bluuebunny/crossref_metadata_2025 huggingface dataset:
IKKβ in recipient myeloid cells promotes lung allograft acceptance and prevents the accumulation of intragraft IL-17+ CD4+ T cells (169.33)
Authors: Huang Howard , Yamamoto Sumiharu , Sugimoto Seiichiro , Zhu Jihong , Krupnick Alexander , Kreisel Daniel , Gelman Andrew
Published: January 2023

Background Mechanisms linking acute solid organ injury to rejection are poorly understood. We previously reported maintenance of IKKβ activity in recipient myeloid cells protects lung grafts from acute injury. We now ask if recipient myeloid cell IKKβ activity regulates lung allograft survival. Methods Balb/c lungs were transplanted into B6 recipients carrying A) Myeloid-specific IKKβ deletion (IKKβΔMye) or B) floxed IKKβ alleles (IKKβfl/fl). Post-surgery, recipients were immunosuppressed (IS) with MR1/anti-CD40L and CTLA4-Ig. In a second cohort, IS-IKKβΔMye and IS-IKKβfl/fl recipients were also treated with control Ig, IL-23p19 or IL-17-specific Abs. Lung allografts were analyzed on POD7 for histopathology and accumulation of IFNγ+ and IL-17+ T cells by FACS analysis. Results Acute rejection occurred in IS-IKKβΔMye, but not IS-IKKβfl/fl recipients. Moreover, IS-IKKβΔMye recipients developed marked intragraft accumulation of IFNγ+CD4+, IFNγ+CD8+ and IL-17+CD4+ T cells. IS-IKKβΔMye recipients treated with IL-23p19 Abs accepted their lungs and had fewer intragraft IL-17+CD4+ T cells with little change in IFNγ+CD4+ and IFNγ+CD8+ T cell abundance. By contrast, IL-17 Ab treatment depleted intragraft IL17+CD4+ T cells, but led to expansion of intragraft Th1 cells and did not prevent rejection. Conclusions IKKβ-regulated signals in recipient myeloid cells couple acute lung graft injury to alloimmunity by preventing Th17-mediated rejection in a IL-23p19-dependent manner.
Crucial roles of mast cells in the development of a murine model of polymyositis (171.20)
Authors: Yokota Masaya , Suzuki Kotaro , Nakagomi Daiki , Tokoyoda Koji , Nakayama Toshinori , Kohsaka Hitoshi , Iwamoto Itsuo , Nakajima Hiroshi
Published: January 2023

Objective: In addition to a pivotal role of mast cells in allergic diseases, recent data suggest that mast cells play a crucial role in a variety of immune responses. However, their role in the pathogenesis of autoimmune skeletal muscle diseases has not been clarified despite their distribution in skeletal muscle. Therefore, the objective of this study is to determine the role of mast cells in the development of a murine model of polymyositis, C protein-induced myositis (CIM). Methods: The susceptibility of mast cell-deficient WBB6F1-KitW/KitWv mice (W/Wv mice) to CIM was compared with that of wild-type (WT) mice. The effect of mast cell reconstitution with bone marrow-derived mast cells (BMMCs) on the susceptibility of W/Wv mice to CIM was also evaluated. Results: W/Wv mice exhibited significantly reduced disease incidence and histological scores of CIM as compared with WT mice. The infiltration of macrophages but not of CD8+ T cells in the skeletal muscle was attenuated in CIM of W/Wv mice. The expression of monocyte chemotactic protein-1 (MCP-1) in skeletal muscle was reduced in W/Wv mice. Vascular permeability in skeletal muscle was elevated in WT mice but not in W/Wv mice upon CIM induction. Engraftment of BMMCs restored the incidence and histological scores of CIM in W/Wv mice. Conclusion: Mast cells play critical roles in the development of CIM.
Non-invasive imaging of islet infiltration reveals an early window of CRIg-dependent diabetes determinism in NOD mice (171.29)
Authors: Fu Wenxian , Wojtkiewicz Gregory , Weissleder Ralph , Benoist Christophe , Mathis Diane
Published: January 2023

All juvenile NOD mice exhibit insulitis, but there is substantial variation in progression to diabetes. We demonstrate that a patient-validated magnetic-resonance-imaging strategy to non-invasively visualize local effects of pancreatic-islet inflammation can predict diabetes onset in NOD mice. MRI signals acquired during a narrow early time-window allowed pre-sorting into disease-progressors and -nonprogressors and an estimate of time-to-diabetes. We exploited this capability to identify novel elements correlated with disease protection. Highlighted was CRIg (complement receptor of the immunoglobulin superfamily), which tagged a subset of macrophages associated with diabetes resistance. Administration of CRIg-Fc to young NODs depressed MRI signals and diabetes incidence. Besides nominating new regulators of disease progression, this study demonstrated that diabetes is set at an early age in NOD mice.
IL-23 induces IRF5 and polarizes inflammatory M1 macrophages to promote inflammation in a mouse model of arthritis (171.5)
Authors: Li Hao , Hsu Hui-Chen , Li Jun , Yang PingAr , Wu Qi , Cua Daniel , Oukka Mohamed , Mountz John
Published: January 2023

