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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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Recognition of native Rv2034 in Mtb lysate by CD4+ T-cell clone.Mtb lysates (10 µg) derived from Mtb grown in log-phase-, starvation- and hypoxic- conditions and recombinant protein Rv2034 (0.4 µg) were analyzed by western blotting for their recognition by specific antibodies using sera of Rv2034-immunized or non-immunized HLA-DR3 transgenic mice (A). Monocyte derived matured DCs were loaded with Rv2034 p81–100, Rv2034 protein and different conditions of Mtb lysate (100 µg/ml) for 24h. Subsequently, the CD4+ T-cell clone was incubated with these antigen-loaded DCs and after 2 hours BFA was added and incubated o/n. CD154 expression was determined using flow cytometry (B). Data represent two independent experiments. An HA-specific T-cell clone was included as a negative control (C). Medium background values were subtracted for each response.
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pone-0099203-g006: Recognition of native Rv2034 in Mtb lysate by CD4+ T-cell clone.Mtb lysates (10 µg) derived from Mtb grown in log-phase-, starvation- and hypoxic- conditions and recombinant protein Rv2034 (0.4 µg) were analyzed by western blotting for their recognition by specific antibodies using sera of Rv2034-immunized or non-immunized HLA-DR3 transgenic mice (A). Monocyte derived matured DCs were loaded with Rv2034 p81–100, Rv2034 protein and different conditions of Mtb lysate (100 µg/ml) for 24h. Subsequently, the CD4+ T-cell clone was incubated with these antigen-loaded DCs and after 2 hours BFA was added and incubated o/n. CD154 expression was determined using flow cytometry (B). Data represent two independent experiments. An HA-specific T-cell clone was included as a negative control (C). Medium background values were subtracted for each response.

Mentions: We next wished to demonstrate recognition of native Mtb expressed Rv2034 protein by the T-cell clone in order to better understand its possible role during Mtb infection. First we examined whether Rv2034 protein was expressed in Mtb lysates by western blot analysis. Using sera of Rv2034-immunized (HLA-DR3 transgenic) mice, expression of Rv2034 was identified in both log phase Mtb lysate and Mtb grown during hypoxic and starvation conditions (Figure 6A). Although the protein size was predicted to be approximately 12 kDa (http://www.sciencegateway.org/tools/proteinmw.htm), the native Rv2034 present in the lysates was larger than expected. The recombinant Rv2034 protein includes a histidine tag and thus was expected to have a molecular weight of ∼14 kDa. Since Rv2034 contains dimerization sites [67], this higher relative molecular mass could be due to formation of multimers. Alternatively but not mutually exclusive, post-translational modifications or binding to other molecules might be responsible for the higher molecular weight. Two bands were clearly visible, the smallest band likely relating to Rv2034 protein monomer, whereas the upper band probably indicates multimer formation. The binding of antibodies present in the sera of Rv2034-immunized mice was specific since no bands were observed using sera from non-immunized HLA-DR3 mice. Indeed, Rv2034 protein was also previously identified in in vitro grown Mtb cultures [10], [68], [69], specifically in lipid (membrane) associated fractions, indicating that the protein is indeed expressed by Mtb.


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)

Recognition of native Rv2034 in Mtb lysate by CD4+ T-cell clone.Mtb lysates (10 µg) derived from Mtb grown in log-phase-, starvation- and hypoxic- conditions and recombinant protein Rv2034 (0.4 µg) were analyzed by western blotting for their recognition by specific antibodies using sera of Rv2034-immunized or non-immunized HLA-DR3 transgenic mice (A). Monocyte derived matured DCs were loaded with Rv2034 p81–100, Rv2034 protein and different conditions of Mtb lysate (100 µg/ml) for 24h. Subsequently, the CD4+ T-cell clone was incubated with these antigen-loaded DCs and after 2 hours BFA was added and incubated o/n. CD154 expression was determined using flow cytometry (B). Data represent two independent experiments. An HA-specific T-cell clone was included as a negative control (C). Medium background values were subtracted for each response.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4048274&req=5

pone-0099203-g006: Recognition of native Rv2034 in Mtb lysate by CD4+ T-cell clone.Mtb lysates (10 µg) derived from Mtb grown in log-phase-, starvation- and hypoxic- conditions and recombinant protein Rv2034 (0.4 µg) were analyzed by western blotting for their recognition by specific antibodies using sera of Rv2034-immunized or non-immunized HLA-DR3 transgenic mice (A). Monocyte derived matured DCs were loaded with Rv2034 p81–100, Rv2034 protein and different conditions of Mtb lysate (100 µg/ml) for 24h. Subsequently, the CD4+ T-cell clone was incubated with these antigen-loaded DCs and after 2 hours BFA was added and incubated o/n. CD154 expression was determined using flow cytometry (B). Data represent two independent experiments. An HA-specific T-cell clone was included as a negative control (C). Medium background values were subtracted for each response.
Mentions: We next wished to demonstrate recognition of native Mtb expressed Rv2034 protein by the T-cell clone in order to better understand its possible role during Mtb infection. First we examined whether Rv2034 protein was expressed in Mtb lysates by western blot analysis. Using sera of Rv2034-immunized (HLA-DR3 transgenic) mice, expression of Rv2034 was identified in both log phase Mtb lysate and Mtb grown during hypoxic and starvation conditions (Figure 6A). Although the protein size was predicted to be approximately 12 kDa (http://www.sciencegateway.org/tools/proteinmw.htm), the native Rv2034 present in the lysates was larger than expected. The recombinant Rv2034 protein includes a histidine tag and thus was expected to have a molecular weight of ∼14 kDa. Since Rv2034 contains dimerization sites [67], this higher relative molecular mass could be due to formation of multimers. Alternatively but not mutually exclusive, post-translational modifications or binding to other molecules might be responsible for the higher molecular weight. Two bands were clearly visible, the smallest band likely relating to Rv2034 protein monomer, whereas the upper band probably indicates multimer formation. The binding of antibodies present in the sera of Rv2034-immunized mice was specific since no bands were observed using sera from non-immunized HLA-DR3 mice. Indeed, Rv2034 protein was also previously identified in in vitro grown Mtb cultures [10], [68], [69], specifically in lipid (membrane) associated fractions, indicating that the protein is indeed expressed by Mtb.

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