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. 2010 Oct 1;185(7):3940-7.
doi: 10.4049/jimmunol.1001222. Epub 2010 Aug 30.

TCR Repertoire, Clonal Dominance, and Pulmonary Trafficking of Mycobacterium-Specific CD4+ and CD8+ T Effector Cells in Immunity Against Tuberculosis

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Free PMC article

TCR Repertoire, Clonal Dominance, and Pulmonary Trafficking of Mycobacterium-Specific CD4+ and CD8+ T Effector Cells in Immunity Against Tuberculosis

George Du et al. J Immunol. .
Free PMC article

Abstract

Clonal responses of Mycobacterium tuberculosis-specific CD4(+) or CD8(+) T effector cells producing antituberculosis cytokine IFN-γ in the context of immune protection against tuberculosis remain poorly characterized in humans. Utilizing decade-long TCR expertise, we previously developed a useful method to isolate clonotypic TCR sequences from Ag-specific IFN-γ-producing T cells and to specifically measure clonotypic TCR frequencies in the T cell pool. In this study, we investigated TCR Vβ repertoires/CDR3 usage, clonal expansion or dominance, and pulmonary trafficking or accumulation for purified protein deritative (PPD)-specific T effector cells producing IFN-γ during bacillus Calmette-Guérin (BCG) vaccination and subsequent M. tuberculosis challenge of macaques. We found that while PPD-specific CD4(+) and CD8(+) T effector clones employed diverse TCR Vβ repertoires, 30-33% of IFN-γ(+)CD4(+) T cell clones from three M. tuberculosis-infected macaques expressed TCR bearing a conserved residue leucine in CDR3. Many Ag-specific IFN-γ(+) CD4(+) and few CD8(+) T effector cells emerged as dominant clones during mycobacterial infections and underwent major recall expansion after pulmonary M. tuberculosis infection of BCG-vaccinated macaques. PPD-specific T cell clones readily trafficked to the airway or lung after BCG vaccination or M. tuberculosis infection, and some of them continuously accumulated in lungs during M. tuberculosis infection even after they became undetectable in the circulation. Importantly, remarkable recall expansion and pulmonary accumulation of T effector cells coincided with BCG-induced protection against tuberculosis. Thus, rapid clonal expansion and pulmonary accumulation of Ag-specific T effector cells appear to be one of the immune mechanisms underlying immunity against tuberculosis.