Increased numbers of macrophages (MΦs) is an early hallmark of active arthritis. Expression of IL-23R has been identified on myeloid cells. We determined if IL-23 can regulate the pathogenesis of collagen II-induced arthritis (CIA) by directly modulating development of MΦs. AdIL-17 was injected to Il23p19-/- and Il23p19+/+ mice to induce equal levels of IL-17 yet Il23p19-/-mice were more resistant to CIA compared to Il23p19+/+ mice. Dramatic expansion of IL-23R+ MΦs, which are the major producers of IL-6 and TNFα in the draining LN of mice with CIA, occurred only in Il23p19+/+ but not Il23p19-/- mice. IL-23 suppressed the expression of anti-inflammatory TGFβ and IL-10 produced by M2 MΦs and promoted expression of inflammatory IL-6 and TNFα produced by M1 MΦs in vitro. IL-23 prevents the ability of M-CSF to promote development of anti-inflammatory M2 MΦs by inducing the expression of Irf5, an M1 specific transcription factor. Using GFP-Foxp3 reporter mice, accumulation of Foxp3+ Tregs were only observed in Il23p19-/- but not Il23p19+/+ mice with CIA. Coculture of IL-23 stimulated MΦs with naïve CD4 T cells prevented the development and function of Treg cells, which could be reversed in the presence of anti-IL-6. Our results suggest that IL-23 induces the expression of IRF5 and promotes M1 MΦs that perpetuate the development of CIA. One important function of IL-23-activated MΦs was to prevent the development of Treg cells via an IL-6 dependent mechanism.
Up-Regulation of Stromal Cell-Derived Factor by IL-17 and IL-18 via Phosphatidylinositol 3-Kinase Dependent Pathway (171.8)
Authors: Cho Mila , Lee Seon-Yeong , Park Jin-Sil , Oh Hye-Joa , Son Hye-Jin , Ryu Jun-Geol , Kim Ho-Youn
Published: January 2023

IL-17 producing Th17 has a key role in the pathogenesis of autoimmune inflammation. Among various cytokines which are interwoven with the IL-17 pathway, members of IL-1beta family including IL-18 have recently gained significance. In the present study, we stimulated synovial fibroblasts with the combination of IL-17 and IL-18, and quantified the cellular production of stromal cell-derived factor-1 (SDF-1) with ELISA and its mRNA with RT-PCR. Both IL-17 and IL-18 significantly increased the level of SDF-1, not only individually but also synergistically (p<0.05). The synergism was effectively suppressed by anti-IL-17, anti-IL-18 antibodies and PI3K inhibitor. In conclusion, PI3K-dependent synergism between IL-18 and IL-17 is first observed in this study, and adds a novel perspective for the role of IL-18 in immune regulation. The two cytokines’ individual effects, mutual interaction and synergism draw a crisscrossing map between IL-1 family and IL-17.
Natural Regulatory T Cell Controls Physiological Tolerance And Regional Immune Privilege: Studies on (C57BL/6xA/J) F1 (B6AF1) DEREG (“depletion of regulatory T cell”) Mice (171.9)
Authors: Harakal Jessica , Rival Claudia , Qiao Hui , Sampson Robert , Tan Steven , Cho Yumee , Sparwasser Timothy , Tung Kenneth
Published: January 2023

Although CD4+CD25+ Foxp3+ regulatory T cell (Treg) prevents autoimmune disease, its depletion in normal mice does not cause organ-specific autoimmunity, thus Treg’s physiological role in tolerance remains unclear. Different outcomes after Treg depletion in two models of Diphtheria toxin (DT) receptor-Foxp3 adult B6 mice have been reported: scurfy syndrome versus no effect. Herein, we report autoimmune disease of testis, ovary, stomach and lacrimal gland in the DEREG B6AF1 mice after Treg depletion. Male B6AF1 DEREG mice show massive immune complex and complement deposition, defective blood-testis barrier, and loss of sperm production. Importantly, immune injury is preceded by defective testis immune privilege, which also occurs reversibly in B6 DEREG mice that lack testis-specific autoAb. AutoAb in male B6AF1 DEREG mice target germ cells but also antigen common to steroidogenic testis (Leydig) cells and ovarian cells. In fact, male CD4 T cells adoptively transfer both orchitis and oophoritis; and autoAb of female B6AF1 DEREG mice with oophoritis targets a different ovarian Ag: the zona pellucida. These findings indicate gender-specific tolerance mechanisms. Notably, the Abs and gonadal failure are part of the human autoimmune polyendocrine syndrome (APS), some with autoimmune regulator (AIRE) gene mutations. Therefore, natural Treg control physiological systemic and regional tolerance to major gonadal Ag and Treg deficiency could explain APS and AIRE-related autoimmunity.
OCT-1 acts as a repressor of C-reactive protein gene expression (172.13)
Authors: Hammond David , Thirumalai Avinash , Voleti Bhavya , Zhang Shuang , Agrawal Alok
Published: January 2023

C-reactive protein (CRP), an ancient host-defense plasma protein, is produced by hepatocytes. In Hep3B cells, CRP expression is induced by IL-6; IL-1β synergistically enhances the IL-6 effect. A critical regulatory segment on the CRP promoter contains binding sites for transcription factors C/EBPβ (-48 to -57) and NF-κB (-63 to -74). The κB site overlaps an octamer motif (-59 to -66) which is the binding site for the constitutively expressed transcription factor OCT-1. OCT-1 is known to function both as a transcriptional repressor and activator depending upon the promoter context. The aim of this study was to investigate the function of OCT-1 on the CRP promoter. In luciferase transactivation assays, overexpressed OCT-1 inhibited cytokine-induced CRP expression. EMSA, mutational analysis of the κB site, and the OCT-1 dose-response assays suggested that OCT-1 and NF-κB compete for binding to their overlapping cognate sites. Deletion of the OCT-1 site drastically reduced the cytokine response because the κB site was altered as a consequence of deleting the OCT-1 site. Surprisingly, overexpressed OCT-1 inhibited the residual cytokine-induced CRP expression through the promoter lacking the OCT-1 site. OCT-1 also inhibited overexpressed C/EBPβ-induced CRP expression. We conclude that OCT-1 acts as a repressor of CRP gene expression and it does so by occupying its cognate site on the promoter and also via other transcription factors by an as yet undefined mechanism.
IL-33 retains mast cells in a selectively unresponsive state to bacterial cell wall products (172.15)
Authors: Sandig Hilary , Jobbings Cath , Orinska Zane , Bulfone-Paus Silvia
Published: January 2023

