bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiplex measurement of inflammatory cytokines in human plasma using a Fluorokine® MAP multiplex assay (124.5)
Authors: Schmidt Jane , Johnson Paige , Anderson Michael
Published: January 2023

Fluorokine® Multianalyte Profiling (MAP) Kits offer the ability to simultaneously measure multiple analytes with less operator time, less sample consumption, and lower cost per result compared to conventional ELISAs. To demonstrate this, we cultured whole blood samples from apparently healthy donors for up to 24 hours without stimulation or stimulated by LPS, PHA, or PMA. Samples were taken from the cultures and processed into plasma. GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, TNF-α, and VEGF were quantified simultaneously using the Human Fluorokine MAP High Sensitivity Cytokine Panel to compare cytokine profiles in stimulated and unstimulated cultures. The Human Fluorokine MAP High Sensitivity Cytokine Panel is a fully validated multiplex assay designed to measure up to twelve inflammation biomarkers in serum or plasma; the specific analytes are selected by the user. Analyte-specific antibodies pre-coated onto color-coded microparticles capture the analytes of interest in standards or samples. After washing away any unbound substances in a filter-bottom microplate, a cocktail of biotinylated antibodies also specific to the analytes of interest is added to each well, followed by a streptavidin-phycoerythrin conjugate. Following a final wash, microparticles are resuspended in buffer and read using a Luminex®-based analyzer. The assay is fully validated for accurate, precise, and reproducible results using serum or plasma samples.
Optimiser™ Microplate enables simultaneous multi-analyte quantitation using ultra-low sample volumes in a 2-hour assay: Validation of a Novel 10-Analyte Th17 Cell Panel (124.7)
Authors: Kai Junhai , Moore Victor , Puntambekar Aniruddha , Sehy David , Lakkis Montaha , Lee Sean , Han Jungyoup , Ahn Chong
Published: January 2023

Optimiser™ Microfluidic ELISA Plates combine microfluidics with 96-well architecture to offer dramatic improvements over conventional ELISA, such as 50fold-reduced sample volume, 2fold-reduced assay time, and ultra-high sensitivity, while still using conventional ELISA reagents. Optimiser™ ELISA Plates are compatible with existing pipetting systems (manual or automated) and ELISA plate readers. Furthermore, the surface area to volume ratio of each microfluidic reaction chamber represents a 50-fold increase when compared to standard plate microwells. The improvement in surface:volume ratio increases binding kinetics dramatically, enabling assays to be run in less than half the time of traditional ELISAs, and providing comparable sensitivity with as little as 2 microliter samples (i.e., 50fold less than required for conventional assay). A multi-analyte assay for detecting products of activated Th17 cells (e.g., IL-6, IL17A, IL-17AF, IL-17FF, IL-21, IL-22, IL-26, G-CSF, CCL20, TNF-α) is used to demonstrate that using only 25 microliter sample volume, the Optimiser™ ELISA can simultaneously measure 10 analytes (with sensitivity comparable to conventional ELISA) in a 2-hour assay, without the need for dedicated multiplexing instruments, multiplexing reagents, or bead-labeling chemistry. The entire 10-analyte assay procedure is fully automated and is run on a Tecan EVO100™ System.
A novel mechanism responsible for IgE regulation involves beta2-adrenergic receptor-mediated regulation of CD23 and ADAM10 localization to B cell-derived exosomes. (125.11)
Authors: Padro Caroline , Sanders Virginia
Published: January 2023

Allergic asthma severity positively correlates with plasma IgE, but physiological mechanisms responsible for regulating IgE production by B cells are unknown. We have shown that beta-2 adrenergic receptor (β2AR) engagement on a CD40L/IL-4-primed B cell increases the level of IgE produced per cell, without affecting class switch recombination. Asthma therapy involves stimulation of the β2AR on bronchiolar smooth muscle cells to relieve bronchoconstriction. This therapy also targets the β2AR expressed on primed B cells to increase IgE production, potentially intensifying symptoms. In this study we show that surface CD23 remained constant on primed B cells exposed to a β2AR agonist, while CD23 expression and soluble CD23 (sCD23), which is a positive regulator of IgE production, increased. We hypothesized that the primary sheddase of CD23, ADAM10, is regulated by β2AR engagement on a primed B cell. We found that CD23 and ADAM10 are expressed on B cell-derived exosomes over time and that blockade of exosome release allows for CD23 and ADAM10 retention, with β2AR engagement augmenting CD23 and ADAM10 retention. Localization of ADAM10 and CD23 to non-lipid raft fractions where CD23 is cleaved by ADAM10 was also increased by β2AR engagement. Thus, ADAM10 may serve as a molecular target that can be blocked during β2AR drug therapy to prevent increased sCD23 and IgE production, while maintaining the therapeutic effect of bronchorelaxation.
Immunotherapy reduces IL-33 and IL-6 expression in the lung of T-bet deficient mice in an experimental model of allergic asthma (125.9)
Authors: Mousset Stephanie , Koch Sonja , Finotto Susetta
Published: January 2023

Immunotherapy (IT) is one of the most effective therapies for increasing allergic diseases worldwide including allergic asthma. Previously, we have established a murine model of subcutaneous IT for experimental allergic asthma. We demonstrated that main components of allergic asthma induced by T-bet deficiency such as airway hyperresponsiveness could be ameliorated. These data suggested that the molecular mechanism induced by IT is T-bet independent. To analyze this yet unknown mechanism we started to investigate IL-6 expression in this model. IL-6 is a cytokine produced by several cell types of the innate as well as adaptive immune system. We previously described that IL-6 is increased in the lung cells of T-bet deficient (T-bet-/-) naïve mice as compared to wild type littermates. In a murine model of allergic asthma we found that IL-6 is significantly down-regulated in the lung of T-bet-/- mice that have been treated according to the IT protocol. Likewise, we found that the newly discovered cytokine IL-33, of the IL-1 family, is significantly up-regulated in the lung of T-bet-/- mice in experimental asthma and down-regulated after IT. In conclusion we discovered that the protective mechanism of the IT comprises down-regulation of IL-6 and IL-33. These findings could be considered for future novel therapy for allergic asthma.
Establishment of nonmyeloablative bone marrow chimerism by double negative Treg cells through inducing T cell clonal deletion and suppressing NK cell function (126.1)
Authors: Zhang Zhu-Xu , Su Ye , Huang Xuyan , Wang Shuang , Min Wei-Ping , Yin Ziqin , Jevnikar Anthony
Published: January 2023

