Cytotoxic response of human regulatory T cells upon T-cell receptor-mediated activation: a matter of purity

Citation: Blood Cancer Journal (2014) 4, e199; doi:10.1038/bcj.2014.20

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LETTER TO THE EDITOR

Cytotoxic response of human regulatory T cells upon T-cell receptor-mediated activation: a matter of purity

Blood Cancer Journal (2014) 4, e199; doi:http://dx.doi.org/10.1038/bcj.2014.20

Web End =10.1038/bcj.2014.20 ; published online 11 April 2014

Regulatory T cells (Tregs) are major players in immune home-ostasis, and defects in their number and/or function are associated with a broad range of immunological disorders including autoimmunity, graft-versus-host disease (GvHD), transplant rejection, chronic infections or malignant diseases.1,2 One way of

how Tregs actively damp an immune response might be direct induction of apoptosis in activated antigen-presenting cells as well as in conventional effector T cells (Teffs) by secretion of lytic granzyme- and perforin-containing granula. Despite the substantial progress made in our understanding of Treg biology, the cytotoxic potential of Tregs is still under debate. As Tregs are under intense investigation as cellular therapeutics for treatment of GvHD and graft rejection, and might also interfere with novel antitumor strategies,3 this question is not a purely academic one but also implies major consequences for the prospective development of clinical treatment strategies involving Tregs. To shed some light on this controversial issue, we decided to analyze this question using peripheral human Tregs, as these cells are one major source for potential therapeutic applications.1,2 As Tregs are

a very rare population in human peripheral blood, it seems currently technically too challenging to isolate sufcient Treg numbers with dened antigen-specicity in a way as it can be done forfor example, virus- or tumor-specic Teffs. Therefore, in this study recombinant bispecic antibodies (bsAb) were applied for antigen-specic redirection of polyclonal human T cells as surrogate for T-cell receptor (TCR)-mediated stimulation. BsAb comprise binding sites for two different target antigens (for example, CD3 and a cell surface antigen) and can directly cross-link T cells and antigen-expressing cells. This results in a major histocompatibility complex (MHC)- and TCR-independent activation and triggering of effector mechanisms of the cross-linked immune cell.4 T-cell-engaging bsAb have been proven to be highly effective in inducing the lytic capacity of both CD4 and CD8 Teffs in vitro, in vivo and even in clinical trials and closely resemble signal cascades induced upon binding of a TCR to its target peptide/MHC complex.46

In the current study, peripheral human Tregs were isolated in a two-step procedure to high purity (Figure 1a). First, CD4CD25

Tregs were enriched using magnetic bead isolation (presort population, 8010% FOXP3). In a second purication step, enriched CD4CD25 T cells were further processed to isolate the CD4CD25CD127low population by ow cytometric cell sorting (postsort population, 953% FOXP3). The cytotoxic potential of isolated Tregs was evaluated in a standard chromium release assay in comparison to autologous CD4 or CD8 Teffs.5 For antigen-specic redirection of T cells, tumor cells pre-decorated with the human La/SS-B antigen and a cross-linking anti-CD3-anti-La bsAb was used as recently established.7 The results demonstrate that target cell killing of bsAb-redirected Tregs is highly dependent on the purity of the isolated cell population (Figure 1b). Whereas highly pure, sorted CD4CD25 CD127low Tregs do not elicit any signicant cytotoxic effect, Tregs isolated only on the basis of CD25 expression display a considerable killing capacity that can most likely be ascribed to contaminating CD25CD127FOXP3 Teffs. The observed killing ability of presort Tregs is as high as the specic lysis provoked by bsAb-engaged autologous CD4 Teffs. One explanation might be that contaminating CD25 Teffs within the presort Treg fraction are highly activated and potent memory T cells. Moreover, isolated CD4CD25 T cells were maintained over night in 300 U/ml interleukin (IL)-2 whereas conventional Teffs were cultured in medium supplemented with 50 U/ml IL-2. It is very likely that, the cytotoxic potential of contaminating CD25CD127FOXP3 Teffs in the presort Treg population was even potentiated due to pre-conditioning with elevated IL-2 concentrations.

