Lymphocyte Lineage Research Articles

Single-cell sequencing analysis of multiple myeloma heterogeneity and identification of new theranostic targets

Multiple myeloma (MM) is a heterogeneous and incurable tumor characterized by the malignant proliferation of plasma cells. It is necessary to clarify the heterogeneity of MM and identify new theranostic targets. We constructed a single-cell transcriptome profile of 48,293 bone marrow cells from MM patients and health donors (HDs) annotated with 7 continuous B lymphocyte lineages. Through CellChat, we discovered that the communication among B lymphocyte lineages between MM and HDs was disrupted, and unique signaling molecules were observed. Through pseudotime analysis, it was found that the differences between MM and HDs were mainly reflected in plasma cells. These differences are primarily related to various biological processes involving mitochondria. Then, we identified the key subpopulation associated with the malignant proliferation of plasma cells. This group of cells exhibited strong proliferation ability, high CNV scores, high expression of frequently mutated genes, and strong glucose metabolic activity. Furthermore, we demonstrated the therapeutic potential of WNK1 as a target. Our study provides new insights into the development of B cells and the heterogeneity of plasma cells in MM and suggests that WNK1 is a potential therapeutic target for MM.

Discovery of an unconventional lamprey lymphocyte lineage highlights divergent features in vertebrate adaptive immune system evolution

Lymphocyte receptors independently evolved in both jawed and jawless vertebrates with similar adaptive immune responses. However, the diversity of functional subtypes and molecular architecture in jawless vertebrate lymphocytes, comparable to jawed species, is not well defined. Here, we profile the gills, intestines, and blood of the lamprey, Lampetra morii, with single-cell RNA sequencing, using a full-length transcriptome as a reference. Our findings reveal higher tissue-specific heterogeneity among T-like cells in contrast to B-like cells. Notably, we identify a unique T-like cell subtype expressing a homolog of the nonlymphoid hematopoietic growth factor receptor, MPL-like (MPL-L). These MPL-L+ T-like cells exhibit features distinct from T cells of jawed vertebrates, particularly in their elevated expression of hematopoietic genes. We further discovered that MPL-L+ VLRA+ T-like cells are widely present in the typhlosole, gill, liver, kidney, and skin of lamprey and they proliferate in response to both a T cell mitogen and recombinant human thrombopoietin. These findings provide new insights into the adaptive immune response in jawless vertebrates, shedding new light on the evolution of adaptive immunity.

BCL6 is required for the thymic development of TCRαβ+CD8αα+ intraepithelial lymphocyte lineage.

TCRαβ+CD8αα+ intraepithelial lymphocytes (CD8αα+ αβ IELs) are a specialized subset of T cells in the gut epithelium that develop from thymic agonist selected IEL precursors (IELps). The molecular mechanisms underlying the selection and differentiation of this T cell type in the thymus are largely unknown. Here, we found that Bcl6 deficiency in αβ T cells resulted in the near absence of CD8αα+ αβ IELs. BCL6 was expressed by approximately 50% of CD8αα+ αβ IELs and by the majority of thymic PD1+ IELps after agonist selection. Bcl6 deficiency blocked early IELp generation in the thymus, and its expression in IELps was induced by thymic TCR signaling in an ERK-dependent manner. As a result of Bcl6 deficiency, the precursors of IELps among CD4+CD8+ double-positive thymocytes exhibited increased apoptosis during agonist selection and impaired IELp differentiation and maturation. Together, our results elucidate BCL6 as a crucial transcription factor during the thymic development of CD8αα+ αβ IELs.

