Two-photon synthetic aperture microscopy for minimally invasive fast 3D imaging of native subcellular behaviors in deep tissue.

Holistic understanding of physio-pathological processes requires noninvasive 3D imaging in deep tissue across multiple spatial and temporal scales to link diverse transient subcellular behaviors with long-term physiogenesis. Despite broad applications of two-photon microscopy (TPM), there remains an inevitable tradeoff among spatiotemporal resolution, imaging volumes, and durations due to the point-scanning scheme, accumulated phototoxicity, and optical aberrations. Here, we harnessed the concept of synthetic aperture radar in TPM to achieve aberration-corrected 3D imaging of subcellular dynamics at a millisecond scale for over 100,000 large volumes in deep tissue, with three orders of magnitude reduction in photobleaching. With its advantages, we identified direct intercellular communications through migrasome generation following traumatic brain injury, visualized the formation process of germinal center in the mouse lymph node, and characterized heterogeneous cellular states in the mouse visual cortex, opening up a horizon for intravital imaging to understand the organizations and functions of biological systems at a holistic level.

Epigenetic recording of stimulation history reveals BLIMP1-BACH2 balance in determining memory B cell fate upon recall challenge.

Memory B cells (MBCs) differentiate into plasma cells (PCs) or germinal centers (GCs) upon antigen recall. How this decision is programmed is not understood. We found that the relative strength between two antagonistic transcription factors, B lymphocyte-induced maturation protein 1 (BLIMP1) and BTB domain and CNC homolog 2 (BACH2), progressively increases in favor of BLIMP1 in antigen-responding B cells through the course of primary responses. MBC subsets that preferentially produce secondary GCs expressed comparatively higher BACH2 but lower BLIMP1 than those predisposed for PC development. Skewing the BLIMP1-BACH2 balance in otherwise fate-predisposed MBC subsets could switch their fate preferences. Underlying the changing BLIMP1-over-BACH2 balance, we observed progressively increased accessibilities at chromatin loci that are specifically opened in PCs, particularly those that contain interferon-sensitive response elements (ISREs) and are controlled by interferon regulatory factor 4 (IRF4). IRF4 is upregulated by B cell receptor, CD40 or innate receptor signaling and it induces graded levels of PC-specifying epigenetic imprints according to the strength of stimulation. By analyzing history-stamped GC B cells, we found progressively increased chromatin accessibilities at PC-specific, IRF4-controlled gene loci over time. Therefore, the cumulative stimulation history of B cells is epigenetically recorded in an IRF4-dependent manner, determines the relative strength between BLIMP1 and BACH2 in individual MBCs and dictates their probabilities to develop into GCs or PCs upon restimulation.

The humoral response and antibodies against SARS-CoV-2 infection.

Two and a half years into the COVID-19 pandemic, we have gained many insights into the human antibody response to the causative SARS-CoV-2 virus. In this Review, we summarize key observations of humoral immune responses in people with COVID-19, discuss key features of infection- and vaccine-induced neutralizing antibodies, and consider vaccine designs for inducing antibodies that are broadly protective against different variants of the SARS-CoV-2 virus.

Heterogeneous plasma cells and long-lived subsets in response to immunization, autoantigen and microbiota.

Durable antibody immunity depends on long-lived plasma cells (LLPCs) that primarily reside in the bone marrow (BM). However, due to LLPC rarity, it has not been possible to define their phenotypes or determine their heterogeneity. By single-cell mRNA sequencing, cytometry and a genetic pulse-chase mouse model, we show that IgG and IgM LLPCs display an EpCAMhiCXCR3- phenotype, whereas IgA LLPCs are Ly6AhiTigit-. While IgG and IgA LLPCs are mainly contributed by somatically hypermutated cells following immunization or infection, cells with innate properties and public antibodies are found in IgA and IgM LLPC compartments. Particularly, IgM LLPCs are highly enriched with public clones shared among different individual animals, differentiated in a T cell-independent manner and have affinity for self-antigens and microbial-derived antigens. Taken together, our work reveals different routes toward LLPC development and paves the way for deeper understanding of cellular and molecular underpinnings of long-term antibody immunity.

High recallability of memory B cells requires ZFP318-dependent transcriptional regulation of mitochondrial function.

