ACK2 hybridoma cell line vials were thawed, washed, centrifuged (150 g, 5 min, 4 °C), and resuspended in ACK2 culture medium at 37 °C, 5% CO₂, and 95% relative humidity. Cells were plated in a range of 0.3-0.5 × 10⁶ cells/mL concentrations to ensure its growth and survival in 50 mm (4mL) and 90 mm (15 mL) culture dishes, and T175 (30 mL) flasks sequentially. During expansion, the FBS concentration was gradually reduced (10%, 5%, and 2.5%) to minimize protein contamination prior to antibody purification. Cell density and viability were assessed using a Neubauer chamber and 0.4% trypan blue exclusion. Cells were passaged upon reaching 80% confluence. Once sufficiently expanded, cells were allowed to undergo slow cell death for 10–15 days to maximize antibody production. When culture viability reached ~10-20%, the medium was collected, centrifuged (150 g, 5 min, 4 °C), filtered using vacuum-driven sterile filters, and stored at -20 °C until protein concentration (Figure 1). The supernatants were tested for the presence of ACK2 antibody (see section 3.6, “ACK2 antibody in vitro competing assay”; Figure 2A).

ACK2 antibody-containing supernatants were concentrated in two rounds using centrifugal filter devices (Figure 1). In the first round, 12 mL of filtered supernatant were centrifuged (3000 g, 7 min, 4 °C). The retained concentrate was collected and subjected to a second round of centrifugation (3500 g, 8 min, 4 °C). ACK2 antibody-containing supernatants must be kept on ice throughout the entire concentration process. The resulting ultra-concentrate was stored at -20 °C until purification.

ACK2 antibody was purified using an IgG affinity purification column, following the manufacturer’s protocol (Figure 1). Briefly, the ACK2 ultra-concentrate was diluted 1:2 in Binding Buffer (pH 5), and 10 mL were incubated (10 min, 25 °C) with end-over-end mixing and centrifuged (1000 g, 1 min, 25 °C) through the column. The non-bound sample components were subjected to another round of incubation and centrifugation to prevent antibody loss. Elution was conducted through addition of 5 mL of Elution Buffer (pH 2.8) and centrifugation (1000 g, 1 min, 25 °C). The different eluted fractions were collected and neutralized in tubes with 0.5 mL of 1 M phosphate buffer (pH 8.4). The pH of all buffers and its dilutions must be checked beforehand using a pH meter. Protein concentration was measured in each fraction with a NanoDrop 2000 spectrophotometer, and purity was assessed by SDS-PAGE (see section 3.4, “Protein electrophoresis”).

The average yield was approximately 6 mg of purified ACK2 per 200 mL of antibody-containing supernatant before concentration. This yield, along with the binding capacity of the purification column, should be considered when determining the number of purification rounds required for a given supernatant volume.

Finally, antibody-containing fractions were desalted using spin desalting columns, centrifuged (1000 g, 2 min, 25 °C) and exchanged into sterile PBS, suitable for in vivo administration (Figure 1). Protein concentration was re-measured, and aliquots were stored at -20 °C. Functional activity was confirmed by in vitro competition assay (section 3.6; Figure 2B).

Purified ACK2 samples were mixed with 4X Laemmli sample buffer and 5% DTT. Samples were boiled at 95 °C for 10 min, vortexed briefly every 2 min, and centrifuged (16,000 g, 1 min). Protein electrophoresis was performed on 12% Tris-glycine gels using 2 µg of protein per well and 5 µL of molecular weight standard. Gels were run for 40 min at 200 V, then stained with Quick Coomassie Stain. Protein bands confirmed purity of ACK2 antibody (Supplementary Figure S).

BM cells were obtained by flushing femurs and tibias with flow cytometry buffer (PBS, 5% FBS, 2 mM EDTA) using a 25 G needle and 10 mL syringe until bones appeared white. Suspensions were filtered through a 70 µm strainer. Splenocytes were obtained by cutting spleens into two pieces and injecting 10 mL of flow cytometry buffer with a 25 G needle and 10 mL syringe until they appeared white. The obtained cell suspension was filtered through a 70 µm strainer and remaining spleen pieces were mashed in the same filter using the piston of a 1 mL sterile syringe. BM and spleen filtered suspensions were centrifuged (450 g, 5 min), incubated with 5 mL or 10 mL of lysis buffer respectively (5 min, 37 °C, 5% CO₂, 95% relative humidity), washed and centrifuged (450 g, 5 min).

ACK2 presence and functionality were evaluated using a flow cytometry-based competition assay (Figures 1, 2).

For detection in supernatants, 2 x 10⁶ RBC-lysed BM cells from C57BL/6 mice were pre-incubated for 15 min at 4 °C with either ACK2 supernatant or RPMI medium (positive control) (Figure 2A).

For functional testing, BM cells were incubated under the same conditions with three dilutions (1/10, 1/100, 1/1000) of purified ACK2 antibody (2 mg/mL) or PBS (positive control) (Figure 2B).

