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Abstract
Background Immunodeficient mice engrafted with peripheral blood mononuclear cells (PBMCs) are models to study new cancer immunotherapy agents. However, this approach is associated with xenograft-versus-host disease (xGVHD), which starts early after PBMC transfer and limits the duration and interpretation of experiments. Here, we explore different approaches to overcome xGVHD and better support the development of cancer immunotherapies.
Methods Immunodeficient NOD-scid IL2Rgnull (NSG) mice were intravenously transferred with human PBMCs and subcutaneously co-engrafted with HT29 human colon carcinoma cells. Diverse strategies to reduce xGVHD while preserving the antitumor activity of human immune cells were evaluated: (1) ex vivo immune graft modification by depleting CD4+ T cells pre-transfer using magnetic beads, (2) post-transplantation cyclophosphamide administration to eliminate proliferating xenoreactive T-cell clones and (3) using major histocompatibility complex (MHC) class I and II-deficient NSG mice: (Kb Db)null (IA)null (MHC-dKO NSG). Body weight and plasma murine alanine aminotransferase levels were measured as indicators of xGVHD and tumor size was measured every 2–3 days to monitor antitumor activity. The antitumor effects and pharmacodynamics of nivolumab plus ipilimumab and an anti-epithelial cell adhesion molecule (EpCAM)/CD3 T-cell engager (αEpCAM/CD3 bispecific antibody (BsAb)) were evaluated in the model.
Results CD4+ T-cell depletion attenuates xGVHD but also abrogates the antitumor activity. Cyclophosphamide limits the antitumor response and does not substantially prevent xGVHD. In contrast, xGVHD was significantly attenuated in MHC-dKO NSG recipients, while the antitumor effect of human PBMCs was preserved. Furthermore, the administration of nivolumab plus ipilimumab caused exacerbated xGVHD in conventional NSG mice, thereby precluding the observation of their antitumor effects. Severe xGVHD did not occur in MHC-dKO NSG mice thus enabling the study of complete and durable tumor rejections. Similarly, NSG mice treated with an αEpCAM/CD3 BsAb showed complete tumor regressions, but died due to xGVHD. In contrast, MHC-dKO NSG mice on treatment with the αEpCAM/CD3 BsAb achieved complete tumor responses without severe xGVHD. A significant proportion of mice rendered tumor-free showed tumor rejection on rechallenge with HT29 cells without further treatment. Finally, tumor-infiltrating CD8+ T-cell number increase, activation and CD137 upregulation were observed on αEpCAM/CD3 BsAb treatment.
Conclusion Humanized MHC-dKO immunodeficient mice allow and refine the preclinical testing of immunotherapy agents for which experimentation is precluded in conventional immunodeficient mice due to severe xGVHD.
- Immune Checkpoint Inhibitors
- Antibodies, Bispecific
- Drug Evaluation, Preclinical
- Immune Reconstitution
- Immunologic Memory
Data availability statement
All data relevant to the study are included in the article or uploaded as supplementary information. Additional data are available upon reasonable request to the corresponding author.
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.
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- Immune Checkpoint Inhibitors
- Antibodies, Bispecific
- Drug Evaluation, Preclinical
- Immune Reconstitution
- Immunologic Memory
WHAT IS ALREADY KNOWN ON THIS TOPIC
It has been observed that major histocompatibility complex (MHC) modifications in mice engrafted with human peripheral blood mononuclear cells (PBMCs) significantly reduce xenograft-versus-host disease (xGVHD) severity, the main limitation of PBMC-humanized mouse models. However, the impact of these modifications on the antitumor immune properties and the suitability of these mice for testing the therapeutic potential of immunotherapeutic strategies remain poorly addressed.
WHAT THIS STUDY ADDS
The absence of severe xGVHD in MHC-dKO mice allows the exploration and characterization of the efficacy of checkpoint inhibitor combinations and T-cell engagers, including long-term immunity.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
This study demonstrates that humanized MHC-dKO immunodeficient mice allow and refine the preclinical testing of immunotherapy agents for which experimentation is precluded in conventional immunodeficient mice, opening up new possibilities for cancer immunotherapy research.
Introduction
The use of preclinical animal models remains a pivotal step in cancer immunotherapy development and is instrumental for choosing the compounds and strategies to move forward into the clinical setting. Animal models provide important preliminary information regarding pharmacodynamics, pharmacokinetics, synergistic/additive effects or mechanisms of resistance. However, in the era of cancer immunotherapy, the shortcomings of preclinical research tools remain a major limitation for the successful translation of scientific research breakthroughs, with regulatory approval rates below 10% for anticancer drugs that enter clinical development in phase 1 clinical trials.1 It is widely admitted that syngeneic mouse models hardly recapitulate many of the very essential features of human cancer such as progressive carcinogenesis and heterogeneity. A classical approach to overcoming these limitations has been the use of human tumor cell lines and patient-derived xenografts, which remain a critical tool for tumor cell-targeted drug development.
The advent of immunotherapy has further highlighted the limitations of current preclinical models for anticancer drug development. Human xenografts require an immunodeficient host in order to avoid rejection. Therefore, if immunotherapeutic strategies are to be tested, the murine host has to be engrafted with a competent human immune compartment, to obtain the so-called humanized murine models. A variety of human immune cell types and sources can be used to humanize mice, each with its advantages and disadvantages in terms of feasibility, experiment duration, recapitulation of the distinct immune compartments and tumor cell recognition.2 Mice humanized with CD34+ hematopoietic stem and progenitor cells (HSPCs) better recapitulate the composition of the different human immune cell types2 and show a significantly delayed xenograft-versus-host disease (xGVHD)-like wasting syndrome.3 Admittedly, mice humanized with HSPCs are today the most complete model to study the human immune system in the preclinical setting. However, the absence of human leukocyte antigen (HLA) expression, necessary for the development of HLA-restricted lymphocytes, requires the implantation in the mouse of human HLA-expressing tissues (most frequently liver and thymus)4 5 or HLA transgenes.6 7 These are possibly the most complete strategies for general immunology research, but the complexity associated with obtaining HSPCs, HLA-expressing healthy tissues and a tumor sample from the same patient makes this strategy hardly scalable8 and neoantigen specificity challenging. Furthermore, additional transfer of human genes is often required to obtain myeloid immune cell engraftment.8 9 Tumor-infiltrating lymphocytes (TILs) are an additional interesting source to humanize mice. As an advantage, tumor-specific T lymphocytes are enriched in the tumor microenvironment (TME) compared with peripheral blood,10 11 although their proportions remain low even in immunogenic tumors.12–14 By definition, TILs are autologous to tumor cells and show reduced xGVHD incidence due to tumor-specificity enrichment.15–17 However, obtaining sufficient cell counts for in vivo experiments requires in/ex vivo TIL expansion procedures.15–17
Humanization by peripheral blood mononuclear cells (PBMCs) is the most straightforward and readily available method of humanization with mature cells that can be autologous to tumor cells and contain a proportion of tumor-specific T-cell clones.18–20 Nevertheless, the administration of mature immune cells to immunodeficient mice consistently leads to the development of xGVHD.21
Murine host modification strategies have been developed to overcome this limitation, mainly the silencing of murine major histocompatibility complex (MHC) molecules. Although this strategy effectively delays the development of xGVHD,21 the characterization of human antitumor immune responses and the exploration of the pharmacodynamics of immunotherapeutic strategies remain limited.22–25 In fact, the vast majority of the research published on mouse models humanized with PBMCs is conducted with conventional NSG or NOD/Shi-scid IL2Rgnull (NOG) mice.26–30
Here we have explored alternative strategies to overcome xGVHD as one of the main limitations of humanized murine models engrafted with mature human immune cells. First, we have explored graft modifications both ex vivo and in vivo, to selectively eliminate cell populations responsible for xenoreactive immune responses. Second, we have explored a previously described MHC class I and II double knock-out NSG host (named “MHC-dKO NSG” hereafter), which consistently shows a marked reduction of severe xGVHD, and tested the impact of this modification on the efficacy of clinical-grade immune checkpoint inhibitors (ICIs) and an anti-epithelial cell adhesion molecule (EpCAM)/CD3 T-cell engager (αEpCAM/CD3 bispecific antibody (BsAb)). The significant extension of the survival of mice allowed for long-term antitumor immune response characterization and opened up new opportunities to better understand the pharmacodynamics of clinical-grade and experimental cancer immunotherapies.
