Establishment of Humanized Mice from Peripheral Blood Mononuclear Cells or Cord Blood CD34+ Hematopoietic Stem Cells for Immune-Oncology Studies Evaluating New Therapeutic Agents

The clinical success of immune checkpoint modulators and the development of next-generation immune-oncology (IO) agents underscore the need for robust preclinical models to evaluate novel IO therapeutics. Human immune system (HIS) mouse models enable in vivo studies in the context of the HIS via a human tumor. The immunodeficient mouse strains NOG (Prkdc^scid Il2rg^tm1Sug) and triple-transgenic NOG-EXL [Prkdc^scid Il2rg^tm1Sug Tg (SV40/HTLV-IL3, CSF2)], which expresses human IL-3 and GM-CSF, allow for human CD34+ hematopoietic stem cell (huCD34+ HSC) engraftment and multilineage immune cell development by 12 to 16 weeks post-transplant and facilitate studies of immunomodulatory agents. A more rapid model of human immune engraftment utilizes peripheral blood mononuclear cells (PBMCs) transplanted into immunodeficient murine hosts, permitting T-cell engraftment within 2 to 3 weeks without outgrowth of other human immune cells. The PBMC-HIS model can be limited due to onset of xenogeneic graft-versus-host disease (xGVHD) within 3 to 5 weeks post-implantation. Host deficiency in MHC class I, as occurs in beta-2 microglobulin knockout in either NOG or NSG mice, results in resistance to xGVHD, which permits a longer therapeutic window. In this article, detailed processes for generating humanized mice by transplantation of HSCs from cord blood–derived huCD34+ HSCs or PBMCs into immunodeficient mouse strains to respectively generate HSC-HIS and PBMC-HIS mouse models are provided. In addition, the co-engraftment and growth kinetics of patient-derived and cell line–derived xenograft tumors in humanized mice and recovery of tumor-infiltrating lymphocytes from growing tumors to evaluate immune cell subsets by flow cytometry are described. © 2020 The Authors.

Basic Protocol 1 : Establishment of patient-derived xenograft tumors in CD34+ hematopoietic stem cell–humanized mice

Basic Protocol 2 : Establishment of patient-derived xenograft tumors in peripheral blood mononuclear cell–humanized mice

Support Protocol 1 : Flow cytometry assessment of humanization in mice

Support Protocol 2 : Flow cytometry assessment of tumor-infiltrating lymphocytes in tumor-bearing humanized mouse models

Human immune system (HIS)–reconstituted mice are a valuable tool to recapitulate the interactions between immune components and tumors of human origin, allowing evaluation of immune-oncology (IO) therapeutic modalities. Advances in developing murine strains with either truncation or complete loss of the IL-2 receptor gene has resulted in immunodeficient mice that lack mature T, B, and NK cells but retain dysfunctional macrophages and dendritic cells (DCs). Use of these mice permits the efficient engraftment of human CD34+ hematopoietic stem cells (huCD34+ HSCs). After transplantation of huCD34+ HSCs, hematopoiesis occurs in situ, leading to immune reconstitution. Lymphoid cells undergo negative selection during differentiation into T and B cells, allowing tolerance to the mouse host, with limited myeloid development (Katano, Ito, Eto, Aiso, & Ito, 2011). Transgenic expression of the human cytokines IL-3 and GM-CSF in NOG (Prkdc^scid Il2rg^tm1Sug) mice can improve engraftment of human progenitor cells, supporting enhanced myeloid lineage development while preserving T-cell development (Ito et al., 2013).

Basic Protocol 1, as described in this article, outlines a protocol for generating humanized mice by transplanting cord blood huCD34+ HSCs. This protocol describes co-implantation of a non-small-cell lung carcinoma (NSCLC) patient-derived xenograft (PDX) into humanized mice, along with evaluation of growth kinetics. Humanization is assessed in Support Protocol 1, and tumor-infiltrating lymphocytes (TILs) are assessed in Support Protocol 2. Basic Protocol 2 describes the humanization of mice using peripheral blood mononuclear cells (PBMCs). This model provides the advantage of faster engraftment (2 to 3 weeks) in comparison to the huCD34+ HSC-HIS model (10 to 12 weeks). Although PBMCs comprise mature T, B, NK, and myeloid cells, only T cells engraft in this model. The mature T cells are not thymically selected in the mouse, ensuring human MHC class I recognition. Further, mice lacking MHC class I have been demonstrated to have a more favorable profile for avoiding the development of xenogeneic graft-versus-host disease (xGVHD) in these models. These models are ideal for short-term studies using cell line–derived xenograft (CDX) tumors or moderate- to fast-growing PDX tumors. In this article, we also provide details on tumor growth kinetics in PBMC-HIS mice as well as infiltration of immune subsets in tumors using the breast cancer cell line MDA-MB-231 and an NSCLC PDX.

