bluuebunny/crossref_metadata_2025 huggingface dataset:
Myeloid-derived suppressor cells mobilized by G-CSF in allografts correlate with low risk of acute graft-versus host disease in patients undergoing unmanipulated allogeneic blood and marrow transplantation (126.18)
Authors: Lv Meng , Zhao Xiang-yu , Wang Ya-zhe , Liu Yan-rong , Xu Lan-ping , Liu Dai-hong , Liu Kai-yan , Huang Xiao-jun
Published: January 2023

Introduction:The aim of this study was to investigate the association of donor myeloid-derived suppressor cells (MDSCs) in allografts with acute graft-versus host disease(aGVHD) in patients who had undergone granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood progenitor cell (G-PBPC) and G-CSF-primed bone marrow (G-BM) transplantation. Methods: Unmanipulated allografts from 71 patients were prospectively analyzed for MDSCs with respect to aGVHD, in which 23 patients got HLA-matched donors and 48 patients had haploidentical donors. Results: Median numbers (range) of monocytic/ granulocytic/ promyelocytic MDSCs were:3.43(0.31-10.65)×106/kg, 8.23(0.85-16.2 )×106/kg , 1.11(0.51-3.53)×107/kg ,respectively in G-BM and these of monocytic/ granulocytic MDSCs were:1.64(0.28-4.3)×107/kg , 1.96(0.62-6.2)×107/kg , respectively in G-PBPC. The incidence of grades II-IV aGVHD was 29.6%, and there was no difference between the HLA-matched and haploidentical transplantation group. Patients who received a higher dose of promyelocytic MDSCs(>0.82×107/kg, p<0.001) in G-BM or a higher total dose of monocytic MDSCs in G-BM and G-PBPC (>1.23×107/kg, p<0.001) exhibited a lower incidence of grades II-IV aGVHD. Conclusion: Our results suggest that a high number of MDSCs infused were associated with decreased risk of aGVHD in allogeneic blood and marrow transplantation, which may reflect the immune suppressive function of MDSCs in allogeneic transplantation.
Calcineurin inhibitor (CNI) use in pediatric liver and kidney transplant (Tx) patients is associated with increased risk of obesity and impaired T-regulatory (Treg) cell function. (126.19)
Authors: Akimova Tatiana , Kamath Binita , Goebel Jens , Meyers Kevin , Rand Elizabeth , Hawkins Andre , Bucuvalas John , Hancock Wayne
Published: January 2023

CNI use can cause hyperlipidemia and new onset diabetes, and obesity may have adverse effects on allograft and patient survival, but the mechanisms are unclear. Design: We assessed if CNI or rapamycin (Rapa) use correlated with the metabolic status of children (n = 62) with stable, long-term liver (26 M, 27 F) or kidney (6 M, 3 F) allografts, and if being overweight or obese (Ow/Ob) was associated with impaired Treg phenotype or function. Results: Most patients had CNI (39) or Rapa (9) monotherapy, though 12 received CNI (8) or Rapa (4) plus MMF, Aza or steroids. 45 children had normal weight, but 7 were overweight and 10 were obese. The CNI group had more Ow/Ob patients (p=0.045) and higher BMI-for age percentile (BMI) than the Rapa group (61.7 vs. 42.9, p=0.047). CNI dose correlated with BMI (0.521, p=0.013), while Rapa use negatively correlated with BMI (-0.888, p=0.044). Also, Ow/Ob patients had reduced Treg suppressive function compared to patients with normal weight (153 vs. 100 units for autologous CD4 responders, 166 vs. 92 for standard CD4 responders, 114 vs. 43 for CD8 responders, p=0.031), and BMI was negatively correlated with CTLA-4 expression in Tregs (-0.555, p=0.011). Conclusions: CNI use is associated with being overweight or obese post-Tx, and the latter are associated with impaired Treg function and phenotype. The metabolic consequences of being overweight or obese post-Tx may include disruption of immune regulation and favor a pro-inflammatory phenotype.
Implication of CD4+ NKG2D+ T cells in the outcome of graft after kidney transplantation. (126.20)
Authors: López-Larrea Carlos , Schlangen Karin , Fernandez-Sanchez Alba , Carbajal Palo Reyes , Garcia-Melendreras Susana , Gómez-Huertas Ernesto , Ortega-Suarez Francisco , Aransay Bañares Ana , Suarez-Alvarez Beatriz
Published: January 2023

The activating receptor NKG2D is mainly expressed on γδ T, αβ CD8+ T, NKT and NK cells but an unusual NKG2D+ CD4+ T subset has been detected in certain pathologies. In this work, we analyzed the presence of CD4+ NKG2D+ T cells in peripheral blood samples from kidney transplanted patients and healthy donors by flow cytometry. Furthermore, whole genome microarray expression studies (Human HT-12 v3, Illumina) and TCR Vβ repertory (TCR Landscape technology) were performed in order to determinate its phenotypic and functional characteristics. We showed that 41% of the patients have a subset of CD4+ NKG2D+ T lymphocytes, which maintains stable during time. These cells presented a phenotype of memory cells with downregulation of costimulatory molecules and an increased expression of cytotoxic markers specific of NK cells. Several pathways associated with immune response, apoptosis, inflammatory response and cellular catabolic process were significant affected. CD4+ NKG2D+ T cells showed resistance to apoptosis and restricted TCR repertoire with oligoclonal expansion of one or two Vβ families, suggesting a limited capacity to respond to different antigens. In short, presence of this subset in kidney transplanted patients could be favorable for the graft acceptance but compromise the immune response against diverse antigens. It could be used as a biomarker to determine the outcome of the allograft and predict the immune response in these patients.
Graft versus host disease impairs vaccine responses via decreased CD4+ and CD8+ T cell proliferation and increased CD8+ T cell apoptosis through the perforin pathway (126.27)
Authors: Nasholm Nicole , Duncan Brynn , Capitini Christian , Fry Terry
Published: January 2023

