Fas/FasL和Bcl-2在人乳腺癌组织中的表达及其与浸润淋巴细胞的关系

目的通过观察乳腺癌组织中凋亡调节蛋白Fas、FasL、Bcl-2的表达及其与肿瘤浸润淋巴细胞(TIL)的关系,探讨Fas、FasL、Bcl-2与乳腺癌发生、发展的关系,为乳腺癌的生物治疗提供实验依据。方法收集手术切除的人乳腺癌组织和癌旁相对正常乳腺组织。用免疫组织化学方法和图像分析技术对21例乳腺癌标本进行检测。结果乳腺癌组织中Fas的阳性表达率明显低于相对正常乳腺组织(P<0.05),且淋巴细胞浸润性乳腺癌组织中Fas的阳性表达明显低于非浸润性乳腺癌组织(P<0.05);乳腺癌组织中FasL、Bcl-2的阳性表达率明显高于相对正常乳腺组织(P<0.05),且淋巴细胞浸润性乳腺癌组织明显高于非淋巴细胞浸润性乳腺癌组织(P<0.05);乳腺癌组织中Fas阳性淋巴细胞的数量与相对正常乳腺组织相比明显增多(P<0.05);乳腺癌组织Bcl-2阳性淋巴细胞的数量与相对正常乳腺组织相比明显减少(P<0.05)。结论①乳腺癌组织中Fas表达下调和FasL的过度表达,逃避了免疫监视,诱导Fas敏感的TIL凋亡,从而导致肿瘤的生长;②乳腺癌组织中癌基因Bcl-2过度表达及Bcl-2阳性淋巴细胞的低表达,使肿瘤抗凋亡机制过度激活,肿瘤细胞对Fas/FasL易感性较淋巴细胞相对低,导致TIL死亡,而不是肿瘤细胞死亡。     

Interleukin-12 up-regulates perforin- and Fas-mediated lymphokine-activated killer activity by intestinal intraepithelial lymphocytes

Human intraepithelial lymphocytes (IELs) comprise a unique compartment of memory T cell receptor (TCR)-alphabeta( +)CD8(+) T lymphocytes interspersed between intestinal epithelial cells. They develop potent lymphokine-activated killer (LAK) activity with interleukin (IL)-15, a cytokine that is found in excess in certain mucosal inflammatory states. IL-12, released by activated antigen-presenting cells, is known to potentiate perforin-induced cytotoxicity. This study evaluates the mechanism by which IL-12 up-regulates LAK activity. When IELs were stimulated with IL-15, the CD94(+) IEL subset expanded and carried out cytotoxic activity in redirected lysis against P815 cells as well as Fas ligand (FL)- and tumour necrosis factor (TNF)-alpha-mediated lysis of Jurkat and WEHI cells, respectively. IL-12 enhanced the perforin- and FL-, but not TNF-alpha-mediated events. In addition, the up-regulated killing of HT-29 cells by IL-12 was reduced by concanamycin (which targets perforin) and antibody neutralizing FL but not by anti-TNF-alpha antibody. Furthermore, IL-12 augmented IL-15-stimulated release of serine esterases as well as expression of perforin and FL by IELs, but not TNF-alpha. This study shows that LAK activity, carried out by the CD94(+) IELs, involves perforin, FL and TNF-alpha. IL-12 up-regulates the first two mechanisms of action, showing for the first time its effect on FL production and lytic activity.     

Stimulation of FACS-analysed CD4+ and CD8+ human tumour-infiltrating lymphocytes with ionomycin + phorbol-12,13-dibutyrate does not overcome their proliferative deficit.

