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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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Subset analysis of the CD4 T-cell clone.The CD4+ T-cell clone was stimulated with Rv2034 p81–100 in presence of BFA and analyzed the expression of CD154 (A-D), IFN-γ and IL-17 (A) and additional markers: the transcriptional factor markers T-bet (Th1), GATA-3 (Th2), RORγt (Th17) and FoxP3 (Treg) (B); T-cell regulatory markers: CD39, IL-10 and CD25 (C); T-cell cytotoxicity markers: Granzyme B, CD107a and Perforin (D). Markers were analyzed using the mAb subset panels described in the materials and methods section. Data is representative for two independent experiments. Stimulated T cells are indicated in black and unstimulated T cells in grey. Dot blots show single live CD14−CD19−CD3+CD4+ T cells. The frequency of all CD3+CD4+ T cell subsets identified upon stimulation are indicated in the corners of each plot.
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pone-0099203-g004: Subset analysis of the CD4 T-cell clone.The CD4+ T-cell clone was stimulated with Rv2034 p81–100 in presence of BFA and analyzed the expression of CD154 (A-D), IFN-γ and IL-17 (A) and additional markers: the transcriptional factor markers T-bet (Th1), GATA-3 (Th2), RORγt (Th17) and FoxP3 (Treg) (B); T-cell regulatory markers: CD39, IL-10 and CD25 (C); T-cell cytotoxicity markers: Granzyme B, CD107a and Perforin (D). Markers were analyzed using the mAb subset panels described in the materials and methods section. Data is representative for two independent experiments. Stimulated T cells are indicated in black and unstimulated T cells in grey. Dot blots show single live CD14−CD19−CD3+CD4+ T cells. The frequency of all CD3+CD4+ T cell subsets identified upon stimulation are indicated in the corners of each plot.

Mentions: The Rv2034 responding CD4+ T-cell clone was subsequently analyzed for Th17, Th2, regulatory and cytotoxicity T cell (CTL) markers to further specify its functional activities. The Rv2034 p81–100 peptide-induced CD154 expression correlated with IFN-γ expression (Figure 4A) while no IL-17 expression was observed. Transcription factor analysis revealed expression of T-bet (Figure 4B), a transcription factor that regulates Th1 development and controls IFN-γ production [42], [43], but there was no expression of GATA-3 (Th2 [44]), RORγt (Th17 [45], [46]) or FOXP3 (Treg [47]). T-bet was constitutively expressed in both p81–100 peptide stimulated and unstimulated T cells. Furthermore, the clone did not reveal any specific combination(s) of other reported T-cell regulatory markers (Figure 4C): no IL-10 could be detected although CD25 and CD39 [48] were constitutively expressed, which, however, are also expressed by activated non-Tregs [49], [50]. Of note, CD25 expressing non-Treg cells mostly express intermediate levels of CD25 (CD25int), whereas Tregs typically express high levels of CD25 (CD25hi) [49]. The expansion of our T-cell clone was mediated by IL-2 which theoretically could have enhanced CD25 expression [51]. Finally, the T-cell clone expressed granzyme B and CD107a but no detectable perforin (Figure 4D). The expression of granzyme B and CD107a was antigen-stimulation dependent. Cytotoxic CD4+ T cells have been reported not only in viral but also in mycobacterial infections [52]–[54]. The expression of the degranulation marker CD107a indicates the release of lysosomal products from granules after antigen-specific activation of the T cell, which likely includes granzyme B. Anti-CD107a antibodies were administered during antigen stimulation of the T-cell clone, because once CD107a is released with its vesicles’ granule contents and becomes integrated in the membrane of the cell, the antibodies will bind to CD107a and are subsequently internalized together with CD107, enabling detection of degranulation. Further studies are required to determine whether granulysin and other granzymes are released by the T-cell clone. These data were obtained by ICS using brefeldin A to accumulate intracellular cytokines by preventing their secretion. All subset conditions were also analyzed using monensin, which acts as protein transport inhibitor via disruption of trans-Golgi protein transport whereas BFA inhibits protein transport between endoplasmic reticulum (ER) and the Golgi apparatus. Monensin, however in our hands did not enhance any of the responses (data not shown). Taken together, these observations show that the Rv2034-specific CD4+ T cell-clone is a pure Th1 clone expressing IFN-γ, TNF-α, IL-2 and several cytotoxic granule markers.


