Mayra A. Carrillo
Anjie Zhen- Department of Medicine, Division of Hematology and Oncology, University of California Los Angeles, Los Angeles, CA, United States
HIV and cancer remain prevailing sources of morbidity and mortality worldwide. There are current efforts to discover novel therapeutic strategies for the treatment or cure of these diseases. Humanized mouse models provide the investigative tool to study the interaction between HIV or cancer and the human immune system in vivo. These humanized models consist of immunodeficient mice transplanted with human cells, tissues, or hematopoietic stem cells that result in reconstitution with a nearly full human immune system. In this review, we discuss preclinical studies evaluating therapeutic approaches in stem cell-based gene therapy and T cell-based immunotherapies for HIV and cancer using a humanized mouse model and some recent advances in using checkpoint inhibitors to improve antiviral or antitumor responses.
Introduction
Humanized mice have emerged as an invaluable tool in providing a model system that enables researchers to study the human immune system and its development and function/dysfunction in vivo (1). The identification of the severe combined immunodeficiency (Prkcdscid or SCID) mouse provided the cornerstone of the development of the humanized mouse model by allowing the xenoengraftment of human (hu) cells [specifically, human peripheral blood lymphocytes (PBLs)] without mouse immune system-mediated rejection (known as the hu-PBL SCID model) (2). This allowed limited examination of components of the human immune system in a manipulatable model system. Further development occurred with the engraftment of SCID mice with human fetal thymus and liver tissue, which is implanted under the kidney capsule of the animals (termed the SCID-hu mouse) (3, 4). The fetal liver tissue provided the hematopoietic cells and the thymus tissue provided the stromal elements to facilitate the engraftment and development of a functional human thymus in these animals. This allowed the closed examination and long-term engraftment of human hematopoietic tissue in vivo. Humanized mouse model development rapidly expanded with the identification and breeding of immunodeficient strains of mice that facilitated a greater engraftment of human cells. SCID mice have been crossed with other mouse strains, such as the nonobese diabetic (NOD) mouse to generate NOD/SCID mice that have defects in innate and adaptive immunity (5). Other mice that have been crossed to SCID mouse strains include those that have genetic mutations in the Rag1, Rag2, or the IL-2 receptor common gamma chain (IL2rγ) genes to generate new strains of immuno-incompetent mice which allow greater human cell and tissue engraftment, particularly the tissues and cells that have a high hematopoietic potential (6). The NOD/SCID and NOD/SCID/IL2rγ-knockout (NSG) strains have been used to generate one of the more recent humanized mouse models that has shown to have the most robust human immune system engraftment, providing long-term human hematopoietic stem/progenitor cell (HSPC) engraftment and functional multilineage hematopoietic differentiation. This model facilitated the engraftment of human CD34+ HSPCs in the bone marrow of the animals and subsequent multilineage hematopoiesis, including B cell production and limited T cell development [termed the CD34-humanized mouse (7)]. More robust T cell reconstitution, which provides a more relevant model for HIV infection and the study of T cell immunity (8), was subsequently developed and involved the intravenous injection of autologous CD34+ human hematopoietic cells from fetal liver tissues, which engraft in the bone marrow (B), along with the transplantation of human fetal liver (L) and thymus (T) tissue under the kidney capsule of the mice, which forms a recapitulated human thymus [known as the bone marrow–liver–thymus (BLT) mouse] (9, 10). New mice strains, such as NOD-SCID IL2Rγnull/IL-3/GM-CSF(NSG-SGM3), are also being adopted for constructing BLT mice for better differentiation of myeloid cells or cancer engraftment (11, 12). Overall, immune-incompetent mouse strains can be humanized by either the transplantation of human peripheral blood mononuclear cells (PBMCs), the transplantation of human HSPCs, or the engraftment of human fetal tissue and HSPCs (Table 1). Among them, the humanized BLT mice are the most robust model in supporting multilineage human immune system development (13). The development of humanized mouse models has been extensively reviewed in Ref. (6, 14, 15) and been utilized in preclinical studies that revealed important discoveries in several fields of research (1).
Table 1. Engraftment of human immune system in the most commonly used immunodeficient mouse models.
In particular, HIV researchers have taken advantage of the humanized mouse model to better understand the pathogenesis of the infection and to examine novel therapeutic strategies to treat and possibly eradicate infection (19). Relatively early in the use of these types of humanized mice, researchers used the SCID-hu mouse as a platform to design and test a gene therapy approach for the treatment of HIV infection. Human HSPCs were transduced with a retroviral vector expressing a reporter gene and were then injected into the human thymus organoid to evaluate the differentiation and development of mature cells carrying the transgene reporter in vivo (20, 21). These studies formed the basis of the development of this approach to protect cells from HIV infection in what was the largest phase II gene therapy trial to that date (22). This sets the stage for the forward progression of other types of HSPC-based gene therapy research involving the development of lentiviral vectors expressing anti-HIV components that result in HIV-resistant immune cells in vivo in humanized mice (23–27). Results for some of these studies enabled stem cell-based gene therapy clinical trials that are currently ongoing (ClinicalTrials.gov Identifier: NCT01734850). Thus, studies such as these performed in humanized mice illustrate the utility of testing new stem cell-based gene therapy approached in humanized mice and highlight the potential therapeutic efficacy and safety of engineering such aspects as HIV resistance through the genetic modification of HSCs with anti-HIV genes (28).
Currently, humanized mouse models are being highly utilized to study human diseases and develop novel therapeutic approaches that can potentially be translated into clinical trials as described above. HIV and cancer are two research fields that have been taking advantage of the humanized mouse model to study stem cell- and T cell-based immunotherapy approaches to treat these chronic diseases. In this review, we highlight important studies using the humanized mouse model in stem cell- and T cell-based immunotherapy using highly potent transgenic T cell receptors (TCRs) and chimeric antigen receptors (CARs). We also discuss utilizing checkpoint inhibitors to overcome common immunosuppression mechanisms used by both diseases that promote disease progression and persistence.
