bluuebunny/crossref_metadata_2025 huggingface dataset:
The RNA virus sensor melanoma differentiation-associated protein 5 mitigates protection from Coxsackievirus B4 and type 1 diabetes in non-obese diabetic mice. (P5211)
Authors: Lincez Pamela , Shanina Iryna , Horwitz Marc
Published: January 2023

Type 1 diabetes is a pancreatic autoimmune disease affecting children and results from both genetic and environmental factors where coxsackievirus B4 (CB4) is the most associated pathogen. Genome-wide association studies have linked variants in MDA5, an RNA virus sensor, with T1D protection. As CB4 is an RNA virus and a likely factor in T1D etiology, we chose to use CB4 to study the role of MDA5 in T1D. CB4 infection enhances T1D in NODs through activation of autoreactive T cells. We hypothesized that MDA5 signals the strength, polarization and regulation of the T cell response after stimulation by agents like CB4 and dampening MDA5 function would alter the T cell response to favour T1D protection. MDA5ko(-/-) were backcrossed to the NOD background and confirmed by SNP analysis. NOD-MDA5ko(10/10) failed to develop spontaneous T1D by 24 weeks when T1D occurred in MDA5het(+/-)(2/10) and wt(+/+)(9/33). While decreased MDA5 expression (confirmed by Western) in MDA5het did not affect spontaneous T1D, it protected MDA5het(4/4 ) from CB4 induced T1D compared to infected wt(2/4). FACS analysis revealed significantly fewer activated effector CD4+ T cells in the spleen and PLNs of MDA5hets. Further, MDA5het PLNs had increased numbers of natural Tregs. These results support our premise that reduced MDA-5 expression alters the T cell response limiting the development of autoreactive effectors and T1D and suggests that dampening MDA-5 function could be a key therapeutic target.
Chemical inhibition of the RORγt-dependent transcriptional network in Th17 cells (P5163)
Authors: Xiao Sheng , Yosef Nir , Kuchroo Vijay
Published: January 2023

RORγt is a master transcription factor required for Th17 development. We identified two novel RORγt-specific inhibitors that suppress Th17 response and Th17-mediated autoimmune disease. To assess their mechanisms of action, we systemically characterize their function within the context of RORγt-directed transcriptional circuitry by integrating RORγt binding data in the presence and absence of drug with corresponding whole-transcriptome sequencing for wildtype and RORγt-deficient cells. RORγt is central in a densely interconnected regulatory network, acting both as a direct activator of genes important for Th17 differentiation and as a direct inhibitor of genes from other T-cell lineages. These inhibitors reversed both of these modes of action, but to varying extents and through distinct mechanisms. While one inhibitor displaced RORγt from its target-loci, the more potent inhibitor affected transcription predominantly without removing DNA-binding. Our work illustrates the power of a system-scale analysis of transcriptional regulation to characterize potential therapeutic compounds that inhibit pathogenic Th17 cells and suppress autoimmunity.
Engineering antigen specific T regulatory cells for transplant tolerance (P2148)
Authors: MacDonald Katherine , Orban Paul , Bramson Jonathan , Levings Megan
Published: January 2023

T regulatory cells (Tregs) are critical for self-tolerance and are being pursued as a potential cell therapy in transplantation and autoimmunity. While polyclonal Tregs can prevent transplant rejection in mice, allo-antigen specific Tregs are more potent. A chimeric antigen receptor (CAR) may be an effective way to make ag-specific Tregs in suitable numbers for the clinic. CARs are created by combining antibody variable domains with T cell receptor signaling domains. However, CARs for Treg cell therapy must a) function within the distinct signaling network of Tregs and b) recognize a transplant-relevant antigen. CAR function in Tregs was assessed by transducing sorted cells with a highly-expressed, humanized HER2 CAR. Cells were labeled with proliferation dye and assessed for their ability to respond to target antigen. Two CARs specific for HLA-A2 were generated by cloning the variable portions of the anti-A2 antibody from a hybridoma, creating a single chain antibody and ligating it in frame in the humanized CAR. These CARs were assessed for expression and activity by flow cytometry, and cytotoxicity and proliferation assays. Preliminary data suggests that CARs can induce strong, ag-specific proliferation in human Tregs and that an ag-specific HLA-A2 CAR has been generated. Further work includes determining the effect of CAR stimulation on Treg phenotype via cytokine staining and suppression assays, and optimizing the surface expression of the HLA-A2 CAR via phage display.
MHC discrepancies between host tissues and allogeneic NK cells expanded by IL-15 and 4-1BBL ex vivo contribute to GVHD but not GVT (P2150)
Authors: Capitini Christian , Kelm Sara , Esposito Thomas , Bates Paul
Published: January 2023

NK cell biology after allogeneic bone marrow transplant (alloBMT) is mainly drawn from studies infusing unmanipulated NK cells. We studied the impact of activating murine NK cells with the co-stimulatory molecule 4-1BBL during expansion with IL-15 ex vivo, and then infused these cells to determine safety and efficacy after alloBMT. NK cells were harvested from Balb/c (H-2d), DBA/2 (H-2d), or CB6F1 (H-2d x H-2b) spleens and cultured with IL-15 in the presence of irradiated artificial APCs transfected with or without 4-1BBL. The addition of 4-1BBL+ cells to IL-15 expanded NK cells decreased yields by up to 50%. The combination of 4-1BBL/IL-15 completely abrogated cytotoxicity against Yac-1 lymphoma, whereas IL-15 alone led to augmented IFNγ production and cytotoxicity. After T cell depleted (TCD) MHC-mismatched alloBMT (CB6F1→Balb/c), recipients of allogeneic NK cell infusions expanded with IL-15/4-1BBL showed weight loss in a dose-dependent fashion and increased lethality from GVHD. Similar results were also seen with CB6F1→C57BL/6. Prior to infusion, IL-15/4-1BBL combination led to less Fas ligand+ NK cells, but after alloBMT, FasL+ NK cells increased. After TCD MHC-matched alloBMT (DBA/2→Balb/c), infusion of NK cells expanded with IL-15 or IL-15/4-1BBL did not cause GVHD, implying MHC discrepancies between donor NK cells and GVHD target tissues contribute to the enhanced alloreactivity induced by 4-1BBL. No differences in graft-versus-tumor effects were observed in vivo.
Penetrating keratoplasty to one eye abolishes immune privilege for corneal allograft placed into the other eye. (P2164)
Authors: Paunicka Kathryn , Niederkorn Jerry
Published: January 2023

