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Studying the relationship of annexin A2, langerin, and Birbeck granules

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Studying the relationship of annexin A2, langerin, and Birbeck granules

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Content
Studying the Relationship of Annexin A2, Langerin, and Birbeck Granules

By

Shantaé M. Thornton

A Thesis Presented to
THE FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA

In Partial Fulfillment of the
Requirements of the Degree

MASTER OF SCIENCE
MOLECULAR MICROBIOLOGY AND IMMUNOLOGY

December 2018
ii
Acknowledgments
This thesis dissertation would not have been possible without the generous support of
the incredible individuals below.

Firstly, I would like to give my utmost appreciation to my mentors Dr. W. Martin Kast
and Dr. Diane Da Silva, both of whom have provided unwavering support and guidance
throughout my research project. I would also like to thank the members of my thesis
committee, Dr. Omid Akbari and Dr. Ralf Langen for providing feedback, time, and
energy in helping me to complete my dissertation.

My work would not have been possible without the significant help of Christopher Buser,
PhD, who provided technical training and troubleshooting, as well as expertise in
developing our methods for transmission electron microscopy. Also, a big thank you to
Varsha Samaratane, who helped in obtaining much of this data and establishing the
systems herein.

Finally, my achievements would not have been possible without the support of my
incredible family. They have always encouraged me in all my endeavors and allowed
me to follow my dreams, and for that I am forever grateful.

iii
List of Abbreviations

A2t Annexin A2/S100A10 Heterotetramer
AnxA2 Annexin A2
APC Antigen Presenting Cell
BG Birbeck Granules
CD Cluster of Differentiation (CD34, CD63, CD207)
CMS Cytomembrane Sandwiching Structures
CRD Carbohydrate Recognition Domain
DC Dendritic cell
DMEM Dulbecco’s Modification of Eagle’s Medium
ERC Endosomal Recycling Compartment
GM-CSF Granulocyte-Macrophage Colony-Stimulating Factor
HIV Human Immunodeficiency Virus
HPV Human Papillomavirus
IEM Immunoelectron Microscopy
LC Langerhans Cell
MEMa Minimum Essential Media Alpha
MHC II Major Histocompatibility Complex Type-II
M-LC MUTZ-3 Derived-Langerhans Cell
PBMC Peripheral Blood Mononucleocytes
PRR Pattern Recognition Receptor
RE Recycling Endosomes
S100A10 S100 Family Protein A10
TEM Transmission Electron Microscopy
TGFb Transforming Growth Factor Beta
TNFa Tumor Necrosis Factor Alpha

iv
Table of Contents

Acknowledgments ii
List of Abbreviations iii
List of Figures v
Abstract 1
Chapter 1 – Introduction 2
1.1 – Birbeck Granules 2
1.2 – Preliminary Findings 10

Chapter 2 – Materials and Methods 16
2.1 – Cell Culture 16
2.2 – Crispr/Cas9 AnxA2 Knock-Out 17
2.3 – Langerin Transfection 18
2.4 – Western Blot and Protein Quantification 18
2.5 – Flow cytometry 18
2.6 – Immunofluorescence Microscopy 19
2.7 – Transmission Electron Microscopy 19
2.8 – Immunoelectron Microscopy 20

Chapter 3 – Results 21
3.1 – Langerin is less abundant in anxA2 knock-down M-LC 21
3.2 – Langerin immunolabeling is not localized to BG in AnxA2 knock-down M-LC 22
3.3 – AnxA2 is required for proper BG formation 24
3.4 – AnxA2 may be involved in langerin recycling in M-LC to form BG 28

Chapter 4 – Discussion and Future Directions 31
References 35

v

List of Figures

Figure 1 – Cytoplasmic Birbeck granules from MUTZ-3 derived Langerhans cells

Figure 2 – Proposed three-dimensional structure of Birbeck granules

Figure 3 – Characteristic Birbeck granule morphology

Figure 4 – Langerin immunolabeling shows localization to BG

Figure 5 – SiMPull showing Langerin and A2t Co-IP in PBMC-LC

Figure 6 – Annexin A2 IEM labeling in MUTZ-3 Derived- Langerhans cells

Figure 7 – Western Blot of M-LC with anxA2 knockdown

Figure 8 – Birbeck granules are less abundant in annexin A2 knock-down M-LC

Figure 9 – BG-like structures in AnxA2 knock-down M-LC

Figure 10 – Quantification of BG in WT and anxA2 knock-down M-LC

Figure 11 – Langerin abundance in pre- and post-differentiated WT and anxA2 knock-
down M-LC

Figure 12 – IEM photos of anxA2 knock-down M-LC labeled for langerin

Figure 13 – S100A10 and anxA2 protein knock-out in HaCaT cells

Figure 14 – Langerin expression in WT and anxA2 knockout HaCaT cells

Figure 15 – TEM image of BG formation in langerin knock-in WT HaCaT cells

Figure 16 – TEM images of BG-like formation in anxA2 knockout HaCaT Cells

Figure 17 – A2t expression in WT M-LC during differentiation

Figure 18 – Surface and internal langerin expression in WT and anxA2 knock-down M-
LC

Figure 19 – Immunofluorescence staining in WT and anxA2 knock-down M-LC
1
Abstract

