HLA-DRB1 *07:01 allele is primarily associated with the Diego a alloimmunization in a...

I M M U N O H E M A T O L O G Y

HLA-DRB1*07:01 allele is primarily associated with the Diego aalloimmunization in a Brazilian population

Wilson Baleotti Jr,1 Marcelo Ortega Ruiz,2 Antonio Fabron Jr,2 Lilian Castilho,3 Silvana Giuliatti,4

and Eduardo Antonio Donadi5

BACKGROUND: The Diego blood group presents amajor polymorphic site at Residue 854, causing aproline (Dib antigen) to leucine (Dia antigen) substitution.Dia alloimmunization has been observed among Asianand Native South American populations. Consideringthat Brazilians represent a genetically diverse popula-tion, and considering that we have observed a high inci-dence of Dia alloimmunization, we typed HLA-DRB1alleles in these patients and performed in silico studiesto investigate the possible associated mechanisms.STUDY DESIGN AND METHODS: We studied 212alloimmunized patients, of whom 24 presented immuno-globulin G anti-Dia, 15 received Di(a+) red blood cellsand were not immunized, and 1008 were healthydonors. HLA typing was performed using commercialkits. In silico analyses were performed using theTEPITOPEpan software to identify Diego-derivedanchor peptide binding to HLA-DRB1 molecules.Residue alignment was performed using the IMGT/HLAfor amino acid identity and homology analyses.RESULTS: HLA-DRB1*07:01 allele was overrepre-sented in Dia-alloimmunized patients compared tononimmunized patients and to healthy donors. Twomotifs were predicted to be potential epitopes for Dia

alloimmunization, the WVVKSTLAS motif was predictedto bind several HLA-DR molecules, and the FVLILTVPLmotif exhibited highest affinity for the HLA-DRB1*07:01molecule. Pocket 4 of the DRB1*07:01 molecule con-tained specific residues not found in other HLA-DRB1molecules, particularly those at Positions 13(Y), 74(Q),and 78(V).CONCLUSION: Individuals carrying the HLA-DRB1*07:01 allele present an increased risk for Dia

alloimmunization. The identification of susceptible indi-viduals and the knowledge of potential sensitizationpeptides are relevant approaches for transfusion care,diagnostic purposes, and desensitization therapies.

The solute carrier family 4, anion exchanger,member 1 (SLC4A1, AE1) gene presents 20 exonsand codes the red blood cell (RBC) membraneprotein band 3, which is one of the major pro-

teins of the RBC membrane, exhibiting 911 amino acidsand 1 million copies per membrane. The 40-kDaN-terminal domain binds to glycolytic enzymes andhemoglobin, whereas the 55-kDa glycosylated transmem-brane C-terminal domain is primarily responsible foranion transport.1-4 The Diego blood group system com-prises 22 potentially immunogenic antigens of Band 3variants: 1) two pairs of antithetical Dia/Dib and Wra/Wrb

antigens, 2) 17 antigens presenting low worldwide fre-quency, and 3) one antigen exhibiting high worldwidefrequency.5-11 The Dia antigen is considered to be the majorantigen of the Diego blood group system due to its impor-tance in hemolytic transfusion reactions and hemolyticdisease of the fetus and newborn. Serologic and molecularstudies have shown that the Dia antigen is rare amongEuropean Caucasians and African Brazilians;12,13 however,the antigen is commonly observed among Native SouthAmerican (up to 5%-65%) and Asian populations (5%-12%), and the Dia antigen is considered to be an important

ABBREVIATION: EF = etiologic fraction.

From the 1Faculty of Medicine of Marília (FAMEMA), Marília,

São Paulo, Brazil; 2Laboratório Diagnósticos do Brasil (DB),

Curitiba, Paraná, Brazil; 3Hemocentro da Universidade Estadual

de Campinas (UNICAMP), Campinas, São Paulo, Brazil;4Department of Genetics, Faculty of Medicine of Ribeirão Preto,

University of São Paulo (USP), Ribeirão Preto, São Paulo, Brazil;5Division of Clinical Immunology, Department of Medicine,

Faculty of Medicine of Ribeirão Preto, University of São Paulo

(USP), Ribeirão Preto, São Paulo, Brazil.

Address reprint requests to: Wilson Baleotti Jr, Hemocentro

da Faculdade de Medicina de Marília, Rua Lourival Freire,

n°240, CEP 17.519-050–Marília, São Paulo, Brasil; e-mail:

[email protected].