Corneal transplants are the most common and successful form of solid tissue transplantation due to the immune privilege (IP) of the eye. In a mouse model of corneal transplantation, 50% of BALB/c recipients normally accept C57BL/6 (B6) corneal allografts. However, BALB/c recipients that previously rejected an A/J or C3H corneal allograft in the right eye experienced 100% graft rejection of the unrelated B6 corneal allograft. This high incidence of rejection was observed when the B6 allograft placed into either the rejected or the unmanipulated eye. Moreover, BALB/c recipients that previously accepted a BALB/c syngeneic corneal graft on the right eye still had a high rejection rate of B6 grafts placed onto the unmanipulated eye. Thus, surgical manipulation performed during the grafting process causes the loss of IP in the unmanipulated eye. A circumferential corneal surface incision that damages the nerves in the left eye resulted in 100% rejection of B6 grafts placed onto the right eye. This incision upregulated the immunoregulatory neuropeptide Substance P (SP) in both eyes. In vivo blockade of SP with Spantide II reduced graft rejection. Direct injection of SP into the eye recapitulated the trephine effect and results in 90% rejection in both eyes. Circumferential incisions of the corneal surface upregulates SP and promotes graft rejection in repeat corneal allograft recipients.
Bone marrow derived mesenchymal stem cell therapy to improve nerve regeneration and function in a rat limb transplant model (P2170)
Authors: Fitzpatrick Emilie , DeHart Mary , Tercero Juan , Brown Tommy , Salgar Shashikumar
Published: January 2023

Introduction: Preventing immune rejection and optimizing nerve regeneration are keys to successful outcomes in hand and limb transplantation (Tx). Our objective was to investigate whether MSC can improve nerve regeneration and function. Methods: Orthotopic syngeneic right hind-limb transplants were performed in Lewis (RT1.Al) rats. Limb recipient rats received syngeneic MSC (5 x106; passage ≤7) or vehicle (saline) locally around nerve, bone and vascular anastamoses sites, and 5x106 MSC intravenously. Results: Rat MSC expanded ex vivo were CD29+, CD90+, CD34-, CD31-, CD45low, MHC Class I+, Class II-, CD80low, and CD86-. MSC used were pluripotent and differentiated in to adipocytes, osteocytes and chondrocytes in ex vivo cultures under specific conditions. At six weeks post-Tx, walking track analysis did not produce clear foot prints to calculate Sciatic Function Index (SFI). However, by six weeks cutaneous pain reaction test demonstrated sensory nerve function; on a scale of Grade 0-3 (0=No function; 3= Normal function) in vehicle treated animals (n=5) it was 2.1±1.3 (tibial), 0.8±0.7 (peroneal) and 1.2±01.0 (sural), and in MSC treated animals (n=5) it was 1.3±1.1 (tibial), 1.2±1.2 (peroneal) and 1.4±0.8 (sural). Laser doppler analysis revealed normal vascularization and limb transplant survival was >95%. Conclusion: The limb transplant procedure was highly successful and awaiting data on long-term effects of MSC therapy which appears to promote functional recovery.
5-Aminolevulinic acid induces permanent acceptance of murine cardiac allografts via induction of regulator dendritic cells and expansion of alloantigen-specific Tregs. (P2176)
Authors: Kitajima Yuya , Cai Songjie , Nishio Yoshiaki , Saito Taro , Ito Hidenori , Takahashi Kiwamu , Abe Fuminori , Nakajima Motowo , Tanaka Tohru , Nagahara Yukitoshi , Li Xiao-Kang
Published: January 2023

5-aminolevulinic acid (5-ALA), an intermediate in heme synthesis is fundamentally important in aerobic energy metabolism. Hemeoxygenase-1 (HO-1) cleaves heme to form biliverdin, carbon monoxide (CO) and iron (Fe2+), and the pathway is accelerated by 5-ALA. In the present study, we examined the effects of 5-ALA on the maturation and function of dendritic cells (DCs), a potent class of APCs, and the downstream cell-cell mediating immunosuppressive activity. 5-ALA inhibited LPS-stimulated maturation of murine bone marrow DCs in vitro as reflected by reduced expression of MHC class-II (I-A/I-E), co-stimulatory molecules (CD80, CD40) and proinflammatory cytokines, IFN-g, IL-12 mRNA expression. In mixed cultures, DCs pretreated with 5-ALA impaired the proliferation of allogeneic T cells while promoting the expansion of CD4+CD25+ regulatory T cells (Tregs). Using an in vitro mixed leukocyte reaction (MLR) assay system, we found that the DCs pretreated with 5-ALA have more regulatory function to suppress the both CD4+ and CD8+ T cells proliferation. Furthermore, 5-ALA treatment impaired T cells proliferation in response to alloantigens and increased Tregs, resulting in permanent acceptance of murine cardiac allografts. Thus we conclude that 5-ALA triggers a tolerogenic state in DCs that allows increasing alloantigen-specific Tregs and the suppression of allograft rejection in vivo.
Both passenger leucocytes and hepatic parenchyma contribute to activation and deletion of graft-reactive CD8 T cells in liver transplantation (P2130)
Authors: Bertolino Patrick , Tay Szun , Lu Bo , Benseler Volker , Sierro Frederic , Roediger Ben , Vo Michelle , McGuffog Claire , McCaughan Geoffrey , Bishop Alex , Cowan Peter , Dwyer Karen , Bowen David
Published: January 2023

