Published online 4 July 2008
Haematologica, Vol 93, Issue 9, 1434-1436 doi:10.3324/haematol.12880
Copyright © 2008 by Ferrata Storti Foundation
Stem Cell Transplantation |
Enumeration of cytomegalovirus-specific interferon
CD8+ and CD4+ T cells early after allogeneic stem cell transplantation may identify patients at risk of active cytomegalovirus infection
Carlos Solano1,2,
Isana Benet1,
María A. Clari3,
José Nieto4,
Rafael de la Cámara5,
Javier López6,
Juan C. Hernández-Boluda1,
María J. Remigia1,
Isidro Jarque7,
María L. Calabuig1,
Ana Garcia-Noblejas5,
Juan Alberola8,
Amparo Tamarit8,
Concepción Gimeno3,8,
David Navarro3,8
1 Hematology and Medical Oncology Service, Hospital Clínico Universitario, Valencia
2 Department of Medicine, School of Medicine University of Valencia
3 Microbiology Service, Hospital Clínico Universitario, Valencia
4 Hematology Service, Hospital Morales Meseguer, Murcia
5 Hematology Service, Hospital de La Princesa, Madrid
6 Hematology Service, Hospital Ramón y Cajal, Madrid
7 Hematology Service, Hospital Universitario La Fe, Valencia
8 Department of Microbiology School of Medicine University of Valencia, Valencia, Spain
Correspondence: David Navarro, Microbiology Service, Hospital Clínico Universitario, and Department of Microbiology, School of Medicine, Valencia, Spain. Av. Blasco Ibáñez 17, 46010 Valencia, Spain. Phone: international +34.96.3864657. Fax: international +34.96.3864173. E-mail:david.navarro{at}uv.es
Key words: cytomegalovirus, IFN
CD8+ and CD4+ T cells, active cytomegalovirus infection, stem cell transplantation.
Recovery of functional cytomegalovirus (CMV)-specific T lymphocytes is critical for protection from active CMV infection and disease in allogeneic stem cell transplant recipients (Allo-SCT).1–6 To date, assessment of CMV-specific T-cell immunity has not had a major impact on the clinical management of CMV infection in these patients, as no widely accepted thresholds in the number of CMV-specific T cells providing protection have been established. In the present study, we optimized a simple intracellular cytokine staining (ICS), and investigated whether enumeration of CMV-specific interferon (IFN) CD8+ and CD4+ T cells early after transplantation could reliably predict the development of active CMV infection within 100 days after transplantation. From January to October 2007, 36 patients undergoing Allo-SCT were included in the study. The study was approved by the Ethics Committees and written informed consent was obtained from all patients. Relevant clinical data of patients are summarized in Table 1. Patients were monitored for active CMV infection once or twice a week by pp65 antigenemia,7 CMV DNAemia (CMV real-time PCR, Abbott Molecular, Des Plaines, IL, USA or AMPLICOR CMV Monitor, Roche Indianapolis, USA) or both. Pre-emptive therapy was initiated upon a positive antigenemia or 2 consecutive positive plasma PCRs, and discontinued upon two consecutive negative results as previously reported.7 CMV pneumonitis was diagnosed and treated on the basis of established protocols.7 Heparinized blood samples from patients were obtained at days +30 (median 34 days; range 30–53) and +60 (median 62 days; range 54 to 85 days). Blood samples were also obtained from healthy CMV-seropositive (n = 7) and CMV-seronegative individuals (n = 5). Enumeration of IFN CD8+ and CD4+ T cells was carried out by ICS (BD Fastimmune, BDBiosciences, San Josè, CA, USA) following the manufacturer’s instructions. A set of overlapping peptides spanning the highly immunogenic pp65 and IE-1 CMV proteins, obtained from JPT peptide Technologies GmbH (Berlin, Germany), was chosen as the antigen (2 µg/peptide/mL).8 Responses >0.1% were considered specific. Control samples and specimens from CMV-seronegative subjects yielded IFN responses <0.07%. IFN responses of CMV-seropositive individuals ranged from 0.20% to 5.5% (median 0.45% of IFN CD8+ T cells and 0.35% of IFN CD4+ T cells).
