We describe a human peripheral blood mononuclear cell (PBMC) — based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.
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Li, Z., Yang, X., Zhang, Y., Yang, X., Cui, X., Zhang, Y., Gong, W., Bai, H., Liu, N., Tang, Z., Guo, M., Li, K., Zhang, T., Wang, L., Song, X. A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research. J. Vis. Exp. (150), e59679, doi:10.3791/59679 (2019).
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The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment challenges persist. Robust and disease-relevant animal models are vital resources for continued preclinical research and development in order to address a range of additional immune checkpoints. Here, we describe a human peripheral blood mononuclear cell (PBMC) — based humanized xenograft model. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody in late-stage clinical development, is used as an example to discuss platform set-up, model characterization and drug efficacy evaluations. These humanized mice support the growth of most human tumors tested, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Once established, our model is comparatively time- and cost-effective, and usually yield highly reproducible results. We suggest that the protocol outlined in this article could serve as a general guideline for establishing mouse models reconstituted with human PBMC and tumors for I-O research.
Immuno-oncology (I-O) is a rapidly expanding field of cancer treatment. Researchers have recently started to appreciate the therapeutic potential of modulating functions of the immune system to attack tumors. Immune checkpoint blockades have demonstrated encouraging activities in a variety of cancer types, including melanoma, renal cell carcinoma, head and neck, lung, bladder and prostate cancers1,2. Contrary to targeted therapies that directly kill cancer cells, I-O therapies potentiate the body’s immune system to attack tumors3.
To date, numerous relevant I-O animal models have been established. These include: 1) mouse tumor cell lines or tumor homograft in syngeneic mice; 2) spontaneous tumors derived from genetically engineered mouse (GEM) or carcinogen-induction; 3) chimeric GEMs with the knock-in of human drug target(s) in a functional murine immune system; and 4) mice with reconstituted human immunity transplanted with human cancer cells or patient-derived xenografts (PDXs). Each of these models have obvious advantages as well as limitations, which have been described and reviewed extensively elsewhere4.
Reconstitution of human immunity in immunodeficient mice have been growingly appreciated as a clinically relevant approach for translational I-O research. This is usually achieved through either 1) engraftment of adult immune cells (e.g., peripheral blood mononuclear cells (PMBC))5,6, or 2) engraftment of hematopoietic stem cells (HSC) from, for example, umbilical cord blood or fetal liver7,8. These humanized mice could support the growth of human tumors, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Despite the advantages, applications of humanized mice in I-O research were usually hindered by several concerns, such as long model development time and considerably high cost.
Here, we describe a human PBMC-based model that could be widely applied for translational I-O studies. This model is comparatively time- and cost-effective with high reproducibility in efficacy studies. It has been used in-house for the evaluations of several I-O therapeutics currently under preclinical and clinical development. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody9 , is used as the example to discuss model development, characterization, and possible applications for anti-tumor efficacy analyses.
All procedures performed in studies involving human participants were in accordance with the ethical standards of BeiGene and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study. All procedures performed in studies involving animals were approved by the Internal Review Board at BeiGene. This protocol has been specifically adjusted for the evaluation of BGB-A317 (Tislelizumab) in humanized NOD/SCID mice.
1. Establishment of human PBMC-based model
- Myeloablation of NOD/SCID mice using cyclophosphamide: determination of optimal doses
- Purchase female NOD/SCID mice (6-8 weeks).
NOTE: All mice involved in this study were female.
- Prepare cyclophosphamide (CP) at different doses (50, 100 and 150 mg/kg) in saline. Prepare disulfiram (DS) in 0.8% Tween-80 in saline at 125 mg/kg.
NOTE: Different concentrations of CP were prepared to enable administration of equal volumes of drug solution to mice getting different doses of CP.
- Treat the animals with CP (i.p.) and DS (p.o.) once a day for 2 days. Give DS (p.o.) 2 h after each dose of CP.
NOTE: DS decreases the urotoxicity of CP in mice, and CP combined with DS has been suggested to have longer-lasting neutropenia than animals treated with CP alone10 The dose regimen of CP might need to be pre-determined prior to actual studies and was found to vary slightly between different immunodeficient mouse strains.