Methods
Mice
NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) and MHC-dKO NSG (NOD.Cg-Prkdcscid H2-K1b-tm1Bpe H2-Ab1g7-em1Mvw H2-D1b-tm1Bpe Il2rgtm1Wjl/SzJ) mice were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA) and maintained under specific pathogen-free conditions.
Animal experiments were conducted in accordance with Spanish laws and approval was obtained from the animal experimentation committee of the University of Navarra (reference: 007–22). 6–8-week-old mice were used for in vivo experiments.
Tumor cell lines
The human colon cancer-derived HT29 cell line was purchased from the American Type Culture Collection (ATCC) in 2019. The human lung adenocarcinoma-derived H358 cell line was kindly provided by Professor Luis Montuenga’s group (CIMA Universidad de Navarra, Pamplona, Spain) in 2023, who originally acquired the cell line from the ATCC. A master cell bank was expanded on arrival and a new ampule was thawed every 4 months for experimentation. Cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin–streptomycin (10,000 U/mL-10,000 μg/mL), all from Life Technologies. The murine 4T1 breast carcinoma cell line of BALB/c origin was provided by Dr Claude Leclerc (Institute Pasteur, Paris, France). This cell line was cultured in complete media containing RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin–streptomycin (10,000 U/mL-10,000 µg/mL) and 2-mercaptoethanol (5×10–5 M), all from Life Technologies. HEK293 cells were obtained from ATCC in 2008. Cells were cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin–streptomycin (10,000 U/mL-10,000 µg/mL), all from Life Technologies.
PBMCs
Blood samples were obtained from healthy donors following informed consent according to a protocol approved by the institutional ethical committee of the Clinica Universidad de Navarra (reference: 2019–76). Blood was obtained in 10 mL EDTA tubes (BD Vacutainer) and stored at room temperature until processed. PBMCs were obtained by Ficoll gradient (GE Healthcare Biosciences).
For cell depletion experiments, CD4+ cells were depleted following negative magnetic selection according to the manufacturer’s instructions (Miltenyi). Adequate CD4+ T-cell depletion was evaluated by flow cytometry.
Antibodies and other drugs
Cyclophosphamide 1000 mg was purchased from Sigma-Aldrich. The powder was reconstituted in 0.9% saline (Braun) and aliquoted at a concentration of 15 µg/µL. The concentration used for intraperitoneal injection in in vivo experiments was 5 µg/µL.
The following antibodies were developed, produced, and quality controlled at Bristol-Myers Squibb facilities: nivolumab (Opdivo—a fully anti-human programmed death-1 (PD-1) human IgG4) and ipilimumab (Yervoy—a fully human anti-human cytotoxic T-lymphocyte antigen-4 (CTLA-4) IgG1). Control mice in experiments where nivolumab and ipilimumab were tested received drug vehicle (saline).
The anti-EpCAM-CD3 BsAb had the following structure: (IgE signal peptide-(Variable Heavy chain CD3)-(GGGGSGGGGSGGGG)-(Variable Light chain CD3)-(GGGGSGGGGSGGGG)-(Variable Light chain EpCAM)-(RATPSHNSHQVPSAGGPTANSGTSGS)-(Variable Heavy chain EpCAM). The variable region sequences targeting CD3 were derived from the anti-human CD3 monoclonal antibody 12F6.31 The variable region sequences targeting EpCAM were obtained from the MOC-31 monoclonal antibody.32 The control irrelevant vector CD3-B12 was obtained by replacing the VH and VL sequences targeting EpCAM with the corresponding sequences from the B12 antibody, which is a murine IgG1-κ neutralizing antibody against a glycoprotein of HIV-1. This glycoprotein is a non-relevant antigen in murine in vivo models.33 The additional control anti-EpCAM/B12 plasmid was obtained by combining the VH and VL sequences targeting EpCAM with the corresponding sequences of the B12 antibody. The sequences were codon-optimized for Mus musculus and synthesized and cloned by GenScript (Nanjing, China) in a pcDNA3.1 backbone. The sequence of the generated construct was verified by direct sequencing and by restriction enzyme digestion. Plasmid productions were made using EndoFree Maxi Kits from Qiagen following the manufacturer’s instructions.
In vitro experiments
Expression plasmids encoding the BsAbs were transfected and expressed in HEK293 cells with the Lipofectamine 3000 Reagent Kit (Invitrogen). HEK293 cells were seeded at a density of 750,000 cells/well the day prior to transfection. The transfection was carried out according to the manufacturer’s instructions and supernatant was collected after 48 hours of culture. Tumor cells were seeded at a density of 5×104 cells/well for HT29 and 2×104 cells/well for 4T1 cells in 96-well plates. After 24 hours, 100 µL of supernatant from transfected HEK293 cells containing the BsAbs were added to the tumor cells, and incubated for 24 hours. After 24 hours, CD3+ cells were isolated from healthy donor blood samples through Ficoll gradient and subsequent magnetic column separation (LS Columns, Miltenyi Biotec). CD3+ cells (105 per well) were added to the tumor cell culture plates and incubated for 72 hours. After 72 hours of incubation, supernatants were collected for human interferon gamma (hIFN-γ) detection by ELISA.
In vivo experiments
6–8-week-old NSG or MHC-dKO NSG mice were intravenously injected with 107 human fresh PBMCs (retro-orbital injection). HT29 cells (2×106 cells/mouse) were subcutaneously injected into the right flank of mice. H358 cells (106 cells/mouse) were subcutaneously injected into the right flank of mice, embedded in a 1:1 solution of CaCl2-free and MgCl2-free phosphate-buffered saline (PBS) (1×; Gibco) and Growth Factor Reduced Phenol-red free Matrigel Matrix (Corning, Ref. 356231). Therapeutic antibodies and cyclophosphamide were intraperitoneally injected according to each experimental design.
For hydrodynamic injections, a volume of 2 mL of saline dilution containing 10 µg of the assigned plasmid was hydrodynamically injected through the tail vein with a 27 G needle at a rate of 0.4 mL/s.
Plasma samples were obtained from submandibular vein punctures and collected in Eppendorf tubes containing 20 µL of sodium heparin. Plasma samples were stored at −80°C until analysis.
Tumor growth (digital caliper) and body weight loss were measured 3 times per week. Baseline body weight was recorded prior to tumor cell administration. Animals that developed clinical signs of xGVHD (≥20% weight loss, hunched posture, reduced mobility, fur loss, tachypnea) were sacrificed.
ELISA assays
Levels of hIFN-γ in mouse plasma samples were measured by a commercial ELISA (Human IFN-γ Elisa Set, BD OptEIA, BD Biosciences) according to the manufacturer’s instructions. All samples were measured in duplicate. The detection cut-off level of the assay is 4.6875 pg/mL.
Murine alanine aminotransferase measurements
Plasma murine alanine aminotransferase (mALT) levels were analyzed using a Roche Cobas C311 analyzer (Roche Diagnostics, Indianapolis, Indiana, USA).
