Figure 5 shows that the reactivity of the mAbs across the HLA-I antigens represented in the SAB's panels of 2 lots is not consistent. MFI differs between HLA-I beads lot 8 and lot 9 in the 3 different loci (HLA-A, -HLA-B, and HLA-Cw). The observations point out that lot-to-lot variability exists in the amount of β2aHC and β2fHC on HLA-I beads.
Specific Characteristics of HC-10 Reactivity Against HLA-I Antigens
HC-10 reactivity against HLA-I are characterized into 4 different groups (Table 1):
Group 1. HC-10-reactive antigens on the different SAB: A shared characteristic of the HC-10-reactive antigens is the presence of the amino acid sequence motif 57PEYWDR62 in the α1 HC.
Group 2. HC-10-non-reactive antigens on iBeads only: HC-10-reactive antigens are found on HLA-I beads and acid-treated beads but not to the same antigens on iBeads. This group also bears the sequence 57PEYWDR62.
Group 3. HC-10-reactive antigens on acid-treated beads only: These antigens become reactive to HC-10 upon acid treatment. Interestingly, R62 is either replaced by glycine (G), leucine (L), or asparagine (Q) in the peptide sequence (57PEYWDR62). Upon alkali treatment, HC-10 failed to recognize the antigens with G62 and Q62 (group 3bis).
Group 4. HC-10 nonreactive antigens on all SAB: Several antigens are not recognized by HC-10 on all SAB. R62 is replaced by aspartic acid (E) or Q. Upon alkali-treatment, HC-10 fails to recognize the antigens with E62 and Q62, as well as antigens with G62 (group 4bis).
HC-10 recognizes, on iBeads, the antigens in group 1 but not in group 2. Because the antigens, in both groups, are recognized by W6/32 but not by TFL-006 on iBeads, HC-10 recognize one of the W6/32-positive variant, namely, pepA-β2aHC or pepF-β2aHC. Because HC-10 binds to the antigens in groups 1 and 2 on acid-treated beads, pepF-β2aHC may be the unique variant recognized by HC-10 on iBeads. Because groups 3 and 4 antigens are not recognized by HC-10 on HLA-I beads and iBeads, they are not included for further characterization of the SAB.
Density of β2fHC Coated on HLA-I Beads and iBeads
HLA-I molecules coated on SAB includes: β2aHC, represented by the sum of pepA-β2aHC and pepF-β2aHC, recognized by W6/32; and β2fHC recognized by TFL-006. Since there is no overlap between the HLA-I variants recognized by these 2 mAbs, the percentage of β2fHC for a specific HLA-I antigen on HLA-I beads and iBeads (Figure 6) is calculated as follows:
Most HLA-I beads harbor detectable amounts (MFI > 1000) of β2fHC on their surface. However, no detectable amounts (MFI < 1000) of β2fHC are observed on the beads coated with A*02:01, A*02:06, A*03:01, A*23:01, A*25:01, A*29:02, A*32:01, A*34:02, A*66:02, A*74:01, B15:01, B*27:05, B*27:08, B*42:01, B*49:01, or B*67:01 (boxed antigens in Figure 6). HLA-Cw antigens coated on HLA-I beads always had a MFI greater than 1000, representing greater than 10% of β2fHC coated on the beads' surface. In contrast, iBeads mostly harbor β2aHC, with nondetectable (MFI < 1000) β2fHC. Only the following HLA-A beads, A*11:01, A*24:02 and A*24:03 (dotted boxed antigens in Figure 6), harbor detectable amounts of β2fHC (1000 < MFI < 1500), with a single exception, A*69:01, that has nondetectable β2fHC (MFI < 1000) with density of 8%.
Table 2 shows that, in HLA-I beads, the median percentages of β2fHC in HLA-A, HLA-B, and HLA-Cw loci is 7% ± 5.7%, 11.7% ± 7.5%, and 28.2% ± 10.6%, respectively, while being lower in iBeads (HLA-A = 1.5% ± 1.9%, HLA-B = 0.1% ± 0.3%, HLA-Cw = 0.2% ± 0.5%) and higher in acid-treated beads (HLA-A = 98.3% ± 3.2%, HLA-B = 98.6% ± 2.7%, HLA-Cw = 98.8% ± 1.2%). In HLA-I beads, the density of β2fHC present on the HLA-A beads is significantly lower compared with HLA-B beads (P < 0.005) and HLA-Cw beads (P < 0.0001).
