bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased susceptibility to colitis in the Vasoactive Intestinal Peptide receptor type 2 (VPAC2) mouse : A negative role for VPAC1 receptor in the induction of intestinal inflammation (50.44)
Authors: Yadav Mahesh , Goetzl Edward J
Published: January 2023

Vasoactive intestinal peptide (VIP) is a prominent neuropeptide with effects on many immune functions which are transduced by VIP G-protein-coupled receptors type 1 (VPAC1) and type 2 (VPAC2) on immune cells. Previous studies have shown that VIP reduces clinical symptoms and inflammation in mouse models of human immune-based diseases such as rheumatoid arthritis, Crohn's Disease, septic shock and multiple sclerosis. We recently demonstrated that VPAC2 knockout (KO) CD4 T cells in the presence of VIP and TGF-b develop into Th17 cells in a cAMP-Protein Kinase A dependent manner. Here we show that increased IL-17 induction in VPAC2-KO CD4 cells is accompanied by a reduced conversion of CD4 T cells into FoxP3+ regulatory T cells. To determine if VPACs play a role in the inflammatory bowel disease (IBD), VPAC2-KO mice were subjected to dextran sulfate sodium (DSS) in drinking water. Compared to wild type (WT), VPAC2-KO mice exhibited rapid and more severe clinical symptoms of colitis and had significantly higher colonic inflammation and increased inflammatory cytokines in colon. Severe colitis in VPAC2-KO mice was ameliorated in the presence of PKA inhibitors. Moreover, VPAC1-KO mice were resistant to the development of DSS-induced colitis. The results demonstrate a new role for VPAC1 which is responsible for increased inflammation in IBD and may provide a new therapeutic target.
The Use of Soluble T Cell Receptors to Identify Ligands for gamma/delta T Cells (78.1)
Authors: Chain Jennifer L , Aydintug M. Kemal , Roark Christina L , Born Willi K , O'Brien Rebecca L
Published: January 2023

Two very distinct types of T lymphocytes, expressing either an alpha/beta (ab) or gamma/delta (gd) T cell receptor (TCR), are involved in immune responses. Knowledge of the antigens that gd T cells recognize and their specific functions lags extensively behind that of ab T cells. The roles of gd cells are poorly defined largely because the antigens recognized by specific gd TCRs are not known. Gd TCRs are typically considered self-reactive, responding to stress-induced host molecules, although some are known to recognize pathogen-derived molecules. Our laboratory has developed a novel technique to detect host-derived ligands on cells using purified, soluble forms of specific gd TCRs as staining reagents. The use of these soluble TCRs (sTCRs) will provide a novel approach to the identification of specific cell surface ligands responsible for activating gd T cells. I propose to use sTCRs to identify the ligands for two common murine gd TCRs, Vg1:Vd6.3 and Vg6:Vd1. sTCRs against these receptors will be used like monoclonal antibodies to screen a retroviral cDNA library made from a ligand-positive cell line. The identification of these ligands will promote further research, leading to a better understanding of the roles of gd cells in a wide variety of immune responses and providing insight into the specificity requirements of the gd TCR.
A cell free antigen processing system identifies immunodominant epitopes (78.19)
Authors: Kim Ae , Hartman Isamu , Sadegh-Nasseri Scheherazade
Published: January 2023

T cell-mediated responses to protein antigen involve recognition of selected antigenic peptides bound to Major Histocompatibility Complex (MHC) molecules. Proteolytic digestion of protein antigens during antigen processing generates many peptide fragments that can potentially bind to MHC molecules. However, only few selected antigenic epitopes induce T cell responses, defined as "immunodominant". Identifying immunodominant epitopes from a given antigen has wide applications in development of peptide vaccines and therapies against infectious diseases, cancer, autoimmune diseases, and allergy. Currently available methods are not efficient in elucidating CD4+ T cell epitopes. Here, we provide a novel approach for identifying the immunodominant epitopes from protein antigens using a cell-free MHC class II antigen-processing assay. Two different novel antigens, Hemagglutinin (HA1) of the A/Vietnam/1203/2004 (H5N1) virus, and a recombinant form of Plasmodium falciparum liver-stage antigen 1 (LSA-NRC) were used to evaluate our assay. HLA-DR1 restricted epitopes from both proteins were identified by mass spectrometry and were tested for their ability to activate T cells in HLA-DR1 tg mice. We observed that the identified epitopes were indeed immunodominant as CD4+ T cells from mice immunized with the proteins proliferated and produced cytokines upon challenge with the identified epitopes at levels comparable to the whole protein. This cell-free assay provides a useful tool for identification of CD4+ T cell epitopes for specific MHC class II alleles.
Vanilloid receptor 1 agonists, capsaicin and resiniferatoxin, induce efficient anti-viral CTL responses by increasing MHC-restricted antigen presentation (78.29)
Authors: Lee Chong-Kil , Lee Young-Hee , Lee Young-Ran , Kang Hyun-Jung , Im Sun-A , Song Sukgil , Kim Kyungjae
Published: January 2023

