Application of the ELISPOT method for comparative analysis of interleukin (IL)-6 and IL-10 secretion in peripheral blood of patients with astroglial tumors

Glioblastoma, (grade IV astrocytoma), is characterized by rapid growth and resistance to treatment. Identification of markers of aggressiveness in this tumor could represent new therapeutic targets. Interleukins (IL)-6 and IL-10 may be considered as possible candidates, regulating cell growth, resistance to chemotherapy and angiogenesis. ELISPOT method provides a useful tool for the determination of the exact cell number of peripheral lymphocytes secreting a specific cytokine. IL-6 and IL-10 secretion levels were determined using ELISPOT methodology in peripheral blood mononuclear cells of 18 patients with astrocytic neoplasms (3 grade II and 15 grade IV), in parallel with 18 healthy controls. Additionally, immunohistochemical expression of these two cytokines was performed in paraffin-embedded neoplastic tissue in 12 of these patients. The secretion of IL-6 from peripheral monocytes was significantly higher in glioma patients compared to controls (P = 0.0003). In addition, IL-10 secretion from peripheral mononuclear and tumor cells of glioma patients was also higher as compared to healthy controls (P = 0.0002). Based on immunohistochemical staining, IL-6 expression was localized in tumor cells and macrophages as well as in areas of large ischemic necrosis, while the major source of IL-10 expression in glioblastomas was the microglia/macrophage cells. It is suggested that IL-10 contributes to the progression of astrocytomas by suppressing the patient’s immune response, whereas IL-6 provides an additional growth advantage. This study demonstrates for the first time the usefulness of ELISPOT in estimating the secretion of IL-6 and IL-10 from peripheral blood and the correlation of their expression in neoplastic cells. Christina Piperi and Penelope Korkolopoulou have equally contributed to this work.     

Weaning induces an increase in the number of specific cytokine-secreting intestinal lymphocytes in mice

Intestinal immunity differs from systemic immunity in several aspects and is frequently studied separately. In this work we have analysed the frequency of mononuclear cells spontaneously secreting the cytokines IL-2, IL-4, IL-5, IL-6, IL-10, interferon gamma (IFN-gamma) and tumour necrosis factor (TNF-alpha), in Peyer's patches and lamina propria of small intestine in mice by enzyme linked immunosorbent spot (ELISPOT) during 1 month after weaning. We have found a high percentage of spontaneous Th(1)as well as Th(2)cytokine-secreting lymphocytes in both populations, Peyer's patches and lamina propria. An increase in the number of the lymphocytes secreting most of the studied cytokines, at 1 and 2 weeks after weaning, was also observed. These results suggest that the increase in the number of cytokine secreting lymphocytes may be one of the potential mechanisms involved in the development of the intestinal immune system at weaning.     

Transforming growth factor β , (TGFβ) secreted by immunogenic ex vivo human carcinoma cells, counteracts the activation and inhibits the function of autologous cytotoxic lymphocytes. Pretreatment with interferon γ and tumor necrosis factor α reduces the production of active TGFβ

 We present the results obtained with an in vitro model system that resembles the in vivo tumour microenvironment, where malignant cells are in close contact with the infiltrating lymphocytes. Unmanipulated blood lymphocytes were cytotoxic against the autologous ex vivo tumour cells in 3/19 patients and this function was generated in 6-day mixed cultures in five additional cases. Production of transforming growth factor β (TGFβ) by the freshly separated tumour cells was determined in parallel. Cytotoxicity was generated by a small number of tumour cells (2–5/100 lymphocytes), while a large number (10–20/100 lymphocytes) inhibited not only the generation but also the existing lytic activity. The presence of a neutralising TGFβ-specific mAb (2G7) potentiated the activation of lymphocytes and suspended the suppression inflicted by the tumour cells. In those tumours, which expressed relatively high levels of MHC class I and ICAM-1 molecules, the quantity of secreted TGFβ interfered with the ability of tumour cells to generate cytotoxic lymphocytes. In the tumours with low expression of class I, such a correlation was not detected, indicating the primordial role of MHC class I expression in the regulation of autologous tumour recognition. Our results demonstrate the involvement of TGFβ in the impaired lymphocyte-mediated reactivity against immunogenic tumours and support a mechanism that contrasts the tolerance or anergy. Since presence of TGFβ in the microenvironment of tumours counteracts the function of cytotoxic T lymphocytes, production of this cytokine can contribute to the failure of immunotherapy. Received: 19 June 1997 / Accepted: 14 August 1997     

