End-stage-renal disease and kidney transplantation
The kidney is responsible for filtering waste and removes excess of water from your body. When the function of the kidneys is below ten percent of the normal function this is referred to as end-stage renal disease (ESRD). Diabetes, hypertension and chronic kidney disease are the most common causes of ESRD (1)(2). Certain autoimmune conditions, like ANCA associated vasculitis (AAV) and systemic lupus erythematodes (SLE) also contribute to the development of ESRD, as a consequence of progressive glomerulonephritis (3)(4).
Two treatments for ESRD are available: dialysis and kidney transplantation. Dialysis filters the waste out of the blood and takes approximately three to four hours, three times a week. Therefore, the preferred method of treatment is renal transplantation, which is replacing the diseased kidney for a healthy donor organ. Transplantation improves the quality of life and increases survival, compared to patients with long-term dialysis treatment(5).
Human leukocyte antigen system and rejection
However, a major problem after transplantation is rejection. The immune system has effective responses to non-self antigens and this mechanism is also involved in the rejection of transplanted organs. The transplanted organ is recognized as foreign by the recipient’s immune system. Rejection is mainly based on an immune response against major histocompatibility complex (MHC) alloantigens, which are antigens of a distinct cell type from the same species. T-cells are able to recognize the MHC allopeptides directly on surface of donor antigen presenting cells (APC), but also indirectly after the peptides are internalized and processed by recipient APCs as self-restricted allopeptides (Figure 1)(6). Eventually, the T-cells will respond and eradicate the donor cells, which is called T cell mediated rejection (TCMR).
In humans, MHC is referred to as the human leukocyte antigen (HLA) system. HLA encodes for self-recognition molecules that are present on the surface of nucleated cells and are essential for the regulation of the immune system. The HLA system is one of the most polymorphic genetic systems and can be divided in different classes(7). The HLA class I gene produces the peptides HLA- A,- B and -C and these are present on every somatic cell. The HLA class II gene produces the peptides HLA-DR, -DQ and -DP and these are present on antigen presenting cells, like B-lymphocytes, dendritic cells and monocytes, meaning that APCs
have six different HLA molecules (Table 1). To prevent rejection, recipients and donors will be matched for their HLA genes. HLA-DR, followed by HLA-B and –A are the strongest determinants for an allogeneic response (9).
Antibody mediated rejection and donor-specific antibodies
The activation of the humoral alloimmuneresponse, also called antibody mediated rejection (AMR), is one of the major causes of acute and chronic allograft dysfunction and graft loss (10). AMR may occur when the recipient is able to produce donor-specific alloantibodies (DSA), most commonly directed against donor HLA (11). Individuals that have been exposed to non-self HLA molecules in the past and developed HLA-specific antibodies are called ‘sensitized’. Risk factors for HLA sensitization are pregnancy, blood transfusions, and previous organ transplantation (12). Previous transplantation has the strongest immunizing effects on both HLA classes, followed by pregnancy and then transfusion(13).
Sensitization following transplant failure is influenced by maintenance of immunosuppression, and transplant nephrectomy. Sensitization increases following removal of a failed kidney allograft(14). A study of 69 patients with graft failure and immediate withdrawal of immunosuppression showed no difference in DSA at the time of failure (~15%)(15). After 150 days 48 patients had an allograft nephrectomy and the presence of DSA in these patients increased to 35%. Within 5 days following nephrectomy, the DSA rate increased to 66% and at last follow-up it was 81%. Compared to the patients without nephrectomy, these increases were significantly higher(52%). Another study showed that decreasing immunosuppression, independent of nephrectomy, leads to HLA sensitization(16).
MHC region Tissue location HLA gene Alleles
HLA class I Nucleated cells A 3356
HLA class II Antigen presenting cells DRA 7
De novo DSA and non-HLA DSA
Up to 30 % of transplant recipients have HLA-antibodies, but development of de novo donor-specific antibodies (dnDSA) also occurs in 8-25% of recipients after transplantation(18)(19). The development of dnDSA is also correlated with poor graft outcomes, and acute and chronic rejection(20). The majority of dnDSA are directed against class II antigens, especially DQ, which also makes a DQ mismatch a risk factor for developing dnDSA(21).
