bluuebunny/crossref_metadata_2025 huggingface dataset:
Defensins block the release but not the processing of IL-1beta and IL-18 from human monocytes (38.13)
Authors: Shi Jishu , Lan Liqiong , Aono Shelly , Morrison Edward , Lu Wuyuan
Published: January 2023

We have previously shown that human defensins can block the release of IL-1beta from LPS-primed human monocytes stimulated with ATP. The objective of this study is to determine whether defensins can inhibit the release IL-1beta and IL-18 from LPS-primed human monocytes stimulated with other inflammasome activators. LPS-activated (20 ng/ml, 2 h), 35S-methionine-labeled (1 h) human monocytes were treated with human defensin HD-5 in the presence or absence of ATP, monosodium urate (MSU), nigericin (Nig) for 90 min. Media and cell-associated fractions were harvested separately. IL-1beta was recovered from each by immunoprecipitation and caspase-1 was examined by western blot analysis. Mature IL-1beta and active caspase-1 were detected in the cytosol fraction from cells treated with HD-5. Thus, HD-5 blocked the release of mature IL-1beta from cells treated with ATP, MSU, or Nig. However, HD-5 did not inhibit ATP-, MSU-, or Nig-induced caspase-1 activation and processing of IL-1beta. In addition, HD-5 also blocked the release of IL-18 from LPS-primed human monocytes stimulated with ATP. To our knowledge, HD-5 is the first known endogenous inhibitor of IL-1beta and IL-18 release. These studies suggest that processing and release of IL-1beta are independent processes.
PGE2 affects the IL-12/IL-23 balance in mature myeloid dendritic cells (38.14)
Authors: Kocieda Virginia Polonia , Khayrullina Tanzilya , Ganea Doina
Published: January 2023

Following TLR signaling dendritic cells (DC) produce pro-inflammatory cytokines including IL-12 and IL-23. IL-12 promotes differentiation of the Th1 subset whereas IL-23 maintains the Th17 subset. PGE2, a lipid mediator, is produced by DC during inflammation. This study is focused on the effects of PGE2 on the regulation of IL-12/IL-23 production in murine DC. PGE2 inhibits LPS-induced IL-12 production through downregulation of p35, and stimulates IL-23 through upregulation of p19 expression. Changes in p35/p19 expression reduce Th1 and promote Th17 differentiation. Similar PGE2 effects on p19/p35 were observed in the presence of other TLR ligands, such as peptidoglycan (TLR2), poly I:C (TLR3) and CpG (TLR9). The effects of PGE2 on p19/p35 were mediated through EP2 and EP4 receptors, and cAMP induction. The involvement of downstream signaling pathways such as PKA and EPAC-induced activation of PI3K and GSK3, as well as the role of specific transcription factors such as CREB, cRel, and IRF5 (for p19 expression) and IRF 1, 3, 7, and RelA (for p35 expression) are currently being investigated.

Research support: NIH2RO1AI052306 & NSF Pre-doctoral Fellowship (VK)
Effects of interferon-a and interferon-b on development of antigen-specific B cells and hybridoma generation (38.28)
Authors: Santulli-Marotto Sandra , Petley Ted , Fisher Jamie , Pitcher Jennifer , Rycyzyn Michael , Giles-Komar Jill
Published: January 2023

The ratio of Interferon-α (IFN-α) and Interferon-β (IFN-β) produced during an immune response can significantly impact the outcome. We find that administration of IFN-α and IFN-β during immunization can alter the fate of antigen-specific B cells with minimum affect on antibody titers in mice. In vitro stimulation with IFN-α and IFN-β either individually or in combination triggered variations in interferon-responsive gene expression that directly correlated to the activity level of the individual interferons as well as the ratio of IFN-α to IFN-β. However, when the activity of IFN-α and IFN-β was normalized according to gene expression profiles the outcome of immunizing mice with the same antigen, using the same protocol but varying relative ratios of IFN-α and IFN-β yielded little to no antigen-specific B cells. Though animals could produce nearly identical antigen-specific antibody titers there was an average difference of almost 7- fold in terms of IgG producing hybridomas and almost 100-fold difference in the number of antigen-specific clones. Importantly, not all genes were affected equally and the in vitro data were generated using a cell line derived from a different mouse strain than used for the immunization studies. Perhaps a subset of genes induced by interferons plays an important role in B cell activation and development or the B cell response to IFN-α and IFN-β varies depending on the strain of mouse.
Selective ablation of the YxxM motif of IL-7R alpha suppresses lymphomagenesis but maintains lymphocyte development (38.5)
Authors: Osborne Lisa , Duthie Kia A , Gascoyne Randy , Abraham Ninan
Published: January 2023

Lymphocyte homeostasis is exquisitely sensitive to the cytokine interleukin (IL)-7. Loss of IL-7 signaling prevents lymphocyte development whereas excess IL-7 causes murine lymphoma and has been detected in several human lymphomas. We have previously shown that targeted disruption of the Y449xxM motif of the IL-7 receptor alpha (IL-7Rα) in a knock-in mouse model (IL-7Rα449F) has minor effects on lymphocyte production, but interferes with activation of survival effectors. We hypothesized that targeted signal ablation would selectively affect lymphocyte transformation. To address this, IL-7Rα449F mice were crossed with two lymphomagenesis models, transgenic (Tg) IL-7 and Eμ Myc mice. We found that loss of IL-7Rα Y449 signaling was sufficient to reverse the developmental aberrations induced by both the IL-7 and Eμ Myc transgenes, prevented Tg IL-7 mediated T and B lymphocyte transformation and significantly decreased development of Eμ Myc-induced B cell tumors. We show that the IL-7Rα449F mutation is essential for Tg IL-7 mediated up-regulation of pro-survival Bcl-2 family members, and for rapid cycling of bone marrow progenitor B cells induced by Eμ Myc. This study highlights the therapeutic potential of targeting the IL-7Rα Y449xxM motif or its downstream effectors in treatment of human lymphocyte malignancies.

