Regulation of CD8� regulatory T cells: Interruption ofthe NKG2A–Qa-1 interaction allows robust suppressiveactivity and resolution of autoimmune diseaseLinrong Lu1, Hye-Jung Kim, Miriam B. F. Werneck, and Harvey Cantor2
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Department of Pathology, Harvard Medical School, Boston, MA 02115
Contributed by Harvey Cantor, October 16, 2008 (sent for review September 12, 2008)
Regulation of autoreactive CD4 T cells is essential to maintain self-tolerance and prevent autoimmune disease. Although CD8 T regula-tory (Treg) cells that recognize self-peptides restricted by Qa-1 (HLA-Ein humans) inhibit autoreactive CD4 cells and attenuate experimentalautoimmune encephalomyelitis (EAE), the mechanism of this inter-action is unclear. We generated Qa-1 mutant knock-in mice thatimpair Qa-1 binding to the T cell receptor (TCR) and CD94/NKG2Areceptors. Analysis of these mice showed that TCR-dependent recog-nition of Qa-1–peptide complexes on target CD4 cells is essential forsuppression by CD8 Treg cells. Further analysis revealed that geneticdisruption of the Qa-1–CD94/NKG2A interaction unleashes robustCD8 Treg cell activity that completely abolishes development of EAE.
autoimmunity � CD8 Treg � suppression
There is considerable evidence that a regulatory sublineage ofCD4 cells can inhibit the development of autoimmune disease
in several murine models (1). However, less attention has beengiven to the potential contribution of regulatory sublineages of CD8cells to the prevention and treatment of autoimmunity. A subpopula-tion of CD8 cells that recognizes the MHC class Ib product Qa-1(HLA-E in man) suppresses the development and relapse of experi-mental autoimmune encephalomyelitis (EAE) in a mouse model ofmultiple sclerosis (MS) (2, 3), and mice lacking this CD8 subpopulationdevelop exaggerated immune responses to self- antigen (4).
Analysis of Qa-1–deficient mice has not fully clarified thecontribution of Qa-1 to immunologic suppression due to the abilityof the Qa-1 molecule to interact with two distinct receptors. First,engagement of the T cell receptor (TCR) by Qa-1–peptide com-plexes can lead to activation and expansion of antigen-specific CD8cells. Second, engagement of the CD94/NKG2A receptor expressedby CD8 cells and natural killer (NK) cells by Qa-1/Qdm peptideligands attenuates the activities of these cells (5–7). Thus, theimmune response phenotype of Qa-1-deficient mice reflects thesefunctionally distinct interactions. Diminished interactions betweenQa-1-restricted CD8 cells and Qa-1-deficient CD4 cells results inenhanced CD4 responses, whereas increased NK cell lysis ofactivated CD4 cells reflects a loss of the inhibitory NKG2A–Qa-1/Qdm interaction.
We isolated Qa-1-dependent engagement of the TCR andNKG2A for analysis and exploited the fact that Qa-1-dependentbinding to each receptor used distinct Qa-1 contact residues. Wethus generated knock-in mice that express a Qa-1 amino acidexchange mutation (Qa-1 D227K) that disrupts binding of Qa-1 tothe CD8 coreceptor (8). Cells that express this Qa-1 D227K pointmutation fail to present Qa-1-restricted peptides to CD8 cells butretain the ability to engage NKG2A receptors on CD8 cells and NKcells. A second Qa-1 knock-in strain (Qa-1 R72A) expresses a Qa-1aa exchange mutation (R72A) that fails to bind to NKG2A recep-tors on NK cells and CD8 cells but spares Qa-1-dependent peptidepresentation to the TCR (7). Expression of this Qa-1 aa exchangemutant by CD4 cells renders them vulnerable to NK and CD8 lysis.These two Qa-1 point mutations were backcrossed to C57BL/6 micefor analysis of CD8 T regulatory (Treg) cell activity.
We report that engagement of Qa-1 by the CD8 coreceptor isessential for Qa-1-restricted Treg activity, as indicated by findingsthat Qa-1 D227K knock-in mice fail to develop regulatory CD8activity and display enhanced proteolipid protein (PLP)-inducedEAE. Analysis of Qa-1 R72A knock-in mice revealed that geneticdisruption of the NKG2A–Qa-1 interaction releases the brakes onCD8-dependent suppressive activity, allowing the development ofrobust CD8� Treg activity, which results in complete resistance tothe development of EAE.