In bone marrow transplantation, T cells and NK cells play important role for graft rejection. In addition, graft-versus-host-disease and establishment of stable chimeric states without complete marrow ablation remain as major obstacles post bone marrow transplantation. In this study, we aimed to establish mixed chimerism in a non-irradiation condition. Our data indicate that adoptive transfer of donor-derived TCRαβ+CD3+CD4-CD8-NK1.1-(double negative, DN) Treg cells prior to C57BL/6 to BALB/c bone marrow transplantation, in combination with cyclophosphamide but not cyclosporine, FK506, or rapamycin, established stable mixed chimerism that led to acceptance of C57BL/6 skin allografts and rejection of 3rd party C3H skin grafts. Adoptive transfer of CD4+ and CD8+ T cells, but not DN-Treg cells, induced graft-versus-host diseases in this regimen. The recipient T cell alloreacitve responsiveness was reduced in the DN-Treg cell-treated group and T cell receptor Vβ2, Vβ7 and Vβ8 clonal deletions were observed in both CD4+ and CD8+ T cells. Furthermore, DN-Treg cell treatment suppressed NK cell-mediated donor bone marrow rejection in perforin-dependent manner. Taken together, our results suggest that adoptive transfer of DN-Treg cells can control both adoptive and innate immunity and promote a stable mixed chimerism and donor-specific tolerance in the non-irradiation regimen.
ICOS promotes T cell activation and function beyond phosphoinositide 3-kinase signaling (126.11)
Authors: Yu Xue-Zhong , Semple Kenrick , Leconte Julien , Suh Woong-Kyung , Li Jun
Published: January 2023

ICOS delivers an important costimulation to promote T-cell activation and function. Using a knock-in mouse strain, termed ICOS-YF, in which the cytoplasmic tail of ICOS cannot activate phosphoinositide 3-kinase (PI3K), we have shown that ICOS-PI3K signaling axis is critical for generation of follicular helper T cells. Although ICOS can potentiate TCR-mediated calcium flux in a PI3K-independent manner, its biological significance is unclear. To address this question, we studied the function of ICOS-YF T cells in comparison with ICOS wild-type (WT) and knock-out (KO) T cells in MHC-mismatched bone marrow transplantation (BMT) models. We found that CD4 T cells from ICOS-YF and KO mice were similarly impaired in their capacity to induce acute graft-versus-host disease (GVHD). In contrast, the pathogenic capacity of CD8 T cells from ICOS-YF mice was comparable to that of WT cells, whereas ICOS KO CD8 T cells were less pathogenic. In vitro, we observed that in both CD4 and CD8 T cells ICOS could potentiate TCR-mediated calcium flux in a PI3K-independent manner. These results suggest that although both CD4 and CD8 T cells depend on ICOS costimulation, the downstream signaling pathways they utilize are distinct: CD4 T cells depend on ICOS-PI3K signaling whereas CD8 T cells are more dependent on PI3K-independent pathways, probably calcium signaling. Taken together, our study reveals a complexity in ICOS signaling mechanisms in GVHD.
Recovery of thymic epithelial cells and immune reconstitution after cord blood transplantation depends on angiogenesis and neovascularization (126.12)
Authors: Politikos Ioannis , Kim Haesook , Cutler Corey , McDonough Sean , Antin Joseph , Ballen Karen , Ritz Jerome , Boussiotis Vassiliki
Published: January 2023

Umbilical cord blood (UCB) is increasingly used as an alternative source for allogeneic hematopoietic stem cell transplantation (HSCT). HSCT compromises thymopoiesis due to injury of thymic epithelial cells (TEC). Human UCB is enriched in endothelial precursors, which promote neovascularization and thymopoiesis in SCID mice transplanted with human thymus. We examined whether markers of angiogenesis, ANG1 and VEGF, might correlate with recovery of TEC, which support T cell development and expansion by producing IL-7 and SCF (a kit ligand). We analyzed 27 patients pre and 1, 2, 3, 6 and 12 months after UCBT. Early after UCBT, IL-7 and SCF levels inversely correlated with TREC, CD4, CD8 and Treg numbers suggesting that uptake of these cytokines by IL-7R and Kit on T cell progenitors induced their expansion. IL-7 and SCF were also inversely correlated with T cell differentiation into pathogen-specific effectors as determined by CMV-specific, IFN-γ ELISpot. Conversely, VEGF and ANG1 positively correlated with CD4, CD4CD25 and CD4CD45RA numbers. Interestingly, VEGF and ANG1 displayed inverse correlation with SCF and more so with IL-7 at early time points after UCBT, which converted to strong positive correlation (r=0.78, p=0.0004) at 12 months, when T cell recovery had occurred. Our results are consistent with a model of TEC recovery supported by VEGF and ANG1, leading to production and uptake of SCF and IL-7 by T cell progenitors, resulting in their differentiation and expansion.
Prior pancreatic islet transplantation mitigated the subsequent skin graft rejection against HY disparity in C57BL/6 with pre-existing immunological memory (126.15)
Authors: Kim Yong-Hee , Lee Sun-Ho , Park Chung-Gyu
Published: January 2023

