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)

<ol>
	<li>Karpel, M. E., Boutwell, C. L., Allen, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BLT+humanized+mice+as+a+small+animal+model+of+HIV+infection.">BLT humanized mice as a small animal model of HIV infection.</a> Current opinion in virology. 13, 75-80 (2015).</li><li>Zhen, A., Kitchen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem-cell-based+gene+therapy+for+HIV+infection.">Stem-cell-based gene therapy for HIV infection.</a> Viruses. 6 (1), 1-12 (2014).</li><li>Goulder, P. J. R., Watkins, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+and+SIV+CTL+escape:+implications+for+vaccine+design.">HIV and SIV CTL escape: implications for vaccine design.</a> Nature Reviews: Immunology. 4 (8), 630-640 (2004).</li><li>Kitchen, S. G., Bennett, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+Antigen-Specific+T+Cells+from+Genetically+Modified+Human+Hematopoietic+Stem+Cells+in+Immunodeficient+Mice.">Engineering Antigen-Specific T Cells from Genetically Modified Human Hematopoietic Stem Cells in Immunodeficient Mice.</a> PloS one. 4 (12), e8208 (2009).</li><li>Goulder, P. J. R., Watkins, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+and+SIV+CTL+escape:+implications+for+vaccine+design.">HIV and SIV CTL escape: implications for vaccine design.</a> Nature Reviews: Immunology. 4 (8), 630-640 (2004).</li><li>Kitchen, S. G. S., Levin, B. R. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+suppression+of+HIV+by+antigen+specific+T+cells+derived+from+engineered+hematopoietic+stem+cells.">In vivo suppression of HIV by antigen specific T cells derived from engineered hematopoietic stem cells.</a> PLoS Pathogens. 8 (4), e1002649 (2012).</li><li>Yang, O. O., Tran, A. C., Kalams, S. A., Johnson, R. P., Roberts, M. R., Walker, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysis+of+HIV-1-infected+cells+and+inhibition+of+viral+replication+by+universal+receptor+T+cells.">Lysis of HIV-1-infected cells and inhibition of viral replication by universal receptor T cells.</a> PNAS. 94 (21), 11478-11483 (1997).</li><li>Zhen, A., Kamata, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-specific+Immunity+Derived+From+Chimeric+Antigen+Receptor-engineered+Stem+Cells.">HIV-specific Immunity Derived From Chimeric Antigen Receptor-engineered Stem Cells.</a> Molecular Therapy. 23 (8), 1358-1367 (2015).</li><li>Barrett, D. M., Singh, N., Porter, D. L., Grupp, S. A., June, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+Antigen+Receptor+Therapy+for+Cancer.">Chimeric Antigen Receptor Therapy for Cancer.</a> Annual Review of Medicine. 65 (1), 333-347 (2014).</li><li>Pejchal, R., Doores, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Potent+and+Broad+Neutralizing+Antibody+Recognizes+and+Penetrates+the+HIV+Glycan+Shield.">A Potent and Broad Neutralizing Antibody Recognizes and Penetrates the HIV Glycan Shield.</a> Science. 334 (6059), 1097-1103 (2011).</li><li>Caskey, M., Klein, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viraemia+suppressed+in+HIV-1-infected+humans+by+broadly+neutralizing+antibody+3BNC117.">Viraemia suppressed in HIV-1-infected humans by broadly neutralizing antibody 3BNC117.</a> Nature. 522 (7557), 487-491 (2015).</li><li>West, A. P., Scharf, L., Scheid, J. F., Klein, F., Bjorkman, P. J., Nussenzweig, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+insights+on+the+role+of+antibodies+in+HIV-1+vaccine+and+therapy.">Structural insights on the role of antibodies in HIV-1 vaccine and therapy.</a> Cell. 156 (4), 633-648 (2014).</li><li>Lan, P., Tonomura, N., Shimizu, A., Wang, S., Yang, Y. -. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+a+functional+human+immune+system+in+immunodeficient+mice+through+combined+human+fetal+thymus/liver+and+CD34++cell+transplantation.">Reconstitution of a functional human immune system in immunodeficient mice through combined human fetal thymus/liver and CD34+ cell transplantation.</a> Blood. 108 (2), 487-492 (2006).</li><li>Melkus, M. W., Estes, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+mice+mount+specific+adaptive+and+innate+immune+responses+to+EBV+and+TSST-1.">Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1.</a> Nature medicine. 12 (11), 1316-1322 (2006).</li><li>Shultz, L. D., Brehm, M. A., Garcia-Martinez, J. V., Greiner, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+mice+for+immune+system+investigation:+progress,+promise+and+challenges.">Humanized mice for immune system investigation: progress, promise and challenges.</a> Nature Reviews: Immunology. 12 (11), 786-798 (2012).</li><li>Vatakis, D. N., Bristol, G. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+BLT+humanized+mouse+as+a+stem+cell+based+gene+therapy+tumor+model.">Using the BLT humanized mouse as a stem cell based gene therapy tumor model.</a> Journal of visualized experiments : JoVE. (70), e4181 (2012).</li><li>De Rosa, S. C., Brenchley, J. M., Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+six+colors:+a+new+era+in+flow+cytometry.">Beyond six colors: a new era in flow cytometry.</a> Nature medicine. 9 (1), 112-117 (2003).</li><li>Shimizu, S., Hong, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+efficient+short+hairpin+RNA+potently+down-regulates+CCR5+expression+in+systemic+lymphoid+organs+in+the+hu-BLT+mouse+model.">A highly efficient short hairpin RNA potently down-regulates CCR5 expression in systemic lymphoid organs in the hu-BLT mouse model.</a> Blood. 115 (8), 1534-1544 (2010).</li><li>Denton, P. W., Olesen, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+HIV+latency+in+humanized+BLT+mice.">Generation of HIV latency in humanized BLT mice.</a> Journal of virology. 86 (1), 630-634 (2012).</li><li>Zhou, J., Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryoid+bodies+formation+and+differentiation+from+mouse+embryonic+stem+cells+in+collagen/Matrigel+scaffolds.">Embryoid bodies formation and differentiation from mouse embryonic stem cells in collagen/Matrigel scaffolds.</a> Journal of Genetics and Genomics. 37 (7), 451-460 (2010).</li><li>Vatakis, D. N., Arumugam, B., Kim, S. G., Bristol, G., Yang, O., Zack, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+Exogenous+T-cell+Receptors+Into+Human+Hematopoietic+Progenitors+Results+in+Exclusion+of+Endogenous+T-cell+Receptor+Expression.">Introduction of Exogenous T-cell Receptors Into Human Hematopoietic Progenitors Results in Exclusion of Endogenous T-cell Receptor Expression.</a> Molecular Therapy. 21 (5), 1055-1063 (2013).</li><li>Ito, R., Takahashi, T., Katano, I., Ito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+advances+in+humanized+mouse+models.">Current advances in humanized mouse models.</a> Cellular &amp; Molecular Immunology. 9 (3), 208-214 (2012).</li><li>Martinez-Torres, F., Nochi, T., Wahl, A., Garcia, J. V., Denton, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypogammaglobulinemia+in+BLT+humanized+mice--an+animal+model+of+primary+antibody+deficiency.">Hypogammaglobulinemia in BLT humanized mice--an animal model of primary antibody deficiency.</a> PloS one. 9 (10), e108663 (2014).</li><li>McCune, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+applications+of+the+SCID-hu+mouse+model.">Development and applications of the SCID-hu mouse model.</a> Seminars in Immunology. 8 (4), 187-196 (1996).</li><li>Srivastava, S., Riddell, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+CAR-T+Cells:+Design+Concepts.">Engineering CAR-T Cells: Design Concepts.</a> Trends in immunology. 36 (8), (2015).</li></ol>

