Thymoglobulin, interferon-γ and interleukin-2 efficiently expand cytokine-induced killer (CIK) cells in clinical-grade cultures
- Giuseppina Bonanno1, 2,
- Paola Iudicone2,
- Andrea Mariotti1,
- Annabella Procoli1,
- Annino Pandolfi2,
- Daniela Fioravanti2,
- Maria Corallo1,
- Alessandro Perillo1,
- Giovanni Scambia1,
- Luca Pierelli†2, 3 and
- Sergio Rutella†4, 5Email author
© Bonanno et al; licensee BioMed Central Ltd. 2010
Received: 31 July 2010
Accepted: 7 December 2010
Published: 7 December 2010
Cytokine-induced killer (CIK) cells are typically differentiated in vitro with interferon (IFN)-γ and αCD3 monoclonal antibodies (mAb), followed by the repeated provision of interleukin (IL)-2. It is presently unknown whether thymoglobulin (TG), a preparation of polyclonal rabbit γ immunoglobulins directed against human thymocytes, can improve the generation efficiency of CIK cells compared with αCD3 mAb in a clinical-grade culture protocol.
Peripheral blood mononuclear cells (PBMC) from 10 healthy donors and 4 patients with solid cancer were primed with IFN-γ on day 0 and low (50 ng/ml), intermediate (250 ng/ml) and high (500 ng/ml) concentrations of either αCD3 mAb or TG on day 1, and were fed with IL-2 every 3 days for 21 days. Aliquots of cells were harvested weekly to monitor the expression of representative members of the killer-like immunoglobulin receptor (KIR), NK inhibitory receptor, NK activating receptor and NK triggering receptor families. We also quantified the frequency of bona fide regulatory T cells (Treg), a T-cell subset implicated in the down-regulation of anti-tumor immunity, and tested the in vitro cytotoxic activity of CIK cells against NK-sensitive, chronic myeloid leukaemia K562 cells.
CIK cells expanded more vigorously in cultures supplemented with intermediate and high concentrations of TG compared with 50 ng/ml αCD3 mAb. TG-driven CIK cells expressed a constellation of NK activating/inhibitory receptors, such as CD158a and CD158b, NKp46, NKG2D and NKG2A/CD94, released high quantities of IL-12p40 and efficiently lysed K562 target cells. Of interest, the frequency of Treg cells was lower at any time-point compared with PBMC cultures nurtured with αCD3 mAb. Cancer patient-derived CIK cells were also expanded after priming with TG, but they expressed lower levels of the NKp46 triggering receptor and NKG2D activating receptor, thus manifesting a reduced ability to lyse K562 cells.
TG fosters the generation of functional CIK cells with no concomitant expansion of tumor-suppressive Treg cells. The culture conditions described herein should be applicable to cancer-bearing individuals, although the differentiation of fully functional CIK cells may be hindered in patients with advanced malignancies.
Adoptive cellular immunotherapy aims at restoring tumour-cell recognition by the immune system, leading to effective tumour cell killing. A major hurdle to the successful immunotherapy of cancer is represented by the difficulty in generating clinically relevant numbers of immune effector cells with potent in vivo anti-tumour activity, especially in heavily pre-treated patients. To date, various populations of cytotoxic effector cells have been expanded using robust cell culture procedures and have been administered in a variety of human cancers. Host effector cells endowed with killing activity against tumour cells were initially described in the early 1980s as lymphokine-activated killer (LAK) cells [1, 2]. The LAK cell population is heterogeneous, being comprised of CD3-CD56+ NK cells, CD3+CD56+ MHC-unrestricted cytotoxic T cells and CD3+CD56- T cells. Over the years, improvements in culture conditions, such as the addition of αCD3 (OKT3) monoclonal antibody (mAb) at the initiation of culture and the provision of cytokines at the end of culture, translated into better expansion of LAK cells. Current protocols to differentiate cytokine-induced killer (CIK) cells are based on a combination of 1,000 IU/ml interferon (IFN)-γ on day 1 of culture, followed 24 hours later by OKT3 at 50 ng/ml and interleukin (IL)-2 at 300 IU/ml . At the end of the 21-28 day culture period, CD3+CD56+ cells, derived from CD3+CD56- cells, acquire cytotoxicity against various tumour cell targets, including acute myeloid leukaemia (AML), chronic myeloid leukaemia (CML), B and T-cell lymphoma. The expression of CD56 on CIK cells is thought to result from IFN-γ priming with IL-12 production from monocytes. CIK cells share phenotypic and functional properties of both T cells and NK cells, insofar they express CD3 and are rapidly expandable in culture like T cells, while not necessitating functional priming for in vivo activity like NK cells. Interestingly, CIK cells do not recognize target cells through the T-cell receptor (TCR) and do not require the presence of major histocompatibility complex (MHC) molecules on target cells, as suggested by the observation that cytotoxicity is not affected by antibody masking of the TCR or MHC class I or class II molecules . Cytotoxicity by CIK cells does not rely on antibody-dependent cell cytotoxicity (ADCC) mechanisms, given the absence of CD16 on their surface membrane, and is not inhibited by the immune suppressive drugs cyclosporine A and FK506 . Conversely, the anti-tumour activity of CIK cells mainly relies on the engagement of NK Group 2, member D (NKG2D) by NKG2D ligands on tumour cells, and on perforin-mediated pathways .