Langerhans cells (LC) are the resident antigen presenting cells of the mucosal
epithelium and play an essential role in initiating immune responses. LC are the only
cells in the body to contain Birbeck granules (BG), unique cytoplasmic organelles
comprised of the c-type lectin langerin. Studies of BG have historically focused on
morphological characterizations, although they have also been implicated in viral
antigen processing which suggests that they may serve a function in antiviral immunity.
This study focused on investigating proteins that may be involved in the biogenesis of
BG to further characterize their structure and potentially function. Here, we report a
novel and essential role for the protein annexin A2 in BG formation. We found langerin,
the primary protein comprising BG, and A2 physically interact, but do not co-localize in
BG. When A2 expression is down-regulated, we found decreased expression of
langerin, a near ablation of cytoplasmic BG, and the presence of disfigured, BG-like
structures. Additionally, in the absence of A2, we also found langerin is no longer
localized to BG or BG-like structures and surface langerin expression is decreased.
Immunofloresence studies suggested an increase in langerin and Rab11a co-
localization with knock-down of A2. Taken together, these results indicate a novel and
essential role for annexin A2 in BG formation, potentially through interaction with
Rab11a.

2
Chapter 1 – Introduction

1.1 – Birbeck Granules

Birbeck granules (BG), first described in 1961 by Michael S. Birbeck, are unique
cytoplasmic organelles which resemble tennis rackets in 2D cross sections (Figure 1)
1
.
Since their discovery over 25 years ago, BG have remained elusive in derivation,
composition, and function. Structurally, they are comprised of two superimposed, joined
pentalamellar membranes separated by regular striations about 5-10nm apart. It has
been suggested that they resemble 1 µm wide and 50nm thick disks with one or more
spherical lobe regions three dimensionally
2
. BG exist in a wide variety of size and
structure and vary visually using Transmission Electron Microscopy (TEM), as this
imaging technique is dependent upon taking ultra-thin cross sections of cells (Figure
2)
3
. However, characteristic images typically contain a translucent head portion
attached to a rod containing a linear striation through its center (Figure 3).

3

Figure 1 – Cytoplasmic Birbeck granules from MUTZ-3 derived Langerhans cells
The characteristic tennis-racket and dumbbell shaped-organelles can be seen outlined
in the white boxes and are in high abundance in this two-dimensional cross section.

500nm
4

Figure 2 – Proposed three-dimensional structure of Birbeck granules
3
.
This three-dimensional model of BG shows a disk-shaped body with a lobe portion was
proposed after visualizing many different 2-dimentional BG morphologies using TEM

Figure 3 – Characteristic Birbeck granule morphology
Arrows indicate the typical translucent head and rod portions with a central linear
striation in a BG cross section.
5
Only a few proteins have been implicated in the biogenesis of BG. Langerin
(CD207), a type-II transmembrane c-type lectin, is the most investigated and arguably
the most critical protein involved in BG formation. The protein structure of langerin is
composed of a small intracellular proline-rich portion, a 20-amino acid transmembrane
portion, and an extracellular lectin portion, termed the neck region
4
. The extracellular
neck region exists as a stable trimer of coiled-coil alpha-helices and has been
characterized as a calcium-dependent carbohydrate recognition domain (CRD) specific
for mannose, fructose, and N-acetylglucosamine
5,6
. It was first identified as a BG-
associated protein 1986, although it had yet to be named or characterized at the time
7
.
The essential role of langerin in BG formation was established through cDNA
expression in fibroblasts, which resulted in the formation of cytoplasmic BG
5
.
Additionally, immunolabeling and imaging via TEM has confirmed that langerin is highly
localized to BG (Figure 4). Langerin has other unique properties which further supports
its role in facilitating the formation of BG structures. It’s been shown to causes formation
of cytomembrane sandwiching structures (CMS), a process which superimposes
membranes
8
. In cells with langerin knock-in, membrane zippering and superimposition
was seen in organelles that do not typically exhibit these characteristics
5
. This further
supports the ability for langerin to induce membrane superimposition, which is a key
characteristic of the BG structure.

6

Figure 4 – Langerin immunolabeling shows localization to BG
10nm gold particles were used to immunolabel langerin and can been seen to highly
localize to BG structures, as indicated by the yellow arrows.

For many years, two conflicting theories regarding BG derivation were widely
contested. The first, termed the secretion/exocytosis theory, suggested BG had an
intracellular origin such as from the Golgi or other endosomes. The second suggested
BG formation occurred via receptor-mediated endocytosis. It postulated BG formed
following membrane invaginations leading to intracellular budding of BG
9,10,11
.
Interestingly, it has been shown that BG formation is dependent upon a high local
concentration of langerin to induce membrane superimposition
5
, which could
hypothetically support both theories. In support of the latter theory, a study using
immunoelectron microcopy (IEM) labeling showed langerin accumulated in deep
striated membrane invaginations after exposure to an anti-CD207 antibody
5,12
. These
conflicting hypotheses were clarified following studies performed by McDermott, et al. in

