Impact of γ-chain cytokines on EBV-specific T cell cultures
- Anna Merlo†1,
- Riccardo Turrini†1,
- Cristina Trento2,
- Paola Zanovello1, 3,
- Riccardo Dolcetti4Email author and
- Antonio Rosato1, 3Email author
© Merlo et al; licensee BioMed Central Ltd. 2010
Received: 17 August 2010
Accepted: 22 November 2010
Published: 22 November 2010
Recent preclinical adoptive immunotherapy studies in murine models prompt to employ "proper" rather than "as many as possible" antigen-specific T cells to gain better therapeutic results. Ideally, "proper" T cells are poorly differentiated in vitro, but retain the capacity to fully differentiate into effector cells in vivo, where they can undergo long-term survival and strong proliferation. Such requirements can be achieved by modifying culture conditions, namely using less "differentiating" cytokines than IL-2.
To evaluate this issue in human T cell cultures, we exploited a well characterized and clinical-grade protocol finalized at generating EBV-specific CTL for adoptive immunotherapy. In particular, we studied the impact of IL-7, IL-15 and IL-21 compared to IL-2 on different aspects of T cell functionality, namely growth kinetics, differentiation/activation marker expression, cytokine production, and short-term and long-term cytotoxicity.
Results disclosed that the culture modifications we introduced in the standard protocol did not improve activity nor induce substantial changes in differentiation marker expression of EBV-specific CTL.
Our data indicated that the addition of γ-chain cytokines other than IL-2 for the generation of EBV-specific T cell cultures did not produce the improvements expected on the basis of recent published literature. This fact was likely due to the intrinsic differences between murine and human models and highlights the need to design ad hoc protocols rather than simply modify the cytokines added in culture.
Infusion of antigen-specific T cells proved to be safe and effective against both virus infections (e.g., CMV ) and cancer, in particular melanoma and EBV-driven malignancies . The vast majority of current protocols rely on the infusion of a high number of effector cells that require long-term in vitro cultures, in particular when dealing with Tumor Infiltrating Lymphocytes (TIL) or clonal cultures. Consequently, this aspect implies labor-intensive and cost-ineffective procedures and, furthermore, has a potential negative impact on the characteristics of cells infused. Indeed, as advanced by Gattinoni and colleagues [3, 4], long-term T cell cultures move toward a differentiated phenotype characterized by a high cytotoxic potential, but also a poor recirculation and in vivo expansion capability. These features are highlighted by a well-defined "marker expression signature", namely CD27low/neg, CD28low/neg, CD62Llow/neg, CCR7low/neg, and CD57high. Thus, the new trend in adoptive cell therapy (ACT) focuses on the infusion of a more limited number of cells, but with the "proper" phenotype and functional characteristics, which can promote prolonged in vivo persistence and expansion, and induction of immunological memory to provide protection against possible relapses. The potentiality to expand and persist in the host also relies on the possibility for the infused cells to find an "immunological space" to colonize. This is "naturally" accomplished in Post Transplant Lymphoproliferative Disease (PTLD) after Haemopoietic Stem Cell Transplantation (HSCT), in which patients are immunocompromised due to the immunosuppressive regimens; in patients with other tumors, it has been achieved by chemotherapy and irradiation  or by immunodepleting (anti-CD45) antibodies . In these conditions, infused T cells have a favourable environment with fewer competitors for and elevated availability of homeostatic cytokines (IL-7 and IL-15), and possibly less numerous T regulatory (Treg) populations.
Although much attention has been paid to shorten the generation protocols in the clinical settings, a stringent correlation between phenotype (and so differentiation) and outcome has been shown mainly in mouse models thus far [4, 7, 8], with few notable exceptions . In this context, several reports have described the impact of different γ-chain cytokines on the differentiation status and functional properties of T-cell cultures in vitro and, more importantly, in vivo. Overall, they suggested that certain γ-chain cytokines, in particular IL-15 and IL-21, are superior to the commonly used IL-2 in maintaining a less differentiated phenotype of cultured T cells, thus possibly resulting in a better therapeutic activity. In this regard, the eradication of large established melanomas (approximately 50 mm2 tumor area) was achieved by the infusion of as little as 5 × 105 IL-21 cultured T cells .
To explore this critical issue in human T cell cultures, we took advantage of a well established and clinical-graded protocol aimed at generating EBV-specific CTL for ACT. We slightly modified the protocol by adding to the cultures IL-7, IL-15 or IL-21 instead of IL-2. Moreover, we separated and maintained in parallel cultures CD4+ and CD8+ T cells to better discriminate the impact of the different cytokines on the two subsets. We therefore compared the proliferative potential, phenotype, cytokine production, and cytotoxic activity of effector cells obtained in different culture conditions. On the whole, addition of different cytokines did not produce any clear improvement or substantial differences between T cell lines. Therefore, to obtain more active T cells for therapy, we infer that several other conditions need to be optimized other than the use of different cytokines, namely ad hoc protocols able to appropriately balance the effector cell expansion and the timing of culture.
