Human leukocyte antigen (HLA) molecules are not only cell-bound, but they can also be identified in a soluble form . These soluble HLA (sHLA) molecules are known to have an immunomodulatory function .
Stable concentrations of sHLA class I (sHLA-I) molecules circulate in all individuals. The sHLA levels in human plasma, measured using enzyme-linked immunosorbent assay (ELISA), have been reported as 1.01±0.72 mg/L for sHLA-I and 1.53±2.44 mg/L for sHLA class II (sHLA-II) . Some individuals have substantially higher concentrations of sHLA-I than others, dividing the general population into high and low sHLA-I groups; HLA-A23 and HLA-A24 (A9) are associated with higher serum concentrations of sHLA-I .
Donor-specific HLA alloantibodies (DSAs) play an important role in graft rejection after solid organ transplantation. In cord blood or hematopoietic stem cell transplantation, the presence of DSAs is also significantly associated with engraftment failure [5, 6].
Since van Rood
In this study, we intended to demonstrate the neutralizing capacity of natural sHLA circulating in donor peripheral blood. Such demonstration would facilitate research using natural sHLA as a therapeutic desensitizing agent in donor plasma or in IVIG preparations.
This study was conducted using 149 HLA crossmatches for kidney transplantation at Kyungpook National University Hospital (Daegu, Republic of Korea) (Table 1). Multiparas were selected as recipients, and previous sensitizers of the recipients (husbands, sons, or daughters) were selected as potential donors who had sensitized the recipients during their pregnancies. In this report, these donors are called “previous sensitizers,” and where donors had not been associated with the participating recipients via any previous sensitizing events, such as pregnancy, transfusion, or transplantation, they are called “non-sensitizers,” and these donors were selected as negative controls (N=6).
Whole blood for FCXM was requested by physicians for kidney transplantation, and was collected in plain and heparin tubes from 149 pairs of recipients and their potential donors after informed consents were obtained. Negative control or third-party sera were obtained from non-transfused healthy blood group AB males. Only discarded samples remaining after conventional FCXM were obtained and used for our study. The samples were anonymized before the data were analyzed. Our study was approved by the Institutional Review Board of our hospital.
The cutoff value of this MFI ratio used to differentiate between positive and negative was 2.0 for both T and B cells.
The MFI ratios of the mixed sera (recipient+donor) were compared to those of the diluted sera (with either RPMI or third-party serum) (Fig. 3). Lower MFI ratios of the mixed sera relative to the diluted sera suggest that the neutralizing effects of the sHLA were superior to the matrix effects. In addition, lower MFI ratios of the diluted sera relative to the mixed sera suggest that the matrix effects were superior to the neutralizing effects.
Statistical analyses were performed using SPSS Statistics, version 23 (IBM Corp., Armonk, NY, USA). Comparisons between the two groups were performed using the Wilcoxon signed-rank test. Statistical significance was defined as a
In the conventional FCXM between multiparous recipients and previous sensitizers, the positivity rate was 51.7% (74/143) (Table 2). The criteria used to differentiate DSAs between class I and class II are also summarized in Table 2.
We initially considered the dilution effects of the recipient sera with RPMI (Fig. 4). The majority of the MFI ratios of the diluted sera in the inhibition FCXM were lower than those of the neat sera in the conventional FCXM. Generally, MFI values linearly reflected the DSA levels in recipient sera. When the MFI ratios of neat sera were more than 10 or less than 2, the dilution effects were not apparent. This may be because the relationship between the MFI ratio and the corresponding DSA level is not linear within these ranges.
To avoid confusion caused by dilution effects, the mixed sera (recipient+donor) were compared only to the diluted sera (with RPMI or third-party serum). The inhibition FCXM experiments for verifying the neutralizing effects of a previous sensitizer’s serum are summarized in Fig. 5 and Table 3.
1. Negative FCXM with non-sensitizers or previous sensitizers as donors: The sera of non-sensitizers (N=6) did not show any neutralizing effects. In negative FCXM, even with previous sensitizers as donors (N=69), matrix effects of RPMI were apparent in both T cell and B cell FCXM.
2. Matrix effects in any positive FCXM (T cell or B cell) with previous sensitizers as donors (N=74): Matrix effects of RPMI were evident in the B cell FCXM with class I DSAs alone (N=14), and in the T cell FCXM with any positive DSA (class I or class II, N=74). Matrix effects of third-party sera were shown in the T cell FCXM with class II DSAs alone (N=27), in the T cell FCXM with any positive DSA (class I or class II, N=74), and in the B cell FCXM for total previous sensitizers (N=143).
3. Neutralizing effects in any positive FCXM (T cell or B cell) with previous sensitizers as donors (N=74): In the group with class I DSAs alone (N=14), neutralizing effects were evident in the T cell FCXM compared to the sera diluted with RPMI. In the group with class II DSAs alone (N=27), no neutralizing effects were seen in the B cell FCXM. In the group with both class I and class II DSAs (N=33), neutralizing effects were apparent in both FCXMs (T cell and B cell) regardless of diluents (RPMI or third-party serum). In the group with any positive DSA (class I or class II DSAs, N=74), neutralizing effects were evident in the B cell FCXM only, regardless of the diluent (RPMI or third-party serum). Overall, the neutralizing effects of sHLA in the serum of a previous sensitizer on DSAs in a recipient serum were confirmed for sHLA-I but not for sHLA-II.
