bluuebunny/crossref_metadata_2025 huggingface dataset:
Intrinsic Interferon Signature in Dendritic Cells in lupus-prone mice. (83.9)
Authors: Gallucci Stefania , Bennett Heather , Shivers Debra , Ning Yue , Behrens Edward , Jog Neelakshi , Caricchio Roberto , Sriram Uma
Published: January 2023

Type I Interferons (IFNs) are pathogenic in Systemic Lupus Erythematosus. PBMCs from lupus patients over-express Type I IFN responsive genes. The cause and cellular source of this "Interferon Signature" are still unclear. Since we found that dendritic cells (DCs) from lupus-prone mice express the IFN Signature, we asked whether this IFN Signature was intrinsic to the DCs. We found that bone marrow-derived dendritic cells (BMDCs) from young pre-diseased Sle1,2,3 lupus-prone mice constitutively over-express Type I IFN responsive genes, analyzed by real-time RT-PCR (IFN-a, IFN-b, STAT1, STAT2, Mx1, IL-6, IL-15, IRF-7), as compared to autoimmune C57BL/6 BMDCs, as well as ELISA and flow cytometry. We also found that STAT1 is constitutively phosphorylated, suggesting an ongoing response to Type I IFN in the Sle1,2,3 DCs. Treatment with ligands for TLR7 and TLR9 showed that lupus BMDCs are hyper-sensitive to nucleic acids. To determine the mechanism of the constitutive hyper-activation of the IFN response in lupus DCs, we treated Sle1,2,3 BMDCs with inhibitors of TLR7 and TLR9 and found a partial role for chronic exposure to nucleic acids. We are now testing other mechanisms to explain the IFN Signature in lupus DCs. We have reported earlier that IL-4 suppresses Type I IFN responses in DCs of normal mice. We have now found that IL-4 inhibits the IFN Signature in Sle1,2,3 BMDCs, suggesting IL-4 as a potential therapeutic strategy for lupus by suppressing the Type I IFN effects.
Resident IL10-competent peritoneal B cells regulate colitis severity (84.18)
Authors: Maseda Damian , Smith Susan , Poe Jonathan , Tedder Thomas
Published: January 2023

B cells mediate multiple functions that influence immune and inflammatory responses. A rare subset of splenic CD1dhiCD5+ B cells has been found to modulate T cell responses, contact hypersensitivity and experimental autoimmune encephalitis by secreting IL10. However, equal numbers of B cells producing IL10 are found in the peritoneal cavity (PC). In this study, the phenotypic and functional characteristics of PC IL10-competent B cells were characterized. PC IL10-competent B cells share several phenotypic markers with their splenic IL10-competent counterparts, and were found within the B1a (47%), B1b (28%), and B2 (5%) subsets. Stimuli that promote in-vitro IL10 production differ between the spleen and PC populations, with PC B1 cells spontaneously producing IL10 without exogenous stimulation, while splenic B cell IL10 production is increased upon TLR stimulation. In a Rag-/- colitis model, the transfer of IL10-competent PC B cells dramatically reduced disease progression, reduced the number of activated and memory CD4+ T cells in the PC and total CD8+ T cells in the mesenteric lymph nodes. Together, these data reveal that IL-10 competent B cells in the peritoneal cavity significantly modulate T cell pathogenicity.
TCR and Fas-mediated apoptosis targets Effector Memory T cells (85.14)
Authors: Ramaswamy Madhu , Cruz Anthony , Price Susan , Rao V Koneti , Siegel Richard
Published: January 2023

The death receptor Fas plays an essential role in TCR-induced apoptosis of activated CD4+ T cells, in a process termed restimulation-induced cell death (RICD). However, within a heterogeneous population of human CD4+ T cells, we have found that cells with an effector memory phenotype (CCR7-CD27-) are specifically programmed for sensitivity to Fas-induced apoptosis and RICD. Similary, mouse effector memory T cells (CD44hiCD62Llo) are the most sensitive to cells are very sensitive to RICD and direct killing by Fas receptor ligation. Fas resistant ‘central memory’ as well as Fas-sensitive ‘effector’ cells had similar levels of induced FasL, surface Fas, as well as pro-survival Bcl2/BCLxL levels. In ALPS patients with Fas mutations, resistance to Fas apoptosis was found predominantly in the effector memory subsets. Biochemical studies show a preponderance of lipid raft localized Fas and highly efficient formation of the death-inducing signaling complex (DISC) in memory and effector T cells. Thus sensitivity to TCR and Fas-mediated apoptoisis is mediated at the level of early signaling events in the Fas pathway. These observations may explain the specificity of the Fas/FasL system in T cell homeostasis toward chronically expressed and endogenous antigens to check autoreactivity and responses to chronic infections.
A programmed gene expression initiated by clearance of infection turns CD4 memory T cells dormant (85.18)
Authors: Sadegh-Nasseri Scheherazade , Khoruzhenko Stanislav , Dalai Sarat
Published: January 2023

Memory T cells survive throughout the lifetime of an individual and are protective upon recall. We have demonstrated that low levels of antigen present at the resolution of infection and upon presentation by B cells induce a state of unresponsiveness in specific CD4+ memory T cells. We have shown that this process occurs naturally during the fall of antigen. The quiescent memory T cells become activated by stimulatory signals generated by a subsequent infection. Here we show that the quiescent memory T cells survive for over a year in the absence of homeostatic proliferation. Microarray gene expression assay of long-term memory CD4 T cells revealed downregulation of proliferation, immune activation and apoptosis prevention genes in unresponsive memory CD4 T cells, compared to the responsive group. Upregulated genes in quiescent memory T cells appear to work synergistically with the downregulated genes in that group. Data from these experiments suggest that memory CD4 T cells survive in the absence of antigen for extended period of time by becoming quiescent naturally as the infection fades and resume their activity once they encounter antigen simultaneously with danger signal, providing quick and efficient immune response.
Age-associated alteration in naive and memory Th17 cell response in humans (85.8)
Authors: Lee Jin Soo , Lee Won-Woo , Kim Sang-Hyun , Kang Seong Wook , Kang Insoo
Published: January 2023

