bluuebunny/crossref_metadata_2025 huggingface dataset:
Autophagosome vaccine cross-protects and breaks tumor immunology paradigm: a p62-dependent mechanism? (41.41)
Authors: Twitty Chris G , Jensen Shawn , Hu Hong-Ming , Fox Bernard A.
Published: January 2023

Vaccination with MCA sarcoma cells protects from a subsequent challenge with the same, but not other MCA sarcomas. This observation of unique tumor-specific protection is a well-established paradigm (Prehn and Main 1957). We postulated that all MCA sarcomas overexpress common mutated gene products with a short half-life (SLiPs), but only the unique tumor-rejection antigens are stable enough to be cross-presented and induce anti-tumor immunity. We recently reported that with proteosomal blockade, SLiPs could be isolated in autophagic-containing (LC-3) vesicles (termed DRibble) that were efficient for cross-presentation of antigens (Li, Y. et al). Here we examined if vaccination with DRibbles from one MCA sarcoma would cross-protect from a challenge with an antigenically distinct MCA sarcoma. While whole tumor cell vaccines provide protection to unrelated tumor in 0 of 9 comparisons, DRibble vaccines provided significant (p<0.05) protection for 8 of 9 tumors (n=10-25 mice/group). Using a model system we showed that SLiPs play a critical role in providing antigen to DRibbles and that siRNA knock down of p62 reduced SLiPs and the capacity of DRibble to stimulate T cells. These results have shaped a model where polyubiquitinated SLiPs spared degradation by the proteasome, are picked up by p62 and delivered to autophagic vesicles. Used as a vaccine, these vesicles can cross-present neo-antigens capable of inducing cross-protection of chemically induced sarcomas. Supported by CA80964 (BAF), CA107243 (HH) the Chiles Foundation and the Murdoch Trust.
Design of artifical tumor cells to induce Natural Killer cell proliferation (42.13)
Authors: Huppert Volker , Müller Sabine , Turkiewicz Anna , Fahrendorf Melanie , Schmitz Jürgen
Published: January 2023

Natural Killer (NK) cells respond to tumor cell contact by cellular cytotoxicity, cytokine release and proliferation upon recognition of the tumor cells by an array of co-activating receptors such as Natural Cytotoxicity Receptors (NKp30, NKp44, NKp46), NKG2D, CD2, NKp80 and CD16 (binding to tumor cell reactive antibodies). Responsiveness of NK cells to tumor cells depends on complicated parameters of the individual's immune system and, if evaluating NK cell function in coculture of NK cells with tumor cell lines, requires maintenance culture of those cell lines and will cause further variation of experimental conditions.

We designed artifical tumor cells to tightly control human NK cell culture conditions. Tumor cells are mimicked by large magnetic spheres ("MACSiBead Particles") coated with ligands to activating NK cell receptors. Combinations of antibodies to NKp46 (CD335), NKG2D (CD314), Nkp80, CD2 have been evaluated as ligands and were feasible to induce a median 30 to 100 fold expansion of MACS isolated human NK cells within 2 to 3 weeks.

We demonstrated that cellular cell culture components such as tumur cell lines or allogeneic peripheral blood mononuclear cells (PBMC) are not required to induce significant NK cell proliferation and that artifical tumor cells (cell sized antibody coated magnetic particles) can be used instead and may provide a tool for NK cell proliferation for either diagnostic / analytical or NK cell therapy production purposes.
Qdot® nanocrystal conjugates in multispectral flow cytometry (42.14)
Authors: Godfrey William L. , Zhang Yu-Zhong , Jaron Shulamit , Buller Gayle M.
Published: January 2023

Qdot® nanocrystal-antibody conjugates allow researchers to detect more parameters in a sample with manageable spectral overlap compensation between fluorophores. These semiconductor nanocrystals optimally excite with ultraviolet or violet light, and provide symmetrical emission peaks with up to 400 nm Stokes shifts. In this study, we review the use of Qdot® nanocrystal and conventional fluorophore conjugates with particular attention to filter selection to minimize the within-laser and cross-laser compensations. All experiments were performed using standard staining protocols with human peripheral blood leucocytes and analyzed using a BD LSR II flow cytometer. Example results: Qdot® 655 and Qdot® 605 nanocrystal conjugates require less than 5% compensation, and the Qdot® 655 nanocrystal can require <20% compensation versus APC. Qdot 705 nanocrystal requires >100% compensation versus RPE-Cy5.5, but filter selection can reduce compensation versus RPE-Alexa Fluor® 700 conjugates to <30%. By optimizing individual channels, 8 to 12 color experiments have been run with good resolution of cell populations using 2 to 4 Qdot® nanocrystal conjugates. The unique spectral characteristics of Qdot® nanocrystals can dramatically facilitate multicolor experimental design when performing multicolor flow cytometry experiments.

Supported by Life Technologies Corporation.
Validation of Bio-Plex Pro™ Human Cytokine Assays on magnetic microspheres (42.26)
Authors: Zhou Hui , Ma Li , Fedynyshyn Joe , Tan Woei , Wang Qian-Shu
Published: January 2023