Received for publication October 15, 2013; revision

received February 12, 2014, and accepted February 14, 2014.

doi: 10.1111/trf.12652

© 2014 AABB

TRANSFUSION 2014;54:2468-2476.

2468 TRANSFUSION Volume 54, October 2014

anthropologic marker.12,13 The Dib antigen, antitheticalpair of Dia antigen, has high prevalence in all populationsstudied. The Dia antigen results from a point mutation ofthe c.2561T>C nucleotide (rs2285644) at Exon 19 of theSLC4A1 gene (17q21.31), which causes a p.Pro854Leu sub-stitution in the Band 3 protein.14 In a previous study, weevaluated the allelic frequencies of DI*01 (Dia) and DI*02(Dib) alleles in several Brazilian populations, identifyingthe DI*01 allele in 3.2% of blood donors.12 Despite the lowprevalence of the DI*01 allele, during the past 10 years, wehave observed in our immunohematology laboratory aquite high incidence of alloimmunization against the Dia

blood group antigen.The production of antibodies against RBC antigens is

considered to be an important adverse effect occurring inindividuals treated with blood transfusion, organ trans-plant, or during pregnancy.15-17 The D antigen of the Rhblood group and K antigen of the Kell blood group areknown to be the most immunogenic RBC antigens, andthe relative immunogenicity of the other RBC antigens hasbeen estimated on the basis of the relative chance of expo-sure to the antigen and the frequency of the specific anti-body. Most studies of RBC alloimmunization haveprimarily focused on the commonest antigens encoun-tered among European, North American and Africanpopulations, that is, K, c, E, k, e, Fya, C, Jka, S, Jkb, and santigens, whereas the study of Diego blood systemalloimmunization has been neglected.18-20

HLA Class II molecules are expressed on antigen-presenting cells and present peptides to T CD4+ lympho-cytes. The heterodimeric HLA Class II molecules possessone α and one β chain, which forms a peptide-bindinggroove, composed of nine pockets (P1 to P9) that accom-modate peptides containing 12 to 25 amino acids inlength.21 Peptide presentation may be influenced byresidues of the peptide itself and by residues of the HLAmolecule, and this relationship has been verified by crys-tallographic studies or amino acid signature of thepeptide-binding pocket analysis, particularly in autoim-mune diseases such as diabetes, thyroiditis, and vit-iligo.22,23 Additional studies have demonstrated thatHLA-DR molecules can influence RBC antigen immuno-genicity, producing posttransfusion alloimmunization.For instance, HLA-DRB1*04 and HLA-DRB1*15 allelegroups code molecules associated with the binding to Fya-derived anchor peptide, while susceptibility to K-derivedpeptide is promiscuous in its HLA binding.24,25

Considering 1) the high incidence of Dia immuniza-tion in our regional Brazilian population, 2) the clinicalrelevance of the Dia immunization, and 3) that the immu-nogenic potential of the Dia antigen has never been previ-ously investigated, in this study we evaluated severalimmunogenic features of Dia alloimmunization, includingassociation with HLA-DRB1 alleles, evaluation of thesubclass of immunoglobulin (Ig)G associated with

alloimmunization, and in silico studies to investigate thepossible associated mechanisms.

MATERIALS AND METHODS

Studied populationA total of 212 individuals alloimmunized against clinicallyrelevant RBC antigens were studied, of whom 24 (13 men)aged 30 to 92 years (median, 49 years) presented anti-Dia.Besides Dia alloimmunization, seven of these patientswere also immunized against Rh (D, E, c, and Cw) antigens.An additional group of 15 patients (six men) aged 11 to53 years (median, 30 years), who received Di(a+) RBCsand were not alloimmunized against Dia antigen was alsostudied. Only one of these patients was immunizedagainst Cw antigen.

Immunized patients were exposed to Di(a+) RBCs byblood transfusion or pregnancy and nonimmunizedpatients by blood transfusion. All patients were followedat the Immunohematology Laboratory of the BloodCenter of the Faculty of Medicine of Marília (22°13′S,49°56′W), SP, Brazil, from 2001 to 2013. Blood transfusions(unique or multiple) were performed for several reasonsincluding chronic renal disease, surgery, hereditary ordeficiency anemia, and cancer.

Blood samples were collected in plastic tubes con-taining 1.2 to 2.0 mg of EDTA/mL (Vacutainer, BectonDickinson, Curitiba, Brazil) after informed writtenconsent was obtained from all individuals. The protocol ofthe study was approved by the institutional review board(Protocol 316/07). The healthy control population studiedwas composed of 1008 unrelated healthy blood donorsfrom the same geographical region of patients.

