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Detection and characterization of cellular immune responses using peptide-MHC microarrays.

Soen Y, Chen DS, Kraft DL, Davis MM, Brown PO - PLoS Biol. (2003)

Bottom Line: The detection and characterization of antigen-specific T cell populations is critical for understanding the development and physiology of the immune system and its responses in health and disease.We have developed and tested a method that uses arrays of peptide-MHC complexes for the rapid identification, isolation, activation, and characterization of multiple antigen-specific populations of T cells.In addition, we were able to use the array to detect a rare population of antigen-specific T cells following vaccination of a normal mouse.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Stanford University, Stanford, California, USA.

ABSTRACT
The detection and characterization of antigen-specific T cell populations is critical for understanding the development and physiology of the immune system and its responses in health and disease. We have developed and tested a method that uses arrays of peptide-MHC complexes for the rapid identification, isolation, activation, and characterization of multiple antigen-specific populations of T cells. CD4(+) or CD8(+) lymphocytes can be captured in accordance with their ligand specificity using an array of peptide-MHC complexes printed on a film-coated glass surface. We have characterized the specificity and sensitivity of a peptide-MHC array using labeled lymphocytes from T cell receptor transgenic mice. In addition, we were able to use the array to detect a rare population of antigen-specific T cells following vaccination of a normal mouse. This approach should be useful for epitope discovery, as well as for characterization and analysis of multiple epitope-specific T cell populations during immune responses associated with viral and bacterial infection, cancer, autoimmunity, and vaccination.

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Related in: MedlinePlus

Activation of OT-1 Lymphocytes Following an Exposure to Preprinted Spots on an MHC ArrayOT-1 lymphocytes were preloaded with Fura-2 dye for 20 min at room temperature prior to exposure to the cellular microarray (time 0). The cells were incubated at 37°C, 5% CO2, and fluorescence intensities at 340 nm and 380 nm were measured every 30 s from each of the spots on the array. Calcium flux signal was triggered in the OVA-specific cells as soon as they reached the OVA/Kb tetramer spots, the activating antibody spots (anti-CD3 and anti-CD3/anti-CD28), but not the irrelevant MCC/Ek tetramer or the nonactivating antibody spots (e.g. anti-CD28).(A) Calcium flux signal (yellow) overlaid onto a 10x DIC image of OT-1 cells on a combined FITC-labeled anti-CD3/anti-CD28 spot. Fura-2 fluorescence intensity ratios are represented in pseudo-color, and the overlaid spot region (blue) is determined from the FITC image. Note that the spot pattern can be inferred from the Fura-2 signal representing activation, even without removing unbound cells.(B) A time-lapsed series of images taken of a representative OT-1 lymphocyte specifically immobilized onto an OVA/Kb spot. The cell undergoes a rapid, transient calcium flux as measured by Fura-2 fluorescence.(C) Averaged calcium flux traces (n = 3) recorded from an OVA/Kb spot (purple circles), anti-CD3/anti-CD28 spot (brown triangles), anti-CD3 spot (green circles), and nonactivating anti-CD28 spot (orange triangles). Error bars represent calculated standard error of mean.
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pbio.0000065-g005: Activation of OT-1 Lymphocytes Following an Exposure to Preprinted Spots on an MHC ArrayOT-1 lymphocytes were preloaded with Fura-2 dye for 20 min at room temperature prior to exposure to the cellular microarray (time 0). The cells were incubated at 37°C, 5% CO2, and fluorescence intensities at 340 nm and 380 nm were measured every 30 s from each of the spots on the array. Calcium flux signal was triggered in the OVA-specific cells as soon as they reached the OVA/Kb tetramer spots, the activating antibody spots (anti-CD3 and anti-CD3/anti-CD28), but not the irrelevant MCC/Ek tetramer or the nonactivating antibody spots (e.g. anti-CD28).(A) Calcium flux signal (yellow) overlaid onto a 10x DIC image of OT-1 cells on a combined FITC-labeled anti-CD3/anti-CD28 spot. Fura-2 fluorescence intensity ratios are represented in pseudo-color, and the overlaid spot region (blue) is determined from the FITC image. Note that the spot pattern can be inferred from the Fura-2 signal representing activation, even without removing unbound cells.(B) A time-lapsed series of images taken of a representative OT-1 lymphocyte specifically immobilized onto an OVA/Kb spot. The cell undergoes a rapid, transient calcium flux as measured by Fura-2 fluorescence.(C) Averaged calcium flux traces (n = 3) recorded from an OVA/Kb spot (purple circles), anti-CD3/anti-CD28 spot (brown triangles), anti-CD3 spot (green circles), and nonactivating anti-CD28 spot (orange triangles). Error bars represent calculated standard error of mean.