Human tumour-infiltrating lymphocytes (TIL) were prepared by enzyme digestion from a series of different tumours and were purified on a fluorescence-activated cell sorter (FACS II) according to their CD4+ and CD8+ phenotype. CD4+ and CD8+ TIL were stimulated separately in a low density microculture system with phytohaemagglutinin (PHA) or with ionomycin plus phorbol-12, 13-dibutyrate (PDBu). The PHA-induced proliferation of TIL was highly decreased when compared with control peripheral blood lymphocytes. A decreased proliferation of TIL was also observed when cells were stimulated with ionomycin plus PDBu, a combination which is thought to circumvent early events associated with lymphocyte activation. Some TIL were also plated in limiting dilution where they showed decreased frequencies of proliferating T cell precursors. The data suggest that one component of the inhibition of TIL must be acting 'downstream' of the early events of lymphocyte activation.     

T cell receptor gene rearrangements and cytotoxic activities of clones isolated from tumour-infiltrating lymphocytes (TIL) from melanoma patients

The lymphocytes which infiltrate tumours and are grown in vitro to be used in adoptive immunotherapy are often characterized by dominant rearrangement of their T cell receptor (TCR) genes. To investigate the frequency and function of cells contributing to the ‘dominant’ rearrangement, we have cloned two bulk cell lines of TIL derived from melanoma patients (TIL-1 and TlL-5). These IL-2-propagaled TIL cell lines had a CD8⁺ phenotype and exerted strong cytotoxic activity against autologous melanoma cells, but not against the natural killer (NK)-sensitive K-562 cell line or LAK targets such as Daudi cells. We derived 40 clones from TIL-1 and 23 from TIL-5. All tested clones were CD3⁺, CD4^?, CD8⁺ and expressed the α/β TCR. From TIL-1.27 of 40 clones, and 13/19 of the TIL-5 clones lysed autologous tumour cells. In contrast to the NK, -negative bulk cultures, K-562 killing was detected in 21 of the TIL-1 clones and 17 of the TIL-5 clones. TIL-1 contained eight clones and TiL-5 two clones with lytic capacity against neither autologous tumour cells nor the K562 cell line, although these clones possessed lytic potential as evidenced in a lectimediated lysis assay. LAK activity was not detected in most clones. Cytotoxic activity against autologous tumour could be inhibited by preincubation with anti-CD3 or anti-HLA class I MoAbs, Of the 34 TlL-1 clones analysed, 15 shared a rearranged TCRβ EcoR1 restriction fragment of approximately 9 5 kb with the bulk culture. Clones sharing the EcoR1 10 5-kb dominant band present in TIL-5 bulk culture were also isolated. When the pattern of TCRβ rearrangement was compared with the cytotoxic functions, the following conclusions could be drawn: (i) clones contributing to the dominant band had heterogeneous functions. Most killed autologous tumour cells, but clones with no cytotoxic activity or even with no proliferative capacity in response lo autologous tumour cells were also detected among those contributing to the dominant rearrangement; (ii) some clones that share an apparently identical rearranged band different from the “dominant” rearrangement, may demonstrate the same cytotoxic function. In addition, our data suggest that many of the clones that share the dominant rearrangement originated from diverse progenitors. The high frequency of clonally diverse anti-tumour reactive TIL is likely to be a reflection of the in vivo selection of the TCR repertoire at the site of tumour. Further study of the TCR gene rearrangements should help to clarify how selection at this level can benefit future immunotherapeutic approaches.     

Regulation of Fas(Apo-1/CD95)- and perforinmediated lytic pathways of primary cytotoxic T lymphocytes by the protooncogene bcl-2

Cytotoxic T cells (CTL) induce cell death of their target cells either by the surface interaction between Fas ligand and Fas or by the release of perforin and granzymes. Both lytic pathways induce apoptosis yet it is not known whether identical or distinct apoptotic pathways are activated. The protooncogene bcl-2 is known to protect various hematopoietic cells from apoptosis induced by diverse agents. Here we show that overexpression of the Bcl-2 protein in the murine mastocytoma line P815 or in concanavalin A-activated splenocytes suppresses apoptotic cell death induced by allospecific primary cytotoxic T lymphocytes (CTL) in which only the Fas lytic pathway was functional. Bcl-2 also reduced target cell killing induced by CTL whose lytic activity was dependent on the perforin/granzyme pathway only. These data provide evidence that, in the target cells studied here, both perforin/granzyme and Fas apoptotic pathways are modulated by Bcl-2 and suggest that these two pathways converge at a step prior to Bcl-2 inhibition.     

