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.
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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.).
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.
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