bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-10-independent regulatory role for B cells in suppressing autoimmunity through GITRL-mediated maintenance of Tregs (176.18)
Authors: Ray Avijit , Basu Sreemanti , Williams Calvin , Salzman Nita , Dittel Bonnie
Published: January 2023

A regulatory role for B cells has been shown in animal models of autoimmunity and several studies suggested an IL-10-dependent mechanism. In our model of experimental autoimmune encephalomyelitis (EAE), WT mice exhibited a monophasic disease and recovered, but B cell deficiency, either genetic (µMT) or anti-CD20-mediated, led to unresolved EAE. Although B cells regulated EAE, we did not detect IL-10-producing B cells. Hence, we investigated IL-10-independent roles for B cells in immune regulation. B cell deficiency resulted in reduced numbers of CD4+Foxp3+ T regulatory cells (Tregs) in the periphery, suggesting involvement of B cells in their peripheral homeostasis. Interestingly, B cell depletion therapy in humans has been associated with the onset of autoimmunity. Likewise, reduction of Tregs post anti-CD20-mediated depletion of B cells resulted in spontaneous EAE and accelerated colitis in genetically susceptible mice. To study B cell-Treg interactions, we transferred B cells into µMT mice, which resulted in the proliferation and partial restoration of Treg numbers, sufficient for the resolution of EAE. Mechanistically, expression of glucocorticoid-induced tumor necrosis factor receptor-ligand (GITRL) by B cells was required, but IL-10 and B7 molecules were dispensable. Our results demonstrate a novel IL-10-independent role for B cells in regulating autoimmunity by maintenance of Tregs via GITRL.
Tolerogenic properties of lymphatic endothelial cells are controlled by the lymph node microenvironment (176.25)
Authors: Cohen Jarish , Fu Yang-Xin , Engelhard Victor
Published: January 2023

Lymphatic endothelial cells that reside in lymph nodes (LN-LEC) express peripheral tissue antigens, including the melanocyte differentiation protein tyrosinase, and induce deletional tolerance of tyrosinase-specific CD8 T cells. We have also recently shown that deletional tolerance is due to PD-L1 expressed by LN-LEC. To determine whether LEC from tissue lymphatic capillaries also have tolerogenic properties, we purified LEC from diaphragm (D-LEC) and colon (C-LEC). D-LEC and C-LEC express significantly less PD-L1 than LN-LEC, and express tyrosinase at levels insufficient to induce proliferation of tyrosinase-specific CD8 T cells in vitro. This suggested that cells and factors in the LN microenvironment induce the tolerogenic properties of LN-LEC. LEC from postnatal day 1 LN, which are severely lymphopenic, do not express PD-L1, but do so by day 7 when lymphocytes have colonized the LN. Adult LN contain 2 distinct LN-LEC subsets: PD-L1hi ICAM-1hi (LN-LEChi) and PD-L1int ICAM-1int (LN-LECint). Interestingly, tyrosinase expression and presentation is restricted to the LN-LEChi population. The representation and absolute number of LN-LEChi cells is reduced in Rag1-/- mice, µMT-/- (B cell-deficient) mice, and mice in which lymphotoxin β receptor (LtβR) signaling is blocked. Thus, LN-LEC are induced to express PD-L1 during the neonatal period, and B cells and LtβR signaling contribute to maintain a LN-LEC population that controls peripheral tolerance to tyrosinase.
Novel tissue regulatory role of IL-22 in human asthma (176.4)
Authors: Pennino Davide
Published: January 2023

BACKGROUND: Asthma is a chronic inflammatory disorder of the airways. Recently, IL-22 has been shown to mediate unclear anti-inflammatory effects in mouse models of lung inflammations. OBJECTIVE: To investigate the anti-inflammatory role of IL-22 in human asthma. METHODS: T cells derived from lung biopsies of asthmatic patients were characterized by flow cytometry. Human bronchial epithelial cells were stimulated with IL-22, IFNg or the combination of both cytokines, and their effects were investigated by whole genome analysis, ELISA and flow cytometry. The functional consequence of cytokine effects was evaluated by in vitro wound repair and T cell mediated cytotoxicity experiments. In vivo cytokine expression was measured by luminex in bronchioalveolar lavage fluids (BAL) of asthmatic patients. RESULTS: Th22 produce IL-22 during asthma. IFNg and IL-22 induced antagonistic responses on epithelial cells. IFNg antagonized IL-22 mediated wound closure while IL-22 impaired the expression of IFNg induced molecules such as MHC-I, MHC-II, CD54/ICAM-1, CCL5/RANTES, CXCL10/IP-10 and the CD8-mediated epithelial cell damage. Consistently, the IL22/IFNg ratio inversely correlated with the expression of CCL5/RANTES and CXCL10/IP-10 in BAL fluids of asthmatic patients. CONCLUSIONS: IL-22 is the first cytokine that act as anti-inflammatory by inhibiting IFNg pro-inflammatory effects on epithelial cells. IL-22 might be used to control the extent of IFNg mediated lung inflammation.
The IgM Fc receptor, FCMR, promotes B cell development and modulates antigen-driven immune responses (176.9)
Authors: Choi Seung-Chul , Wang Hongsheng , Tian Linjie , Murakami Yousuke , Shin Dong-Mi , Borrego Francisco , Morse Herbert , Coligan John
Published: January 2023