Research supported by the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada and the Michael Smith Foundation for Health Research.
Insight into Mechanism of IL-2-Induced Toxicity Provides Rationale for Improved Treatment Strategy using IL-2/mAb Complexes (38.8)
Authors: Boyman Onur , Krieg Carsten , Létourneau Sven , Pantaleo Giuseppe
Published: January 2023

IL-2 immunotherapy is used against certain metastatic cancers and for patients infected with human immunodeficiency virus. However, administration of IL-2 can lead to serious side effects including pulmonary edema, the pathogenesis of which is ill-defined. Here, we provide data showing that IL-2 may bind directly to CD25 on lung endothelial cells and thus cause toxicity and pulmonary edema. Thus, lung endothelial cells express CD25 under steady-state conditions, and expression levels of CD25 on these cells increase in vivo upon injection of mice with IL-2. Activation of CD25+ lung endothelial cells and of IL-2 receptor-positive NK cells and T cells leads to pulmonary edema upon IL-2 treatment. Significantly, co-administration of anti-CD25 antibody along with IL-2 treatment is able to completely abrogate IL-2-induced pulmonary edema despite considerable proliferation of NK cells and T cells. Similarly, knocking out CD25 on non-immune cells or obscuring the CD25-binding epitope of IL-2 by the use of selective immune complexes of IL-2 plus anti-IL-2 antibody (IL-2/mAb) is able to prevent IL-2-induced pulmonary edema. Notably, IL-2-mediated toxic effects are also evident on a human lung cell line and can be abrogated using the anti-CD25 antibody daclizumab or IL-2/mAb complexes. Moreover, IL-2/mAb complexes are very efficient in generating anti-tumor responses in vivo against syngeneic local or metastatic tumor nodules. Thus, CD25+ endothelial cells play a crucial role in IL-2-mediated vascular leak syndrome and this can be circumvented by the use of IL-2/mAb complexes.
Inhibition of IFN-γ responses in human choriocarcinoma cells by PTPs (166.18)
Authors: Bushway Meghan , Weise Paige , Grace Daniel , Olsen Keith , Choi Jason , Murphy Shawn
Published: January 2023

Placental trophoblast cells (TBCs) form the fetal component of the placenta, and are the only blastocyst-derived cells in direct contact with maternal blood and thus immune system. Interferon-γ (IFN-γ) is a pro-inflammatory cytokine that is critical for the activation of both innate and adaptive immune responses, inhibition of cell proliferation, and induction of apoptosis. Interestingly, IFN-γ is present in the uterus during normal pregnancy and levels can be further enhanced upon intrauterine infection. Despite the presence of IFN-γ in the uterus, human TBCs do not express either MHC class Ia or class II antigens and are resistant to IFN-γ-mediated apoptosis. We previously demonstrated that TBCs are hypo-responsive to IFN-γ due to protein tyrosine phosphatase (PTP)-mediated inhibition of the JAK-STAT-1 pathway. These results suggest that inhibition of IFN-γ signaling in TBCs is due to differential regulation of PTP activity. In most cell types, PTPs are constitutively active and are transiently inactivated by reactive oxygen species that are generated in response to ligand binding. Our current studies suggest that SHP-2 and/or PTP-1B are responsible for inhibiting IFN-γ responses in human TBCs. Moreover, our data demonstrate that human TBC-derived choriocarcinoma cells produce H2O2 in response to IFN-γ. Furthermore, studies in which TBCs were exposed to exogenous H2O2 or antioxidants suggest that lack of H2O2 production is not the basis for differential PTP activity.
IL-17 and IL-22 induced inhibitor of nuclear factor κB kinase-α (IKKα) expression in HaCaT human keratinocyte cell line (166.19)
Authors: Lee Kyung Ho , Cho Kyung-Ah , Kim Jin-Young , Woo So-Youn
Published: January 2023

The pathogenesis of psoriasis may involve the IL-23 and Th17-mediated immune responses. Th17 cells secret IL-17 and IL-22 which mediates dermal inflammation and acanthosis. As IKKα has been previously identified as a primary regulator of keratinocyte differentiation and proliferation, we proposed that IL-17 and IL-22 might affect keratinocyte differentiation by changing the expression of IKKα. We employed HaCaT cells maintained culture medium at a low calcium concentration (0.06 mM) and induced differentiation by switching high concentration (2.8 mM) media with IL-17 or IL-22, then compared IKKα expression and the cell cycle. We employed reconstituted human epidermal skin (Neoderm) and mice ears for the in vivo studies. Elevated calcium concentration induced IKKα expression and terminal differentiation with cell cycle arrest in HaCaT cell cultures. Moreover, IL-17 and IL-22 treatment also induced IKKα in HaCaT cells and reconstituted human epidermis. IKKα induction was also noted following the injection of IL-17 and IL-22 into mice ears. Although the induction of IKKα was accompanied by keratinocyte differentiation, IL-17 and IL-22 did not affect calcium-mediated differentiation or the cell cycle. Rather, IL-17 and IL-22 appear to contribute to inflammation occurring via the induction of IKKα from keratinocytes or skin layers.
Generation of memory TCRγδ T cells following oral L. monocytogenes infection (166.4)
Authors: Sheridan Brian , Pham Quynh-Mai , Lefrançois Leo
Published: January 2023

