bluuebunny/crossref_metadata_2025_split huggingface dataset:
PLZF is essential for iNKT cell development (82.5)
Authors: Uche Olisambu U. , Sant’Angelo Derek B. 
Published: January 2023

Abstract
The promyelocytic leukaemia zinc finger (PLZF) protein is a member of the BTB/POZ zinc finger family of transcription factors. This family of transcription factors also include Th-Pok, BCL-6 and MAZR which are known to play important roles in the immune system. Here we demonstrate that PLZF deficient mice have a severe impairment in iNKT cell lineage development leading to a lack of iNKT cells in the thymus and periphery. Real-time PCR showed that PLZF mRNA is highly expressed in iNKT cells. This correlates with flow cytometry analysis which revealed exclusive expression of PLZF protein in iNKT cells within the immune system. Radiation chimeras showed that PLZF is required for iNKT cell development in a cell intrinsic manner. These results identify PLZF as a key factor in iNKT development. They also underline the BTB/POZ family of transcription factors as important factors in the immune system.
Natural killer dendritic cells are an intermediate of developing dendritic cells (82.6)
Authors: Gao Jian-Xin , Chen Li , Calomeni Edward , Wen Jing , Shen Rulong 
Published: January 2023

Abstract
Natural killer dendritic cells (NKDCs) appear to emerge as a distinct dendritic cell (DC) subset in human and rodents, which have the functions of both natural killer (NK) cells and DCs. However, the developmental relationship of NKDCs (CD11c+K1.1+) to CD11c+NK1.1− DCs has not been addressed. Herein, we show that NKDCs exclusively exist in the compartment of CD11c+MHC II− cells in the steady state, and express variable levels of DC subset markers, such as Interferon-producing killer dendritic cells (IKDC) marker B220, in a tissue-dependent manner. They can differentiate into NK1.1− DCs accompanied by the up-regulation of major histocompatibility complex (MHC) class II molecules and down-regulation of NK1.1 upon adoptive transfer. However, NK cells (NK1.1+CD11c−) did not differentiate into NK1.1+CD11c+ cells upon adoptive transfer. Bone marrow (BM)-derived Ly6C+ monocytes can be a potential progenitor of NKDCs, as some of them can differentiate into CD11c+NK1.1+ as well as CD11c+NK1.1− cells in vivo. The steady NKDCs have great capacity for lysis of tumor cells, but little capability to present antigens. Our studies suggest that NKDCs are an intermediate of developing DCs with the unique surface phenotype of CD11c+NK1.1+MHC-II−, strong cytotoxic function, and poor capability of antigen presentation in the steady state, and they probably play a critical role in linking innate and adaptive immunity.
Defects in early DN cell development and CD4 SP cell selection in Foxn1Lacz mice (82.7)
Authors: Xiao Shiyun , Chen Lizhen , Manley Nancy R 
Published: January 2023

Abstract
The Foxn1 gene is required for thymic epithelial cell (TEC) development and is expressed in the thymus from the fetal to adult stages. To track Foxn1 gene expression, we inserted an IRES-lacZ gene into the 3′UTR in exon 9 of the Foxn1 gene. We found that the postnatal thymus was progressively atrophic in Foxn1Lacz/LacZ mice and displayed a gradual disorganization. The total thymocyte cell number was reduced 10%, 56% and 80% at postnatal day 8, 14, and 28. The percent of CD4 SP cells in Foxn1Lacz/LacZ mice was significant reduced, but not CD8 SP cells compared to control Foxn1+/Lacz mice. The percent of DP cell was slightly increased, and the percent of DN cell was significant increased 2 to 3 fold in day 21 to 28 mice. The percent of DN1 cells was dramatically increased to more than 60% (around 20% in control mice), and more than 80% of these cells were CD19+ cells. The percent of DN2, DN3 cells were correspondingly reduced to 15% (around 60% in control mice), and the percent of DN4 cells was also reduced in Foxn1Lacz/LacZ mice during the period. Our preliminary data suggests that the expression of MHCII on surface of TSC and Foxn1 mRNA from the Foxn1Lacz allele was reduced; furthermore, the phenotype was more severe in Foxn1Lacz/nu mice, indicating that the defects depended on the dosage of the Foxn1 gene. Taken together, our results suggest that reduction of Foxn1expression in the postnatal thymus caused defects in TEC maintenance, resulting in defects in DN1 cells and in positive selection for CD4 SP cells, which ultimately greatly reduces production of T cells in the thymus. Furthermore, the resulting thymic microenvironment is favorable for B cells development in the postnatal thymus.
Supported by grant AI055001 NIH.
Categorical selection of the Ig CDR-H3 repertoire in splenic B cells (83.14)
Authors: Schroeder Harry W , Tanner Jason M , Zemlin Michael , Schelonka Robert L 
Published: January 2023

