Primary Immune Deficiencies

When you meet with Dr. Luigi Notarangelo, the first sign that he is at work today comes from hearing the sounds of frantic typing emanating out of his office at Children’s Hospital Boston. Without breaking stride, he senses your presence, greets you with a “welcome!” and punctuates his last thought in an email. If the speed at which Dr. Notarangelo works isn’t impressive enough, his broad and comprehensive knowledge of immunology truly leaves one’s head spinning after even a short conversation.

Dr. Notarangelo, or “Gigi” as he is affectionately known by his colleagues, began working on primary immunodeficiencies (PIDs) while attending medical school at the University of Pavia in Lombardy, Italy. PIDs are a spectrum of genetically inherited diseases that affect at least one of the major arms of the immune system used to combat microbes. Going into medical school, Gigi already had a predilection towards studying immunology, but a fortuitous roommate placement with Dr. Antonio Lanzavecchia really set the rest of his career in motion.

“I met Antonio, of course before he became a famous immunologist, as my roommate, and he really thought that PIDs made a great window into understanding immunology in humans,” he recalls. He goes on to explain how, by studying these inborn errors of immunity, one can essentially learn the inner-workings of the human immune system by learning the underlying genetic and cellular mechanisms that predispose certain patients to be more susceptible to certain classes of viruses, bacteria, or fungi.

One type of PID on which Gigi has spent a large part of his career working on is known as severe combined immunodeficiency (SCID). SCID, as the name suggests, is a life-threatening disease, which impacts children within the first few months of life. The clinical presentation is an extreme susceptibility to ordinarily nonpathogenic or poorly pathogenic viruses, bacteria, or fungi, which would otherwise be cleared by the immune system.

The only treatment widely available is to find a bone marrow transplant (BMT) donor fast enough. Bone marrow is where hematopoietic stem cells (HSCs) live and divide to form their offspring. Typically, HSCs of healthy humans have the capacity to give rise to all of the circulating blood cells but not to other cells such as neurons. Blood cells have a variety of functions. For example, some deliver oxygen to tissues, as is the case with red blood cells. On the other hand, other blood cells fight infection by rapidly mobilizing to a site where a pathogen has entered. In particular, T cells, B cells, and NK cells are extremely important for combating an infection and are also uniquely endowed with the ability to remember a pathogen and how they defeated it to form long-lasting memory. (This is the basis for how vaccines work). The extreme susceptibility to infection in children with SCID is a result of absent or poorly functional T-cells: cells which are responsible for detecting infected cells and directly eliminating them, or providing help to other branches of the immune system to clear infection

In children with SCID, T cells and either B cells, NK cells, or both are absent from circulation. A bone marrow transplant can be curative for a child because, through a transplant, the HSCs incapable of producing these cell types become replaced with HSCs from a healthy donor that are known to be able to give rise to T, B, and NK cells. Of course, while a BMT can be curative, it is fraught with difficulties and hurdles including finding a genetically compatible donor for the child.

A Genetic Understanding of PIDs

When Gigi started to work in earnest on SCID in the late 1980s, he happened to be working in a medical center in Italy that treated many SCID patients. Since SCID diseases are rare (~1 in 100,000 live births depending on the population), many physicians can go an entire career without encountering a child presenting with SCID. As such, few physicians are as intimately familiar with the full spectrum of disease and clinical presentation as Gigi is.

Gigi explains how he used to spend hours under the microscope individually analyzing a patient’s cells to see if certain kinds were absent from the blood. Before 1990, the diagnosis of SCID was almost purely based on the clinical presentation and crude analyses of the presence or absence of T cells and other cell types circulating in the blood.

The 1990s brought the advent of molecular diagnoses, making those years an incredibly exciting time to be working in the field.  With the elucidation of several genetic defects responsible for the clinical presentations, one might think that this would be the whole story. But as any great scientist would do, Gigi began to take one answer and ask several new questions that would push the field even further.

“How is it that mutations in the same gene can lead to diseases that are so distinct from each other?” Gigi asks rhetorically. While most immunologists who study mechanisms revert to mouse models and an approach of “knocking out” defined genes to study their function by removing the ability of the mouse to produce a certain protein, humans don’t always have mutations that are all-or-none. In a type of PID known as Omenn syndrome, children actually present with both immune deficiency and allergic, or autoimmune, features. One might think that this could not be due to the same gene responsible for a type of SCID, but in fact, this is the case. Rather than having a complete absence of the protein that is characteristic of SCID, patients with Omenn syndrome have just enough residual protein activity to make some T and B cells. However, because of the reduced activity of the protein, instead of protecting against infection, these cells turn on the child and begin to attack the body’s own tissues.