DCs, like the sensory neurons, express vanilloid receptor 1 (VR1). Here we demonstrate that VR1 agonists, capsaicin (CP) and resiniferatoxin (RTX), increase MHC-restricted antigen presentation in DCs. Treatment of DCs, that were infected with recombinant vaccinia virus (VV) expressing OVA (VV-OVA), with CP or RTX significantly increased MHC class I-restricted OVA presentation. In accordance with the in vitro results, oral administration of CP into VV-OVA-infected mice significantly enhanced MHC class I-restricted OVA presentation, and consequently elicited potent OVA-specific CTL activity compared to untreated controls. The effects of VR1 agonists on the exogenous antigen presentation were also examined in DCs. CP and RTX enhanced MHC class I-restricted, but not MHC class II-restricted, presentation of exogenous OVA. CP and RTX did not affect phagocytic activity, the expression level of total MHC molecules and co-stimulatory molecules on DC, suggesting that they enhance the intracellular processing events of phagocytosed antigens. These results demonstrate that VR1 agonists preferentially increase MHC class I-restricted antigen processing pathways. The present study also shows that oral consumption of CP, a common component of pungent foods, could enhance the efficacy of DC-based anti-viral and anti-tumor vaccines.
Skin Delivery of the JVRS-100 Immunostimulant with and without Fluzone using a Protrusion Array Device (PAD) Results in Potent Adaptive Immune Responses (78.34)
Authors: Chang Stella , Gonzalez-Gonzalez Emilio , Contag Christopher H. , Fairman Jeff , Kaspar Roger , Speaker Tycho
Published: January 2023

Background: A protrusion array device (PAD) configured with hollow dissolvable microneedles was loaded with the JVRS-100 adjuvant or JVRS-100 combined with commercial Fluzone. The studies were designed to establish if adjuvant or adjuvant/vaccine could be administered via PAD patches. Methods: JVRS-100, Fluzone, or JVRS-100/Fluzone were loaded onto PAD patches and administered at day 0 and 14. At day 28 responses were assessed by HAI and ELISA (IgG, IgG1, and IgG2). Separate cohorts of mice were vaccinated intramuscularly (IM) with Fluzone and JVRS-100-containing patches were placed over the site of vaccination to see if intradermal administration of JVRS-100 would adjuvant IM vaccination. Results: The administration of JVRS-100 in addition to Fluzone stimulated a response greater than Fluzone alone patches as measured by geometric mean titer (EC50) for IgG (4.8x104 vs. 1.6x104) and IgG2 (4.5x103 vs 6.0x102). IgG1 titers were elevated in the Fluzone only group compared with JVRS-100/Fluzone (8.3x103 vs. 1.9x103). Administration of JVRS-100-containing patches over IM Fluzone vaccination increased IgG (3.7x105 vs. 2.2x105) and IgG2a (1.5x105 vs. 9.0x104). IgG1 titers were elevated in the Fluzone-only group compared with JVRS-100/Fluzone (1.5x105 vs. 2.9x105). HAI levels correlated with antibody titer levels for all groups. Conclusion: The antibody response from both studies indicate that the TH1 bias for JVRS-100 is retained as a dry PAD patch and intradermal administration over IM vaccination or combined for ID vaccination.
Tissue restricted activation of basophils mediates primary immunity against helminthes (79.22)
Authors: Sullivan Brandon M , Wu Davina , Liang Hong-Erh , Loke P'ng , McKerrow James H , Voehringer David , Locksley Richard M
Published: January 2023

CD4 T cells and the cytokines IL-4 and IL-13 are required for primary immunity to helminthes and for allergic responses. Although T-helper-2 (Th2) cells are believed to provide protection, genetic deletion of IL-4/13 from CD4 T cells revealed that cytokines from innate cells mediate Nippostrongylus brasiliensis clearance and survival after Schistosoma mansoni infection. Basophils were the only IL-4-secreting innate cells identified. Basophils were recruited to numerous sites, but basophil cytokine secretion occurred only in affected tissues, and was independent of CD4 T cell-derived IL-4/13 and IgE. Depletion of basophils compromised the ability to expel intestinal helminthes. Thus, basophils serve as innate effector cells during primary helminth infection by secreting cytokines at sites of tissue involvement, a process amplified during adaptive immunity by IgE through IL-4-mediated T cell help.
Basophils play an important role in the resolution of inflammation by inducing alternative activation of macrophages through Th2 cytokine release (80.4)
Authors: Karasuyama Hajime , Mukai Kaori
Published: January 2023

Basophils have long been thought to be an initiator or effector of allergic inflammation. Here we report that basophils can function as a terminator of inflammation in IgE-mediated chronic allergic inflammation. In this allergy model, mice were passively sensitized with hapten TNP-specific IgE and then challenged with subcutaneous injection of TNP-OVA in the ear. The allergen challenge induced not only immediate-type ear swelling (mast cell-dependent) but also delayed-onset ear swelling (basophil-dependent). Analyses with flow cytometry and RT-PCR revealed that alternatively activated macrophages (AAMs) appeared in the skin lesions as the inflammation proceeded. The AAMs were induced in T cell-deficient but not IL-4/IL-13-deficient mice, indicating that AAM-inducing Th2 cytokines were produced by non-T cells. Basophil depletion abolished the development of AAMs. The cell transfer experiment revealed that basophil-derived IL-4 was essential for the alternative activation of macrophages. Depletion or developmental block of AAMs resulted in deterioration and prolongation of inflammation with massive accumulation of inflammatory cells. Taken together, basophils induce alternative activation of macrophages through Th2 cytokine release, leading to the resolution of inflammation.
Nitration of aldolase: a critical post-translational modification in nitric oxide-mediated regulation of mast cell function (80.6)
Authors: Sekar Yokananth , Moon Tae Chul , Slupsky Carolyn M , Befus A. Dean
Published: January 2023

