Identiﬁcation,immunomodulatoryactivity ...€¦ · TRANSFUSION MEDICINE Identiﬁcation,immunomodulatoryactivity,andimmunogenicityofthemajor helperT-cellepitopeontheKbloodgroupantigen

TRANSFUSION MEDICINE

Identification, immunomodulatory activity, and immunogenicity of the majorhelper T-cell epitope on the K blood group antigen*Jillian Stephen,1 *Lindsay S. Cairns,1 Wendy J. Pickford,1 Mark A. Vickers,1,2 †Stanislaw J. Urbaniak,2 and†Robert N. Barker1

1Division of Applied Medicine, University of Aberdeen, Aberdeen, United Kingdom; and 2Academic Transfusion Medicine Unit, Scottish National BloodTransfusion Service, Aberdeen, United Kingdom

The K blood group remains an importanttarget in hemolytic disease of the new-born (HDN), with no immune prophylaxisavailable. The aim was to characterize theTh response to K as a key step in design-ing specific immunotherapy and under-standing the immunogenicity of the Ag.PBMCs from K-negative women who hadanti-K Abs after incompatible pregnancy,and PBMCs from unimmunized controls,were screened for proliferative responsesto peptide panels spanning the K or

k single amino acid polymorphism.A dominant K peptide with the polymor-phism at the C terminus elicited prolifera-tion in 90% of alloimmunized women, andit was confirmed that responding cellsexpressed helper CD3�CD4� and“memory” CD45RO� phenotypes, andwere MHC class II restricted. A relativelyhigh prevalence of background peptideresponses independent of alloimmuniza-tion may contribute to K immunogenicity.First, cross-reactive environmental Ag(s)

pre-prime Kell-reactive Th cells, and,second, the K substitution disrupts anN-glycosylation motif, allowing the ex-posed amino acid chain to stimulate aTh repertoire that is unconstrained byself-tolerance in K-negative individuals.The dominant K peptide was effective ininducing linked suppression in HLA-transgenic mice and can now be takenforward for immunotherapy to preventHDN because of anti-K responses.(Blood. 2012;119(23):5563-5574)

Introduction

K is the most immunogenic and clinically important Kell bloodgroup Ag and a target for harmful alloantibody responses whenthere is either transfusion of mismatched blood, or incompatibilitybetween mother and fetus.1 Maternal anti-K Abs that cross theplacenta during an incompatible pregnancy can suppress fetalerythropoiesis and cause hemolytic disease of the newborn (HDN).2,3

Unlike the RhD blood group Ag, which was the most frequentblood group targeted in HDN until the advent of effectiveprophylaxis with anti-D Ab, there are no specific immune-basedtreatments to prevent or control K alloimmunization in K-negativewomen. The management of pregnancies affected by anti-K Abstherefore remains unsatisfactory and reliant on invasive proceduressuch as fetal blood sampling and transfusion.4 Advances in theunderstanding of immune tolerance now raise the prospect ofeffective immunotherapy,5-7 but the exploitation of such an ap-proach first requires detailed characterization of the pathogenicanti-K response.

Definition of the molecular basis of the Kell Ags now providesthe opportunity to analyze the immune responses they elicit. Kell isa 93-kDa type II glycoprotein peptidase that is expressed primarilyon erythroid tissues and encodes at least 20 blood group Ags. K iscarried by 9% of the white population and differs from theantithetical k because of a single nucleotide polymorphism, C to T,in exon 6 at position 698.8,9 This substitution results in theexpression of methionine, rather than threonine, at residue aminoacid 193 to create the K Ag.8,9 The loss of threonine also disrupts an

N-glycosylation site at amino acid 191,9 but this is not thought tocontribute directly to the formation of anti-K Ab binding sites.Characterization of the immune response to K may lead not only toa therapeutic benefit, but also help to explain the surprisingimmunogenicity of such blood groups that arise from only singleamino acid substitutions.

Conventional IgG Ab responses are helper dependent,10 includ-ing all those studied that are specific for RBC Ags.11 It follows thatthe differentiation of B cells to produce IgG maternal anti-Kalloantibodies is also likely to require stimulation from CD4�

Th cells responding to the same or a linked Ag. Unlike B cells,which often recognize conformational epitopes dependent onsecondary or tertiary protein structure, Th cells are specific forshort sequences of amino acids processed from the Ag anddisplayed bound to MHC class II molecules on the surface ofAPCs.5-7 Given that the K Ag is determined by a single amino acidsubstitution, the Th cells responsible for providing help for anti-Kalloantibodies could well recognize peptide(s) spanning the poly-morphic site. However, despite the advent of predictive algorithms,mapping experiments are required to identify Th epitope(s) defini-tively12,13 and, in the case of K, the precise register(s) of the M193

residue for optimum immunogenicity.Peptide(s) that contain alloreactive Th epitope(s) spanning the

K polymorphism would represent potential immunotherapeutics toprevent or switch off anti-K Ab responses. There is abundantevidence from rodent models,6,14-16 and support from early human

Submitted February 9, 2012; accepted February 24, 2012. Prepublished online asBlood First Edition paper, April 5, 2012; DOI 10.1182/blood-2012-02-410324.

