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Y-shaped DNA based pMHC nonamers for detecting low-affinity T cells
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Y-shaped DNA based pMHC nonamers for detecting low-affinity T cells
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Content
Y-shaped DNA based pMHC nonamers for detecting low-affinity T cells
By
Ze Wang
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)
August 2016
Table of Contents
Acknowledgement I
List of Figures II
Abbreviation III
Abstract IV
1. Introduction 1
1.1 Antigen recognition by T cells 1
1.2 Structure and function of MHC molecules 2
1.3 T cell repertoire 4
1.4 The importance of low-affinity T cells 4
1.5 The vital role of low-affinity T cells in cancer and autoimmune diseases 5
1.6 Detection of T cells by pMHC tetramers 7
1.7 Further development of the pMHC tetramer staining method 8
1.8 Other forms of pMHC multimers 8
1.9 The limitation of current pMHC multimers 9
1.10 The design of a novel pMHC nonamer 10
2. Results and Discussion 13
2.1 Expression, in vitro refolding, and purification of pMHC complexes 13
2.1.1 Expression, in vitro refolding and purification of peptide/H-2Kb complexes 13
2.1.2 Expression, in vitro refolding and purification of HLA-A2 17
2.1.3 Expression, in vitro refolding and purification of I-Ek 21
2.2 Expression, in vitro refolding and purification of BirA 24
2.3 Expression, in vitro refolding and purification of streptavidin 28
2.4 Biotinylation of MHC molecules 30
2.4.1 Biotinylation of H-2K
b
30
2.4.2 Biotinylation of HLA-A2 36
2.5. Concluding remarks 40
2.6. Future work 45
3. Methods and Materials 48
3.1 Expression, in vitro refolding, and purification of MHC molecules 48
3.1.1 MHCs sequence 48
3.1.2 Expression 49
3.1.3 In vitro refolding 50
3.1.4 Purification 51
3.2 Expression, in vitro refolding, and purification of the BirA enzyme 52
3.3 Expression, in vitro refolding, and purification of streptavidin 53
3.4 Biotinylation of MHC molecules 53
References 55
I
Acknowledgement
I have received much support and help from many individuals in writing this thesis. I would like
to extend my sincere gratitude to all of them.
First of all, I would like to express my special gratitude and thanks to my advisor, Dr. Jianming
Xie, for giving me the opportunity to join his lab. I appreciate his patience, understanding,
motivation, generous guidance, and valuable advice during this year.
I am grateful to my committee members, Dr. Stiles and Dr. Okamoto, for providing me with
valuable suggestions and helpful assistance for this thesis, as well as my graduate study.
I also would like to thank all lab members, especially Dr. Liang Rong and Rebecca Lim, for
teaching me, guiding me in my experiments, and revising this thesis.
Finally, I must express my very profound gratitude to my parents and friends for providing me
with unfailing support and continuous encouragement throughout my years of study and
research.
II
List of Figures
Figure 1. The pMHC–TCR interaction and the structure of MHC molecule 3
Figure 2. Schematic illustration of a pMHC nonamer 11
Figure 3. Technology roadmap 12
Figure 4. Purification of H-2K
b
by the Superdex 200 10/300 GL column 15
Figure 5. Purification of HLA-A2 by the Superdex 200 16/600 column 19
Figure 6. Purification of I-E
k
by the Superdex 200 10/300 GL column 22
Figure 7. Purification of BirA by the Ni-NTA and Superdex 200 16/600 columns 26
Figure 8. Purification of streptavidin by the Q column 29
Figure 9. Exploration of optimal conditions for biotinylation of H-2K
b
34
Figure 10. Biotinylation of HLA-A2 38
Figure 11. Illustration of crosslink between streptavidin and the Y-shaped DNA scaffold 42
III
Abbreviation
TCR, T Cell Receptor
MHC, Major Histocompatibility Complex
HLA, Human Leukocyte Antigen
pMHC, peptide-Major Histocompatibility Complex
K
D
, Dissociation Constant
LTD, Limiting Dilution Analysis
CyTOF, Cytometry by Time-of-Flight Mass Spectrometry
UV , Ultraviolet
E.coli, Escherichia coli
MCC, Moth Cytochrome c
β-ME, β-Mercaptoethanol
IPTG, Isopropyl β-D-1-thiogalactopyranoside
SA V , Streptavidin
SDS-PAGE, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
PBS, Phosphate Buffered Saline
EDTA, Ethylenediaminetetraacetic Acid
pI, Isoelectric Point
FPLC, Fast Protein Liquid Chromatography
IRF4, Interferon Regulatory Factor 4
DMSO, Dimethyl Sulfoxide
IV
Abstract
The detection, quantification, and characterization of antigen-specific T cells from a mixed
population have been limited for many years because of the low affinity and short disassociation
half-life between T cell receptors (TCR) and peptides bound to major histocompatibility complex
(MHC) molecules. This problem has been overcome by the development of peptide-MHC
(pMHC) tetramers and other forms of multimers, which are able to bind to multiple TCRs
simultaneously and thus have significantly enhanced avidity to the latter. However, it has been
reported that some types of T cells, such as cancerous T cells and auto-reactive T cells, can
express extremely low-affinity TCRs that are beyond the detection limit of pMHC tetramers. To
overcome this limitation, pMHC dextramers have been recently developed to further increase the
valency between TCRs and pMHCs. Although the pMHC dextramer has been shown to exhibit a
higher sensitivity in detecting low-affinity T cells, it can lead to high background staining and
inaccurate determination of the total number of cognate T cells. In order to further enhance the
detection of low-affinity T cells, we aim to develop a novel pMHC derivative in which a
Y-shaped DNA is used to orchestrate pMHC nonamers. A potential advantage of utilizing a
Y-shaped DNA scaffold is that the number of and the distance between pMHCs can be easily
controlled, thereby providing an additional means to optimize the binding between pMHC
nonamers and the TCRs on the surface of T cells. So far, I have accomplished the expression,
purification, and modification of the various proteins and protein complexes required for the
V
generation of the Y-shaped-DNA based pMHC nonamers. First of all, I have expressed various
MHC complexes loaded with different peptides, including the human MHC class I molecule
HLA-A2, the mouse MHC class I molecule H-2K
b
, and the mouse MHC class II molecule I-E
k
. I
have also expressed the BirA biotin ligase, and utilized it to successfully add biotin moieties onto
pMHC complexes using an optimized protocol. Furthermore, I have expressed, refolded, and
purified streptavidin which will be used to strongly bind the nine pMHC complexes onto the
Y-shaped DNA scaffold. Once this pMHC nonamer design is completed, its detection sensitivity
for low-affinity T cells will be evaluated and compared with other forms of pMHC multimers. If
successful, the novel pMHC nonamer developed in this project can help enhance our
understanding of the functions of T cells in cancer and autoimmune diseases, and also provide
valuable information for vaccine development and disease targeting.
1
1. Introduction
1.1 Antigen recognition by T cells
The recognition of specific external peptides in the context of major histocompatibility
complex (MHC) molecules by T cell receptors (TCRs) is vital for activating the adaptive
immune response (Pancer and Cooper, 2006). The human body contains three primary forms of
defense against invading pathogens— the first is a physical barrier consisting of a layer of skin
epithelial cells, the second is the innate immune system which utilizes cells such as macrophages
and neutrophils to non-specifically eradicate pathogens, and the third is the adaptive immune
system which utilizes B and T cells to specifically target pathogens. If invading pathogens
succeed in crossing the physical skin barrier and in bypassing the innate immune cells, then the
adaptive immune system will be activated to eradicate the pathogen. Specifically, extracellular
pathogens are taken up by antigen-presenting cells (APCs), and the protein antigens are
processed into small peptides in endocytic compartments. Then a specific peptide antigen is
loaded onto the MHC class II molecule in late endosomes or lysosomes to form a peptide-MHC
class II (pMHC-II) complex. Similarly, intracellular antigens are processed into short peptides by
the ubiquitin-proteasome pathway, and the resulting peptides are transported into the
endoplasmic reticulum where they are further digested and then loaded onto MHC class I
molecules to form pMHC class I (pMHC-I) complexes. Once formed, the pMHC complexes
(both class I and II) are transported to the cell surface for recognition by TCRs on the surfaces of
2
T cells. (Leone et al., 2013) (Figure 1A). Once antigen-specific T cells recognize specific pMHC
complexes by their TCRs, these naï ve T cells will be activated to proliferate and differentiate into
effector T cells (Malissen and Bongrand, 2015). There are two main types of effector T cells:
cytotoxic T cells and helper T cells. Cytotoxic T cells will directly kill pathogen-infected cells
while helper T cells recruit and activate other cells of the immune system, such as B cells and
macrophages, to collectively target the invading pathogen.
