Epigenetic modification of the PD-1 (Pdcd1) promoter in effector CD4+ T cells tolerized by peptide immunotherapy
Antigen-based immunotherapy remains the ‘holy grail’ of immune tolerance because it should target only those pathogenic lymphocytes driving autoimmune , allergic , or alloreactive immunopathology , whilst leaving beneficial immune surveillance unaltered . Peptide immunotherapy ( PIT ) is the subject of clinical trials in autoimmune and allergic disease ( Larche and Wraith , 2005; Larché , 2007 ) . Despite this the molecular basis for effects of PIT , particularly on T effector ( Teff ) cells , which is the clinical imperative , remains to be fully understood . The co-inhibitory receptor PD-1 ( encoded by the Pdcd1 locus ) is transiently upregulated on both CD4+ and CD8+ T cells upon activation in order to restrain primary immune responses ( Agata et al . , 1996; Keir et al . , 2006 , 2008 ) . Its role in maintaining peripheral tolerance under steady state conditions is illustrated by the spontaneous development of autoimmune pathology in mice that lack PD-1 ( Nishimura et al . , 1999 ) . PD-1 is also highly-expressed on exhausted CD8+ T cells ( Barber et al . , 2006; Youngblood et al . , 2013 ) . PD-1 contains an immunoreceptor tyrosine-switch motif ( ITSM ) that is thought to recruit SHP-2 , a phosphatase that can inhibit the PI3K pathway ( Zhang et al . , 2002; Chemnitz et al . , 2004 ) . Signalling through PD-1 upon TCR stimulation has been shown to inhibit proliferation and the production of IL-2 and effector cytokines by T cells ( Freeman et al . , 2000; Sandner et al . , 2005; Keir et al . , 2006 ) . The importance of PD-1 signalling in PIT has been unclear . Reversal of unresponsiveness has been reported in CD8+ T cells upon blockade of PD-1 signalling ( Tsushima et al . , 2007; Chikuma et al . , 2009 ) , but PD-1 was dispensable for both the induction and maintenance of tolerance in PIT-exposed naïve CD4+ T cells ( Konkel et al . , 2010 ) . In the clinical setting , PIT is required to control activated Teff cells during ongoing inflammation . Although PIT has been reported to reverse clinical signs of disease ( Leech et al . , 2007 ) , this scenario has been seldom explored mechanistically . An understanding of this is clearly of major importance to successful clinical translation . Here we used a peptide of myelin basic protein ( MBP ) and MBP-responsive TCR transgenic cells to show that PIT was capable of silencing Teff cells , thereby preventing murine experimental autoimmune encephalomyelitis ( EAE ) . PD-L1hi CD4+ dendritic cells ( DC ) were uniquely capable of providing sustained presentation of peptide-MHC ( pMHC ) complexes following PIT . PD-1-deficient T cells were resistant to PIT . In PD-1-sufficient Teff , PIT drove demethylation of the Pdcd1 promoter , correlating with loss of 5-hydroxymethylation ( a potential DNA demethylation intermediate ) and lasting PD-1 expression . These data help define an epigenetic signature of T cell tolerance following PIT and therefore have implications for the development of protein biomarkers for clinical efficacy in current and anticipated tolerogenic modalities .
The Ac1-9 ( 4Tyr ) peptide of MBP , containing a Lys→Tyr substitution at residue 4 of the peptide , is a potent tolerogen when administered in soluble form either to wildtype ( WT ) H-2u mice or to Tg4 mice expressing a transgenic TCR responsive to this peptide ( Liu and Wraith , 1995; Burkhart et al . , 1999 ) . To trace a defined antigen-responsive cohort of T cells we adapted these protocols by prior transfer of naïve CD4+ Tg4 . CD45 . 1 T cells into B10 . PL ( H-2u ) , or B10 . PLxC57BL/6 ( H-2u , b ) mice . These F1 mice are resistant to EAE induced with the MBP peptide , unless first seeded with a cohort of Tg4 T cells ( Ryan et al . , 2005 ) . Tracing the presence and function of the transferred Tg4 cells is therefore of direct relevance as they are the pathogenic T cell population in these experiments . A single i . v . injection of the MBP peptide protected against subsequent efforts to induce EAE by immunization ( Figure 1A ) . Donor T cells persisted in the spleen ( Figure 1B , C ) , but there was reduced production of IFN-γ and IL-17 , in splenic recall assays amongst PIT-treated mice ( Figure 1C and Figure 1—figure supplement 1 ) . Of note , we found no evidence for an elevation in the frequency of Foxp3+ donor Tg4 cells , nor in IL-10 production in response to PIT ( Figure 1C ) . We concluded from these initial studies that a single exposure to the MBP peptide was sufficient for successful PIT , without enhanced induction of cell death , or establishment of Treg-mediated suppression , but rather an intrinsic unresponsiveness in the persisting Tg4 cells . 10 . 7554/eLife . 03416 . 003Figure 1 . PIT induces unresponsiveness in naïve Tg4 cells . ( A , B ) B10 . PLxC57BL/6 mice received PBS or PIT i . v . 1 day after transfer of naïve CD4+ Tg4 cells . EAE was induced 7 days later by immunization with Ac1-9 . ( A ) Mean clinical scores ± SEM . ( B ) Frequency of CD4+ Tg4 cells in the spleen at day 19 post-EAE induction ( six mice per group , from one of three experiments giving consistent results ) . ( C ) Spleens were sampled four and 7 days after PIT/PBS for analysis of CD4+ Tg4 numbers and Foxp3 expression in host and donor CD4+ T cells ( 3–4 mice per group , from one of three experiments giving consistent results ) . A separate cohort were immunized on day 7 after PIT/PBS and spleens analyzed 10 days later for CD4+ Tg4 cell numbers and the production of IFN-γ , IL-17 and IL-10 by splenocytes in response to stimulation with 100 μM Ac1-9 ( dotted lines represent cytokine levels for unstimulated controls ) ( four mice per group , from one of three experiments giving consistent results ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03416 . 00310 . 7554/eLife . 03416 . 004Figure 1—figure supplement 1 . PIT reduces the frequency and number of pro-inflammatory cytokine producing Tg4 cells . B10 . PLxC57BL/6 mice received PBS/PIT 1 day after transfer of naïve CD4+Tg4 cells . 7 days later mice were immunized and spleens were taken for analysis 10 days after immunization . ( A ) Percentage and ( B ) numbers of IL-17+ and IFN-γ+ Tg4 donor cells upon recall stimulation with 20 μM Ac1-9 . Data are from one of three experiments giving consistent results ( four mice per group ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03416 . 004 Splenocytes isolated from mice administered Ac1-9 ( 4Tyr ) were potent in vitro stimulators of a Tg4 T cell line ( Tg4 . TCL ) and remained so beyond 72 hr after peptide injection ( Figure 2A ) . Splenic populations were MACS-sorted into B cell ( CD11c−CD19+ ) and DC ( CD11c+ ) populations . Whilst DC were more efficient presenters on a per cell basis than unfractionated splenocytes , B cells were very poor presenters ( Figure 2A ) . Although the B cell populations used contained fewer than 2% of CD11c+ cells , it is plausible that this level of contamination accounted for the Tg4 . TCL stimulation evident with high numbers of B cells isolated at the 2-hr time-point . The ability of DC to present the peptide-MHC complex in vivo was underlined by immunofluorescent staining showing transferred Tg4 cells in areas of the spleen rich in CD11c+ cells ( Figure 2—figure supplement 1 ) . 10 . 7554/eLife . 03416 . 005Figure 2 . PD -L1hiCD4+ DC present tolerogenic pMHC complexes to T cells . ( A ) B10 . PL mice received PIT i . v . and splenocytes were isolated at the indicated times . B cells ( CD11c−CD19+ ) and DC ( CD11c+ ) were MACS-sorted and their ability to stimulate a Tg4 . TCL was measured compared to whole splenocytes . Positive controls ( solid symbols ) show proliferative responses of Tg4 . TCL to DC , B cells and splenocytes in the presence of 20 μM Ac1-9 . Data are from one of three experiments giving consistent results . ( B ) Gating strategy for FACS-sorting of DC sub-sets from spleen isolated 1 day after PIT administration . ( C ) Proliferative responses of Tg4 . TCL to increasing numbers of DC . Data are from one of three experiments giving consistent results . ( D ) Representative histograms of steady state expression of PD-L1 , gated on CD11chiCD4+ and CD11chiCD8+ splenic DC . ( E ) MFI ± SEM of staining with mAb to co-stimulatory and co-inhibitory molecules on steady state splenic APC populations ( four mice per group , , from one of two experiments giving consistent results , dotted lines represent MFI of isotype control staining ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03416 . 