Enhanced T cell apoptosis in common variable immunodeficiency: negative role of the fas/fasligand system and of the Bcl-2 family proteins and possible role of TNF-RS

CVI is a primary immunodeficiency characterized by a failure of B cell differentiation associated with an array of T cell defects, such as enhanced T cell apoptosis. In this study we investigated the mechanisms underlying CVI enhanced T cell death. We analysed both the expression of Fas using flow cytometry techniques and the expression of FasL mRNA using RT-PCR in CVI T cells. We could not find any significant differences between CVI and normal subjects with regard to Fas expression, although there was a subgroup of CVI patients with very high Fas expression which was accompanied by an up-regulation of FasL mRNA. However, attempts to induce Fas-mediated apoptosis in these high Fas expressing cells, as evaluated by propidium iodide staining and APO2.7 staining, were unsuccessful. We also investigated intracellular levels of Bcl-2, bcl-xl and bax in CD4(+) and CD8(+) CVI T cells, as well as the bax/Bcl-2 ratio, using flow cytometry techniques but could not detect any differences between CVI and normal subjects. Finally we analysed TNF-RI and TNF-RII mRNA expression in CD4(+) and CD8(+) CVI T cells using semiquantitative RT-PCR and found a significant increase in expression of both TNF-Rs in CD4(+) T cells from CVI patients. Our data suggest that the increased expression of both TNF-Rs on T cells may be one of the mechanisms responsible for the accelerated T cell apoptosis in CVI.     

Characterization of fresh (uncultured) tumour-infiltrating lymphocytes (TIL) and TIL-derived T cell lines from patients with renal cell carcinoma.

Fresh (uncultured) TIL from 12 untreated patients with primary renal cell carcinoma were prepared from tumour specimens by enzymatic digestion, and were characterized by immunofluorescence using MoAbs recognizing leucocyte differentiation antigens or particular V alpha or V beta segments of the T cell receptor (TCR). These fresh TIL comprised CD3+ (20-84%); CD4+ (3-15%); CD8+ (13-35%); alpha beta TCR+ (20-50%); gamma delta TCR+ (3-17%); CD16+ (1-18%) and CD56+ (3-10%) cells. Significant proportions of V alpha 2+, V beta 5.1+ and V beta 6+ cells were found in TIL of certain patients with renal cell carcinoma, suggesting that they comprised oligoclonal T cells. T cell lines were developed in low concentrations of rIL-2 (200 U/ml) from TIL from 11 patients with renal cell carcinoma, and were characterized by immunofluorescence and cell-mediated cytotoxicity. These T cell lines consisted primarily of CD3+ (51-94%); CD4+ (1-80%); CD8+ (0-84%); alpha beta TCR+ (65-87%); gamma delta TCR+ (0-25%); CD16+ (0-16%) and CD56+ (2-57%) cells. These T cell lines exhibited non-specific cytotoxicity against autologous and allogeneic renal tumour cells, with the exception of one T cell line that exhibited preferential cytotoxicity against autologous renal tumour cells. These results suggest that fresh TIL from patients with renal cell carcinoma contain significant proportions of oligoclonal T cells that may have accumulated at the tumour site as a result of a clonal expansion.     

Antigen(s)-specific tumour-infiltrating lymphocytes from tumour induced by human herpes virus-6 (HHV-6) DNA transfected NIH 3T3 transformants.

