Image courtesy of Anusha Bishop
The doctors were stumped. The nine-year-old girl in front of them, who would later be code-named patient A.1, was anemic and fatigued. She had a chronic cough and difficulty breathing, and her ears, skin, and urinary tract were constantly infected. In the coming years, her situation would worsen as she developed colitis, inflammation in her digestive tract. Clinical blood tests found abnormal white blood cell counts, a hint that her disease was related to immune system dysfunction. To reconcile this clue with her diverse range of symptoms, her team of physicians at the National Institutes of Health (NIH) turned to Carrie Lucas, now assistant professor of immunobiology at the Yale School of Medicine, for help.
Ten years after the onset of patient A.1’s symptoms, researchers in the Lucas lab and their collaborators have finally uncovered the cause of her illness: a defective immune system protein called PI3Kγ. This previously unseen disease has now been named Inactivated PI3K-gamma Syndrome (IPGS). By elucidating the molecular mechanisms behind IPGS, the researchers both enabled improved treatment for patient A.1 and clarified the role of PI3Kγ in immune system regulation, overthrowing previous assumptions from mouse model studies. This information is particularly valuable because PI3Kγ is a promising new target for immunotherapy, which uses patients’ own immune systems to fight disease.
A needle in a haystack of genes
The Lucas lab at Yale studies rare genetic diseases of the immune system: “not the standard allergies or colds like kids normally get, but those patients who get really severe infections really easily, or who have fevers and feel really sick, but show no signs of an infection,” Lucas said. The researchers’ subjects are generally children because early onset of disease is an indicator of a possible mutation in a single gene. Diseases that appear in adulthood, conversely, are more likely to be caused by complex genetic and environmental triggers. Understanding a single mutation that causes a disease reveals specific insight into the affected gene.
Patient A.1’s symptoms and young age led the researchers to hypothesize that she had a single-gene mutation. To find it, the researchers needed to sequence her entire exome, the portion of the genome that encodes for functional protein, with consent from the patient and her family. They examined the unique variants in her genetic code. However, there are thousands of variants between any two individual’s exomes, most of which do not cause disease. The task now was to comb through all these variants in search of the single mutation underlying patient A.1’s symptoms.
To help whittle down their initial list, the researchers analyzed the genes of patient A.1’s mother and father, who were healthy. As with everyone else, patient A.1 inherited half of her genome—that is, one of two alleles of each gene—from each of her parents. Thus, any combination of variants she shared with her parents could be excluded. “We can filter the list and say, if only the kid is sick, then we can rule out all these variants,” Lucas said.
After further refining their list, the researchers identified a prime suspect: a mutated PIK3CG gene, encoding the PI3Kγ protein. Patient A.1’s healthy parents both possessed one mutated PIK3CG allele and one normal PIK3CG allele. A single copy of the defective gene was not enough to cause disease, but by chance, both parents passed their mutated allele on to patient A.1, leaving her with two defective alleles. This double mutation provided strong evidence that defective PI3Kγ could be to blame.
Figuring out the pathway
Previous research had implicated PI3Kγ as an actor in the immune system. As part of the phosphatidylinositol 3-kinase (PI3K) family, PI3Kγ initiates immune signaling pathways by appending phosphate groups to lipids in the cell membrane. Because of its central role in signaling, PI3Kγ has become a potential target for immunotherapy. Expecting abnormalities in her immune system, the researchers analyzed patient A.1’s blood to determine the types and amounts of white blood cells present. They used flow cytometry, a method of counting cells in a mixture using a laser that detects fluorescently stained markers specific to a given type.
Our immune system comprises two arms. The adaptive immune system, which includes T- and B-cells, develops to respond in a highly specific manner to pathogens—foreign disease-causing agents—as we encounter them. Meanwhile, the innate immune system comprises non-specific “first responders,” which recognize common molecular patterns on pathogens to kick off the adaptive response by, for example, promoting inflammation. In her adaptive immune system, patient A.1 had a low count of regulatory T-cells, which stop the immune system from attacking our own cells, and memory B-cells, which remember past pathogens to prevent future re-infection. This finding explained her recurrent ear, skin, and urinary tract illness. Conversely, she had a high count of T cells expressing CXCR3, a marker that allows them to infiltrate nose, gut, and lung tissue, in line with her difficulty breathing and colitis.