FCMR is a Fc receptor specific for pentameric IgM expressed at high levels by B cells. Although circulating IgM has profound effects on responses to pathogens, autoimmunity and B cell homeostasis, the biologic consequences of its binding to FCMR are poorly understood. We interrogated FCMR contributions to B cell function by studying mice lacking FCMR. FCMR transcripts are expressed at highest levels by follicular (FO) B cells and at much lower levels by developing B cells and other mature B cell subsets. FCMR-deficient mice have reduced numbers of developing B cells, splenic FO and peritoneal B-2 cells, but increased levels of peritoneal B-1a cells and autoantibodies. Following immunization, germinal center B cell and plasma cell numbers are increased. FCMR-deficient B cells are resistant to apoptosis induced by BCR ligation. Our studies demonstrate that FCMR functions are critical for B cell homeostasis, the prevention of autoreactive B cells and responsiveness to antigenic challenge.
Detection of auto-IgE in pediatric atopic eczema indicates autosensitization as an early immunological event. (177.12)
Authors: Seher Tanja , Mayer Johannes U. , Beck Helen , Brockow Knut , Traidl-Hoffmann Claudia , Ollert Markus , Ring Johannes , Gutermuth Jan , Schmidt-Weber Carsten B.
Published: January 2023

IgE-mediated allergies (e.g. asthma, atopic eczema [AE]) affect about 25% of western populations. Autoreactive IgE was detected in AE-patients and suspected to aggravate and chronify the disease. It is unclear, if autosensitization is a complication due to chronic allergic inflammation in adults or whether it is already present in children. Aims of this study were to develop a high-throughput assay for serologic detection of auto-IgE and to analyze if auto-IgE is organ-specific. We established an immunoassay to screen autoreactivity against whole cell protein extracts from primary keratinocytes (pKc) from non-AE and AE-patients, intestinal- and airway epithelial cells. After defining optimal conditions for all protein-extracts, we established a 384 well plate based immunoassay. We compared IgE-reactivity of 106 AE-patients (10mo.-70y.) and healthy controls on protein-extracts of all four cell types. 25% of AE-pts. showed IgE-autoreactivity against pKc from non-AE patients, 29% against pKc of AE-pts. and 33% against FHs74 Int. cells. Interestingly, the highest reactivity was detected in children. pKc from non-AE and AE-pts. bound more auto-IgE than HaCaT cells. This study identified auto-IgE at a similar level as reported previously. The IgE-specificity did not show organ-specificity, but auto-IgE seems to be an early immunological feature of AE in children. Current studies in birth-cohorts address the kinetics of auto-IgE-levels in relation to age and disease activity.
Global microRNA expression is essential for mast cell development in vivo (177.18)
Authors: Oh Sun-Young , Brandal Stephanie , Roers Axel , Zhu Zhou , Takemoto Clifford
Published: January 2023

microRNAs (miRNAs) are small, non-coding RNAs that have been shown to play a critical role in both normal physiology and disease, such as hematopoietic development and cancer. However, their role in mast cell function and development is unexplored. The RNaseIII endonuclease, Dicer, is required for the processing of pre-miRNAs into mature miRNAs. To investigate the role of global miRNA depletion on mast cells in vivo, we generated a mast cell-specific knock out of Dicer in mice. Transgenic mice (Mcpt5-Cre) that express Cre selectively in mast cells were crossed with mice containing the floxed conditional Dicer allele (Dicer f/f). Mice with both homozygous (Dicer -/-) and heterozygous (Dicer f/-) mast cell-specific deletion of Dicer were viable and healthy. However, Dicer -/- mice were found to have a profound mast cell deficiency with near complete loss of peritoneal, gastrointestinal, and skin mast cells. We examined the in vivo functional consequence of mast cell-specific Dicer deletion using an IgE-dependent passive systemic anaphylaxis (PSA) murine model. IgE sensitized wild type (Dicer f/f) and heterozygous (Dicer f/-) mice show marked hypothermia with antigen; however, homozygous (Dicer -/-) mice were completely unreactive to antigen challenge. These studies suggest a critical role for Dicer and miRNA expression for establishment of mast cell tissue compartments in vivo.
Mast cell specific negative regulation of LPS induced pro-inflammatory response by Itk and Btk (177.20)
Authors: Huang Weishan , Morales J. Luis , Gazivoda Victor P. , August Avery
Published: January 2023

Gram-negative bacterial endotoxin lipopolysaccharide (LPS) induces mast cell secretion of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, which is responsible for septic hypothermia. The non receptor tyrosine kinases Itk and Btk are both expressed in mast cells, and act as signaling regulators, but their roles in the response of mast cells to LPS are unclear. Here we show that Itk/Btk double knockout (DKO) mice suffered exacerbated and prolonged LPS induced hypothermia with increased TNF-α production by mast cells, compared to WT, Itk-/- and Btk-/- mice. This aggravated LPS induced hypothermia was recapitulated in mast cell deficient Sash mice reconstituted with DKO bone marrow derived mast cells (BMMCs). LPS (TLR4 ligand) stimulated DKO BMMCs exhibited hyperactive pro-inflammatory cytokine production with severely altered NF-κB signaling, which was not observed with Pam3CSK4 (TLR1/2 ligand). By contrast, DKO bone marrow derived macrophage and dendritic cells were not hyper-responsive to LPS as observed in DKO BMMCs, suggesting that Itk and Btk act as mast cell specific negative regulators in LPS/TLR4 signaling. Microarray analysis further revealed that the absence of Itk and Btk in mast cells significantly enhanced LPS induced pro-inflammatory program, which could potentially deteriorate endotoxin induced mast cell mediated inflammatory diseases.
Expression of ADAM10 on mast cells: effects on IgE and stem cell factor activation (177.9)
Authors: Faber Travis , Fernando Josephine , Pullen Nicholas , Morales Johanna , Conrad Daniel , Ryan John
Published: January 2023