*J.S. and L.S.C. are joint first authors.

†S.J.U. and R.N.B. are joint senior authors.

There is an Inside Blood commentary on this article in this issue.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.

© 2012 by The American Society of Hematology

5563BLOOD, 7 JUNE 2012 � VOLUME 119, NUMBER 23

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trials,17,18 that damaging immune responses can be prevented orameliorated by tolerogenic delivery of peptides containing domi-nant Th epitopes. Such tolerization of specific Th cells can beexploited to inhibit pathogenic Ab production, and the applicationof this approach to prevent HDN caused by anti-K would representan important therapeutic advance. The aim of this work, therefore,was to characterize the Th cells that drive the alloresponse to K,and, in particular, to identify the epitope(s) that they recognizespanning the polymorphic site on the Kell protein and any featurescontributing to immunogenicity. It was then confirmed that apeptide containing the dominant epitope was capable of inducinglinked suppression in a humanized mouse model of immunization.

Methods

Donors

Approval for the study was received from the Grampian Local and RegionalEthics Committee, and informed consent was obtained from all donors inaccordance with the Declaration of Helsinki. PBMCs were isolated from11 healthy K-negative women who had K alloantibodies in their sera as aresult of incompatible pregnancy (Table 1). Control PBMCs were alsoobtained from 11 healthy K-negative unimmunized donors with nodetectable anti-K serum alloantibodies (Table 1). None of the 22 individualsstudied had Abs to any other blood group Ags. Blood samples for PBMCsand serum separation were taken by venepuncture, respectively, into lithiumheparin and plain Vacutainers (BD Biosciences).

Ags and mitogens

Two panels of 15-mer peptides were synthesized (Pepceuticals Limited),corresponding to the Kell protein sequence spanning polymorphic residueamino acid 193, with either M193 (K) or T193 (k) included at every possibleposition (Table 2), together with a control peptide of the reverse sequence to1M(179-193) (MRNFNLSTWKGSIRW). A glycosylated version of Kellpeptide 1T(179-193), WRISGKWTSLNF-Asn(AcNH-b-Glc)-RT, was alsosynthesized by Cambridge Research Biochemicals using solid-phase chem-istry, with a single Fmoc-Asn (Ac3-AcNH-b-Glc)-OH residue coupled

manually. Peptides were checked for purity by mass spectrometry and usedto stimulate PBMC cultures at a final concentration of 20 �g/mL.

Table 1. Details of K-negative alloimmunized and control donors

Donor Sex Age, y Kell genotype HLA-DR genotype Anti-K Ab in serum, titer

Alloimmune

1 F 43 KEL*2,KEL*2 DRB1*13, DRB1*14 40

2 F 54 KEL*2,KEL*2 DRB1*15, DRB1*07 Positive†

3 F 35 KEL*2,KEL*2 DRB1*01, DRB1*11 40

4 F 62 KEL*2,KEL*2 DRB1*03, DRB1*15 5120

5 F 42 KEL*2,KEL*2 DRB1*13, DRB1*04 40

6 F 38 KEL*2,KEL*2 NT 1


8 F 44 KEL*2,KEL*2 DRB1*04, DRB1*15 2560

9 F 53 KEL*2,KEL*2 DRB1*07, DRB1*07 160


Control

1 F 43 KEL*2,KEL*2 NT Negative

2 M 43 KEL*2,KEL*2 DRB1*01, DRB1*04 Negative

3 F 41 KEL*2,KEL*2 DRB1*07, DRB1*07 Negative






9 M 42 KEL*2,KEL*2 NT Negative


NT indicates not tested.†Donors having anti-K alloantibodies but levels too low for accurate Ab titer.

Table 2. Sequences of the Kell peptide panels

Peptide Sequence Residues

1M WRISGKWTSLNFNRM 179-193

2M RISGKWTSLNFNRML 180-194

3M ISGKWTSLNFNRMLR 181-195

4M SGKWTSLNFNRMLRL 182-196

5M GKWTSLNFNRMLRLL 183-197

6M KWTSLNFNRMLRLLM 184-198

7M WTSLNFNRMLRLLMS 185-199

8M TSLNFNRMLRLLMSQ 186-200

9M SLNFNRMLRLLMSQY 187-201

10M LNFNRMLRLLMSQYG 188-202

11M NFNRMLRLLMSQYGH 189-203

12M FNRMLRLLMSQYGHF 190-204

13M NRMLRLLMSQYGHFP 191-205

14M RMLRLLMSQYGHFPF 192-206

15M MLRLLMSQYGHFPFF 193-207

1T WRISGKWTSLNFNRT 179-193

2T RISGKWTSLNFNRTL 180-194

3T ISGKWTSLNFNRTLR 181-195

4T SGKWTSLNFNRTLRL 182-196

5T GKWTSLNFNRTLRLL 183-197

6T KWTSLNFNRTLRLLM 184-198

7T WTSLNFNRTLRLLMS 185-199

8T TSLNFNRTLRLLMSQ 186-200

9T SLNFNRTLRLLMSQY 187-201

10T LNFNRTLRLLMSQYG 188-202

11T NFNRTLRLLMSQYGH 189-203

12T FNRTLRLLMSQYGHF 190-204

13T NRTLRLLMSQYGHFP 191-205

14T RTLRLLMSQYGHFPF 192-206

15T TLRLLMSQYGHFPFF 192-207

The Kell peptide panels consisted of 2 sets of 15-mer peptides spanning the K/kpolymorphism. Peptides 1-15M and 1-15T include the K and k polymorphism,respectively (polymorphic site shown in bold).