Expression of the transcriptional regulator ZFP318 is induced in germinal center (GC)-exiting memory B cell precursors and memory B cells (MBCs). Using a conditional ZFP318 fluorescence reporter that also enables ablation of ZFP318-expressing cells, we found that ZFP318-expressing MBCs were highly enriched with GC-derived cells. Although ZFP318-expressing MBCs constituted only a minority of the antigen-specific MBC compartment, their ablation severely impaired recall responses. Deletion of Zfp318 did not alter the magnitude of primary responses but markedly reduced MBC participation in recall. CD40 ligation promoted Zfp318 expression, whereas B cell receptor (BCR) signaling was inhibitory. Enforced ZFP318 expression enhanced recall performance of MBCs that otherwise responded poorly. ZFP318-deficient MBCs expressed less mitochondrial genes, had structurally compromised mitochondria, and were susceptible to reactivation-induced cell death. The abundance of ZFP318-expressing MBCs, instead of the number of antigen-specific MBCs, correlated with the potency of prime-boost vaccination. Therefore, ZFP318 controls the MBC recallability and represents a quality checkpoint of humoral immune memory.

BCL6 controls contact-dependent help delivery during follicular T-B cell interactions.

BCL6 is required for development of follicular T helper (Tfh) cells to support germinal center (GC) formation. However, it is not clear what unique functions programmed by BCL6 can explain its absolute essentiality in T cells for GC formation. We found that ablation of one Bcl6 allele did not appreciably alter early T cell activation and follicular localization but inhibited GC formation and Tfh cell maintenance. BCL6 impinged on Tfh calcium signaling and also controlled Tfh entanglement with and CD40L delivery to B cells. Amounts of BCL6 protein and nominal frequencies of Tfh cells markedly changed within hours after strengths of T-B cell interactions were altered in vivo, while CD40L overexpression rectified both defective GC formation and Tfh cell maintenance because of the BCL6 haploinsufficiency. Our results reveal BCL6 functions in Tfh cells that are essential for GC formation and suggest that BCL6 helps maintain Tfh cell phenotypes in a T cell non-autonomous manner.

Germinal-center development of memory B cells driven by IL-9 from follicular helper T cells.

Germinal centers (GCs) support high-affinity, long-lived humoral immunity. How memory B cells develop in GCs is not clear. Through the use of a cell-cycle-reporting system, we identified GC-derived memory precursor cells (GC-MP cells) that had quit cycling and reached G0 phase while in the GC, exhibited memory-associated phenotypes with signs of affinity maturation and localized toward the GC border. After being transferred into adoptive hosts, GC-MP cells reconstituted a secondary response like genuine memory B cells. GC-MP cells expressed the interleukin 9 (IL-9) receptor and responded to IL-9. Acute treatment with IL-9 or antibody to IL-9 accelerated or retarded the positioning of GC-MP cells toward the GC edge and exit from the GC, and enhanced or inhibited the development of memory B cells, which required B cell-intrinsic responsiveness to IL-9. Follicular helper T cells (TFH cells) produced IL-9, and deletion of IL-9 from T cells or, more specifically, from GC TFH cells led to impaired memory formation of B cells. Therefore, the GC development of memory B cells is promoted by TFH cell-derived IL-9.

Spatiotemporal basis of innate and adaptive immunity in secondary lymphoid tissue.

Secondary lymphoid tissues are the sites of both innate and adaptive host defense. Aside from the relatively static nonhematopoietic stromal elements and some macrophages and dendritic cells, most of the cells in these tissues are in constant movement, but the organs maintain a defined microanatomy with preferred locations for the bulk of T cells, B cells, and other lymphocytes and subsets of myeloid cells. Here we describe both the cell dynamics and spatial organization of lymph nodes and review how both physical features and molecular cues guide cell movement to optimize host defense. We emphasize the role of locality in improving the efficiency of a system requiring rare cells to find each other and interact productively through membrane-bound or short-range secreted mediators and highlight how changes in steady-state cell positioning during an infectious challenge contribute to rapid generation of productive responses.

PD-1 Controls Follicular T Helper Cell Positioning and Function.

