Biol. Cell (2010) 102, 581–591 (Printed in Great Britain) doi:10.

1042/BC20100046
Research article

Spatial organization of the

transforming MHC class II
compartment
Hezder E. van Nispen tot Pannerden*†, Willie J. Geerts*‡, Monique J. Kleijmeer*† and Harry F.G. Heijnen*§1
*Institute of Biomembranes, Padualaan 8, 3584 CH Utrecht, The Netherlands, †Cell Microscopy Center, Department of Cell Biology,
University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands, ‡Department of Molecular Cell Biology, Utrecht
University, Utrecht, The Netherlands, and §Laboratory of Clinical Chemistry and Haematology, University Medical Center Utrecht,
Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
www.biolcell.org

Background information. DC (dendritic cells) continuously capture pathogens and process them into small pep-
tides within the endolysosomal compartment, the MIIC (MHC class II-containing compartment). In MIICs peptides
are loaded on to MHC class II and rapidly redistributed to the cell surface. This redistribution is accompanied
by profound changes of the MIICs into tubular structures. An emerging concept is that MIIC tubulation provides
a means to transport MHC class II–peptide complexes to the cell surface, either directly or through vesicular
intermediates. To obtain spatial information on the reorganization of the MIICs during DC maturation, we per-
formed electron tomography on cryo-immobilized and freeze-substituted mouse DCs after stimulation with LPS
(lipopolysaccharide).
Results. In non-stimulated DCs, MIICs are mostly spherical. After 3 h of LPS stimulation, individual MIICs transform
into tubular structures. Three-dimensional reconstruction showed that the MIICs frequently display fusion profiles
and after 6 h of LPS stimulation, MIICs become more interconnected, thereby creating large MIIC reticula. Micro-
tubules and microfilaments align these MIICs and reveal physical connections. In our tomograms we also identified a
separate population of MIIC-like intermediates, particularly at extended ends of MIIC tubules and in close proximity
to the trans-Golgi network. No fusion events were captured between reticular MIICs and the plasma membrane.
Conclusions. Our results indicate that MIICs have the capacity to fuse together, whereby the cytoskeleton possibly
provides a scaffold for the MIIC shape change and directionality. MIIC-like intermediates may represent MHC class
Biology of the Cell

II carriers.

Introduction oid organs and interact with antigen-specific CD4+

Immature DCs (dendritic cells) principally reside in
peripheral tissues and continuously survey for patho-
gens (e.g. Sallusto et al., 1995). On activation by
pathogen-derived antigens, DCs migrate to lymph-
1 To whom correspondence should be addressed (email
h.f.g.heijnen@umcutrecht.nl).
Key words: dendritic cell, electron microscopy, MHC class II-containing
compartment (MIIC), spatial organization, three-dimensional analysis,
tomography.
Abbreviations used: APC, antigen-presenting cell; DC, dendritic cell; ET,
electron tomography; HPF-FS, high-pressure freezing and freeze substitution;
IEM, immunoelectron microscopy; IF, immunofluorescence; LPS,
lipopolysaccharide; MIIC, MHC class II-containing compartment; MT,
microtubule; TGN, trans -Golgi network.
T-cells to induce an immune response. It has been well
established that the major sites of antigen processing
and peptide loading in APCs (antigen-presenting
cells) are the late-endosomal and lysosomal com-
partments, collectively termed MIICs (MHC class
II-containing compartments) (Neefjes et al., 1990;
Watts, 1997; Mellman et al., 1998; Trombetta and
Mellman, 2005). MIICs have been extensively charac-
terized in several APCs, including B-cells (Kleijmeer
et al., 1997) and DCs (Kleijmeer et al., 2001).
They appear as spherical multivesicular bodies, but
may also have a multilaminar appearance. Previous
three-dimensional analyses showed that the luminal