Tumour infiltrating lymphocytes (TIL) have recently been shown to mediate potent therapeutic effects in certain malignancies in mice and in humans. To understand the mechanism of TIL immunotherapy it would be advantageous to generate tumour-specific TIL and to study a defined system of TIL and target cells in which the tumour epitope(s) recognized by TIL might be identified. We have established tumourigenic cell lines by transfection of NIH 3T3 cells with the entire genome of human herpesvirus-6 (HHV-6) and its small fragment (about 5% of the viral DNA sequence). Injection of these cells into nude mice produced tumours termed G-2T and 14-2T, respectively. Cell lines derived from these tumours when injected in NIH Swiss mice produced tumours, G-2TS and 14-2TS, respectively. We have generated TIL from G-2TS tumour that can kill G-2TS tumour cells in vitro but not other related tumours (14-2TS or MCA-106). These TIL can be expanded between 2-6.5 every 3-5 days. The TIL proliferated in tissue culture in response to recombinant interleukin-2 and interleukin-4 and maintained their tumor specificity for up to 6 months in vitro. Their phenotype was Thy 1.2+, Lyt-2+ and L3T4-. The availability of such tumour-specific stable TIL lines and specific viral-transformed targets will provide an opportunity to characterize the tumour-associated antigen critical for the specific cytotoxicity in this system and thereby to clarify the mechanism of this promising immunological approach to cancer therapy.     

Essential role for hematopoietic Fas ligand (FasL) in the suppression of melanoma lung metastasis revealed in bone marrow chimeric mice

The outcome of cancer metastasis depends on multiple interactions between the malignant cell and the host environment. Such interactions can influence primary cancer growth and metastasis by altering the balance between tumor cell proliferation and death. We have previously reported that the pro-apoptotic protein FasL could potently suppress spontaneous lung metastasis of the Fas-sensitive melanoma, K1735-P. In this report, we have constructed bone marrow chimeric mice using wt and FasL-deficient animals to delineate the source of FasL (hematopoietic or nonhematopoietic) required to control spontaneous metastatic spread from a subcutaneous tumor. Using FasL-deficient animals (gld) reconstituted with wt FasL bone marrow (wt --> gld), and wt animals reconstituted with FasL-derived bone marrow (gld --> wt), we show, for the first time, an essential role for hematopoietic-derived FasL in the suppression of K1735-P metastasis. When FasL was expressed only in the nonhematopoietic compartment (gld --> wt), K1735-P spread was ineffectively controlled with a metastatic phenotype similar to that observed in animals completely lacking FasL (gld --> gld or gld controls). These studies provide evidence for the indispensable role for FasL+ bone marrow-derived cells in the control of melanoma and offer a strategy to target FasL expression in the prevention or therapy of metastatic disease.     

Activated status of tumour-infiltrating lymphocytes and apoptosis in testicular seminoma.

Testicular seminoma is characterized by a prominent lymphoid infiltrate and an excellent prognosis. Cytotoxic T-lymphocytes (CTLs) infiltrating seminoma tumour nests constitute a major subset of the lymphoid infiltrate. The objective of this study was to determine whether CTLs express markers of cytotoxic potential and activity and whether the number of activated CTLs correlates with the extent of apoptosis in testicular seminomas, as opposed to non-seminomatous testicular germ cell tumours (NSTGCTs). Twenty cases of pure seminoma as well as 20 cases of NSTGCTs including 16 mixed germ cell tumours (MGCTs) were studied. Immunohistochemistry for the cytotoxic markers TIA-1 (cytotoxic potential) and granzyme B (cytotoxic activity) and the T-cell markers CD3 and CD8 was performed on formalin-fixed, paraffin-embedded sections. The apoptotic index (AI) was determined by the TUNEL method. The number of CD3(+), CD8(+), TIA-1(+), and granzyme B(+) cells in tumour cell nests was markedly increased in testicular seminomas, compared with NSTGCTs (p<0.01). Activated granzyme B(+) cells numbered 25.6+/-5.2 per high power field in seminomas and 8.9+/-3.2, 8.1+/-3.9, and 0.4+/-0.2 for embryonal carcinomas, yolk sac tumours, and immature teratomas, respectively. Double immunohistochemical staining for granzyme B and CD8 revealed that 82.6+/-8.5% of granzyme B-expressing cells were CD8(+). The tumour cell AI was significantly increased in embryonal carcinoma, compared with the seminoma, yolk sac tumour, and immature teratoma subgroups (6.7+/-1.3, 2.3+/-0.3, 3.0+/-1.1, and 2.3+/-1.1, respectively, p<0.001). TUNEL/CD3 double immunostaining revealed that a significant proportion of the apoptotic seminomatous tumour cells were in direct contact with one or more CD3(+) lymphocytes (47.2+/-6.2%). The number of activated granzyme B(+) CTLs showed a strong linear correlation with the AI in the seminoma group (r=0.71, p<0.0001) but not in other subgroups. TUNEL/granzyme B double immunolabelling revealed that a proportion of activated granzyme B(+) lymphocytes (20%) were often seen in close contact with apoptotic tumour cells. The presence of increased numbers of activated cytotoxic lymphocytes in testicular seminomas suggests that apoptotic tumour cell death in this neoplasm may be triggered by cytotoxic granule effectors. This phenomenon may be one of the key host immune mechanisms leading to the excellent prognosis in this tumour.     