Th17 cells play an important role in clearing extracellular microorganisms and developing autoimmune diseases. T cell immunity changes with aging, likely contributing to the increased risk of infection and malignancy in elderly people. However, it is not known whether aging affects Th17 cell response in humans. Thus, we investigated this issue. The healthy elderly (age ≥ 65) had a decreased frequency of IL-17-producing CD4+ T cells in peripheral blood after 4 hour PMA/ionomycin stimulation compared to the healthy young (age ≤ 40). In contrast, the frequency of IFN-γ-producing CD4+ T cells was not different between the two groups. We next purified naive and memory CD4+ T cell subsets from the peripheral blood of both groups and stimulated them for 7 days with T cell receptor triggering in the presence or absence of Th17-polarizing cytokines. The frequency of IL-17-producing cells in the memory subset was still lower in the elderly than in the young. However, the frequency of naive CD4+ T cells producing IL-17 was higher in the elderly than in the young. ELISA measuring IL-17 and IFN-γ levels in tissue culture supernatants showed similar results. These findings indicate that the elderly have a defect in maintaining Th17 memory cells but not in differentiating these cells from naive CD4+ T cells. Such a defect could potentially contribute to the increased risk of infection with aging.
Circadian clock gene expression in bone marrow derived mast cells and effects on FcϵRI expression (86.12)
Authors: Wang Xiaojia , Van Scott Michael , Brown Jared
Published: January 2023

Many functions in the immune system are associated with time-of-day. For instance, blood histamine levels in mastocytosis patients display a diurnal variation. In addition, pulmonary function and inflammation in patients with nocturnal asthma is more severe during the night. However, it is largely unknown if local circadian clocks in immune cells directly control physiological outcomes. We hypothesized that a circadian clock in murine bone marrow derived mast cells (BMMCs) modulates their activation in vitro. Mature BMMCs were synchronized with serum rich media (50% horse serum). Total RNA was harvested at 4 hour intervals for 72 hours following synchronization and expression of circadian genes (mPer1, mPer2, Bmal1, Rev-erbα, and Dbp) and fcer1a were amplified by qPCR. The surface expression of FcϵRIα was analyzed by flow cytometry every 4 hours for 72 hours following synchronization. Serum shock induced rhythmic expression of circadian genes (mPer2, Bmal1, Rev-erbα, and Dbp) with a mean period of 25.9 hours in BMMCs. The expression of fcer1a gene and FcϵRIα protein displayed expression changes following serum shock with mean periods of 26.3 and 28.6 hours respectively as calculated by Single Cosinor Time Series Analysis. These findings demonstrate that BMMCs express circadian clock genes and FcϵRI expression is modulated by synchronization with serum rich media. Serum shock of BMMCs may provide an in vitro model to study the circadian biology of allergic disease.
CCR3-mediated degranulation and eosinophil-associated RNases secretion from murine eosinophils; capacity of intact cells and free granules (86.21)
Authors: Shamri Revital , Melo Rossana , Weller Peter
Published: January 2023

Piecemeal degranulation (PMD), in which intact eosinophils completely or partially empty granules and selectively secrete performed mediators, is the principal mechanism for human eosinophil degranulation. Unlike extensively studied human eosinophils, both the capacity and mechanisms underlying eosinophil degranulation responses of murine eosinophils have been poorly investigated and remain controversial. We applied a new methodology to detect degranulation by measuring secretion of eosinophil-associated RNases (mEARs) and found that murine eosinophils “degranulated” following stimulation with CCL11 and CCL24, as detected by an increase in RNase A and EPO activities in cell supernatants. TEM revealed signs of PMD including significant increase in the number of emptying granules. CCL11 and CCL24-mediated mouse eosinophil degranulation was Gαi and PI3K-dependent. Moreover, intact beta 1 and 2 integrins as well as dynamic cytoskeleton are required for this PMD. Unexpectedly, we found that inhibition of Rho-GTPase kinase increases PMD. Moreover, cell-free granules purified from mouse eosinophils were found to express functional CCR3 and secreted mEARs as well as EPO in response to CCL11, suggesting a unique mechanism of degranulation in cell-free context. Collectively, these data demonstrate that murine eosinophils undergo PMD in response to physiological stimulation with CCL11 and CCL24, findings that are pertinent to murine models of eosinophil-associated diseases.
Phenotypic features and differentiation of human interleukin 9-producing CD8+ T cells (86.25)
Authors: Wang Shengjun , Yang Xianzhi , Shi Ye , Tong Jia , Su Zhaoliang , Tian Jie , Liu Qingling , Liu Yan , Lu Liwei , Xu Huaxi
Published: January 2023

