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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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Identification of immunogenic epitope(s) of Rv2034 recognized by CD4 T-cell clone.To identify the immunogenic epitope(s) in Rv2034, the CD4+ T-cell clone was stimulated with all individual Rv2034 20-mer peptides with 10 aa overlap; Rv2034 recombinant protein; Rv2034 peptide pool; control ESAT-6/CFP10 fusion protein; an Ag85B/ESAT-6/Rv2034 trimeric fusion protein; and negative and positive control conditions. Autologous irradiated PBMC were used as APCs. Both IFN-γ (open bars) and T-cell proliferation (black bars) were determined. CPM bars represent median ranging the highest and lowest value (n = 3) (A). To determine the Rv2034 p81–100 specific response by flow cytometry, the CD4+ T-cell clone was stimulated with Rv2034 p81–100 (B), Rv2034 protein (C) and Rv2034 p11–30 (D) using autologous irradiated PBMC, in the presence of BFA. Intracellular CD154 and Th1 cytokine expression was determined. Data is representative of three independent experiments. Flow cytometry plots show single live CD14−CD19−CD3+CD4+ T cells, the frequency of all subsets of CD3+CD4+ T cells are indicated in the corners of each plot.
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pone-0099203-g003: Identification of immunogenic epitope(s) of Rv2034 recognized by CD4 T-cell clone.To identify the immunogenic epitope(s) in Rv2034, the CD4+ T-cell clone was stimulated with all individual Rv2034 20-mer peptides with 10 aa overlap; Rv2034 recombinant protein; Rv2034 peptide pool; control ESAT-6/CFP10 fusion protein; an Ag85B/ESAT-6/Rv2034 trimeric fusion protein; and negative and positive control conditions. Autologous irradiated PBMC were used as APCs. Both IFN-γ (open bars) and T-cell proliferation (black bars) were determined. CPM bars represent median ranging the highest and lowest value (n = 3) (A). To determine the Rv2034 p81–100 specific response by flow cytometry, the CD4+ T-cell clone was stimulated with Rv2034 p81–100 (B), Rv2034 protein (C) and Rv2034 p11–30 (D) using autologous irradiated PBMC, in the presence of BFA. Intracellular CD154 and Th1 cytokine expression was determined. Data is representative of three independent experiments. Flow cytometry plots show single live CD14−CD19−CD3+CD4+ T cells, the frequency of all subsets of CD3+CD4+ T cells are indicated in the corners of each plot.

Mentions: To identify the specific T cell epitope(s) of Rv2034, the CD4+ T-cell clone was restimulated with single 20-mer peptides from Rv2034. As expected, the Rv2034 peptide pool was strongly recognized by the clone as measured by IFN-γ production and T-cell proliferation (Figure 3A). Rv2034 p81–100 was identified as the dominant immunogenic epitope, while variable responses were observed to Rv2034 p88–107, which overlapped 13 amino acids with Rv2034 p81–100. In addition, recombinant protein Rv2034 and fusion protein Ag85B/ESAT-6/Rv2034 were both recognized, suggesting that Rv2034 epitope is adequately processed from this protein. The fusion protein Ag85B/ESAT-6/Rv2034 includes two early infection phase expressed Mtb proteins (Ag85B and ESAT-6; together designated as H1), fused to the in vivo expressed Rv2034 protein. Inclusion of multiple infection phase related Mtb proteins in a single fusion construct has been shown to improve vaccine efficacy in both mouse and non-human primate models of TB [40], [41], especially if the antigens are expressed during different phases of Mtb infection. It is therefore relevant that the immunogenic epitope present in Rv2034 p81–100 is efficiently processed and presented from both the Rv2034 protein and the trimeric Ag85B/ESAT-6/Rv2034 fusion protein. The negative control proteins HPV16E6, Ag85B and ESAT-6/CFP10 fusion protein, and the negative control peptide HIV-GAG were not recognized, in agreement with the strict Rv2034-specificity. As expected, expression of CD154 and Th1 cytokines was detected upon stimulation of the CD4+ T-cell clone with Rv2034 p81–100 (Figure 3B) and Rv2034 protein (Figure 3C) whereas no activation was observed upon stimulation with negative control peptide p11–30 of Rv2034 (Figure 3D), further demonstrating the specificity of this CD4+ T cell-clone for Rv2034 p81–100.