Successful generation of humanized mouse models with human tumors (CDX or PDX) requires careful planning of variables of interest (please refer to Table 1). Additional details are discussed in the Critical Parameters section.

NOTE : A barrier facility is required to house immunodeficient mice in a pathogen-free environment, using sterile techniques, microisolator caging, and appropriate husbandry practices (National Research Council Committee on Immunologically Compromised Rodents, 1989). All studies described here were conducted in an AALAC-certified facility with full IACUC approval, under the review of a laboratory veterinarian.

NOTE : Humanized mice, tumor-bearing humanized mice, and mouse bedding and cages should be considered potential biological biohazards and handled with appropriate personal protective equipment at animal biosafety level 2 in accordance with institutional biosafety guidelines.

HIS reconstitution using cord blood–derived huCD34+ HSCs in sublethally irradiated immunodeficient mouse strains is described in detail in this protocol. After huCD34+ HSC engraftment into NOG or NOG-EXL mice, subcutaneous implantation of PDX tumors into these mice generates a tumor-bearing humanized mouse model (please refer to Fig. 1 for a schema).

Materials

• 4- to 7-week-old female NOG (NOD.Cg-Prkdc^scid Il2rg^tm1Sug /JicTac) or NOG-EXL [NOD.Cg-Prkdc^scid Il2rg^tm1Sug Tg (SV40/HTLV-IL3, CSF2)10-7Jic/JicTac] mice (Taconic), housed in individually ventilated microisolator cages

• Cryopreserved cord blood huCD34+ HSCs, 1 × 10⁶/vial (Lonza or equivalent)

• 1× phosphate-buffered saline (PBS; Invitrogen)

• RPMI-1640 medium (ATCC, cat. no. 30-2001)

• Diet gel or other suitable feed (Tekland)

• Cryopreserved PDX tumors

• Nude mice: Hsd: Athymic Nude-Foxn1 ^nu (Envigo; optional)

• Irradiation cages

• Irradiation source (e.g., RS 2000 Biological Research Irradiator, Rad Source Technologies)

• 37°C water bath

• 15-ml polypropylene tubes

• Class II biosafety cabinet (BSC)

• Standard tabletop centrifuge

• Hematocytometer, trypan blue, and light microscope or automated cell counter (e.g., Cellometer Auto 2000, Nexcelom Bioscience)

• 1-cc tuberculin syringes with 25-G × ⅝-in. needle

• Scale

• Scalpel

• Forceps

• Isoflurane anesthesia machine

• Vernier or digital calipers

• K₂EDTA tubes

• Scissors

• Digital calipers

• Additional reagents and equipment for flow cytometry and phenotyping (see Support Protocol 1), subcutaneous tumor implantation, and mouse euthanasia

1.Approximately 4 hr prior to inoculation, sublethally irradiate 4- to 7-week-old female NOG or NOG-EXL mice in irradiation cages with 175 cGy whole-body irradiation from an irradiation source.

2.Thaw cryopreserved cord blood huCD34+ HSCs (1 × 10⁶ CB-CD34+ cells per vial for 8 to 10 mice) in a 37°C water bath until a sliver of ice remains. Then, transfer cells to a 15-ml polypropylene tube in a class II BSC and wash twice in 10 ml of 1× PBS at room temperature, with centrifugation for 5 min at 300 × g. Resuspend cells in 0.5 to 1 ml RPMI-1640 medium and count viable cells either manually, using a hematocytometer, trypan blue, and a light microscope, or with an automated cell counter.

3.Adjust concentration of huCD34+ HSCs by resuspension to 6 × 10⁵ cells/ml in RPMI-1460 and store cells briefly on ice prior to injection. Using a 1-cc tuberculin syringe with a 25-G × ⅝-in. needle, inject 0.2 ml (1.2 × 10⁵ HSCs) into lateral tail vein of each irradiated mouse by intravenous (IV) injection.

4.Monitor animals via clinical observation and record body weight at days 0, 3, 6, and 8 of the humanization period and once weekly thereafter for 3 months.

5.Provide mice with diet gel or other suitable feed for 2 weeks during recovery after irradiation. Provide water ad libitum.

