综述与专论: 免疫突触形成的生物学特点及其光学成像研究

免疫突触(immunological synapse,IS)是抗原提呈细胞与T细胞免疫识别时,多种分子参与、分阶段不断变化的过程,涉及黏附分子、细胞因子、信号传导分子、细胞骨架蛋白等多分子的聚集或离散．其形成不仅促进T细胞和抗原提呈细胞的稳定接触,而且激活T细胞信号传导途径,促进T细胞的活化和增殖．对IS的研究可以从分子水平解释免疫激活、免疫耐受、病原微生物感染与免疫细胞相互作用的机制,为进一步揭示疾病发生的分子机制,寻求疾病防治的靶向分子提供新的思路．近年来,光学成像的发展为可视化研究IS形成与T细胞活化的关系提供了有力帮助,为研究生理病理状态下的免疫应答提供了有力工具．     

Human immunodeficiency virus type 1 envelope gp120 induces a stop signal and virological synapse formation in noninfected CD4+ T cells

Human immunodeficiency virus type 1 (HIV-1)-infected T cells form a virological synapse with noninfected CD4⁺ T cells in order to efficiently transfer HIV-1 virions from cell to cell. The virological synapse is a specialized cellular junction that is similar in some respects to the immunological synapse involved in T-cell activation and effector functions mediated by the T-cell antigen receptor. The immunological synapse stops T-cell migration to allow a sustained interaction between T-cells and antigen-presenting cells. Here, we have asked whether HIV-1 envelope gp120 presented on a surface to mimic an HIV-1-infected cell also delivers a stop signal and if this is sufficient to induce a virological synapse. We demonstrate that HIV-1 gp120-presenting surfaces arrested the migration of primary activated CD4 T cells that occurs spontaneously in the presence of ICAM-1 and induced the formation of a virological synapse, which was characterized by segregated supramolecular structures with a central cluster of envelope surrounded by a ring of ICAM-1. The virological synapse was formed transiently, with the initiation of migration within 30 min. Thus, HIV-1 gp120-presenting surfaces induce a transient stop signal and supramolecular segregation in noninfected CD4⁺ T cells.     

Comparison of results using the gel microdrop cytokine secretion assay with ELISPOT and intracellular cytokine staining assay

The gel microdrop (GMD) secretion assay involves encapsulating single cells in a biotinylated agarose matrix, addition of a streptavidin bridge, diffusion of a biotinylated capture antibody, and detection of secreted molecules using a fluorescently labeled reporter antibody. Using flow cytometry, encapsulated cells can be analyzed or recovered based on cell type and secretory profile. Using murine Th2 cell line D10.G4.1 as a model, we recently demonstrated the feasibility of using the GMD cytokine secretion assay combined with flow cytometry to detect IL-4-producing cells after stimulation with the mitogen, Con A. In addition, subpopulations of encapsulated cells secreting IL-4 were simultaneously characterized by immunophenotyping. We found good correlation between results using the GMD cytokine secretion assay and results with the standard ELISPOT and intracellular cytokine (ICC) assays. The GMD cytokine secretion assay permits simultaneous detection of secreted cytokine and determination of cell surface phenotype on viable, single cells. Moreover, using fluorescence activated cell sorting (FACS), secreting cells of interest can be sorted, recovered, and cultured for further studies.     

Internalization of CD4 molecules in human T-cells demonstrated by immuno-electron microscopy.

Internalization of CD4 molecules on human CD4-enriched T-cells was demonstrated by immunocytochemical electron microscopy. CD4+ T-cell subclones were obtained from normal human peripheral blood, followed by one-way MLC screening and co-culturing with IL-2. Fixed and non-fixed T-cell samples were indirectly immunolabeled with mouse anti-human CD4 monoclonal antibody and goat anti-mouse IgG conjugated with peroxidase. Unfixed T-cells were immunolabeled at 4 degrees C and then re-incubated for 5-45 min at 37 degrees C. The selected CD4+ T-cell subclones showed strong CD4 binding on the cell surface after IL-2 incubation. However, fresh T-cells, monocytes, bone marrow cells and CD8+ T-cells all stained negative for CD4. The distribution of CD4 molecules on the fixed cell surface showed a homogeneous pattern. Capping and internalization of CD4-antibody-peroxidase complexes from the cell surfaces were observed follow a pathway of receptor-mediated endocytosis in unfixed T cells. Endocytotic vesicles, vacuoles of diverse sizes and shapes near the cell membrane or deep in the cell center were found to contain CD4 molecules. Negatively stained Golgi saccules were observed up to 45 min after re-incubation. These results suggest that increased CD4 molecules can be induced on the surface of normal human T-cells in vitro. Internalization and accumulation of CD4 molecules occurred in CD4-enriched T-cells with IL-2 pretreatment.     

