bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced cardiac allograft survival by TNFRSF25 expanded natural Treg cells in an allogeneic mouse model (169.9)
Authors: Wolf Dietlinde , Li Sen , Schreiber Taylor , Tryphonopoulos Panagiotis , Tzakis Andreas , Ruiz Phillip , Podack Eckhard
Published: January 2023

Thymic derived Foxp3+CD4+ regulatory T cells (Treg) - also called natural Treg or nTreg - are critical for the induction and maintenance of transplantation tolerance. Using an agonistic TNFRSF25 antibody, clone 4C12, we recently showed that TNFRSF25 is a powerful regulator of Treg proliferation in vivo mediating expansion of natural Treg cells in vivo to 30-35% of the CD4+ cells. In the present study we investigate the role of TNFRSF25 expanded Treg cells on cardiac allograft survival in a fully MHC mismatched heterotopic heart transplant mouse model. A single administration of 20 μg of 4C12 antibody four days prior to cardiac transplantation resulted in a significant prolongation of median graft survival to 18 days. On day 7 after transplant - the timepoint when controls reject the graft - the grafts of 4C12 treated animals beat strongly, which was associated with the infiltration of 40% FoxP3+ cells to the graft compared to only 10% FoxP3+ cells out of all CD4+ cells in rejecting controls. A 4-fold upregulation of Foxp3 mRNA in the graft of 4C12 treated mice compared to controls confirmed the increased presence of regulatory cells. We further detected a 3-fold increase of TNFRSF25 mRNA in grafts of 4C12 treated animals. Finally, the combination of 4C12 with the costimulation blocker CTLA4-Ig has synergistic effects on cardiac graft survival. These data highlight the therapeutic potential of preventing allograft rejection using TNFRSF25 agonists to modulate Treg function.
Low molecular weight forms of chicken IgM (170.6)
Authors: Koppenheffer Thomas
Published: January 2023

Low MW Igs with truncated heavy chains have been described in a small number of reptilian species and anseriform birds. While these shortened Abs can bind Ag, their immunological significance is not understood since they are unable to mediate effector functions performed by full-length Ig. We isolated a low MW form of IgM from chicken serum by gel filtration chromatography. The low MW IgM was present in both adult heparinized plasma and EDTA-plasma containing protease inhibitors, but was not detected in sera of 2-3 week old birds. Thus it is unlikely that low MW IgM was derived from the native pentamer/tetramer by proteolytic fragmentation. Low MW IgM retained biologic activity as a partially purified isolate prepared from anti-DNP antisera exhibited hapten binding as determined by ELISA. SDS-PAGE of serum under non-reducing conditions followed by Western Blotting using a polyclonal goat anti-mu chain Ab revealed two low MWspecies of about 260 and 160KD. Both low MW forms were detected by Western Blotting with a monoclonal anti-mu chain and anti-L chain Abs, but only the larger form was detected with a second anti-mu chain Ab. These observations suggest the possibility that at least the smaller of the low MW IgM species contains a truncated heavy chain.
pDCs and IL-4+ T-cells in scleroderma as novel targets of imatinib mesylate (44.18)
Authors: Divekar Anagha , Khanna Dinesh , Abtin Fereidoun , Maranian Paul , Saggar Rajeev , Saggar Rajan , Furst Daniel , Singh Ram
Published: January 2023

Fibrosis is the end-result of many inflammatory conditions. Using scleroderma-associated interstitial lung disease (SLD) as a model fibrotic disease, we investigated cellular mediators of fibrosis in an open-label trial of imatinib mesylate (imatinib), a tyrosine kinase inhibitor. At baseline, high-resolution computer tomography (HRCT) findings of SLD strongly correlated with plasmacytoid dendritic cells (pDC) and IL-4-producing (IL-4+) T-cells in bronchoalveolar lavage (BAL) from individual lung lobes. pDC and IL-4+ and CD4+ T-cells in BAL correlated with each other, and with molecules CCL24, Nampt, PAI1-active, and PDGF-AB, which can recruit/activate leukocytes, smooth muscle cells and collagen-producing cells. pDCs also correlated with IL-3 and M-CSF. One-year post-imatinib, lung pDC and IL-4+ T-cells were significantly reduced. Interestingly, CD4+ T-cells increased along with soluble ICAM-3 and PECAM-1, except in one patient who showed worsened SLD. Thus we envision a role for pDCs in SLD, and propose that IL-3/M-CSF induce differentiation and maturation of pDCs that mediate local T-cell infiltration and differentiation into IL-4+ T-cells and promote fibrosis. Further elucidation of this axis and how drugs such as imatinib intercept it may identify targets to counter fibrosis that invariably leads to end-organ failure.
Exosome-like microparticles can stimulate both innate and adaptive autoimmunity in non-obese diabetes mice (44.20)
Authors: Dai Yang , Sheng Huiming , Hassanali Saleema , Nugent Courtney , Wen Li , Hamilton-Williams Emma , Dias Peter
Published: January 2023