6.After 10 to 12 weeks, evaluate HIS reconstitution by flow cytometry every 2 weeks (see Support Protocol 1).

7.At the time of humanization, thaw cryopreserved PDX tumors in a 37°C water bath, implant subcutaneously (SC) into a parallel cohort of immunocompromised mice (nude or NOG) bilaterally while under isoflurane anesthesia, and monitor for growth using Vernier or digital calipers (see Current Protocols article; Verma, Ritchie, & Mancini, 2017).

8.At 10 and 12 weeks post-transplant of huCD34+ HSCs, assess human chimerism in circulating cells by collecting ∼100 µl peripheral blood from mice via the facial vein into K₂EDTA tubes and prepare cells for flow cytometry phenotyping (refer to Figs. 2 and 3 for data and to Support Protocol 1 for phenotyping details).

9.To generate humanized PDX mice, harvest PDX tumors from stock mice (see step 7) using a scalpel. Generate 5 × 5 × 5–mm fragments using scissors and implant SC into flank of the humanized mice under isoflurane anesthesia.

10.Following subcutaneous tumor implantation, monitor tumor growth kinetics by digital caliper measurements of length and width to calculate volume, with the first measurements taken on the seventh day of tumor implantation. Record tumor volume measurements (TVMs) at two timepoints to ensure that tumors are growing prior to randomization into study groups, performed when tumor volumes range from 80 to 150 mm³, as shown in Figure 4.Continue TVMs twice weekly throughout duration of the study (see Fig. 5 for example data). Calculate tumor volume using the following formula:

11.At the endpoint of the study, euthanize mice as per IACUC-approved protocol and collect tumors for downstream analysis.

This protocol describes generation of humanized mice by injecting PBMCs isolated from healthy adult donors. After a PDX or CDX is implanted SC, PBMCs are transplanted within several days via IV injection. Tumor growth kinetics are recorded, and PBMC engraftment is monitored as described in Support Protocol 1.For PBMC-humanized mice, no irradiation is used, as this would enhance the onset and severity of xGVHD.

Additional Materials (also see Basic Protocol 1)

• Cryopreserved PDX or CDX tumors
• 4- to 7-week-old female NOG (NOD.Cg-Prkdc^scid Il2rg^tm1Sug /JicTac) or NOG-B2M (Taconic) mice or NSG (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl /SzJ) or NSG-B2M (NOD.Cg-B2m^tm1Unc Prkdc^scid Il2rg^tm1Wjl /SzJ) mice (The Jackson Laboratory), housed in individually ventilated microisolator cages
• Cryopreserved PBMCs, isolated from normal human donor leukopaks by density gradient centrifugation with Ficoll-Paque Premium (Thomas Scientific) as per manufacturer's recommendation

1.Implant cryopreserved PDX or CDX tumors SC into 4- to 7-week-old female NOG, NOG-B2M, NSG, or NSG-B2M mice prior to humanization with PBMCs.

2.Thaw cryopreserved PBMCs in a 37°C water bath and transfer cells to a 15-ml polypropylene tube in a class II BSC. Wash twice in 10 ml of 1× PBS by centrifugation for 5 min at 350 × g , room temperature. Resuspend cells in 0.5 to 1 ml RPMI-1640 medium and enumerate viable cells either manually, using a hematocytometer, trypan blue, and a light microscope, or with an automated cell counter.

3.Adjust PBMC concentration by resuspension to 5 × 10⁷ cells/ml in RPMI-1640 and store on ice briefly prior to injection. Using a 1-cc tuberculin syringe with a 25-G × ⅝-in. needle, inject 0.2 ml (1 × 10⁷ PBMCs) into lateral tail vein of each mouse by IV injection.

4.Monitor animals via clinical observation and record body weight twice weekly for signs of xGVHD throughout duration of the study.

5.Collect 100 µl peripheral blood from mice via the facial vein into K₂EDTA tubes weekly or at least every 2 weeks post–PBMC implant for assessment of humanization by flow cytometry phenotyping (see Figs. 9 to 11 and Support Protocol 1).

6.Following subcutaneous tumor implantation, monitor tumor growth kinetics by digital caliper measurements of length and width to calculate tumor volume (typical results are shown in Fig. 12). Randomize mice into study groups within 7 days of CDX implantation or when PDX tumor volumes are in the range of 80 to 150 mm³. Continue TVMs twice weekly throughout duration of the study. Calculate tumor volume using the following formula:

7.At the study endpoint or another predetermined time point, euthanize tumor-bearing humanized mice as per IACUC-approved protocol and collect tumors for downstream analysis.

