bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of chemotaxis and cell invasion assays in microfluidic flow cells for use with real-time microscopy. (140.9)
Authors: Conant Carolyn , Kendall Stephen , Schwartz Michael , Ionescu-Zanetti Cristian
Published: January 2023

Many immunological processes in health and disease involve migration, homing, invasion and/or transmigration of cells in response to chemoattractants. In vitro analysis of these phenomena typically involves use of a transwell plate insert to create a two environment experiment. This allows quantitation of either migration or invasion, but the inserts can be cumbersome to handle and are normally processed at the endpoint of the experiment. Here, we demonstrated a microfluidic based method to follow chemotaxis and cell invasion in real time. The method enabled acquisition of high content data by microscopy using microfluidic flow cells which are optimized for imaging. Up to 16 conditions were tested simultaneously. The channels were filled with chemokine or matrix in one half of the channel while the other half was maintained with a neutral buffer. Using this technique, we tested differentiated HL-60 cells’ response to fMLP in real time. We also investigated invasion of serum starved HT1080 and MCF-7 cells into FBS containing matrix and found that as predicted only HT1080 cells successfully invaded the matrix. Using these experimental conditions, the method has the potential to yield insights into the early and ongoing processes that occur during homing and invasion of cells within the vasculature.
Reassessment of innate and adaptive immune pathways in fungal-dependent allergic lung disease (141.1)
Authors: Porter Paul , Abramson Stuart , Delclos George , Kheradmand Farrah , Corry David
Published: January 2023

Genes relevant to allergic lung disease were re-evaluated using our fungal inhalation model. We focused on the asthma phenotype as well as fungal clearance in IL-13, IL-5, Stat6, Rag1, CD1, and TSLP deficient mice. For asthma phenotyping, genetic knock-out (or anti-IL-5 treated) mice were intra-nasally inoculated with 400,000 Aspergillus niger conidia every other day for 16 days, and endpoints measured 24 hours after the final dose. The endpoints measured were airway hyper-reactivity (AHR), bronchial lavage fluid differential cell analysis, and lung IL-4 and IFNγ elispot analysis. For fungal clearance, mice were intra-nasally inoculated with 400,000 Aspergillus niger conidia, and lungs removed at various time points to determine the number of viable conidia remaining. Our results confirmed the requirement of IL-13, Stat6, Rag1, and TSLP for AHR induction; the requirement of Stat6, Rag1, IL-13, IL-5 and TSLP for eosinophil recruitment; the requirement of IL-13, Stat6, Rag1, and TSLP for IL-4 producing cells recruitment. CD1 KO mice showed an exaggerated phenotype with increased AHR and IL-4 production. IL-13, Rag1, and IL-5 were required for optimal fungal clearance. Thus, similar to non-infectious asthma models, conventional T cells, the IL-13 signaling pathway and TSLP were important for disease expression induced by viable fungi. In contrast, non-conventional CD1-resrticted NKT cells were found to be potent suppressors of fungal-dependent allergic lung disease.
Notch ligand Delta-like 4 regulates development and pathogenesis of allergic airway responses by modulating IL-2 production and Th2 immunity (141.15)
Authors: Jang Sihyug , Schaller Matthew , Lukacs Nicholas
Published: January 2023

Activation of the conical Notch pathways have been implicated in Th cell differentiation, but the role of specific Notch ligands in Th2 mediated allergic airway responses has not been completely elucidated. In this study, we show that delta-like 4 (Dll4) was up-regulated on dendritic cells in response to cockroach allergen. Blocking Dll4 in vivo at either the afferent or efferent phases of the immune response enhanced allergen-induced pathogenic responses including airway hyperresponsiveness (AHR) and mucus production via increased Th2 cytokines. In vitro assays demonstrated that Dll4 regulates IL-2 in T cells from established Th2 responses as well as during primary stimulation. Interestingly, Dll4 blockade during the afferent, but not the efferent phase, increased IL-2 levels in lung and lymph node of allergic mice. Moreover, Dll4-mediated Notch activation of T cells during primary stimulation in vitro increased apoptosis during the contraction/resting phase of the response, which could be rescued by exogenous IL-2. Consistent with the role for Dll4-mediated IL-2 regulation in overall T cell function, the frequency of IL-4 producing cells were also significantly altered by Dll4 both in vivo and in vitro. These data demonstrate a regulatory role of Dll4 on both Th2 differentiation as well as on Th2 cytokine production in established Th2 responses.
Artemisinin inhibits lipopolysaccharide-induced nitric oxide production by blocking IFN-β production and STAT-1 signaling in macrophages (142.2)
Authors: Kang Jong Soon , Park Ki Hwan , Lee Hyunju , Han Sang-Bae , Lee Kiho , Park Song-Kyu , Kim Hwan Mook
Published: January 2023

Artemisinin is a well-known anti-malarial drug and has been reported to exert anti-inflammatory and anti-tumor effects. In this study, we investigated the effect of artemisinin on lipopolysaccharide (LPS)-induced NO production in macrophages and the molecular mechanisms responsible for its effects. Artemisinin significantly suppressed NO production and inducible nitric oxide synthase (iNOS) mRNA expression in LPS-stimulated macrophages. NF-κB activity, which is known to be important for NO production and iNOS expression, was minimally inhibited only by highest concentration of artemisinin used and MAPKs were not affected by artemisinin treatment. In contrast, LPS-induced activation of STAT-1 was suppressed by artemisinin treatment in a concentration-dependent manner. Further studies demonstrated that the inhibition of STAT-1 activation by artemisinin might be mediated by blocking interferon-β (IFN-β) production. These results suggest that artemisinin suppresses NO production and iNOS expression, at least in part, by blocking IFN-β production and concomitant down-regulation of STAT-1 signaling.
Long lasting elimination of B cells in NZB/W F1 mice can be induced by NZB but not NZW parental CD8 CTL. (143.12)
Authors: Via Charles , Puliaeva Irina , Soloviova Kateryna , Puliaiev Maksym , Puliaiev Roman
Published: January 2023