Results and DiscussionAnalysis of B6.Qa-1 D227K and R72A Knock-In Mice. A genomic 4-KbQa-1 fragment containing a D3K amino acid exchange mutationat position 227 after site-directed mutagenesis (Fig. 1A) was clonedinto a replacement vector and transfected into the TC1 ES cell line.After injection of positive homologous recombinant clones intoblastocysts to produce germline chimeras (Fig. 1B), and deletion ofthe Neor gene after crossing to B6-EII�-CRE mice, progeny werebackcrossed to C57BL/6 (B6) for seven generations and inter-crossed to produce homozygous B6.Qa-1 D227K mutant mice.Expression of cell surface Qa-1 by activated (ConA-stimulated)CD4 cells from B6.Qa-1 D227K knock-in mice was indistinguish-able from Qa-1 WT T cells (Fig. 1C). Expression of the Qa-1D227K mutation by L cells (Fig. 1D, Left) or activated CD4 cells(Fig. 1D, Right) failed to target these cells for lysis by Qa-1-restrictedcytolytic T cells (CTLs). Qa-1/Qdm-dependent resistance of acti-vated B6.Qa-1 D227K CD4 cells to lysis by NKG2A� NK cells wasunimpaired in vitro (Fig. 1E, Left) and in vivo, as judged byhomeostatic expansion in Rag-2�/� hosts (Fig. 1E, Right).
Cells from B6 Qa-1 knock-in mice that expressed the Qa-1 R72Amutation were unable to bind to NKG2A and, as a consequence,displayed increased susceptibility to NK lysis in vitro (Fig. 1E, Left)and failed to expand in Rag-2�/� hosts in vivo (Fig. 1E, Right).Taken together, these observations indicate that the interaction ofQa-1 with the TCR–CD8 complex can be experimentally separatedfrom its interaction with NKG2A using B6.Qa-1 D227K andB6.Qa-1 R72A knock-in mice.
We next monitored the effect of the Qa-1 D227K point mutationon the inhibitory interaction between CD8 Treg cells and CD4 Thcells (4). CD8 cells obtained from B6 mice immunized withirradiated activated OT-2 CD4 T cells 2 weeks earlier were trans-ferred along with OT-2 CD4 cells into Rag2�/�Prf1�/� hostsimmunized with OT-2 peptide. Expansion of Qa-1 WT and Qa-1R72A OT2� CD4 cells was substantially inhibited by CD8 cells,whereas expansion of D227K OT-2 cells was unimpaired (Fig. 1F).These findings indicate that the Qa-1-restricted suppressive inter-
Author contributions: L.L. and H.C. designed research; L.L., H.-J.K., and M.B.W. performedresearch; L.L. contributed new reagents/analytic tools; L.L., H.-J.K., M.B.W., and H.C.analyzed data; and L.L. and H.C. wrote the paper.
The authors declare no conflict of interest.
1Current affiliation: Institute of Immunology, Zhejiang University, Zhangzhou, P.R. China.
2To whom correspondence should be addressed. E-mail: harvey�[email protected].
action between CD8 and CD4 cells requires CD8 coreceptorbinding to the Qa-1 molecule on CD4 cells, but not binding to theNKG2A receptor.
Enhanced Susceptibility of Qa-1 D227K Knock-In Mice to EAE. As notedpreviously (4), preimmunization of mice with myelin oligodendro-cyte glycoprotein (MOG) peptide before induction of EAE withMOG/complete Freund’s adjuvant (CFA) and pertussis toxin in-duces CD8� Treg cells, which inhibit the development of EAE (Fig.2A). In contrast, preimmunization of B6.Qa-1 D227K mice withMOG peptide is followed by robust disease development (Fig. 2A).Moreover, CD4 cells from B6.Qa-1 D227K mice, but not from B6control littermates, mediate vigorous anti-MOG recall responses, asindicated by production of the IFN-� and IL-17 proinflammatorycytokines (Fig. 2B). Enhanced IFN-�/IL-17 responses were notaccompanied by diminished levels of inhibitory cytokines, includingIL-10 and IL-4, which were not detectable in these supernatants(not shown).
We then investigated whether the Qa-1 D227K mutation pre-vented the development of CD8 Treg cells to a second self-antigen,
PLP. Preimmunization of B6 mice with PLP peptide (withoutpertussis toxin) induces PLP-specific CD8� Treg cells, which inhibitEAE on a subsequent challenge with PLP/CFA and pertussis toxin(4). In contrast, preimmunization of B6.Qa-1 D227K littermateswith PLP/CFA failed to prevent the development of severe EAE onchallenge with PLP/CFA plus pertussis toxin (Fig. 2C). Loss ofprotection by Qa-1 D227K knock-in mice was associated withsubstantially increased anti-PLP IFN-� responses by CD4 cells onchallenge with PLP (Fig. 2D). In summary, expression of the Qa-1D227K point mutation that prevents Qa-1-restricted recognition byCD8 cells results in markedly enhanced CD4 response to PLPself-antigens and the consequent development of EAE.