This protocol provides a detailed procedure for assessment of engraftment levels of chimerism in immunodeficient mice implanted with huCD34+ HSCs (Basic Protocol 1) or PBMCs (Basic Protocol 2). The percent chimerism is calculated by flow cytometric assessment of levels of human pan-leukocyte marker (CD45) in the peripheral blood in comparison to total live cells, as follows:

Materials

• Mouse whole blood, freshly collected via facial vein into K₂EDTA tubes (see Basic Protocols 1 and 2)

• Streck CD-Chex™ Plus Immunophenotyping Control (Fisher Scientific, cat. no. 23-046-500D)

• Human BD Fc Block (BD Biosciences, cat. no. 564220)

• FACS buffer: MACSQuant Running Buffer (Miltenyi Biotec, cat. no. 130-092-747) + 5% (v/v) fetal bovine serum (FBS)

• Fluorochrome-labeled antibodies: antibodies to murine CD45 and human CD45 (clone HI30), CD3 (clone UCHT1), CD4 (clone RM4-5), CD8 (clone SK1), CD33 (clone p67.6), and CD19 (clone HIB19; all Biolegend; see Table 2)

• 1× red blood cell–lysing buffer (diluted from 10×; BD Pharm Lyse, BD Biosciences, cat. no. 555899)

• 7-Aminoactinomycin D (7-AAD; Biolegend, cat. no. 420403)

• 96-deep-well plates (Analytical Sales and Services, cat. no. 59623-23)

• 5-ml Falcon round-bottom tubes (VWR, cat. no. 60819-820)

• Tube rocker

• Aluminum foil

• 5°C centrifuge with plate rotor (Eppendorf 5810 or equivalent)

• Flow cytometer [MACSQuant® Analyzer 10 (Miltenyi Biotec) or equivalent flow analyzer with three-laser (405/488/635 nm) configuration]

• Flow cytometry analysis software (FlowJo V8 or equivalent)

NOTE : A maximum of 150 µl blood can be drawn weekly from each mouse, as per IACUC guidelines.

1.Aliquot 60 μl mouse whole blood per sample into each well of a 96-deep-well plate. Include 50 μl Streck CD-Chex™ Plus Immunophenotyping Control as a positive control in first well and pool small aliquots of blood for a 60-μl unstained negative control.

2.Incubate samples with 0.6 μl Human BD Fc Block + 0.14 μl FACS buffer (2 μl total; 2.5 μg/million cells) for 10 min.

3.Prepare a flow cytometry antibody staining cocktail in a 5-ml Falcon round-bottom tube using fluorochrome-labeled antibodies at the manufacturer's recommended concentration in FACS buffer as per the flow panel being tested. Add antibody staining cocktail to all whole-blood samples, followed by incubation for 30 min at room temperature in the dark.

4.Add 1.25 ml of 1× red blood cell–lysing buffer per sample. Incubate samples at room temperature on a tube rocker for 20 min, covered with aluminum foil.

5.Centrifuge samples for 5 min at 388 × g , 5°C, and discard supernatant.

6.Wash off excess antibody and lysing buffer by adding 1.25 ml FACS buffer per sample, centrifuging for 5 min at 388 × g , 5°C, and discarding supernatant. Repeat this step for a total of two washes. After the last wash, discard supernatant and resuspend each cell pellet in a final total volume of 150 μl FACS buffer.

7.Add 1 μl viability stain (7-AAD) per sample. Incubate for 5 min at room temperature in dark.

8.Acquire samples using a flow cytometer, ensuring collection of 20,000 gated live-cell events.

9.Perform flow cytometry data analysis with flow cytometry analysis software (Fig. 3).

Flow cytometric assessment of TILs in tumor-bearing humanized mice (Basic Protocols 1 and 2) is described in detail in this support protocol. At the study endpoint, tumors are harvested from the humanized mice and dissociated to obtain a single-cell suspension for staining and marker assessment.

