Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mycobacterium tuberculosis -induced BAL gene expression signature in latent tuberculosis infection is dominated by pleiotropic effects of CD4+ T cell-dependent IFNγ production despite the presence of polyfunctional T cells within the airways

doi: 10.4049/jimmunol.1900230

Figure Lengend Snippet: BAL cell parameters in LTBI and Mtb -naïve subjects

Article Snippet: For T-cell subset depletion, BAL cells were incubated with magnetized EasySep antibodies (CD4 #18052, CD8 #18053 StemCell Technologies, Cambridge MA) and collected following passage through EasySep magnet using EasySep human cell depletion protocols.

Techniques: Cell Counting

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mycobacterium tuberculosis -induced BAL gene expression signature in latent tuberculosis infection is dominated by pleiotropic effects of CD4+ T cell-dependent IFNγ production despite the presence of polyfunctional T cells within the airways

doi: 10.4049/jimmunol.1900230

Figure Lengend Snippet: Canonical pathways associated with Mtb- induced BAL cell gene expression in LTBI subjects

Article Snippet: For T-cell subset depletion, BAL cells were incubated with magnetized EasySep antibodies (CD4 #18052, CD8 #18053 StemCell Technologies, Cambridge MA) and collected following passage through EasySep magnet using EasySep human cell depletion protocols.

Techniques: Expressing, Activation Assay, Cell Differentiation, Inhibition

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mycobacterium tuberculosis -induced BAL gene expression signature in latent tuberculosis infection is dominated by pleiotropic effects of CD4+ T cell-dependent IFNγ production despite the presence of polyfunctional T cells within the airways

doi: 10.4049/jimmunol.1900230

Figure Lengend Snippet: CD4-depletion of BAL cells from LTBI subjects results in loss of significance of several Mtb-induced pathways associated with the impact of IFNγ production (1A); in contrast, the significance of pathways associated with IL-17 production are not reduced in CD4-depleted LTBI BAL samples (1B). In both figures, significance is expressed as -log (p-value). Dark blue bars represent findings from unsorted BAL cells whereas light blue bars represent p-values of Mtb-induced gene expression in BAL cells from which CD4+ T cells had been removed by magnetic bead sorting.

Article Snippet: For T-cell subset depletion, BAL cells were incubated with magnetized EasySep antibodies (CD4 #18052, CD8 #18053 StemCell Technologies, Cambridge MA) and collected following passage through EasySep magnet using EasySep human cell depletion protocols.

Techniques: Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mycobacterium tuberculosis -induced BAL gene expression signature in latent tuberculosis infection is dominated by pleiotropic effects of CD4+ T cell-dependent IFNγ production despite the presence of polyfunctional T cells within the airways

doi: 10.4049/jimmunol.1900230

Figure Lengend Snippet: BAL cell Mtb-induced canonical pathways that demonstrated a reduction in p-value of at least 2 logs following depletion of CD4+ T cells. Heat maps of -log (p-value) in unsorted BAL samples from LTBI subjects, CD4-depleted BAL cells from LTBI subjects, and unsorted BAL from Mtb-naïve subjects are presented in Figure 2A. For the 12 “signature” pathways (top), pathways significance was reduced by >2 logs in both LTBI BAL from which CD4+ T cells had been depleted and in Mtb-naïve subjects. For 22 “non-signature” pathways, decrease in -log (p-value) of >2 log was observed following CD4 depletion from LTBI BAL, but not in that of Mtb-naïve subjects. The -log p-values for 12 “signature” pathways are presented graphically in 2B. -log (p-values) for LTBI-unsorted BAL cells are presented in dark blue bars. Bars for LTBI CD4-depleted BAL cells are light blue and red bars represent -log (p-values) for unsorted BAL cells of Mtb-naïve subjects.

Article Snippet: For T-cell subset depletion, BAL cells were incubated with magnetized EasySep antibodies (CD4 #18052, CD8 #18053 StemCell Technologies, Cambridge MA) and collected following passage through EasySep magnet using EasySep human cell depletion protocols.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mycobacterium tuberculosis -induced BAL gene expression signature in latent tuberculosis infection is dominated by pleiotropic effects of CD4+ T cell-dependent IFNγ production despite the presence of polyfunctional T cells within the airways

doi: 10.4049/jimmunol.1900230

Figure Lengend Snippet: Mtb -induced gene expression signature in baseline bronchoalveolar lavage cells from individuals with LTBI

Article Snippet: For T-cell subset depletion, BAL cells were incubated with magnetized EasySep antibodies (CD4 #18052, CD8 #18053 StemCell Technologies, Cambridge MA) and collected following passage through EasySep magnet using EasySep human cell depletion protocols.

