N.M. van Besouw

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.

The direct and indirect allogeneic presentation pathway during acute rejection after human cardiac transplantation

Alloreactive T cells may be activated via a direct or an indirect antigen presentation pathway. We questioned whether the frequency of interferon (IFN)‐γ producing cells determined by enzyme‐linked immunospot (ELISPOT) assay is an effective tool to monitor the direct and/or indirect presentation pathway. Secondly, we wondered whether early and late acute rejection (AR) are associated with both pathways. Before (n = 15), during (n = 18) and after (n = 16) a period of AR, peripheral blood mononuclear cell (PBMC) samples were tested from 13 heart transplant recipients. The direct presentation pathway was always present. The number of IFN‐γ producing cells reactive to this pathway increased significantly (P = 0·04) during AR and the number decreased (P = 0·005) after AR therapy. In contrast, the indirect allogeneic presentation pathway was present in only eight of 18 AR samples. When the indirect presentation pathway was detectable, it increased significantly during AR. Five of eight of these AR occurred more than 6 months after transplantation. The ELISPOT assay, enumerating alloreactive IFN‐γ producing cells, is a valuable tool to determine the reactivity via both the direct and the indirect presentation pathway. The direct presentation pathway always plays a role in AR, while the indirect pathway contributes especially to late AR.

Down-regulated donor-specific T-cell reactivity during successful tapering of immunosuppression after kidney transplantation

Stable cadaveric renal transplant patients were routinely converted from cyclosporin A (CsA) to either azathioprine (AZA) or mycophenolate mofetil (MMF) 1 year after transplantation to reduce the side effects of long‐term immunosuppressive therapy. Thereafter, the AZA and MMF dose was gradually tapered to 50% at 2 years after transplantation. We questioned whether a reduction of immunosuppressive treatment results in a rise of donor‐specific T‐cell reactivity. Before transplantation (no immunosuppression), 1 year (high dose immunosuppression) and 2 years (low dose immunosuppression) after transplantation, the T‐cell reactivity of peripheral blood mononuclear cells (PBMC) against donor and third‐party spleen cells was tested in mixed lymphocyte cultures (MLC) and against tetanus toxoid (TET) to test the general immune response. We also measured the frequency of donor and third‐party reactive helper (HTLpf) and cytotoxic (CTLpf) T‐lymphocyte precursors in a limiting dilution assay. Donor‐specific responses, calculated by relative responses (RR = donor/third‐party reactivity), were determined. Comparing responses after transplantation during high dose immunosuppression with responses before transplantation (no immmunosuppression), the donor‐specific MLC‐RR (P = 0·04), HTLp‐RR (P = 0·04) and CTLp‐RR (P = 0·09) decreased, while the TET‐reactivity did not change. Comparing the responses during low dose with high dose immunosuppression, no donor‐ specific differences were found in the MLC‐RR, HTLp‐RR and CTLp‐RR, although TET‐reactivity increased considerably (P = 0·0005). We observed a reduction in donor‐specific T‐cell reactivity in stable patients after renal transplantation during in vivo high dose immunosuppression. Tapering of the immunosuppressive load had no rebound effect on the donor‐specific reactivity, while it allowed recovery of the response to nominal antigens.

A Multiplex Bead Array Analysis to Monitor Donor-Specific Cytokine Responses After Withdrawal of Immunosuppression in HLA-Identical living Related Kidney Transplant Patients

Immune reactivity after HLA-identical living related (LR) kidney transplantation can be caused by minor histocompatibility antigen and non-HLA antigen mismatches between donor and recipient. In our center, HLA-identical LR kidney transplant recipients receive azathioprine (AZA) or mycophenolate mofetil (MMF) in combination with corticosteroids for 1 year after transplantation. Thereafter, AZA or MMF was withdrawn, and the patients were treated with steroid monotherapy as maintenance therapy. We questioned whether withdrawal of AZA or MMF affected the donor-specific lymphocyte proliferation and cytokine production. Donor and third-party T-cell reactivities were determined by mixed lymphocyte reactions and by cytokine production using multiplex bead array technique. The donor and third-party proliferative capacities were not affected after withdrawal of AZA or MMF. Thirteen of 17 cytokines were detected by the multiplex bead array technique. No differences were observed after third-party induced cytokine production after withdrawal of AZA or MMF. However, production of donor-specific interferon-gamma and macrophage inflammatory protein-1beta increased after discontinuation of AZA or MMF, but no clinically relevant acute rejection was observed. In conclusion, after HLA-identical LR kidney transplantation, donor-specific cytokine responses can be found when AZA or MMF therapy is discontinued. The clinical relevance of this phenomenon is still not evident.

