FACS Analysis Of Blood Samples Research Articles

Abstract A005: Preclinical evaluation of novel immune cell therapies, check point inhibitors, and immune cell engagers in humanized mouse models

Abstract Background The preclinical evaluation of novel immune therapies requires humanized mouse models with functional human immune cells. In previous studies we have demonstrated, that either peripheral blood mononuclear cells (PBMC), subsets of PBMCs like T- and NK-cells or hematopoietic stem cells (HSC) can be used to establish a humanized immune system with functional T-, B-, and NK cells, as well as monocytes and dendritic cells in immunodeficient mice. By xenotransplantation of cell-line-derived (CDX) or patient-derived (PDX) tumors on humanized mice, we successfully generated a full human tumor-immune-cell model for different disease entities. Finally, we validated the functionality of these models using checkpoint inhibitors like Ipilimumab (Ipi), Nivolumab (Nivo), Pembrolizumab (Pembro), cell therapies, and immune cell engagers. Methods HSC-humanized mice were generated by i.v. transplantation of CD34+ stem cells to immunodeficient 1st and 2nd generation NOG mice. Engraftment of immune cells was monitored by FACS analysis of blood samples. PBMC or isolated T- or NK-cell preparations were used to humanize mice by single or multiple i.v. injections. CDX and PDX from different entities were s.c. (i.e. lymphoma) and/or i.v. (leukemia) transplanted on those humanized mice. Tumor growth of orthotopic models was followed up by BLI measurement. Blood and tumor samples were analysed by for immune cell infiltration and activation. Results The transplanted HSCs engrafted in mice and established a functional human immune system with proliferation and differentiation. The 2nd generation NOG mice are showing an increased total engraftment and lineage specific differentiation of cells. The selected CDX and PDX tumors successfully engrafted on humanized mice without significant differences in tumor growth compared to non-humanized mice. Check point inhibitor treatments (Ipi, Nivo, or Pembro) induced tumor growth delay in selected models. BLI measurement is a suitable method to follow up systemic disease models and treatment effects over time. We identified a set of CDX and PDX models without interference with parallel injection of PBMC, T- or NK-cell preparations for the evaluation of immune cell engagers and other cell therapies. Conclusions We established human tumor-immune-cell models of different entities using CDX or PDX in combination with different donor derived immune cell subsets as effector cells. We demonstrated successful engraftment of HSC on different immunodeficient mouse strains, generating mice with a functional human hematopoiesis. These models have been successfully used for preclinical evaluation of novel check point inhibitors, cell therapies and immune cell engagers. Our human tumor-immune-cell models allow preclinical translational research on tumor immune biology as well as evaluation of new therapies, drug combinations, as well as biomarker identification and validation in subcutaneous and orthotopic models. Citation Format: Maria Stecklum, Annika Wulf-Goldenberg, Bernadette Brzezicha, Joshua Alcaniz, Glenn Smits, Wolfgang Walther, Jens Hoffmann. Preclinical evaluation of novel immune cell therapies, check point inhibitors, and immune cell engagers in humanized mouse models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A005.

32 (PB022) - Humanized mouse models for preclinical evaluation of novel immune therapies, checkpoint inhibitors and immune cell engagers

