Name: retroviral overexpression of cxcr4 on murine b-1a cells and adoptive transfer for targeted b-1a cell migration to the bone marrow and igm production
Uploaded: 2020-05-31T13:00:00.000+00:00
Duration: 8 min 22 s
Description: Watch this Scientific Journal Video about Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production at JoVE.c

This work was supported by 1R01 HL107490, 1R01 HL136098, Project 3 of P01 HL055798, P01 HL136275-01 (C.A. McNamara), and R01GM100776 (T.P. Bender). A. Upadhye was supported by American Heart Association Pre-doctoral fellowship 16PRE30300002 and 5T32AI007496-20. We thank Joanne Lannigan, Mike Solga, and Claude Chew from the University of Virginia Flow Cytometry Core for their excellent technical assistance.

All animal protocols were approved by the Animal Care and Use Committee at the University of Virginia.
1. Magnetic separation and enrichment of peritoneal B-1 cells
<ol>
	<li>Euthanize a 12&#8722;14-week-old, male, CD45.1+ApoE-/- mouse using CO2.</li>
	<li>Make a superficial cut in the abdomen using straight surgical scissors and peel back skin using curved scissors to expose the peritoneal wall. Flush peritoneal cavity with 10 mL of 37 &#176;C RPMI-1640 medium using a 10 mL syringe and 25 G needle. Shake mouse to disengage cells by grasping the tail and moving the mouse side to side thoroughly for 15&#8722;20 s. 
	NOTE: Massaging the lavaged peritoneum can also maximize cell recovery.</li>
	<li>Collect peritoneal fluid using a 10 mL syringe and 25 G needle by drawing up fluid at the lower right side of the peritoneum just above the level of the hip, near the intestines. Avoid disrupting epidydimal fat depots and underlying organs. Avoid drawing fluid from the mouse&#8217;s left side as the omental fat can easily be drawn into the syringe.</li>
	<li>Once ~6&#8722;7 mL of fluid is collected, dispense into a 50 mL conical tube placed on ice. Next, elevate the mouse vertically by holding the peritoneal wall above the diaphragm using forceps so that any remaining fluid remains at the bottom of the peritoneal cavity. Make a small cut in the peritoneal wall using surgical scissors above the liver (make sure not to cut the liver) and collect any remaining peritoneal fluid using a glass pipet and bulb.</li>
	<li>Pool peritoneal washout cells from all CD45.1+ApoE-/- mice (n = 15&#8722;20) into 50 mL conical tubes, and store on ice. 
	NOTE: For calculating the number of mice needed: B-1a cells comprise 5&#8722;10% of the total peritoneal population and yield roughly 2.5&#8722;5 x 105 B-1a cells per mouse in authors&#8217; hands. Transduction efficiency is ~30&#8722;40%, as shown below.</li>
	<li>Count live cells using a viability dye such as trypan blue and a hemocytometer (dilute samples 1:5 in trypan blue and load 10 &#181;L into the hemocytometer chamber). Pool cells at up to 1 x 108 cells per tube. Centrifuge cells at 400 x g for 5 min at 4 &#176;C, then aspirate supernatant.</li>
	<li>Resuspend up to 1 x 108 cells in 1 mL of anti-CD16/CD32 antibody (Table of Materials) diluted 1:50 in assay buffer (1x phosphate buffered saline [PBS], 0.5% bovine serum albumin [BSA], 2 mM EDTA) in order to block Fc receptors. Scale as necessary based on cell count. Incubate on ice for 10 min at 4 &#176;C.</li>
	<li>Prepare a 2x master mix of the biotinylated antibodies (Table 1) in assay buffer for depletion. The 2x master mix accounts for the volume of the liquid the cells are already in when incubating with anti-CD16/CD32. For example, if cells are incubating in 500 &#181;L of diluted anti-CD16/CD32, then add 500 &#181;L of 2x master mix containing 10 &#181;L of biotinylated Ter119, Gr-1, CD23, and NK1.1 antibodies, and 25 &#181;L of biotinylated F4/80 antibody to achieve the final concentrations given in Table 1. Add 2x master mix to cells and stain for 20 min at 4 &#176;C.</li>