Exosomes (EXO) are secreted intracellular microparticles that can trigger inflammation and induce antigen-specific immune responses. To test possible roles of EXO in autoimmunity, we isolated EXO from mouse insulinoma and examined their activities to stimulate the autoimmune responses in NOD mice, a model for human type 1 diabetes. We demonstrate that the EXO contains strong innate stimuli and expresses candidate diabetes autoantigens; it can activate splenocytes or purified APC through MyD88-dependent pathways, and result in T cell proliferation. To address whether exosomal antigens could be targets of the islet-specific autoimmunity, we monitored the spontaneously developed memory response in NOD mice for its reactivity to the EXO and compared the response with resistant strains. We found that older NOD females, which have advanced islet destruction, accumulated more EXO-reactive, IFN-gamma-producing lymphocytes than younger females or age-matched males, and that pancreatic lymph nodes of NOD but not resistant strains were enriched with EXO-reactive Th1 cells. Importantly, high titer of EXO-binding antibodies was also detected in serum of the NOD females but absent in resistant strains. Thus, certain exosomal antigens can be recognized by the autoreactive T cells and autoantibodies that are spontaneously developed in prediabetic NOD mice, supporting that EXO might be a possible autoimmune target in this diabetes-susceptible strain.
Anti-U1 autoantibody interferes with the U1RNP-mediated splicing of Angiopoietin-1 gene and induces functionally defective Ang-1/ins variant in the patients with MCTD (44.3)
Authors: Komai Koichiro , Hashiramoto Akira , Shiozawa Kazuko , Shiozawa Shunichi
Published: January 2023

Purpose: Angiopoietin-1 (Ang1) is the ligand of endothelial tyrosine kinase receptor Tie2. We showed that the frequency of the splicing variant of Ang1 with nt805GGT insertion (Ang1/ins) was significantly increased in the Japanese MCTD and those with PH (Arth. Rheum. 52: S283, ‘05). We also found that Ang1/ins proliferate pulmonary endothelial cells and potentially induce hyperplasia of smooth muscle cell in relation to the pathogenesis of PH as compared with Ang1/del (Arth. Rheum. 60: S8, ‘09). We have now studied the contribution of subclass of Abs against each U1 compoments to the induction of Ang1/ins. Methods: Anti-U1A, C and 70K Abs were introduced into human pulmonary artery smooth muscle cells using PULS-in (Polyplus) with IL6 and TNFα (20ng/ml each). Ang1 mRNA was obtained by RT-PCR. Anti-U1RNP Ab titer in sera of Japanese MCTD was determined by ELISA. Results: Anti-U1C and 70K Abs significantly induced Ang1/ins (P<0.05). Frequency of the patients having anti-U1RNP Ab titer exceeding 200 was 81.3% (39/48) and 50.0% (6/12) (P<0.05) among those with Ang1/ins dominant and Ang1/del dominant. The titer of each subclass of anti-U1RNP Ab in the sera of patients among those with Ang1/hetero (n=13) and Ang1/ins dominant (n=18) was: 11.8±23.6 and 47±58.9 (P<0.05) for anti-U1A, 13.6±25.7 and 122.1±124.5 (P<0.005); for anti-U1C, 406.2±599.5 and 5512.9±12484.7 (n.s.); for anti-U1-70K. Conclusions: It is suggested that anti-U1C Ab specifically affect the splicing of Ang1.
Antigen-independent recruitment of CTL to pancreatic islets expressing an NKG2D ligand (44.38)
Authors: Markiewicz Mary , Wise Erica , Buchwald Zachary , Pinto Amelia , Shaw Andrey
Published: January 2023

The mechanisms by which CTLs enter and are retained in non-lymphoid tissue are not well-characterized. Using a novel transgenic mouse expressing the NKG2D ligand RAE1ϵ in β-islet cells of the pancreas, we found RAE1 expression was sufficient to induce the recruitment of adoptively transferred CTLs to islets. This was dependent on NKG2D expression by the CTLs and independent of antigen recognition. While transgenic mice did not develop diabetes, RAE1 expression was sufficient to induce insulitis in older, unmanipulated transgenic mice that was enhanced by viral infection and pancreatic inflammation. Surprisingly, the recruitment of CTLs resulted in the subsequent recruitment of a large number of endogenous lymphocytes. These results demonstrate that the expression of NKG2D ligands in islets is sufficient to recruit CTLs and induce a significant insulitis.
IFNα causes systemic lupus erythematosus and possibly mediates self-organized criticality theory of autoimmunity (44.47)
Authors: Shiozawa Shunichi , Akiyama Chieri , Honda Eriko , Miyazaki Yumi , Tsumiyama Ken
Published: January 2023

Objectives: The pathogenesis of SLE is unclear. Overstimulation of CD4 T cells by antigen led to the development of autoantibody-inducing CD4 T (aiCD4 T) cell capable of inducing autoantibodies and lupus tissue disease. In this novel ‘Self-organized criticality theory of autoimmunity’, we noted that aiCD4 T cell is induced via de novo TCR revision. We now test IFNα as a mediator for the induction of aiCD4 T cell to cause SLE. Methods: Mouse IFNα gene controlled under TetOp promoter was microinjected into eggs of C57BL/6 to generate TetOp-mIFNα Tg mice. They were mated with EμSR-tTA Tg mice, expressing doxycycline-transactivator (tTA) gene under the control of IgH chain enhancer and SRα promoter, to obtain double Tg mice. Results: By increasing IFNα, anti-dsDNA antibody and immune complex (IC) were generated. Pathological lesions consisted of IC-deposited glomerulonephritis, interstitial lung disease, alopecia, liquefaction and positive skin lupus band test, onion skin splenic lesion, and inflammatory infiltrates to salivary gland and bile duct. There was an increase in IFNγ-producing effector CD4+, full-matured CD8+ CTL, and activated CD3+CD4-CD8- double negative (DN) T cells. The DN T cells not only infiltrated to the glomerular lesions of mice, but also induced de novo glomerulonephritis and alopecia when transferred into naïve recipients. Conclusions: IFNα is responsible for the core manifestation of SLE, and may induce lupus tissue injury via DN T cells.
A role for CD8+ T cells in an autoantibody response (44.7)
Authors: Hoyer Katrina , Isakson Sara , Abbas Abul
Published: January 2023

