Bone marrow basophils provide survival signals to immature B cells in vitro but are dispensable in vivo

Immature B cells are the first B cell progenitors to express a fully formed B cell receptor and are therefore subject to extensive selection processes that act to mitigate the emergence of autoreactive clones. While it is well appreciated that most B cell generation in the bone marrow is highly dependent on access to molecules present in the local milieu, the existence of extrinsically provided factors that modulate immature B cell biology is ambiguous. Nonetheless, a population of CD49b+CD90lo cells has demonstrated in vitro potential to promote immature B cell survival. Using a mouse basophil reporter strain we confirmed the identity of these CD49b+CD90lo supportive cells as basophils. However, analysis of bone marrow B cell populations following lineage specific basophil depletion demonstrates that basophils do not have a significant role in vivo in modulating immature B cell biology during steady-state conditions.


Introduction
The bone marrow microenvironment is critical in supporting B lymphopoiesis and mature B cell function. Several defined cellular niches have been identified in this organ corresponding to the localization pattern of marrow B cells and progenitors [1][2][3]. Moreover, the cells comprising these niches express various molecules, such as IL-7, CXCL12, and MIF, conducive to B cell survival or differentiation [1,2,4,5]. While immature B cells are found enriched within and around the bone marrow sinusoids, a definitive cellular niche supportive of their biology has not been characterized in vivo [6,7]. This issue is of particular significance because it is at the immature stage that central tolerance is enforced though negative selection of autoreactive B cell receptors (BCR) [8]. Maturing B cells expressing an autoreactive BCR are able to reexpress the recombinase genes RAG1 and RAG2; in doing so such cells have the opportunity to undergo a secondary rearrangement process termed receptor editing [9][10][11]. B cells successfully producing a non autroreactive BCR at this juncture are able to mature normally while unsuccessful cells are deleted and unable to contribute to the repertoire. Thus any cellular and PLOS ONE | https://doi.org/10.1371/journal.pone.0185509 September 28, 2017 1 / 12 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 molecular factors comprising a niche specific to immature B cells would have the potential to act as regulators of receptor editing and thereby contribute to the peripheral repertoire [8,11]. While a specific bone marrow niche for immature B cells has not been identified, a few lines of evidence hint to the existence of such a feature and its potential importance to central tolerance. Immature B cells from mice expressing a transgenic autoreactive BCR had low expression levels of BAFF receptor while non autoreactive cells maintained receptor expression levels sufficient to induce signaling [12]. Moreover, BAFF was found to aid the generation of CD23 + transitional cells from non autoreactive immature cells, but not autoreactive one [12]. This is further substantiated by experiments demonstrating that IL-4 works synergistically with BAFF to promote immature B cell maturation into CD23 + transitional cells [13]. Other experiments have indicated that autoreactive and non-autoreactive immature B cells differentially localize within the marrow dictated by responsiveness to the chemoattractant S1P [14]. It has also been observed that co-culturing immature B cells with bone marrow cells provided protection against anti-BCR induced apoptosis and enhanced RAG expression [15,16]. This response involved contact dependent signals and was narrowed down to a non-lymphocyte cellular fraction contained within the CD90 lo CD49b + flow cytometry gate [15,16]. Subsequent work has noted the similar phenotype of these cells to basophils, including expression of CD90, CD49b, and asialo-GM1 [17].
As basophils are known to express high levels of both BAFF and IL-4, have been shown to support plasma cell survival, and exhibit a cell surface phenotype consistent with a CD90 lo CD49b + cell population we hypothesized that this cell type comprises part of the immature B cell niche in vivo [17][18][19][20][21]. Using Basoph8 lineage specific reporter mice we demonstrate that the in vitro effect of bone marrow CD90 lo CD49b + cells on B cells is indeed attributable to basophils [22]. However, lineage specific ablation of basophils by crossing Basoph8 mice to ROSA-DTA mice failed to yield any obvious abnormalities in B cell development or receptor editing. Thus our data indicates that while basophils are capable of supporting B cell survival they are expendable for modifying immature B cell biology in vivo.

