bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role for Blimp-1 in the generation and function of cytotoxic CD4 effector T cells identified by NKG2A/C/E expression (P6046)
Authors: Marshall Nikki , Brauner Matthew , Kuang Yi , Berg Leslie , Swain Susan 
Published: January 2023

Abstract
CD4 T cells are best known for helper roles in immunity, but they can also acquire cytotoxic activity (ThCTL) and kill MHC class II+ targets presenting cognate antigen. We have identified a subset of CD4 effectors in the lung of influenza A/PR8 (IAV)-infected mice that peak at 8 days post-infection. They mediate potent perforin-dependent, Fas-independent antigen-specific cytotoxic activity and express the NK cell-associated receptors NKG2A/C/E. CD4 T cells from lung, spleen and draining lymph nodes express mRNA for the NKG2A inhibitory receptor, while mRNA for the activating receptors, NKG2C and NKG2E, are only detected in the lung. Interestingly, blocking NKG2A/C/E receptors or their ligand, non-classical MHC class I molecule Qa-1, significantly inhibits ThCTL-mediated specific killing of target cells. The NKG2A/C/E+ ThCTL subset co-expresses PD-1, CD27, PSGL-1 and Ly6c, but not CD103, as well as transcription factors Blimp-1 and Eomes. We analyzed the role of transcriptional repressor Blimp-1 in the CD4 response and found that although the conditional KO of Blimp-1 in T cells did not affect total numbers of CD4 effectors in IAV-infected lung, it significantly reduced the number of ThCTL, their expression of the aforementioned phenotype and their cytotolytic function. NKG2A/C/E is a valuable marker for identifying ThCTL that are generated during an acute anti-viral response through a unique program that involves Blimp-1.
Inflammatory cytokines extend the division of activated CD8 T cells in vivo by maintaining high affinity IL-2 signaling (P6050)
Authors: Starbeck-Miller Gabriel , Xue Hai-Hui , Harty John 
Published: January 2023

Abstract
Activation of naïve CD8 T cells requires several signals including TCR ligation and costimulation. Although these signals induce division, accumulation of activated CD8 T cells is dependent on a third signal provided by inflammatory cytokines. In vitro studies suggest that “signal 3”specifically enhances survival; however, the mechanisms for optimal accumulation of activated CD8 T cells in vivo is unknown. To explore these mechanisms, we transfer ~600 OT-I tg-CD8 T cells into congenic hosts and immunize hosts with OVA257-coated dendritic cells in the absence (DC) or presence of inflammation (CpG; DC+CpG). OT-I numbers were similar in DC and DC+CpG recipients at day 5 p.i. However, the presence of inflammation during immunization increased accumulation and extended division of OT-I at day 6-7 p.i. while not altering survival. Extended division of DC+CpG OT-I depended on the maintenance of IL-2/PI3K signaling that was regulated by early/direct IL-12 signaling. These findings indicate that "signal 3" enhances accumulation by maintaining division (not survival) of activated CD8 T cells in a manner dependent on a temporally orchestrated sequence of cytokine signals.
IRF-1 is a key transcriptional regulator of Tr1 differentiation (P1135)
Authors: Karwacz Katarzyna , Yosef Nir , Kuchroo Vijay 
Published: January 2023

Abstract
IL-10 producing Type 1 regulatory T cells (Tr1) are crucial for maintenance of peripheral tolerance and prevention of autoimmune inflammation. Using IL-27 as the differentiation factor, we undertook a detailed whole genome transcriptional network analysis of differentiating Tr1 cells. We identified IRF-1 as a key transcriptional regulator of Tr1 differentiation. Our studies demonstrate a critical requirement for IRF1 in the generation of Tr1 cells both in vitro and in vivo. IRF1-/- mice immunized for the development of Experimental autoimmune encephalomyelitis (EAE) develop very severe clinical disease and fail to recover. Furthermore, CD4+ T cells in IRF1-deficient mice with active disease showed increased number of IL-17 secreting cells in the CNS. In fact, over-expression of IRF1 during Th17 differentiation suppresses induction of both IL17 and IL23 receptor, the role opposite to the one played by another IRF family member - IRF4. Interestingly, loss of IRF-1 enhances IRF4 binding to the key DNA-binding sites involved in Th17 differentiation and functional assays further support that IRF1 can actively repress IRF4-mediated transactivation of IL17A, pointing to a cross-competition of IRF1 and IRF4 for the same DNA-binding sites in CD4+ T cells. Collectively these data demonstrate a critical role of IRF-1 in the generation of Tr1 cells, but at the same time, the induction of IRF-1 represses Th17 differentiation by actively inhibiting IRF-4 mediated Th17 differentiation.
Cross-talk between T cell receptor and cytokine signaling is mediated by Itk: implications for the balance between Th17 and regulatory T cells (P1141)
Authors: Gomez-Rodriguez Julio , Wohlfert Elizabeth , Handon Robin , Wu Julie , Belkaid Yasmine , Schwartzberg Pamela 
Published: January 2023

