bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential localization of T-bet and Eomes within distinct human CD8 T-cell memory populations (110.10)
Authors: McLane Laura , Cosma Gabriela , Paley Michael , Wherry E. , Makedonas George , Betts Michael 
Published: January 2023

Abstract
In mice, two T-box transcription factors, T-bet and Eomes, drive the differentiation of CD8 T-cell lineages; however, little is known regarding their role in human CD8 T-cell differentiation. Here, we characterized T-bet and Eomes expression and localization within human CD8 memory T-cell populations. We find T-bet and Eomes are broadly expressed in human memory CD8 T cells, with increasing levels of T-bet and Eomes strongly correlating with differentiation from central memory to effector memory and effector subpopulations. Interestingly, T-bet levels directly correlate to subcellular localization, with a higher propensity for nuclear expression of T-bet within T-bethi cells and predominately cytoplasmic expression in T-betlo cells. In contrast, Eomes appears to be localized solely to the nucleus. In murine CD8 T cells, T-bet can be nuclear, cytoplasmic, or both; however, Eomes is detectable in both compartments suggesting differential regulation compared to humans. Additionally, in humans, Eomes tightly correlated with nuclear T-bet suggesting T-bet localization might impact Eomes expression. Together, our data provide a novel mechanism for CD8 T-cell differentiation in humans and mice based on subcellular localization of T-bet, and reveal the likely presence of previously unknown regulation mechanisms for these critical lineage defining transcription factors.
Defining the early effector CD8 T cell population: plastic cells capable of populating the short-lived and memory pools. (110.11)
Authors: Plumlee Courtney , Obar Joshua , Lefrançois Leo 
Published: January 2023

Abstract
Naïve antigen specific CD8 T cells expand in response to infection and can be phenotypically separated into distinct effector populations at the peak of the response, which include memory precursor effector cells (MPEC) and short-lived effector cells (SLEC). Shortly after infection, a third population called early effector cells (EEC) predominate in the antigen specific response and give rise to both the MPEC and SLEC populations. To understand if EEC populations were pre-committed to either an MPEC or SLEC fate, we purified EEC from mice infected with Listeria monocytogenes (LM) or vesicular stomatitis virus (VSV), in which the ratio of EEC/SLEC/MPEC at the peak of the response is known to be different. Transfers of sorted EEC into uninfected hosts revealed that EEC were pre-programmed to differentiate based on early signals received from the different infection settings. Additionally, gene expression analysis revealed that a subset of genes was differentially regulated between EEC derived from VSV or LM infections. Surprisingly, when these same EEC were transferred early into mismatched infected hosts, the transferred EEC were diverted from their original commitment programming. These results delineate a model of differentiation where EEC are programmed to form MPEC or SLEC, but where additional inflammatory signals can alter this program. Understanding the molecular regulation of the development of an EEC to a memory cell will facilitate rational vaccine design.
The CD8+ T cell response to infection is dominated by the progeny of few naïve T cells (110.12)
Authors: Gerlach Carmen , Rohr Jan , Perié Leila , van Heijst Jeroen , van Rooij Nienke , Velds Arno , Urbanus Jos , Hauptmann Michael , Jacobs Heinz , de Boer Rob , Schumacher Ton 
Published: January 2023

Abstract
Following infection, antigen-specific CD8+ T cell numbers increase dramatically. Analysis of antigen-specific T cell responses at the bulk cell level has provided insight into the average number of cell divisions that T cells undergo upon activation. However, it is unclear whether all naïve T cells of a given affinity for antigen produce equal numbers of progeny, or whether the total effector T cell pool is predominantly created by the output of few. To quantify how many daughter cells individual naïve T cells produce, we have generated naïve OT-I T cells harboring unique genetic tags (barcodes) and have measured the number of progeny per labeled cell by second-generation sequencing of barcode sequences after infection. Our data reveal that upon infection, individual OT-I T cells that are recruited into the response produce highly variable numbers of daughter cells. These data indicate that the bulk of pathogen protection provided by an antigen-specific T cell response is due to the progeny of only a handful of cells.
Dissociating markers of senescence and protective ability in memory T cells (110.16)
Authors: Prlic Martin , Sacks Jilian , Bevan Michael 
Published: January 2023

