/boost regimen. We show that IgG2c responses towards flagellin are TLR5-, and inflammasome-dependent. While IgG1 levels were partially dependent on TLR5 and the inflammasome, a substantial IgG1 response was induced through an undefined pathway. Conversely, the adjuvant effects of flagellin extrinsic antigen OVA are TLR5 and inflammasome dependent. Our results demonstrate a novel third pathway of flagellin recognition that contributes to antibody production. Further characterization of this pathway will be useful for flagellin-based vaccine design.
STING, the sensing of cytosolic DNA and innate immune-mediated inflammatory disease (P1256)
Authors: Ahn Jeonghyun , Gutman Delia , Saijo Shinobu , Barber Glen
Published: January 2023

Autoimmune diseases such as systemic lupus erythematosus (SLE) affect millions worldwide, the cause of which remains unknown. SLE and diseases such as Aicardi-Goutieres syndrome (AGS) are characterized by the overproduction of cytokines such as type I interferon (IFN), speculatively by chronic infection or self nucleic acids. Mice lacking DNAse II die during embryonic development through comparable autoimmune disease since phagocytosed DNA from apoptotic cells cannot be adequately digested and intracellular host DNA sensors are activated resulting in the production of a variety of cytokines including type I IFN. The cellular sensor responsible for triggering DNA-mediated inflammation aggravated autoimmune disease remains to be determined. However, we report here that STING (Stimulator of Interferon Genes) complexes with phagocytosed undigested DNA and controls innate immune signaling events that facilitate such events. DNase II-dependent autoimmune embryonic lethality was rescued by loss of STING function and polyarthritis completely prevented since cytosolic DNA failed to trigger cytokine production. Accordingly, loss of STING similarly alleviated Trex1-dependent lethal inflammatory myocarditis in mice, a model for AGS. Our data provides molecular insight into the causes of DNA-mediated inflammation-dependent autoimmune disorders and affords a new target that could plausibly be therapeutically controlled, to help prevent such diseases.
Non-canonical inflammasome activation of Caspase-11 protects against cytosolic bacteria (P1262)
Authors: Aachoui Youssef , Leaf Irina , Hagar Jon , Fontana Mary , Campos Cristine , Zak Daniel , Tan Michael , Cotter Peggy , Vance Russell , Aderem Alan , Miao Edward
Published: January 2023

Caspases are either apoptotic or inflammatory. The inflammatory Caspases-1 and -11 trigger pyroptosis, a form of programmed cell death. Whereas both can be detrimental in inflammatory disease, only Caspase-1 has an established protective role during infection. Here, we report that Caspase-11 is required for innate immunity to cytosolic, but not vacuolar, bacteria. Although Salmonella typhimurium and Legionella pneumophila normally reside in the vacuole, specific mutants (sifA and sdhA, respectively) that aberrantly enter the cytosol triggered Caspase-11 in vitro. This detection enhanced clearance of S. typhimurium sifA in vivo in infected mice. Similarly, Caspase-11 triggered pyroptosis after infection by Burkholderia species, which naturally invade the cytosol. Burkholderia mutants that lack the type III secretion system remain in the vacuole and are not detected through Caspase-11. While C57BL/6 mice could survive infection with B. pseudomallei, Casp11-/- mice rapidly succumbed. B. thailandensis is related to B. pseudomallei, but is normally avirulent as C57BL/6 mice survive infection with 10^7 cfu intraperitoneally; strikingly, Caspase-11 deficient mice succumbed to only 100 bacteria within 3 days. Thus, Caspase-11 is critical to surviving exposure to ubiquitous environmental pathogens that escape into the cytosolic compartment.
A role of Zeb2 in the development of terminally differentiated effector T cells (P1434)
Authors: Best John , Seuntjens Eve , Huylebroeck Danny , Goldrath Ananda
Published: January 2023