Abstract
H chain CDR-H3 serves as the focus of the diveristy of the antibody repertoire. Repertoire development in the bone marrow is associated with a reduction of specific categories of CDR-H3, particularly those with excessive hydrophobic or polar (charged) amino acids and those with a length of eight or fewer amino acids. To test whether CDR-H3 selection continues within the spleen, we sorted B cells into transitional (T1), marginal zone (MZ) and follicular (FO) populations; cloned 364 unique, in-frame, open V7183DJCμ transcripts; and compared them to 509 previously obtained sequences from immature (IM) and mature, recirculating (MA) bone marrow B cells. The T1 CDR-H3 repertoire shared extensive similarity of length and loop amino acid hydrophobicity to that of IM CDR-H3s, whereas FO CDR-H3s most resembled that of MA B cells. MZ CDR-H3s averaged one half-codon shorter than IM and T1 CDR-H3s, and a full codon shorter than FO and MA CDR-H3s (p&lt;0.05 and p&lt;0.005, respectively). 13% (20/151) of MZ CDR-H3s contained 8 or fewer amino acids, versus only 4% (9/254) MB CDR-H3s [p&lt;0.001]. MZ CDR-H3s were also enriched for highly charged CDR-H3s, representing 5.3% (8/151) of the transcripts versu only 0.4% (1/254)among MB CDR-H3s. Unlike the splenic F0 and bone marrow MA CDR-H3 repertoires, the MZ appears to be a reservoir for short and highly charged CDR-H3s amino acid motifs, including those with two or more arginines. The latter in particular has been associated with self-reactive specificities. Our findings suggest that specific categories of CDR-H3 sequence content may inhibit, permit, or even facilitate passage of the host B cell through critical checkpoints in central as well as peripheral development.
Supported by HD043327, AI42732, and AI48115.
Ag-engaged B cells express CD83, a sensitive marker for B cell activation (83.15)
Authors: Prazma Charlene M. , Tedder Thomas F. 
Published: January 2023

Abstract
CD83 is predominantly known as a surface marker that defines mature DCs. The generation of CD83−/− mice further demonstrates that thymic epithelial expression of CD83 plays a significant role in the efficient generation of CD4 single positive thymocytes. The altered phenotype and reduced number of peripheral CD4+ T cells in CD83−/− mice further suggests a role for CD83 in the peripheral lymphocyte function. With regards to the surface expression pattern of CD83, CD83 was detected in the thymic medullary compartment and was specifically detected on thymic DCs and B cells in the mouse. Peripheral CD83 expression is limited to low level expression by resting follicular B cells but is quickly upregulated and transiently expressed on nearly 90% of spleen B220+ cells within 4 h of in vivo stimulation. Additionally, the majority (&gt; 70%) of adoptively transferred B cells, expressing a BCR specific to hen egg lysozyme (HEL), expressed CD83 within 4 h of administration of a low dose of soluble HEL. At this timepoint, and low dose of antigen, less than 20% of B220+ splenocytes expressed CD69. Thus, the restricted expression pattern of CD83 and its near immediate upregulation on B cells in response to various stimuli suggest that CD83 is a sensitive, early activation marker of B cells.
Complement C2 and CRIT are involved in the terminal differentiation of monocytes (83.17)
Authors: Inal Jameel Malhador , Eva Wintergerst , Lange Sigrun , Shamshiev Adijapar , Schifferli Juerg 
Published: January 2023

Abstract
The protein expression of the C2/FB binding complement regulatory receptor CRIT was monitored in myeloid cell differentiation using primary monocytes or pluropotentiated promonocytes. CRIT expression was markedly reduced in HL60 cells differentiated with retinoic acid or DMSO into neutrophils but was maintained on differentiation using vitamin C3 or phorbol esters into monocytes. Macrophages derived from primary monocytes have downregulated CRIT unlike monocyte-derived dendritic cells which retain expression levels. Coimunoprecipitation experiments show that CRIT associates with c-fes, a cytoplasmic tyrosine kinase involved in the terminal differentiation of monocytes and neutrophils, in THP-1 cells (but not Jurkat cells which lack c-fes). Blocking the binding of C2/FB to CRIT on THP-1 cells either by adding excess of CRIT-ed1 peptide or anti-C2 polyclonal antibody significantly limits growth and increases nitroblue tetrazolium reducing activity from 4 to 8 (A650/107 cells). With primary monocytes (but not with monocytes from a C2 deficient patient) ed1 blockage of C2/FB induced terminal differentiation to macrophages as observed by the increase in % CD11b+ cells from 25% to 40%, increase in % CD14+ cells from 32% to 40% and reduction in the relative mean fluorescence index (CD14+) from 108 to 80. Throughout, interferon-γ was used as a positive control for induction of monocyte to macrophage differentiation.
Multiple effects of Wnt3a on lymphoid progenitors and their environment (83.4)
Authors: Malhotra Sachin , Kincade Paul W 
Published: January 2023

