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TRAV1-2+ CD8+ T-cells including oligoconal expansions of MAIT cells are enriched in the airways in human tuberculosis
Emily B. Wong ORCID: orcid.org/0000-0003-4755-53801,2,3,4 na1,
Marielle C. Gold ORCID: orcid.org/0000-0003-2505-32915,6,7 na1,
Erin W. Meermeier ORCID: orcid.org/0000-0002-9709-76245,
Bongiwe Z. Xulu1,
Sharon Khuzwayo ORCID: orcid.org/0000-0003-4482-59551,
Zuri A. Sullivan ORCID: orcid.org/0000-0002-6111-65121,
Eisa Mahyari8,
Zoe Rogers1,
Hénrik Kløverpris1,4,9,
Prabhat K. Sharma6,
Aneta H. Worley6,
Umesh Lalloo10,
Prinita Baijnath10,11,
Anish Ambaram11,
Leon Naidoo11,
Moosa Suleman11,12,
Rajhmun Madansein13,14,
James E. McLaren ORCID: orcid.org/0000-0002-7021-593415,
Kristin Ladell ORCID: orcid.org/0000-0002-9856-293815,
Kelly L. Miners15,
David A. Price ORCID: orcid.org/0000-0001-9416-273715,16,
Samuel M. Behar ORCID: orcid.org/0000-0002-3374-669917,
Morten Nielsen ORCID: orcid.org/0000-0001-7885-431118,19,
Victoria O. Kasprowicz1,20,21,
Alasdair Leslie1,4,
William R. Bishai22,
Thumbi Ndung'u1,19,20,21,23 &
David M. Lewinsohn ORCID: orcid.org/0000-0001-9906-94945,6,7
Communications Biologyvolume 2, Article number: 203 (2019) | Download Citation
T-cell receptor
Mucosal-associated invariant T (MAIT) cells typically express a TRAV1-2+ semi-invariant TCRα that enables recognition of bacterial, mycobacterial, and fungal riboflavin metabolites presented by MR1. MAIT cells are associated with immune control of bacterial and mycobacterial infections in murine models. Here, we report that a population of pro-inflammatory TRAV1-2+ CD8+ T cells are present in the airways and lungs of healthy individuals and are enriched in bronchoalveolar fluid of patients with active pulmonary tuberculosis (TB). High-throughput T cell receptor analysis reveals oligoclonal expansions of canonical and donor-unique TRAV1-2+ MAIT-consistent TCRα sequences within this population. Some of these cells demonstrate MR1-restricted mycobacterial reactivity and phenotypes suggestive of MAIT cell identity. These findings demonstrate enrichment of TRAV1-2+ CD8+ T cells with MAIT or MAIT-like features in the airways during active TB and suggest a role for these cells in the human pulmonary immune response to Mycobacterium tuberculosis.
Mucosal-associated invariant T (MAIT) cells are unconventional lymphocytes that use semi-invariant T cell receptor-alpha (TCRα) chains to recognize non-peptide small molecule ligands presented by the HLA-Ib molecule MR11,2,3,4,5,6. In mice, MAIT cells have been shown to play a protective role in models of respiratory infection7,8,9,10. In humans, MAIT cells are abundant in the peripheral blood of healthy individuals, where they produce cytolytic enzymes and pro-inflammatory cytokines and typically express a TRAV1-2+ TCRα chain and the CD8 coreceptor1,2,3,4,11,12,13. MAIT cells are depleted in the blood of humans with TB4,5,14. However, little is known about the function and phenotype of MAIT cells in the human lung, especially in the setting of pulmonary tuberculosis (TB). We postulated that MAIT cells are recruited to and/or expand at sites where Mycobacterium tuberculosis (Mtb) antigens are present, potentially acting as sentinels of infection in the respiratory mucosa.
Here we report that a population of pro-inflammatory TRAV1-2+ CD8+ T cells are present in the airways and lungs of healthy individuals and are enriched in bronchoalveolar fluid of patients with active pulmonary TB. Some of these cells demonstrate MR1-restricted mycobacterial reactivity, phenotypic features and/or TCRα chain usage suggestive of MAIT cell identity. We conclude that TRAV1-2+ CD8+ T cells with MAIT or MAIT-like features are oligoclonally expanded in the airways during active TB, suggesting that they play a role in the human pulmonary immune response to Mycobacterium tuberculosis.
TRAV1-2+ CD8+ T-cells in human lung and intestine tissues
To explore the role of MAIT cells in healthy mucosal tissues, we first determined the frequency of TRAV1-2+ CD8+ cells in the respiratory tract of an individual organ donor (Fig. 1a). Dramatic enrichment was observed in the trachea, where nearly half of all CD8+ T cells expressed TRAV1-2 (Fig. 1a). TRAV1-2+ cells were also enriched in the proximal and distal bronchi (35 and 22% of CD8+ T cells, respectively) and in the lung parenchyma (17% of CD8+ T cells), relative to the draining mediastinal lymph node where the frequency (6% of CD8+ T cells) approximated levels typically found in peripheral blood11,15. To determine the anatomical localization of TRAV1-2+ CD8+ cells in the airway, we used immunohistochemistry to quantify CD8+ and TRAV1-2+ cells in 1st and 2nd order bronchial sections from three additional organ donors (Fig. 1b, left). Although the number of CD8+ cells was similar in tissue sections from the proximal and distal airways, TRAV1-2+ cells were more frequent in the proximal compared to distal airway (Fig. 1b, right). As expression of TRAV1-2+ TCRs is insufficient to define MAIT cells, we also performed ex vivo functional assays in which cytokine-production by TRAV1-2+ CD8+ cells upon exposure to HLA mismatched M. smegmatis-infected antigen-presenting cells is used to define mycobacterial-reactive MAIT cells4,12,16,17. In a single donor for whom paired tissues were available, we evaluated lymphocytes from lung parenchyma, the small intestinal lamina propria (LP), and the small intestinal intraepithelial lymphocytes (IEL) for M. smegmatis-dependent release of the pro-inflammatory cytokine TNF. Interestingly, TNF-producing TRAV1-2+ cells were found only in the lung (Fig. 1c, left). It is also notable that CD161, a C-type lectin highly expressed on peripheral MAIT cells11,13,17, was not detected on TRAV1-2+ CD8+ T cells from the lung but was found in abundance on small intestinal TRAV1-2+ CD8+ T cells (Fig. 1c, right). We next compared the frequencies of TRAV1-2+ and TNF-producing cells in the lung (n = 9) and intestinal mucosa (n = 8, unmatched samples) where MAIT cells were initially found to be enriched3. The frequencies of TRAV1-2+ CD8+ T cells were similar across mucosal sites, associated lymphoid tissues and unmatched peripheral blood samples (n = 6) (Fig. 1d). In contrast, significantly higher frequencies of TRAV1-2+ cells from the lung produced TNF in response to M. smegmatis-infected cells compared with TRAV1-2+ cells from lymphoid tissues, small intestine, or peripheral blood (P = 0.035, 0.0025, 0.0023 and 0.0005 (Mann–Whitney U test), Fig. 1e). Cell yields from these tissues were insufficient to establish functional dependence on MR1 as has been shown previously with this assay4. Nonetheless, these data demonstrate that mycobacterial stimulation results in TNF production by donor-unrestricted, lung resident TRAV1-2+ CD8+ T cells.
TRAV1-2+ CD8+ T cells from the lung but not the intestine of healthy organ donors respond to mycobacterial infection by producing TNF. a Dot plots showing the frequency of TRAV1-2+ CD8+ T cells among live CD3+ cells in the indicated tissue samples from one donor. b Tissue sections from the 1st and 2nd order bronchi were obtained from healthy individuals (n = 3 biologically independent samples). Immunohistochemistry was performed to quantify CD8+ (median 1.6 × 104 vs. 2 × 104 cells /mm3) and TRAV1-2+ cells (7,000 vs. 4,000 cells/mm3, Supplementary Data). Representative sections from 1st and 2nd order bronchi are depicted (left), showing CD8+ cells (red), TRAV1-2+ cells (green) and cell nuclei (DAPI; blue). c Histograms depicting TNF production (left) and CD161 expression (right) by TRAV1-2+ CD8+ T cells from matched lung parenchyma (green), small intestine lamina propria (LP; blue) and the small intestinal intraepithelial layer (IEL; violet) after overnight stimulation with M. smegmatis-infected antigen-presenting cells (dotted black line indicates the unstimulated control). d Frequency of TRAV1-2+ cells among CD8+ T cells from lung (n = 9 biologically independent samples), mediastinal lymph node (Med LN; n = 11 biologically independent samples), IEL (n = 7 biologically independent samples), LP (n = 8 biologically independent samples), mesenteric lymph node (Mes LN, n = 5 biologically independent samples), and peripheral blood (PBMC; n = 6 biologically independent samples, Supplementary Data). Medians and interquartile ranges are displayed. e Frequency of TNF-producing TRAV1-2+ CD8+ T cells after exposure to M. smegmatis-infected antigen-presenting cells: lung (n = 7 biologically independent samples), Med LN (n = 6 biologically independent samples), IEL (n = 5 biologically independent samples), LP (n = 6 biologically independent samples), Mes LN (n = 2 biologically independent samples), PBMC (n = 12 biologically independent samples, Supplementary Data). From top to bottom, P = 0.035, 0.0025, 0.0023 and 0.0005 (Mann–Whitney U test). Medians and interquartile ranges are displayed
TRAV1-2+ CDR3α usage in Mtb-infected lung tissue
On the basis of these results, we hypothesized that pulmonary infection with Mtb leads to the migration to and/or expansion of TRAV1-2+ CD8+ cells in the lung, potentially driven by Mtb-derived MR1 ligands. A hallmark of the human immune response to Mtb is the formation of lung granulomas. We therefore sought to determine the relevance of TRAV1-2+ T cell receptor (TCR) usage in lung granulomas from patients with TB. Single cell suspensions were prepared from diseased lung parenchyma from individuals (n = 5) undergoing clinically indicated surgical resection for complications of TB18. The most highly diseased lung granuloma (LG) tissues were designated "A" and the least diseased tissues designated "C." CD4- T cells from these samples were sorted by flow cytometry and subjected to high-throughput repertoire analysis using the bias-controlled immunoSEQ TCR sequencing platform19. In the 12 samples that yielded the minimal necessary sequencing data for analysis (>104 productive reads, yielding a median of 3,919 unique productive TCRα reads (range 397–28,792) and a median of 167 TRAV1-2-utilizing unique productive TCRα reads (range 19–1081), the overall frequency of TRAV1-2+ TCR sequences in granulomas ranged from 3.1 to 5.9% across all donors and tissue samples (Fig. 2a and Supplementary Table 1). These frequencies are similar to those observed in peripheral blood and lymph nodes. We then developed an algorithm based on published MAIT CDR3α amino acid (aa) sequences16,20 to determine which of these TRAV1-2+ CDR3α sequences represented MAIT cell-consistent TCRαs. A CDR3α sequence similarity analysis was performed using "MAIT Match" (http://www.cbs.dtu.dk/services/MAIT_Match), a tool based on the method described by Shen et al.21, where a score of 1 reflects a perfect match and a score of 0 a perfect mismatch with published MAIT cell CDR3α sequences. To determine the validity of this tool, we compared the proportion of TRAV1-2+ sequences with the proportion of TRAV12-2+ sequences (an unrelated control) for TCRs with scores ranging from 0.85 to 1. MAIT Match scores of 0.95 to 1 were significantly increased among the in TRAV1-2+ but not TRAV12-2+ TCR sequences (P = 0.0035, P = 0.00046, t test; Fig. 2b). We therefore chose a MAIT Match score of 0.95 as a conservative threshold to define MAIT cell-consistent TCRs (Fig. 2b). In one individual with paired samples from the lung and mediastinal lymph node (LN), TRAV1-2 usage was comparable at both sites, but similarity analysis revealed MAIT cell-consistent TCR enrichment in the lung (P < 0.0001; 2-way ANOVA; Fig. 2c).
Expansions of MAIT cell-consistent CDR3α's are present in tuberculous lung granulomas. a Frequency of TRAV1-2+ sequences as a percentage of all productive TCRα sequences. In some cases, multiple areas of tissue were sampled, ranging from closest (A) to furthest (C) from the site of disease. b MAIT cell TCRα sequences are consistent with similarity scores of 0.95 and 1. Each symbol represents the frequency of TRAV1-2+ or TRAV12-2+ sequences within each similarity score for each donor sample (n = 12 biologically independent samples). c Frequency of total TRAV1-2+ sequences or those with similarity scores of 0.95 and 1 in the lung (n = 3 biologically independent samples) and mediastinal lymph node (LN, 1 sample) from donor 23. Height represents mean, error bars represent standard error. d Frequencies among TRAV1-2+ sequences of the top 10 public and private MAIT cell CDR3α sequences (MAIT Match score ≥0.95) across individual donors and lung samples. e Variation in the number of synonymous nucleotide sequences encoding the five most frequent private (left) and public (right) MAIT cell CDR3α amino acid (aa) sequences from all samples displayed in Fig. 3d. For each aa sequence, each colored bar represents a different nucleotide sequence. The 13 different nucleotide sequences used to generate the shared MAIT cell CDR3α aa sequence CAVLDSNYQLIW are displayed. Text color represents nucleotide origin: purple (TRAV), black (TRAD or n insertion), red (TRAJ). LG lung granuloma, LN lymph node. Source data are provided in Supplementary Data