Zeb2 (also known as Zfhx1b and Sip1) is a DNA-binding multi-zinc-finger transcription factor found to play a significant role in the development of cells both early in neuronal differentiation and in cancer metastasis. By binding dual E2 box enhancer elements, Zeb2 acts primarily as a transcriptional repressor and may inhibit E-protein DNA binding, although it is known to act also as a transcriptional activator. Zeb2 also binds activated Smads, the co-repressor CtBP and the NuRD chromatin remodeling complex but not all of its functions may necessarily depend on each of these interactions. While the closely related family member, dEG1/Zfhx1a/Zeb1, is known to plays an early role in lymphocyte development, the function of Sip1 in T lymphocytes remains unknown. We find that Zeb2 is upregulated in activated T cells and that antigen specific T cells show diminished expansion during an immune response. Zeb2 is selectively expressed and necessary for the development of a subset of terminally differentiated CD8+ T cells. We are currently assessing the interplay between Zeb2 and E-proteins in differentiation of naïve CD8+ T cells to cytotoxic effector cells.
Identification of eomesodermin-expressing innate-like T cells in adult humans (P1436)
Authors: Gombert Jean-Marc , Jacomet Florence , Basbous Sara , Levescot Anais , Robin Aurélie , Roy Lydia , Giraud Christine , Barra Anne , Guilhot François , Herbelin André
Published: January 2023

Innate-like CD8(+) T cells have been recently described in mice. These cells express NK cell markers with high levels of the transcription factor Eomesodermin (Eomes(br)) and rapidly produce IFN-γ in response to IL-12 and IL-18 stimulation without previous exposure to antigen. To date, only a single study in human has investigated Eomes(br) innate-like T cells which were found in small numbers in fetal spleen and cord blood. In mice, their generation depends on IL-4 expressing invariant NKT (iNKT) cells. Here, we identified a similar subset of T cells, mainly CD8(+) T cells, in PBMC from healthy adult individuals that have increased expression of Eomes. They harbor a marked innate phenotype as attested by a higher frequency of perforin(+) and NK-like (CD158(+) and/or CD159(+)) cells, as compared with total T cells. Likewise, they preferentially produce IFN-γ in response to stimulation by IL-12 and IL-18. Remarkably, in patients with chronic myeloid leukemia (CML), a disease known to be associated with a deficiency in IL-4 expression by iNKT cells, the size of Eomes(br) innate-like T cells is severely impaired. and they have lost the capacity to produce IFN-γ after stimulation with IL-12 and IL-18. These impairments together with the deficiency in IL-4 expression by iNKT cells are corrected in CML patients having achieved complete remission. Altogether, these results demonstrate the presence of Eomes expressing innate-like T cells in adult human peripheral blood.
Hyaluronic acid/CD44 interactions control CD8 T cell memory development to influenza virus infection. (P1450)
Authors: Carrette Florent , Mueller Erin , Bradley Linda
Published: January 2023

CD44 is upregulated upon T cell activation during an immune response and remains highly expressed on memory T cells, which contribute to protection against challenge infection. However, its role in CD8 T cell response during infection is not well understood. Its main ligand is hyaluronic acid (HA), a glycosaminoglycan component of the extracellular matrix. In the steady state, HA exists at high molecular weight. Upon local inflammation, it is transiently broken down to low molecular weight forms. During influenza infection, we found that CD44-/- CD8 T cells accumulate normally during the expansion phase, but have an intrinsic defect in becoming memory cells. Furthermore, upon reinfection, the few CD44-/- memory CD8 T cells expand less than WT cells. CD44/HA binding is transiently induced in activated T cells, and we show that this interaction is crucial for effector CD8 T cell accumulation during infection. In addition, treatment of infected mice with IRAWB14, an agonist antibody promoting HA/CD44 binding, increased the accumulation of effector CD8 T cells. Correlating with an important role for low molecular weight HA, we found that IRAWB14 treatment is beneficial only if administered during the first phase of the infection, when low molecular weight HA is produced. Our data reveal that HA/CD44 interaction is an early essential and major regulator of the CD8 T cell immune response that controls the development of memory to influenza infection.
Myeloid-derived suppressor cell subsets inhibit T- and B-cell responses in murine Retrovirus-induced immunodeficiency (P6179)
Authors: Green Kathy , Fu Whitney , Cook W. , Green William
Published: January 2023

