We would like to thank Ms. Jessica Selander in providing artistic assistant in making our figures. This work was funded by grants from the NIAID/NIH, grant no. RO1AI078806, the UCLA Center for AIDS Research (CFAR), grant no. P30AI28697, the California Institute for Regenerative Medicine, grant no. TR4-06845, the American Federation for AIDS Research (amfAR), grant no. #108929-54-RGRL, and the UC Multi-campus Research Program and Initiatives, California Center for Antiviral Drug discovery (CCADD)

Ethic Statement:&#160;Human fetal tissue was obtained from Advanced Biosciences Resources or from Novogenix and was obtained without identifying information and did not require IRB approval for its use. Animal research described in this manuscript was performed under the written approval of the University of California, Los Angeles, and (UCLA) Animal Research Committee (ARC) in accordance to all federal, state, and local guidelines. Specifically, these studies were carried out under strict accordance to ...

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With CAR and HSC-based engineered immunity gaining momentum towards clinical studies, it is important to have a proper animal model to closely examine the differentiation and function of these engineered cells. In this protocol we described the methods for constructing and testing humanized mice with genetically modified stem-cells engineered against HIV. It is important to have efficient transduction of stem cells prior to transplant. However, due to the ability of T cell to proliferate upon recognition of target cells,...

Despite the success of combined anti-retroviral therapy, HIV infection is still a lifelong disease. The cellular immune response against HIV plays highly important role in controlling HIV replication. Recent advances in stem cell manipulation has allowed for the rapid development of gene therapy approaches for HIV treatment1-3. As a result, it is important to have a proper animal model that allows in vivo study of the efficacy of cell-based therapies against HIV.
Working with HIV in animal models is complicated by the fact that the virus only infects human cells. To circumvent this limitation, scientists have resorted to using disease models like the Simian Immunodeficiency Virus (SIV) in Rhesus macaques4,5. Unfortunately, there are major limitations in this model due to the inherent differences across species and the differences between SIV and HIV. Additionally, only highly specialized facilities are capable of supporting work with non-human primates and each macaque requires a large investment. Thus, there is a pressing need for a model that utilizes the human immune system, which is susceptible to HIV infection/pathogenesis, and is less financially prohibitive.
The non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-) (or NSG) Blood/Liver/Thymus (BLT) humanized mouse model is increasingly proven to be an important tool to study HIV infection. By implanting hematopoietic stem cells (HSCs) and fetal thymus, the mice are able to develop and recapitulate a human immune system1-3. One type of stem cell based gene therapy involves &#39;redirecting&#39; peripheral T cells to target HIV by reprogramming Hematopoietic Stem Cells (HSCs) to differentiate into antigen specific T cells. We have shown previously that engineering HSCs with a molecular cloned anti-HIV specific T cell receptor (TCR) against the SL9 epitope (amino acid 77-85; SLYNTVATL) of HIV-1 Gag can redirect stem cells into forming mature T cells that suppress HIV replication in the humanized NSG-BLT mouse model6. The caveat of using a molecular cloned TCR is that it is restricted to a specific human leukocyte antigen (HLA) subtype that will limit the application of this therapy. Chimeric antigen receptors (CAR), on the other hand, can be universally applied to all HLA subtypes. Initial studies were performed utilizing a CAR constructed with the extracellular and transmembrane domains of human CD4 fused to the intracellular &#950; signaling domain of CD3 (termed the CD4&#950;CAR). CD4&#950;CAR expressed on CD8 T cells can recognize HIV envelope and trigger a cytotoxic T cell response that is similar to that mediated by a T cell receptor7. We have recently demonstrated that human HSCs can be modified with CD4&#950;CAR, which can then differentiate into multiple hematopoietic lineages, including functional T cells capable of suppressing HIV replication in the humanized mouse model8. With the rapid advancement in chimeric antigen receptor therapies