International Immunology, Vol. 26, No. 3, pp. 173–181 doi:10.1093/intimm/dxt054 Advance Access publication 12 November 2013
© The Japanese Society for Immunology. 2013. All rights reserved. For permissions, please e-mail: [email protected]
Mycophenolic acid-treated dendritic cells generate regulatory CD4+ T cells that suppress CD8+ T cells’ allocytotoxicity Ihab Kazma1*, Roxane Lemoine1*, Florence Herr1, Stephanie Chadet1, Daniel Meley1, Florence Velge-Roussel1, Yvon Lebranchu1,2 and Christophe Baron1,2 UPRES EA 4245 « Cellules Dendritiques, Immunomodulation et Greffes », UFR de Médecine, Université François-Rabelais, 10 Boulevard Tonnellé, 37000 Tours, France 2 Service de Néphrologie et d’Immunologie Clinique, CHRU de Tours, 2 bis Boulevard Tonnellé, 37000 Tours, France 1
*These authors have equally participated to this work. Received 24 June 2013, accepted 7 October 2013
Abstract Regulatory T cells (Treg) play a crucial role in controlling immunity and transplant rejection. Two main groups of Treg have been described: antigen-induced Treg (iTreg) and natural Treg (nTreg). The ways to induce and the mechanisms of action of Treg subsets remained ill defined, particularly for their effects on CD8+ T cells. CD8+ T cells are major agents in the rejection of allografts; the aim of this study is to investigate the effects exerted on CD8+ T cells by human CD4+ iTreg induced by mycophenolic acid-treated dendritic cells. iTreg suppress the proliferation of CD8+ T cells by allogeneic cell–cell interaction with mature dendritic cells and irrespectively of the TCR specificity of the CD8+ T cells and cell–cell contact of iTreg with CD8+ T cells. In our model, this suppression is independent of the action of IL-10 and TGF-β1. iTreg were able to modify phenotype and inhibited IFN-γ and TNF-α secretion by CD8+ T cells. Most interestingly, iTreg inhibit the synthesis of perforin and of granzymes A and B by CD8+ T cells and impaired their cytotoxicity against allogeneic targets. In summary, our study showed the involvement of iTreg in the down-regulation of cytotoxic responses mediated by CD8+ T cells in an allospecific context. Following studies that have shown the existence of a regulation control exerted by iTreg on CD4+ T cells and dendritic cells, this work ultimately shows that this regulation can reach CD8+ T-cell functions. Keywords: cytotoxicity, immune regulation, organ transplantation
Introduction Regulatory T cells (Treg) play a key role in controlling antigen-specific immune responses and autoimmune diseases as well (1, 2). Treg are able to maintain peripheral tolerance by suppression of proliferating effector T cells (Teff) (3–6). Furthermore, Treg appear to be able to inhibit multiple stages of T-cell activity: proliferation, differentiation, as well as effector function (7). Mechanisms used by Treg to mediate suppression of T-cell functions are not fully understood. Most of the experimental data have addressed the capacity of Treg to regulate CD4+ effector T cells. However, evidence also exists that murine natural Treg (nTreg) can also regulate the responses of CD8+ T cells (8–10). Piccirillo et al. (2) clearly illustrated that the proliferation of transgenic CD8+ T cells in response to interactions of cognate TCR with tetrameric MHC class I–peptide complexes was inhibited in the
presence of CD4+ CD25+ Treg. Similarly, in neonatal mice, depletion of Treg before herpes simplex virus (HSV) infection significantly enhanced granzyme B and cytotoxicity of HSV-specific CD8+ T cell in vivo (11). Furthermore in murine transplantation, Treg can also suppress heart allograft rejection mediated by CD8+ T cells (12) and in a skin transplant model, Treg inhibited allograft rejection but not the proliferation of alloreactive CD8+ effector T cells (13). Lin et al. (14) reported that tolerance mediated by Treg acts by censoring immune effector functions rather than by limiting the induction of T-cell expansion. Mempel et al. (15) reported that granule exocytosis by human CTLs was markedly impaired in the presence of nTreg and Dieckmann et al. (16) showed that nTreg inhibited CD8+ T-cell proliferation in response to allogeneic stimulation.
