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May 12, 2017

Lights, Camera, T-Cell Tentacle Action!



    Time-resolved lattice light-sheet microscopy and quantum dot–enabled synaptic contact mapping microscopy show how microvilli on the surface of T cells search opposing cells and surfaces before and during antigen recognition. [Krummel Lab/UCSF]


  • “All right, Dr. Krummel, I'm ready for my close-up." That’s what the T cell might have said to the director of a video project focused on microvillar dynamics. As you would guess from the word “dynamics,” the T cell isn’t playing the everyman or romantic lead. It’s more of an action hero, sensing danger, leaping into action, and ultimately besting microscopic villains.

    The videomaker who would make the T cell an A-list science celebrity is Matthew Krummel, Ph.D., associate professor of pathology at the University of California, San Francisco (UCSF). Dissatisfied with earlier treatments of the T-cell microvilli story, Dr. Krummel and his team decided to record microvilli in motion, not just in static poses. To achieve this feat, they used lattice light-sheet and quantum dot–enabled synaptic contact mapping microscopy.

    "Previous techniques had allowed us to take snapshots of the surface of T cells," said Dr. Krummel. "Now we can watch these amazing little fingers of membrane move around in real-time—and it turns out they're incredibly efficient."

    Among other potential benefits, notes Dr. Krummel, understanding how T cells efficiently sample their environment to search for invasive pathogens opens up new questions about what countermeasures infectious organisms or even cancer cells may have evolved as a way of avoiding detection, and could suggest new ways for researchers to help T cells see through such a ruse.

    Dr. Krummel’s videos premiered May 12 in the journal Science, in an article entitled “Visualizing Dynamic Microvillar Search and Stabilization during Ligand Detection by T Cells.” The article describes how Dr. Krummel’s UCSF team was able to study how T cells efficiently interrogate antigen-presenting cells (APCs) in real time.

    “It has long been supposed that microvilli on T cells act as sensory organs to enable search, but their strategy has been unknown,” wrote the article’s authors. “We showed that anomalous diffusion and fractal organization of microvilli survey the majority of opposing surfaces within 1 minute.”

    Essentially, the USCF team studied mouse T cells exploring simulated patches of APC membrane in laboratory dishes and found that the T-cell microvilli move independently of one another in a fractal-like geometry, such as is often seen in nature as a way of optimizing efficient use of space, such as by plant roots or foraging animals.

    The researchers calculated that, thanks to this efficient search pattern, in an average minute-long encounter win an APC, T-cell microvilli can thoroughly explore 98% of the contact surface between the two cells—called an "immunological synapse" after the neuronal synapses of the nervous system. This suggests that T cells are tuned to spend the minimum time necessary to get a clear read on the information available at each APC before moving on, the authors indicated.

    “Based on these findings,” the authors continued, “the palpation of opposing cell surfaces by dynamic microvilli on T cells underlies TCR recognition. These microvillar dynamics impose a time pressure for ligands to solidify interactions with an opposing surface.”

    To study the details of threat detection by microvilli, the authors devised a new approach that allowed them to simultaneously track microvilli as well as the T-cell receptor (TCR) proteins T cells use to detect their target antigens. To do this, the team covered simulated patches of APC membrane with tiny fluorescent particles called quantum dots, which questing T-cell microvilli had to push out of the way to reach the membrane surface. This technique, dubbed synaptic contact mapping, allowed the researchers to visualize the microvilli as holes of negative space in the quantum dot fluorescence, while at the same time visualizing TCRs with a different-colored fluorescent marker.

    They found that, normally, individual microvilli poke and prod at the APC membrane for an average of about 4 seconds at a time. But when the microvilli found the antigen they were searching for, they stayed in contact with the APC membrane for 20 seconds or more and accumulated large rafts of TCRs, suggesting that they were likely signaling the T cell to trigger its immune response.

    "These videos give me a much more visceral understanding of what's happening when T cells and APCs come into contact," asserted Dr. Krummel. "T cells have these anemone-like sensory organs, and when they want to get information from another cell, their only chance appears to be during this short period of intimate contact. If they don't detect a strong signal during that contact, they move on."

    Dr. Krummel's team also briefly studied the surfaces of other types of immune cells, such as dendritic cells and B cells, which play different roles in pathogen detection and immune response. They found that each cell type appears to use distinct patterns of surface protrusions—such as tentacles, waves, or curtain-like ripples—to probe and communicate with their environments, though more research is needed to understand these diverse patterns and how they interact with one another.

    "Understanding how the immune system reliably detects and responds to the huge range of potential threats it has to deal with is one of the key questions we still face as immunologists," stated Dr. Krummel. "Of course, the immune system also makes mistakes—like when it attacks the body's own cells in autoimmune disease or fails to recognize cancerous cells as a threat. Understanding the mechanics and constraints of how the immune system recognizes threats in the first place could potentially help us correct those errors."

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