To address the possibility that Mtb drives the recruitment and/or expansion of TRAV1-2+ T cells with MAIT-consistent CDR3α's in granulomatous tissue, we analyzed the MAIT cell-consistent CDR3α sequences (MAIT Match score 0.95-1) found in diseased lung parenchyma (n = 5 individuals, 11 samples). It is established that certain MAIT cell TCRα chains can be shared among individuals (public sequences)22, while donor-unique (private) CDR3α sequences can be selected in response to distinct microbes16. As shown in Fig. 2d, both private and public CDR3α sequences were detected among the MAIT cell-consistent CDR3α sequences present in granulomatous lung tissue isolated from patients with TB. Notably, public MAIT cell-consistent CDR3α were frequently encoded by multiple synonymous nucleotide sequences within individuals suggesting the expansion of multiple clones with the same CDR3α amino acid sequences (Fig. 2e, right). In contrast, private MAIT cell-consistent CDR3α sequences were encoded by individual nucleotide sequences suggesting that these were the result of expansions of a single MAIT cell clone in each donor (Fig. 2e, left). Private CDR3α sequences were not restricted to infrequent clonotypes and in some tissue samples occurred as the dominant MAIT cell-consistent TCR.
Bronchoalveolar TRAV1-2+ CD8+ T cells in active pulmonary TB
Diminished frequencies of circulating MAIT cells have consistently been observed in people with TB4,5. This apparent peripheral depletion may occur as a consequence of selective MAIT cell migration to the lung or may reflect increased host vulnerability to infection with Mtb. Having found that TRAV1-2+ CD8+ cells are enriched in healthy airways and respond to mycobacteria (Fig. 1e)4, we hypothesized that pulmonary infection with Mtb drives the accumulation and expansion of TRAV1-2+ CD8+ cells in the lung in response to Mtb-derived MR1 ligands. To address this possibility, we measured the frequency of TRAV1-2+ CD8+ T cells in bronchoalveolar (BAL) fluid samples obtained from individuals with untreated, active pulmonary TB and controls with no evidence of infectious or inflammatory pulmonary disease (Supplementary Table 2). In BAL fluid, TRAV1-2+ CD8+ T cells were significantly enriched in patients with TB at frequencies approximately 3-fold higher than controls (P = 0.0022, Mann–Whitney U test, Fig. 3a). Conversely, in matched peripheral blood samples, TRAV1-2+ CD8+ T cells were significantly diminished in patients with TB at frequencies approximately 2-fold lower compared to healthy controls (P = 0.0028, Mann–Whitney U test, Fig. 3a). To assess the functional capacity of TRAV1-2+ CD8+ T cells in the BAL fluid and matched peripheral blood samples, we utilized α-CD2/CD3/CD28 beads as a stimulant to trigger responses via the TCR. Cell yields were insufficient to explore ligand-specific activation, which may also be subject to bias arising from compartment-specific differences in MR1-expression by antigen-presenting cells23. MAIT cells have been reported to produce IFN-γ, TNF, granzymes, granulysin, IL-17 and IL-2224,25,26. Among these, we chose to measure TNF, a representative Th1 effector cytokine essential for immune control of Mtb27 and IL-17, an immunomodulatory cytokine reportedly produced in a TCR-independent manner by MAIT cells28. A significantly greater proportion of TRAV1-2+ CD8+ T cells in BAL fluid produced TNF (median 40%, range 36–91%) compared with TRAV1-2+ CD8+ T cells in matched peripheral blood samples (median 15%, range 4.7–27%) (P = 0.004, Mann–Whitney U test, Fig. 3b, c and Supplementary Fig. 1). In contrast fewer than 1% of TRAV1-2+ CD8+ T cells in the BAL fluid and only 2% in matched peripheral blood samples produced IL-17 (Supplementary Fig. 2). We therefore concluded that TCR triggering of these BAL-resident TRAV1-2+ CD8+ T cells does not evoke IL-17 production, though other mitogenic or cytokine-associated stimulations may do so. Next, we characterized the phenotype of BAL-resident TRAV1-2+ CD8+ T cells. MAIT cells can be defined in peripheral blood by TRAV1-2 usage in conjunction with high-level expression of the c-type lectin CD161, and the di-peptidase CD2613,26. In BAL fluid obtained from patients with TB, TRAV1-2+ CD8+ T cells expressed low levels of CD161 compared with peripheral blood TRAV1-2+ CD8+ T cells (Fig. 3d), which is consistent with the data from healthy lung tissue (Fig. 1c) and the prior demonstration that CD161 can be down-regulated as a result of MAIT cell activation17,28,29. In contrast, TRAV1-2+ CD8+ T cells in the BAL fluid more consistently expressed CD26, which is abundantly present on all functional MR1-restricted MAIT cells in peripheral blood17,24. CD103, the αE integrin associated with tissue-resident memory T cells30 was expressed variably but exclusively on BAL TRAV1-2+ CD8+ T cells.
TNF-producing TRAV1-2+ CD8+ cells including oligoclonally expanded MAIT cells are enriched in bronchoalveolar lavage fluid from patients with TB. a Frequency of TRAV1-2+ cells among CD8+ T cells from the bronchoalveolar lavage (BAL) fluid from patients with TB (n = 6 biologically independent samples) and cancer controls (n = 6 biologically independent samples), and among CD8+ T cells in matched peripheral blood samples (PBMC) from patients with TB (n = 5 biologically independent samples) and unmatched peripheral blood samples from healthy controls (n = 13 biologically independent samples). Medians and interquartile ranges are displayed. **P < 0.01; Mann–Whitney U test. b Dot plots showing TNF production by TRAV1-2+ CD8+ T cells in matched BAL and peripheral blood samples (PBMC) from a patient with TB. Cells were stimulated with α−CD2/CD3/CD28 beads. c Frequency of TNF or IL-17 production by TRAV1-2+ CD8+ T cells in matched BAL and peripheral blood samples (PBMC; n = 5 biologically independent samples). Medians and interquartile ranges are displayed. **P < 0.01; Mann–Whitney U test. d Expression of CD161, CD26 and CD103 on TRAV1-2+ CD8+ T cells in matched BAL and peripheral blood samples (PBMC) from patients with TB (n = 4 biologically independent samples). Histograms are mode-normalized. e Frequency of MAIT cell-consistent CDR3α sequences within TRAV1-2+ CD4- T cells in BAL fluid and peripheral blood samples (PBMC) from patients with TB (n = 3 biologically independent samples). Height represents the mean, error bars represent the range. f Relative frequency of CDR3α sequences by MAIT Match Score category in BAL fluid vs. matched peripheral blood (PBMC; n = 3 biologically independent samples). g Depiction of the top 10 most frequent MAIT cell-consistent CDR3α sequences (MAIT Match score ≥0.95) among TRAV1-2+ sequences in each compartment. Legend format: CDR3α aa (# of synonymous nucleotide sequences in peripheral blood, # of synonymous nucleotide sequences in BAL fluid). h IFNγ spot-forming units (SFU) produced by BAL T cell clones stimulated with M. smegmatis-infected or Mtb-infected wildtype (WT) or MR1-KO A549 cells (n=8 biologically independent clones). Height represents the mean of two independent replicates per stimulation, error bars represent the standard deviation. Source data are available in Supplementary Data.
Although TRAV1-2 usage is a defining feature of MAIT cells, the same gene segment can be expressed by T cells recognizing mycobacterial ligands presented in the context of HLA-Ia molecules and CD1b31. On the basis that TRAV1-2+ CD8+ T cells display a surface phenotype suggestive of tissue-resident MAIT cells in BAL fluid isolated from patients with active TB, we postulated that the corresponding CDR3α sequences would provide a molecular signature reflecting MAIT cell enrichment relative to TRAV1-2+ CD8+ T cells in matched peripheral blood samples. To test this hypothesis, we performed high-throughput TCR repertoire analysis of TRAV1-2+ CD4− T cells sorted by flow cytometry from cryopreserved BAL fluid and matched peripheral blood specimens obtained from three donors with active TB (Supplementary Table 3). MAIT cell-consistent CDR3α sequences comprised a higher percentage of the TRAV1-2+ repertoire in BAL fluid compared with peripheral blood, irrespective of the parameter used to define MAIT cell-consistent CDR3α sequences, including assessment of similarity to published MAIT cell CDR3α sequences (MAIT Match score = 0.95 or 1) or according to usage of TRAJ12, TRAJ20 or TRAJ33 (Fig. 3e; P = 0.0036; 2-way ANOVA). Among the patients with TB, CDR3α sequences with the highest MAIT Match scores (≥0.95) were enriched in BAL fluid, while those with the lowest MAIT Match scores (<0.85), were more frequent in peripheral blood (Fig. 3f ).
To determine the extent to which individual MAIT cell-consistent CDR3α sequences (MAIT Match Score ≥0.95) were shared between these two anatomical compartments, we created a TCR Enrichment Analysis (TEA) webtool (https://github.com/eisascience/Wong-Gold-Lewinsohn) to enable visualization and weighted frequency analysis of the most common MAIT cell-consistent CDR3α sequences in matched samples (Fig. 3g and Supplementary Table 4). In all three patients, the most frequent MAIT cell-consistent CDR3α sequences were present in both compartments, with disproportionate expansion in the BAL fluid compared with the peripheral blood. In contrast, CDR3α sequences with low MAIT Match scores (<0.85) were generally expanded only in one anatomical compartment (Supplementary Fig. 3). The selective expansion of MAIT cell-consistent CDR3α sequences in the lung compartment relative to peripheral blood suggests antigen-driven clonal expansion in response to pulmonary infection with Mtb.
To determine if TRAV1-2+ CD8+ T cells present in BAL fluid contained MAIT cells, we examined the MR1-restricted function of T cell clones generated from a BAL fluid sample obtained from a patient with TB. Six of these TRAV1-2+ clones (D0033-A1, A2, A3, A6, A8 and A10) expressed MAIT cell-consistent CDR3α sequences (MAIT Match score = 0.98-1; Table 1). Stimulation of these clones with HLA-mismatched M. smegmatis-infected or Mtb-infected antigen-presenting cells induced robust IFN-γ, while two control clones from the same patient (D0033-D7 and E7) failed to produce IFNγ under identical conditions. In contrast, stimulation of the TRAV1-2+ clones with HLA-mismatched M. smegmatis-infected or Mtb-infected MR1-KO antigen-presenting cells32 resulted in negligible IFN-γ-production, thereby demonstrating MR1-dependent cytokine production consistent with MAIT cell function (Fig. 3h).
Table 1 TCRα/β sequences and MAIT Match scores for six MAIT cell clones and two control clones derived from bronchoalveolar fluid from a patient with tuberculosis
MR1 tetramer loaded with 5-OP-RU ligand has been shown to identify functional MAIT cells in the human peripheral circulation13. To evaluate whether TRAV1-2+ CD8+ T cells in the BAL of humans with TB could be stained by MR1/5-OP-RU tetramer, as well as the relationship between MAIT cell-consistent CDR3α usage and MR1/5-OP-RU tetramer staining, we took advantage of two donors with TB with available cryopreserved specimens. We stained cells from paired BAL and peripheral blood samples with TRAV1-2 antibody, MR1/5-OP-RU tetramer and MR1/6-FP tetramer (negative control). As shown in Fig. 4a, BAL cells from donor 1020 demonstrated MR1/5-OP-RU tetramer staining of 33.7% of the TRAV1-2+ cells, supporting the TCRα sequencing analysis that found that 40.7% of BAL TRAV1-2 CDR3α sequences were MAIT cell-consistent (MAIT Match score >.95). In the peripheral blood of this participant, only 3.06% of the TRAV1-2+ peripheral cells demonstrated MR1/5-OP-RU tetramer staining, in line with the TCRα sequencing analysis that had found that 5.09% of peripheral TRAV1-2 CDR3α's were MAIT cell-consistent.
Heterogeneous MR1/5-OP-RU staining of bronchoalveolar TRAV1-2+ CD8+ T cells with MAIT cell-consistent CDR3α's and MR1-restricted function. a Frequency of MR1-tetramer+ cells (loaded with active (5-OP-RU) and control (6FP) ligand) in TRAV1-2+ T cells (gated on live, CD3+, CD8+ lymphocytes) from the BAL fluid and peripheral blood of a patient with TB. The proportion of cells utilizing MAIT cell-consistent CDR3α's (MAIT Match Score ≥.95) in MR1/5-OP-RU tetramer positive and negative populations are shown. b IFNγ spot-forming units (SFU) produced by four T cell clones generated from BAL fluid and stimulated with M. smegmatis-infected wildtype (WT) or MR1-KO A549 cells, Supplementary Data. c α-TRAV1-2 staining of four T cell clones generated from BAL fluid demonstrates consistent staining. Histograms are mode-normalized. d Binding of MR1/5-OP-RU tetramer on the same four T cell clones generated from BAL fluid demonstrates heterogenous MR1/5-OPRU tetramer staining (left). Binding of MR1/6-FP (control) and MR1/5-OPRU tetramer is shown for two clones (right). Histograms are mode-normalized
