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. 2004 Oct;138(1):21-9.
doi: 10.1111/j.1365-2249.2004.02573.x.

CD4 T cells producing IFN-gamma in the lungs of mice challenged with mycobacteria express a CD27-negative phenotype

I V Lyadova  1 , S Oberdorf, M A Kapina, A S Apt, S L Swain, P C Sayles
Affiliations

CD4 T cells producing IFN-gamma in the lungs of mice challenged with mycobacteria express a CD27-negative phenotype

I V Lyadova et al. Clin Exp Immunol. 2004 Oct.

Abstract

Protection against tuberculosis depends upon the generation of CD4(+) T cell effectors capable of producing IFN-gamma and stimulating macrophage antimycobacterial function. Effector CD4(+) T cells are known to express CD44(hi)CD62L(lo) surface phenotype. In this paper we demonstrate that a population of CD44(hi)CD62L(lo) CD4(+) effectors generated in response to Mycobacterium bovis BCG or M. tuberculosis infection in C57BL/6 mice is heterogeneous and consists of CD27(hi) and CD27(lo) T cell subsets. These subsets exhibit a similar degree of in vivo proliferation, but differ by the capacity for IFN-gamma production. Ex vivo isolated CD27(lo) T cells express higher amounts of IFN-gamma RNA and contain higher frequencies of IFN-gamma producers compared to CD27(hi) subset, as shown by real-time PCR, intracellular staining for IFN-gamma and ELISPOT assays. In addition, CD27(lo) CD4(+) T cells uniformly express CD44(hi)CD62L(lo) phenotype. We propose that CD27(lo) CD44(hi)CD62L(lo) CD4(+) T cells represent highly differentiated effector cells with a high capacity for IFN-gamma secretion and antimycobacterial protection at the site of infection.

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Figures

Fig. 1
Fig. 1
Activated CD4+ T cells transiently accumulate in the lungs of BCG infected mice. Suspensions of lung cells were isolated from the lungs of control (week 0) or BCG-infected mice, and stained with PerCP-anti-CD4 mAbs combined with: FITC-anti-CD44, FITC-anti-CD43, or FITC-anti-CD29; PE-anti-CD27 or PE-anti-CD49d; and APC-anti-CD62L mAbs. Cells were analysed by Flow cytometry (2–3 independent experiments, 2–4 mice analysed individually per group per time-point in each experiment).
Fig. 2
Fig. 2
Heterogeneity of CD44hiCD62Llo lung CD4+ T cells with respect to CD27 expression and accumulation of CD27lo subset in the lungs during BCG infection. Lung cells from individual control or BCG challenged mice were stained with FITC-anti-CD44, PE-anti-CD27, PerCP-anti-CD4, and APC-anti-CD62L mAbs. (a, c) Representative patterns of CD44 versus CD62L (a), CD62L versus CD27, and CD44 versus CD27 (c) expression by CD4+ T cells. (b) CD27 expression by distinct subsets of CD4+ T cells. Cells were obtained from the lungs of the same mouse, as in (a, c). Histograms were generated after gating on the indicated subsets of CD4+ T cells. (d, e) Percentages (d) and absolute numbers (e) of CD27hi and CD27lo subsets of CD44hiCD62Llo cells in the lungs. Depicted are results of one out of 3 independent experiments performed at week 2 post challenge with BCG (▪) (3 mice per group per experiment analysed individually). Control (□) Similar results were obtained at week 4. *P < 0·05; **P < 0·01; ***P < 0·001.
Fig. 3
Fig. 3
Cells producing IFN-γ in response to stimulation in vitro belong to CD27lo subset of CD62Llo cells. Lung cell suspensions were obtained from control or BCG- infected mice and stimulated in vitro with PPD; plate-bound anti-CD3 and anti-CD28 mAbs; or were left unstimulated. After overnight culture in the presence of brefeldin, cells were harvested and stained with labelled mAbs. (a) CD62L and CD27 expression by lung cells isolated from BCG challenged mouse and cultured in vitro in different conditions (pictures were generated after gating on CD4+ cells). Note down-regulation of CD62L by T cells stimulated via CD3 receptor. (b) Typical staining for the IFN-γ after gating on distinct subsets of CD4+ T cells (the same cells as in (a)). (c, d) Percentages of IFN-γ producing cells within distinct subsets of lung CD4+ T cells stimulated with PPD or via CD3 receptor (summarized results). Data are mean ± SD from 2 independent experiments performed at week 2 postinfection (total of 6–10 mice per group analysed individually). At week 4 postinfection similar results were obtained. *P < 0·05 as compared to cells isolated from control mice.
Fig. 4
Fig. 4
CD62Llo CD4+ T cells with CD27lo, but not CD27hi phenotype, are the major cells producing IFN-γ as detected by Elispot assay. Mice were challenged with BCG i.t. (a, b) or i.v. (c). Cells were isolated from the lungs (a, b) or spleen (c) 3 weeks later, stained with FITC-anti-CD4, PE-anti-CD27, and APC-anti-CD62L mAbs, and sorted. Sorted cells were cultured at different concentrations in the presence of syngeneic irradiated spleen cells and PPD and frequencies of IFN-γ-producing cells were determined using ELISPOT assay. (a) Pictures of representative wells, in which 2·5 × 104 lung cells of indicated phenotype were cultured. (b, c) Frequencies of IFN-γ producing cells within distinct subsets of CD4+ T cells isolated from the lungs of mice infected with BCG i.t. (b) or from the spleen of mice infected with BCG i.v. (c). In the absence of PPD numbers of IFN-γ-producing cells were equal to those in CD62Lhi subset and never exceeded 1/103 (data not shown).
Fig. 5
Fig. 5
CD4+ cells proliferating in vivo in response to BCG have up-regulated CD44 expression, but do not differ by the expression of CD27. Control (□) and BCG-infected (formula image) mice were pulsed with BrdU at week 2 postinfection. 24 h later lung cells were stained with FITC-anti-BrdU, PE-anti-CD27, PerCP-anti-CD4 and APC-anti-CD44 mAbs and analysed by Flow cytometry. (a) Percent of BrdU+ cells within CD4+ subset of lung cells. (b) Percent of BrdU+ cells within subsets of CD4+ T cells with different expression of CD44 and CD27 molecules. Data are mean ± SD from 2 independent experiments (total of 6 mice per group analysed individually). *P < 0·05; **P < 0·01; as compared to control mice.
Fig. 6
Fig. 6
CD27lo subset of lung CD62Llo CD4 T cells is the major source of IFN-γ during infection with virulent M. tuberculosis. Lung cells were obtained from control mice (data not shown) or mice infected with M. tuberculosis strain H37Rv 3 weeks prior the experiment. Cells were stimulated with H37Rv sonicate (8 µg/ml), cultured in the presence of brefeldin, stained and analysed by Flow cytometry (see Legend to Fig. 3 for details). No significant production of IFN-γ was detected in cells isolated from control mice.
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