Abstract
The Wnt family of secreted glycoproteins has been implicated in many aspects of development, but its contribution to blood cell formation is poorly understood. We over-expressed Wnt3a in stromal cells of osteopetrotic mice and used them in co-culture experiments with highly enriched stem and progenitor cells. The objective was to learn if and how particular stages of B lymphopoiesis are responsive to this canonical Wnt ligand. We found that functional receptors were present on all populations tested, and that Wnt signaling generally inhibited B lineage progression. However, this effect was less in long term cultures. Other results suggest that lineage instability results from Wnt signaling and there was even evidence for loss of differentiation. That is, RAG1+ cells down-regulated expression of this gene and acquired some characteristics of stem cells. At the same time, short term exposure to Wnt3a increased myeloid colony forming potential and retained the ability of progenitors to be transplanted. We also found a surprising effect on expression of cell adhesion molecules thought to retain cells in the bone marrow. VCAM-1 on stromal cells, stem cells or an endothelial cell line was dramatically down-regulated when the cells were exposed to Wnt3a. We conclude that in addition to effects on differentiation, canonical Wnt signaling may control positioning of stem and progenitor cells within the bone marrow.
Regulation of B cell fate determination by EBF (83.7)
Authors: Medina Kay L. 
Published: January 2023

Abstract
B cell fate specification is associated with the expression of genes encoding components of the B cell antigen receptor and the ordered rearrangement of the immunoglobulin heavy-chain locus. Accompanying activation of the early program of B lineage gene expression is the concerted repression of lineage inappropriate genes. EBF is a transcription factor expressed exclusively in the B lineage and evidence is mounting that it is the limiting factor directing the B cell fate choice. To gain insight into mechanisms of EBF directed B cell fate specification, we generated EBF−/− cell lines. The cell surface phenotype, growth factor requirements, gene expression profile, and in vivo functional capabilities of EBF−/− clonal cell lines is consistent with a lymphoid-biased multipotential progenitor. To identify and discriminate novel EBF target genes involved in B cell fate specification, we performed comparative gene expression profiling of EBF−/−, Pax-5−/−, and RAG2−/− cell lines. ANOVA analysis of variance revealed that the EBF−/− lines overexpress several genes associated with early hematopoietic differentiation and stem cell function, notably a subset of HoxA genes. Conversely, the EBF−/− cells are defective in expression of a subset of early B lineage genes including lymphocyte enhancer factor-1, LEF-1. Consistent with the deficiency in LEF-1, EBF−/− lines display elevated levels of fas and increased sensitivity to apoptosis. Taken together, these data suggest novel regulatory circuits involving EBF during B cell fate specification, including inhibition of HoxA genes and activation of the Wnt signaling pathway.
Shnurri-2 controls memory Th1 and Th2 cell numbers in vivo (84.3)
Authors: Nakayama Toshinori , Kimura Motoko Y , Iwamura Chiaki , Hasegawa Akihiro , Yamashita Masakatsu 
Published: January 2023

