Expression and cytokine mediated modulation of adhesion/costimulation molecules ICAM-1(CD54) and LFA-3(CD58) in human ovarian cancer

Previous studies have shown that paracrine delivery of helper cytokines by transduced tumor cells can bypass the lack of the B7 costimulation pathway, which when engaged induces secretion of a number of cytokines, most notably IL-2 and which results in clonal T-cell proliferation and effector function. However, other costimulation signals such as those mediated by ICAM-1 and LFA-3 molecules remain crucial to increase antigen independent conjugate formation and therefore synergize with TcR activation pathways. In this study we have analyzed tumor cells from eight freshly isolated and histologically similar human ovarian tumors for their surface antigen expression of the adhesion/costimulation molecules ICAM-1 and LFA-3 before and after exposure to the cytokines TNF-α plus IFN-γ. All eight fresh tumors expressed variable levels of ICAM-1 antigens and these levels were markedly upregulated after exposure to TNF-α plus IFN-γ. Similarly, LFA-3 antigens were shown to be expressed in all fresh tumor evaluated. However, in contrast to the ICAM-1/LFA-1 pathway of costimulatory molecules, LFA-3/CD2 pathway was shown to be resistant to modulation with these cytokines. Such findings suggest that fresh ovarian tumor cells pretreated with TNF-α plus IFN-γ could significantly increase their adhesion/costimulation activity for T cell recognition when admixed with genetically manipulated tumor cells to be used as tumor vaccines.     

Immunization with adenoviral vectors carrying recombinant IL-12 and E7 enhanced the antitumor immunity to human papillomavirus 16-associated tumor

OBJECTIVE: Human papillomavirus (HPV) infection play a significant role in cervical carcinogenesis, and HPV oncoprotein E7 has important functions in the formation and maintenance of cervical cancers. Interleukin-12 (IL-12) has been reported to induce cellular immune responses, and has also been demonstrated to suppress the growth of tumors and the expression of E7. Here, we investigate the utility of adenovirus E7 (AdE7) and adenovirus IL-12 (AdIL-12) for protection against TC-1 tumor using an animal model. METHODS: The antitumor effects induced by AdIL-12 and/or E7 were assessed by measurements of tumor size. E7-specific antibody and INF-gamma production in sera were measured, as were T-helper cell proliferative responses. Cytotoxic T-lymphocytes (CTL) and T cell subset depletion studies were also performed. RESULTS: Infection of tumor sites with a combination of AdIL-12 and AdE7 resulted in an antitumor effect which was significantly more profound than that which resulted from singular infections with either AdIL-12 or AdE7. Combined infection resulted in regression of 9-mm-sized tumors in approximately 80% of our experimental animals as compared to the PBS group. Serum levels of E7-specific antibody and INF-gamma production, as well as T-helper cell proliferative responses, were found to be significantly higher in coinfected with AdIL-12 and AdE7 group than in single infection with either AdIL-12 or AdE7 group. CTL responses only exhibited by the AdIL-12 and AdE7 coinjected group suggested that these tumor suppression effects were mediated primarily by CD8+ and, to a lesser degree, by CD4+ T cells. CONCLUSION: Combined injection with adenovirus carrying IL-12 and E7 induced significant antitumor immunity against TC-1 tumors. They may prove useful in clinical applications for the treatment of HPV-associated tumors.     

Cervical carcinoma antigen and the relationship to HSV-2

Two antigens associated with invasive squamous cell carcinoma of the cervix have been demonstrated by immunological techniques. All tumors tested contained one antigen which appeared to be specific for cervical carcinoma. A second antigen which cross-reacted with ovarian and vulvar carcinoma was found in 80% of the tumors tested. Cervical carcinoma antigen (CCA) and its antibody were tested for cross-reactivity with Herpes Virus Simplex Type-1 and Type-2 and their antiserum utilizing immuno-diffusion, counter-immunoelectrophoresis and immuno-fluorescent techniques.CCA prepared by either saline or perchloric acid extraction from cervical carcinoma showed a line of identity with a Herpes Virus Simplex Type-2 antigen when reacted against anti-HSV-2. Extracts of normal cervical tissue, breast and ovarian tissue as well as extracts of breast and colon cancer did not form precipitin lines. However, anti-CCA did not show a precipitin reaction when reacted with HSV-2 preparations.The significance of the findings lies in the demonstration that a HSV-2 induced antigen has been shown to be immunologically identical to associated tumor antigen extracted from cervical carcinomas.     

