Increased expression of Fas (APO-1, CD95) on CD34+ haematopoietic progenitor cells after allogeneic bone marrow transplantation

Up-regulation of Fas/APO-1 (CD95) on haematopoietic progenitors and Fas-mediated apoptosis have been suggested to occur in a possible pathological mechanism in some bone marrow failure syndromes. We examined the expression of Fas antigen and susceptibility to Fas-mediated suppression of donor-derived haematopoietic cells of allogeneic bone marrow transplantation (BMT) recipients. Cytofluorometric analysis revealed low expression of Fas on CD34+ bone marrow cells from marrow donors or healthy controls. However, significantly higher expression of Fas antigen was observed on CD34+ bone marrow cells of BMT recipients, in whom engraftment of donor bone marrow (BM) cells was confirmed. The addition of an agonistic anti-Fas antibody (Ab) (CH-11) to haematopoietic stem cell culture of BM cells more strongly suppressed colony formation from granulocyte-macrophage colony-forming units (GM-CFU) and erythroid burst-forming units (BFU-E) after BMT. Pretreatment by blocking anti-Fas Ab (ZB4) abrogated the Fas-mediated GM-CFU and BFU-E suppression. Purified marrow CD34+ cells from BMT recipients were also susceptible to the Fas-mediated colony suppression. Thus, donor-derived CD34+ haematopoietic cells increased their expression of Fas antigen and were susceptible to Fas-mediated haematopoietic suppression. These findings provide new insight for understanding the haematological condition after BMT.     

Increased surface receptor Fas (CD95) levels on CD4+ lymphocytes in patients with primary intestinal lymphangiectasia

Objective. Exudative enteropathy secondary to primary intestinal lymphangiectasia (PIL) is characterized by lymphopenia, hypogammaglobulinemia and hypoalbuminemia resulting from leakage of lymph fluid into the intestinal tract. The objective of this study was to better characterize the lymphopenia of PIL-confirmed patients. Material and methods. T-cell markers and T-cell proliferation/capacities (differentiation, activation and death) were analyzed for phenotype in 9 patients (6 F, 3 M, aged from 18 to 72 years). Results. Mean counts of CD4 and CD8 subsets were significantly decreased, 174±123/µl and 134±77/µl compared with controls, 858±260/µl and 482±164/µl, respectively (p<0.0001). Significant depletion of naïve (CD45RA⁺ CD62L⁺) CD4⁺ T cells was noted, with a mean expression of 7±4% compared with controls, 45±1% (p<0.0001). Both CD4⁺ and CD8⁺ T-cell mean subsets were activated as assessed by their proportion expressing the late activation markers HLA-DR, 18±7% and 19±9% compared with controls, 6±3% and 10±6%, respectively (p<0.0001 and p<0.01). The mean expression of CD95/Fas on CD4⁺ T cells was significantly higher in patients than in controls, 83±16% versus 45±13% (p<0.0001). No major abnormality of T-cell proliferation/capacities was observed. Conclusions. Our results suggest that the T-cell loss in PIL patients is probably due to various mechanisms including enteric lymphocytes loss and activation of residual T cells, leading to death. Moreover, this loss is not compensated by a sufficient increase in T-cell thymic production.     

Defective expression of the apoptosis-inducing CD95 (Fas/APO-1) molecule on T and B cells in IDDM

