Chantal Eijsink

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.

Human monoclonal HLA antibodies reveal interspecies crossreactive swine MHC class I epitopes relevant for xenotransplantation.

Crossreactivity of anti-HLA antibodies with SLA alleles may limit the use of pig xenografts in some highly sensitized patients. An understanding of the molecular basis for this crossreactivity may allow better selection of xenograft donors. We have tested 68 human monoclonal HLA class I antibodies (mAbs) for reactivity with pig lymphocytes from SLA defined pigs and found nine to be crossreactive. Eight of nine were broadly HLA reactive IgM-mAbs. The putative HLA epitopes for seven mAbs. were conserved in the aminoacid sequence of the SLA alleles studied. The lack of reactivity of a large number of mAbs largely correlated with the absence of the putative epitopes in the SLA alleles studied. We conclude that most patients with anti-HLA class I antibodies should be able to find pig donors lacking SLA antigens that cross react with their antibodies and that many of the crossreacting epitopes can be defined by analysis of shared epitopes in the aminoacid sequence of human and pig MHC antigens.

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.

Identification, Isolation, and Culture of HLA-A2-Specific B Lymphocytes Using MHC Class I Tetramers

Characterizing the individual B cells that participate in the production of anti-HLA Abs requires isolation and culture of these cells and a suitable assay for detection of Abs produced in these B cell cultures. We previously showed that B cell precursors, programmed for anti-HLA Ab secretion, are present at measurable frequencies in peripheral blood of women immunized by pregnancy. In this study, we show that tetrameric HLA-A2, although designed for characterization of CTLs, provides a suitable affinity ligand for isolation of allospecific B cells, which subsequently can be induced to produce HLA-A2 Ab in a CD40-driven culture system. The validity of this concept was established by assaying human hybridomas, producing anti-HLA Abs, for specific tetrameric HLA-A2 binding. The availability of anti-HLA Ab-producing B cell cultures that are established without immortalization will be of value when T-B cell interaction is studied at an alloantigen-specific level.

Impact of Peptides on the Recognition of HLA Class I Molecules by Human HLA Antibodies

MHC class I molecules expressed on cell surfaces are composed of H chain, β2-microglobulin and any of a vast array of peptides. The role of peptide in the recognition of HLA class I by serum HLA Abs is unknown. In this study, the solid-phase assay of a series (n = 11) of HLA-A2-reactive, pregnancy-induced, human mAbs on a panel (n = 12) of recombinant monomeric HLA-A2 molecules, each containing a single peptide, revealed peptide selectivity of the mAbs. The flow cytometry membrane staining intensities on the HLA-A2-transduced cell line K562, caused by these mAbs, correlated with the number of monomer species detected by the mAbs. Flow cytometry staining on HLA-A2-bearing cell lines of a variety of lineages was indicative of tissue selectivity of these HLA-A2 mAbs. This tissue selectivity suggests that the deleterious effect on allografts is confined to alloantibodies recognizing only HLA class I loaded with peptides that are derived from tissue-specific and household proteins. Since Abs that are only reactive with HLA loaded with irrelevant peptides are expected to be harmless toward allografts, the practice of HLA Ab determination on lymphocyte-derived HLA deserves reconsideration.

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.

HLA-C Expression on Platelets: Studies with an HLA-Cw1-Specific Human Monoclonal Antibody

Background and Objectives: The expression of HLA-C on the surface of platelets is rarely studied due to the lack of proper alloantisera. We addressed this question using an IgM human monoclonal antibody directed against HLA-Cw1 (VP6G3). Material and Methods: Both flow cytometry and complement dependent cytotoxicity studies were used in the current analysis. Results: The expression of the HLA-Cw1 antigen on platelets is lower than on peripheral blood lymphocytes as shown by flow cytometry. Variation in expression levels between individuals is also observed. Using this antibody in a complement-dependent cytotoxicity assay, we did not observe lysis using platelets as targets, whereas peripheral blood lymphocytes of the same blood donors were adequately lysed. Conclusions: These results confirm that platelets indeed express HLA-C. Furthermore, the results support the insignificant role of HLA-C in immunological platelet refractoriness.