Downloaded from http://intimm.oxfordjournals.org/ at Selcuk University on January 14, 2015
Correspondence to: I. Kazma; E-mail: [email protected]
174 Regulation of CD8+ T cells’ cytotoxicity by iTreg Generation of two different T-cell subsets in long-term culture CD4+ T cells were co-cultured with allogeneic mDC or MPA-DC during the first incubation at a density of 2 × 106 T cells with 0.6 × 06 DC without any exogenous cytokines. At day 7, T cells were re-stimulated with 0.6 × 106 mDC or MPA-DC generated from the same donor as for the first coculture (stored at −80°C in RPMI-10% FCS + 10% dimethyl sulfoxide until use), as described by Lagaraine et al. (19), in culture medium containing 2 IU ml−1 IL-2 and 25 ng ml−1 IL-4. Two more cycles of mDC or MPA-DC re-stimulations were performed after intervals of 7 days. As previously described (19), MPA-DC induced human antigen-specific CD4+ iTreg contrary to mDC, which induced activated CD4+ T cells designed by the term Teff. At the end of longterm culture, MPA-DC- and mDC-induced T cells were harvested, counted and used in V-bottomed culture wells (NUNC A/S, Roskilde, Denmark) to carry out a mixed leukocyte reaction with mDC from the same donor as used in the first activation. An equal number of MPA-DC-induced T cells or mDC-induced T cells and a 1:3 DC:T-cell ratio were used in the tests. Cell proliferation was assessed from the incorporation of 1 µCi [3H]-thymidine (Amersham Pharmacia Biotech, Little Chalfont, England) during the last 18 h of 5 days’ culture and measured by liquid scintillation counting (Tri-Carb 2550 TR/LL, Packard). Transwell membranes (0.2 µm, NUNCA/S) were used in some experiments. MPA-DC- or mDC-induced T cells were above the Transwell membrane, while CD8+ T cells with mDC were in the bottom well. When specified, blocking anti-IL-10 (clone 23738) and anti-IL-10R (clone 90220) or blocking anti-TGF-β1 antibodies (clone 9016) were added to the wells at 20, 5 and 20 µg ml−1, respectively (R&D Systems).
Methods Isolation of CD4+ and CD8+ T lymphocytes
Treg–CD8 co-culture
Blood from healthy donors was obtained by cytapheresis (informed consent was obtained from volunteers). Human PBMC were then isolated by centrifuging over Ficoll hypaque (Lymphoprep; Abcys, France). PBMC were first depleted of adherent cells by two 45-min adhesion cycles on plastic. CD4+ and CD8+ T lymphocytes were then isolated using anti-CD4- or anti-CD8-coated Dynabeads®, followed by Detachabeads® (Dynal, Compiegne, France) according to the manufacturer’s instructions. The purity of CD4+ and CD8+ T populations was >95% (data not shown).
mDC were used to stimulate allogeneic CD8+ T lymphocytes in the presence or absence of CD4+ T-cell subsets at a DC:CD8 ratio of 1:3 and a CD8:CD4 ratio of 1:1 for 96 h in P96 plates (Falcon, Becton Dickinson, Mountain View, CA, USA). After 4 days of co-culture, cells were washed, harvested and used for phenotypic analysis, cytokine production and cytotoxic activity.
Generation of DC Human PBMC from healthy blood donors were isolated with Ficoll Hypaque, as described above. Monocyte-derived DC were differentiated with IL-4 and GM-CSF as previously described (20). On day 5, immature DC were harvested, washed and re-suspended in culture medium with IL-4 and GM-CSF. TNF-α (R&D Systems, Minneapolis, MN, USA) was added at 20 ng ml−1 for 2 days, with or without 100 µM MPA (Sigma-Aldrich, St Quentin Fallavier, France), to obtain MPA-DC or mature DC (mDC), respectively. On day 7, DC were harvested, extensively washed and used for subsequent experiments.