To obtain sufcient Treg material for a clinical application, in vitro expansion of this rare cell population is inevitable.However, previous studies pointed out that the only population allowing for an efcient in vitro expansion over a long-lasting period without losing phenotypic and functional Treg characteristics is the CD4CD25CD127lowCD45RA population.8 Therefore, we expanded highly pure, sorted CD4CD25CD127lowCD45RA Tregs over a 12-day expansion period and subsequently analyzed their cytotoxic potential. The purity of the expanded Treg population was conrmed by ow cytometry analysis to be on average 953% FOXP3 cells (Figure 1c). Redirecting expanded Tregs with a bsAb does not result in tumor-cell elimination. By contrast, bsAb-redirected expanded CD4 and CD8 Teffs efciently lyse antigen-positive target cells (Figures 1d and f). To conrm these results, we repeated the chromium release assays using another bsAb, which

Figure 1. Highly pure, freshly isolated or expanded human Tregs do not display a cytotoxic potential. (a) Representative example of the purity of freshly isolated cells. CD4CD25 Tregs were isolated by magnetic activated cell sorting technology (presort). A fraction of cells was further sorted to obtain CD4CD25CD127dim Tregs (postsort). Cell fractions were maintained over night in complete RPMI 1640 medium in the presence of 300 U/ml IL-2 and purity was conrmed by ow cytometry analysis. (b) Presort or postsort Tregs were incubated with chromium-labeled PC3 cells at a 5:1 or 10:1 ratio in the presence or absence of either the La protein or 6 pmol of the cross-linking bsAb CD3-La for 48 h. As positive control, freshly isolated autologous CD4 or CD8 Teffs were used. (c) Representative example of the purity of expanded T cells. Sorted CD4CD25highCD127lowCD45RA Tregs were expanded as recently established in the absence of rapamycin.3 Isolated CD4 and CD8 Teffs were stimulated for 4 days with aCD3/CD28-coated beads in the presence of 200 U/ml recombinant IL-2.

(d, f) Expanded T-cell populations were incubated with chromium-labeled PC3 cells at a 5:1 or 10:1 ratio in the presence or absence of La protein or 6 pmol of the bsAb CD3-La. After 24 h, tumor-cell lysis was determined. (e, g) Cocultivation of expanded Teffs or Tregs with PSCA tumor cells and 6 pmol of a cross-linking CD3-PSCA bsAb. As control, cells were stimulated with conventional aCD3/CD28-coated beads at a ratio of 1:5 beads per cell. One representative donor (d, e) as well as summary of four (g) or ve (f) independent donors (T cell to tumor cell ratio 10:1) is shown. Statistical signicance was determined using one-way analysis of variance with Bonferroni multiple comparison test. Error bars represent means.d. *Po0.05, **Po0.01, ***Po0.001 (n.s., not signicant).

Letter to the Editor

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targets the prostate stem cell antigen (PSCA) and mediates efcient cancer cell lysis upon cross-linkage of Teffs to PSCA tumor cells.5 Again, Tregs are not capable of eliminating target

cells (Figures 1e and g), proving that the lack of cytotoxicity is independent of the chosen target antigen or bsAb. Next, we wanted to exclude that an additional costimulatory signal is

Figure 1. For caption see page 1.

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Teff + Teff Teff + Tregs

eFluor670

CD25

85.1 58.3

38.4

CD4+

CD8+

64.3

Figure 2. For caption see page 4.

necessary to trigger the cytotoxic potential of Tregs. Therefore, we used conventional aCD3/CD28-coated beads for T-cell activation.

However, as opposed to autologous CD4 and CD8 Teffs, Tregs do not kill cocultured tumor cells even upon polyclonal bead stimulation (Figures 1e and g).

To determine whether Tregs release cytotoxic molecules including granzymes and perforin upon activation, we stimulated T cells either with conventional aCD3/CD28-coated beads or with bsAb in the presence of target cells and assessed expression of the activation marker CD69 and degranulation marker CD107a.