Additional Materials (also see Support Protocol 1)

• Surgically excised PDX or CDX tumors (see Basic Protocols 1 and 2), collected from mice in tubes with MACS Tissue Storage Solution (Miltenyi Biotec, cat. no. 130-100-008) and kept on ice until processing

• Tumor Dissociation Kit, human (Miltenyi Biotec, cat. no. 130-095-929), including Enzymes A, H, and R (see recipes)

• RPMI-1640 medium (ATCC, cat. no. 30-2001)

• 1× red blood cell–lysing buffer (diluted from 10×; Miltenyi Biotec, cat. no. 130-094-183)

• Class II BSC

• Small petri dishes

• Forceps

• Scalpel

• gentleMACS C Tubes (Miltenyi Biotec, cat. no. 130-096-334)

• gentleMACS Dissociator (Miltenyi Biotec, cat. no. 130-093-235) or gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec, cat. no. 130-096-427)

• 15- and 50-ml conical tubes (Thermo Fisher, cat. no. 339650 and cat. no. 339652, respectively)

• MACS SmartStrainers (30 µm; Miltenyi Biotec, cat. no. 130-098-458)

• Room-temperature centrifuge (Eppendorf 5810 or equivalent)

• Hematocytometer, trypan blue, and light microscope or automated cell counter (e.g., Cellometer Auto 2000, Nexcelom Bioscience)

1.In a class II BSC, transfer each surgically excised PDX or CDX tumor to a small petri dish. Using a forceps and scalpel, dissect each tumor into pieces of 2 to 4 mm³ in the dish.

2.Transfer tumor fragments into a gentleMACS C Tube and add Enzymes A, H, and R from Tumor Dissociation Kit according to the manufacturer's instructions. Place tube in a gentleMACS Dissociator or gentleMACS Octo Dissociator with Heaters and use appropriate setting based on tumor toughness according to the manufacturer's instructions.

3.After completion of tumor digestion, empty contents of the C Tube into a 15-ml conical tube, strain into a 50-ml conical tube using a MACS SmartStrainer, and add 10 ml RPMI-1640 medium.

4.When filtration has stopped, cap tube and centrifuge 10 min at 300 × g. Remove supernatant and add 2 ml of 1× 1× red blood cell–lysing buffer per sample to dissociated tumor cells.

5.Incubate cells for 20 min on a tube rocker at room temperature. Centrifuge 5 min at 350 × g , 5°C, and discard supernatant.

6.Add 2 ml FACS buffer, centrifuge as in step 5, discard supernatant, and resuspend cells in 0.5 to 1 ml FACS buffer. Count cells with a hematocytometer, trypan blue, and light microscope or an automated cell counter and aliquot 500,000 live cells in 60 μl per sample per well of a 96-deep-well plate.

7.Incubate cells with 0.3 μl Human BD Fc Block + 0.7 μl FACS buffer (1 μl total; 2.5 μg/million cells) for 10 min.

8.Prepare a flow cytometry antibody staining cocktail in a 5-ml Falcon round-bottom tube using fluorochrome-labeled antibodies at the manufacturer's recommended concentration in FACS buffer as per the flow panel being tested. Add antibody staining cocktail to cells, followed by incubation for 30 min at 4°C in the dark.

9.Wash cells twice with 1.25 ml FACS buffer by centrifugation for 5 min at 350 × g , 5°C. After the last wash, discard supernatant and resuspend each cell pellet in a final total volume of 150 μl FACS buffer.

10.Add 1.5 μl viability stain (7-AAD) and incubate at room temperature in dark for 5 min.

11.Acquire samples using a flow cytometer, ensuring collection of a minimum of 200,000 total events.

12.Perform flow cytometry data analysis with flow cytometry analysis software as per defined gating strategy (Figs. 7 and 8).

Enzyme A

Reconstitute Enzyme A (lyophilized powder from Tumor Dissociation Kit, human, Miltenyi Biotec, cat. no. 130-095-929) by adding 1 ml Buffer A (provided with kit) to vial and mixing gently (do not vortex). Prepare 0.5-ml aliquots of Enzyme A solution in 1.5-ml tubes to avoid multiple freeze-thaw cycles. Store aliquots ≤6 months at −20°C, until ready for use.

Enzyme H

Reconstitute Enzyme H (lyophilized powder from Tumor Dissociation Kit, human, Miltenyi Biotec, cat. no. 130-095-929) by adding 3 ml RPMI-1640 medium (ATCC, cat. no. 30-2001) to vial and mixing gently (do not vortex). Prepare 1-ml aliquots of Enzyme H solution in 1.5-ml tubes to avoid too many freeze-thaw cycles. Store aliquots ≤6 months at −20°C, until ready for use.

Enzyme R

Reconstitute Enzyme R (lyophilized powder from Tumor Dissociation Kit, human, Miltenyi Biotec, cat. no. 130-095-929) by adding 2.7 ml RPMI-1640 medium (ATCC, cat. no. 30-2001) to vial and mixing gently (do not vortex). Prepare 1-ml aliquots of Enzyme R solution in 1.5-ml tubes to avoid multiple freeze-thaw cycles. Store aliquots ≤6 months at −20°C, until ready for use. Mix Enzyme R very well by pipetting up and down immediately before use in a reaction.