1.2 Structure and function of MHC molecules
MHC molecules play an important role in T-cell activation by binding to peptide fragments
and presenting them on the cell surface for recognition by T cells. There are two different classes
of MHCs involved in antigen presentation: class I and class II. The class I and II MHCs have
similar structures but differ in subunit composition (Madden, 1995). Both these complexes are
composed of three domains: two Ig-like domains and a peptide-binding site constructed by one
α-helix/ β-sheet (αβ) domain. In MHC class I, the peptide-binding groove is only formed by the
heavy chain, and the α3 subunit of heavy chain associates with a light chain called
β-microglobulin (β2m). In contrast, the peptide-binding site for MHC class II molecule is
assembled by two heavy chains (Rudolph et al., 2006) (Figure 1B). In addition to their structural
differences, the MHC class I and II complexes differ in immune effector function. The MHC I
complexes activate the cytotoxic CD8+ T cells upon intracellular viral. On the other hand, MHC
II complexes activate the helper CD4+ T cells upon recognition of extracellular antigens.
3
A B
Figure 1. The pMHC- TCR interaction and the structure of MHC molecules
(A) This figure illustrates the interaction between pMHC (shown in purple, yellow, and red) and
TCR (shown in blue). (B) Comparison of the structures of MHC class I molecule and MHC class
II molecule (Modified from Kuby Immunology(Kindt et al., 2007))
4
1.3 T cell repertoire
A well-established immune system requires a highly diverse T cell repertoire to provide the
host with an efficient defense against virus and other pathogens (Laydon et al., 2015). The T cell
repertoire depends on the diversity and population of different TCRs. The huge diversity of
TCRs is generated by V(D)J recombination, a process which involves the random assembly of
different gene segments— the variable (V), diversity (D), and joining (J) genes— within the
variable region of the TCR. The TCR diversity is further enhanced when the host continues to
generate T cells against an unknown, evolving pathogen (Johnson et al., 2014). The overall
population of T cells is estimated as 10
12
, in which there are at least 10
7
different clonotypes
defined by their respective TCRs (Arstila et al., 1999). Maintenance of the T cell repertoire is
crucial to human health. Thus, identification of different antigen-specific T cell clonotypes can
enhance our understanding of T cell-mediated immunity, vaccine development, and some
diseases such as cancer (Laydon et al., 2015).
1.4 The importance of low-affinity T cells
The TCR–pMHC interaction has been reported to be much weaker than many other
protein-protein interactions. The dissociation constant (K
D
) is estimated to be between 1-100 μM,
and the half-life is about 0.5 -10 seconds (Xie et al., 2012). Interestingly, within this
physiological range, the TCR affinity for MHC complexes has been demonstrated to exhibit a
normal distribution (Hebeisen et al., 2013). Current hypotheses indicate that the T cells bearing
5
high affinity TCRs play a more important role in the immune system because these cells are
assumed to trigger a stronger and prolonged immune response than T cells bearing low affinity
TCRs. The low affinity T cells were thought to have a limited probability to initiate T cell
signaling (Klein et al., 2014)
However, evidence is emerging that highlights the importance of low-affinity TCRs (Martinez
and Evavold, 2015). In the naive T cell repertoire, the low-affinity T-cell population is more
prominent than that of the high-affinity T cells, indicating that the critical early-phase of the
immune response relies primarily on the low-affinity T cells (Zehn et al., 2009). Additionally, the
high-affinity T cells have been reported to initially divide faster than low-affinity T cells upon
activation, but the number of low-affinity T cells in the final population will reach the same
value as that of the higher-affinity T cells after several rounds of division. Therefore, the
lower-affinity T cells behave similarly to the higher-affinity T cells upon activation (Rosenthal et
al., 2012). It has also been shown that long-lived memory cells can be produced by lower-affinity
TCR-pMHC interactions (Baumgartner et al., 2012). Together, these findings demonstrate that
low-affinity T cells also play an essential role in mediating the adaptive immunity and in
maintaining homeostasis essential for human health.
1.5 The vital role of low-affinity T cells in cancer and autoimmune diseases
More importantly, the lower-affinity TCR plays a vital role in cancer and autoimmune
diseases. The immune response to viruses and bacteria mainly requires CD8+ T cells bearing
6
high-affinity TCRs, which recognize pathogen-derived (non-self) antigens. In contrast, more
evidence has demonstrated that self/tumor-specific T cells express low-affinity TCRs and are
capable of targeting and destroying cancer cells (Zhong et al., 2013). A potential explanation for
the prominence of low-affinity tumor-specific T cells is that most of the high affinity
self/tumor-antigen specific T cells are eliminated by both thymus negative selection and
peripheral tolerance mechanisms, while the cytotoxic CD8+ T cells with lower-affinity TCRs
can escape from this selection mechanism (Turner et al., 2008). Although it was assumed that
low affinity T cells do not proliferate and expand during immune system activation, it is also
observed in cancer patients that the self/tumor antigen specific T cells go through huge clonal
expansion and differentiate into memory and effector T cells which can last for many years in
high frequencies (Boon et al., 2006). Additionally, it has been verified quantitatively that TCRs
recognizing pathogen-derived antigens exhibit a much higher affinity (K
D
=0.18-0.25 M) than
TCRs recognizing tumor-associated antigens (K
D
=11-387 M) (Aleksic et al., 2012).
Moreover, many studies have revealed that T cell activation requires a given affinity threshold
for the pMHC-TCR interaction. T cells will not be triggered and induce downstream signaling if
the affinity between TCRs and pMHC is above the affinity threshold (Hebeisen et al., 2013).
Krogsgaard et al. assessed the TCR-affinity threshold for anti-tumor activity and autoimmunity
in vivo and found that the affinity threshold for maximal anti-tumoral activity and autoimmunity
is around 10 M. This data demonstrates that a low affinity threshold is required to prevent
7
autoimmune self-destruction (Zhong et al., 2013). Therefore, the detection and characterization
of low-affinity T cells is essential for future vaccine development and for targeting cancer and
autoimmune disease.
1.6 Detection of T cells by pMHC tetramers
The techniques to identify antigen-specific T cells have been very limited for many years due
to the low binding affinity between pMHCs and TCRs and the low frequency of antigen-specific
T cells in a mixed population (Novak et al., 1999). The old standard method for quantitatively
analyzing T cells is limiting dilution analysis (LTD), which can identify a certain cell type by its
response to an activation signal, such as release of cytokines. However, LTD will lead to a
significant underestimation of the number of T cells (Altman et al., 1996). The development of
pMHC tetramers has revolutionized the direct detection, quantification, and purification of
antigen-specific T cells by flow cytometry (Altman et al., 1996). Additionally, the pMHC
tetramers have enhanced the measurement of the kinetics of TCR binding (Choi et al., 2003).
The pMHC tetramer is generated by nucleating four biotinylated pMHC monomers with one
fluorescently-labeled streptavidin. Having four MHC molecules in one complex significantly
enhance the avidity of pMHCs to cognate T cells (Davis et al., 2011).
8
1.7 Further development of the pMHC tetramer staining method
The application of the pMHC tetramer staining method has recently been further expanded.
