This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer’s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer’s patch.
This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer's patch (PP). Common dendritic cell progenitors (CDPs, lin– c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.
Here, we demonstrate a protocol to assess whether common dendritic cell progenitors (CDPs) are capable of giving rise to the plasmacytoid dendritic cell (pDC) population in Peyer's patch (PP). The overall goal of using this method was to evaluate the developmental regulation of pDCs in Peyer's patch (PP pDCs). The reason this is important is that PP pDCs differ from pDCs found in other tissues, including bone marrow, blood and spleen, and therefore it is unclear whether PP pDCs and other pDC populations are developmentally and/or functionally related. Specifically, pDCs are widely known for being the principal type I interferon (IFN) producers within the hematopoietic system, responding to Toll-like receptor 7 and 9 (TLR7/9) stimulation by rapid IFN secretion1-3. However, PP pDCs are deficient in producing type I IFN in response to TLR agonist stimulation4,5. Moreover, PP pDCs also differ from pDCs found in bone marrow and spleen in requiring signals from the type I interferon (IFN) receptor (IFNAR1) or the IFN signaling molecule STAT1 for their development and/or accumulation5. These data have suggested the possibility that distinct regulatory mechanisms control PP pDCs versus pDCs in other organs (e.g. bone marrow, spleen)5.
The rationale that led to the development of this method was based on recent advances in understanding dendritic cell (DC) biology. Most, if not all, DC subsets derive from hematopoietic progenitors that express the FMS-like tyrosine kinase 3 receptor (Flt3)6-10; however, DC development is not restricted to the classic myeloid and lymphoid pathways. For example, Flt3+ common myeloid progenitors (CMPs, lin– IL-7R– Sca-1– c-kit+ CD34+ FcγRlo/-) give rise to CDPs (lin– c-kitlo CD115+ Flt3+), which further differentiate into pDCs and conventional DCs (cDCs)9,10. By contrast, Flt3+ common lymphoid progenitors (CLPs, lin– IL-7R+ Sca-1lo c-kitlo) develop primarily into pDCs11. Therefore, prior studies indicate pDCs arise from at least 2 distinct hematopoietic progenitor populations under the regulation of Flt3L, although the typical analysis has been restricted to the bone marrow, spleen and/or blood pDC subsets. Thus, the progenitor population(s) that generates PP pDCs required investigation. Understanding the origins of PP pDCs will shed light on whether they share common developmental pathways with other pDC populations, or utilize distinct mechanisms during their generation in PP.
A unique advantage of the approach described herein is the use of mice that show a severe deficiency in PP pDCs as recipients for the adoptive transfer of hematopoietic progenitors. Mice with genetic deletion in the gene encoding IFNAR1 (Ifnar-/- mice) or STAT1 (Stat1-/-) revealed a striking depletion in PP pDCs5. Therefore, these strains provide an environment in which PP pDCs are reduced, allowing adoptive transfer studies to be performed in the absence of potent cell ablation regimes such as lethal irradiation. An additional strength of the method presented here is the use of hydrodynamic gene transfer (HGT) to stimulate elevated circulating amounts of Flt3L. This provides a cost effective approach to induce Flt3L in vivo, versus injection of recombinant protein. Numerous studies, including those in our lab, have employed HGT to induce cytokine amounts in a variety of experimental conditions5,12,13.
The division of labor and precise immune functions for DCs is of major interest in immunology. In particular, pDCs are important mediators of oral tolerance and systemic anti-viral responses, yet they also appear to contribute to the development and persistence of autoimmunity and cancer14-17. The protocol described herein will allow the developmental mechanisms regulating PP pDCs to be more fully explored. In addition, this approach may allow studies to assess PP pDC function, and may be extended to understanding the regulation and function of other immune populations within PPs.
Institutional approval must be obtained in advance for all experimental manipulations described herein involving mice. These include the use of C57BL6 mice for isolation of bone marrow progenitor cells, Ifnar-/- mice as recipients for adoptive transfer of hematopoietic progenitors and use of HGT for cytokine overexpression in vivo. Appropriate housing and animal care must also be provided by the investigator or institution. Furthermore, institutional approval may be required for the plasmids used in hydrodynamic gene transfer (i.e. recombinant DNA approval). The studies described here were approved by the Institutional Animal Care and Use Committee at UT MD Anderson Cancer Center.