Diego system RBC genotyping andantibody identificationDI*01/DI*02 genotyping was performed using polymerasechain reaction (PCR)-amplified DNA followed by restric-tive enzyme digestion to confirm previous serologicanalyses.12

Identification of antibody against Dia antigen wasperformed in gel tests using commercial kits, containing11 RBC suspensions in low-ionic-strength saline solution(ID-Card LISS/Coombs, Bio-Rad, DiaMed Latino América,Lagoa Santa, Minas Gerais, Brazil).

IgG and IgG subclasses against Dia antigensDi(a+) RBCs were incubated with patient serum present-ing antibody against the Dia antigen for 15 minutes at37°C. After centrifugation (1000 × g for 10 min), sensitizedRBCs were tested for the presence of total IgG or specificIgG subclasses (IgG1, IgG3, or IgG2 and/or IgG4), usingID-card direct antiglobulin test IgG1/IgG3 (DiaMedGmbH, Cressier, FR Switzerland).

HLA-DRB1 ALLELES IN DIA ALLOIMMUNIZATION

Volume 54, October 2014 TRANSFUSION 2469

HLA typingHLA allele typing was performed using PCR-amplifiedDNA hybridized with sequence specific oligonucleotideprobes, using commercial kits, following the manufactur-er’s instructions (One Lambda, Inc., Canoga Park, CA).

Prediction of HLA-DRB1–binding sitesThe prediction of HLA-DR epitope–binding regions wasperformed using quantitative matrices provided by theTEPITOPEpan software (http://www.biokdd.fudan.edu.cn/Service/TEPITOPEpan/index.html), setting thresholdvalues at 1% (high stringency) and 3% (low stringency) todifferentiate between binders and nonbinders. Consider-ing that the Dia antigen is located at the C-terminal regionof the protein, we evaluated the peptide profiles spanningaround Residue 854, where the major variation site islocated. Using this strategy, and considering that the HLAmolecules are able to bind peptides of approximatelybetween 12 and 25 residues, the 36-mer sequence836FTGIQIICLAVLWVVKSTLASLALPFVLILTVPLRR871was selected to be input, where the central 854Leu is themajor characteristic of the Dia antigen and the central854Pro is typical of the Dib antigen. High- and low-affinitypeptide binding was defined from the results obtained bythe TEPITOPEpan software, which is based on thresholdvalues (1% is defined as high stringency and 3% as lowstringency) and on the magnitude of the binding scores.

HLA-DRB1 molecule alignmentHLA-DRB1 molecule alignment was performed using theIMGT/HLA (http://www.ebi.ac.uk/cgi-bin/ipd/imgt/hla/align.cgi) database, and HLA-DR pocket assignmentwas performed as previously reported by Zhang andcolleagues.26

Statistical analysisAssociations between HLA-DRB1 allele and Dia alloim-munization were performed by the Fisher exact text, usingthe MedCalc software (http://www.medcalc.org). Theetiologic fraction (EF) that represents the attributable riskat the population level was also estimated.27

RESULTS

Diego genotyping and IgG and IgGsubclasses identificationAll 24 patients presenting with antibodies against Dia andall 15 nonalloimmunized patients exhibited the DI*02/DI*02 genotype. The DI genotype analyses were in agree-ment with previous phenotyping performed during thepretransfusion tests. All alloimmunized patients pre-sented total IgG against Dia antigen. IgG1, IgG3, or both

subclasses were identified in 14 of the 24 (58%) patientsand 10 patients (42%) were negative for IgG1 and IgG3 andsupposedly exhibited IgG2, IgG4, or both subclasses.

HLA genotypingOnly the HLA-DRB1*07:01 allele was overrepresented inDia-alloimmunized patients (f = 0.3750) when comparedto healthy control individuals (f = 0.1299, p < 0.0001), con-ferring an odds ratio (OR) of 4.0168 (95% confidence inter-val [CI], 2.2075-7.3089) and an EF of 0.2816. All patientsexhibiting the HLA-DRB1*07:01 allele (n = 18) were het-erozygous, being accompanied by the DRB1*01:01, DRB1*01:02, DRB1*04:07, DRB1*04:23, DRB1*08:02, DRB1*11:03,DRB1*11:01, DRB1*15:01, DRB1*15:03, and DRB1*16:01alleles. Non-DRB1*07:01 patients exhibited the followinggenotypes: DRB1*03:01/13:02, DRB1*01:04/16:01, DRB1*03:01/11:02, DRB1*03:02/16:02, DRB1*04:03/11:04, andDRB1*08:03/13:02.