Analysis of cytokine production Stimulated -CD8+ T cells were co-cultured for 4 days with iTreg or Teff as described above. At the end of short-term coculture, supernatants were harvested and stored at −20°C until analysis. Cytokine levels of IL-4, IL-5, IL-10, IL-2, TNF-α and IFN-γ were determined using appropriate human ELISA kits (eBiosciences, Montrouge, France) according to the manufacturer’s protocols. Analysis of molecule expression by flow cytometry CD8–Treg co-cultures were harvested and 1 × 105 cells per sample were re-suspended in PBS. Cells were then incubated with saturating concentrations of the different fluorochrome-conjugated mAbs for 30 min at 4°C. The stained cells
Downloaded from http://intimm.oxfordjournals.org/ at Selcuk University on January 14, 2015
It is important to note that in humans, most of the effects of Treg on CD8 + T cells were analyzed with nTreg. Besides nTreg, another type of Treg, generated in the periphery from CD25 − CD4 + T cells, has been recognized and referred to as induced Treg (iTreg). Several studies have shown that iTreg and nTreg have different characteristics, especially in their antigen specificities and in the TCR signal strength and costimulatory requirements needed for their generation (17). iTreg are essential in mucosal immune tolerance and in the control of severe chronic allergic inflammation. Their development is driven by the need to maintain a non-inflammatory environment in the gut, to control immune responses to environmental and food allergens, and to eventually decrease chronic inflammation, whereas nTreg mainly prevent autoimmunity (18). However, we investigate here for the first time the effect of allospecific iTreg on CD8+ T cells in a human model. We have previously demonstrated that human dendritic cells (DC) treated by mycophenolic acid (MPA-DC) induced antigen-specific CD4+ iTreg (19). These iTreg expressed CD25, GITR, CTLA-4, CD95 and Foxp3 markers and secreted high levels of IL-10 and TGF-β1 and low levels of IFN-γ. In this study, we investigated the interactions between CD8+ T cells and either CD4+ Teff or CD4+ iTreg. The present study reports for the first time that human iTreg lead to the inhibition of CD8+ T-cell proliferation in response to allogeneic stimulation. Suppression was mediated by cell–cell interactions and occurred irrespective of the TCR specificity of the CD8+ T cells. Most interestingly, iTreg were able to impair cytotoxicity of CD8+ T cells through the inhibition of IFN-γ and of cytotoxicity-associated markers such as perforin, CD107 and both granzymes A and B.
Regulation of CD8+ T cells’ cytotoxicity by iTreg 175
Cytotoxicity assay by flow cytometry, CFSE/7-amino actinomycin D PBMC target cells (106) were labeled with carboxyfluorescein succinimidyl ester (CFSE) at a final concentration of 5 µM (Sigma-Aldrich) for 10 min at 37°C in 1 ml of PBS. After quenching the labeling reaction by addition of FCS, cells were washed extensively and immediately used in the cytotoxicity assay. Effector cells were seeded with a constant number of CFSE-labeled target cells at different E:T ratios (4:1, 2:1, 1:1, 1:2, 1:4 and 1:10). In parallel, target cells were incubated alone to measure basal lysis. Cells were incubated in 96-well microplates in a total volume of 200 µl complete medium for 16 h in a 5% CO2 atmosphere at 37°C. Cell mixtures were then washed in PBS and incubated in PBS containing 1 μg of 7-amino actinomycin D (7-AAD; Sigma-Aldrich). CFSE fluorescence and 7-AAD emission were detected in the FL-1 and FL-3 channels, respectively. For each E:T ratio, 30 000 target cells were acquired. Analysis was performed with the Diva Software. The percentage of specific lysis (PSL) was determined by the formula: PSL = (100 × (% sample lysis − % basal lysis))/(100 − % basal lysis). Statistical analysis Statistical significance of parametric data was calculated using standard methods. Results are reported as the mean ± SD. The statistical significances of results were evaluated by
the Mann–Whitney U test or Wilcoxon test. For all tests, a P value of