Histology
Tumors and livers were formalin-fixed (3.7–4%, PanReac AppliChem, ITW Reagents) for 72 hours and maintained in 70% ethanol, dehydrated, and paraffin-embedded according to standard protocols conducted at the Morphology Core Facility at CIMA. Five-micrometer sections were stained in H&E. For CD3 immunohistochemistry, a rabbit anti-human CD3 antibody (clone SP7—Epredia RM-9107-S Thermo Fisher Scientific) was used. Image acquisition was performed on an Aperio CS2 slide scanner and processed with the ScanScope software (Leica Biosystems).
Multiplex immunofluorescence staining and analysis
Multiplex immunofluorescence staining was performed on a Bond RX autostainer (Leica Biosystems), as previously described.34 35 Briefly, 4 μm-thick formalin-fixed, paraffin-embedded (FFPE) tissue sections were deparaffinized (Bond DeWax, Leica Biosystems) and rehydrated per standard protocols. Antigen retrieval was performed with BOND Epitope Retrieval Solution 1 (ER1, Leica Biosystems) or 2 (ER2, Leica Biosystems, product number AR9640), followed by sequential cycles of staining with each cycle including a 30 min combined block and primary antibody incubation (Akoya antibody diluent/block), followed by a secondary horseradish peroxidase- (HRP)-conjugated polymer. Signal amplification was achieved with TSA-Opal fluorophores. Between cycles of staining, tissue sections underwent heat-induced epitope retrieval to remove the primary/secondary-HRP antibody complexes before staining with the next antibody. Two multiplex immunofluorescence panels were performed. Primary antibodies and corresponding fluorophores were:
Panel 1: anti-CD3 (rabbit polyclonal, IgG, ready-to-use, Agilent, product number IR503) in Opal 520; anti-CD8 (mouse monoclonal, clone C8/144B, ready-to-use, Agilent, product number GA62361-2) in Opal 480; and anti-cytokeratin (mouse monoclonal, clone AE1/AE3, ready-to-use, Leica Biosystems, product number NCL- L-AE1/AE) in Opal 780. Panel 2: anti-CD137 (TNFRSF9 or 4-1BB, mouse monoclonal, clone BBK-2, 1:80, Thermo Fisher, product number MA5-13736) in Opal 520; anti-CD8 (mouse monoclonal, clone C8/144B, ready-to-use, Agilent, product number GA62361-2) in Opal 480; and anti-cytokeratin (mouse monoclonal, clone AE1/AE3, ready-to-use, Leica Biosystems, product number NCL-L-AE1/AE) in Opal 780. Nuclei were counterstained with Spectral DAPI (Akoya Biosciences, FP1490). Stained slides were then mounted in a ProLong Diamond Antifade mounting medium (Thermo Fisher Scientific). Slides were further scanned using the PhenoImager HT Automated Quantitative Pathology Imaging System (Akoya Biosciences). After image acquisition, unmixing of the spectral libraries was performed with inForm software (Akoya Biosciences). Unmixed images were then imported into the open-source digital pathology software QuPath V.0.4.4 for stitching, cell segmentation and cell phenotyping. A supervised machine learning algorithm trained on expert-provided examples was used for classifying cells as: CD3+, CD8+, CD137+, and tumorous (AE1/AE3+). CD4+ T cells were defined as CD3+CD8–. The densities of each cell population were quantified and expressed as the number of cells per mm2.
Flow cytometry
Liver and tumor tissue were harvested for flow cytometry analysis. Tumors were minced and digested with 400 U/mL collagenase D and 50 µg/mL DNase-I (Roche) for 20 min at 37°C. Tissue digestion was stopped by adding 12.5 µL of 0.5 M EDTA (Invitrogen) to each sample. Single-cell suspensions were obtained by passing samples through 70 µm cell strainers (Falcon). For peripheral blood staining, whole blood (125–150 µL/sample, depending on availability) was centrifuged and washed in PBS twice (5,000 rcf, 5 min). Afterwards, corresponding surface-staining antibody mixes were added to each sample and incubated for 10 min at 4°C. For red cell lysis, FACS Lysing Solution 10× (BD Biosciences, Catalog #349202, diluted to 1× in distilled water) was added to each sample for 5 min. Samples were then centrifuged at 5,000 rcf for 1 min and cleaned in sorting buffer (PBS 1× with 0.5% fetal bovine serum(FBS), 0.5% of 0.5 M EDTA and 1% of penicillin/streptomycin 104 U/µg/mL (Gibco)) prior to intracellular protein staining.
Samples were treated with 10 mg/mL of beriglobin (CSL Behring, Marburg, Germany) and surface stained with suitable combinations of the following fluorochrome-labeled antibodies: mouse anti-hCD3 (Clone: UCHT1)-AF647 (BioLegend), mouse anti-hCD3 (Clone: UCHT1)-AF488 (BioLegend), mouse anti-hCD8 (Clone: SK-1)-BV510 (BioLegend), mouse anti-hCD4 (Clone: OKT-4)-BV650 (BioLegend), mouse anti-hCD4 (Clone: RPA-T4)-BV605 (BD Biosciences), mouse anti-hCD14 (Clone: M5E2)-PECy7 (BioLegend), mouse anti-hCD16 (Clone: 3G8)-BV510, (BioLegend), mouse anti-hCD19 (Clone: HIB19)-BV650 (BioLegend), mouse anti-hCD45 (Clone: HI30)-PB (BioLegend), mouse anti-hCD45RO (Clone: UCHL1)-PECy7 (BD Biosciences), mouse anti-hCD56 (Clone: HCD56)-PE/Dazzle594 (BioLegend), mouse anti-hPD-1 (Clone: EH12.2H7)-PerCP-Cy5.5, mouse anti-hCCR7 (Clone: 3D12)-AF647 (BD Biosciences), mouse anti-hPD-1 (Clone: EH12-2H7)-PECy7 (BioLegend), mouse anti-hCD25 (Clone: BC96)-BV421 (BioLegend), mouse anti-hCD137 (Clone: 4B4-1)-PECy7 (BioLegend), mouse anti-hCD137 (Clone: 4B4-1)-BV421 (BioLegend), mouse anti-human granzyme B (Clone: GB11)-PE (BD Biosciences), mouse anti-hKi67 (Clone: Ki67)-AF488 (BioLegend), mouse anti-hFoxP3 (Clone: PCH101)-PE (Invitrogen), mouse anti-hEpCAM (Clone: 9C4)-PerCP-Cy5.5 (BioLegend) and rat anti-mEpCAM (Clone: G8.8)-PE (BioLegend). Cell viability was analyzed with Zombie-NIR Dye (1:1,000 dilution, BioLegend) or Live/Dead Near IR 876 nm marker (1:1,000 dilution, Invitrogen). The CytoFLEX S and CytoFLEX LX cytometers were used for data acquisition and the CytExpert V.2.5 software was used for data analysis.
Reverse transcription-quantitative PCR
Total RNA was obtained with a Maxwell RSC simply RNA Tissue kit, following the manufacturer’s instructions. Subsequently, a specific reverse transcription (RT) reaction was performed for the generation of complementary DNA (cDNA). RNA was pretreated with DNAse I using the TURBO DNAfree Kit (Ambion, #1908) and RT was carried out from 500 ng of total RNA in a final volume of 20 µL. To carry out the reaction, a mix with 10 mM dNTPs, 250 ng/mL random primers, 40 U/µL of RNase Inhibitor, 1× Buffer and 200 U/µL of M-MLV Reverse Transcriptase (Invitrogen, #28025013) were prepared. After adding the mix, samples were incubated for 1 hour at 37°C and 1 min at 95°C. Then, each cDNA was diluted with nuclease-free water to a final concentration of 50 ng/4.6 µL.