- Collect blood samples from the orbital venous sinus and transfer to EDTA-K coated tubes on ice on day 0 (1 h before the 1st dose), day 2 (24 h after the 2nd dose) and day 4 (72 h after the 2nd dose).
- Examine the myeloablation effect after CP and DS treatment by FACS. Use rat anti-mouse CD11b (M1/70), rat anti-mouse Ly6C (HK1.4, ) and rat anti-mouse Ly6G (1A8) for gating CD11b+ Ly6Ghigh as neutrophils, CD11b+Ly6Chigh as monocytes11,12.
- Record body weight and health conditions of the mice daily for one week. The optimal dose of CP and DS is determined as the regimen that results in maximum depletion of neutrophils and monocytes without causing severe toxicity to mice.
- Purchase female NOD/SCID mice (6-8 weeks).
- Human PBMC transplantation and tumor engraftment: model set-up
- Isolate human PBMCs from healthy donors by density gradient centrifugation according to the manufacturer’s instructions.
- Pre-treat the mice with CP and DS as indicated by step 1.1.2 and 1.1.3 to increase transplantation efficiency.
- 20 to 24 h after the second dose of CP and DS, inject human tumor cell line such as A431 cells (ATCC, 2.5 x 106) and 5 x 106 isolated PBMCs (mixed in a total of 200 μL phosphate-buffered saline (PBS) containing 50% Matrigel), or tumor fragments (3 x 3 x 3 mm3, in a total volume of 200 μL PBS containing 50% Matrigel) and 200 μL of 5 x 106 PBMCs (100 μL each to the left and right side of engrafted tumor fragment) (s.c.) subcutaneously in the right flank of the animals.
- Measure primary tumor volume and record twice a week for 4-6 weeks.
NOTE: The mice will be euthanized once their body weights lose over 20% or their tumor volume reaches 2,000 mm3 or the tumor is ulcerated.
- Euthanize the mice in gas chambers with carbon dioxide. Collect the whole tumor tissues in sacrificed mice with ophthalmic scissors and process them for histology and immunohistochemistry (IHC) analysis. Examine the Human CD8, PD-1 and PD-L1 expressions in these tissues. See protocol step 4.
2. PBMC Donor Screen
- Screen a panel of PBMC donors due to the anticipated variations resulted from PBMCs collected from individuals. Use A431 cells co-injecting with PBMCs from different donors according to the procedures as indicated by step 1.2.
NOTE: Over 50 healthy PBMC donors were screened in the study in order to obtain enough number of suitable donors. Researchers who would like to adopt this protocol might decide on their own how many healthy PBMC donors to be screened, based on the design of the planned studies.
- Monitor tumor volume twice a week by measuring with a caliper.
NOTE: Tumor growth rate may vary with PBMC from different donors.
- Collect the tumor tissues at an average volume of 200-500 mm3 and process them for histology and immunohistochemistry (IHC) analysis. Examine human CD8, PD-1 and PD-L1 expressions. See step 4 for detailed protocol.
- Select PBMC donors that result in moderate tumor growth (tumor volume > 200 mm3 14 days post inoculation) and relatively high PD-1, PD-L1 and CD8 expressions (mean IHC scores > 2). See step 4 for detailed IHC scoring protocol.
3. Human Cancer Cell Line and PDX Screen
- Screen cell lines and PDXs according to the procedures stated in step 1.2, to evaluate tumor growth rate, human PD-L1 expression of the tumors and immune cell infiltrations.
NOTE: Over 30 human cancer cell lines and over 20 PDXs of different cancer types were screened by the authors. Data of selected tumor models were shown in the results section.
4. Immunohistochemistry (IHC)
- Harvest as indicated by step 1.2.5 and fix tumor tissues by immersing in formalin. Dehydrate and embed fixed tissues in paraffin. Section the fixed tissues at 3 μm and place them on polylysine-coated slides.
- Deparaffinize in xylenes three times 7 min each. Hydrate the sections through graded alcohols: 100% ethanol twice for 3 min each, followed by 90%, 80% and 70% ethanol in turn for 3 min each. Rinse by deionized H2O three times and remove excess liquid from the slides.
