High frequency of autoreactive myelin proteolipid protein-specific T cells in the periphery of naive mice: mechanisms of selection of the self-reactive repertoire

Abstract

The autoreactive T cells that escape central tolerance and form the peripheral self-reactive repertoire determine both susceptibility to autoimmune disease and the epitope dominance of a specific autoantigen. SJL (H-2(s)) mice are highly susceptible to the induction of experimental autoimmune encephalomyelitis (EAE) with myelin proteolipid protein (PLP). The two major encephalitogenic epitopes of PLP (PLP 139-151 and PLP 178-191) bind to IA(s) with similar affinity; however, the immune response to the PLP 139-151 epitope is always dominant. The immunodominance of the PLP 139-151 epitope in SJL mice appears to be due to the presence of expanded numbers of T cells (frequency of 1/20,000 CD4(+) cells) reactive to PLP 139-151 in the peripheral repertoire of naive mice. Neither the PLP autoantigen nor infectious environmental agents appear to be responsible for this expanded repertoire, as endogenous PLP 139-151 reactivity is found in both PLP-deficient and germ-free mice. The high frequency of PLP 139-151-reactive T cells in SJL mice is partly due to lack of thymic deletion to PLP 139-151, as the DM20 isoform of PLP (which lacks residues 116-150) is more abundantly expressed in the thymus than full-length PLP. Reexpression of PLP 139-151 in the embryonic thymus results in a significant reduction of PLP 139-151-reactive precursors in naive mice. Thus, escape from central tolerance, combined with peripheral expansion by cross-reactive antigen(s), appears to be responsible for the high frequency of PLP 139-151-reactive T cells.

Figure 1

Naive SJL mice show a significant T cell response to PLP 139–151 but not to other myelin antigens. LNCs were harvested from 9-wk-old naive SJL mice and tested in triplicate for reactivity to various myelin antigens over a dose–response of 0.1–100 μg/ml of peptide. [³H]Thymidine was added at 48 h, and plates were harvested 16 h later. The data is shown as mean Δcpm (CPM) of triplicate wells, where ΔCPM = mean CPM in test wells − mean CPM in wells with media only. An experiment representative of at least four independent experiments is shown.

Figure 2

Endogenous PLP 139–151-reactive repertoire in different mouse strains. LNCs from naive SJL, Balb/s, B10.S (all H-2^s), C57Bl/6 (H-2^b), and Balb/c (H-2^d) mice were harvested and tested in triplicate for reactivity to PLP 139–151 and to a control antigen, NASE, over a dose–response of 0.1–100 μg/ml of peptide. [³H]Thymidine was added at 48 h, and plates were harvested 16 h later. The data is shown as mean ΔCPM of triplicate wells, where ΔCPM = mean CPM in test wells − mean CPM in wells with media only.

Figure 3

PLP 139–151 reactivity is enriched in the memory subset. LNCs from naive SJL mice were harvested. LNCs were then fractionated into CD3⁺CD44^lo and CD3⁺CD44^hi populations (as described in Materials and Methods) and tested for a proliferative response to PLP 139–151 and to the control antigen, NASE, over a dose–response of 0.1–100 μg/ml of peptide. T cells were cultured in triplicate. [³H]Thymidine was added at 48 h, and plates were harvested 16 h later. The data is shown as mean ΔCPM of triplicate wells, where ΔCPM = mean CPM in test wells − mean CPM in wells with media only.

Figure 4

Endogenous PLP 139–151-reactive repertoire is present in PLP-deficient mice. LNCs were harvested from PLP^+/+ and PLP^−/Y mice on the Balb/s background and tested in triplicate for reactivity to PLP 139–151 and to a control antigen, NASE, over a dose–response of 0.1–100 μg/ml of peptide. [³H]Thymidine was added at 48 h, and plates were harvested 16 h later. The data is shown as mean ΔCPM of triplicate wells, where ΔCPM = mean CPM in test wells − mean CPM in wells with media only.

