Nature Immunology

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CD11c
CD205
a
CD8
CD103
dDC
CD326
LC
CD8α +
DC
CD103 +
DC
Divided gBT-I/well (×10 3 )
Divided gBT-I/well (×10 3 )
b
c
25
20
15
10
0
DC/well (×10 3 0 1.9 3.8 7.5 15
)
50
40
20
LC
dDC
CD8α + CD103
DC
+ DC
analysis of presentation in the brachial lymph nodes on day 5 showed
presentation by dermal DCs (Fig. 3e) that was not evident on day 4
(Fig. 3c). In this case, presentation by CD8a + DCs was minimal and
presentation by dermal DCs was somewhat less than in the axillary
lymph nodes, possibly because effector CTLs were already actively
killing antigen-presenting cells in this site.
CD103 + DCs present after viral recrudescence
Several groups have reported a third population of skin-derived DCs
that, like Langerhans cells, express langerin 26–28 but, unlike other skinderived
DCs, express CD103 (ref. 26). These cells are restricted mainly
to the dermis and have therefore been called ‘langerin-positive dermal
DCs’, but for simplicity, we will call them ‘CD103 + DCs’ here. These
DCs are resident in the skin and migrate to the lymph nodes after the
skin is painted with tetramethylrhodamine isothiocyanate plus irritant
26 . Both dermal DCs and Langerhans cells migrate after skin is
painted with fluorescein isothiocyanate (FITC) 9 ; here we used painting
with FITC to show that CD103 + DCs migrated in the context of
irritant or infection with HSV-1 (Supplementary Fig. 5 online). These
studies also confirmed the migratory nature of Langerhans cells and
the classical CD103 – dermal DCs and showed a lack of migration by
lymph node–resident CD8a + DCs.
Given the strong presentation by dermal DCs on day 5 reported
above (Fig. 3d), it became imperative to examine the antigenpresenting
activity of contaminating CD103 + DCs in these groups.
To achieve this, we included CD103 staining in our sorting protocol.
Because of limitations in the number of groups that could be sorted,
in subsequent studies we did not include the double-negative DCs
identified above, which represented mainly lymph node–resident
CD8a – DCs that did not seem to contribute to HSV-1-specific
5
30
10
0
Day 2, br LN
Day 5, ax LN
DC/well (×10 3 0 1.9 3.8 7.5 15
)
Figure 5 Many DC subsets present MHC class II–restricted viral antigen to
gDT-II CD4 + T cells. (a,b) Proliferation of 5 10 4 CFSE-labeled HSV-1specific
CD4 + T cells (gDT-II) after 60 h of culture together with serial
dilutions of DC subsets isolated (as described in Fig. 4a) frombrachial
lymph nodes (a) or axillary lymph nodes (b) of mice infected 2 d earlier.
(c,d) Proliferation analysis as described in a,b for DC subsets isolated
from brachial lymph nodes (c) or axillary lymph nodes (d) of mice infected
5 d earlier. Data are pooled from two to four individual experiments
(mean ± s.e.m.).
Figure 4 CD103 + DCs present HSV-1 antigens to CD8 + T cells after
secondary viral infection of the skin. (a) Gating strategy for isolation of
CD103 + DCs: after DC enrichment, CD8a + DCs were purified on the
basis of expression of CD11c and CD8a (top; right gate); expression of
CD205 and CD103 (middle) was assessed in CD11c + CD8a – cells (top; left
gate) for the isolation of CD103 + DCs (middle; right gate); and CD103 –
CD11c + CD8a – DCs (middle; left gate) were categorized as Langerhans cells
(CD326 hi ) and dermal DCs (CD326 – ) on the basis of CD326 expression
(bottom; sort gates outlined). Cells of DC subsets purified from lymph
nodes of naive and infected mice are counted in Supplementary Figure 8.
(b,c) Proliferation of 5 10 4 CFSE-labeled gBT-I cells cultured for 60 h
together with serial dilutions of DC subsets (identified as in a) sorted from
brachial lymph nodes 2 d after infection (b) or from axillary lymph nodes
5 d after infection (c). Data are pooled from two to four individual
experiments (mean ± s.e.m.).
CD8 + T cell activation. In this sorting scheme (Fig. 4a), we isolated
CD8a + DCs directly from the CD11c + cells, then isolated CD103 + cells
and separated Langerhans cells and dermal DCs on the basis of CD205
expression and differences in expression of CD326 (Ep-CAM;
expressed by Langerhans cells). We isolated CD8a + DCs first, as
they had low expression of CD103 (Supplementary Fig. 6 online)
that would otherwise potentially result in contamination of the
CD103 + population by this subset.
After sorting the cells as described above, we examined these DC
subsets for their presentation of antigen on day 2 in the brachial
lymph nodes (Fig. 4b) and on day 5 in the axillary lymph nodes
(Fig. 4c), which represent the primary and secondary sites, respectively.
This showed that CD103 + DCs were the dominant migratory
DCs that presented viral antigen on day 5 in the axillary lymph nodes,
whereas little activity by any migratory DC was evident on day 2 in the
brachial lymph nodes. In contrast, CD8a + DCs presented viral antigen
during both phases. Some presentation by dermal DCs and, to a lesser
extent, Langerhans cells was also present on day 5 in the axillary
lymph nodes, but it is difficult to exclude the possibility that this was
not due to contaminating CD103 + DCs, which showed detectable
presentation even with as few as 467 cells (data not shown). These
findings suggest that the CD103 + DCs were dominant among migratory
DCs for the ability to cross-present.
To formally address whether CD103 + DCs that had migrated from
the skin presented viral antigen, we examined presentation on day 5 in
the axillary lymph nodes after labeling the flank skin with FITC at 2 d
Divided gDT-II/well (×10 3 )
Divided gDT-II/well (×10 3 )
a
c
Day 2, br LN
25
20
15
10
5
0
0 1.9 3.8 7.5 15
Day 5, br LN
40
30
20
10
DC/well (×10 3 )
LC
dDC
CD8α + CD103
DC
+ DC
0 1.9 3.8 7.5 15
DC/well (×10 3 )
0
0 1.9 3.8 7.5 15 0 1.9 3.8 7.5 15
DC/well (×10 3 ) DC/well (×10 3 0
)
Divided gDT-II/well (×10 3 )
Divided gDT-II/well (×10 3 )
b
d
25
20
15
10
5
40
30
20
10
Day 2, ax LN
Day 5, ax LN
ARTICLES
NATURE IMMUNOLOGY VOLUME 10 NUMBER 5 MAY 2009 491