Higher Expression Of CD95 Research Articles

Optimizing NK-92 serial killers: gamma irradiation, CD95/Fas-ligation, and NK or LAK attack limit cytotoxic efficacy

BackgroundThe NK cell line NK-92 and its genetically modified variants are receiving attention as immunotherapies to treat a range of malignancies. However, since NK-92 cells are themselves tumors, they require irradiation prior to transfer and are potentially susceptible to attack by patients’ immune systems. Here, we investigated NK-92 cell-mediated serial killing for the effects of gamma-irradiation and ligation of the death receptor Fas (CD95), and NK-92 cell susceptibility to attack by activated primary blood NK cells.MethodsTo evaluate serial killing, we used 51Cr-release assays with low NK-92 effector cell to target Raji, Daudi or K562 tumor cell (E:T) ratios to determine killing frequencies at 2-, 4-, 6-, and 8-h.ResultsNK-92 cells were able to kill up to 14 Raji cells per NK-92 cell in 8 h. NK-92 cells retained high cytotoxic activity immediately after irradiation with 10 Gy but the cells surviving irradiation lost > 50% activity 1 day after irradiation. Despite high expression of CD95, NK-92 cells maintained their viability following overnight Fas/CD95-ligation but lost some cytotoxic activity. However, 1 day after irradiation, NK-92 cells were more susceptible to Fas ligation, resulting in decreased cytotoxic activity of the cells surviving irradiation. Irradiated NK-92 cells were also susceptible to killing by both unstimulated and IL-2 activated primary NK cells (LAK). In contrast, non-irradiated NK-92 cells were more resistant to attack by NK and LAK cells.ConclusionsIrradiation is deleterious to both the survival and cytotoxicity mediated by NK-92 cells and renders the NK-92 cells susceptible to Fas-initiated death and death initiated by primary blood NK cells. Therefore, replacement of irradiation as an antiproliferative pretreatment and genetic deletion of Fas and/or NK activation ligands from adoptively transferred cell lines are indicated as new approaches to increase therapeutic efficacy.

Increased CD95 (Fas) and PD-1 expression in peripheral blood T lymphocytes in COVID-19 patients.

SummaryA low count of CD4+ and CD8+ lymphocytes is a hallmark laboratory finding in the coronavirus disease 2019 (COVID‐19). Using flow cytometry, we observed significantly higher CD95 (Fas) and PD‐1 expression on both CD4+ T and CD8+ T cells in 42 COVID‐19 patients when compared to controls. Higher CD95 expression in CD4+ cells correlated with lower CD4+ counts. A higher expression of CD95 in CD4+ and CD8+ lymphocytes correlated with a lower percentage of naive events. Our results might suggest a shift to antigen‐activated T cells, expressing molecules increasing their propensity to apoptosis and exhaustion during COVID‐19 infection.

Novel Simultaneous Single Cell mRNA and Protein Expression Profiling Identifies Distinct Treg and T Effector Signatures in the Bone Marrow of MDS Patients