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Figure 2. Highly pure, expanded Tregs are not cytotoxic in vivo and display a potent suppressive capacity. (a) Expanded T cells were cocultured together with PC3 cells either without or with 6 pmol of the CD3-La bsAb. Polyclonal stimulation with aCD3/CD28-coated beads was included as positive control. After 20 h, cells were harvested and stained for CD69 and CD107a. Relative percentage of CD69/CD107a double-positive, CD69 or CD107a single-positive and double-negative cells were summarized for three different donors. (b) NMRInu/nu mice were injected

subcutaneously with PC3-PSCA cells. Animals received either CD4 Teffs or expanded Tregs alone with a CD3xPSCA bsAb or a mixture of both T-cell populations with or without the cross-linking bsAb. Tumor size was measured weekly. The in vivo experiment was performed in parallel with previously presented work.3 Statistical signicance was determined using one-way analysis of variance with Bonferroni multiple comparison test. (ce) eFluor670 proliferation dye-labeled CD4 or CD8 Teffs (5 104) were cultured in the presence of La-decorated target

cells and 6 pmol bsAb CD3-La together with either 5 104 (1:1) or 12.5 104 (4:1) unlabeled autologous Teffs or expanded Tregs. (c) CD25

surface expression and dilution of proliferation dye was assessed after 96 h. Numbers in upper right quadrant refer to percentage of CD25 Teffs related to total eFluor670-labeled cells. One representative donor out of ve independently performed experiments is depicted. (d) Absolute cell number of bsAb- or bead-activated, eFluor670-labeled CD4 or CD8 Teffs was measured using a MACSQuant Analyzer (Miltenyi Biotec, Bergisch-Gladbach, Germany) at days 0 and 4. Overall expansion in the presence of unlabeled Teffs was determined, equalized to 100% and relative expansion in the presence of Tregs was calculated. (e) Culture supernatants were collected after 48 h and analyzed by enzyme-linked immunosorbent assay. Secreted interferon (IFN)-g, tumor necrosis factor (TNF) and IL-2 by 5 104 bsAb- or bead-

activated CD4 or CD8 Teffs were set to 100% (black bars) and relative cytokine levels detected in the supernatant in cocultures with autologous Teffs (gray bars) or Tregs (patterned bars) at indicated ratios were calculated. (d, e) Five independent donors were summarized and statistical signicance was determined using one-sample t-test. Error bars represent means.d. *Po0.05, **Po0.01, ***Po0.001.

Analysis of three different donors (Figure 2a) revealed that although Tregs become activated (45.59.7% and 60.326.6% CD69 cells with bsAb and bead activation, respectively), they only marginally upregulate the degranulation marker CD107a(9.41.4% and 16.17.0% upon stimulation with bsAb or beads, respectively) in accordance with published results.9 This suggests that Tregs are not capable of directly inducing apoptosis via release of cytotoxic granula. However, we cannot rule out the possibility that apoptosis in Teffs is induced indirectly for example, via cytokine deprivation. In order to verify our results in vivo, athymic nude mice were inoculated with PSCA tumor cells and a cross-linking CD3xPSCA bsAb as well as either one of the two T-cell populations or a mixture of both CD4 Teffs and Tregs. As shown in Figure 2b, injection of expanded Tregs in combination with tumor cells and bsAb did not reduce tumor outgrowth in mice, substantiating that Tregs are not capable of eliminating co-injected cancer cells. Nevertheless, despite the lack of cytotoxic activity bsAb-redirected Tregs hold a great suppressive potential. They are efciently diminishing CD25 upregulation, proliferation and overall expansion of cocultured autologous CD4 and CD8 Teffs (Figures 2c and d). Moreover, bsAb-engaged Tregs substantially inhibit interferon-g, tumor necrosis factor and IL-2 cytokine release of autologous Teffs (Figure 2e). The suppressive potency of bsAb-redirected Tregs was further conrmed in vivo, as Treg administration rather suppressed the antitumor effect of co-injected CD4 Teffs and signicantly enhanced tumor growth (Figure 2b). The same observation holds true for Tregs antigen-specically engineered with chimeric immune receptors, which instead of contributing to tumor-cell killing efciently suppress the antitumor reaction of Teffs in vivo.10

