bluuebunny/crossref_metadata_2025 huggingface dataset:
Validation of markers for phenotyping naïve and memory T-cells by multicolor flow cytometry. (138.22)
Authors: Hingorani Ravi , Cheung Jocelyn , Ernst David , Lane Cynthia , Rohrer Jurg
Published: January 2023

The antigen experienced T-cell population can be broadly divided into the effector and memory cell compartments, both of which are needed for sustaining a responsive immune system. Simplistically, the effector T lymphocytes are described as cells needing active antigenic stimulation to eliminate pathogens. Memory T lymphocytes, on the other hand are described as cells which remain present in the absence of antigenic stimulation and have the capacity to expand rapidly upon secondary challenge. Recently, with the identification of central and effector memory T-cell subsets, tremendous effort has been devoted to characterize markers on the surface of these cells. Though various markers have been used to identify the subsets, no single marker that segregates one subset from the other has been described. Thus, multiple markers are needed to subset the cells in order to characterize them. Here we report the verification of a 9-color panel that can successfully identify 5 distinct CD4 and CD8 T cell populations within the naïve and effector cell subsets. In addition, functionality of the memory subsets was validated by stimulating the cells and using the production of IFN-γ as a read-out. Avoiding potential pitfalls when setting up 9 color panels for use in flow cytometry will be discussed.
Investigating molecular bases for mucosal mast cell motility and interactions in the gut (140.13)
Authors: Lee Jinmin , Williams Rebecca , Blum Lisa , Appleton Judith , Baird Barbara , Holowka David
Published: January 2023

Mast cells function as effector and immunomodulatory cells in innate and adaptive immune responses, and they are the primary mediators of immunoglobulin E (IgE)-dependent allergic disorders. Although homing of mast cell progenitors is relatively well studied, little is known about mature mast cell motility and migration in tissue. Our recent studies have characterized novel polarization and motility of mucosal mast cells in culture, including the RBL-2H3 cell line and rat bone marrow-derived mast cells. Motility of these cells depends on several properties, including Ca2+ homeostasis. Preliminary evidence indicates a role for store-operated Ca2+ entry in this process. RBL-2H3 mast cells undergo adhesion and motility on cultured monolayers of rat intestinal epithelial cells, and they exhibit occasional transepithelial migration under these conditions. Intestinal infection of animals by parasitic nematodes, including Trichinella spiralis, induces local mastocytosis. Using multiphoton confocal microscopy of ex vivo intestinal tissue from infected rats, we have been able to visualize fluorescently labeled and adoptively transferred mast cells in the epithelial layer of jejunal villi.
Cyclophilin A synergizes with MIP-2 to augment pro-inflammatory leukocyte recruitment (140.5)
Authors: Heine Shannon , Olive Denise , Okwumabua Ifeanyi , Bukrinsky Michael , Constant Stephanie
Published: January 2023

Chemokines contribute to inflammatory responses by inducing leukocyte migration and extravasation. Extracellular cyclophilins are a group of alternative chemokines that can be elevated during inflammation, and we have previously shown that their inhibition reduces neutrophil infiltration by >40% in a mouse model of LPS-induced acute lung inflammation. Given the presence of many other chemokines during this inflammatory response, the observed potent contribution of cyclophilins led us to investigate whether they might function in a synergistic partnership with concurrent chemokines. To test whether cyclophilin A (CypA) can work with other chemokines in vivo, recombinant extracellular CypA was co-delivered with intranasal LPS and lung neutrophil numbers were examined at a time point when two classical chemokines, MIP-2 and KC, but not CypA, are present. The addition of CypA induced a significant increase in early neutrophil recruitment that was greater than the additive recruitment induced by LPS and CypA alone. Boyden chamber chemotaxis assays in which purified mouse neutrophils were incubated with various combinations of CypA plus MIP-2 or KC were used to show that CypA had the capacity to synergize with MIP-2, but not KC, to induce augmented neutrophil migration. These findings provide a potential explanation for why the inhibition of cyclophilin activity in vivo has such a great impact on cell recruitment.
Particulate Allergens Exacerbate Allergic Asthma via Extended Localization within Lipid Raft Enriched Compartments in Mast Cells (141.19)
Authors: Jin Cong , Shelburne Christopher , Li Guojie , Potts Erin , Riebe Kristina , Sempowski Gregory , Foster Michael , Abraham Soman
Published: January 2023

Numerous epidemiological studies have related the exacerbation of allergic asthma with exposure to increased ambient particles from air pollutants. However, the mechanism by which particulate allergens (pAg) exacerbate allergic asthma remains undefined. To evaluate this, we modeled environmental pAg induced allergic asthma by exposing mice to polystyrene beads coated with natural allergen extracts. Compared to equal amounts of soluble allergen extracts (sAg), pAg triggered markedly enhanced airway hyper-responsiveness and pulmonary eosinophila in allergen sensitized mice. The cellular basis for this effect was determined to be mast cells (MCs), as both airway allergic responses were attenuated in MC deficient mice compared to MC sufficient mice. The divergent responses of MCs to pAg versus sAg were due to differences in the termination rate of IgE/FcϵRI initiated signaling. Following ligation of sAg, IgE/FcϵRI rapidly shuttled into a degradative endosome/lysosome pathway. However, following ligation by pAg, IgE/FcϵRI migrated into lipid raft enriched compartments and subsequently failed to follow a degradative pathway, which resulted in a prolonged signaling and heightened synthesis of proinflammatory mediators. These observations highlight the overlooked contributions of the particulate nature of allergens and mast cell endocytic circuitry to the aggravation of allergic asthma.
Suppression of liver injury through inhibition of MyD88 signaling in a mouse model of fulminant hepatitis by 18β-glycyrrhetinic acid (142.11)
Authors: Xu Jingwei , Zhang Yanyun
Published: January 2023

