bluuebunny/crossref_metadata_2025_split huggingface dataset:
In situ visualization of CD8 T cell-DC interactions reveals distinct features of the early and late response to antigen after influenza virus infection (87.27)
Authors: Cauley Linda S , Khanna Kamal M , Lefrancois Leo 
Published: January 2023

Abstract
The anatomical characteristics of T cell activation following infection are poorly described. Using transferred CD8 T cells and confocal microscopy we show that small clusters of virus-specific T cells formed transiently in the lung-draining lymph nodes (DLN) early in the response to influenza virus infection. Several weeks after infection naïve nucleoprotein (NP)-specific CD8 T cells still responded to antigen in the DLN, but remained predominantly in large stable clusters that included CD11c+ APCs. Many activated cells upregulated CD69 and PD-1, but did not lose CD62L-expression and remained clustered with CD11c+ APCs in the paracortex of the DLN until at least 7 days after transfer. Some central/memory cells also entered the DLN after transfer, but did not colocalize with the clustered naïve cells or show any signs of a response to the antigen. These data indicated a change in the response to antigen stimulation in the DLN as the time interval after infection increased and suggested that naïve and memory F5 CD8 T cells used different mechanisms to locate antigen-bearing APCs within the pLN. Some endogenous NP366-374/Db-specific CD8 T cells were also retained in the MLN and showed signs of a continuing response to antigen stimulation, while PA324-332/Db-specific cells were predominantly found in non-lymphoid tissues. Overall all the data revealed a dichotomy in the response to late antigen presentation which could influence epitope selection during reinfection by favoring preferential expansion of the NP366-374/Db-specific cells in the DLN.
This work was supported by the American Lung Association RG1119 (L.S.C.), the Damon Runyon foundation (K.M.K.) and National Institutes of Health grants; R21-AI065895 (L.S.C.) and DK45260, AI41576 and AI56172 (LL).
Negative Regulation of NIP45 Function by Peptidylarginine Deminase 4 (87.28)
Authors: Mowen Kerri A , Hemmers Saskia , Schurter Brandon , Fathman John W. , Glimcher Laurie H. , David Michael 
Published: January 2023

Abstract
IL-4 production in Th2 lymphocytes is regulated by the nuclear factor of activated T-cells (NFAT) and NFAT-interacting protein 45kDa (NIP45). Recently, we showed that arginine methylation of NIP45 modulates its interaction with NFAT and augments IL-4 transcription. Here we report that NIP45 methylation is negatively regulated by peptidylarginine-deiminase 4 (PAD4) via deimination of arginine residues and generation of the atypical amino acid citrulline. NIP45 loses its ability to become methylated after deimination by PAD4. Ectopic expression of PAD4 impairs the association of NIP45 with NFAT and profoundly inhibits IL-4 promoter activity. Whereas, knockdown of PAD4 expression in Th2 cells by a PAD4-directed shRNA-expressing retrovirus leads to elevated IL-4 production. Interestingly, a PAD4 variant is association with rheumatoid arthritis (RA) in a Japanese population. Indeed, many autoantibodies in RA are directed against citrullinated proteins. It has been proposed that PAD4 is linked to RA because citrullination of peptides leads to a breakdown of self-tolerance to self-antigens. Our data suggest that PAD4 may also influence RA development through regulation of the cytokine milieu. Modification of NIP45 by PAD4 provides a novel negative regulatory mechanism whereby NFAT and NIP45 control IL-4 and potentially other cytokines production.
Negative regulation of T cell receptor signaling by NFkappaB2/p100 (87.4)
Authors: Legarda-Addison Diana , Ting Adrian T. 
Published: January 2023

Abstract
The positive regulation of the NFκB signaling pathway in response to T cell receptor (TCR) stimulation has been well studied. However, little is known about the negative regulation of this pathway in T cells. This negative regulation is crucial in controlling the duration of TCR signaling and preventing abnormal lymphocyte activation and proliferation. Therefore, understanding the negative regulation of TCR-mediated NFκB signaling is essential in understanding the mechanisms involved in T cell function and homeostasis. TCR stimulation of human CD4+ T cells resulted in an increase in NFκB2/p100 expression with minimal increase in p52, its cleavage product. Due to the presence of inhibitory ankyrin-repeats in the unprocessed p100, this observation suggests that p100 may function as a negative regulator of the NFκB pathway. Consistent with this hypothesis, ectopic expression of p100 inhibited TCR-mediated NFκB activity and IL-2 production in Jurkat T cells. Conversely, knockdown of p100 expression enhanced NFκB transcriptional activity and IL-2 production upon TCR activation. p100 inhibited the pathway by binding and sequestering Rel transcription factors in the cytoplasm without affecting the activity of the upstream IκB kinase (IKK). Taken together, this study indicates that NFκB2/p100 can act as an inhibitory molecule in the TCR-mediated NFκB signaling pathway.
This work was supported by a grant from the National Institutes of Health to A.T.T.
A biochemical signature for rapid recall of memory CD4 T cells (87.41)
Authors: Chandok Meena R , Okoye Francesca I , Ndejembi Modesta P , Farber Donna L 
Published: January 2023

