bluuebunny/crossref_metadata_2025 huggingface dataset:
A crucial role of IL-6 in B cell tolerance induction after antigen activation (167.6)
Authors: Yan Yi , Diamond Betty
Published: January 2023

Receptor editing, clonal deletion and anergy are the key components of the central tolerance. It is well characterized that receptor editing plays the most important role among the three mechanisms. We recently identified a post germinal center tolerance check point, where receptor editing is re-induced to diminish the autoreactive B cells generated by class switching and somatic hypermutation. Here we demonstrate that this de-novo re-induction of the recombinase RAG gene in antigen-activated B cells is reduced by neutralization of IL-6. Serum level of autoreactive anti-dsDNA antibodies is increased in mice immunized with a peptide mimetope of dsDNA if they are given antibodies to IL-6 to reduce receptor editing. The same effect is observed in mice haploid for IL-6 when compared to WT mice. This dependence on IL-6 is B cell intrinsic, as demonstrated in adoptive transfer experiments when B cells from either WT mice or IL-6+/- mice were reconstituted in uMT mice. Our data reveal a novel but crucial role of IL-6 in regulation of the B cell autoimmunity, and show that Il-6 serves a regulatory function as well as being proinflammatory.
Estrogen upregulates cyclic AMP response element modulator alpha expression and downregulates interleukin-2 production by human T lymphocytes (167.8)
Authors: Moulton Vaishali , Holcomb Dana , Tsokos George
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease with a complex multifactorial etiopathogenesis and primarily afflicts women. T lymphocytes play a critical role in disease pathogenesis and display abnormal gene expression and function. A major defect is their inability to produce the vital cytokine interleukin-2 (IL-2) due to defects in IL2 transcription. We previously showed that the expression of the transcriptional repressor cyclic AMP response element modulator alpha (CREMα) is increased in SLE T cells, and contributes to reduced IL-2 production. While estrogen is implicated in the onset and exacerbation of SLE, the precise nature of molecular events regulated by estrogen in immune cell function is not well understood. Here we asked whether estrogen regulates the expression of CREMα in human T lymphocytes. We show that exposure of human T cells to 17- β-estradiol leads to increased CREMα mRNA expression, and this increase appears to be estrogen receptor dependent. We show that the increased expression of CREMα is due to increased transcriptional activity of the CREM promoter, and is mediated by increased expression and binding of the Sp1 transcriptional activator. We further show that estrogen treatment leads to decreased IL-2 production by T cells. Our results show that estrogen can modulate the expression of CREMα and lead to IL-2 suppression in human T lymphocytes, thus revealing a molecular link between hormones and the immune system in SLE.
Foxp3+ regulatory T cells promote Th17 development in vivo through regulation of IL-2 (168.16)
Authors: McGeachy Mandy , Cua Daniel , Hammerling Gunther , Li Ming , Hochweller Kristin , Gutcher Ilona , Haines Christopher , Chen Yi
Published: January 2023

Th17 development is driven by multiple cytokines, including TGF-β. Regulatory T cells (Tregs) promote Th17 development through production of TGF-β in vitro. However their role in vivo remains unclear, particularly since Tregs inhibit inflammation in many models of autoimmunity where Th17 cells are active. Using mice expressing Diphtheria toxin receptor under control of the Foxp3 promoter, Foxp3+ Tregs were depleted in adult mice during in vivo Th17 priming. Treg depletion resulted in a reduced frequency of antigen-specific IL-17 producers in draining lymph nodes and blood, correlating with reduced inflammatory skin responses. In contrast, Tregs did not promote IL-17 after initial activation stages. Treg production of TGF-β was not required for Th17 promotion, neither was suppression of Th1-associated cytokines. Rather, regulation of IL-2 availability and resultant signaling through CD25 and pSTAT5 by Tregs was found to play an important role.
A role of TGFβ in p53-induced CD28-dependent T cell apoptosis (168.18)
Authors: Takami Mariko , Iwashima Makio
Published: January 2023

Naturally arising CD4+CD25+ Tregs (nTregs) are essential for maintaining immune homeostasis and peripheral tolerance. The balance between nTregs and conventional T cells appears to play a significant role in immune regulation. The majority of nTregs are considered to recognize antigen peptides derived from self tissues and are predicted to receive continuous antigen stimulation, but regulation of Treg growth and survival against activation-induced cell death (AICD) in vivo is not well-understood. Recently, we reported a novel form of apoptosis (p53-dependent CD28- induced T cell apoptosis: PICA) which is distinguishable from classical AICD. We demonstrated that conventional T cells undergo massive p53- induced apoptosis when stimulated by anti-CD3 and anti-CD28 antibodies coated on flat surface. Most importantly, nTregs survive and expand effectively under the same culture conditions, showing that there is a different survival/ apoptosis mechanism between nTregs and non-Tregs in regards to PICA. Here, we report that transforming growth factor β (TGFβ) signaling is required for survival of Tregs against PICA and the presence of the exogenous active form of TGFβ renders conventional T cells resistant to PICA. TGFβ-mediated survival of nTregs is in part mediated by downmodulation of a pro-apoptotic protein, Bim, by a posttranscriptional mechanism. Together, the data show that TGFβ plays a critical role in PICA and may contribute to the maintenance of Tregs: non-Tregs balance.
Interleukin-17 overcomes inhibition of Th1 immune responses following mycobacterial exposure (168.2)
Authors: Gopal Radha , Lin Yin-Yao , Obermajer Nataša , Kalinski Pawel , Khader Shabaana
Published: January 2023