Materials and methods
Mice Basoph8 (C.129S4(B6)-Mcpt8 tm1(cre)Lky /J) and ROSA-DTA (C.129P2(B6)-Gt(ROSA) 26Sor tm1(DTA)Lky /J) mice were purchased from The Jackson Laboratory. Mice were housed and bred under specific pathogen-free conditions in the animal facilities of the Princess Margaret Cancer Centre, University Health Network (UHN). Experiments were performed on 7-20 week old male and female mice (as indicated) according to protocols approved by the UHN Animal Care Committee. Euthanasia was carried out by isoflurane overdose followed with cervical dislocation. Individual experiments were always sex and age matched and compared littermates. In vivo sinusoidal labeling was accomplished by IV injection of 1 μg Armenian hamster anti-mouse FcεRIα (MAR-1; Biolegend) or rat anti-mouse B220 (RA3-6B2; eBioscience) 2 minutes prior to euthanasia.

Enumeration of total organ cell numbers
To obtain organ cell counts isolated cell suspensions from a single mouse leg was diluted in Trypan Blue (Sigma) and live cells counted using a hemocytometer. The number of live cells was multiplied by 10.6 since radiographic isotype distribution studies have found that one set of mouse femur and tibia contain 9.4% of the total marrow [23].

Real-time PCR
B cells were purified by magnetic cell selection using a mouse CD19 positive selection kit (STEMCELL Technologies). Single cell suspensions were lysed in TRIzol (ThermoFisher Scientific) and RNA extracted by phenol/chloroform ethanol precipitation. cDNA was prepared using RT 2 First Strand Kit (Qiagen), while qPCR was performed using RT 2 SYBR Green Mastermixes (Qiagen) both according to manufacturer protocols with a mouse specific RAG1 primers (cat. PPM24586F Qiagen) or mouse specific B-actin primers: 5'-ACGGCCAGGTCA TCACTATTG-3'; 5'-CAAGAAGGAAGGCTGGAAAAGAG-3'. Samples were run on an ABI SDS 7900HT (Applied Biosystems) using a standard protocol for RAG1 expression: 2 min at 50˚C; 95˚C; 40 cycles of 15 s at 95˚C and 1 min at 60˚C. Serial dilutions for each sample were tested for linearity in amplification. Differences in expression levels were calculated using the comparative CT method.

Statistical analysis
Statistical analysis was conducted using GraphPad Prisim v6 (GraphPad Software). Significance levels were defined as Ã p<0.05; ÃÃ p<0.01; ÃÃÃ p<0.001 as determined by one-way ANOVA with a Dunnett's multiple comparisons test; one-way ANOVA with a Tukey's multiple comparisons test; two-way ANOVA with a Sidak's multiple comparisons test.