IL-2-deficient mice develop a spontaneous systemic autoimmune disease and on the BALB/c background die at 3-4 weeks due to hypoxia from autoimmune hemolytic anemia (AIHA). These mice develop progressive lymphadenopathy due to the accumulation of CD4+ and CD8+ T cells. It is known that CD4+ T cells are necessary for disease progression and we have previously shown that IL-2-KO CD4+ T cells from sick mice can transfer AIHA and lymphadenopathy to recipient mice. As CD8+ T cells also accumulate during disease, we asked whether these cells play a role in the development of autoimmune disease. To address this, we depleted CD8+ T cells by treating mice twice a week with anti-CD8α (clone 2.43). Elimination of CD8+ T cells for two weeks resulted in an augmentation of survival from 3-4 weeks to 3 months. The number and activation state of the CD4+ T cells and B cells was decreased, and the development of red blood cell (RBC)-specific antibodies delayed. Our results suggest that autoreactive CD8+ T cells contribute to the initiation and/or propagation of lymphoproliferation and pathogenesis of autoimmunity. Both CD4+ and CD8+ T cells from the IL-2-deficient mice produce IFNγ in response to RBC antigens. We are currently addressing the importance of these autoreactive CD8+ T cells relative to that of the self-reactive CD4+ T cells. We are also evaluating the possible mechanisms by which CD8+ T cells influence the disease process.
Human Fc receptor-like 5 promotes B cell proliferation and the development of cells displaying surface IgG and IgA (45.10)
Authors: Tolnay Mate , Dement-Brown Jessica , Newton Christopher , Ise Tomoko , Nagata Satoshi
Published: January 2023

The functional roles of B cell membrane proteins belonging to the Fc receptor-like (FCRL) family are unclear. However, the demonstrated signaling capacities of FCRL suggest co-receptor functions that modulate B cell receptor (BCR) signaling. Nevertheless, the subsequent functional consequences of FCRL engagement are unknown. Using naive B cells obtained from peripheral blood of human subjects, we show that while FCRL5 surface protein is not present on resting cells, it is temporarily induced following BCR stimulation. Co-stimulation of FCRL5 and the BCR on B cells induced to express FCRL5 significantly enhanced proliferation in the presence of toll-like receptor 9 agonists. The same combination of stimuli led to the development of high proporsion of cells expressing the switched isotypes IgG and IgA on their surface. Optimal development of cells expressing downstream isotypes also required T cell help. Unexpectedly, IgG and IgA expression was most prominent on cells that underwent 1-3 cell divisions, suggesting an unusual mechanism. We suggest that FCRL5-dependent enhancement of proliferation and downstream isotype expression could play roles in the physiological expansion and development of antigen-primed B cells. Additionally, FCRL5 may contribute to tumor progression in hairy cell leukemia and other FCRL5 expressing B cell malignancies.
Non-steroidal anti-inflammatory drugs reduce optimal antibody production in response to vaccination and vaccinia virus infection (45.18)
Authors: Phipps Richard , Bancos Simona , Topham David , Bernard Matthew
Published: January 2023

Protective humoral immune responses generated by vaccination, depends on optimal production of antibodies. Non-steroidal anti-inflammatory drugs (NSAIDs) are used to curb undesirable effects of vaccination such as fever. We previously demonstrated that B cells express the “proinflammatory” enzyme cyclooxygenase-2 (Cox-2) in response to activation stimuli such as the TLR9 ligand CpG DNA. Using genetic approaches and small molecule Cox-2 selective inhibitors, we demonstrated that Cox-2 is crucial for optimal antibody production. Expression of plasma cell transcriptional regulatory factors, including Blimp-1 and Xbp-1, were dependent on Cox-2 activity. Significantly fewer antibody secreting plasma cell precursors were generated in the presence of Cox-2 inhibitors, indicating that Cox-2 is essential for human B cell terminal differentiation. Mice chronically treated with Cox-2 inhibitors produced less neutralizing IgG following live virus infection with vaccinia virus. In vitro studies using human B cells show that all commonly used NSAIDs, as well as Tylenol (acetaminophen), at pharmacologic doses, blunt antibody production. Interestingly, a recent clinical trial showed that Tylenol use in infants blunted antibody responses to a variety of vaccines. These new data support the concept that the Cox pathway is necessary for optimal antibody production. The use of NSAIDs, may impair antibody production to vaccination and to certain infections.
Plasticity and commitment of memory CD8+ T cell progenitors early after infection (46.13)
Authors: Plumlee Courtney , Obar Joshua , Lefrancois Leo
Published: January 2023

Naïve antigen specific CD8+ T cells expand in response to antigenic stimulation to form a large effector T cell population. Following expansion, the majority of the antigen specific CD8+ T cells die, leaving behind a memory population capable of protecting the host. CD8+ T cell memory formation has been extensively studied, but little is understood about the early events in memory commitment. At the peak of the T cell response, effector T cells can be separated using the markers CD127 (IL-7Rα) and KLRG1. Memory precursor effector cells (MPECs) express CD127 while short-lived effector cells (SLECs) express KLRG1. CD8+ T cells lacking expression of both markers are called early effector cells (EECs) and can give rise to both the MPECs and SLECs. To understand if EEC populations are committed to either an MPEC or SLEC fate prior to differentiation, we used EECs generated from Listeria monocytogenes and vesicular stomatitis virus infections, where the ratio of SLECs/MPECs at the peak of the T cell response is known to be different. EECs were sorted and transferred into infection-matched or mismatched hosts at various days post infection. As late as 7 days post-infection the EECs were not committed to an MPEC or SLEC fate and could be driven in either direction based on the inflammatory environment of the host. Based on these results we conclude that commitment of a CD8 T cell toward a memory lineage does not occur until after CD127 expression has been established.
The priming stimulus dictates senescence of memory T cells (46.15)
Authors: Prlic Martin , Bevan Michael
Published: January 2023