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)

Subset analysis of the CD4 T-cell clone.The CD4+ T-cell clone was stimulated with Rv2034 p81–100 in presence of BFA and analyzed the expression of CD154 (A-D), IFN-γ and IL-17 (A) and additional markers: the transcriptional factor markers T-bet (Th1), GATA-3 (Th2), RORγt (Th17) and FoxP3 (Treg) (B); T-cell regulatory markers: CD39, IL-10 and CD25 (C); T-cell cytotoxicity markers: Granzyme B, CD107a and Perforin (D). Markers were analyzed using the mAb subset panels described in the materials and methods section. Data is representative for two independent experiments. Stimulated T cells are indicated in black and unstimulated T cells in grey. Dot blots show single live CD14−CD19−CD3+CD4+ T cells. The frequency of all CD3+CD4+ T cell subsets identified upon stimulation are indicated in the corners of each plot.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099203-g004: Subset analysis of the CD4 T-cell clone.The CD4+ T-cell clone was stimulated with Rv2034 p81–100 in presence of BFA and analyzed the expression of CD154 (A-D), IFN-γ and IL-17 (A) and additional markers: the transcriptional factor markers T-bet (Th1), GATA-3 (Th2), RORγt (Th17) and FoxP3 (Treg) (B); T-cell regulatory markers: CD39, IL-10 and CD25 (C); T-cell cytotoxicity markers: Granzyme B, CD107a and Perforin (D). Markers were analyzed using the mAb subset panels described in the materials and methods section. Data is representative for two independent experiments. Stimulated T cells are indicated in black and unstimulated T cells in grey. Dot blots show single live CD14−CD19−CD3+CD4+ T cells. The frequency of all CD3+CD4+ T cell subsets identified upon stimulation are indicated in the corners of each plot.
Mentions: The Rv2034 responding CD4+ T-cell clone was subsequently analyzed for Th17, Th2, regulatory and cytotoxicity T cell (CTL) markers to further specify its functional activities. The Rv2034 p81–100 peptide-induced CD154 expression correlated with IFN-γ expression (Figure 4A) while no IL-17 expression was observed. Transcription factor analysis revealed expression of T-bet (Figure 4B), a transcription factor that regulates Th1 development and controls IFN-γ production [42], [43], but there was no expression of GATA-3 (Th2 [44]), RORγt (Th17 [45], [46]) or FOXP3 (Treg [47]). T-bet was constitutively expressed in both p81–100 peptide stimulated and unstimulated T cells. Furthermore, the clone did not reveal any specific combination(s) of other reported T-cell regulatory markers (Figure 4C): no IL-10 could be detected although CD25 and CD39 [48] were constitutively expressed, which, however, are also expressed by activated non-Tregs [49], [50]. Of note, CD25 expressing non-Treg cells mostly express intermediate levels of CD25 (CD25int), whereas Tregs typically express high levels of CD25 (CD25hi) [49]. The expansion of our T-cell clone was mediated by IL-2 which theoretically could have enhanced CD25 expression [51]. Finally, the T-cell clone expressed granzyme B and CD107a but no detectable perforin (Figure 4D). The expression of granzyme B and CD107a was antigen-stimulation dependent. Cytotoxic CD4+ T cells have been reported not only in viral but also in mycobacterial infections [52]–[54]. The expression of the degranulation marker CD107a indicates the release of lysosomal products from granules after antigen-specific activation of the T cell, which likely includes granzyme B. Anti-CD107a antibodies were administered during antigen stimulation of the T-cell clone, because once CD107a is released with its vesicles’ granule contents and becomes integrated in the membrane of the cell, the antibodies will bind to CD107a and are subsequently internalized together with CD107, enabling detection of degranulation. Further studies are required to determine whether granulysin and other granzymes are released by the T-cell clone. These data were obtained by ICS using brefeldin A to accumulate intracellular cytokines by preventing their secretion. All subset conditions were also analyzed using monensin, which acts as protein transport inhibitor via disruption of trans-Golgi protein transport whereas BFA inhibits protein transport between endoplasmic reticulum (ER) and the Golgi apparatus. Monensin, however in our hands did not enhance any of the responses (data not shown). Taken together, these observations show that the Rv2034-specific CD4+ T cell-clone is a pure Th1 clone expressing IFN-γ, TNF-α, IL-2 and several cytotoxic granule markers.

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