Abstract
A proper balance between Th17 and T regulatory cells (Tregs) is critical for the generation of immune protective immune responses while minimizing autoimmunity. We show that the Tec family kinase Itk influences the balance between Th17 and T regulatory cells by regulating crosstalk between T Cell Receptor (TCR) and cytokine signaling. Under both Th17 and Tregs differentiation conditions, Itk-/- CD4 T cells develop higher percentages of functional Foxp3+ cells, associated with increased responsiveness to IL-2. Itk-deficient CD4 cells show reduced TCR-induced phosphorylation of mTOR targets, including ribosomal S6 and AKT, accompanied by changes in metabolic signatures that are affected by mTOR pathways. Surprisingly, iTreg Itk-/- cells also show reduced IL-2-induced mTOR pathways, despite increased STAT5 phosphorylation. We find that TCR stimulation in WT CD4+ T cells leads to a dose-dependent repression of expression of Pten; however, at low TCR stimulation or in the absence of Itk, Pten is not effectively repressed, thereby uncoupling STAT5 phosphorylation and PI3K/mTOR pathways and increasing Foxp3 expression. Functional studies show that naïve Itk-/- CD4 T cells also preferentially develop into Tregs in an in vivo colitis model. Furthermore Itk-deficient Tregs are capable to inhibit the development of colitis demonstrating that Treg function is independent of Itk. Our results suggest that Itk is a potential target for altering the balance between Th17 and Treg cells.
Immune memory in the CNS (P1177)
Authors: Garcia Samantha , Portocarrero Carla , Kean Rhonda , Schutsky Keith , Hooper Douglas 
Published: January 2023

Abstract
Due to specialized neurovasculature, referred to as the blood-brain barrier (BBB), CNS tissues are immunologically privileged and only infiltrated by lymphocytes under certain circumstances. One of these is the clearance of attenuated rabies virus (RABV) from the CNS which is dependent upon CD4 T and B cell entry into CNS tissues and local production of virus neutralizing antibody (VNA). Despite spread to the CNS, these processes do not occur during wild-type RABV infection. Differences in the spread and induction of immunity to different RABV in mice allow us to determine if immune effector accumulation in CNS tissues results in the establishment of immune memory in this location. The attenuated recombinant RABV TriGAS has a severely curtailed ability to spread and does not reach the CNS when administered to the gastrocnemius. Mice infected with TriGAS via this route are fully protected against a peripheral challenge with wild-type RABV but not against intra-nasal infection with the same virus. Protection against a CNS challenge with wild-type RABV is only established by vaccination regimens that deliver RABV-specific immune effectors to the CNS. Lymphocytes remain in the CNS for extended periods of time following clearance of attenuated virus and appear to rapidly divide upon local challenge with wild-type RABV. This suggests that these cells may represent CNS resident immune memory cells capable of reactivating to produce VNA upon local challenge.
A mechanism for inheritance of active chromatin in dividing T-helper cells (P1195)
Authors: Josefowicz Steven , Li Charles , Allis C. 
Published: January 2023

Abstract
Immune cell lineage specification and phenotypic stability is defined by distinct transcription factor activity and cell type-specific heritable chromatin signatures, which collectively control the transcriptional program. Heritable acquisition of chromatin states is likely to be especially relevant in activated, rapidly dividing immune cells during the initial stages of the adaptive immune response. To evaluate how chromatin signatures are propagated through the cell cycle in CD4+ T cells we have combined single cell quantitative global chromatin modification analysis with cell cycle analysis and chromatin immunoprecipitation (ChIP). We have identified novel combinatorial histone H3 tail modifications that are enriched in mitotic chromatin and are selectively retained at lineage specific regulatory elements. Biochemical approaches have identified chromatin-binding proteins, including bromodomain-containing (BRD) factors whose recruitment to these motifs is being explored for a role in heritable lineage specific chromatin characteristics. Our data indicate a model in which signaling to chromatin and “writing” of a combinatorial H3 motif at open chromatin during T-helper cell activation and proliferation results in heritable maintenance of lineage specific active chromatin through the cell cycle by recruitment of specific chromatin “readers” which may act as mitotic bookmarks and subsequently facilitate transcription.
MeCP2 reinforces STAT3 signaling and effector T cell differentiation by controlling miR-124-mediated suppression of SOCS5 (P1125)
Authors: Jiang Shan , Li Chaoran , McRae Gabrielle , Lykken Erik , Wan Ying , Sevilla Jose , Li Qi-jing 
Published: January 2023