Cross-talk between TGF-β and IL-4 can direct the differentiation of CD4+ T cells into interleukin 9-producing cells, which are termed Th9 cells. In this study, we show that CD8+ T cells also express IL-9. TGF-β and IL-4 could stimulate CD8+ T cells to differentiate into IL-9 producing cells in plate bound-anti-CD3 mAb (pbCD3) activated peripheral blood mononuclear cells (PBMCs). Soluble-anti-CD28 mAb (sCD28) decreased the expression of IL-9 which induced by IL-4 and TGF-β with pbCD3. Interleukin 9-producing CD8+ T cells display greatly suppressed cytotoxic function and low expression IFN-γ. The mRNA of Th1/Tc1 transcription factors T-bet is decreased in CD8+ cells from IL-4 and TGF-β with pbCD3 activated PBMCs. However, the expression of Th2/Tc2 transcription factors GATA3 is increased in human IL-9+ CD8+ T cells. Additionally, the proportion of IL-9 producing CD8+ T cells was higher in the allergic rhinitis as compared with the health controls. Taken together these data show that the existence of a human IL-9 producing CD8+ subset and can be categorized as a T cytotoxic 9 (Tc9) subset.
Sepsis-induced immunosuppression is mediated by TRAIL-expressing CD8+ regulatory T cells (87.13)
Authors: Griffith Thomas , Unsinger Jacqueline , Kazama Hirotaka , McDonough Jacqueline , Hotchkiss Richard , Ferguson Thomas
Published: January 2023

Patients who survive severe sepsis often display severely compromised immune function. One hallmark of such immune suppression in septic patients is an impaired delayed-type hypersensitivity (DTH) response, manifested by a loss of skin testing to recall antigens. Since sepsis induces significant apoptosis in lymphoid and myeloid cells and apoptotic cells are themselves tolerogenic, we tested the hypothesis that suppression of DTH is mediated by tolerogenic properties of the apoptotic cells generated during sepsis. Mice subjected to cecal ligation and puncture (CLP) demonstrated a loss of DTH for the 7 days following CLP; however, the immune response returned to normal by day 10. Blocking sepsis-induced apoptosis via Bcl-2 overexpression or Bim deficiency prevented the loss of DTH. Importantly, injection of apoptotic cells into Bim-/- mice prevented an effective DTH response, thereby suggesting a causal link between apoptotic cells and immune suppression. Surprisingly, when Trail-/- mice were examined, we found that these animals had significant apoptosis but retained their DTH responses. Further studies revealed that apoptotic cells generated during sepsis induced a CD8+ regulatory T cell (Treg) that mediated suppressed DTH by TRAIL production. These results establish a link between apoptotic cells and immune suppression during sepsis, and suggest TRAIL may be a viable therapeutic target for boosting the adaptive immune response following sepsis.
Oxidized phospholopids induce a tolerogenic response that is protective in a Streptozotocin-induced model of type 1 diabetes (T1D) (87.19)
Authors: Sharma Rahul , Sharma Poonam , Du Zhongmin , Leitinger Norbert
Published: January 2023

Oxidized phospholipids (OxPL) derived from oxidized lipoproteins or from membranes of apoptotic cells accumulate in damaged tissue. We hypothesized that OxPL generated during apoptosis may protect from inflammation through inducing a tolerogenic response. Our data shows that i.v. injection of C57BL/6 (B6) mice with OxPAPC (oxidation product of the membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-3-glycero-phosphoryl-choline) results in a reduced proportion of the CD11b-Hi CD11c+ dendritic cells (DC) in the spleen with much lower expression of MHC II and co-stimulatory molecules CD80 and CD86 as compared to saline injected controls. Furthermore, the number of CD4+Foxp3+ regulatory T cells (Tregs) was significantly higher in the spleens of mice treated with OxPAPC. Mechanistically, both the splenic CD11c+ DC purified from OxPAPC-treated mice and the OxPAPC-treated bone marrow-derived DC induced a more potent in vitro differentiation of naïve T cells to inducible Tregs as compared to the untreated controls. In a multiple low dose streptozotocin (MLD-STZ)-induced model of T1D, the B6 mice pre-treated with OxPAPC had a higher proportion of Tregs and were protected against MLD-STZ induced hyperglycemia as compared to saline injected controls. The data suggests that OxPAPC has therapeutic potential for treatment of T1D and other autoimmune diseases by inducing a DC-mediated increase of Treg differentiation.
Resistance training and aerobic exercise alters immune function (87.25)
Authors: Kumaraguru Uday , Nandakumar Subhadra , Chi David , Stone Michael , Krishnaswamy Guha
Published: January 2023

Exercise affects various components of the immune system that could be clinically beneficial or deleterious. Accordingly, moderate exercise stimulates the immune system, while intense exercise suppresses certain immune cell activities. Some of the immune-regulation observed may be due to the regulatory T cells. This could also explain the difference between moderate and strenuous exercisers and the probable reason for lower incidence of autoimmunity in active individuals. Intense exercisers (Trained -regular) and Moderate exercisers (untrained- occasional) were recruited as volunteers from of our university community after IRB approval. Blood samples were collected. PBMCs were used for immune cell profile analysis and the plasma was used for cytokine estimation. Results suggest recruitment of all lymphocyte subpopulations to the vascular compartment: CD4, CD8, B, and Natural killer (NK) cells. Depending on intensity of exercise, the CD4-to-CD8 ratio decreased, reflecting the greater increase in CD8s than CD4s. Both memory and naive phenotype were found, while memory type prevailed. The number of Tregs was increased in all subjects and inversely correlated with IL-6 concentration. Resistant training associated initiation of inflammatory cytokine suggest metabolic link between skeletal muscles and the lymphoid system. It is therefore to be expected that moderate exercise protects against malignancy or infection whereas exhaustive exercise is linked to increased pathology.
Overexpression of B7-H1 on epidermal keratinocytes promotes squamous cell carcinoma formation. (87.28)
Authors: Cao Yujia , Zhang Lu , Ritprajak Patcharee , Hashiguchi Masaaki , Azuma Miyuki
Published: January 2023

