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Characterization of effector and memory T cell subsets in the immune response to bovine tuberculosis in cattle.

Maggioli MF, Palmer MV, Thacker TC, Vordermeier HM, Waters WR - PLoS ONE (2015)

Bottom Line: The objective of the present study was to characterize the relative contribution of Tcm (CCR7+, CD62Lhi, CD45RO+), T effector memory (Tem, defined as: CCR7-, CD62Llow/int, CD45RO+), and T effector cells (CCR7-, CD62L-/low, CD45RO-), in the immune response to Mycobacterium bovis.These findings suggest that proliferative responses of Tcm cells to some extent occurs simultaneously with reversion to effector phenotypes (mostly Tem).The present study characterizes Tcm cells of cattle and their participation in the response to M. bovis infection.

View Article: PubMed Central - PubMed

Affiliation: Infectious Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Ames, IA, United States of America; Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, IA, United States of America.

ABSTRACT
Cultured IFN-γ ELISPOT assays are primarily a measure of central memory T cell (Tcm) responses with humans; however, this important subset of lymphocytes is poorly characterized in cattle. Vaccine-elicited cultured IFN-γ ELISPOT responses correlate with protection against bovine tuberculosis in cattle. However, whether this assay measures cattle Tcm responses or not is uncertain. The objective of the present study was to characterize the relative contribution of Tcm (CCR7+, CD62Lhi, CD45RO+), T effector memory (Tem, defined as: CCR7-, CD62Llow/int, CD45RO+), and T effector cells (CCR7-, CD62L-/low, CD45RO-), in the immune response to Mycobacterium bovis. Peripheral blood mononuclear cells (PBMC) from infected cattle were stimulated with a cocktail of M. bovis purified protein derivative, rTb10.4 and rAg85A for 13 days with periodic addition of fresh media and rIL-2. On day 13, cultured PBMC were re-stimulated with medium alone, rESAT-6:CFP10 or PPDb with fresh autologous adherent cells for antigen presentation. Cultured cells (13 days) or fresh PBMCs (ex vivo response) from the same calves were analyzed for IFN-γ production, proliferation, and CD4, CD45RO, CD62L, CD44, and CCR7 expression via flow cytometry after overnight stimulation. In response to mycobacterial antigens, ~75% of CD4+ IFN-γ+ cells in long-term cultures expressed a Tcm phenotype while less than 10% of the ex vivo response consisted of Tcm cells. Upon re-exposure to antigen, long-term cultured cells were highly proliferative, a distinctive characteristic of Tcm, and the predominant phenotype within the long-term cultures switched from Tcm to Tem. These findings suggest that proliferative responses of Tcm cells to some extent occurs simultaneously with reversion to effector phenotypes (mostly Tem). The present study characterizes Tcm cells of cattle and their participation in the response to M. bovis infection.

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Frequencies of Tcm, Tem and effector cells producing IFN-γ in response to mycobacterial antigens in long-term and ex vivo assays.Peripheral blood mononuclear cells were isolated from calves ~ 8 weeks after challenge with virulent M. bovis. Cells were stimulated with a cocktail of rAg85A (1 μg/ml), rTB10.4 (1 μg/ml), and rESAT-6:CFP10 (1 μg/ml) as well as PPDb (5 μg/ml) for 13 days followed by transfer to 96 well round bottom plates with APCs and addition of media alone, PPDb or rESAT-6:CFP10 for an additional 16h. For ex vivo culture, PBMC were stimulated with media alone, PPDb or rESAT-6:CFP10 for 16 h. (A) Relative contribution of Tcm, Tem, and T effector cells to IFN-γ production in response to PPDb by long-term (i.e., 14-day) (left) and ex vivo (i.e., 16 h) (right) cultures, 8 weeks after M. bovis challenge. Data are presented in percentages (pies) and as mean (± SEM) number of cells producing IFN-γ / 104 cells (histograms) (n = 16). Relative contribution of Tcm, Tem and T effector cells to IFN-γ production in response to PPDb (B) or to rESAT-6:CFP10 (C) in long-term cultures at three, six, eight or 12 weeks post-infection (WPI, n = 6). Tcm, Tem and effector cell phenotypes were as defined in Fig 2 and S3 Fig Tcm and Effector T cell contribution to IFN-γ production differs (*P < 0.05; **P < 0.01, paired Student's t-tests) between short- and long-term cultures.
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pone.0122571.g003: Frequencies of Tcm, Tem and effector cells producing IFN-γ in response to mycobacterial antigens in long-term and ex vivo assays.Peripheral blood mononuclear cells were isolated from calves ~ 8 weeks after challenge with virulent M. bovis. Cells were stimulated with a cocktail of rAg85A (1 μg/ml), rTB10.4 (1 μg/ml), and rESAT-6:CFP10 (1 μg/ml) as well as PPDb (5 μg/ml) for 13 days followed by transfer to 96 well round bottom plates with APCs and addition of media alone, PPDb or rESAT-6:CFP10 for an additional 16h. For ex vivo culture, PBMC were stimulated with media alone, PPDb or rESAT-6:CFP10 for 16 h. (A) Relative contribution of Tcm, Tem, and T effector cells to IFN-γ production in response to PPDb by long-term (i.e., 14-day) (left) and ex vivo (i.e., 16 h) (right) cultures, 8 weeks after M. bovis challenge. Data are presented in percentages (pies) and as mean (± SEM) number of cells producing IFN-γ / 104 cells (histograms) (n = 16). Relative contribution of Tcm, Tem and T effector cells to IFN-γ production in response to PPDb (B) or to rESAT-6:CFP10 (C) in long-term cultures at three, six, eight or 12 weeks post-infection (WPI, n = 6). Tcm, Tem and effector cell phenotypes were as defined in Fig 2 and S3 Fig Tcm and Effector T cell contribution to IFN-γ production differs (*P < 0.05; **P < 0.01, paired Student's t-tests) between short- and long-term cultures.