HLA-DRB1*07:01 allele was also overrepresented inDia-alloimmunized patients (f = 0.3750) when comparedto nonimmunized patients (f = 0.1333, p < 0.0267), confer-ring an OR of 3.900 (95% CI, 1.1700-13.0004). One patientwas homozygous for HLA-DRB1*07:01 allele and the othertwo were heterozygous, being accompanied by theDRB1*03: 01 and DRB1*11:01 alleles. The comparison ofthe frequency of HLA-DRB1*07:01 allele between nonim-munized patients with healthy controls showed no signifi-cant difference (p = 0.9565). Table 1 shows all theseresults.

Prediction of HLA-DRB1–binding sitesProvided the 36-mer Diego sequence, the TEPITOPEpansoftware identified four DI-derived anchor peptidesequences exhibiting nine amino acids each one: 1)839IQIICLAVL847 (I839-L847), 2) 848WVVKSTLAS856(W848-S856), 3) 861FVLILTVPL869 (F861-L869), and 4)862VLILTVPLR870 (V862-R870). When DRB1 alleles foundamong samples were input into analysis, the I839-L847and V862-R870 anchor peptide sequences were predictedto bind no DRB1 molecules, while the W848-S856 andF861-L869 sequences were predicted to bind several DRB1molecules.

The analysis at 3% threshold showed that W848-S856peptide was predicted to bind to molecules encoded bythe DRB1*08:02, DRB1*08:03, DRB1*11:01, DRB1*11:02,DRB1*11:03, DRB1*11:04, and DRB1*13:02 alleles. Incontrast, the F861-L869 peptide was predicted to bindmolecules coded by the DRB1*01:01, DRB1*01:02,DRB1*01:04, DRB1*03:02, DRB1*04:03, DRB1*04:07,DRB1*04:23, DRB1*07:01, DRB1*16:01, and DRB1*16:02alleles. The strongest binding was observed betweenthe F861-L869 peptide, located four residues upstreamto 854Leu residue, and the molecule encoded by the

BALEOTTI ET AL.


HLA-DRB1*07:01 allele (affinity score, 9.0869/percentagescore, 0.15). When analyzed at 1% threshold (high strin-gency), the W848-S856 peptide did not bind moleculesencoded by the DRB1*11:02 and DRB1*11:04 alleles, andthe F861-L869 peptide was excluded to the binding toDRB1*01:02, DRB1*01:04, DRB1*03:02, DRB1*04:03, andDRB1*04:23 allele encoded molecules (Table 2).

HLA-DRB1 peptide alignment and analyses ofresidue propertiesThe HLA-DRB1 peptide alignment according to the 22binding residues (at Positions 9, 11, 13, 26, 28, 30, 37, 47,57, 60, 61, 67, 70, 71, 74, 77, 78, 81, 82, 85, 86, and 89)observed along the β1 domain,26 encompassing Pockets 1to 9, disclosed two groups of molecules with high binding

TABLE 1. HLA-DRB1 alleles observed in Dia-alloimmunized patients (Group A), in patients who received Di(a+)RBCs and were not alloimmunized (Group B), and in healthy blood donors (Group C)*

Alleles

Alloimmunized patients (A) Nonalloimmunized patients (B) Healthy blood donors (C)

(n = 24) (n = 15) (n = 1008)

DRB1*01 4 (0.0833) 2 (0.0667) 210 (0.1042)DRB1*03 4 (0.0833) 3 (0.1000) 193 (0.0957)DRB1*04 5 (0.1042) 3 (0.1000) 239 (0.1186)DRB1*07† 18 (0.3750)† 4 (0.1333)† 262 (0.1299)†DRB1*08 2 (0.0417) 4 (0.1333) 118 (0.0585)DRB1*09 37 (0.0183)DRB1*10 1 (0.0333) 43 (0.0213)DRB1*11 6 (0.1250) 3 (0.1000) 288 (0.1428)DRB1*12 27 (0.0134)DRB1*13 4 (0.0833) 4 (0.1333) 260 (0.1290)DRB1*14 75 (0.0372)DRB1*15 2 (0.0417) 4 (0.1333) 185 (0.0918)DRB1*16 3 (0.0625) 2 (0.0667) 79 (0.0392)Total 48 (1) 30 (1) 2016 (1)

* The comparisons of the frequencies of HLA-DRB1 alleles among these groups showed that only HLA-DRB1*07 alleles were significantlydifferent in Dia-alloimmunized patients.

