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Signal 3 determines tolerance versus full activation of naive CD8 T cells: dissociating proliferation and development of effector function.

Curtsinger JM, Lins DC, Mescher MF - J. Exp. Med. (2003)

Bottom Line: The requirement for the third signal to stimulate Ag-dependent proliferation is variable, making the greatest contribution when Ag levels are low.Thus, proliferation and development of cytolytic function can be fully uncoupled.Thus, the presence or absence of the third signal appears to be a critical variable in determining whether stimulation by Ag results in tolerance versus development of effector function and establishment of a responsive memory population.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine and Pathology, Center for Immunology, University of Minnesota, MN 55455, USA.

ABSTRACT
Activation of naive CD8 T cells to undergo clonal expansion and develop effector function requires three signals: (a) Ag, (b) costimulation, and (c) IL-12 or adjuvant. The requirement for the third signal to stimulate Ag-dependent proliferation is variable, making the greatest contribution when Ag levels are low. At high Ag levels, extensive proliferation can occur in vitro or in vivo in the absence of a third signal. However, despite having undergone the same number of divisions, cells that expand in the absence of a third signal fail to develop cytolytic effector function. Thus, proliferation and development of cytolytic function can be fully uncoupled. Furthermore, these cells are rendered functionally tolerant in vivo, in that subsequent restimulation with a potent stimulus results in limited clonal expansion, impaired IFN-gamma production, and no cytolytic function. Thus, the presence or absence of the third signal appears to be a critical variable in determining whether stimulation by Ag results in tolerance versus development of effector function and establishment of a responsive memory population.

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Related in: MedlinePlus

In vivo activation of CD8+ cells with high Ag dose stimulates proliferation but not lytic effector function. (A) C57BL/6 mice received OT-I/PL LN cells by adoptive transfer on day −1 and were challenged on day 0 with the indicated amounts of OVA257–264 peptide either alone or with 1 μg/ mouse of IL-12. Spleens were harvested on day 4, and the number of OT-I/PL cells was determined by flow cytometry analysis as described in Materials and Methods. The values shown are averages of duplicate mice and the error bars represent the ranges. (B) C57BL/6 mice received OT-I/PL LN cells by adoptive transfer on day −1 and were challenged on day 0 with the indicated amounts of OVA257–264 peptide either alone or with 50 μg/mouse of LPS. Spleens were harvested on day 3, and the number of OT-I/PL cells was determined by flow cytometry. The values shown are averages of duplicate mice and the error bars represent the ranges. (C) Spleen cells from the animals described in A that received 10 μg of peptide, with and without IL-12, were assayed for lytic activity against 51Cr-labeled E.G7 targets at spleen cell/target ratios of 200, 100, 50, and 25:1. These ratios were converted to OT-I/PL target ratios by multiplying by the percent OT-I/PL cells in each spleen cell population. The results shown are for one of the two animals in each treatment group; splenocytes from the other animal in each group showed essentially identical lytic activity. (D) C57BL/6 mice received OT-I/PL LN cells by adoptive transfer on day −1 and were left unchallenged (transfer only) or challenged with 10 μg OVA257–264/mouse alone (peptide only) or with 1 μg/mouse of IL-12 (peptide + IL-12). On day 3, mice were injected with equal numbers of unpulsed and OVA257–264-pulsed C57BL/6 spleen cells that were labeled with low and high concentrations of CFSE, respectively. Spleens were harvested after 3 h and the cells were analyzed for the preferential loss of peptide-pulsed, CFSEhigh versus unpulsed, CFSElow target cells. Histograms show the CFSEhigh and CFSElow cells from one of two identically treated mice; the percent lysis, calculated as described in Materials and Methods, is indicated.
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fig3: In vivo activation of CD8+ cells with high Ag dose stimulates proliferation but not lytic effector function. (A) C57BL/6 mice received OT-I/PL LN cells by adoptive transfer on day −1 and were challenged on day 0 with the indicated amounts of OVA257–264 peptide either alone or with 1 μg/ mouse of IL-12. Spleens were harvested on day 4, and the number of OT-I/PL cells was determined by flow cytometry analysis as described in Materials and Methods. The values shown are averages of duplicate mice and the error bars represent the ranges. (B) C57BL/6 mice received OT-I/PL LN cells by adoptive transfer on day −1 and were challenged on day 0 with the indicated amounts of OVA257–264 peptide either alone or with 50 μg/mouse of LPS. Spleens were harvested on day 3, and the number of OT-I/PL cells was determined by flow cytometry. The values shown are averages of duplicate mice and the error bars represent the ranges. (C) Spleen cells from the animals described in A that received 10 μg of peptide, with and without IL-12, were assayed for lytic activity against 51Cr-labeled E.G7 targets at spleen cell/target ratios of 200, 100, 50, and 25:1. These ratios were converted to OT-I/PL target ratios by multiplying by the percent OT-I/PL cells in each spleen cell population. The results shown are for one of the two animals in each treatment group; splenocytes from the other animal in each group showed essentially identical lytic activity. (D) C57BL/6 mice received OT-I/PL LN cells by adoptive transfer on day −1 and were left unchallenged (transfer only) or challenged with 10 μg OVA257–264/mouse alone (peptide only) or with 1 μg/mouse of IL-12 (peptide + IL-12). On day 3, mice were injected with equal numbers of unpulsed and OVA257–264-pulsed C57BL/6 spleen cells that were labeled with low and high concentrations of CFSE, respectively. Spleens were harvested after 3 h and the cells were analyzed for the preferential loss of peptide-pulsed, CFSEhigh versus unpulsed, CFSElow target cells. Histograms show the CFSEhigh and CFSElow cells from one of two identically treated mice; the percent lysis, calculated as described in Materials and Methods, is indicated.

