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Review
. 2015 Nov;136(5):1150-62.
doi: 10.1016/j.jaci.2015.03.049. Epub 2015 Jun 13.

Insights into primary immune deficiency from quantitative microscopy

Emily M Mace  1 , Jordan S Orange  2
Affiliations
Review

Insights into primary immune deficiency from quantitative microscopy

Emily M Mace et al. J Allergy Clin Immunol. 2015 Nov.

Abstract

Recent advances in genomics-based technology have resulted in an increase in our understanding of the molecular basis of many primary immune deficiencies. Along with this increased knowledge comes an increased responsibility to understand the underlying mechanism of disease, and thus increasingly sophisticated technologies are being used to investigate the cell biology of human immune deficiencies. One such technology, which has itself undergone a recent explosion in innovation, is that of high-resolution microscopy and image analysis. These advances complement innovative studies that have previously shed light on critical cell biological processes that are perturbed by single-gene mutations in primary immune deficiency. Here we highlight advances made specifically in the following cell biological processes: (1) cytoskeletal-related processes; (2) cell signaling; (3) intercellular trafficking; and (4) cellular host defense.

Keywords: Primary immune deficiency; cell biology; cytoskeleton; host defense; microscopy.

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Figures

Figure 1
Figure 1. Effect of primary immune deficiency on the actin cytoskeleton
A) Pathway leading to F-actin polymerization and structural rearrangement at the immune synapse. Upon activation, autoinhibition of WASP by WIP is relieved. DOCK8 promotes both WASP recruitment and Cdc42 function. WASP and enables activation of Arp2/3, which forms de novo branched actin filaments that are required for firm adhesion and receptor clustering at the immune synapse. Existing branched filaments undergo depolymerization and/or rearrangement by Coronin 1A. B) Effect of primary immune deficiency affecting the actin cytoskeleton as measured by high-resolution microscopy. i) transmission electron micrograph of F-actin at the NK cell immune synapse from a healthy donor (left) or WAS patient (right). Note the absence of F-actin filaments and lack of branched network as a result of the loss of WASp-driven arp2/3 function. ii) Lytic granule polarization in NK cells from a healthy donor (left) or Myosin IIA-deficient cell line (right) conjugated to susceptible target cells. MYH9-RD patients suffer NK cell deficiency due to an inability of NK cells to appropriately polarize to the immune synapse and subsequently degranulate and lyse virally infected target cells. Conjugates were fixed and stained for perforin (blue) or tubulin (green). iii) F-actin accumulation at the immune synapse formed between a WASp-deficient (left) or WASp-expressing (right) NK cell isolated from the peripheral blood of a WAS patient treated by lentiviral gene therapy. Note the restored F-actin accumulation at the immune synapse (white arrow) that accompanies restored WASp expression. Conjugates were fixed and stained for F-actin (red). iv) Defective F-actin accumulation at the immune synapse (white arrow) in NK cells isolated from a DOCK8-deficient patient when compared to a healthy donor (left). Conjugates were fixed and stained for F-actin (red). v) impaired lytic granule access to the plasma membrane in the absence of Coro1A function in a Coro1AKD human NK cell line (bottom) compared to the control line (top). Cells were fixed and stained for F-actin (green) and lytic granules (red) then visualized in the XZ plane by STED microscopy.
Figure 2
Figure 2. Intracellular trafficking leads to lytic granule exocytosis
A) Lytic granules from NK cells expressing the degranulation indicator LAMP1-pHlourin were labeled with lysotracker red for visualization prior to degranulation. Shown is a single granule undergoing exocytosis as marked by a transition from acidified organelle (red) to neutralization and exposure of the pH sensitive GFP variant to the neutral extracellular environment. Image was acquired by TIRF microscopy at the plasma membrane of an NK cell activated on glass. B) Table of primary immune deficiencies affecting intracellular trafficking and their effect on hair pigmentation and lytic granule polarization. Microscopy references refer to those cited in this review that have examined the specific effect on granule behavior through quantitative microscopy.
Figure 3
Figure 3. Cellular host defense through the formation of NETs
A) NET formation requires both phagosome formation and reactive oxygen species (ROS). B) NET formation is impaired in neutrophils from CGD patients. Control (left) or patient (right) neutrophils were stimulated with PMA and ionomycin and imaged 5h later by scanning electron microscopy. Scale bar=10 mm. Image courtesy of Dr. Janine Reichenbach, University Children's Hospital Zurich.
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