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

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

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

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

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

Tetramer was able to specifically stain P65-specific CD4 T cells in <i>M. tuberculosis</i>-infected <i>Mamu-DRB*W201⁺</i> macaques.

<p>(a) PBL collected at day 42 from the <i>M. tuberculosis</i>-infected <i>Mamu-DRB*W201⁺</i> macaque was stimulated with P65 for different days in CFSE-incorporated culture; the proliferating cells were assessed for the ability to be stained by Mamu-DR*W201/P65 tetramer. The upper panel shows CD3-gated flow cytometry histograms indicating proliferating and non-proliferating CD4 T cells as determined by CFSE dilution, with the numbers illustrated as percentages of P65-expanded CD4 T cells. The middle panel histograms indicate that majority of P65-proliferating CD4 cells as gated on CD4 and CFSE could be stained by Mamu-DR*W201/P65 tetramer. The lower panel shows CD3-gated flow histograms indicating the percentages of the tetramer-bound CD4⁺ T cells in the cultures stimulated with for 7 and 12 days, respectively. The PBL not stimulated with P65 stimulation was denoted as day 0. (b) The CD3-gated flow cytometric data show that Mamu-DR*W201/P65 tetramer specifically stains P65-proliferating CD4 T cells from the <i>M. tuberculosis</i>-infected <i>Mamu-DRB*W201⁺</i> macaque but not the naïve macaque or the <i>M. tuberculosis</i>-infected <i>Mamu-DRB*W201</i>^- animal (lower panel).</p

Tetramer-based enrichment conferred high-fidelity to measure P65-specific CD4 cells in comparisons of standard tetramer staining.

<p>(a) Left bar graph shows the frequencies of Mamu-DR*W201/P65 tetramer-bound epitope-specific CD4 T cells detected by standard tetramer staining in the different samples after <i>M. tuberculosis</i> infection. Standard tetramer staining detected significant increases in percentages of the P65-specific CD4 T cells in the blood, lymph nodes, spleens, and lungs compared to base line levels in blood or to those of the infected <i>Mamu-DRB*W201</i>^- macaques at day 63 after the infection (**, p<0.01), but no significance at days 28 and 42 post-infection. Right bar graph shows that ICS does not detect significantly-increased numbers of P65-specific IFNγ-producing CD4 T cells after <i>M. tuberculosis</i> infection. (b) The bar graph shows that the tetramer staining after P65 stimulation detects about 10-fold greater numbers of DR*W201 tetramer-bound CD4 T cells in PBL or tissue lymphocytes compared to standard tetramer staining without P65 stimulation (**,p<0.01; *, p<0.05). (c) Flow cytometry histograms show that the tetramer-based enrichment approach confers the enhanced ability to enumerate P65-specific CD4 T cells and to distinguish from background staining in 1×10⁷ PBL or tissue lymphocytes from individual <i>M. tuberculosis</i>-infected monkeys. Tetramer-unbound cells washing out from the microbead column were shown in CD4 versus CD3 or CD45 events in the flow cytometry analysis. The bead-enriched tetramer-bound cells were counted and displayed in the contour plot in the upper right CD4 quadruple, with the total numbers shown in the upper left CD4 quadruple. (d) Bar graph shows absolute numbers of Mamu-DR*W201/P65 tetramer-bound epitope-specific CD4 T cells in 10⁷ total cells detected by the tetramer-based enrichment approach in different samples. This enriched approach readily detects significant increases in numbers of the tetramer-bound CD4 T cells in blood at days 28 and 42 post-infection (P = 0.016, P = 0.008 as indicated) thanks to the much lower nonspecific staining for control samples. This is in sharp contrast to the standard tetramer staining that fails to reveal significant increases in the tetramer-bound cells at these time points due to the relatively-high background staining (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006905#pone-0006905-g004" target="_blank">Fig. 4a</a>). Also, at day 63 after the infection, the tetramer-based enrichment approach can more dramatically distinguish the tetramer-bound cells from control nonspecific cells (P values 0.0004–0.0005) than the standard tetramer staining (P values 0.008–0.005).</p

This tetramer-enriched approach detected BCG-elicited resting memory P65-specific CD4 T cells at a frequency of 2-3/10,000 in PBL.