Investigate the impact of laser dosages on the regeneration of peripheral blood leukocytes after gamma radiation exposure

This research aims to examine how treatment with lasers affects the regeneration of peripheral blood leukocytes following ionizing radiation exposure. Furthermore, how may laser treatment be administered to people who have inadvertently come into contact with ionizing radiation? After being subjected to one dose of five Gy, non-lethal (1.5 Gy/min) from gamma radiation (cobalt-60), the mice developed radiation-induced sickness, as evidenced by a drop in white blood cell count and hematological symptoms. The mice were subsequently subjected to non-ionizing radiation using a 20 W power laser (Nd: YAG 1064 nm). Within the first two weeks after the application of gamma rays for 30 min, there were a total of five laser treatments. Laser energy yielded a total of 60,000 J/cm2. The energy density in each treatment was 12,000 J/cm2. Based on the peripheral blood examination, the study establishes that the use of lasers had an important influence on the number of lymphocytes two weeks after irradiation. Furthermore, following laser treatment, the lymphocyte lineage of white blood cells recovered more quickly. In gamma-irradiated mice, the study showed that the treatment laser under examination increased the overall counts of several types of white blood cells, indicating an indefinite effect. The investigation's findings may have an impact on how radiation illness is treated for specific patients. The results of this investigation may have a substantial influence on the treatment of radiation sickness in people.

John Joseph Thomas Hayes Owen. 7 January 1934—5 January 2023

John Owen was an anatomist and immunologist who conducted pioneering work on the origins and development of the two major classes of lymphocytes, the cells that are the central players in the adaptive immune systems of vertebrates. He was a meticulous biological experimenter who liked to keep questions simple and precise. His observations contributed to two key discoveries: that mouse and avian stem cells migrate into the embryonic thymus and give rise to T lymphocytes; and that in birds the bursa of Fabricius is the tissue in which stem cells develop into B lymphocytes. He also helped to establish that in mammals the earliest B lymphocytes develop in the liver of the fetus. He and his colleagues went on to define how combinations of several cell populations in the thymus provide the settings in which mammals first generate and then select from the variety of T lymphocyte lineages and immune specificities that will equip them with healthy immune surveillance and responses. His wry Liverpudlian sense of humour let him engage easily with everyone, from any walk of life, and he was an inspiration to young colleagues, many of whom would go on to flourish in the research areas that he pioneered. John died on 5 January 2023, from prostate cancer and other ailments: but he was in the gym only a couple of weeks before he died. He is buried in Kirkdale cemetery, Lancashire, under a family tombstone that also celebrates his grandfather, his father and his mother: his epitaph reads ‘One push-up too many’.

Clinical and genetic analysis of essential hypertension with mitochondrial tRNAMet 4435A>G and YARS2 mutation.

To investigate the role of m.4435A>G and YARS2 c.572G>T (p.G191V) mutations in the development of essential hypertension. A hypertensive patient with m.4435A>G and YARS2 p.G191V mutations was identified from previously collected mitochondrial genome and exon sequencing data. Clinical data were collected, and a molecular genetic study was conducted in the proband and his family members. Peripheral venous blood was collected, and immortalized lymphocyte lines constructed. The mitochondrial transfer RNA (tRNA), mitochondrial protein, adenosine triphosphate (ATP), mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) in the constructed lymphocyte cell lines were measured. Mitochondrial genome sequencing showed that all maternal members carried a highly conserved m.4435A>G mutation. The m.4435A>G mutation might affect the secondary structure and folding free energy of mitochondrial tRNA and change its stability, which may influence the anticodon ring structure. Compared with the control group, the cell lines carrying m.4435A>G and YARS2 p.G191V mutations had decreased mitochondrial tRNA homeostasis, mitochondrial protein expression, ATP production and MMP levels, as well as increased ROS levels (all P<0.05). The YARS2 p.G191V mutation aggravates the changes in mitochondrial translation and mitochondrial function caused by m.4435A>G through affecting the steady-state level of mitochondrial tRNA and further leads to cell dysfunction, indicating that YARS2 p.G191V and m.4435A>G mutations have a synergistic effect in this family and jointly participate in the occurrence and development of essential hypertension.

Against all odds: The road to success in the development of human immune reconstitution mice.

