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 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 (cγ-/-)-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.
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 (cγ-/-) (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 'redirecting' 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 ζ signaling domain of CD3 (termed the CD4ζCAR). CD4ζCAR 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 CD4ζCAR, 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 CD4ζCAR, 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 CD4ζCAR; and 2) characterization of the differentiation of the genetically modified cells; and 3) characterization of the functionality of the genetically modified cells.
Ethic Statement: 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 the guidelines in The Guide for the Care and Use of Laboratory Animals of the National Research Council and the accreditation and guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care (AALAC) International under UCLA ARC Protocol Number 2010-038-02B. All surgeries were performed under ketamine/xylazine and isoflurane anesthesia and all efforts were made to minimize animal pain and discomfort.
1. Construction of Humanized Mice Engineered with CD4 Chimeric Antigen Receptor
2. Characterization of Differentiation and Development of Gene Modified Cells
3. Functional Characterization of Gene Modified Cells
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 CD4ζCAR. The CD4ζCAR used in this protocol contains CD4 chimeric antigen receptor and GFP that can be detected by anti-CD4 antibody and the expression of GFP8. Cells were isolated and stained with antibody against human CD45 as well as anti-CD4 antibody and analyzed by flow cytometry. GFP and CD4 double positive cells were detected, indicating the presence of CD4CAR+ cells in multiple lymphoid tissues.
To investigate the differentiation of the gene modified cells, splenocytes were stained with antibodies against human CD45 (lymphocyte), CD3 (T cells), CD19 (B cells), CD14 (monocyte and macrophages) and CD337 (NK cells). As shown in Figure 3, CD4ζCAR hematopoietic stem cells differentiate into multiple lineages.
To investigate if CD4CAR modified cells are functional, we coincubated splenocytes with target cells that CD4CAR cells would recognize (HIV infected T1 cells or uninfected T1 cells as control). Cells were co-incubated overnight and the protein transport inhibitor was added for an additional 6 hours. Afterwards, cells were fixed and permeabilized to stain for intracellular expression of cytokines such as IFNΓ and TNFα. As shown in Figure 4, CAR expressing cells produced higher amount of IFNΓ and TNFα with infected T1 cells.
Figure 1: Outline of construction of humanized BLT mice with modified stem cells. FT: Fetal thymus. FL: Fetal liver. Please click here to view a larger version of this figure.
Figure 2: CD4 chimeric antigen receptor modified cells can be detected in multiple lymphoid tissues. Mouse with CD4CAR modified HSCs were sacrificed 10 weeks after surgery and multiple lymphoid tissues were harvested and the cells were stained with anti-human CD45 and anti-human CD4 antibodies and analyzed by flow cytometry. Please click here to view a larger version of this figure.
Figure 3: CD4 chimeric antigen receptor modified cells can differentiate into multiple lineages. Splenocytes from CD4CAR modified mice were harvested and stained with antibodies against human CD45, CD3, CD19, CD14 and CD337 and analyzed by flow cytometry. Please click here to view a larger version of this figure.
Figure 4: Ex vivo cytokine assay of CD4 CAR+ T cells. Splenocytes from HIV infected CD4CAR mice were stimulated with either HIV infected or uninfected T1 cells and their intracellular production of cytokine is shown. Please click here to view a larger version of this figure.
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, low levels of stem cell modification were sufficient to generate a robust response against HIV replication8.
Nonetheless, to achieve high level of stem cell modification, we recommend using transduced CD34+ cells in gelatinous protein mixture with autologous thymus instead of liver and thymus chunks for mice surgery that had been described elsewhere13-15. Gelatinous protein mixture is a solubilized tissue basement membrane rich in extracellular matrix proteins. Under normal physiological conditions, gelatinous protein mixture polymerize to produce a reconstituted, biologically active and stable matrix that would allow effective attachment and differentiation of the stem cell20. Human cell reconstitution can be checked by retro-orbital bleeding and flow cytometry 6 – 8 weeks post-surgery. To ensure that the vector is not toxic for stem cell survival and renewal, it is recommended to titer the vector in CD34+ cells prior to the experiment as described in 1.2.6.