Bio-Plex Pro(tm) cytokine, chemokine, and growth factors assays are magnetic bead-based multiplex assays designed to measure these cell signaling proteins in diverse matrices like serum, plasma and tissue culture supernatants. The abstract describes the development and validation of a Bio-Plex Pro(tm) Human Cytokine Assay Group II Panel. The assays are based on the Luminex® xMAP® technology capable of simultaneously quantifying 23 targets. Performance characteristics, including assay sensitivity, working ranges, precision, accuracy, linearity of dilution and parallelism are reported. Three assays were compared with commercially available ELISA kits using human serum samples (N=25). A good correlation was observed between the Bio-Plex Pro(tm) assays and the commercially available ELISA kits. Cross-panel-plexing between Bio-Plex Pro(tm) Human Cytokine Assay Group II and Group I panel was also investigated. The study showed that 16 of the 23 targets in Group II panel could be multiplexed with the 27-plex Group I panel. Analysis of normal and disease human serum samples using the Bio-Plex Pro(tm) human cytokine assays reported data consistent with published literatures.
Contribution of CD137 signaling to Hepatic Fibrosis via T cell and Macrophage Mediated Chronic Liver Inflammation (43.5)
Authors: Wang Shengdian , Wang Shengdian
Published: January 2023

It is accepted that viral clearance and disease pathogenesis during HBV infection are mediated by the immune responses. Molecular mechanisms of the emergence or regulation of the immune response in this process remain largely unknown. Here we characterized that CD137 signaling acted as a potential pathogenic factor in progression of chronic hepatitis B. CD137 signaling successively stimulated by agonist anti-CD137 mAb could induce persistent hepatic infiltration and IFN-¦Ã production of CD8 T cells in HBV tansgenic mice. Through secreted IFN-¦Ã, the activated CD8 T cells subsequently recruited macrophage into liver to produce pro-inflammatory cytokines, which caused chronic liver inflammation and hepatic fibrogenesis, and promoted hepatocarcinoma development. Moreover, expression of CD137L was indeed up-regulated in peripheral monocytes of CHB patients and closely correlated with liver cirrhosis and the plasma level of IL-8, but not with ALT. Stimulation of monocytes from cirrhotic patients with recombinant human CD137 ligand induced IL-8 secretion in vitro, a chemokine closely associated with enhanced liver fibrosis in chronic HBV patients.

These resutls support an important role of CD137 signaling in liver inflammation and fibrosis, indicating that CD137 pathway may be a target for immunotherapy of chronic hepatitis B. Our findings also suggest that IFN-¦Ã,secreted by CD8 T cells, plays pivotal roles in mediating chronic liver inflammation and fibrosis. Further elucidation on this question will be of great significance for the immunotherapy of chronic hepatitis B.
Effector T-cells control lung inflammation during acute respiratory virus infection by producing interleukin-10 (43.7)
Authors: Sun Jie , Madan Rajat , Karp Christopher L , Braciale Thomas J.
Published: January 2023

Activated antigen-specific T cells produce a variety of effector molecules for clearing infection, but also contribute significantly to inflammation and tissue injury. Here we report an anti-inflammatory property of anti-viral CD8+ and CD4+ effector T cells (Te) in the infected periphery during acute pulmonary virus infection. We find that, during acute influenza and other respiratory virus infection, IL-10 is produced in the infected lungs at high levels -- exclusively by infiltrating virus-specific Te, with CD8+ Te contributing a larger fraction of the IL-10 produced. These Te in the periphery simultaneously produce IL-10 and proinflammatory cytokines, and express lineage markers characteristic of conventional Th/c1 cells. Importantly, blocking the action of the Te-derived IL-10 results in enhanced pulmonary inflammation and injury associated with the viral infection. Our results demonstrate that anti-viral Te exert regulatory functions --

that is, fine-tune the extent of lung inflammation/injury associated with respiratory virus infection by the production of an anti-inflammatory cytokine.
Plasmacytoid dendritic cells control influenza-specific CD8 T cells numbers through regulated FasL expression during lethal influenza virus infections (44.3)
Authors: Langlois Ryan A , Legge Kevin L
Published: January 2023

Previously we have demonstrated that the difference in survival of high versus low dose conventional influenza infections is linked to a severe T cell lymphopenia in high dose infections similar to what has been described in lethal H5N1 infections. This difference in T cell numbers is mediated by a novel regulatory mechanism where lymph node dendritic cells (LNDC) eliminate influenza-specific CD8 T cells in a FasL/Fas dependent manner within the LN in high but not low dose infections. Currently, it is unknown which LNDC subsets and what additional factors are required for driving this CD8 T cell apoptosis. Our results demonstrate that high dose pDC directly induce apoptosis of influenza-specific CD8 T cells ex vivo. This elimination occurs in the absence of influenza antigen presentation as peptide pulsing of pDC abrogates the induction of apoptosis. Further pDC downmodulate FasL expression, a process that appears to be dependent upon rDC migration and reduced rDC production of IL-12 p40 homodimer during low dose infections, therein allowing influenza-specific CD8 T cells to escape pDC induced apoptosis. Our results detail a heretofore undescribed role for pDC during influenza virus infections whereby pDC control the overall magnitude of the influenza-specific CD8 T cell response within the LN and ultimately, therein contribute to the outcome of influenza virus infections.
Novel IL-17–producing memory cells are key to vaccine-based protective immunity against M.tuberculosis challenge (45.12)
Authors: Lin Yinyao , Guglani Lokesh , Strawbridge Heather , Ritchea Shane , Logar Alison , Khader Shabaana
Published: January 2023

Interferon-gamma (IFNγ) is essential for limiting Mycobacterium tuberculosis infection. Using the mouse model, we have recently shown that vaccination triggered accelerated Interleukin-17 (IL-17) and IFNγ responses by CD4+ T cells in the lung during M. tuberculosis aerosol challenge. We propose that vaccination induces IL-17- producing CD4+ T cells that populate the lung and, after challenge, trigger the production of chemokines. The induction of chemokines results in recruitment of IFNγ-producing CD4+ T cells from the lymphoid compartment and ultimately restricts bacterial growth. The differential ability of IL-17-producing memory cells to populate the lung compared to the IFN-γ-producing cells suggests that these two cell types differ in their ability to migrate in response to chemokines. In support of this, our data using adoptive transfer models and chemotaxis assays suggests that differential responsiveness to chemokines and retention in different organs may provide the basis for the differential tissue distribution of these two cell types. During progression from effector to memory T cells, we also show differential expression of activation markers on IL17-producing and IFNγ-producing T cells. This information will lead us to determine whether altering the nature of the lung-resident IL-17 producing population impacts protective efficacy of the recall response to M.tuberculosis challenge.