00510 . 7554/eLife . 03416 . 006Figure 2—figure supplement 1 . Colocalization of transferred naïve Tg4 cells with CD11c+ splenocytes . Representative images of immunofluorescent localization of Tg4 cells . CD4+ Tg4 ( CD45 . 1+ ) cells were transferred into CD45 . 2+ B10 . PLxC57BL/6 hosts 1 day after PIT administration . Spleens were isolated and frozen 48 hr after cell transfer . Frozen sections were stained with CD4 ( green ) , CD11c ( white ) and CD45 . 1 ( red ) . Scale bars represent 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 03416 . 006 Splenic CD11c+ cells from MBP peptide-treated mice were sorted into CD11cintPDCA-1+ ( pDC ) CD11chiCD4+CD8− ( CD4+ DC ) , CD11chiCD4−CD8+ DC ( CD8+ DC ) and CD11chiCD4−CD8− populations ( Figure 2B ) . The ability to stimulate the Tg4 . TCL was only maintained in the CD4+ DC ( Figure 2C ) . Isolation of splenic macrophage populations from peptide-treated mice provided no evidence that these cells could maintain the pMHC complex ( data not shown ) . The CD4+ DC population is located in the T zone of the spleen and so is ideally placed to present tolerogenic peptide to naive T cells ( McLellan et al . , 2002 ) . CD4+ DC from steady state mice had particularly high expression of PD-L1 in comparison to other DC populations ( Figure 2D , E ) . Transferred Tg4 T cells expressed high levels of PD-1 in response to PIT , in contrast to those from PBS-treated mice ( Figure 3A , B ) . Addition of an anti-PD-1 blocking antibody to ex vivo rechallenge cultures restored the ability of splenocytes from PIT-treated mice to produce IFN-γ and IL-17 ( Figure 3C ) . 10 . 7554/eLife . 03416 . 007Figure 3 . PD-1 is required for the establishment and maintenance of tolerance in naïve CD4+ Tg4 cells . ( A–C ) B10 . PL mice received PBS/PIT 1 day after transfer of naïve CD4+ Tg4 cells . ( A ) Representative histograms of PD-1 expression gated on CD4+ Tg4 donor cells in spleen 4 days after PBS/PIT . ( B ) MFI of PD-1 staining gated on CD4+ Tg4 donor cells in spleen 4 and 7 days after PBS/PIT ( 4–6 mice per group , from one of three experiments giving consistent results , dotted line represents MFI of isotype control staining ) . ( C ) IFN-γ and IL-17 production in response to Ac1-9 by splenocytes isolated 7 days after PIT and cultured in the presence of anti-PD-1 or isotype ( four mice per group , from one of three experiments giving consistent results , dotted lines represent cytokine levels for unstimulated cultures ) . ( D ) EAE in B10 . PLxC57BL/6 mice that received PBS/PIT 1 day after transfer of naïve CD4+ cells from Tg4 . PD-1+/+ or Tg4 . PD-1−/− donors . EAE was induced 7 days after PBS/PIT by immunization with Ac1-9 ( five mice per group , from one of three experiments giving consistent results ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03416 . 007 Collectively , the above data indicated that sustained pMHC presentation during PIT can drive high expression of PD-1 in the responding T cells . This , coupled with pMHC presentation specifically by PD-L1hi CD4+ DC renders the T cells unable to produce pro-inflammatory cytokines . To determine the functional importance of PD-1 in this tolerogenic process , we generated Tg4 . PD-1−/− mice . Transferred naïve Tg4 cells from these mice were insensitive to PIT , going on to cause a typical course of EAE upon subsequent immunization that was indistinguishable from that induced with Tg4 . PD-1+/+ cells that had not been subjected to PIT ( Figure 3D ) . We therefore conclude that PD-1 plays a non-redundant role in this model of CD4+ T cell tolerance . Although PIT can inhibit ongoing EAE ( Leech et al . , 2007 ) , the mechanisms behind this have been less well explored than for the effects of PIT on naïve T cells . We therefore determined the effects of PIT using a previously established passive EAE model in which Teff cells are generated from naïve Tg4 T cells prior to transfer into WT hosts ( O'Connor et al . , 2010 ) . PIT rendered the host mice profoundly resistant to disease ( Figure 4A ) . As with naïve T cells shown above , the Teff cells persisted after PIT-treatment , but importantly these cells failed to populate the CNS efficiently ( Figure 4B ) . Instead , they were present in the spleen in sufficient numbers to allow detailed analysis following retrieval by FACS-sorting ( Figure 4C ) . PIT did not drive Foxp3-expression in Tg4 Teff cells ( Figure 4D ) , nor was IL-10 production evident ( not shown ) . Rather , within 4 days of PIT , proinflammatory cytokine production by the transferred Teff cells was profoundly diminished ( Figure 4E , F ) . The loss of IFN-γ producing capacity in PIT-exposed Teff was not reflected in any loss in T-bet expression ( Figure 4—figure supplement 1 ) . 10 . 7554/eLife . 03416 . 008Figure 4 . PIT induces tolerance in pathogenic Tg4 Teff cells . ( A ) EAE in B10 . PL mice that received PBS/PIT 1 day after transfer of Tg4 Teff cells ( six mice per group , from one of five experiments giving consistent results ) . ( B ) Numbers of CD4+ Tg4 donor cells in spleen and CNS of PBS/PIT-treated mice sampled at the peak of EAE ( six mice per group , from one of two experiments giving consistent results ) . ( C ) Tg4 Teff cells were transferred and spleens were sampled 4 days after PBS/PIT . ( D ) The frequencies of Foxp3+ cells amongst the donor Tg4 CD4+ Teff cells on day 4 after PBS or PIT . ( E ) Ac1-9-induced IL-2 and IFN-γ production by retrieved CD4+ Tg4 donor cells ( FACS-sorted 4 days after PBS/PIT and cultured with irradiated B10 . PLxC57BL/6 splenic APC ) ( four mice per group , from one of two experiments giving consistent results , dotted lines represent cytokine levels for unstimulated controls ) . ( F ) GM-CSF and IFN-γ production following overnight culture of splenocytes with Ac1-9 ( gated on CD4+ Tg4 donor cells ) ( n = 4 per group , from one of four experiments giving consistent results , dotted lines represent cytokine levels for unstimulated controls ) . ( G ) Modulation of EAE by anti-PSGL-1 ( 50 μg given i . v . on alternate days from day 1 after Tg4 Teff cells ) . Teff cells were also incubated with 20 μg/ml of the relevant antibody for one hour prior to transfer . ( n = 10 per group , from one of three experiments giving consistent results ) . ( H ) PSGL-1 expression by CD4+ host and Tg4 donor cells 4 days after PBS/PIT ( four mice per group , from one of two experiments giving consistent results , dotted line represents MFI of isotype control staining ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03416 . 00810 . 7554/eLife . 03416 . 009Figure 4—figure supplement 1 . Pathogenic Tg4 Teff cells maintain T-bet after PIT . ( A , B ) B10 . PLxC57BL/6 mice received PBS/PIT 1 day after transfer of naïve CD4+ Tg4 cells . ( A ) Representative histograms of T-bet expression gated on CD4+ host or Tg4 donor cells in spleen 4 days after PBS/PIT . ( B ) MFI of T-bet staining gated on CD4+ host or Tg4 donors within the spleen 4 days after PBS/PIT ( 4 mice per group , from one of four experiments giving consistent results , dotted line represents MFI of isotype control staining ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03416 . 00910 . 7554/eLife . 03416 . 010Figure 4—figure supplement 2 . PIT reduces active PSGL-1 levels on Tg4 Teff cells . B10 . PLxC57BL/6 mice received PBS/PIT 1 day after transfer of Tg4 Teff cells . Splenocytes were isolated 4 days after PBS/PIT ( n = 6–8 per group ) . Active PSGL-1 was detected using a P-selectin-hIgG fusion protein , followed by PE-anti-human IgG staining . Events were gated on CD4+ host or Tg4 donor cells . Dotted line represents staining with PE-anti-human IgG alone . Data are from one of two experiments giving consistent results . DOI: http://dx . doi . org/10 . 