Mast cells are located at sites of microbial entry into the host therefore it is important that their response to pathogen associated molecular patterns (PAMPs) is tightly regulated to avoid unnecessary activation and pathology. Here we show that treatment of mast cells with picomolar concentrations of the stromal cell product IL-33 renders the cells insensitive to bacterial cell wall products. Responsiveness to antigen, cytoplasmic dsDNA and TLR7/8 agonists is unaffected or enhanced by IL-33. The IL-33 induced selective tolerance is dependent on the IL-33 receptor ST2 and independent of c-kit. The LPS insensitivity cannot be explained by up-regulation of the TLR4 signalling inhibitors A20 or Tollip nor does IL-33 affect the levels of TLR4, MyD88 or TIRAP, rather the cytokine potently causes rapid IRAK1 degradation which is sustained for at least 24 h. Unlike its redundant role in macrophages, we show that IRAK1 is a key component of TLR4 signalling in mast cells. LPS treatment also resulted in down-regulation of IRAK1 protein levels which may explain the mechanism behind endotoxin tolerance in mast cells. We propose a hitherto unidentified role for IL-33 in maintaining immune homeostasis by retaining mast cells in a selectively responsive state.
IL-33 is processed into mature bioactive forms by neutrophil serine proteases (172.26)
Authors: Lefrançais Emma , Roga Stéphane , Gautier Violette , Gonzales de Peredo Anne , Monsarrat Bernard , Girard Jean Philippe , Cayrol Corinne
Published: January 2023

IL-33 is a chromatin-associated nuclear cytokine from the IL-1 family, which has been linked to many inflammatory diseases. IL-33 signals through the receptor ST2 and drives production of cytokines in innate effector cells and T helper type 2 lymphocytes. IL-33 is constitutively expressed in the nuclei of endothelial cells and in epithelial cells of tissues exposed to the environment. It was initially believed that IL-33, like IL-1β and IL-18, requires processing by caspase-1 for biological activity. On the contrary, we reported that full length IL-33 is biologically active, and that processing by apoptotic caspases results in IL-33 inactivation, rather than activation. We also found that full length IL-33 is released upon cellular damage and we proposed that IL-33 may function, similarly to HMGB1, as an endogenous danger signal. Because IL-33 plays important roles in inflammatory diseases, we hypothesized that IL-33 could be cleaved by proteases released during inflammation. We analyzed the effects of inflammatory proteases from innate effector cells, on IL-33 processing and biological activity. We found that IL-33 can be cleaved and activated by several inflammatory proteases. Interestingly, all forms generated by these proteases are “super-active” IL-33 forms that contain an intact IL1-like cytokine domain. We propose that the inflammatory microenvironment exacerbates disease-associated functions of IL-33 through the generation of “super-active” IL-33 forms.
Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 Regulates Macrophage-Mediated Inflammatory Responses (172.35)
Authors: Racioppi Luigi , Noeldner Pamela , Lin Fumin , Arvai Stephanie , Means Anthony
Published: January 2023

Calcium/calmodulin-dependent Kinase Kinase 2 (CaMKK2) plays a key role in regulating food intake and energy expenditure at least in part by its actions in hypothalamic neurons. Previously we showed that loss of CaMKK2 protected mice from high fat diet (HFD)-induced obesity and glucose intolerance. However, whereas pair-feeding HFD to WT mice to match food consumption of CAMKK2-null mice slowed weight gain, it failed to protect from glucose intolerance. Here we show that relative to WT mice, HFD-fed CaMKK2-null mice are protected from inflammation in adipose and remain glucose tolerant. Moreover, loss of CaMKK2 also protected mice from endotoxin shock and fulminant hepatitis. We explored the expression of CaMKK2 in immune cells, and found it to be restricted to those of the monocyte/macrophage lineage. CaMKK2-null macrophages exhibited a remarkable deficiency to spread, phagocytize bacteria and synthesize cytokines in response to the Toll Like Receptor 4 (TLR4) agonist lipopolysaccharide (LPS). Mechanistically, loss of CaMKK2 uncoupled the TLR4 cascade from activation of protein tyrosine kinase 2 (PYK2; also known as PTK2B). Our findings uncover an important function for CaMKK2 in mediating mechanisms that control the amplitude of macrophage inflammatory responses to excess nutrients or pathogen derivatives.
Altered humoral immune responses in mice deficient for the Fc receptor for IgM (FcμR) (172.37)
Authors: Honjo Kazuhito , Kubagawa Yoshiki , Jones Dewitt , Dizon Brian , Ohno Hiroshi , Kearney John , Kubagawa Hiromi
Published: January 2023

Unlike humans, FcμR expression in mice is restricted to B cells with a hierarchy of cell surface expression level: follicular > marginal zone (MZ) > newly formed in spleen and B-2 = B-1a > B-1b in peritoneal cavity. To define the in vivo function of FcμR, Fcmr-deficient (KO) mice were generated by gene targeting. While total splenic CD19+ B and CD3+ T cell-numbers were comparable in KO and littermate control (WT) mice, MZ B cells were reduced ~4 fold and B-1 cells were increased ~2 fold in KO as compared with WT mice. Both serum IgM and IgG3 antibody (Ab) levels in naïve animals were significantly elevated in KO than WT mice. IgM and IgG3 natural Abs reacting with the nucleus or cytoplasm of HEp-2 cells were also elevated in KO mice. When immunized i.p. with a live avirulent strain of S. pneumoniae (R36A), much more robust and long-lasting IgM, IgG3 and IgA Ab-responses to phosphorylcholine developed in KO at low doses of R36A compared with WT. By contrast, when immunized i.p. with nitrophenyl (NP)-coupled chicken γ-globulin in alum, primary IgM anti-NP responses were comparable in both groups of mice, but secondary IgM responses were significantly impaired in KO mice. In contrast, the primary IgG1 anti-NP responses were impaired in KO, however secondary IgG1 responses were indistinguishable in both KO and WT mice irrespective of the antigen dose. These results show that Fcmr ablation results in altered Ab responses to both T-independent and -dependent antigens.
γδ T Cells and diet-induced inflammation in adipose tissue (173.2)
Authors: Mehta Pooja , Smith C. Wayne
Published: January 2023