B cells are a target in current lupus therapy. In the parent-into-F1 (P->F1) model of induced murine lupus, lupus is prevented by donor anti-host cytotoxic T lymphocytes (CTL) elimination of B cells. To determine the therapeutic feasibility of CTL in a spontaneous lupus model, we transferred NZB or NZW parental splenocytes (60 or 90 x 106 cells) into 8 week old female NZB/W F1 mice. 2 weeks after transfer, NZB->F1 mice exhibited significant host B cell reduction and upregulation of B cell Fas consistent with in vivo CTL activity, better seen at the higher dose. By contrast, neither dose of NZW donor cells induced elimination of host B cells below control despite significant donor CD4/CD8 T cell engraftment and donor CD4 upregulation of CD44. At 12 weeks after transfer, NZB->F1 mice exhibited persistent reduction of host B cells significantly below control at both doses whereas B cells were not eliminated in NZW->F1 mice at either dose. No mice developed proteinuria >2+. Thus, NZW splenocytes fail to induce detectable B cell elimination short or long term despite engraftment and activation of donor T cells, indicative of defective effector CTL function. In contrast, NZB->F1 mice support the feasibility of CD8 CTL induction and B cell elimination both short and long term. Studies are in progress to determine if lupus-like disease is also attenuated. If so, more narrowly targeted CD8 CTL may be useful in selectively targeting pathogenic B cells in lupus.
PD-1 expression is required in recent thymic emigrants to establish systemic self-tolerance (143.15)
Authors: Anderson Colin , Uwiera Richard , Thangavelu Govindarajan
Published: January 2023

Homeostatic activation of T cells occurs in both natural and artificially induced states of lymphopenia. Control of this process may be particularly important during the period when the first T cells are exported from the thymus, as recent thymic emigrants (RTE) have a heightened sensitivity to homeostatic cytokines. We tested whether PD-1 is required to prevent lymphopenia-induced immunopathology, and assessed its importance in recent thymic emigrants vs. established peripheral T cells. Established T cells express low levels of PD-1 and established PD-1-/- T cells did not cause overt disease in lymphopenic Rag-/- recipients. In contrast, PD-1 was expressed at high levels on RTE, and PD-1-/- RTE produced from hematopoietic stem cells (HSC) generated a rapidly lethal multi-organ inflammatory disease in the Rag-/- recipients. Disease could be blocked by increasing competition through co-transfer of wild type HSC or by using cytokine receptor deficient or neonatal recipients that have reduced lymphoid space for homeostatic activation. Thus, the PD-1 pathway has a broadly important role in self-tolerance, controlling a key checkpoint in the prevention of lymphopenia driven autoimmunity by RTE. Clinical therapies for autoimmune disease employing lymphoablation and hematopoietic stem cell transplantation will need to take into account functional polymorphisms in the PD-1 pathway, if the treatment is to ameliorate rather than exacerbate autoimmunity.
Oligoclonality and innate-like features in the TCR repertoire of type II NKT cells reactive to a β-linked self-glycolipid (143.16)
Authors: Arrenberg Philomena , Halder Ramesh , Dai Yang , Maricic Igor , Kumar Vipin
Published: January 2023

CD1d-restricted type II NKT cells are distinct from the invariant type I NKT cells and are poorly characterized. A major subset recognizes the β-linked self-glycolipid sulfatide, playing a role in autoimmunity and anti-tumor immunity. Here, we analyzed the TCR repertoire of this type II NKT cell subset in mouse, using flowcytometry, TCR gene sequencing and spectratyping analysis of sorted sulfatide/CD1d-tetramer+ cells. For the TCR β-chain, we found predominant usage of the Vβ8.1/Vβ3.1 and Jβ2.7 gene segments with few dominant clonotypes of restricted CDR3 lengths and conserved acidic residues. The TCR α-chain was predominantly encoded by Vα1/Vα3 and Jα7/Jα9 gene segments and showed defined CDR3α regions of 8 or 9 a.a. lengths comprising conserved basic residues. CDR3 regions of both TCR α- and β-chains were encoded by germline sequences or N-additions. Though potential CD1d-binding tyrosine residues in the CDR2β region are conserved between most type I and type II NKT TCRs, CDR1α and 3α regions differ significantly between the two subsets. Collectively, these findings emphasize innate-like features of sulfatide-reactive type II NKT cells as well as oligoclonal TCR Vα-Jα/Vβ-Jβ gene usage characteristic of the antigen-specific responses of conventional MHC-restricted T cells. These data provide important clues to the recognition of β-linked glycolipids by CD1d-restricted T cells in general. (Supported by grants from NIH, JDRF, MSNRC and DNRG (to VK) and DFG (to PA).)
Cross Talk Between Pathogenic CD4+T Cells and B Cells (143.35)
Authors: Xiang Yufei , Peng Jian , Tai Ningwen , Hu Changyun , Zhou Zhiguang , Wen Li
Published: January 2023

