CD4(+) T cells from lupus-prone mice are hyperresponsive to T cell receptor engagement with low and high affinity peptide antigens: a model to explain spontaneous T cell activation in lupus

Abstract

Polyclonal CD4(+) T cell activation is characteristic of spontaneous lupus. As a potential explanation for this phenotype, we hypothesized that T cells from lupus-prone mice are intrinsically hyperresponsive to stimulation with antigen, particularly to those peptide ligands having a low affinity for the T cell receptor (TCR). To test this hypothesis, we backcrossed the alpha and beta chain genes of the AND TCR specific for amino acids 88-104 of pigeon cytochrome C (PCC) to the Fas-intact MRL/Mp(+)(Fas-lpr) and to the H-2(k)-matched control backgrounds B10.BR and CBA/CaJ (MRL.AND, B10.AND, and CBA.AND, respectively), and assessed naive CD4(+) TCR transgenic T cell activation in vitro after its encounter with cognate antigen and lower affinity altered peptide ligands (APLs). MRL.AND T cells, compared with control B10.AND and CBA.AND cells, proliferated more when stimulated with agonist antigen. More strikingly, MRL.AND T cells proliferated significantly more and produced more interleukin 2 when stimulated with the APLs of PCC 88-104, having lower affinity for the transgenic TCR. These results imply that one of the forces driving polyclonal activation of alpha/beta T cells in lupus is an intrinsically heightened response to peptide antigen, particularly those with low affinity for the TCR, independent of the nature of the antigen-presenting cell and degree of costimulation.

Figure 1

CD4⁺ AND transgenic T cells from MRL mice proliferate more after agonist peptide and mitogenic stimulation than CD4⁺ AND transgenic T cells from nonautoimmune strains. (A) Naive CD4⁺ transgenic T cells were labeled with CFSE and stimulated with 0.05 μM PCC 88–104 agonist peptide using CH27 cells as APCs. The number of cell divisions at 72 h was determined by CFSE labeling. CH27 and dead T cells were excluded from the analysis on the basis of forward/side scatter. The mean number of cell divisions at 72 h is indicated in the bar graph at the right. *P < 0.005 for comparison of MRL.AND to B10.AND or CBA.AND cells. Arrows indicate evidence of cell division. (B) Naive AND transgenic T cells were stimulated in vitro by I-E^k bearing B7.1⁺/B7.2⁺ CH27 cells pulsed with varying concentrations of PCC 88–104. Proliferation was measured at 72 h by [³H]thymidine incorporation. Results are means of quadruplicate measurements. Error bars represent SEM. *P < 0.05 for comparison of MRL.AND to B10.AND or CBA.AND cells. (C) Naive AND transgenic T cells were stimulated in vitro with 10 ng/ml of PMA and 500 ng/ml of ionomycin. Proliferation was measured at 72 h by [³H]thymidine incorporation. Results are means of quadruplicate measurements. *P < 0.05 for comparison of MRL.AND to B10.AND or CBA.AND cells.

Figure 1

CD4⁺ AND transgenic T cells from MRL mice proliferate more after agonist peptide and mitogenic stimulation than CD4⁺ AND transgenic T cells from nonautoimmune strains. (A) Naive CD4⁺ transgenic T cells were labeled with CFSE and stimulated with 0.05 μM PCC 88–104 agonist peptide using CH27 cells as APCs. The number of cell divisions at 72 h was determined by CFSE labeling. CH27 and dead T cells were excluded from the analysis on the basis of forward/side scatter. The mean number of cell divisions at 72 h is indicated in the bar graph at the right. *P < 0.005 for comparison of MRL.AND to B10.AND or CBA.AND cells. Arrows indicate evidence of cell division. (B) Naive AND transgenic T cells were stimulated in vitro by I-E^k bearing B7.1⁺/B7.2⁺ CH27 cells pulsed with varying concentrations of PCC 88–104. Proliferation was measured at 72 h by [³H]thymidine incorporation. Results are means of quadruplicate measurements. Error bars represent SEM. *P < 0.05 for comparison of MRL.AND to B10.AND or CBA.AND cells. (C) Naive AND transgenic T cells were stimulated in vitro with 10 ng/ml of PMA and 500 ng/ml of ionomycin. Proliferation was measured at 72 h by [³H]thymidine incorporation. Results are means of quadruplicate measurements. *P < 0.05 for comparison of MRL.AND to B10.AND or CBA.AND cells.