Unlike most solid organs, liver transplants are spontaneously accepted across MHC mismatch and induce donor-specific tolerance. The mechanisms and site of tolerance induction remain unclear. Both recipient lymphoid tissues (RLT) where donor passenger leukocytes (PL) migrate, and the liver parenchyma itself are thought to contribute to this process by inducing abortive activation of alloreactive T cells. To determine the relative contribution of these compartments to alloreactive T cell fate, we developed a murine liver transplant model in which we traced the fate of PL and a naïve alloreactive CD8 T cell reporter population specific for donor MHC. Ly5.2+C57BL/6 livers were transplanted into allogeneic Ly5.1+B10.BR recipients. Directly allograft reactive Des-TCR transgenic T cells specific for donor H-2Kb were adoptively transferred as a reporter population. Donor and recipient leukocyte location and fate were traced by flow cytometry and radiolabelling. All Des T cells were rapidly activated in RLT and liver but numbers dropped dramatically within 48h, with cells resident in RLT predominantly dying in situ, while most circulating Des T cells were deleted in the liver. Intrahepatic clearance was associated with degradation of Des T cells in hepatocyte lysosomal compartments. In conclusion, these results show that both PL and the hepatic parenchyma contribute to deletion of graft reactive T cells, and reveal a novel mechanism of tolerance induction within the hepatic allograft.
Development of gammadelta T-cell receptor repertoire after TCRab/CD19 depleted haploidentical stem cell transplantation in children (P2190)
Authors: Schlegel Paul , Wölfl Matthias , Braun Matthias , Winkler Beate , Eyrich Matthias
Published: January 2023

Recent advances in stem cell manipulation techniques have allowed to optimize cellular composition of HLA-mismatched haploidentical stem cell grafts. We analyzed immune and haematopoietic reconstitution in 4 children transplanted with TCRab/CD19-depleted haploidentical stem cell grafts with a special emphasis on gammadelta-T-cell reconstitution. Grafts consisted of 8.5x10e6 CD34/kg, 8.8x10e4 CD3TCRab+/kg, 8.8x10e6 CD3TCRgd+/kg, and 17.5x10e6 CD16+CD56+/kg BW. Haematopoietic reconstitution was rapid in all patients. T-cell counts reached >100/µl after a median of 34 days, 75% of these expressing the gd-TCR. The predominance of gd-T-cells decreased and by day +100 50% of T-cells were positive for the gd-TCR and 46% for the ab-TCR. Gd-TCR Immunoscope analysis revealed the molecular composition of the gd-population: the Vg9-chain was expressed by a median of 54%, Vg2 by 17.6% of gd-T-cells, the remaining Vg-chains by less than 10%, respectively. Vd4-expressing subclones made up 53% of the whole gd-TCR-repertoire, followed by 33.4% Vd2+ subclones. Distribution of gd-subclones was relatively stable over time, so that the contraction of the gd-T-cell population could not be attributed to the disappearance of specific Vg- or Vd families. These data provide new insight into the T-cell reconstitution kinetics after TCRab/CD19-depleted transplantation.
IVIg treatment reduces catalytic antibody titers of renal transplanted patients (P2208)
Authors: Mahendra Ankit , Peyron Ivan , Dollinger Cécile , Gilardin Laurent , Wootla Bharath , Padiolleau-Lefevre Séverine , Friboulet Alain , Legendre Christophe , Thaunat Olivier , Kaveri Srini , Lacroix-Desmazes Sébastien
Published: January 2023

Catalytic antibodies are immunoglobulins endowed with enzymatic activity. Catalytic IgG has been reported in several human autoimmune and inflammatory diseases. In particular, low levels of catalytic IgG have been proposed as a prognostic marker for chronic allograft rejection in patients undergoing kidney transplant. Kidney allograft is treatment of choice for patients with end-stage renal failure. Intravenous immunoglobulins, a therapeutic pool of normal human IgG, is used in patients with donor-specific antibodies, alone or in conjunction with other immunosuppressive treatments, in order to desensitize the patients and prevent the development of acute graft rejection. In the present study, we followed for a period of 24 months the levels of catalytic IgG in a large cohort of patients undergoing kidney transplantation. Twenty-four percent of the patients received IVIg at the time of transplantation. Our results demonstrate a marked reduction in the levels of catalytic antibodies in all patients three months following kidney transplant. The decrease was significantly more pronounced in patients receiving adjunct IVIg therapy. The results suggests that prevention of acute graft rejection using intravenous immunoglobulins induces a transient reduction in the levels of catalytic IgG, thus potentially jeopardizing the use of levels of catalytic antibodies as a prognosis marker for chronic allograft nephropathy.
Study of alarmin release during ischemia reperfusion injury after human renal transplantation (P2214)
Authors: Gombert Jean-Marc , Thierry Antoine , Robin Aurelie , Barra Anne , Hauet Thierry , Touchard Guy , Herbelin Andre
Published: January 2023

