FLT3 Ligand Is Dispensable for the Final Stage of Type 1 Conventional Dendritic Cell Differentiation

Cindy Audiger and Sylvie Lesage
J Immunol October 15, 2020, 205 (8) 2117-2127; DOI: https://doi.org/10.4049/jimmunol.2000742

Key Points

  • cDC subset differentiation from precommitted precursors is not proportional.

  • CD135 is differentially expressed in the cDC1 and cDC2 lineages.

  • Late stages of cDC1 differentiation are independent of FLT3L.

Abstract

Conventional dendritic cells (cDCs) are comprised of two major subsets, type 1 cDC (cDC1) and type 2 cDC (cDC2). As each cDC subset differentially influences the nature of immune responses, we sought factors that would allow the manipulation of their relative abundance. Notably, cDC1 are less abundant than cDC2 in both lymphoid and nonlymphoid organs. We demonstrate that this bias is already apparent in bone marrow precommitted precursors. However, comparison of five common inbred strains revealed a disparity in precursor–product relationship, in which mice with fewer precursors to cDC1 had more cDC1. This disparity associated with contrasting variations in CD135 (FLT3) expression on cDC subsets. Hence, we characterized the response to FLT3 ligand during cDC1 and cDC2 lineage differentiation and find that although FLT3 ligand is required throughout cDC2 differentiation, it is surprisingly dispensable during late-stage cDC1 differentiation. Overall, we find that tight regulation of FLT3 ligand levels throughout cDC differentiation dictates the cDC1 to cDC2 ratio in lymphoid organs.

This article is featured in Top Reads, p.1979

Introduction

Conventional dendritic cells (cDCs) are key players in the initiation of an immune response and are divided into two main subsets, namely type 1 cDC (cDC1) and type 2 cDC (cDC2), which are distinct in terms of phenotype and function (1). cDC1 are characterized by the expression of CD8α, CD24, and XCR1 cell surface markers in lymphoid organs and are specialized in Ag cross-presentation, whereas cDC2 express CD11b and CD172a (also named SIRPα) and primarily stimulate CD4+ T cells (2, 3). Interestingly, although they arise from a common precursor, cDC subsets are not equally distributed, and cDC2 predominate in both lymphoid and nonlymphoid tissues (4, 5). In vivo quantification of cellular proliferation and cDC subset homeostasis in parabiotic pairs suggest that the preferential cDC2 bias is unlikely because of proliferation or turnover (6, 7) but may, instead, result from a bias in cDC differentiation.

cDC differentiation is a stepwise and highly regulated process that initiates in the bone marrow. cDC commitment is imprinted during hematopoietic differentiation as early as hematopoietic stem cell progenitors (8, 9). Lymphoid-primed multipotent progenitors derive from hematopoietic stem cell progenitors and yield macrophage–DC progenitors, which differentiate into common myeloid progenitors to subsequently produce common DC progenitors that give rise to precursors to cDCs (pre-cDCs) (10–13). The pre-cDC population is composed of specific precursors to cDC1 (pre-cDC1) and precursors to cDC2 (pre-cDC2), as well as uncommitted precursors that can yield to both cDC1 and cDC2 (14). These pre-cDC1 and pre-cDC2 populations can migrate to the spleen via the blood for the final differentiation stages (5, 14). In addition, a cDC1-committed precursor, expressing CD24 but not CD8αhigh (named CD24+ cDC1 splenic precursors hereafter), was identified in the spleen and has the capacity to differentiate into functional cDC1 (15).

FLT3 ligand (FLT3L) is clearly one of the primary factors that drives cDC differentiation. Hematopoietic precursor cells expressing CD135, the receptor for FLT3L, effectively generate cDCs in response to FLT3L (16, 17). In addition, mouse models lacking FLT3L or CD135 show a drastic reduction in cDCs (18–20). Of interest, cDC differentiation in the spleen is particularly dependent on hematopoietic production of FLT3L (21). Whether FLT3L contributes to determining the cDC1 to cDC2 ratio in both lymphoid and nonlymphoid tissues is unclear.

We have recently shown that the low cDC1 to cDC2 ratio in the spleen is generalizable to eight genetically distinct inbred mouse strains (common, C57BL/6 carrying the CD45.2 allele [B6], A/J, NZO/HILtJ [NZO], 129S, and NOD and wild-derived, CAST, PWK, and WSB) (22). To explore the factors that contribute to cDC subset distribution in the spleen, we characterize cDC differentiation in five common inbred strains. Investigating the most variable cDC differentiation phenotypes among these strains has led us to determine that FLT3L is not necessary for all stages of cDC differentiation. Indeed, in the absence of FLT3L, splenic pre-cDCs can differentiate into cDC1 but not into cDC2. Altogether, this study reveals that the late stages of cDC1 and cDC2 differentiation are differentially dependent on FLT3L, which likely contributes to defining the size of their respective niches.

Materials and Methods

Animals

The following mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and subsequently bred in-house at the animal facility of the Maisonneuve-Rosemont Hospital (Montreal, Canada): A/J (no. 000646), C3H/HeJ (C3H, no. 000659), B6 (no. 000664), B6.NOD.Idd1 (B6g7; no. 003300), B6.SJL carrying the CD45.1 allele (B6.1, no. 002014), NOD (no. 001976), and NZO (no. 002105). B6 and B6.1 mice were mated to obtain B6 mice carrying both CD45.1 and CD45.2 alleles (referred to as B6.1.2). F1g7 mice are the offspring of a first-generation cross between B6g7 and NOD mice. Six- to fourteen-week-old mice were used for all phenotypic analyses. No differences were observed between male and female mice, and data from both sexes were pooled. Within individual experiments, mice were age and sex matched to littermates whenever possible. The Maisonneuve-Rosemont Hospital Ethics Committee, overseen by the Canadian Council for Animal Care, approved all experimental procedures.