Fas/FasL、Bcl-2/Bax蛋白在母胎界面的表达

目的: 探讨在牦牛妊娠早期的功能及与其它动物的异同.方法: 取妊娠早期牦牛不同阶段的胎盘组织, 采用SP免疫组化方法检测牦牛妊娠早期胎盘中Fas/FasL、 Bcl-2/Bax蛋白表达特征.结果: 在牦牛妊娠早期, 子宫内膜上皮和滋养层中Fas/FasL呈阳性表达, 而在子宫内膜中的淋巴细胞和浆细胞也呈Fas/FasL阳性.Fas蛋白在子宫内膜上皮中的表达量随妊娠进行有下降趋势, 而在滋养层中几乎均为强阳性表达.FasL的表达量在滋养层中没有发生变化, 一直呈强阳性表达;而在子宫内膜中, 几乎均为中等表达. Bcl-2蛋白在子宫内膜中的表达量16～20 d较高, 之后表达量呈下降趋势;在滋养层中没有发生变化.Bax蛋白在妊娠16 d的子宫内膜中的表达量较高, 其余均为低表达;在滋养层中的表达量变化不大, 一直较高.在凋亡的细胞中, Bax着色较深.结论: 在牦牛妊娠过程中, 子宫内膜上皮和滋养层中Fas/FasL呈阳性表达, 可能与牦牛妊娠过程中的免疫赦免有关.     

Mechanisms of villous atrophy in autoimmune enteropathy and coeliac disease

Since in coeliac disease mucosal flattening has been suggested to result from an increased enterocyte apoptosis triggered by Fas/Fas ligand system and perforin cytolytic granules, we looked for a similar mechanism in autoimmune enteropathy. Moreover, we tried to assess whether enterocyte autoantibodies, which are the hallmark of autoimmune enteropathy, may be involved in triggering enterocyte apoptosis in this condition. Immunohistochemical staining with anti-Fas, -FasL and -perforin MoAb, and TUNEL technique were applied on endoscopic duodenal biopsies of two autoimmune enteropathy patients, two untreated coeliac patients and two biopsied controls. Cytotoxicity assays were carried out by incubating peripheral blood mononuclear cells from a healthy subject (effectors) with enterocytes primed with patient or control sera (targets). In autoimmune enteropathy a large number of enterocytes were apoptotic, as in coeliac disease, whereas neither Fas/Fas ligand or perforin expressions were up-regulated. On the other hand, antibody-dependent cellular cytotoxicity assay revealed the ability of sera from patients with autoimmune enteropathy to mediate enterocyte death through apoptosis. These results point to enterocyte autoantibody-dependent cellular cytotoxicity as the prevalent mechanism of increased enterocyte apoptosis in autoimmune enteropathy but not in coeliac disease.     