B7-H1 is inducible in various tumor cells and inflammatory tissue cells. We established B7-H1 transgenic mice (Tg), which overexpressed B7-H1 in keratinocytes (KCs). KC-associated B7-H1 directly regulated effector CD8+ T cells at the local inflammatory sites in contact hypersensitivity. Malignant tumor-formation is closely correlated with inflammatory responses. In this study, we investigate the roles of KC-associated B7-H1 in MCA-induced squamous cell carcinoma (SCC) formation. Intradermal injection of MCA preferentially induces SCC. We compared histology and inflammatory status between control and B7-H1Tg mice. Seven days after MCA injection, control BALB/c mice showed severe inflammatory responses with rapid expansion of epidermal cells, more infiltrations in the dermis, and higher cytokine expression. In contrast, B7-H1Tg mice showed less infiltration and expansion of epidermal cells, and exhibited poor alignment and higher chromatin condensation in the basal cells. After 7 wks, B7-H1Tg showed significantly higher incidence of SCC. Our results demonstrated that overexpression of B7-H1 on KCs promotes SCC formation, although the B7-H1/PD-1-mediated interactions between infiltrated cells and epidermal cells regulates inflammatory responses at early time points. The exhibition of higher incidence of SCC in B7-H1Tg mice suggests intrinsic changes in epidermal KCs by B7-H1-transduction rather than PD-1-dependent action.
Phenotypic analysis of spinal cord infiltrating CD4+ T lymphocytes during nerve injury induced neuropathic pain (87.4)
Authors: Cao Ling , Draleau Kyle , Slaiby Aaron , Minzy Courtney
Published: January 2023

We previously demonstrated that CD4+ T cells contribute to the maintenance of L5 spinal nerve transection (L5Tx) induced neuropathic pain in BALB/c mice. It is proposed that a particular subtype(s) of the L5Tx-induced spinal cord infiltrating CD4+ T cells mediates this maintenance of neuropathic pain via T cell - glial interactions. Here, we determined the subtype of L5Tx-induced spinal cord infiltrating CD4+ T cells. There were significantly higher numbers of T-Bet+ lumbar spinal cord infiltrating CD4+ T cells in the L5Tx group compared to the sham group at day 7 post-L5Tx, the peak time for detecting lumbar infiltrating CD4+ T cells, while no significant GATA3+ CD4+ T cells were observed. There were no significant differences in the levels of T-Bet+ or GATA3+ CD4+ T cells in the spleen or lumbar lymph nodes detected at this time. Interestingly, although no significantly higher levels of IFNγ were found in L5Tx mice in the lumbar spinal cord post-surgery, there was a notable increase in splenic levels of IFNγ in the L5Tx group at day 3 post-L5Tx compared to both sham and naïve mice. As we also have data indicating no differences in Th17 responses between groups, altogether our data suggests the involvement of Th1 cells in L5Tx-induced neuropathic pain and that the maintenance of neuropathic pain is likely a Th1-mediated process. Specific downstream responses mediated by these infiltrating Th1 cells require further investigation.
MicroRNAs (miR16 and miR146a) regulation of lupus development in NZB/W F1 mouse model (87.9)
Authors: Yuan Yao , Kotenko Sergei , Laurindo Fernanda , Salerno Erica , Raveche Elizabeth
Published: January 2023

SLE is a heterogeneous autoimmune disease and the specific cause remains unknown. B and T cell abnormalities are considered to play a central role in SLE. More recently, microRNAs (miRNAs), a group of small (~22nt) non-coding RNAs have been studied in SLE patients. miR16, targeting Bcl-2, regulates cell apoptosis.miR-146a, which plays a role in downregulation of type I interferon signaling, is found to be decreased in SLE. In NZB/W F1 mouse lupus model, these two particular microRNAs will be investigated in order to identify the microRNAs in regulation of balancing of B and T cell immunity. In preliminary studies, we treated B/W mice with IFNα or IFNλ for 12 week period and IFNα treated group, but not the IFNλ group developed much earlier and more severe lupus-disease than others. We found that spleen cells from IFNα or IFNλ treated B/W mice had similar level of miR16 and miR146a with control B/W, and all the B/W mice showed a pronounced underexpression of these two miRs comparing to C57 mice. By flow cytometry, only IFNα treated group cells showed hyper-activation of B and T cells, and decreased regulatory B. Thus, it is likely that due to the failure to turn off activation of type I interferon signaling in B/W mice by miR146a and anti-apoptotic effects in activated B cells resulting from low level miR16 may drive the acceleration of lupus-disease in the lupus-prone B/W mice.
Altered response of CD4+ T cells to TGF-β and IL-6 in the absence of the vitamin D receptor (88.9)
Authors: Yu Sanhong , Cantorna Margherita
Published: January 2023

Th17 cells play critical roles in autoimmunity. The active form of vitamin D, 1,25(OH)2D3, has been shown to directly regulate T cell function, but the role of vitamin D and vitamin D receptor (VDR) in the differentiation of Th17 cells is not very clear. Induction of Th17 cells using TGF-β and IL-6 resulted in twice as many Th17 cells in VDR KO cultures as WT. The increased frequency of Th17 cells in VDRKO cultures was not a result of higher expression of retinoic acid receptor-related orphan receptor-γT (ROR-γT). 1,25(OH)2D3 deficient CD4+ T cells also overproduced IL-17. Treatment of WT CD4+ T cells with 1,25(OH)2D3 significantly inhibited the generation of Th17 cells. The number of Treg cells in Th0 cultures of CD4+ cells from VDR KO mice was higher, compared to WT. TGF-β addition to the cultures induced Tregs in both the VDR KO and WT cultures, but there were significantly more Tregs in WT than VDR KO cultures. Addition of both TGF-β and IL-6 to WT CD4 cultures caused a 4-fold reduction in Treg cells. The reduction of Tregs by TGF-β and IL-6 in VDR KO cultures was only 2-fold. VDR KO T cells cultured with TGF-β and IL-6 produced significantly more IL-10 than their WT counterparts. Our data suggest the 1,25(OH)2D3 and signaling through the VDR are important regulators of Th17 differentiation. Furthermore, VDR KO T cells have altered responses to TGF-β and IL-6 that resulted in increased numbers of both Th17 and Treg cells.
Epithelial STAT3 activation is associated with expression of the antimicrobial peptide S100A7 (89.14)
Authors: Hulse Kathryn , Norton James , Harris Kathleen , Conley David , Chandra Rakesh , Kern Robert , Peters Anju , Grammer Leslie , Cohen Elyse , Singh Rashmi , Carter Roderick , Suh Lydia , Schleimer Robert
Published: January 2023

