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bacteria with engaged TLR4 or both. The

report by the authors that their cells lack-

ing Rab11a have no defects in phagocy-

tosis supports their model, but it is also

counter to much previous literature on

the role of recycling endosomes in

controlling the very formation of phago-

somes (Bajno et al., 2000; Cox et al.,

2000). Future studies will need to be per-

formed to definitively implicate this novel

route of transport. Despite this potential

caveat, it is clear that Rab11a regulates

TLR4 transport in several cell types and

thus plays an important role in controlling

LPS-induced signal transduction.

As with any provocative study, several

questions arise. What is the signal that

originates from phagosomes that induce

the recruitment of recycling endosomes

to this bacterial containing compartment?

Do all cells that undergo TLR signaling

deliver TLR4 to phagosomes by the

580 Immunity 33, October 29, 2010 ª2010 El

same process? Is there any function for

the delivery of recycling endosomes to

phagosomes in terms of the control of

antigen presentation?

In summary, the study by Husebye et al.

adds to the idea that pattern recognition

receptors have been integrated in a well-

oiled cell biological machine, which

ensures rapid ignition of innate immune

signaling, and (we presume), rapid inacti-

vation of signaling when needed. Future

work will need to be done to build upon

this rapidly burgeoning field of innate

immune cell biology.

REFERENCES

Bajno, L., Peng, X.R., Schreiber, A.D., Moore, H.P.,Trimble, W.S., and Grinstein, S. (2000). J. Cell Biol.149, 697–706.

Barton, G.M., and Kagan, J.C. (2009). Nat. Rev.Immunol. 9, 535–542.

sevier Inc.

Cox, D., Lee, D.J., Dale, B.M., Calafat, J., andGreenberg, S. (2000). Proc. Natl. Acad. Sci. USA97, 680–685.

Ea, C.K., Deng, L., Xia, Z.P., Pineda, G., and Chen,Z.J. (2006). Mol. Cell 22, 245–257.

Husebye, H., Halaas, O., Stenmark, H., Tunheim,G., Sandanger, O., Bogen, B., Brech, A., Latz, E.,and Espevik, T. (2006). EMBO J. 25, 683–692.

Husebye, H., Aune, M.H., Stenvik, J., Samstad,E., Skjeldal, F., Halaas, Ø., Nilsen, N.J., Stenmark,H., Latz, E., Lien, E., et al. (2010). Immunity 33,this issue, 583–596.

Kagan, J.C., and Medzhitov, R. (2006). Cell 125,943–955.

Kagan, J.C., Su, T., Horng, T., Chow, A., Akira, S.,and Medzhitov, R. (2008). Nat. Immunol. 9,361–368.

Tseng, P.H., Matsuzawa, A., Zhang, W., Mino, T.,Vignali, D.A., and Karin, M. (2010). Nat. Immunol.11, 70–75.

Underhill, D.M., Ozinsky, A., Hajjar, A.M., Stevens,A., Wilson, C.B., Bassetti, M., and Aderem, A.(1999). Nature 401, 811–815.

A Flt3L Encounter:mTOR Signaling in Dendritic Cells

Li Wu1,2,*1Immunology Division, Walter and Eliza Hall Institute, Melbourne, Parkville Victoria 3052, Australia2Tsinghua University School of Medicine, Beijing 100084, China*Correspondence: wu@wehi.edu.auDOI 10.1016/j.immuni.2010.10.001

The signaling pathway of the cytokine Flt3L in dendritic cells (DCs) is poorly defined. In this issue of Immunity,Sathaliyawala et al. (2010) report that the kinasemTOR functions as amediator of Flt3L signaling in the devel-opment and homeostasis of DCs, particularly of the CD8+ and CD103+ DCs.