Expression of the costimulatory molecule CD86, but not CD80, in keratinocytes of normal cervical epithelium and human papillomavirus-16 positive low squamous intraepithelial lesions

Keratinocytes have been traditionally considered as nonprofessional antigen presenting cells, since multipassaged cells from skin biopsies of healthy individuals do not constitutively express major histocompatibility complex (MHC) class II or costimulatory molecules, but can be induced to do so after exposure to interferon-gamma. In normal and human papillomavirus (HPV)-infected cervical epithelium, keratinocytes are affected by a variety of soluble mediators that could modulate the expression of molecules including costimulatory proteins; however, the presence of these molecules within the cervix has been poorly studied. Therefore, our aim was to further explore the presence of costimulatory molecules on normal cervical epithelium and HPV-16 positive low squamous intraepithelial lesions (LSIL). We found in situ CD86 (but not CD80) displayed on the surface of normal keratinocytes from the spinous layer of human cervical epithelium. The presence of the protein and its messenger RNA level (evaluated by in situ hybridization) was diminished in HPV-16 positive LSILs. Although downregulation of costimulatory molecules is frequently related to cytokines expression, we did not observe differences in the presence of interleukin-10, the main cytokine that inhibits CD86 expression. Expression of CD86 on keratinocytes from normal cervical epithelium could indicate the potentiality of these cells to activate cytotoxic T cells, while the shut-off of this molecule in HPV-16 positive lesions could be a mechanism for evading host immune surveillance, resulting in the persistent HPV infection and probable progression of cervical lesions.     

Experimental testicular teratoma promotes formation of humoral immune responses in the host testis

The testis is an immunologically privileged site. Very little is known about the factors regulating formation of immune responses elicited by a neoplasm in the testis. We have studied the immune response of the host testis against experimental testicular teratoma in mouse by localizing adhesion molecules (CD106, CD54, CD49d/CD29, CD44, CD18, CD8 and CD4), cytokines (IL-2, IL-4, IL-6, IL-10 and IL-12), T-cell costimulators (CD80, CD86) and the lipid antigen presenting molecule CD1d in the testis of 129/SvJ mice with and without experimental testicular teratoma. The testicular teratomas were induced by grafting male gonadal ridges from 12-day-old 129/SvJ mouse fetuses into testes of adult mice from the same strain. The tumors cultured intratesticularly for 2, 3, 4 and 8 weeks (three animals per time point) were used for immunocytochemistry. CD1d was detected in Sertoli cells and in some degenerated tubules of the host testis surrounding the graft. In the tumor, CD1d was detected in glandular epithelia, smooth muscle and in thin fibers of neural origin. IL-2 was observed in some blood vessels of the host testis and of the tumor and in occasional cell infiltrates around these vessels. Some tubular structures of the tumor were also positive for IL-2. IL-6 was detected in Sertoli cells of the normal testis and in Sertoli cells and in solitaryinterstitial cells as well as in the walls of some blood vessels of the host testis. The reaction for IL-6 was more prominent in the tubules apparently damaged by the growing tumor. In the tumor IL-6 was detected in epithelial structures, muscle cells, in thin fibers of neural origin and in some blood vessels. IL-10 was detected in individual cells in the interstitium and in degenerating tubules of the host testis. In the tumor the epithelial structures were positive for IL-10. The interstitium of the host testis was positive for CD106 and the embryonic testicular cords in the graft were also positive, but the tumor was negative. CD44 and CD18 were observed in some blood vessels and in degenerated tubules of the host testis. In the tumor CD44 and CD18 were occasionally observed in cartilage and in epithelial structures. The results of the present study suggest that cytokine microenvironment in the testis containing neoplastic tissue promotes activation of humoral immune responses. In addition, as the damaged seminiferous tubules expressed increased amounts of two cytokines promoting humoral immune responses, IL-6 and IL-10, it is possible that also in other conditions with damage to the tubules, humoral immune responses predominate.     