Summary Triggering of CD95 (Fas/APO-1) cell surface receptors regulates the elimination of autoreactive T and B lymphocytes through a mechanism of cell suicide called apoptosis. Three different mutations involving CD95 or its ligand are responsible for induction of autoimmunity in susceptible mouse strains. To determine whether a defect involving the CD95 receptor is associated with human insulin-dependent diabetes mellitus (IDDM), we have studied the expression of CD95 on peripheral blood mononuclear cells from IDDM patients at different stages of the disease. Three-colour flow cytometry and mean fluorescence analysis showed that T and B lymphocytes from newly diagnosed IDDM and patients with long-standing disease, and subjects at high risk of developing the disease were highly defective in CD95 expression (p<0.001), whereas monocytes from all the groups studied expressed normal amounts of CD95 molecules on their cell surface. T-cell subset analysis showed that the impairment of CD95 expression in IDDM patients and high-risk subjects involved both CD3⁺ CD4⁺ (p<0.001) and CD3⁺ CD8⁺ cells (p range: <0.01–0.001), suggesting that this alteration concerns both helper and cytotoxic T cells. Moreover, after activation in vitro with anti-CD3 monoclonal antibody, T cells from newly diagnosed IDDM patients maintained a reduced CD95 expression during the entire cell culture period (24–72 h) in comparison to the control population (p<0.001). In conclusion, we found a reduced expression of the apoptosis-inducing CD95 receptor on T and B lymphocytes of individuals with clinical and preclinical IDDM. We hypothesize that this defective expression may impair the capacity of autoreactive lymphocytes to undergo CD95-mediated apoptosis, contributing to the lack of control on beta-cell specific B- and T-cell clones.Abbreviations MFI Mean fluorescence intensity - mAb monoclonal antibody - ICA islet cell autoantibody - IAA insulin autoantibody - PE phycoerythrin - FITC fluorescein - Per-CP peridinid chlorophyll protein - PBMC peripheral blood mononuclear cells - AICD activation-induced cell death     

Plasma-soluble Fas (APO-1, CD95) and soluble Fas ligand in immune thrombocytopenic purpura

We investigated the levels of various cytokines and soluble factors in ITP patients, in order to determine the influence of these factors on the pathogenesis of ITP. We found increases in IL-2, IL-6, IFN-gamma, and M-CSF levels in ITP patients compared with those in healthy individuals. On lymphocyte phenotype analysis, we found no clear difference in total T cell population (CD2+ CD19- cells) or cytotoxic T cell frequency (CD8+ CD11b- cells) between these two groups. The frequency of helper/inducer T cells (CD4+ CD8- cells) was decreased in ITP patients. There was a significant increase in activated T cells (CD3+ HLA-DR+ cells) in ITP patients. Furthermore, frequencies of NK cells of potent activity (CD16+ CD56+ cells) were significantly elevated in ITP patients. Seventeen of the 54 ITP patients (31.5%) had elevated levels of sFas, and 11 of the 54 patients (20.4%) of sFasL. In addition, a significant increase of sFasL was observed in sFas-positive ITP patients, and in these patients the sFasL level was correlated with that of sFas (r = 0.687, p < 0.01). We found significant increases in IL-2 and sIL-2R levels in sFas-positive ITP patients. For other factors examined, however, there were no differences in level between sFas-positive and -negative ITP patients. Percentages of activated T cells (CD3+ and HLA-DR+ cells) and NK cells (CD16+ and CD56+ cells) were significantly higher in sFas-positive ITP patients than in sFas-negative ITP patients. These findings suggests that the pathogenesis of ITP includes alteration of the Fas/FasL pathway.     

Fas (CD95) expression and death-mediating function are induced by CD4 cross-linking on CD4+ T cells.

The CD4 receptor contributes to T-cell activation by coligating major histocompatibility complex class II on antigen presenting cells with the T-cell receptor (TCR)/CD3 complex, and triggering a cascade of signaling events including tyrosine phosphorylation of intracellular proteins. Paradoxically, CD3 cross-linking prior to TCR stimulation results in apoptotic cell death, as does injection of anti-CD4 antibodies in vivo of CD4 ligation by HIV glycoprotein (gp) 120. In this report we investigate the mechanism by which CD4 cross-linking induces cell death. We have found that CD4 cross-linking results in a small but rapid increase in levels of cell surface Fas, a member of the tumor necrosis factor receptor family implicated in apoptotic death and maintenance of immune homeostasis. Importantly, CD4 cross-linking triggered the ability of Fas to function as a death molecule. Subsequent to CD4 cross-linking, CD4+ splenocytes cultured overnight became sensitive to Fas-mediated death. Death was Fas-dependent, as demonstrated by cell survival in the absence of plate-bound anti-Fas antibody, and by the lack of CD4-induced death in cells from Fas-defective lymphoproliferative (lpr) mice. We demonstrate here that CD4 regulates the ability of Fas to induce cell death in Cd4+ T cells.     

