Immunologic risk stratification of pediatric heart transplant patients by combining HLA-EMMA and PIRCHE-II.

Human leukocyte antigen (HLA) molecular mismatch is a powerful biomarker of rejection. Few studies have explored its use in assessing rejection risk in heart transplant recipients. We tested the hypothesis that a combination of HLA Epitope Mismatch Algorithm (HLA-EMMA) and Predicted Indirectly Recognizable HLA Epitopes (PIRCHE-II) algorithms can improve risk stratification of pediatric heart transplant recipients. Class I and II HLA genotyping were performed by next-generation sequencing on 274 recipient/donor pairs enrolled in the Clinical Trials in Organ Transplantation in Children (CTOTC). Using high-resolution genotypes, we performed HLA molecular mismatch analysis with HLA-EMMA and PIRCHE-II, and correlated these findings with clinical outcomes. Patients without pre-formed donor specific antibody (DSA) (n=100) were used for correlations with post-transplant DSA and antibody mediated rejection (ABMR). Risk cut-offs were determined for DSA and ABMR using both algorithms. HLA-EMMA cut-offs alone predict the risk of DSA and ABMR; however, if used in combination with PIRCHE-II, the population could be further stratified into low-, intermediate-, and high-risk groups. The combination of HLA-EMMA and PIRCHE-II enables more granular immunological risk stratification. Intermediate-risk cases, like low-risk cases, are at a lower risk of DSA and ABMR. This new way of risk evaluation may facilitate individualized immunosuppression and surveillance.

Evaluation of 19 years of international external proficiency testing for high-resolution HLA typing.

The international high-resolution external proficiency testing (EPT) started in 2004 with high-resolution typing of human leucocyte antigen (HLA) class I (HLA-A,B,C) and HLA class II (HLA-DRB1, DRB345, DQB1, and DPB1) alleles, since possibilities for such an EPT within Europe were limited and all existing EPTs at that time made use of the comparison of HLA typing results without a reference. This EPT was set up as a collaboration between the HLA laboratory of Leiden, providing DNA samples to the participants, and the laboratory of Maastricht, performing the high-resolution typing as the reference result and evaluating the results of all participants according to the prevailing European Federation for Immunogenetics (EFI) standards. Once a year, 12 samples were sent to the participating laboratories, and evaluation and certificates were provided at the end of that same year. During the years, the EPT was extended to low-resolution HLA class I and II typing, high-resolution typing including DQA1 and DPA1, and allelic resolution typing for HLA class I, the latter one being unique in this field. Evaluation of the high-resolution typing results of the last 19 years showed a clear increase in the number of loci tested by the participating laboratories and a clear change of method from Sanger sequencing with additional other techniques (SSO/SSP) to the nowadays widely used next-generation sequencing method. By strictly using the EFI rules for high-resolution HLA typing, the participants were made aware of the ambiguities within exons 2 and 3 for class I and exon 2 for class II and the presence of null alleles even in a two-field HLA typing. There was an impressive learning curve, resulting in >98% correctly typed samples since 2017 and a 100% fulfillment of EFI rules for all laboratories for all loci submitted in the last 2 years. Overall, this EPT meets the need of an EPT for high-resolution typing for EFI accreditation.

Soluble HLA-G blood levels are not increased during ongoing pregnancy in women with a history of recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) affects 1-2 % of couples who are trying to conceive. At some point, some couples do maintain a healthy pregnancy to term, but the underlying mechanism of RPL remains elusive. Human leukocyte antigen (HLA)-G is an immune modulatory molecule. Our group previously showed increased HLA-G levels in the decidua of term pregnancies after RPL, while other studies showed reduced soluble HLA-G (sHLA-G) blood levels in women with RPL. This led us to investigate sHLA-G levels in blood of women with RPL who had either a subsequent pregnancy loss (RPL-pregnancy loss) or a healthy term pregnancy (RPL-live birth), and compare these to healthy control pregnancies and non-pregnant controls. Soluble HLA-G concentrations were quantified by ELISA. Women with healthy term pregnancy had increased sHLA-G levels compared to non-pregnant controls. In contrast, RPL-live birth women at term did not have increased blood sHLA-G levels. Soluble HLA-G levels remained stable between first and third trimester. Interestingly, when comparing first trimester samples of RPL-live birth to RPL-pregnancy loss, sHLA-G levels also did not significantly differ. High sHLA-G levels in blood seem not to be crucial for an ongoing healthy pregnancy after RPL. However, since it was previously shown that women with RPL-live birth have increased HLA-G levels in term decidua compared to control pregnancies, the current data suggest that local and systemic immune regulation are not necessarily in concert. Further study of the contribution of fetus-derived HLA-G and HLA-G of maternal origin may provide more insight in the pathophysiology of RPL.