Listeria monocytogenes (LM) has been used as a model organism for studying T cell responses. However, the vast majority of LM immunological studies utilize inoculation routes other than oral. The oral infection studies here utilize a recombinant LM that expresses an internalin A protein with high affinity for mouse E-cadherin thus mimicking the physiologic route of infection in humans. Our findings reveal a remarkable mucosal TCRγδ T cell response to oral LM infection, whose kinetics mimicked an adaptive T cell response. The responding TCRγδ subset was polyfunctional and was comprised of both IL-17 and IFNγ producers and notably, IL-17/IFNγ double producers. They appeared distinct from “innate” TCRγδ IL-17 producers which are present early after infection and in peripheral lymph nodes of naïve mice. Moreover, this phenotypically and functionally distinct subset of mucosal TCRγδ T cells were retained long-term and underwent a recall response upon oral challenge similar to that of a TCRαβ T cell recall response. However, memory TCRγδ T cells failed to expand following iv rechallenge, suggesting tissue-specific mechanisms regulate their ability to mount a secondary response. Very little is known regarding the anatomy of TCRγδ T cell responses and our results identify a bona-fide mucosal memory TCRγδ T cell population following oral LM infection. With this model we can address novel questions regarding the induction of specialized TCRγδ subsets and the development of memory.
Negative regulation of autoreactive B cell activation by integrin CD11b molecule (167.13)
Authors: Ding Chuanlin , Yan Jun
Published: January 2023

The immunological hallmark of systemic lupus erythematosus (SLE) is a loss of immune tolerance and B cell hyperactivity. Recent studies demonstrated an association between the variant of the integrin-α-M (ITGAM, also called CD11b) gene and SLE susceptibility. However, how CD11b regulates disease pathogenesis remains elusive. We found surprisingly that B cells from CD11b knock out (KO) mice exhibited hyperproliferative responsiveness to B cell receptor (BCR) cross-linking. CD11b KO autoreactive B cells had the decreased tyrosine phosphorylation including Lyn and PLCγ2 and increased p38 MAKP. Moreover, CD11b deficiency promoted B cell survival which was associated with increased BCL-xl expression. In vivo engagement of BCR by apoptotic cells resulted in significantly increased autoAb production in CD11b KO mice. Furthermore, we found that BCR cross-linking could induce CD11b activation and modulate CD11b-medited phagocytosis, suggesting ‘out-side-in’ activation of CD11b integrin by BCR crosslinking. Taken together, these results reveal the interaction between CD11b integrin and BCR signaling pathways and suggest the critical role for CD11b molecule in maintaining autoreactive B cell tolerance (This work was supported by the Alliance for Lupus Research).
Exosomes from mouse bone marrow-derived mesenchymal stem cells (mMSCs) mediate a potent immunosuppressive function (167.15)
Authors: Kim Sueon , Shin Jin-Young , Kim Yong-Hee , Kim Chung-Gyu
Published: January 2023

Mouse mesenchymal stem cells (mMSCs) have been reported to control the inflammation in the central nervous system (CNS) and alleviate the disease severity in the neurodegenerative disease models such as experimental autoimmune encephalitis (EAE). Although increasing evidences suggest that these cells can contribute to modulate immune responses, the underlying mechanisms are not fully understood. In this study, exosomes were purified from the culture supernatant of mMSCs using ultracentrifugation under sucrose cushion and tested for their immunosuppressive effects toward activated T cells. Exosomes exhibited potent suppressive effects on T cell proliferation through cell cycle arrest and apoptosis as revealed by thymidine incorporation and flow cytometric analyses. Importantly, exosome-depleted culture supernatant (EDCS) had no such effect. To prove exosome's effect in vivo, EAE was induced in four cohorts of mice and exosome, exosome-enriched, exosome-lowered by methyl-β-cyclodextrin (MβCD) culture supernatant or saline was treated to each group from disease initiation phase. Results showed that exosome and exosome-enriched culture supernatant, but not exosome-lowered culture supernatant can ameliorate autoimmune symptoms. In conclusion, our results suggest that exosomes secreted from mMSCs mediate immuno suppressive function.
MicroRNA-29 controls Th1 bias of CD4+ T cells in multiple sclerosis (167.5)
Authors: Smith Kristen , Guerau-de-Arellano Mireia , Williams Jessica , Cox Gina , Croce Carlo , Racke Michael , Lovett-Racke Amy , Whitacre Caroline
Published: January 2023

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system initiated by autoreactive CD4+ T cells of a T-helper (Th)1 and Th17 phenotype. Identifying novel factors that control Th differentiation will lead to a better understanding of MS pathogenesis and the development of new therapeutic strategies. MicroRNAs (miRs) are small non-coding RNAs that negatively regulate post-transcriptional gene expression. Dicer-deficient CD4+ T cells that lack miRs produce more of the Th1-associated cytokine IFN-γ; however, the specific miR(s) responsible for controlling Th1 programming have not been defined. Using in silico target prediction algorithms, we identified miR-29 as a factor that could potentially regulate multiple genes in the Th1 pathway. We confirmed that miR-29 directly targets T-bet and IFN-γ, and gain- and loss-of-function experiments demonstrated the ability of miR-29 to modulate Th1 polarization. Importantly, we found that naïve CD4+ T cells from MS patients express lower basal levels of miR-29 than controls. We further established an inverse relationship between basal expression of miR-29 and expression of T-bet/IFN-γ following activation of MS patient T cells. These results identify miR-29 as a novel regulator of Th1 differentiation and function, and implicate low miR-29 as an MS susceptibility factor. Restoring miR-29 may therefore be a powerful therapeutic modality in MS and other Th1-driven autoimmune diseases.
Differential requirements for Th1 and Th17 responses to a systemic self antigen (167.7)
Authors: Katzman Shoshana , Gallo Eugenio , Hoyer Katrina , Abbas Abul
Published: January 2023