7
2002 and Uzan-Gafsou, et al. in 2007. Taken together, it was found that although BG
formation does contain some aspects from both theories, their biogenesis is a unique
and previously undescribed process. It is now understood that BG are subdomains of
the endosomal recycling compartment (ERC)
13,14
.
BG are only found in Langerhans cells (LC), the antigen presenting cells (APC)
of the mucosal epithelium. LC are a distinct subset of dendritic cells (DC) and are most
notably characterized by their presence of BG. APC are highly specialized cells of the
innate immune system with the primary function of sampling the environment for foreign
pathogens
15
. Once antigen is recognized and processed, LC become mature, which is
marked by upregulation of co-stimulatory molecules, cytokine secretion, and migration
from the epithelium to the lymph nodes. Interestingly, it’s been shown that langerin acts
as a ligand to induce internalization, suggesting it may act as a pattern recognition
receptor (PRR) in LC and play a role in antigen processing. The first investigations of
binding, endocytosis, and antigen delivery via langerin were studied using the anti-
CD207 antibody DCGM4. Following antibody treatment, endocytosis was found to be
receptor-mediated, highly rapid, and independent of the major histocompatibility
complex type-II (MHC II)
12
. Subsequent studies found langerin entry is facilitated
through classic clathrin-mediated endocytosis
13
. Post-internalization, there was no
routing of langerin to HLA-DR
+
compartments, indicating langerin is not involved in
antigen delivery via the MHC II pathway
12
. Additionally, langerin did not co-localize with
EEA1, a marker for early endosomes, or Lamp 2 and CD63, lysosomal markers.
However, it was found that langerin strongly co-localized with Rab11a, a marker for
recycling endosomes (RE)
16
.
8
Following this critical finding, studies regarding the recycling pathway of langerin
became the new focus of BG research. It is known that RE have a dual role as a sorting
compartment for internalized proteins and as a storage reservoir for regulated protein
delivery to the plasma membrane
17–19
. Taken together, we now know langerin is
internalized from the cell surface and transported to the ERC where it accumulates.
When the concentration of langerin is optimal, a budding event is likely induced which
facilitates the formation of cytoplasmic BG. An event such as antigen capture could
likely promote large amounts of langerin accumulation.
Evidence that langerin and BG in are involved in antigen capture and processing
has continued to build in the past few years
20
. De Witte, et al. found langerin binds to
human immunodeficiency virus (HIV) via its CRD, implicating it further as an important
PRR for antigen processing. They found that HIV was endocytosed through langerin
binding and is then trafficked to BG. Surprisingly, it was found that the BG may actually
have a protective role as they were able to prevent HIV transmission by sequestering
and trafficking the virus to lysosomal compartments
21
. These results indicate the role
BG have in fighting HIV, and potentially other viral infections as well.
These findings are particularly interesting in the context of viral manipulation of
innate immune cells such as LC. Many viruses are known to manipulate their host cell
to establish infection by promoting immune evasion. One example of this is the Human
Papillomavirus (HPV) which has been shown to have drastic effects on infected LC
such as delay in maturation, altered antigen presentation, and a reduction in an
activated phenotype
22
. Interestingly, these effects were found to be facilitated by and
dependent upon the presence of the annexin A2/S100A10 heterotetramer protein
9
complex (A2t)
23
. A2t is a multi-function protein comprised of two monomeric annexin A2
(anxA2) subunits bridged by an S100A10 dimer. S100A10 is stabilized by anxA2 and is
ubiquitinated and rapidly degraded in its absence
24
. This protein complex is involved in
other immunological contexts such as regulating infection-initiated inflammation and
promoting viral infection
25,26
. A2t also been shown to be involved in proper intracellular
distribution of RE
27
. Given that both A2t and langerin have been implicated in innate
immune events, interaction with antigens, localization to RE, and have membrane
bending and trafficking abilities, we initially sought to investigate if A2t could be involved
in BG formation.
Research on LC remains limited and is difficult due to inefficient isolation from
peripheral blood mononucleocytes (PBMCs). Additionally, these cells only live in culture
for at most 7 days, which prevent long-term experiments and there can be variability
between PBMC donors which is difficult to control for. In order to facilitate LC studies,
the CD34+ human acute myeloid leukemia cell line MUTZ-3 was generated. Following
treatment with the cytokines GM-CSF, TNFa, and TGFb, these immortalized cells
phenotypically resemble primary LC. They express high amounts of langerin, can
induce anti-tumor T-cell immunity, and are transcriptionally similar to that of human-
derived LC. Additionally, differentiated MUTZ-3 derived LC (M-LC) contain anxA2, while
undifferentiated MUTZ-3 do not. Most importantly, M-LC have a high abundance of
cytoplasmic BG, and are therefore a great candidate cell line for our studies of the
involvement of A2t and langerin in BG
28
.

10
1.2 – Preliminary Findings
The novel findings regarding HPV and A2 in LC manipulation first led us to
investigate any interactions A2 and langerin may have. We utilized the Single Molecule
Pulldown assay (SiMPull) to see if langerin and A2 physically interact. It was found that
langerin co-immunoprecipitates with A2 in PBMC-LC (Figure 5). Based on these
findings, we postulated A2 and langerin could co-localize in BG and sought to further
define the role of A2 in BG formation.
Many of our studies are dependent upon TEM as this is the only imaging
technique which provides the resolution to properly study BG structure. To determine
protein localization within the BG and cells, we utilized 10nm gold particle
immunolabeling in conjunction with TEM. We began our investigations into the
relationship between A2 and BG by immunolabeling M-LC for anxA2 and found that
anxA2 does not localize to BG (Figure 6). We then proceeded to knock-down anxA2 in
MUTZ-3 and create clonal cell populations (Figure 7). Strikingly, we found that in the
absence of anxA2, BG formation is nearly ablated (Figure 8). We also observed the
presence of misshapen BG structures, which we termed BG-like, that often lacked a
translucent head portion and did not contain the central striation in the rod portion
(Figure 9). Quantification of BG in WT, anxA2 knock-down, and mock-infected M-LC
confirmed our observations and found that BG formation is significantly decreased in
the absence of anxA2 (Figure 10). Following these novel and exciting results, we then
sought to investigate how anxA2 is involved in BG formation. Based on these findings,
we hypothesized that anxA2 is required for proper langerin localization to BG.
11