Peripheral Cell-Based Immunotherapy Modeling in Humanized Mice
Transgenic TCRs in Humanized Mice
One of the earliest attempts for treating HIV through an immunotherapy-based approach using peripheral T cells was to isolate HIV-specific CTLs from HIV patients, expand ex vivo, and infuse them back into the patients (29–32). However, these studies demonstrated that this approach had very little impact on antiviral efficacy in treated individuals. There are current attempts to improve the efficiency of this approach through the “redirection” of peripheral T cells to target HIV infection through the genetic modification of cells with HIV-specific, molecularly cloned TCRs [for review on transgenic TCRs, see Ref. (33, 34)]. Proof of principle studies were conducted in the humanized mouse model wherein Joseph et al. produced a lentiviral vector encoding the TCR that recognizes the HIV-1 gag epitope SL9, which elicits a potent antiviral response by CTLs carrying the SL9-specific TCR (35). Using the SCID-hu mouse model, transduced CD8+ T cells carrying the SL9-specific TCR were co-injected with human leukocyte antigen (HLA)-matched HIV-1-infected PBMCs and tested for in vivo suppression of HIV-1. Isolated spleens of the mice treated with transduced HIV TCR CD8 T cells showed no signs of HIV-1-infected PBMCs; thus, peripheral CD8+ T cells modified with this potent anti-HIV TCR were capable of controlling and clearing HIV-1 infection in vivo. Although TCR-based immunotherapy has been shown to be effective in nonhumanized mouse models (36–38), there are rising safety concerns with using cloned TCRs in adoptive immunotherapy because of the possibility of exogenous TCR mispairing with an endogenous TCR chain, generating a new TCR that can have lethal off-target toxicity (39, 40). However, other studies conducted in humanized mice suggest that this may not be a significant issue (see below).
CAR-Based Immunotherapy in Humanized Mice
An ever-present issue with the use of molecularly cloned TCRs in therapy is that they have to be used in HLA-matched individuals, lessening their potential use to a limited number of people. CARs, which combine antigen-recognizing, HLA-independent extracellular domains with the TCR-zeta chain intracellular signaling domain, broaden these molecules’ potential use as a T cell redirection/engineering therapeutic approach [for a review on CAR T cell design, see Ref. (41)]. There have been numerous preclinical studies and clinical trials that have tested or are currently testing the effectiveness of CAR T cell therapy against certain cancers, reviewed in Ref. (42). In many preclinical studies, humanized mice were used to test the antitumor efficacy of various CAR designs: for example, second- or third-generation CARs which contain immune-enhancing costimulatory domains (43–45). Humanized mice can also be used to study the effect of combination therapy with CAR T cells and antibody-targeting immune checkpoint inhibitors such as PD-1 and CTLA-4 (46). A combinatorial therapeutic approach using CAR T cells and an immune checkpoint inhibitor has recently been studied in a humanized mouse model of metastatic clear-cell renal cell carcinoma (47). These CAR T cells targeting human anti-carbonic anhydrase are also equipped to secrete human anti-programmed death ligand 1 (PD-L1) antibodies to overcome checkpoint inhibition mediated by PD-1 and PD-L1 interactions. This approach to immune-checkpoint blockade resulted in an enhanced antitumor efficacy compared to mice treated with CAR T cells alone. Continuous efforts to study the behavior of CAR T cells in vivo using humanized mice can provide important understandings into overcoming the immunosuppressive properties of the tumor microenvironment.
With the success of CAR T cell therapy against B cell malignancies, HIV researchers are revisiting the CAR T cell approach for the treatment of HIV infection (48–50). Very recently, peripheral anti-HIV CAR T cells have been tested for antiviral efficacy using a humanized mouse model of HIV infection (51). The study’s approach was to redesign a CD4-based CAR vector used previously in clinical trials to augment expression and CAR T cell performance. Anti-HIV CAR T cells that contained the costimulatory 4-1BB domain outperformed those that contained the CD28 costimulatory domain in reducing viral rebound after ART treatment and prolonged persistence in vivo in the absence of antigen. Thus, opposed to the minimal clinical efficacy seen with the first-generation CD4-based CAR, newer generation of anti-HIV CARs can potentially have a more promising outcome in clinical trials. Future studies using humanized mouse models of HIV infection can provide more information on differences in anti-HIV responses and the clearance of HIV infection in vivo using anti-HIV CAR T cells containing different combinations of costimulatory domains.
Stem Cell-Based Gene Therapy in Humanized Mice
Recent developments of new humanized mouse models have opened opportunities in efforts to modify human stem cells to generate an immune system designed to mount a more efficient, targeted immune response against a specific pathogen or a disease. Humanized mice are being employed to test the therapeutic efficacies of stem cell-based gene therapies involving the modification of HSPCs with potent antigen-specific TCRs and CARs, and engineering a human immune system equipped to specifically target HIV or cancer antigens in vivo. Below, we discuss key studies that have utilized the humanized mouse model system for stem cell-based therapy for HIV and cancer.
Stem Cell-Based Gene Therapy Using TCRs Against HIV and Cancer
To enhance the immune response to HIV infection, studies have used HSPCs to introduce HIV-specific TCRs into immunodeficient mice to reconstitute a human immune system that contains a population of T cells carrying an HIV-specific TCR. The testing of this concept initially utilized the SCID-hu mouse model (52). CD34+ HSPCs were isolated from a human fetal liver, transduced with a molecularly cloned anti-HIV TCR, and transplanted into irradiated HLA-matched SCID-hu mice. This resulted in the generation of mature CD8+ T cells carrying the transgenic anti-HIV TCR. These anti-HIV TCR+ T cells were functional in response to peptide stimulation ex vivo, differentiating into effector cells, producing interferon (IFN)-gamma, and lysing targeted cells. To test the functionality of anti-HIV TCR+ T cells generated from transduced HSCs in vivo, a follow up study used the NSG strain mouse that is engrafted with human liver/thymus and injected with transduced fetal liver CD34+ cells. Using this NSG-CTL mouse model, the injected transduced HSCs were able to differentiate into mature human CD8+ T cells carrying the transgenic anti-HIV TCR (16). More importantly, anti-HIV TCR+ CD8 T cells were found to migrate into multiple tissues including the spleen, bone marrow, and the implanted human thymus. Following an HIV-1 challenge into these mice, these anti-HIV TCR+ CD8 T cells were able to suppress viral load at 2 weeks and 6 weeks post infection in the peripheral blood. In addition, mice carrying the anti-HIV TCR T cells were protected against CD4 T cell depletion and had lower levels of infected cells by 6 weeks post infection. Other key outcomes observed in this study were the reduced viral burden in anti-HIV TCR mice in lymphoid tissues and the expansion and differentiation of anti-HIV TCR+ T cells in response to an active HIV infection. These studies using two different humanized mouse models showed the feasibility and therapeutic potential of modifying HSCs with a potent anti-HIV TCR to produce a functional antiviral immune response to HIV.