In contrast to MR1/5-OP-RU tetramer staining in the peripheral blood where positive and negative populations were clearly discernable, the MR1/5-OP-RU tetramer staining of BAL cells was of heterogeneous intensity and did not allow unambiguous delineation of MR1/5-OP-RU tetramer negative and positive populations. As a result, we sorted TRAV1-2+ cells based on MR1/5-OP-RU tetramer staining, subjected both positive and negative subsets to TCR sequencing, and analyzed MAIT cell-consistent CDR3α usage in each population (Table 2). CDR3α chain sequencing of MR1/5-OP-RU tetramer positive cells from BAL and peripheral blood revealed that 93.9% and 89.2% of these respectively utilized MAIT cell-consistent TCRs. CDR3α chain sequencing of the MR1/5-OP-RU tetramer negative TRAV1-2+ populations demonstrated that a substantial proportion (13.7%) of the MR1/5-OP-RU tetramer negative cells in the BAL utilized MAIT cell-consistent CDR3α chains. In contrast, only 2.7% of MR1/5-OP-RU tetramer negative cells from the peripheral blood utilized MAIT cell-consistent CDR3α chains. These data suggest that MR1/5-OP-RU tetramer may perform less efficiently in BAL fluid than in peripheral blood. Notably, in the other donor (91), in whom 28.5% of the TRAV1-2+ cells had a MAIT cell-consistent CDR3α, only 5.09% of the TRAV1-2+ cells from the BAL stained MR1/5-OP-RU positive. In this donor, 24.7% of the MR1/5-OP-RU tetramer negative cells had MAIT cell-consistent CDR3α chains, suggesting that MR1/5-OP-RU tetramer staining of BAL cells may underestimate the presence of MAIT cells as determined by CDR3α usage.
Table 2 Comparison of MR1/5-OPRU tetramer staining and usage of MAIT cell-consistent CDR3α sequences within TRAV1-2+ CD8+ T cells in the bronchoalveolar (BAL) and peripheral blood (PBMC) compartments from two patients with active TB
To better understand the relationship between MR1/5-OP-RU tetramer staining, CDR3α usage and MR1-dependent T cell activity, we sorted MR1/5-OP-RU positive cells from the BAL of an available individual with non-TB pneumonia and performed limiting dilution cloning using anti-CD3 and IL-2 stimulation. Following rapid expansion33, each clone was characterized functionally for MR1-restriction and antigenic specificity. As shown in Fig. 4c, four clones (D1004-B3, E1, E5, and H3) produced IFN-γ when stimulated with M. smegmatis-infected antigen-presenting cells (A549) and abrogated IFN-γ production when stimulated with identically infected MR1-KO antigen-presenting cells32. TCR sequencing demonstrated that each of these clones utilized a MAIT cell-consistent CDR3α (Fig. 4b). Surprisingly, despite clear evidence of MR-1 restricted function, usage of MAIT cell-consistent CDR3α's (Table 3) and TRAV1-2 staining of similar intensity (Fig. 4c), these clones demonstrated considerable heterogeneity in MR1/5-OP-RU tetramer staining, with two of the four clones staining weakly (Fig. 4d).
Table 3 TCRα/β sequences and MAIT Match scores for four MAIT cell clones derived from bronchoalveolar cells
Collectively, our data indicate that donor-unrestricted mycobacterial-reactive TRAV1-2+ CD8+ T cells are present in the human respiratory mucosa and that pulmonary infection with Mtb leads to an enrichment of airway resident, pro-inflammatory TRAV1-2+ CD8+ cells including oligoclonal expansions of MAIT cells.
In lung tissue explanted from healthy organ donors, we find that TRAV1-2+ CD8+ T cells localize to the respiratory tract mucosal surface. In contrast to their counterparts in the gut mucosa, TRAV1-2+ CD8+ T cells from the respiratory mucosa produce TNF in response to mycobacterial stimulation by donor-unrestricted antigen-presenting cells. This suggests that TRAV1-2+ CD8+ T cells in the airway mucosa may play a role in anti-Mtb immunity by initiating a local pro-inflammatory response upon exposure to aerosolized Mtb.
In the setting of active pulmonary tuberculosis, we observed striking expansions of TRAV1-2+ CD8+ T cells in the bronchoalveolar compartment. Compared to paired peripheral blood TRAV1-2+ CD8+ T cells, the bronchoalveolar TRAV1-2+ CD8+ T cells produced significantly more TNF. Some, but not all, of these expanded bronchoalveolar TRAV1-2+ CD8+ T cells could be identified as MAIT cells based on their utilization of MAIT cell-consistent CDR3α chains, demonstration of MR1-restricted function or selective binding of the MR1/5-OP-RU tetramer. It should be noted that among the TRAV1-2+ CD8+ T cells that could not be unequivocally confirmed as MAIT cells we identified subpopulations that displayed certain "MAIT-like" features, such as high-level expression of CD26 or oligoclonal expansions of TRAV1-2+ TCRα chains with features similar to MAIT cell CDR3α sequences (incorporation of the TRAJ12, TRAJ20 or TRAJ33 segments, or the presence of the Tyr95 which is known to be critical for MAIT cell TCR binding to MR1-restricted ligands34). Our attempts to clone these populations have been unsuccessful to date, such that further work will be required to determine if these TRAV1-2+ CD8+ T cells with "MAIT-like" features are restricted by MR1. It is also notable that MR1/5-OP-RU tetramers identified only a subset of TRAV1-2+ CD8+ T cells with MAIT cell-consistent CDR3a's.
We postulate that variable MR1-tetramer staining observed on bronchoalveolar TRAV1-2+ CD8+ cells could reflect a state of activation among tissue-resident cells. Supporting this, we note that differential tetramer staining can be observed following expansion of MAIT cell clones with activating cytokines (Supplementary Figure 4). Alternatively, we postulate that TRAV1-2+ CD8+ T cells with MAIT cell-consistent CD3α's may have altered tetramer-binding avidity as a result of differential affinity of their TCRs for MR1-ligands. This possibility is suggested by the variable magnitude of response to M. smegmatis in the functional assay, and has recently been demonstrated for the photolumazine I ligand35. Further work will be required to better understand the relationship between TCR-dependent MR1-dependent activation, MR1/5-OP-RU tetramer staining, and ligand selectivity among bronchoalveolar TRAV1-2+ CD8+ T cells. At this point we conclude that MR1/5-OP-RU tetramer staining of bronchoalveolar MAIT cells is weaker and more variable than MR1/5-OP-RU tetramer staining of peripheral blood MAIT cells and hence may underestimate MAIT cell prevalence in the BAL.
In contrast to the bronchoalveolar fluid of active TB patients, analysis of TCRα chain usage in granulomas of patients undergoing lung-resection for clinically complicated tuberculosis did not demonstrate dramatic expansions of TRAV1-2+TCRα's. The contrast between the enrichment of TRAV1-2+ CD8+ T cells observed in bronchoalveolar lavage fluid and the relatively low frequencies of TRAV1-2+TCRα's found in the lung granuloma tissue may be due to differences between cells present in the airway mucosal environment and in lung parenchymal tissue. It is also possible that the kinetics of expansion of TRAV1-2+ CD8+ cells with MAIT cell-consistent CDR3α's varied during the long course of TB disease and anti-tuberculosis therapy that preceded surgical treatment in these medically-complex lung-resection patients.
It is therefore notable that even in the resected granuloma tissue, the subset of TCRα's with MAIT cell-consistent sequences was enriched among the TRAV1-2+ CDR3α's in lung granuloma tissue compared to paired mediastinal lymph node tissue. We postulate that this relative enrichment of MAIT cell-consistent TCRs among TRAV1-2+ sequences from the lung was driven by local antigen exposure, while acknowledging that tissue-specific non-antigen stimuli could also lead to the independent expansion of clones in the lung compartment. Further understanding of this will require additional organ-specific datasets to allow comparison of diseased and reference TCR repertoires. We found both public and private MAT cell-consistent CDR3α chains in the TB-infected human lung tissues we analyzed. Interestingly, public MAIT cell-consistent CDR3α chains were frequently encoded by multiple synonymous nucleotide sequences within an individual sample. This finding is consistent with a previous report implicating convergent recombination as a determinative process in the generation of public MAIT cell CDR3α sequences22, and suggests that tissue-resident public MAIT cell-consistent CDR3α expansions are the result of multiple individual MAIT cells clonally expanding in infected tissues. The significance of cells with private MAIT cell-consistent CDR3α chains in the context of Mtb-infected tissue remains uncertain. One possibility is that public and private MAIT cell-consistent CDR3α chains have similar ligand-binding properties, such that utilization and expansion of specific clonotypes in individual hosts is the result of differences in the naive TCR repertoire and is not driven by specific microbial exposures. Alternatively, the observed clonal expansion of private MAIT cell-consistent clonotypes within Mtb-infected tissue may reflect selective expansions in response to the local presence of microbe-derived ligands presented by MR135. A third possibility is that because our sample set is small, sequences that appear to be private in this analysis could in fact turn out to be public when larger numbers of individual donors are sampled. In order to determine the significance of private and public MAIT cell-consistent TCRs in the context of mycobacterial infection, further study of selective ligand specificity in larger numbers of donors is needed. Nonetheless, the convergence of multiple nucleotide rearrangements on expanded public MAIT cell-consistent TCRα chains suggests that in some instances, multiple MAIT cells with genetically unique but functionally similar TCRα chains clonally expand in the TB-infected lung, potentially in response to microbe-derived antigenic-stimulation.
In line with recent studies that have found MAIT cell expansions in the lungs of mice experimentally infected with Mtb36 and S. enterica serovar Typhimurium (S. Typhimurium)37, we found expansions of TRAV1-2+ CD8+ T cells with MAIT or MAIT-like features in the BAL and lung parenchyma in patients with TB. Supported by the findings Chen et al. who found that accumulation of MAIT cells in the lungs of mice following challenge with S. Typhimurium is dependent on antigen derived from the microbial riboflavin synthesis pathway37, we postulate that these TRAV1-2+ CD8+ cell enrichments contain MAIT cells and are driven by Mtb-derived small molecular ligands. Howson et al. recently reported that MAIT cell clones with more avid ligand-binding expand during S. enterica serovar Paratyphi A infection and that these clones remain expanded after treatment of the infection38. This finding supports the idea that exposure to microbe-derived MR1 ligands alters the human MAIT cell TCR repertoire and suggests a role for MR1-ligand vaccine strategies. Overall our findings suggest a previously unrecognized and potentially important role for TRAV1-2+ CD8+ T cells with MAIT or MAIT-like features in the immune response to aerosolized Mtb infection, and would support exploration of these cells as targets of either vaccination or immunotherapeutic strategy.
Samples from Portland, Oregon, USA. Airway, lung, small intestine and associated lymph node tissues ineligible for transplantation were obtained from the Pacific Northwest Transplant Bank under a protocol approved by the Institutional Review Board at Oregon Health & Science University. Limited clinical information was available for these individuals, who were generally considered healthy prior to demise. For comparison with the organ samples, PBMCs were obtained by apheresis from healthy adult donors providing informed consent.
Samples from Durban, South Africa. Explanted granulomatous lung tissue and associated lymph nodes were obtained under a protocol approved by the University of KwaZulu Natal Human Biomedical Research Ethics Commitee (UKZN BREC) allowing adults undergoing clinically indicated lung resection for complicated tuberculosis at Inkosi Albert Luthuli Central Hospital (IALCH) to donate excess tissue for scientific research18. Tissue was isolated from different areas of resected lungs based on the experience of the operating surgeon and the preoperative radiological data. Clinical characteristics of the individuals and samples have been described18. All donors provided written informed consent prior to surgery. BAL fluid and paired peripheral blood samples were obtained under a protocol approved by the UKZN BREC and Partners Institutional Review Board allowing collection of excess fluid from adult patients undergoing clinically indicated diagnostic bronchoscopies at IALCH. Active tuberculosis was defined microbiologically (positive BAL Mtb culture or BAL Mtb PCR by GeneXpert) and/or histologically (Ziehl-Neelsen positive necrotizing granulomas on transbronchial biopsy obtained at the time of BAL). Uninfected controls were defined as individuals with no evidence of either infectious or inflammatory lung disease, as determined by a committee of study physicians on the basis of clinical history, chest x-rays, computerized tomography scans, and negative BAL microbiology (mycobacterial, bacterial and fungal cultures, and Mtb PCR). Most controls underwent bronchoscopy for suspected lung cancer, and a non-cancerous segment was lavaged in these cases. All donors provided written informed consent prior to bronchoscopy. Cryopreserved peripheral blood mononuclear cells (PBMCs) from healthy donors (defined as asymptomatic and HIV-negative with no evidence of Mtb by ELISPOT) were available from the iThimba Cohort which was approved by the UKZN BREC and Partners Institutional Review Board39. All participants provided written informed consent.