The mouse genome has a high degree of homology with the human genome, and its physiological, biochemical, and developmental regulation mechanisms are similar to those of humans; therefore, mice are widely used as experimental animals. However, it is undeniable that interspecies differences between humans and mice can lead to experimental errors. The differences in the immune system have become an important factor limiting current immunological research. The application of immunodeficient mice provides a possible solution to these problems. By transplanting human immune cells or tissues, such as peripheral blood mononuclear cells or hematopoietic stem cells, into immunodeficient mice, a human immune system can be reconstituted in the mouse body, and the engrafted immune cells can elicit human-specific immune responses. Researchers have been actively exploring the development and differentiation conditions of host recipient animals and grafts in order to achieve better immune reconstitution. Through genetic engineering methods, immunodeficient mice can be further modified to provide a favorable developmental and differentiation microenvironment for the grafts. From initially only being able to reconstruct single T lymphocyte lineages, it is now possible to reconstruct lymphoid and myeloid cells, providing important research tools for immunology-related studies. In this review, we compare the differences in immune systems of humans and mice, describe the development history of human immune reconstitution from the perspectives of immunodeficient mice and grafts, and discuss the latest advances in enhancing the efficiency of human immune cell reconstitution, aiming to provide important references for immunological related researches.

Radioreparative Effect of Diode Laser on Leukopoiesis Recovery: A Pilot Study.

The aim of the present study was to investigate the effect of laser therapy on leukopoiesis recovery after irradiation with ionizing radiation. A dose of ionizing radiation was used that induced the hematological form of radiation sickness, reducing the number of blood cells. Subsequently, mice were treated with non-ionizing laser radiation. Based on the examination of the peripheral blood, the study found that laser therapy significantly impacted the number of eosinophils and basophils two weeks after irradiation. Laser therapy also led to the faster reparation of the lymphocyte lineage of white blood cells (WBCs). The research showed that the examined therapeutic laser had a long-term radioreparative effect on gamma-irradiated mice, improving the absolute counts of different lines of WBCs. The results of this study could have implications for the treatment of radiation sickness in humans.

Maintenance of caecal homeostasis by diverse adaptive immune cells in the rhesus macaque.

The caecum bridges the small and large intestine and plays a front-line role in discriminating gastrointestinal antigens. Although dysregulated in acute and chronic conditions, the tissue is often overlooked immunologically. To address this issue, we applied single-cell transcriptomic-V(D)J sequencing to FACS-isolated CD45+ caecal patch/lamina propria leukocytes from a healthy (5-year-old) female rhesus macaque ex vivo and coupled these data to VDJ deep sequencing reads from haematopoietic tissues. We found caecal NK cells and ILC3s to co-exist with a spectrum of effector Tcells partially derived from SOX4 + recent thymic emigrants. Tolerogenic Vγ8Vδ1-T cells, plastic CD4+ T helper cells and GZMK + EOMES + and TMIGD2 + tissue-resident memory CD8+ Tcells were present and differed metabolically. An IL13 + GATA3 + Th2 subset expressing eicosanoid pathway enzymes was accompanied by IL1RL1 + GATA3 + regulatory Tcells and a minorproportion of IgE+ plasma cells (PCs), illustrating tightly regulated type 2 immunity devoid of ILC2s. In terms of B lymphocyte lineages, caecal patch antigen-presenting memory B cells sat alongside germinal centre cells undergoing somatic hypermutation and differentiation into IGF1 + PCs. Prototypic geneexpression signatures decreased across PC clusters, and notably, expanded IgA clonotypes could be traced in VDJ deep sequencing reads from additional compartments, including the bone marrow, supporting that these cells contribute a steady stream of systemic antibodies. The data advance our understanding of caecal immunological function, revealing processes involved in barrier maintenance and molecular networks relevant to disease.