Cytokine production by peripheral lymphocytes in melanoma

BACKGROUND: The differentiation of T cells towards a T helper 1 (Th1) or Th2 phenotype based on their profile of cytokine production, is of great relevance in the regulation of immune responses. We have determined by flow cytometry, the expression of selected Th1 and Th2 cytokines by activated T cells in whole blood samples (WB) from normal donors and from patients with different clinical stages of melanoma in different clinical stages. METHODS: WB samples from 6 normal donors and 19 patients with melanoma were activated over 4 hours with PMA + ionomycin in presence or absence of a protein secretion inhibitor. Following surface staining (CD3-Cy5+CD8-FITC), fixation and permeabilization, cells were stained with PE-labelled antibodies against Th1 cytokines (IL-2, IFN-gamma, TNF-alpha) and Th2 cytokines (IL-4, IL-10). RESULTS: The most relevant results were related to IFN-gamma and IL-10 production. The percentage of IFN-gamma producer cells was significantly lower in melanoma patients, independent of the stage, than in controls. IL-10 production was significantly increased in melanoma patients with respect to normal donors. CONCLUSIONS: Our data support the notion that the pattern of cytokines produced by lymphocytes from melanoma patients may help to explain the impairment in their T cell immune response. More extensive studies regarding the pattern of cytokines, not only in peripheral blood, but also in tumour tissue and sentinel lymph nodes, are needed to confirm these data.     

体外制备和增殖烟曲霉特异性T细胞的研究

目的体外制备和增殖烟曲霉特异性T淋巴细胞。方法从健康志愿者外周抗凝血中分离并体外扩增DC,利用加热灭活的烟曲霉孢子作为抗原,体外共孵育制备烟曲霉孢子负载的DC,进一步将此成熟DC与源自同一个体的去除了DC细胞的外周血细胞共培养,体外诱导并扩增烟曲霉特异性T淋巴细胞。应用ELISPOT(酶联免疫斑点)技术检测活化T细胞IFN-γ的分泌情况,流式细胞仪检测细胞因子胞内合成情况,并分析功能细胞的类型和比例。结果 ELISPOT分析显示:PBMC+DC+Conidia实验组IFN-γ分泌(87.33±1.33/4.0×105)高于其他对照组,具有统计学意义(P0.05)。细胞因子流式细胞仪分析显示:PBMC+DC+Conidia组中,2.76%的细胞分泌IFN-γ,其中1.61%为CD4+T细胞,与各对照组相比具有统计学意义(P0.05)。获得的烟曲霉特异性T细胞可以在体外可进行大量增殖。结论本文结果显示烟曲霉孢子在体外可以作为变应原诱导产生烟曲霉特异性CD4+T细胞介导的Th1型免疫反应,为未来制备和扩增烟曲霉特异性T细胞及过继免疫治疗侵袭性曲霉病提供实验基础。     

