ABSTRACT
The target of an anti‐HLA antibody is an epitope on the surface of the antigen, and in particular the polymorphic eplet of its core, which contains one or a few polymorphic residues accessible to the molecule surface. The HLA Eplet Registry database references more than 550 HLA eplets thus deduced from AA sequence alignments. However, not all eplets have yet been verified and this list is not exhaustive. We performed a systematic analysis of sequence alignment of DQ antigens, to identify polymorphic amino acids so far not proposed as candidate eplets. From this, we describe and validate 3 DQB1 eplets targeted by sera of organ‐transplanted patients and explore a new eplet overlapping the DQA1*03 and DQB1*03 chains. Serum antibody profiles using LABScreen, Lifecodes and a complementary DQ Luminex single antigen bead panel all showed a concordant pattern incriminating these residues. Moreover, antibodies were adsorbed and eluted using human splenic mononuclear cells and HLA‐DQ transfected murine cell clones, thereby validating these four eplets. Their localisation by 3D modelling revealed uncertain accessibility of some residues implicated. The protrusion and accessibility of an eplet on the HLA surface may ultimately not always be sufficient to predict antibody binding, since the dynamic flexibility of the molecule can modify these parameters. Our strategy, combining Luminex single antigen assays, cell adsorption/elution and in silico prediction of surface exposure, altogether concur to ascertaining the realness of candidate eplets in the highly complex HLA system.
Keywords: DQ, HLA, new eplet, transfection, transplantation
1. Introduction
Anti‐HLA antibodies directed against donor HLA mismatches lead to antibody‐mediated rejection [1]. These donor specific antibodies (DSA) are routinely detected and identified using the Luminex Single Antigen (LSA) bead assays. DSA recognise polymorphic amino acids (AA) on mismatched HLA molecules defined as eplets [2]. An eplet is composed of one or a few AA in close proximity, within 3.5 Å radius [3], and is proposed to be the functional part of an epitope which represents the whole surface area that interacts with the antibody and determines affinity [4].
Several studies demonstrated a major role for HLA‐DQ in humoral alloreactivity, being by far the main locus contributing to DSA [5, 6, 7]. HLA‐DQ is constituted of two polymorphic alpha and beta chains, increasing the number of possibly immunogenic eplets. Eplets are described in the HLA Eplet Registry database (Epregistry) after aligning the AA sequences of HLA alleles and highlighting the polymorphic residues exposed at the surface, as only these can be accessible to antibodies [8, 9]. However, this list is not exhaustive and probably not all eplets can be identified with this method as (1) some eplets seem to involve the peptide together with the HLA molecules that present it [10, 11] and (2) Epregistry does not include the potential eplets involving both the alpha and beta chains. Finally, the vast majority of these eplets have not yet been reliably experimentally verified [12].
We identified polymorphic residues exposed at the surface of the DQ protein that were surprisingly not referenced in Epregistry. In addition, we also identified others located in hidden regions of the protein and therefore not expected to be referenced. Furthermore, we found LSA patterns obtained with human sera that could correspond to an eplet incorporating both the DQA1 and DQB1 chains of the DQ protein. Using a strategy combining (1) search in our local database for LSA profiles corresponding to these so far not reported eplets, (2) experimental verification by adsorption of the corresponding patient's sera on spleen mononuclear cells (SMCs) from deceased donors and HLA‐DQ transfected murine cell clones and (3) in silico analysis of 3D models of the relevant HLA molecules, we report in the present article the identification of five new eplets, located for 3 of them on DQB1, for one on DQA1 and for the last one on selective DQA1/DQB1 combinations.
2. Materials and Methods
2.1. In Silico Identification of Candidate New Eplets
AA sequence alignments of the DQA1 and DQB1 antigens represented in the LABScreen LSA kit were performed using the IPD‐IMG/HLA database version 3.56 (2024–04) [13] and surface accessibility of the residues was established using the HLA‐EMMA website [14]. Only LABScreen antigens were analysed because this kit is the one used in our laboratory for routine patient follow‐up, making possible the search among a large database for sera with antibodies targeting the potential new eplet. As this alignment strategy is unable to pick up candidates involving both chains, we added a recurrent LSA profile pattern that we could not explain with Epregistry.
2.2. Selection of Patients' Sera
Sera were selected from our local database gathering the LSA MFI values of 303,638 LABScreen LSA assays performed for the patients' routine follow‐up between September 15th, 2015 and June 7th, 2024 at the Histocompatibility Laboratory of the Saint‐Louis Hospital in Paris, France. September 15th, 2015 represents the day when systematic EDTA pretreatment of serum was implemented to circumvent the complement interference phenomenon. The sera that were selected had an MFI of at least 1000 for the DQ beads bearing the candidate eplet, and an MFI of less than 500 for the other DQ beads. No selection criterion was applied to beads of the DR, DP and HLA class I loci.
2.3. LSA Assays
The LABScreen LSA HLA Class II (LS2A01, lot 16, One Lambda, West Hills, California) was used according to the manufacturer's instructions, but with half the recommended dose of beads, as is performed for our routine activity. The Lifecodes LSA class II assay (LSA2 lots 3,012,775 and 3,014,206, Werfen, Barcelona, Spain) was modified as previously described [15]. For both assays, microtitration filter plates (Merck, MSBVN1250, 1.2 μm) were used for the incubation and washing steps. Fluorescence data were acquired on a LABScan 200 apparatus (Luminex, Austin, Texas). A complementary DQ panel, developed and provided by One Lambda, was performed with the LABScreen LSA protocol used in the laboratory and read on a LABScan 3D flow analyser.