7554/eLife . 03416 . 010 The poor accumulation of Tg4 Teff cells within the CNS after PIT ( Figure 4B ) suggested that their ability to home to the target organ might also have been affected . Although the molecular requirements for T cell entry into the CNS during EAE are complex and controversial ( Prendergast and Anderton , 2009 ) , others have described a requirement for P-selectin glycoprotein ligand-1 ( PSGL-1 ) in the pathogenic process ( Deshpande et al . , 2006 ) . We have also found that encephalitogenic function of Tg4 Teff populations correlates with their elevated expression of PSGL-1 and that an antibody to PSGL-1 can inhibit Tg4 Teff-driven EAE ( Figure 4G ) , presumably by interfering with the interaction of PSGL-1 on the Teff cells with P-selectin and/or E-selectin on the CNS endothelium . Whilst transferred Tg4 Teff cells exposed to PBS showed elevated PSGL-1 expression , their counterparts that had been exposed to PIT showed PSGL-1 expression more comparable with that seen on the EAE-irrelevant CD4+ cells of the host mice ( Figure 4H ) , PSGL-1 glycosylation mediated by 2 β-1 , 6-N-acetyl glucosaminyltransferase and α ( 1 , 3 ) -fucosyltransferase-VII ) is required for functionality ( binding to selectins ) ( Yu et al . , 2000; Smithson et al . , 2001; Sperandio et al . , 2001; Deshpande et al . , 2006 ) . Staining with a mouse P-selectin-human IgG fusion protein indicated that active PSGL-1 was also reduced on Tg4 Teff cells in response to PIT ( Figure 4—figure supplement 2 ) . These data suggest that one consequence of PIT is disrupted PSGL-1-dependent T cell trafficking . We also observed that PD-1 expression was high on Teff cells retrieved from PIT-treated mice compared to PBS-treated controls and that this difference was maintained beyond 2 weeks from peptide administration ( Figure 5A–C ) . This was not a consequence of any long-term pMHC presentation in the lymphoid system , because PIT-treated Teff cells that had been retrieved from their first hosts maintained PD-1 ( and did not induce EAE ) when transferred into secondary hosts that were not exposed to PIT ( Figure 5D–F ) . The tolerant phenotype was therefore stable in T cells exposed to PIT . 10 . 7554/eLife . 03416 . 011Figure 5 . PD-1 expression is maintained by PIT and is required for tolerance . ( A ) Representative histograms of PD-1 expression by CD4+ Tg4 cells sampled as naïve cells , Teff on day of transfer and Teff cells retrieved 4 days after PBS/PIT . ( B ) PD-1 expression ( MFI ) gated on CD4+ host cells and Tg4 donor cells from spleen 4 days after treatment ( four mice per group , from one of four experiments giving consistent results , dotted line represents MFI of isotype control staining ) . ( C ) Time course of PD-1 expression on CD4+ Tg4 donor cells from PBS/PIT-treated mice ( four mice per group ) . ( D ) B10 . PLxC57BL/6 mice received PBS/PIT 1 day after transfer of Tg4 Teff cells . 4 days later CD4+ Tg4 donor cells were FACS-sorted and 2 × 106 were transferred into secondary hosts that were not exposed to PIT ( PTX was given on the same day ) . ( E ) EAE in secondary hosts ( n = 4 for PBS; 21 for PIT , pooled from two experiments ) . ( F ) PD-1 expression gated on CD4+ Tg4 donor cells from spleens isolated 16 days after secondary transfer ( dotted line represents MFI of isotype control staining ) . ( G ) EAE in B10 . PLxC57BL/6 mice that received PBS/PIT 1 day after transfer of Teff generated from Tg4 . PD-1+/+ or Tg4 . PD-1−/− donors ( n = 20–36 mice per group , pooled from three experiments ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03416 . 01110 . 7554/eLife . 03416 . 012Figure 5—figure supplement 1 . Elevated accumulation of Tg4 . PD-1−/− Teff cells following PIT . B10 . PLxC57BL/6 mice received PBS or PIT 1 day after transfer of Tg4 . PD-1+/+ or Tg4 . PD-1−/− Teff cells . ( A ) Percentage and ( B ) numbers of CD4+ Tg4 cells within the spleen 4 days after PBS or PIT ( 12 mice per group ) , data are pooled from three experiments giving consistent results . ( C ) Percentage of Ki-67+ cells within the Tg4 CD4+ population ( 4 mice per group , from one of two experiments giving consistent results ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03416 . 01210 . 7554/eLife . 03416 . 013Figure 5—figure supplement 2 . PIT does not increase the frequency of Foxp3+ Tg4 Teff cells . B10 . PLxC57BL/6 mice received PBS/PIT 1 day after transfer of Tg4 . PD-1+/+ or Tg4 . PD-1−/− Teff cells . ( A ) Percentage and ( B ) numbers of Foxp3+CD4+ Tg4 cells within the spleen 4 days after PBS/PIT . Data are pooled from three experiments giving consistent results ( 12 mice per group ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03416 . 01310 . 7554/eLife . 03416 . 014Figure 5—figure supplement 3 . PIT limits the frequency of cytokine+ Tg4 Teff cells independently of PD-1 . B10 . PLxC57BL/6 mice received PBS/PIT 1 day after transfer of Tg4 . PD-1+/+ or Tg4 . PD-1−/− Teff cells . Spleens were taken 4 days later for analysis ( A ) Percentage and ( B ) numbers of IFN-γ+ and GM-CSF+ CD4+ Tg4 cells following recall stimulation with 20 μM Ac1-9 . Data are pooled from three experiments giving consistent results ( 12 mice per group ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03416 . 01410 . 7554/eLife . 03416 . 015Figure 5—figure supplement 4 . PIT-exposed Tg4 . PD-1−/− Teff maintain their ability to produce IL-2 . B10 . PLxC57BL/6 mice received PBS or PIT 1 day after transfer of Tg4 . PD-1+/+ or Tg4 . PD-1−/− Teff cells . Spleens were taken for analysis 4 days after PIT for retrieval of CD4+ Tg4 Teff ( FACS-sorting ) . IL-2 production was measured after 48 hr culture with irradiated B10 . PLxC57BL/6 splenic APC and MBP peptide , ( dotted lines represent IL-2 levels from unstimulated cultures ) . Data ( 4 mice per group ) are from one of two experiments giving consistent results ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03416 . 01510 . 7554/eLife . 03416 . 016Figure 5—figure supplement 5 . PD-1 limits CD25 up-regulation following recall stimulation of PIT-exposed Tg4 Teff . B10 . PLxC57BL/6 mice received PBS/PIT 1 day after transfer of Tg4 . PD-1+/+ or Tg4 . PD-1−/− Teff cells . Spleens were taken 4 days later for analysis . CD25 expression within the Tg4 CD4+ population ( A ) directly ex vivo and ( B ) 12 hr after recall stimulation with 20 μM Ac1-9 . DOI: http://dx . doi . org/10 . 7554/eLife . 03416 . 01610 . 7554/eLife . 03416 . 017Figure 5—figure supplement 6 . PD-1 limits phosphorylation of STAT5 in Tg4 Teff cells following PIT . B10 . PLxC57BL/6 mice received PBS/PIT 1 day after transfer of Tg4 . PD-1+/+ or Tg4 . PD-1−/− Teff cells . Spleens were taken 4 days later for analysis . Cells were stimulated for 12 hr with 20 μM Ac1-9 , to allow upregulation of CD25 , before 15-min exposure to IL-2 and detection of pSTAT5 . ( A ) Representative plots of pSTAT5 staining . ( B ) Percentage of pSTAT5+ cells within the Tg4 CD4+ population . Data ( 4 mice per group ) are from one of two experiments giving consistent results . DOI: http://dx . doi . org/10 . 7554/eLife . 03416 . 01710 . 7554/eLife . 03416 . 018Figure 5—figure supplement 7 . PIT limits effector cytokine production by polyclonal Teff cells . ( A ) CD45 . 1 mice were immunized with pOVA/CFA and Teff cells generated by restimulation of draining lymph node cells with pOVA + cytokines . Teff cells were transferred into C57BL/6 ( CD45 . 2 ) host mice prior to administration of pOVA or PBS and splenocytes were retrieved on day 4 ( B ) Gating strategy for identification of CD45 . 1+ donor cells within the spleen . ( C ) Representative plots of intracellular detection of GM-CSF , IFN-γ , TNF-α and IL-17 within the donor CD4+CD45 . 1+ population following recall stimulation with pOVA . ( D ) Percentage of cytokine+ cells within the donor Teff population . Data ( 6 mice per group ) are from one of two experiments giving consistent results . DOI: http://dx . doi . org/10 . 7554/eLife . 03416 . 01810 . 7554/eLife . 03416 . 