Clonal immune responses of Mycobacterium-specific gammadelta T cells in tuberculous and non-tuberculous tissues during M. tuberculosis infection

Background: We previously demonstrated that unvaccinated macaques infected with large-dose M.tuberculosis(Mtb) exhibited delays for pulmonary trafficking of Ag-specific alphabeta and gammadelta T effector cells, and developed severe lung tuberculosis(TB) and "secondary" Mtb infection in remote organs such as liver and kidney. Despite delays in lungs, local immunity in remote organs may accumulate since progressive immune activation after pulmonary Mtb infection may allow IFNgamma-producing gammadelta T cells to adequately develop and traffic to lately-infected remote organs. As initial efforts to test this hypothesis, we comparatively examined TCR repertoire/clonality, tissue trafficking and effector function of Vgamma2Vdelta2 T cells in lung with severe TB and in liver/kidney without apparent TB. Methodology/Principal Findings: We utilized conventional infection-immunity approaches in macaque TB model, and employed our decades-long expertise for TCR repertoire analyses. TCR repertoires in Vgamma2Vdelta2 T-cell subpopulation were broad during primary Mtb infection as most TCR clones found in lymphoid system, lung, kidney and liver were distinct. Polyclonally-expanded Vgamma2Vdelta2 T-cell clones from lymphoid tissues appeared to distribute and localize in lung TB granuloms at the endpoint after Mtb infection by aerosol. Interestingly, some TCR clones appeared to be more predominant than others in lymphocytes from liver or kidney without apparent TB lesions. TCR CDR3 spetratyping revealed such clonal dominance, and the clonal dominance of expanded Vgamma2Vdelta2 T cells in kidney/liver tissues was associated with undetectable or low-level TB burdens. Furthermore, Vgamma2Vdelta2 T cells from tissue compartments could mount effector function for producing anti-mycobacterium cytokine. Conclusion: We were the first to demonstrate clonal immune responses of mycobacterium-specific Vgamma2Vdelta2 T cells in the lymphoid system, heavily-infected lungs and lately subtly-infected kidneys or livers during primary Mtb infection. While clonally-expanded Vgamma2Vdelta2 T cells accumulated in lately-infected kidneys/livers without apparent TB lesions, TB burdens or lesions appeared to impact TCR repertoires and tissue trafficking patterns of activated Vgamma2Vdelta2 T cells

Vγ2Vδ2 T cells that accumulated in tissue compartments could mount effector function and produce anti-TB cytokine.

<p>Shown are ELISPOT data for IPP-driven IFNγ+ cellular response in lymphocytes from blood, lung and liver collected from four Mtb-infected macaques at 4–6 weeks after the infection. Data were subtracted from values of glucose/medium control and expressed here as IFNγ+ Vγ2Vδ2 T cells in 10∧6 lymphocytes. IPP stimulates activation of only Vγ2Vδ2 T cells but not other immune cells.</p

Broad T cell repertoire in Vγ2Vδ2 T-cell subpopulation in lymphoid system during primary Mtb infection of macaques.

<p>Shown are individual Vδ2 TCR clones isolated from PBL (left) and lymphocytes of spleen tissues (right) from 5 Mtb-infected macaques. The flow cytometry data indicating cellular expansion of Vγ2Vδ2 T cells in spleen were described in the text. Note that spleen lymphocytes in which major expansion of Vγ2Vδ2 T cells was seen were used for RNA isolation, cDNA synthesis and Vδ2 TCR sequence analyses. Note polyclonal sequences of Vδ2 TCR in cDNA derived from spleen lymphocytes and PBLs. Frequencies were expressed as the number of individual clones among the total analyzed clones. Similar data indicating polyclonal representation of Vγ2Vδ2 T cells in PBL before Mtb infection were also seen (data not shown). CDR3 were presumably indicated based on the definition for TCR β CDR3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030631#pone.0030631-Du1" target="_blank">[9]</a>. D indicates diversity region; N indicates non-determining region of TCR receptor genes. Clones marked by ‘♦’ were present in the blood, lung and kidney (2717). Clones marked by ‘’ and ‘•’ were present in the blood, lung and spleen(3055 and 2823). Clones marked by ‘▴’ and ‘▪’ were present in the blood and lung(2722).</p

Polyclonally-expanded Vγ2Vδ2 T cells from lymphoid tissues appeared to distribute and localize in lung TB granuloms after Mtb infection by aerosol.

<p>Shown are individual Vδ2 TCR clones isolated from lymphocytes of lung tissues from five Mtb-infected macaques. The immunohistochenistry data showing infiltration and distribution of Vγ2Vδ2 T cells in TB granulomas were shown in the previous publication <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030631#pone.0030631-Huang2" target="_blank">[18]</a>. Flow cytometry data indicating cellular expansion of Vγ2Vδ2 T cells in CD3+ T cells isolated from the lung tissues were described in the text. Note polyclonal Vδ2 TCR sequences and sub-dominant clones in cDNA derived from lung lymphocytes in which expansion of Vγ2Vδ2 T cells was detected. Clones present in blood and spleen were marked as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030631#pone-0030631-g001" target="_blank">Fig. 1</a>.</p

P values derived from statistical analyses of frequencies of dominant Vδ2 clonotypes between different tissues compartments (n = 5).

