bluuebunny/crossref_metadata_2025 huggingface dataset:
Endothelial-like cells differentiated from bone marrow-derived mesenchymal stem cells preserve T-cell suppressive function (147.13)
Authors: Kim Jung-Sik , Park Min Jung , Lim Hyun-Ju , Kim Young-Hee , Kim Sang-Joon , Park Chung-Gyu
Published: January 2023

Endothelial-like mesenchymal stem cells (EMSCs) differentiated from human mesenchymal stem cells (MSCs) have great potential in cell therapy for ischemic diseases and in tissue engineering for vascular grafts. Although they was proven to stimulate functional blood vessel formation in vivo, it still need to be investigated whether EMSCs harbor immunomodulatory capacity for applying immune-mediated vascular disorders. In this study, therefore, we compared in vivo angiogenic potential of EMSCs with MSCs and studied their immunosuppressive functions. In the phenotype analysis, differentiated EMSC showed several endothelial cell markers including vWF, TGF-beta and Flt-1. In in vitro angiogenesis analysis by using Matrigel and fibrin, they have augmented angiogeneic potential compared to MSCs. Moreover, EMSCs showed neovascularization potential superior to MSCs in subcutaneous transplantation of fibrin-islet-cell composite. In allogeneic mixed lymphocyte reaction, EMSCs showed preserved immunosuppressive function on T cell comparable to that of MSCs. Apoptosis induction and cell-cycle arrest mediated by cell free-culture supernatant were associated with EMSC-mediated T-cell immunosuppression, as evidenced by the increased P27kip and decreased cdk2, cyclin D2 and Bcl-2. These results suggest that ex-vivo expanded EMSC could be a useful source for postnatal neovascularization and for cell-based therapies of tissue damages involving immune reactions.
Topical application of mesenchymal stem cells effectively reduces corneal surface inflammation by inhibiting Th17 T cells proliferation in mouse chemical burn model (147.16)
Authors: Kim Mee Kum , Han Eui Seok , Jeong Hyun Jeong , Lee Hyun Ju , Jeong So Hee , Wee Won Ryang
Published: January 2023

Mesenchymal stem cells(MSCs) has known to have antiinflammatory properties in various inflammatory disease models, however the in vivo effect of MSCs on the inflammation is variable depending on the administration site for the in vivo delivery. To investigate the mechanism of antiinflammatory effect of topical application of MSCs on chemcal burn model, and to compare whether the effect of systemic application of MSCs is the same as topical introduction of MSCs, we made mouse chemical burn model, delivered MSCs, and evaluated local and systemic changes. It had been conducted in conformance with the FASEB Statement of Principles for the use of animals in research and education. We found that topical application of MSCs reduced corneal inflammation in mouse chemical burn model, however, systemic administration of MSCs could not decrease corneal inflammation in the same model. Direct application of MSCs to inflammatory corneal surface diminished not only infiltration of inflammatory cells in the cornea but also Th17 cells in the draining lymph nodes, on the other hand, systemic administration of MSCs did not. It suggested that systemic application of MSC might not effectively reach the target organ (eye) or the draining lymph nodes, and implied that adequate administration of MSCs might be crucial to maximize efficient antiinflammatory property of MSCs in various inflammatory disesase models.
Modulation of MHC II and costimulatory molecules expression on monocytes by intravenous immunoglobulins (147.17)
Authors: Padet Lauriane , Bazin Renée
Published: January 2023

Intravenous immunoglobulins (IVIg) are therapeutic preparations of human IgG isolated from thousands of plasma donations and are known to have anti-inflammatory effects in many autoimmune diseases. IVIg are also used as prophylactic agent to reduce episodes of allograft rejection but the mechanisms responsible for this effect remain to be determined. In the present work, we showed by using a mixed lymphocyte reaction as an in vitro model of allograft rejection and GvHD, that IVIg strongly inhibited T cell activation. To explain this inhibition, we postulated that IVIg modulated the expression of MHC II and costimulatory molecules expressed on APC and involved in antigen presentation and regulation of T cell activation. To study this hypothesis, PBMC were cultured with or without 10 mg/ml IVIg for 24 hours, followed by flow cytometry analysis of cell surface expression of 8 different molecules. Among these, the expression of 5 was significantly modulated on monocytes. IVIg decreased the expression of MHC II (50%), CD80 (80%), CD86 (52%) and L-ICOS (50%). Interestingly, preliminary results showed a 5-fold increase in the expression of PD-L1, a negative regulator of T cell activation. These results suggest that IVIg impair the presentation of allopeptides to T cells as well as induce negative signalling in these cells, leading to their reduced activation. Altogether these findings could help explain the induction of peripheral tolerance and improvement of graft survival by IVIg.
Osteopontin induced inhibition of dendritic cell polarization towards pDC cells is not exclusively mediated by NKT cells (147.28)
Authors: Habeeb Keith , Durre Kainath , Beal Brooks , Qureshi Mahboob
Published: January 2023

Pediatric post-RSV infection associated bronchial asthma is known to be mediated via an enhanced pulmonary Th2 response. However, adult RSV infection is not known to be associated with this pathology. Further, osteopontin and NKT cell have been reported to be involved with induction of Th1 and Th2 type host-immune responses, respectively. The effect of NKT cells on naïve T helper cells is suggested to be a result of NKT cells’ subsequent modulation of naïve dendritic cells (DCs) towards plasmacytoid DCs (pDCs). So, in our present studies, using an NKT cell KO (CD1d-/-) mouse model, we have studied the role of osteopontin and NKT cells in modulating the polarization of DCs towards pDC in RSV-infected neonatal lungs. Flowcytometry analysis determined that there was an enhanced NKT cell (CD19-CD4+CD1d+) response in the RSV-infected neonatal lungs compared to that in the infected adult lungs. There was a decrease in the NKT cell response to RSV infection in the neonatal lungs upon concomitant administration of exogenous osteopontin as compared to those without osteopontin. Furthermore, in the absence of NKT cells (CD1d-/-), the administration of exogenous osteopontin induced inhibition of DC polarization towards pDC. Thus, differential pDC responses to RSV infection in neonates with exogenous osteopontin administration, even in the absence of NKT cells, warrants investigation into involvement of other factors in the development of RSV-induced bronchial asthma in later life.
Suppression of VαiNKT cell activation by Kupffer cells. (147.5)
Authors: Tyznik Aaron , Ichikawa Shintaro , Cheroutre Hilde , Kronenberg Mitchell
Published: January 2023