Figure 5 – SiMPull showing Langerin and A2t Co-IP in PBMC-LCs. (A) SiMPull slides
are passivated with biotin-conjugated polyethylene glycol (PEG-biotin). Flow chambers
are then delineated and epoxy-sealed with a coverslip. Biotin conjugated capture
antibodies against the protein of interest are then immobilized on the surface of the slide
by successively flowing them through with NeutrAvidin. LC lystate is added to the slides
and the protein of interest along with its interaction partner is immobilized through binding
to the capture antibody. Presence of interacting proteins is detected with fluorescently
tagged detection antibodies and Total Internal Reflection Microscopy(TIRF). (B)
Quantification of A2t-langerin SIMPull complexes.

A
B
12

Figure 6 – Annexin A2 IEM labeling in MUTZ-3 Derived- Langerhans cells
Solid yellow triangles indicate cytoplasmic BG and yellow arrow indicate IEM anxA2
labeling.

13

Figure 7 – Western Blot of M-LC with anxA2 knockdown
Prior to differentiation, WT and anxA2 knock-down MUTZ-3 do not express A2t. Lane 4
shows that following differentiation, anxA2 knock-down M-LC do not express A2t.

14

Figure 8 – Birbeck granules are less abundant in annexin A2 knock-down M-LC
(A) WT M-LC have abundant cytoplasmic BG, outlined in white boxes. (B) AnxA2
knock-down M-LC do not have a large abundance of cytoplasmic BG and exhibit BG-
like structures, outlined in white boxes.

A
B
15

Figure 9 – BG-like structures in AnxA2 knock-down M-LC
(A) A BG-like structure is missing a central striation in the rod portion and has a missing
or misshapen head portion. (B) WT BG contain central striation within the rod portion.
A membrane structure was counted as BG-like if:
1. sheet-shaped membrane =
2. or elongated sheet-shaped invagination of the plasma membrane
3. Not ER (no ribosomes)
4. Not Golgi (not part of a Golgi stack)
5. Not vesicular
A membrane was counted as BG if as BG-like but with a clear membrane-membrane
boundary (“zipper”) anywhere within the membrane =

Figure 10 – Quantification of BG in WT and anxA2 knock-down M-LC
(A) PBG/Pcytoplasm ratios are plotted for each of the 20 M-LCs and 20 anxA2 KD M-LCs.
(B) IBG/Pcytoplasm ratios are plotted for each of the 20 M-LCs and 20 anxA2 KD M-LCs.
Significance was assigned to a value of p≤0.05.

A B
16
Chapter 2 – Materials and Methods

2.1 – Cell Culture
The CD34+ human acute myeloid leukemia cell line, MUTZ-3, was a generous gift from
Rik J. Scheper at VU Medical Center in Amsterdam, The Netherlands. MUTZ-3 cells
were cultured at a density of 2x10
5
cells/mL in Minimum Essential Media, Alpha 1X
(MEMa) with Earle’s salts, ribonucleosides, deoxyribonucleosides, and L-glutamine
(Corning 10-022-CV, NY) supplemented with 20% heat-inactivated human fetal bovine
serum (Omega Scientific, Tarzana, CA), 10% conditioned medium from the renal
carcinoma cell line 5637, and 50µM 2-mercaptoethanol (Gibco, Grand Island, NY) at
37°C with 5% CO2. (Santegoets SJAM) (Masterson AJ). To induce a Langerhans cell
phenotype, MUTZ-3 cells were seeded at a density of 1x10
5
cells/mL and were cultured
for 14 days in the medium conditions described above. On days 0, 4, and 8, the cells
were treated with a cytokine regimen containing 100ng/mL GM-CSF (Sanofi,
Bridgewater, NJ), 2.5ng/mL TNFa (PeproTech, Rocky Hill, NJ), and 10ng/mL human
TGFb (Invitrogen, Carlsbad, CA). Medium was replenished on day 8 of differentiation
(Santegoets SJAM).

To generate 5637 conditioned medium, 5637 cells were cultured in RMPI 1640, 1X with
L-glutamine (Corning 10-040-CV, NY) supplemented with 10% heat-inactivated human
fetal bovine serum (Omega Scientific, Tarzana, CA), 50µM 2-mercaptoethanol (Gibco,
Grand Island, NY), and 1X Gentamycin (Lonza, Walkersville, MD) at 37°C with 5% CO2.
At 80% confluency, cells were harvested, seeded at a density of 15x10
6
in a 175cm
2
17
tissue culture flask (Corning 353112, NY), and were allowed to reach confluency
overnight. The medium was replaced at 24 and collected at 72 hours post-seeding.

The spontaneously immortalized keratinocyte cell line, HaCaT, was cultured in
Dulbecco’s Modification of Eagle’s Medium (DMEM) with 4.5g/L glucose, L-glutamine,
and sodium pyruvate (Corning, 10-013CV, NY) supplemented with 10% heat-inactivated
human fetal bovine serum (Omega Scientific, Tarzana, CA), and 1X Gentamycin
(Lonza, Walkersville, MD) at 37°C with 5% CO2. Cells were passaged at 80%
confluency.