for cancer9, and the ongoing characterization of potent broad neutralizing antibodies10-12 against HIV that allow the construction of single chain antibody CARs, it is perceivable that many new candidate constructs, in addition to CD4&#950;CAR, will be generated and tested for stem-cell based gene therapy of HIV diseases and other diseases. In addition, the humanized NSG-BLT mouse model containing these antigen-specific CARs can also provide a useful tool to closely examine human T cell responses in vivo. Importantly, our protocol differs from previous described methods for construction of humanized of BLT mice13-15 in that the HSCs in gelatinous protein mixture is used in place of fetal liver trunks16. This protocol describes: 1) construction of humanized BLT mice engineered with CD4&#950;CAR; and 2) characterization of the differentiation of the genetically modified cells; and 3) characterization of the functionality of the genetically modified cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CD34 microbead kit</td>
			<td>miltenyi</td>
			<td>130-046-702</td>
			<td>For sorting human CD34+ progenitor cells</td>
		</tr>
		<tr>
			<td>Bambanker</td>
			<td>Wako</td>
			<td>302-14681</td>
			<td>for freezing cells</td>
		</tr>
		<tr>
			<td>QIAamp Viral RNA kit&#160;</td>
			<td>Qiagen</td>
			<td>52904</td>
			<td>For measuring viral load in the serum</td>
		</tr>
		<tr>
			<td>MACSQuant Flow Cytometer</td>
			<td>Miltenyi</td>
			<td></td>
			<td>For flow analysis</td>
		</tr>
		<tr>
			<td>BD LSRFortessa&#8482;</td>
			<td>BD biosciences</td>
			<td></td>
			<td>For flow analysis</td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma</td>
			<td>H6254-500MG&#160;</td>
			<td>For tissue digestion</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Worthington</td>
			<td>LS002006&#160;</td>
			<td>for tissue digestion</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Life technology</td>
			<td>17104-019&#160;</td>
			<td>for tissue digestion</td>
		</tr>
		<tr>
			<td>CFX Real time PCR detection system</td>
			<td>Biorad</td>
			<td></td>
			<td>For measuring viral load and gene expression</td>
		</tr>
		<tr>
			<td>Mice, strain NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ</td>
			<td>The Jackson Laboratory</td>
			<td>5557</td>
			<td>For constructing the humanized mice</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin (Pen Strep)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10378016</td>
			<td>For culturing cells</td>
		</tr>
		<tr>
			<td>piperacillin/tazobactam</td>
			<td>Pfizer</td>
			<td>Zosyn</td>
			<td>Anti-fungal</td>
		</tr>
		<tr>
			<td>Amphotericin B (Fungizone antimycotic)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15290-018</td>
			<td>Anti-fungal</td>
		</tr>
		<tr>
			<td>AUTOCLIP Wound Clips, 9 mm - 1000 units&#160;&#160;&#160;&#160;</td>
			<td>Becton Dickinson</td>
			<td>427631&#160;</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>Sterile Poly-Reinforced Aurora Surgical Gowns, 30 per case&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Medline</td>
			<td>DYNJP2707&#160;</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>sutures, 4-0, vicryl&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Owens and Minor</td>
			<td>23000J304H&#160;&#160;</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>Alcohol prep pads&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Owens and Minor</td>
			<td>3583006818</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>Gloves, surgical, 6 1/2</td>
			<td>Owens and Minor</td>
			<td>4075711102</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>Yssel&#8217;s Serum-Free T-Cell Medium</td>
			<td>Gemini Bio-products</td>
			<td>400-102</td>
			<td>For CD34+ cell transduction</td>
		</tr>
		<tr>
			<td>Human Serum Albumin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A9511</td>
			<td>For CD34+ cell transduction</td>
		</tr>
	</tbody>
</table>

stem-cell based engineered immunity against hiv infection in the humanized mouse model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

Figure 1 shows an outline of constructing humanized BLT mice with modified stem cell. 10 weeks after the implant surgery, the mice were sacrificed to evaluate the differentiation and development of gene modified cells. As shown in Figure 2, multiple lymphoid tissues (blood, spleen, thymus and bone marrow) were harvested from a mouse that was modified with CD4&#950;CAR. The CD4&#950;CAR used in this protocol contains CD4 chimeric antigen receptor and GFP t...

