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Absence of Galectin-1 accelerates CD8+ T cell-mediated graft rejection

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Absence of Galectin-1 accelerates CD8+ T cell-mediated graft rejection
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  Eur. J. Immunol. 2012. 42: 2881–2888  Cellular immune response DOI: 10.1002/eji.201142325  2881 S HORT  C OMMUNICATION Absence of Galectin-1 accelerates CD8 + T cell-mediatedgraft rejection  Aur´ elie Moreau 1  , Alistair Noble  2  , Kulachelvy Ratnasothy  1  , Jian-Guo Chai 3  , Louise Deltour  4  , Maria-Cristina Cuturi 5  , Elizabeth Simpson 3  , Robert Lechler 1 and Giovanna Lombardi 1 1 MRC Centre for Transplantation, King’s College London, Guy’s Hospital, London, UK  2 MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, King’s College London, Guy’sHospital, London, UK  3 Section of Immunobiology, Division of Immunology and Inflammation, Faculty of Medicine,Imperial College London, London, UK  4 Institut Jacques Monod, UMR 7592 CNRS, Universit´e Paris Diderot, Paris, France 5 ITUN/INSERM U643, CHU Hotel-Dieu, Nantes, France Galectin-1 (Gal-1) is a member of a family of endogenous  β -galactose-binding proteinswith a role in preventing autoimmune diseases and chronic inflammation. In this study,the involvement of Gal-1 in graft rejection was investigated by using Gal-1-deficient mice(Gal-1 −  / −  ). We demonstrate that in the absence of Gal-1, skin grafts are rejected earliercompared with those of WT mice, and that this is due to the role played by CD8 + T cells ingraft rejection. The difference in graft survival observed between Gal-1 −  / − and WT micewas explained by both an increase in the percentage of antigen-specific CD8 + T cells andby preferential secretion of IFN- γ  and IL-17 by CD8 + T cells in Gal-1 −  / − mice comparedwith WT mice. This study suggests that endogenous expression of Gal-1 contributes tograft survival. The results obtained from the use of mice deficient in Gal-1 also confirm akey role for CD8 + T cells in graft rejection. Keywords:  Cytokines   Galectin-1   Knockoutmice   Tc1CD8 + Tcells   Transplantimmunology  Introduction Galectin-1 (Gal-1) is a member of a family of endogenous β -galactose-binding proteins that modulate immune functions by controlling activation, proliferation, and survival of immune cells[1]. Expression of Gal-1 on different cells of the immune systemappears to be an important tolerogenic mechanism. Indeed, onT cells, Gal-1 has been shown to contribute to central toleranceby inhibiting positive selection and promoting negative selectionof conventional CD8 + T cells [2]. Furthermore, we and othershave demonstrated that Gal-1 expressed by regulatory T cells con-tributes to their function [3–5]. Gal-1 also regulates CD4 + T-cell Correspondence:  Prof. Giovanna Lombardie-mail: giovanna.lombardi@kcl.ac.uk polarization as ligation of Gal-1 by CD4 + T cells favors Th2-typeresponses by inducing apoptosis of Th1 and Th17 cells and pro-moting Th2 cytokine secretion (IL-4, IL-5, IL-10) [6,7].Together these data suggest that the role of Gal-1 in CD4 + T cells is relatively well defined. In contrast, very few reports haveinvestigated the function of Gal-1 in CD8 + T cells. The data thathavebeenpublishedsofarhavesuggestedthatGal-1issecretedby activatedCD8 + Tcells[8].Inaddition,recombinantGal-1inducedapoptosis of CD8 + T cells, while neutralizing anti-Gal-1 antibod-ies inhibited cell death [9]. Recently, the use of Gal-1-deficientmice made transgenic for the OT1 TCR has demonstrated thatthe OT1 cells from Gal-1 −  / − mice proliferated more in responseto OVA compared with OT1 cells from WT animals [10]. This was due to cell death of the proliferating OT1 T cells in WT ani-mals [10]. Furthermore, in the same study, the authors demon-strated that the increased production of IFN- γ  by OT1 cells from C  2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim  www.eji-journal.eu  2882  Aur´ elie Moreau et al.  Eur. J. Immunol. 2012. 