Flow cytometry

Spleens were minced and treated with collagenase (1 mg/ml in PBS, type V from Clostridium histolyticum; Sigma Aldrich, St-Louis, MO) for 15 min at 37°C. Bone marrow was flushed from the femurs, tibias, and iliac crests. Digested spleen and bone marrow were passed through a 70-μm cell strainer (BD Biosciences, Mississauga, ON, Canada) to yield single-cell suspensions prior to RBCs lysis with NH4Cl solution. Cell suspensions were labeled with Abs from BioLegend (San Diego, CA) listed as Ag (clone): CD3 (17A2), CD8α (53-6.7), CD11b (M1/70), CD11c (N418), CD19 (6D5), CD24 (M1/69), CD26 (H194-112), CD45.1 (A20), CD45.2 (104), CD115 (AFS98), CD117 (2B8), CD135 (A2F10), CD172a (P84), B220 (RA3-6B2), I-Ab (AF6-120.1), IA/IE (M5/114.15.2), I-Ad (39-10-08), Ly6C (HK1.4), Siglec-H (551), XCR1 (ZET), and Zombie Aqua Viability Dye. CD45RA (14.8) is from BD Biosciences. All samples were acquired on a Fortessa (BD Biosciences) and were analyzed with the FlowJo software (FlowJo, BD Biosciences). Cells were sorted using a FACSAria III (BD Biosciences). For all flow cytometry analyses, doublets were carefully excluded using forward side scatter width versus height, and dead cells were excluded based on Zombie Aqua Viability Dye. Intracellular expression of CD135 on splenic cDC subsets was determined by saturating cell surface epitopes with CD135 PE followed by intracellular staining with CD135 Pacific Blue using BD Cytofix/Cytoperm (no. 554714; BD Biosciences), as per the manufacturer’s instructions.

In vivo FLT3L in competitive bone marrow chimeras

F1g7 recipients were lethally irradiated (11 Gy) and injected i.v. with a 1:1 mixture of 2 × 106 bone marrow cells from B6g7 (CD45.2) and NOD (CD45.1) mice. At least 6 wk postreconstitution, the bone marrow and spleens were harvested and stained for flow cytometry analysis. Alternatively, F1g7 bone marrow chimeric mice were s.c. injected with the B16F10-FLT3L cell line (23) to increase the systemic level of FLT3L. Mice were sacrificed when tumors were palpable, at approximately day 10 post–tumor graft. Bone marrow and spleens were harvested, prepared, and stained for flow cytometry analysis.

In vitro differentiation of pre-cDC subsets

Bone marrow pre-cDCs were identified as CD3−CD19−B220−CD11c+MHC class II (MHC-II)−/intCD172aintCD135+ cells and further separated as pre-cDC1 (CD117+), pre-cDC2 (CD115+), and CD117− pre-cDC2 (CD117−CD115−) as noted. Pre-cDC subsets were sorted from the pooled bone marrows of three B6.1 (CD45.1) mice and added to feeder cells that had been prepared three days prior. The feeder cells consisted of 4.5 × 106 B6 (CD45.2) bone marrow cells cultured with 100 ng/ml of FLT3L (Bio X Cell, Lebanon, NH) (14, 20). After 4 d of coculture with 100 ng/ml of FLT3L, the cells were harvested and labeled for flow cytometry analysis (14, 20). cDC commitment was assessed based on the expression of CD11c, as well as either CD24 or CD172a, for commitment to the cDC1 and cDC2 lineages, respectively. This protocol was also applied to assess the differentiation potential of CD24+ cDC1 splenic precursors (CD3−CD19−B220−CD11c+MHC-II+CD24+CD8α−/int) isolated either from a B6 (CD45.2) or NOD (CD45.1) mice except that the bone marrow feeder were isolated from B6.1.2 mice (CD45.1.2). The requirement for FLT3L during splenic pre-cDC (CD45.1+CD3−CD19−B220−CD11c+MHC-II−/intCD135+CD172aint) differentiation to cDC1 and cDC2 was evaluated following the same approach, except that the feeder cells (CD45.2+) were cultured in either the absence or the presence of FLT3L (100 ng/ml), and cDC commitment was assessed by flow cytometry after 3 d of coculture (14, 20).

Quantification and statistical analysis

Significance was tested using a two-way ANOVA for differences between more than two groups or a one-way ANOVA with a Bonferroni post hoc test. A Mann–Whitney U test was applied when testing for significant differences between two groups. All tests were performed with GraphPad. The p values <0.05 were considered to be significant. Statistical details of the experiments are noted in the figure legends.

Results

The precursor–product relationship between committed pre-cDC and their corresponding cDC subset is not linear

In B6 mice, the most commonly studied inbred strain, the proportion of cDC1 and cDC2 among cDCs in the spleen is ∼20 and 60%, respectively (Fig. 1A, Supplemental Fig. 1A). This bias in favor of cDC2 is observed in other common inbred strains, with NOD mice presenting the lowest cDC1 to cDC2 ratio (Fig. 1A) (22). To determine if the higher proportion of cDC2 relative to cDC1 in the spleen resulted from a preferential differentiation bias toward cDC2 in bone marrow cDC precursors, we quantified bone marrow pre-cDC1 (CD117+) and pre-cDC2 (CD115+) precursors committed to the cDC1 and cDC2 lineages, respectively (5). In contrast with the lower number of cDC1 relative to cDC2, we found a higher proportion and number of pre-cDC1 relative to pre-cDC2 in the bone marrow in all five common inbred strains (Fig. 1B, Supplemental Fig. 1B) (5). B6 mice, which showed the highest cDC1 to cDC2 ratio, also had the lowest pre-cDC1 to pre-cDC2 ratio (Fig. 1A, 1B), suggesting an inverse quantitative relationship for the cellular precursor-to-product differentiation. Moreover, most mouse strains seem to lack pre-cDC2, although they had a high proportion of cDC2. This intriguing observation prompted us to validate that pre-cDC1 and pre-cDC2 effectively differentiate toward cDC1 and cDC2, respectively. We decided to simultaneously test the differentiation potential of CD117−CD115− pre-cDCs, reported to differentiate into plasmacytoid DCs as well as cDCs, with a bias toward cDC2 (5). Expectedly, sorted pre-cDC1 primarily yielded cDC1 and sorted pre-cDC2 generated cDC2 following in vitro culture in the presence of FLT3L (Fig. 1C, Supplemental Fig. 1C, 1D). The CD117−CD115− pre-cDC subset also primarily differentiated toward a cDC2 phenotype (Fig. 1C, Supplemental Fig. 1C, 1D). Therefore, both the CD117−CD115+ pre-cDC2 and the CD117−CD115− pre-cDC precursors contribute to the cDC2 pool. As such, we decided to include all CD117− pre-cDCs as cDC2 precursors (Fig. 1D).