Invariant NKT (VαiNKT) cells are a unique subset of T lymphocytes that rapidly produce and secrete large amounts of cytokines following activation. These cytokines have a profound impact on a variety of different immune reactions. However, little is known how VαiNKT cell activation is regulated at steady state or curtailed to avoid the escalation of an out of control inflammatory response. Our preliminary data indicates that Kupffer cells, liver resident macrophages, produce soluble factors that can block the cytokine production of primary VαiNKT cells. Surprisingly, this inhibition acts directly on VαiNKT cells stimulated either by cytokine mediated activation or antigen-specific TCR activation. The Cytokine suppression is unique to Kupffer cells as macrophages from the spleen are able to stimulate VαiNKT cells and have no inhibitory action. These studies have revealed a unique mechanism of VαiNKT cell regulation in the liver.
CD11blo Gr1+ osteoclast precursors are increased in inflammatory arthritis and have myeloid derived suppressor cell function. (148.1)
Authors: Charles Julia , Hsu Lih-Yun , Niemi Erene , Weiss Arthur , Nakamura Mary
Published: January 2023

We have identified a CD11blo Gr1+ bone marrow population of osteoclast precursors (OCP). This population is further defined by the markers Ly6Chi CX3CR1+ CCR2+ CD117- CD11c- and is distinct from previously characterized myeloid precursors and monocyte subsets, suggesting that osteoclasts derive from a distinct precursor analogous to the common dendritic cell precursor. OCP increase in inflammatory arthritis in mice and humans. We find that CD11blo Ly6Chi OCP increase significantly prior to the development of synovitis in zymosan-induced SKG arthritis and remain elevated throughout the course of disease. Like the OCP we identified, monocytic myeloid derived suppressor cells (M-MDSC) are Ly6Chi. We tested our OCP for suppressive activity in vivo using an adoptive transfer model of SKG arthritis. Co-transfer of OCP with SKG CD4+ T-cells into RAG-/- recipients significantly decreased development of inflammatory arthritis compared to transfer of CD4+ T-cells alone, although less dramatically than co-transfer of CD4+ T-cells with wild-type Treg cells. We tested for in vitro suppression of T-cell proliferation by OCP and found that CD11blo Ly6Chi OCP suppress both CD4+ and CD8+ T-cell proliferation. Suppression by OCP is dependent on IFNγ and NOS2, similar to the mechanism described for M-MDSC. Our findings suggest that a myeloid precursor population with both T-cell suppressive activity and the potential to become osteoclasts is expanded in inflammatory arthritis.
Differential requirement for STAT4 in IL-18R expression by CD4 T cells during EAE (148.11)
Authors: McWilliams Ian , Harrington Laurie
Published: January 2023

Multiple Sclerosis (MS) is an autoimmune disease characterized by demyelination of neurons in the central nervous system. In order to study the mechanisms underlying MS, we use the well-described mouse model, experimental autoimmune encephalomyelitis (EAE), during which, the CD4 T cell subsets, TH1 and TH17, are associated with immunopathology. STAT4 is an important TH1 transcription factor that, when activated by IL-12, results in the production of IFNγ. Deletion of STAT4 protects mice from EAE; in contrast, deletion of IL-12 and IFNγ does not ameliorate disease. This disconnect raises the question about the role of STAT4 in EAE. The IL-18R complex is thought to be regulated by STAT4, and it has been shown that IL-18R-/- mice are not susceptible to EAE indicating the importance of this receptor during disease. Due to the association between STAT4 and IL-18R, we have investigated the regulation of the IL-18R complex in vitro as well as during disease and the requirement of STAT4 for its expression. Interestingly, we find that in vitro maximal IL-18R levels are detected on WT TH1 CD4 T cells and that this expression is dependent on STAT4. However, in vivo, we find the IL-18Rα expression is similar on WT and STAT4-/- CD4 T cells during EAE. Taken together, these data indicate an important role for STAT4 in IL-18R expression. Nevertheless, during autoimmune chronic inflammation, other factors promote IL-18R independent of STAT4 signaling.
Autoantibodies targeting phosphatidylserine, cardiolipin, β2-glycoprotein I, annexin V and prothrombin in patients with recurrent spontaneous miscarriage (148.18)
Authors: Sater Mai , Almawi Wassim
Published: January 2023

