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Use of modified peptides for live tracking of peptide-MHCII complexes during antigenspecific T cell-dendritic cell interactions

Jafar A Al Souz, Munir Akkaya, Rahul Kamdar, Olena Kamenyeva, Juraj Kabat, Ethan M Shevach and Billur Akkaya
J Immunol May 1, 2019, 202 (1 Supplement) 130.2;
Jafar A Al Souz
1LISB, NIAID, NIH
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Munir Akkaya
2LIG, NIAID, NIH
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Rahul Kamdar
1LISB, NIAID, NIH
3Univ. of Maryland, Baltimore County
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Olena Kamenyeva
4RTB, NIAID, NIH
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Juraj Kabat
4RTB, NIAID, NIH
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Ethan M Shevach
1LISB, NIAID, NIH
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Billur Akkaya
1LISB, NIAID, NIH
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Abstract

The recognition of peptide-MHC class II complexes (pMHCII) presented by dendritic cells (DCs) is critical for the activation of CD4+ T cells, and proper detection of such complexes is essential for understanding T cell-DC interactions. However, current detection methods for pMHCII complexes using antibodies may block cellular interactions, limiting their use, while conventional tags, such as GFP, are too large to be loaded onto MHCII without disrupting its function. To probe pMHCII complexes, we constructed a variant of the ovalbumin peptide (OVA323–339) linked to a tetracysteine tag (CCPGCC), which can be treated with organoarsenic compounds to induce fluorescence. This modified peptide retained its functionality as it was able to stimulate T cells to the same degree as the unmodified peptide. We also analyzed signal specificity both at the pMHCII level, by using MHCII−/− APCs, and at a broader level by quenching endogenous tetracysteine expression, and confirmed that our fluorescent signal was derived from the peptide modification. We then used microscopy to visualize pMHCII movement after interaction of pulsed DCs with OVA-specific T cells in vitro and subsequently in vivo, and detected a gradual increase of pMHCII complexes at the synapse. Interestingly, we also observed a transfer of fluorescent signal from DCs to antigen-specific T cells, but not to polyclonal T cells, indicating acquisition of pMHCII, which we were able to quantify. Taken together, our results show a non-disruptive approach to assay pMHCII, and provide a window to observe intricate T cell-DC interactions in real-time.

  • Copyright © 2019 by The American Association of Immunologists, Inc.
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The Journal of Immunology
Vol. 202, Issue 1 Supplement
1 May 2019
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Use of modified peptides for live tracking of peptide-MHCII complexes during antigenspecific T cell-dendritic cell interactions
Jafar A Al Souz, Munir Akkaya, Rahul Kamdar, Olena Kamenyeva, Juraj Kabat, Ethan M Shevach, Billur Akkaya
The Journal of Immunology May 1, 2019, 202 (1 Supplement) 130.2;

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Use of modified peptides for live tracking of peptide-MHCII complexes during antigenspecific T cell-dendritic cell interactions
Jafar A Al Souz, Munir Akkaya, Rahul Kamdar, Olena Kamenyeva, Juraj Kabat, Ethan M Shevach, Billur Akkaya
The Journal of Immunology May 1, 2019, 202 (1 Supplement) 130.2;
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Print ISSN 0022-1767        Online ISSN 1550-6606