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Metazoan cell death mechanisms are diverse and include numerous non-apoptotic programs. One program called entosis involves the invasion of live cells into their neighbors and is known to occur in cancers. Here, we identify a developmental function for entosis: to clear the male-specific linker cell in C. elegans. The linker cell leads migration to shape the gonad and is removed to facilitate fusion of the gonad to the cloaca. We find that the linker cell is cleared in a manner involving cell-cell adhesions and cell-autonomous control of uptake through linker cell actin. Linker cell entosis generates a lobe structure that is deposited at the site of gonad-to-cloaca fusion and is removed during mating. Inhibition of lobe scission inhibits linker cell death, demonstrating that the linker cell invades its host while alive. Our findings demonstrate a developmental function for entosis: to eliminate a migrating cell and facilitate gonad-to-cloaca fusion, which is required for fertility.

The origin of the autophagosomal membrane started to be debated by scientists working in the field within one year of the modern definition of autophagy in 1963. There is now converging evidence from older and newer studies that the endoplasmic reticulum is involved in formation of autophagosomes. Thus, it is possible to trace from early morphological work - done without the benefit of molecular descriptions - to recent studies - dissecting how specific proteins nucleate autophagosome biogenesis - a long series of experimental findings that are beginning to answer the 55-year old question with some confidence. The view that has emerged is that specialised regions of the endoplasmic reticulum, in dynamic cross talk with most intracellular organelles via membrane contact sites, provide a platform for autophagosome biogenesis.

Nemiralisib (GSK2269557) is a potent inhaled inhibitor of phosphoinositide 3-kinase delta (PI3Kδ) which is being developed for the treatment of respiratory disorders including COPD (Chronic Obstructive Pulmonary Disease). Determining the pharmacokinetic (PK) and pharmacodynamic (PD) responses of inhaled drugs early during drug development is key to informing the appropriate dose and preferred dose regimen in patients. We set out to measure PD changes in induced sputum in combination with drug concentrations in plasma and bronchoalveolar lavage (BAL) taken from healthy smokers (n=56) treated for up to 14 days with increasing doses of inhaled nemiralisib (0.1 mg to 6.4 mg). Induced sputum analysis demonstrated a dose-dependent reduction in phosphatidylinositol-trisphosphate (PIP3, the product of PI3K activation), with a maximum placebo-corrected reduction of 23% (90% CI 11-34%) and 36% (90% CI 11-64%) following single dose or 14 days of treatment with nemiralisib respectively (2 mg, once daily). Plasma analysis suggested a linear PK relationship with an observed accumulation of ~3-4.5-fold (peak vs. trough) in plasma exposure following 14 days of nemiralisib treatment. BAL analysis at trough confirmed higher levels of drug in lung vs. plasma (32-fold in the BAL fluid component, and 214-fold in the BAL cellular fraction). Comparison of drug levels in plasma and reductions in sputum PIP3 show a direct relationship between exposure and PIP3 reduction. In conclusion, these results demonstrate target engagement upon treatment with inhaled nemiralisib and provide confidence for a once-daily dosing regimen.

Juvenile idiopathic arthritis (JIA) is the most common class of childhood rheumatic diseases, with distinct disease subsets that may have diverging pathophysiological origins. Both adaptive and innate immune processes have been proposed as primary drivers, which may account for the observed clinical heterogeneity, but few high-depth studies have been performed.

In mammals, the B-cell lineage arises from pluripotent progenitors in the bone marrow. During their development, B-cells undergo lineage specification and commitment, followed by expansion and selection. These processes are mediated by regulated changes in gene expression programmes, rearrangements of immunoglobulin (Ig) genes, and well-timed rounds of proliferation and apoptosis. Many of these processes are initiated by environmental factors including cytokines, chemokines, and cell-cell contacts. Developing B-cells process these environmental cues into stage-specific functions via signalling pathways including the PI3K, MAPK, or JAK-STAT pathway. The cytokines FLT3-Ligand and c-Kit-Ligand are important for the early expansion of the B-cell precursors at different developmental stages and conditions. Interleukin 7 is essential for commitment to the B-cell lineage and for orchestrating the Ig recombination machinery. After rearrangement of the immunoglobulin heavy chain, proliferation and apoptosis, and thus selection, are mediated by the clonal pre-B-cell receptor, and, following light chain rearrangement, by the B-cell receptor.