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membrane sheets and vesicles are not connected to the
limiting membrane of the MIIC (Murk et al., 2003b;
Murk et al., 2004). Most of the intracellular pool of
MHC class II molecules in immature DCs is localized
to the MIIC internal vesicles (Kleijmeer et al., 2001).
On DC activation, peptide-loaded MHC class II mo-
lecules are redistributed to the cell surface, together
with a set of co-stimulatory molecules, thereby en-
abling their interaction with CD4+ T-cells (Hart,
1997; Kleijmeer et al., 2001).
The exact pathway by which MHC class II–peptide
complexes are transported to the cell surface is
still not completely understood. Several independent
studies have shown that during activation and on in-
teraction of DCs with CD4+ T-cells, MIICs undergo
major structural changes and transform into tubular
compartments (Kleijmeer et al., 2001; Boes et al.,
2002; Chow et al., 2002). These tubules are believed
to be formed in an MT (microtubule)-dependent way
(Boes et al., 2002) and contain high amounts of MHC
class II on their limiting membranes. Back-fusion
of the MIIC internal membranes with the limit-
ing membrane was suggested to facilitate peptide
loading and transport of the MHC class II–peptide
complex towards the plasma membrane (Kleijmeer
et al., 2001). Back-fusion may promote the associ-
ation of MHC class II with the chaperone protein
HLA-DM, which resides predominantly on the lim-
iting membrane of MIICs (Kleijmeer et al., 2001).
This would facilitate the release of the CLIP (MHC
class II-associated invariant-chain peptide) from the
peptide-binding cleft of MHC class II (Kropshofer
et al., 1999; Alfonso and Karlsson, 2000), thus en-
abling peptide loading. Back-fusion also generates
the membrane supply necessary for the MIIC tubula-
tion and outgrowth towards the plasma membrane.
The final step in MHC class II transport would then
be fusion of the MIIC tubules with the plasma mem-
brane and/or the budding and subsequent fusion of
transport vesicles with the plasma membrane. Part
of the MIIC luminal membranes are also secreted as
exosomes when MIICs fuse with the plasma mem-
brane, a phenomenon that has been documented as a
constitutive process in B-cells (Raposo et al., 1996)
and DCs (Buschow et al., 2005).
We present here the three-dimensional reconstruc-
tion of transformed MIICs in maturating DCs. On
stimulation with LPS (lipopolysaccharide), MIICs be-
come tubular and frequently reveal fusion profiles,
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C 2010 Portland Press Limited
H.E. van Nispen tot Pannerden and others
indicating that MIICs fuse together. Increased ex-
posure to LPS causes the MIIC profiles to become
more interconnected, thereby creating large MIIC
networks. The reticular MIICs are surrounded by a
complex network of MTs and microfilaments with
which physical connections exist, thereby providing a
scaffold for their structural alteration. We also found
isolated MIIC intermediates, distinct from the re-
ticulum, which may represent MHC class II carrier
vesicles en route to the cell surface.
Results
To obtain a detailed three-dimensional view on the
membrane reorganization of transforming MIICs,
cultured D1 cells were left untreated or stimu-
lated with LPS for 3 and 6 h respectively and
subjected to HPF-FS (high-pressure freezing and
freeze substitution), followed by plastic embed-
ding and ET (electron tomography) analysis. Stim-
ulated cells were carefully selected on 200–300 nm
thick serial sections on the basis of their character-
istic morphology (i.e. dendritic profile). The stim-
ulation response of the D1 cells was verified by
determining the cell surface expression of MHC
class II molecules using confocal microscopy and IEM
(immunoelectron microscopy) analyses (see Supple-
mentary Figure S1 and S2 at http://www.biolcell.
org/boc/102/boc1020581add.htm). ET does not al-
low identification of MIICs by immunolabelling.
Our knowledge of the recognition of MIICs in APCs
comes from a previous detailed IEM analysis, carried
out in our laboratory. From these studies we know
that the MHC class II compartments belong to the
regular endocytic pathway and include late endo-
somes and lysosomes (Geuze, 1998). It is based on
this knowledge that we have selected the structures
as MIICs in our three-dimensional reconstructions.
As a rule we simultaneously carried out IEM
experiments and compared immunolabelled MIICs
in thin frozen sections with those in our tomo-
grams. Two distinct markers of early stages (invari-
ant chain) and intermediate and late MIICs (HLA-
DM) were used (Geuze, 1998). DCs were stimu-
lated for 6 h with LPS, the same conditions as those
used for the three-dimensional analysis. Labelling
of HLA-DM was absent from early compartments
(low vesicle number) and abundant in multivesicu-
lar (intermediate and late) MIICs (see Supplementary
Three-dimensional organization of the transforming MIIC
Figures S3A and S3B at http://www.biolcell.org/
boc/102/boc1020581add.htm). Vice versa, invariant
chain was abundant in the early compartments and
absent from multivesicular MIICs (Supplementary
Figures S3C and S3D). Based on the ultrastructural
criteria and the immunogold labelling, we conclude
that the multivesicular and multilaminar structures
in our tomograms represent intermediate and late
stages of MIICs. To maximally capture membrane
continuities, three-dimensional reconstructions were
carried out in large-volume tomograms, covering
areas where MIICs are positioned both at TGN (trans-
Golgi network) exit sites and in close proximity to
the plasma membrane. Figures 1(A) and 1(B) repres-
ent an example of such a tomogram after 6 h of LPS
stimulation, covering a total volume of 23.6 μm3 .
The MIICs in our tomograms appear as spher-
ical and elongated tubular organelles (Figure 1B,
coloured asterisks, highlighted in Figures 1C–1E).
Tubular MIICs were more regularly shaped in the
HPF-FS samples (Figures 1C and 1F) as com-
pared with those observed in ultrathin cryosec-
tions from chemically fixed cells (Figure 4D in
Kleijmeer et al., 2001). Fusion/fission profiles of
the internal membranes with the limiting mem-
brane of the tubular MIICs were occasionally ob-
served (results not shown). Tubular MIICs are dis-
tinct from the tubular elements emerging from
early endosomes, which are also encountered in LPS-
stimulated cells, albeit at low frequency (see Sup-
plementary Figure S4 at http://www.biolcell.org/
boc/102/boc1020581add.htm). These early endo-
somes exhibit a characteristic spherical morphology
with low number of luminal vesicles and abundant
invariant chain (see also Supplementary Figure S3).
The tubular extensions are significantly smaller than
the MIIC tubules detected after LPS stimulation (av-
erage diameter 50+ −11 nm, n = 11 nm versus 153 + −
57 nm, n = 6, P = 0.015). The tubular MIICs cap-
tured in our tomograms could reach lengths of at
least 3 μm.
MIICs become organized into a reticular network
When membranes of MIICs were traced manually, it
became apparent that the individual MIIC profiles are
often interconnected and form reticular membrane
networks (Figures 1D, 1E, 1G and 1H; Figures 2A
and 2B; see Supplementary Movie 1 at http://www.
biolcell.org/boc/102/boc1020581add.htm). Most of
Research article
the networks analysed exhibit internal membranes
that have the morphological characteristics of late
MIICs. Within these networks we detected in-
tact, but also partially degraded vesicles, tubular
membrane structures and isolated membrane sheets
(Figures 2C and 2D; see Supplementary Movie 2 at
http://www.biolcell.org/boc/102/boc1020581add.
htm). The last two features are reminiscent of the
morphology of internal membranes in multilam-
inar lysosomes. This suggests that MIIC networks
fuse with multilaminar lysosomes. Representative ex-
amples of fusion profiles and neck regions are shown
in Figure 3. In Figures 3(A)–3(C), two interconnect-
ing MIICs are shown after 3 h of LPS stimulation (see
also Supplementary Movie 3 at http://www.biolcell.
org/boc/102/boc1020581add.htm). The compart-
ment comprises two distinct MIIC profiles, con-
nected through a ∼ 25 nm wide fusion pore,
and is completely captured within the tomo-
gram. Interconnecting MIICs are generally larger
after 6 h LPS (Figures 3D–3H; see Supplement-
ary Movie 4 at http://www.biolcell.org/boc/102/
boc1020581add.htm), and reveal a multi-domain
structure and multiple neck regions (inset to
Figure 3H; see Supplementary Movie 5 at http://
www.biolcell.org/boc/102/boc1020581add.htm).
Semi-quantitative analysis of the MIIC profiles
within our tomograms revealed that after 3 and
6 h of LPS stimulation respectively, 12% (n = 23)
and 22% (n = 23) of the MIIC profiles are con-
nected to a network (Figure 3I). MIICs in non-
stimulated cells are often completely captured within
our tomograms, and no fusion profiles or inter-
connections between them were observed (see Sup-
plementary Figure S5 at http://www.biolcell.org/
boc/102/boc1020581add.htm). Their average vol-
ume was 1.6×107 nm3 (n = 18). In contrast, the
MIIC networks in LPS-stimulated cells were much
larger (>1.4×108 nm3 , n = 26) and were never cap-
tured completely in our tomograms. We therefore
conclude that MIICs must fuse together on LPS stim-
ulation.
Fusion of MIICs is crucial for MHC class II
transport to the cell surface
Bafilomycin is known to inhibit lysosomal acidifica-
tion and thereby the fusion of late endosomes with
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 H.E. van Nispen tot Pannerden and others