Figure 1

CD4⁺ AND transgenic T cells from MRL mice proliferate more after agonist peptide and mitogenic stimulation than CD4⁺ AND transgenic T cells from nonautoimmune strains. (A) Naive CD4⁺ transgenic T cells were labeled with CFSE and stimulated with 0.05 μM PCC 88–104 agonist peptide using CH27 cells as APCs. The number of cell divisions at 72 h was determined by CFSE labeling. CH27 and dead T cells were excluded from the analysis on the basis of forward/side scatter. The mean number of cell divisions at 72 h is indicated in the bar graph at the right. *P < 0.005 for comparison of MRL.AND to B10.AND or CBA.AND cells. Arrows indicate evidence of cell division. (B) Naive AND transgenic T cells were stimulated in vitro by I-E^k bearing B7.1⁺/B7.2⁺ CH27 cells pulsed with varying concentrations of PCC 88–104. Proliferation was measured at 72 h by [³H]thymidine incorporation. Results are means of quadruplicate measurements. Error bars represent SEM. *P < 0.05 for comparison of MRL.AND to B10.AND or CBA.AND cells. (C) Naive AND transgenic T cells were stimulated in vitro with 10 ng/ml of PMA and 500 ng/ml of ionomycin. Proliferation was measured at 72 h by [³H]thymidine incorporation. Results are means of quadruplicate measurements. *P < 0.05 for comparison of MRL.AND to B10.AND or CBA.AND cells.

Figure 2

CD4⁺ AND transgenic T cells from MRL mice proliferate more and differentially express activation markers in response to low affinity TCR ligands compared with CD4⁺ transgenic T cells from nonautoimmune mice. (A and B) Naive splenic CD4⁺ transgenic T cells were stimulated with varying concentrations of K99Q (A) or A96I (B) presented by I-E^k bearing B7.1⁺/B7.2⁺ CH27 cells. (C) Naive CD44^loCD62L^hi AND transgenic T cells from lymph nodes were stimulated with varying concentrations of K99R presented by CH27 cells. Proliferation was measured at 72 h by [³H]thymidine incorporation. Results are means of quadruplicate measurements. *P < 0.005 for comparison of MRL.AND to B10.AND or CBA.AND cells in A–C. (D) Naive CD44^loCD62L^hi splenic CD4⁺ transgenic T cells were stimulated with 25 μM K99Q or 0.05 μM PCC 88–104 presented by CH27 cells. CD44 and CD62L expression were determined by flow cytometry 48 h after stimulation, with CD44^hi cells designated as activated cells and CD44^hiCD62L^lo cells as memory cells. Arrows indicate the population of activated T cells that have upregulated CD44.

Figure 2

CD4⁺ AND transgenic T cells from MRL mice proliferate more and differentially express activation markers in response to low affinity TCR ligands compared with CD4⁺ transgenic T cells from nonautoimmune mice. (A and B) Naive splenic CD4⁺ transgenic T cells were stimulated with varying concentrations of K99Q (A) or A96I (B) presented by I-E^k bearing B7.1⁺/B7.2⁺ CH27 cells. (C) Naive CD44^loCD62L^hi AND transgenic T cells from lymph nodes were stimulated with varying concentrations of K99R presented by CH27 cells. Proliferation was measured at 72 h by [³H]thymidine incorporation. Results are means of quadruplicate measurements. *P < 0.005 for comparison of MRL.AND to B10.AND or CBA.AND cells in A–C. (D) Naive CD44^loCD62L^hi splenic CD4⁺ transgenic T cells were stimulated with 25 μM K99Q or 0.05 μM PCC 88–104 presented by CH27 cells. CD44 and CD62L expression were determined by flow cytometry 48 h after stimulation, with CD44^hi cells designated as activated cells and CD44^hiCD62L^lo cells as memory cells. Arrows indicate the population of activated T cells that have upregulated CD44.

Figure 2

CD4⁺ AND transgenic T cells from MRL mice proliferate more and differentially express activation markers in response to low affinity TCR ligands compared with CD4⁺ transgenic T cells from nonautoimmune mice. (A and B) Naive splenic CD4⁺ transgenic T cells were stimulated with varying concentrations of K99Q (A) or A96I (B) presented by I-E^k bearing B7.1⁺/B7.2⁺ CH27 cells. (C) Naive CD44^loCD62L^hi AND transgenic T cells from lymph nodes were stimulated with varying concentrations of K99R presented by CH27 cells. Proliferation was measured at 72 h by [³H]thymidine incorporation. Results are means of quadruplicate measurements. *P < 0.005 for comparison of MRL.AND to B10.AND or CBA.AND cells in A–C. (D) Naive CD44^loCD62L^hi splenic CD4⁺ transgenic T cells were stimulated with 25 μM K99Q or 0.05 μM PCC 88–104 presented by CH27 cells. CD44 and CD62L expression were determined by flow cytometry 48 h after stimulation, with CD44^hi cells designated as activated cells and CD44^hiCD62L^lo cells as memory cells. Arrows indicate the population of activated T cells that have upregulated CD44.