Nitric oxide (NO) is a short-lived free radical that regulates mast cell (MC) activities including inhibition of MC degranulation. To elucidate the molecular mechanisms underlying the effects of NO in MC, we investigated protein tyrosine nitration in human mast cell lines treated with the NO donor S-Nitrosoglutathione. Using 2D gel western blot analysis with an anti-nitrotyrosine antibody together with mass spectroscopy, we identified aldolase A, an enzyme of the glycolytic pathway as a target for tyrosine nitration in MC. Aldolase A nitration was associated with reduction in the Km and Vmax of aldolase in HMC-1 and LAD-2. Nuclear Magnetic Resonance analysis showed that despite these changes in activity of a critical enzyme in glycolysis, there was no significant change in total cellular ATP content, although the AMP/ATP ratio was increased. Elevated levels of lactate and pyruvate suggested that NO treatment enhanced glycolysis in MC. Reduced MC aldolase activity was associated with increased intracellular levels of its substrate, fructose 1,6 bisphosphate (FBP). Interestingly, FBP inhibited IgE-mediated MC degranulation in LAD-2 cells. Thus for the first time we report evidence of protein tyrosine nitration in two human MC lines and identify aldolase A as a prominent target that may regulate MC phenotype and function.

Funded by: Canadian Institutes of Health Research, The Lung Association, Alberta & NWT and 75th Anniversary Award, University of Alberta.
Imbalance of effector and regulatory T cells in the pathogenesis of experimental eosinophilic esophagitis (80.8)
Authors: Mishra Anil , Rothenberg Marc
Published: January 2023

RATIONALE: Eosinophilic esophagitis (EE) is an emerging disease and we previously demonstrated that adaptive T cell immunity is critical in the disease pathogenesis. However, the specific subsets of T cells that promote esophageal eosinophilia are not yet understood. Therefore, we focused our studies to examine the role of CD45RB T subsets, as the pathogenic and regulatory activity of T cells depends on the expression levels of CD45RB. METHODS: A flow cytometric analysis was performed to examine the frequency of CD45RBhigh and CD45RBlow T cell subsets in the mouse esophagus. A real time PCR analysis was performed to examine the pathogenic and anti-inflammatory characteristics of both subsets of T cells. In addition, we adoptively transferred these purified cell subsets in mice with experimental EE and examined their role in the disease pathogenesis.

RESULTS: An increase of CD4+CD45RBhigh and a decrease of CD4+CD45RBlow cells were observed in mouse esophagus following the induction of experimental EE. A relatively high transcript level of IL-5 and IL-13 in CD4+CD45RBhigh and IL-10 and TGF-β1 in CD4+CD45RBlow spleen purified cells was observed. Further, our initial experimentation revealed that mice adoptively transferred with 4X105 CD4+CD45RBlow T cells/week were protected from the induction of esophageal eosinophilia compared to control mice that did not received adoptively transferred cells. Mice receiving CD4+CD45RBhigh T cell had no significant change in the number of eosinophils in the esophagus compared to control mice following allergen challenge. CONCLUSIONS: CD4+CD45RBlow T cells have a critical role in down-regulating allergen-induced eosinophil recruitment to the mouse esophagus.
Evaluation of granulysin and perforin as candidate biomarkers for protection following vaccination with Mycobacterium bovis BCG or M. bovis delta-RD1 (81.10)
Authors: Scherer Charles C , Endsley Janice J , deAguiar Juliana , Jacobs Jr. William R , Larsen Michelle H , Palmer Mitchell V , Nonnecke Brian J , Golde William T , Waters W Ray , Estes D Mark
Published: January 2023

Development of improved vaccines against tuberculosis (TB) is directly correlated to the investigation of new and better correlates of protection after vaccination. We examined the kinetics of granulysin, perforin, IFN-gamma and Fas-L responses to M. bovis purified protein derivative (PPD) stimulation by peripheral blood mononuclear cells from BCG- and non-vaccinated cattle. Gene expression profiles following PPD stimulation showed significant increases in transcripts for granulysin and IFN-gamma in both CD4+ and CD8+ T cells in BCG-vaccinated as compared to non-vaccinated animals. Perforin & IFN-gamma examined by flow cytometry, showed a difference of 1-2% more PPD-specific cells in BCG-vaccinated than non-vaccinated animals. In a second trial, M. bovis BCG vaccinated animals had lower disease and pathological scores as compared to M. bovis Delta-RD1(a new vaccinate candidate) and the control group. Gene expression of granulysin and perforin were significantly increased in both vaccine groups (M. bovis BCG and M. bovis Delta-RD1 vaccinated) as compared to control after vaccination and challenge. IFN-gamma expression was increased only after vaccination and secretion was higher in the control, as compared to both vaccine groups demonstrating no correlation with protection upon vaccination.
Chinook Salmon (Oncorhynchus tshawytscha) Have Multiple Interferon-gamma Genes

(81.7)
Authors: Rhodes Linda D , Demlow S. Ellen
Published: January 2023

Interferon-gamma (IFNg) is a central cytokine in modulating innate and adaptive immunity in a wide range of vertebrates. A study of genes associated with an effective host response by Chinook salmon (Oncorhynchus tshawytscha) to a persistent Gram-positive bacterial pathogen (Renibacterium salmoninarum) suggested that an interferon-inducible signal was abrogated during early stages of exposure. Splenic DNA was amplified with IFNg primers from a related species, rainbow trout (Oncorhynchus mykiss), and multiple clones from individual fish were sequenced. Up to five genotypes per fish were found, and sequence variations included microsatellites within introns and single nucleotide polymorphisms (SNPs) within exons. Southern hybridizations supported the prediction that Chinook salmon possess at least three related IFNg loci. Multiple clones of cDNA, synthesized from RNA isolated from pronephros of fish exposed to an attenuated strain of R. salmoninarum, displayed a surprising abundance of SNPs. These findings are consistent with the phenomenon of genome duplication known to have occurred in teleosts. Although the presence of multiple loci complicates the analysis of IFNg gene expression, it may also offer a mode of immune modulation not found among mammals.

