bluuebunny/crossref_metadata_2025_split huggingface dataset:
V cells: A novel antigen specific immunoglobulin expressing cell lineage in immunized animals distinct from classical B cell populations (P4386)
Authors: Chronopoulou Efthalia , Uribe-Benninghoff Alejandro , Elia Jeanne , Nguyen Andrea , Rabenstein Jacob , Fisher Janean , Berry Jody 
Published: January 2023

Abstract
Following immunization with protein antigens, an uncharacterized immunoglobulin bearing antigen specific cell population was identified and named as "V cells". This cell population lacks surface expression of B cell lineage specific markers such as B220, CD19, as well as T cell, NK, NKT, basophil and stem cell specific markers as determined by flow cytometric analysis. V cells are characterized as being positive for CD24, CD43, CD45, CD49b, CD48, CD79b and IgG. They are observed in the spleen, bone marrow, and blood of immunized mice but were not detected in other tissues such as the thymus, lymph nodes and peritoneal exudate cells. Antigen specific V cells were generated following immunization of at least 6 different protein antigens and appeared as early as 7 to 14 days following a single immunization. Molecular analysis of isolated mRNA from bulk sorted V cells proved that this novel cell population produces antigen specific Ig. Molecular analysis was also performed on the cDNA of individually and bulk sorted V cells extracted from the bone marrow and spleen. Immunoglobulin VH genes were then sequenced from each cDNA pool with results indicating a higher diversity of VH genes in the bone marrow when compared to the spleen. In addition, presence of shared VH genes between the spleen and bone marrow suggests migration of the V cells between the two compartments. The role of this novel cell population in innate and/or adaptive immunity is currently under investigation.
ZFP521 transcription factor modulates B cell development through interactions with EBF1 (P4392)
Authors: Lei Hong , Lukin Kara , Warming Soren , Hagman James 
Published: January 2023

Abstract
Early B cell factor 1 (EBF1) determines B cell lineage specification and commitment. ZFP521, a transcription factor with 30 zinc fingers, negatively regulates EBF1. We determined that EBF1 accelerates B cell development in the absence of ZFP521. Pre-pro-B and pro-B cells are increased substantially, while late pro-B, pre-B and immature B cells are reduced significantly in Zfp521 KO mice. Interestingly, mature IgM+IgD+ B cells, which accumulate in the bone marrow and spleens of 4-5 week old WT mice, are readily detected in 2-3-week old Zfp521 KO mice. Additionally, RUNX1 plays a role in ZFP521-mediated regulation of EBF1 activity. ZFP521 is detected in the nuclei of B cells and interacts with EBF1 in vivo. In contrast with published reports, ZFP521 and EBF1 co-localize on B cell-specific promoters. This indicates that ZFP521 regulates B cell development through functional interactions with EBF1 on target gene chromatin. Using ChIPseq data we are differentiating between two possible models concerning functions of ZFP521: 1) ZFP521 may modulate EBF1 function through direct protein:protein interactions, and/or 2) ZFP521 may direct transcription of cognate target genes independently of EBF1. Together, our studies provide exciting new information concerning functions of EBF1 and ZFP521 in the B cell regulatory network.
The RAG2 acidic hinge regulates pathway choice, maintains genomic stability, and prevents lymphomagenesis (P4398)
Authors: Coussens Marc , Wendland Rebecca , Lindsay Cory , Deriano Ludovic , Arnal Suzzett , Roth David 
Published: January 2023

Abstract
VDJ recombination-associated DNA double-stranded breaks are normally repaired by the high fidelity, classical NHEJ machinery. Previous studies have implicated the RAG/DNA post-cleavage complex (PCC) in regulating pathway choice by preventing access to inappropriate alternative repair mechanisms such as HR and alternative NHEJ (aNHEJ). We report that RAG2’s acidic hinge is critical for several steps. Mutations reducing the negative charge in this region destabilize the PCC, disrupt pathway choice, permit repair of RAG-mediated DSBs by the low-fidelity, translocation-prone aNHEJ machinery, and reduce genomic stability in developing lymphocytes. Structural predictions and experimental data support our hypothesis that flexibility of the acidic hinge is necessary for these functions. Similar human RAG2 sequence variants alter the predicted flexibility and permit repair by aNHEJ, indicating the potential for hazardous physiological consequences. Mice bearing similar neutralizing mutations (RAG2Neut), in the absence of p53, have an increased incidence of lymphomagenesis with hallmarks of VDJ recombination errors. Thus, the acidic hinge, along with stabilizing the PCC and ensuring proper pathway choice, aids in the prevention of lymphomagenesis by maintaining genomic integrity. This novel regulatory role that helps to ensure the safe development of a diverse immune repertoire, is to the best of our knowledge, the first function ascribed to this conserved region of RAG2.
Analysis of illegitimate Notch1 and Bcl11b V(D)J deletion events during mouse development (P4400)
Authors: Champagne Devin , Shockett Penny 
Published: January 2023