The effect of B cell depletion on diabetogenic T cells is not fully understood. We generated hCD20BDC2.5 NOD mice to specifically address this question. We treated hCD20BDC2.5 NOD mice with 2H7 or control IgG.Our results showed that dividing BDC2.5 cells from 2H7 treated mice produced more IFNg than their counterparts from IgG treated mice (P=0.0035). Interestingly, circulating IFNg in serum of IgG treated mice was higher than that in 2H7 treated mice (p=0.0029). To investigate effect of B cell depletion on diabetogenicity of BDC2.5 T cells, we transferred purified CD4+ BDC2.5 T cells, from either 2H7 or IgG treated mice, into NOD/SCID mice. The overall incidence of diabetes was much higher in NOD/SCID mice received BDC2.5 CD4+ cells from 2H7 treated donors than that from IgG treated donors. To investigate the direct effect of B cells on diabetogenic CD4+ T cells, we co-transferred BDC2.5 CD4+ T cells with purified B cells from NOD mice. Interestingly, we found that B cells may have a protective effect as diabetes development was delayed in the NOD/SCID mice received more B cells. In vitro, the addition of B cells inhibited the proliferation of BDC2.5 CD4+ T cells, supporting the in vivo diabetes development results. In summary, B cell depletion affects pathogenic T cells and there is a dichotomy in the role of B cells in diabetes development.
Protection against lupus nephritis in MRL-lpr mice expressing a natural autoantibody transgene (143.39)
Authors: Chen Qing , Mannoor Kaiissar , Matejuk Agata
Published: January 2023

Natural autoantibodies (NAA) are antibodies present in normal sera without exogenous antigen stimulation. They often have relatively low yet specific reactivity towards multiple self and non-self antigens. Despite their potential importance, the functional role of NAA is poorly understood. We recently generated an Ig site-directed transgenic (sd-tg) mouse model carrying a prototypic NAA that binds a variety of antigens including DNA and phosphocholine. Using this model, we have shown previously that the NAA-producing B-cells are positively selected in normal mice. In the present study, we show that expression of NAA in autoimmune MRL-lpr mice results in nearly complete protection from autoimmune nephritis as assessed by proteinuria, kidney histopathology and immune complex deposition. Accordingly, the NAA sd-tg mice have significantly improved survival. The non-tg and tg MRL-lpr mice have very different IgG subclasses: while the non-tg MRL-lpr mice have high titers of IgG2 and IgG3, both of which are highly pathogenic in lupus nephritis, the sd-tg mice have a predominance of IgG1, the least pathogenic IgG subclass. In addition, the tg MRL-lpr mice have fewer CD44hiCD62L+/- activated T-cells and more CTLA-4+ T-cells as compared with the non-tg mice. These results indicate that NAA and NAA-producing B-cells play an important role in protection from lupus nephritis, and suggest that NAA B-cells may have regulatory effects on CD4+ T cells.
A rapid and efficient method for the isolation of distinct human regulatory T cell populations using combinations of antibody mediated buoyant density centrifugation and/or column-free immunomagnetic cell separation (143.41)
Authors: Kokaji Andy , MacDonald Neil , Fairhurst Maureen , Thomas Terry
Published: January 2023

Regulatory T cells (Tregs) are a specialized subset of T cells that play a key role in immune regulation. Harnessing the suppressive function of Tregs is a major area of interest as they hold great potential for the treatment of autoimmune disorders. The first step towards quality Treg research is the isolation of highly purified, functional Tregs. With unique cell isolation platforms, STEMCELL has developed a full range of products for the rapid and efficient isolation of highly functional Tregs from virtually any peripheral blood sample. RosetteSep is an antibody mediated buoyant density centrifugation method used to isolate unlabelled cells specifically from whole blood or buffy coat samples. EasySep is an immunomagnetic cell separation method used to isolate cells from fresh or previously frozen PBMCs. Treg pre-enrichment is achieved by antibody mediated crosslinking of unwanted cells to either red blood cells (RosetteSep) or magnetic particles (EasySep) allowing their removal by Ficoll centrifugation or magnetic separation, respectively. RosetteSep or EasySep pre-enriched Treg populations consist of CD4+, CD4+CD127low or CD4+CD127lowCD49d- T cells. Pre-enriched Tregs can be further purified using EasySep positive selection to isolate Tregs expressing high levels of cell surface CD25. Purities of 85% +/- 10% CD4+CD25highFOXP3+ human Tregs can be achieved depending on the Treg population. From start to finish, Treg isolations can be completed in less than 3 hours.
Sulfatide-mediated activation of type II NKT cells prevents hepatic ischemic reperfusion injury (143.43)
Authors: Arrenberg Philomena , Maricic Igor , Kumar Vipin
Published: January 2023

Hepatic ischemic reperfusion injury (IRI) is a major complication of liver transplantation and resectional hepatic surgeries. Recent studies suggested involvement of NKT cells in the inflammatory response leading to hepatocellular damage. However, no detailed analysis on the role of the distinct type I and type II CD1d-restricted NKT cells has been carried out. We have identified a major subset of type II NKT cells reactive to the self-glycolipid sulfatide. Here, we show that mice lacking type I NKT cells (Jα18-/-) are protected from hepatic IRI, as reflected by reduced hepatocellular necroses and serum ALT levels. In contrast, sulfatide-mediated activation of type II NKT cells is protective, associated with reduced intracellular IFN-γ expression by type I NKT cells. These data are consistent with our earlier report of anergy induction in type I NKT cells following sulfatide administration. Prevention of hepatic IRI also involved inhibition of hepatic recruitment of myeloid cell subsets, especially the CD11b+Gr-1int and Gr-1- subsets, and of NK cells. Our findings clearly indicate a pathological role for type I NKT cells and a protective role for sulfatide-reactive type II NKT cells in hepatic IRI. Our data have important implications for the development of novel strategy for amelioration of clinical problems associated with hepatic reperfusion, including liver transplantation. (Supported by grants from NIH, JDRF, MSNRC and DNRG (to VK) and DFG and YAEL Foundation (to PA)).
Breaking B cell tolerance by multimerized self antigens on follicular dendritic cells (143.7)
Authors: El Shikh Mohey Eldin , El Sayed Rania , Szakal Andras , Tew John
Published: January 2023