Research supported by National Marine Fisheries Service, NOAA Fisheries, U.S. Department of Commerce.
Peripheral blood and jejunal intraepithelial immune cell phenotypes associated with body weight of newly weaned pigs (81.9)
Authors: Davis Ellen , Rehberger Joshua , King Michael , Brown Dari C. , Maxwell Charles V. , Rehberger Tom
Published: January 2023

Early immune development has been purported to have ramifications on metabolic functions related to subsequent growth. To determine if specific immune cell populations were associated with pigs with heavy and light body weight (BW), piglets were weaned to an on-site conventional or a segregated weaning facility and immune cell phenotypes were characterized and correlated to piglet BW. Pig BW was measured and flow cytometry analyses were performed on PBMC and jejunal intraepithelial lymphocytes (IEL) on day 11, 18, and 25 after weaning. Ordination methods were performed to determine the relationship of specific immune cell phenotypes to piglet BW. Cell phenotypes consistent with γδ (CD4+γδ+CD8-, CD8+γδ+CD4-) and activated (CD25+) IEL were positively correlated to pig BW on day 11, whereas PBMC expressing MHC2 (MHC2+CD3-) resulted in a negative correlation. On day 18, CD25+CD8+CD4+ IEL were positively correlated and PBMC expressing CD8+CD4+ were negatively correlated to pig BW. CD25+ and CD4+CD8+ IEL populations were positively correlated to pig BW on day 25, whereas IEL γδ populations (γδ+CD8+CD4-, CD4+CD8+γδ+) and PBMC expressing MHC2 (MHC2+CD3-) were negatively correlated to BW. These data indicate that specific immune cell phenotypes correlate to pig BW after weaning. Specifically, IEL expressing CD25 are associated with pigs with heavier BW after weaning, whereas IEL expressing the γδ T cell receptor are associated with heavy BW pigs early after weaning but are correlated to low BW in older pigs.
CD4+ recent thymic emigrants are biased to the TH2 effector lineage (82.15)
Authors: Hendricks Deborah W , Boursalian Tamar , Fink Pamela J
Published: January 2023

Following intra-thymic development, T cells exit the thymus and join the peripheral T cell pool. These newly generated cells, termed recent thymic emigrants (RTEs), undergo phenotypic and functional maturation for the first 3 weeks they reside in the periphery. Using mice transgenic (Tg) for green fluorescent protein (GFP) driven by the Rag2 promoter (RAG2p-GFP Tg), in which GFP reliably distinguishes RTEs from their more mature naïve (MN) counterparts, our lab is able to isolate untouched RTEs for functional and phenotypic analysis.

CD4+ RTEs exhibit defects in proliferation, IL-2Rα upregulation, and IL-2 production when stimulated through the TCR. We now show that CD4+ RTEs also exhibit defects in Th0, Th1, Th17 and Treg lineage commitment, with diminished cytokine production and transcription factor expression. In contrast to these diminished functions, RTEs are enriched for IL-4 producers when differentiated under Th2, but not Th0, conditions. This bias to the Th2 lineage may be due to regulated expression of T-bet, a key Th1 transcription factor.

These data show that CD4+ RTEs represent a unique subset of the peripheral T cell pool, defined by a distinct interpretation of and response to immunological cues. These characteristics may be beneficial during the post-thymic maturation period to avoid inappropriate immune responses.

This work was supported by R01AI064318 from the NIAID (with a supplement to DWH).
Memory-like CD8+ T cells generated during homeostatic proliferation defer to antigen-experienced memory cells (82.18)
Authors: Cheung Kitty P , Yang Edward , Goldrath Ananda W
Published: January 2023

Naïve T cells proliferate in response to lymphopenia and acquire the phenotypic and functional qualities of memory T cells, providing enhanced protection against infection. How well 'memory-like' T cells generated during lymphopenia-induced homeostatic proliferation (HP-memory) differentiate into secondary memory cells and compete with antigen-experienced 'true-memory' cells is unknown. We found that CD8+ HP-memory T cells generated robust responses upon infection and produced a secondary memory population comparable to true-memory cells in the absence of competition. However, when true-memory and HP-memory T cells competed during infection, HP-memory cells contributed less to the effector population, contracted earlier and formed fewer secondary memory cells. Furthermore, HP- and true-memory cells demonstrated aberrant chemokine receptor expression and distinct localization within the spleen during infection, indicating differential access to signals necessary for secondary memory formation. Thus, HP-memory T cells provide protection without compromising the true-memory population. Differences in HP-and true-memory T cells may reveal the basis for competition for limited resources within the memory T cell compartment.
Role of TOSO in activation induced cell death of human T- and NK cells (82.2)
Authors: Narayanan Sriram , Borrego Francisco , Coligan John E.
Published: January 2023

