The investigation of peripheral blood cellular immune responses during infection with Mycobacterium Tuberculosis

Veenstra, Hannelore F. U. (2007-03)

Thesis (PhD (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2007.

Thesis

Despite the ongoing global tuberculosis (TB) problem and extensive research into protective immunity against this intracellular pathogen, mechanisms of protective immunity against Mycobacterium tuberculosis (Mtb) in humans have not been fully clarified. Numerous reports have addressed the potential immunological defect(s) in infected individuals that have developed active TB in comparison to those who have remained healthy in spite of infection. Markers of treatment response phenotypes are still elusive. The aims of this study were to define lymphocyte subsets in the peripheral blood of TB patients and controls, to determine intracellular interferon-γ (IFN-γ) and interleukin-4 (IL-4) production and to find correlations of these data with microbiologically-defined treatment response. Methods Whole blood tests were done on 30 HIV-negative, smear-positive pulmonary TB patients and 18 healthy skin test positive volunteers resident in the same community. Immunophenotyping was performed by flow cytometry, combined with routine haematology, for the enumeration of peripheral blood immune cell subtypes. Whole blood was also stimulated in vitro with anti-CD3 monoclonal antibody and intracellular IFN-γ and IL-4 determined by flow cytometry. Lymphocyte proliferation in response to heat-killed Mtb was determined by tritiated thymidine incorporation. Routine microbiological monitoring by sputum smears and culture was done throughout the patients’ 26 weeks of treatment. Results Compared to healthy controls, absolute numbers of peripheral blood lymphocytes and lymphocyte subsets were significantly depressed in patients at diagnosis but normalized during treatment with the exception of natural killer (NK) cells and natural killer T (NKT) cells. A novel subset of the latter was found to correlate significantly with treatment response. IFN-γ-producing T cells after a 4-hour T cell receptor stimulation were significantly higher in patients at diagnosis and normalized during treatment. Supplementary kinetic experiments showed that IFN-γ production in patients at diagnosis seemed to be accelerated. Lymphocyte proliferation was lower in patients at diagnosis and normalized during treatment. Neither IFN-γ production nor lymphocyte proliferation correlated with treatment response. Low intracellular IL-4 production was constitutive in patients and controls, was insignificantly lower in patients at diagnosis than in controls and, in the slow responder patient group, it was significantly lower than in the fast responder group. High IL-4 expression was found in low numbers of T cells in patients and controls and supplementary experiments showed co-expression of active caspase-3 in these cells, which signified apoptosis. Conclusions Lymphocyte subset phenotypes associated with TB are largely abnormal only during active infection and only a novel subset of NKT cells showed correlation with treatment response. Intracellular IFN-γ production and lymphocyte proliferation is increased and decreased, respectively, only during active infection and does not correlate with treatment response. The T helper 1/T helper 2 (Th1/Th2) hypothesis could not be confirmed in the context of tuberculosis but instead constitutive IL-4 production may play a role as a growth factor.

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