Background Regulatory T cells (Tregs) are a non-redundant, suppressive population of CD4+ T cells that are essential for the prevention of autoimmune diseases in humans. They may also contribute to overall immune suppression in malignancies and prevent effective immune surveillance against malignant cells. The correlation between the increased number of Tregs and higher-risk MDS has been shown before (Kordasti, Blood 2007). Expansion of effector T cells (Teff) also signifies the lower-risk disease (Kordasti, BJH, 2009). Using deep phenotyping and unbiased automated clustering we have previously identified novel populations of Tregs with distinct functional properties (Kordasti, Blood 2016), including a CD161-expressing subpopulation (Povoleri, Nature Imm, 2018). The aim of this study was to interrogate Treg and Teff subpopulations in more depth and identify transcriptomic profiles of Tregs and Teff in bone marrow (BM) and peripheral blood (PB) of MDS patients, and potential differences between PB and BM residing cells. Single-cell RNA sequencing (scRNAseq) in combination with oligonucleotide-tagged antibodies and an in-house pipeline utilizing Seurat for QC, data integration and analysis were used for this purpose. Methods PB and BM were collected from a low (del(5q)) and a high-risk (monosomy 7) MDS patient. These samples were magnetically enriched for CD4+ T cells, followed by flow sorting of Tregs (CD4+, CD25high, and CD127low) and Teff (CD4+, CD25low, and CD127high). In total, 8 cell fractions were used for downstream scRNAseq and concomitant protein expression profiling using the BD Rhapsody Single-Cell Analysis System (Becton Dickinson, CA, USA). Cell fractions were first labelled by oligo-tagged antibodies (CD95, CD45RO, CD38, CTLA-4, PD-1, HLA-DR) and a sample tag (for sample multiplexing). After labelling, all samples were combined for cartridge-based single-cell capture and molecular indexing of mRNA transcripts on magnetic barcoded beads. Following bead retrieval and on-bead cDNA synthesis, parallel targeted mRNA (259 T cell-related genes) and Abseq sequencing libraries were generated. Deep sequencing was performed using the HiSeq400 platform. Nonlinear dimensionality reduction was carried out using uniform manifold approximation and projection (UMAP), which produced UMAP layouts and heatmaps to identify populations. Tregs were further stratified according to high/low expression of the CD95 protein, CD161, and CD150 mRNA expression. The Wilcoxon Rank Sum test was used for statistics followed by Bonferroni corrections. The Gene Set Variation Analysis (GSVA) package was used for pathway enrichment analysis. Results and discussion Unbiased analysis by UMAP was able to clearly separate the high- and low-risk patients based on the Treg and Teff signature in BM, which was not the case in PB (Figure 1a and 1b). At protein level, BM Tregs from the high-risk patient had a significantly higher expression of CD95 (p = 7.23 x 10-17), CD45RO (p = 1.06 x 10-51), CTLA4 (p = 4.47 x 10-83) PD-1(p = 5.42 x 10-81), and CD38 (p = 4.16 x 10-63) (Figure 1c), suggesting a more functional Treg profile. GSVA analysis of transcriptomes showed significant changes between high- and low-risk patients, for both Tregs (102 pathways p≤0.05) and Teffs (89 pathways p≤0.05) in BM. Specifically, Tregs from high-risk MDS were more enriched with non-TCR-mediated activation pathways, including leukocyte differentiation, Jak-STAT signaling, and positive regulation of leukocyte cell-cell adhesion (Figure 1d). A deeper investigation of Tregs showed that subsets of interest, in particular the IL-17 secreting CD161+ Tregs and the hematopoietic stem cell quiescence-maintaining CD150+ Tregs (Hirata, 2018) were found in low abundance in the BM of both high- and low-risk patients; 16% and 10%; and, 1% and 7%, respectively. In summary, we have developed an analytical pipeline and provide evidence for the ability of simultaneous single-cell transcriptomics and protein expression to identify different BM-specific T cell signatures in high- versus low-risk MDS. Interestingly, Tregs from high risk MDS BM show higher expression of Treg-related proteins and are more enriched in non-TCR-specific activation pathways, which may reflect a myeloid derived inflammatory environment. Overall, these findings suggest a bone marrow-specific Treg signature in MDS, which could distinguish low- and high-risk disease. Disclosures McLornan: Jazz Pharmaceuticals: Honoraria, Speakers Bureau; Novartis: Honoraria. Platzbecker:Celgene: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria. Kordasti:Celgene: Research Funding; Novartis: Research Funding; Boston Biomed: Consultancy; API: Consultancy.

CD95 ligand induces senescence in mismatch repair-deficient human colon cancer via chronic caspase-mediated induction of DNA damage

CD95 is best known for its ability to induce apoptosis via a well-characterized pathway involving caspase-mediated proteolytic events. However, in apoptosis-resistant cell lines of diverse cancer types stimulation of CD95 primarily has pro-tumorigenic effects that affect many of the hallmarks of cancer. For instance, in colon cancer cells with a mutant KRAS gene CD95 primarily promotes invasion and metastasis. In the current study, we further investigated the context dependency of the consequences of CD95 activation in colon cancer. We used a series of patient-derived three-dimensional colon cancer cultures and studied their response to stimulation with CD95 ligand (CD95L). CD95L had a strong inhibitory effect on the clone-forming capacity of five out of nine cultures. In line with previous work, these cultures all had a wild-type KRAS gene and expressed high levels of CD95. Furthermore, the most sensitive cultures were characterized by microsatellite instability (MSI) and deficient mismatch repair. The reduced clonogenic growth of MSI-type colonospheres resulting from chronic CD95 stimulation was only partly due to apoptosis as many tumor cells survived treatment, yet were unable to regenerate clones. CD95 stimulation caused an irreversible cell cycle arrest, which was associated with cytokine secretion, similar to the senescence-associated secretory phenotype (SASP), and expression of senescence-associated β-galactosidase. In human colon cancer cohorts, CD95 expression was strongly correlated with the recently identified consensus molecular subtype 1 (CMS1), which mainly consists of MSI-high tumors, and with two independent SASP signatures. Mechanistically, CD95-induced senescence was caused by chronic DNA damage via caspase-activated DNAse resulting in p53 activation and p21 expression, with a minor contribution of the SASP. We conclude that induction of senescence is a hitherto unrecognized consequence of high CD95 expression, which appears to be most relevant for CMS1.

