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Peptide-MHC cellular microarray with innovative data analysis system for simultaneously detecting multiple CD4 T-cell responses.

Ge X, Gebe JA, Bollyky PL, James EA, Yang J, Stern LJ, Kwok WW - PLoS ONE (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Benaroya Research Institute at Virginia Mason, Seattle, Washington, USA.

ABSTRACT

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.

Methodology/principal findings: 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/significance: 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.

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Related in: MedlinePlus

Characteristics of peptide:MHC cellular microarrays.(A) A part of a freshly fabricated microarray image with magnification of 15.75× (left panel), 100× (middle panel) and 200× (right panel). (B) A raw fluorescence image of a 4-supergrid 8×12 microarray probed with Cy3-conjugated anti-Rat-IgG. The absolute fluorescence of all 96 spots for the low-right supergrid was quantified and analyzed for the variance. (C) A microarray loaded with 6×106 primary human CD4 T cells. (Left panel, 25× magnification) image taken immediately after 6 hours of incubation; (middle panel, 200× magnification) image taken after aspirating culture medium; (right panel) image taken after washing the slide with 1xPBS for three times. The numbers at the low-right corners of middle and right panels indicate cell counts. (D) The box/whisker plots represent cell counts of 20 randomly selected spots before (loose attached) and after (tightly attached) washing procedure.
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pone-0011355-g001: Characteristics of peptide:MHC cellular microarrays.(A) A part of a freshly fabricated microarray image with magnification of 15.75× (left panel), 100× (middle panel) and 200× (right panel). (B) A raw fluorescence image of a 4-supergrid 8×12 microarray probed with Cy3-conjugated anti-Rat-IgG. The absolute fluorescence of all 96 spots for the low-right supergrid was quantified and analyzed for the variance. (C) A microarray loaded with 6×106 primary human CD4 T cells. (Left panel, 25× magnification) image taken immediately after 6 hours of incubation; (middle panel, 200× magnification) image taken after aspirating culture medium; (right panel) image taken after washing the slide with 1xPBS for three times. The numbers at the low-right corners of middle and right panels indicate cell counts. (D) The box/whisker plots represent cell counts of 20 randomly selected spots before (loose attached) and after (tightly attached) washing procedure.

Mentions: We used a contact-printing robot to fabricate our cellular microarray. The pin repeatedly delivered ∼12.5 nl/spots of sample solution onto the glass surface of a chamber slide in a pre-arranged format (Figure 1A far-left panel), yielding a series of spots with the size ∼0.5 mm×0.5 mm (Figure 1A far-right panel). For a typical 1-chamber slide, the size ratio of printable surface (18×47 mm) to a single spot was approximately 3,400:1. The distance between the centers of two adjacent spots was 0.8 mm (Figure 1A middle panel). The size and amount of proteins immobilized on the surface was highly uniform, with 0.50% co-efficient of variation (Figure 1B). The spot size and the cell density determined the number of cells possibly settling on each spot. Since only the cells “settling down” on the top of a spot had an opportunity to be activated, the number of cells “settling” on top of the spots was theoretically relevant to assay sensitivity. In practice, when we loaded 6×106 cells on a 1-chamber slide, the average number of cells attaching to each individual spot was 897±106 (loose attached) (Figure 1C, left and middle panel; Figure 1D). Around 519±68 cells were firmly attached, even after two rounds of washing with 1xPBS (tightly attached) (Figure 1C, right panel and Figure 1D). Given the fact that at least one single Ag-specific T cell is required to produce cytokine upon activation, based on the cell number/spot as we counted, the best estimate of the detection limit for Ag-specific T cells is as low as 0.1–0.2% among all the cells loaded on the slide. Taken together, by using this setting, we can easily print an array of 20 rows×50 columns in a 1-chamber slide. With 10 replicates for each peptide:MHC/cytokine feature, this format allows us to evaluate up to 100 features, simultaneously. The lowest number of Ag-specific T cells (among those total 6×106 T cells loaded onto the chamber slide) required for generating detectable signal range from 6,000 to 12,000. Increasing the total number of cells input improved the number of cells “landing” on those spots, but it negatively influenced signal/noise ratio of fluorescence measurements.