Figure 1 Tubular and reticular MIICs in a stimulated D1 cell

(A) Overview of an HPF-FS, 6 h LPS-stimulated D1 cell. Boxes indicate the area where tomograms have been taken. PM, plasma
membrane; G, Golgi area. (B) Slice of the montaged tomogram. The asterisks indicate MIIC profiles, all containing internal
membranes. The MIICs marked with bright green asterisks represent tubular profiles; all other coloured asterisks belong to MIIC
networks. Bright green, pink and yellow indicate the MIICs that are shown in larger magnification in (C–E). (C, F) Tomographic
slice and reconstructed model of two tubular-shaped MIICs. (D, E) Interconnected MIIC profiles, corresponding to the models
shown in (G, H). (G, H) Reconstructed models of the two reticular MIICs shown in (D, E). Scale bars: (A, B) 1 μm and (C–E)
500 nm.

lysosomes in other cell types (Yamamoto et al., 1998). of bafilomycin A1 . As expected, LPS alone induces
To check the importance of MIIC fusion for trans- transport of MHC class II from intracellular stores
port of MHC class II to the cell surface, we incub- to the cell surface (Supplementary Figure S1 and
ated D1 cells with LPS in the presence or absence S2). On treatment with bafilomycin A1 , most MHC

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Three-dimensional organization of the transforming MIIC Research article
Figure 2 Examples of MIICs containing various shapes of internal membranes
Cells were stimulated for 6 h with LPS. (A) A reticular MIIC with traced membranes. Next to the network an intermediate MIIC
can be seen (iMIIC). (B) Reconstructed model of the reticular MIIC in (A). (C) Intact luminal membranes and membrane sheets
(arrowheads) are detected. The later ones are suggestive of membrane degradation activity. (D) Tomographic slices through an
MIIC displaying tubular-shaped luminal membranes. The white arrowheads point to a tubular membrane in cross section. Scale
bars: (A, B) 250 nm and (C, D) 100 nm.