Abstract
A set of surface markers is commonly used to predict the potential of CD8 effector T cells to differentiate into memory cells. Similarly, these surface markers together with TNF family member CD27 are frequently used to predict a memory T cell’s ability to mount a recall response. Expression of these markers changes every time a memory cell is stimulated and repeated stimulation can lead to T cell senescence and loss of memory T cell responsiveness. This is a concern for prime-boost vaccine strategies which repeatedly stimulate T cells to increase memory T cell frequency. The molecular cues that cause senescence are still unknown, but cell division history is likely to play a major role. We sought to dissect the roles of inflammation and cell division history in developing T cell senescence and their impact on the expression pattern of commonly used markers of senescence. We developed a system that allows priming of CD8 T cells with minimal inflammation and without acquisition of maximal effector function, such as granzyme expression, but a cell division history similar to priming with systemic inflammation. Memory cells derived from minimal effector T cells are fully functional upon rechallenge, have full access to non-lymphoid tissue and appear to be less senescent by phenotype upon rechallenge. However, these currently used biomarkers to measure senescence do not predict proliferative potential or protective ability, but merely reflect initial priming conditions.
B7-H1 limits the entry of effector CD8 T cells to the memory pool by up-regulating Bim (110.2)
Authors: Gibbons Rachel , Liu Xin , Dong Haidong 
Published: January 2023

Abstract
Studies from cancer and chronic infections show that B7-H1 (PD-L1) engagement with its receptor PD-1 enhances apoptosis of effector T cells. It is not clear how B7-H1 regulates T cell apoptosis and the subsequent impact of B7-H1 on T cell memory generation. In immunized B7-H1 deficient mice we observed increased expansion of effector CD8 T cells and delayed T cell contraction followed by protective CD8 T cell memory capable of completely rejecting tumor metastases in lung tissue. Intracellular staining revealed that antigen-primed CD8 T cells in B7-H1 deficient mice express lower levels of the pro-apoptotic molecule Bim. Injection of B7-H1 or PD-1 blocking antibody into immunized mice reduced Bim levels in effector CD8 T cells. In vitro studies show that engagement of activated CD8 T cells by plate-bound B7-H1 fusion protein led to increased cell death and higher levels of Bim expression. Our results suggest that B7-H1 may negatively regulate CD8 T cell memory by enhancing the depletion of effector CD8 T cells through up-regulation of Bim. Our findings would provide a new strategy to target B7-H1 signaling in effector CD8 T cells to achieve protective immunity in the treatment of cancer and infectious diseases.
Differential roles of CD95L and TRAIL in activation-induced cell death of cytotoxic T lymphocytes (110.22)
Authors: Zhang Jimin , Devadas Satish , Shi Yufang 
Published: January 2023

Abstract
Cytotoxic T lymphocytes can be differentiated into type 1 (Tc1) and type 2 (Tc2) subsets as their T helper counterparts. Interestingly, our investigation in activation-induced cell death mechanisms reveals that CD95L mediates cell death in Tc1 lymphocytes and that Tc2 lymphocytes require TRAIL. Cell death in both subsets is also dependent on caspases. Inhibiting CD95L binding to its receptors in Tc1 lymphocytes from wild type mice displays altered Tc1 lymphocyte death, while Inhibiting TRAIL binding to its receptors in Tc2 lymphocytes shows altered Tc2 lymphocyte death. Activation-induced cell death in both Tc1 and Tc2 lymphocyte subsets can be blocked by pan caspase inhibitors. Moreover, Tc2 lymphocytes differentiated from TRAIL knockout mice show alleviated cell death. Rescued Tc2 lymphocytes secrete significantly higher amounts of IL-4, IL-5 and IL-10 while Tc1 lymphocytes produce higher IFN-γ and TNF-α. Our results indicate that CD95L and TRAIL play differential seminal roles in activation-induced cell death of these two cytotoxic T lymphocyte subsets, and that their different sensitivity to activation-induced cell death can alter CD8+ phenotype. With our study we conclude that Tc1 lymphocyte activation-induced cell death is mediated by CD95L while Tc2 lymphocytes undergo TRAIL-dependent activation-induced cell death.
Regulatory T cells are critical for effector-to-memory transition of CD8 T cells (110.24)
Authors: Kalia Vandana , Khan Arif , Penny Laura , Sarkar Surojit 
Published: January 2023

