Polymorphisms in CD1a and susceptibility to mTb

Emily Ford5/29/2012

Acknowledgments:

• Thomas Hawn Chetan SeshadriMeera ShenoyGlenna PetersonRick Wells

NA 1/34McMichael 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 analysisof 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

• 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.

CD1D CD1A CD1C CD1B CD1E

MOLT-4(CD1)

IDP2 IDP2

IDP2

IDP2

IDP2

Brenner MB, J McLean et al. (1987.) Nature 325: 389-394

CytotoxicCD4-8- T cells

CD1-transfected cells

CD1cPorcelli S, MB Brenner et al. (1989). Nature 341 (6241): 447-450.

IDP2

BK6

CD1-expressing cells can be recognized and lysed by specific CTLs

CD1a

CTLs will respond to MTb in the presence of CD1

Porcelli, S, CT Morita, MB Brenner. (1992). Nature 360: 593-597

APC

DN1

DN1

DN1

DN1

?

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.

Sugita, M, DB Moody et al. 1998. Clinical Immunology and Immunopathology 87 (1): 8-14.

Overview and homology w/ known MHC presenting processes

Tb: How is it so good at getting around our immune system?

Coding region

Untranslated region

Are SNPs in CD1a Associated with Expression?

• Experimental Method– Collect blood from healthy individuals in Seattle (N=21)– Genotype for CD1a polymorphisms– Generate dendritic cells and analyze surface expression of CD1a

rs31

8102

9

rs85

8998

rs41

1089

rs22

6971

4

rs44

0419

rs38

9293

rs12

3071

6

Chromosome 1158,223,927 158,228,059

Peripheral BloodMononuclear Cells

CD1- Monocytes

CD1+

dendritic cellsGM-CSFIL-4

C Seshadri, not yet published

Distribution of CD1a expression in Seattle cohort

Donor 3

Donor 4

Donor 5

CD1a CD1c

C Seshadri, not yet published

CD1A Expression Stratified by SNP

Genotypic Recessive

p = 0.02 p = 0.006

Genotypic Recessive

p = 0.764 p = 0.472

Genotypic Test = Kruskal-Wallis rank testRecessive Test = Wilcoxon rank sums testC Seshadri, not yet published

Is CD1a-deficiency Associated with Tuberculosis?

• Combined Cohort Analysis– 762 Cord Blood Controls– 385 Pulmonary TB– 307 Meningeal TB

SNP Cohort Genotypic Analysis Recessive Analysis

    TT CT CC P value TT+CT CC OR P value

rs858998 control 372(49%) 327(43%) 59(8%)   699(92%) 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

Promoter Intron Exon

Chromosome 1 – 5000kb

PGL4

1. Make plasmids containing CD1a variants

CD1a “HI”

CD1a “LO”

1kb

1kb

2. Try to get it into a cell

Lipofection Electroporation

cellcell

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

Human thymocytesNA 1/34

NA 1/34

HTA1/CD1

Human thymocytes

Other T-cell types also recognize CD1

• Reminder of T cell subsets

CD4-8-

CD4+

CD8+