T cell-APC interactions are essential for the initiation of effector responses against foreign and self-antigens, but the role of these interactions in generating different populations of effector T cells in vivo remains unclear. Using a model of CD4+ T cell responses to a systemic self-antigen without adjuvants or infection, we demonstrate that activation of APCs augments Th17 responses much more than Th1 responses. Recognition of systemic antigen induces tolerance in self-reactive CD4+ T cells, but induction of CD40 signaling, even under tolerogenic conditions, results in a strong, antigen-specific IL-17 response without large numbers of IFN-γ-producing cells. Transfer of the same CD4+ T cells into lymphopenic recipients expressing the self-antigen results in uncontrolled production of IL-17, IFN-γ, and systemic inflammation. If the antigen-specific T cells lack CD40L, production of IL-17 but not IFN-γ is decreased and the survival time of recipient mice is significantly increased. In addition, transient blockade of the initial MHC Class II-dependent T-APC interaction results in a greater reduction of IL-17 than of IFN-γ production. These data suggest that Th17 differentiation is more sensitive to T cell interactions with APCs than is the Th1 response, and interrupting this interaction, specifically the CD40 pathway, may be key to controlling Th17-mediated autoimmunity.
Granzyme B regulates antigen-specific CD8 cell responses to Sendai virus (168.10)
Authors: Salti Suzan , Hammelev Erin , Grewal Jenny , Grossman William , Grayson Mitchell , Verbsky James
Published: January 2023

Introduction. Human regulatory T (T reg) cells express functional granzymes and perforin (Gzm/Prf) and can induce autologous target cell death in vitro. Prf1-/- mice die from exaggerated immune responses after viral challenges, implicating a potential role for this pathway in immune regulation. To further investigate the role of GzmB in immune regulation of viral infections, we infected wild-type (WT) mice, Gzmb-/- mice and Prf1-/- mice with Sendai virus (SeV) and characterized the magnitude of the immune response. Methods. Mice were infected with SeV and Antigen (Ag)-specific CD8 responses were measured by flow cytometry. T reg cell depletion was performed with the anti-CD25 mAb PC61. Results. Gzmb-/- mice exhibited a significant increase in the number of Ag-specific CD8 cells in the lungs and draining lymph nodes in response to SeV infection. This increase was not the result of failure in viral clearance since viral titers in Gzmb-/- mice were similar to WT mice and significantly less than Prf1-/- mice. T reg cells from WT mice expressed high levels of GzmB, and depletion of T reg cells from these mice resulted in an increase in the number of Ag-specific CD8 cells, similar to that observed in Gzmb-/- mice. Furthermore, Gzmb-/- T reg cells displayed a clear defect in their ability to suppress CD8 cell proliferation in vitro. Conclusion. Taken together, these results suggest a possible role for GzmB in the T reg cell compartment in immune regulation of viral infections.
Regulatory T cells halt islet destruction by cytotoxic T cells as an initial step in protecting against progression of autoimmune diabetes. (168.11)
Authors: Mahne Ashley , Tang Qizhi
Published: January 2023

In the NOD mouse model of type 1 diabetes, transfer of islet antigen-specific regulatory T cells (Tregs) isolated and expanded from BDC2.5 TCR transgenic mice halts disease progression. Having observed that BDC2.5 Tregs home to the inflammatory site of the pancreatic islets of Langerhans and that they are capable of protecting against disease development in the absence of the draining pancreatic lymph node, we sought to elucidate the mechanisms by which BDC2.5 Tregs suppress autoimmune destruction within the islets. We began with a transcriptional profiling approach to assess islet expression levels of a large panel of inflammatory mediators, chemokines, leukocyte population markers and markers of β-cell health at one week post-Treg transfer as compared to age-matched controls. We found that BDC2.5 Treg treatment induces the downregulation of a cytotoxic T lymphocyte (CTL) signature. IFNγ, Cxcl9 and granzymes A and B were among the genes most downregulated in Treg-treated islets. By flow cytometric analyses, we observed that BDC2.5 Tregs suppress IFNγ production by both CD4 and CD8 T cells within the islets. Current studies are investigating the effects of Tregs on islet CD8 T cell dynamics using multi-photon time-lapse imaging. Taken together, our results indicate that an early and critical step towards halting the progression of autoimmune diabetes by islet antigen-specific Tregs is the reigning in of β-cell destruction exerted by islet CTLs.
Characterization of the immunological synapses formed by induced T regulatory cells (168.13)
Authors: Thauland Timothy , Parker David
Published: January 2023

T regulatory cells (Tregs) play a vital role in the maintenance of self-tolerance and in the suppression of active immune responses. FoxP3+ Tregs arise naturally in the thymus, but can also be induced both in vivo and in vitro. The immunological synapses (IS) formed by induced Tregs (iTregs) have not been studied. We show that iTregs form either immunological kinapses or stable IS depending on the level of CD80 on the surface of APCs. As has been shown for natural Tregs, iTregs are capable of downmodulating the costimulatory ligand CD80 on the surface of dendritic cells (DCs) in an antigen-specific, CTLA-4-dependent manner. On the same timescale as CD80 downmodulation, iTregs transition from stable to motile contacts with DCs. Experiments utilizing supported planar bilayers containing pMHC, ICAM-1 and CD80 show that iTregs form motile immunological kinapses with a highly polarized cell shape in the presence of low levels of CD80. However, iTregs arrest and form stable IS when interacting with bilayers containing high levels of CD80. In experiments with blocking antibodies, we demonstrate that CD28-CD80, but not CTLA-4-CD80, interactions modulate iTreg motility. Thus, iTregs are capable of downmodulating costimulatory molecules on the surface of DCs, which in turn alters their motility.
Transient TCR engagement with an anti-TCRβ monoclonal antibody induces tolerogenic effects to allografts as well as prevents the development and reverses the onset of type 1 diabetes (169.1)
Authors: Khattar Mithun , Miyahara Yoshihiro , Schroder Paul , Mierzejewska Beata , Chen Wenhao , Stepkowski Stanislaw
Published: January 2023

