Immunophenotyping of patients with oral squamous cell carcinoma in peripheral blood and associated tumor tissue.

The immune system is important for elimination of cancer cells. Tumors including oral squamous cell carcinoma (OSCC) are capable of escaping detection by host immune cells through apoptotic depletion of tumor-infiltrating lymphocytes (TILs). Circulating peripheral blood lymphocytes (PBLs) and corresponding TILs of tumor specimen were evaluated before and after curative tumor resection (n = 30) compared with PBLs of controls (n = 87). PBLs were characterized for the total number of T cells (CD3(+)), T helper cells (Th, CD3(+)/CD4(+)), regulatory T cells (Treg, CD4(+)/CD25(+)/CD127(low)), cytotoxic T cells (Tc, CD3(+)/CD8(+)), activated T cells (CD3(+)/HLA-DR(+)), and natural killer (NK) cells (CD3(-)/CD16(+)/CD56(+)). In tumor tissue, the prevalence of CD3(+), CD4(+), and CD8(+) TILs was assessed using immunohistochemistry, whereas the incidence of apoptosis was assessed using terminal deoxynucleotidyl transferase deoxyuridinetriphosphate nick-end labeling (TUNEL) assay. In PBLs of pretreated OSCC patients, a highly significant decrease in total number of T cells (p = 0.0001), Th cells (p < 0.0001), Treg cells (p < 0.0001), Tc cells (p < 0.0001), and NK cells (p = 0.0037) were found compared with controls. Decreased PBLs of OSCC patients were correlated with decreased numbers of corresponding TILs, which were associated with increased detection of apoptosis in the tumor tissue. Compared with the controls, the total number of T cells remained unchanged after surgery but the total number of NK cells significantly increased. Standardized immunophenotyping of OSCC may help to identify patients likely to benefit from cancer immunotherapy strategies and/or chemoradiation. Finally, future attempts to enhance an effective tumor-reactive immune response by immunotherapy or vaccination should be made by promoting tumor-specific Th and/or Tc cell/NK cell responses.

A biomarker based detection and characterization of carcinomas exploiting two fundamental biophysical mechanisms in mammalian cells.

BACKGROUND: Biomarkers allowing the characterization of malignancy and therapy response of oral squamous cell carcinomas (OSCC) or other types of carcinomas are still outstanding. The biochemical suicide molecule endonuclease DNaseX (DNaseI-like 1) has been used to identify the Apo10 protein epitope that marks tumor cells with abnormal apoptosis and proliferation. The transketolase-like protein 1 (TKTL1) represents the enzymatic basis for an anaerobic glucose metabolism even in the presence of oxygen (aerobic glycolysis/Warburg effect), which is concomitant with a more malignant phenotype due to invasive growth/metastasis and resistance to radical and apoptosis inducing therapies. METHODS: Expression of Apo10 and TKTL1 was analysed retrospectively in OSCC specimen (n = 161) by immunohistochemistry. Both markers represent independent markers for poor survival. Furthermore Apo10 and TKTL1 have been used prospectively for epitope detection in monocytes (EDIM)-blood test in patients with OSCC (n = 50), breast cancer (n = 48), prostate cancer (n = 115), and blood donors/controls (n = 74). RESULTS: Positive Apo10 and TKTL1 expression were associated with recurrence of the tumor. Multivariate analysis demonstrated Apo10 and TKTL1 expression as an independent prognostic factor for reduced tumor-specific survival. Apo10+/TKTL1+ subgroup showed the worst disease-free survival rate in OSCC.EDIM-Apo10 and EDIM-TKTL1 blood tests allowed a sensitive and specific detection of patients with OSCC, breast cancer and prostate cancer before surgery and in after care. A combined score of Apo10+/TKTL1+ led to a sensitivity of 95.8% and a specificity of 97.3% for the detection of carcinomas independent of the tumor entity. CONCLUSIONS: The combined detection of two independent fundamental biophysical processes by the two biomarkers Apo10 and TKTL1 allows a sensitive and specific detection of neoplasia in a noninvasive and cost-effective way. Further prospective trials are warranted to validate this new concept for the diagnosis of neoplasia and tumor recurrence.

EDIM-TKTL1 blood test: a noninvasive method to detect upregulated glucose metabolism in patients with malignancies.