The in vivo activity of CIK cells was initially demonstrated in a murine SCID/human lymphoma model, where the co-administration of CIK cells with B lymphoma cells exerted a favorable effect on mice survival, with a 1.5-2-log cell kill and minimal toxicity against normal hematopoietic precursors . CIK cells were subsequently shown to protect against syngeneic and allogeneic tumors in other experimental models, including nude mice xenografted with human cervical carcinoma cells [7–9]. An international registry (IRCC) has been recently established with the aim to report results from current clinical trials using CIK cells, either as such or additionally manipulated . Eleven clinical trials with autologous or allogeneic CIK cells were identified, with 426 patients enrolled. Most trials included male patients with hepatocellular carcinoma, gastric cancer and relapsed lymphoma [11, 12]. A clinical response was reported in 384 patients who received up to 40 infusions of CIK cells. The total response rate was 24% and a decrease of tumour volume was documented in 3 patients. However, disease-free survival rates were significantly higher in patients treated with CIK cells than in a control group without CIK treatment.
Thymoglobulin® (TG) is a purified, pasteurized preparation of polyclonal γ immunoglobulin raised in rabbits against human thymocytes . TG is currently indicated for the prevention and/or treatment of renal transplant rejection, and displays specificity towards a wide variety of surface antigens on both immune system and endothelial cells. The precise mechanism(s) of action underlying its immunosuppressive efficacy are unclear, although T-cell depletion is considered to play a prominent role. Other mechanisms include lymphocyte surface antigen modulation, transcription factor activation, and interference with processes of immune system cells, such as cytokine production, chemotaxis, endocytosis, stimulation and proliferation (reviewed in ref. ). TG may also induce apoptosis, antibody-dependent lysis or complement-mediated lysis of various immune system cells, thus negating leukocyte-endothelial cell adhesion. Intriguingly, anti-lymphocyte globulin therapy in patients with aplastic anemia enhanced the function of MHC-unrestricted lymphocytes . It is presently unknown whether TG can expand CIK cells more efficiently than αCD3 mAb in clinical-grade cultures.
We report herein the results of an in vitro study where TG was confronted with αCD3 mAb for its ability to promote the expansion and acquisition of cytotoxicity by CIK cells. We show that TG amplifies the number of CIK cells with greater efficiency than αCD3 after 21 days in culture. CIK cells generated in this fashion express a constellation of NK cell-associated inhibitory/activating receptors, release considerable amounts of IL-12p40 and lyse the NK-sensitive K562 cell line. The above culture conditions were also applied to PBMC from heavily pre-treated cancer patients, to ascertain whether TG can be a candidate drug for the optimization of CIK expansion protocols in preparation for clinical trials.
