CXCR4 is a G protein-coupled receptor with excellent potential as a therapeutic target for a range of clinical conditions including stem cell mobilization, cancer prognosis and treatment, fibrosis therapy and HIV. We report here the development of a fully human single-domain antibody-like scaffold termed an i-body, the engineering of which produces an i-body library possessing a long complementarity determining region (CDR) binding loop, and the isolation and characterisation of a panel of i-bodies with activity against human CXCR4. The CXCR4-specific i-bodies show antagonistic activity in a range of in vitro and in vivo assays including inhibition of HIV infection, cell migration and leukocyte recruitment but, importantly, not mobilization of hematopoietic stem cells. Epitope mapping of three CXCR4 i-bodies AM3-114, AM4-272 and AM3-523 revealed binding deep in the binding pocket of the receptor.

The inherent disadvantages of using granulocyte colony-stimulating factor (G-CSF) for hematopoietic stem cell (HSC) mobilization have driven efforts to identify alternate strategies based on single doses of small molecules. Here, we show targeting α₉β₁/α₄β₁ integrins with a single dose of a small molecule antagonist (BOP (N-(benzenesulfonyl)-L-prolyl-L-O-(1-pyrrolidinylcarbonyl)tyrosine)) rapidly mobilizes long-term multi-lineage reconstituting HSC. Synergistic engraftment augmentation is observed when BOP is co-administered with AMD3100. Impressively, HSC in equal volumes of peripheral blood (PB) mobilized with this combination effectively out-competes PB mobilized with G-CSF. The enhanced mobilization observed using BOP and AMD3100 is recapitulated in a humanized NODSCIDIL2Rγ^−/− model, demonstrated by a significant increase in PB CD34⁺ cells. Using a related fluorescent analogue of BOP (R-BC154), we show that this class of antagonists preferentially bind human and mouse HSC and progenitors via endogenously primed/activated α₉β₁/α₄β₁ within the endosteal niche. These results support using dual α₉β₁/α₄β₁ inhibitors as effective, rapid and transient mobilization agents with promising clinical applications.

Regulatory T cells (Tregs) play a crucial role in the maintenance of peripheral tolerance. Quantitative and/or qualitative defects in Tregs result in diseases such as autoimmunity, allergy, malignancy, and graft-versus-host disease (GVHD), a serious complication of allogeneic stem cell transplantation (SCT). We recently reported increased expression of autophagy-related genes ( Atg ) in association with enhanced survival of Tregs after SCT. Autophagy is a self-degradative process for cytosolic components that promotes cell homeostasis and survival. Here, we demonstrate that the disruption of autophagy within FoxP3 ⁺ Tregs (B6. Atg7 ^fl/fl -FoxP3cre ⁺ ) resulted in a profound loss of Tregs, particularly within the bone marrow (BM). This resulted in dysregulated effector T cell activation and expansion, and the development of enterocolitis and scleroderma in aged mice. We show that the BM compartment is highly enriched in TIGIT ⁺ Tregs and that this subset is differentially depleted in the absence of autophagy. Moreover, following allogeneic SCT, recipients of grafts from B6. Atg7 ^fl/fl -FoxP3cre ⁺ donors exhibited reduced Treg reconstitution, exacerbated GVHD, and reduced survival compared with recipients of B6. WT-FoxP3cre ⁺ grafts. Collectively, these data indicate that autophagy-dependent Tregs are critical for the maintenance of tolerance after SCT and that the promotion of autophagy represents an attractive immune-restorative therapeutic strategy after allogeneic SCT.

Megakaryocytes (MK) generate platelets. Recently, we and others, have reported MK also regulate hematopoietic stem cells (HSC). Here we show high ploidy large cytoplasmic megakaryocytes (LCM) are critical negative regulators of HSC and critical for platelet formation. Using a mouse knockout model (Pf4-Srsf3^Δ/Δ) with normal MK numbers, but essentially devoid of LCM, we demonstrate a pronounced increase in BM HSC concurrent with endogenous mobilization and extramedullary hematopoiesis. Severe thrombocytopenia is observed in animals with diminished LCM, although there is no change in MK ploidy distribution, uncoupling endoreduplication and platelet production. When HSC isolated from a microenvironment essentially devoid of LCM reconstitute hematopoiesis in lethally irradiated mice, the absence of LCM increases HSC in BM, blood and spleen, and the recapitulation of thrombocytopenia. In contrast, following a competitive transplant using minimal numbers of WT HSC together with HSC from a microenvironment with diminished LCM, sufficient WT HSC-generated LCM regulates a normal HSC pool and prevents thrombocytopenia. Importantly, LCM are conserved in humans.