Immunization drives the proliferation and differentiation of effector CD8 T cells followed by a contraction phase after the peak of the T cell response. The survivors of this contraction phase differentiate into long-lived memory cells, which can provide life-long protection. Generating long lived T cell memory through vaccination holds great promise to provide protection in situations where antibody-inducing vaccines fail. Prime-boost strategies have been used to repeatedly stimulate T cells with the aim of increasing memory T cell frequency. However, repeated stimulation of the memory T cell pool leads to T cell senescence and loss of responsiveness. We sought to determine whether a prime-boost vaccination strategy can be developed that does not suffer from this drawback. We report that the extent of senescence at the memory stage is determined during the primary response. We developed a system that allows priming of CD8 T cells without acquisition of maximal effector function and show that the ensuing memory population is fully functional upon rechallenge. Importantly, these memory cells derived from minimal effector T cells have full access to non-lymphoid tissue and are less senescent upon rechallenge than memory cells that did acquire maximal effector markers and function during priming. Understanding how the nature of the priming stimulus shapes memory T cell differentiation allows situation dependent tailoring of vaccines to achieve maximal protection efficacy.
Genome wide association study identifies many novel susceptibility genes for illumne related skin complex diseases (47.11)
Authors: Sun Liangdan , Yang Sen , Zhang Xuejun
Published: January 2023

With the completion of HGP and HapMap, the genome wide association study (GWAS) has become feasible and been proven to be a very useful and efficient approach to identify the susceptibility genes for complex diseases as compared to previous method in terms of linkage study. Psoriasis, vitiligo,SLE and atopic dermatitis are common immune related skin complex diseases with unknown pathogenesis. To identify the susceptibility genes predispoing to these dieases in Chinese Han populations, we carried out a seris GWAS for psoriasis, vitiligo, SLE and atopic dermatitis in Chinese populations and identified many novel genetic factors influencing disease susceiptibilty as well as found the genetic heterogenetity of these complex diseases within different ethinic populations(Nat Genet 2009, 2010). Our reserach should provide novel insights into the disease pathoghensis and lay a solid fundation for further functional research.
Congenic mouse strains identify a novel gene for which allelic variation affects susceptibility to type 1 diabetes (47.9)
Authors: Tan Iris , Mackin Leanne , Wang Nancy , Papenfuss Anthony , Elso Colleen , Ashton Michelle , Quirk Fiona , Phipson Belinda , Bahlo Melanie , Speed Terence , Smyth Gordon , Morahan Grant , Brodnicki Thomas
Published: January 2023

Type 1 diabetes (T1D) is an autoimmune disease resulting from the complex interaction of multiple genetic and environmental factors. The nonobese diabetic (NOD) mouse has proven to be a useful model for investigating T1D susceptibility loci and their immunological effects upon disease progression. We have mapped a NOD diabetes susceptibility locus, termed Idd11, on chromosome 4 (Chr4). A panel of congenic NOD mouse strains, harbouring different C57BL/6 (B6, a diabetes-resistant strain)-derived intervals for Chr4, localized Idd11 to an ~7kb interval. Comparison of diabetes incidence and crossover breakpoints for these strains suggested that a haplotype for this locus underlies T1D susceptibility in NOD mice. Based on publicly available databases, the Idd11 haplotype is located within a predicted gene of unknown function (AK005651). Genomic characterization of AK005651 reveals that this novel gene encodes multiple splice isoforms and open-reading frames, none of which has homology with known proteins. Preliminary expression analysis also indicates that AK005651 is differentially expressed in tissues relevant to T1D, with NOD mice demonstrating altered splicing and reduced expression compared to diabetes-resistant strains. Ongoing studies aim to determine the effect of Idd11 / AK005651 on T1D pathogenesis.
Indirect stimulation of human Vγ2Vδ2 T cells through alterations in isoprenoid metabolism: implications for cancer immunotherapy (48.14)
Authors: Morita Craig , Sarikonda Ghanashyam , Puan Kia-Joo , Feng Ju , Giner José-Luis , Cao Rong , Oldfield Eric , Wang Hong
Published: January 2023