An association between human reproductive failure and the presence of anti-phospholipid autoantibodies (APA) was suggested; both traditional and nontraditional APAs were associated with increased risk of recurrent spontaneous miscarriage (RSM). We measured levels of antibodies to phosphatidylserine (anti-PS), cardiolipin (ACA), β2-glycoprotein-I (anti-β2GPI), annexin V (anti-Anx V) and prothrombin (anti-PT) in 277 women with idiopathic RSM, and 288 control women by ELISA. Anti-PS IgG, ACA IgM/IgG, anti-Anx V IgM/IgG, and anti-PT IgM positivity were significantly associated with higher RSM risk, after controlling for age, BMI, menarche, and gravida. RSM was independently associated with high levels of ACA IgM (OR, 6.5; 95% CI, 2.5-17.1) and IgG (OR, 12.1; 95% CI, 6.6-22.2), anti-PS IgG (OR, 13.3; 95% CI, 6.4-27.4), anti-AnxV IgM (OR, 31.1; 95% CI, 15.7-56.6) and IgG (OR, 23.3; 95% CI, 8.8-51.7), and anti-PT IgM (OR, 2.8; 95% CI, 1.3-5.9). Levels of autoantibodies were categorized into 5 strata (percentiles 1-75, 76-99, 91-95, 96-99, >99 among control patients) and analyzed in logistic regression models. The adjusted OR increased as the percentile value of ACA IgM/IgG ,anti-PS IgG and anti-Anx V IgM/IgG, and anti-PT IgM, increased. In conclusion, the presence of ACA IgM/IgG, anti-Anx IgM/IgG, anti-PS IgG, and anti-PT IgM antibodies are risk factors for RSM. The strongest association was seen for anti-Anx V IgM and IgG, followed by ACA IgG, and anti-PS IgG antibodies.
AMPK, a metabolic enzyme, negatively regulate EAE by modulating IL17 response (148.20)
Authors: Giri Shailendra , Mangalam Ashutosh , Rattan Ramandeep , Shridhar Viji , Rodriguez Moses
Published: January 2023

AMPK is an energy sensing metabolic switch in mammalian cells and regulates diverse metabolic pathway in cells. Recently, we observed that AMPK is lost in all immune cells of Experimental autoimmune encephalomyelitis (EAE), widely used animal model for MS. AMPK is lost at protein level in spleen macrophages, total T cells and their subsets (CD4, CD8 and regulatory T cells) isolated from EAE afflicted animals compared to control. To examine the role of AMPK, EAE was induced in Wt (+/+) and AMPKalpha1 null mice (-/-) and found that AMPKalpha1 (-/-) mice exhibited severe EAE disease. AMPKalpha2 is not involved in enhancing the severity of the disease. Moreover, AMPKalpha1 null mice significantly exhibited severe inflammation and demyelination in CNS. Loss of AMPKalpha1 leads to higher production of IL17 cytokine and reduced production of IFNgamma and IL4 under recall response with MOG35-55 as antigen. AMPKalpha1 KO mice exhibited higher number of infiltrated mononuclear cells in CNS and produced higher amount of IL17 significantly compared to Wt mice. Moreover, adoptive transfer of MOG35-55 specific T cells from AMPKalpha1 KO in Wt mice (KO to Wt) induced significantly higher degree of clinical disease compared to adoptive transfer of MOG specific T cells from Wt to Wt mice suggesting loss of AMPK negatively regulate clinical disease of EAE and identify AMPK as a novel therapeutic target for MS.
At-retinal differentially regulates Foxp3+ regulatory and IL-17+ pathogenic T cell in autoimmune arthritis (148.7)
Authors: Cho Mi-LA , Park Mi-Kyung , Oh Hye-Jwa , Park Eun-Mi , Lee Dong-Gun , Park Sung-Hwan
Published: January 2023

Retinoids can regulate immune responses. The aim of this study was to determine whether all-trans retinal (at-retinal) improves the inflammation responses and destruction of joints on collagen-induced arthritis (CIA). The arthritis score and incidence of arthritis were lower in the mice treated with at-retinal. Histopathologic evidence of joint damage was lower, and the infiltrations of immune cell were reduced in the mice treated with at-retinal. CII-stimulated proliferation of spleen cells, the expression of cytokines and oxidative stress proteins the serum levels of total IgG and IgG2a anti-CII antibodies, and the expression of osteoclast markers were significantly reduced in the mice treated with at-retinal. In vitro, at-retinal increased Foxp3 expression. At-retinal also inhibits inhibits Th17 development. The proportion of Foxp3+ Treg cells was increased in the spleen of mice treated with at-retinal, whereas the proportion of Th17 cells was reduced. In both mice and human culture system, TRAP positive mononuclear cells and multinucleated cells were significantly reduced in the presence of at-retinal. Taken together, these findings indicate that at-retinal has profound immunoregulatory functions and potential protective values for the treatment of autoimmune inflammatory disorders.
The CXCL13-CXCR5 axis contributes to atherosclerosis development in LDLr-/- mice by regulating myeloid development (149.1)
Authors: Daissormont Isabelle , Seijkens Tom , Halvorsen Bente , Hopken Uta , Lipp Martin , Aukrust Pal , Lutgens Esther , Biessen Erik
Published: January 2023

CXCL13 is a homeostatic chemokine instrumental in guided lymphocyte and dendritic cell trafficking towards lymphoid organs and stromal tissue. Evidence is culminating that in various chronic immune disorders extranodal CXCL13 expression at the site of inflammation contributes to the disease. Here we addressed the role of CXCL13 and its receptor CXCR5 in atherosclerosis. Bone marrow ablated LDLr-/- mice were repopulated with CXCL13 KO and CXCR5 KO bone marrow cells and after recovery fed a high-fat diet for 14 weeks. Carotid artery lesions were induced by perivascular collar placement. Both CXCL13 and CXCR5 deficiency resulted in significant attenuation of atherosclerosis, characterized by a decreased infiltration of granulocytes into the lesions. Haematopoietic deficiency of CXCR5 resulted in impaired homing of B cells and regulatory T cells towards peripheral lymph nodes. However, we didn’t observe this effect in the CXCL13 KO group. Surprisingly, FACS analysis showed a significant reduction in circulating CD11b+Ly6C+ pro-inflammatory monocytes and granulocytes both in the CXCR5 KO and CXCL13 KO group. In addition, plasma levels of M-CSF, G-CSF and RANK-L were strongly reduced. To conclude, haematopoietic deficiency of CXCL13/CXCR5 attenuates atherogenesis, probably attributable to the effects seen on Ly6Chighmonocyte and granulocyte levels in circulation. This study is the first to describe a critical role for the CXCL13/CXCR5 axis in myeloid development.
CCR4:CCL17 interaction supports clonal cell survival and complements BCR- and TLR-9- mediated cell proliferation in chronic lymphocytic leukemia (149.14)
Authors: Damle Rajendra , Temburni Sonal , Andersen Ryon , Kolitz Jonathan , Allen Steven , Rai Kanti , Chiorazzi Nicholas
Published: January 2023