1. Hydrodynamic Gene Transfer (HGT)
This step should be performed 2 days prior to the adoptive transfer of CDPs to induce circulating Flt3L for DC expansion in vivo5. Prepare at least 5 recipient mice/group.
2. Isolation of Hematopoietic Progenitors from Mouse Bone Marrow
This step should be performed 2 days after HGT. Use congenic CD45.1+ mice as the source of bone marrow progenitors for adoptive transfer into recipient Ifnar-/- animals (CD45.2+). In this protocol, congenic strains are required to distinguish donor and recipient-derived DCs, as well as to avoid immune-mediated depletion due to MHC mismatch. Approximately 10-20 mice will be required to provide sufficient numbers of hematopoietic progenitor cells (105 cells/recipient mouse) for the transfer experiments.
3. Fluorescence-activated Cell Sorting (FACS) to Isolate CDPs
This step requires access to a MidiMACS cell separator and MACS LD columns for an initial negative selection procedure, as well as a FACS machine with at least 3 lasers to purify the multicolor progenitor subset after staining with fluorescently conjugated antibodies.
4. Adoptive transfer of CDPs
This step is typically done in the animal facility where the recipient mice are housed. Depending on the location of the FACS machine, it may involve transport of the purified progenitor cell populations into the animal facility prior to adoptive transfer. Progenitor cell suspensions should be kept sterile and transported on ice.
5. Isolation of PP and Measurement of pDC Amounts
Our results show the gating strategy for the isolation of CDPs from mouse bone marrow cells (Figure 1), as detailed in Protocols 2 and 3. CDPs comprise approximately 0.1% of total bone marrow cells, and roughly 4-6 x 104 CDPs can be isolated from one mouse. Upon adoptive transfer, CDPs differentiate into pDCs and cDCs10.
Figure 1. Gating strategy for FACS purification of CDPs. Bone marrow cells were collected from C57BL6 mice. Lineage-positive cells were depleted with Abs against lineage markers (CD3, CD19, CD11b, CD11c, Ter119) using MACS microbead-mediated selection. The enriched lineage-negative bone marrow population was stained with fluorescently-labeled Abs for CDP and lineage markers, and purified by FACS as shown. Please click here to view a larger version of this figure.
To identify pDCs in PPs, we use an initial forward and side scatter gating strategy, followed by gating for CD11c+ Siglec-H+ cells (Figures 2A and 2B). Ifnar-/- mice have a significant reduction in pDCs in PPs relative to wild type mice (Figure 2B)5. By contrast, CD11c+ Siglec-H– cDCs are found at similar amounts in both genotypes (Figure 2B). Hence, Ifnar-/- mice provide an opportunity to examine PP pDC reconstitution without effects of lethal irradiation5. For example, the adoptive transfer of wild type CDPs into Ifnar-/- mice, as described in step 4, stimulates an increase in PP pDCs (Figure 2B). Moreover, pretreatment with Flt3L HGT (step 1) further enhanced pDC expansion in PPs (Figure 2B), implying transferred CDPs and possibility endogenous Flt3+ progenitors respond to Flt3L by inducing PP pDCs. Both conditions also stimulated CD11c+ Siglec-H– cDCs amounts, consistent with the developmental origin of cDCs6. pDCs in PPs express traditional pDC markers including PDCA-1, Siglec-H and B220, and lack CD11b (Figures 2B-D)4,5. In addition, analysis of PPs from Ifnar-/- mice that received both Flt3L HGT and transferred CDPs showed that the majority (~70%) of pDCs were derived from donor (CD45.1+) mice (Figure 2E). Collectively, these data demonstrate that adoptive transfer of CDPs induces the PP pDC population in response to Flt3L-mediated signals in vivo.
Figure 2. Analysis of PP pDC in Ifnar-/- mice upon Flt3L HGT and adoptive transfer of CDPs. Ifnar-/- mice were injected intravenously with 5 μg of plasmid encoding Flt3L or an empty vector (pORF) by HGT. Two days later, 105 FACS-purified CDPs were adoptively transferred via tail vein injection. Seven days post CDP transfer, PPs were collected and analyzed for pDC amounts (A, B). CD11c+ Siglec-H+ pDCs in mice that received CDPs + Flt3L HGT were further analyzed for PDCA-1, B220 (C), CD11b (D), CD45.1 and CD45.2 (E) expression. The expression pattern of PDCA-1, B220 and CD11b was similar in pDCs in all 3 groups (data not shown). Please click here to view a larger version of this figure.