Mentions: There are a number of works describing CD8 T cell populations that have expanded in vivo but lack effector function (32, 38–42). To determine if a high level of signal 1 might stimulate in vivo clonal expansion without leading to development of effector function, experiments were done using adoptive transfer of OT-I T cells into normal C57BL/6 recipients to allow quantitation of the Ag-specific T cell response in a relatively normal immune system (51). OT-I cells were adoptively transferred by intravenous (tail vein) injection and allowed to equilibrate in the recipient's immune system for 1 d. Mice were challenged with varying doses of OVA257–264 peptide alone, or along with IL-12. 3 d later, at the peak of the response (34), the numbers of OT-I cells in the LN and spleen were determined and cytotoxicity was measured for the populations. At low peptide concentrations, clonal expansion was minimal in the absence of IL-12, but was at maximal levels when IL-12 was coadministered (Fig. 3 A). Clonal expansion in response to peptide in the absence of IL-12 increased in a dose-dependent manner, whereas increasing the peptide dose above 2 μg in the presence of IL-12 did not increase the number of OT-I T cells. Very similar results were obtained when peptide was administered along with LPS as an adjuvant (Fig. 3 B).


Signal 3 determines tolerance versus full activation of naive CD8 T cells: dissociating proliferation and development of effector function.

Curtsinger JM, Lins DC, Mescher MF - J. Exp. Med. (2003)

In vivo activation of CD8+ cells with high Ag dose stimulates proliferation but not lytic effector function. (A) C57BL/6 mice received OT-I/PL LN cells by adoptive transfer on day −1 and were challenged on day 0 with the indicated amounts of OVA257–264 peptide either alone or with 1 μg/ mouse of IL-12. Spleens were harvested on day 4, and the number of OT-I/PL cells was determined by flow cytometry analysis as described in Materials and Methods. The values shown are averages of duplicate mice and the error bars represent the ranges. (B) C57BL/6 mice received OT-I/PL LN cells by adoptive transfer on day −1 and were challenged on day 0 with the indicated amounts of OVA257–264 peptide either alone or with 50 μg/mouse of LPS. Spleens were harvested on day 3, and the number of OT-I/PL cells was determined by flow cytometry. The values shown are averages of duplicate mice and the error bars represent the ranges. (C) Spleen cells from the animals described in A that received 10 μg of peptide, with and without IL-12, were assayed for lytic activity against 51Cr-labeled E.G7 targets at spleen cell/target ratios of 200, 100, 50, and 25:1. These ratios were converted to OT-I/PL target ratios by multiplying by the percent OT-I/PL cells in each spleen cell population. The results shown are for one of the two animals in each treatment group; splenocytes from the other animal in each group showed essentially identical lytic activity. (D) C57BL/6 mice received OT-I/PL LN cells by adoptive transfer on day −1 and were left unchallenged (transfer only) or challenged with 10 μg OVA257–264/mouse alone (peptide only) or with 1 μg/mouse of IL-12 (peptide + IL-12). On day 3, mice were injected with equal numbers of unpulsed and OVA257–264-pulsed C57BL/6 spleen cells that were labeled with low and high concentrations of CFSE, respectively. Spleens were harvested after 3 h and the cells were analyzed for the preferential loss of peptide-pulsed, CFSEhigh versus unpulsed, CFSElow target cells. Histograms show the CFSEhigh and CFSElow cells from one of two identically treated mice; the percent lysis, calculated as described in Materials and Methods, is indicated.
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Related In: Results  -  Collection