The mechanism of action of anti-CD20 monoclonal antibodies used in the treatment of multiple sclerosis

CD20 is a transmembrane molecule located on the surface of a major population of cells belonging to the B lymphocyte lineage. Although the significance of this protein remains unknown, the presence of CD20 is a marker for mature circulating B cells. Monoclonal antibodies against CD20 are able to quickly and effectively eliminate circulating B lymphocytes. Four different anti-CD20 antibodies have been studied in the treatment of multiple sclerosis: rituximab, ocrelizumab, ofatumumab and ublituximab. Although all these monoclonal antibodies bind to the same protein, they have different molecular and pharmacological characteristics. One of the important differences between these molecules is the degree of humanisation of their molecular structure, as well as the mechanism of B cell removal. Phase III multicentre clinical trials conducted with each of these antibodies consistently confirmed that the elimination of B cells is an effective method of slowing down the progression of multiple sclerosis. Thus, B cells are considered as main group of immune cells involved in the development and course of multiple sclerosis. Numerous in vitro and in vivo data confirm the involvement of these cells in the pathogenesis of multiple sclerosis, despite the lack of evidence for the presence of pathogenic autoantibodies in this disease. Nevertheless, B cells represent a mixture of cell populations with different immunomodulatory properties. Therefore, future multiple sclerosis therapies should target the pathogenic groups of B lymphocytes, rather than non-selectively eliminating the entire population of these cells.

Ефективність вирощування молоді осетрових (Acipenseridae) видів риб за різної твердості води

Ефективність вирощування молоді осетрових (Acipenseridae) видів риб за різної твердості води

Resveratrol alleviates imidacloprid-induced mitochondrial apoptosis, necroptosis, and immune dysfunction in chicken lymphocyte lines by inhibiting the ROS/MAPK signaling pathway.

Imidacloprid (IMI) is a neonicotinoid insecticide with the highest global market share, and IMI exposure in the environment can negatively affect many nontarget organisms (a general term for organisms affected by drugs other than target organisms). Resveratrol (RSV), a non-flavonoid polyphenolic organic compound derived from peanuts, grapes, and other plants, has anti-inflammatory and antioxidant effects. It is currently unclear how RSV protects against cell damage caused by IMI. Therefore, we established an experimental model of chicken lymphocyte lines exposed to 110 μg/mL IMI and/or 0.5 μM RSV for 24 h. According to the experimental results, IMI markedly raised intracellular reactive oxygen species levels and diminished the activity of the cellular antioxidant enzymes (CAT, SOD, and GPx), leading to MDA accumulation and decreased T-AOC. JNK, ERK, and P38, the essential components of the mitogen-activated protein kinase (MAPK) signaling pathway, were also expressed more when IMI was present. Additionally, IMI resulted in upregulation of mitochondrial apoptosis (Caspase 3, Caspase 9, Bax, and Cyt-c) and necroptosis (Caspase 8, RIPK1, RIPK3, and MLKL) related factors expression, downregulation of Bcl-2 expression, induction of upregulation of cytokine IL-6 and TNF-α expression, and downregulation of IFN-γ expression. The combined treatment of RSV and IMI significantly reduced cellular oxidative stress levels, inhibited the MAPK signaling pathway, and alleviated IMI-induced mitochondrial apoptosis, necroptosis, and immune dysfunction. To summarize, RSV antagonized IMI-induced mitochondrial apoptosis, necroptosis, and immune dysfunction in chicken lymphocyte lines by inhibiting the ROS/MAPK signaling pathway.

Single-Cell Revelation of Extensive Heterogeneity in the Human Lymphocyte and Neutrophil/Monocyte Lineage Restriction Process Despite Highly Co-Regulated Changes in Cell Cycle Control