† p values: A versus B, p = 0.0267 (OR, 3.9000); A versus C, p < 0.0001 (OR, 4.0170); B versus C, p = 0.9565.

TABLE 2. In silico analysis of a 36-mer linear fragment, encompassing the major polymorphic residue thatdifferentiates Dia (854Leu) from Dib (854Pro) antigens (underlined)

DI fragment:Linear (36-mer) FTGIQIICLAVLWVVKSTLASLALPFVLILTVPLRR

Anchor peptide: IQIICLAVL WVVKSTLAS FVLILTVPL VLILTVPLR

Threshold: 3% 1% 3% 1% 3% 1% 3% 1%

DRB1*01:01 3.5804/0.25† 3.5804/0.25†DRB1*01:02 2.5886/2.85† 2.5886/2.85DRB1*01:04 2.5996/2.65† 2.5996/2.65DRB1*03:01 4.3018/4.35 4.3018/4.35DRB1*03:02 4.2798/1.95† 4.2798/1.95DRB1*04:03 4.0486/2.05† 4.0486/2.05DRB1*04:07 5.0294/0.25† 5.0294/0.25†DRB1*04:23 4.0303/2.05† 4.0303/2.05DRB1*07:01 9.0869/0.15† 9.0869/0.15†DRB1*08:02 6.5357/0.15† 6.5357/0.15†DRB1*08:03 6.6308/0.15† 6.6308/0.15†DRB1*11:01 4.8872/0.15† 4.8872/0.15†DRB1*11:02 4.1869/1.05† 4.1869/1.05DRB1*11:03 4.3777/0.65† 4.3777/0.65†DRB1*11:04 3.9064/1.65† 3.9064/1.65DRB1*13:02 5.4671/0.15† 5.4671/0.15†DRB1*15:01 3.9425/9.24 3.9425/9.24DRB1*15:03 3.9724/9.24 3.9724/9.24DRB1*16:01 3.6156/0.65† 3.6156/0.65†DRB1*16:02 4.0893/0.35† 4.0893/0.35†

* The TEPITOPEpan software generated four predicted 9-mer peptides (shown in square brackets) that bind to molecules encoded by theHLA-DRB1 alleles observed in Dia-alloimmunized patients. Affinity scores/percentage scores at high (1%) and low (3%) stringency thresh-olds are also shown. The strongest binding was observed between the F861-L869 peptide, located four residues upstream to 854Leuresidue, and the molecule encoded by the HLA-DRB1*07:01 allele.

† Represents all possible alleles that bind to DI anchor peptide sequences.



affinity to the major anchor peptides (848W-856S and861F-869L) defined by TEPITOPEpan software as shown inTable 2.

The first group encompassed seven DRB1 (*08:02,08:03, 11:01, 11:02, 11:03, 11:04, and 13:02) molecules thatwere predicted to bind to the 848W-856S anchor peptideat 3% threshold. Among these molecules, we observedthat 14 of the 22 positions presented the same amino acids(residue identity), reaching 64% of identity (Residues 9, 11,26, 28, 30, 60, 61, 70, 77, 78, 81, 82, and 85). When thephysical-chemical properties of these amino acids weretaken into account (residue homology), homology amongthe seven DRB1 molecules reached 82%, that is, 18 of the22 amino acids (Residues 9, 11, 26, 28, 30, 37, 60, 61, 67, 70,74, 77, 78, 81, 82, 85, 86, and 89). Even at 1% threshold, thepercentages of residue identity and residue homologywere identical as shown in Fig. 1 and Table 3.

The second group of molecules was predicted to bindthe 861F869L anchor peptide, including 10 DRB1 (*01:01,*01:02, *01:04, *03:02, *04:03, *04:07, *04:23, *16:01, *16:02,and *07:01) molecules at 3% and three DRB1 molecules at1% threshold (*01:01, *04:07, and *07:01). Among these 10molecules, 23% presented amino acid identity (Residues

47, 61, 81, 82, and 89) and 59% homology (Residues 26, 28,47, 60, 61, 67, 71, 77, 81, 82, 85, 86, and 89), as shown inFig. 1 and Table 3.