2.2 – Crispr/Cas9 AnxA2 Knock-Out
HaCaT cells were cultured in DMEM with 4.5g/L glucose, L-glutamine, and sodium
pyruvate (Corning, 10-013CV, NY) supplemented with 10% heat-inactivated human
fetal bovine serum (Omega Scientific, Tarzana, CA), and 1X Gentamycin (Lonza,
Walkersville, MD) at 37°C with 5% CO2. Cells were subcultured in antibiotic-free media
the night before treatment. Transfection was preformed according to the manufactures
protocol (Lipofectamine2000, Thermo). 2µg of plasmid encoding for anxA2 KO and 2µg
of plasmid encoding for puromycin resistance were added per 1x10
6
cells. Cells were
transfected using Lipofectamine2000 for 6 hours. 48hrs-post transfection, 40µg/mL of
puromycin was added to the cells for 72hrs. Cells were then seeded in 96-well plates
using limiting dilution to grow single-cell colonies. Protein knock-outs were confirmed via
western blot analysis.

18
2.3 – Langerin Transfection
HaCaT cells were transfected according to the manufactures protocol
(Lipofectamine2000, Thermo). Briefly, 1x10
6
cells were subcultured in 6 well plates the
night before transfection in antibiotic-free media. Plasmid encoding CD207 was added
to Lipofectamine2000 reagent and 5µg DNA was added per 1x10
6
cells. Cells were
treated for 6 hours and collected 24hrs post-transfection for analysis. Langerin
expression was verified via flow cytometry.

2.4 – Western Blot and Protein Quantification
Cells were lysed using RIPA buffer (Pierce) with protease inhibitor (Halt). Protein was
quantified using a NanoDrop2000 Spectrophotometer. Samples were run in 10% Bis-
Tris agarose gels with MES running buffer for 35min at 200v. Gels were transferred
using the iBlot2 mini system and were blocked in Starting Block (PBS) Blocking Buffer
(Thermo) prior to primary antibody staining overnight at 4°C. Secondary antibodies were
added for 1 hour at room temperature. Membranes were imaged using LiCOR imaging
system and protein images were analyzed and quantitated using ImageStudioLite
software.

2.5 – Flow cytometry
Flow cytometry samples were analyzed using a Cytomics FC500 flow cytometer and
data was collected using CXP software (BeckmanCoulter, Indianapolis, IN). The
following antibodies were purchased from BioLegend (San Diego, CA): FITC anti-
human CD80, PE/Cy7 anti-human CD86, PE anti mouse/human CD207, PerCP/Cy5.5
anti-human CD1a, PE/Cy7 mouse IgG1, PE mouse IgG1, PerCP/Cy5.5 mouse IgG1,
19
PE/Cy7 anti-human CD4, PE anti-human CD4, PerCP/Cy5.5 anti-human CD4.
Additionally, FITC mouse IgG1 and FITC anti-human CD4 were purchased from BD
Biosciences (San Jose, CA).

2.6 – Immunofluorescence Microscopy
Fully differentiated WT and A2 knock-down MUTZ-derived Langerhans cells were
suspended in PBS at a concentration of 2.5x10
5
cells/mL in 3 well chamber glass slides
(Ibidi 80381, Madison, WI). The cells were affixed to the slides by spinning at 300xg and
fixed with 4% paraformaldehyde. The cells were blocked using PBS, 5% goat serum,
and 1X Triton X-100. Primary antibodies were diluted in buffer containing PBS, 1% goat
serum, and 1x Trition X-100 and added overnight at 4°C. Secondary antibodies were
added for 1 hour in the dark. The cover glass slips were fixed using ProLong Gold
antifade reagent with DAPI (Life Technologies, Eugene, OR. Images were taken using
the Nikon Eclipse Ti-E laser scanning confocal microscope.

2.7 – Transmission Electron Microscopy
Cells were pelleted by and fixed in 2.5% glutaraldehyde. Cells were post fixed with 1%
osmium tetroxide in de-ionized water for 1 hour at room temperature. The pellets were
treated with 1% uranyl acetate 1 hour. The pellet was dehydrated through a graded
addition of ethanol for 5 minutes each. Finally, the samples were embedded in epoxy
resin of Eponate, NMA, DDSA, and DMP-30. The pellets were then sectioned into 70nm
ultrathin sections. Samples were imaged using a Jeol 2000 Transmission Electron
Microscope.

20
2.8 – Immunoelectron Microscopy
Cells were processed for immuno-gold labelling as previously described
29–31
. They were
high-pressure frozen in PBS containing 20% BSA (Sigma-Aldrich) using an EMPact2
with RTS (Leica Microsystems, Vienna, Austria). Freeze-substitution was performed in
acetone containing 0.1% uranyl acetate and 2% water in a Leica AFS2 (Leica
Microsystems, Vienna, Austria). Cells were then embedded in HM20 and UV
polymerized at -50°C for 24h. Samples were then sectioned into 70nm sections, picked
up on formvar-coated copper grids and blocked for 10 min in blocking buffer (0.5% BSA
in PBS). Primary antibodies were diluted in blocking buffer. After centrifugation at
14,000rpm for 2 min, the supernatant was used to label the blocked sections for 30 min
at RT, followed by five washes for 2 min each in 0.01% PBS-Tween 20. Then a rabbit
anti-goat bridging antibody was diluted at 1:50 in blocking buffer. After centrifugation at
14,000rpm for 2 min, the supernatant was used to label the sections for 30 min at RT,
followed by five washes for 2 min each in 0.01% PBS-Tween 20. Finally, 10nm protein
A gold was diluted to 1:50 in blocking buffer and used to label sections for 30min at RT,
followed by three washes for 2 min each with PBS and two washes 2 min each with
deionized water. The antibody-labeled sections were examined at 80kV on a Jeol 2000
TEM.