For the One Lambda LSA assay, raw MFI data were normalised for each bead by subtracting the MFI of the negative control bead. The positive threshold was set at MFI 500 for the LABScreen and MFI 1000 for the complementary DQ panel as MFI values achieved higher values with this DQ LSA due to the use of a 3D flow analyser. For Lifecodes LSA, the raw MFI values were used and the positive cut‐off was set at 750 according to the manufacturer's recommendations. All sera were pretreated for 10 min at room temperature with EDTA at a final concentration of 0.01 M.
2.4. Cells Used for Adsorption/Elution Assays
We established P815 mouse cell clones expressing only one combination of HLA‐DQA1 and DQB1 alleles [16]. The cDNAs were provided by Dr. H. Miyadera [17] and sequenced by Sanger sequencing.
Remnants of spleens from deceased organ donors were used within 48 h following the pretransplant emergency cross‐matches for which they were sent to the laboratory. Spleen mononuclear cells (SMCs) were isolated by Ficoll density gradient (reference CMSMSL01‐01, Eurobio, Les Ulis, France). Donor HLA intermediate resolution typing was performed with LinkSeq SureTyper RT‐PCR (One Lambda) for HLA‐A, B, C, DRB1, DRB3/4/5, DQB1, DQA1, DPB1 and DPA1 genes. HLA typing to the second field was assigned according to the most likely allele given by the technique report and by extrapolation using the HaploSFHI tool [18, 19].
2.5. Adsorption/Elution of Patients' Sera on Cells
The adsorption assay was performed as already described [16]. After centrifugation, the supernatant was collected to perform the LABScreen LSA assay, and the cell pellet was frozen. Depending on the results of the adsorbed serum in LSA, elution was carried out or not on frozen cell pellets. After washes, the cell pellet was incubated for 15 min at room temperature with an acid‐based eluting solution (ELU KIT Plus, Immucor). Then the mix was centrifuged, and the eluate was collected and neutralised with the alkaline buffering solution before running the LSA assay.
A small volume of an internal standard serum (ISS) was added to the patient's serum to be analysed, containing an anti‐HLA‐DR53 or DR52 reactivity chosen according to SMC's donor typing to not interfere with the cells to be used but that could be detected with the class II LABScreen LSA. The ISS allowed the MFI values of the patient's serum, and therefore, the adsorption efficiency, to be normalised.
A percentage of adsorption (%ads) was calculated for each LSA bead expressing the eplet of interest according to the following formula: %ads = (MFIads– MFINA)/MFINA *100, with MFIads representing the MFI for the adsorbed serum and MFINA the MFI for the non‐adsorbed serum. Then, this percentage was normalised with the mean ratios (MFIads/MFINA) of the ISS beads, as already described [16]. A significant adsorption was defined by an average %ads of all beads bearing the eplet below −20%.
We also calculated a percentage of elution (%elu) when elution was performed, according to the following formula: %elu = (MFIelu/MFINA/9)*100 with MFIelu representing the MFI of the eluted serum for a given bead and MFINA/9 the MFI for the non‐adsorbed serum diluted 1 to 9, which corresponded to the dilution of the serum antibody retrieved in the neutralised eluate.
2.6. Molecular Modelling of Antigens
Structures for the DQA1*01:01/DQB1*06:03, DQA1*05:01/DQB1*02:01 and DQA1*03:01/DQB1*03:01 antigens were generated using the default Alphafold v3 protocol including an Amber relaxation procedure [20]. This protocol uses as input solely the AA sequences of the extracellular and globular part of the two chains that form each antigen (186 AA for the DQA1 chain and 192 AA for the DQB1 chain).
2.7. Statistical Analysis
Statistical analysis and graphs were performed using GraphPad software version 8.0 (San Diego, California). All MFINA and MFIads of each LSA bead bearing the eplet for all sera were studied and compared as a global average and standard deviation by one‐way analysis of variance (ANOVA).
3. Results
3.1. Candidate DQ Eplets Not Listed in Epregistry
We compared the polymorphic and exposed AAs according to HLA‐EMMA, to all the DQ eplets already described in Epregistry, and identified eight candidate polymorphic residues that were absent from the list (Table 1), and which could therefore represent so far unknown eplets. Four were on DQA1, which were 26 T/187 A, 52 R, 54 F and 215 F, and four were on DQB1, which were 14 M/116 V, 126 Q, 203 I and 203 V. 26 T/187 A and 14 M/116 V are pairs of residues present on the same antigens but too distant from each other to constitute the same eplet [3]. In addition, the LSA assay would not be able to pinpoint either residue in each pair. We decided to explore them as potentially real new eplets.
TABLE 1.