Age-associated decline in effective immune synapse formation of CD4(+) T cells is reversed by vitamin E supplementation

Aging is associated with reduced IL-2 production and T cell proliferation. Vitamin E supplementation, in aged animals and humans, increases cell division and IL-2 production by naive T cells. The immune synapse forms at the site of contact between a T cell and an APC and participates in T cell activation. We evaluated whether vitamin E affects the redistribution of signaling proteins to the immune synapse. Purified CD4(+) T cells, from the spleens of young and old mice, were treated with vitamin E before stimulation with a surrogate APC expressing anti-CD3. Using confocal fluorescent microscopy, we observed that CD4(+) T cells from old mice were significantly less likely to recruit signaling proteins to the immune synapse than cells from young mice. Vitamin E increased the percentage of old CD4(+) T cells capable of forming an effective immune synapse. Similar results were found following in vivo supplementation with vitamin E. When compared with memory cells, naive T cells from aged mice were more defective in immune synapse formation and were more responsive to vitamin E supplementation. These data show, for the first time, that vitamin E significantly improves age-related early T cell signaling events in naive CD4(+) T cells.     

Cloning of Human T and Natural Killer Cells

The discovery of the lymphokine interleukin 2 (IL-2), which induces growth of T cells, set the stage for establishing methods forin vitrocloning of human T cells. More recently, it has become clear that in addition to IL-2, other T-cell growth factors, such as IL-4 and IL-7, can be used for generation and expansion of T-cell clones. In the past it was shown that it is possible to grow T cells differing in function and phenotype.In vitro-expanded T-cell clones have been instrumental in studies on the antigen specificities and biological properties of these cells. Cloned lines of natural killer (NK) cells can also be established using IL-2, permitting detailed studies on the biology of these cells. Here we describe the methods we use to generate and to expand human T-cell and NK cell clones. We discuss the differences between IL-2 and IL-4 as growth factors to expand T-cell clones. In addition, we summarize some recent findings of studies using T- and NK cell clones.     

Interleukin 1 secretion is not required for human macrophage support of T-cell proliferation

Interleukin 1 (IL-1) is a soluble factor secreted by stimulated monocytes (Mo) and animal macrophages (Mx). We have previously demonstrated that human Mo cultured in vitro for 1-6 days transform to Mx, and retain their ability to support concanavalin A (Con A)-driven T-cell proliferation. We have also shown that, paradoxically, these Mx do not secrete IL-1, when stimulated by endotoxin (LPS). In this study we examined two alternative hypotheses: T cells plus mitogen induce Mx IL-1 production, and human Mx deliver a second signal to T cells via a non-IL-1 mechanism. IL-1 was assayed in a mouse CD-1 thymocyte system without concanavalin A. Mo/Mx were cultured with T cells at low (2 X 10(4)/200 microliters) or high (1 X 10(5)/200 microliters) concentrations for 2 or 4 days, in the presence of Con A. Six hours prior to quantitation of proliferation, 50 microliters of supernatant was removed and assayed for IL-1. As expected both Mo and Mx enhanced T-cell proliferation eight- to tenfold. Mo secreted large amounts of IL-1; there was no demonstrable IL-1 activity present in supernatants from cultures containing either T cells and Mx, or Mx alone. Similar results were obtained by preincubating the cells (Mo, Mx, and T cells) with Con A for 12 hr and removing Con A prior to a 36-hr coculture. We examined the possibility that a small amount of IL-1 may be able to support Con A-stimulated T-cell proliferation and yet may not induce thymocyte proliferation. The highest dilutions of Mo supernatant (1:125) which supported T-cell proliferation also caused a fivefold increase in thymocyte proliferation. Supernatants from Mx failed to stimulate thymocyte proliferation or support Con A-driven T-cell proliferation. However, Mo and Mx lysates contain Il-1 activity. We conclude that human Mx support Con A-induced T-cell proliferation in the absence of IL-1 secretion. Mx may support T-cell proliferation by cell-bound IL-1 or by a non-IL-1 mechanism.     