Effect of seminal plasma on dendritic cell differentiation in vitro depends on the serum source in the culture medium.

Dendritic cells (DCs) are key in shaping immune responses and are recruited to the human cervix after coitus by seminal plasma (SP). SP has been shown to skew the differentiation of monocyte-derived DCs towards an anti-inflammatory profile when cultured in medium containing fetal calf serum (FCS). Here, we confirmed that SP skewed DCs cultured in fetal bovine serum (FBS) towards a tolerogenic profile. To create a setting more similar to the in vivo situations in humans, we tested the immune regulatory effect of SP on DCs in cell cultures containing human serum (HS). SP-DCs cultured in HS did show increased CD14 and decreased CD1a, indicating an inhibited maturation phenotype. Gene expression of TGF-ß and IL-10 and IL-10 protein expression were elevated in LPS-activated SP-DCs, whereas IL-12p70 protein levels were decreased compared to LPS-activated control DCs. In contrast to FBS culture conditions, in the presence of HS co-cultures of SP-DCs with allogeneic peripheral blood mononuclear cells (PBMCs) did not result in decreased T cell proliferation and inflammatory cytokine production. Thus, under HS culture conditions SP can skew the differentiation of monocyte-derived DCs phenotypically towards alternatively activated DCs, but this immune regulatory phenotype is functionally less pronounced compared to SP-treated DCs cultured in FBS containing medium. These findings highlight the importance of the source of the serum that is used in SP treated cell cultures in vitro.

The development of preeclampsia in oocyte donation pregnancies is related to the number of fetal-maternal HLA class II mismatches.

In oocyte donation (OD) pregnancy, a fetus can be completely allogeneic to the recipient. Consequently, the maternal immune system has to cope with greater immunogenetic dissimilarity compared to naturally conceived pregnancy. Previously, we showed an association between successful OD pregnancy and lower immunogenetic dissimilarity, reflected by the number of fetal-maternal Human Leukocyte Antigen (HLA) mismatches, than expected by chance. In this study we aimed to determine whether the development of preeclampsia in OD pregnancies is related to the number of fetal-maternal HLA mismatches. A retrospective, nested case-control study was performed within a cohort of 76 singleton OD pregnancies. Maternal and fetal umbilical cord blood was typed for HLA-A, -B, -C, -DR and -DQ, and the number of fetal-maternal HLA mismatches was calculated. In addition, the incidence of child-specific HLA antibodies was determined. 13 pregnancies were complicated by preeclampsia. To demonstrate an influence of HLA mismatches on the development of preeclampsia, a univariate logistic regression analysis was performed adjusted for maternal age and socio-economic status. A significant association between the number of fetal-maternal HLA class II mismatches and the development of preeclampsia was observed (OR = 3.8, 95 % CI: 1.6-9.0; p = 0.003). This association was not linked to the development of HLA class II antibodies. According to our findings, an increased number of HLA class II mismatches is a risk factor for the development of preeclampsia in OD pregnancies. The effect of HLA class II mismatches might be explained by the induction of a cellular rather than a humoral immune response.

Reciprocal HLA-DR allogenicity between mother and child affects pregnancy outcome parameters.