Graft versus leukemia (GVL) responses contribute to the curative potential of allogeneic blood or marrow transplantation (alloBMT) but can be associated with graft versus host disease (GVHD). Tumor-targeted vaccines have the potential to enhance the potency and specificity of GVL, but we have shown that GVHD can limit responses. Using a minor histocompatibility-mismatched BMT (B6 ->C3H.SW) with donor lymphocyte infusions (DLI), we examined the mechanism of reduced vaccine responses. The proliferative capacity, persistence and survival of vaccine-responding T cells were monitored following adoptive transfer of HY-specific T cells and HY-expressing dendritic cells in mice with GVHD. Both CD4+ and CD8+ HY specific T cells undergo less vaccine-driven proliferation in recipients with GVHD. Furthermore, HY-specific CD8+ T cells demonstrated increased apoptosis, whereas HY-specific CD4+ T cells did not. To determine a mechanism of apoptosis, we used a Fas Ligand-, TRAIL-, or perforin-deficient DLI to induce GVHD. Fas ligand- and TRAIL-deficient DLI did not affect apoptosis of the CD8+ HY-specific T cell population. However, a perforin-deficient DLI resulted in significantly less apoptosis. Finally, perforin-deficient DLI allowed for enhanced HY-expressing tumor protection. Thus, diminished vaccine responses seen during GVHD result from reduced proliferation and perforin-mediated CD8+ T cell apoptosis, factors to be considered when optimizing vaccine strategies following alloBMT.
Tolerance-promoting regimens permit extensive T cell activation but alter differentiation in vivo (126.29)
Authors: Wang Szu-I , Gill Ronald
Published: January 2023

Short-term perturbation of the costimulatory molecule CD154 and/or the adhesion molecule LFA-1 can induce allograft tolerance. However, the effect of these agents on antigen-specific T cells is unclear. Therefore, we determined how anti-CD154 or anti-LFA-1 monoclonal antibody (mAb) therapy impacted the reactivity of CD4 and CD8 T cells in vivo. We monitored both antigen-specific responses of ovalbumin (OVA)-specific CD8 (OT-I) or CD4 (OT-II) TCR transgenic T cells and alloreactivity by polyclonal T cells (C57BL/6 anti-BALB/c). CD45.1 OT-I, OT-II or B6 T cells were transferred into CD45.2 B6 hosts locally challenged with mAct-OVA or BALB/c APCs, respectively, in the presence of mAb therapy. Anti-CD154 and anti-LFA-1 each inhibited the magnitude of OT-I and OT-II responses but permitted extensive proliferation and conversion to an antigen-experienced CD44hiCD62Llo phenotype. Both treatments decreased expression of Gzm B in OT-I T cells and IFN-gamma in OT-II T cells. Polyclonal alloreactivity also was markedly inhibited by either anti-LFA-1 or anti-CD154 therapy, though activation and proliferation still occurred in both cases. Importantly, anti-LFA-1 but not anti-CD154 treatment resulted in a striking increase in Treg/ Teff cell ratio in the reactive lymph node. Overall, neither blocking CD154 nor LFA-1 prevented T cell recognition/activation in vivo. However, LFA-1 blockade exhibited a more pronounced ability to skew alloreactivity towards a Treg phenotype in vivo.
Reconstitution of protective immune responses against CMV and VZV does not require disease development in paediatric recipients of umbilical cord blood transplantation. (126.4)
Authors: Soudeyns Hugo , Salem Fourati Insaf , Brito Rose-Marie , Charrier Emily , Cordeiro Paulo , Caty Martine , Mezziani Samira , Malette Brigitte , Duval Michel , Alfieri Carolina , Ovetchkine Philippe , Le Deist Françoise , Merindol Natacha
Published: January 2023

Cytomegalovirus (CMV) and varicella-zoster virus (VZV) are significant causes of morbidity and mortality following umbilical cord blood transplant (UCBT). Yet the kinetics of reconstitution and protective potential of antiviral cell-mediated immune responses following UCBT remain poorly characterized. Here, the reconstitution of CMV and VZV-specific T cell responses was assessed from 1 to 36 months post-transplant using IFN-γ ELISpot and MHC-peptide tetramers in 28 children who underwent UCBT to treat haematological or inherited disorders. Barely detectable in the first 3 months post-UCBT, antiviral T cell responses directed against CMV and VZV were observed in 30.4% and 40.3% of subjects after 36 months. Four CMV-seronegative subjects developed IFN-γ responses against CMV, and 4 subjects developed VZV-specific IFN-γ responses without clinical signs of VZV infection. No CMV- or VZV-related events were observed in subjects following the development of CMV- or VZV-specific responses >150 SFU per 10E6 PBMC, consistent with T cell-mediated protection. Famciclovir prophylaxis did not strictly prevent the reconstitution of the VZV-specific T cell repertoire. Lastly, the magnitude of VZV- and CMV-specific responses was correlated with the frequency of naïve CD8+ T cells, underlining the importance of immune reconstitution to achieve antiviral protection. Monitoring of cell-mediated immunity during the post-transplant period could guide the tailoring of antiherpetic prophylaxis.
Allogeneic inducible regulatory T cells allow the generation of mixed chimerism via nonmyeloablative conditioning in mice models (126.5)
Authors: Ruiz Paulina , Maldonado Paula , Hidalgo Yessia , Silva Cinthia , Ramirez Valerie , Rosemblatt Mario , Bono Maria
Published: January 2023