Suppression by CD8� Treg Cells Is Attenuated by Engagement ofNKG2A. In contrast to B6 mice, which express the Qa-1 D227Kmutation, B6.Qa-1 R72A knock-in mice were completely protectedfrom induction of EAE by preimmunization with PLP. Protectionwas accompanied by markedly reduced anti-PLP IFN-� responseson restimulation of CD4 cells in vitro (Fig. 2D), suggesting thatengagement of Qa-1/Qdm on CD4 cells by NKG2A on CD8 Tregcells impairs their suppressive activity.
Fig. 1. GenerationandanalysisofQa-1D227Kmutantknock-inmice. (A)Qa-1genomic locusandtargetingstrategy.Boxesrepresentexons;exon4(graybox) indicatesthe mutation site. The loxP sites are represented by black triangles. TK, thymidine kinase gene; neor, neomycin-resistance gene. (B) Southern blot analysis of ES cellgenomic DNA. The upper band (12.5 kb) corresponds to the WT allele; the lower band (7.2 kb) represents the knock-in allele. Right: PCR genotyping of knock-in mice.WT (280 bp) and knock-in (430 bp) products represent the addition of base pairs from the remaining loxP site and surrounding sequence (WT/WT, Qa-1 WT; DK/DK,homozygous mutant knock-in mice; WT/DK, heterozygous). (C) ConA-induced Qa-1 expression of WT and D227K mutant. Splenocytes from littermates (Qa-1-deficient[KO], Qa-1 WT [WT], and Qa-1 D227K knock-in [D227K]) were individually stimulated with ConA for 40 h and analyzed for surface Qa-1 expression by FACS analysisusing anti-Qa-1 Ab (BD Bioscience). (D) L cells transfected with GFP, WT Qa-1 or Qa-1 D227K, and ConA blasts from WT or Qa-1 D227K mice were used as targets in aCTL assay by CD8 cells from B6.Tla mice immunized with Qa-1 ConA blasts. Percent lysis is shown at the indicated E:T ratios. (E) Left: NK cell susceptibility of Qa-1 R72Amutant CD4 cells. CD4� T cells from Qa-1 WT, Qa-1-deficient, Qa-1 D227K, and Qa-1-R72A mutant mice were activated by ConA for 48 h; labeled with 51Cr; and usedas targets for IL-2-activated NKG2A� NK cells in a standard 4-h killing assay. Percent lysis at an E:T ratio is shown. Data shown represents mean � SD. (n � 3). Right:Homeostatic expansion of Qa-1 mutant CD4 cells. A total of 106 CD4� T cells isolated from Qa-1 WT, Qa-1-deficient, Qa-1 D227K, and Qa-1 R72A mice were transferredinto syngeneic Rag2�/� hosts. Fourteen days later, CD4� T cells from spleen and lymph nodes were enumerated. Data, shown as mean � SD, represent one of twoindependent experiments (n � 3). (F) CD4 T cells from OT-2 TCR-transgenic B6 mice activated with ConA were used to vaccinate B6 mice. Fourteen days later, purifiedCD8 T cells (1.5 �106) from OT-2-immunized mice were transferred with OT-2 CD4 T cells (from Qa-1 WT, Qa-1�/�, Qa-1 D227K, and Qa-1 R72A; 1 � 106) intoRag2�/�Prf1�/� mice before challenge with OVA peptide (50 �g) in CFA. Expansion of OT-2 T cells was quantitated by enumerating OT-2 cells in both lymph nodes andspleen 2 weeks after transfer. Data are representative of two independent experiments (n � 3).
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To directly test this hypothesis, we investigated the susceptibilityof MOG-immune CD4 cells expressing the R72A mutation to Qa-1-restricted inhibition by CD8� Treg cells in adoptive Rag2�/�Prf1�/�
hosts. Because activated CD4 T cells that fail to engage inhibitoryNKG2A receptors on NK cells are also susceptible to NK cell lysis (7),we eliminated NK activity using adoptive Rag2�/�Prf1�/� hosts. Co-transfer of CD8 cells with MOG-immune Qa-1 WT CD4 cells intoRag2�/�Prf1�/� hosts resulted in modest inhibition of EAE (Fig. 3A,Left). In contrast, transfer of EAE by MOG-immune CD4 cells fromR72A donors was completely abolished by cotransfer of CD8 cells,despite the fact that R72A CD4 cells alone induced EAE levels thatwere at least as robust as those induced by CD4 cells from Qa-1 WTlittermate donors (Fig. 3B, Left). Analysis of the in vitro recall responseto MOG at 3 weeks revealed that cotransfer of CD8 cells with R72ACD4cellsvirtuallyabolishedtheanti-MOGrecall response,as indicatedby IFN-� secretion (Fig. 3A and B, Right).