Abstract
Memory differentiation involves a coordinate downregulation of effector functions, and progressive acquisition of memory properties. It is believed that abrogation of antigenic stimulation of T cells following pathogen clearance serves as a passive means for effector CD8 T cells to shut-off their effector functions, and transition into memory. Based on the well-established suppressive functions of Tregs on CD4 T cells, we hypothesized that Tregs aid memory CD8 T cell differentiation by actively suppressing the effector program in memory-fated cells. To test this hypothesis, we evaluated effector-memory transition of Ag-specific cells in the presence or absence of Tregs. Tregs were transiently deleted after Ag clearance using the FOXP3-DTR transgenic mice, following which key memory properties of recall proliferation upon secondary challenge, polyfunctionality and homeostatic proliferation were assessed. Consistent with our hypothesis, these studies revealed that effector program failed to downregulate in the absence of Tregs, and acquisition of all three functional memory properties was significantly hampered in the absence of Tregs. Removal of Tregs even after acquisition of optimal memory properties also led to a decline in memory function. Collectively, these studies present a novel mechanism by which Tregs serve to generate as well as maintain a functional memory CD8 T cell compartment by actively suppressing the effector program following antigen clearance.
Retinoic acid modulation of CD8 effector and memory differentiation. (110.4)
Authors: Allie S. , Zhang Weijun , Tsai Ching-Yi , Lee Yu-Chi , Noelle Randolph , Usherwood Edward 
Published: January 2023

Abstract
A plethora of work implicates important effects of the Vitamin A derivative, retinoic acid (RA), in myeloid differentiation, while fewer studies explore the role of RA on lymphoid cells. Most work has looked at the role of RA on CD4 T cells, focusing on regulatory CD4 T cell differentiation and maintenance of this phenotype along with some Th1 functions. There is little information about the role of RA in CD8 T cell differentiation, and even less on cell-intrinsic effects in the CD8 T cell. In this study we explored the role of RA on effector and memory differentiation in a direct and cell intrinsic manner in the context of vaccinia virus infection. We used dominant negative RA alpha-receptor (RARα DN) expressing mice and bone marrow chimeric mice containing both WT and RARα DN compartments. We observed the loss of the short-lived effector cell phenotype and an enhanced memory precursor phenotype at the effector and contraction phase of the CD8 response in the absence of RA signaling. Further, the lack of RA increased the proportion of central memory CD8s. Vitamin A is used in conjunction with various vaccines (e.g. Measles vaccine), these results will elucidate the effect of RA in shaping the effector and memory response helping to tailor the supplement to better enhance the outcome of vaccination.
In vitro generated human microglia derived from blood precursors (111.14)
Authors: Etemad Samar , Ruitenberg Marc , Filgueira Luis 
Published: January 2023

Abstract
Microglia as the resident innate immune cells of the brain share common characteristics with monocyte-macrophage lineage such as expression of surface markers and chemokine/cytokine receptors. To date, there is no standardized simple model available to investigate the biology of human microglia. The aim of this study is to establish a new in vitro microglia model using blood-derived precursor cells. For that purpose, human peripheral blood monocytes were cultured in serum free medium in the presence of M-CSF, GM-CSF, NGF and CCL2 to generate monocyte-derived microglia (M-MG).M-MG were clearly different in morphology, phenotype and function from freshly isolated monocyte-derived dendritic cells (M-DC).M-MG acquired a ramified morphology with primary and secondary processes .M-MG displayed a comparable phenotype to the human microglia cell line HMC3, expressing very low levels of CD45, CD14 and HLA-DR, CD11b and CD11c; and a distinct pattern of chemokine receptors (CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR3, CX3CR1).In comparison with M-DC, M-MG displayed lower T-lymphocyte stimulatory capacity as well as lower phagocytosis activity.The described protocol for the generation of human monocyte-derived microglia is feasible, well standardized and reliable, as it uses well defined culture medium and recombinant cytokines, but no serum or conditioned medium. This protocol will certainly be very helpful for future studies investigating the biology and pathology of human microglia.
Development of a novel method for in vitro analysis of γc-cytokine effects on CD8 T cells positive selection (111.15)
Authors: Rafei Moutih , Rouette Alexandre , Ruiz Vanegas Juan , Perreault Claude 
Published: January 2023

