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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

Detection of a Weak Immune Response to VaccinationCTLs from OVA-vaccinated and mock-vaccinated mice were analyzed in parallel using an MHC array and flow cytometry analysis. Mice received base-of-tail injections of OVA/Freund's adjuvant emulsions or PBS (mock)/Freund's adjuvant on day 0 and day 7. Draining lymph nodes were harvested on day 11, and dissociated into a single-cell suspension.(A) CTLs were enriched on an anti-CD8-bead column. Cells (2 × 106 and 3.2 × 106) from the OVA- and mock-vaccinated mice, respectively, were incubated on identical but separate arrays printed with OVA/Kb tetramer, LCMV/Kd (control) tetramer, and various antibodies. The three panels show the relevant array results. A rare population of cells from the OVA-vaccinated mouse was captured on the OVA/Kb (left), but not on the LCMV/Kd (right), tetramer spot. The cells captured on the OVA/Kb tetramer spot were CD8+ (as determined by counter-staining using an anti-CD8–FITC mAb on the array). An arrowhead points to some of the cells that were bound to that spot. The cells from the mock-vaccinated mouse did not bind the OVA tetramer (middle spot) or the LCMV/Kd tetramer (data not shown). Spot regions are marked with a blue color by overlaying the tetramer's PE fluorescent signal onto the DIC image.(B) FACS analysis of the cells from the OVA-vaccinated (left panel) and mock-vaccinated mice (right). Of the total CD8+ cells from the vaccinated mouse, 0.27% co-stain with OVA/Kb, compared with 0.01% in the mock-vaccinated mouse.
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pbio.0000065-g004: Detection of a Weak Immune Response to VaccinationCTLs from OVA-vaccinated and mock-vaccinated mice were analyzed in parallel using an MHC array and flow cytometry analysis. Mice received base-of-tail injections of OVA/Freund's adjuvant emulsions or PBS (mock)/Freund's adjuvant on day 0 and day 7. Draining lymph nodes were harvested on day 11, and dissociated into a single-cell suspension.(A) CTLs were enriched on an anti-CD8-bead column. Cells (2 × 106 and 3.2 × 106) from the OVA- and mock-vaccinated mice, respectively, were incubated on identical but separate arrays printed with OVA/Kb tetramer, LCMV/Kd (control) tetramer, and various antibodies. The three panels show the relevant array results. A rare population of cells from the OVA-vaccinated mouse was captured on the OVA/Kb (left), but not on the LCMV/Kd (right), tetramer spot. The cells captured on the OVA/Kb tetramer spot were CD8+ (as determined by counter-staining using an anti-CD8–FITC mAb on the array). An arrowhead points to some of the cells that were bound to that spot. The cells from the mock-vaccinated mouse did not bind the OVA tetramer (middle spot) or the LCMV/Kd tetramer (data not shown). Spot regions are marked with a blue color by overlaying the tetramer's PE fluorescent signal onto the DIC image.(B) FACS analysis of the cells from the OVA-vaccinated (left panel) and mock-vaccinated mice (right). Of the total CD8+ cells from the vaccinated mouse, 0.27% co-stain with OVA/Kb, compared with 0.01% in the mock-vaccinated mouse.

Mentions: An important potential application of this technology is in systematically monitoring the T cell repertoire in vivo and its response to immunization, infection, and other stimuli. We tested the feasibility of detecting a weak immune response by monitoring the response of C57BL/6 mice to vaccination with OVA (Figure 4). CD8+ lymphocytes (2 × 106 and 3.2 × 106) from the OVA-vaccinated and mock-vaccinated mice, respectively, were incubated separately with duplicate peptide–MHC arrays (on the same slide) containing OVA/kb tetramer, LCMV/kd (control) tetramer, and various antibodies. The arrays were incubated at room temperature for 30 min and then washed with RPMI to remove free-floating cells. The cells were also analyzed by flow cytometry after staining with anti-CD8–fluorescein isothiocyanate (FITC) mAb and OVA/kb streptavidin (SA)–PE tetramer. FACS analysis revealed that a small fraction (0.27%) of the CTLs from the vaccinated mouse are OVA-specific, whereas the control mouse did not exhibit an OVA-specific response (Figure 4B). A small but significant number of cells from the OVA-vaccinated mouse were captured on the OVA/Kb spots, while none were bound by the LCMV/Kd tetramer spot (Figure 4A). Cells from the mock-vaccinated mouse did not bind to either of the peptide–MHC tetramers. These results demonstrate that these arrays can provide a way to detect even very weak immune responses in normal mice. The ability to use this approach to detect postvaccination populations of peptide-specific T cells in wild-type immune responses has been further confirmed with different peptide–MHC systems (unpublished data).