Monoclonal anti-idiotype antibodies in immunotherapy of ovarian carcinoma (MAb ACA125) and breast carcinoma (MAb ACA14C5)]

Anti-idiotypic antibodies, which imitate a tumor-associated antigen by their variable region, offer an elegant method for the induction of a specific immune response, when used as a surrogate antigen for immunization. We generated anti-idiotypic antibodies imitating 2 different tumor-associated antigens. I. CA125 for ovarian carcinomas and II. 14C5, a tumor-associated cell substrate adhesion molecule on breast cancer cells, whereas the first approach could be introduced in a first clinical trial and the second was evaluated in an immunocompetent animal model. For the induction of an immune response against CA125, 18 patients with advanced ovarian cancer (n = 6) or heavily pretreated recurrences (n = 12) were immunized with the anti-idiotypic antibody MAb ACA125. Patients were treated with 2 mg anti-idiotype antibody every two weeks for 4 injections i.m. and then monthly. 12 of 18 patients demonstrated an anti-anti-idiotypic (Ab3) response, which was to a lower extent also directed against CA125 and 9 of 18 patients developed a CA125 specific cellular immune response by their peripheral blood lymphocytes. Based on this data a follow-up clinical trial in advanced ovarian cancer patients with minimal residual disease in an adjuvant approach after primary therapy was started to evaluate the effect of the immune response on the progression free survival. For immunotherapy of breast cancer, we generated a murine monoclonal anti-idiotypic antibody (MAb ACA14C5), which imitates a cell substrate adhesion molecule on breast cancer cells. The anti-idiotype was introduced in an immunocompetent animal to prove his capability on induction of an immune and tumor response. The results showed a highly significant difference in the tumor growth of the ACA14C5 treated group in contrast to the controls starting the immunization on day 6 after tumor cell application with 10 of 12 animals being cured from their tumor burden. Prophylactic immunization against the invasion antigen of breast cancer by anti-idiotypic antibodies showed protection against increasing tumor burden. However, in the situation of established tumors only minor responses could be detected. Vaccination with anti-idiotypic antibodies comprises an effective method for induction of a specific immune response against non-immunogenic tumor-associated antigens and should be therefore considered in immunological approaches to tumor therapy, where the primary structure and sequence of the antigen, e.g. CA125, is up to now not available.     

树突状细胞体外诱导抗卵巢癌免疫的实验研究

目的 观察人外周血树突状细胞(Dendritic cells,DC),体外能否诱导抗卵巢癌免疫应答。方法 用粒细胞-巨噬细胞集落刺激因子(GM-CSF)、白介素-4(IL-4)和肿瘤坏死因子α(TNF-α)从健康女性外周血分化诱导DC,以源于人卵巢癌细胞系HO-8910的肿瘤抗原粗提物冲击致敏DC,将致敏DC、同源淋巴细胞和卵巢癌细胞共育,观察负载抗原DC体外诱导淋巴细胞对HO-8910细胞的杀伤作用,同时设不同类型肿瘤细胞(Eca-109和PC-12)作为对照。MTT法测定细胞杀伤活性。结果 经卵巢癌细胞HO-8910肿瘤抗原脉冲致敏的DC能诱导淋巴细胞特异性地杀伤卵巢癌细胞。结论 用GM-CSF、IL-4和TNF-α从人外周血诱生的DC能从卵巢癌细胞HO-8910冻融物有效递呈抗原并诱导出高效而特异的抗卵巢癌免疫反应。     

Expressions of HLA class Ⅰ and CD80 in human epithelial ovarian carcinomas

Ｈｕｍａｎｅｐｉｔｈｅｌｉａｌｏｖａｒｉａｎｃａｒｃｉｎｏｍａｓ(ＥＯＣ)ａｒｅｖｅｒｙａｇｇｒｅｓｓｉｖｅｔｕｍｏｒｓｗｉｔｈｐｏｏｒｐｒｏｇｎｏｓｉｓ .Ｉｔｉｓａｓｓｕｍｅｄｔｈａｔｔｕｍｏｒｃｅｌｌｓｍｉｇｈｔｓｕｒｖｉｖｅｂｙｅｓｃａｐｉｎｇｔｈｅｉｍｍｕｎｅｓｕｒｖｅｉｌ ｌａｎｃｅ .Ｍａｊｏｒｈｉｓｔｏｃｏｍｐａｔｉｂｉｌｉｔｙｃｏｍｐｌｅｘ(ＭＨＣ)ｍｏｌｅｃｕｌｅｓａｓｗｅｌｌａｓｔｈｅｃｏ ｓｔｉｍｕｌａｔｏｒｙｍｏｌｅｃｕｌｅＣＤ80ａｐｐｅａｒｔｏｐｌａｙａｎｉｍｐｏｒｔａｎｔｒｏ…     