Increase in CD95 (Fas/APO-1)-positive CD4+ and CD8+ T cells in peripheral blood derived from patients with autoimmune hepatitis or chronic hepatitis C with autoimmune phenomena

BACKGROUND: The expressions of CD95 (Fas/APO-1) and Bcl-2 are determinants of apoptosis in normal lymphocytes, and abnormalities in their expressions might contribute to the induction of autoimmunity. In this study, we examined the expressions of CD95 and Bcl-2 on freshly isolated T and B cells from patients with autoimmune hepatitis (AIH) or chronic hepatitis C associated with autoimmune phenomena (CH-C(AI)). METHODS: The CD95 and Bcl-2 expressions within CD4+ T, CD8+ T, and CD19+ B cell subsets were analysed by two-colour flow cytometry. RESULTS: The surface expression of CD95 was significantly high in both the CD4+ T and CD8+ T cell subsets derived from the patients with AIH and those with CH-C(AI), compared with expression in patients with CH-C and normal subjects. The increase in CD95 expression was associated with the phenotypic conversion of naive CD45RO- to primed CD45RO+ CD4+ T cells. Bcl-2 was detected in the vast majority of peripheral T and B cells. There was no significant difference in the percentage of Bcl-2-positive cells in the CD4+ T cell, CD8+ T cell and CD19+ B cell subsets among the patient groups and normal subjects. CONCLUSIONS: These results indicate that an increase in CD4+ T cells expressing CD45RO and CD95 marks an important subset of AIH and CH-C(AI) patients. These expanded CD95+ CD45RO+ primed T cells most likely reflect a continuous antigen-specific or non-specific activation of T lymphocytes, and/or the persistent presence of activated lymphocytes as a consequence of abnormalities in the peripheral deletion of activated lymphocytes. These persistently activated lymphocytes might play a role in the induction of autoimmunity in AIH and CH-C(AI).     

Resistance to Fas (APO-1/CD95)-mediated apoptosis and expression of Fas ligand in esophageal cancer: the Fas counterattack

The mechanisms by which esophageal tumors escape immunologic recognition and clearance are only partly understood at the molecular level. Esophageal cancers have been shown to evade host recognition by down-regulation of antigen presentation and production of immunosuppressive factors. Recently, two independent reports have shown that esophageal tumor cells abundantly express Fas ligand (FasL) in vivo. As the triggering agonist for Fas receptor (Fas or APO-1/CD95)-mediated apoptosis of lymphocytes, FasL normally plays immune down-regulatory roles, including activation-induced cell death of T and B cells, as well as maintaining immune privilege in certain organs. Fas ligand expressed by esophageal cell lines has been shown to induce apoptosis of cocultured Fas-sensitive lymphoid cells in vitro. FasL expression by esophageal carcinomas in vivo has been associated with significantly reduced tumor-infiltrating lymphocytes (TILs) in FasL-positive tumor nests, concomitant with significantly increased TIL apoptosis in these nests. These studies support a 'Fas counterattack' mechanism of immune escape in esophageal cancer. By expressing functional Fas ligand, esophageal cancer cells can deplete antitumor lymphocytes by inducing apoptosis. To express functional FasL, esophageal carcinomas also acquire molecular mechanisms to resist autocrine Fas-mediated apoptosis of tumor cells.     

Effect of long-term infection with nef-defective attenuated HIV type 1 on CD4+ and CD8+ T lymphocytes: increased CD45RO+CD4+ T lymphocytes and limited activation of CD8+ T lymphocytes