019Figure 5—figure supplement 8 . PIT drives PD-1 expression in polyclonal Teff cells . Splenocytes from host mice as described in Figure 5—figure supplement 6 and analysed on day 4 . ( A ) Representative plots of PD-1 staining on CD4+ donor Teff cells after PIT/PBS . ( B ) Percentage of PD-1+ cells within the donor CD4+ population . ( C ) MFI of PD-1 staining within the PD-1+ population . Data ( six mice per group ) are from one of two experiments giving consistent results . DOI: http://dx . doi . org/10 . 7554/eLife . 03416 . 019 As was the case with the earlier experiments using naïve T cells ( Figure 3D ) , Tg4 . PD-1−/− Teff cells were not tolerized and were able to cause disease in the presence of PIT ( Figure 5G ) , also establishing PD-1 as a required component of tolerance in this more therapeutic setting . To understand how the absence of PD-1 signaling might allow the maintenance of pathogenic function in spite of PIT administration , we further compared the function of Tg4 . PD-1+/+ versus Tg4 . PD-1−/− Teff cells . There were no gross differences in the numbers or frequencies of donor cells in PBS-treated hosts ( Figure 5—figure supplement 1 ) , indicating that PD-1 expression does not constrain the accumulation of donor cells in the absence of PIT . However , the PIT-driven elevation in donor cell numbers seen with Tg4 . PD-1+/+ Teff cells was accentuated in Tg4 . PD-1−/− Teff cells and a high frequency of these cells showed expression of the cell cycle marker Ki-67 ( Figure 5—figure supplement 1 ) . A consequence of this overall increase in the number of Tg4 . PD-1−/− Teff cells was increased numbers of Foxp3+ cells as well as cytokine+ cells in this group . However , as seen with Tg4 . PD-1+/+ Teff cells , the frequencies of Foxp3+ or cytokine+ Tg4 . PD-1−/− Teff cells were reduced following PIT ( Figure 5—figure supplements 2 , 3 ) . In contrast to their reduced effector cytokine production , PIT did not block the ability of Tg4 . PD-1−/− Teff cells to produce IL-2 upon ex vivo re-challenge ( Figure 5—figure supplement 4 ) . Tg4 Teff cells retrieved from PBS-treated mice had low expression of CD25 and this was absent in their PIT-treated counterparts ( Figure 5—figure supplement 5A ) . After 12 hr culture with MBP peptide , CD25 expression was increased in PBS-exposed Teff cells , irrespective of their PD-1-status , but there was a notable impairment in this upregulation of CD25 in PIT-treated Tg4 . PD-1+/+ Teff cells . This impairment was not evident in PIT-exposed Tg4 . PD-1−/− Teff cells ( Figure 5—figure supplement 5B ) . The deficit in CD25-upregulation seen in PIT-treated Tg4 . PD-1+/+ Teff cells was reflected by a low frequency of cells with phosphorylated STAT5 in response to IL-2 . Notably however , PIT-treated Tg4 . PD-1−/− Teff cells did not show such abrogated STAT5 phosphorylation ( Figure 5—figure supplement 6 ) . We conclude that the ability of PD-1-deficient Teff cells to drive EAE despite administration of PIT is associated with their heightened capacity for clonal expansion . To assess whether PIT-driven PD-1 expression was evident in a non-transgenic system , we studied ovalbumin ( OVA ) -responsive Teff in mice on the C57BL/6 background . Lymph nodes from CD45 . 1 mice that had been immunized with the OVA ( 323–339 ) peptide ( pOVA ) in CFA provided a source of Teff , which were then transferred into CD45 . 2 hosts prior to administration of pOVA or PBS ( Figure 5—figure supplement 7A , B ) . Reduced frequencies of cytokine+ donor cells following in vitro restimulation were indicative of PIT-induced unresponsiveness ( Figure 5—figure supplement 7C , D ) . Ex vivo analysis showed clear PD-1hi CD4+ populations in donor cells from the PIT-treated group ( Figure 5—figure supplement 8A , B ) . Focusing on cells within the PD-1+ gate , PIT-exposed donor cells showed higher levels of PD-1 expression than PBS-exposed donor cells ( Figure 5—figure supplement 8C ) . We conclude that elevated expression of PD-1 associates with abrogation of effector function in response to PIT in this non-transgenic system . PD-1 is rapidly upregulated by T cells upon initial TCR stimulation but is then lost over time ( Riley , 2009 ) . This is exemplified by the fact that our pathogenic Tg4 Teff cells expressed PD-1 on the day of transfer , but this was lost within 3 days of transfer in PBS-treated hosts ( Figure 5A , C ) . In contrast , PIT drove the long-term expression of PD-1 by potentially pathogenic CD4+ T cells and this was a required component of PIT . Sustained expression of PD-1 is important in another scenario in which T cells develop antigen-unresponsiveness; CD8+ T cell ‘exhaustion’ in the face of chronic viral infection ( Barber et al . , 2006; Wherry et al . , 2007 ) . So , are similar mechanisms , leading to long-term expression of PD-1 , at the heart of these two states of T cell unresponsiveness ? Stable gene expression is often associated with a variety of epigenetic changes within promoter regions , including alterations in the levels of active histone post-translational modifications ( PTMs ) such as trimethylation of H3K4 ( H3K4me3 ) , or repressive histone PTMs such as H3K27me3 . Indeed , CD8+ Teff cells with low PD-1 expression were reported to have elevated levels of the repressive H3K27me3 PTM at the Pdcd1 promoter , compared to either PD-1− naive cells or PD-1hi CD8+ cells from chronically infected mice ( Youngblood et al . , 2011 ) . We measured active ( H3K4me3 ) and repressive ( H3K27me3 ) histone marks at the Pdcd1 promoter by ChIP-PCR and did not identify similar changes that could distinguish pathogenic Tg4 Teff , from PIT-tolerized Teff . In naïve Tg4 cells and PBS-treated Tg4 Teff cells ( neither of which expressed PD-1 ) , neither modification ( H3K27me3 or H3K4me3 ) was enriched at the two conserved regions , CR-B and CR-C ( Youngblood et al . , 2011 ) , of the Pdcd1 promoter ( Figure 6A , B ) . In contrast , both the PD-1 expressing groups ( Teff on the day of transfer and PIT-treated Teff ) exhibited elevated levels of H3K4me3 , but no enrichment for H3K27me3 ( Figure 6A , B ) . Importantly , functional ( pathogenic ) PBS-treated Teff that lacked PD-1 expression did not show elevated levels of the repressive H3K27me3 PTM , thereby distinguishing their behaviour from that reported for PD-1lo CD8+ Teff cells ( Youngblood et al . , 2011 ) . Moreover , it was not possible to distinguish PD-1hi pathogenic Teff ( on the day of transfer ) from PD-1hi PIT-tolerized Teff based on their relative abundance of H3K4me3 versus H3K27me3 at the Pdcd1 promoter . 10 . 7554/eLife . 03416 . 020Figure 6 . PIT induces epigenetic modification of the Pdcd1 promoter . CD4+ Tg4 T cells were isolated as naïve cells , Teff on day of transfer and cells retrieved 4 days after either PBS or PIT for analysis of the CR-C and CR-B regions . ( A , B ) Histone modifications ( H3K4me3 and H3K27me3 ) were analysed at CR-C and CR-B by ChIP and qPCR . Data in ( B ) show the ratio of H3K4me3 over H3K27me3 fold-enrichment over background and are representative of two independent experiments . ( C , D ) DNA methylation status determined by pyrosequencing ( C ) and bisulfite sequencing ( D ) ( data are from two ( C ) or one ( D ) bisulfite conversions , four or more mice per group ) . ( D ) Methylated ( black ) and unmethylated ( white ) CpGs . Vertical columns represent individual CpGs ( 1–4 for CR-C and 7–14 for CR-B ) . Horizontal rows represent individual cloned alleles . ( E ) 5hmC enrichment at CR-C and CR-B as measured by 5hmC DNA Immunoprecipitation and qPCR . Data are from two experiments using pooled samples . ( F ) Total RNA was extracted from the isolated cell populations and expression levels of TET1 , TET2 and TET3 were measured by qPCR . Values are expressed relative to housekeeping gene ( GAPDH ) and are representative of three experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 03416 . 