HLA-DRB1 peptide properties according tomolecule pocketsConsidering the W848-S856 anchor peptide, the percent-ages of amino acid identity and homology were closelysimilar for all pockets (P1 to P9). On the other hand, whenwe analyzed the F861-L869 anchor peptide, the patternsof amino acid identity and homology among pockets weredistinct; that is, the pockets P1, P2, and P8 exhibited highidentity and homology (identity 50% and homology75%-100%), whereas P6, P7, and P9 presented moderateidentity and homology (identity 17%-25% and homology33%-62.5%), and P3, P4, and P5 exhibited no residue iden-tity and only 0% to 37.5% of homology. Considering thatP4 amino acids have been pointed to be critical for thedevelopment of autoimmune disease,22 given the pres-ence of five polymorphic residues at Positions 13, 70, 71,74, and 78, we analyzed the particular features of thispocket. Noteworthy, the molecule encoded by the

Fig. 1. HLA-DRB1 residue alignment taking into account the 22 binding sites at Positions 9, 11, 13, 26, 28, 30, 37, 47, 57, 60, 61, 67,

70, 71, 74, 77, 78, 81, 82, 85, 86, and 89 (underlined residues) observed along the β1 domain, encompassing Pockets 1-9 (P1 to P9),

on the basis of the binding of the major anchor peptides (W848-S856 and F861-L869) to HLA-DRB1 alleles observed Dia-

alloimmunized patients. Major amino acid differences were observed for the DRB1*07:01 sequence (Residues 13 tyrosine [Y], 74

glutamine [Q], and 78 valine [V]). The DRB1*01:01 amino acid sequence is shown only as a reference protein. Residues that

compose P1 to P9 and residue identity among pockets are also shown.

BALEOTTI ET AL.


TAB

LE

3.A

lign

men

to

fre

sid

ues

that

com

po

seth

eH

LA

-DR

po

cket

s(1

-9)

and

ph

ysic

al-c

hem

ical

pro

per

ties

of

the

bin

din

gre

sid

ues

that

acco

mm

od

ate

the

anch

or

pep

tid

ese

qu

ence

sW

848-

S85

6an

dF

861-

L86

9A

.W

848-

S85

6an

chor

pept

ide

Res

idue

s9

1113

2628

3037

4757

6061

6770

7174

7778

8182

8586

89D

RB

1*08

:02

AP

PN

NA

PP

PP

AP

PN

NA

PB

PN

PP

BP

PN

NN

DR

B1*

08:0

3A

PP

NN

AP

PP

PP

PN

NA

PB

PN

PP

BP

PN

NN

DR

B1*

11:0

1A

PP

PN

AP

PP

NA

PP

NN

AP

BP

NP

PB

PP

NN

ND

RB

1*11

:02

AP

PP

NA

PP

PN

AP

PN

NA

PA

PN

PP

BP

PN

NN

DR

B1*

11:0

3A

PP

PN

AP

PP

NA

PP

NN

AP

AP

NP

PB

PP

NN

ND

RB

1*11

:04

AP

PP

NA

PP

PN

AP

PN

NA

PB

PN

PP

BP

PN

NN

DR

B1*

13:0

2A

PP

PN

AP

PP

NA

PP

NN

AP

AP

NP

PB

PP

NN

NP

ocke

tsho

mol

ogy:

P1

(100

%);

P2

(100

%);

P3

(100

%);

P4

(78%

);P

5(6

7%);

P6

(67%

);P

7(7

5%);

P8

(100

%);

P9

(83%

)B

.F

861-

L869

anch

orpe

ptid

eR

esid

ues

911

1326

2830

3747

5760

6167

7071

7477

7881

8285

8689

DR

B1*

01:0

1N

NN

NA

PN

PP

AP

PN

NP

BP

NP

PB

PP

NN

ND

RB

1*01

:02

NN

NN

AP

NP

PA

PP

NN

PB

PN

PP

BP

PN

NN

DR

B1*

01:0

4N

NN

NA

PN

PP

AP

PN

NP

BP

NP

PB

PP

NN

ND

RB

1*03

:02

AP

PP

NA

PP

PP

AP

PN

NP

BP

BP

PP

BP

PN

NN

DR

B1*

04:0

3A

PN

BP

NA

PP

PP

AP

PN

NP

BP

AP

PP

BP

PN

NN

DR

B1*

04:0

7A

PN

BP

NA

PP

PP

AP

PN

NP

BP

AP

PP

BP

PN

NN

DR

B1*

04:2

3A

PN

BP

NA

PP

PP

AP

PN

NP

BP

NP

PB

PP

NN

ND

RB

1*16

:01

NN

BP

NA

PP

PP

AP

PN

NA

PB

PN

PP

BP

PN

NN

DR

B1*

16:0

2N

NB

PN

AP

PP

PA

PP

NN

AP

BP

NP

PB

PP

NN

ND

RB

1*07

:01

NN

PN

AP

NN

PN

PN

NA

PB

PP

PN

BP

PN

NN

Poc

kets

hom

olog

y:P

1(1

00%

);P

2(7

5%);

P3

(0);

P4

(37.