The frequency and avidity of committed cytotoxic T lymphocytes (cCTL) for donor HLA class I and class II antigens and their relation with graft vascular disease

Cellular immune processes may trigger the development of graft vascular disease (GVD). CD4 and CD8 cytotoxic T lymphocytes that infiltrate the allograft could play a role in the development of GVD. We studied the presence of in vivo primed or committed CTL (cCTL) and their avidity for donor HLA class I and class II antigens in graft‐infiltrating lymphocyte cultures propagated from endomyocardial biopsies derived from patients with and without signs of GVD. The fraction of cCTL with high avidity for HLA class I or class II antigens was estimated by the addition of anti‐CD8 or anti‐CD4 MoAbs to the cytotoxic phase of the limiting dilution analysis. In the first year after transplantation no difference in the frequency of donor‐specific class I cCTL between patients with and without GVD was found. Addition of anti‐CD8 MoAb revealed that most cultures predominantly consisted of cCTL with low avidity for donor HLA class I antigens, irrespective of the development of GVD at 1 year after transplantation. However, in patients who did not develop GVD, the frequency of cCTL with donor HLA class II specificity was significantly higher than in patients who did develop GVD. The avidity for donor HLA class II antigens was comparable in both groups. A high frequency of donor‐specific cCTL for HLA class II antigens seems to be a protective factor against the development of GVD. These cCTL might be cytotoxic for cells involved in GVD development, e.g. activated endothelium and smooth muscle cells of donor origin.

Nonspecific immune reactivity of peripheral blood mononuclear cells is related to graft vascular disease

I N HEART transplant patients, high responses of donorspecific mixed lymphocyte culture (MLC) reactivity’ --’ or incrcascd numbers of IL-2 producing helper T-lymphocytcs (HTL)’ have been reported in relation to acute rcjcction. chronic rejection or in patients with stable graft. Thcsc tests also have been used to identify patients with donor-specific proliferative hypeor hypcr-reactivity.? 7 We I-eport hcrc on the relation of graft vascular disease (GVD) to donor-specific reactivity of peripheral blood mononuclear cells (PBMC) in the MLC and the HTL-assay as well as following ;I nonspecitic stimulation with tetanus toxoid (TET).