The immunological synapse

T-cell activation requires interaction of T-cell antigen receptors with proteins of the major histocompatibility complex (antigen). This interaction takes place in a specialized cell-cell junction referred to as an immunological synapse. The immunological synapse contains at least two functional domains: a central cluster of engaged antigen receptors and a surrounding ring of adhesion molecules. The segregation of the T-cell antigen receptor (TCR) and adhesion molecules is based on size, with the TCR interaction spanning 15 nm and the lymphocyte-function-associated antigen-1 (LFA-1) interaction spanning 30-40 nm between the two cells. Therefore, the synapse is not an empty gap, but a space populated by both adhesion and signaling molecules. This chapter considers four aspects of the immunological synapse: the role of migration and stop signals, the role of the cytoskeleton, the role of self-antigenic complexes, and the role of second signals.     

Immunoreactive growth hormone production by human lymphocyte cell lines

1. Two human lymphocyte cell lines, a T-cell line and a B-cell line, were shown to produce and secrete immunoreactive growth hormone (irGH). The irGH molecules secreted by the two cell lines appeared to be de novo synthesized and their molecular size was similar to that of pituitary GH as well as irGH secreted by peripheral blood lymphocytes. 2. Affinity-purified irGH molecules had human growth hormone (hGH)-like mitogenic activity on Nb2 cells. These findings indicate that the irGH molecules produced by H9 and IM9 were similar to hGH in structure. 3. However, the irGH messages could not be amplified by polymerase chain reaction (PCR) primers which had been demonstrated to be able to amplify reverse-transcribed hGH messenger RNA successfully, suggesting that the lymphocyte-derived irGH and pituitary hGH are not exactly identical molecules. 4. We conclude that the H9 and IM9 cells produce a growth hormone-related molecule whose structure is different from that in the anterior pituitary.     

IL-4 plays a dominant role in the differential development of Tho into Th1 and Th2 cells.

We have analyzed the evolution of the pattern of lymphokine secretion by Th cell lines specific for either the synthetic terpolymer Glu60Ala30Tyr10 (GAT) or killed bacillus Calmette Guérin. When cultured in the presence of exogenous rIL-2 as a growth factor, GAT-specific Th cell lines secreted mainly IL-4, whereas bacillus Calmette Guérin-specific lines produced predominantly IL-2. However, culturing in the presence of rIL-4 or of anti-IL-4 mAb and rIL-2 led to the establishment of Th2-like and Th1-like lines, respectively, regardless of their Ag specificity. Inasmuch as we show that the proliferative response of mature Th1 and Th2 cells was identical in the presence of IL-4, these results indicate that IL-4 influences the development of Th cell subsets. To understand the mode of IL-4 action, we isolated immature GAT-specific Tho clones able to secrete IL-2 and IL-4. Two types of Tho cells were isolated: ThoA cells that secreted IL-2 and IL-4, but not IFN-gamma, and ThoB cells that secreted IL-2, IL-4, and IFN-gamma. We show for the first time that such cells are indeed Th precursors able to differentiate into Th1 or Th2 cells. We demonstrate that IL-4 positively and negatively controls the differentiation of Tho cells into Th2 and Th1 cells, respectively. When cultured in rIL-4, Tho cells stop secreting IL-2 and IFN-gamma, but maintain IL-4 secretion. Moreover, endogenous IL-4 produced by Tho cells has similar effects: when cultured in rIL-2 alone, Tho cells either keep their immature phenotype or become Th2 cells, but do not become Th1 cells. In contrast, neutralization of secreted IL-4 completely prevents the differentiation of Tho into Th2 cells, but permits the development of Th1 cells. The presence of exogenous IFN-gamma does not affect the development of Tho into Th1 and Th2 cells, because it does not modify either mode of IL-4 action. However, it influences the ratio between the two types of Tho cells: when IL-4 is neutralized, added IFN-gamma can induce IFN-gamma secretion by ThoA cells and thereby facilitate their passage into ThoB cells. Taken together, our results demonstrate that IL-4, in addition to mediating T cell growth, is a principal factor that controls the differential development of Tho cells into Th1 and Th2 cells.     