Mast cells are innate immune cells best characterized for their role in allergy. In response to cross-linking of the high-affinity IgE receptor, FcϵRI, mast cells release mediators such as histamine, leukotrienes, IL-6, and TNF-α. Additionally, mast cells are widely noted for their secreted proteases like chymases and cathepsins. We have decided to investigate the role that the protease A Disintegrin and Metalloproteinase 10 (ADAM10) plays on murine mast cells. To date, we have discovered mast cells express ADAM10 in vitro and in vivo, and that Treg cytokines TGF-β1 and IL-10 down regulate mast cell ADAM10. IL-10 effects depend on STAT3. ADAM10 deletion results in abrogated mast cell migration through Collagen IV to stem cell factor, but not to antigen. Also, ADAM10 deletion decreases IL-6 release post cross-linking, but does not alter other commonly released cytokines such as TNF-α, IL-13, and MIP-1α. Because of these findings, we hypothesize that ADAM10 plays a pro-inflammatory role in mast cell migration and also in allergic disease.
2P Imaging Reveals that PD-1 Induces Functional Exhaustion by Stabilizing Synapse Formation during Viral Persistence (178.16)
Authors: Zinselmeyer Bernd , Heydari Sara , Nayak Debasis , McGavern Dorian
Published: January 2023

Restoration of T cell function mediated by PD-1 blockade is a finding of great clinical relevance for the treatment of persistent infections and tumors. Presently, it is not known how PD-1 blockade improves the function of exhausted T cells. Infection of mice with an immunosuppressive strain of lymphocytic choriomeningitis virus (LCMV) is a well described model for T cell exhaustion. Here, we set out to gain novel insights into the mechanisms that underlie T cell exhaustion by imaging virus-specific CD8 and CD4 T cell responses to an acute versus persistent LCMV infection in the spleen using two-photon microscopy. Analysis of T cell dynamics in these two distinct environments revealed that T cell exhaustion during viral persistence is mediated by prolonged motility paralysis in the splenic marginal zone and red pulp - two regions where an abundance of viral antigen and PD-L1 was found. Importantly, we observed that intravenous injection anti-PD-1 enhanced anti-viral T cell motility in 30 minutes, which coincided with increased phosphorylation of ZAP-70 and ERK. Once T cells were released from infected targets, IFN-γ production significantly increased (within 1 hr), viral loads dropped by 70% (within 16 hrs), and mice ultimately developed fatal immunopathology (with 2-4 days). These data indicate that the PD-1 disables anti-viral CD8 and CD4 T cell signaling and function by stabilizing the synapse and holding them in a state of motility paralysis.
Effects of PD-1 signaling on the molecular control of T cell expansion (178.17)
Authors: Patsoukis Nikolaos , Brown Julia , Petkova Victoria , Liu Fang , Li Lequn , Boussiotis Vassiliki
Published: January 2023

Programmed death (PD)-1 is a potent T cell inhibitor critical for peripheral tolerance. Currently, it is unclear whether PD-1 has a global or a selective inhibitory effect on TCR signaling pathways involved in T cell expansion. It is also unclear how PD-1 affects the molecular machinery of cell cycle progression. Using primary human T cells we determined that PD-1 blocked cell cycle progression in the G1 phase. PD-1 did not alter the expression of G1 phase cyclins or cyclin-dependent kinases (Cdks) but, instead, suppressed transcription of the SCFSkp2 ubiquitin ligase that leads p27kip1 to degradation and resulted in accumulation of p27kip1. T cells receiving PD-1 signals displayed impaired Cdk2 activation and failed to phosphorylate Rb and Smad3 leading to suppression of E2F target genes but enhanced Smad3 transactivation. These events resulted in abrogation of the Cdk-activating phosphatase Cdc25A and upregulation of the Cdk4/6 inhibitor p15INK4. Suppression of Skp2 by PD-1 was mediated by inhibition of both PI3K/Akt and Ras/MEK/Erk pathways and was only partially reversed by IL-2, which restored activation of MEK/Erk but not Akt. In contrast to the inhibition of Ras and MEK/Erk pathway, PD-1 did not affect activation of Rap1, p38 and JNK MAP kinases. Thus, PD-1 selectively targets Ras/MEK/Erk and PI3K/Akt signaling to inhibit transcription of Skp2 and to activate Smad3 as an integral component of a pathway regulating blockade of cell cycle progression in T lymphocytes.
Vitamin D promotes Tr1 differentiation through the CD46 pathway (178.18)
Authors: Astier Anne , Kickler Karoline , Ni Choileain Siobhan
Published: January 2023

The complement regulator CD46 induces a regulatory Tr1 phenotype, characterized by large amounts of IL-10 secretion. Secretion of IL-10 upon CD46 activation is largely impaired in T cells from patients with multiple sclerosis (MS). Vitamin D has an immunomodulatory role and may be beneficial in several pathologies, including MS. Herein, we show that, upon CD46 costimulation, Vitamin D strongly increased the IL-10:IFNγ ratio, characteristic of the CD46-induced Tr1 phenotype. This was correlated with the modulation of several markers known to control T cell activation. Vitamin D strongly promoted the expression level of CD28, CD25 and CTLA-4 on CD46-activated T cells, while it concomitantly decreased CD46 expression. Importantly, addition of Vitamin D could restore the defective IL-10 production observed in T cells from patients with MS. Hence, Vitamin D promotes anti-inflammatory responses by mainly acting on CD46, and might be beneficial for T cell responses in MS.
Adoptive transfer of Regulatory T cells from burn-injured rats depressed T cell responses in the recipient sham rats (179.1)
Authors: Fazal Nadeem , Shelip Alla , Melaku Kalkidan , Bougher Ryan
Published: January 2023