This work was supported by AI075106-01 and Children's Hospital of Pittsburgh.
The Lifespan of Naïve CD4 T cells is a Critical Determinant for the Cellular Aging in Periphery (46.2)
Authors: Tsukamoto Hirotake , Clise-Dwyer Karen , Huston Gail E. , Buck Amanda L. , Duso Debra K. , Haynes Laura , Swain Susan L.
Published: January 2023

Cell intrinsic functional defect in aged naive CD4 T cells is a key cause of an age-associated increase in susceptibility to infectious diseases. However the causes of the CD4 T cell defects and how they develop are largely unknown. We have suggested that many defects develop during the sojourn of the naïve CD4 T cells in the periphery. We report here that aged naïve CD4 T cells have a longer lifespan than young cells even when transferred to young intact hosts. In vitro studies suggest that their increased longevity is regulated by an intrinsic property for survival. Consistent with this, aged naïve CD4 T cells express reduced level of a pro-apoptotic molecule, Bim, and the reduction of Bim in naïve CD4 T cells is sufficient for longer lifespan in vivo. This longer lifespan of aged naïve T cells could contribute to the maintenance of T cell homeostasis in aged mice. On the other hand, shutdown of new T cell emigration from the thymus by thymectomy leads to long-term persistence of remaining naïve T cells in the periphery and accelerates development both of the functional defects and of increased lifespan in naive CD4 T cells. Neither of these is observed when new naïve T cells are derived from aged hematopoietic stem cells. Thus, these results suggest that development of functional defects in aged naive CD4 T cells is associated with long-term persistence of aged naïve T cells due to their increased lifespan in periphery. The authors have no financial conflict of interest. This work was supported by National Institutes of Health Grants AG025805, AG021600 and the Trudeau Institute.
BCL6 Is Up-regulated in Th17 cells and Is Required to Repress GATA3 Activity During Th17 Differentiation (47.27)
Authors: Dent Alexander Lloyd , Mondal Arpita , Sawant Deepali
Published: January 2023

One important goal for immunology is a complete understanding of the regulatory mechanisms controlling the differentiation of CD4 T helper cells. The BCL6 gene, originally identified as an oncogene for B cell lymphoma, encodes a transcriptional repressor protein. We have shown previously that BCL6 is a potent inhibitor of Th2 cell differentiation, and BCL6-deficient mice develop greatly exaggerated Th2 responses and Th2-type inflammation. We have recently found that BCL6-deficient T cells are severely impaired in their ability to undergo Th17 differentiation, indicating that BCL6 function is required for normal Th17 differentiation. The cytokine IL-6 can promote Th17 differentiation, but the Th2 cytokine IL-4 strongly blocks Th17 differentiation by promoting Th2 differentiation. We have found that BCL6 is necessary to repress GATA3-mediated Th2 differentiation induced by IL-6 during Th17 differentiation. Further, we have found that BCL6 is significantly up-regulated in T cells stimulated under Th17 conditions, indicating a unique requirement for BCL6 in Th17 differentiation. These data show a novel, critical role for BCL6 in promoting Th17 responses.
Insulin reactive Type B T cells escape negative selection and are diabetogenic (48.11)
Authors: Mohan James F , Herzog Jeremy , Levisetti Matteo , Unanue Emil R
Published: January 2023

Several autoantigens thought to play a role in type 1 diabetes pathogenesis have been implicated, one of which, insulin, has been extensively associated with onset of disease. In particular, the peptide spanning the residues 9-23 of the insulin beta chain bound to the MHC class II allele, I-Ag7, has been characterized as the dominant epitope in NOD mice for islet infiltrating insulin specific CD4+ T cells. Intriguingly, the majority but not all of autoreactive T cells that recognize insulin are deleted during development in the thymus. In an effort to figure out why certain autoreactive T cells escape selection we have a found a unique population of insulin reactive T cells that recognize the peptide fragment 9-23 given exogenously (type B) but are unable to recognize processed native protein (type A). These type B insulin reactive T cells escape negative selection during development and can be elicited by peptide immunization. Primary lines derived from these T cells proliferated when cocultured with ex vivo isolated beta cells suggesting that dendritic cells present in islets constituitively present the type B conformer of the 9-23 epitope of insulin. Finally, primary lines were capable of inducing diabetes when adoptively transferred into NOD.scid recipients. These results suggest T cells that recognize type B conformers of self proteins escape negative selection and have the capacity to induce autoimmunity.
Alternative Splicing Factor/Splicing Factor 2 (ASF/SF2) regulates the expression of human T cell receptor CD3ζ chain (48.8)
Authors: Moulton Vaishali R , Perl Marcel A , Tsokos George C
Published: January 2023