Secretion of mature mouse interleukin-2 by Saccharomyces cerevisiae: use of a general secretion vector containing promoter and leader sequences of the mating pheromone alpha-factor

We have constructed a general expression vector which allows the synthesis and secretion of processed gene products in Saccharomyces cerevisiae. This vector contains yeast DNA, including the promoter of the mating pheromone (alpha-factor), its downstream leader sequence, and the TRP5 terminator. A cDNA [encoding mature mouse interleukin-2 (IL-2); Yokota et al., Proc. Natl. Acad. Sci. USA 82 (1984) 68-72] was fused immediately downstream to the alpha-factor leader sequence. The resulting recombinant plasmid directed the synthesis of mature mouse IL-2 in S. cerevisiae, with most of the T-cell growth-factor (TCGF) activity secreted into the culture fluid and extracellular space. TCGF activities in the cell extract, as well as in the culture fluid, increased in parallel with cell growth. Production of mature mouse IL-2 was inhibited by tunicamycin (TM), with precursor molecules accumulating in the cell extract. The precursor was processed accurately at the junction between the alpha-factor peptide leader sequence and the coding sequence downstream, yielding mature IL-2. The Mr of the secreted mouse IL-2 determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was 17 kDal, a value expected for the mature mouse IL-2 polypeptide based on the nucleotide (nt) sequence.     

Internalization of CD4 molecules in human T-cells demonstrated by immuno-electron microscopy

Summary Internalization of CD4 molecules on human CD4-enriched T-cells was demonstrated by immunocytochemical electron microscopy. CD4⁺ T-cell subclones were obtained from normal human peripheral blood, followed by one-way MLC screening and co-culturing with IL-2. Fixed and non-fixed T-cell samples were indirectly immunolabeled with mouse anti-human CD4 monoclonal antibody and goat anti-mouse IgG conjugated with peroxidase. Unfixed T-cells were immunolabeled at 4° C and then re-incubated for 5–45 min at 37° C. The selected CD4⁺ T-cell subclones showed strong CD4 binding on the cell surface after IL-2 incubation. However, fresh T-cells, monocytes, bone marrow cells and CD8⁺ T-cells all stained negative for CD4. The distribution of CD4 molecules on the fixed cell surface showed a homogeneous pattern. Capping and internalization of CD4-antibody-peroxidase complexes from the cell surfaces were observed follow a pathway of receptor-mediated endocytosis in unfixed T cells. Endocytotic vesicles, vacuoles of diverse sizes and shapes near the cell membrane or deep in the cell center were found to contain CD4 molecules. Negatively stained Golgi saccules were observed up to 45 min after re-incubation. These results suggest that increased CD4 molecules can be induced on the surface of normal human T-cells in vitro. Internalization and accumulation of CD4 molecules occurred in CD4-enriched T-cells with IL-2 pretreatment.     

Modulating HIV-1 replication by RNA interference directed against human transcription elongation factor SPT5

Background

Dendritic cell (DC) transmission of human immunodeficiency virus (HIV) to CD4+ T cells occurs across a point of cell-cell contact referred to as the infectious synapse. The relationship between the infectious synapse and the classically defined immunological synapse is not currently understood. We have recently demonstrated that human B cells expressing exogenous DC-SIGN, DC-specific intercellular adhesion molecule-3 (ICAM-3)-grabbing nonintegrin, efficiently transmit captured HIV type 1 (HIV-1) to CD4+ T cells. K562, another human cell line of hematopoietic origin that has been extensively used in functional analyses of DC-SIGN and related molecules, lacks the principal molecules involved in the formation of immunological synaptic junctions, namely major histocompatibility complex (MHC) class II molecules and leukocyte function-associated antigen-1 (LFA-1). We thus examined whether K562 erythroleukemic cells could recapitulate efficient DC-SIGN-mediated HIV-1 transmission (DMHT).

