Regulatory activity of azabisphosphonate-capped dendrimers on human CD4+ T cell proliferation enhances ex-vivo expansion of NK cells from PBMCs for immunotherapy
© Portevin et al; licensee BioMed Central Ltd. 2009
Received: 27 May 2009
Accepted: 24 September 2009
Published: 24 September 2009
Adoptive cell therapy with allogenic NK cells constitutes a promising approach for the treatment of certain malignancies. Such strategies are currently limited by the requirement of an efficient protocol for NK cell expansion. We have developed a method using synthetic nanosized phosphonate-capped dendrimers allowing such expansion. We are showing here that this is due to a specific inhibitory activity towards CD4+ T cell which could lead to further medical applications of this dendrimer.
Mononuclear cells from human peripheral blood were used to investigate the immunomodulatory effects of nanosized phosphonate-capped dendrimers on interleukin-2 driven CD4+T cell expansion. Proliferation status was investigated using flow cytometry analysis of CFSE dilution and PI incorporation experiments. Magnetic bead cell sorting was used to address activity towards individual or mixed cell sub-populations. We performed equilibrium binding assay to assess the interaction of fluorescent dendrimers with pure CD4+ T cells.
Phosphonate-capped dendrimers are inhibiting the activation, and therefore the proliferation; of CD4+ T cells in IL-2 stimulated PBMCs, without affecting their viability. This allows a rapid enrichment of NK cells and further expansion. We found that dendrimer acts directly on T cells, as their regulatory property is maintained when stimulating purified CD4+ T cells with anti-CD3/CD28 microbeads. Performing equilibrium binding assays using a fluorescent analogue, we show that the phosphonate capped-dendrimers are specifically interacting with purified CD4+ T cells. Ultimately, we found that our protocol prevents the IL-2 related expansion of regulatory T cells that would be deleterious for the activity of infused NK cells.
High yield expansion of NK cells from human PBMCs by phosphonate-capped dendrimers and IL-2 occurs through the specific inhibition of the CD4+ lymphocyte compartment. Given the specificity of the interaction of dendrimers with CD4+ T cell, we hypothesize that regulatory activity may signal through a specific receptor that remains to be indentified. Therefore phosphonate-capped dendrimers constitute not only tools for the ex-vivo expansion of NK cells in immunotherapy of cancers but their mode of action could also lead to further medical applications where T cell activation and proliferation need to be dampened.
Natural Killer cells constitute a heterogeneous and multi-functional population of the innate immune system. Although the CD56dim/bright functional dichotomy has been revised recently , NK cells are generally divided in two subsets that differ in their anatomic distribution, cytotoxic potential and ability to proliferate and produce cytokines [2, 3]. NK cells initially-obtained their name due to their natural cytotoxicity against tumor cells requiring no prior sensitization, unlike T cells . It is well established that the cytotoxicity of NK cells relies notably on their ability to sense the decrease/absent expression of MHC-I molecules on their target ("missing-self model") [5, 6]. In humans, this sensing is controlled by a set of inhibitory receptors belonging to the Killer immunoglobulin-like receptor (KIR) family and/or the heterodimer CD94/NKG2A: each receptor having variable specificity for allotypic variants of MHC-I molecules . The NK cell repertoire of inhibitory receptors is qualitatively and quantitatively variable between humans due to the inherited set of genes coding for these receptors, but also within the same individual, due to the stochastic expression of these genes . This has important implications particularly during the treatment of acute leukemias which require a Stem Cell Transplantation (SCT). Indeed, alloreaction mediated by NK cells could occur between haploidentical individuals presenting a functional mismatch in the NK cell repertoire towards recipients MHC-I ligands. In this context, NK cell alloreactivity has been shown to increase prognosis by enhancing anti-tumor activity (GvL effect) and decrease side effects of immune reconstitution (GvHD) by depleting recipients' DCs [9, 10]. In mice, infusion of alloreactive NK cells in the context of SCT also induces potent antitumor effects [9, 11] and such therapeutic approaches are now realistic in humans . More generally, adoptive transfer of ex-vivo expanded NK cells constitutes a promising approach in immunotherapy