Mentions: The expression of CD45RO, CD4, CCR7 and intracellular expression of IFN-γ by PBMC cells was evaluated following long-term or ex vivo culture (Fig 2). CD4 T cells producing IFN-γ following long-term culture predominantly co-expressed CD45RO and CCR7 surface antigens (Fig 3), consistent with the Tcm phenotype described for humans and mice (S3 Fig) [26],[46]. The phenotype of cells responding to PPDb (CD4+ IFN-γ+) was compared under ex vivo versus long-term culture conditions (Fig 3A and S4A Fig). The predominant cell phenotype responding to antigenic stimulation in long-term cultures was that of Tcm cells, whereas few Tcm were present under ex vivo conditions (P < 0.01, % Tcm in long-term versus ex vivo cultures). In contrast, effector cells contributed to ex vivo IFN-γ production, but only minimally to the long-term culture response (P < 0.05). Tem cells contributed to IFN-γ production in both ex vivo (~50%) and long-term cultures (~25%). The respective overall effector/memory CD4 T cells (i.e. CD4+ IFN-γ+/- cells) proportions under both long- and short-term conditions are shown in S4B Fig The relative contribution of Tcm, Tem and effector CD4+ T cells in the response to PPDb (Fig 3B) and to rESAT-6:CFP10 (Fig 3C) remained the same over the course of infection (i.e., at 6, 8, and 12 weeks after challenge). In general a greater number of responding cells (IFN-γ+) were observed in the long-term cultured assay as compared to the ex vivo assay (Fig 3), perhaps due to the greater percentage of CD4 cells within long-term cultures (S5 Fig).


Characterization of effector and memory T cell subsets in the immune response to bovine tuberculosis in cattle.

Maggioli MF, Palmer MV, Thacker TC, Vordermeier HM, Waters WR - PLoS ONE (2015)