Human Vγ2Vδ2 T cells monitor isoprenoid metabolism by recognizing (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate in the foreign MEP pathway used by protozoa and bacteria, and isopentenyl pyrophosphate (IPP), an intermediate in the self mevalonate pathway. Synthetic bisphosphonates (BPH), used for cancer immunotherapy, and alkylamines indirectly stimulate Vγ2Vδ2 T cells by inhibiting self farnesyl diphosphate synthase (FDPS), thereby increasing IPP which is directly stimulatory. Here, we further characterize stimulation by these compounds, and define pathways used by new classes of compounds. Consistent with FDPS inhibition, stimulation of Vγ2Vδ2 cells by BPH and alkylamines was much more sensitive to upstream statin inhibition than stimulation by phosphoantigens or mitogens. However, the continuous presence of BPH or statins was toxic, blocking Vγ2Vδ2 T cell proliferation but not cytokine release. SiRNA downregulation of FDPS in APC increased IPP and rendered them stimulatory for Vγ2Vδ2 cells. SiRNAs downregulation of isopentenyl diphosphate isomerase functioned similarly, albeit less potently. New stimulatory compounds-mevalonate and the alcohol of HMBPP-may also stimulate indirectly, but required high statin doses for inhibition. The critical chemical feature of BPH is the amino moiety, since BPH lacking amino moieties acted as direct antigens. Thus, a variety of manipulations affecting isoprenoid metabolism lead to stimulation of Vγ2Vδ2 T cells.
Reduced redox-regulating thiol levels render effector memory like T cells obtained after repetitive TCR stimulation more susceptible to activation-induced cell death (48.17)
Authors: Al-Khami Amir , Norell Håkan , Kaur Navtej , Naga Osama , Dinkins De’Angelo , Kesarwani Pravin , Voelkel-Johnson Christina , Mukherji Bijay , Nishimura Michael , Mehrotra Shikhar
Published: January 2023

Human antigen-specific CD8+ T lymphocytes have a heterogeneous phenotype that includes functionally distinct populations. Ex vivo-expanded CD8+ T cells used for adoptive immunotherapy generally acquire an effector memory-like phenotype (TEM cells) and exhibit impaired in vivo persistence and anti-tumor efficacy. Our data show that TEM cells (CD62LloCCR7loCD45ROhi) obtained after repetitive T cell receptor (TCR) stimulation show increased sensitivity to activation-induced cell death (AICD) induced by cognate antigen re-exposure compared to cells with a central memory-like phenotype (TCM cells; CD62LhiCCR7hiCD45ROlo). Of interest, TEM cells have reduced intracellular glutathione levels compared to TCM cells. This decrease in key redox-regulating thiol proteins inversely correlated to the extent of T cell proliferation and coincided with a decrease in the antioxidant protein thioredoxin. It was also associated with lower expression of the anti-apoptotic proteins bcl2 and bclxl and higher frequency of the T cell exhaustion markers CD57 and Tim-3. Treatment of tumor-specific T cells with the thiol donor N-acetyl cysteine increased cellular thiols and rescued T cells from AICD, without impairing their function. These data suggest that T cell expansion after antigen encounter could affect CD8+ T cell quality by decreasing its antioxidant capacity. Increasing the antioxidant capacity may increase the persistence of T cells and has translational implications for immunotherapy.
TCR transduced CD4+ T cells exhibit a TH1 phenotype while inhibiting priming of naïve CD8+ T cells by peptide-loaded mature dendritic cells (48.19)
Authors: Lesher Aaron , Norell Håkan , da Palma Telma , Graves Michael , Mehrotra Shikhar , Nishimura Michael
Published: January 2023

Adoptive immunotherapy is a promising new treatment modality for patients with advanced malignancies. A key limitation to adoptive T cell transfer is generating sufficient numbers of tumor reactive T cells for patient treatment. We have developed the ability to engineer T cells with TCR genes to redirect the specificity of PBL-derived T cells. A novel population we can produce are MHC class I restricted CD4+ T cells. CD4+ T cells have been implicated in the priming and maintenance of the CD8+ T cell response, in addition to having cytolytic properties in experimental models. In this study, tyrosinase-reactive human CD4+ T cells were created by retroviral transduction using a CD8-independent T cell receptor. Tyrosinase-reactive gene-modifed CD4+ cells produced IFN-γ, GM-CSF, and IL-2 in an HLA-A2 restricted, antigen-specific manner. Following tumor antigen recognition in vitro, TCR-transduced CD4+ T cells upregulated secondary costimulatory molecules, including OX-40 and 4-1BB. Despite their type I phenotype, priming of CD8+ cells by MART-1-loaded dendritic cells was impaired by these TCR-transduced CD4+ cells. Therefore, simultaneous engraftment of genetically-engineered CD4+ and CD8+ effector T cells may inhibit tumor killing by suppressing both CD8+ T cell induction and maintenance.
High throughput sequencing identified specific T cells from breast cancer tissue and peripheral blood (48.37)
Authors: Han Jian , Wang Chunlin , Sanders Catherine , Yang Qunying , Byrne-Steele Miranda , Lu Chiungmei
Published: January 2023

Tumor specific antigens may trigger an immune response that leads to T lymphocytes infiltrating the tumor tissue. We have developed methods using high throughput sequencing technology to study the immune repertoire within a sample (Wang et al., PNAS 2010). Using this method, we studied a patient’s surgically removed breast cancer tissue, the nearby normal tissue, and sorted peripheral blood. Dominant T cell clones with specific CDR3 sequences were identified from the breast cancer tissue. The same clones were found expressed at high levels in the nearby normal tissue and peripheral CD8+ cells. The clones were also expressed at low levels in other subsets of T cells in peripheral blood. Peripheral blood was collected after lumpectomy and radiation therapy from the same patient and subsets of T cells were sequenced. The study demonstrated the capability to identify specific T cells associated with a patient’s cancer tissue. These specific T cells can serve as personalized biomarkers for prognosis, treatment evaluations and early detection of recurrent tumors. Identification of these cancer specific T cells also make it possible to design personalized treatment strategies.
TLR-4 dependence of COX-2 and VEGF in prostate cancer (48.38)
Authors: Priyadarshini Anjali , Singh Shrawan , Kakkar Nandita , Chakraborti Anuradha
Published: January 2023