Syntaxin 17 (Stx17) has been implicated in autophagosome-lysosome fusion. Here, we report that Stx17 functions in assembly of protein complexes during autophagy initiation. Stx17 is phosphorylated by TBK1 whereby phospho-Stx17 controls the formation of the ATG13FIP200 mammalian pre-autophagosomal structure (mPAS) in response to induction of autophagy. TBK1 phosphorylates Stx17 at S202. During autophagy induction, Stx17 transfers from the Golgi, where its steady-state pools localize, to the ATG13FIP200 mPAS. Stx17 was in complexes with ATG13 and FIP200, whereas its non-phosphorylatable mutant Stx17 was not. Stx17 or TBK1 knockouts blocked ATG13 and FIP200 puncta formation. Stx17 or TBK1 knockouts reduced the formation of ATG13 protein complexes with FIP200 and ULK1. Endogenous Stx17 colocalized with LC3B following induction of autophagy. Stx17 knockout diminished LC3 response and reduced sequestration of the prototypical bulk autophagy cargo lactate dehydrogenase. We conclude that Stx17 is a TBK1 substrate and that together they orchestrate assembly of mPAS.

Following publication of the original article [1], it was reported that the incorrect "Additional file 3" was published. The correct additional file is given below.

Complex multicellular organisms, such as mammals, express two complete sets of chromosomes per nucleus, combining the genetic material of both parents. However, epigenetic studies have demonstrated violations to this rule that are necessary for mammalian physiology; the most notable parental allele expression phenomenon is genomic imprinting. With the identification of endogenous imprinted genes, genomic imprinting became well-established as an epigenetic mechanism in which the expression pattern of a parental allele influences phenotypic expression. The expanding study of genomic imprinting is revealing a significant impact on brain functions and associated diseases. Here, we review key milestones in the field of imprinting and discuss mechanisms and systems in which imprinted genes exert a significant role.

In this contribution, we describe a multi-omics systems biology study of the metabolic changes that occur during aging in . Sampling several time points from young adulthood until early old age, our study covers the full duration of aging and include transcriptomics, and targeted MS-based metabolomics. In order to focus on the metabolic changes due to age we used two strains that are metabolically close to wild-type, yet are conditionally non-reproductive. Using these data in combination with a whole-genome model of the metabolism of and mathematical modeling, we predicted metabolic fluxes during early aging. We find that standard Flux Balance Analysis does not accurately predict measured fluxes nor age-related changes associated with the Citric Acid cycle. We present a novel Flux Balance Analysis method where we combined biomass production and targeted metabolomics information to generate an objective function that is more suitable for aging studies. We validated this approach with a detailed case study of the age-associated changes in the Citric Acid cycle. Our approach provides a comprehensive time-resolved multi-omics and modeling resource for studying the metabolic changes during normal aging in .

Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1 chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function. Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine.

Stem cells are maintained by transcriptional programs that promote self-renewal and repress differentiation. Here, we found that the transcription factor c-Myb was essential for generating and maintaining stem cells in the CD8 T cell memory compartment. Following viral infection, CD8 T cells lacking Myb underwent terminal differentiation and generated fewer stem cell-like central memory cells than did Myb-sufficient T cells. c-Myb acted both as a transcriptional activator of Tcf7 (which encodes the transcription factor Tcf1) to enhance memory development and as a repressor of Zeb2 (which encodes the transcription factor Zeb2) to hinder effector differentiation. Domain-mutagenesis experiments revealed that the transactivation domain of c-Myb was necessary for restraining differentiation, whereas its negative regulatory domain was critical for cell survival. Myb overexpression enhanced CD8 T cell memory formation, polyfunctionality and recall responses that promoted curative antitumor immunity after adoptive transfer. These findings identify c-Myb as a pivotal regulator of CD8 T cell stemness and highlight its therapeutic potential.