Isolation and stimulation of lung and gut T cells
Lymphocytes were isolated from fresh lung tissue as described previously4. A two-step process was used to extract cells from the small intestine. For collection of lymphocytes from the intraepithelial (IEL) layer, the tissue was washed in HBSS, stripped of muscle, and incubated with agitation for 30 min in 0.15% dithiothreitol (Sigma-Aldrich). IEL lymphocytes were then harvested, and the remaining tissue was incubated for 30 min in PBS. Lamina propria (LP) lymphocytes were released by digestion with 0.1% collagenase (CLS-3, Worthington) and 0.3% DNAse (Roche) for 30 min at 37 °C. IEL and LP preparations were further enriched over a discontinuous Percoll gradient. Lymphocyte stimulations were performed as described previously4,12,16. Briefly, lymphocytes were incubated for 16 h with uninfected (control) or M. smegmatis strain mc2122-infected (multiplicity of infection = 3) A549 cells (ATCC CCL-185) at a ratio of 3:1 in the presence of α-CD28 and α-CD49d (Biolegend), together with an α-TNF mAb (Beckman Coulter) and the TNF-Processing Inhibitor 0 (TAPI-0, 10 μM) (Calbiochem). Cells were then stained as described above for surface expression of CD45, CD3, CD8, TRAV1-2, and CD1614. Dead cells were excluded using Aqua LIVE⁄DEAD (Invitrogen). Stained samples were acquired on a Fortessa flow cytometer (BD Biosciences) and data were analyzed with FlowJo software version 10.6 (Tree Star).
Immunohistochemistry of airway tissues
Cryosections (10 µM) of frozen airway tissues were treated with acetone and air-dried prior to incubation with α-TRAV1-2 antibody (clone-3C10; Biolegend) followed by goat α-mouse IgG1-Alexa Fluor 488 (1:1000), and then α-CD8 antibody (1:50; LSBio) followed by goat α-mouse IgG1-Alexa Fluor 568 (1:1000). Sections were washed and stained with DAPI. Images were acquired using an Olympus FluoView FV1000 laser scanning confocal microscope system with a 40 × 1.3 Oil Plan Fluorite objective. Confocal images were analyzed using Imaris Analysis Software.
Isolation and TCR sequencing of T cells from lung granulomas
Diseased lung tissue (approximately 3 cm3) was isolated from surgically resected explants. Each sample was washed in multiple changes of Hank's Balanced Salt Solution (HBSS), diced into smaller pieces (approximately 1 mm3), strained, resuspended in pre-warmed R10 supplemented with 0.5 mg/ml collagenase D (Roche) and 40 U/ml DNaseI (Roche), and transferred to GentleMACS C-tubes (Miltenyi Biotec) for mechanical digestion per the manufacturer's instructions. The resulting suspension was incubated for 60 min at 37 °C, subjected to an additional mechanical digestion step, strained through a 70 μm filter, washed twice in HBSS, and stained prior to sorting CD4− T-cells using a FACSARIA flow cytometer (BD Biosciences). Cells were gated as live (nearIR−, Invitrogen), single lymphocytes (determined on the basis of light scatter), then sorted as CD45+, CD3+, CD4− events directly into RLT buffer. Genomic DNA was extracted using a DNeasy Minikit (Qiagen) and high-throughput TCRα sequencing was performed using the ImmunoSEQ assay (Adaptive Biotechnologies Corp.)40. Data were analyzed using the ImmunoSEQ Analyser.
CDR3α sequence similarity
Similarity between CDR3α sequences was calculated as described previously21. This method allows similarities to be assigned between sequences of different length in an alignment-free manner. An implementation of the similarity matching between CDR3 sequences is publicly available at http://www.cbs.dtu.dk/services/MAIT_Match. The server takes as input a list of CDR3α sequences, and returns for each a score based on the maximal sequence similarity with a reference database of MAIT cell CDR3α sequences. A perfect match has a similarity score of 1, and a perfect mismatch a similarity score of 0.
Collection, staining and stimulation of BAL lymphocytes
Bronchoscopies were performed by pulmonologists at IALCH. Patients received sedation and bronchodilators according to the local standard of care; Normal saline (200 mL) was lavaged into the lobe with the highest burden of pathology or, in patients with diffuse disease, the right middle lobe. Available BAL fluid was combined in a 1:1 ratio with R10 (RPMI 1640 supplemented with 10% fetal bovine serum, L-glutamine, penicillin and streptomycin) and stored on ice. All samples were processed within 3 h of collection, BAL fluid was filtered through a 40 µm strainer (BD Pharmingen) and centrifuged. Resuspended BAL cells were aliquoted for staining with Aqua LIVE⁄DEAD (Invitrogen) and some or all of the following antibodies: α-CD3-PE-CF594 (BD Horizon, clone UCHT1), α-CD8-APC-H7 (BD Pharmingen, clone SK1), α-CD14-PerCP-Cy5.5 (BioLegend, clone HCD14), α-CD235a-PerCP-Cy5.5 (BioLegend, clone HIR2), α-TRAV1-2-APC (clone OF-5A1212), α-CD161-PE-Cy7 (BioLegend, clone HP-3G10), α-CD26-PE (BioLegend, clone BA5b). All stains were performed at 4 °C. Cells were then fixed with 4% paraformaldehyde. Functional studies were performed if sufficient numbers of BAL lymphocytes were available. After depletion of macrophages via plastic adherence for 1 h, 1 × 106 lymphocytes were stimulated for 18 h at 37 °C with α-CD2/CD3/CD28-loaded Anti-Biotin MACSiBead Particles (Miltenyi Biotec) at a ratio of 2:1 in RPMI 1640 supplemented with 10% fetal bovine serum, L-glutamine, HEPES, penicillin, and streptomycin. Brefeldin A was added after the first hour to inhibit protein transport from the endoplasmic reticulum. Stimulated and unstimulated cells were then stained with Aqua LIVE⁄DEAD (Invitrogen) and the following antibodies: α-CD235a-PerCP-Cy5.5, α-CD14-PerCP-Cy5.5, α-CD8-APC-H7, α-TRAV1-2-APC, α-CD161-PE-Cy7. After a wash step, cells were fixed with PERM/FIX Medium A (Invitrogen), permeabilized with PERM/FIX Medium B (Invitrogen), and stained with the following antibodies: α-CD3-PE-CF594, α-TNFa-PE (Beckman Coulter, clone IPM2), and α-IL-17-BV421 (BioLegend, clone BL168). Stained samples were acquired using a Fortessa flow cytometer (BD Bioscience). Rainbow Fluorescent Particles (BD Bioscience) and applications settings in FACSDiva7 were used to correct for day-to-day variations in instrument performance. Cells were gated as live (aqua viability dye negative) lymphocytes (determined on the basis of light scatter), and CD14++ cells were excluded prior to selecting CD3+ cells for analysis. Paired peripheral blood samples were collected where possible and freshly isolated PBMC were processed in parallel with matched BAL cells. Data were analyzed with FlowJo10.6 (Treestar). Background cytokine production was subtracted to calculate percentage of cells producing cytokine in response to stimulation. When available, paired cryopreserved BAL and PBMC cells were thawed and stained with some or all of the above antibodies and MR1/5-OP-RU or MR1/6-FP tetramers (courtesy of the McCluskey Laboratory). Cell suspensions were acquired and sorted on a FACSARIA flow cytometer (BD Biosciences) into TRIzol (Invitrogen). Genomic DNA was extracted utilizing the phenol-chloroform method according to manufacturer protocol, using linear acrylamide (Invitrogen) as a carrier. High-throughput TCRα sequencing was performed using the ImmunoSEQ assay (Adaptive Biotechnologies Corp)40.
Visualization of MAIT cell CDR3α sequences
Data were coded in R using the packages RColorBrewer, Shiny, data.table, ggplot2, and dplyr. Synonymous nucleotide sequences within a tissue were counted, and the associated frequencies are summed. These frequencies were visualized using the TCR Enrichment Analysis (TEA) webtool the code for which is archived at https://github.com/eisascience/Wong-Gold-Lewinsohn/tree/v1.0.0
Generation and characterization of T cell clones
Cells from BAL samples were stained with Aqua LIVE/DEAD (Invitrogen), MR1/5-OP-RU tetramer (0.3 nM, McCluskey Laboratory), α-CD4-FITC (clone OKT4; BioLegend), and α-CD8-APC-Cy7 (clone SK8; BioLegend). Live tetramer-binding cells were sorted by the basis of co-receptor expression using an Influx flow cytometer (BD Biosciences), rested overnight in RPMI 1640 supplemented with 10% heat-inactivated pooled human serum and 0.5 ng/ml rhIL-2, and then distributed in limiting dilution format with irradiated PBMCs (150 × 105/well) and irradiated B-lymphoblastoid cells (3 × 104/well) in a 96-well round bottom plate. The cultures were stimulated with rhIL-2 (5 ng/ml), rhIL-12 (0.5 ng/ml), rhIL-7 (0.5 ng/ml), rhIL-15 (0.5 ng/ml) and α-CD3 (0.03 µg/ml). Clones were harvested after incubation for 20 days at 37 °C and assessed for clonality by flow cytometry, TCR sequencing, and MR1-restricted function by ELISPOT.
Nitrocellulose-backed multiscreen 96-well plates (Millipore) were coated overnight at 4 °C with a 10 µg/ml solution of α-IFNγ antibody (clone 1-D1K; Mabtech) in 0.1 M Na2CO3, 0.1 M NaHCO3, pH 9.6. The plate was washed three times with sterile PBS and blocked for 1 h at room temperature with RPMI 1640 containing 10% heat-inactivated pooled human serum. Uninfected, M. smegmatis mc2122-infected (multiplicity of infection = 3), or M. tuberculosis H37Rv-infected (multiplicity of infection = 30) wildtype or MR1-null32 A549 cells (1 × 104/well) and clonal T cells (1 × 104/well) were added and incubated overnight at 37 °C. The plates were then washed six times in PBS containing 0.05% Tween-20, incubated for 2 h at room temperature with a 1 µg/ml solution of α-IFNγ-biotin antibody (clone 7-B6-1; Mabtech) in PBS containing 0.5% bovine serum albumin and 0.05% Tween-20, washed again six times in PBS containing 0.05% Tween-20 followed by PBS alone, and developed using an AEC Vectastain Kit (Vector Laboratories). Spots were counted using an automated ELISPOT Reader System (Autoimmun Diagnostika GmbH).
TCR sequence analysis of CD8+ T cell clones isolated from BAL fluid
For some clones, total RNA was extracted using an RNeasy Mini Kit (Qiagen). Unbiased amplification of all expressed TRA and TRB gene products was then conducted using a template-switch anchored RT-PCR with chain-specific constant region primers41. Amplicons were sub-cloned, sampled, sequenced and analyzed as described previously42. Gene usage was assigned according to the IMGT nomenclature. For other clones, genomic DNA was extracted using a DNeasy Mini Kit (Qiagen) and high-throughput TCRα and TCRβ sequencing was performed using the ImmunoSEQ assay (Adaptive Biotechnologies Corp)40. Data were analyzed using the ImmunoSEQ Analyser.
Statistics and reproducibility
Statistical analyses were performed using Prism 6 (GraphPad Software Inc). The non-parametric Mann–Whitney U test was used to assess differences between groups unless indicated otherwise. All statistical tests were two-sided unless indicated otherwise. P values < 0.05 were considered significant for direct comparisons. In cases of multiple comparisons the Bonferonni correction was applied. Experiments were repeated with as many biologically indpendent samples as were available; when possible a minimum of two experimental replicates were performed.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
The datasets generated during and/or analyzed during the current study are archived at https://github.com/eisascience/Wong-Gold-Lewinsohn or available from the corresponding author on reasonable request.
Code availability
Custom code for the MAIT Match tool is available at http://www.cbs.dtu.dk/services/MAIT_Match/. Custom code for the visualization of frequencies of specific TCRα's in the TB granuloma tissues and the TCR Enrichment Analysis (TEA) webtool are archived at https://github.com/eisascience/Wong-Gold-Lewinsohn
Porcelli, S., Yockey, C. E., Brenner, M. B. & Balk, S. P. Analysis of T cell antigen receptor (TCR) expression by human peripheral blood CD4-8- alpha/beta T cells demonstrates preferential use of several V beta genes and an invariant TCR alpha chain. J. Exp. Med. 178, 1–16 (1993).
Tilloy, F. et al. An invariant T cell receptor alpha chain defines a novel TAP-independent major histocompatibility complex class Ib-restricted alpha/beta T cell subpopulation in mammals. J. Exp. Med. 189, 1907–1921 (1999).
Treiner, E. et al. Selection of evolutionarily conserved mucosal-associated invariant T cells by MR1. Nature 422, 164–169 (2003).
Gold, M. C. et al. Human mucosal associated invariant T cells detect bacterially infected cells. PLoS Biol. 8, e1000407 (2010).