Fulminant hepatitis, develops secondary to infection, toxin or immune mediated attack, is a rare but potentially fatal disease. However, the molecular mechanisms underlying are not well known. 18β-glycyrrhetinic acid (GA), the major bioactive component of licorice root extract, has a protective effect on hepatic injury and exhibits anti-inflammatory activity. Here we investigate the potential role of GA in a mouse model of fulminant hepatitis induced by injection of Propionibacterium acnes (P. acnes) and LPS. GA or vehicle control was administered intraperitoneally daily 1 d after P. acnes priming. The results revealed that GA treatment significantly improved mouse mortality and ameliorated liver injury through inhibition of the activation and proliferation of liver-infiltrating CD4+ T cells and the production of serum pro-inflammatory cytokines such as IFN-γ and TNF-α. However, GA exhibited no direct effect on the proliferation of liver-infiltrating CD4+ T cells, but by reduced recruitment of CD11c+B220- DC precursors into the liver, which is mediated by the inhibition of MIP-1α expression in Kupffer cells by GA. More importantly, GA inhibited the activation of Kupffer cells by down-regulating MyD88 expression and its down-stream signal NF-κB activation. Taken together, our results suggest that GA exhibits anti-inflammatory effects through inhibition of MyD88 signaling in a mouse model of fulminant hepatitis.
Beclin-1 is required for T cell-mediated immune responses (143.19)
Authors: Kovacs Jeffrey , Li Changyou , Lu Binfeng
Published: January 2023

Beclin 1, the mammalian homologue of the yeast Atg6, regulates macroautophagy (autophagy) in eukaryotic cells and plays a critical role in the regulation of cell death and survival in various types of cells. Beclin 1 is expressed in T cells. However, its role in T cell-mediated immune responses remains unknown. We have found that mice with Beclin-1 deletion in T cells have profound deficiencies in mounting T dependent immune responses. Autophagy is abolished in Beclin 1 deficient T cells. We observed that, upon activation, Beclin 1 deficient T cells undergo programmed cell death. Mice with Beclin 1 deletion in T cells fail to mount T cell-dependent humoral immune responses to KLH and are resistant to experimental autoimmune encephalomyelitis. Our work establishes a crucial role of Beclin 1 in autoimmunity.
Self reactive B cells that do not exhibit higher BAFF dependency for survival (143.42)
Authors: Nikbakht Neda , Manser Timothy
Published: January 2023

The pro-survival cytokine BAFF was recently proposed to regulate peripheral immunological tolerance within the B cell lineage. Studies conducted in two different model systems of B cell autoimmunity demonstrated a selective elimination of autoreactive B cells upon BAFF depletion. These observations led to a model positing a higher dependency of autoreactive B cells on BAFF. We tested this model by exploiting our experimental system in which targeted Ig heavy chain knock-in transgenic mice (HKI) produce large numbers of B cells that are dually reactive to foreign (Ars) and self (DNA) antigens. HKI B cells do not exhibit the classical features of anergy, escape central tolerance mechanisms, and gain access to normal follicular locales. We show that mature, naïve, autoreactive HKI B cells are out-competed in the presence of a polyclonal repertoire. However, this is not due to a higher dependency of HKI B cells on BAFF for survival. In addition, excess BAFF does not rescue HKI B cells from selective elimination. These findings suggest that some autoreactive B cells can fully develop while in competition with non-autoreactive cells for normal levels of BAFF, but remain at a competitive disadvantage for other trophic factors that regulate peripheral stability. As such, our data indicate the existence of parallel peripheral tolerogenic mechanisms that may act through BCR signaling and at further stages of differentiation such as those that take place in germinal centers.
IL-17 downregulates p50 and p105 to sustain efficient active NF-κB signaling responses in the splenic B cells of autoimmune BXD2 mice (143.48)
Authors: Xie Shutao , Hsu Hui-Chen , Wu Qi , Li Jun , Yang PingAr , Ding Yanna , Mountz John
Published: January 2023

Constitutively active NF-κB signaling with increased expression of p50 is associated with many autoimmune diseases. In autoimmune BXD2 mice, there is dramatically higher p50 in splenic B cells, compared to B6 B cells (>10.0-fold, p<0.01). Surprisingly, both p50 and p105 in BXD2 B cells are significantly lower than those in B cells from BXD2-Il17ra-/- mice, indicating IL-17 downregulates them in B cells. Consistent with it, administration of AdIL-17RA:Fc which blocks IL-17RA signaling, resulted in significantly increased p50 and p105 and decreased phospho-p65 in splenic B cells, leading to decreased autoantibody production in BXD2 mice. In contrast, administration of AdIL-17A exhibited opposite effects. Stimulation of BXD2 B cells with IL-17 led to cytoplasmic translocation of p50 from the nucleus while p65 was phosphorylated and translocated to the nucleus. In BXD2-Il17ra-/- B cells, there is increased localization of p50 and p105 in the nucleus, compared to BXD2 B cells. The enhanced nuclear p50 and p105 in BXD2-Il17ra-/- B cells are associated with dramatically decreased LPS induction of NF-κB response genes including Rgs which are essential for development of autoreactive germinal centers, indicating higher p50 and p105 inhibit these genes expression in BXD2-Il17ra-/- B cells. The results suggest IL-17 downregulate p50 and p105 to sustain efficient active NF-κB signaling responses in autoreactive BXD2 B cells and result in increased production of pathogenic autoantibodies.
Expansion of CD3+/CD4+/CD25+/Foxp3+ T cells in rapamycin-treated lupus patients (143.52)
Authors: Perl Andras , Lai Zhi-Wei , Telarico Tiffany , Bartos Adam , Miklossy Gabriella , Hanczko Robert , Francis Lisa , Tily Hajra , Ramos Irene , Garcia Ricardo , Phillips Paul , Jimah John , Doherty Edward
Published: January 2023

