Central development: from HSCs to immature B cells

CXCR4 is already involved at a very early stage of hematopoiesis in the BM, maintaining a pool of quiescent HSCs. The CXCL12/CXCR4 axis, throughout contacts with the so-called CXCL12-abundant reticular (CAR) cells, maintains retention of HSCs within specialized niches (Sugiyama et al, 2006; Tzeng et al, 2011). Indeed, the induced deletion of CXCR4 in adult mice leads to a reduction in the number of HSCs within the BM. HSCs, in the absence of CXCR4, demonstrate heightened vulnerability to myelosuppressive stress triggered by 5-fluorouracil and exit the G0 quiescent phase (Sugiyama et al, 2006). In accordance, the in vitro addition of CXCL12 inhibits the entry of murine HSCs into the cell cycle in a dose-dependent manner, confirming the central role of CXCR4 in governing HSC proliferation (Nie et al, 2008). Depletion of CAR cells in mice results in a reduced number of HSCs and an up-regulation of early myeloid selector genes, resembling the phenotype observed in wild-type HSCs cultured without a niche (Omatsu et al, 2010). Conversely, aberrant CXCR4 signaling is also deleterious to generate early lymphoid progenitors, as the increased quiescence of short-term HSCs observed in CXCR4 GOF Cxcr4^+/1013 mice impairs transition to multipotent progenitors and to the common lymphoid progenitor (CLP) (Freitas et al, 2017). Hence, both CAR cells and a fine regulation of CXCR4 signaling are essential for the generation of lymphoid progenitors and for the maintenance of HSCs in an undifferentiated state (Omatsu et al, 2010).

Furthermore, CXCR4 enables CLP to position in the vicinity of IL7⁺CXCL12⁺ stromal cells, allowing the commitment of CLP in the B-lineage (Tokoyoda et al, 2004; Cordeiro Gomes et al, 2016; Kaiser et al, 2023). Subsequently, pro-B cells in contact with the IL-7–expressing cells proliferate, while remaining anchored within these niches (Tokoyoda et al, 2004) (Fig 1). During this stage, the IL-7 signaling pathway sustains the expression of CXCR4 and the adhesion protein FAK augmenting adherence to the stromal environment (Clark et al, 2014; Fistonich et al, 2018). VDJ recombination ensues, leading to the formation of a functional immunoglobulin heavy chain (Igµ), constitutive of the pre-BCR (Clark et al, 2014). Observations from high-power field confocal microscopy of mouse BM have revealed that small pre-B cells, which cease proliferating, tend to localize in proximity to stromal cells exhibiting low levels of IL-7 but high levels of CXCL12 (Mandal et al, 2019). This strategic localization is attributed to the effect of the pre-BCR, which induces an up-regulation of CXCR4 expression through IRF4, concurrently down-regulating the expression of adhesion factors (Mandal et al, 2019). This orchestrated response enables pre-B cells to disengage from IL-7–rich niches within the BM (Johnson et al, 2008; Fistonich et al, 2018). Between pre-B cells and immature/mature B cells, a twofold down-regulation of CXCR4 leads to a decreased response to CXCL12 in mice and allows the egress of these cells from the BM (Honczarenko et al, 1999; Beck et al, 2014) (Fig 1). Immature and mature B cells increase simultaneously CCR7 expression favoring the formation of CXCR4-CCR7 heterodimers, which may also impair CXCR4 signaling and induce BM exit (Mcheik et al, 2019).