FIGURE 1.
FIGURE 1.

The precursor–product relationship between committed pre-cDC and their corresponding cDC subset is not linear. (A) Representative flow cytometry plot of CD8α and CD11b expression on B6 splenic CD11chiMHC-IIhi cDCs (top left). The ratio of the percentage of cDC1 to cDC2 among total cDCs (top right) as well as the compilation of cDC subset proportions and absolute numbers (bottom) for the indicated mouse strains. cDC1 are gated as CD8α+CD11b− (orange), and cDC2 are CD8α−CD11b+ (green) among total cDCs. (B) Representative flow cytometry plot of CD117 and CD115 expression on B6 bone marrow pre-cDCs (top left). The log of the ratio of the percentage of bone marrow pre-cDC1 to pre-cDC2 among bone marrow pre-cDCs (top right) as well as the compilation of bone marrow pre-cDC subset proportion and absolute numbers (bottom) for the indicated mouse strains. Pre-cDC1 are gated as CD117+CD115− (orange), and pre-cDC2 are CD117−CD115+ (green) among total bone marrow pre-cDCs. (C) Representative flow cytometry plot of CD24 and CD172a expression on cDCs (gated as CD11c+CD45RA−) derived from B6 pre-cDC1 (left), pre-cDC2 (middle), and CD115−CD117− pre-cDCs (right) cultured 4 d in vitro. Data are representative of three independent experiments. (D) Representative flow cytometry plot of CD117 and CD115 expression on B6 bone marrow pre-cDCs. The ratio of the percentage of bone marrow pre-cDC1 to CD117− redefined pre-cDC2 among bone marrow pre-cDCs (top left) as well as the compilation of bone marrow pre-cDC subsets and absolute numbers (bottom) for the indicated mouse strains. Pre-cDC1 are gated as CD117+CD115− (orange) and CD117− redefined pre-cDC2 (green) among total bone marrow pre-cDCs. (A–D) Numbers near the gates indicate the corresponding percentage of cells. (A, B, and D) Each symbol represents the data for one mouse. Dash represents the mean. Data were collected in at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, based on a one-way ANOVA multiple comparison test.

Using this strategy, we re-evaluated the proportion and numbers of pre-cDC subsets among the five inbred mouse strains. In line with the heavy bias toward cDC2 over cDC1 in the spleen, we found that a high proportion of bone marrow pre-cDC precursors were committed to the cDC2 lineage in all five common inbred strains (Fig. 1D). This result suggests that a pre-cDC commitment bias in the bone marrow likely contributes to the low cDC1 to cDC2 ratio in the spleen. Still, the ratio of pre-cDC1 to pre-cDC2 is generally lower than that of cDC1 to cDC2 for most mouse strains, suggesting that cDC1 lineage differentiation is slightly favored over cDC2. In addition, there are significant variations in cDC subset proportions among the different mouse strains. For instance, NOD mice have the lowest cDC1 to cDC2 ratio (Fig. 1A) and the highest pre-cDC1 to pre-cDC2 ratio relative to the other mouse strains (Fig. 1D). Altogether, we show that the proportion of pre-cDC1 and pre-cDC2 in the bone marrow do not parallel the relative proportion of cDC1 and cDC2 in the spleen. This observation prompted us to further study cDC differentiation to identify factors that could influence the relative abundance of cDC1 and cDC2 in the spleen.

CD24+ cDC1 splenic precursor do not explain the strain to strain variation in cDC subset distribution

In addition to precommitted bone marrow precursors, an intermediate cDC1 precursor has been defined in the spleen, expressing a CD11chighMHC-II+CD24+CD8α−/int phenotype (Fig. 2A, Supplemental Fig. 2A) (15). Variations in the proportion and number of these CD24+ cDC1 splenic precursors could contribute to the variations observed in the cDC1 to cDC2 ratios among the different inbred strains. Quantification of CD24+ cDC1 splenic precursors showed a modest increase in proportion, but not in absolute number, in C3H mice relative to B6, A/J, and NOD (Fig. 2B). B6 mice showed an increase in absolute number of CD24+ cDC1 splenic precursors relative to the four other mouse strains (Fig. 2B). These differences did not translate into a higher relative abundance of cDC1 over cDC2 as B6, A/J, NZO, and C3H have a comparable proportion of cDC1 as well as a comparable cDC1 to cDC2 ratio (Fig. 1A). As we had done for precommitted pre-cDC precursors, we also validated the cDC1 lineage commitment of CD24+ cDC1 splenic precursors. Sorted CD24+ cDC1 splenic precursors lack XCR1 expression, a cDC1-specific cell surface marker (24) (Fig. 2C, Supplemental Fig. 2B). When cultured in the presence of FLT3L, CD24+ cDC1 splenic precursors upregulate the expression of XCR1, in line with differentiation to cDC1 (Fig. 2D, Supplemental Fig. 2C). As both B6 and NOD showed the most contrasting cDC differentiation phenotypes regarding the relative proportions of pre-cDC1 to pre-cDC2 and of cDC1 to cDC2 (Fig. 1), we compared the differentiation potential of CD24+ cDC1 splenic precursors to cDC1 from these two strains. CD24+ cDC1 splenic precursors from both B6 and NOD mouse strains yielded a comparable proportion of XCR1-expressing cDC1 (Fig. 2E). Altogether, our data show that the variations in cDC1 to cDC2 ratio in the spleen of common inbred strains is not the result of an imbalance in CD24+ cDC1 splenic precursors. Considering that the low cDC1 to cDC2 ratio is also unlikely because of proliferation or turnover (6, 7), these results suggest a nonlinear cDC subset differentiation process that is regulated by unknown factors.