Abstract
Methyl-CpG binding protein 2 is an X-linked multifunctional epigenetic regulator whose heterozygous mutation causes the neurological disorder Rett Syndrome in 1:10,000 women, and has also been linked to multiple autoimmune disorders. Speculating that loss of Methyl-CpG binding protein 2 might result in mechanistically similar defects in the nervous and immune systems, we examined Methyl-CpG binding protein 2’s role in regulating CD4+ T cells’ antigen response. We showed that Methyl-CpG binding protein 2 was indispensable for Th1 and Th17 differentiation and Th1- or Th17-mediated pathology both in vitro and in vivo. Loss of Methyl-CpG binding protein 2 in CD4+ T cells resulted in impaired transcription of microRNA miR-124, and consequently relieved miR-124-mediated repression of SOCS5 translation. The accumulation of SOCS5 inhibited cytokine-induced STAT1 and STAT3 activation that was required for Th1 and Th17 differentiation. Primary neurons and astrocytes also failed to respond properly to STAT3-dependent neurotrophic factor signaling when MeCP2 was silenced. Thus, regulation of STAT3 signaling could represent a common etiology underpinning MeCP2’s importance in both the nervous and immune systems.
Epigenetic environment, transcriptional elements, and RSS sequences collectively regulate non-random VH gene usage at the mouse Igh locus. (P4394)
Authors: Choi Nancy , Verma-Gaur Jiyoti , Torkamani Ali , Feeney Ann 
Published: January 2023

Abstract
A diverse antibody repertoire is formed through the rearrangement of V, D, and J segments of the immunoglobulin heavy and light chain loci. The mouse heavy chain (Igh) locus has 110 functional V gene segments that can recombine to a rearranged DJ segment. While the unequal usage of V genes has been well described, it is not understood what factors determine the frequency of gene segment utilization. To answer this question we conducted deep sequencing of 5’-RACE products amplified in an unbiased manner. The results quantitatively represented the entire expressed repertoire of Igh genes in pro-B cells. We compared this data with ChIP-seq results for several histone modifications that indicate different states of chromatin accessibility to determine whether the degree of ‘openness’ was important for differential V gene usage. RNA pol II binding and RNA-seq for both sense and antisense transcripts were examined to evaluate the role of non-coding transcription in rearrangement. ChIP-seq for transcription factors EBF and E2A were also considered, as well as insulator binding factors CTCF and Rad21, both of which have been described to influence the 3D structure and the degree of compaction at the Igh locus. The RSS sequences for each V gene family were evaluated as well. Our study describes how the collective effect of all of the above influences the decision of which V gene is recombined and emphasizes that V gene segment usage is not determined by a single factor.
A role for CD3 conformation in the preTCR/CD3 complex (P4428)
Authors: Hoffmann Michele , Parks Christopher , Gil Pages Diana 
Published: January 2023

Abstract
Mature T cells recognize and respond to antigens through the T cell antigen receptor (TCR). We have established that upon TCR antigenic engagement, the TCR/CD3 complex undergoes a conformational change (CD3dc) of its CD3 epsilon tails to expose a proline-rich sequence and allow downstream signaling to occur. CD3dc has been shown to be required for full TCR/CD3 signaling and function of mature T cells. Pre-TCR assembly with the CD3 complex serves as a developmental checkpoint that ensures the expression of a function variant TCRβ chain in thymocytes. Interestingly, stimulation of the preTCR complex has been found to be ligand-independent; assembly is sufficient. Knowing this, we first determined the stoichiometry of the preTCR/CD3 complex and asked the question, how does assembly impact its ability to undergo conformational change? To answer this question, we employed various novel biochemical methods developed in our laboratory including a flow-cytommetry based immunoprecipitation (IP-FCM) as well as a CD3-pull down assay (CD3-PD). We aim to study the conformation of CD3 in the preTCR/CD3 complex and establish whether or not the assembly of this receptor is enough to induce the active CD3 conformation that supports functional CD3 signaling in mature cells. With our data, we are further able to characterize previously unstudied requirements for CD3 conformational change in the preTCR/CD3 complex with the potential of using this knowledge to manipulate CD3dc in the future.
TCF-1 and LEF-1 promote CD4+ T cell lineage choice by upregulating Myb (P4439)
Authors: Steinke Farrah , Zhou Xinyuan , Yu Shuyang , Xue Hai-Hui 
Published: January 2023