To perform the quantitative PCR (qPCR), the GoTaq qPCR Master Mix was used with 50 ng of cDNA. qPCR was carried out using the CFX96 Real-Time Detection System (BioRad). The results were normalized to the human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) gene transcripts for each sample. The relative value for the expression level of hIFN-γ was calculated by the equation Y=2−∆Ct, where Ct is the point at which the fluorescence rises significantly above baseline, and ∆Ct is the difference between control and target products (∆Ct=Ct hINF-γ – Ct GAPDH). Forward and reverse primer sequences for hIFN-γ were 5’-CTCTGCATCGTTTTGGGTTC-3’ and 5’-GCGTTGGACATTCAAGTCAG-3’, respectively. Forward and reverse primer sequences for hGAPDH were 5’- GGTCGGAGTCAACGGATTT −3’ and 5’- CCAGCATCGCCCACTTGA-3’, respectively.
Statistical analysis
Statistical analyses were performed with the Prism V.8.0.2 version software (GraphPad). Continuous unpaired variables were analyzed with the Mann-Whitney test. Continuous paired variables were analyzed with the Wilcoxon matched-pairs signed-rank test. Correlations between continuous variables were calculated with the Spearman test and linear regression lines were represented on correlation graphs. Survival was described following the Kaplan-Meier method and analyzed between groups with the log-rank (Mantel-Cox) test. Event-free survival analyses for xGVHD included ≥20% weight loss or death from any cause (except humane sacrifice due to excessive tumor burden) preceded by a ≥10% weight loss as events. A two-tailed p<0.05 was considered as statistically significant. Tumor growth differences were analyzed by two-way analysis of variance tests. When differences are statistically significant, the significance is represented with asterisks (*): *for a p value<0.05, **for a p value<0.01, ***for a p value<0.001, ****for a p value<0.0001. Error bars indicate the SEM unless otherwise indicated. In grouped body weight loss and tumor growth graphs, curves were not further represented when ≥50% of the individuals within a group were missing to avoid misleading representations.
Patients and public involvement
Patients were not involved in the conduction of the experiments presented in this document.
Results
CD4+ T-cell depletion from the PBMC inoculum significantly reduces xGHVD but also abrogates antitumor activity
Previous data from our group explored the influence of diverse ex vivo graft modifications on xGVHD-associated weight loss. We observed that mice engrafted with PBMCs devoid of CD4+ T cells and treated with a combination of nivolumab and urelumab did not develop significant xGVHD as compared with their counterparts humanized with total PBMCs.36 This finding has been confirmed by others.37 In keeping with these notions, we explored the effect of human total PBMCs or PBMCs devoid of CD4+ T cells once engrafted in NSG mice bearing tumors derived from a human colon cancer cell line (HT29) (figure 1A). CD4+ T-cell depletion was carried out by negative magnetic selection and verified by flow cytometry (online supplemental figure 1A). Results confirmed that xGVHD development was highly dependent on the presence of CD4+ T cells, both in terms of body weight loss (figure 1B,C) and liver toxicity (figure 1D). Proportionally, the antitumor effect mediated by PBMCs was completely lost in the absence of human CD4+ T cells (figure 1E,F). As a measure of human immune cell activation, hIFN-γ was assessed in the plasma of humanized mice. Plasmatic hIFN-γ was clearly observed in mice engrafted with total PBMCs, but was significantly lower in mice engrafted with PBMCs devoid of CD4+ T cells over time (figure 1G). These results indicate that in these humanized experimental conditions, human CD4+ T cells are critical for xGVHD, but also for antitumor activity.
Supplemental material
Ex vivo graft modification by CD4+ T-cell depletion significantly attenuates xGVHD but also abrogates antitumor activity. (A) Schematic representation of the experiment. (B) Individual relative body weight loss curves by experimental groups. The dashed line at −20% represents the threshold for a humane endpoint. (C) EFS analysis by experimental groups. At day +54 all mice had reached the endpoint criteria for sacrifice due to tumor burden or body weight loss. (D) mALT plasma levels throughout the experiment. The dashed line represents the upper limit of normality (50 U/L). (E) Individual tumor growth curves. The dashed line at 15 mm represents the tumor burden limit for a humane endpoint. (F) Tumor growth curves of the experimental groups. The dashed line at 15 mm represents the tumor burden limit for a humane endpoint. (G) hIFN-γ plasma concentrations throughout the experiment. Body weight ∆, relative percentage of body weight loss; EFS, ≥20% body weight loss event-free survival; hIFN-γ, human interferon gamma; i.v., intravenous; mALT, mouse alanine aminotransferase; PBMCs∆CD4, CD4-depleted peripheral blood mononuclear cells; s.c., subcutaneous; xGVHD, xenograft-versus-host disease; Φ, diameter.
Post-transplantation cyclophosphamide does not significantly reduce xGVHD and curbs antitumor activity
Recent data have confirmed in clinical practice that high-dose cyclophosphamide administration a few days after allogeneic bone marrow transplantation significantly reduces GVHD incidence, affecting preferentially the most proliferative and alloreactive T-cell clones.38 39 Following this experience, we decided to explore if intraperitoneal cyclophosphamide administration post-PBMC engraftment was able to selectively deplete xenoreactive T-cell clones thereby limiting xGVHD and sparing the antitumor effect. First, we identified a 50 mg/kg cyclophosphamide dose as tolerable and active to modify xGVHD (data not shown). Second, we explored different schedules of intraperitoneal cyclophosphamide administration: day +3, +5 or +7 post-PBMC engraftment (figure 2A). We injected the human HT29 colorectal carcinoma cell line subcutaneously on day +8. Previously, we tested that cyclophosphamide administration 24 hours prior to tumor inoculation did not show a significant impact on tumor growth (data not shown). Results confirmed that early cyclophosphamide administration (day +3) significantly reduced xGVHD-associated body weight loss (figure 2B,C) and led to a numerically lower incidence of plasma mALT elevations over the upper limit of normality (online supplemental figure 2A). However, this schema also abrogated the antitumor effect of PBMCs (figure 2D,E). Intermediate-term cyclophosphamide (day +5) preserved antitumor efficacy but did not significantly delay xGVHD-associated body weight loss (figure 2B–E). Finally, late cyclophosphamide administration (day +7) did not show any significant effect on weight loss or tumor growth control, suggesting that the proliferation peak of all T-cell clones occurs earlier (figure 2B–E). Consistently, plasma hIFN-γ levels showed a significant drop in mice treated with early cyclophosphamide (day+3), but no significant impact at the other two time points (figure 2F). These data suggest that even though xGVHD and antitumor effects appear to be differentially affected by cyclophosphamide, the window of opportunity to explore antitumor activity is too narrow to be exploited experimentally.
Supplemental material
Post-transplantation cyclophosphamide does not significantly delay xenograft-versus-host disease and curbs antitumor activity. (A) Schematic representation of the experiment. (B) Individual relative body weight loss curves. The dashed line at −20% represents the threshold for a humane endpoint. (C) Relative body weight loss of the experimental groups. The dashed line at −20% represents the threshold for a humane endpoint. Statistical significances of comparisons with the HT29 control group are not shown for simplification. (D) Individual tumor growth curves. The dashed line at 15 mm represents the tumor burden limit for a humane endpoint. (E) Tumor growth curves of the experimental groups. The dashed line at 15 mm represents the tumor burden limit for a humane endpoint. Statistical significances of comparisons with the HT29 control group are not shown for simplification. (F) hIFN-γ plasma levels throughout the experiment. Body weight ∆, relative percentage of body weight loss; Cy, cyclophosphamide; D3, day +3 of the experiment; D5, day +5 of the experiment; D7, day +7 of the experiment; hIFN-γ, human interferon gamma; i.p., intraperitoneal; i.v., intravenous; PBMC, peripheral blood mononuclear cell; s.c., subcutaneous; Φ, diameter.