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)

Identification of immunogenic epitope(s) of Rv2034 recognized by CD4 T-cell clone.To identify the immunogenic epitope(s) in Rv2034, the CD4+ T-cell clone was stimulated with all individual Rv2034 20-mer peptides with 10 aa overlap; Rv2034 recombinant protein; Rv2034 peptide pool; control ESAT-6/CFP10 fusion protein; an Ag85B/ESAT-6/Rv2034 trimeric fusion protein; and negative and positive control conditions. Autologous irradiated PBMC were used as APCs. Both IFN-γ (open bars) and T-cell proliferation (black bars) were determined. CPM bars represent median ranging the highest and lowest value (n = 3) (A). To determine the Rv2034 p81–100 specific response by flow cytometry, the CD4+ T-cell clone was stimulated with Rv2034 p81–100 (B), Rv2034 protein (C) and Rv2034 p11–30 (D) using autologous irradiated PBMC, in the presence of BFA. Intracellular CD154 and Th1 cytokine expression was determined. Data is representative of three independent experiments. Flow cytometry plots show single live CD14−CD19−CD3+CD4+ T cells, the frequency of all subsets of CD3+CD4+ T cells 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-g003: Identification of immunogenic epitope(s) of Rv2034 recognized by CD4 T-cell clone.To identify the immunogenic epitope(s) in Rv2034, the CD4+ T-cell clone was stimulated with all individual Rv2034 20-mer peptides with 10 aa overlap; Rv2034 recombinant protein; Rv2034 peptide pool; control ESAT-6/CFP10 fusion protein; an Ag85B/ESAT-6/Rv2034 trimeric fusion protein; and negative and positive control conditions. Autologous irradiated PBMC were used as APCs. Both IFN-γ (open bars) and T-cell proliferation (black bars) were determined. CPM bars represent median ranging the highest and lowest value (n = 3) (A). To determine the Rv2034 p81–100 specific response by flow cytometry, the CD4+ T-cell clone was stimulated with Rv2034 p81–100 (B), Rv2034 protein (C) and Rv2034 p11–30 (D) using autologous irradiated PBMC, in the presence of BFA. Intracellular CD154 and Th1 cytokine expression was determined. Data is representative of three independent experiments. Flow cytometry plots show single live CD14−CD19−CD3+CD4+ T cells, the frequency of all subsets of CD3+CD4+ T cells are indicated in the corners of each plot.
Mentions: To identify the specific T cell epitope(s) of Rv2034, the CD4+ T-cell clone was restimulated with single 20-mer peptides from Rv2034. As expected, the Rv2034 peptide pool was strongly recognized by the clone as measured by IFN-γ production and T-cell proliferation (Figure 3A). Rv2034 p81–100 was identified as the dominant immunogenic epitope, while variable responses were observed to Rv2034 p88–107, which overlapped 13 amino acids with Rv2034 p81–100. In addition, recombinant protein Rv2034 and fusion protein Ag85B/ESAT-6/Rv2034 were both recognized, suggesting that Rv2034 epitope is adequately processed from this protein. The fusion protein Ag85B/ESAT-6/Rv2034 includes two early infection phase expressed Mtb proteins (Ag85B and ESAT-6; together designated as H1), fused to the in vivo expressed Rv2034 protein. Inclusion of multiple infection phase related Mtb proteins in a single fusion construct has been shown to improve vaccine efficacy in both mouse and non-human primate models of TB [40], [41], especially if the antigens are expressed during different phases of Mtb infection. It is therefore relevant that the immunogenic epitope present in Rv2034 p81–100 is efficiently processed and presented from both the Rv2034 protein and the trimeric Ag85B/ESAT-6/Rv2034 fusion protein. The negative control proteins HPV16E6, Ag85B and ESAT-6/CFP10 fusion protein, and the negative control peptide HIV-GAG were not recognized, in agreement with the strict Rv2034-specificity. As expected, expression of CD154 and Th1 cytokines was detected upon stimulation of the CD4+ T-cell clone with Rv2034 p81–100 (Figure 3B) and Rv2034 protein (Figure 3C) whereas no activation was observed upon stimulation with negative control peptide p11–30 of Rv2034 (Figure 3D), further demonstrating the specificity of this CD4+ T cell-clone for Rv2034 p81–100.

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