Homeostasis of both NK and T cells in the periphery is regulated by activation induced cell death (AICD) mediated by signaling through Fas, and other death receptors. It has been shown that IL-2 is required for priming cells for this type of apoptosis. Cytokine primed NK cells, in addition to killing their target cells, also undergo AICD upon activation by activating ligands expressed by the target cells. TOSO is an anti-apoptotic molecule known to inhibit apoptosis mediated by the death receptor pathway and we investigated the role of TOSO in regulating AICD in NK and T cells. We observed by realtime PCR that stimulation of human NK cells and T cells in vitro with IL-2 resulted in down regulation of TOSO. We further analyzed TOSO expression levels on sorted human CD4 T cells and found that cells with an effector/memory phenotype have markedly lower levels of TOSO than naïve cells, which suggests that activation in vivo correlates with down-regulation of TOSO. In agreement, we found that activation of CD45RO+ cells in vitro dramatically down regulated TOSO compared to naïve CD4 T cells. Further, overexpression of Toso in Jurkat cells was able to inhibit Fas-mediated cell death. We hypothesize that IL-2 has a dual action: first, it activates and induces the proliferation of NK and T-cells and second, it down-regulates TOSO, thereby sensitizing or priming them for apoptosis upon re-stimulation. Considering that IL-2 is being used as a therapeutic agent for cancer treatment and the recent study showing that HIV envelope protein can down regulate TOSO levels on CD4 T cells, unraveling how TOSO functions and is regulated might have potential for treating cancer and other diseases.
Advanced age does not impair the development of functional memory CD8+ T cells following West Nile virus infection (83.4)
Authors: Lelic Alina , Parsons Robin , Hayes Lisa , Bramson Jonathan L
Published: January 2023

West Nile virus (WNV) infection of aged patients typically leads to the development of severe neuroencephalitis. Studies in mice have elucidated an important role for CD8+ T cell function in controlling WNV infection and we hypothesized that the increased severity of WNV illness in the aged results from impaired CD8+ T cell function due to immunosenescence. We examined the functional profile of WNV-specific CD8+ T cell memory responses from a cohort of young and aged WNV infected patients using polychromatic flow cytometry. We quantified production of IFN-γ, TNF-α, IL-2 and CD107a mobilization by WNV-specific CD8+ T cells. We did not observe any relationship between advanced age and either the breadth or the magnitude of CD8+ T cell function. The magnitude of IFN-γ and TNF-α responses to WNV epitope stimulation did not decline with advanced age. Production of IL-2 was not detected from either young or aged CD8+ T cells. The ability to degranulate in response to virus peptide was also the same in young and aged patients. Collectively these data fail to support our hypothesis and suggest that, contrary to current models of immunosenescence, aged patients do develop functional CD8+ T cell immunity following exposure to a novel virus infection. This work was funded by a contract from NIAID (N01-AI-40066).
Extrafollicular foci-derived B cells provide long-lived mucosal immunity (84.16)
Authors: Baumgarth Nicole , Rothaeusler Kristina
Published: January 2023

Antibodies to influenza are contributed by extrafollicular foci and germinal centers. The latter induce long-lived immunity via bone marrow plasma cells and memory B cells. Extrafollicular foci are thought to only provide early, short-lived responses generated from high-affinity B cells. The selection of the highest affine B cells into the early response might ensure survival from acute infection, but it seems counterintuitive that they would be excluded from the long-lived response. Here we assessed the contributions of the extrafollicular foci response to long-term immunity. We tracked a prototypic early B cell response to hemagglutinin (HA) of influenza A/PR8 in BALB/c mice by multicolor flow cytometry using an idiotype-specific mAb to the C12 idiotype (Id) together with fluorescent-labeled HA. C12Id+ HA-specific antibodies were measured by ELISA and ELISPOT. The results demonstrate the extrafollicular nature of the C12Id response and the lack of C12Id+ bone marrow plasma cells. Nonetheless, following infection HA-specific serum C12Id+ antibodies were measurable for the life of mice, secreted presumably by antibody-secreting C12Id+ plasma cells present in the lung. BrDU-labeling experiments suggested continuous turnover of these cells. Thus, extrafollicular foci induce long-term mucosal antibody responses via short-lived plasma cells. Supported by NIH AI051354 (N.B) and T32 HL007013 (K.R.).
Characterization of CD44-high memory phenotype T cells induced by enforced expression of the transcription factor PLZF (85.12)
Authors: Raberger Julia , Grabner Beatrice , Sakaguchi Shinya , Schebesta Alexandra , Boucheron Nicole , Blomberg K. Emelie M. , Berglöf Anna , Kolbe Thomas , Smith Edvard , Rülicke Thomas , Ellmeier Wilfried
Published: January 2023

We and others have shown that transgenic expression of the transcription factor PLZF in the T cell lineage induces the appearance of CD44-high memory-phenotype (MP) T-cells (Savage et al. Immunity 2008; Raberger et al. PNAS, 2008). These MP T cells arise already in the thymus and produce IFNã upon PMA/ionomycin stimulation, thus sharing certain features with innate-like T cells. Furthermore, we observed that PLZF expression is up-regulated in Itk-deficient T cells, which are enriched with innate-like T cell subsets. To study the PLZF-induced MP T cell population in more detail, we are investigating whether these cells, like other innate-like T cells, are selected on classical or non-classical MHC molecules. Furthermore, PLZF transgenic mice are intercrossed with several knockout mice (e.g. Itk-/- or IL-15-/- mice) to determine signaling pathways/molecules required for their development and maintenance. Our studies will provide more insight into transcription pathways controlling the development of MP T cells with "innate-like" functions.