Several successful immunomodulatory strategies in specific pathogen-free rodents have failed in outbred larger animals by pre-existing memories. In our previous report, we showed that the islet transplantation across the minor histocompatibility of HY (male antigen) could induce immunological tolerance to male antigen in female C57BL/6 mice. In this study, we tested whether islet-transplantation-induced immunological tolerance could be achieved against the barrier of pre-existing immunological memory. Splenocytes isolated from male C57BL/6 were injected into syngeneic female mouse intraperitoneally. The induction of memory was confirmed by accelerated male skin rejection from 27 days (naïve control mouse) to 19 days. The islets isolated from male C57BL/6 mice were transplanted into renal subcapsule of the sensitized mice. 37.5% of sensitized recipients rejected male islets, whereas 100% of naïve recipients accepted the grafts. Remaining 62.5% of sensitized recipients that accepted male islets did not accept subsequent male skin graft. Although male skin graft failed to be accepted in the male islet-accepted recipients, the intensity of inflammatory response was significantly lower than that shown in presensitized mice without male islet graft. In conclusion, the islet-transplantation-induced immunological tolerance could not circumvent the barrier of immunological memory, however, it could mitigate the intensity of inflammatory rejection process.
Regulatory-B cell and regulatory-T cell collaborate to promote transplantation tolerance (126.2)
Authors: Lee Kang Mi , Kim James , Yeh Heidi , Stott Ryan , Soohoo Julie , Deng Shaoping , Markmann James
Published: January 2023

Backgrounds: The role of regulatory-B cells (Bregs) in transplant tolerance remains unclear. We found that the anti-CD45RB/anti-TIM1 induced tolerance depends on both Bregs and regulatory-T cells (Tregs). This finding offers a unique opportunity to explore the interplay of Bregs and Tregs in a transplant setting. Methods: Islet allograft were prepared from BALB/c donors and transplanted into B6. Recipients were treated with anti-CD45RB and anti-TIM1 antibodies for tolerance induction. Naive B cells or B cells isolated from mice that have been transplanted and treated in combination antibody therapy transferred into µMT-KO and assessed Treg expression by FACS. Anti-CD25 antibody, PC61, was used for Treg depletion. Anti-IL-10 or anti-TGF-β was used to neutralizing cytokine activity. Results: Treg numbers significantly increase following adoptive transfer of B cells purified from recipients bearing long-term surviving grafts (LTS). Moreover, adoptive transfer of LTS B cells suppressed allograft rejection upon transfer to untreated B cell-deficient secondary host, but this effect was not observed in Treg-deleted recipients. The absence of IL-10 by using anti-IL10 antibody did not affect on induction of long-term islet allograft by anti-CD45RB/anti-TIM1, however anti-TGF-β treatment abrogated the graft survival. Conclusions: B cell regulation induced by anti-CD45RB/anti-TIM1 is associated with Treg generation. This effect may be mediated by Treg promoting cytokines such as TGF-β.
Persistence in graft of Foxp3,TGFβ and IL-10 is hallmark of successful retransplantation of grafts after primary transplantation under cover of CD200 expression (126.21)
Authors: Gorczynski Reg , Chen Zhiqi , Yu Kai
Published: January 2023

We showed that overexpression of CD200, a molecule of the immunoglobulin supergene family, increases graft survival and suppresses inflammation/acquired immunity after binding to its receptors (CD200Rs). Skin/cardiac allograft survival is increased in transgenic mice over-expressing CD200 under control of a doxycycline-inducible promoter, (CD200tg), or following transplantation of CD200tg grafts to control mice, along with increased intra-graft expression of mRNAs implicated in Treg differentiation, including TGFβ, IL-10 and Foxp3. We have investigated whether C57BL/6 CD200tg grafts taken from control BALB/c mice at 14 days post transplantation can be successfully re-grafted into secondary control BALB/c mice, and whether such animals show perturbation of specific alloimmunity, and/or changes in graft gene expression in comparison to mice receiving control C57BL/6 grafts. Control grafts taken from mice at d14 and re-transplanted to control BALB/c mice were rejected rapidly, and with mice developing the expected CTL response in splenocytes detected by lysis of EL4 tumor targets in 4-hour 51Cr-release assays. In contrast, CD200tg grafts survived on secondary transfer to control BALB/c mice, with specific suppression of the cytotoxicitv response to EL4 targets. In these secondary recipients only, persistently high expression of Foxp3, IL-10 and TGFβ gene expression was seen. We conclude that expression of these latter genes is crucial for adoptive transfer of tolerance.
Role of MHC class II in Treg-mediated tolerance to allogeneic transplants (126.26)
Authors: LeGuern Christian , Martins Paulo , Germana Sharon
Published: January 2023

Transfer of donor MHC class II genes (MHCII) in the bone marrow of recipients of allografts induced tolerance spreading to all alloantigens. Tolerance was donor MHCII-specific, transferable only via host CD4 Tregs and selectively induced by MHCII gene therapy. Studies on the fate of transgenic MHCII molecules revealed that both the α and β chains provided peptides presented on the MHCII of bone marrow-derived antigen-presenting cells (APCs). Transgene-matched graft MHCII was critical for survival as donor MHCII-treated recipients promptly rejected MHCII-deficient transplants. Next, the role of thymic transgenic MHCII peptides on Treg development was studied in a model recapitulating MHCII peptide presentation: the IEα 52-68 peptide/IAb complexes recognized by the YA-e mAb and TEa transgenic TCR. With this model we showed that 1) the highly conserved IEα sequence was dominantly exposed on all dendritic cells located in the thymic medulla, the birthplace of Tregs, and 2) transfer of the IEα peptide into IEneg mice via bone marrow transplantation, converted host IEα-specific CD4 T cells into CD4+ CD25+ Foxp3+ Tregs. These data are consistent with a dominant role of MHCII peptides in shaping Treg TCR specificity and differentiation. They also propose a mechanism for MHCII-tolerance: MHCII-specific Tregs require activation by cognate MHCII peptide provided by MHCII-matched transplants prior to suppressing alloreactive T cells of different TCR specificity.
Rag1 and Rag2 Knockout rats to drive xenografts and oncology studies (126.30)
Authors: Xue Jeff , Keys Kelly , McCoy Aaron , Weinstein Edward
Published: January 2023