42: 2881–2888 Gal-1 −  / − mice correlated with their augmented proliferation. Inhumans, a role for Gal-1 in CD8 + T cells was demonstrated inHodgkin’s disease. Indeed, patients with Gal-1 hi expression had asignificant reduction in the number of infiltrating CD8 + T cells inthe tumor site, and this correlated with a selective loss of IFN- γ expression by CD8 + T cells in these patients [11].Gal-1 has been used therapeutically to prevent the develop-ment of autoimmune diseases and chronic inflammation, suchas EAE [7], diabetes [6], retinal disease [5], colitis [12], sys-temic lupus [13], and arthritis [14,15]. During transplantation,treatment with exogenous recombinant Gal-1 has been shown toincrease survival following bone marrow transplantation and toinhibit GvHD [16]. More recently, the same treatment describedabove with exogenous recombinant Gal-1 was shown to prolongkidney allograft survival in the rat [17].Here, we investigated the role of endogenous Gal-1 in graftrejection by comparing skin allograft outcome between WT andGal-1 −  / − mice. We demonstrate that the absence of Gal-1 in recip-ient mice accelerates skin graft rejection and this process is inhib-ited by the depletion of CD8 + T cells. The difference in the time of skin graft rejection between Gal-1 −  / − and WT mice is explainedby an increased number of antigen-specific CD8 + T cells and by anaugmented production of proinflammatory cytokines by the samecells in Gal-1 −  / − mice. Results and discussion In the absence of Gal-1 skin grafts are rejected earlierand in a CD8 + T cell-dependent manner To investigate the role of Gal-1 in transplantation, WT andGal-1 −  / − mice on a B6 background were compared. First, weconfirmed the genetic histocompatibility of these two strains of mice, by transplanting gender-matched WT and Gal-1 −  / − recipi-ents with Gal-1 −  / − and WT skin grafts, respectively. All the grafts were accepted indefinitely confirming that the two strains weregenetically histocompatible (data not shown).Gal-1 −  / − and WT recipients were then transplanted with skinfrom B6.K  d mice (mice transgenic for K  d on a B6 background)[18]. Gal-1 −  / − mice rejected the B6.K  d skin grafts significantly earlier (Mean Survival Time — MST 11 days) compared with WTrecipients (MST 14.5 days) (Fig. 1A).To understand which T-cell subset was responsible in Gal-1 −  / − mice for the accelerated rejection observed, CD4 + or CD8 + T cells were depleted in recipient animals (WT or Gal-1 −  / − ) that receivedB6.K  d skin grafts (Fig. 1A and B). Injection of anti-CD4 and anti-CD8 antibodies in vivo led to more than 90% depletion of thetargeted cells for at least 7 days. Depletion of CD4 + T cells didnot alter the kinetics of allograft rejection in Gal-1 −  / − mice, whileshortened allograft survival was apparent in WT recipients. How-ever, this difference was not statistically significant. One possi-ble explanation is that by using anti-CD4 antibody, Tregs weredepleted and the lack of this population of cells was responsiblefor accelerated graft rejection. Indeed Gal-1 has been implicatedin Treg-cell function [3–5]. An alternative explanation is that thetreatment with anti-CD4 antibody depleted Th2-type cells, whichare known to expand in the presence of Gal-1, and without theirprotective effect, the graft is rejected earlier [6,7].In contrast, while depletion of CD8 + T cells in WT mice didnot modify the course of rejection, the absence of CD8 + T cellsincreased the time of survival in Gal-1 −  / − recipients to valuessimilartoB6.K  d graftrejectioninWTmice.Theseresultssuggestedthat CD8 + T cells are responsible for the earlier rejection observedin Gal-1 −  / − recipients. Higher production of proinflammatory cytokines at the time of rejection by Gal-1 −  / − CD8 + T cells To understand the mechanisms behind the early skin graft rejec-tion mediated by CD8 + T cells in Gal-1 −  / − mice, B6.K  d skin grafts were transplanted into Gal-1 −  / − and WT recipients. The percent-age of CD8 + T cells and their cytokine profile were evaluated inthedraininglymphnodesandinthespleenatthetimeofrejection,in particular at days 13 and 16 posttransplantation for Gal-1 −  / − and WT mice, respectively. As shown in Fig. 1C, a similar percent-age of CD8 + T cells was measured in the two strains of mice inthe spleens and in the draining LNs. However, the percentage of IFN- γ -producing CD8 + T cells was significantly higher in Gal-1 −  / − compared with WT mice, both in the spleen and draining LNs(Fig. 1D). These results are in agreement with a previous study showingthatCD8 + TcellsfromimmunizedGal-1 −  / − miceproducemore IFN- γ  compared with CD8 + T cells from WT animals [10].In addition, a higher percentage of IL-17 producing CD8 + T cells was observed in the LNs but not in the spleens of Gal-1 −  / − micecompared with WT mice  ( Fig. 1E). In contrast, no differences inTNF- α  production were obtained between Gal-1 −  / − and WT mice(Fig. 1F) indicating