of cancer [13, 14]. Unfortunately, NK cell expansion remains tedious to achieve, using protocols with purification steps, clonal dilution and/or monoclonal antibodies limiting the outcome of NK cell-based immunotherapy . Dendrimers are versatile tree-like branched synthetic polymers with very promising medical applications such as chemotherapeutic agent delivery . More remarkably, it was shown that a N-acetyl-glucosamine-coated poly-amido-amine (PAMAM) dendrimer stimulates an antitumor immune response involving enhancement of the functions of CD4 T cells and NK cells . A mannosylated dendrimer of the same PAMAM family conjugated to ovalbumin (OVA) has been shown to induce, in vitro and in vivo, a very potent immune response against OVA highlighting their adjuvanticity . We have recently reported that a group of nanosized synthetic phosphonate-capped dendrimers (especially 3a-G1) activate human monocytes toward an anti-inflammatory and immunosuppressive pathway [19–21]. We also described an innovative protocol using dendrimer 3a-G1 that allows high yield expansion human NK cells from PBMCs . Expanded NK cells are fully functional and can efficiently lyse a broad spectrum of tumor cell lines. Prospecting the transfer from bench to clinic of such expanded NK cells, we had to decipher the origin of this expansion process. Here, we show that 3a-G1 driven expansion of NK cells from PBMCs is not occurring through a direct activation of the NK cell reservoir but actually through the regulation of CD4+ T cell expansion. Ultimately, we found that our protocol prevents the IL-2 related expansion of CD4+/CD25+/CD127low/FoxP3+ regulatory T cells. Given the fact that regulatory T cells might affect NK cell functions in vivo [23, 24], this last finding supports the use of our expansion protocol for NK cell-based adoptive immunotherapy of cancers.
Blood samples, cells and cell cultures
Fresh blood samples were collected from healthy adult donors, and PBMCs were prepared on a Ficoll-Paque density gradient (Amersham Biosciences AB, Uppsala, Sweden) by centrifugation (800 g, 30 min at room temperature). Collected PBMCs were washed twice and finally diluted at 1.5 million cells/ml in complete RPMI 1640 medium, i.e., supplemented with penicillin and streptomycin, both at 100 U/ml (Cambrex Bio Science, Verviers, Belgium), 1 mM sodium pyruvate, and 10% heat-inactivated fetal calf serum (both from Invitrogen Corporation, Paisley, UK) and when specified recombinant IL-2 (400 U/ml) and dendrimers solution (20 μM). Detailed chemical synthesis of dendrimers could be found here [19, 20, 22]. NK cells, CD4 T cells, and monocytes were selected from PBMC by magnetic cell sorting using respectively the NK isolation kit II, the CD4 T cell isolation kit and CD14 microbeads (Miltenyi Biotec, Auburn, CA, USA) according to manufacturer's recommendations. Cell purity checked by flow cytometry was always >95% for NK cells and >98% for CD4 T cells and monocytes.
Flow cytometry and cell surface staining
Flow cytometry was performed using a LSR-II cytometer, BD biosciences, San Jose, CA, USA. Data treatment and analysis were performed using Flowjo or BD FacsDiva software. Anti-CD3 FITC or PE (UCHT1), anti-CD4 PE or PC5 (13B8.2), anti-CD56 PC5 (N901), anti-CD127 PE (R34.34) (Beckman Coulter Immunotech), anti-CD14 PE or PC7 (clone M5E2), anti-CD56 PC7 (clone B159) (BD biosciences) and anti-FoxP3 PE (PCH101) (eBioscience) were used according to manufacturer's recommendations. For surface staining, cells were incubated with fluorochrome-conjugated monoclonal antibodies in cold PBS containing 5% of fetal bovine serum at 4°C for 15 min in the dark, then washed before analysis. Eventually, intracellular staining of FoxP3 was done using Foxp3 Staining Buffer Set (eBioscience) following manufacturer's instructions.
CFSE dilution, NK cell amplification and cell cycle analysis
For carboxyfluorescein succinimidyl ester (CFSE) cell staining, a 250 μM stock solution in DMSO was freshly diluted in PBS and immediately used to resuspend cells at 5.106 cells/ml for 8 min at 37°C. Reaction was stopped after adding one volume of fetal calf serum and cells were washed twice with PBS before culture. For anti-CD3/CD28 stimulation of PBMCs or purified CD4+ T cells, 5.104 CFSE labelled cells were mixed 1.2.103 anti-CD3/CD28 mAb-coated Dynabeads (Invitrogen) and displayed in U-shaped 96 well plates. CFSE dilution was favourably analyzed after 7 days of culture. In experiments aimed at measuring the NK cell amplification, cultures were maintained during 12 to 14 days to enhance the effect of the inhibition of CD4+ T cell proliferation on the subsequent amplification of NK cells.