Background Information

There is an increasing demand for improved preclinical animal models to support mechanistic and therapeutic evaluation of novel agents targeting activation of the immune system against cancer. Data obtained from immunocompetent mice do not always support translation into human therapies due to species-specific differences (Mestas & Hughes, 2004), underscoring the need for humanized mouse models comprising both human immune cells and a human tumor compartment. Generation of immunodeficient mouse strains carrying either mutation or truncation of IL-2rγ [e.g., NOD/Shi-scid-IL2rγ^null (NOG) or NOD/LtSz-scid IL2rγ^null (NSG)] results in mice deficient in T, B, and NK cells and allows for efficient immune reconstitution (Ito et al., 2002; see Current Protocols article; Pearson, Greiner, & Shultz, 2008). Next-generation strains include immunodeficient transgenic mice expressing human cytokines, namely GM-CSF and IL-3 [NOG-EXL: Prkdc^scid Il2rg^tm1Sug Tg (SV40/HTLV-IL3, CSF2)] or cKIT-L, GM-CSF, and IL-3 (NSG-SGM3), which further support engraftment of CD34 cells, and particularly the development of myeloid cells (Bryce et al., 2016; Ito et al., 2013). Such models have also been used as an improved platform for preclinical efficacy studies of checkpoint inhibitors (Wang et al., 2018). Adult human PBMCs engraft readily and efficiently in NOG and NSG mice. However, due to human T-cell recognition of MHC class I and MHC class II on murine host cells, xGVHD has somewhat limited human PBMCs’ application in preclinical oncology studies with PDX tumors that require longer timelines. Development of mice that lack MHC antigens has led to reduced xGVHD (Brehm, Wiles, Greiner, & Shultz, 2014). An MHC class I and MHC class II double-knockout mouse strain developed on the NOG background was evaluated for anti-PD-1 efficacy in a PBMC-CDX model system. TIL profiling showed that more exhausted (PD-1+TIM3+LAG3+) T cells were maintained in anti-PD-1-antibody-treated tumors. A greater number of CD8+ and granzyme-producing T cells infiltrated the tumors in mice treated with the anti-PD-1 antibody (Ashizawa et al., 2017).

PDX models, which preserve the histology of patient tumors and exhibit similar gene expression patterns, represent an asset for testing therapeutic agents in development (Izumchenko et al., 2017). A comprehensive review of PDX model generation and use in humanized mouse models was published earlier by our group (see Current Protocols article; Verma et al., 2017).

Critical Parameters and Troubleshooting

The HIS-PDX platform is a valuable translational tool to study and modulate the interactions between immune components and tumors of human origin, allowing for evaluation of IO therapeutics. However, its success is dependent on several critical parameters, understanding of the limitations, and careful planning of studies.

Humanized model system

The basic protocols described in the article (Basic Protocols 1 and 2) offer two different methods for reconstitution of the HIS in immunodeficient mice. Selection of either one is dependent on the experimental design and the hypothesis being tested (please refer to Fig. 14). The huCD34+ HSC engraftment model (Basic Protocol 1) provides the advantage of stable and long-term humanization with minimal risk of xGVHD, allowing for complex biological evaluations of humanized tumor-bearing models in which long-term analysis is required (Wang et al., 2017). HuCD34+ HSC engraftment leads to development of various immune lineages, such as T cells, B cells, DCs, and myeloid cells (Audigé et al., 2017). One major caveat is that human T-cell education occurs in the mouse thymus, and hence, tumor recognition is mediated by alloreactive or xenoreactive T cells (Walsh et al., 2017). PBMC engraftment (Basic Protocol 2) facilitates adult human T-cell investigation and is well suited for examining the function of the mature immune system and even antigen-specific responses if the donor and tumor are HLA matched. The study timeline is considerably shorter for PBMC-HIS than for huCD34+ HSC-HIS models. However, the PBMC-HIS platform supports investigation of immune checkpoint drugs and their combinations (Sanmamed et al., 2015; Spranger, Frankenberger, & Schendel, 2012) and therapeutic assessment of bispecific antibodies. In particular, Bacac et al. (2016) showed in vivo therapeutic efficacy using carcinoembryonic antigen (CEA) T-cell bispecific antibody against CEA-expressing xenograft tumors with variable amounts of immune infiltrate.