A more stringent test of the affect of the Qa-1 R72A mutationon the susceptibility of CD4 cells to CD8 Treg cell activity was basedon an analysis of the ability of CD8 cells to inhibit CD4 cells thatexpress the MOG-specific 2D2 TCR transgene. Transfer of 2D2�
CD4 cells into Rag2�/� Prf1�/� hosts initiates a progressive andlethal form of EAE that results in death from fulminant diseasewithin 14–16 days after transfer (Fig. 3C). If CD8-dependentsuppression of 2D2� CD4 cells is normally attenuated by aninhibitory interaction between Qa-1/Qdm expressed by CD4 targetcells and NKG2A receptors expressed by CD8 Treg cells, thengenetic disruption of this interaction might be expected to suppressvirulent EAE. Transfer of 106 2D2� CD4 cells (five times the lethaldose) from B6 or B6 Qa-1 R72A donors into Rag2�/�Prf1�/� hostsprovoked lethal EAE by day 15 (Fig. 3C, Left); however, cotransferof CD8� Treg cells completely abrogated disease induced by R72ACD4 cells, whereas cotransfer of CD8� Treg cells had no effect onQa-1 WT CD4 cells (Fig. 3C, Right).
We next determined whether abolition of EAE development bycotransfer of CD8 cells was associated with suppression of 2D2�
CD4 T cell expansion in Rag2�/�Prf1�/� hosts. Expansion of R72A2D2 CD4 T cells was almost completely (� 95%) suppressed bycotransfer of CD8 cells, whereas expansion of Qa-1 WT 2D2 CD4
T cells was reduced only slightly by MOG-immune CD8 T cells (Fig.3D). Moreover, a residual amount (5%) of Qa-1 R72A 2D2� CD4cells displayed almost no response to MOG in vitro (Fig. 3E). Thus,failure of EAE development is attributed to CD8-dependent sup-pression of the anti-MOG response of 2D2� CD4 cells bearing theMOG-specific TCR.
Interruption of the Qa-1–NKG2A Interaction Unmasks Potent CD8�
Treg Cell Activity In Vitro. The analysis summarized in Fig. 3 suggeststhat CD4 T cells expressing a Qa-1 point mutation that impairsengagement of NKG2A expressed by CD8� Treg cells releases thebrakes on suppressive activity inflicted by CD8� Treg cells. Analysisof the interaction between Qa-1 R72A mutant CD4 cells and CD8Treg cells in vitro revealed that the MOG-specific response of Qa-1R72A 2D2� CD4 T cells was highly susceptible to dose-dependentsuppression by CD8 Treg cells in vitro, as indicated by diminishedproliferation and IL-2 secretion. In contrast, CD4 cells bearing the Qa-1D227K mutation were fully resistant to CD8 Treg cell activity (Fig. 4A).The dependence of CD8 Treg cell activity on Qa-1 recognition wasconfirmed by the finding that anti-Qa-1 Ab blocked CD8-dependentsuppression of the CD4 cell IL-2 response (Fig. 4B).
Perforin Expression Contributes to Suppressive Activity. We testedthe contribution of perforin and Fas ligand to Treg-mediatedsuppression. CD8 T cells from immunized perforin-deficient(Prf1�/�) or Fas ligand-deficient generalized lymphoproliferativedisease (FasL�/�) mice were cotransferred with Qa-1 R72A 2D2CD4 T cells. Expansion of R72A 2D2 CD4 T cells was inhibited byFasL�/� CD8 cells but not by Prf1�/� CD8 cells.
The requirement for perforin (but not Fas ligand) for CD8� Tregcell activity also was noted in vitro. CD8 T cells from immunizedPrf1�/� mice were unable to inhibit the response of R72A 2D2�
CD4 cells, in contrast to CD8 T cells from FasL�/� mice (Fig. 5B).Although Qa-1 R72A CD4 cells may be lysed by CD8 cells fromimmunized mice (Fig. 5C), these observations do not rule out anonlytic mechanism of suppression. Indeed, a cardinal feature of‘‘suppressed’’ CD4 cells is their failure to express effector (Th1)cytokines, including IFN-�, on stimulation with antigen.
Fig. 2. MOG-induced EAE in Qa-1 D227K mice. (A) EAE was induced into WT (n � 5) and D227K (n � 5) mice by injecting 100 �g of MOG in CFA (50 ng of MTb) withpertussis toxin on days 0 and 2. The mice were reimmunized with 100 �g of MOG in CFA on day 7. EAE disease score was determined as described in Methods. (B) Recallresponse.AttheendoftheEAEexperiments, spleenandlymphnodecellswerepooledfromthreemice ineachgroup,andthecellswererestimulatedwiththe indicateddoses of MOG peptides in vitro. IFN-� and IL-17 production was measured by ELISA using 48 h culture supernatants. Data shown are representative of two independentexperiments. (C and D) Effects of preimmunization in PLP-induced EAE development. B6 Qa-1 WT and B6 Qa-1 D227K mice were preimmunized with PLP peptide (20�g) in CFA and boosted 10 days later with PLP peptide in CFA plus pertussis toxin. Clinical score (C, Left) and incidence (C, Right) were determined after the boost (time,horizontal axes; 5–8 mice per group). (D) Lymph nodes and spleen were harvested from mice at the end of the EAE experiments, and single-cell suspensions wereprepared; 4 � 105 pooled draining lymph node and splenic cells were restimulated in vitro with the indicated concentrations of PLP peptide. Production of IFN-� wasmeasured 48 h after culture. Data shown represent two independent experiments.