Suppression of apoptosis in normal and neoplastic human B lymphocytes by CD40 ligand is independent of Bcl-2 induction

The tendency of isolated germinal center (GC) B cells to undergo apoptosis was suppressed by recombinant cell-bound CD40 ligand (CD40L): after 2 days at 37°C, > 80 % of cells remained viable in the presence of CD40L as compared to < 1 % in control cultures. CD40L sustained a high rate of DNA synthesis in GC cells and was more effective than monoclonal antibody to CD40 in this regard. Group I Burkitt lymphoma (BL) cell lines induced to undergo apoptosis with anti-immunoglobulin or calcium ionophore were also protected by CD40L. In BL cells, this route of rescue was not accompanied by induction of Bcl-2 protein, the expression of which has been linked to hemopoietic cell survival. Bcl-2 was induced in GC cells responding to CD40L, but its appearance was a relatively late event not reaching significant levels over controls until day 2 of culture. Thus induction of Bcl-2 appears to be secondary to the survival signal imparted by CD40L. These findings are discussed in relation to a potential role for CD40L in supporting B cell tumors in vivo and the discovery that the molecular defect in the X-linked Hyper-IgM syndrome is targeted to the CD40L gene.     

Perforin and Fas/FasL cytolytic pathways at the maternal-fetal interface

The immunogenetic enigma of maternal acceptance of the fetal semiallograft has been termed an immunological paradox. The first trimester decidua is heavily infiltrated with CD56(bright) CD16- uterine natural killer (uNK) cells which must be prepared to respond to potential pathogen challenges and still be able to control immune responses that allow the development of the fetus. The significant presence of cytolytic mediators, perforin and Fas/Fas ligand (FasL), at the maternal-fetal interface raises a question of their role(s) in the immunological interrelations between maternal tissues and trophoblast cells. As uNK cells in vitro lyse target cell lines (K562, P815 and P815Fas) using these effector molecules, it seems that, although immunocompetent, their cytotoxicity is not directed against trophoblast during normal pregnancy. Therefore, it is generally believed that the hormonal and Th1/Th2 cytokine balance plays an important role in the tolerance and maintenance of pregnancy. This paper gives an overview of the recent findings on the complex immunological events that occur at the maternal-fetal interface.     

Cytolytic mechanisms of intraepithelial lymphocytes in coeliac disease (CoD)

The effector arm of the mucosal immune system comprises lymphocytes scattered at intraepithelial and lamina propria levels. Intraepithelial lymphocytes (IEL) are a large population of oligoclonal resting cells which exhibit phenotypic and functional characteristics of cytolytic T cells when activated. Several mechanisms have been demonstrated to account for their cytotoxicity. Among them, one is mediated by perforin and granzyme molecules, another is mediated by Fas ligand (FasL) which delivers apoptotic signals through Fas receptor on target cells. There is good evidence that a flat intestinal mucosa may be produced by activated T cells. The aim of our study was to evaluate FasL and perforin expression by IEL, and its possible correlation with the increased enterocyte apoptosis in coeliac mucosa. Endoscopic duodenal biopsy specimens from 10 untreated coeliac patients, 10 treated coeliac patients, and 10 biopsied controls were evaluated for enterocyte apoptosis by terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine triphosphate nick end label method, for perforin expression by immunohistochemistry, and for FasL expression by immunocytochemistry. In untreated CoD there was a significant increase of percentage of both FasL+ and perforin+ IEL which positively correlated with enterocyte apoptosis in comparison with controls. All these parameters were significantly lower in treated CoD, even though they did not normalize. Our study demonstrates that in untreated CoD FasL and perforin expression by IEL is increased, and significantly correlates with the level of enterocyte apoptosis.     

