Limits...
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.

Show MeSH

Related in: MedlinePlus

Sensitivity of Peptide–MHC Tetramer-Mediated Detection of OVA-Specific CTLs and MCC-Specific Helper T Cells(A) DiD-labeled OT-1 OVA-specific cells (red) were diluted 1:100 (top panels) or 1:1,000 (bottom panels) in DiO-labeled, monocyte-depleted C57BL/6 mouse lymph node cells (green). DiD-labeled OT-1 cells of 2.7 × 104 (top panels) and 4.41 × 103 (bottom panels) were mixed, respectively, with 2.7 × 106 and 4.41 × 106 of DiO-labeled CD11b-depleted lymph node cells. The different dilutions were applied to identical but separate arrays printed with the OVA/Kb tetramer, the LCMV/Kd control tetramer, and three different antibody spots (mouse anti-CD8, mouse anti-CD4, and mouse anti-CD28). Following 10-min incubation at 37°C, the cells were washed in RPMI and the slide was scanned. Shown are the OT-1 (red) and lymph node (green) cells that were captured by the OVA tetramer (left), LCMV tetramer (second left), and the antibody spots.(B) Class II MHC-mediated detection and sorting of helper T cells. DiD-labeled 5c.c7 lymphocytes (3.5 × 104) (red) were diluted 100-fold in 3.6 × 106 B10A DiO splenocytes (green). The mixed suspension was incubated at 37°C for 10 min prior to wash with RPMI. Shown are the 5c.c7 sells (red) and splenocytes (green) captured by the MCC (left) and anti-CD28 (right) spots.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC300678&req=5

pbio.0000065-g003: Sensitivity of Peptide–MHC Tetramer-Mediated Detection of OVA-Specific CTLs and MCC-Specific Helper T Cells(A) DiD-labeled OT-1 OVA-specific cells (red) were diluted 1:100 (top panels) or 1:1,000 (bottom panels) in DiO-labeled, monocyte-depleted C57BL/6 mouse lymph node cells (green). DiD-labeled OT-1 cells of 2.7 × 104 (top panels) and 4.41 × 103 (bottom panels) were mixed, respectively, with 2.7 × 106 and 4.41 × 106 of DiO-labeled CD11b-depleted lymph node cells. The different dilutions were applied to identical but separate arrays printed with the OVA/Kb tetramer, the LCMV/Kd control tetramer, and three different antibody spots (mouse anti-CD8, mouse anti-CD4, and mouse anti-CD28). Following 10-min incubation at 37°C, the cells were washed in RPMI and the slide was scanned. Shown are the OT-1 (red) and lymph node (green) cells that were captured by the OVA tetramer (left), LCMV tetramer (second left), and the antibody spots.(B) Class II MHC-mediated detection and sorting of helper T cells. DiD-labeled 5c.c7 lymphocytes (3.5 × 104) (red) were diluted 100-fold in 3.6 × 106 B10A DiO splenocytes (green). The mixed suspension was incubated at 37°C for 10 min prior to wash with RPMI. Shown are the 5c.c7 sells (red) and splenocytes (green) captured by the MCC (left) and anti-CD28 (right) spots.

Mentions: To directly assess the feasibility of detecting rare T cell specificities in a complex population, we prepared synthetic mixtures of differentially labeled, antigen-specific CTLs and T helper cells, diluted with syngeneic wild-type lymph node cells (Figure 3A) or splenocytes (Figure 3B). DiD-labeled OT-1 cells were diluted 1:100 (Figure 3A, top panels) and 1:1,000 (bottom panels) in DiO-labeled C57BL/6 mouse lymph node cells that had been depleted of monocytes using anti-CD11b beads (to remove cells capable of peptide-independent binding to the MHC complex). OT-1 cells of 2.7 × 104 (Figure 3A, top panels) and 4.41 × 103 (Figure 3A, bottom panels) were mixed with 2.7 × 106 and 4.41 × 106 of CD11b-depleted lymph node cells, respectively. The two different mixtures were then separately incubated with identical peptide–MHC arrays, each printed with OVA/Kb tetramer, LCMV/Kd control tetramer (lymphocytic choriomengingitis virus gp peptide 33–41, H-2Kd class I MHC molecule, β2-microglobulin), MCC/Ek tetramer, and three different antibody spots ( mouse anti-CD8, mouse anti-CD4, and mouse anti-CD28). Following a 10–30 min incubation at 20°C or 37°C (similar results were obtained), the cells were washed twice in RPMI and the slide was scanned.


