Grants Awarded

Principal Investigator
Michelle Hermiston MD, PhD
University of California, San Francisco – San Francisco, California USA

Date of Award
September 2008

Amount of Award
$50,000

Layperson Summary
Hemophagocytic syndromes, including hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS), encompass a spectrum of life-threatening disorders characterized by severe inflammation. The mechanistic basis for these syndromes remains incompletely understood. We unexpectedly generated a novel mouse model that shares many characteristics of these hyperinflammatory conditions. We first generated mice expressing a mutation in CD45, an enzyme called a phosphatase that functions as a key regulator of communication within blood cells. This mutation, termed CD45E613R, results in unrestrained phosphatase activity. Stimulation of blood cells from CD45E613R mice shows that they are hyperactive.

However, despite this hyperactivity, CD45E613R mice on C57Bl/6 (a type of mouse strain) genetic background are healthy until at least a year of age. To study how this mutation affects T-cells, we then introduced a T-cell receptor (TCR) called OT1 into CD45E613R mice. This TCR is widely used to study CD8 T-cells and is not known to affect animal health. Surprisingly, the CD45E613R/OT1 mice develop a severe inflammatory disorder that is uniformly fatal by 6 weeks of age. Using cell transplant approaches, we found that CD45E613R/OT1 CD8 T-cells initiate disease and that normal CD8 T-cells can prevent or reverse disease.

The goal of this proposal is to define the mechanism(s) by which the CD45E613R/OT1 T-cells drive disease. We will also study how normal CD8 T-cells reverse disease. An advantage of mouse disease models is that large numbers of genetically identical animals can be generated to test hypotheses and to evaluate potential novel therapeutic approaches. This is particularly important for diseases as rare as HLH where accruing sufficient patients for clinical studies is challenging. A second advantage of our model is that it arose in an unbiased and incidental manner. As such, it may provide novel insights into pathogenesis of HLH/MAS by highlighting new pathways that can be interrogated in patient samples and targeted therapeutically.

Twelve Month Report
Hemophagocytic syndromes, including hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS), encompass a spectrum of life-threatening disorders characterized by severe inflammation. The mechanistic basis for these syndromes remains incompletely understood. We unexpectedly generated a novel mouse model that shares many characteristics of these hyperinflammatory conditions. We first generated mice expressing a mutation in CD45, an enzyme called a phosphatase that functions as a key regulator of communication within blood cells. This mutation, termed CD45E613R, results in unrestrained phosphatase activity. Stimulation of blood cells from CD45E613R mice shows that they are hyperactive. However, despite this hyperactivity, CD45E613R mice on C57Bl/6 (a type of mouse strain) genetic background are healthy until at least a year of age. To study how this mutation affects T-cells, we then introduced a T-cell receptor (TCR) called OT1 into CD45E613R mice. This TCR is widely used to study CD8 T-cells and is not known to affect animal health. Surprisingly, the CD45E613R/OT1 mice develop a severe inflammatory disorder that is uniformly fatal by 6 weeks of age.

The phenotype of these mice was highly reminiscent of many of the characteristics I see in patients with HLH while caring for children on our clinical Pediatric Hematology-Oncology service. A review of the literature revealed that abnormal expression of CD45 had been reported in several children with HLH, although disease-causing mutations in CD45 have yet to be identified. We proposed to exploit the CD45E613R/OT1 mouse model to better understand potential mechanisms contributing to HLH. An advantage of mouse disease models is that large numbers of genetically identical animals can be generated to test hypotheses and to evaluate potential novel therapeutic approaches. This is particularly important for diseases as rare as HLH where accruing sufficient patients for clinical studies is challenging. A second advantage of our model is that it arose in an unbiased and incidental manner. As such, it may provide novel insights into pathogenesis of HLH/MAS by highlighting new pathways that can be interrogated in patient samples and targeted therapeutically.

In our preliminary studies we used cell transplant approaches to test which cell types in the blood were necessary for disease in CD45E613R/OT1 mice. We found that CD45E613R/OT1 CD8 T-cells initiate disease and that normal CD8 T-cells can prevent or reverse disease. The goal of our proposal was to define the mechanism(s) by which the CD45E613R/OT1 T-cells drive disease. With the support of the HAA, we have made significant progress towards this goal. An overactive and uncontrolled immune response is a central feature of HLH. We hypothesized that CD8 T-cells could contribute to the excessive inflammation due to an inability to turn off the immune response (a key function of CD8 T-cells is the cytolytic T-cell (or CTL) response in which they kill macrophages that have engulfed infectious organisms). Persistence of infected macrophages and activated CD8 T-cells results in excessive production of chemicals into the blood called cytokines. Our preliminary studies had shown a 30-fold increase in the levels of one of these cytokines, interferon gamma (IFNg) that we hypothesized might contribute to disease in these animals.

To test these hypotheses we first compared CTL function in CD8 T-cells from OT1 mice with abnormal or normal CD45 phosphatase activity. We found that the CD45 mutant CD8 T-cells were completely defective in their ability to mount a CTL response. This is an exciting result because it suggests a new signaling pathway to explore for mutations in children with HLH that have no known genetic cause. In a second line of experiments, we tested whether IFNg played a causative role in disease in our model system. We bred CD45E613R/OT1 mice to mice containing a genetic mutation that prevents IFNg production. Confirming a causative role for this cytokine in disease, CD45E613R/OT1 mice lacking IFNg had a normal lifespan and no evidence of disease. These data suggest that IFNg may serve as a novel therapeutic target for children with HLH.