Activation and death pathway selection of T cells are regulated via the mitochondrial transmembrane potential (Δψm). Systemic lupus erythematosus (SLE) patients’ T cells exhibit persistent Δψm elevation or mitochondrial hyperpolarization (MHP) and ATP depletion which predispose them to pro-inflammatory death via necrosis. Here, we examined the role of the mammalian target of rapamycin (mTOR), which serves as a sensor of MHP, in T cell activation in 34 female SLE patients and 30 female controls matched for age and ethnicity. MHP and mitochondrial mass was most robustly (1.6-2.0-fold; p= 0.0027) increased in CD3+/CD4-/CD8- cells. The prevalence of Foxp3+ cells within the CD4+/CD25+ compartment was reduced in SLE patients (37.05 ± 3.0 %) relative to controls (48.37 ± 2.5 %; p = 0.0014). mTOR activity, as measured by the accumulation of pS6 protein, was increased > 1.5-fold in CD3+ T cells (p=0.012) and CD19+ B cells (p=0.006). Relative to healthy controls (3.18 ± 0.39%), the frequency of Foxp3+/CD4+/CD25+ cells was reduced in 6 lupus patients (1.70 ± 0.42%; p=0.021) prior to rapamycin treatment. In vivo treatment with rapamycin increased the frequency of Tregs from 1.50 ± 0.45% to 2.8 ± 0.59% (p=0.040) while the SLEDAI scores were reduced in 5 SLE patients from 22.4 ± 5.7 to 16.7 ± 2.8 (p=0.017).These data identify the in vivo expansion of Tregs as a potential mechanism of action that may contribute to the therapeutic efficacy of rapamycin in SLE.
Therapeutic Potential of in vitro Expansion of Foxp3+ Tregs Utilizing GM-CSF-treated Bone Marrow Dentritic Cell Precusors in Experimental Myathenia Gravis (143.6)
Authors: Sheng Jianrong , Prabhakar Bellur , Meriggioli Matthew
Published: January 2023

We have previously shown that regulatory T cells (Tregs) generated in vivo by the selective activation of particular subsets of dendritic cells (DCs) in response to low-dose granulocyte-macrophage colony-stimulating factor (GM-CSF) have the capacity to prevent and reverse experiemental autoimmune myasthenia gravis (EAMG). To further understand the underlying mechanisms of GM-CSF-induced Treg mobilization, we studied the effects of GM-CSF on T cell/DC co-cultures using matured splenic DCs as well bone marrow (BM) - derived DC precursors in vitro. Compared to splenic DCs, bone marrow-derived DC precursors, when cultured in the presence of GM-CSF, gave rise to a population of CD11c+CD11bhighCD8α- DCs (BMDCs) which were able to expand Foxp3+ regulatory T cells (Tregs) in the absence of TCR stimulation. Additionally, the BMDCs secreted high levels of TGF-β which was required for adaptive differentiation of naive T cells to Foxp3+ Tregs upon TCR stimulation. Adoptive transfer of expanded Tregs effectively suppressed ongoing EAMG. These results strongly indicate that BMDCs differentiated in the presence of GM-CSF can expand natural Tregs and induce adaptive Tregs through different mechanisms. Expansion of polyclonal and antigen-specific Tregs in vitro using this strategy may represent an effective treatment for restoring immune tolerance and ameliorating EAMG.
Immunostimulatory characteristics and in vivo trafficking of a primary dendritic cell-based vaccine for coccidioidomycosis. (144.23)
Authors: Awasthi Shanjana , Vilekar Prachi , Lagisetty Pallavi , Awasthi Vibhudutta
Published: January 2023

Dendritic cell (DC)-based vaccines have shown promising results in cancer and infectious diseases. Success of the DC-vaccine depends on multiple factors including host genetic background, trafficking through tissues, interaction and activation of naïve immune cells. We studied the immunostimulatory ability and trafficking of DC-vaccine in BALB/c mice, extremely-susceptible to Coccidioides infection. DC-vaccine was prepared by transfecting DCs with a plasmid DNA encoding Coccidioides-Ag2/PRA-cDNA (protective epitope). Secreted cytokine/chemokines were measured by cytometric bead array, and T cell activation was studied by flow-cytometry after DC-vaccine-lymphocyte co-culture. For trafficking studies, syngeneic BALB/c mice were intranasally injected with DC-vaccine expressing Coccidioides-Ag2/PRA and HSV-1-thymidine kinase (PET-reporter gene). On day 2 and 7 of DC-vaccination, PET imaging was performed 2h after intravenous injection of radiolabeled probe F-18-labeled 2'-fluoro-2'-deoxy-1β-D-arabinofuranosyl-5-iodouracil. Mice were sacrificed, and major organs and blood were collected. Organs- and blood-associated F-18 radioactivity was determined. Our results suggest that DC-vaccine secretes MCP-1 chemokine, activates T cells and traffics through lung, thymus and liver. Biodistribution data suggest an accumulation of F-18 radioactivity in all organs (except intestine) with more pronounced increase in lung, thymus and blood (p<0.005) of DC-vaccinated mice as compared to control.
Conventional splenic dendritic cells regulate development of graft-versus-host disease. (145.10)
Authors: Askew David , Eid Saada , Guardia-Wolff Rocio , Pierce Elizabeth , Cooke Kenneth
Published: January 2023

