Xin Chen

The extraction process of total flavonoids in Chinese kale was studied. The mathematical models of extraction rate of total flavonoids related to ultrasonic time, ultrasonic temperature, liquid-to-material ratio and ethanol concentration were established by response surface methodology. Through the mathematical model, ultrasound assisted method is the optimum conditions to extract the total flavonoids from Chinese kale. The results showed that the optimum conditions for the extraction was: ultrasonic time 29 min, ultrasonic temperature 71 °C, liquid-to-material ratio 27 mL/g and ethanol concentration 77%. Based on these conditions, the extraction rate of total flavonoids in Chinese kale was 18.47 mg/g, with a relative error of 0.75% compared with the predicted value. The mathematical model had a high fitting degree and a better predictability about the extraction rate of total flavonoids in Chinese kale. The obtained conditions were reliable and could be used to optimize the extraction process of total flavonoids in Chinese kale.

A distinct population of Foxp3⁺CD4⁺regulatory T (Treg) cells promotes repair of acutely or chronically injured skeletal muscle. The accumulation of these cells depends critically on interleukin (IL)-33 produced by local mesenchymal stromal cells (mSCs). An intriguing physical association among muscle nerves, IL-33⁺mSCs, and Tregs has been reported, and invites a deeper exploration of this cell triumvirate. Here we evidence a striking proximity between IL-33⁺muscle mSCs and both large-fiber nerve bundles and small-fiber sensory neurons; report that muscle mSCs transcribe an array of genes encoding neuropeptides, neuropeptide receptors, and other nerve-related proteins; define muscle mSC subtypes that express both IL-33 and the receptor for the calcitonin-gene–related peptide (CGRP); and demonstrate that up- or down-tuning of CGRP signals augments or diminishes, respectively, IL-33 production by muscle mSCs and later accumulation of muscle Tregs. Indeed, a single injection of CGRP induced much of the genetic program elicited in mSCs early after acute skeletal muscle injury. These findings highlight neural/stromal/immune-cell crosstalk in tissue repair, suggesting future therapeutic approaches.

Humoral immune responses to immunization and infection and susceptibilities to antibody-mediated autoimmunity are generally lower in males^1,2,3. 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.

T cells play critical roles in anti-tumor immunity. Up-regulation of immune checkpoint molecules (PD-1, PD-L1, CTLA-4, TIM-3, Lag-3, TIGIT, CD73, VISTA, B7-H3) in the tumor microenvironment is an important mechanism that restrains effector T cells from the anti-tumor activity. To date, immune checkpoint antibodies have demonstrated significant clinical benefits for cancer patients treated with mono- or combination immunotherapies. However, many tumors do not respond to the treatment well, and merely blocking the immune suppression pathways by checkpoint-regulatory antibodies may not render optimal tumor growth inhibition. Binding of the antibody Fc-hinge region to Fc gamma receptors (FcγRs) has been shown to exert a profound impact on antibody function and in vivo efficacy. Investigation of immune checkpoint antibodies regarding their effector functions and impact on therapeutic efficacy has gained more attention in recent years. In this review, we discuss Fc variants of antibodies against immune checkpoint targets and the potential mechanisms of how FcγR-binding could influence the anti-tumor activity of these antibodies.

How follicular T-helper (Tfh) cells develop is incompletely understood. We find that, upon antigen exposure in vivo, both naïve and antigen-experienced T cells sequentially upregulate CXCR5 and Bcl6 within the first 24 h, relocate to the T-B border, and give rise to phenotypic Bcl6⁺CXCR5⁺ Tfh cells before the first cell division. CXCR5 upregulation is more dependent on ICOS costimulation than that of Bcl6, and early Bcl6 induction requires T-cell expression of CXCR5 and, presumably, relocation toward the follicle. This early and rapid upregulation of CXCR5 and Bcl6 depends on IL-6 produced by radiation-resistant cells. These results suggest that a Bcl6^hiCXCR5^hi phenotype does not automatically define a Tfh lineage but might reflect a state of antigen exposure and non-commitment to terminal effector fates and that niches in the T-B border and/or the follicle are important for optimal Bcl6 induction and maintenance.

