Interleukin-16 as a Marker of Sézary Syndrome Onset and Stage

Abstract

Introduction

Sézary syndrome is one of the most common forms of cutaneous T cell lymphoma (CTCL). It is characterized by skin infiltration of malignant T cells. We examined interleukin-16, a potent T cell chemoattractant and cell-cycle regulator, as a prospective marker of disease onset and stage.

Methods

The correlation of total intracellular interleukin-16 and surface CD26 was studied by flow cytometry. Confocal microscopy was performed to determine localization of interleukin-16 at different stages of the disease. The levels of interleukin-16 in plasma and culture supernatants were examined by enzyme-linked immunoassay. Additionally, lymphocytes from stage IB patients were cultured in the presence of interleukin-16 alone and in combination with interleukin-15, and their ability to survive and proliferate was determined by cell counts and [3H]TdR incorporation.

Results

The data indicate that loss of both nuclear and intracellular pro-interleukin-16 highly correspond to disease stage, with a concomitant increase in secreted mature interleukin-16 in both culture supernatants and patients’ plasma that peaks at stage IB. Loss of intracellular interleukin-16 strongly corresponded to loss of surface CD26, which has been shown to occur with more advanced stage of CTCL. Nuclear translocation of pro-interleukin-16 was not observed in late stages of Sézary syndrome, indicating this loss is not reversible.

Conclusions

We propose that it is feasible to use plasma levels of IL-16 as a potential diagnostic marker of Sézary syndrome and to use loss of intracellular IL-16 as a prognostic indicator of disease severity and stage.

Appendix

We began examining the consequences of the elevated IL-16 levels we found in patients’ plasma by determining whether or not IL-16 could serve as a survival factor or mitogen for SS malignancies. A dose–response curve of IL-16 demonstrated that SS patients’ T cells did proliferate following IL-16 treatment with a maximal effect at 1–10 nM depending upon stage; normal donor cells did not respond to IL-16 at any dose (Fig. 5a).

Fig. 5

Proliferation and survival of SS cultures in response to IL-16. a Dose titration assays at 1, 10, or 100 nM IL-16 show that most stages of malignant T cells proliferate in response to 10 nM IL-16. Cells were cultured for 3 days in the presence of cytokines before addition of 1 μCi [3H]TdR. Cultures were harvested 16 h post-pulse, and cpm from five replicates were expressed as stimulation index of media control (baseline; normal = 1 donor, stage 1A = 2 patients, stage 1B = 6 patients, stage IV = 3 patients). b CTCL mitogenic cytokine cocktails were tested in different types of T cell lymphomas. IL-2, IL-15, and IL-7 (10 ng/ml) were used separately or in combination with IL-16 (10 nM). SS cultures responded best to the combinations of IL-15/IL-2, or IL-15/IL-16, as demonstrated by this stage IB patient. c CTCL mitogenic cytokine cocktails were tested for their ability to support SS long-term cultures. Cells from various stages all responded best to a combination of IL-15 and IL-16, as demonstrated by these three representative patients. d SS stages above IA were maintained with 10 nM IL-16 in culture for 2 weeks. Cells were cultured in triplicate and counted on day 14 by Trypan blue exclusion; data are expressed as fold above media control wells (n = 1 representative patient per stage, except n = 2 for stage IB)

We also wanted to determine if combinations of IL-16 and other known CTCL mitogens could enhance this proliferative response. SS cells were cultured with combinations of IL-2, IL-7, IL-15, and IL-16. Cells that had been cultured for 1 to 2 weeks were assessed for proliferative index. We found that the combination of IL-15 and IL-16 seemed to induce the best proliferative responses (Fig. 5b, c; the most representative experiments).

Finally, we performed long-term cultures (2 to 3 weeks) of SS cells with 10 nM IL-16 and determined if it could serve as a survival factor. We found that IL-16 promoted survival for malignant cells from different disease stages (Fig. 5d).