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Although the regulatory lineage of CD4� T cells has been theobject of intense study, regulatory cells within the CD8� lineagehave received much less attention. Analyses of normal and Qa-1-deficient mice have defined a subpopulation of CD8 cells thatmediate Treg activity through recognition of the class Ib MHCmolecule Qa-1 expressed on activated CD4 cells. The interactionsbetween CD8� Treg cells and target CD4 cells that regulate thisprocess are not well understood, however. The present studyestablishes that this inhibitory interaction requires CD8 corecog-nition of peptides complexed to the Qa-1 MHC molecule expressedby activated target CD4 cells. These findings also provide newinsight into the molecular constraints that dampen CD8� Treg cellactivity.
We find that Qa-1/Qdm engagement of CD94/NKG2A on CD8�
Treg cells transmits an inhibitory signal that attenuates CD8 Tregsuppressive activity. The CD94/NKG2A receptors belong to theinhibitory NK receptor family (iNKRs) that inhibits cellular acti-vation through dephosphorylation of signaling molecules. Althoughthe CD94 chain can pair with other members of the NKG2 family,including NKG2C and NKG2E (9), the high levels of expressionand strong binding affinity of CD94/NKG2A for Qa-1/Qdm dom-inate binding. As a result, engagement of CD94/NKG2A receptorsexpressed by activated CD8 cells during viral infection down-
regulates cytotoxicity and production of proinflammatory cyto-kines (6, 10). Although only a small proportion of CD8 cells expressNKG2A according to immunofluorescence with available antibod-ies, most CD8 cells specifically bind Qa-1/Qdm tetramers and thismay account for the substantial affect of NKG2A ligation (10).
Autoreactive CD4 cells that express the Qa-1 R72A pointmutation fail to transmit an inhibitory signal through NKG2A toCD8� Treg cells. As a result, these CD4 cells received maximalinhibitory signals from CD8 Treg cells and failed to induce EAE.These findings suggest that releasing the brakes on CD8 Tregactivity by blockade of NKG2A with Ab may represent a new CD8�
Treg-based approach to ameliorating autoimmune disease.The timing of CD8�-dependent inhibitory responses can be
contrasted with that of naturally occurring CD4�CD25� Treg cells,which interrupt expansion of self-reactive T cells during the initialstages of primary responses. Regulatory CD8� T cells arise later inthe immune response and become apparent experimentally onlyafter restimulation by antigen (11). The development of Qa-1-restricted Treg activity more closely resembles the kinetics ofCD8�-dependent memory/effector activity rather thanCD4�CD25� regulatory activity. CD8�-dependent suppressiveand memory/effector responses both require primary immuniza-tion to generate TCR-dependent lysis or Qa-1-restricted inhibitionof target cells (4, 12).
Fig. 3. MOG-induced EAE development in Qa-1 R72A mice. Left: CD4 cells were purified from MOG-immunized EAE mice [WT (A) and R72A (B)] and transferred intoRag2�/�Prf1�/� hosts with or without CD8 cells from the EAE mice. Mice were then immunized with MOG peptide with pertussis toxin, and the development of EAEwas monitored daily. Data shown represent two independent experiments (n � 5). Right: Draining lymph nodes and spleens were collected 35 days after EAE induction.Single-cell suspensions were pooled and stimulated with the indicated concentrations of MOG peptide. IFN secretion was measured by ELISA after 48 h of culture. Toensure complete removal of NK cells from donor T cell populations, NK cells were depleted as described in Methods. (C–E) 2D2-induced EAE. (C) 2D2 CD4� T cells (106)enriched from Qa-1 WT and R72A 2D2 TCR-transgenic mice were transferred into syngeneic Rag2�/�Prf1�/� hosts (n � 5) with or without CD8 Treg cells generated asdescribed in Methods. Hosts were then immunized with 10 �g of MOG/CFA and 200 �g of pertussis toxin (on days 0 and 2). Development of EAE was scored daily asdescribed in Methods. (D) In vivo suppression of R72A 2D2 expansion by CD8 regulatory cells. 2D2 CD4� T cells (106) from Qa-1 WT or R72A mice were transferred intosyngeneic Rag2�/�Prf1�/� hosts (n � 3) with or without CD8 Treg cells generated as described in Methods. Hosts were then immunized with 10 �g of MOG/CFA. 2D2CD4 T cells in the draining lymph nodes and spleens were enumerated after 14 days. Data are shown as mean � SD (n � 3). (E) Single-cell suspensions were preparedfrom draining lymph nodes collected as described above and pooled according to each group. The lymph node cells were stimulated with the indicated concentrationsof MOG peptide in the presence of irradiated splenocytes as antigen-presenting cells. IL-2 secretion was measured by ELISA 48 h after culture.