Figure 2

CD4⁺ AND transgenic T cells from MRL mice proliferate more and differentially express activation markers in response to low affinity TCR ligands compared with CD4⁺ transgenic T cells from nonautoimmune mice. (A and B) Naive splenic CD4⁺ transgenic T cells were stimulated with varying concentrations of K99Q (A) or A96I (B) presented by I-E^k bearing B7.1⁺/B7.2⁺ CH27 cells. (C) Naive CD44^loCD62L^hi AND transgenic T cells from lymph nodes were stimulated with varying concentrations of K99R presented by CH27 cells. Proliferation was measured at 72 h by [³H]thymidine incorporation. Results are means of quadruplicate measurements. *P < 0.005 for comparison of MRL.AND to B10.AND or CBA.AND cells in A–C. (D) Naive CD44^loCD62L^hi splenic CD4⁺ transgenic T cells were stimulated with 25 μM K99Q or 0.05 μM PCC 88–104 presented by CH27 cells. CD44 and CD62L expression were determined by flow cytometry 48 h after stimulation, with CD44^hi cells designated as activated cells and CD44^hiCD62L^lo cells as memory cells. Arrows indicate the population of activated T cells that have upregulated CD44.

Figure 3

(A) AND transgenic T cells labeled with CFSE and stimulated in vitro with CH27 cells alone (pink line) or pulsed with either 5 μM (red line) or 25 μM (blue line) K99Q. Fluorescence of T cells cultured without APCs is indicated in green with analysis performed at 96 h. Arrows indicate evidence of cell division. The mean number of cell divisions at 96 h is indicated in the bar graph at the right. *P < 0.005 for comparison of MRL.AND to B10.AND or CBA.AND cells. The change in scale used in MRL plot is indicative of the increased number of T cells. CH27 and dead T cells were excluded from the analysis on the basis of forward/side scatter. (B) AND transgenic T cells were labeled with CFSE and stimulated with 25 μM antagonist K99R. Arrows indicate cell division at 48 h. The probability of cell division is shown in the bar graph at right. Error bar represents SEM. *P < 0.0001 for comparison of MRL.AND to CBA.AND T cells.

Figure 3

(A) AND transgenic T cells labeled with CFSE and stimulated in vitro with CH27 cells alone (pink line) or pulsed with either 5 μM (red line) or 25 μM (blue line) K99Q. Fluorescence of T cells cultured without APCs is indicated in green with analysis performed at 96 h. Arrows indicate evidence of cell division. The mean number of cell divisions at 96 h is indicated in the bar graph at the right. *P < 0.005 for comparison of MRL.AND to B10.AND or CBA.AND cells. The change in scale used in MRL plot is indicative of the increased number of T cells. CH27 and dead T cells were excluded from the analysis on the basis of forward/side scatter. (B) AND transgenic T cells were labeled with CFSE and stimulated with 25 μM antagonist K99R. Arrows indicate cell division at 48 h. The probability of cell division is shown in the bar graph at right. Error bar represents SEM. *P < 0.0001 for comparison of MRL.AND to CBA.AND T cells.

Figure 4

MRL.AND transgenic T cells produce more IL-2 after stimulation with APLs than control transgenic T cells. (A) Negatively selected naive CD4⁺ AND transgenic T cells were stimulated in vitro with varying concentrations of a low affinity APL of PCC 88–104 (K99Q) presented by CH27 cells. IL-2 was determined by indirect ELISA from culture supernatant 24 h after stimulation. Error bars represent SEM of triplicate wells. *P < 0.05 for comparison of MRL.AND to control nonautoimmune transgenic strains. (B) The percentage of transgenic cells producing IL-2 was estimated using intracellular cytokine staining with anti–IL-2 by gating on CD4⁺Vα11^hi double-positive cells. *P < 0.05 for comparison of MRL.AND T cells to CBA.AND and B10.AND T cells.

Figure 4

MRL.AND transgenic T cells produce more IL-2 after stimulation with APLs than control transgenic T cells. (A) Negatively selected naive CD4⁺ AND transgenic T cells were stimulated in vitro with varying concentrations of a low affinity APL of PCC 88–104 (K99Q) presented by CH27 cells. IL-2 was determined by indirect ELISA from culture supernatant 24 h after stimulation. Error bars represent SEM of triplicate wells. *P < 0.05 for comparison of MRL.AND to control nonautoimmune transgenic strains. (B) The percentage of transgenic cells producing IL-2 was estimated using intracellular cytokine staining with anti–IL-2 by gating on CD4⁺Vα11^hi double-positive cells. *P < 0.05 for comparison of MRL.AND T cells to CBA.AND and B10.AND T cells.