An array of techniques including optical tweezers have been used to find the “switch” that can swiftly change a T cell – a white blood cell that patrols the bloodstream and organs for signs of disease – from jury to executioner. Revealing this “missing link” in immune response to disease may lead to more effective, precisely targeted therapies for cancers and infections. The immune system’s T cells have unique responsibilities. They examine other cells for signs of disease, including cancers or infections. When they encounter another cell, they “frisk” it to determine whether it is normal or infected, cancerous or foreign to the body. If such evidence is found, the T cells rid the defective cells from the body. Precisely how T cells shift so swiftly from one role to another, however, has been a mystery. A highly focused laser beam (at right) is used to apply mechanical force (shown as a double-headed arrow) to a microsphere (white) coated with histocompatibility protein. The microsphere abuts the surface of a single T cell, shown in gray (top). Activation of the T cell is measured by a change in calcium levels within the cell, which are shown by green colorization (left, prior to force application; bottom, after force application). The direction of force must be tangential, rather than perpendicular, to the T cell surface to trigger a rise in calcium levels. Without an application of force, the binding of the histocompatibility protein produces no such rise. (Image: Dana-Farber Cancer Institute)In a new study, investigators at Dana-Farber Cancer Institute, Harvard Medical School and the Massachusetts Institute of Technology used an array of techniques – including optical tweezers that exploit laser light to press molecules against surface structures found on T cells – to find out what operates the switch. Their answer: sheer mechanical force, making the T cell receptor a mechanosensor. Structural biologists led by Harvard Medical School’s Gerhard Wagner, PhD, used nuclear magnetic resonance techniques to determine the shape of the T cell receptor (TCR) and the arrangement of its component molecules. Biomechanics scientists led by Matthew Lang, PhD, of MIT then devised a set of experiments involving mAbs (monoclonal antibodies) and the molecules of histocompatibility proteins, or pMHCs. The T cell’s surface bristles with receptors – intricate webs of proteins designed to snag specific antigens, much as a lock accepts only certain keys. When a T cell’s receptors lock onto their targeted structures – antigens – on the surface of a diseased cell, parts of the receptors bend in a way that signals the T cell to change from disease-scanning to disease-fighting mode, the researchers report. (Antigens are made of peptides bound to pMHCs.) They also found that after TCRs and antigens meet, an additional force generated during scanning triggers the T cell’s response to disease. The experiments sought to mimic, under controlled conditions, what normally happens when the TCR encounters an antigen from a diseased cell. The mAbs or pMHCs were mounted on tiny beads called microspheres that can be guided into place by laser beams. The mAbs and pMHCs were brought into contact with TCRs on T cells. By adjusting the angle of the laser beams, researchers could subtly alter the strength and direction with which the TCR and mAb or TCR and pMHC were brought together. They found that, although certain mAbs may bind quite well to the TCR, they were unable to activate the T cells if they bound in a perpendicular fashion – that is, in a mode similar to pMHC binding to the TCR. The activation occurred only after the mAb or pMHC bound to the TCR was dragged along the T cell surface with optical tweezers. Application of force to other surface molecules, including the co-receptor molecule CD8, failed to activate T cells. The authors also observed that when certain anti-CD3 mAbs attached diagonally beneath a leverlike portion of the TCR, the T cell was signaled to activate without any additional force application. These mAbs bind to the most sensitive part of the TCR, suggesting how the relay of TCR signals operates via its various component parts.“Our findings with mAbs demonstrate that TCR activation function depends on the angle at which anti-CD3 mAb binding takes place,” says the study’s lead author, Sun Taek Kim, PhD, of Dana-Farber and Harvard. “The mechanical energy generated by diagonal binding is converted into a signal for activating the T cell.” The work will be published in the Nov. 6 issue of the Journal of Biological Chemistry and is currently available on the journal’s Web site. “The study fills a major gap in our understanding of the molecules that make up the TCR – the role they play in recognizing abnormal antigens and in subsequently activating a T cell to attack diseased cells,” said senior author Ellis L. Reinherz, MD, of Dana-Farber and Harvard Medical School. “Our findings explain how TCRs can detect ‘a needle in a haystack,’ enabling T cells to identify infected or cancerous cells that may look very similar to normal cells, and destroy the diseased cells for the good of the body. Distinguishing between cells that belong in the body from those that don’t is the key function of T cells, a discriminative task mediated by their TCRs.” Understanding the details of T cell activation opens the way to development of better immune-based therapies against viral infections and cancers, the authors say. “Vaccines have shown a great deal of promise as cancer treatments, but they need to be made more efficient,” said Reinherz. “This fundamental discovery offers important insights that may make it possible to target such vaccines precisely, destroying cancer cells without the harsh side effects of more traditional therapies.” A broader range of tumor antigens can be selected as potential targets because of the intrinsic sensitivity of the TCR triggering mechanism revealed by this study, Reinherz said. Likewise, the discovery offers promise for the development of T cell-based vaccines for infectious disease prevention, currently an area almost exclusively restricted to antibody-based approaches. Antibodies target regions of viruses that vary substantially in many cases, requiring alterations of vaccines, as in the annual flu vaccines. This is not the case for T cell-based therapies, since they can target antigens that don’t vary among diverse strains of the same type of virus. “Our findings concerning the mechanosensor function of the TCR imply that specific target antigens can be expressed at very low levels on tumor cells and still be recognized efficiently by these T cells. With this insight, the number of tumor target antigens for cancer-based vaccine therapies can be increased,” Reinherz said. The study was supported by grants from the National Institutes of Allergy and Infectious Diseases. For more information, visit: www.dana-farber.org