Cells were washed, resuspended and analysed by FACSCalibur (Becton Dickinson). For cytokine studies, PBMCs (1 × 106 /ml) were activated with anti-CD3 (100 ng/ml) plus anti-CD28 PD-0332991 research buy (200 ng/ml) for 48 h, and supernatants were collected for the analysis of cytokines [interferon (IFN)-γ and interleukin (IL)-5] by enzyme-linked immunosorbent assay (ELISA) (BD Pharmingen, San Diego, CA, USA). Most of the data, including total IgG, IgG subclasses, lymphocyte subsets, lymphocyte proliferation assays and specific antibody responses, were obtained at the time of diagnosis, prior

to the start of IVIG. Studies of NK cytotoxicity, neutrophil oxidative burst and cytokine levels were measured later while patients were receiving IVIG; however, blood samples were drawn immediately prior to receiving the next scheduled IVIG dose (at trough level). All laboratory tests listed above were performed by a California State and CLIA (Clinical Laboratory Improvement Amendments)-certified laboratory, which requires validation and reproducibility of data. Demographic and clinical features of 17 adult patients with selective IgG3 deficiency are listed in Table 1. There was a significant

female predominance (female : male, 3:1), and the mean age at diagnosis was 47 years. The majority of patients presented with recurrent upper respiratory infection, sinusitis and pneumonia. In addition, 10 of 17 patients had concurrent allergic rhinitis and/or asthma. This was based upon patients’ history and statement that radioallergosorbent tests (RAST) and GS-1101 in vivo skin tests were performed by the referring allergists. Lymphocyte subpopulations. Figure 1 show proportions of CD3+ T cells, CD3+CD4+ helper/inducer T cells, CD3+CD8+ cytotoxic T cells, CD3–CD19+ B cells and CD3–CD16+CD56+ NK cells. The majority of patients had percentages of subsets within the range of age- and sex-matched controls (Fig. 1, top panel). When data were analysed for absolute numbers, two patients each had low CD8+ T cells and low B cells (Fig. 1, bottom panel). DNA synthesis GBA3 in lymphocytes. 

Data for lymphocyte proliferation are shown in Fig. 2. Low response to at least two of three mitogens or two of three antigens was considered abnormal. Four of 12 patients (33%) on whom mitogen studies were performed had low mitogen responses, and four of 10 patients (40%) had low antigen responses. Specific antibody responses.  The pneumococcal antibody responses were recorded in 11 patients, five of whom had protective prevaccination titres greater than 1·0 IU/ml for at least half of the 14 serotypes. Of the six patients who had low prevaccination titres, two patients had no response to vaccination with Pneumovax-23. The most common unprotective antibody levels were observed against serotypes 3, 8, 9N and 12F, and the least common impairment was observed against serotypes 4, 5, 7F, 18C and 23F. Specific antibody responses to tetanus toxoid were recorded in 10 of 17 patients.

In addition, these data also support the notion that the secondary CD8+ T-cell response exhibits elements of “programming” [[43]] since the NP118-specific CD8+ T-cell expansion after LCMV infection is proportional to the initial memory levels in PKO mice, suggesting all recruited cells underwent a similar number of divisions (Fig. 3D). We observed minor differences in the phenotype of Ag-specific CD8+ T cells between DC- and att LM-primed PKO mice at memory

time points. For example, the frequency of KLRG-1-expressing memory CD8+ T cells is higher in LM-infected compared with DC-primed mice. The extent to which such phenotypic differences influence the ability of memory cells to respond to LCMV infection may be minimal, since we observed the same massive expansion of that NP118-specific Deforolimus manufacturer memory cells in both groups. In addition, recent data suggested that KLRG-1 was dispensable for normal CD8+ T-cell differentiation and function after viral infections [[44]]. Tight regulation of

cytolysis and cytokine production by effector and memory CD8+ T cells in the presence of antigen has been proposed as a likely mechanism to minimize immunopathology [[8, 45]]. IFN-γ production by wild-type NP118-specific CD8+ T cells from LCMV-infected mice is not detected in direct ex vivo assays at any time postinfection see more without addition of antigen [[46, 47]]. In addition, IFN-γ production by these cells is rapidly extinguished by removal of antigen [[46, 47]]. Thus, it is likely that failure to clear LCMV in vaccinated

PKO mice causes chronic stimulation of the massively expanded NP118-specific CD8+ T-cell population, resulting in dysregulated production of cytokines and mortality. Interestingly, we observed significant reduction of LCMV viral titer in the spleen of NP118-vaccinated PKO mice at day 5 post-LCMV infection compared with control mice (Fig. 5). We would have predicted that lower viral titer would correspond with lower systemic cytokine levels. However, in this case, lower viral titer may be the result of increased systemic cytokine (i.e. cytokine storm) that potentially interferes Adenosine with viral replication. The inability to clear the virus leads to rebound of LCMV titer in these vaccinated PKO mice suggesting that despite enormous number of Ag-specific CD8+ T cells perforin-mediated cytolysis is absolutely required to control LCMV infection and provide sterilizing immunity. Thus, the early substantial reduction in viral titers is still associated with mortality in these PKO mice. In addition, this result also suggested that cytokine dysregulation is a property inherent to PKO-derived memory CD8+ T-cell response as has been suggested from in vitro studies [[48]]. Naïve BALB/c-PKO mice (H-2d) survive LCMV-Arm infection by exhausting their NP118-specific CD8+ T cells [[16]].