5564 STEPHEN et al BLOOD, 7 JUNE 2012 � VOLUME 119, NUMBER 23




The mitogen Con A (Sigma-Aldrich) and the nominal Ag Mycobacteriumtuberculosis purified protein derivative (PPD; Statens Seruminstitut) were used aspositive control T-cell stimuli and added to cultures at a final concentration of10 �g/mL. PPD readily invokes recall T-cell responses in a high proportion ofUnited Kingdom citizens19 as most have been immunized with the BacilliCalmette-Guerin vaccine.

A conjugate immunogen was a gift from Prof R. Fraser and Dr R. Drake(Scottish National Blood Transfusion Service). It was made by cross-linking a synthetic peptide corresponding to Kell protein residues aminoacids 184-197, and spanning the K polymorphic residue M193, to the carrierkeyhole limpet hemocyanin (KLH). This conjugate was prepared forimmunization by emulsification in IFA and 2% Tween 80 in saline.

Isolation and culture of PBMCs

PBMCs were isolated from fresh whole blood by density gradientcentrifugation (Lymphoprep 1077; Nycomed). The viability of the PBMCswas � 90%, as confirmed by trypan blue staining. As previously de-scribed,13,19-21 PBMCs were cultured at a final concentration of 1.25 � 106

cells/mL in � modification of Eagle medium (�-MEM; Invitrogen)supplemented with 1% 2mM L-glutamine (Invitrogen), 2% 20mM HEPESbuffer (Sigma-Aldrich), 2% penicillin streptomycin (Invitrogen), and 5%autologous sera. PBMCs were incubated with peptides or control stimuli for5 days at 37°C in a humidified atmosphere of 5% CO2/95% air.

T-cell proliferation assay

The rate of proliferation of cell cultures was estimated by the incorporationof 3H-thymidine into newly synthesized DNA after 5 days of stimulation, asdescribed previously.13,19-21 The proliferation by the cells was recorded asmean cpm � SD of the triplicate samples, or as a stimulation index (SI)expressing the rate of mean cpm in stimulated versus unstimulated controlcultures. An SI � 3 was interpreted as a proliferative response.21

Characterization of responding cells

The phenotype of the cells responding in cultures was determined usingflow cytometry. Cells were stained with anti-CD3 PE Texas Red, anti-CD4FITC, anti-CD71 PE (all from Beckman Coulter) and CFSE (Invitrogen).Stained cells were analyzed on a Beckman Coulter Epics XL flowcytometer with a minimum of 10 000 events counted. The flow cytometricdata were processed using Expo V2 analysis software (Applied CytometrySystems).

HLA-blocking Abs

As described elsewhere,13,19,20 to determine the HLA class II restriction ofthe cells responding in cultures, blocking Abs specific for HLA-DP/DQ/DRor HLA-DR (all from BD Biosciences) were added at a final concentrationof 5 �g/mL.

Preparation of CD45RO� and CD45RA� depleted PBMCs

PBMCs depleted of CD45RO� or CD45RA� T cells were prepared aspreviously described.19,21-23 Briefly, PBMCs were incubated with murinemAb UCHL1 (specific for human CD45RO) or SN130 (specific for humanCD45RA; gifts from Profs P. C. L. Beverley (University College London,London, United Kingdom) and G. Janossy (Royal Free Hospital, London,United Kingdom). Cells that bound mAb were removed by immunomag-netic separation using ferrous beads coated with Ab specific for mouse IgG(Biomag; PerSeptive Biosystems). Each depleted population contained� 10% cells expressing the respective CD45 isoform, as determined byflow cytometry (FACSCalibur).

Administration of Ag to HLA transgenic mice

All animal experiments were carried out under a project license granted bythe United Kingdom Home Office and approved by the University ofAberdeen Local Ethical Committee. The generation of HLA-DR15 trans-genic mice by Professor D. Altmann (Imperial College London) isdescribed in detail elsewhere.24,25 Each mouse was positive for the genetic

modification by PCR of DNA samples extracted from tail tips or earpunches, and transgenic expression of the human DR15 MHC class IImolecule on APC was confirmed by flow cytometry. To induce responses,100 �g of Kell-KLH conjugate or control unlinked KLH was injectedsubcutaneously in IFA to mice aged 12 weeks, followed at 14-day intervalsby 2 IP boosters of the respective immunogen. For the induction ofsuppression, 100 �g/mL candidate dominant K1 peptide or control peptidewith reverse sequence was administered to the nasal mucosa of HLA-DR15transgenic mice either 2 weeks before immunization or 2 weeks after thelast booster.

