Limits...
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.

Show MeSH

Related in: MedlinePlus

Long-term cultured cells have higher proliferative responses than short-term cells.Long-term and short-term cultured PBMCs were analyzed ~ 7 weeks after aerosol challenge with virulent M. bovis. Long-term cells consist of PBMC from M. bovis aerosol infected cattle cultured in the presence of rAg85A, rTB10.4, rESAT-6:CFP10 and PPDb for 13 days and then CellTrace violet-stained and re-stimulated with either rESAT-6:CFP10, PPDb or medium in the presence of fresh autologous adherent cells for an additional six days. Short-term cells consist of CellTrace violet-stained PBMC from M. bovis aerosol infected cattle cultured for six days in the presence of either rESAT-6:CFP10, PPDb or medium. The gating strategy was performed in accordance with procedures described in Fig 2A for single cells, and as shown in S3 Fig for lymphocytes, and CD4+ cells. (A) Percentages of CD4+ cells proliferating (low Celltrace dye MFI) in response to rESAT-6:CFP10 within long or short-term cultures. (B) Percentages of CD4+ cells proliferating (low Celltrace dye MFI) in response to PPDb within long or short-term cultures. (C) Memory/effector phenotype of proliferating CD4+ cells within long-term or short-term cultures in response to rESAT-6:CFP10. (D) Memory/effector phenotype of proliferating CD4+ cells within long-term or short-term cultures in response to PPDb. For panels A and B, cell proliferation differs (**P < 0.01, n = 6 paired Student's t-tests) between long and short-term cultures to either rESAT-6:CFP10 or PPDb. For panels C and D, Tcm and effector cell content differs (*P < 0.05; **P < 0.01, n = 4 paired Student's t-tests) between short-term and long-term cultures to either rESAT-6:CFP10 or PPDb.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4400046&req=5

pone.0122571.g006: Long-term cultured cells have higher proliferative responses than short-term cells.Long-term and short-term cultured PBMCs were analyzed ~ 7 weeks after aerosol challenge with virulent M. bovis. Long-term cells consist of PBMC from M. bovis aerosol infected cattle cultured in the presence of rAg85A, rTB10.4, rESAT-6:CFP10 and PPDb for 13 days and then CellTrace violet-stained and re-stimulated with either rESAT-6:CFP10, PPDb or medium in the presence of fresh autologous adherent cells for an additional six days. Short-term cells consist of CellTrace violet-stained PBMC from M. bovis aerosol infected cattle cultured for six days in the presence of either rESAT-6:CFP10, PPDb or medium. The gating strategy was performed in accordance with procedures described in Fig 2A for single cells, and as shown in S3 Fig for lymphocytes, and CD4+ cells. (A) Percentages of CD4+ cells proliferating (low Celltrace dye MFI) in response to rESAT-6:CFP10 within long or short-term cultures. (B) Percentages of CD4+ cells proliferating (low Celltrace dye MFI) in response to PPDb within long or short-term cultures. (C) Memory/effector phenotype of proliferating CD4+ cells within long-term or short-term cultures in response to rESAT-6:CFP10. (D) Memory/effector phenotype of proliferating CD4+ cells within long-term or short-term cultures in response to PPDb. For panels A and B, cell proliferation differs (**P < 0.01, n = 6 paired Student's t-tests) between long and short-term cultures to either rESAT-6:CFP10 or PPDb. For panels C and D, Tcm and effector cell content differs (*P < 0.05; **P < 0.01, n = 4 paired Student's t-tests) between short-term and long-term cultures to either rESAT-6:CFP10 or PPDb.

Mentions: Human CD4 memory T cells, predominantly those exhibiting Tcm phenotype, proliferate in response to cytokine and antigenic stimulations, differentiating into Tem or effector T cells in vitro [30,47]. To assess the proliferative capacity of bovine Tcm cells following long-term culture, cells were harvested at day 13 and stained with CellTrace Violet. CellTrace Violet stained cells were re-stimulated with rESAT-6:CFP10 or PPDb for additional six days, without IL-2 (S5 Fig). For comparative purposes, freshly isolated PBMC were isolated and stained with CellTrace Violet and cultured for six days (short-term culture). Cells proliferated in response to antigenic stimulation under both long- and short-term conditions (Table 2). CD4 T cells were the most proliferative fraction (P < 0.05), followed by γδ, CD8 T cells, and CD8 expressing γδ T cells. In response to rESAT-6:CFP10 stimulation, the number of CD4 T cells proliferating in long-term cultures exceeded (P < 0.05) that of short-term cultures. Similarly, the CellTrace Violet mean fluorescence intensity (MFI) was significantly lower (P = 0.003), indicating greater cell proliferation in rESAT-6:CFP10-stimulated CD4 T cells in long- vs short-term cultures (Fig 5 and Fig 6). Greater percentages of CD4 T cells (P < 0.05) proliferated under long-term culture in response to either rESAT-6:CFP10 (Fig 6A) or PPDb (Fig 6B). These findings demonstrate that bovine Tcm cells are highly proliferative in response to repeated stimulation with recall antigen.


