MS-Based HLA-II Peptidomics Combined With Multiomics Will Aid the Development of Future Immunotherapies

Abstract

Immunotherapies have emerged to treat diseases by selectively modulating a patient's immune response. Although the roles of T and B cells in adaptive immunity have been well studied, it remains difficult to select targets for immunotherapeutic strategies. Because human leukocyte antigen class II (HLA-II) peptides activate CD4+ T cells and regulate B cell activation, proliferation, and differentiation, these peptide antigens represent a class of potential immunotherapy targets and biomarkers. To better understand the molecular basis of how HLA-II antigen presentation is involved in disease progression and treatment, systematic HLA-II peptidomics combined with multiomic analyses of diverse cell types in healthy and diseased states is required. For this reason, MS-based innovations that facilitate investigations into the interplay between disease pathologies and the presentation of HLA-II peptides to CD4+ T cells will aid in the development of patient-focused immunotherapies.

Keywords: CD4 T cells; HLA-II; antigen; immunopeptidome; neoantigen.

Graphical abstract

Fig. 1

Overview schematic of HLA-II presentation by HLA-DR, HLA-DP, and HLA-DQ heterodimers.A, APCs, such as dendritic cells (DCs), B cells, and macrophages, express HLA-II heterodimers that present peptide antigens to CD4+ T cells. Upon T cell receptor (TCR)-mediated antigen recognition, CD4+ T cells release cytokines and chemokines that activate both B cells and CD8+ T cells. These cytokines can induce B cell class switching to plasma cells to promote antibody production. Simultaneously, this cytokine and chemokine release can cause upregulation of HLA-I expression on APCs such as DCs. B, example of HLA-II heterodimer pairing between alpha and beta chains expressed from HLA-DR, HLA-DP, and HLA-DQ alleles. HLA-DPA and HLA-DQA chains can pair with multiple HLA-DQB and HLA-DPB chains. Conversely, a monomorphic HLA-DRA chain binds to multiple DRB1 chains. DRB3/4/5 alleles in linkage with DRB1 can also be expressed and bind to the same DRA chain. Shown is an example heterodimer, DRB1∗11:01/DRA1∗01:01, to demonstrate possible binding motif registers and an example HLA-II-binding motif with anchor residues (colored) in positions P1, P4, P6, and P9. C, HLA-II allele frequency plots for HLA-DR (23) (http://www.allelefrequencies.net/) (24). APCs, antigen-presenting cells; HLA-II, human leukocyte antigen class II.

Fig. 2

Profiling the HLA-II processing and presentation pathway by LC-MS/MS. HLA-II heterodimers are formed in the endoplasmic reticulum when alpha and beta chains are paired and loaded with CD74 to prevent heterodimer dissociation. The MHC-II heterodimers are then trafficked to the MIIC complex, where CD74 is trimmed via proteases such as cathepsin S into CLIP peptides that act as placeholders to block peptide loading. Concurrently, antigen source proteins enter the MIIC by both endocytosis (exogenous proteins–purple) and autophagy (endogenous proteins–green). Before trafficking to the MIIC, source proteins are digested by proteases in endosomal/lysosomal compartments. Once cleaved by proteases, they are transported to the MIIC and loaded onto the HLA-II heterodimers with the help of the HLA-DM and HLA-DO chaperones, which removes CLIP peptides from the binding groove and facilitates the binding of antigen-derived peptides. Loaded HLA-II heterodimers are transported to the cell surface where circulating CD4+ T cells can recognize MHC-II-bound peptides via their TCR. As shown, HLA-II heterodimers on APCs tend to present peptides from endogenous proteins (autophagy) and exogenous source proteins (endocytosis). The repertoire of HLA-II-bound peptides can be profiled directly using LC-MS/MS to determine peptide sequences and abundances to retrospectively learn the rules of endogenous peptide processing and presentation and identify disease-specific CD4+ T cell targets. APCs, antigen-presenting cells; CLIP, class II–associated invariant chain peptide; HLA-II, human leukocyte antigen class II; MIIC, MHC class II compartment; TCR, T cell receptor.