WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome is a rare, combined, primary immunodeficiency, and chronic neutropenic disorder predominantly caused by autosomal dominant gain-of-function (GOF) variants of the chemokine receptor CXCR4, resulting in impaired receptor internalization, hyperactive signaling, and enhanced chemotaxis to CXCL12. This signaling dysregulation underlies the clinical and hematologic manifestations of WHIM syndrome, such as enhanced bone marrow leukocyte retention. While effects of CXCR4 antagonists on GOF signaling in cells from individuals with WHIM syndrome have been reported, comprehensive analyses from larger patient cohorts are lacking. We assessed CXCR4 molecular functions in T lymphocytes from individuals with WHIM syndrome enrolled in the mavorixafor phase 3 trial (NCT03995108) and evaluated the potential of mavorixafor, an oral CXCR4 antagonist, to counteract molecular GOF phenotypes in vitro. For this, we first investigated the most conserved hallmark of CXCR4 GOF variants, impaired CXCR4 receptor internalization, in primary T lymphocytes from peripheral blood mononuclear cells collected pre dose. Second, ligand-induced CXCR4 signaling and chemotaxis of expanded T lymphocytes were assessed after in vitro pretreatment with mavorixafor or control. T lymphocytes from patients with WHIM syndrome showed defective receptor internalization and hyperactive signaling, including enhanced CXCL12-induced calcium flux, increased ERK, and prolonged AKT phosphorylation, consistent with enhanced chemotaxis observed in a CXCL12 gradient. Pretreatment with mavorixafor in vitro inhibited CXCL12-induced hyperactive signaling and migration of T lymphocytes. This study represents the first comprehensive characterization of CXCR4 GOF phenotypes in T lymphocytes from a large cohort of individuals with WHIM syndrome. The correction of underlying molecular defects may be linked to improved leukocyte mobilization observed in participants in the mavorixafor WHIM syndrome phase 3 trial.
