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An AXL/LRP-1/RANBP9 complex mediates DC efferocytosis and antigen cross-presentation in vivo
Manikandan Subramanian, … , Madepalli Lakshmana, Ira Tabas
Manikandan Subramanian, … , Madepalli Lakshmana, Ira Tabas
Published February 10, 2014
Citation Information: J Clin Invest. 2014;124(3):1296-1308. https://doi.org/10.1172/JCI72051.
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Research Article Immunology

An AXL/LRP-1/RANBP9 complex mediates DC efferocytosis and antigen cross-presentation in vivo

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Abstract

The phagocytosis of apoptotic cells (ACs), or efferocytosis, by DCs is critical for self-tolerance and host defense. Although many efferocytosis-associated receptors have been described in vitro, the functionality of these receptors in vivo has not been explored in depth. Using a spleen efferocytosis assay and targeted genetic deletion in mice, we identified a multiprotein complex — composed of the receptor tyrosine kinase AXL, LDL receptor–related protein–1 (LRP-1), and RAN-binding protein 9 (RANBP9) — that mediates DC efferocytosis and antigen cross-presentation. We found that AXL bound ACs, but required LRP-1 to trigger internalization, in murine CD8α+ DCs and human-derived DCs. AXL and LRP-1 did not interact directly, but relied on RANBP9, which bound both AXL and LRP-1, to form the complex. In a coculture model of antigen presentation, the AXL/LRP-1/RANBP9 complex was used by DCs to cross-present AC-associated antigens to T cells. Furthermore, in a murine model of herpes simplex virus–1 infection, mice lacking DC-specific LRP-1, AXL, or RANBP9 had increased AC accumulation, defective viral antigen-specific CD8+ T cell activation, enhanced viral load, and decreased survival. The discovery of this multiprotein complex that mediates functionally important DC efferocytosis in vivo may have implications for future studies related to host defense and DC-based vaccines.

Authors

Manikandan Subramanian, Crystal D. Hayes, Joseph J. Thome, Edward Thorp, Glenn K. Matsushima, Joachim Herz, Donna L. Farber, Kang Liu, Madepalli Lakshmana, Ira Tabas

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Figure 1

In vivo splenic DC efferocytosis assay.

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In vivo splenic DC efferocytosis assay.
(A) Flow cytometric detection of...
(A) Flow cytometric detection of cells that costained for PKH67 (square gate) in the splenocytes of mice injected with PKH67-labeled ACs. (B) Analysis and quantification (n = 3 per group) of the AC-gated region in A to determine the proportion of ACs that were internalized by splenic DCs (CD11c+) in C57BL/6J mice injected with vehicle control (Con) or the DC maturation stimulus CpG-DOTAP. Numbers represent the percentage of cells in the respective quadrants. (C) Flow cytometric analysis and quantification (n = 3 mice per group) of the distribution of internalized ACs between the CD8α+ and CD8α– subsets of CD11c+PKH67+-gated splenocytes. (D) Top: Confocal microscopy z section sequence (top-to-bottom at 2-μm intervals) of FACS-sorted CD11c+PKH67+ splenic DCs (representative of cells in the top-right quadrant of B), demonstrated the presence of ACs inside the DCs. Red, PE staining of splenic CD11c+ cells; green, PKH67-labeled ACs. Bottom: z section sequence of BMDCs (red) treated with 5 μg/ml cytochalasin D (CytoD) prior to addition of ACs (green) to demonstrate what a DC would look if there was AC binding, but not engulfment. Scale bars: 5 μm. (E) Comparison of in vivo splenic DC efferocytosis in mice injected with ACs labeled with PKH67 or Cypher-5E (n = 3 per group). *P < 0.05 vs. respective control.

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