Immunohistochemistry, TD and TI-II LY294002 datasheet immunizations, TNP-specific and total Ig subclass ELISA assays were performed as described 28, 41. Levels of anti-nucleosome antibodies were measured by ELISA (using coated oligonucleosomes and peroxidase-coupled anti-mouse Ig isotype-specific antibodies for subsequent detection). For ELISPOT assays, 96-well Multiscreen plates (MAHAN4550; Millipore) were coated overnight at 4°C with 1 μg/mL anti-Ig subclass antibodies (BD Pharmingen) and subsequently blocked in PBS/1% BSA at r.t. for 1 h. Serial dilutions of splenic cell suspensions were incubated at 37°C for 3 h. Production was detected with corresponding biotin-labeled anti-Ig isotype-specific
antibodies, streptavidin-peroxidase (BD Pharmingen) and 3-amino-9-ethylcarbazole. Antibody secreting cells were counted under the microscope. Statistical significance was calculated using the Mann–Whitney U test. The authors thank the people from the Erasmus MC Animal Care facility for their assistance. This work was supported by the Netherlands Organization for Scientific Research, the Dutch Cancer Society and the
Dutch Arthritis Association. Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. “
obliterans syndrome (BOS) is associated with lack Daporinad supplier of immunosuppression of T cell proinflammatory cytokines and increased T cell granzyme B. Repeated antigen-driven proliferation down-regulates T cell CD28. We hypothesized that down-regulation of CD28 and up-regulation of alternate co-stimulatory molecules (CD134, CD137, CD152 and CD154) on T cells may be associated with BOS. Co-stimulatory molecules, granzyme B, perforin and intracellular cytokines were measured by flow cytometry on T cells from stable lung transplant patients (n = 38), patients with BOS (n = 20) and healthy controls (n = 10). There was a significant increase in the percentage of CD4/28null and CD8/28null T cells producing Ketotifen granzyme B, interferon (IFN)-γ and tumour necrosis factor (TNF)-α in BOS compared with stable patients. Down-regulation of CD28 was associated with steroid resistance and up-regulation of CD134, CD137, CD152 and CD154 on CD4+ T cells and CD137 and CD152 on CD8+ T cells. There was a significant correlation between increased CD28null/CD137 T cells producing IFN-γ, TNF-α with BOS grade (r = 0·861, P < 0·001 for CD28null/CD137 IFN-γ/CD8) and time post-transplant (r = 0·698, P < 0·001 for CD28null/CD137 IFN-γ/CD8). BOS is associated with down-regulation of CD28 and up-regulation of alternate co-stimulatory molecules on steroid-resistant peripheral blood proinflammatory CD4+ and CD8+ T cells.