NK Cell-Depleting Anti-Asialo GM1 Antibody Exhibits a Lethal Off-Target Effect on Basophils In Vivo

Natural killer cells provide innate defense against viruses and tumor cells by killing target cells and producing immunoregulatory cytokines (1–3). Two types of NK cell-depleting Abs, a polyclonal Ab specific to asialo GM1 (ASGM1) (4–6) and an mAb specific to NK1.1 (7), have commonly been used to elucidate in vivo functions of NK cells in mice. Anti-ASGM1–mediated NK cell depletion is effective in a variety of mouse strains, whereas anti-NK1.1–mediated NK cell depletion works only in certain strains such as C57BL/6 and SJL and not in many other strains lacking the NK1.1 allotype, including BALB/c, C3H, and A/J mice (8). Anti-ASGM1 activity was first identified >30 y ago in antiserum produced by immunizing rabbits with mouse brain tissues and shown to be responsible for antiserum-mediated elimination of NK activity in the spleen from various mouse strains (4). The expression of ASGM1 is not strictly confined to NK cells among hematopoietic cells and is detected on a subpopulation of NKT, CD8⁺ T, and γδ T cells (9, 10) and some activated form of CD4⁺ T cells, macrophages, and eosinophils under certain experimental conditions (11–13). Nevertheless, anti-ASGM1–mediated NK cell depletion still remains a powerful tool to analyze in vivo functions of NK cells.

Basophils are basophilic granulocytes and represent <1% of peripheral blood leukocytes (14). Interestingly, basophils in mice express typical NK markers CD49b (DX5) and NKR2B4 (2B4) on their surface (15–17) and produce granzyme B-like serine protease, mouse mast cell protease-8 (18, 19), even though cytotoxic activity of basophils remains to be investigated. Basophils also display several characteristics shared by tissue-resident mast cells, including surface expression of the FcεRI, and the release of allergy-related chemical mediators such as histamine in response to various stimuli (14). Because of these mast cell-like phenotypes and their rarity, basophils have often erroneously been considered as minor relatives of mast cells or as blood-circulating precursors of tissue-resident mast cells (20). However, recent studies using basophil-depleting Abs (21–23) and genetically engineered mice deficient only in basophils (24, 25) have illuminated nonredundant roles for basophils in acquired immunity regulation, protective immunity to pathogens, and immunological disorders such as allergy and autoimmunity (26–36).

In the current study, we found that the whole population of basophils in mice homogeneously expresses high levels of ASGM1 and that in vivo administration of anti-ASGM1 readily ablates most of basophils, in addition to NK cells, in a variety of mouse strains, regardless of NK1 allotype and MHC H2 haplotype. These unexpected findings raise concern about the possible contribution of basophils, rather than or in addition to NK cells, to some of phenotypes observed in anti-ASGM1–treated mice. Indeed, treatment of mice with anti-ASGM1 abolished the development of IgE-mediated chronic cutaneous allergic inflammation as efficiently as did the treatment with basophil-depleting Ab.

C57BL/6, BALB/c, C3H mice (6–10 wk old) were purchased from CLEA Japan (Tokyo, Japan). A/J mice were from Japan SLC (Hamamatsu, Japan), and rag2^−/−γc^−/− C57BL/6 mice were from Taconic Farms (Germantown, NY). All animal studies were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University.

FITC-conjugated anti-CD3ε (145-2C11) and anti-rabbit IgG, FITC-conjugated streptavidin, PE-conjugated anti-TCRγδ (GL3), PE-conjugated streptavidin, allophycocyanin (APCy)-conjugated anti-CD8α (53-6.7) and CD49b (HMα2), and APCy-conjugated streptavidin were purchased from BD Pharmingen (San Diego, CA). PE-conjugated anti-FcεRIα (MAR-1) and biotinylated anti-CD3ε (145-2C11) were purchased from eBioscience (San Diego, CA). Unconjugated anti-FcεRIα (MAR-1), APCy-Cy7–conjugated streptavidin, PE-Cy7–conjugated anti-CD3ε (145-2C11), and PE-Cy7–conjugated streptavidin were purchased from Biolegend (San Diego, CA). Anti-CD16/32 (2.4G2) and anti-NK1.1 (PK136) were prepared from hybridoma culture supernatants in our laboratories. Anti-CD200R3 (Ba91) was established as reported previously (21). Rabbit anti-ASGM1 was purchased from Wako Pure Chemicals (Osaka, Japan), and control rabbit serum, mouse IgG2a, monosialoganglioside GM1, and ASGM1 were from Sigma-Aldrich (St. Louis, MO).

