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).

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.&#160;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.
<ol>
	<li>To achieve efficient HGT, the blood vessels of the recipient Ifnar-/- mice (CD45.2+) should be dilated by exposing mice to a heating lamp for 5-10 min.</li>
	<li>Place the mouse in a restrainer device and disinfect its tail with 70% ethanol.</li>
	<li>Inject 5 &#956;g of plasmid encoding Flt3L in 2 ml of sterile PBS into the tail vein using a 27 G&#160;needle. For the control cohort, inject 5 &#956;g of empty vector (pORF) via the tail vein as indicated.</li>
	<li>Monitor mice for 15-30 min to ensure there are no deleterious effects of the tail vein injection. Return mice to housing facility for 2 days.</li>
</ol>
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.
<ol>
	<li>Euthanize 10 congenic CD45.1+ mice by CO2 asphyxiation and cervical dislocation.</li>
	<li>Place each carcass on a dissection tray and sterilize the abdomen and legs with 70% ethanol.</li>
	<li>Make an incision at the mid-abdomen and cut through the skin from the abdomen to each leg, cutting skin down the length of the leg.</li>
	<li>Gently remove the skin from each leg, and cut and remove the legs from the carcass at the hip joint using sharp scissors.</li>
	<li>Carefully remove the muscles from the femur and tibia of each leg using a sharp blade.</li>
	<li>Remove both ends of the femur and tibia with a sharp scalpel and place bones in a culture dish containing complete RPMI (RMPI with 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin, 1 mM sodium pyruvate and 50 &#956;M &#946;-mercaptoethanol).</li>
	<li>Prepare a syringe containing 1 ml of complete RPMI, connected to a 27 G&#160;needle.</li>
	<li>Insert the 27 G&#160;needle into one end of the femur or tibia. Flush the bone marrow from the femur or tibia by gently injecting the complete RPMI into the bone. Ensure that bone marrow cells are being removed from the bone; this can be accomplished by visually detecting expelled media that appears cloudy.</li>
	<li>Repeat flushing each femur and tibia 3x to thoroughly remove bone marrow cells.</li>
	<li>Prepare a single cell suspension by gently pipetting cells up and down 3-5x in the culture dish.</li>
	<li>Remove debris by passing the bone marrow cells through a 40 &#956;M cell strainer, placing the exuded cells into a new culture dish. Disrupt any cell clumps that appear on the strainer by gentle pressure with the end of a sterile syringe plunger.</li>
	<li>Lyse the red blood cells (RBC) present in the total bone marrow cell suspension with commercial RBC lysis buffer per the manufacturer&#39;s instructions. Use 2 ml RBC lysis buffer/4-6 x 107 cells (generally cells from one mouse) and an incubation time of 5 min at RT.</li>
	<li>Wash the bone marrow cells by pelleting cells by centrifugation for 4 min at 500 x g, gently aspirating the culture medium, resuspending in 10 ml FACS buffer (1x PBS + 2 mM EDTA + 1% FBS), pelleting cells by centrifugation and gently aspirating wash buffer.</li>
	<li>Repeat the wash, as described in step 2.13, for a total of 2 washes.</li>
</ol>
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.
<ol>
	<li>After the second wash in step 2.14, count the bone marrow cells. Pellet the cells by centrifugation as described in step 2.13 and resuspend in FACS buffer to achieve a final concentration of 4 x 107 cells per 30 &#956;l of FACS buffer.</li>
	<li>To prepare cells for FACS, lineage-negative cells are first enriched by a negative selection technique that removes lineage-positive cells from the bone marrow mixture using magnetic bead column chromatography. For the negative selection procedure, add 1 &#956;g of each of the following commercial rat anti-mouse antibodies (Abs) that recognize hematopoietic lineage markers: CD3, CD19, CD11c, CD11b, and Ter-119 Abs. The Abs should be added in a volume of 2 &#956;l/Ab to the 30 &#956;l cell suspension in FACS buffer.</li>
	<li>Incubate the cell suspension at 4 &#176;C for 30 min. Following the incubation, wash the cells twice with 10 ml of FACS buffer as described in step 2.13.</li>
	<li>Resuspend cells to achieve a final concentration of 4 x 107 cells/40 &#956;l of FACS buffer. Add 20 &#956;l of goat anti-rat IgG magnetic microbeads per each 40 &#956;l of cell suspension. Mix gently and incubate at 4 &#176;C for 30 min. Wash cells with 10 ml of FACS buffer as described in step 2.13.</li>
	<li>Resuspend up to 108 bone marrow cells in 500 &#956;l of FACS buffer.</li>
	<li>Load a MACS LD column onto a MidiMACS cell separator per the manufacturer&#39;s instructions and prerinse the column with 2 ml FACS buffer.</li>
