不同大鼠胶质瘤抗原致敏的DC体外诱导CTL活性的研究

目的: 比较大鼠骨髓来源的树突状细胞(dendritic cells, DC)体外经由大鼠C6胶质瘤细胞由不同方式制备的不同抗原致敏后,对特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte, CTL)的诱导作用。方法: 自大鼠骨髓分离DC前体细胞,经重组大鼠粒细胞巨噬细胞集落刺激因子(rrGM-CSF)+白细胞介素4(rrIL-4)诱导培养、扩增;由C6胶质瘤细胞经由反复冻融、煮沸灭活及超声破碎细胞抽提其总蛋白的方法制备各种不同抗原致敏DC,致敏的DC与T淋巴细胞进行共培养诱导CTL;以ELISA法检测CTL诱导过程中淋巴细胞趋化因子(lymphocyte chemoattractant factor)及细胞因子IFN-γ分泌水平:以 -TdR掺入法检测DC诱导T细胞增殖及其特异性CTL杀伤活性。 结果: 体外应用煮沸灭活瘤细胞制备的肿瘤抗原致敏DC,能诱导更强的刺激T细胞增殖的能力、并且可以诱导杀伤活性更强的CTL。结论: 应用煮沸灭活的瘤细胞制备瘤抗原负载DC获得瘤苗可获得更强的抗肿瘤保护作用。     

Modification of tumor cells by a low dose of Newcastle disease virus. Augmentation of the tumor-specific T cell response in the absence of an anti-viral response

The present study elucidates the mechanism whereby viral xenogenization of highly metastatic ESb lymphoid tumor cells increases tumor immunogenicity and syngeneic tumor-specific T cell responses in comparison to nonmodified tumor cells. It was found that the frequency of cytotoxic T lymphocytes specific for the Esb tumor-associated transplantation antigen (TATA) and the cytotoxic anti-tumor activity in bulk cultures of immune spleen cells were significantly increased (by factor 3 and 25, respectively) when using virus-modified tumor cells. An amplified response was observed both in vivo and in vitro which might explain the demonstrated effectiveness of this approach for postoperative immunotherapy of ESb metastases. For the stimulation of tumor-specific cytolytic T lymphocytes (CTL) the ESb tumor cells which are highly metastatic were infected with an avirulent strain of the paramyxovirus Newcastle Disease Virus (NDV). Infection of ESb cells with low amounts of NDV was sufficient to lead to an increase in cytolytic activity of tumor-specific CTL after sensitization in vivo and restimulation in vitro. In a sensitive limiting dilution mixed leukocyte-tumor cell microculture system the direct effect of viral modification on the frequency and specificity of CTL was investigated. The number of ESb-specific CTL per spleen could be raised from about 3300 (without modification) to 9100 by both in vivo and in vitro application of ESb-NDV. One application of ESb-NDV (in vivo or in vitro) increased the number of CTL to 4900 and 4600, respectively. In split-type experiments it could be shown at the clonal level that viral modification did not alter the specificity of ESb-specific CTL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cross-presentation of antigens from apoptotic tumor cells by liver sinusoidal endothelial cells leads to tumor-specific CD8+ T cell tolerance

Development of tumor-specific T cell tolerance contributes to the failure of the immune system to eliminate tumor cells. Here we report that hematogenous dissemination of tumor cells followed by their elimination and local removal of apoptotic tumor cells in the liver leads to subsequent development of T cell tolerance towards antigens associated with apoptotic tumor cells. We provide evidence that liver sinusoidal endothelial cells (LSEC) remove apoptotic cell fragments generated by induction of tumor cell apoptosis through hepatic NK1.1+ cells. Antigen associated with apoptotic cell material is processed and cross-presented by LSEC to CD8+ T cells, leading to induction of CD8+ T cell tolerance. Adoptive transfer of LSEC isolated from mice challenged previously with tumor cells promotes development of CD8+ T cell tolerance towards tumor-associated antigen in vivo. Our results indicate that hematogenous dissemination of tumor cells, followed by hepatic tumor cell elimination and local cross-presentation of apoptotic tumor cells by LSEC and subsequent CD8+ T cell tolerance induction, represents a novel mechanism operative in tumor immune escape.     

Induction of T cell responses against autologous ovarian tumors with whole tumor cell lysate-pulsed dendritic cells.