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)

Long-term cultured cells have higher proliferative responses than short-term cells.Long-term and short-term cultured PBMCs were analyzed ~ 7 weeks after aerosol challenge with virulent M. bovis. Long-term cells consist of PBMC from M. bovis aerosol infected cattle cultured in the presence of rAg85A, rTB10.4, rESAT-6:CFP10 and PPDb for 13 days and then CellTrace violet-stained and re-stimulated with either rESAT-6:CFP10, PPDb or medium in the presence of fresh autologous adherent cells for an additional six days. Short-term cells consist of CellTrace violet-stained PBMC from M. bovis aerosol infected cattle cultured for six days in the presence of either rESAT-6:CFP10, PPDb or medium. The gating strategy was performed in accordance with procedures described in Fig 2A for single cells, and as shown in S3 Fig for lymphocytes, and CD4+ cells. (A) Percentages of CD4+ cells proliferating (low Celltrace dye MFI) in response to rESAT-6:CFP10 within long or short-term cultures. (B) Percentages of CD4+ cells proliferating (low Celltrace dye MFI) in response to PPDb within long or short-term cultures. (C) Memory/effector phenotype of proliferating CD4+ cells within long-term or short-term cultures in response to rESAT-6:CFP10. (D) Memory/effector phenotype of proliferating CD4+ cells within long-term or short-term cultures in response to PPDb. For panels A and B, cell proliferation differs (**P < 0.01, n = 6 paired Student's t-tests) between long and short-term cultures to either rESAT-6:CFP10 or PPDb. For panels C and D, Tcm and effector cell content differs (*P < 0.05; **P < 0.01, n = 4 paired Student's t-tests) between short-term and long-term cultures to either rESAT-6:CFP10 or PPDb.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400046&req=5

pone.0122571.g006: Long-term cultured cells have higher proliferative responses than short-term cells.Long-term and short-term cultured PBMCs were analyzed ~ 7 weeks after aerosol challenge with virulent M. bovis. Long-term cells consist of PBMC from M. bovis aerosol infected cattle cultured in the presence of rAg85A, rTB10.4, rESAT-6:CFP10 and PPDb for 13 days and then CellTrace violet-stained and re-stimulated with either rESAT-6:CFP10, PPDb or medium in the presence of fresh autologous adherent cells for an additional six days. Short-term cells consist of CellTrace violet-stained PBMC from M. bovis aerosol infected cattle cultured for six days in the presence of either rESAT-6:CFP10, PPDb or medium. The gating strategy was performed in accordance with procedures described in Fig 2A for single cells, and as shown in S3 Fig for lymphocytes, and CD4+ cells. (A) Percentages of CD4+ cells proliferating (low Celltrace dye MFI) in response to rESAT-6:CFP10 within long or short-term cultures. (B) Percentages of CD4+ cells proliferating (low Celltrace dye MFI) in response to PPDb within long or short-term cultures. (C) Memory/effector phenotype of proliferating CD4+ cells within long-term or short-term cultures in response to rESAT-6:CFP10. (D) Memory/effector phenotype of proliferating CD4+ cells within long-term or short-term cultures in response to PPDb. For panels A and B, cell proliferation differs (**P < 0.01, n = 6 paired Student's t-tests) between long and short-term cultures to either rESAT-6:CFP10 or PPDb. For panels C and D, Tcm and effector cell content differs (*P < 0.05; **P < 0.01, n = 4 paired Student's t-tests) between short-term and long-term cultures to either rESAT-6:CFP10 or PPDb.
Mentions: Human CD4 memory T cells, predominantly those exhibiting Tcm phenotype, proliferate in response to cytokine and antigenic stimulations, differentiating into Tem or effector T cells in vitro [30,47]. To assess the proliferative capacity of bovine Tcm cells following long-term culture, cells were harvested at day 13 and stained with CellTrace Violet. CellTrace Violet stained cells were re-stimulated with rESAT-6:CFP10 or PPDb for additional six days, without IL-2 (S5 Fig). For comparative purposes, freshly isolated PBMC were isolated and stained with CellTrace Violet and cultured for six days (short-term culture). Cells proliferated in response to antigenic stimulation under both long- and short-term conditions (Table 2). CD4 T cells were the most proliferative fraction (P < 0.05), followed by γδ, CD8 T cells, and CD8 expressing γδ T cells. In response to rESAT-6:CFP10 stimulation, the number of CD4 T cells proliferating in long-term cultures exceeded (P < 0.05) that of short-term cultures. Similarly, the CellTrace Violet mean fluorescence intensity (MFI) was significantly lower (P = 0.003), indicating greater cell proliferation in rESAT-6:CFP10-stimulated CD4 T cells in long- vs short-term cultures (Fig 5 and Fig 6). Greater percentages of CD4 T cells (P < 0.05) proliferated under long-term culture in response to either rESAT-6:CFP10 (Fig 6A) or PPDb (Fig 6B). These findings demonstrate that bovine Tcm cells are highly proliferative in response to repeated stimulation with recall antigen.

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