Techniques: Expressing, Binding Assay, Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mycobacterium tuberculosis -induced BAL gene expression signature in latent tuberculosis infection is dominated by pleiotropic effects of CD4+ T cell-dependent IFNγ production despite the presence of polyfunctional T cells within the airways

doi: 10.4049/jimmunol.1900230

Figure Lengend Snippet: Type I IFN-associated genes identified as impacted CD4+ and CD8+ T cells on Mtb-induced BAL cell gene expression in LTBI. The figure shows 30 Type I IFN-associated upstream regulators and the degree to which the significance of their associations with the dataset were altered by depletion of CD4+ (black) or CD8+ (gray) T cells, expressed as the change in -log of p-values. As indicated, although both T-cell subset depletion impacted the significance of multiple regulators, the magnitude of this effect was far greater for CD4+ T-cells that for CD8+ cells.

Article Snippet: For T-cell subset depletion, BAL cells were incubated with magnetized EasySep antibodies (CD4 #18052, CD8 #18053 StemCell Technologies, Cambridge MA) and collected following passage through EasySep magnet using EasySep human cell depletion protocols.

Techniques: Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mycobacterium tuberculosis -induced BAL gene expression signature in latent tuberculosis infection is dominated by pleiotropic effects of CD4+ T cell-dependent IFNγ production despite the presence of polyfunctional T cells within the airways

doi: 10.4049/jimmunol.1900230

Figure Lengend Snippet: Top 20 upstream regulators not associated with Type I IFN, as based on impact of T-cell depletion on –log (adjusted p-values)

Article Snippet: For T-cell subset depletion, BAL cells were incubated with magnetized EasySep antibodies (CD4 #18052, CD8 #18053 StemCell Technologies, Cambridge MA) and collected following passage through EasySep magnet using EasySep human cell depletion protocols.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mycobacterium tuberculosis -induced BAL gene expression signature in latent tuberculosis infection is dominated by pleiotropic effects of CD4+ T cell-dependent IFNγ production despite the presence of polyfunctional T cells within the airways

doi: 10.4049/jimmunol.1900230

Figure Lengend Snippet: Cluster analysis of expression of the 47 signature genes in Mtb-naïve subjects (4A) and individuals with LTBI (4B). Clustering was performed on a blinded basis according to patterns of responses of the 47-gene signature without regard to the study conditions of each sample. As illustrated in the y-axis of each figure, the clustering of the signature genes was substantially different in Mtb-naïve and LTBI subject groups, leading to the need to present the results in separate heat maps. As indicated on the x-axes, samples from each subject group were segregated into two clusters. In Mtb-naïve subjects (4A), uninfected samples are represented by blue rectangles, whereas Mtb-infected samples are indicated in red. This clustering effectively separates responses of uninfected vs Mtb-infected BAL cells, with the exception of one subject whose uninfected cells gave responses more typical of the Mtb-infected cells (green arrow). For each LTBI subject, three samples are represented, with blue again indicating unsorted, uninfected cells and red unsorted, Mtb-infected cells, whereas black rectangles indicate CD4-depleted, Mtb-infected BAL cells (4B). Despite the representation of three sample types, these responses remain classified into two clusters, reflecting that finding that Mtb-induced gene expression of the signature 47 genes following CD4+ T cell depletion was largely the same as that observed in unsorted, uninfected BAL cells of the same individuals. Unsorted Mtb-infected BAL samples from all 11 LTBI subjects segregated to the same cluster, which also included a single uninfected sample, as well as a single sample infected following CD4 T-cell depletion (green arrows). Of note, both of these “outlier” samples were from the same individual.

Article Snippet: For T-cell subset depletion, BAL cells were incubated with magnetized EasySep antibodies (CD4 #18052, CD8 #18053 StemCell Technologies, Cambridge MA) and collected following passage through EasySep magnet using EasySep human cell depletion protocols.

Techniques: Expressing, Infection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mycobacterium tuberculosis -induced BAL gene expression signature in latent tuberculosis infection is dominated by pleiotropic effects of CD4+ T cell-dependent IFNγ production despite the presence of polyfunctional T cells within the airways

doi: 10.4049/jimmunol.1900230

Figure Lengend Snippet: The top networks identified through analysis of Mtb-induced BAL cell gene expression in LTBI subjects. Schematic figures illustrate gene interactions in these top 3 networks, A. “infectious diseases; endocrine system disorders; gastrointestinal diseases”, B. antimicrobial response; inflammatory response; cancer”, and C. “cellular function and maintenance, hematological system development and function; cellular development”. For each network figure (left), shading indicates degrees of significant upregulation (red) and downregulation (green) of gene expression in unsorted BAL cells from LTBI subjects. Heat maps to the right of each figure indicate Mtb-induced expression of all network genes in this condition, as well as in CD4-depleted cells of LTBI subjects and unsorted BAL cells of Mtb-naïve controls.

