Using a new form of microscopy to penetrate living lymph nodes, UCSF scientists have for the first time viewed immune cells at work, helping clarify how T cells control autoimmunity.
The technique, known as two-photon laser-scanning microscopy, was able to focus deep within the lymph node of a diabetic mouse, allowing the researchers to show that immune cells known as T regulatory, or Treg, cells control the destructive action of rogue autoimmune cells when each of the two cell types interact with a third kind of cell.
The role of the third cell type -- the antigen-presenting dendritic cell -- in preventing autoimmune attacks of healthy tissue has been a focus of intense research over the last 15 years. The new study supports one contending hypothesis: It is not the interaction between the two types of T cells, but rather the interaction of each with the dendritic cells that leads to protection from autoimmune assaults.
Analyzing these physical interactions experimentally in living organs or even in cell cultures has been impossible before now, the scientists note. Their long-term aim is to use such imaging to diagnose immune diseases such as type 1 diabetes, and to further develop therapies that act at the level of T cell interactions.
The research, published online this week in Nature Immunology, includes online videos of these immune cell interactions -- the first time this has been accomplished.
The scientists used fluorescent dye to mark different types of cells so they could directly observe the interactions of the key cell types involved in initiating autoimmune attacks and the control of their actions by therapeutic regulatory T cells.
The "nonobese diabetic" (nob) mouse model used in the research is considered the "prototypic" model for human type 1 diabetes, said Qizhi Tang, PhD, UCSF adjunct assistant professor in the UCSF Diabetes Center and lead author of the paper. The researchers expect the findings from this study to be consistent in humans.
The UCSF team has already developed a regulatory T cell-based therapy that can prevent and even reverse the course of autoimmune diabetes in mouse models, and this study begins to analyze how such protection occurs in vivo, she explained.
"By understanding the interplay between pathogenic cells and protective cells, we hope to be able to refine the therapy to enhance its efficacy."
The microscopy technique is a vital new tool, Tang said. "The function of the immune system involves multiple cell types interacting dynamically in three dimensions, which is very difficult to analyze in vivo -- and nearly impossible to authentically reproduce in vitro."
The team of immunologists and diabetes researchers used the new microscope to show that when Treg cells are absent, the potentially destructive autoreactive T cells, known as T helper cells, swarm around the dendritic cells where they are primed to attack the body's own tissue -- the cause of type 1 diabetes, arthritis and other autoimmune diseases.
The scientists showed that Treg cells prevent this destructive response after they and the T helper cells independently interact with dendritic cells. The mechanism of this protective effect remains a major immunology puzzle. The new study suggests that regulation may occur through direct or indirect modification of the critical antigen-presenting dendritic cells -- the scavenger cells of the immune system that pick up and display self proteins that trigger the auto-aggressive T cell response, said Jeffrey Bluestone, PhD, distinguished professor of metabolism and endocrinology at UCSF and senior author on the paper. Bluestone directs the UCSF Diabetes Center.
The research provides a "blueprint" of what immune regulation looks like, says study co-author Max Krummel, PhD, UCSF assistant professor of pathology whose lab adapted the new two-photon microscopy technique for visualizing cell-cell interactions within the lymph node.
"We now have a pattern to look for when we try to boost or prevent immune responses. 'Clusters' of cells in a lymph node such as we have seen may be indicative of certain unbridled T cell responses. The ultimate hope here is that seeing patterns of T cell activation may ultimately allow similar imaging to be used for diagnostic purposes," Krummel said.
Immunologists have long sought to harness the potent immunosuppressive properties of the regulatory T cells to treat autoimmune diseases and organ transplant rejection. By pinpointing where and how regulatory T cells work in vivo in mouse models, the researchers hope to better adapt the regulatory T cells for therapeutic use in the future. For example, Tang said, one can imagine that at the early stage of an autoimmune attack, it may be very helpful to direct the therapeutic regulatory T cells to the lymph nodes so they interact with dendritic cells before the autoimmune T cells are able to, and thereby "stamp out the initial sparks" before the disease spreads to the tissue.
Krummel notes that the research dispels some assumptions about cellular movement and interaction. Many had assumed that T cells move very little inside the lymph node. The video microscopy shows that they move about one body length per minute, and that much of the movement is quite directed, for example toward the dendritic cells, rather than random activity. These directed movements are followed by prolonged interaction between autoimmune T cells and dendritic cells that lead to proliferation of autoimmune cells and eventually tissue destruction.
Co-authors on the paper and collaborators in the research along with Tang, Krummel and Bluestone are Jason Y. Adams, a medical student at UCSF; Mingying Bi, MS, staff research associate; and Brian Fife PhD, a postdoctoral fellow, all in the UCSF Diabetes Center; Aaron Tooley, a UCSF graduate student in pathology; and Richard Locksley, PhD, professor of medicine and an investigator in the Howard Hughes Medical Institute at UCSF.
Other co-authors are Pau Serra, a graduate student, and Pere Santamaria, MD, PhD, professor of microbiology and infectious diseases, both at the University of Calgary.
The research is supported in part by the Juvenile Diabetes Research Fund, the National Institutes of Health, the Sandler New Technology fund, and philanthropic support to the UCSF Diabetes Center.
UCSF is a leading university that consistently defines health care worldwide by conducting advanced biomedical research, educating graduate students in the life sciences, and providing complex patient care.
The journal article can be viewed at: http://www.nature.com/ni/journal/vaop/ncurrent/abs/ni1289.html
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