Interleukin-3 (IL-3) is overexpressed by T lymphocytes in multiple myeloma patients

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Abstract
In order to clarify this issue, in the present study we have investigated IL-3 mRNA expression and IL-3 secretion by human myeloma cell lines (HMCLs), total BM mononuclear cells (BMMCs), and isolated stromal cells (BMSCs) as well as by CD138+ cells, CD14+ cells, CD20+ cells, and CD3+ (CD4+ and CD8+) cells obtained from BM aspirates of MM patients (n = 20) and MGUS subjects (n = 10). The purification of the different cell types was performed by an immunomagnetic method (magnetic activated cell sorting [MACS]; Miltenyi, Auburn, CA) using anti-CD138, anti-CD14, anti-CD20, anti-CD3, anti-CD4, and anti-CD8 mAbs coated with microbeads, and purity was checked by flow cytometry. Only samples with purity more than 90% were analyzed. Using a reverse-transcription-polymerase chain reaction (RT-PCR) as previously published2 (IL-3: primer pairs: forward, 5′-CTTCAACAACCTCAATGGGG-3′ and reverse, 5′-AATTCATCTGATGCCGCAGG-3′; IL-3 receptor: forward, 5′-TCT CCA GCG GTT CTC AAA GTT CCC ACA TCC-3′ and reverse, 5′-CCC AGA CCA CCA GCT TGT CGT TTT GGA AGC-3′), we found that RPMI-8226, XG-1, XG-6, U266, OPM-2, JJN3, ARP-1, and H929 did not express IL-3 mRNA, whereas IL-3 receptor was expressed only on RPMI and OPM2 (Figure 1A). Thereafter, we analyzed freshly purified CD138+ cells and we found that all samples tested were negative for IL-3 mRNA expression as shown for 12 representative MM patients (Figure 1A). On the other hand, we found that BMMCs of MM patients were positive for IL-3 mRNA, whereas BMMCs of MGUS subjects were negative. Among the BMMCs, we show that CD14+, CD20+ cells as well as BMSCs of both MM patients and MGUS subjects were negative for IL-3 mRNA (Figure 1B). On the contrary, we found that CD3+ cells of MM patients strongly expressed IL-3 mRNA, whereas CD3+ cells in MGUS subjects were negative as shown for 4 representative MM patients (Figure 1B). Among CD3+ cells, we found that CD4 T cells expressed IL-3 mRNA in MM patients (Figure 1B). Using flow cytometry to detect intracytoplasmic IL-3 (clone: BVD3-1F9; BD Biosciences, San Jose, CA), we confirmed that CD4+ T cells produce IL-3 in MM patients but not in MGUS subjects (data not shown). Consistently, we found significantly higher IL-3 levels, detected by enzyme-linked immunosorbent assay (ELISA; Biosource International, Camarillo, CA), in the supernatants of CD3+ (106 cells/mL) cells of MM patients compared with MGUS (mean ± SD/106 CD3+ cells: 86.6 ± 15.4 pg/mL vs 9.9 ± 2.3 pg/mL; P = .01) (Figure 1C). No detectable IL-3 levels were measured in the conditioned medium of CD138+, CD14+, CD20+, and BMSCs in both groups analyzed (data not shown).