Article Snippet: For T-cell subset depletion, BAL cells were incubated with magnetized EasySep antibodies (CD4 #18052, CD8 #18053 StemCell Technologies, Cambridge MA) and collected following passage through EasySep magnet using EasySep human cell depletion protocols.

Techniques: Expressing, Cell Function Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mycobacterium tuberculosis -induced BAL gene expression signature in latent tuberculosis infection is dominated by pleiotropic effects of CD4+ T cell-dependent IFNγ production despite the presence of polyfunctional T cells within the airways

doi: 10.4049/jimmunol.1900230

Figure Lengend Snippet: Novel gene expression network representing the interrelationships of genes identified as the signature of CD4+ T-cell associated Mtb-induced gene expression in BAL cells from individuals with LTBI. Again, the figure represents gene expression in unsorted BAL samples from LTBI subjects; upregulated genes are represented with red shading and downregulated ones in green; as illustrated, all but 2 of the 47 genes within the identified signature have defined connections to a network that is centered on IFNγ. The associated heat map schematically compares expression of all network genes in this condition and in CD4+-depleted BAL cells from LTBI subjects and unsorted BAL cells from Mtb-naïve individuals using the same shading key. As indicted, significant Mtb-induced gene expression is almost completely absent within this network in these additional conditions.

Article Snippet: For T-cell subset depletion, BAL cells were incubated with magnetized EasySep antibodies (CD4 #18052, CD8 #18053 StemCell Technologies, Cambridge MA) and collected following passage through EasySep magnet using EasySep human cell depletion protocols.

Techniques: Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mycobacterium tuberculosis -induced BAL gene expression signature in latent tuberculosis infection is dominated by pleiotropic effects of CD4+ T cell-dependent IFNγ production despite the presence of polyfunctional T cells within the airways

doi: 10.4049/jimmunol.1900230

Figure Lengend Snippet: Mtb-responsive resident airway CD4+ T-cells of LTBI individuals produce multiple cytokines. Supernatants of Mtb-infected BAL cells from cultures used in our gene expression studies were evaluated for production of IFNγ, TNFα, IL-2 and IL-17. As illustrated in Figure 7A, levels of IFNγ, IL-2, and IL-17 were all significantly reduced in cultures of BAL cells from which CD4+ T cells were depleted as compared to those of unsorted BAL cells (based on paired t-tests); reduction in TNFα was observed as well but was not statistically significant. In contrast, depletion of CD8+ T cells had no significant impact on production on any of these cytokines by BAL cell of LTBI subjects (not shown). In all figures, results are represented as cytokine levels for Mtb-infected cultures subtracted from cultures of uninfected, unsorted BAL cells from the same individual. Cytokine levels are reported as pg/mL of culture supernatants, with the exception of TNFα, which is reported as ng/mL. Based on these findings, we also examined polyfunctionality of BAL CD4+ TEM from LTBI subjects using intracellular cytokine staining for these same four cytokines in response in vitro stimulation with PPD. Figure 7B shows the percentage of effector-memory (CD45RA-/CCR7-) CD4+ T cells (TEM) in each subject’s samples that produced each of the possible 15 cytokine combinations in response to PPD. As illustrated, the most commonly observed PPD-induced cytokine profiles were single production of TNFα and dual production of TNFα and IFNγ, with polyfunctional production of TNFα, IFNγ and IL-2 being the next most common. These findings are represented in pie chart form in Figure 7C. The very low number of CD4+ TEM producing all 4 cytokines are indicated by the thin black slice pointing straight upward; from there, the remaining combinations of three, two and single cytokines proceed clockwise in the same order displayed in 7B. The colored arcs indicate production of each of the measured cytokines. As indicated, TNFα (dark blue arc) is the most frequently observed cytokine produced by PPD-responsive BAL CD4+ TEM, followed by IFNγ (red arc), IL-2 (green arc), and IL-17 (light blue arc).

Article Snippet: For T-cell subset depletion, BAL cells were incubated with magnetized EasySep antibodies (CD4 #18052, CD8 #18053 StemCell Technologies, Cambridge MA) and collected following passage through EasySep magnet using EasySep human cell depletion protocols.

Techniques: Infection, Expressing, Staining, In Vitro, Produced