Introduction-Cancer is a complex multifactorial disease with many players including TLR-4, COX-2 and VEGF associated with inflammation and angiogenesis. Though some information is there regarding their individual role in prostate cancer, but their interdependence in disease pathogenesis has not been well studied. This study was done to relate the expression of COX-2 and VEGF with the expression of TLR-4. Method-The expression study was done in pathogenic malignant and benign prostatic tissue (Prostate cancer and BPH) by RT-PCR. Further, study of dependence of VEGF and COX-2 expression on TLR-4 was done on Prostate Cancer cell line LNCaP which was stimulated with Lipopolysaccharide (LPS). Results- Expression of TLR-4 and COX-2 was found to be significantly high in cancer tissue (p<0.05). Expression of COX-2 and VEGF was found to be enhanced by stimulation of TLR-4 by LPS. Conclusions- Since upregulation of COX-2 and VEGF in Prostate cancer has already been well documented, a better understanding of the underlying biological mechanism of this may unveil new targets for therapy in patients of Prostate Cancer.
Upregulation of cytolytic functions of human Vd2neg gd T lymphocytes through engagement of ILT2/CD85j expressed by tumor target cells. (48.5)
Authors: Bonneville Marc , Harly Christelle , Scotet Emmanuel
Published: January 2023

In human adults, the majority of peripheral blood gd T cells expresses T cell receptors (TCR) using the Vg9 and Vd2 chains and recognize small nonpeptidic phosphorylated antigens. In contrast, most tissue-derived gd T cells, which are located mainly in spleen and epithelia, preferentially use Vd1 or Vd3 chains paired with diverse Vg chains to form their TCR. Our knowledge about the antigenic specificity and costimulation requirements of human Vd2neg gd T cells remains limited. To address this, we characterized the specificity of a murine mAb (#256), screened for its ability to specifically inhibit cytolytic responses of several human Vd2neg gd T cell clones (such as the Vg8Vd3 T cell clone #73R9) against transformed B cells (e.g. B-LCLs and Burkitt’s lymphomas). Using gd#73R9-derived TCR transfectants and comparative gene expression arrays, we show that mAb#256 does not target a gdTCR ligand, but blocks a key activation pathway involving interaction between non-TCR molecules on effector gd T cells and ILT2 (LILRB1/CD85j) molecule, expressed by tumor targets. In line with the previously reported specificity of this NK receptor for classical and non-classical MHC-I molecules, blockade of MHCgd class I / ILT2 interactions using MHC I- or ILT2-specific mAbs and ILT2-Fc molecules, gdinhibited tumor-induced activation of Vg8Vd3 T cell clones. Therefore, this study describes a new CTL activation pathway involving MHC class I engagement on gd T cells.
Immunotherapy of pancreatic adenocarcinoma using adoptive cell transfer of redirected T lymphocytes (48.6)
Authors: Eshhar Zelig , Maliar Amit , Servais-Mavor Charlotte , Waks Tova
Published: January 2023

Pancreatic adenocarcinoma (PAC) is a common malignancy that is often diagnosed at an advanced stage, when surgery is not feasible. Here we investigated in an animal model whether adoptive cell transfer using redirected, tumor-specific T cells could be applied for the treatment of primary and metastatic PAC. To this end we genetically engineered. human effector T cells (‘T-bodies’) to express antibody-based chimeric receptor against pancreatic tumor markers CD24 (putatively defined as PAC stem cell antigen) or Her2/neu. In in-vitro co-culture studies we demonstrated that both CD24 and Her2/neu specific T-bodies were specifically stimulated and lysed PAC cells. In-vivo experiments showed that T-body treatment of SCID mice bearing Capan-1, PAC cell-line was highly effective in reducing tumor size and prolongation of survival. Moreover, in an orthotopic model, most mice systemically receiving human T-bodies, remained disease-free for several months whereas all control animals developed large tumors. Using luciferase-labeled PAC demonstrated that these treatments resulted in the elimination of metastases in the liver and draining lymph nodes. Repeated course of T-bodies resulted in complete responses in more than 50% of the treated mice. Intratumoral injection of T-bodies eliminated more than 80% of the tumors. Taking together, we suggest redirected lymphocytes as an efficient cell-therapy modality for systemic treatment of localized and disseminated pancreatic adenocarcinoma.
Cytokine expression pattern differences between conserved versus non-conserved influenza pandemic H1N1-specific CD4 T cells (49.15)
Authors: Weaver Jason , Deng Nan , Bishop Courtney , Mosmann Tim
Published: January 2023

The T cell response to conserved epitopes of influenza, which have been primed and repeatedly boosted in most adults, is a strongly biased Th1 response in which most responding cells produce TNFα, IFNγ and IL2. We have now analyzed the cytokine specificity of the T cell response to non-conserved peptides from the pandemic H1N1 influenza virus. This response should be enriched for non-cross-reactive responses that have been primed by a single exposure, but not boosted. Pepsets spanning the H1N1 proteome of a seasonal flu, pandemic flu, and vaccine components were sorted according to amino acid sequence comparisons into identical and non-identical groups. PBMC from healthy donors were stimulated with the different peptide pools and cytokine expression patterns were analyzed by flow cytometry. Peptides identical in all three strains or identical between the seasonal H1N1 and pandemic H1N1 induced responses biased toward expression of TNFα+IFNγ+IL2+/-. In contrast, the pool of peptides unique to the pandemic H1N1 induced more TNFα+IFNγ-IL2+ producing T cells. These data suggest that responses against unique peptides of H1N1 (some of which will be non-cross-reactive with other influenza strains) include relatively more IL2-producing cells, possibly including the IFNγ-IL2+ non-polarized (Thpp) cells that are typically induced by protein vaccination. These cells may be further polarized by repeated stimulation during subsequent infections.
Enhanced induction of CD4+CD25+FOXP3+ suppressive function in memory versus naïve CD4+CD25-FOXP3- T-cells is proportional to strength of activating stimulus. (50.2)
Authors: Mohiuddin Imran , Pillai Vinodh , Mendoza Jason , Lee Thomas , Karandikar Nitin
Published: January 2023

