Ex vivo pleural effusion cultures to study chimeric antigen receptor T cell cytotoxicity in an immunocompetent environment

Summary

Current in vitro and in vivo assays used to study immunotherapeutic interventions lack human immune components that mimic the tumor microenvironment to investigate drug potency and limitations of efficacy. Herein, we describe an ex vivo pleural effusion culture (ePEC) assay, using malignant pleural-effusion-derived soluble and cellular factors that differentially affected the cytotoxicity of chimeric antigen receptor (CAR) T cells. Following identification of CAR T cell-suppressive factors, blocking of individual factors reveals their contribution to compromising T cell efficacy. ePEC is a human component assay that can be utilized for developing next-generation cell and antibody therapies that counteract immunosuppression.

Graphical abstract

Highlights

• •Malignant pleural effusion (MPE) is a rich source of cellular and soluble factors
• •Soluble inhibitory factors in MPEs influence CAR T cell cytotoxicity
• •Ex vivo pleural effusion cultures (ePECs) aid in determining CAR T cell cytotoxicity
• •ePECs allow for the identification of active inhibitory factors

Motivation

In vitro assays that are currently being used in a pre-clinical setting to test CAR T cell functional activity do not reflect the activity seen when used in a patient’s immunosuppressive environment. We used patient-derived malignant pleural effusions to provide an ex vivo milieu that allowed us to determine CAR T cell functional activity in an immunosuppressive environment.

Malignant pleural effusions (MPEs) are drained from patients with cancer to provide symptomatic relief. In this report, Tano et al. describe an assay utilizing MPE to exploit its immunosuppressive properties to test immunotherapeutic agents’ potency in ex vivo cultures.

Introduction

Adoptive cell therapy with the transfer of T cells expressing chimeric antigen receptors (CARs) has been successful in treating hematological malignancies. To successfully translate CAR T cell therapy to solid tumors, an assay that provides human tumor immune milieu is essential. The solid tumor microenvironment consists of immune cells, cancer cells, and fibroblasts that also secrete immune-modulating cytokines.¹ The balance between pro-tumor and anti-tumor immune cells and cytokines influences cytotoxic T cell efficacy in the tumor²; therefore, it is critical to investigate CAR T cells in an immunocompetent microenvironment for the development of next-generation, patient-specific cell therapy agents.

Our laboratory developed and translated mesothelin (MSLN)-targeted, CD28 co-stimulated CAR T cells for solid tumors,^3,4 resulting in three ongoing clinical trials (ClinicalTrials.gov: NCT02414269, NCT02792114, and NCT04577326). To investigate next-generation CAR constructs in human tumor immune milieu, we have developed and optimized a patient-derived ex vivo pleural effusion culture (ePEC). Malignant pleural effusion (MPE), which occurs in 150,000 patients from solid tumors each year in the United States, is composed of infiltrating immune cells and cell-derived soluble factors that form an immunosuppressive environment.^5,6 Herein, we show that the composition of MPE possesses substantial patient-to-patient heterogeneity; this offers a unique opportunity to study CAR T cells in a translationally relevant environment that can provide guidance for the design of next-generation immunotherapies.

Results

We assessed the antigen-specific cytotoxicity of CAR T cells against MSLN-expressing lung cancer and mesothelioma cells co-cultured in 15 cell-free MPEs that were derived from patients with stage IV lung cancer. The efficacy of the CAR T cells varied among the MPEs over multiple effector-to-target (E:T) ratios, as shown in Figure 1A. We also observed the suppression of CAR T cell-mediated cytotoxicity when the cellular MPE component (macrophages) was introduced into the assay (Figure 1B). Based on the inhibition of cytotoxicity on multiple donor CAR T cells, MPEs were stratified by a group-based trajectory model (Figure 1C), and a Nanostring assay outlined the differential gene expression profile in CAR T cells (Figure 1D) in these groups. These results indicate that the immunomodulatory properties of MPE differentially affect CAR T cell cytotoxicity.

Figure 1.

Chimeric antigen receptor (CAR) T cell cytotoxicity varies following co-culture with malignant pleural effusion (MPE)

(A) Cytotoxicity of CAR T cells targeting mesothelin-positive cancer cells following co-culture with varied MPEs derived from multiple patients.