Existence and degree of somatic mutation of expressed Ig V genes stratify CLL cases into unmutated (U-CLL) and mutated (M-CLL), clinically distinct subgroups. CLL cells likely receive survival cues via interactions through their BCR, TLR, and cytokine/chemokine receptors. We reported ex vivo expression of CCR4 and its induction on CLL cells stimulated in vitro. In assays of chemotactic behavior in response to CCL17, migrated cells showed significantly higher percentages and densities of CD38 expression than the non-migrated cells suggesting a role for CD38 (a known marker of CLL prognosis) in the CCR4-mediated downstream pathway. Dendritic cell-derived CCL17 may exert an accessory role in BCR- and TLR-9-mediated immune responses in B cells in peripheral organs. CCL17 augmented BCR-mediated B-cell proliferation in 9/16 (56%) U-CLL cases, and in 3/15 (20%) M-CLL cases, and promoted TLR-9-mediated cell proliferation in 13/15(87%) M-CLL cases and 6/16 U-CLL cases. In addition, CCL17 promoted cell survival and down-regulated pro-apoptotic molecules Bid and PUMA in 6/16 cases studied. These pro-survival effects of CCL17 were partially abrogated by the blocking anti-CCR4 mAb (1G1). Taken together, these findings suggest that CCL17 plays a role in modulating BCR- and TLR-9-mediated signaling and migration in CLL. Inhibition of CCR4:CCL17 interaction in vivo could represent a novel therapy by preventing migration of CLL cells towards an environment that promotes their survival.
Lipid phosphate phosphatase 3 maintains low thymic sphingosine-1-phosphate and enables efficient thymic egress (149.8)
Authors: Schwab Susan , Bréart Béatrice , Ramos-Pérez Willy , Mendoza Alejandra , Huang Yong , Adams Ralf , Escalante-Alcalde Diana
Published: January 2023

The signaling lipid sphingosine-1-phosphate (S1P) stabilizes the vasculature, directs lymphocyte circulation, and shapes inflammatory responses. However, little is known about how S1P distribution is controlled in vivo, and it is not clear how a ubiquitously made lipid functions as a signal that requires precise spatial and temporal control. Here we have found that lipid phosphate phosphatase 3 (LPP3) maintains low thymic S1P and efficient egress of mature T cells from the thymus into circulation. Although five additional S1P-degrading enzymes are expressed in the thymus, they cannot compensate for the loss of LPP3. Moreover, conditional deletion of LPP3 in either epithelial cells or endothelial cells is sufficient to increase thymic S1P. These results suggest that S1P generation and destruction are tightly regulated, and LPP3 is essential to establish the balance.
Additional loss of Bim rescues viral-specific effector and memory T cells, but not endogenous memory T cells or NK cells in IL-15-deficient mice (150.10)
Authors: Kurtulus Sema , Sholl Allyson , Hildeman David
Published: January 2023

IL-15 is γc cytokine well appreciated for its role in the homeostasis of memory CD8+ T cells and survival of NK cells. IL-15 drives homeostatic proliferation of memory phenotype (CD44hi CD122hi) and antigen specific memory CD8+ T cells. We and others have recently shown that IL-15 regulates survival of a subset of effector CD8+ T cells (KLRG1hi CD127lo) and acts redundantly with IL-7 for the survival of KLRG1lo CD127hi effector CD8+ T cells. Mechanistically, IL-15 is thought to maintain T cells by elevating Bcl-2 expression. Further, enforced expression of Bcl-2 can prevent the loss of effector CD8+ T cells in IL-15-deficient mice. Here, we examined whether deficiency of the pro-apoptotic BH3-only molecule Bim could prevent the loss of NK cells and memory CD8+ T cells in IL-15-deficient mice. We found that lack of Bim did not prevent the loss of NK cells and endogenous memory (CD44hi CD122hi) CD8+ T cells in the absence of infection. However, when we analyzed antigen-specific responses following infection with lymphocytic choriomeningitis virus (LCMV), we found that deletion of Bim partially rescued the survival of KLRG1hi CD127lo effector CD8+ T cells in the absence of IL-15. In addition, deletion of Bim completely rescued the loss of functional LCMV-specific memory CD8+ T cells. Our results indicate that IL-15 exerts its homeostatic effects through different mechanisms for the maintenance of endogenous memory phenotype and antigen-specific CD8+ T cells.
Inflammatory chemokine receptors regulate CD8 T cell contraction and memory generation following infection (150.3)
Authors: Kohlmeier Jacob , Reiley William , Perona-Wright Georgia , Freeman Michael , Eric Yager , Connor Lisa , Brincks Erik , Cookenham Tres , Roberts Alan , Burkum Claire , Winslow Gary , Blackman Marcia , Mohrs Markus , Woodland David
Published: January 2023

The development of T cell memory from naïve precursors is a complex process influenced by molecular cues received during T cell activation and differentiation. In the present study, we describe a novel role for the chemokine receptors CCR5 and CXCR3 in regulating effector CD8 T cell contraction and memory generation following influenza virus infection. Using mixed bone marrow chimeras, we find that Ccr5-/- Cxcr3-/- cells show markedly decreased contraction following viral clearance, leading to the establishment of massive numbers of memory CD8 T cells. Ccr5-/- Cxcr3-/- cells show preferential localization to the non-inflamed interstitial spaces of the infected lung, which coincides with reduced expression of the activation marker CD69 and the rapid appearance of CD127hi KLRG1lo memory precursor cells after viral clearance. Importantly, influenza-specific effector Ccr5-/- Cxcr3-/- CD8 T cells in the lung could be activated in vivo by the addition of exogenous antigen, which restored effector T cell contraction and significantly reduced memory T cell generation following viral clearance. Together, the data support a model of memory CD8 T cell generation where the chemokine-directed localization of T cells within infected tissues regulates antigen encounter and controls the extent of CD8 T cell activation and differentiation, which ultimately regulates effector versus memory cell-fate decisions.
The role of basophils in skin Th2 response using newly generated basophil-specific conditional depletion model (151.1)
Authors: Otsuka Atsushi , Kubo Masato , Miyachi Yoshiki , Kabashima Kenji
Published: January 2023