Adjuvants and the promotion of Th1-type cytokines in tumour immunotherapy

Immunotherapy includes both active and passive mechanisms that have the potential to treat many tumour types. Whereas monoclonal antibodies may kill cells by merely binding to them, 'cancer vaccines' involve the induction of an active immune response. The activation of tumour antigen-specific T-helper and cytotoxic T lymphocytes or non-specific macrophages and natural killer (NK) cells using immunotherapeutic approaches may lead to the subsequent destruction of tumour tissue. Administration of a tumour antigen alone is often not sufficient to stimulate an appropriate immune response. However, incorporating an immunological adjuvant into a vaccine regime often improves anti-tumour immunity. There are various types of adjuvants used in immunotherapy, ranging from microbial, chemical, and cellular components to proteins and cytokines. Previous reports have demonstrated that the induction of Th1-promoting cytokines, using specific adjuvants, can enhance anti-tumour immunity and can reduce or even prevent tumour growth. There is also increasing evidence that many adjuvants induce Th1-type cytokines, which correlates with the induction anti-tumour immunity. Th1-type responses which comprise cell-mediated immunity are characterised by the secretion of interferon-gamma by T cells, which is induced by antigen-presenting cell (APC)-derived IL-12. This review describes immunoadjuvants that are currently undergoing preclinical investigation, and emerging clinical data revealing that adjuvants which induce Th1-type responses can improve the efficacy of cancer vaccines. Therefore, the use of Th1-inducing adjuvants may provide an essential strategy for the future success of immunotherapy.     

Antigen-dependent cytokine mRNA expression by individual rhesus macaque T helper cells by flow cytometry

Specific patterns of cytokine secretion by CD4(+) T helper (Th) cells determine the nature of immune effector responses. Using a multiparameter, flow cytometric fluorescent in situ hybridization (FISH) assay that detected cytoplasmic mRNA within intact cells, we assessed antigen-specific cytokine expression in rhesus macaque Th cells. In the peripheral lymphocytes of immunized rhesus macaques, FISH detected antigen-induced cytokine gene expression in single Th cells. Analysis of simultaneous cytokine expression by single cells demonstrated that the recall immune response consisted of Th cells expressing either a Th1 (IL-2(+)/IFN-gamma(+)) or a Th2 (IL-4(+)/IL-6(+)) cytokine pattern. In addition to the classic Th subsets, Th cells expressing only one of two Th1 or Th2 defining cytokines were common following antigen restimulation. The data gathered with the FISH assay suggest that, in primates, the immune response to recall antigens consists of nonclassic Th cells, as well as a mixture of polarized Th1 and Th2 T cells.     

Troglitazone exhibits immunomodulatory activity on the cytokine production of activated human lymphocytes.

Troglitazone (TGL), a thiazolidinedione compound that improves the response of peripheral target tissue to insulin, also has anti-inflammatory properties, a potential means of protection from Type 1 (insulin dependent) diabetes. In order to test the ability of TGL to affect cytokine production, peripheral blood mononuclear cells from healthy donors were exposed to TGL in the presence or absence of a polyclonal activator (PHA) and the production of cytokines assayed. TGL enhanced PHA response, promoted secretion of the cytokines granulocyte and macrophage colony-stimulating factor and leukaemia inhibitory factor and inhibited tumour necrosis factor-alpha secretion, consistent with causing Th-2 differentiation in T-cells. These results suggest that TGL is capable of modulating cytokine production and could therefore influence Th1/Th2 differentiation.     

Dysregulation of proinflammatory and regulatory cytokines in HIV infected persons with active tuberculosis

Proinflammatory and regulatory cytokines have been implicated to play important role in immunopathology of HIV and tuberculosis (TB) infection. Capacity of unstimulated and mitogen-stimulated peripheral blood mononuclear cells (PBMCs) to secrete cytokines (interleukin (IL)-2, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), IL-4, IL-10 and IL-6) was estimated for 15 HIV-TB coinfected patients, 22 HIV seropositives without TB, 32 HIV negative TB patients, and 36 healthy subjects. Dually infected patients had suppression of both Th1 and Th2 cytokine secretion as evidenced by significantly lower production of IL-2, IFN-gamma and TNF-alpha as well as IL-4 and IL-10. Production of IL-2 and TNF-alpha was significantly decreased only in case of HIV infection. Significantly higher IL-6 secretion was found in unstimulated cultures in dually infected patients. The mitogen induced cytokine secretion was generally lower in HIV-TB coinfected patients indicating profound perturbation of both Th1 and Th2 responses.     