	<li>Wash cells with 5 mL of assay buffer and centrifuge at 400 x g for 5 min at 4 &#176;C and aspirate supernatant.</li>
	<li>Resuspend and incubate cells with anti-biotin microbeads (Table of Materials) diluted in assay buffer as per the concentration and protocol recommended by the manufacturer.</li>
	<li>Wash with 5 mL of assay buffer and centrifuge at 400 x g for 5 min at 4 &#176;C. Aspirate supernatant and resuspend up to 1 x 108 cells in 500 &#181;L of assay buffer.</li>
	<li>Prime magnetic selection columns (Table of Materials) with 3 mL of assay buffer. Transfer cells onto primed magnetic selection columns and collect the eluent containing enriched B-1 cells in a 15 mL conical tube on ice. Wash the magnetic selection column with additional assay buffer till the overall volume collected is 10 mL. 
	NOTE: An aliquot of the pre-purified and post-purified cell fractions should be separately analyzed by flow cytometry for purification efficiency by staining for CD19+ B cells, F480+ macrophages, and CD5+ T cells as shown in Figure 2.</li>
	<li>Count the post-purified cell fraction (the eluent containing enriched B-1 cells) by counting live cells as in step 1.6, and resuspend at 1 x 106 cells/mL in B cell culture medium (RPMI-1640, 10% heat-inactivated fetal bovine serum [FBS], 10 mM HEPES, 1x non-essential amino acids, 1 mM sodium pyruvate, 50 &#181;g/mL gentamicin, 55 &#181;M &#946;-mercaptoethanol).</li>
</ol>
2. Peritoneal B-1 cell stimulation
<ol>
	<li>Split pooled cells for the two transduced conditions (Ctl-GFP and CXCR4-GFP), while setting aside and plating at least 10 x 106 cells for a non-transduced control.</li>
	<li>Plate up to 150 &#181;L (150,000 cells) per well into 96-well round-bottom plates.</li>
	<li>Add 100 nM TLR9 agonist ODN1668 to all wells to stimulate cell proliferation.</li>
	<li>Incubate for 16&#8722;18 h at 37 &#176;C, 5% CO2.</li>
</ol>
3. Retroviral transduction of peritoneal B cells
<ol>
	<li>Generate Ctl-GFP (MigR1) and CXCR4-GFP retroviral particles using calcium phosphate transfection as described in previously published protocols14. 
	NOTE: This will take several days, so prepare and titer retroviral stocks and store aliquots at -80 &#176;C prior to starting this protocol.</li>
	<li>Thaw retrovirus stocks on ice. Use immediately and do not re-freeze as virus titer significantly diminishes during freeze-thaw cycles. Add Ctl-GFP or CXCR4-GFP retroviral supernatants at 20:1 multiplicity of infection (MOI) to cells, in the presence of 8 &#181;g/mL polybrene and fresh &#946;-mercaptoethanol at 55 &#181;M final concentration. Do not add viral stocks to the cells set aside for the non-transduced control.</li>
	<li>Perform spinfection by centrifuging plates at 800 x g for 90 min at room temperature.</li>
	<li>Incubate plates with retrovirus at 37 &#176;C, 5% CO2 for an additional 3 h.</li>
	<li>Harvest and replate cells in fresh B cell medium and incubate at 37 &#176;C, 5% CO2 overnight.</li>
</ol>
4. Cell sorting of transduced peritoneal B-1a cells
<ol>
	<li>Harvest cultured cells and pool into three separate 50 mL conical tubes for each condition: non-transduced, Ctl-GFP transduced, and CXCR4-GFP transduced.</li>
	<li>Count live cells as in step 1.6, then centrifuge at 400 x g for 5 min at 4 &#176;C and aspirate supernatant.</li>
	<li>Resuspend cells at 100,000 cells/&#181;L in sort buffer (PBS + 1% BSA) containing 1:50 anti-CD16/CD32 antibody (Table of Materials) in order to block Fc receptors. Incubate on ice for 10 min at 4 &#176;C.</li>