It has been demonstrated that the generation of mixed chimerism induces stable tolerance in allogeneic transplant organs. Current protocols require myeloablative or nonmyeloablative conditioning, both involving physical, pharmacological and/or cellular conditioning in order to produce general immunosuppression. Here, we have successfully developed a protocol that produces mixed chimerism using inducible allogeneic regulatory T cells (iTreg), low dose irradiation (3 Gy) and the FDA approved immunosuppressive drugs Rapamycin and Abatacept. Under experimental conditions, allogeneic iTreg treated mice produced mixed chimerism whereas, control mice lacking iTreg, rejected allogeneic bone marrow transplantation. Additionally, chimeric mice are able to accept secondary allogeneic skin transplants. Therefore we propose that generation of mixed chimerism is an improved specific therapy designed for stable alloinjert tolerance induction.
TNF but not IL-12 or interferon-alpha (IFN-a) plays a critical signal 3 role in in vivo CD8 cytotoxic T lymphocyte (CTL) maturation in the parent-into-F1 (P→F1) model. (126.7)
Authors: Via Charles , Soloviova Kateryna , Puliaiev Maksym
Published: January 2023

In addition to TcR signaling and costimulation, CD8 T cells require a third signal to mature into CTL. In pathogen models, IFN-a and IL-12 serve as signal 3 however in the P→F1 model, B6→B6D2F1 mice exhibit robust donor anti-host CD8 CTL in the absence of exogenous pathogens resulting in >90% elimination of host splenocytes i.e. acute graft-vs.host disease (GVHD). Selective inhibition of donor CD8 (but not donor CD4) activation converts acute GVHD to chronic GVHD with splenocyte expansion. To determine whether signal 3 molecules IL-12 or IFN-a are required to directly act on donor T cells in signal 3 manner, we transferred IL-12 or IFN-a receptor defective B6 donor cells into normal B6D2F1 mice. In neither transfer was acute GVHD phenotype significantly impaired compared to wild type (WT) B6→F1 mice. Moreover, peak CD8 CTL effectors were not impaired quantitatively or qualitatively. Similarly, blocking of WT B6→F1 with anti-IL-12 mAb did not alter phenotype. Significant IL-12 or IFN-a related gene expression was not detected by RT-PCR in WT B6→F1 mice prior to day 7. By contrast, TNF receptor 2 KO (TNFR2KO)→F1 but not TNFR1KO→F1 mice convert phenotype from acute to chronic GVHD. Thus, in the absence of pathogens, IFN-a and IL-12 play no significant signal 3 role alone. Instead, endogenous TNF signaling through TNFR2 plays a critical signal 3 role. These results have implications for CTL formation in the non-pathogen setting i.e. tumors, autoantigens and transplantation.
Infiltration of mixture of immune cells is associated with good prognosis in patients with differentiated thyroid cancer. (127.1)
Authors: Cunha Lucas , Morari Elaine , Razolli Daniela , Nonogaki Suely , Soares Fernando , Vassallo José , Ward Laura
Published: January 2023

Immune responses against differentiated thyroid carcinomas (DTC) have long been recognized but its effect on patients prognosis is a matter of debate. In order to investigate the role of immune cell infiltration in the progression of DTC, we studied 398 patients - 253 with papillary and 13 with follicular thyroid cancers, as well as 132 with nonmalignant tissues. Immune cell infiltration was identified using CD3, CD4, CD8, CD20, CD68, and FoxP3 immunohistochemical markers. We colocalized CD4/IL-17 in order to identify Th17 lymphocytic infiltration. We also colocalized CD33/CD11b in order to identify myeloid derived suppressor cells (MDSCs) infiltration. Immune cells infiltrated malignant tissues more often than benign lesions. We observed a close correlation among CLT, tumor infiltrating lymphocytes, tumor-associated macrophages and cell molecular profile, suggesting this profile may be related to antitumor immune response. The presence of chronic lymphocytic thyroiditis (CLT), CD68+, CD4+, CD8+, CD20+, FoxP3+, Th17 and MDSCs was associated with clinical and pathological features of lower tumor aggressiveness and a more favorable patient outcome. A log-rank test confirmed an association between CLT, tumor-associated macrophage infiltration, and CD8+ lymphocytes and an increased disease-free survival. In conclusion, immune cell infiltration and the presence of concurrent CLT helped characterize specific tumor histotypes and were associated with favorable prognostic features.
Inducible costimulator (ICOS) regulates the immune response in a mouse brain tumor model (127.12)
Authors: Nigam Pragati , Auffinger Brenda , Han Yu , Lesniak Maciej
Published: January 2023

Glioblastoma multiforme (GBM) is the most invasive form of brain tumors and average survival less than 14.6 months. Tumors in the central nervous system (CNS) are thought to be poorly immunogenic due to compartmentalization and immune specialization. However, presence of CD4+IL-17+ (Th17) and CD4+Foxp3+ regulatory T cells (Tregs) in GBM indicates that immune responses occur within the context of CNS-resident tumors. Inducible costimulator (ICOS) belongs to the CD28 family of costimulatory molecules. ICOS is upregulated on the surface of activated T cells and its interaction with ICOS ligand can shape diverse immune responses, such as differentiation of helper T cells and IL-10-secreting Tregs. Here, we hypothesized that ICOS plays a role in regulating T cell responses involved in tumor progression. To study the role of ICOS in tumor progression, wild-type (WT) and ICOS-deficient (ICOS-/-) mice were intracranially-implanted with murine GL261 glioma cells. The median survival of tumor-bearing ICOS-/- mice was reduced to 25 days from 30 days in WT mice. The data shows a two-fold reduction in frequency of intracranial Tregs in tumor-bearing ICOS-/- mice (p<0.01). Additionally, the frequency of intracranial CD4+IFN-g+ T cells increased 3-fold (p<0.02), accompanied by a trend for increased in Th17 cells in tumor-bearing ICOS-/- mice. Collectively this indicates a role for ICOS in regulation of brain tumor-specific immune response and provides rationale for further investigation.
Characterization of human monocyte-derived dendritic cells in the presence of leukemic cell soluble products (127.14)
Authors: Motta Juliana , Castelo-Branco Morgana , Rumjanek Vivian
Published: January 2023