Abstract
Mechanisms for the rapid effector responses mediated by memory T cells remain unknown. In this study, we used multiparameter analysis of intracellular signaling and function in naive and memory T cells in vitro and in vivo to determine a biochemical basis for immunological recall. We identify here a striking and specific elevation in expression of the ZAP-70 protein tyrosine kinase in resting memory compared to naive CD4 T cells that is stably maintained independent of protein synthesis, whereas low ZAP-70 expression in naive cells increases with sustained antigenic stimulation requiring new protein synthesis. We demonstrate that ZAP-70 levels control effector function, as specific downregulation of ZAP-70 expression in memory CD4 T cells abrogates rapid effector function, and acquisition of effector function occurs exclusively from activated T cells that have upregulated ZAP-70. Moreover, rapid IFN-γ production from ZAP-70hi memory CD4 T cells occurs via a quantitative bypass in distal signaling in vitro and in vivo, contrasting the increased distal phosphorylation events that precede late IFN-γ production from activated naive CD4 T cells. Our findings reveal a novel biochemical signature imparted to memory CD4 T cells enabling efficient functional coupling through increased ZAP-70 expression and reduced downstream signaling events.
This work was supported by NIH AI42092
In vivo and in vitro zinc (Zn) deficiency diminished T lymphocyte activity through the modulation of PKC isoenzymes. (87.46)
Authors: Cremaschi Graciela Alicia , Klecha Alicia Juana , Orqueda Andres , Kaliski María Alejandra , Arcos María Laura Barreiro , Zubillaga Marcela , Wald Miriam , Genaro Ana María 
Published: January 2023

Abstract
Zn is essential to numerous signaling proteins that share Zn-finger structures as a common motif. It is crucial for immunocompetent cells, as its deficiency leads to immune function alterations. The exact mechanisms involved in Zn regulation of lymphocyte activity are still unclear. Here we analyzed both the direct and the in vivo effect of Zn deficiency on normal T cells and the involvement of protein kinase C (PKC) (an enzyme with cysteine-rich domains) and its isoenzyme profile. For this purpose mitogen stimulated normal T cells were cultured in the absence or presence of specific intra- or extracellular Zn chelators. These effects were compared with those obtained in mitogen-stimulated lymphocytes purified from mice fed with Zn deficient diets. Proliferation, PKC activity and the pattern of PKC isoenzymes were evaluated. Zn chelators inhibited normal cell proliferation and their effects were reverted by in vitro Zn addition. Similar effects were observed in lymphocytes from Zn deficient mice. In both experimental conditions, a decrease in PKC activity and in mitogen-induced translocation of the enzyme to cell membranes were observed. These effects were accompanied by a decrease in PKC α and 𝛉 isoforms.
These results show that Zn plays an important role in T cell growth and that its deficiency would alter the expression of key PKC isoenzymes that are essential signalling molecules involved in lymphocyte activation.
HSF1 is necessary for reactive oxygen species (ROS) homeostasis and proliferation in T cells at fever temperatures. (87.47)
Authors: Woodward Jerold G. , Murapa Patience , Gandhapudi Siva , Skaggs Hollie , Sarge Kevin 
Published: January 2023

Abstract
HSF1 is a major heat and stress inducible transcription factor in eukaryotic cells responsible for the regulation of a variety of genes involved in maintenance of cellular homeostasis. We have previously shown that HSF1 is activated at physiologic fever temperatures (39oC) in T cells vs. much higher, non-physiologic temperatures (42oC) in most other cell types. T cells from HSF1−/− mice proliferate normally at 37oC, but are severely inhibited at 39oC. These T cells are blocked at the G1-S phase transition of the cell cycle. T cells normally increase ROS levels as a result of activation. Antioxidants which block ROS increases also inhibit proliferation. We found that fever temperatures also inhibited the normal activation induced increase of ROS in T cells within 2h. Wild type T cells can overcome this inhibition in ROS generation within 5h and go on to proliferate normally. In contrast, HSF1−/− T cells were unable to overcome this dysregulation and failed to proliferate. Thus, fever temperature causes a dysregulation in ROS homeostasis in T cells and HSF1 is critical in restoring this regulation.
Prolonged FRC-DC interaction negatively affects DC-T cell crosstalk (88.10)
Authors: Kronenberger Konrad , Liwski Robert S. , Trenholm Tara , West Kenneth A. 
Published: January 2023