Tuberculosis (TB) caused by the intracellular pathogen Mycobacterium tuberculosis, remains a worldwide health problem. Generation of effective T helper cell type 1 (Th1) responses are required for immunity against intracellular bacteria. However, multiple bacteria successfully inhibit the direct pathway of induction of Th1 responses, promoting the production of factors such as prostaglandin E2 (PGE2) and interleukin (IL-10), that suppress IL-12 and interferon gamma (IFNγ) production. Here, in a model of Mycobacterium bovis Bacille Calmette Guerin (BCG) vaccination, we demonstrate that a nominal Th1-suppresive factor, PGE2, can promote an alternative pathway of induction of Th1 immunity that is mediated by IL-23-dependent IL-17 induction. We show that PGE2 limits early Th1 responses via IL-10 production but also drives Th17 responses via IL-23 production. IL-17 then functions to overcome PGE2-IL-10-mediated Th1 inhibition and drive Th1 immunity following BCG exposure. Therefore, we demonstrate that PGE2-induced IL-17 response allows overcoming BCG-induced PGE2- and IL-10-mediated inhibition of IL-12 production and Th1 responses. The currently described alternative IL-17-dependent pathway of induction of Th1 immunity helps to understand the complex role of PGE2 in the regulation of immune responses and helps to design new therapeutic approaches in chronic intracellular infections.
Neuropilin-1 is a potent negative regulator of CD4 T cell responses (168.21)
Authors: Bynoe Margaret , Solomon Benjamin , Mueller Cynthia , Chae Wook-Jin , Alabanza Leah
Published: January 2023

Neuropilin-1 (Nrp1) is a type-1 transmembrane cell surface molecule originally identified for its role in neuronal development. We have found that Nrp1 is highly expressed on CD4 T cells from mice that had been epicutaneously immunized with a myelin antigen, myelinoligodendrocyte glycoprotein (MOG), and confer dominant tolerance against experimental autoimmune encephalitis (EAE, the animal model for multiple sclerosis). Recently, Nrp1 has been implicated in several aspects of immune function including maintenance of the immune synapse and development of regulatory T cells (Treg). Here, we identify Nrp1 as a critical negative regulator of CD4 T cell effector function. We demonstrate that CD4 T cells lacking Nrp1 (via conditional deletion) exhibit preferential Th17 lineage commitment, are hyperproliferative and produce high levels of proinflammatory cytokines. Mice with conditional deletion of Nrp1 in CD4 T cells develop fulminant EAE that can be inhibited by blocking Th17 cell development. Conversely, mice receiving CD4 T cells that are retrovirally transduced with Nrp1 do not develop EAE. Further, Nrp1-expressing CD4 T cells are potent suppressors of CD4 T cell effector function. This suppression is independent of FoxP3 and can be inhibited by blockade of TGF-beta but not by IL-10 blockade. These findings are the first to demonstrate a specific regulatory role for Nrp1 in immune regulation and provide the impetus for the use of Nrp1 as a therapeutic immune modulator.
Regulatory T cells suppress immune activation under homeostatic conditions in adult mice by antigen-independent and -dependent mechanisms (168.22)
Authors: Punkosdy George , Shevach Ethan
Published: January 2023

Regulatory T cells (Treg) maintain immune homeostasis by preventing excessive immune activation. This is clear in neonates since mice lacking Treg (scurfy) develop multi-organ autoimmunity early in life. However, the mechanism by which Treg maintain homeostasis in adults is less clear. We show, using “depletion of Treg” (DEREG) mice, that Treg depletion in adult mice results in the immediate acquisition of a CD44hi phenotype on the vast majority of conventional (CD4+Foxp3-) T cells. CD44 expression is a characteristic of newly activated as well as memory phenotype (MP) T cells, so we sought to determine whether the increase in CD44hi cells was the result of naïve cell activation or expansion of MP cells. The proliferative rate of MP cells increased dramatically during the 72-hour period following Treg depletion as determined by Ki-67 positivity and BrdU incorporation. This expansion was not inhibited by the administration of anti-MHC Class II antibodies, suggesting that the mechanism was antigen-independent. Naïve T cells also developed a CD44hi phenotype following Treg depletion but via an antigen-dependent mechanism. Activation of OT-II T cells transferred into DEREG-RIP/OVAlo mice was markedly enhanced by Treg depletion while transfer into wild-type DEREG mice did not result in activation regardless of Treg depletion. Collectively, these results demonstrate that Treg control immune activation under homeostatic conditions by antigen-independent and -dependent mechanisms.
B7-CTLA4 reverse signaling is responsible for regulatory T cell-induced suppression of dendritic cells (168.23)
Authors: Kowalczyk Aleksandra , Zhang Li
Published: January 2023

CD4+CD25+Foxp3+ regulatory T cells (Treg) are essential for maintaining immune tolerance and homeostasis. It has been shown that natural Treg can inhibit up-regulation of costimulatory molecules on dendritic cells (DC). However, the mechanism leading to this phenomenon has not been elucidated. Whether inducible Treg (iTreg) also possess this function has not been previously investigated. Here, we demonstrate that iTreg could inhibit LPS-induced DC maturation and mediate down-regulation of CD80 and CD86 on mature DC. This process is CTLA4-dependent as Treg isolated from CTLA4-/- mice did not down-modulate B7 molecules on DC. Furthermore, CTLA4Ig, but not CD28Ig, mimicked iTreg action. Down-regulation of CD80/CD86 was not due to endocytosis or protease-dependent shedding, but rather to a reverse signaling through B7. CTLA4Ig treatment decreased CD80 and CD86 transcription, as well as NFκB activity upon LPS stimulation. Interestingly, both CTLA4Ig and co-incubation with iTreg induced a profound STAT3 phosphorylation in DC, whereas no significant effect on the proximal NFκB pathway was observed. Inhibition of STAT3 activation partially abolished CTLA4Ig-induced CD80/CD86 down-modulation suggesting involvement of this molecule in the observed process. Our data reveal a novel signaling pathway by which Treg can suppress DC via reverse signaling, and may provide new opportunities for therapies targeting costimulatory pathways.
The regulatory function of lpr TCRαβ+ CD4-CD8- double negative T cells requires autocrine interferon gamma (168.24)
Authors: Juvet Stephen , Han Mei , Kim Edward , Vanama Ramesh , Joe Betty , Jeon Caroline , Zhao Fei , Amash Alaa , Adeyi Oyedele , Zhang Li
Published: January 2023