Successful pregnancy outcome depends on local immunoregulatory mechanisms preventing a detrimental immune response towards the semi-allogeneic fetus. We investigated the influence of HLA-DR (in)compatibility on pregnancy outcome parameters in 480 women. The parameters tested were birth weight, individualized birthweight ratio (IBR), gestational age, and maternal highest diastolic blood pressure. Irrespective of pregnancy complications, maternal-fetal HLA-DR incompatibility resulted in increased IBR. We conclude that reciprocal HLA-DR allogenicity between mother and child positively affect pregnancy outcome parameters.

Presence of intragraft B cells during acute renal allograft rejection is accompanied by changes in peripheral blood B cell subsets.

B cells have various functions, besides being plasma cell precursors. We determined the presence of intragraft B cells at time of acute rejection (AR) and looked for correlates of B cell involvement in peripheral blood. Renal biopsies at time of AR or stable graft function were analysed for the presence of B cells and B cell-related gene expression, as well as C4d staining. Peripheral blood B cell subset distribution was analysed at various time-points in patients with AR and controls, alongside serum human leucocyte antigen (HLA) antibodies. AR was accompanied by intragraft CD20+ B cells, as well as elevated CD20 (MS4A1) and CD19 gene expression compared to controls. B cell infiltrates were proportional to T cells, and accompanied by the chemokine pair C-X-C motif chemokine ligand 13 (CXCL13)-C-X-C motif chemokine receptor 5 (CXCR5) and B cell activating factor (BAFF). Peripheral blood memory B cells were decreased and naive B cells increased at AR, in contrast to controls. While 22% of patients with AR and 5% of controls showed de-novo donor-specific antibodies (DSA), all biopsies were C4d-negative. These results suggest a role for B cells in AR by infiltrating the graft alongside T cells. We hypothesize that the shift in peripheral blood B cell composition is related to the graft infiltration at time of AR.

Differential effects of donor-specific HLA antibodies in living versus deceased donor transplant.

The presence of donor-specific anti-HLA antibodies (DSAs) is associated with increased risk of graft failure after kidney transplant. We hypothesized that DSAs against HLA class I, class II, or both classes indicate a different risk for graft loss between deceased and living donor transplant. In this study, we investigated the impact of pretransplant DSAs, by using single antigen bead assays, on long-term graft survival in 3237 deceased and 1487 living donor kidney transplants with a negative complement-dependent crossmatch. In living donor transplants, we found a limited effect on graft survival of DSAs against class I or II antigens after transplant. Class I and II DSAs combined resulted in decreased 10-year graft survival (84% to 75%). In contrast, after deceased donor transplant, patients with class I or class II DSAs had a 10-year graft survival of 59% and 60%, respectively, both significantly lower than the survival for patients without DSAs (76%). The combination of class I and II DSAs resulted in a 10-year survival of 54% in deceased donor transplants. In conclusion, class I and II DSAs are a clear risk factor for graft loss in deceased donor transplants, while in living donor transplants, class I and II DSAs seem to be associated with an increased risk for graft failure, but this could not be assessed due to their low prevalence.

A Memory B Cell Crossmatch Assay for Quantification of Donor-Specific Memory B Cells in the Peripheral Blood of HLA-Immunized Individuals.

Humoral responses against mismatched donor HLA are routinely measured as serum HLA antibodies, which are mainly produced by bone marrow-residing plasma cells. Individuals with a history of alloimmunization but lacking serum antibodies may harbor circulating dormant memory B cells, which may rapidly become plasma cells on antigen reencounter. Currently available methods to detect HLA-specific memory B cells are scarce and insufficient in quantifying the complete donor-specific memory B cell response due to their dependence on synthetic HLA molecules. We present a highly sensitive and specific tool for quantifying donor-specific memory B cells in peripheral blood of individuals using cell lysates covering the complete HLA class I and class II repertoire of an individual. Using this enzyme-linked immunospot (ELISpot) assay, we found a median frequency of 31 HLA class I and 89 HLA class II-specific memory B cells per million IgG-producing cells directed at paternal HLA in peripheral blood samples from women (n = 22) with a history of pregnancy, using cell lysates from spouses. The donor-specific memory B cell ELISpot can be used in HLA diagnostic laboratories as a cross-match assay to quantify donor-specific memory B cells in patients with a history of sensitizing events.