Follicular T helper (Tfh) cells highly express the programmed cell death-1 (PD-1) molecule. Whereas inhibition of T cell receptor (TCR) signaling and CD28 co-stimulation is thought to be the primary mode of PD-1 functions, whether and how PD-1 regulates Tfh cell development and function is unclear. Here we showed that, when engaged by the ensemble of bystander B cells constitutively expressing PD-1 ligand 1 (PD-L1), PD-1 inhibited T cell recruitment into the follicle. This inhibition involved suppression of PI3K activities downstream of the follicle-guidance receptor CXCR5, was independent of co-signaling with the TCR, and necessitated ICOS signaling to overcome. PD-1 further restricted CXCR3 upregulation on Tfh cells, serving to concentrate these cells toward the germinal center territory, where PD-L1-PD-1 interactions between individual Tfh and B cells optimized B cell competition and affinity maturation. Therefore, operating in both costimulation-independent and -dependent manners, PD-1 controls tissue positioning and function of Tfh cells.

Affinity-coupled CCL22 promotes positive selection in germinal centres.

Antibody affinity maturation depends on positive selection in germinal centres (GCs) of rare B cell clones that acquire higher-affinity B cell receptors via somatic hypermutation, present more antigen to follicular helper T (TFH) cells and, consequently, receive more contact-dependent T cell help1. As these GC B cells and TFH cells do not maintain long-lasting contacts in the chaotic GC environment2-4, it is unclear how sufficient T cell help is cumulatively focused onto those rare clones. Here we show that, upon stimulation of CD40, GC B cells upregulate the chemokine CCL22 and to a lesser extent CCL17. By engaging the chemokine receptor CCR4 on TFH cells, CCL22 and CCL17 can attract multiple helper cells from a distance, thus increasing the chance of productive help. During a GC response, B cells that acquire higher antigen-binding affinities express higher levels of CCL22, which in turn 'highlight' these high-affinity GC B cells. Acute increase or blockade of TFH cells helps to rapidly increase or decrease CCL22 expression by GC B cells, respectively. Therefore, a chemokine-based intercellular reaction circuit links the amount of T cell help that individual B cells have received recently to their subsequent ability to attract more help. When CCL22 and CCL17 are ablated in B cells, GCs form but B cells are not affinity-matured efficiently. When competing with wild-type B cells in the same reaction, B cells lacking CCL22 and CCL17 receive less T cell help to maintain GC participation or develop into bone-marrow plasma cells. By uncovering a chemokine-mediated mechanism that highlights affinity-improved B cells for preferential help from TFH cells, our study reveals a principle of spatiotemporal orchestration of GC positive selection.

Brain control of humoral immune responses amenable to behavioural modulation.

It has been speculated that brain activities might directly control adaptive immune responses in lymphoid organs, although there is little evidence for this. Here we show that splenic denervation in mice specifically compromises the formation of plasma cells during a T cell-dependent but not T cell-independent immune response. Splenic nerve activity enhances plasma cell production in a manner that requires B-cell responsiveness to acetylcholine mediated by the α9 nicotinic receptor, and T cells that express choline acetyl transferase1,2 probably act as a relay between the noradrenergic nerve and acetylcholine-responding B cells. We show that neurons in the central nucleus of the amygdala (CeA) and the paraventricular nucleus (PVN) that express corticotropin-releasing hormone (CRH) are connected to the splenic nerve; ablation or pharmacogenetic inhibition of these neurons reduces plasma cell formation, whereas pharmacogenetic activation of these neurons increases plasma cell abundance after immunization. In a newly developed behaviour regimen, mice are made to stand on an elevated platform, leading to activation of CeA and PVN CRH neurons and increased plasma cell formation. In immunized mice, the elevated platform regimen induces an increase in antigen-specific IgG antibodies in a manner that depends on CRH neurons in the CeA and PVN, an intact splenic nerve, and B cell expression of the α9 acetylcholine receptor. By identifying a specific brain-spleen neural connection that autonomically enhances humoral responses and demonstrating immune stimulation by a bodily behaviour, our study reveals brain control of adaptive immunity and suggests the possibility to enhance immunocompetency by behavioural intervention.

A GPR174-CCL21 module imparts sexual dimorphism to humoral immunity.