Measurement of murine T-cell responses

To measure murine T-cell responses, spleens were taken from immunizedtransgenic mice 2 weeks after the last boosting or suppressing treatment andsingle-cell suspensions were generated by homogenization. The spleno-cytes were incubated in �-MEM (Invitrogen) at a concentration of 2 � 106

cells/mL under conditions described above for isolation and culture ofPBMCs, but supplemented with 0.5% complement inactivated murineserum and 50�M �-mercaptoethanol (Sigma-Aldrich).24 T-cell prolifera-tion was estimated from the incorporation of 3H-thymidine in triplicatemicrotiter wells 5 days after stimulation. These experiments focused onsplenocytes because T cells from a selection of lymph nodes demonstratedno consistent responsiveness to the immunization regimen.

Statistical analysis

All statistical analysis was carried out using SPSS 15.0 for Windows andSigmaStat 2.0 (Jandel Scientific). The 2, Mann-Whitney U, Wilcoxonrank-sum, or the Student t test on log-transformed data, were used whereappropriate. P � .05 was taken as being significant.

Results

Proliferative responses to the Kell peptide panels by PBMCsfrom alloimmune K-negative donors

The initial aim was to map the epitope(s) containing the M193

K polymorphism that are recognized by alloreactive Th cells fromK-negative women who have produced anti-K alloantibodies afterincompatible pregnancy with a K-positive fetus. PBMCs wereobtained from 10 alloimmunized women (Table 1) and used toscreen 2 panels of 15-mer linear peptides spanning the polymor-phic region, with either M193 (K) or T193 (k) at each possibleposition (Table 2), for the ability to stimulate in vitro proliferation.

The results are summarized in Figure 1A, with representativeproliferative responses from 3 anti-K alloantibody positive womenillustrated in Figure 1B. Responses to one or more K peptides wereobserved in each alloimmune individual, and peptide 1M(179-193)

with the polymorphic residue at the C terminus was the mostcommonly stimulatory sequence, eliciting proliferation in 90% ofthe donors. Furthermore, where multiple sequences were stimula-tory, responses induced by the peptide 1M(179-193) were typically thestrongest. Other, less dominant, peptides that elicited proliferationin several of the women included 3M(181-195), 5M(183-197), and8M(186-200).

In addition to proliferating against K sequences, PBMCs fromalloimmune donors also demonstrated some responsiveness, albeitsignificantly less commonly, to the panel of control k peptides(2 test P � .001, comparing total number of K and k peptideresponses in all alloimmune donors). Unlike the K sequences,among which 1M(179-193) elicited strong proliferation by PBMCsfrom a large majority of the alloimmune women, there was no cleardominant k peptide.

T-CELL SPECIFICITY FOR K 5565BLOOD, 7 JUNE 2012 � VOLUME 119, NUMBER 23




Proliferative responses to the Kell peptide panels by PBMCsfrom control unimmunized K-negative donors

To test whether the proliferative responses to K peptides, particu-larly 1M(179-193), were associated with exposure to K-positive RBCsand the development of alloantibody, PBMC samples were alsotested from 10 K-negative control unimmunized donors, none of

whom had any detectable K alloantibodies in their sera (Table 1).PBMCs were again stimulated with the K and k peptide panels, andthe proliferative responses are summarized in Figure 2A withrepresentative examples shown in Figure 2B. Proliferation to thepeptide panels was not uncommon in the control group, despite thelack of exposure to mismatched RBCs, but the pattern of responses

Figure 1. Proliferative responses to the panel of K and k Kell peptides by PBMCs from K-alloimmunized women are focused on K sequences. (A) Summary map ofresponses by PBMCs from 10 K-alloimmunized women to the panel of peptides spanning the M193 (K) and T193 (k) polymorphism. Filled boxes represent positive proliferativeresponses (SI � 3). K peptides elicited more responses than k (2 test P � .001), and K peptide 1M(179-193) was the predominant stimulatory sequence (90% donors werepositive). (B) Representative proliferative responses to the Kell peptide panel by PBMCs from alloimmunized donors 1, 2, and 5. Dotted line denotes the level of proliferationtaken as representing a positive response (SI � 3). These examples illustrate that, despite the predominance of responses to peptide 1M(179-193), other peptides can elicitproliferation, including k sequences. PBMCs from all donors proliferated significantly in response to the control recall Ag PPD.





was less focused than that seen in the alloimmunized donors. Inparticular, there was no dominant peptide that elicited responses byPBMCs from many of the control donors. K peptide 1M(179-193),which was the dominant sequence identified in the alloimmunized

group, stimulated significantly less responsiveness in the controldonors (Mann-Whitney U, P .036). No other peptide demon-strated significant differences in stimulatory ability between the2 donor groups.

Figure 2. Proliferative responses to the panel of K and k Kell peptides by PBMCs from unimmunized control donors are not focused on particular sequences.(A) Summary map of responses by PBMCs from 10 unimmunized control donors to the panel of peptides spanning the M193 (K) and T193 (k) polymorphism. Filled boxesrepresent positive proliferative responses (SI � 3). There is no significant difference between the numbers of responses to K or k peptides and no peptide elicited responses in� 60% of donors. (B) Representative proliferative responses to the Kell peptide panel by PBMCs from unimmunized control donors 2, 6, and 8. Dotted line denotes the level ofproliferation taken as representing a positive response (SI � 3). These examples illustrate that responses to peptide 1M(179-193) do not predominate, while other peptides canelicit proliferation, including k sequences. PBMCs from all donors proliferated significantly in response to the control recall Ag PPD.