CD49b + CD90 lo bone marrow cells are highly enriched for basophils
Previous studies have noted a bone marrow derived cell population that protected immature B cells from BCR crosslinking induced apoptosis [15,16]. These cells were found to be CD49b + CD90 lo and asialo-GM1 + while negative for B220, MHC class II, CD11c, CD4, CD5, and CD8 [15,16]. Further, the supportive cells could be isolated from Rag-2 -/and IL-2RU -/mice [16]. To test the hypothesis that these cells were basophils we obtained Basoph8 mice from the Jackson Laboratory. Basoph8 is a reporter mouse stain where the mast cell protease 8 (Mcpt8) gene has been replaced with a cassette containing sequences encoding YFP and humanized Cre recombinase [22]. Mcpt8 is a basophil specific protein found in mice but not humans and does not appear to be required for basophil development [22,24]. Staining bone marrow from Basoph8 mice for CD49b, CD90, CD200R3, and the high affinity IgE receptor revealed a dramatic enrichment of basophils (YFP + CD200R3 + F-cεRIα + ) in the CD49b + CD90 lo flow cytometry gate as compared to other cell populations (Fig 1A). The CD49b + CD90 lo population represented roughly half of cells doubly expressing YFP and CD200R3, while the rest were CD49b + CD90 - (Fig 1B). To further confirm the identity of the CD49b + CD90 lo YFP + population we FACS sorted these as well as CD49b + CD90 lo YFPcells and made cytospins. Giemsa staining reveled that the YFP + fraction was homogenous with most cells exhibiting bilobed nuclei and light granule staining typical of mouse basophils [25,26]. In contrast, the YFPpopulation appeared more lymphocytic with a large nuclear to cytoplasmic ratio ( Fig 1C).
C CD YFP Bone marrow basophils provide in vitro support for immature B cell survival As the CD49b + CD90 lo bone marrow cell population is heterogeneous, we sought to determine whether the basophil fraction among CD49b + CD90 lo cells was responsible for protecting B cells from BCR crosslinking induced apoptosis, in vitro. To accomplish this bone marrow from Basoph8 mice was collected and stained for both CD49b and CD90. CD49b + CD90 lo cells were then sorted by FACS into YFP + (basophils) and YFPfractions. We co-cultured these populations overnight with CD2 + IgD -B cell progenitors at a 1:25 ratio in the presence or absence of BCR crosslinking anti-μ antibody. The frequency of live B cells was then measured with a Nicoletti assay to gate out apoptotic cells [27]. As shown in Fig 2A,   CD49b + CD90 lo YFPcells were sorted and co-cultured with CD2 + IgM -IgD -B cells, as before. Two days later the frequency and absolute numbers of IgD + CD23and IgD + CD23 + cells were assessed by flow cytometry. These markers indicate the maturation of B cell progenitors into the early transitional and late transitional/mature stages respectively [28]. The addition of YFP + cells to these cultures greatly enhanced the recovery of IgD + CD23 + cells (Fig 2B). Collectively, these data indicate that basophils are in fact the CD49b + CD90 lo bone marrow cell population that provides support to developing B cells, in vitro.

Basophils localize to the bone marrow sinusoids
Having seen that basophils are capable of providing a survival advantage to immature B cells when co-cultured, we aimed to determine whether basophils provide the same support in vivo. Previous work has determined that CD49b + cells maintain close contact with B cells in the bone marrow tissue [16]. Since immature B cells are known to be preferentially distributed near bone marrow sinusoids we examined if basophils are similarly localized near the sinusoids [4,7]. To assess this, basoph8 mice were intravenously injected with anti-FcεRIα antibody conjugated to R-phycoerythrin and euthanized two minutes later. The large molecular weight of this flurophore prevents it from rapidly diffusing into the parenchymal tissue and therefore when conjugated to a specific antibody has been shown to preferentially label sinusoidal cells [7]. A large majority of YFP + bone marrow cells were found to be FcεRIα-PE + indicating that like immature B cells basophils tend to accumulate in and around the marrow vasculature (Fig 3).

Lineage specific depletion of basophils does not alter B cell development in vivo
To test if basophils influence B cell development in vivo, we crossed Basoph8 and ROSA-DTA mouse strains and analyzed the bone marrow B cell compartment in the resulting offspring. This cross produces lineage specific expression of diphtheria toxin and therefore efficient deletion of basophils [22] (Fig 4A). Surprisingly, examination of the bone marrow of Basoph8 x ROSA-DT mice did not reveal any notable anomalies despite thorough basophil deletion when compared to age and sex matched ROSA-DT controls. Total marrow cellularity, pro-B and pre-B, immature-B, transitional, and mature B cell populations were normal (Fig 4B-4D).
In addition there was no difference in the usage of λ light chain among immature, transitional and mature bone marrow B cells (Fig 4E). Immature B cells, especially non autoreactive ones, are known to collect in and around the bone marrow sinusoids [6,7,14]. To determine if basophils could influence B cell localization to the sinusoids we intravenously injected mice with anti-B220 conjugated to R-phycoerythin and euthanized the mice two minutes later, as   5).
A key finding of earlier work examining the potential for CD49b + CD90 lo cells to support immature B cells was that when these cells were co-cultured together and treated with an anti-IgM BCR crossing antibody B cells were induced to express RAG genes [15]. We sought to determine if basophil deletion would mirror these in vitro experiments and produce a reduction of B cell RAG expression. To test this we measured RAG1 expression by real-time PCR in purified B cells derived from Basoph8 x ROSA-DT or Basoph8 X B6 mice. No significant difference in expression level was discernable (Fig 6). Overall, these analyses indicate that lineage specific deletion of basophils does not have a significant impact on the normal development of B cells in vivo.