Increased TIM3+CD8+T cells in Myelodysplastic Syndrome patients displayed less perforin and granzyme B secretion and higher CD95 expression

T cell immunoglobulin and mucin domain 3(TIM3) is a negative regulator of cellular immunity and it is highly expressed on CD8+T cells in persistent viral infection and cancer setting as report. However, how TIM3 expressed on CD8+T cells in myelodysplastic syndrome (MDS), that is a malignant disorder, has not been clarified. Here, decreased CD8+T cells, less IFN-γ secretion in CD8+T cells and increased TIM3 on CD8+T cells had been seen. Increased TIM3+CD8+T cells with lower perforin and granzyme B expression and higher CD95 expression in MDS patients had been observed. These findings suggested that TIM3 might be related to CD8+T cells defect. Therefore, further explorations about mechanism of TIM3+CD8+T cells defect are needed, which might be helpful for adoptive T-cell therapy in MDS.

Deep phenotyping of Tregs identifies an immune signature for idiopathic aplastic anemia and predicts response to treatment

AbstractIdiopathic aplastic anemia (AA) is an immune-mediated and serious form of bone marrow failure. Akin to other autoimmune diseases, we have previously shown that in AA regulatory T cells (Tregs) are reduced in number and function. The aim of this study was to further characterize Treg subpopulations in AA and investigate the potential correlation between specific Treg subsets and response to immunosuppressive therapy (IST) as well as their in vitro expandability for potential clinical use. Using mass cytometry and an unbiased multidimensional analytical approach, we identified 2 specific human Treg subpopulations (Treg A and Treg B) with distinct phenotypes, gene expression, expandability, and function. Treg B predominates in IST responder patients, has a memory/activated phenotype (with higher expression of CD95, CCR4, and CD45RO within FOXP3hi, CD127lo Tregs), expresses the interleukin-2 (IL-2)/STAT5 pathway and cell-cycle commitment genes. Furthermore, in vitro–expanded Tregs become functional and take on the characteristics of Treg B. Collectively, this study identifies human Treg subpopulations that can be used as predictive biomarkers for response to IST in AA and potentially other autoimmune diseases. We also show that Tregs from AA patients are IL-2–sensitive and expandable in vitro, suggesting novel therapeutic approaches such as low-dose IL-2 therapy and/or expanded autologous Tregs and meriting further exploration.

Induction of death receptor CD95 and co-stimulatory molecules CD80 and CD86 by meningococcal capsular polysaccharide-loaded vaccine nanoparticles.

Neisseria meningitidis is a leading cause of bacterial meningitis and sepsis, and its capsular polysaccharides (CPS) are a major virulence factor in meningococcal infections and form the basis for serogroup designation and protective vaccines. We formulated a novel nanovaccine containing meningococcal CPS as an antigen encapsulated in albumin-based nanoparticles (NPs) that does not require chemical conjugation to a protein carrier. These nanoparticles are taken up by antigen-presenting cells and act as antigen depot by slowly releasing the antigen. In this study, we determined the ability of CPS-loaded vaccine nanoparticles to induce co-stimulatory molecules, namely CD80, CD86, and CD95 that impact effective antigen presentation. Co-stimulatory molecule gene induction and surface expression on macrophages and dendritic cells pulsed with meningococcal CPS-loaded nanoparticles were investigated using gene array and flow cytometry methods. Meningococcal CPS-loaded NP significantly induced the surface protein expression of CD80 and CD86, markers of dendritic cell maturation, in human THP-1 macrophages and in murine dendritic cells DC2.4 in a dose-dependent manner. The massive upregulation was also observed at the gene expression. However, high dose of CPS-loaded NP, but not empty NP, induced the expression of death receptor CD95 (Fas) leading to reduced TNF-α release and reduction in cell viability. The data suggest that high expression of CD95 may lead to death of antigen-presenting cells and consequently suboptimal immune responses to vaccine. The CPS-loaded NP induces the expression of co-stimulatory molecules and acts as antigen depot and can spare antigen dose, highly desirable criteria for vaccine formulations.