(B) Cytotoxicity of CAR T cells targeting mesothelin-positive cancer cells following co-culture in presence or absence of MPE cells in Roswell Park Memorial Institute (RPMI) medium. Cytotoxicity was determined by ⁵¹Cr-release assay after 18 h co-culture.

(C) Cytotoxicity curves from multiple donor CAR T cells in multiple MPEs were stratified by a group-based trajectory model.

(D) Nanostring analyses of CAR T cells following culture in RPMI medium or MPEs compared with untransduced T cells showed a differential gene expression pattern. cRPMI, complete RPMI.

Next, we assessed the cytokine environment in MPEs that may have influenced the cytotoxicity of CAR T cells. We observed that—out of 34 cytokines analyzed—members of the transforming growth factor β (TGF-β) family, among others (Figure 2A), were found to be elevated in inhibitory MPEs, and CAR T cells cultured in MPEs exhibited enhanced expression of TGF-β receptors (Figure 2B). Based on its described immunosuppressive properties on CAR T cell therapy,⁷ we used TGF-β to validate the utility of ePECs. First, we demonstrated that MPE-derived TGF-β was biologically active, as evidenced by its ability to induce phosphorylation of the Smad2/3 signaling pathway, similar to its recombinant counterpart (Figure 2C). Second, we showed that Smad2/3 phosphorylation could be blocked by adding an anti-TGFβ-1,2,3 neutralizing antibody (Figure 2D). Third, we confirmed that combining CAR T cells with the anti-TGF-β1,2,3-blocking antibody increased tumor cytolysis toward MSLN-expressing target cells co-cultured in MPE (Figure 2E). Similarly, in MPEs obtained from patients treated with checkpoint blockade agents, elevated soluble PD-1 and PD-L1 (Figure 2F) was observed, resulting in corresponding cytotoxicity inhibition (Figure 2G). By analyzing MPE composition (Figures 3 and S1) and the differential gene expression of CAR T cells (Figure 4) co-cultured with target cells in MPEs, further insights were provided into effector and suppressor pathways that were being activated.

Figure 2.

Ex vivo pleural effusion culture (ePEC) allows identification of soluble factors suppressing CAR T cell efficacy

(A) MPE-contained soluble factors were biologically active, as exemplified by transforming growth factor β (TGF-β), and associated with suppression of CAR T cell cytotoxicity ex vivo.

(B) CAR T cells co-cultured in MPEs high in soluble TGF-β demonstrated upregulated TGF-β receptor expression.

(C) MPEs with high TGF-β-induced activation of the pSmad2/3 signaling pathway in CAR T cells cultured in either cell-free MPE or RPMI medium supplemented with recombinant TGF-β1 that can be rescued by using anti-TGF-β1,2,3 blocking antibodies.

(D) Corresponding flow cytometry plots of the data that are shown in (C).

(E) MPE-induced suppression of cytotoxicity was measured using an impedance-based cytotoxicity assay upon repeated co-culture of CAR T cells with tumor cells in MPE at an effector-to-target ratio of 1:1. Addition of an anti-TGF-β1,2,3 blocking antibody ablated pSmad2/3 signaling and restored CAR T cell cytotoxicity. MFI, median fluorescence intensity.

(F) MPEs collected from patients receiving checkpoint blockade agents showed elevated soluble PD-1 or PD-L1 levels corresponding to the anti-PD-1 or anti-PD-L1 treatment.

(G) CAR T cells co-cultured with MPEs with highly soluble PD-1 demonstrated significant cytotoxicity inhibition, depending upon the soluble PD-1 concentration in the MPE. CPB, checkpoint blockade; cRPMI, complete RPMI; IL, interleukin; MFI, mean fluorescence intensity; RANTES, regulated on activation, normal T cell expressed and secreted.

Figure 3.

Heterogeneity in MPE cellular and soluble factors

(A) Stacked bar graph shows heterogeneity in the proportion of cellular phenotypes characterized from MPEs collected from patients, which were classified as non-inhibitory, mildly inhibitory, or strongly inhibitory based on their inferred ability to inhibit cytolytic activity of CAR T cells using ePECs.

(B) Heatmap display of 34 immunomodulatory soluble factors evaluated from MPEs collected from patients showing heterogeneity in their individual expression levels.

Figure 4.

