免疫小鼠脾脏中抗原提呈细胞的快速分离

应用胶原酶消化脾脏、 Ｂ Ｓ Ａ 梯度离心富集低浮密度细胞和 Ｆ Ａ Ｃ Ｓ分选细胞法,自小鼠脾脏中分离出高纯度树突细胞、吞噬细胞和 Ｂ淋巴细胞。分离的 ３ 种细胞样品中分别含 ９７％ 树突细胞、９６％ 吞噬细胞和 ９９％ Ｂ淋巴细胞。而纯化前,树突细胞和吞噬细胞在低浮密度细胞中仅分别占 ６５％ 和 ３５％ , Ｂ 淋巴细胞占脾细胞的 ４２％ 左右。该程序简便、快速、准确,在一个工作日内可制备大量细胞, 并适用于其他淋巴组织中抗原提呈细胞的纯化。     

A role for CD40 expression on CD8+ T cells in the generation of CD8+ T cell memory

The delivery of CD4 help to CD8+ T cell responses requires interactions between CD40 and CD40 ligand and is thought to occur through antigen-presenting cell (APC) activation. Here we show that generation of memory CD8+ T cells displaying an enhanced capacity for cell division and cytokine secretion required CD4 help but not CD40 expression by the APCs. Activated CD4+ and CD8+ T cells expressed CD40; and in the absence of this protein, CD8+ T cells were unable to differentiate into memory cells or receive CD4 help. These results suggest that, like B cells, CD8+ T cells receive CD4 help directly through CD40 and that this interaction is fundamental for CD8+ T cell memory generation.     

Distinct pathways of antigen uptake and intracellular routing in CD4 and CD8 T cell activation

The mechanisms that allow antigen-presenting cells (APCs) to selectively present extracellular antigen to CD8+ effector T cells (cross-presentation) or to CD4+ T helper cells are not fully resolved. We demonstrated that APCs use distinct endocytosis mechanisms to simultaneously introduce soluble antigen into separate intracellular compartments, which were dedicated to presentation to CD8+ or CD4+ T cells. Specifically, the mannose receptor supplied an early endosomal compartment distinct from lysosomes, which was committed to cross-presentation. These findings imply that antigen does not require intracellular diversion to access the cross-presentation pathway, because it can enter the pathway already during endocytosis.     

Differential antigen processing by dendritic cell subsets in vivo

Dendritic cells (DCs) process and present self and foreign antigens to induce tolerance or immunity. In vitro models suggest that induction of immunity is controlled by regulating the presentation of antigen, but little is known about how DCs control antigen presentation in vivo. To examine antigen processing and presentation in vivo, we specifically targeted antigens to two major subsets of DCs by using chimeric monoclonal antibodies. Unlike CD8+ DCs that express the cell surface protein CD205, CD8- DCs, which are positive for the 33D1 antigen, are specialized for presentation on major histocompatibility complex (MHC) class II. This difference in antigen processing is intrinsic to the DC subsets and is associated with increased expression of proteins involved in MHC processing.     

Differential lysosomal proteolysis in antigen-presenting cells determines antigen fate

Antigen-presenting cells (APCs) internalize antigens and present antigen-derived peptides to T cells. Although APCs have been thought to exhibit a well-developed capacity for lysosomal proteolysis, here we found that they can exhibit two distinct strategies upon antigen encounter. Whereas macrophages contained high levels of lysosomal proteases and rapidly degraded internalized proteins, dendritic cells (DCs) and B lymphocytes were protease-poor, resulting in a limited capacity for lysosomal degradation. Consistent with these findings, DCs in vivo degraded internalized antigens slowly and thus retained antigen in lymphoid organs for extended periods. Limited lysosomal proteolysis also favored antigen presentation. These results help explain why DCs are able to efficiently accumulate, process, and disseminate antigens and microbes systemically for purposes of tolerance and immunity.     