Basophils have been reported to promote Th2 skewing by antigen presentation in a certain condition without necessity of dendritic cells (DCs). On the other hand, recent studies also showed that DCs play a pivotal role of Th2 development. Therefore, it is controversial about the roles of basophils in induction of Th2-type immune responses. We have previously reported that HS4 region of IL-4 gene is essential for basophils. Taking advantage of this system, we have newly generated BaS TRECK transgenic (Tg) mice in which a diphtheria toxin (DT) receptor is knocked-in to the HS4 region to deplete basophils conditionally and specifically by injection of DT. We first evaluated the effect of basophils on repeated hapten application model as a representative of cutaneous Th2 skewing responses. Skin inflammation and serum IgE levels were significantly decreased by depletion of basophils. On the other hand, in ovalbumin patch induced-cutaneous Th2 skewing responses, skin reactions and the serum IgE level were not affected by basophil-deficiency.In addition, bone marrow derived-basophils induced IL-4 producing CD4+T cells only by OVA-peptide but not by OVA-proteins. Furthermore basophils did not uptake and process OVA-proteins. These data suggest that basophils elicited antigen presentation but did not uptake or process protein allergens themselves. Basophils seemed to induce Th2-type immune responses only to the exposure to haptens or protein peptides but not to protein allergens.
Stat5 is serine phosphorylated during IgE- and IgG-mediated mast cell activation (151.16)
Authors: Barnstein Brian , Falanga Yves , Ryan John
Published: January 2023

Stat5 ablation results in defective cytokine production as well as survival, supporting a critical role for this transcription factor in mast cell biology. We have reported the Stat5 tyrosine phosphorylation following IgE-mediated mast cell activation. We now find that Stat5 is also serine phosphorylated following activation by either IgE- or IgG-mediated signals. Both Stat5A and Stat5B were serine phosphorylated. The pathways leading to this event appear to differ based upon the initiating signal. While IgG-mediated activation appears to flow through the Fyn kinase and is strongly JNK-dependent, IgE-mediated Stat5 serine phosphorylation was Fyn-independent and showed a requirement for PI-3 kinase activation. These data demonstrate that Stat5 is activated by both tyrosine and serine modification through pathways that can be selectively regulated based on initiating signals.
T helper 9 cell development is dependent on the T cell maturation stage (152.13)
Authors: Ramming Andreas , Schulze-Koops Hendrik , Skapenko Alla
Published: January 2023

T helper 9 (Th9) cells, a novel CD4 T cell subset, seem to play a complex role in the outcome of specific immune responses. Whilst they may enhance a pro-inflammatory Th17 cell-driven immune response they are also able to prevent allograft rejection by intermediating T cell tolerance. Here, we have analyzed the Th9 cell differentiation requirements from human naive and memory CD4 T cells. Neonatal CD4 T cells required more stringent Th9 differentiation conditions when compared to adult CD4 T cells reflecting a different cytokine requirement for the expression of the Th9 cell master transcription factor PU.1. The requirements for Th9 cell differentiation weakened from neonatal through adult naive to memory T cells. Moreover, the phenotype of developing cells became gradually more heterogeneous even under stringent Th9-inducing conditions from neonatal through adult naive to memory CD4 T cells. Whereas neonatal CD4 T cells differentiated into “pure” Th9 cells producing exclusively IL-9, adult naive or memory CD4 T cells developed into effectors consisting of IL-9-producing cells as well as of cells capable of IL-4, IFN-γ, IL-5, or IL-17 co-production together with IL-9. The data suggest that the conditions for Th9 cell development and therefore for IL-9 production are continuously changing during life which may indicate a growing contributive role of IL-9 in specific immune responses with advancing age.
IL-27 mediated silencing of Foxp3 gene expression via enhanced DNA methylation (152.26)
Authors: Kim Gisen , Shinnakasu Ryo , Roh Sujin , Morris Venetia , Cheroutre Hilde , Kronenberg Mitchell
Published: January 2023

Foxp3 expression is regulated by multiple cytokine signals. While TGFβ and IL-2 are required for the induction and the maintenance of Foxp3 expression, others such as IL-4, IL-6 and IL-27 have been reported to inhibit Foxp3 induction. Despite the previous reports that demonstrated the indispensable role of the Stat3 signaling pathway for IL-6 and IL-27 mediated inhibition of Foxp3 expression, we have found that Stat3 was dispensable for the IL-27 mediated Foxp3 inhibition. Interestingly, while the IL-6 mediated inhibition was effectively reversed by retinoic acid (RA) in Stat3-/- T cells stimulated in the presence of TGFβ, RA failed to reverse IL-27 mediated Foxp3 inhibition. During Foxp3 induction, IL-27 significantly counteracted TGFβ-mediated acetylation of chromosomal histones in the Foxp3 regulatory elements, which was reversed by the addition of DNA methyltransferase inhibitor RG108. In addition to the increased expression of Dnmt1 and Dnmt3a, IL-27 potently enhanced the recruitment of at least Dnmt1 to the Foxp3 loci. Finally, the addition of inhibitors of DNA methyltransferase and histone deacetylases reversed the inhibitory effect by IL-27. These data indicate that IL-27 enhances DNA methylation and subsequent histone deacetylation in the Foxp3 loci, which results in silencing of Foxp3 gene expression. In addition, IL-27 may inhibit Foxp3 expression via a different mechanism from those mediated by IL-6.
B7-H1 (CD274) and sialoadhesin (CD169) on human dendritic cells induce IL-35 producing regulatory T cells (152.27)
Authors: Seyerl Maria , Kirchberger Stefanie , Majdic Otto , Seipelt Joachim , Jindra Christoph , Schrauf Catharina , Stöckl Johannes
Published: January 2023