AIM: To determine whether the TKTL1 protein epitope detection in monocytes (EDIM) test allows detection of upregulated glucose metabolism in malignancies. MATERIALS & METHODS: The EDIM-TKTL1 blood test was conducted in 240 patients with 17 different confirmed or suspected malignancies. Test scores were compared with (18)F-fluoro-2-deoxy-D-glucose (FDG)-PET/computed tomography (CT) results. RESULTS: EDIM-TKTL1 score and FDG-PET results showed a concordance of 90% with a sensitivity of 94% and specificity of 81%. Including CT data, all values were enhanced. A subgroup analysis of non-small-cell lung cancer patients showed a significant correlation between the EDIM-TKTL1 score and the primary tumor size determined by FDG-PET/CT. CONCLUSION: EDIM-TKTL1 blood test revealed good concordance with FDG-PET/CT results in patients with malignancies demonstrating its efficacy to detect upregulation of glucose metabolism in primary tumors or metastases.

An immunodeficiency disease with RAG mutations and granulomas.

We describe three unrelated girls who had an immunodeficiency disease with granulomas in the skin, mucous membranes, and internal organs. All three girls had severe complications after viral infections, including B-cell lymphoma associated with Epstein-Barr virus (EBV). Other findings were hypogammaglobulinemia, a diminished number of T and B cells, and sparse thymic tissue on ultrasonography. Molecular analysis revealed that the patients were compound heterozygotes for mutations in recombination activating gene 1 or 2 (RAG1 or RAG2). In each case, both parents were heterozygous carriers of a RAG mutation. The mutations were associated with reduced function of RAG in vitro (3 to 30% of normal activity). The parents and one sibling in the three families were healthy.

Addition of Multimodal Immunotherapy to Combination Treatment Strategies for Children with DIPG: A Single Institution Experience.

Background: The prognosis of children with diffuse intrinsic pontine glioma (DIPG) remains dismal despite radio- and chemotherapy or molecular-targeted therapy. Immunotherapy is a powerful and promising approach for improving the overall survival (OS) of children with DIPG. Methods: A retrospective analysis for feasibility, immune responsiveness, and OS was performed on 41 children treated in compassionate use with multimodal therapy consisting of Newcastle disease virus, hyperthermia, and autologous dendritic cell vaccines as part of an individualized combinatorial treatment approach for DIPG patients. Results: Patients were treated at diagnosis (n = 28) or at the time of progression (n = 13). In the case of 16 patients, histone H3K27M mutation was confirmed by analysis of biopsy (n = 9) or liquid biopsy (n = 9) specimens. PDL1 mRNA expression was detected in circulating tumor cells of ten patients at diagnosis. Multimodal immunotherapy was feasible as scheduled, until progression, in all patients without major toxicity. When immunotherapy was part of primary treatment, median PFS and OS were 8.4 m and 14.4 m from the time of diagnosis, respectively, with a 2-year OS of 10.7%. When immunotherapy was given at the time of progression, median PFS and OS were 6.5 m and 9.1 m, respectively. A longer OS was associated with a Th1 shift and rise in PanTum Detect test scores. Conclusions: Multimodal immunotherapy is feasible without major toxicity, and warrants further investigation as part of a combinatorial treatment approach for children diagnosed with DIPG.

Memory B-cells in healthy and antibody-deficient children.

Recently it has become clear that a reduction of IgD-CD27+ memory B-cells in adult CVID patients correlates with clinical aspects of the disease. However, little is known about B-cell dysregulation in pediatric antibody deficiency. Reference values are essential for the interpretation of B-cell subpopulations in children. We present the clinical and immunophenotypical characterization of 16 children and adolescents with CVID and hypogammaglobulinemia. Reference values for IgD+CD27-, IgD+CD27+ and IgD-CD27+ B-cells in healthy children were established for five age groups. In healthy controls we found a continuous increase in IgD-CD27+ B-cell percentage with age from 1.35-5% of B-cells in the second year of life to 4.1-18.7% in adolescents. Interestingly, in 12/14 antibody-deficient patients memory B-cells are significantly below the age-related 10th percentile. We conclude that the reduction of memory B-cells is a useful additional marker for the detection of children with CVID hypogammaglobulinemia and may contribute to the early presentation.

Saving the red baby: successful allogeneic cord blood transplantation in Omenn syndrome.