Abstract
Previously, others identified hotspots for deletional illegitimate V(D)J rearrangements in normal mouse thymus involving cryptic RSSs in Notch1 and Bcl11b, onco/tumor suppressor genes involved in T cell development. These deletions are associated with radiation-induced and other T cell malignancies. We are conducting expanded frequency and junctional analyses of deletions during mouse development to examine and compare age- and gender-specific detection and occurrence of these events. Deletions in thymus increase in frequency between fetal and neonatal stages, are seen through at least 7 months of age, and are found in spleen. Deletions are more frequent at Bcl11b than Notch1 as previously observed, but both genes show clonality of deletion bearing cells. Junctional processing analysis indicates that nucleotide loss is more frequent and extensive at Bcl11b than Notch1, thus P nucleotide insertions are more abundant in Notch1 junctions. N nucleotide insertions increase between fetal and juvenile stages, with recessed palindromes (Pr nucleotides) corresponding mostly to single-base additions consistent with normal TdT activity. An in depth age and gender junctional processing analysis is in progress as are determinations of postnatal inducibility of deletions using inducible RAG transgenic mice. We present our results in the context of similar studies of V(D)J deletions at the nonimmune HPRT locus and normal V(D)J recombination at antigen receptor loci.
Direct interaction between E47 and p21 controls LT-HSC proliferation and functional longevity (P4471)
Authors: Santos Patricia , Borghesi Lisa 
Published: January 2023

Abstract
Long-term hematopoietic stem cells (LT-HSCs) are important multipotent cells that replenish all immune cells lost due to natural turnover, infection or injury. LT-HSC proliferation is controlled by cell cycle regulators and upstream transcription factors but direct links between the two have not been established. Here, we demonstrate a mechanistic link between the transcription factor E47 and the major cell cycle regulator p21 in controlling LT-HSC proliferation, self-renewal, and functional competence. Gain-of-function and loss-of-function studies show that E47 directly regulates p21 transcription in HSCs. Using E47/p21 compound heterozygotes, a powerful approach for analyzing HSC activity in vivo, we show a 2-fold increase in BrdU+ LT-HSCs compared to WT or single HET controls in both steady state conditions and under transplantation stress. E47HETp21HET HSCs are also more sensitive to mitotoxic challenge with 5-FU resulting in a 45% decrease in HSC numbers compared to treated controls. In serial adoptive transfer assays, a rigorous challenge to HSC self-renewal, E47HETp21HET HSCs were decreased by 40% and 60% in secondary and tertiary recipients, respectively. In addition to hyperproliferation and poor self-renewal, E47HETp21HET adoptive transfer recipients had selective myeloid bias indicating HSC skewing. Together, these data suggest a developmental stage-specific, cell-intrinsic collaboration between E47 and p21 in the regulation of LT- HSC competence in vivo.
Foxp1 and Foxp4 regulate Foxp3+ nTreg generation in the thymus (P4420)
Authors: Wiehagen Karla , Staub Elizabeth , Tucker Haley , Morrisey Edward , Maltzman Jonathan 
Published: January 2023

Abstract
CD4+ Foxp3+ regulatory T cells (Tregs) are fundamental for transplantation tolerance and prevention of autoimmunity. nTreg generation occurs in the thymus and requires both T cell receptor- and cytokine receptor-generated signals. Foxp1 and Foxp4 are closely related transcription factors expressed in both Tregs and conventional T cells. We sought to determine whether Foxp1 and Foxp4 have a role in nTreg development. Using a CD4Cre transgenic conditional knockout approach, we deleted both Foxp1 and Foxp4 in the T lineage. Mice conditionally deficient for both Foxp1 and Foxp4 (cDKO) have normal thymic cellularity. CD4SP and CD8SP thymocytes are present in normal ratios. CD4+Foxp3+ cells are present but express higher levels of receptors for IL-2 and IL-7. Markers of activation CD44, CD69, and GITR are also found at elevated levels on the cell surface. In the competitive setting of mixed bone marrow chimeras, there is a significant reduction in cDKO-derived chimerism in the Foxp3+ nTreg population. The competitive disadvantage occurs despite expression of the high affinity IL-2 receptor alpha chain, CD25. Together, these results suggest that Foxp1 and Foxp4 are necessary for the normal generation of nTreg cells. Ongoing experiments to understand the underlying molecular basis of these findings will be discussed.
Maternal methyl-donor supplementation increases regulatory T-cell-mediated redox remodeling (P4422)
Authors: Yung Raymond , Delaney Colin , Garg Sanjay , Banerjee Ruma 
Published: January 2023