Adipose tissues contain several subsets of immune cells. Relatively little is known about their contributions to adipose tissue inflammation induced by feeding mice a diet rich in saturated fats. Using a model of diet-induced obesity in male C57BL/6J mice, we investigated the possible contributions of γδ T cells to this inflammation. γδ T cells make up about one-third of the total T cell population in the epididymal adipose tissue. The γδ T cell receptor was blocked in wild-type mice using an anti-TCRδ antibody. A decrease in macrophage marker F4/80 was observed in these mice as compared to saline-treated control mice on high fat diet (HFD). TCRδ-/- mice adipose tissue failed to show an increase in mRNA expression of cytokines such as CCL-2, IL-6, IFN- γ even though they ate similar amounts of food and showed similar weight gain pattern as wild-type mice. A fraction of adipose tissue γδ T cells were shown to produce IFN-γ in obese mice by flow cytometry and were CD27+, a marker for IFN-γ-producing γδ T cells in the periphery. Obesity also results in inflammation in the skeletal muscle and liver, along with adipose tissue inflammation. Obese TCRδ-/- mice have reduced expression of markers of inflammation in the muscle after 5 weeks on HFD and liver after 6 months on HFD as compared to wild-type mice. These results are consistent with the conclusion that adipose tissue, liver and skeletal muscle inflammation during high saturated fat feeding is dependent in part on γδ T cells.
Opsonizing antibodies against influenza virus proteins mediate activation of plasmacytoid dendritic cells and are broadly cross-reactive. (P6194)
Authors: Ricklin Meret , Zimmer Gert , Summerfield Artur
Published: January 2023

Cross-protective immunity are observed between different strains of influenza viruses within one subtype and even between certain subtypes in the absence of neutralizing antibodies. So far most studies have focused on T cell immunity as the main mechanism of heterologous protection. The aim of this study was to test and characterize the functional and cross-reactive activity of antibodies against nucleoprotein (NP), hemagglutinin (HA) and neuraminidase (NA) of swine influenza viruses in the pig model. Our results demonstrate that antibodies against the internal well-conserved NP of distant strains can mediate NP-RNA complex uptake by plasmacytoid DC resulting in a robust IFN-alpha response. This function can mainly be observed at low concentrations of NP-RNA complexes released from dying infected cells. Furthermore, antibodies against the external HA and NA can mediate efficient uptake of immune complexed virus resulting in a robust IFN-alpha response at low concentrations of virus and antibodies. This reactivity is found with both, neutralizing and non-neutralizing antibodies. The latter are highly cross-reactive within on subtype and even between subtypes. In addition to T-cell immunity, such antibodies could play a role in promoting subtype cross-reactive (partial) immunity after influenza virus infection or following vaccination. Studies to test the impact of opsonizing antibodies in vivo are ongoing.
mTOR regulates protection against a lethal H5N1 influenza infection by modulating the antibody response (P6184)
Authors: McGargill Maureen , Keating Rachael , Hertz Tomer , Lukens John , Wehenkel Marie , Harris Tarsha , Edwards Benjamin , McClaren Jennifer , Brown Scott , Surman Sherri , Wilson Zachary , Hurwitz Julia , Chi Hongbo , Doherty Peter , Thomas Paul
Published: January 2023

Highly pathogenic H5N1 avian influenza A viruses pose a serious threat for global catastrophe. Due to frequent mutations in the influenza genome, current vaccination protocols promote only strain-specific responses, and therefore fail to provide protection against novel pandemic strains. Antibodies that protect against multiple subtypes of influenza have been identified, however, these antibodies are extremely rare, and the immunological mechanisms that lead to their development are not clear. We show here that rapamycin, an immunosuppressive drug that inhibits mTOR, promoted cross-strain protection against lethal H5N1 infection when administered during H3N2 vaccination. Surprisingly, this protection did not require memory CD8 T cells, but rather required CD4 T cells and B cells, suggesting the protection was antibody-mediated. In support of this, serum from rapamycin-treated mice immunized with a H3N2 strain protected naïve mice from a lethal H5N1 infection. Our data further reveal that rapamycin reduced germinal center formation and B cell class-switching, yielding a unique repertoire of antibodies that was more protective against a lethal, heterosubtypic influenza infection. These data not only reveal a requirement for mTOR in B cell class-switching, but also indicate that a broader antibody repertoire, which provides better protection against multiple strains of influenza, can be achieved by reducing germinal center formation during the primary infection.
Induction of memory immunity to Mycobacterium tuberculosis is independent of Toll-like receptor 9 signaling (IRC9P.706)
Authors: Gopalakrishnan Archana , Dietzold Jillian , Salgame Padmini
Published: January 2023

