Avi Kupfer stares at his computer monitor, intently watching a movie of immune cells mingling with each other. He points to one particular cell, which glows with a soft green. “This is a CD4 T-cell,” he says. “They are like the head housekeepers of the immune system. They go around and probe the other cells in our body to make sure everyone who’s there belongs in the neighborhood.”

This ability to properly distinguish intruders and then destroy them is vitally important. Without CD4 cells doing their thing, disease-causing bacteria or malignant tumor cells can spread like wildfire. On the other hand, if these cells get too aggressive, they start ravaging our own body, leading to autoimmune disease. The attacks can occur anywhere, from our joints (rheumatoid arthritis) to our spinal cord (multiple sclerosis) or even multiple tissues (lupus). Nearly one fifth of the world’s population suffers with one of the 18 known autoimmune conditions. “So this,” says Kupfer, “is not a minor concern.”

While what happens inside these T-cells is pretty well defined, Kupfer says, the question of what happens between the cells is not. Knowing that the immune system is always just one mixed-up message from disaster, Kupfer is on a quest to get ever better pictures of this intercellular communication process in action.

A little more than a decade ago, Kupfer radically changed the prevailing view of how immune cells “talk” when he provided the first visual evidence showing that CD4 cells communicate with each other via what he came to dub immune “synapses.” Now, in his third year at Johns Hopkins, as co-director of the Immunology Program at the Institute for Cell Engineering, Kupfer is building rapidly on that work. He’s using the latest in 4-D imaging technology to offer unparalleled insights into just how the immune synapse functions.

The potential for healing human suffering is vast: Beyond finding better ways to prevent autoimmune disease, understanding how the synapse works could lead to improved control over immune suppression. “For example, CD4 cells might be reconfigured so they won’t turn on following a transplant operation,” Kupfer says excitedly. “We could help prevent organ rejection without the use of any drugs.” 

Born and educated in Israel, Avi Kupfer still recalls the quiet elation he felt that afternoon back in 1995 when he stood before an auditorium of his colleagues at a meeting in Cold Spring Harbor. They sat mesmerized as he flashed before them the first 3-D microscopical images of the immune synapse in action.

“The term immunological synapse had become a mantra; this was the secret we were trying to figure out,” recalls Michael Dustin, a colleague of Kupfer’s who studies T-cell activation at NYU’s Skirball Institute of Biomolecular Medicine. “Not long afterwards I saw Avi’s images in Charles Janeway’s immunology book; this might be the only result to make it into the ‘textbooks’ before it appeared in a peer-reviewed journal.”

To most scientists, the word synapse conjures up images of the body’s nervous system. Synapses are formed in the gaps between two nerve cells or a nerve cell and its action target, such as a muscle cell. These adhesive junctions ensure quick and efficient transfer of every message traveling through the neural network. Without them, these messages might take wayward detours or end up getting scrambled like a game of telephone.

The notion of an immune synapse was first suggested in 1984 by National Institutes of Health researcher Michael Norcross. He also suggested that the immune and nervous systems may both have arisen from the same ancestral signaling pathway. Over the next decade, the idea began to pick up steam. After all, both systems contain specialized cell types that communicate with each other using a wide array of chemical signals, both rely on a method of memory storage and retrieval in order to function well, and both require cell-to-cell contact to acquire information.

Plus, recalls Kupfer, there was more: “On any given CD4, the exact same surface receptor can produce wildly different end results depending on which signal hits it,” he notes. “It could be a productive outcome like rapid T-cell division, or it could be the exact opposite: cell death. I, and others, thought, There must be some unique events on the surface that tell immune cells what to do.”

But how to watch those events unfold? To do so, Kupfer built on pioneering work in immunofluorescence he’d done as a postdoc in the lab of Jonathan Singer at the University of California, San Diego. Under normal viewing, cells are fairly transparent. But Singer had been using antibodies to coat proteins in cells to bring out more miscroscopic detail and better distinguish internal components.

Kupfer first used immunofluorescence to observe the movement of a fibroblast, a cell that travels to sites of injury to help repair damaged connective tissue. He’d found that as cells approached the wound site, two internal structures—the Golgi and microtubules—in each fibroblast rotated like the hands of a clock to both point toward the edge of the wound.

Kupfer understood the significance right away. The Golgi acts like a processing plant, sorting and packaging all the proteins that need to go to the cell surface or outside of the cell; microtubules, among other roles, serve as molecular conveyor belts that help transport these packaged proteins to their destination. Together, this implied that the fibroblasts had designed a directed flow of traffic to maximize the delivery of their repair proteins.

