Grants Awarded

Principal Investigator
Kimberly Risma MD, PhD
Cincinnati Children’s Hospital Medical Center – Cincinnati, Ohio USA

Date of Award
September 2008

Amount of Award
$50,000

Layperson Summary
Cytotoxic T-cells (CTL) and natural killer (NK) cells contain specialized cell compartments, called secretory granules, which release their contents upon contact with an abnormal cell in the body, such as a cell infected by a virus. These secretory granules contain perforin, a toxic molecule that can damage target cells, and another granule protein, granzyme B, that work together to cause death of a target cell. Perforin-mediated, granzyme B delivery to target cells is critical for the normal immune response to viruses, tumors, and transplanted tissue grafts.

However, very little is known about how perforin delivers granzyme B to target cells and the molecular requirements for cytotoxic function. Importantly, defects in the perforin gene have been identified as a genetic cause of a severe inflammatory disease of early childhood: familial hemophagocytic lymphohistiocytosis (FHLH2). The disease is fatal unless treated with immune suppression and ultimately bone marrow transplant. The Risma laboratory has successfully established cellular models to address how mutations in perforin lead to absent protein detection and impaired NK function.

Assessment of the pathogenicity of perforin mutations is critical in the assessment of patients with FHLH2, as not all mutations may be equally deleterious. Since many nucleotide changes represent benign polymorphisms, we and others have been eager to determine which missense mutations in perforin are indeed pathogenic. The aim of the current study is to test whether perforin mutations that interfere with perforin maturation and storage in secretory granules also abolish perforin function. These studies will advance our current understanding of the pathogenesis of FHLH2 and the basic cellular mechanisms underlying perforin-mediated, lymphocyte cytotoxicity.

Twelve Month Report
The immune system utilizes killer white blood cells to eliminate virally-infected cells and tumor cells from the body by secreting two proteins that work together to eliminate abnormal cells: perforin and granzyme B. Perforin and granzyme B are both stored together in the cell in a special location called a secretory granule. When the cell receives a signal to secrete these two proteins, the granule delivers its contents to the target cell. Perforin “delivers” the lethal weapon, granzyme B, to the targeted cell by a mechanism that is not clearly defined, but may involve formation of a pore. When perforin expression and/or function are defective due to a mutation in the gene, affected children are at risk for a severe inflammatory disease called hemophagocytic lymphohistiocytosis (HLH). When mutations in the perforin gene have been described in children and adults, some of them are disease causing, but others may not actually cause HLH. The Risma laboratory has successfully established cellular models to address how mutations in perforin lead to absent protein detection and impaired NK function. We have been eager to determine why some perforin mutations lead to HLH, while others lead to milder clinical disorders or no symptoms at all. We and others have shown that the perforin protein is processed from a precursor form to a mature form that is stored inside the cell. Some mutations interfere with this processing step, but new data suggest that the processing may not be an absolute requirement for function of perforin. The focus of our 2008 proposal was to define how perforin processing impacts perforin secretion and function. We are so thankful for funding from the HAA in 2008 and the award of a second year of funding (2009) to continue our research.

What we have learned from our research this year:

1. A new and unusual mutation in perforin was identified causing a change in an amino acid at location 438. We published our findings regarding this mutation in the Blood Journal in 2009. Briefly, the amino acid change at 438 in perforin impairs the processing of perforin but doesn’t prevent perforin from working. This contradicts what we thought we knew about perforin- that only the processed, mature form could work. Now we need to determine if this is true of other mutant perforins we have identified as having defective processing. It is possible that the unusual mutation at 438 is exactly that- not typical of other mutations. This is important as we consider the role of processing in perforin function.

2. In the same journal article we also published our findings that a previously disputed mutation at amino acid 435 was a serious mutation causing absent perforin function and therefore, likely to contribute to the development of HLH. Other investigators weren’t sure if this mutation was important or not, until we published our findings. The reason this mutation is so serious is due to the fact that it prevents perforin from binding to the membranes of target cells. It is secreted by killer lymphocytes but it does not cause the death of target cells. We hope to learn more about other mutations that have a similar effect on perforin function.

3. Finally we have learned that the unprocessed form of perforin does not go to the secretory granule for storage and yet is still secreted from the killer white blood cells. We want to know if there are conditions that favor the pathway that leads to immediate secretion of the unprocessed form, versus sending it on the pathway for processing and storage in the granule. We want to know if granzyme B could still work if it is secreted from the granule when perforin is secreted from another pathway. If it doesn’t, then we need to determine how to get perforins to the granule instead of the non-productive pathway.