Pulmonary Immune Regulation of Lung Injury after Pediatric Hematopoietic Cell Transplant

Project Summary/Abstract Allogeneic hematopoietic cell transplantation (HCT) can be a curative treatment for children with leukemia, immunodeficiencies, and other life-threatening conditions. However, infection, chemotherapy toxicity, and alloreactivity can lead to post-HCT lung injury in up to 40% of children, which is a major driver of morbidity and mortality. Paradoxically, patients exhibit high levels of systemic inflammation, yet also show limited ability to contain pulmonary infections, suggesting a multifaceted state of both immune activation and dysfunction. Despite this complexity, the clinical diagnosis of post-HCT lung injury largely relies on indirect or non-specific signs and symptoms such as cough, fever, radiographic opacities, and blood-based measures of inflammation. Thus, a major gap in the field is the limited understanding of lung-specific immune signaling that drives the progression and hinders the resolution of post-HCT lung injury. The goal of this proposal is to elucidate lung-specific immune pathways driving post-HCT lung injury in order to identify cellular and molecular targets for precision diagnostics and therapeutics. To do this, we will leverage bronchoalveolar lavage (BAL) fluid and paired blood samples obtained from pediatric HCT patients as well as non-HCT controls. In Specific Aim 1, we will elucidate peripheral blood correlates of post-HCT lung injury. Here we will contrast cross-site transcriptomes and identify pulmonary vs. extrapulmonary contributors to patient mortality, with each patient serving as their own control. To support the development of a diagnostic test, we will measure alveolar peptidomes and test whether key BAL hub proteins can discriminate lung injury subtypes when measured in blood. In Specific Aim 2, we will determine key pulmonary immune cell populations associated with subtypes of post-HCT lung injury. We will use flow cytometry and single cell RNA sequencing of BAL cells to determine key lineages and transcriptional states that define lung injury subtypes and predict outcomes. Immune cells will be further characterized as marrow-derived vs tissue-resident and of patient vs. allograft origin to unravel the specific cellular drivers of post-HCT lung injury. Finally, in Specific Aim 3, we will evaluate the functional capacity of pulmonary mononuclear phagocytes and test for reversibility of hypo- and hyper-activation using ex vivo stimulation. Here, patient mononuclear phagocytes will be stimulated with lipopolysaccharide and interferon gamma and response will be assessed by cytokine production levels and single cell transcriptomes. We will test for the reversibility of hypo and hyper- secretory response with addition of GM-CSF and IL-10, respectively. Together these experiments will elucidate the pulmonary-specific immune mechanisms that drive post-HCT lung injury. These results will support the development of the next generation of BAL and blood-based diagnostic tests and will identify cellular and molecular targets that may be leveraged for precision therapeutics in this vulnerable population.