Frequencies of Tcm, Tem and effector cells producing IFN-γ in response to mycobacterial antigens in long-term and ex vivo assays.Peripheral blood mononuclear cells were isolated from calves ~ 8 weeks after challenge with virulent M. bovis. Cells were stimulated with a cocktail of rAg85A (1 μg/ml), rTB10.4 (1 μg/ml), and rESAT-6:CFP10 (1 μg/ml) as well as PPDb (5 μg/ml) for 13 days followed by transfer to 96 well round bottom plates with APCs and addition of media alone, PPDb or rESAT-6:CFP10 for an additional 16h. For ex vivo culture, PBMC were stimulated with media alone, PPDb or rESAT-6:CFP10 for 16 h. (A) Relative contribution of Tcm, Tem, and T effector cells to IFN-γ production in response to PPDb by long-term (i.e., 14-day) (left) and ex vivo (i.e., 16 h) (right) cultures, 8 weeks after M. bovis challenge. Data are presented in percentages (pies) and as mean (± SEM) number of cells producing IFN-γ / 104 cells (histograms) (n = 16). Relative contribution of Tcm, Tem and T effector cells to IFN-γ production in response to PPDb (B) or to rESAT-6:CFP10 (C) in long-term cultures at three, six, eight or 12 weeks post-infection (WPI, n = 6). Tcm, Tem and effector cell phenotypes were as defined in Fig 2 and S3 Fig Tcm and Effector T cell contribution to IFN-γ production differs (*P < 0.05; **P < 0.01, paired Student's t-tests) between short- and long-term cultures.
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pone.0122571.g003: Frequencies of Tcm, Tem and effector cells producing IFN-γ in response to mycobacterial antigens in long-term and ex vivo assays.Peripheral blood mononuclear cells were isolated from calves ~ 8 weeks after challenge with virulent M. bovis. Cells were stimulated with a cocktail of rAg85A (1 μg/ml), rTB10.4 (1 μg/ml), and rESAT-6:CFP10 (1 μg/ml) as well as PPDb (5 μg/ml) for 13 days followed by transfer to 96 well round bottom plates with APCs and addition of media alone, PPDb or rESAT-6:CFP10 for an additional 16h. For ex vivo culture, PBMC were stimulated with media alone, PPDb or rESAT-6:CFP10 for 16 h. (A) Relative contribution of Tcm, Tem, and T effector cells to IFN-γ production in response to PPDb by long-term (i.e., 14-day) (left) and ex vivo (i.e., 16 h) (right) cultures, 8 weeks after M. bovis challenge. Data are presented in percentages (pies) and as mean (± SEM) number of cells producing IFN-γ / 104 cells (histograms) (n = 16). Relative contribution of Tcm, Tem and T effector cells to IFN-γ production in response to PPDb (B) or to rESAT-6:CFP10 (C) in long-term cultures at three, six, eight or 12 weeks post-infection (WPI, n = 6). Tcm, Tem and effector cell phenotypes were as defined in Fig 2 and S3 Fig Tcm and Effector T cell contribution to IFN-γ production differs (*P < 0.05; **P < 0.01, paired Student's t-tests) between short- and long-term cultures.
Mentions: The expression of CD45RO, CD4, CCR7 and intracellular expression of IFN-γ by PBMC cells was evaluated following long-term or ex vivo culture (Fig 2). CD4 T cells producing IFN-γ following long-term culture predominantly co-expressed CD45RO and CCR7 surface antigens (Fig 3), consistent with the Tcm phenotype described for humans and mice (S3 Fig) [26],[46]. The phenotype of cells responding to PPDb (CD4+ IFN-γ+) was compared under ex vivo versus long-term culture conditions (Fig 3A and S4A Fig). The predominant cell phenotype responding to antigenic stimulation in long-term cultures was that of Tcm cells, whereas few Tcm were present under ex vivo conditions (P < 0.01, % Tcm in long-term versus ex vivo cultures). In contrast, effector cells contributed to ex vivo IFN-γ production, but only minimally to the long-term culture response (P < 0.05). Tem cells contributed to IFN-γ production in both ex vivo (~50%) and long-term cultures (~25%). The respective overall effector/memory CD4 T cells (i.e. CD4+ IFN-γ+/- cells) proportions under both long- and short-term conditions are shown in S4B Fig The relative contribution of Tcm, Tem and effector CD4+ T cells in the response to PPDb (Fig 3B) and to rESAT-6:CFP10 (Fig 3C) remained the same over the course of infection (i.e., at 6, 8, and 12 weeks after challenge). In general a greater number of responding cells (IFN-γ+) were observed in the long-term cultured assay as compared to the ex vivo assay (Fig 3), perhaps due to the greater percentage of CD4 cells within long-term cultures (S5 Fig).

Bottom Line: The objective of the present study was to characterize the relative contribution of Tcm (CCR7+, CD62Lhi, CD45RO+), T effector memory (Tem, defined as: CCR7-, CD62Llow/int, CD45RO+), and T effector cells (CCR7-, CD62L-/low, CD45RO-), in the immune response to Mycobacterium bovis.These findings suggest that proliferative responses of Tcm cells to some extent occurs simultaneously with reversion to effector phenotypes (mostly Tem).The present study characterizes Tcm cells of cattle and their participation in the response to M. bovis infection.

View Article: PubMed Central - PubMed

Affiliation: Infectious Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Ames, IA, United States of America; Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, IA, United States of America.

ABSTRACT
Cultured IFN-γ ELISPOT assays are primarily a measure of central memory T cell (Tcm) responses with humans; however, this important subset of lymphocytes is poorly characterized in cattle. Vaccine-elicited cultured IFN-γ ELISPOT responses correlate with protection against bovine tuberculosis in cattle. However, whether this assay measures cattle Tcm responses or not is uncertain. The objective of the present study was to characterize the relative contribution of Tcm (CCR7+, CD62Lhi, CD45RO+), T effector memory (Tem, defined as: CCR7-, CD62Llow/int, CD45RO+), and T effector cells (CCR7-, CD62L-/low, CD45RO-), in the immune response to Mycobacterium bovis. Peripheral blood mononuclear cells (PBMC) from infected cattle were stimulated with a cocktail of M. bovis purified protein derivative, rTb10.4 and rAg85A for 13 days with periodic addition of fresh media and rIL-2. On day 13, cultured PBMC were re-stimulated with medium alone, rESAT-6:CFP10 or PPDb with fresh autologous adherent cells for antigen presentation. Cultured cells (13 days) or fresh PBMCs (ex vivo response) from the same calves were analyzed for IFN-γ production, proliferation, and CD4, CD45RO, CD62L, CD44, and CCR7 expression via flow cytometry after overnight stimulation. In response to mycobacterial antigens, ~75% of CD4+ IFN-γ+ cells in long-term cultures expressed a Tcm phenotype while less than 10% of the ex vivo response consisted of Tcm cells. Upon re-exposure to antigen, long-term cultured cells were highly proliferative, a distinctive characteristic of Tcm, and the predominant phenotype within the long-term cultures switched from Tcm to Tem. These findings suggest that proliferative responses of Tcm cells to some extent occurs simultaneously with reversion to effector phenotypes (mostly Tem). The present study characterizes Tcm cells of cattle and their participation in the response to M. bovis infection.

Show MeSH
Related in: MedlinePlus