The use of allogeneic hematopoietic stem cell transplant (HSCT) to treat hematologic malignancies is associated with the development of graft-versus-host disease (GVHD), a frequently fatal complication that limits successful outcomes. The treatment of GVHD often requires potent immunosuppressive drugs and can predispose to relapse of the original cancer and the development of opportunistic infection. Understanding how GVHD is initiated may help to identify targets for therapeutic interventions that do not require wholesale suppression of the immune response. Prior studies have shown that host antigen presenting cells (APCs) are critical to GVHD induction. We examined how functionally distinct host splenic DC subpopulations (CD4+ vs. CD8+) regulate the development of GVHD mediated by donor CD4+ T cells. When compared to purified Balb/c CD4+ DCs, we found that Balb/c CD8+ DCs significantly increased C57BL/6 CD4+ T cell proliferation and cytolytic activity in vitro. To control the source of T cell stimulation for in vivo HSCT studies, we created chimeric Balb/c mice that have C57BL/6 APCs (C57BL/6 → Balb/c). Subsequently, we found that transplantation of C57BL/6 CD4+ T cells stimulated ex vivo by CD8+ Balb/c DCs resulted in more robust GVHD (survival, clinical score, pathology) compared to T cells activated by CD4+ DCs. Targeting specific DC-T cell interactions early in HSCT may help to control the severity of GVHD without requiring wholesale suppression of the immune system.
Cytotoxic class I tetramers inhibit CTL priming and alter immunodominance hierarchies in the HY model system (145.33)
Authors: Murray Sabrina , Miller Keith , Young Ellen , Vincent Benjamin , Steele Shaun , Buntzman Adam , Collins Edward , Frelinger Jeffrey , Hess Paul
Published: January 2023

The CD8+ cytotoxic T lymphocyte (CTL) response is a major effector mechanism in allograft rejection. In this study, we used the well-established murine HY-reactive immune response as a rejection model. Sensitization of B6 females with male bone marrow generated robust Db-Uty and Db-Smcy tetramer-positive CTLs that produced IFN-gamma, TNF-alpha, and granzyme B. We hypothesized that treatment of female mice with Db-Uty or Db-Smcy tetramers coupled to the ribosome-inactivating toxin, saporin, would selectively delete naïve cognate T cells and prevent priming of CTL responses. As expected, administration of the cytotoxic tetramers prior to immunization dramatically reduced the HY -specific CTL population at the peak of expansion and permitted the survival of adoptively-transferred, HY peptide-pulsed female cells. Interestingly, elimination of one CTL population magnified T cell reactivity against the remaining dominant epitope, demonstrating that the relative amplitude of these responses depends, in part, on competition between CD8+ T cells during priming. These data suggest that cytotoxic class I tetramers could be useful to dissect or manipulate T cell immunodominance hierarchies.
Over-expression of FGL2 leads to indefinite graft survival without the need for immunosuppression. (145.37)
Authors: Bartczak Agata , He William , Shalev Itay , Mendicino Michael , Urbanellis Peter , Zhang Jianhua , Ma Max , Khattar Ramzi , Adeyi Oyedele , Phillips James , Grant David , Levy Gary
Published: January 2023

FGL2 is an effector molecule of CD4+CD25+FoxP3+ T regulatory cells (Treg) which exerts its effect through binding to FcγRIIb and FcγRIII on APC. Mice deficient in fgl2 show impaired Treg activity associated with increased immune reactivity and development of autoimmune glomerulonephritis. In a fully mismatched model of heart allotransplantation where recipients were treated with recombinant FGL2, graft survival was extended significantly but grafts were rejected following cessation of treatment. To further explore the function of FGL2, we generated an FGL2 over-expressing mouse (fgl2Tg) using a conditional, double reporter cre/lox expression system. These mice express 1000-fold higher levels of FGL2 in plasma compared to wildtype mice. The high levels of FGL2 do not appear to adversely affect mouse development. Treg isolated from the fgl2Tg mice have increased suppressive activity in vitro and effector T cells isolated from fgl2Tg mice are hyporesponsive to alloantigens. In vivo, fully mismatched hearts transplanted into fgl2Tg mice survived indefinitely without the need for immunosuppression. These data demonstrate that FGL2 is an important effector immunosuppressive molecule of Treg which has therapeutic implications for transplantation and autoimmune disease.
central tolerance induction by adoptive transfer of donor dendritic cells (145.5)
Authors: Yamano Tomoyoshi , Goto Masafumi , Abe Ryo , Kishimoto Hidehiro
Published: January 2023

The principal function of DCs is to promote T cell activation and differentiation into effecter T cells by presenting foreign Ags and providing costimulatory signals. DCs also play a critical role in maintaining not only peripheral but also central tolerance. Central tolerance is established by negative selection of developing T cells that have high-affinity TCRs for self-peptide-MHC complexes expressing on DCs or mTECs. We studied whether i.v. injection of allogeneic DCs that are obtained BM culture with Flt3L (FLDCs), GM-CSF(GMDCs) or allogeneic B cells could home to the recipient thymus. We found that only FLDCs could traffic to the recipient thymus. In MLR assay, splenic T cells and mature thymocytes which were recovered from donor FLDCs infused mice, have poor proliferative activity in response to donor APCs, whereas the responses of T cells against third party APCs were as same as control mice. The MHC classI Ld specific 2C thymocytes were selectively deleted after intravenous injection of BALB/c FLDCs. Furthermore, adoptive transfer of donor FLDCs showed significant prolonged donor skin graft. FLDCs transfer was also able to prolong the donor islet graft and prevented the mice from streptozotocin induced diabete mellitus. These results show that i.v injection of FLDCs induces donor specific both central and peripheral tolerance.
Inhibition of the primary antibody response to transfused allogeneic platelets (145.8)
Authors: Gilson Christopher , Zimring James
Published: January 2023

