CD4⁺CD25⁺FOXP3⁺Treg (tTreg) protect against induction of autoimmunity, but fresh naïve or ex vivo expanded tTreg have limited ability to control established autoimmunity.  We have been examining pathways of naïve tTreg activation by antigen and Th2 cytokines IL-4 and IL-5.  First, IL-4 and antigen induces expression of the IL-5 receptor (IL-5Ra) inducing Ts2 Treg.  In a second step, IL-5, not IL-4, and antigen promotes induction of Th2-like Treg.

EAE was induced in Lewis rats by immunization with myelin basic protein (MBP) and Freund’s complete adjuvant. Naïve CD4⁺CD25⁺FOXP3⁺Treg were cultured with MBP and IL-4 for 4d to produce Ts2 cells. To produce Th2-like Treg, Ts2 cells were cultured with IL-5 and MBP for 4d.  Cell populations were assessed by FACS and RT-PCR. 

To examine their capacity to accelerate recovery from EAE, 5x10⁶ Ts2 or 5x10⁵ Th2-like Treg were given ivi 9d post-immunization.  Ts2 cells activated by MBP, but not those activated by renal tubular antigen suppressed EAE between 11 and 16d post-immunization; clinical score 0.1±0.5(n=6) in treated vs 1.5±2.7(n=13) in controls(p<0.01) at 12d; 1.3±0.7 vs 2.5±0.8(p<0.001) at 13d.  Fresh naïve tTreg had no effect on EAE. Thus, Ts2 cells were more potent than naïve tTreg and tTreg activated by IL-4 and a third-party antigen.

Th2-like Treg had no effect on the onset until 14d when there was rapid recovery with no weakness from 15-21d.  Controls did not recover until 22d.

The Ts2 and Th2-like Treg were 99%CD4⁺, 98% CD25⁺ and 70-80% FOXP3⁺.  Naïve tTreg expressed no il5ra whereas Ts2 and Th2-like Treg expressed Il5ra.  Th2-like Treg had higher Foxp3, irf4 and gata3.

Ex vivo culture of naïve tTreg with IL-4 and autoantigen induces antigen specific Treg that can control established autoimmune responses.  These Ts2 cells further activated by culture with IL-5 and MBP also control autoimmunity and hence may have therapeutic potential.