class II remained intracellular (see Supplementary
Figures S6A and S6B at http://www.biolcell.
org/boc/102/boc1020581add.htm), associated with
accumulated luminal membranes in MIICs (Supple-
mentary Figure S6C). These results suggest that the
luminal pH of MIICs and fusion of MIICs are im-
portant determinants for the translocation of MHC
class II molecules to the cell surface.
Spatial organization of MIIC with cytoskeleton
elements
Recent studies in DCs using IF (immunofluores-
cence) have established that the profound changes
from spherical MIIC into long tubular structures
highly depend on intact MTs. A close association
of MIICs with the cytoskeleton was confirmed
after three-dimensional reconstruction analysis. MTs
(blue arrowheads in Figure 4A) and ∼ 7 nm wide
microfilamentous structures (pink double arrow-
heads in Figures 4A and 4B) appear in close
proximity to the MIIC networks, thereby form-
ing a scaffold of cytoskeleton elements (see the
model in Figure 4C and Supplementary Movie 6
at http://www.biolcell.org/boc/102/boc1020581add.
htm). Short and long MTs appeared arranged par-
allel to the tubular MIIC profiles. Also in IF
(results not shown) and in immunogold-labelled
cryosections of LPS-stimulated DCs, we found close
association of α-tubulin with the MIICs (Figure 4D).
The MTs showed closed capped ends (Figure 4E)
as well as frayed open ends (Figure 4F) and some
revealed a kinked appearance (Figure 3G, arrows).
Physical linkages were detected between the MIIC
limiting membrane and microfilamentous structures

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 H.E. van Nispen tot Pannerden and others

Figure 3 Fusion and neck profiles within the MIIC network

Cells were stimulated for 3 h (A–C) and 6 h (D–H) with LPS. (A, D, G) Tomographic slices through fusing MIICs. Arrowheads mark
small membrane continuities between adjacent MIICs. The arrows in (G) point out to a kinked MT. (B, E) Series of tomographic
slices showing the small connecting pore depicted in (A, D). (C, F) Reconstructed models and the view through the fusion pore
(arrow, view inset from the right to left). (H) Detail of a neck region in the MIIC network (double arrowhead), and complete model
of the MIIC network (inset). Note the multidomain structure of the MIIC with multiple interconnections (see also Supplementary
Movie 5 at http://www.biolcell.org/boc/102/boc1020581add.htm). (I) Relative number of MIIC profiles that represent individual
MIICs or that are connected to a network, control, n = 18; 3 h LPS, n = 23; 6 h LPS, n = 73. Scale bars, 100 nm.

(Figure 4B, black arrowheads, average distance of
filament to MIIC was 16+
−5 nm, n = 20), as well as
the MTs (Figures 4G and 4H, black arrowheads).
Small MIIC intermediates
In our tomograms, we also encountered a large
number of isolated MIIC profiles, which appeared
not connected to the tubular and reticular MIIC
networks (‘iMIIC’ in Figure 2A; Figure 5A–5D;
see Supplementary Movie 7 at http://www.biolcell.
org/boc/102/boc1020581add.htm). These MIIC pro-
files had diameters ranging from 50 to 200 nm (av-
erage volume 2.1×106 nm3 , n = 37) and were fre-
quently found positioned in the close vicinity of the
extended poles of the reticular MIICs (Figure 5E) and
appeared also in the Golgi area. They often contained
internal membranes and partially degraded material
such as short isolated membrane sheets (Figures 5A–
5D). The small MIIC structures were also detected in
thin frozen sections of chemically fixed D1 cells, as

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Three-dimensional organization of the transforming MIIC Research article
Figure 4 Physical linkages connect transforming MIICs with cytoskeleton elements
Cells were stimulated for 6 h with LPS. (A) A tubular part of the MIIC (yellow asterisks) is flanked by an MT (blue arrowheads)
and microfilaments (pink double arrowheads). (B) Highlighted subdomain of the same MIIC showing distinct connections (black
arrowheads) between microfilaments and the MIICs limiting membrane. (C) Reconstructed side view of the MIIC reticulum
(yellow asterisks in A) with associated MTs and microfilaments. (D) Chemically fixed cryosection of a stimulated D1 cell (6 h
LPS). Immunogold labelling of α-tubulin associated with an MIIC. (E) MT with a capped end (arrow). (F) MT with a frayed end
(arrows). (G, H) Tomographic slices through longitudinal (G) and cross-sectioned (H) orientated MT displaying physical linkages
to the limiting membranes of MIICs (black arrowheads). Scale bars: (A) 250 nm, (B, E–H) 100 nm and (D) 200 nm.