Professional antigen-presenting cells, dendritic cells (DCs) play an important role in controlling tumors, since they are able to initiate an antitumoral immune response. Some works demonstrate that products secreted by solid tumor cells can modulate DCs, however, leukemic cell products are not well studied. Considering that leukemic cells are present in the same place as monocytes (DCs precursor), we analyzed a possible influence of leukemic cell products on DC development and function. For this, monocytes from healthy donors were separated by density gradient and cultured, for 5 days, in the presence of IL-4 and GM-CSF and also with K562 supernatant (SN). In another experiment, immature DCs were cultured with TNF-α for a further 2 days. During a control differentiation, monocytes down regulate CD14, CD16 and CD68 expression and start to express CD1a. In this stage, immature DCs present high endocytic ability. In the presence of SN K562, CD14, CD16 and CD68 expression remained high and CD1a expression was low. However, the addition of SN K562 did not interfere with dextran endocytosis. If activated, DCs increase CD83, CD80 and CD86 expression. It was observed that monocytes differentiated in the presence of SN K562 and then activated expressed less CD83, although CD80 and CD86 expression were not modulated. Finally, these results suggest that products released by leukemic cells affect DC differentiation and these cells become unable to complete their development.
A novel p56lck-interacting protein expressed in CD8+ memory cells and tumor infiltrating T cells that inhibits proximal TCR-mediated signaling (127.16)
Authors: Frey Alan , Burns Jeremy , Vazquez-Cintron Edwin
Published: January 2023

The presence of tumor infiltrating CD8+ T cells (‘TIL’) demonstrates that cancers elicit immune response, however, TIL lack lytic function demonstrating tumor-induced suppression. In MCA38 adenocarcinoma, defective TIL lytic function is due to blockade of TCR signal transduction in turn due to inactivation of p56lck. We have identified an interacting partner for p56lck in nonlytic TIL: Protocadherin-18 (‘pcdh18’). In the hematopoietic system long-term, antigen-specific central memory CD8+ T cells all express pcdh18 protein. pcdh18 gene is robustly transcribed upon activation of CD8+CD44+CD62LhiCD127+ cells in vitro coincident with conversion into Em cells (CD44+CD62LloCD127+). After pcdh18 transfection into primary CD8+ effector T cells, activation causes defective: proximal TCR signaling, cytokine secretion, and lytic function, and also enhanced AICD, a phenotype that mimics that of freshly-isolated nonlytic TIL. pcdh18 has a motif at Y842 (QGQY*QP) which is highly homologous to the inhibitory tyrosine motif found in p56lck at Y505 (EGQY*QP). Y842 is phosphorylated in nonlytic TIL upon activation, but not in lytic TIL implying an association between Y842 phosphorylation and lytic function. Y842 was shown by site-directed mutagenesis to be required for the pcdh18 inhibitory phenotype in transfected T cells. Thus, we have identified an activation marker of CD8+ central memory T cells which functions as a TCR inhibitory signaling receptor and therein effector phase function.
IL-17-producing CD8+ T cells induce myeloid-derived suppressor cell chemotaxis and correlate with human cancer progression (127.2)
Authors: Zou Quanming , Zhuang Yuan , Zhao Yongliang , Chen Weisan , Pang Ken C.
Published: January 2023

Tumor progression is a multistage process involving cross-talk between tumor-infiltrating immune cells and tumor cells within the tumor microenvironment. IL-17-producing CD8+ T cells (Tc17 cells) represent a newly discovered T-cell subset, but their nature, regulation, function, and clinical relevance in human tumors are presently unknown. We found that Tc17 cells accumulated in tumors of 103 gastric cancer patients, and that increased Tc17 cell percentage independently predicted reduced overall survival. Tumor-activated macrophages secreted IL-6, IL-1β, and IL-23, which together favored Tc17 cell polarization, and this process was negatively regulated by tumor-infiltrating regulatory T cells (Tregs) through a cell contact-dependent mechanism. Supernatants from Tc17 cell cultures induced CXCL12 chemokine production by tumor cells, which in turn promoted CXCR4-dependent migration of myeloid-derived suppressor cells (MDSCs) and subsequent impairment of anti-tumor CD8+ cytotoxic T cell (CTL) function via a cell contact-dependent mechanism. Collectively, this study identifies a novel regulatory network involving Tc17 cells, which in conjunction with Macrophage, tumor cells, and MDSCs exert an immunosuppressive effect within the tumor microenvironment. Efforts to inhibit Tc17 cell activity might therefore prove a valuable strategy in anti-cancer therapy.
A new potential target for enhancing the anti-tumor T cell response: Lysophosphatidic acid signaling inhibits CD8+ T cell activation (127.29)
Authors: Oda Shannon , Fujiwara Yuko , Oravecz Tamas , Tigyi Gabor , Pelanda Roberta , Torres Raul
Published: January 2023

Lysophosphatidic acid (LPA) is a lysophospholipid that is present at low nanomolar concentrations in the plasma of healthy individuals. It has been well documented that many cancers aberrantly produce LPA and the increased level of LPA has been shown to be beneficial to the tumor, promoting tumorigenesis, invasion, metastases, and vascularization. However the effects of elevated levels of LPA on the adaptive immune response to the tumor have not been addressed. The immune system, and T cells in particular, are important for their ability to recognize and eliminate nascent tumors in cancer immunosurveillance and tumor rejection. However multiple inhibitory mechanisms within the tumor microenvironment have been described that protect the tumor from T cells. Work in our laboratory has found that LPA inhibits CD8+ T cell activation in vitro and in vivo. Specifically, we have demonstrated that CD8+ T cells express an LPA receptor that, in the presence of LPA, is able to inhibit T cell receptor (TCR) signaling and subsequent cell activation, proliferation and function. These data document that LPA is able to negatively regulate T cell activation and function. We have preliminary data that increased LPA production by tumors functions to protect the tumor from CD8+ T cell-mediated eradication. If true, these findings would represent the first report of lysophospholipid-mediated protection of tumor from adaptive immunity and highlight a potential target for cancer treatment.
Specific recruitment of gammadelta regulatory T cells by tumor cells in human breast cancer (127.38)
Authors: Ye Jian , Ma Chunling , Peng Guangyong
Published: January 2023