IMPAIRED VARICELLA ZOSTER VIRUS SPECIFIC MEMORY T-CELL RESPONSES IN KIDNEY TRANSPLANT PATIENTS: 976

Introduction: Primary infection with varicella zoster virus (VZV) causes varicella, and VZV reactivation results in herpes zoster. Compared to healthy individuals, the incidence and severity of herpes zoster is higher in transplant recipients. VZV-specific memory T-cells are considered to prevent VZV reactivation. DCs are essential to present the cognate VZV antigens to T-cells. We questioned whether mature monocyte-derived dendritic cells (moDCs) from renal transplant patients are more susceptible to VZV than those from healthy controls, and whether phenotype and frequency of circulating VZV-specific T-cells and VZV-specific IgG titres in kidney transplant recipients are comparable with controls. Methods: Patients and controls had experienced varicella during childhood and had no signs of herpes zoster. CD3+ T-cells and monocytes (CD14+) were isolated from PBMC from adult kidney transplant recipients, 2 years after transplantation and on Tacro and MMF therapy, and healthy gender and age matched volunteers served as controls. Monocytes were differentiated into mature moDCs and infected with VZV. T-cells were co-cultured with autologous VZV-infected moDCs and subjected to flowcytometric analysis to identify the phenotype (i.e. naïve (NA: CCR7+CD45RO-), central (CM: CCR7+CD45RO+) and effector memory (EM: CCR7-CD45RO+) T-cells) and the frequency of VZV reactive T-cell subsets (i.e. IFN-g+). Results: The VZV-specific IgG titres were comparable between transplant recipients and healthy individuals. In whole blood the number of CD4 (p=0.006) and CD8 (p=0.03) T-cells were significantly lower in transplant recipients compared to healthy individuals. The proportion of CD4 and CD8 cells within the CD3+ T-cells were comparable between transplant recipients and controls. Also, the proportion NA, CM and EM within the CD4 and CD8 cells were comparable between transplant recipients and controls. There was no difference in grade of VZV infection between moDCs from transplant recipients and controls. No difference was found in the percentage of VZV-specific CD4 NA, CM and EM T-cells between transplant recipients and controls. However, the percentage of VZV-specific CD8 memory T-cells was lower in patients compared to controls (p=0.07). This impairment was significant in the CD8 EM T-cell pool (p=0.05). Remarkably, lower number of transplant patients (5 of 11) had circulating VZV-specific CD8 EM T-cells compared to controls (10 of 13). In contrast to the CD8 CM T-cells (rs=0.42, p=0.16), the frequency of VZV-specific CD8 EM T-cells population was inversely correlated with the age of healthy individuals (rs=-0.53, p=0.06), suggesting that transplant patients have ‘aged’ VZV-specific CD8 EM T-cells. Conclusion: Mature moDCs from transplant recipients and healthy individuals can be similarly infected by VZV. The VZV related complications after transplantation results from an impaired VZV-specific memory T-cell response. Prophylactic VZV vaccination before transplantation might boost the patient’s memory T-cell repertoire and thereby reduces the morbidity associated with herpes zoster after transplantation.

Donor-specific CTL frequencies in peripheral blood in relation to graft vascular disease after clinical heart transplantation

Abstract Cellular mechanisms may play a role in the development of graft vascular disease (GVD). We previously demonstrated that GVD correlated with an increase of donor‐specific T‐helper 1 cytokine production by graft‐infiltrating lymphocytes but not by peripheral blood mononuclear cells (PBMC). These T‐helper 1 cytokines aid the generation of cytotoxic T‐lymphocytes (CTL). In the present report, we investigated whether there is a relationship between the frequency of donor‐specific CTL precursors (pCTL) in PBMC and the development of GVD. We tested PBMC samples of five patients with GVD and five patients without GVD in the periods 3–6 months, 1 year, and 3 years after heart transplantation. At all time points, GVD was not related to the number of pCTL. In conclusion, donor‐specific cellular tests in peripheral blood could not be related to GVD. Apparently, donor‐specific reactions associated with the induction of GVD can only be monitored in the graft.

Patterns in donor-specific mRNA and protein production of Th1 and Th2 cytokines by graft-infiltrating lymphocytes and PBMC after heart transplantation

Abstract  We used RT‐PCR and ELISA to study the kinetics of IL‐2 (Thl) and IL‐4 (Th2) both on mRNA and protein level from “naive” PBMC and “primed” graft‐infiltrating lymphocytes (GIL) obtained from a heart transplant recipient. For this purpose, these cells were stimulated for 1–48 h with donor and control third‐party antigens. Only stimulation of GIL with donor‐specific antigen resulted in early detectable IL‐2 and IL‐4 mRNA and protein levels. Maximal relative IL‐2 mRNA levels were significantly higher than maximal relative IL‐4 mRNA levels (100‐fold) in both GIL and PBMC after donor‐specific stimulation. This was accompanied by a maximum protein production of 908 pg/ml IL‐2 and 19 pg/ml IL‐4 by GIL, and of 82 pg/ml IL‐2 and undetectable IL‐4 production by PBMC. These results suggest that, after stimulation donor‐specific “primed” GIL, and not “naive” PBMC, rapidly produce abundant levels of IL‐2 (Thl) and IL‐4 (Th2) at both the transcriptional and protein level.