Poster Presentation 31st Annual Lorne Proteomics Symposium 2026

Defining the spatial immunopeptidome of mouse spleen (#139)

Seyed Mohammad SJ Javadzadeh 1 , Patricia Illing 1 , Hong Ngoc Le 1 , Mohammadreza Dorvash 1 , Asolina Braun 1 , Shanzou Chung 1 , Anthony Purcell 1
  1. Monash University, Clayton, VIC, Australia

Background:
The immune system relies on major histocompatibility (MHC) molecules presenting peptide antigens on the cell surface (referred to as the immunopeptidome) for recognition by T cells (1-3). Immunopeptidomics refers to mass spectrometry–based identification and quantification of the immunopeptidome (4). Conventional immunopeptidomics isolates MHC from homogenised tissues or cells, losing spatial and cell type–specific information. Although spatial-omics has mapped proteins and/or transcripts within splenic microenvironments, including white pulp (WP), marginal zone (MZ), and red pulp (RP), no immunopeptidomics approach existed to define the spatial distribution of specific MHC-peptide complexes.

Aim:
To establish an immunopeptidomics workflow that resolves spatial presentation of specific MHC-peptide complexes across mouse spleen.

Methods:
The Leica LMD7 enabled isolation of different zonally distinct regions from healthy adult mouse spleen. 

In parallel, splenic B and T cells were enriched by Magnetic-Activated Cell Sorting (MACS) and MHC class I–bound peptides were isolated from all microdissected regions and the extracted peptides were analysed by LC–MS/MS using an EvoSEP One LC and Bruker timsTOF Pro2 system. MS data was acquired by DDA-PASEF methodologies and searched using PEAKS Online 10 (Bioinformatic Solutions Inc) against the reviewed mouse proteome (Swiss-Prot/UniProt). The flow through from each
immunoprecipitation was subjected to S-Trap-based tryptic digestion and the corresponding MHC-depleted proteomes acquired by ZenoSWATH-DIA using a SCIEX 7600 ZenoTOF MS equipped with an ACQUITY M Class UHPLC system. Data was analysed using DIA-NN 2.3 against an in-silico generated library from the reviewed mouse proteome (Swiss-Prot/UniProt).

Key findings:
LMD enabled isolation of WP, MZ, and RP, and successful regional differentiation was confirmed by comparison to adjacent H&E-stained sections. Peptides identified were predominantly 8–9 amino acids in length, and sequence motif analysis aligned with expected H-2Db and H-2Kb binding motifs. Region-specific antigen presentation was observed across spleen microenvironments, alongside shared peptides corresponding to the distribution of resident immune cells. MACS enrichment achieved high-purity B and T cell populations, and their immunopeptidomes closely matched those from MZ and WP, respectively, consistent with lymphocyte localisation in the spleen (5). DIA proteomics highlighted spatially distinct protein expression, with cell-type markers enriched within their anatomical niches, supporting the identity and spatial discrimination of the microdissected sections.
Conclusion
This study establishes a foundational spatial immunopeptidomics workflow integrating LMD with immunopeptidomics/proteomics to map regional antigen peptide presentation in the mouse spleen, enabling future application to diverse tissues to elucidate how microenvironmental context shapes immune surveillance.

