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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

Specificity of Lymphocyte Immobilization by Peptide–MHC ArraysPrelabeled, OVA-specific OT-1 (green) and MCC-specific 5c.c7 (red) lymphocytes (6 ×105) were mixed at a 1:1 ratio and added to the preprinted peptide–MHC array. While anti-CD3 and anti-CD28 mAb spots (left and right top panels) immobilize both lymphocyte populations, the OVA and MCC tetramers (left and right bottom panels) capture only the OVA-specific and MCC-specific cells, respectively. Cells were labeled with DiO and DiD lipophilic tracers that incorporate into the cell membrane and do not affect the specificity of cell capture. The printed peptide–MHC tetramer spots contain the fluorescent dye, which allows visualization of peptide–MHC spot borders (data not shown).
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pbio.0000065-g002: Specificity of Lymphocyte Immobilization by Peptide–MHC ArraysPrelabeled, OVA-specific OT-1 (green) and MCC-specific 5c.c7 (red) lymphocytes (6 ×105) were mixed at a 1:1 ratio and added to the preprinted peptide–MHC array. While anti-CD3 and anti-CD28 mAb spots (left and right top panels) immobilize both lymphocyte populations, the OVA and MCC tetramers (left and right bottom panels) capture only the OVA-specific and MCC-specific cells, respectively. Cells were labeled with DiO and DiD lipophilic tracers that incorporate into the cell membrane and do not affect the specificity of cell capture. The printed peptide–MHC tetramer spots contain the fluorescent dye, which allows visualization of peptide–MHC spot borders (data not shown).

Mentions: We tested the specificity of binding to individual spots by using pure populations of differentially labeled, antigen-specific CTLs and helper T cells from transgenic mice. Antigen-specific lymphocytes from OT-1 and 5c.c7 transgenic mice were activated and expanded by adding 1 μM OVA and MCC peptides to the respective single-cell suspensions on day 0, and IL-2 (30 U/ml) was supplemented on day 1. On day 7, active OT-1 (CD8+) and 5c.c7 (CD4+) lymphocytes were labeled with DiO or DiD–lipophilic tracers, respectively. The two differentially labeled lymphocyte suspensions were mixed in a 1:1 ratio and coincubated on the peptide–MHC array. Following a 10-min incubation at 20°C, the array was washed twice in RPMI medium and examined by fluorescence microscopy (Figure 2). While anti-CD3 and anti-CD28 mAb spots captured both lymphocyte populations, the arrayed MCC/Ek and OVA/Kb tetramers exclusively captured the MCC-specific (red) and OVA-specific (green) cells, respectively. The nearly complete segregation of OT-1 and 5c.c7 cells on the appropriate spots illustrates the highly specific nature of lymphocyte capture and the feasibility of array-based T cell sorting and analysis.


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

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

Specificity of Lymphocyte Immobilization by Peptide–MHC ArraysPrelabeled, OVA-specific OT-1 (green) and MCC-specific 5c.c7 (red) lymphocytes (6 ×105) were mixed at a 1:1 ratio and added to the preprinted peptide–MHC array. While anti-CD3 and anti-CD28 mAb spots (left and right top panels) immobilize both lymphocyte populations, the OVA and MCC tetramers (left and right bottom panels) capture only the OVA-specific and MCC-specific cells, respectively. Cells were labeled with DiO and DiD lipophilic tracers that incorporate into the cell membrane and do not affect the specificity of cell capture. The printed peptide–MHC tetramer spots contain the fluorescent dye, which allows visualization of peptide–MHC spot borders (data not shown).
© Copyright Policy
Related In: Results  -  Collection

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

pbio.0000065-g002: Specificity of Lymphocyte Immobilization by Peptide–MHC ArraysPrelabeled, OVA-specific OT-1 (green) and MCC-specific 5c.c7 (red) lymphocytes (6 ×105) were mixed at a 1:1 ratio and added to the preprinted peptide–MHC array. While anti-CD3 and anti-CD28 mAb spots (left and right top panels) immobilize both lymphocyte populations, the OVA and MCC tetramers (left and right bottom panels) capture only the OVA-specific and MCC-specific cells, respectively. Cells were labeled with DiO and DiD lipophilic tracers that incorporate into the cell membrane and do not affect the specificity of cell capture. The printed peptide–MHC tetramer spots contain the fluorescent dye, which allows visualization of peptide–MHC spot borders (data not shown).
Mentions: We tested the specificity of binding to individual spots by using pure populations of differentially labeled, antigen-specific CTLs and helper T cells from transgenic mice. Antigen-specific lymphocytes from OT-1 and 5c.c7 transgenic mice were activated and expanded by adding 1 μM OVA and MCC peptides to the respective single-cell suspensions on day 0, and IL-2 (30 U/ml) was supplemented on day 1. On day 7, active OT-1 (CD8+) and 5c.c7 (CD4+) lymphocytes were labeled with DiO or DiD–lipophilic tracers, respectively. The two differentially labeled lymphocyte suspensions were mixed in a 1:1 ratio and coincubated on the peptide–MHC array. Following a 10-min incubation at 20°C, the array was washed twice in RPMI medium and examined by fluorescence microscopy (Figure 2). While anti-CD3 and anti-CD28 mAb spots captured both lymphocyte populations, the arrayed MCC/Ek and OVA/Kb tetramers exclusively captured the MCC-specific (red) and OVA-specific (green) cells, respectively. The nearly complete segregation of OT-1 and 5c.c7 cells on the appropriate spots illustrates the highly specific nature of lymphocyte capture and the feasibility of array-based T cell sorting and analysis.

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