The production of mature blood lineages in humans has been viewed as a hierarchical process in which the primitive cells with multi-lineage potential generate a series of progenitors with increasingly reduced self-renewal abilities and restricted lineage potentials. This concept has been largely supported by phenotypic characterization of starting CD34+ subsets of human CD45+ cells isolated from different hematopoietic tissues and assayed for their mature cell outputs in supportive systems in vitro or post-transplant and more recently by tracking changes in their molecular composition. Such experiments have suggested that cells that give rise to the lymphocyte lineages (L) share a common origin almost exclusively with the neutrophil and monocyte (macrophage) lineages (NM). However, the mechanisms that regulate the subsequent separation of these lineages starting from cells with presumed restricted L+NM potential have remained elusive. To address this question, we first developed a culture system that supports outputs of differentiated B (CD19+) and NM (CD15+/CD14+) from ≥50% of single CD34+ cells plated on a stromal layer of MS-5 feeders in a medium containing human IL-7, SCF and FLT3L. Use of this clonal assay revealed that cells with B+NM bi-potential constitute 20% of the CD45RA-CLEC12A- (RA-C-) cell subset of the CB “P-Mix” phenotype (CD34+CD38medCD71-CD10-, Blood 133:927, 2019); with the CD45RA+CLEC12A- (RA+C-) and CLEC12A+ (C+) cells displaying largely restricted outputs of only B- (30%) or only NM- (50%) progeny phenotypes, respectively. These same 3 “progenitor” phenotypes (RA-C-, RA+C-, C+) were also found to be regenerated in xenografted immunodeficient mice as well as in a further optimized in vitro system and their clonal output types and numbers consistent with the outputs of those isolated from fresh CB. To analyze T-cell lineage (T) restriction in parallel, we then further modified this culture system to include adherent DLL4, that we found allows B, T, N and M progeny to be produced from single input CB cells at a 40-60% efficiency, with numerous clones derived from single RA+C- cells containing B+T+M, or B+M, or B+T or T-only; a few with B- or M- only, and none with N. In contrast, under the same conditions, half of the documented C+ outputs were exclusively N with much lower lymphoid outputs of T-only or T+N/M. Together, these findings reveal the unanticipated importance of the external stimuli in revealing lineage potentials when these are undergoing restrictive changes. We then asked if and how these identified early phenotypes might change their cycling properties during the course of their progression. For this, the rate and progeny content of sequential cell divisions during the stimulated expansion of the CFSE labelled RA-C- input cells were measured under a supportive liquid culture condition. Throughout a 6-day period, almost all of the C+ progeny and a slightly lower proportion of the RA+C- outputs were generated from cells that transit through the cell cycle within 24 hours, whereas cells maintaining an undifferentiated RA-C- phenotype divided at significantly slower rates, suggesting a simultaneously initiated shortening of the cell cycle transit time during both L- and NM- restriction. In agreement with these functional results, a recent single-cell CITE-seq dataset generated in parallel showed higher expression of genes involved in cycling activities in the early L- and NM-restricted subsets as compared to the unrestricted RA-C- cells. Taken together, these results reveal important and disconnected features in the cell cycle control and previously unrecognized complex patterns in the biological outputs and molecular changes during the steps of L and NM restriction. In addition, they set the stage to enable preferred sequences and molecular changes responsible for normal and regenerative needs to be identified and their potential relevance to the formation of human lymphoblastic and myeloid leukemias to be characterized.

Polycomb Group Protein Yin Yang 1 Controls DNA Methylation in Murine T Lymphocyte Development

