CXCR4 antagonism ameliorates leukocyte abnormalities in a preclinical model of WHIM syndrome

Abstract

Background: WHIM (Warts, Hypogammaglobulinemia, Infections, and Myelokathexis) syndrome is an ultra-rare, combined primary immunodeficiency and chronic neutropenic disorder characterized by a range of clinical presentations, including peripheral neutropenia, lymphopenia, and recurrent infections. WHIM syndrome is most often caused by gain-of-function mutations in the gene encoding C-X-C chemokine receptor 4 (CXCR4). As such, inhibition of CXCR4 with XOLREMDI^® (mavorixafor), an orally bioavailable CXCR4 antagonist, demonstrated clinically meaningful increases in absolute neutrophil and lymphocyte counts and concomitant reduction in infections in patients with WHIM syndrome, resulting in its recent U.S. Food and Drug Administration approval. The impact of CXCR4 antagonism on other aspects of the pathobiology in WHIM syndrome, such as lymphopoiesis and leukocyte trafficking between primary and secondary lymphoid organs, is less understood.

Methods: In the current study, the effects of CXCR4 antagonism on leukocyte trafficking and distribution in primary and secondary lymphoid organs were investigated in a mouse model of WHIM syndrome carrying the heterozygous Cxcr4¹⁰¹³ mutation. Cxcr4^+/1013 and Cxcr4 wild-type mice received the orally bioavailable CXCR4 antagonist X4-185. Blood, spleen and bone marrow samples were collected for numeration, flow cytometry, and functional studies.

Results: Cxcr4^+/1013 mice exhibited profound peripheral blood leukopenia as seen in patients with WHIM syndrome. CXCR4 antagonism corrected circulating leukopenia and mobilized functional neutrophils without disrupting granulopoiesis in the bone marrow of Cxcr4^+/1013 mice. Furthermore, Cxcr4^+/1013 displayed aberrant splenic T and B-cell counts and frequency. Treatment with X4-185 normalized splenic T-cell abnormalities, correcting the reduced CD8⁺ T-cell numbers, restoring the CD4/CD8 T-cell ratio, and ameliorating peripheral blood T-cell lymphopenia. In addition, CXCR4 antagonism was able to correct the abnormal frequencies and numbers of splenic marginal zone and follicular B cells in Cxcr4^+/1013 mice, and ultimately normalize B-cell lymphopenia in the peripheral circulation.

Conclusions: Our study provides comprehensive evidence that oral dosing with a CXCR4 antagonist can effectively correct WHIM-associated neutrophil and lymphocyte abnormalities in a mouse model of WHIM syndrome. These findings extend our understanding of how targeting the dysregulated CXCR4 signaling pathway can ameliorate the pathogenesis of WHIM syndrome.

Keywords: CXCR4 antagonism; WHIM syndrome; chronic neutropenia; neutrophil function; preclinical study; primary immunodeficiency.

Figure 1

CXCR4 antagonism corrects the peripheral panleukopenia in Cxcr4^+/1013 mice. (A) Experimental design for CXCR4 signaling inhibition by CXCR4 antagonist X4-185 in WT and Cxcr4^+/1013 mice. Absolute (B) leucocyte, (C) lymphocyte, (D) granulocyte, and (E) monocyte counts were determined in the blood of WT and Cxcr4^+/1013 mice 3 hours after the last dose on day 7. Data (mean + SEM) were from two independent experiments with 6–10 mice per group. Statistics were calculated using the nonparametric Mann-Whitney test, two-sided. *p < 0.05, **p < 0.01, ***p < 0.0001. CXCR4, C-X-C chemokine receptor 4; d, day; SEM, standard error of the mean; Veh., vehicle; WT, wild-type.

Figure 2

CXCR4 antagonism mobilizes neutrophils in the peripheral blood and preserves their function. (A) Absolute counts of mature neutrophils in the blood and (B) granulocyte subset cell counts in BM were determined 3 hours after the last dose on day 7. (C) Blood neutrophils were harvested 3 hours after the last dose on day 7, incubated with pHrodo BioParticles, and phagocytotic uptake was measured. (D) Blood neutrophils were harvested at 3 hours after the last dose on day 7, stimulated with phorbol 12-myristate 13-acetate, and ROS formation was measured. Data (mean + SEM) were from two independent experiments with 6–10 mice per group (A, B) and from one experiment with three to four mice per group (C, D). Statistics were calculated using the nonparametric Mann-Whitney test, two-sided. *p < 0.05, **p < 0.01, *** p < 0.001. BM, bone marrow; CXCR4, C-X-C chemokine receptor 4; ROS, reactive oxygen species; SEM, standard error of the mean; Veh., vehicle; WT, wild-type.

Figure 3

CXCR4 antagonism normalizes the splenic T-cell compartment in Cxcr4^+/1013 mice. Absolute (A) CD4⁺ T-cell counts, (B) CD8⁺ T-cell counts, and (C) CD4/CD8 T-cell ratio were determined in the blood 3 hours after the last dose on day 28. Total (D) CD4 T-cell numbers, (E) CD8 T-cell numbers, and (F) CD4/CD8 T-cell ratio were determined in the spleen of WT and Cxcr4^+/1013 mice after 28 days of X4-185 treatment. Data (mean + SEM) were from two independent experiments with 6–10 mice per group. Statistics were calculated using the nonparametric Mann-Whitney test, two-sided. *p < 0.05, **p < 0.01. CXCR4, C-X-C chemokine receptor 4; SEM, standard error of the mean; Veh., vehicle; WT, wild-type.

Figure 4

CXCR4 antagonism normalizes the splenic B-cell compartment in Cxcr4^+/1013 mice. (A) Absolute B-cell counts were determined in the blood of WT and Cxcr4^+/1013 mice 3 hours after the last dose on day 7. (B) Total splenic B-cell number in WT and Cxcr4^+/1013 mice after 28 days of X4-185 treatment. The frequency of (C) FO or (D) MZ B cells and the total numbers of (E) FO or (F) MZ B cells were determined in the spleen of WT and Cxcr4^+/1013 mice after 28 days of X4-185 treatment. Data (mean + SEM) were from two independent experiments with 6–10 mice per group. Statistics were calculated using the nonparametric Mann-Whitney test, two-sided. *p < 0.05, **p < 0.01, ***p < 0.001. CXCR4, C-X-C chemokine receptor 4; FO, follicular; MZ, marginal zone; SEM, standard error of the mean; Veh., vehicle; WT, wild-type.