verified by immunogold labelling using anti-MHC
class II antibodies (Figure 5F). However, due to the
limited Z resolution of thin frozen sections, we can-
not exclude that at least a part of them represent
cross-sectioned MIIC tubules. The positioning of a
population of small MIIC profiles within the Golgi
area could point towards early stages in the forma-
tion of MIICs. Although MIIC intermediates were
sometimes closely positioned to trans-Golgi stacks,
we have not found direct membrane continuities with
the TGN.
Discussion
In the present study we have generated large-scale
three-dimensional cell volumes from HPF-FS LPS-
stimulated D1 cells to define the spatial membrane
architecture of transformed MIICs. The HPF-FS tech-
nology preserves the cellular fine structure in a close-
to-live condition, and has previously been used to
study the three-dimensional architecture of the endo-
lysosomal system in DCs. In immature D1 cells, early
endosomes and MIICs are globular shaped, some-
times with small tubular extensions (Murk et al.,
2004). Based on the morphological criteria (i.e. mul-
tivesicular and multilaminar morphology), and the
characteristic immunolabelling patterns of invariant
chain, HLA-DM and MHC class II, we here show
that the multivesicular and multilaminar structures
presented in our tomograms are intermediate/late
MIICs. Our three-dimensional analysis revealed that
after LPS stimulation, MIICs form large intercon-
necting membrane networks with alternating spher-
ical and tubular domains. This typical spatial mor-
phology of the MIIC, the large increase in their size
and the presence of small membrane openings con-
necting them indicate that MIICs have the capacity
to fuse with each other during DC maturation. This
fusion of individual MIICs contributes to the forma-
tion of large extended and reticular MIICs. Whether