Self antigen-bearing follicular dendritic cells (FDCs) appear to be involved in the pathogenesis of autoimmunity, and interfering with the development of FDC-reticula attenuates ectopic germinal centers (GCs) & auto-Ab production. FDCs multimerize monomeric antigens in immune complexes (ICs) and present them polyvalently in an array that extensively cross-links BCRs and induces T cell independent Ig responses with FDC-BAFF & -C4bBP providing co-signals. We hypothesized that self antigens presented as multimerized ICs on FDCs would break B cell tolerance and that would be indicated by induction of autoreactive GCs & auto-Ab responses. To test this, mice were challenged with murine TNF-α or IgE in the form of ICs. Sera were assessed for auto-Ab production, and draining lymph nodes were examined for autoreactive GCs and plasma cells. Murine TNF-α & IgE ICs were retained on well-developed FDC reticula and numerous GCs & plasma cells were induced. Murine TNF-α & IgE-specific IgM auto-Abs were detectable in 48 hrs, and switched to IgG by day 6. This is the first report indicating that B cell tolerance can be broken by presenting self antigens and co-stimulatory molecules on FDCs. The ability to deliberately break B cell tolerance and induce self-regulated auto-Abs provide potential novel therapies targeting endogenous mediators of chronic inflammation & hypersensitivity diseases.
ABfinity™ Antibodies - A novel approach to antibody development (144.20)
Authors: Allen Michael , Kim Yun , Dubbels Bradley , Low Quentin , Leong Louis , Huuskonen Jarkko
Published: January 2023

Antibody development falls into two major categories. Polyclonal antibodies are simple and inexpensive to produce but suffer from high variability and low reproducibility. Monoclonal antibodies developed through traditional hybridoma technologies are more expensive and time consuming to produce but have less variability. We have developed ABfinity™ technology which allows us to combine the advantages of these technologies to develop species-independent recombinant monoclonal antibodies from peripheral blood of immunized animals in less than 3 months. ABfinity™ technology allows us to culture and select antigen-specific B cells, clone the corresponding IgG heavy and light chain genes and express them in mammalian cells in a rapid and high-throughput manner. ABfinity™ antibodies are tested in a variety of applications including western blot, imaging, flow cytometry, and immunoassay. Lot-to-lot variability is virtually zero and many antibodies are extremely potent compared to currently available antibodies. In addition to de novo antibody development, ABfinity™ technology can be used to recover IgG genes from existing hybridomas to rapidly develop a recombinant antibody. Finally, because of the recombinant nature of ABfinity™ antibodies we can add affinity tags and other modifications that greatly enhance their use in downstream applications. Through ABfinity™ technology we can mix-and-match specificities, isotypes, species, and modifications to create truly modular antibodies.
Tumor cells engineered to codisplay on their surface 4-1BBL and LIGHT costimulatory proteins as a novel approach for cancer immunotherapy (144.24)
Authors: Sharma Rajesh , Yolcu Esma , Elpek Kutlu , Shirwan Haval
Published: January 2023

Tumor cells genetically modified to express immunomodulatory molecules as vaccine have shown efficacy against cancer. However, such vaccines are not only cost/labor and time intensive, but also have safety repercussions. As an alternative, we developed the ProtEx™ technology that involves modification of the cell membrane with biotin and engineering with proteins of interest chimeric with core streptavidin. We herein demonstrate that tumor cells can be engineered simultaneously with two SA-chimeric costimulatory ligands, SA-4-1BBL and SA-LIGHT. The choice of 4-1BBL and LIGHT were due to their immune activating pleiotropic functions and stimulatory effects on tumor stroma, respectively. Using TC-1 tumor cells as a transplantable cervical cancer mouse model, we demonstrated that these cells can be engineered simultaneously with both molecules. Vaccination with irradiated TC-1 cells engineered with both costimulatory proteins resulted in 100% efficacy in prophylactic and >57% efficacy in therapeutic TC-1 settings. In marked contrast, TC-1 cells displaying these molecules singly showed limited efficacy. Vaccine efficacy was associated with the generation of primary and memory T cell, IFN-γ, and Ab responses, and enhanced CD8+ T cell infiltration into the tumor. Engineering tumor cells in a rapid and effective manner to simultaneously display on their surface a collection of immunostimulatory molecules with distinct functions presents a novel approach for cancer immunotherapy.
A novel M cell-based strategy to boost immune responses to oral vaccines: induction of intestinal villous M cells by RANKL pretreatment leads to enhanced vaccine uptake and improved antibody responses (144.26)
Authors: Knoop Kathryn , Butler Betsy , Kumar Nachiket , Williams Ifor
Published: January 2023

An established strategy for improving immune responses to mucosal vaccines relies on targeting ligands to promote vaccine uptake by microfold (M) cells. M cells are antigen-sampling epithelial cells mostly found in the epithelium covering Peyer’s patches and other organized mucosal lymphoid tissues, but also present on a subset (~7%) of small intestinal villi as villous M cells (vM). Recently we showed that 4 days of systemic treatment (50 μg s.c. per day) with RANKL, a TNF superfamily cytokine, induces differentiation of vM on 100% of small intestinal villi, dramatically increasing the total number of available vM and leading to large increases in uptake of fluorescently labeled bacteria (30-fold) and 200 nm microspheres (100-fold). The present study was done to test a novel strategy for enhancing mucosal vaccine responses to oral vaccines based on RANKL pretreatment to induce supraphysiologic numbers of vM. Mice treated daily with RANKL beginning 4 days prior to a single oral immunization with 2 x 108 fixed OVA-expressing E. coli had an average 8-fold increase in the serum IgM anti-OVA response measured 7 to 11 days later. This effect of RANKL on the antibody response also synergized with the use of LPS as a systemic adjuvant given i.p. at the time of oral immunization. These results provide a proof of principle demonstration that RANKL-mediated induction of vM prior to oral immunization is a novel and effective strategy capable of enhancing the potency of mucosal vaccines.
Distinct role of ICOS in the generation of regulatory T cells induced by CD40-activated B cells (145.12)
Authors: Zheng Jian , Liu Yinping , Lau Yu-Lung , Tu Wenwei
Published: January 2023

