[Analysis of ESAT-6-specific multifunctional CD4+; T cells in pleural fluid from patients with tuberculous pleurisy].

AIM: To evaluate cytokine production by CD4(+); T cells and frequency of multifunctional CD4(+); T cells after stimulation with ESAT-6 peptide pool. To understand the role of ESAT-6 specific CD4(+); T cells in the control of local TB infection. METHODS: PFCs were isolated from patients with tuberculous pleurisy, and assessed by flow cytometry for cytokine production, subpopulation, frequency and function of multifunctional CD4(+);T cells after stimulation with ESAT-6 peptide pool. RESULTS: Following stimulation with BCG, ESAT-6 peptide pool and recombinant ESAT-6 protein, CD4(+); but not CD8(+); T cells expressed IFN-γ, IL-2 and TNF-α. Distinct from BCG, ESAT-6 peptide pool and recombinant ESAT-6 protein induced similar frequencies of IFN-γ, IL-2 and TNF-α. High frequency of multifunctional CD4(+);T cells was observed. Analysis of mean fluorescence intensity (MFI) indicated that triple positive cells produced most amounts of cytokines on single cell level compared with double positive and single positive cells. CONCLUSION: ESAT-6 peptide pool predominantly induced Th1 cytokine production by CD4(+);T cells in PFCs. Multifunctional CD4(+); T cells were observed and secreted most amounts of cytokines on single cell level, suggesting that these cells probably played essential role in local TB infection.

[Cytokine production and clonal analysis of memory γδ T cells from patients with tuberculous pleurisy induced by bacille calmette guerin].

OBJECTIVE: To evaluate cytokine production and expression of γδ T cells within pleural fluid cells (PFCs) from patients with tuberculous pleurisy following bacille calmette guerin (BCG) stimulation. METHODS: PFCs were isolated from patients with tuberculous pleurisy, and assessed for cytokine production, cell subpopulation, phenotype and characterization of T cell receptors after stimulation with BCG. The positive PCR products were further labeled with fluorescence and analyzed by genescan technique to determine the CDR3 size and evaluate the clonality of the detectable TCR Vγ and Vδ T cells. RESULTS: Following stimulation with BCG, the positivity of interferon-γ (IFN-γ)-producing CD(4) T cells and γδ T cells were 0.38% and 5.35%, respectively. Phenotypic analysis indicated that the majority of IFN-γ(+)γδ(+) T cells expressed CD(45RO)(+) (73.5%). In addition, δ(2) T cells produced IFN-γ (11.1%) and TNF-α (25.5%). After expansion with BCG for 3 weeks, cells were harvested and mRNA extracted and RT-PCR conducted to amplify cDNA with 3 primers for Vγ and 8 primers for Vδ. The results indicated that BCG selectively expanded δ(2) T cells with oligoclonal peak in Vδ(2) cells. CONCLUSIONS: BCG induced memory γδ and δ(2) T cells to produce cytokines in PFCs. Genescan analysis showed that Vδ(2) displayed oligoclonality.

Increased frequency of CD4(+)CD25(high) Treg cells inhibit BCG-specific induction of IFN-gamma by CD4(+) T cells from TB patients.

Cell-mediated immunity plays a considerable role in the protection against Mycobacterium tuberculosis infection. The immune response to tuberculosis (TB) was dominated by both CD4(+) T cells with the T helper 1 type cytokines and CD8(+) T cells. Recent studies have suggested that the circumstances in which protective or tissue-damaging T cell responses to microbes are affected by the activity of Treg (CD4(+)CD25(high)) cells. In the present study, we demonstrated that the frequencies of CD4(+)CD25(+) and CD4(+)CD25(high) T cells in TB patients were significantly higher compared to normal individuals. These Treg cells expressed CTLA-4 and Foxp3 at protein level and displayed activation and memory phenotypes as assessed by flow cytometric analysis. The frequencies of CD4(+)CD25(high)CTLA-4(+) and CD4(+)CD25(high)Foxp3(+) T cells within the total CD4(+) T cell population were significantly increased in the blood of TB patients compared to healthy donors. Moreover, the expression of GITR on Treg cells was higher in TB patients than in normal donors. The phenotypic analysis demonstrated that CD4(+)CD25(high) Treg expressed higher levels of CD45RO and HLA-DR, and lower levels of CD45RA compared to CD4(+)CD25(low) and CD4(+)CD25(-) T cells. The addition of CD4(+)CD25(high) T cells back to cultures could significantly suppress the antigen-specific production of IFN-gamma induced by BCG-stimulated CD4(+)CD25(-) T cells, suggesting that Treg might play a key role in the control of cellular immune responses in TB infection.

[A retrospective study of one case of X-linked agammaglobulinemia].

OBJECTIVE: To investigate the clinical manifestations, diagnostic methods and treatment of X-linked agammaglobulinemia (XLA). METHODS: Flow cytometric analysis of the peripheral monocytes using the anti-BTK antibody was used to characterize the expression of BTK in a 21 year old male patient and his mother. The patient suffered from frequent pneumonia, and was found to be complicated with lymphocytopenia in the B cell populations, hypogammaglobulinemia (IgG 1.38 g/L, IgA 0.25 g/L, IgM 0.17 g/L) and angiotelectasis (which had not been reported in XLA patients). Sequencing of the BTK cDNA obtained from the peripheral monocytes of the patient and his mother was performed to confirm the genetic defect. RESULTS: The BTK expressions in peripheral monocytes of the patient and his mother were 96.9% and 97.8% respectively. Sequencing of the BTK gene revealed a missense mutation of R525Q in exon 16, and his mother was confirmed to be an XLA carrier. The patient was treated with immunoglobulin replacement therapy (2 g/kg). One month later, the serum IgG level of the patient was elevated to 5.79 g/L, and the clinical symptoms (included angiotelectasis), lung function and the CT scan results significantly improved. CONCLUSION: Genetic diagnosis was made for one Chinese XLA adult patient complicated with angiotelectasis. This case suggests that some XLA cases may present angiotelectasis. High dose intravenous immunoglobulin given at 2 g/kg may be of efficacy in severe XLA cases. More attention should be paid to the disease in China.