Supported by the Austrian Science Fund FWF (SFB-F23, P19930).
ADAM protease-mediated cleavage of Delta-like-1 regulates the degree of Notch signaling during T cell development (85.18)
Authors: Gravano David M , Manilay Jennifer O
Published: January 2023

Hematopoietic progenitor cells require Notch signaling in the thymus to develop into mature T cells. Members of the Delta-like family of Notch ligands expressed on thymic epithelial cells (TECs) initiate this signaling. Delta-like-1 (Dll1) is proteolytically processed by ADAM (a disintegrin and metalloprotease) proteases, but the effect of this cleavage on T cell development has not yet been analyzed. Mice lacking ADAM17 in TECs display blocks in T cell development (Li, N. et al. J Immunol 2007. 178(7) p. 4214-21), leading to speculation that Notch signaling between TECs and thymocytes is aberrant in these animals. The objective of our study is to determine the functional significance of proteolytic cleavage of the Delta-like ligands on T cell development. Using the well-established OP9-Dll1 in vitro T cell differentiation system, we observed that Dll1 cleavage on OP9 cells does occur in the presence of thymocyte progenitors. A non-cleavable Delta-like-1 construct (NC-Dll1) (Six, E. et al. Proc Natl Acad Sci USA 2003. 100(13) p. 7638-43) was utilized to elucidate the functionality of Dll1 cleavage on thymocyte differentiation. Lack of Dll1 cleavage did not affect T lineage fate or development. However, Notch signaling was potentiated in hematopoietic progenitor cells cultured on OP9-NC-Dll1, as measured by higher expression of the Notch target gene Hes1. These data indicate that proteolytic cleavage of Dll1 may function to reduce the level of Notch signaling in developing thymocytes. In conclusion, ADAM mediated cleavage of Dll1 occurs during the process of T cell development, but blocking it does not appear to stunt development toward the T cell lineage and rather, increases the strength of Notch signaling.
Real time multi-photon imaging reveals coordination between thymocyte development, location and migration through intrathymic microenvironments (85.6)
Authors: Ehrlich Lauren I Richie , Oh David Y , Lewis Richard S , Weissman Irving L
Published: January 2023

As they develop, CD4-CD8- (DN) thymocytes are found progressively from the corticomedullary junction towards the capsule. Following beta selection, CD4 and CD8 are expressed, generating double positive (DP) thymocytes that are found throughout the cortex. If a DP thymoycte expresses T cell receptor (TCR) and passes positive-selection, it becomes a single positive (SP) thymocyte, which is located in the medulla. Static images have revealed this intrathymic localization, suggesting a model in which DN thymocytes migrate directionally towards the capsule, while DP thymocytes move towards the medulla, which they enter as SP thymocytes.

Using multi-photon imaging of thymocyte subsets in thick thymic slices, we directly test this model. Using thymuses expressing EGFP, we identify the precise microenvironments in which thymocyte subsets are localized. For the first time, we now image within the thymic medulla. We find that DN and DP thymocytes migrate without directional bias throughout the cortex, but DP velocities are influenced by cortical micorenvironmental cues. Interestingly, only SP thymocytes enter the medulla, where they migrate very rapidly. SPs also migrate rapidly in the cortex, where their movement is biased towards the medulla. Importantly, medullary entry is strictly dependent on G-protein-coupled receptor activity, though CCR7 signaling cannot entirely account for this requirement.

L.I.R.E is a Leukemia and Lymphoma Society Special Fellow
Age-dependent T and B lymphocyte subsets defined by multicolor flow cytometry (86.13)
Authors: Crabtree Matthew Jude , DeRosa Stephen C. , Cohen Gordon , Wiedeman Alice , Stevens Anne M
Published: January 2023

Immune dysregulation in children due to primary immunodeficiency, autoimmune disease or immunomosuppressive therapy may be characterized in detail by multiparametric flow cytometry. To determine age-dependent reference values for healthy children, we developed four panels: aß and γδ T lymphocytes; regulatory T lymphocytes; B lymphocytes; NK and NK T cells. Detailed analyses performed on 18 healthy subjects aged 8 to 46 years revealed differences between adult and juvenile phenotypes in 66 of 301 distinct subsets. Among naïve aß T lymphocytes (CD45RA+CCR7+CD27+) we found a 60% decrease in the CD4+ subset but no change in the CD8+ subset in adults compared to children. Expression of CD31, an aß T lymphocyte marker of recent thymic emigration, decreased 50% with age. T regulatory cells (CD4+CD127loCD25+FoxP3+) were 30% lower in adults. No differences were found in major γδ T lymphocyte, NK, or NK T cell subsets. Memory B cells (CD38loCD24+) increased 2-fold with age, while naïve B cells (CD24hiCD38+IgM+IgD-) decreased by 50%. Thus, the influence of aging greatly effects subset frequencies, and may reflect decreasing thymus function and acute/chronic stimulation of memory B and T lymphocytes. Phenotypic analysis of leukocyte subsets may serve as a valuable tool for detecting and monitoring immune dysregulation in children, but age-matched controls are essential.

Supported by the McMillen Foundation
Functional competence of T cells and resistance to thymic involution in long-lived mice deficient in pregnancy-associated plasma protein A (PAPPA) (86.2)
Authors: Vallejo Abbe N , Michel Joshua J , Bale Laurie K , Lemster Bonnie H , Borghesi Lisa A , Conover Cheryl A
Published: January 2023

Thymus organogenesis is dependent on insulin-like growth factor (IGF), but involuted thymi of old mice cannot be reconstituted with infusion of IGF. In adult humans, tumor growth is associated with local upregulation of IGF, but increased incidence of cancer with aging is associated with decreasing levels of serum IGF. We hypothesized that suppression of tissue availability of IGF prevents/delays thymic atrophy and maintains a pool of functionally competent T cells throughout life. Hence, we examined mice with homozygous deletion of PAPPA, a gene encoding for a protease that degrades inhibitory IGF-binding proteins (IGFBP) that limit IGF levels in tissues. PAPPA-/- mice have normal serum IGF levels, indicating maintenance of the endocrine IGF reservoir. They have 40% extension of lifespan; and have reduced burden of spontaneous tumors, the major cause of early mortality of wildtype mice. Strikingly, PAPPA-/- mice aged =18 months have histologically organized thymi densely populated by CD4+CD8+ thymocytes with high TREC levels. Bone marrows of old mutants are enriched with thymus-seeding progenitors. Old mutant mice have a diverse repertoire of T cells that are functionally indistinguishable from young wildtype mice. These data indicate an IGF-immune axis of longevity. We suggest that PAPPA-mediated proteolysis is a potential target for intervention to maintain thymic function and promote healthy aging.
Human thymic epithelial cell (TEC) culture system for promoting T lineage commitment and development of T cells from cord blood and bone marrow stem cells (86.6)
Authors: Le Phong T , Zhang Shubin , Beaudette-Zlatanova Britte , Stiff Patrick J , Knight Katherine L
Published: January 2023