For example, a magnetized column has been utilized to conveniently purify antigen-specific T
cells if the pMHC tetramer is conjugated to a magnetic particle (Davis et al., 2011). Additionally,
the combination of pMHC tetramers with Cytometry by Time-Of-Flight Mass Spectrometry
(CyTOF) has allowed simultaneous measurement of up to 100 protein markers of
antigen-specific T cells. Another new technology (which will be utilized for this project) is the
ultraviolet (UV)-mediated peptide exchange technique. Specifically, a pMHC complex
consisting of an MHC and a UV-cleavable peptide is produced using the standard in vitro
refolding method. Upon UV irradiation, the bound peptide will be cleaved into two pieces and
subsequently be replaced with a new peptide (Rodenko et al., 2006). This method has allowed
the convenient production of hundreds of different pMHCs from one common pMHC reagent,
dramatically reducing the time and labor required in generating a large amount of different
pMHC class I tetramers.
1.8 Other forms of pMHC multimers
Although pMHC tetramers have been very successful in detecting many T cell subsets, they
still have limitations. One major limitation is that they show poor sensitivity to T cells specific
for self-derived peptides (e.g., tumor and autoimmune antigens), which often have lower affinity
than T cells recognizing viral antigens (Tungatt et al., 2015). This limitation has stimulated
9
scientists to develop other forms of MHC multimers, all based on the principle of further
improving the binding valency of pMHC complexes. For example, the pMHC pentamer from
Proimmune has demonstrated a better binding affinity than the pMHC tetramer (Davis et al.,
2011). In addition, the streptavidin-coated quantum dot was also applied on the pMHC tetramer
to further enhance the staining efficiency (Chattopadhyay et al., 2006). Recently, the pMHC
dextramer was developed, in which about 180 pMHC molecules are conjugated to a linear
polydextran backbone (Massilamany et al., 2015). Compared with the pMHC tetramer, the
pMHC dextramer has four to five fold higher detection sensitivity for T cells (Dolton et al.,
2014).
1.9 The limitation of current pMHC multimers
Most of the current pMHC multimers, including tetramers, pentamers, and quantum-dot based
multimers, still show a limited capacity to detect T cells of extremely low-affinity (Huang et al.,
2016). While the pMHC dextramer has allowed the staining of low-affinity T cells, it is
associated with two fundamental limitations. First, the pMHC dextramer is not economically
attractive and can result in high background staining. Second, the pMHC dextramer may bind to
more than one T cells simultaneously. Therefore, it is of critical importance to develop a new
technique that can efficiently detect low-affinity T cells.
10
1.10 The design of a novel pMHC nonamer
Here we propose to develop a novel pMHC nonamer based on a Y-shaped DNA scaffold,
which can be conveniently assembled, able to bind to nine TCRs simultaneously, yet still
relatively small in size and may have less background staining than the dextramer (Figure 2).
The self-assembly of DNA nanostructures, including the Y-shaped DNA and others, has
previously been widely used in the design of novel nanomaterials (Chhabra et al., 2010). We
speculate that the Y-shaped DNA would enable the arrangement of pMHC molecules in a
controllable manner, because the length of each arm can be easily adjusted (Rinker et al., 2008).
The structure of pMHC nonamer is highly stable because the pMHCs and the Y-shaped DNA
scaffold are connected by the streptavidin-biotin conjugation, one of the strongest non-covalent
bonds found in nature (K
D
=10
-14
M). Unlike the pMHC pentamer or dextramer, the pMHC
nonamer has a flat structure, enabling for all nine pMHCs to bind to TCRs at the same time. This
may enhance the signal/noise ratio in detecting low-affinity T cells. Shown in Figure 3 is the
technology roadmap for this research project.
11
Figure 2. Schematic illustration of pMHC nonamer
The Y-shaped DNA acts as a scaffold in the center. Three biotins (shown in green circles) are
attached to each arm of the Y-shaped DNA scaffold. Three streptavidin proteins (SAV, shown in
blue) can non-covalently bind to the three biotins on the DNA scaffold. Nine pMHC complexes
(shown in orange and grey) bind to the nine vacant binding pockets on the three streptavidin
protein to form the final pMHC nonamer.
12
Figure 3. Technology roadmap
13
2. Results and Discussion
2.1 Expression, in vitro refolding, and purification of pMHC complexes
In order to test the detection sensitivity of the pMHC nonamer for different subtypes of T cells,
different MHC molecules were expressed in this project, including H-2K
b
(a mouse class I
MHC), HLA-A2 (a human class I MHC), and I-E
k
(a mouse class II MHC). All of them were
expressed in the E.coli strain BL21 (DE3), and protein expression was induced by 1mM IPTG
when the OD600 value reached 0.6. The MHC molecules were refolded together with the
antigenic peptide by using the dilution refolding method. The correctly folded pMHC complexes
were obtained using gel filtration (Superdex 200 column). In addition, all MHC molecules
possess an Avi-tag at the C-terminus for the introduction of a biotin.
2.1.1 Expression, in vitro refolding and purification of peptide/H-2Kb complexes
H-2K
b
is a mouse MHC class I molecule that has been widely used in immunological research.
It consists of two chains: a heavy α-chain (34 kDa) and a light β2m chain (13.3 kDa). Both
chains were expressed as inclusion bodies in E. coli BL21 (DE3), and refolded with the OVA
(257-264) peptide (SIINFEKL) using the dilution refolding method (H-2K
b
: β2m molar ratio:
1:2). Fast protein liquid chromatography (FPLC) with gel filtration column (Superdex 200
column 10/300 GL) was used to purify the OV A/H-2K
b
complex. As shown in Figure 4A, two
main peaks were observed from the FPLC spectrum. Based on the calibration curve, the
14
calculated molecular weight of the protein in the first peak (15.32 ml) is about 38 kDa,
corresponding to the OVA/H-2K
b
complex which has a calculated molecular weight of 34 kDa.
The second peak (17.79 ml) has a molecular weight of 16 kDa, corresponding to the free β2M
chain. The identities of both proteins were further verified by the SDS-PAGE analysis.
Specifically, both the H-2K
b
α chain and β2M chain were observed in the first peak fraction, and
only the β2m chain was detected in the second peak fraction (Figure 4B).
In order to easily produce a large batch of H-2K
b
loaded with different peptides that can be
recognized by different T cells, the UV-cleavable peptide OV Auv (SIINFEXL, X is
3-amino-3-(2-nitrophenyl)propanoic acid (ANP)) was also used to form a complex with H-2K
b
.
The method for refolding the OVAuv/H-2K
b
complex was identical to that of OV A/H-2K
b
,
except that the protein needed to be protected from light strictly during the refolding and
concentration process. Similar to the previous refolding using the conventional OV A (257-264)
peptide, there were two peaks on the FPLC spectrum (Figure 3C). It was confirmed by
SDS-PAGE that the first peak was the pMHC complex and the second peak was the excessive
β2M chain alone (Figure 4D). Therefore, both OV A/H-2K
b
and OVAuv/H-2K
b
have been
successfully expressed. Both proteins were purified to homogeneity, and the yield from 1L of LB
medium was about 3 mg.
15
A
B
C
D
16
Figure 4. Purification of H-2Kb by Superdex 200 10/300 GL column
(A) Elution curve of OV A (257-264) in complex with H-2K
b
. The blue line represents the
absorption at 280 nm. Elution buffer: Phosphate Buffered Saline, pH 7.4 (PBS). (B) SDS-PAGE
analysis of fractions #29-#31 and #35-#37. Lanes #2 to #7 are the fractions #29, #30, #31 and
Lanes #3, #5, #7 are the fractions that heated at 95° C. Lanes #8 to #13 are the fractions #35 to #
37 and the lane #9, lane #11, lane #12 are the fractions that were heated at 95° C. (C) Elution
curve of H-2K
b
with the peptide SIINFEXL. The blue line stands for the A280 absorption.
Elution buffer: PBS. (D) SDS-PAGE analysis of fractions #28 to #35 (Elution volumes from 14.5
ml to 18 ml). Lanes #2 to #10 correspond to the fractions from #28 to #35. The first lane is the
protein marker.
17
2.1.2 Expression, in vitro refolding and purification of HLA-A2
In humans, the MHC is called human leukocyte antigen (HLA). Our study is focused on
peptides complexed with HLA-A2, one of the most common alleles of human class I MHC.