Defining the immunogenicity and antigenicity of HLA epitopes is crucial for optimal epitope matching in clinical renal transplantation.

Transplantation of an human leukocyte antigen (HLA) mismatched graft can lead to the development of donor-specific antibodies (DSA), which can result in antibody mediated rejection and graft loss as well as complicate repeat transplantation. These DSA are induced by foreign epitopes present on the mismatched HLA antigens of the donor. However, not all epitopes appear to be equally effective in their ability to induce DSA. Understanding the characteristics of HLA epitopes is crucial for optimal epitope matching in clinical transplantation. In this review, the latest insights on HLA epitopes are described with a special focus on the definition of immunogenicity and antigenicity of HLA epitopes. Furthermore, the use of this knowledge to prevent HLA antibody formation and to select the optimal donor for sensitised transplant candidates will be discussed.

Regulatory B cells: Phenotype, function and role in transplantation.

While B cells are traditionally known for their roles in antibody production, antigen presentation and cytokine production, recent studies have highlighted the existence of B cells with regulatory properties, which have been termed Bregs, analogous to regulatory T cells (Tregs). Bregs have been found to play a role in autoimmune disease, malignancies, infections, and may also be involved in solid organ transplantation. Their main mechanism of action is by promoting the development of Tregs while suppressing effector CD4+ and CD8+ T cells, primarily by IL-10 secretion. In the field of transplantation evidence for an active role of Bregs is scarce. While the presence of Bregs has been associated with improved graft survival and operational tolerance in kidney transplant recipients, these findings are not without controversy. Since the majority of fundamental research on Bregs has been performed in the fields in autoimmunity and infectious diseases, we will first focus on what these fields taught us on basic Breg biology, after which the relevance for the transplant setting is discussed.

How can we reduce costs of solid-phase multiplex-bead assays used to determine anti-HLA antibodies?

Solid-phase multiplex-bead assays are widely used in transplantation to detect anti-human leukocyte antigen (HLA) antibodies. These assays enable high resolution detection of low levels of HLA antibodies. However, multiplex-bead assays are costly and yield variable measurements that limit the comparison of results between laboratories. In the context of a Dutch national Consortium study we aimed to determine the inter-assay and inter-machine variability of multiplex-bead assays, and we assessed how to reduce the assay reagents costs. Fifteen sera containing a variety of HLA antibodies were used yielding in total 7092 median fluorescence intensities (MFI) values. The inter-assay and inter-machine mean absolute relative differences (MARD) of the screening assay were 12% and 13%, respectively. The single antigen bead (SAB) inter-assay MARD was comparable, but showed a higher lot-to-lot variability. Reduction of screening assay reagents to 50% or 40% of manufacturers' recommendations resulted in MFI values comparable to 100% of the reagents, with an MARD of 12% or 14%, respectively. The MARD of the 50% and 40% SAB assay reagent reductions were 11% and 22%, respectively. From this study, we conclude that the reagents can be reliably reduced at least to 50% of manufacturers' recommendations with virtually no differences in HLA antibody assignments.

What is wrong with the regulatory T cells and foetomaternal tolerance in women with recurrent miscarriages?

Couples of whom the woman has had a miscarriage have two major concerns: the cause and possible risk of recurrence. Unfortunately, a significant proportion of cases of recurrent miscarriage (RM) remain unexplained despite detailed investigation. Because data suggest that regulatory T cells (Treg) are involved in the maternal acceptance of the allogeneic foetus, RM could possibly be explained by a disturbance of the Treg network. The possible role of Tregs in RM is described in this review, as well as their potential application in diagnostics and therapeutic intervention trials.

Beneficial Immune Effects of Myeloid-Related Proteins in Kidney Transplant Rejection.