21
Chapter 3 – Results
3.1 – Langerin is less abundant in anxA2 knock-down M-LC
After determining the significant decrease of BG formation with anxA2 knock-
down, we wondered if this could be attributed to an overall decrease in langerin
expression. It has been well established that large pools of langerin are necessary to
induce BG formation, and decreased expression or accumulation of langerin could
explain the decrease in BG abundance. To quantify langerin in WT and anxA2 knock-
down M-LC, we performed western blot analysis on 3 biological replicates of whole cell
lysates (n=9) (Figure 11). These results clearly show that langerin expression is indeed
decreased in anxA2 M-LC compared to WT M-LC and could explain the decrease in BG
formation.

Figure 11 – Langerin abundance in pre- and post-differentiated WT and anxA2
knock-down M-LC
Pre-differentiation, WT and anxA2 knock-down MUTZ-3 do not contain langerin. After
14 days of differentiation, WT M-LC contain significantly more langerin than anxA2
knock-down M-LC. Western blot image is cropped to show bands of interest.

22
3.2 – Langerin immunolabeling is not localized to BG in AnxA2 knock-down M-LC
In WT M-LC, langerin immunolabeling is very clearly localized to BG structures in
the cytoplasm. With the decreased abundance of BG seen in anxA2 knock-down, we
wanted to investigate if BG-like structures still contained langerin. The very low
abundance of BG in anxA2 knock-down makes langerin localization studies challenging,
as finding BG can be difficult. However, we still saw langerin immunolabeling, even in
the absence of BG (Figure 12). Upon visualization, the difference in labeling pattern in
the knock-down M-LC was immediately evident. This labeling pattern was markedly
different than observed in the WT cells which is typically linear as it is located in the BG.
Here, we observed significant langerin labeling in circular patterns (Figure 12A). In
structures that may resemble BG, we did not find langerin immunolabeling, suggesting
that langerin is no longer localized to BG in the absence of anxA2 (Figure 12B).

23

Figure 12 – IEM photos of anxA2 knock-down M-LC labeled for langerin
(A) The lower magnification shows clusters of langerin labeling within the cytoplasm
(B) Higher magnification shows a potential BG-like structure, outlined in the white box,
with little langerin immunolabeling. Surrounding the BG-like structure is more clusters of
langerin immunolabeling.
500
A
B
24
3.3 – AnxA2 is required for proper BG formation
To insure the decrease of BG seen in anxA2 knock-down was not an artifact of
the system used to decrease protein expression, we preformed anxA2 rescue
experiments. This initially presented a unique challenge as our fully differentiated M-LC
were also terminally differentiated, meaning we were unable to re-express proteins. As
the literature reports langerin expression induces BG formation, we created a new
langerin expressing model system to study the effects of anxA2 knock-down. We
utilized the spontaneously immortalized keratinocyte cell line HaCaT as they are easily
manipulated for protein knock in and knock out and rapidly proliferate. We first created
clonal populations of S100A10 and anxA2 knockout cells using CRISPR/Cas9. Since
S100A10 is stabilized by anxA2, anxA2 knockout is also a full A2t knockout (Figure 13).
HaCaT cells are very amenable to CRISPR-mediated full protein knockout which
provided a more reliable system than the previous knock-down MUTZ-3 system. We
then transiently expressed langerin in the WT and anxA2 knockout cells using a high-
copy plasmid. Langerin expression was analyzed via flow cytometry 24-hours post-
transfection (Figure 14). Almost 40% of WT HaCaT cells expressed langerin (Figure
14B) and around 30% of anxA2 knockout HaCaT cells expressed langerin (Figure
14D). After verifying langerin expression, we processed them for TEM to look for BG
formation. In WT HaCaT cells expressing langerin, we see a WT BG phenotype (Figure
15). In anxA2 knockout HaCaT cells expressing langerin, we saw BG-like structures
(Figure 16). These results indicate that the decreased abundance and appearance of
BG-like structures is caused by anxA2 deficiency and is not an artifact of the knock-
down MUTZ-3 system.
25

Figure 13 – S100A10 and anxA2 protein knock-out in HaCaT cells
Western blot from whole cell lysates showing protein expression for anxA2 and
S100A10 in CRISPR/Cas9 knockout clonal populations of each repective cell type.

Figure 14 – Langerin expression in WT and anxA2 knockout HaCaT cells
Langerin expression via flow cytometry analysis. (A) Isotype control in WT HaCaT. (B)
Langerin expression in WT HaCaT. (C) Isotype control in anxA2 knockout HaCaT. (D)
Langerin expression in anxA2 knockout HaCaT.
A B
D C
26

Figure 15 – TEM image of BG formation in langerin knock-in WT HaCaT cells
Various cross sections of cytoplasmic BG can be seen outlined in the white boxes
displaying characteristic tennis racket and dumbbell shapes. Cells were processed and
imaged 24-hours post langerin transfection.
27

Figure 16 – TEM images of BG-like formation in anxA2 knockout HaCaT Cells
Decreased abundance of cytoplasmic BG is observed in anxA2 knockout HaCaT cells.
BG-like structures are outlined in white boxes and are missing rod striations.