Inducible co-stimulator (ICOS) belongs to CD28 and CTLA-4 cell-surface receptor family and is broadly expressed in activated T cells. ICOS has been shown to regulate the interactions between T and B cells. However, the role of ICOS in the induction of regulatory T cells (Tregs) by allogeneic CD40-activated B cells remains to be clarified. In this study, we assayed the expressions of ICOS and ICOSL in CD4+ and CD8+ Tregs induced by CD40-activated B cells and examined the roles of them in the generation and function of these Tregs. With the blockade of ICOS-ICOSL interaction by ICOS-Ig, we found that the generation of both CD4+ and CD8+ Tregs was significantly decreased as compared to control. Interestingly, this blockade during the induction of Tregs only abolished the suppressive capacity of CD4+ Tregs but did not affect the suppressive function of CD8+ Tregs. The loss of suppression in CD4+ Tregs was related to the decrease of the surface expression of CTLA-4 in CD4+ Tregs, which resulted from the impaired transfer of CTLA-4 from cytoplasma to cell surface. In conclusion, ICOS-ICOSL interaction plays an important role in the generation of CD4+ and CD8+ Tregs, and mediates the suppressive functions of CD4+ Tregs by regulating CTLA-4 expression on cell surface induced by allogeneic CD40-activated B cells.
Tumor-sensitized, Vβ spectratype-selected CD8+ T cells promote graft-versus-leukemia responses in the absence of severe lethal graft-versus-host disease in a murine model of allogeneic bone marrow transplantation. (145.20)
Authors: Fanning Stacey , Berger Stephanie , Korngold Robert , Friedman Thea
Published: January 2023

The successful use of allogeneic blood and marrow transplantation (BMT) in the treatment of hematological malignancies is hampered by the fact that many donor T cells responsible for mediating graft-versus-leukemia (GVL) effects are also involved in development of graft-versus-host disease (GVHD). Optimizing outcomes for allo-BMT depends upon successful separation of GVHD and GVL responses. Our work focuses on identifying host reactive and tumor reactive T cells based on TCR Vβ CDR3-size spectratype analysis. In this study, we used a MHC-matched, miHA-mismatched murine model of BMT (B10.BR→CBA) and a CBA-derived myeloid leukemia, MMC6. Previous studies have shown this GVHD model to be mediated by CD4+-independent CD8+ T cells. Here, we show that the Vβ13 family is uniquely skewed in the B10.BR anti-MMC6 CD8+ T cell response. Transplantation of CD8+Vβ13+ T cells at the dose equivalent of 10x106 CD8+ T cells, the dose at which recipient mice develop severe lethal GVHD, did not mediate GVHD or GVL. Increased doses of Vβ13+ T cells led to increased GVL responses, however, recipient mice began to exhibit symptoms of GVHD. Of most interest, when recipients were given MMC6 presensitized Vβ13+ donor T cells, they displayed a significant GVL response in the absence of lethal GVHD. These results indicate that Vβ spectratyping can be used to identify T cells uniquely skewed in GVL responses and suggest that tumor-presensitized T cells can promote GVL in the absence of GVHD.
De novo induction of antigen-specific regulatory T cells by islet transplantation (145.34)
Authors: Kim Yong-Hee , Yoon Il-Hee , Hong So-Hee , Cho Hyoung-Su , Nam Hye-Young , Park Chung-Gyu
Published: January 2023

T cell receptor transgenic mouse on RAG knock out background does not have regulatory T cells (Tregs). We could induce adaptive Tregs in one of such mouse strain Marilyn by HY-antigen-mismatched islet transplantation. We transplanted male C57BL/6 islet to streptozotocin-induced diabetic female Marilyn mouse. Marilyn mouse is HY(minor histocompatibility antigen in Y chromosome)-specific TCR transgenic mouse on RAG2 knock out C57BL/6 background. Naïve Marilyn mouse rejects male skin. But, male islets were accepted by Marilyn mouse and induced adaptive Tregs. After acceptance of islet grafts, we grafted male C57BL/6 skin to confirm the tolerance to male antigen. In that skin graft, induced Tregs could prevent rejection to male’s skin. We could also detect Tregs after female islet transplantation. But, they were donor-origin Tregs, and those Tregs could not protect the male skin from rejection. In conclusion, we can make de novo induction of Tregs by islet transplantation, and those Tregs are antigen-specific Tregs.
Drak2 is required for T cell mediated allogeneic transplant rejection (145.9)
Authors: Weist Brian , Walsh Craig
Published: January 2023

Drak2 is a serine threonine kinase that has previously been shown to negatively regulate activation of T cells through the T cell receptor. Additionally, T cells lacking Drak2 are hypersensitive to low levels of stimulation, but are surprisingly resistant to autoimmunity in an EAE model. Since it has been hypothesized that Drak2 deficient T cells die after activation, we tested the ability of these T cells to respond to allogeneic stimulation, which would mimic transplantation from an allogeneic donor. Using in vitro mixed lymphocyte reactions, Drak2-/- CD8+ T cells responded less to allogeneic stimulation compared to wildtype T cells when analyzed by CFSE dilution. Additionally, allogeneic tumors were used to determine the role of Drak2 during an in vivo allogeneic transplant. Rag2-/- gamma C-/-mice adoptively transferred with purified Drak2-/- T cells were not able to clear allogeneic tumors contrary to wildtype T cell recipients. Additionally, transgenic Bcl-xL expression in Drak2-/- T cells rescued the alloresponse, illustrated by restored ability to clear allogeneic tumors. Next, we aim to determine if Drak2 blockade is sufficient to prevent rejection in a pancreatic islet transplant model.
TNF promoter region allele frequencies in the White population and their expression levels (34.11)
Authors: Selby Sandra , Posch Phillip
Published: January 2023