Psoriasin (S100A7) is a potent antimicrobial peptide produced by epithelial cells. Previous work by our group has demonstrated a significant reduction in both the expression of S100A7, and the activation of STAT3 (pSTAT3) in tissue samples from patients with chronic rhinosinusitis with nasal polyps (CRSwNP) compared controls. To investigate whether these two independent observations were linked, we examined the relationship between pSTAT3 and S100A7 expression in primary human bronchial (NHBE) and nasal epithelial cells (NEC). NHBEs or NECs were stimulated with a variety of known STAT3 activators in the presence or absence of TNF. STAT3 activation was assessed by western blot analysis of phospho-STAT3, while S100A7 gene and protein expression were analyzed by qRT-PCR and ELISA, respectively. Among the stimuli tested, IL-22 was the most effective inducer of S100A7 in NHBEs and NECs, and this response was enhanced by TNF (22.3 ng/mg protein and 321.2 ng/mg protein, respectively). Moreover, IL-22 induced phosphorylation of STAT3, and co-incubation with STAT3 inhibitor V resulted in > 75% inhibition of S100A7 protein expression. Interestingly, the TLR3 activator dsRNA, also potently induced S100A7 expression, but this induction was only slightly attenuated by inhibition of STAT3. These data suggest that STAT3 plays a role in the activation and expression of S100A7 in airway epithelial cells, and may be relevant to innate immunity in the airways.
Salivary gland NK cells are phenotypically and functionally unique (89.20)
Authors: Reilly Emma , Tessmer Marlowe , Brossay Laurent
Published: January 2023

NK cells and CD8+ T cells play vital roles in containing and eliminating systemic cytomegalovirus. However, CMV has a tropism for the salivary gland acinar epithelial cells and persists in this organ for several weeks after primary infection. Here we characterize a distinct NK cell population that resides in the salivary gland, uncommon to any described to date, expressing both mature and immature NK cell markers. Notably, naïve salivary gland NK cells have a reduced expression of CD27, are nearly absent of the inhibitory receptor KLRG1, yet are mostly CD11b positive. Using RORγt reporter mice and nude mice, we also show that the salivary gland NK cells are not lymphoid tissue inducer (LTi) NK-like cells and are not thymic derived. During the course of MCMV infection, we found that salivary gland NK cells detect the infection and acquire activation markers, but have limited effector functions in vivo. Salivary gland NK cell effector functions are not regulated by iNKT or Treg cells, which are mostly absent in the salivary gland. Additionally, we demonstrate that peripheral NK cells are not recruited to this organ even after the systemic infection has been controlled. Altogether, these results indicate that latency in SMG may result from both the presence of unfit NK cells and the lack of recruitment of peripheral NK cells.
Immunomodulation of the epithelial cell responses by CD80 blockade and disease amelioration in experimental colitis. (89.3)
Authors: Srinivasan Mythily , Dudhgaonkar Shailesh
Published: January 2023

Under physiological conditions the intestinal epithelium adapts to a constantly changing environment of the intestinal luminal content including the enteric bacteria. The colonic epithelial cells (CEC) respond to microbes through a family of tightly regulated toll like receptors (TLR) that constantly survey their surroundings for potential threats. In inflammatory bowel diseases (IBD) abnormal response to bacteria mobilizes immune effector cells to the intestinal mucosa, chiefly the CD4+ T cells which perpetuate sustained inflammation. Successful therapeutic strategies for IBD should therefore suppress lymphocytic responses; promote epithelial healing and re-establishment of intestinal homeostasis. Inhibition of T cell activation suppresses inflammatory cytokines in animal models of IBD. Here we investigated the effects of a synthetic peptide that blocks T cell costimulation, the CD80-CAP, on the CEC responses in experimental colitis. We observed that the CEC from CD80-CAP treated mice exhibited significantly reduced pro-inflammatory responses and a skewing towards anti-inflammatory cytokine response. Importantly, the CD80-CAP treatment also resulted in concomitant restoration of TLR-2, TLR-3, TLR-4 and TLR-5 mRNA in the CEC of colitic mice to physiologic levels. Taken together our data suggest that the CD80-CAP administration suppress inflammation and modulate epithelial cell responses potentially facilitating re-establishment of mucosal homeostasis in experimental colitis.
Inflammation-induced Expression of Immunoreceptor Tyrosine-based Inhibition Motif (ITIM)-less SIRPα Enhances Neutrophil Transmigration (89.39)
Authors: Guo Ya-Lan , Chen Yi-Tien , Soto Ileana , Zen Ke , Liu Yuan
Published: January 2023