FIGURE 2.
FIGURE 2.

CD24+ cDC1 splenic precursors do not explain the strain to strain variation in cDC subset distribution. (A) Representative flow cytometry plots of CD8α and CD24 expression on splenic cDCs from B6 mice. The gate selects CD24+ cDC1 splenic precursor as CD8α−/intCD24+ cells among the splenic cDCs. The number in the plot indicates the corresponding percentage of gated cells. (B) Compilation of the percentage of CD24+ cDC1 splenic precursors (CD8α−/intCD24+) proportion among splenic cDCs and absolute number for each mouse strain. (C) Representative histograms of XCR1 expression on CD24+ cDC1 splenic precursors and splenic cDC1 (CD8α+CD11b−) from B6 mice. Numbers near the histograms indicate the corresponding mean fluorescence intensity. (D) Representative flow cytometry plots of CD24 and XCR1 expression on CD24+ cDC1 splenic precursors from B6 mice, after in vitro differentiation. The number near the gate indicates the corresponding percentage of cells. (E) Compilation of the percentage of cDC1 (CD24+XCR1+) differentiated from B6 or NOD CD24+ cDC1 splenic precursors in vitro. (B and E) Each symbol represents the data for one mouse. Dash represents the mean. *p < 0.05, **p < 0.01, ***p < 0.001 and p ≥ 0.05 is noted as ns, for nonsignificant, based on a one-way ANOVA multiple comparison test (B) or a Mann–Whitney U test (E). The data were obtained in at least three independent experiments.

The cDC traits are bone marrow intrinsic

The factors that contribute to defining variations in the low cDC1 to cDC2 ratio could be influenced by both hematopoietic-intrinsic or -extrinsic factors. To address this, we generated competitive bone marrow chimeras. As B6 and NOD mice represented the extreme phenotypes among the five strains studied, we used mice bearing these genetic backgrounds to generate the chimeric mice. Specifically, we replaced B6 with B6g7 mice as B6g7 mice carry the NOD MHC locus, which allows for competitive bone marrow engraftment. Moreover, both B6 and B6g7 mice had comparable proportions of cDC1 and cDC2 (Fig. 3), demonstrating that the MHC locus does not influence cDC subset proportion in these mice. B6g7 and NOD competitive bone marrow into F1(B6g7 × NOD) recipients confirmed that both strains exhibit a lower proportion of cDC1 relative to cDC2 in the spleen and that the cDC1 to cDC2 ratio is highest in cells of B6g7 origin (Fig. 4A–D). In addition, these competitive bone marrow chimeras showed a lower pre-cDC1 to pre-cDC2 ratio in the bone marrow for cells of B6g7 origin relative to the NOD, whereas the proportion of CD24+ cDC1 splenic precursors remains comparable for both strains (Fig. 4E–I). These results confirm that the proportion of precommitted cDC subsets in the bone marrow and spleen, as well as the proportion of cDC subsets in the spleen, are all regulated in a hematopoietic cell–intrinsic manner.

FIGURE 3.
FIGURE 3.

The relative proportion of cDC1 and cDC2 is MHC independent. Compilation of splenic (A) cDC1 and (B) cDC2 proportions among cDCs and absolute numbers from B6 (dot), B6g7 (square), and NOD (triangle) mice. cDC1 and cDC2 are, respectively, gated as CD8α+CD11b− and CD8α−CD11b+ among the CD3−CD19−B220−CD11chiMHC-II+ cDCs. Each symbol represents the data for one mouse. Dash represents the mean. The data were acquired in three independent experiments. *p < 0.05, **p < 0.01 based on a two-way ANOVA multiple comparison test. p ≥ 0.05 is noted as ns, for nonsignificant.

FIGURE 4.
FIGURE 4.

The cDC traits are bone marrow intrinsic. (A) Schematic representation of the competitive bone marrow chimeras. Bone marrow cells (2 × 106) isolated from B6g7 (CD45.2) and NOD (CD45.1) mice were injected at a 1:1 ratio into a lethally irradiated F1g7 (B6g7 × NOD, CD45.1.2) mouse. cDC reconstitution was analyzed in the spleen after at least 6 wk postreconstitution. (B) Representative flow cytometry plots of CD8α and CD11b expression on splenic cDCs differentiated either from the B6g7 or the NOD bone marrow in the F1g7 recipient mice. The gates define cDC1 as CD8α+CD11b− (orange) and cDC2 as CD8α−CD11b+ (green), among the CD3−CD19−B220−CD11chiMHC-II+ cDCs. (C) Data compilation from (B) showing the percentage of cDC1 (orange) and cDC2 (green) among total splenic cDCs differentiated from the B6g7 or the NOD bone marrow precursors, as indicated. (D) Data compilation of the ratio of cDC1 to cDC2 for genetic background. (E) Representative flow cytometry plots of CD117 and CD115 expression on B6g7- or NOD-derived bone marrow pre-cDCs from the F1g7 chimeric mice. Pre-cDC1 (orange) and CD117− pre-cDC2 (green) are gated, respectively, as CD115−CD117+ and CD117− among the CD3−CD19−B220−CD11c+MHC-II−/intCD135+CD172aint total pre-cDCs. (F) Data compilation from (E) showing the percentage of pre-cDC1 (orange) and CD117− pre-cDC2 (green) among the total bone marrow pre-cDCs derived from B6g7 and NOD bone marrow in the chimeric mice. (G) Data compilation of the ratio of pre-cDC1 to CD117− pre-cDC2 derived from each bone marrow precursor. (H) Representative flow cytometry plots of CD8α and CD24 expression on B6g7- or NOD-derived bone marrow splenic cDCs. The gate selects CD24+ cDC1 splenic precursors as CD8α−/intCD24+ cells among the CD3−CD19−B220−CD11chiMHC-II+ cDCs. (I) Data compilation from (H) showing the percentage of CD24+ cDC1 splenic precursors among splenic cDCs derived from B6g7 or NOD bone marrow in the chimeric mice. (B, E, and H) Numbers near the gates indicate the corresponding percentage of cells. (C, D, F, G, and I) Each symbol represents the data for one mouse. Dash represents the mean. Data were collected in at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and p ≥0.05 is noted as ns, for nonsignificant, based on a one-way ANOVA multiple comparison test (C and F) or a Mann–Whitney U test (D, G, and I).