Taken together, our data provide strong evidence that human CD4CD25CD127low T cells harbor no considerable cytotoxic potential, neither freshly isolated nor after in vitro expansion and prolonged culture, but have substantial suppressive capacity and therefore fulll the criteria to be indeed Tregs. Likewise, human CD4CD25CD127low Tregs isolated from synovial uid of juvenile idiopathic arthritis patients were able to efciently inhibit Teff activation, but no elevated apoptosis rate of Teffs was observed in cocultures.11 As shown by others, Tregs do not have to rely on active apoptosis induction in Teffs by release of cytotoxic granula as other suppressive mechanisms known to be utilized by Tregs such as cytokine deprivation, IL-10 secretion or presentation of cell surface-bound TGF-b are sufcient to suppress

Teff activation.1113 On the other hand, we demonstrate that

human T cells isolated on the basis of surface markers CD4CD25 exhibit considerable cellular cytotoxicity, most likely due to contamination with CD25-activated CD4 Teffs. Our observation might explain previous reports on cellular cytotoxicity of human Tregs, which were based either on

isolated CD4CD25 T-cell populations or ex vivo-induced Treg-like populations.14,15 Interestingly, recent observations from a transgenic mouse model allowing discrimination between peripherally derived induced Tregs (pTregs) and thymus-derived Tregs (tTregs) indicate that, pTregs, but not tTregs, express granzyme B and therefore are capable of mediating cytotoxic effector mechanisms (Kretschmer, personal communication). One can speculate that CD4CD25CD127low Tregs in human peripheral blood represent the human analog to tTregs in mice. Human pTregs might be hidden in the remaining CD4CD25 T-cell population, but so far they have not been distinguished from activated CD25-upregulating CD4 Teffs.

Collectively, at present the CD4CD25CD127lowCD45RA population is the Treg subset of choice for clinical use as these cells have the highest capacity to maintain FOXP3 expression and preserve their suppressive capacity even after prolonged in vitro cultivation.8 In this article, we clearly demonstrate that this population does not have any cytotoxic capacity upon CD3 triggering via conventional activator beads or recombinant antibody derivatives. Our ndings indicate that the risk of harming recognized target cells by bsAb-redirected CD4CD25CD127low human Tregs is negligible. This should encourage attempts to use bsAb-mediated Treg activation within inamed tissues for the treatment of autoimmunity, transplant rejection or GvHD.

CONFLICT OF INTEREST

MB and GE hold patents related to the antibodies directed to La and PSCA. They both have founded the start-up company GEMoaB. The remaining authors declare no conict of interest.

ACKNOWLEDGEMENTS

We acknowledge Dr Sonja Schallenberg and Dr Karsten Kretschmer for helpful discussion and communication of unpublished data. We also thank Barbara Ute, Livia Schulze, Christine Grafe and Susan Hfner for outstanding technical assistance. The lentiviral vector system for transduction of bsAb-expressing stable cell lines was kindly provided by Professor Dr Dirk Lindemann (Institute of Virology, Technical University Dresden, Germany). This work was supported by a MeDDrive grant from the Medical Faculty of TU Dresden to MC and a seed grant from the Center for Regenerative Therapies Dresden (CRTD) to MB.

S Koristka1,6, M Cartellieri1,2,6, C Arndt1, A Feldmann1,2, K Tpfer3, I Michalk1, A Temme3, G Ehninger4 and M Bachmann1,2,5

1Institute of Immunology, Medical Faculty Carl Gustav Carus, Technical University Dresden, Dresden, Germany;

2Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany;

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3Department of Neurosurgery, University Hospital Carl Gustav Carus, Technical University Dresden, Dresden, Germany;

4Medical Clinic and Policlinic I, University Hospital Carl Gustav Carus, Technical University Dresden, Dresden, Germany and

5Center for Regenerative Therapies Dresden, Cluster of Excellence, Technical University Dresden, Dresden, Germany

E-mail: mailto:[email protected]

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6These authors contributed equally to this work.

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