Cellular immune reconstitution after subcutaneous alemtuzumab (anti-CD52 monoclonal antibody, CAMPATH-1H) treatment as first-line therapy for B-cell chronic lymphocytic leukaemia.

Little information is available on long-term immune reconstitution after therapy with alemtuzumab in B-CLL patients. We present long-term follow-up data for blood lymphocyte subsets analysed by flow cytometry in previously untreated B-CLL patients who received alemtuzumab subcutaneously as first-line therapy. All lymphoid subsets were significantly (P<0.001) and profoundly reduced; the median end-of-treatment counts for CD4(+), CD8(+), CD3(-)56(+) (natural killer (NK)), CD3(+)56(+) (NK-T) and CD19(+)5(-) (normal B) cells were 43, 20, 4, 1 and 8 cells/microl, respectively. The median cell count of all subsets remained at <25% of the baseline values for >9 months post-treatment. CD4(+) and CD8(+) levels in blood had reached >100 cells/microl in >50% of the patients at 4 months after the end of treatment. One patient had a cytomegalovirus reactivation and one patient developed Pneumocystis carinii pneumonia during therapy. No opportunistic or other major infections were recorded during unmaintained, long-term follow-up. There was no correlation between the cumulative dose of alemtuzumab and the severity or length of immunosuppression. CD52(-) T-cell subsets occurred during the treatment and comprised >80% of all CD4(+) and CD8(+) cells in the blood at the end of therapy. These subpopulations declined gradually during unmaintained follow-up. The relationship between these observations and the safety/antitumour effects of alemtuzumab is discussed.

Variability in B-cell antigen expression: implications for the treatment of B-cell lymphomas and leukemias with monoclonal antibodies.

OBJECTIVES: Antigen expression intensity is becoming important for decision-making in relation to monoclonal antibody therapy. By quantifying CD20, CD22 and CD52 expression on chronic lymphocytic leukemia and normal (control) B cells, over time. The effect of Interleukin-4 therapy on CD20 antigen intensity on B-CLL cells in vivo was also determined. METHODS: Lymphocytes were purified at weeks 0, 4 and 8 from five B-CLL patients, five healthy volunteers and seven B-CLL patients receiving IL-4 therapy. The number of antigen receptor sites was calculated in molecules of equivalent soluble fluorochrome using flow cytometry. RESULTS: The mean number of CD20 receptors at baseline was significantly lower on B-CLL cells compared to normal B cells (8160 vs 87 046; P<0.0001). Similar results were obtained for CD22 (8630 vs 27 647; P<0.01), but not for CD52 (371 303 vs 409 484; P = 0.54). When soluble fluorochrome values at weeks 4 and 8 were analysed as change in per cent from baseline (delta%), there was <10 delta% variability in CD20 expression on control B cells, but considerable variability (22.5-67.5 delta%) on B-CLL cells. Expression of CD22 in CLL and control B cells varied by <15 delta%. CD52 on CLL B cells showed slightly greater variability (+/-35 delta%) than that of CD22 (+/-15delta%), but less than that of CD20. IL-4 therapy did not consistently increase the CD20 expression on B-CLL cells in vivo. CONCLUSION: Our data confirm differences in intensity between different target antigens on B-CLL cells, and draws attention to the fact that a substantial variability may occur over time, which may influence clinical decision-making. Caution must be taken when interpreting in vitro results on cytokine-mediated receptor intensity up-regulation.

Kinetics of cytokine gene expression in human CD4+ and CD8+ T-lymphocyte subsets using quantitative real-time PCR.

The time kinetics of five cytokines [interleukin-2 (IL-2), IL-5, interferon-gamma (IFN-gamma), granulocyte macrophage-colony stimulating factor (GM-CSF) and tumour necrosis factor-alpha (TNF-alpha)] and one cytotoxic effector protein (granzyme B) was analysed by real-time quantitative polymerase chain reaction (PCR) following in vitro stimulation of human CD4 and CD8 T lymphocytes. Two stimuli were used, a mitogen [phytohemagglutinin (PHA)] and a recall antigen [purified protein derivative (PPD)]. The pattern of cytokine mRNA expression was found to be dependent on the T-cell subset and stimulus used. A wide interindividual variability in the cytokine gene expression pattern was demonstrated. Two expression patterns were observed. A bell-shaped expression profile was seen for most cytokines upon PHA activation in both subsets and PPD-activated CD4 T cells, whereas a biphasic/multiphasic expression pattern was noted in CD8 T cells upon PPD stimulation. For most cytokines, the time to induction was within 30 min of activation, and maximum accumulation seemed to be obtained after 4-8 h of activation. A sustained high level could, however, be noticed for up to 24 h. Granzyme B gene expression was also induced within 30 min of activation but showed a continuous gradual increase and late maximal accumulation (48-72 h). The findings of the present study are of importance when designing studies using the cytokine gene expression profile as a marker for antigen-specific T lymphocytes. It might be recommended that cytokine gene expression (IL-2, IL-5 and IFN-gamma) should be measured after 4-8 h of specific activation but also up to 24 h of stimulation is acceptable. Granzyme B should preferentially be measured after 48-72 h of activation.