Discerning the direct and indirect effects of CXCR4 signaling in vivo poses a considerable challenge. Numerous data from studies conducted in artificial BM progenitor culture systems indicate that CXCR4 exerts effects beyond mere chemotaxis. In vitro, the expression of pre-BCR and evasion of IL-7 only result in minor effects on B-cell developmental transcriptional and epigenetic programs without CXCL12 (Mandal et al, 2019). B220⁺IgM⁻ progenitors from WT mice, cultivated with a reduced IL-7 dose in the presence of CXCL12, have a transcriptomic profile of cell cycle repression and tend toward differentiation programs involving Irf4, Irf8, and Ikzf3 (encoding the transcription factor Aiolos) in an ERK-dependent manner (Mandal et al, 2019). Consistently, ATAC-seq experiments reveal that CXCR4 opens binding sites for transcription factors involved in late B lymphopoiesis (e.g., FOXO1, E2A) while preventing the binding of factors such as MYC and STAT5, implicated in earlier processes (Mandal et al, 2019). CXCR4 is also involved in light-chain recombination in small pre-B cells, favoring Rag expression and Igk transcription (Mandal et al, 2019; McLean & Mandal, 2020).

In addition to the pivotal role of CXCR4 in B-cell ontogeny, recent data highlight its involvement in central B-cell tolerance. In the 3–83Igi,H-2^b mb1Cre mouse model, autoreactive immature B cells, characterized by high avidity for the self-antigen MHC-I H-2K^b, have a 1.5-fold increase in CXCR4 expression compared with non-autoreactive cells, preventing them from migrating to the periphery (Greaves et al, 2019; Pelanda et al, 2022). This CXCR4 higher expression in immature autoreactive B cells is also observed in a human immune system humanized mouse (HIS hu-mice), in which all mouse cells express a self-antigen that reacts with developing human Igκ⁺ B cells (Alves da Costa et al, 2021). In this model, autoreactive immature B cells show a higher migration potential in response to CXCL12 than non–self-reactive immature B cells. Furthermore, treatment of these mice with a CXCR4 antagonist (AMD3100) results in a twofold increase in the frequency of autoreactive B cells in the spleen after 48 h, confirming that CXCR4 plays functionally a key role in the retention of autoreactive B cells in the BM (Alves da Costa et al, 2021).

More recently, Okoreeh et al suggested, with in vitro culture of B220⁺IgM⁻ progenitors, the direct requirement for CXCR4 signaling in orchestrating receptor editing and Igλ recombination (Okoreeh et al, 2022). Indeed, CXCR4 deficiency impairs Igλ+ B-cell development, as CXCR4 signaling promotes chromatin accessibility of the Igλ locus and modulates accessibility at binding motifs for transcription factors critical in receptor editing (Okoreeh et al, 2022).

Altogether, CXCR4 not only plays a role in B-cell development in BM but also contributes to central B-cell tolerance mechanisms.

From mature B cells to antibody response

In the periphery, CXCR4 function on mature naive B cells requires the expression of the IgD-BCR (Becker et al, 2017). In mice, the absence of IgD results in the abolition of CXCL12-induced migration, underlining the pivotal role of IgD in this migratory process. CXCR4 signaling further activates the ERK and AKT pathways in an IgD-dependent manner, and intriguingly, CD19 emerges as a potential intermediary between CXCR4 and IgD-BCR. This is supported by the restoration of CXCR4 signaling upon in vitro stimulation of CD19 in IgD-deficient B cells, indicating a functional connection between CXCR4, CD19, and IgD-BCR during B-cell activation (Becker et al, 2017).

Furthermore, CXCR4 is essential for the T-dependent immune response notably through its role in the mobilization of centroblasts in the germinal center (GC) of secondary lymphoid organs. Centroblasts located in the dark zone of the GC, which contains a network of CXCL12-expressing reticular cells, exhibit strong CXCR4 expression in both mice and humans (Allen et al, 2004; Caron et al, 2009; Rodda et al, 2015). In contrast, centrocytes down-regulate CXCR4, enabling them to leave the dark zone and to enter the light zone directed by the CXCL13/CXCR5 axis (Allen et al, 2004, 2007; Caron et al, 2009; Victora et al, 2010) (Fig 1). Multiple mechanisms may contribute to the differential CXCR4 expression between the light and dark zones. In the light zone, follicular dendritic cells express CD161, which is the binding partner for the C-type lectin-like receptor LLT1 found on the membranes of GC B cells. When human tonsillar GC B cells are exposed to recombinant human CD161 in culture, a decrease in CXCR4 expression is observed, indicating that CD161/LLT1 interactions could potentially trigger the phenotypic transition from the dark to the light zone (Llibre et al, 2016). Another potential mechanism for explaining this transition comes from murine data, which demonstrate that IL-21 produced by follicular helper T cells, along with an increase in CD63 expression, facilitates internalization and may contribute to the down-regulation of CXCR4 in centrocytes (Yoshida et al, 2011). As a result, altered CXCR4 expression could have an impact on the homeostasis of the GC. In accordance with this, altered lymphoid follicle architecture has been reported in CXCR4 GOF Cxcr4^+/1013 mice and in two histopathological reports of lymph nodes from WHIM patients, but the origin of these abnormalities is difficult to interpret in the context of WHIM-associated lymphopenia (Zuelzer, 1964; Mentzer et al, 1977; Balabanian et al, 2012).