Aligned polymorphic and exposed amino acids of HLA‐DQA1 and DQB1 antigens used in the present study.
| DQA1 antigens | DQB1 antigens | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Position | 26 | 52 | 54 | 187 | 215 | Position | 14 | 116 | 126 | 203 |
| 01:01 | T | S | F | A | F | 02:01 | M | V | Q | I |
| 01:02 | T | S | F | A | F | 02:02 | M | V | Q | I |
| 01:03 | T | S | F | A | F | 02:07 | M | V | Q | I |
| 01:04 | T | S | F | A | F | 02:180 | M | V | Q | I |
| 02:01 | T | H | L | A | L | 03:01 | M | V | Q | I |
| 03:01 | S | R | F | T | L | 03:02 | M | V | Q | I |
| 03:02 | S | R | F | T | L | 03:03 | M | V | Q | I |
| 03:03 | S | R | F | T | L | 03:13 | M | V | Q | I |
| 04:01 | T | R | F | A | F | 03:16 | M | V | Q | I |
| 05:01 | T | R | F | A | F | 03:19 | M | V | Q | I |
| 05:02 | T | R | F | A | F | 03:42 | M | V | Q | I |
| 05:03 | T | R | F | A | F | 04:01 | M | V | Q | I |
| 05:05 | T | R | F | A | F | 04:02 | M | V | Q | I |
| 06:01 | T | R | F | A | F | 05:01 | L | I | Q | V |
| 05:02 | L | I | H | V | ||||||
| 05:03 | L | I | Q | V | ||||||
| 05:10 | L | I | Q | V | ||||||
| 05:23 | L | I | Q | V | ||||||
| 06:01 | M | V | Q | I | ||||||
| 06:02 | M | V | Q | V | ||||||
| 06:03 | M | V | Q | V | ||||||
| 06:04 | M | V | Q | V | ||||||
| 06:09 | M | V | Q | V | ||||||
| 06:49 | M | V | Q | V | ||||||
| 06:110 | M | V | Q | V | ||||||
Note: In bold, the LABScreen antigens.
As shown in Table 1, Threonine at position 26 and Alanine 187 on DQA1 are both shared by DQA1*01/02/04/05/06 alleles, whereas the DQA1*03 series has a Serine and a Threonine, respectively. Arginine 52 is restricted to DQA1*03/04/05/06 alleles, whereas the DQA1*01 and DQA1*02 series have a Serine (52SK verified eplet) and a Histidine (47KHL verified eplet), respectively. Phenylalanine 54 is shared by all DQA1 alleles except DQA1*02, which has a Leucine, a residue also part of the 47KHL eplet. Finally, Phenylalanine 215 is encoded by DQA1*01/04/05/06 alleles, whereas DQA1*02/03 share a Leucine.
On DQB1, residues Methionine 14 and Valine 116 are shared by DQB1*02/03/04/06 alleles and correspond to Leucine and Isoleucine respectively on DQB1*05 alleles (part of the 116I verified eplet). Glutamine at position 126 is shared by all DQB1 alleles except DQB1*05:02. Finally, position 203 is an Isoleucine on DQB1*02/03/04/06:01 alleles versus a Valine on DQB1*05/06:02/06:03/06:04/06:09 alleles, but they have not been described as eplets.
3.2. Analysis of Patient's Sera Potentially Targeting the Candidate Eplets
Patient's sera were identified from our database for which LABScreen LSA assays were performed for their follow‐up, using the strict selection criteria described in the Materials and Methods section. No sera were found for DQA1 26 T/187A and 54F, and for DQB1 126Q. For the remaining 14 M/116 V, 203I, 203 V and 215F, we sorted 2, 2, 2 and 1 sera respectively positive for these DQ patterns with average MFI values ± standard deviation (SD) at 18749 ± 3490, 17553 ± 1950, 6130 ± 4381 and 11,386 respectively (Figure 1). We identified several patients' sera for 52R, but the peculiar behaviour of the eplet/sera combinations in LSA profiles called for additional experiments that justified a dedicated report (manuscript in preparation).
FIGURE 1.
Distribution of LABSCreen LSA MFI values of DQ beads, for each patient serum (one symbol per patient). Antigens tested are on the vertical axis, with A and B representing DQA1 and DQB1 loci respectively. Raw MFI—NC refers to raw MFI data after subtraction of the MFI of the negative HLA‐free control bead. The dotted line depicts the positive MFI threshold set at 500. Beads bearing the eplet are depicted in bold text.
All patients had received an organ transplant, all with a different donor, and the DQ pattern was consistent with a DSA in all cases (Table 2). For two patients no serum had been sent to the laboratory pretransplant, and for the remaining 5, the DSA appeared de novo between 5 months and 12 years after transplantation and remained positive (MFI > 1000 for all beads) all along the subsequent follow‐up that ranged from 1–14 years. No clinical information was available that could explain the presence of anti‐HLA antibodies in patients without DSA.
TABLE 2.
Characteristics of the patients' sera targeting the new HLA‐DQ eplets.