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that plays a variety of roles in immune regulation and disease. The TNF regulatory region contains numerous SNPs some of which affect expression. We previously identified nine alleles of TNF promoter region from random samples, but allele frequencies in different populations and diversity of the gene, as well as expression levels driven by the promoter alleles are not known. The TNF promoter and gene were sequenced from 82 random healthy White individuals. Three novel promoter alleles and two novel SNPs were identified. Novel SNPs also were found in intron 1 and the 3’ UTR. Promoter allele p*001 was the most common allele (55.49%). Allele p*002 (15.85%), p*006 (14.02%) and p*003 (7.32%) also were frequent, while all other alleles were found at less than 5% frequency. Differences were noted in an initial analysis of expression levels in T cells driven by the TNF promoter alleles. Allele p*004 was significantly increased in unstimulated and PMA/ionomycin conditions when compared to allele p*001. Expression was decreased for alleles p*005, p*006, and p*008 in comparison to allele p*001 in stimulated and unstimulated conditions. Allele p*007 showed decreased expression only in unstimulated conditions, while allele p*002 showed reduced expression only under stimulated conditions. These data confirm some of the previously reported impacts of SNPs on TNF expression and suggest an impact of additional SNPs on TNF levels.
IL-17A plays a pro-atherogenic role in atherosclerosis. (34.8)
Authors: Butcher Matthew , Smith Emily , Phillips Tracy , Prasad Konkall-Matt , Kolls Jay , Ley Klaus , Galkina Elena
Published: January 2023

Atherosclerosis is a disease of large arteries that is characterized by the formation of atherosclerotic plaques within the arterial wall and the immune response accompanies this process. To date, the contribution of IL-17A+ T cells in atherogenesis remains unclear. To test whether atherosclerotic conditions induce IL-17+ cell generation, we analyzed atherosclerosis-prone apolipoprotein E-deficient (Apoe-/-) and control C57BL/6 mice for IL-17A. CD4+ and γσ+ IL-17A+ T cells were significantly increased in the aortas, spleen and lamina propria of Apoe-/- vs. C57BL/6 mice. To analyze the role of IL-17A in vivo, Apoe-/- mice were transfected with adenovirus constructs expressing soluble IL-17 receptor (Ad-IL-17RA:Fc) or adenovirus-Luciferase controls. After a 15 week western diet, Ad-IL17RA:Fc-treated mice had reduced plaque burden, macrophage infiltration, and CXCL1 expression compared to controls. Atherosclerosis was attenuated in newly generated IL-17A-deficient Apoe-/- (Il-17a-/-Apoe-/-) mice vs. Apoe-/- mice. The expression of CX3CL1, CXCL1, CXCL2, CXCL5, IFNγ, IL-6 and TNFα was also reduced within the aortas of Il-17a-/-Apoe-/- mice. The adhesion of Monocytes to Il-17a-/-Apoe-/- aortas were diminished vs. Apoe-/- controls in ex vivo assays. Together, these results suggest that atherosclerotic-prone conditions induce Th17 and γσ+ IL-17A+ T cells, and accelerate atherosclerosis through the induction of aortic chemokines and recruitment of monocytes to atherosclerotic lesions.
Plasma membrane-derived vesicles (PMVs) carry TGF-β and promote adhesion and differentiation of THP-1 monocytes to macrophages (35.15)
Authors: Ansa-Addo Ephraim , Inal Jameel
Published: January 2023

Microvesiculation is a ubiquitous cellular mechanism, which occurs as a result of exocytosis, to release exosomes (between 50-100nm) or by direct release of vesicles from the cell surface membrane, known as plasma membrane-derived vesicles (PMVs) (0.1-1μm). Plasma membrane-derived vesicles are small, intact membrane vesicles, which are released by most cells upon activation with various extracellular stimuli and contain numerous proteins and lipids characteristic of the cell of origin. Among these are cytokines, for example transforming growth factor-β (TGF-β), a multifunctional protein, well documented to function in the regulation of cell proliferation, differentiation, morphogenesis and phenotype expression. Since TGF-β plays an important role in cell regulation, we tested whether TGF-β-bearing PMVs derived from THP-1 cells could themselves regulate the growth and differentiation of monocytes and their leukaemic counterparts. We show that addition of PMVs to monocytes or THP-1 cells did indeed cause significant reductions in the growth rate and induced their terminal differentiation into macrophages. Further investigations also showed that PMVs isolated from cells with high TGF-β content, caused significantly higher reductions in the growth rate of THP-1 cells as compared to PMVs from cells, which express less TGF-β. These results present a possible autocrine-like mechanism involving monocyte-derived PMVs by which monocytes terminally differentiate into macrophages.
Didox (3,4-Dihydroxybenzohydroxamic acid) is a potent anti-oxidant and anti-inflammatory compound (35.17)
Authors: Rice Charles , Gallicchio Vincent , Matsebatlela Thabe
Published: January 2023