Mentions: The ability to observe highly specific interactions between live lymphocytes and arrays of proteins raises the possibility of conducting systematic parallel studies of responses to diverse signals arrayed on the surface. We therefore tested the possibility of using a protein array to investigate dynamic responses of lymphocytes to diverse signals, including specific peptide–MHC complexes and other receptor-specific binding molecules. OT-1 lymphocytes were loaded with the calcium-sensitive dye Fura-2 and were subsequently exposed to an MHC array containing OVA/Kb, LCMV/Kd, MCC/Ek, anti-CD3, anti-CD28, and anti-CD3/anti-CD28 spots. Throughout the experiment, the array was kept at 37°C and 5% CO2, and intracellular calcium levels were monitored every 30 s by measuring the ratio of the fluorescence intensity at 340 nm and 380 nm. Binding of OT-1 lymphocytes to the OVA/Kb, anti-CD3, and anti-CD3/anti-CD28 MHC array spots, but not to LCMV/Kd, MCC/Ek, or anti-CD28, led to transient spikes in intracellular calcium levels (Figure 5).


Detection and characterization of cellular immune responses using peptide-MHC microarrays.

Soen Y, Chen DS, Kraft DL, Davis MM, Brown PO - PLoS Biol. (2003)

Activation of OT-1 Lymphocytes Following an Exposure to Preprinted Spots on an MHC ArrayOT-1 lymphocytes were preloaded with Fura-2 dye for 20 min at room temperature prior to exposure to the cellular microarray (time 0). The cells were incubated at 37°C, 5% CO2, and fluorescence intensities at 340 nm and 380 nm were measured every 30 s from each of the spots on the array. Calcium flux signal was triggered in the OVA-specific cells as soon as they reached the OVA/Kb tetramer spots, the activating antibody spots (anti-CD3 and anti-CD3/anti-CD28), but not the irrelevant MCC/Ek tetramer or the nonactivating antibody spots (e.g. anti-CD28).(A) Calcium flux signal (yellow) overlaid onto a 10x DIC image of OT-1 cells on a combined FITC-labeled anti-CD3/anti-CD28 spot. Fura-2 fluorescence intensity ratios are represented in pseudo-color, and the overlaid spot region (blue) is determined from the FITC image. Note that the spot pattern can be inferred from the Fura-2 signal representing activation, even without removing unbound cells.(B) A time-lapsed series of images taken of a representative OT-1 lymphocyte specifically immobilized onto an OVA/Kb spot. The cell undergoes a rapid, transient calcium flux as measured by Fura-2 fluorescence.(C) Averaged calcium flux traces (n = 3) recorded from an OVA/Kb spot (purple circles), anti-CD3/anti-CD28 spot (brown triangles), anti-CD3 spot (green circles), and nonactivating anti-CD28 spot (orange triangles). Error bars represent calculated standard error of mean.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC300678&req=5

