Floor Pietersma

Immune surveillance of EBV-infected B cells and the development of non-Hodgkin lymphomas in immunocompromised patients.

After infection with the Epstein – Barr virus, a common gammaherpes virus which infects and persists in the B cells, an equilibrium is established in which newly infected and differentiating B cells are controlled by cytotoxic T cell (CTL) responses. Disturbance of this equilibrium, which can occur in immunocompromised situations, can lead to uncontrolled lymphoproliferation and subsequent development of non-Hodgkin Lymphomas (NHL). Here, we review the role of immunesurveillance of EBV-infected B cells and two situations where immunesurveillance is altered because of immunodeficiencies, transplantation recipients and HIV infection, which can lead to EBV-mediated NHL. In transplant recipients, immunosuppression prior and during transplantation can lead to lack of immunesurveillance and results in proliferation of infected B cells, which would normally be controlled by CTL responses. Interestingly, in HIV infection both deregulation of the normal B cell biology and a reduction in immunity play a role in developing NHL. Therefore, the nature of EBV infection in HIV-positive subjects is very different from that in transplanted individuals, in whom (re-)appearance of EBV-specific CD8+ T cells – either by a decrease in immune suppression or infusion of donor lymphocytes – immediately leads to a decrease in EBV load.

Rituximab Treatment before Reduced-Intensity Conditioning Transplantation Associates with a Decreased Incidence of Extensive Chronic GVHD

Chronic graft-versus-host-disease (cGVHD) is the major cause of late morbidity and mortality after allogeneic stem cell transplantation. B cells have been reported to be involved in mediating cGVHD. To assess whether preemptive host B cell depletion prevents extensive cGVHD after allogeneic reduced-intensity conditioning transplantation (RICT), 173 patients treated with RICT for various hematologic diseases, who had or had not received Rituximab (Rtx) within 6 month prior to RICT, were analyzed retrospectively. Rtx treatment within 6 months prior to RICT reduced extensive cGVHD significantly from 45.8% to 20.1%. We hypothesize that most likely host B cells initiate cGVHD, and thus, host B cell depletion prior to RICT by Rtx might be a valuable strategy to reduce extensive cGVHD after RICT.

Influence of Donor Cytomegalovirus (CMV) Status on Severity of Viral Reactivation after Allogeneic Stem Cell Transplantation in CMV-Seropositive Recipients

We investigated the role of donor cytomegalovirus (CMV) serostatus on reactivation of CMV infection in CMV-infected transplant recipients. Reactivation of CMV infection occurred more frequently in patients receiving a CMV-positive graft but was less severe than in patients receiving a CMV-negative graft. These data suggest roles for both virus as well as CMV-specific immunity present in the graft.

The immunological phenotype of rituximab-sensitive chronic graft-versus-host disease: a phase II study

Chronic graft-versus-host disease is the major long-term complication after allogeneic stem cell transplantation with a suboptimal response rate to current treatments. Therefore, clinical efficacy and changes in lymphocyte subsets before and after rituximab treatment were evaluated in a prospective phase II study in patients with steroid-refractory chronic graft-versus-host disease. Overall response rate was 61%. Only responding patients were found to have increased B-cell numbers prior to treatment. B cells had a naïve-antigen-presenting phenotype and were mainly CD5 negative or had a low CD5 expression. Normal B-cell homeostasis was reestablished in responding patients one year after ritxumab treatment and associated with a significant decline in skin-infiltrating CD8+ T cells, suggesting that host B cells play a role in maintaining pathological CD8+ T-cell responses. Imbalances in B-cell homeostasis could be used to identify patients a priori with a higher chance of response to rituximab treatment (Eudra-CT 2008-004125-42).

High level of perforin expression in T cells: an early prognostic marker of the severity of herpesvirus reactivation after allogeneic stem cell transplantation in adults.

BACKGROUND Epstein-Barr virus (EBV) and cytomegalovirus reactivations are frequent complications of hematopoeitic allogeneic stem cell transplantation (SCT) because of a lack of T cell control after immunosuppression. Early diagnosis of reactivation and subsequent preemptive therapy relies on frequent viral load measurement. Additional virus-specific T cell reconstitution data could improve the predictive value of viral load detection for viral complications after transplantation. Here, we studied perforin expression in CD8(+) T cells as a measure of cytotoxic T cell capacity in relation to the occurrence of viral reactivation. METHODS In a prospective study, we monitored 40 patients during the first 3 months after transplantation and measured viral loads in combination with intracellular perforin expression in CD8(+) T cells. RESULTS Median perforin expression in CD8(+) T cells throughout follow-up was higher in patients with viral reactivations than in patients without viral reactivations (4.9% vs 2.3%; P = .001). The median percentage of perforin-expressing CD8(+) T cells in patients with high viral reactivations exceeding 1000 copies/mL (10.7%) was statistically significantly higher than that in patients with minor reactivations of 50-1000 copies (4.0%), that in patients with detectable EBV loads that did not exceed the detection limit of 50 copies/mL (2.9%), and that in patients without reactivations (0.8%). Patients with high viral reactivations reached a high percentage of perforin-expressing CD8(+) T cells (>10.2%) more often and faster than did patients with low viral loads (1000 copies/mL) or without viral reactivations. High perforin expression preceded high viral loads. CONCLUSION Perforin-expressing CD8(+) T cells may be useful as an easy-to-measure prognostic marker for identifying patients at risk for severe viral reactivation very soon after SCT.