Investigators have turned to the humanized mouse model to test the proof of principle of this type of stem cell-based gene therapy against cancer. Similar to the HIV-based studies, stem cell-based gene therapy for cancer is also being examined as a potential therapeutic strategy to provide a long-lasting immune surveillance against tumor cells using human HSPCs modified with an antitumor TCR (53). Using the BLT-humanized mouse model, Vatakis et al. transplanted HSPCs modified with a HLA-A*0201-restricted anti-melanoma TCR (54, 55). The transduced HSPCs were able to differentiate and produce high levels of naïve CD8+ T cells carrying the anti-melanoma TCR. Upon challenging these mice with HLA-matched tumors, mice treated with anti-melanoma TCRs were able to control tumor growth, and in some mice, clear the tumor compared to control mice carrying nonmodified T cells. Further analysis on the functionality of these anti-melanoma-specific T cells showed that they can differentiate into different subsets of effector and memory phenotype and infiltrate into tumors. Moreover, analysis of the bone marrow of these mice carrying transgenic HSCs showed continued expression of the integrated vector in isolated bone marrow samples. Thus, transgenic HSPCs can repopulate the bone marrow and provide a long-lasting supply of modified mature immune cells, including T and natural killer (NK) cells, directed against a specific pathogen. Other studies have also utilized the CD34-humanized mouse model in examining stem cell gene therapy using candidate antitumor specific TCRs which exhibited similar and new informative outcomes (56–58). In particular, these studies found that the introduction of the TCR transgene in HSPCs could inhibit endogenous TCR rearrangement in T cells (56, 57, 59). This is an important discovery as it can overcome the potential of off-target toxicities from transgene expression and endogenous TCR chains rearrangement and alpha and beta chain receptor mixing. Hence, humanized mouse models enabled investigators to study the development and dynamics of an immune system with unlimited replenishment of immune cells carrying a disease-specific receptor which can provide key aspects of its therapeutic potential in clearing a persistent infection or a disease.
Stem Cell-Based CAR T Cell Studies in HIV and Cancer
To test the safety and efficacy of a stem cell-based CAR approach in HIV infection, Zhen et al. used the BLT-humanized mouse model and modified HSPCs with a lentiviral vector expressing an anti-HIV CD4-based CAR to determine whether this can result in the generation of mature anti-HIV CAR+ CTLs (17). This anti-HIV CAR is based on utilizing the HIV receptor CD4 molecule that is fused to an internal TCR-signaling domain (60). Stem cells from fetal liver were modified with anti-HIV CAR-expressing lentiviral vector and infused into NSG mice transplanted with fetal liver and thymus. Investigators observed subsequent maturation of CAR+ T cells, NK cells, B cells, and myeloid cells in vivo. In addition, cells carrying the CAR-expressing lentiviral vectors were protected from HIV infection by coexpressing protective anti-HIV shRNAs and were able to functionally suppress HIV replication in vivo through CTL activity. Also, similar to the TCR-modified HSPC-based studies, developing T cells carrying the anti-HIV CAR receptor can successfully go through positive selection in a human thymus, and the expression of the anti-HIV CAR resulted in the suppression of endogenous TCR rearrangement. This observation that developing T cells expressing an anti-HIV CD4-based CAR suppressed endogenous TCR rearrangement suggests that the CD4-based CAR can act as the sole natural TCR during development. This could be a beneficial trait in the long term, as emerging T cells expressing CD4-based CARs will be specific to HIV antigen and chances of off-target activation will be minimal. A similar approach was also done examining the development of CD19CAR-expressing cells in the CD34-humanized mouse model (61, 62). They found that the introduction of a lentiviral vector expressing either a CD19CAR or a second-generation CD19CD28CAR into HSPCs and engrafting into NSG mice led to the differentiation of different hematopoietic lineages expressing CAR including T cells, B cells, and myeloid cells and produced potent antitumor responses in the CD19CD28CAR-treated mice (61, 62). It remains to be seen if the therapeutic effects of stem cell-based CAR T cell therapy performed on humanized mice will be translated into human clinical trials.
PD-1 and IFN-I Blockade Therapy for HIV and Cancer
While humanized mice have been useful in the examination of human immunotherapeutic approaches involving gene therapies, their use in examining antiviral or antimalignancy responses and immunotherapies is at a relatively nascent stage. More sensitive immune-based assays and improvements in humanized mice now allow the examination of antitumor and antiviral immune responses and show great promise in the development of novel immunotherapies to treat these conditions. In recent studies, humanized mouse models were used to examine the effects of blocking key immune and antiviral factors in chronic HIV infection. Chronic viral infections can persist by upregulating immune checkpoint receptors that can functionally compromise virus-specific T cells and prevent them from clearing the infection (63). HIV infection has been shown to upregulate T cell exhaustion markers that enable the virus to chronically persist, which includes PD-1, Tim-3, LAG-3 among others (64–69). To investigate whether T cell exhaustion can be reversed and rescue function in exhausted T cells, these recent studies closely examined immune factors in chronically HIV-infected mice and found elevated PD-1 levels on T cells, similar to that seen in infected individuals. These chronically infected mice were treated with an antibody that blocks the PD-1/PD-L1 pathway and found reduced viral loads and increased CD4+ and CD8+ T cell levels (70, 71). In addition, PD-L1 blockade increased the percentages of naïve and central memory T cells and increased Th1 cytokines IFN-gamma and IL-12 during treatment (70). Thus, blocking the PD-1/PL-1 pathway during chronic HIV leading to reduced viral loads has now been shown in two different humanized mouse models and supports results seen in a study applying PD-1 blockade during chronic SIV infection in a macaque model, which reduced SIV levels (72). It remains to be seen whether PD-1/PD-L1 blockade can have clinical success in antiviral therapy in chronically HIV-infected individuals as it has already been observed in individuals treated for human cancer (73–75). PD-1 blockade treatment for cancer therapy has been shown to have therapeutic benefits in patients with certain types of malignancies (76). Recently, preclinical studies have utilized humanized mice either transplanted with human CD34+ HSPCs (HuNSG) or mice containing a double knockout of MHC class I or class II (NOG-dKO) to show the therapeutic potential of utilizing PD-1 blockade for cancer therapy (77, 78). These studies highlight the usefulness of humanized mice to study not only the antitumor effects of anti-PD-1 blockade but also the human immune responses to human tumors, as these studies revealed significant tumor growth suppression and antitumor CD8+ T cell responses following PD-1 blockade treatment.