Similar to the mouse MHC class I H-2K
b
produced above, HLA-A2 consists of two chains: an
α-chain (A2, 34 kDa) and a β chain (β2m, 13.3 kDa). The two chains were expressed as inclusion
bodies in E. coli BL21 (DE3), separately. They were then used together with the UV-cleavable
derivative of HIVgag peptide (SLYNTV AXL, X = ANP) in an in vitro refolding reaction to
produce the HIVgag/HLA-A2 complex. In the refolding the A2 chain and the β2m chain were
used at a molar ratio of 1:2. The refolded product was purified using the Superdex S200 16/600
gel filtration column. As shown in Figure 5A, there are three peaks in the elution curve, and the
elution volumes are 74.48 ml, 83.80 ml and 94.19 ml, respectively. Based on the calibration
curve and calculation, the size of the protein eluted at 83.80 ml (i.e., the 2
nd
peak fraction) is
approximately 40 kDa, suggesting that it is the HIVgag/HLA-A2 complex. The protein was
further verified by SDS-PAGE analysis. On the protein gel, there were two bands located on 34
kDa (A2 chain) and 13 kDa (β chain) in the second peak. In addition, there are more aggregates
in the first peak, and the third peak is the excess of the β chain after refolding (Figure 5B).
Unexpectedly, there is also a strong band located between the A2 chain and the β chain. This is
likely either the degradation of the A2 chain or the dimer of the β chain. The assumption was
verified by adding the reducing agent β-ME to the fractions #20 to #23 (Second peak). The bands
disappeared after the addition of the reducing agent, indicating that it is the β chain dimer formed
18
during the refolding process but not the A2 chain degradation (Figure 5C). The prevention of
HLA-A2 β-chain dimerization is yet to be elucidated, but the dimerization has been found to not
affect the next experiments to be conducted with this complex.
19
A
B C
20
C
Figure 5. Purification of HLA-A2 by Superdex 200 16/600 column
(A) Superdex 200 16/600 elution curve of H-2Kb. The blue line represents the A280 absorption.
Elution buffer: 10 mM Tris-HCl pH 8.0, 75 mM NaCl. (B) 4%-12% Bis-Tris gel electrophoresis.
Lanes #1 to #11 correspond to the collections #19 to #29 from FPLC and Lane #12 is the protein
marker. (C) SDS-PAGE analysis of collections #20 to #23 (Second peak) after adding reducing
agent β-ME.
21
2.1.3 Expression, in vitro refolding and purification of I-Ek
I-E
K
is a mouse MHC class II molecule that presents a peptide antigen to CD4+ T cells. It is
formed by two chains that are similar in size— I-E
k
α chain (23.8 kDa) and β chain (22.9 kDa).
Both of the two chains were expressed in E. coli BL21 (DE3). The peptide that was chosen for
refolding is the MCC peptide. The Ni-NTA column was used to purify I-E
k
after refolding
because there is a His6 tag at the C-terminus of the I-E
k
α subunit.
However, no I-E
k
peptide-MHC complex was detected in the Superdex 200 column
chromatogram after refolding for the first time (Data not shown). The possible cause is that the
refolding condition did not allow I-E
K
to form proper disulfide bonds. In order to let I-E
k
form
disulfide bonds, the I-E
k
was denatured by guanidinium hydrochloride (GuHCl) and exposed to
air for 3 days at room temperature, allowing it to form random disulfide bonds. Then the I-E
K
complex was refolded and purified by the Ni-NTA column. This time, the I-E
k
subunits were
detected in the 4%-12% Bis-Tris gel (Figure 6A). However, we found many protein bands in the
upper part of the lanes, suggesting that I-E
k
might have aggregated during the refolding process.
The MCC/I-E
k
complex was further purified by the Superdex 200 10/300 GL column. Based on
the calculation, the peak that eluted at 14.79 ml should be the I-E
k
, and it was verified by a
4%-12% Bis-Tris gel. The protein aggregates were also observed from the FPLC and gel results
(Figure 6C). However, compared with H-2K
b
and HLA-A2, the yield of I-E
k
was significantly
lower. The refolding and purification methods should be further improved in future to increase
the yield of I-E
k
.
22
A
B
C
23
Figure 6. Purification of I-E
k
by Superdex 200 10/300 GL
(A) Superdex 200 column elution curve of I-E
k
. The blue line represents the A280 absorption.
Elution buffer: PBS. Blue line represents the A280 absorbance. (B) 4%-12% Bis-Tris gel
analysis of collection from Ni-NTA column. F.T represents the flow through after loading the
I-E
k
sample to column. W1-W3 are the collections after washing column by using wash buffer
(25 mM Tris-HCl pH 8.0). E1-E6 are the elution collections after adding elution buffer (1 M
Tris-HCl pH 8.0). (C) SDS-PAGE analysis of collection #13 to #35 (Second peak). M is the
protein marker.
24
2.2 Expression, in vitro refolding and purification of BirA
BirA is a biotin-protein ligase that adds biotin covalently to biotin-acceptor peptides/proteins
in a highly efficient and specific manner. In this project, BirA was used to attach biotin to MHC
molecules. The BirA construct used in our research has a GST tag at the N-terminus and a His6
tag at the C-terminus. It was expressed in a soluble form in E.coli BL21 (DE3), and purified by
the Ni-NTA column and the Superdex 200 16/600 column. The principle of the Ni-NTA based
metal affinity chromatography is that proteins containing the His6 tag, typically six consecutive
histidine residues, can efficiently bind to the immobilized metal ion matrices. By washing the
column matrix with free imidazole, the His6-tagged protein can be easily eluted (Bornhorst and
Falke, 2000). BirA was eluted out from the Ni-NTA column (Figure 7B), but still contained
many impurities as demonstrated by the presence of several bands on the protein gel. Therefore,
the protein was further purified by gel filtration. When purifying BirA with the Superdex 200
16/600 column, the protein was eluted directly by elution buffer (50 mM NaH
2
PO
4
, 300 mM
NaCl, 250 mM Imidazole). The second peak that eluted at 67.41 ml was BirA based on the
calibration curve (Figure 7A, 7C). However, there were still too many impurities that may affect
the activity of BirA. In order to further remove the impurities, a packed Ni-NTA column and a
gradient elution method using the AKTA system was then applied to purify BirA with gradient
elution (from 20 mM imidazole to 1 imidazole, in 13 min). By this way much more of the
25
impurities were successfully removed after gradient elution compared with the previous results
(Figure 7D, 7E).
26
A
B C
D
E
27
Figure 7. Purification of BirA by Ni-NTA column, Superdex 200 16/600 column
(A) The elution curve of BirA on the Superdex 200 16/600 column. The blue line represents the
A280 absorption. Elution buffer: PBS. (B) 4%-12% Bis-Tris gel analysis of collection from
Ni-NTA column. F.T. represents the flow through sample after loading the I-E
k
sample to the
column. W1 to W3 are the collections after washing column by using wash buffer (25 mM
Tris-HCl pH 8.0). E1 to E6 are the elution collections after adding elution buffer (1 M Tris-HCl
pH 8.0). (C) 4%-12% Bis-Tris gel analysis analysis of collections #13 to #35 (Second peak) (D)
Ni-NTA column elution curve of BirA when using gradient elution method. Blue line stands for
A280 absorbance. (E) 4%-12% Bis-Tris gel analysis analysis of collections from #4 to #14, M is
the protein marker.
28
2.3 Expression, in vitro refolding and purification of streptavidin
Streptavidin is a 52-kDa homotetramer formed by four 13-kDa subunits. Four biotins can be
attached to four binding pockets in the streptavidin with high affinity, which is one of the
strongest non-covalent bonds found in nature (K
D
=10
-14
M). The streptavidin monomer was
expressed first in E.coli BL21 (DE3) and refolded to tetramers by using the quick dilution
method. The refolded streptavidin was purified by the Q column, an anion exchange column that
can purify proteins containing a negative charge. The protein can bind to Q column in the low
salt-containing environment and be eluted out slowly by gradually increasing the salt
concentration in the elution buffer. Streptavidin carries negative charge in the binding buffer
(25mM Tris, pH 8.0) (pI=5.52) so that it can be separated by the Q column.