Haematopoietic stem cell transplantation is the treatment of choice for severe primary immunodeficiencies, but only has moderate prognosis in Omenn syndrome as it is complicated by highly activated Omenn T-cells resulting in delayed T-cell engraftment and a high rate of graft failure. A 6 1/2 months old patient with a previously unknown compound heterozygous defect within the RAG1 gene (R474C; R975W) underwent 8/10 HLA-matched cord blood transplantation after myeloablative conditioning. Immune reconstitution was impressive with T-, B- and NK-cells reaching the median of age-dependent reference values within twelve, four and two months respectively. With a continuous decrease of activated Omenn T-cells there was a steady increase of naive, probably thymus-derived T-cells. Polyclonal B-cell activation and hypergammaglobulinaemia disappeared with B-cell engraftment. This case emphasizes that, despite their naive status and HLA-barriers, cord blood T-cells were apparently able to achieve T-effector function resulting in the elimination of all activated Omenn T-cells.

Memory B cell function in HIV-infected children-decreased memory B cells despite ART.

B cell dysfunction is a well-studied complication of HIV infection in adults. Data on B cell differentiation in normal and HIV-infected children are lacking. We show the distribution of B cell subsets and immunoglobulin levels in HIV-infected children compared with controls. Furthermore, we observe the long-term B cell reconstitution of vaccine-specific immunity after antiretroviral therapy (ART). Phenotype of B cells (naive, non-switched memory, switched memory) was analyzed in 48 infected children and 62 controls. In nine HIV-infected children, functional reconstitution was quantified by tetanus-specific antibodies and by performing a lymphocyte transformation test (LTT) in a longitudinal approach. Switched memory B cells are significantly reduced in HIV-infected children. Vaccine-specific antibodies and response to LTT increase after initiation of ART. Our data indicate a significant dysfunction in the B cell system, despite effective ART. Partial reconstitution of humoral immunity may have therapeutic implications in a subset of HIV-infected children.

Molecular genetics of maple syrup urine disease in the Turkish population.

In maple syrup urine disease (MSUD), disease-causing mutations can affect the BCKDHA, BCKDHB or DBT genes encoding for the E1alpha, E1beta and E2 subunits of the multienzyme branched-chain alpha-keto acid dehydrogenase (BCKDH) complex. Here we summarize the MSUD genotypes of a cohort of 32 unrelated Turkish patients in whom both alleles at a single gene locus harbored presumable disease-causing nucleotide changes. The patients had different forms of MSUD, ranging from the severe classical form (26 patients) to severe and mild variants (6 patients). In all except two patients (92%), the mutations occurred homozygously. The mutational spectrum included 27 different sequence variations--12 changes in the BCKDHA, 10 in the BCKDHB, and 5 in the DBT genes. In 37% (12 patients) of a total of 64 alleles, the supposed disease-causing mutations were located in the BCKDHA gene, in 44% (14 patients) in the BCKDHB gene and in 19% (6 patients) in the DBT gene. The mutational profile is heterogeneous, although two mutations occurred three times and five mutations occurred twice. There was no cluster for a single mutation except for c.773G>A (p.Cys258Tyr) in the BCKDHA gene, a hypothetical founder mutation in the Camlidere population.

Off-target activity of TNF-alpha inhibitors characterized by protein biochips.

Tumor necrosis factor-alpha inhibitors are widely and successfully used to treat rheumatic diseases. However, significant side effects have been reported. To detect the potential off-target activities of such inhibitors we characterized two therapeutic antibodies (adalimumab, infliximab) and one receptor fusion protein (etanercept) on protein biochips (UNIchip AV-400) containing a printed serial dilution of tumor necrosis factor-alpha and about 384 different human proteins. Etanercept binds to ten proteins (affinity: 20-33% of tumor necrosis factor-alpha recognition), and six of these proteins are related to ribosomal proteins. Interestingly, adalimumab binds to the same six proteins related to ribosomal proteins (affinity: 12-18%) as well as to four proteins crucially involved in ribosomal protein synthesis. Alignment of protein sequences indicates no significant sequence homology between these ten proteins bound by the biological drugs with the highest off-target activities. Taken together, our in vitro results demonstrate that a significant number of proteins are recognized by tumor necrosis factor-alpha inhibitors and are related to ribosome biogenesis.

Monitoring carcinogenesis in a case of oral squamous cell carcinoma using a panel of new metabolic blood biomarkers as liquid biopsies.