	<li>Apply the bone marrow cell suspension to the column, loading up to 5 x 108 cells in 2.5 ml of FACS buffer. Ensure a collection tube (15 ml conical tube) is placed under the column. Wash the column 3x with 2 ml of FACS buffer/wash, and collect the cells that pass through the column, which will be enriched for lineage-negative cells. Count the cells in the material eluted (wash-through) from the column.</li>
	<li>To perform positive selection of hematopoietic progenitor subsets by FACS, stain cells with the following fluorescently conjugated Abs: IL-7R (Pacific Blue), Flt3 (PE), Sca-1 (PE.Cy7), CD115 (APC), c-kit (APC.Cy7) Abs, plus a mixture of lineage marker Abs directed against CD3, CD19, CD11c, CD11b, F4/80 and Ter119 (all labeled with PerCP Cy5.5). Use 0.5-1 &#956;l of each Ab in a total volume of 100 &#956;l. Incubate cell suspension at 4 &#176;C for 20-30 min.</li>
	<li>Wash cells as indicated in step 2.13 and resuspend in FACS buffer at a concentration of 2-3 x 107 cells/ml. Filter cells through a 35 &#956;M cell strainer cap tube to remove cell clumps. This last step is crucial to avoid clogging the FACS machine.</li>
	<li>Place the cell suspension in a FACS tube on the FACS stage and sort CDPs (lin- c-kitlo CD115+ Flt3+) on the basis of the indicated marker profile. Collect purified cells in a 15 ml tube containing 5 ml of complete RPMI.</li>
	<li>Record the absolute number of sorted cells at the end of the FACS run. Pellet the cells by centrifugation as indicated in step 2.13 and resuspend in sterile PBS for adoptive transfer experiments.</li>
</ol>
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.
<ol>
	<li>Prepare cell suspensions for injection by diluting 105 purified CDPs in a total volume of 100 &#956;l sterile PBS. Place cell suspension in a syringe attached to a 27 G&#160;needle.</li>
	<li>Expose the recipient Ifnar-/- mice (CD45.2+) to a heating lamp for 5-10 min to achieve efficient injection via the tail vein.</li>
	<li>Place the mouse in a restrainer device and disinfect its tail with 70% ethanol.</li>
	<li>Inject 105 FACS-purified CDPs in 100 &#956;l PBS into the tail vein using a 27 G&#160;needle.</li>
	<li>Monitor mice for 15-30 min to ensure there are no deleterious effects of the tail vein injection. Return mice to housing facility.</li>
</ol>
5. Isolation of PP and Measurement of pDC Amounts
<ol>
	<li>At 7-10 days following adoptive transfer, euthanize the recipient mice. Gently open the mouse by dissection and expose the intestine.</li>
	<li>Remove the entire intestine and place it on PBS-soaked paper towels. Collect all visible PPs along the wall of the small intestine using fine forceps and scissors. C57BL6 and Ifnar-/- mice typically have 5-10 PP/mouse, which are different from isolated lymphoid follicles found along the small intestine18. Note that the PPs are often structurally distinct even within a single mouse, so carefully ensure all PPs are identified and dissected.</li>
	<li>Place PPs in a Petri dish containing PBS, and use forceps to remove feces. Repeat this procedure twice to clean PPs.</li>
	<li>Digest PPs with 1 mg/ml collagenase IV in 10 ml 1x Hank&#39;s balanced salt solution (HBSS) in a 50 ml flask with vigorous stirring for 1 hr at 37 &#176;C.</li>
	<li>Place the digested PP in the top compartment of a 40 &#956;m cell strainer and force cells through strainer with a sterile syringe plunger. Collect the PP cell suspension in a 15 ml conical tube.</li>
	<li>Pellet strained PP cells by centrifugation at 500 x g for 5 min and resuspend in 6 ml of 37% Percoll solution (37% Percoll in RPMI medium). Gently place 6 ml of 70% Percoll solution (70% Percoll in RPMI medium) underneath the cell suspension, to form a Percoll step gradient.</li>
	<li>Centrifuge cells for 20 min at 800 x g with the centrifuge break off. Mononuclear cells will migrate to the 37/70% interface. After centrifugation, the mononuclear cell population should be collected by careful pipetting at the interface region. Pellet collected cells by centrifugation and wash twice in 50 ml of complete RPMI/wash. A large volume is used to ensure complete Percoll removal.</li>
	<li>Stain the collected PP cells with the following antibodies to detect murine pDCs that derive from the adoptively transferred progenitors (CD45.1+) or recipient mice (CD45.2+): CD45.1 (APC.Cy7), CD45.2 (PE.Cy7), CD11c (Pacific Blue), CD11b (PerCP Cy5.5), B220 (APC), Siglec-H (PE) and PDCA-1 (FITC) Abs. Perform flow cytometry analysis of PP cells as indicated in steps 3.9 and 3.10. Identify pDCs by their CD11c+ CD11b- B220+ Siglec-H+ PDCA-1+ phenotype. Absolute pDC numbers can be determined by the following equation: % pDCs x total PP cell number = PP pDCs.</li>
</ol>