Streptavidin can be successfully separated by Q column using a gradient elution method
(From 25 mM Tris, 0 mM NaCl to 25 mM Tris, 1 M NaCl within 10 min at pH 8.0). There is
only one relatively sharp peak in the FPLC spectrum, and the fractions corresponding to this
peak was assessed by an 8%-12% Tris-Glycine gel (Figure 8A). As demonstrated on the
Tris-Glycine gel, highly pure streptavidin at 52 kDa was attained, and it dissociated into
streptavidin monomers (14 KDa) after heating for 10 min at 95 ° C (Figure 8B, lane #1 to lane
#6). As expected, the Q column showed a great capacity for removing most of the impurities
when compared with the sample before column purification (Figure 8B, lane #7).
29
A
B
Figure 8. Purification of streptavidin by Q column
(A) Q column elution curve for streptavidin using gradient elution method. Elution buffer was
gradually changed from 0 to 1 M NaCl in 10 min. (B) 8%-12% Tris-Glycine gel electrophoresis
of streptavidin. Lanes #1 to #6 correspond to fractions 5 to 7. Lanes #7 and #8 represent the
30
samples that were directly concentrated.
2.4 Biotinylation of MHC molecules
Biotinylation is the process in which proteins, nucleic acids or other molecules are attached
with a biotin covalently, either chemically of enzymatically (Hofmann et al., 1980). It is a fast,
specific reaction and typically does not affect the normal function of the protein. Biotinylation
has been widely used in biochemistry research. For example, proteins tagged with biotin can be
purified by affinity chromatography together with a column that has avidin or streptavidin. And
the biotin tag can be also utilized to detect the protein via anti-biotin antibodies. In this project,
biotin was attached to the C-terminus of MHC molecules by the BirA enzyme. All of the MHC
molecules that will be utilized for this project were tagged with an Avi-tag
(GLNDIFEAQKIEWHE). BirA will attach a biotin to the lysine residue within the Avi-tag in the
presence of biotin and ATP. So far, both H-2K
b
and HLA-A2 have been successfully biotinylated,
while I-E
k
will be biotinylated in future because the yield of I-E
k
still needs to be improved.
2.4.1 Biotinylation of H-2K
b
The first step for biotinylation is exchanging the buffer of the purified H-2K
b
from PBS to the
biotinylation reaction buffer (10 mM Tris-HCl, 75 mM NaCl). The H-2K
b
was then incubated
with Biomix A (10X concentration, 0.5 M bicine buffer PH8.3), Biomix B (10X concentration,
100 mM ATP, 100 mM Mg(OAc)
2
, 500 M D-biotin, PH 7.0), Bio200 (10X concentration, 500
M D-biotin) and BirA at 37 ℃ for 3 hours. After the reaction, the excess of the biotin and
31
small molecules were removed by the Zeba spin column (Thermo Fisher). In order to test the
efficiency of biotinylation, the biotinylated H-2K
b
was then incubated with streptavidin at room
temperature for 10 min, and both of the free biotinylated H-2K
b
and the biotinylated H-2K
b
conjugated with streptavidin were verified by SDS-PAGE. The method to evaluate the
biotinylation efficiency on the SDS-PAGE gel is to observe the band shift of biotinylated H-2K
b
on the gel. If the biotinylation works successfully, the bands of biotinylated H-2K
b
(34 kDa) will
shift to an upper location (~86kDa) after reacting with streptavidin.
Since our lab will need to produce many biotinylated pMHC complexes and the commercial
biotinylation kit from Avidity is quite expensive, we attempted to use our homemade BirA
enzymes and reaction buffers instead. For the first time, our home-made biotinylation buffers
(including BirA enzyme, Biomix A, Biomix B and Bio200) were used to conjugate biotin to
H-2K
b
. However, the efficiency of biotinylation did not work as expected. Compared with the
negative control (biotinylated H-2Kb alone), the band representing the biotinylated H-2K
b
did
not shift to the upper location in the gel (Figure 9A, lane#5), indicating that the biotinylation for
H-2K
b
was not successful so that it could not bind to streptavidin. The potential reason for this
failure was that there might be some problems with either the H-2K
b
substrate or the homemade
buffers.
Then, the commercial biotinylation kit from Avidity was used to biotinylate H-2K
b
in order
to test whether the H-2K
b
that we expressed can be biotinylated. Compare with our homemade
biotinylation kit, the Avidity Kit worked significantly better, and the efficiency of the
32
biotinylation is almost 100%. There was a clear band-shift for the biotinylated H-2Kb on the gel
when it bound to streptavidin (Figure 9A, lane #8). Therefore, the H-2Kb expressed was pure
and capable of biotinylation.
The quality of the three buffers – Bio 200, Biomix A, and Biomix B – was then evaluated. In
order to assess which homemade buffer hindered biotinylation, each buffer in the Avidity kit was
replaced with homemade buffer and tested for the biotinylation of the H-2Kb substrate. We
found that the biotinylation reaction worked well when we replaced the Bio 200 and Biomix A in
the Avidity kit with the homemade ones (Figure 9B, lane #3 and lane #4). Specifically, the
band-shifts on the gel for these samples were as strong as when we used all the buffers from the
original Avidity kit. On the contrary, there is no band-shift observed when the BiomixB buffer
was exchanged (Figure 9B, lane #6), indicating that Biomix B is the main factor that attenuated
the efficiency of biotinylation for H-2K
b
. We additionally found that there was also a slight band
left at 34 kDa (H-2K
b
α-chain) when we used the homemade BirA, suggesting that it was not as
efficient as the BirA in the Avidity Kit.
In order to find the difference between the homemade and the commercial Biomix B buffer,
we tested the pH of the two buffers. Surprisingly, the pH of the two buffers were significantly
different. The pH of Biomix B in the Avidity Kit was around 7 and the pH of the home-made
Biomix B was just 4-5. The biotinylation reaction for H-2K
b
was then re-attempted using the
new homemade Biomix B, from which the pH was adjusted to 7.0 using 1 M NaOH. Compared
with the old Biomix B, the new Biomix B (pH 7.0) demonstrated an increased efficiency in
33
H-2K
b
biotinylation (Figure 9C, lane #3 and lane #5). In addition, the band-shift of biotinylated
H-2K
b
was just as strong as when the Avidity Kit was completely utilized, indicating that the
biotinylation efficiency of the homemade kit was comparable to the Avidity Kit (Figure 9C, lane
#4 and lane #5).
From all these results, we found that three bands appeared when biotinylated H-2K
b
reacted
with streptavidin (from 80 kDa to 160 kDa). After heating at 95° C, these three bands disappeared,
and a new band appeared at 13 kDa, suggesting that all of the three bands are the H-2K
b
–
streptavidin complexes. Based on the molecular weight, we concluded that the top two bands
may represent streptavidin conjugated to 2 and 3 biotinylated H-2K
b
(~120 kDa and 162 kDa)
respectively, and the band at ~86 kDa may represent the streptavidin conjugated to only one
biotinylated H-2K
b
.
A
34
A
B
C
Figure 9. Exploration of optimal conditions for biotinylation of H-2K
b
35
(A) Comparison of biotinylation of H-2K
b
using the homemade and the Avidity commercial
biotinylation kit. The first lane is the protein marker. The second lane is the unbiotinylated H-2K
b
as a negative control. Lane #3 and lane #4 are the biotinylated H-2K
b
using the homemade and
the Avidity kits. Lanes #5, #6, #7 are the results after the biotinylated H-2K
b
reacted with
streptavidin when using the homemade buffers. Lane #8, #9, #10 are the results after the
biotinylated H-2K
b
reacted with streptavidin when using the buffers from the Avidity Kit. (B)
Biotinylation of H-2K
b
using the modified Avidity Kit. Lane #1 is the protein marker. Lane #2 is
the biotinylated H-2K
b
as a negative control. Lane #3 is the result after exchanging the Biomix A
in the Avidity Kit with the homemade Biomix A. Lane #4 is the result after exchanging the
Biomix B in the Avidity Kit with the homemade Biomix B. Lane #5 is the result after exchanging
the BirA in the Avidity Kit with the homemade BirA. Lane #6 is the result after exchanging the
Bio200 in Avidity Kit with the homemade Bio200. (C) Comparison of biotinylation of H-2K
b
using the old Biomix B (pH 4~5) and the new Biomix B (pH 7.0). Lane #1 is the protein marker.