The fms-like thyrosine kinase 3 receptor

(Flt3) and its ligand (Flt3L) are key regula-

tors of dendritic cell (DC) commitment

and development. Flt3 is expressed

on the surface of early mutlipotent hea-

matopoietic progenitors, committed DC

precursors, and differentiated DC in

mouse lymphoid tissues. Flt3 expression

and its signaling has been shown to be

essential for steady-state DC develop-

ment and homeostasis as loss of Flt3 or

its ligand resulted in a substantial reduc-

tion in DC numbers in mouse lymphoid

tissues (Schmid et al., 2010). The ligand

for Flt3 can promote DC differentiation

from both mouse and human hematopoi-

etic progenitors. Administration of Flt3L

induces marked expansion in numbers

of all DC subsets including the plasmacy-

toid DC (pDC) and both CD8+ and CD8�

conventional DC (cDC) in mouse spleen,

albeit with a strong bias toward the

expansion of the CD8+ cDC subset

(Schmid et al., 2010). Flt3L-supplemented

bone marrow (BM) cultures support the

generation of pDCs and the two cDC

subsets phenotypically and functionally

equivalent to those identified in mouse

spleen (Naik et al., 2005). Despite the

importance of Flt3 and its ligand in DC

development, the signaling pathway

downstream of Flt3 in DC differentiation

and homeostasis remains poorly charac-

terized.

The mammalian target of rapamycin

(mTOR) is a serine-threonine kinase that

acts as a central regulator for protein

synthesis and cell growth. mTOR activa-

tion is induced by PI3K-Akt signaling

pathway upon receptor binding to cyto-

kine or growth factors, which leads to

phosphorylation of ribosomal protein S6

that regulates ribosomal biogenesis,

protein translation, and cell growth

(Engelman et al., 2006). The PI3K-mTOR

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Previews

signaling pathway has been shown to play

multiple roles in regulating both innate

and adaptive immunity. Within the DC

lineage, activation of mTOR can induce

the production of type-I IFN by pDCs but

suppress lipopolysacchride induced

interleukin-12 (IL-12) production by bone

marrow-derived cDCs (Weichhart and

Saemann, 2009). The activity of mTOR is

tightly regulated by several negative regu-

lators. Rapamycin, a bacterial protein, is

a specific inhibitor of mTOR signaling.

The phosphatase and tensin homolog

(Pten) is a key inhibitor of Akt signaling

that prevents Akt activation through de-

phosphorylating PI3K-generated inositol

phospholipids. Deletion of Pten results in

constitutive activation of PI3K-Akt

signaling. Although the roles of mTOR in

regulating DC functions have been

demonstrated, it is not clear whether

mTOR signaling also plays a role in DC

development. The link between Flt3 and

mTOR signaling has been suggested in

acute myeloid leukemia (AML) in which

Flt3 mutations result in ligand-indepen-

dent activation of the receptor and its

downstream signaling pathways

including the PI3K-mTOR pathway (Stire-

walt and Radich, 2003). However, the

potential role of Flt3L-induced PI3K-

AKT-mTOR signaling in normal hemato-

poietic cell development, particularly in

DC development, has not been clearly

defined.

