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Campath-1H, an anti-CD52 monoclonal antibody, is therapeutically active in lymphoproliferative and autoimmune diseases. After Campath-1H therapy, lymphocytes with a paroxysmal nocturnal haemoglobinuria (PNH) phenotype have been reported to emerge. We characterized a PNH-like lymphocyte population emerging after Campath-1H therapy, in a patient with fludarabine refractory B-cell chronic lymphocytic leukaemia (B-CLL). We demonstrated a reduction in PIG-A mRNA levels compared with controls, and of all cytokines tested [interleukin (IL)-4, IL-13, IL-2, interferon(IFN)-γ, IL-6, IL-10, and tumour necrosis factor (TNF)-α], except transforming growth factor (TGF)-β. Given the inhibitory activity of TGF-β, its elevated levels may contribute to the selective pressure of Campath-1H, leading to the emergence of PNH-like lymphocytes.
Campath-1H, a humanized monoclonal antibody against CD52, is increasingly used in the treatment of lymphoid malignancies, autoimmune disease and bone marrow transplantation. After Campath-1H treatment, the emergence of lymphocytes deficient in glycosylphosphatidylinositol (GPI)-anchored proteins has been described (Hertenstein et al, 1995; Brett et al, 1996; Taylor et al, 1997). The phenotype of these CD52-negative B and T cells closely resembles that of lymphocytes from paroxysmal nocturnal haemoglobinuria (PNH) patients, in which the first step of the GPI-biosynthetic pathway is blocked (Rowan et al, 1998). The molecular involvement by mutation of the PIG-A gene in these PNH-like lymphocytes is controversial (Hertenstein et al, 1995; Taylor et al, 1997) and has been described in one B cell chronic lymphocytic leukaemia (B-CLL) patient who developed PNH-like T lymphocytes after treatment with Campath-1H (Rawstron et al, 1999).
We characterized the PNH-like lymphocyte population that emerged in a patient affected by fludarabine refractory B-CLL after treatment with Campath-1H, using molecular analysis and determination of cytokine production.
The patient, a 57-year-old man, was diagnosed with B-CLL (SmIgM, Gλ) Binet stage C in 1991. He was initially treated with alkylating agents, followed by α-interferon (IFN) maintenance, obtaining a partial remission. A progression of disease occurred in 1997 and fludarabine treatment was started with no response. In October 1998, Campath-1H treatment (30 mg every other day for 12 weeks) was started for disease progression. Post-treatment evaluation revealed a complete remission in the peripheral blood and bone marrow (CD5/CD19 double-positive cells < 3% using flow cytometry). After Campath-1H treatment, the great majority of the patient's lymphoid cells were represented by PNH-like T-lymphocytes, with loss of CD52 (Fig 1A,B), 55, and 59 expression (data not shown). Notwithstanding the lymphocyte PNH phenotype, the study of the entire codifying region and flanking intronic sequences of the PIG-A gene, using single-stranded conformation polymorphism (SSCP) analysis, showed no abnormal electrophoretic patterns suggestive of gene mutation. To test the hypothesis that the loss of GPI-anchored proteins may be the result of reduced levels of PIG-A mRNA, we assessed the PIG-A mRNA levels using quantitative-competitive reverse transcription-polymerase chain reaction (RT-PCR). Interestingly, the expression of PIG-A mRNA was reduced compared with normal controls (5 × 103−104 molecules in four consecutive samples during Campath-1H treatment, versus 105 molecules in three normal controls (see Fig 1C). These findings may suggest a possible role for defective PIG-A mRNA production in the genesis of PNH-like lymphocytes, even in the absence of detectable PIG-A gene mutations (Rawstron et al, 1999). Regarding cytokine production (Table I), we found that all cytokines, except transforming growth factor (TGF)-β, were reduced compared with CLL control-patients and to a greater extent than normal controls. Interleukin (IL)-4 and IL-13 production was very low and IL-2, IFN-γ, IL-6, IL-10 and tumour necrosis factor (TNF)-α were, on average, 20–30% of normal controls and 50% of CLL patients. Interestingly, TGF-β production was disproportionately high compared with the other cytokines; furthermore, it was comparable to normal controls and threefold greater than the production occurring in CLL patients.