CAR T cell gene expression signatures post-activation in different culture conditions

Volcano plots showing differential gene expression (DEG) profiles of M28z CAR T cells 24 h post-activation by mesothelin-expressing target cells in the presence of (A) cRPMI, (B) mildly inhibitory MPE, and (C) strongly inhibitory MPE. The top 20 DEGs are displayed as a heatmap next to each volcano plot, showing upregulated and downregulated transcripts compared with their pre-activation baseline levels.

Discussion

In contrast to traditional cytotoxicity assays that use commercially available, chemically defined culture media employing a single cell or factor, we employed a patient-derived culture that is rich in a complex milieu of immunomodulatory factors to assess CAR T cells in a translationally relevant environment. Our study indicated that MPE components differentially alter CAR T cell function that is dependent on the unique composition of cellular and soluble factors, and the influence of dominant immunosuppressive factors can be tested in the background of complex immune milieu. As exemplified by TGF-β, the investigation of CAR T cells in MPE enabled the identification of factors associated with immunosuppression and helped implement blocking strategies to counteract immunosuppression. More importantly, our investigation of MPEs obtained from patients being treated with checkpoint blockade agents provided novel observations that can guide mitigation strategies for next-generation therapies to combat immunosuppression.

In consideration of response rates to immunotherapy in 20%–30% in patients,⁸ it has become apparent that a “one size fits all” approach does not fit. Current CAR T cell development programs, along with other immunotherapies, have failed to assess efficacy in the context of a heterogeneous human tumor immune environment. By incorporating the MPE-derived fraction, ePECs represents a first step toward a multifactor readout system to identify broad-spectrum responses across a heterogeneous patient-derived culture. We believe that ePEC is a novel tool to augment current pre-clinical investigations, elaborate tailored combination approaches, and support the development of next-generation CAR T cells and other immunotherapies.

Limitations of the study

The limitations of the study included the heterogeneity among MPEs and multiple soluble factors that can influence CAR T cell efficacy. However, the large volume of the MPE available from each patient provided an opportunity for repeat experimentation with blockade of individual factors. Heterogeneity provided a clinically relevant tumor milieu to investigate the efficacy of immunotherapeutic agents. Another limitation was that the yield of viable cells in some MPEs were low; implementation of our protocol increased the yield of cellular viability (Figure 3A). Ongoing confirmatory investigations are exploring the differential gene signaling pathway observations between CAR T cells co-cultured in different MPEs; this is beyond the scope of this study.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
PE anti-MSLN Rat IgG2A	R&D Systems	FAB32652P
AF700 Mouse anti-human CD45	Biolegend	368513
FITC anti-human CD3; Clone HIT3A	Biolegend	300305
APC anti-human CD326 EpCAM; Clone 9C4	Biolegend	324207
TGF beta-1,2,3 Monoclonal Antibody (1D11)	Invitrogen	MA5-23795
eBioscience™ Fixable Viability Dye eFluor™ 506	Invitrogen	65-0866-14
Bacterial and virus strains
SFG-gamma retroviral plasmid vector	Cell Therapy and Cell Engineering Facility, Memorial Sloan Kettering Cancer Center	N/A
Biological samples
Malignant pleural effusions	Patient derived, Memorial Sloan Kettering Cancer Center	N/A
Experimental models: Cell lines
MSTO-211H (Mesothelioma)	American Type Culture Collection	CRL-2081
A549 (Lung Cancer)	American Type Culture Collection	CCL-185

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Prasad S. Adusumilli (adusumip@mskcc.org).

Materials availability

No unique reagents were generated in this study. All materials used in the study were resourced from commercial vendors, except for malignant pleural effusions, which were derived from patients at Memorial Sloan Kettering Cancer Center upon consent. Details about the materials can be provided upon request by the lead contact, Prasad S. Adusumilli (adusumip@mskcc.org).

Experimental model and subject details

Human participants

A total of 30 malignant pleural effusions (MPEs) collected between 2016 and 2018 from patients with various malignancies at Memorial Sloan Kettering Cancer Center (MSK) were included in this study. Eighteen out of 30 MPEs contained sufficient cellular fraction to conduct flow cytometry characterization and cytotoxicity assessments. Malignant pleural effusions collected from both male and female sexes between 35 and 85 years of age were represented, with no influence on the study outcomes.