Mycobacterium tuberculosis (Mtb) is known to engage Toll-like receptors (TLR) 2 and 9 on macrophages and dendritic cells to initiate an inflammatory response. Nonetheless, studies in the murine model of tuberculosis revealed minimal involvement of TLR2 and TLR9 in host resistance during acute infection. We have published that the induction of memory immunity to Mtb is not compromised in the absence of TLR2. Given that the inclusion of TLR9 ligands in vaccine preparations enhances their efficacy, we hypothesized that TLR9 adjuvanticity is necessary for the induction of memory immunity to Mtb. Mice were immunized with an Mtb auxotroph. Upon challenge with Mtb, WT and TLR9-/- immunized mice demonstrated similarly enhanced control of bacterial burden in the lungs compared to their unimmunized counterparts. The granulomatous response, IFN-gamma production and the frequency of immune cells recruited to the lungs was similar in the two genotypes. These findings indicate that the absence of TLR9 signaling during priming does not affect T cell memory response to Mtb infection. Together, our studies point that induction of effector and memory T cell responses and resistance to acute Mtb infection are independent of TLR2 and TLR9. In a recent study we showed that TLR2 signaling, although nonessential in acute infection, was critical to host resistance during chronic Mtb infection. In ongoing studies we are investigating whether TLR9, like TLR2, has a role in chronic Mtb infection.
Identifying novel regulators of TIM-3 expression in exhausted T cells (IRC9P.707)
Authors: Avery Lyndsay , Kane Lawrence
Published: January 2023

Chronic viral infection and the tumor microenvironment provide settings where T cells are persistently stimulated by their cognate antigen. These T cells are often ‘exhausted’ and exhibit decreased effector function and proliferative potential. Tim-3 is an inhibitory receptor upregulated on the surface of exhausted T cells, and Tim-3 blockade has shown potential for reversal of this phenotype. Understanding mechanisms that regulate Tim-3 expression and signaling can provide insight into therapeutics to rescue exhausted T cell function. Upon T cell activation, the unfolded protein response (UPR) is initiated to resolve a build-up of newly translated protein in the endoplasmic reticulum (ER). Bat3, an ER chaperone upregulated during the UPR, was suggested to be a repressor of Tim-3 cytoplasmic signaling. XBP1, a transcription factor activated during the UPR, is predicted to bind the Tim-3 gene. We aim to confirm the interaction of XBP1 with the Tim-3 gene, as well as understand its functional significance. Linking the UPR and Tim-3 could provide novel information on pathways that drive exhaustion and offer insight into therapies for diseases where chronic antigen stimulation occurs.
Protein N-myristoylation is indispensable for T-cell development and activation (LYM7P.720)
Authors: Rampoldi Francesca , Boehm Martin , Lehmann Wolf , Gröne Hermann-Josef , Porubsky Stefan
Published: January 2023

Protein N-myristoylation refers to an irreversible attachment of myristic acid to the N-terminal glycine of proteins. This modification promotes protein-protein and protein-membrane interactions. Thymus represents an organ with a high degree of protein N-myristoylation. Our aim was to elucidate the role of protein N-myristoylation in T-cell development and in T-cell receptor (TCR) signaling in vivo. To this end, mice with a T-cell specific deletion of both genes responsible for protein N-myristoylation (N-myristoyltransferase, Nmt, 1 and 2) were generated. Abrogation of protein N-myristoylation led to a defective maturation of thymocytes. The transition from the double negative (DN) 3 to DN4 stage and the transition from the double positive to both single positive stages were impeded. Functional analyses of T-cells deficient in Nmt1 and Nmt2 showed that N-myristoylation of the proteins Lck, Fyn and Marcks, which are involved in early and late TCR signalling events, was essential for a proper transduction of TCR signals. We could demonstrate that the absence of myristoylation caused a mislocalization and malfunction of these proteins. These findings demonstrate for the first time in vivo the indispensable role of protein N-myristoylation during T-cell development and in the transmission of TCR signals.
Therapeutic treatment with cryoablation and Meriva improves T cell responses to tumor-associated antigens and is highly effective against metastatic breast cancer (TUM2P.910)
Authors: Chandra Dinesh , Jahangir Arthee , Meheus Lydie , Rombauts Klara , Gravekamp Claudia
Published: January 2023

Cryoablation of tumors involve the insertion of a probe into a tumor nodule to administer tissue-ablative cold temperatures. Its mechanism of action has been attributed to the mechanical forces of crystallization, the osmotic changes due to crystallization, and the ischemic effects of microvascular injury. This leads to immunogenic tumor cell death resulting in cross presentation of tumor-associated antigens (TAA) of the cryo-killed tumor cells by antigen-presenting cells (APC) to the immune system. This in turn leads to the recruitment of TAA-specific T cells, which are powerless because of the strong immune suppression in the tumor microenvironment. We found that Meriva, a Curcumin derivate with high bioavailability, reduced immune suppression by IL-6, resulting in the activation of T cell responses to TAA Mage-b and Survivin (highly expressed by tumors and metastaess), and a nearly complete elimination of metastatic breast cancer (4T1 model) when combined with Cryoablation. In addition, the overall survival of mice that received Cryoablation and Meriva was significantly increased compared to the saline group. Our results demonstrate that Meriva is a potent agent to reduce immune suppression by IL-6 and to stimulate TAA-specific T cells in a tumor microenvironment. Moreover, Meriva might be useful in other combination therapies that induce immunogenic tumor cell death like adriamycin or radiation, or with vaccines that are hampered by immune suppression in the TME.
Qualitative and quantitative effects of 4-1BBL in boosting pre-existing influenza immunity (VAC2P.923)
Authors: Zhou Angela , Wagar Lisa , Wortzman Michael , Watts Tania
Published: January 2023