Taken together, the results from alloimmunized and controldonors demonstrate that PBMC responsiveness to one K se-quence, 1M(179-193), is associated with alloimmunization with K,consistent with this peptide recapitulating a dominant Thepitope. However, there was a higher prevalence of backgroundof responses, both to peptides incorporating the alternativek polymorphism, and among unimmunized control donors, thanhas been reported in studies of Th specificity for other bloodgroups.13,26

Characterization of lymphocytes that proliferated in responseto Kell peptides

To test whether the PBMCs proliferating in response to Kellpeptides were of the CD3�CD4� Th phenotype, respondingcultures were analyzed by flow cytometry, using the marker CD71to identify activated cells. Figure 3A illustrates representative flowcytometric analyses of PBMCs from an alloimmune donor (n 5),either resting, or responding to dominant K peptide 1M(179-193) or

Figure 3. Proliferation of PBMCs from alloimmunized and unimmunized donors in response to K peptides is mediated by cells with the CD3�CD4� Thphenotype. Flow cytometric analyses of PBMCs from (A) alloimmunized donor 5 or (B) control donor 4 left unstimulated, or stimulated with K peptides 1M(179-193) or3M(181-195) and stained for expression of CD3, CD4, and the activation marker CD71. Tables next to each pair of plots indicate the percentage of cells in quadrants. Theresults are representative of 5 alloimmunized and 3 control donors. In all cases, � 90% of the increase in the CD71� activated population after peptide stimulation isaccounted for by cells that are CD3� and CD4�. (C) Flow cytometric analyses of CFSE-stained PBMC from control donor 5 left unstimulated, or stimulated withK peptide 1M(179-193) and stained for expression of CD4.





peptide 3M(181-195). As expected, proliferation is accompanied byexpansion of the activated CD71� population, and in all cases� 90% of these responding cells were CD3�CD4�. Similar resultswere obtained when stimulatory k peptides were tested (notshown), and when PBMCs from unimmunized control donors(n 3) proliferated to K sequences (representative analyses Figure3B). Flow cytometric analyses of responding PBMCs that had beenstained with CFSE before stimulation with Kell peptides confirmeddivision of cells within the CD4� population (Figure 3C).

To further demonstrate that PBMCs responding to Kell peptidescame from the helper subset which is restricted by MHC class IImolecules, blocking Abs with pan-reactivity to HLA-DP, -DQ, and-DR, or specific for HLA-DR alone, were tested for their ability toinhibit proliferation. Representative results from alloimmunized(n 4) and unimmunized control (n 4) donors are depicted inFigure 4 and demonstrate that addition of the anti-HLA Abs topeptide-stimulated cultures markedly inhibited all proliferative

responses to either 1M(179-193) or other K peptides. Similarly,consistent inhibitory effects were seen when proliferative re-sponses to k peptides were investigated (results not shown).Addition of isotype-matched Ab with irrelevant specificity had noconsistent or significant effects on proliferation to Kell peptides.

The above results indicate that the proliferative responses to Kellpeptides, whether against the dominant sequence 1M(179-193), or otherpeptides, or by PBMCs from alloimmunized or control donors, aremediated by Th cells that are MHC class II restricted, largely by DR.When a web-based algorithm (www.imtech.res.in/raghava/propred)27

was interrogated for HLA-DR–restricted motifs spanning the M193

K polymorphism, several candidates were proposed. These included thecore sequence amino acids 185-193, which was predicted as a relativelypromiscuous binder to a panel of HLA-DR molecules, with W185

forming the key anchor residue in pocket one of the MHC class IIbinding groove, and K-specific residue M193 located at the C terminus,corresponding to the dominant K peptide 1M(179-193).

Figure 4. The proliferation of T cells against Kellpeptides is dependent on HLA-class II molecules.Cultures of PBMCs from (A-B) alloimmunized donor 3 or(C) control donor 5 were stimulated with Kell peptide1M(179-193) or 3M(181-195), and class II–restricted re-sponses were blocked by addition of Ab against allHLA-DP, DQ, and DR molecules, or specific only for DR.Isotype control Ab was added in selected experiments.P � .05 was taken as significant inhibition (t test onlog-transformed data). The results are representative of4 alloimmunized and 4 control donors.