Acute rejection is a risk factor for inferior long-term kidney transplant survival. Although T cell immunity is considered the main effector in clinical acute rejection, the role of myeloid cells is less clear. Expression of S100 calcium-binding protein A8 (S100A8) and S100A9 was evaluated in 303 biopsies before and after transplantation from 190 patients. In two independent cohorts of patients with acute rejection (n = 98 and n = 11; mostly cellular rejections), high expression of S100 calcium-binding protein A8 (S100A8) and A9 (S100A9) was related to improved graft outcome. Mechanisms of action of the S100 molecules were investigated. In the graft and peripheral blood cells, S100A8 and S100A9 expression correlated with myeloid-derived suppressor markers. In line with this finding, recombinant S100A8 and S100A9 proteins inhibited maturation and the allogeneic T cell stimulatory capacity of dendritic cells. S100A9 enhanced the production of reactive oxygen species by macrophages, which suppressed T cell activity at low concentrations in the form of hydrogen peroxide. Intragraft S100A8 and S100A9 expression linked to reduced expression of T cell immunity and tissue injury markers and higher expression of immune regulatory molecules. This study sheds new light on the importance of myeloid cell subsets in directing the outcome of T cell-mediated acute rejection.

Polyclonal B cell activation for accurate analysis of pre-existing antigen-specific memory B cells.

The enzyme-linked immunospot (ELISPOT) assay is a widely used tool for enumeration of antigen-specific memory B cells in several disciplines, such as vaccination, cancer immunotherapy and transplantation. For the accurate estimation of antigen-specific memory B cell frequencies, a well-defined B cell activation protocol is pivotal. In this study, we aimed to characterize a polyclonal B cell activation protocol to facilitate optimal monitoring of antigen-specific memory B cell frequencies. Total, naive and memory B cells were activated polyclonally with an α-CD40 monoclonal antibody, cytosine-phosphate-guanine (CPG) oligodeoxynucleotide (ODN) 2006, interleukin (IL)-2, IL-10 and IL-21. Polyclonal activation of B cells resulted in equal cell death ratios in naive and memory B cells. When tested in an antigen-specific system, immunoglobulin (Ig)G spots were detected only in the memory fraction. There was no change in B cell polyclonality due to in-vitro activation. Our data show that the current polyclonal activation protocol may be used reliably to estimate the frequency of memory B cells in ELISPOT assays.

B cell repopulation after alemtuzumab induction-transient increase in transitional B cells and long-term dominance of naïve B cells.

In organ transplantation, the composition of the B-cell compartment is increasingly identified as an important determinant for graft outcome. Whereas naïve and transitional B cells have been associated with long-term allograft survival and operational tolerance, memory B cells have been linked to decreased allograft survival. Alemtuzumab induction therapy effectively depletes B cells, but is followed by rapid repopulation up to levels exceeding base line. The characteristics of the repopulating B cells are currently unknown. We studied the phenotypic and functional characteristics of B cells longitudinally in 19 kidney transplant recipients, before and at 6, 9 and 12 months after alemtuzumab induction therapy. A transient increase in transitional B cells and cells with phenotypic characteristics of regulatory B cells, as well as a long-term dominance in naïve B cells was found in alemtuzumab-treated kidney transplant recipients, which was not influenced by conversion from tacrolimus to sirolimus. At all time-points after treatment, B cells showed unaltered proliferative and IgM-producing capacity as compared to pretransplant samples, whereas the ability to produce IgG was inhibited long-term. In conclusion, induction therapy with alemtuzumab results in a long-term shift toward naïve B cells with altered phenotypic and functional characteristics.

A NOVel ELISPOT assay to quantify HLA-specific B cells in HLA-immunized individuals.

Quantification of the humoral alloimmune response is generally achieved by measuring serum HLA antibodies, which provides no information about the cells involved in the humoral immune response. Therefore, we have developed an HLA-specific B-cell ELISPOT assay allowing for quantification of B cells producing HLA antibodies. We used recombinant HLA monomers as target in the ELISPOT assay. Validation was performed with human B-cell hybridomas producing HLA antibodies. Subsequently, we quantified B cells producing HLA antibodies in HLA-immunized individuals, non-HLA-immunized individuals and transplant patients with serum HLA antibodies. B-cell hybridomas exclusively formed spots against HLA molecules of corresponding specificity with the sensitivity similar to that found in total IgG ELISPOT assays. HLA-immunized healthy individuals showed up to 182 HLA-specific B cells per million total B cells while nonimmunized individuals had none. Patients who were immunized by an HLA-A2-mismatched graft had up to 143 HLA-A2-specific B cells per million total B cells. In conclusion, we have developed and validated a highly specific and sensitive HLA-specific B-cell ELISPOT assay, which needs further validation in a larger series of transplant patients. This technique constitutes a new tool for quantifying humoral immune responses.