The endogenous molecules high-mobility group box 1 (HMGB1) and interleukin-33 (IL-33) have been recognized as alarmins, capable of mediating danger signals during tissue damage. Here, we address their possible role as innate-immune mediators in ischemia-reperfusion injury (IRI) following human kidney transplantation. Urinary HMGB1 and IL-33 levels were significantly increased as soon as 30 min after reperfusion, as compared to prior transplantation. Moreover, a significant correlation was shown between both serum and urinary IL-33 (but not HMGB1) levels and cold ischemia time, from 30 min to 3 days post-tranplantation. In vitro, human umbilical vein endothelial cells submitted to hypoxia-reoxygenation significantly released IL-33. Finally, we can propose that PBMC from renal recipient patients are targeted by both HMGB1 and IL-33 as their receptor (TLR2/4 and ST2-L, respectively) transcripts were enhanced early after transplantation. Consistent with this view, by analyzing iNKT cells, which are been suggested to play a crucial role in IRI and we recently reported to be targeted by IL-33, we showed an early activation state of this innate-like T cell subset in kidney transplant recipients, as attested by the upregulation of CD69 surface expression 1 hour after transplantation. Altogether, these results underline the possible role of IL-33 as an innate-immune mediator during IRI in humans.
Induction of cell transplant tolerance: a roadmap from bench to bedside (P2220)
Authors: Qian Shiguang , Chou Hong-Shiue , Charles Ronald , Fung John , Lu Lina
Published: January 2023

Organ transplants have worked for decades, but outcomes of cell transplants remain disappointing. Enlightened by mouse liver allografts being spontaneously accepted, but hepatocyte transplants acutely rejected (suggesting lack of non-parenchymal cell protection), we identified strong regulatory activity of hepatic stellate cells (HpSC). Islet allografts (300) mixed with HpSC (3x105) achieved long survival without immunosuppression, associated with apoptosis of CD8 T cells, enhancement of Tregs, which depends on IFNγ/B7-H1 pathway. However, co-transplanted HpSC have to be syngeneic to recipients - a hurdle for clinical application. We revealed that HpSC did not directly induce Tregs, but induce MDSC. Addition of HpSC in DC culture (1:40) generated MDSC (instead of DC) with high iNOS and argenase 1 and potent suppressive activity. The induction of MDSC was found to be mediated by soluble factors, particularly retinoic acid and iC3b. Islet allografts mixed with (or followed by iv injection of) generated MDSC (3x106) were protected as effectively as HpSC, associated with elimination of effector T and enhancement of Tregs, which also required intact IFNγ/B7-H1 signaling. Human HpSC were tested showing markedly T cell suppression in MLR, which was reversed by B7-H1 blocking Ab. Human HpSC also induced MDSC in vitro. We will reexamined these in vivo in a humanized NOD/SCID mouse model before pursue to clinical trial.
Profiling class II HLA alloantibodies in transplant patient sera (P2222)
Authors: McMurtrey Curtis , Lowe Dave , Buchli Rico , Royer Derek , Cate Steven , Osborn Sean , Mojsilovic Aleksandar , VanGundy Rod , Bardet Wilfried , Mojsilovic Danijela , Stastny Peter , Briggs David , Zehnder Daniel , Higgins Rob , Hildebrand William
Published: January 2023

Allogeneic immunizing events including pregnancy, blood transfusion, and transplantation promote strong antibody responses to HLA. Such anti-HLA antibodies preclude organ transplantation, foster hyperacute rejection, and contribute to chronic transplant failure. Diagnostic antibody-screening assays can detect alloreactive antibodies and determine their HLA specificity, yet a number of key antibody attributes remain unexplored. The goal here was to provide the first detailed profile of antibodies directed to allogeneic HLA. Methodologically, sixteen sensitized patients were identified with alloantibodies to HLA-DR11, antibodies reactive with this class II HLA antigen were purified using a novel immunoaffinity column constructed with milligram quantities of native DR11, and these purified DR11 specific antibodies were categorized. Results show that allogeneic antibodies to DR11 were found in the serum at a median concentration of 2.3ug/ml and consisted of the IgM, IgG, IgA, and IgE isotypes. IgG2, IgM, and IgE were elevated while IgG1 was decreased in sensitized patients and, in most patients, these antibodies fixed complement. In conclusion, we show that HLA alloantibody responses are consistently comprised of multiple isotypes, that these alloantibodies reach an appreciable serum concentration, and that these alloantibodies fix complement.
Isolation, expansion and function of cord blood natural killer cells. (P2226)
Authors: Tomchuck Suzanne , Leung Wing , Dallas Mari
Published: January 2023

Rate of immune reconstitution (IR) directly correlates with the number of hematopoietic stem cells (HSC) infused and is particularly delayed in patients undergoing cord blood (CB) transplantation (CBT). Methods to increase the number of CB progenitors have the potential to accelerate IR after CBT. NK cells are the first lymphocyte subset to recover after HSC transplant (HCT) and are crucial in preventing early relapse. Unfortunately, NK cells in CB are low in number, incomplete in maturation and require activation for effective function. Here, we report a clinically relevant ex vivo expansion method that optimizes maturation as well as activation of CB NK cells. CB NK cells cultured 14-21 days with CB mononuclear cells, IL-2 and IL-15 result in a multi-log increase in cell number. Potential killer cell Ig-like receptor (KIR) ligand and adhesion molecule(s) responsible for NK cell cytotoxicity were examined prior to and after culture. Phenotypic analysis showed that the percent of NK cells expressing activating receptors and adhesion molecules increased (CD336 +39.11%) while inhibitory receptor expression decreased (CD159a -25.09%). Furthermore, cytotoxic function of CB NK cells against NK cell sensitive (K562) and resistant (MV4-11) cancer cells lines were enhanced after culture. The data suggest that this ex vivo expansion method of matured and activated NK cells is a potential clinically relevant method to prevent early relapse after CBT.
STAT2 is required for TLR-induced cross-presentation (APP3P.109)
Authors: Xu Jun , Chain Robert , Gamero Ana , Gallucci Stefania
Published: January 2023

