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MHC class II tetramers engineered for enhanced binding to CD4 improve detection of antigen-specific T cells


The ability to identify T cells that recognize specific peptide antigens bound to major histocompatibility complex (MHC) molecules has enabled enumeration and molecular characterization of the lymphocytes responsible for cell-mediated immunity. Fluorophore-labeled peptide:MHC class I (p:MHCI) tetramers are well-established reagents for identifying antigen-specific CD8+ T cells by flow cytometry, but efforts to extend the approach to CD4+ T cells have been less successful, perhaps owing to lower binding strength between CD4 and MHC class II (MHCII) molecules. Here we show that p:MHCII tetramers engineered by directed evolution for enhanced CD4 binding outperform conventional tetramers for the detection of cognate T cells. Using the engineered tetramers, we identified about twice as many antigen-specific CD4+ T cells in mice immunized against multiple peptides than when using traditional tetramers. CD4 affinity-enhanced p:MHCII tetramers, therefore, allow direct sampling of antigen-specific CD4+ T cells that cannot be accessed with conventional p:MHCII tetramer technology. These new reagents could provide a deeper understanding of the T cell repertoire.

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Fig. 1: Identification of I-Ab molecules with enhanced binding to CD4.
Fig. 2: Detection of polyclonal CD4+ T cells from immunized mice using p:I-Ab-4E tetramers.
Fig. 3: Detection of polyclonal CD4+ effector T cell p:I-Ab-4E tetramers is peptide specific.


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This work was supported by National Institutes of Health grants R01 AI143826 and R01 AI039614 to M.K.J., F32 AI114050 to D.M., T32 AI083196 and T32 AI007313 to D.I.K. and R01 AI096879 to B.D.E.

Author information




T.D., D.M., D.I.K., P.D.K., and E.M.K. designed and performed experiments and edited the manuscript. B.D.E. designed experiments and edited the manuscript. M.K.J. designed experiments and wrote the manuscript.

Corresponding author

Correspondence to Marc K. Jenkins.

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Experiments were approved by the University of Minnesota Institutional Animal Care and Use Committee and conducted in accordance with its policies.

Competing interests

M.K.J, T.D. and D.M. are co-inventors on a patent application covering CD4 affinity enhanced p:MHCII tetramers owned by Regents of the University of Minnesota (#PCT/US19/44605 – Co-receptor affinity enhanced major histocompatibility class II molecules).

Additional information

Peer review information Nature Biotechnology thanks Lawrence Stern and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Dileepan, T., Malhotra, D., Kotov, D.I. et al. MHC class II tetramers engineered for enhanced binding to CD4 improve detection of antigen-specific T cells. Nat Biotechnol (2021).

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