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fig3: In vivo activation of CD8+ cells with high Ag dose stimulates proliferation but not lytic effector function. (A) C57BL/6 mice received OT-I/PL LN cells by adoptive transfer on day −1 and were challenged on day 0 with the indicated amounts of OVA257–264 peptide either alone or with 1 μg/ mouse of IL-12. Spleens were harvested on day 4, and the number of OT-I/PL cells was determined by flow cytometry analysis as described in Materials and Methods. The values shown are averages of duplicate mice and the error bars represent the ranges. (B) C57BL/6 mice received OT-I/PL LN cells by adoptive transfer on day −1 and were challenged on day 0 with the indicated amounts of OVA257–264 peptide either alone or with 50 μg/mouse of LPS. Spleens were harvested on day 3, and the number of OT-I/PL cells was determined by flow cytometry. The values shown are averages of duplicate mice and the error bars represent the ranges. (C) Spleen cells from the animals described in A that received 10 μg of peptide, with and without IL-12, were assayed for lytic activity against 51Cr-labeled E.G7 targets at spleen cell/target ratios of 200, 100, 50, and 25:1. These ratios were converted to OT-I/PL target ratios by multiplying by the percent OT-I/PL cells in each spleen cell population. The results shown are for one of the two animals in each treatment group; splenocytes from the other animal in each group showed essentially identical lytic activity. (D) C57BL/6 mice received OT-I/PL LN cells by adoptive transfer on day −1 and were left unchallenged (transfer only) or challenged with 10 μg OVA257–264/mouse alone (peptide only) or with 1 μg/mouse of IL-12 (peptide + IL-12). On day 3, mice were injected with equal numbers of unpulsed and OVA257–264-pulsed C57BL/6 spleen cells that were labeled with low and high concentrations of CFSE, respectively. Spleens were harvested after 3 h and the cells were analyzed for the preferential loss of peptide-pulsed, CFSEhigh versus unpulsed, CFSElow target cells. Histograms show the CFSEhigh and CFSElow cells from one of two identically treated mice; the percent lysis, calculated as described in Materials and Methods, is indicated.
Mentions: There are a number of works describing CD8 T cell populations that have expanded in vivo but lack effector function (32, 38–42). To determine if a high level of signal 1 might stimulate in vivo clonal expansion without leading to development of effector function, experiments were done using adoptive transfer of OT-I T cells into normal C57BL/6 recipients to allow quantitation of the Ag-specific T cell response in a relatively normal immune system (51). OT-I cells were adoptively transferred by intravenous (tail vein) injection and allowed to equilibrate in the recipient's immune system for 1 d. Mice were challenged with varying doses of OVA257–264 peptide alone, or along with IL-12. 3 d later, at the peak of the response (34), the numbers of OT-I cells in the LN and spleen were determined and cytotoxicity was measured for the populations. At low peptide concentrations, clonal expansion was minimal in the absence of IL-12, but was at maximal levels when IL-12 was coadministered (Fig. 3 A). Clonal expansion in response to peptide in the absence of IL-12 increased in a dose-dependent manner, whereas increasing the peptide dose above 2 μg in the presence of IL-12 did not increase the number of OT-I T cells. Very similar results were obtained when peptide was administered along with LPS as an adjuvant (Fig. 3 B).

Bottom Line: The requirement for the third signal to stimulate Ag-dependent proliferation is variable, making the greatest contribution when Ag levels are low.Thus, proliferation and development of cytolytic function can be fully uncoupled.Thus, the presence or absence of the third signal appears to be a critical variable in determining whether stimulation by Ag results in tolerance versus development of effector function and establishment of a responsive memory population.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine and Pathology, Center for Immunology, University of Minnesota, MN 55455, USA.

ABSTRACT
Activation of naive CD8 T cells to undergo clonal expansion and develop effector function requires three signals: (a) Ag, (b) costimulation, and (c) IL-12 or adjuvant. The requirement for the third signal to stimulate Ag-dependent proliferation is variable, making the greatest contribution when Ag levels are low. At high Ag levels, extensive proliferation can occur in vitro or in vivo in the absence of a third signal. However, despite having undergone the same number of divisions, cells that expand in the absence of a third signal fail to develop cytolytic effector function. Thus, proliferation and development of cytolytic function can be fully uncoupled. Furthermore, these cells are rendered functionally tolerant in vivo, in that subsequent restimulation with a potent stimulus results in limited clonal expansion, impaired IFN-gamma production, and no cytolytic function. Thus, the presence or absence of the third signal appears to be a critical variable in determining whether stimulation by Ag results in tolerance versus development of effector function and establishment of a responsive memory population.

Show MeSH
Related in: MedlinePlus