Interpretation of the HLA epitopes—Continuous dilution method for detection of high‐titer IgG HLA antibody

Abstract Background The presence or absence of human leukocyte antigen (HLA) antibodies, especially the strength of donor‐specific HLA antibodies (DSAs), has important roles in clinical evaluation and diagnostic decision‐making for solid‐organ transplantation. Dilution patterns help to give a new sight of HLA epitopes. “Epitope matching” is likely to lower the risk of developing DSA and increase the likelihood of matching a compatible donor. Methods We collected data evaluating HLA antibodies with a titration study using mean fluorescence intensity. Results Diluting the serum of recipients can reduce potential inhibitory effects, accurately evaluate the intensity of donor‐specific HLA antibodies, and guide surgeons to take or not take intervention measures. Dilution patterns also help to give a new sight of HLA epitopes. Conclusion We believe that from the viewpoint of HLA antibodies, the dilution model can provide new tools and insights for the study of HLA epitopes.


| INTRODUC TI ON
Donor-specific human leukocyte antigen (HLA) antibodies before or after transplantation may have different effects based on antibody strength. This feature may increase the risk of antibody-mediated rejection and lose valuable opportunities for transplantation, affect graft survival, and even lead to graft failure. A history of sensitization before renal transplantation, including a history of blood transfusion, pregnancy, secondary transplantation, and other risk factors, may lead to the production of donor-specific HLA antibodies (DSAs) in the body. 1 Why some recipients develop DSAs to their HLA-mismatched donor and others do not is not known. Recently, "epitope matching" has entered the lexicon, but what contributes to antigenic epitopes is incompletely understood.
HLAMatchmaker identifies the number of differences in amino acid sequences between the donor and recipient HLA antigens, which are called "eplets". HLAMatchmaker has been used in several studies to calculate "eplet loads" (ie, the overall difference in HLA class-II amino acid sequences between donors and recipients). Tambur   Human leukocyte antigen typing was undertaken using a LABType ® kit (One Lambda). Testing of HLA antibody was undertaken using a LifeCodes ® Single Antigen kit (Immucor). Both methodologies followed manufacturer guidelines, as described previously. 4 The MFI of most of the SABs of our patient was >15 000, and the pattern of the cross-reaction group was disordered ( Figure 1).

Testing of the neat antibody indicated strong interference effects,
with MFI values not correlating with the true strength of the antibody. In the specific reaction of an antigen and antibody, only when the proportion of antigen and antibody is appropriate can an antigen-antibody complex be formed. If the amount of antibody is much larger than that of antigen, the binding of antigen and antibody is not at an optimal proportion. The continuous dilution method was used to describe a peak MFI > 5000 in which 24 HLA-A and -B-coated SABs were positive. Different antibodies have different dilution patterns, which may be of higher or lower strength than the initial test.
These factors led to the prozone phenomenon in serum to have distinct effects on different antibodies.
We divided all loci into several specific clusters, which are revealed separately in Figure 2A-D. The latter shows quite unique dilution patterns for each cross-reactive group. Figure Figure 2B shows the patient developing antibodies to a HLA-A2 cross-reactive group.
The four quite similar dilution curves reached a peak MFI of titer 1:4.
During titration, the antibody strength weakened gradually. There were several types of decomposition products of A*19, including A*29, A*31, and A*32. All antibodies in Figure 2C illustrated seemingly similar inhibitory effects, and all had a peak MFI of ~23 000.  44Q, 44K, 45m, and 46e), 149A + 150V + 151H and 158V. The epitope symbol is 44KM3, which indicates the possibility of three different epitopes. These epitopes can be predicted as recognition sites of specific antibodies and have been verified by experiments with informative specific antibodies. Each HLA antigen has its own unique combination of epitopes, but at the same time, many of these epitopes are shared with other HLA antigens. Through interpretation of the cross-reaction group and antibody-dilution model, the antigenic epitopes described in Table 1 located in the same cross-reaction group were found to be quite similar.   An almost identical dilution pattern indicated that these sites had a common epitope. Figure 4D shows that two HLA-DQ alleles had strong inhibition in undiluted samples. The peak MFI of DQA1*01:03/ DQB1*06:01 and DQA1*01:03/DQB1*06:03 was ~13 000, both.
When the antibody was diluted to a certain concentration, the antibody titer out (turned negative). Some features seemed to follow the current convention of naming HLA-DQ antibodies.

TA B L E 1
The epitope specificity of HLA antibodies

| D ISCUSS I ON
In recent years, explaining what contributes to an HLA B-cell epitope has proved to be limited and inadequate, as has understanding of the immunogenicity and antigenicity of mismatched HLA epitopes. The concept of epitopes on different HLA antigens has been extended to structural eplets. These epitopes themselves are determined by a small number of adjacent amino acids. 5 The high level of sharing of antigenic epitopes among HLA molecules is the reason why HLA antibodies produced by individuals exposed to a single HLA antigen can also react with other, unexposed HLA antigens. Compared with a single different antigen, it is more accurate to regard any given HLA molecule as a common and unique set of antigenic epitopes. 6 We provided data suggesting that MFI values for neat antibody Dilution methods are recommended to detect HLA antibodies in highly sensitized patients undergoing a secondary transplant.
Kosmoliaptsis and colleagues 7 pointed out that the exact mechanism of the interference effects observed in detection of monoclonal antigens using the Luminex platform is not completely clear, and that it may be multifactorial. Compared with serum-pretreatment methods such as ethylenediamine tetraacetic acid and C1q status, 8 the dilution method can better solve the prozone phenomenon.
Epitope matching minimizes the risk of developing de novo HLA-DSAs after transplantation. Therefore, solving the mystery of HLA B-cell epitopes, understanding HLA epitopes, and deciphering the factors that determine the antigenicity and immunogenicity of epitopes may elicit substantial changes to solid-organ transplantation.
We believe that from the viewpoint of HLA antibodies, the dilution model can provide new tools and insights for the study of HLA epitopes.

E TH I C A L A PPROVA L
Written informed consent was obtained from the patient to use his data. Approval from the ethics board of Tianjin First Central Hospital was not required.