Abstract
While Fibroblastic Reticulum Cells (FRC) of the lymph node (LN) are known to produce chemokines responsible for the migration of naïve T cells and DC, little is known about their interactions with these cells and their influence on DC and T cell activation. Therefore, we have evaluated the interaction between DC, FRC and T cells in an in vitro model.
For the generation of FRC cells lymph nodes of BALB/c mice were isolated and cultured ex vivo. After co culture with FRC DCs were characterized by FACS analysis concerning their survival rate, maturation state and expression of cell surface molecules as well as for their production of soluble cytokines. The ability of DC to induce T cell proliferation was evaluated using the D011.10 TCR transgenic system and CFSE labeling.
Our in vitro culture method leads to a highly homogeneous FRC population regarding expression of gp38, ER-TR 7, CD44 and V-CAM. Confocal microscopy analysis demonstrated that DC migrated to FRC where they made physical contact leading to stable associations in vitro as well as in vivo in the LN after DC were injected s.c..
Co incubation of DC with FRCs resulted in a complete down regulation of CD40 and partial decrease of co stimulatory molecules. Furthermore there was a strongly diminished ability of these DC to induce a peptide specific proliferation of naïve T cells although a clear CD25 up regulation could be detected. It remains to be investigated whether these CD25 positive T cells show a regulatory phenotype.
Our results demonstrate that LN-FRC provide a milieu that may induce tolerogenic DCs which then drive naïve T cells to a rather regulatory phenotype.
In the tumor microenvironment of head and neck cancer patients (HNSCC), suppression is mediated by a unique subset of CD4+CD25highFoxp3+ T cells secreting IL-10 and TGF-beta1 (88.47)
Authors: Strauss Laura , Bergmann Christoph , Johnson Jonas T. , Whiteside Theresa L. 
Published: January 2023

Abstract
Objective: CD4+CD25highFoxp3+T cells (nTreg) are a characteristic feature of HNSCC. Phenotype and function of nTreg in tumor-infiltrating (TIL) vs. peripheral blood mononuclear (PBMC) T cells remain controversial.
Methods: Flow cytometry was used to study Treg in TIL of 15 HNSCC patients, PBMC of 25 HNSCC patients and PBMC of 15 normal controls (NC). CD4+CD25high T cells were sorted from TIL or PBMC by FACS and tested for function by co-culture with CFSE-labeled CD4+CD25− T responder cells (R). Transwell inserts and neutralizing IL-10 or TGFβ1 Ab were used to evaluate the potential suppressor mechanisms of Treg.
Results: Treg were enriched in TIL (13±3%) relative to PBMC (5±3%) (p ≤ 0.01). The mean % of Treg in NC was 2±1.5% (p ≤ 0.001). Treg in TIL were positive for GITR, IL-10 and TGFβ1, while Treg in PBMC up-regulated CD62L and CCR7 but not GITR, IL-10 or TGFβ1. Treg in TIL mediated stronger suppression (85±7%) of R than Treg in PBMC (20±5%) (p ≤ 0.001). Suppression mediated by Treg in TIL was decreased by transwell inserts (20±3% vs. 80±5%) and anti-IL-10 or TGFβ1 Abs (10±3%). In co-cultures, where the number of R exceeded the number of Treg, suppression was not blocked by the use of transwell inserts (58±1.2% vs. 68±5.13%), although the addition of anti- IL-10 or TGFβ1 Abs abrogated suppression (5±2.51%).
Conclusions: nTreg in the tumor suppress R by IL-10 and TGFβ1. Cell-to-cell contact is not needed, but optimizes suppression.
Regulation by SHPS-1 of α-GalCer-induced prevention of cancer metastasis (89.14)
Authors: Okajo Jun , Kaneko Yoriaki , Tomizawa Takeshi , Okuzawa Chie , Saito Yasuyuki , Sekigami Tomomi , Matozaki Takashi , Nojima Yoshihisa 
Published: January 2023