Abstract
Interleukin (IL)7 is the only γc-cytokine known to support double-positive thymocytes differentiation to the single-positive (SP) stage. To determine if additional γc-cytokines can support T-cell development, we studied their differential effects on positive selection in vitro using a monolayer of OP9 bone marrow-derived stromal cells. We chose the OTI transgenic TCR as our working model due to the previous identification of positively selecting peptides in FTOC assays. In addition to rIL7, rIL4 and rIL13 (but not rIL2, rIL9, rIL15 or rIL21) were able to induce SP CD8 T-cell development. Interestingly, rIL4 induces the de novo expression of EOMES and triggers the development of 2 distinct SP CD8 T cell populations, which we termed CD8int and CD8hi. Flow-cytometry analysis showed that ex vivo generated CD8int, but not CD8hi, expressed high levels of CD69, PD-L1 and CD44 whereas rIL7 or rIL13 treatments did not up regulate these markers. Furthermore, rIL4 has a unique ability to enhance expression of FOXO1, KLF2 and S1PR1. Flow-cytometry analysis of signaling events demonstrated that every γc-cytokine leads to differential activation of AKT, ERK, STAT3, STAT5 and STAT6. Taken together, our findings show that CD8 T-cell positive selection can be studied with an in vitro model that is more convenient than FTOC assays and can be customized to a high-throughput format. Notably, we validate a novel role for rIL4 and rIL13 in supporting positive selection of thymocytes.
A Pre-BCR/NFκB Pathways Suppresses STAT5-dependent Pre-B Cell Leukemia (111.18)
Authors: Katerndahl Casey , Willette Mark , Heltemes-Harris Lynn , Ramsey Laura , Koeuth Thearith , Baechler Emily , Farrar Michael 
Published: January 2023

Abstract
Mice expressing a constitutively active form of STAT5b (called Stat5b-CA mice) develop pre-B leukemia with low frequency (~1-2%). Microarray analysis of these leukemias shows decreased expression of genes involved in pre-BCR signaling. Stat5b-CA mice crossed to mice with defects in a coherent signaling pathway downstream of the pre-BCR involving BLNK, BTK, or PKCβ developed pre-B leukemia in &gt; 75% of progeny. These leukemias exhibited reduced expression of a subset of NFκB target genes, which was important for transformation as Stat5b-CA x Nfkb1-/- mice also developed pre-B leukemia. Our results point to an unexpected role for NFκB1 as a tumor suppressor in pre-B cells. We found that STAT5 cooperates with defects in this pre-BCR-dependent pathway to repress expression of the potential tumor suppressor genes Foxo1, Foxo3a, Ikzf1, and Ikzf3. Our findings demonstrate that STAT5 activation antagonizes a pre-BCR-dependent pathway that limits pre-B cell proliferation, promote pre-B cell differentiation, and inhibit pre-B cell transformation.
Specialized role of thymic transendothelial dendritic cells in antigen acquisition. (111.23)
Authors: Vollmann Elisabeth , von Andrian Ulrich 
Published: January 2023

Abstract
A functional immune system has to protect the body from pathogens while being tolerant to self-antigens (sAg). To achieve this goal, developing thymocytes are subjected to tolerance mechanisms. Central tolerance takes place within the thymus and removes the bulk of auto-reactive thymocytes by either clonal deletion (negative selection) or redirection into a regulatory T cell lineage (Treg). Dendritic cells (DCs) are key players in this process. Thymic DCs can be distinguished and subdivided into distinct subsets according to their surface markers as well as by their origin. Thymus resident DCs differentiate locally from a progenitor cell, while homed DCs enter the thymus fully differentiated from the blood. Here, we report a functional sub-specialization between thymus resident and homed periphery-derived DCs. While the latter can deliver peripheral sAg to the thymus as their cargo, some resident DCs are strategically positioned in the thymus to extend processes across the endothelial barrier of capillaries and are able to acquire, process and present circulating Ag.
Enumeration and characterization of self-reactive T cells in polyclonal populations (111.25)
Authors: Stritesky Gretta , Hogquist Kristin 
Published: January 2023

