Peptide-MHC cellular microarray with innovative data analysis system for simultaneously detecting multiple CD4 T-cell responses.
BACKGROUND
Peptide:MHC cellular microarrays have been proposed to simultaneously characterize multiple Ag-specific populations of T cells. The practice of studying immune responses to complicated pathogens with this tool demands extensive knowledge of T cell epitopes and the availability of peptide:MHC complexes for array fabrication as well as a specialized data analysis approach for result interpretation.


RESULTS
We co-immobilized peptide:DR0401 complexes, anti-CD28, anti-CD11a and cytokine capture antibodies on the surface of chamber slides to generate a functional array that was able to detect rare Ag-specific T cell populations from previously primed in vitro T cell cultures. A novel statistical methodology was also developed to facilitate batch processing of raw array-like data into standardized endpoint scores, which linearly correlated with total Ag-specific T cell inputs. Applying these methods to analyze Influenza A viral antigen-specific T cell responses, we not only revealed the most prominent viral epitopes, but also demonstrated the heterogeneity of anti-viral cellular responses in healthy individuals. Applying these methods to examine the insulin producing beta-cell autoantigen specific T cell responses, we observed little difference between autoimmune diabetic patients and healthy individuals, suggesting a more subtle association between diabetes status and peripheral autoreactive T cells.


CONCLUSIONS
The data analysis system is reliable for T cell specificity and functional testing. Peptide:MHC cellular microarrays can be used to obtain multi-parametric results using limited blood samples in a variety of translational settings.
Ge
Xinhui
X
Benaroya Research Institute at Virginia Mason, Seattle, Washington, USA.
Gebe
John A
JA
Bollyky
Paul L
PL
James
Eddie A
EA
Yang
Junbao
J
Stern
Lawrence J
LJ
Kwok
William W
WW
D000595:Amino Acid Sequence; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D006680:HLA Antigens / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D009980:Influenza A virus / Q000276:immunology; D007371:Interferon-gamma / Q000096:biosynthesis; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D040081:Protein Array Analysis
D018984:Epitopes, T-Lymphocyte; D006680:HLA Antigens; D010455:Peptides; D007371:Interferon-gamma