Understanding the role of different subtypes of tumor-infiltrating lymphocytes (TILs) in the tumor immunosuppressive microenvironment is essential to cancer treatment and elimination. We recently discovered that enriched gammadelta1 T cell populations in breast cancer TILs can suppress naïve and effector T cell responses and block the maturation and activities of dendritic cells. However, the mechanism(s) governing the gammadelta regulatory T (Treg) cell increase in breast cancer patients remains elusive. In this study, we showed that IP-10 secreted by breast cancer cells significantly attracted the migration of gammadelta Treg cells. Using the loss-of-function assay with neutralizing antibodies against the chemokines secreted by breast cancer cells, we demonstrated that IP-10 was the only functional chemokine that attracts the migration of gammadelta Treg cells by breast cancer cells. We further demonstrated that human breast tumor cells also can attract gammadelta Treg cells in vivo in NOD-SCID mice using live cell imaging systems. In addition, neutralization of IP-10 and CXCR3 significantly inhibited the migration and trafficking of gammadelta Treg cells to the breast tumor sites in vivo. These studies identify the mechanism responsible for the accumulation of gammadelta Treg cells in breast cancer patients and provide novel immunologic approaches capable of preventing the trafficking and generation of gammadelta Treg cells in breast tumor suppressive microenvironments.
Natural cytotoxic anti-tumor antibodies against NeuGcGM3 in healthy donors (127.4)
Authors: Hernandez Ana , Rodríguez Nely , Martínez Darel , Rondón Teresa , Griñán Tania
Published: January 2023

N-glycolylated (NeuGc) gangliosides are not naturally expressed in normal human tissues but are overexpressed in several tumors and have immunosuppressive capacity, contributing to cancer progression. The present work shows the existence of a natural antibody response against NeuGcGM3 in healthy donors. 64 of the 100 donors tested had antibodies that specifically recognized NeuGcGM3 and killed tumor cells expressing the antigen by a complement mediated mechanism. Interestingly, even after complement inactivation 17% of the positive sera showed a direct cytotoxic effect on the tumor cells. This cytotoxicity was dependent on the presence of the antigen on the tumor cells and resembles an oncotic necrosis kind of cell death. The levels of anti-NeuGcGM3 antibodies in the sera of healthy donors and the percentage of donors with this natural immunity decreases with age. Furthermore we could only detect this reactivity in the sera of 5 from 51 lung cancer patients that matched in sex and age the assessed healthy donors. Non-small cell lung cancer patients vaccinated with an idiotypic antibody that mimics NeuGcGM3 elicited specific anti-tumor cytotoxic antibodies and showed longer survival times than non-responder patients. These results suggest the existence of natural antibodies against NeuGcGM3, with anti-tumor immune surveillance functions, that can be boosted in cancer patients, reinforcing the importance of N-glycolylated ganglioside as anti-tumor targets.
Ovarian cancer progression is controlled by phenotypic changes in dendritic cells (127.7)
Authors: Conejo-Garcia Jose , Scarlett Uciane , Rutkowski Melanie , Baird Jason , Fiering Stephen
Published: January 2023

We have characterized the initiation and evolution of the immune response against a new p53-dependent model of aggressive ovarian carcinoma. In these previously healthy tumor-bearing hosts, we detect measurable anti-tumor immunity from very early stages, which is driven by infiltrating dendritic cells (DCs) and prevents steady tumor growth for prolonged periods. Coinciding with a phenotypic switch in expanding DC infiltrates, tumors become noticeable and aggressively progress to terminal disease in a comparatively much shorter time. Notably, tumor cells remain immunogenic at advanced stages, but the responsiveness of anti-tumor T cells decreases during tumor progression. Most importantly, their residual protective immunity is abrogated by functionally different microenvironmental DCs massively recruited to advanced tumors. Correspondingly, depleting DCs early in the disease course accelerates tumor initiation, but DC depletion at advanced stages significantly delays aggressive malignant progression. Our results indicate that aggressive ovarian cancer progression after a comparatively much longer latency period is primarily driven by both the activity of immunosuppressive microenvironmental DCs and intrinsic T cell paralysis, rather than loss of immunogenicity.
Autoantibody profiling from ANA positive population to identify individual at increased risk for systemic lupus erythematosus (159.1)
Authors: Wang Ling , Raj Prithvi , Karp David , Wakeland Ward , Olsen Nancy , Li Quanzhen
Published: January 2023

Antinuclear antibodies (ANA) are positive in over 90% systemic lupus erythematosus (SLE) patients but are also present in about 20% normal population. In order to delineate the correlation between ANA expression and the risk for lupus, we investigated the autoantibody profiles that are best associated with ANA in a cohort of 106 samples with different ANA titers from SLE, incomplete lupus (ILE) and normal controls (NC), using a slide-based autoantigen array bearing 80 specificities. The antibodies which showed best correlation with ANA were the IgG AutoAbs against RNA-associated antigens (Ro, La, Sm, snRNP) and histones (H3, H1, H2A, H4). There are only 3 autoAbs (H1, H2B, Scl-70) showed significant increase in ANA+ NC group compared with ANA- NC (p<0.05), while more than a dozen IgG AutoAbs, including Ro, La, Sm/RNP, H1, H2B, H4, dsDNA, chromatin, collagen VI, CENP, were significantly increased in SLE compared with ANA+ NC (p<0.05). ILE patients have similar autoAb profiles to SLE with the exception of anti-chromatin and anti-U1snRNP. More IgM autoAbs were expressed in ILE than SLE but none of them were correlated with ANA. This data suggested that ANA+ individuals are usually accompanied by an increased number of IgG autoAbs. When more than 5 AutoAbs appeared, especially the transition from anti-histone to anti-RNA and anti-DNA antibodies may indicate an increased risk for SLE. Monitoring the autoAbs in ANA+ population may be beneficial for early diagnosis of lupus.
Anti-U1C autoantibody interferes with the U1C-mediated splicing of both Ang1 and Tie2 genes in human pulmonary artery smooth muscle cells (159.10)
Authors: Komai Koichiro , Okuyama Tatsuki , Sasaki Yohei , Hashiramoto Akira , Shiozawa Kazuko , Shiozawa Shunichi
Published: January 2023