IL-35 is a heterodimer of EBV-induced gene 3 and of the p35 subunit of IL-12, and was identified as an inhibitory cytokine produced by natural regulatory T cells in mice, but not in humans. Here we demonstrate that DC activated by human rhinoviruses (R-DC) induce IL-35 production and release, as well as a suppressor function in CD4+ and CD8+ T cells derived from human peripheral blood but not in naïve T cells from cord blood. The induction of IL-35-producing T cells by R-DC was FOXP3-independent. We observed that, when we blocked B7-H1 and sialoadhesin on the R-DC side with specific mAb against both receptors, the induction of IL-35 was prevented. Thus, the combinatorial signal delivered by R-DC to T cells via B7-H1 and sialoadhesin is crucial for the induction of human IL-35 producing regulatory T cells. These results demonstrate a novel pathway and its components for the induction of immune-inhibitory T cells.
The next step in the analysis of lymphocyte proliferation (152.7)
Authors: Langsdorf Chris , Bradford Jolene , Zhang Yu-Zhong
Published: January 2023

The mixed lymphocyte reaction (MLR), in which lymphocytes from two donors are combined and co-cultured, has significantly contributed to our understanding of immune function. Initially, the proliferation of responder cells was evaluated by measuring the uptake of radioactive 3H-thymidine. As technology improved, this method gave way to a technique measuring the incorporation of 5'-bromo-2'-deoxyuridine. Further advances led to assays for detection of incorporated 5'-ethynyl-2'-deoxyuridine and dye-dilution assays using carboxyfluorescein-diacetate succinimidyl ester (CFSE). Here we evaluate another advance in the measurement of lymphocyte proliferation kinetics using CFSE in combination with immunophenotyping and a novel fluorescent cell proliferation tracing dye. In the current study mononuclear cells were isolated from the peripheral blood of healthy human donors using a density gradient and labeled with CFSE or a novel violet cell proliferation dye. One-way MLRs were performed by inhibiting proliferation of antigen-presenting cells with Mitomycin-C. Two-way MLRs were also performed by co-culturing lymphocytes from allogeneic donors. The phenotype, viability, and proliferative index of stimulator and responder cells were evaluated using multicolor flow cytometry.
Effect of age related changes in the bone marrow stroma on B lymphopoiesis (153.19)
Authors: Bilwani Fareena , Knight Katherine
Published: January 2023

B lymphopoiesis arrests in rabbits by 16 wk-of-age, and we hypothesize that the arrest is due to age-related changes in the BM stroma. We determined the number of BM stromal cell progenitors (BMSCPs) using limiting dilution analysis and found a nearly 90% decrease in the number of BMSCPs by 6 wk-of-age. This decrease in BMSCPs is reflective of the age-related decline in proB and preB cell progenitors in BM. We tested the capability of BMSCPs to differentiate into adipocytes or osteoblasts in vitro, and found that BMSCPs from 4-8 wk-old rabbits displayed a propensity to form osteoblastic colonies while BMSCPs of rabbits over 8 wk-of-age readily formed adipocytic colonies. Histology of frozen BM tissue sections revealed trabecular bone in rabbits less than 8 wk-of-age. In contrast, BM from rabbits 8 wk-of- age or older showed large amounts of adipose tissue and almost no trabecular bone formation. Using a transwell co-culture system we found that adipocytes secrete a soluble factor that inhibits production of proB cells. We conclude that the decline in B lymphopoiesis of rabbits with age is due both to a decrease in the number of BMSCPs and also to the preferential differentiation of BMSCPs into adipocytes which in turn, secrete a molecule that inhibits B lymphopoiesis.
Expression of KIT isoforms in systemic mastocytosis: correlation with disease severity and KITD816V (153.32)
Authors: Chan Ching , Bai Yun , Maric Irina , Klion Amy , Metcalfe Dean , Wilson Todd
Published: January 2023

Human KIT has several variants as the result of alternative mRNA splicing. Two major isoforms differ by the presence or absence of four amino acids (GNNK) at the juxta-membrane region of the extracellular domain. Somatic gain of function mutations in the tyrosine kinase domain of c-kit, most notably KITD816V, are detected in the majority of adult patients with systemic mastocytosis. We hypothesized that the expression pattern of GNNK variants may differ with disease severity, and transcripts containing the KITD816V mutation may segregate preferentially to one GNNK variant. A quantitative real-time PCR assay to assess GNNK- and GNNK+ transcripts was developed. We analyzed the ratio of GNNK- to GNNK+ transcripts in bone marrow mononuclear cells from 25 mastocytosis patients and 16 healthy controls. The GNNK- variant was more abundant in both patient and control samples. In advanced forms of mastocytosis, we observed a significant lower GNNK-/+ ratio compared to indolent systemic mastocytosis (p=0.0005). The KITD816V mutation was detected in both variants from KITD816V positive patients (n=21) and the KITD816V GNNK-/+ ratio was significantly higher than the total KIT GNNK-/+ ratio. Altogether, a relative increase in the GNNK+ variant was observed in advanced disease, and KITD816V specific transcripts displayed a relative GNNK- predominance. These data suggest that the determination of the GNNK-/+ ratio may be used to help determine disease prognosis.
Thymus-derived innate CD8+CD44hi T cells display features of antigen-experienced T cells with bystander helper properties (153.43)
Authors: Rafei Moutih , Williams Patrick , Ruiz Juan , Forner Kathy-ann , Dulube Gael , Galipeau Jacques , Perreault Claude
Published: January 2023