Background responses to Kell peptides by Th cells fromunimmunized control donors

One explanation for the relatively high number of backgroundresponses to Kell peptides by PBMCs from the unimmunizedcontrol donors would be the detection of primary proliferation bynaive Th cells in vitro, independent of Ag exposure in vivo. Theexperiments were designed to minimize this possibility becausePBMC proliferation was assessed relatively early (on day 5 afterstimulation), and cell culture was performed in microtiter plates.These conditions strongly favor recall, rather than primary, Th-cell

responses.12,13,19 However, to confirm that any Th cells proliferat-ing against Kell peptides in vitro had been activated in vivo,depletion experiments determined the isoform of the CD45 mol-ecules they express because primary and recall responses aremediated by T cells bearing CD45RA and CD45RO, respec-tively.19,22,23 Results from alloimmunized and unimmunized indi-viduals are illustrated in Figure 5 and show that the CD45RO�

subset containing previously activated T cells is a major contribu-tor to Kell peptide-induced proliferation in vitro, regardless of thehistory of exposure to K-mismatched RBCs in vivo. The additional

Figure 5. T cells from alloimmunized and unimmu-nized donors that proliferate in response to K pep-tides have previously been activated in vivo. Prolifera-tive responses of CD45RO� (previously activated/memory) and CD45RA� (previously inactive/naive) T-cellfractions from alloimmunized donors 1 and 5 and unimmu-nized control donors 4 and 5 against selected Kellpeptides. The results are representative of 4 alloimmu-nized and 4 control donors.





contributions made by the CD45RA� subset to proliferationagainst Kell peptides are typical of in vitro responses to recallAgs13,19 and may reflect either naive T-cell production after theexposure of donors to Ag, the presence of T cells with low affinitythat ignore Ag in vivo, or partial reversion of the memorypopulation to the CD45RA� phenotype.19 Thus, despite anyproliferation by the CD45RA� subset, the presence of responsiveCD45RO� T cells is a reliable indicator of prior primingin vivo.19,22,23

The confirmation of Th memory for particular Kell peptides inunimmunized individuals raises the possibility that the cells areprimed in vivo by another Ag. Searches for homology between thesequence of Kell and microbial Ags have already reported severalkey matches28,29 and, to further illustrate the potential for suchcross-reactivity, we used the basic local alignment search tool(BLAST)30 to interrogate the SwissProt database. The searchrevealed extensive homologies between the Kell protein sequencesspanned by the peptide panels and many bacterial, parasite, viral,and fungal sequences (examples illustrated in supplemental Table1, available on the Blood Web site; see the Supplemental Materialslink at the top of the online article).

Effects of glycosylation on background Th responses tok self-peptides

In addition to the background responses to K sequences exhibitedby unimmunized control donors, and in contrast to parallel studiesof other blood group Ags,13,26 Th cells from both the alloimmu-nized and unimmunized donors commonly proliferated against thek peptide panel. In such k-positive individuals, these responsespotentially represent autoreactivity and a failure of self-tolerance.However, substitution of T193 for M193 is believed to disrupt anN-glycosylation consensus sequence at amino acid 191 of the Kellprotein,9,31 which therefore displays carbohydrate residues at thisposition in the k, but not K, type. One explanation for the relativelyhigh background of responses to k peptides is that self-tolerance isestablished to the glycosylated k sequence spanning T193 and istherefore less secure to the exposed amino acid chain of thesynthetic k peptides used for epitope mapping, and of the homolo-gous K sequence. To test this possibility, a glycosylated version ofpeptide 1T(179-193), with a nominal sugar residue at amino acid 191,was synthesized and compared with the unglycosylated sequencefor the ability to stimulate in vitro proliferation by PBMCs fromunimmunized control donors (n 3). The results in Figure 6

Figure 6. Glycosylation of k peptide determines theability to stimulate T-cell proliferation. The k peptide1T(179-193) was synthesized in glycosylated (GLYCO P1T)and unglycosylated (P1T) forms and used to stimulateT cells from unimmunized donors 4-6. P � .05 was takenas significant inhibition (t test on log-transformed data).





demonstrate that the glycosylated form, which reflects the naturalstate of the sequence, does fail to elicit proliferative responses.

In vivo suppression established by dominant K peptide1M(179-193)

Despite the relatively common background responses to Kellsequences in unimmunized controls, in vitro screening identifiedK peptide 1M(179-193) as containing a dominant helper epitope. Toconfirm the dominance of 1M(179-193) in vivo, and its potential as aspecific immunomodulator of responses, we sought to determinewhether this peptide could induce suppression to T-cell epitopes ona carrier protein in mice immunized with a conjugate of KLH witha short sequence of the Kell protein spanning the K polymorphicsite. We used a strain that had been genetically modified to expresshuman, rather than murine, MHC class II molecules24,25 to modelthe presentation and restriction of peptides in humans. Micetransgenic for HLA-DR15 were chosen because DR was identifiedin Figure 4 as the predominant restricting locus for Kell peptides,and our typing of alloimmunized women with anti-K Abs revealedthat DR15 was the most common allele they expressed (12 of25 women were positive). Figure 7 demonstrates that when peptide1M(179-193) was administered via the nasal mucosa (a recognizedsuppressive route)6,32 either before or after immunization, the T-cellresponse to the conjugate (including the linked KLH carrierprotein) was significantly reduced. Although incomplete, thereductions are comparable with those seen in parallel work testingthe suppressive properties of immunodominant peptides from otherAgs including the RhD protein.24 The inhibition by 1M(179-193) wasspecific and not the result of any intrinsic immunosuppressiveactivity because treatment of mice with a control peptide of the

reverse sequence failed to replicate the effect. Administration of1M(179-193) did not reduce the response to immunization with thecarrier protein KLH alone. An apparently spontaneous T-cellresponse to peptide 1M(179-193) in control unimmunized mice wasalso abrogated by the suppressive regimen (result not shown).