Metastatic progression is a major cause of mortality in cervical cancers, but factors regulating migratory and pre-metastatic cell populations remain poorly understood. Here, we sought to assess whether a SUV39H1-low chromatin state promotes migratory cell populations in cervical cancers, using meta-analysis of data from The Cancer Genome Atlas (TCGA), immunohistochemistry, genomics and functional assays. Cervical cancer cells sorted based on migratory ability in vitro have low levels of SUV39H1 protein, and SUV39H1 knockdown in vitro enhanced cervical cancer cell migration. Further, TCGA SUV39H1-low tumours correlated with poor clinical outcomes and showed gene expression signatures of cell migration. SUV39H1 expression was examined within biopsies, and SUV39H1 cells within tumours also demonstrated migratory features. Next, to understand genome scale transcriptional and chromatin changes in migratory populations, cell populations sorted based on migration in vitro were examined using RNA-Seq, along with ChIP-Seq for H3K9me3, the histone mark associated with SUV39H1. Migrated populations showed SUV39H1-linked migratory gene expression signatures, along with broad depletion of H3K9me3 across gene promoters. We show for the first time that a SUV39H1-low chromatin state associates with, and promotes, migratory populations in cervical cancers. Our results posit SUV39H1-low cells as key populations for prognosis estimation and as targets for novel therapies.

Low temperatures are required to regulate the transition from vegetative to reproductive growth via a pathway called vernalization. In wheat, vernalization predominantly involves the cold upregulation of the floral activator (). Here, we have used an extreme vernalization response, identified through studying ambient temperature responses, to reveal the complexity of temperature inputs into , with allelic inter-copy variation at a gene expansion of modulating these effects. We find that the repressors of the reproductive transition, () and , are re-activated when plants experience high temperatures during and after vernalization. In addition, this re-activation is regulated by photoperiod for but was independent of photoperiod for We also find this warm temperature interruption affects flowering time and floret number and is stage specific. This research highlights the important balance between floral activators and repressors in coordinating the response of a plant to temperature, and that the absence of warmth is essential for the completion of vernalization. This knowledge can be used to develop agricultural germplasm with more predictable vernalization responses that will be more resilient to variable growth temperatures.

Chronic graft-versus-host disease is a leading cause of morbidity and mortality following allotransplant. Activated donor effector T-cells can differentiate into pathogenic T helper (Th)-17 cells and germinal center -promoting Tfollicular helper cells, resulting in cGVHD. Phosphoinositide-3-kinase-δ, a lipid kinase, is critical for activated T-cell survival, proliferation, differentiation, and metabolism. We demonstrate PI3Kδ activity in donor T-cells that become Tfhs is required for cGVHD in a non-sclerodermatous multi-organ system disease model that includes bronchiolitis obliterans, dependent upon GC B-cells, Tfhs, and counterbalanced by Tfollicular regulatory cells, each requiring PI3Kδ signaling for function and survival. Although B-cells rely on PI3Kδ pathway signaling and GC formation is disrupted resulting in a substantial decrease in Ig production, PI3Kδ kinase-dead mutant donor bone marrow derived GC B-cells still supported BO cGVHD generation. A PI3Kδ-specific inhibitor, compound GS-649443 that has superior potency to idelalisib while maintaining selectivity, reduced cGVHD in mice with active disease. In a Th1-dependent and Th17-associated scleroderma model, GS-649443 effectively treated mice with active cGVHD. These data provide a foundation for clinical trials of FDA-approved PI3Kδ inhibitors for cGVHD therapy in patients. This article is protected by copyright. All rights reserved.

Alternative splicing is a key regulatory mechanism in eukaryotic cells and increases the effective number of functionally distinct gene products. Using bulk RNA sequencing, splicing variation has been studied across human tissues and in genetically diverse populations. This has identified disease-relevant splicing events, as well as associations between splicing and genomic features, including sequence composition and conservation. However, variability in splicing between single cells from the same tissue or cell type and its determinants remains poorly understood.