Abstract
Regulatory T cells (Treg) maintain peripheral tolerance by inhibiting the proliferation of effector T (Teff) cells. Previously, we exposed dams to a diet supplemented with methyl donors (MS) or not (control) during mating and pregnancy, and found the MS diet suppresses selected T cell pro-inflammatory chemokines and cytokines in F1 mice. Here, we studied the effects of these prenatal diets on Treg function in F1 mice. Dams were fed either MS or control diets prior to mating, and during pregnancy and lactation. At 4 weeks of age, F1 mice from both groups were either analyzed or placed on the standard NIH-31 chow and allowed to age. We found that prenatal MS diet exposure is associated with lower Treg numbers in F1 mice, and this decrease is sustained into late life while on the standard NIH-31 chow. Both FoxP3 mRNA and protein were decreased in mice on the MS diet, compared to the control diet. The difference in Treg numbers is not due to differences in food consumption or weight gain between dams exposed to either prenatal diet. Lower Treg cell numbers are associated with hyper-methylation of the FoxP3 promoter and enhancer regions. Despite their lower numbers, Tregs from F1-MS mice produce increased IL-10 and suppress Teff proliferation more, compared to Treg from F1-control mice. Together, these results show that a methyl-donor-rich prenatal diet lowers total Treg numbers, though these cells maintain an anti-inflammatory phenotype.
Homeostatic properties and phenotypic maturation of murine CD4+ pre-thymic emigrants in the thymus (P4436)
Authors: Ge Qing 
Published: January 2023

Abstract
After a tightly regulated developmental program in the thymus, “mature” single positive (SP) thymocytes leave the thymus and enter the periphery. These newly arrived recent thymic emigrants (RTEs) are phenotypically and functionally immature, and will complete a dynamic maturation in the peripheral lymphoid organs before being licensed to be resident naïve T cells. To study the early events occurring in the RTE maturation process, we identified the phenotype of CD4+ pre-RTEs, a population of CD4+ SP thymocytes that have acquired the thymus egress capability. Compared to peripheral naïve T cells, CD4+ pre-RTEs displayed superior survival capability in lymphoreplete mice and faster proliferation under lymphopenic condition. The differences in Bcl2/Bim expression and/or heightened IL-7 signaling pathway may account for the pre-RTEs’ better responsiveness to homeostatic signals. Qa2, the expression of which indicates the phenotypic maturation of SPs and RTEs, was found to be upregulated in CD4+ pre-RTEs in thymic perivascular space. Migratory dendritic cells surround this region contribute to Qa2 expression in pre-RTEs. The dendritic cell-driven Qa2 induction of CD4+ pre-RTEs is independent of MHC class II and Aire molecules.
TSLP-mediated extramedullary hematopoiesis promotes allergic inflammation (P4473)
Authors: Siracusa Mark , Tait Wojno Elia , Osborne Lisa , Saenz Steven , Kim Brian , Spergel Jonathan , Comeau Michael , Artis David 
Published: January 2023

Abstract
Extramedullary hematopoiesis (EMH) refers to the differentiation of hematopoietic stem cells (HSCs) into effector cells that occurs in compartments outside of the bone marrow. Previous studies established a link between pattern recognition receptor-expressing HSCs, EMH and innate immune responses to bacterial or viral pathogens. However, the factors that regulate EMH and whether EMH is a conserved mechanism of innate immunity to diverse stimuli remain poorly defined. Here, we demonstrate a previously unrecognized role for thymic stromal lymphopoietin (TSLP) in promoting the population expansion of Lineage-, CD34+, c-Kit+ granulocyte-monocyte progenitor (GMP)-like cells in the periphery. TSLP-elicited progenitors possess the capacity to differentiate into macrophages, mast cells and basophils. Critically, adoptive transfer of TSLP-elicited GMP-like cells was sufficient to promote Th2 cytokine-dependent immunity to an intestinal helminth parasite, demonstrating a role for TSLP-elicited GMP-like cells in promoting inflammation. Further, circulating progenitor cells were also increased in allergic patients with a gain-of-function polymorphism in TSLP, suggesting that the TSLP-EMH pathway may be operational in human disease. These data demonstrate a previously unrecognized role for TSLP in promoting EMH that contributes to the development of Th2-cytokine-mediated inflammation and identify the TSLP-EMH pathway as a possible therapeutic target to treat allergic inflammation.
Human peripheral Vδ1+ γδ T cells can develop into αβ T cells (P4460)
Authors: Schilbach Karin , Ziegler Hendrik , Haarer Jan , Sterk Marco , Rammensee Hans-Georg , Handgretinger Rupert 
Published: January 2023