小鼠骨髓源CD103+DC分离培养及LPS对其形态与功能特征的影响

旨在建立C57BL/6小鼠骨髓源CD103⁺树突状细胞(CD103⁺ dendritic cell,CD103⁺DC)分离培养方法,阐述LPS对其形态与功能特征的影响。在无菌条件下分离C57BL/6小鼠骨髓细胞,并用重组GM-CSF和FLT3L对其进行体外联合诱导培养;利用光镜、扫描电镜、荧光显微镜和流式细胞术,分别对LPS作用前后细胞形态、表型及功能进行了分析。结果表明,细胞培养至第3天有零星集落出现,第13天后集落开始分散,可见典型的树突状突起,第15天后可得到大量的CD103⁺DC,加LPS刺激培养24 h后细胞表面树突样结构更加明显;分离培养的骨髓细胞能够表达表面分子CD103,其表达率达90%以上。RPMI-1640组(LPS未刺激组)可吞噬VOA的CD103⁺DC比例为25.70%,能够表达MHC-Ⅱ和CD83阳性细胞分别为41.31%和13.79%;LPS刺激组可吞噬VOA的CD103⁺DC比例为10.33%,能够表达MHC-Ⅱ和CD83的阳性细胞分别为68.10%和24.71%。MTT法检测结果显示,经LPS处理的CD103⁺DC刺激T细胞增殖的能力明显增强。综上所述,分离于C57BL/6小鼠的骨髓细胞,可在体外经FLT3L和GM-CSF共同诱导培养出CD103⁺DC,LPS可促进CD103⁺DC的成熟。     

Development of CD8alpha-positive dendritic cells from a common myeloid progenitor

Dendritic cells (DCs) are critical in both initiating adaptive immune responses and maintaining tolerance to self antigens. These apparently contradictory roles have been suggested to depend on different subsets of DCs that arise from either myeloid or lymphoid hematopoietic origins, respectively. Although DC expression of CD8alpha is attributed to a lymphoid origin, here we show that both CD8alpha+ and CD8alpha- DCs can arise from clonogenic common myeloid progenitors in both thymus and spleen. Thus, expression of CD8alpha is not indicative of a lymphoid origin, and phenotypic and functional differences among DC subsets are likely to reflect maturation status rather than ontogeny.     

TLR7配体Loxoribine对调节性T细胞抑制作用的解除

调节性T细胞是机体免疫功能的重要调节因素,近年来的研究提示Toll样受体可以直接或间接的调控调节性T细胞的抑制功能。作者的研究显示:在CD4、Treg和DC反应体系中加入TLR7配体Loxoribine后CD4 T细胞的增殖增强,Treg的抑制功能丧失;进一步用Loxoribine分别处理CD4 T细胞、Treg、和DC细胞后发现,Loxoribine处理的DC,一方面使CD4 T细胞增殖增强,一方面使Treg抑制功能丧失;且通过trans-well试验证明Loxoribine作用于DC使Treg抑制功能丧失是通过DC释放的可溶性分子介导的,而不是通过细胞与细胞之间的接触来实现的。     

Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells

Toll-like receptors (TLRs) control activation of adaptive immune responses by antigen-presenting cells (APCs). However, initiation of adaptive immune responses is also controlled by regulatory T cells (TR cells), which act to prevent activation of autoreactive T cells. Here we describe a second mechanism of immune induction by TLRs, which is independent of effects on costimulation. Microbial induction of the Toll pathway blocked the suppressive effect of CD4+CD25+ TR cells, allowing activation of pathogen-specific adaptive immune responses. This block of suppressor activity was dependent in part on interleukin-6, which was induced by TLRs upon recognition of microbial products.     

Split anergy in a CD8+ T cell: receptor-dependent cytolysis in the absence of interleukin-2 production

Engagement of the antigen-specific receptor (TCR) of CD4+ T lymphocytes without a second (costimulatory) signal prevents the subsequent production of interleukin-2 (IL-2) by these cells. Because IL-2 is a key immunoregulatory lymphokine and is also produced by a subset of CD8+ T cells that are able to kill target cells, the effect of engaging the TCR of one such clone in the absence of costimulatory signals was examined. The capacity for TCR-dependent IL-2 production was lost, indicating comparable costimulator-dependent signaling requirements for IL-2 production in CD4+ and CD8+ T cells. However, TCR-mediated cytotoxicity was not impaired, implying that costimulation is required for only certain TCR-dependent effector functions.     

Reciprocal control of T helper cell and dendritic cell differentiation

It is not known whether subsets of dendritic cells provide different cytokine microenvironments that determine the differentiation of either type-1 T helper (TH1) or TH2 cells. Human monocyte (pDC1)-derived dendritic cells (DC1) were found to induce TH1 differentiation, whereas dendritic cells (DC2) derived from CD4+CD3-CD11c- plasmacytoid cells (pDC2) induced TH2 differentiation by use of a mechanism unaffected by interleukin-4 (IL-4) or IL-12. The TH2 cytokine IL-4 enhanced DC1 maturation and killed pDC2, an effect potentiated by IL-10 but blocked by CD40 ligand and interferon-gamma. Thus, a negative feedback loop from the mature T helper cells may selectively inhibit prolonged TH1 or TH2 responses by regulating survival of the appropriate dendritic cell subset.     