28
3.4 – AnxA2 may be involved in langerin recycling in M-LC to form BG

Following the confirmation of the role of anxA2 in BG formation, we began to
investigate the stage at which it might be involved. Since the MUTZ-3 system does not
express anxA2 pre-differentiation, we wanted to see when these cells began to express
it. Using whole cell lysates, we probed for A2t expression on several days throughout
the differentiation process (Figure 17). Here we see that A2t is only detectable starting
at day 10 and 13 of differentiation. Based on these findings, we then wondered if there
were any notable differences in langerin localization once anxA2 is expressed in the M-
LC system which might indicate if anxA2 is involved in langerin trafficking. We
measured extracellular and intracellular langerin expression via flow cytometry on the
same days we analyzed M-LC via western blot (Figure 18). We see that in the anxA2
knock-down M-LC, overall langerin expression is decreased. Interestingly, after day 7 of
differentiation, the WT M-LC have much higher levels of surface langerin expression
compared to the anxA2 knock-down M-LC. This could indicate that anxA2 expression is
required for langerin trafficking from the ERC back to the cell surface. To investigate this
further, we preformed immunofluorescence studies to see if there was an increase in
langerin co-localization with Rab11a in anxA2 knock-down M-LC (Figure 19). Although
we were unable to generate enough images to preform statistical analysis, visual
comparison of WT and anxA2 M-LC appear to show differences in co-localization. Of
interest, knock-down M-LC seem to have larger, more punctate yellow staining,
compared to the WT cells in which the yellow staining is more diffuse. This could
indicate that in the absence of anxA2, langerin remains in Rab11a compartments and
could explain the decrease in BG formation.
29

Figure 17 – A2t expression in WT M-LC during differentiation
(A) Expression of A2t was analyzed via western blot in WT M-LC lysates at days 1, 4, 7,
10, and 13 of differentiation and compared to WT HaCaT A2t expression. Western blots
shown here are cropped from the original image to show proteins of interest.

Figure 18 – Surface and internal langerin expression in WT and AnxA2 knock-
down M-LC. Differentiating WT and anxA2 knock-down M-LC were collected and
analyzed on days 1, 4, 7, 10, and 13 for surface and internal langerin expression. (A)
Mean florescent intensity of langerin expressed on the surface. (B) Mean florescent
intensity of intracellular langerin.

Day 1
Day 4
Day 7
Day 10
Day 13
0
100
200
300
Surface Langerin Expression
Mean Floresence Intensity (MFI)
Day 1
Day 4
Day 7
Day 10
Day 13
0
500
1000
1500
2000
2500
Internal Langerin Expression
Mean Floresence Intensity (MFI)
30

Figure 19 – Immunofluorescence staining in WT and anxA2 knock-down M-LC
WT and anxA2 knock-down M-LC stained for langerin and Rab11a to determine any
differences in co-localization between the two cell types. Langerin is stained in the FIT-
C channel and Rab11a is stained in the TRIT-C channel.