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Figure 5 Small MIIC profiles not connected to a network
Cells were stimulated for 6 h with LPS. (A–D) Gallery of small
isolated MIICs. The diameter of these MIICs varies from 50
to 200 nm (average volume 2.1×106 nm3 , n = 37). Note that
some of the internal membrane structures are incompletely
closed vesicles. (E) Series of tomographic slices showing an
isolated MIIC profile in close apposition to an MIIC reticulum.
(F) MHC class II immunogold-labelled cryosection of a small
MIIC intermediate. G, Golgi. Scale bars (A–D) 100 nm and
(E–F) 200 nm.
the reticular MIICs are further interconnected and
form a large reticulum throughout the cell requires
whole cell reconstruction analysis, which was not es-
tablished in the present study.
Previous studies have suggested that back-fusion
of the luminal membranes with the limiting mem-
brane of the MIICs is responsible for MIIC tubu-
lation and timed translocation of MHC class II to
the cell surface (Kleijmeer et al., 2001). Continuit-
ies between luminal vesicles and the limiting mem-
brane of the MIIC tubular reticulum were occasion-
ally observed in tomograms of LPS-stimulated D1
cells. However, we could not establish if these profiles
represent inward budding of newly formed vesicles
or back-fusion of pre-existing vesicles as suggested
previously (Kleijmeer et al., 2001).
Using GFP (green fluorescent protein)-tagged
MHC class II, fusion of class II-containing vesicles
with the cell surface has been reported in cultured
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H.E. van Nispen tot Pannerden and others
cells (Wubbolts et al., 1996), B-cells and mouse DCs
(Boes et al., 2002; Chow et al., 2002). It was pro-
posed that tubular MIICs extending towards the cell
periphery could facilitate the transfer of MHC class II
to the plasma membrane, either directly or through
vesicular intermediates budding from the tubules.
An important reason to use HPF-FS and ET analyses
is the ability to capture the rapid membrane dynam-
ics that occur during MIIC transformation. When
analysing large-scale cell volumes in the cell peri-
phery, we found no membrane connections of reticu-
lar MIICs with the cell surface. In a previous study,
we reported on MHC II-containing structures prox-
imate to the tips of MIIC tubules. These structures
exhibited a diameter similar to that of the tubules,
suggesting that these could derive from the tubules
(Figure 3D in Kleijmeer et al., 2001). We here con-
firm the existence of such isolated vesicular MIIC
profiles in maturating D1 cells, and their close posi-
tioning to tubular/reticular MIICs. The fact that they
contain signs of internal membrane degradation may
indeed be an indication of their origin from the MIIC
tubules/reticula and represent a population of MHC
class II carrier vesicles en route to the cell surface. Be-
sides at the polar ends of tubular profiles of the MIIC
reticulum, isolated MIIC vesicles were also detected
in the vicinity of TGN exit sites, where they often
exhibited close membrane apposition to trans-Golgi
stacks. The isolated MIIC vesicles may thus also rep-
resent early stages in the formation of the MIICs.
However, when analysing many TGN exit sites, we
were not able to detect any membrane continuities
between MIIC intermediates and trans-Golgi stacks.
It is of course possible that despite our large-scale
volume analysis, such continuities have still escaped
our detection.
The question arises what is the advantage of MIIC
fusion and the formation of an elaborate MIIC net-
work, for the transport of MHC class II to the cell sur-
face. MIIC-derived tubules are long and directional
when DCs are co-cultured with T-cells (Boes et al.,
2002; Vyas et al., 2007). Shorter and more randomly
oriented MIIC tubules are observed on Toll-like re-
ceptor ligation (Vyas et al., 2007). This may suggest
that formation of a reticulum such as observed here in
cultured D1 cells represents a transitional stage and
requires other ‘extracellular’ stimuli (i.e. from inter-
acting T-cells) to ultimately form the long tubules
observed in co-cultures (Boes et al., 2002; Bertho
Three-dimensional organization of the transforming MIIC
et al., 2003; Vyas et al., 2007). Fusion between MIICs
may also be important for ‘temporal’ regulation of the
internal pH required for optimal processing of anti-
gens. Both in macrophages and DCs, fusion of pha-
gosomes with lysosome-related organelles has been
described as a mechanism to prevent full degradation
of antigens (Savina et al., 2006; Jancic et al., 2007;
Mantegazza et al., 2008). Absence of fusion could lead
to accelerated acidification and degradation of anti-
genic peptides. It is well established that treatment of
APCs with alkalinization drugs results in impaired
peptide loading and T-cell response (Savina et al.,
2006; Jancic et al., 2007; Mantegazza et al., 2008).
Stimulation of D1 cells with LPS in the presence of
bafilomycin A1 completely blocked the delivery of
MHC class II to the cell surface and resulted in the
intracellular accumulation of MIICs. These observa-
tions indicate that MIIC fusion and MHC class II
transport to the cell surface are related to the luminal
pH of the MIICs. Formation of MIIC tubules has
been demonstrated to depend on intact MTs (Vyas
et al., 2007). In our electron tomograms we found
MTs and intermediate filaments flanking the reticu-
lar MIIC. These MTs were often kinked and fragmen-
ted, and exhibited flaired and capped ends, suggest-
ing that they may have been severed during MIIC
transformation. Our three-dimensional analysis also
revealed that physical linkages exist between the lim-
iting membrane of the reticular MIICs and MTs and
intermediate filaments, suggesting that fusion and
tubulation of MIICs depend on these cytoskeleton
elements.
In summary, our ET analysis provides insight into
the mechanism of MIIC reorganization during DC
maturation. We have shown that intermediate and
late MIICs are capable of fusing together to form re-
ticular membrane networks. We also show that phys-
ical linkages exist between the transforming MIICs
and MTs and intermediate filaments. Furthermore,
we have confirmed the existence of vesicular MIIC
profiles, possibly representing MHC class II carriers
en route to the plasma membrane or early stages in
MIIC formation.
Materials and methods
Cell culture and cell treatments
The mouse DC cell line D1 was cultured as described previously
(Winzler et al., 1997). The cells were grown in Petri dishes.
For IF experiments the Petri dishes contained glass coverslips.
Research article
For HPF-FS experiments the Petri dishes contained golden grids
layered with a Formvar film and a thin layer of gelatin (Murk
et al., 2003b). To stimulate the D1 cells, LPS (Sigma–Aldrich,
St. Louis, MO, U.S.A.) was added to the medium (final concen-
tration 10 μg/ml) for 3 or 6 h as described previously (Kleijmeer
et al., 2001). The maturation state of the cells was verified mor-
phologically and after determination of the cell surface expression
of MHC class II by IF and IEM analyses. In some experiments, the
cells were exposed to 100 nM bafilomycin A1 (Sigma–Aldrich)
in DMSO (final concentration 0.1%) half an hour before and
during LPS stimulation.
HPF-FS
Cryo-immobilization and freeze substitution were performed es-
sentially as described before (Murk et al., 2003b). In short,
cells adhering to the grids were sandwiched between two alu-
minium cups (flat side towards the grid) and immediately cryo-
immobilized using a high-pressure freezer (Leica HPF; Leica