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These data also suggest that the relative ratio of the two classesof Qa-1 peptide ligand— Qdm peptide versus non-Qdm (e.g.,HSP60) peptide—on the surface of activated CD4 cells maydetermine the susceptibility of these CD4� T cells to suppression byCD8� Treg cells. According to this view, engagement of NKG2Aon CD8� Treg cells by the Qdm/Qa-1 ligand on target CD4 cellsinhibits Treg activity, whereas engagement of the TCR expressed byCD8� Treg cells by Qa-1 peptide ligands, such as HSP60 peptides(13), activates suppressive activity. Because the majority of murineCD8� T cells bind to Qa-1/Qdm tetramer, differential expression ofQa-1/Qdm and Qa-1/HSP60 peptide ligands on activated Qa-1 CD4target cells may determine the cell’s fate.
Although MS is characterized by a clinical sequence of exacer-bations followed by remissions, the clinical course is generallyprogressive. Analysis of disease in a murine model of MS (i.e.,EAE) has begun to define the immunologic mechanisms respon-sible for this process. Immunologic susceptibility to disease anddisease remission depends in part on inhibition of pathogenicautoreactive CD4 T cells by CD8� Treg cells, resulting in decreasedtissue destruction and inhibition of disease progression (4, 7, 14, 15).These findings provide the basis for development of new therapeu-tic approaches to autoimmune diseases, including MS. Althoughcurrent approaches have generated CD8� Treg cell activity beforedisease induction (16, 17), these cells are not potent enough to shutdown disease progression.
Recent analysis of T cells from MS patients also indicates thatCD94/NKG2A inhibitory receptors expressed by CD8 Treg cellsrestrain their HLA-E-restricted suppressive activity, and that theseHLA-E-restricted CD8 Treg cells cloned from patients duringdisease exacerbation express higher levels of CD94/NKG2A com-pared with CD8 cells cloned from MS patients in remission or CD8cells obtained from healthy controls (18). Moreover, Ab-dependentblockade of the HLA-E–CD94/NKG2A interaction enhances lysisof autoreactive CD4 T cell clones by HLA-E-restricted CD8 cellsfrom MS patients (18), consistent with our findings using a murinemodel of MS. Additional studies are currently underway usingcombination therapy of CD8� Treg cells with blocking anti-NKG2A Ab to treat ongoing MOG-induced EAE in B6 mice, in aneffort to increase the therapeutic potential of this approach to MS.
MethodsAnimals and Reagents. C57BL/6 (B6) and Rag2�/�Prf1�/� (perforin) mice werepurchased from Taconic. Qa-1 D227K and Qa-1 R72A mice were generated asdescribed previously and backcrossed onto B6 for eight generations. OT-2 TCR-transgenic mice (provided by H. Ploegh, MIT) and 2D2 TCR-transgenic mice thatrecognize the MOG autoantigen (19) were crossed with Qa-1 D227K and Qa-1R72A mice. Mice were housed in a specific pathogen-free, viral Ab-free animalfacility at the Dana-Farber Cancer Institute. All experiments were done in com-pliance with federal laws and institutional guidelines and were approved by theDFCI Animal Care and Use Committee.
PeptidesMOG35–55(MEVGWYRSPFSRVVHLYRNGK),OVA323–339(ISQAVHAA-HAEINEAGR), and PLP 172–183 (PVYIYFNTWTTC) were synthesized by New EnglandPeptide. Mouse CD4 and CD8 T lymphocyte enrichment kits, ELISA kits for cytokines,and other purified antibodies were purchased from BD Biosciences.
Generation of Qa-1 Mutant Knock-In Mice. A BAC clone containing a 11-kb DNAfragment including the Qa-1 gene was identified and fully mapped. A 4-kbfragment containing exons 1–5 of Qa-1 (from NdeI to AvrII) was cloned into acloning vector, and the mutation of D227 to K was introduced by site-directedmutagenesis. After confirmation by sequencing, the mutated DNA fragment wascloned into the SalI site of pLNTK (20). The 2.3-kb short arm (AvrII to NdeI) wascloned into the XhoI site of pLNTK to complete the replacement vector. Thetargeting vector was linearized by NotI and used for ES cell electroporation. AfterTC1 ES cells were transfected with targeting vector, positively selected recombi-nants were identified by long-range PCR screening and Southern blot analysis.The Neor gene was deleted by crossing germline-transmitted litters to EII�-CREmice, followed by backcrossing to B6 mice for eight generations. HomozygousQa-1-D227K mutant mice were obtained by intercrossing heterozygouslittermates.