INTRODUCTION: One of the common malignant tumors of the head and neck worldwide with generally unfavorable prognosis is squamous cell carcinoma (OSCC) of the oral cavity. Early detection of primary, secondary, or recurrent OSCC by liquid biopsy tools is much needed. CASE PRESENTATION: Twelve blood biomarkers were used for monitoring a case of OSCC suffering from precancerous oral lichen ruber planus mucosae (OLP). After curative R0 tumor resection of primary OSCC (buccal mucosa), elevated epitope detection in monocytes (EDIM)-Apo10, EDIM-transketolase-like-1 (TKTL1), squamous cell carcinoma antigen (SCC-Ag), total serum lactate dehydrogenase (LDH), and its anaerobic isoforms (LDH-4, LDH-5) decreased to normal levels. Three and six months after surgery, transformation of suspicious mucosal lesions has been accompanied with an increase of EDIM scores, total serum LDH values, and a metabolic shift from aerobic (decrease of LDH-1, LDH-2) to anaerobic (increase of LDH-4, LDH-5) conditions. Two months later, secondary OSCC was histopathologically analyzed after tissue biopsy. Cytokeratin fraction 21-1 (CYFRA 21-1), carcinoembryonic antigen (CEA), and carbohydrate antigen 19-9 (CA19-9) were not affected during the clinical course of carcinogenesis. CONCLUSIONS: A combination strategy using a standardized panel of established (metabolic) blood biomarkers (TKTL1, LDH, LDH isoenzymes) is worth and can be recommended among others (apoptosis resistance-related Apo10, SCC-Ag) for early detection and diagnosis of primary, secondary, and recurrent OSCC. A tandem strategy utilizing (metabolic pronounced) routine liquid biopsies with imaging techniques may enhance diagnosis of OSCC in the future. Although we demonstrated the diagnostic utility of separated liquid biopsies in our previous study cohorts, further investigations in a larger patient cohort are necessary to recommend this combination strategy (EDIM blood test, LDH value, metabolic shift of LDH isoenzymes, and others, e.g., SCC-Ag or immunophenotyping) as a diagnostic tool for the addition to the OSCC staging system and as a routine procedure in the aftercare.

Analysis of circulating CD14+/CD16+ monocyte-derived macrophages (MDMs) in the peripheral blood of patients with oral squamous cell carcinoma.

OBJECTIVES: Monocytes/macrophages are regarded as the first line of defense in tumors. Therefore, analyzing monocyte subtypes in oral squamous cell carcinoma (OSCC) may be of value in disease monitoring and to explore immunotherapeutic strategies for cancer patients. STUDY DESIGN: Circulating peripheral blood CD14+/CD16+ monocyte-derived macrophages (MDMs) were evaluated in OSCC patients with oral squamous cell carcinoma (n = 44) compared with controls (n = 85). Moreover, epitope detection in monocytes (EDIM) technology was used to detect biomarkers Apo10 and transketolase-like-1 in CD14+/CD16+ MDMs. RESULTS: Compared with controls, no significant (P = .3646) difference (control group 9.8%, OSCC group 8.8%) in CD14+/CD16+ MDM were noted in OSCC. However, EDIM-Apo10 and EDIM-TKTL1 scores detected in the CD14+/CD16+ MDMs were increased in OSCC compared with controls (P < .0001). CONCLUSIONS: Analyzing CD14+/CD16+ MDMs represents a stable cell population for detecting biomarkers in cancer disease monitoring.

A novel mutation in the interleukin-2 receptor gamma gene as the cause of lymphopenia in a neonate vertically exposed to human immunodeficiency virus.

Perinatal transmission prophylaxis has led to a significant reduction of vertical human immunodeficiency virus (HIV) infection. Antiretroviral drugs are being widely used before and during pregnancy, although these drugs can have possible adverse effects on the fetus and newborn. In this context, we report a unique case of X-linked severe combined immunodeficiency in a neonate vertically exposed to HIV.

Effect of age on homeostasis of lymphocytes in an interleukin-7-deficient mouse model.