CD135 surface expression is differentially regulated on cDC subsets from B6 and NOD mice

Hematopoietic cDC differentiation is primarily driven by cellular response to FLT3L (16–20). Previous studies suggest that FLT3L also influences cDC differentiation in the spleen (21, 25), but the impact of FLT3L in determining the relative proportion of cDC1 and cDC2 subsets has not been investigated. We thus postulated that the cell-intrinsic expression of CD135, the receptor for FLT3L, was a likely candidate in defining the cDC1 to cDC2 ratio. We quantified CD135 throughout cDC ontogeny. In the bone marrow, total pre-cDCs as well as both pre-cDC1 and pre-cDC2 expressed comparable levels of CD135 in both B6 and NOD mice (Fig. 5A, 5B). Splenic total pre-cDCs also expressed similar levels of CD135 (Fig. 5C, 5D). However, CD24+ cDC1 splenic precursors, cDC1 and cDC2, expressed lower levels of CD135 in NOD mice as compared with B6 mice (Fig. 5C, 5D). To determine if the levels of CD135 were hematopoietic cell intrinsic, we quantified CD135 expression throughout cDC differentiation in the B6g7 to NOD competitive bone marrow into F1(B6g7 × NOD). Of note, CD135 expression is similar on cDC subsets from B6 and B6g7 mice (Fig. 6A). In the competitive chimeras, we find that CD135 expression levels throughout cDC differentiation is regulated in a hematopoietic-intrinsic manner (Fig. 6B, 6C). Therefore, the differential expression of CD135 in cDCs from the two strains is not due to a difference in systemic FLT3L levels. From these experiments, we can additionally conclude that CD135 expression levels in CD24+ cDC1 splenic precursors cDC1 and cDC2 as well as the ratio of cDC1 to cDC2 are all regulated in a hematopoietic cell–intrinsic manner.

FIGURE 5.
FIGURE 5.

CD135 surface expression is differentially regulated on cDC subsets from B6 and NOD mice. (A) Representative histograms of CD135 expression on total bone marrow pre-cDCs (CD3−CD19−B220−CD11c+MHC-II−/intCD135+CD172aint) as well as bone marrow pre-cDC1 (CD117+CD115−) and bone marrow CD117− pre-cDC2 (CD117−) selected among pre-cDCs from (filled gray) B6 and (black line) NOD bone marrow, with the staining control population (T and B cells, gated as CD3+CD19+B220+) (filled light gray) for B6 and (black dashed) for NOD mice, respectively. (B) Compilation of CD135 mean fluorescence intensity (MFI) for each cell subset depicted in (A). (C) Representative histograms of CD135 expression on total splenic pre-cDCs (CD11c+MHC-II−/intCD135+CD172aint), CD24+ cDC1 splenic precursors (CD11c+MHC-II+CD24+CD8α−/int), splenic cDC1 (CD11c+MHC-II+CD8α+CD11b−), and cDC2 (CD11c+MHC-II+CD8α−CD11b+) subsets from the spleen of (filled gray) B6 or (black line) NOD mice, with the staining control population (T and B cells, gated as CD3+CD19+B220+) (filled light gray) for B6 and (black dashed) for NOD mice, respectively. (D) Compilation of CD135 MFI for the respective cell subsets depicted in (C). (A and C) Numbers near the histogram denote the MFI. Note that pre-cDC1 and pre-cDC2 as well as cDC1 and cDC2 are analyzed from the same sample and thus have the same control staining profile. (B and D) Each symbol represents the data for one mouse. Dash represents the mean. Data were collected in at least three independent experiments. **p < 0.01, ***p < 0.001 based on a Mann–Whitney U test. p ≥ 0.05 is noted as ns for nonsignificant.

FIGURE 6.
FIGURE 6.

CD135 surface expression is regulated in a bone marrow–intrinsic manner. (A) Compilation of CD135 mean fluorescence intensity (MFI) on splenic cDC1 and cDC2 isolated from a B6 (dot), B6g7 (square), and NOD (triangle) mice. (B) Representative histograms of CD135 expression on total bone marrow pre-cDCs (CD3−CD19−B220−CD11c+MHC-II−/intCD135+CD172aint), CD24+ cDC1 splenic precursors (CD3−CD19−B220−CD11c+MHC-II+CD24+CD8α−/int), cDC1 (CD3−CD19−B220−CD11c+MHC-II+CD8α+CD11b−), and cDC2 (CD3−CD19−B220−CD11c+MHC-II+CD8α−CD11b+) subsets derived from (filled gray) B6g7 or (black line) NOD bone marrow precursors in the F1g7 recipient mice, with the staining control population (T and B cells, gated as CD3+CD19+B220+) (filled light gray) for B6g7 and (black dashed) for NOD mice, respectively. Numbers near the histograms denote the MFI. Note that CD24+ cDC1 splenic precursors cDC1 and cDC2 are analyzed from the same sample and thus have the same negative control staining profile. (C) Compilation of CD135 MFI on the respective cell subsets depicted in (B). (A and C) Each symbol represents the data for one mouse. Dash represents the mean. Data were acquired in at least three independent experiments. **p < 0.01, ***p < 0.001, based on Mann–Whitney U test.