Heterogeneity of helper/inducer T lymphocytes. IV. Stimulation of resting and activated B cells by Th1 and Th2 clones

To test the hypothesis that resting and previously activated B lymphocytes differ in their proliferative and differentiative responses to various Th cell-derived stimuli, we have examined the interactions of purified small (resting) and large (activated) murine B cells with rabbit Ig-specific Th1 and Th2 clones in the presence of the Ag analogue, rabbit anti-mouse Ig antibody. Small numbers of Th2 cells induce strong Ag-dependent proliferation of and Ig secretion by both resting and activated B lymphocytes. In contrast, Th1 clones stimulate lower responses of activated B cells and fail to stimulate small resting B cells. An interaction with Th1 clones does make small B cells responsive to the Th2-derived cytokine, IL-4, indicating that Th1 clones are capable of delivering some but not all the stimuli necessary for the induction of humoral immunity. Finally, in order to compare the responses of small and large B cells to cognate interactions and secreted cytokines, we used an autoreactive I-Ak-specific Th2 line. This line induces proliferation of and Ig secretion by I-Ak expressing but not H-2d resting and activated B cells as a result of cognate interactions. However, when the H-2d B cells are bystanders in the presence of cytokine secretion by this Th2 line, or are directly exposed to Th2-derived cytokines, both small and large B cells are induced to proliferate but only the large B cells secrete antibody. These results indicate that the magnitude and nature of antibody responses depend on three principal factors: the cytokines produced by Th cells, the state of activation of the responding B lymphocytes, and whether the B cells are recipients of cognate help or are bystanders at the site of T cell stimulation. Our findings also confirm the view that cognate T-B interactions are most efficient for initiating B cell responses and may allow B cells to subsequently respond to a variety of T cell-derived cytokines.     

Modulation by biologic response modifiers of hepatitis C virus antigen-independent cytokine secretion in blood mononuclear cells.

The systemic hepatitis C virus (HCV) antigen-non-specific cytokine responses were investigated in cultures of peripheral blood mononuclear cells. Cytokines of the T helper (Th) 1 [interferon (IFN)gamma and interleukin (IL)-2] and Th2 (IL-4 and IL-10) phenotype, and the pro-inflammatory cytokines tumour necrosis factor alphaIL-1beta and IL-6, were secreted by the cells activated by the HCV antigen-independent pathway. Furthermore, these cytokine responses were shifted towards a Th1 predominance by treatment with IFN-beta and with IL-2, whereas cytokine responses were selectively amplified towards a combined Th1-Th2 profile by granulocyte-macrophage- but not by macrophage colony-stimulating factor. Moreover, pro-inflammatory cytokine production was significantly enhanced by IFN-beta and augmented dose-dependently with GM-CSF and IL-2. Therefore, these immune mediators can promote unique-as well as overlapping-systemic cytokine responses that may be relevant for immunotherapeutic interventions to control HCV infection.     

Vdelta1 T lymphocytes expressing a Th1 phenotype are the major gammadelta T cell subset infiltrating the liver of HCV-infected persons

BACKGROUND: Hepatitis C infection induces an acute and chronic liver inflammation that may lead to cirrhosis, liver failure, or hepatocarcinoma. Since the role of alphabeta T lymphocytes in hepatitis C virus (HCV) immunopathology has been analyzed extensively, we investigated the distribution and functional activation of gammadelta T cell subsets in chronically HCV-infected patients. MATERIALS AND METHODS: Blood samples and liver biopsies from 35 patients with compensated chronic HCV infection were compared in terms of T cell subset distribution, expression of activation markers, gammadelta T cell receptor (TCR) repertoire, and pattern of cytokine production. Moreover, we analyzed whether these immunological parameters were associated with other clinical observations (plasma viremia, ALT levels, Ishak index). RESULTS: Differing from peripheral blood distribution, a specific compartmentalization of Vdelta1 T cells (p < 0.001) was observed in the liver of HCV patients. These cells represented a relevant fraction of intrahepatic T lymphocytes (1.8-8.7%) and expressed the memory/effector phenotype (CD62-L- CD45-RO+CD95+). This phenotype was consistent with selective homing upon antigen recognition. Mitogenic stimulation of Vdelta1 + T lymphocytes recruited in the liver revealed the T helper cell type 1 (Th1) pattern of cytokine secretion. Interestingly, the frequency of interferon-gamma (IFN-gamma)-producing Vdelta1 T cells was associated with an higher degree of liver necroinflammation, measured by the Ishak index. Finally, the T-cell repertoire analysis revealed the absence of Vgamma selection in the TCR repertoire of intrahepatic Vdelta1 T cells. CONCLUSIONS: gammadelta T cell distribution in the peripheral blood differs from the Vdelta1 T cell subset because it is policlonally activated and recruited in the liver of chronic HCV-infected patients. During HCV-infection, this T cell subset may release Th1 cytokines and contribute to the necroinflammatory liver disease.     