T cells in patients with systemic lupus erythematosus (SLE) fail to control the aberrant autoimmune response and also infiltrate target organs. SLE T cells express low levels of the T cell receptor (TCR) CD3ζ chain, the main intracellular signal transducer upon antigen encounter. This deficiency is partially due to altered splicing of the CD3ζ mRNA. SLE T cells express a short alternatively spliced (AS) isoform of CD3ζ which is missing 562 nucleotides within its 3` untranslated region (UTR). This transcript is unstable and leads to reduced CD3ζ protein expression. We hypothesized that proteins binding to the splice-deleted region of the 3`UTR of CD3ζ play a role in regulating post transcriptional processing. Using an RNA oligonucleotide pulldown assay and mass spectrometry, we identified the serine/arginine-rich (SR) protein alternative splicing factor/ splicing factor 2 (ASF/SF2) as a putative protein binding to the CD3ζ 3`UTR. Specific knockdown of ASF/SF2 in Jurkat T cells led to a significant reduction in CD3ζ protein expression. Blocking of ASF/SF2 in human T cells with di-erythro-C6-ceramide enhanced the alternative splicing of CD3ζ, while overexpression of ASF/SF2 repressed this splicing. ASF/SF2 did not alter the RNA stability of CD3ζ. These data reveal a novel factor in the regulation of the post transcriptional processing of CD3ζ that may lead to altered CD3ζ chain expression in SLE.
Investigation of MICA*A5.1 Allele in Ankylosing Spondylitis (49.23)
Authors: huo zhi , Zou Yizhou , Hu Fengping , Yu Ping
Published: January 2023

MICA (major histocompatibility complex class I chain-related genes A) is known to be associated with susceptibility to various autoimmune diseases. Recent studies have also demonstrated that MICA*A5.1, one allele of MICA, contributed to the resistance of microorganism infection. The aim of this study was to investigate the association between MICA*A5.1 allele and ankylosing spondylitis (AS). A total of 144 AS patients selected from south China and 110 ethnically matched unrelated healthy controls were typed for MICA*A5.1 polymorphism in addition to HLA-B27 typing by polymerase chain reaction using sequence-specific primers. Statistical analysis revealed: (1) a significantly higher frequency of HLA-B27 in the patient group (96.5%) than in the control group (8.2%) (P < 0.0001); (2) the distribution of MICA*A5.1 genotype was significantly different between the patients and controls (P = 0.029), the frequency of MICA*A5.1 homozygote was significantly decreased in the patient group (2.1%) as compared with the controls (8.9%) (P = 0.023); (3) the association between MICA*A5.1 genotype and AS is not independent of HLA-B27 status (P = 0.94). These results suggest that MICA*A5.1 allele may play a potential role in the protection against AS.
Unique antibody gene signature in Multiple Sclerosis (49.25)
Authors: Monson Nancy L , Cameron Elizabeth , Spencer Sade , Lazarini Jonathan , Harp Christopher , Ward E. Sally , Burgoon Mark , Owens Gregory P. , Racke Michael K , Bennett Jeffrey L. , Frohman Elliot M.
Published: January 2023

Patients with multiple sclerosis (MS), an inflammatory, demyelinating disease of the Central Nervous System (CNS), derive the most benefit from disease modifying pharmacotherapies the earlier they are treated. Patients who have had a single episode of demyelination are at greater risk to develop clinically definite MS, but are not treated for MS, even though diagnosis of clinically definite MS could be significantly delayed if currently approved treatments are initiated immediately after the first attack. Defining biomarkers that identify patients with one demyelinating event who will convert to MS may provide a tool to allow earlier treatment of this "at risk" patient group. We have discovered a unique pattern of somatic mutations in the antibody genes of B cells isolated from the cerebrospinal fluid of patients with MS that can accurately and independently predict patients that subsequently develop definite MS after a single episode of demyelination. This antibody signature represents the first genetic biomarker candidate for MS outside of the MHC locus. If independently confirmed, our observations will have a major impact as a new diagnostic tool in patients with very early demyelinating disease of the CNS.

This work was supported by the National Institutes of Health (NIH) to NLM (RO1 NS 40993) and MKR (RO1 NS 37513, RO1 AI 47133, and K24 NS 44250), the National Multiple Sclerosis Society (NMSS) to NLM (RG3267) and JLB (RG3908), the Yellow Rose Foundation (NLM and MKR), the Wadsworth Foundation (to NLM) and Howson funds (to NLM).
n-3 PUFA ameliorates experimental autoimmune encephalomyelitis by modulating dendritic cell function (50.11)
Authors: Switzer Kirsten , Zheng Biao
Published: January 2023