Relative Density of pepF-β2aHC on iBeads
W6/32 binds to both pepA-β2aHC and pepF-β2aHC and HC-10 binds to pepF-β2aHC and β2fHC, suggesting that there is an overlap between the HLA-I conformation recognized by both mAbs. Therefore, the percentage of pepF-β2aHC coated on iBeads is derived only for the HLA-I antigens expressing the antigenic determinant R62 recognized by HC-10 (Table 1, groups 1 and 2), because these antigens on iBeads are free of β2fHC and are coated with HLA-I variants restricted to pepA-β2aHC and pepF-β2aHC. The percentage of pepF-β2aHC for a specific antigen on iBeads (Figure 7) was calculated as follows:
Most beads found in the iBeads' panel have detectable (MFI > 1000) pepF-β2aHC. Only 19 HLA-B and 6 HLA-Cw have nondetectable (MFI < 1000) pepF-β2aHC (boxed-antigens in Figure 7). All HC-10-reactive HLA-A antigens had MFI greater than 1000, demonstrating greater than 10% of β2fHC coated on the beads. Table 2 shows that the median percentage of pepF-β2aHC on HLA-A, HLA-B, and HLA-Cw loci is 21.9% ± 21%, 7.4% ± 9.5%, and 13.2% ± 8.1% respectively. HLA-A antigens have significantly higher density of pepF-β2aHC compared with HLA-B (P < 0.001) and HLA-Cw (P < 0.005).
Ranking of HLA-I Antigen-Coated Beads Based on β2fHC Density in HLA-I Beads
Table 3 shows the ranking of all HLA-I antigens (n = 97) in the HLA-I beads' panel, based on TFL-006 reactivity and categorized in 1000 MFI increment (from 1000 to 9000). This stratification points out the heterogeneous density of β2fHC coated on HLA-I beads and shows how the use of a standard cutoff across all beads may not distinguish anti-β2aHC from anti-β2fHC. Indeed, if 1000 is used, then only 16% of the beads will truly detect anti-β2aHC, the remaining beads may detect both anti-β2aHC and -β2fHC. If 5000 is used, then 88% of the beads will truly detect anti-β2aHC, whereas 72% of the beads may be false negative for anti-β2aHC and 12% may detect both anti-β2aHC and anti-β2fHC. This ranking suggests that each individual bead should have a bead-specific MFI cutoff to critically evaluate and distinguish anti-β2aHC from anti-β2fHC when using HLA-I beads.
Ranking of HLA-I Antigen-Coated Beads Based on pepF-β2aHC Density in iBeads
Table 4 shows the ranking of all HC-10–reactive antigens (n = 74) in the iBeads' panel, based on HC-10 reactivity and categorized in 1000 MFI increments (from 1000 to 8000). The heterogeneity of pepF-β2aHC density on iBeads is obvious, as the MFI of HC-10 against antigens on iBeads ranged from 175 to 7760. Such a categorization is useful for assessing meaningful cutoffs to evaluate the HLA antibodies against pepA-β2aHC or pepF-β2aHC.
T-cell FCXM Using the Anti–HLA-I mAbs
Although W6/32 and TFL-006 can bind to all of the donor's HLA-I antigens and HC-10 only to HLA-B and HLA-Cw ones, Table 5 reveals that W6/32 but not TFL-006 and HC-10 showed reactivity to HLA-I expressed on the surface of resting T cell, confirming the prevalence of pepA-β2aHC on the on the surface of resting T cells (CD4+ or CD8+). Explicitly resting T cells express neither β2fHC nor pepF-β2aHC.
This investigation reveals the distribution and relative density of the conformational variants of the HLA-I on SAB by comparing the reactivities of mAbs, W6/32, TFL-006, and HC-10 on HLA-I beads, iBeads, acid, and alkali-treated beads. Figure 8 elucidates the specificity of the mAbs used, the relative density of the different conformational variants of HLA-I present on the surface of SAB and the variant that is associated with positive FCXM. The assessment of the intrinsic characteristics of the beads is critical for the reliability of virtual XMs in predicting CDCXM and FCXM outcomes and to increase accessibility of donor organs for highly sensitized patients on the waiting list.