Compared to tumor microenvironments, myeloid-derived suppressor cells (MDSCs) have been less well studied in infectious diseases, particularly for retroviruses inducing immunodeficiency. We have found the development of a large population of immunosuppressive MDSCs after LP-BM5 retrovirus infection which causes murine acquired immunodeficiency. These MDSCs express a cell-surface marker signature (CD11b+Gr-1+Ly6C+) and inhibit not only T-cell, but B-cell, responsiveness by a substantially iNOS-dependent mechanism. MDSC immunosuppressiveness parallels the extent of disease in LP-BM5-infected w.t. vs. k.o. mice, differentially susceptible to disease. Retroviral load indicate that the suppressive Ly6C+CD11b+MDSC subset is LP-BM5 positive, although significantly lower than non-fractionated splenocytes from infected mice. Further fractionation of the MDSCs by FACS indicated, whereas all four subsets defined by Ly6C versus CD11b expression suppressed B-cell responses, only the Ly6Cbright CD11b+ subset suppressed T-cells, indicating distinct versus overlapping targeting of the two adaptive immune arms. These results, including the strong, direct MDSC inhibition of B-cell responsiveness, are novel for murine retrovirus-induced immunosuppression, and, as the total MDSC suppressive function mirrors that of LP-BM5-induced disease, support a pathogenic effector role for these retrovirus-induced MDSCs.
Identification of human endogenous retrovirus (HERV-K(HML-2))-specific mucosal CD4+ T cell responses in HIV-1-exposed, seronegative individuals (P6195)
Authors: SenGupta Devi , Ribeiro Susan , Michaud Henri-Alexandre , Loh Liyen , Tandon Ravi , Jones R. , Garrison Keith , York Vanessa , Cunha-Neto Edecio , Ostrowski Mario , Pilcher Christopher , Hecht Frederick , Martin Jeff , Deeks Steven , Hunt Peter , Nixon Douglas
Published: January 2023

The transcriptional silence of human endogenous retroviruses (HERV) can be disrupted in HIV-1 infection. We have reported that the strength of the HERV-specific CD8+ T cell response predicts lower HIV-1 viral load in untreated adults. To determine whether HERV-K immunity plays a role in inhibiting HIV-1 replication at a major site of transmission and CD4+ T cell depletion, we assessed these responses in gut-associated lymphoid tissue (GALT) from rectosigmoid biopsies and blood of HIV-1 exposed, seronegative (HESN) individuals (n=6) by flow cytometry. CMV pp65- and HIV-1 Gag-specific T cells were also measured, and all responses were compared with peripheral antigen-specific responses in uninfected, low-risk controls (n=11). With the exception of stronger CMV-specific CD8+ T cell responses in the HESN group (p=0.024), there were no significant differences between the magnitudes of peripheral antigen-specific T cell responses in the HESN vs. controls. However, HESN subjects had remarkably robust mucosal HIV-1- (median %CD4+cytokine+cells=2.67) and HERV-specific (median %CD4+cytokine+cells=3.41) CD4+ responses, which were much stronger than the corresponding PBMC responses (p=0.0079 and p<0.0001, respectively). These findings suggest that the GALT is an important site of HERV-K expression and immunity in individuals exposed to HIV-1, and should be further investigated in the context of novel vaccine strategies that target conserved antigens such as HERV.
Exploitation of cross-reactive CTL “help” as a potential immune escape strategy for HIV-1 (P6201)
Authors: Mailliard Robbie , Smith Kellie , Fecek Ronald , Rappocciolo Giovanna , Watkins Simon , Mullins James , Rinaldo Charles
Published: January 2023

The ability of HIV-1 to rapidly accumulate mutations provides the virus with an effective means of escaping CD8+ cytotoxic T lymphocyte (CTL) responses. Here we describe how subtle alterations in CTL epitopes expressed by naturally occurring HIV-1 variants can result in an incomplete escape from CTL recognition, providing the virus with a selective advantage. Rather than paralyzing the CTL response, these epitope modifications are capable of “baiting” pre-existing cross-reactive CTL to partially respond by producing pro-inflammatory cytokines in the absence of target killing. Importantly, instead of dampening the immune response through CTL elimination of variant antigen-expressing immature dendritic cells (iDC), a positive CTL-to-DC immune feedback loop dominates whereby iDC differentiate into pro-inflammatory mature DC that have a superior capacity to mediate T cell trans-infection. This discordant induction of CTL “helper” activity in the absence of killing likely contributes to the chronic immune activation associated with HIV-1 infection, and can be utilized by HIV-1 to promote viral dissemination and persistence. Our findings highlight the need to address the detrimental potential of eliciting dysfunctional cross-reactive memory CTL responses when designing and implementing anti-HIV-1 immunotherapies.
Shp1 regulates T cell homeostasis by antagonizing IL-4 signalling (P1312)
Authors: Johnson Dylan , Pao Lily , Dhanji Salim , Murakami Kiichi , Neel Ben , Ohashi Pam
Published: January 2023

