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Antigen-specific precursor frequency impacts T cell proliferation, differentiation, and requirement for costimulation.

Ford ML, Koehn BH, Wagener ME, Jiang W, Gangappa S, Pearson TC, Larsen CP - J. Exp. Med. (2007)

Bottom Line: Using an adoptive transfer system to incrementally raise the precursor frequency of antigen-specific CD8(+) T cells, we found that donor-reactive T cells primed at low frequency exhibited increased cellular division, decreased development of multifunctional effector activity, and an increased requirement for CD28- and CD154-mediated costimulation relative to those primed at high frequency.The results demonstrated that recipients with low CD4(+) and CD8(+) donor-reactive T cell frequencies exhibited long-term skin graft survival upon CD28/CD154 blockade, whereas simultaneously raising the frequency of CD4(+) T cells to approximately 0.5% and CD8(+) T cells to approximately 5% precipitated graft rejection despite CD28/CD154 blockade.These results demonstrate a critical role for initial precursor frequency in determining the CD8(+) T cell requirement for CD28- and CD154-mediated costimulatory signals during graft rejection.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Emory Transplant Center, Emory University, Atlanta, GA 30322, USA.

ABSTRACT
After a brief period of antigenic stimulation, T cells become committed to a program of autonomous expansion and differentiation. We investigated the role of antigen-specific T cell precursor frequency as a possible cell-extrinsic factor impacting T cell programming in a model of allogeneic tissue transplantation. Using an adoptive transfer system to incrementally raise the precursor frequency of antigen-specific CD8(+) T cells, we found that donor-reactive T cells primed at low frequency exhibited increased cellular division, decreased development of multifunctional effector activity, and an increased requirement for CD28- and CD154-mediated costimulation relative to those primed at high frequency. The results demonstrated that recipients with low CD4(+) and CD8(+) donor-reactive T cell frequencies exhibited long-term skin graft survival upon CD28/CD154 blockade, whereas simultaneously raising the frequency of CD4(+) T cells to approximately 0.5% and CD8(+) T cells to approximately 5% precipitated graft rejection despite CD28/CD154 blockade. Antigenic rechallenge of equal numbers of cells stimulated at high or low frequency revealed that cells retained an imprint of the frequency at which they were primed. These results demonstrate a critical role for initial precursor frequency in determining the CD8(+) T cell requirement for CD28- and CD154-mediated costimulatory signals during graft rejection.

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CD8+ T cells stimulated at initial high or low frequency retain the imprint of the conditions under which they were primed after secondary antigenic challenge. OT-I T cells were CFSE labeled and stimulated with OVA peptide at high (3 × 106 cells/well) or low frequency (3 × 105 cells/well) in the presence of 100 μg/ml CTLA-4 Ig/anti-CD154. B6 splenocytes were added to the low frequency cultures to keep the total number of cells per well consistent. (A) 3 d later, CFSE dilution of live CD8+ Thy1.1+ cells was measured by flow cytometry. Cells stimulated at low frequency underwent more rounds of division than those primed at high frequency, and CTLA-4 Ig/anti-CD154 did not inhibit cell division in the 72-h culture period (data shown are representative examples from three independent experiments). (B) 5 × 106 T cells from cultures primed at an initial high or low antigen-specific T cell frequency were adoptively transferred into P14xRAG−/− recipients. Mice received an mOVA skin graft 48 h later and were monitored for signs of rejection (data shown are cumulative results from three independent experiments, with the total number of mice indicated in the legend). (C) Analysis of peripheral blood of recipients of cells stimulated in vitro at low or high frequency at day 40 after transplantation revealed similar frequencies of CD8+ Thy1.1+ cells.
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fig6: CD8+ T cells stimulated at initial high or low frequency retain the imprint of the conditions under which they were primed after secondary antigenic challenge. OT-I T cells were CFSE labeled and stimulated with OVA peptide at high (3 × 106 cells/well) or low frequency (3 × 105 cells/well) in the presence of 100 μg/ml CTLA-4 Ig/anti-CD154. B6 splenocytes were added to the low frequency cultures to keep the total number of cells per well consistent. (A) 3 d later, CFSE dilution of live CD8+ Thy1.1+ cells was measured by flow cytometry. Cells stimulated at low frequency underwent more rounds of division than those primed at high frequency, and CTLA-4 Ig/anti-CD154 did not inhibit cell division in the 72-h culture period (data shown are representative examples from three independent experiments). (B) 5 × 106 T cells from cultures primed at an initial high or low antigen-specific T cell frequency were adoptively transferred into P14xRAG−/− recipients. Mice received an mOVA skin graft 48 h later and were monitored for signs of rejection (data shown are cumulative results from three independent experiments, with the total number of mice indicated in the legend). (C) Analysis of peripheral blood of recipients of cells stimulated in vitro at low or high frequency at day 40 after transplantation revealed similar frequencies of CD8+ Thy1.1+ cells.