Humoral alloimmunity is now recognized as a major determinant of transplant outcome. MHC glycoprotein is considered a typical T-dependent antigen, but the nature of the T cell alloresponse that underpins alloantibody generation remains poorly understood. Here, we examine how the relative frequencies of alloantigen-specific B cells and helper CD4 T cells influence the humoral alloimmune response and how this relates to antibody-mediated rejection (AMR). An MHC-mismatched murine model of cardiac AMR was developed, in which T cell help for alloantibody responses in T cell deficient () C57BL/6 recipients against donor H-2K MHC class I alloantigen was provided by adoptively transferred "TCR75" CD4 T cells that recognize processed H-2K allopeptide via the indirect-pathway. Transfer of large numbers (5 × 10) of TCR75 CD4 T cells was associated with rapid development of robust class-switched anti-H-2K humoral alloimmunity and BALB/c heart grafts were rejected promptly (MST 9 days). Grafts were not rejected in T and B cell deficient recipients that were reconstituted with TCR75 CD4 T cells or in control (non-reconstituted) recipients, suggesting that the transferred TCR75 CD4 T cells were mediating graft rejection principally by providing help for effector alloantibody responses. In support, acutely rejecting BALB/c heart grafts exhibited hallmark features of acute AMR, with widespread complement C4d deposition, whereas cellular rejection was not evident. In addition, passive transfer of immune serum from rejecting mice to recipients resulted in eventual BALB/c heart allograft rejection (MST 20 days). Despite being long-lived, the alloantibody responses observed at rejection of the BALB/c heart grafts were predominantly generated by extrafollicular foci: splenic germinal center (GC) activity had not yet developed; IgG secreting cells were confined to the splenic red pulp and bridging channels; and, most convincingly, rapid graft rejection still occurred when recipients were reconstituted with similar numbers of TCR75 CD4 T cells that are genetically incapable of providing T follicular helper cell function for generating GC alloimmunity. Similarly, alloantibody responses generated in recipients reconstituted with smaller number of wild-type TCR75 CD4 T cells (10), although long-lasting, did not have a discernible extrafollicular component, and grafts were rejected much more slowly (MST 50 days). By modeling antibody responses to Hen Egg Lysozyme protein, we confirm that a high ratio of antigen-specific helper T cells to B cells favors development of the extrafollicular response, whereas GC activity is favored by a relatively high ratio of B cells. In summary, a relative abundance of helper CD4 T cells favors development of strong extrafollicular alloantibody responses that mediate acute humoral rejection, without requirement for GC activity. This work is composed of two parts, of which this is Part I. Please read also Part II: Chhabra et al., 2019.

Different profiles of alloantibody responses are observed in the clinic, with those that persist, often despite targeted treatment, associated with poorer long-term transplant outcomes. Although such responses would suggest an underlying germinal center (GC) response, the relationship to cellular events within the allospecific B cell population is unclear. Here we examine the contribution of germinal center (GC) humoral alloimmunity to chronic antibody mediated rejection (AMR). A murine model of chronic AMR was developed in which T cell deficient () C57BL/6 recipients were challenged with MHC-mismatched BALB/c heart allografts and T cell help provided by reconstituting with 10 "TCR75" CD4 T cells that recognize self-restricted allopeptide derived from the H-2K MHC class I alloantigen. Reconstituted recipients developed Ig-switched anti-K alloantibody responses that were slow to develop, but long-lived, with confocal immunofluorescence and flow cytometric characterization of responding H-2K-allospecific B cells confirming persistent splenic GC activity. This was associated with T follicular helper (T) cell differentiation of the transferred TCR75 CD4 T cells. Heart grafts developed progressive allograft vasculopathy, and were rejected chronically (MST 50 days), with explanted allografts displaying features of humoral vascular rejection. Critically, late alloantibody responses were abolished, and heart grafts survived indefinitely, in recipients reconstituted with TCR75 CD4 T cells that were genetically incapable of providing T cell function. The GC response was associated with affinity maturation of the anti-K alloantibody response, and its contribution to progression of allograft vasculopathy related principally to secretion of alloantibody, rather than to enhanced alloreactive T cell priming, because grafts survived long-term when B cells could present alloantigen, but not secrete alloantibody. Similarly, sera sampled at late time points from chronically-rejecting recipients induced more vigorous donor endothelial responses than sera sampled earlier after transplantation. In summary, our results suggest that chronic AMR and progression of allograft vasculopathy is dependent upon allospecific GC activity, with critical help provided by T cells. Clinical strategies that target the T cell subset may hold therapeutic potential. This work is composed of two parts, of which this is Part II. Please read also Part I: Alsughayyir et al., 2019.