Purpose: We previously showed that the frequency of the splicing variant of Angiopoietin-1 gene with nt805GGT insertion (Ang1/ins) was significantly increased in the Japanese patients with MCTD and those with PH (Shiozawa K et al., Arth. Rheum. 52: S283, ‘05). Because anti-U1RNP Ab is highly associated with the patients with MCTD, we investigated the correlation between anti-U1RNP Ab and the splicing of Ang1. Although we found that anti-U1C Ab specifically affects the splicing of Ang1 (Komai K et al., J. Immunol. 186: 44.3 (Abstr) '11), other genes also might be affected because U1RNP is the general splicing factor. We have now studied the contribution of anti-U1C Ab to the splicing of endothelial tyrosine kinase receptor Tie2 gene which codes the receptor of Ang1. Methods: Anti-U1C Abs were introduced into human pulmonary artery smooth muscle cells using PULS-in (Polyplus) with recombinant proteins of U1A, U1C or U1-70k respectively. After 24hrs, Tie2 mRNAs were obtained by RT-PCR. Results: Several Tie2 variants were induced with anti-U1C Ab only in the case with the introduction of U1C recombinant protein. Major products were 1100, 900, 600, 550, 450 and 400bp fragments. The 550 bp fragment was the Tie2-468 variant which was identified previously (Jin P et al., Arth. Res. & Ther. 10: R73, '08). Conclusions: Our results suggest that anti-U1C Ab interferes with the splicing of both Ang1 and Tie2 genes and induced variants may affect the pathogenesis of MCTD or those with PH.
Anti-dengue virus nonstructural protein 1 antibodies cause endothelial cell damage via ceramide-regulated GSK-3 and NF-kappaB activation (159.19)
Authors: Chen Chia-Ling , Lin Chiou-Feng , Wan Shu-Wen , Wei Li-Shiung , Lin Yee-Shin
Published: January 2023

Immunopathogenesis of dengue virus (DV) infection is involved in hemorrhagic syndrome resulted from coagulopathy and vasculopathy. We have proposed a mechanism of molecular mimicry in which antibodies (Abs) against DV nonstructural protein 1 (NS1) cross-react with human endothelial cells and cause NF-κB-regulated inflammation and NO-regulated apoptosis. However, the involvement of membrane sphingolipid signaling following the binding of anti-DV NS1 Abs to endothelial cells is unresolved. We found that anti-DV NS1 caused the formation of lipid raft-like structure and that disrupting the lipid raft formation by methyl-β-cyclodextrin decreased NO production and apoptosis. Treatment with anti-DV NS1 Abs elevated ceramide generation in lipid rafts. Pharmacologically inhibiting acid sphingomyelinase (ASMase) decreased anti-DV NS1-mediated ceramide and NO production as well as apoptosis. Exogenous ceramide treatment caused biogenesis of inducible NO synthase (iNOS)/NO and apoptosis through a NF-κB-regulated manner. Furthermore, activation of glycogen synthase kinase (GSK)-3 was required for ceramide-induced NF-κB activation and iNOS expression. Notably, anti-DV NS1 Abs caused GSK-3-activated NF-κB and iNOS expression which was regulated by ASMase. These results suggest that anti-DV NS1 Abs bind to endothelial cell membrane and cause inflammation and apoptosis via a mechanism involving the ASMase/ceramide/GSK-3/NF-κB/iNOS/NO signaling pathway.
B lineage-specific deletion of Bim leads to lymphoproliferative and autoimmune disease (159.23)
Authors: Wright Jacqueline , Clark Emily , Takeuchi Osamu , Zinkel Sandra , Satterthwaite Anne , Khan Wasif
Published: January 2023

Autoreactive B cells are known to contribute to the onset and progression of a variety of autoimmune diseases including systemic lupus erythematosus (SLE). To prevent this, the B cell receptor (BCR) exerts specificity-based quality control and B cell activating factor receptor (BAFF-R) maintains homeostasis primarily through controlling apoptosis. An appropriate balance in the levels of the pro- and anti-apoptotic members of the Bcl-2 family is central to this process. Using mice with B lineage-specific deletion of the pro-apoptotic gene Bim (B-bim-/-), we showed that B cell autonomous dysregulation of apoptosis is sufficient to cause systemic autoimmune disease with lymphoproliferation. B-bim-/- mice display progressive splenomegally and lymphoadenopathy with a significant increase in B cells as well as an overall increase in innate and T cells which display markers of activation. B-bim-/- mice had higher serum levels of autoantibodies and proinflammatory cytokines. These mice also displayed increased lymphocyte proliferation and infiltration of the salivary gland, lung and liver. Furthermore, accumulation of lymphocytes in the B-bim-/- mice results in the formation of tertiary lymphoid structures. These results suggest that apoptosis resistant B-bim-/- B cells can acquire an activated phenotype and become pathogenic, culminating in the activation of T cells and the generation of a pro-inflammatory microenvironment leading to systemic autoimmunity. In addition, massive lymphoproliferation and inflammation may predispose B-bim-/- mice to B cell and other hematopoietic malignancies.
Interaction of TAPP adapter proteins with phosphatidylinositol (3,4)-bisphosphate regulates B cell activation and autoantibody production (159.5)
Authors: Jayachandran Nipun , Landego Ivan , Gibson Ian , Miller Angela , Wullschleger Stephan , Alessi Dario , Marshall Aaron
Published: January 2023