020 Stable PD-1 expression in exhausted CD8+ T cells is associated with DNA demethylation within the CR-B and CR-C regions of the Pdcd1 promoter ( Youngblood et al . , 2011 ) . We retrieved Tg4 cells at various stages of the Teff PIT model for DNA methylation analysis . DNA modification was quantified , after bisulfite conversion , using pyrosequencing to assess individual CpG sites within the whole cell population . Cloning and sequencing after bisulfite conversion provided complementary information on the pattern of DNA methylation at CpG sites across individual cloned alleles ( Figure 6C , D ) . Consistent with previous analyses of naive CD8+ T cells ( Youngblood et al . , 2011 ) , CpGs within the CR-B region were largely modified ( ∼80% methylation across most CpG sites ) in naïve CD4+ Tg4 cells ( Figure 6C–D ) , whereas DNA methylation levels were considerably lower across the CR-C region . Despite expression of PD-1 by Tg4 Teff cells on the day of transfer ( Figure 5A ) , there were no differences in DNA methylation at the Pdcd1 promoter of these pathogenic cells compared to naïve Tg4 cells ( Figure 6C , D ) , indicating that demethylation is not a pre-requisite for transient PD-1 expression following initial CD4+ T cell activation . As would be expected based on this result , DNA methylation was maintained at CR-B and CR-C in the Teff cells retrieved from PBS-treated hosts , where PD-1 expression had returned to levels observed in naïve T cells ( Figure 5A ) . In marked contrast , PIT-exposed Teff , which had prolonged PD-1 expression ( Figure 5A–F ) , showed complete demethylation at CR-C and significantly reduced DNA methylation at the CR-B region in the Pdcd1 promoter ( Figure 6C , D ) . Therefore only PIT was capable of altering DNA methylation patterns at the Pdcd1 promoter . This enables PD-1hi tolerant cells to be distinguished from fully functional Teff , which only transiently express PD-1 . The precise processes that lead to demethylation of 5-methylcytosine ( 5mC ) are not fully understood , but direct conversion of 5mC to cytosine seems unlikely . Rather , the favoured model is that 5mC is progressively oxidized to generate intermediates , facilitated by the Ten-Eleven-Translocation ( TET ) proteins , and these intermediates are subsequently converted either by DNA repair or replication dilution to cytosine ( Kohli and Zhang , 2013 ) . The first intermediate is 5-hydroxymethylcytosine ( 5hmC ) , a stable epigenetic mark with widespread tissue distribution ( Kriaucionis and Heintz , 2009; Tahiliani et al . , 2009; Song et al . , 2011; Nestor et al . , 2012; Thomson et al . , 2012 ) . Reciprocal changes for both 5mC and 5hmC at gene loci imply that demethylation may be occurring via a 5hmC intermediate ( Thomson et al . , 2013 ) . Of note , standard bisulfite conversion techniques cannot distinguish 5mC from 5hmC . Using an affinity based technique ( Thomson et al . , 2012 ) , we identified considerable enrichment of 5hmC at the Pdcd1 promoter CR-B and CR-C regions in naïve Tg4 cells ( Figure 6E ) . 5hmC was also present in Teff cells generated by 3-day culture ( particularly within the CR-B region ) and in Teff cells retrieved from PBS-treated mice . In contrast , 5hmC was undetectable at CR-B and CR-C in cells retrieved from PIT-treated mice ( Figure 6E ) . We tested whether changes in the relative expression of 5hmC at the Pdcd1 promoter were reflected by changes in the global expression of TET genes ( as assessed by qPCR ) . All three known mammalian TETs were expressed in naïve CD4+ Tg4 cells , but these levels were substantially reduced in Teff cells ( Figure 6F ) . Expression of each TET was partially restored in Tg4 Teff cells retrieved from either PIT or PBS-treated hosts , suggesting these that cells were equipped for the oxidation of both 5mC and 5hmC to occur . Collectively our data indicate that stable expression of PD-1 is coincident with demethylation of the Pdcd1 promoter and that this requires a combination of permissive histone changes and oxidation of 5mC and 5hmC involving the activity of TETs . In the system used here , these two requirements only coincide following TCR engagement in response to PIT .
Our data provide key insights into the mechanisms of Teff cell unresponsiveness induced by PIT , which are pertinent to its clinical translation . A role for PD-1 in the establishment , or maintenance , of T cell unresponsiveness has been reported in various experimental models of autoimmune and allergic inflammation ( Probst et al . , 2005; Cheng et al . , 2007; Francisco et al . , 2010 ) . Our previous work had excluded such a role for PD-1 in PIT-driven tolerance for naive CD4+ T cells , because PIT remained effective irrespective of antibody blockade or genetic deletion of PD-1 in the peptide-responsive T cells ( Konkel et al . , 2010 ) . In the two experimental models used in that study , PIT appeared to drive the abortive activation and apoptosis of peptide-responsive naïve T cells within 4 days , which would override any potential for PD-1-mediated long-term unresponsiveness . In contrast to those previous studies using naïve T cells , we found here that Teff persisted following therapeutic application of PIT . In a recent study using PIT to silence allergic airways inflammation , we found an analogous accumulation of allergic Teff cells , indicating that persistence following PIT is common to Teff with different inflammatory functions ( Mackenzie et al . , 2014 ) . The observations that pMHC complexes were specifically maintained on CD4+ DC in vivo , and that these DC showed the highest constitutive expression of PD-L1 , led us to assess the role of PD-1 in PIT here . A recent study transgenically expressed autoantigen on all DC subsets and found PD-1-dependent elevation in the frequencies of autoreactive Foxp3+ T cells as a result ( Yogev et al . , 2012 ) . That result may well have reflected an influence of CD8+ DC , which can drive de novo Foxp3–expression due to their production of TGF-β ( Yamazaki et al . , 2008 ) . CD4+ DC do not have that capacity , accounting for the absence of increased Foxp3-expression here , in Tg4 cells exposed to PIT . This , together with the lack of evidence for IL-10 production in response to PIT suggests that , in this study , the predominant effect of PIT is to suppress pathogenic effector function rather than to drive gain of regulatory function . PD-1 has been reported to co-locate to TCR microclusters at the immunological synapse , where , following Lck-mediated phosphorylation of its ITSM , PD-1 recruits the SHP-2 phosphatase to counteract phosphorylation of TCR-proximal signalling machinery ( Yokosuka et al . , 2012 ) . Use of Tg4 . PD-1−/− Teff cells indicated a key role for PD-1 in limiting clonal expansion following PIT . There are several ways in which this inhibition could be achieved . Inhibition of PI3K/Akt and MEK/ERK pathways can lower cell proliferation by reducing transcription of SKP2 . This gene encodes a crucial component of the ubiquitin ligase SCFSkp2 , which is required for cyclin-dependent kinase 2 activation and cell cycle progression from G1 into S phase ( Parry et al . , 2005; Appleman et al . , 2006; Patsoukis et al . , 2012; Pauken et al . , 2013 ) . Exogenous IL-2 only partially restores cell proliferation by activating the MEK/ERK pathway but not the PI3K/Akt pathway ( Patsoukis et al . , 2012 ) , suggesting that IL-2 cannot reverse PD-1-dependent unresponsiveness . Recruitment SHP-2 to the cytoplasmic tail of PD-1 is thought to function primarily by inhibiting the PI3K pathway ( Chemnitz et al . , 2004 ) . However , SHP-2 has also been implicated in the dephosphorylation and inactivation of pSTAT5 ( Yu et al . , 2000; Chen et al . , 2003 ) and PD-1 signalling has been associated with a decrease in both the expression and phosphorylation of STAT5 ( Iliopoulos et al . , 2011 ) . These observations correlate with the decreased levels of pSTAT5 we see in Tg4 Teff cells ( with intact PD-1 signalling ) following PIT . Furthermore , inactivation of STAT5 can lead to a reduction in cell-surface CD25 expression ( Kim et al . , 2001 ) . This , coupled with the inhibition of TCR signalling pathways by PD-1 , would likely account for the impaired CD25 expression that we saw in