Cell lines

MSTO-211H (human pleural mesothelioma) and A549 (human non-small cell lung cancer) cell lines were obtained from the American Type Culture Collection (ATCC). Both cell lines were retrovirally transduced to express green fluorescent protein (G) and human mesothelin (M) to produce MGM and A549GM cell lines, respectively, that were used in ePECs assays.

Ethics statement

Malignant pleural effusions were obtained upon patients’ consent in accordance with the protocol that was approved by the MSK Institutional Review Board (IRB) (IRB# 16–047).

Method details

Pleural effusion collection, processing, and storge

Malignant pleural effusion (MPE) was drained via insertion of a chest tube or pleural catheter (PleurX Catheter, Becton, Dickinson and Company, Franklin Lakes, NJ) upon patients’ consent in accordance with an institutional review board approved protocol (IRB# 16–047). Freshly collected MPEs from the operating room were transferred to lab with 5000 IU of heparin added to prevent clotting in cases of hemorrhagic effusion and processed within 2 h to separate cellular (cell-pellet) and cell-free (supernatant) components via centrifugation at 450g for 5 min at room temperature. Separated cell-pellets were treated with ammonium-chloride-potassium lysis buffer, washed (3x) in phosphate buffered saline, and passed through a 100μm cell-strainer to remove debris before storing at −80°C in cell-freezing medium (heat-inactivated fetal bovine serum +10% dimethyl sulfoxide) at a concentration of 5e6-10e6 cells/ml. Cell-free MPEs were aliquoted into smaller volumes of 1mL, 15mL and 50mL conicals for Luminex/ELISA, co-culture, and back-up assays respectively and transferred to −80°C until further use. Frozen aliquots were thawed by placing in 37°C water bath for 1 min. Immediately upon thawing, contents of cell-pellet aliquots were added, drop-by-drop, to 25mL of Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum, 100units/mL of penicillin and 100ug/mL of streptomycin, followed by centrifugation at 450g for 5 min at room temperature. Next, supernatants were discarded, and cell-pellets were washed in phosphate buffered saline (3x) and brought to a volume of desired concentration after final wash step. Upon thawing, contents from aliquots of cell-free components (supernatants) were passed through a 0.22μm filter to remove any microbial contaminants present prior to their use. Typically, 100mL–300mL of pre-processed MPE obtained from a patient should allow performing ePECs assay 1–3 times, depending on the concentration of cells present in effusion.

γ-Retroviral vector Construction and viral production

To generate MSLN-specific CARs, we engineered a fusion protein encoding a fully human scFv, m912 [provided by D. Dimitrov, National Cancer Institute (NCI)–Frederick], tagged with myc and linked to the human CD8 leader peptide and the CD28/CD3ζ sequences. The CAR sequences were inserted into the SFG γ-retroviral vector backbone (provided by I. Riviere, Memorial Sloan Kettering Cancer Center). The M28z-encoding plasmids were transfected into 293T H29 producer cell lines using a calcium phosphate mediated, mammalian transfection method (Promega ProFection) to produce DNA encoded cell supernatant, as per the manufacturer’s instructions. The viral supernatant was used for further transduction into 293Vec RD114 packaging cell lines to produce stable retroviral supernatant encoding the CARs. The 293T H29 retroviral producer cells and 293Vec RD114 cells were maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal calf serum and 2 mmol/L glutamine.

T cell isolation and gene transfer

Peripheral blood mononuclear cells (PBMC’s) from healthy donors were isolated by low-density centrifugation using Lymphocyte separation medium or Ficoll (Corning) and activated with phytohemagglutinin (2 μg/ml; Remel). Two days after activation of the T-cells, the PBMC’s were spinoculated with 293Vec RD114 produced retroviral particles encoding M28z CAR for 1h at 3000 rpm on non-tissue culture plates coated with retronectin (15 μg/mL; r-Fibronectin, Takara). Transduced cells were kept in culture in RPMI 1640 supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin, and 2 mmol/L glutamine. Transduced CAR T cells were used for in-vitro and in-vivo experiments 5–6 days after transfection and supplemented with 20 units/mL IL-2 (Novartis) every 2 days for maintenance. Transduction efficiencies were tested by flow cytometric analysis.

