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

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

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

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

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

Intermittent Picostim/IL-2 administration during early pulmonary Mtb infection induced major expansion of Vγ2Vδ2 T cells and accumulation of them in pulmonary compartment, and led to reduction in Mtb bacterial burdens in lungs.

<p>(a) Representative flow histograms of percentages of Vγ2Vδ2 T cells gated on CD3(left) at day 1 postinfection (day 4 after 1^st treatment) and graph data (right) of absolute numbers of Vγ2Vδ2 T cells over time after treatments, respectively, in the blood. Percentages of Vγ2Vδ2 T cells in CD3 T cells are indicated in right upper quadruples. Graph data are means with SEM from nine macaques/per group. Vertical arrows denote the times for treatment. **p<0.01, *p<0.05, by both parametric and nonparametric analyses. (b) BAL fluid (BALF) data in a similar fashion as (a), except that flow data are on day 8 post infection(day 11 after treatments) and that numbers in graphs (right) are percentages of Vγ2Vδ2 T cells(n = 9 for each group). Increases in Vγ2Vδ2 T cells in BALF consist with accumulation of them in lung tissues of phosphoantigen/IL-2-treated macaques (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003501#ppat.1003501-Huang1" target="_blank">[14]</a> and data not shown). The data suggest changes in Vγ2Vδ2 T cells in the airway of the pulmonary compartment, not precisely in lung tissues. Please see Fig. S3c in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003501#ppat.1003501.s001" target="_blank">Text S1</a> for immunohistocheminstry <i>in situ</i> analysis of γδ T cells in lung parenchyma and granulomatous tissues). (c) Mean CFU counts of bacilli in lung tissue homogenates of different lung lobes collected at the end point (at day 65) from Picostim/IL-2-treated and control groups. Lung tissue homogenates generated from each lobe was serially diluted and used for CFU enumeration(CFU/ml homogenates). **p<0.01, *p<0.05, respectively, by parametric and nonparametric comparisons between Picostim/IL-2-treated and control groups(n = 9/group). Data from control groups <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003501#ppat.1003501-Chen3" target="_blank">[22]</a> were shown here again to prove the concept that phosphoantigen/IL2 expansion of Vγ2Vδ2 T cells can more apparently limit Mtb growth compared to saline/BSA and IL2 alone controls as all groups were evaluated simultaneously.</p

Vγ2Vδ2 T effector cells and macaque granulysin/perforin restricted Mtb growth.

<p>(a). Shown on left are real time quantitative PCR data for mRNA expression levels of perforin and granulysin expressed by macaque Vγ2Vδ2 T effector cells capable of restricting intracellular Mtb growth. The quantitation was done using the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003501#s4" target="_blank">methods</a> as we previously described <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003501#ppat.1003501-Du1" target="_blank">[40]</a>. Data are means with SEM from phosphoantigen-activated Vγ2Vδ2 T effector cells from 3 Mtb-infected macaques in 2 independent experiments. Control B cells were purified using CD20 mAb and immunemagnetic beads <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003501#ppat.1003501-Zeng1" target="_blank">[29]</a>. Right panel shows that co-culturing of Mtb-infected monocytes with Vγ2Vδ2 T effector cells expressing perforin/granulysin led to reduction in CFU bacterial counts. (b). Graph curves show that recombinant granulysin(Gnl), but not perforin(PC), killed extracellular Mtb (left); Gnl+PC inhibited intracellular Mtb growth(right). Data are the killing or inhibiting percentage of extracellular or intracellular Mtb CFUs relative to Mtb CFUs in medium-only control(see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003501#s4" target="_blank">Methods</a>). Data are means derived from Mtb-infected monocytes from three macaques in 3 experiments. ** p<0.001; ***p<0.0001; ****p<0.00001, by student t test.</p

Picostim/IL2-treated group showed immune resistance to TB lesions in lungs compared to control groups.

<p>(a). Graph data of mean gross pathology scores for Picostim/IL-2-treated and control groups. The scores were calculated and compared as we previously described <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003501#ppat.1003501-Chen2" target="_blank">[21]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003501#ppat.1003501-Wei2" target="_blank">[43]</a>. n = 9/group. Control groups <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003501#ppat.1003501-Chen3" target="_blank">[22]</a> were included here to prove the concept that phosphoantigen/IL2 expansion of Vγ2Vδ2 T cells can lead to increased resistance to TB. (b). Representative histopathology photos for Picostim/IL-2-treated and control groups. Also see Fig. S2 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003501#ppat.1003501.s001" target="_blank">Text S1</a> in the supplementary materials for histopathology from other macaques of three groups. Overall, saline/BSA-treated control macaques (represented in upper panel) exhibited widespread necrosis, fulminant TB pneumonia, and TB hemorrhage/thrombosis lesions. The alveolar septa are destroyed or indistinct. Most TB granulomas from IL2-treated macaques tended to be a lack of widespread necrosis, TB pneumonia, or TB hemorrhage in the sections from the right caudal lung lobe (infection site) and other lobes. In the Picostim/IL-2-treated group (lower panel), three macaques (ID: 8022,8014,8013) exhibited no detectable TB granulomas, four macaques predominantly showed well-contained TB granulomas with lymphocyte cuff, without widespread necrosis. Two of them exhibited histology similar to what were seen in IL-2-treated macaques. Original magnification ×100 for all photos. See Fig. S2 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003501#ppat.1003501.s001" target="_blank">Text S1</a> for similar contrasting data in other controls and IL-2–treated macaques.</p

Picostim/IL2 treatment, while expanding Vγ2Vδ2 T cells, could confer immune resistance to TB lesions in lungs after pulmonary Mtb infection.

<p>Shown are digital photos of cut sections of lung lobes from 12 representatives of 27 macaques (9 for each group). Lungs and other organs were obtained in complete necropsy at day 65 after Mtb infection. Macaque ID numbers are shown in upper left corners, with the right caudal(RC) lobe (infection site) indicated in each photo. Extent and severity of the lesions could be adjudged based on the examples pointed by large arrows for caseation pneumonia or extensive coalescing granulomas and by small arrows for less coalescing or small granulomas. Yellow arrows indicate the enlarged hilar lymph nodes with caseation. Note that four Picostim/IL-2–treated macaques (ID 8022, 8014, 8013, 8006: first two animals shown in this figure and last two shown in Fig. S1 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003501#ppat.1003501.s001" target="_blank">Text S1</a>) did not show any detectable gross TB lesions. Other five Picostim/IL-2–treated macaques displayed more focal, less coalescing or less caseating TB granulomas than control groups. (see Fig. S1 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003501#ppat.1003501.s001" target="_blank">Text S1</a>). Vertical/horizontal bars at upper right corner of each photo represent the 1-cm scale derived from the fluorescence rulers of original photos. >50% of saline/BSA and 10% of IL-2–treated controls had extrathoracic TB dissemination, which was not seen in Picostim/IL-2-treated macaques. The efficacy evaluation was done sequentially in three separate trials each involving Picostim/IL-2 group and control groups of a total of 27 macaques.</p