Materials and methods
Generation of CIK cells
Stage/grade at diagnosis
Advanced, metastatic disease
Cervical cancer (squamous)
Neoadjuvant radiochemotherapy, radical surgery, chemotherapy (2 lines)
Cervical cancer (squamous)
Radical surgery, adjuvant radiochemotherapy, chemotherapy (4 lines)
Cervical cancer (squamous)
Radiochemotherapy, chemotherapy (3 lines)
Peripheral blood samples collected by venipuncture were layered over Ficoll-Paque® (GE Healthcare Life Sciences; Milan, Italy) and peripheral blood mononuclear cells (PBMC) were separated by centrifugation at 1,400 rpm for 30 minutes, as already detailed . After washings with PBS, PBMC were grown in serum-free medium (X-VIVO 10; Bio-Whittaker Europe, Belgium) supplemented with 80 mg/L gentamycin (Schering Plough, Milan, Italy) and incubated at 37°C in a 5% CO2 atmosphere. Cells were seeded at 2.0 × 106 cells/ml in 25 cm2 cell culture flasks (Corning, NY 14831, USA). On day 0, cells were activated with recombinant human IFN-γ (1,000 IU/ml; Imukin®, Boehringer Ingelheim, Ingelheim, Germany). The following day, cells were stimulated with either αCD3 mAb (UCHT1 clone; 50-500 ng/ml, BD Biosciences, San Diego, CA) or Thymoglobulin® (50-500 ng/ml, Genzyme Corp., Cambridge, MA) and recombinant human IL-2 (rHuIL-2, 300 IU/ml; Proleukin®, Novartis Pharma, Milan, Italy). Cell suspensions were maintained in subculture with fresh medium supplemented with rHuIL-2 every 3 days for 3 weeks. For quality control, aliquots of cells were harvested weekly and used for automatic cell counting, phenotypic analysis, and microbiologic testing. Cell viability was evaluated at the end of the culture period by flow cytometry, after labeling with 7-amino-actinomycin-D (7-AAD; Sigma-Aldrich, Milan, Italy) .
Flow cytometry and immunofluorescence
At baseline (day 0) and after 7, 14 and 21 days in culture, aliquots of cells were incubated for 30 minutes at 4°C with fluorochrome-conjugated mAb to CD3, CD8, CD45, CD16+CD56 (BD Multitest™IMK Kit; BD Biosciences, Mountain View, CA), CD94, CD158a (KIR2DL1), CD158b (KIR2DL2/DL3; BD Biosciences), NKG2A (KLRC1 or CD159a; R&D Systems, Oxon, UK), NKp46 (CD335), NKG2D (CD314; Beckman Coulter, Milan, Italy). Isotype-matched, fluorochrome-conjugated mAb from the same manufacturers were used to control for background fluorescence. The intracellular expression of the FoxP3 transcription factor was detected in fixed/permeabilized T cells that were initially labeled with anti-CD4 and anti-CD25 mAb (both from BD Biosciences), followed by Alexa Fluor 488-conjugated rat anti-human FoxP3 mAb (PCH101 clone; Human Regulatory T Cell Staining Kit; eBioscience, San Diego, CA). Cells were run through a FACS Canto® flow cytometer (BD Biosciences) with standard equipment . Samples were analyzed with the FACS Diva® software package (BD Biosciences).
Measurement of IL-12p40
After 21 days, supernatants from CIK cell cultures were collected and used to quantify IL-12p40 production by enzyme-linked immunosorbent assay (ELISA; R&D Systems, Oxon, UK), as reported . The limit of detection was <15 pg/ml IL-12p40.
Data distribution was preliminarily tested with kurtosis and symmetry. Data were presented as median and inter-quartile range. All comparisons were performed with the Mann-Whitney or the Wilcoxon signed-rank tests for paired or unpaired determinations, as appropriate. The criterion for statistical significance was defined as p < 0.05.
Generation of CIK cells with TG
TG-induced expansion (fold-increase) of PBMC from healthy donors.
T = 7d
T = 14d
T = 21d
Phenotype and effector functions of in vitro-generated CIK cells
IL-12 is a T helper type 1 (Th1) cytokine that augments NK-cell proliferation in vitro and enhances their cytotoxicity in vivo . The expression of IL-12p40 subunit is known to be restricted to cells that produce the biologically active IL-12 heterodimer . As shown in Figure 6C, IL-12p40 levels were significantly higher in day 21-cultures differentiated with hiTG compared with either lower doses of TG or αCD3 mAb. Taken together, these experiments suggest that hiTG-differentiated CIK cells may be particularly suitable for adoptive immunotherapy approaches to cancer.
Generation and function of CIK cells from cancer patients
Phenotypic features of patient-derived effector cells after 21 days in culture.
