Transcript The Journal of Immunology, 2010

The Journal of Immunology, 2010
GILT Accelerates Autoimmunity to the Melanoma
Antigen Tyrosinase-Related Protein 1
Xu Zhenjie
2011,08,31
1. Background
GILT ( Gamma-IFN inducible lysosomal thiol reductase) is
expressed in APCs, where it localizes to MHC class II-loading
compartments.
GILT can facilitates the generation of MHC class II-restricted
epitopes from disulfide bond containing Ags.
Melanocyte differentiation Ags are melanosomal integral membrane
proteins involved in melanin pigment synthesis. These Ags contain a
dileucine-based sorting signal that targets them to the endosomal
system where they can be processed for MHC class II-restricted
presentation.
However, it is not known how GILT would influence the development
of immune responses to these Ags in vivo.
2. Materials
95-10: an I-Ab-restricted T cell hybridoma recognizing
murine TRP1
Cell lines
B16.F10: Mel (Melanoma line)
PVD: SCC (squamous cell carcinoma) line
C57BL/6 : wild-type mice
GILT-/- mice
mice
TRP1tg mice
TRP1Bw RAG-/- TRP1tg mice
3. Methods and results
GILT is required for efficient MHC class II-restricted processing
of a TRP1 epitope in vitro.
GILT accelerates the onset of vitiligo in TRP1-specific TCR
transgenic mice.
Increased percentage of TRP1-specific transgenic T cells in GILTdeficient mice.
Effector memory cells are increased in the presence of GILT and
following development of vitiligo.
GILT facilitated class II-restricted processing of TRP1, thereby
enhancing T cell activation and accelerating vitiligo.
3.1. GILT is required for efficient MHC class II-restricted processing
of TRP1
B16 cells
Wild-type mice
freeze-thaw
splenic B cells
Stained by
CD45R and I-Ab
lysates
Immunoblotted
with anti-TRP1
mAb
control
GILT-/- mice
Flow
cytometry
Coculture
with B16 cells
lysates and
95-10 cells
Measuring IL-2
production by ELISA
Positive control,
TRP1 peptide
Negative control,
SCC lysates
There was a statistically significant difference between the ability of
wild-type and GILT-/- B cells to present the TRP1 epitope.
Wild-type mice
GILT-/- mice
DCs
Flow
cytometry
Stained by CD11c and I-Ab
Coculture with B16
cells lysates and
95-10 cells
Measuring IL-2
production by ELISA
control
There was a statistically significant difference between the ability of
wild-type and GILT-/- DCs to present the TRP1 epitope.
3. 2. GILT accelerates the onset of vitiligo mediated by TRP1-specific
CD4+ T cells
3.2.1 To determine whether GILT would impact the development
of a TRP1-specific autoimmune response in vivo,
3.2.2 To demonstrate that GILT in APCs is necessary for the
accelerated onset of vitiligo,
Wild type (n=127)
TRP1tg mice
GILT+/- (n = 56)
GILT-/- (n =76)
A
A
Transfer
CD4+
into
TRP1specific
T cells
RAG-/- mice
GILT-/- RAG-/mice
GILT in APCs increases the severity and accelerates the onset of vitiligo.
3. 3. Increased percentage of TRP1-specific transgenic T cells in GILTdeficient mice
CD4+ TRP1specific T cells
Transfer into
Total cell numbers
isolated from thymi,
spleens, and lymph
nodes of the GILTpositive and -negative
strains were identical.
CD4:CD8 T cell ratio were
observed in the thymus,
spleen, or lymph node.
RAG-/- mice
GILT-/- RAG-/- mice
Transgenic T cells were identified by
Vβ14 and CD4 staining.
The percentage of
transgenic T cells
in GILT-/-TRP1tg
mice increased.
To exclude the absence of GILT in T cells as
the cause of increased T cells, we evaluated
the percentage of nontransgenic T cells.
?
TCR was expressed in
both GILT-/-TRP1tg
mice and TRP1tg mice.
The influence of T regulatory (Treg)
cell was exclude.
The expansion of T cells was limited to the TRP1-specific CD4+Vb14+ T
cells.
3.4 Effector memory cells are increased in the presence of GILT and
following development of vitiligo.
GILT-/-TRP1tg mice
and TRP1tg mice
CD4+Vβ14+ T cells
CD62L-CD44+ (effector memory)
CD62L+CD44+ (central memory)
CD62L+CD44- (naive)
GILT facilitated class II-restricted processing of TRP1, thereby
enhancing T cell activation and accelerating vitiligo.