Rejection of grafted tissues by host immune systems is problematic during xenotransplantation. To help alleviate this and provide models amenable to the xenograft process, we created rats with genetic modifications of genes: Rag1 or Rag2. Rag1 and Rag2 play an important role in the rearrangement and recombination of genes of immunoglobulin and T-cell receptor molecules during the process of VDJ recombination. These rats were created via zinc finger nuclease (ZFN) targeted editing of Sprague-Dawley and F344 rat genomes. ZFN creates a double stranded break at the target locus. This lesion is repaired by the low fidelity repair mechanism non-homologous end-joining which commonly yields out of frame mutations in the gene, leading to non-functional proteins. Phenotypic characterization of these animals occurs when homozygosity is achieved. Rag1 and Rag2 protein ablation was demonstrated by western blot analysis. Flow cytometric analysis of peripheral blood cells in Rag1 KO animals at 4, 10 and 20 weeks demonstrated a significant decrease in mature T and B lymphocytes relative to age-matched wild type rats. Rag2 KO rats demonstrate a non-leaky depletion of mature T and B lymphocytes at 4 and 10 weeks. By employing the ZFN technique, we developed Rag1 and Rag2 KO rats with compromised immunology systems, which we believe will be helpful in the studies of xenografts, cancer, vaccine development, autoimmune, infectious and GVHD diseases.
Inhibition of the Heat Shock Protein receptor CD91 abrogate concomitant anti-tumor immunity (127.24)
Authors: Zhou Yu , Binder Robert
Published: January 2023

Most tumors are capable of generating immune response in the bearing host, a phenomenon described as concomitant immunity. Since tumors lack the typical (foreign) PAMPs that provide signal 2 for T cell priming, the mechanism of how concomitant immunity is established remains a mystery. We provide evidence here that, heat shock proteins (HSPs), when released into the tumor microenvironment, is the immunogen necessary for priming concomitant tumor specific immunity. These responses require CD91- the endocytic and signaling receptor for the immunogenic HSPs. Our study abrogates the interaction of tumor-derived HSP with CD91 in vivo by over-expression of a natural inhibitor of CD91. The data shows that: first, tumors expressing CD91 inhibitor grew with significantly faster kinetics than the non-inhibitor expressing counterparts in wild type mice, although this difference was non-existent in immunocompromised mice and the inherent proliferative rates of tumors with or without inhibitor were identical; second, inhibitor-expressing tumors, when used as an immunogen in tumor prophylaxis, were significantly less efficient in priming immune responses. We also examined the competition for CD91 between the inhibitor and tumor-derived HSP in vivo. In summary, our study demonstrates that the HSP-CD91 pathway is critical for establishment of concomitant immunity. We propose a novel mechanism of immune evasion for tumors expressing competing ligands for immune receptors such as CD91.
Immunogenetic Hypothesis for Cancer Survival (127.26)
Authors: Williams R.Michael
Published: January 2023

Immunosurveillance reflects an ongoing war between the immune system and the specific cancer. If the honeymoon of no detectable cancer lasts a lifetime, we say there was a cure by primary therapy. Adjuvant therapy alters the outcome statistically. Adjuvant trials are balanced for known relevant patient and tumor characteristics but NOT for the genetics of the host or the tumor. Small absolute differences seen in adjuvant trials likely reflect an imbalance in these important characteristics, and adjuvant drugs or radiation alter the immune response in addition to killing some tumor cells. In short, survival probably depends as much on the immune system as it does on the cancer treatment. We proved that survival with a histocompatible tumor was genetic and immunological. Single AA differences in H-2 can change survival. Adaptive immune responses are antigen or epitope specific and almost always involve class I and class II MHC genes. T-cells are involved and the immune response is regulated so that decreasing T-suppressor cells (Tregs) can result in increased survival (ipilumumab in melanoma). Immunization, even after the cancer and its antigens are in abundant supply, can prolong survival (Spiruleucel-T in prostate cancer). We propose that the clinical oncology community investigate the Immunogenetic Hypothesis for Cancer Survival, and predict that HLA genes and T-cell immunoregulation effects will participate importantly in how long a cancer patient will survive.
Evaluation of the serum level of soluble E -cadherin and cell surface expression of α5β1 integrin in patients with acute leukemia whether lymphocytic or myeloid ,in order to extrapolate their prognostic value (127.3)
Authors: Abdelrahman Zakia , Abo shamaa Lobna , Sadek Nadia , El sayed Amina , Mekway Nada
Published: January 2023

Background:E-cadherin is expressed on CD34+ stem cell,where it likely contributes to intracellular interactions during hematopoiesis. Also integrin such as α5β1 integrin represent an essential component of the bone marrow microenvironment that regulates cell survival .In leukemia, immature blasts are not retained within the marrow, suggesting a breakdown of adhesive mechanisms and cell migration. Objective: this study was done to evaluate serum soluble E -cadherin and cell surface expression of α5β1 integrin on PBMCs in patients with acute leukemia whether lymphocytic or myeloid Twenty patients (11 AML and 9 ALL)and ten age and sex matched normal individuals were studied. soluble E -cadherin were assayed using ELISA and cell surface expression of α5β1 integrin on PBMCs by indirect immunoflourescence . Results: Soluble E-cadherin was significantly lower in all leukemic and AML patients than control ( p=.0108,p=.0049 respectively).However no significant difference between AML and ALL. α5β1 integrin cell surface expression was significantly higher in both AML,ALL groups than control(p=.0001,0.0038 respectively). E-cadherin has negative significant correlation (r= -.573, p= .008) with bone marrow blast percentage after the induction of chemotherapy. Conclusion this suggests that a dual double way interaction between increased expression of α5β1 integrin and loss or decreased expression of E-cadherin that governs the process of tumour progression and metastasis.
Characterization of a novel mouse model for human B-cell lymphomas (127.31)
Authors: Bommireddy Ramireddy , Besselsen David , Rimsza Lisa
Published: January 2023