NK Cell Purification and NK Cytotoxic Assay. To purify NKG2A� NK cells,splenocytes from Rag2�/� mice were incubated with biotinylated NKG2AB6 mAb(eBioscience), followed by incubation with antibiotin Ab-coated magnetic mi-crobeads (Mytech) before cell separation using magnetic-activated cell sorting.Purified NKG2A� NK cells were cultured in RPMI 1640 complete medium with10% FCS and 1000 U/ml of human recombinant IL-2 (BD Biosciences) for 5 days.Then 106 target cells were labeled with 100 �Ci of Na2(51Cr)O4 for 1 h at 37 °C.After three washes with PBS, 104 target cells were mixed with NK cells at differentE:T ratios and incubated for 4 h. Cell-free supernatants were collected, and theirradioactivity was measured with a Wallac Microbeta counter. The percentage oflysis was calculated by the following formula: (sample release � spontaneousrelease)/(maximum release � spontaneous release) � 100%.
Preimmunization with Peptides and Induction of EAE. To analyze EAE resistance,mice were injected s.c. on the back with 10–25 �g of PLP peptide in CFA withoutpertussis toxin. Fourteen days later, the mice were immunized s.c. with 150 �g ofPLP peptide and 200 �g of killed Mycobacterium tuberculosis (H37ev) in CFA onthe flanks. In addition, 200 ng of pertussis toxin was injected i.p. on days 0 and 2.Mice were monitored daily and scored from 0–5: 0, normal mouse with no signof disease; 1, limp tail; 2, limp tail and partial hind limb weakness; 3, completehind limb paralysis; 4, complete hind limb and partial front limb paralysis; or 5,moribund state or death.
Induction of EAE by MOG Peptide Immunization. For active EAE induction, WTand Qa-1 D227K mice were immunized s.c. at two sites on the flank with a totalof 100 �g of MOG35–55 peptide emulsified in an equal volume of CFA containing1 mg/ml of M. tuberculosis on days 0 and 7. The mice also received i.p. injectionsof 200 ng of pertussis toxin on days 0 and 2 after immunization.
Fig. 4. Susceptibility of Qa-1 R72A 2D2 cells to CD8� Treg cells. CD8 cells werepurified from the draining lymph nodes of R72A 2D2 and CD8 suppressor cellcotransferred mice. 2D2 CD4 T cells were isolated from Qa-1 WT, D227K, andR72A mice and stimulated in vitro with different doses of MOG peptide in thepresence of irradiated splenocytes as antigen-presenting cells with or withoutpurified CD8 suppressor cells. (A) Proliferation of CD4 T cells was measured 60 hlater by 3H-thymidine incorporation. Different CD4:CD8 ratios were used insimilar experiments, and proliferation of CD4 T cells on stimulation with 30 �g ofMOG peptide was measured (Lower). (B) IL-2 secretion was measured by ELISA48 h after culture; relative IL-2 secretion is shown (normalized to no CD8 controlcultures). To confirm specificity of Qa-1-restricted suppression, anti-Qa-1 Ab wasincluded in one group at a concentration of 10 �g/ml. The data shown representthree independent experiments.
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Generation of CD8 Suppressor Cells and In Vivo Suppression Assay. CD4 T cellswere purified and activated in vitro by ConA (1 �g/ml) or peptide antigen for 2days before irradiation (3,000 rads) and injection into B6 mice (2 � 106 cells i.v.).After 14 days, CD8 T cells purified from the spleen were adoptively transferredinto Rag2�/�Prf1�/� mice along with purified target CD4 T cells. Adoptive hostswere challenged with antigen peptide (50 �g) in CFA, and the expansion of CD4T cells and CD8 cells was analyzed 14 days later. CD8� Treg cells also weregenerated after injection of 106 2D2� TCR-transgenic CD4 cells i.v. into B6 mice,followed by immunization with 50 �g of MOG peptide to activate and expand2D2 cells. CD8 cells were purified 14 days later from both draining lymph nodesand spleen and tested for suppressive activity against 2D2 CD4 T cells inRag2�/�Prf1�/� hosts, as described above. CD8 cells expressing suppressive activ-ity against polyclonal MOG-reactive CD4 T cells were purified from draininglymph nodes of MOG-induced EAE mice.
