Transcript Polymorphisms in CD1a and susceptibility to mTb

Polymorphisms in CD1a and
susceptibility to mTb
Emily Ford
5/29/2012
Acknowledgments:
• Thomas Hawn
Chetan Seshadri
Meera Shenoy
Glenna Peterson
Rick Wells
NA 1/34
McMichael AJ, JR Pilch, G Galfre, DY Mason, JW Fabre, C Milstein. 1979. Eur J Immunol 9 (3):
205-210. A human thymocyte antigen defined by a hybrid myeloma monoclonal antibody.
1984 – Nobel Prize - Niels K. Jerne, Georges J.F. Kohler, Cesar Milstein
“The dissection
of the immune
response by the
hybridoma
technique”
Milstein C. Nobel prize lecture. 1984.
Cluster of Differentiation (CD)
Cluster analysis
of the original 139 human antibody/antigen complexes
Bernard, A and L Boumsell (1984). Human Immunology 11: 1-10.
IUIS-WHO Nomenclature Subcommittee (1984). Bull WHO 62 (5): 809-811.
CD1
CD1D
•
•
•
•
CD1A
CD1C
CD1B
CD1E
Sequence lies on chromosome 1
Significant sequence homology with MHC-I
Associates with beta 2-microglobulin
Expressed on antigen presenting cells (APCs)
Porcelli S, MB Brenner et al. (1989). Nature 341 (6241): 447-450.
Brigl M and MB Brenner. (2004). Annu Rev Immunol 22: 817-890.
MOLT-4
(CD1)
IDP2
IDP2
IDP2
IDP2
IDP2
Brenner MB, J McLean et al. (1987.) Nature 325: 389-394
CD1-expressing cells can be recognized and lysed by specific CTLs
IDP2
BK6
CD1-transfected cells
Cytotoxic
CD4-8- T cells
CD1a
CD1c
Porcelli S, MB Brenner et al. (1989). Nature 341 (6241): 447-450.
CTLs will respond to MTb in the
presence of CD1
DN1
DN1
DN1
?
APC
DN1
Porcelli, S, CT Morita, MB Brenner. (1992). Nature 360: 593-597
CD1 presents lipid
antigens
Beckman EM, SA Porcelli et al. 1994. Nature 372: 691-4
CD8+ CD1-responsive T cells can cause
intracellular lysis of mycobacteria
Stenger S, RJ Mazzaccaro et al. 1997. Science 276: 1684-1687.
CD1 family structure
Van Rhijn I, DM Zajonc et al. (2005). Curr Opin Immunol 17(3): 22-29.
Overview and homology w/ known MHC presenting processes
Sugita, M, DB Moody et al. 1998. Clinical Immunology and Immunopathology 87 (1): 8-14.
Tb: How is it so good at getting
around our immune system?
Are SNPs in CD1a Associated with Expression?
rs1230716
rs389293
rs440419
rs2269714
rs411089
158,228,059
rs858998
rs3181029
Chromosome 1
158,223,927
Untranslated region
Coding region
• Experimental Method
– Collect blood from healthy individuals in Seattle (N=21)
– Genotype for CD1a polymorphisms
– Generate dendritic cells and analyze surface expression of CD1a
Peripheral Blood
Mononuclear Cells
C Seshadri, not yet published
CD1Monocytes
GM-CSF
IL-4
CD1+
dendritic cells
Distribution of CD1a expression in Seattle cohort
CD1a
Donor 3
Donor 4
Donor 5
C Seshadri, not yet published
CD1c
CD1A Expression Stratified by SNP
Genotypic
Recessive
Genotypic
Recessive
p = 0.764
p = 0.472
p = 0.02
p = 0.006
C Seshadri, not yet published
Genotypic Test = Kruskal-Wallis rank test
Recessive Test = Wilcoxon rank sums test
Is CD1a-deficiency Associated with Tuberculosis?
• Combined Cohort Analysis
– 762 Cord Blood Controls
– 385 Pulmonary TB
– 307 Meningeal TB
SNP
Cohort
Genotypic Analysis
TT
CT
CC
Recessive Analysis
P value
TT+CT
CC
699(92%)
59(8%)
OR
P value
rs858998
control
372(49%)
327(43%)
59(8%)
T/C
TB all
337(49%)
271(39%)
82(12%)
0.023
608(88%)
82(12%)
1.60
0.009
pTB
188(49%)
149(39%)
47(12%)
0.037
337(88%)
47(12%)
1.65
0.014
Hypothesis:
differences in the promoter region of
CD1a may affect its expression
Chromosome 1 – 5000kb
Promoter
Intron Exon
1. Make plasmids containing CD1a variants
CD1a “HI”
1kb
PGL4
CD1a “LO”
1kb
2. Try to get it into a cell
Lipofection
cell
Electroporation
cell
Results
The end!
• If there’s time – mysterious CD1d
• How did they move into dendritic cells?
• They first had to make their own CD1 expressing
cells, then technology developed
• Ralph Steinman – PNAS from 1980s?
– Dendritic cells were very hard to study! (rare, difficult
to isolate)
– Wesley Van Voorhees was his phd student
– GM-CSF IL-4 method to make dendritic cells (legend of
figure 1) Nature paper
NA 1/34
Human thymocytes
NA 1/34
Human thymocytes
HTA1/CD1
Other T-cell types also recognize CD1
• Reminder of T cell subsets
CD4+
CD4-8-
CD8+