Humoral immune responses to immunization and infection and susceptibilities to antibody-mediated autoimmunity are generally lower in males1-3. However, the mechanisms underlying such sexual dimorphism are not well understood. Here we show that there are intrinsic differences between the B cells that produce germinal centres in male and female mice. We find that antigen-activated male B cells do not position themselves as efficiently as female B cells in the centre of follicles in secondary lymphoid organs, in which germinal centres normally develop. Moreover, GPR174-an X-chromosome-encoded G-protein-coupled receptor-suppresses the formation of germinal centres in male, but not female, mice. This effect is intrinsic to B cells, and correlates with the GPR174-enhanced positioning of B cells towards the T-cell-B-cell border of follicles, and the distraction of male, but not female, B cells from S1PR2-driven follicle-centre localization. Biochemical fractionation of conditioned media that induce B-cell migration in a GPR174-dependent manner identifies CCL21 as a GPR174 ligand. In response to CCL21, GPR174 triggers a calcium flux and preferentially induces the migration of male B cells; GPR174 also becomes associated with more Gαi protein in male than in female B cells. Male B cells from orchidectomized mice exhibit impaired GPR174-mediated migration to CCL21, and testosterone treatment rescues this defect. Female B cells from testosterone-treated mice exhibit male-like GPR174-Gαi association and GPR174-mediated migration. Deleting GPR174 from male B cells causes more efficient positioning towards the follicular centre, the formation of more germinal centres and an increased susceptibility to B-cell-dependent experimental autoimmune encephalomyelitis. By identifying GPR174 as a receptor for CCL21 and demonstrating its sex-dependent control of B-cell positioning and participation in germinal centres, we have revealed a mechanism by which B-cell physiology is fine-tuned to impart sexual dimorphism to humoral immunity.

MicroRNAs of the miR-17∼92 family are critical regulators of T(FH) differentiation.

Follicular helper T cells (T(FH) cells) provide critical help to B cells during humoral immune responses. Here we report that mice with T cell-specific deletion of the miR-17∼92 family of microRNAs (miRNAs) had substantially compromised T(FH) differentiation, germinal-center formation and antibody responses and failed to control chronic viral infection. Conversely, mice with T cell-specific expression of a transgene encoding miR-17∼92 spontaneously accumulated T(FH) cells and developed a fatal immunopathology. Mechanistically, the miR-17∼92 family controlled the migration of CD4(+) T cells into B cell follicles by regulating signaling intensity from the inducible costimulator ICOS and kinase PI(3)K by suppressing expression of the phosphatase PHLPP2. Our findings demonstrate an essential role for the miR-17∼92 family in T(FH) differentiation and establish PHLPP2 as an important mediator of their function in this process.

Multi-omics study reveals different pathogenesis of the generation of skin lesions in SLE and IDLE patients.

Lupus erythematosus (LE) is a heterogeneous, antibody-mediated autoimmune disease. Isolate discoid LE (IDLE) and systematic LE (SLE) are traditionally regarded as the two ends of the spectrum, ranging from skin-limited damage to life-threatening multi-organ involvement. Both belong to LE, but IDLE and SLE differ in appearance of skin lesions, autoantibody panels, pathological changes, treatments, and immunopathogenesis. Is discoid lupus truly a form of LE or is it a completely separate entity? This question has not been fully elucidated. We compared the clinical data of IDLE and SLE from our center, applied multi-omics technology, such as immune repertoire sequencing, high-resolution HLA alleles sequencing and multi-spectrum pathological system to explore cellular and molecular phenotypes in skin and peripheral blood from LE patients. Based on the data from 136 LE patients from 8 hospitals in China, we observed higher damage scores and fewer LE specific autoantibodies in IDLE than SLE patients, more uCDR3 sharing between PBMCs and skin lesion from SLE than IDLE patients, elevated diversity of V-J recombination in IDLE skin lesion and SLE PBMCs, increased SHM frequency and class switch ratio in IDLE skin lesion, decreased SHM frequency but increased class switch ratio in SLE PBMCs, HLA-DRB1*03:01:01:01, HLA-B*58:01:01:01, HLA-C*03:02:02:01, and HLA-DQB1*02:01:01:01 positively associated with SLE patients, and expanded Tfh-like cells with ectopic germinal center structures in IDLE skin lesions. These findings suggest a significant difference in the immunopathogenesis of skin lesions between SLE and IDLE patients. SLE is a B cell-predominate systemic immune disorder, while IDLE appears limited to the skin. Our findings provide novel insights into the pathogenesis of IDLE and other types of LE, which may direct more accurate diagnosis and novel therapeutic strategies.