Cells were then washed and stained with biotin-labeled lineage (Lin) markers followed by streptavidin-APC to distinguish Lin⁺ (majority) from Lin^– (HSPC-containing) fractions. Cells were co-stained with PE–anti-c-Kit (clone ACK2) or BUV395–anti-c-Kit (clone 2B8). Flow cytometric analysis showed that pre-incubation with ACK2 supernatant or purified antibody blocked detection of Lin^– c-Kit⁺ HSPCs by the PE-labelled clone ACK2, confirming antibody presence and activity. Clone 2B8, recognizing a non-competing c-Kit epitope, remained capable of detecting HSPCs.

Cell suspensions were stained with specific antibody combinations in flow cytometry buffer for 15 min at 4 °C. Fc receptors were blocked prior to incubation using FcBlock (anti-CD16/CD32). Samples were analyzed on an LSRFortessa cytometer, and data were processed with FlowJo v10 software. See Table 1 for used antibody specifications.

Purified ACK2 antibody (500 µg in 250 µL PBS/mouse) was administered intraperitoneally (Figure 3).

For antibody clearance kinetics, blood from ACK2- or PBS-injected mice was collected from the maxillary vein, centrifuged twice (3000 g, 5 min) to obtain cell-freeserum, and incubated with 2 x 10⁶ RBC-lysed BM cells from untreated mice for 15 min at 4 °C. Cells were fluorescently stained with lineage markers and anti-c-Kit (clone ACK2) and analyzed by flow cytometry (Figure 3A).

For HSPC depletion kinetics, RBC-lysed BM cells from ACK2-injected mice were fluorescently stained with lineage markers and anti-c-Kit antibodies (clones 2B8 or 3C11) and analyzed as above (Figure 3B). BM from PBS-injected mice served as control.

HSPCs were obtained from DsRed.T3 donors by flushing femurs and tibias with PBS using a 25 G needle and 10 mL syringe until bones appeared white. Suspensions were filtered through a 70 µm strainer, extensively washed and centrifuged (450 g, 5 min). Lin^– HSPC-enriched cells were isolated by immunomagnetic depletion using the Lineage Cell Depletion Kit and AutoMACS Pro Separator, according to the manufacturer’s instructions. The Lin^– HSPC-enriched fraction was washed, centrifuged, resuspended in PBS, and transplanted intravenously into recipient C57BL/6 mice (1 x 10⁶ Lin^– cells in 0.1 mL PBS per mouse).

C. albicans PCA2 (low-virulence, non-germinative strain) or the virulent strain ATCC 26555 were cultured in endotoxin-free YPD medium (1% yeast extract, 2% peptone, 2% dextrose) at 28 °C with gentle agitation until exponential growth (A₆₀₀ = 0.6-0.8). Yeasts were pelleted (7200 g, 5 min), resuspended in sterile water, and incubated for 3 h at 28 °C with shaking. Cultures were then starved at 4 °C for 24 h before use. Yeast cells were quantified by microscopic counting using a Neubauer chamber, washed, and diluted in PBS to the desired concentration.

Peritoneal cells were isolated as previously described (18). Mice were euthanized by cervical dislocation and placed supine and disinfected with 70% ethanol. A midline skin incision was made from the abdomen to the neck without penetrating the peritoneum, then extended to hind- and forelimbs to expose the cavity. 10 mL of cold flow cytometry buffer were injected into the peritoneal cavity using a 25 G needle. The abdomen was gently massaged to release resident and recruited cells. An incision was made in the peritoneal wall, and the lavage fluid was collected into a 15 mL tube via funnel.

All statistical analyses were performed with the use of GraphPad Prism v8.4.2 software. Statistical differences for dual comparisons (ACK2-injected vs. PBS-injected or PCA2-infected vs. PBS-treated) were determined using two-tailed Mann-Whitney U test. Statistical differences for independent multiple comparisons (PBS-injected vs. day 3, 4, 5 post-ACK2-injected) were determined using Kruskal-Wallis test followed by Dunn’s post-hoc pairwise comparisons. Data are expressed as median with interquartile range (IQR). Significance was accepted at *P < 0.05, **P < 0.01, and ***P < 0.001 levels.

Previous studies demonstrated that administration of the monoclonal anti-c-Kit antibody (clone ACK2) in Rag2^-/- mice induces HSPC ablation, thereby enhancing hematopoietic reconstitution (2). In this study, we aimed to clear BM niches to improve the adoptive transfer of donor HSPCs. To accomplish this, it was necessary to account for the residual ACK2 antibody remaining in circulation, which could otherwise deplete transplanted donor HSPCs.

To determine the in vivo kinetics of antibody clearance, we administered 500 µg of ACK2 intraperitoneally and tested serum samples collected at days 3, 4, and 5 post-injection. The presence of circulating ACK2 was assessed by an in vitro competing assay: BM cells were incubated with serum samples, then stained with a fluorescently labelled lineage markers cocktail and anti-c-Kit clone ACK2 antibody, and analyzed by flow cytometry (Figure 3A). The results showed that ACK2 was detectable in serum on day 3, as HSPCs could not be labelled with the fluorescent ACK2 anti-c-Kit antibody, but was absent by days 4 and 5 (Figure 3A).