In the current study, we investigated the immune regulatory effects of TCR engagement simultaneously by an anti-TCRβ chain mAb (H57-597) without or with antigens. We found that administration of anti-mouse TCRβ mAb resulted in a preferential reduction of antigen-reactive T cells with simultaneous enrichment (~3-fold increase) of CD4+Foxp3+ Treg cells. Moreover, administration of H57-597 mAb did not produce high levels of TNF-α, IFN-γ, IL-2 and IL-6 cytokines in serum in contrast to anti-CD3 mAb (145-2C11). Strikingly, a single injection of H57-597 mAb into RIP-OVAhi mice completely inhibited the development of type 1 diabetes (T1D) that was induced by adoptively transferred OVA-specific T cells. In NOD mice, a short course treatment with H57-597, not only prevented development of T1D (90% disease free treated at 8-weeks), but also induced remission in 7 of 7 mice treated at onset. In a transplantation model, transient H57-597 mAb treatment alone produced long-term Balb/c cardiac allograft survivals (>100 days; n=9) in C57BL/6 mice. Adoptive transfer of splenocytes from the heart allograft-accepting recipients to Rag1-/- recipients (B6 background, heart grafted) showed significantly extended survival of donor-specific (Balb/c) but not third-party (C3H) heart allografts. Thus, transient modulation of TCRβ chain during an ongoing immune response exhibits potent tolerogenic effects applicable for the therapy of T cell-mediated diseases and transplant rejection.
Return of lymphocyte immunoreactivity after discontinuation of a brief course of Prograf, Cyclosporine, or Sirolimus in normal subjects. (169.13)
Authors: Lwin Wint , Park Ken , Khanna Ajai , Verma Meenakshi , Perkins David
Published: January 2023

The mainstay of immunosuppression is Prograf (FK), Cyclosporine (CSA), and Sirolimus (SRL). These drugs target T cells which are pivotal in transplant rejection. Little is known about their effects on length of T cell suppression once stopped. We hypothesized that T cell reactivity would return later with SRL compared to CSA or FK. 18 health volunteers were recruited. 8 received FK, 3 received CSA, and 7 received SRL. FK and CSA were taken twice daily for 3.5 days and SRL once daily for 4 days. Drugs were titrated to respective troughs (FK 8-10 mg/L, CSA 150-200 mcg/L, SRL 10-15 ng/ml). Blood were drawn at 0, 12, 24, & 48 hrs after last dose. CD4 cells were isolated using CD4 isolation beads, stimulated with CD3/28 beads, and cultured in 96 well plates. Cell proliferation and ATP concentration were measured using BrdU labeling and Cylex assay respectively. At 24 hrs, ATP concentration fold change between FK and SRL by Cylex was not statistically significant (1.0 vs. 0.8, p=0.2). T cell proliferation measured by BrdU compared to time 0 was significant in 2/8 patients at 12 & 24 hrs and 4/8 patients at 48 hrs in FK group, 1/3 patients at 12 & 48 hrs in CSA group, and 6/7 patients at 12 & 48 hrs in the SRL group (p<0.05). Proportions were not significant but did trend towards significance in SRL compared to FK at 12 hrs (p=0.07). In conclusion, SRL was not associated with longer immunosuppression of T cells stimulated in-vitro compared to patients taking FK and CSA.
Allograft survival is prolonged in the absence of NKG2D (169.19)
Authors: Wai Lu-En , Wang Mouer , Piard-Ruster Karine , Martinez Olivia , Krams Sheri
Published: January 2023

NKG2D is expressed by Natural Killer (NK cells) and subsets of T cells. On NK cells, NKG2D functions as a stimulatory receptor that induces effector functions. We have demonstrated increased expression of NKG2D ligands on rejecting grafts and enhanced infiltration of NKG2D-expressing cells in allografts during rejection. To further examine the role of NKG2D in graft survival we utilized Klrk1 mice that are deficient for NKG2D as recipients of cardiac or small intestine allografts. Groups (n=4-6) of C57BL/6 (H-2b) or NKG2D deficient Klrk1 mice (H-2b) received heterotopic donor hearts or vascularized, orthotopic small intestine grafts from BALB/c (H-2d) or BALB/c x C57BL/6 F1 mice (H-2d/b). Some groups of mice also received 200μg (heart) or 500μg (intestine) anti-CD40L mAb (MR1) at transplant. Cardiac allograft survival was similar in wild-type and Klrk1 mice although intestinal allograft survival was prolonged in Klrk1 mice. Treatment with MR1 prolonged cardiac allograft survival that was further enhanced in the absence of NKG2D. Treatment with MR1 did not prolong survival of intestine allografts in wild-type recipients. Quite dramatically, however, treatment with MR1, significantly (p=0.007) prolonged intestinal allograft survival in Klrk1 mice. Prolongation of intestinal graft survival in the absence of NKG2D and CD40-CD40L interactions suggest that the addition of therapeutics to block NKG2D has great potential for improving costimulation blockade based therapies
Janus kinase 2 offers a precise therapeutic target against DC stimulated alloreactivity in transplantation (169.21)
Authors: Betts Brian , Abdel-Wahab Omar , Hanash Alan , St. Angelo Erin , van den Brink Marcel , Levine Ross , Young James
Published: January 2023