Microsystems, Vienna, Austria; now M. Wohlwend, Sannwald,
Switzerland). The samples were transferred to the freeze sub-
stitution apparatus (Leica AFS; Leica Microsystems), containing
precooled anhydrous acetone as the substitution fluid, and fixed
with 0.5% osmium tetroxide and 0.25% glutaraldehyde in an-
hydrous acetone at a temperature of − 90◦ C for 48 h. The tem-
perature was increased by 1◦ C/h to − 30◦ C and this temperature
was further maintained for 8 h. The substitution medium was
washed away by anhydrous acetone and the cells and grids were
embedded in Epon.
Tomography and data analysis
Sections of 200–400 nm thickness were collected on 1 hole
Formvar-coated copper grids, and post-stained with uranyl acet-
ate and lead citrate. Fiducial markers (10 or 15 nm gold particles)
were applied to both sides of the section and one extra layer of
Formvar was applied on top of the section for stability. Cells
displaying the typical activated dendritic phenotype were pre-
selected using a Jeol 1010 electron microscope (Jeol). Dual-
axis tilt series of selected MIICs were then recorded using
a Tecnai 20 transmission electron microscope (FEI) equipped
with a slow scan CCD camera (charge-coupled-device camera;
Temcam F214; TVIPS, Gauting, Germany), as previously de-
scribed (Murk et al., 2003a, 2004). Tilt series were acquired
using a single-axis high-tilt tomography holder (model 670;
Gatan, München, Germany) or a Fischione tomography holder
(model 2020; Fischione Instruments, K-Vision BV, Huizen,
The Netherlands). After the first tilt series the specimens were
manually rotated over an angle of 90◦ , and the second tilt
series were acquired. Then, 1000×1000 images were recor-
ded automatically with Xplore 3D software (FEI) with an
angular range from − 60◦ to +60◦ with 1◦ increment. Tilt
series were aligned with ETomo, a program from the IMOD
program package (Kremer et al., 1996; Mastronarde, 1997;
http://bio3d.colorado.edu/imod/), using the fiducial markers,
and combined into one three-dimensional volume. First, aligned
single-axis tomograms were computed by resolution-weighted
back-projection. These two volumes were combined into one
final volume. In some cases (e.g. Figure 1), tomograms of serial
sections were stitched together using the ETomo join applic-
ation. After reconstruction each tomogram was unpacked into
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tiff format and manually stitched together with adjacent tomo-
grams using Adobe Photoshop CS3. The tiff files were stacked
back into a three-dimensional volume (per section) and the three
serial sections were joined together using ETomo join. For ex-
ample, the tomogram presented in Figure 1 is composed of
one single-axis and 12 dual-axis tilt series spread over three
serial sections, resulting in a reconstructed volume of 23.6 μm3 .
Within the IMOD program, MIIC membranes were manually
traced to form a three-dimensional representation of the mem-
branes. The MIICs were identified by morphological criteria, i.e.
membrane-bound organelles containing luminal membranes, as
also recognized by MHC class II immunolabelling in thin frozen
sections.
Volumes of the modelled organelles were measured using the
IMOD info option in IMOD. Quantification of MIIC profiles
was done on randomly selected tomographic slices from sev-
eral tomograms and different maturation stages (3 and 6 h LPS
stimulation) of the D1 cells. For each MIIC profile we analysed
whether the structure was connected to an MIIC network or
represented an isolated spherical/tubular MIIC.
The statistical significance for the quantification of tubule
diameter was assessed by a two-tailed unpaired Student’s t test.
The difference in tubule diameter was considered statistically
significant when P < 0.05.
IEM
For detection of MHC class II, monoclonal rat anti-mouse MHC
class II antibody was used [M5/114 (Bhattacharya et al., 1981);
BD PharMingen, San Diego, CA, U.S.A.]. Mouse anti-α-tubulin
was obtained from Sigma–Aldrich. Rat anti-mouse H2DM
(2E5A) directed to the HLA-DM β-chain and rat anti-mouse
invariant chain (In-1) have been described previously (Kleijmeer
et al., 2001). For immunogold labelling, cells were fixed in a mix-
ture of 2% (w/v) paraformaldehyde and 0.2% glutaraldehyde in
0.1 M phosphate buffer (0.1 M NaH2 PO4 and 0.1 M Na2 HPO4
in aqua dest). The cells were washed with PBS/lysine to quench
free aldehydes, then embedded in gelatin (Liou et al., 1997) and
infiltrated in 2.3 M sucrose, followed by rapid freezing in liquid
N2 . Then, 50 nm thick cryosections were cut at − 120◦ C us-
ing an Ultracut-S ultra microtome (Leica Microsystems, Vienna,
Austria). The sections were collected on Formvar-coated grids
using a mixture of 1.8% methylcellulose and 2.3 M sucrose (Liou
et al., 1996), and incubated with primary antibodies and Protein
A–gold (Slot et al., 1991). As bridging antibodies, rabbit anti-
mouse IgG and rabbit anti-rat IgG were used. Immunogold
labelling was performed using 10 nm gold particles (Slot and
Geuze, 1985). After labelling, the sections were fixed with 1%
glutaraldehyde, counterstained with uranyl acetate, and embed-
ded in methylcellulose-uranyl acetate. The sections were viewed
in a Jeol 1200CX electron microscope (Jeol).
Immunofluorescence
Control and LPS-stimulated D1 cells were fixed with 4% para-
formaldehyde in PBS. The cells were washed with PBS and
quenched with NaBH4 (final concentration 1 μg/ml in PBS) fol-
lowed by permeabilization in 0.5% Triton X-100 and blocked
with fish skin gelatin (0.2%). The samples were incubated with
rat anti-mouse MHC class II antibody, followed by secondary
anti-rat IgG coupled with Alexa Fluor® 568 (Molecular Probes).
Images were acquired using a Zeiss LSM-510 confocal micro-
590 
C The Authors Journal compilation 
C 2010 Portland Press Limited
H.E. van Nispen tot Pannerden and others
scope. The acquisition settings were kept the same for the dif-
ferent conditions within each experiment. The subcellular dis-
tribution pattern of MHC class II was quantified in control and
LPS-stimulated cells (+
−bafilomycin A1 ) by counting five to eight
random fields reaching a total number of ∼ 100 cells per con-
dition with respectively intracellular, mixed and predominantly
surface-expressed MHC class II (n = 100 cells).
Author contribution
The concept of the research was created by Monique
Kleijmeer and Harry Heijnen, and the design and
supervision of the research was by Harry Heijnen.
Experimental work was carried out by Hezder van
Nispen tot Pannerden, and Willie Geerts participated
in the acquisition of the three-dimensional data. Data
analysis, interpretation and writing of the manuscript
were carried out by Hezder van Nispen tot Pannerden
and Harry Heijnen.
Acknowledgements
We are grateful to V. Kondylis for his help with con-
focal microscopy, G. Posthuma for useful discussions
on quantification and S. van Dijk for her assistance
with ultrathin sectioning and immunolabelling. We
also thank M. van Peski and R. Scriwanek for their
help with preparation of the Movies and Figures and
J.L. Murk for his help with sample preparation. We
thank Dr P. Ricciardi-Castagnoli (Department of Bio-
technology and Bioscience, University of Milano-
Bicocca, Milan, Italy) for providing us with D1 cell
line.
Funding
This work was supported by The Netherlands Organ-
isation for Scientific Research [grant number ALW
813.08.001 (to H.E.v.N.t.P.)].
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Biol. Cell (2010) 102, 581–591 (Printed in Great Britain) doi:10.1042/BC20100046