Activation of PI3-kinase enzymes through the BCR is essential for B cell development and function. Active PI3Ks generate D3 phosphoinositide PI(3,4)P2 that bind to signaling molecules such as TAPP (Tandem PH domain containing protein) adaptor via their C terminal PH domain. This interaction leads to translocation of TAPPs to the plasma membrane. However, the role of TAPP adaptors in regulating PI3K signaling in B cell is not completely understood. We have performed studies on TAPP mutant mice, where a mutation was introduced to the C terminal domain of both TAPP1 and TAPP2 to determine the effect of uncoupling TAPPs from PI(3,4)P2. The studies showed an enhanced B cell activation and elevated serum antibody levels in these mice, suggesting an inhibitory role associated with TAPPs in B cells. To test whether the aberrant responses observed in B cells leads to autoimmunity in these animals, we analyzed their serum and observed a progressive increase in anti-dsDNA antibodies in the aging cohort. This along with other observed characteristics such as the presence of anti-nuclear antibodies within the serum, progressive development of lymphopenia and glomerulonephritis in aged mice is reminiscent of lupus. We are currently working to define the cellular interactions involved in this autoimmune phenotype and the signaling mechanism associated with the inhibitory role of TAPPs.
Lipoteichoic acid induces B cell activation in female MRL/lpr (fas) mice: implications for lupus cerebritis (159.6)
Authors: Dasgupta Subhajit , Molano Ivan , Bandyopadhyay Mausumi , Kindy Mark
Published: January 2023

Activation of B cells, production of autoantibodies and formation of immune complex with complements are hallmark immune aberrations in lupus. The female MRL/MpJ (Faslpr/ Faslpr) mice develop spontaneous lupus-like symptoms and distinct brain specific neuronal dysfunction. These mice are recognized to study mechanism of immune responses in lupus and cerebritis. We found, elevated mRNA expression of TLR2, IL6 and IL17F in spleen tissues from 22 weeks old MRL mice with progression of disease. The flow cytometry analysis demonstrated an expansion of activated CD19+CD86+ B cells in LTA injected MRL/lpr mice. The expanded B cell pool was found less IgM+B220+ cells in 22 weeks old MRL/lpr-LTA mice than the age matched untreated mice. The IgM-IgG+B220+ cells were found higher in LTA treated mice. The LTA injection also induced an expansion of CD21+CD23+ B cells in 22 weeks old MRL/lpr mice. Studies are underway to determine mechanism of TLR2 and IL17F mediated signals on auto reactive B cell activation and switch in balance from tolerance to antigen specific effector immune responses during onset of lupus as well as in distinct brain specific cerebritis.
CD45RBlo 122lo autoantibody-inducing CD4 T cell (aiCD4 T cell) causes systemic lupus erytematosus (SLE) (159.7)
Authors: Miyazaki Yumi , Tsumiyama Ken , Shiozawa Shunichi
Published: January 2023

Objective: We have repeatedly immunized non-autoimmune-prone mice with the antigen to show that a novel T cell type which we termed an autoantibody-inducing CD4 T (aiCD4 T) cell was generated via TCR revision, and this aiCD4 T cells induced autoimmunity, i.e., SLE. We show here that the aiCD4 T cell, required for the generation of not only varieties of autoantibodies but also CTL and tissue injuries, belongs to CD45RBlo 122lo CD4 subpopulation. Methods: BALB/c mice were immunized 12x with OVA, and were depleted of CD4 T cells by anti-CD4 antibodies. The aiCD4 T cell of the mice immunized 12x with KLH was transferred into the mice immunized 8x with OVA to prove aiCD4 T cell-help. Proteinuria and IFNγ-producing CD8 T cell in spleen were examined. To assign CD number on aiCD4 T cell, surface markers were examined after 12x immunization with OVA. Results: In the BALB/c mice, CTL remained immature after immunization 8x with OVA. However, the transfer of CD4 T cells of the mice immunized 12x with KLH matured CTL and induced lupus nephritis. This was due to the aiCD4 T cells generated after repeated immunization capable of inducing autoantibodies. We then adoptively transferred subfractions of CD4 T cells into naïve recipients to identify the CD marker of aiCD4 T cell. We found that the aiCD4 T cells capable of inducing RF and anti-dsDNA antibody existed in CD45RBlo 122lo subfraction. Conclusion: The aiCD4 T cell that causes SLE belongs to CD45RBlo 122lo CD4 T subpopulation.
Thymocyte- and B-cell derived vesicles, incorporated into IgG2a-bound complexes, activate autoreactive B cells through a BCR/TLR-dependent mechanism (159.8)
Authors: McCahan John , Bossaller Lukas , Marshak-Rothstein Ann
Published: January 2023

B-cells from AM14 transgenic mice are a model of self reactive B-cells. They express an antigen receptor specific for IgG2a and make a class of autoantibody known as rheumatoid factor (RF). We had previously reported that immune complexes (ICs) composed of IgG2a-chromatin or IgG2a-RNA can stimulate AM14 B-cells by the synergistic engagement of the antigen receptor and a member of the Toll-like receptor (TLR) family. The source of the antigen in this system is derived from the co-culture itself but it is unclear in what form the auto-antigen exists. Using biotinylated syngeneic cells and an IgG2a anti-biotin monoclonal antibody (mAb) we found that the mAb together with remarkably low numbers of biotinylated B-cells or biotinylated-thymocytes induced substantial AM14 B-cell activation and proliferation. This effect was mostly dependent on the synergistic engagement of the BCR and TLR9, and to a lesser extent on TLR7. Interestingly, we saw even stronger proliferative responses with the cell free portion of the biotinylated-B-cell or biotinylated-thymocyte culture supernatants in this system. Confocal microscopy showed, that vesicles released from biotinylated cells are delivered via anti-biotin IgG2a antibodies to AM14 B- cells. Our findings suggest that self-reactive B-cells can be stimulated by auto-antigen in association with cell-derived vesicles through a TLR 7/9 mediated process.
Dual infections with Mycobacterium avium ss. paratuberculosis and bovine leukemia virus alter regulatory T cell prevalence and activity in cattle. (160.12)
Authors: Coussens Paul , Roussey Jonathon , Murphy Brooke
Published: January 2023