The risk of developing lymphoma is markedly increased after solid organ transplantation. Up to 20% of people who receive a solid organ transplant develop a lymphoma. Calcineurin (CN) A beta knockout (Cnab-/-) mice do not reject allogeneic tumors suggesting an immunosuppressive state, is also a unique model for reverse immunosurveillance. Currently, there is no mouse model for low grade lymphomas based on immune dysregulation. Since we observed a spontaneous activation of T cells, and B-cell lymphomas in CNAβ deficient mice, we hypothesize that CN is involved in T-cell regulation, the lack of regulation causes smoldering inflammation which in turn activates B cells and results in lymphomagenesis. The lymphomas in Cnab-/- mice are positive for B cell surface marker (CD21) but negative for T cell (TCRβ) markers and they are clonal in origin and exhibit malignant (destroying the normal architecture of adjoining area) phenotype. In addition, we do see spontaneous activation of T cells in Cnab-/- mice early in life and continue to accumulate, and B cells later when they are old with mild inflammatory lesions in the gut and liver in Helicobacter-free (clean) colony. Since inflammation is considered to be one of the hallmarks of cancer and also the root cause of cancer, this is a novel mouse model for understanding a slow growing, difficult to cure form of lymphomas.
Tumor-targeted T cells secreting TLR5 ligand effectively treat melanoma-bearing mice (127.34)
Authors: Geng Degui , Srivastava Ratika , Li Chaoyang , Davila Eduardo
Published: January 2023

T-cell-based therapies can kill tumor cells expressing immunodominant tumor antigens (TAg). However, this process allows the escape and metastases of tumor cells expressing low levels of TAg or expressing subdominant TAgs. Our preliminary data and published reports indicate that stimulating TLR2 or TLR5 directly on tumor-reactive T-cells reduces the activation threshold to poorly immunogenic TAgs. Genetically engineering T-cells expressing the MART-1 TCR and producing the TLR5 ligand flagellin (Fg) will generate potent and long-lived antitumor activity by costimulating T-cells and antigen-presenting cells at the tumor site. Human T-cells engineered to express the MART-1 TCR-Fg showed significantly greater proliferation, survival, and cytolytic activity than MART-1 T-cells. TLR5-stimulated T-cells responded to 10-to-50 times lower levels of TCR stimulation. TLR5 stimulation also augmented the production of various cytokines including IL-2, GM-CSF, IFN and TNF. Unexpectedly, we found that TLR5 stimulation on melanoma tumors induced the production of cytokines and chemokines which further promoted the recruitment of CD4 and CD8 T-cells, and NK cells. We are currently examining the therapeutic efficacy of using TCR-Fg T cells to treat NOD-scid IL2Rgammanull (NSG) mice bearing melanoma tumors.
Thymosin beta 4 is involved in interleukin 18 enhanced interferon gamma secretion in human NK cell line, NK-92MI (127.8)
Authors: Lee Ha-Reum , Cho Daeho
Published: January 2023

Thymosin beta 4 (Tβ4), which is the major G-actin sequestering protein, has been shown to have ubiquitous distribution and multiple biological activities. However, what regulates Tβ4 expression and what its function is on natural killer (NK) cells are still unknown. In this study, recombinant human IL-18 (rhIL-18) enhanced the endogenous level of Tβ4 through p38 MAPK and JNK signaling pathway in the human NK cell line, NK-92MI. IFN-γ production is stimulated by over-expression of endogeneous Tβ4. Furthermore, pretreatment with an inhibitor for Tβ4 decreased IL-18-enhanced IFN-γ secretion, and transfection with Tβ4-specific short hairpin RNA resulted in reduction of IFN-γ production in primary NK cells as well as in the human NK cell line. This suggests that Tβ4 is regulated by IL-18 and is involved in IL-18-enhanced IFN-γ secretion in NK cells.
Protection of Irf5-deficient mice from pristane-induced lupus involves altered cytokine production and class switching (159.11)
Authors: Barnes Betsy , Feng Di , Yang Lisong , Bi Xiaohui , Stone Rivka , Patel Priya
Published: January 2023

Polymorphisms in the transcription factor interferon (IFN) regulatory factor 5 (IRF5) have been identified that show strong association with increased risk of developing the autoimmune disease systemic lupus erythematosus (SLE). A potential pathologic role for IRF5 in SLE development is supported by the fact that increased IRF5 mRNA and protein abundance are observed in primary blood cells of SLE patients that correlate with increased risk of developing the disease. Here, we demonstrate in clean littermates that IRF5 is required for pristane-induced lupus via its ability to control multiple facets of autoimmunity. We show that IRF5 has a distinct influence on pathological hypergammaglobulinemia and provide evidence for its role in regulating IgG1 class switching and antigen specificity. Examination of in vivo cytokine expression (and autoantibody production) identified an imbalance in Irf5-/- mice favoring Th2 polarization. In addition, we provide clear evidence that loss of Irf5 significantly weakens the in vivo type I IFN signature that is critical for disease pathogenesis in this model of murine lupus. Together, these findings demonstrate the global effect that IRF5 has on autoimmunity and provides significant new insight into how overexpression of IRF5 in blood cells of SLE patients may contribute to disease pathogenesis.
The immunodominant CD4+ T cell epitope of myeloperoxidase induces cell mediated injury in murine anti-myeloperoxidase glomerulonephritis (159.14)
Authors: Kitching A Richard , Ooi Joshua , Chang Janet , Holdsworth Stephen
Published: January 2023