Severe xGVHD is abrogated in MHC-dKO NSG mice ((KbDb)null (IA)null), while the antitumor effect is preserved
It has been previously described that xGVHD is preferentially mediated by major mismatches between transferred human T-cell receptors (TCR) and murine H-2 molecules.40 Furthermore, it has been observed that murine MHC modifications significantly influence xGVHD incidence and severity in mice engrafted with human PBMCs.21 However, the impact of these modifications on the antitumor immune properties in this model has been poorly addressed. We first evaluated the impact of silencing MHC class I and II on the engraftment of human PBMCs. Both strains of mice, NSG and MHC-dKO NSG, were co-engrafted with human HT29 carcinoma cells and human PBMCs. Human PBMCs were analyzed longitudinally over time in peripheral blood and in the tumor at the end of the experiment (online supplemental figure 3A). As previously described,21 we observed a rapid shift toward a T-cell enriched composition in peripheral blood (online supplemental figure 3B). From day +8, there was a marked peripheral blood human CD45+ cell expansion, in particular in NSG mice (online supplemental figure 3C), although no major immune cell type composition differences were observed between the two mouse strains in blood or tumor tissue (online supplemental figure 3D–G), with the exception of a higher abundance of circulating regulatory T cells in NSG mice (online supplemental figure 3H). Interestingly, and consistently with a previous report,21 we observed that circulating naive (CCR7+CD45RO–) and effector (CCR7–CD45RO–) CD8+ T cells persisted longer in MHC-dKO NSG mice as compared with NSG counterparts (online supplemental figure 3I–K). We then tested the impact of silencing MHC class I and II on both xGVHD and tumor growth control, engrafting human PBMCs in NSG and MHC-dKO NSG mice. Both groups of mice were co-engrafted with human HT29 carcinoma cells and human PBMCs (figure 3A). NSG mice showed significant weight loss (figure 3B,C) and significant plasma mALT increase compared with controls with no PBMCs as early as day+2, and more remarkably on day+22 (figure 3D), both indicators of xGVHD. In contrast, MHC-dKO NSG mice did not show weight loss (figure 3B,C), or any significant changes in plasma mALT levels on day +2 or +22 (figure 3D). Antitumor activity was observed in both groups, NSG and MHC-dKO NSG mice engrafted with PBMCs, when compared with control groups (figure 3E,F). Nevertheless, the duration and characterization of responses in NSG mice were limited due to xGVHD (figure 3E,F), while in the MHC-dKO NSG group, three out of eight mice achieved complete responses which were maintained in two of them at day +53 (figure 3E,F). Consistently, detectable plasma hIFN-γ levels were observed in both NSG and MHC-dKO NSG mice, but NSG individuals showed on average fivefold higher concentrations than MHC-dKO NSG mice (figure 3G). These data indicate that MHC class I and II gene deletions uncouple antitumor efficacy and xGVHD toxicity phenomena.
Supplemental material
Xenograft-versus-host disease is significantly attenuated in MHC I/II-dKO NSG mice, while the antitumor effect of PBMCs is preserved. (A) Schematic representation of the experiment. (B) Individual relative body weight loss curves. The dashed line at −20% represents the threshold for a humane endpoint. (C) Relative body weight loss curves by experimental groups. The dashed line at −20% represents the threshold for a humane endpoint. (D) mALT plasma levels throughout the experiment. The dashed line represents the upper limit of normality (50 U/L). (E) Individual tumor growth curves. The dashed line at 15 mm represents the tumor burden limit for a humane endpoint. (F) Tumor growth curves of the experimental groups. The dashed line at 15 mm represents the tumor burden limit for a humane endpoint. (G) hIFN-γ plasma levels throughout the experiment. Both NSG and dKO mice with no PBMC administration showed negligible plasma hIFN-γ levels (mean of 5.6 and 12.1 pg/mL, respectively) and were not represented for simplification. Body weight ∆, relative percentage of body weight loss; CR, complete response rate; dKO, MHC-dKO NSG mice; hIFN-γ, human interferon gamma; mALT, mouse alanine aminotransferase; MHC, major histocompatibility complex; PBMC, peripheral blood mononuclear cells; s.c., subcutaneous; Φ, diameter; .
T-cell density and activation are attenuated in MHC-dKO NSG mice livers, while the tumor immune infiltrate remains comparable
We explored whether MHC class I and II silencing produced significant changes in terms of immune infiltration of the liver (as an xGVHD target organ) and of tumors. Following an experimental design as in figure 3, we again observed significant body weight decrease in NSG mice engrafted with human PBMCs starting on day +20 (online supplemental figure 4A). We then decided to sacrifice mice on day +25 to analyze liver and tumor immune infiltrates (figure 4A). Liver human CD3+ (figure 4B–D), CD8+ (figure 4E) and CD4+ (figure 4F) T-cell infiltration was significantly higher in NSG mice compared with MHC-dKO NSG mice. Additionally, both CD8+ (figure 4G,H) and CD4+ (figure 4I,J) T cells showed a more intense expression of the cytotoxic marker granzyme b. Accordingly, plasma hIFN-γ levels were significantly higher in NSG mice (online supplemental figure 4B). In contrast, tumor human CD3+ (figure 4B,C,K), CD8+ (figure 4L) and CD4+ (figure 4M) T-cell infiltration was comparable in NSG and MHC-dKO NSG groups. Activation markers in TILs did not show consistent differences between NSG and MHC-dKO NSG groups (online supplemental figure 4C–H), with the exception of CD137, which was significantly higher in tumor-infiltrating CD8+ T cells in NSG mice (online supplemental figure 4I,J). Further exploration of the tumor microenvironment showed a higher proportion of CD4+CD25+ cells in tumors compared with livers, in both NSG and MHC-dKO NSG mice (online supplemental figure 4K,L). Overall, these data show that MHC silencing in the NSG mouse model is associated with a significant decrease in liver inflammation, while the abundance and overall phenotypic characteristics of TILs are preserved.
Supplemental material
Liver as a target organ of xenograft-versus-host disease showed significantly higher human T-cell density and activation in NSG mice as compared with MHC-dKO NSG mice, while the tumor immune infiltrate remains comparable. (A) Schematic representation of the experiment. (B) Representative CD3 immunohistochemistry and H&E microphotographs of liver and tumor sections from NSG and MHC-dKO NSG mice. The bar in low-power magnification images represents 2 mm except for the “Tumor NSG” sample, where it represents 600 µm. Dashed squares represent the area amplified in high-power magnification images. The bar in high-power magnification images represents 200 µm (C). Percentage of CD3+ T cells among parental live cell events in cell suspensions from livers and tumors from NSG and MHC-dKO NSG mice. The gate for CD3+ cells with their proportion among total live events is shown. (D) CD3+ T cells in livers (proportions of live events and counts per mg of tissue). (E) CD8+ T cells in livers (proportions of live events and counts per mg of tissue). (F) CD4+ T cells in livers (proportions of live events and counts per mg of tissue). (G) GzmB expression on CD8+ cells in livers (proportions and MFIs). (H) Representative histograms of GzmB expression on CD8+ T cells in liver samples. The vertical line in the histograms represents the positivity threshold established with the FMO control. (I) GzmB expression on CD4+ cells in livers (proportions and MFIs). (J) Representative histograms of GzmB expression on CD4+ T cells in liver samples. The vertical line in the histograms represents the positivity threshold established with the FMO control. (K) CD3+ T cells in tumors (proportions of live events and counts per mg of tissue). (L) CD8+ T cells in tumors (proportions of live events and counts per mg of tissue). (M) CD4+ T cells in tumors (proportions of live events and counts per mg of tissue). dKO, MHC-dKO NSG; FMO, fluorescence minus one; FSC, forward scatter; GzmB, granzyme B; i.v., intravenous; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; PBMC, peripheral blood mononuclear cell; s.c., subcutaneous.