IL-12 elispot assays to detect and enumerate IL-12 secreting cells

The cytokine IL-12 promotes Th(1)type immune responses and plays a key role in immune regulation. The complex nature of IL-12 hampered its detection without use of stimulants that might give less relevant information. To detect circulating IL-12 p40, we developed enzyme-linked immunospot (ELISPOT) assays that allow enumeration of IL-12 p40 secreting cells without prior in vitro stimulation of the cells. In parallel, intracellular staining of IL-12 p40 by flow cytometry was performed to compare the two methods. IL-12 p40 secreting cells were detected in healthy subjects at a mean number of 103+/-155 per 10(5)blood mononuclear cells (MNC). Numbers of IL-12 p40 secreting blood MNC correlated with IL-12 p40 positive blood MNC detected by flow cytometry. Bacterial endotoxins and the inflammatory cytokines TNF-alpha and IFN-gamma control IL-12 production by antigen presenting cells. Utilizing IL-12 p40 ELISPOT assays, we could confirm occurrence of elevated numbers of IL-12 p40 secreting blood MNC after stimulation with TNF-alpha, IFN-gamma, LPS, LPS+TNF-alpha or LPS+IFN-gamma, compared to cultures without stimulant. Due to its central role in inflammation and autoimmunity, IL-12 is an attractive target for immunotherapy. IL-12 p40 ELISPOT assays represent a sensitive, specific and reliable tool for investigating the role of IL-12 in both health and disease.     

Tumour-reactive lymphocytes stimulated in mixed lymphocyte and tumour culture

Summary Lymphocytes from cancer patients were stimulated in mixed culture with autologous tumour (MLTC) or pooled allogeneic lymphocytes (MLC). Both protocols induced increased uptake of ³H-thymidine at 5 days and the appearance of lymphoblasts. Blasts were isolated on discontinuous Percoll gradients and either expanded as bulk cultures or cloned directly under limiting dilution conditions in the presence of conditioned medium containing IL-2. Results with MLTC-blast-CTC have been reported elsewhere. MLC-activated cultures lysed autologous tumour but not autologous lymphoblasts. Lysis of some allogeneic tumours, lymphoblasts from members of the inducing pool, and K562 was also apparent. MLC activated cultures did not undergo restimulation in response to autologous tumour or lymphocytes but were restimulated by leukocytes from pool members.MLTC clones showed autologous tumour-specific cytotoxic activity or cross-reactive proliferative responses with tumours of the same site and histology. The majority of MLC clones cytotoxic for autologous tumour were also specific and did not lyse allogeneic tumour, K562, or lymphoblasts from the inducing pool. Two clones lysed autologous tumour and pool members. None of the clones tested proliferated in response to autologous tumour following MLC activation but some were responsive to pool members and one clone was restimulated by autologous monocytes. No association was found between clone phenotype and function. The implication of these data is that the effector cells with activity against autologous tumour induced in MLC arose largely by transstimulation of in vivo-activated tumour reactive lymphocytes by IL-2 release rather than expansion of NK-like effectors or sharing of antigenic specificities between tumour and allogeneic lymphocytes. Since MLC activation of cancer patients lymphocytes does not induce proliferative responses to autologous tumour it is unlikely to be a useful procedure in preparing cells for immunotherapy protocols. Abbreviations used in this paper: PBL, peripheral blood lymphocytes; TIL, tumour infiltrating lymphocytes; MLTC, mixed lymphocyte tumour culture; IL-2, interleukin-2; MLC, mixed lymphocyte culture; LSM, lymphocyte separation medium; BSS, balanced salt solution; HuSe, human serum; PBS, phosphate-buffered saline; CTC, cultured T cells; PHA, phytohaemagglutinin; CM, cultured medium; NK, natural killer; FcR, receptor for the Fc portion of IgG     