5%);

P5

(33%

);P

6(5

0%);

P7

(62.

5%);

P8

(100

%);

P9

(33%

)

AP

=ac

idic

pola

r;B

P=

basi

cpo

lar;

N=

nonp

olar

;P

=po

lar.



DRB1*07:01 allele did not exhibit identity or homology atResidues 13, 70, 74, and 78, when compared to other DRB1molecules that bind the F861-L869 anchor peptide,whereas Residue 71 exhibited only homology.

DISCUSSION

Dia is the major antigen of the Diego blood groupsystem and it is observed in 3.2% of the Brazilian blooddonors; however, this frequency may be increased in someBrazilian regions where interracial mixture is observed,including Japanese descendants and Native South Ameri-cans.12,13 Dia alloimmunization is relatively common inBrazil, but is not common in European and North Ameri-can countries. Anti-Dia is considered to be clinicallyimportant for its immunologic features, that is, immuno-globulin classes and subclasses, ability to activate thecomplement system, potential to produce hemolytictransfusion reactions, and hemolytic disease of the fetusand newborn.

In this study, we identified a strong associationbetween alloimmunization against the Dia antigen andthe HLA-DRB1*07:01 allele, which was present in 75% ofalloimmunized individuals, when compared to nonal-loimmunized (20%) patients and to controls (15%), con-ferring high ORs (3.900 and 4.0170, respectively) and ahigh EF (0.2816). These findings mean that individualscarrying the DRB1*07:01 allele exhibit a fourfold increasedrisk to be immunized against the Dia antigen, onceexposed. At the population level, the DRB1*07:01 allele isresponsible for 28.2% of the attributable genetic risk forthe development of Dia alloimmunization.

Specific DRB1 alleles have been associated withalloimmunization against particular RBC antigens afterblood transfusion, particularly the Fya, which is associatedwith HLA-DRB1*04 and HLA-DRB1*15 allele groups.24

Alloimmunization against Jka of the Kidd blood groupsystem has also been associated with the presence of HLA-DRB1*01 and DRB1*10 allele groups, which present ashared sequence in the beta chain.28 In contrast, the Kantigen has been pointed to be one of the most immuno-genic RBC antigens, exception made to RhD, andalloimmunization to the K reveals high promiscuity ofHLA Class II molecule presentation.25 Similarly to the Fya

and Jka alloimmunization, Dia alloimmunization is associ-ated with a specific HLA allele, as reported in this study.

Besides alloimmunization with RBC antigens,patients with autoimmune diseases have specific aminoacid signatures for HLA molecules associated with predis-position to Type 1 diabetes, thyroiditis,22 and vitiligo,23 andcertain residues, particularly the one located at Position74 of HLA-DRB1 molecules, have been considered to becritical to development of Type 1 diabetes and thyroid-itis.22 To understand the possible mechanisms implicatedon the association of the DRB1*07 alleles with the devel-

opment of anti-Dia, and given the availability of thestructure of the Diego antigens in public databases, weperformed several in silico studies to: 1) predict majorpeptide binding sites of the Band 3 protein with HLA-DRDB1 molecules, 2) analyze the properties of the resi-dues that make the structure of the HLA-DRB1 molecules,and 3) determine the HLA-DRB1 pockets that accommo-date Band 3 peptides during antigen presentation.