Didox (3,4-Dihydroxybenzohydroxamic acid) is a synthetic antioxidant and one of the most potent ribonucleotide reductase inhibitors developed to date. Little is known about the potential for Didox as an anti-inflammatory agent. After an analysis of more than 200 oxidative stress- and inflammatory-associated genes in mouse RAW264.7 macrophages we show that Didox significantly lowers LPS-induced expression of inflammatory genes (iNOS, IL-1α, IL-1β, IL-6, TNF-α, NF-KB, Cox-2, p38α, TLR4, GM-CSF, G-CSF, CCL2, CCL4, others), while upregulating expression levels of anti-oxidant and anti-inflammatory genes (Catalase, glutathione peroxidase, IL-10, superoxide dismutase, and peroxiredoxins, others). In addition, Didox dose-dependently inhibits LPS-induced IL-6 secretion and nitric oxide production. Buthionine Sulfoximine- and LPS-induced ROS production are also suppressed by Didox. We also show that phagocytosis of yeast particles is decreased as well. In terms of cellular signaling, Didox prevents NF-KB activation and translocation of NF-kappa beta p65 subunits in LPS-treated cells. These results suggest that the mechanism behind anti-oxidant and anti-inflammatory properties of Didox may be mediated, at least in part, via modulation of NF-kappa beta signaling. Didox may, therefore, may hold promise in managing chronic diseases exacerbated by LPS-induced oxidative stress and inflammation in macrophage-mediated inflammation.
Macrophage migration inhibitory factor promotes central nervous system pathology in a model of neuroinflammation (35.8)
Authors: Cox Gina , Alexander J. , Kithcart A. , Williams J. , Smith K. , Shawler T. , Satoskar A. , Whitacre C.
Published: January 2023

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Cerebrospinal fluid collected from MS patients during relapse was found to contain greater levels of macrophage migration inhibitory factor (MIF) than samples collected during remission. We have shown that induction of experimental autoimmune encephalomyelitis (EAE) in MIF-deficient mice results in reduced clinical signs and CNS inflammatory infiltrates relative to wild type controls. However, there was no difference in T cell function in the periphery between the two groups. These findings suggest that MIF acts within the CNS to potentiate disease progression. The potent pro-inflammatory properties of MIF are well established; however, the precise contribution of MIF to neuroinflammation is not well defined. MIF is expressed by many cell types in the CNS including infiltrating macrophages and resident microglia, suggesting that MIF may enhance the inflammatory environment of MS lesions. To assess the contribution of MIF to CNS inflammation, primary microglial cultures were treated with increasing doses of rMIF, which upregulated inflammatory mediators associated with MS and EAE: IL-1β, iNOS, and CCL2. We are currently exploring the role of MIF in vivo using an intraspinal model of neuroinflammation. Taken together, these data suggest that inhibition of MIF may serve as a therapeutic strategy for resolving CNS inflammation.
Paneth cell activation after acute kidney injury causes liver and intestine injury and systemic inflammation in mice (35.9)
Authors: Park Sang Won , Kim Mihwa , Chen Sean , Brown Kevin , Kolls Jay , Lee H. Thomas
Published: January 2023

Patients who develop acute kidney injury (AKI) frequently suffer from extra-renal organ dysfunction and systemic inflammation syndrome. We aimed to determine the mechanisms of ischemic and non-ischemic AKI induced systemic complications. C57BL/6 mice were subjected to sham surgery, 20 min renal ischemia or unilateral nephrectomy. In addition to plasma Cr, plasma ALT markedly increased 5 hrs after both ischemic and non-ischemic AKI. These rises were correlated with peri-portal hepatocyte necrosis, vacuolization, neutrophil infiltration and pro-inflammatory mRNA upregulation. Plasma levels of TNF-α and IL-17 were also elevated after AKI. Small intestine histology after AKI showed massive epithelial necrosis and apoptosis as well as profound endothelial apoptosis. Neutralization or genetic deletion of TNF-α or IL-17 prevented AKI induced hepatic and intestine injury. Remarkable hyperplasia and degranulation of Paneth cells were observed. Laser capture microscopy of Paneth cells showed significant TNF-α and IL-17 mRNA induction after ischemic or non-ischemic AKI. Finally, Paneth cell depletion with dithizone prevented both hepatic and intestinal injury after AKI. Taken together, we propose that Paneth cell activation/degranulation after AKI produces systemic inflammation and hepatic damage via cytokine induction and intestinal barrier dysfunction. Modulating Paneth cell dysregulation may have important therapeutic implications in reducing systemic complications arising from AKI.
Downregulation of intracellular apolipoprotein D in DNA-PKcs-independent V(D)J recombination (36.16)
Authors: Franco Daniel , Reardon Christopher
Published: January 2023

Recombination-inducible mouse pre-B cell lines are models for intracellular events that occur during lymphocyte V(D)J recombination. Using the ts-abl transformed pre-B cell lines with wild type DNA-PKcs or lines from SCID mice that have a DNA-PKcs deficiency, they can be induced to recombine their V(D)J genes segments. One of the events coincident with recombination is G1 cell cycle arrest that is much more dramatic in the SCID pre-B cells. We have recently become interested in intracellular apolipoprotein D (ApoD), an ancient intracellular lipocalin. ApoD has been described to have a role in extending lifespan by modulating cellular stress responses. ApoD would be expected to be upregulated in cells undergoing cell cycle arrest as this is a potentially stressful event for dividing cells. However, when assessing the effects of cell cycle arrest in the pre-B cell lines, we found that the intracellular Apo D actually decreases. The SCID pre-B cells, having a more severe G1 cycle arrest from recombination induction, also show a more dramatic reduction in ApoD levels. It is unclear whether the down-regulation of ApoD is associated with the recombination induction or the cell cycle arrest events. Since the SCID pre-B cells are deficient in DNA-PKcs function, it is tempting to speculate that ApoD gene expression may be influenced by this protein kinase. Nevertheless, these data suggest a previously undescribed role for intracellular ApoD in B-cell V(D)J recombination events.
Predominance of an unusual natural killer phenotype during recovery after natural killer cell depletion (36.23)
Authors: Alvarez Maite , Barao Isabel , Orr Mark , Lanier Lewis , Redelman Dog , Murphy William
Published: January 2023