The authors have nothing to disclose.

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><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&amp;nbsp;</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&amp;trade;</td><td>BD biosciences</td><td></td><td>For flow analysis</td></tr><tr><td>Hyaluronidase</td><td>Sigma</td><td>H6254-500MG&amp;nbsp;</td><td>For tissue digestion</td></tr><tr><td>Deoxyribonuclease I&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Worthington</td><td>LS002006&amp;nbsp;</td><td>for tissue digestion</td></tr><tr><td>Collagenase</td><td>Life technology</td><td>17104-019&amp;nbsp;</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 - 1,000 units</td><td>Becton Dickinson</td><td>427631&amp;nbsp;</td><td>For surgery</td></tr><tr><td>Sterile Poly-Reinforced Aurora Surgical Gowns, 30 per case&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Medline</td><td>DYNJP2707&amp;nbsp;</td><td>For surgery</td></tr><tr><td>Sutures, 4-0, vicryl&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Owens and Minor</td><td>23000J304H&amp;nbsp;&amp;nbsp;</td><td>For surgery</td></tr><tr><td>Alcohol prep pads&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</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&amp;rsquo;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&amp;nbsp;</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 ...

stem-cell-based-engineered-immunity-against-hiv-infection-humanized

Research

JoVE Journal

Medicine

10.1K Views.  University of California, Los Angeles. This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection. 