	<li>Aliquot cells for compensation controls (~30,000 cells per compensation control): for unstained and single stain controls aliquot from non-transduced sample and for GFP single stain control aliquot from transduced samples. 
	NOTE: Commercially available compensation beads can alternatively be used for single stain controls if non-transduced cell number is low.</li>
	<li>Prepare a 2x antibody master mix containing the fluorophore-conjugated antibodies in sort buffer (Table 1). Add 2x master mix to non-transduced, CTL-GFP transduced, and CXCR4-GFP transduced samples. Add individual antibodies to single stain controls. Incubate for 20 min at 4 &#176;C in the dark. 
	NOTE: The 2x master mix accounts for the volume of liquid the cells are already in when incubating with anti-CD16/CD32, as in step 1.8. An aliquot of cells from each condition can separately be stained for CXCR4 to confirm CXCR4 overexpression.</li>
	<li>Wash samples with 1 mL of sort buffer and strain through 70 &#181;m filters into polypropylene tubes.</li>
	<li>Centrifuge at 400 x g for 5 min at 4 &#176;C and aspirate supernatant. Resuspend samples at 50,000 cells/&#181;L in sort buffer.</li>
	<li>Prior to cell sorting prepare labeled fluorescence-activated cell sorting (FACS) collection tubes containing 1 mL of collection medium (RPMI-1640, 20% heat-inactivated FBS, 10 mM HEPES, 1x non-essential amino acids, 1 mM sodium pyruvate, 50 &#181;g/mL gentamicin, 55 &#181;M &#946;-mercaptoethanol) for each population to be sorted.</li>
	<li>Prior to running samples on the cell sorter add 2x DAPI (prepared as 1:5000 dilution in sort buffer) for dead cell discrimination.</li>
	<li>Sort GFP+ B-1a cells into FACS tubes containing collection medium as DAPI- CD19+ GFP+ B220mid-lo CD23- IgM+ CD5+ cells from the CTL-GFP and CXCR4-GFP samples. Use the non-transduced sample to set the GFP+ gate and to sort DAPI- CD19+ GFP- B220mid-lo CD23- IgM+ CD5+ non-transduced B-1a cells. 
	NOTE: Alternatively, non-transduced cells can also be sorted from the CTL-GFP and CXCR4-GFP samples by separately gating B-1a cells within the GFP- fraction. Transduced or non-transduced B-1b cells can also be sorted using this sort strategy as DAPI- CD19+ GFP+/- B220mid-lo CD23- IgM+ CD5- cells.</li>
</ol>
5. Adoptive transfer
<ol>
	<li>After cell sorting, centrifuge cells at 400 x g for 5 min at 4 &#176;C and aspirate supernatant carefully.</li>
	<li>Resuspend cells in cold sterile 1x PBS at 1,000 cells/&#181;L.</li>
	<li>Anesthetize male Rag1-/- ApoE-/- mice using isoflurane and inject 100 &#181;L (100,000 cells) per mouse via intravenous retro-orbital or tail vein injection using an ultra-fine insulin syringe. Inject a few mice with 1x PBS as a control.</li>
</ol>
6. Quantification of donor cells and plasma IgM
<ol>
	<li>At the desired time post-adoptive transfer, analyze transferred cells in recipient mice by bone marrow and spleen tissue harvest4 and flow cytometry. Quantify donor cell localization and CXCR4 overexpression by staining for CD45.1, CD45.2, and CXCR4 and analyze flow cytometry results using a software such as Flowjo. 
	NOTE: See Table 1 for antibodies used in this step. Ensure not to use an antibody conjugate that fluoresces in the FITC channel as GFP will be present on transduced donor cells.</li>
	<li>Isolate plasma by adding 10 &#181;L of 0.5 M EDTA to whole blood collected via cardiac puncture at the time of animal sacrifice. Centrifuge whole blood at 7,000 x g and aliquot plasma into separate 1.5 mL centrifuge tubes. Store at -80 &#176;C until performing analysis of secreted factors such as IgM by ELISA4.</li>
</ol>

