CD200 Is Preferentially Expressed in CD138 ^dim Multiple Myeloma Cells: Implications for a Myeloma Stem Cell Subpopulation with Potential Immune-Resistance.

The CD138^negCD20⁺ cell population has been suggested as the putative multiple myeloma (MM) cancer stem cells, both in MM cell lines and in patient specimens. CD200 is an immunosuppressive membrane glycoprotein reported to be co-expressed with other stem-cell markers in prostate, breast, brain, and colon cancers. Also, CD200 is a negative prognostic factor for MM. It remains unknown whether CD200 is expressed in the CD138^negCD20⁺ MM cell population, the putative MM cancer stem cells. Here we addressed this question by assessing CD200 expression in different subpopulations of MM cells according to their CD138 expression levels.

Two MM cell lines (RPMI8226 and NCI-H929) were co-stained with FITC-labeled anti-CD200, PE-labeled anti-CD20, and APC-labeled anti-CD138 antibodies for multicolor flow cytometry analysis. The cells were gated into 4 subpopulations (h1>h2>h3>h4 corresponding to CD138 expression levels: high>dim>low>negative). In each gated cell population, CD200 expression was assessed, and the CD200⁺ and CD200^neg cell subpopulations were further gated for analysis of their CD20 expression levels.

In the RPMI 8226 cell line, the distribution of gated h1, h2, h3, and h4 populations was 87.51%, 5.86%, 5.26%, and 1.29%, respectively. In the h2 (CD138^dim) population, 22.88% of the cells were CD200+. In contrast, CD200 was expressed at much lower levels in the other three populations, ranging from 7.28% (h1), 6.65% (h3), to 0.48% (h4). In CD200⁺ cells from the gated h1, h2, h3, and h4 population, CD20⁺ cells were 19.50%, 23.03%, 27.67%, and 18.75%, respectively, while in the CD200^neg cells, CD20⁺ cells were 20.79%, 22.50%, 25.08%, and 28.02%, respectively. In the NCI-H929 cell line, 18.59% of the cells in the h2 population were CD200⁺, whereas only 1.61%, 1.69%, and 0.47% of the cells in the h1, h3, and h4 populations were CD200⁺. In each population, there were more CD20⁺ cells in CD200⁺ cells than in CD200^neg cells, which were 24.52% vs 0.04% (h1), 41.94% vs 11.34% (h2), 11.11% vs 3.06% (h3), and 50.00% vs 2.25% (h4), respectively.

These results demonstrate that the immunosuppressive molecule CD200 is preferentially expressed in a CD138^dim subpopulation of multiple myeloma cells. Depending on cell line, the putative myeloma stem cell marker CD20 is either preferentially, or not preferentially, expressed in the CD200⁺ cells, suggesting that an immune-resistant subpopulation of MM stem cells might exist, which may have important implications for designing immunotherapeutic approaches to treat this disease.

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