Intracerebral Human Regulatory T Cells: Analysis of CD4+CD25+FOXP3+ T Cells in Brain Lesions and Cerebrospinal Fluid of Multiple Sclerosis Patients

Impaired suppressive capacity of CD4+CD25+FOXP3+ regulatory T cells (Treg) from peripheral blood of patients with multiple sclerosis (MS) has been reported by multiple laboratories. It is, however, currently unresolved whether Treg dysfunction in MS patients is limited to reduced control of peripheral T cell activation since most studies analyzed peripheral blood samples only. Here, we assessed early active MS lesions in brain biopsies obtained from 16 patients with MS by FOXP3 immunohistochemistry. In addition, we used six-color flow cytometry to determine numbers of Treg by analysis of FOXP3/CD127 expression in peripheral blood and cerebrospinal fluid (CSF) of 17 treatment-naïve MS patients as well as quantities of apoptosis sensitive CD45ROhiCD95hi cells in circulating and CSF Treg subsets. Absolute numbers of FOXP3+ and CD4+ cells were rather low in MS brain lesions and Treg were not detectable in 30% of MS biopsies despite the presence of CD4+ cell infiltrates. In contrast, Treg were detectable in all CSF samples and Treg with a CD45ROhiCD95hi phenotype previously shown to be highly apoptosis sensitive were found to be enriched in the CSF compared to peripheral blood of MS patients. We suggest a hypothetical model of intracerebral elimination of Treg by CD95L-mediated apoptosis within the MS lesion

Soluble Serum CD81 Is Elevated in Patients with Chronic Hepatitis C and Correlates with Alanine Aminotransferase Serum Activity

Aim: Cellular CD81 is a well characterized hepatitis C virus (HCV) entry factor, while the relevance of soluble exosomal CD81 in HCV pathogenesis is poorly defined. We performed a case-control study to investigate whether soluble CD81 in the exosomal serum fraction is associated with HCV replication and inflammatory activity. Patients and Methods: Four cohorts were investigated, patients with chronic hepatitis C (n = 37), patients with chronic HCV infection and persistently normal ALT levels (n = 24), patients with long term sustained virologic response (SVR, n = 7), and healthy volunteers (n = 23). Concentration of soluble CD81 was assessed semi-quantitatively after differential centrifugation ranging from 200 g to 100,000 g in the fifth centrifugation fraction by immunoblotting and densitometry. Results: Soluble CD81 was increased in patients with chronic hepatitis C compared to healthy subjects (p = 0.03) and cured patients (p = 0.017). Patients with chronic HCV infection and persistently normal ALT levels and patients with long term SVR had similar soluble CD81 levels as healthy controls (p>0.2). Overall, soluble CD81 levels were associated with ALT levels (r = 0.334, p = 0.016) and severe liver fibrosis (p = 0.027). Conclusion: CD81 is increased in the exosomal serum fraction in patients with chronic hepatitis C and appears to be associated with inflammatory activity and severity of fibrosis

Representative six-color FACS analysis of lymphocytes derived from CSF and peripheral blood.

<p>Isolated lymphocytes were stained with monoclonal antibodies against CD4, CD25, CD127, FOXP3, CD45RO and CD95. CD4⁺ cells were gated (G1) and analyzed for FOXP3, CD127 and CD25 <b>(A)</b>. CD4⁺ cells are shown in all other dot plots of Fig. 2A, B in blue and represent 48% of peripheral lymphocytes. <b>(B)</b> CD4 positive cells with significant FOXP3 expression and low to high CD25 expression were included in gate (G2). These cells are shown in all other dot plots of Fig. 2A, B in red and represent 7.1% of CD4 positive cells. Multicolor analysis confirms that the majority of FOXP3 positive cells co-segregate with CD127^loCD25^lo-hi expression. Note: CD127 negative cells accumulate in the first few channels by conventional dot plotting. <b>(C)</b> Pairwise analysis from CSF and PBMC of a treatment-naïve MS patient. Isolated lymphocytes were stained with monoclonal antibodies against CD4, CD25, CD127, FOXP3, CD45RO and CD95. Lymphocytes were gated for CD4⁺CD25⁺CD127^loFOXP3⁺ cells and the percentage of CD95^hiCD45RO^hi was determined.</p

Chemotactic activities for CD45RA^hi Treg and CD45RO^hi Treg subsets.

<p>Chemotactic indices (CI) of Treg from a healthy donor towards CSF supernatants of 11 MS patients as determined by transwell migration assays. Original input PBMC and migrated cells were stained for CD4, CD25, FOXP3, CD95, CD45RO, and CD45RA, and CIs were calculated by dividing percentages of Treg subsets in CD4⁺ T-cells of migrated cells by percentages of Treg in CD4⁺ T-cells in original input PBMCs. Symbols indicate CIs of individual patients. Means are indicated, ** indicates p<0.001.</p

Six-color FACS analysis of lymphocytes derived from CSF and peripheral blood.

<p>Analysis of CSF and PBMC from 17 treatment-naïve MS patients. Isolated lymphocytes were stained with monoclonal antibodies against CD4, CD25, CD127, FOXP3, CD45RO and CD95. <b>(A)</b> Lymphocytes were gated for Treg and the percentage of CD95^hiCD45RO^hi was determined. Treg were increased in CSF when compared to peripheral blood, * indicates p<0.05. <b>(B)</b> Expression of CD95 on Treg from CSF and PBMC derived from the same MS patient on the same day, representative figure.</p

Prevalence of CD4⁺ and FOXP3⁺ cells in MS lesions.

<p>Brain biopsies of 16 MS patients were analyzed by immunohistochemistry for the presence of CD4⁺ and FOXP3⁺ cells. Sections of each biopsy were stained with CD4 mAb, FOXP3 mAb or isotype control mAbs. Whole-slide analysis by complete scanning of the section and computer-aided analysis of positive cells was performed. (<b>A</b>) Representative CD4 immunohistochemistry of MS lesion. Positive cells show red cell surface staining. Bar indicates µm scale in two different magnifications. Tissue was hematoxylin counterstained prior to analysis. (<b>B</b>) Representative FOXP3 immunohistochemistry of MS lesion. Positive cells show brown nuclear staining. Bar indicates µm scale in two different magnifications. Tissue was hematoxylin counterstained prior to analysis. (<b>C</b>) Number of patients with CD4⁺ T cells in the section (0% negative, 100% positive) and number of patients with FOXP3⁺ cells in the analyzed section (31% negative with 69% positive for FOXP3). (<b>D</b>) Number of patients grouped by their content of FOXP3⁺ cells and CD4⁺ cells respectively in the analyzed section: “-“ indicates 0 cells/mm², “(+)” 0-3 cells/mm², “+” >4-15 cells/mm², “++” >15 cells/mm². (<b>E</b>) Number of patients with (+) or without detection of FOXP3⁺ cells (-) in active or inactive MS lesions.</p