Background: The preclinical evaluation of novel immune therapies demands humanized mouse models with functional human immune cells. In previous studies we have established a humanized immune system with functional T- B- and NK cells, as well as monocytes in immunodeficient mice by transfer of hematopoietic stem cells (HSCs) or peripheral blood mononuclear cells (PBMC). By transplantation of cell-line-derived (CDX) or patient-derived (PDX) tumor xenografts on humanized mice, we successfully generated a full human tumor-immune-cell model for different tumor entities. Finally, we validated the functionality of these models using checkpoint inhibitors or immune cell engagers. Methods: HSC-humanized mice were generated by single i.v. transplantation of CD34+ stem cells to immunodeficient NOG mice. Engraftment of immune cells was monitored by FACS analysis of blood samples. CDX and PDX from different entities were transplanted on the humanized mice. PBMC or isolated T- or NK-cell preparations were used to humanize mice by single or multiple i.v. injections into tumor-bearing mice or with simultaneous tumor cell transplantation. These models were used to evaluate immune checkpoint inhibitors. Blood and tumor samples were analysed by FACS and immunohistochemistry for immune cell infiltration and activation. Results: The transplanted HSCs engrafted in mice and established a functional human differentiated immune system with proliferating immune cells. Up to 20% of the human immune cells in the blood were functional T cells, characterized by a high PD-1 expression 14 weeks after HSC inoculation. Selected CDX and PDX tumors successfully engrafted on humanized mice without significant differences in tumor growth compared to non-humanized mice. Checkpoint inhibitor treatments induced tumor growth delay in selected models. FACS analysis of xenograft tumors revealed an increased percentage of tumor-infiltrating T cells. In addition, we identified a set of CDX and PDX models without interference with parallel injection of PBMC, T- or NK-cell preparations for the evaluation of immune cell engagers and other immune therapeutics. Conclusions: We established human tumor-immune-cell models of different entities using CDX or PDX in combination with different donor derived immune cell subsets as effector cells. We demonstrated successful engraftment of HSCs on immunodeficient mouse strains generating mice with a functional human hematopoiesis. These models have been employed for preclinical evaluation of novel checkpoint inhibitors and immune cell engagers. Our human tumor-immune-cell models allow preclinical, translational studies on tumor immune biology as well as evaluation of new therapies, drug combinations and biomarker identification and validation. No conflict of interest.

Abstract 5631: Leukaemia and lymphoma patient-derived xenografts (PDX) engraft on humanized mice and respond to immune therapy with check point inhibitors

Abstract Background The preclinical evaluation of novel immune therapies for cancer remains a challenge, as models require both, engraftment of human tumor cells and a matching human immune cell population. In previous experiments, we have demonstrated, that we can use either peripheral blood mononuclear cells (PBMC) or hematopoietic stem cells (HSC) to establish a humanized immune system on highly immunodeficient mice. In this study, we transplanted patient-derived xenografts (PDX) from leukaemia and lymphoma on these humanized mice. Further, we validated the models by evaluating treatments with checkpoint inhibitors Nivolumab (Nivo) and Ipilimumab (Ipi) or standard of care therapies. Methods HSC were transplanted i.v. on immunodeficient mice. Engraftment of human immune cells was monitored by FACS analysis of blood samples. Three lymphoma PDX and one acute myeloid leukaemia (AML) model were s.c. transplanted on humanized mice. To model systemic leukaemia, three AML models were transplanted i.v. on humanized mice. Engraftment of the PDX was compared to growth on non-humanized mice. After successful engraftment, mice were treated with Nivo, Ipi or in combination with standard therapies and irradiation. Results We confirmed successful engraftment of HSC in the humanized mice. Within 14 weeks after HSC inoculation, FACS analysis revealed an established functional human immune system with up to 20% T-cells, characterized by a high PD-1 expression. The s.c. transplanted leukaemia and lymphoma PDX engrafted on the humanized mice and tumor growth was measured with calipers. Engraftment of i.v. transplanted AML models was confirmed by FACS measurement of human CD33+ AML cells in the blood of the humanized mice. Treatment with Ipi or Nivo alone or in combination led to a minor growth delay. FACS analysis confirmed an increased percentage of activated T-cells in the blood and in the s.c. tumors. Response to checkpoint inhibitors was in correlation to PD-L1 expression on the lymphoma and AML cells. Combination of check point inhibitors with radiotherapy provided additive effects in our models. Conclusions We developed a humanized immune-PDX model for different blood cancers enabling appropriate preclinical translational research on tumor immune biology and the evaluation of new therapies and combinations, as well as the identification and validation of biomarkers for immune therapy. These novel model are currently optimized for the preclinical evaluation of new bispecific immune cell engagers (BITE) and cell therapies (CART). Citation Format: Maria Stecklum, Annika Wulf-Goldenberg, Antje Siegert, Bernadette Brzezicha, Anja Sterner-Kock, Wolfgang Walther, Jens Hoffmann. Leukaemia and lymphoma patient-derived xenografts (PDX) engraft on humanized mice and respond to immune therapy with check point inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5631.