| Eplet | Luminex HLA alleles | Nb of LABScreen beads | Patient | Graft | Recipient typing | Donor typing | DSA | dnDSA appearance (year) | Follow‐up (year) | ||
|---|---|---|---|---|---|---|---|---|---|---|---|
| DQA1 | DQB1 | DQA1 | DQB1 | ||||||||
| 14 M/116 V | DQB1*02:01, DQB1*02:02, DQB1*03:01, DQB1*03:02, DQB1*03:03, DQB1*03:19, DQB1*04:01, DQB1*04:02, DQB1*06:01, DQB1*06:02, DQB1*06:03, DQB1*06:04, DQB1*06:09 | 26 | 1 (●) | Kidney | 01:02, 01:04 | 05:02, 05:03 | 01:04, 05:05 | 05:02, 03:01 | Y | / | 14 |
2 ( ) |
Kidney | 01:02 | 05:02 | 03:01, 03:03 | 03:01, 03:02 | Y (dn) | 6 | 2 | |||
| 203I | DQB1*02:01, DQB1*02:02, DQB1*03:01, DQB1*03:02, DQB1*03:03, DQB1*03:19, DQB1*04:01, DQB1*04:02, DQB1*06:01 | 21 | 1 (●) | Kidney | 01:01, 01:02 | 05:01, 06:03 | 02:01, 03:03 | 02:02, 03:01 | Y (dn) | 0.4 | 8 |
2 ( ) |
Heart | 01:01, 01:02 | 05:01, 06:03 | 03:01, 05:01 | 02:01, 02:02 | Y (dn) | 12 | 12 | |||
| 203 V | DQB1*05:01, DQB1*05:02, DQB1*06:02, DQB1*06:03, DQB1*06:04, DQB1*06:09 | 7 | 1 (●) | Heart | 03:03, 04:01 | 03:01, 03:02 | 01:02, 03:02 | 03:03, 05:02 | Y (dn) | 0.7 | 10 |
2 ( ) |
Kidney | 01:03, 05:01 | 02:01, 06:01 | 01:02, 05:01 | 02:01, 06:02 | Y (dn) | 4 | 12 | |||
| 215F | DQA1*01:01, DQA1*01:02, DQA1*01:03, DQA1*04:01, DQA1*05:01, DQA1*05:03, DQA1*05:05, DQA1*06:01 | 14 | 1 (●) | Liver | 02:01, 03:03 | 03:01, 03:03 | 01:01, 05:05 | 03:01, 05:01 | Y | / | 1 |
|
α76V β55PP |
DQA1*03:01, DQA1*03:02, DQA1*03:03/DQB1*03:01, DQB1*03:02, DQB1*03:03 | 1 (●) | / | 01:02 | 06:02, 06:04 | / | / | / | / | 1 | |
| 5 | 2 (×) | Kidney | / | / | 01:04, 02:01 | 02:02, 05:03 | N | / | 4 | ||
3 ( ) |
Heart | 01:02 | 05:02 | 01:02, 02:01 | 02:02, 06:02 | N | / | 13 | |||
4 ( ) |
Kidney | 01:02 | 05:01, 06:09 | 01:04, 03:01 | 03:02, 05:03 | Y | / | 10 | |||
5 ( ) |
Heart | 05:01 | 02:01 | 01:04, 01:05 | 05:01, 05:03 | N | / | 2 | |||
6 ( ) |
/ | 02:01, 05:01 | 02:01, 02:02 | / | / | / | / | 6 | |||
7 ( ) |
Liver | 02:01, 03:03 | 02:02 | 02:01, 05:01 | 02:01, 02:02 | N | / | 7 | |||
Note: /, data not available. N, no. Y, Yes. dn, de novo. In bold, the donor's immunising HLA. Symbols are the same as those used for each patient in the Figures.
3.3. Detection of a Novel DQA1/DQB1 Eplet
An intriguing LSA pattern observed during a patient's immunological follow‐up displayed positivity among five DQ beads specific to the DQA1*03/DQB1*03 combinations. We named this eplet α76Vβ55PP which corresponded to the association of the DQA1 76 V and DQB1 55PP verified eplets targeting the DQA1*03 and DQB1*03 alleles respectively. We identified a total of seven patients in our database presenting this isolated DQA1/DQB1 pattern (Figure 1) in the DQ locus, with an average MFI ± SD at 4117 ± 781. Notably, only five of these patients had been transplanted, and among them, this pattern was a DSA for one (Table 2). For one patient, no HLA typing was available.
3.4. Consistency of Additional LSA Assays
The LSA pattern of each new eplet was confirmed for all the sera with the complementary DQ One Lambda LSA panel, with the exception of 203 V (Figure 2A). For 203 V, one of the two sera was in agreement with the regular panel for the DQ5 and DQ6 beads, but displayed additional positivity against the three DQ2 beads of the complementary DQ panel. Regarding the second serum, it was unexpectedly negative for two beads supposed to express the eplet, but which were associated with a DQA1 chain different from that in the regular panel (DQA1*01:03, with DQB1*05:02 or DQB1*06:04).
FIGURE 2.
Distribution of additional LSA MFI values of DQ beads, for each patient's serum (one symbol per patient). MFI values of LSA beads from the One Lambda complementary DQ (Panel A) and the Lifecodes (Panel B) panels are represented for each patient's serum which displayed the 14 M/116 V, 203I, 203 V, 215F and α76Vβ55PP eplet. Antigens tested are on the vertical axis. Raw MFI—NC refers to raw MFI data after subtraction of the MFI of the negative HLA‐free control bead. The dotted line depicts the positive MFI threshold set at 1000 or 750 units for the complementary DQ and Lifecodes LSA respectively. Beads bearing the eplet are in bold.
With the Lifecodes LSA assay, all the sera targeting 14 M/116 V, 203I and 215F were positive with this reagent (Figure 2B). For 203I, one of the two sera was unexpectedly positive with lower MFI, for the DQA1*01:03/DQB1*06:03 and DQA1*01:02/DQB1*06:04 beads although this eplet was supposed to only target the DQB1*06:01 antigen. Only one serum among the two and seven tested for 203 V and α76Vβ55PP, respectively, was positive.
3.5. Validation of the New Eplets by Serum Adsorption and Elution on Cells
The patients' sera were adsorbed on the HLA‐DQ transfected cell clones or SMCs bearing the eplet of interest, C+ and S+ respectively. The HLA typings of the SMCs used are reported in Table S1. Among the five eplets studied, adsorption for 215F showed that the sole serum we could identify actually contained a combination of antibodies targeting the 52SK and 40GR antibody‐verified eplets that altogether displayed the very same pattern as that of the eplet under study (data not shown).