Figure 1. (A and B) Flow cytometric analysis of the patient's lymphocytes after Campath-1H treatment. Mononuclear cells were separated from peripheral blood using a Ficoll-Hypaque density gradient. The cells were analysed by two-colour immunofluorescence staining with the indicated fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-labelled monoclonal antibodies. The analysis was performed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA). A portion of each sample was incubated with the appropriate isotype control-antibodies to establish the background level of non-specific staining, and positivity was defined as staining greater than non-specific background staining. (A) left, gated event; right, 1·84% of B lymphocytes co-expressed CD52 antigen. (B) left, gated events; right, 2·47% of T-lymphocytes co-expressed CD52 antigen. Before Campath-1H treatment, > 90% of the patient's T-lymphocytes and > 90% of the B-lymphocytes co-expressed CD52 antigen (data not shown). (C) Evaluation of the expression of PIG-A gene in the patient and in normal controls by quantitative-competitive PCR. The competitor was obtained using a 36-bp PCR internal deletion of the PIG-A cDNA fragment to be quantified. In the first PCR round forward 1 (5′-CTGTACTAAGAGCAGCACTG-3′) and reverse (5′-CTGTCATGCAGCTGGTATCT-3′) primers were used, generating a fragment of 274 bp; in the second PCR round, the 274 bp fragment was amplified in the presence of reverse primer and forward 2 primer (5′-CTGTACTAAGAGCAGCACTGGATCCTACTGACTTCACTCC-3′), a hybrid represented by forward 1 sequence fused to a cDNA PIG-A sequence 36 bp downstream. Therefore, a PCR fragment of 238 bp derived from PIG-A cDNA, delimited by forward 1 and reverse sequences was generated. Total RNA (1 µg) was reverse transcribed to cDNA and for quantitative-competitive PCR, 100 ng of product were amplified in the presence of scalar amounts of competitor molecules using forward 1 and reverse primers. PCR conditions were as follows: hot start, 5 min at 95°C; 35 cycles at 95°C for 50 s, 55°C for 50 s and 72°C for 60 s, the same used to generate the competitor. Equivalence (indicated by an arrow) was 5 × 103 molecules/100 ng of total RNA in the patient, and 105 molecules/100 ng of total RNA in controls. M, molecular markers (Amplisize Molecular Ruler 50–2000 bp, Biorad, Hercules, California, USA).
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Table I. Cytokine production by mitogen-stimulated blood cultures.
| ||Patient||Normal controls||CLL|
|IL-2||35 ± 11||237 ± 17||117 ± 28|
|IFN-γ||926 ± 265||5147 ± 304||3234 ± 519|
|IL-4||1·9 ± 0·5||21·5 ± 1·9||9·7 ± 2·9|
|IL-6||540 + 235||2245 ± 120||939 ± 161|
|IL-10||115 ± 37||435 ± 44||218 ± 43|
|IL-13||1·2 ± 0·5||133 ± 14||129 ± 24|
|TNF-α||53 ± 11||147 ± 18||96 ± 22|
|TGF-β||1649 ± 317||1394 ± 78||630 ± 134|
Several abnormalities of cytokine production have been described in CLL regarding both the B and T cell compartment, including reduced IL-4, IFN-γ (Hill et al, 1999) and IL-6 (Hulkkonen et al, 1998), which have been related to the cellular and humoral immune defects of the disease. However, little is known about cytokine production by PNH lymphocytes, except for reduced IL-2 release in patients with aplastic anaemia who progressed to clinical PNH (Nissen et al, 1999). Compared with the CLL group, we found increased production of TGF-β, a well-known inhibitory cytokine. Excessive production of TGF-β by bone marrow stromal cells has been hypothesized to be responsible for the inhibitory activity on haematopoietic precursors in CLL (Lagneaux et al, 1993). Given the inhibitory activity of TGF-β, our results may suggest that elevated levels of this cytokine could contribute to alterations of the micro-environment and synergize with the selective pressure of Campath-1H, leading to expansion of pre-existing PNH-like lymphocytes (Rawstron et al, 1999).
The study of larger series, and in particular of classical PNH lymphocytes, will be necessary to demonstrate if this cytokine production pattern is characteristic of the PNH-like lymphocytes emerging after Campath-1H treatment.