Therapeutic implications of variable expression of CD52 on clonal cytotoxic T cells in CD8+ large granular lymphocyte leukemia

Abstract

Background: T-cell large granular lymphocytic leukemia is a clonal proliferation of cytotoxic T-lymphocytes which often results in severe cytopenia. Current treatment options favor chronic immunosuppression. Alemtuzumab, a humanized monoclonal antibody against glycophosphatidylinositol-anchored CD52, is approved for patients refractory to therapy in other lymphoid malignancies.

Design and methods: We retrospectively examined treatment outcomes in 59 patients with CD8+ T-cell large granular lymphocytic leukemia, 41 of whom required therapy. Eight patients with severe refractory cytopenia despite multiple treatment regimens had been treated with subcutaneous alemtuzumab as salvage therapy. Flow cytometry was used to monitor expression of glycophosphatidylinositol-anchored CD52, CD55, and CD59 as well as to characterize T-cell clonal expansions by T-cell receptor variable beta-chain (Vbeta) repertoire.

Results: Analysis of the effects of alemtuzumab revealed remissions with restoration of platelets in one of one patient, red blood cell transfusion independence in three of five patients and improvement of neutropenia in one of three, resulting in an overall response rate of 50% (4/8 patients). Clonal large granular lymphocytes exhibited decreased CD52 expression post-therapy in patients refractory to treatment. Samples of large granular lymphocytes collected prior to therapy also unexpectedly had a significant proportion of CD52-negative cells while a healthy control population had no such CD52 deficiency (p=0.026).

Conclusions: While alemtuzumab may be highly effective in large granular lymphocytic leukemia, prospective serial monitoring for the presence of CD52-deficient clonal cytotoxic T-lymphocytes should be a component of clinical trials investigating the efficacy of this drug. CD52 deficiency may explain lack of response to alemtuzumab, and such therapy may confer a survival advantage to glycophosphatidylinositol-negative clonal cytotoxic T-lymphocytes.

Figure 1.

Response to alemtuzumab (Campath) in patients with LGL leukemia may depend on expression of CD52 on CD8 T cells. (A) Exemplary recovery of absolute reticulocyte count in a patient refractory to multiple treatments (early time points) but responsive to alemtuzumab. (B) Gating strategy for flow cytometric analysis of patient in A. This patient’s CD8 cells were all positive for NKG2D 38.42% were positive for CD57 and 98.62% were positive for CD52. (C) LGL leukemia patients show significantly reduced levels of CD52 on CD8 cells when compared to healthy volunteers (p=0.026, n=12 in each group, Wilcoxon’s rank sums test). (D) Representative flow cytometry results of CD52 expression on CD8 cells. Top panels are data from LGL patients prior to treatment, bottom panels are data from control volunteers.

Figure 2.

Following alemtuzumab treatment, both the percentage of lymphocytes (left) and the absolute clone counts (right) decreased more in responders (circles, solid lines) than in non-responders (squares, dashed lines). Mean follow-up time was 75±31 days after initiation of therapy. Absolute clone counts were calculated by multiplying the absolute lymphocyte count by the proportion of CD8 cells, then by the proportion of clonal cells as determined by Vβ flow cytometry.

Figure 3.

(A) In patients with CD52-deficient CD8 cells, alemtuzumab treatment may result in selection of CD52-deficient cells. Left panels are pretreatment, right panels are post-treatment. Top panels are gated on CD8⁺ Vβ 7.1 as shown below. After alemtuzumab treatment there was a marked increase in the proportion of Vβ 7.1 cells that then stained negative for CD52. (B) CD8-positive, CD52-negative cells from LGL patients were also deficient in other GPI-anchored molecules such as CD55 and CD59. In contrast, neutrophils from LGL patients displayed a normal GPI phenotype. Left panels present data from LGL patients with CD52 deficiency, middle panels present data from LGL patients without CD52 deficiency, and right panels present data from healthy controls. For the upper panels gates were set on lymphocyte and CD8 populations, whereas for the lower panels gates were set on neutrophil populations by forward and side scatter.