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.

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.

Pretransplantation Donor-Recipient Pair Seroreactivity Against BK Polyomavirus Predicts Viremia and Nephropathy After Kidney Transplantation.

Kidney transplant donors are not currently implicated in predicting BK polyomavirus (BKPyV) infection in kidney transplant recipients. It has been postulated, however, that BKPyV infection originates from the kidney allograft. Because BKPyV seroreactivity correlates with BKPyV replication and thus might mirror the infectious load, we investigated whether BKPyV seroreactivity of the donor predicts viremia and BKPyV-associated nephropathy (BKPyVAN) in the recipient. In a retrospective cohort of 407 living kidney donor-recipient pairs, pretransplantation donor and recipient sera were tested for BKPyV IgG levels and correlated with the occurrence of recipient BKPyV viremia and BKPyVAN within 1 year after transplantation. Donor BKPyV IgG level was strongly associated with BKPyV viremia and BKPyVAN (p < 0.001), whereas recipient BKPyV seroreactivity showed a nonsignificant inverse trend. Pairing of high-BKPyV-seroreactive donors with low-seroreactive recipients resulted in a 10-fold increased risk of BKPyV viremia (hazard ratio 10.1, 95% CI 3.5-29.0, p < 0.001). In multivariate analysis, donor BKPyV seroreactivity was the strongest pretransplantation factor associated with viremia (p < 0.001) and BKPyVAN (p = 0.007). The proportional relationship between donor BKPyV seroreactivity and recipient infection suggests that donor BKPyV seroreactivity reflects the infectious load of the kidney allograft and calls for the use of pretransplantation BKPyV serological testing of (potential) donors and recipients.

Allo-HLA Cross-Reactivities of Cytomegalovirus-, Influenza-, and Varicella Zoster Virus-Specific Memory T Cells Are Shared by Different Healthy Individuals.

Virus-specific T cells can recognize allogeneic HLA (allo-HLA) through TCR cross-reactivity. The allospecificity often differs by individual (private cross-reactivity) but also can be shared by multiple individuals (public cross-reactivity); however, only a few examples of the latter have been described. Because these could facilitate alloreactivity prediction in transplantation, we aimed to identify novel public cross-reactivities of human virus-specific CD8+ T cells directed against allo-HLA by assessing their reactivity in mixed-lymphocyte reactions. Further characterization was done by studying TCR usage with primer-based DNA sequencing, cytokine production with ELISAs, and cytotoxicity with 51 chromium-release assays. We identified three novel public allo-HLA cross-reactivities of human virus-specific CD8+ T cells. CMV B35/IPS CD8+ T cells cross-reacted with HLA-B51 and/or HLA-B58/B57 (23% of tetramer-positive individuals), FLU A2/GIL (influenza IMP[58-66] HLA-A*02:01/GILGFVFTL) CD8+ T cells with HLA-B38 (90% of tetramer-positive individuals), and VZV A2/ALW (varicella zoster virus IE62[593-601] HLA-A*02:01/ALWALPHAA) CD8+ T cells with HLA-B55 (two unrelated individuals). Cross-reactivity was tested against different cell types including endothelial and epithelial cells. All cross-reactive T cells expressed a memory phenotype, emphasizing the importance for transplantation. We conclude that public allo-HLA cross-reactivity of virus-specific memory T cells is not uncommon and may create novel opportunities for alloreactivity prediction and risk estimation in transplantation.

Virus-Specific CD8(+) T Cells Cross-Reactive to Donor-Alloantigen Are Transiently Present in the Circulation of Kidney Transplant Recipients Infected With CMV and/or EBV.

T cells play a dual role in transplantation: They mediate transplant rejection and are crucial for virus control. Memory T cells generated in response to pathogens can cross-react to alloantigen, a phenomenon called heterologous immunity. Virus-specific CD8(+) T cells cross-reacting to donor-alloantigen might affect alloimmune responses and hamper tolerance induction following transplantation. Here, we longitudinally studied these cross-reactive cells in peripheral blood of 25 kidney transplant recipients with a cytomegalovirus and/or Epstein-Barr virus infection. Cross-reactive T cells were identified by flow cytometry as virus-specific T cells that proliferate in response to donor cells in a mixed-lymphocyte reaction. In 13 of 25 patients, we found cross-reactivity to donor cells for at least 1 viral epitope before (n = 7) and/or after transplantation (n = 8). Cross-reactive T cells were transiently present in the circulation, and their precursor frequency did not increase following transplantation or viral infection. Cross-reactive T cells expressed interferon-γ and CD107a in response to both alloantigen and viral peptide and resembled virus-specific T cells in phenotype and function. Their presence was not associated with impaired renal function, proteinuria, or rejection. In conclusion, virus-specific T cells that cross-react to donor-alloantigen are transiently detectable in the circulation of kidney transplant recipients.