Adoptive CD4 T Cell Transfer and Induction and Assessment of EAE. CD4 T cells(106) from either MOG-immunized mice or 2D2 TCR-transgenic mice were trans-ferred i.v. into Rag2�/�Prf1�/� hosts with or without CD8 suppressor cells (1.5 �106). Because incomplete removal of NK cells from donor T cell populationsobtainedafterCD4orCD8enrichmentcanaffectexpansionofQa-1�/� CD4Tcellsin adoptive hosts (7), we depleted NK cells from donors of CD4 or CD8 cells byinjecting anti-NK1.1 Ab (200 �g/mouse, i.v.) 48 h before harvesting draininglymph nodes and spleen and treated again with anti-NK1.1-coated beads in vitroto ensure depletion of NK cells. EAE was induced by s.c. immunization with MOGpeptide emulsified in CFA supplemented with 4 mg/ml of M. tuberculosis in theflanks.Atotalof150 �gofpeptidewasusedforpolyclonalMOG-immunizedCD4transfer, and only 10 �g of peptide was used for 2D2 CD4 transfer. Mice wereinjected i.p. on days 0 and 2 with 200 ng of pertussis toxin. Clinical assessment ofEAE was performed daily and scored as described above.
In Vitro T Cell Stimulation and Suppression Assay. CD4 T cells were purified fromdraining lymph nodes of immunized mice or TCR-transgenic mice (2 � 104 to 1 �
105 cells/well) and cultured with Ag and irradiated splenocytes from B6 mice (4 �
105 cells/well) in RPMI 1640 complete medium, 10% FCS, and 50 �M �-mercap-toethanol. Proliferation was measured by [3H]thymidine incorporation (1 �Ci/well) during the last 18 h of culture. To test the suppressive activity of CD8 Tregcells, CD8 T cells were titrated into cultures at different CD4:CD8 ratios beforesupernatants were collected at 48 h and cytokine concentrations determined byELISA (BD PharMingen).
Generation of CTLs and In Vitro Lysis. To generate anti Qa-1 alloreactive CTLs,CD4 T cells were purified from B6 mice and activated in vitro by ConA (1 �g/ml)for 2 days before irradiation (3,000 rads) and injection into B6.Tla mice (2 �106
cells i.v.). Fourteen days later, 5 � 106 purified CD8 T cells from spleen wererestimulated in vitro with 107 irradiated ConA-activated B6 lymphocytes with 20U/ml of IL-2 in RPMI 1640 complete medium, 10% FCS, and 50 �M �-mercapto-ethanol. Cytotoxic activity was measured after 5 days of culture. First, 106 targetcells in 100 �l of RPMI 1640 complete medium were labeled with 100 �Ci ofNa2(51Cr)O4 for 1 h at 37 °C and washed three times with PBS. After resuspensionin RPMI 1640 complete medium, 104 labeled target cells were mixed with effectorcells at different E:T ratios (5:1, 10:1, and 20:1) in triplicate and then incubated for4 h at 37 °C. Cell-free supernatants were collected, and their radioactivity wasmeasured with a Wallac Microbeta counter. The percentage of lysis was calcu-lated by the following formula: (sample release � spontaneous release)/(maximum release � spontaneous release) �100%.
ACKNOWLEDGMENTS. This work was supported by grants from the NationalInstitutes of Health (AI 37562) and the National Multiple Sclerosis Society and agift from the Schecter Family Research Foundation (to H.C.). L. L. is a ClaudiaAdams Barr Investigator and NRSA Fellow (T32 AI07386). We thank V. K. Kuchroofor providing the 2D2 TCR transgenic mice, D. Laznik and X. Sun for providingtechnical assistance, and A. Angel for assisting with manuscript and graphicspreparation.
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Fig. 5. (A) Suppression of Qa-1 R72A 2D2 cell expansion by CD8 Treg cells requires perforin. First, 2D2 CD4� T cells (106) from Qa-1 R72A mice were transferred intosyngeneic Rag2�/�Prf1�/� hosts (n � 3) with CD8 suppressor cells generated in B6 mice or mice deficient in perforin and Fas ligand. Hosts were then immunized with10 �g of MOG/CFA. 2D2 CD4 T cells in draining lymph nodes and spleen were enumerated after 14 days. Data are shown as mean � SD (n � 3). (B) In vitro suppressionof Qa-1 R72A 2D2 CD4 cell response by CD8 Treg cells. CD8 cells were purified from the draining lymph nodes of Qa-1 R72A 2D2 and CD8 suppressor cells cotransferredinto mice. 2D2 CD4 T cells were isolated from Qa-1 R72A mice and stimulated in vitro with different doses of MOG peptide in the presence of irradiated splenocytesas antigen-presenting cells with or without different purified CD8 suppressor cells, as indicated. Proliferation of CD4 T cells was measured 60 h later by 3H-thymidineincorporation. Data shown represent two independent experiments. (C) In vitro lysis of Qa-1 R72A CD4 targets by CD8 Treg cells from 2D2-immunized mice. Qa-1 WTand Qa-1 R72A 2D2 CD4 T cells were activated by MOG peptide for 48 h and used as target cells in a lysis assay by CD8 T cells purified from OT2-immunized mice. CD8cells from naïve B6 mice were used as controls. Percent lysis is shown at the indicated E:T ratios.
Lu et al. PNAS � December 9, 2008 � vol. 105 � no. 49 � 19425