Transcription factors and epigenetic regulators play key functions in T lymphocyte lineage commitment and development. Mammalian PcG proteins are major negative regulators of gene expression that typically bind as large complexes (such as Polycomb Repressive Complex 1 (PRC1) and PRC2) to gene regulatory regions selectively targeted by an as yet unknown mechanism. While nearly all mammalian PcG proteins are lack of specific DNA binding capacities, transcription factor Yin Yang 1 (YY1) can bind at the DNA sites specifically and recruit other PcG proteins to specific DNA sequences. A small 25 amino acid YY1 domain (the REPO domain) is necessary and sufficient for the recruitment of PcG proteins, PcG-mediated stable transcriptional repression and histone modification. While YY1ΔREPO is competent for DNA binding, transcriptional activation, transient transcriptional repression and interaction with transcriptional coregulators such as HDACs, YY1ΔREPO is defective in all YY1 PcG functions. Our prior study demonstrated that deletion of YY1 in the hematopoietic system leads to T cell development blockage at the double-negative 3 (DN3) stage. Interestingly, ectopic expression of YY1∆REPO in YY1 null background by retroviral bone marrow transplantation could not rescue YY1 deficiency mediated T cell survival defect. This suggests that the YY1 REPO domain/PcG function plays a vital role in T cell development. Herein, we generated the YY1 REPO domain conditional knockout mouse model ( Yy1 f/∆REPO Vav-Cre) by CRISPR/Cas9 to further dissect the YY1 REPO domain/PcG function in T cell development. Yy1 f/∆REPO Vav-Cre ( Yy1 -/∆REPO) mice have reduced thymus weight and cellularity with medulla aplasia. Compared with wild-type (WT) and YY1 heterozygous ( Yy1 +/-) mice, Yy1 -/∆REPO mice have an increased percentage of DN3 cells with decreased DN4 percentage supporting a developmental blockage at the DN3 stage. Yy1 -/∆REPO DN T cells fail to go through TCRβ rearrangement with increased apoptosis and reduction of TCRβ + cells in the thymus compared with WT and Yy1 +/-cells. Interestingly, RNA-seq analysis in sorted primary DN3 cells revealed the genetic network governing DNA methylation, histone methylation, chromosome maintenance, and translation initiation complex formation are deregulated in Yy1 -/∆REPO mice compared to WT and Yy1 +/-mice. DN T cell development requires precise and dynamic regulation of histone and DNA methylation and demethylation. Interestingly, the DNA demethylase enzymes ten-eleven translocation (TET) family proteins Tet1 and Tet2 are downregulated in Yy1 -/∆REPO DN3 thymocytes compared with WT and Yy1 +/- DN3 cells. Our data show that the YY1 REPO domain/PcG function is required for DN3-DN4 transition via regulating TCRβ rearrangement and the YY1 REPO domain/PcG function plays a critical role in proper DNA methylation status in early T cell development. As PcG proteins modify histone via trimethylation of histone H3 on lysine 27, we will further assess the impact of YY1 REPO domain/PcG function on histone methylation in T cell development. By utilizing a powerful mouse model for identifying YY1 functions as PcG domain-dependent or PcG domain-independent, our study has generated fundamental new insights into the epigenetic landscape in T cell development.

Lymphocyte subset phenotyping for the prediction of progression to inflammatory arthritis in anti-citrullinated-peptide antibody-positive at-risk individuals.

Inflammatory arthritis (IA) is considered the last stage of a disease continuum, where features of systemic autoimmunity can appear years before clinical synovitis. Time to progression to IA varies considerably between at-risk individuals, therefore the identification of biomarkers predictive of progression is of major importance. We previously reported on the value of three CD4+T cell subsets as biomarkers of progression. Here, we aim to establish the value of 18 lymphocyte subsets (LS) for predicting progression to IA. Participants were recruited based on a new musculoskeletal complaint and being positive for anti-citrullinated-peptide antibody. Progression (over 10 years) was defined as the development of clinical synovitis. LS analysis was performed for lymphocyte lineages, naive/memory subsets, inflammation-related cells (IRC) and regulatory cells (Treg/B-reg). Modelling used logistic/Cox regressions. Of 210 patients included, 93 (44%) progressed to IA, 41/93 (44%) within 12 months (rapid progressors). A total of 5/18 LS were associated with progression [Treg/CD4-naïve/IRC (adjusted P < 0.0001), CD8 (P = 0.021), B-reg (P = 0.015)] and three trends (NK-cells/memory-B-cells/plasmablasts). Unsupervised hierarchical clustering using these eight subsets segregated three clusters of patients, one cluster being enriched [63/109(58%)] and one poor [10/45(22%)] in progressors. Combining all clinical and LS variables, forward logistic regression predicted progression with accuracy = 85.7% and AUC = 0.911, selecting smoking/rheumatoid-factor/HLA-shared-epitope/tender-joint-count-78 and Treg/CD4-naive/CD8/NK-cells/B-reg/plasmablasts. To predict rapid progression, a Cox regression was performed resulting in a model combining smoking/rheumatoid factor and IRC/CD4-naive/Treg/NK-cells/CD8+T cells (AUC = 0.794). Overall, progression was predicted by specific LS, suggesting potential triggers for events leading to the development of IA, while rapid progression was associated with a different set of subsets.