Besides the GC, CXCR4 regulation is also important to control extrafollicular response, that is, the T-independent immune response, which serves as the primary line of defense during infections. In CXCR4 GOF Cxcr4^+/1013 mice, the defect of CXCR4 desensitization leads to an exacerbated extrafollicular B-cell response, marked by an increase in IgM⁺ splenic plasmablasts and an elevation in total IgM serum titers after T-independent antigen immunization (Alouche et al, 2021). Accordingly, in vitro plasmablast differentiation, with TLR4 ligands, of splenic B cells from Cxcr4^+/1013 mice is increased compared with controls (Alouche et al, 2021). Mechanistically, the persistence of CXCR4 signaling favors B-cell entry into the cell cycle through the mTORC1 pathway and promotes differentiation into extrafollicular plasmablasts (Alouche et al, 2021). Furthermore, Cxcr4^+/1013 mice exhibit a fivefold increase in BM plasmablast cells compared with WT mice 3 d after immunization (Alouche et al, 2021). Interestingly, this finding is corroborated in humans, as WHIM patients display a significant increase in CD19⁺CD138⁻ plasmablasts in the BM compared with healthy donors (Alouche et al, 2021). Regulation of CXCR4 expression is therefore necessary to contain the extrafollicular response and the medullary tropism of plasmablasts.

Antigen-specific antibody-producing cells that emerge from the GC also re-express CXCR4, enabling them to accumulate within the BM, where some mature into long-lived plasma cells (Hargreaves et al, 2001; Hauser et al, 2002) (Fig 1). Immunohistological analysis of WT mouse BMs confirms that almost all plasma cells cluster in niches with cells expressing CXCL12 (Tokoyoda et al, 2004). This localization is necessary for plasma cells to benefit from a favorable microenvironment, in particular, to be exposed to survival factors such as APRIL (Cassese et al, 2003; Belnoue et al, 2012). A defect in CXCR4 expression in plasma cells results in their accumulation in the spleen and peripheral blood in mice (Hargreaves et al, 2001; Nie et al, 2004; Tokoyoda et al, 2004), and through time-lapse intravital tibial imaging, Benet et al demonstrate that blocking CXCR4 with an antagonist, AMD3100, inhibits intra-BM plasma cell dynamics (Benet et al, 2021). Paradoxically, in CXCR4 GOF mice, GC-derived antigen-specific plasma cells fail to accumulate in the BM and to induce a long-term immune response (Biajoux et al, 2016). This highlights our incomplete understanding of plasma cell homing and survival, both within and outside the BM environment. However, this counter-intuitive finding could be explained by the exacerbated extrafollicular reaction in this mouse model, leading to the accumulation of low-affinity extrafollicular plasmablasts, which competes with antibody-producing cells from the GC reaction for the BM niches (Alouche et al, 2021). This is consistent with a defect of durable humoral T-dependent immune response (i.e., after vaccination) observed in some WHIM patients (Gulino et al, 2004; Handisurya et al, 2010).

Altogether, a fine regulation of CXCR4 signaling is necessary to obtain a specific and durable humoral response. The apparent contradiction between its roles in plasma cell differentiation and HSC quiescence underscores CXCR4’s context-dependent functions across cell types.