Hyper-immune activation is a hallmark of chronic HIV infection, and arising evidence is suggesting that chronic type I (IFN-I) is driving this continuous immune activation that may be leading to disease progression (79). To investigate the role IFN-I plays in driving chronic HIV infection, investigators have turned to BLT-humanized mouse models of HIV infection to study this (80, 81). In the study by Zhen et al., after establishing a chronic HIV infection, blocking IFN-1 signaling using an anti-interferon alpha receptor 2(IFNR2)-blocking antibody resulted in a decreased immune activation, a decreased expression of T exhaustion markers and reversal of T cell exhaustion, and reduced plasma viral loads. In addition, treatment with the anti-IFNR2-blocking antibody in combination with ART resulted in a rapid viral suppression and reduced viral reservoirs. Cheng et al. found similar results using IFNR1-blocking antibody in combination with ART treatment throughout their study (80). These results shed light on the role IFN-I signaling plays during chronic HIV infection in maintaining chronic immune activation and T cell exhaustion that leads to uncontrolled HIV infection in vivo. Findings from these and future studies may lead to the application of IFN-I blockade treatment in combination with ART during chronic HIV infection that could alleviate residual immune activation and reduce viral reservoirs in HIV-positive individuals. It remains to be seen whether IFN-I blockade will have a beneficial antitumor efficacy during tumor progression since IFN-I is important in inducing antitumor responses such as promoting CD8 T cell priming. However, continuous IFN-I signaling can also have immunosuppressive properties that may play a role in promoting tumor growth (82). It has been recently shown that continuous IFN signaling drives PD-L1-dependent and -independent resistance to radiation therapy and checkpoint blockade, and blocking IFN-I signaling restores tumor cell response to checkpoint blockade treatment (83). Whether IFN-I blockade treatment can restore response to treatment in tumors that are resistant to PD-1 blockade or other immune checkpoint blockade in a humanized mouse model of cancer remains to be determined.
Future Directions
Although humanized mice have been an essential tool in several fields of research to better understand the mechanisms of disease progression and develop therapeutic strategies, these mouse models do come with their own limitations that need to be addressed to create more optimized models that will fit the needs of each research field (84). Currently, SCID mice engrafted with human PBMCs develop graft-versus-host disease (GVHD) within 4 weeks of engraftment, limiting the time of experimentation to just a few short weeks. The humanized BLT mouse model also has its own limitations for use. BLT mice can have poor B cell development, limited antibody class switching following activation, and lymphocyte homing in lymph nodes and germinal centers, limiting their antibody responses. In addition, these mice also typically develop a GVHD-like condition after around 20 weeks post engraftment of fetal tissue and HSCs, putting a limitation on the duration of a given study (84, 85). Therefore, there is a pressing need to develop new mouse strains with genetic properties that will eliminate the generation of this GVHD-like condition. Recently, a new modification of the BLT mouse model was made by transplanting fetal thymus, liver, and autologous CD34+ HSCs into a C57BL/6 mouse strain that contain a triple knockout of Rag2, IL-2Υc, and CD47 genes (TKO-BLT) (18). These mice were observed to be healthy with no signs of GVHD for 45 weeks post transplantation, which is months longer than that of the current BLT models. In addition, they retained high reconstitution of human cells throughout the 45 weeks. They also found this model to establish HIV latency, respond well to orally fed and subcutaneously injected ART treatment, and upon ART interruption, can generate rapid viral rebound. Thus, this new humanized TKO-BLT mouse model can provide an extended duration of a variety of studies that will be useful for addressing issues requiring longer periods of infection or disease progression.
Because of the variety of humanized mouse models currently available, it is important for investigators to be knowledgeable on the different mouse models and which one will be the more appropriate model to answer the questions they are investigating. Differences in the background mutations of the immunocompromised strains can have an impact on the engraftment of human cells and the development of peripheral lymph nodes and germinal centers (14). Therefore, results using humanized mice must be carefully interpreted. It is also important to include proper controls, particularly for immune-based studies, such as uninfected and unmanipulated animals, to control for any potential changes/interference by GVHD or specific effects pertaining to the individual tissues.
Humanized mouse models are also currently being improved upon for cancer research (86). Cancer therapy studies evaluating the immune response to tumors would benefit from a humanized BLT model where the human reconstituted immune system is compatible with the transplanted tumor tissue. One possibility will be to acquire HSPCs from a patient and transplant autologous tumor cells or HLA-matched tumor cells into the mice. This will generate a closer representative of the patient’s antitumor response without the interference of alloreactive T cells resulting from the mismatch of the reconstituted immune system and engrafted tumor cells. Further advances in generating humanized mouse models that overcome current limitations will be highly beneficial for HIV and cancer researchers to advance stem cell-based gene therapy, T cell immunotherapy, and other immunological studies such as T cell exhaustion and tumor immunosuppressive microenvironment for eradicating HIV and cancer.