For cell cycle analysis, 105 cells were resuspended on ice with cold PBS containing 2% fetal calf serum and fixed with 3 volumes of absolute ethanol overnight at 4°C. Pelleted cells were resuspended with 50 μl propidium iodide 10 μg/ml in PBS and 18 μl of a RNAse solution for 30 min RT and washed with PBS containing 5% fetal calf serum before flow cytometry analysis.
Equilibrium binding assay
Cells in triplicates were incubated for 15 min on ice with detailed concentration of dendrimer solution in PBS containing 5% fetal calf serum and washed before flow cytometry analysis. Progression of cellular mean fluorescence intensity was analysed using modelling software (SAAMII, v1.2, University of Washington).
Statistical analyses were carried out using the biostatistic software GraphPad Prism (GraphPad Software, Inc). Wilcoxon signed-rank test was performed to compare amplification rate and cell proportion between 3a-G1 treated and untreated samples (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).
Azabisphosphonate branched dendrimers specifically inhibit IL-2 driven proliferation of CD4+ T cell among human PBMCs
3a-G1 interferes with CD4+ T cell activation and proliferation inducing NK cell enrichment
Regulatory activity of 3a-G1 is direct and does not require monocytes
Cellular interaction of azabisphosphonate branched dendrimers using a fluorescent analogue of 3a-G1
3a-G1 inhibits IL-2 related expansion of CD4+/CD25+/CD127-/FoxP3+ regulatory T cells
In this report, we elucidate the origin of the enrichment and subsequent expansion of NK cells from human PBMCs using 3a-G1 phosphonate-capped dendrimers . Therefore, we focused our analysis on the first two weeks of culture although the expansion procedure requires 4 weeks to provide suitable amounts of cells for clinical purposes. Such amplified NK cells are perfectly cytotoxic against the K562 cell line but also a broad range of other tumor cell line. Although this has not been checked systematically, we did found that mid-term amplified NK are also cytotoxic against the K562 cell line and that 3a-G1 doesn't affect their cytotoxicity when compared with untreated cells [see Additional file 1]. Contrary to expectation, we could not demonstrate any significant activation of proliferation of pure NK cells exposed to 3a-G1. Conversely, we showed that during the first week of culture, 3a-G1 mainly acts by inhibiting CD4+ T cell proliferation without affecting NK cells. In terms of cell expansion, we found that NK cells are normally competing with CD4+ T cells when PBMCs are exposed to interleukin-2 and that 3a-G1 cancels this competition. Therefore, the decreased CD4+ T cell representation results in more nutrients and cytokines for the expansion of NK cells. We propose that the higher proliferation status of NK cells when PBMCs are exposed to 3a-G1 (Fig. 1) is mainly due to an increase in the availability of IL-2 that has not been consumed by proliferating T cells. Supporting our hypothesis, other investigators have described the use of anti-CD3 antibodies and IL-2 as a method for the in vitro expansion of human NK cells from PBMCs . No clues were provided about the origin of this process but it suggests that targeting T cells to some extent sustains the expansion of NK cells from PBMCs. Interestingly; we demonstrated that like such antibodies, 3a-G1 dendrimers specifically interacts with CD4+ T cells. We believe that this interaction might drive the inhibition of CD4+ T cell proliferation observed not only among PBMCs but also when pure CD4+ T cells were stimulated with anti-CD3/CD28 coated beads. Molecular determinants are still needed regarding the mode of action of 3a-G1 but given its structural features, it is tempting to speculate that 3a-G1 could act by triggering Sphingosine 1-phosphate (S1P) receptors. Indeed, there is some evidence that S1P regulates T cell proliferation . Interestingly, the phosphate moiety was shown to be important for this effect. To address that point, we are now concentrating our effort in the synthesis of a biotin analogue of 3a-G1 to perform pull-down experiment on CD4+ T cell protein extracts with the aim of identifying by proteomics the molecular determinants of 3a-G1 regulatory activity. Furthermore, Miller and colleagues have described the importance of monocytes in the expansion of human NK cells from IL-2 treated PBMCs . We have shown that depleting monocytes from PBMCs prevents CD4+ T cell