CD4(+) T cells prevent spontaneous experimental autoimmune encephalomyelitis in anti-myelin basic protein T cell receptor transgenic mice

Abstract

Autoimmune diseases result from a failure of tolerance. Although many self-reactive T cells are present in animals and humans, their activation appears to be prevented normally by regulatory T cells. In this study, we show that regulatory CD4(+) T cells do protect mice against the spontaneous occurrence of experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis. Anti-myelin basic protein (MBP) TCR transgenic mice (T/R+) do not spontaneously develop EAE although many self-reactive T cells are present in their thymi and peripheral lymphoid organs. However, the disease develops in all crosses of T/R+ mice with recombination-activating gene (RAG)-1 knockout mice in which transgenic TCR-expressing cells are the only lymphocytes present (T/R- mice). In this study, crosses of T/R+ mice with mice deficient for B cells, CD8(+) T cells, NK1.1 CD4(+) T (NKT) cells, gamma/delta T cells, or alpha/beta T cells indicated that alpha/beta CD4(+) T cells were the only cell population capable of controlling the self-reactive T cells. To confirm the protective role of CD4(+) T cells, we performed adoptive transfer experiments. CD4(+) T cells purified from thymi or lymph nodes of normal mice prevented the occurrence of spontaneous EAE in T/R- mice. To achieve full protection, the cells had to be transferred before the recipient mice manifested any symptoms of the disease. Transfer of CD4(+) T cells after the appearance of symptoms of EAE had no protective effect. These results indicate that at least some CD4(+) T cells have a regulatory function that prevent the activation of self-reactive T cells.

Figure 1

Incidence of spontaneous EAE in anti-MBP TCR transgenic RAG-1^+/− (T/R⁺) and RAG-1^−/− (T/R⁻) mice. (a) Percentage of mice with EAE among T/R⁺ (white bar, n = 45), and T/R⁻ (striped bar, n = 30) mice. All mice were monitored for EAE symptoms for at least 5 mo starting at 1 mo of age unless they developed EAE and died. (b) Time course of the development of spontaneous EAE in T/R⁻ mice.

Figure 2

Incidence of spontaneous EAE in various KO mice carrying the anti-MBP TCR transgene. The incidence of EAE was monitored in H-2^u/u anti-MBP TCR transgenic mice that were homozygous (−/−) or heterozygous (+/−) for the disruption of the gene indicated on top of each figure. All mice were monitored for signs of disease for at least 5 mo starting at 1 mo of age unless they developed EAE and died. The number of mice observed with EAE as well as the total number of mice in each group is indicated at the top of each bar. Disease incidences between each +/− and −/− group were compared using the normal-theory method. The only statistical difference observed was between the TCR α +/− and −/− groups with a P < 0.001.

Figure 3

Protection of T/R⁻ mice against EAE by adoptive transfer of CD4⁺ T cells. (a) 29–41-d-old T/R⁻ mice were injected with 2.5 × 10⁶ CD4⁺ T cells isolated from lymph nodes (open circle) or from thymi (filled circle). One mouse injected with thymocytes showed mild symptoms of EAE (level 1) but quickly recovered. The control noninjected mouse (filled square) developed severe EAE (level 3.5). (b) Transfer of 5 × 10⁶ CD4⁺ T cells in T/R⁻ mice grouped by age. Cells isolated from either lymph nodes or thymi were injected into five T/R⁻ mice, 30–40 d old (filled square), seven T/R⁻ mice, 41–45 d old (open diamond), and seven T/R⁻ mice, >45 d old (cross). Among the three groups of 19 treated animals, 8 and 11 mice received cells isolated from lymph nodes and thymi, respectively. 13 control mice received no injection (open circle).

Figure 4

Flow cytometry analysis of the donor CD4⁺ T cells. (A) CD4/CD3 profile of aliquots of the donor cells isolated from lymph nodes (a) or from thymi (b) of female B10.PL mice. The percentage of CD4⁺CD3⁺ T cells is indicated at the upper right corner of each panel. (B) Histograms of the expression of the indicated markers on the surface of the CD4⁺-gated donor cells from lymph nodes (a) or thymi (b).

Figure 5

Flow cytometry analysis of lymphocytes recovered from a T/R⁻ recipient of CD4⁺ T cells and from a T/R⁺ littermate. (A) CD4/Vβ8.2 profile of mesenteric lymph node lymphocytes from a T/R⁻ recipient mouse 9 wk after the transfer of CD4⁺ T cells from lymph nodes of B10.PL mice (a), and from an age-matched T/R⁺ littermate that received no injection (b). The percentage of CD4⁺Vβ8.2⁻ (i.e., CD4⁺ nontransgenic cells) and CD4⁺ Vβ8.2⁺ (i.e., mostly CD4⁺ transgenic cells) is indicated on each panel at the upper left and right quadrants, respectively. (B) Histograms (filled area) of the expression of the indicated markers on the surface of the CD4⁺Vβ8.2⁻-gated mesenteric lymph node lymphocytes from a T/R⁻ recipient mouse 9 wk after the transfer of CD4⁺ T cells from lymph node cells, or from a T/R⁺ littermate (b). In B (a), the histograms of the donor cells shown in Fig. 4 B, a are reproduced for comparison (open area). The experiments were carried out with 11 T/R⁻ recipient mice and with 5 T/R⁺ littermates. The data shown are representative of these experiments. Similar experiments were also carried out with mesenteric lymph node lymphocytes from T/R⁻ mice that received CD4⁺ T cells from B10.PL thymi. The results were similar to those shown here in part a of A and B.