Mentions: It is becoming increasingly apparent that T cells retain an imprint of their initial priming conditions, which may then influence characteristics of the response upon subsequent encounters with antigen (11–13, 31–33). To test the hypothesis that precursor frequency during the priming phase of T cell activation is an important factor influencing the T cell program, we established a system where OT-I T cells were stimulated at high or low frequency with OVA peptide in vitro in the presence of CTLA-4 Ig/anti-CD154, and later rechallenged with an mOVA skin graft in vivo. B6 splenocytes were added to the low frequency in vitro cultures to keep the total number of cells per well constant. Consistent with our in vivo studies, OT-I T cells primed at low frequency in vitro underwent more extensive division than cells primed at high frequency (Fig. 6 A). 3 d later, equal numbers (5 × 106) of live T cells primed at an initial high or low antigen-specific T cell frequency were adoptively transferred into P14xRAG−/− recipients. These mice have a T cell compartment restricted to the recognition of LCMV gp33-41 and therefore cannot reject an mOVA skin graft without the provision of exogenous antigen-specific T cells (unpublished data). In contrast to polyclonal CD4+ and CD8+ T cell populations (34 and unpublished data), OT-I T cells do not undergo significant homeostatic expansion in these hosts (unpublished data), thus allowing analysis of the fate and function of the adoptively transferred cells in this system, relatively free from the effects of homeostatic proliferation.


Antigen-specific precursor frequency impacts T cell proliferation, differentiation, and requirement for costimulation.

Ford ML, Koehn BH, Wagener ME, Jiang W, Gangappa S, Pearson TC, Larsen CP - J. Exp. Med. (2007)

CD8+ T cells stimulated at initial high or low frequency retain the imprint of the conditions under which they were primed after secondary antigenic challenge. OT-I T cells were CFSE labeled and stimulated with OVA peptide at high (3 × 106 cells/well) or low frequency (3 × 105 cells/well) in the presence of 100 μg/ml CTLA-4 Ig/anti-CD154. B6 splenocytes were added to the low frequency cultures to keep the total number of cells per well consistent. (A) 3 d later, CFSE dilution of live CD8+ Thy1.1+ cells was measured by flow cytometry. Cells stimulated at low frequency underwent more rounds of division than those primed at high frequency, and CTLA-4 Ig/anti-CD154 did not inhibit cell division in the 72-h culture period (data shown are representative examples from three independent experiments). (B) 5 × 106 T cells from cultures primed at an initial high or low antigen-specific T cell frequency were adoptively transferred into P14xRAG−/− recipients. Mice received an mOVA skin graft 48 h later and were monitored for signs of rejection (data shown are cumulative results from three independent experiments, with the total number of mice indicated in the legend). (C) Analysis of peripheral blood of recipients of cells stimulated in vitro at low or high frequency at day 40 after transplantation revealed similar frequencies of CD8+ Thy1.1+ cells.
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Related In: Results  -  Collection

