Summer Undergraduate Research Program - Fred Hutchinson ...

Analyzing the Antibody Response against H-Y Antigens
Nathan Ord1 ; Takazu Kawase, M.D., Ph.D2 ; Edus Warren, M.D., Ph.D. 2
1 Whitman College, Walla Walla, WA; 2 Fred Hutchinson Cancer Research Center, Seattle, WA
Results
Abstract
Background
2011 Lee Hartwell Poster Award
XY
Female to male (F→M) hematopoietic stem cell transplant (HSCT) exhibits:
- Increased GVHD and GVL due to donor T-cell targeting mHAs
- Recently demonstrated antibody against H-Y antigens (Miklos et al.)
Hypothesis: KDM5D is the most important T-cell H-Y antigen and it will be highly
immunogenic for B-cells as well
To test this we obtained blood plasma from:
- 39 HSCT F→M transplant patients
- 16 HSCT patients with other donor/recipient gender combinations
- 12 healthy female donors
- 9 healthy male donors
We tested for IgG antibody against:
- 36 overlapping pentadecapeptide fragments representing residues 197-383 of KDM5D,
peptides for a unique region of isoform 1, and several X-homologue peptides
- Recombinant proteins DDX3Y, UTY, ZFY RPS4Y and E1F1AY, their X-homologues,
and KDM5D-f (the one fragment currently purified) comprising residues 1320 to 1570
We found:
- 15 of 39 (38%) F→M HSCT samples were reactive with ≥1 KDM5D peptide while 7 of
39 (18%) samples reacted with ≥3 KDM5D peptides.
- 9 of 16 (56%) samples from other donor/recipient gender combinations were reactive
with ≥1 KDM5D peptide and 2 of 16 (13%) were reactive with ≥3 KDM5D peptides.
- Peptide specific results suggest that a potential target region for H-Y antibody exists
around KDM5D281-375 - Strong antibody responses to KDM5D19 (VSSTLLKQHL--------SLEPC) were only
observed in male recipients of female grafts (7 of 39) and responses to peptide 32
were also primarily observed in F→M transplant patients.
- Recombinant protein responses were detected most frequently for UTY, followed by
KDM5D-f.
- Antibodies to UTY were observed in 11 of 39 (28%) F→M patients and in 1 of 16
(6%) patients with other donor/recipient gender combinations.
Hematopoietic Stem Cell Transplant
Donor Stem Cells
GVHD
No Rxn
GVL
The Bone marrow transplant replaces the blood forming cells of the patient with those of the donor. This
reconstitutes the patient’s immune system and the engrafted T-cells interact with the host.
Immune Cell Recognition
148
DNA polymorphisms result in differences in host/donor proteins, the
donor immune system may recognize host peptide fragments displayed by
MHC molecules as foreign resulting GVH or GVL effect against these
minor histocompatibility antigens (mHAs).
Minor Histocompatibility Antigens and the H-Y antigen system
:
XX XY
Experimental Approach
Coat 96 well plates with protein (protein fragments or peptides) in
coating buffer
Add plasma sample. If specific antibody exists in the plasma,
the antibody will bind to the protein
Add secondary antibody (anti human IgG antibody) conjugated with AP
Add color development buffer and measure absorbance at 405 nm
The female to male transplant scenario is a unique opportunity to study mHAs and their clinical significance
because it restricts the search to just the Y-chromosome. Additionally, the gene must be expressed widely
outside the testes, have a similar but non-identical X homolog, escape X inactivation, and reside outside the
ampliconic region.
Conclusions and Further Directions
But what about the B cells?
Conclusions
1. Results suggest that KDM5D is an important target for post-transplant
antibody
2. There appears to be an antibody binding cluster near KDM5D281-375 3. The antibody response to H-Y antigens, particularly DDX3Y may not be
as strong as previously suggested
Further Directions
1. Assay more samples. It is interesting that the healthy samples exhibit
high antibody responses, does everyone have similar antibody?
2. Does Rituximab benefit patients with ≥1 H-Y antibody response?
3. And what about the rest of SMCY? Currently we are synthesizing the full
recombinant protein, does an antibody epitope reside outside the region
we assayed?
4. How much antibody is present? Conduct sequential dilution to determine
the antibody titer
Y
Y
Y
Y
Y Y
Y
Y
Y
Y Y
Y
Y Y Y
Y
Y Y
YY Y Y Y
Y Y
Y
Y Y YY Y
Y Y
YY Y Y
YY
Y
Y
GVHD
No Rxn ?
Acknowledgements and References
GVL
I would like thank Hootie and everyone in the Warren lab; David Miklos for supplying recombinant
protein; Jim Russo, Doug Juers, and Marilyn Gotz for helping me here; Jennifer Anderson, Jorden
Canas, and the countless people who supported the internship program; and the Howard Hughes
Medical Institute and Whitman College along with Fred Hutchinson Cancer Research Center for
sponsoring this opportunity.
Miklos, D et al. 2004. “Antibody response to DBY minor histocompatibility antigen is induced
after allogeneic stem cell transplantation and in healthy female donors.” Blood. 103: 353-
359
Miklos, D et al. 2005. “Antibody responses to H-Y minor histocompatibility antigens
correlate with chronic graft-versus-host disease and disease remission.” Blood. 105:
2973-2978
Murphy, K et al. Janeway’s Immuno Biology 7th Edition. New York: Garland Science, 2008.
Although T-cells are the most relevant to both GVL and GVH, the efficacy of
Rituximab in GVHD (a monoclonal CD20 antibody specific for B cells) and
accumulating evidence has shown the presence and significance of antibodies in
the H-Y transplant setting