Influenza virus induces an acute respiratory infection responsible for up to 500,000 annual deaths, primarily in older persons. CD8+ T cells against conserved influenza proteins confer heterotypic protection, potentially providing a “universal vaccine.” However, live infection only transiently boosts influenza-specific T cell memory, and immunity further declines with age. To improve the longevity of T cell memory, we investigated the effects of stimulating the TNFR-family member 4-1BB. To mimic vaccination of previously flu-immune adults, mice were infected with influenza A/HK-X31 and boosted intranasally one-month later with control replication-deficient adenovirus or adenovirus encoding influenza nucleoprotein (NP) alone (Ad-NP) or NP with 4-1BBL (Ad-NP-4-1BBL). 4-1BBL dramatically enhanced NP-specific CD8+ T cell responses at a dose where Ad-NP had minimal effects. 4-1BBL induced a remarkably long-lived effector-memory population that protects mice against lethal challenge into old age (>1 year old). Increasing the Ad-NP dose did not replicate these effects, and instead caused partial functional exhaustion. Preliminary analysis indicates that the 4-1BBL-induced effector memory T cells have increased expression of IL-7R as well as the transcriptional factor TCF-1 and an increased ratio of T-bet to Eomesodermin. Thus, 4-1BBL in a vaccine vector administered in flu-immune mice induces a potentially unique protective subset of memory CD8+ T cells.
A cationic lipid-DNA complex adjuvant (JVRS-100) enhances the immunogenicity and cross-protective efficacy of influenza pre-pandemic H5N1 vaccine in ferrets (VAC2P.931)
Authors: Liu Feng , Tumpey Terrence , Sun Xiangjie , Fairman Jeff , Levine Min , Katz Jacqueline , Lu Xiuhua
Published: January 2023

Highly pathogenic avian influenza A H5N1 viruses continue to pose a public health threat. Adjuvants are needed to improve the immunogenicity of H5N1 vaccine in humans. JVRS-100 is a cationic lipid-DNA complex adjuvant. We investigated the immunogenicity and cross-protective efficacy of JVRS-100-adjuvanted H5N1 (clade 2.2) vaccine in ferrets. Four weeks after the first vaccination, significantly higher levels of serum hemagglutination-inhibition (HI), neutralizing and H5 IgG antibody were detected in ferrets immunized with adjuvanted vaccine compared to the unadjuvanted vaccine group. Following a second dose of JVRS-100 adjuvanted vaccine, cross-reactive HI antibody titers of ≥40 were detected against viruses from multiple H5N1 clades. Ferrets were challenged with a high lethal dose of heterologous H5N1 virus (clade 2.1.3.2) 2 months after the second vaccination. Ferrets that received JVRS-100 adjuvanted vaccine had significantly reduced virus titers in the nasal washes and were completely protected from severe disease and death. In contrast, ferrets that received unadjuvanted vaccine had high viral titers through day 6 and 3 of 6 animals succumbed to the lethal infection. Our results indicate that the addition of JVRS-100 to H5N1 vaccine enhanced immunogenicity and cross-protection against lethal H5N1 virus disease in ferrets. JVRS-100 warrants further investigation as a potential adjuvant for use in human vaccination against avian influenza viruses with pandemic potential.
Sphingosine kinase 1 and its role in modulation of anti-tumor T-cell responses (VAC3P.951)
Authors: Thyagarajan Krishnamurthy , Al-Khami Amir , Ponnusamy Suriyan , Guo Beichu , Ogretmen Besim , Mehrotra Shikhar
Published: January 2023

Sphingosine kinase 1(Sphk1) is a key enzyme in sphingolipid metabolism that is involved in the production of sphingosine-1-phosphate (S1P). S1P controls many cellular processes such as cell proliferation, migration and cytokine production. We sought to address the role of Sphk1 in regulating T cell responses to a tumor specific ‘self’ antigen gp100, in a mouse model of melanoma by generating TCR transgenic mice pMel on Sphk1 deficient background. We found that Sphk1-/-pMel mice showed reduced tumor progression on subcutaneous administration of B16-melanoma. Tumor progression was slower when T-cells from Sphk1-/-pMel mice were adoptively transferred to treat Rag-/- mice with established subcutaneous tumors. There was an increased infiltration of Sphk1-/-pMel T-cells at the tumor site as compared to pMel cells. This suggests that loss of Sphk1 from T-cells enhanced their ability to target the tumor site. We also found TGF-β mediated suppression of IFN-γ secretion was poorer in Sphk1-/- T-cells suggesting better functionality of effector T cells infiltrating the tumor site. A comparative evaluation of T cells from WT and Sphk1-/-pMel showed similar cell surface markers, activation status and proliferation and Tc1 cytokine response. However, we found that Sphk1-/- T-cells showed greater IL-17 secretion. It is possible that the enhanced anti-tumor T cell immunity noticed in Sphk1-/- mice is dependent on the innate Tc17 phenotype and lesser susceptibility to suppression.
Interferon-α augments the clinical efficacy of regulatory T cell depletion in ovarian cancer through direct and indirect T cell effects (VAC3P.941)
Authors: Thibodeaux Suzanne , Hurez Vincent , Wall Shawna , Pandeswara Sri Lakshmi , Sun Lishi , Daniel Benjamin , Liu Aijie , Wood Leslie , Zou Weiping , Curiel Tyler
Published: January 2023

Tregs blunt anti-tumor immunity, and their depletion effectively treats tumor in mouse cancer models. However, Treg depletion in human trials is only partially effective. In our phase II ovarian cancer trial, denileukin diftitox (DT) effected significant Treg depletion in blood and the tumor microenvironment but did not produce significant clinical efficacy. Interferon-α is not an effective ovarian cancer treatment, but we show here that it significantly augments immune and clinical DT-mediated Treg depletion efficacy in human ovarian cancer. In a mouse ovarian cancer model, DT modestly improved anti-tumor immunity and survival. Interferon-α alone did not alter Treg numbers or function, but boosted CD8+ T cell anti-tumor immunity. Interferon-α plus DT significantly prolonged mouse survival over either individual agent. Using type I IFNR-/- mice that cannot mediate interferon-α signals, we showed that interferon-α directly boosted CD8+ T cell function independent of CD4+ T cell help, and in combination with DT reduced Treg function (without further numerical reduction) through indirect effects on tumor microenvironmental dendritic cells. When three ovarian cancer patients failed DT alone, two experienced immunologic and clinical benefit by adding pegylated interferon-α2a, with manageable toxicities. These studies demonstrate novel immune and clinical interferon-α anti-cancer benefits that augment Treg depletion using FDA-approved agents for rapid clinical translation.
LPS and HIV-1 induce a mature phenotype in human plasmacytoid dendritic cells and enhance their programmed death ligand-1 expression (VIR1P.970)
Authors: Patel Meher , Dai Jihong , Singh Sukhwinder , Fitzgerald-Bocarsly Patricia
Published: January 2023