The tyrosine phosphatase Shp1 is expressed ubiquitously in hematopoietic cells and is generally viewed as a negative regulator. Null mutations in Ptpn6, which encodes Shp1, result in the motheaten phenotype, characterized by widespread inflammation that causes death by 3-4 weeks of age. Previous studies of motheaten mice identified Shp1 as a negative regulator of TCR signaling, but the severe inflammation in these mice could have confounded our understanding of Shp1 function in T cell biology. To define the T cell intrinsic role of Shp1, we have characterized T cell-specific conditional knockout mice. Surprisingly, we detect no changes in TCR responsiveness in Shp1-deficient thymocytes or peripheral T cells. Instead, T cell Shp1 KO mice are enriched for memory-phenotype T-cells that express elevated levels of CD44 in the periphery. In addition, activated Shp1-deficient CD4+ T cells skew to Th2 and produce elevated levels of IL-4. Following IL-4 stimulation, Stat 6 activation is sustained in Shp1 deficient T-cells, leading to enhanced IL-4 production. Elimination of IL-4, through in vivo administration of a neutralizing antibody or by crossing to IL-4 KO mice, results in marked reduction of the excessive memory-like population in T cell specific Shp1 KO mice. Therefore, Shp1 is an essential negative regulator of IL-4 signaling in and is critical for the maintainance of T cell homeostasis.
Remarkably intact antiviral CD8 T cell response despite the absence of caspase 8 apoptotic and RIP3 necrotic pathways (P1318)
Authors: Livingston-Rosanoff Devon , Daley-Bauer Lisa , Mocarski Edward
Published: January 2023

Two programmed cell death pathways, apoptosis and necrosis, impact T cell function in mice and humans. Germ line disruption of pro-apoptotic caspase 8 (Casp8) activity in T cells triggers receptor interacting protein kinase (RIP)3-dependent necrosis upon TCR stimulation. This absolute requirement for Casp8 repression of RIP3-dependent pathways overshadows potential contributions of extrinsic apoptosis to T cell expansion and contraction. Mice deficient in both Casp8 and RIP3 (Casp8-/-Rip3-/-) have an intact immune compartment and maintain naïve CD8 T cells comparable to Rip3-/- and WT controls. Based on CD44 expression, aging mice show evidence of CD8 T cell proliferation and lymphadenopathy. Despite this, CD8 T cells in Casp8-/-Rip3-/- mice proliferate normally in response to TCR stimulation. Upon infection with murine CMV, Ag-specific CD8 T cells from Casp8-/-Rip3-/- mice expand, produce cytokines, kill cells expressing viral peptide, and control infection similarly to controls. Memory CD8 T cells develop in Casp8-/-Rip3-/- mice with appropriate KLRG1 and CD127 expression. These memory cells expand in response to and protect from secondary challenge. Additionally, memory inflation, a characteristic of the response to murine CMV, is preserved. Thus, when RIP3 pathways are eliminated, Casp8 restricts homeostatic lymphoproliferation but plays no direct role in the CD8 T cell response to infection.
Peripheral T cell-specific role of CDC42 of Rho GTPase family in T cell homeostasis and activation (P1286)
Authors: Guo Fukun , Yang Jun-Qi , Zhang Shuangmin , Zheng Yi
Published: January 2023

We have reported that deletion of CDC42 of Rho GTPase family in T cells at early developmental stage (CD4-CD8-CD44-CD25+) affects T cell homeostasis by the use of CDC42flox/flox/LckCre mice. Here, we generated CDC42-deficient mice with selective expression of the Cre recombinase beginning at a late stage of T cell development (after positive selection of CD4+CD8+) using the distal promoter of the Lck (dLck) gene. As expected, CDC42 deficiency (CDC42flox/flox/dLckCre) did not affect T cell development. However, splenic CD8+, but not CD4+, T cells were significantly reduced in CDC42- deficient mice. Further analysis revealed that there was a marked increase of both proliferation and apoptosis in vivo in naïve CD8+ T cells from CDC42-deficient mice. IL-7/IL-7R signaling is critical for T cell survival. The expression of IL-7R in naïve CDC42-deficient CD8+ T cells was down-regulated. These changes were not significant in naïve CDC42-deficient CD4+ T cells. CDC42 deficiency impaired in vitro activation of both CD4+ and CD8+ T cells. Furthermore, CDC42 deficiency significantly reduced Th1 and Th2 cytokine secretion, but increased Th17 cytokine secretion from CD4+ T cells under non-polarized culture conditions. These results are significantly different from that obtained by using CDC42flox/flox/LckCre mice and suggest a peripheral T cell-specific role of CDC42 in T cell homeostasis and activation.
Blocking IL-1β prevents M2 muscarinic receptor dysfunction in parainfluenza infected guinea pigs. (P6025)
Authors: Rynko Abby , Fryer Allison , Jacoby David
Published: January 2023