CD3+CD8+ (T cells)
CD3+CD16+CD56+ (CIK cells)
The present study aimed at dissecting the role of TG in the differentiation of CIK cells, a heterogeneous population of immune effector cells sharing T-cell and NK-cell characteristics. The relationship between in vivo circulating CD3+CD56+ T cells and in vitro-generated CIK cells is poorly understood. Human CD3+CD56+ T cells can be detected within peripheral blood CD8+ T cells and express CD16, CD161, NKG2D and KIR such as CD158a, CD158b and CD94 . The most extensively characterized human NK antigen-expressing CD3+ T-cell subset is represented by CD56+ T cells that account for ~5% of peripheral blood T cells. CD56+ T cells lyse NK-sensitive target cell lines in vitro, can be selectively expanded by IL-2 and IL-15, but require cell activation to trigger the secretion of effector cytokines such as IFN-γ and TNF-α. It has been recently shown that CIK cells expanded with IFN-γ, OKT3 and IL-2 resemble activated effector-memory CD8+ T cells and likely derive from CD56- T cells, as suggested by gene expression profiling . In this respect, only 50 differentially expressed genes were identified when comparing CIK cells and CD56- T cells, whereas 115 genes were either up-regulated or down-regulated in CIK cells compared with CD56- T cells . Collectively, it is now recognized that CIK cells have undisputed advantages over other cell therapy products that make them particularly attractive, such as ease of in vitro expansion, superior in vivo activity than LAK cells, and no need for exogenous administration of IL-2 for in vivo priming [33, 34]. Current laboratory protocols dictate that CIK cells should be differentiated with IFN-γ and the OKT3 mAb to CD3, followed by repeated additions of IL-2 for a maximum of 21-28 days [3, 11, 12, 33].
Our interest in TG as a candidate drug to expand CIK cells in preparation for clinical trials originated from reports indicating that binding of TG to CD16, CD18 and NKp46 on NK cells potentiates their activation and degranulation, and enhances IFN-γ production, although this translated into the decrease of NK cytotoxicity against K562 cells . When selecting the optimal TG concentration to be used in culture, we took advantage of previously published papers showing the following points. First, TG may induce ~ 15% NK cell apoptosis in vitro, when added at concentrations ranging from 1 μg/ml to 100 μg/ml [35, 36]. Second, TG directly affects CD4+ T-cell function and cytokine release when used at 10 μg/ml, transiently up-regulating CD25, FoxP3 and CTLA-4 mRNA and protein, and increasing IL-2, IL-4, IL-10 and IFN-γ secretion in culture supernatants. Third, CD4+ T cells pre-treated with 10 μg/ml TG inhibit the proliferation of autologous CD4+ T cells to allogeneic PBMC, suggesting the acquisition of a regulatory phenotype . We therefore elected to provide TG at relatively low concentrations (from 50 to 500 ng/ml) to the PBMC cultures, in order to minimize both NK and possibly CIK-cell apoptosis as well as the amplification of Treg cell numbers. TG significantly expanded PBMC compared with lowαCD3 mAb, leading to the in vitro generation of a heterogeneous population comprised of CD8+ T cells, NK cells and CIK cells. Especially when used at 500 ng/ml, TG augmented the proliferation of PBMC with subsequent enhanced generation of CD8+ T cells, NK and CIK cells, compared both with an equally high concentration of αCD3 mAb and with lowTG or intTG. This implies that hiTG may be particularly effective at the concurrent expansion of all three types of immune effector cells, namely, CD8+ cytotoxic T cells, NK cells and CIK cells, at variance with αCD3 mAb. Of potential importance for the design of clinical trials with TG/IL-2-expanded CIK cells, the frequency of bona fide Treg cells at any time-point in culture was similar when comparing PBMC preparations activated with IL-2 and TG or αCD3 mAb, thus reassuring against the infusion of excessive numbers of tumor-suppressive Treg cells .
NK cells express a wide array of inhibitory and activating receptors such as KIR, NKG2A/CD94, NKG2D, NKp46 and others, which recognize both foreign and self antigens expressed by target cells, and finely regulate NK cytotoxicity against virus-infected and tumor cells . NK receptors play a crucial role in innate immunity against infections and in anti-tumor immune responses. It is presently unknown whether TG modulates the expression of NK receptors on CIK cells, a finding with important implications for their cytotoxic activity and for their ability to combat infections. The KIR family consists of 11 highly polymorphic receptors that are clonally distributed on NK cells and bind directly to classical MHC molecules such as particular HLA-Cw alleles. KIR may be expressed at low levels (i.e., < 10%) on CIK cells differentiated with standard protocols . In our study, both CD158a (KIR2DL1) and CD158b (KIR2DL2/DL3) were readily detected on CIK cells expanded with hiTG, with expression levels ranging from ~15% to ~65% of CD3+CD56+ cells for CD158a and CD158b, respectively. Although KIR-expressing CD8+ T cells exist in human peripheral blood , the stimuli that regulate KIR induction in T cells are poorly defined , and may include demethylation events . Interestingly, engagement of CD158b by MHC ligands on human CD8+ effector T cells hinders TCR signaling and limits T-cell proliferation . Based on our findings, it is tempting to speculate that TG provided an in vitro signal orchestrating the expression of KIR on CIK cells. Conceivably, the TG-driven expression of KIR might represent a feedback signal to limit excessive CIK expansion and/or uncontrolled in vitro cell death. Although the nature of the signal(s) delivered to CIK cells through TG remains to be identified, it is unlikely that cytokine stimuli such as IL-15 are implicated, based on our observation that IL-15 provision to CIK cultures did not translate into any further induction of KIR (Rutella S, unpublished observations, 2010). Our statement is also supported by a previous report demonstrating the inability of IL-15 and IL-21 to induce KIR expression on cord blood-derived NK progenitor cells .