Alloantibodies are a clinically significant sequela of platelet (PLT) transfusion, potentially rendering patients refractory to ongoing transfusion support. PLTs are restricted to the peripheral circulation, where antigens are primarily sampled by the spleen and liver. We hypothesized that the primary antibody (Ab) response to allogeneic PLTs depends upon CD4 T cell help in the splenic microenvironment. C57BL/6 recipients (H-2b) transfused four times weekly with 1x108 leukoreduced BALB/c (H-2d) PLTs developed anti-donor Ab by day 28 (15/15 mice). Adoptive transfer of CFSE labeled CD4+ TCR75 cells, a TCR transgenic specific for a peptide from Kd presented by I-Ab, augmented the Ab response by day 14 to a single transfusion (9/9). TCR75 division by day 5 was exclusive to the spleen and not the liver or lymph nodes. Anti-donor Ab was undetectable in CD4 depleted recipients (0/15). Splenectomized recipients did not develop detectable anti-donor Ab (0/13) and adoptively transferred TCR75 cells did not divide or rescue the Ab response (0/9). The non-responsiveness in splenectomized animals was not due to donor specific tolerance as these recipients responded robustly to subsequent donor whole blood (6/6). Enhanced alloimmunization by adoptive transfer and abrogation by depletion supports the critical role for CD4 T cell help. Failure of TCR75 proliferation in splenectomized recipients suggests that an intact spleen is required, but the liver insufficient, for PLT alloimmunization.
Signaling through the TNF-R2 (p75) but not TNF-R1 (p55) is absolutely required for in vivo CTL maturation in parent-into-F1 mice. (34.4)
Authors: Soloviova Kateryna , Puliaeva Irina , Lang Tom , Puliaev Roman , Via Charles
Published: January 2023

The transfer of B6 splenocytes into normal BDF1 mice results in donor cytotoxic T lymphocytes (CTL) that eliminate host B cells by two weeks and is a useful model of in vivo CTL generation. We previously demonstrated that in vivo anti-TNF mAb treatment prevents CTL and B cell elimination, shifting disease phenotype to a lupus-like disease. To determine if TNF acts directly on naïve donor T cells or whether TNF acts indirectly to promote CTL, we transferred p55 and/or p75 TNF-R defective or wild type (WT) B6 donor cells into WT F1 hosts. At two weeks after transfer host splenocyte populations were assessed by flow cytometry. P55->F1 mice exhibited host B cell elimination comparable to WT->F1 mice (~10% of control F1 values) whereas p75 and p55/p75->F1 mice exhibited a significant increase in host B cells (>150% control) consistent with CTL failure and lupus-like disease. Host DC (CD11c+) and macrophage (CD11b+) values paralleled those of B cells for p55 or p75->F1 mice. Compared to WT->F1 mice, peak day 7 serum IFN-g was significantly reduced only for p75->F1 mice. Defective elimination of host populations in p75->F1 mice was not due to failure of donor CD8 T cells to engraft or to express an effector phenotype (CD44hi, CD62Llo). These results support the conclusion that TNF signaling through the p75 receptor is critical for in vivo CD8 CTL maturation in normal unirradiated mice whereas p55 signaling has a minimal if any role.
IL-10 desensitizes Th17 cells memorized with Candida antigen (34.9)
Authors: Inatsu Akihito , Jeschke Marc , Yoshida Shohei , Asai Akira , Kobayashi Makiko , Herndon David , Suzuki Fujio
Published: January 2023

Th17 cells are important effector cells in host resistance against C. albicans infection. Here, we demonstrated the desensitization of Th17 cells memorized with C. albicans in severely burned patients. Under stimulation with the Candida antigen, T cells with abilities to produce IL-17, to express RORγt and to be proliferative were considered to be Th17 cells memorized with Candida antigen. PBMC (2 x 106 cells/ml), isolated from burn patients (> 30% TBSA burn), were stimulated with Candida antigen (heat-killed C. albicans) for 3 days. Culture fluids obtained were assayed for IL-17, and cells harvested were analyzed for expression of RORγt. The proliferative response of these PBMC stimulated with the antigen was tested by BrdU proliferation kit. In the results, as compared with healthy donor PBMC, the decreased amounts of IL-17 were produced, and RORγt was not expressed by patient PBMC. The proliferation of burn patient PBMC stimulated with the antigen was greatly decreased as compared with that of controls (326 ± 32% vs. 86 ± 15%). Burn patient serum (15%) inhibited the proliferation of healthy donor PBMC stimulated with the antigen. IL-10 was detected in burn patient serum, and burn patient serum treated with anti-IL-10 mAb did not inhibit the proliferation of Th17 cells stimulated with the antigen. These results indicate that T cells memorized with Candida antigen are desensitized in severely burned patients. IL-10 is indicated as an effector of this desensitization.
The role of cyclophilins in regulating leukocyte recruitment in chronic allergic asthma. (35.14)
Authors: Stemmy Erik , Constant Stephanie
Published: January 2023