#<p>p value = 0.0041(**, very significant) when frequencies of dominant Vδ2 clonotypes in blood were compared with those in spleen(Blood vs Spleen). Individual dominant Vδ2 clonotypes were defined if they comprised >20% of the clones identified in a tissue compartment or blood from a macaque. Frequencies of dominant Vδ2 clonotypes among total TCR clones in a compartment from five macaques (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030631#pone-0030631-g001" target="_blank">Figs. 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030631#pone-0030631-g002" target="_blank">2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030631#pone-0030631-g003" target="_blank">3</a>) were calculated and analyzed for statistical significance between different tissue compartments using two-tailed Fisher exact test. We also statistically compared percentage numbers for total distinct Vδ2-bearing clones between different tissues compartments (n = 5), and found similar trends of results suggesting that Vδ2 repertoires in blood and lung were significantly broader than those in liver and kidney(data not shown).</p

Some Vδ2 T cell clones of Vγ2Vδ2 T-cell subpopulation appeared to be more predominant than others in lately Mtb-infected liver or kidney.

<p>The localization of Vγ2Vδ2 T cells in interstitial tissues of kidney or liver were shown in the previous publication <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030631#pone.0030631-Huang2" target="_blank">[18]</a>. Expansion of Vγ2Vδ2 T cells in CD3+ T cells isolated from the kidney or liver tissues were described in the text. Note that three macaques(2717, 3055, 2935) exhibited dominance of a single clone or oligo-clones bearing a same length of CDR3 in TCR cDNA derived from kidney lymphocytes in which expansion of Vγ2Vδ2 T cells was detected. In cDNA derived from liver lymphocytes, a dominance of a single TCR clone or clones with a restricted CDR3 length was also noted in three macaques (2717, 2823, 2935). Clones marked by ‘♦’were present in the blood,lung and kidney(2717).</p

Depletion of CD8 lymphocytes in CD8 Ab-treated macaques resulted in a significant decrease in BCG vaccine-induced immunity against tuberculosis after <i>M. tuberculosis</i> infection.

<p>(A) Top-panel photos show that the isotype IgG-treated BCG-vaccinated macaque (7406) exhibited limited numbers of granulomas (small arrows) in the cut-section of the right caudal lobe; the CD8 Ab-treated macaque (7348) showed extensive tuberculosis granulomas (large arrows) in the right caudal lobe, with a unilaterally enlarged hilar lymph node (a large green arrow). A naïve macaque (7419) showed large extensive tuberculosis lesions with caseation (large arrows), and much larger bilateral hilar lymph nodes (large green arrows). Middle- and bottom-panel photos show tuberculosis lesions in cut-sections of lungs of six monkeys in three respective groups. Extent and severity of the lesions could be adjudged based on the examples pointed by arrows at the top panel photos. Overall, CD8 Ab-treated macaques exhibited greater numbers of lung lobes displaying extensive coalescing granulomas than isotype IgG-treated control animals (p<0.05, by nonparametric <i>t</i> test). The CD8 Ab-treated macaques also showed more lobes with extensive caseating and miliary lesions or caseation pneumonia than the isotype IgG-treated controls (p<0.05, by nonparametric <i>t</i> test). Tuberculosis lesions in the CD8 Ab-treated macaques were more likely distributed or disseminated in other lobes or opposite lungs and hilar nodes/pleural. Small vertical/horizontal bars at bottom-left corner of each photo represent the 1-cm scale derived from the fluorescence rulers of each original photo. (B) Gross pathology scores of tuberculosis lesions in CD8 Ab-treated group, Isotype control IgG-treated group and naïve saline-treated group of macaques. Each macaque was given a total gross pathology scores based on the tuberculosis lesions in thoracic and extrathoracic organs. The mean gross pathology score was then calculated for each group of macaques and subjected to statistical analysis. *, P<0.05 (by ANOVA and nonparametric <i>t</i> test) for comparison between CD8 Ab-treated and isotype control IgG-treated groups, and for comparison between CD8 Ab-treated and naïve groups. (C) Histology evaluation of lung tissue sections of CD8 Ab-treated, isotype control IgG-treated and naïve saline-treated macaques. Shown are H&E stained sections taken from four representative macaques for each group, with macaque ID and magnification indicated for each image. Note that the isotype control IgG-treated macaques exhibited well-contained granulomas, which were generally infiltrated by numerous lymphocytes and some neutrophils. The CD8-depleted macaques displayed less-contained granulomas that were more likely to be necrotic (arrows). Naïve macaques showed less lymphocytic and more necrotic tuberculosis lesions than the other two groups.</p

Depletion of CD8 lymphocytes in macaques immunized by previous <i>M. tuberculosis</i> infection resulted in a loss of immune control of tuberculosis following re-infection.