n-3 polyunsaturated fatty acids (PUFA) have been shown to significantly attenuate T cell-mediated inflammatory diseases. Although evidence from epidemiological observations has long supported that dietary intake of n-3 PUFA may provide protection against multiple sclerosis (MS), available data about the effects of n-3 PUFA on the development and pathogenesis of MS or experimental autoimmune encephalomyelitis (EAE) remain very limited. We have found that treatment of mice with n-3 PUFA significantly prevented the development and severity of EAE. In addition, n-3 PUFA inhibited antigen-specific T cell proliferation and IFNγ production. Our preliminary studies suggest that n-3 PUFA exert their anti-encephalitogenic effects by interfering with multiple processes in the immunopathological cascade. Dendritic cells (DCs) are the most potent antigen-presenting cells, critical in the initiation of effective T cell immunity, therefore we investigated whether n-3 PUFA could interfere with DC function. We found that n-3 PUFA impaired DC migration from antigen entry sites to lymphoid tissue. In vitro treatment of BMDCs with docosahexanoic acid (DHA) resulted in suppression of DC maturation as assessed by reduced MHCII and CD86 expression. In addition, IL-12 cytokine production was decreased in a dose-dependant manner. Finally, pretreatment of DCs with DHA reduced their ability to present peptide for T cell activation. The findings from these projects may provide important information regarding therapeutic intervention for MS and other chronic inflammatory disorders.
Tolerization of BWF1 lupus mice with pConsensus peptide results in a distinct gene signature in WBC, CD4+ and CD8+T cells: Role of Foxp3 and IFI202b in suppression of SLE (50.22)
Authors: Singh Ram P , Dinesh Ravi , La Cava Antonio , Hahn Bevra H
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease associated with autoantibodies, including IgG anti-DNA and immune complexes. Following tolerization with an artificial peptide (pCONSENSUS, pCons) that is based on anti-DNA IgG sequences containing MHC class I and class II T cell determinants, NZB/NZW F1 female (BWF1) mice develop CD4+CD25+ regulatory T cells and CD8+ Foxp3+ inhibitory T cells, both of which suppress anti-DNA Ig production. In this study, we analyzed 45,000 murine genes using Affymetrix Gene Chip 430, 2.0 arrays, in a comparison of total white blood cells (WBC), CD4, and CD8 spleen cell subsets from tolerized vs. non-tolerized mice. Results showed 448, 174 and 60 genes that were differentially expressed by at least two-fold in the two groups of mice. From the CD8+T cell arrays, we confirmed up regulation of several genes in cells from tolerized mice using real-time PCR. Increased expression (more than two-fold) in the CD8+Tcells from tolerized mice was repeatedly found for six genes: IFI202B, Bcl2, Foxp3, Trp-53, CCR7 and IFNar1. Silencing with siRNA of IFI202b and bcl2 abrogated the suppressive capacity of the tolerized CD8+T cells. In contrast, silencing of IFNar1 and CCR7 had no effect on the ability of the CD8+T cells to suppress autoantibody production. Further silencing of combination of multiple genes affected other genes in addition to silenced gene/genes in tolerized CD8+T cells. These data indicate the roles of IFI202b, Foxp3, bcl2, and further demonstrate that silencing of one gene or combinations may affect other genes and or their functions.
GM-CSF induces differentiation of bone marrow precursors into tolerogenic CD8a- dendritic cells (50.32)
Authors: Bhattacharya Palash , Gopisetty Anupama , Ganesh Balaji B , Prabhakar Bellur S
Published: January 2023

Experimental autoimmune thyroiditis, a murine model of Hashimoto's thyroiditis is characterized by the infiltration of the thyroid by self-reactive CD4+ T cells. Our earlier studies have shown that GM-CSF treatment of experimental animals can suppress ongoing thyroiditis through the induction of tolerogenic CD8a- dendritic cells (DCs) expressing low levels of pro-inflammatory cytokines. Antigen presentation by these tolerogenic CD8a- DCs resulted in an expansion of IL-10 secreting CD4+Foxp3+ regulatory T-cells (T-regs) which suppressed the effector T-cell function. To understand the underlying mechanism we studied the effect of GM-CSF on matured splenic DCs as well as on bone marrow (BM) DC precursors in vitro. In spite of increased surface expression of programmed death ligand 2 (PDL-2), a negative regulator of T-cell activation, and increased indole-deoxygenase (IDO) secretion, splenic DCs exposed to GM-CSF induced only a modest increase in Foxp3 expression in T-cells. In contrast, BM cells when cultured in the presence of GM-CSF gave rise to a population of CD11c+CD11bhigh DCs with lower levels of expression of pro-inflammatory cytokines IL-12 and IL-6 which induced Foxp3 expression in a very high percentage of T-cells in a contact dependent manner. Our studies strongly suggest that the tolerogenic effect of GM-CSF is mediated by a special class of CD8a- DCs that differentiate from BM precursors. NIH 1RO1AI058190
The VR1 ligand capsaicin confers protection from autoimmune diabetes by suppressing autoreactive T cells in an APC dependent manner (50.38)
Authors: Nevius Erin , Basu Sreyashi
Published: January 2023

Sensory nerve fibers innervating the pancreas play a critical role in the etiology of autoimmune diabetes. These sensory neurons express a nonselective cation channel vanilloid receptor 1 (VR1) which has been identified as a receptor for acid, temperatures > 43°C and Capsaicin (CP), the principal pungent ingredient in chili peppers. We have reported that dendritic cells (DCs), a key cell type in immune responses, have the receptor for CP, and engagement of this receptor has powerful immune consequences. However very little is known about the function of VR1 expressing pancreas resident antigen presenting cells (APCs) including DCs in Type 1 diabetes. Here we have explored the immunomodulatory activity of CP in the non-obese diabetic (NOD) mouse and found that oral administration of CP delays the onset of diabetes and reduces the incidence of diabetes in NOD mice in a dose dependent manner. Moreover, splenocytes from CP treated mice are able to transfer protection in recipient NOD mice. CP administration attenuates the proliferation of pancreatic antigen-specific autoreactive T cells in pancreatic lymph nodes (PLN) both in vivo and in vitro in two distinct mouse models of diabetes, NOD and RIP-OVA. We have further found that this CP dependent modulation of autoimmunity requires both VR1 expression and a population of myeloid CD11b+ cells present in the PLN. These results reveal that CP is an immunologically potent ligand that when administered orally suppresses autoimmune diabetes by inhibiting the proliferation of autoreactive T cells in an APC dependent manner.
MR1 antigen presentation to mucosal-associated invariant T cells is highly conserved in mammals (78.7)
Authors: Huang Shouxiong , Martin Emmanuel , Kim Sojung , Yu Lawrence , Soudais Claire , Fremont Daved H , Lantz Olivier , Hansen Ted H
Published: January 2023

The development/expansion of mucosal associated invariant T cells or MAIT cells is dependent upon the class Ib molecule MR1, B cells and commensal flora. Additionally, MAIT cells express a canonical TCRα, a memory phenotype and rapidly secrete cytokines upon TCR ligation. Although these combined properties suggest that MAIT cells function as innate T cells in regulating mucosal immunity, this model has been difficult to test due to i) the paucity of MAIT cells that display MR1 specific activation in vitro, and ii) not knowing whether MR1 presents antigen. Here we show both mouse clones and human polyclonal MAIT cells display a high level of cross-reactivity on mammalian MR1 orthologs, but with differences consistent with limited ligand discrimination. Furthermore, acid eluates from recombinant or cellular MR1 proteins enhance MAIT cell activation in an MR1 specific and cross-species manner. Our combined findings demonstrate that the presentation pathway of MR1 to MAIT cells is highly physiologically conserved. These properties are reminiscent of pattern-like stimuli presented to previously described innate T cell populations and likely underlies the activated or memory phenotypes of MAIT cells.