Members of the Sydney Blood Bank Cohort (SBBC) have been infected with an attenuated strain of HIV-1 with a natural nef/LTR mutation and have maintained relatively stable CD4+ T lymphocyte counts for 14-18 years. Flow cytometric analysis was used to examine the phenotype of CD4+ and CD8+ T lymphocytes in these subjects, including the immunologically important naive (CD45RA+CD62L+), primed (CD45RO+), and activated (CD38+HLA-DR+ and CD28-) subsets. The median values were compared between the SBBC and control groups, comprising age-, sex-, and transfusion-matched HIV-1-uninfected subjects; transfusion-acquired HIV-1-positive LTNPs; and sexually acquired HIV-1-positive LTNPs. Members of the SBBC not only had normal levels of naive CD4+ and CD8+ T lymphocytes, but had primed CD45RO+ CD4+ T lymphocytes at or above normal levels. Furthermore, these primed cells expressed markers suggesting recent exposure to specific antigen. SBBC members exhibited variable activation of CD8+ T lymphocytes. In particular, SBBC members with undetectable plasma HIV-1 RNA had normal levels of activated CD8+ T lymphocytes. Therefore, the result of long-term infection with natural nef/LTR mutant HIV-1 in these subjects suggests a decreased cytopathic effect of attenuated HIV-1 on susceptible activated CD4+ T lymphocyte subsets in vivo, and minimal activation of CD8+ T lymphocytes.     

Th1 CD4+ lymphocytes delete activated macrophages through the Fas/APO-1 antigen pathway.

The Fas/APO-1 cytotoxic pathway plays an important role in the regulation of peripheral immunity. Recent evidence indicates that this regulatory function operates through deletion of activated T and B lymphocytes by CD4+ T cells expressing the Fas ligand. Because macrophages play a key role in peripheral immunity, we asked whether Fas was involved in T-cell-macrophage interactions. Two-color flow cytometry revealed that Fas receptor (FasR) was expressed on resting murine peritoneal macrophages. FasR expression was upregulated after activation of macrophages with cytokines or lipopolysaccharide, although only tumor necrosis factor-alpha rendered macrophages sensitive to anti-FasR antibody-mediated death. To determine the consequence of antigen presentation by macrophages to CD4+ T cells, macrophages were pulsed with antigen and then incubated with either Th1 or Th2 cell lines or clones. Th1, but not Th2, T cells induced lysis of 60-80% of normal macrophages, whereas macrophages obtained from mice with mutations in the FasR were totally resistant to Th1-mediated cytotoxicity. Macrophage cytotoxicity depended upon specific antigen recognition by T cells and was major histocompatibility complex restricted. These findings indicate that, in addition to deletion of activated lymphocytes, Fas plays an important role in deletion of activated macrophages after antigen presentation to Th1 CD4+ T cells. Failure to delete macrophages that constitutively present self-antigens may contribute to the expression of autoimmunity in mice deficient in FasR (lpr) or Fas ligand (gld).     

Prognostic relevance of Fas (APO-1/CD95) ligand in human colorectal cancer

OBJECTIVE: Fas ligand (FasL) is an important mediator of immune function and induces apoptosis by binding to its receptor Fas on sensitized cells. It has recently been shown that malignancies may express FasL and acquire immune privilege by inducing apoptosis of lymphocytes. Acquired resistance to Fas mediated apoptosis is known to be an early event in carcinogenesis. The aim of this study was to determine the extent of FasL expression in patients with colorectal cancer and examine its relationship with several prognostic pathological features and survival. DESIGN AND METHODS: Sixty-eight patients (median age 66 years) with colorectal cancer, whose diagnosis was made between 1988 and 1991 and in whom long-term follow-up was available, were evaluated. The tumours were of varying stages at diagnosis (eight Dukes' A, 28 Dukes' B, 23 Dukes' C and nine Dukes' D). The expression of FasL was detected immunohistochemically with a rabbit polyclonal IgG using the DAKO EnVision+ System. The specificity of FasL binding was confirmed by pre-incubation of the antibody with the immunizing peptide prior to staining. The relationship with several pathological features was determined using Kendall's tau-b correlation. Overall survival was estimated using the Kaplan-Meier product limit curves. Differences in observed survival were tested for statistical significance using the Mantel-Haenszel log rank test. Both the extent and intensity of staining were graded by a blinded observer. RESULTS: FasL was predominantly expressed in tumour epithelial cells in 88% of the cases. The positive staining of tumours varied in extent. FasL staining was higher in earlier Dukes' stage tumours in that the extent of FasL staining negatively correlated with Dukes' stage (Kendall tau-b = -0.22, P = 0.038). Consistent with this, the overall survival was better with a greater extent of FasL expression (log rank chi2 = 5.68, P = 0.017). There was a lower extent of FasL expression in mucinous adenocarcinomas (Kendall tau-b = 0.288, P = 0.01) and in those tumours with neural invasion (Kendall tau-b = -0.26, P = 0.03). No relationship was detected between FasL and tumour site, size, margin, differentiation, vascular invasion, necrosis or Crohn's-like reaction. CONCLUSIONS: FasL is widely expressed in colorectal cancers. This finding suggests that the extent of FasL expression in colorectal tumours is directly related to patients' survival.     