Le Bourhis, L. et al. Antimicrobial activity of mucosal-associated invariant T cells. Nat. Immunol. 11, 701–708 (2010).
Kjer-Nielsen, L. et al. MR1 presents microbial vitamin B metabolites to MAIT cells. Nature 491, 717–723 (2012).
Georgel, P., Radosavljevic, M., Macquin, C. & Bahram, S. The non-conventional MHC class I MR1 molecule controls infection by Klebsiella pneumoniae in mice. Mol. Immunol. 48, 769–775 (2011).
Chua, W. J. et al. Polyclonal mucosa-associated invariant T cells have unique innate functions in bacterial infection. Infect. Immun. 80, 3256–3267 (2012).
Meierovics, A., Yankelevich, W. J. & Cowley, S. C. MAIT cells are critical for optimal mucosal immune responses during in vivo pulmonary bacterial infection. Proc. Natl Acad. Sci. USA 110, E3119–E3128 (2013).
Meierovics, A. I. & Cowley, S. C. MAIT cells promote inflammatory monocyte differentiation into dendritic cells during pulmonary intracellular infection. J. Exp. Med. 213, 2793–2809 (2016).
Martin, E. et al. Stepwise development of MAIT cells in mouse and human. PLoS Biol. 7, e54 (2009).
Gold, M. C. et al. Human thymic MR1-restricted MAIT cells are innate pathogen-reactive effectors that adapt following thymic egress. Mucosal Immunol. 6, 35–44 (2013).
Reantragoon, R. et al. Antigen-loaded MR1 tetramers define T cell receptor heterogeneity in mucosal-associated invariant T cells. J. Exp. Med. 210, 2305–2320 (2013).
Jiang, J. et al. MAIT cell function is modulated by PD-1 signaling in patients with active tuberculosis. Am. J. Respir. Crit. Care Med. https://doi.org/10.1164/rccm.201401-0106OC (2014).
Gold, M. C. & Lewinsohn, D. M. Co-dependents: MR1-restricted MAIT cells and their antimicrobial function. Nat. Rev. Microbiol. https://doi.org/10.1038/nrmicro2918 (2012).
Gold, M. C. et al. MR1-restricted MAIT cells display ligand discrimination and pathogen selectivity through distinct T cell receptor usage. J. Exp. Med. https://doi.org/10.1084/jem.20140507 (2014).
Sharma, P. K. et al. High expression of CD26 accurately identifies human bacterial-reactive MR1-restricted MAIT cells. Immunology https://doi.org/10.1111/imm.12461 (2015).
Nunes-Alves, C. et al. Human and murine clonal CD8+ T cell expansions arise during tuberculosis because of TCR selection. PLoS Pathog. 11, e1004849 (2015).
Carlson, C. S. et al. Using synthetic templates to design an unbiased multiplex PCR assay. Nat. Commun. 4, 2680 (2013).
Greenaway, H. Y. et al. NKT and MAIT invariant TCRalpha sequences can be produced efficiently by VJ gene recombination. Immunobiology https://doi.org/10.1016/j.imbio.2012.04.003 (2012).
Shen, W.-J. W., Hau-San, Xiao, Quan-Wu, Guo & Xin, Smale,Stephen Towards a mathematical foundation of immunology and amino acid chains. eprint arXiv 1205, 6031 (2012).
Greenaway, H. Y. et al. NKT and MAIT invariant TCRalpha sequences can be produced efficiently by VJ gene recombination. Immunobiology 218, 213–224 (2013).
McWilliam, H. E. et al. The intracellular pathway for the presentation of vitamin B-related antigens by the antigen-presenting molecule MR1. Nat. Immunol. 17, 531–537 (2016).
Dusseaux, M. et al. Human MAIT cells are xenobiotic-resistant, tissue-targeted, CD161hi IL-17-secreting T cells. Blood 117, 1250–1259 (2011).
Gibbs, A. et al. MAIT cells reside in the female genital mucosa and are biased towards IL-17 and IL-22 production in response to bacterial stimulation. Mucosal Immunol. https://doi.org/10.1038/mi.2016.30 (2016).
Sharma, P. K. et al. High expression of CD26 accurately identifies human bacteria-reactive MR1-restricted MAIT cells. Immunology https://doi.org/10.1111/imm.12461 (2015).
Tobin, D. M. et al. Host genotype-specific therapies can optimize the inflammatory response to mycobacterial infections. Cell 148, 434–446 (2012).
Leeansyah, E. et al. Activation, exhaustion and persistent decline of the anti-microbial MR1-restricted MAIT cell population in chronic HIV-1 infection. Blood https://doi.org/10.1182/blood-2012-07-445429 (2012).
Eberhard, J. M. et al. CD161+ MAIT cells are severely reduced in peripheral blood and lymph nodes of HIV-infected individuals independently of disease progression. PloS ONE 9, e111323 (2014).
Shane, H. L. & Klonowski, K. D. Every breath you take: the impact of environment on resident memory CD8 T cells in the lung. Front. Immunol. 5, 320 (2014).
Van Rhijn, I. et al. A conserved human T cell population targets mycobacterial antigens presented by CD1b. Nat. Immunol. 14, 706–713 (2013).
Laugel, B. et al. Engineering of isogenic cells deficient for MR1 with a CRISPR/Cas9 Lentiviral system: tools to study microbial antigen processing and presentation to human MR1-restricted T cells. J. Immunol. 197, 971–982 (2016).
Riddell, S. R. et al. MHC-restricted cytotoxic T lymphocyte recognition of cells infected with human cytomegalovirus does not require endogenous viral gene expression. J. Immunol. 146, 2795–2804 (1991).
Reantragoon, R. et al. Structural insight into MR1-mediated recognition of the mucosal associated invariant T cell receptor. J. Exp. Med. 209, 761–774 (2012).
Harriff, M. J. et al. MR1 displays the microbial metabolome driving selective MR1-restricted T cell receptor usage. Sci. Immunol. 3, https://doi.org/10.1126/sciimmunol.aao2556 (2018).
Sakala, I. G. et al. Functional heterogeneity and antimycobacterial effects of mouse mucosal-associated invariant T cells specific for riboflavin metabolites. J. Immunol. https://doi.org/10.4049/jimmunol.1402545 (2015).
Chen, Z. et al. Mucosal-associated invariant T-cell activation and accumulation after in vivo infection depends on microbial riboflavin synthesis and co-stimulatory signals. Mucosal Immunol. 10, 58–68 (2017).
Howson, L. J. et al. MAIT cell clonal expansion and TCR repertoire shaping in human volunteers challenged with Salmonella Paratyphi A. Nat. Commun. 9, 253 (2018).
Mitchell, J. E. et al. Prospective monitoring reveals dynamic levels of T cell immunity to Mycobacterium tuberculosis in HIV infected individuals. PloS one 7, e37920 (2012).
Lepore, M. et al. Parallel T-cell cloning and deep sequencing of human MAIT cells reveal stable oligoclonal TCRbeta repertoire. Nat. Commun. 5, 3866 (2014).
Quigley, M. F. et al. Unbiased molecular analysis of T cell receptor expression using template‐switch anchored RT‐PCR. Curr. Protoc. Immunol. https://doi.org/10.1002/0471142735.im1033s94 (2011).
Price, D. A. et al. Avidity for antigen shapes clonal dominance in CD8+ T cell populations specific for persistent DNA viruses. J. Exp. Med. 202, 1349–1361 (2005).
We would like to thank Hollis Shen of the AHRI Immunology Core for technical assistance, Kamini Gounder for HLA genotyping, James McCluskey and his laboratory for use of the MR1 tetramers, the Pacific Northwest Transplant Bank for ongoing provision of research tissue, the HIV Pathogenesis Programme Processing Laboratory staff, the AHRI Clinical Core, the staff of Inkosi Albert Luthuli Central Hospital, and the study participants. This work was funded in part by a Burroughs-Wellcome Fund/American Society of Tropical Medicine and Hygiene fellowship (EBW), a Fulbright Award (ZAS), the National Institutes of Health (grants T32 AI007387 and K08 AI118538 to E.B.W., grant R01AI078965 to M.C.G., grant R01AI048090 to D.M.L., grants R01AI37856 and R01AI97138 to W.R.B., grant R01AI106725 to S.M.B.), the National Institute of Allergy and Infectious Diseases Mucosal Immunology Studies Team (grant U01AI09577 to M.C.G. and D.M.L.), and Merit Review Awards # I01 BX001231 and I01 BX000533 from the United States Department of Veterans Affairs (VA) Biomedical Laboratory Research and Development, supported by use of the facilities and resources at the VA Portland Health Care System. T.N. received funding from the South African DST/NRF Research Chairs Initiative and the Victor Daitz Foundation. D.A.P. is a Wellcome Trust Senior Investigator. Collection of samples from the iThimba Cohort was supported by the Harvard University Center for AIDS Research (grant P30 AI060354). Research reported in this publication was supported by the Strategic Health Innovation Partnerships (SHIP) Unit of the South African Medical Research Council (SA MRC) with funds received from the South African Department of Science and Technology as part of a bilateral research collaboration agreement with the Government of India; and through a SA MRC Collaborating Centre (ACT4TB/HIV).This work was also supported in part through the Sub-Saharan African Network for TB/HIV Research Excellence (SANTHE), a DELTAS Africa Initiative [grant # DEL-15-006]. The DELTAS Africa Initiative is an independent funding scheme of the African Academy of Sciences (AAS)'s Alliance for Accelerating Excellence in Science in Africa (AESA) and supported by the New Partnership for Africa's Development Planning and Coordinating Agency (NEPAD Agency) with funding from the Wellcome Trust [grant # 107752/Z/15/Z] and the UK government. Open access publication of this article has been made possible through support from the Victor Daitz Information Gateway, an initiative of the Victor Daitz Foundation and the University of KwaZulu-Natal. The views expressed in this publication are those of the authors and do not represent the views of the Unites States Department of Veterans Affairs, the United States Government, AAS, NEPAD Agency, Wellcome Trust or the UK government.
These authors contributed equally: Emily B. Wong, Marielle C. Gold.
Africa Health Research Institute, KwaZulu-Natal, South Africa
Emily B. Wong
, Bongiwe Z. Xulu
, Sharon Khuzwayo
, Zuri A. Sullivan
, Zoe Rogers
, Hénrik Kløverpris
, Victoria O. Kasprowicz
, Alasdair Leslie
& Thumbi Ndung'u
Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA, USA
Harvard Medical School, Boston, MA, USA
Division of Infection and Immunity, University College London, London, UK
& Alasdair Leslie
Department of Pulmonary & Critical Care Medicine, Oregon Health & Science University, Portland, OR, USA
Marielle C. Gold
, Erin W. Meermeier
& David M. Lewinsohn
VA Portland Health Care System, Portland, OR, USA
, Prabhat K. Sharma
, Aneta H. Worley
Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, OR, USA
Division of Bioinformatics and Computational Biology (BCB), Department of Medical Informatics and Clinical Epidemiology (DMICE), Oregon Health & Science University, Portland, OR, USA
Eisa Mahyari
Institute for Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark
Hénrik Kløverpris
Durban University of Technology, Durban, South Africa
Umesh Lalloo
& Prinita Baijnath
Department of Pulmonology, Inkosi Albert Luthuli Hospital, Durban, South Africa
Prinita Baijnath
, Anish Ambaram
, Leon Naidoo
& Moosa Suleman
Department of Pulmonology & Critical Care, Nelson R. Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa
Moosa Suleman
Department of Cardiothoracic Surgery, Nelson R. Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa
Rajhmun Madansein
Centre for AIDS Programme of Research in South Africa (CAPRISA), Durban, South Africa
Institute of Infection & Immunity, Cardiff University School of Medicine, Cardiff, Wales, UK
James E. McLaren
, Kristin Ladell
, Kelly L. Miners
& David A. Price
Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
David A. Price
Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, USA
Samuel M. Behar
Center for Biological Sequence Analysis, Department of Bio and Health Informatics, Technical University of Denmark, Lyngby, Denmark
Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, Buenos Aires, Argentina
HIV Pathogenesis Programme, Doris Duke Medical Research Institute, Nelson R. Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa
Victoria O. Kasprowicz
The Ragon Institute of MGH, MIT, and Harvard, Harvard Medical School, Cambridge, MA, USA
Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, MD, USA
William R. Bishai
Max Planck Institute for Infection Biology, Berlin, Germany
Thumbi Ndung'u
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E.B.W., M.C.G., S.M.B., A.L., T.N. and D.M.L. designed the experiments; E.B.W., M.C.G., B.Z.X., EW.M., S.K., Z.A.S., H.K., P.K.S., A.H.W., J.E.M., K.L., K.L.M. and M.N. performed the experiments; E.B.W., M.C.G., E.W.M., S.K., J.E.M., D.A.P., S.M.B., A.L., T.N., E.M. and D.M.L. analyzed the results. U.L., Z.R., P.B., A.A., L.N., R.M., M.S., V.O.K. and W.R.B. enrolled human subjects and performed procedures; E.B.W., M.C.G., D.A.P., E.M., T.N. and D.M.L. wrote the manuscript. All co-authors provided comments and approved the content.
Correspondence to David M. Lewinsohn.
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Would you like to help blind, partially sighted and people with learning disabilities and complex needs in your community? Do you enjoy driving and meeting new people? If so, RNIB wants you to be a Volunteer Driver.
Our volunteer drivers provide a friendly face and that personal touch to brighten their day and help get them to where they need to be. Our drivers can become a regular part of someone's life. If you are friendly, courteous and reliable then this is the opportunity for you.