JAK2 is critical to cytokine signaling and has a specific role in mediating alloreactivity. We have demonstrated that specific JAK2 inhibition with TG101348 suppresses alloimmunity stimulated by human dendritic cells in vitro, while maintaining immunity against viral antigens. JAK2 inhibition also extends survival in a mouse graft-versus-host disease (GvHD) model. Methods: Major MHC-mismatch models were used in human and mouse experiments. Human moDC and moDC:T cell cultures were treated with either TG101348 (1uM) or vehicle. Mice underwent 850 cGy irradiation followed by T cell-depleted marrow and splenic T cells. TG101348 (60mg/kg) or vehicle was administered per oral gavage daily. Results: JAK2 inhibition reduced the expression of CD83 and CCR7 in 2-day cytokine-matured moDCs (p<0.05) and suppressed T cell proliferation in a 5-day alloMLR (p<0.005). JAK2 blockade preserved allogeneic Tregs (p=NS), and enhanced the Treg:CD8+CD25+ T cell ratio (p<0.05). T cells exposed to JAK2-inhibited DCs maintained tolerance to alloantigen restimulation, with preserved response to flu-loaded autologous moDCs. JAK2 inhibition delayed the onset of GvHD and improved survival in BALB/c mice that received B6 marrow and T cells (median survival JAK2 inhibitor 35 days vs vehicle 25 days, p<0.005). All mice engrafted. Conclusions: JAK2 offers a novel, specific therapeutic target against GvHD and allograft rejection by suppressing DC allostimulation of T cells, without broader immune impairment.
Expression of microRNA miR-182 is induced in alloimmune responses (169.23)
Authors: Wei Liang , Pham Betty , Wang Mouer , Harris Aleishia , Phillips Lori , Piard-Ruster Karine , Martinez Olivia , Krams Sheri
Published: January 2023

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by translational repression or mRNA degradation. We defined a profile of miRNAs expression during graft rejection and suggested that the miRNA miR-182, which is a member of the mir-183-miR-96-miR-182 cluster, is significantly increased in the allograft, graft infiltrating cells, spleen and peripheral blood lymphocytes during rejection. To further define the function of miR-182 in alloimmune responses, splenocytes isolated from C57BL/6 or BALB/c mice were used as responders or stimulators in a one-way MLR. Proliferation was measured by [3H]-thymidine uptake on days 0 to 7 and cells were collected at each time point and RNA isolated to detect kinetic expression of miR-182 and its potential targets. Expression of miR-182 increased specifically in allo MLR between days 2-3 and reached peak expression on day 5. Computational target prediction suggested the genes Foxo1, Rac1, Bcl2 and Grb2 as putative targets of miR-182. The four genes all decreased from days 4 to 6 specifically in the allo MLR group supporting that increased expression of miR-182 may inhibit expression of these genes. CONCLUSION. Our results suggest an important role of miR-182 in alloimmune responses. Further studies may result in novel therapeutics to control the proliferation of alloreactive cells.
NK cells mediate chronic kidney allograft injury (169.26)
Authors: Zhang Zhu-Xu , Jiang Jifu , Huang Xuyan , Yin Ziqin , Liu Weihua , Garcia Bertha , Jevnikar Anthony
Published: January 2023

Chronic allograft injury remains the leading cause of kidney graft loss after transplantation and no-specific therapy currently exist. We have recently demonstrated that NK cells can kill syngeneic tubular epithelial cells (TEC) and participate in kidney ischemia-reperfusion injury. In this study, we investigated the capacity of NK cells to mediate kidney injury in transplantion. C57BL/6 (B6, H-2b) kidneys were transplanted into nephrectomized F1 (B6 x BALB/c, CB6F1) mice. Serum creatinine levels increased from baseline (22+5 uM, n=8) to 38±6 uM (n=6, P<0.001) at 60 days post transplant, demonstrating a clear loss of kidney function. Infiltrates and low grade renal tubular cell injury were consistently present in B6-to-CB6F1transplants suggesting a non T cell pathway. This was supported by results using a B6-Rag-/--to- CB6F1Rag-/- (B6Rag-/-xBALB/cRag-/-) F1 kidney transplant that eliminates T and B cell but not NK cell participation. Similar levels of kidney dysfunction (creatinine: 34+8 uM, n=6, p<0.01) and histological injury were observed 65 days post transplant. Finally depletion of NK cells prevented kidney injury (25±6 vs 34+8 uM in no antibody injection, n=6, p<0.05) as well as improved histology. In conclusion, these data demonstrate for the first time a critical role for NK cells in mediating chronic kidney injury, which is independent of T and B cells. NK cells are newly appreciated and formidable effectors to chronic kidney injury and graft loss.
Regulatory T cells establish and maintain chimerism after in utero hematopoietic cell transplantation (169.30)
Authors: Nijagal Amar , Le Tom , Wegorzewska Marta , Tucker Andrew , Tang Qizhi , MacKenzie Tippi
Published: January 2023

Background: In utero hematopoietic cell transplantation (IUHCTx) is a promising strategy to treat congenital diseases and induce donor-specific tolerance. The role of regulatory T cells (Tregs) in the establishment and maintenance of chimerism after IUHCTx has not been established. We characterized the expansion of Tregs in a wildtype (wt) and antigen-specific setting after IUHCTx. Methods: We analyzed chimerism in BALB/c fetuses 5 weeks after allogeneic IUHCTx and characterized their T cell and APC subsets. To quantify antigen-specific Tregs, BALB/c cells were transplanted into T cell receptor transgenic fetuses that recognize BALB/c antigen via the direct (4C) or indirect (TCR75) pathway. Results: In wt animals, we found an equivalent percentage of Tregs among uninjected animals, chimeras, and injected non-chimeras. The percentage of activated CD44+ Tregs was significantly higher among chimeras compared to uninjected controls. APC maturation markers (B7 and CD40) were elevated in donor and host-derived pDC and B cells of chimeric animals. In both 4C and TCR75 recipients, chimeric mice showed deletion of T effector cells and expansion of Tregs in the thymus and spleen. 4C animals losing chimerism showed a decrease in the number of Tregs. Conclusions: Our data suggests that Tregs interact with both host and donor-derived APC and undergo activation. Successful Treg induction is important for establishing and maintaining chimerism after IUHCTx.
Critical role of STAT3 in IL-17-mediated survival of fibroblast-like synoviocytes in rheumatoid arthritis. (171.16)
Authors: Cho Mila , Lee Seon-Yeong , Son Hye-Jin , Ryu Jun-Geol , Kim Eun-Kyung , Kim Ho-Youn
Published: January 2023