Lane #2 is the biotinylated H-2K
b
as a negative control. Lane #3 is the result after using old
Biomix B (pH 4~5). Lane #4 is the result using all buffers from the Avidity Kit.
36
2.4.2 Biotinylation of HLA-A2
Since we have already established the optimal protocol for the biotinylation of H-2K
b
, the
HLA-A2 complex was next biotinylated using this standard method. The HLA-A2 was also
incubated with BirA and the rest of the biotinylation buffers at 37° C for 3 hours, and the
excessive biotin was removed by the centrifuge filter (Amicon, 10K). However, the biotinylation
for HLA-A2 did not work as efficiently as expected for the first time (Figure 9A). A potential
reason for this failure was that the enzymatic activity of the BirA decreased significantly during
the long period storage in the fridge. Therefore, the BirA was expressed again and utilized to
conduct the biotinylation. Compared with the first time, the biotinylation for HLA-A2 using the
fresh BirA was significantly enhanced. When evaluating the biotinylation results by SDS-PAGE,
it was observed that there was an increased level of biotinylated HLA-A2 complex which has
shifted higher on the gel upon interaction with streptavidin. This shift was more significant with
the addition of the newly-expressed BirA compared to the last biotinylation result (Figure 10C
and 10A). However, we found the biotinylation reaction was still not 100% efficient for HLA-A2
when using the fresh BirA (Figure 10A and 9C).
In an attempt to further enhance the biotinylation of HLA-A2, we tried to repeat the
biotinylation reaction for a total of two times. The first biotinylation reaction was conducted as
previously mentioned, and the second reaction—which involved adding more BirA and biotin
and incubating the reaction for an additional hour, was performed 10 hours after the first reaction.
The biotinylation efficiency, however, remained the same (Figure 10C and 10D). In another
37
attempt to improve the biotinylation of HLA-A2, the commercial biotinylation kit from Avidity
was utilized. Nevertheless, some of the H-2K
b
α-chain still did not shift upon interaction with
streptavidin, demonstrating that the biotinylation of HLA-A2 was still not 100% efficient (Figure
10B). As a result, it was hypothesized that the HLA-A2 itself might have some problems which
may have attenuated the biotinylation reaction. A potential problem is that the structural
conformation of HLA-A2, may occlude the Avi-tag, preventing access by the BirA enzyme for
biotinylation.
38
A B
C D
Figure 10. Biotinylation of HLA-A2
(A) Biotinylation of HLA-A2 using the homemade kit. The first lane is protein marker. Lane #2
is the biotinylated HLA-A2 alone. Lane #3 and #4 are the results after the biotinylated HLA-A2
39
interacts with streptavidin. The sample for Lane #4 was heated at 95° C for 10 min before loading
onto the gel. (B) Biotinylation of HLA-A2 using the commercial Avidity Kit. The first lane is the
protein marker. Lane #2 is the biotinylated HLA-A2 alone. Lane #3 and #4 are the results after
the biotinylated H-2K
b
interacts with streptavidin. The sample for Lane #4 was heated at 95° C
for 10 min before loading to onto the gel. (C) Biotinylation of H-2K
b
using homemade buffers
with fresh BirA enzyme. The first lane is protein marker. Lane #2 is the control. Lane #3 and #4
are the results after biotinylated H-2K
b
interacts with streptavidin. The sample for Lane #4 was
heated at 95° C for 10 minutes before loading onto the gel. (D) More BirA enzyme and biotin
were added into the biotinylation system 10 hours after the first reaction, and the system was
rotated at 37° C for another hour in order to complete the reaction. The first lane is the protein
marker. Lane #2 is the biotinylated H-2K
b
. Lane #3 and #4 are the results after the biotinylated
H-2K
b
interacts with streptavidin. The sample for Lane #4 was heated at 95° C for 10 minutes
before loading onto the gel.
40
2.5. Concluding remarks
pMHC tetramers have had a huge impact on the detection and characterization of low-affinity
T cells. Even though some new forms of pMHC multimers are continuously being developed
(Doherty, 2011), pMHC tetramers are still the most widely used for the following reasons: 1) the
pMHC tetramer is easy to construct and economical attractive, and 2) the pMHC tetramer
exhibits low non-specific binding. However, pMHC tetramers showed a limited detection
capacity when it was used to stain low-affinity T cells. The development of the pMHC dextramer
overcame this problem to some extent and showed better binding affinity to low-affinity T cells
(Dolton et al., 2014). Nevertheless, the drawbacks of the pMHC dextramer are also obvious. It is
difficult to make, costly, and exhibits high non-specific binding.
In order to further improve the detection of T cells, especially of low-affinity T cells which are
dominant in the T cell population in cancer and autoimmune diseases, we proposed to build a
novel pMHC nonamer. This pMHC multimer is designed based on a Y-shaped DNA scaffold,
which enables the display of nine pMHC complexes on a relatively stable and flat structure.
Therefore, the presence of these nine pMHCs will likely enhance the detection sensitivity of
low-affinity T cells compared to the pMHC tetramer not only because of its increased valency,
but also because the flat structure of this nonamer enables the presentation of all nine pMHCs at
the same time. So far, streptavidin has been successfully conjugated to a single oligonucleotide
(Tao Ma et al., unpublished), and the MHC complexes have been biotinylated. The next step
41
involves annealing the three streptavidin-oligonucleotide complexes together and labeling the
conjugate with fluorophores such as fluorescein. The final step entails loading of the biotinylated
pMHCs onto the nine vacant binding sites on the three streptavidin molecules displayed on the
Y-shaped DNA scaffold. The detection capacity of the pMHC nonamer will then be evaluated by
staining different T cell lines.
Originally, our plan was to build the pMHC nonamer by annealing the three oligonucleotides
first to generate the full Y-shaped DNA scaffold before conjugation to streptavidin. However, we
have revised our plan and adopted another method, that is, conjugating streptavidin with a single
oligonucleotide first and then annealing the three streptavidin-oligonucleotide complexes
together subsequently. The main reason for this change is that the streptavidin and the Y-shaped
DNA scaffold may crosslink to form aggregates if we used the original design (Figure 11).
42
Figure 11. Illustration of crosslink between streptavidin and Y shaped DNA scaffold
43
Even though we have obtained the class I pMHC complexes (including the mouse MHC class
I H-2K
b
and the human class I HLA-A2 complexes) with good yields, we found that there are
still impurities in the produced proteins, especially in the HLA-A2 product. The main impurity is
likely the β2m dimer. Previous studies have shown that the α chain and β2m chain of the MHC
class I molecule formed unwanted multimers and self-refolding products during the refolding
process, resulting in the low yield of the correctly refolded MHC class I molecules (Parker et al.,
1992). A potential reason for the incorrect refolding is the refolding condition, especially the
ratio of the redox reagents (including the reduced glutathione and the oxidized glutathione), is
still not optimal. Some strategies have been utilized to improve the expression system of human
MHC class I molecule, including changing vectors, the inducing reagents, and the refolding
conditions (Ferre et al., 2003; Piao et al., 2004). Recently, Ding Ren et al. found that the yield of
MHC class I molecule will be significantly increased and the unwanted multimers can be
avoided if the genes for the α and β2m chains were tandem-cloned into a plasmid and expressed
as a fusion protein in E. coli (Ren et al., 2006). This method will be examined in the future.
For the expression and refolding of peptide/I-E
k
complexes, the yield is currently too low to
carry on further experiments. Indeed, there is a consensus that the expression and refolding of the
MHC class II molecule is much more difficult than that of MHC class I molecule (Altman et al.,
1993). The reason for the difficulty probably results from the structural difference between the
MHC class I molecule and the MHC class II molecule. The peptide-binding groove for the MHC
44
class I molecule is formed only by the heavy chain, whereas the peptide-binding site for the
MHC class II molecule is formed by two chains, which causes more complexity. Many other
previous studies have attempted to refold the MHC class II molecule by dissolving the inclusion
bodies in a denaturing solution with reducing agents before dilution of the inclusion body into a
refolding buffer containing other redox reagents. However, all these attempts have resulted in a
low yield of the MHC class II molecule. We also used this refolding method to refold MCC–I-E
k
for the first time, but little to no I-Eᵏ was attained. It has been reported that the pre-oxidized
MHC class II molecule refolded more efficiently than the fully reduced MHC class II molecule
(Justesen et al., 2009). This observation may explain why I-E
k
was successfully refolded for the
second time when the denatured MHC class II complex was pre-exposed to the air for several
days. However, exploring the optimal conditions to maintain the oxidation status of the MHC
class II molecule is necessary for increasing its yield.