Genetic imprints of grafting in wild iron walnut populations in southwestern China.

BACKGROUND: Anthropogenic activities are causing unprecedented loss of genetic diversity in many species. However, the effects on genetic diversity from large-scale grafting onto wild plants of crop species are largely undetermined. Iron walnut (Juglans sigillata Dode) is a deciduous nut tree crop endemic to southwestern China with a long history of cultivation. Due to the rapid expansion of the walnut industry, many natural populations are now being replaced by cultivars grafted onto wild rootstocks. However, little is known about the potential genetic consequences of such action on natural populations. RESULTS: We sampled the scion and the rootstock from each of 149 grafted individuals within nine wild populations of J. sigillata from Yunnan Province which is the center of walnut diversity and cultivation in China, and examined their genetic diversity and population structure using 31 microsatellite loci. Scions had lower genetic diversity than rootstocks, and this pattern was repeated in seven of the nine examined populations. Among those seven populations, AMOVA and clustering analyses showed a clear genetic separation between all rootstocks and all scions. However, the two remaining populations, both from northern Yunnan, showed genetic similarity between scions and rootstocks, possibly indicating that wild populations here are derived from feralized local cultivars. Moreover, our data indicated probable crop-to-wild gene flow between scions and rootstocks, across all populations. CONCLUSIONS: Our results indicate that large-scale grafting has been causing genetic diversity erosion and genetic structure breakdown in the wild material of J. sigillata within Yunnan. To mitigate these effects, we caution against the overuse of grafting in wild populations of iron walnut and other crop species and recommend the preservation of natural genotypes through in situ  and ex situ conservation.

Unravelling the genetic diversity and population structure of common walnut in the Iranian Plateau.

BACKGROUND: Common walnut (Juglans regia L.) has a long cultivation history, given its highly valuable wood and rich nutritious nuts. The Iranian Plateau has been considered as one of the last glaciation refugia and a centre of origin and domestication for the common walnut. However, a prerequisite to conserve or utilize the genetic resources of J. regia in the plateau is a comprehensive evaluation of the genetic diversity that is conspicuously lacking. In this regard, we used 31 polymorphic simple sequence repeat (SSR) markers to delineate the genetic variation and population structure of 508 J. regia individuals among 27 populations from the Iranian Plateau. RESULTS: The SSR markers expressed a high level of genetic diversity (HO = 0.438, and HE = 0.437). Genetic differentiation among the populations was moderate (FST = 0.124), and genetic variation within the populations (79%) significantly surpassed among populations (21%). The gene flow (Nm = 1.840) may have remarkably influenced the population genetic structure of J. regia, which can be attributed to anthropological activities and wind dispersal of pollen. The STRUCTURE analysis divided the 27 populations into two main clusters. Comparing the neighbor-joining and principal coordinate analysis dendrograms and the Bayesian STRUCTURE analysis revealed the general agreement between the population subdivisions and the genetic relationships among the populations. However, a few geographically close populations dispersed into different clusters. Further, the low genetic diversity of the Sulaymaniyah (SMR) population of Iraq necessitates urgent conservation by propagation and seedling management or tissue culture methods; additionally, we recommend the indispensable preservation of the Gonabad (RGR) and Arak (AKR) populations in Iran. CONCLUSIONS: These results reflected consistent high geographical affinity of the accession across the plateau. Our findings suggest that gene flow is a driving factor influencing the genetic structure of J. regia populations, whereas ecological and geological variables did not act as strong barriers. Moreover, the data reported herein provide new insights into the population structure of J. regia germplasm, which will help conserve genetic resources for the future, hence improving walnut breeding programs' efficiency.

Rapid and sensitive single-cell RNA sequencing with SHERRY2.