Human pleural B-cells regulate IFN-γ production by local T-cells and NK cells in aMycobacterium tuberculosis-induced delayed hypersensitivity reaction

DTH (delayed type hypersensitivity) reactions are secondary cellular immune responses that appear 24-72 h after antigen exposure. Tuberculous pleurisy is a common manifestation of extrapulmonary TB (tuberculosis) and is considered a human model of Th1-mediated DTH. In order to identify functional cross-talk among cellular populations sited in this inflammatory microenvironment, we analysed phenotypic and functional features of human B-cells isolated from the PF (pleural fluid) of TB patients. Freshly isolated PF-B-cells displayed a lower expression of CD20, CD1d and HLA-DR, and a higher expression of CD95, CD38, CD25, CXCR3 (CXC chemokine receptor 3) and CXCR4 (CXC chemokine receptor 4) than their PB (peripheral blood) counterparts, suggesting a non-classical in situ activation. Although memory PF-T-cell frequencies were increased, the frequencies of memory PF-B-cells were not. We demonstrated that, upon stimulation with γ-irradiated M. tuberculosis, mycobacterially secreted proteins or a lectin mitogen, PF-B-cells had a strong activation and produced IL-10 by a mechanism that was dependent on bystander activation of CD19(-) PF cells. Besides, within PF cells, B-cells diminished in vitro M. tuberculosis-induced IFN (interferon)-γ production by T-cells and NK (natural killer) cells in an IL-10-dependent manner. Finally, we found that the lower the frequency of B-cells, the higher the ratio of IFN-γ/IL-10 within PF. Thus our results suggest that B-cells can regulate a human DTH reaction induced by M. tuberculosis.

Apoptotic Pathways of Human Regulatory T Cells in Chronic Graft Versus Host Disease (cGVHD) After Allogeneic Hematopoietic Stem Cell Transplantation (HSCT)