Discussion

This study determined, for the first time, the specificity ofalloreactive Th cells that recognize the sequence of the Kell proteincontaining the M193 K polymorphism. The characterization of thehelper response that drives anti-K Ab production in K-negativeindividuals reveals properties of the Ag that contribute to itsimmunogenicity, and now opens the way to develop novel strate-gies to prevent or treat alloimmunization, based on immunomodu-lation of specific T cells. Such approaches would be of clinicalbenefit given that current HDN prophylaxis targets only RhDallommunization, with no effective measures available to preventalloresponses to other blood groups such as K.

Mapping of Th epitopes in women who had produced anti-KAbs after incompatible pregnancy identified several stimulatoryKell sequences, most notably the K peptide 1M(179-193), with thepolymorphic residue M193 located at the C terminus. Phenotypicand functional analyses confirmed that the responses, includingthose against 1M(179-193), were mediated by CD3�CD4� MHC classII–restricted Th cells. Several lines of evidence together supportthe view that the sequence 1M(179-193) recapitulates a dominanthelper epitope that drives responses in vivo after exposure to K.First, Th proliferation to 1M(179-193) was detected in 90% of

Figure 7. Tolerogenic administration of dominant K peptide 1m(179-193)to HLA-DR transgenic mice suppresses T-cell responsiveness to aKell sequence immunogen and linked carrier protein. Proliferativeresponses of splenocytes against a Kell sequence-KLH conjugate whenHLA-DR15 transgenic mice were given K peptide 1M(179-193) or controlpeptide with reverse sequence via the nasal mucosa either before or afterimmunization with the conjugate or the unlinked carrier protein KLH.P � .05 was taken as significant inhibition (t test on log-transformed data,n 3-9/group). Splenocytes from all mice proliferated significantly inresponse to the control stimulus Con A.





K-negative women who had produced anti-K Abs, and wastypically the strongest evoked by any of the Kell peptides in thesedonors. Peptide 1M(179-193) was also the only Kell sequence tostimulate responses that were significantly stronger in the alloimmu-nized versus the unimmunized control group. Second, alloimmu-nized donor Th cells that were responsive to 1M(179-193) in vitro hadbeen activated in vivo because the culture conditions were de-signed to minimize primary responses, and it was demonstratedthat a major proportion of the responding cells was drawn from theCD45RO� memory population. Finally, a dominant role forpeptide 1M(179-193) was confirmed (and its potential for immuno-therapy illustrated) by the ability of the sequence to inducetolerance in vivo to a linked carrier protein in a humanized mousemodel24,25 of DR-restricted Th responses.

The use of blocking Abs specific for MHC class II moleculesdemonstrated that the predominant locus restricting responses to1M(179-193) and other peptides was HLA-DR. The relatively highfrequency of HLA-DR15 among K alloimmunized women sug-gests that this allele restricts many of the specific Th cells. Thepitopes from other blood groups, including RhD and HPA-1a, arealso predominantly restricted by HLA-DR.13,26 Dominant peptide1M(179-193) corresponded to one of a number of potential DR-binding motifs spanning the K M193 polymorphism that werepredicted by computer algorithm, and was modeled with W185

forming the key anchor residue in pocket one of the MHC classII–binding groove, and K-specific residue M193 located at theC terminus. There are several explanations as to why the dominantTh-cell epitope requires M193 in this terminal position. It is possiblethat peptides extending beyond the polymorphism M193 either formmotifs with high affinity for MHC molecules that do not restrict thealloreactive response, or create a secondary structure that directlyinterferes with TCR recognition.33 K peptides expressing M193 inother registers may represent further epitopes, but the responsesthey stimulated were similar in frequency in both alloimmunizedand control donors.

The immunogenicity of blood groups such as K is striking,given that simple exposure to a foreign protein alone is typicallyinsufficient for induction of immune responses, and, in the case ofAgs arising from single amino acid polymorphisms, a highlyhomologous self sequence would be expected to tolerize manypotentially reactive Th cells. Clinically important blood groups willtherefore be predicted to exhibit features that enhance their abilityto induce Th-cell responses, and the current work identifies 2 suchcharacteristics in the case of K. First, the relatively high back-ground of memory Th responses to K peptides among unimmu-nized control donors, compared with parallel work on other bloodgroups such as RhD and HPA-1a,13,26 now confirms that priming iscommon by environmental Ags cross-reactive with sequencesspanning the Kell polymorphic site. The apparently spontaneousT-cell responses to peptide 1M(179-193) in HLA-DR transgenic miceis a further demonstration of such activation. It has long beenrecognized that certain bacteria—such as Escherichia coli orShigella sonnei—are capable of expressing K-like Ags on their cellsurface,29 and there are more recent suggestions of potentialmolecular mimicry between K Th epitopes and microbial peptidesfrom Haemophilus influenzae, Bacteroides fragilis, and Vibriospecies.28 Our own homology searches also illustrate that thepotential for mimicry is extensive, and while it will be a majorchallenge to identify the particular environmental Ag(s) respon-sible for priming, animal models confirm that preexisting cross-reactive Th memory is a powerful factor in increasing theimmunogenicity of both auto-34 and allo-28 RBC Ags. Such effects

may be common in the induction of blood group responses. Theirpotency is illustrated by the finding that murine Ab responses to anRBC-bound complex of foreign Th- and B-cell determinants wereincreased 100- to 1000-fold after prior infection with virusexpressing the helper epitope.28

