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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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Long-term cultured and ex vivo IFN- γ responses by cattle after M. bovis aerosol challenge.Cultured ELISPOT analysis was performed ~3 weeks after challenge with virulent M. bovis. Long-term cultured cells were generated by stimulating PBMC with a cocktail of rAg85A (1 μg/ml), rTB10.4 (1 μg/ml), and rESAT-6:CFP10 (1 μg/ml) antigens as well as PPDb (5 μg/ml) for 13 days followed by transfer to ELISPOT plates with APCs and addition of either rESAT-6:CFP10, PPDb or medium alone. For the ex vivo response, freshly isolated PBMCs were stimulated with rESAT-6:CFP10, PPDb or medium alone for 16h. Medium control responses were subtracted from antigen-stimulated responses and results are presented as mean spot forming cells (SFC)/million cells (± SEM, n = 8) for (A) long-term culture or (B)ex vivo conditions. (C) The kinetics of the response is shown as the percent of CD4+ cells producing IFN-γ in long-term cultures at 3, 6, 8, and 12 weeks post infection (WPI n = 6). Two-way ANOVA (Šídák’s multiple comparison post-test).
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pone.0122571.g001: Long-term cultured and ex vivo IFN- γ responses by cattle after M. bovis aerosol challenge.Cultured ELISPOT analysis was performed ~3 weeks after challenge with virulent M. bovis. Long-term cultured cells were generated by stimulating PBMC with a cocktail of rAg85A (1 μg/ml), rTB10.4 (1 μg/ml), and rESAT-6:CFP10 (1 μg/ml) antigens as well as PPDb (5 μg/ml) for 13 days followed by transfer to ELISPOT plates with APCs and addition of either rESAT-6:CFP10, PPDb or medium alone. For the ex vivo response, freshly isolated PBMCs were stimulated with rESAT-6:CFP10, PPDb or medium alone for 16h. Medium control responses were subtracted from antigen-stimulated responses and results are presented as mean spot forming cells (SFC)/million cells (± SEM, n = 8) for (A) long-term culture or (B)ex vivo conditions. (C) The kinetics of the response is shown as the percent of CD4+ cells producing IFN-γ in long-term cultures at 3, 6, 8, and 12 weeks post infection (WPI n = 6). Two-way ANOVA (Šídák’s multiple comparison post-test).

Mentions: With bTB vaccine efficacy studies, long-term cultured (i.e., 14 days) IFN-γ ELISPOT responses to vaccination (i.e., BCG, M. bovis ΔRD1, and viral-vectored Ag85) negatively correlates with mycobacterial burden and TB-associated pathology and positively correlates with vaccine-induced protection [23–25]. As with vaccination, M. bovis infection also elicited long-term cultured IFN-γ ELISPOT responses in cattle (Fig 1). Three weeks after infection, long-term cultured IFN-γ ELISPOT responses by PBMCs from infected cattle to rESAT-6:CFP10 and PPDb exceeded (P < 0.05) respective responses by PBMCs from non-infected calves (Fig 1A). Similar results were detected with ex vivo (i.e., short-term) responses (Fig 1B). Also, Tcm and ex vivo responses did not differ (P > 0.05) between M. bovis 95-1315- and 10-7428-infected groups (S1 Fig). The weak response detected to PPDb by non-infected cattle in both ex vivo and long-term cultures was likely due to prior exposure to non-tuberculous mycobacteria (NTM, a common occurrence in US dairy cattle) as pre-infection whole blood (18 h stimulation) IFN-γ responses to M. avium PPD (PPDa) exceeded (P < 0.05) respective responses to PPDb (S2 Fig). Using intracellular cytokine staining, IFN-γ responses to PPDb and to rESAT-6:CFP10 were also detected in long-term PBMC cultures at 3, 6, 8, and 12 weeks after aerosol infection (Fig 1C). Responses increased (P < 0.05) from 3 and 6 to 12 weeks after infection. These findings demonstrate that infection of cattle with virulent M. bovis elicits long-term cultured IFN-γ ELISPOT responses, which are considered a surrogate of Tcm responses in humans [34,38,45].


