Unexpected developments in immune organs in WHIM syndrome

In this issue of Blood, Balabanian and colleagues describe a mouse model of WHIM syndrome, a combined myeloid and lymphoid immunodeficiency disorder.¹  The results provide a high-definition histopathologic picture of the effects of WHIM mutation CXCR4¹⁰¹³ on immune organs and suggest complex, unexpected defects in both leukocyte trafficking and development.

Consider a patient with recurrent bacterial sino-pulmonary infections since early childhood. During infection the absolute neutrophil count (ANC) is elevated but falls below normal with recovery. Immunoglobulin levels and circulating B cells are also decreased; CD4⁺ T lymphocytes are as low as in AIDS. In fact, almost all circulating leukocyte subsets are low. Yet classic opportunistic infections do not occur, bacterial infections usually do not become life-threatening, and the main problematic virus is human papillomavirus (HPV), which causes large numbers of skin and anogenital warts. The mother, brother, and 2 of 3 children of this patient have a similar condition.

This patient with deficiency of both myeloid and lymphoid immunity is a classic case of a rare and fascinating disease known as WHIM syndrome.²  Despite neutropenia, the bone marrow contains abundant mature neutrophils, an almost pathognomonic finding known as myelokathexis (Greek for marrow retention, and the “M” in the acronym WHIM). W, H, and I refer to warts, hypogammaglobulinemia, and infections, respectively. Infections transiently elevate the ANC, promoting clinical recovery and survival into adulthood. Thus, neutropenia in WHIM syndrome is considered in part a problem of neutrophil egress from bone marrow, not neutrophil production.

This idea gained traction in 2003 when Hernandez et al discovered inherited autosomal dominant mutations in the chemokine receptor CXCR4 as the cause of WHIM syndrome.³  The mutations truncate the C-terminal domain, but because this region normally mediates GRK- and arrestin-dependent desensitization, they result in increased signaling. Because CXCR4 normally mediates bone marrow homing and retention of neutrophils,⁴  gain-of-function WHIM mutations may cause myelokathexis by simply increasing these activities. Because CXCR4 is widely expressed on all leukocyte subtypes, other cytopenias could arise from a similar mechanism in immune organs. If so, immune organs should contain increased numbers of all leukocytes deficient in blood. This hypothesis is difficult to test in patients and direct data have been lacking. In fact, apart from bone marrow biopsies, knowledge of immune organ histopathology in WHIM syndrome is limited to lymph nodes from 2 patients. In this regard, Balabanian et al blaze an important new trail with their mouse, in which the WHIM allele CXCR4¹⁰¹³ replaces 1 wild-type mouse Cxcr4 allele, allowing study of the mutation in any tissue at any time under both homeostatic conditions and specific stresses.¹  The mouse developed normally and phenocopied severe panleukopenia, validating the model in the blood. However, the tissue findings are complex, lineage-specific, and unexpected, and suggest effects of the mutation on both leukocyte trafficking and development in immune organs (see figure).

In primary immune organs (bone marrow and thymus), the tissue architecture was normal. Myelopoiesis was also normal; however, unexpectedly, the numbers of both total and apoptotic neutrophils were not increased in bone marrow. The authors concluded that CXCR4 desensitization is therefore unlikely to be the primary cause of myelokathexis. However, total bone marrow neutrophils cannot be readily measured in humans and could be normal or even decreased in WHIM patients, too. More likely, multiple mechanisms, including increased neutrophil retention in and accelerated homing to bone marrow plus a relative decrease in myelopoiesis, account for neutropenia.

Unlike myelopoiesis, B and T lymphopoiesis both appeared to be suppressed. Single- and double-positive T cells were deficient in thymus, and bone marrow contained reduced B-cell precursors without increased apoptosis. This is surprising because the Cxcr4 knock-out mouse identified Cxcr4 as a positive regulator of hematopoiesis.⁵  Together, these results suggest that lymphopoiesis may depend on fine-tuning of CXCR4 signal strength. Despite reduced precursors, mature B-cell number was normal in bone marrow. Thus, B lymphopenia, like neutropenia, probably also depends on defective trafficking and egress.

Unlike primary immune organs, secondary immune organ architecture was grossly abnormal in the model, including absent or reduced numbers of B-cell follicles. Naive but not effector/memory CD4⁺ and CD8⁺ T cells were deficient in spleen. In contrast, T-cell numbers were normal or increased in lymph nodes. In addition, there were fewer and abnormally localized lymphatic vessels in lymph node, suggesting impaired egress as a potential mechanism.

In spleen, where immature B cells from bone marrow mature further into type 1 and 2 transitional B cells and then become mature marginal zone and follicular B cells, only marginal zone B-cell number was not decreased. Interestingly, in spite of severe B lymphopenia and absence of follicles, immunoglobulin levels were normal or even high, raising questions about where B cells are being activated. In WHIM patients, hypogammaglobulinemia is the least penetrant feature, and when present may be mild. However, the durability of protective memory responses could still be affected. In this regard, there are several reports of short-lived antibody responses to vaccines and vaccination failures in WHIM patients.⁶  Importantly, the CXCR4¹⁰¹³ knock-in mouse will now allow systematic modeling of primary and memory vaccine responses, germinal center formation, and the relative importance of lymphoid versus myeloid defects to infection susceptibility in WHIM syndrome.

Leukocytes can be mobilized to blood in WHIM syndrome not only by infection but also by plerixafor (Mozobil, AMD3100),^7,8  a CXCR4 antagonist approved by the US Food and Drug Administration for HSC mobilization in the setting of transplantation for specific lymphoid malignancies. Clinical trials are under way to test the safety and efficacy of plerixafor as mechanism-based therapy in WHIM syndrome; however, the source of mobilized cells has remained undefined. In this regard, plerixafor readily mobilized both neutrophils and B cells to the blood in the WHIM mouse, as it does in patients, but, surprisingly, without significantly affecting the number of these cells in bone marrow, suggesting the existence of other mobilizable storage sites. Alternatively, a small transient change in bone marrow could still potentially cause a large change in blood. Future in vivo trafficking studies may help define the source of plerixafor-mobilized cells in the model.

The CXCR4¹⁰¹³ knock-in mouse, like transgenic zebrafish and xenotransplant mouse models of WHIM that preceded it,^9,10  is actually a model of “M,” not WHIM. Immunoglobulins were normal, spontaneous infections were not reported, and mice are not permissive for HPV. Nevertheless, this mouse is clearly an important tool for future studies of hematopoiesis and infection susceptibility, particularly for viruses other than HPV. It also provides a unique opportunity to test the phenotypic effects of homozygous CXCR4¹⁰¹³ and CXCR4¹⁰¹³ expression specifically in hematopoietic versus nonhematopoietic cells. In this regard, wart keratinocytes up-regulate CXCR4 in both immunologically healthy individuals and patients with WHIM syndrome, and a recent study revealed that WHIM receptors may directly drive keratinocyte transformation in vivo in mice, independently of immunodeficiency.¹¹  This suggests CXCR4 may play a direct role in HPV disease, and could be a target for local treatment.

For patients, the CXCR4¹⁰¹³ knock-in mouse has confirmed that CXCR4 is the correct therapeutic target in WHIM syndrome and that dialing down CXCR4 signaling is an ideal therapeutic strategy. In the future, it may help accelerate preclinical development of drug candidates while continuing to reveal surprising new concepts about pathogenesis and the normal role of CXCR4 in hematopoiesis.