Sebastiaan Heidt

Effects of Immunosuppressive Drugs On Purified Human B Cells: Evidence Supporting the Use of MMF and Rapamycin

Background. Humoral immunity is increasingly recognized as an important factor in the rejection of organ transplants. In general, humoral rejection is treated with standard immunosuppressive drugs. The direct effect of these immunosuppressive drugs on B cells is not well known. Methods. Purified human B cells devoid of T cells were stimulated with CD40L expressing L cells, or by anti-CD40 mAb with or without Toll-like receptor triggering, all in the presence of B-cell activating cytokines. These three protocols resulted in various degrees of B-cell stimulation. We added four commonly used immunosuppressive drugs (tacrolimus, cyclosporin, mycophenolic acid [MPA], and rapamycin) to these cultures and tested a variety of parameters of B-cell activity including proliferation, apoptosis induction, and both IgM and IgG production. Results. Tacrolimus and cyclosporin marginally inhibited B-cell proliferation and immunoglobulin production, and the extent of inhibition depended on the degree of the B-cell stimulation. In contrast, MPA and rapamycin profoundly inhibited both B-cell proliferation and immunoglobulin production, which was independent of the degree of B-cell stimulation. Both drugs induced B-cell apoptosis. Moreover, rapamycin caused a reduction in the number of B cells capable of producing immunoglobulins. Conclusions. Our data show that MPA and rapamycin are capable of strongly inhibiting B cells responses. This provides a rationale for the use of both MPA and rapamycin to prevent or counteract humoral 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.

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.

Peripheral Blood Sampling for the Detection of Allograft Rejection: Biomarker Identification and Validation

Currently, acute allograft rejection can only be detected reliably by deterioration of graft function confirmed by allograft biopsy. A huge drawback of this method of diagnosis is that substantial organ damage has already taken place at the time that rejection is diagnosed. Discovering and validating noninvasive biomarkers that predict acute rejection, and chronic allograft dysfunction, is of great importance. Many studies have investigated changes in the peripheral blood in an attempt to find biomarkers that reflect changes in the graft directly or indirectly. Herein, we will review the promises and limitations of the peripheral blood biomarkers that have been described in the literature so far.

Th17: contributors to allograft rejection and a barrier to the induction of transplantation tolerance?

T helper (Th) type 17 cells are a recently described CD4+ T-cell subset that may contribute to allograft rejection and act as a barrier to the induction of transplant tolerance. This review examines the involvement of Th17 cells in transplant rejection, how immunosuppressive medication may affect their induction and maintenance and the potential plasticity of developing Th17 cells. It also addresses the complex interplay between the Th17 and regulatory T-cell developmental pathways and the susceptibility of Th17 cells to regulation. Despite accumulating evidence, the precise impact of Th17 cells on transplant rejection and the induction of tolerance require further clarification.

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 25th anniversary of the Eurotransplant Acceptable Mismatch program for highly sensitized patients

In 2014, the Eurotransplant Acceptable Mismatch (AM) program celebrated its 25th anniversary. The AM program was initiated to enhance transplantation of highly sensitized patients awaiting a renal transplant within the Eurotransplant region. Unlike the regular renal transplant allocation, in which the histocompatibility parameters consist of the degree of compatibility with the patients human leucocyte antigen (HLA) type and the absence of unacceptable antigens, the AM program is based on compatibility of the possible donor with the combination of the patients HLA type and the acceptable antigens. These acceptable antigens are defined as HLA antigens to which the patient has never made antibodies. This strategy aims at the prediction of a negative cross match. Since the start of the program almost 2000 patients participated and more than 1000 patients were transplanted with excellent transplant outcome, comparable to that of non-immunized transplant recipients within Eurotransplant. Progressive insights have led to fine-tuning of the AM program through the years, as well as to novel initiatives, including a recent consortium study to determine the feasibility of a Europe-wide AM program. The current review will tell the story of the AM program in a historical perspective, but will also provide an open-minded look into the future of transplanting highly sensitized patients.

Blood cell mRNAs and microRNAs: optimized protocols for extraction and preservation

Assessing messenger RNA (mRNA) and microRNA levels in peripheral blood cells may complement conventional parameters in clinical practice. Working with small, precious samples requires optimal RNA yields and minimal RNA degradation. Several procedures for RNA extraction and complementary DNA (cDNA) synthesis were compared for their efficiency. The effect on RNA quality of freeze-thawing peripheral blood cells and storage in preserving reagents was investigated. In terms of RNA yield and convenience, quality quantitative polymerase chain reaction signals per nanogram of total RNA and using NucleoSpin and mirVana columns is preferable. The SuperScript III protocol results in the highest cDNA yields. During conventional procedures of storing peripheral blood cells at -180°C and thawing them thereafter, RNA integrity is maintained. TRIzol preserves RNA in cells stored at -20°C. Detection of mRNA levels significantly decreases in degraded RNA samples, whereas microRNA molecules remain relatively stable. When standardized to reference targets, mRNA transcripts and microRNAs can be reliably quantified in moderately degraded (quality index 4-7) and severely degraded (quality index <4) RNA samples, respectively. We describe a strategy for obtaining high-quality and quantity RNA from fresh and stored cells from blood. The results serve as a guideline for sensitive mRNA and microRNA expression assessment in clinical material.

BIOMARKERS OF OPERATIONAL TOLERANCE IN SOLID ORGAN TRANSPLANTATION.

INTRODUCTION: Long-term immunosuppressive therapy represents a huge burden on transplant recipients, but currently cannot be omitted. Improving long-term transplant outcome by immunosuppressive drug withdrawal may be achieved in patients who have developed (partial) immunological unresponsiveness towards their graft, either spontaneously or through tolerance induction. Reliable biomarkers are essential to define such immunological unresponsiveness and will facilitate controlled immunosuppressive drug weaning as well as provide surrogate end-points for tolerance induction trials. AREAS COVERED: Tolerance biomarkers have been defined for both liver and kidney transplantation and can accurately identify operationally tolerant transplant recipients retrospectively. These two tolerance fingerprints are remarkably different, indicating the involvement of distinct mechanisms. Limited data suggest that tolerance biomarkers can be detected in immunosuppressed transplant recipients. Whether these patients can safely have their immunosuppressive drugs withdrawn needs to be established. EXPERT OPINION: Mechanistic interpretation of the kidney transplant tolerance biomarker profile dominated by B cell markers remains a challenge in light of experimental evidence suggesting the pivotal involvement of regulatory T cells. Therefore, defining animal models that resemble human transplant tolerance is crucial in understanding the underlying mechanisms. Additionally, to ensure patient safety while monitoring for tolerance, it is essential to develop biomarkers to non-invasively detect early signs of rejection as well.

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.