In addition to detecting low-affinity T cells, the pMHC nonamer may also be potentially
valuable in other aspects. For example, researchers recently have started to apply the pMHC
tetramer in detecting antigen-specific T cells in situ. Instead of staining the cells isolated from
blood or semen, staining the T cells in situ will help determine the T cell location in tissues and
provide an enhanced understanding of the cellular biology of T cell responses in vivo. Pamela J.
Skinner et al. developed a technique—which involves generating the MHC class I tetramer with
FITC and then amplifying the tetramer signal using rabbit anti-FITC antibody—to detect the T
45
cells in the spleen section in the 2C TCR transgenic mouse. However, there was a concern that
the signal amplification from the anti-FITC antibody can potentially cause background staining.
Then, it is reported that MHC class II dextramers can be successfully applied to detect
auto-reactive CD4 T cells with a great specificity and sensitivity in the brain and heart in situ.
Their strategy does not require the need to amplify the signals with fluorophore antibodies. All of
the studies mentioned above demonstrate that the pMHC multimer can be successfully used in
staining of T cells in situ. The advantageous design of the pMHC nonamer compared to the
dextramer—especially its relatively small size—can potentially enable this complex to achieve
an enhanced signal-to-noise ratio in the in situ staining of antigen-specific T cells. Therefore, we
believe that the pMHC nonamer will exhibit enhanced in situ staining.
In summary, our preliminary data in the development of the Y-shaped DNA based pMHC
nonamers is promising. If successful, we believe that the pMHC nonamer can provide a powerful
new method to detect low-affinity T cells in cancer and autoimmune diseases, and help the
development of new immunotherapies to these diseases.
2.6. Future work
So far, the biotinylated peptide-MHC class I complexes have been produced. The wild-type
streptavidin has also been successfully prepared and conjugated to each of the oligonucleotides
in a 1:1 ratio. My colleagues, Ting Fu and Tao Ma, have successfully produced and purified these
novel streptavidin-DNA complexes. The next step for this project is to anneal the three
46
streptavidin-oligonucleotide complexes together to form the Y-shaped DNA scaffold. Fluorescein
or other fluorophores will then be conjugated to the complex. Subsequently, nine biotinylated
pMHC complexes will be loaded onto the nine vacant binding pockets in the three streptavidins.
Once the final pMHC nonamer is obtained, the evaluation of its detection capacity then can be
conducted using mouse or human T cells.
Different T cell lines that recognize different peptide antigens will be adopted in this project to
test the detection sensitivity of pMHC nonamer, including both mouse and human blood samples.
MHC nonamers loaded with different kinds of peptides will be produced using the UV-mediated
peptide exchange technology, and flow cytometry will be utilized to visualize and quantify T
cells specific for these pMHCs. This detection capacity will be compared to those of other
pMHC multimers, including the pMHC tetramer and dextramer. The expected result is that
pMHC nonamer will show enhanced detection sensitivity compared to other forms of pMHC
multimers and may also have less background staining due to its relative compact structure
compared to that of the pMHC dextramer.
In addition, if these results look promising, some other improvements for the pMHC nonamer
can be further studied. The important advantage of a DNA scaffold is the flexible length of each
arm. Therefore, in order to potentially enhance the detection capacity of the pMHC nonamers,
pMHC nonamers with varying DNA-arm lengths will be generated, and the optimal arm length
for detecting low-affinity T cells will be determined.
47
The final goal for this project is to successfully detect and separate the low-affinity T cells in
cancer and autoimmune disease by using this novel pMHC nonamer. The separation of these
low-affinity T cells can help enhance our understanding of these diseases and generate new
insight for vaccine and immunotherapy development.
48
3. Methods and Materials
3.1 Expression, in vitro refolding, and purification of MHC molecules
3.1.1 MHCs sequence
The amino acid sequence of H-2K
b
and HLA-A2 are aligned as below.
There is an Avi-tag added to the C-terminal of both H-2K
b
and HLA-A2 in order to conjugate
biotin to H-2K
b
and HLA-A2.
49
The amino acid sequence of I-E
k
α chain and β chain are showed below:
N’-MIKEEHTIIQAEFYLLPDKRGEFMFDFDGDEIFHVDIEKSETIWRLEEFAKFASFEAQGAL
ANIAVDKANLDVMKERSNNTPDANVAPEVTVLSRSPVNLGEPNILICFIDKFSPPVVNVTWL
RNGRPVTEGVSETVFLPRDDHLFRKFHYLTFLPSTDDFYDCEVDHWGLEEPLRKHWEFEEK
TLLEFGGLNDIFEAQKIEWHEGTGLEVLFQ GPGTSHHHHHHHHHHHH -C’
N’-MASLVRDSRPWFLEYCKSECHFYNGTQRVRLLVRYFYNLEENLRFDSDVGEFRAVTELG
RPDAENWNSQPEFLEQKRAEVDTVCRHNYEIFDNFLVPRRVEPTVTVYPTKTQPLEHHNLLV
CSVSDFYPGNIEVRWFRNGKEEKTGIVSTGLVRNGDWTFQTLVMLETVPQSGEVYTCQVEH
PSLTDPVTVEWKAQST-C’
A His-Tag was added to the C-terminal of I-E
k
in order to purify it with Ni-NTA column.
The amino acid sequence of BirA:
N’-MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYS
LPEPIQLLNAEQILGQLDGGSVTVLPVIDSTNQYLLDRIGELKSGDACVAEYQQAGRGRRGR
KWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLR KLGADKVRVKWPNDLYLQDR
KLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGWITLQEAGINLDRNTLAAMLIR
ELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGIIKP
WMGGEISLRSAEK-C’
A His-tag was added to the C-terminal of BirA sequence in order to purify it with Ni-NTA
column.
3.1.2 Expression
A colony was picked and incubated in 5 ml Luria-Bertani (LB) broth at 37 ° C, 225 RPM
(Revolutions per minute) for 8 hrs. 25 l bacteria from the small culture was next transferred to a
50
50 mL medium culture and incubated at 28 ° C, 225 rpm overnight. A 10 ml bacteria from the
medium culture were then transferred to 1 L LB broth. The protein expression was induced by 1
mM IPTG when the OD600 value reached 0.6. The protein expression proceeded at 37° C for 4
hrs. The bacteria were then collected and transferred into 5 ml lysis buffer (50 mM NaH
2
PO
4,
300 mM NaCl, 20 mM Imidazole) plus 5 mM DTT, 5 mg lysozyme, 50 M MgCl
2,
50 g/ml
Dnase 1, 0.5 g/ml leupeptin and 5 g/ml pepstatin. The cell pellet was kept at RT for 1 hour
before sonication. The bacteria were lysed by an ultrasonic liquid processor (Misonix) (Pulse on:
10s; Pulse off: 20s) and the inclusion body was obtained after removing the supernatant. The
inclusion body was then washed by detergent buffer (1% sodium deoxycholate, 1% NP-40, 50
mM Tris pH8.0, 200 mM NaCl, 1 mM EDTA) 2 times, wash buffer #1(5% Triton X-100, 50 mM
Tris pH 8.0, 100 mM NaCl, 0.1% sodium azide, 1 mM EDTA) 3 times, and wash buffer #2(50
mM Tris pH8.0, 100 mM NaCl, 0.1% azide, 1 mM EDTA ) 3 times to remove all the impurities.
For every wash, the inclusion body was intensely vortexed in wash buffers and then centrifuged
at 1300 rpm for 10 minutes to remove the supernatant.