Discussion
Immature and transitional B cells represent a crucial stage in B lineage development [29]. These are the first B cell progenitors to express a fully formed, lineage defining BCR; nonetheless, by virtue of the necessity to regulate autoreactive potential within the mature B cell repertoire they are highly sensitive to selection processes. Crosslinking of immature and transitional BCRs by self-antigen is likely to induce receptor editing, deletion, or anergy [8, 10,29]. Passing such selection events promotes survival, further differentiation, and relocation to mature immune environments [1,8]. An intriguing possibility is that cell extrinsic signals present in the local environment in which an immature B cell encounters antigen could act to modify the selection processes of immature and transitional cells [8,11]. By extension, cellular or In the current study, we sought to examine the hypothesis that bone marrow basophils contribute to supporting immature and transitional B cell development. Previous work research demonstrated that a peculiar CD49b + CD90 lo bone marrow cellular fraction is capable of promoting the survival of immature and transitional B cells during exposure to BCR crosslinking [15,16]. Using Basoph8 lineage reporter mice we have unambiguously identified these cells as basopholis and validated that they are capable of providing B cells beneficial signals during in vitro co-culture. However, when we crossed Basoph8 with ROSA-DTA mice so as to generate basophil ablated mice we could find no defects in the populations numbers of bone marrow B cells, the usage of the λ light chain, or the accumulation of B cells within the marrow sinusoids. In addition, there was no difference in the expression level of RAG1 in basophil deleted mice. We interpret these findings to imply that while basophils possess the molecular machinery to influence immature and transitional B cells, they are dispensable or redundant in vivo.
Given that we have previously documented an accumulation of CD49b + CD90 lo cells in the bone marrow in response to adjuvant induced inflammation it tempting to speculate that the basophils may gain functional relevance to B cells during non steady-state conditions such infection [30]. Potentially, in vitro culture conditions are relatively skewed towards an environment conducive to basophil help based upon the balance of various nutrients, inflammatory factors, and cellular stress. The feasibility of this idea is supported by a discrepancy seen between this and previous work: when CD49b + CD90 lo cells were targeted for deletion by repetitive administration of antibodies against asialo-GM1 a concurrent loss of immature B cells was observed [16]. While this result may have been due to off target effects of the antiasialo-GM1, possibly the punctuated apoptosis of marrow resident cells induced by this treatment may have created an environment where immature B cells were dependent on basophil availability.
Through the experiments presented we have failed to find evidence that implicates basophils as major regulators of immature B cell biology during steady-state marrow conditions. However, it is clear that in the conditions of in vitro co-culture basophils efficiently support B cell survival and possibly maturation. In this, regard our experiments are in agreement with previous studies as well as the profile of basophil as IL-4 + BAFF + CD40L + cells with a known propensity to support B cells in a variety of contexts [18,19,21]. Since our main aim was to determine if there is a role for basophils in vivo, we did not explore the extent to which any of these or other cell supplied molecules may contribute to B cell survival. Deeper investigation of basophil-B cell behavior during co-culture has potential to serve as a useful vehicle for the discovery of B cell modulating factors. Similarly, the very fact that basophils enhance B cell survival in vitro but not in vivo bolsters the initial rational for our study: environment matters in B cell biology.