BACKGROUND: Prevalent single-cell transcriptomic profiling (scRNA-seq) methods are mainly based on the synthesis and enrichment of full-length double-stranded complementary DNA. These approaches are challenging to generate accurate quantification of transcripts when their abundance is low or their full-length amplifications are difficult. RESULTS: Based on our previous finding that Tn5 transposase can directly cut-and-tag DNA/RNA hetero-duplexes, we present SHERRY2, a specifically optimized protocol for scRNA-seq without second-strand cDNA synthesis. SHERRY2 is free of pre-amplification and eliminates the sequence-dependent bias. In comparison with other widely used scRNA-seq methods, SHERRY2 exhibits significantly higher sensitivity and accuracy even for single nuclei. Besides, SHERRY2 is simple and robust and can be easily scaled up to high-throughput experiments. When testing single lymphocytes and neuron nuclei, SHERRY2 not only obtained accurate countings of transcription factors and long non-coding RNAs, but also provided bias-free results that enriched genes in specific cellular components or functions, which outperformed other protocols. With a few thousand cells sequenced by SHERRY2, we confirmed the expression and dynamics of Myc in different cell types of germinal centers, which were previously only revealed by gene-specific amplification methods. CONCLUSIONS: SHERRY2 is able to provide high sensitivity, high accuracy, and high throughput for those applications that require a high number of genes identified in each cell. It can reveal the subtle transcriptomic difference between cells and facilitate important biological discoveries.

Xanthones from securidaca inappendiculata antagonizes the anti-rheumatic effect of methotrexate by inhibiting reduced folate carrier 1.

BACKGROUND: The first-line anti-rheumatic drug methotrexate (MTX) is used in the combination. Because of the unpredictable adverse reactions, optimization of relevant regimens is necessary and meaningful. This study aimed to study the possible interaction between Securidaca inappendiculate Hassk. Derived xanthones and MTX. METHODS: We established adjuvant-induced arthritis (AIA) model, which was treated with MTX and MTX + xanthone-rich fraction (XRF). The clinical efficacy was evaluated by histopathological examination, and LC-MS was used to monitor the blood concentration of MTX. Western blotting and immunohistochemistry were used to detect protein expression. In vitro, we assessed the activity of related transporters by cellular uptake assay based on HEK-293T cells. RESULTS: Compared with MTX-treated rats, inflammation in the immunized rats in the MTX + XRF group was obvious, indicating that XRF antagonized the anti-rheumatic effect of MTX. Meanwhile, XRF reduced liver and kidney injuries caused by MTX in addition to MTX. Results from immunohistochemical and nappendiculat assays suggested that XRF may reduce uptake of MTX by down-regulating reduced folate carrier 1 (RFC1). CONCLUSION: This study indicated that XRF could reduce the plasma concentration of MTX by inhibiting the expression of RFC1, antagonize the therapeutic effect of MTX on AIA rats, and reduce its oral bioavailability. The combination of S. inappendiculate and MTX should be further optimized to achieve the goal of increasing efficiency and reducing toxicity.

TFH cells in bystander and cognate interactions with B cells.

Follicular T-helper (TFH ) cells play a crucial role in three aspects of the germinal center (GC) response. They promote GC formation, arbitrate competition among GC B cells to determine the outcome of affinity maturation, and regulate GC output of memory and plasma cells to shape the long-lived humoral immune memory. Of fundamental importance are dynamic physical interactions between TFH and B cells, which are the main platform for TFH cells to deliver "help" factors to B cells and also for reciprocal signaling from B cells to maintain the helper state of TFH cells. Recent work has significantly expanded our understanding of how T-B interactions are spatiotemporally regulated and molecularly orchestrated to fulfill those TFH functions. In this review, we elaborate two modes of T-B interactions, the antigen-specific or cognate mode in which TFH cells engage individual antigen-presenting B cells and the antigen nonspecific bystander mode in which TFH cells are engaged with the ensemble of follicular B cells. We discuss findings that indicate how short-lived cognate T-B contacts coupled with an intercellular positive feedback drive affinity-based selection and how bystander interactions between T and B cells regulate follicular T-cell recruitment and maintenance of an appropriate helper state. We argue that this combination of bystander and cognate interactions with B cells constantly shapes the internal state of TFH cells and provides the platform to execute their helper functions.