The discovery of Delta-like-1 (Dl1)/Notch signaling as the earliest requirement for T lineage commitment and development makes it possible to generate T cells from hematopoietic stem cells in vitro. The mouse bone marrow stroma OP9 cells that overexpress Dl1is the first stroma cell line that is capable of promoting the development of both mouse and human T cells; however, a similar approach with human stroma cells has not been established. To develop a similar culture system to closely emulate human T cell development, we have generated a human TEC line from a pediatric thymus and expressed the mouse Dl1 in this line (TEC84-Dl1). The TEC84-Dl1 expresses CD104, CD29, CD49f, CD54 (ICAM-1), and CD106 (VCAM-1); these cells also express HLA-ABC and HLA-DR. When TEC84-Dl1 were co-cultured with cord blood CD34posCD45RAposCD38neg cells, we were able to generate preT/NK (CD34posCD7posCD1aneg), preT (CD34posCD7posCD1apos), immature single positive and double positive subsets; the co-culture system also generated a CD3pos subset. Similar results were also obtained with CD34pos cells isolated from adult bone marrow. Thus, our co-culture system with the human TEC provides a complete human system for replicating thymopoiesis in vitro and offers a novel tool to optimize the development of T lineage cells that would benefit cord blood and bone marrow transplant recipients (Supports: AG023809 to PL, IDPH to PS, AI068390 to KK).
Potential role for serum soluble CD200 in human Chronic Lymphocytic Leukemia (88.15)
Authors: Wong Karrie K , Shaha Suchinta , Spaner David , Gorczynski Reg
Published: January 2023

CD200, a transmembrane protein expressed on multiple cell types, regulates anti-tumor immunity in animal models. Its overexpression is associated with a poor prognosis in human AML, MM, and melanoma. Blockade of CD200 expression on CD200-expressing tumor cells using CD200-specific monoclonal antibodies or siRNA enhanced anti-tumor immunity against tumor cells both in vitro and in vivo. A soluble variant of CD200 (sCD200) is detectable in human serum, and patients with Chronic lymphocytic leukemia show elevated serum levels.

We investigated the correlation between serum sCD200 levels and clinical disease in CLL patients. sCD200 levels are associated with Rai disease stage and lymphocyte-doubling time. Shedding of cell surface CD200 from CLL cells in vitro can be induced by PMA and TLR7 agonists, with patient-to-patient variability. Shedding of CD200 is correlated with patient serum sCD200 levels, suggesting that inherent differences of CLL cells in responses to shedding stimuli may have clinical significance.

sCD200 levels in serum may provide prognostic and/or diagnostic information in CLL, and may contribute to the efficacy of treatments targeting cell-surface CD200.
Identification of IL-17-producing FOXP3+ regulatory T cells in humans (89.1)
Authors: voo kui S , Wang Yui-Hsi , Santori Fabio R , Boggiano Cesar , Wang Yi-Hong , Arima Kazuhik , Bover Laura , Hanabuchi Shino , Khalili Jahan , Marinova Kkaterina , Zheng Biao Q , Littman Dan R , Liu Yong-Jun
Published: January 2023

IL-17-producing CD4+ T cells (Th17) have recently been defined as a unique subset of pro-inflammatory helper cells whose development depends on signaling initiated by IL-6 and TGF-β, autocrine activity of IL-21, activation of STAT3, and induction of the orphan nuclear receptor RORγt. The maintenance, expansion and further differentiation of the committed Th17 cells depends on IL-1β and IL-23. IL-17 was originally found produced by human CD45RO+ memory T cells. We report that human peripheral blood and lymphoid tissue contain a significant number of CD4+FOXP3+ T cells that express CCR6 and produce IL-17 upon activation. These cells co-express FOXP3 and RORγt transcription factors and strongly inhibit the proliferation of CD4+ responder T cells. CD4+CD25high-derived T cell clones express FOXP3, RORγt and IL-17, and maintain their suppressive function. We further show that human CD4+FOXP3+CCR6- Treg cells differentiate into IL-17 producer cells upon TCR stimulation in the presence of IL-1β, IL-2, IL-21, IL-23 and human serum. This, together with the finding that human thymus does not contain IL-17-producing Treg cells, suggests that the IL-17+FOXP3+ Treg cells are generated in the periphery. IL-17-producing Treg cells may play critical roles in anti-microbial defense while controlling autoimmunity and inflammation.