Abstract 225CD4+CD25+Foxp3+ regulatory T cells (Treg) are known to play a central role in the maintenance of self-tolerance and immune homeostasis. After allogeneic stem cell transplantation, impaired recovery of Treg is associated with the development of cGVHD. Interleukin-2 (IL-2) is a critical regulator of Treg development, expansion and survival and lack of IL-2 results in Treg deficiency. In patients with cGVHD, we previously demonstrated that Treg proliferate at high levels but this subset is also highly susceptible to apoptosis leading to inadequate Treg numbers (Matsuoka et al. JCI 2010). We also reported that low-dose IL-2 administration resulted in selective expansion of Treg in vivo and clinical improvement of cGVHD (Koreth et al. NEJM 2011). To identify mechanisms responsible for increased Treg susceptibility to apoptosis in cGVHD we used a new flow cytometry-based assay to measure mitochondrial membrane depolarization in response to a panel of pro-apoptotic BH3 peptides (BIM, BID, BAD, NOXA, PUMA, BMF, HRK). This assessment allowed us to compare BH3 peptide-induced mitochondrial membrane depolarization (“priming”) in different T cell subsets, including CD4 Treg, conventional CD4 T cells (CD4 Tcon), and CD8 T cells. Expression of Bcl-2, CD95 and Ki67 were also studied in each T cell subset. We studied peripheral blood samples from 36 patients with hematologic malignancies (median age 59 yr) who are > 2 years post HSCT (27 patients with cGVHD and 9 patients without cGVHD) and 15 patients who received daily subcutaneous IL-2 for 8 weeks for treatment of steroid-refractory cGvHD. Severity of cGVHD was classified according to NIH criteria.In patients without cGVHD, BH3 priming was similar in all 3 T cell subsets (CD4 Treg, CD4 Tcon and CD8). In patients with cGVHD, CD4 Treg were more primed than CD4 Tcon when challenged with BIM, BAD, PUMA, BMF and the combination of BAD + NOXA peptides (p<0.01 – 0.0001). Treg were more primed than CD8 T cells when challenged with PUMA peptide (p<0.0001), but priming in Treg and CD8 T cells was similar for other BH3 peptides in patients with cGVHD. We also compared BH3 priming of each T cell subset in patients with different grades of cGVHD. When challenged with BH3 peptides, Treg, Tcon and CD8 T cells were less primed in patients with severe cGVHD. In patients with cGVHD, Treg expressed higher levels of Ki-67, higher levels of CD95 and lower levels of Bcl-2 than Tcon. Expression of CD95 did not vary with severity of GVHD in any T cell subset, but expression of Bcl-2 was significantly increased in all subsets in patients with severe cGVHD. Increased BH3 priming and high expression of CD95 indicate that Treg are more susceptible to apoptosis than Tcon in cGVHD. However, both Treg and Tcon become less primed and Bcl-2 levels increase in severe cGVHD suggesting that these cells are less susceptible to mitochondrial pathway apoptosis. Since the total number of Treg and Tcon are significantly reduced in patients with cGVHD, these findings suggest that the remaining circulating cells are relatively resistant to mitochondrial pathway apoptosis. CD95 expression in Treg remains high indicating no change in death receptor pathway apoptosis. Daily treatment with low-dose IL-2 for 8 weeks selectively expands Treg in vivo in patients with severe cGVHD. As the number of Treg increase, BH3 profiling shows that these cells gradually become more primed and therefore more susceptible to mitochondrial pathway apoptosis. Taken together, these studies help define the complex and distinct pathways that regulate survival in different T cell subsets and changes in these pathways that occur in patients with chronic GVHD. These pathways play important roles in the maintenance of T cell homeostasis and targeting these complex pathways can provide new opportunities to promote immune tolerance after allogeneic HSCT. Disclosures:No relevant conflicts of interest to declare.

Classical Ataxia Telangiectasia Patients Have a Congenitally Aged Immune System with High Expression of CD95

Ataxia-telangiectasia (A-T) is a rare neurodegenerative immunodeficiency disorder caused by mutations in the ataxia telangiectasia mutated gene. Patients commonly have lymphopenia and Ig-production abnormalities. We used multicolor flow cytometry and IL-7 ELISA to investigate the effect of A-T and age on the proportions of major lymphocyte subsets and their pattern of CD95 expression in relation to IL-7 levels in 15 classical A-T patients. We also analyzed the sensitivity of T cells from four classical A-T patients to CD95-mediated apoptosis using TUNEL and caspase-activation assays. Our results confirmed lymphopenia and a deficiency in naive T and B cells in A-T patients. In contrast to controls, the proportions of naive and memory T and B cell subsets in A-T patients did not vary in relation to age. There was no evidence of a deficiency in plasma IL-7 or IL-7R expression, and IL-7 concentration correlated positively with CD95 expression on CD4(+) T cells. CD95 expression on unstimulated A-T lymphocytes was high, and the apoptotic sensitivity of activated naive and central memory T cells was increased. These findings show that the immunodeficiency in A-T patients may be described as congenitally aged and is not progressive. The naive cell deficiency is not related to a deficiency in IL-7 or its receptor. However, IL-7 may upregulate CD95 on A-T lymphocytes. High CD95 expression and increased apoptotic sensitivity of activated naive and central memory T cells may result in an increased level of CD95-mediated apoptosis, which could contribute to the congenital lymphopenia in A-T.

IL-7 promotes CD95-induced apoptosis in B cells via the IFN-γ/STAT1 pathway.

Interleukin-7 (IL-7) concentrations are increased in the blood of CD4+ T cell depleted individuals, including HIV-1 infected patients. High IL-7 levels might stimulate T cell activation and, as we have shown earlier, IL-7 can prime resting T cell to CD95 induced apoptosis as well. HIV-1 infection leads to B cell abnormalities including increased apoptosis via the CD95 (Fas) death receptor pathway and loss of memory B cells. Peripheral B cells are not sensitive for IL-7, due to the lack of IL-7Ra expression on their surface; however, here we demonstrate that high IL-7 concentration can prime resting B cells to CD95-mediated apoptosis via an indirect mechanism. T cells cultured with IL-7 induced high CD95 expression on resting B cells together with an increased sensitivity to CD95 mediated apoptosis. As the mediator molecule responsible for B cell priming to CD95 mediated apoptosis we identified the cytokine IFN-γ that T cells secreted in high amounts in response to IL-7. These results suggest that the lymphopenia induced cytokine IL-7 can contribute to the increased B cell apoptosis observed in HIV-1 infected individuals.