Chronic allergic asthma is an airway disease characterized by persistent inflammation. Elevated numbers of eosinophils, activated lymphocytes, and mast cells, as well as increased levels of IL-5, are detectable in the airways and lung tissues of asthmatic patients even in the absence of allergen challenge. The factors regulating this persistent inflammation are not known. While classical chemokines such as eotaxins 1-3, RANTES, MIP-1α, and MCP-1 are highly elevated after acute allergen challenge, they return to baseline levels by 24h after exposure. Although better known as intracellular chaperone proteins or as the target of the immunosuppressive drug cyclosporine A, extracellular cyclophilins are also potent chemoattractants that are elevated during lung inflammation. We propose that extracellular cyclophilins could function as important chemoattractants regulating the leukocyte recruitment and persistence observed during the chronic phase of allergic asthma. To test this possibility, we made use of a mouse model of chronic asthma and inhibited the function of cyclophilins during the chronic phase of the disease using the drug NIM811, a non-immunosuppressive analog of cyclosporine A (provided by Novartis). Mice treated with NIM811 demonstrated a significant reduction (~50%) in airway and tissue inflammation upon acute allergen challenge. These findings suggest a potential role for cyclophilins in maintaining inflammation in chronic asthma.
Inhibition of T cell proliferation decreases atherosclerotic lesion size revealing a role of IL-17 in mediating aortic CD11b+CD11c+ cell accumulation (35.4)
Authors: von Vietinghoff Sibylle , Koltsova Ekaterina , Mestas Javier , Ley Klaus
Published: January 2023

Atherosclerosis is a chronic inflammatory disease of the artery wall with innate and adaptive immune components, however, immunosuppressive therapy is not uniformely beneficial. Mycophenolate suppresses lymphocyte proliferation by inhibiting inosine-monophosphate dehydrogenase. In this study, we tested whether mycophenolate inhibited atherosclerosis in apolipoprotein-E-deficient (Apoe-/-) mice. Apoe-/- mice treated with 30 mg/kg/day mycophenolate during 12 weeks of high fat diet showed significantly decreased aortic leukocyte infiltration. Flow cytometry at 3 and 12 weeks of high fat diet revealed significantly reduced aortic αβTCR+ cell content and reduced αβTCR+ cell proliferation as measured by BrdU incorporation. Aortic atherosclerotic lesion sizes were significantly reduced. Among T cell cytokines, IL-17 was significantly reduced in plasma of mycophenolate-treated mice. IL-17 receptor A was expressed on both blood monocytes and aortic CD11b+CD11c+ cells. Proliferation and aortic content of CD11b+CD11c+ cells and plasma levels of IL-6 and TNF-α were reduced. CD11b+CD11c+ cells in aortas of IL-17 receptor deficient mice after 12 weeks of high fat diet were significantly reduced compared to wild-type mice. Our data suggest that that mycophenolate curbs IL-17 production, which in turn limits myeloid cell accumulation in atherosclerosis. Since mycophenolate is approved for human use, this suggests its potential application for prevention and/or therapy.
Anti-inflammatory and anti-arthritic effect of essential oil isolated from Curcuma wenyujin (35.7)
Authors: Kim Hwan Mook , Kang Jong Soon , Lee Ki Hoon , Lee Chang Woo , Lee Kiho , Park Song-Kyu
Published: January 2023

Curcuma species has been reported to exert anti-inflammatory and anti-tumor effects. Curcuma wenyujin is mainly cultivated in wenzhou region of China and it was reported that the main constituents of Curcuma wenyujin is different from that of other Curcuma species. In this study, we investigated the effect of essential oil isolated from Curcuma wenyujin (CW) on inflammation and arthritis in rodents. CW dose-dependently suppressed lipopolysaccharide (LPS)-induced TNF-α production in mice. CW also inhibited zymosan-induced inflammation in concentration-dependent manner. Further studies demonstrated that CW attenuated the development of arthritis in adjuvant- and collagen-induced arthritis model and the anti-arthritic effect of the highest dose of CW was comparable to that of 0.1 mg/kg of dexamethasone. Collectively, the results presented in this report demonstrate that CW exerts anti-inflammatory and anti-arthritic effects. Our results suggest that CW might be a potential therapeutic agent for the treatment of inflammatory diseases, including rheumatoid arthritis.
Aicda inhibits B-cell development (36.12)
Authors: Kuraoka Masayuki , Kelsoe Garnett
Published: January 2023

Activation-induced cytidine deaminase (AID) is required for B lymphocytes to undergo somatic hypermutation (SHM) and class-switch recombination (CSR) in the immunoglobulin genes. Whereas AID expression, SHM and CSR are crucial for effective humoral immunity, these processes exact substantial costs in genomic damage that often result in programmed cell death and occasionally, lead to oncogenesis. In mice and humans, AID expression, SHM, and CSR are thought to be largely restricted to the mature, antigen-activated B cells in germinal centers (GC). In amphibians, birds, and some mammals, however, AID expression and activity is present both in GC B cells and in distinct subsets of developmentally immature B cells. Whether this developmentally regulated expression of AID and activity is also present in mice and humans is controversial. Here we quantify AID expression in developing mouse B cells and demonstrate its substantial effects on B lymphopoiesis. Bone marrow cells from AID deficient mice exhibit a significant advantage over normal congenic hematopoietic competitors in the production of B lymphocytes but not any other leukocytic lineage. This early lymphopoietic advantage in AID deficient cells is also manifested by an increased tempo of B-cell development. We conclude that early, developmentally regulated AID expression represents a fundamental and conserved lymphopoietic pattern that is widely distributed among the vertebrate species for diversification of immunoglobulin.
miRNAs are required for the epidermal Langerhans cell development (36.18)
Authors: Zhou Li , Wang He , Li Kai , Qi Ruiqun , Zhang Zhenggun , Weiland Matthew , Kaplan Daniel , Mi Qing-Sheng
Published: January 2023