The adoptive transfer technique described herein assessed the contribution of CDPs to the PP pDC population in recipient mice that are deficient in PP pDCs (e.g. Ifnar-/- mice). In future experiments, it will be important to evaluate the potential of other progenitor subsets in generating PP pDCs, in particular whether PP pDCs derive from Flt3+ CLPs. This question is significant since it remains unclear why PP pDCs are uniquely sensitive to IFNAR-STAT1 signals for PP accrual5, and if PP pDCs follow similar developmental cues as pDCs in other organs.
In theory, this method might also be performed in mice that lack pDC populations in all organs. For example, mice with deficiency in the transcription factor E2-2, the pDC "master regulator" (i.e. Tcf4-/- mice), show striking pDC depletion19. However, Tcf4-/- mice are embryonic lethal, and thus Tcf4-/- bone marrow chimeric mice would need to be used as recipients for adoptive transfer experiments. However, it remains unknown whether PP pDCs are sensitive to irradiation and would be effectively depleted in Tcf4-/- chimeras. Moreover, lethal irradiation has wide effects on the hematopoietic system and supporting stromal populations that might impact PP pDC reconstitution. Thus, the use of Ifnar-/- mice as recipients to assess the capability of adoptively transferred progenitor subsets to generate PP pDCs might be preferred to Tcf4-/- chimeras. Stat1-/- mice also show a striking reduction in PP pDCs and could be employed as recipients for adoptive transfer studies to study PP pDC developmental origins5. A caveat to the use of Ifnar-/- or Stat1-/- mice is their immunodeficient status, which could impact PP pDC reconstitution or function in unknown ways. Thus, independent approaches to assess PP pDC developmental origins would enhance confidence in data interpretation.
Technically, it should be noted that the CDP population is found at a very low frequency in total bone marrow, thus depletion of lineage marker-positive cells by magnetic bead-mediated column chromatography prior to FACS is important for efficient purification of the progenitor cells by FACS. This depletion step enriches lineage-negative cells, resulting in decreased FACS time (and cost) for progenitor purification. The depleting procedure described herein utilizes a mixture of lineage-specific antibodies that is combined for each experiment. Commercial lineage antibody cocktails are also available and can be used for removal of lineage-positive cells, in place of the mixture that we describe. The advantage of the described approach is its flexibility in being adaptable for different depletion purposes, by adjusting the antibodies that are present in the mixture.
In the preparation of single cell suspensions from PPs, it is important to remove as much of the intestinal tissue surrounding the PPs as possible. This can be accomplished by careful dissection of the PPs. Sufficient digestion of PPs with collagenase is a key step to release leukocytes from the intestinal tissue. The Percoll density-gradient centrifugation technique described is a preferred method to enrich leukocytes from digested PP samples. In addition, it is important to note that the pDC marker protein PDCA-1 may be regulated by type I IFN and other stimuli20. Therefore, Siglec-H is preferred as a pDC marker for studies involving cytokine manipulation.
The use of HGT has a clear advantage in terms of being highly cost effective. While Flt3L HGT was utilized in this study, the ability of other cytokines or soluble factors to regulate PP pDCs could be tested using HGT, in the absence or presence of adoptive cell transfer of hematopoietic progenitors. However, HGT results in sustained production of cytokines from the transferred plasmid versus the more transient increases observed during recombinant cytokine injection, which depend on cytokine half-life5,13. The extended elevation of circulating cytokine may not reflect physiological amounts achieved during emergency hematopoietic or infection responses. This caveat should be kept in mind during experimental planning stages and assessment of data.