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

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

Immunology and Infection

Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine cancer models are extremely limited in their diversity and suffer from poor translation to the clinic. To serve as a more physiological preclinical model for immunotherapy studies, this protocol has been developed to evaluate the treatment of human tumors in a mouse reconstituted with a human immune system. This unique protocol demonstrates the development of human immune system (HIS, &#34;humanized&#34;) mice, followed by implantation of a human tumor, either a cell-line derived xenograft (CDX) or a patient derived xenograft (PDX). HIS mice are generated by injecting CD34+ human hematopoietic stem cells isolated from umbilical cord blood into neonatal BRGS (BALB/c Rag2-/- IL2R&#947;C-/- NODSIRP&#945;) highly immunodeficient mice that are also capable of accepting a xenogeneic tumor. The importance of the kinetics and characteristics of the human immune system development and tumor implantation is emphasized. Finally, an in-depth evaluation of the tumor microenvironment using flow cytometry is described. In numerous studies using this protocol, it was found that the tumor microenvironment of individual tumors is recapitulated in HIS-PDX mice; &#34;hot&#34; tumors exhibit large immune infiltration while &#34;cold&#34; tumors do not. This model serves as a testing ground for combination immunotherapies for a wide range of human tumors and represents an important tool in the quest for personalized medicine.

This protocol outlines the generation of human immune system (HIS) mice for immuno-oncology studies. Instructions and considerations in the use of this model for testing human immunotherapeutics on human tumors implanted in this model are presented with an emphasis on characterizing the response of the human immune system to the tumor.

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine ...

Cancer Research

Humanized NOD/SCID/IL2r&#947;null (hu-NSG) Mouse Model for HIV Replication and Latency Studies

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in high demand. With a close genetic and behavioral resemblance to humans, small animals such as the mouse are good candidates for human disease models, through which human-like symptoms and responses can be recapitulated. Further, the mouse genetic background can be altered to accommodate diverse demands. The NOD/SCID/IL2r&#947;null (NSG) mouse is one of the most widely used immunocompromised mouse strains; it allows engraftment with human hematopoietic stem cells and/or human tissues and the subsequent development of a functional human immune system. This is a critical milestone in understanding the prognosis and pathophysiology of human-specific diseases such as HIV/AIDS and aiding the search for a cure. Herein, we report a detailed protocol for generating a humanized NSG mouse model (hu-NSG) by hematopoietic stem cell transplantation into a radiation-conditioned neonatal NSG mouse. The hu-NSG mouse model shows multi-lineage development of transplanted human stem cells and susceptibility to HIV-1 viral infection. It also recapitulates key biological characteristics in response to combinatorial antiretroviral therapy (cART).

This protocol provides a method to establish humanized mice (hu-NSG) via intrahepatic injection of human hematopoietic stem cells into radiation-conditioned neonatal NSG mice. The hu-NSG mouse is susceptible to HIV infection and combinatorial antiretroviral therapy (cART) and serves as a suitable pathophysiological model for HIV replication and latency investigations.

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in ...

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...

Chronic, Acute, and Reactivated HIV Infection in Humanized Immunodeficient Mouse Models

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical research on infectious diseases. Described here are three models of humanized immunodeficient mice for studying the dynamics of HIV infection. The first is based on the intrahepatic injection of CD34+ hematopoietic stem cells in newborn mice, which allows for the reconstitution of several blood and lymphoid tissue-confined cells, followed by infection with a reference HIV strain. This model allows monitoring for up to 36 weeks post-infection and is hence called the chronic model. The second and third models are referred to as the acute and reactivation models, in which peripheral blood mononuclear cells are intraperitoneally injected in adult mice. In the acute model, cells from a healthy donor are engrafted through the intraperitoneal route, followed by infection with a reference HIV strain. Finally, in the reactivation model, cells from an HIV-infected donor under antiretroviral therapy are engrafted via the intraperitoneal route. In this case, a drug-free environment in the mouse allows for virus reactivation and an increase in viral load. The protocols provided here describe the conventional experimental approach for humanized, immunodeficient mouse models of HIV infection.

Described here are three experimental approaches for studying the dynamics of HIV infection in humanized mice. The first permits the study of chronic infection events, whereas the two latter allows for the study of acute events after primary infection or viral reactivation.

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical ...

Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes the establishment of a human immune system in adult NOG mice. Here, we explain all the practical steps from isolation of umbilical cord blood derived human CD34+ cells and their subsequent intravenous transplantation into the mice, to the manipulation of the model through HIV infection, combination antiretroviral therapy (cART), and blood sampling. Approximately 75,000 hCD34+ cells are injected intravenously into the mice and the level of human chimerism, also known as humanization, in the peripheral blood is estimated longitudinally for months by flow cytometry. A total of 75,000 hCD34+ cells yields 20%&#8211;50% human CD45+ cells in the peripheral blood. The mice are susceptible to intravaginal infection with HIV and blood can be sampled once weekly for analysis, and twice monthly for extended periods. This protocol describes an assay for quantification of plasma viral load using droplet digital PCR (ddPCR). We show how the mice can be effectively treated with a standard-of-care cART regimen in the diet. The delivery of cART in the form of regular mouse chow is a significant refinement of the experimental model. This model can be used for preclinical analysis of both systemic and topical pre-exposure prophylaxis compounds as well as for testing of novel treatments and HIV cure strategies.