PIT-exposed Tg4 Teff cells . An impaired ability to upregulate CD25 , and therefore to respond to IL-2 , is distinct from the classical profile for clonal anergy , in which unresponsive T cells can upregulate CD25 but cannot produce their own IL-2 ( anergy is broken by addition of exogenous IL-2 ) ( Schwartz , 2003 ) . An unresponsive state that blocks CD25 upregulation can be viewed as more secure , since it removes the risk of IL-2 from a bystander source overcoming that unresponsiveness . Consistent with their lack of encephalitogenicity , Teff that had been tolerized by PIT had an impaired ability to establish themselves in the CNS . This might reflect their impaired ability to undergo clonal expansion upon subsequent exposure to MBP in the CNS , as discussed above . A recent report has indicated an additional role for PD-1 in inhibiting autoimmune T cell infiltration into the pancreas in diabetes ( Pauken et al . , 2013 ) . Here we observed that tolerized Teff had lower levels of active PSGL-1 relative to pathogenic T cells ( PBS group ) , which is an important component of pathogenic T cell entry into the CNS in this model . To our knowledge , no direct causal relationship between PD-1 and PSGL-1 has been established . T follicular helper ( TFH ) cells are PD-1hi and PSGL-1lo , but we do not believe that PIT drives Teff towards a TFH fate , since we did not observe any production of the TFH-associated cytokine IL-21 , nor did we find evidence for any gain of the TFH-associated transcription factor BCL-6 following PIT ( data not shown ) . The transcription factor T-bet can inhibit PD-1 expression in CD8+ cells ( Kao et al . , 2011 ) , but here we found that this was not the case for CD4+ Teff cells , which were T-bet+ PD-1hi following PIT , indicating that T-bet and PD-1 are not necessarily mutually antagonistic . Our data allow us to develop a model to explain the pronounced demethylation of the Pdcd1 promoter that , in the experimental system used here , is a unique characteristic of Tg4 Teff that have been tolerized in response to PIT . The Pdcd1 promoter of Tg4 cells in their naïve state appears to be fully methylated , although this includes a significant level of 5-hydroxymethylation . This , together with the availability of TETs 1–3 and the absence of the repressive H3K27me3 histone mark , suggests that the gene is poised for transcriptional activation and PD-1 expression . This would fit with the known early expression of PD-1 following initial TCR ligation ( as seen here in Teff on the day of transfer ) . This PD-1 expression during Teff generation was mirrored by chromatin changes associated with gene transcription ( H3K4me3 enrichment ) , but without a contemporaneous demethylation of the Pdcd1 promoter . The considerable reduction in TET expression observed at the Teff stage should impede any active demethylation through the oxidation of either 5mC or 5hmC ( a process that involves TET activity ) , thereby averting long-term PD-1 expression that would affect Teff function . As these cells transition to a PD-1-negative Teff state ( pathogenic Tg4 cells retrieved from PBS-treated hosts ) , histone modifications associated with active transcription are no longer evident and , although TET expression has been restored , this repressive chromatin state prevents the TET ( and other ) proteins from accessing the Pdcd1 promoter to allow demethylation . However , a key feature of the tolerogenic properties of PIT is to provide long-term PD-1 expression by tolerant CD4+ T cells , which is associated with the presence of stable histone modifications characteristic of active transcription ( thus enabling access of the TET proteins , amongst others ) , and therefore loss of both 5mC and 5hmC at the Pdcd1 promoter . These data indicate that analysis of the methylation/hydroxymethylation status of the Pdcd1 promoters may facilitate the discrimination of tolerant versus recently activated PD-1+ CD4+ T cells . Juvenile arthritis patients who responded to PIT using peptide from the DNAj antigen were reported to have an ‘immune signature’ that included elevated global PD-1 expression , relative to non-responders ( Koffeman et al . , 2009 ) . However , that work was based on assessment of the patients before undergoing PIT , rather than following PIT . It would therefore be important to understand the relative expression of PD-1 before and after treatment in individual patients in future studies . A limitation is the need to identify T cells that are relevant to the peptide ( s ) used for PIT . As the range of pMHC tetramers available expands , this should become more feasible . Furthermore , our study highlights the need for analysis of the Pdcd1 promoter to discriminate PD-1hi pathogenic Teff from the PD-1hi Teff rendered tolerant by PIT . This would be challenging to combine with tetramer-based isolation of relatively small T cell populations , although perhaps not impossible . Therefore , whilst locus-specific DNA methylation-status would not prove a viable biomarker in itself , it might aid the identification of a surrogate phenotypic profile amenable to flow cytometric analysis . The demethylation of the Pdcd1 promoter seen in PIT-tolerized Teff is reminiscent of the changes reported to occur in CD8+ T cells exhausted in response to chronic viral infection ( Youngblood et al . , 2011 ) . So , do these two situations of CD4+ T cell tolerance and CD8+ T cell exhaustion have the same underlying mechanism ( s ) ? In some ways yes , but there are some pronounced differences . In both scenarios Teff cells express PD-1 but this is lost , either as they transition to memory cells ( Youngblood et al . , 2011 ) , or here within 2 days of transfer in the absence of PIT . Conversely , exhausted or peptide-tolerized Teff cells maintain PD-1 expression . Youngblood et al reported that CD8+ Teff cells that transiently expressed PD-1 already have demethylated Pdcd1 promoters and their transition towards PD-1-negative memory cells is driven by the specific remethylation of their Pdcd1 promoters ( Youngblood et al . , 2011 ) . In contrast , the methylation status of the Pdcd1 promoters within our CD4+ Teff cells that transiently expressed PD-1 appeared unaltered from that of naïve CD4+ T cells . It was only in the PIT-treated Teff that we saw significant demethylation of the Pdcd1 promoter , consistent with their sustained PD-1 expression . It will be important to determine whether these differences reflect intrinsic differences in how PD-1 expression is controlled in CD4+ versus CD8+ T cells . In addition , we are not aware of any information on 5hmC status of the Pdcd1 promoter in CD8+ cells . We found marked enrichment of 5hmC at the Pdcd1 promoter in naïve CD4+ T cells . This is intriguing because the biological significance of 5hmC has chiefly been explored in the context of embryonic development . Its appearance in the promoter of a key gene controlling T cell activation suggests that DNA hydroxymethylation may play a broader role in maintaining the ‘pluripotent’ state of naïve CD4+ T cells . We suggest that this warrants global analyses to search for 5hmC and other intermediates of 5mC oxidation within the CD4+ T cell genome and also for the TET proteins with direct roles in the oxidation process ( Deplus et al . , 2013; Koh and Rao , 2013 ) .