RATIONALE: Viral infections cause asthma attacks. Viruses cause loss of function of inhibitory M2 receptors on parasympathetic nerves. Blocking IL-1β prevents M2 receptor dysfunction in antigen-challenged guinea pigs. Here we investigated whether blocking IL-1β would prevent M2 receptor dysfunction is virus infected guinea pigs. METHODS: Guinea pigs were infected with parainfluenza. In some animals, IL-1β was blocked with anakinra (30mg/kg, i.p.) 30min before infection and every 24 hours after infection. Four days after infection, guinea pigs were mechanically ventilated and M2 receptor function was measured as the ability of gallamine to increase vagally stimulated bronchoconstriction. Viral titers were determined by measuring viral RNA in the lungs. White cells in blood and lung lavage were counted. RESULTS: Anakinra prevented M2 receptor dysfunction in infected animals. White cell counts were increased in lavage and decreased in the blood of infected animals. Anakinra did not affect viral titers or white cell counts. CONCLUSIONS: Treatment with an IL-1β receptor antagonist prevents M2 receptor dysfunction in parainfluenza infected guinea pigs.
Characterization of IgE+ B cells during primary and secondary immune responses (P6035)
Authors: Allen Christopher , Yang Zhiyong
Published: January 2023

IgE antibodies may have a protective role in parasite immunity, but they are best known for contributing to the pathology of allergic diseases. Exaggerated IgE responses can have dangerous consequences such as systemic anaphylaxis. However, direct studies of the B cells that express IgE have been hindered by technical challenges. We have generated fluorescent IgE reporter mice and also developed an improved flow cytometry procedure, enabling specific detection of IgE+ B cells in vivo. With these tools, we have analyzed various types of immune responses and determined general features of the genesis and fate of IgE+ B cells. The frequency of IgE+ B cells was typically very low compared with other isotypes such as IgG1. The majority of IgE+ B cells differentiated into short-lived plasma cells. IgE+ B cells transiently participated in germinal centers, and showed limited affinity maturation. Consistent with these findings, IgE+ B cells expressing memory cell markers were virtually undetectable, and no persistent population of IgE+ B cells could be identified several weeks after immunization. However, secondary immunizations generated faster IgE+ plasma cell responses compared with primary immunizations, suggesting that other memory precursors likely give rise to IgE plasma cells in recall responses.
Blockade of JAK1/3 signaling with a selective inhibitor, R256, attenuates Th2 differentiation and development of allergen-induced airway hyperresponsiveness (AHR) and airway inflammation (P6047)
Authors: Ashino Shigeru , Takeda Katsuyuki , Pine Polly , Gelfand Erwin
Published: January 2023

JAK are major downstream regulators of signaling through cytokine receptors, activating STAT. Effective targeting of the Th2-associated JAK-STAT pathway would represent a new therapeutic approach for the treatment of asthma. Here, we studied the effects of a novel selective JAK1/3 inhibitor, R256, on Th1, Th2, and Th17 differentiation in vitro and on the development of ovalubmin-induced AHR and airway inflammation in vivo in mice. R256 prevented only Th2 differentiation in vitro and was associated with the downregulation of STAT5 and STAT6 phosphorylation. In contrast, Th2 cells were unaffected by the inhibitor when added after polarization was complete. In vivo, R256 administered during the allergen sensitization phase prevented the development of AHR, airway eosinophilia, mucus hypersecretion, and Th2 cytokine production without altering Th1 and Th17 cytokine production, indicating that selective blockade of Th2 differentiation was critical. Inhibitor administration after allergen sensitization but during the challenge phase also suppressed AHR, airway eosinophilia, and mucus hypersecretion but no effect on Th2 cytokine levels in BAL could be detected, suggesting effects on downstream cytokine receptor signaling pathways. Thus, R256-mediated inhibition of JAK1/3 activation was effective when administered both prior to and after Th2 polarization to attenuate allergen-induced Th2 dominant airway inflammation and AHR.