The discrepancy between virtual XMs and actual XMs is influenced by the conventionally used SAB (HLA-I beads) for monitoring HLA-I antibodies. HLA-I antibodies in an allograft recipient can detect different conformations of HLA-I,9,11,27 namely, β2aHC and β2fHC. Anti-β2aHC IgGs are associated with AMR and/or graft loss,28,29 whereas anti-β2fHC IgGs may not have a deleterious effect on graft function.9,12,14 These observations influence the assumption that SAB only detect and identify clinically relevant HLA-I antibodies,30 without examining the HLA variants (antigen integrity, conformation, and orientation) on the beads.31
Anti-β2fHC IgGs were documented in nonalloimmunized men, cord blood, and in CDCXM-negative transplant candidates without prior immunizations.8,10,32 Similarly, HLA-I–sensitized kidney recipients had anti-β2fHC IgGs without positive FCXM.11,14 Therefore, HLA-I beads can detect antibodies that may not be clinically relevant, such as anti-β2fHC not associated with a positive FCXM.27,30,33,34
Strategies to minimize the “false SAB reactions” (as implicated by Gombos et al32) are critical in maximizing the specificity of SAB that correlate with positive FCXM results. One approach is to define an MFI cutoff that is associated with transplant outcome, which is challenging to reach.7,30 The 2 aspects of MFI cutoffs are: a cutoff for a positive reaction (or sensitivity) and a cutoff for clinical relevance (or specificity); both contributing to the divergence and nonagreement of a specific “standard” MFI cutoff. Indeed, the MFI cutoffs used can range from 30035 to 6000,36 while 1000 is most common,30,32,37-39 particularly when the sera are used neat or minimally diluted, or in the presence of IgM and other interfering serum factors. Both the sensitivity and the specificity of SAB for detecting serum antibodies is related to the antigenic density of the HLA-I proteins displayed on their surface.40 Our results emphasize that the cutoff should be based on the density of β2fHC, as indicated by the MFI of the mAb TFL-006. Because the MFIs obtained with TFL-006 vary among different antigen-coated beads, a specific cutoff should be attributed for each antigen in the panel of HLA-I beads. Therefore, when an anti–HLA-I DSA is identified, the raw MFI obtained for a DSA should be normalized not only against the negative control bead and a negative control sample, but also against a positive control for DSA reacting to β2fHC, monitored with TFL-006. Only the β2fHC-normalized MFI would represent IgG directed against β2aHC. This approach using a bead-specific MFI cutoff is supported by the findings in literature, where the levels of anti-β2fHC were documented to vary from low (MFI < 1000) to high (MFI > 9000) levels.14,32 In the case of low MFI DSA, when TFL-006 MFI is higher than the DSA's MFI, then there is a possibility of underestimating the DSA, if it recognizes an epitope present in both β2aHC and β2fHC, as described earlier.8,11,14 This limitation is applicable only to HLA-I beads, because this issue is eliminated if iBeads are used because they express predominantly β2aHC.
The other strategy to reduce the amount of “false SAB reactions” is to revive the production of iBeads (by the vendor of HLA-I beads), which eliminates “false positive reactions” caused by the presence of β2fHC on SAB.13 iBeads are not routinely used in clinical settings; however, promising data suggest that iBeads mainly detect anti-β2aHC9,41 associated with positive FCXM.11,14,42 Otten et al9 has described low levels of “denatured” HLA on iBeads, using mAb HC-10. However, our study shows that iBeads bear an even lower density of β2fHC, as assessed by β2fHC-specific mAb TFL-006. Assessing the reactivities of TFL-006 and HC-10 to iBeads shows evidence that HC-10 reacts to the pepF-β2aHC on iBeads. Visentin et al11,14,42 have also noted that DSA against “denatured” HLA can be iBeads-positive, and such DSAs were not associated with AMR, graft loss, and/or positive FCXM. In this context, our results clarify the DSA detected by Visentin et al may be directed against pepF-β2aHC, equivalent to HC-10–positive DSA on iBeads that are FCXM negative. Therefore, the HC-10 reactivity against specific antigens on iBeads will provide the cutoff for monitoring a DSA reacting to the pepF-β2aHC. Therefore, this study recommends the use of iBeads, when made available, in clinical monitoring of anti–HLA-I DSA, because the DSA detected with iBeads is more reliable than those detected with conventional HLA-I beads. Owing to the work of Visentin et al,11,14,42 DSA identified with iBeads have a propensity for allograft rejection and positive FCXM, and this DSA can be considered as pathogenic DSA.
Another aspect, emerging from the results, is the impact of a peptide, loaded in the peptide-binding groove of HLA-I HC, on the ability of HC-10 to bind to HLA-I antigens. It is known that R62 is the antigenic determinant of HC-1020 which is located in the second helix of the α1 domain.43 More precisely, R62 establishes interactions inside the A pocket anchoring the peptide N-terminus (P1), and its orientation could be strongly influenced by the P1 residues of a peptide.44 Although HC-10 recognizes both β2fHC and pepF-β2aHC, these variants are not expressed on resting T cells. Indeed, the deleterious effect of DSA is confined to the recognition of HLA-I HC loaded with peptides by anti–pepA-β2aHC,45 which is supported by the flexibility of the peptide-binding groove in the presence or absence of a peptide26,45,46 and their implication for immune recognition.46
In conclusion, while using HLA-I beads for monitoring DSA, it is important to be aware that the DSA detected by the SAB may or may not correlate with positive FCXM and rejection. Two strategies can reduce the discrepancy between SAB and FCXM results while increasing the concordance of virtual and actual XMs. Table 6 illustrates the strategies to follow while seeking “pathogenic” DSA using HLA-I beads or iBeads: a bead-specific MFI cutoff based on either TFL-006 reactivity with HLA-I beads or that based on HC-10 reactivity with iBeads. Essentially, using a cutoff based on TFL-006 and/or HC-10 MFI enables the normalization of DSA “strength” against β2aHC by removing “false SAB reactions” triggered by the possible presence of HLA antibodies against β2fHC and/or pepF-β2aHC, which are not associated with positive FCXM.
The authors wish to express their sincere thanks to Mrs. Judy Hopfield for her meticulous editing of the manuscript. The authors also wish to thank Dr. Mathew Everly and Dr. Junchao Cai for their comments and criticisms during laboratory meetings and presentation.
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