Increased CD4+CD8+ Double Positive T Cells during Hantaan Virus Infection

Hantaan virus (HTNV) infection causes an epidemic of hemorrhagic fever with renal syndrome (HFRS) mainly in Asia. It is well known that T cells mediated anti-viral immune response. Although previous studies showed that double positive T (DP T) cells, a little portion of T lymphocytes, were involved in adaptive immune response during virus infection, their kinetic changes and roles in HTNV infection have not yet been explored. In this study, we characterized DP T cells from HFRS patients based on flow cytometry data combined with scRNA-seq data. We showed that HTNV infection caused the upregulation of DP T cells in the peripheral blood, which were correlated with disease stage. The scRNA-seq data clustered DP T cells, unraveled their gene expression profile, and estimated the ordering of these cells. The production of granzyme B and CD107a from DP T cells and the abundant TCR distribution indicated the anti-viral property of DP T cells. In conclusion, this study identified, for the first time, an accumulation of DP T cells in the peripheral blood of HFRS patients and suggested these DP T cells belonging to CD8⁺T cells lineage. The DP T cells shared the similar characteristics with cytotoxic T cells (CTL) and exerted an anti-viral role in HFRS.     

Mutation analysis of CD95 (APO-1/Fas) in childhood B-lineage acute lymphoblastic leukaemia

The CD95 system plays an important role in lymphocyte homeostasis, has been implicated in the development of lymphoid malignancies, exerts a tumour suppressor function, and contributes to drug-induced cytotoxicity. We hypothesized that mutations of CD95 may occur in childhood B-lineage acute lymphoblastic leukaemia (ALL), a disease known for its constitutive resistance towards CD95-mediated apoptosis. We investigated 32 primary B-lineage ALL of childhood and five B-lineage ALL cell lines. All primary leukaemias expressed CD95 and bcl-2 to a variable degree. Most of the leukaemias were resistant towards CD95-mediated apoptosis. However, using SSCP analysis, no mutations in the coding and proximal promoter region could be detected. We conclude that the resistance towards CD95-mediated apoptosis observed in most de novo B-lineage ALL is not caused by mutations of the CD95 death receptor.     

Intrathyroidal CD4+ T lymphocytes express high levels of Fas and CD4+ CD8+ macrophages/dendritic cells express Fas ligand in autoimmune thyroid disease.

In autoimmune thyroid disease (AITD), the proportion of CD4 lymphocytes is lower in the thyroid than in the peripheral blood. We examined both Fas and Fas ligand (FasL) expression in lymphocyte subsets and nonlymphoid mononuclear cells including monocytes, macrophages, and dendritic cells (M/DCs) in both peripheral blood and thyroid specimens from 11 patients with Graves' disease and 1 with Hashimoto's disease by three-color flow cytometry. Proportions and intensities of Fas expression were increased in CD4 single-positive (SP) (CD4(+) CD8(-)), CD8 SP (CD8(+) CD4(-) ), and CD4(+) CD8(+) double-positive (DP) lymphocytes in AITD thyroids compared to those in blood, and were much higher in CD4(+) (CD4 SP and DP) lymphocytes than in CD8 SP lymphocytes in the thyroid. In the blood, most M/DCs expressed only CD4, but approximately 60% of M/DCs expressed both CD4 and CD8 in AITD thyroid. The proportion of DP M/DCs expressing FasL was higher in thyroid than in blood; proportion and intensity of FasL expression were much higher in DP M/DCs than in CD4 SP and CD8 SP M/DCs in the thyroid. These data indicate that increased Fas expression in intrathyroidal CD4(+) T lymphocytes may be the cause of CD4 lymphocyte reduction in AITD thyroid, and that intrathyroid DP M/DCs with high FasL expression may be related to the reduction in AITD.