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Keywords:

  • post-transplant lymphoproliferative disorders;
  • Epstein-Barr virus;
  • rituximab;
  • T cell

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results and discussion
  5. Financial support
  6. Declaration of commercial interests
  7. Acknowledgements
  8. References

Immunosuppression following solid organ transplantation results in impaired T-cell immunity and risk of Epstein–Barr virus (EBV)-positive post-transplant lymphoproliferative disorders (PTLD). The B-cell targeting antibody rituximab has efficacy in PTLD. As B cells are the principle reservoir for EBV, we investigated the effect of rituximab on the persistence of EBV-specific CD8+ T-cell immunity. To avoid the confounding factor of concurrent immunosuppression to prevent transplant rejection, immunity was analysed in non-transplanted lymphoma patients (i.e. a non-PTLD setting). Cytomegalovirus-specific T-cell immunity was assessed as an internal control. Our data demonstrated that circulating B cells were not critical for maintaining EBV-specific T-cell immunity.

Following solid-organ transplantation, recipients require immunosuppression to prevent organ rejection. This results in an increased risk of Epstein–Barr virus (EBV)-positive post-transplant lymphoproliferative disorders (PTLD) (Young & Rickinson, 2004). Although the anti-CD20 antibody rituximab has efficacy in PTLD, relapse is not infrequent. It remains unclear what the effect of rituximab is on EBV-specific CD8+ T-cell effector function. B cells express high levels of class II major histocompatibility complex (MHC) and co-stimulatory molecules and have effective antigen-presenting capabilities, and in animal models they are necessary for the priming and persistence of CD4+ and CD8+ T-cell memory (Shen et al, 2003). In healthy seropositive individuals, the principle reservoir of EBV is in circulating B cells, with typically 1–50/106 B cells infected. It has been put forward that the high level of EBV-specific T-cell immunity maintained in the periphery of healthy seropositive subjects is the consequence of frequent antigenic challenge induced by EBV latently infected B cells, which undergo periodic viral reactivation (Young & Rickinson, 2004). Data in six liver transplant recipients with PTLD showed that, following rituximab monotherapy, the magnitude of EBV-specific cellular immunity was impaired, and that this was associated with a high rate of relapse (Savoldo et al, 2005). The authors suggested that the relapse rate was a result of the re-institution of immunosuppression used to prevent graft rejection. Ongoing immunosuppression prevents any conclusions regarding the potential alteration of EBV-specific cellular immunity as a consequence of rituximab. To date, there remains little data regarding the importance of B cells on the maintenance of T-cell immunity in the human setting. However, in a randomised trial of CHOP (cyclophosphamide, doxorubicin, vincristine, prednisolone) versus CHOP + rituximab (CHOP-R) chemotherapy for human immunodeficiency virus (HIV)-associated non-Hodgkin lymphoma (NHL), there was a significant increase in opportunistic infections [25% cytomegalovirus (CMV) related] and infectious mortality in the CHOP-R arm (Kaplan et al, 2005). Conversely, cellular immunity induced by idiotypic vaccination for lymphoma was not impaired by prior rituximab (Neelapu et al, 2005).

This study aimed to determine the effect (if any) of rituximab on the persistence of EBV-specific CD8+ effector T-cell immunity. Although the intention was to provide data that will be of practical value to the management of patients with EBV-positive PTLD, we specifically confined the study to patients receiving therapy for NHL in the non-PTLD setting, i.e. patients with lymphomas who had not received prior solid organ transplant. In this way, we were able to study immunity without the confounding factor of administration of concurrent immunosuppression to prevent transplant rejection.