脐血中树突状细胞的体外诱导和抗肿瘤效应的实验研究

探讨脐血来源的树突状细胞（ＤＣ）的体外诱导及其在抗肿瘤免疫中的作用。方法无菌条件下常规要脐血,采用淋巴细胞分离液分离脐血单个核细胞,去除悬浮细胞后获得单核细胞。将获得的单核细胞分为两组,实验组１在含有细胞因子白介素４（ＩＬ－４）、粒单细胞集落刺激因子（ＧＭ－ＣＳＦ）和肿瘤坏死因子（ＴＮＦ－α）的综合成培养液ＤＭＥＭ中培养,对照组１培养液中未加上述细胞因子,第１０天收集部分细胞进行细胞表型分析,并     

Recruitment of CD4 T lymphocytes and macrophages into the cervical epithelium of women after coitus

OBJECTIVE: T lymphocytes and macrophages are considered essential components of the immune response. Many factors are known to influence the presence and distribution these cells in genital mucosa. This study investigated the effect of sexual intercourse on cervical intraepithelial T lymphocytes and macrophages in healthy uninfected women. STUDY DESIGN: Cervical intraepithelial samples were obtained with an endocervical brush from 31 women; the cervical T lymphocytes and macrophages were analyzed by flow cytometry. Eleven women with a history of last sexual intercourse at <3 days were compared against 20 women with last sexual intercourse of >3 days. Furthermore, cellular activation markers (CD69, CD25, HLA-DR) on T lymphocytes and costimulatory molecules (CD80, CD86) on macrophages were studied. RESULTS: Women with last sexual intercourse at <3 days showed predominance of CD4(+) T lymphocytes compared with women with last sexual intercourse of >3 days (P <.02); the numbers of macrophages were higher in the latter (P <.005). No difference was found in the density of T-lymphocyte activation and macrophage costimulatory markers between the two cohorts. CONCLUSION: Within cervical epithelium, the distribution of mononuclear leucocytes may be altered after coitus. The higher proportion of cervical intraepithelial CD4(+) T cells that were observed in the early postcoital period suggests a mechanism by which the relative risk of the acquisition of human immunodeficiency virus infection is increased in women.     

卵巢上皮癌组织中HLAⅠ、CD54和CD80的表达及其临床意义

目的 :研究卵巢上皮癌 (OEC)组织中人类白细胞抗原Ⅰ (HLAⅠ )、CD54和CD80的表达及其临床意义。方法 :应用免疫组化法检测HLAⅠ和CD54的表达 ,用逆转录多聚酶链反应 (RT -PCR)检测CD80mRNA的表达。结果 :4 4份OEC组织中 ,HLAⅠ、CD54的阳性表达率分别为 59.0 9%和 4 5.4 5%。CD80mRNA的表达率为 9.0 9%。HLAⅠ类分子的阳性表达在Ⅲ～Ⅳ期显著低于Ⅰ～Ⅱ期 (P <0 .0 5) ,有淋巴结转移组显著低于无淋巴结转移组 (P <0 .0 5) ,肿瘤复发组显著低于未复发组 (P <0 .0 1)。CD54的表达率与临床指标及复发均无关 ,但在无HLAⅠ表达组 ,CD54表达患者的复发率显著升高 (P <0 .0 5)。CD80mRNA阳性 4例均未复发 ,而CD80mRNA阴性者复发率为 57.5% ,两组差异有显著性 (P <0 .0 5)。HLAⅠ与CD80同时缺乏时 ,患者复发率较HLAⅠ与CD80共表达时复发率显著升高 (P <0 .0 1)。结论 :OEC可能通过HLAⅠ、CD80、及CD54的异常表达逃避机体的免疫监视。检测上述调节分子的表达 ,可能对判断OEC患者的预后及指导免疫治疗有一定的意义     