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The authors declare&#160;no competing financial interests.

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.&#160;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 accrual<s...

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&#39;s patch (PP). The overall goal of using this method was to evaluate the developmental regulation of pDCs in Peyer&#39;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.&#160;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+ Fc&#947;Rlo/-) 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.

<table border="0" cellpadding="0" cellspacing="0" width="867">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">C57BL/6J</td>
			<td style="width:201px;">JAX</td>
			<td style="width:177px;">664</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">B6.SJL</td>
			<td style="width:201px;">JAX</td>
			<td style="width:177px;">2014</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">RPMI</td>
			<td style="width:201px;">Invitrogen</td>
			<td style="width:177px;">11875-093</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">15 ml conical tubes</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">352095</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">50 ml conical tubes</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">352070</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Sterile surgical tweezers</td>
			<td style="width:201px;"></td>
			<td style="width:177px;"></td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Sterile small pair scissors</td>
			<td style="width:201px;"></td>
			<td style="width:177px;"></td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Sterile large pair scissors</td>
			<td style="width:201px;"></td>
			<td style="width:177px;"></td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">40 &#956;m cell strainer</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">352340</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">35 &#956;m cell strainer cap tubes</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">352235</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">RBC lysing buffer</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:177px;">R7757</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:260px;">FACS buffer</td>
			<td style="width:201px;"></td>
			<td style="width:177px;"></td>
			<td style="width:229px;">PBS, 2 mM EDTA, 1% FCS, filter sterilized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Percoll</td>
			<td style="width:201px;">GE Healthcare</td>
			<td style="width:177px;">17089102</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">10XHBSS</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:177px;">H4641</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Collagenase IV</td>
			<td style="width:201px;">Worthington</td>
			<td style="width:177px;">LS004188</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Goat ant-rat IgG microbeads</td>
			<td style="width:201px;">Milteyni Biotec</td>
			<td style="width:177px;">130-048-501</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">LD column</td>
			<td style="width:201px;">Milteyni Biotec</td>
			<td style="width:177px;">130-042-901</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Rat anti-D3</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">555273</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Rat anti-CD19</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">553783</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Rat anti-CD11b</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">553308</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Rat anti-CD11c</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">553799</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Rat anti-Ter119</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">553671</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD3 (PerCP)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">45-0031</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD19 (PerCP)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">45-0193</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD11b (PerCP)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">45-0112</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD11c (PerCP)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">45-0114</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-F4/80 (PerCP)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">45-4801</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-Ter119 (PerCP)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">45-5921</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-Sca-1 (PE.Cy7)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">25-5981</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD115 (APC)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">17-1152</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-c-kit (APC.Cy7)&#160;</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">47-1171</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-IL-7R (Pacific Blue)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">48-1271</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-Flt3 (PE)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">12-1351</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD45.1 (APC.Cy7)</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">560579</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD45.2 (PE.Cy7)</td>
			<td style="width:201px;">Biolegend</td>
			<td style="width:177px;">109830</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD11c (Pacific Blue)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">48-0114</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-B220 (APC)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">25-0452</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-Siglec-H (PE)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">12-0333</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-PDCA-1 (FITC)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">Nov-72</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Cell sorter</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;"></td>
			<td style="width:229px;">e.g. BD Fortessa</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Heat lamp</td>
			<td style="width:201px;"></td>
			<td style="width:177px;"></td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Mouse restrainer</td>
			<td style="width:201px;"></td>
			<td style="width:177px;"></td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">1 ml syringes</td>
			<td style="width:201px;">Becton Dickinson</td>
			<td style="width:177px;">309602</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">27&#189; G needles (sterile)</td>
			<td style="width:201px;">Becton Dickinson</td>
			<td style="width:177px;">305109</td>
			<td style="width:229px;"></td>
		</tr>
	</tbody>
</table>

assessing the development of murine plasmacytoid dendritic cells in peyer's patches using adoptive transfer of hematopoietic progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;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.

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.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51189/51189f...

Watch this Scientific Journal Video about Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors at JoVE.com

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;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&#8217;s patch.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

assessing-development-murine-plasmacytoid-dendritic-cells-peyer-s

Research

JoVE Journal

Immunology and Infection

11.1K ビュー.  The University of Texas MD Anderson Cancer Center. このプロトコルは、FACSを介した細胞の分離、流体力学的遺伝子導入、およびパイエル板の免疫サブセットのフロー分析を含む技術を使用して、一般的な樹状細胞前駆体からのパイエル板での形質細胞様樹状細胞の分化を評価するための実験手順について説明します。 

ビデオ: Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer&#039;s Patches

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during inflammatory responses. Unfortunately, study of pDC function is hindered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation predominantly favor the production of cDCs over pDCs. Here we present a unique approach to efficiently generate pDCs from common lymphoid progenitors (CLPs) in vitro. Specifically, the protocol described details how to purify CLPs from bone marrow and generate pDCs by coculturing with &#947;-irradiated AC-6 feeder cells in the presence of Flt3 ligand. A unique characteristic of this culture system is that the CLPs migrate underneath the AC-6 cells and become cobblestone area-forming cells, a critical step for expanding pDCs. Morphologically distinct DCs, namely pDCs and cDCs, were generated after approximately 2 weeks with a composition of 70-90% pDCs under optimal conditions. Typically, the number of pDCs generated by this method is roughly 100-fold of the number of CLPs seeded. Therefore, this is a novel system with which to robustly generate the large numbers of pDCs required to facilitate further studies into the development and function of these cells.

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...