Lymphocyte pathway analysis using naturally lymphocyte-deficient fish.

Comparative phylogenetic analyses are of potential value to establish the essential components of genetic networks underlying physiological traits. For species that naturally lack particular lymphocyte lineages, we show here that this strategy readily distinguishes trait-specific actors from pleiotropic components of the genetic network governing lymphocyte differentiation. Previously, three of the four members of the DNA polymerase X family have been implicated in the junctional diversification process during the somatic assembly of antigen receptors. Our phylogenetic analysis indicates that the presence of terminal deoxynucleotidyl transferase is strictly associated with the facility of V(D)J recombination, whereas PolL and PolM genes are retained even in species lacking Rag-mediated somatic diversification of antigen receptor genes.

NK cells in peripheral blood carry trogocytosed tumor antigens from solid cancer cells.

The innate immune lymphocyte lineage natural killer (NK) cell infiltrates tumor environment where it can recognize and eliminate tumor cells. NK cell tumor infiltration is linked to patient prognosis. However, it is unknown if some of these antitumor NK cells leave the tumor environment. In blood-borne cancers, NK cells that have interacted with leukemic cells are recognized by the co-expression of two CD45 isoforms (CD45RARO cells) and/or the plasma membrane presence of tumor antigens (Ag), which NK cells acquire by trogocytosis. We evaluated solid tumor Ag uptake by trogocytosis on NK cells by performing co-cultures in vitro. We analyzed NK population subsets by unsupervised dimensional reduction techniques in blood samples from breast tumor (BC) patients and healthy donors (HD). We confirmed that NK cells perform trogocytosis from solid cancer cells in vitro. The extent of trogocytosis depends on the target cell and the antigen, but not on the amount of Ag expressed by the target cell or the sensitivity to NK cell killing. We identified by FlowSOM (Self-Organizing Maps) several NK cell clusters differentially abundant between BC patients and HD, including anti-tumor NK subsets with phenotype CD45RARO+CD107a+. These analyses showed that bona-fide NK cells that have degranulated were increased in patients and, additionally, these NK cells exhibit trogocytosis of solid tumor Ag on their surface. However, the frequency of NK cells that have trogocytosed is very low and much lower than that found in hematological cancer patients, suggesting that the number of NK cells that exit the tumor environment is scarce. To our knowledge, this is the first report describing the presence of solid tumor markers on circulating NK subsets from breast tumor patients. This NK cell immune profiling could lead to generate novel strategies to complement established therapies for BC patients or to the use of peripheral blood NK cells in the theranostic of solid cancer patients after treatment.

Jiedu-Quyu-Ziyin Fang (JQZF) inhibits the proliferation and activation of B cells in MRL/lpr mice via modulating the AKT/mTOR/c-Myc signaling pathway

Ethnopharmacological relevanceJiedu-Quyu-Ziyin Fang (JQZF) is a new herbal formula improved based on “Sheng Ma Bie Jia Tang” in the Golden Chamber, has been proved to be effective in the treatment of SLE. The ability of JQZF to prevent lymphocyte growth and survival has been demonstrated in earlier investigations. However, the specific mechanism of JQZF on SLE has not been fully investigated. Aim of the studyTo reveal the potential mechanisms of JQZF inhibiting B cell proliferation and activation in MRL/lpr mice. Materials and methodsMRL/lpr mice were treated with low-dose, high-dose JQZF and normal saline for 6 weeks. The effect of JQZF on disease improvement in MRL/lpr mice was studied using enzyme-linked immunosorbent assay (ELISA), histopathological staining, serum biochemical parameters and urinary protein levels. The changes of B lymphocyte subsets in the spleen were analyzed by flow cytometry. The contents of ATP and PA in B lymphocytes from the spleens of mice were determined by ATP content assay kit and PA assay kit. Raji cells (a B lymphocyte line) were selected as the cell model in vitro. The effects of JQZF on the proliferation and apoptosis of B cells were detected by flow cytometry and CCK8. The effect of JQZF on the AKT/mTOR/c-Myc signaling pathway in B cells were detected via western blot. ResultsJQZF, especially at high dose, significantly improved the disease development of MRL/lpr mice. Flow cytometry results showed that JQZF affected the proliferation and activation of B cells. In addition, JQZF inhibited the production of ATP and PA in B lymphocytes. In vitro cell experiments further confirmed that JQZF can inhibit Raji proliferation and promote cell apoptosis through AKT/mTOR/c-Myc signaling pathway. ConclusionJQZF may affect the proliferation and activation of B cells by inhibiting the AKT/mTOR/c-Myc signaling pathway.