Immunosuppression occurs after burn injury with or without septic complications. We hypothesize that CD4+CD25+ T cells (Treg) are responsible for transmitting inhibitory signals to the IL-2-producing T cells in burn related inflammatory/infectious conditions. Enriched Treg were obtained from day-3 burn rats by flow cytometry and magnetic microbead cell sorting and given intravenously to sham rats. The rats were then sacrificed day 1 and day 3 post-adoptive transfer and T cell proliferation responses were assessed by thymidine assay. The results indicated that when burn Treg were adoptively transferred to sham rats, T cell proliferation (CD4+, CD4+CD25+ and CD4+CD25-) of sham rats was significantly depressed. Furthermore, the results showed that adoptive transfer of Treg did not influence the phenotypic expression of CD4+ T cells of the recipient rats. The percentage expressions of CD25, CD11a (LFA), and CD62L of the recipient rats remained same as before adoptive transfer. Experiments are currently underway to expand these findings to burn plus septic rats. These preliminary findings however allow us to hypothesize that while burn injury alone can produce attenuated CD4+ T cell mediated responses as a result of actions of Treg; burn injury with septic complications may further cause an irreversible loss of T cell-mediated responses. The latter happening could be responsible for high morbidity and mortality in the injured host afflicted with burn plus a critical infection.
The protective effects of bee venom on cisplatin-induced nephrotoxicity in mice (179.6)
Authors: Kim Hyunseong , Lee Hyojung , Lee Gihyun , Park Soojin , Shin Minkyu , Bae Hyunsu
Published: January 2023

Cisplatin is widely used as a potent effective anticancer drug. However it causes acute renal injury and dysfuntion, mainly nephrotoxicity. Bee venom (BV) has traditionally been used for pain reduction and known to have anti-inflammatory activities. This study designed to investigate the effects of BV on cisplatin-induced nephrotoxicity in mice. BV was administrated once a day for 5 days. After 2 days from BV injections, all groups of mice received a single injection of cisplatin. Mice were sacrificed at 72 hours after cisplatin administration for kidney histological examination and cytokine production analysis. BV treated group showed significantly reduced levels of creatinine, blood urea nitrogen (BUN) and renal tissue damage compared with control. In addition, CD4+CD25+Foxp3+ regulatory T cells (CD4+CD25+Foxp3+Tregs) were increased by BV administration. Moreover, depletion of Treg cells by anti-CD25 antibody injection abolished the renoprotective effects of BV in cisplatin induced nephrotoxicity. These results demonstrated that BV possessed remarkable renoprotective effects in cisplatin-induced nephrotoxicity through regulation of CD4+CD25+FoxP3+Tregs without interfering cisplatin anti-cancer effects.
Defective nuclear IKKα function in patients with ectodermal dysplasia with immune deficiency (180.19)
Authors: Jain Ashish , Ma Chi , Zhao Yongge , Temmerman Stephane
Published: January 2023

Ectodermal dysplasia with immune deficiency (EDI) is an immunological and developmental disorder caused by alterations in the gene encoding NF-κB essential modulator (NEMO; also known as IκB kinase γ subunit [IKKγ]). Missense mutations in the gene encoding NEMO are associated with reduced signal-induced nuclear translocation of NF-κB proteins, resulting in defective expression of NF-κB target genes. Here, we report 2 unrelated male patients with EDI, both of whom have normal NEMO coding sequences, but exhibit a marked reduction in expression of full-length NEMO protein. TLR4 stimulation of APCs from these patients induced normal cytoplasmic activation and nuclear translocation of NF-κB. However, cells deficient in full-length NEMO were defective in expression of NF-κB-regulated cytokines, such as IL-12, suggesting a downstream defect in chromatin accessibility for NF-κB transcription factors. TLR4-stimulated APCs from the patients were defective in IKKα-dependent H3 histone phosphorylation at the IL-12 promoter and recruitment of NF-κB heterodimers RelA and cRel to the promoter. Expression of a super-active form of IKKα restored IL-12 production in a NEMO knockdown human monocytic cell line following LPS treatment. Our findings suggest that NEMO regulates the nuclear function of IKKα and offer new insights into the mechanisms underlying diminished NF-κB signaling in patients with EDI.
Characterization of Chicken Dendritic Cell Markers (181.1)
Authors: Lillehoj Hyun , Lee Sung
Published: January 2023

New mouse monoclonal antibodies which detect CD80 and CD83 were developed to characterize chicken dendritic cells (DCs). The characteristics of these molecules have been studied in human, swine, ovine, feline, and canine but not in chickens. MAbs to chCD80 showed staining of chicken macrophage cell line (HD11), bursa, and spleen. MAbs against chCD80 reduced chCD80’s ability inducing T cell proliferation and IFN-γ production in blast chicken spleen cells, and NO secretion in the HD11. Two mAbs, CD83-159 and CD83-22, reacted with a HD11 and recognized a single 53 kDa protein on Western blots of lysates from LPS-stimulated spleen mononuclear cells. Immunostaining of chicken secondary lymphoid organs identified CD80+ and CD83+cells with morphologic and subtissue localization properties comparable to mammalian DCs. In vitro stimulation of spleen monocytes with concanavalin A (Con A) decreased the percentage of CD83+ cells compared with cells treated with medium alone. Interestingly, spleen cells treated with Con A in the presence of chCD83-227 mAb exhibited decreased percentage of MHCII+ cells compared with cells treated with plus an isotype-matched negative control mAb. These mAbs will be useful for future investigations of DC maturation and mechanisms of action in chickens.
Molecular characterization of antigen-specific B1 and B2 B-cells populations in Cryptococcus neoformans infection in mice (43.1)
Authors: Rohatgi Soma , Pirofski Liise-anne
Published: January 2023