The loading of dendritic cells (DCs) with whole tumor cell lysates may circumvent the facts that few tumor-specific antigens have been identified in human solid tumors. The present study was designed to investigate whether ovarian cancer cells lysate-pulsed DCs activate T cell responses against autologous ovarian tumors. Incubation of T cells with autologous tumor cell lysate-pulsed DCs stimulated proliferation of autologous T cells. T cells primed by autologous tumor cell lysate-pulsed DCs showed significant killing activity against autologous tumor cells, which could be blocked by anti-MHC-class-I and anti-CD8 mAb. By contrast, T cells primed by autologous unpulsed DCs alone or tumor lysates alone failed to exhibit significant killing activity. In addition, T cells primed by DCs pulsed with allogeneic tumor cell lysates or with autologous normal cell lysate or by these cell lysates alone did not induce the increase in the autologous tumor killing activity. As additional controls, T cells stimulated with autologous tumor lysate-pulsed DCs express no increase in the lysis of autologous monocytes, allogeneic ovarian tumor cells and other cell lines including K562, Daudi and Molt-4. Furthermore, T cells stimulated with autologous tumor lysate-pulsed DCs could produce the considerable amounts of cytokines such as GM-CSF, TNF-alpha and IFN-gamma. The data in the present study suggest that whole tumor cell lysates-pulsed DCs could activate T cell responses against autologous ovarian tumor cells, and that these pulsed DCs may be used as a new approach for the specific immunotherapy of ovarian cancer patients.     

Induction of Th1 and Th2 CD4+ T cell responses by oral or parenteral immunization with ISCOMS

We examined the ability of oral or parenteral immunization with immune stimulating complexes containing ovalbumin (ISCOMS-OVA) to prime T cell proliferative and cytokine responses. A single subcutaneous immunization with ISCOMS-OVA primed potent antigen-specific proliferative responses in the draining popliteal lymph node, which were entirely dependent on the presence of CD4⁺ T cells. CD8⁺ T cells did not proliferate in vitro even in the presence of the appropriate peptide epitope and exogenous interleukin (IL)-2. Primed popliteal lymph node cells produced IL-2, IL-5 and interferon (IFN)-γ, but not IL-4 when restimulated with OVA in vitro. Serum antigen-specific IgG1 and IgG2a antibody responses were also primed by subcutaneous immunization with ISCOMS-OVA, confirming the stimulation of both Th1 and Th2 cells in vivo. Spleen cells from subcutaneously primed mice produced a similar pattern of cytokines, indicating that disseminated priming had occurred. Oral immunization with ISCOMS-OVA also primed local antigen-specific proliferative responses in the mesenteric lymph node and primed an identical pattern of systemic cytokine responses in the spleen. The ability of ISCOMS to prime both Th1 and Th2 CD4⁺ T cell responses may be central to their potent adjuvant activities and confirm the potential of ISCOMS as future oral vaccine vectors.     

Induction of a Th17 cell response by Helicobacter pylori Urease subunit B

Th17 cells represent a novel subset of CD4⁺ T cells, which is associated with Helicobacter pylori infection. In the present study, we investigated the potential role of Urease subunit B (UreB) in the induction of Th17 cell response. Co-cultured splenic lymphocytes from H. pylori-infected mice with the recombinant UreB (rUreB) elevated IL-17 secretion and caused an increase in the number of Th17 cells. The expression of IL-6 and IL-23 p19 was significantly increased in rUreB-stimulated macrophages. Whole cell protein (WCP) of UreB-deficient strain (UreB⁻ strain) induced less Th17 cell responses than that of wild-type strain. In addition, subcutaneous and intranasal immunization of rUreB elicited antigen-specific Th17 cell responses. Intranasal immunization of rUreB reduced H. pylori colonization in the stomach, which was closely related with the increased rUreB-specific Th17 cell responses. These results suggest that UreB is an important protein which is able to elicit Th17 cell responses against H. pylori both in vivo and in vitro.     

Consequences of antigen self-presentation by tumor-specific cytotoxic T cells

CD8-positive cytotoxic T cells (CTL) recognize antigenic peptides in combination with major histocompatibility complex (MHC) class I molecules on the surface of syngeneic antigen presenting cells (APC). In the present paper we show that cells from tumor antigen-specific CTL clones present their cognate antigenic peptide to other CTL from the same clone. Inter-CTL peptide presentation resulted in activation of the cells of one CTL clone to MHC-unrestricted lysis of bystander cells. In contrast to the behaviour of this clone, another CTL clone did not lyse bystander cells after incubation with the cognate peptide, but was activated to self-destruction. The human herpes virus Epstein-Barr virus is involved in the pathogenesis of a broad spectrum of human neoplasias. Using freshly established non-clonal T cells with specificity for a peptide derived from an Epstein-Barr virus encoded antigen we found again lysis of MHC mismatched bystander cells as a consequence of inter-CTL peptide presentation, indicating that bystander lysis following antigen self-presentation is not a phenomenon restricted to long-term in vitro cultured T cell clones. The potential implications for immunosurveillance against cancer and for tumor escape mechanisms are discussed.     