Author Contributions
MC, AZ, and SK contributed equally to the preparation of this manuscript.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Funding
This work was funded by NIH grants AI078806 and AI110306-01 (to SK); NIH/NIAID 1U19AI117941—01; AmfAR 108929-56-RGRL, 108688-54-RGRL, 109577-62-RGRL (Kitchen-PI), the UCLA Center for AIDS Research (P30AI28697); the California Institute for Regenerative Medicine (TR4-06845, DISC2-10748), and California HIV/AIDS Research Program (F12-LA-215 to AZ); NIH grant T32-AI060567 (to AZ and MC); the UCLA AIDS Institute and UCLA Center for AIDS Research (AI28697 to AZ), and the UCLA AIDS Institute and UCLA Center for AIDS Research (AI028697 to MC), and the UPLIFT: UCLA Postdocs’ Longitudinal Investment in Faculty (K12 GM106996 to MC).
References
1. Walsh NC, Kenney LL, Jangalwe S, Aryee KE, Greiner DL, Brehm MA, et al. Humanized mouse models of clinical disease. Annu Rev Pathol (2017) 12:187–215. doi:10.1146/annurev-pathol-052016-100332
2. Mosier DE, Gulizia RJ, Baird SM, Wilson DB. Transfer of a functional human immune system to mice with severe combined immunodeficiency. Nature (1988) 335:256–9. doi:10.1038/335256a0
3. McCune JM, Namikawa R, Kaneshima H, Shultz LD, Lieberman M, Weissman IL. The SCID-hu mouse: murine model for the analysis of human hematolymphoid differentiation and function. Science (1988) 241:1632–9. doi:10.1126/science.2971269
4. Namikawa R, Weilbaecher KN, Kaneshima H, Yee EJ, McCune JM. Long-term human hematopoiesis in the SCID-hu mouse. J Exp Med (1990) 172:1055–63. doi:10.1084/jem.172.4.1055
5. Shultz LD, Schweitzer PA, Christianson SW, Gott B, Schweitzer IB, Tennent B, et al. Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice. J Immunol (1995) 154:180–91.
6. Shultz LD, Brehm MA, Bavari S, Greiner DL. Humanized mice as a preclinical tool for infectious disease and biomedical research. Ann N Y Acad Sci (2011) 1245:50–4. doi:10.1111/j.1749-6632.2011.06310.x
7. Watanabe Y, Takahashi T, Okajima A, Shiokawa M, Ishii N, Katano I, et al. The analysis of the functions of human B and T cells in humanized NOD/shi-scid/gammac(null) (NOG) mice (hu-HSC NOG mice). Int Immunol (2009) 21:843–58. doi:10.1093/intimm/dxp050
8. Karpel ME, Boutwell CL, Allen TM. BLT humanized mice as a small animal model of HIV infection. Curr Opin Virol (2015) 13:75–80. doi:10.1016/j.coviro.2015.05.002
9. Lan P, Tonomura N, Shimizu A, Wang S, Yang YG. Reconstitution of a functional human immune system in immunodeficient mice through combined human fetal thymus/liver and CD34+ cell transplantation. Blood (2006) 108:487–92. doi:10.1182/blood-2005-11-4388
10. Melkus MW, Estes JD, Padgett-Thomas A, Gatlin J, Denton PW, Othieno FA, et al. Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1. Nat Med (2006) 12:1316–22. doi:10.1038/nm1431
11. Bryce PJ, Falahati R, Kenney LL, Leung J, Bebbington C, Tomasevic N, et al. Humanized mouse model of mast cell-mediated passive cutaneous anaphylaxis and passive systemic anaphylaxis. J Allergy Clin Immunol (2016) 138:769–79. doi:10.1016/j.jaci.2016.01.049
12. Jangalwe S, Shultz LD, Mathew A, Brehm MA. Improved B cell development in humanized NOD-SCID IL2Rgamma(null) mice transgenically expressing human stem cell factor, granulocyte-macrophage colony-stimulating factor and interleukin-3. Immun Inflamm Dis (2016) 4:427–40. doi:10.1002/iid3.124
13. Wege AK, Melkus MW, Denton PW, Estes JD, Garcia JV. Functional and phenotypic characterization of the humanized BLT mouse model. Curr Top Microbiol Immunol (2008) 324:149–65. doi:10.1007/978-3-540-75647-7_10
14. Shultz LD, Brehm MA, Garcia-Martinez JV, Greiner DL. Humanized mice for immune system investigation: progress, promise and challenges. Nat Rev Immunol (2012) 12:786–98. doi:10.1038/nri3311
15. Shultz LD, Ishikawa F, Greiner DL. Humanized mice in translational biomedical research. Nat Rev Immunol (2007) 7:118–30. doi:10.1038/nri2017
16. Kitchen SG, Levin BR, Bristol G, Rezek V, Kim S, Aguilera-Sandoval C, et al. In vivo suppression of HIV by antigen specific T cells derived from engineered hematopoietic stem cells. PLoS Pathog (2012) 8:e1002649. doi:10.1371/journal.ppat.1002649
17. Zhen A, Kamata M, Rezek V, Rick J, Levin B, Kasparian S, et al. HIV-specific immunity derived from chimeric antigen receptor-engineered stem cells. Mol Ther (2015) 23:1358–67. doi:10.1038/mt.2015.102
18. Lavender KJ, Pace C, Sutter K, Messer RJ, Pouncey DL, Cummins NW, et al. An advanced BLT-humanized mouse model for extended HIV-1 cure studies. AIDS (2018) 32:1–10. doi:10.1097/QAD.0000000000001674
19. Marsden MD, Zack JA. Mouse models for human immunodeficiency virus infection. Annu Rev Virol (2017) 4:393–412. doi:10.1146/annurev-virology-101416-041703
20. Akkina RK, Rosenblatt JD, Campbell AG, Chen IS, Zack JA. Modeling human lymphoid precursor cell gene therapy in the SCID-hu mouse. Blood (1994) 84:1393–8.
21. An DS, Koyanagi Y, Zhao JQ, Akkina R, Bristol G, Yamamoto N, et al. High-efficiency transduction of human lymphoid progenitor cells and expression in differentiated T cells. J Virol (1997) 71:1397–404.
