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 associations of loss of function mutations in the corresponding human genes in human disease cohorts. 1.) In FY18, we reported in the Journal of Clinical Immunology the mechanisms responsible for sustained neutrophilia in patient WHIM-09, who was cured of WHIM Syndrome by chromothripsis. WHIM-09 is the first patient described with WHIM syndrome, an autosomal dominant form of neutropenia related to bone marrow retention of neutrophils. Originally diagnosed incorrectly with autoimmune neutropenia, the patient underwent splenectomy at age 9, but the absolute neutrophil count (ANC) did not rise. Subsequently, she was spontaneously cured by chromothripsis (chromosome shattering), which deleted the disease allele CXCR4 R334X , and 163 other genes, on chromosome 2 in a single hematopoietic stem cell (HSC), as we reported in Cell in FY2015. Chromothriptic CXCR4 +/o HSCs replaced CXCR4 +/R334X WHIM HSCs, and the ANC rose to a new sustained and benign baseline 2-3-fold above normal that had remained unexplained. In FY18, we reported that splenectomized Cxcr4 +/o mice had sustained and benign neutrophilia, phenocopying neutrophilia in WHIM-09. In addition, WHIM-09's granulocyte-macrophage precursor cells possessed increased granulocyte colony-forming activity ex vivo. Thus, WHIM-09's neutrophilia may be multifactorial, involving neutrophil-extrinsic factors (splenectomy), as well as CXCR4 haploinsufficiency-dependent neutrophil-intrinsic factors (increased myeloid precursor cell differentiation). The strong bone marrow retention signal for neutrophils conferred by the WHIM mutation may have prevented neutrophilia after splenectomy until the mutation was deleted by chromothripsis. 2.) In FY18, we reported in the Journal of Clinical Investigation that Cxcr4-haploinsufficient bone marrow transplantation corrects leukopenia in an unconditioned WHIM Syndrome model. As mentioned above, WHIM syndrome is caused by autosomal dominant gain-of-function mutations. We have been investigating both treatment and cure strategies for this disease. Cure strategies under investigation include gene therapy and allotransplantation. For gene therapy of gain-of-function autosomal dominant diseases, either correcting or deleting the disease allele is potentially curative. To test whether there may be an advantage of one approach over the other for WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome, we performed competitive transplantation experiments using both lethally irradiated WT (Cxcr4+/+) and unconditioned WHIM (Cxcr4+/w) recipient mice. In both models, hematopoietic reconstitution was markedly superior using BM cells from donors hemizygous for Cxcr4 (Cxcr4+/o) compared with BM cells from Cxcr4+/+ donors. Remarkably, only approximately 6% Cxcr4+/o hematopoietic stem cell (HSC) chimerism after transplantation in unconditioned Cxcr4+/w recipient BM supported more than 70% long-term donor myeloid chimerism in blood and corrected myeloid cell deficiency in blood. Donor Cxcr4+/o HSCs differentiated normally and did not undergo exhaustion as late as 465 days after transplantation. Thus, disease allele deletion resulting in Cxcr4 haploinsufficiency was superior to disease allele repair in a mouse model of gene therapy for WHIM syndrome, allowing correction of leukopenia without recipient conditioning. The mechanism was different in the two models. In lethally irradiated recipients, the Cxcr4+/o engraftment advantage was apparent in both blood and bone marrow where mature and progenitor cells alike were markedly skewed towards that donor. In non-conditioned recipients, the frequency of Cxcr4+/o cells in the bone marrow was similar to the frequency of Cxcr4+/W cells in the same animal, whereas the Cxcr4+/o donor-derived cells in the blood greatly exceeded the Cxcr4+/w donor-derived cells. It is unclear why the bone marrow results differ. Nevertheless, the blood results are consistent in the two models and reflect both superior bone marrow engraftment and mature cell release to blood in the irradiated recipients, and superior release in the non-conditioned recipients. This paper is an important extension of our previous work published in Cell in 2015 in that provides a comprehensive descriptive and mechanistic analysis of the irradiated model, and extends the results to the more clinically relevant situation in the non-conditioned recipients. The latter results suggest a cure strategy involving editing of the WHIM allele and syngeneic transplantation of the edited cells to non-conditioned or lightly conditioned WHIM recipients. The work also highlights the ability of CXCR4 signaling to inhibit HSC proliferation. The work was Highlighted by a Commentary published by JCI, June 25, 2018. www.jci.org/articles/view/121857 H. Broxmeyer. Enhancement of stem cell engraftment on a WHIM. 3.) In FY18, we submitted an application for patent protection to the PTO for a method of universal allotransplantation. The method uses an antibody drug conjugate (ADC) involving the ribosome toxin saporin complexed to anti-CD117 to selectively deplete hematopoietic stem cells in mice. When mice are transiently immunosuppressed after ADC conditioning they engraft durably and at high levels when given bone marrow from a completely immunologically mismatched donor mouse. Chimeric mice thereby acquire robust donor specific tolerance for skin allografts. Importantly, we did not observe acute toxicity in the ADC conditioned animals, probably because mature leukocytes are mainly unaffected by the immunotoxin. Thus, the patent describes proof of principle for a safe method of universal allotransplantation, which could abrogate the donor shortage that limits the application of allotransplantation in humans.
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