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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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Restriction of Rv2034 p81–100 and p88–107 presentation by HLA-DR and -DQ molecules.To determine the HLA-DR and -DQ restriction of Rv2034 p81–100 responding CD4+ T-cell clone, T cells were incubated with Rv2034 p81–100 (A), Rv2034 protein (B) or Ag85B/ESAT-6/Rv2034 fusion protein (C), in the presence of irradiated PBMC with (mis)matched HLA-DR and/or DQ molecules. Both IFN-γ production (white bars) and proliferation (CPM) (black bars) was determined. CPM bars represent median ranging the highest and lowest value (n = 3). Matching HLA alleles are indicated in bold on the x-axis. HLA-DR and HLA-DQ molecules of APCs pulsed with either Rv2034 protein, Rv2034 p81–100, Rv2034 p88–107 (5 µg/ml) or control conditions, were blocked using monoclonal antibodies directed against HLA-DR or -DQ [21], and T-cell proliferation was analyzed (CPM) (D). Data is representative for three independent experiments.
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pone-0099203-g005: Restriction of Rv2034 p81–100 and p88–107 presentation by HLA-DR and -DQ molecules.To determine the HLA-DR and -DQ restriction of Rv2034 p81–100 responding CD4+ T-cell clone, T cells were incubated with Rv2034 p81–100 (A), Rv2034 protein (B) or Ag85B/ESAT-6/Rv2034 fusion protein (C), in the presence of irradiated PBMC with (mis)matched HLA-DR and/or DQ molecules. Both IFN-γ production (white bars) and proliferation (CPM) (black bars) was determined. CPM bars represent median ranging the highest and lowest value (n = 3). Matching HLA alleles are indicated in bold on the x-axis. HLA-DR and HLA-DQ molecules of APCs pulsed with either Rv2034 protein, Rv2034 p81–100, Rv2034 p88–107 (5 µg/ml) or control conditions, were blocked using monoclonal antibodies directed against HLA-DR or -DQ [21], and T-cell proliferation was analyzed (CPM) (D). Data is representative for three independent experiments.

Mentions: To identify the genetic HLA-restriction of T-cell recognition of Rv2034 p81–100, the T-cell clone was cultured with (irradiated) PBMC from donors expressing various matching or mismatching HLA class II alleles in the presence of Rv2034 p81–100 (Figure 5A), Rv2034 protein (Figure 5B) and Ag85B/ESAT-6/Rv2034 fusion protein (Figure 5C). The autologous HLA genotype was HLA-DR2(15),-DR3(17),-DQ1(6),-DQ2,-DR51,-DR52. As expected, completely HLA-DRB1 and HLA-DQ mismatched PBMC (HLA-DR11,13 and HLA-DQ3(7)) failed to present peptide or protein antigens to the T-cell clone, even though these cells were matched for HLA-DRB3 (DR52), indicating that DR52 was not involved in Rv2034 p81–100 presentation. Both HLA-DR2(15)+/DQ1(6)+ and HLA-DR3(17)+/DQ1(5)+ PBMC induced strong T-cell activation. Since HLA-DR2(15) is in strong genetic linkage disequilibrium with HLA-DQ1(6) it was impossible to match for HLA-DR2(15) while mismatching for HLA-DQ1(6). HLA-DQ1(5,6)+ PBMC, which were mismatched for HLA-DR3(17) and HLA-DR2(15), also induced T-cell proliferation and IFN-γ in response to Rv2034 (fusion) protein and p81–100, whereas HLA-DQ2+ PBMC failed to present antigen. While the results were suggestive of HLA-DR3 and HLA-DQ1 presentation, the addition of purified monoclonal antibodies specific for only HLA-DR but not HLA-DQ backbone determinants resulted in a strong reduction of autologous APC presented Rv2034 p81–100 induced T-cell proliferation (Figure 5D). Thus the autologous responses towards Rv2034 p81–100 is predominantly HLA-DR and not HLA-DQ-restricted.