In future work, selective manipulation of the PP pDC population, as described herein, may aid in addressing PP pDC function. PP pDCs are conditioned by mediators present in the mucosal environment and are deficient in type I IFN production upon TLR activation4,5. Analysis of PP pDCs reconstituted within Stat1-/- mice demonstrated these cells have a comparably low ability to induce type I IFN upon TLR9 triggering relative to PP pDCs arising naturally (not shown), indicating PP pDCs derived from transferred CDPs retain at least this property of natural PP pDCs. The reduced type I IFN production of PP pDCs contrasts with the robust type I IFN secretion of other pDC populations and raises a question regarding the functional role of PP pDCs. Moreover, PP pDCs resemble pDCs that develop in the presence of type I IFN, a pDC population that demonstrates efficient stimulation of IL-17-producing CD4+ T lymphocytes (Th17 cells)5. While Th17 cells are widely considered to be an inflammatory-inducing population, they have both protective and pathogenic roles in the gut21. Separately, pDCs have been reported to mediate systemic tolerance to orally administered antigen15. The role of PP pDCs in local and systemic immunity is of significant interest, as understanding this point may reveal new approaches to manipulate intestinal immune and inflammatory responses in disease therapy.
In conclusion, the method presented herein enables the assessment of the developmental potential of hematopoietic progenitor subsets for generating PP pDCs. This procedure provides mice with reconstituted PP pDCs. Thus, this approach may be used not only for evaluating PP pDC hematopoietic origins but also for understanding the contribution of PP pDCs to immune functions within the intestinal environment.
The authors have nothing to disclose.
We thank Drs. Alex Gelbard and Willem Overwijk for advice on hydrodynamic gene transfer. This work was supported by grants from the NIH (AI098099, SSW), the MD Anderson Center for Cancer Epigenetics, the MD Anderson Center for Inflammation and Cancer (SSW), and the R.E. Bob Smith Education Fund (HSL).
C57BL/6J | JAX | 664 | |
B6.SJL | JAX | 2014 | |
RPMI | Invitrogen | 11875-093 | |
15 ml conical tubes | BD Biosciences | 352095 | |
50 ml conical tubes | BD Biosciences | 352070 | |
Sterile surgical tweezers | |||
Sterile small pair scissors | |||
Sterile large pair scissors | |||
40 μm cell strainer | BD Biosciences | 352340 | |
35 μm cell strainer cap tubes | BD Biosciences | 352235 | |
RBC lysing buffer | Sigma | R7757 | |
FACS buffer | PBS, 2 mM EDTA, 1% FCS, filter sterilized | ||
Percoll | GE Healthcare | 17089102 | |
10XHBSS | Sigma | H4641 | |
Collagenase IV | Worthington | LS004188 | |
Goat ant-rat IgG microbeads | Milteyni Biotec | 130-048-501 | |
LD column | Milteyni Biotec | 130-042-901 | |
Rat anti-D3 | BD Biosciences | 555273 | |
Rat anti-CD19 | BD Biosciences | 553783 | |
Rat anti-CD11b | BD Biosciences | 553308 | |
Rat anti-CD11c | BD Biosciences | 553799 | |
Rat anti-Ter119 | BD Biosciences | 553671 | |
Anti-CD3 (PerCP) | eBiosciences | 45-0031 | |
Anti-CD19 (PerCP) | eBiosciences | 45-0193 | |
Anti-CD11b (PerCP) | eBiosciences | 45-0112 | |
Anti-CD11c (PerCP) | eBiosciences | 45-0114 | |
Anti-F4/80 (PerCP) | eBiosciences | 45-4801 | |
Anti-Ter119 (PerCP) | eBiosciences | 45-5921 | |
Anti-Sca-1 (PE.Cy7) | eBiosciences | 25-5981 | |
Anti-CD115 (APC) | eBiosciences | 17-1152 | |
Anti-c-kit (APC.Cy7) | eBiosciences | 47-1171 | |
Anti-IL-7R (Pacific Blue) | eBiosciences | 48-1271 | |
Anti-Flt3 (PE) | eBiosciences | 12-1351 | |
Anti-CD45.1 (APC.Cy7) | BD Biosciences | 560579 | |
Anti-CD45.2 (PE.Cy7) | Biolegend | 109830 | |
Anti-CD11c (Pacific Blue) | eBiosciences | 48-0114 | |
Anti-B220 (APC) | eBiosciences | 25-0452 | |
Anti-Siglec-H (PE) | eBiosciences | 12-0333 | |
Anti-PDCA-1 (FITC) | eBiosciences | Nov-72 | |
Cell sorter | BD Biosciences | e.g. BD Fortessa | |
Heat lamp | |||
Mouse restrainer | |||
1 ml syringes | Becton Dickinson | 309602 | |
27½ G needles (sterile) | Becton Dickinson | 305109 |