ACK2 treatment could be inducing a transient depletion of host HSPCs, potentially creating a short time window during which recipient mice might be receptive to donor HSPC transplantation. We therefore sought to define this “transplantation window”, characterized by both the absence of circulating ACK2 and depletion of endogenous HSPCs in C57BL/6 mice. To this end, BM cells were collected at days 3, 4, and 5 after ACK2 administration (Figure 3B). HSPCs were identified as Lin^– c-Kit⁺ cells using two distinct anti-c-Kit clones, 2B8 and 3C11. The use of clones 2B8 and 3C11 allowed us to distinguish between ACK2 antibody bound to c-Kit and true HSPC depletion, as these antibodies recognize non-competing epitopes. Flow cytometric analysis revealed that HSPCs were undetectable in BM at days 3 and 4 post-injection but had reappeared by day 5 (Figure 3B).

Together, these data define day 4 post-ACK2 treatment as the optimal transplantation window, coinciding with maximal endogenous HSPC depletion and complete clearance of circulating antibody.

We next evaluated whether ACK2-mediated depletion of host HSPCs could improve donor engraftment in C57BL/6 mice. For this purpose, recipient C57BL/6 mice were i.p. injected with either 500 µg of purified ACK2 or PBS. Four days later, Lin^– HSPC-enriched cells were isolated from the BM of DsRed donor mice and a total of 1 x 10⁶ donor cells were transplanted i.v. into either PBS- or ACK2-conditioned recipients. We have previously determined that functional imprinting and enhanced responses are reliably detected at day 7 post-transplantation (15, 16). Therefore, seven days after transplantation, host BM and spleen were analyzed for the presence of donor-derived DsRed⁺ cells by flow cytometry (Figure 4A).

The results demonstrated a significant fold increase in donor cell numbers in ACK2-conditioned mice compared with PBS controls, with a much greater difference observed in BM than in spleen (Figures 4B, C). Engraftment was assessed in both tissues because transplanted HSPCs have been reported to localize in BM and spleen (19, 20).

Together, these findings demonstrate that host conditioning with the ACK2 antibody enhances the engraftment efficiency of adoptively transferred donor HSPCs in C57BL/6 mice. Moreover, we verified the optimal transplantation window, a time point when circulating ACK2 antibody is cleared, and BM niches are transiently available for donor cells.

To exemplify the applicability of the ACK2 antibody-based HSPC depletion for transplantation studies, we designed a specific in vivo scenario.

Our group previously demonstrated that HSPCs from mice infected with the low-virulence, non-germinative C. albicans strain PCA2 are reprogrammed to generate trained macrophages with enhanced proinflammatory cytokine production, conferring protection against secondary infection (15). More recently, we showed that HSPCs exposed in vitro to C. albicans give rise to trained neutrophils, characterized not only by elevated cytokine production but also by superior microbicidal activity due to increased mitochondrial ROS (mtROS) generation (16). Beyond cytokine secretion and microbicidal activity, additional neutrophil functions such as recruitment to infection sites are critical for pathogen clearance. Based on this, we hypothesized that C. albicans-reprogrammed HSPCs could generate neutrophils with improved recruitment capacity in vivo.

To test this hypothesis, HSPCs isolated from DsRed donor mice either infected for 24 h with PCA2 or treated with PBS were adoptively transferred into C57BL/6 recipients preconditioned with ACK2 antibody (500 µg, i.p.) four days earlier. Seven days post-transplantation, recipient mice were i.p. challenged with the virulent C. albicans strain ATCC 26555, and four hours later, peritoneal lavage was performed to recover recruited immune cells (Figure 5A). Under steady-state conditions, neutrophils are absent from the peritoneal cavity, and four hours following C. albicans challenge is characterized by a selective and robust recruitment of neutrophils.

Flow cytometric analysis of peritoneal cells revealed comparable numbers of host-derived (DsRed^–) neutrophils (CD11b⁺ Ly6G^hi) (Figure 5C), indicating that the treatment of adoptively transferred HSPCs did not influence host-derived myeloid responses. In contrast, significantly higher numbers of donor-derived (DsRed⁺) neutrophils originating from PCA2-infected HSPCs were recruited to the peritoneal cavity compared with neutrophils from PBS-treated HSPCs (Figures 5B, C).

These findings demonstrate that neutrophils derived from C. albicans-reprogrammed HSPCs exhibit an enhanced capacity for recruitment to the site of infection during a secondary C. albicans challenge. This experimental model exemplifies how antibody-mediated HSPC depletion with ACK2 facilitates efficient adoptive transfer, enabling the detection and functional assessment of donor-derived cells in remote regions of the BM that would otherwise remain undetectable in non-conditioned hosts.