Watch this Scientific Journal Video about Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model at JoVE.com

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

The overall goal of this procedure is to create humanized mouse modified with chimeric antigen receptor. This method can help answer key questions in the HIV and immunology fields in regards to testing the efficacy of stem-cell based engineered immunity against HIV infection and other chronic infections and malignancies. Such as a mechanism of immune failure and ways to enhance the immune system and develop a therapy to create better anti-viral or anti-tumor immunity.The main advantage of this technique is that it is capable of a large amount of experimental manipulations. The humanized BLT mice model mimics human immunity and in this case allows for pre-clinical testings for cell based gene therapies against HIV infection. Demonstrating the procedure will be Valerie Rezek and Brianna Lam, they are technicians form my laboratory.Begin by using scalpels to cut the thymus into small pieces of about one millimeter cubed in a dish containing supplemented RPMI medium. Then used curved blunt forceps to place each single thymus piece into a single well on a 96 well plate. Add 100-200 microliters of media to all of the wells so that the tissue does not dry.Visualize the tissue under the microscope to eliminate any tissue that may not be thymus or that has connective tissue attached. Next, remove the confirmed pieces of thymus, combine them into a T25 cell culture flask. Add seven milliliters of antibiotic supplemented RPMI media to the flask and gently rock to mix.Then place the flask in the tissue culture incubator. This step prevents bacterial contamination of the tissue. Begin the isolation of CD34 positive HSCs from fetal liver by using two scalpels to cut the liver into small pieces of about three millimeters cubed in a dish containing IMDM.Remove and discard any white connective tissue. Then use a 10 milliliter syringe fitted with a 16 gauge blunt needle to take out the liver pieces and media and transfer to a 50 milliliter conical tube. Gently take up and expel five to seven more times to completely homogenize the tissue.After preparing 10 milliliters of IMDM media, supplement it with collagenase, hyaluronidase, antibiotics, Dnase, and anti fungal. Pass the media through a 0.22 micron filter. Then add the filtered media to the liver suspension.After capping the 50 milliliter conical tube containing the liver suspension, seal tightly with self sealing film such as parafilm to prevent leaks. Rotate in a tube rotator in the 37 degrees celsius incubator for 90 minutes. Following the incubation, filter the digested cell suspension through a 100 micron cell strainer into a fresh 50 milliliter tube.Add PBS to the suspension to bring the total volume up to 50 milliliters. Split this into two 50 milliliter tubes, each containing 25 milliliters of cell suspension. Slowing and gently underlay the cells in each tube with 10 milliliters of a density centrifugation media, such as Ficoll.Spin at 1200 times g for 20 minutes without break. Following the centrifugation, carefully remove the interface from each tube and transfer it into two separate 50 milliliter tubes. Bring the volume of each tube of interface up to 50 milliliters with PBS.And then centrifuge at 300 times g for seven to 10 minutes. Combine and wash the cell pellets three times with 50 milliliters of PBS containing two percent FBS, spinning at 300 times g for seven to 10 minutes each time. Following the final spin, re-suspend the pellet in 50 milliliters of RPMI media plus 10 percent FBS.Count the cells using a hemocytometer. Next, sort CD34 positive cells immediately using a CD34 sorting kit according to the manufacturer's protocol, and save both CD34 positive and CD34 negative fraction. Pour the thymus pieces and medium from the flask into a 60 millimeter dish.Chill some positive displacement pipette tips by putting them in open 1.5 milliliter sterile screw-cap tubes on ice. Also, place the cell pellets and a volume of gelatinous protein mixture such as matrigel on ice. Anesthetize the recipient mice with an injectible anesthetic according to an approved animal protocol.Check the anesthesia level of the mouse by squeezing a paw. The absence of a reaction to the pinch indicates a surgical plane of anesthesia. Shave the left side of each mouse from hip to shoulder between the center of the back and the stomach.Subcutaneously inject six milligrams per kilogram of diluted Carprofen into the animal's shoulder or inguinal triangle. Flush the cannula of the trocar with PBS in a 60 millimeter