bluuebunny/crossref_metadata_2025_split huggingface dataset:
Engineering antigen specific T regulatory cells for transplant tolerance (P2148)
Authors: MacDonald Katherine , Orban Paul , Bramson Jonathan , Levings Megan 
Published: January 2023

Abstract
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

Abstract
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

Abstract
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

Abstract
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 &gt;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

Abstract
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

Abstract
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

Abstract
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 &gt;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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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.