We have developed a protocol for the generation and evaluation of a humanized and human immunodeficiency virus-infected NOG mouse model based on stem cell transplant, intravaginal human immunodeficiency virus exposure, and droplet digital PCR RNA quantification.

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes ...

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation of human systems, thereby allowing for a more accurate study of pathogenesis, biomarkers, and many other scientific queries. In this method described, immune-deficient NOD-scid IL2R&#947;null (NSG) mice are implanted with autologous thymus, injected with liver-derived CD34+ cells followed by a series of injected cytokine deliveries. In contrast to other models of a similar nature, the model described here promotes an improved reconstitution of immune cells by delivering cytokines and growth factors via transgenes encoded in AAV8 or pMV101 DNA-based vectors. Moreover, it offers long-term stability with reconstituted mice having an average lifespan of 30 weeks after CD34+ injections. Through this model, we hope to provide a stable and impactful method of studying immunotherapy and human disease in a murine model, thus demonstrating the need for predictive preclinical models.

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Humanized mouse models provide a more accurate representation of the human immune microenvironment. This manuscript describes the process in which these models are created through a renal graft of human thymus, injection of human CD34+ cells, and the targeted delivery of human cytokine transgenes to promote CD34+ cell proliferation and differentiation.

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation ...

Oral Combinational Antiretroviral Treatment in HIV-1 Infected Humanized Mice

The human immunodeficiency virus (HIV-1) pandemic continues to spread unabated worldwide, and currently, there is no vaccine available against HIV. Although combinational antiretroviral therapy (cART) has been successful in suppressing viral replication, it cannot completely eradicate the reservoir from HIV-infected individuals. A safe and effective cure strategy for HIV infection will require multipronged methods, and therefore the advancements of animal models for HIV-1 infection are pivotal for the development of HIV cure research. Humanized mice recapitulate key features of HIV-1 infection. The humanized mouse model can be infected by HIV-1 and viral replication can be controlled with cART regimens. Moreover, cART interruption results in a prompt viral rebound in humanized mice. However, administration of cART to the animal can be ineffective, difficult, or toxic, and many clinically relevant cART regimens are unable to be optimally utilized. Along with being potentially unsafe for researchers, administration of cART by a commonly used intensive daily injection procedure induces stress by physical restraint of the animal. The novel oral cART method to treat HIV-1 infected humanized mice described in this article resulted in suppression of viremia below the detection level, increased rate of CD4+ restoration, and improved overall health in HIV-1 infected humanized mice.

This protocol describes a novel method to deliver oral combinational antiretroviral drugs that successfully suppress HIV-1 RNA replication in humanized mice.

The human immunodeficiency virus (HIV-1) pandemic continues to spread unabated worldwide, and currently, there is no vaccine available against HIV. ...

Isolating Cord Blood Hematopoietic Stem Cells to Generate Human Immune System Mice

Source: Lanis, J. M., et al. <a href="https://app.jove.com/v/64606">Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors.</a> J. Vis. Exp. (2022)
This video demonstrates a technique for generating human immune system (HIS) mice. CD34+ hematopoietic stem cells (HSCs) are isolated from human cord blood buffy coat cells using antibody-coated magnetic beads. The CD34+ HSCs are expanded in a growth medium enriched with cytokines and cryopreserved. Finally, the thawed HSCs are injected into an immunodeficient mouse pup to establish the HIS mouse model.

Izolowanie hematopoetycznych komórek macierzystych krwi pępowinowej w celu wytworzenia myszy z ludzkim układem odpornościowym

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines. All procedures involving animal ...

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Model Development and Disease Study

Developing a Humanized Mouse Model with Human Liver Cells and Immune Cells

Source: Dagur, R. S. et al., <a href="https://app.jove.com/v/58645">Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis.</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates a method for generating a dual humanized mouse model with a human immune system and liver cells. It involves transplanting human liver cells and hematopoietic stem and progenitor cells into a transgenic immunodeficient mouse via intrasplenic injection, leading to engraftment of liver cells and development of human immune cells.

Opracowanie humanizowanego modelu myszy z ludzkimi komórkami wątroby i komórkami odpornościowymi

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

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

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo