Affinity-matured HLA class II dimers for robust staining of antigen-specific CD4<sup>+</sup> T cells thumbnail

Affinity-matured HLA class II dimers for robust staining of antigen-specific CD4+ T cells

Abstract

Peptide–major histocompatibility complex (pMHC) multimers enable the detection of antigen-specific T cells in studies ranging from vaccine efficacy to cancer immunotherapy. However, this technology is unreliable when applied to pMHC class II for the detection of CD4+ T cells. Here, using a combination of molecular biological and immunological techniques, we cloned sequences encoding human leukocyte antigen (HLA)-DP, HLA-DQ and HLA-DR molecules with enhanced CD4 binding affinity (with a Kd of 8.9 ± 1.1 µM between CD4 and affinity-matured HLA-DP4) and produced affinity-matured class II dimers that stain antigen-specific T cells better than conventional multimers in both in vitro and ex vivo analyses. Using a comprehensive library of dimers for HLA-DP4, which is the most frequent HLA allele in many ancestry groups, we mapped 103 HLA-DP4-restricted epitopes derived from diverse tumor-associated antigens and cloned the cognate T-cell antigen receptor (TCR) genes from in vitro-stimulated CD4+ T cells. The availability of affinity-matured class II dimers across HLA-DP, HLA-DQ and HLA-DR alleles will aid in the investigation of human CD4+ T-cell responses.

Data availability

The main data of this study are available within the article and its Supplementary Figures. Source data are provided with this paper. All other data are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by the Ontario Institute for Cancer Research Clinical Investigator Award IA-039 (N.H.), the Princess Margaret Cancer Centre Innovation Accelerator Fund (N.H.), the Ira Schneider Memorial Cancer Research Foundation (N.H.), the Princess Margaret Cancer Foundation (N.H. and M.O.B.), the Uehara Memorial Foundation Research Fellowship Program (K. Sugata), the Mitacs Internship (K.M.), the Japan Society for the Promotion of Science Postdoctoral Fellowship for Overseas Researchers and the Guglietti fellowship (Y.K.), the Province of Ontario (T.G. and M.A.), the Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship (T.G.) and the Frederick Banting and Charles Best Canada Graduate Scholarship (C.-H.W.).

Author information

Author notes

  1. These authors contributed equally: Kenji Sugata, Yukiko Matsunaga.

Affiliations

  1. Tumor Immunotherapy Program, Campbell Family Institute for Breast Cancer Research, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada

    Kenji Sugata, Yukiko Matsunaga, Yuki Yamashita, Munehide Nakatsugawa, Tingxi Guo, Yota Ohashi, Kayoko Saso, Muhammed A. Rahman, Mark Anczurowski, Chung-Hsi Wang, Kenji Murata, Hiroshi Saijo, Yuki Kagoya, Dalam Ly, Brian D. Burt, Marcus O. Butler, Tak W. Mak & Naoto Hirano

  2. Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada

    Levon Halabelian

  3. Department of Immunology, University of Toronto, Toronto, Ontario, Canada

    Yota Ohashi, Mark Anczurowski, Chung-Hsi Wang, Marcus O. Butler, Tak W. Mak & Naoto Hirano

  4. Department of Medicine, University of Toronto, Toronto, Ontario, Canada

    Marcus O. Butler

Contributions

K. Sugata, Y.M., Y.Y., M.N. and N.H. designed the project. K. Sugata, Y.M., Y.Y., M.N., T.G., L.H., K. Saso, M.A.R., M.A., C.-H.W., K.M., H.S., Y.K., Y.O., D.L. and B.D.B. performed the experiments. M.O.B. provided critical human samples. T.W.M. provided critical resources. N.H. administered and supervised the project. K. Sugata and N.H. analyzed the results and wrote the manuscript.

Corresponding author

Correspondence to
Naoto Hirano.

Ethics declarations

Competing interests

M.O.B. has served on advisory boards for Merck, BMS, Novartis, GSK, Immunocore, Immunovaccine, Sanofi and EMD Serono and received research funding for investigator-initiated clinical trials from Merck and Takara Bio. N.H. has received research funding from Takara Bio and served as a consultant for Takara Bio. The University Health Network has filed a patent application related to this study on which N.H., K. Sugata, Y.Y., M.N., K. Saso, M.A.R. and T.G. are named as inventors. T.W.M. and N.H. are cofounders and have equity in TCRyption to which the technologies used in this study have been licensed.

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

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Sugata, K., Matsunaga, Y., Yamashita, Y. et al. Affinity-matured HLA class II dimers for robust staining of antigen-specific CD4+ T cells.
Nat Biotechnol (2021). https://doi.org/10.1038/s41587-021-00836-4

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