Abstract

Type 1 conventional dendritic cells (cDC1s) are a subset of antigen-presenting cells required for the activation of T cells during anti-viral and anti-tumoral immune responses. cDC1s specialize in cross-presentation (XP), or the MHC-I presentation of exogenous antigens, which allows them to prime antigen-specific CD8+ T cells. In classical MHC-I presentation, cytosolic antigens are processed through the endoplasmic reticulum (ER), relying on both proteasomes and transport associated with antigen processing (TAP). In contrast, the molecular mechanism of XP remains less understood, especially in regard to how the antigens are processed, how the peptides are loaded, and why this function is specialized by cDC1s. WDFY4, a member of the beige and Chédiak-Higashi (BEACH) domain protein family, is absolutely required in cDC1s for XP of cell-associated antigens present during viral and tumor challenges. Indeed, mice deficient in Wdfy4 fail to induce antigen-specific CD8+ T cells due to the inability of cDC1s to cross-present cell-associated antigens. Structural modeling indicates that WDFY4 consists of two extensive alpha-solenoid regions (armadillo repeats) that flank an extended Concanavalin A-like lectin domain, followed by a Pleckstrin homology and BEACH domain and a C-terminal WD40 repeat, suggesting a potential scaffolding role in vesicular trafficking. High resolution Airyscan imaging of endogenous 3xFLAG-tagged WDFY4 reveals its constitutive sequestration in a juxtanuclear cleft of cDC1s. This compartment colocalizes with RAB43, a small GTPase required for optimal XP, and is architecturally distinct from classical trafficking organelles. Furthermore, unlike cDC2s and macrophages, an intracellular pool of mature MHC-I molecules can be observed at this compartment in cDC1s. Upon antigen uptake, both WDFY4 and RAB43 are enriched around the antigen-containing phagosomes. To identify WDFY4 interactors, we utilized proximity labeling and in situ cross-link mass spectrometry in MutuDC1940 cell line. This identified VAC14-PIKFYVE-FIG4 complex as a primary interacting partner. Additionally, VAC14 colocalized with WDFY4, suggesting endogenous interaction between VAC14 and WDFY4 in cDC1s. The multidomain nature of WDFY4 and its cellular localization and interactions suggest that WDFY4 may act as a scaffold along with VAC14 to facilitate the specialized transport of internalized antigen towards the MHC-I-containing compartment, providing a new molecular framework for understanding XP in cDC1s.

Committee Chair

Kenneth Murphy

Committee Members

Daved Fremont; David Pagliarini; Gwendalyn Randolph; Takeshi Egawa

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Immunology)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

4-28-2026

Language

English (en)

Author's ORCID

https://orcid.org/0000-0001-7897-3169

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