Johne’s disease (paratuberculosis) is a chronic disease of domestic ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP) that currently ranks as one of the most costly diseases in the U.S. Dairy Industry. We proposed that the chronic nature of Johne’s disease would result in development of regulatory T cells (Tregs). Tregs would shift immune responses away from a pro-inflammatory Th1 to an unproductive Th2 response. In the course of studies on Tregs and MAP, we uncovered a confounding effect due to bovine leukemia virus (BLV) infection. Healthy control cattle displayed significant FoxP3 (Tregs) staining in mesenteric lymph nodes, but lymph nodes from BLV infected cattle were devoid of FoxP3 expressing cells. Concurrent infection with MAP partially restores FoxP3 expressing cell populations. BLV infection also reduces TGFβ (2-fold reduction) mRNA abundance in mesenteric lymph nodes, while concurrent infection with MAP partially restores TGFβ mRNA levels. In peripheral blood mononuclear cells, BLV infection reduces abundance of TGFβ, but dramatically enhances abundance of IL-10 mRNA. Taken together, our results suggest that BLV alters immune profiles to favor progression of MAP infection. Recent estimates suggest over 83% of U.S. dairy operations are currently positive for BLV and approximately 68% are positive for MAP. If concurrent infection with these two pathogens increases losses due to MAP, then the economic consequences could be staggering.
Studies on the biological function of virus-like particles of foot and mouth disease (160.13)
Authors: Chang Yu-Ching , Cheng Yung-Chih , Wang Ting-Fang , Liang Shu-Mei
Published: January 2023

Foot and mouth disease (FMD), a severe and highly contagious viral disease of cloven-hoofed animals, is the most economically devastating livestock disease in the world. The development of a cheap and noninfectious virus-like particle (VLP) vaccine has now attracted worldwide interest. In this study, we used SUMO fusion technology to produce a VLP of FMD virus (FMDV). The purified FMD-VLPs morphologically resembled FMD virions and were able to interact with host receptors in a manner similar to FMDV. Immunization of guinea pig or swine with FMD-VLPs resulted in the production of anti-FMDV neutralizing antibodies. RT-PCR analysis of peripheral blood mononuclear cells (PBMC) from the FMD-VLPs-immunized swine revealed that FMD-VLPs enhanced the transcription of Th1-like cytokines (IFN- and IL-2) and Th2-like cytokines (IL-4 and IL-10). Further investigation of the immune mechanisms revealed that FMD-VLPs induced maturation of dendritic cells (DCs) via toll-like receptors 2 (TLR2) to induce CD4+ and CD8+ T proliferation. These results suggest that FMD-VLPs could be used as an effective vaccine candidate against FMDV.
The canine MHC class I allele, DLA-88*50801, presents peptides of varying lengths with three key anchor residues (160.14)
Authors: Ross Peter , Buntzman Adam , Holmes Jennifer , Gojanovich Greg , Miller Keith , Collins Edward , Frelinger Jeffrey , Hess Paul
Published: January 2023

The binding of a peptide to a major histocompatibility (MHC) class I allele depends upon a few key residues within the peptide sequence, which is present only in ~1-2% of peptides in an average protein. Identification of this motif allows one to select candidate epitopes from a protein that may be recognized by cytotoxic T lymphocytes (CTL). While several studies have determined the binding specificities of numerous class I alleles in both humans and mice, no such study has been performed in the domestic dog. The principal aim of this study was to determine the peptide binding motif of a prevalent MHC class I allele, Dog Leukocyte Antigen (DLA) -88*50801. We hypothesized that a conserved binding motif could be identified through sequencing of endogenously presented peptides eluted from DLA-88*50801. To accomplish this goal, a plasmid encoding the DLA-88*50801 gene with an attached flag tag was stably transfected into a canine cell line (DH82). DLA-88*50801 protein was obtained through affinity purification of lysate from ~8 x 109 cells. Bound peptides were eluted from the MHC binding groove utilizing low pH and heat, isolated by size exclusion and affinity purification, and subjected to tandem mass spectrometry analysis to obtain individual peptide sequences. Sequences were manually aligned to determine the binding motif. This study provides essential information to study CTL responses in dogs.
Chemokine-mediated B cell trafficking in rabbit GALT during diversification of the primary antibody repertoire (160.15)
Authors: Lanning Dennis , Knight Katherine
Published: January 2023

Rabbits preferentially rearrange the 3’-most VH gene segment during VDJ gene rearrangement. Early in life, rabbit B cells generate a diversified primary antibody repertoire by mutating their VDJ genes in gut-associated lymphoid tissue (GALT) in response to signals acquired from intestinal commensals. To gain insight into the cell-cell and cell-microbe interactions required for VDJ gene diversification, we examined B cell surface expression levels of four chemokine receptors that mediate trafficking to important sites within GALT during repertoire diversification. We stained the chemokine receptors CXCR4, CCR6, CXCR5 and CCR7 on appendix B cells from two- through seven-day-old rabbits with fluorescence-labeled antibodies or Ig-fusion proteins of their ligands and analyzed them by flow cytometry. The changes in relative chemokine receptor expression levels we observed during the first week of life suggest that B cells, upon entering appendix follicles, first traffic to the follicle-associated epithelium, where they likely encounter commensal bacteria sampled from the intestinal microbiota. B cells subsequently migrate to, and likely receive additional signals from, the follicular dendritic cell network. Upon becoming activated, B cells then traffic to the basal region of the follicle, where they proliferate and diversify their VDJ genes to generate the primary antibody repertoire.