In this issue of Immunity, Sathaliyawala

et al. (2010) examined the role of mTOR

signaling in Flt3L-induced DC develop-

ment and demonstrated that mTOR can

act as a mediator of Flt3L signaling in DC

development and homeostasis. Using

the Flt3L-supplemented BM cultures that

generate pDCs and the CD11bhiCD24lo

and CD11bloCD24hi cDCs, equivalent to

the CD8� and CD8+ cDC subsets found

in mouse spleen, respectively (Naik et al.,

2005), Sathaliyawala et al. (2010) found

that addition of rapamycin into these

cultures markedly reduced the number of

all DC subsets generated and the pDCs

and CD11bloCD24hi cDCs appeared to

be more sensitive to rapamycin treatment

than theCD11bhiCD24lo cDCs. Analysis of

CFSE-labeledBM inday3Flt3L cultures in

the presence of rapamycin revealed

a marked reduction in the numbers of

proliferating DC progenitors. In contrast,

addition of rapamycin did not affect DC

development in GM-CSF-supplemented

BM cultures, suggesting a specific role of

mTOR signaling in Flt3L-induced DC

development. Measurement of phosphor-

ylated S6 (p-S6) as a readout of mTOR

signaling after injection of Flt3L revealed

a robust p-S6 signal in splenic DCs,

confirming that Flt3L could induce

mTOR signaling in DCs in vivo. Moreover,

Flt3L-induced p-S6 signal was found to

be higher and more sustained in splenic

CD8+ than CD8� cDCs, indicating a

higher activity of mTOR in CD8+ cDCs

thatmaypartially account for thepreferen-

tial expansion of CD8+ cDC numbers by

Flt3L.

Deletion of Pten causes hyperactivation

of PI3K-Akt pathway. For further testing of

the effects of PI3K-mTOR signaling in DC

development, Pten-deleted adult BM

were subjected into Flt3L cultures. An

accelerated and marked increase in the

development of CD11bloCD24hi cDC

was observed in these cultures. Further-

more, Pten deletion partially reversed

the inhibition of DC development by rapa-

mycin. Again, GM-CSF-induced DC

development was not affected by Pten

deletion, indicating a specific role of

PI3K-mTOR signaling in Flt3L-induced

DCdevelopment. Analysis of BMchimeric

mice established with BM from mice with

either global Pten deletion or DC-specific

Pten deletion showed similar strong

biased expansion of splenic CD8+ cDC

numbers, whereas the numbers of CD8�

cDCs and pDCs were not affected. The

expansion of CD8+ cDC numbers in these

mice could be reversed to almost normal

amounts by administration of rapamycin,

confirming the requirement of mTOR

activation in the expansion of Pten-

deleted CD8+ cDC numbers. In addition,

by using the Cx3cr1-EGFP reporter

mice, Sathaliyawala et al. (2010) con-

firmed that the expansion of CD8+ cDC

numbers in Pten-deleted mice is mainly

within the Cx3cr1-EGFP�CD8+ subset

but not the recently identified Cx3cr1-

EGFP+CD8+ cDCs, the development of

latter is Flt3 independent (Bar-On et al.,

2010).

In nonlymphoid tissues, the CD103+ DC

have been shown to have similar func-

tional capacities and developmental

origin to that of CD8+ cDCs in lymphoid

tissues. The development of these tissue

CD103+ DCs is also dependent on Flt3L

signaling (del Rio et al., 2010). In line

with this, increased number of the

Immunity 33

CD103+CD11b� cDCs was also observed

in various tissues of the Pten-deleted

mice, again demonstrating a regulatory

role of Pten in PI3K-mTOR signaling in

Flt3L-induced DC development.

The maintenance of DC subset homeo-

stasis is important for a balanced

immune system and for efficient protec-

tive immune responses against certain

pathogens. Sathaliyawala et al. (2010)

showed that the imbalanced expansion

of CD8+ cDC in Pten-deleted mice is

detrimental. An impaired immunity to

the intracellular bacteria Listeria was

observed in Pten-deleted mice. This

could be partially due to the immaturity

of Flt3L expanded DCs. It is also possible

that the increased number of DCs served

as a reservoir for intracellular bacterial

replication.

The transcription factor Stat3 has been

reported as an essential factor for Flt3L-

dependent DC development (Laouar

et al., 2003). Loss of Stat3 activity

completely blocked Flt3L-induced devel-

opment of all DC subsets in lymphoid

tissues. It is not clear whether Stat3 is

involved in PI3k-mTOR-mediated Flt3L

signaling as described by Sathaliyawala

et al. (2010). It has been suggested that

Stat3 activity can be regulated by mTOR

pathway in immune cells. Further investi-

gation is required to define the molecular

events downstream of mTOR signaling.