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Training/Onboarding
Enter the Chief Supply Chain Officer
At CSCO Summit virtual conference, leading executive practitioners and industry analysts explore the evolving role of the supply chain and top supply chain leaders
The role of the chief supply chain officer is becoming a reality and a necessity within companies, and the role is helping to elevate visibility of the supply chain to the C-level within organizations.
That was a key takeaway from the "Chief Supply Chain Officer Strategic Agenda" report presented at the CSCO Summit, held June 15-16 online with registered participants that included more than 2,500 senior global supply chain, operations, procurement and logistics executives, including many at C-level.
The event, free for registrants, offered a no-cost venue in which to hear leading practitioners and analysts share their thoughts on the progress of supply chain as a function, and to interact with other executives in a virtual environment. (Supply & Demand Chain Executive was a media partner to the event.) Presentations during the event were recorded and are available, along with slide decks and podcasts of the speeches, at www.CSCOSummit.com.
Top Pressures for SCM Improvement
Didier Chenneveau, executive vice president and chief supply chain officer at Korea's LG Electronics, joined top industry observer Nari Viswanathan, vice president and principal analyst in the Supply Chain Management Practice at Boston-based Aberdeen Group, in presenting the results of the CSCO summit survey. The final report, which was available to participants in the CSCO Summit and is posted to the summit Web site, featured insights from the survey of 391 supply chain professionals, including 50 C-level executives.
The study found that the leading pressure forcing companies to focus on improving their supply chain management was the increase in customer service demands and increased demand volatility, cited by 56 percent of all respondents to the survey. That was a hopeful sign for the economy, in a sense, when contrasted to the low number of respondents — just 9 percent — who said that they were under pressure due to a significant decrease in demand for their company's products.
Four in ten cited the rising business complexity of managing increasing global business networks. Interestingly, while 21 percent of the total respondents saw economic and financial volatility as a leading pressure for SCM improvement, nearly one-third (31 percent) of C-level respondents checked that box, suggesting the financial issues still resonate more strongly in the C-suites than further down the corporate ladder.
Chenneveau noted that LG Electronics, which had revenues of $50 billion in 2009, began focusing more heavily on improving its supply chain performance as part of its initiative to improve its market share position to the top three in each of the key markets that it served between 2006 and 2010. That involved moving from a "push-driven" supply chain that was totally in-sourced to a more demand-driven, technology-enabled model. The result was that by this year, LG in fact was in the top three in most of its markets. "Supply chain has played a critical role" in this transformation, Chenneveau said.
Collaboration as Culture
In a session called "Supply Chain Transformation Enabled through Collaboration," Jon Giacomin, executive vice president of pharmaceutical operations at Cardinal Health, joined Mike Fleming, corporate vice president of supply chain at Motorola Mobile Devices, in discussing how their respective organizations transformed and realigned their supply chain strategies in order to adapt to changes in the marketplace and ensure their companies' future growth.
Cardinal Health was faced with a changing business model, rising costs and higher customer expectations. Meanwhile, Motorola Mobile Devices had made the decision to focus its portfolio on the growing smartphone market. Both companies successfully leveraged end-to-end collaboration to transform their respective value chains, and each has been able to engage customers, increase visibility and reduce inventory through the pursuit of world-class supply chain performance for their respective customers and shareholders.
"Our business has transitioned from being very capital intensive to now being one where we're driven by the efficient use of capital," said Giacomin of Cardinal Health's business transformation. "We've gone from siloed information to information sharing up and down the supply chain. And, with that, we've gone from an insular focus to an end-to-end focus starting with our customers and going all the way back to the manufacturers."
For his part, Fleming noted that a key success factor for transformation was defining a high-level direction of the supply chain, focused around simplification, velocity and deeper supplier relationships. "I found it important to get the team thinking about [those three factors] to the extent that it became almost embedded in their thinking," he said. "Once it becomes embedded in their thinking, it becomes almost part of their culture, [and then] things like build-to-order, direct ship or range-based forecasting initiatives really become a natural output of this culture."
Supply Chain in the Cloud
Aberdeen's Nari Viswanathan returned on the second day of the conference to participate in a session called "Cloud Logistics: What It Is and Why It Is Poised to Transform the Supply Chain Technology Landscape." Viswanathan joined Peter Weis, vice president and chief information officer with Matson Navigation Company, and Greg Johnsen, executive vice president and co-founder of GT Nexus, in discussing the rapid advance of Cloud-based models in the logistics and supply chain space.
Cloud computing is rapidly taking hold in all areas of business IT because of what its proponents view as its superior economics and its lower risk profile. Companies typically can take advantage of Cloud-based solutions without making significant investments in IT infrastructure, and often with minimal involvement of their own IT personnel. Advocates of the Cloud say that finally makes possible the inter-company collaboration and information sharing that is so foundational to transforming global supply chains. In this panel discussion, the three participants discussed how Cloud is transforming operations and creating real, sustainable value for companies today.
Other sessions during the summit covered topics ranging from supply chain visibility and supply chain risk management to leveraging banks as effective supply chain partners and moving to design for supply chain. More information on the CSCO Summit, along with recorded presentations (available for viewing on-demand) and other materials, is available at www.CSCOSummit.com.
Matson Navigation Company
GT Nexus
Latest in Professional Development
Investing in the Development of Your Logistics Employees
Out-of-Stock Items Will be Biggest Challenge for Retailers in 2023
High Salary, Annual Raise Not Enough to Retain Supply Chain Workers: Study
78% of respondents reported a higher turnover rate in the previous 12 months, and 61% expect this to continue or get worse in the year to come.
Bastian Consulting
A Little Bit About The New Associate Editor: Emry Lloyd
Emry Lloyd introduces herself as the new associate editor for Food Logistics and Supply and Demand Chain Executive. She is a South Jersey native and graduated from Rutgers University in 2021.
5 Ways to Improve the Industrial Industry's Labor Shortage
By taking the necessary steps to address it through harnessing today's digital solutions, companies can empower their existing employees and perhaps attract newer generations to change their point of view to see factories as the workplace of the future.
Fero Labs
Aligning Customer Service with Leadership Priorities
One of the most effective ways to ensure that KPIs align with strategic priorities from leaders is to ask them directly. These responses are helpful for getting a sense of broader goals for your customer service team.
APQC
Need for Job Shops to Modernize U.S. Supply Chains
Results from a Paperless Parts survey indicates a need for job shops and contract manufacturers to modernize.
Paperless Parts
Over Half of Supply Chain Managerial Roles Don't Require Software Experience
Only one-third (30%) of all roles require ERP experience and nearly half (42%) of managerial jobs require no software experience at all, demonstrating a lack of technology adoption across supply chain facilities and operations.
Nominations Close Dec. 30 for Supply & Demand Chain Executive's 2023 Pros to Know Award
Are you or someone in your organization an outstanding leader, reinventing what it means to be a supply chain professional? Get them nominated by Dec. 30.
Women in Supply Chain: How to Manage Work-Life Balance
Holly Tran, director of account management for ALOM, and Taylor Houser, account manager for ALOM, both winners of this year's Women in Supply Chain award, talk about the ongoing challenge of balancing work and life.
ALOM Technologies Corp.
5 Trends Affecting Supply Chains in New Year
Adaptability has been key as companies looked to their supply chain teams to overcome a steady stream of disruptive events. Looking ahead to 2023, the end is not yet in sight.
Women in Supply Chain: Not About Being the Loudest in the Room, but the Most Effective
Kathleen Smith, global services operations leader for Converge, an Arrow company, and one of Supply & Demand Chain Executive's 2022 Women in Supply Chain award winners, explains why it's not always about being the loudest voice in the room.
Converge, an Arrow company
Women in Supply Chain: Be Comfortable with the Uncomfortable
Abigail Nawrocki, network GM of specialty fulfillment for Shopify Logistics and one of the winners of this year's Women in Supply Chain award, details the importance of getting comfortable with uncomfortable situations.
Deliverr, division of Shopify Logistics
SCN Summit: Future of Supply Chains Kicks off Today
SCN Summit features industry experts and thought leaders from the supply chain space to talk future of supply chains, what's in store for logistics companies, trends and technologies in logistics and more.
Women in Supply Chain: There's a Place in Supply Chain for Everyone
Paige VanFossen, VP, e-commerce operations, DHL Supply Chain, North America and one of this year's Women in Supply Chain award winners, details why the many layers to the supply chain industry make it so everyone can be involved.
Women in Supply Chain: More Gender-Diverse Workplace Produces Higher Performance
Susan Estes, Senior Supply Chain Consultant, Client Services for Open Sky Group and one of this year's Women in Supply Chain award winners, details how a more gender-diverse workplace environment produces higher performance.
Open Sky Group
3 Personal Insights for Women Breaking Through the Trucking Industry
The demand for drivers and the competitive pay that comes with the job make the trucking industry an untapped resource for women. This is an exciting time to level the playing field and build a revolutionary system that supports startups owned by women.
Davis & Davis Transportation LLC
Suicide Prevention and Mental Wellness in the Workplace
For many companies, safety is often thought of in terms of the physical instead of being seen as both physical and psychological.
Avetta
Women in Supply Chain: Don't Fear Speaking Up
Smitha Raphael, Chief Product and Delivery Officer for SnapFulfil and one of this year's Women in Supply Chain award winners, details why it's easy to question what women know about the supply chain, but that goes away once women speak up.
SnapFulfil
Last-Mile
U.S. Drivers Deliver 9 Packages per Hour: Survey
67% of respondents state they now have to complete deliveries to different types of drop-off points, 66% are now expected to work faster, and the same amount (66%) reported an increase in new tasks like identity verification at the door.
Scandit
5 Lessons Learned from the Stage at This Year's Women in Supply Chain Forum
If I could chat with my 25-year-old self and say, one day, you will help create an event where you will not be the only female in the room, I wonder how my 25-year-old self would respond.
Manifest Celebrates Women in LogisticsTech During Women's Lunch
The Women's Lunch will celebrate women in LogisticsTech in partnership with DHL Supply Chain, Daimler Truck North America, Food Logistics and Supply & Demand Chain Executive.
Women in Supply Chain: Step Back and Understand the Big Picture
Amanda Hall, Director of Quality Assurance at DHL Supply Chain, North America and one of this year's Women in Supply Chain winners, advises newcomers to the supply chain industry to take a step back, understand processes and look for areas to improve.

