Towards cell therapies to treat Type I Diabetes

“Can we walk around the lab while we talk?” asks UW Carbone Cancer Center researcher, Rahul Das, PhD, with a big grin. He grabs an ice bucket with test tubes and heads from the cell culture room towards his lab’s chemical room, where on the bench opposite the chemicals is a computer connected to the lab’s workhorse instrument.

Das is a new postdoctoral fellow in the laboratory of Jacques Galipeau, MD. Galipeau himself started as faculty in UW’s Department of Medicine the fall of 2016, bringing to this campus expertise in translational immunology. The lab’s focus is in using cell therapy to treat auto-immune diseases and cancer.

The immune system deals with the invasion of bacteria, viruses, other microorganisms, and even cancer cells by mounting an inflammatory response with a dedicated set of specialized cells and proteins. B cells and T cells work together to recognize invading foreign entities from the other cells of the body. B cells produce antibodies that bind to specific regions on the intruding cells, or antigens. A protein called HLA then binds to the antigen, and the antigen-HLA complex serves as a signal for activated T cells to target these antigen-presenting cells for destruction.

The study of the immune system has led to life-saving treatments for patients. For example, one type of cancer immunotherapy, CAR-T cell therapy, focuses on better directing T cells towards killing cancer cells. The treatment involves isolating T cells from the patient’s blood and engineering the patient’s T-cells to display chimeric antigen receptors (CAR) on their cell surface which combine the antigen-recognition capabilities of B cell receptor proteins with the signaling portion of T cell receptor proteins.

Rather than studying the T cells, the Galipeau lab has focused on B cells. In a healthy immune system, some B cells release small proteins called cytokines that in this case signal to T cells to stop the inflammatory response. On the other hand, people with chronic inflammatory diseases are thought to have deficiencies in the number and activity of these suppressor or regulatory B cells (Bregs).

 “Bregs hold tremendous therapeutic potential since they selectively affect activated T cells,” says Das, still setting up for his experiment by testing different acquisition parameters on the instrument.

Finding the right environment to promote Breg development has been a major area of research within the Galipeau lab. Initially, the lab was interested in studying signaling events between cells triggered by cytokines, and their work uncovered how the fusion of different cytokines into one bioactive molecule can dramatically affect the immune response. In bringing together two different types cell surface receptors, these GIFT fusokines (granulocyte-macrophage colony-stimulating factor [GM-CSF] interleukin fusion transgenes) change the way proteins within the cell interact with each other after the receptors are activated. These changes in cellular signaling events are responsible for changes in the immune response. Surprisingly, one of the early GIFT fusokines developed had the unexpected property of being able to convert B cells into Bregs. Bregs made using this approach have been used successfully in mouse models of the autoimmune disease multiple sclerosis.

This research made Das excited to join the Galipeau lab with more ideas for applications of these cell therapy approaches. Bridging his background studying insulin signaling and the genetic factors underlying Type 2 diabetes with the lab’s interests in autoimmune diseases, Das’s central research focus is now Type 1 diabetes (T1D).

T1D, also known as juvenile diabetes, is an autoimmune disease in which immune cells attack the insulin-producing beta cells of the pancreas, resulting in the body being unable to properly regulate blood sugar levels. Because they are not able to naturally produce insulin, Type I diabetics rely on synthetic insulin administered via injection or pump, and these patients need to constantly check their blood sugar levels to avoid dangerously low or high levels. Insulin therapy helps T1D patients survive, but it does not cure them of the disease. Even with diligent monitoring of blood sugar levels, patients still have difficulties striking a balance between diet, exercise, and their insulin dosage.

“There is a huge need for cell therapeutic approaches in the prevention and treatment of T1D,” says Das, who is has now started acquiring data from his samples and is explaining to what all the different plots on the computer screen mean.

Currently, he is currently using cell culture and mouse models of T1D to test the efficacy of Bregs in suppressing the immune response. Having only been in the lab six months, his project is in its early stages, but he hopes that his work could form the foundation for future studies in human patients. The overarching goal of this research would be that in the future Bregs prepared from a patient’s own blood could be used to prevent the infiltration of pancreatic beta cells by immune cells.

For the time being, though, Das is happy to spend his days in “a productive and encouraging environment,” where he can continue doing research he finds fulfilling and pursuing his dream to come a professor.