Calcineurin inhibitors affect B cell antibody responses indirectly by interfering with T cell help.

In general, humoral immune responses depend critically upon T cell help. In transplantation, prevention or treatment of humoral rejection therefore require drugs that ideally inhibit both B cell and T helper cell activity. Here, we studied the effects of commonly used immunosuppressive drugs [tacrolimus, cyclosporin, mycophenolic acid (MPA) and rapamycin] on T cell helper activity and on T cell-dependent B cell responses. T cells were activated polyclonally in the presence of immunosuppressive drugs in order to analyse the effect of these drugs on T cell proliferation, co-stimulatory ligand expression and cytokines. The impact of immunosuppressive drugs on T cell-dependent immunoglobulin production by B cells was addressed in T-B cell co-cultures. All drugs affected T cell proliferation and attenuated T cell co-stimulatory ligand (CD154 and CD278) expression when T cells were activated polyclonally. Tacrolimus, cyclosporin and rapamycin also attenuated B cell stimulatory cytokine mRNA levels in T cells. As a consequence, a decrease in immunoglobulin levels was observed in autologous T-B cell co-cultures, where T cell help is essential for immunoglobulin production. In contrast, when pre-activated T cells were used to stimulate autologous B cells, calcineurin inhibitors failed to inhibit B cell immunoglobulin production, whereas MPA and rapamycin did show inhibition. From these studies, it is evident that calcineurin inhibitors affect the humoral immune response by interfering with T helper signals, but not by targeting B cells directly. Furthermore, our studies support the necessity of intervening in T cell helper function to attenuate humoral responses.

Intravenous immunoglobulin preparations have no direct effect on B cell proliferation and immunoglobulin production.

Intravenous immunoglobulin (IVIg) is used for treatment of a variety of immunological disorders and in transplantation. As one of its applications in transplantation is the reduction of donor specific antibodies in the circulation, we examined the direct effect of IVIg on essential parameters of human B cell responses in vitro. Purified human B cells, human B cell hybridomas and T cells were cultured in the presence of graded concentrations of IVIg to test its effect on their proliferative capacity. To address the effect of IVIg on immunoglobulin production, we designed a novel technique making use of quantitative polymerase chain reaction to assess IgM and IgG levels. IVIg failed to inhibit proliferation of human B cells and human B cell hybridomas. In contrast, when IVIg was added to T cell cultures, a dose-dependent reduction of the proliferative capacity was observed. IVIg did not affect the levels of IgM and IgG mRNA of activated B cells. Our data show that IVIg is not capable of directly inhibiting key B cell responses. Direct B cell inhibition by IVIg seems therefore unlikely, implying that alteration in humoral immunity by IVIg is due to indirect effects on T cells and/or interactions with circulating antibodies and complement factors.

Bortezomib affects the function of human B cells: possible implications for desensitization protocols.

Bortezomib is a potent inducer of apoptosis in malignant as well as non-malignant human plasma cells. Recently, bortezomib has come to attention for the treatment of humoral rejection. As bortezomib is a proteasome inhibitor, it likely affects other cell types, such as activated B cells, as well. Since additional anti-B cell effects could be beneficial for the treatment of humoral rejection, we tested whether bortezomib inhibited human B cell function. When B cells were activated in a CD40 mAb driven culture system, bortezomib dose-dependently abrogated their IgM and IgG production as well as their proliferation. This bortezomib induced inhibition was caused by induction of apoptosis, since levels of caspase 3/7 activity were increased. In addition to its effects on plasma cells, bortezomib profoundly inhibits activated human B cells. This finding suggests that when bortezomib is used for desensitization or for the treatment of humoral rejection, there is no need for additional anti-B cell therapy, such as anti-CD20 mAb (Rituximab) treatment.