Type I Interferons (IFNs) are one of the principal mediators in cross-presentation of exogenous antigens to prime CD8+ T cells. Toll-like receptor (TLR) stimulation enhances cross-presentation in dendritic cells (DCs), however, the molecular mediators involved in TLR-induced cross-presentation remain to be elucidated. Since STAT2 is a critical signaling component in the canonical signaling pathway downstream of type I IFN receptor, we hypothesized that TLR-induced cross-presentation may require STAT2. We generated an in vitro model of murine conventional DCs, the bone marrow derived DCs (BMDCs) from WT and STAT2KO mice, and found that STAT2 deficiency did not affect GMCSF-dependent DC development in vitro. Yet upon TLR stimulation, we found STAT2 deficiency impaired the type I IFN response as postulated, including reduction in costimulatory molecules (MHC Class I and CD86), and in the up-regulation of IFN-stimulated genes. Furthermore, STAT2KO DCs were impaired in their expression of signal 3 cytokines, such as IL-12, upon TLR stimuli. To complement our findings, we performed in vitro cross-presentation assays using OT-I TCR transgenic (Tg) T cells, and found that both proliferation and IFN-γ production of CD8+ Tg T cells were reduced when primed with LPS- or CpG-induced STAT2KO BMDCs cross-presenting OVA. Hence, our current data demonstrate that STAT2/type I IFNs axis is essential for TLR4 and 9-induced cross-presentation.
Direct functional interaction between HLA-DM and human immunoglobulin. (APP3P.102)
Authors: Mellins Elizabeth , Strohman Michael , Macmillan Henriette , Rajasekaran Narendiran , Ayyangar Sashi
Published: January 2023

B cells internalize extracellular antigen into endosomes using the immunoglobulin (Ig) component of the B cell receptor. In endosomes, antigen-derived peptides are loaded onto MHC class II proteins (MHC-II). How these pathways intersect remains unclear. We previously found that HLA-DM (DM), a catalyst for MHC-II peptide loading, co-precipitates with Ig in lysates from human B cell lines. We also detected DM/Ig complexes in intact cells by proximity ligation assay. Here, we report that DM/Ig complexes co-localize with endosomal markers in intact cells and that a larger fraction of Ig precipitates with DM after BCR crosslinking. In vitro, in the endosomal pH range, soluble HLA-DM (sDM) directly binds the Ig Fab domain, interacting with greater kinetic stability when Ig is immobilized onto a surface. Interestingly, sDM increases levels of free antigen released from immune complexes. Together, our results reveal a novel, direct HLA-DM/Ig interaction that may contribute to Ig-mediated antigen presentation by MHC-II.
Dissecting a highly immunogenic antigen presentation process triggered by a natural pathogenic antigen: listeriolysin O (APP3P.104)
Authors: Valderrama Adriana , Unanue Emil , Carrero Javier
Published: January 2023

Listeriolysin O (LLO) is the major immunogen and virulence factor secreted by L. monocytogenes. Peptides processed from the LLO are presented at low nanomolar concentration to CD4 T cells. The mechanism for the efficiency of this antigen presentation process remains unknown and may provide novel insight into antigen processing and presentation. We tested LLO mutants with impaired molecular properties, such as membrane binding, cytolytic activity, and catabolism. All LLO variants triggered T cell responses at low nanomolar concentrations. Because LLO presentation was independent of any known activity intrinsic to LLO, we investigated cellular mechanisms that may increase its antigenicity. All LLO mutants were taken up into cells at high amounts in large undefined vesicles. Based on this, we hypothesized that bulk protein uptake of LLO could cause a novel cellular stress mechanism that would explain its immunogenicity. Endoplasmic reticulum stress (ERS) is a cellular response that deals with large amounts of unfolded protein. We investigated if the entry of LLO into APC triggered the ERS. Treatment with nanomolar concentrations of LLO caused an increase in Chop and Gadd34 expression, as well as other canonical markers of the ERS. However, it did not induce Xbp1 splicing or the ER-chaperone response. In conclusion, LLO may be activating the Perk pathway leading to an ERS in APC. The biological and antigen presentation outcomes of this response remain under investigation.
Statins induce microautophagy in RAW 264.7 cell line. (APP3P.107)
Authors: Sahu Ranjit , Singh Satwinder , Davidson Anne
Published: January 2023

Autophagy is process by which cells recycle cellular components to overcome starvation. This process is constitutive in antigen presenting cells such as dendritic cells and macrophages. Among the various autophagy pathways, microautophagy (Mi) has a unique significance as it is implicated in the biogenesis of exosomes which are relevant to the design of therapeutics. Statins have been shown to up regulate macroautophagy and alleviate the effect of LPS apart from inhibiting cholesterol synthesis. However their role in microautophagy has not been reported. In the present study we investigated the effect of statins on microautophagy using the RAW264.7 cell line as a model system. Treatment with statins resulted in an up regulation of the genes for MA as well as Mi as determined by qPCR. Further studies showed that the levels of proteins TSG101 and VPS4 were increased during statin treatment. Pretreatment with statins led to a lowering of LAMP2A levels and decreased LAMP2A on the late endosomes, mimicking a LAMP2A knock down. Pretreatment with statins not only lowered the transcription of iNOS and TNF-alpha genes but also prevented the effect of LPS by decreasing initial levels of LAMP2A and increasing levels of TSG101. We propose that Mi can be a surrogate for Chaperone mediated autophagy (CMA) under conditions where the components of CMA are rendered non-functional due to disruption in normal cellular metabolism.
Trogocytosis efficiency is dependent upon TCR affinity for MHC: Peptide ligands (APP4P.115)
Authors: Wetzel Scott , Kreitinger Joanna , Crane Naomi , Hayes Timmon
Published: January 2023

