Adoptive Cell Transfer: Introducing Donor Mouse Splenocytes to a Host Mouse and Assessing Success via FACS

Overview

Source: Meunier Sylvain^1,2,3, Perchet Thibaut^1,2,3, Sophie Novault⁴, Rachel Golub^1,2,3
¹ Unit for Lymphopoiesis, Department of Immunology, Pasteur Institute, Paris, France
² INSERM U1223, Paris, France
³ Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Paris, France
⁴ Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France

Adoptive cell transfer is a method for introducing cells into a patient or study organism in order to treat a disease or study a biological process, such as haematopoiesis. Aims of adoptive transfer are various; it can be used in fundamental biology as well as in medical sciences (1, 2). In mouse models, migration and distribution of transferred cells can be studied and followed by a tracking system (cell surface marker, staining by CFSE, etc.). In cancer studies on mouse models, transfer of specific cell populations can be used as experimental treatment against tumors. Another example for this technique is creation of chimeric mice by transfer of bone marrow cells to irradiated mice or mice with a severe immunodeficiency phenotype. This mouse model can be used to assess the impact of gene deletion on a specific cell population for instance. Transfer of bone borrow cells is also used in human medical treatment. When patients are irradiated in case of cancer therapy, adoptive transfer of bone marrow allows immune system reconstitution.

The first step in this technique is to obtain the cell population of interest. The technique chosen to isolate this population depends on the level of specificity of the targeted population. The largest level of selection is the whole organ, in which all cell populations present in the organ are taken. A more precise method is selection of a target cell population, often selected by one cell surface marker. The ideal method to sort cells in this case is by magnetic sorting. Finally, the most stringent level is the selection of cells by several cell surface markers to sort very specific cell populations. Flow cytometry sorting is the most popular method for this level of selection. Once population of interest is obtained, it can be transferred to the host. Before adoptive transfer it is essential to ensure compatibility between host and donor. Indeed, regardless of transfer goal, compatibility is crucial to assure cells adoption by the host without cells rejection.

In this lab exercise, we demonstrate the adoptive cell transfer technique by transferring splenocytes from a CD45.2 mouse into a CD45.1 Ragγc mouse (lacking lymphocytes) and four days later confirm the splenocytes transfer using flow cytometry (see Figure 1).

Figure 1: Schematic representation of adoptive transfer. (1) Splenocytes are isolated from CD45.2 mice and (2) transferred in CD45.1 Ragγc mouse, control mouse is injected with PBS only. (3) 4 days after adoptive transfer, splenocytes are recovered from mice and (4) analyzed by flow cytometry. Please click here to view a larger version of this figure.

Procedure

1. Preparation

Before beginning, put on laboratory gloves and the appropriate protective clothing.
Sterilize all the dissection tools, first with a detergent and then with 70% ethanol and then dry thoroughly.
Prepare 50 mL of Hank's balanced salt solution (HBSS) containing 2% fetal calf serum (FCS).

2. Dissection

Using a carbon dioxide delivery system, euthanize the mouse by hypoxia. Secure the euthanized mouse on a dissection plate in the supine position and perform a longitudinal laparotomy using scissors and forceps.
Using forceps move the intestines and stomach on the right side of the abdomen to expose the stomach and spleen. The spleen is attached to the stomach.
Using forceps carefully detach the spleen from the stomach and place it on the Petri dish containing 5 mL of HBSS 2% FCS.

3. Immune Cell Isolation

Place the spleen on a 40 µm cell strainer over the Petri dish. Crush the spleen with a plunger to dissociate it.
Recover the adhered cells by rinsing the plunger and the strainer with 1 mL of HBSS 2% FCS.
Transfer the dissociated spleen and the fluid into a 15 mL centrifuge tube.
Wash the Petri dish with 5 milliliters of HBSS 2% FCS and transfer the fluid to the 15 mL tube.
Centrifuge the tube at 370 x g for 7 min at 10°C and discard the supernatant avoiding the pellet.
Resuspend the pellet in 2 mL of potassium ammonium chloride pipet up and down to resuspend the pellet and lyse the erythrocytes.
Wait for 2 min and add HBSS 2% FCS to the resuspended pellet to obtain the volume up to 14 mL.
Centrifuge the tube again at 370 x g for 7 min at 10°C. Discard the supernatant and resuspend the pellet in 5 mL of HBSS 2% FCS by pipetting up and down.
Count the cells using trypan blue staining assay and adjust the final cell concentration to 10⁷ cells/mL using appropriate volume of HBSS 2% FCS.

4. Adoptive Transfer

Transfer 2 mL of cell suspension obtained in a 5 mL collection tube.
Centrifuge the tube at 370 x g for 7 min at 10°C and then discard the supernatant.
Resuspend the pellet in 2 mL of PBS and centrifuge the tube at 370 x g for 7 min at 10°C.
Discard supernatant and resuspend pellet in 200 µL of PBS.
Using a 0.5 mL syringe with a 29G needle inject 200 µL of cell suspension into the experimental mouse intravenously into the retro-orbital blood sinus.
As a control, inject a second mouse in the same blood sinus with 200 µL of phosphate buffered saline.

