# Defining the four major routes of MHC class II antigen processing and presentation with influenza antigens

> **NIH NIH R01** · CHILDREN'S HOSP OF PHILADELPHIA · 2024 · $614,983

## Abstract

PROJECT SUMMARY
CD4+ T cells (TCD4) orchestrate adaptive immune responses to infectious agents and cancers and instigate most
autoimmune diseases. Thus, greater mechanistic insight into the drivers of TCD4 activation will be broadly bene-
ficial. TCD4 are stimulated by antigen-derived peptides (“epitopes”) displayed at the surfaces of antigen-presenting
cells (APCs) in combination with major histocompatibility complex class II (MHCII) molecules. By convention,
peptide display entails internalization and proteolysis of extracellular antigen, loading of the resultant peptides
onto MHCII in the late endosome, and transit of peptide:MHCII (p:MHCII) complexes to the cell surface. In truth,
p:MHCII production is far more complex. The classical route is made up of many distinct pathways, and there
are 3 multifaceted, nonclassical routes: 1) a recycling route: antigens that unfold in the early endosome are
captured by MHCII in that compartment. 2) an endogenous network: antigens located to the APC cytoplasm,
typically through biosynthesis, are converted to p:MHCII via an array of intracellular pathways, and 3) an “indi-
rect” route: material from an infected non-APC is transferred to an uninfected APC. Despite their obscurity, these
non-classical routes can play essential roles in TCD4 activation. Poor mechanistic understanding of all 4 routes,
even the classical, has precluded their incorporation into many models of adaptive immunity or strategies to
modulate TCD4+ responses. Here we propose to transform the MHCII processing and presentation landscape by
testing the hypotheses that: (A) the degree to which cellular components contribute to epitope production and
display is highly variable - from far reaching (impacting many epitopes) to highly focused (impacting only one
epitope or a small set), and (B) processing and presentation pathways within one route or within functionally
adjacent routes (e.g., classical and recycling), share more cellular components than disparate routes (e.g., clas-
sical and endogenous). Drawing from our influenza (flu) antigen system, we have carried out an siRNA-based
high throughput screen (HTS) for proteins involved in generation of one classical (“S1”) and one endogenous
(“NA79”) epitope. Consistent with our hypothesis, hits are largely non-overlapping. Aim 1 of this project is to
solidify this screen by validating key hits. Aim 2 is to expand the landscape by screening 6 flu epitopes that
comprehensively encompass the 4 major routes. Instead of an siRNA screen, in Aim 2 we will use genome-wide
CRISPR-Cas9 knockout and select hits via FACS using a panel of T cell receptor-like antibodies in concert with
single-cell illumina sequencing. Outcomes from both aims will contribute to Gene Set Enrichment Analysis that
will substantially revise the MHCII processing and presentation landscape with respect to both scope and reso-
lution. Outcomes will launch many new avenues of investigation that explore: a) additional epitopes fro...

## Key facts

- **NIH application ID:** 10981093
- **Project number:** 1R01AI180250-01A1
- **Recipient organization:** CHILDREN'S HOSP OF PHILADELPHIA
- **Principal Investigator:** Laurence Crane Eisenlohr
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $614,983
- **Award type:** 1
- **Project period:** 2024-06-01 → 2029-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10981093

## Citation

> US National Institutes of Health, RePORTER application 10981093, Defining the four major routes of MHC class II antigen processing and presentation with influenza antigens (1R01AI180250-01A1). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10981093. Licensed CC0.

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