For the remaining 4 eplets, the HLA antigens expressed on cells for adsorption and average %ads obtained for each eplet were reported in Table 3. Average %ads for 203 V and α76Vβ55PP were significant at −78% and −47% respectively on C+ and at −74% and −82% on S+. Adsorption was not conclusive for C+ or S+ bearing the 14 M/116 V or 203I, because of the saturating MFI values of the sera. For these sera, elutions performed post adsorption on the cell pellets revealed the adsorption of the antibody. As expected for these four eplets, average %ads on clones or SMCs not bearing the eplets were not significant (i.e., above the −20% threshold).
TABLE 3.
Efficiency of the adsorption/elution step for each eplet.
| Eplet | Patients' sera | S+ | Se | S− | C+ | Ce | C− | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| HLA‐DQ | %ads | %elu | HLA‐DQ | %ads | HLA‐DQ | %ads | %elu | HLA‐DQ | %ads | ||
| 14 M/116 V | 1 | A03:01 B03:02 | −14% | 93% | A01:01 B05:01 | −2% | A01:02 B06:02 | −10% | 81% | NT | NT |
| A03:01 B02:01 | −6% | 94% | NT | NT | |||||||
| 2 | −47% | NT | −10% | A01:02 B06:02 | −29% | NT | NT | NT | |||
| A03:01 B02:01 | −20% | 78% | NT | NT | |||||||
| 203I | 1 | A01:03 B06:01, A05:05 B03:01 | −9% | 63% | A01:02 B06:02 | −2% | A02:01 B03:02 | −26% | NT | A01:02 B06:02 | −2% |
| 2 | −20% | 47% | −10% | A03:01 B02:01 | −20% | 70% | −11% | ||||
| A02:01 B03:02 | −28% | NT | |||||||||
| 203 V | 1 | A01:02 B06:02 | −55% | NT | A01:03 B06:01, A05:05 B03:01 | −2% | A01:02 B06:02 | −63% | NT | NT | NT |
| 2 | −93% | NT | −3% | −93% | NT | NT | NT | ||||
| α76Vβ55PP | 1 | A03:03 B03:01, A03:03 B03:02 | −89% | NT | A01:01 B05:01, A05:05 B03:01 | 3% | A03:01 B03:01 | −64% | NT | A03:01/B02:01 | 27% |
| A01:01 B05:01, A03:02 B03:03 | −110% | A05:01 B03:01 | 25% | ||||||||
| 2 | A03:03 B03:01, A03:03 B03:02 | −97% | NT | −19% | −70% | NT | A03:01 B02:01 | 38% | |||
| A05:01 B03:01 | 35% | ||||||||||
| 3 | A03:03 B03:01, A03:03 B03:02 | −86% | NT | −8% | −96% | NT | A03:01 B02:01 | 12% | |||
| A05:01 B03:01 | 42% | ||||||||||
| 4 | A03:03 B03:01, A03:03 B03:02 | −45% | NT | 28% | −36% | NT | A03:01 B02:01 | 54% | |||
| A05:01 B03:01 | 42% | ||||||||||
| 5 | A03:03 B03:01, A03:03 B03:02 | −65% | NT | 14% | −29% | NT | A03:01 B02:01 | 25% | |||
| A05:01 B03:01 | 13% | ||||||||||
| 6 | A03:03 B03:01, A03:03 B03:02 | −82% | NT | −13% | −55% | NT | A03:01 B02:01 | 34% | |||
| A05:01 B03:01 | 21% | ||||||||||
| 7 | A03:03 B03:01, A03:03 B03:02 | −79% | NT | −4% | −61% | NT | A03:01 B02:01 | −2% | |||
| A05:01 B03:01 | −3% | ||||||||||
Note: S+/S−, represents the average percentage of adsorption on SMCs bearing/not bearing the eplet. C+/C−, represented the average percentage of adsorption on clones bearing/not bearing the eplet. Se/Ce, represented the average percentage of elution on SMCs or clones bearing the eplet.
Abbreviation: NT, not tested.
The MFI values after adsorption/elution assays were compared (Figure 3, Figures [Link], [Link], [Link] and S4). A significant difference was observed between average MFINA and MFIads for C+ and S+ for 14 M/116 V (p = 0.0003 and p < 0.0001), 203I (p < 0.0001 and p < 0.0001) and α76Vβ55PP (p < 0.0001 and p < 0.0001). For 203 V, adsorption was significant with the C+ (p = 0.009) but not with S+ (p = 0.45). As expected, no difference occurred after adsorption on clones or SMCS not bearing the eplet for all of them, with the exception of 203I for clones not bearing the eplet (p < 0.0001).
FIGURE 3.
New DQ eplets validated by adsorption/elution assay. MFI values, expressed as Raw MFI—NC, between non‐adsorbed sera (MFINA) and adsorbed or eluted (e) sera on clones (C) or SMCs (S) bearing (+) or not (−) the indicated eplet (C+/S+, Ce/Se and C−/S− respectively). The asterisk represents p < 0.05 for C+ and S+ (One‐Way ANOVA).
