download report - Istituto Pasteur

P a r t i c i p a n t s :
Marzia Soligo, Cristiano Scottà, post-doc fellows; Cristina
Camperio, PhD student.
C o l l a b o r a t o r s :
Dipartimento di Dermatologia, Università di Roma Tor Vergata,
(Prof. Antonio Costanzo).
Report of activity
Both in mice and in humans CD4 + CD25 + T cells
with regulatory functions are important components
of peripheral tolerance that is responsible in the control
of autoreactive T cells in normal individuals.
FOXP3 transcription factor is a critical regulator of
these cells. Among the signals necessary to generate
CD4 + CD25 + FOXP3 + T cells, a pivotal role is
played by CD28. However, while in mice it was possible
to study the role played by the interaction of
CD28 with its ligand B7 in the activation and maintenance
of CD4 + CD25 + FOXP3 + T cells, in
humans similar results are lacking. Therefore we
verified whether CD28 signaling independently of
TCR promoted FOXP3 expression and regulates
CD4 + CD25 + FOXP3 + T cell functions in humans.
Specific tasks of the project are: i) the analysis of the
effects of the unique signal delivered by CD28 in
CD4 + CD25 - T cells on FOXP3 gene activation and
ii) the influence of FOXP3 on the phenotypic
and functional properties of CD28-activated
CD4 + CD25 - T cells.
The analysis of the effects of CD28 signal provided
for the first time the experimental evidence that
CD28 signals independent from TCR and dependent
on PI3K/Akt pathways are sufficient to induce the
transcriptional activation of FOXP3, in primary
CD4 + CD25 - T cells. We reached this conclusion by
activating CD28 either by the natural ligand B7 or by
cross-linking with specific mAb. However, despite
FOXP3 was rapidly induced, less than 30% of the
Principal investigator: Enza Piccolella
Professor of Immunology
Dipartimento di Biologia Cellulare e dello Sviluppo
Tel: (+39) 06 49917584; Fax: (+39) 06 49917584
enza.piccolella@uniroma1.it
99
Cellular and molecular immunology - AREA 5
Analysis of the molecular mechanisms regulating FOXP3 gene and
protein expression in TCR- and CD28-activated CD4 + CD25 - T cells
and their influence on regulatory functions
CD4 + CD25 - T cells expressed FOXP3 after CD28mediated
activation, supporting the view that among
CD4 + CD25 - T cells only a small number of cells are
pre-committed to express FOXP3. Starting from
these results, we went on to study: i) the translocation
of CD28-induced FOXP3 in the nucleus, ii) its binding
to FOXP3 consensus regions on target genes, and
iii) cell proliferation and regulatory functions of
CD4 + CD25 - T cells become CD4 + CD25 + FOXP3 + .
To verify whether CD28 signal could induce posttranslational
modification of FOXP3, we prepared
cytoplasmic and nuclear extracts from CD28-stimulated
CD4 + C25 - T cells, and FOXP3 was revealed by
immunoblotting. Figure 1 shows that the maximum
expression of FOXP3 occurred in the cytoplasm
after 24h from CD28-mediated activation, but only
after 48h in the nucleus.
The recruitment of FOXP3 on target genes has been
analyzed by chromatin immunoprecipitation (ChIP)
experiments. Indeed it has been demonstrated that
FOXP3 could occupy CD25, Il-2 and Ctla4 promoters
in both murine CD4 + CD25 + T regulatory cells
and Jurkat T cells retrovirally transduced with
FOXP3, and stimulated by TCR plus CD28 and/or
ionomycin. To verify this phenomenon also in primary
CD4 + CD25 - T cells, we have activated the
cells for 24 and 48 h by Dap3/B7. The results
reported in Figure 2 show for the first time, at the
best of our knowledge, that the activatory pathways
mediated by CD28 in CD4 + CD25 - T cells favour not
only the transcription and translation of FOXP3 but
also its occupancy of CD25, Il-2 and Ctla4 promoters.
Interestingly, after 24 h FOXP3 was recruited on
all studied promoters, but the occupancy of CD25
and Il-2 promoters (Panels A, C) was maintained for
all the observation time, while FOXP3 left Ctla4 promoter
between the 24 and 48 h from the activation
(Panel B). To analyze the sensitivity of FOXP3
recruitment on Il-2 promoter to CsA, we stimulated
CD4 + CD25 - T cells with anti-CD28 mAbs for 24 h