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)

Restriction of Rv2034 p81–100 and p88–107 presentation by HLA-DR and -DQ molecules.To determine the HLA-DR and -DQ restriction of Rv2034 p81–100 responding CD4+ T-cell clone, T cells were incubated with Rv2034 p81–100 (A), Rv2034 protein (B) or Ag85B/ESAT-6/Rv2034 fusion protein (C), in the presence of irradiated PBMC with (mis)matched HLA-DR and/or DQ molecules. Both IFN-γ production (white bars) and proliferation (CPM) (black bars) was determined. CPM bars represent median ranging the highest and lowest value (n = 3). Matching HLA alleles are indicated in bold on the x-axis. HLA-DR and HLA-DQ molecules of APCs pulsed with either Rv2034 protein, Rv2034 p81–100, Rv2034 p88–107 (5 µg/ml) or control conditions, were blocked using monoclonal antibodies directed against HLA-DR or -DQ [21], and T-cell proliferation was analyzed (CPM) (D). Data is representative for three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099203-g005: Restriction of Rv2034 p81–100 and p88–107 presentation by HLA-DR and -DQ molecules.To determine the HLA-DR and -DQ restriction of Rv2034 p81–100 responding CD4+ T-cell clone, T cells were incubated with Rv2034 p81–100 (A), Rv2034 protein (B) or Ag85B/ESAT-6/Rv2034 fusion protein (C), in the presence of irradiated PBMC with (mis)matched HLA-DR and/or DQ molecules. Both IFN-γ production (white bars) and proliferation (CPM) (black bars) was determined. CPM bars represent median ranging the highest and lowest value (n = 3). Matching HLA alleles are indicated in bold on the x-axis. HLA-DR and HLA-DQ molecules of APCs pulsed with either Rv2034 protein, Rv2034 p81–100, Rv2034 p88–107 (5 µg/ml) or control conditions, were blocked using monoclonal antibodies directed against HLA-DR or -DQ [21], and T-cell proliferation was analyzed (CPM) (D). Data is representative for three independent experiments.
Mentions: To identify the genetic HLA-restriction of T-cell recognition of Rv2034 p81–100, the T-cell clone was cultured with (irradiated) PBMC from donors expressing various matching or mismatching HLA class II alleles in the presence of Rv2034 p81–100 (Figure 5A), Rv2034 protein (Figure 5B) and Ag85B/ESAT-6/Rv2034 fusion protein (Figure 5C). The autologous HLA genotype was HLA-DR2(15),-DR3(17),-DQ1(6),-DQ2,-DR51,-DR52. As expected, completely HLA-DRB1 and HLA-DQ mismatched PBMC (HLA-DR11,13 and HLA-DQ3(7)) failed to present peptide or protein antigens to the T-cell clone, even though these cells were matched for HLA-DRB3 (DR52), indicating that DR52 was not involved in Rv2034 p81–100 presentation. Both HLA-DR2(15)+/DQ1(6)+ and HLA-DR3(17)+/DQ1(5)+ PBMC induced strong T-cell activation. Since HLA-DR2(15) is in strong genetic linkage disequilibrium with HLA-DQ1(6) it was impossible to match for HLA-DR2(15) while mismatching for HLA-DQ1(6). HLA-DQ1(5,6)+ PBMC, which were mismatched for HLA-DR3(17) and HLA-DR2(15), also induced T-cell proliferation and IFN-γ in response to Rv2034 (fusion) protein and p81–100, whereas HLA-DQ2+ PBMC failed to present antigen. While the results were suggestive of HLA-DR3 and HLA-DQ1 presentation, the addition of purified monoclonal antibodies specific for only HLA-DR but not HLA-DQ backbone determinants resulted in a strong reduction of autologous APC presented Rv2034 p81–100 induced T-cell proliferation (Figure 5D). Thus the autologous responses towards Rv2034 p81–100 is predominantly HLA-DR and not HLA-DQ-restricted.

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