Intrigued, Kupfer began adapting immunofluorescence toward immune cells, largely unheard of at the time. “Immunology was studied as more of a systems process,” says Kupfer. “Researchers would look at how all the different immune cells worked together to respond to infection. Not many set their sights at the level of a single cell.”

Collaborating with Gunther Dennert, a researcher at the nearby Salk Institute, he took on T-cells—shock troops that destroy virus- or bacteria- infected cells by hitting them with a potent barrage of toxins—and found the same clockwork shifting of the Golgi and microtubules shortly after they made contact with their target cells.

Dennert recalls the finding as “a giant step forward.”

Kupfer had been bitten by the imaging bug and now, with the internal motions of the immune cell visualized, he was ready to look externally—to figure out how two immune cells interact with each other. Because of the limited area on the surface, though, the flat, two-dimensional cell images of the past wouldn’t suffice. Kupfer had to go 3-D.

It was a daunting task, one that required the young scientist, newly installed in his own lab at the national Jewish Medical and Research Center in Denver, to design and build a 3-D imaging machine from scratch. Kupfer’s first pictures of CD4 surface activity, captured in 1995, vindicated the labor and proffered an unexpected bonus. He had correctly guessed that surface proteins would converge on the contact area to further guide the microtubule highway that was being formed. What he didn’t anticipate was that different proteins segregated into a well-defined inner and outer ring, creating a bull’s-eye pattern that remarkably resembled a cross-section view of a neuronal synapse!

At first, he says, “I didn’t believe it. This had to be some kind of experimental artifact.”

Additional studies, though, confirmed that this organized ring of receptors, structural proteins and other molecules was indeed a genuine biological process. When Kupfer slightly altered the receptors on the target cell so they could still bind CD4 cells but not activate them, he could not see the distinct bull’s-eye pattern form. “So this clustering is critical for turning T-cells on,” he concluded.

He dubbed the two rings the central and peripheral supramolecular activation clusters (c-SMAC and p-SMAC). When a colleague then suggested the phrase immune synapse as a possible nickname, Kupfer was hesitant at first, he says, “because we really were looking at it before any signaling took place.” But, he says with a smile, “I ended up liking it pretty quickly.”

> The inner and outer rings of receptors so critical for turning T-cells "on"

On Kupfer’s computer monitor at Hopkins’ Institute for Cell Engineering, the hazy green CD4 cell touches a neighboring cell, defining Kupfer’s discovery in a matter of seconds. As the two cells momentarily stick together, the diffuse green glow coalesces and methodically travels to the point of contact, creating a bright, triangular mass before gradually dissipating. “That,” he says, “is an immune synapse in action.”

Action may be an appropriate word, for as this short demonstration highlights, Kupfer has gone Hollywood. Previously, his work examined cell events over time through a series of still images. But in an immune system that reacts extremely quickly and unfolds its events in seconds, still imaging can’t tell the full story.

Smitten by Kupfer’s discoveries and imaging breakthroughs, ICE Director Steve Desiderio recruited him to join the Hopkins institute in 2004, giving him a grand opportunity to pursue his real-time movie ambitions. With the help of Hopkins experts in fields such as physics and biomedical engineering, Kupfer has managed to develop the necessary software and tools for his new enterprise—multidimensional live cell imaging. “What our technique essentially does is capture individual slices of a cell and stack them together to reconstruct a 3-D image,” he explains. “We then repeat this process every second to create our movie.”

With this technique, as well as continued use of still images and good old molecular biology, Kupfer is trying to unravel both the functional significance of the immune synapse and how it is activated. Just before coming to Hopkins, Kupfer had discovered that a protein called PKC-theta is a key “master switch”; it needs to turn on in order for the other components of the SMAC to organize. Currently, he is looking for other proteins that regulate synapse formation. 

Beyond that, Kupfer hopes to share his imaging technology with the rest of Hopkins. He conducts weekly informal brainstorming sessions with the other professors in ICE’s immunology department to find new research avenues, and recently he has initiated a pilot project that spans multiple researchers and several Hopkins campuses to thoroughly describe T-cell communication in health and disease. He hopes to follow suit with colleagues investigating neuronal communication as well.

 “It’s been a long-standing dream of mine,” he says, “to expand my imaging studies to look at our big brother, the neuronal synapse.”