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

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

Detection of a Weak Immune Response to VaccinationCTLs from OVA-vaccinated and mock-vaccinated mice were analyzed in parallel using an MHC array and flow cytometry analysis. Mice received base-of-tail injections of OVA/Freund's adjuvant emulsions or PBS (mock)/Freund's adjuvant on day 0 and day 7. Draining lymph nodes were harvested on day 11, and dissociated into a single-cell suspension.(A) CTLs were enriched on an anti-CD8-bead column. Cells (2 × 106 and 3.2 × 106) from the OVA- and mock-vaccinated mice, respectively, were incubated on identical but separate arrays printed with OVA/Kb tetramer, LCMV/Kd (control) tetramer, and various antibodies. The three panels show the relevant array results. A rare population of cells from the OVA-vaccinated mouse was captured on the OVA/Kb (left), but not on the LCMV/Kd (right), tetramer spot. The cells captured on the OVA/Kb tetramer spot were CD8+ (as determined by counter-staining using an anti-CD8–FITC mAb on the array). An arrowhead points to some of the cells that were bound to that spot. The cells from the mock-vaccinated mouse did not bind the OVA tetramer (middle spot) or the LCMV/Kd tetramer (data not shown). Spot regions are marked with a blue color by overlaying the tetramer's PE fluorescent signal onto the DIC image.(B) FACS analysis of the cells from the OVA-vaccinated (left panel) and mock-vaccinated mice (right). Of the total CD8+ cells from the vaccinated mouse, 0.27% co-stain with OVA/Kb, compared with 0.01% in the mock-vaccinated mouse.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC300678&req=5

pbio.0000065-g004: Detection of a Weak Immune Response to VaccinationCTLs from OVA-vaccinated and mock-vaccinated mice were analyzed in parallel using an MHC array and flow cytometry analysis. Mice received base-of-tail injections of OVA/Freund's adjuvant emulsions or PBS (mock)/Freund's adjuvant on day 0 and day 7. Draining lymph nodes were harvested on day 11, and dissociated into a single-cell suspension.(A) CTLs were enriched on an anti-CD8-bead column. Cells (2 × 106 and 3.2 × 106) from the OVA- and mock-vaccinated mice, respectively, were incubated on identical but separate arrays printed with OVA/Kb tetramer, LCMV/Kd (control) tetramer, and various antibodies. The three panels show the relevant array results. A rare population of cells from the OVA-vaccinated mouse was captured on the OVA/Kb (left), but not on the LCMV/Kd (right), tetramer spot. The cells captured on the OVA/Kb tetramer spot were CD8+ (as determined by counter-staining using an anti-CD8–FITC mAb on the array). An arrowhead points to some of the cells that were bound to that spot. The cells from the mock-vaccinated mouse did not bind the OVA tetramer (middle spot) or the LCMV/Kd tetramer (data not shown). Spot regions are marked with a blue color by overlaying the tetramer's PE fluorescent signal onto the DIC image.(B) FACS analysis of the cells from the OVA-vaccinated (left panel) and mock-vaccinated mice (right). Of the total CD8+ cells from the vaccinated mouse, 0.27% co-stain with OVA/Kb, compared with 0.01% in the mock-vaccinated mouse.
Mentions: An important potential application of this technology is in systematically monitoring the T cell repertoire in vivo and its response to immunization, infection, and other stimuli. We tested the feasibility of detecting a weak immune response by monitoring the response of C57BL/6 mice to vaccination with OVA (Figure 4). CD8+ lymphocytes (2 × 106 and 3.2 × 106) from the OVA-vaccinated and mock-vaccinated mice, respectively, were incubated separately with duplicate peptide–MHC arrays (on the same slide) containing OVA/kb tetramer, LCMV/kd (control) tetramer, and various antibodies. The arrays were incubated at room temperature for 30 min and then washed with RPMI to remove free-floating cells. The cells were also analyzed by flow cytometry after staining with anti-CD8–fluorescein isothiocyanate (FITC) mAb and OVA/kb streptavidin (SA)–PE tetramer. FACS analysis revealed that a small fraction (0.27%) of the CTLs from the vaccinated mouse are OVA-specific, whereas the control mouse did not exhibit an OVA-specific response (Figure 4B). A small but significant number of cells from the OVA-vaccinated mouse were captured on the OVA/Kb spots, while none were bound by the LCMV/Kd tetramer spot (Figure 4A). Cells from the mock-vaccinated mouse did not bind to either of the peptide–MHC tetramers. These results demonstrate that these arrays can provide a way to detect even very weak immune responses in normal mice. The ability to use this approach to detect postvaccination populations of peptide-specific T cells in wild-type immune responses has been further confirmed with different peptide–MHC systems (unpublished data).

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