CD4+CD25+FOXP3+ regulatory T-cells (Tregs) play a vital role in suppressing autoimmune responses. We have previously shown that, in humans, all activated T-cells attain a state of transient FOXP3 expression that correlates with transient suppressive ability. In fact, peripheral generation of induced Tregs (iTregs) is likely the dominant source of regulatory cells in healthy adults. Through the use of a sensitive flow-based suppression assay, we observed that both memory and naïve CD4+CD25-FOXP3- T-cells developed regulatory ability following a variety of activating stimuli. This suppressive ability was not due to competition for nutrients or antigen presenting cells. Interestingly, memory T-cells displayed significantly greater FOXP3 induction compared to naïve counterparts, and this phenotype correlated to significantly enhanced suppressive function. Furthermore, acquisition of suppressive ability in both the memory and naïve iTreg compartments was proportional to the strength of activating stimulus. While blockade of activation using anti-IL-2, CTLA-4 Ig, anti-TGF-B, indomethacin or cyclosporin A did not affect iTreg generation, methotrexate drastically preempted the induction of regulatory ability. Moreover, irradiation of iTregs also abrogated regulatory function. These studies suggest that iTreg development and function may vary dependent on precursor origin and emphasize the importance of characterizing the iTreg compartment prior to therapeutic application.
Resveratrol and curcumin: effects on human T cells (50.7)
Authors: Valenzuela Hector , Molgora Brenda , Bateman Riley , Wu Jennifer , Sweeney Greg , Effros Rita
Published: January 2023

Various natural products have been proposed to both increase health at the organismic level, and delay the onset of cellular aging. Of particular interest is resveratrol, a phytoalexin that has been demonstrated to have cardioprotective effects and to slow the progression of a variety of illnesses, including various types of cancers. More recently, resveratrol has been demonstrated to increase the maximum lifespan of model organisms, such as C. elegans, D. melanogaster and N. furzeri. However, the potential health benefits of resveratrol and other similar compounds on the human immune response have not been adequately addressed. We have, therefore, initiated research to determine the in vitro effects of the nutraceuticals resveratrol, and its analogs (polydatin and acetyl-resveratrol), as well as curcumin on a variety of human T cell functions. In this study we evaluated the effect of these compounds on proliferative potential, telomerase activity, expression of costimulatory and memory surface markers and sirtuin gene expression profiles of both human CD4 and CD8 T cells. Our data show that these compounds stimulated strong dose-dependent anti-inflammatory responses, and upregulated surface protein markers associated with the suppression of T cell activity. Our preliminary results suggest that these compounds may function to down-modulate ongoing CD4 and CD8 immune activity, while, nevertheless, poising the T cells for a subsequent immune response.
The Young Scientist Program: successful use of a volunteer based outreach program created by graduate and medical students to improve science education in the St Louis Public School system (51.10)
Authors: Lynch Jennifer , Moss Britney , Chiappinelli Katherine , Mosher Jennifer , Woolsey Thomas
Published: January 2023

YSP aims to supplement science curricula and attract young students to scientific careers through a series of programs designed for underserved middle and high school students and their teachers. Teaching Teams develop and lead inquiry-based science modules in classrooms, after school programs, and to community organizations throughout the year. In the summer, high school students and teachers perform independent research at Washington University under the guidance of graduate student or post doctoral mentors through our Summer Focus (SF) and Teacher Researcher Partnership (TRP) programs. SF culminates in written and oral presentations of laboratory findings. TRP participants develop tools to bring bench work into their classrooms. Analysis of the past five years elucidates the immense success YSP has had within the St Louis community. Students report more interest and aptitude in science due to YSP. Applications for SF and TRP have reached another record high and come from an expanding demographic. Participants of both programs often pursue advanced degrees in science or education. Analyses of SF writing samples show remarkable improvement in YSP’s ability to teach scientific writing and critical thinking skills. YSP continues to increase the number of volunteer hours and reach new community venues. We present YSP as a model for the successful use of a volunteer based approach to improve science education and inspire future generations of science professionals.
Integrating immunology into a case based medical curriculum (51.3)
Authors: Moticka Edward
Published: January 2023

Several innovations in the delivery of the pre-clinical medical curriculum have focused on non-discipline approaches. These include Problem Based Learning, Team Based Learning and the Clinical Presentation (CP) Curriculum. All these approaches require the integration of various science disciplines into clinical scenarios organized by organ systems. The CP Curriculum involves integrating didactic material in the context of approximately 125 clinical presentations (i.e. chest pain, hematuria, shortness of breath). During the pre-clinical years, a clinician provides an overview of the clinical problems which might be seen followed by a series of basic science presentations relevant to the clinical presentation. Incorporation of immunology into a CP curriculum requires coverage of both basic concepts and pathophysiology of immunologic diseases in each organ system. Our approach is to use a small number of clinical presentations (sore throat, recurrent infections) to cover basic concepts. This is followed by presentations in each organ system during which disease mechanisms and additional immunological concepts are introduced. This presentation will review the ability to include topics identified by USMLE as well as those identified by ASM in such a curriculum along with data on the performance of students enrolled in a medical school using this approach.
Using differentiated instruction to teach immunological concepts to a diverse group of learners. (51.6)
Authors: Rodriguez Victor , Havran Wendy
Published: January 2023