This study was supported by grant AI046553 (Ted H. Hansen) from NIH, and in part by ANR MIME 2006 (Olivier Lantz) from the Inserm and ligue contre le cancer.
Aerosol allergen challenge results in airway hyperresponsiveness, inflammation, goblet cell metaplasia, and fibrosis in a murine model of fungal allergic airways disease (79.15)
Authors: Schuh Jane M. , Hoselton Scott A.
Published: January 2023

Allergic asthma is a chronic syndrome punctuated by acute respiratory distress that can result in irreversible remodeling of the airways. Experimental models that represent both acute and chronic dysfunction may provide insights into the pathology of asthma and a platform to test therapeutics. OBJECTIVE: To provide an allergic airway disease model that more closely reflects the human asthma syndrome. METHODS: BALB/c mice were sensitized with soluble Aspergillus fumigatus antigens over 5 weeks and were then challenged with a 10-min nose-only aerosol exposure to A. fumigatus spores. RESULTS: Airway hyperresponsiveness was observed through day 28 following allergen challenge. Goblet cell metaplasia and mucus hypersecretion peaked at day 7 after challenge. Eosinophil and lymphocyte recruitment peaked 7 and 14 days after allergen challenge, respectively, although persistent inflammation was still observed 35 days after challenge. Increased peribronchial and perivascular collagen deposition was first noted at day 14 after challenge in some animals and was consistently noted in lungs by day 35 post-challenge as compared to naïve animals. Visual evidence suggested that smooth muscle mass may also be increased at these time points. CONCLUSION: Aerosol delivery of fungal conidia provides a model that reflects both the acute and chronic outcomes of the allergic airways syndrome.
Soybean isoflavones regulate dendritic cell function: implications in food allergy (79.16)
Authors: Masilamani Madhan , Ruiter Bert , Shreffler Wayne G , Sampson Hugh A
Published: January 2023

Peanuts and tree nuts are the most allergenic foods in children and adults in the U.S. Several allergens in peanut and soy share amino acid similarity by as much as 60%, yet soy allergy is less common and less severe compared to peanut allergy. Unlike peanuts, soybeans are rich in isoflavones such as genistein, daidzein and glycitein and are the most common source of isoflavones in the human food supply. Isoflavones are known to have anti-inflammatory and anti-oxidant properties. The maximum bioavailability of isoflavones is from the gut since they are quickly converted to inactive forms before entering the circulation. Therefore, we hypothesize that the active-isoflavones in the gut milieu are capable of modulating the initiation of immune responses to dietary-antigens by regulating DC function. In vitro, isoflavones inhibited lipopolysachharide (LPS) or cholera toxin (CT)-induced cell-surface expression of CD83, CD80 and CD86 in a dose dependent manner in human monocyte derived dendritic cells (MDDC). Isoflavone treatment augmented CT induced up-regulation of CXCR4 and CCR7. Isoflavone treated MDDC inhibited T cell proliferation in a mixed lymphocyte reaction and inhibited the secretion of Th2 cytokines. We hypothesize that these changes in DC function could induce tolerance to food allergens. Studies are underway to test this hypothesis in a peanut allergic mouse model.
Toll-like receptor 2 and 4 in cockroach antigen-induced asthma: a potential mechanism for establishing a Th1 phenotype (79.23)
Authors: Durham Carolyn G. , Lorenz Robin G.
Published: January 2023

According to the hygiene hypothesis, sensing bacteria in the gut is imperative to switching the immune system from Th2 to Th1, thus reducing potential for an asthma-like phenotype developing in the lungs. In the gut toll-like receptors 2 and 4 (TLR2, TLR4) are critical for sensing Gram positive and negative bacteria, respectively. Previous data indicate in the first two weeks of life when the immune system switch generally occurs and a microbial repertoire is established, these two receptors are significantly upregulated. This project's hypothesis is TLR2 and 4 are critical for microbial sensing in the gut, thus C57BL/6 mice deficient in TLR2, TLR4, or both (double knockout) should have a more asthmatic phenotype. Interestingly, after inducing asthma with cockroach antigen (CRA) in a specific pathogen-free environment, the TLR2-/-/4-/- treated with CRA had a significant increase in serum IgE compared to the C57BL/6 treated with CRA, while the TLR4-/- had increased IL-13 mRNA levels compared to the C57BL/6 treated with CRA. This indicates the lack of interplay of TLR2 and 4 may play a role in maintaining a Th2 phenotype in the lungs; consequently preventing the immune switch to a Th1 phenotype.