Abstract
During late stages of T cell development, positively selected CD4+CD8+ (DP) thymocytes must make a lineage choice decision to become MHC-I-restricted cytotoxic CD8+ T cells or MHC-II-restricted helper CD4+ T cells. This lineage choice process is determined by extrinsic signals such as TCR and γc cytokines and by intrinsic transcriptional factors such as Myb, Gata3, ThPOK and Runx. The canonical Wnt effector transcription factors, TCF-1 and LEF-1, are essential for the transition from CD4-CD8- (DN) to the DP stage, but their requirement beyond the DP stage is unknown. Here we show that deficiency in TCF-1 resulted in a preferential loss of CD4+ T cells and reduced CD4/CD8 ratio, and this phenotype was exacerbated upon loss of both TCF-1 and LEF-1. By crossing TCF-1-/- mice to MHC-I-deficient strain or OT-2 CD4+ TCR transgenic strain, we found that DP thymocytes selected on MHC-II were re-directed to the CD8 lineage in the absence of TCF-1 and/or LEF-1. Mechanistically, we observed that Myb expression is greatly diminished in post-select DP thymocytes lacking TCF-1 and/or LEF-1, and that activation of the Wnt signaling pathway in post-select DP thymocytes induced Myb expression. Collectively, these findings revealed a novel role of Wnt effectors TCF-1 and LEF-1 in promoting CD4+ lineage choice, which is likely mediated by Wnt-dependent upregulation of Myb.
Identification of a novel, developmental-stage-specific enhancer in the Cd4 gene (P4442)
Authors: Swan Gregory , Sarafova Sophia 
Published: January 2023

Abstract
CD4 helper T cells coordinate the immune response and are highly dependent on the expression of the Cd4 gene for proper development and function. The function of a promoter, proximal enhancer, and a silencer have been well documented and together explain how the Cd4 gene gets turned on in CD4 T cells and off in CD8 T cells. However once turned on, the amount and timing of Cd4 expression varies during T cell development and activation. This modulation of CD4 surface levels is essential for proper lineage specification and T cell function. Yet, how subtle changes of CD4 expression are regulated remains unclear. We have recently identified a novel positive cis-acting transcriptional regulatory element (NCE) in the Cd4 locus. Here we demonstrate that NCE enhances Cd4 promoter function in position and orientation independent manner using a transient transfection assay with an eGFP reporter construct in RLM11 murine thymoma cell line. Using cell lines at different developmental stages, we determined that NCE functions well at the intermediate, but not the DP stage of development. This formally demonstrates that NCE functions as an enhancer and may have developmental specificity. We are in the process of confirming these findings in vivo by generating BAC transgenic mice that lack the NCE in the Cd4 locus.
NKAP is required for iNKT cell development (P4450)
Authors: Thapa Puspa , Das Joy , McWilliams Douglas , Shapiro Michael , Sundsbak Rhianna , Nelson-Holte Molly , Tangen Sarah , Anderson Joshua , Desiderio Stephen , Hiebert Scott , San'tAngelo Derek , Shapiro Virginia 
Published: January 2023

Abstract
Invariant Natural Killer T cells (iNKT) are innate lineage of lymphocytes with distinct developmental pathway than conventional αβ T cells. The transcriptional repressor NKAP is required for early T cell development and maturation of conventional αβ T cells. Using CD4-cre NKAP cKO mice, we show that NKAP is required for positive selection of thymocytes into the iNKT cells lineage. Deletion of NKAP using CD4-cre leads to an early block in iNKT cell development at the DP stage. This cell intrinsic defect was not due to decreased DP thymocyte survival or inability of NKAP deficient thymocytes to rearrange their Vα14-Jα18 TCR. Moreover, ectopic over-expression of Vα14-Jα18 invariant TCR α chain rescued the development of iNKT cells. This suggests that NKAP plays a role in the positive selection of thymocytes into the iNKT cell lineage. Interestingly, conditional deletion of NKAP associated protein Hdac3 using CD4-cre also leads to a similar block in iNKT cell development, but not conventional T cell development. This is first evidence demonstrating for an Hdac in T cell development. Whether NKAP and Hdac3 work together functionally to regulate the positive selection of DP thymocytes into iNKT cell lineage is currently being investigated.
Ly108 enhances PLZF expression in developing thymocytes (P4452)
Authors: Kraus Zachary , Dutta Mala , Schwartzberg Pamela 
Published: January 2023