Abstract
[Background] α-GalCer activates NKT cells through the interaction between NKT cells and DCs. SHPS-1 is a transmembrane protein predominantly expressed on DCs. The functional roles of SHPS-1 in the regulation of NKT are investigated by the use of mice bearing mutant SHPS-1, which lack most of its cytoplasmic region.
[Methods] B16 melanoma cells were injected into WT or SHPS-1 mutant mice, and α-GalCer or the vehicle was administered. For adoptive transfer of DCs, α-GalCer pulsed DCs were prepared from spleens from WT or SHPS-1 mutant mice, and such cells were injected together with B16 melanoma cells. The mice were sacrificed and the number of tumor colonies in the lung was counted. Splenic or hepatic MNCs were cultured with α-GalCer, and the amounts of IFN-γ and IL-4 in culture supernatants were determined by ELISA.
[Results] The prevention of metastasis by α-GalCer was attenuated in the SHPS-1 mutant mice. The antimetastatic effect was induced in WT recipient mice by adoptive transfer of DCs from WT mice, while such effect by that of DCs from SHPS-1 mutant mice was reduced. The production of either INF-γ or IL-4 in response to α-GalCer was reduced in MNCs from SHPS-1 mutant mice.
[Conclusion] SHPS-1 on DCs is crucial for the α-GalCer-induced prevention of metastasis and Th1-and Th2-responses by NKT cells.
Dendritic Cells from Male and Female Rodents Respond Differentially to Progesterone (90.3)
Authors: Butts Cherie L , Belyavskaya Elena , Sternberg Esther M 
Published: January 2023

Abstract
Females show a 2- to 10-fold higher incidence of autoimmune diseases compared to males, indicating a role for sex hormones. In this study, we determined the role of progesterone (P) in modulating male and female rodent dendritic cell (DC) responses. We found differences both in male and female DC responses to P and differences in DC responses throughout the female estrus cycle. Male LPS-matured bone marrow-derived DCs (BMDCs) secreted higher levels of TNF-? and IL-10 compared to females; however P suppressed BMDC TNF-? and IL-12 secretion in females more than males. Reversal by the PR antagonist RU486 indicated mediation through PR. Females also showed greater progesterone downregulation of BMDC CD80 and MHC Class II expression than males. Female BMDCs differed in P responsiveness depending on the estrus cycle phase, with similar responses to males in proestrus and estrus but greater sensitivity to P during metaestrus and diestrus. This effect is consistent with increased levels of PR in BMDCs during these phases. Thus, depending on estrus cycle phase and gender, P differentially affects mature BMDC function. This could indicate that differences in susceptibility to infection and disease between males and females and at specific estrus cycle phases in females could be related to differential BMDC function.
This work was supported by the NIMH and NIAID Intramural Research Programs.
Regionalized combinatorial diversity provides an evolutionary link between innate and adaptive immunity (90.7)
Authors: Ostrov David A. , Hernández Prada Jose A. , Haire Robert , Cannon John , Litman Gary W. 
Published: January 2023

Abstract
Adaptive immunity emerged in jawed vertebrates exhibiting the ability to express somatically rearranged antigen receptor genes with combinatorial binding sites consisting of residues from each of the paired v-type (variable) Ig domains (i.e., VαVβ). The origins of contemporary antigen receptors have been described by a model in which an ancestral Ig gene diversified to form polymorphic variants consisting of an antigen binding domain linked to an effector module (i.e., VC). The high degree of clustering of polymorphic residues at the combining site between v-type Ig domains throughout evolution of adaptive immunity suggests that this feature confers a powerful advantage in creating a diverse repertoire of antigen receptors. To address the origins of adaptive immunity, we solved the crystal structure of VCBP3 D1 and D2 from the cephalochordate Branchiostoma floridae, which has characteristics of a primordial antigen receptor: a high degree of germline encoded polymorphism and paired v-type Ig domains with polymorphism clustered at the combining site. In addition to finding combinatorial recognition in a jawless invertebrate, we solved the structure of a primordial V domain to 1.15 Å, a level of refinement that permits new insight into the atomic basis for Ig domain organization and stability.
Dendritic cells pulsed with either secretory antigens of Mycobacterium tuberculosis or live mycobacteria show a differential expansion of Th1, Th2 and Th3 type T cells in immune mice (92.10)
Authors: Jagannath Chinnaswamy , Roche Cherie Michelle , Arora Ashish 
Published: January 2023