pbio.0000065-g005: Activation of OT-1 Lymphocytes Following an Exposure to Preprinted Spots on an MHC ArrayOT-1 lymphocytes were preloaded with Fura-2 dye for 20 min at room temperature prior to exposure to the cellular microarray (time 0). The cells were incubated at 37°C, 5% CO2, and fluorescence intensities at 340 nm and 380 nm were measured every 30 s from each of the spots on the array. Calcium flux signal was triggered in the OVA-specific cells as soon as they reached the OVA/Kb tetramer spots, the activating antibody spots (anti-CD3 and anti-CD3/anti-CD28), but not the irrelevant MCC/Ek tetramer or the nonactivating antibody spots (e.g. anti-CD28).(A) Calcium flux signal (yellow) overlaid onto a 10x DIC image of OT-1 cells on a combined FITC-labeled anti-CD3/anti-CD28 spot. Fura-2 fluorescence intensity ratios are represented in pseudo-color, and the overlaid spot region (blue) is determined from the FITC image. Note that the spot pattern can be inferred from the Fura-2 signal representing activation, even without removing unbound cells.(B) A time-lapsed series of images taken of a representative OT-1 lymphocyte specifically immobilized onto an OVA/Kb spot. The cell undergoes a rapid, transient calcium flux as measured by Fura-2 fluorescence.(C) Averaged calcium flux traces (n = 3) recorded from an OVA/Kb spot (purple circles), anti-CD3/anti-CD28 spot (brown triangles), anti-CD3 spot (green circles), and nonactivating anti-CD28 spot (orange triangles). Error bars represent calculated standard error of mean.
Mentions: The ability to observe highly specific interactions between live lymphocytes and arrays of proteins raises the possibility of conducting systematic parallel studies of responses to diverse signals arrayed on the surface. We therefore tested the possibility of using a protein array to investigate dynamic responses of lymphocytes to diverse signals, including specific peptide–MHC complexes and other receptor-specific binding molecules. OT-1 lymphocytes were loaded with the calcium-sensitive dye Fura-2 and were subsequently exposed to an MHC array containing OVA/Kb, LCMV/Kd, MCC/Ek, anti-CD3, anti-CD28, and anti-CD3/anti-CD28 spots. Throughout the experiment, the array was kept at 37°C and 5% CO2, and intracellular calcium levels were monitored every 30 s by measuring the ratio of the fluorescence intensity at 340 nm and 380 nm. Binding of OT-1 lymphocytes to the OVA/Kb, anti-CD3, and anti-CD3/anti-CD28 MHC array spots, but not to LCMV/Kd, MCC/Ek, or anti-CD28, led to transient spikes in intracellular calcium levels (Figure 5).

Bottom Line: The detection and characterization of antigen-specific T cell populations is critical for understanding the development and physiology of the immune system and its responses in health and disease.We have developed and tested a method that uses arrays of peptide-MHC complexes for the rapid identification, isolation, activation, and characterization of multiple antigen-specific populations of T cells.In addition, we were able to use the array to detect a rare population of antigen-specific T cells following vaccination of a normal mouse.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Stanford University, Stanford, California, USA.

ABSTRACT
The detection and characterization of antigen-specific T cell populations is critical for understanding the development and physiology of the immune system and its responses in health and disease. We have developed and tested a method that uses arrays of peptide-MHC complexes for the rapid identification, isolation, activation, and characterization of multiple antigen-specific populations of T cells. CD4(+) or CD8(+) lymphocytes can be captured in accordance with their ligand specificity using an array of peptide-MHC complexes printed on a film-coated glass surface. We have characterized the specificity and sensitivity of a peptide-MHC array using labeled lymphocytes from T cell receptor transgenic mice. In addition, we were able to use the array to detect a rare population of antigen-specific T cells following vaccination of a normal mouse. This approach should be useful for epitope discovery, as well as for characterization and analysis of multiple epitope-specific T cell populations during immune responses associated with viral and bacterial infection, cancer, autoimmunity, and vaccination.

Show MeSH
Related in: MedlinePlus