Abstract
Schnurri-2 (Shn-2) is a large zinc-finger containing protein, and it plays a critical role in cell growth, signal transduction and lymphocyte development. In Shn-2-deficient CD4 T cells, the activation of NF-kB was upregulated and their ability to differentiate into Th2 cells was enhanced. We herein demonstrate that Th1 and Th2 memory cells are not properly generated from Shn-2-deficient effector Th1/Th2 cells. Even a week after the transfer of effector Th1/Th2 cells into syngeneic mice, a dramatic decrease in the number of Shn-2-deficient donor T cells was detected particularly in the lymphoid organs. The transferred Shn-2-deficient Th1/Th2 cells express higher levels of the activation marker CD69. No significant defect in the BrdU incorporation in the Shn-2-deficient transferred CD4 T cells was observed. The numbers of apoptotic cells were selectively higher in Shn-2-deficient donor Th1/Th2 cell population. Moreover, Shn-2-deficient effector Th1 and Th2 cells showed an increased susceptibility to cell death in in vitro cultures with increased expression of FasL. Transfer of Th2 effector cells over-expressing the p65 subunit of NF-kB resulted in a decreased number of p65-expressing cells in the lymphoid organs. As expected, T cell-dependent antibody responses after in vivo immunization of Shn-2-deficient mice were significantly reduced. Thus, Shn-2 appears to control the generation of memory Th1/Th2 cells through a change in their susceptibility to cell death.
Combined Photometric and Morphometric Analysis of Apoptosis Using the ImageStream Imaging Flow cytometer (84.8)
Authors: Henery Shannon , George Thaddeus , Hall Brian , Basiji David , Ortyn William , Morrissey Phillip 
Published: January 2023

Abstract
Apoptosis involves multiple biochemical pathways resulting in caspase activation, membrane blebbing and phosphatidylserine (PS) ‘flipping’, nuclear condensation, and nuclear fragmentation. While morphological characterization by microscopy remains the gold standard for accurately identifying apoptotic cells, a variety of photometric assays are employed to quantify apoptosis by flow cytometry. These assays target hallmarks of apoptosis, such as DNA fragmentation (TUNEL assay), surface PS exposure (AnnexinV assay), and caspase activation (caspase 3 substrates). Here we present a combined method for quantifying apoptosis that directly measures nuclear fragmentation using ImageStream imaging cytometry in addition to photometric parameters.
To investigate both
the temporal relationship between nuclear fragmentation and activation of other apoptotic pathways andpossible false positive and negative events associated with each measurement method, samples were analyzed on the ImageStream using both fluorometric and morphometric classifiers for measuring the apoptotic index.
Ramos, JurkatO, and Colo205 cells were incubated with three mechanistically distinct apoptosis inducers (Camptothecin, Staurosporine, and TNFα/IFNγ), and stained for nuclear morphology and AnnexinV, TUNEL, or PhiPhiLux. Acquisition of multi-spectral imagery on large numbers of cells couples the quantitative advantage of flow cytometry with the accuracy of morphology-based algorithms that can exclude artifacts common to these assays and provide a more accurate and complete analysis of apoptosis.
Aberrant Innate Immune Functions of Dendritic Cells in Aged Humans (85.12)
Authors: Agrawal Anshu , Agrawal Sudhanshu , Cao Jia-ning , Su Houfen , Gupta Sudhir 
Published: January 2023

Abstract
The molecular mechanisms underlying the chronic inflammation and reduced immunity observed during aging are not well understood. Comparison of the innate immune functions of monocyte-derived DCs from aging subjects with DCs from young individuals showed that MDDCs from aging individuals display
significantly reduced capacity to capture antigens via macropinocytosis and receptor-mediated endocytosis as determined by flow cytometry;impaired capacity to migrate in vitro in response to the chemokine MIP3-beta; andsignificantly increased LPS induced secretion of TNF-alpha and IL-6.
Investigations of intracellular signaling revealed reduced phosphorylation of AKT in MDDCs from aging, suggesting decreased activation of PI3kinase signaling pathway. Since PI3K signaling pathway plays a positive regulatory role in phagocytosis and migration, and it also functions as a negative regulator of TLR signaling by inducing activation of p38 MAP kinase, this may explain the aberrant innate immune functioning of DCs from aged subjects. Results further revealed an increased expression of PTEN, a negative regulator of PI3Kinase signaling pathway, in MDDCs from aged subjects. Increased PTEN may thus be responsible for the defect in AKT phosphorylation and therefore, altered innate immune response of DCs from aged humans.
This study is supported in part by a grant AG027512 from NIH and partly by new scholar grant from the Ellison Medical Foundation.
Early CD8 T cell proliferative heterogeneity entails diverse memory differentiation programs (85.19)
Authors: Kalia Vandana , Sarkar Surojit , Kaech Susan M. , Ahmed Rafi 
Published: January 2023