22. Mitsuyasu RT, Merigan TC, Carr A, Zack JA, Winters MA, Workman C, et al. Phase 2 gene therapy trial of an anti-HIV ribozyme in autologous CD34+ cells. Nat Med (2009) 15:285–92. doi:10.1038/nm.1932
23. Burke BP, Levin BR, Zhang J, Sahakyan A, Boyer J, Carroll MV, et al. Engineering cellular resistance to HIV-1 infection in vivo using a dual therapeutic lentiviral vector. Mol Ther Nucleic Acids (2015) 4:e236. doi:10.1038/mtna.2015.10
24. Ringpis GE, Shimizu S, Arokium H, Camba-Colon J, Carroll MV, Cortado R, et al. Engineering HIV-1-resistant T-cells from short-hairpin RNA-expressing hematopoietic stem/progenitor cells in humanized BLT mice. PLoS One (2012) 7:e53492. doi:10.1371/journal.pone.0053492
25. Walker JE, Chen RX, McGee J, Nacey C, Pollard RB, Abedi M, et al. Generation of an HIV-1-resistant immune system with CD34(+) hematopoietic stem cells transduced with a triple-combination anti-HIV lentiviral vector. J Virol (2012) 86:5719–29. doi:10.1128/JVI.06300-11
26. Shimizu S, Hong P, Arumugam B, Pokomo L, Boyer J, Koizumi N, et al. A highly efficient short hairpin RNA potently down-regulates CCR5 expression in systemic lymphoid organs in the hu-BLT mouse model. Blood (2010) 115:1534–44. doi:10.1182/blood-2009-04-215855
27. Shimizu S, Ringpis GE, Marsden MD, Cortado RV, Wilhalme HM, Elashoff D, et al. RNAi-mediated CCR5 knockdown provides HIV-1 resistance to memory T cells in humanized BLT mice. Mol Ther Nucleic Acids (2015) 4:e227. doi:10.1038/mtna.2015.3
28. Pernet O, Yadav SS, An DS. Stem cell-based therapies for HIV/AIDS. Adv Drug Deliv Rev (2016) 103:187–201. doi:10.1016/j.addr.2016.04.027
29. Lieberman J, Skolnik PR, Parkerson GR III, Fabry JA, Landry B, Bethel J, et al. Safety of autologous, ex vivo-expanded human immunodeficiency virus (HIV)-specific cytotoxic T-lymphocyte infusion in HIV-infected patients. Blood (1997) 90:2196–206.
30. Koenig S, Conley AJ, Brewah YA, Jones GM, Leath S, Boots LJ, et al. Transfer of HIV-1-specific cytotoxic T lymphocytes to an AIDS patient leads to selection for mutant HIV variants and subsequent disease progression. Nat Med (1995) 1:330–6. doi:10.1038/nm0495-330
31. Brodie SJ, Lewinsohn DA, Patterson BK, Jiyamapa D, Krieger J, Corey L, et al. In vivo migration and function of transferred HIV-1-specific cytotoxic T cells. Nat Med (1999) 5:34–41. doi:10.1038/4716
32. McKinney DM, Lewinsohn DA, Riddell SR, Greenberg PD, Mosier DE. The antiviral activity of HIV-specific CD8+ CTL clones is limited by elimination due to encounter with HIV-infected targets. J Immunol (1999) 163:861–7.
33. Ping Y, Liu C, Zhang Y. T-cell receptor-engineered T cells for cancer treatment: current status and future directions. Protein Cell (2018) 9:254–66. doi:10.1007/s13238-016-0367-1
34. Patel S, Jones RB, Nixon DF, Bollard CM. T-cell therapies for HIV: preclinical successes and current clinical strategies. Cytotherapy (2016) 18:931–42. doi:10.1016/j.jcyt.2016.04.007
35. Joseph A, Zheng JH, Follenzi A, Dilorenzo T, Sango K, Hyman J, et al. Lentiviral vectors encoding human immunodeficiency virus type 1 (HIV-1)-specific T-cell receptor genes efficiently convert peripheral blood CD8 T lymphocytes into cytotoxic T lymphocytes with potent in vitro and in vivo HIV-1-specific inhibitory activity. J Virol (2008) 82:3078–89. doi:10.1128/JVI.01812-07
36. Morris EC, Tsallios A, Bendle GM, Xue SA, Stauss HJ. A critical role of T cell antigen receptor-transduced MHC class I-restricted helper T cells in tumor protection. Proc Natl Acad Sci U S A (2005) 102:7934–9. doi:10.1073/pnas.0500357102
37. de Witte MA, Bendle GM, van den Boom MD, Coccoris M, Schell TD, Tevethia SS, et al. TCR gene therapy of spontaneous prostate carcinoma requires in vivo T cell activation. J Immunol (2008) 181:2563–71. doi:10.4049/jimmunol.181.4.2563
38. de Witte MA, Jorritsma A, Kaiser A, van den Boom MD, Dokter M, Bendle GM, et al. Requirements for effective antitumor responses of TCR transduced T cells. J Immunol (2008) 181:5128–36. doi:10.4049/jimmunol.181.7.5128
39. Bendle GM, Linnemann C, Hooijkaas AI, Bies L, de Witte MA, Jorritsma A, et al. Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy. Nat Med (2010) 16:565–70. doi:10.1038/nm.2128
40. Cameron BJ, Gerry AB, Dukes J, Harper JV, Kannan V, Bianchi FC, et al. Identification of a Titin-derived HLA-A1-presented peptide as a cross-reactive target for engineered MAGE A3-directed T cells. Sci Transl Med (2013) 5:197ra03. doi:10.1126/scitranslmed.3006034
41. Oldham RAA, Medin JA. Practical considerations for chimeric antigen receptor design and delivery. Expert Opin Biol Ther (2017) 17:961–78. doi:10.1080/14712598.2017.1339687
42. Fesnak AD, June CH, Levine BL. Engineered T cells: the promise and challenges of cancer immunotherapy. Nat Rev Cancer (2016) 16:566–81. doi:10.1038/nrc.2016.97
43. Guedan S, Chen X, Madar A, Carpenito C, McGettigan SE, Frigault MJ, et al. ICOS-based chimeric antigen receptors program bipolar TH17/TH1 cells. Blood (2014) 124:1070–80. doi:10.1182/blood-2013-10-535245
44. Song DG, Powell DJ. Pro-survival signaling via CD27 costimulation drives effective CAR T-cell therapy. Oncoimmunology (2012) 1(4):547–9. doi:10.4161/onci.19458
45. Carpenito C, Milone MC, Hassan R, Simonet JC, Lakhal M, Suhoski MM, et al. Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains. Proc Natl Acad Sci U S A (2009) 106(9):3360–5. doi:10.1073/pnas.0813101106
46. Gay F, D’Agostino M, Giaccone L, Genuardi M, Festuccia M, Boccadoro M, et al. Immuno-oncologic approaches: CAR-T cells and checkpoint inhibitors. Clin Lymphoma Myeloma Leuk (2017) 17:471–8. doi:10.1016/j.clml.2017.06.014
47. Suarez ER, Chang de K, Sun J, Sui J, Freeman GJ, Signoretti S, et al. Chimeric antigen receptor T cells secreting anti-PD-L1 antibodies more effectively regress renal cell carcinoma in a humanized mouse model. Oncotarget (2016) 7:34341–55. doi:10.18632/oncotarget.9114
48. Deeks SG, Wagner B, Anton PA, Mitsuyasu RT, Scadden DT, Huang C, et al. A phase II randomized study of HIV-specific T-cell gene therapy in subjects with undetectable plasma viremia on combination antiretroviral therapy. Mol Ther (2002) 5:788–97. doi:10.1006/mthe.2002.0611
49. Mitsuyasu RT, Anton PA, Deeks SG, Scadden DT, Connick E, Downs MT, et al. Prolonged survival and tissue trafficking following adoptive transfer of CD4zeta gene-modified autologous CD4(+) and CD8(+) T cells in human immunodeficiency virus-infected subjects. Blood (2000) 96:785–93.