T细胞凋亡受体和共刺激分子的表达变化与终末期肾功能衰竭患者细胞免疫功能的关系研究

目的：探究终末期肾功能衰竭患者T细胞亚群的凋亡受体CD95分子与共刺激分子CD28、CDl52（CTLA-4）的表达与细胞免疫功能的关系。方法：采用流式细胞术检测外周血T细胞的凋亡受体CD95（Fas）与共刺激分子CD28／CDl52表达。结果：终末期肾功能衰竭患者CD3^＋T细胞和CD4^＋T细胞比例明显高于健康对照组，CD4／CD8比值增加（P=0．008），CD4^＋T细胞和CD8^＋T细胞上CD95分子表达均上调（P=0．001），以CD8^＋T细胞上CD95分子增加更为明显；CD28和CD152分子在不同T细胞亚群上的表达均上调（P〈0．05），然而，CD4^＋T细胞以CD28分子表达增加为主，而CD8^＋T细胞则CD152分子表达增加为主。结论：终末期肾功能衰竭患者的细胞亚群失衡，共刺激分子CD28和CD152表达异常增加，提示T细胞活化与抑制性调节发生紊乱。T细胞亚群上凋亡受体CD95分子表达增加，以CD8^＋T细胞为主，说明终末期肾功能衰竭者淋巴细胞的减少可能通过两种途径——受体配体途径和负性共刺激分子CD152抑制信号途径，其中以CD8^＋T细胞减少为主，造成患者细胞免疫缺陷。     

Concentrations of Soluble CD95 and CD8 Antigens in the Plasma and Levels of CD8+CD95+, CD8+CD38+, and CD4+CD95+ T Cells Are Markers for HIV-1 Infection and Clinical Status

Apoptosis mediated via the CD95 (FAS/APO-1) receptor is thought to play a role in the depletion of CD4+ T cells in HIV infection. In the present study expression of the CD95 antigen on lymphocyte subsets and the plasma level of soluble CD95 (sCD95) were determined in HIV-1-infected adults. The expression of CD95 was increased on CD8 cells in all groups of HIV+ individuals, while increased expression of CD95+ cells on CD4 cells was limited to individuals with CD4 counts of <200 mm³. The proportion of CD4+ that expressed CD95 was inversely correlated with the percentage of CD4+ PBL. The concentration of sCD95 was significantly higher in the plasma of HIV-infected individuals than in normal controls. The level of sCD95 in HIV-infected subjects showed no correlation with the percentage of PBL expressing CD95, indicating that the increased level of sCD95 did not reflect release from CD95+ PBL. The plasma sCD95 concentration was significantly correlated with the percentage of CD8+ cells and, particularly, with CD8+CD38– cells. A striking inverse correlation was found between the sCD95 plasma concentration and the proportion of CD4+CD95+ cells out of the total CD4+ population. There was no correlation between the serum level of sCD95 and that of soluble CD8 (sCD8), both of which were increased in the plasma of HIV+ individuals. Unlike the level of sCD95, the level of sCD8 in the plasma of HIV+ individuals. Unlike the level of sCD95, the level of sCD8 in the plasma of HIV+ individuals was correlated with the percentage of CD95+ and CD8+CD38+ cells. The present study indicates that plasma sCD95 may be one of the factors that regulate apoptotic death of lymphocytes in HIV infection.     

Comparison of Fas(Apo-1/CD95)- and perforin-mediated cytotoxicity in primary T lymphocytes

Cytolytic T lymphocytes kill target cells by two independentcytolytic mechanisms. One pathway depends on the polarized secretionof granule-stored proteins including perform and granzymes,causing target cell death through membrane and DNA damage. Thesecond cytolytic effector system relies on the interaction ofthe Fas ligand (FasL) on the effector cell with its receptor(Fas) on the target cell, leading to apoptotic cell death. Usingmixed lymphocyte culture (MLC)-derived primary T lymphocytesof perforin-knockout and gld (with non-functional FasL) mice,the molecular basis of the two killing mechanisms was compared.The activity of both pathways was dependent on extracellularCa²⁺. Incubation of MLC-stimulated primary T cells with proteinsynthesis inhibitors prior to TCR triggering impaired FasL cellsurface expression and abolished cytolytic activity, althoughthe cells exhibited an intracellular pool of FasL. The perforin-dependentmechanism induced cell death more rapidly, although both pathwaysultimately showed similar killing efficiencies. Both pathwaysinduced comparable levels of DNA degradation, but Fas-inducedmembrane damage was less pronounced. We conclude that upon TCRtriggering FasL may be recruited in part from pre-existing intracellularstores. However, efficient induction of target cell death stilldepends on the continuous biosynthesis of FasL molecules.