Ectopic lymphoid structures form in a wide range of inflammatory conditions, including infection, autoimmune disease, and cancer. In the context of infection, this response can be beneficial for the host: influenza A virus infection-induced pulmonary ectopic germinal centers give rise to more broadly cross-reactive antibody responses, thereby generating cross-strain protection. However, despite the ubiquity of ectopic lymphoid structures and their role in both health and disease, little is known about the mechanisms by which inflammation is able to convert a peripheral tissue into one that resembles a secondary lymphoid organ. Here, we show that type I IFN produced after viral infection can induce CXCL13 expression in a phenotypically distinct population of lung fibroblasts, driving CXCR5-dependent recruitment of B cells and initiating ectopic germinal center formation. This identifies type I IFN as a novel inducer of CXCL13, which, in combination with other stimuli, can promote lung remodeling, converting a nonlymphoid tissue into one permissive to functional tertiary lymphoid structure formation.

Circulating neutrophils are, by necessity, quiescent and relatively unresponsive to acute stimuli. In regions of inflammation, mediators can prime neutrophils to react to acute stimuli with stronger proinflammatory, pathogen-killing responses. In neutrophils G protein-coupled receptor (GPCR)-driven proinflammatory responses, such as reactive oxygen species (ROS) formation and accumulation of the key intracellular messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP ), are highly dependent on PI3K-γ, a Ras-GTP, and Gβγ coincidence detector. In unprimed cells, the major GPCR-triggered activator of Ras is the Ras guanine nucleotide exchange factor (GEF), Ras guanine nucleotide releasing protein 4 (RasGRP4). Although priming is known to increase GPCR-PIP signaling, the mechanisms underlying this augmentation remain unclear. We used genetically modified mice to address the role of the 2 RasGEFs, RasGRP4 and son of sevenless (SOS)1/2, in neutrophil priming. We found that following GM-CSF/TNFα priming, RasGRP4 had only a minor role in the enhanced responses. In contrast, SOS1/2 acquired a substantial role in ROS formation, PIP accumulation, and ERK activation in primed cells. These results suggest that SOS1/2 signaling plays a key role in determining the responsiveness of neutrophils in regions of inflammation.

R-loops are three-stranded nucleic acid structures that form during transcription, especially over unmethylated CpG-rich promoters of active genes. In mouse embryonic stem cells (mESCs), CpG-rich developmental regulator genes are repressed by the Polycomb complexes PRC1 and PRC2. Here, we show that R-loops form at a subset of Polycomb target genes, and we investigate their contribution to Polycomb repression. At R-loop-positive genes, R-loop removal leads to decreased PRC1 and PRC2 recruitment and Pol II activation into a productive elongation state, accompanied by gene derepression at nascent and processed transcript levels. Stable removal of PRC2 derepresses R-loop-negative genes, as expected, but does not affect R-loops, PRC1 recruitment, or transcriptional repression of R-loop-positive genes. Our results highlight that Polycomb repression does not occur via one mechanism but consists of different layers of repression, some of which are gene specific. We uncover that one such mechanism is mediated by an interplay between R-loops and RING1B recruitment.

In chronic lymphocytic leukemia (CLL), signaling through several prosurvival B cell surface receptors activates the PI3K signaling pathway. Idelalisib is a highly selective PI3K (PI3Kδ) isoform-specific inhibitor effective in relapsed/refractory CLL and follicular lymphoma. However, severe autoimmune adverse effects in association with the use of idelalisib in the treatment of CLL, particularly as a first-line therapy, gave indications that idelalisib may preferentially target the suppressive function of regulatory T cells (Tregs). On this background, we examined the effect of idelalisib on the function of human Tregs ex vivo with respect to proliferation, TCR signaling, phenotype, and suppressive function. Our results show that human Tregs are highly susceptible to PI3Kδ inactivation using idelalisib compared with CD4 and CD8 effector T cells (Teffs) as evident from effects on anti-CD3/CD28/CD2-induced proliferation (order of susceptibility [IC]: Treg [.5 μM] > CD4 Teff [2.0 μM] > CD8 Teff [6.5 μM]) and acting at the level of AKT and NF-κB phosphorylation. Moreover, idelalisib treatment of Tregs altered their phenotype and reduced their suppressive function against CD4 and CD8 Teffs. Phenotyping Tregs from CLL patients treated with idelalisib supported our in vitro findings. Collectively, our data show that human Tregs are more dependent on PI3Kδ-mediated signaling compared with CD4 and CD8 Teffs. This Treg-preferential effect could explain why idelalisib produces adverse autoimmune effects by breaking Treg-mediated tolerance. However, balancing effects on Treg sensitivity versus CD8 Teff insensitivity to idelalisib could still potentially be exploited to enhance inherent antitumor immune responses in patients.