Cells prepared from the spleen, bone marrow, and peripheral blood were preincubated with anti-CD16/32 mAb and normal rat serum on ice for 15 min prior to incubation with the indicated combination of Abs to prevent the nonspecific binding of irrelevant Abs. For ASGM1 staining, cells were incubated with rabbit anti-ASGM1 (1:500 diluted) on ice for 20 min, followed by FITC-conjugated goat anti-rabbit IgG (1:500 diluted). In the experiments shown in Fig. 3B, anti-ASGM1 was preincubated in 100 μl with 10 μg ASGM1 or control GM1 before cell staining.

For NK cell depletion, mice were given an i.p. injection of anti-ASGM1 (10 μl) or anti-NK1.1 (300 μg). For basophil depletion, mice were treated with a total of 30 μg anti-FcεRIα (MAR-1) that was administered i.p. twice a day (5 μg each time) for 3 consecutive d (23). Stained cells were analyzed on an FACSCanto II (BD Biosciences, Palo Alto, CA), and data were analyzed with FACSDiva software (BD Biosciences) and FlowJo software (Tree Star, Ashland, OR).

IgE-mediated chronic allergic skin inflammation was elicited as described (17). In brief, mice were passively sensitized with IgE by an i.v. injection of 300 μg 2,4,6-trinitrophenol (TNP)-specific IgE (IGELb4). A day later, 10 μg TNP-conjugated OVA (Sigma-Aldrich) in 10 μl PBS was injected s.c. into the left ear of the mice under light anesthesia with diethyl ether, and an equal amount of OVA was injected into the right ear using a microsyringe. Ear thickness was measured with a dial thickness gauge (G-1A; Ozaki, Tokyo, Japan) at the indicated time points. The difference in ear thickness (left − right) was calculated at each time point.

The statistical significance of differences between groups was calculated with a two-tailed Student t test or ANOVA followed by Ryan’s test. A p value <0.05 was considered statistically significant.

Preparation of a highly purified basophil population for functional analysis is extremely difficult, particularly in mice, due to the rarity of basophils and the lack of murine basophil-specific Ab that leaves basophils unstimulated. Although enrichment of basophils (CD49b⁺FcεRIα⁺) without their activation can be achieved in mice by magnetic sorting using anti-CD49b–bound beads, basophils represent only 5–20% of the CD49b⁺ cells in the spleen, bone marrow, and peripheral blood, whereas the rest of cells are mainly NK and NKT cells. We thought that Ab-mediated depletion of NK/NKT cells in vivo before the cell preparation could increase the efficiency of basophil enrichment. When administered i.p., both anti-ASGM1 and anti-NK1.1 were indeed effective in reducing number of CD49b^highCD3ε⁻ cells in the spleen with higher efficacy by anti-ASGM1 than by anti-NK1.1 (Fig. 1A, upper panels). The CD49b^highCD3ε⁻ fraction of splenocytes includes FcεRIα⁻ NK cells (90–95%) and FcεRIα⁺ basophils (5–10%). Unexpectedly, anti-ASGM1 but not anti-NK1.1 drastically reduced the number of basophils in the spleen, whereas both anti-ASGM1 and anti-NK1.1 efficiently depleted NK cells (Fig. 1A, lower panels, and summarized in Fig. 1B). As many as 90% of basophils are depleted by the anti-ASGM1 treatment as compared with those in mice treated with control rabbit serum (Fig. 2A). The efficacy of basophil depletion by anti-ASGM1 was comparable to that mediated by a basophil-depleting anti-FcεRIα mAb MAR-1 that did not deplete NK cells, unlike anti-ASGM1 (Fig. 2A). One shot of anti-ASGM1 injection reduced the number of basophils in the spleen, for 4 d after the injection, to less than a quarter of that in the control mice treated with normal rabbit serum (Fig. 2B, upper panel). The basophil number gradually increased thereafter and returned to the normal level by 14 d after the injection. These kinetics of recovery were faster than those of NK cells (Fig. 2B), most likely due to the quicker turnover of basophils than NK cells (37, 38).

We next examined whether the basophil depletion by the anti-ASGM1 polyclonal Ab stems from the expression of ASGM1 on the surface of basophils or due to an ASGM1-independent, off-target effect of the polyclonal Ab. Flow cytometric analysis revealed that virtually all basophils in the spleen, peripheral blood, and bone marrow were homogeneously stained with anti-ASGM1 but not anti-NK1.1 (Fig. 3A). The ASGM1 staining of basophils was blocked by preincubation of anti-ASGM1 with ASGM1 but not GM1, as observed in the staining of NK cells (Fig. 3B), demonstrating the specificity of the Ab and expression of ASGM1 on basophils. The expression of ASGM1 was also detected on NKT cells, CD8⁺ T cells, and γδ T cells, but the expression was confined to a subpopulation of them and heterogeneous in its level even among the same cell lineage (Fig. 3A), in accordance with previous reports (9, 10). No significant expression of ASGM1 was detected on other hematopoietic cells, including neutrophils and eosinophils in the spleen, peripheral blood, and bone marrow as well as mast cells in the peritoneum (data not shown).