We examined B1 and B2 B-cell repertoires in Cryptococcus neoformans infection in mice. Flow-cytometry based antigen-specific staining for B-cell populations generated in response to pulmonary infection was performed using labeled un-encapsulated and encapsulated C. neoformans strains. FACS sorting and single-cell nested PCR was employed to characterize the IgH repertoire. An increase in total peritoneal B1a, B1b and B2 cell numbers was observed as early as day 3 post-infection. Frequencies of both acapsular and capsular antigen-specific cells were increased in the B1a subpopulation (18%, 5%) in infected compared to naïve mice (11%, 2%). The heavy chains of antigen-specific B2 and B1b cells recognizing acapsular and capsular cryptococcus belonged predominantly to the VH1 family. Interestingly, increased VH11 (22%) and VH12 (33%) usage was observed in antigen-specific B1a cells recognizing acapsular and encapsulated cryptococcus, respectively, compared to the conventional B1a population. Although all acapsular (VH11+) and capsular (VH12+) B1a cells used the same VH11.2.53 and VH12.1.78 genes, 60% and 80% rearrangements were unique, respectively. VH genes in both cases were essentially germline, however 40% of acapsular specific B1a cells had 4 or more N-nucleotide additions compared to capsular specific B1a cells (20%). These results indicate that naturally occurring phosphatidyl choline reactive B1a cells have broad specificities and can bind to encapsulated cryptococcus.
A2B adenosine receptors are essential for effective worm expulsion and enhanced Th2 type immune response to the intestinal parasite Heligmosomoides polygyrus (43.12)
Authors: Patel Nirav , Mishra Pankaj , Wu Wenhui , Hasko Gyorgy , Gause William
Published: January 2023

A2B adenosine receptor (A2B) signaling can modulate lymphocyte function.A2B receptors can control proinflammatory immune responses by enhancing IL-10 and down regulating IFN-γproduction.A2B receptors can activate mast cells to secrete IL-4,a Th2 cytokine that can trigger alternatively activated (M2) macrophages and expulsion of the intestinal nematode parasite, H. polygyrus (Hp).To examine whether A2B receptors contribute to the in vivo protective Th2-type immune response to helminthes, A2B-/- mice were infected with Hp.Worm burden and egg production were increased while serum IgE and IgG1 were reduced in A2B-/- mice compared to WT mice at day 14 after Hp inoculation. Worms recovered from A2B-/- mice at day 11 after secondary H. polygyrus inoculation exhibited higher protein and ATP levels,suggesting impaired host protective responses. Th2 cytokine expression and M2 macrophages markers were also inhibited in small intestines and in mesenteric lymph nodes (MLN) in A2B-/- mice at day11 after secondary Hp infection. Absence of A2B receptor signaling reduced IL-4 but not IFN-γ secreting MLN CD4+T cells after Hp inoculation. Eosinophil and M2 macrophage accumulation in the small intestine and increases in mucous producing intestinal epithelial cells were markedly inhibited in A2B-/- mice, after Hp secondary inoculation. These studies suggest that A2B receptors play an essential role in the development of the host protective Th2 type immune response during helminth infection.
Differential roles for Th1 transcription factors STAT1 and STAT4 during visceral leishmaniasis (43.22)
Authors: Snider Heidi , Gupta Gaurav , Satoskar Abhay
Published: January 2023

Leishmaniasis is a tropical disease caused by protozoan parasites belonging to the genus Leishmania. Leishmania donovani causes visceral disease (VL) with tropism for reticuloendothelial organs. Left untreated, L. donovani infections are fatal, often due to secondary bacterial infections and complications resulting from profound hepatosplenomegaly. Although Leishmania species are capable of parasitizing several different mammalian cell types, the definitive host cell and site of parasite replication is tissue macrophages. Proper macrophage activation and parasite clearance is contingent upon hosts mounting Th1 CD4+ T cell responses associated with the production of IL-12 and IFN-gamma. While IL-12 primarily signals upstream of the transcription factor STAT4, IFN-gamma signaling primarily leads to the activation of STAT1. Our laboratory has already shown that STAT1-/- mice are unexpectedly resistant to VL, despite its critical role in driving Th1 responses. We therefore wanted to investigate the phenotype of STAT4-/- mice. While both STAT1-/- and STAT4-/- mice were unable to mount effective Th1 cytokine responses following infection, STAT4-/- mice were highly susceptible to VL and exhibited significant tissue immunopathology compared to WT and STAT1-/- animals. These studies suggest that early STAT1 signaling is actually required for disease establishment prior to initiation of a Th1 response, while STAT4 signaling is indispensible for Th1-driven resistance.
Severe tuberculosis induces high degree of CD4 T cell differentiation (43.25)
Authors: Lyadova Irina , Nikitina Irina , Vasilyeva Irina , Ganusov Vitaly
Published: January 2023

Effector Th1 cells represent a key component of anti-TB defense. Until now it has been unclear whether active TB promotes or alters the differentiation of functionally active effector CD4 T cells. Our previous observations in mice have revealed that Mtb infection leads to the formation of highly differentiated Th1-biased effectors expressing CD27low phenotype. Here, we addressed the generation of CD27low effectors during pulmonary TB in humans. We have analyzed the percents of CD27low cells within Mtb-specific CD4 T lymphocytes (cells producing IFN-γ after stimulation with Mtb sonicate) located in the focus of lung Mtb infection or circulating in peripheral blood. In the lungs, CD27low subset formed a vast majority (>70%) of Mtb-specific CD4 T cells. In blood, the percents of CD27low cells varied from low (12%) to very high (92%). The major correlates for the accumulation of CD27low cells in the blood were lung tissue destruction and clinical TB severity (r>0.5; p<0.0001). Following the therapy, the content of CD27low cells declined in parallel with lung tissue repair. Compared to patients, the content of CD27low cells in healthy donors and Mtb-exposed individuals was significantly lower (p<0.001). Overall, there was no deficiency in the generation of CD4 effector cells during TB. Instead, active TB promoted the accumulation of highly differentiated CD27low effectors, which may serve as a valuable marker for TB severity and lung tissue destruction.
Tuberculosis-specific CD4+ and CD8+ T cells maintained in older latently infected individuals (43.27)
Authors: Canaday David , Tavera Gloria , Banks Richard , Aung Htin
Published: January 2023