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 and ex vivo IFN- γ responses by cattle after M. bovis aerosol challenge.Cultured ELISPOT analysis was performed ~3 weeks after challenge with virulent M. bovis. Long-term cultured cells were generated by stimulating PBMC with a cocktail of rAg85A (1 μg/ml), rTB10.4 (1 μg/ml), and rESAT-6:CFP10 (1 μg/ml) antigens as well as PPDb (5 μg/ml) for 13 days followed by transfer to ELISPOT plates with APCs and addition of either rESAT-6:CFP10, PPDb or medium alone. For the ex vivo response, freshly isolated PBMCs were stimulated with rESAT-6:CFP10, PPDb or medium alone for 16h. Medium control responses were subtracted from antigen-stimulated responses and results are presented as mean spot forming cells (SFC)/million cells (± SEM, n = 8) for (A) long-term culture or (B)ex vivo conditions. (C) The kinetics of the response is shown as the percent of CD4+ cells producing IFN-γ in long-term cultures at 3, 6, 8, and 12 weeks post infection (WPI n = 6). Two-way ANOVA (Šídák’s multiple comparison post-test).
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Related In: Results  -  Collection

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

pone.0122571.g001: Long-term cultured and ex vivo IFN- γ responses by cattle after M. bovis aerosol challenge.Cultured ELISPOT analysis was performed ~3 weeks after challenge with virulent M. bovis. Long-term cultured cells were generated by stimulating PBMC with a cocktail of rAg85A (1 μg/ml), rTB10.4 (1 μg/ml), and rESAT-6:CFP10 (1 μg/ml) antigens as well as PPDb (5 μg/ml) for 13 days followed by transfer to ELISPOT plates with APCs and addition of either rESAT-6:CFP10, PPDb or medium alone. For the ex vivo response, freshly isolated PBMCs were stimulated with rESAT-6:CFP10, PPDb or medium alone for 16h. Medium control responses were subtracted from antigen-stimulated responses and results are presented as mean spot forming cells (SFC)/million cells (± SEM, n = 8) for (A) long-term culture or (B)ex vivo conditions. (C) The kinetics of the response is shown as the percent of CD4+ cells producing IFN-γ in long-term cultures at 3, 6, 8, and 12 weeks post infection (WPI n = 6). Two-way ANOVA (Šídák’s multiple comparison post-test).
Mentions: With bTB vaccine efficacy studies, long-term cultured (i.e., 14 days) IFN-γ ELISPOT responses to vaccination (i.e., BCG, M. bovis ΔRD1, and viral-vectored Ag85) negatively correlates with mycobacterial burden and TB-associated pathology and positively correlates with vaccine-induced protection [23–25]. As with vaccination, M. bovis infection also elicited long-term cultured IFN-γ ELISPOT responses in cattle (Fig 1). Three weeks after infection, long-term cultured IFN-γ ELISPOT responses by PBMCs from infected cattle to rESAT-6:CFP10 and PPDb exceeded (P < 0.05) respective responses by PBMCs from non-infected calves (Fig 1A). Similar results were detected with ex vivo (i.e., short-term) responses (Fig 1B). Also, Tcm and ex vivo responses did not differ (P > 0.05) between M. bovis 95-1315- and 10-7428-infected groups (S1 Fig). The weak response detected to PPDb by non-infected cattle in both ex vivo and long-term cultures was likely due to prior exposure to non-tuberculous mycobacteria (NTM, a common occurrence in US dairy cattle) as pre-infection whole blood (18 h stimulation) IFN-γ responses to M. avium PPD (PPDa) exceeded (P < 0.05) respective responses to PPDb (S2 Fig). Using intracellular cytokine staining, IFN-γ responses to PPDb and to rESAT-6:CFP10 were also detected in long-term PBMC cultures at 3, 6, 8, and 12 weeks after aerosol infection (Fig 1C). Responses increased (P < 0.05) from 3 and 6 to 12 weeks after infection. These findings demonstrate that infection of cattle with virulent M. bovis elicits long-term cultured IFN-γ ELISPOT responses, which are considered a surrogate of Tcm responses in humans [34,38,45].

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