5. Cells Harvest and Staining

Four days after the adoptive transfer, euthanize mice and remove the spleen.
Harvest the splenocytes as described in section 3.
Transfer 100 µL of cell suspensions from each mouse into two FACS tubes, labeled "control" and "transferred".
Centrifuge the tube at 370 x g for 7 min at 10°C and then discard the supernatants.
Prepare a mix containing the four antibodies at the dilutions listed in Table 1.

Antibodies	Fluorochrome	Dilution
CD45.1	BV711	1/200
CD45.2	APCCy7	1/400
CD4	BV786	1/1600
CD3	BV421	1/200

Table 1: Antibodies mix composition. Four antibodies cocktails preparation using concentrated antibody-fluorescent conjugates and HBSS.

Add 100 µL of the mix to each tube, and then incubate for 20 min on ice in the dark.
Add 1 mL of HBSS 2%FCS and then centrifuge the tubes at 370 x g for 3 min at 10°C.
Discard the supernatants and resuspend the pellets in 200 µL of HBSS 2% FCS.
Transfer the resuspended cells to new, labelled FACS tubes.
Using flow cytometry, as shown in FACS protocol, evaluate the presence of CD45.2 positive lymphocytes.

6. Data Analysis

Open the "FlowJo" software and drag the files for each tube in "All sample" window.
Double click on the "transferred" file to display the cells recorded from that sample on a dot plot that displays forward scatter "SSC-A" on the Y axis and side scatter "FSC-A" on the X axis.
Click on "polygon" and create a gating strategy to select lymphocytes, then distinguish donor and host cells using cell surface markers (CD45.1, CD45.2), and then characterize CD45.2⁺ cell population (CD3, CD4).
Repeat the analysis steps with "control mouse" file.
To visualize a cell population, click on "Layout editor".
Drag the "transferred cells" and the "transferred CD4 cells" population from "transferred" and "control" files into the "Layout Editor tab".
A dot plot representing CD45.2⁺ cells and CD4 lymphocytes will appear.
CD45.2 transferred cells should only appear in the "transferred mouse" dot plot.

Results

Ragγc mice have an altered immune system composition, mainly lacking lymphocytes. Adoptive transfer of splenocytes allows introduction of lacking population such as T and B cells. Our staining included cell surface markers CD45.1 and CD45.2 to distinguish host and donor cells respectively (Figure 2A). It also included other cell surface marker to highlight cell populations absent in Ragγc mice, such as CD4 T cells (Figure 2B). As expected, control mouse did not have CD45.2-positive cells (Figure 2B, top panels) and transferred mouse did (Figure 2B, bottom panels, 71.2% of total cells). We could also specifically detect CD4 T cells within transferred cells (22.1% of CD45.2 cells).

Figure 2: Representative results of adoptive transfer. (A) Histograms of CD45.2 cells from mice injected with PBS (control group) (dashed) and mice injected with CD45.2 splenocytes (test group) (solid line). (B) Gating strategy of CD45.2-positive cells in control mice injected with PBS (top panels) and mice injected with CD45.2 splenocytes (bottom panels). Donor and host cells are distinguished using cell surface markers (CD45.1, CD45.2), then CD45.2-positive cell population are characterized (CD3, CD4). Please click here to view a larger version of this figure.

Application and Summary

Adoptive transfer is a translational technique in different fields of science, with applications in medicine. This technique can be used to study cell migration and tropism or incidence of protein deficiency in specific cell populations. In the last case, different technologies can be used, especially GMO mice where specific cell populations are intrinsically deficient. However, genetic construction to obtain GMO mice can be a very complex and long process. In this case, adoptive transfer of deficient cell population is easier and faster.

Adoptive transfers have direct applications in medicine. For instance, bone marrow grafts in irradiated patients during cancer therapy are used to reconstitute the immune system. Recently, other applications of adoptive transfer have been used in the medical field. Artificial T cells (called CAR T cell) are designed to recognize and eliminate some cancers. In addition, these engineered cells are built to dampen rejection risk by the host. Transfer of CAR T cells is currently tested in clinical trials.

References

Restifo, N. P., Dudley, M. E. and Rosenberg., S. A. Adoptive immunotherapy for cancer: harnessing the T cell response. Nature reviews. Immunology, 12 (4): 269-281, (2012).
Bonini, C., and Mondino, A. Adoptive T-cell therapy for cancer: The era of engineered T cells. European journal of immunology, 45 (9): 2457-69, (2015).