One risk factor for reactivation of latent M. tuberculosis (MTB) is old age. Although there are significant data in elderly and nursing home patients on clinical matters such as use of two-step PPD testing, there are a severe paucity of data on changes in MTB-specific immune responses that occur with aging. We have studied MTB-specific immune responses using 13-color intracytoplasmic flow cytometry analysis of CD4+ and CD8+ memory T cell subsets simultaneously examining their expression of IFN-gamma, IL-2, MIP1-alpha, and perforin after stimulation with the MTB-antigen PPD. Data from 13 latently MTB-infected PPD+ older subjects (mean age 77 range 61-89) showed that MTB-specific responses from CD4+ and CD8+ T cells were maintained compared to 8 younger PPD+ subjects (mean age 31 range 23-40). Surprisingly, frequencies of MTB-specific IL-2 secreting effector and central memory CD4+ T cell were higher in older subjects (mean 1.1%) than in younger persons (mean 0.22%, p<0.05). In older subjects, many of these MTB-specific CD4+ T cells only secreted IL-2 after stimulation while there was no defect in their ability to produce the full range of cytokines studied when stimulated with mitogen. Overall, as there was no defect in MTB-specific T cells in older persons, these data support a need to further broaden the T cell studies as well to expand to other areas such as potential deficits in macrophage function.
Regulation of T-cell dependent IgG response to soluble protein antigens in the presence and absence of intact bacteria (43.9)
Authors: Saumyaa Saumyaa , Arjunaraja Swadhinya , Torres Raul , Snapper Clifford
Published: January 2023

During bacterial infections the immune system encounters Ags associated with the intact bacterium simultaneously with those that are released in soluble form, although the immunologic consequences of this on IgG responses to the latter are unknown. We previously demonstrated that intact inactivated Streptococcus pneumoniae (strain R36A) inhibits IgG responses to co-immunized soluble Ags. We investigated the mechanism of this inhibition using intact R36A and soluble chicken ovalbumin (OVA). Using the i.v. route, no inhibition was observed if R36A was given 24h before or after immunization with OVA, indicating an early and transient inhibitory effect. This inhibition could be partially overcome by co-immunization with a strong adjuvant. Using transgenic OVA-specific CD4+ T cells, we observed that R36A had no significant effect on OVA-induced T-cell activation (24h) or generation of regulatory T cells (d7), and only a modest effect on proliferation (48-96h). However, R36A mediated a significant reduction in PD1+GL7+ OVA-specific T cells (germinal center T follicular helper cells) suggesting an inhibitory effect on the GC reaction. Of note, co-immunization of OVA with either intact inactivated Streptococcus agalactiae or Neisseria meningitidis resulted in a significant enhancement of the OVA-specific IgG response. These results strongly suggest that Streptococcus pneumoniae may uniquely express a component capable of inhibiting humoral immune responses to soluble antigens.
Understanding the effects of IL-18 and IL-1β in a complex pro-inflammatory milieu. (44.11)
Authors: Ferguson John , Sleeman Matt , Briend Emmanuel , Finch Donna
Published: January 2023

Disease association studies suggest that the cytokines IL1 and IL18 play a role in exacerbations of Chronic Obstructive Pulmonary Disease. A classical role of IL18 is to stimulate NK and T-Cells to produce IFNγ, a cytokine associated with viral exacerbations of COPD. IL1β, which is processed by the same mechanism as IL18, can have a similar effect on IFNγ. We sought to understand their respective role in IFNγ secretion in situations where they both could be released. Treatment of PBMCs with IL18 or IL1β (in the presence of IL12) was able to induce IFNγ release. This response was most sensitive to IL1β but IL18 was able to stimulate a greater maximal response. When LPS was used as a trigger of IFNγ release, a large amount of IL1β (11ng/mL) and a low amount of IL18 (27pg/mL) could be detected in the supernatant. Co-incubation with an IL18 neutralizing antibody, or the IL1 receptor antagonist anakinra, was able to inhibit the IFNγ response by 50% in both cases. In a second system, relevant to COPD exacerbations, the infection of bronchial epithelial cells with a human Rhinovirus induced the release of both IL18 (2.5ng/mL) and IL1β (435pg/mL). Transfer of the supernatant to PBMCs or NK cells induced IFNγ production which was largely IL1-dependent; inhibition of IL18 only had a small effect on IFNγ. This data showed that while they could induce IFNγ following an inflammatory stimulus, it is the context of their release that defines a predominant role for either IL18 or IL1β.
G protein-coupled receptor kinase 6 mediated phosphorylation of activator of G protein signaling 3: role in CXCR2 activation and regulation (44.12)
Authors: Singh Vandana , Richardson Ricardo
Published: January 2023