CAR T cell cytotoxicity assays

The cytotoxicity of CAR and untransduced donor T cells cultured in presence of cell-free MPE or complete RPMI-1640 medium supplemented with 10% fetal bovine serum, 100units/mL of penicillin and 100ug/mL of streptomycin was determined by ⁵¹Cr-release assay against mesothelin-expressing lung cancer (A549GM) or mesothelioma (MGM) target cells. Resulting cytotoxicity curves were analyzed by group-based trajectory modeling⁹ to stratify MPEs by level of inhibition. CAR T cell or cell-free MPE mediated cytolysis of tumor cells (either independently or as a combination in 1:1 ratio) was assessed using the xCELLigence RTCA system (ACEA Biosciences, San Diego, CA).

Flow cytometry analysis

Cellular phenotypes present in MPEs were broadly characterized as dead cells (LIVE/DEAD fixable viability dye+), non-immune cells (CD45⁻), and two subsets of immune cells (CD45⁺ CD3⁺ and CD45⁺ CD3⁻) by flow cytometry. We used fluorochrome-conjugated antibodies to MSLN (anti-MSLN PE-conjugated Rat IgG2A FAB32652P; R&D Systems), CD3 (FITC anti-human CD3 Clone HIT3A, 300305; Biolegend), CD45 (AF700 anti-human CD45, 368513; Biolegend), EpCAM (APC anti-human CD326, 324207; Biolegend), Fixable viability dye (AmCyan/eFluor506, 65-0866-14; Invitrogen). All flow cytometric analyses were performed on Attune NxT Flow cytometer (Thermo Fisher Scientific) and data were analyzed using FCS Express 7 Research software (version 7.18, De Novo Software).

Luminex/ELISA Binding assays

Pleural effusion soluble factors levels were determined using 30-plex human cytokine/chemokine (HCYTMAG-60K-PX30, Millipore sigma), and 3-plex TGFβ-1, 2, 3 (TGFBMAG-64K-03, Millipore sigma) Luminex assay kits and were run on a Luminex IS100 system (version 2.3) as per manufacturer’s instructions. Human PD1 DuoSet (DY1086, R&D systems), and human PDL1 DuoSet (DY156, R&D systems) ELISA kits were run on the MSD Mesoscale Quickplex SQ120 platform (MSD Mesoscale Diagnostics, Rockville, ML) as per manufacturer’s instructions. Values represent the mean of triplicates ±standard deviation. These data were analyzed using IS 2.3 software (Luminex), Microsoft Excel, and GraphPad Prism.

TGFb neutralization assay

Phosphorylation of Smad2/3 was detected by flow cytometry after serum starvation of CAR T cells and incubation in MPE alone or RPMI medium supplemented with recombinant transforming growth factor beta-1 (TGFβ1, 10 ng/mL) in presence or absence of a TGFβ 1,2,3 blocking antibody (10 μg/mL, clone 1D11).

Gene expression analysis

Gene expression analysis was performed using nCounter assay by Nanostring Technologies (Seattle, WA). CAR T cells were isolated by flow sorting, followed by isolation of RNA using RNEasy Mini kit (Qiagen), ensuring that adequate (>100 ng) RNA was obtained. Samples were then prepared as outlined by the manufacturer for the Nanostring nCounter cartridge using the CAR T cell Characterization Panel—a 770-plex gene expression panel that profiles CAR T cells. Samples were run in duplicates. Analysis was performed with Nanostring nSolver software (version 3.0, Nanostring Technologies).

Quantification and statistical analysis

The group-based trajectory modeling approach was conducted by use of SAS (version 9.4, Cary, NC, USA) software through the Proc Traj procedure. Proc Traj uses the maximum likelihood method to estimate parameters, including group sizes and shapes of trajectories. Trajectory memberships were then used as categorical variables in a multinomial logistic regression model to identify predictors of trajectory membership; outcomes are presented as odds ratios (ORs) and 95% confidence intervals (CI). To determine if the differences in cytokine levels measured between non-inhibitory vs. strongly inhibitory MPE groups were significant, we used two-tailed Ratio paired t test for statistical analysis. Since the differences in the levels of measured cytokines were not expected to be consistent but a difference in the ratio of these levels between the groups could be consistent, a Ratio paired t test was chosen. Data were analyzed using GraphPad Prism 9 (v9.4.1) and presented as mean ± standard deviation. Statistical significance was defined as p < 0.05.

Published: October 23, 2023

Data and code availability

The data generated in this study are available within the article and its supplementary data files. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact, Prasad S. Adusumilli (adusumip@mskcc.org), upon request.