The second characteristic of the K polymorphism that may enhanceits ability to induce Th responses is the loss of the glycosylation motifencoded by the antithetical k sequence.9,31 The Kell protein displayscarbohydrate residues at amino acid 191 in the k, but not K, type becausesubstitution of T193 for M193 ablates the N-glycosylation site.9,31 Al-though this loss does not appear to affectAb binding,9,31 there is growingevidence that posttranslational modifications such as glycosylation playan important role in T-cell recognition of Ags.35 There was a relativelyhigh background of Th responses to self-k peptides among alloimmu-nized donors and unimmunized control volunteers when these peptideswere synthesized by standard chemistry with no glycosylation, indicat-ing a lack of tolerance to the primary amino acid sequence. However, wereasoned that self-tolerance would naturally be directed to the glycosy-lated form of the k sequence9,31 available in vivo, and this was borne outby the demonstration that addition of N-linked carbohydrate at residue191 abolished background responses to k peptide. These results reveal anew mechanism to explain blood group immunogenicity, whereby anextended Th repertoire is available to drive K alloresponses because offailure of glycosylated k sequences to censor cells specific only for theunderlying amino acid chain.Alloimmunization with the K blood grouptherefore challenges Th cells with anAg that is “foreign” due not only tothe single substitution, but also to the exposure an amino acid sequencethat is cryptic in the homologous self-protein. This model has someparallel with immunogenicity of the platelet blood group HPA-1a,where the responsive Th repertoire is thought to be enhanced becausethe amino acid substitution in the antithetical HPA-1b sequence directlyprevents binding to the restricting MHC molecule and thus induction ofself-tolerance.26,36 However, although addition of carbohydrate to keyresidues may reduce the ability of peptides to bind restricting MHCmolecules,37,38 in the case of k sequences, the glycosylation site N191 isnot predicted to be such an anchor. We, therefore, consider it more likelythat the N-glycosylation interferes with T-cell recognition of theMHC-peptide complex.

The therapeutic use of peptides containing dominant helper epitopesfrom target Ags to suppress damaging immune responses has provedvery successful in rodent models.6,7,11,14-18 Although major hurdlesremain in translating this approach to patients, most notably determiningthe optimum dosing regimen, human trials are ongoing in severaldiseases.39 In the field of transfusion medicine, the mapping ofalloreactive helper epitopes on Rh proteins13 led to the development of apeptide product to suppress responses to the D Ag,24 which has nowbeen modified to facilitate large-scale manufacture and is ready to enterclinical trials. The identification of peptide 1M(179-193) as containing animmunodominant epitope in the alloresponse to K, therefore, hasimplications for the future development of novel peptide immunothera-pies. The administration of the immunodominant peptide 1M(179-193) viaa suppressive route, such as the nasal mucosa, has the potential to inhibitalloresponses to the K Ag and therefore reduce or prevent K alloanti-body production in susceptible individuals. Furthermore, the results inhumanized mice encourage the possibility that such peptide immuno-therapy could also have the potential to reverse fully establishedimmune responses in individuals who have been previously alloimmu-nized with the K Ag. This would represent the first specific preventivemeasure for HDN because of anti-K responses and provide an approachthat could be replicated for other blood groups encoded by single aminoacid polymorphisms.





Acknowledgments

This work was supported by the Scottish National Blood Transfu-sion Service.

Authorship

Contribution: J.S. performed research, analyzed data, and wrote themanuscript; L.S.C. and W.J.P. performed research and analyzed

data; M.A.V. collected data; and S.J.U. and R.N.B. designedresearch, collected and analyzed data, and wrote the manuscript.

Conflict-of-interest disclosure: S.J.U. and R.N.B. are co-inventers on patents covering the identification and therapeuticapplication of blood group derived peptides that are recognized byalloreactive Th cells, with the intention of securing commercialinvestment to fund clinical trials. The remaining authors declare nocompeting financial interests.

Correspondence: Prof Robert N. Barker, Section of Immunol-ogy and Infection, Division of Applied Medicine, Institute ofMedical Sciences, University of Aberdeen, Foresterhill, AberdeenAB25 2ZD United Kingdom; e-mail: [email protected].

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online April 5, 2012 originally publisheddoi:10.1182/blood-2012-02-410324

2012 119: 5563-5574

Robert N. BarkerJillian Stephen, Lindsay S. Cairns, Wendy J. Pickford, Mark A. Vickers, Stanislaw J. Urbaniak and major helper T-cell epitope on the K blood group antigenIdentification, immunomodulatory activity, and immunogenicity of the

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Identiﬁcation,immunomodulatoryactivity ...€¦ · TRANSFUSION MEDICINE Identiﬁcation,immunomodulatoryactivity,andimmunogenicityofthemajor helperT-cellepitopeontheKbloodgroupantigen