Research supported by the Strategic Program for Asthma Research
Human embryonic stem cells as a source of human mast cells for studies of allergies and inflammatory diseases (80.3)
Authors: Kovarova Martina , Latour Anne M , Koller Beverly H
Published: January 2023

Human mast cells are principal cells initiating allergic reaction and asthma exacerbations. They are isolated either as mature cells from human tissues including skin and lung or differentiated from hematopoietic progenitors isolated from peripheral or cord blood. Isolation is accompanied with several disadvantages and difficulties including necessity of continuous access to human samples, low proliferation capacity and low efficiency in genetic modifications of isolated mast cells. In order to overcome these problems we developed a new strategy for differentiation of human mast cells from human embryonic stem (hES) cells. First, we differentiated hES cells to hematopoietic progenitors (CD34+ CD43+ cells) by co-culture of hES cells with stromal cells or by direct differentiation from embryonic bodies. Mast cells were subsequently differentiated in mast cell medium. Both approaches result in population of cells stained for mast cell specific proteins tryptase and chymase. They express a broad range of receptors and are able to be activated by their ligands. However, only mast cells differentiated directly from embryonic bodies express functional FcεRI. Together with techniques for genetic manipulation of hES cells, human ES cell-derived mast cells represent a unique model to analyze the effects of specific genetic alterations on human mast cell development, phenotype, and function.
T cell-intrinsic B7-homolog-1 (B7-H1) regulates effector CD8 T cell survival and memory cell generation (83.13)
Authors: Dong Haidong , Pulko Vesna , Liu Xin , Harris Kimberley
Published: January 2023

B7 homolog-1 ( B7-H1) expressed by antigen-presenting cells or tumor cells is a negative regulator of T cell responses, however its function on T cells is not clear. Here we show that B7-H1 was upregulated in CD8 T cells during immunization and its expression was maintained on memory CD8 T cells. Although B7-H1 deficient naïve CD8 T cells responded normally to antigen stimulation and underwent normal antigen-independent homeostatic proliferation, the maximal expansion of CD8 T cells was blocked due to their high susceptibility to apoptosis. B7-H1-deficient effector CD8 T cells showed weaker effector function and cytotoxicity against tumor cells compared to that of normal CD8 T cells. Once activated, B7-H1-deficient CD8 T cells generated less memory T cells. This effect was independent of the primary expansion of the antigen-specific CD8 T cell population. Our results revealed previously unknown function of B7-H1 on CD8 T cells in mediating survival and differentiation of effector CD8 T cells into memory T cells.
An allelic series of CD45 dosages uncouples basal and inducible TCR signaling during thymic development (84.13)
Authors: Zikherman Julie , Watson Susan , Jenne Craig , Raschke William C. , Goodnow Christopher , Weiss Arthur
Published: January 2023

CD45 is a receptor-like tyrosine phosphatase expressed at very high levels on all nucleated hematopoietic cells. CD45 serves as a critical positive regulator of TCR signal transduction by dephosphorylating the inhibitory tyrosine of the Src family kinases, Lck and Fyn. Previously, CD45 deficient animals have revealed a critical positive regulatory role in beta-selection and positive selection during thymic development.

Here we describe a novel allele of CD45, lightning, in which a single residue substitution in the extracellular domain produces low protein expression levels but normal isoform splicing. An allelic series of CD45 dosages reveals differential requirements for CD45 in basal and inducible TCR signaling during thymic development. While low levels of CD45 are sufficient to rescue T cell development and TCR inducible signaling, high doses of CD45 are required to fully reconstitute beta-selection and basal signaling. Furthermore, a negative regulatory role for CD45 is identified during inducible but not basal signaling. Hyperphosphorylation of the activation loop tyrosine of Lck but not Fyn at intermediate doses of CD45 correlates with hyper-responsive inducible signaling and demonstrates differential regulation of the src family kinases. Further, distinct thymic subsets exhibit differential requirements for CD45, Fyn and co-receptor-associated Lck in TCR signaling.
c-Myb is a novel regulator of Bcl-xL in the CD4+ and CD8+ double positive stage of T cell development (85.2)
Authors: Yuan Joan , Crittenden Rowena B , Rajewsky Klaus , Bender Timothy P
Published: January 2023

The lifespan of quiescent CD4+ and CD8+ double positive (DP) thymocytes regulates the composition of the peripheral T cell repertoire. However, the molecular mechanisms controlling DP thymocyte survival remain poorly understood. The c-Myb transcription factor is highly expressed in quiescent pre-selection DP thymocytes. Using a conditional deletion mouse model, we demonstrate that pre-selection DP thymocytes lacking c-Myb undergo premature apoptosis due to decreased expression of the key anti-apoptotic factor Bcl-xL. Moreover, c-Myb deficient DP thymocytes display a skewed TCRα repertoire, with a bias towards 3' Jα?usage, as a consequence of premature apoptosis occurring shortly after the initiation of TCRα rearrangements in vivo. Finally, re-introduction of c-Myb to c-Myb deficient DP thymocytes restored control levels of Bcl-xL mRNA and protein. Taken together, our study identifies c-Myb as a novel regulator of Bcl-xL expression in the DP stage of T cell development. We demonstrate that c-Myb acts through a novel pathway, independently of RORγt and TCF-1, two transcription factors previously shown to promote Bcl-xL expression in DP thymocytes. This study sheds new light on the regulation of the survival of pre-selection DP thymocytes and provides a novel mechanism by which c-Myb guards the balance between life, death and differentiation during hematopoiesis.
Id1 attenuates Notch signaling and impairs T cell commitment by elevating Deltex1 expression (85.3)
Authors: Wang Hong-Cheng , Sun Xiao-Hong
Published: January 2023

Complete inhibition of E protein transcription factors by Id1 blocks the developmental transition of CD4/CD8 double negative 1 (DN1, CD44+CD25-) thymocytes to the DN2 (CD44+CD25+) stage. To understand the underlying mechanisms, we observed that mRNA levels of Deltex1, as well as Deltex4, were elevated in Id1-expressing thymocytes, which could result in developmental arrest by attenuating Notch function. In support of this hypothesis, we found that Deltex1 ablation enabled Id1-expressing progenitors to differentiate to the DN3 (CD44-CD25+) stage, which was accompanied by enhanced Notch1 expression in T cell progenitors. Consistently, constitutive activation of Notch1 drove the differentiation of Id1-expressing progenitors to the DN3 stage. Furthermore, we showed that Gfi1b levels decreased whereas GATA3 levels increased in Id1 transgenic thymocytes, and GATA3 expression up-regulated Deltex1 transcription. Thus, T cell commitment may be controlled by the interplay among E proteins, Gfi1b and GATA3 transcription regulators, which influence Notch function through Deltex1 expression.