The anti-NK1.1–mediated NK cell depletion, unlike the anti-ASGM1–mediated one, can be applied to only certain mouse strains with NK1.1 allotype, such as C57BL/6 (H2^b) (8). For this reason, anti-ASGM1 is commonly used for the investigation of in vivo NK cell function in NK1.1⁻ mouse strains. Accordingly, we examined whether basophils express ASGM1 and are depleted by anti-ASGM1 treatment in NK1.1⁻ mouse strains BALB/c (H2^d), C3H (H2^k), and A/J (H2^a). Basophils as well as NK cells from all the mouse strains expressed ASGM1 (Fig. 4A), and the anti-ASGM1 treatment drastically reduced the number of both cell types in these mice, irrespective of NK1 allotype and MHC H2 haplotype (Fig. 4B). This finding raised concern about the possible contribution of basophils, rather than or in addition to NK cells, to some of phenotypes that have been reported in anti-ASGM1–treated mice with various genetic backgrounds.

To address the issue of whether a certain phenotype observed in anti-ASGM1–treated mice could indeed be attributed to basophil depletion, we examined the effect of anti-ASGM1 treatment on IgE-mediated chronic allergic inflammation (IgE-CAI) in the skin. IgE-CAI is elicited by passive sensitization of mice with hapten TNP-specific IgE and subsequent challenge with s.c. injection of TNP-conjugated OVA, and basophils play an essential role in the development of IgE-CAI (17, 21, 25). Treatment of C57BL/6 mice with anti-ASGM1 but not control rabbit serum 1 d after the Ag challenge completely abolished the development of IgE-CAI as judged by the inhibition of ear swelling (Fig. 5A, left panel), as observed in mice treated with basophil-depleting Ab (21). In contrast, treatment with anti-NK1.1 showed no significant effect on IgE-CAI (Fig. 5A, right panel). Furthermore, treatment with anti-ASGM1 but not control rabbit serum abolished the development of IgE-CAI in rag2/γc doubly deficient mice that lack NK and NKT cells as well as T and B cells (Fig. 5B). These results demonstrated that the inhibitory effect of anti-ASGM1 on IgE-CAI was not due to the secondary effect of NK/NKT cell depletion. Thus, the anti-ASGM1 treatment abolishes basophil function in vivo.

Anti-ASGM1 is commonly used as an NK cell-depleting Ab in mice for study on NK cell functions in diverse immune responses. In the current study, we revealed a previously unrecognized and lethal off-target effect of anti-ASGM1 on basophils. Among hematopoietic cells examined, only NK cells and basophils, at least under homeostatic conditions, show constitutive and homogeneous expression of ASGM1 at high levels. Although ASGM1 expression is also detected on NKT, CD8⁺ T, and γδ T cells, the expression is confined to a subpopulation of them and is heterogeneous even among the same cell lineage. In accordance with this, in vivo administration of anti-ASGM1 ablated the vast majority of both NK cells and basophils with no significant reduction in the number of other lineage cells, including NKT, CD8⁺ T, and γδ T cells, as far as examined (Supplemental Fig. 1).

Basophils are as sensitive as NK cells to anti-ASGM1–mediated ablation, and treatment of C57BL/6 mice with as little as 2.5 μl anti-ASGM1 still eliminates three quarters of basophils (Supplemental Fig. 2). Although anti-ASGM1–mediated basophil depletion is transient, repeated administration of anti-ASGM1 can induce a long-term depletion of basophils (Supplemental Fig. 3). Although both NK cells and basophils are depleted in vivo by anti-ASGM1, the mechanism underlying the Ab-mediated depletion appears to differ between them. Treatment of IgFcR-deficient Fcrg^−/− mice with anti-ASGM1 resulted in depletion of NK cells but not basophils (Supplemental Fig. 4). Anti-ASGM1–mediated basophil depletion was observed even in the absence of NK cells (Fig. 5B), ruling out the depletion through the Ab-dependent cellular cytotoxicity by NK cells. Basophil depletion by anti-CD200R3 is also defective in Fcrg^−/− mice (21), suggesting that Ab-mediated basophil depletion is dependent on FcRs, probably through FcR-mediated phagocytosis.