- Perform antigen retrieval by placing the slides in a container and cover with 10 mM sodium citrate buffer (pH 6.0), or Tris-EDTA (pH 9.0). Heat the slides container by microwave for 3 min. Boil in a water bath at 95 °C for 30 min and then cool down to room temperature. Rinse by deionized H2O three times and aspirate excess liquid from the slides.
- Block the sections by 3% bovine serum albumin in PBS for 1 h and 0.3% H2O2 solution in PBS for 10 min. Stain by antibodies against human CD8 (EP334), PD-1 (NAT105,) and PD-L1 (E1L3N) at 4 °C overnight, and HRP conjugated 2nd antibodies at RT for 1 h. Drop the substrate DAB (3,3'-diaminobenzidine) onto the slides and control the reaction time (seconds to minutes) by monitoring the brown color from microscope.
- Cover the slides with neutral balsam after immersing the slides in 0.5% hydrochloric acid alcohol and 0.5% ammonia water in turn for 5 s each, then in 80%, 90% and 100% ethanol in sequence for 3 min each, and finally in xylenes using three changes for 5 min each. Detect the antibodies by observing the brown color of DAB using microscope.
NOTE: Human CD8 and PD-1 expression on tumor-infiltrating leucocytes (TIL) were assessed by assigning an expression score on a 5-point scale (IHC score, range 0-4) at high objective magnification (20x, 40x). 0, absent; 1, weak intensity/ less than 20% cells; 2, weak-to-moderate intensity/ 20%-50% cells; 3, moderate-to-strong intensity/ 50%-80% cells; 4, strong intensity/ more than 80% cells. Human PD-L1 staining within tumor cells was scored using an adjusted scoring system on a 5-point scale (IHC score, range 0-4) because of its relatively diffused signal. 0, absent; 1, weak intensity/ less than 10% cells; 2, weak-to-moderate intensity/10%-30% cells; 3, moderate/ 30%-50% cells; 4, strong intensity/ more than 50% cells.
5. In Vivo efficacy and Pharmacodynamics Studies in Humanized PBMC-NOD/SCID Xenograft Models
- Pre-treat NOD/SCID mice as indicated by step 1.1.3. In brief, treat the mice with 100 mg/kg CP (i.p.) and 125 mg/kg DS (p.o.) once a day for 2 days.
- 20 to 24 h after the second dose, inject subcutaneously (s.c.) with indicated number of human cancer cells and 2.5-5 x 106 PBMCs (a total of 200 μL cell mixture in 50% Matrigel) in the right front flank of animals.
NOTE: The number of PBMC used for any individual mouse in one single study should be the same. However, due to variations in the availability of total isolated PBMC at the time of each study, the authors have chosen to use 2.5 x 106, 4 x 106, or 5 x 106 PBMC at different studies. Although this 2-fold difference in the administered amount of PBMCs might affect the degree of humanization, the authors do not observe significant differences in evaluating anti-tumor efficacies of the tested immunotherapies.
- For PDXs engraftment, inject subcutaneously tumor fragments (3 x 3 x 3 mm3) in the right front flank of animals. Inject subcutaneously 200 μL of 5 x 106 PBMCs (100 μL each side) to the left and right of engrafted tumor fragment.
NOTE: PDX tumor tissues were administered in a Matrigel solution, same as described for the cell line models.
- On the day of cell inoculation, randomly group the animals and treat as the planned study protocol. Assess the anti-tumor activity of candidate drugs, BGB-A317 (QW, i.p.) in this case, at the indicated doses in various tumor models.
NOTE: The three human cancer cell lines (i.e., A431 (epidemoid carcinoma), SKOV3 (ovarian cancer) and SK-MES-1 (lung cancer)), as well as two PDX models (i.e., BCLU-054 (lung cancer) and BCCO-028 (colon cancer)), are considered good tumor models for I-O therapy evaluation in this humanized mouse model.
- Measure primary tumor volume twice every week, using a caliper.
NOTE: Gand body weights loss were observed around 4-6 weeks post PBMC engraftment in our studies, allowing a 1-2 months window for therapeutic efficacy evaluations.