The fact that Flt3L administration in vivo

induced marked expansion of all DC

subsets, albeit with greater extent in

CD8+ cDCs in normal mice, whereas

Pten deletion only caused expansion of

CD8+ cDCs in vivo, suggests that this

PIK3-mTOR pathway is mainly operating

in CD8+ cDCs or their precursors in vivo

and may represent an additional pathway

downstream of Flt3L signaling, which

parallels or synergizes with Flt3L-Stat3

pathway in the development or expansion

of CD8+ cDC numbers (Figure 1).

The importance of mTOR in regulating

human DC differentiation and functions

has also been recognized. A recent study

demonstrated a divergent role of mTOR

during activation and differentiation of

human myeloid DCs and monocyte-

derived DCs (Haidinger et al., 2010).

The human equivalent of mouse CD8+

cDC has also been identified by their

expression of BDCA-3 (CD141), CLEC9A

(a C-type lectin-like receptor), and XCR1

(a chemokine receptor) and their capacity

, October 29, 2010 ª2010 Elsevier Inc. 581

Figure 1. mTOR Mediates Flt3L Signaling in Dendritic Cell Development and HomeostasisBinding of Flt3L to its receptor expressed on the surface of DC precursors or DC induces activation of itsdownstream PI3K-Akt-mTOR signaling pathway that leads to DC differentiation and expansion. The PI3K-Akt-mTOR pathway can be suppressed by Pten, a key inhibitor of Akt signaling, and by rapamycin, thespecific inhibitor of mTOR, which leads to restrained DC development and expansion. Flt3L signalingmay directly activate Stat3 pathway and lead to DC development.

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to produce IL-12 and to cross-present

exogenous antigen. They can also be

generated in culture in the presence of

582 Immunity 33, October 29, 2010 ª2010 El

Flt3L. It is important to determine whether

the PI3K-mTOR pathway also functions in

a similar way during the development and

sevier Inc.

homeostasis of human BDCA-3+ cDCs.

These studies should provide important

information for the potential effects of ra-

pamycin treatment on the development

and functions of DCs in human patients

that is crucial for developing novel strate-

gies for improved therapeutic applica-

tions of rapamycin in the treatment of

various human diseases.

REFERENCES

Bar-On, L., Birnberg, T., Lewis, K.L., Edelson, B.T.,Bruder, D., Hildner, K., Buer, J., Murphy, K.M.,Reizis, B., and Jung, S. (2010). Proc. Natl. Acad.Sci. USA 107, 14745–14750.

del Rio, M.L., Bernhardt, G., Rodriguez-Barbosa,J.I., and Forster, R. (2010). Immunol. Rev 234,268–281.

Engelman, J.A., Luo, J., and Cantley, L.C. (2006).Nat. Rev. Genet. 7, 606–619.

Haidinger, M., Poglitsch, M., Geyeregger, R., Kas-turi, S., Zeyda, M., Zlabinger, G.J., Pulendran, B.,Horl, W.H., Saemann, M.D., and Weichhart, T.(2010). J. Immunol. 185, 3919–3931.

Laouar, Y., Welte, T., Fu, X.Y., and Flavell, R.A.(2003). Immunity 19, 903–912.

Naik, S.H., Proietto, A.I., Wilson, N.S., Dakic, A.,Schnorrer, P., Fuchsberger, M., Lahoud, M.H.,O’Keeffe, M., Shao, Q.X., Chen, W.F., et al.(2005). J. Immunol. 174, 6592–6597.

Sathaliyawala, T., O’Gorman, W.E., Greter, M.,Bogunovic, M., Konjufca, V., Hou, Z.E., Nolan,G.P., Miller, M.J., Merad, M., and Reizis, B.(2010). Immunity 33, this issue, 597–606.

Schmid, M.A., Kingston, D., Boddupalli, S., andManz, M.G. (2010). Immunol. Rev. 234, 32–44.

Stirewalt, D.L., and Radich, J.P. (2003). Nat. Rev.Cancer 3, 650–665.

Weichhart, T., and Saemann, M.D. (2009). TrendsImmunol. 30, 218–226.