Materials and methods

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results and discussion
  5. Financial support
  6. Declaration of commercial interests
  7. Acknowledgements
  8. References

Any newly diagnosed (n = 14), relapsed (n = 2) or partial remission (n = 1) patients with histologically confirmed NHL were eligible. Exclusion criteria were previous purine analogue therapy, prior stem cell transplantation, seronegativity to EBV and CMV, and previous rituximab treatment. Seventeen patients were recruited: 10 chemotherapy/rituximab: seven chemotherapy alone; females:male ratio 7:10; 70% in each arm had stage III–IV disease and the mean age was identical at 62 years. Three of 10 patients in the chemotherapy/rituximab group received rituximab alone (four times, at weekly intervals). Mean CD19+ lymphocytes were 3·5% (SE 0·5%), 0·026% (SE 0·01%) and 8% (SE 4·24%) prior to, during and following chemotherapy/rituximab therapy respectively. The corresponding values in the chemotherapy alone group were 4·0% (SE 0·58%), 1·46% (SE 0·41%) and 3·0% (SE 2·0%). The difference between during therapy values was significant (P = 0·023). Seven patients had diffuse large B-cell lymphoma, and 10 had indolent lymphomas. The study conformed to the Declaration of Helsinki and patients provided informed consent. Blood was collected immediately prior to the first (pre) and fourth cycle (during), and then at a late time-point following the final cycle (chemotherapy/rituximab: mean 5 months, range 1–10; chemotherapy alone: mean 5 months, range 1–8, P = NS). To minimise intra-assay variability, all samples from a particular subject were assayed at one time by the same operator.

Results and discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results and discussion
  5. Financial support
  6. Declaration of commercial interests
  7. Acknowledgements
  8. References

In the EBV-related herpes virus CMV, monocytes are the major circulating reservoir (Gandhi & Khanna, 2004). Monocytes do not express CD20, nor does chemotherapy result in long-term suppression of the monocyte lineage. If the circulating level of virus-specific T-cell immunity were dependent on the circulating antigenic viral load, we would expect levels of peripheral EBV-specific, but not CMV-specific, CD8+ T cells to be affected. Using intracellular gamma-interferon intracellular cytokine staining (ICS), we characterised ex-vivo herpes virus peptide-specific CD8+ effector T-cell responses prior to, during and following therapy in the chemotherapy/rituximab and chemotherapy alone groups (Table I). The proportion of EBV and/or CMV-specific T cells was determined by establishing the mean for each patient at that time-point. Only those peptides relevant to the particular human leucocyte antigen (HLA) type of each patient were used. We minimised this limitation by using 29 different EBV peptides (from latent and lytic antigens known to be relevant in PTLD) presented by a wide array of MHC class I alleles, such that nine of 10 chemotherapy/rituximab patients and six of seven chemotherapy alone patients had (at least two) informative alleles. Between cohorts, there was no difference in EBV peptide-specific CD8+ T-cell effector function at pretherapy (P = 0·60). Within cohorts, results were compared between each patient at paired time-points using the Wilcoxon matched pairs test. Within cohorts, there was no change in EBV peptide-specific CD8+ T-cell effector function between prior, during and following therapy time-points. When T-cell responses were sub-divided to those against either the immuno-dominant lytic, immuno-dominant latent [EBV nuclear antigen (EBNA) 3/4/6] and immuno-subdominant (latent membrane proteins 1/2) antigens, again no changes over time was observed. Similarly, using MHC class I EBV peptide-specific pentamers, no change was observed between time-points. Consistent with previous reports, peptide-specific T-cell frequency was higher by direct visualisation (pentamer) than functional (ICS) assays (Gandhi & Khanna, 2004; Gandhi et al, 2006a). Mean CD8+ EBNA3/4/6 and lytic T cells from pretherapy samples from both cohorts (1·28%, range 0–7) were similar to those observed in healthy individuals (Tan et al, 1999), suggesting that lymphoma does not impair EBV-specific CD8+ T-cell immunity. Both functional and direct visualisation assays showed CMV-specific T-cell immunity did not fluctuate over time. These data suggest that circulating cellular virus is not critical in maintaining virus-specific T-cell immunity.