Supplementary online data

Spatial organization of the transforming MHC class II
compartment
Hezder E. van Nispen tot Pannerden*†, Willie J. Geerts*‡, Monique J. Kleijmeer*† and Harry F.G. Heijnen*§1
*Institute of Biomembranes, Padualaan 8, 3584 CH Utrecht, The Netherlands, †Cell Microscopy Center, Department of Cell Biology,
University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands, ‡Department of Molecular Cell Biology, Utrecht
University, Utrecht, The Netherlands, and §Laboratory of Clinical Chemistry and Haematology, University Medical Center Utrecht,
Heidelberglaan 100, 3584 CX Utrecht, The Netherlands

Figure S1 MHCII transport to the cell surface after LPS

stimulation
Representative confocal images showing immunostaining of
MHC class II in untreated D1 cells (A) and cells stimulated for
6 h with LPS (B). After 6 h of LPS stimulation, much of the
MHC class II has reached the plasma membrane. Scale bar,
10 μm.

1 To whom correspondence should be addressed (email

h.f.g.heijnen@umcutrecht.nl).

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 H.E. van Nispen tot Pannerden and others

Figure S2 Identification of MIICs by immunogold labelling

(A) Non-stimulated control D1 cell. MHC class II localizes to the luminal and limiting membranes of the spherical MIIC, and is
virtually absent from the plasma membrane (PM). (B) After 6 h of LPS treatment, MHC class II is both found on the plasma
membrane and associated with irregularly shaped MIICs. Scale bars, 200 nm.


C The Authors Journal compilation 
C 2010 Portland Press Limited
Three-dimensional organization of the transforming MIIC

Figure S3 Identification of early and late MIICs

D1 cells were stimulated for 6 h with LPS. (A, B) Two subsequent cryosections showing abundant labelling of HLA-DM on the
limiting membrane of multivesicular phenotypes representing intermediate and late MIICs. Note the absence of label in the early
MIICs with low vesicle number. Arrows indicate interconnecting MIICs. (C, D) Vice versa, early compartments with sparse luminal
vesicles (EE) contain abundant invariant chain (Ii), whereas multivesicular MIIC phenotypes, representing the late stages, are
devoid of invariant chain. Note also the abundant labelling of invariant chain in the ER (arrowheads). Two subsequent cryosections
are shown. PM, plasma membrane; MIIC, MHC class II compartment; ER, endoplasmic reticulum; EE, early endosomes. Scale
bars: (A, B) 500 nm and (C, D) 200 nm.

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 H.E. van Nispen tot Pannerden and others

Figure S4 Early endosomes

(A) Tubular structures emerging from early endosomes (EE) in a 3 h LPS-stimulated D1 cell. (B) Two models of early endosomes
[the pink model represents the early endosomes in (A)]. Gold colour indicates internal vesicles. Arrowheads indicate tubules
emerging from the early endosomes. Scale bars, 100 nm.

Figure S5 Series of tomographic slices through two MIICs in non-stimulated D1 cells

The closely positioned MIICs are not interconnected and are both completely captured within the tomogram. Inset: 3D model.
Scale bar, 100 nm.


C The Authors Journal compilation 
C 2010 Portland Press Limited
Three-dimensional organization of the transforming MIIC

Figure S6 Effect of bafilomycin A1 on MHCII transport

(A) Representative confocal image showing immunostaining of MHC class II in D1 cells stimulated for 6 h with LPS in the
presence of bafilomycin A1 . MHC class II remains mostly intracellular (compare with non-stimulated cells in Supplementary
Figure S2A). (B) Semi-quantitative evaluation of the MHC class II distribution in control and LPS-stimulated D1 cells (in the
presence or absence of bafilomycin A1 ). The percentages of D1 cells exhibiting the indicated staining patterns of MHC class II
are shown: mostly intracellular, partially transported to the cell surface, or mostly expressed on the cell surface (n = 100 cells
per condition). (C) Example of MIIC profile after 6 h of LPS stimulation in the presence of bafilomycin A1 . Accumulated luminal
vesicles contain large numbers of MHC class II (10 nm gold). Scale bars: (A) 10 μm and (C) 200 nm.

Received 13 April 2010/13 August 2010; accepted 16 August 2010

Published as Immediate Publication 16 August 2010, doi:10.1042/BC20100046

www.biolcell.org | Volume 102 (11) | Pages 581–591