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Clonal analysis of the T-cell response to in vivo expressed Mycobacterium tuberculosis protein Rv2034, using a CD154 expression based T-cell cloning method.

Commandeur S, Coppola M, Dijkman K, Friggen AH, van Meijgaarden KE, van den Eeden SJ, Wilson L, van der Ploeg-van Schip JJ, Franken KL, Geluk A, Ottenhoff TH - PLoS ONE (2014)

Bottom Line: Importantly, the T-cell clone was able to inhibit Mtb outgrowth from infected monocytes significantly.The characterization of the polyfunctional and Mtb inhibitory T-cell response to IVE-TB Rv2034 at the clonal level provides detailed further insights into the potential of IVE-TB antigens as new vaccine candidate antigens in TB.Our new approach allowed the identification of T-cell subsets that likely play a significant role in controlling Mtb infection, and can be applied to the analysis of T-cell responses in patient populations.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.

ABSTRACT
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of death worldwide. A better understanding of the role of CD4+ and CD8+ T cells, which are both important to TB protection, is essential to unravel the mechanisms of protection and to identify the key antigens seen by these T cells. We have recently identified a set of in vivo expressed Mtb genes (IVE-TB) which is expressed during in vivo pulmonary infection in mice, and shown that their encoded antigens are potently recognized by polyclonal T cells from tuberculin skin test-positive, in vitro ESAT-6/CFP10-responsive individuals. Here we have cloned T cells specific for one of these newly identified in vivo expressed Mtb (IVE-TB) antigens, Rv2034. T cells were enriched based on the expression of CD154 (CD40L), which represents a new method for selecting antigen-specific (low frequency) T cells independent of their specific function. An Rv2034-specific CD4+ T-cell clone expressed the Th1 markers T-bet, IFN-γ, TNF-α, IL-2 and the cytotoxicity related markers granzyme B and CD107a as measured by flow cytometry. The clone specifically recognized Rv2034 protein, Rv2034 peptide p81-100 and Mtb lysate. Remarkably, while the recognition of the dominant p81-100 epitope was HLA-DR restricted, the T-cell clone also recognized a neighboring epitope (p88-107) in an HLA-DR- as well as HLA-DQ1-restricted fashion. Importantly, the T-cell clone was able to inhibit Mtb outgrowth from infected monocytes significantly. The characterization of the polyfunctional and Mtb inhibitory T-cell response to IVE-TB Rv2034 at the clonal level provides detailed further insights into the potential of IVE-TB antigens as new vaccine candidate antigens in TB. Our new approach allowed the identification of T-cell subsets that likely play a significant role in controlling Mtb infection, and can be applied to the analysis of T-cell responses in patient populations.

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PBMC recognition of TB10.4 and IVE-TB antigen Rv2034.PBMC from a PPD+ donor were stimulated with different stimuli for 6 days and IFN-γ production (pg/ml) was determined in the supernatants. Both TB10.4 protein (10 µg/ml) (A) and Rv2034 protein (10 µg/ml) (B) were analyzed as well as control mitogen PHA and Mtb derived PPD (A and B). Medium values (unstimulated PBMC) were subtracted. IFN-γ concentrations were determined from triplicate-pooled supernatant. A cut-off value was set arbitrarily at 100 pg/ml.
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pone-0099203-g001: PBMC recognition of TB10.4 and IVE-TB antigen Rv2034.PBMC from a PPD+ donor were stimulated with different stimuli for 6 days and IFN-γ production (pg/ml) was determined in the supernatants. Both TB10.4 protein (10 µg/ml) (A) and Rv2034 protein (10 µg/ml) (B) were analyzed as well as control mitogen PHA and Mtb derived PPD (A and B). Medium values (unstimulated PBMC) were subtracted. IFN-γ concentrations were determined from triplicate-pooled supernatant. A cut-off value was set arbitrarily at 100 pg/ml.

Mentions: First, PBMC of an in vitro PPD+ donor known to respond to these antigens (Figure 1A and 1B) were stimulated with peptide pools from TB10.4 or Rv2034 for one week in the presence of IL-2, followed by re-stimulation with the peptide pool for 16 hours, in the presence of anti-CD40 antibodies to inhibit CD154-CD40 interactions [6], preventing loss of surface expressed CD154 [28], [29]. CD154 positive cells were then sorted using Magnetic-activated Cell Sorting (MACS) and seeded at 0.3 or 1 cell per well densities as described in detail in the materials and methods section. To analyze the antigen specificities of the expanded T-cell clones, cell populations were gated based on live CD14−CD19−CD3+ T cells. For TB10.4, eight clones showed responses towards TB10.4 peptide pool stimulation (Table 1). Out of these, five T-cell clones were CD154+CD4+ and produced either IFN-γ, TNF-α and IL-2, or TNF-α and IL-2. Two clones appeared to be CD8+ T cells that produced IFN-γ, with one of these producing a relatively low amount of IL-2 as well. CD154 is known to be expressed on activated CD4+ T cells, but can also be upregulated on a fraction of CD8+ T cells [30]. However, none of the CD8+ T cells expressed CD154. Somewhat surprisingly, one T-cell clone was CD3+/CD4−/CD8−, (double negative (DN) T cells) and did not express CD154 either, nevertheless, CD3+ CD4−/CD8− are able to express CD154 [31]. This DN T-cell clone produced IFN-γ in response to TB10.4 antigen. The remaining expanded T-cell cultures were TB10.4 non-responsive and are indicated in Table 1.