Mice and humans with Fas mutations develop lymphoproliferative (lpr) disease and accumulate a large number of lymphocytes including TCRαβ+ CD4-CD8- non-NK double negative (DN) T cells. In these settings, CD4+ T cells and B cells are implicated in causing lupus-like autoimmunity; in contrast, the function of the DN T cells is largely unknown. DN T cells in other situations have been shown to secrete interferon gamma (IFNγ) and possess FasL-dependent regulatory functions. Therefore, we sought to determine whether lpr DN T cells can exert regulatory effects on syngeneic T cells and to what extent these phenomena might depend on IFNγ secretion. We found that lpr DN T cells produce IFNγ after antigen stimulation and can suppress and kill syngeneic Fas+ CD4+ T cells in responding to alloantigens. Lpr DN T cells deficient in either IFNγ or its receptor exhibited reduced suppressive and cytotoxic function in vitro and a decreased ability to suppress CD4+ T cells in vivo during graft-versus-host disease (GVHD). FasL-deficient DN T cells from gld mice also failed to suppress GVHD. These findings demonstrate that lpr DN T cells are able to suppress Fas+ syngeneic CD4+ T cells in vitro and in vivo. Expression of both FasL and the IFNγ receptor on lpr DN T cells is critical to their regulatory function. These studies have revealed a previously unrecognized IFNγ-IFNγ receptor autocrine loop that is central to DN T cell mediated immune suppression.
Differential gene expression and suppressive properties of ex vivo-generated vs. naturally-occurring regulatory T-cells (168.25)
Authors: Karlsson Fridrik , Gray Laura , Zhang Songlin , Grisham Matthew
Published: January 2023

We have recently reported a novel ex vivo method to convert large numbers of CD4+Foxp3- T-cells to induced regulatory T-cells (iTregs; CD4+Foxp3+) via their incubation with plate-bound CD3 mAb, IL-2, TGFβ and retinoic acid (RA) for 4 days. These iTregs possess enhanced immune-suppressive activity in vitro and in vivo when compared to naturally-occurring Tregs (nTregs; CD4+Foxp3+). The objectives of this study were to: a) quantify and compare mRNA transcripts for genes known to be associated with Treg function using flow-purified iTregs and nTregs obtained from Foxp3GFP reporter mice and b) evaluate the stability of iTreg-mediated suppression in vitro and in vivo. We found 8 genes associated with iTregs that were significantly up-regulated when compared with nTregs with CCL4 (132-fold), Granzyme A (18-fold) and Runx1 (14-fold) showing the most dramatic increases. We also observed significant downregulation of 19, Treg-associated genes with IL-18 (600-fold), CD7 (300-fold), and IL-7 (100-fold) showing the most dramatic reductions in iTregs vs. nTregs. In addition, we found that when ex vivo-generated iTregs were “rested” by culturing iTregs for an additional 3 days in the absence of CD3 mAb, TGFβ and RA but in the presence of IL-2, Foxp3 expression, proliferative activity and in vitro suppressive activity were maintained. Unexpectedly, we found that these cells were more potent than nTregs in reversing preexisting colitis in a mouse model of chronic gut inflammation.
BCL6 in control of the Th2 inflammatory activity of regulatory T cells (168.3)
Authors: Sawant Deepali , Sehra Sarita , Kaplan Mark , Dent Alexander
Published: January 2023

The transcriptional repressor Bcl6 is a critical arbiter of T helper cell fate, promoting the follicular helper (Tfh) lineage while repressing other T helper cell lineages. Bcl6-deficient (Bcl6-/-) mice develop a spontaneous and severe Th2-type inflammatory disease early in life, thus warranting assessment of the role of Bcl6 in Treg cell function. Bcl6-/- Tregs displayed enhanced suppressor function in vitro and were competent at suppressing Th1-mediated colitogenic inflammation in vivo. In contrast, Bcl6-/- Treg cells were uniquely defective in their ability to control Th2-type airway inflammation, and in fact, actively exacerbated the disease. Bcl6 deficiency promoted the expression of Th2 cytokines by Tregs, in part due to increased transcriptional activity of Gata3, and also due to increased expression of microRNA-21 (miR-21). Ectopic expression of miR-21 in naïve T cells in vitro induced polarization towards Th2, while antagomir-mediated miR-21 repression was able to specifically suppress Th2 differentiation. Our results define novel roles for Bcl6 in regulating Treg gene expression and in dictating the ability of Treg cells to control Th2 inflammation. Thus, Bcl6 represents a crucial regulatory layer in the Treg functional program.
Column-free isolation of untouched CD4+CD127lowCD49d- human regulatory T cells in 45 minutes (168.4)
Authors: Kokaji Andy , MacDonald G. , Thomas Terry , Fairhurst Maureen
Published: January 2023