<p>(A) Anti-CD8 Ab treatment of the <i>M. tuberculosis</i>-immunized macaques resulted in profound depletion of CD8 lymphocytes in <i>M. tuberculosis</i> re-infection. Shown are absolute levels of blood CD8 cells (/ul) and BAL fluid CD8 cells (×50,000). (B) Depletion of CD8 lymphocytes in the <i>M. tuberculosis</i>-immunized macaques led to higher levels of bacilli in BAL fluids and increased <i>M. tuberculosis</i> RNA in tissues following <i>M. tuberculosis</i> re-infection by aerosol. Numbers of bacilli are shown as CFU counts in 10 ml of BAL fluid; <i>M. tuberculosis</i> RNA was determined as Ag85B RNA copy numbers in 10 mg of tissue <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000392#ppat.1000392-Shen1" target="_blank">[6]</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000392#ppat.1000392-Huang1" target="_blank">[29]</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000392#ppat.1000392-Shen3" target="_blank">[36]</a>. (C) The CD8 Ab-treated macaques with CD8 depletion developed severe forms of tuberculosis after <i>M. tuberculosis</i> re-infection by aerosol. Macaque ID are indicated for CD8 Ab-treated (right) and isotype control IgG-treated (left) macaques. Shown are the lung surface images of two representative macaques (partially cut in the lung of macaque 2762). Note that the CD8 Ab-treated macaques exhibited pale-colored lungs with dissemination of >0.5 cm coalescing or caseating granulomas or tubercle nodules (the lesion-containing areas are pointed out by large arrows on surface view from 3050) and apparent caseation necrosis. The control macaques displayed no or few small non-caseating granulomas as indicated by small arrows in the lungs. When >0.5 cm coalescing or caseating tubercle nodules both lungs were scored using the scoring system as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000392#s4" target="_blank">Methods</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000392#ppat.1000392-Barclay1" target="_blank">[41]</a> for the individual macaques, the pathology scores (mean±SD = 72±24) of the CD8-depleted macaques were significantly worse than those (mean±SD = 6±2) of the control animals (p<0.01, by nonparametric <i>t</i> test). (D) Histology of representative granulomas seen in CD8 Ab-treated (right panel) and isotype control IgG-treated (left panel) macaques. Shown are H&E stained lung sections, with macaque ID and magnification indicated in each slide. Note that granulomas in isotype IgG-treated macaques were small, and highly lymphocytic without apparent necrosis. Granulomas in CD8 Ab-treated macaques were large and less lymphocytic, with necrosis seen in the center as pointed out by arrows.</p

CD8 Ab treatment of BCG-vaccinated macaques resulted in profound depletion of CD8 lymphocytes and mycobacterium-specific CD8 T effector cells during <i>M. tuberculosis</i> infection.

<p>(A) Flow cytometry data showed that anti-CD8 Ab-treated BCG-vaccinated macaques, but not isotype control IgG-treated BCG-vaccinated or naïve saline-treated macaques, exhibited marked decreases in percentages and absolute (data not shown) numbers of CD3+CD8+ and CD3−CD8+ lymphocytes in blood (upper left) and BAL fluid (upper right) during <i>M. tuberculosis</i> infection. Means for six animals are shown for each group, and error bars represent SEM. Arrows indicate the times for the treatment with anti-CD8 Ab or control antibodies. (B) Intracellular cytokine staining data showed that anti-CD8 Ab-treated BCG-vaccinated macaques exhibited an absence of PPD-specific IFNγ-producing CD8 T cells early after <i>M. tuberculosis</i> infection.</p

Depletion of CD8 lymphocytes resulted in a significant decrease in BCG-induced immune control of <i>M. tuberculosis</i>.

<p>(A) CD8 Ab-treated macaques exhibited higher levels of bacilli in BAL fluid than isotype IgG-treated macaques after pulmonary <i>M. tuberculosis</i> infection. Data are means +/− SEM of CFU counts/10 ml BAL fluid derived from 6 macaques for each group; * indicates p<0.05 for a comparison between the CD8 Ab-treated and isotype IgG-treated group. (B) CD8 Ab-treated macaques showed significantly higher levels of bacilli organisms in lung tissues than isotype control macaques after pulmonary <i>M. tuberculosis</i> infection. Data are mean values with SEM error bars of CFU counts/1 ml lung tissue homogenates derived from 6 macaques for each group. Rt, right; Lt, left. Note that right caudal lobe was the <i>M. tuberculosis</i> infection site for each macaque; *, p<0.05 (by both ANOVA and nonparametric <i>t</i> test) for a comparison between the CD8 Ab-treated and isotype control IgG-treated groups.</p