The humanized MHC-dKO NSG model enables the antitumor activity characterization of clinical-grade immune checkpoint inhibitors nivolumab plus ipilimumab
Despite the advent and success of the so-called ICIs in the clinic, we are largely missing useful biomarkers guided by a precise understanding of the mechanism of action of these agents.41 One important limitation is the absence of in vivo models where these agents can be tested and characterized. We decided to explore the antitumor effect of clinical-grade immunotherapy in our models testing the combination of nivolumab (anti-PD-1 monoclonal antibody (mAb)) and ipilimumab (anti-CTLA-4 mAb) (figure 5A). We observed that the combined immunotherapy significantly accelerated xGVHD in NSG mice engrafted with human PBMCs in terms of body weight loss (figure 5B). It also produced a tendency toward higher plasma mALT levels (figure 5C). This precluded the characterization of antitumor responses and any long-term follow-up in this group (online supplemental figure 5A,B). In contrast, early events of weight loss ≥20% were not observed in MHC-dKO NSG mice regardless of immunotherapy administration, enabling a longer follow-up (figure 5B). MHC-dKO NSG mice treated with ipilimumab plus nivolumab showed a non-significant plasma mALT elevation at day +15 of the experiment (figure 5C), but this hypertransaminasemia did not have any impact on body weight loss or survival in this group. Of note, mALT elevation was significantly higher in NSG mice as compared with MHC-dKO NSG mice treated with immunotherapy (figure 5C). PBMCs significantly controlled tumor growth in both MHC-dKO NSG (figure 5D,E) and NSG (online supplemental figure 5A,B) mice. This effect was significantly improved with nivolumab and ipilimumab in MHC dKO NSG mice (figure 5D,E), but was not evaluable due to xGVHD in NSG mice (online supplemental figure 5A,B). Plasma hIFN-γ levels showed significantly higher levels in mice treated with nivolumab plus ipilimumab when compared with control groups (figure 5F; online supplemental figure 5C). Similar results were observed using the human H358 lung adenocarcinoma cell line (online supplemental figure 6). Overall, MHC-dKO NSG mice enabled the observation of profound and durable antitumor immune responses to nivolumab plus ipilimumab that were not assessable in NSG mice due to accelerated xGVHD.
Supplemental material
Supplemental material
The humanized MHC-dKO NSG model enables the evaluation of the antitumor activity of the nivolumab plus ipilimumab combination. (A) Schematic representation of the experiment. (B) EFS analysis by experimental groups. Control mice with HT29 tumors with no PBMCs were not represented for simplification. (C) mALT plasma levels at day +15 of the experiment. The dashed line represents the upper limit of normality (50 U/L). Control mice with HT29 tumors with no PBMCs were not represented for simplification (no mALT levels >50 U/L detected). (D) Individual tumor growth curves. The dashed line at 15 mm represents the tumor burden limit for a humane endpoint. (E) Tumor growth curves of the experimental groups. The dashed line at 15 mm represents the tumor burden limit for a humane endpoint. (F) hIFN-γ plasma levels at day +8 of the experiment. dKO mice with no PBMC administration showed undetectable plasma hIFN-γ levels and were not represented for simplification. CR, complete response rate; dKO, MHC-dKO NSG mice; EFS, ≥20% body weight loss event-free survival; hIFN-γ, human interferon gamma; Ipi, ipilimumab; i.p., intraperitoneal; i.v., intravenous; mALT, mouse alanine aminotransferase; MHC, major histocompatibility complex; Nivo, nivolumab; PBMC, peripheral blood mononuclear cell; s.c., subcutaneous; Φ, diameter.
The humanized MHC-dKO NSG model enables long-term examination of antitumor effects of a T-cell engager
Bispecific anti-CD3 agents (αCD3 BsAbs) are a promising strategy to exploit the cytotoxic potential of T lymphocytes and have already been approved for some hematologic malignancies and metastatic uveal melanoma and small cell lung cancer.42 43 In order to refine our knowledge of the mechanism of action of αCD3 BsAbs, it is critical to develop suitable preclinical in vivo models. We evaluated the efficacy of an αEpCAM/CD3 BsAb in NSG and MHC-dKO NSG mice (figure 6A). We confirmed that murine EpCAM was spared by the BsAb in vitro (online supplemental figure 7A,B), as well as we confirmed that none of the two Fab regions of the BsAb showed an antitumor activity when the specificity of the second Fab region was modified (online supplemental figure 7C). As we observed before, NSG mice showed a significantly shorter ≥20% weight loss-free survival (figure 6B) and a tendency to plasma mALT increase 4 days after being treated with the αEpCAM/CD3 BsAb (figure 6C), which therefore limited the experimental capabilities of the model. In contrast, MHC-dKO NSG mice did not show weight loss or mALT elevation enhancement attributable to the αEpCAM/CD3 BsAb (figure 6B,C). Tumor response rates were similar in NSG and MHC-dKO NSG mice (figure 6D; online supplemental figure 8A,B). However, the limited survival of NSG mice precluded long-term study of tumor responses (figure 6D,E; online supplemental figure 8A,B). Similar results were observed in experiments using the H358 human lung adenocarcinoma cell line (online supplemental figure 9). To further characterize responses to the αEpCAM/CD3 BsAb, we repeated the same experiment only with MHC-dKO NSG mice. Consistent with the first experiment, we observed three complete responses out of five treated mice (online supplemental figure 8C,D). At day +54 of the experiment, longer than 30 days from the confirmed complete response, the three mice were rechallenged with HT29 tumor cells. Tumor-naive mice engrafted with the same PBMCs from day −7, but not previously exposed to HT29 tumor cells, were used as a control group. HT29 tumor growth was observed in control mice, while two out of three rechallenged mice spontaneously rejected the new tumor inoculi (figure 6F). As late as at day +86 of the experiment, circulating human T-cell counts were not significantly different between the two mice groups (online supplemental figure 8E). We also checked that tumor progression was not caused by a lack of tumor infiltration by lymphocytes at late time points, since persistent CD8+ and CD4+ TILs were identified by flow cytometry at day +90 (online supplemental figure 8F).
Supplemental material
Supplemental material
Supplemental material
The humanized MHC-dKO NSG model is suitable for testing the long-term efficacy of T-cell engagers, including tumor-rechallenge experiments. (A) Schematic representation of the experiment. The irrelevant control plasmid contained the sequence for an anti-HIV-1/CD3 antibody, considered irrelevant in this model. (B) EFS analysis by experimental groups. Control mice with HT29 tumors with no PBMCs were not represented for simplification. (C) mALT plasma levels at day +11 of the experiment. The dashed line represents the upper limit of normality (50 U/L). Control mice with HT29 tumors with no PBMCs were not represented for simplification (no >50 U/L mALT levels detected). (D) Individual tumor growth curves. The dashed line at 15 mm represents the tumor burden limit for a humane endpoint. (1): one of five mice in the HT29+PBMCs + αEpCAM/CD3 pl. group was found dead at day +8 with no previous signs of xenograft-versus-host disease. (E) Tumor growth curves of the experimental groups. The dashed line at 15 mm represents the tumor burden limit for a humane endpoint. (F) Individual tumor growth curves in mice rechallenged with HT29 tumor cells. “Tumor-naive” represents control mice engrafted with the same PBMCs as tumor-rechallenged mice at the same day but not exposed previously to HT29 tumor cells. αEpCAM/CD3, anti-epithelial cell adhesion molecule/CD3; CR, complete response rate; dKO, MHC-dKO NSG mice; EFS, ≥20% body weight loss event-free survival; h.d., hydrodynamic administration; hIFN-γ, human interferon gamma; i.v, intravenous; Irr. pl., irrelevant plasmid (B12/CD3 bispecific antibody); mALT, mouse alanine aminotransferase; MHC, major histocompatibility complex; PBMC, peripheral blood mononuclear cells; pl., plasmid; s.c., subcutaneous; Φ, diameter.