Regulatory T cells (Tregs) are a subset of lymphocytes that play a key role in maintaining immune homeostasis. The isolation of highly purified Tregs is essential for advancing this exciting field of research. FOXP3 remains the best marker for the identification of Tregs, but its intracellular localization currently precludes its use for the isolation of viable human Tregs. To overcome these technical difficulties, researchers have exploited the expression of other surface markers to isolate Treg populations. Typically, the expression of CD127 inversely correlates with FOXP3 expression, while CD49d, is expressed on the majority of pro-inflammatory effector cells but is absent on Tregs. Removal of CD127high cells from the CD4+ T-cell population enriches for Tregs with high levels of FOXP3 expression. Additional depletion of CD49d expressing cells further removes contaminating IFNγ and IL-17 secreting cells. Together this strategy permits selective isolation of highly enriched Tregs. We have developed a new kit for the negative enrichment of highly purified human Tregs. Our new EasySep™ CD4+CD127lowCD49d- kit isolates untouched Tregs by by column-free magnetic separation in only 45 minutes. Mean CD4+ T-cell purity is 94.3% +/- 5.5% of which 65.5% +/- 11.7% express high levels of FOXP3 (n=7). This new product will add to the array of powerful yet convenient tools available for the isolation of functional highly purified human Tregs.
Unique function of B cells in immune regulation through CD86-mediated maintenance of Foxp3 (168.6)
Authors: Ray Avijit , Basu Sreemanti , Dittel Bonnie
Published: January 2023

In recent years, B cells have emerged as an important player in the immune regulatory network. Using the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we observed a lack of resolution of disease in B cell-deficient (μMT) mice. Whereas, their WT counterparts recovered, indicating a regulatory role for B cells in central nervous system (CNS) autoimmunity. CD4+Foxp3+ T regulatory cells (Tregs) also mediate resolution of EAE. Absence of CD80/CD86 (B7) on B cells correlated with late emergence of Tregs in the CNS during EAE, suggesting an interaction between B cells and Tregs through B7 molecules. Anti-CD20 mediated B cell depletion disrupted this interaction resulting in significant reduction in the number of Tregs and like μMT, manifested in unresolved EAE. Interestingly, in vitro studies identified a unique role for B cells in maintaining Treg Foxp3 expression, which was enhanced upon CD86 upregulation following anti-IgM activation. Adoptive transfer of B cells into μMT mice resulted in partial restoration of Treg numbers leading to resolution of EAE. When deficient in CD86, B cells had a significantly reduced ability to restore Treg numbers in μMT. These data identify a distinct role for B cells in immune regulation through CD86-mediated interaction with Tregs and maintenance of Foxp3. This novel immune regulatory property of B cells may be harnessed to develop therapies for autoimmune diseases.
Evidence for NK cell subset licensing in anti-viral and anti-leukemic responses after syngeneic hematopoietic stem cell transplantation in mice (169.10)
Authors: Sungur Can , Alvarez Maite , Tang-Feldman Yajarayma , Lanier Lewis , Pomeroy Claire , Murphy William
Published: January 2023

Natural killer (NK) cells are an initial defense against cancers and viruses and can be classified into subsets based on their expression of receptors. These subsets are considered “licensed” if inhibitory receptors are capable of binding host MHC I and “unlicensed” if not. Since in vivo data on licensing and subsets are limited, our studies aim to show licensing in tumor and viral models as well as elucidate the roles of NK subsets in a post-hematopoietic stem cell transplantation (HSCT) setting. We have recently observed that cells bearing the Ly49G2 inhibitory receptor predominated post-HSCT regardless of MHC I haplotype. Additionally, studies in resting mice indicated that the unlicensed subset is the major mediator of murine cytomegalovirus (MCMV) protection. Surprisingly, we have found that depleting unlicensed, Ly49G2+, NK cells from leukemia (C1498) bearing, H2b strain mice after syngeneic HSCT significantly improved survival of mice, while depleting the licensed subset, Ly49C/I+, decreased survival. Furthermore, depletion of the Ly49C/I+ subset resulted in significantly increased viral loads in the liver, spleen, and salivary glands in H2b strain mice infected with MCMV 14 days post-HSCT. Conversely, depletion of the unlicensed ly49G2+ subset resulted in only a minor increase in viral load. Collectively, these data support the importance of NK licensing in tumor and viral models post-HSCT and suggest a regulatory role for the unlicensed subsets.
Graft versus host disease is associated with overproduction of TNFα and not with interleukin 17 (169.14)
Authors: Moreno-Lafont Martha , Gutiérrez-Hoya Adriana , Montiel-Cervantes Laura , Vela-Ojeda Jorge , Rodríguez-Cortés Octavio , López-Santiago Rubén
Published: January 2023

Allogeneic transplantation of hematopoietic cells (HCT) is a therapy used to treat a number of malignant and nonmalignant diseases, including acute and chronic leukemia, myelomas, lymphomas, aplastic anemia, solid tumors and severe immunodeficiencies. Unfortunately, a main complication of this treatment is the development of graft versus host disease (GVHD). GVHD is characterized by events caused by the reaction of immunocompetent cells from the graft against host tissues, developing an inflammatory reaction. In this work we studied 6 patients undergoing HCT, and one patient undergoing syngeneic transplantation of hematopoietic stem cells; 6 healthy donors were included as controls. In contrast to studies in mice showing that proinflammatory cytokine Th17 correlated with the occurrence and severity of GVHD, we did not observe overproduction of IL-17 in any patient that developed GVHD, in comparison to IL-17 production in controls and before transplantation. Furthermore, patients whose lymphocytes did not respond to polyclonal activators were of poor prognosis, correlating with graft loss, infections by opportunistic microorganisms and relapse. Remarkably, patients with GVHD showed exacerbated TNFα production, which correlated with the occurrence and severity of GVHD. These data show the importance of the TNFα and its contribution to the widespread damage experienced by patients who develop GVHD, and the importance of the use of specific immunosuppression of TNFα.
Pretransplant conditioning is required for minor but not major mismatch graft-versus-host disease: a vital role for intestinal damage (169.15)
Authors: Nalle Sam , Joseph Nora , Savage Peter , Turner Jerrold
Published: January 2023