Generation and immunologic functions of Th17 cells in malignant gliomas

Th17 cells, a recently discovered inflammatory T cell subtype, have been implicated with autoimmune disorders. However, mechanism of generation or functions of intratumoral Th17 cells are still unclear. We have been investigating the mechanism of induction and role of Th17 cells in malignant gliomas using primary tumor as well as cell lines. We report here that: (1) a higher frequency of Th17 cells in gliomas were associated with higher number of myeloid (CD11b) cells as well as the expression of TGF-β1 or IL-6; (2) conditioned medium from glioma cells (Gl CM) induced Th17 cell differentiation, which was inhibited by anti-TGF-β1 and anti-IL-6; (3) glioma-associated monocytes secreted Th17-promoting cytokines IL-1β and IL-23; (4) CM from glioma and monocyte co-culture (Gl+Mo CM) induced high frequency of Th17 cells in naïve T cell culture, which was abrogated by anti-IL-1β and anti-IL-23 antibodies; (5) In vitro Gl+Mo CM-mediated Th17 generation was associated with a decrease in IFN-γ and a concomitant increase in IL-10 secretion. Anti-TGF-β1, but not anti-IL-6, significantly reversed this cytokine profile. These results demonstrate prevalence of Th17 cells in gliomas and implicate the cytokines derived from the tumor as well as infiltrating myeloid cells in the induction of Th17 cells in glioma microenvironment. Moreover, the data also suggest that glioma-associated Th17 cells may contribute to immune-suppression via TGF-β1-induced IL-10 secretion. Further studies on the mechanism of tumor-infiltration, developmental pathways, and pro-/anti-tumor functions of Th17 cells will provide rationale for developing novel adjuvant immunotherapeutic strategies for malignant gliomas.     

TH1/TH2 cytokine secretion of first degree relatives of T1DM patients

Th1/Th2 cytokine imbalance has been demonstrated in Type 1 diabetes (T1DM) patients. We characterized the peak levels, secretory pattern and total cytokine production of the Th1 cytokines (IL-2 and IFN gamma) and Th2 cytokines (IL-4 and IL-10), by stimulated peripheral blood mononuclear cells of twenty six first-degree relatives of T1DM patients, and eleven matched controls. At enrollment, first degree relatives demonstrated a significant increase in peak and overall secretion of IL-2; P<0.01 and P<0.005 respectively and IL-4 cytokine; P<0.05 and P<0.01 respectively, as compared to normal controls. Their mean IFN gamma secretion increased significantly, P<0.05, after one year while their higher IL-2 and IL-4 secretion remained unchanged. Ab-negative and Ab-positive relatives demonstrated a similar cytokine secretion pattern. Four relatives all Ab positive, developed diabetes: Peak IL-4 levels were low in three and markedly decreased within one year in one of these relatives, while peak IL-2 and IFN gamma levels were elevated in all of them. These data demonstrate that secretion of both Th1 and Th2 cytokines is increased in first-degree relatives of T1DM patients independently of their diabetes-associated autoantibodies. The presence of low IL-4 and elevated IL-2 and IFN gamma levels in autoAb positive relatives is associated with progression to overt disease.     

The human cytokine response to Leishmania major early after exposure to the parasite in vitro

Leishmaniasis is caused by the protozoan parasite Leishmania spp. In murine leishmaniasis, a T helper cell type-I (Th1) response, characterized by the secretion of interferon (IFN)-gamma is necessary for clearing the infection. whereas a Th2 response, accompanied by the production of interleukin (IL)-5, can exacerbate the disease. Moreover, the early cytokine milieu is thought to play an important role in determining the outcome of infection. In human leishmaniasis little is known about this early cytokine response. Because of this, we cocultured human peripheral blood mononuclear cells (PBMC) with Leishmania major in vitro and measured the production of IFN-gamma, IL-5, and IL-10. We also treated PBMC cultures with various cytokines and neutralizing anticytokines. We found that the principal cytokine produced was IFN-gamma and that its production was regulated by IL-10 and IL-12. In contrast, only low levels of Th2 cytokines such as IL-5 were produced. Therefore, the Th1-Th2 dichotomy that exists in inbred strains of mice does not appear to apply to the response of humans to L. major. Rather, Th2 cytokines may play a role in regulating IFN-gamma production.     