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The authors have nothing to disclose.

The method provided here enables stable and relatively efficient primary B-1a cell gene delivery, in vivo adoptive transfer, and identification and localization of injected cells. Cells were able to be detected 17 weeks post-cell transfer and retained increased CXCR4 expression. Retrovirus-mediated delivery yielded 30-40% transduction efficiency of primary murine B-1a cells with minimal impact on cell viability in our hands (Figure 4e). This is in line with results from a previous study by M...

Recent studies have demonstrated considerable immune cell, and specifically B cell, phenotypic and functional heterogeneity depending on cell localization1,2,3,4,5. B-1a cells are one such population with heterogeneous capacity to produce protective IgM antibodies; bone marrow B-1a cells secrete IgM constitutively and contribute significantly to plasma IgM titers6, while peritoneal B-1a cells have low-level IgM secretion at homeostasis and instead can be activated through innate toll-like receptor (TLR) or cytokine-mediated signaling to rapidly proliferate, migrate, and secrete IgM7,8,9,10. B-1a cell IgM antibodies recognize oxidation-specific epitopes (OSE) that are present on pathogens, apoptotic cells, and oxidized LDL, and IgM binding to OSE can prevent inflammatory downstream signaling in diseases like atherosclerosis11. Therefore, strategies to increase IgM production via increasing peritoneal B-1a cell migration to sites like the bone marrow may be therapeutically useful. However, it is important for such strategies to be targeted and cell-type specific, as off-target effects may negatively impact immune function or health.
Here we describe a method for targeted and long-term overexpression of CXCR4 in primary murine B-1a cells and subsequent adoptive transfer to visualize cell migration and functional IgM antibody production (Figure 1). Genetic manipulation of primary B cells is limited by low transfection efficiencies compared to transfection of transformed cell lines. However, as transformed cell lines can significantly deviate from primary cells12,13, the use of primary cells is likely to provide results that more closely align to normal physiology. Several techniques have been described for gene transfer in primary murine B cells, including retroviral transduction, adenoviral transduction, lipofection, or electroporation-based transfection, which have varying levels of efficiency, transience, and impact on cell health13,14,15. The following method utilized retroviral transduction as it yielded adequate gene transfer efficiency of &#62;30% while minimally impacting cell viability. The CXCR4-expressing retrovirus was generated using the previously described retroviral construct murine stem cell virus-internal ribosomal entry site-green fluorescent protein (MSCV-IRES-GFP; MigR1)16, into which the mouse CXCR4 gene was sub-cloned4. MigR1 (control(Ctl)-GFP) and CXCR4-GFP retroviral particles were generated using calcium phosphate transfection as described in previously published protocols4,14.
Successfully transduced B-1a cells were then intravenously transferred into lymphocyte-deficient Rag1-/- mice. Both donor and recipient mice additionally contained knockout of the apolipoprotein E (ApoE) gene, which results in increased OSE accumulation and atherosclerosis, thereby providing a model for in vivo B-1 cell activation and IgM production. Moreover, donor and recipient mice differed in CD45 allotype; donor B-1 cells came from CD45.1+ ApoE-/- mice and were transferred into Rag1-/- CD45.2+ ApoE-/- recipients. This allowed differentiation of donor CD45.1 from recipient CD45.2 B cells post-transfer without the need to additionally stain for B cell markers during flow cytometry analysis. The results provided here demonstrate that targeted CXCR4 overexpression on B-1a cells associates with increased ability of B-1a cells to migrate to the bone marrow, which associates with increased plasma anti-OSE IgM. We additionally provide a method for the enrichment of peritoneal B-1 cells through negative selection and demonstrate the requirement of B-1 cell activation for efficient transduction. This method can be adapted for other retroviral constructs to study the effect of protein overexpression on B-1a cell migration, phenotype, or function. Moreover, the use of CD45.1 versus CD45.2 allotype distinction could theoretically allow transfer into other immune-sufficient murine models containing endogenous B cells.