Graft-versus-host disease (GVHD) is a major complication of allogeneic bone marrow transplantation (BMT). Previous studies suggest that the magnitude of the pre-BMT conditioning influences the severity of GVHD. To test this, we developed minor mismatch (129→B6) and major mismatch (Balb/c→B6) models of GVHD. In lethally irradiated WT recipients, major mismatch caused severe GVHD that was fatal in all recipients by 24 days after BMT. Minor mismatch led to a milder GVHD, where 50% of recipients survived to 35 days after BMT. We examined the role of irradiation in these models by minor or major mismatch adoptive transfer (AT) into unconditioned immunodeficient recipients. Major mismatch AT alone resulted in prominent GVHD. In contrast, minor mismatch AT required irradiation conditioning to cause GVHD. Conditioning and minor mismatch AT lead to elevated IFNγ, TNF, and IL-6, and an increased ability of donor cells to kill allogeneic targets in vivo (63±7% vs. 26±6%) compared to AT alone. As the intestine is very sensitive to irradiation, it was possible intestinal damage from the irradiation conditioning was facilitating minor mismatch GVHD. When we induced transient colonic damage with dextran sodium sulfate prior to minor mismatch AT, we observed systemic target organ histopathology characteristic of GVHD. Thus, we conclude that pretransplant conditioning, particularly intestinal damage, is required for minor mismatch GVHD.
IL-17 expression is inversely correlated with luminal narrowing in transplant vasculopathy: potential involvement of eNOS induction in vascular endothelial cells (169.20)
Authors: Choy Jonathan , Enns Winnie , Lee Martin , Escalante Nichole , von Rossum Anna , McManus Bruce
Published: January 2023

The role of IL-17 in transplant vasculopathy (TV), which is a leading reflection of chronic allograft rejection, remains poorly defined. The pathogenesis of TV involves luminal narrowing of allograft arteries caused by immune-driven thickening of the intima as well as insufficient compensatory vasodilation or remodeling. The vasodilatory process involves reduced expression and/or activity of the nitric oxide-producing enzyme eNOS by the endothelium. We examined the expression of IL-17 by immunohistochemistry in formalin-fixed, paraffin-embedded human specimens of TV (n=8). IL-17 was expressed by infiltrating leukocytes in the intima of arteries with TV and the majority of IL-17-positive cells were T cells (67.2 ± 11.0% of IL-17A-positive cells were also CD3-positive). Unexpectedly, the number of IL-17-positive cells (when normalized to total T cells) was inversely correlated with the amount of luminal narrowing (Pearson’s r = -0.709, p<0.05), but was not correlated with the intima/media ratio (r = 0.069, p = NS). Because our findings suggest that IL-17 may be involved in compensatory vasodilation, the effect of IL-17 on eNOS expression was examined. IL-17 dose-dependently increased eNOS protein expression in cultured endothelial cells, as determined by Western blotting. These findings suggest that IL-17 may limit luminal narrowing in arteries with TV by contributing to vasodilation through induction of eNOS expression in the endothelium.
Acute rejection results in a unique signature of microRNAs (169.22)
Authors: Wei Liang , Wang Mouer , Harris Aleishia , Pham Betty , Martinez Olivia , Krams Sheri
Published: January 2023

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate the post-transcriptional expression of genes. Little is known about the roles of specific miRNAs in alloimmune responses. Groups of syngeneic and allogeneic recipients of heart transplants were sacrificed on day 7 post-transplant and heart grafts and spleens obtained. Graft infiltrating lymphocytes (GIL) and peripheral blood lymphocytes (PBL) were isolated on post-transplant day 5. MicroRNAS were profiled using TaqMan microfluidic cards. Forty-two miRNAs were increased and 32 were decreased (p<0.05) in allogeneic heart grafts as compared to syngeneic. To determine if these miRNAs were expressed by lymphocytes or heart tissue, we profiled the GIL and determined that 22 miRNAs were increased and 52 decreased. There was less differential expression seen in spleen and PBL with 11 and 9 miRNAs changed respectively. Seventeen miRNAs were selected for validation by qPCR. The miRNAs, miR-7a and miR-182 were significantly increased in the heart graft, GIL, and PBL indicating that acute rejection results in specific profile of miRNAs in both the graft and the periphery. Further studies analyzing the targets of the miRNAs associated with graft rejection may lead to novel therapeutics and diagnostics.
Heme-oxygenase-1 in host tissues prevents visceral GVHD by regulating donor T cell expansion. (169.24)
Authors: Varelias Antiopi , Kreijveld Ellen , Olver Stuart , Koyama Motoko , Robb Renee , Raffelt Neil , Wilson Yana , Kuns Rachel , Don Alistair , Markey Kate , Anderson Greg , Clouston Andrew , MacDonald Kelli , Hill Geoff
Published: January 2023