Aberrant immune activation and immune exhaustion are features characteristic of chronic HIV infection. Elevated plasma LPS levels resulting from disruption of gut mucosal integrity during HIV infection have been implicated as a major factor contributing to immune activation. In chronic HIV-1 infection immune exhaustion molecules PD-1 and its ligand PD-L1 negatively regulate the function of exhausted CD8 T cells. The pDC constitute only a minor portion of the PBMC population, but are crucial contributors to the innate as well as adaptive immune response. We have confirmed our previous results that pDC express low levels of TLR4 and respond to LPS. We undertook this study to examine the influence of LPS on virally stimulated pDC maturation and PD-L1 expression and mechanisms thereof. In order to do so we primed pDC with LPS prior to stimulation with HIV. We observed that LPS enhanced HIV-1-induced pDC maturation (CD83 and CD40 expression) and PD-L1 expression. PD-L1 was up-regulated on mature pDC specifically by LPS and HIV combination treatment versus individual treatment with LPS or HIV. The combination treatment specifically induced co-expression of PD-L1 with positive co-stimulatory molecule CD86 but not CD80. Additionally, LPS enhanced CD4 expression on pDC but depressed HIV-1 induced CXCR4 and CCR5 expression. Together, these results indicate that in response to HIV stimulation, pDC adopt a tolerogenic phenotype (i.e. PD-L1-positive) and LPS helps drive this.
Galectin-3 translocates to virological synapse and promotes HIV-1 transfer (VIR1P.1000)
Authors: Chen Huan-Yuan , Wang Sheng-Fan , Hsu Daniel K Hsu , Chen Yi-Ming Arthur , Liu Fu-Tong
Published: January 2023

Galectin-3 contains β-galactoside-binding domain and is mainly expressed in immune cells and epithelial cells. Galectin-3 is known to play regulatory roles in the immune system to defend against pathogens. Previous studies showed that Alix is a host factor employed by HIV-1 for its replication. Here, we report that galectin-3 is another such factor. We have reported that galectin-3 is translocated to the immunological synapse and affects T cell cytokine production. We have now studied the role of galectin-3 in the virological synapse (VS). Our data showed that HIV-1 infection induces galectin-3 expression in primary CD4 T cells. Immunofluorescence imaging showed that galectin-3 is translocted to the virological synapse and co-localized with Gag and Env at the cell-to-cell junction of HIV-1-infected cells. We have studied the effects of galectin-3 in the VS by using fluorescent-tagged HIV-1 and incubation of fluorescent-labeled T cells with galectin-3 knockeddown (KD). Our analysis indicates that HIV-1 transmission efficacy is significantly attenuated in galectin-3 KD cells comparing to galectin-3-expressing wild type cells. Our analysis also shows that Alix plays a role in promoting cell-to-cell transfer of HIV-1. Further analysis indicate that effects of galectin-3 in effector cells is likely more important than expression of galectin-3 in target cells. We conclude that endogenous galectin-3 translocates to VS and promotes cell-to-cell viral transfer upon HIV-1 infection.
IFNγ attenuates of yellow fever virus 17D vaccine strain in vivo and its implication for severe adverse events (VIR1P.966)
Authors: Lam Long Kwan Metthew , Watson Alan , Klimstra William , Ryman Kate
Published: January 2023

The yellow fever virus (YFV) vaccine strain 17D elicits long-lasting immunity against wild-type YFV infection. The success of 17D has led to vaccine candidates using 17D as a vector to deliver heterologous antigen against other pathogens. However, the attenuation mechanism for 17D remains elusive and reports of rare but potentially lethal severe adverse events (SAE) post-vaccination raise questions about the continued use of 17D. Type I IFN receptor-deficient mice (AB6) are susceptible to subcutaneous infection with WT virus and display clinical signs of disease similar to humans whereas 17D infection is self-limiting. In mice lacking both type I and type II IFN receptors (AGB6), 17D results in a lethal infection, suggesting that the vaccine virus is attenuated by IFNγ. Intact IFNγ responses restrict 17D replication to lymphoid organs whereas 17D spreads viscerally and neuroinvades leading to paralysis in the absence of IFNγ. Interestingly, IFNγ production is largely restricted to draining lymph nodes (DLN) but antiviral gene induction is observed globally. In the presence of IFNγ, we observe more robust expression of MIG, MCP-1 and IFIT1, indicative of potential downstream effectors against 17D. We are investigating the DLN immune infiltrates and identifying the IFNγ producing cells. Our data suggest that host-specific variations in the IFNγ signaling network may increase susceptibility to SAE following vaccination.
NK cells modify anti-influenza CD8 T cell responses and memory formation (VIR2P.1014)
Authors: Rose David , Tompkins Stephen , Tripp Ralph , Klonowski Kimberly
Published: January 2023

