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

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.

Figure 1.

OT-I T cells primed at high precursor frequency undergo less division and take on characteristics of higher quality effector cells. 10⁶ or 10⁷ OT-I T cells (along with 10⁶ OT-II T cells) were adoptively transferred into naive B6 recipients, which then received an mOVA skin graft. (A) DLN OT-I T cells primed at low frequency underwent more rounds of division and with earlier kinetics at days 3, 7, and 14 after transplantation as measured by CFSE analysis. (B) Absolute numbers of OT-I T cells isolated from the DLN showed a convergence at the peak of expansion in both the low and high frequency groups. (C) Cytokine-producing ability of cells primed at low versus high frequency was measured as the percentage of cells that stained positive for intracellular anti–IFN-γ after a 4-h in vitro peptide stimulation. (D) The absolute number of multifunctional effector cells that secreted both IFN-γ and TNF-α was calculated by multiplying the percentage of cytokine-positive cells by the total number of OT-I T cells as determined by TruCount analysis in B.

Figure 2.

High frequency graft-specific CD4⁺ and CD8⁺ T cells precipitate costimulation blockade-resistant rejection. Increasing numbers (10⁵, 10⁶, or 10⁷) of mOVA-specific OT-I and OT-II T cells were adoptively transferred into naive B6 recipients. (A) Mice were analyzed 48 h after transfer to determine the precursor frequency of graft-specific T cells in the axillary LNs resulting from the transfer of each number of cells. Data are representative of three mice per group. (B) Recipients of the indicated numbers of graft-specific T cells received mOVA skin grafts and CTLA-4 Ig and anti-CD154 where indicated (n = 6 mice group, two independent experiments). Results demonstrate that raising the precursor frequency of graft-specific T cells to the level observed in fully allogeneic situations resulted in costimulation blockade-resistant rejection (P < 0.001 for mice receiving 10⁷ OT-I/OT-II T cells as compared with all other groups treated with CTLA-4 Ig and anti-CD154).

Figure 3.

Decreased OT-I T cell expansion in the presence of CTLA-4 Ig/anti-CD154 after priming at low, but not high, antigen-specific precursor frequency. 10⁶ or 10⁷ OT-I T cells (along with 10⁶ OT-II cells) were adoptively transferred into naive B6 recipients that then received an mOVA skin graft. (A) Results showed that mice receiving a low frequency (10⁶) of OT-I T cells were protected from rejection by CTLA-4 Ig/anti-CD154, whereas mice receiving a high frequency (10⁷) rapidly rejected their grafts (P < 0.001). (B) Analysis of the percentage of Thy1.1⁺ CD8⁺ T cells in the DLN revealed that costimulation blockade inhibited the expansion/accumulation of OT-I T cells in mice responding from low frequency, whereas the expansion/accumulation of OT-I cells responding from high frequency was not as potently affected. (C) Absolute numbers of OT-I T cells isolated from the DLN showed a convergence at the peak of expansion at day 10 in both the low and high frequency untreated groups as well as the CTLA-4 Ig/anti-CD154–treated high frequency group. In contrast, the low frequency, CTLA-4 Ig/anti-CD154–treated group exhibited minimal expansion of OT-I T cell numbers. These data are representative examples of four independent experiments, with two to three mice per group, per time point, per experiment.

Figure 4.

OT-I T cells primed at low frequency in the presence of CTLA-4 Ig and anti-CD154 fail to accumulate in later rounds of cell division. 10⁶ or 10⁷ OT-I T cells (along with 10⁶ OT-II cells) were CFSE labeled and adoptively transferred into naive B6 recipients, which then received an mOVA skin graft. (A) Analysis of Thy1.1⁺ CD8⁺ DLN cells demonstrated reduced rounds of cell division in OT-I T cells responding from low frequency in the presence of CTLA-4 Ig/anti-CD154 relative to those left untreated (top two rows). In contrast, the cell division profile of OT-I cells responding from high frequency was not as potently affected by costimulation blockade (bottom two rows). (B) Absolute numbers of DLN OT-I T cells in each cell generation (0, no cell division) were calculated from the percentage of each population in peaks 0–7 or >7 (generation 8+). Data show that CTLA-4 Ig/anti-CD154–treated graft-specific cells responding from high frequency exhibited a division profile similar to those in the untreated controls, whereas cells responding from low frequency divided but failed to accumulate in the presence of CTLA-4 Ig/anti-CD154. These data are representative examples of four independent experiments, with two to three mice per group per experiment.

Figure 5.

Reduced numbers of functional effector cells in CTLA-4 Ig– and anti-CD154–treated mice with low precursor frequency of graft-specific cells. 10⁶ or 10⁷ OT-I T cells (along with 10⁶ OT-II cells) were adoptively transferred into naive B6 recipients before transplantation with an mOVA skin graft. (A) IFN-γ production by cells primed at low versus high frequency in the presence or absence of CTLA-4 Ig/ anti-CD154 was measured as the percentage of Thy1.1⁺ CD8⁺ T cells that stained positive for intracellular anti–IFN-γ after a 4-h in vitro peptide stimulation. (B) The absolute number of cytokine-producing effector cells that secreted IFN-γ or TNF-α was calculated by multiplying the percentage of cytokine-positive cells by the total number of OT-I T cells as determined by TruCount analysis. Results indicate that both the percentage (A and not depicted) and absolute numbers (B) of IFN-γ– and TNF-α–secreting cells were diminished in mice primed at low but not high frequency in the presence of CTLA-4 Ig/anti-CD154. These data are representative examples of four independent experiments, with two to three mice per group per experiment.

Figure 6.

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.