Increasing systemic FLT3L concentration favors cDC1 differentiation

CD135 is internalized upon FLT3L binding (26). The differential surface expression of CD135 on cDCs of B6 and NOD origin is thus likely due to cell-intrinsic differences in the sensitivity of response to FLT3L. Indeed, cDC1 and cDC2 from both B6 and NOD express similar intracellular levels of CD135 (Fig. 7A, 7B), suggesting that cDCs from these strains have the same potential to express CD135 at the cell surface and thus to respond to FLT3L. Considering the variations in cell surface expression of CD135 between the strains, we generated additional competitive bone marrow chimeras and grafted FLT3L-producing B16 melanoma to determine the impact of enhanced systemic levels of FLT3L on cDC subset differentiation (Fig. 8A). The successful engraftment of the FLT3L-producing B16 melanoma led to a significant increase in the number of all cDCs (Fig. 8B), whereas the engraftment of non–FLT3L-producing B16F10 melanoma in B6g7 mice do not (data not shown) (27). In these B16-FLT3L–grafted chimeras, CD24+ cDC1 splenic precursors expanded efficiently in both strains (Fig. 8C, 8D). In addition, as previously shown by others (17, 19, 28), FLT3L administration in vivo biased cDC differentiation toward cDC1 to the detriment of cDC2 (Fig. 8C, 8D). This result suggests that cDC1 precursors are more sensitive to FLT3L than cDC2 precursors, favoring cDC1 differentiation in the context of an overabundance of the cytokine. The low FLT3L availability in the bone marrow and spleen (21) may be responsible, at least in part, for the elevated proportion of cDC2 relative to cDC1 observed in mouse spleens.

FIGURE 7.
FIGURE 7.

CD135 intracellular expression on cDC subsets is comparable for B6 and NOD mice. Intracellular expression of CD135 expression was assessed on splenic cDC1 and cDC2 from B6 and NOD mice. (A) Representative histograms of CD135 intracellular expression on cDC subsets from B6 and NOD mice as indicated, with the intracellular staining on a control cell population (T and B cells, gated as CD3+CD19+B220+) (filled light gray) for B6 and (black dashed) for NOD mice, respectively. Numbers near the histogram denote the mean fluorescence intensity (MFI). Note that cDC1 and cDC2 are analyzed from the same sample and thus have the same control staining profile. (B) The ratio of intracellular CD135 MFI of B6 and NOD for cDC1 (left) or cDC2 (right) over the MFI of the control cell population is shown. Each symbol represents the data for one mouse. Dash represents the mean. Data were collected in at least three independent experiments. p ≥ 0.05 is noted as ns, nonsignificant.

FIGURE 8.
FIGURE 8.

Increasing systemic FLT3L concentration favors cDC1 differentiation. (A) Schematic representation of the competitive bone marrow chimeras. Bone marrow isolated from B6g7 (CD45.2) and NOD (CD45.1) mice were injected, at a 1:1 ratio, into a lethally irradiated F1g7 (CD45.1.2) mouse. At least 6 wk after reconstitution, the B16-secreting FLT3L cell line (B16FLT3L) was injected s.c., and splenic cDCs proportion were analyzed 10 d later. (B) Data compilation of the absolute number of splenic total cDCs (CD11chiMHC-IIhi) in the absence (filled diamond) or presence (empty diamond) of B16FLT3L. (C) Representative flow cytometry plots of (left) CD8α and CD24 expression to select CD24+ cDC1 splenic precursors as well as (right) CD8α and CD11b expression to select cDC1 and cDC2 of both B6g7 and NOD hematopoietic origin, among CD11chi MHC-II+ cDCs (top) in the absence or (bottom) in the presence of B16FLT3L. Numbers near the gates indicate the corresponding percentage of cells. (D) A compilation of the percentage of CD24+ cDC1 splenic precursors, cDC1 and cDC2, among splenic cDCs differentiated from B6g7 or NOD bone marrow (white) in the presence or (black) in the absence of B16FLT3L is shown. (B and D) Each symbol represents the data for one mouse. Dash represents the mean. Data were acquired in at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, based on a one-way ANOVA multiple comparison test.

Differential CD135 expression levels throughout cDC differentiation reveals a dispensable role for FLT3L in the late stages of cDC1 commitment

Increasing the systemic concentration of FLT3L favored cDC1 differentiation over cDC2, yet cDC2 typically predominates over cDC1 in the spleen. We thus more carefully compared CD135 expression between cDC1 and cDC2 subsets and observed that cDC2 expressed much lower levels of CD135 than cDC1 (Figs. 5C, 5D, 6). We further investigated the differential expression of CD135 throughout cDC differentiation, starting from the pre-cDC1 and pre-cDC2 bone marrow precursors. Because levels of expression can be sensitive to the number of cells stained, we pooled bone marrow and spleen from B6 mice expressing either the CD45.1 or the CD45.2 allele. As such, cDC precursors and cDC subsets from the bone marrow and spleen are tested in the same well, and CD135 expression levels can be directly compared (Supplemental Fig. 3). Interestingly, CD135 is differentially expressed throughout cDC1 and cDC2 differentiation. Although pre-cDC1 and pre-cDC2 express comparable levels of CD135 in the bone marrow, as they transit to the spleen, the level of expression decreases more drastically on pre-cDC1 than on pre-cDC2 (Fig. 9). Following early-stage differentiation, CD135 is re-expressed in the cDC1 lineage to slightly higher levels than in the bone marrow, whereas CD135 expression continues to decrease in the cDC2 lineage (Fig. 9). As CD135 is internalized following FLT3L binding (26), these data suggest that the late stages of cDC1 and cDC2 commitment in the spleen may have a distinct sensitivity of response for FLT3L.