New insight into the cellular pathways that control hormone secretion

 As UW-Madison researcher Xingmin Zhang finishes his lunch and heads back into lab, the cells in his pancreas are already hard at work sending out insulin to convert that food into the energy his body needs to finish up the day’s experiments.

Hormones, like insulin, are secreted from cells in a tightly regulated manner with the help of many proteins. Zhang, a graduate student from Dr. Tom Martin’s biochemistry lab, identified and studied a new protein involved in the processes that transport proteins to the plasma membrane for secretion. The results, published this month in The Journal of Cell Biology, highlight how the proper flow of traffic both to and from the plasma membrane is necessary for efficient secretion. Impaired insulin secretion ultimately leads to diabetes.

“It helps to have mechanistic insight into a cellular process to then begin to understand what is going wrong in a disease state,” said Zhang.

On their way to the plasma membrane, proteins destined for secretion make a few stops in different compartments within the cell. Like all other proteins, they are synthesized in the cytoplasm, but then are immediately move into the endoplasmic reticulum. Then they travel to the Golgi apparatus, which functions as the post office for cell, sorting proteins into small cellular transport boxes called vesicles destined for specific locations. Hormones are packaged into dense core vesicles that bud off the Golgi apparatus.

Key steps must occur to make these vesicles ready for secretion as they travel to the plasma membrane. First, the cargo contained within the vesicles needs to be refined as a quality control measure, ensuring that all those proteins are in the appropriate “box.” This cargo refinement occurs via a class of smaller cellular transport boxes called endosomes. Additionally, the inside of the vesicle becomes more acidic, making the proteins biologically active.

Once the vesicles reach the plasma membrane, a group of proteins that respond to calcium help the vesicle fuse to the plasma membrane, resulting in the dumping of the vesicle’s contents outside the cell. This subset of proteins then needs to be recycled in endosomes back to the Golgi apparatus for successive waves of vesicle fusion to occur.

Cell biologists have established that many of the proteins mediating vesicle fusion rely on the same region of the protein, so Zhang designed a method in a cell line to assess the ability of the cells to secrete in the absence of each protein containing this region. Several proteins identified in this manner already had known roles in protein trafficking and secretion, but many others did not.

Zhang focused his efforts on ascertaining the role of one of those proteins of unknown function, BAIAP3.  BAIAP3 seemed to be a promising candidate for follow-up study because it was abundantly present in the regions of the brain which signal to other tissues via secretion of hormones and other signaling proteins. Zhang also discovered that BAIAP3 was expressed in the beta cells of the pancreas and that eliminating its expression in a beta cell line blunted insulin secretion, making him more confident in its role in protein trafficking.

Zhang knew that to figure out how BAIAP3 was involved in protein trafficking he had to know its address in the cell. But, naturally, this turned out to be the hardest aspect of the study.

Microscopes enable scientists to see where their protein is within a cell. Proteins known to reside in a specific compartment are landmarks of that location. Scientists color-code different proteins within the cell using markers to identify either the protein itself or a small region of the protein called a tag. Zhang encountered a problem many researchers face at some point: finding a marker that faithfully labeled BAIAP3. He tried adding a tag onto the protein since the markers that recognize tags are more accurate, only to find that the tag was interfering with the protein’s function.

After months of troubleshooting, Zhang finally found that BAIAP3 was in endosomes. He now needed another completely different method to be sure of this finding, but again, he ran into difficulties. He struggled with published protocols detailing the steps required to crack open cells and separate the different subcellular compartments, having to methodically optimize his own protocol before confirming that BAIAP3 is indeed localized on endosomes.

Zhang used this information to infer that BAIAP3 was involved in recycling proteins from the plasma membrane to the Golgi apparatus to support continuing levels of secretion. He then demonstrated that BAIAP3 interacts with proteins known to mediate the fusion of endosomes with the Golgi apparatus in a calcium dependent manner.

Zhang’s work emphasizes that protein trafficking back from the plasma membrane is critical to maintaining a steady flow of traffic towards it. This study adds to the growing body of literature describing the significance of this trafficking route, outlining another possible avenue through which hormone and neuropeptide secretion can go awry.

Having just defended his PhD thesis in April, Zhang is now busily interviewing for postdoctoral fellowship positions across the country, looking to do more translational research.

“Secretion mediates a lot of cellular processes, so I’m glad to have gotten a good training in cell biology,” said Zhang. “It’s a great foundation upon which to learn new skills.”