Overall, data show that MHC-dKO NSG mice can be used to characterize T-cell engager strategies and suggest that αCD3BsAb might be capable of inducing protective long-term immune responses.
The PBMC-humanized MHC-dKO NSG model enables the pharmacodynamic characterization of a T-cell engager
To characterize the pharmacodynamics of αEpCAM/CD3 BsAb, we performed an experiment similar to the one presented in figure 6. In this case, mice treated with BsAb at day +10 and sacrificed at the time tumors started to shrink (day +20 post-PBMC engraftment and +72-hour post-αEpCAM/CD3 BsAb treatment) (figure 7A and online supplemental figure 10A). Tumors and livers were collected from mice and analyzed by flow cytometry and multiple immunofluorescence. As expected by the mechanism of action of T-cell engagers, mice treated with the αEpCAM/CD3 BsAb showed a significantly higher abundance of CD3+ TILs as compared with control mice (figure 7B), which was predominantly dependent on CD8+ T cells (online supplemental figure 10B). In contrast, no differences were observed in the abundance of lymphocytes between livers from both mice groups (figure 7B and online supplemental figure 10B). Additionally, we studied the change in CD137 expression, a membrane receptor upregulated by CD3+ T-cell activation signaling. A higher CD137 membrane expression within the tumor-infiltrating CD8+ T-cell compartment was observed in treated tumors but not in the liver (figure 7C and online supplemental figure 10C). Tumor-infiltrating CD4+ T cells showed a tendency that did not reach statistical significance (online supplemental figure 10D). A transient and significant elevation of plasma hIFN-γ was observed 24 hours after plasmid administration only in αEpCAM/CD3 treated mice (figure 7D). When local hIFN-γ expression was studied by RT-qPCR, a significant increase in the tumor tissue, but not in the liver, of treated mice was observed (figure 7E). Finally, multiple immunofluorescence analyses of tumor tissue showed a significant decrease in cytokeratin-positive (CK+) epithelial tumor cell density (figure 7F) and a visual tumor clearance in treated mice when compared with the control group (online supplemental figure 10E). This analysis also showed CD137 surface expression on tumor-infiltrating CD8+ T cells, which were spatially in contact with tumor cells in mice treated with the αEpCAM/CD3 BsAb, but not in control mice (figure 7G and online supplemental figure 10F).
Supplemental material
EThe PBMC-humanized MHC-dKO NSG model enables the pharmacodynamic characterization of a T-cell engager. (A) Schematic representation of the experiment. The irrelevant control plasmid contained the sequence for an anti-HIV-1/CD3 BsAb (B12/CD3), which is irrelevant to the epitopes in this model. Tumors were treated 3 days later compared with the experiment represented in figure 6 to have a sufficient residual tumor mass for downstream analyses. (B) CD3+ T-cell infiltration of tumors and livers as a percentage of total live cells by flow cytometry. (C) CD137 expression in the CD8+ T-cell compartment in tumors and livers by flow cytometry. (D) Plasma hIFN-γ levels 24 and 72 hours after plasmid delivery (days +11 and +13 of the experiment). Intergroup differences were analyzed with the Mann-Whitney unpaired test, while intragroup differences were analyzed with the Wilcoxon paired test. (E) Tumor and liver hIFN-γ transcriptional activity normalized to hGAPDH (housekeeping gene) transcripts by reverse transcription quantitative PCR. (F) CK-positive cell quantification per mm2. Note that one out of six tumor samples in the αEpCAM/CD3 group is missing because flow cytometry analysis was prioritized due to the small residual tumor size. (G) Representative multiple immunofluorescence images of tumors in each treatment group. Scale bars in low-magnification and high-magnification boxes represent 150 and 50 µm, respectively. The white dashed-line boxes indicate high-magnification areas. BsAb, bispecific antibody; CK, broad-spectrum anti-cytokeratin (AE1/AE3); DAPI, 4′,6-diamidino-2-phenylindole; EpCAM, epithelial cell adhesion molecule; hGAPDH, human glyceraldehyde-3-phosphate dehydrogenase; hIFN-γ, human interferon gamma; i.v., intravenous; Irr. pl., irrelevant plasmid (B12/CD3 BsAb); PBMC, peripheral blood mononuclear cells; s.c., subcutaneous.
Overall, we have observed an increase in the number and activation of tumor-infiltrating T cells, which confirms the mode of action of these agents; and reveals CD8+ T-cell upregulation of the co-stimulatory receptor CD137, which as a result may offer a potential target for new-generation tri-specific T-cell engagers.44 These data illustrate how PBMC-humanized MHC-deficient models are suitable to characterize the mode of action of novel cancer immunotherapies.
Discussion
Preclinical models to shape and modulate an antitumor immune response in a human context are an urgent unmet need to support the development of new cancer immunotherapy strategies. One of the most feasible models, in this regard, are murine models humanized with mature PBMCs. Circulating PBMCs are readily available in high counts from healthy donors and patients with cancer with a preserved functional status and can contain a representation of tumor-specific T-cell clones in the latter.18–20 Nevertheless, the xenoreactivity of mature human T cells against mouse cells, which is reportedly less dependent on antigen-specificity than MHC-matched interactions,40 typically limits the utility and interpretation of this approach.40
Overall, we have explored three different strategies to reduce xGVHD. First, an ex vivo PBMC graft modification by depleting CD4+ T cells. This approach demonstrated that both xGVHD and antitumor effect are CD4-dependent in this model. Second, high-dose cyclophosphamide post-PBMC transplantation was attempted to selectively target xenoreactive lymphocyte clones that presumably enter the cell cycle faster and more intensely than other clones. Nevertheless, the interventional window appears too narrow to attenuate xenoreactivity while preserving the antitumor effect in this model. Finally, recipient mouse deficiency of MHC class I and II was the most effective approach to mitigate xGVHD while preserving the antitumor potential of PBMCs.
Like others before,21 we have observed that MHC class I-deficient and II-deficient mice have a significantly longer survival than their MHC-proficient counterparts on PBMC transfer. We have analyzed the liver as an xGVHD target organ and observed progressive liver damage over time in MHC-proficient mice with a concomitant elevation of plasma mALT levels. Furthermore, when these mice started to show weight loss (a major sign of xGVHD), we observed significantly more abundant liver infiltration by human T cells with more intense granzyme B expression. These clinical, analytical and pathological signs of xGVHD were significantly diminished in MHC-deficient mice, enabling the performance of more durable and refined experiments.
For the first time, tumor immune infiltration and efficacy of cancer immunotherapy strategies were compared between MHC-deficient and proficient mice on PBMC transfer. The abundance of TILs and the CD4/CD8 composition were similar in both mouse strains, suggesting that xenoreactivity is not a crucial factor in the tumor immune microenvironment in this model.
More importantly, the absence of murine MHC did not preclude the antitumor effect of the ICI combination of nivolumab plus ipilimumab, or the αEpCAM/CD3 BsAb. Additionally, the longer survival of mice in the absence of MHC allowed a longer follow-up and a better characterization of complete responses to these therapies. Of note, we used a hydrodynamic gene transfer approach to convert hepatocytes into endogenous producers of the αEpCAM/CD3 BsAb.
Complete responses to ipilimumab plus nivolumab were not observed in NSG mice, because this treatment markedly accelerated xGVHD and mice died before responses occurred. Furthermore, plasma hIFN-γ elevations on treatment with nivolumab and ipilimumab were more significant in MHC-deficient mice as compared with MHC-proficient mice. NSG mice consistently showed higher baseline plasma hIFN-γ levels, which are presumably of xenoreactive origin to a higher degree and hinder the observation of pharmacodynamic differences induced by enhancer cancer immunotherapies.