PAXBP1 SERVES AS A NOVEL REGULATOR OF T HELPER SPECIFICATION BY RESTRAINING THE TYPE 1 PROGRAM

Abstract To properly extinguish a diverse number of pathogens naïve T cells speciate into several T helper subsets in a highly regulated manner. In order to identify novel regulators of T helper specification we explored single cell RNA-seq datasets of T cells exposed to several different challenges. From this analysis, we found Paxbp1, a poorly described nuclear factor, was highly expressed in Th2 cells with its expression correlating well with Gata3. To understand the role Paxbp1 plays in T helper differentiation we generated a mouse model where Paxbp1 was deleted in T cells using a CD4cre model. Analysis from scRNA-seq reveal that Paxbp1 deficiency leads to a decrease in mature naïve T cells in both the thymus and periphery. Moreover, loss of Paxbp1 results in an aberrant expression of a Type I program, despite being exposed to a Type 2 environment in a reductionist approach; specifically, Paxbp1 deficiency results in the expression of what appears to be a terminal cytotoxic program. During in vivo challenges, T cells from Paxbp1 deficient animals recapitulated these results showing an increase in expression of the Type 1 program. Current work is aimed at determining the function of Paxbp1 by identifying binding partners through mass spectrometry. Additionally, we will use scATAC-seq to define transcription factors with differential activity in Paxbp1 deficient T cells. Altogether, our data suggests that Paxbp1 may be a previously unrecognized player in T helper specification and development that constrains inappropriate expression and accessibility of programs employed by alternative lymphocyte lineages.

Sensing and alarm function of vaccine-elicited SIV-gag specific CD8 TRM in the reproductive mucosa of rhesus macaques

Abstract Resident memory T cells (TRM) constitute a recently identified lymphocyte lineage that occupies non-lymphoid tissues (NLT) without recirculating. In murine models, upon antigenic rechallenge, TRM trigger antiviral responses in neighboring innate and adaptive immune cells, and recruit immune effectors from circulation. Collectively this is referred to as a ‘sensing and alarm’ function. However, the full range of TRM functions has not been assessed, and non-human primate/human TRM function remains almost entirely uncharacterized. Here we established a prime-boost vaccine modality in rhesus macaques to generate abundant SIV-gag-specific CD8 TRM in the female reproductive tract (FRT) and 14 other NLTs. Upon vaginal challenge with CM9 peptide from the SIV gag protein, CD8 TRM reactivation induced expression of CD69 and granzyme B in SIV-gag specific CD8 T cells throughout the rhesus FRT, in situ proliferation (Ki67 expression), and rapid antiviral and IFN response gene expression in essentially all hematopoietic and non-hematopoietic cells. Upregulation of effector genes in CD8 T cells, CD4 T cells, NK cells, and ILCs peaked at 24h and persisted at 48h. CITE-Seq analysis revealed that mucosal CD4 T cells expressed antiviral genes including HIV restriction factors and had reduced expression of the HIV-co-receptor CCR5. Increased numbers of vaginal T cells, memory B cells, and plasma cells at 24h coincided with increased expression of chemokines and VCAM-1 on endothelial and stromal cells and a concomitant reduction in circulating T and B cells. These data demonstrate that CD8 TRM trigger local activation and the mobilization of innate, cellular, and humoral immune responses to the site of antigen exposure in non-human primates.