Microscopic polyangiitis, characterized by pathogenic autoantibodies to neutrophil cytoplasmic proteins (ANCA), including myeloperoxidase (MPO), frequently targets the glomerulus. Although the pathogenicity of ANCA has been established, the role of CD4+ cells, cell mediated effector responses and MPO T cell epitopes are not known. By screening overlapping peptides of 20aa spanning the entire MPO molecule, we identified an immunodominant MPO CD4+ cell epitope (MPO409-428). Immunization with MPO409-428 induced disease similar to that seen with whole MPO when disease was triggered with anti-glomerular basement membrane antibodies. A CD4+ MPO409-428 specific clone transferred into Rag1-/- mice induced necrotizing glomerulonephritis with renal impairment when MPO409-428 was planted in glomeruli, or when whole MPO was deposited in glomeruli after neutrophil recruitment induced by transfer of anti-MPO antibodies with LPS. MPO409-428 also induced biologically active anti-MPO antibodies in mice. The MPO409-428 T cell epitope has a minimum immunogenic core region of 11 aa, MPO415-426, with several critical residues. These studies identify an immunodominant MPO T cell epitope and redefine how effector responses induce injury in MPO-ANCA associated microscopic polyangiitis. ANCA activate neutrophils not only induce injury, but also lodge the autoantigen MPO in glomeruli, where autoreactive CD4+ cells act to induce delayed-type hypersensitivity like necrotizing glomerular lesions.
IL-10-producing regulatory B10 cells ameliorate collagen-induced arthritis via suppressing Th17 cell generation (159.27)
Authors: Yang Min , Lu Liwei
Published: January 2023

IL-10-producing CD1dhiCD5+ B cells, namely B10 cells, have been shown to possess a regulatory function in inhibiting immune responses, but whether and how B10 cells suppress the development of autoimmune arthritis remains largely unclear. In this study, we detected significantly decreased IL-10-producing B cells but increased Th17 cells in both spleen and draining lymph nodes of mice at the acute stage of collagen-induced arthritis (CIA) when compared with adjuvant-treated control mice. Upon adoptive transfer of in vitro-expanded B10 cells, collagen-immunized mice exhibited a marked delay of arthritis onset with reduced severity of clinical symptoms and joint damage, accompanied by a substantial reduction of Th17 cells. To determine whether B10 cells directly inhibit Th17 cell generation in culture, CFSE-labeled naïve CD4+ T cells were cocultured with B10 cells. It was found that B10 cells suppressed Th17 cell differentiation via reducing STAT3 phosphorylation and RORγt expression. Moreover, Th17 cells exhibited significantly decreased proliferation when cocultured with B10 cells. Although adoptive transfer of Th17 cells triggered the development of CIA in IL-17-/- DBA mice, co-transfer of B10 cells with Th17 cells profoundly delayed the onset of arthritis. Thus, our findings have demonstrated a novel regulatory role of B10 cells in arthritic progression via suppressing Th17 cell generation.
Cellular and molecular mechanisms for lupus nephritis in a mouse model of systemic lupus erythematosus (159.4)
Authors: Hai Si-Han , Lam Viola , Cook Brian , Hermiston Michelle
Published: January 2023

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disorder with a range of presentations. Lupus nephritis is a particularly severe feature of SLE that predicts a poor outcome. Currently, it is unclear why some patients develop severe end organ disease while others have a relatively benign course. The CD45E613R mouse is a tractable model of SLE to study the transition from autoreactivity to autoimmunity. These mice express a single amino acid substitution (E613R) in the juxtamembrane wedge of CD45 that results in dysregulated phosphatase activity in all hematopoietic cells. Mirroring the variable presentation of human SLE, the phenotype of CD45E613R mice varies with genetic background. CD45E613R mice backcrossed to C57BL/6 (B6) or 129/Sv backgrounds fail to develop autoantibodies or end organ damage while F1 B6-129 CD45E613R mice develop high titer auto-antibodies and severe nephritis. Interestingly, BALB/c CD45E613R mice develop auto-antibodies but no nephritis. Using cytokine arrays and flow cytometry, we identified dysregulated cytokines and immune cell subsets in F1 B6-129 CD45E613R mice relative to the parental strains and wild-type controls. Classical inflammatory cytokines IL-1a and TNF-a were elevated in B6-129 F1 wedge mice. In contrast, MIP-1a and GM-CSF were most elevated in BALB/c wedge mice, implicating a protective effect. Uncovering new biomarkers and understanding disease pathogenesis should provide new insights into therapies for lupus nephritis.
Epigenetic contribution to between-animal variation in their fibroblast response to lipopolysaccharide (160.11)
Authors: Green Benjamin , Kerr David
Published: January 2023