Expression and prognostic relevance of the death receptor CD95 (Fas/APO1) in renal cell carcinomas

CD95 (Fas/APO1) is one of the best known members of the death receptor family which can either mediate apoptosis or activate tumor-promoting pathways. Using a tissue microarray we investigated the association between the expression of CD95 and prognosis in 617 patients with renal cell carcinomas (RCCs). CD95 was expressed in the vast majority of RCCs. High CD95 expression was associated with lymph node metastasis and correlated negatively with disease-specific survival. Multivariate Cox regression analysis confirmed CD95 expression as an independent prognostic factor. In conclusion, high CD95 expression is a negative independent prognostic factor in RCCs which could be used to identify high-risk patients with a poor clinical prognosis.

High CD95 expression of BAL lymphocytes predicts chronic course in patients with sarcoidosis

The prognosis of sarcoidosis is highly variable, with spontaneous remission in some patients. Apoptosis may be associated with spontaneous resolution of the granulomata. CD95 (Fas), an apoptotic molecule, and CD29 and CD45RO (T-cell memory markers) are expressed at higher levels on T lymphocytes from sarcoid patients compared with normal subjects. However, the prognostic significance of CD95, CD29 and CD45RO expression in sarcoidosis is not clear. It was hypothesized that expression of CD95 would correlate with spontaneous remission. CD29, CD45, CD45RO and CD95 expression of BAL fluid and peripheral blood (PB) lymphocytes was studied with flow cytometry in 50 patients with sarcoidosis. Results of the 15 chronic patients and 21 patients who remitted spontaneously were compared. BAL CD95 (59 vs 8, P = 0.002), and PB CD95 (48 vs 18, P = 0.004) and PB CD45RO (50 vs 41, P = 0.003) expression was significantly higher in patients with chronic disease compared with those with spontaneous remission. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy for these markers were: BAL CD95 (cut-off: 42.5%) 73.3%, 85.7%, 78.6%, 81.8% and 80.6%; PB CD95 (cut-off: 25%) 86.7%, 66.7%, 65%, 87.5% and 75%; and PB CD45RO (cut-off: 44.5%) 80%, 61.9%, 60%, 81.3% and 69.4%, respectively. Levels of BAL and PB CD95 and PB CD45RO were unexpectedly elevated in patients with chronic disease and may be useful in predicting prognosis in patients with sarcoidosis. Further studies with more patients are necessary to confirm the prognostic role and cut-off value for these markers.

Up-regulation of c-FLIPS+R upon CD40 stimulation is associated with inhibition of CD95-induced apoptosis in primary precursor B-ALL

Previous studies on apoptosis defects in acute lymphoblastic leukemia (ALL) have focused on chemotherapy-induced, primarily mitochondrial death pathways. Yet, immunologic surveillance mechanisms including sensitization to apoptotic signals mediated via the death receptor CD95 might contribute to leukemic control. Here, we show that primary B-cell precursor ALL cells from children escape from receptor-dependent cell death in 2 ways: Resting ALL blasts are protected from receptor-mediated apoptosis due to the absence of CD95 surface expression. However, even though CD40 ligation results in up-regulation of CD95, ALL blasts, unlike normal B cells, remain resistant to apoptosis. We show that this apoptosis resistance involves the selective up-regulation of the short isoforms of the caspase-8 inhibitor c-FLIP acting directly at the CD95 receptor level. Treatment with cycloheximide during CD40 activation prevents up-regulation of those c-FLIP isoforms and sensitizes ALL cells toward CD95-mediated apoptosis. We therefore propose that induction of the short c-FLIP isoforms inhibits the onset of CD95-induced apoptosis in primary CD40-stimulated ALL cells despite high CD95 expression.