Innate CD8 T cells are abundant in several transcription factors (TFs)-deficient mouse models, but little is known about their development in wild-type animals. We used the RAG2-GFP mouse model to discriminate between memory, naïve classic and innate TCRαβ CD8 cells in the thymus. Relative to classic T cells, innate thymocytes display increased levels of numerous cell surface molecules (TCR, CD2, CD3, CD5, CD38, CD62L, B7H1) and enhanced expression of the TFs T-bet, EOMES, Id3 and KFL2. Hematopoietic chimera studies revealed that non-hematopoietic MHCI+ cells are required for innate CD8 T cell selection in the thymus. Mice expressing the transgenic OTI TCR generate both classic and innate CD8 thymocytes suggesting that both subsets have overlapping TCR repertoires. Innate CD8 T cells were also found to rapidly produce high levels of IFNγ upon stimulation and to readily proliferate in response to IL2 and IL4. Under lymphopenic conditions, classic CD8 T cell expansion was 2-3 fold greater than that of innate T cells, which constitutively express B7H1 making them unduly susceptible to apoptosis. To test their bystander properties, we added classic or innate T cells as third-party cells to an in vitro antigen presentation assay. Innate T cells were capable of promoting the proliferation and activation of responding T cells in a CD44-dependent manner. These observations show that innate CD8 T cells represent a subset of thymocytes that differ in many ways from classic CD8 T cells.
CD28 costimulation is not required for the development and differentiation of γδ T cell effectors (153.45)
Authors: Laird Renee , Wolf Benjamin , Princiotta Michael , Hayes Sandra
Published: January 2023

Both antigen recognition and CD28 costimulation are required for the activation of naïve αβ T cells and their subsequent differentiation into cytokine-producing or cytotoxic effectors. However, because of contradictory results, it remains unresolved as to whether CD28 costimulation is also necessary for the activation and differentiation of γδ T cell effectors. Recently, it has been shown that γδ T cells acquire effector functions in the thymus following encounter with self-ligands. This is in contrast to αβ T cells, which acquire effector functions in secondary lymphoid tissues following encounter with foreign antigens. In light of these newly identified requirements for γδ effector cell development, we re-examined the role of CD28 in γδ T cell effector fate specification and function. We report that, although IFNγ-producing γδ effectors (γδ-IFNγ) express higher levels of CD28 than IL-17-producing γδ effectors (γδ-17), CD28-deficiency has no effect on the development of either γδ-17 or γδ-IFNγ cells. Also, following infection with Listeria monocytogenes, we found that the activation, expansion and differentiation of γδ-17 and γδ-IFNγ cells were normal in CD28-/- mice. Thus, these data not only indicate that CD28 is not essential for γδ T cell effector development and differentiation but also highlight differences in the requirements for the generation of αβ and γδ T cell effectors.
NFIL3/E4BP4 is a key transcription factor for CD8α+ dendritic cell development (153.47)
Authors: Kashiwada Masaki , Pham Nhat-Long , Pewe Lecia , Harty John , Rothman Paul
Published: January 2023

Antigen presentation by mature dendritic cells (DCs) is the first step for initiating adaptive immune responses. DCs are comprised of heterogeneous functional subsets. However, the molecular mechanisms regulating differentiation of specific DC subsets are not understood. Here, we report that the basic leucine zipper transcription factor NFIL3/E4BP4 is essential for the development of CD8α+ conventional DC (cDC). Nfil3-/- mice specifically lack CD8α+ cDCs but not CD8α- cDCs or plasmacytoid DCs in lymphoid tissues. Flt3 ligand-dependent generation of CD8α+ cDCs in lymphoid tissues and CD8α+-equivalent cDCs from Nfil3-/- bone marrow cells was also impaired. NFIL3 regulates CD8α+ cDC development in part throuth Batf3 expression. Importantly, Nfil3-/- mice exhibit impaired cross-priming of CD8+ T cells against cell-associated antigen, a process normally carried out by CD8α+ cDCs, and failed to produce IL-12 after TLR3 stimulation. Thus, NFIL3 plays an essential role in the development of CD8α+ cDCs.
Human thrombopoietin knockin mice efficiently support human hematopoiesis in vivo (153.5)
Authors: Rongvaux Anthony , Willinger Tim , Takizawa Hitoshi , Rathinam Chozhavendan , Eynon Elizabeth , Stevens Sean , Manz Markus , Flavell Richard
Published: January 2023

Hematopoietic stem cells (HSCs) both self-renew and give rise to all blood cells for the lifetime of an individual. Xenogeneic mouse models are broadly used to study human hematopoietic stem and progenitor cell biology in vivo. However, maintenance, differentiation, and function of human hematopoietic cells is suboptimal in these hosts. We hypothesized that this defect was due to reduced cross-reactivity of mouse cytokines on the human receptor. Thrombopoietin (TPO) has been demonstrated as a crucial cytokine supporting maintenance and self-renewal of HSCs. We generated RAG2-/-γc-/- mice in which we replaced the gene encoding mouse TPO by its human homologue, and we transplanted human CD34+ cells in these recipients. Homozygous humanization of TPO (TPOh/h) led to increased levels (higher than 80%) and longer maintenance (more than 6 months) of human cell engraftment in the bone marrow of the hosts. Multilineage differentiation of hematopoietic cells was also improved, with an increased ratio of myelomonocytic verus lymphoid lineages. Moreover, maintenance of human stem and progenitor cells was improved : we observed a significant increase in the fraction of human Lin-CD34+CD38-CD90+CD45RA- cells in TPOh/h compared to control recipients; and CD34+ cells purified from TPOh/h primary recipient had a significantly increased capacity to efficiently engraft secondary recipients. Therefore, RAG2-/-γc-/- TPO-humanized mice represent a novel model to study human hematopoiesis in vivo.
Increased lethality but decreased encephalitis in IRF8-deficient mice following West Nile Virus infection (154.1)
Authors: Minten Carsten , Terry Rachael , King Nicholas , Campbell Iain
Published: January 2023