redpajama_set_name: RedPajamaCommonCrawl


With eye health so crucial to quality of life, no patient should have to decline treatment based on cost. Our loan process is fast and efficient, with affordable options – so your patients can get the vision correction procedures they need. All without hidden fees, costs, or hassles to your practice.
You'll also have free technical and marketing support, plus a dedicated Relationship Manager always on-hand to provide expert guidance and help you reach your business goals. It's everything you need to help grow your practice.


text:
Zyn / D'Arenberg GSM Ironstone Pressings
D'Arenberg GSM Ironstone Pressings
Qty : 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 132 144 156 168 180 192 204 216 228 240 252 264 276 288 300 312 324 336 348 360 372 384 396 408 420 432 444 456 468 480
1. Free In-Store Pick Up. Please allow 1- 4 business days.
Most of McLaren Vale's acclaimed and historic vineyards are impregnated with ancient, decomposed laterite granite known as ironstone. Its extraordinary rusty redbrown colour is derived from the iron oxides present in the stone. The larger pieces were cleared from the vineyards in the late 1880's and utilised in many of the buildings at d'Arenberg, most notably the old stables. From entry level to iconic, all d'Arenberg wines have been basket pressed, reds and whites, with all red ferments foot trod during fermentation. d'Arenberg's red wines are typically sophisticated, fragrant, fruit flavoured wines with excellent palate texture, free of excess oak. Long, lively, gritty and youthful tannins full of fragrant fruit and mineral notes, melding seamlessly with complex spice, game and earthy body.
The GSM Ironstone Pressings is opulent, generous and full flavoured, dark fruits and earthy notes are blended with spicy, floral highlights.
Alpha Box & Dice 'Tarot' Rosso 2019
South Australia, Australia
Alpha Box & Dice 'Rebel Rebel' Montepulciano 2016
Alpha Box & Dice 'O' Gruner Veltliner 2021
Alpha Box & Dice 'Dead Winemakers Society' Dolcetto 2016
78 Degrees Sunset Gin
I would like to know the price of this product "D'Arenberg GSM Ironstone Pressings".


It has been manufactured by Avio Interiors, an italian aircraft furniture company, for Airbus 300 and 340 range as Business and First Class seating solution. The back rests, the leg support and the inflatable cushions are all controlled electronically for ease of use.
Some Skyline First Class Aircraft Passenger seats have motorise controls and they work just as they work on an aircraft. The built in power source works with 12V, 24V, 115V or 220V current. Please choose this feature from product options.
140 Degrees motorised recline, lower back inflatable cushion (motorised), electric controlled foot rest.
SkyLine Airbus 340 First Class Seat is in stock and ready to dispacth.


The Journey Tank and Dress is your reminder that life is all about the journey, not the destination. I've always had the motto that if you look good, you feel good…and if you feel good, you play good (how's that for bad grammar??). It doesn't matter what stage of life you're in, this tank and dress has options that will help you have the most fun while you're dealing with all of the curve balls life throws at you. You can use the tank for low-impact workouts such as weight lifting, yoga, or wrangling kids. You can make the perfect summer dress (in two lengths!).
No matter what kind of "play" you're doing, this is the pattern for you.
Information for fabric and notions and size chart can be seen in one of the pictures. This pattern is intended for medium weight 4-way stretch knit with at least 50% stretch horizontally, such as lycra blends with cotton, nylon, supplex, or polyester. Notions include polyester thread to match, ballpoint needles for jersey or stretch needles for swim knits, 1" non-roll elastic for band, 1/4" knit elastic for bust ruching, 3/8" clear elastic for arms/neck/back, Wash Away Wonder Tape (optional but recommended), Removable swim cups or sew-in bra cups (optional), and 1/2″ or 5/8″ Nursing clips or snaps (optional).
This PDF sewing pattern comes in sizes XXS – 3XL. Included in your purchase is a professional tutorial that includes pictures and drawings to help you sew up your new favorite top. The files also include professionally drawn pattern pieces formatted for 8.5×11, A4, and A0 printers. PLEASE only use the A4 formatted pattern pieces if you live in a country where the standard paper size is A4!
Made this in the morning, and wore it to work. Modest neckline with a sweater in the office and cool for summer. Quick sew, flattering and versatility!
In love with this pattern!


text:
Q: How to get HBase IP address for Phoenix URL I can ssh to the Hadoop Cluster and can run the hbase command. But I need to connect using the Phoenix JDBC driver which needs the IP address of the HBase server.
I tried the IP address I used for the cluster with no luck.
This is probably just a generic Hadoop question but where are the IP addresses configured?