<p>(a) Left bar graph shows that the frequency of ≤0.03% tetramer-bound CD4⁺ T-cells detected by tetramer direct staining in the BCG-vaccinated <i>Mamu-DRB*W201⁺</i> macaques was difficult to distinguish from background staining (p>0.05). The bar graph on right shows that intracellular IFN-γ staining could not detect the epitope-specific IFN-γ-producing memory CD4 T cells. (b) The flow cytometry histograms on left show the total numbers of tetramer-bound epitope specific CD4 T cells detected by the tetramer-based enrichment approach in 10⁷ PBL from each macaque. The enriched tetramer⁺ CD4⁺ T-cell population is counted by flow cytometry and displayed in the contour plot in the upper right of the CD4 quadruple, with the total numbers shown in the upper left CD4 quadruple. The bar graph on right shows that this tetramer enriched approach can detect significantly greater numbers of P65-specific CD4 T cells in BCG-vaccinated <i>Mamu-DRB*W201⁺</i> macaques than those in the vaccinated <i>Mamu-DRB*W201^-</i> animals. (c) The bar graph shows that the tetramer staining after P65 stimulation detects about ten-fold greater numbers of the BCG-elicited tetramer-bound CD4 T memory cells than without stimulation. The numbers of tetramer-bound cells in PBL from <i>Mamu-DRB*W201⁺</i> macaques were significantly greater than those in naïve or BCG-vaccinated <i>Mamu-DRB*W201^-</i> animals (**, p<0.01).</p

Ag85B peptide 65 induced apparent proliferation in PBL from BCG-vaccinated <i>Mamu-DRB*W201⁺</i> macaques.

<p>(a) Sixty nine peptides spanning entire Ag85B protein were divided as 10 groups to examine PBL proliferation in the BCG-vaccinated macaques. The proliferation index data indicate that Ag85B Group 10 peptide pool comprised 6 overlapping peptides induced significant PBL proliferation than other peptide groups (*, p<0.05). Data were mean values derived from PBL of four BCG-vaccinated macaques, with error bars indicating standard errors of means (SEM). (b) PBL from four BCG-vaccinated macaques were further tested for their proliferation to individual peptides in the Group 10 peptide pool, and the proliferation index data reveal that PBL had stronger proliferation to the peptide #65 (P65) than other peptides (**,p<0.01; *, p<0.05). P65 bears the sequence of PNGTHSWEYWGAQLN that corresponds to 258∼272 amino acid of Ag85B. (c) Nest-PCR were used to amplify full-length <i>Mamu-DRB</i> cDNA (left gel) and, subsequently, the exon 2 (right gel) of β1 domain in <i>DRB*W201</i>. As illustrated, each lane represents a sample from one animal; ∼250 bp DNA fragments from the lanes 3 and 5 were excised for direct sequencing. The <i>Mamu-DRB*W201⁺</i> allele was determined by sequencing alignments through Blast research of GeneBank data base. Representative DNA sequence shows that the nucleotide sequences from a rhesus macaques were identical to the <i>Mamu-DRB*W201</i> prototype sequence except for one base substitution (T→A).</p

Production and characterization of soluble Mamu-DR αβ monomer and Mamu-DR*W201/P65 tetramer.

<p>(a) Schematic presentation (cartoon) of the covalent peptide approach for developing class II MHC/peptide complex constructs. The epitope-coding sequence is linked to the 5′ <i>Mamu-DRβ</i> cDNA; <i>Jun</i> and Fos-BSP are introduced by linking the extracellular domains of DR-α and -β chain, respectively. The recombinant Mamu-DR αβ monomer is stabilized by leucine zipper (LZ) formed through Jun-Fos interaction. (b) Protein samples after 1^st round (lane 1) and second round (lane 2) purifications were separated in the SDS-PAGE gel in reduction conditions and stained. Lane 1, the (His)₆ tagged recombinant protein purified from Ni-NTA agarose; lane 2, the biotinylated Mamu-DR recombinants purified further through an avidin column after biotinylation. The arrow indicates two closely-separated protein bands in lane 2 (∼34, 36 kD) that correspond to predicted molecular weights of Mamu-DR α and β recombinants, respectively. (c) Dot blot assay indicates that anti-HLA-DR antibody (L243) bound to the soluble recombinant Mamu-DR αβ monomer purified by Ni-NTA affinity column (loading 1) and further by an avidin column (loading 2), but not to the denatured Mamu-DR αβ sample (loadings 3). The supernatant of non-transfected S2 cells served as a negative control (loading 4). (d) FPLC graph shows that the unbound Mamu-DR αβ molecules and free fluorescents were washed out, and the assembled Mamu-DR*W201-P65 tetramer was collected and marked as tetramer.</p