G protein-coupled receptor (GPCR) kinases (GRKs) are kinases that phosphorylate activated GPCRs as their primary targets. Recent studies suggest that GRKs may have more diverse protein/protein interactions in cells. A screen to find GRK6 binding partners revealed activator of G protein signaling 3 (AGS3) as an interacting protein. Co-immunoprecipitation studies in RBL-2H3 cells stably expressing CXCR2 (RBL-CXCR2) showed that, upon stimulation with interleukin 8 (IL-8/CXCL8), GRK6 directly interact with and phosphorylate AGS3. The GRK6/AGS3 complex is time-dependent and peaks within 2-3 minutes. Over-expression of AGS3 in RBL-CXCR2 significantly inhibited intracellular Ca2+ mobilization, phosphoinositide (PI) hydrolysis and chemotaxis. Suppression of AGS3 by shRNA, in contrast, inhibited GRK6/AGS3 association and increased CXCR2 resistance to desensitization. Altogether, these data identify AGS3 as a GRK6 substrate and indicate that phosphorylation of AGS3 by GRK6 likely play an important role in modulating CXCR2 mediated cellular responses.
MicroRNAs modulate chemokine expression in naïve CD8 T cells (44.19)
Authors: Cheng Nai-Lin , Chen Xiaochun , Nguyen Cuong , Weng Nan-ping
Published: January 2023

Chemokines play a pivotal role in regulating T cell function and migration in a space- and time-tight fashion. With aging, the levels of several chemokines alter in serum and have detrimental impact on the immune function. However, the mechanisms that regulate chemokine expressions and their age-associated alteration are not fully understood. MicroRNAs (miRNA) are key regulators for gene expression at the posttranscriptional level but their roles in the age-associated change of gene expression in T cells have not been examined. We sought to understand the role of miRNA in regulation of chemokine expression in CD8 T cells by profiling chemokines and miRNAs in naïve and memory CD8 T cells. Six miRNAs (miR-99a, -125b, -186, -335, -424, and -542-5p) were highly expressed in naïve CD8 T cells. In addition, we found that miR-125b, -335, and -186 have a putative binding site in the 3’ UTR of CC chemokine ligand 4 (CCL4), CCL5, and CXC chemokine ligand 5 (CXCL5), respectively. These chemokines are highly expressed in activated T cells and increased serum levels with age. Currently, we are testing these individual miRNA in the chemokine-reporter transfected Jurkat cells and in human primary CD8 T cells from young (≤ 30 yrs) and old (≥ 70yrs) donors. Our preliminary findings suggest that miRNA regulates chemokine expression in CD8 T cells and such regulation may change with age.
IL-21 promotes the pathologic immune response to Pneumovirus infection (44.2)
Authors: Spolski Rosanne , Wang Lu , Wang Chi-Keung , Bonville Cynthia , Domachowske Joseph , Kim Hyoung-Pyo , Yu Zuxi , Leonard Warren
Published: January 2023

IL-21 is a type I cytokine whose targets include both lymphoid and myeloid populations and whose role in host responses to pulmonary pathogens has not been evaluated. Pneumonia virus of mice (PVM) is a natural rodent pathogen that initiates a respiratory infection in mice that resembles severe human respiratory syncytial virus (RSV) disease. We demonstrate that IL-21 is expressed by CD4+ T cells in the lung following infection of animals with PVM. Examination of PVM-infected Il21r-/- mice revealed much less lung infiltration by neutrophils than was seen in WT mice, and consistent with this, Il21r-/- mice also had reduced levels of the chemokine CXCL1 in both bronchoalveolar lavage fluid and lung parenchyma. Both CD8+ and CD4+ T cells as well as γδ T cells were also reduced in PVM-infected Il21r-/- mice, even though CD8+ T cell cytokine responses were not diminished. Th17 cytokines were expressed at the same levels as in WT mice, but IL-6 production in response to PVM was diminished in Il21r-/- mice. Although virus recovery was similar in WT and Il21r-/- mice, the absence of IL-21 signaling conferred a survival advantage to Il21r-/- mice, indicating that IL-21 can contribute to a detrimental inflammatory state in the lung. Overall, our findings suggest that therapeutic manipulation of IL-21 signaling, such as IL-21 receptor blockade, may represent an immunomodulatory strategy for averting the negative sequelae of this and potentially other respiratory virus infections.
CCL18 does not require CCR6 for functional activity in mice (44.3)
Authors: Atamas Sergei , Luzina Irina
Published: January 2023

CCL18 is an orphan chemokine present in primates but not in mice. Human CCL18 is fully functional in mice in vivo: it is chemotactic to mouse T lymphocytes similar to its effect on human T cells, suggesting that although mice have lost CCL18 after evolutionary separation from primates, its receptor remains preserved and functional. Attempts to identify CCL18 receptor have been unsuccessful, but a recent study (Am J Respir Crit Care Med 2011, 183:A5574) suggested that CCR6, the receptor for CCL20, is also used by CCL18. To explore this possibility, we administered a replication-deficient recombinant adenoviral construct encoding human CCL18 (AdV-CCL18) to mouse lungs in wild type (WT) and CCR6-deficient (-/-) animals. Non-manipulated as well as AdV-NULL-infected control WT mice had 2-4% of lymphocytes in their bronchoalveolar lavage (BAL), whereas AdV-CCL18-infected WT mice had 20-25 % of BAL lymphocytes. Flowcytometric analyses showed that less than 5% of CD3+ BAL cells in CCL18-expressing WT mice were also CCR6+. Histological changes caused by CCL18 delivery were similar in WT and CCR6 -/- mice, with characteristic perivascular and peribronchial infiltration of T cells which was not observed in AdV-NULL-treated mice. Thus, it appears that human CCL18 utilizes a receptor different than CCR6 in mice. A possibility exists that mice and humans utilize different CCL18 receptors, but it is unlikely considering the remarkable similarity of CCL18 effects on human and mouse cells.
TGF-β dramatically reduces the numbers of short-lived effector CD8 T cells during primary and secondary influenza virus infections. (44.4)
Authors: Hu Yinghong , Wu Tao , Bouchard Keith , Cauley Linda
Published: January 2023