The transcription factor IRF8 is crucial in myeloid and dendritic cell development and the response of these cells to the cytokine IFN-γ. The absence of IRF8 is associated with specific morphological and functional changes in peripheral macrophages as well as CNS resident myeloid cells known as microglia. These cells have been shown to be pivotal in the pathogenesis of West Nile Virus (WNV) encephalitis in mice. Therefore, we examined the role of IRF8 in the host response to infection of the brain with WNV. IRF8 KO mice showed increased lethality following intranasal challenge with low dose WNV compared with similarly infected WT mice. In the brain, WNV loads were higher and virus more disseminated, however, there was a dramatic reduction of infiltrating T-cells and macrophages in WNV-infected IRF8 KO mice compared with similarly infected WT animals. While the cerebral expression of many proinflammatory chemokine (CCL2, CCL4, CCL5, CXCL10) and cytokine (TNF and IL-6) genes was significantly increased in both WT and IRF8 KO mice infected with WNV, expression of the IL-12p40 and IFN-γ genes was increased in WT but not IRF8 KO mice. In conclusion, IRF8 is apparently dispensable in the localised innate inflammatory response to WNV infection, however, IRF8 has a key role in the antiviral response involved in controlling virus replication and spread within the CNS. Furthermore, our data demonstrates that IRF8 may be crucial for the adaptive immune response against WNV in the CNS.
Cationic Lipid DNA Complex (JVRS-100) treatment of woodchucks with Hepatitis B virus induced Hepatocellular Carcinoma (154.12)
Authors: Fairman Jeffery , Liu Katherine , Menne Stephan
Published: January 2023

To determine the dependence of responsiveness to immune stimulation on the woodchuck hepatitis B viral (wHBV) load, we evaluated cytokine and cell surface marker mRNA expression following dosing of JVRS-100 in chronic woodchuck carriers with low (mean: 2.2x1010 ge/ml), medium (4.0x1010 ge/ml) and high (4.2x1011 ge/ml) serum viral loads. Our hypothesis was that high viral loads seen in chronic wHBV infection (10x human) result in immune suppression to external immune stimuli. Woodchucks were dosed intravenously with JVRS-100 and evaluated for immune activation. Woodchucks with low viral load had increased expression of cytokine and cell surface markers in contrast to animals with moderate or high viral loads with no change in expression. To evaluate anti-tumor effects of JVRS-100 we dosed groups of woodchucks with 100µg or 300µg JVRS-100. When normalized for pre-treatment levels of wHBV serum DNA, there was a steady decrease in viral load of treated woodchucks. There was no difference in the tumor burden; however, woodchucks in the high dose JVRS-100 group developed no new tumors over the 24 weeks in which they were monitored (12 weeks Rx followed by 12 weeks observation). This is unusual for this disease model and we believe highly significant to the development of an intervention for HBV induced HCC. Our working hypothesis is that the administration of JVRS-100 blocks the conversion of chronic HBV infection to HCC.
Reduction of HIV-1 innate immune activation by protease inhibitors (154.15)
Authors: Wallet Mark , Appelberg Sofia , Reist Caroline , Guiulfo Giorgio , Sleasman John , Goodenow Maureen
Published: January 2023

HIV-1-associated innate immune activation is caused by viral replication and/or microbial substances that leak from the gut lumen into the circulation (microbial translocation). Consequences of the ensuing HIV-1-associated inflammation range from neurocognitive impairment to cardiovascular disease where macrophages play a central role. HIV-1 therapy is evaluated based on suppression of viremia and restoration of CD4 T cells, while the effects of therapy on innate immune activation are rarely considered. The current study assessed the effects of protease inhibitors on macrophage activation ex vivo and in vivo. Nelfinavir [NFV] and tipranavir [TPV] inhibited LPS activation of human macrophages ex vivo as measured by reduced secretion of LPS-binding protein soluble CD14 [sCD14] and pro-inflammatory cytokines IL-6 and TNF. Cytokine secretion was inhibited prior to transcription by modulation of toll-like receptor signaling pathways. In vivo, NFV-treated HIV-1-infected patients presented with significantly reduced monocyte/macrophage activation as measured by a greater reduction in sCD14 plasma levels compared to those treated with indinavir or ritonavir. These data suggest that anti-inflammatory effects of NFV or TPV may augment efficacy of HIV-1 treatment by targeting HIV-1-assocated innate immune activation. Delineation of cellular targets of NFV or TPV will lead to novel approaches to suppress innate immune activation in HIV-1, other infectious diseases or autoimmunity.
Epstein-Barr Virus LMP2A modulates B cell proliferation and cytokine production (154.16)
Authors: Swanson-Mungerson Michelle , Incrocci Ryan , Bultema Rebecca
Published: January 2023

Despite the association of Epstein-Barr Virus (EBV) with numerous malignancies and autoimmune diseases, the mechanism by which EBV promotes disease is unknown. After initial infection, EBV transitions to a latent state in which few viral transcripts, such as Latent Membrane Protetein 2A (LMP2A), are expressed. While LMP2A is known to promote B cell survival, our data using primary LMP2A-expressing B cells indicate that LMP2A increases B cell proliferation in response to mitogens. Since cytokines are critical in promoting B cell survival and proliferation, we hypothesized that LMP2A increases B cell cytokine production to increase their survival and proliferation. Our data indicate that LMP2A does not globally increase B cell-produced cytokine levels, but rather targets a limited number of cytokines. These findings implicate that LMP2A-expressing B cells not only have the ability to influence B cell survival and proliferation in an autocrine manner, but may also secrete cytokines to modulate immune cells in their environment. This work is funded by NIH grant 1 R15 CA149690-01 and Midwestern University Intramural Funds.