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

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

Sensitivity of Peptide–MHC Tetramer-Mediated Detection of OVA-Specific CTLs and MCC-Specific Helper T Cells(A) DiD-labeled OT-1 OVA-specific cells (red) were diluted 1:100 (top panels) or 1:1,000 (bottom panels) in DiO-labeled, monocyte-depleted C57BL/6 mouse lymph node cells (green). DiD-labeled OT-1 cells of 2.7 × 104 (top panels) and 4.41 × 103 (bottom panels) were mixed, respectively, with 2.7 × 106 and 4.41 × 106 of DiO-labeled CD11b-depleted lymph node cells. The different dilutions were applied to identical but separate arrays printed with the OVA/Kb tetramer, the LCMV/Kd control tetramer, and three different antibody spots (mouse anti-CD8, mouse anti-CD4, and mouse anti-CD28). Following 10-min incubation at 37°C, the cells were washed in RPMI and the slide was scanned. Shown are the OT-1 (red) and lymph node (green) cells that were captured by the OVA tetramer (left), LCMV tetramer (second left), and the antibody spots.(B) Class II MHC-mediated detection and sorting of helper T cells. DiD-labeled 5c.c7 lymphocytes (3.5 × 104) (red) were diluted 100-fold in 3.6 × 106 B10A DiO splenocytes (green). The mixed suspension was incubated at 37°C for 10 min prior to wash with RPMI. Shown are the 5c.c7 sells (red) and splenocytes (green) captured by the MCC (left) and anti-CD28 (right) spots.
© Copyright Policy
Related In: Results  -  Collection

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

pbio.0000065-g003: Sensitivity of Peptide–MHC Tetramer-Mediated Detection of OVA-Specific CTLs and MCC-Specific Helper T Cells(A) DiD-labeled OT-1 OVA-specific cells (red) were diluted 1:100 (top panels) or 1:1,000 (bottom panels) in DiO-labeled, monocyte-depleted C57BL/6 mouse lymph node cells (green). DiD-labeled OT-1 cells of 2.7 × 104 (top panels) and 4.41 × 103 (bottom panels) were mixed, respectively, with 2.7 × 106 and 4.41 × 106 of DiO-labeled CD11b-depleted lymph node cells. The different dilutions were applied to identical but separate arrays printed with the OVA/Kb tetramer, the LCMV/Kd control tetramer, and three different antibody spots (mouse anti-CD8, mouse anti-CD4, and mouse anti-CD28). Following 10-min incubation at 37°C, the cells were washed in RPMI and the slide was scanned. Shown are the OT-1 (red) and lymph node (green) cells that were captured by the OVA tetramer (left), LCMV tetramer (second left), and the antibody spots.(B) Class II MHC-mediated detection and sorting of helper T cells. DiD-labeled 5c.c7 lymphocytes (3.5 × 104) (red) were diluted 100-fold in 3.6 × 106 B10A DiO splenocytes (green). The mixed suspension was incubated at 37°C for 10 min prior to wash with RPMI. Shown are the 5c.c7 sells (red) and splenocytes (green) captured by the MCC (left) and anti-CD28 (right) spots.
Mentions: To directly assess the feasibility of detecting rare T cell specificities in a complex population, we prepared synthetic mixtures of differentially labeled, antigen-specific CTLs and T helper cells, diluted with syngeneic wild-type lymph node cells (Figure 3A) or splenocytes (Figure 3B). DiD-labeled OT-1 cells were diluted 1:100 (Figure 3A, top panels) and 1:1,000 (bottom panels) in DiO-labeled C57BL/6 mouse lymph node cells that had been depleted of monocytes using anti-CD11b beads (to remove cells capable of peptide-independent binding to the MHC complex). OT-1 cells of 2.7 × 104 (Figure 3A, top panels) and 4.41 × 103 (Figure 3A, bottom panels) were mixed with 2.7 × 106 and 4.41 × 106 of CD11b-depleted lymph node cells, respectively. The two different mixtures were then separately incubated with identical peptide–MHC arrays, each printed with OVA/Kb tetramer, LCMV/Kd control tetramer (lymphocytic choriomengingitis virus gp peptide 33–41, H-2Kd class I MHC molecule, β2-microglobulin), MCC/Ek tetramer, and three different antibody spots ( mouse anti-CD8, mouse anti-CD4, and mouse anti-CD28). Following a 10–30 min incubation at 20°C or 37°C (similar results were obtained), the cells were washed twice in RPMI and the slide was scanned.

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