Fibroblast-like synoviocytes (FLS) are major cell population of the pannus that invades adjacent cartilage and bone in rheumatoid arthritis (RA). The study was undertaken to determine the effect of interleukin-17 (IL-17) on the survival and/or proliferation of FLS from RA patients and to investigate whether signal tranducer and activator of transcription 3 (STAT3) is implicated in this process. The expressions of Bcl-2 and Bax in FLS were determined using real-time PCR and western blot analysis. The expression of CD55, Bcl-2, Bax, IL-17 and phosphoSTAT3 in synovial tissues were investigated by immunohistochemistry. Apoptosis of FLS was detected by Annexin V/propidium iodide staining and/or phase contrast microscopy. The proliferation of FLS was determined by CCK-8 ELISA assay. The pro-apoptotic Bax is decreased and anti-apoptotic Bcl-2 is increased in FLS from RA patients compared with in that from OA (Osteoarthritis) patients. IL-17 upregulates the expression of Bcl-2 in FLS from RA patients, not in FLS from OA patients. STAT3 was found to mediate IL-17-induced Bcl-2 upregulation in FLS from RA patients. In addition, IL-17 promotes the survival and proliferation of FLS from RA patients. Most importantly, treatment with STAT3 inhibitor reverses the protective effect of IL-17 on FLS apoptosis induced by SNP.
Macrophage scavenger receptor 1 (Msr1) promotes autoantibody-mediated arthritis by impairing B cell tolerance (171.18)
Authors: Haasken Stefanie , Mueller Daniel , Binstadt Bryce
Published: January 2023

Peripheral tolerance is achieved through several mechanisms, including induction of the hyporesponsive state of anergy in potentially autoreactive lymphocytes. The class A macrophage scavenger receptor, Msr1, is reported to participate in the maintenance of immunological tolerance by performing functions such as the clearance of apoptotic debris and the tonic sampling of self-antigens. We evaluated the role of Msr1 in a mouse model of autoantibody-dependent arthritis. Genetic deficiency of Msr1 in the K/BxN TCR transgenic model (Msr1-/- K/BxN) decreased the incidence and severity of arthritis. However, arthritis induced by transfer of arthritogenic autoantibodies was unimpaired in naïve mice lacking Msr1, suggesting that Msr1 deficiency in K/BxN mice affected the pathogenic events preceding autoantibody synthesis. Indeed, antibodies against the key autoantigen in this model, glucose 6-phosphate isomerase (GPI), were undetectable in Msr1-/- K/BxN mice, despite the presence of an expanded population of GPI-specific B cells. We did not observe any defect in T cell numbers or function. We hypothesized that Msr1 might control the availability of autoantigen and found that Msr1-/- macrophages displayed impaired uptake of GPI. Taken together, these data suggest a model in which deficiency of Msr1 can lead to a higher concentration of soluble autoantigens, resulting in B cell anergy and protection from autoimmune disease.
Complement Orchestrates T cell response during Mesenteric Ischemia Reperfusion Injury in Autoimmune Prone Mice. (171.22)
Authors: Ioannou Antonis , Lapchak Peter , Kannan Lakshmi , Rani Poonam , Dalle Lucca Jurandir , Lambris John , George Tsokos
Published: January 2023

Ischemia reperfusion injury is a type of injury seen in clinical conditions, such as trauma, hemorrhagic shock, organ transplantation, revascularization processes, and autoimmune diseases like systemic lupus erythematosus. MRL/lpr mice due to their autoimmune background present with accelerated mesenteric ischemia reperfusion injury (IR/I). To evaluate the effect of complement inhibition on tissue damage after mesenteric IR in MRL/lpr mice, we depleted complement using Cobra Venom factor (CVF) or blocked C5a receptor with a C5aR antagonist, and performed intestinal ischemia reperfusion. First, we confirmed that CVF treatment completely depleted C3 from the mice serum. Next, we found that the treatment with either CVF or C5aR antagonist attenuated intestinal injury following intestinal IR. Notably, depletion of complement increased the presence of IL-10 producing CD4+ cells (Tr1) and IL-10 producing Th17 cells in the intestine. These results suggest that complement may regulate the T-cell response during intestinal ischemia reperfusion injury.
Intersection of Complement C4 and Interferon Alpha pathways in B cell tolerance (171.32)
Authors: Chatterjee Priyadarshini , Agyemang Amma , Alimzhanov Marat , Degn Soren , Herbst Ronald , Tan Tze , Alicot Elizabeth , Carroll Michael
Published: January 2023