Besides HLA-specific DSA, there are also antibodies directed toward other donor specific antigens, like MHC class I related chain A (MICA) and B (MICB) (10,11). In recipients who were otherwise well-matched for HLA, these antibodies might contribute to allograft loss(22).
The mechanism primarily responsible for AMR is the activation of the classical pathway of the complement system by the antigen-antibody complex (23). Binding of DSAs to HLA on the donor cell can initiate this pathway. The classical pathway is triggered by activation of the C1-complex, which is composed of one C1q molecule, two C1r molecules and two C1s molecules. C1q is able to bind the Fc region of Immunoglobulin M (IgM) or IgG subclasses 1 and 3 antibodies, which are the subclasses considered to have the strongest complement activating properties (24). When these Fc regions bind the C1 complex, conformational changes in the C1q molecule lead to the activation of C1r molecules, which can cleave C1s. C1s splits C4 and then C2, producing C4a, C4b, C2a, and C2b. A degradation product of C4 is C4d, which binds at the site of the complement activation, usually the vascular endothelium in the renal allograft (Figure 2). C4b and C2a together form the C3-convertase, which cleavages C3 into C3a and C3b. C3b together with the C4bC2a complex forms C5-convertase, which converts C5 into C5a and C5b. Eventually the clustering of complement factors C5b- C9 leads to the formation of a membrane attack complex (MAC), which causes osmotic lysis of the donor cell(23). This will damage the endothelium and eventually the donor organ. The Fc regions of the DSA can also be recognized by Natural Killer (NK)-cells, which can induce cell lysis via antibody dependent cell-mediated cytotoxicity.
Patients with complement-binding DSA after transplantation have the highest risk of graft loss, compared to patients with non-complement binding DSA and patients without DSA (25). However, DSAs are also associated with non-AMR rejection(26). Nevertheless, the presence of DSAs are related to chronic graft dysfunction, graft loss and recipient death (26). The role of IgM DSA in rejection is not clear. They appear to be associated with decreased survival of the allograft (27). However, another study found that an IgM response alone did not affect allograft loss, but rejections appeared to be more severe(28). For this aspect, additional research have to be done.
Since DSAs are associated with a high risk of transplant rejection, screening of DSA in the recipients serum will be done before transplantation. Several assays are developed for DSA screening. The complement-dependent cytotoxicity cross-match (CDC-XM) assay was established for highly sensitive detection of DSAs to recipient HLAs(29). The serum of the recipient will be crossmatched with donor lymphocytes. Then complement will be added and in this way complement fixing DSAs in the serum can be detected. Although the complement fixation is used as read-out, due to the use of rabbit serum as a source of complement, the results are not fully reflective for the human complement fixing capacity of DSAs(7). Because human IgG2 is highly effective in the activation of rabbit complement, DSA subclass and CDC-XM results may not always correlate(30).
The development of high-throughput, single-antigen, bead-based assays is an important tool for identifying patients with complement-fixing DSAs(25). Luminex single antigen bead (SAB) technology is a more sensitive method, whereby purified HLA antigens are bound to inert beads, impregnated with fluoropores (31). If DSAs are present in the serum, they will bind to the coated HLA antigens on the appropriate beads and to complement factor C1q. This binding can be detected with a conjugated secondary antibody specific for human IgG and C4d, that will precipitate on the bead, if C1q is bound. The combination of the fluorescence signals from each bead, indicating the HLA specificity, and the secondary reagent, indicating the bound HLA specific antibodies, can be measured with the Luminex system (32).