Antigen recognition by CD4+ T cells leads to large-scale spatial and temporal molecular redistributions, forming the immunological synapse. We have previously shown that upon dissociation from APC, T cells capture large membrane fragments from the antigen presenting cell containing both MHC:peptide ligands and costimulatory molecules directly from the immunological synapse in a process termed trogocytosis. The transferred molecules are retained on the surface of the T cell and sustain intracellular signaling and are associated with the selective survival of the trogocytosis positive cells after removal of the antigen presenting cells. To further characterize trogocytosis, we have examined the impact of TCR affinity for cognate MHC:peptide ligands on the efficiency of trogocytosis. Using moth cytochrome C 88-103 and the altered peptide ligands MCC K99A and MCC-A, we show here that the extent of trogocytosis by primary murine CD4+ T cells correlates with the affinity of the TCR for the MHC:peptide ligands. Preliminary studies also show that sustained intracellular signaling and selective survival of the trogocytosis+ cells varies based upon the TCR affinity for the specific MHC:peptide ligands captured by the T cell.
Requirement for PD-1 in the induction and maintenance of tolerance differs depending on tolerogenic stimulus (BA8P.118)
Authors: Pauken Kristen , Martinov Tijana , Heffernan James , Spanier Justin , Sahli Nathanael , Jenkins Marc , Vezys Vaiva , Fife Brian
Published: January 2023

The inhibitory receptor Programed Death (PD)-1 interacting with PD-L1 is important for tolerance since blocking this pathway accelerates autoimmunity. However, the mechanisms by which PD-1/PD-L1 contribute to tolerance remain unclear. In this study we tracked endogenous insulin-specific CD4 T cells in mice that are susceptible (NOD) or resistant (B6.g7) to Type 1 Diabetes (T1D). Despite the presence of insulin-reactive CD4 T cells in both strains, anti-PD-L1 precipitated T1D in NOD but not B6.g7 mice. Accelerated T1D in NOD mice correlated with increased numbers of insulin-specific CD4 T cells in the spleen, pancreatic LN, and pancreas. The number of cells increased in the pancreatic LN of B6.g7, but not the spleen or pancreas, suggesting that loss of PD-1 signaling was insufficient to promote trafficking to the pancreas in these mice. Unexpectedly, the breakdown of self tolerance in NOD mice did not rely on PD-1 signals as anergic cells did not change with PD-1 blockade. Rather, PD-1 was more important for regulating effector T cells. This contrasts data using antigen-coupled ECDI-fixed cells to induce anergy in potently activated effector cells during T1D in NOD, where PD-1/PD-L1 ligation is required for both the induction and maintenance of tolerance. This work reveals the role PD-1 signaling has for different contexts of tolerance, and has important therapeutic implications for use of PD-1 inhibitors in patients.
Disruption of tolerance by ER stress in type 1 diabetes (BA8P.128)
Authors: Marre Meghan , Profozich Jennifer , Coneybeer Jorge , Geng Xuehui , Bertera Suzanne , Trucco Massimo , Piganelli Jon
Published: January 2023

Type 1 diabetes (T1D) is an autoimmune disease, largely mediated by autoreactive T cells, in which islet β cells are destroyed. The events that break peripheral tolerance in individuals genetically predisposed to autoimmunity are poorly understood. Many physiological and environmental triggers associated with T1D, such as viral infection, chemicals, reactive oxygen species, and dynamic glucose sensing, result in endoplasmic reticulum (ER) stress. ER stress increases the potential for abnormal post-translational modification (PTM) of proteins. We hypothesized that β cell ER stress generates neo-antigens that activate autoreactive T cells and worsen T1D. Indeed, chemical (Thapsigargin) induction of ER stress in murine islets or insulinomas increased IFNγ secretion from diabetogenic BDC2.5 T cells (28-1500 fold). Chelation of stress-induced calcium (Ca2+) flux reduced this response (27-64%), supporting a role for Ca2+-dependent enzymes such as tissue transglutaminase 2 (Tgase). Tgase activity is higher (18 fold) in β cells under stress, and reduced Tgase expression decreased the BDC2.5 response to β cells under stress (58%). Finally, nonantigenic insulinomas were exposed to normal physiological conditions in vivo. At harvest, these cells had increased ER stress (300 fold), increased Tgase activity (44 fold), and elicited higher BDC2.5 IFNγ responses (22 fold) compared to cultured cells. Thus, β cell ER stress causes formation of neo-antigens that activate autoreactive T cells.
Activation of autoreactive T cells by endogenous microflora induces spontaneous autoimmunity in the immunologically privileged retina (BA8P.130)
Authors: Zarate-Blades Carlos , Horai Reiko , Chen Jun , Silver Phyllis , Dillenburg-Pilla Patricia , Yamane Hidehiro , Chan Chi-Chao , Honda Kenya , Caspi Rachel
Published: January 2023

Autoimmune uveitis (AU) has an incidence similar to multiple sclerosis, it develops spontaneously and is a major cause of blindness. We hypothesized that AU could be triggered by endogenous microbiota in view of anecdotal evidence linking AU to systemic infections. We tested this in R161H mice, a new model of AU developed in our lab. R161H mice express a transgenic T cell receptor (TCR) specific to retinal protein IRBP and develop spontaneous uveitis starting at 4 weeks of age. R161H mice treated with an antibiotic cocktail, or reared under germ free conditions, had a significant delay in disease onset and intensity compared to controls, as well as fewer IL-17-secreting T cells in the gut LP. Notably, conventionally housed R161H mice consistently displayed 2-5x more Th17 cells in their LP compared to WT littermates, even when crossed onto an IRBP-deficient background. Compared to polyclonal T cells, R161H TCR transgenic T cells were more readily activated by extracts of gut contents in vitro and adopted the Th17 phenotype in the gut with higher frequency in vivo. Experiments utilizing Nur77 reporter mice and pZap70, revealed clear evidence that IRBP-specific T cells receive an activation signal through their TCR in the gut. These data not only have implications for etiology of human uveitis, but also raise the possibility that activation of autoreactive TCRs by commensal microflora may be a more common trigger of autoimmune diseases than is currently appreciated
Temporal regeneration of aire-expressing medullary epithelial cells and its impacts on reestablishing central immune tolerance (BA8P.122)
Authors: Tajima Asako , Coppola Antonina , Grupillo Maria , Gualtierotti Giulio , Piccirillo Ann , Geng Xuehui , Rudert William , Trucco Massimo , Fan Yong
Published: January 2023