CD8alphaalpha-mediated survival and differentiation of CD8 memory T cell precursors

Memory T cells are long-lived antigen-experienced T cells that are generally accepted to be direct descendants of proliferating primary effector cells. However, the factors that permit selective survival of these T cells are not well established. We show that homodimeric alpha chains of the CD8 molecule (CD8alphaalpha) are transiently induced on a selected subset of CD8alphabeta+ T cells upon antigenic stimulation. These CD8alphaalpha molecules promote the survival and differentiation of activated lymphocytes into memory CD8 T cells. Thus, memory precursors can be identified among primary effector cells and are selected for survival and differentiation by CD8alphaalpha.     

Thymic origin of intestinal alphabeta T cells revealed by fate mapping of RORgammat+ cells

Intestinal intraepithelial T lymphocytes (IELs) are likely to play a key role in host mucosal immunity and, unlike other T cells, have been proposed to differentiate from local precursors rather than from thymocytes. We show here that IELs expressing the alphabeta T cell receptor are derived from precursors that express RORgammat, an orphan nuclear hormone receptor detected only in immature CD4+CD8+ thymocytes, fetal lymphoid tissue-inducer (LTi) cells, and LTi-like cells in cryptopatches within the adult intestinal lamina propria. Using cell fate mapping, we found that all intestinal alphabeta T cells are progeny of CD4+CD8+ thymocytes, indicating that the adult intestine is not a significant site for alphabeta T cell development. Our results suggest that intestinal RORgammat+ cells are local organizers of mucosal lymphoid tissue.     

Epidermal viral immunity induced by CD8alpha+ dendritic cells but not by Langerhans cells

The classical paradigm for dendritic cell function derives from the study of Langerhans cells, which predominate within skin epidermis. After an encounter with foreign agents, Langerhans cells are thought to migrate to draining lymph nodes, where they initiate T cell priming. Contrary to this, we show here that infection of murine epidermis by herpes simplex virus did not result in the priming of virus-specific cytotoxic T lymphocytes by Langerhans cells. Rather, the priming response required a distinct CD8alpha+ dendritic cell subset. Thus, the traditional view of Langerhans cells in epidermal immunity needs to be revisited to accommodate a requirement for other dendritic cells in this response.     

Transport of peptide-MHC class II complexes in developing dendritic cells

Major histocompatibility complex class II (MHC II) molecules capture peptides within the endocytic pathway to generate T cell receptor (TCR) ligands. Immature dendritic cells (DCs) sequester intact antigens in lysosomes, processing and converting antigens into peptide-MHC II complexes upon induction of DC maturation. The complexes then accumulate in distinctive, nonlysosomal MHC II+ vesicles that appear to migrate to the cell surface. Although the vesicles exclude soluble lysosomal contents and antigen-processing machinery, many contain MHC I and B7 costimulatory molecules. After arrival at the cell surface, the MHC and costimulatory molecules remain clustered. Thus, transport of peptide-MHC II complexes by DCs not only accomplishes transfer from late endocytic compartments to the plasma membrane, but does so in a manner that selectively concentrates TCR ligands and costimulatory molecules for T cell contact.     

Regulation of antigen-specific CD8+ T cell homeostasis by perforin and interferon-gamma

T cell memory depends on factors that regulate expansion and death of these cells after antigenic stimulation. Mice deficient in perforin and interferon-gamma (IFN-gamma) exhibited increased expansion, altered immunodominance, and decreased death of antigen-specific CD8+ T cells after infection with an attenuated strain of Listeria monocytogenes, which was cleared from these mice. Expansion of CD8+ T cells was controlled by perforin, whereas IFN-gamma regulated immunodominance and the death phase. Thus, perforin and IFN-gamma regulate distinct elements of CD8+ T cell homeostasis independently of their role as antimicrobial effector molecules.     

Use of CD134 as a primary receptor by the feline immunodeficiency virus

Feline immunodeficiency virus (FIV) induces a disease similar to acquired immunodeficiency syndrome (AIDS) in cats, yet in contrast to human immunodeficiency virus (HIV), CD4 is not the viral receptor. We identified a primary receptor for FIV as CD134 (OX40), a T cell activation antigen and costimulatory molecule. CD134 expression promotes viral binding and renders cells permissive for viral entry, productive infection, and syncytium formation. Infection is CXCR4-dependent, analogous to infection with X4 strains of HIV. Thus, despite the evolutionary divergence of the feline and human lentiviruses, both viruses use receptors that target the virus to a subset of cells that are pivotal to the acquired immune response.