Memory and other B cells
Despite screening assays, in some cases patients experience allograft rejection when no alloantibodies were visible during screening. Han et al. determined HLA antibody specificities of ex vivo activated B cells. Supernatants of purified B-cells, cultured with a murine cell line expressing human CD40L, were concentrated and assessed with Luminex SAB assays(33). DSAs were found in 13 of 16 transplant recipients, whereby a total of 50 DSA found by Luminex, matched 35 found in the serum. 11 DSAs were detected in serum, but not in the supernatant. Similar experiments were performed by other research groups(34)(35)(36). These studies raised the idea that memory B cells directed against HLA antigens can be present, despite undetectable levels of serum antibodies towards these antigens. These memory B cells may become activated if a transplantation is performed with an organ expressing these mismatched antigens.
New DSA screening assay
When no DSAs were detected, especially in previous transplanted patients, more than serum antibody screening is needed. Analysis of alloantigen-specific B cells may provide new insights into the capacities of the recipients to produce donor specific antibodies. Therefore, in this study, the first aim is to develop such a screening assay. Lepse et al., developed an antibody stimulating assay, using peripheral blood mononuclear cells (PBMC) from patients with Granulomatosis with polyangiitis (GPA), which is small-vessel vasculitis characterized by circulating anti neutrofilic cytoplasmatic antibodies (ANCA) against proteinase 3 (PR3)(37). In this assay PBMCs were stimulated with IL-21, B cell activating factor (BAFF) and oligodeoxynucleotides containing CpG motifs (CpG-ODN), which is a Toll-like receptor (TLR) 9 ligand to promote autoantibody production in vitro. For this study, this assay will be translated to the transplantation context. Here we try to induce DSA production in vitro by stimulating PBMCs. Thus, instead of exclusively assessing circulating antibodies, alloantigen-specific B-cells producing alloantibodies could be measured, whereby immune monitoring tools before transplantation could be refined.
Toll-like receptors are a family of pattern recognition receptors that recognize various ligands expressed by potentially invading pathogens. All TLRs mediate signal transduction through the MyD88 adaptor, except TLR3, which depends on the TRIF adapter. TLR4 signals via both pathways (38). Most TLRs are expressed on the cell surface, except TLR3, 7/8, and 9. In response to stimulation of TLRs, B cells can be activated and differentiate into antibody secreting cells (ASCs). TLR9 is the best known TLR member expressed in human B cells. TLR9 binds bacterial and viral CpG motifs(39). Besides TLR9, human peripheral blood B cells express high levels of TLR1, TLR6, and TLR10, intermediate levels of TLR7, and low levels of TLR2 and TLR4 (40).
Hanten et al. compared human B cell activation by TLR7, TLR7/8 and TLR9 agonists. TLR7 binds single-stranded RNA and TLR8 recognizes imidazoquinoline compounds and single-stranded RNA(41)(42). All agonists induced IgM and IgG antibody production in human B cells. Interestingly, at a higher concentration the TLR7/8 agonist induced significantly more IgG production than the TLR9 agonist (43). Therefore, it might be interesting to stimulate PBMCs with TLR7 and TLR7/8 agonists for producing DSAs. Activation of PBMCs, using TLR7 and TLR9 agonists, expanded IgM+ memory B cells and plasma cell populations, which resulted in both IgM and IgG production, but favored secretion of IgM (44). Plasmacytoid dendritic cell derived type I IFN is required for TLR7-mediated polyclonal B cell expansion, TLR7 up-regulation, and B cell differentiation towards immunoglobulin-producing plasma cells, but not for TLR9-mediated B cell activation (45).
Although B cells from healthy donors express little to no surface TLR4, analysis of samples from inflammatory disease patients demonstrated that TLR4 expression modestly increased on circulating B cells from periodontal disease and type 2 diabetes patients, and more dramatically increased on B cells from Crohn’s Disease (CD) (46). TLR4 binds lipopolysaccharides (LPS) of Gram-negative bacteria(47), but also endogenous ligands like high-mobility group box-1 (HMGB1)(48). TLR-4 signaling has been shown to play a role in the pathogenesis of renal ischemia-reperfusion injury (IRI), which is an inevitable event during kidney transplantation(49). Another study suggested that activation of innate immunity through TLR4 in the donor kidney contributes to the development of acute rejection after renal transplantation(50), whereby donor TLR4 contributes to graft inflammation and injury following transplantation(51). Thus, IRI enhances the immunogenicity of the graft and TLR stimulation can directly and/or indirectly affect the alloimmuneresponse(52), Via indirect pathways TLR4 stimulation might induce DSA production.