The essential role of tissue specific antigen (TSA) expression in medullary thymic epithelial cells (mTECs) in mediating central tolerance has been well-established. We have previously shown that mice with mTEC-specific insulin deletion develop autoimmune diabetes within 3 weeks postnatal. To further understand the mechanisms of mTEC-mediated self-tolerance, we generated the Aire-YFP mice by crossing R26R-YFP reporters with Aire-Cre transgenic mice. Three subsets of mTECs were isolated from these mice: the YFPLOWCD80LOWClass IILOW pre-Aire cells; the YFPHICD80HIClass IIHI Aire+ cells; and the YFPHICD80INTClass IIINT post-Aire cells. Diverse TSA transcripts were detected in Aire+ mTECs, but not, or more restricted in pre- and post-Aire subsets, respectively. To investigate the temporal differentiation of mTECs, we generated the Aire-Cre:R26R-DTR (Aire-DTR) mice, which marked both Aire+ and post-Aire mTECs with diphtheria toxin (DT) receptors. Consecutive administration of DT resulted in ablation of both subsets, whereas pre-Aire mTECs remained intact. Upon DT withdrawal, transcripts of Aire and TSAs remained absent at day 3, but became detectable at day 5. Interestingly, no autoimmunity was observed in Aire-DTR mice treated weekly with a single dose of DT, suggesting that the newly generated Aire+ mTECs could reestablish self-tolerance in a timely manner. Our data provide new insights into the temporal regeneration of mTECs and its importance in maintaining central tolerance.
Priming of myelin-reactive CD4+ T cells in the absence of CD11c+ dendritic cells (BA8P.123)
Authors: Waldner Hanspeter , Cheung Julie , Wexler Aaron
Published: January 2023

Experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis, is predominantly mediated by pro-inflammatory CD4+ T cell responses to myelin antigens, including myelin proteolipid protein (PLP). Dendritic cells (DCs) are considered critical for inducing T cell responses against infectious agents, but their role in priming of myelin-specific CD4+ T cells is still unclear. To investigate their importance in promoting PLP-specific CD4+ T cell responses, we have established novel SJL mice that express the simian diphtheria toxin receptor (DTR) on CD11c+ DCs, allowing ablation of DCs following diphtheria toxin administration (CD11c-DTR SJL mice). PLP-pulsed spleen cells from DC-depleted mice promoted significantly diminished proliferation and pro-inflammatory cytokine responses of CD4+ T cells from PLP TCR transgenic mice in vitro compared to DC-sufficient splenocytes. However, CFSE-labeled PLP-specific CD4+ T cells transferred into DC-depleted mice showed undiminished proliferation in response to PLP/CFA immunization, suggesting that non-DCs were capable of myelin-specific T cell priming. We determined the importance of DCs in EAE development by transferring PLP TCR transgenic CD4+ T cells into DC-depleted RAG2-deficient mice lacking B cells. Interestingly, PLP-specific CD4+ T cells mediated severe EAE even in the persistent absence of DCs, suggesting that differentiation of encephalitogenic CD4+ T cells required neither B cells nor CD11c+ DCs.
A novel function of RGS10 in effector T lymphocytes to augment mouse EAE (BA8P.126)
Authors: Lee Jae-Kyung , Chung Jaegwon , Kannarkat George , Graham Kareem , Tansey Malu
Published: January 2023

A broad range of immune-based therapeutic drugs have been available for treatment of various autoimmune diseases but many have had limited success. Therefore, it is critical to identify additional cellular targets that can regulate the pathogenic response of immune cells. We hypothesize that GPCR modulator RGS proteins are important modulators of the immune responses involved in the development of multiple sclerosis (MS). Our hypothesis is based on reports that various SNPs in RGS proteins is highly correlated with the diagnosis of MS. Especially, an in-depth search of the GEO profiles database also revealed higher levels of RGS10 transcripts in MS patients’ PBMCs. Here, we showed RGS10-null mice displayed significantly milder clinical symptoms of EAE with reduced incidence and delayed disease onset. We observed that there were less numbers of CD45+ cells and CD4+ T cells in MOG35-55-immunized RGS10-null mice in the CNS. RGS10-null LN T cells were less proliferatative and produce less IFN-γ and IL-17 cytokines in response to MOG. Our data suggest a critical role for RGS10 in modulating disease by attenuating lymphocyte infiltration and/or peripheral immune responses during EAE. This is the first study ever conducted to elucidate the function of RGS10 in effector lymphocytes in the context of EAE. The identification of RGS10 as a central regulator of the inflammatory processes will open a possibility for developing more specific targeted therapy for the treatment of MS.
Cutaneous B-1 B cells can secrete IL-10 and require integrin α4β1 (VLA-4) for migration into skin (CAM3P.201)
Authors: Debes Gudrun , Wilson R. , Lauer Uta , Hamann Alf , Geherin Skye
Published: January 2023