Taken together, our data indicate that most SS patients would have malignancies that respond to IL-16. This supports our hypothesis that IL-16 can serve as a marker for SS disease progression, as it demonstrates possible biological effects of secreted IL-16 that link it to cancer growth and survival.

We tested other CTCL subtypes to determine whether or not our observations were unique to the SS subtype. The samples used for this analysis are represented in Tables II and III. We began by examining intracellular IL-16 and surface CD26 levels from a PTCL patient and two MF patients. Seventy percent of the PTCL patient’s cells were CD26+ and 8.85% were IL-16 positive, a stage IB MF patient had 4.46% CD26+ and 62.31% IL-16+, and a stage II MF patient had 24.74% CD26+ and 46.75% IL-16+ lymphocytes. (Fig. 6a). Flow data show that the IL-16 and CD26 levels exhibit patterns distinct from SS samples.

Table II Clinical characteristics of MF CTCL patients

Table III Clinical characteristics of other CTCL subtypes

Fig. 6

Other CTCL subtypes do not exhibit the same patterns of IL-16 loss or responsiveness. a Intracellular IL-16 and surface CD26 levels for a PTCL patient and two MF patients. Patterns of loss are distinct from that of the SS subtype. b Secreted IL-16 levels from cultures of MF stage IA (n = 4), MF stage IB (n = 5), MF stage II (n = 1), and CD30+ anaplastic large cell (n = 1) CTCL subtypes are higher than normal donor cell culture supernatants (n = 12) (left). Plasma IL-16 levels from MF stage IA (n = 2), stage IA/B (n = 1), stage IB (n = 6), stage II (n = 1), and stage III (n = 2) are higher than normal donor levels (n = 31; middle). Plasma IL-16 levels from CD8+ (n = 2), CD30+ (n = 1), PTCL (n = 1), and non-Hodgkin’s (n = 1) lymphoma subtypes are also higher than normal donor levels (n = 31; right). c CTCL mitogenic cytokine cocktails were tested in different types of T cell lymphomas. IL-2, IL-15, and IL-7 (10 ng/ml) were used separately or in combination with IL-16 (10 nM). No other CTCL subtypes exhibited increased responsiveness to IL-16 alone or in combination with other mitogens, except for a PTCL that responded to IL-16 in combination with IL-7 (representative donor for each subtype). d CTCL mitogenic cytokine cocktails were tested for their ability to support MF cells in long-term culture. IL-16 treatment was comparable to media controls, and combinations with IL-2 and IL-15 resulted in decreased proliferative responses (representative donor)

Secreted IL-16 levels from plasma and culture supernatant samples from other CTCL subtypes were measured by ELISA. We found increases in IL-16 levels when compared to normal donors for most CTCL patients (Fig. 6b). We also tested the ability of several different CTCL subtypes to proliferate in response to IL-16 treatment (Fig. 6c). We found that a PTCL patient seemed to respond best to a combination of IL-7 and IL-16, though the cells did not proliferate in response to IL-16 treatment alone. Cells from a MF patient and cells from a CD30+ anaplastic large cell lymphoma did not proliferate in response to IL-16, and combinations of IL-16 with IL-2, IL-7, or IL-15 seemed to yield decreased responses when compared to known mitogens by themselves. A CD8+ lymphoma patient’s cells did not respond to any cytokines or combinations. Finally, cells from a combined T and B cell lymphoma patient did seem to require endogenous IL-16 to achieve maximal proliferative responses, as addition of a neutralizing antibody against IL-16 (clone 14.1) [30] resulted in decreased proliferative profiles. However, these levels were still above basal proliferative levels determined from media control wells. We also performed a long-term culture analysis on cells from a MF patient and found that they did not respond to IL-16 alone or in combination with other mitogens (Fig. 5d). This indicates that while IL-16 is secreted from other CTCL subtypes, it may not have a direct link to disease onset and progression since tumors are non-responsive.