The PI3K p110δ regulates expression of CD38 on regulatory T cells

Daniel T Patton, Marcus D Wilson, Wendy C Rowan, Dalya R Soond, Klaus Okkenhaug, Daniel T Patton, Marcus D Wilson, Wendy C Rowan, Dalya R Soond, Klaus Okkenhaug

Abstract

The PI3K pathway has emerged as a key regulator of regulatory T cell (Treg) development and homeostasis and is required for full Treg-mediated suppression. To identify new genes involved in PI3K-dependent suppression, we compared the transcriptome of WT and p110δ(D910A) Tregs. Among the genes that were differentially expressed was the gene for the transmembrane cyclic ADP ribose hydrolase CD38. Here we show that CD38 is expressed mainly by a subset of Foxp3(+)CD25(+)CD4(+) T cells originating in the thymus and on Tregs in the spleen. CD38(high) WT Tregs showed superior suppressive activity to CD38(low) Tregs, which failed to upregulate CD73, a surface protein which is important for suppression. However, Tregs from heterozygous CD38(+/-) mice were unimpaired despite lower levels of CD38 expression. Therefore, CD38 can be used as a marker for Tregs with high suppressive activity and the impaired Treg function in p110δ(D910A) mice can in part be explained by the failure of CD38(high) cells to develop.

Conflict of interest statement

Competing Interests: Klaus Okkenhaug is a paid consultant for GSK. Klaus Okkenhaug is employed by the BBSRC and has received research funding from the BBSRC, the Wellcome Trust and GSK. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1. Genome-wide expression-profiling of WT and…
Figure 1. Genome-wide expression-profiling of WT and p110δD910A CD4+CD25+cells.
CD4+CD25+ cells were isolated from WT and p110δD910A mice and RNA expression analysed by gene arrays. A. Shown is data from all 45,002 probe sets plotted as expression in WT Treg against expression in p110δD910A Treg (n = 3 for each genotype). Black dots represent genes that were significantly differentially expressed between WT and p110δD910A (greater than 2-fold difference and p<0.01). B. Differentially expressed genes from A were then compared against a previously published set of Treg signature genes . The numbers of probe sets belonging in each section of the Venn diagram are shown. C. Comparison of expression data from selected probe sets found to be significantly different in A with qRT-PCR performed on RNA from separate preparations of WT and p110δD910A Treg.
Figure 2. Expression of CD38 by WT…
Figure 2. Expression of CD38 by WT and p110δD910A CD4+Foxp3+ Treg cells.
A. Expression of CD38 on DN, DP and CD4 and CD8 single-positive cells from the thymus of WT and p110δD910A mice. Mean percentages of the cells within the CD38+ gate are for WT (bold typeface) and p110δD910A mice (italic). B. Summary of statistics shown in A (n = 3 for each genotype). C. Expression of CD38 on Foxp3+ and Foxp3− cells from the spleen of WT and p110δD910A mice. D. Representative FACS plots of CD38 on Foxp3+CD4+ T cells from the spleens of WT∶B6.SJL or p110δD910A∶B6.SJL bone marrow chimeras. E. Mean expression of CD38 on Foxp3+ and Foxp3− cells from WT∶B6.SJL or p110δD910A∶B6.SJL bone marrow chimeras, n = 4 for WT∶B6.SJL and n = 3 for p110δD910A∶B6.SJL.
Figure 3. In vitro suppression of T…
Figure 3. In vitro suppression of T cell proliferation by CD38high and CD38low Treg.
A. Proportion of undivided CD4+CD25− after three days culture with CD38high or CD38low CD4+CD25+ Tregs. B. Expression of CD73 by CD38high and CD38low Tregs after suppression. C. Expression of CD73 by CD4+CD25− cells after suppression by either CD38high or CD38low Treg. D. Expression of CD38 versus CD73, CTLA-4 and Granzyme B on CD4+CD25+Foxp3+ cells from WT and p110δD910A Treg cells.
Figure 4. Intact development and function of…
Figure 4. Intact development and function of Treg development in CD38+/− mice.
Percentage of Foxp3+ cells and mean linear fluorescence (MLF) of anti-CD38 antibody staining on CD4+Foxp3+ in the thymus (A) and spleen (B) of WT, CD38+/−, CD38−/− and p110δD910A mice. (C) Comparison of suppression of CD4+CD25− proliferation by WT, p110δD910A or CD38+/− Tregs.
Figure 5. Induction of CD38 on induced…
Figure 5. Induction of CD38 on induced Treg cells is mildly enhanced by IC87114 and ATRA.
TGF-β-dependent conversion of CD4+CD25− cells in presence of different concentrations of ATRA and the PI3K inhibitors IC87114 or PI103. (A) Percentage of Foxp3+ cells after 3 days of stimulation with anti-CD3, TGF-β and indicated drugs. (B) Mean expression of CD38 on Foxp3+ cells induced in A. (C) Mean expression of CD73 on Foxp3+ cells induced in A.

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