B1 B cells are innate-like B cells that provide key effector functions during infection and inflammation. Despite their importance, the target organs, function and trafficking of this B cell subset are largely unknown. Here we show that, unexpectedly, B cells, including B1(-like) cells with the potential to secrete IL-10, were part of the normal cutaneous immune system of mice and humans. In the mouse, we found steady-state migration of peritoneal B1 B cells into skin. In addition, B1, but not follicular B2, B cells efficiently migrated into inflamed skin by virtue of α4β1 integrin (VLA-4) expression. Importantly, B1 B cells expressed high affinity VLA-4 at steady state, indicating that B1 cells were poised to migrate into inflamed sites. Our data suggest that IL-10+ innate-like B cells are recruited into skin inflammation to dampen cutaneous inflammation, and we propose that dysregulation of the recruitment or function of B1(-like) B cells contributes to inflammatory skin diseases, such as psoriasis.
TNFR2 expression by CD4 effector T cells is required to induce full-fledged experimental colitis (CCR3P.204)
Authors: Chen Xin , Xiao Haitao , Howard O.M.Zack , Oppenheim Joost
Published: January 2023

There is now compelling evidence that TNFR2 is constitutively expressed on Tregs and TNF-TNFR2 interaction is critical for the activation, expansion and functional stability of CD4+Foxp3+ regulatory T cells (Tregs). However, we also showed that the expression of TNFR2 could be up-regulated on CD4+Foxp3- effector T cells (Teffs) upon TCR stimulation, and TNFR2-expressing Teff cells were more activated and more resistant to Treg-mediated inhibition. In order to further define the role of TNFR2 in the proliferative expansion of pathogenic CD4 T cells in a setting of autoimmune disorder, we transferred naïve CD4 cells from WT mice and TNFR2-/- mice into Rag1-/- recipients. The results showed that TNFR2-deficient Teff cells failed to induce full-fledged colitis unlike WT Teffs. This was due to defective proliferative expansion of TNFR2-deficient Teff cells in the lymphopenic mice, as well as their reduced capacity to produce proinflammatory Th1 cytokines on a per cell basis. In vitro, TNF blockade inhibited the up-regulation of TNFR2 expression, as well as proliferative responses and cytokine production, by TCR-stimulated naïve CD4 cells. This effect of TNF blockade can be replicated by anti-TNFR2 Ab, but not by anti-TNFR1 Ab. Therefore, TNFR2 is critical for the proliferative expansion of pathogenic CD4 T cells, and thus may be a therapeutic target in the treatment of CD4 T cell-mediated autoimmune inflammation, although at a cost of attenuation of Treg activity.
IFN-α induces soluble IL-15 complexes via ADAM17 mediated cleavage (CCR3P.206)
Authors: Anthony Scott , Howard Megan , Stonier Spencer , Dai Zhimin , Khong Hiep , Overwijk Willem , Schluns Kimberly
Published: January 2023

Type I Interferons (IFN) are potent inducers of IL-15 transcription and cell surface expression. Previous studies have shown that sIL-15 complexes, which act as IL-15 agonists, are generated by TLR stimulation and after non-myeloablative lymphodepletion. Since TLR stimulation induces type I IFNs, we investigated the role of type I IFNs in generating sIL-15 complexes. We report that treatment with a plasmid inducing IFN-α production potently increased serum sIL-15 complexes levels in mice. Production of sIL-15 complexes by bone marrow derived dendritic cells (BMDC) also increased after treatment with IFN-α in an IFNAR-dependent manner. Increases in sIL-15 complexes after total body irradiation coincided with appearance of serum IFN-α and were significantly impaired in the absence of Type I IFN signaling. In addition, we found that IFN-α induced activity of the metalloprotease, ADAM17 in BMDCs, which was critical as induction of sIL-15 complexes by IFN-α was abolished after deleting ADAM17. We also determined whether sIL-15 complexes are generated during a Vesicular stomatitis virus (VSV) infection as VSV induces Type I IFNs. One day after infection, sIL-15 complexes are transiently induced, which was surprisingly unabated in the absence of Type I IFN signaling. Overall, our findings show that Type I IFNs induce production of sIL-15 complexes by activating ADAM17-mediated cleavage, thus identifying a new mechanism regulating sIL-15 complexes during inflammation.
The association of ROS levels in blood cells with age, past radiation exposure, and IL6R gene polymorphisms (CCR3P.211)
Authors: Hayashi Tomonori , Hu Yiqun , Furukawa Kyoji , Ohishi Waka , Geyer Susan , Weng Nan-ping , Hayashi Ikue , Yoshida Kengo , Kajimura Junko , Kyoizumi Seishi , Kusunoki Yoichiro , Nakachi Kei
Published: January 2023

Reactive oxygen species (ROS) play an important role in cell-mediated immune responses, although overproduction and excessive accumulation of ROS may also enhance risks of inflammation-related diseases. To address these concerns, we have developed an assay system to determine ROS (H2O2 and O2.-) levels in blood cells to investigate the effects age and of radiation exposure on ROS levels in the blood cells obtained from 2,789 Japanese atomic-bomb survivors, as well as associations between intracellular ROS levels and immune-/inflammation-related gene polymorphisms. The results showed that H2O2 levels in monocyte and granulocyte fractions increased with age (P<0.001) but not with radiation dose. No effects of age or radiation dose on H2O2 levels were observed for T-cell subsets. On the other hand, O2.- levels in both lymphocyte and granulocyte fractions increased with age and radiation dose (P<0.05). In addition, O2.- levels in T cells, especially in CD8+ T cells, increased with age and radiation dose (P<0.05). Analysis of the association between intracellular ROS levels and gene polymorphisms revealed a significant difference in O2.- levels in T cells by IL6R genotypes. These results suggest that ROS levels in selected types of immune cells are affected by aging, radiation, and genetic factor. Thus, the intracellular ROS levels may serve as a biomarker for age of the cells as well as for estimating the risk of inflammation-related disease development.