Lymphoblastoid cell lines (LCL)
EBV-transformed lymphoblastoid cells were generated from peripheral blood mononuclear cells of HLA-typed healthy donors using culture supernatant from the EBV-producing marmoset cell line B95.8 (American Type Culture Collection). Signed informed consent was obtained from the donors and the research protocol was approved by the institutional ethical review board of the Istituto Oncologico Veneto, in accordance with the ethical standards of Helsinki Declaration.
Cyclosporin A (CsA, Sandoz Pharmaceuticals AG; Cham, Switzerland) was initially added to the cultures to inhibit T cell growth (final concentration, 700 ng/ml). LCL were maintained in RPMI 1640 (Euroclone, Pero, Milan, Italy) supplemented with 10% heat-inactivated type AB Human Serum (HS, Lonza BioWhittaker; Basel, Switzerland), 1 mM Na Pyruvate, 10 mM Hepes Buffer, 2 mM Ultraglutamine (all from Lonza BioWhittaker), 1% Antibiotic/antimycotic (Gibco, Invitrogen Corporation), hereafter referred to as HS complete medium.
Generation of EBV-specific CD4+ and CD8+ T-cell lines
EBV-specific T cells were established as previously described , with modifications. Briefly, PBMC were co-cultivated with irradiated (40 Gy) autologous LCL at a ratio of 40:1 in 24-well plates (Corning Incorporated; Corning, NY) in HS complete medium. PBMC were seeded at a concentration of 2 × 106 cells/ml and maintained at 37°C in a 6.5% CO2 humidified atmosphere. On day 10 and weekly thereafter, CTL were re-stimulated with irradiated LCL at a 4:1 ratio. Recombinant IL-2 (35 I.U./ml, Proleukin, Chiron Corporation; Emeryville, CA) or IL-7 (10 ng/ml; Peprotech; Rocky Hill, NJ) or IL-15 (10 ng/ml; Peprotech) or IL-21 (10 ng/ml; eBioscience; San Diego, CA) were added on day 14 and replenished every 2 days. On day 14, before cytokine addition, CD4+ T cells were immunomagnetically sorted using the CD4+ T cell Isolation Kit II (Miltenyi Biotec; Bergisch Gladbach, Germany), and both CD8+ and CD4+ T cells were cultured in parallel. At each subsequent re-stimulation with LCL, CD4+ T cells were adjusted to 1.5 × 106 cells/ml and CD8+ T cells to 2 × 106 cells/ml.
Cytotoxic activity of CD4+ and CD8+ T cells was determined in a standard 4-h 51Cr release assay, as previously reported . Autologous LCL were used as target cells, while K562 cell line served as indicator of NK-like activity. All tests were carried out with an excess of unmarked ("cold") K562 (5:1 ratio between "cold" and "hot" target). Where indicated, CD4+ T cells were pre-treated for 2 h at 37°C with either 20 μM brefeldin A (BFA, Sigma-Aldrich; St. Louis, MO) or 100 nM concanamycin A (CMA, Sigma-Aldrich) and assayed in the presence of the drugs. To assess calcium-dependence of cytolytic activity, 4 mM EGTA (Sigma-Aldrich) was added to the assay. For antibody blocking experiments, T cells were pre-incubated with 10 μg/ml of anti-FasLigand mAb (clone NOK-1; BioLegend; San Diego, CA).
Surface markers were determined by staining with FITC- or PE-conjugated antibodies and the respective isotypes. CTL lines were stained with antibodies to CD3, CD16, CD56 (BD-Pharmingen; San Diego, CA), CD4 and CD8 (BD Biosciences; San Diego, CA), CCR7 (eBioscience), CD27, CD28, CD57, CD62L and CD127 (IL7Rα; BioLegend). Cells (2 × 105) were washed with phosphate-buffered saline (PBS; Sigma-Aldrich) and re-suspended in 50 μl of staining solution (PBS, 3% FBS and 0,1% NaN3) containing an optimal concentration of antibody. After a 20-minute incubation in ice, cells were washed again and analyzed using a FacsCalibur (BD) flow cytometer. Flow cytometry data were analyzed with FlowJo software (Tree Star, Inc.; Ashland, OR).
Cytokine ELISA tests were performed using Human TNFα Screening Set and Human IFNγ Screening Set (Thermo Scientific, Rockford, IL), according to the manufacturer's instructions. Briefly, 2 × 105 effector cells and 2 × 105 autologous LCL were seeded in 96-well round-bottom plates. Positive controls were represented by effector T cells incubated with PMA-ionomycin (40 ng/ml and 4 μg/ml, respectively; Sigma-Aldrich). Baseline cytokine production was determined in supernatants from unstimulated T cells, or LCL only. Cytokine secretion was measured after 5h-incubation.