that not all proinflammatory cytokines wereincreased in CD8 + T cells from Gal-1 −  / − mice during graft rejec-tion.Takentogethertheseresultssuggestthatthehigherproductionof some of the inflammatory cytokines by CD8 + T cells obtainedfromGal-1 −  / − ,comparedwithWTmice,maycontributetotheear-lier skin graft rejection observed. The importance of these resultsis further supported by previously published data showing thatboth Tc1 and Tc17 cells were involved in transplant rejection[19,20]. Preferential differentiation of CD8 + T cells fromGal-1 −  / − to Tc1 after cell polarization in vitro Next, we addressed the question of whether CD8 + T cells fromGal-1 −  / − mice have an intrinsically enhanced capacity to polarizetoward Tc1 or Tc17 cells. Resting CD8 + T cells isolated from na¨ıvemice of both strains were cultured with anti-CD3/CD28 antibod-ies for 5 days. CD8 + T cells from Gal-1 −  / − mice produced signif-icantly more IFN- γ  than those from WT mice after restimulation(Fig. 2A). Similar results were obtained when TNF- α  production C  2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim  www.eji-journal.eu  Eur. J. Immunol. 2012. 42: 2881–2888  Cellular immune response  2883 Figure 1.  CD8 + T cells mediate skin graft rejection in Gal-1 −  / − mice and produce more proinflammatory cytokines in vivo at the time of transplantrejectioncomparedwithWTmice.(A,B)Gal-1 −  / − andWTrecipientmiceweretransplantedwithB6.K d skingraftswithouttreatment,with(A)anti-CD4 antibody treatment or (B) anti-CD8 antibody treatment. The percentage of allograft survival is shown; data are pooled from three experimentsperformed.  n = 8 for Gal-1 −  / − mice without treatment,  n = 6 for WT mice without treatment and for Gal-1 −  / − mice with anti-CD4 treatment, and n = 5 mice for the other groups. Statistics were calculated by Log Rank Mantel–Cox test, **  p ≤ 0.01 and ***  p ≤ 0.001. (C) The percentage of CD3 + CD8 + T cells in spleens and draining LNs of Gal-1 −  / − and WT recipients at the time of rejection is shown. Data are shown as mean  +  SEM of   n  =  5Gal-1 −  / − mice and  n = 3 WT mice and are pooled from three experiments performed. Statistics were calculated by Student’s  t -test. (D–F) Cytokineproduction in CD8 + T cells from spleens and draining LNs of Gal-1 −  / − and WT mice at the time of B6.K d skin graft rejection was evaluated byflow cytometry. Cells from individual mice were stimulated for 5 h with PMA (50 ng/mL), ionomycin (1  µ M), and brefeldin A (1 × ). Analysis wasperformed at 13-days postgraft for Gal-1 −  / − mice and 16-days postgraft for WT mice by intracellular staining. (D) IFN- γ ,  n  =  7 mice; (E) IL-17, n = 4 mice; and (F) TNF- α ,  n = 4 mice were evaluated and data are shown as mean + SEM pooled from two experiments performed. Statistics werecalculated by Student’s  t -test, *  p ≤ 0.05.  was measured (Fig. 2B). To induce polarization of CD8 + T cells toTc17, CD8 + T cells were cultured with anti-CD3/CD28 antibod-ies, in the presence of TGF- β , IL-1 β , IL-23, and IL-6. No significantdifference between the two strains was observed (Fig. 2C). Theseresults demonstrate that CD8 + T cells from Gal-1 −  / − mice have ahigherintrinsic ability todifferentiate toTc1and toproduceIFN- γ and TNF- α , but not to Tc17. Higher production of IFN- γ  and increased Ag-specificCD8 + T cells in Gal-1 −  / − mice in response to HY We have shown in Fig. 1C that the total number of CD8 + T cells was not different in the two strains of mice at thetime of MHC-mismatched skin transplant rejection. However, thisresult does not exclude the possibility that antigen-specific T cells C  2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim  www.eji-journal.eu  2884  Aur´ elie Moreau et al.  Eur. J. Immunol. 2012. 42: 2881–2888 Figure 2.  CD8 + T cells from Gal-1 −  / − mice polarize toward a Tc1/Tc17 phenotype. (A and B) Purified resting CD8 + T cells from individual naivemice (Gal-1 −  / − and WT) were polarized to Tc1 cells using anti-CD3 and anti-CD28 antibodies. After 5-days stimulation, the frequencies of (A) IFN- γ and (B) TNF- α -producing CD8 + T cells were analyzed. Results with isotype controls are shown as negative controls. Dot plots were derived from arepresentative mouse while graphs show mean + SEM from nine Gal-1 −  / − mice and eight WT mice. Data shown are pooled from four experimentsperformed. Statistics were calculated by Student’s  t -test, *  p ≤ 0.05 and **  p ≤ 0.01. (C) CD8 + T cells from naive mice (Gal-1 −  / − and