Langerhans cells (LCs) are skin-resident DCs that express the C-type lectin Langerin and have a life cycle distinct from many other types of DCs. Even though LCs were first described more than 100 years ago, their development and immunological functions still remain enigmatic. MicroRNAs (miRNAs), a class of 21-25 nt single-stranded non-coding small RNAs, are increasingly being recognized as important regulators of gene expression through the inhibition of effective mRNA translation. Using Cre-loxP tissue-specific Dicer deletion, our laboratory and others have reported that deletion of miRNAs significantly affects the development and function of B, T, and NKT cells. To test the roles of miRNAs in the development of LCs, we generated a mouse strain with tissue-specific disruption of Dicer in the late stage of LC development mediated by Langerin-Cre. We found that Dicerfl/fl.langerinCre+ mice had significantly reduced number of epidermal LCs and the expression of Langerin was significantly reduced in miRNA-deficient I-Ab positive LCs. miRNA-deficient LCs have a defect on their maturation in vitro, but have normal capacity for antigen uptaking. Using BRDU staining, we found that increased both LC proliferation and apoptosis appear in Dicerfl/fl.langerinCre+ mice compared to WT mice. Thus, our findings highly suggest that miRNAs are required for the LC development and homeostasis.
Neutrophil density in bone marrow controls granulopoiesis by feedback inhibition (36.21)
Authors: Cain Derek , Snowden Pilar , Sempowski Gregory , Kelsoe Garnett
Published: January 2023

Normally, neutrophil pools are maintained by “steady-state” granulopoiesis. Inflammation, however, is thought to elicit growth factors that accelerate neutrophil production through a distinct developmental program known as “emergency” granulopoiesis. In IL-1RI-/- mice, adjuvant-induced emergency granulopoiesis is defective due to impaired progenitor proliferation. Interestingly, this hematopoietic defect is associated with impaired neutrophil mobilization from bone marrow (BM), raising the possibility that increased proliferation by hematopoietic progenitors is a response to reduced BM neutrophil density rather than pro-inflammatory signals. Consistent with this potential feedback mechanism, non-inflammatory depletions of mature BM neutrophils by passive antibody elicit emergency granulopoietic responses similar to those induced by adjuvant, and progenitor proliferation fluctuates inversely with BM neutrophil numbers. Furthermore, mice bearing a genetic defect in neutrophil survival exhibit constitutive emergency granulopoiesis. Whereas adjuvant potently induces serum G-CSF, a neutrophil growth and mobilization factor, neutrophil depletion elicits equally robust granulopoietic responses with substantially reduced levels of G-CSF. These observations suggest that other - perhaps unknown - factors mediate accelerated granulopoiesis, and we propose a common model for the feedback regulation of both steady-state and emergency granulopoiesis.
Dendritic cell expression of CD24 inhibits autoantigen-mediated deletion of T lymphocytes in the thymus (36.30)
Authors: Bai Xue-Feng , Liu Jin-Qing , Zhang Xuejun , Reid Hugh , Boyd Richard , Khattabi Mazin , El-Omrani Hani , Zheng Pan , Liu Yang
Published: January 2023

Despite negative selection, significant numbers of autoreactive T cells can be easily detected and expanded even in normal individuals. While lack of self antigen expression has been largely attributed as a key factor, we have reported that CD24, a GPI-anchored glycoprotein, regulates thymic deletion of autoreactive T lymphocytes. CD24-deficient 2D2 TCR transgenic mice (2D2+CD24-/-), whose TCR recognize myelin antigen MOG, failed to generate functional 2D2 T cells. Here we further show that elimination of MOG antigen expression in 2D2+CD24-/- mice (through creation of 2D2+CD24-/-MOG-/- mice), completely restored thymic cellularity and function of 2D2 T cells. Restoration of CD24 expression on dendritic cells, but not on thymocytes also restored 2D2 T cell generation in 2D2+CD24-/- mice. Taken together, we propose that CD24 expression on dendritic cells inhibits autoantigen-mediated deletion of autoreactive thymocytes.
A novel role of IκBα and WWOX in promoting lymphoid Molt-4 and Jurkat T cell differentiation (36.42)
Authors: Huang Shenq Shyang , Chang Nan-Shan
Published: January 2023

Human common fragile site gene WWOX, encoding WW domain-containing oxidoreductase (designated WWOX, FOR or WOX1), plays critical roles in the pathogenesis of cancer and maintenance of normal physiology. To explore the role of WOX1 in lymphoid cancer, here we determined that transient expression of WOX1 induced the expression of a differentiation marker CD3 in an acute lymphoblastic leukemia cell line Molt-4, but the induction was less effective in better-differentiated Jurkat T cells. Upregulation of RORγ, one of the Th17 cell markers, was shown in Molt-4, but not Jurkat T cells, overexpressing WOX1. Mapping analysis revealed that the N-terminal WW domain of WOX1 induced T lymphoid cell differentiation, in which a dominant negative and phosphorylation mutants (i.e. Y33R and Y61R) of WOX1 blocked the effect. We determined that the N-terminal WW domain of WOX1 physically interacts with IκBα (an inhibitor of NF-κB), as determined by co-immunoprecipitaiton and Fluorescence/Förster resonance energy transfer (FRET). Ectopic IκBα, via its N-terminal ankyrin-repeat region, induced Molt-4 differentiation. Both WOX1 and IκBα did not enhance T cell differentiation synergistically, suggesting that they may nullify each other’s function. In positive controls, phorbol and ionophore A23187 induced cell differentiation by upregulating IκBα, CD3 and WOX1 in Molt-4 T-cells. Together, our finding demonstrates a novel WOX1/IκBα regulatory mechanism for T-cell differentiation.
An unexpected role for the Src family kinase Blk in early T cell development (36.44)
Authors: Laird Renee , Hayes Sandra
Published: January 2023