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.

HLA epitopes as viewed by antibodies: what is it all about?

The need for new approaches to define HLA antibodies, in the context of organ transplantation, is intensely debated among HLA professionals. In this review, we sought to provide background and perspective to current understanding of the immunogenicity of HLA mismatches with respect to the humoral alloimmune response and the definition of B cell epitopes. Initial data suggest that epitope matching not only assists in defining better matches for the current transplant, but also minimizes the risk of developing de novo HLA-donor-specific-antibodies posttransplant. In other words, other than lowering the risk of current graft rejection, epitope matching is likely to lower overall future sensitization levels and thus increases the likelihood of finding a compatible donor when the need for a retransplantation arises. More detailed knowledge of epitopes makes it possible to investigate what constitutes permissible versus non-permissible HLA mismatches. The currently available evidence suggest that epitope matching is the most rational way to decrease the risk of HLA-linked transplant rejection. This review is aimed at stimulating further and more intense collaborative effort in this field.

Cell-Free DNA: An Upcoming Biomarker in Transplantation.

After organ transplantation, donor-derived cell-free DNA (ddcfDNA) can be detected in the recipient's blood and urine. Different ddcfDNA quantification techniques have been investigated but a major breakthrough was made with the introduction of digital droplet PCR and massive parallel sequencing creating the opportunity to increase the understanding of ddcfDNA kinetics after transplantation. The observations of increased levels of ddcfDNA during acute rejection and even weeks to months before histologic features of graft rejection point to a possible role of ddcfDNA as an early, noninvasive rejection marker. In this review, we summarize published research on ddcfDNA in the transplantation field thereby elaborating on its clinical utility.

Should HLA mismatch acceptability for sensitized transplant candidates be determined at the high-resolution rather than the antigen level?

Defining HLA mismatch acceptability of organ transplant donors for sensitized recipients has traditionally been based on serologically defined HLA antigens. Now, however, it is well accepted that HLA antibodies specifically recognize a wide range of epitopes present on HLA antigens and that molecularly defined high resolution alleles corresponding to the same low resolution antigen can possess different epitope repertoires. Hence, determination of HLA compatibility at the allele level represents a more accurate approach to identify suitable donors for sensitized patients. This approach would offer opportunities for increased transplant rates and improved long term graft survivals.

Follicular T helper cells and humoral reactivity in kidney transplant patients.

Memory B cells play a pivotal role in alloreactivity in kidney transplantation. Follicular T helper (Tfh) cells play an important role in the differentiation of B cells into immunoglobulin-producing plasmablasts [through interleukin (IL)-21]. It is unclear to what extent this T cell subset regulates humoral alloreactivity in kidney transplant patients, therefore we investigated the absolute numbers and function of peripheral Tfh cells (CD4(POS) CXCR5(POS) T cells) in patients before and after transplantation. In addition, we studied their relationship with the presence of donor-specific anti-human leucocyte antigen (HLA) antibodies (DSA), and the presence of Tfh cells in rejection biopsies. After transplantation peripheral Tfh cell numbers remained stable, while their IL-21-producing capacity decreased under immunosuppression. When isolated after transplantation, peripheral Tfh cells still had the capacity to induce B cell differentiation and immunoglobulin production, which could be inhibited by an IL-21-receptor-antagonist. After transplantation the quantity of Tfh cells was the highest in patients with pre-existent DSA. In kidney biopsies taken during rejection, Tfh cells co-localized with B cells and immunoglobulins in follicular-like structures. Our data on Tfh cells in kidney transplantation demonstrate that Tfh cells may mediate humoral alloreactivity, which is also seen in the immunosuppressed milieu.