Abstract
The T cell compartment can be divided in two large classes: αβ and γδ T cells. It is believed that they develop in the thymus from a common progenitor and that the choice between αβ and γδ T cell fate is the first lineage decision made by progenitors after they commit to the T-cell lineage. However, here we show that peripheral Vδ1+ γδ T cells in an inflammatory environment can transdifferentiate into αβ T cells. Upon their extrathymic route of differentiation, that resembles well-characterized molecular program of thymic αβ lineage development, Vδ1+ T cells upregulate CD4+ coreceptor, develop Vδ1+ CD4+CD8+ double positive cells, show heterodimeric CD8αβ, transcribe RAG, preTα and express a particular Vβchain on their surface. Simultaneously inflammation confers controlled initiation of rearrangement in the TCRα locus. Transdifferentiation of Vδ1+ T cells at the clonal and bulk-culture level into functional CD4+ or CD8+ αβ T cells via a Vδ1+TCR/αβ+TCR double positive stage suggests that, upon inflammatory stimuli, Vδ1+ T-cell conversion participates in the induction of adaptive immune responses. Identifying an innate T cell as an αβ T-cell progenitor and showing the developmental steps linking the progenitor to adaptive, thymus-independant αβ T-cell responses as inflammatory conditions is of utmost relevance and will deeply impact evaluation of immune responses in infection, malignancy and autoimmune processes.
Enhancing T lineage production in aged mice: a novel function of Foxn1 in the bone marrow niche (P4475)
Authors: Le Phong , Zook Erin , Zhang Shubin , Gerstein Rachel , Witte Pamela 
Published: January 2023

Abstract
Foxn1 is essential for thymic organogenesis and T lymphopoiesis. While reduced Foxn1 expression results in a decline in T lymphopoiesis, overexpression of Foxn1 in the thymus of a transgenic mouse model (Foxn1Tg) attenuates the age-associated decline in T lymphopoiesis. T lymphopoiesis begins with ETP, derived from MPP in the BM. A decline in MPP and ETP numbers with age is thought to contribute to reduced T lymphopoiesis. Previously, we showed that reduced ETP number with age is attenuated in Foxn1Tg; whether the effect is initiated in the BM with MPP is not known. Here, we report that Foxn1 is expressed in Wt BM and over expressed in Foxn1Tg. With age, the number of MPP in Foxn1Tg was not reduced, and Foxn1Tg also have a larger pool of HSC. Furthermore, the Foxn1Tg BM is more efficient in generating MPP. In contrast to MPP, CLP and B lineage cell numbers were significantly lower in both young and aged Foxn1Tg compared to Wt. We identified a novel population of lineageneg/low, EpCAMpos, CD138neg cells as Foxn1-expressing BM cells that also express Delta-like 4. Thus, Foxn1 affects both T lymphopoiesis and hematopoiesis, suggesting that the Foxn1 BM niches function in skewing MPP development toward T lineage progenitors.
Nr4a1 regulates thymic resident macrophage development and function (P4468)
Authors: Tacke Robert , Nowyhed Heba , Wu Amy , Hedrick Catherine 
Published: January 2023