31
Chapter 4 – Discussion and Future Directions
This study sought to investigate the relationship between annexin A2, langerin,
and Birbeck granules. Although not much is known about the proteins involved in BG
formation, they likely play a key role in antigen processing, and are therefore important
to understand and characterize. Here we found strong data to implicate that AnxA2, in
addition to langerin, is critical for BG formation. We have demonstrated a novel role of
anxA2 in BG formation and show it may be facilitating BG formation through interaction
with Rab11a to promote membrane budding from recycling endosomes. This is the first
study to implicate another protein involved in BG biogenesis aside from langerin, which
is critically important for future characterization and functional studies.
Following our promising preliminary findings, we continued to investigate how
anxA2 has an effect on BG formation. We began by characterizing phenotypic changes
observed in the anxA2 knock-down M-LC. We first found that langerin expression is
decreased in the knock-down M-LC (Figure 11). Since it is postulated that abundant
langerin accumulation is necessary to form BG, the loss of BG could likely be explained
by this decrease in langerin expression. Additionally, these findings suggest anxA2
could play a role in proper gene regulation for langerin expression, although we did not
investigate this regulation further in this study.
Since BG formation was ablated in anxA2 knock-down, we were surprised to see
that langerin was still being expressed. This led us to question how langerin localization
in the cell may be altered without anxA2. BGs are sparse in the anxA2 knock-down, we
had a difficult time seeing if langerin localized to any of their structures. However,
having looked at many WT cells immuno-labeled for langerin, we immediately noticed a
32
significantly altered staining pattern in the knock-down cells. Whereas in the WT M-LC
we see linear labeling consistent with the BG structure, the knock-down langerin
staining was found in large circular patches distributed throughout the cytoplasm of the
cells (Figure 12A). We were able to visualize some BG-like structures and did not see
langerin labeling (Figure 12B). Previous findings reported BG are generated from
Rab11a+ recycling endosomes and that langerin cycles from the surface into the
endosomal recycling pathway. The intracellular langerin accumulation is likely what
would occur prior to BG formation from the endosomal recycling compartments. This
could indicate that the recycling endosomal pathway could be disturbed in the absence
of anxA2, leading to langerin accumulating in the RE and preventing BG formation.
We also generated data to support the knock-down phenotype was, in fact, an
effect of anxA2 depletion. This simple proof of concept experiment led us to creating
two new cell lines as a model system. Following differentiation treatment, M-LC no
longer divide and therefore can’t be used to study phenotype rescue. Instead, we used
epithelial cells with high wild type anxA2 expression, which was knocked-out to
generate another anxA2 knock-out cell line (Figure 13). This is actually favorable to the
M-LC knock-down system as genome editing via CRISPR/Cas9 is more efficient than
siRNA in inhibiting gene expression. These cells also maintained viability and mitosis
following the knock-out, allowing us to then knock-in langerin. At around 30-40%, the
transfection efficiency was suitable for these studies (Figure 14). Future studies should
seek to quantify the amount of langerin expressed in these knock-out cells to ensure it
is comparable to that in PBMC-derived and M-LC cells.
33
We processed these langerin-expressing anxA2 WT and KO cells for TEM
imaging to look for BG formation. In the WT cells, we saw a higher frequency of BG
formation, all of which appeared to have a normal morphology (Figure 15).
Comparatively, the anxA2 KO cells only showed the occasional BG-like structure with
no WT BG found within the sample (Figure 16). This supports our findings in the M-LC
system and indicates that the loss of BG can be attributed to anxA2 expression. One
limitation of this study is that we were unable to immunolabel these cells, which would
allow us to ensure we are able to only examine the langerin-expression cells. Such
immunolabeling would also allow for a thorough quantification of BG in these new cells
to further strengthen the M-LC data.
To further investigate the involvement of anxA2 in langerin recycling, we
compared expression levels within the M-LC system. Flow cytometry studies measured
surface levels of langerin expression and compared them to intracellular expression in
WT and anxA2 knock-down M-LC (Figure 18). Indeed, we found that surface langerin
expression is much decreased in anxA2 knock-out cells. We also see that anxA2 starts
being expressed around day 4-7 of differentiation (Figure 17), and the levels of langerin
surface expression rapidly climb in the WT but do not in the anxA2 knock-out. This
supports the hypothesis that anxA2 is necessary in langerin trafficking back to the
surface. Furthermore, co-localization via immunofluorescence labeling of langerin and
Rab11a appears more punctate and concentrated in the absence of anxA2 (Figure 19).
Though a statistical quantity of photos has not yet been obtained and analyzed, the
differences in co-localization just observed with the naked eye seem promising as a
route to explore in support our findings.
34
Taken together, we have strong evidence which supports a critical role of anxA2
in facilitating BG formation from the recycling endosomes, likely through interaction with
Rab11a. Many studies have previously implicated anxA2 as having membrane curving
properties. This supports our conclusion that anxA2 is involved in facilitating the
budding events from the RE to form BG. Additionally, anxA2 has many interacting
partners and could likely interact with Rab11a to achieve BG formation. Although much
of our conclusions remain speculative, we are excited for the future of these studies, as
these results are very promising.
Moving forward, we will continue to validate the effects of anxA2 in our systems
through robust BG quantification in the M-LC and HaCaT systems. Additionally, we will
continue with IF studies to determine the degree of co-localization, and we can also use
IEM to visualize any Rab11a staining that is localized to BG. Furthermore, it would be of
interest to determine if anxA2 and Rab11a physically interact. Overall, we have laid a
solid foundation regarding the involvement of anxA2 in BG formation, and have added a
significant set of knowledge to the characterization of BG. In a broader context, future
studies can utilize our findings to help study how anxA2 may be involved in viral
processing in LC and BG.

35
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Abstract (if available)

Abstract Langerhans cells (LC) are the resident antigen presenting cells of the mucosal epithelium and play an essential role in initiating immune responses. LC are the only cells in the body to contain Birbeck granules (BG), unique cytoplasmic organelles comprised of the c-type lectin langerin. Studies of BG have historically focused on morphological characterizations, although they have also been implicated in viral antigen processing which suggests that they may serve a function in antiviral immunity. This study focused on investigating proteins that may be involved in the biogenesis of BG to further characterize their structure and potentially function. Here, we report a novel and essential role for the protein annexin A2 in BG formation. We found langerin, the primary protein comprising BG, and A2 physically interact, but do not co-localize in BG. When A2 expression is down-regulated, we found decreased expression of langerin, a near ablation of cytoplasmic BG, and the presence of disfigured, BG-like structures. Additionally, in the absence of A2, we also found langerin is no longer localized to BG or BG-like structures and surface langerin expression is decreased. Immunofloresence studies suggested an increase in langerin and Rab11a co-localization with knock-down of A2. Taken together, these results indicate a novel and essential role for annexin A2 in BG formation, potentially through interaction with Rab11a.

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Asset Metadata

Creator Thornton, Shantaé Marie (author)

Core Title Studying the relationship of annexin A2, langerin, and Birbeck granules

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 11/12/2018

Defense Date 07/17/2018

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag annexin A2, Birbeck granules, langerin, Langerhans cell,OAI-PMH Harvest

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Language English

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Advisor Kast, W. Martin (committee chair), Akbari, Omid (committee member), Langen, Ralf (committee member)

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Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-103643

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