3.6. Surface Localisation of These New DQ Eplets by 3D Modelling
A 3D analysis of these eplets on DQ molecules was performed (Figure 4). Residue 14 M appeared near the junction of the two chains and the peptide binding groove. Residue 116 V was not well exposed on the surface, but the dynamic flexibility of the molecule could potentially allow the residue's side chain to be alternately hidden or exposed [21]. However, 116 V was distant from 14 M, suggesting that most probably only one of these residues is responsible for the antibody reactivity.
FIGURE 4.
Localisation of the five candidate eplets on the DQ molecule surface. The DQα chain is coloured in green and the DQβ chain in blue. Each eplet is depicted by a unique colour (orange for 14 M, brown for 116 V, purple for 203 V, yellow for 203I and red for 215F).
Positions 203 and 215 were embedded in the membrane, revealing that they were not exposed in physiological conditions on the surface. However, the 203 residues were located in the upper section of the transmembrane domain, and no other eplet could have explained these two LSA patterns that were observed for a total of four patients.
For the eplet involving both DQ chains, the DQA1 76 and DQB1 55 and 56 positions are very close to each other, strongly suggesting that a single antibody could bind these 3 residues simultaneously through its paratope. Alternatively, 76 V may impact the peptide that could be presented, which could itself contribute to the epitope, instead of directly being in contact with the paratope.
4. Discussion
In this work, we described and validated 3 new DQB1 eplets not referenced in Epregistry to date, and 1 DQA1/DQB1 eplet, by exploiting our previously reported strategy relying on adsorption on cells [16]. Sequence alignments and identification of patients' sera in our database of more than 45,000 patients suggested that these polymorphic residues could represent eplets, although they did not always seem to be reachable at the protein surface according to 3D analysis. However, a clue in favour of the relevance of this modelling approach came from 215F, our fifth candidate. Indeed, although LSA analyses incriminated this residue, despite it being localised deeply in the HLA transmembrane domain, adsorption experiments showed that its LSA pattern was explained by the combination of two distinct antibodies recapitulating the 215F LSA pattern. On the contrary, residue 203, in the upper part of the transmembrane region, was confirmed in all approaches. Overall, only the use of multiple approaches, as we perform according to the strategy we designed, will provide elements solid enough to ascertain the eplet character of an HLA polymorphism.
An eplet is usually larger than one polymorphism [22], and an epitope is even wider; therefore, we cannot exclude that other AAs in the vicinity of the main implicated AA could also contribute, notably for the transmembrane eplets at position 203. Site‐directed mutagenesis should help delineate the polymorphisms localised in this position by focusing on residues close to the cell membrane but not incorporated into it.
For 52R, a dozen sera were identified in our database and we observed that MFI values among the different antigens expressing the eplet were highly but reproducibly heterogeneous depending on the DQ alloantigen having triggered the sensitisation, for all patients. A deeper analysis was therefore performed notably investigating the impact of antibody affinity on the efficiency of antigen binding. This parameter is also capable of impacting our capacity to identify eplets. Given the peculiarity of 52R, and the additional experimental approaches required, we will report its specific features in another manuscript (in preparation).
We attempted to verify the One Lambda LSA results with the Lifecodes LSA assay. However, discrepancies existed for two eplets. For 203 V, only one serum among the two showed positivity, which may be attributed to the relatively low One Lambda LSA MFI values of the serum found negative with Lifecodes. This low signal could possibly be linked to serum dilution as used in this assay. For α76Vβ55P, the limited reactivity (only one positive serum) could stem from structural differences in HLA antigen conformation on the bead surface, as different production methods are used by both manufacturers. Additionally, a variation in the peptide repertoire presented by the HLA molecules, because of the different cellular backgrounds used for antigen production, could also contribute to this discrepancy [11]. We underscore the importance of considering kit‐specific characteristics when interpreting LSA patterns, due to the diversity of the parameters involved (among which are antigen conformation, peptide repertoire, alpha/beta chains combinations…). In addition, differences in affinity of antibodies for the epitopes encompassing the eplets that they target will generate heterogeneity in adsorption efficiency according to the DQ expressed by the cell, and in MFI values among the eplet‐positive beads. Therefore, considering average rather than individual bead results appeared to us more relevant. However, the impact of affinity on antibody/epitope interaction is a major point, which was beyond the scope of the present study, but will be analysed with the DQA1 52R eplet as mentioned above.
Other methods can detect DSA, such as FlowPRA (One Lambda) assay based on beads (i.e., with several HLA alleles per bead, purified from normal human cells), and the classical cross‐match assay performed on cells, especially in its flow cytometry design that can reach a detection level close to that of LSA assays. However, FlowPRA is limited in the panel of alleles available, and flow cross‐match depends on the availability of a large panel of cells to analyse a potentially wide variety of patterns, or of clones expressing only one recombinant HLA antigen. However, as the clones we raised were not in a human background, nonspecific binding of human serum due to so‐called xenoreactive and/or heterophilic antibodies renders the interpretation of the results poorly reliable. Moreover, for eplet validation, adsorption experiments are to us more convincing than testing many LSA assays as each of them will suffer technical limitations that can impact antigen recognition by the antibody. Adsorptions also clarify the possible situation of a mixture of antibodies in serum (as exemplified by 215F) and demonstrate it was not the case for the sera found discordant between the two LSA providers.