WT) were polarizedto Tc17 cells using anti-CD3 and anti-CD28 antibodies, IL-6, IL-23, IL-1 β , and TGF- β . After 5-days stimulation, IL-17 cytokine production by CD8 + T cells was analyzed. Results with isotype controls are shown as negative controls. Dot plots are derived from a representative mouse while thegraph shows mean + SEM from 12 mice per group. Data shown are pooled from six experiments performed. Statistics were calculated by Student’s t -test. are increased in Gal-1 −  / − compared with WT mice that couldexplain at least in part the augmented IFN- γ  and IL-17 productionobserved in Fig. 1D and E. To test this possibility, we grafted maleskin onto syngeneic female recipients. In this model, Palmowski etal.haveshownpreviouslythatCD8 + TcellsspecificforUtypeptide(derived from HY) and restricted by H2D b (identified in recipientmice by tetramer staining) control skin graft rejection [21].First, we tested whether female Gal-1 −  / − recipients rejectedmale skin grafts earlier than female WT mice (Fig. 3A). Indeed,Gal-1 −  / − mice rejected HY incompatible skins after an MST of  C  2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim  www.eji-journal.eu  Eur. J. Immunol. 2012. 42: 2881–2888  Cellular immune response  2885 Figure 3.  HighernumbersofHY-specificCD8 + TcellsinGal-1 −  / − femalemicecomparedwithfemaleWTmicefollowingmaleskintransplantation.(A) Male skin grafts were transplanted onto Gal-1 −  / − and WT female recipients without or with anti-CD8 antibody treatment. The percentage of allograftsurvivalisshownandispooledfromthreeexperimentsperformed.  n = 10forGal-1 −  / − micewithouttreatment, n = 9forWTmicewithouttreatment,  n = 4 for Gal-1 −  / − mice with anti-CD8 treatment, and  n = 5 for WT mice with anti-CD8 treatment. Statistics were calculated by Log RankMantel–Cox test, *  p ≤ 0.05, **  p ≤ 0.01, and ***  p ≤ 0.001. (B) Percentage of tetramer-positive HY-specific CD8 + T cells in the PBLs of Gal-1 −  / − and WTrecipients during the course of graft rejection. Skin grafts were performed at day 0. At different time points, the PBL from individual mice weresampled for the presence of HY-specific CD8 + T cells by tetramer staining. Data shown are shown as mean + SEM of   n = 5 mice per group and arepooled from five experiments performed. Statistics were calculated by Student’s  t -test, *  p  ≤  0.05. (C) Percentage of tetramer-positive HY-specificCD8 + T cells in spleens and draining LNs from Gal-1 −  / − and WT mice. Cells from individual mice were analyzed at day 14 postgraft (left) and againafter 1-week stimulation with irradiated syngeneic B6 male splenocytes (right). Data are shown as mean + SEM of   n = 5 mice per group on the leftpanel,  n  =  3 Gal-1 −  / − mice, and  n  =  4 WT mice on the right panel. Data are pooled from two experiments performed. Statistics were calculatedby Student’s  t -test, *  p ≤ 0.05 and **  p ≤ 0.01. (D, E) After in vitro expansion, (D) IFN- γ -producing CD8 + T cells and (E) IL-17-producing CD8 + T cellsfrom transplanted Gal-1 −  / − and WT mice were quantified, either after 5-h restimulation with PMA/ionomycin in the presence of brefeldin A (left),or after 5-h restimulation with HY Uty peptide and female WT DCs in the presence of brefeldin A (right). (D) Data are shown as mean + SEM of   n =  3 Gal-1 −  / − mice and  n  =  4 WT mice. (E) Data are shown as mean  +  SEM of   n  =  5 mice per group. Statistics were calculated by Student’s  t -test,*  p ≤ 0.05, **  p ≤ 0.01, and ***  p ≤ 0.001. 23 days while rejection in female WT mice occurred after 37 days.In addition and consistent with Palmowski’s results, we observeda delay in the time of graft rejection when WT mice receivedanti-CD8 antibody treatment compared with that in untreatedWT animals (  p < 0.05). Furthermore, this delay was even longer when CD8 + T cells were depleted in Gal-1-deficient compared with untreated mice (  p < 0.01). These results further support thekey role of CD8 + T cells in graft rejection in the absence of Gal-1in a different mouse graft model.Next, to quantify the numbers of CD8 + T cells specific for HY,CD8 + T cells in PBL from Gal-1 −  / − and WT female mice thatreceived male skin grafts were stained with an HY D b Uty tetramerat different time points after transplantation. Tetramer-positiveHY-specific CD8 + T cells in PBLs were increased in Gal-1 −  / − mice C  2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim  www.eji-journal.eu
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