The capacity of the NSG-BLT mouse model to support mucosal infection, consistent viremia and cellular immune responses makes it a highly useful model to study HIV immune pathology and cell-based therapies to treat HIV infection1. Most importantly, T cells generated from the NSG-BLT mice are selected in the autologous thymic tissue, allowing the researcher to study the fate of gene modified stem cells after thymic selection8,21. With the described method, we have been able to get 40%-90% human immune cell reconstitution consistently. Low levels of human cell reconstitution can result from multiple factors, including the skills of the person performing the surgery and the quality of tissues for transplant. To achieve high level of human cell reconstitution, it is important to ensure the transplants are securely placed underneath the kidney capsule. In addition, it is highly recommended to examine each thymic implant prepared for surgery under light microscope and discard any questionable pieces.
Although the humanized BLT mouse model is a promising tool for studying engineered immunity against HIV (reviewed in 1,15,22), it has its own limitations. Namely, this model does not perfectly mimic a human peripheral immune system. Studies have shown impaired development of hyper-mutated, class-switched IgG antibody1,23. Additionally, using immune-deficient mice is technically challenging and keeping these mice healthy requires considerable resources and training. Subtle opportunistic infections can manifest as significant differences across samples and potentially have negative consequences on the experimentation. Therefore it is important to have well prepared facilities and appropriately trained staff to maintain integrity of the future data1,24. With these limitations in mind, the humanized NSG-BLT mouse model still provides an important tool for the study of stem cell based engineered immunity, as is demonstrated by these examples4,8.
With the trend of developing chimeric antigen receptors based on HIV broad neutralizing antibodies10 and modification of the signaling domain for more efficient CAR25, this model and protocol can be used to characterize and investigate the functionality of the gene modified cells with a new generation of CARs. In addition, this model can potentially accommodate studies on immune based therapy (such as inhibitory receptor blockade) in conjunction with engineered immunity.
The authors have nothing to disclose.
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)
CD34 microbead kit | miltenyi | 130-046-702 | For sorting human CD34+ progenitor cells |
Bambanker | Wako | 302-14681 | for freezing cells |
QIAamp Viral RNA kit | Qiagen | 52904 | For measuring viral load in the serum |
MACSQuant Flow Cytometer | Miltenyi | For flow analysis | |
BD LSRFortessa™ | BD biosciences | For flow analysis | |
Hyaluronidase | Sigma | H6254-500MG | For tissue digestion |
Deoxyribonuclease I | Worthington | LS002006 | for tissue digestion |
Collagenase | Life technology | 17104-019 | for tissue digestion |
CFX Real time PCR detection system | Biorad | For measuring viral load and gene expression | |
Mice, strain NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ | The Jackson Laboratory | 5557 | For constructing the humanized mice |
Penicillin Streptomycin (Pen Strep) | Thermo Fisher Scientific | 10378016 | For culturing cells |
piperacillin/tazobactam | Pfizer | Zosyn | Anti-fungal |
Amphotericin B (Fungizone antimycotic) | Thermo Fisher Scientific | 15290-018 | Anti-fungal |
AUTOCLIP Wound Clips, 9 mm – 1000 units | Becton Dickinson | 427631 | For surgery |
Sterile Poly-Reinforced Aurora Surgical Gowns, 30 per case | Medline | DYNJP2707 | For surgery |
sutures, 4-0, vicryl | Owens and Minor | 23000J304H | For surgery |
Alcohol prep pads | Owens and Minor | 3583006818 | For surgery |
Gloves, surgical, 6 1/2 | Owens and Minor | 4075711102 | For surgery |
Yssel’s Serum-Free T-Cell Medium | Gemini Bio-products | 400-102 | For CD34+ cell transduction |
Human Serum Albumin | Sigma-Aldrich | A9511 | For CD34+ cell transduction |