Abstract
The thymic processes of positive and negative selection are crucial for the formation of a functional T cell repertoire. TCR transgenic models have provided crucial insight into these processes, but it has not been possible to quantitate positive and negative selection in normal diverse polyclonal populations. We developed a Nur77GFP transgenic reporter mouse that specifically expresses GFP in T cells in response to TCR stimulation. Using Nur77GFP mice crossed to mice deficient in the pro-apoptotic molecule Bim, we found that two times more DP thymocytes are negatively selected by high affinity interactions than are positively selected by low affinity interactions. We also identified the fraction of SP thymocytes undergoing further deletion in the medulla, after positive selection. Cells that are rescued from negative selection have increased Nur77GFP compared to foxP3+ T regulatory cells, suggesting that T cells undergoing negative selection have TCRs with higher affinity for self compared to developing T regulatory cells. Finally, our results identify a population of self-reactive CD25-CD44+ CD4 T cells in mesenteric LNs, which have a phenotype consistent with clonal anergy. Future studies using this model will allow us to identify and quantitate selection defects in various mutant or autoimmune prone animals.
Infection-induced dysregulation of the hematopoietic stem cell niche (111.36)
Authors: McCabe Amanda , Zhang Lu , Jones Maura , MacNamara Katherine 
Published: January 2023

Abstract
Hematopoietic stem cells (HSCs) are critical in replenishing all cells of the blood. At homeostasis, HSCs reside in close proximity to bone-lining osteoblasts within the endosteal niche. Osteoblasts produce CXCL12, a chemokine that supports HSC retention in the bone marrow (BM). BM-resident macrophages have recently been shown to retain HSCs in the endosteum through regulation of osteoblast function. How BM-resident macrophages contribute to HSC function during infection has not been addressed. Ehrlichiosis, a tick-born disease, results in anemia and thrombocytopenia, stemming, in part, from alterations in hematopoiesis. Using Ehrlichia muris infection of C57BL/6 mice we observed an IFNγ-dependent increase in F480+Ly6G+CD169+ BM-resident macrophages. These macrophages exhibited increased expression of MHCII and CD80 during infection indicating an activated phenotype. Infection also resulted in reduced expression of osteoblast-specific genes within cells of the endosteum, including osteocalcin and Cxcl12. By histology, we observed a diminished osteoblast lining. Furthermore, HSCs were mobilized into the bloodstream, indicating a dysregulation of the endosteal HSC niche. Our current hypothesis is that during ehrlichial infection, expansion and activation of resident macrophages results in altered osteoblast function and subsequent HSC mobilization from the BM. Future experiments will address mechanisms of macrophage-mediated alterations to the HSC niche during infection.
Development of CD4+ PLZF+ T Cells in the Absence of Itk (111.41)
Authors: Prince Amanda , Evans John , Berg Leslie 
Published: January 2023

Abstract
Innate-like lymphocytes develop in the thymus and share characteristics of the innate and adaptive immune systems. These lymphocytes undergo antigen receptor rearrangement and are able to exert their effector function immediately upon ex vivo stimulation. Our lab is interested in how TCR signaling affects the development of conventional versus innate-like lymphocytes. Itk is a nonreceptor protein tyrosine kinase that signals downstream of the TCR. Previously, our lab has shown that in the absence of Itk, CD8+ Eomesodermin+ T cells develop that have a diverse TCR repertoire and produce IFNγ immediately upon ex vivo stimulation. Recently, several labs have demonstrated that the development of these CD8+ Eomesodermin+ T cells is dependent upon CD4+ PLZF+ thymocytes producing IL-4. We have determined that there are two CD4+ PLZF+ populations consisting of αβ and γδ T cells in Itk-/- thymi. In the absence of γδ T cells, Itk-/- CD4+ PLZF+ αβ T cells retain the ability to induce Eomesodermin expression in CD8+ T cells. Furthermore, Itk-/- CD4+ PLZF+ αβ T cells do not bind CD1d tetramer and develop independently of IL-15, class II MHC, and β2m. We have also found that Itk-/- CD4+ PLZF+ αβ T cells are found in the spleen and mesenteric lymph node in the periphery and have a restricted TCR repertoire. Thus, in the absence of Itk, a non-canonical innate-like population of CD4+ PLZF+ αβ T cells develop that influences the development of conventional T cells.