Tags

Adoptive Cell Transfer Donor Mouse Splenocytes Host Mouse FACS Immune Cells Biological Mechanisms Bone Marrow Transplants Cancer Treatments T cells Animal Models CD45 1 Rag Gamma c Knockout Mice Cytokines Lymphocyte Development Natural Killer Cells NK Cells B cells Spleen Cells Erythrocytes Red Blood Cells

1. Preparation

Before beginning, put on laboratory gloves and the appropriate protective clothing.
Sterilize all the dissection tools, first with a detergent and then with 70% ethanol and then dry thoroughly.
Prepare 50 mL of Hank's balanced salt solution (HBSS) containing 2% fetal calf serum (FCS).

2. Dissection

Using a carbon dioxide delivery system, euthanize the mouse by hypoxia. Secure the euthanized mouse on a dissection plate in the supine position and perform a longitudinal laparotomy using scissors and forceps.
Using forceps move the intestines and stomach on the right side of the abdomen to expose the stomach and spleen. The spleen is attached to the stomach.
Using forceps carefully detach the spleen from the stomach and place it on the Petri dish containing 5 mL of HBSS 2% FCS.

3. Immune Cell Isolation

Place the spleen on a 40 µm cell strainer over the Petri dish. Crush the spleen with a plunger to dissociate it.
Recover the adhered cells by rinsing the plunger and the strainer with 1 mL of HBSS 2% FCS.
Transfer the dissociated spleen and the fluid into a 15 mL centrifuge tube.
Wash the Petri dish with 5 milliliters of HBSS 2% FCS and transfer the fluid to the 15 mL tube.
Centrifuge the tube at 370 x g for 7 min at 10°C and discard the supernatant avoiding the pellet.
Resuspend the pellet in 2 mL of potassium ammonium chloride pipet up and down to resuspend the pellet and lyse the erythrocytes.
Wait for 2 min and add HBSS 2% FCS to the resuspended pellet to obtain the volume up to 14 mL.
Centrifuge the tube again at 370 x g for 7 min at 10°C. Discard the supernatant and resuspend the pellet in 5 mL of HBSS 2% FCS by pipetting up and down.
Count the cells using trypan blue staining assay and adjust the final cell concentration to 10⁷ cells/mL using appropriate volume of HBSS 2% FCS.

4. Adoptive Transfer

Transfer 2 mL of cell suspension obtained in a 5 mL collection tube.
Centrifuge the tube at 370 x g for 7 min at 10°C and then discard the supernatant.
Resuspend the pellet in 2 mL of PBS and centrifuge the tube at 370 x g for 7 min at 10°C.
Discard supernatant and resuspend pellet in 200 µL of PBS.
Using a 0.5 mL syringe with a 29G needle inject 200 µL of cell suspension into the experimental mouse intravenously into the retro-orbital blood sinus.
As a control, inject a second mouse in the same blood sinus with 200 µL of phosphate buffered saline.

5. Cells Harvest and Staining

Four days after the adoptive transfer, euthanize mice and remove the spleen.
Harvest the splenocytes as described in section 3.
Transfer 100 µL of cell suspensions from each mouse into two FACS tubes, labeled "control" and "transferred".
Centrifuge the tube at 370 x g for 7 min at 10°C and then discard the supernatants.
Prepare a mix containing the four antibodies at the dilutions listed in Table 1.

Antibodies	Fluorochrome	Dilution
CD45.1	BV711	1/200
CD45.2	APCCy7	1/400
CD4	BV786	1/1600
CD3	BV421	1/200

Table 1: Antibodies mix composition. Four antibodies cocktails preparation using concentrated antibody-fluorescent conjugates and HBSS.

Add 100 µL of the mix to each tube, and then incubate for 20 min on ice in the dark.
Add 1 mL of HBSS 2%FCS and then centrifuge the tubes at 370 x g for 3 min at 10°C.
Discard the supernatants and resuspend the pellets in 200 µL of HBSS 2% FCS.
Transfer the resuspended cells to new, labelled FACS tubes.
Using flow cytometry, as shown in FACS protocol, evaluate the presence of CD45.2 positive lymphocytes.

6. Data Analysis

Open the "FlowJo" software and drag the files for each tube in "All sample" window.
Double click on the "transferred" file to display the cells recorded from that sample on a dot plot that displays forward scatter "SSC-A" on the Y axis and side scatter "FSC-A" on the X axis.
Click on "polygon" and create a gating strategy to select lymphocytes, then distinguish donor and host cells using cell surface markers (CD45.1, CD45.2), and then characterize CD45.2⁺ cell population (CD3, CD4).
Repeat the analysis steps with "control mouse" file.
To visualize a cell population, click on "Layout editor".
Drag the "transferred cells" and the "transferred CD4 cells" population from "transferred" and "control" files into the "Layout Editor tab".
A dot plot representing CD45.2⁺ cells and CD4 lymphocytes will appear.
CD45.2 transferred cells should only appear in the "transferred mouse" dot plot.

Skip to...

0:01

Concepts

2:26

Preparation of Materials

3:15

Dissection and Immune Cell Isolation

5:39

Adoptive Cell Transfer

6:35

Cell Harvest and Staining

8:04

Data Analysis and Results

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