Outgrowth assay was carried out as previously described . Briefly, target LCL were seeded as replicates in U-bottom 96-well plates at doubling dilution, starting from 104 cells/well to 78 cells/well. T cells were added to half of the replicates at 104 cells/well in HS complete medium without IL-2. Plates were then incubated at 37°C in 6.5% CO2 and re-feeded weekly by replacing half of the medium. LCL outgrowth was scored after 4 weeks by visual examination with an inverted microscope. Results are expressed as the minimum number of LCL required for successful outgrowth in 50% of replicate wells.
Analysis of in vitro growth kinetics
Assessment of phenotype
Evaluation of cytokine production
Analysis of in vitro functional activity
Recent advances in immunotherapeutic approaches have highlighted the importance of infusing antigen-specific T cells that have ideally a poorly differentiated phenotype and are characterized by a strong proliferative potential upon in vivo transfer. These conditions have been partially met by acting on recipient patients with lymphodepleting strategies or by proposing the shortening of T cell in vitro expansion protocols with the use of "less differentiating" cytokines. With regard to this latter issue, we exploited a protocol successfully used in immunotherapeutic approaches for EBV-related malignancies to compare the impact of different γ-chain cytokines on phenotype and functionality of cultured T cells, as suggested by recent studies [4, 7, 8]. We analyzed purified CD8+ and CD4+ T cells to avoid potential influence of one population on the other one; indeed, despite a trend toward a "natural" expansion of CD8+T cells, the percentage of CD4+T cells in cultures turns out to be quite various among different donors and different preparations from the same donor. Our choice furthermore took into account the increasing attention paid on the CD4+T cells as actual effector cells in immunotherapeutic approaches [23, 24].
Intriguingly, the results presented herein are profoundly different from those of recently published studies. Previous reports, in fact, mainly rely on murine T cells derived from mice expressing transgenic TCR specific for the antigen of interest. All T cells have therefore the desired specificity and hence they only need to be activated in vitro, bypassing a potentially long selection phase. Conversely, this phase was absolutely required by our protocol, and covered the first 14 days of culture. Moreover, our protocol envisages the addition of cytokine only after this phase. During this gap, EBV-specific T cells that are present in PBMC of seropositive donors respond to the viral antigens presented by LCL, very likely producing IL-2 that in turn can influence the culture. In this regard, IL-21 has been reported to be capable of reverting the IL-2-induced differentiation , but no information is available for IL-7 and IL-15. In addition, it must be noted that in vitro expansion selectively involved EBV-specific precursors belonging to the memory compartment and therefore the obtainment of less differentiated cells is expected to be difficult. The long and likely confounding selection phase could be bypassed by performing faster (e.g., overnight) peptide mix stimulation followed by immunomagnetic isolation of cytokine-producing T cells, as recently proposed , or by introducing the wanted antigen specificity through CAR- or transgenic TCR-coding vector transduction [26, 27]. In these cases, the alternatively chosen cytokines could be added in a less preconditioned milieu, thus driving a less pronounced differentiation of responding T lymphocytes, or, in the case of CAR or TCR transfer, of the whole population of transduced peripheral T cells.
Overall, although the use of γ-chain cytokines other than IL-2 did not produce any substantial in vitro improvement, a realistic and clear-cut description of the activity of a determined T cell population should be derived by in vivo studies. In this regard, however, we could not produce definitive results since we only had the possibility to test those cultures that reach a sufficient number for infusion. Moreover, the PTLD-SCID mouse model suffers from different intrinsic biases that might have frustrated the purpose of our study. In fact, we have evidence that human T cells survive no longer than 24 hr after in vivo transfer , even when this follows irradiation or cyclophosphamide treatment of recipient mice. Moreover, this poor survival was verified not only for EBV-specific T cells, but also for less differentiated, CAR-transduced antigen-specific T cells (data not shown). In addition, due to the intrinsic differences between mouse and human adhesion molecules and receptors, it is hard to evaluate the lymph node homing and recirculation capacity that have a fundamental role in the more physiological model described by Gattinoni et al., which envisages the transfer of mouse T cells into a syngeneic murine microenvironment. In such experimental context, moreover, the concomitant vaccination strategies make the lymph node homing properties even more relevant, as they dramatically contribute to the improvement of the final outcome . Thus, it is left to be verified in a human context the impact of different lymphoid homing marker expression on the outcome of adoptive transfer strategies.
As a whole, our results indicate the need to design ad hoc protocols to appreciate the impact of γ-chain cytokines other than IL-2 on the functionality of CTL for adoptive cell therapy.
This study was partly supported by grants from the Italian Ministry of Health (Progetto oncologico di medicina molecolare: i tumori femminili; Progetto strategico: Farmaci cellulari, vaccini e bioterapie innovative dei tumori; Alleanza Contro il Cancro, ACC-4), the European Community (FP6 VITAL, Contract no. 037874) and the Italian Association for Cancer Research (AIRC).
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