Therefore, in this study stimulation with TLR agonists 4, 7, 7/8 and 9 will be examined for the production of antibodies.
It is also of interest to predict clinical outcomes based on B-cell phenotypes of transplant to phenotypes. Several studies have investigated the role of different B-cell subsets in transplantation patients. Switched B-cells were assessed as predicting factor for acute rejection. Hereby a decrease in naïve B-cell subsets was related to risk of acute rejection(53). In patients with ESRD was found that all B-cell populations significantly reduced, except for transitional B cells(54). Recently, transitional B-cells were associated with protection from rejection (55). Transitional B-cells are immature B-cells that migrate to secondary lymphoid organs from the bone marrow. In lymphoid organs they will maturate into follicular naïve B-cells. If these B-cells become activated by an antigen, they will undergo class-switching and develop into memory B-cells and plasma cells.
Also was found that increased activated naïve B cells are associated with pretransplant HLA immunization and development of posttransplant DSA(56).
Cytomegalovirus (CMV) is one of the most important opportunistic infections in transplant recipients. Donor seropositive and recipient seronegative has the highest risk of CMV infection after transplantation(57)
For this study, flowcytometry will be used to determine the B-cell subsets of patients before transplantation compared to the sub sequential events of rejection or non-rejection. Therefore, the second aim of this study is to prospectively link B-cell phenotyping of patient PBMCs to clinical outcomes, like rejection and infection (CMV). Expected is that patients with more transitional B-cells are longer free of rejection.
Methods & Materials
Cell culture and stimulation
Blood was obtained from healthy controls, renal transplant patients with antibody mediated rejection, cellular rejection and patients with no rejection. PBMCs were isolated from these by centrifugation over Lymphoprep (….) and stored in liquid nitrogen until analysis.
TLR agonists include ultrapure LPS for TLR 4 stimulation, CL264 for TLR7 stimulation, CL097 for TLR7/8 stimulation (Invivogen), and CpG-B DNA (ODN 2006) for TLR9 stimulation (Hycult Biotech). Stimulation was performed in concentrations varying from 0.05- 100 uM. Additional stimulation factors included recombinant human IL-21 (Immunotools) and recombinant human BAFF (Peprotech). Culture media consisted of RPMI 1640 (…) supplemented with gentamycine (..) and 5% FCS (…). PBMCs were cultured in a 24 wells plate at a density of 5 x 10^6/ml. To promote antibody production IL-21, BAFF and a TLR agonist was added and cultured for 12 days.
Total IgG levels in the supernatant after 12 days were quantified using IgG ELISA, developed with TMB and stopped with sulfuric acid. The plate was read using the SoftMax Pro
DSA levels in the supernatant were measured using Luminex single-bead antigen assay. The amount of antibodies with specificity to HLA antigens and complement fixing antibodies were quantified.
B-cell phenotyping and Flow cytometry
PBMCs B-cells were stained with surface markers. Cells were analyzed using an LSR…. The following flow cytometry panel was used to define: B lymphocytes (CD19+); naïve B cells (CD19+CD27-CD38+/-); activated naïve B cells (CD19+CD27-CD38+); resting naïve B-cells (CD19+CD27-CD38-); transitional B-cells (CD19+CD24hiCD38hi) and (CD19+CD27-CD38hi); Transitional 1 B-cells (CD19+CD24hiCD38hi); Transitional 2 B-cells (CD19+CD24++CD38++); non-class-switched memory B-cells (CD19+CD27+IgD+IgM+); class-switched memory B cells (CD19+CD27+IgD-IgM-CD38+/-); Plasmablasts and plasma cells (CD19+CD27+CD38hi)
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