Heme oxygenase-1 (HO-1), a ubiquitously expressed enzyme that degrades heme, has anti-inflammatory, anti-apoptotic and anti-proliferative actions yet its role in alloreactivity is unclear. Previously we demonstrated that HO-1 mRNA levels were elevated in recipient tissues of IFNγR-/- animals resistant to acute GVHD of the GI tract. Here we demonstrate that HO-1 expression in host tissues is critical for the suppression of acute GVHD as B6.HO-1-/- recipients of allogeneic BALB/c.WT grafts developed severe acute GVHD with rapid mortality (0% survival at d10) while WT mice survived longer term (median survival 42d). This was T cell mediated since mice transplanted with T cell depleted grafts showed no evidence of acute GVHD. Histological analysis revealed B6.HO-1-/- recipients developed severe acute GVHD of the GI tract and liver. TNF and IFNγ were elevated in the sera of B6.HO-1-/- recipients and systemic IFNγ but not TNF neutralization prevented hyperacute gut GVHD. The transplantation of grafts containing luciferase+ donor T cells demonstrated increased bioluminence signals in the mesenteric lymph node, spleen and GI tract of HO-1-/- recipients. Furthermore, the absolute number of donor CD4+ and CD8+ T cells co-producing the pro-inflammatory cytokines TNF and IFNγ were increased early after BMT in the lymph nodes of B6.HO-1-/- recipients, demonstrating that HO-1 within recipient tissue controls the acquisition and expansion of donor T cell effector function after BMT.
SRL-derived Tregs blocking alloresponses, but its generation is impaired by inflammatory cytokines (169.29)
Authors: Xu Luting , La Monica Giovanna , Leventhal Joseph , Gallon Lorenzo
Published: January 2023

Subpopulations of T cells play important roles in promoting transplant rejection or in maintaining transplantation tolerance. Little is known about the impact of immunosuppressive drugs calcineurin inhibitor Tacrolimus (Tac) and mTOR inhibitor Sirolimus (SRL) on the generation/expansion and functions of allospecific T cell subpopulations. Alloreactive CD4 T cells generated with naive T cells and allogeneic APCs were re-stimulated with autologous APCs and anti-CD3 in the presence of SRL/Tac and cytokines. Tac significantly blocked the productions of IFNgamma (Th1) and IL-17 (Th17) while SRL had minimal effect. FOXP3 expression (Treg) was markedly increased in SRL compared to Tac. These also confirmed at transcriptional levels by determining the T-bet, RORgt, and FOXP3 mRNA expressions. When used together, Tac at 2-5ng/ml with SRL at 2.5-10ng/ml achieved the maximal effect in inhibiting the productions of IFNgamma and IL-17 while maintaining a high level of FOXP3 expression. SRL-derived Tregs expressed normal Treg surface markers, were anergic to allostimulation, and functioned normally and specifically suppressed proliferation of allogenic effector T cells and Th1 and Th17 responses. When TNFalpha, IL-6, IL-1beta were added with SRL, there was a markedly decrease of SRL-induced Tregs. SRL significantly decreased DNA methyltransferase1, and thus increased stability of FOXP3 expression, which in part accounted for increased Treg conversion in alloreactive CD4 T cells.
Regulatory T cell therapy combined with donor-specific T cell deletion promotes long-term survival of islet allografts (169.32)
Authors: Lee Karim , Nguyen Vinh , Miao Gang , Scholz Hanne , Brennan Todd , Kang Sang-Mo , Tang Qizhi
Published: January 2023

Although Tregs are vital for peripheral tolerance, adoptive transfer of Tregs is insufficient in inducing long-term allograft survival in lymphoreplete hosts without preconditioning or adjunct immunosuppression. We hypothesize that reducing donor-reactive T cells may create a therapeutic window for Tregs to induce allograft tolerance without additional immunosuppression. We preconditioned recipient mice using donor-specific transfusion followed by cyclophosphamide treatment to selectively kill proliferating donor-reactive cells. This approach deleted 70-90% donor-reactive T cells, but failed to prolong islet allograft survival. Addition of Tregs with direct donor reactivity prior to transplantation led to long-term survival of BALB/c islets in 70% of C57BL/6 recipients. Equal numbers of polyclonal Tregs significantly prolonged graft survival, but 90% of the recipients rejected their grafts by 100 days. Increasing the numbers of polyclonal Tregs was able to confer long-term graft protection. Similar strategy of combining donor-reactive T cell deletion and polyclonal Tregs did not protect C3H islets in autoimmune NOD recipients. Further addition of Tregs specific for islet autoantigen was required to prolong graft survival in NOD mice. Together, these results demonstrate a clinically viable approach to harness the tolerogenic property of Tregs for inducing long-term allograft acceptance without additional immunosuppression.
Inhibition of Activated Protein C Attenuated Experimental Autoimmune Encephalomyelitis (171.13)
Authors: Alabanza Leah , Bynoe Margaret
Published: January 2023

Activated Protein C (APC) is an anticoagulant that is involved in the complex two-way interactions between the coagulation and immune systems. APC is widely known to be anti-inflammatory; most notably it has been clinically developed for treatment of severe sepsis. The anti-inflammatory properties of APC include conferring endothelial barrier protection, thus limiting the extravasation of immune cells into tissues. We investigated whether APC can affect the progression of experimental autoimmune encephalomyelitis (EAE), the mouse model for multiple sclerosis. The major pathological component of EAE is blood brain barrier (BBB) dysfunction resulting in the extravasation of autoreactive immune cells into the central nervous system (CNS). We observed that inhibition of endogenous APC during EAE resulted in considerable immune cell infiltration in the brain. Interestingly, however, these mice exhibited mild to no outward signs of EAE as assessed by paralysis severity. The anti-APC treated mice also had significantly higher percentage of CD4+Foxp3+ cells both in the CNS and in the periphery compared to controls. Moreover, the frequency of cells characterized as myeloid derived suppressor cells are increased in anti-APC treated EAE mice. Our study shows that lowered APC levels during EAE can lead to divergent effects on EAE pathogenesis, specifically leading to increased BBB dysfunction while simultaneously inducing immunological conditions that are non-pathogenic in EAE.
IFNβ promotes the conversion of pro-inflammatory M1-like into anti-inflammatory M2-like microglia (171.14)
Authors: Yen Jui-Hung , Ganea Doina
Published: January 2023