Mouse strains for humanization

Currently, there are many immunodeficient mouse strains available on the market that can support reconstitution of the HIS using both huCD34+ HSC (Basic Protocol 1) and PBMC (Basic Protocol 2) platforms. This article focuses on the utility of NOG mice [and transgenic NOG-EXL mice: Prkdc^scid Il2rg^tm1Sug Tg (SV40/HTLV-IL3, CSF2] expressing human IL-3 and GM-CSF for humanization using huCD34+ HSCs (Basic Protocol 1). NOG mice engrafted with huCD34+ HSCs reach the acceptable threshold of 25% circulating huCD45+ cells by 10 to 12 weeks, with reconstitution of B, T, and myeloid cells. The frequency of T cells circulating in the periphery varies between 10% and 30%, depending on donor engraftment efficiency (as in Fig. 2). This model is suitable for evaluation of T cell–based therapeutics. NOG-EXL mice expressing huIL-3 and huGM-CSF have a greater ability to undergo myeloid reconstitution and are suitable for testing therapeutic agents that require or may modulate the myeloid compartment (Ito et al., 2013). Engraftment of mice using PBMCs (Basic Protocol 2) can be successfully achieved using NOG mice; however, these mice will succumb to xGVHD by ∼4 weeks, providing a very narrow therapeutic window for assessment, or may also require PBMC dose optimization for optimal engraftment levels. Alternatively, MHC class I–null B2M knockout mice on the NOG or NSG background provide a longer therapeutic window, namely up to 8 weeks (Fig. 10). The PBMC-HIS model is exclusively a T cell–based model, with both CD4+ and CD8+ T-cell subsets represented (Fig. 11).

Human donor cells

HuCD34+ HSCs

The major sources of huCD34+ HSCs (Basic Protocol 1) include umbilical cord blood, fetal liver tissue, bone marrow, and mobilized peripheral blood. Umbilical cord blood–derived huCD34+ HSCs have several advantages, including that they a) are readily available and b) demonstrate high pluripotency and reconstitution potential. Fetal liver–derived huCD34+ HSCs result in similarly robust engraftment. However, ethical issues associated with their use necessitate IRB approval and oversight (Drake, Chen, & Chen, 2012). Substituting different sources of huCD34+ HSCs may impact the overall engraftment rates and kinetics, and hence, cell doses should be titrated when the source of cells differs. Donor-to-donor variability in engraftment and response to therapeutic efficacy is an important factor to be considered, and thus, it is recommended to have mice engrafted separately with cells from at least three or four donors in parallel cohorts (mixed donor populations should be avoided to prevent graft-versus-graft allogeneic interactions). We have also evaluated tumor responses to the checkpoint inhibitor nivolumab (10 mg/kg) utilizing two CB-CD34 donors. Dosing was initiated at a tumor volume of 80 to 150 mm³. Donor-dependent antitumor efficacy was observed: CB-CD34 donor 1's cells inhibited tumor growth in the nivolumab group in comparison to the control group, whereas for the CB-CD34 donor 2's cells, no efficacy was observed in the nivolumab group (data shown in Fig. 5).

Cell purity and huCD34+ HSC viability (>70%) are critical parameters to be monitored for optimal study performance.

PBMCs

Leukopaks are the ideal source for preparation of adult PBMCs (Basic Protocol 2), ensuring that large enough numbers of humanized mice with cells from a single donor can be generated for a study. As for HSCs, post-thaw viabilities of >70% are required. In some instances, the possibility of recalling healthy donors for repeat blood draws can lend more consistency across studies. Intrinsic donor-to-donor variability is expected in the model system, and hence, two or four donors should be used (see example data in Fig. 10).

Tumor model system

Both humanization protocols (Basic Protocols 1 and 2) support the evaluation of immunotherapeutic agents using CDX or PDX. The huCD34+ HSC-HIS platform (Basic Protocol 1) allows for evaluation of slow-growing tumors, whereas the PBMC-HIS platform (Basic Protocol 2) is applicable to moderate- to fast-growing tumors due to the limited therapeutic window. These parameters should be considered while designing a study for implantation of tumors in humanized mice.