- For the pharmacodynamics analysis of tumor infiltrated immune cells, cut the tumor tissues into small pieces and digest them with collagenase type I (1 mg/mL) and DNase I (100 μg/mL) in RPMI1640 plus 5% fetal bovine serum (FBS) for 30 min at 37 °C. Pass the digested tissues through 40 μm cell strainers to obtain single cell suspensions.
- Wash the cells and adjust cell number to a concentration of 1 x 107 cells/mL in ice cold FACS Buffer (PBS, 1% FBS) in 96-well round bottom plates. Wash the cells by centrifuging and block them by adding 20 μg/mL human IgG for 30 min, followed by staining with anti-human CD3 (HIT3a), CD8 (OKT8) and PD-1 (MIH4) antibodies at 4 °C for 30 min. Then subject the stained samples to flow cytometry and analyze using guavaSoft 3.1.1.
Following the procedures presented here, a PBMC-based humanized xenograft model was successfully established. In brief, CP myeloablation effects in NOD/SCID mice was determined by flow cytometry analysis of neutrophil and monocyte populations post CP and DS treatment (Figure 1). 100 mg/kg CP plus 125 mg/kg DS was determined as the optimal dose and used in later studies as the regimen results in maximum depletion of neutrophils and monocytes without causing severe toxicity to mice. Next, human PBMC and tumor transplantation was performed. Presence of human immune cell infiltrates in the tumor microenvironment was verified by IHC (Figure 2).
A panel of PBMC donors were screened in vivo to ensure relatively high immune cell infiltrations in the tumor microenvironment and acceptable tumor growth rate (section 2, Figure 3A). Meanwhile, over 30 human cancer cell lines as well as over 20 PDXs of different cancer types were screened to evaluate tumor growth rate, tumor PD-L1 expression and immune cell infiltrations (section 3). Representative results were shown in Figure 3B.
These PBMC-engrafted humanized mice were then used to examine the anti-tumor activity of BGB-A317. Human PBMCs from selected healthy donors were co-injected with human tumor cells (A431, SKOV3 and SK-MES-1) or primary tumor tissue fragments derived from cancer patients (BCCO-028 and BCLU-054) subcutaneously. The mice were treated, as indicated in Figure 4, with BGB-A317 or PBS intraperitoneally once a week from the day of tumor implantation. In all abovementioned models, BGB-A317 demonstrated significant anti-tumor activities (Figure 4).
Figure 1: Myeloablation of NOD/SCID mice using cyclophosphamide (CP) and disulfiram (DS). (A) Gating strategy used to identify myeloid cell subsets including neutrophils and monocytes. (B) Representative results of myeloid cell (CD11b+), neutrophil (CD11b+Ly6G+) and monocyte (CD11b+Ly6C+) numbers upon different dosages of CP treatment. The boxes represent the 75th, 50th and 25th percentile of the values. The top and bottom lines represent maximum and minimal data points within the 1.5x IQ (inter quarter) range, respectively. n = 3 for the vehicle group and n = 6 for CP and/or DS treated groups. Please click here to view a larger version of this figure.
Figure 2: Human PBMC transplantation and tumor engraftment. (A) Schematic diagram showing the general workflow of PBMC-based humanized xenograft model. (B) Tumor growth of A431 cells upon subcutaneous co-injection with donor PBMC with the indicated conditions (data represents mean tumor volume ± SEM, n = 6). (C) IHC analysis of tumors developed in mice treated with or without CP+DS. Please click here to view a larger version of this figure.
Figure 3: PBMC donor and human cancer cell line screen. (A) Representative summary data from PBMC donor screen. PBMC were mixed with A431 cells and inoculated subcutaneously in humanized NOD/SCID mice (see step 2). Each dot represents the mean data value of 3 mice engrafted with PBMCs from 1 donor. (B) Representative results from human cancer cell line screen. PBMC from selected donors were co-injected with A431, SK-MES-1 or SKOV3 cells. Data represents mean tumor volume ± SEM collected from 3 mice, 14 day post inoculation of the indicated cell lines. Mean IHC score ± SEM represents the average expression of human CD8, PD-1, and PD-L1 of all 3 mice. Please click here to view a larger version of this figure.