Results

Here we demonstrate that DMHT requires cell-cell contact. Despite similar expression of functional DC-SIGN, K562/DC-SIGN cells were inefficient in the transmission of HIV-1 to CD4+ T cells when compared with Raji/DC-SIGN cells. Expression of MHC class II molecules or LFA-1 on K562/DC-SIGN cells was insufficient to rescue HIV-1 transmission efficiency. Strikingly, we observed that co-culture of K562 cells with Raji/DC-SIGN cells impaired DMHT to CD4+ T cells. The K562 cell inhibition of transmission was not directly exerted on the CD4+ T cell targets and required contact between K562 and Raji/DC-SIGN cells.

Conclusions

DMHT is cell type dependent and requires cell-cell contact. We also find that the cellular milieu can negatively regulate DC-SIGN transmission of HIV-1 in trans.     

Characterization of a CD4-positive T-cell line derived from an athymic (nu/nu) mouse.

We have isolated a Thy-1+, CD3+, CD4+ T-cell line from the spleen of a 12-week-old nu/nu (nude) BALB/c mouse. The cell line is clonal, and it expresses an alpha beta T-cell antigen receptor. Upon activation, these cells secrete IL-2 but not IL-4, putting them in the Th1 category. The cells can be triggered to proliferate and secrete lymphokines in the presence of irradiated syngeneic or allogeneic splenic feeder cells that express a variety of MHC haplotypes. This response is MHC class II-specific, because it can be blocked by either anti-Ia or anti-CD4 antibodies. From the response pattern of this T-cell line, we conclude that it recognizes a common determinant on class II MHC antigens. This nude mouse T-lymphocyte presumably has not undergone thymic selection. Therefore its unique specificity may reflect both the bias of T-cell antigen receptor genes for encoding receptors that recognize MHC molecules and the requirement for functional thymic epithelial cells for the efficient education of a self-MHC-restricted repertoire.     

CTLA-4 differentially regulates the immunological synapse in CD4 T cell subsets

Primary murine Th1 and Th2 cells differ in the organization of the immunological synapse, with Th1 cells, but not Th2 cells, clustering signaling molecules at the T cell/B cell synapse site. We sought to determine whether differential costimulatory signals could account for the differences observed. We found that Th2 cells express higher levels of CTLA-4 than Th1 cells, and demonstrated that Th2 cells lacking CTLA-4 are now able to cluster the TCR with the same frequency as Th1 cells. Furthermore, reconstitution of CTLA-4 into CTLA-4-deficient Th2 cells, or into Th1 cells, inhibits the clustering of the TCR. We have also shown that Th2 cells, but not Th1 cells, show variations in the organization of the immunological synapse depending on levels of expression of CD80/CD86 on the APC. These studies demonstrate a unique role for CTLA-4 as a critical regulator of Th2 cells and the immunological synapse.     

Constitutive expression of TGF-bêta1, interleukin-6 and interleukin-8 by tumor cells as a major component of immune escape in human ovarian carcinoma

Tumors could use several mechanisms to coexist with the host's immune system or to protect themselves from an immune response. Thus, insufficient expression of cell surface molecules on tumor cells, which are important for T cell recognition or activation, could lead to induction of a state of tolerance. Tumor cells could also produce cytokines that would inhibit the immune response and allow tumor progression. Here, we studied, in vitro, the cell surface expression of immunologically important molecules in seven ovarian carcinoma (OVCA) cell lines and the constitutive expression of cytokines. All OVCA cell lines expressed MHC class I molecules, ICAM-1 and LFA-3 adhesion molecules, necessary to induce a specific cytotoxic T-cell response, as well as the CD40 costimulatory molecules. Conversely, the lack of the dominant costimulatory molecules, CD80 (B7.1) and CD86 (B7.2) could be a possible explanation of poor immunogenicity of OVCA tumors. Immunosuppressive TGF-beta1 was detected at the mRNA level in all cell lines but was weakly secreted in supernatants. By contrast, IL-10 was never found. Most of them constitutively produced IL-8 and IL-6, two cytokines known as tumor promoting factors whereas the proinflammatory cytokines TNF-alpha, IL-1beta and GM-CSF were rarely produced. Data from this study could be useful for designing new strategies of immunotherapy to improve immunogenicity and/or limit protumor cytokine production.     