Implementing Technology

This brings Gigi to his next point about embracing technology in the right way. He explains how, on the clinical side, PIDs have always been a fascinating area to work in due to the rarity of the disease and the ability to try cutting-edge technologies on small sets of patients. Modern technology has allowed for cost-effective whole genome sequencing, but recent papers suggest that even the ability to read the entire genetic code can sometimes leave one with more questions than answers and an inability to pinpoint the precise genetic defect. As a result, the diagnosis and treatment of PID is still an artform that cannot simply be resolved by sheer computing power. Gigi says how the difficult and most puzzling cases are still the most rewarding, because “even if we cannot come up with good answers, patients always teach us something.”

Gigi explains that he was drawn to the technology and cutting-edge work at Children’s Hospital where, again a lucky “roommate” placement brought him in touch with the right person at the right time. He moved to Children’s shortly after the method for producing induced pluripotent stem cells (iPSCs) was published which was received both with acclaim and a healthy dose of skepticism from the scientific community. While previously it had been thought that cells could only move forward, from embryo to a mature cell in an adult, the discovery that a defined set of only four proteins introduced into cells could take an adult cell back to a stem-cell like state was truly game-changing. Gigi’s new lab space was located in close proximity to Dr. George Daley’s lab, where, in 2007, Dr. In-Hyun Park was one of the first scientists in the world to generate human iPSCs. While HSCs have the capacity to generate all of the circulating blood cells, iPSCs have the capacity to give rise to all cell types in an embryo including tissues as diverse as the nervous system, skin, muscle, gut and blood.

Shortly after their discovery, iPSCs were touted as a panacea for almost any inherited genetic condition. It was thought that scientists could take skin cells from a patient, turn them into iPSCs, correct known underlying genetic defects in a stem cell, and then grow the genetically identical tissue (except for the corrected mutation), before transplanting it back into a patient. One could imagine how for patients with SCID this could greatly facilitate the difficulties and complications of bone marrow transplantation by overcoming the issues inherent in transplanting genetically mismatched HSCs from different individuals. The way that iPSC research has typically been portrayed in the media led to some premature hopes on the side of families struggling with otherwise incurable diseases. However, when Gigi finishes telling this part of the story, he closes by saying that, “One should never blame the patient for seeing stem cells as a dream, if that is how it is communicated.” For the time being, refined bone marrow transplantation protocols (which replace a patient’s HSCs) remain the standard of care for SCID and other PIDs.

Several scientists including Gigi embraced iPSCs not for their potential curative properties but rather for the ability to gain a greater understanding of cell types that they had never been able to explore previously. While mouse models of human disease have their limits, they do give scientists the ability to probe and explore otherwise inaccessible tissues such as the brain and the nervous system. While many classical PIDs affect circulating blood cells, some manifest as a specific susceptibility to infection by cell types that do not arise from HSCs, such as neurons. One particular case is a subset of patients with Herpes Simplex Encephalitis (HSE) in which infection with the virus HSV-1 leads to a severe disseminated infection of the brain instead of just an unsightly cold sore. Tapping into his broad network of international collaborators and also close neighbors, Gigi helped orchestrate one of the first studies to use iPSCs to model a human disease in a dish, which enabled researchers to discover that neurons and oligodendrocytes (the cells which produce the insulation for neurons) are particularly susceptible to infection by HSV-1 in patients lacking a specific immune pathway. Future work utilizing this platform could lead to novel drug targets to block viral replication or entry in otherwise susceptible cell types.

During a normal meeting with Gigi, his email alerts fire at a rather alarming rate, but his attention remains focused on the task at hand. During this particular meeting, we happened to touch on not only several aspects of immunology but also on the intricacies of the active sites of enzymes and basic biochemistry. It’s evident that PIDs aren’t just a way to learn about human immunology, but also about the latest technologies and discoveries in biology, chemistry, and medicine. In the near future, patients may benefit from the ability of iPSCs to serve as a screening platform for drugs before the cells are actually transplanted. When I ask him about how he wants to use iPSCs next in the lab, Gigi gets a twinkle in his eye tells me another story of how by screening a chemical library they have discovered a compound which could be used to treat a severe PID and ends by saying, “Without iPSCs I would have never been able to do this.”

Jose Ordovas-Montanes is a graduate student in the Harvard Immunology program and a member of the von Andrian lab.

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