Abstract
Dendritic cells (DCs) can prime naïve or immune T cells and expand Th1, Th2 and Th3 type of T cells that determine protection against pathogens. While the Th1 immunity has been thought to be protective against tuberculosis, the ability of vaccines to induce Th1-Th3 immunity is unclear. Emerging tuberculosis vaccines include DNA vaccines encoding 85 complex (A, B, C), ESAT-6 and CFP-10 (ES antigens) or live attenuated Mycobacterium tuberculosis (MTB) in comparison with BCG vaccine. Interestingly, MTB H37Rv secretes all three ES antigens while BCG secretes Ag85 but lacks ESAT-6 and CFP-10. We therefore hypothesized that ES antigens and live bacteria may induce different types of T cell responses and the efficacy of vaccines delivering these would vary in mice.
METHODS: Bone marrow derived C57Bl/6 DCs were infected with live H37Rv or BCG or were pulsed with Ag85(ABC), ESAT-6 or CFP-10 antigens. After 24 h, they were cocultured with naïve T cells or immune T cells from heat killed MTB immunized mice. T cells were then stained for intracellular T-bet/IFN-&#947;, GATA3/IL-4 and Foxp3/IL-10 to determine the expansion of Th1, Th2 and Th3 response respectively.
RESULTS: ES antigens induced a strong Th1 response with very little Th2 or Th3 responses in immune T cells. Paradoxically, H37Rv and BCG induced a Th1 response as well as significant levels of Th2 and Th3 type of T cells.
CONCLUSIONS: We suggest that live mycobacterial vaccines concurrently induce deleterious Th2 and Th3 T cells during vaccination or infection that may affect the protective function of Th1 T cells.
Motif structure of minor histocompatibility antigen for MHC class II-restricted presentation (93.19)
Authors: Sahara Hiroeki , Imai Akihito , Mori Yoko , Mita Hiroaki , Tamura Yasuaki , Takahashi Nobuaki , Shastri Nilabh , Sato Noriyuki 
Published: January 2023

Abstract
Unlike major histocompatibility complex (MHC) class I, the processing pathway of endogenous MHC class II-restricted antigen has been poorly understood. We previously reported that endogenous MHC class II-restricted minor histocompatibility antigen that encoded murine H46 locus on chromosome 7 was IL-4-inducible gene 1 (IL4i1), which was continually expressed in bone marrow dendritic cells (BMDCs) (J.Exp.Med. 2003, 197: 375). In this study, we investigated the processing pathway of endogenous MHC class II-restricted IL4i1 antigen in BMDCs. IL4i1 antigen localized at RhoB-localized endosome, and MHC class II-restricted presentations were inhibited by endosomal vesicle trafficking inhibitor, such as bafilomycin. Whereas, lysosomal enzyme inhibitors, such as chloroquine, could not influence the presentations, suggesting that seem to be different from autophagy processing pathway. Next, we focused on the IL4i1 gene structure that does arrangement of tri-leucine-based motif in the downstream of tyrosine-based motif, designated YL motif. When YL motif was mutated, the antigen presentations were dramatically inhibited and this mutated-IL4i1 did not colocalize with RhoB-localized vesicle. These data suggested that YL motif would be a sorting signal for it where might occur antigen processing.
In the absence of IGF-1 signaling, IFN-gamma induces T cell apoptosis (94.14)
Authors: Novelli Francesco , Conti Laura , Regis Gabriella , Bernabei Paola , Chiarle Roberto , Giovarelli Mirella 
Published: January 2023

Abstract
IFNg exerts its biological activities by interacting with its cell surface receptor (IFNgR), which consists of two binding chains (R1) and two signal transducing chains (R2).The binding of IFNg to its receptor complex activates the JAK/STAT signal transduction pathway and induces the transcription of IFNg-inducible apoptotic genes. T cells that undergo Th1 polarization or malignant transformation become resistant to the antiproliferative effects of the IFNg/STAT1 pathway.This refractoriness is mainly due to R2 downregulation, due to ligand-independent internalization within clathrin-coated pits. We have previously demonstrated that, by inducing R2 internalization, IGF1 is a critical factors in limiting the IFNg/STAT1 pathway in T cells. Thus, the blockade of its signaling in T cells could hinder their intracellular R2 trafficking and reinstate their sensitivity to IFNg/STAT1 apoptotic signaling. To address this point, a retrovirus-based approach was exploited to stably express a dominant negative form of the IGF1R (IGF1R DN) in ST4 T cell line. Expression of IGF1R DN induced R2 cell surface accumulation and reinstated ST4 cell sensitivity to the IFNg/STAT1 apoptotic pathway in vitro and in vivo. Moreover, the combined exposure to an inhibitor of IGF1R tyrosine kinase activity (picropodophyllin, PPP) and IFNg induced STAT1-dependent apoptosis of three T cell lines (Jurkat ST4 and PF382).These results could be used in the elaboration of new therapeutic approaches based on IGF1 signaling blockade to overcome the resistance of malignant or autoreactive T cells to the apoptotic effect of IFNg.