T-cell exhaustion is a state of T-cell dysfunction that arises during many chronic infections and cancer. One immunotherapy that revitalizes T cells by blocking an immune checkpoint protein called LAG3 was recently approved by federal regulators. However, how LAG3 works has not been fully understood. Now, new research by a team at the University of Pittsburgh School of Medicine reveals how LAG3 regulates T-cell activity, and may pave the way for other LAG3-blocking therapies for cancer and autoimmune disorders.

The findings are published in the journal Nature Immunology in a paper titled, “LAG3 associates with TCR–CD3 complexes and suppresses signaling by driving co-receptor–Lck dissociation.”

“LAG3 is an inhibitory receptor that is highly expressed on exhausted T cells. Although LAG3-targeting immunotherapeutics are currently in clinical trials, how LAG3 inhibits T-cell function remains unclear,” the researchers wrote. “Here, we show that LAG3 moved to the immunological synapse and associated with the T-cell receptor (TCR)-CD3 complex in CD4+ and CD8+ T cells, in the absence of binding to major histocompatibility complex class II—its canonical ligand.”

Dario A.A. Vignali, Ph.D., distinguished professor and vice chair of immunology at the University of Pittsburgh School of Medicine and associate director for scientific strategy and co-leader of the Cancer Immunology & Immunotherapy Program at UPMC Hillman Cancer Center. [University of Pittsburgh]
“Although LAG3 has been studied extensively as a potential immunomodulatory target, there hadn’t been an awful lot known about how it works. It truly was a black box,” said co-senior author Dario A.A. Vignali, PhD, distinguished professor and vice chair of immunology at the University of Pittsburgh School of Medicine. “I believe this is a landmark study because we finally know how LAG3 works. This will help the development of new LAG3-targeting therapeutics.”

In March 2022, the FDA approved the first LAG3 inhibitor for advanced melanoma, and 18 other LAG3-inhibitors are currently in clinical trials.

To “look under the hood” of LAG3, Vignali started this research in his previous position at St. Jude Children’s Research Hospital along with first author Clifford Guy, PhD, who was a postdoc in his lab at the time, and co-senior author Creg Workman, PhD, who is now a research assistant professor of immunology in Vignali’s lab at Pitt.

Creg Workman, PhD, research assistant professor of immunology at the University of Pittsburgh School of Medicine.
[Courtesy of Creg Workman]
When T cells engage with an antigen-presenting cell, they form a contact point called an immunological synapse that gets flooded with T-cell receptors.

“We found that LAG3 binds to T-cell receptors, but it doesn’t inhibit them directly,” explained Workman. “Instead, LAG3 uses T-cell receptors to hitch a ride to the immunological synapse.”

When using a special dye that measures acidity, the researchers observed accumulation of LAG3 in the synapse creates acidic conditions that disrupt the association between coreceptors—CD4 on helper T cells or CD8 on killer T cells—and a signaling enzyme called Lck, thwarting a key requirement for T-cell activation and signaling.

In mice that express LAG3 (left image), accumulation of LAG3 at the immunological synapse in T cells lowers the pH, making conditions more acidic (green and blue) compared with less acidic (red) conditions in T cells from mice that do not express LAG3 (right image). [Guy, C. et al. Nature Immunology, (2022)]

“This research calls into the question the design of LAG-blocking therapeutics in development,” said Vignali, who is also associate director for scientific strategy and co-leader of the cancer immunology and immunotherapy program at UPMC Hillman Cancer Center. “They obviously work, but do they work optimally? They’ve been designed to block Class II interactions, but this study shows that LAG3 can function in absence of Class II.”

“Now that we know LAG3 binds to the T-cell receptor, we might be able to generate optimal blockers that target this interaction,” he added.

The new findings may pave the way for treatment approaches for autoimmune and inflammatory disorders.

“To help the body fight cancer, you want to release the immune system brakes,” added Vignali. “But for autoimmunity, you want to pump the brakes harder.”

“These observations indicated that LAG3 functioned as a signal disruptor in a major histocompatibility complex class II-independent manner, and provide insight into the mechanism of action of LAG3-targeting immunotherapies,” concluded the researchers.

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