C57BL/6 ( CD45 . 1 or CD45 . 2 ) , B10 . PL ( CD45 . 2 ) , B10 . PLxC57BL/6 ( CD45 . 2 ) and Tg4 ( CD45 . 1 or CD90 . 1 ) mice ( Liu et al . , 1995 ) were used . PD-1−/− mice ( kindly provided by Prof Tasuku Honjo , Kyoto University ) were crossed with Tg4 ( CD90 . 1 ) mice to obtain Tg4 . PD-1−/− ( CD90 . 1 ) mice . All mice were bred under specific pathogen-free conditions at the University of Edinburgh . All experiments were approved by the University of Edinburgh Ethical Review Committee and were performed in accordance with UK legislation . Acetylated MBP peptides Ac1-9 ( Ac-ASQKRPSQR ) and Ac1-9 ( 4Tyr ) ( Ac-ASQYRPSQR ) and the ovalbumin peptide 323–339 ( pOVA ) ( ISQAVHAAHAEINEAGR ) were synthesised with C-terminal amides by Cambridge Research Biochemicals , Billingham , UK . Tissue culture medium ( RPMI 1640 medium ) was supplemented with 2 mM L-glutamine , 100 U/ml penicillin , 100 µg/ml streptomycin , and 5 × 10−5 M 2-ME ( all from Invitrogen Life Technologies , Paisley , UK ) and 10% FCS ( Sigma , Poole , UK ) . Cells isolated from immunized mice were cultured in X-VIVO15 serum-free medium ( Lonza , Walkersville , MD , USA ) supplemented with 2 mM L-glutamine and 5 × 10−5 M 2-ME . Naïve CD4+ T cells were isolated from Tg4 or Tg4 . PD1−/− mice by magnetic activated cell-sorting ( MACS ) according to manufacturer's instructions ( Miltenyi Biotec , Teterow , Germany ) , and 2 × 106 cells were transferred in 200 µl PBS i . v . Pathogenic MBP-responsive Teff cells were generated by stimulation of Tg4 or Tg4 . PD1−/− splenocytes with Ac1-9 in the presence of IL-12 , IL-18 and IL-2 as described previously ( O'Connor et al . , 2010 ) . pOVA-responsive Teff cells were generated by immunization of C57BL/6 ( CD45 . 1 ) mice with pOVA , followed by restimulation of draining lymph node cells with pOVA and the above cytokine cocktail . Cells were harvested after 72 hr culture and 2 × 106 blasts transferred i . v . in 200 µl PBS . For the retrieval of donor Tg4 cells , CD4+ T cells were isolated from splenocytes by MACS and donor cells ( CD4+ CD45 . 1+ or CD90 . 1+ ) were identified and isolated by surface staining and FACS using a FACSAria II ( Becton Dickinson , Franklin Lakes , NJ , USA ) . On the day indicated , mice received Ac1-9 or pOVA emulsified in CFA containing 50 μg heat-killed Mycobacterium tuberculosis H37Ra ( Sigma ) in a total volume of 100 μl injected subcutaneously ( s . c . ) into the hind legs . For PIT , mice received 200 µg of Ac1-9 ( 4Tyr ) , or pOVA , in 200 µl PBS ( or PBS alone ) i . v . at the indicated time . For the induction of active EAE , mice were immunized as above and received 200 ng of pertussis toxin ( Health Protection Agency , Dorset , U . K . ) in 0 . 5 ml PBS i . p on the day of immunization and 48 hr later . Clinical signs of EAE were assessed using the following scoring index: 0 , no signs; 1 , flaccid tail; 2 , impaired righting reflex and/or gait; 3 , partial hind limb paralysis; 4 , total hind limb paralysis; 5 , hind limb paralysis with partial front limb paralysis; 6 , moribund or dead . For the induction of passive EAE , mice received 2 × 106 Tg4 effector cells via i . v . injection . On the same day , or the day following cell transfer , mice received 200 ng pertussis toxin in 0 . 5 ml PBS i . p . and clinical signs were assessed as described above . For some experiments , an anti-PSGL-1 antibody ( 4RA10 , rat IgG1 , BioXCell , West Lebanon , NH , USA ) , or purified rat IgG ( Sigma ) were administered , as indicated . Mice received the Ac1-9 ( 4Tyr ) peptide as described above for the induction of PIT . Spleens were isolated at the indicated times , injected with 100 µl of 8 mg/ml of collagenase IV ( Worthington Biochemical Corp , Lakewood , NJ , USA ) and incubated at 37°C for 20 min before manual disaggregation . In some experiments , APC populations were purified by positive selection using MACS and anti-CD11c beads ( DC ) , or by negative selection with anti-CD11c beads , followed by positive selection using anti-CD19 beads . For isolation of DC populations , CD3+ and CD19+ cells were depleted by MACS prior to staining and FACS for pDC ( PDCA-1+CD11cint ) , CD4+DC ( CD11chiPDCA-1−CD4+CD8− ) , CD8+DC ( CD11chiPDCA-1−CD8+CD4− ) , and CD4−CD8−DC ( CD11chiPDCA-1−CD8−CD4− ) . APC populations were used to stimulate a Tg4 T cell line ( Tg4 . TCL; generated as previously described [Anderton et al . , 1998] ) , with or without the addition of exogenous Ac1-9 peptide to the cultures . Cells ( 2 × 104 Tg4 . TCL with a range of APC numbers per well ) were cultured in flat-bottom 96-well microtitre plates ( Becton Dickinson ) for 72 hr [3H]thymidine incorporation over the final 16 hr was measured using a liquid scintillation β-counter ( LKB Wallac , Turku , Finland ) . Splenocyte suspensions were cultured in 96-well flat-bottom microtitre plates ( Becton Dickinson ) at 8 × 105 spleenocytes/well . For purified donor T cells , 2 × 104 T cells were cultured with 2 × 105 irradiated ( 30 Gy ) splenic APC's/well . Cultures were stimulated with a dose range of Ac1-9 and supernatants were tested for production of IFN-γ and IL-17 at 72 hr by ELISA . Where stated , neutralizing anti-PD-1 antibody ( RMP1-14 , rat IgG2a ) , or rat IgG ( Sigma ) were added to cultures at 10 µg/ml . The RMP1-14 antibody was a gift from Dr Hideo Yagata ( Juntendo University , Japan ) . Cells were stained using the following Abs and isotype controls ( all from eBioscience , Hatfield , UK , except where stated ) ; anti-CD4-AF700 ( Invitrogen ) , anti-CD4-eFluor450 , anti-CD8- ( APC/PE ) , anti-CD45 . 1- ( FITC/PE ) , anti-CD90 . 1-FITC , anti-CD11b-APC , anti-CD11c-PE-Cy7 , anti-PDCA-1-eFluor450 , anti-PD-1-PE , anti-CD162-PE , anti-CD80 biotin , anti-CD86 PE , anti-PD-L1 APC , anti-PD-L2 APC , anti-OX-40L biotin , andti-CD40 PE , anti-IFN-γ-APC , anti-GM-CSF-PE ( Becton Dickinson ) , anti-Ki-67-PerCP-eFluor710 , anti-Foxp3-eFlour450 , Streptavidin APC , rat IgG1- ( FITC/APC/PerCPCy5 . 5 ) , rat IgG2a-PE , Rat IgG2a-PerCP-eFluor710 , rat IgG2b- ( PE/biotin ) , Armenian hamster IgG-PE . Active PSGL-1 levels were detected by staining cells with a P-Selectin-human IgG fusion protein ( BD ) and anti-human IgG-PE . Flow cytometric data were acquired using a BD LSRFortessa cell analyzer ( Becton Dickinson ) and data analysed using FlowJo software ( Treestar version 3 . 2 . 1 , Ashland , OR , USA ) . For intracellular staining in response to peptide , cells were re-suspended at 8 × 106/ml in the presence or absence of 20 μM Ac1-9 , or pOVA , as appropriate . After overnight culture , 1 μl/ml of brefeldin A ( eBioscience , 1000× stock ) was added for the last four hours of culture . Cells were washed once in FACS buffer ( PBS , 2% FCS , 0 . 01% NaN3 ) and surface stained prior to processing for intracellular staining using proprietary buffers according to the manufacturer's instructions ( e-bioscience for transcription factor staining or Becton Dickinson for cytokine staining ) . After incubation in fix/perm buffers , cells were stained for intracellular antigens . Detection of pSTAT5 was performed as previously described ( Leech et al . , 2013 ) following 12 hr culture in the presence of 20 μM Ac1-9 . 4 × 106 Tg4 cells were transferred to mice 1 day after PBS/PIT . Spleens were isolated 48 hr later and embedded and frozen in OCT ( Cellpath , Newtown , UK ) . 6 µm thick sections were fixed in ice-cold acetone . Non-specific binding and endogenous biotin were blocked with 3% BSA ( Sigma ) and an avidin-biotin blocking kit ( Vector Laboratories , Peterborough , UK ) respectively . Tissue sections were stained with hamster anti-CD11c-biotin ( Biolegend , San Diego , CA , USA ) , rat anti-CD4-FITC and mouse anti-CD45 . 1-APC ( eBioscience ) . Biotinylated antibodies were detected with streptavidin-Alexa Fluor 555 ( Invitrogen ) . Sections were mounted in Permafluor mounting medium ( Thermo Scientific , Hemel Hempstead , UK ) . To determine specific binding , secondary antibodies alone or isotype controls ( IgG-Biotin [Biolegend] , IgG2a-FITC , IgG2a-APC [eBioscience] ) were used . Images were acquired with a Leica confocal laser scanning microscope SP5 and LAS AF software ( Leica , Wetzlar , Germany ) and processed with ImageJ/Fiji software ( NIH , Bethesda , MD , USA ) . Genomic DNA was isolated from frozen cell pellets of CD4+ T cells using the DNeasy blood and tissue kit ( Qiagen , Crawley , UK ) according to manufacturer's instructions , and ≤1 µg of DNA was subjected to bisulfite conversion using the Epi Tect Bisulfite Kit ( Qiagen ) . Primers for Pyrosequencing ( Eurofins MWG Operon , Ebersberg , Germany ) were designed for conserved regions C and B ( CR-C and CR-B ) ( Table 1 ) within the Pdcd1 ( PD-1 encoding ) gene using PyroMark Assay Design 2 . 0 software ( Qiagen ) . Pyrosequencing was performed using PyroMark reagents with PyroMark Q24 instrumentation , and data were analysed using PyroMark Q24 software 2 . 