Activating T regulatory cells for tolerance in early pregnancy — the contribution of seminal fluid

A state of active tolerance mediated by T regulatory (Treg) cells must be functional from the time of embryo implantation to prevent the conceptus from maternal immune attack. Male seminal fluid and ovarian steroid hormones are implicated in regulating the size and suppressive function of the Treg cell pool during the peri-implantation phase of early pregnancy. Evidence that antigens and cytokine signals in seminal fluid regulate the maternal immune response includes the following: (1) the Treg cell-inducing cytokine TGFβ and male alloantigens are present in seminal fluid; (2) seminal fluid delivery at coitus is sufficient to induce a state of active immune tolerance to paternal alloantigen, even in the absence of conceptus tissue; (3) female dendritic cells can cross-present seminal fluid antigens to activate both CD8⁺ and CD4⁺ T cells, and (4) mating events deficient in either sperm or seminal plasma result in diminished CD4⁺ CD25⁺ Foxp3⁺ Treg cell populations at the time of embryo implantation. Ongoing studies indicate that the cytokine environment during priming to male seminal fluid antigens influences the phenotype of responding T cells, and impacts fetal survival in later gestation. Collectively, these observations implicate factors in the peri-conceptual environment of both male and female origin as important determinants of maternal immune tolerance. Defining the mechanisms controlling tolerance induction will be helpful for developing new therapies for immune-mediated pathologies of pregnancy such as miscarriage and pre-eclampsia.     

CTLA4Ig gene transfer alleviates abortion in mice by expanding CD4+CD25+ regulatory T cells and inducing indoleamine 2,3-dioxygenase

Successful pregnancy requires a state of immunological tolerance since normally the maternal immune system does not reject the semi-allogeneic conceptus. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), a ligand for B7, delivers negative signals to antigen presenting cells (APCs) to compete with CD28 for binding to B7 molecules and down-regulate proinflammatory responses, thus inhibiting T cell activation. Using CBA/J × DBA/2 matings as an abortion-prone model, we observed that adenovirus-mediated CTLA4Ig (Ad-CTLA4Ig) gene transfer improves pregnancy outcome. Ad-CTLA4Ig therapy skewed the ability of serum cytokine production toward a Th2 bias. Flow cytometry revealed that Ad-CTLA4Ig administration expanded peripheral CD4⁺CD25⁺ regulatory T cell populations in CBA/J × DBA/2 matings. Furthermore, Ad-CTLA4Ig administration induced indoleamine 2,3-dioxygenase (IDO) and Foxp3 mRNA expression at the materno-fetal interface. Our results demonstrate that adenovirus-mediated CTLA4Ig gene transfer improves pregnancy outcome in a murine model of abortion by expanding the CD4⁺CD25⁺ regulatory T cell population and inducing IDO mRNA expression.     

Induction of class II major histocompatibility complex antigen expression in human granulosa cells by interferon gamma: a potential mechanism contributing to autoimmune ovarian failure

We investigated the possibility that major histocompatibility complex (MHC) class II antigens and interferon gamma, a product of activated T lymphocytes, play a role in human autoimmune oophoritis. MHC class II molecules initiate immune responses by presenting antigens to T-helper lymphocytes; interferon gamma can induce class II antigen expression at ectopic sites and has been implicated in the cause of various autoimmune disorders. We studied the expression of class II MHC antigens in ovaries from normal women of reproductive-age and from women with premature autoimmune ovarian failure. Immunoperoxidase technique applied to tissue sections of nine normal human ovaries revealed class II MHC antigen expression only on occasional cells of macrophage morphology; granulosa cells were negative regardless of stage of follicular maturation. In contrast, extensive and intense class II antigen expression was observed in granulosa cells in ovarian biopsy sections from four patients with premature autoimmune ovarian failure. Immunofluorescence and radioimmunoassay techniques used to detect and quantitate MHC antigens revealed that class II antigen expression could be induced and class I MHC antigen expression was enhanced in granulosa cell cultures after the addition of interferon gamma. These data provide evidence that autoimmune oophoritis is associated with ectopic expression of MHC class II antigens by ovarian granulosa cells, and that this phenomenon can be induced by the immunologic cytokine interferon gamma.     

