1) Naive CD4+ T cells maintain a pro-inflammatory cytokine profile in dendritic cells, increasing IL-6 mRNA. 2) Regulatory CD4+CD25+ T cells induce an anti-inflammatory dendritic cell cytokine profile, sharply increasing IL-10 mRNA but not IL-6. 3) Memory/activated CD4+ T cells have little influence on dendritic cell cytokine production and reduce IL-10 mRNA levels induced by LPS.

1. Modulation of dendritic cell
function by naive and
regulatory CD4+ T cells.
Marc Veldhoen, Halima Moncrieffe, Richard J Hocking, Christopher J
Atkins, Brigitta Stockinger
J. Immunol. 2006 vol. 176 pp. 6202-10

2. Immune Interactions
• Dendritic cells control the activation of T-
cells
• Cytokines used to determine type of
immune responses
• Pro-inflammatory e.g. IL6
• Anti-inflammatory e.g. IL10

3. CD4+ T-Cells
• Naive T cells [CD4+]
• Regulatory T cells [CD4+CD25+]
• inhibit T-cell activation, proliferation
• prevent autoimmunty
• attenuate anti-tumor immunity
• limit chronic immune pathology

4. Dendritic Cells
• Antigen presenting cells
• Many ways to detect foreign antigens
• Toll-Like Receptors for LPS/CpG
• Sensing receptors
• Expression of antigen on MHCII
• Co-stimulatory molecules e.g. CD40,
CD80/86

5. Experimental Setup
Bone Marrow BMDCs
Spleen Splenic DCs (CD11c+)
Lymph Node CD4+ T-cells
•CD25+
•CD25-CD44lo
•CD25-CD44hi
Cytokines
Isolate mRNA FACS
Luminex

6. MODULATION OF DC FU
Cytokine mRNA Expression of DCs Only
6204
FIGURE 1. Stimulation of DC results in pro- and anti-inflammatory cytokines. BMDC were gen
C57BL/6 (D–F). A and D, Staining for CD11c and MHC class II of the isolated DC populations. DC
mRNA for IL-6 (B and E) and IL-10 (C and F) was assessed at 1- to 2-h intervals. Values are plotte
point 0. The experiments shown were repeated three times with similar results.4
effects of T cells vs
Naive CD4 T cells maintain a proinflammatory DC cytokine
profile by DC. It should be
focusing on, we ar
Cell– cell interactions play an important role in immune regulation,
although the cocult
providing bidirectional stimulatory signals that are important in the
analysis. Stimulatio
activation of specific T cells and in the regulation of bystander
anti-CD3 and anti-
cells. This cross-talk between DC and T cell not only influences
detectable levels of
the surface expression of costimulatory molecules, but also regu-
Stimulation of DC results in pro- and anti-inflammatory cytokines.lates the production of generated from C57BL/6 (A–C) and splenic
BMDC were cytokines (35). period (data not sho
In the absence of
We, therefore, investigated the kinetics of cytokine expression

7. anti-CD3 and anti-CD28 in the absence of DC did not result in
C and T cell not only influences
detectable levels of IL-6 or IL-10 mRNA during the 14-h culture
ulatory molecules, but also regu-
period (data not shown).
s (35).
Cytokine mRNA Expression of DC + naive Tcells
In the absence of an inflammatory signal, coculture of BMDC
he kinetics of cytokine expression
with FACS-sorted naive CD4 T cells was found to marginally
D62LhighCD44lowCD25 ) CD4
increase levels of IL-6 mRNA (Fig. 2A), while no IL-10 mRNA
nti-CD3. Control cultures contain-
parallel to allow comparison of the could be found (Fig. 2B). T cell activation in the context of
lls
o-
ry
l-
ce
T
n-
on
nd
to
as
in
nt
ith

8. Effects of CD8+ T cells on Cytokine mRNA
Production
6205
,
.

9. mRNA levels when cocultured with BMDC in the absence or pres- the absence of an inflammatory signal increased levels of IL-6 mRNA
ence of LPS (Fig. 3). (Fig. 4C), but lacked the sharp increase of IL-10 seen with cocultures
containing CD4 CD25 T cells (cf. Fig. 4, D vs B). The increase in
CD4 CD25 T cells induce an anti-inflammatory DC cytokine IL-6 mRNA is largely T cell derived, because stimulation of memory/
profile activated T cells with anti-CD3/anti-CD28-coated beads in the ab-
sence of DC resulted in detectable IL-6 mRNA (data not shown).
Next, we asked whether CD4 CD25 T cells differentially influence
Activated/memory CD4 T cells, like naive CD4 T cells, but in con-
the expression of DC-derived pro- and anti-inflammatory cytokines.
trast with CD4 CD25 T cells, did not strongly influence IL-6
In the absence of an inflammatory signal but with anti-CD3, cocul-
mRNA induction in the presence of LPS and reduced the levels of
tures of BMDC and CD4 CD25 T cells failed to induce any IL-6
mRNA (Fig. 4A), but a sharp increase in IL-10 mRNA could be IL-10 mRNA (Fig. 4, G and H).
FIGURE 4. CD25 CD4 T cells, but not memory/activated CD4 T cells, induce an anti-inflammatory DC cytokine profile and suppress proinflam-
matory IL-6. Top panels, C57BL/6 BMDC cultured with anti-CD3 alone ( ), in the presence of naive CD4 T cells (Œ), or in the presence of CD25
CD4 T cells (f) (A and B) or memory/activated CD44 CD4 T cells (!) (C and D). Bottom panels, The same experimental set up with the addition
of LPS. mRNA for IL-6 (A, C, E, and G) and IL-10 (B, D, F, and H) was assessed at 1- to 2-h intervals. Values are plotted as fold increase over mRNA
levels in DC at time point 0. The experiments shown in A, B, E, and F were repeated three times; C, D, G, and H twice with similar results.

10. MODULATION
BMDC were cocultured with CD4 CD25 T cells and anti-CD3
without any addition of LPS, and IL-10 mRNA levels were deter-
mined during the first 10 h. Fig. 6A shows that CD4 CD25 T
s of
cells are capable of inducing IL-10 mRNA with similar efficiency
ular
in wild-type and MyD88 / BMDC.
-10
In a series of in vitro studies, it has been shown that
s of
CD4 CD25 T cells require TCR triggering to suppress T cell
dds
proliferation of naive T cells (16, 37). We, therefore, determined
ion.
cocultures with CD25 T cells require TCR stimulation CD4 CD25
BMDC were cocultured with to induce
0 of
in whether CD4

11. /
IL-10 in DC. To test this, C57BL/6 or C57BL/6 MyD88 with an inflammatory s

12. of Immunology
DC cytokine profiles during cocultures of naive and regulatory CD4 T cells. A and B, C57BL/6 BMDC cultured with an
aive CD4 T cells on their own (Œ), Tregs on their own (f), or naive and regulatory CD4 together (F). C and D, The same
e addition of LPS. mRNA for IL-6 (A and C) and IL-10 (B and D) was assessed at 1- to 2-h intervals. Values are plotted as
levels in DC at time point 0. The experiment shown was repeated three times with similar results.

13. . 7A). In con- populations had so far been determined on the level of gene ex-
CD4 T cells pression in our study. Although this method is highly sensitive, it
cannot account for potential posttranslational modification of cy-
D25 T cells
Cytokine Expression Levels
een in cultures tokine expression. We, therefore, also determined protein levels
for IL-6 and IL-10 in supernatants of DC that had been cultured in
res containing

14. Discussions