dish. Using a pair of blunt curved forceps, place a piece of thymus from the 60 millimeter dish just inside the opening of the cannula, then pull back on the trocar to aspirate the tissue into the cannula.Next, use a positive displacement pipette and a chilled tip to add five microliters of cold gelatinous protein mixture into the tube with the cell pellet. And gently stir to generate the cell suspension. Pipette the cell suspension into the opening of the cannula and slowly pull back on the trocar to load the needle.Swab the shaved area of the mouse with povidone iodine followed by isopropenyl three times. Determine the darkest spot under the skin which indicates the location of the spleen. The kidney is approximately five millimeters dorsal to the spleen.After ensuring a suitable level of anesthesia by the absence of a reaction of the paw pinch, use curved forceps to lift the skin and use surgical scissors to make a 15 millimeter incision in the skin parallel to the spleen. Then make a similar cut in the peritoneum layer below. In males, the kidney is easily visible, thus extruded simply by pressing on the abdomen.Support the kidney with a hemostat or a pair of curved blunt forceps. In females, the ovaries tend to block the kidney from easy extraction. Using a hemostat, pick up the ovary and carefully expose the kidney.Use needle-tipped forceps to pluck a tiny hole at the posterior end of the kidney capsule. Slide the trocar into this hole and along the kidney until the opening of the cannula is completely covered by the kidney capsule. Gently extrude the tissue under the kidney capsule and pull the trocar back out.Use the curved forceps to make sure the thymus piece does not come back out with the needle. Next, lift up the peritoneum with the forceps and gently use the hemostat to push the kidney back into place. Tie one double knotted stitch with surgical suture in the peritoneum.Also place two Autoclips Wound clips to close the skin. Now mix the transduced CD34 positive cells and aspirate 0.5 times 10 to the six cells in a 100 microliter volume into an insulin syringe. Inject these cells into the mouse through retro orbital vein injection.After injection, swab a small drop of eye lubricant onto each eye, and lay the mouse on its side back in a cage. After implanting all the mice, confirm that the animals have regained conscientiousness and are ambulatory before returning them to the housing room. Mice were sacrificed 10 weeks after surgery and blood, spleen, thymus and bone marrow were harvested.The resulting cells were stained with anti-human CD45 and anti-human CD4 antibodies and analyzed by flow cytometry. As seen here, CD4 chimeric antigen receptor modified cells can be detected in multiple lymphoid tissues by flow cytometry. The CD4 chimeric antigen receptor modified cells can differentiate into multiple lineages.Here, splenocytes from CD4 chimeric antigen receptor modified mice were harvested and stained for markers of T and B cells. Cells negative for T and B cell markers were positive for markers of monocytes and macrophages and for natural killer cells. Splenocytes from HIV infected CD4 chimeric antigen receptor mice were stimulated with either HIV infected or uninfected T one cells and their intracellular production of cytokine is shown.After watching this video, you should have a good understanding of how to construct humanized BLT mice modified with chimeric antigen receptors. Once mastered, this technique can be done in 48 hours if it is performed properly. While attempting this procedure, it is important to remember to work at a steady pace and pay close attention to the state of the mouse being operated on.For beginners it is important to practice surgery and eye injections before working with tissues and stem cells that are more precious. After its development, this technique paved the way for researchers in the field of HIV and other infectious diseases to explore disease pathogenesis and immune based therapies in the humanized mouse model.

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Research

JoVE Journal

Medicine

10.0K visualizações.  University of California, Los Angeles. The overall goal of this procedure is to create humanized mouse modified with chimeric antigen receptor. This method can help answer key questions in the HIV and immunology fields in regards to testing the efficacy of stem-cell based engineered immunity against HIV infection and other chronic infections and malignancies. Such as a mechanism of immune failure and ways to enhance the immune system and develop a therapy to create better anti-viral or anti-tumor immunity.The main advantage...