Considerable variation exists between cows in the ability of their fibroblasts to produce IL-8 in response to LPS in vitro, and there is some evidence in support of an association with in vivo responsiveness to LPS or E. coli infection. Dermal fibroblast cultures were established from 43 cows and challenged with LPS for 24 h. A 4.5 fold difference (P<0.05) in the production of IL-8 was found between the 4 highest (HR) and 4 lowest (LR) ranked cultures. Preconditioning the cultures with DNA demethylating (5-aza-2'-deoxycytidine) and hyper-acetylating (trichostatin A) agents (AZA-TSA) potentiated the IL-8 response to LPS and eliminated the difference between HR and LR cultures. Subsequent gene expression studies on cultures with or with out AZA-TSA, and with or without 3 h LPS treatment indicated that IL-8, but not TNF, IL-6, SAA3, CCL20, or TLR4 was increased following AZA-TSA in the absence of LPS. After LPS treatment all genes studied except for TLR4 were up-regulated in response to LPS exposure under control conditions, with HR cultures displaying higher expression than LR cultures of IL-8, IL-6, and TNF (4.1, 2.8 and 6.8 fold, respectively). Preconditioning with AZA-TSA prior to LPS increased expression, more so in LR cultures, resulting in lower HR to LR differences of 1.1, 1.7 and 2.6 fold, respectively. The reduction in differential response to LPS due to AZA-TSA suggests a role for epigenetic modification in contributing to between-animal variation in immune response.
The identification and expression of a new light chain isotype, Igλ, in rainbow trout, Oncorhynchus mykiss (160.17)
Authors: Bromage Erin , Rego Kate , Ramirez-Gomez Francisco , Hansen John
Published: January 2023

The discovery of a trout EST bearing similarity to Igλ prompted an investigation of this previously uncharacterized isotype. Cloning and sequencing of gene-specific mRNA allowed the identification of two products. While the smaller product represented a fully spliced form, containing a variable segment (V), the joining segment (J), the constant region (C), and the 3’ UTR, the larger product was not fully spliced and contained a V segment, a 194bp intron, and a spliced J-C segment followed by the 3’ UTR. Phylogenetic analysis of the constant region confirmed that this new antibody isotype clustered with Igλ sequences of other species. Analysis of the recombination signaling sequences revealed that like Igλ in other species, the V and J segments are in the same transcriptional orientation with the V segment is followed by a heptamer-23bp spacer-nonamer while the J segment is preceded by a nonamer-12bp spacer-heptamer. In contrast to the Igκ and Igσ isotypes in trout that possess multiple VJC clusters, genomic analysis suggest that only a single Igλ VJC segment exists. Igλ accounts for only 1% to 3% of the total light chain expressions (IgκLC1 ~70%; IgσLC2 ~20%; IgκLC3~3%; IgλLC4~2%). Our identification of Igλ in rainbow trout shows that it is conserved throughout the teleost lineage. However, the role of the various Igλ splicing forms and whether the three Ig Heavy isotypes (IgM, IgD, IgT) demonstrate preferential light chain utilization still requires investigation.
Ontogenic analysis of Xenopus γδ T cells: expressed T cell receptor (TCR) γ and δ chains and IL-17 family members (160.8)
Authors: Jackson Shawn , Undieh Ada , Steiner Harold , Parra Zuly , Miller Robert , Ohta Yuko , Flajnik Martin
Published: January 2023

γδ TCR that have structural differences from conventional γδ TCR in mammals have been observed in non-eutherian vertebrates. Three different types of Vδ regions are present in Xenopus tropicalis, similar to Vδ, Vα, and VHδ (derived from the V regions of Ig H chains). Semi-quantitative PCR analyses in adult and larval Xenopus laevis show that expression levels of TCRδC and TCRγC, as well as of TCRδV and TCRγV repertoires, vary between tissues. Comparative analyses of adult and larval CDR3 sequences suggest ontogenic-, tissue- and perhaps V family-specific expression. We have detected changes at the VDJ junctions including N-nucleotide addition, nucleotide trimming, and homologous recombination events. γδ T cell populations in mammals differentially express either IL-17 or IFNγ. In mammals, six IL-17 forms have been described (A-E), the least well characterized of which is IL-17D. Most non-mammalian IL-17 homologs, including those in chicken, fish, lamprey, nematode, oyster, sea urchin, and tunicate, and especially, Xenopus, are the IL-17D form. We have cloned two types of IL-17A/F, IL-17B, and IL-17D from X. laevis. Semi-quantitative PCR analyses in adult and larval Xenopus show that the expression of these IL-17 family members vary during ontogeny and between tissues. Study of γδ TCR and IL-17 family expression in Xenopus will illuminate evolutionary and developmental changes in γδ T cell antigen recognition and effector strategies.
Analysis of the mechanism underlying contact hypersensitivity-induced hair growth (161.13)
Authors: Kitoh Akihiko , Nakamizo Satoshi , Natsuaki Yohei , Miyachi Yoshiki , Kabashima Kenji
Published: January 2023

Alopecia areata (AA) is believed to be a tissue-specific autoimmune disease mediated by T cells. Topical immunotherapy using contact sensitizer such as squaric acid dibutylester (SADBE) is one of the most effective treatments for intractable AA. However, the precise mechanism under which topical immunotherapy, i.e., contact hypersensitivity (CHS) reaction, induces hair regrowth in AA remains to be elucidated. It has been reported that CHS in the skin lesion of human and murine AA modulates the composition of the peribulbar mononuclear cell infiltrate, which may abrogate autoimmune reaction and promote hair regrowth. In fact, we have revealed that CHS induces predominant accumulation of CD4+Foxp3+ regulatory T cells and elevated expression of Il10 in the skin, which might contribute to tolerance induction in the AA lesion after topical immunotherapy. In addition, interestingly, CHS induces hair growth not only in bald skin of AA mouse but also in telogen skin of non-alopetic mice, implying the possibility that some mechanism other than abrogating autoimmunity may underlie the effect of topical immunotherapy in AA. In this study, we have disclosed the underlying mechanism of CHS-induced hair growth in alopetic and non-alopetic skin, which may provide a fundamental finding for the development of a better topical immunotherapy for AA.
Expression and histopathological correlation of CXCR5 and its natural ligand CXCL13 in ovarian cancer (161.14)
Authors: Singh Rajesh , Grizzle William , Singh Shailesh , Lillard, Jr. James
Published: January 2023

