Molecular Analysis Of Leukocyte Activation By Chemoattractants

National Institute Of Allergy And Infectious Diseases

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Abstract

The aim of this project is to define the molecular mechanisms and biological contexts for blood leukocyte migration to specific tissue sites that are inflamed or infected. We have focused on chemoattractant proteins that mediate this process and have identified members of a large family of chemoattractant receptors that are deployed on the leukocyte cell surface. We have also identified members of a diverse group of chemoattractant and chemoattractant receptor mimics made by viruses, including herpesviruses, poxviruses and HIV. We use genomics, molecular biology, cell biology and epidemiology as the principle methods for analyzing these molecules. A major goal is to identify specific disease associations of individual chemoattractants and chemoattractant receptors, in order to identify potential new therapeutic targets. A key strategy is to analyze phenotypes of gene knockout mice in disease models as well as phenotypes in patients with mutations in human chemoattractant system genes. 1.) In FY23, we reported a case of severe neutropenia in a 6-year-old boy. The patient has a classic CXCR4 mutation consistent with WHIM syndrome. The case illustrates the heterogeneity of presentation in this disorder. 2.) In FY23, we reported that WHIM mutations cause more severe CD8 than CD4 lymphopenia in WHIM patients and WHIM model mice. Mechanistic studies in mice revealed selective and WHIM allele dose-dependent accumulation of mature CD8 single-positive cells in thymus in a cell-intrinsic manner due to prolonged intrathymic residence, associated with increased CD8 single-positive thymocyte chemotactic responses in vitro toward the CXCR4 ligand CXCL12. In addition, mature WHIM CD8+ T cells preferentially home to and are retained in the bone marrow in mice in a cell-intrinsic manner. Administration of the specific CXCR4 antagonist AMD3100 (plerixafor) in mice rapidly and transiently corrected T cell lymphopenia and the CD4/CD8 ratio. After lymphocytic choriomeningitis virus infection, we found no difference in memory CD8+ T cell differentiation or viral load between wild-type and WHIM model mice. Thus, lymphopenia in WHIM syndrome may involve severe CXCR4-dependent CD8+ T cell deficiency resulting in part from sequestration in the primary lymphoid organs, thymus, and bone marrow. 3.) In FY23, we reported in Blood a two-step preclinical gene therapy protocol involving autologous HSPC transplantation. First, one copy of Cxcr4 in HSCs was inactivated ex vivo by CRISPR/Cas9 editing with a single guide RNA (sgRNA) that does not discriminate between WHIM and wildtype Cxcr4 alleles. Then, through in vivo natural selection, WHIM allele-inactivated cells were enriched over wildtype allele-inactivated cells. The WHIM allele-inactivated HSCs retained long-term pluripotency and selective hematopoietic reconstitution advantages. The paper was accompanied by a Perspective in Blood. To our knowledge this is the first example of gene therapy for an autosomal dominant gain-of-function disease using a disease allele inactivation strategy in place of the less efficient disease allele repair approach. This paper extends to a gene therapy platform our previously published discovery that Cxcr4+/o HSCs have superior engraftment potential in congenic mouse transplantation over wild type HSCs as well as our characterization of chromothriptic cure of WHIM syndrome by deletion of the disease allele in a single HSC. The results provide proof of principle that WHIM allele silencing of patient HSCs is a viable and more feasible gene therapy strategy than WHIM allele correction.