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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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CD4+ T-cell clone has Mtb inhibitory properties.Autologous PBMC were loaded with Mtb (10 MOI) and the Rv2034 T-cell clone was added at an effector/target (E/T) ratio of 20∶1 and 50∶1 as indicated on the x-axis. CFU were determined after o/n incubation. Eight replicates from two independent experiments were included for 50∶1 and only target cells condition, four replicates were included for 20∶1 condition (A). The HA-specific clone was included as a negative control (n = 4) (B). The horizontal line indicates the median value and the outer boundaries of the box represents the 25th and 75th percentiles. The whiskers indicate the highest and lowest values. A Mann-Whitney U test was performed to analyze the difference between CFU. A p-value≤0.05 was considered significant and indicated with an asterisk.
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pone-0099203-g007: CD4+ T-cell clone has Mtb inhibitory properties.Autologous PBMC were loaded with Mtb (10 MOI) and the Rv2034 T-cell clone was added at an effector/target (E/T) ratio of 20∶1 and 50∶1 as indicated on the x-axis. CFU were determined after o/n incubation. Eight replicates from two independent experiments were included for 50∶1 and only target cells condition, four replicates were included for 20∶1 condition (A). The HA-specific clone was included as a negative control (n = 4) (B). The horizontal line indicates the median value and the outer boundaries of the box represents the 25th and 75th percentiles. The whiskers indicate the highest and lowest values. A Mann-Whitney U test was performed to analyze the difference between CFU. A p-value≤0.05 was considered significant and indicated with an asterisk.

Mentions: The T-cell clone thus expressed cytotoxic markers (Figure 4) and recognized native Rv2034 in Mtb lysate (Figure 6B). To test whether the T-cell clone could directly inhibit Mtb outgrowth from infected APCs, autologous monocytes were infected with Mtb and the T-cell clone was added in an effector/target ratio of 20∶1 and 50∶1. Interestingly, addition of the T-cell clone resulted in a significant decrease of CFU in a dose-dependent fashion (p = 0.0038, Figure 7A), whereas this was not observed when the HA-specific T-cell clone was added to the Mtb loaded APCs (Figure 7B). Thus the T-cell clone is able to inhibit the outgrowth of Mtb from infected cells directly.


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)

CD4+ T-cell clone has Mtb inhibitory properties.Autologous PBMC were loaded with Mtb (10 MOI) and the Rv2034 T-cell clone was added at an effector/target (E/T) ratio of 20∶1 and 50∶1 as indicated on the x-axis. CFU were determined after o/n incubation. Eight replicates from two independent experiments were included for 50∶1 and only target cells condition, four replicates were included for 20∶1 condition (A). The HA-specific clone was included as a negative control (n = 4) (B). The horizontal line indicates the median value and the outer boundaries of the box represents the 25th and 75th percentiles. The whiskers indicate the highest and lowest values. A Mann-Whitney U test was performed to analyze the difference between CFU. A p-value≤0.05 was considered significant and indicated with an asterisk.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099203-g007: CD4+ T-cell clone has Mtb inhibitory properties.Autologous PBMC were loaded with Mtb (10 MOI) and the Rv2034 T-cell clone was added at an effector/target (E/T) ratio of 20∶1 and 50∶1 as indicated on the x-axis. CFU were determined after o/n incubation. Eight replicates from two independent experiments were included for 50∶1 and only target cells condition, four replicates were included for 20∶1 condition (A). The HA-specific clone was included as a negative control (n = 4) (B). The horizontal line indicates the median value and the outer boundaries of the box represents the 25th and 75th percentiles. The whiskers indicate the highest and lowest values. A Mann-Whitney U test was performed to analyze the difference between CFU. A p-value≤0.05 was considered significant and indicated with an asterisk.
Mentions: The T-cell clone thus expressed cytotoxic markers (Figure 4) and recognized native Rv2034 in Mtb lysate (Figure 6B). To test whether the T-cell clone could directly inhibit Mtb outgrowth from infected APCs, autologous monocytes were infected with Mtb and the T-cell clone was added in an effector/target ratio of 20∶1 and 50∶1. Interestingly, addition of the T-cell clone resulted in a significant decrease of CFU in a dose-dependent fashion (p = 0.0038, Figure 7A), whereas this was not observed when the HA-specific T-cell clone was added to the Mtb loaded APCs (Figure 7B). Thus the T-cell clone is able to inhibit the outgrowth of Mtb from infected cells directly.

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