Expression of heat-stable antigen on tumor cells provides co-stimulation for tumor-specific T cell proliferation and cytotoxicity in mice

Heat-stable antigen (HSA/J11d/possibly homologous to CD24), a cell adhesion molecule capable of providing a co-stimulatory signal for T cell proliferation, is expressed on B cells, activated T cells, monocytes, granulocytes, Langerhans cells and thymocytes. Recent studies have demonstrated that co-stimulatory signals provided by cell adhesion molecules such as B7-1 play an essential role in generation of an anti-tumor immune response. To examine whether the co-stimulatory signal provided by HSA can induce an anti-tumor immune response, we have transfected HSA cDNA into the murine melanoma cell line K1735M2, and examined the ability of this transfected cell line to induce tumor-specific T cell responses. The results demonstrate that spleen cells from mice immunized with HSA-transfected K1735M2 cells showed enhanced T cell proliferation in a mixed lymphocyte tumor reaction (MLTR) assay and also demonstrated a significant anti-tumor cytotoxicity to the parent tumor cell (K1735M2). This anti-tumor cytolytic activity could be abrogated by pretreatment of effector cells with anti-mouse CD8 monoclonal antibody and complement. Under similar conditions, spleen cells from C3H mice immunized with vector-transfected K1735M2 cells neither actively proliferate in an MLTR assay, nor did they exert significant cytolytic activity against the respective tumor cells. In summary, our study demonstrated that HSA can provide a co-stimulatory signal for the T cell immune response against tumor cells in a murine model.     

Lack of Th1 or Th2 polarization of CD4+ T cell response induced by particulate antigen targeted to phagocytic cells

Several factors are involved in the selective activation of Th1 or Th2 subset of CD4+ T cells, such as the type of antigen-presenting cells, the dose of antigen, the route of immunization, etc. To analyze the influence of accessory cells on Th1/Th2 cell differentiation, we used a particulate antigen prepared by covalent linkage of hemocyanin (LH) to 1 microns synthetic microspheres. This particulate antigen was efficiently presented to T cells by macrophages but not by B lymphocytes. BALB/c mice immunized either with soluble LH in alum or with particulate LH without adjuvant produced both Th1 (IL-2 and IFN- gamma) and Th2 (IL-4 and IL-5) cytokines. Moreover, mice primed either with soluble or particulate LH secreted higher levels of IgG1- than of IgG2a-specific antibodies. The induction of this cytokine profile response was independent of the route of administration of the antigen, and was observed both in BALB/c and C57BL/6 mice. In contrast, immunization of mice with particulate LH in the presence of poly(I):(C) or of IL-12 induced a strong activation of Th1 cells, as shown by an up- regulated IFN-gamma production, and by decreased IL-4 and IL-5 levels associated to a greatly enhanced IgG2a antibody response. These results therefore demonstrate that targeting the antigen to phagocytic cells is not sufficient to stimulate a polarized Th response and that environmental cytokines play the major role in the selective activation of Th1 cells. This study provides important conclusions for the development of new vaccines and shows that particulate antigen associated with appropriate cofactor can selectively activate Th1 cells.      

Homing phenotypes of tumor-specific CD8 T cells are predetermined at the tumor site by crosspresenting APCs

Expression of tissue-specific homing molecules directs antigen-experienced T cells to particular peripheral tissues. In studies using soluble antigens that focused on skin and gut, antigen-presenting cells (APCs) within regional lymphoid tissues were proposed to be responsible for imprinting homing phenotypes. Whether this occurs in other sites and after physiologic antigen processing and presentation is unknown. We define in vivo imprinting of distinct homing phenotypes on monospecific T cells responding to antigens expressed by tumors in intracerebral, subcutaneous, and intraperitoneal sites with efficient brain-tropism of CD8 T cells crossprimed in the cervical lymph nodes (LNs). Multiple imprinting programs could occur simultaneously in the same LN when tumors were present in more than one site. Thus, the identity of the LN is not paramount in determining the homing phenotype; this critical functional parameter is dictated upstream at the site of antigen capture by crosspresenting APCs.     