Previous studies by us and others have demonstrated that SIRPα, an immunoreceptor tyrosine-based inhibition motif (ITIMs)-containing cell surface receptor, regulates neutrophil (PMN) and monocyte transmigration in response to inflammatory stimuli. In this study, we further characterized leukocyte SIRPα and found that PMN express multiple forms of SIRPα including the typical SIRPα with a long cytoplasmic domain containing the full ITIMs (ITIM+SIRPα) and shorter, ITIM-depleted SIRPα (ITIM-SIRPα). Importantly, our studies in both human and inflammatory animals revealed that, while ITIM+SIRPα is generally expressed in PMN of healthy donors, active inflammatory conditions induce diminishing of ITIM+SIRPα but a dominant expression of ITIM-SIRPα. In vitro and in vivo functional assays indicate that, compared to PMN from healthy donors bearing ITIM+SIRPα, PMN with chiefly ITIM-SIRPα display a higher tendency of activation and exhibit enhanced transmigration under inflammation. However, despite the differences in the intracellular domains, purified ITIM+SIRPα and ITIM-SIRPα from healthy and inflammatory donors had similar extracellular binding with the ligand CD47. In addition, our results suggest that conversion of SIRPα into ITIM-less receptor is through inflammation-induced proteolytic cleavage in PMN and such process serves as an important mechanism to enhance inflammatory response and dynamically modulate PMN transmigration.
Complement anaphylatoxin function and signaling in the CNS (89.48)
Authors: Ingersoll Sarah , Martin Carol , Martin Brian
Published: January 2023

Demyelination of the CNS in autoimmune diseases is known to involve several immune effector mechanisms, including complement. The brain is considered an immunoprivileged site; however, complement proteins are produced locally by glial cells. We are investigating the roles of C3a and C5a on glial cell activation and their overall role in demyelination and remyelination pathology. Using the cuprizone model to induce demyelination, we found that transgenic mice expressing C3a or C5a in the CNS had exacerbated demyelination and slightly delayed remyelination in the corpus callosum compared to wild type mice. C3a and C5a transgenics had increased cellularity in the corpus callosum. Using immunofluorescence, it was shown that microglia and astrocyte levels were increased in the brains of transgenic mice during peak demyelination. To study the role of anaphylatoxins on individual glial subsets, we created novel adenoviral vectors to express murine C3a or C5a. ERK1/2, were phosphorylated when BV-2 microglia were stimulated with C5a, but not C3a. JNK and ERK1/2 were phosphorylated in primary astrocytes treated with C3a or C5a. Additionally, C5a was able to induce chemtoaxis in J774 macrophages; however, we did not see significant migration of astrocytes or BV-2 microglia in vitro. Overall, our findings show that anaphylatoxin production in the brain plays a negative role during demyelination, most likely due to an increase in inflammatory cells in demyelinated areas of the brain.
Human innate immune cells that engage soluble beta-1,3/1,6 glucans: Role for complement receptor 3 (CR3, CD11b/CD18) (89.53)
Authors: Vasilakos John , Bose Nandita , Chan Anissa , Danielson Michael , Dudney Christine , Elmasry Natalie , Gorden Keith , Guerrero Faimola , Magee Andy , Maristany Carolyn , Walsh Richard , Will Paul , Wurst Lindsay
Published: January 2023

Beta-1,3/1,6 glucans (BGs) are a diverse class of PAMPs. Structurally, BGs can vary on the basis of chain length, types of linkages, branching, and tertiary structure. BGs have been reported to activate various types of innate immune cells; most studies demonstrate the use of particulate BGs activating murine immune cells. Unlike particulate BGs, most soluble BGs demonstrated minimal activities typically associated with innate immune cell activation, such as induction of cytokines. Hence, little is known regarding the types of human immune cells or receptors that engage soluble BGs, and if structurally distinct BGs differ in their cell selectivity or receptor requirements. The objective of this study was to determine the types of human immune cells that soluble BGs bind to, and to determine the receptors required for binding. Since BGs have been implicated in interacting with Dectin-1 and CR3 expressing cells, we hypothesize that human innate immune cells that express these receptors will bind soluble BGs. Specific binding of BGs was evaluated by flow cytometry on primary human immune cells and cell lines using BG specific antibody and DTAF-labeled BGs. In addition, these studies were conducted using three structurally distinct, purified, and chemically characterized BGs. Key findings demonstrate that on cells that express both CR3 and Dectin-1, CR3 is a critical receptor for binding soluble BGs and not all BGs bind equivalently to human innate immune cells.
Galectin expression and modulation by viruses in fish cell lines (89.54)
Authors: Jackson Shawn , de las Heras Sanchez Ana , Ahmed Hafiz , Vakharia Vikram , Vasta Gerardo
Published: January 2023

The galectin family of β-galactoside-binding proteins is evolutionarily conserved. In homeotherm vertebrates, galectin-virus interactions can affect viral infectivity and/or pathogenicity. We hypothesize that in teleost fish, galectin interactions with viral proteins modulate viral infectivity. We examined fish cell lines for expression of galectin-like molecules, and assessed changes in expression of identified galectins as well as of cytokines in response to viral infection. Minnow (EPC), trout (RTG-2), bluegill (BF-2), salmon (CHSE-214, ASK), zebrafish (ZFL), and catfish (COO, 42TA, G14D, 28S.3, 1G8, and 3B11) cells were tested. A proto-type galectin-like protein is expressed in EPC, RTG-2, and ZFL cell lines, and a chimera-type in EPC and RTG-2 cell lines. All tested cell lines express mRNA that exhibits high conservation with the zebrafish Drgal1-L2 proto-type galectin. Ten of the 12 cell lines express Drgal1-L3-like mRNA, 7 express Drgal1-L1-like mRNA, and 6 express Drgal1-L4-like mRNA. With the exception of 3B11, all tested cell lines express Drgal3-L1-like mRNA. Eight of the 12 express Drgal3-L2-like mRNA. Two of the 12 cell lines expressed Drgal9-like mRNA. Following infection of EPC cells with IHNV or IPNV, we observe modulations in expression of proto-type and tandem-repeat-type galectin-like molecules. We see modulations in expression of various cytokines, including type I and II interferons and TNF family members.
KIR2DS1 positive Natural Killer cells from individuals expressing cognate HLA ligand are tolerant. (89.58)
Authors: Pittari Gianfranco , Selvakumar Annamalai , Liu Xiao-Rong , Dupont Bo
Published: January 2023