This work was supported by grant from the Nation Institute of Health to X.-H. S. (CA77553).
The CD3 ε subunit of the T cell receptor contains a basic-rich stretch important for multiple T cell functions (85.4)
Authors: Watts Laura M , Tassin Tara C , Becker Amy M , Medeiros Jennifer J , Albanesi Joseph P , Love Paul E , Wülfing Christoph , van Oers Nicolai S C
Published: January 2023

The TCR is a multimeric complex, consisting of a ligand-binding α/β heterodimer and four associated signaling chains (CD3γ, δ, ε, and ζ). The CD3 subunits each possess one or more copies of a signaling motif termed an ITAM, which is rapidly phosphorylated on tyrosine residues following TCR engagement. The cytoplasmic tails of CD3 ε and ζ also contain one or more clusters of basic amino acids. We report that these basic-rich stretches (BRS) can complex a subset of acidic phospholipids, including PI(4)P, PI(4,5)P2, and PI(3,4,5)P3. To determine how the BRS of CD3 ε contributes to T cell functions, we generated several CD3 ε constructs in which the positively charged residues of the BRS were eliminated. Analysis of these constructs in transfected cells revealed a role for the BRS in regulating both ITAM phosphorylation and TCR translocation to the immunological synapse. In transgenic mice, the introduction of modifications to the CD3 ε BRS resulted in a significant reduction in surface TCR expression and thymic cellularity. Furthermore, T cells from the BRS-mutant mice exhibited a diminished tyrosine phosphorylation of several signaling intermediates following TCR ligation. Based on these findings, we propose a model whereby electrostatic interactions between the BRS and phospholipids expressed on intracellular membranes regulate multiple T cell functions, including TCR expression and signaling.
Foxn1 is Required for Thymic Vascularization (86.7)
Authors: Manley Nancy R , Bryson Jerrod L , Griffith Ann V , Takahama Yousuke , Richie Ellen R
Published: January 2023

Development of a functional thymic microenvironment depends on crosstalk between multiple cell types. Recent studies suggest that crosstalk between developing vasculature and thymic epithelial cells (TEC) actively contributes to the complex thymic architecture, but little is known about initial immigration of endothelial precursor cells (EPC) into the thymus or subsequent vasculature formation. In a Foxn1 hypomorphic allele, Foxn1ΔΔ, TEC differentiation is arrested and cortical and medullary regions fail to form. We used wild-type, Foxn1Δ, and nude mice to define a role for Foxn1 in thymic vascularization. We show that EPCs first enter the wild-type thymus at E13.5, PDGFR-β + NCC perivascular mesenchyme follows at E14.5, and the vasculature is functional at E15.5 by the detection of intrathymic FITC-dextran after intraocular injection. EPC entry was delayed by one day in Foxn1ΔΔ mice, associated with down regulation of VEGF-A, PDGF-β, and CCL25 by 28%, 49%, and 98% at E13.5 compared to controls. In Foxn1Δ/nu mice, EPC entry was delayed until E15.5, and VEGF-A, PDGF-β, and CCL25 were more severely down regulated. In nude mice, endothelial cells could not be detected in the thymus rudiment. These data suggest that Foxn1 is required in a dose-dependent manner in TECs to regulate VEGF-A and PDGF-β expression necessary for normal thymic vascularization, and CCL25 for recruitment of LPCs in the embryonic thymus.
Bcl3 protects against lung graft injury by limiting emergency granulopoiesis (87.7)
Authors: Sugimoto Seiichiro , Kreisel Daniel , Carmody Ruaihdrhi , Gelman Andrew E
Published: January 2023

Excessive granulocyte accumulation in solid organ grafts leads to severe tissue injury and dysfunction. However, the mechanisms that differentially regulate emergency granulopoiesis from homeostatic granulopoiesis are poorly understood. The IκB family member Bcl3 is thought to mainly control cell survival and toll-like receptor mediated inflammatory gene expression. Here we report Bcl3 expression is required to protect against lung graft injury in a mouse orthotopic lung transplantation model by attenuating emergency granulopoiesis. Notably, Bcl3 was not required for homeostatic granulopoiesis as mice reconstituted with Bcl3-/- bone marrow (B6 (Bcl3-/-)) had equivalent numbers of granulocytes as compared to control wildtype B6 (B6) mice but following lung transplantation B6 → B6 (Bcl3-/-) recipients accumulated significantly more intragraft and peripheral blood granulocytes which exacerbated graft injury relative to B6 → B6 (B6) recipients. However, graft injury and granulocyte accumulation could be significantly attenuated with G-CSF neutralizing antibodies administered to B6 (Bcl3-/-) recipients. Moreover, G-CSF but not GM-CSF or IL-3 administration to resting B6 (Bcl3-/-) mice produced 2-fold more peripheral blood granulocytes than control wildtype B6 (B6) mice. Analysis of common myeloid progenitors revealed that Bcl3 negatively regulates the proliferation and the capacity of these cells to support emergency granulopoiesis following lung transplantation as well as following G-CSF but not GM-CSF or IL-3 stimulation. These data demonstrate a critical negative regulatory role for Bcl3 in G-CSF mediated emergency granulopoiesis and suggest that modulating signals that control the differentiation of myeloid progenitors can help prevent solid organ injury.