© The Japanese Society for Immunology. 2013. All rights reserved. For permissions, please e-mail: [email protected]
Mycophenolic acid-treated dendritic cells generate regulatory CD4+ T cells that suppress CD8+ T cells’ allocytotoxicity Ihab Kazma1*, Roxane Lemoine1*, Florence Herr1, Stephanie Chadet1, Daniel Meley1, Florence Velge-Roussel1, Yvon Lebranchu1,2 and Christophe Baron1,2 UPRES EA 4245 « Cellules Dendritiques, Immunomodulation et Greffes », UFR de Médecine, Université François-Rabelais, 10 Boulevard Tonnellé, 37000 Tours, France 2 Service de Néphrologie et d’Immunologie Clinique, CHRU de Tours, 2 bis Boulevard Tonnellé, 37000 Tours, France 1
*These authors have equally participated to this work. Received 24 June 2013, accepted 7 October 2013
Abstract Regulatory T cells (Treg) play a crucial role in controlling immunity and transplant rejection. Two main groups of Treg have been described: antigen-induced Treg (iTreg) and natural Treg (nTreg). The ways to induce and the mechanisms of action of Treg subsets remained ill defined, particularly for their effects on CD8+ T cells. CD8+ T cells are major agents in the rejection of allografts; the aim of this study is to investigate the effects exerted on CD8+ T cells by human CD4+ iTreg induced by mycophenolic acid-treated dendritic cells. iTreg suppress the proliferation of CD8+ T cells by allogeneic cell–cell interaction with mature dendritic cells and irrespectively of the TCR specificity of the CD8+ T cells and cell–cell contact of iTreg with CD8+ T cells. In our model, this suppression is independent of the action of IL-10 and TGF-β1. iTreg were able to modify phenotype and inhibited IFN-γ and TNF-α secretion by CD8+ T cells. Most interestingly, iTreg inhibit the synthesis of perforin and of granzymes A and B by CD8+ T cells and impaired their cytotoxicity against allogeneic targets. In summary, our study showed the involvement of iTreg in the down-regulation of cytotoxic responses mediated by CD8+ T cells in an allospecific context. Following studies that have shown the existence of a regulation control exerted by iTreg on CD4+ T cells and dendritic cells, this work ultimately shows that this regulation can reach CD8+ T-cell functions. Keywords: cytotoxicity, immune regulation, organ transplantation
Introduction Regulatory T cells (Treg) play a key role in controlling antigen-specific immune responses and autoimmune diseases as well (1, 2). Treg are able to maintain peripheral tolerance by suppression of proliferating effector T cells (Teff) (3–6). Furthermore, Treg appear to be able to inhibit multiple stages of T-cell activity: proliferation, differentiation, as well as effector function (7). Mechanisms used by Treg to mediate suppression of T-cell functions are not fully understood. Most of the experimental data have addressed the capacity of Treg to regulate CD4+ effector T cells. However, evidence also exists that murine natural Treg (nTreg) can also regulate the responses of CD8+ T cells (8–10). Piccirillo et al. (2) clearly illustrated that the proliferation of transgenic CD8+ T cells in response to interactions of cognate TCR with tetrameric MHC class I–peptide complexes was inhibited in the
presence of CD4+ CD25+ Treg. Similarly, in neonatal mice, depletion of Treg before herpes simplex virus (HSV) infection significantly enhanced granzyme B and cytotoxicity of HSV-specific CD8+ T cell in vivo (11). Furthermore in murine transplantation, Treg can also suppress heart allograft rejection mediated by CD8+ T cells (12) and in a skin transplant model, Treg inhibited allograft rejection but not the proliferation of alloreactive CD8+ effector T cells (13). Lin et al. (14) reported that tolerance mediated by Treg acts by censoring immune effector functions rather than by limiting the induction of T-cell expansion. Mempel et al. (15) reported that granule exocytosis by human CTLs was markedly impaired in the presence of nTreg and Dieckmann et al. (16) showed that nTreg inhibited CD8+ T-cell proliferation in response to allogeneic stimulation.