Figure 5

Endogenous PLP 139–151-reactive repertoire is present in germ-free and defined flora SJL mice. LNCs were harvested from naive wild-type, defined flora, and germ-free SJL mice and tested in triplicate for reactivity to PLP 139–151 and to a control antigen, NASE, over a dose–response of 0.1–100 μg/ml of peptide. [³H]Thymidine was added at 48 h, and plates were harvested 16 h later. The data is shown as mean ΔCPM of triplicate wells, where ΔCPM = mean CPM in test wells − mean CPM in wells with media only.

Figure 6

PLP/DM20 expression in the brains and thymi of SJL mice. (A) Schematic diagram of the PLP/DM20 gene. (B) PLP/DM20 cDNA was amplified by RT-PCR from brains and thymi of SJL mice of different ages using primers that amplify both PLP and DM20 (top row). Amplification of β-actin from the same cDNA samples is shown as a control for quality of the cDNA (bottom row). Embryonic day 16 (lanes 1 and 7), neonate (lanes 2 and 8), 1 wk (lanes 3 and 9), 9 wk (lane 4), 11 wk (lane 10), 20 wk (lanes 5 and 11), 36 wk (lanes 6 and 12), and double distilled H₂0 control (lane 13).

Figure 6

PLP/DM20 expression in the brains and thymi of SJL mice. (A) Schematic diagram of the PLP/DM20 gene. (B) PLP/DM20 cDNA was amplified by RT-PCR from brains and thymi of SJL mice of different ages using primers that amplify both PLP and DM20 (top row). Amplification of β-actin from the same cDNA samples is shown as a control for quality of the cDNA (bottom row). Embryonic day 16 (lanes 1 and 7), neonate (lanes 2 and 8), 1 wk (lanes 3 and 9), 9 wk (lane 4), 11 wk (lane 10), 20 wk (lanes 5 and 11), 36 wk (lanes 6 and 12), and double distilled H₂0 control (lane 13).

Figure 7

Introduction of the PLP 139–151 epitope into the thymi of SJL mice reduces the endogenous PLP 139–151-reactive repertoire. (A) Thymocytes were purified from neonatal mice that had received IgPLP 139–151, IgCTRL, or no treatment in utero and activated by PLP 139–151 or control peptide (HA) in vitro. Treatment with IgPLP 139–151 but not IgCTRL or no treatment abolished PLP 139–151-specific thymocyte proliferation. (B) LNCs were harvested from naive 8-wk-old SJL mice (male and female) that were exposed in utero to IgPLP 139–151 or IgCTRL. LNCs were tested in triplicate for reactivity to PLP 139–151 and to a control antigen, NASE, over a dose–response of 0.1–100 μg/ml of peptide. [³H]Thymidine was added at 48 h, and plates were harvested 16 h later. The data is shown as mean ΔCPM of triplicate wells, where ΔCPM = mean CPM in test wells − mean CPM in wells with media only.

Figure 7

Introduction of the PLP 139–151 epitope into the thymi of SJL mice reduces the endogenous PLP 139–151-reactive repertoire. (A) Thymocytes were purified from neonatal mice that had received IgPLP 139–151, IgCTRL, or no treatment in utero and activated by PLP 139–151 or control peptide (HA) in vitro. Treatment with IgPLP 139–151 but not IgCTRL or no treatment abolished PLP 139–151-specific thymocyte proliferation. (B) LNCs were harvested from naive 8-wk-old SJL mice (male and female) that were exposed in utero to IgPLP 139–151 or IgCTRL. LNCs were tested in triplicate for reactivity to PLP 139–151 and to a control antigen, NASE, over a dose–response of 0.1–100 μg/ml of peptide. [³H]Thymidine was added at 48 h, and plates were harvested 16 h later. The data is shown as mean ΔCPM of triplicate wells, where ΔCPM = mean CPM in test wells − mean CPM in wells with media only.