FIGURE 9.
FIGURE 9.

FLT3L response and CD135 expression in cDC subset differentiation. Bone marrow (BM) and spleen (SP) cells from B6 mice expressing either the CD45.1 or the CD45.2 allele were pooled in the same well and stained for CD135 expression along with markers to define the specified cell types. (A) Representative flow cytometry plots of CD135 expression on the different subsets of the (top) cDC1 and (bottom) cDC2 lineages. Numbers near the histogram indicate the corresponding mean fluorescence intensity (MFI). The different cell subsets are gated as follows: pre-cDC1, CD11c+MHC-II−/intCD135+CD172aintLy6C−Siglec-H−; pre-cDC2, CD11c+MHC-II−/intCD135+CD172aintLy6C+Siglec-H−; CD24+ cDC1 splenic precursors, CD11c+MHC-II+CD24+CD8α−/int; cDC1, CD11c+MHC-II+CD8α+CD11b−; and cDC2, CD11c+MHC-II+CD8α−CD11b+. (B) CD135 expression for (orange) cDC1 and (green) cDC2 lineages. Symbols represents the mean ± SEM. *p < 0.05, ***p < 0.001, based on a Mann–Whitney U test. Data were collected for eight mice in three independent experiments.

To test this possibility, we sorted total splenic pre-cDCs from a B6 mouse and cultured the cells for 3 d with or without FLT3L in the presence of feeder cells (Fig. 10A, Supplemental Fig. 4A, 4B). Total pre-cDCs lack both CD8α and CD11b expression (Fig. 10B), and in contrast to bone marrow precursors (29), pre-cDCs upregulate CD8α and CD11b expression as they differentiate toward cDC1 and cDC2, respectively. Therefore, we used CD8α and CD11b expression to define cDC1 and cDC2 output in culture. Expectedly, in the presence of FLT3L, splenic pre-cDCs differentiated in vitro into cDC1 and cDC2, with a slight bias toward cDC2 (14) (Fig. 10C, Supplemental Fig. 4C). As previously shown by others (20), cDC2 differentiation is severely impaired in the absence of FLT3L (Fig. 10C). This confirms that bone marrow feeder cells cultured without FLT3L do not secrete a sufficient amount of FLT3L to support cDC2 differentiation (20). In stark contrast to cDC2, pre-cDCs differentiate into cDC1 in absence of FLT3L (Fig. 10C). This finding demonstrates that FLT3L is not required for the differentiation of splenic pre-cDC into cDC1. Altogether, these data demonstrate that CD135 expression is highly regulated during cDC differentiation, and in contrast to cDC2, FLT3L is dispensable for cDC1 commitment in the late phase of differentiation.

FIGURE 10.
FIGURE 10.

FLT3L is dispensable in the late stages of cDC1 commitment. Total splenic pre-cDCs (CD11c+MHC-II−/intCD135+CD172aint) were sorted from B6 mice and cultured with or without FLT3L for 3 d. (A) Representative flow cytometry plots before and after sorting of splenic pre-cDCs. (B) Representative flow cytometry plots showing lack of CD8α and CD11b expression on splenic pre-cDCs (CD11c+MHC-II−/intCD135+CD172aint; black line). cDC1 (CD11c+MHC-II+CD8α+CD11b−; filled gray) and cDC2 (CD11c+MHC-II+CD8α−CD11b+; black dotted) are used as positive controls for CD8α and CD11b expression, respectively. (C) Representative cytometry plot of CD8α and CD11b expression on cDCs (CD11c+MHC-II+) differentiated from splenic pre-cDCs (left) in the presence or (right) in the absence of FLT3L. Splenic pre-cDCs–derived cDC1 are gated as CD8α+CD11b− and cDC2 as CD8α−CD11b+. Numbers near the gates indicate the corresponding percentage of cells. Data were acquired in two independent experiments.

Discussion

cDCs are heterogeneous in terms of their ontogeny, phenotype, location, and function. For instance, each cDC subset has different Ag presentation potential, produces a distinct set of cytokines, and uniquely impacts the adaptive immune system responses (1, 3). To eventually exploit cDCs in clinical contexts, such as cancer therapy or vaccine development, it is critical that we carefully define the specific attributes of each cDC subset (30).

We previously showed that cDC2 is the predominant subset in the spleen of eight genetically distinct inbred strains (22). Among the common inbred strains, B6 and NOD mice exhibit the highest and lowest ratio of cDC1 to cDC2, respectively. Additional investigation of cDC differentiation in five mouse strains revealed a nonlinear differentiation potential for committed cDC precursors. To identify parameters that determine cDC1 to cDC2 proportion, we quantified CD135 expression throughout cDC differentiation. We observed a differential expression of CD135 on cDC subsets. By characterizing the sensitivity to FLT3L throughout cDC differentiation, we find that FLT3L impacts the relative proportions of cDC1 to cDC2.

cDC differentiate from bone marrow precursors, namely pre-cDC1 and pre-cDC2 that are specifically committed to cDC1 and cDC2 subsets, respectively (5, 14). In addition to pre-cDC1 and pre-cDC2, the pre-cDCs include a subset of uncommitted precursors with a differentiation potential toward both plasmacytoid and cDCs (5, 14). By using CD115 and CD117 to define pre-cDC subsets, we and others (5) demonstrate that the uncommitted CD115−CD117− pre-cDC subset almost exclusively yielded cDC2. Pre-cDC2 precursors are thus more abundant than pre-cDC1 in the bone marrow, contributing in part to the heightened proportion and number of cDC2 over cDC1 in the periphery. Nevertheless, the relative proportions of pre-cDC1 to pre-cDC2 do not parallel that of cDC1 to cDC2, suggesting that differentiation to committed cDC subsets from pre-cDC is not linear and influenced by additional factors.