proliferation. In agreement with Miller's report, we also found that NK cells are less able to proliferate when monocytes are depleted from PBMCs. Therefore, monocytes are supporting the ex-vivo expansion of both cell types. Interestingly, we showed that monocytes rapidly engulfed phosphorus-containing dendrimers and consequently become activated [19, 20]. We have addressed the particular mode of activation of these monocytes highlighting an immune-suppressive phenotype on mixed leukocyte reaction  that could sustain the inhibition of T cell proliferation although we have shown here, using anti-CD3/CD28 microbeads, that monocytes are not required for regulatory activity of phosphonate-capped dendrimers. Again, Miller and colleagues showed that CD5+ and CD8+ cell depletion led to higher NK cell expansion yield providing support that T cells constitute a barrier for the expansion of NK cells. IL-2 stimulation of PBMCs was shown to elicit absolute expansion of NK cells and CD56+ T cells, e.g. NK-T cells, γδ T cells and some αβ/CD8+ T cells . The combination of IL-2 and 3a-G1 in our hands also led to a generally slightly higher representation of γδ-T cells (data not shown) but we were never able to detect any NKT (Vα 24+) cell or CD8+ T cell expansion under our conditions. In contrast, we found that a proportion of CD4+ T cells that became activated under IL-2 stimulation were presenting a regulatory T cell phenotype e.g. CD25+/FoxP3+/CD127 low , the best up to date combination to characterise regulatory T cells . Such in vitro induction of T regulatory activity by stimulated human CD4+/CD25- has already been described . In vivo, regulatory T cells play an important role in maintaining peripheral tolerance and preventing auto-immunity but they could also affect anti-tumor immunity by notably acting on NK cell activity [23, 24]. Then, the presence of regulatory T cells during the process of NK cell expansion by 3a-G1 would have had a highly detrimental effect. Interestingly, the inhibition of CD4+ T cell activation by 3a-G1 is global and also affects the accumulation of these phenotypically related regulatory T cells. Although it can't be excluded that the presence of few remaining regulatory T cells could have a detrimental effect for the activity of infused NK cells in vivo, it does not affect the cytotoxic property of the expanded NK cells in vitro against classical tumor cell lines .
On the wave of tetramer technology , this project was initiated to use dendrimer plasticity to chemically build a platform of bi-phosphate entities that would promote γδ-T cell expansion . This contemporary attempt of building and testing a sophisticated hypothesis actually ended with unexpected results. Indeed, it turned to favour the expansion of NK cells another subset of cytotoxic lymphocytes and we show here that this is happening by selectively inhibiting the activation and proliferation of CD4+ T lymphocytes. Having set the expansion protocol using good manufacturing practice (GMP)-compliant components, we are now planning to translate from bench to clinic the use of such ex-vivo amplified NK cells as a conditioning treatment for patients undergoing leukemia therapy. The therapeutic relevance of our method does have some limitation as we did observe variation in NK cell expansion between donors at the term of the amplification process . However, deciphering the molecular determinants of phosphonate-capped dendrimer activity in the regulation of T cell proliferation could also lead to further applications in the treatment of pathologies where T cell proliferation is undesirable, such as cutaneous T-cell lymphoma and/or auto-immune diseases for which efficient treatments are still needed .
Peripheral Blood Mononuclear Cells
CarboxyFluorescein Succinimidyl Ester
We thank Dr. Frederic Pont (IFR30, Toulouse, France) for valuable support in the Akaike's criterion determination. We are grateful to Dr. Ludovic Martinet (U563, Toulouse, France) for crucial discussion and helpful overview and Dr. Anna Cousse for critical reading of this manuscript. D. P. was the recipient of a Fellowship from the Association pour la Recherche contre le Cancer (ARC). This work was supported by the Institut National de la Sante et de la Recherche Medicale (INSERM, France), Paul Sabatier University and the Institut National du Cancer (INCa) and the Region Midi-Pyrenees.
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