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Flowcytometric assessment of intracellular interferon -γ production in human CD4 + ve T-cells on mycobacterial antigen stimulation

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Abstract

Interferon-(IFN-γ) has been considered to be a critical protective immunomodulatory component against Mycobacterium tuberculosis (M. tb.) infection. In this study T-cell proliferation and IFN-γ production upon stimulation with M. tb. were assessed in patients of pulmonary tuberculosis and healthy contacts. The studies were based on lymphocyte transformation test and detection of intracellular IFN-γ production by CD4 + ve T-cells by flowcytometry. Patients showed lower levels of proliferation, the stimulation index being in the range of 2.17 1.1 (mean + SD) compared to the contacts (SI = 4 59±1.6) (P < 0.01). The kinetics of intracellular induction of IFN-γ on M. tb. stimulation showed a proportional increase in the CD4 + ve T-cell population. The increase was maximal between 96–120 h of culture. In healthy contacts the number of IFN-γ expressing CD + ve T-cells increased to 2.5 to 41 × 104 cells/ml in M. tb. stimulated cultures compared to control cultures (0.1 – 15 × 104). In contrast patients showed no/marginal increase in CD4 + ve T-cell population expressing intracellular IFN-γ Thus the lack of induction of IFN in CD4 + ve T-cells in patients could be a critical shortcoming in their ability to combat tubercle bacilli infection.

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Abbreviations

M. tb:

Mycobacterium tuberculosis (H37Rv) IFN γ interferon-γ

LTT:

lymphocyte transformation test

S.I:

stimulation index

PBS:

phosphate buffer saline

DPBS:

Dulbecco’s phosphate buffer saline

PFA:

paraformaldehyde

FACS:

fluorescence activated cell sorter

PHA:

phytohaemaglutinin

PBMC:

peripheral blood mononuclear cell

BD:

Becton Dickinson

FCS:

fetal calf serum

SA-PE:

streptavidin-phycoerythrin

FITC:

fluorescein isothiocyanate

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Authors and Affiliations

  1. Department of Biotechnology, All India Institute of Medical Sciences, 110029, New Delhi, India

    S. Bhattacharyya, S. N. Das, A. B. Dey, K. Nagarkar, J. Lobo, S. K. Kapoor & H. K. Prasad

  2. Department of Medicine, All India Institute of Medical Sciences, 110029, New Delhi, India

    A. B. Dey & K. Nagarkar

  3. Center for Community Medicine, All India Institute of Medical Sciences, 110029, New Delhi, India

    J. Lobo & S. K. Kapoor

Authors
  1. S. Bhattacharyya
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  2. S. N. Das
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  3. A. B. Dey
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  4. K. Nagarkar
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  5. J. Lobo
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  6. S. K. Kapoor
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  7. H. K. Prasad
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Correspondence to H. K. Prasad.

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Bhattacharyya, S., Das, S.N., Dey, A.B. et al. Flowcytometric assessment of intracellular interferon -γ production in human CD4 + ve T-cells on mycobacterial antigen stimulation. J. Biosci. 22, 99–109 (1997). https://doi.org/10.1007/BF02703622

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  • DOI: https://doi.org/10.1007/BF02703622

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