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation.

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Abstract 3660: Blocking the DC-HIL receptor reverses the T-cell suppression induced by proliferating myeloid-derived suppressor cells in common cancer types

Abstract Myeloid-derived suppressor cells (MDSC) are the most potent suppressors of T-cell function, and their exponential proliferation in cancer states counteracts the benefits of immunotherapy given to these patients. Having discovered the T cell-inhibitory DC-HIL receptor, we showed that DC-HIL is responsible for MDSC’s T-cell suppressive function. We found that melanoma patients (but not healthy controls) harbor in the blood an expanded population of DC-HIL+ MDSC, whose suppressor effects in vitro can be blocked by our 3D5 anti-DC-HIL mAb. To determine whether similar outcomes apply to non-melanoma cancers, we recruited patients with metastases from bladder (BL, n=4), breast (BR, n=11), colon (CO, n=44), kidney (KI, n=6), lung (LU, n=20), pancreatic (PA, n=17), and prostate (PR, n=9) cancer. FACS analysis of blood samples showed all cancer types (except LU) were associated with elevated blood HLA-DRno/low CD14+ MDSC vs. age-matched controls (n=21, median of 0.5%). % DC-HIL+ MDSC in PBMC was also significantly high in all cancer types; median of 2.5% for BL; 1.7% for BR; 3.5% for CO; 3.6% for KI; 0.3% for LU; 1.8% for PA; and 4.8% for PR, vs. 0.04% for healthy controls. MDSC in all cancer types showed high DC-HIL-positivity (20-90% vs. 8% for controls). We assayed T-cell suppressor activity by purifying MDSC and T-cells from the same patient and co-culturing them at varying cell ratios, with co-stimulators. Activity was determined by % suppression in IFN-γ secretion, which was correlated with % DC-HIL-positivity of MDSC. MDSC from KI (n=3) were more suppressive than those from CO and PA, with suppressor activity correlating with DC-HIL-positivity: Pearson’s r for 0.5 for CO (n=13) and 0.73 for PA (n=8). We evaluated effects of anti-DC-HIL mAb on co-cultures of MDSC/ T-cells from cancer patients (n=25). The mAb restored IFN-γ secretion by >30% in 8/15 cases of CO; 4/8 of PA, and 2/2 of KI. To determine whether DC-HIL+ MDSC migrate into cancer sites, lesional specimens from CO patients (n=5) with high blood counts of DC-HIL+ MDSC were immunohistochemically stained for CD14 and DC-HIL expression: DC-HIL was absent from all cancer cells, but present strongly in many CD14+ cells surrounding the cancers. This outcome contrasts starkly with melanoma, in which DC-HIL was expressed highly by the cancer cells surrounded by only a few DC-HIL+ CD14+ cells. Finally we evaluated in vivo effects of blocking DC-HIL using a mouse model of MC38 colon cancer, which is DC-HILneg. MC38 tumor challenge induced DC-HIL+ Gr1lowLy6Chigh MDSC in the tumor site. Intravenous infusion of anti-DC-HIL mAb into mice with pre-established MC38 tumor significantly inhibited tumor growth by 50-70%, reduced blood levels of MDSC, and enhanced cytotoxic T-lymphocyte response. Our findings support DC-HIL blockade as a potential treatment for metastatic cancers, with high blood levels of DC-HIL+ MDSC as a prognostic marker for the best responders. Citation Format: Masato Kobayashi, Jin-Sung Chung, Muhammad Beg, Yull Arriaga, Udit Verma, Kevin Courtney, John Mansour, Barbara Haley, Saad Khan, Yutaka Horiuchi, David Harker, Purva Gopal, Ponciano D. Cruz, Kiyoshi Ariizumi. Blocking the DC-HIL receptor reverses the T-cell suppression induced by proliferating myeloid-derived suppressor cells in common cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3660. doi:10.1158/1538-7445.AM2017-3660