The molecular regulation of zygotic genome activation (ZGA) in mammals remains an exciting area of research. Primed mouse embryonic stem cells contain a rare subset of "2C-like" cells that are epigenetically and transcriptionally similar to the two-cell embryo and thus represent an in vitro approximation for studying ZGA transcription regulation. Recently, the transcription factor Dux, expressed in the minor wave of ZGA, was described to activate many downstream ZGA transcripts. However, it remains unknown what upstream maternal factors initiate ZGA in either a Dux-dependent or Dux-independent manner. Here we performed a candidate-based overexpression screen, identifying, among others, developmental pluripotency-associated 2 (Dppa2) and Dppa4 as positive regulators of 2C-like cells and transcription of ZGA genes. In the germline, promoter DNA demethylation coincides with expression of Dppa2 and Dppa4, which remain expressed until embryonic day 7.5 (E7.5), when their promoters are remethylated. Furthermore, Dppa2 and Dppa4 are also expressed during induced pluripotent stem cell (iPSC) reprogramming at the time that 2C-like transcription transiently peaks. Through a combination of overexpression, knockdown, knockout, and rescue experiments together with transcriptional analyses, we show that Dppa2 and Dppa4 directly regulate the 2C-like cell population and associated transcripts, including Dux and the Zscan4 cluster. Importantly, we teased apart the molecular hierarchy in which the 2C-like transcriptional program is initiated and stabilized. Dppa2 and Dppa4 require Dux to initiate 2C-like transcription, suggesting that they act upstream by directly regulating Dux. Supporting this, ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) analysis revealed that Dppa2 and Dppa4 bind to the Dux promoter and gene body and drive its expression. Zscan4c is also able to induce 2C-like cells in wild-type cells but, in contrast to Dux, can no longer do so in Dppa2/4 double-knockout cells, suggesting that it may act to stabilize rather than drive the transcriptional network. Our findings suggest a model in which Dppa2/4 binding to the Dux promoter leads to Dux up-regulation and activation of the 2C-like transcriptional program, which is subsequently reinforced by Zscan4c.

Conventional human embryonic stem cells are considered to be primed pluripotent but can be induced to enter a naive state. However, the transcriptional features associated with naive and primed pluripotency are still not fully understood. Here we used single-cell RNA sequencing to characterize the differences between these conditions. We observed that both naive and primed populations were mostly homogeneous with no clear lineage-related structure and identified an intermediate subpopulation of naive cells with primed-like expression. We found that the naive-primed pluripotency axis is preserved across species, although the timing of the transition to a primed state is species specific. We also identified markers for distinguishing human naive and primed pluripotency as well as strong co-regulatory relationships between lineage markers and epigenetic regulators that were exclusive to naive cells. Our data provide valuable insights into the transcriptional landscape of human pluripotency at a cellular and genome-wide resolution.

This data is related to the research article entitled "Germinal center humoral autoimmunity independently mediates progression of allograft vasculopathy" (Harper et al., 2016) [2]. The data presented here focuses on the humoral autoimmune response triggered by transferred allogeneic CD4 T cells and includes details on: (a) the recipient splenic germinal center (GC) response; (b) augmentation of humoral autoimmunity and accelerated heart allograft rejection following transplantation from donors primed against recipient; (c) flow cytometric analysis of donor and recipient CD4 T cells for signature markers of T follicular helper cell differentiation; (d) donor endothelial cell migration in response to column purified autoantibody from recipient sera; (e) analysis of development of humoral responses in recipients following adoptive transfer of donor CD4 T cells and; (f) the development of humoral autoimmunity in mixed haematopoietic chimeric mice.