Chemokine-guided lymphocyte positioning in tissues is crucial for normal operation of the immune system. Direct, real-time manipulation and measurement of single-cell responses to chemokines is highly desired for investigating the cell biology of lymphocyte migration in vivo. Here we report the development of the first two-photon-activatable chemokine CCL5 through efficient one-pot total chemical synthesis in milligram scale. By spatiotemporally controlled photoactivation, we show at the single-cell level that T cells perceive the directional cue without relying on PI3K activities, which are nonetheless required for persistent migration over an extended period of time. By intravital imaging, we demonstrate artificial T-cell positioning in cutaneous tissues and lymph nodes. This work establishes a general strategy to develop high-quality photo-activatable protein agents through tailor-designed caging of multiple residues and highlights the potential of photo-activatable chemokines for understanding and potential therapeutic manipulation of cell positioning and position-controlled cell behaviours in vivo.

Follicular helper T cells (Tfh) are a newly defined helper T-cell subset that is specialized in facilitating B-cell responses. These cells have a unique tissue localization pattern and a distinct transcriptional program suited for the B-cell helper function. Co-opting of the follicular program affords regulatory T cells, NK T cells, and γδ T cells with opportunities to participate in the regulation of humoral immunity. Abnormal Tfh development and function can lead to immunodeficiencies, autoimmune inflammation, and tumors. Detailed understanding of Tfh cell differentiation and function in animal models and the human system promises better strategies toward vaccine development and therapies for inflammatory diseases.

A new synthetic method for peptide thioesters is described using Fmoc solid-phase peptide synthesis (Fmoc-SPPS). This method employs a novel enamide motif to facilitate irreversible intramolecular N-to-S acyl migration, which can efficiently afford the desired peptide thioesters (3 h, 30 °C) under the final trifluoroacetic acid (TFA) cleavage conditions. The acyl-transfer-mediated approach for synthesis of peptide thioesters tolerated different C-terminal residues and was used to synthesize human C–C motif chemokine 11 (hCCL11) via native chemical ligation.

Here, the helix–loop–helix transcription factor Ascl2 is shown to be critically important for the initiation of follicular T-helper-cell development and the germinal centre response. TFH (follicular T-helper) cells are specialized in helping B cells in the germinal centres — lymphoid organs involved in the proliferation, development and maturation of B lymphocytes. The transcription factor Bcl6 is required for TFH cell development but is thought not to control CXCR5 upregulation or T-cell migration to follicles. This study shows that a second transcription factor, Ascl2, directly regulates CXCR5 upregulation and CCR7 downregulation and is critically important for the initiation of TFH cell development and the germinal centre response. In immune responses, activated T cells migrate to B-cell follicles and develop into follicular T-helper (TFH) cells, a recently identified subset of CD4+ T cells specialized in providing help to B lymphocytes in the induction of germinal centres1,2. Although Bcl6 has been shown to be essential in TFH-cell function, it may not regulate the initial migration of T cells3 or the induction of the TFH program, as exemplified by C-X-C chemokine receptor type 5 (CXCR5) upregulation4. Here we show that expression of achaete-scute homologue 2 (Ascl2)—a basic helix–loop–helix (bHLH) transcription factor5—is selectively upregulated in TFH cells. Ectopic expression of Ascl2 upregulates CXCR5 but not Bcl6, and downregulates C-C chemokine receptor 7 (CCR7) expression in T cells in vitro, as well as accelerating T-cell migration to the follicles and TFH-cell development in vivo in mice. Genome-wide analysis indicates that Ascl2 directly regulates TFH-related genes whereas it inhibits expression of T-helper cell 1 (TH1) and TH17 signature genes. Acute deletion of Ascl2, as well as blockade of its function with the Id3 protein in CD4+ T cells, results in impaired TFH-cell development and germinal centre response. Conversely, mutation of Id3, known to cause antibody-mediated autoimmunity, greatly enhances TFH-cell generation. Thus, Ascl2 directly initiates TFH-cell development.