<table><tbody><tr><td>70 micron filter caps</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>anti-biotin microbeads</td><td>Miltenyi Biotec</td><td>130-090-485</td><td></td></tr><tr><td>anti-CD16/CD32, or Fc block</td><td>Life Technologies</td><td>MFCR00</td><td></td></tr><tr><td>B220 APC</td><td>eBioscience</td><td>17-0452-83</td><td>Clone: RA3-6B2</td></tr><tr><td>Beta-mercaptoethanol</td><td>Gibco</td><td>21985-023</td><td></td></tr><tr><td>CD19 APCef780</td><td>eBioscience</td><td>47-0193-82</td><td>Clone: eBio1D3</td></tr><tr><td>CD23 biotin</td><td>eBioscience</td><td>13-0232-81</td><td>Clone: B3B4</td></tr><tr><td>CD23 PECy7</td><td>eBioscience</td><td>25-0232-82</td><td>Clone: B3B4</td></tr><tr><td>CD3e biotin</td><td>eBioscience</td><td>13-0033-85</td><td>Clone: eBio500A2</td></tr><tr><td>CD45.1 ApoE-/- mice</td><td>N/A</td><td>N/A</td><td>Bred in house</td></tr><tr><td>CD45.1 PerCP-Cy5.5</td><td>BD Biosciences</td><td>560580</td><td>Clone: A20</td></tr><tr><td>CD45.2 BV421</td><td>BD Biosciences</td><td>562895</td><td>Clone: 104</td></tr><tr><td>CD45.2 Rag1-/- ApoE-/- mice</td><td>N/A</td><td>N/A</td><td>Bred in house</td></tr><tr><td>CD5 PE</td><td>eBioscience</td><td>12-0051-83</td><td>Clone: 53-7.3</td></tr><tr><td>Ctl-GFP retrovirus</td><td>N/A</td><td>N/A</td><td>Generated in house using GFP-expressing retroviral plasmid MigR1 provided by Dr. T.P. Bender</td></tr><tr><td>CXCR4 APC</td><td>eBioscience</td><td>17-9991-82</td><td>Clone: 2B11</td></tr><tr><td>CXCR4-GFP retrovirus</td><td>N/A</td><td>N/A</td><td>Generated in house by cloning mouse CXCR4 into MigR1 retroviral plasmid</td></tr><tr><td>F4/80 biotin</td><td>Life Technologies</td><td>MF48015</td><td>Clone: BM8</td></tr><tr><td>Flowjo Software v. 9.9.6</td><td>Treestar Inc.</td><td>License required</td><td></td></tr><tr><td>Gentamicin</td><td>Gibco</td><td>15710-064</td><td></td></tr><tr><td>Gr-1 biotin</td><td>eBioscience</td><td>13-5931-82</td><td>Clone: RB6-8C5</td></tr><tr><td>heat-inactivated fetal bovine serum</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>IgM PECF594</td><td>BD Biosciences</td><td>562565</td><td>Clone: R6-60.2</td></tr><tr><td>Insulin syringes</td><td>BD Biosciences</td><td>329461</td><td></td></tr><tr><td>Isoflurane</td><td>Henry Schein Animal Health</td><td>029405</td><td></td></tr><tr><td>Live/Dead Yellow</td><td>Life Technologies</td><td>L34968</td><td></td></tr><tr><td>LS columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>NK1.1 biotin</td><td>BD Biosciences</td><td>553163</td><td>Clone: PK136</td></tr><tr><td>Non-essential amino acids</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>ODN 1668</td><td>InvivoGen</td><td>tlrl-1668</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>RPMI-1640</td><td>Gibco</td><td>11875-093</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Gibco</td><td>11360-070</td><td></td></tr><tr><td>Ter119 biotin</td><td>eBioscience</td><td>13-5921-82</td><td>Clone: Ter119</td></tr></tbody></table>

retroviral overexpression of cxcr4 on murine b-1a cells and adoptive transfer for targeted b-1a cell migration to the bone marrow and igm production

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

An overview of the protocol is given in Figure 1. Figure 2 displays enrichment of peritoneal B-1a cells after magnetic depletion of other peritoneal cell types. Live singlet cells in the post-depletion fraction have a greater proportion of CD19+ B cells compared to F4/80+ macrophages, lack CD5hi CD19- T cells, and contain an increased frequency of CD19+ CD5mid B-1a cells compared to the pre-depletion fractio...