In this study, natural sHLA in donor serum was demonstrated to neutralize DSAs in recipient serum. In particular, the neutralizing effects were verified for sHLA-I but not for sHLA-II, although it has been reported that both sHLA-I and sHLA-II can be measured using ELISA . However, our finding is consistent with the previous observation that plasma exchange with IVIG can eliminate class I DSAs more easily than class II DSAs . To the best of our knowledge, this is the first report where FCXM has been used to demonstrate the neutralizing effects of sHLA. These potentially beneficial effects of sHLA infused via blood-derived products should be considered for desensitization of highly HLA-sensitized patients.
We considered that the most efficient way of revealing neutralization effects of sHLA would be by comparing the diluted serum with third-party serum (inducing minimal matrix effects) and the mixed serum. Thus, attention was given to the following five FCXM measurement conditions in the current study: 1, 2) T cell and B cell FCXM for sera with DSA class I alone; 3) B cell FCXM for sera with DSA class II alone; and 4, 5) T cell and B cell FCXM for sera with DSA of both classes. As expected, both the mean and median values of MFI ratios for the mixed sera were always lower than those for the corresponding diluted sera (Table 3, bold fonts) in all five cases; two cases showed statistically significant differences.
An MFI ratio in FCXM reflects the DSA level in the recipient serum, and this is more accurate in T cells than in B cells . Class II DSAs can be detected in B cell FCXM only. This may explain why neutralizing effects could not be demonstrated for sHLA-II in our study. In other words, we failed to measure and show the effects of sHLA-II in B cell FCXM, whereas sHLA-II should be present at any level in donor serum, and thus neutralize class II DSAs in recipient serum.
In our study, diluent matrix effects were seen with third-party serum as well as RPMI. This may be because we used third-party sera of blood group AB that had low total IgG levels. Indeed, individuals with blood group AB have been reported to often have relatively low total IgG levels .
For histocompatibility antigen-mismatched skin grafts in rats, pretransplant infusion of donor serum has been shown to significantly prolong graft survival . In human liver transplantation, some studies have suggested that tolerance may be associated with the formation of immune complexes involving sHLA-I shed from the transplanted liver . The donor sHLA in liver transplant recipients may be capable of removing the alloreactive T cells from circulation , since sHLA-I is capable of inhibiting all core active CD8+ T cells . Liver-kidney transplant recipients with high concentrations of soluble HLA-G in their sera have been reported to have low numbers of acute rejection episodes, potentially requiring less immunosuppressive therapy . In blood transfusions, sHLA-I could contribute to the immunosuppressive effects .
Fortunately, HLA molecules per se are very poor immunogens, unless they are presented on live lymphocytes . If the T cell receptor or CD4/CD8 of a given T cell is simply blocked or engaged without receiving the second signal (co-stimulation) to become fully activated, the T cell leads to cell apoptosis and sHLA-II promotes suppressive activity in CD4+ T cells [20, 21]. Good evidence is being found using the desensitization protocol (plasma exchange with IVIG) for ABO-incompatible organ transplantation. When fresh frozen plasma (FFP) of group AB is used as a replacement fluid in ongoing immunosuppressive drug therapy, soluble ABH substance in the FFP can neutralize recipient ABO antibodies without evoking
Pretransplant donor-specific transfusion (DST) has either a beneficial effect of improving graft survival or a detrimental effect of inducing sensitization . The exact mechanism by which DST induces the beneficial effect has remained a mystery . One possibility is that pre-exposure to alloantigen results in the induction of regulatory cells with the capacity to control the effector arm of the immune response . Alternatively, as deduced from our study, sHLA in donor plasma may have the capacity to neutralize DSAs and eliminate alloreactive T cells. Therefore, pretransplant or posttransplant infusion of donor plasma, from which cellular components have been completely removed, might be an efficient approach to antigen-specific immunomodulatory therapy under concomitant administration of immunosuppressive drugs.
IVIG preparations block cytotoxic DSAs
As sHLA-I are detectable in blood components , both sHLA-I and sHLA-II are also detectable in blood-derived products, such as IVIG, factor VIII, albumin, and prothrombin complex concentrate. However, the sHLA-I and sHLA-II concentrations in these products are unequivocally below the mean sHLA concentrations in human plasma . For example, the concentration of sHLA in albumin and IVIG is more than 50-fold lower than that in normal human serum, but considerable differences exist between products of different manufacturers [19, 27]. Therefore, if given in a sufficient volume, these preparations may trigger immunomodulatory activities [3, 27, 28].
In a previous report, sHLA-II concentration in IVIG preparations exceeded that found in the plasma of healthy humans, suggesting that the extraction procedure may concentrate not only immunoglobulins but also sHLA-II molecules . Therefore, the process of manufacturing IVIG preparations from pooled human plasma should be improved to maintain or concentrate sHLA molecules in IVIG to enhance their therapeutic efficacy for desensitization protocols.
Maternal formation of anti‐HLA alloantibodies to paternal HLAs is estimated to occur in 7–39% of pregnant women . In our study, the positivity rate of multipara FCXM with previous sensitizers was 51.7%. Currently, neither FFP nor platelet concentrates from multiparous donors, which are associated with a high risk of inducing transfusion-related acute lung injury, are transfused anymore. To avoid the neutralization of sHLA by these maternal antibodies, we suggest that plasma from males or from non-multiparous non-sensitized females should be pooled to prepare IVIG as a desensitizing agent.
In conclusion, our study, using inhibition FCXM, demonstrated that natural sHLA-I in donor serum neutralizes DSAs in recipient serum. To use sHLA as an antigen-specific immunomodulatory or desensitizing agent, further clinical trials of pretransplant or posttransplant donor plasma infusion are required, along with further research to enhance sHLA concentrations in IVIG manufactured from pooled human plasma.
No potential conflicts of interest relevant to this article were reported.