Clonal analysis of the T-cell response to in vivo expressed Mycobacterium tuberculosis protein Rv2034, using a CD154 expression based T-cell cloning method.

Commandeur S, Coppola M, Dijkman K, Friggen AH, van Meijgaarden KE, van den Eeden SJ, Wilson L, van der Ploeg-van Schip JJ, Franken KL, Geluk A, Ottenhoff TH - PLoS ONE (2014)

PBMC recognition of TB10.4 and IVE-TB antigen Rv2034.PBMC from a PPD+ donor were stimulated with different stimuli for 6 days and IFN-γ production (pg/ml) was determined in the supernatants. Both TB10.4 protein (10 µg/ml) (A) and Rv2034 protein (10 µg/ml) (B) were analyzed as well as control mitogen PHA and Mtb derived PPD (A and B). Medium values (unstimulated PBMC) were subtracted. IFN-γ concentrations were determined from triplicate-pooled supernatant. A cut-off value was set arbitrarily at 100 pg/ml.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048274&req=5

pone-0099203-g001: PBMC recognition of TB10.4 and IVE-TB antigen Rv2034.PBMC from a PPD+ donor were stimulated with different stimuli for 6 days and IFN-γ production (pg/ml) was determined in the supernatants. Both TB10.4 protein (10 µg/ml) (A) and Rv2034 protein (10 µg/ml) (B) were analyzed as well as control mitogen PHA and Mtb derived PPD (A and B). Medium values (unstimulated PBMC) were subtracted. IFN-γ concentrations were determined from triplicate-pooled supernatant. A cut-off value was set arbitrarily at 100 pg/ml.
Mentions: First, PBMC of an in vitro PPD+ donor known to respond to these antigens (Figure 1A and 1B) were stimulated with peptide pools from TB10.4 or Rv2034 for one week in the presence of IL-2, followed by re-stimulation with the peptide pool for 16 hours, in the presence of anti-CD40 antibodies to inhibit CD154-CD40 interactions [6], preventing loss of surface expressed CD154 [28], [29]. CD154 positive cells were then sorted using Magnetic-activated Cell Sorting (MACS) and seeded at 0.3 or 1 cell per well densities as described in detail in the materials and methods section. To analyze the antigen specificities of the expanded T-cell clones, cell populations were gated based on live CD14−CD19−CD3+ T cells. For TB10.4, eight clones showed responses towards TB10.4 peptide pool stimulation (Table 1). Out of these, five T-cell clones were CD154+CD4+ and produced either IFN-γ, TNF-α and IL-2, or TNF-α and IL-2. Two clones appeared to be CD8+ T cells that produced IFN-γ, with one of these producing a relatively low amount of IL-2 as well. CD154 is known to be expressed on activated CD4+ T cells, but can also be upregulated on a fraction of CD8+ T cells [30]. However, none of the CD8+ T cells expressed CD154. Somewhat surprisingly, one T-cell clone was CD3+/CD4−/CD8−, (double negative (DN) T cells) and did not express CD154 either, nevertheless, CD3+ CD4−/CD8− are able to express CD154 [31]. This DN T-cell clone produced IFN-γ in response to TB10.4 antigen. The remaining expanded T-cell cultures were TB10.4 non-responsive and are indicated in Table 1.

Bottom Line: Importantly, the T-cell clone was able to inhibit Mtb outgrowth from infected monocytes significantly.The characterization of the polyfunctional and Mtb inhibitory T-cell response to IVE-TB Rv2034 at the clonal level provides detailed further insights into the potential of IVE-TB antigens as new vaccine candidate antigens in TB.Our new approach allowed the identification of T-cell subsets that likely play a significant role in controlling Mtb infection, and can be applied to the analysis of T-cell responses in patient populations.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.

ABSTRACT
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of death worldwide. A better understanding of the role of CD4+ and CD8+ T cells, which are both important to TB protection, is essential to unravel the mechanisms of protection and to identify the key antigens seen by these T cells. We have recently identified a set of in vivo expressed Mtb genes (IVE-TB) which is expressed during in vivo pulmonary infection in mice, and shown that their encoded antigens are potently recognized by polyclonal T cells from tuberculin skin test-positive, in vitro ESAT-6/CFP10-responsive individuals. Here we have cloned T cells specific for one of these newly identified in vivo expressed Mtb (IVE-TB) antigens, Rv2034. T cells were enriched based on the expression of CD154 (CD40L), which represents a new method for selecting antigen-specific (low frequency) T cells independent of their specific function. An Rv2034-specific CD4+ T-cell clone expressed the Th1 markers T-bet, IFN-γ, TNF-α, IL-2 and the cytotoxicity related markers granzyme B and CD107a as measured by flow cytometry. The clone specifically recognized Rv2034 protein, Rv2034 peptide p81-100 and Mtb lysate. Remarkably, while the recognition of the dominant p81-100 epitope was HLA-DR restricted, the T-cell clone also recognized a neighboring epitope (p88-107) in an HLA-DR- as well as HLA-DQ1-restricted fashion. Importantly, the T-cell clone was able to inhibit Mtb outgrowth from infected monocytes significantly. The characterization of the polyfunctional and Mtb inhibitory T-cell response to IVE-TB Rv2034 at the clonal level provides detailed further insights into the potential of IVE-TB antigens as new vaccine candidate antigens in TB. Our new approach allowed the identification of T-cell subsets that likely play a significant role in controlling Mtb infection, and can be applied to the analysis of T-cell responses in patient populations.

Show MeSH
Related in: MedlinePlus