An IL-2-dependent switch between CD95 signaling pathways sensitizes primary human T cells toward CD95-mediated activation-induced cell death.

The CD95 (APO-1/Fas) system plays a critical role in activation-induced cell death (AICD) of T cells. We previously described two distinct CD95 (APO-1/Fas) signaling pathways: 1) type I cells show strong death-inducing signaling complex (DISC) formation and mitochondria-independent apoptosis and 2) DISC formation is reduced in type II cells, leading to mitochondria-dependent apoptosis. To investigate the relevance of these pathways, we set up an in vitro model that mimics the initiation and the down phase of an immune response, respectively. Freshly activated human T cells (initiation) are resistant toward CD95-mediated AICD despite high expression of CD95. We previously reported that these T cells show reduced DISC formation. In this study, we show that freshly activated T cells are CD95-type II cells that show high expression levels of Bcl-x(L) and display a block in the mitochondrial apoptosis pathway. Furthermore, we show that, upon prolonged culture (down phase), human T cells undergo a switch from type II to type I cells that renders T cells sensitive to CD95-mediated AICD. Finally, we demonstrate that this switch is dependent on the presence of IL-2. Our observations reveal for the first time that the existence of coexisting CD95 signaling pathways is of physiological relevance.

Cell surface expression of CD25, CD54, and CD95 on B- and T-cells in chronic lymphocytic leukaemia in relation to trisomy 12, atypical morphology and clinical course.

Surface antigen expression can be used to define subgroups of patients with different clinical courses in chronic lymphocytic leukaemia of the B-cell type (CLL). PURPOSE-METHODS: To study the clinical significance of functional markers linked to proliferation (CD25), adhesion (CD54), and apoptosis (CD95) on B- and T-cells in 68 patients with CLL using dual colour flow cytometry (FCM). The mean proportion of CD19+ B-cells expressing CD25 was significantly higher in CLL patients compared to controls (P=0.02), while CD54+ and CD95+ B-cells did not differ significantly. In CLL with atypical morphology and in patients with trisomy 12, the mean percentage of CD25+ B-cells was lower than in typical CLL (P<0.02) and in patients with disomic tumor cells (P<0.03). Patients with 30% of CD25+ B-cells had a shorter median time to treatment than CD25-negative cases (P=0.01). A low CD54 expression was associated with a prolonged median time to treatment (P=0.004), low WBC counts (P<0.05), and low S-LDH (P=0.03). A high CD95 expression was correlated with elevated S-LDH (P=0.02) and a finding of lymphadenopathy (P=0.02). In individual patients there was a strong correlation between B- and T-cell expression of CD25 (P<0.0001), CD54 (P=0.0002), and CD95 (P=0.0002), respectively. CD25 and CD54 expression on CD19+ cells seems to give prognostic information. The strong correlation between the expression of CD25, CD54 and CD95 on B-and T-cells suggests that the expression of these antigens is not an inherent characteristic of the malignant B-cell clone.

HELICOBACTER PYLORI ORGANISMS INDUCE EXPRESSION OF ACTIVATION AND APOPTOTIC SURFACE MARKERS ON HUMAN LYMPHOCYTES AND AGS CELLS:A CYTOFLUORIMETRIC EVALUATION

ABSTRACTHuman peripheral blood mononuclear cells (PBMCs) were treated with Helicobacter pylori (Hp) organisms alone or with Hp-stimulated AGS cells (a gastric adenocarcinoma cell line). Hp organisms were able per se to increase the percentage of CD8+CD95+ cells, while number of CD25+ cells and HLA-DR molecule expression increased following pretreatment with Hp-stimulated AGS cells.A comparison was made with a test system in which PBMCs were stimulated with Escherichia coli (Ec) organisms and colo-cells (a colon carcinoma cell line). In this case, CD95+ cells and CD25+ cells increased when the combination Ec organisms/colo-cells was present in the culture. On the other hand, Hp bacteria in combination with colo-cells were not able to induce activation and/or apoptotic surface markers on PBMCs, while Ec-stimulated AGS cells increased the expression of CD95 on PBMC.Finally, the direct interaction of AGS cells with Hp was able to induce higher expression of CD95 on gastric epithelial cells than Hp-stimulated PBMCs. Taken together, these data support the interplay between bacteria and epithelial cells in the course of Hp-mediated gastropathy.