Natural killer (NK) cells are considered as a front line defense against cancer and viral infection. Despite multiple studies based on NK cell development, the regulation of the maturation and differentiation of NK cells is still poorly understood. Shortly after bone marrow transplantation (BMT) we observed rapid appearance of an NK cell subset characterized by increased Ly49G2 median fluorescence intensity (MFI) along with the activation marker Thy1.2. This subset was no longer detectable at 28 days post-BMT. To determine if this activated mature subset appeared as a result of the cytoreductive conditioning versus normal differentiation, we assessed NK cell recovery on mice depleted with anti-NK1.1. We found a significant increase in the percentage of NK cells that express higher Ly49G2 MFI as well as Thy1.2, an indication of their activated state, on day 3 and 9 post-depletion which decreases to levels similar to resting mice on day 14. These results suggest that lymphopenic conditions after irradiation as well as cell depletion lead to a reduction of NK cells suggesting the lack of competition between the remaining cells may make endogenous cytokines available for developing NK cells that become highly activated.
Visualization of type 2 NKT cell development in the thymus (36.55)
Authors: Hamad Abdel , Xiao Zuoxiang
Published: January 2023

Beside CD4 and CD8 αβT cells, several minor but important T cell subsets develop in the thymus. These include NKT cells, which are selected on MHC class 1b molecule CD1d. NKT cells are broadly divided into type 1 NKT cells, which express an invariant TCR and bind αGalCer-CD1d tetramer and type 2 NKT cells, which express non-invariant TCRs and do not bind αGalCer-CD1d tetramer. Significance advances in development of type 1 NKT cells have been made by using αGalCer-CD1d tetramer for visualization of their thymic development. However, very little is known about development of type 2 NKT cells due to the lack of a specific marker for their identification in the thymus. Here we show that the highly conserved heparan sulphate proteoglycan syndecan-1 (sdc1) is selectively expressed by a small subset of TCRαβ thymocytes. Unlike mainstream i.e sdc1-TCRβ+ thymocytes, the majority of sdc1+ TCRβ+ thymocytes are CD4-CD8-(DN) T cells whose development is CD1d-dependent and are not reactive to αGalCer-CD1d tetramer and hence are type 2 NKT cells. Functional maturity of this subset is indicated by their production of Th2 cytokines in vitro stimulation with CD3/CD28 or CD1d beads. Sdc1 appears to play an important role in regulation of cytokine production and homing of type 2 NK T cells. Identification of sdc1 as a specific marker for type 2 NKT cells in the thymus will facilitate understanding their development.
Requirement of MMP2 and MMP9 for lung innate immune defense against Streptococcus pneumoniae infection (37.34)
Authors: Hong Jeong Soo , Greenlee Kendra , Lee Seung-Hyo , Song Li-Zhen , Shan Ming , Pitchumani Ramanan , Chang Seon Hee , Park Pyong Woo , Bidani Akhil , Dong Chen , Werb Zena , Corry David , Kheradmand Farrah
Published: January 2023

The first wave of host innate immune response to bacterial invasion is central to protection against microbial pathogens. Identification of the critical local mediators in the lung that provide early protection against bacteria is essential for a complete understanding of the molecular mechanism(s) underlying host defense. In an acute model of Streptococcus pneumoniae TIGR4 (SP-TIGR4) lung infection, we found MMP2 and MMP9 double null (MMP2/9-/-) were more susceptible to death when compared to wild type (WT) mice. In response to SP-TIGR4 challenge, MMP2/9-/- mice recruited more polymorphonuclear cells (PMNs) to the lung, however lungs and spleens isolated from the infected mice showed higher systemic bacterial load in the absence of MMP2 and MMP9. Consistent with an increase in lung PMNs, MMP2/9-/- mice showed significant increase in levels of IL-17, IP-10 and RANTES in BAL fluid. In vitro studies showed MMP2 mediated inhibited function of IL-17A proinflammation and MMP9 was related to ineffective bacterial phagocytosis and opsonization of PMNs. Taken together, these data support a protective role for MMP2 and MMP9 in host immune responses in early phase of bacterial infection.
An attenuated innate immune response to a clinical strain of respiratory syncytial virus (RSV) in human monocyte derived dendritic cells (MDDC) (37.43)
Authors: Hillyer Philippa , Chen Aaron , Schramm Lynnsie , Mane Viraj , Navarro Maria , Luongo Cindy , Raviv Nataly , Collins Peter , Rabin Ronald
Published: January 2023

RSV clinical strain 19 was isolated from a severely affected infant and was characterized in a model in which mice develop severe disease defined by enhanced mucous secretion, IL-13 expression, and low expression of IFN-α compared to infection with the lab strain A2. We compared the innate response induced by RSV strain 19 with strain A2 in human MDDC using a qRT-PCR based array and a novel qRT-PCR assay we developed that discriminates among all IFN-α and IFN-λ subtypes and also includes IFN-β. Human MDDC expressed a similar profile of inflammatory and IFN response genes (IRG) to both RSV strains. The response to strain 19, however, was attenuated for many IRG and inflammatory genes, including CXCL9, CXCL10, CCL2 and genes associated with the RIG-I pathway. Similarly, MDDC expressed IFN-α1, IFN-β, IFN-λ1, and -λ2 in response to either strain, but strain 19 expressed lower levels of each. Another IFN subtype, IFN-α14, was expressed by 5 donors in response to strain A2 but was undetectable in response to strain 19. IFN-α protein, however, was undetectable in 9 of 10 supernatants from strain 19 in contrast to 1 of 10 supernatants from A2 infected cells. Evasion or active suppression of the IFN-α, -β or -λ response may explain the enhanced pathogenicity of strain 19. The qualitative difference in IFN-α14 expression in response to A2 but not 19 suggests a potentially important role for this subtype.