Abstract
Promyelocytic zinc finger transcription factor (PLZF) is an important transcription factor expressed by natural killer T (NKT) cells and a variety of other innate T cells. Development of these PLZF positive cells require the adaptor molecule SLAM- associated protein (SAP) and the interaction of the signaling lymphocyte activation molecule (SLAM) family member receptors in addition to TCR stimulation. However, it remains unclear how these interactions influence the development of innate thymocytes. We have found that costimulation of pre-selection double positive (PS-DP) thymocytes with an agonist to the SLAM family receptor Ly108 greatly enhances PLZF expression compared to TCR stimulation alone. Furthermore, PLZF expressing cells were reduced in Ly108 deficient mice and increased in transgenic mice expressing a hyperactive Ly108 isoform. Costimulaiton with Ly108 also increased the expression of Egr-2 and the binding of Egr-2 to the PLZF (Zbtb16) promoter. Surprisingly costimulation with CD28 was unable to enhance Egr-2 nor PLZF expression in vitro suggesting that Ly108 has a unique role in PLZF expression. This work provides a novel mechanism by which innate thymocyte development is regulated via Ly108 and possibly other SLAM family receptor interactions.
Differential education of human fetal natural killer cells provides a mechanism for fetal-maternal tolerance. (P4458)
Authors: Ivarsson Martin , Loh Liyen , Marquardt Nicole , Björkström Niklas , Westgren Magnus , Nixon Douglas , Michaelsson Jakob 
Published: January 2023

Abstract
Little is known about the human fetal immune system, but it has for long been considered as immature and unresponsive. Here we have investigated the development and function of human fetal natural killer (NK) cells. Using advanced flow cytometry, the phenotype and function of fetal NK cells was examined. In contrast to the notion that the fetal immune system is immature, we found that human fetal NK cells differentiate as early as gestational week 15 in utero, evidenced by the presence of NK cells with decrease in CD94/NKG2A and increase in CD16 and KIR expression. NK cells with varying degrees of differentiation were found in the fetal lung, liver, spleen, lymph nodes and bone marrow. Moreover, we show that fetal NK cells are highly responsive to cytokine stimulation and are capable of killing target cells both via natural cytotoxicity and by antibody-dependent cellular cytotoxicity. Strikingly, and in contrast to the education of adult NK cells, we demonstrate that while education via CD94/NKG2A was established in fetal NK cells, both self- and non-self KIR expression rendered fetal NK cells hyporesponsive, thus potentially blunting the response to maternal allogeneic cells. Fetal NK cells were also found to be highly sensitive to TGF-β-mediated inhibition as compared to adult NK cells. Taken together, our data indicate a fundamental difference in the education and regulation of fetal compared to adult NK cells, which have implications for fetal-maternal tolerance in utero.
Critical role of histone deacetylase 11 in lineage differentiation of immune cells (P4378)
Authors: Sahakian Eva , Powers John , Rock-Klotz Jennifer , Deng Susan , Woods David , Woan Karrune , Nguyen Michael , Gabrilovich Dmitry , Sotomayor Eduardo , Pinilla Javier 
Published: January 2023

Abstract
Previously our group reported HDAC11 as a regulator of IL-10 expression in immune cells; however the functions of HDAC11 still remain largely unknown. Here we reveal the novel role of HDAC11 as an epigenetic regulator of lineage differentiation in leukocytes. To “visualize” dynamic changes in HDAC11 transcriptional activation, we utilized HDAC11 promoter-driven eGFP reporter mice, where eGFP expression is indicative of HDAC11 transcription. Using this system, we found that hematopoietic stem cells are devoid of eGFP expression. However, eGFP expression increases from CD4-/CD8- T-cells to single positive CD4+ and CD8+, and decreases in effector compared to naïve T-cells. In the bone marrow, expression of eGFP moderately increases in the transition from Pro- to Pre-B-cells, however in the periphery eGFP significantly increases in the B-1 stage of differentiation. Remarkably, in plasma cells eGFP expression appears to be at its highest. Moreover, less plasma cells and more B-1 cells were observed when HDAC11KO were compared to wild-type mice. Expression of eGFP in myeloblasts appears to be absent, however it increases to 50% in the promyelocytes and to 98% in the granulocytes. Strikingly, monocytes showed no eGFP expression. These results unveiled a previously unknown role of HDAC11 in hematopoiesis, regulating leukocyte lineage differentiation and expansion. These key findings will potentially lead to targeted epigenetic therapies to influence immune cell differentiation.