A: If you are aware of the hadoop cluster namenodes, then you can try pinging them or send a curl request like below
curl 'http://my-namenode-lv-101:50070/jmx?qry=Hadoop:service=NameNode,name=NameNodeStatus'
{
  "beans" : [ {
    "name" : "Hadoop:service=NameNode,name=NameNodeStatus",
    "modelerType" : "org.apache.hadoop.hdfs.server.namenode.NameNode",
    "SecurityEnabled" : false,
    "NNRole" : "NameNode",
    "HostAndPort" : "my-namenode-lv-101:8020",
    "LastHATransitionTime" : 1561605051455,
    "State" : "standby"
  } ]
}

If the state is Standby, then that is the current inactive node, you have to try the other nodes to find for which the State says 'active' ... example below:
curl 'http://my-namenode-lv-102:50070/jmx?qry=Hadoop:service=NameNode,name=NameNodeStatus'
{
  "beans" : [ {
    "name" : "Hadoop:service=NameNode,name=NameNodeStatus",
    "modelerType" : "org.apache.hadoop.hdfs.server.namenode.NameNode",
    "State" : "active",
    "SecurityEnabled" : false,
    "NNRole" : "NameNode",
    "HostAndPort" : "my-namenode-lv-102:8020",
    "LastHATransitionTime" : 1561605054944
  } ]
}

A: To connect to phoenix-hbase  use the zookeeper address, port & zookeeper.znode.parent configuration's value which is configured in your cluster. (it can be found in your hbase-site.xml file)

redpajama_set_name: RedPajamaStackExchange


It has been awhile since my last post. I have written but not published and I am not sure why.
However I must share this.
I look across the street to a little old man waving madly at me.
"Miss, hello, that was me." He pants excitedly, "That was me as you were walking right up the stairs.". He can hardly catch his breath even though he just got out of his 4-door blue Honda Civic.
"You were walking up the stairs," he repeats.
I nod and glare at him. I was aware of the honk. If he had of looked closely my right leg had momentarily stopped mid-step when he honked his car horn. I had done my best to not show my surprise at the mad honking and kept walking up the stairs without turning around.
Now he was across the street out of his car talking a mile a minute with his European accent.
I heard him get in his car and drive slowly up behind me.
His window rolled down. I am thanking my lucky stars for my martial arts – at least I may stand a chance.
Yes I got it the first time as I forced a sneering smile – stop that car again buddy and you'll be damn sorry.
He sees me dialing my friend as he drives away, yelling out his window.
I get it – bugger off before I hurt you.
Time for a can of mace, a hand gun permit, a car or all three?


text:
Free tuition at Brown/Trinity Rep nurtures inclusive theater training
March 7, 2018, 12:54 a.m.
David L. Ryan/Globe Staff/File
THERE'S NO BUSINESS like show business. Or rather, there's no business that encourages burnout, turnover, and persistent debt among holders of its terminal degree quite like show business.
Brown University took a tremendous step forward on Feb. 26, when it announced it would cover full tuition for students studying for a master of fine arts degree in acting and directing in the school's training program with Trinity Repertory Company. The advocacy of faculty and students was crucial. "I think the tone shifted after we, as a community, started interrogating the way in which Brown/Trinity was not an inclusive environment," said Alexis Green, who graduated from the acting program in 2017. "This breaks down a whole wall for those from lower-income backgrounds," said Green, who helped form the Brown/Trinity Student Resource Board, focused on greater inclusion at Brown. The university deserves credit for responding to criticism and for financially supporting its current and future students.
To be clear: Tuition is far from the entire cost of training a young artist. Brown estimates graduate students' nine-month cost of living to be $22,095. The university cited stipends and positions as teaching assistants and proctors as steps to close that gap.
"Some of my mentors and professors are still in debt now, 20 years after graduating," said Anita Castillo-Halvorssen, a candidate for an MFA in acting at Brown/Trinity. More often than not, modern theater rests upon students' ability to pay for access to major training programs. If an aspiring artist can't take on the burden of debt to enter such programs, their opportunities are sharply limited. And even for those new graduates who do find jobs and parts, the pay can be extremely low.
Financial barriers don't affect everyone equally. "In the theater world, diversity is often hampered by the inability of low-income artists and artists of color to afford to be creators," wrote Brown University theater arts and performance studies department chair Patricia Ybarra in an online statement.
Brown's decision is especially noteworthy because university programs have helped define the place the arts hold in the American public square. "This change in funding . . . signals the idea that art-making, like arts scholarship, is a public good, and that as such it deserves to be supported as part of the mission of a research university," wrote Ybarra.
It's heartening to see private institutions leading the way, especially while the federal government neglects its responsibility. The same nation that once funded the extensive Federal Theatre Project during the New Deal could now lose the National Endowment for the Arts and the National Endowment for the Humanities under President Trump's budget proposal.
Brown/Trinity isn't the only school offering this level of support; the University of California at San Diego also covers full tuition. Harvard's ART Institute offered a graduate program in theater but suspended admissions in 2017, after the US Department of Education gave the program a "failing" grade for burdening its graduates with unmanageable levels of student debt. Perhaps when its training program reopens, the ART Institute will be able to offer prospective students more support. In the meantime, Brown/Trinity meets a higher standard by breaking the cycle.


text:
	Author: PicoleDeLimao
	Date: 05.01.2016
	Add fuuton resistance when kazekage armor is equipped
]]
function Equip(event)
	local caster = event.caster
	local ability = event.ability
	local fuutonDef = ability:GetLevelSpecialValueFor("fuuton_resistance", ability:GetLevel() - 1)
	caster.fuutonDef = caster.fuutonDef + fuutonDef
end

function Unequip(event)
	local caster = event.caster
	local ability = event.ability
	local fuutonDef = ability:GetLevelSpecialValueFor("fuuton_resistance", ability:GetLevel() - 1)
	caster.fuutonDef = caster.fuutonDef - fuutonDef
end

redpajama_set_name: RedPajamaGithub


text:
Check out the latest finds!
Monastery Dining Table
Decrease quantity for Monastery Dining Table
Increase quantity for Monastery Dining Table
Also known as a refectory table, as a refectory was the name of the dining room in monasteries and convents. This style of table was used in monasteries in the middle ages, and over time, the style grew in popularity and was seen commonly in castles, châteaus, and family homes.
It is common for this type of table to have cylindrical bases with a stretcher running the length of the table between them.
This beautiful table was found locally, and has an antique base in a darker finish, with a new top that has been sealed in its natural color. This would make a lovely dining table or a large desk or conference table for an office.
Gorgeous, sturdy pedestals
Naturally finished table top
Seats 10 - 12
84 in L x 44.75 in W x 30.75 in T
Vist us
4238 Frost Street
Marshall, VA 20115
OPEN: Thursday - Monday 11:30 - 5:30
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HONG KONG. The "Tech Trends Symposium 2019 - Roadmap to the Future" gathered an array of industry experts to share exciting advancements.
UAE. A duo of white-hat hackers have earned themselves a brand new Tesla Model 3 after exposing a vulnerability in the car's integrated browser; Tesla is working to release a fix.
Who deserves to win the title of Best Content Creator of Sadeem for 2019?
What are feeds? What is RSS?
Below is a short list of our feeds.
To see all of our feeds click here.
INTERNATIONAL. Episode 1 of new video series discusses just why it is that an industry in a period of unprecedented activity is struggling to capitalise on this demand.


Segment 1: In our Success Interview we talk with entrepreneur and "gratitude specialist," Tina Torres. Tina has built her business by using partnerships to extend her reach and expand the services she provides to clients. Tina talks with us about how she has leveraged partnerships to grow her business.
Segment 2: Our Business Builder Segment focuses on what makes a great course stand out from all the others. Not all courses are created equal, and we give you our tips for making your courses ... See More both valuable and engaging.


text:
Q: Calculating REM for elements other than font-size #sign-up-cta .striking {
  font-family: "Rubik", sans-serif;
  padding-bottom: .75rem;
}

If we use rem for padding or margin, where is it taking the default values from? Then if we do declare padding: 0 and margin: 0 then where is it taking the value from?
The example above .75rem is for the position of the text. What would .75rem be if we don't declare padding or margin?

A: The rem unit is equal to the font-size of the html element, which you can define in your project or is defined by the user's browser settings. The default value in all browsers is 16px (as far as I know).
Rem is used if you want to use the same unit anywhere in your project while still having a responsive design by accomodating to the user's custom set browser font-size.


text:
The ACEC Research Institute has pledge levels that provide opportunities for members, firms and affiliates from all sectors of the industry to engage.
Pledges can be spread over multiple years, making it possible for members at every level to participate in the great work of the Institute while enjoying the benefits of their participation.
See the full list of contributors
why you should support the ACEC Research Institute
ACEC Research Institute one-time gift
The ACEC Research Institute has pledge levels that accommodate leaders from all sectors of the industry and at every stage of their leadership career. Pledges can be spread over 3 – 5 years, making it possible for field leaders at every level to participate in the great work of the Institute while enjoying the benefits of their participation.
ACEC Research Institute Donor Pledge Form
Including the ACEC Research Institute in your charitable estate plans, through a gift from your will or trust, a gift of life insurance, or a gift or assignment of qualified retirement plan assets leaves a lasting legacy for the engineering profession. For information on how to leave a legacy to the Institute, contact Daphne Bryant at dbryant@acec.org.
ACEC Research Institute is a nonprofit organization exempt from income tax under Section 501(c)(3) of the Internal Revenue Code. EIN: 52-1140340. Donations are tax deductible to the extent allowable by law.
Support the ACEC Research Institute When You Shop at Amazon
Do you shop at Amazon? You can support the ACEC Research Institute through Amazon Smile! Every time you make an eligible purchase, Amazon will donate 0.5% of the price to the ACEC Research Institute. Simply login to Amazon Smile where the Institute is already selected for you, and click the "Start Shopping" button. This will add the ACEC Research Institute as the non-profit organization your shopping with support when you buy from Amazon on smile.amazon.com. In order for purchases to qualify for donations, you must start your shopping at smile.amazon.com.
In the Amazon App:
Make sure to have the latest version of the Amazon shopping app. Then go to the bottom right corner, and select the triple bar ( ≡ ). Scroll down to the second to last option, "settings", and then to the third option, "AmazonSmile". If you previously registered with Amazon Smile, it should remember ACEC Research Institute as your non-profit of choice.
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Washington DC, 20005-2605
E-mail: institute@acec.org
© 2022 - ACEC Research Institute / American Council of Engineering Companies - All rights reserved.


Robert Edric is one of our most prolific contemporary novelists. Besides his recent trilogy of crime novels set in Hull, this is his fourteenth novel; the last two have been longlisted for the Man Booker Prize. His work is serious but highly readable. Moral confusions animate it, often in the context of a collapsed society or one on the brink of collapse. His historical backgrounds range widely: the Arctic of the 1845 Franklin Expedition (Broken Lands, 1992); Conrad's Congo (The Book of the Heathen, 2000); Tasmania in 1864 (Elysium, 1995); the First World War (In Desolate Heaven, 1997). They are vividly portrayed, with a lightness of touch that makes one hesitate to call them historical novels. No detail is there just for period flavour.


Hint water is a delicious flavored water that has no sugar or artificial sweeteners. It features just a hint of natural flavoring, just enough, but not too much. Hint's water is really delicious and refreshing. We had the opportunity to try several of their flavors. I loved many of the flavors, including mango-grapefruit and raspberry-lime, but the watermelon was my favorite. My husband really loved the blackberry and my kids' favorites were strawberry-kiwi and watermelon as well. Their other flavors are pomegranate-tangerine, cucumber, pear, honeydew-hibiscus, and lime. There weren't any we didn't like, we just preferred some over others.
Hint is a great alternative to sodas or juices, which contain a lot of sugar and/or high fructose corn syrup. My children really enjoyed it and asked for it at meals. My kids love water and drink a lot of it, but this would really help encourage children who usually don't drink water to drink it. Plus, parents can feel good about it's ingredients.
You can buy Hint Water online at DrinkHint.com, Amazon.com, or any major health food stores, such as Whole Foods, The Fresh Market, or Henry's. You may also be able to find it at your local Starbucks. Check their website to find a retailer near you and don't forget to visit Drink Hint on Facebook.
Thank you to Hint for sending us sample product free of charge to base this review upon.