Src family kinases are involved in the initiation of antigen receptor signaling, and each lymphocyte lineage expresses different family members. B cells express Lyn, Fyn and Blk, while T cells express Lck and Fyn. To determine whether there are differences in the expression of Src family kinases between αβ and γδ T cells, we used gene expression profiling and found that γδ T cells express Blk in addition to Lck and Fyn. Next, we developed an intracellular flow cytometric assay to examine Blk expression in T lineage cells. Surprisingly, we detected Blk in progenitor populations in both the thymus and bone marrow. To determine the significance of Blk expression in these progenitors, we compared T cell development in various aged blk+/+ and blk-/- mice. We found that during ontogeny the cellularity of the blk-/- thymus did not reach wild-type numbers until 4 weeks of age, whereas the cellularity of the blk-/- lymph nodes, which was always less than that of blk+/+ mice, reached a level that was not significantly different from blk+/+ mice at 5 weeks of age. Given that thymus size and output are dependent on CD25-CD44-CD4-CD8- (DN4) thymocyte proliferation, we compared the proliferation status of DN4 thymocytes from blk+/+ and blk-/- mice and noted significantly less proliferating DN4 thymocytes in blk-/- mice than in blk+/+ mice. Together, these data demonstrate that Blk is expressed in T cell progenitor populations and suggest a role for Blk in regulating thymus cellularity.
Obesity induces premature thymic involution and restricts T cell repertoire diversity (36.47)
Authors: Youm Yun-Hee , Yang Hyunwon , Vandanmagsar Bolormaa , Rood Jennifer , Kumar K.Gannesh , Butler Andrew , Dixit Vishwa
Published: January 2023

Yun-Hee Youm, Hyunwon Yang, Bolormaa Vandanmagsar, Jennifer Rood, K. Ganesh Kumar, Andrew A. Butler and Vishwa Deep Dixit Obesity increases risk of infections and cancer, suggesting adverse effects on immune-surveillance. Here we report that obesity compromises the mechanisms regulating T cell generation via inducing premature thymic involution. Diet-induced obesity reduced the total number of thymocytes and significantly increased apoptosis in developing T cell populations. Obesity accelerated the age-related reduction of naïve T cells and decreased T cell receptor excision circle bearing peripheral T cells, an index of recently generated T cells from thymus. Defects in thymopoiesis in obese mice were due to a reduction in the lymphoid progenitor pool and deterioration in the thymic stromal microenvironment. Interestingly, obesity also increased the frequency of thymic fibroblasts and reduced the number of thymic epithelial cells (TECs). Furthermore, the TECs of DIO mice displayed reduced expression of IL-7 and auto immune regulator (Aire) mRNA. Obesity induced via melanocortin 4 receptor (Mc4r) deficiency also constricted the T cell receptor repertoire diversity, recapitulating the thymic defects observed with diet-induced obesity. In humans, progressive adiposity with or without type-2-diabetes also compromised thymic output. Collectively, these findings establish that obesity constricts T cell diversity by accelerating age-related thymic involution.
Impairment in the strength and duration of ERK activation in Gads deficient mice (36.51)
Authors: Hassaballa Ashraf , Yankee Thomas
Published: January 2023

The Gads adaptor protein regulates TCR-induced calcium mobilization by mediating the interaction between SLP-76 and LAT. Gads-/- mice had a defect in the transition from double positive (DP) thymocytes to single positive (SP) cells, suggesting a role for Gads in positive and negative selection. This function of Gads was supported by crossing Gads-/- mice with TCR transgenic mice. Our goal is to understand the biochemical basis of the defect in thymic selection seen in Gads-/- mice. To address this we investigated signaling molecules upstream and downstream of Gads. First, we examined whether the magnitude and duration of TCR-dependent ERK phosphorylation in Gads+/+ and Gads-/- thymocytes. The peak level of ERK phosphorylation was reduced in Gads-/- mice, but the signal was prolonged. We then examined CD69 upregulation in vitro. In DP thymocytes, TCR-induced CD69 upregulation was comparable in wild-type and Gads-/- cells, indicating that all the DP thymocytes received TCR stimulation, even though ERK phosphorylation was reduced. To test the mechanism by which Gads might regulate ERK phosphorylation, we tested whether Gads might regulate the expression or phosphorylation of TCRζ. We found that the activation of TCRζ is impaired in Gads-/- thymocytes. We conclude that Gads regulates thymic positive selection by regulating TCRζ phosphorylation and ERK activation. This research is supported by NIH grant P20 RR016443 and American Cancer Society RSG-08-182-01-LIB.
Developmental targets of IL-7 and Flt ligand in human B cell production (36.8)
Authors: Milford Terry-Ann , Baez Ineavely , Dovat Sinisa , Payne Kimberly
Published: January 2023

Signals induced by IL-7 and Flt ligand (FL) are critical components of the regulatory network that governs early B cell development in mice. The role of these cytokines in human B lymphopoiesis is less clear. We previously reported that IL-7 is required for in vitro human B cell production from adult bone marrow (Parrish et al. JI 2009 182:4255). Here, we use a human-only culture model to examine the effects of IL-7 and FL, separately and together, on the production of developmental subsets of human B lineage cells from hematopoietic stem cells in cord blood. While B cell production was essentially absent in cultures without IL-7 or FL, small populations of CD19+ B lineage cells were produced with either IL-7 or FL alone. The combination of IL-7 and FL increased B cell production up to 75 fold. To identify the developmental targets of IL-7 and FL we assessed 3 subsets that include developmentally sequential populations of human B cell precursors. The production of CD19-IL-7Rα+CD34+ progenitors was dependent on FL, but not IL7, and the addition of IL-7 did not expand this population. CD34+ pro-B cells showed variable responses to both IL-7 and FL while IL-7 primarily targeted CD19+CD34- pro-B cells for expansion. The intensity of CD19 staining was approximately a log higher in all cultures with IL-7. These data suggest that FL targets CD19-CD34+IL-7Rα+ progenitors that respond to IL-7 to upregulate CD19 and then expand as CD34 is lost during human B cell differentiation.