Interleukin (IL)-7 is thought to be a non-redundant cytokine for lymphopoiesis as there is a reduction of T and B cells in peripheral blood (PB) and a loss of TCRγδ+ cells in PB and bone marrow (BM) in IL-7⁻/⁻ mice. To investigate whether the absence of IL-7 influences the organ-dependent distribution of the lymphocytes, we analyzed single cell suspensions of several organs (BM, lung, liver, small intestine, and spleen) at different ages (three and 12 months) of IL-7+/+ and IL-7⁻/⁻ mice using flow cytometry; immunohistochemical staining was performed on frozen sections of various organs. We observed lymphocytopenia in almost all organs of IL-7⁻/⁻ mice, but normal counts in the liver and the lung of three-month-old IL-7⁻/⁻ mice. CD4+ and CD8+ cell numbers were decreased in the spleen and the BM. With aging, we found a greater increase in CD4+ and CD8+ cells in the BM of IL-7⁻/⁻ than in IL-7+/+ mice, particularly of memory cells. The spleen of IL-7⁻/⁻ mice was characterized by lymphocytopenia. We challenge the view that IL-7 is a non-redundant cytokine for lymphocyte development. Some of the changes observed, e.g. partial absence of TCRγδ+ T cells in the PB, BM and small intestine and complete loss in liver, lung and spleen, may be due to the altered organ distribution instead of a defect in γδ+ T cell lymphopoiesis. In this model, aging leads to a significantly altered composition of lymphocyte subsets, and the lack of IL-7 seems to accelerate this process.

Fas-induced apoptosis of renal cell carcinoma is mediated by apoptosis signal-regulating kinase 1 via mitochondrial damage-dependent caspase-8 activation.

Renal cell carcinoma (RCC) is a prototype of a chemo refractory tumour. It remains the most lethal of the common urologic cancers and is highly resistant to conventional therapy. Here, we confirmed the efficiency of anti-Fas monoclonal antibody (CH11) as alternative therapeutic approach for the treatment of RCC and investigated the molecular mechanism(s), whereby CH11 induces apoptosis of RCC cells. The present study shows an essential role for apoptosis signal-regulating kinase 1 (ASK1), together with both c-jun-N-terminal kinase (JNK) and p38 pathways, and caspase-8 in this process. Furthermore, CH11-dependent induction of the ASK1-JNK/p38 pathways was found to activate the transcription factors AP-1 and ATF-2, and FADD-caspase-8-Bid signalling, resulting in the translocation of both Bax and Bak proteins, and subsequently mitochondrial dysregulation that is characterized by the loss of mitochondrial membrane potential (DeltaPsim), cytochrome c release and cleavage of caspase-9, caspase-3 and PARP. Thus, the described molecular mechanisms of CH11-induced apoptosis suggest the reliability of Fas activation as an alternative therapeutic approach for the treatment of patients with advanced renal cell carcinoma.

Girls homozygous for an IL-2-inducible T cell kinase mutation that leads to protein deficiency develop fatal EBV-associated lymphoproliferation.

The fatal immune dysregulation that sometimes follows EBV infection in boys has been linked to mutations in two X chromosome-encoded genes, SLAM-associated protein (SAP) and X-linked inhibitor of apoptosis (XIAP). In this study we describe 2 girls from a consanguineous Turkish family who died after developing severe immune dysregulation and therapy-resistant EBV-positive B cell proliferation following EBV infection. SNP array-based genome-wide linkage analysis revealed IL-2-inducible T cell kinase (ITK) as a candidate gene for this immunodeficiency syndrome. Both girls harbored a homozygous missense mutation that led to substitution of a highly conserved residue (R335W) in the SH2 domain of ITK. Characteristics of ITK deficiency in mouse models, such as absence of NKT cells and high levels of eomesodermin in CD8+ cells, were seen in either one or both of the girls. Two lines of evidence suggested that R335W caused instability of the ITK protein. First, in silico modeling of the mutant protein predicted destabilization of the SH2 domain. Additionally, Western blot analysis revealed that, unlike wild-type ITK, the R335W mutant was nearly undetectable when expressed in 293 T cells. Our results suggest that ITK deficiency causes what we believe to be a novel immunodeficiency syndrome that leads to a fatal inadequate immune response to EBV. Because ITK deficiency resembles EBV-associated lymphoproliferative disorders in boys, we suggest that this molecular cause should be considered during diagnosis and treatment.

A single intravenous dose of prednisolone induces phosphatidylserine externalization, loss of surface marker expression and a 24-h net increase in human peripheral blood lymphocytes ex vivo.