Abstract
Mononuclear phagocytes, including monocytes, macrophages, and dendritic cells, function in tissue homeostasis, and also regulate immune responses. Tissue resident macrophages in a variety of tissues arise from the yolk sac, and are generated independently of hematopoietic stem cells. However, the origin of thymic macrophages remains poorly understood. Nr4a1 is an orphan nuclear receptor that limits pro-inflammatory cytokine production in macrophages, and functions as the master regulator of Ly6C- patrolling monocyte development. Here, we report a significant defect in the development of thymic resident macrophages in Nr4a1-deficient animals. Thymic macrophage levels were reduced over two-fold compared with wild-type controls. However, Nr4a1 does not globally regulate the development of tissue macrophages, as macrophages resident in the spleen, lung, brain, pancreas, and peritoneum from Nr4a1-deficient mice develop normally. Moreover, Nr4a1-deficient thymic macrophages fail to clear apoptotic thymocytes in vivo, and express reduced levels of genes important for apoptotic cell engulfment, including engulfment receptors Mertk, and Axl. Defective thymic macrophage development likely impairs proper T cell selection, thus altering the immune system as a whole. Together, these data show that Nr4a1 regulates the development and functional capacity of tissue resident macrophages in the thymus, and provide a novel mechanism for maintenance of thymic homeostasis.
Muc1 regulates MDSC expansion and suppressive activity in healthy and tumor-bearing mice (P2049)
Authors: Mukherjee Pinku , Sahraei Mahnaz , Curry Jennifer , Das Roy Lopamudra , Gendler Sandra 
Published: January 2023

Abstract
We demonstrate that a membrane-tethered epithelial mucin, Muc1 affects the differentiation of immature myeloid derived cells (IMCs) in the bone marrow (BM). First, in wild-type (WT) mice, BM-derived Lin-Sca1+ hematopoietic progenitor cells (HPCs) express Muc1. Second, in Muc1-/- mice, the percentage of IMCs is higher and these cells express higher levels of c-MYC as compared to IMCs from WT mice. Interestingly, the myeloid derived suppressor cells (MDSCs) in the Muc1-/- tumor-bearing mice are less differentiated and have higher suppressor function as compared to MDSCs from WT mice. Because of the higher suppressive functions of the MDSCs in the Muc1-/- mice, we confirm that subcutaneous tumors develop significantly faster in these mice as compared to the WT mice (Poh 2009). Further, Muc1-/- mice have decreased NK cell function possibly due to increased suppressive function of their MDSCs. Decreased cytotoxic function of NK cells can partially explain the increased susceptibility of Muc1-/- mice to tumor growth. Lastly, we show that MDSCs from Muc1-/- mice have higher levels of phospho-STAT3, which is known to be critical for MDSC expansion. This finding is of great importance since systemic targeting of Muc1 in several carcinomas is being actively explored. It becomes clear that therapeutic targeting of Muc1 must be localized to the epithelial tumors because down-regulating Muc1 expression in hematopoietic progenitor cells may lead to increased MDSCs.
Medroxiprogesterone acetate drives expansion of CD11b+Gr1high myeloid-derived suppressor cells which suppress NK cell effector functions in tumor-bearing mice (P2059)
Authors: Zwirner Norberto , Spallanzani Raul , Dalotto Tomas , Rossi Lucas , Avila Damian , Ziblat Andrea , Domaica Carolina , Fuertes Mercedes , Raffo Iraolagoitia Ximera , Rabinovich Gabriel , Salatino Mariana 
Published: January 2023

Abstract
Hormone replacement therapy with the progesterone analogue medroxyprogesterone acetate (MPA) is widely used in postmenopausal women for the treatment of endometrial conditions, and as a contraceptive. However, prolonged administration of MPA is associated with increased incidence of breast cancer through ill-defined mechanisms. Our aim was to explore if MPA threats immunosurveillance to tumors by effecting myeloid-derived suppressor (MDSCs; CD11b+Gr1+) and NK cells in mammary tumor-bearing mice. We used the highly metastatic 4T1 breast tumor which does not express classical progesterone or glucocorticoid receptors. MPA did not affect primary tumor growth in 4T1-tumor bearing mice but promoted lung metastasis burden. This effect was accompanied by expansion of splenic CD11b+Gr1+ cells (mostly CD11b+Gr1high cells). Sorted CD11b+Gr1+ cells from MPA-treated tumor bearing mice showed a more pronounced inhibitory activity of NK cell degranulation in response to YAC-1 cells and IFN-γ production in response to cytokines than those isolated from untreated tumor-bearing mice. Also, MPA significantly increased the percentage of spleen NK cells in tumor-bearing mice with similar lung infiltration of CD11b+Gr1+ and NK cells as compared to untreated tumor-bearing mice. We conclude that in breast cancer-bearing hosts MPA promotes the accumulation of CD11b+Gr1+ which suppress NK-cell mediated anti-tumor activity potentially contributing to tumor progression and metastasis.