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fig6: CD8+ T cells stimulated at initial high or low frequency retain the imprint of the conditions under which they were primed after secondary antigenic challenge. OT-I T cells were CFSE labeled and stimulated with OVA peptide at high (3 × 106 cells/well) or low frequency (3 × 105 cells/well) in the presence of 100 μg/ml CTLA-4 Ig/anti-CD154. B6 splenocytes were added to the low frequency cultures to keep the total number of cells per well consistent. (A) 3 d later, CFSE dilution of live CD8+ Thy1.1+ cells was measured by flow cytometry. Cells stimulated at low frequency underwent more rounds of division than those primed at high frequency, and CTLA-4 Ig/anti-CD154 did not inhibit cell division in the 72-h culture period (data shown are representative examples from three independent experiments). (B) 5 × 106 T cells from cultures primed at an initial high or low antigen-specific T cell frequency were adoptively transferred into P14xRAG−/− recipients. Mice received an mOVA skin graft 48 h later and were monitored for signs of rejection (data shown are cumulative results from three independent experiments, with the total number of mice indicated in the legend). (C) Analysis of peripheral blood of recipients of cells stimulated in vitro at low or high frequency at day 40 after transplantation revealed similar frequencies of CD8+ Thy1.1+ cells.
Mentions: It is becoming increasingly apparent that T cells retain an imprint of their initial priming conditions, which may then influence characteristics of the response upon subsequent encounters with antigen (11–13, 31–33). To test the hypothesis that precursor frequency during the priming phase of T cell activation is an important factor influencing the T cell program, we established a system where OT-I T cells were stimulated at high or low frequency with OVA peptide in vitro in the presence of CTLA-4 Ig/anti-CD154, and later rechallenged with an mOVA skin graft in vivo. B6 splenocytes were added to the low frequency in vitro cultures to keep the total number of cells per well constant. Consistent with our in vivo studies, OT-I T cells primed at low frequency in vitro underwent more extensive division than cells primed at high frequency (Fig. 6 A). 3 d later, equal numbers (5 × 106) of live T cells primed at an initial high or low antigen-specific T cell frequency were adoptively transferred into P14xRAG−/− recipients. These mice have a T cell compartment restricted to the recognition of LCMV gp33-41 and therefore cannot reject an mOVA skin graft without the provision of exogenous antigen-specific T cells (unpublished data). In contrast to polyclonal CD4+ and CD8+ T cell populations (34 and unpublished data), OT-I T cells do not undergo significant homeostatic expansion in these hosts (unpublished data), thus allowing analysis of the fate and function of the adoptively transferred cells in this system, relatively free from the effects of homeostatic proliferation.

Bottom Line: Using an adoptive transfer system to incrementally raise the precursor frequency of antigen-specific CD8(+) T cells, we found that donor-reactive T cells primed at low frequency exhibited increased cellular division, decreased development of multifunctional effector activity, and an increased requirement for CD28- and CD154-mediated costimulation relative to those primed at high frequency.The results demonstrated that recipients with low CD4(+) and CD8(+) donor-reactive T cell frequencies exhibited long-term skin graft survival upon CD28/CD154 blockade, whereas simultaneously raising the frequency of CD4(+) T cells to approximately 0.5% and CD8(+) T cells to approximately 5% precipitated graft rejection despite CD28/CD154 blockade.These results demonstrate a critical role for initial precursor frequency in determining the CD8(+) T cell requirement for CD28- and CD154-mediated costimulatory signals during graft rejection.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Emory Transplant Center, Emory University, Atlanta, GA 30322, USA.

ABSTRACT
After a brief period of antigenic stimulation, T cells become committed to a program of autonomous expansion and differentiation. We investigated the role of antigen-specific T cell precursor frequency as a possible cell-extrinsic factor impacting T cell programming in a model of allogeneic tissue transplantation. Using an adoptive transfer system to incrementally raise the precursor frequency of antigen-specific CD8(+) T cells, we found that donor-reactive T cells primed at low frequency exhibited increased cellular division, decreased development of multifunctional effector activity, and an increased requirement for CD28- and CD154-mediated costimulation relative to those primed at high frequency. The results demonstrated that recipients with low CD4(+) and CD8(+) donor-reactive T cell frequencies exhibited long-term skin graft survival upon CD28/CD154 blockade, whereas simultaneously raising the frequency of CD4(+) T cells to approximately 0.5% and CD8(+) T cells to approximately 5% precipitated graft rejection despite CD28/CD154 blockade. Antigenic rechallenge of equal numbers of cells stimulated at high or low frequency revealed that cells retained an imprint of the frequency at which they were primed. These results demonstrate a critical role for initial precursor frequency in determining the CD8(+) T cell requirement for CD28- and CD154-mediated costimulatory signals during graft rejection.

Show MeSH
Related in: MedlinePlus