Differences in signaling molecule organization between naive and memory CD4+ T lymphocytes

The immunological synapse is a highly organized complex formed at the junction between Ag-specific T cells and APCs as a prelude to cell activation. Although its exact role in modulating T cell signaling is unknown, it is commonly believed that the immunological synapse is the site of cross-talk between the T cell and APC (or target). We have examined the synapses formed by naive and memory CD4 cells during Ag-specific cognate interactions with APCs. We show that the mature immunological synapse forms more quickly during memory T cell activation. We further show that the composition of the synapse found in naive or memory cell conjugates with APCs is distinct with the tyrosine phosphatase, CD45, being a more integral component of the mature synapses formed by memory cells. Finally, we show that signaling molecules, including CD45, are preassociated in discrete, lipid-raft microdomains in resting memory cells but not in naive cells. Thus, enhanced memory cell responses may be due to intrinsic properties of signaling molecule organization.     

T-cell activation. IV. Evidence for a functional linkage between MHC class I, interleukin-2 receptor, and interleukin-4 receptor molecules.

The aim of the present work was to study regulatory interactions between MHC class I molecules and the interleukin (IL)-2, IL-3, and IL-4 receptors and functional interactions between the receptors for IL-2 and IL-4. Our major observations were: (1) quiescent splenic T cells exposed to specific anti-MHC class I antibodies become responsive to IL-2 and IL-4 stimulation; (2) T-cell clones (CTLL-2 and HT-1) grown at high cell density or low IL-2 concentrations become refractory to IL-2 and IL-4 stimulation. After exposure to anti-class I antibodies the refractory cells recover responsiveness to lymphokine-induced proliferation; (3) IL-2 receptor expression is non-inducible in class I-negative T-lymphoma cells, but is inducible following class I gene transfection of the cells; (4) exposure of T-cells and clones to IL-2 receptor antibody increases the responsiveness to IL-4 stimulation; (5) IL-2 and IL-4 act synergistically at low and substimulatory lymphokine levels; and (6) IL-3 responsiveness of hemopoietic cells is not influenced by exposure to anti-MHC class I antibody. It is concluded that class I molecules are of importance for the functional expression of the receptors for IL-2 and IL-4 and that these receptors are functionally interrelated.     

Fish oil disrupts MHC class II lateral organization on the B-cell side of the immunological synapse independent of B-T cell adhesion

Fish oil-enriched long chain n-3 polyunsaturated fatty acids disrupt the molecular organization of T-cell proteins in the immunological synapse. The impact of fish oil derived n-3 fatty acids on antigen-presenting cells, particularly at the animal level, is unknown. We previously demonstrated B-cells isolated from mice fed with fish oil-suppressed naïve CD4⁺ T-cell activation. Therefore, here we determined the mechanistic effects of fish oil on murine B-cell major histocompatibility complex (MHC) class II molecular distribution using a combination of total internal reflection fluorescence, Förster resonance energy transfer and confocal imaging. Fish oil had no impact on presynaptic B-cell MHC II clustering. Upon conjugation with transgenic T-cells, fish-oil suppressed MHC II accumulation at the immunological synapse. As a consequence, T-cell protein kinase C theta (PKCθ) recruitment to the synapse was also diminished. The effects were independent of changes in B-T cell adhesion, as measured with microscopy, flow cytometry and static cell adhesion assays with select immune ligands. Given that fish oil can reorganize the membrane by lowering membrane cholesterol levels, we then compared the results with fish oil to cholesterol depletion using methyl-B-cyclodextrin (MβCD). MβCD treatment of B-cells suppressed MHC II and T-cell PKCθ recruitment to the immunological synapse, similar to fish oil. Overall, the results reveal commonality in the mechanism by which fish oil manipulates protein lateral organization of B-cells compared to T-cells. Furthermore, the data establish MHC class II lateral organization on the B-cell side of the immunological synapse as a novel molecular target of fish oil.