Inhibitory KIR-HLA interactions are critical for NK tolerance to self. KIR2DS1 is the only activating counterpart of inhibitory KIRs to have ligand specificity for HLA class I, recognizing HLA-C group 2. Since KIR and HLA genes segregate independently, individuals that carry KIR2DS1 may have HLA-C antigens of the HLA-KIR ligand group C2. We here report that 2DS1 positive NK cells from such individuals are tolerant to such self HLA antigens. We demonstrate that the mechanism for tolerance is different for individuals homozygous or heterozygous for the HLA ligand. NK clones have been generated from KIR2DS1-positive and HLA-C2 homozygous, HLA-C1/C2 heterozygous or HLA-C1 homozygous individuals. Their response against HLA-KIR ligand group C2-expressing targets and their KIR repertoire have been assessed. NK clones expressing KIR2DS1 display anti-HLA-C2 alloreactivity but are tolerant to HLA-C2 if this is a self antigen. This hyporesponsiveness to C2 in C2/C2 homozygous donors is also observed for NK clones lacking 2DL1. In turn, the co-expression of inhibitory KIRs with ligand specificity for self HLA class I including C2 is an important mechanism for tolerance in HLA-C1/C2 heterozygous individuals. This is the first demonstration of activating NK receptor tolerance to cognate self MHC ligand. NK clones expressing only the activating receptor and not the homologous inhibitory receptor are readily identified. Remarkably, NK clones with self HLA ligand specificity are not deleted.
Human gastrointestinal stem cell (hGISC) derived non-transformed epithelial cells but not malignant colonic adenocarcinoma cell line HT29 respond to combined innate and adaptive immunostimulation by producing high levels of nitric oxide. (90.14)
Authors: Panja Asit , Joseph Ansamma , Panja Aparna , Harrison David
Published: January 2023

Gastrointestinal (GI) inflammation is a global health burden. Yet the mechanism of GI inflammation remains undefined. Among proposed mechanisms, increased nitric oxide (NO) production by intestinal epithelial cells (EC) is thought to play a crucial role in inflammatory conditions such as IBD. However, the regulation of NO production in human intestinal epithelium are undefined, thus treatments for these diseases remain inadequate. Therefore, to gain insight about NO production regulation by intestinal EC, we studied the effects of innate (LPS) and adaptive (IFNγ) immunostimulatory agents on a novel hGISC cell system derived non-transformed primary epithelial cells (EC) and compared it to a colonic adenocarcinoma EC line HT29. A macrophage cell line MHS served as a positive control. Cells were cultured with/without LPS (1μg/ml), IFNγ (200 u/ml), or in combination for 24 hours. Culture supernatants were analyzed for NO content by Griess reaction assay. Our results show that neither LPS nor IFNγ alone affect NO production by intestinal ECs. However, NO production by hGISC derived primary ECs was dramatically increased (comparable to the NO level seen in MHS cells) when LPS and IFNγ were added in combination. In contrast, malignant HT29 cells do not show any difference in NO synthesis even when LPS (innate stimuli) and IFNγ (adaptive stimuli) were combined. These findings may help understand the intestinal inflammatory disease mechanisms and development of new treatments.
Cooperation of retinoic acid and Toll-like receptor signaling in induction and function of mucosal dendritic cells (90.5)
Authors: Feng Ting , Qin Hongwei , Benveniste Etty , Cong Yingzi , Elson Charles
Published: January 2023

Mucosal dendritic cells (DCs) play a crucial role in the maintenance of intestinal immune homeostasis. However, how DCs become mucosal DCs is still unclear. In this study, we investigated whether retinoic acid (RA) induced the mucosal DC phenotype and function, and the mechanisms involved in this process. We report here that bone marrow cells expressed RA-synthesizing enzyme aldehyde dehydrogenase family 1a2 (ALDH1a2) and provided RA to DC precursors in the bone marrow microenvironment. RA induced bone marrow-derived DCs (BMDCs) to express gut-homing receptor CCR9 and ALDH1a2. Similar to mucosal DCs, RA-educated DCs (RA-DCs) promoted conversion of Foxp3+ regulatory T cells from naïve Foxp3- precursors, induced B cell IgA secretion, and enhanced lymphocyte gut-homing receptor expression. The effect of RA on educating BMDCs to acquire mucosal DC functionality was dependent on a narrow time window and tight dose. RA inhibited DC SOCS3 expression through direct interference with SOCS3 promoter activity thus enhanced STAT3 activation, which promoted bioactive TGF-β production. In cooperation with RA signaling, commensal microbiota stimulation further enhanced RA-DC ALDH1a2 expression and was required for RA-DC TGF-β production. Our data reveal a mechanism for RA to instruct bone marrow DC precursors to differentiate into mucosal-like DCs. Cooperation of RA signaling and microbiota stimulation is required for the development of fully functional mucosal DCs.
HVEM-LIGHT interactions promote generation of memory Th2 cells that mediate lung inflammation (91.8)
Authors: Soroosh Pejman , Doherty Taylor , So Takanori , Fukuyama Satoshi , Ware Carl , Croft Michael
Published: January 2023