Natural Killer (NK) cells are part of the first line of defense against viral infection as they quickly mobilize and eliminate infected cells independent of prior activation. However, recent evidence also suggests that NK cell activity can influence subsequent adaptive immune responses via contact dependent and independent interactions with APCs and effector T cells. Despite the importance of NK cells in coordinating these early responses, little is known regarding how this NK cell-mediated tuning regulates long-term anti-viral immunity. Using an antibody depletion method, we studied how loss of NK cells impacts the formation and maintenance of influenza-specific memory CD8 T cells. We find that in the absence of NK cells, there are increased numbers of influenza-specific CD8 T cells at the peak of the response and persisting into memory, with a skewed memory phenotype apparent earlier than observed in non-depleted mice. Loss of NK cells also reduced levels of lung-derived IFNγ and indoleamine 2,3-dioxygenase (IDO), known T cell modulators. Moreover, the anti-influenza response after heterologous challenge was also altered in the NK cell depleted cohort. Together these data suggest that NK-derived IFNγ may regulate local immunity by suppressing anti-viral memory CD8 T cell development. Importantly, loss of NK cell-derived signals has long-term consequences suggesting that differential regulation of NK cell activation should be considered in rational vaccine design.
The role of unanchored polyubiquitin chains and the TRIM E3-ubiquitin ligase family of proteins in the innate immune response to influenza virus infection (INM3P.402)
Authors: Rajsbaum Ricardo , Bharaj Preeti , Ellis Jane , Versteeg Gijs , Schmid Sonja , Maestre Ana , Belicha-Villanueva Alan , Patel Jenish , Morrison Juliet , Giuseppe Pisanelli Giuseppe , Miorin Lisa , Martínez-Romero Carles , Laurent-Rolle Maudry , Moulton Hong , Stein David , Fernandez-Sesma Ana , tenOever Benjamin , Garcia-Sastre Adolfo
Published: January 2023

Intracellular innate immune responses are essential to protect host cells against pathogens. Upon infection, pattern recognition receptors detect incoming microbes and trigger downstream signaling pathways leading to production of antiviral type-I interferons (IFNs).These signaling pathways are regulated by different posttranslational modifications including the ubiquitin (Ub) system. Proteins covalently attached to K48-linked polyUb are targeted for degradation by the proteasome, whereas protein modification with K63-linked polyUb may have non-proteolytic activating functions. Unanchored Ub chains that are not covalently attached to any protein are also important for kinase activation. Tripartite motif (TRIM) proteins, which function as E3-ubiquitin ligases, have been implicated in antiviral activity. We found that TRIM6 is important in the synthesis of unanchored K48-linked polyUb chains, which activate the IKKε kinase for STAT1 phosphorylation and induction of an antiviral response. These polyUb chains and IKKε interact in vivo in an IFN-I signaling dependent manner upon virus infection in mice. To better understand the physiological role of unanchored polyUb during virus infection, we developed a system to isolate ubiquitin-interacting proteins from the lungs of influenza-infected mice. Using a proteomics approach we identified new cellular factors that interact with unanchored polyUb chains in vivo and may be involved in the innate immune antiviral response.
The tumor suppressor PTEN enforces Treg stability in control of TH1 and TFH cell responses (IRC7P.427)
Authors: Shrestha Sharad , Yang Kai , Guy Cliff , Vogel Peter , Neale Geoffrey , Chi Hongbo
Published: January 2023

Regulatory T cells (Tregs) suppress CD4+ T cell responses during homeostasis and inflammation to prevent autoimmunity and other immune disorders. Although the transcriptional and epigenetic programs regulating Treg function have been extensively studied, the signaling and metabolic pathways underlying Treg stability and function are not fully understood. In this study, we determined the role of the phosphatase PTEN in Tregs. We found that specific depletion of Pten in Tregs results in excessive TH1 and T follicular helper cells (TFH) responses, associated with elevated germinal center (GC) B cells and spontaneous development of autoimmune and lymphoproliferative disease in vivo. Interestingly, the exaggerated TFH and GC responses and autoimmune symptoms are reduced when IFNγ expression is abrogated in mice containing Pten-deficient Tregs. Thus, the uncontrolled TH1-mediated inflammation in these mice drives aberrant TFH responses and autoimmune and lymphoproliferative disease. Mechanistically, we linked PTEN to mTORC2-mediated control of transcriptional and metabolic programs that enforce Treg stability and function. Consistent with this notion, deletion of Rictor, the obligate component for mTORC2, restores Treg function and stability in the absence of Pten. Together, these results establish that Tregs rely on the PTEN-mTORC2 axis to maintain their stability and suppressive activity in controlling TH1 and TFH cell responses.
Regulation of T cell function by butyrophilin 3A1 (CD277) (IRC8P.453)
Authors: Wang Jinghua , Manick Brian , Wu Guoping , Kalabokis Vassili
Published: January 2023

The butyrophilins have similar immunomodulatory functions to B7 family, and may represent a novel subset of co-regulatory molecules. However, the functions of these butyrophilin molecules remain to be identified. We expressed the human butyrophilin 3A1 (BTN3A1) extracellular domain human IgG1 Fc Chimeras in a mammalian cell line and investigated the biological functions of the purified recombinant BTN3A1 protein in vitro. In this study, human T cells were treated with plate-bound anti-CD3 and either BTN3A1/Fc, or control human IgG1 Fc. The levels of multiple cytokines were measured in cell culture supernates using R&D Systems Proteome Profiler™ Human Cytokine Array, and confirmed by measuring individual cytokines using cytokine-specific Quantikine® ELISA kits. BTN3A1 significantly reduced T-cell derived cytokines including IL-2 and IL-5. BTN3A1 also markedly inhibited anti-CD3-induced human T cell proliferation. Furthermore, BTN3A1 showed binding to anti-CD3 activated T cells, but not inactivated T cells. Moreover, recombinant human BTN3A1 modified TCR signaling to inhibit Foxp3 expression and regulatory T cell development in naïve CD4+ T cells. Taken together, our data suggest that BTN3A1 acts as a co-inhibitory molecule to modulate T cells through unknown receptor on the surface of activated T cells.