Watch this Scientific Journal Video about Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production at JoVE.c

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide ...

retroviral-overexpression-cxcr4-on-murine-b-1a-cells-adoptive

Research

JoVE Journal

Immunology and Infection

5.0K Views.  University of Virginia. Dit protocol kan evalueren hoe gerichte genlevering b-1a-cellokalisatie en -functie beïnvloedt en kan een nuttige proof of concept-benadering zijn voor het onderzoeken van gentherapeutische potentialen in vivo. Deze methode biedt een stabiele en relatief efficiënte genlevering aan primaire B-1a-cellen en maakt de identificatie van donorcelfenotype en functie na adoptieve overdracht mogelijk. Deze methode kan worden gebruikt om diverse donor- en gastheercelprocessen in vivo te onderzoeken, w...

Video: Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the gene of interest in hematopoietic cells and can be used to study the gene's role in normal and/or malignant hematopoiesis. This protocol provides a detailed description on how to perform transduction-transplantation using calreticulin (CALR) mutations recently identified in myeloproliferative neoplasm (MPN) as an example. In this protocol whole bone marrow cells from 5-flurouracil (5-FU) treated donor mice are transduced with a retrovirus encoding mutant CALR and transplanted into lethally irradiated syngeneic hosts. Donor cells expressing mutant CALR are marked with green fluorescent protein (GFP). Transplanted mice develop an MPN phenotype including elevated platelets in the peripheral blood, expansion of megakaryocytes in the bone marrow, and bone marrow fibrosis. We provide a step-by-step account of how to generate retrovirus, calculate viral titer, transduce whole bone marrow cells, and transplant into irradiated recipient mice.

This manuscript provides a description of the methodology used to establish transduction-transplantation mouse models. A detailed account is given of technical errors to avoid when performing bone marrow transplants. A clear understanding should be gained of the importance of high viral titer, transfection/transduction, and irradiation.

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the ...

Cancer Research

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.

We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality.

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible animal models mirror common components of human disease, including the bm12 transfer model. Upon transfer of bm12 splenocytes or purified CD4 T cells, C57BL/6 mice rapidly develop large frequencies of T follicular helper cells (Tfh), germinal center (GC) B cells, and plasma cells followed by high levels of circulating anti-nuclear antibodies. Since this model utilizes mice on a pure C57BL/6 background, researchers can quickly and easily study disease progression in transgenic or knockout mouse strains in a relatively short period of time. Here we describe protocols for the induction of the model and the quantitation Tfh, GC B cells, and plasma cells by multi-color flow cytometry. Importantly, these protocols can also be used to characterize disease in most mouse models of SLE and identify Tfh, GC B cells, and plasma cells in other disease models.

The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible ...

Medicine

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 ...

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...

Deciphering Early Follicular Helper T-Cell Commitment in Acute LCMV Infection

Accessing Early Differentiation of Virus-Specific Follicular Helper CD4+ T Cell in Acute LCMV-Infected Mice

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, thus establishing long-term humoral immunity. During acute viral infection, the fate commitment of virus-specific TFH cells is determined in the early infection phase, and investigations of the early-differentiated TFH cells are crucial in understanding T cell-dependent humoral immunity and optimizing vaccine design. In the study, using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection and the TCR-transgenic SMARTA (SM) mouse with CD4+ T cells specifically recognizing LCMV glycoprotein epitope I-AbGP66-77, we described procedures to access the early fate commitment of virus-specific TFH cells based on flow cytometry stainings. Furthermore, by exploiting retroviral transduction of SM CD4+ T cells, methods to manipulate gene expression in early-differentiated virus-specific TFH cells are also provided. Hence, these methods will help in studies exploring the mechanism(s) underlying the early commitment of virus-specific TFH cells.

Author Spotlight: Elucidating the Pathways of TFH Cell Differentiation in Acute LCMV Challenges

The current study showcases protocols for assessing the early fate commitment of virus-specific TFH cells and manipulating gene expression in these cells.

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, ...

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

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