Complete responses to the αEpCAM/CD3 BsAb were observed in both MHC-deficient and proficient mice, because this compound did not significantly accelerate xGVHD in our experiments. This may be related to the selective expression of human EpCAM in the human tumor xenograft and the lack of cross-reactivity with its murine counterpart. Nevertheless, MHC-proficient mice eventually developed xGVHD, which limited the survival of this group despite tumor control. MHC-deficient mice did not show signs of severe xGVHD, which allowed a durable observation and characterization of complete responses. In this regard, for the first time with a T-cell engager in humanized mice to the best of our knowledge, a tumor rechallenge experiment was feasible 54 days after the first tumor-cell inoculation. Interestingly, two out of three mice that had previously experienced complete responses spontaneously rejected the tumor on rechallenge. Which clones and which type of human T cells remain to control this rechallenge warrant further investigations, but these results indicate long-term memory (longer than 40 days after the first complete response was observed). Furthermore, tumor-infiltrating T cells expanded in numbers and underwent activation on treatment with an αEpCAM/CD3 BsAb treatment, observations that are consistent with the mode of action of T-cell engagers. Interestingly, CD137 upregulation was observed specifically on tumor-infiltrating CD8+ T cells. This finding is of interest since CD137 induction is downstream of the TCR/CD3 signaling pathway and it is a potential target for new-generation T-cell engagers.44 This is an example of how the refinement of this model, with lower baseline activation levels in untreated MHC-dKO NSG mice, facilitates the observation of immune pharmacodynamic changes.
Here we demonstrate a significant improvement regarding one of the most important limitations of murine models humanized with mature human PBMC, which encourages the use of MHC-deficient mice to reduce the confounding impact of xenoreactivity, especially on experiments with survival and T-cell activation readouts.
Nevertheless, it is important to remember that there are significant limitations to this model that need to be considered regarding the recapitulation of the tumor-immune system interaction in patients. Regarding humanization with PBMCs, it has been reported that tumor-specific lymphocytes, a key population to modulate antitumor responses, are present in peripheral blood.18 19 However, proportions are lower than for TILs and recent evidence indicates that TCRs with the highest affinity for neoantigens are particularly enriched in tumors and less represented in PBMCs.10 11 Furthermore, IL2rgnull mice show a deficient development of lymph nodes,45–47 which may limit the T-cell priming phase and the construction of complete adaptive and memory responses. In this regard, thymic-stromal-cell-derived lymphopoietin knock-in mice and mice expressing IL2rg specifically in lymphoid tissue inducer cells have been developed and should contribute to overcoming this limitation.46 47 Additionally, immunodeficient mice engrafted with PBMCs favor the survival of human lymphocytes,2 21 but B cells are underrepresented2 21 and the survival of myeloid cells is limited to the first few days following engraftment.2 Human knock-ins of myeloid cell-promoting cytokines into the murine host and the incorporation of HSPCs for humanization might be some of the steps to further refine humanized mouse models, in particular, to test the potential influence of myeloid cells on tested therapeutic strategies. In addition, experiments with various donors and/or patients might be required to account for the inherent inter-donor variability of human samples. Finally, our model lacks the autologous interaction that is essential to appropriately recapitulate the cornerstone of TCR-mediated immune recognition of tumors, that is, the neoantigens produced during the carcinogenic process.48 We do consider, however, that murine models humanized with mature PBMCs are, for the time being, the most reasonably valid, scalable and potentially autologous method for the preclinical testing of immunotherapy agents and combination strategies. Results presented in this work need to be validated in an autologous setting, since TCR-mediated signaling might be significantly different with these two approaches. Furthermore, concordance antitumor studies between PBMC-humanized autologous models and matched patients are missing. Lastly, evidence of tumor-driven T-cell clonal expansion would contribute to confirm the validity of these approaches to test cancer immunotherapy strategies. Further work is warranted in the direction of progressively achieving a more accurate replication of the immune tumor microenvironment of patients with cancer.
Conclusion
MHC-deficient mice are more advantageous than MHC-proficient mice for mouse humanization with mature PBMCs for testing cancer immunotherapies. More durable and refined experiments permitted the long-term characterization of complete antitumor immune responses in a way that is not feasible due to the xGVHD in MHC-proficient mice.
Data availability statement
All data relevant to the study are included in the article or uploaded as supplementary information. Additional data are available upon reasonable request to the corresponding author.
Ethics statements
Patient consent for publication
Ethics approval
Human blood samples for PBMC isolation were obtained from healthy donors following informed consent according to a protocol approved by the institutional ethical committee of the Clínica Universidad de Navarra (reference: 2019-76). Participants gave informed consent to participate in the study before taking part.
Acknowledgments
We are grateful to other members of the Melero Laboratory for their helpful discussions and comments; Elixabet Bolaños and Arantza Azpilikueta for their kind technical assistance and drug supply. Saray Garasa and María Carmen Ochoa for providing us the training on the hydrodynamic delivery of plasmids for in vivo experiments. Eva Santamaría and Sara Arcelus for their kind help with plasma analyses. Laura Guembe and the rest of the CIMA Morphology Core Facility for their assistance with pathology sample preparation. Javier Glez-Vaz for his inspiring assistance throughout the project.
References
Supplementary materials
Supplementary Data
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Footnotes
X @inma_aguilera6
Contributors Project conception: IE-S, IM and MFS. Experiments: IE-S, EFdP, GC, PM-M, MC, DC, RA, CEDA and IA-B. Data interpretation: IE-S, EFdP, GC, IRL, RS-V, CEDA, LÁ-G, PB, IM and MFS. Manuscript writing: IE-S. and MFS. Manuscript review and editing: IE-S, EFdP, GC, PM-M, MC, DC, IA-B, IRL, RS-V, CEDA, LÁ-G, PB, IM and MFS. Guarantor of the study: MFS.
Funding IE-S is supported by a Fundación Científica Asociación Española Contra el Cáncer (AECC) Clinico Junior 2020 grant (ID: CLJUN20011EGUR). MFS is supported by a Fundación Científica AECC Lab AECC grant (ID: LABAE211756FERN) and a CRIS Cancer Foundation Excellence Program grant (ID: PR_EX_22-36). IM is supported by a Spanish Ministry of Science, Innovation and Universities/Spanish Research Agency (MICIU/AEI) grant (ID: PID:2020-112892RB), the Mark Foundation (ASPIRE Award), a Fundación la Caixa grant (ID: LCF/PR/HR21/00083), a Fundació Marató de TV3 grant (ID: 488/C/2019), a Fundación Fero grant (ID: BBASELGAFERO2022-01) and a Instituto de Salud Carlos III/Fondo Europeo de Desarrollo Regional (ISCIII/FEDER) grant (ID: PI21/01547[CEDA]).
Competing interests IE-S, EFdP, GC, PM-M, MC, DC, IA-B, IRL, RS-V, CEDA, LÁ-G and PB declare no conflicts of interest. IM reports grants and personal fees from Genmab during the conduct of the study, as well as grants and personal fees from Bristol Myers Squibb, Roche, AstraZeneca, and Pharmamar and personal fees from F-Star, Numab, Pieris, Boehringer Ingelheim, Gossamer, Alligator, Hotspot, Biolinerx, Bioncotech, Dompe, Highlight Therapeutics, Bright Peaks and Boston Therapeutics outside the submitted work. MFS reports grants from Bristol Myers Squibb and Roche during the conduct of the study, as well as grants and personal fees from Roche and Bristol Myers Squibb, and personal fees from Numab outside the submitted work.
Provenance and peer review Not commissioned; externally peer reviewed.
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