CD4+ Perforin+ T Cells in B-CLL: MHC Class II Restriction and Anti-Cytomegalovirus Reactivity.

Abstract

Introduction: An expansion of CD4+ T cells expressing perforin (PF) has recently been described in B-CLL and we have previously demonstrated anti-cytomegalovirus (CMV) reactivity within this population (Walton et al; 2004; Blood [ASH annual meeting abstracts] 104:4787). Here we further characterise the anti-CMV response of CD4+PF+ T cells in B-CLL and investigate the role of CMV in CD4+PF+ T cell expansion. Methods: Peripheral blood mononuclear cells (PBMC’s) from 24 untreated B-CLL patients (17 CMV seropositive [SP], 7 CMV seronegative [SN]), 2 SP treated (Campath) patients and 12 healthy age-matched control individuals (8 SP, 4 SN) were fixed, permeabilised and stained with anti-CD4PerCP, anti-IFN-γ-APC and anti-PF-FITC monoclonal antibodies (mABs) (BD). PBMC were cultured for 18 hrs with DOWNE cell lysate (Dade Behring) containing CMV-antigen or lysate alone and with anti-CD28 and anti-CD49d mAbs (BD), in the presence of Brefeldin A (eBiosciences). In blocking experiments PBMC were pre-incubated with anti-HLA DR,DP,DQ mAb (BD) for 1 hour. The CMV specific response was assessed by flow cytometry (Dako Cyan, Summit software) as the percentage of IFN-γ+ cells in PF+ and PF− CD4+ T cell populations. Statistical analysis was performed using the Mann-Whitney U test and Spearman rank correlation. Results: The proportion of CD4+ T cells expressing PF directly ex vivo was significantly higher in SP B-CLL patients (17.5±18.6%) compared to SN patients (2.0±2.3%, p=0.019). In seropositive aged matched controls the percentage of CD4+ cells expressing perforin was positively correlated with the percentage of CMV-reactive CD4+ cells (r=0.976, p<0.01). In contrast, there was no significant correlation in the patient group. However, two patients with relatively large expansions of CD4+PF+ cells (37.7±3.39%) post-Campath treatment had high percentages of CMV-reactive CD4+ cells (10.93±0.62%) compared to SP B-CLL patients (1.34±1.19%) and SP controls (1.31±1.14%), implying Campath related CMV reactivation. The addition of anti-HLA-DR,DP,DQ mAb to patients’ PBMCs, prior to CMV stimulation, led to an 80% (from 3.26% to 0.79%) and 90% (from 3.9% to 0.45%) reduction in the proportion of antigen reactive CD4+ and CD4+PF+ cells respectively. Conclusions: A population of major histocompatibility complex (MHC) class II restricted, CMV reactive, CD4+PF+ T cells exists peripherally, in a large group of CMV SP B-CLL patients. Furthermore, CMV is associated with CD4+PF+ T cell expansion in patients and controls. Our data implies that high numbers of B-CLL cells inhibit anti-viral effector function, leading to increased viral activity and chronic antigenic exposure, potentially driving CD4+PF+ T cell expansion.