Understanding Results

HuCD34+ HSC-HIS model

Both NOG and NOG-EXL mice successfully undergo engraftment of CD34+ cells, resulting in circulating levels of huCD45+ cells that are detectable 8 to 10 weeks after transfer. Doses of 1–1.2 × 10⁵ cord blood CD34+ cells are enough to achieve a 25% level of engraftment in mice 10 weeks post-implant. Head-to-head comparison of NOG and NOG-EXL strains revealed that NOG-EXL mice are superior in engraftment efficiency. In particular, 100% engraftment was observed across four huCD34+ HSC donors, in comparison to ∼80% engraftment efficiency in NOG mice across five donors, with some variation by donor in terms of the overall level of engraftment (Fig. 2). Overall high levels of circulating huCD45+ cells were observed in NOG-EXL mice (50% to 75%) and were stable for up to 15 weeks. Typically, engraftment can be expected to be maintained for up to 25 weeks or more (data not shown). T-cell lineage development was equivalent in the two strains at 12 weeks post-HSC transplantation. Improved myeloid lineage (CD33+) development was found in NOG-EXL animals (Fig. 3). Although minimal impact on tumor growth rate was observed with both humanization techniques (data not shown), in some cases, donor-mediated effects can impede tumor growth rates. HuCD45+ lymphocytes were detected in tumors collected from humanized mice at 19 to 20 weeks post-humanization. Further analysis of TILs revealed ∼35% CD3+ T cells, with greater frequencies of CD4+ T cells detected in comparison to CD8+ cells for this model and these donors. CD8+ T cells appeared to be functional, as evidenced by production of granzyme B. High PD-1 expression was observed in T cells (> 60%); however, few regulatory T cells (CD4+CD25+FOXP3+) were present. Among human cell infiltrates in the tumor, ∼35% of these were myeloid cells. Myeloid subpopulations were characterized by marker expression through gating on CD45+CD11b+CD33+ cells. Flow cytometric analysis revealed the presence of CD14+HLA-DR+ macrophages (10%), CD14+HLA-DR- monocytic myeloid-derived suppressor cells (M-MDSCs; <20%), and CD14-HLA-DR- granulocytic MDSCs (G-MDSCs)/immature MDSCs (<10%) in humanized NOG-EXL tumors (Fig. 6). The NSCLC PDX model (CTG-1932) was infiltrated with various myeloid populations in this humanized model using NOG-EXL mice; however, the exact subsets present in a tumor model (CDX or PDX) may vary based on factors produced by each tumor.

PBMC-HIS model

A single IV dose of PBMCs in NOG or NSG mice leads to 20% to 60% human chimerism for up to 5 weeks (as shown in Fig. 9). The majority (>95%) of huCD45+ cells in circulation are CD3+ T cells, with a phenotype ratio of CD4/CD8 (40:60), which is stable throughout the 5 weeks. NOG mice succumb to xGVHD (characterized by body weight loss, dehydration, and worsening body condition) within ∼40 days post-implant. Transplant of the same donor PBMCs in NOG-B2M mice results in slower engraftment, with 10% circulating huCD45+ cell levels at 4 weeks and stable increased levels of huCD45+ cells (30%) at up to 8 weeks. An increased CD4/CD8 ratio is observed in B2M-deficient mice (data shown in Fig. 11). Similar trends were observed when multiple donors were evaluated using cell engraftment into NOG or NSG-B2M mice, as shown in Figure 10, with some donor-dependent effects on the magnitude and kinetics of engraftment. A donor-dependent effect on tumor growth kinetics was observed after 5 weeks with the CDX MDA-MB-231 tumor cell line using three different donors (please refer to Fig. 12 for data).

Time Considerations

Establishment of humanized PDX models using huCD34+ HSCs, as described in Basic Protocol 1, requires up to 12 weeks for engraftment post–-HSC implant. Tumors are then SC implanted into humanized mice, requiring an additional 2 to 3 weeks to establish the tumor prior to initiation of dosing for a therapeutic model. Dosing schedule and study endpoints define the length of the study post-randomization of the tumor-bearing humanized mice. A total of 19 to 20 weeks is thus required for completion of Basic Protocol 1.

The PBMC-HIS model (Basic Protocol 2) has an abbreviated timeline as compared to the huCD34+ HSC-HIS model (Basic Protocol 1). The timing of PBMC implantation is dictated by the study design and the tumor model selected (see Current Protocols article; Verma et al., 2017). A PDX tumor study requires subcutaneous PDX implantation prior to PBMC implantation, followed by randomization and dosing, whereas in a CDX study, PBMCs can be implanted before or after tumor cell engraftment. A total of 6 to 9 weeks is required for completion of Basic Protocol 2, depending on the duration of dosing and the tumor growth kinetics.

For Support Protocols 1 and 2, a total of 2 to 3 days are required for processing and acquisition followed by data analysis.

Acknowledgments

Special thanks to Linda St. John for excellent technical support and Dr. Geoffery Cole for review of the manuscript.

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