Table I. Ex vivo herpes-virus peptide-specific T cells in non-Hodgkin lymphoma patients.
VirusSub-group/time-point% IFN-γ D8+ cells Mean (SE)P-value (versus pretherapy)P-value (versus during)
  1. Ex vivo T-cell analysis was performed as previously described (Gandhi et al, 2006a). As post-transplant lymphoproliferative disorder is known to express a restricted set of latent and early lytic cycle Epstein–Barr virus (EBV) proteins (Young & Rickinson, 2004), only relevant defined EBV-specific HLA class I peptides, derived from latent (EBV nuclear antigens 3, 4, 6 and latent membrane proteins 1 and 2) and early lytic cycle (BamHI-Z left reading frame number 1) antigens, were used (Gandhi et al, 2006a). Cytomegalovirus (CMV)-specific HLA class I peptides against phosphoproteins 65 and 50, and immediate early-1 and 2 were used as previously listed (Gandhi & Khanna, 2004; Gandhi et al, 2006a). Arithmetic mean values of frequency of interferon-γ (IFN-γ) producing CD8+ T cells in response to EBV and CMV HLA class I antigenic peptides in each study group at each time-point, as assessed by intracellular cytokine staining. Only those peptides relevant to the particular HLA type of each patient were used. Nine of 10 chemotherapy/R (rituximab) patients and six of seven chemotherapy alone patients had (at least two) informative alleles. P-values were generated on paired values from each patient using the Wilcoxon matched pairs test. SE indicates standard error. Chemotherapy/R regimens used were either rituximab alone (i.v. rituximab 375 mg/m2 weekly for four cycles in three patients), or CHOP-R (in seven patients): 6–8 cycles of three weekly i.v. cyclophosphamide 750 mg/m2, i.v. doxorubicin 50 mg/m2, i.v. vincristine 1·4 mg/m2 all on day 1 and oral prednisolone 100 mg days 1–5, in combination with i.v. rituximab 375 mg/m2 on day 1; the chemotherapy alone regimen used was CVP = 3 weekly cycles of i.v. cyclophosphamide 1 g/m2, i.v. vincristine 1·4 mg/m2 and oral prednisolone 100 mg days 1–5 (in seven patients).

EBVChemotherapy/R pretherapy0·05 (0·02) 
Chemotherapy/R during0·03 (0·01)0·50
Chemotherapy/R post0·03 (0·01)0·720·91
CMVChemotherapy/R pretherapy0·36 (0·19) 
Chemotherapy/R during0·67 (0·51)1·0
Chemotherapy/R post0·54 (0·36)0·430·31
EBVChemotherapy alone pretherapy0·18 (0·09) 
Chemotherapy alone during0·17 (0·08)0·29
Chemotherapy alone post0·10 (0·06)0·830·31
CMVChemotherapy alone pretherapy0·24 (0·13) 
Chemotherapy alone during0·32 (0·21)0·43
Chemotherapy alone post0·13 (0·05)0·30·3

The role of antigen in the maintenance of T-cell memory remains contentious (Antia et al, 2005), and our findings should not be over interpreted. Dendritic cells effectively present antigen to T cells. Virus residing within the oropharynx may provide an additional source of antigen. Although rituximab depletes B cells in lymphoid organs, it is likely that EBV-infected cells persist, albeit at a reduced frequency. We quantified peripheral cellular EBV-DNA using primers to amplify the single copy viral gene BALF5 (Gandhi et al, 2006b). In 76% of patients viral load remained undetectable at all time-points. In the remaining patients, low level virus was occasionally observed. Targeting the BAMH1-W region (which contains large internal repeats and might have enhanced sensitivity) produced identical results. This effect was seen irrespective of whether rituximab was received. As expected, plasma EBV-DNA was generally undetectable in both sub-groups, indicating that sub-clinical viral reactivation was not present. These results are consistent with the observation that the widespread use of rituximab did not result in an increase in the incidence of EBV-related diseases.

CD20 is expressed on a small subset of T cells present in blood and marrow (Algino et al, 1996). Following transduction of human T cells with human CD20 cDNA, EBV-specific T cells can be lysed by rituximab and complement (Serafini et al, 2004). As in-vivo depletion of CD20+ herpes-virus specific T lymphocytes may have important implications we evaluated CD20 expression on T cells. In five healthy subjects (mean age 41 years, range 29–62 years), we found 1–2% of circulating CD3+ T cells were CD20dim, and 0·74% CD20hi (range 0·1–0·9%). Within CD20hi CD3+ T cells, 40% were CD4+ (range 34–59%) and 23% CD8+ (13–38%). A mean 78% (range 58–96) utilised the αβT-cell receptor (TCR) and 16% (0–29) the γδTCR. In patients prior to therapy the mean value of CD20hi T cells was only 0·84% of pentamer positive T cells. Further, T-cell function prior to and following chemotherapy/rituximab or after in-vitro administration of rituximab was not impaired (Fig 1). Rituximab appears to have no direct inhibitory activity on herpes virus-specific T cells.