This work was supported by KECK Foundation 01, PP-4 and National Institute of Health Grant AI091130 (to Y.-J.L.)
Antigen-specific suppression of non-lymphoid tissue auto-antibody production by CD25+ FoxP3+ regulatory T cells (89.22)
Authors: Kurts Christian , Ludwig-Portugall Isis , Hamilton-Williams Emma E , Gottschalk Catherine , Gotot Janine
Published: January 2023

To study how peripheral B cell tolerance against non-lymphoid tissue autoantigens is maintained, we generated transgenic RIP-OVA/HEL (ROH) mice expressing the model antigens, OVA and HEL, in pancreatic islet beta cells. Immunization with OVA/HEL/aluminiumhydroxide induced IgG auto-Ab titers that were much lower than in non-transgenic controls. Depletion of CD25+ cells during immunization completely restored auto-Ab production but did not affect titers against foreign antigens, indicating regulatory tolerance. Purified CD25+ FoxP3+ CD4+ T cells from ROH mice transferred B cell suppression into non-transgenic recipients. CD25+ cells also suppressed naïve transgenic HEL-specific B cells adoptively transferred into ROH mice, confirming peripheral B cell tolerance. B cell suppression mechanistically involved inhibiting the proliferation of autoreactive B cells, inducing their apoptosis after immunization with autoantigen, suppressing antibody secretion per B cell and downregulating IgMa and MHC II B cell surface expression in the autoantigen-draining lymph node. We conclude that CD25+ FoxP3+ regulatory T cells were necessary and sufficient to specifically suppress auto-Ab production against pancreatic islet cell antigens.
Suppression of the Immune Responses to Factor VIII in the Hemophilia A Mice by Delivery of Transgene Modified Apoptotic Syngeneic Fibroblasts (89.36)
Authors: Su Rui-Jun , Epp Angela , Latchman Yvette , Josephson Neil
Published: January 2023

The development of anti-FVIII inhibitory antibodies is a major obstacle to treatment of hemophilia A patients. Here, we employ the immunosuppressive properties of FVIII gene modified apoptotic syngeneic fibroblasts to reduce immune response to FVIII in hemophilic mice. We generated a fibroblast cell line from the 129Sv-FVIII KO mouse that expresses both FVIII and zeocin resistance gene. A cell line expressing only the zeocin resistance gene was also generated. Both vector modified cell lines were maintained under selective pressure and induced to apoptose just prior to infusion into 129Sv-FVIII KO mice. Mice treated with 2 intravenous infusions of FVIII expressing apoptotic fibroblasts showed up to a 79.5% reduction in Bethesda titers and significantly lower T cell responses after subsequent challenge with 4 doses of rhFVIII. Moreover, hemophilic mice pre-immunized with rhFVIII showed a 65% reduction in Bethesda titers following treatment with 4 infusions of FVIII expressing apoptotic cells. To investigate the antigen specificity of the induced immunosuppression, apoptotic fibroblast treated mice were challenged with an unrelated protein OVA. The immune response to OVA was not affected by the infusion of apoptotic cells. In adoptive transfer studies, a blunted immune response to rhFVIII could be induced in naïve mice by infusion of CD4+ splenocytes from FVIII expressing apoptotic cell treated animals. Moreover, in vitro Treg suppression assays showed that CD4+CD25+ regulatory T cells from FVIII expressing apoptotic cell treated mice were capable of inhibiting the T cell response to rhFVIII, but not to OVA. These data demonstrate that the CD4+CD25+ regulatory T cells in these mice direct antigen specific immune suppression to human FVIII.
Costimulation through ICAM-1 induces the differentiation of human naïve CD4+ T cells to regulatory T cells: acquisition of distinct signaling and homing profiles during differentiation (89.39)
Authors: Cool Kelli M. , Dotson Abby L. , Benedict Stephen H.
Published: January 2023

Inducible regulatory T (Treg) cells are important mediators of peripheral tolerance that arise by mechanisms not fully defined. We have previously shown that a subset of human naïve CD4+ T cells that are costimulated through Intercellular Adhesion Molecule-1 (ICAM-1) will differentiate into cells with a Foxp3+CD25+CD127(-) phenotype and suppressive function characteristic of inducible Treg cells. Our present work identifies some of the signaling pathways that are activated during this differentiation process. We also characterize some of the chemokine receptors and other homing markers that are displayed on these Treg cells, including CCR7, CD62L, and specific integrins. These results help clarify the signaling requirements for in vitro differentiation of human naïve CD4+ T cells to Treg cells after costimulation through ICAM-1 and suggest possible microenvironments in which these cells could migrate and function during immune responses in vivo.

This work was supported by the National Institute on Aging/NIH.
Bone marrow-derived dendritic cells generated in the presence of resolvin E1 induce apoptosis of activated CD4+T cells (90.1)
Authors: Vassiliou Evros K , Tadros James H , Kessler Olga M , Ganea Doina
Published: January 2023

In contrast to the role of dendritic cells (DC) in immunity and tolerance, little is known about their possible role in the resolution of inflammatory processes. Elimination of effector T cells present at the inflammatory site represents an essential step toward resolution. Recently, lipid mediators such as the omega-3 fatty acids and their metabolites, including resolvin E1 (RvE1), have been shown to accumulate in inflammatory foci during the resolution phase. RvE1 has been reported to reduce immune cell infiltration and proinflammatory cytokine production. In this study we report that DC exposed to RvE1, especially during differentiation, induce apoptosis of activated T cells through expression of indoleamine 2,3-dioxygenase (IDO). To our knowledge, this study is the first to report on an omega-3 fatty acid derivative inducing IDO expression in DC. RvE1-DC maintain an immature chemokine receptor expression pattern even following TLR stimulation, with high CCR5 and no CCR7 expression. This effect implies that DC exposed to RvE1 and pathogens remain at the inflammatory site where they induce apoptosis in infiltrating effector T cells. To our knowledge, the DC described in this study represent a new functional DC subtype, whose essential function resides in the resolution of inflammation.

Support: 2R01AI052306 (D.G.) and a grant from the Office of Research and Sponsored Programs of Kean University (E.K.V.).