3.1.3 In vitro refolding
Since MHC molecules are expressed as insoluble proteins, proper in vitro refolding methods
are needed to refold MHC chains into the correct conformation. For MHC molecule refolding,
the dilution method was adopted. The inclusion bodies of the MHC α and β chains were first
denatured by 5 ml denature buffer (10M urea, 62.5 mM MES pH6.5, 0.125 mM EDTA, 0.125
51
mM DTT) and filtered using a 0.45 μm syringe filter (ACRODISC
®
) to remove the impurities
that cannot be dissolved. Then the concentration was measured by Nanodrop 2000 (Thermo
Scientific), and 1 mol α chain and 2 mol β chain were diluted into injection buffer (3M
guanidine-HCl, 10 mM sodium acetate, pH 4.2, 10 mM EDTA) and utilized for refolding the
MHC complex. 1 L refolding buffer (400 mM L-arginine, 100 mM Tris pH 8.0, 2 mM EDTA, 5
mM reduced glutathione, 0.5 mM oxidized glutathione, 0.5 mM PMSF, 1ug/ml leupeptin, 1ug/ml
pepstatin) was prepared and precooled in the 4 ° C fridge. 10 mg peptide was dissolved in DMSO
and injected to refolding buffer firstly. Then the α and β chains were then injected into the
refolding buffer at a very slow speed while the refolding buffer was stirring at a considerably
high speed. The second injection of the MHC α and β chains was conducted the next morning
using the same amount of inclusion body. After the injection, the stirring speed was adjusted to a
very slow speed and the refolding lasted for an additional 2 days.
3.1.4 Purification
The refolding buffer for the MHC was filtered by both a filter paper and a 0.45 μm syringe
filter (ACRODISC
®)
. The protein was then concentrated from 1 L to 50 ml by using a tangential
flow device (ULTRASETTE
TM
) before further concentration of the protein from 50 ml to ~2 ml
by using the 50mL Vivaspin centrifuge concentrators (GE Healthcare). Afterwards, the protein
was purified by gel filtration using the Superdex 200 10-300GL column (GE Healthcare). All
columns utilized for MHC protein purification were controlled by the AKTA FPLC (GE
52
Healthcare), utilizing phosphate buffered saline (PBS) as the running buffer. SDS-PAGE
(Bis-Tris gel, Thermo Fisher Scientific) was then used to confirm the quality of the proteins.
After confirmation, the protein was collected, concentrated, and mixed with 50% glycerol final
concentration before storage in the -20 ° C freezer.
3.2 Expression, in vitro refolding, and purification of the BirA enzyme
Since the BirA enzyme is a soluble protein, refolding is not required for BirA preparation. The
procedure for BirA expression is exactly the same as that of MHC molecules. For BirA
purification, the Ni-NTA column (GE healthcare) was used because there is a His6-tag at the
C-terminus of BirA. After loading the protein sample onto the Ni-NTA column, the column was
washed by wash buffer (50 mM NaH
2
PO
4
, 300 mM NaCl, 20 mM Imidazole) and elution buffer
(50 mM NaH
2
PO
4
, 300 mM NaCl, 250 mM Imidazole). The eluates were collected and further
purified by Fast Protein liquid chromatography with the Superdex 200 column. The gradient
elution method is adopted when there are impurities in the sample after Ni-NTA purification. For
gradient elution (From 20 mM imidazole to 1 M imidazole, 13 min), packed Ni-NTA column
(GE Healthcare) and the AKTA system was used. The protein was next concentrated and mixed
with 50% glycerol before storage in the -20 ° C freezer for future use.
53
3.3 Expression, in vitro refolding, and purification of streptavidin
Since streptavidin is also expressed in E.coli as an inclusion body, the procedure for MHC
expression was also applied to streptavidin. For the refolding process, the dilution method was
also utilized, and the refolding buffer for streptavidin conatins 50 mM Tris pH 8.0, 150 mM
NaCl, and 2 mM EDTA. The Q column was applied to purify streptavidin. After binding to the
column (25 mM Tris-HCl, 100 mM NaCl), streptavidin was eluted out by gradient elution from
25 mM Tris-HCl pH 8.0 to 1M Tris-HCl pH 8.0 within 30 min. The Tris-glycine SDS-PAGE gel
was used to test the quality of streptavidin.
3.4 Biotinylation of MHC molecules
Both biotinylation substrates—the MHC molecules—and BirA enzyme were prepared as
mentioned above. The buffers are made according to the following recipe: Biomix A (10X
concentration, 0.5 M bicine buffer PH8.3) Biomix B (100 mM ATP, 100 mM Mg(OAc)
2
, 500uM
d-biotin PH 7.0) and Bio200 (500 uM d-biotin). When every component is ready, the MHC
buffer was exchanged into biotinylation reaction buffer (10 mM Tris-HCl, 75 mM NaCl). The
concentration of MHC molecules was then diluted to 60 mM, which is recommended by the
Avidity kit. Next, the Biomix A (10X concentration), Biomix B (10X concentration) and Bio200
(10X concentration) were added to the reaction system. As for BirA, for every 10 nmol of
substrate, 2.5 ug is required for complete biotinylation. The whole system was then rotated at
37 ° C for 3 hrs. The excess biotin was removed afterwards either by the centrifuge filter
54
(Amicon), Zeba spin column, or FPLC.
The efficiency of biotinylation was then evaluated by SDS-PAGE analysis. 5 ug of
biotinylated MHC was used to react with 5 times amount of commercial streptavidin (AnaSpec.
Inc) at RT for 10 minutes. The band-shift of biotinylated MHC molecules will be observed if the
biotinylation works successfully.
55
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Abstract (if available)
Abstract
The detection, quantification, and characterization of antigen-specific T cells from a mixed population have been limited for many years because of the low affinity and short disassociation half-life between T cell receptors (TCR) and peptides bound to major histocompatibility complex (MHC) molecules. This problem has been overcome by the development of peptide-MHC (pMHC) tetramers and other forms of multimers, which are able to bind to multiple TCRs simultaneously and thus have significantly enhanced avidity to the latter. However, it has been reported that some types of T cells, such as cancerous T cells and auto-reactive T cells, can express extremely low-affinity TCRs that are beyond the detection limit of pMHC tetramers. To overcome this limitation, pMHC dextramers have been recently developed to further increase the valency between TCRs and pMHCs. Although the pMHC dextramer has been shown to exhibit a higher sensitivity in detecting low-affinity T cells, it can lead to high background staining and inaccurate determination of the total number of cognate T cells. In order to further enhance the detection of low-affinity T cells, we aim to develop a novel pMHC derivative in which a Y-shaped DNA is used to orchestrate pMHC nonamers. A potential advantage of utilizing a Y-shaped DNA scaffold is that the number of and the distance between pMHCs can be easily controlled, thereby providing an additional means to optimize the binding between pMHC nonamers and the TCRs on the surface of T cells. So far, I have accomplished the expression, purification, and modification of the various proteins and protein complexes required for the generation of the Y-shaped-DNA based pMHC nonamers. First of all, I have expressed various MHC complexes loaded with different peptides, including the human MHC class I molecule HLA-A2, the mouse MHC class I molecule H-2Kᵇ, and the mouse MHC class II molecule I-Eᵏ. I have also expressed the BirA biotin ligase, and utilized it to successfully add biotin moieties onto pMHC complexes using an optimized protocol. Furthermore, I have expressed, refolded, and purified streptavidin which will be used to strongly bind the nine pMHC complexes onto the Y-shaped DNA scaffold. Once this pMHC nonamer design is completed, its detection sensitivity for low-affinity T cells will be evaluated and compared with other forms of pMHC multimers. If successful, the novel pMHC nonamer developed in this project can help enhance our understanding of the functions of T cells in cancer and autoimmune diseases, and also provide valuable information for vaccine development and disease targeting.
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Asset Metadata
Creator
Wang, Ze
(author)
Core Title
Y-shaped DNA based pMHC nonamers for detecting low-affinity T cells
School
School of Pharmacy
Degree
Master of Science
Degree Program
Pharmaceutical Sciences
Publication Date
07/28/2016
Defense Date
06/24/2016
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low affinity T cells,OAI-PMH Harvest,pMHC multimer,T cell staining
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Xie, Jianming (
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), Okamoto, Curtis T. (
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), Stiles, Bangyan L. (
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wangze@usc.edu,wangze19910316@gmail.com
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low affinity T cells
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