To understand how corticosteroids act; a characterization of their effects on lymphocytes is necessary. The effect of in vivo corticosteroids on lymphocyte subpopulations, their surface molecules and externalization of phosphatidylserine (apoptosis) is examined. In a crossover study, a single, intravenous dose of 2 mg/kg prednisolone or saline was given to six male adult human volunteers. Blood samples were withdrawn before and 30 min, 2, 5, 23 and 29 h thereafter. Lymphocyte subsets were determined by FACS analysis. Externalization of phosphatidylserine was measured by Annexin-V; cell fragments were excluded by propidium iodide staining. Lymphocyte number decreased from 2,007 +/- 473 to 634 +/- 119 microl after 5 h and rose to 3,112 +/- 436 microl after 23 h. CD4, CD8 and B cell counts declined significantly after 5 h (P < or = 0.01). The expression of CD28 or CD95 on T cells and the natural killer cells were unaffected. There was a significant rebound of lymphocyte numbers above baseline 23 h after prednisolone. At baseline 9.9 +/- 3.8% of cells in the lymphocyte gate did not stain for CD3, CD20 or CD56 (referred to as "null cells"). 5 h after application of prednisolone, there was a significant increase of "null cells" (28 +/- 12%, P = 0.018). The percentage of phosphatidylserine positive CD4 cells rose from 8.1 +/- 3.3 to 19.8 +/- 8% after intravenous prednisolone, while the percentage of phosphatidylserine positive CD8, B and NK cells remained largely unchanged. Prednisolone induces a most significant depletion of CD4 cells, which to some degree is associated with apoptosis. The net increase of lymphocyte numbers 23 h after prednisolone application may be a beneficial late effect of a single i.v. prednisolone shot.

Primary necrotizing lymphocytic central nervous system vasculitis due to perforin deficiency in a four-year-old girl.

We report the case of a 4-year-old girl who presented with headaches, ataxia, and visual disturbances. Cranial magnetic resonance imaging showed multiple supra- and infratentorial lesions with peripheral contrast enhancement and central necrosis. Brain biopsy revealed necrotizing lymphocytic vasculitis of undetermined etiology. Perforin expression was found to be significantly reduced in the patient's peripheral blood cells, and sequence analysis of the patient's perforin gene showed a compound heterozygous state with 1 nonsense mutation and 2 missense alterations in exon 2. Central nervous system (CNS) vasculitis was thus attributed to the perforin deficiency, and the patient was successfully treated by transplantation of stem cells from an HLA-identical brother. The findings described herein indicate that, even in the absence of classic non-neurologic symptoms of hemophagocytic lymphohistiocytosis, measurement of perforin expression should be one of the diagnostic tests used to identify the cause of unexplained CNS vasculitis, since this may have profound implications regarding therapy.

Ex vivo apoptosis, CD95 and CD28 expression in T cells of children with juvenile idiopathic arthritis.

We hypothesise that T-cell apoptosis and the percentage of T cells expressing molecules involved in apoptosis modulation (CD95, CD28) are altered at the inflammation site and in peripheral blood (PB) of children with juvenile idiopathic arthritis (JIA). Paired JIA samples of synovial fluid (SF) and PB ( n=7) and PB samples from age-matched normal children ( n=23) were analysed immediately ex vivo. Apoptosis was measured by detection of phophatidylserine (PS) externalisation on T cells. CD95 or CD28 was detected by FACS, and soluble CD95 and CD95 ligand levels were detected by enzyme-linked immunosorbent assay (ELISA). In SF, the mean percentage of apoptotic T cells was somewhat higher than in PB. However, the percentages of T cells expressing CD95 and soluble CD95 levels were markedly higher in SF (CD4 cells 96+/-2%, CD8 91+/-6%, soluble CD95 6,420+/-2,571 pg/ml) than in PB (CD4 32+/-10%, CD8 36+/-9%, soluble CD95 4,296+/-2,142 pg/ml). Peripheral blood T-cell apoptosis in JIA (CD4 20+/-8%, CD8 42+/-19%) was higher than in the control group (CD4 5+/-2%, CD8 9+/-6%). Interestingly, the percentage of PB CD4 cells expressing CD28 was lower in JIA than controls. In conclusion, systemic T-cell apoptosis was higher in JIA while a substantial number of SF T cells survived locally, despite the fact that almost all cells express CD95.