IFNβ is an approved therapeutic option for the treatment of certain autoimmune diseases such as MS/EAE. However, the molecular mechanisms underlying the effects of IFNβ in MS/EAE are not fully understood. In this study, we identified a novel mechanism for the beneficial effect of IFNβ in MS/EAE through the conversion of microglia (MG) from pro-inflammatory M1-like (MG1) to anti-inflammatory M2-like (MG2). Our results show that IFNβ inhibits expression and production of proinflammatory M1-type cytokines, such as IL-12p40, IL-23 and TNFα and promotes the anti-inflammatory M2 cytokine IL-10 in LPS-stimulated primary MG. In addition, IFNβ treatment also enhanced the expression of specific M2 makers, such as arginase and YM-1. Through this phenotype switch, IFNβ-treated MG suppressed Th1 and Th17 activation leading to the downregulation of IFNγ and IL-17. In vivo, IFNβ-treated EAE mice exhibit lower clinical scores, reduced Iba-1, IFNγ and IL-17 expression, and increased expression of the M2 markers, arginase, YM-1 and IL-10 in the CNS. Taken altogether, our results demonstrate that IFNβ favors the generation of MG2 leading to the secretion of the anti-inflammatory cytokine IL-10 and reduced expression of the pro-inflammatory cytokines, IL-12, IL-23, IFNγ and IL-17 in vitro and in vivo.
Kinetics of knee synovial lavage inflammation in rodent models of collagen induced arthritis. (171.15)
Authors: Goess Christian , McCarthy Richard , Rundell Lian , Mathieu Suzanne , Wu Fei , Cuff Carolyn , Long Andrew
Published: January 2023

Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation, swelling, and destruction of joints. In this study we used intra-articular lavage of the knee joint to easily and rapidly assess the kinetics of cellular infiltration and cytokine production within the local joint synovial environment during disease in rodent models of arthritis. In collagen induced arthritis in rats, knees of arthritic animals had increased cellularity composed of neutrophils, monocytes and T cells as well as marked increases in pro-inflammatory cytokines and chemokines including IL-1b, IL-6, and KC. The kinetics of infiltration showed an initial infiltration of monocytes shortly followed by a significant increase in neutrophil infiltration that strongly correlated with paw swelling. CD4+ T cells increased as well, but were not found in high numbers in the joint until after maximal paw swelling had been reached. We also found that the influx of monocytes into the knee joint space correlated with increased levels of KC, IL-1b, and IL-6, with KC levels preceding the subsequent neutrophil influx. Mouse models show similar disease course in regards to both inflammation and cytokine production in the synovial knee lavage. Understanding the kinetics of disease in the local joint presents new opportunities for biomarker development, target identification, and improvement of mechanistic understanding of disease processes in rodent models of arthritis.
Natural killer cells suppress cardiac eosinophilia in autoimmune myocarditis (171.21)
Authors: Ong SuFey , Ligons Davinna , Baldeviano Christian , Barin Jobert , Talor Monica , Wu Lei , Legault Jillian , Rose Noel , Cihakova Daniela
Published: January 2023

Eosinophilic myocarditis is a likely fatal form of myocarditis characterized by high mortality, compromised cardiac function, and severe cardiac infiltration dominated by eosinophils. We have demonstrated that eosinophils are present in the heart and contribute to disease severity during a murine model of experimental autoimmune myocarditis (EAM). We now show that natural killer (NK) cells exert control of cardiac eosinophilic inflammation and recruitment. Animals were depleted in vivo of DX5+CD3- NK cells by i.p. injection of anti-asialo GM1 antibody throughout EAM, resulting in significantly more severe disease (pathology score of 3.7 versus 1.5) and cardiac fibrosis. Depleted animals demonstrated greater numbers of SiglecF+Ly6Gmid cardiac eosinophils (6.9% versus 0.56%, p<0.001) during the peak of disease at day 21. The observed increase in number of eosinophils after NK cell depletion was not seen for Ly6GhiSiglecF- neutrophils. Analysis of cardiac supernatants revealed increased levels of eotaxin (CCL11) and IL5, while flow cytometry demonstrated an expansion of CD4+ T cells producing either IL13 or IL17A in the heart. From these data, we propose a mechanism by which NK cells impact CD4+ cell polarization and downstream production of eosinophil growth factors and chemokines, thereby suppressing the development of eosinophilic myocarditis. Therefore, we provide the first direct in vivo evidence of NK cells regulating eosinophilic inflammation.
Antigen-targeting to dendritic cells is required for engraftment of antigen-encoding bone marrow and long-term tolerance under non-myeloablative conditions (171.27)
Authors: Coleman Miranda , Thomas Ranjeny , Steptoe Ray
Published: January 2023