0 ( all Qiagen ) . Background non-conversion levels were between 2–3% . For bisulfite sequencing , genomic DNA was subjected to bisulphite conversion using the EZ DNA Methylation Gold Kit ( Zymo Research , Irvine , CA , USA ) . Bisulfite sequencing was conducted as previously described ( Hackett et al . , 2012; Reddington et al . , 2013 ) . Primers specified in Table 2 were used to amplify specific regions of interest with Platinum Taq ( Invitrogen ) . A single band was excised , gel extracted , and cloned into pGEM-T-easy ( Promega , Madison , WI , USA ) for sequencing . 10 . 7554/eLife . 03416 . 021Table 1 . Primer sequences for pyrosequencing analysisDOI: http://dx . doi . org/10 . 7554/eLife . 03416 . 021PDCD1 regionAssayCpGPCR primers 5′-3′Sequencing primer 5′-3′Product length ( bp ) CR-C11 and 2F: AGGTATAAAGGAGGTTTTGTAATAGTGAGGTTTTGTAATAGTTAGG186R: CCTCAACCACCCAAATTCAAA-BIO23 , 4 , 5F: TGGGTGGTTGAGGTAGTTGTTGAGGTAGTTGTTAGAT253R: CACCTCACCTCCTACTTATCTCT-BIO36F: TGTTTATTTTAGGGTGGTGAGATTTATGTTAGGTATTATGTATGTATATAAG221R: TAAAAACCCACCTCACCTCCTACTT-BIOCR-B18 , 9 , 10 , 11F: AAAGGAAGAAAAGTTTTAAGAGAAAGTAAGCTATCCCACATACTCC167R: ACCCAACTATCCCACATACT-BIO212 , 13 , 14 , 15F: GGGTTTTTGTTTTTTAGTAGTAAAGGATTAATTAAGGTATAGTTTAGGGTA164R: AAAACCAAACTCTTATCCCTTTAAAA-BIO316 and 17F: GGTAGGGGAGGGTTTAGTGTTTTGGGAGTTAAGG193R: TCCTCTCCATTTCTAACCCCTCTTATA-BIO47F: AGGGTAGTAGAGTTAGTAAATTTAAGATAAGTAGAGAAAATAGTGAGAT103R: CTCTTAAAACTTTTCTTCCTTTCAAAATAC-BIO10 . 7554/eLife . 03416 . 022Table 2 . Primer sequences for bisulfite sequencing and RT-PCRDOI: http://dx . doi . org/10 . 7554/eLife . 03416 . 022RegionAssayCpGPCR primers 5′-3′Product length ( bp ) CR-C*0–4F: ATAAAGGTATAAAGGAGGTTTTG363R: CCTAACTAACTAATCATTTCTCR-C ( nested ) *1–4F: GAGGGGATTTTTTTAGTTTTTGT289R: AATTTAATCATTCTACTCTCTCR-B*7–14F: GAAAGGAAGAAAAGTTTTAAG332R: AAACTAAAACCAAACTCTTATCCCR-B ( nested ) *7–14F: GTTTTTTTGAATTTATAGGGGTG276R: ACTCTTATCCCTTTAAAAAATCR-C†13–5F: AGTTGCCAGATGGTTTCCAG154R: CTGGGGCATTCTGATGATTTCR-B†110–14F: CGGGTCCTAGGAAATGTTCA236R: GCCAGACTCTTGTCCCTTTG27–10F: TACAGGGGTGTCTGGAGAGG156R: ATGCCCTGAGCTATGCCTTAGAPDH†F: CCACTCCCCTTCCCAGTT147R: CCTATAAATACGGACTGC*Primer sequences for bisulfite sequencing . †Primer sequences for RT-PCR . Genomic DNA was extracted as above , and 1 μg of DNA was sonicated ( Bioruptor , Diagenode , Liege , Belgium ) to produce fragments 200–500 bp ( mean 300 bp ) in length . Enrichment of 5hmC DNA was carried out using the Active Motif Hydroxymethyl Collector kit ( Active Motif , La Hulpe , Belgium ) according to manufacturer's instructions . Cross-linked ChIP was conducted essentially as previously described ( Hackett et al . , 2012 ) using 1–3 × 106 cells per IP . In brief; after fixation in 1% formaldehyde , cells were lysed and chromatin was sonicated ( Bioruptor , Diagenode ) to produce fragment sizes 200–1000 bp in length . Chromatin was pre-cleared by incubation with Protein A magnetic beads ( Invitrogen ) , before incubation with antibody bound beads . Antibodies used were anti-H3K27me3 , anti-H3K4me3 and the isotype control rabbit IgG ( Merck Millipore , Billerica , MA , USA and Santa Cruz Biotechnology , Dallas , TX , USA , respectively ) . Beads were washed , DNA was eluted and cross-linking was reversed before DNA was purified using the QIAquick PCR purification kit according to manufacturer's instructions ( Qiagen ) . Eluted DNA was quantified using real-time PCR . qPCR was conducted on DNA obtained after ChIP or 5hmC enrichment using SYBR Green Real-time PCR master mix ( Roche Diagnostics , Burgess Hill , UK ) and the primers listed in Table 2 . Reactions were performed on the Roche LightCycler 480 . For ChIP analysis , fold enrichment of specific enrichment over background was calculated using values normalized to input . For 5hmC analysis , relative enrichment was calculated to a known amount of input DNA and all data normalised to a negative control region at the gapdh promoter . Levels of 5hmC were tested over one region at the CR-C and two at the CR-B which were combined to generate an average value of 5hmC enrichment . For detection of Tet1 , Tet2 and Tet3 mRNA , total RNA was isolated using the RNeasy kit and cDNA was generated using the QuantiTect Reverse Transcription kit ( both Qiagen ) according to manufacturer's instructions . Quantification of mRNA was conducted by qPCR using pre-designed TaqMan assays from Applied Biosystems ( Life technologies , Catalogue no's: Mm01169087_m1 , Mm00524395_m1 , Mm00805756_m1 ) as previously described ( Drake et al . , 2010 ) . Results were normalized to the expression of the housekeeping gene GAPDH ( Applied Biosystems gene expression assay Mm99999915_g1 ) . Statistical analysis of results was performed using the Mann–Whitney U test or the Kruskal–Wallis with Dunn's post-hoc test ( p values: *<0 . 05 , **<0 . 01 , ***<0 . 005 ) .
Clinically effective antigen-based immunotherapy must silence antigen-experienced effector T cells ( Teff ) driving ongoing immune pathology . Using CD4+ autoimmune Teff cells , we demonstrate that peptide immunotherapy ( PIT ) is strictly dependent upon sustained T cell expression of the co-inhibitory molecule PD-1 . We found high levels of 5-hydroxymethylcytosine ( 5hmC ) at the PD-1 ( Pdcd1 ) promoter of non-tolerant T cells . 5hmC was lost in response to PIT , with DNA hypomethylation of the promoter . We identified dynamic changes in expression of the genes encoding the Ten-Eleven-Translocation ( TET ) proteins that are associated with the oxidative conversion 5-methylcytosine and 5hmC , during cytosine demethylation . We describe a model whereby promoter demethylation requires the co-incident expression of permissive histone modifications at the Pdcd1 promoter together with TET availability . This combination was only seen in tolerant Teff cells following PIT , but not in Teff that transiently express PD-1 . Epigenetic changes at the Pdcd1 locus therefore determine the tolerizing potential of TCR-ligation .
The immune system protects the body from dangerous microbes and removes damaged cells . However , in some cases , the immune system can malfunction and attack healthy tissues , which can lead to type-1 diabetes , multiple sclerosis , and other autoimmune diseases . Many of the current treatments for these disorders suppress the immune system , which can make the individuals more susceptible to infections . It may be possible to treat autoimmune diseases using small pieces of protein—called peptides—that are based on proteins found on the cells that the immune system attacks by mistake . This strategy would target the specific immune cells that are malfunctioning , but allow the rest of the immune system to continue to work as normal . Peptide-based therapies for autoimmune diseases are currently being tested in clinical trials , and although the results look promising , it is not known precisely how they work . McPherson et al . used mice that develop a disease similar to multiple sclerosis because some of their immune cells , known as effector T cells , attack a protein found in the mouse brain called MBP . The mice were treated with a peptide based on part of MBP , which prevented them from developing the autoimmune disease . The success of the peptide therapy depended on the T cells producing large amounts of a protein called PD-1 . This protein stops the T-cells from activating immune responses when they detect the MBP protein . The gene that makes PD-1 can have a methyl-tag—a chemical modification to DNA—which alters how much PD-1 is made in the T cells . When the gene has this methyl-tag , it can only be switched on for a short time to make a small amount of PD-1 , which helps to control the immune responses activated by the T cell . However , when the methyl-tag was removed as a result of the peptide therapy the gene could be switched on for much longer , so that much more PD-1 was produced . This work helps us to understand how peptide therapy works and should improve the chances of using this therapy to successfully treat patients with autoimmune diseases .