Because of their discrepant cDC1 to cDC2 ratios, we took advantage of B6 and NOD mice to gain a clearer understanding of the factors regulating the cDC1/cDC2 ratio. Of note, NOD mice are autoimmune prone and spontaneously develop type 1 diabetes (31). However, the reduced number of cDC1 in this strain is not due to the underlying pathology, as the phenotype is conserved in both competitive bone marrow chimeric mice and NOD.Rag−/− mice (32), two models which do not progress to diabetes (33). Comparison of cDC differentiation traits in common inbred strains has allowed us to pinpoint key factors in cDC differentiation. Namely, the most divergent traits between the two strains are the relative ratios of pre-cDC and cDC subsets as well as CD135 expression in late-stage cDC differentiation. We find that studying phenotypes among various mouse strains is a useful strategy to decipher the regulation of cell niches (22, 34–38), and it has empowered our approach to define novel aspects relative to cDC commitment.

Our study finds that whereas increasing the systemic concentration of FLT3L promotes cDC1 differentiation, the late stages of cDC1 differentiation are FLT3L independent. These seemingly paradoxical results can be reconciled by examining the level of CD135 expression throughout cDC differentiation. In the spleen, pre-cDC1 exhibit a lower expression of CD135 relative to pre-cDC2. As CD135 is internalized following FLT3L binding (26), this suggests that pre-cDC1 are more responsive to FLT3L than pre-cDC2. In conditions of excess FLT3L availability, we indeed find that cDC differentiation is biased toward cDC1. Therefore, the early stages of cDC1 and cDC2 differentiation are differentially affected by FLT3L availability. Importantly, the concentration of FLT3L in the bone marrow and spleen is relatively low (21), which may explain, in part, the increased abundance of cDC2 relative to cDC1 observed in all mouse strains. In addition to the early stages of cDC differentiation, we observed that CD135 expression was highly regulated throughout late stages of cDC differentiation, in which late-stage cDC1 differentiation can proceed in the absence of FLT3L, whereas cDC2s require FLT3L for their differentiation. As such, in NOD mice, the relatively lower levels of CD135 expression in comparison with B6 may favor cDC2 differentiation. Altogether, without excluding the possibility that FLT3L may be important for cDC survival and/or proliferation, following differentiation (18), these data suggest that there is a differential sensitivity to FLT3L throughout cDC differentiation, which most likely contributes to the cDC1/cDC2 ratio.

We are not the first to demonstrate that the late-stage cDC2 differentiation is FLT3L dependent (20, 39). Indeed, ADAM10 expression in cDC2 allows for autocrine FLT3L activity for the maintenance of cDC2 in the spleen (39). In contrast to cDC2, we observed that late-stage cDC1 differentiation does not require FLT3L. Accordingly, IRF8 is a key transcription factor for early-stage cDC1 commitment up to the pre-cDC1 stage, which becomes dependent on BATF3 expression for the final differentiation to cDC1 (5, 40). It is tempting to suggest that the FLT3L-independent cDC1 differentiation is driven by the factors that promote BATF3 expression. Our work adds to the growing body of evidence suggesting that cDC differentiation from pre-cDC1 and pre-cDC2 is dependent on distinct cytokines and transcription factors.

FLT3L is an important cytokine for cDC differentiation and survival (16–21, 25). Still, the consequence of FLT3L signaling and how this influences cDC lineage commitment is not fully understood. For instance, FLT3 signaling is involved, either directly or indirectly, in the expression of cDC-specific transcription factors, including PU.1, STAT-3, or DC-SCRIPT (41–43). These transcription factors are required for cDC lineage commitment and differentiation at least up to the pre-cDC stage (41–43). Nevertheless, the impact of FLT3L/FLT3 in preferentially driving either cDC1 or cDC2 differentiation had not been previously investigated.

In conclusion, cDC subsets are clearly heterogeneous in terms of their function, phenotype, and transcriptomic signature, as well as their requirement for differentiation factors in various tissues. Additional studies are therefore required to identify the external factors that can imprint pre-cDC commitment and cDC subset differentiation. Understanding the regulation and differentiation of cDC subsets may offer new strategies to modulate their specific proportion and guide the development of relevant functional cDC subset–based therapeutic strategies.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Drs. Heather Melichar, Salix Boulet, Nathalie Labrecque, Martin Guimond, and Mary M. Stevenson for a critical review of the manuscript and all laboratory members for helpful discussions. We are grateful for Martine Dupuis from the flow cytometry facility as well as all animal house staff for technical support. Dr. Martin Guimond provided some FLT3L.

Footnotes

  • This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada to S.L. (2014-06531 and 2019-05047). C.A. held scholarships from the Lucie Besner Foundation, the Montreal Diabetes Research Center, and the Département de Microbiologie, Infectiologie et Immunologie at the Université de Montréal. S.L. is a Research Scholars Emeritus awardee from the Fonds de Recherche Québec Santé.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    B6
    C57BL/6 carrying the CD45.2 allele
    B6.1
    B6.SJL carrying the CD45.1 allele
    B6g7
    B6.NOD.Idd1
    cDC
    conventional DC
    cDC1
    type 1 cDC
    cDC2
    type 2 cDC
    C3H
    C3H/HeJ
    DC
    dendritic cell
    FLT3L
    FLT3 ligand
    MHC-II
    MHC class II
    NZO
    NZO/HILtJ
    pre-cDC
    precursors to cDC
    pre-cDC1
    precursors to cDC1
    pre-cDC2
    precursors to cDC2.

  • Received June 22, 2020.
  • Accepted August 10, 2020.

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