Humoral autoimmunity is an important hallmark of autoimmune diseases like lupus. Factors that allow self-reactive B cells to escape negative selection stages and become activated remain poorly defined. Using a novel B cell receptor knock-in mouse strain 564Igi, we define a complement-dependent pathway by which B cell tolerance is upheld. Interestingly, the 564-autoantibody could bind SSB/La, a common lupus antigen, establishing the physiological relevance of this model. Deficiency in complement C4 caused a breakdown in the elimination of autoreactive B cell clones at the transitional stage, as demonstrated by their continued maturation, their ability to respond to a range of stimuli, entry into follicles (lack of follicular exclusion) and formation of germinal centers with a higher propensity. All results taken together indicate that the complement system must be intact for B cell tolerance to be upheld. Strikingly, the dysregulation of autoreactive B cells in C4-deficient mice was not B cell intrinsic, as a C4-sufficient myeloid compartment could re-establish efficient B cell selection. Our current model holds that poor clearance of apoptotic debris in the absence of C4 chronically activates myeloid cells to release cytokines, like IFNα, in excess and reduce the stringency of tolerance at the transitional stage. In support of our hypothesis, IFNα receptor blockade was found to restore negative selection of B cells in C4-deficient 564Igi mice.
Breaking self-tolerance in MyD88-deficient mice following intracellular bacterial infection (171.35)
Authors: al-Ramadi Basel , Issac Jincy , Rabah Maryem , El-Ojali Samia , Fernandez-Cabezudo Maria , Conca Walter
Published: January 2023

Activation of the innate immune system is a prerequisite for the induction of adaptive immunity to infectious and non-infectious agents. Signaling through TLRs, crucial components of the innate immune system that recognize conserved microbial ligands, takes place mainly through MyD88 and lead to the activation of NF-κB and induction of inflammatory responses. Previously, we demonstrated that MyD88-deficient mice are extremely susceptible to infection by Salmonella typhimurium, succumbing to infection with doses 10,000-fold lower than the LD50 in wild-type counterparts. Curiously, despite their heightened susceptibilities, infected MyD88-/- mice produce elevated serum levels of anti-salmonella antibodies, not only of expected Th1-driven antibody isotypes (IgG2c and IgG3) but also of Th2-associated IgG2b and IgG1 isotypes, suggesting a dysregulation in antibody production. Interestingly, this phenotype was not observed in mice infected with other Gram-negative bacteria, including E. coli and Acinetobacter baumanii. In order to further test this hypothesis, sera from infected mice were tested for reactivity to self antigens by determining titers of anti dsDNA antibodies, thyroglobulin, RF and extent of reactivity to Hep2 cells. Our data revealed the existence of high levels of these autoantibodies in Salmonella-infected but not in normal MyD88-/- mice. These findings suggest that TLR-MyD88 signaling pathway may be critical for maintaining self tolerance in B lymphocytes.
Gene expression and sub-congenic analyses of the Idd9/11 locus that regulates the diabetogenic activity of CD4 T-cells in NOD mice (171.43)
Authors: Chen Yi-Guang , Jia Shuang , Kaldunski Mary , Serreze David , Hessner Martin
Published: January 2023

Despite sharing the H2g7 MHC, the closely NOD-related NOR strain remains type 1 diabetes (T1D) resistant due to the presence of protective Idd5.2, Idd9/11, and Idd13 loci. We previously showed that CD4-T cells isolated from the Idd9/11 congenic strain (NOD.Chr4NOR) were less diabetogenic than those from standard NOD mice. To further determine the underlying mechanism, we asked if CD4-T cells from the NOD.Chr4NOR strain have enhanced suppressive activity compared to those from NOD mice. We used a co-transfer system to show that CD4-T cells isolated from NOD.Chr4NOR but not standard NOD mice (both expressing CD45.1) could actively suppress the diabetogenic activity of those from CD45.2 congenic NOD mice. We further demonstrated that the T1D suppressive activity of the NOR-derived Idd9/11 region was completely lost in the CD28-deficient mice that had greatly reduced numbers of Foxp3+ regulatory T-cells (Tregs). These results suggest that an enhanced T1D suppressive Treg activity contributes to NOR Idd9/11-mediated disease resistance. We have also generated two subcongenic lines carrying distinct intervals of the NOR-derived Idd9/11 region. T1D incidence studies indicated that more than one gene within the NOR-derived Idd9/11 region conferred T1D resistance. The region within the Idd9/11 interval suppressing the diabetogenic activity of CD4 T-cells was further refined from 44.3Mb to 27.1Mb. Microarray gene expression analysis was used to identify Idd9/11 candidate genes.
Genetic control of antibody production during collagen induced arthritis development in heterogeneous stock mice (171.44)
Authors: Foerster Michael , Raposo Bruno , Ekman Diana , Klaczkowska Dorota , Popovic Marjan , Kutty Selva Nandakumar , Lindvall Therese , Hultqvist Malin , Teneva Ivanka , Johannesson Martina , Ahlqvist Emma , Holmdahl Rikard
Published: January 2023

The aim of this study was to identify genetic factors driving pathogenic autoantibody formation in collagen induced arthritis (CIA), a mouse model for rheumatoid arthritis (RA) in order to understand the etiology of the disease and improve possibilities for therapeutic intervention. To this end we made a genome wide analysis of quantitative trait loci (QTL) controlling autoantibodies towards type II collagen (AC2A), anti-citrullinated protein antibodies (ACPA) and rheumatoid factors (RF). To identify loci controlling autoantibody production, we induced CIA in a heterogeneous stock (HS) derived mouse cohort. Serum samples of 1843 mice were collected before onset and at peak of the disease. All antibody concentrations were measured by ELISA and linkage analysis was performed using a linear regression based method. We identified loci controlling formation of AC2A of different IgG isotypes, antibodies to major type II collagen (CII) epitopes, to a citrullinated CII peptide and RF. The AC2A, ACPA and RF responses were all found to be controlled by distinct genes, one of the most important loci being the Immunoglobulin heavy chain (IgH) locus. Here, we provide a comprehensive genetic analysis of autoantibody formation in CIA. Our study demonstrates that not only AC2A, but interestingly also ACPA and RF are associated with arthritis development in mice. These results underscore the importance of non-MHC genes controlling the formation of clinically relevant autoantibodies.