Induction of regular cytolytic T cell synapses by bispecific single-chain antibody constructs on MHC class I-negative tumor cells

Certain bispecific single-chain antibody constructs exhibit an extraordinary potency for polyclonal T cell engagement and target cell lysis. Here we studied the structural basis for this potency, using laser scanning confocal microscopy. Cytolytic human T cell synapses could be triggered either by addition of a specific peptide antigen or an Ep-CAM-/CD3-bispecific T cell engager (BiTE). Both kinds of synapses showed a comparable distribution of all protein markers investigated. Two other BiTEs constructed from different Ep-CAM-specific antibodies gave similar results. BiTEs could also induce lytic synapses between human T cells and a MHC class I-negative, Ep-CAM cDNA-transfected cell line resulting in potent target cell lysis. This shows that certain T cell recognition molecules on target cells are dispensable for synapse formation and BiTE activity, and suggests that BiTE-activated polyclonal T cells may ignore major immune evasion mechanisms of tumor cells in vivo, such as loss of MHC class I expression.     

In vivo tracking of tumor-specific T cells

Novel immunologic assays now enable visualization of the antigen-specific response to an extent not previously possible. Assessment of not only the numeric frequency but also the functional properties of individual tumor-specific T cells in the endogenous and manipulated immune response has provided insights that will facilitate the development of immunotherapeutic strategies.     

Defective lymphokine production by most CD8+ and CD4+ tumor-specific T cell clones derived from human melanoma-infiltrating lymphocytes in response to autologous tumor cells in vitro

Human melanomas are infiltrated by tumor-reactive T lymphocytes. However, the ability of these cells to elicit a specific anti-tumor response in vivo remains to be established. Because lymphokine production is critical for T cell functions, we have analyzed the capacity of melanoma-specific tumor-infiltrating lymphocyte (TIL) clones to produce major lymphokines: interleukin-2 (IL-2), interferon-γ (IFN-γ) and interleukin-4 (IL-4), as well as tumor necrosis factor (TNF), in response to direct antigen presentation by autologous and allogeneic tumor cells. We report here that, upon stimulation by autologous melanoma cells, all TIL clones secreted TNF but only a few of them produced significant amounts of IL-2, IL-4 or IFN-γ. Nonetheless, all these clones consistently produced two or three of these last lymphokines upon stimulation with phorbol myristate acetate and calcium ionophore, as well as IL-2 upon CD3 stimulation, showing the existence of three lymphokine profiles among them: Th1, Th0 and a profile characterized by IL-2 and IL-4, but not IFN-γ secretion. Stimulation of TIL clones by allogeneic melanoma lines sharing the appropriate HLA-peptide complexes revealed that defective IL-2 production seemed to be a constant feature for some clones, while it was, for other clones, dependent on the antigen-presenting tumor cells. For this last type of clone, we further showed that defective IL-2 induction resulted from an LFA-3 defect of some melanoma cells or from distinct yet undefined defects of other melanoma lines. Our data suggest that defective lymphokine secretion may be an essential component of the in vivo failure of melanoma-reactive TIL to control tumor development. Interestingly both CD4⁺ and CD8⁺ TIL clones from one patient were fully activated by the autologous melanoma cells in vitro, supporting a potential role of such TIL in spontaneous or induced tumor rejection.     

Induction of Th2 cell differentiation in the primary immune response: dendritic cells isolated from adherent cell culture treated with IL-10 prime naive CD4+ T cells to secrete IL-4

A number of observations indicate that exposure to IL-4 is essential for the priming of Th2-type effector T cells and that exposure to IL-12 is essential for the priming of Th1-type effector T cells. However, the initial source of IL-4 in the early immune response has not been clearly identified. Dendritic cells (DC) are the most potent antigen- presenting cells (APC) in priming naive T cells. In this report, we show that DC exposed to IL-10 may play an important role in the priming of IL-4-secreting cells in the early immune response. DC isolated from splenic adherent cell cultures treated with rIL-10 (IL-10-DC) primed naive ovalbumin (OVA)-TCR transgenic T cells to secrete IL-4 upon re- stimulation with OVA and splenic APC. By contrast, DC isolated from rIL- 12, rIL-4 or control treated cultures induced almost exclusively Th1- type effector T cells. IL-4 secretion was detected in the primary cultures of IL-10-DC plus naive CD4+ T cells and the priming of IL-4- secreting T cells by IL-10-DC was dependent on endogenous IL-4 production in the priming culture since anti-IL-4 neutralizing antibody completely abrogated the priming of IL-4-secreting cells. Anti-B7-2 but not anti-B7-1 inhibited the ability of IL-10-DC to prime T cells to secrete IL-4. Furthermore, the ability of IL-10 DC to prime for IL-4- secreting T cells was closely related to the down-regulation of CD40 ligand-mediated IL-12 p70 production by DC in the primary cultures and was markedly reduced by adding exogenous IL-12 to the priming cultures. Thus, our findings indicate that early immunologic events that drive Th2 differentiation involve the effects of IL-10 on DC.      

