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

[摘要] Despite the ongoing global tuberculosis (TB) problem and extensive research intoprotective immunity against this intracellular pathogen, mechanisms of protectiveimmunity against Mycobacterium tuberculosis (Mtb) in humans have not been fullyclarified. Numerous reports have addressed the potential immunological defect(s) ininfected individuals that have developed active TB in comparison to those who haveremained healthy in spite of infection. Markers of treatment response phenotypes arestill elusive. The aims of this study were to define lymphocyte subsets in theperipheral 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 datawith microbiologically-defined treatment response.MethodsWhole blood tests were done on 30 HIV-negative, smear-positive pulmonary TBpatients and 18 healthy skin test positive volunteers resident in the same community.Immunophenotyping was performed by flow cytometry, combined with routinehaematology, for the enumeration of peripheral blood immune cell subtypes. Wholeblood was also stimulated in vitro with anti-CD3 monoclonal antibody andintracellular IFN-γ and IL-4 determined by flow cytometry. Lymphocyteproliferation in response to heat-killed Mtb was determined by tritiated thymidineincorporation. Routine microbiological monitoring by sputum smears and culturewas done throughout the patients' 26 weeks of treatment. ResultsCompared to healthy controls, absolute numbers of peripheral blood lymphocytesand lymphocyte subsets were significantly depressed in patients at diagnosis butnormalized during treatment with the exception of natural killer (NK) cells andnatural killer T (NKT) cells. A novel subset of the latter was found to correlatesignificantly with treatment response. IFN-γ-producing T cells after a 4-hour T cellreceptor stimulation were significantly higher in patients at diagnosis and normalizedduring treatment. Supplementary kinetic experiments showed that IFN-γ productionin patients at diagnosis seemed to be accelerated. Lymphocyte proliferation waslower in patients at diagnosis and normalized during treatment. Neither IFN-γproduction nor lymphocyte proliferation correlated with treatment response. Lowintracellular IL-4 production was constitutive in patients and controls, wasinsignificantly lower in patients at diagnosis than in controls and, in the slowresponder 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 controlsand supplementary experiments showed co-expression of active caspase-3 in thesecells, which signified apoptosis.ConclusionsLymphocyte subset phenotypes associated with TB are largely abnormal only duringactive infection and only a novel subset of NKT cells showed correlation withtreatment response. Intracellular IFN-γ production and lymphocyte proliferation isincreased and decreased, respectively, only during active infection and does notcorrelate with treatment response. The T helper 1/T helper 2 (Th1/Th2) hypothesiscould not be confirmed in the context of tuberculosis but instead constitutive IL-4production may play a role as a growth factor.

[发布机构] Stellenbosch University