Fifteen patients (44%) experienced active CMV infection (11 D+/R+, 3 D–/R+ and 1 D+/R–), 8 before day +30 (median 19.5 days; range, 8–41 days) and 7 beyond day 30 (median 44.0 days; range 35–56 days). Twenty-one patients (18 D+/R+, 3 D–/R+) did not. Two patients died during the study period (one due to CMV pneumonitis on day +68, and the other due to a proven invasive pulmonary aspergillosis on day +25). Of the 28 patients free of active CMV infection at the first sampling time, IFN responses were detected in all but one patient. Individual data are shown in Figure 1. The median counts of either cell subset were significantly higher in patients not developing active CMV infection (1.69 cells/µL of CD8+ and 1.29 cells/µL of CD4+ T cells) than in those who experienced it later (0.32 cell/µL and 0.24 cell/µL respectively). A threshold in the number of either IFN subset predicting protection against active CMV infection was established: 1 cell/µL for CD8+ T cells (specificity 100%; sensibility 76%, positive predictive value 100%, and negative predictive value 54%) and 1.2 cells/µL for CD4+ T cells (specificity 100%, sensibility 62.5%, positive predictive value 100%, and negative predictive value 43.8%). Enumeration of absolute CD8+ and CD4+ T cells did not allow the prediction of development of active CMV infection (data not shown). Our data are in keeping with those published by other groups.4–6,9,10 In these studies, however, no discrimination between patients who eventually developed viremia and those who did not could be ascertained as early as around 30 days after transplantation.
Reconstitution of CMV-specific T cell immunity may have proceeded at a faster rate in our patients, as most of them were CMV-seropositive, were treated with a non-myeloablative conditioning regimen and received a non-T-cell depleted graft from a CMV-seropositive donor. Some patients not developing active CMV infection displayed early IFN CD8+ and CD4 T-cell counts below the established cut-off levels, indicating that other CMV proteins may also elicit protective immune responses.11
Of the 7 patients developing active CMV infection after the first immunological control, 5 resolved the episode within the study period. The number of IFN CD8+ T cells increased significantly (p = 0.008) by around day +60 in these patients (median increase of 1.8 cells/µL; range 0.4 to 6.2 cells/µL). In contrast, no increase was observed in the 2 patients who failed to clear the episode. Similarly, in patients experiencing active CMV infection before the first immunological control (n = 8), either failure to clear the episode (n = 2), or occurrence of a relapsing episode (n = 4) were associated significantly (p = 0.003) with lower IFN CD8+ T cell-counts at the first immunological sampling (4.9, 1.9, 0.1, 0.2, 1.9, and 0.6 cells/µL), compared to those found in patients resolving the episode (n = 2; 9,3 and 11.5 cells/µL). These data support previous observations.2,3,5,6
One patient died of CMV disease (in the setting of a grade IV GvHD) despite earlier detection of IFN CD8+ and CD4+ T cells (1.9/µL and 0.6/µL). As no samples obtained either immediately before or within the disease period were available from this patient, no major conclusions can be drawn from this case.
Reconstitution of both IFN cell subsets occurred in the absence of detectable active CMV infection. Indeed, median counts of either cell subset at day +60 in patients developing active CMV beyond day +30 (0.89 cell/µL of IFN CD8+ and 0.65 cell/µL of CD4+ T cells) and those found in patients not developing it (2.15 cells/µL and 1.0 cell/µL respectively) were not significantly different (p = 0.18 and p = 0.25 respectively).
These data are in accordance with a previous report.12 The impact of clinical variables on early reconstitution of IFN cell subsets was not evaluated here given the limited sample size of our cohort. In summary, our data suggest that quantification of IFN CD8+ and CD4+ T cells at around day +30 may help to stratify patients according to the risk of developing active CMV infection.
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Acknowledgments
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we thank all the internal fellow staff of the Microbiology Service of Hospital Clínico Universitario for technical assistance.
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Footnotes
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Funding: this research was supported by a grant from FIS 06/1738 from Fondo de Investigaciones Sanitarias (Ministerio de Sanidad y Consumo, Spain).
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References
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