Figure 4: Anti-tumor activities and pharmacodynamics analysis of BGB-A317 in PBMC-based humanized xenograft model. The anti-tumor activity of BGB-A317 at indicated doses (i.p., QW) was assessed using human cancer cell lines (A) A431 (with 5 x 106 PBMC), (C) SKOV3 (with 5 x 106 PBMC), (D) SK-MES-1 (with 5 x 106 PBMC), and patient-derived xenografts (PDXs) (E) BCLU-054 (with 5 x 106 PBMC) and (F) BCCO-028 (with 5 x 106 PBMC). PBMC from selected healthy donors and corresponding tumor cells were co-injected subcutaneously into humanized NOD/SCID mice (n = 8 to 10). (B) Quantification of tumor-infiltrating hCD8+ and hCD8+hPD-1+ cells in BGB-A317 treated A431 tumors. n = 8-10 animals per group in A, and C to F, n = 4-6 animals per group in B; data represents mean ± SEM. The significance was evaluated using a two-tailed unpaired Student's t-test under the assumption of unequal variance. Please click here to view a larger version of this figure.
Our knowledge of cancer development and progression has advanced significantly in recent years, with focus on a comprehensive understanding of both the tumor cells and its associated stroma. Harnessing the host immune mechanisms could induce a greater impact against cancer cells, representing a promising treatment strategy. Murine models with intact mouse immune systems, such as syngeneic and GEM models, have been widely used to study checkpoint-mediated immunity. Efficacy assessments using these models depend largely on surrogate anti-mouse target antibodies13,14. However, inherent differences between human and murine immune systems and the lack of some human targets in murine models limit preclinical studies of I-O anti-tumor effects15,16. Therefore, robust mouse models that include both human immune cells and human tumors are urgently desired, which will significantly improve the translation and development of novel I-O therapeutics.
Here, we describe a human PBMC-based xenograft mouse model that could potentially be widely used for translational I-O studies. PBMC donor as well as cancer cell line/PDX screens are critical to ensure robustness and reproducibility of in vivo efficacy studies. PBMC donors were screened in vivo to ensure successful human tumor and immune cell engraftment. Meanwhile, over 50 cell lines and PDXs of various human cancer origins were screened to evaluate tumor growth rate, human PD-L1 expression, and immune cell infiltrations. Our analyses suggest that about 20% of cancer cell lines and PDXs examined demonstrate acceptable tumor growth rate while at the same time having relatively high TILs and PD-L1 staining, which are considered good models for I-O efficacy evaluations.
HLA matching is routinely used in the clinic to match patients and donors for organ or marrow transplants17. The authors, however, have only performed limited characterization on HLA typing, and this remains an interesting topic to be investigated in future studies. The authors would like to note that a PBMC donor might be suitable for one cancer cell line/ PDX but not ideal for others. Therefore, PBMC donors might need to be screened for each cancer model to ensure optimal results.
Engraftment of human PBMC into NOD/SCID or NSG mice invariably leads to a xenogeneic graft-vs-host disease (xGvHD), a post-transplant disorder that results from immune-mediated attack of recipient tissue by donor T cells18,19. Clinical observations commonly associated with xGVHD have been observed in our humanized model, such as erythema, hunched posture, weight loss and mortality (data not shown). These phenotypes were usually observed towards the end of our studies, usually at 1-2 months post engraftment, indicating the propagation and infiltration of human T cells in xGVHD target organs. This allows a 1-2 months window for therapeutic I-O therapy evaluations. Several approaches have been utilized to decrease mouse innate immunity and enhance human immune cell engraftment20,21. For example, NSG mice defective in murine MHC Class I and Class II expression support engraftment of functional human T cells in the absence of acute xGvHD following injection of PBMC22.
NOD/SCID mice were used in this protocol. Mice homozygous for the SCID mutation have impaired T and B cell lymphocyte development and the NOD background additionally results in deficient natural killer (NK) cell function20. Other more highly immunodeficient mice, such as the NSG (The Jackson Laboratory), NCG (Charles River) and NOG (CIEA) strains, have been established. When engrafted with PBMCs, these mice have been shown develop human immune cells and form an environment that resembles the human immune system23,24. Alternatively, these mice could be engrafted with CD34+ human hematopoietic progenitor cells (HPCs) and display more sustained T cell differentiation and maturation25. In addition, next generation immunodeficient mouse models with further genetic modifications have been established to support better human myeloid lineage development and increased engraftment efficiency (refer to the variants portfolio webpage of The Jackson Laboratory and Taconic Biosciences).