These new eplets triggered antibodies that could be adsorbed by two different experimental models: SMCs from deceased donors and a panel of transfected cell clones each expressing only one combination of DQA1 and DQB1 alleles. The former cell source is the gold standard because of the physiological expression of the HLA molecules under study but usually displays two haplotypes, complicating data interpretation and/or being potentially rare and delaying serum analysis until cells with the adequate typing become available, which may not always happen in a reasonable time frame. Therefore, transfected cells represent a valuable option, provided that antigen conformation is adequate and that the antigen expression level is high enough. Although these parameters cannot be controlled a priori, they need to be verified, for example, with sera targeting known verified eplets, as we did in a previous study [16]. Nevertheless, some combinations of DQ alleles were not expressed on the cell surface and therefore several clones were not available for adsorption/elution experiments. Notably, the selection of these cells relied on the eplet‐positive beads for the cells expressing the eplet and the most relevant cell not expressing the eplet that was the closest to the eplet‐expressing antigens. In consequence, combining both cell sources was the option we applied.
In some cases, adsorption was not able to strongly deplete the serum antibody. For the transfected cell clones, this could not be explained by parameters associated with the cells, since their validation had been carried out previously, confirming the correct conformation of the antigen and the correct quantity of cells used for the experiments [16]. The most probable explanation was the very high MFI of the antibody in the serum. Indeed, %ads was underestimated for such sera due to the saturation of the Luminex beads staining and MFI reading. Several rounds of successive adsorption on fresh cells or serial dilutions of pre and post adsorption samples would be required, which we did not perform. Instead, we carried out elution from cells after the adsorption step to confirm the eplet specificity of the adsorbed antibody. However, unlike other studies [23, 24] we do not favour this alternative, as it cannot be excluded that acid elution could alter the bound antibody, possibly leading to annihilate its ability to bind its epitope in the subsequent LSA assay. This did not occur in the present case, but a negative post elution LSA result might not necessarily mean that the antibody had not been successfully adsorbed on the cell and therefore that the eplet does not exist.
At this stage, it is unlikely that new candidate DQ eplets will be discovered through the systematic alignment of AA sequences of LSA antigens, as all possible positions have already been considered by Epregistry and with the present work. Despite this, it is yet possible to improve the delineation of the eplet we already know. Updates will be required as new DQ antigens will probably modify the current list. In addition, despite having a large cohort of sera analysed with LSA, that has already allowed us to validate a significant number of DQ eplets [16], sera from other populations of patients could be very useful. Indeed, additional sera could be identified using databases from other countries, made up of patients from ethnic groups different from our own, and thus presenting different frequencies of the same polymorphisms. Therefore, depending on the patient database studied in a laboratory, the likelihood of identifying patients immunised against eplets considered rare in our population may vary.
Besides a population bias, although the Paris area one is quite cosmopolitan, it is possible that the putative eplets against which no sera were identified are not immunogenic. It is well known that the Epregistry database is not accurate because it collects information obtained in suboptimal experimental conditions [12]. Alternatively, we focused on “pure” sera, but it is not known whether sensitisation against a given eplet could come along with sensitisation against a second one, leaving the first one undetectable with our selection strategy.
Among our candidate eplets was one that we reported as depending on specific DQA1*03 and DQB1*03 combinations. Case reports have been published describing antibodies selectively reacting with specific DQA1/DQB1 combinations [25, 26, 27]. However, to our knowledge, no study has yet performed adsorption assays nor compared different LSA assays to assess the realness of such putative combined eplets. By using several cells expressing either the DQA1*03 chain or the DQB1*03 chain with different combinations of beta or alpha chains, respectively, we were able to ensure that these sera were not targeting the 76 V and 55PP eplet separately, but on the contrary that these two eplets together formed part of the antibody's specificity. 3D modelling also strongly favoured the involvement of these residues in a unique eplet. Consequently, the present work is the first study that investigates this particular DQA1/DQB1 eplet configuration in depth. However, this is not surprising given that the DQA1 and DQB1 loci are distinct from other HLA loci. This uniqueness arises from the presence of two polymorphic chains, which allows for the formation of eplets and epitopes involving both chains as described in this study. Additionally, it has been reported that certain DQ epitopes may also involve the embedded peptide [11], further contributing to the peculiar nature of the DQ antigens.
In conclusion, we identified and verified 3 DQB1 eplets that we named 14 M/116 V, 203I and 203 V respectively, not described so far in Epregistry. A new eplet overlapping the DQA1*03/DQB1*03 allelic combinations was also validated and named α76Vβ55PP according to the verified eplet of each associated chain.
Author Contributions
M.D., T.M. and L.G. performed the research. D.A.R. and C.U. performed the 3D modelling and analysed the structures of the HLA‐DQ proteins. M.D. and J.L.T. designed the research study and wrote the manuscript. T.M., H.M., O.T. and D.L. provided reagents for the study. T.M., L.T., H.M., C.U., M.S.T. and D.L. were involved in critical revision of the article.
Disclosure
This manuscript is the authors' own original work. We ensure the quality and integrity of our research. The paper is not currently being considered for publication elsewhere. The paper reflects the authors' own research and analysis in a truthful and complete manner. All authors have approved the manuscript to publish.
Conflicts of Interest
The authors declare no conflicts of interest.
Supporting information
Figure S1: Distribution of LABScreen LSA MFI values of 14 M/116 V‐positive beads, for each patient serum (numbered 1 and 2). Antigen expressing the eplet are on the horizontal axis, with A and B representing DQA1 and DQB1 loci respectively. MFI values, expressed as Raw MFI—NC, between non‐adsorbed sera (MFINA) and adsorbed or eluted (e) sera on clones (C) or SMCs (S) bearing (+) or not (−) the eplet (C+/S+, Ce/Se and C−/S− respectively) are depicted for each bead (one symbol per condition). The dotted line depicts the positive MFI threshold set at 500.