Induction of mast cell interactions with blood vessel wall components by direct contact with intact T cells or T cell membranes in vitro

BACKGROUND: Mast cells exert profound pleiotropic effects on immune cell reactions at inflammatory sites, where they are most likely influenced not only by the extracellular matrix (ECM) and inflammatory mediators but also by the proximity of activated T lymphocytes. We recently reported that activated T cells induce mast cell degranulation with the release of TNF-alpha, and that this activation pathway is mediated by lymphocyte function-associated antigen-1 (LFA-1)/intercellular adhesion molecule-1 (ICAM-1) binding. OBJECTIVE: To determine how this contact between the two cell types can modulate mast cell behaviour in an inflammatory milieu by examining the adhesion of mast cells to endothelial cells and ECM ligands in an integrin-dependent manner. METHODS: Human mast cells (HMC-1) were co-cultured with resting or activated T cells followed by testing their adhesion to endothelial cell and ECM ligands, stromal derived factor-1alpha (SDF-1alpha)-induced migration, and western blotting. RESULTS: Co-culturing HMC-1 with activated, but not with resting T cells resulted in marked stimulation of mast cell adhesion to vascular cell adhesion molecule-1 and ICAM-1 in a very late antigen-4- and LFA-1-dependent fashion. In addition, activated T cells or T cell membranes promoted HMC-1 adhesion to fibronectin (FN) and laminin. This effect was accompanied by the phosphorylation of extracellular regulated kinase and p38, but not of c-Jun N-terminal kinase. Importantly, the adhesive property of mast cells depended exclusively on the direct contact between the two cell types, since neither supernatants from activated T cells nor separation of the two cell populations with a porous membrane affected mast cell adhesion to FN. Furthermore, similar results were obtained when mast cells were incubated with purified membranes from activated T cells. These results suggest that, in addition to stimulating mast cell degranulation, the proximity of activated T lymphocytes to mast cells can mediate the adhesion of mast cell precursors to the endothelial ligands and ECM. Activated T cells also stimulated SDF-1alpha-induced mast cell migration. CONCLUSION: This symbiotic relationship between the two types of immune cells may serve to direct mast cells to specific sites of inflammation where their effector functions are required.     

Mucosal-associated invariant T cells regulate Th1 response in multiple sclerosis

Mucosal-associated invariant T (MAIT) cells are innate T cells expressing an invariant Vα7.2-Jα33 T-cell antigen receptor α chain and are enriched in mucosal-associated lymphoid tissues. Although the regulatory role of MAIT cells in experimental autoimmune encephalomyelitis has been determined, their role in multiple sclerosis (MS) has not been elucidated. In the present study, the character of MAIT cells in the peripheral blood of MS patients was analyzed. Compared with healthy controls, the frequency of MAIT cells in peripheral blood was significantly reduced in MS patients in remission and even more profoundly reduced in those with relapse. The frequency of MAIT cells reflected the disease activity, as they were reduced significantly in patients with active disease compared with stable patients, and when blood samples from patients undergoing attack were analyzed 2-3 months later, the frequency significantly increased in parallel with clinical recovery. The frequency of MAIT cells positively correlated with the frequency of CD4(+) invariant NKT cells and of CD56(bright) NK cells in healthy controls but not in MS patients. This suggests the existence of an immune-regulatory link between MAIT cells and these other cell populations with disruption of this cross talk in MS. Moreover, MAIT cells showed a suppressive activity against IFN-γ production by T cells in vitro. This suppression required cell contact but was independent of IL-10, inducible co-stimulator or the presence of B cells. Taken together, these results suggest an immune-regulatory role of MAIT cells in MS through suppression of pathogenic T(h)1 cells.     

HLA-A匹配的人胃癌细胞系诱生胃癌特异性T淋巴细胞

目的 用ＨＬＡ－Ａ匹配的人胃癌细胞系诱导对胃癌细胞具有特异性杀伤作用的Ｔ淋巴细胞。方法 分离胃癌患者及正常人外周血淋巴细胞（ＰＢＬ）,用ＨＬＡⅠ类型别已知、经照射的异体胃癌细胞系刺激,进行筛选性的混合淋巴细胞肿瘤细胞培养（ＭＬＴＣ）,初步获得ＨＬＡ－Ａ可能匹配的淋巴细胞供体,血清法测定其ＨＬＡ－Ａ、Ｂ分子确认匹配后,进一步进行ＭＬＴＣ,＾３Ｈ掺入法评估其增殖能力,直接免疫荧光法分析其细胞表型,＾５