Downloaded from http://intimm.oxfordjournals.org/ at Selcuk University on January 14, 2015
Correspondence to: I. Kazma; E-mail: [email protected]
174 Regulation of CD8+ T cells’ cytotoxicity by iTreg Generation of two different T-cell subsets in long-term culture CD4+ T cells were co-cultured with allogeneic mDC or MPA-DC during the first incubation at a density of 2 × 106 T cells with 0.6 × 06 DC without any exogenous cytokines. At day 7, T cells were re-stimulated with 0.6 × 106 mDC or MPA-DC generated from the same donor as for the first coculture (stored at −80°C in RPMI-10% FCS + 10% dimethyl sulfoxide until use), as described by Lagaraine et al. (19), in culture medium containing 2 IU ml−1 IL-2 and 25 ng ml−1 IL-4. Two more cycles of mDC or MPA-DC re-stimulations were performed after intervals of 7 days. As previously described (19), MPA-DC induced human antigen-specific CD4+ iTreg contrary to mDC, which induced activated CD4+ T cells designed by the term Teff. At the end of longterm culture, MPA-DC- and mDC-induced T cells were harvested, counted and used in V-bottomed culture wells (NUNC A/S, Roskilde, Denmark) to carry out a mixed leukocyte reaction with mDC from the same donor as used in the first activation. An equal number of MPA-DC-induced T cells or mDC-induced T cells and a 1:3 DC:T-cell ratio were used in the tests. Cell proliferation was assessed from the incorporation of 1 µCi [3H]-thymidine (Amersham Pharmacia Biotech, Little Chalfont, England) during the last 18 h of 5 days’ culture and measured by liquid scintillation counting (Tri-Carb 2550 TR/LL, Packard). Transwell membranes (0.2 µm, NUNCA/S) were used in some experiments. MPA-DC- or mDC-induced T cells were above the Transwell membrane, while CD8+ T cells with mDC were in the bottom well. When specified, blocking anti-IL-10 (clone 23738) and anti-IL-10R (clone 90220) or blocking anti-TGF-β1 antibodies (clone 9016) were added to the wells at 20, 5 and 20 µg ml−1, respectively (R&D Systems).
Methods Isolation of CD4+ and CD8+ T lymphocytes
Treg–CD8 co-culture
Blood from healthy donors was obtained by cytapheresis (informed consent was obtained from volunteers). Human PBMC were then isolated by centrifuging over Ficoll hypaque (Lymphoprep; Abcys, France). PBMC were first depleted of adherent cells by two 45-min adhesion cycles on plastic. CD4+ and CD8+ T lymphocytes were then isolated using anti-CD4- or anti-CD8-coated Dynabeads®, followed by Detachabeads® (Dynal, Compiegne, France) according to the manufacturer’s instructions. The purity of CD4+ and CD8+ T populations was >95% (data not shown).
mDC were used to stimulate allogeneic CD8+ T lymphocytes in the presence or absence of CD4+ T-cell subsets at a DC:CD8 ratio of 1:3 and a CD8:CD4 ratio of 1:1 for 96 h in P96 plates (Falcon, Becton Dickinson, Mountain View, CA, USA). After 4 days of co-culture, cells were washed, harvested and used for phenotypic analysis, cytokine production and cytotoxic activity.
Generation of DC Human PBMC from healthy blood donors were isolated with Ficoll Hypaque, as described above. Monocyte-derived DC were differentiated with IL-4 and GM-CSF as previously described (20). On day 5, immature DC were harvested, washed and re-suspended in culture medium with IL-4 and GM-CSF. TNF-α (R&D Systems, Minneapolis, MN, USA) was added at 20 ng ml−1 for 2 days, with or without 100 µM MPA (Sigma-Aldrich, St Quentin Fallavier, France), to obtain MPA-DC or mature DC (mDC), respectively. On day 7, DC were harvested, extensively washed and used for subsequent experiments.
