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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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Rv2034 responsive CD4+ T-cell clone phenotype.The shown CD4+ T-cell clone that had been expanded was restimulated with the Rv2034 peptide pool and analyzed for the expression of CD154 expression, IFN-γ, TNF-α and IL-2 (black dots). Data is representative of over three independent experiments. CD154 and Th1 cytokine expression of non-activated T cells is indicated in grey dots. 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-g002: Rv2034 responsive CD4+ T-cell clone phenotype.The shown CD4+ T-cell clone that had been expanded was restimulated with the Rv2034 peptide pool and analyzed for the expression of CD154 expression, IFN-γ, TNF-α and IL-2 (black dots). Data is representative of over three independent experiments. CD154 and Th1 cytokine expression of non-activated T cells is indicated in grey dots. 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: Thus, both TB10.4 and Rv2034 T-cell clones could be generated using the CD154 sorting method. As the main aim of this work was the analysis of IVE-TB specific T-cell responses, we further studied a CD4+ T-cell clone that responded to Rv2034 peptide pool stimulation by CD154, IFN-γ, TNF-α and IL-2 expression (Figure 2). First, its clonality was further confirmed by PCR for both TCRα and TCRβ. The detected variable regions consisted of Vα13, Vα27 and Vβ14, following Arden nomenclature [39] (data not shown). Vα27 was predicted to be an unproductive TCRA rearranged sequence due to an out-of-frame junction, whereas Vα13 was successfully rearranged (the international ImMunoGeneTics database (IMTG)). Thus the clone represents a truly clonal population based on TCR genotyping.


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)

Rv2034 responsive CD4+ T-cell clone phenotype.The shown CD4+ T-cell clone that had been expanded was restimulated with the Rv2034 peptide pool and analyzed for the expression of CD154 expression, IFN-γ, TNF-α and IL-2 (black dots). Data is representative of over three independent experiments. CD154 and Th1 cytokine expression of non-activated T cells is indicated in grey dots. 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-g002: Rv2034 responsive CD4+ T-cell clone phenotype.The shown CD4+ T-cell clone that had been expanded was restimulated with the Rv2034 peptide pool and analyzed for the expression of CD154 expression, IFN-γ, TNF-α and IL-2 (black dots). Data is representative of over three independent experiments. CD154 and Th1 cytokine expression of non-activated T cells is indicated in grey dots. 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: Thus, both TB10.4 and Rv2034 T-cell clones could be generated using the CD154 sorting method. As the main aim of this work was the analysis of IVE-TB specific T-cell responses, we further studied a CD4+ T-cell clone that responded to Rv2034 peptide pool stimulation by CD154, IFN-γ, TNF-α and IL-2 expression (Figure 2). First, its clonality was further confirmed by PCR for both TCRα and TCRβ. The detected variable regions consisted of Vα13, Vα27 and Vβ14, following Arden nomenclature [39] (data not shown). Vα27 was predicted to be an unproductive TCRA rearranged sequence due to an out-of-frame junction, whereas Vα13 was successfully rearranged (the international ImMunoGeneTics database (IMTG)). Thus the clone represents a truly clonal population based on TCR genotyping.

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