NK cells and basophils share CD49b (DX5) expression in addition to ASGM1 expression. NK cell depletion by anti-ASGM1 treatment has often been verified by the disappearance of the CD49b⁺CD3⁻ population, particularly in NK1.1⁻ mouse strains. As long as CD49b is considered as a selective maker of NK cells, the presence of basophils among this population would never be noticed. Moreover, in flow cytometric analysis, basophils, like NK cells, display the side and forward scatter profile closer to that of lymphoid cells rather than myeloid cells. These might be reasons among others why the important off-target effect of anti-ASGM1 treatment on basophils has long been overlooked by researchers. Our findings underscore the need to carefully interpret data obtained from experiments with anti-ASGM1–mediated NK cell depletion. The phenotype observed in anti-ASGM1–treated mice may be attributed to NK cell depletion, the off-target effect on basophils, or both. Indeed, the impaired IgE-CAI response in anti-ASGM1–treated mice was attributed to the depletion of basophils but not NK cells (Fig. 5B).

Recent studies have illuminated previously unappreciated roles for basophils in various immune responses (26–36). Basophils play an important role in the initiation of Th2-type immune responses by providing IL-4 (22) and even by functioning as APCs under certain conditions (39–41). They also enhance humoral memory responses by promoting B cell activation and differentiation (23, 42). Basophils play crucial roles in protective immunity to parasitic infections (24, 25) and also contribute to the development of immunological disorders, including allergy and autoimmunity (17, 43, 44). We also appreciate that NK cells play a key role in the immune response to certain infections and malignancies by direct cytolysis of infected or transformed cells and by secretion of potent immune mediators (1–3). NK cell-derived cytokines, such as IFN-γ and TNF, enhance the innate immune response and shape the subsequent adaptive immune response. Thus, anti-ASGM1–mediated depletion of both NK cells and basophils could have greater impact on various immune responses compared with the depletion of either NK cells or basophils alone. Accordingly, it is important to dissect the contribution of basophils and NK cells to the immunological phenotype observed in anti-ASGM1–treated mice.

NK cells have been suggested to suppress the development of protective immunity against mycoplasma infection in a mouse model in that IFN-γ promotes the protection, whereas IL-4 inhibits it (45, 46). Intriguingly, treatment of BALB/c mice with anti-ASGM1 before immunization with mycoplasma Ags resulted in enhancement rather than suppression of the protection from subsequent mycoplasma infection. Lymphocytes isolated from draining lymph nodes of anti-ASGM1–treated immunized mice produced higher IFN-γ and lower IL-4 compared with those from untreated immunized mice, suggesting a shift toward a more Th1-dominant response in the anti-ASGM1–treated mice. These observations are quite puzzling, given that anti-ASGM1 treatment depleted NK cells alone, as the authors judged on the basis of the finding that all ASGM1⁺ cells expressed a typical NK cell marker CD49b. However, this puzzle might be solved when we take basophils into account in this type of immune response. Basophils might contribute to Th2 differentiation, and therefore anti-ASGM1–mediated basophil depletion results in a shift toward a more Th1-dominant response that is favorable for protection from mycoplasma infection.

A peculiar Thy1^dullCD49b⁺ cell population in the bone marrow, distinct from NK, NKT, and T cells, was identified as a cell type that displays the ability to protect immature B cells from apoptosis induced by BCR engagement and to redirect them toward receptor editing (47, 48). This population, designated as bone marrow protective cells (BMPCs), constitutes only 0.5–1% of the bone marrow, and homogenously expresses ASGM1. Treatment of mice with anti-ASGM1 eliminates most of this population, leading to the loss of B cell-protective activity in the bone marrow. These features of BMPCs are all shared by basophils. Basophils produce B cell-stimulating molecules such as IL-4, IL-6, and BAFF (23, 42, 44). Therefore, BMPCs probably represent basophils.

In conclusion, the current study identified basophils as a previously unrecognized target of anti-ASGM1–mediated cell depletion. This finding raises concern about the possibility that certain functions of basophils might have been erroneously interpreted as those of NK cells in studies using anti-ASGM1 for NK cell depletion. Therefore, in the functional study of NK cells with use of anti-ASGM1, it is essential to dissect the role of NK cells and basophils, in conjunction with the use of more specialized tools, including anti-NK1.1 and basophil-depleting Abs (21–23), engineered mice deficient only in NK cells (49, 50), and those deficient only in basophils (24, 25).

We thank the members of the Karasuyama laboratory for helpful discussions and Michiko Miki for administrative and secretarial assistance.

The authors have no financial conflicts of interest.