50. Scholler J, Brady TL, Binder-Scholl G, Hwang WT, Plesa G, Hege KM, et al. Decade-long safety and function of retroviral-modified chimeric antigen receptor T cells. Sci Transl Med (2012) 4:132ra53. doi:10.1126/scitranslmed.3003761
51. Leibman RS, Richardson MW, Ellebrecht CT, Maldini CR, Glover JA, Secreto AJ, et al. Supraphysiologic control over HIV-1 replication mediated by CD8 T cells expressing a re-engineered CD4-based chimeric antigen receptor. PLoS Pathog (2017) 13:e1006613. doi:10.1371/journal.ppat.1006613
52. Kitchen SG, Bennett M, Galic Z, Kim J, Xu Q, Young A, et al. Engineering antigen-specific T cells from genetically modified human hematopoietic stem cells in immunodeficient mice. PLoS One (2009) 4:e8208. doi:10.1371/journal.pone.0008208
53. Gschweng E, De Oliveira S, Kohn DB. Hematopoietic stem cells for cancer immunotherapy. Immunol Rev (2014) 257:237–49. doi:10.1111/imr.12128
54. Vatakis DN, Koya RC, Nixon CC, Wei L, Kim SG, Avancena P, et al. Antitumor activity from antigen-specific CD8 T cells generated in vivo from genetically engineered human hematopoietic stem cells. Proc Natl Acad Sci U S A (2011) 108:E1408–16. doi:10.1073/pnas.1115050108
55. Vatakis DN, Bristol GC, Kim SG, Levin B, Liu W, Radu CG, et al. Using the BLT humanized mouse as a stem cell based gene therapy tumor model. J Vis Exp (2012) (70):4181. doi:10.3791/4181
56. Giannoni F, Hardee CL, Wherley J, Gschweng E, Senadheera S, Kaufman ML, et al. Allelic exclusion and peripheral reconstitution by TCR transgenic T cells arising from transduced human hematopoietic stem/progenitor cells. Mol Ther (2013) 21:1044–54. doi:10.1038/mt.2013.8
57. Najima Y, Tomizawa-Murasawa M, Saito Y, Watanabe T, Ono R, Ochi T, et al. Induction of WT1-specific human CD8+ T cells from human HSCs in HLA class I Tg NOD/SCID/IL2rgKO mice. Blood (2016) 127:722–34. doi:10.1182/blood-2014-10-604777
58. Hu Z, Xia J, Fan W, Wargo J, Yang YG. Human melanoma immunotherapy using tumor antigen-specific T cells generated in humanized mice. Oncotarget (2016) 7:6448–59. doi:10.18632/oncotarget.7044
59. Vatakis DN, Arumugam B, Kim SG, Bristol G, Yang O, Zack JA. Introduction of exogenous T-cell receptors into human hematopoietic progenitors results in exclusion of endogenous T-cell receptor expression. Mol Ther (2013) 21:1055–63. doi:10.1038/mt.2013.28
60. Kitchen SG, Zack JA. Engineering HIV-specific immunity with chimeric antigen receptors. AIDS Patient Care STDS (2016) 30:556–61. doi:10.1089/apc.2016.0239
61. De Oliveira SN, Ryan C, Giannoni F, Hardee CL, Tremcinska I, Katebian B, et al. Modification of hematopoietic stem/progenitor cells with CD19-specific chimeric antigen receptors as a novel approach for cancer immunotherapy. Hum Gene Ther (2013) 24:824–39. doi:10.1089/hum.2012.202
62. Larson SM, Truscott LC, Chiou TT, Patel A, Kao R, Tu A, et al. Pre-clinical development of gene modification of haematopoietic stem cells with chimeric antigen receptors for cancer immunotherapy. Hum Vaccin Immunother (2017) 13:1094–104. doi:10.1080/21645515.2016.1268745
63. Kahan SM, Wherry EJ, Zajac AJ. T cell exhaustion during persistent viral infections. Virology (2015) 47(9–480):180–93. doi:10.1016/j.virol.2014.12.033
64. Day CL, Kaufmann DE, Kiepiela P, Brown JA, Moodley ES, Reddy S, et al. PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature (2006) 443:350–4. doi:10.1038/nature05115
65. Trautmann L, Janbazian L, Chomont N, Said EA, Gimmig S, Bessette B, et al. Upregulation of PD-1 expression on HIV-specific CD8+ T cells leads to reversible immune dysfunction. Nat Med (2006) 12:1198–202. doi:10.1038/nm1106-1329b
66. Chew GM, Fujita T, Webb GM, Burwitz BJ, Wu HL, Reed JS, et al. TIGIT marks exhausted T cells, correlates with disease progression, and serves as a target for immune restoration in HIV and SIV infection. PLoS Pathog (2016) 12:e1005349. doi:10.1371/journal.ppat.1005349
67. Eichbaum Q. PD-1 signaling in HIV and chronic viral infection—potential for therapeutic intervention? Curr Med Chem (2011) 18:3971–80. doi:10.2174/092986711796957239
68. Hoffmann M, Pantazis N, Martin GE, Hickling S, Hurst J, Meyerowitz J, et al. Exhaustion of activated CD8 T cells predicts disease progression in primary HIV-1 infection. PLoS Pathog (2016) 12:e1005661. doi:10.1371/journal.ppat.1005661