  1. 1. Wieczorek M, Abualrous ET, Sticht J, Álvaro-Benito M, Stolzenberg S, Noé F, et al. Major Histocompatibility Complex (MHC) Class I and MHC Class II Proteins: Conformational Plasticity in Antigen Presentation. Frontiers in Immunology. 2017;Volume 8 - 2017.
  2. 2. Garstka MA, Fish A, Celie PHN, Joosten RP, Janssen GMC, Berlin I, et al. The first step of peptide selection in antigen presentation by MHC class I molecules. Proceedings of the National Academy of Sciences. 2015;112(5):1505-10.
  3. 3. Yewdell JW. MHC Class I Immunopeptidome: Past, Present, and Future. Mol Cell Proteomics. 2022;21(7):100230.
  4. 4. Abelin JG, Cox AL. Innovations Toward Immunopeptidomics. Mol Cell Proteomics. 2024;23(9):100823.
  5. 5. Schuster H, Shao W, Weiss T, Pedrioli PGA, Roth P, Weller M, et al. A tissue-based draft map of the murine MHC class I immunopeptidome. Sci Data. 2018;5:180157.
  6. 6. Schuster H, Shao W, Weiss T, Pedrioli PGA, Roth P, Weller M, et al. A tissue-based draft map of the murine MHC class I immunopeptidome. Sci Data. 2018;5:180157.
  7. 7. Marcu A, Bichmann L, Kuchenbecker L, Kowalewski DJ, Freudenmann LK, Backert L, et al. HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy. Journal for ImmunoTherapy of Cancer. 2021;9(4):e002071.
  8. 8. Shao W, Pedrioli PGA, Wolski W, Scurtescu C, Schmid E, Vizcaíno JA, et al. The SysteMHC Atlas project. Nucleic Acids Res. 2018;46(D1):D1237-d47.
  9. 9. Becker JP, Riemer AB. The Importance of Being Presented: Target Validation by Immunopeptidomics for Epitope-Specific Immunotherapies. Front Immunol. 2022;13:883989.
  10. 10. Mayer RL, Mechtler K. Immunopeptidomics in the Era of Single-Cell Proteomics. Biology. 2023;12(12):1514.
  11. 11. Liu S, Iorgulescu JB, Li S, Borji M, Barrera-Lopez IA, Shanmugam V, et al. Spatial maps of T cell receptors and transcriptomes reveal distinct immune niches and interactions in the adaptive immune response. Immunity. 2022;55(10):1940-52.e5.
  12. 12. Williams CG, Moreira ML, Asatsuma T, Skinner OP, Lee HJ, Li S, et al. Spatial transcriptomics maps molecular and cellular requirements for CD4<sup>+</sup> T cell-dependent immunity to malaria. bioRxiv. 2023:2023.02.23.529309.
  13. 13. Balogh P, Horváth G, Szakal AK. Immunoarchitecture of Distinct Reticular Fibroblastic Domains in the White Pulp of Mouse Spleen. Journal of Histochemistry & Cytochemistry. 2004;52(10):1287-98.
  14. 14. Liu Y, DiStasio M, Su G, Asashima H, Enninful A, Qin X, et al. High-plex protein and whole transcriptome co-mapping at cellular resolution with spatial CITE-seq. Nat Biotechnol. 2023;41(10):1405-9.
  15. 15. Goltsev Y, Samusik N, Kennedy-Darling J, Bhate S, Hale M, Vazquez G, et al. Deep Profiling of Mouse Splenic Architecture with CODEX Multiplexed Imaging. Cell. 2018;174(4):968-81.e15.
  16. 16. Zhu Y, Dou M, Piehowski PD, Liang Y, Wang F, Chu RK, et al. Spatially Resolved Proteome Mapping of Laser Capture Microdissected Tissue with Automated Sample Transfer to Nanodroplets*. Molecular & Cellular Proteomics. 2018;17(9):1864-74.
  17. 17. Lewis SM, Williams A, Eisenbarth SC. Structure and function of the immune system in the spleen. Sci Immunol. 2019;4(33).
  18. 5. Schuster H, Shao W, Weiss T, Pedrioli PGA, Roth P, Weller M, et al. A tissue-based draft map of the murine MHC class I immunopeptidome. Sci Data. 2018;5:180157.
  19. 5. Schuster H, Shao W, Weiss T, Pedrioli PGA, Roth P, Weller M, et al. A tissue-based draft map of the murine MHC class I immunopeptidome. Sci Data. 2018;5:180157.
  20. 6. Schuster H, Shao W, Weiss T, Pedrioli PGA, Roth P, Weller M, et al. A tissue-based draft map of the murine MHC class I immunopeptidome. Sci Data. 2018;5:180157.