Analysis of cytokine production Stimulated -CD8+ T cells were co-cultured for 4 days with iTreg or Teff as described above. At the end of short-term coculture, supernatants were harvested and stored at −20°C until analysis. Cytokine levels of IL-4, IL-5, IL-10, IL-2, TNF-α and IFN-γ were determined using appropriate human ELISA kits (eBiosciences, Montrouge, France) according to the manufacturer’s protocols. Analysis of molecule expression by flow cytometry CD8–Treg co-cultures were harvested and 1 × 105 cells per sample were re-suspended in PBS. Cells were then incubated with saturating concentrations of the different fluorochrome-conjugated mAbs for 30 min at 4°C. The stained cells
Downloaded from http://intimm.oxfordjournals.org/ at Selcuk University on January 14, 2015
It is important to note that in humans, most of the effects of Treg on CD8 + T cells were analyzed with nTreg. Besides nTreg, another type of Treg, generated in the periphery from CD25 − CD4 + T cells, has been recognized and referred to as induced Treg (iTreg). Several studies have shown that iTreg and nTreg have different characteristics, especially in their antigen specificities and in the TCR signal strength and costimulatory requirements needed for their generation (17). iTreg are essential in mucosal immune tolerance and in the control of severe chronic allergic inflammation. Their development is driven by the need to maintain a non-inflammatory environment in the gut, to control immune responses to environmental and food allergens, and to eventually decrease chronic inflammation, whereas nTreg mainly prevent autoimmunity (18). However, we investigate here for the first time the effect of allospecific iTreg on CD8+ T cells in a human model. We have previously demonstrated that human dendritic cells (DC) treated by mycophenolic acid (MPA-DC) induced antigen-specific CD4+ iTreg (19). These iTreg expressed CD25, GITR, CTLA-4, CD95 and Foxp3 markers and secreted high levels of IL-10 and TGF-β1 and low levels of IFN-γ. In this study, we investigated the interactions between CD8+ T cells and either CD4+ Teff or CD4+ iTreg. The present study reports for the first time that human iTreg lead to the inhibition of CD8+ T-cell proliferation in response to allogeneic stimulation. Suppression was mediated by cell–cell interactions and occurred irrespective of the TCR specificity of the CD8+ T cells. Most interestingly, iTreg were able to impair cytotoxicity of CD8+ T cells through the inhibition of IFN-γ and of cytotoxicity-associated markers such as perforin, CD107 and both granzymes A and B.
Regulation of CD8+ T cells’ cytotoxicity by iTreg 175
Cytotoxicity assay by flow cytometry, CFSE/7-amino actinomycin D PBMC target cells (106) were labeled with carboxyfluorescein succinimidyl ester (CFSE) at a final concentration of 5 µM (Sigma-Aldrich) for 10 min at 37°C in 1 ml of PBS. After quenching the labeling reaction by addition of FCS, cells were washed extensively and immediately used in the cytotoxicity assay. Effector cells were seeded with a constant number of CFSE-labeled target cells at different E:T ratios (4:1, 2:1, 1:1, 1:2, 1:4 and 1:10). In parallel, target cells were incubated alone to measure basal lysis. Cells were incubated in 96-well microplates in a total volume of 200 µl complete medium for 16 h in a 5% CO2 atmosphere at 37°C. Cell mixtures were then washed in PBS and incubated in PBS containing 1 μg of 7-amino actinomycin D (7-AAD; Sigma-Aldrich). CFSE fluorescence and 7-AAD emission were detected in the FL-1 and FL-3 channels, respectively. For each E:T ratio, 30 000 target cells were acquired. Analysis was performed with the Diva Software. The percentage of specific lysis (PSL) was determined by the formula: PSL = (100 × (% sample lysis − % basal lysis))/(100 − % basal lysis). Statistical analysis Statistical significance of parametric data was calculated using standard methods. Results are reported as the mean ± SD. The statistical significances of results were evaluated by
the Mann–Whitney U test or Wilcoxon test. For all tests, a P value of
