Figure 4. Viral infection promotes the differentiation of CD44^hiId3^hi to CD44^hiId3^lo Treg cells.

(a) Id3 expression in splenic Treg cells (CD4⁺CD25⁺CD45RB^lo) from naive Id3^GFP/+ mice and Id3^GFP/+ mice at the indicated times upon 200 pfu LCMV WE infection using flow cytometry (left) and quantification of the percentage of Id3^lo cells among splenic Treg cells (right). Each symbol represents an individual mouse and horizontal lines are the mean. (b) Cell number of Id3^hi (left) or Id3^lo (right) Treg cells (CD4⁺CD25⁺CD45RB^lo) in spleens of naive, d8 or d35 LCMV WE (200 pfu) infected Id3^GFP/+ mice. Each symbol represents an individual mouse and horizontal lines are the mean. ^**p < 0.01; ns = not significant (unpaired Student’s t test). (c) Id3 and CD44 expression in splenic Treg cells (CD4⁺CD25⁺) and quantification of the percentages of CD44^loId3^hi, CD44^hiId3^hi and CD44^hiId3^lo cells among splenic Treg cells of naive or d8 LCMV WE infected mice. Mean ± SEM from at least 3 individual mice are shown. (d) Id3^hi Treg cells (CD4⁺CD25⁺CD45RB^lo) from naive Id3^GFP/+ mice (Thy1.2) were transferred into wt mice (Thy1.1). One day after, mice were left untreated or infected with 200 pfu LCMV WE. Id3 expression of purified Id3^hi Treg cells immediately before transfer and of transferred cells (Thy1.2⁺) 7 days post-transfer in spleens of naive or LCMV WE infected mice (left). Quantification of the percentage of Id3^lo cells among splenic transferred cells (right). Each symbol represents the mean of 1-3 mice obtained from 4 independent experiments and horizontal lines are the mean. ^*p < 0.05 (unpaired Student’s t test). (e) CD44^loId3^hi and CD44^hiId3^hi Treg cells (CD4⁺CD25⁺CD45RB^lo) from naive Id3^GFP/+ mice (Thy1.2) were transferred into wt mice (Thy1.1). One day after, mice were infected with 200 pfu LCMV Docile. Id3 and CD44 expression of transferred cells (Thy1.2⁺) in spleens were analyzed 7 days post-transfer (left). Quantification of the percentage of Id3^lo cells among splenic transferred cells (right). For clarity the quadrant of the transferred cell population is marked in red and red arrows indicated the differentiation pathway. Each symbol represents one individual mouse obtained from 2 independent experiments and horizontal lines are the mean.