Induction of CD95 ligand expression on T lymphocytes and B lymphocytes and its contribution to apoptosis of CD95-up-regulated CD4+ T lymphocytes in macaques by infection with a pathogenic simian/human immunodeficiency virus.

Using an established SIV/HIV-C2/1-infected cynomolgus monkey model displaying stable CD4+ T cell depletion, the kinetics of apoptosis and the levels of expression of CD95 membrane-associated CD95L on lymphocytes were investigated to test the involvement of the CD95/CD95L system in CD4+ T lymphocyte loss in vivo. Rapid depletion of CD4+ T cells occurred up to 2 weeks after infection, with chronic CD4+ T lymphopenia thereafter. During the initial CD4+ T cell loss, which was accompanied by viraemia, about 90% of the peripheral CD4+ T cell subset underwent spontaneous apoptotic cell death during 24 h of culture. Increased expression of CD95 was observed on both CD4+ and CD8+ T cell subsets, with CD95 expression on CD8+ cells declining rapidly, but high CD95 expression being maintained on CD4+ cells. Since CD95L was expressed on CD8+ T cells, B cells and to a lesser extent on CD4+ T cells, this suggests that CD95-mediated apoptosis might be controlled in an autocrine/paracrine fashion.

Natural killer type 2 bias in remission of multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune disease characterized by clinical relapse and remission. Because of the potential role of natural killer (NK) cells in the regulation of autoimmunity, we have examined cytokine profile and surface phenotype of NK cells in the peripheral blood of MS. Here we demonstrate that NK cells in the remission of MS are characterized by a remarkable elevation of IL-5 mRNA and a decreased expression of IL-12Rbeta2 mRNA, as well as a higher expression of CD95. Moreover, the NK cells from MS in remission produced much larger amounts of IL-5 than did those from controls after stimulation with phorbol myristate acetate (PMA) and ionomycin. These features are reminiscent of those of NK type 2 (NK2) cells that can be induced in a condition favoring functional deviation of T cells toward Th2. Remarkably, the NK cells lose the NK2-like property when relapse of MS occurs, but regain it after recovery. We also found that NK2 cells induced in vitro inhibit induction of Th1 cells, suggesting that the NK2-like cells in vivo may also prohibit autoimmune effector T cells. Taken together, it is possible that NK cells play an active role in maintaining the remission of MS.

T Cells of Dialysis And Praedialysis Patients Are Undergoing CD9S(Fas) Mediated Apoptosis

Purpose of Study: T cells of Left Ventricular Assist Device (LVAD) recipients are aberrantly activated via the CD95 pathway with high levels of apoptosis in vivo and low lymphocyte counts. Since LVAD recipients and Dialysis patients are reported to have high incidence of infections we sought to investigate whether similar activation pathways are induced. Methods: Thus we explored 20 Predialysis (PD) and 20 Dialysis (D) patients. 10 Healthy Humans (HH) served as controls. By FACS analysis we stained T cells for CD95, HLA DR expression, and utilized HIV batch analysis to evaluate absolute counts of subpopulations. For detecting apoptosis we utilized annexin V mABs, an AB binding to phosphatidylserine, a phospholipid present on cell membranes undergoing apoptosis. Results: D vs PD patients demonstrated in a cross sectional analysis signifcantly lower CD4 T cells (mean 420/mm3 vs 668/mm3, p < 0.05). In contrast CD8 T cells showed no difference (334/mm3 vs 363/mm3, p < NS). This reduction of lymphocyte levels was accompanied with high expression of CD95(Fas) of CD3 cells on both groves studied (mean 72.2% vs 80% - HH 33.5%). HLA-DR expression as sign of T cells activation was not increased in both groups studied (mean 10% vs 10%). As CD95 triggering is initiating programmed cell death, we compared the levels of annexin V D and PD patients demonstrated a 7 fold increase in annexin binding in comparison to HH (72% vs 74% vs 10%). Togehter, these results suggest that dialysis and predialysis patients are susceptible to undergo AICD (Activation Induced Cell Death) and are likely to be responsible for decreased T lymphocyte counts in the patients evaluated. This increase of this apoptotic pathway might suggest another mechanism of immunosuppresion besides uremic sideproducts and might suggest an additional explanation for the increased incidence of systemic infections in the patient populations studied.