Peptide-MHC cellular microarray with innovative data analysis system for simultaneously detecting multiple CD4 T-cell responses.

Ge X, Gebe JA, Bollyky PL, James EA, Yang J, Stern LJ, Kwok WW - PLoS ONE (2010)

Characteristics of peptide:MHC cellular microarrays.(A) A part of a freshly fabricated microarray image with magnification of 15.75× (left panel), 100× (middle panel) and 200× (right panel). (B) A raw fluorescence image of a 4-supergrid 8×12 microarray probed with Cy3-conjugated anti-Rat-IgG. The absolute fluorescence of all 96 spots for the low-right supergrid was quantified and analyzed for the variance. (C) A microarray loaded with 6×106 primary human CD4 T cells. (Left panel, 25× magnification) image taken immediately after 6 hours of incubation; (middle panel, 200× magnification) image taken after aspirating culture medium; (right panel) image taken after washing the slide with 1xPBS for three times. The numbers at the low-right corners of middle and right panels indicate cell counts. (D) The box/whisker plots represent cell counts of 20 randomly selected spots before (loose attached) and after (tightly attached) washing procedure.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2902358&req=5

pone-0011355-g001: Characteristics of peptide:MHC cellular microarrays.(A) A part of a freshly fabricated microarray image with magnification of 15.75× (left panel), 100× (middle panel) and 200× (right panel). (B) A raw fluorescence image of a 4-supergrid 8×12 microarray probed with Cy3-conjugated anti-Rat-IgG. The absolute fluorescence of all 96 spots for the low-right supergrid was quantified and analyzed for the variance. (C) A microarray loaded with 6×106 primary human CD4 T cells. (Left panel, 25× magnification) image taken immediately after 6 hours of incubation; (middle panel, 200× magnification) image taken after aspirating culture medium; (right panel) image taken after washing the slide with 1xPBS for three times. The numbers at the low-right corners of middle and right panels indicate cell counts. (D) The box/whisker plots represent cell counts of 20 randomly selected spots before (loose attached) and after (tightly attached) washing procedure.
Mentions: We used a contact-printing robot to fabricate our cellular microarray. The pin repeatedly delivered ∼12.5 nl/spots of sample solution onto the glass surface of a chamber slide in a pre-arranged format (Figure 1A far-left panel), yielding a series of spots with the size ∼0.5 mm×0.5 mm (Figure 1A far-right panel). For a typical 1-chamber slide, the size ratio of printable surface (18×47 mm) to a single spot was approximately 3,400:1. The distance between the centers of two adjacent spots was 0.8 mm (Figure 1A middle panel). The size and amount of proteins immobilized on the surface was highly uniform, with 0.50% co-efficient of variation (Figure 1B). The spot size and the cell density determined the number of cells possibly settling on each spot. Since only the cells “settling down” on the top of a spot had an opportunity to be activated, the number of cells “settling” on top of the spots was theoretically relevant to assay sensitivity. In practice, when we loaded 6×106 cells on a 1-chamber slide, the average number of cells attaching to each individual spot was 897±106 (loose attached) (Figure 1C, left and middle panel; Figure 1D). Around 519±68 cells were firmly attached, even after two rounds of washing with 1xPBS (tightly attached) (Figure 1C, right panel and Figure 1D). Given the fact that at least one single Ag-specific T cell is required to produce cytokine upon activation, based on the cell number/spot as we counted, the best estimate of the detection limit for Ag-specific T cells is as low as 0.1–0.2% among all the cells loaded on the slide. Taken together, by using this setting, we can easily print an array of 20 rows×50 columns in a 1-chamber slide. With 10 replicates for each peptide:MHC/cytokine feature, this format allows us to evaluate up to 100 features, simultaneously. The lowest number of Ag-specific T cells (among those total 6×106 T cells loaded onto the chamber slide) required for generating detectable signal range from 6,000 to 12,000. Increasing the total number of cells input improved the number of cells “landing” on those spots, but it negatively influenced signal/noise ratio of fluorescence measurements.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Benaroya Research Institute at Virginia Mason, Seattle, Washington, USA.

ABSTRACT

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.

Methodology/principal findings: 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/significance: 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.

Show MeSH
Related in: MedlinePlus