Blockade of CD80 and CD86 at the time of implantation inhibits maternal rejection to the allogeneic fetus in abortion-prone matings

CD28/CTLA-4 interactions with their specific B7-ligands (CD80 and CD86) play a decisive role in antigenic and allogenic responses. Recently, experimental transplant studies demonstrated that donor-specific tolerance was achieved by blocking these interactions. However, the role of blockade of CD28/B7 costimulatory pathway in the maintenance of materno-fetal tolerance has received little attention. In the present study, abortion-prone CBA/J females mated with DBA/2 males were administered with anti-CD80 and anti-CD86 monoclonal antibodies (mAbs) on day 4 of gestation (time of murine implantation). We demonstrated that the combined use of anti-CD80 and anti-CD86 mAbs induced maternal tolerance to the fetus in the abortion-prone CBA/J mice, and displayed expansion of the maternal CD4(+)CD25+ regulatory T cell population and up-regulated expression of CTLA-4, suggesting an active mechanism of regulatory T cells in suppressing maternal rejection to the fetus. In addition, the anti-CD80/86 mAbs treatment enhanced Th2 and reduced Th1 cytokine production in mice, implying that the development of Th2 cells might contribute to maternal tolerance to her fetus. Together, these findings indicated that blocking CD80 and CD86 enhanced maternal tolerance to her fetus in mice by increasing regulatory T cell function and skewing toward a Th2 response. Our data might provide an enhanced understanding of the maternal-fetal immune relationship and be helpful in clinical trials for immunotherapy of recurrent spontaneous abortion.     

Altered dendritic cell function in normal pregnancy

In pregnancy, maternal immunity is skewed to favour maintenance of gestation and immune tolerance of a semi-allogeneic fetus. Dendritic cells are thought to play a crucial role in mediating the balance between immunity and tolerance, and determining the type of T helper cell response. We postulated that myeloid dendritic cells would be modified in pregnancy to favour type 2 T helper cell responses. We show that the proportion of circulating myeloid dendritic cells expressing CD86 and staining for HLA-DR were significantly lower in the third trimester of pregnancy compared with non-pregnant women. As pregnancy progressed through the third trimester to term, CD86 expression increased. Furthermore, monocytes from pregnant women differentiated into less phenotypically mature dendritic cells which expressed lower levels of CD80, CD86 and HLA-DR molecules compared with non-pregnant women. In response to inflammatory stimuli, monocyte-derived dendritic cells, from pregnant women up-regulated CD86 more than CD80, and secreted less IL-12p70 but more IL-10, compared with monocyte-derived dendritic cells from non-pregnant controls. Our results demonstrate that, in pregnancy, the dendritic cell system is modified to favour type 2 T helper cell responses.     

Human embryonic stem cells are immunogenic in allogeneic and xenogeneic settings

Recent studies have suggested that human embryonic stem cells (HESC) are immune-privileged and may thereby circumvent rejection. The expression of immunologically active molecules was studied by DNA microarray analysis and by flow cytometry. HESC were transplanted into immunologically competent mice and traced by fluorescence in-situ hybridization (FISH) and immunohistochemistry. The ability of HESC to directly and indirectly induce immune responses in CD4+ T-cells from naive and transplanted mice was studied. Their ability to induce immune responses of human CD4+ T-cells, when cultured in the presence of dendritic cells (DC) syngeneic to responder T-cells, was also analysed. HESC demonstrated expression of HLA class I and HLA class II genes, but the cell surface expression of HLA class II molecules was low even after incubation with IFNgamma. In wild-type mice, HESC could be demonstrated by FISH until 3 days after transplantation and were surrounded by heavy infiltrates of T-cells and macrophages. HESC induced a similar immune response as human fibroblast cells (HFib) on naive and immunized T-cells, both directly and in the presence of syngeneic DC. A similar response was observed in the allogeneic setting. It is concluded that HESC are immunologically inert and do not inhibit immune responses during direct or indirect antigen presentation, and they were acutely rejected in a xenogeneic setting.