Immunotherapy of human colon cancer by antibody-targeted superantigens

T lymphocytes generally fail to recognize human colon carcinomas, suggesting that the tumour is beyond reach of immunotherapy. Bacterial superantigens are the most potent known activators of human T lymphocytes and induce T cell cytotoxicity and cytokine production. In order to develop a T-cell-based therapy for colon cancer, the superantigen staphylococcal enterotoxin A (SEA) was given tumour reactivity by genetic fusion with a Fab fragment of the monoclonal antibody C242 reacting with human colon carcinomas. The C242Fab-SEA fusion protein targeted SEA-reactive T cells against MHC-class-II-negative human colon carcinoma cells in vitro at nanomolar concentrations. Treatment of disseminated human colon carcinomas growing in humanized SCID mice resulted in marked inhibition of tumour growth and the apparent cure of the animals. Therapeutic efficiency was dependent on the tumour specificity of the fusion protein and human T cells. Immunohistochemistry demonstrated massive infiltration of human T cells in C242Fab-SEA-treated tumours. The results merit further evaluation of C242Fab-SEA fusion proteins as immunotherapy in patients suffering from colon carcinoma.     

Interleukin-13 induces thymus and activation-regulated chemokine (CCL17) in human peripheral blood mononuclear cells

BACKGROUND: In allergic inflammation involving allergic rhinitis, the predominance of Th(2) lymphocytes is one of the primary causal agents in promotion of the allergic condition. Thymus and activation-regulated chemokine (TARC/CCL17) is a recently identified chemokine that induces the development of Th(2) lymphocytes. One of the sources of TARC has been reported to be peripheral blood mononuclear cells (PBMCs). OBJECTIVE: We investigated TARC production from PBMCs by the stimulation of specific antigens and Th(2) type cytokines. METHOD: PBMCs were isolated from both allergic rhinitis patients and healthy volunteers. PBMCs were incubated with cytokine. TARC mRNA expression was examined by real time PCR methods and the amount of TARC production was examined by ELISA. RESULTS: IL-13 was found to be the most potent inducer for TARC mRNA expression and protein production in PBMCs. Furthermore, tumour necrosis factor alpha and IL-13 synergistically induce TARC. The amount of TARC from allergic rhinitis patients was significantly larger than that from healthy volunteers. Moreover, TARC was induced by a specific antigen, and was 35% inhibited by an anti-IL-13 neutralizing antibody. CONCLUSION: These results indicate that IL-13 is important in TARC mediated Th(2) lymphocytes infiltration in the nasal mucosa.     

正常妊娠大鼠的Th2型免疫反应

采用流式细胞仪,^3H-TdR掺入和酶联免疫打点(enzyme-linked immunospot,ELISPOT)方法研究妊娠免疫学指标的改变。妊娠晚期大鼠脾脏单个核细胞表面分子主要组织相容性抗原Ⅱ(MHCⅡ)明显下调,外周血单个核细胞表达CD11c明显减少,共激活分子B7-1和B7-2未见改变;脾脏和外周血单个核细胞中Th2细胞因子IL-10、IL-4表达增多,TGFD阳性细胞数也明显增加,而Th1细胞因子IFNγ的产生未受抑制。此外,脾脏和外周血中单个核细胞的抗原特异性增殖未见改变,而腹腔淋巴结细胞的增殖明显升高。脾脏单个核细胞在妊娠晚期分泌较少的抗原特异性抗体。提示妊娠期性激素具有免疫调节作用,可能与怀孕时Th1细胞介导的自身免疫性疾病得到缓解有关。