image

Figure 1.  Effect of in vivo and in vitro rituximab on herpes virus peptide-specific T cells. (A) Representative data showing the effect of in vivo rituximab on EBNA3FLRGRAYGL peptide-specific CD8+ effector T cells as enumerated ex vivo using intracellular cytokine staining, in a patient treated with chemotherapy/rituximab; samples were taken prior to and following completion of therapy. A sample from the same patient, without peptide stimulation is shown as a negative control. (B) Effect of in vivo rituximab on latent EBV (Epstein–Barr virus)-peptide specific CD3+ CD8+ T cells as enumerated ex vivo using EBV-specific major histocompatibility complex-class I peptide pentamers, in chemotherapy/rituximab patients. Samples were taken prior to pre-, during- and following post-therapy. Histograms showing mean and SE of directly visualised latent EBV-peptide specific CD3+ CD8+ T cells. Numbers of pentamer assays performed for chemotherapy/rituximab were nine, 13 and 13 (pre, during and post) and chemotherapy/alone 13, nine, and nine (pre, during and post) respectively. (C) EBV-peptide specific CD3+ CD8+ T cells directly visualised in chemotherapy alone patients. (D) In each panel, peptide-specific cytotoxic T-lymphocytes (CTL) were generated in parallel from blood taken at two time-points from a patient with diffuse large B-cell lymphoma: immediately prior to initiation of (preCHOP-R) and then following a course of CHOP-R chemotherapy (postCHOP-R). The left-panel shows CTL recognition of the EBV-BZLF1 RAK epitope by polyclonal RAK peptide-stimulated CTL against a range of effector to target (E:T) ratios, as determined by a standard chromium-release assay. Targets were autologous phytohaemagglutinin (PHA) blasts presensitised with either RAK or a no peptide control. Right-panel shows data for CMV-pp65 TPR peptide-specific CTL. Standard deviation was <10% at all ratios. (E) In each panel, peptide-specific CTL were generated in parallel from healthy EBV/CMV seropositive subjects. CTL lines had been pre-exposed for 16 h to rituximab 20 mg/ml suspended in serum (CTL + rituximab) or a serum alone control (CTL alone). The rituximab/serum culture conditions were known to induce lysis of CD20 expressing cells in vitro. The left-panel shows CTL recognition of the EBV-LMP2 FLY epitope by polyclonal FLY peptide-stimulated CTL against a range of effector to target ratios. Targets were autologous PHA blasts presensitised with either FLY or a no peptide control. Similar data (not shown) were obtained in this subject with CLG-specific CTL. Right-panel shows data in a healthy CMV-seropositive subject for CMV-pp65NLV peptide-specific CTL. SD was <10% at all ratios.

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We conclude that severe B-cell depletion does not impede persistence of EBV-specific CD8+ T-cell immunity. This is relevant for individuals with PTLD, who are increasingly treated with rituximab, and in whom immunotherapeutic strategies to restore EBV-specific cellular immunity are being actively pursued (Young & Rickinson, 2004).

Financial support

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results and discussion
  5. Financial support
  6. Declaration of commercial interests
  7. Acknowledgements
  8. References

The study was supported by the National Health and Medical Research Council NHMRC (Australia), the British Society for Haematology and the Queensland Department of State Development and Innovation. M.K. Gandhi has an NHMRC Clinical Career Development Award and R. Khanna is an NHMRC Principal Research Fellow.

Acknowledgements

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results and discussion
  5. Financial support
  6. Declaration of commercial interests
  7. Acknowledgements
  8. References

We would like to thank Kristina Harej, Sue Godwin and Tanya Graham for their kind help in coordinating the clinical aspects of this study.

References

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results and discussion
  5. Financial support
  6. Declaration of commercial interests
  7. Acknowledgements
  8. References
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