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tetramers Peptide-MHC

2. Introduction
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Figure 1. Model for the interaction of an MHC-peptide complexon one cell with a T cell
receptor on another cell.

3.  TCRs recognize and bind to complexes composed of MHC molecules
and specific peptides expressed on the surface of antigen-presenting
cells. antigen-specific T cells could be detected using soluble MHC/
peptide complexes, monomeric MHC/peptide complexes proved to be
impractical due to their instability and low affinity to the TCR.
 To overcome this difficulty, MHC/ peptide monomers are biotinylated
and tetramerized with streptavidin to maintain stable binding to
multiple TCR, enabling MHC/peptide Tetramers to be used as
detection tools. MHC Tetramers are labeled with fluorescent
molecules including phycoerythrin (PE) or allophycocyanin (APC)
and thus allow detection of antigenspecific T cells by flow cytometry
or fluorescence microscopy.
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Fluorochrome-conjugated peptide–MHC (pMHC)
multimers are commonly used in combination with flow
cytometry for direct exvivo visualization and
characterization of Ag-specific T cells, but these reagents
can fail to stain cells when TCR affinity and/or TCR cell-
surface density are low. pMHC multimer staining of
tumor-specific, autoimmune, or MHC class II–restricted T
cells can be particularly challenging, as these T cells tend
to express relatively low-affinity TCRs.

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to improve staining using anti-fluorochrome
unconjugated primary Abs followed by secondary staining
with anti-Ab fluorochrome conjugated Abs to amplify
fluorescence intensity. an anti-fluorochrome unconjugated
Ab during staining resulted in considerably improved
fluorescence intensity with both pMHC tetramers and
dextramers and with phycoerythrin (PE) ,
allophycocyanin (APC), or FITC-based reagents.

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FIGURE 2. Schematic representation of the test and control conditions used in this study. Alongside
a standard pMHC multimer (tetramer or dextramer) staining protocol (test 1), the binding of a mouse
anti-fluorochrome unconjugated 1° Ab to the pMHC multimer associated fluorochrome followed by a
goat anti-mouse conjugated 2° Ab (test 2).

8. Recombinant a and b Chains of MHC Class II
Tetramers with Affinity-Tagged Peptides
Targeting CD4+ T cells through their unique antigen-
specific, MHC class II-restricted T cell receptor makes
MHC class II tetramers an attractive strategy to identify,
validate and manipulate these cells at the single cell level.
Currently, Using class II chains expressed individually in
E. coli as versatile recombinant reagents, we have
previously generated peptide-MHC class II monomers, but
failed to generate functional class II tetramers.
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9. Adding a monomer purification principle based upon
affinity-tagged peptides, we here provide a robust method
to produce class II tetramers and demonstrate staining of
antigen-specific CD4+ T cells. We also provide evidence
that both MHC class II and T cell receptor molecules
largely accept affinity-tagged peptides. As a general
approach to class II tetramer generation, this method
should support rational CD4+ T cell epitope discovery as
well as enable specific monitoring and manipulation of
CD4+ T cell responses.
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10. As an approach to the preparation of peptide-HLA class II
monomers, the antigenic peptides were synthetically
extended with a C-terminal H6 sequence, which
subsequently allowed functional, monomeric peptide-HLA
complexes to be concentrated and purified by IMAC
chromatography. This raises the questionwhether HLA
class II in general will accept this kind of extension.
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11. A reasonable explanation of this apparent discrepancy is
that the a and b chains were kept together by the leucine
zipper in a soluble, but inactive, peptide-free form, and
that this complex was bound to the Ni2+-IDA-matrix
through the weak b chain interaction. In contrast, for the
molecules found in the later eluting peak 2 , the presence of
a and b chains in a 1:1 stoichiometry, and of L243
staining. demonstrated the presence of properly folded
monomer.
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12. Applications of tetramers
1. T cell epitope identification
2. Monitoring vaccine studies
3. Monitoring effect of Immunotherapy
4. Monitoring T-cell responses in infectious disease,
cancer, autoimmunity and other malignancies
5. Detection of antigen-specific T-cell responses in
immunological studies
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13. Epitope mapping
Epitope identification using a tetramer guided
mapping strategy. Large peptide libraries, separated into
sets of pooled sequences, are loaded into the binding
groove of recombinant class II molecules and assembled
into tetramers. Direct flow cytometry analysis of T cell
populations identifies peptides containing
actual epitopes representative of the natural immune
response
.
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15. MHC tetramers assay for analysis of virus-
specific T cells
 MHC tetramer production (A) Soluble MHC I heavy chain tagged at
the carboxyl terminus with BirA Substrate Peptide (BSP) is expressed
in E. coli. (B) The heavy chain is refolded with β2-microglobulin and
the specific peptide. This refolded MHC monomer is biotinylated with
the BirA enzyme at BSP site. (C) A tetramer is assembled from four
MHC–biotin complexes through biotin-streptavidin interaction. The
streptavidin is conjugated to a fluorochrome that allows it to be
detected on the flow cytometer. (D) Tetramer when added to the
lymphocytes binds to specific T-cell receptors on CD8 T cells, and with
the anti-CD8 antibody the antigen-specific T cell population can be
identified.
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17. Tetramer profiling of low-avidity T cells in
autoimmunity
class II pMHC tetramers containing autoantigenic
specificities Have been used to study T cell responses in a
variety of diseases, including type 1 diabetes (T1D), celiac
disease, pemphigus vulgaris, rheumatoid arthritis, multiple
sclerosis, and uveitis.
 tetramer analyses in the infectious pathogen and allergen
studies cited above, in which most of the responding T cells
have a high-avidity binding to appropriate pMHC
tetramers, tetramer binding to peripheral T cells in the
context of autoimmunity displays a much wider spectrum
of relative strength of interaction.
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18. In vivo tracking of tumor-specific T cells
The intensity of tetramer staining can also be correlated
with TCR affinity, which may be critical when vaccination
strategies to induce high-affinity tumor-reactive T cells
are evaluated. Furthermore, following sorting of T cells,
TCR usage or cDNA microarray analysis of specific T cells
can be performed Recently, the addition of confocal laser
scanning microscopy to tetramer analysis has enabled
precise anatomic localization of antigen-specific T cells in
tissues.
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19. Reference
 1-cellular and molecular immunology, 6th
ed, 2007
 2-MHC Class II Tetramers. Gerald T. Nepom.
doi:10.4049/jimmunol.1102398. J Immunol 2012; 188:
 3-Antibody Stabilization of Peptide–MHC Multimers Reveals Functional T
Cells Bearing Extremely Low-Affinity TCRs. The Journal of Immunology,
2015, 194: 463–474.
 4-MHC Class II Tetramers Made from Isolated Recombinant a and b Chains
Refolded with Affinity-Tagged Peptides. Francesco Dieli, University of
Palermo, Italy. June 3, 2013; Accepted July 22, 2013; Published September 2,
2013
 5-MHC tetramers assay for analysis of virus-specific T cells.Pervaiz A. Dar
and Sabrin H. Beituni
 6- Class II major histocompatibility complex tetramer staining: progress,
problems, and prospects. Sabrina S. Vollers and Lawrence J. Stern. Received
5 December 2007; revised 10, 1 December 2007; accepted 13 December 2007.
 7- In vivo tracking of tumor-specific T cells. Cassian Yee, Stanley R Riddell
and Philip D Greenberg
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