Figure S2: Distribution of LABScreen LSA MFI values of 203I‐positive beads, for each patient serum (numbered 1 and 2). Antigen expressing the eplet are on the horizontal axis, with A and B representing DQA1 and DQB1 loci respectively. MFI values, expressed as Raw MFI—NC, between non‐adsorbed sera (MFINA) and adsorbed or eluted (e) sera on clones (C) or SMCs (S) bearing (+) or not (−) the eplet (C+/S+, Ce/Se and C−/S− respectively) are depicted for each bead (one symbol per condition). The dotted line depicts the positive MFI threshold set at 500.
Figure S3: Distribution of LABScreen LSA MFI values of 203 V‐positive beads, for each patient serum (numbered 1 and 2). Antigen expressing the eplet are on the horizontal axis, with A and B representing DQA1 and DQB1 loci respectively. MFI values, expressed as Raw MFI—NC, between non‐adsorbed sera (MFINA) and adsorbed or eluted (e) sera on clones (C) or SMCs (S) bearing (+) or not (−) the eplet (C+/S+, Ce/Se and C−/S− respectively) are depicted for each bead (one symbol per condition). The dotted line depicts the positive MFI threshold set at 500.
Figure S4: Distribution of LABScreen LSA MFI values of α76Vβ55PP‐positive beads, for each patient serum (numbered 1 to 7). Antigen expressing the eplet are on the horizontal axis, with A and B representing DQA1 and DQB1 loci respectively. MFI values, expressed as Raw MFI—NC, between non‐adsorbed sera (MFINA) and adsorbed or eluted (e) sera on clones (C) or SMCs (S) bearing (+) or not (−) the eplet (C+/S+, Ce/Se and C−/S− respectively) are depicted for each bead (one symbol per condition). The dotted line depicts the positive MFI threshold set at 500.
Table S1: HLA‐DR and DQ typing of SMCs.
Acknowledgements
We thank the biologists of the St Louis hospital HLA laboratory for their expertise.
Devriese M., Amaya‐Ramirez D., Meng T., et al., “The Hunt for HLA‐DQ Allogeneic Eplets Is Not Over: Four New Ones, Including a Cross‐Chain Eplet,” HLA 106, no. 3 (2025): e70386, 10.1111/tan.70386.
Funding: This work was supported by public funding agencies (ANR EPIHLA AAPG2022 and PHRC‐N ACORGHLA AAP2016 NCTT03861962 to JLT), French Agence de la Biomédecine (AOR 2022), nonprofit organisations (FRM 2022 and Fondation Victor and Erminia Mescle 2022 to MD) and the commercial sector (Research Services Agreement between One Lambda Inc. and the French Law 1901 affiliated Association Alloimmunity and Inflammation).
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Figure S1: Distribution of LABScreen LSA MFI values of 14 M/116 V‐positive beads, for each patient serum (numbered 1 and 2). Antigen expressing the eplet are on the horizontal axis, with A and B representing DQA1 and DQB1 loci respectively. MFI values, expressed as Raw MFI—NC, between non‐adsorbed sera (MFINA) and adsorbed or eluted (e) sera on clones (C) or SMCs (S) bearing (+) or not (−) the eplet (C+/S+, Ce/Se and C−/S− respectively) are depicted for each bead (one symbol per condition). The dotted line depicts the positive MFI threshold set at 500.
Figure S2: Distribution of LABScreen LSA MFI values of 203I‐positive beads, for each patient serum (numbered 1 and 2). Antigen expressing the eplet are on the horizontal axis, with A and B representing DQA1 and DQB1 loci respectively. MFI values, expressed as Raw MFI—NC, between non‐adsorbed sera (MFINA) and adsorbed or eluted (e) sera on clones (C) or SMCs (S) bearing (+) or not (−) the eplet (C+/S+, Ce/Se and C−/S− respectively) are depicted for each bead (one symbol per condition). The dotted line depicts the positive MFI threshold set at 500.
Figure S3: Distribution of LABScreen LSA MFI values of 203 V‐positive beads, for each patient serum (numbered 1 and 2). Antigen expressing the eplet are on the horizontal axis, with A and B representing DQA1 and DQB1 loci respectively. MFI values, expressed as Raw MFI—NC, between non‐adsorbed sera (MFINA) and adsorbed or eluted (e) sera on clones (C) or SMCs (S) bearing (+) or not (−) the eplet (C+/S+, Ce/Se and C−/S− respectively) are depicted for each bead (one symbol per condition). The dotted line depicts the positive MFI threshold set at 500.
Figure S4: Distribution of LABScreen LSA MFI values of α76Vβ55PP‐positive beads, for each patient serum (numbered 1 to 7). Antigen expressing the eplet are on the horizontal axis, with A and B representing DQA1 and DQB1 loci respectively. MFI values, expressed as Raw MFI—NC, between non‐adsorbed sera (MFINA) and adsorbed or eluted (e) sera on clones (C) or SMCs (S) bearing (+) or not (−) the eplet (C+/S+, Ce/Se and C−/S− respectively) are depicted for each bead (one symbol per condition). The dotted line depicts the positive MFI threshold set at 500.
Table S1: HLA‐DR and DQ typing of SMCs.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.