Chemokines and their corresponding receptors play an important role in metastasis and organ specific homing of cancer cells including ovarian cancer (OvCa). CXCL13 is a homeostatic chemokine that acts through its only known receptor, CXCR5. In this study, we provide the first evidence that in OvCa CXCR5 and CXCL13 are highly elevated in OvCa cell lines and clinical samples. Flow cytometry analysis of OvCa cell lines (OVCAR3 and SKOV3) show significantly higher expression of CXCR5 in comparison to normal adult ovarian epithelial cells. To determine the clinical significance of CXCR5 expressed by OvCa cell lines, ovarian cancer tissue microarrays were stained for CXCR5 and CXCL13. Aperio ScanScope scanning system was used to quantify the immunohistochemical staining of CXCR5 and CXCL13. Our results show significantly higher expression of CXCR5 and CXCL13 (p < 0.001) in cancerous tissue than compared to non-neoplastic ovarian tissue. Among cancerous tissues, CXCR5 and CXCL13 expression was highest in serous adenocarcinoma followed by serous papillary cystadenoma, endometrioid adenocarcinoma, mucinous adenocarcinoma, cystadenoma, mucinous boderline adenocarcinoma, clear cell carcinoma, granulosa cell tumor, dysgerminoma, transitional cell carcinoma, brenner tumor, yolk sac tumor, adenocarcinoma and fibroma. These data demonstrate biological and clinical significance of CXCR5 and CXCL13 as an OvCa biomarker.
Co-expression of IL-15 and IL-15Rα on pancreatic β islet cells induced insulin dependent diabetes mellitus in mice (161.2)
Authors: Chen Jing , Feigenbaum Lionel , Awasthi Parirokh , Butcher Donna , Anver Miriam , Bamford Richard , Thomas Craig , Waldmann Thomas
Published: January 2023

Interleukin-15 (IL-15) is a pro-inflammatory cytokine that stimulates T and natural killer (NK) cells. Increased serum levels of IL-15 are found in type 1 diabetes (T1D). Here we showed increased serum levels of soluble IL-15Rα in T1D. Moreover, co-expression of IL-15 and IL-15Rα on pancreatic beta islet cells induced insulin dependent diabetes mellitus in mice. Inhibiting IL-15 signaling by blocking IL-15 trans-presentation with TMβ1 (anti-CD122) or by Jak3 inhibitor Tasocitinib completely reversed diabetes in the mice. Islets purified from the double transgenic mice demonstrated increased expression of pro-inflammatory cytokines/chemokines and increased surface expression of MHC class I/II and ICAM-1. Depletion of CD4 cell at the diabetes onset reversed hyperglycemia while CD8 and NK cell depletion had no effect. Furthermore, CD4 cells purified from the double transgenic mice proliferated in response to wild type islets in vitro. Moreover, in non-obese diabetic (NOD) mice, inhibiting IL-15 signaling delayed diabetes development at pre-diabetic stage. Taken together, our data suggested that disordered IL-15 and IL-15Rα may be involved in the pathogenesis of type 1 diabetes and IL-15/IL15Rα pathway may be a rational therapeutic target for type 1 diabetes.
Development of IGF-1R signaling antibody arrays for the identification of hepatocellular carcinoma biomarkers (161.9)
Authors: Mao Ying-Qing , Zhou Qi , Jiang Wei-Dong , Chen Yun-Ru , Zhou Xiang-Bing , Wang Ya-Feng , Shi Zhi , Liu Yan-Li , Huang Ruo-Pan
Published: January 2023

A novel IGF-1R signaling antibody array was created through immobilizing the antibodies against ten IGF family proteins (IGF-1, IGF-1R, IGF-2, IGF-2R, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, and Insulin) on the surface of glass slide in array format. Tissue lysates prepared from patient’s liver cancer tissues and adjacent tissues were then applied to the arrays. The proteins captured by antibodies on the arrays were then incubated with a cocktail of biotinylated detection antibodies and visualized through fluorescence detection system. By comparison with standard protein amount, the exact protein concentrations in the samples can be determined. Using this novel antibody array technology, the expression levels of the ten IGF family proteins in 25 pairs of hepatocellular carcinoma (HCC) and adjacent tissues were quantitatively measured. The different expressions between cancer tissues and adjacent tissues were then statistically analyzed. Using this approach, we found that the levels of IGF-2R and IGFBP-2 in HCC tissues were higher than those in adjacent tissues.
IRF8-dependent regulation of MMP3 influences tumor progression (162.1)
Authors: Banik Debarati , Abrams Scott
Published: January 2023

Matrix metalloproteinases (MMP) are involved in tissue remodeling under physiologic or pathologic conditions, including neoplasia. Although the stromelysin member, MMP3, has been implicated in early stages of tumorigenesis, its role during later stages of tumor progression are poorly understood. Thus, we tested the hypothesis that tumor-derived MMP3 influences the neoplastic process beyond the level of tumor initiation. We employed two mouse tumor models, CMS4 sarcoma and 4T1 mammary carcinoma, in which endogenous MMP3 levels could be experimentally altered. Using loss-of-function approaches, we showed that MMP3 downregulation reduced CMS4 or 4T1 tumor growth rate in vivo, but not in vitro. Moreover, MMP3 downregulation attenuated CMS4 experimental lung metastasis. Conversely using gain-of-function approaches, we found that MMP3 overexpression enhanced CMS4 tumor growth. MMP3 levels also directly correlated with invasion capacity in vitro, consistent with MMP mode of action. Lastly, we examined potential mechanisms of MMP3 regulation in our CMS4 model. Interestingly, we identified interferon regulatory factor-8 (IRF8), previously portrayed as a leukemic suppressor, as a negative regulator of MMP3 expression. Altogether, our data show that tumor-derived MMP3 impacts the neoplastic process at stages beyond initiation and may do so via novel IRF8-dependent mechanisms. Thus, these data unveil new factors of tumor progression, which may have implications for cancer therapy.