More details of using these new strains for human immunity reconstitution are pending further investigations. Nevertheless, the protocol outlined in this article could serve as a general guideline of establishing and characterizing immunodeficient mouse models reconstituted with human PBMC. Three human cancer cell lines and two human patient-derived xenografts, covering a range of cancer types, are demonstrated in this article, suggesting the potential broad applications of our protocol in translational I-O studies. Most humanized PBMC models, to our knowledge, have chosen IV or IP as the route of injection26,27. Our models instead provide partially reconstituted human immunity in human tumor bearing mice through subcutaneously admixing of human PBMC with cancer xenografts. This approach provides a rapid and cost-effective, yet highly reproducible alternative to full stem cell reconstitution (e.g., CD34+ hematopoietic stem cell-engrafted humanized mice). Our model has been proved to be useful for evaluating T cell-engaging cancer immunotherapies, particularly when working on short timelines or to select agents before moving to a more complex multi-lineage immunity model.
All authors have ownership interest in BeiGene. Tong Zhang and Kang Li are inventors on a patent covering BGB-A317 described in this study.
We thank members of our laboratories for helpful discussions. This work was partially supported by the Biomedical and Life Science Innovation and Cultivation Research Program of the Beijing Municipal Science and Technology Commission under Grant Agreement No. Z151100003915070 (project "Preclinical study on a novel immune oncology anti-tumor drug BGB-A317"), and it was also partially supported by internal company funding for preclinical research.
|PBMC separation /cell culture|
|Penicillin-Streptomycin, Liquid||Gibco||15140-163||Cell culture|
|Trypsin-EDTA (0.25%), phenol red||Gibco||25200-114||Cell culture|
|Deoxyribonuclease I from bovine pancreas||Sigma||DN25||Sample preparation|
|Collagenase Type I||Sigma||C0130||Sample preparation|
|Anti-mouse/human CD11b (M1/70) antibody||BioLegend||101206||FACS|
|Anti-mouse Ly-6C (HK1.4) antibody||BioLegend||128008||FACS|
|Anti-mouse Ly-6G (1A8) antibody||BioLegend||127614||FACS|
|Anti-human CD8 (OKT8) antibody||Sungene Biotech||H10082-11H||FACS|
|Anti-human CD279 (MIH4) antibody||eBioscience||12-9969-42||FACS|
|Anti-human CD3 (HIT3a) antibody||4A Biotech||--||FACS|
|Guava easyCyte 8HT Benchtop Flow Cytometer||Millipore||0500-4008||FACS|
|Tumor/PDX implantation /dosing / measurement|
|Cyclophosphamide||J&K||Cat#419656, CAS#6055-19-2||In vivo efficacy|
|Disulfiram||J&K||Cat#591123, CAS#97-77-8||In vivo efficacy|
|Hypodermic needles (14 G)||Shanghai SA Mediciall & Plastic Instruments Co., Ltd.||0.7*32 TW SB||PDX inoculation|
|Vernier Caliper (MarCal)||Mahr||16ER||Tumor measurement|
|IVC individual ventilated cages||Lingyunboji Ltd.||IVC-128||Animal facility|
|Leica ASP200 Vacuum tissue processor||Leica||ASP200||IHC|
|Leica RM2235 Manual Rotary Microtome for Routine Sectioning||Leica||RM2235||IHC|
|Leica EG1150 H Heated Paraffin Embedding Module||Leica||EG1150 H||IHC|
|Ariol-Clinical IHC and FISH Scanner||Leica||Ariol||IHC|
|Anti-human CD8 (EP334) antibody||ZSGB-Bio||ZA-0508||IHC|
|Anti-human PD1 [NAT105] antibody||Abcam||ab52587||IHC|
|Anti-human PD-L1 (E1L3N) antibody||Cell Signaling Technology||13684S||IHC|
|Polink-2 plus Polymer HRP Detection System||ZSGB-Bio||PV-9001/9002||IHC|
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