Memory Th2 cells are important in the pathogenesis of allergic responses including asthma, orchestrating recruitment of eosinophils and other inflammatory cells. Members of the tumor necrosis factor (TNF) receptor superfamily have emerged as key contributors to T cell activity, and therefore we investigated whether the engagement of the herpesvirus entry mediator (HVEM) on T cells by the TNF family molecule LIGHT controls Th2 mediated lung inflammation. Using systems where either HVEM or LIGHT are only defective on responding antigen-specific T cells, we demonstrated that the presence of HVEM on T cells is indispensible for the generation and persistence of Th2 memory cells and for allergen-induced Th2 lung inflammation, despite having a limited effect on early Th2 effector responses. Using adoptive co-transfer experiments we demonstrate that HVEM-LIGHT interaction on CD4 T cells can support longevity of T cells and promote memory generation. We also observed reduced activity of protein kinase B (PKB/Akt) and NF-κB in HVEM-deficient effector CD4 T cells, correlating with their poor survival. Furthermore, expression of an active form of PKB in HVEM-deficient T cells rescued Th2 cell survival in vivo, and restored lung inflammation in recall responses. Collectively, our results demonstrate a critical role of HVEM signals in the pathogenesis of Th2 derived lung inflammation and suggest that blocking of HVEM-LIGHT interactions could be a novel therapeutic target for asthma
Temporal and spatial distribution of antigen following influenza virus infection (92.16)
Authors: Turner Damian , Lefrancois Leo
Published: January 2023

Memory CD8 T cells generated by vaccines are important for prevention of certain viral infections. However, our understanding of the factors that contribute to the development of memory CD8 T cell remains unclear. We hypothesized that, in response to a localized influenza virus (IAV) infection, CD8 T cells are recruited to the local draining lymph node (dLN) at different times and thus acquire different fates. We therefore studied the kinetics of recruitment and activation of naïve IAV-specific CD8 T cells in the dLN of the respiratory tract following IAV infection. We observed early antigen presentation in the dLN; however, we also observed evidence of antigen encounter in the spleen. To determine the differentiation outcome of CD8 T cells primed in disparate sites following a localized infection we adoptively transferred spleen-primed or dLN-primed cells into naïve mice and determined their survival and ability to respond upon recall. Surprisingly we found that CD8 T cells primed in the spleen differentiate into memory cells of equal longevity and with similar capacity to respond to recall as CD8 T cells that are primed in the dLN. Thus, in a model of respiratory IAV infection, virus-specific memory CD8 T cells are generated in the spleen in addition to the dLN. Antigen presentation in the spleen may therefore serve to ensure the involvement of a greater fraction of the virus-specific CD8 T cell repertoire after a local infection.
Characterization of a second C-type lectin-like gene in rat cytomegalovirus (92.8)
Authors: Voigt Sebastian , Friedrich Robert , Kutschenko Lara
Published: January 2023

Gene capture has been a prominent feature in herpesvirus evolution, and many of these hijacked genes serve immunomodulatory purposes. Recently, we characterised a lectin-like gene (RCTL) in the English isolate of rat cytomegalovirus (RCMV) that interacts with an inhibitory receptor on Natural Killer (NK) cells. Here, we show that RCMV encodes an additional, spliced lectin-like gene (RCTL2) that, compared to RCTL, is located at the opposite end of the viral genome. RCTL2 consists of four exons and is expressed in the early phase of viral replication. To analyse if RCTL2 can be expressed as a protein, we tagged the ORF with the FLAG sequence. After transfection of 293 cells, RCTL2 was detected by fluorescent microscopy and Western blot analysis. Treatment with tunicamycin reduced the protein size, thereby confirming predicted N-linked glycosylation sites. The sequence similarity of RCTL2 with RCTL and the reported interaction with NK cells of the latter suggest that RCTL2 is similarly involved in the modulation of the innate immune response and that the capture of lectin-like genes is beneficial for viral replication. This notion is supported by the existence of lectin-like genes in poxviruses.
CIKS/Act1 is required for collagen-induced arthritis (93.3)
Authors: Pisitkun Prapaporn , Claudio Estefania , Ren Nina , Wang Hongshan , Bolland Silvia , Siebenlist Ulrich
Published: January 2023

Collagen-induced arthritis (CIA) is a widely used animal model for human rheumatoid arthritis (RA). Th17 cells are thought to be critical for development of RA and CIA, mediated in part via production of IL-17. CIKS/Act1 is an important adaptor molecule for signaling by IL-17 and related cytokines. Although evidence suggests a role for IL-17 in RA, the physiologic relevance of CIKS/Act1 in RA and the CIA model has not been evaluated. We have applied the CIA model to CIKS deficient and wild-type (WT) B6 mice. Based on clinical scores and histochemical analyses, arthritis could be induced in WT, but not CIKS deficient mice. The production of collagen type 2-specific antibodies (CII-Abs) and neutrophil recruitment to the joint were also blocked in CIKS deficient mice. We also performed CIA model studies in mice with compound deficiency in CIKS and FcgammaRIIb (B6). Although the loss of FcgammaRIIb greatly exacerbated arthritis in the CIA model in CIKS-sufficient mice, arthritis and production of CII-Abs were still blocked in the absence of CIKS. In addition we determined that CIKS is not required in the collagen antibody-induced arthritis (CAIA) model, in contrast to this adaptor’s role in CIA. These data suggest a crucial role of CIKS during the generation of CII-Abs and clearly implicate the CIKS adaptor as essential for development of CIA and thus a potential therapeutic target in RA.