Figures

FIGURE 1
FIGURE 1
PPD-specific T effector cells producing IFN-γ upon stimulation were detectable after BCG vaccination and M. tuberculosis infection. Shown are ELISPOT data (A) indicating IFN-γ+ cellular responses in PBMCs after BCG vaccination and M. tuberculosis infection of juvenile Indian rhesus macaques (A) and representative flow histogram data (B) displaying PPD-specific IFN-γ+ CD8+ and CD8 (CD4+) T cells measured by intracellular cytokine staining after stimulation with PPD or medium control. Data were gated on CD3, with percentages indicated in quadruples. Both IFN-γ+ CD8+ and CD4+ T cell populations were purified by flow cytometry sorting (15). ELSPOT data are mean values with SEM from four macaques.
FIGURE 2
FIGURE 2
PPD-specific CD4+ and CD8+ T effector clones employed diverse TCR Vβ repertoires; 30–33% of IFN-γ+CD4+ T cell clones from three M. tuberculosis-infected macaques expressed TCR bearing a conserved residue leucine in CDR3. Shown are TCR sequences in V-D-J junctional regions for CD4+ and CD8+ T cell clones. Clone numbers (#) from TCR isolation, clone ID (Vβ-Jβ usage), amino acid sequences of Vβ (3′ end), D+N and Jβ, and CDR3 lengths are indicated. CDR3 lengths were defined as previously described (23, 27, 29). Note a conserved residue leucine that is bold and underlined in the TCR N+D region of the clones isolated from macaques 3055, 2995, and 3050. To maximize the detection of M. tuberculosis-specific IFN-γ+ T effector cells using the ICS method, PBLs collected at weeks 5 or 7 after M. tuberculosis infection were stimulated with PPD, stained for IFN-γ and surface CD3, CD4, and CD8, and subjected to isolation of IFN-γ+ CD4+ or CD8+ T effector cells by flow cytometry sorting as we previously described (15, 23). Thirty to 55% of the clones were isolated from PBLs obtained at both weeks 5 and 7 after M. tuberculosis infection.
FIGURE 3
FIGURE 3
Many Ag-specific IFN-γ+ CD4+ and few CD8+ T effector cells emerged as dominant clones during mycobacterial infections and underwent major recall expansion after pulmonary M. tuberculosis infection of BCG-vaccinated macaques. Shown are the frequencies for individual clonotypic TCR clones in the blood T cell pool after i.v. BCG vaccination (four macaques) and pulmonary M. tuberculosis infection (five macaques). Data are expressed as clonotypic TCR copy numbers in 105 total TCR β (Cβ) transcripts. The time for BCG vaccination or M. tuberculosis infection is pointed to by an arrow in each panel. CD8+ T effector clones were indicated; otherwise, they were CD4+ T effector clones. Note that the quantitatable and unquantitatable TCR clones are grouped, respectively, for each macaque. The real-time quantitation method confers a detection limit for TCR transcripts corresponding to 10–5 Ag-specific T cells (15). Most clones became unquantitatable around the time point of anti-TB chemotherapy (denoted by drug), which might be attributed to the vaccine-induced immune control of acute M. tuberculosis infection. Note that Vβ23Jβ2.5 (◇) and Vβ16Jβ1.2 (*) clones from macaque 2995 exhibited similar frequencies early after M. tuberculosis infection, whereas Vβ16Jβ1.1 clone (marked by #) became unquantitatable after M. tuberculosis infection. The low-level recall clonal expansion for macaque 2762 appeared to correlate with extremely low bacterial burden after M. tuberculosis infection (16).
FIGURE 4
FIGURE 4
PPD-specific IFN-γ–producing cells were detectable in the airway in M. tuberculosis-infected macaques. Shown are ELSPOT data for PPD-specific IFN-γ+ cell responses in BAL fluid collected from three M. tuberculosis-infected macaques at day 41 after the infection, as well as from three uninfected healthy macaques (control). Data were subtracted from values of medium alone and expressed as IFN-γ+ cells in 106 BAL cells. From Student t test analysis, p < 0.05 between the groups.
FIGURE 5
FIGURE 5
PPD-specific CD4+ and CD8+ T cell clones readily trafficked to the airway after M. tuberculosis infection, and some of them continuously accumulated in lungs even after they became undetectable in the circulation. Shown are the frequencies (clonotypic TCR copy numbers in 105 total TCR β (Cβ) transcripts) of clonotypic TCR clones detected both in blood (PBLs) and airway (BAL) at different days (D) after M. tuberculosis infection of macaques. Data for BAL are highlighted in light black. Only those dominant clones quantitatable in PBLs were subjected to real-time quantitation in BAL samples due to the limited numbers of BAL cells.
FIGURE 6
FIGURE 6
PPD-specific IFN-γ–producing CD4+ T cell clones readily trafficked to the airway as well after the second i.v. BCG vaccination. Shown are the frequencies of clonotypic TCR clones detected both in PBLs and BAL fluid after the second BCG vaccination of macaques. The first BCG vaccination was done 4 mo earlier. The primary BCG infection is usually resolved within 2 mo after the first i.v. BCG inoculation. No BCG mycobacteria were detected in BAL samples after the second BCG vaccination. # indicates that no BCG bacteria were isolated from the blood and BAL fluid collected from denoted times (#) after the second BCG vaccination. BCG CFUs were measured using 7H10 agar plates (16). There were no or few neutrophils seen over time in BAL fluid.
FIGURE 7
FIGURE 7
Accumulation of T effector cells in the airway was also induced by intradermal BCG vaccination. Shown are ICS data indicating mean frequencies of PPD-specific IFN-γ+ T cells in BAL cells from four rhesus macaques vaccinated intradermally with BCG (18).
FIGURE 8
FIGURE 8
Rapid major expansion of BCG-primed T effector cells after M. tuberculosis infection was associated with protection against fatal TB in juvenile Indian rhesus macaques. Shown are mean comparative ELI-SPOT data from BCG-vaccinated and naive control groups at 3 wk after M. tuberculosis infection. All vaccinated macaques survived with very low-level M. tuberculosis burden, whereas all naive controls died (16).

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