Adequate control of primary EBV infection and subsequent reactivations after cardiac transplantation in an EBV seronegative patient

EBV seronegative recipients of cardiac transplantation are at risk for development of post transplant lymphoproliferative disease following primary EBV infection due to the ongoing treatment with immunosuppressive drugs. Here we present detailed kinetics of the EBV-specific T-cell response following cardiac transplantation in an EBV seronegative recipient who developed a primary EBV infection 15weeks post transplantation and subsequent viral reactivations throughout follow up. The patient developed an EBV-specific CD8(+) T-cell response within 24days after first detection of the primary infection. Subsequently, an increased EBV-specific CD8(+) T-cell response developed upon viral reactivation, indicated by a threefold increase of EBV-specific CD8(+) T cells and increased IFNy production after stimulation with EBV-specific peptide pools. These data indicate that an EBV-specific T-cell response capable of adequate control of a primary EBV-infection and subsequent viral reactivations can develop in an EBV seronegative cardiac transplant recipient in the presence of severe immunosuppression.

Epstein-Barr Virus Reactivation does not Impact Total T-Cell Reconstitution but is associated with Decreased Functional T Cells after Stem Cell Transplantation

Epstein-Barr virus reactivations are frequently observed after allogeneic stem cell transplantation (SCT). We investigated the role of total as well as EBV-specific T-cell reconstitution in relation to onset and severity of EBV reactivation. To this end, 116 patients were prospectively sampled for viral load and absolute T-cell numbers weekly post-SCT during the first 3 months and thereafter monthly until 6 months post-SCT. In addition, we retrospectively analysed EBV-specific T-cell responses in 12 patients during the first year after SCT.In contrast to the general belief, we found that early T-cell reconstitution after SCT does not play a role in the onset of EBV reactivation as numbers of CD4+ and CD8+ T cells during the first 3 months post SCT are similar in patients with or without viral reactivation. However, functional T-cell responses after non-specific in-vitro restimulation were impaired in patients with high-level EBV-reactivation. Although EBV-specific CD8+ T-cell responses were readily detected from 2 months onward, EBV-specific CD4+ T cells remained low throughout followup, and especially EBNA-1-specific CD4+ T cells did not normalize to healthy control levels one year post-SCT.In conclusion, EBV-reactivation does not influence total T-cell reconstitution, but functional capacity is impaired in patients with high-level EBV-reactivation.

Cytomegalovirus (CMV)-Reactivation Influences T-Cell Differentiation and CMV-Specific T-Cell Reconstitution after Stem Cell Transplantation

CMV-specific T cells were shown to be important for protection against CMV-disease in SCT recipients. Here we investigated specific T-cell features like effector cell differentiation and perforin-expression as well as CMV-specific T cells after SCT in relation to CMV-reactivation. To this end, CD4+ and CD8+ T-cell characteristics (differentiation, activation and functional CMV-specific immunity) of SCT patients with (n=13) or without (n=8) CMV-reactivation were analysed longitudinally by flow cytometry. CMV-specific IFNγ-production as measured by intracellular staining and proliferation as measured by CFSE dye dilution were analysed after stimulation with overlapping peptide pools of the tegument protein pp65 and immediate early antigen 1. A more differentiated phenotype, up-regulation of the activation markers CD38 and HLA-DR on CD4+ T cells and increased expression of perforin on CD8+ T cells was more frequently observed in patients with CMV-reactivation compared to patients without reactivation. Interestingly, these T-cell features were often already different early after SCT. In addition, CMV-specific CD8+ T-cell responses, both based on IFNγ-production as well as proliferation, directed against both pp65 and IE1 tended to be present more frequently in patients with CMV-reactivation compared to patients without reactivation. These data suggest that CMV-reactivation influences CMV-specific T-cell reconstitution after SCT and that early T-cell differentiation differences may be helpful in predicting viral reactivations.