Defining the spatial immunopeptidome of mouse spleen — ASN Events
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  2. Lunch & Poster Session 2
  3. Defining the spatial immunopeptidome of mouse spleen

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.

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