The TEPITOPEpan software identified four anchorpeptides predicted to be presented by HLA-DRB1molecules; two of them were considered to be major DIanchors, one located at the polymorphic site that differ-entiates Dia from Dib antigens (W848-S856) and the otherlocated four residues upstream of the 854Leu residue(F861-L869). The greatest prediction score was observedbetween the F861-L869 anchor peptide and the moleculeencoded by the HLA-DRB1*07:01 allele. Although thisanchor peptide does not contain the specific 854Leu poly-morphism of the Dia antigen, this peptide seems to be themajor motif that the DRB1*07:01 molecule presents to TCD4 cells to induce Dia alloimmunization. Similar resultshave been reported regarding the relative immunogenic-ity of Fya and K antigens, that is, peptide flanking the poly-morphic residues may influence the immunogenicity ofthe antigen, since antibodies may recognize conforma-tional protein epitopes.29,30

The limited number of different HLA Class IImolecules turns these molecules highly promiscuous,given the enormous quantity of peptides that antigen-presenting cells must process and present to T CD4+lymphocytes. Notwithstanding, a particular antigen pre-sented in the context of a particular HLA Class II mol-ecules may yield a very specific immune response, as isthe case of alloimmunization against epitopes of the RBCantigens. In this context, the high frequency of HLA-DRB1*07 molecule group in Dia alloimmunization indi-cates that this molecule may possess particular featuresthat enable it to present such response. We observed thatthe peptide exhibiting the DI polymorphic site (W848-S856) can be presented by several HLA-DRB1 molecules,some of them with high-affinity scores, as presented inTable 2; however, this peptide is not presented by theHLA-DRB1*07 molecule. In addition, the frequency of thealleles that accommodates the W848-S856 anchor peptidewas closely similar between patients and controls, and thegroove structure of these molecules showed a great iden-tity and homology of the residues in all DR pockets. There-fore, the motif WVVKSTLAS present in the W848-S856DI anchor peptide seems to be less relevant for Dia

alloimmunization.Noteworthy, the molecule encoded by the HLA-

DRB1*07:01 allele exhibited the strongest binding affinityscore (9.0869/0.15) for the F861-L870 anchor peptide, fol-lowed by the DRB1*04:07 molecule (5.0294/0.25). Consid-ering that the frequency of the DRB1*04:07 allele between

BALEOTTI ET AL.


patients and controls was closely similar, and consideringthe overrepresentation of the DRB1*07:01, the epitopeFVLILTVPL present in this anchor peptide seems to be ofhigh relevance for Dia alloimmunization. The study of therelative importance of HLA-DR pockets in Dia-alloim-munized patients showed that P1, P2, and P8 exhibitedhigh identity and homology, and P6, P7, and P9 presentedlow identity and moderate homology. In contrast, P3, P4,and P5 exhibited by the molecule encoded by the HLA-DRB1*07:01 allele showed no identity and low homologyin relation to other molecules. Taken together, these find-ings indicate that P3 (Residue 78), P4 (Residues 11, 13, 26,28, 70, 71, and 78), and P5 (Residues 11, 13, 28, 70, 71, and74) are highly relevant for Dia alloimmunization. Impor-tantly, residues at Positions 13 (Y = tyrosine), 74 (Q = glu-tamine), and 78 (V = valine) were exclusively observed forthe molecule encoded by the HLA-DRB1*07:01 allele.Indeed, most of these residues have been reported to becritical for the susceptibility to Type 1 diabetes and thy-roiditis22 and alloimmunization against the Jka,28 Fya,24 andK31 RBC antigens. Particularly, residue exchange at Posi-tion 74 may modify the three-dimensional structure of P4of the groove-binding peptide in autoimmune disorders.22

Major conclusions of this study included: 1) HLA-DRB1*07:01 was the major allele associated with suscep-tibility to Dia alloimmunization, conferring a high EF; 2)four peptide motifs flanking the polymorphic site of theDia antigen (854Leu) were considered to be potentialanchor peptides for HLA-DR molecules, two of them withhigh affinity (the WVVKSTLAS and FVLILTVPL motifs); 3)the FVLILTVPL motif exhibited the highest affinity for theHLA-DRB1*07:01 molecule; 4) Pocket 4 of the HLA-DRB1*07 contained specific residues not found in otherHLA-DRB1 molecules, particularly those at Positions 13Y,74Q, and 78V; 5) since the residue at Position 74 has beenimplicated on alloimmunization against self tissue anti-gens as well as for other RBC antigens, this residue may becritical for Dia alloimmunization. The identification ofepitopes associated with Dia alloimmunization is of greatinterest for potential use in diagnosis and possible desen-sitization therapies.

ACKNOWLEDGMENTS

We thank Doralice Marvule Tan, Rodrigo Buzinaro Suzuki, and

Joana Ribeiro de Oliveira for their technical support.

CONFLICT OF INTEREST

The authors report no conflicts of interest or funding sources.

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