Last summer, I was a teacher intern in the Havran laboratory at The Scripps Research Institute where the research focused on how to define the mechanisms by which γδ T cells interact with neighboring epithelial cells, how they recognize and respond to self produced antigen, and ultimately how they accelerate the repair of damaged tissue. Upon completion of my internship, I developed a curriculum unit with a Biology Action Model (BAM) and a double diffusion test that demonstrates the interaction between antibodies and antigen. Students will be introduced to the role of γδ T cells by interpreting pictures of wild type and γδ knock out epidermal mouse tissue that have received identical wounds. Students will observe delayed wound closure and healing in the tissue that comes from the γδ knock out mouse. In addition to the content, students will apply laboratory skills such as microscopy and tissue staining. This unit is aligned with the California Content Standards for the Immune System and is designed to be implemented in a college prep biology class. This unit will incorporate a variety of teaching strategies that engage students in active learning and will foster a better understanding of immunology.
Real-time tracking ATAK cells in vivo (52.1)
Authors: Lin Lin , Ibrahim Ashraf S. , Baquir Beverlie , Palosaari Andrew , Spellberg Brad
Published: January 2023

Background: Activated Targeted Killer (ATAK) cells are a cellular immunotherapy which protects neutropenic mice from invasive candidiasis and aspergillosis. However,the lifespan and distribution of ATAK cells into organs has not been determined in vivo. Methods: ATAK cells were stably transfected with a luminescence reporter system. Neutropenic mice were imaged by IVIS after administration of coelentrazine substrate. RT-PCR was used to confirm the luminescence results. Results: On day 1 after ATAK cell administration, livers, kidneys, and spleens were infiltrated by the ATAK cells. By day 3, cells had infiltrated more significantly into the spleen, and also spread into the lungs. By day 5, cell signal began to wane. By day 8, the day after neutrophil recovery, there was no detectable signal. By RT-PCR, viable cells were detected at 6 hours and 3 days after treatment. By day 5, no mRNA was detected. Mouse immunity to ATAK cells was detectable by ELISA and flow cytometry post-neutrophil recovery. Discussion: ATAK cells are viable and circulate widely into visceral organs in neutropenic mice, commensurate with their ability to protect mice from tissue invasive fungal infections. Hence the cells can provide protection during the critical neutropenic period yet the risk of engraftment is minimized by host-versus-graft affect after marrow recovery.
Memory CD8+ T cell proliferation after anti-CD40/IL-2 treatment is independent of CD4+ T cells (52.17)
Authors: Tietze Julia , Hsiao Hui-Hua , Koehn Brent , Wilkins Danice , Sckisel Gail , Blazar Bruce , Murphy William
Published: January 2023

Systemic cancer immunotherapy (IT) with anti-CD40/IL-2 in mice leads to marked antigen-independent proliferation of CD44high CD8+ cells, which are NKG2D+/CD25- and do not upregulate PD1 significantly. Here we investigated the role of CD4+ T cells on the memory CD8+ expansion in vivo using CD4 knockout mice and in mice whose CD4+ T cells were depleted with antibody. We observed that CD4 depletion significantly increased total numbers of CD44highCD8+ cells in untreated mice due to homeostatic proliferation. After IT there was no difference in the number of CD44highCD8+ cells or in the expression of NKG2D compared to IT treated control mice. However, we observed upregulation of PD1 on the CD8+ T cells in CD4-depleted mice after IT compared to control mice. Treatment of IL21 RKO mice with IT also did not affect the expansion of the CD44highCD8+ T cells compared to WT mice. We conclude that the effect of anti-CD40/IL-2 treatment on CD44highCD8+ T cell proliferation is independent of CD4 help or IL21. However, the absence of CD4+ T cells appears to affect the phenotype of the expanded CD8+ T cell population resulting in the upregulation of PD-1.
Targeting and manipulating mIgD-expressing B cells by anti-migis-δ monoclonal antibodies (52.18)
Authors: Chen Nien-Yi , Hung Alfur , Yu Hui-Ming , Chang Hwan-You , Chang Tse-Wen
Published: January 2023

Membrane IgD and IgM double positive cells are the major population of B cells in the peripheral circulation. The δ chain of mIgD (mδ) differs from the δ chain of IgD in that mδ contains an extra C-terminal transmembrane-anchor peptide, of which the N-terminal extracellular segment of 26 a. a. residues, also referred as migis-δ segment, potentially provides as a unique antigenic site for isotype-specific targeting mIgD+ B cells. Here we report the preparation of mouse monoclonal antibodies (mAbs) specific for migis-δ using hybridoma methodology with ovalbumin conjugated with synthetic migis-δ peptide as the immunogen. The mAbs bound to recombinant IgD.Fc-migis-δ fusion protein in ELISA and to mIgD-expressing transfectants of a CHO cell line in fluorescence flow cytometric analysis. Only mAb 20E6, which binds to an epitope toward the N-terminal of migis-δ, was found to stain human B cell line MC116, which expressed surface IgD and IgM. MC116 cells could be induced to undergo apoptosis by the treatment of 20E6, in the absence or presence of crosslinking second antibody. These results encourage further investigation on the potential of anti-migis-δ mAbs to target or control mIgD+ B cells.