Highly differentiated CTLs make large quantities of proinflammatory cytokines and contribute to high mortality rates during influenza virus infections. Some pathogens trigger robust responses by short lived effector CD8 T cells (SLECs) which express KLRG1 but not CD127. Other less differentiated CD8 T cells which lack KLRG1 and quickly regain CD127 expression are better equipped to avoid deletion which led to the designation memory precursor cells (MPECs). Recent studies have shown that influenza virus-specific CTLs do not adhere to this pattern, since very small numbers of virus-specific CTLs acquired the KLRG1+CD127- phenotype during the peak of the effector response. We show that exposure to TGF-β during antigen stimulation greatly increases the size of the CTL response and changes the balance between the numbers of MPECs and SLECs during primary and secondary influenza virus infections. We reveal a dichotomy in the response to TGF-β which develops as the virus-specific CTLs become more differentiated. Our data suggest that TGF-β protects the lungs from immune damage through widespread depletion of SLECs, while simultaneously helping less pathogenic virus-specific CTLs colonize the mucosa where they can enhance to response to repeated viral infection.
Distinct requirements for the cytokine-mediated activation of NKT and NK cells during viral infection. (45.16)
Authors: Tyznik Aaron , Verma Shilpi , Kronenberg Mitchell , Benedict Chris
Published: January 2023

Innate immune cells respond rapidly and provide protection early following infections. We have studied the roles of two innate lymphocyte cell types, natural killer (NK) cells and natural killer T cells (NKT cells) in the protective response to mouse cytomegalovirus (MCMV), a β herpes virus infection. The importance of NK cells for host protection following this infection had been known for many years, but here we establish that NKT cells are also important and that the two cell types have different cytokine requirements for optimal induction of IFNγ. NKT cells are most sensitive to IL-12, when a small amount of IL-18 is present. NK cells, by contrast, are more responsive to IFNβ combined with either IL-18 or IL-12. We further demonstrate that the two cell types are not redundant by identifying dendritic cells, that when infected with MCMV, activates NK cells but does not necessarily activate NKT cells. Consistent with these findings, the activation of NKT cells following MCMV infection was more drastically decreased; compared to NK cells, in mice lacking the ability to produce IL-12. BALB/c mice deficient for NKT cells had increased viral titers, and depletion of NK cells led to a further increase. Our data indicate that NKT and NK cells are both required for maximal MCMV protection, and that they have different requirements for cytokine mediated activation and may reflect the activity of different types of infected antigen presenting cells.
The role of autophagy and amphisomes in virus recognition, TLR 9 recruitment and virus-stimulated IFN-α production by human plasmacytoid dendritic cells (pDC) (45.19)
Authors: Deng Jing , Singh Sukhwinder , Dai Jihong , Fitzgerald-Bocarsly Patricia
Published: January 2023

Human pDC produce large amounts of IFN-α in response to viral nucleic acid stimulation via Toll-like receptor 7/9. However, the mechanisms of virus trafficking and virus-stimulated IFN-α production without infection in pDC remain unclear. In recent studies, we observed double-membrane autophagosomes formed within 2hr stimulation of pDC with HSV, Flu, HIV-1 or CpGA. We also found early endosomal marker Rab5 colocalized with autophagosomal marker LC3 in pDC after stimulation with HSV-GFP virus, indicating amphisomes were induced. These amphisomes were accumulated with chloroquine treatment and included internalized HSV-GFP particles. In this study, we knocked down autophagy gene Beclin 1 by siRNA and found virus-induced IFN-α production was significantly suppressed. We further investigated the interactions between early endosomes, autophagosomes and TLR9 after HSV stimulation using imaging flow cytometry: HSV-GFP particles were internalized into early endosomes by endocytosis and are subsequently transported to amphisomes; Concurrent with the trafficking of HSV-GFP, TLR9 rapidly redistributed to HSV-accumulating amphisomes by colocalizing with LC3 within 30min for ligand binding and signal transduction. Collectively, our studies continue to unveil a previously unknown mechanism of autophagy and amphisome formation in virus-induced IFN-α production possibly involving the recognition of virus and the recruitment of TLR9 to sites of virus uptake in human pDC.
The innate function of T cells in control of inflammation (45.21)
Authors: Tang Hong , Du Jie , Feng Jin , Ma Feifei
Published: January 2023

We have previously shown that T cells are both necessary and sufficient to temper Toll like receptor (TLR) mediated inflammatory response to pathogens, in a TCR-independent manner. In addition, CD8 gene targeting (CD8 KO) or CD8+ T cells depletion by antibody significantly reduce cardiac interstitial fibrosis induced by angiotensin II (Ang II) infusion, whereas CD8 KO mice reconstituted with CD8+ T cells become sensitive to Ang II. Most importantly, CD8+ T cells are required for macrophage infiltration and expression of monocyte chemoattractant protein 1 (MCP-1) that are responsible to Ang II-induced cardiac fibrosis. Furthermore, in vitro transwell experiments show that MCP-1 production by macrophages and its chemotaxis effect on macrophage motility require direct contact with activated CD8+ T cells, but independent of T cell receptor (TCR). A similar cardiac fibrotic response to Ang II is observed in both OT-I transgenic mice that expresses a surrogate TCR specific to ovalbumin and in wild type mice. Thus, CD8+ T cells are both necessary and sufficient for macrophage-induced hypertensive cardiac fibrosis. Therefore, TCR-independent activation of macrophages by CD8+ T cells casts yet a novel innate function of T cells that, in addition to suppressing the innate inflammatory response to infections, CD8+ T cells are required to activate inflammatory response of macrophages to danger signals.