NKG2D encodes for a lectin-related protein expressed as a homodimer and functioning as an activating receptor for ligands often expressed by tumor cells, namely, class I MHC-related molecules such as MICA, MICB, and UL16-binding proteins . The NKG2A/CD94 receptor contains C-type lectin ectodomains, binds to HLA-E, a non-classical MHC protein important for viral surveillance, and functions as an inhibitory receptor by signaling through ITIM motifs [44, 45]. As recently proposed, high surface levels of NKG2A/CD94 may be required to avoid excessive NK cell-mediated killing of HLA-E-bearing normal target cells . Of interest, CD94/NKG2A expression on CD8+ T cells may protect from apoptosis and favor the eventual emergence of memory T-cell responses . In light of these findings, it is conceivable that high levels of CD94/NKG2A and KIR on TG-differentiated CIK cells promote cell survival, leading to protection from CIK-mediated killing of normal cells.
NKp46 belongs to a family of activating natural cytotoxicity receptors (NCR) for tumor cells , also including NKp30 and NKp44, that enables a precise identification of all NK cells. Upon engagement by specific ligands, NCR induce a strong activation of NK-mediated cytotoxicity, thus playing a pivotal role in tumor cell killing . To date, NCR have been detected on NK cells in a restricted fashion and regardless of NK-cell activation status. Notably, NKp46 was found on ~15-20% of CD3+ CIK cells differentiated with hiTG, and lower expression levels of NKp46 correlated with lower TG concentrations in the culture medium. These data are backed by a recent study documenting a 10-20% expression of NKp30, NKp44 and NKp46 on CIK cells driven by IFN-γ, OKT3 and IL-2 . Overall, these observations question the specificity of NCR for cells of the NK lineage and suggest that NCR may also contribute to the killing activity of CIK cells. When evaluated for their ability to lyse tumor targets, CIK effectors differentiated with TG were significantly more effective at killing K562 cells compared with those nurtured with αCD3 mAb. It should be noted that patient-derived CIK cells expressed lower levels of activating/inhibitory NK receptors and manifested a reduced lytic activity in vitro in 2 out of 4 cases. Although the very small number of patients under study precludes any sensible conclusion, it is likely that the generation of fully functional CIK cells by TG was hindered by immune suppressive circuits in patients with advanced metastatic disease.
IL-12, a prototype member of a family of IL-12-related cytokines that includes IL-23 and IL-27, is an instigator of Th1 immune responses and possesses in vivo anti-tumor activities . IL-12 is a heterodimer formed by a 35-kDa light chain (known as p35 or IL-12α) and a 40-kDa heavy chain (known as p40 or IL-12β). Messenger RNA encoding IL-12p35 is present in many cell types, whereas mRNA encoding IL-12p40 is restricted to cells that produce the biologically active heterodimer . Importantly, CIK cells generated with hiTG released higher quantities of IL-12p40 compared with the other culture conditions here established. This finding portends favorable implications for the use of hiTG in the generation of CIK cells, given the established role of IL-12 in the promotion of anti-tumor immunity .
In conclusion, we propose that TG is an attractive drug to maximize the yield and anti-tumor potency of CIK cell preparations. The expansion of immune effector cells in response to a combination of IFN-γ, TG and IL-2 occurred in the absence of a significant induction of Treg cells, whose infusion into cancer-bearing patients would be highly undesirable. From a clinical standpoint, CIK cells are likely to be efficacious at disease stages where the tumor burden is relatively low or in an adjuvant setting, rather than in advanced disease . It is presently unknown whether the overall survival rate is significantly affected by this type of adoptive cellular therapy. Future studies will have to address whether CIK cells differentiated with TG offer advantages over those obtained with αCD3-based protocols and whether they may be integrated into current cancer treatments.
These studies were supported by a research grant from Fondazione Roma, Rome, Italy (to S.R. and G.S.) and from Associazione Italiana per la Ricerca sul Cancro (AIRC; grant #8556 to S.R.).
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