69. Tian X, Zhang A, Qiu C, Wang W, Yang Y, Liu A, et al. The upregulation of LAG-3 on T cells defines a subpopulation with functional exhaustion and correlates with disease progression in HIV-infected subjects. J Immunol (2015) 194:3873–82. doi:10.4049/jimmunol.1402176
70. Palmer BE, Neff CP, Lecureux J, Ehler A, Dsouza M, Remling-Mulder L, et al. In vivo blockade of the PD-1 receptor suppresses HIV-1 viral loads and improves CD4+ T cell levels in humanized mice. J Immunol (2013) 190:211–9. doi:10.4049/jimmunol.1201108
71. Seung E, Dudek TE, Allen TM, Freeman GJ, Luster AD, Tager AM. PD-1 blockade in chronically HIV-1-infected humanized mice suppresses viral loads. PLoS One (2013) 8:e77780. doi:10.1371/journal.pone.0077780
72. Velu V, Titanji K, Zhu B, Husain S, Pladevega A, Lai L, et al. Enhancing SIV-specific immunity in vivo by PD-1 blockade. Nature (2009) 458:206–10. doi:10.1038/nature07662
73. Feng D, Hui X, Shi-Chun L, Yan-Hua B, Li C, Xiao-Hui L, et al. Initial experience of anti-PD1 therapy with nivolumab in advanced hepatocellular carcinoma. Oncotarget (2017) 8:96649–55. doi:10.18632/oncotarget.20029
74. Overman MJ, McDermott R, Leach JL, Lonardi S, Lenz HJ, Morse MA, et al. Nivolumab in patients with metastatic DNA mismatch repair-deficient or microsatellite instability-high colorectal cancer (CheckMate 142): an open-label, multicentre, phase 2 study. Lancet Oncol (2017) 18:1182–91. doi:10.1016/S1470-2045(17)30422-9
75. Guo L, Zhang H, Chen B. Nivolumab as programmed death-1 (PD-1) inhibitor for targeted immunotherapy in tumor. J Cancer (2017) 8:410–6. doi:10.7150/jca.17144
76. Wang Y, Wu L, Tian C, Zhang Y. PD-1–PD-L1 immune-checkpoint blockade in malignant lymphomas. Ann Hematol (2017) 97(2):229–37. doi:10.1007/s00277-017-3176-6
77. Wang M, Yao LC, Cheng M, Cai D, Martinek J, Pan CX, et al. Humanized mice in studying efficacy and mechanisms of PD-1-targeted cancer immunotherapy. FASEB J (2018) 32(3):1537–49. doi:10.1096/fj.201700740R
78. Ashizawa T, Iizuka A, Nonomura C, Kondou R, Maeda C, Miyata H, et al. Antitumor effect of programmed death-1 (PD-1) blockade in humanized the NOG-MHC double knockout mouse. Clin Cancer Res (2017) 23:149–58. doi:10.1158/1078-0432.CCR-16-0122
79. Snell LM, Brooks DG. New insights into type I interferon and the immunopathogenesis of persistent viral infections. Curr Opin Immunol (2015) 34:91–8. doi:10.1016/j.coi.2015.03.002
80. Cheng L, Ma J, Li J, Li D, Li G, Li F, et al. Blocking type I interferon signaling enhances T cell recovery and reduces HIV-1 reservoirs. J Clin Invest (2017) 127:269–79. doi:10.1172/JCI90745
81. Zhen A, Rezek V, Youn C, Lam B, Chang N, Rick J, et al. Targeting type I interferon-mediated activation restores immune function in chronic HIV infection. J Clin Invest (2017) 127:260–8. doi:10.1172/JCI89488
82. Snell LM, McGaha TL, Brooks DG. Type I interferon in chronic virus infection and cancer. Trends Immunol (2017) 38:542–57. doi:10.1016/j.it.2017.05.005
83. Benci JL, Xu B, Qiu Y, Wu TJ, Dada H, Twyman-Saint Victor C, et al. Tumor interferon signaling regulates a multigenic resistance program to immune checkpoint blockade. Cell (2016) 167:1540–54.e12. doi:10.1016/j.cell.2016.11.022
84. Brehm MA, Shultz LD, Luban J, Greiner DL. Overcoming current limitations in humanized mouse research. J Infect Dis (2013) 208(Suppl 2):S125–30. doi:10.1093/infdis/jit319
85. Akkina R, Allam A, Balazs AB, Blankson JN, Burnett JC, Casares S, et al. Improvements and limitations of humanized mouse models for HIV research: NIH/NIAID "Meet the Experts" 2015 Workshop Summary. AIDS Res Hum Retroviruses (2016) 32:109–19. doi:10.1089/aid.2015.0258
Keywords: HIV, cancer, humanized mice, gene therapy, immunotherapy, T cell receptor, chimeric antigen receptor, hematopoietic stem cells
Citation: Carrillo MA, Zhen A and Kitchen SG (2018) The Use of the Humanized Mouse Model in Gene Therapy and Immunotherapy for HIV and Cancer. Front. Immunol. 9:746. doi: 10.3389/fimmu.2018.00746
Received: 23 December 2017; Accepted: 26 March 2018;
Published: 20 April 2018
Edited by:
Ramesh Akkina, Colorado State University, United StatesReviewed by:
James Riley, University of Pennsylvania, United StatesSylvie Fournel, Université de Strasbourg, France
Copyright: © 2018 Carrillo, Zhen and Kitchen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Scott G. Kitchen, skitchen@ucla.edu