Abstract
Initial events governing delineation of the fraction of effector cells that advance into the long-lived memory lineage following antigen clearance remain unclear. We hypothesized that the order of recruitment of naïve CD8 T cells into the immune response regulates the memory potential of an effector cell. To address this hypothesis, we adoptively transferred naïve transgenic P14 CD8 T cells that encode a TCR specific for the GP33-41 epitope of lymphocytic choriomeningitis virus (LCMV) at different days post-infection. As predicted, cells transferred later during an infection demonstrated accelerated acquisition of memory cell phenotype (IL-7RaHi, L-SelectinHi), and proliferated to a greater extent upon secondary challenge compared to cells that were adoptively transferred earlier. Importantly, in a physiologically relevant setting, when cells that had divided &lt; or &gt; 4 divisions (based on CFSE dilution at day 2.5 post-infection) were FACS sorted and their potential to differentiate into long-lived memory cells evaluated, we observed that cells that entered into division later exhibited faster reversion to central memory phenotype, relative to cells that had entered cell division earlier. Memory cells arising from Ag-specific cells that entered proliferation later, also mounted more robust recall proliferative potential compared to those arising from cells that entered proliferation earlier. These data present a model that heterogeneity in early priming events likely dictate heterogeneity in initial proliferation, which in turn regulates their ultimate memory fate.
Supported by Elizabeth Glaser Pediatric AIDS Foundation awards to VK & SS, and NIH & Gates Foundation Grants to RA.
Accelerated T Cell Memory Development in the Peritoneal Cavity (85.24)
Authors: Olson Matthew R. , Varga Steven M. 
Published: January 2023

Abstract
Acute virus infection results in the expansion of the number of virus-specific CD8 T cells, virus clearance, followed by the death (contraction) of 90% of the expanded antigen-specific CD8 T cell population ultimately resulting in a stable memory population. Memory cells are characterized by their enhanced ability to expand in number after secondary infection, enhanced cytokine production (IL-2, TNF-α) and surface expression of the IL-7Rα (CD127), CD27, and the lymph node homing molecule CD62L. Most of what’s known about the kinetics of memory development is from data acquired from the spleen, however it is unclear if other peripheral tissues share those same kinetics. We demonstrate here that CD8 T cells isolated from the peritoneal cavity (PECs) after intraperitoneal or intravenous infection with lymphocytic choriomeningitis virus (LCMV) display enhanced IL-2 and TNF-α production as well as a greater frequency of cells expressing the memory markers CD127 and CD27 compared to their splenic counterparts. Interestingly, PECs demonstrated prolonged expression of the activation marker CD43 and similar levels of CD62L compared to splenic memory cells. PEC cells adoptively transferred into naïve mice maintain their phenotype in all tissues found, whereas splenic memory cells adopt the phenotype of their host tissue. These data suggest that the generation of memory CD8 T cells is controlled by tissue-specific signals.
Ability of aged transgenic B cells to mount both TH1 and TH2 responses (85.25)
Authors: Panwar Akanksha , Porter Katrina , Vogel Dr Laura 
Published: January 2023

Abstract
Humoral immunity has long been known to deteriorate with aging, however the cause is unclear. Previous research has focused on decreased T cell function or changes in microenvironment rather than aging-associated intrinsic alteration in B cells. Our previous in vivo research found that aged B cells can respond to T cell-dependent antigens when appropriate T cell help and a young microenvironment are provided. To examine this interaction in further detail, we investigated if aged B cells had a preference for TH1 or TH2 helper cells. OTII transgenic CD4+ cells were differentiated in vitro into TH1 or TH2 and co-transferred to young normal recipients with aged or young QM B cells. Analysis of expansion and activation phenotype of QM B cells that are induced following immunization was then analyzed using flow cytometry and confocal microscopy. Specific serum antibody amount, isotype and relative affinity were measured by ELISA. The results of the experiment showed that B cells from young and aged QM mice showed characteristic responses to the help provided by both young TH1 and TH2 helper cells in a young microenvironment. These data suggest aged B cells are able to respond to both TH1 and TH2 help.
Funded by NIH grant
Age Related Induction of Functional Transplantation Tolerance to Rat Skin Xenografts in Mice Treated with Co-stimulation Blockade and Donor Antigen (85.28)
Authors: Gordon Ethel J. 
Published: January 2023

Abstract
Our laboratory has developed a protocol to induce functional transplantation tolerance to rat xenografts in mice. Our method requires a single transfusion of donor derived spleen cells (DST) plus four treatments with an antibody to block T lymphocyte activation. Our preliminary analysis of DST donors demonstrated an advantage for using older donors. Median survival time (MST) was longer when older vs. very young DST donors were used. Other variables were constant. We will test the hypothesis that the age of the spleen cell donor has a significant affect on graft survival in xenograft and allograft models. Future studies will analyze cellular and molecular mechanisms responsible for this observation.
This research was supported in part by grant # 2P20RR016477 NCRR/NIH.