Transplantation of hematopoietic stem cells (HSC) or bone marrow (BM) engineered to encode antigens is a means of inducing antigen-specific tolerance that might be suitable for ‘turning off’ pathogenic effector and memory T-cell responses. However, transplantation into primed hosts under non-myeloablative conditions could lead to rejection of engineered HSC or BM. We hypothesized targeting antigen expression to mature APC would alleviate rejection of engineered HSC while still permitting expression in the ‘tolerogenic’ cells that arise after transplantation. Using non-myeloablative conditions, we compared engraftment and ‘tolerogenicity’ in naïve or immune hosts of BM encoding an antigen that was ubiquitously expressed, targeted to diverse APC, or targeted to DC. When antigen was expressed ubiquitously, engraftment failed (<1%) but targeting antigen to DC, permitted high levels of BM engraftment (≈40%). Unexpectedly, BM failed to engraft (<1%) when antigen was targeted using a MHC class II promoter. This was due low level MHC class II expression by HSC leading to antigen expression and subsequent immune rejection of engrafting HSC. Long-term tolerance of antigen-specific memory T cells occurred only when engraftment was facilitated by targeting antigen to DC. For clinical translation, careful selection for a promoter that optimally targets DC will be imperative to ensure engraftment of antigen-encoding cells and long-term tolerance.
Steroid Resistant Autoimmune Inner Ear Disease: An Autoinflammatory Disease Instead? (171.28)
Authors: Pathak Shresh , Hatam Lynda , Bonagura Vincent , Vambutas Andrea
Published: January 2023

Autoimmune Inner Ear Disease (AIED) is characterized by bilateral, fluctuating sensorineural hearing loss that has periods of exacerbation triggered by unknown stimuli. We have previously shown that IL-1β is aberrantly expressed in steroid unresponsive AIED patients. These patients exhibit low titer antibodies to mixed molds, at dilutions 1:256 (p<0.005) and 1:512 (p < 0.05) and the inner ear protein, cochlin at dilution 1:256 (p < 0.05), compared to control subjects. PBMC exposure to mold induces an up-regulation of IL-1β mRNA expression, enhanced IL-1β secretion and the generation of IL-17 expressing cells. Mold enriched T-cell cultures from AIED patients showed a five-fold induction of IL-17 producing cells in response to cochlin, suggesting that AIED patients share cross-reactive epitope(s). These results suggest that AIED may, in fact, be better characterized as an auto-inflammatory disease rather than autoimmune, as dysregulation of IL-1β and low-titer autoantibodies are observed in these patients. To further investigate the hypothesis that these patients have autoinflammatory disease, we asked whether LPS alone would be sufficient to induce IL-1β or whether the second stimulus of ATP was necessary. Monocytes from AIED patients secrete higher levels of IL-1β in response to LPS alone than healthy controls, and that the addition of ATP fails to augment IL-1β secretion. Further studies are underway to characterize the inflammasome defect in these patients.
BCR/TLR7 coligation uniquely drives plasma cell differentiation of autoreactive B cells (171.34)
Authors: Kiefer Kerstin , Green Nathaniel , Oropallo Michael , Cancro Michael , Marshak-Rothstein Ann
Published: January 2023

Endosomal Toll-like receptors (TLRs) play an important role in the manifestation of systemic autoimmune diseases such as SLE. In vitro, DNA-/RNA-associated autoantigens have been reported to activate B cells, pDCs, and other APCs, through TLR9/TLR7-dependent pathways. However, in vivo, TLR9 appears to negatively regulate TLR7-dependent responses. Hence, TLR9-/- autoimmune prone mice develop more severe disease and increased autoantibody titers against RNA associated proteins, while TLR7-/- and TLR7/9-/- autoimmune-prone mice develop less severe disease. AM14 BCR side-directed transgenic B cells recognize IgG2a-bound immune complexes (ICs) and initially proliferate in response to both DNA and RNA autoantigens. However, the TLR9-dependent DNA responses eventually promote a form of programmed cell death that can only be rescued by B cell survival factors such as BLyS. By contrast, the TLR7-dependent RNA responses promote differentiation to the plasma cell lineage. The RNA-driven phenotype is characterized by increased levels of plasma cell associated proteins such as IRF-4 and CD138 and antibody secretion. Even in the presence of BLyS, DNA containing ICs did not promote plasma cell development. Our results identify B cell intrinsic mechanisms whereby TLR9 activation restrains B cell responses and TLR7 activation induces a unique signal for plasma cell development.
The unique properties of neonatal NK cells predispose newborns to autoimmune ovarian disease (nAOD) induced by maternal autoantibody (171.37)
Authors: Rival Claudia , Samy Eileen , Setiady Yulius , Sun Yuefang , Sparks Joshua , Yagita Hideo , Tung Kenneth
Published: January 2023

The transfer of maternal autoantibody (autoAb) to the progeny can cause neonatal autoimmune diseases. An example is the fatal congenital heart block elicited by maternal SSA/SSB antibodies in Sjogren’s patients. Disease mechanism is unknown but can now be investigated in a nAOD model developed in our laboratory. Ovarian inflammation and atrophy occurs in C57BL/6 Rag ko pups after the exposure to maternal autoAb against ovarian zona pellucida 3 within 1-5 days of life. Resistance to nAOD induction in both NK cell-depleted Rag ko pups, and Rag/common gamma chain (Rag/γc) double ko pups demonstrates the critical role of NK cells. Notably, neonatal but not adult NK cells restore nAOD in Rag/γc ko pups. Compared to adult NK cells, neonatal NK cells have lower expression of Ly49 receptors and higher frequency of the CD27+ subset. We now show that the expression of CD27, CXCR3 and IFNγ by neonatal NK cells are crucial for nAOD. Furthermore, Ly49C/I-depleted adult NK cells acquire the ability to induce nAOD. This suggests that the low expression of Ly49 reduces the activation threshold and enhances the pathogenic capacity of neonatal NK cells. Lastly, FcgRIII expression on both neonatal NK cells and ovarian resident macrophages and dendritic cells (APC) is obligatory for nAOD induction. However, ovarian APC requirement is not ontogenetically regulated. We conclude that the unique properties of neonatal NK cell predispose newborns to autoimmune disease by maternal autoAb.