In her innate immune system, patient A.1 exhibited abnormally high levels of small signaling proteins called cytokines, which induce inflammation. Accordingly, when the researchers treated healthy cells with a chemical that inhibits PI3Kγ, they found increased expression of the IL12B gene, which encodes the IL-12 and IL-23 cytokines. This further aligned with patient A.1’s breathing and digestive tract issues. “Her [innate immune white blood cells] make a lot of pro-inflammatory cytokines—more than they should. One of [PI3Kγ’s] functions is to tamp down that inflammation,” Lucas said.
In essence, the researchers found that normal-functioning PI3Kγ has both adaptive and innate immunity functions, supporting regular T-cell activity while preventing inflammatory cytokine activity.
“Dirty” mice, better results
While the researchers’ findings held promise, they were also confusing: laboratory mice previously engineered to lack PI3Kγ did not display the same symptoms as patient A.1. To rationalize the discrepancy, the researchers looked to past work by the Masopust and Jameson labs at the University of Minnesota, where blood samples from pet-store mice and laboratory mice were shown to express vastly different immune cell profiles. “Laboratory mice are kept in super clean environments… they don’t even get exposure to the normal air, and so they have very few infections or challenges throughout their full developmental spectrum,” Lucas said. In contrast, pet-store mice, having been exposed to various environmental pathogens, have more mature immune systems. The researchers predicted that the immune systems of conventional laboratory mice could not accurately reflect PI3Kγ activity.
To circumvent this issue, they decided to co-house laboratory mice that were deficient in PI3Kγ with “dirty” mice they bought from pet stores. Andrew Takeda and Timothy Maher, co-first authors on the paper, noted that this method presented unique challenges as the “dirty” pet store mice were accompanied by a host of microbes that threatened the sterile lab environment. “We can screen for some specific pathogens normally kept out of the lab, but the pet store mice could be carrying additional viruses, bacteria, and parasites that we haven’t identified,” Takeda said. For this reason, the researchers had to conduct their research in a special facility, typically reserved for research with more dangerous pathogens.
This unconventional setup introduced complications, as pet store mice may bring lethal pathogens that can lead to experiments being cut short. The researchers’ pathogen screens once had missed Mycoplasma pulmonis, a deadly bacterial infection towards which pet store mice have developed resistance, which resulted in their experiment ending just three weeks in. “The twenty-one days, that’s just starting the experiment. There’s four to six weeks of preparing the lab mice before,” Maher said. “So that’s three months, just gone.”
Ultimately, the researchers found that when incubated with the “dirty” mice and their associated pathogens, the immune systems of PI3Kγ-deficient laboratory mice became activated to behave in a similar manner to that of patient A.1. This behavior included decreased regulatory T-cell counts, poor B-cell responses, and increased production of IL-12 and IL-23, supporting their hypothesis that PI3Kγ regulates T- and B-cells and cytokine activity. Their experiment also highlighted potential drawbacks of modeling human immune processes in sterile lab organisms.
Insights into the immune system
Having identified a double PI3Kγ-mutation underlying patient A.1’s illness, the researchers, in collaboration with her physicians, improved patient A.1’s treatment. For instance, to combat her inflammation, she was prescribed an IL-12 and IL-23 inhibitor called ustekinumab, an FDA-approved drug for psoriasis and Crohn’s disease. Beyond patient A.1’s specific Inactivated PI3K-gamma Syndrome, the researchers’ work has uncovered fundamental knowledge about the PI3Kγ protein’s role in immune response, with broad medical implications. For example, PI3Kγ inhibitors have shown promise as immunotherapy treatments for cancers. By understanding the effects of PI3Kγ deficiency, doctors can now anticipate and manage potential side effects when targeting the protein.
While the Lucas lab remains interested in refining their understanding of PI3Kγ, they have also expanded their scope, using the same methodology to study additional rare genetic diseases. By working backwards from symptoms to their genetic causes, the researchers are able to investigate lesser-known genes. “In a lab, you don’t just think up these different rare mutations. [In a rare genetic disease] these mutations just happen, and it helps you get a better understanding of basic immunology and how cells really work in humans,” Maher said. Takeda believes in the dual impacts of this line of research: “the project starts out because there’s a patient who’s sick and they don’t know why. It’s really rewarding to be able to help figure out why someone has this mystery disease while understanding something new about the immune system.”
Takeda, A. J., Maher, T. J., Zhang, Y., Lanahan, S. M., Bucklin, M. L., Compton, S. R., … Lucas, C. L. (2019). Human PI3Kγ deficiency and its microbiota-dependent mouse model reveal immunodeficiency and tissue immunopathology. Nat. Commun., 10(4364).