Abstract
Introduction: Perforin (PF) expressing CD4+ T cells are expanded in B-CLL patients [1] and are able to induce PF-mediated apoptosis of autologous leukaemic cells in the presence of bispecific anti-CD3/CD19 antibodies [2]. However, the role of the expanded PF+CD4+ T population in B-CLL remains unclear. In this study we have examined possible involvement of this cell subset in immune responses to cytomegalovirus (CMV).
Methods: Blood mononuclear cells from 11 CMV seropositive (SP) and 6 seronegative (SN) B-CLL patients were cultured for 4 and 18 hours in presence of Downe cell lysates containing CMV-antigen or cell lysates alone (control) and brefeldin and co-stimulated with anti-CD28 and anti-CD49 monoclonal antibodies. Anti-CMV response was assessed by flow cytometry as percentages of CD69+ and IFNγ+ cells in PF+ and PF- CD4+T cell populations.
Results: CD4+ T cells from 9 of 11 SP patients showed a strong increase in percentages of IFNγ+ cells after 18h, but not 4h, in culture with CMV antigens, compared to the control lysates and SN patients (Table) and healthy age-matched SP subjects (1.78±0.45, p=0.012). In contrast, none of the 6 SN patients as well as 2 out of 11 SP patients responded to CMV by increased IFNγ expression. In responders IFNγ+ cells were proportionally distributed between PF+ and PF- CD4+ T cells. High levels of cell activation measured by CD69 expression, especially in PF+ population, were mainly due to the Downe cell lysate since expression was significantly lower in lysate free cell cultures (8.3±11.5%, p=0.0018 for SP B-CLL patients). Anti-CMV response was accompanied by a decrease in percentages of CD4+PF+ cells suggesting antigen-induced degranulation.
Conclusion: PF+CD4+ T cells respond to CMV antigens suggesting an expansion of mature CMV specific cytotoxic CD4+ T cells in the majority of CMV seropositive B-CLL patients.
IFN-γ and CD69 expression by CD4+PF+ and PF- T cells after 18h exposure to CMV+ and CMV- lysates in CMV seropositive and seronegative B-CLL patients
| . | CD4+ . | PF+ CD4+ . | PF-CD4+ . | |||
|---|---|---|---|---|---|---|
| . | SN . | SP . | SN . | SP . | SN . | SP . |
| IFN- γ, % | ||||||
| CMV+lys | 0.7±0.58 | 9.1±7.9 | 2.0±3.0 p=0.013 | 14.4±13.6 | 0.61±0.56 p=0.008 | 9.0±8.1 |
| CMV−lys | 0.26±0.34 | 0.9±1.7 p=0.007 | 1.1±1.7 | 3.9±6.9 p=0.029 | 0.24±0.27 | 1.46±2.55 p=0.013 |
| CD69+cells, % | ||||||
| CMV+lys | 16.3±14.2 | 21.4±13.0 | 41.9±35.1 | 51.2±30.3 | 14.9±13.4 | 20.4±13.2 |
| CMV−lys | 14.4±12.8 | 9.7±7.8 | 28.6±34.8 | 39.8±33.0 | 13.8±12.1 | 12.9±15.0 |
| . | CD4+ . | PF+ CD4+ . | PF-CD4+ . | |||
|---|---|---|---|---|---|---|
| . | SN . | SP . | SN . | SP . | SN . | SP . |
| IFN- γ, % | ||||||
| CMV+lys | 0.7±0.58 | 9.1±7.9 | 2.0±3.0 p=0.013 | 14.4±13.6 | 0.61±0.56 p=0.008 | 9.0±8.1 |
| CMV−lys | 0.26±0.34 | 0.9±1.7 p=0.007 | 1.1±1.7 | 3.9±6.9 p=0.029 | 0.24±0.27 | 1.46±2.55 p=0.013 |
| CD69+cells, % | ||||||
| CMV+lys | 16.3±14.2 | 21.4±13.0 | 41.9±35.1 | 51.2±30.3 | 14.9±13.4 | 20.4±13.2 |
| CMV−lys | 14.4±12.8 | 9.7±7.8 | 28.6±34.8 | 39.8±33.0 | 13.8±12.1 | 12.9±15.0 |
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