Immune regulation in plasma cell myeloma
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The pathogenesis of monoclonal gammopathies, particularly plasma cell myeloma (MM), is multifaceted and complex. In recent years, Treg and Th17 cells have emerged as key factors in the development and progression of malignancies including MM. However, there are still conflicting reports on whether regulatory T (Treg) cells and Th17 cells are increased or decreased in the peripheral blood (PB) of patients with MM. This is partly due to technical difficulties associated with the use of the transcriptional repressor, forkhead box P3 (FoxP3) to identify Treg cells. Studies have shown FoxP3 results to be dependent on the clones, fluorochromes attached and fixation/permeabilisation methods used. More recent studies have defined Treg cells as CD4⁺CD25ͪͥ cells which do not express CD127, an IL-7 receptor. This methodology was used to determine Treg cell number and develop assays for the assessment of Treg cell function. The study also extends to exploring Th17 cell number in plasma cell dyscrasias and the overall effect of the Treg and Th17 cell equilibrium on the survival of patients with MM. CD4⁺CD25ͪͥCD127⁻ expression was used to quantitate Treg cell numbers and an intracellular IL-17 assay on CD3⁺CD4⁺ cells was used for Th17 cell enumeration. Treg cell function was determined using carboxyfluorescein succinimidyl ester (CFSE) tracking of Treg depleted lymphocyte preparations stimulated by anti-CD3,CD2,CD28 beads at a 1:1 ratio for 4 days ± 1:1 fluorescence-activated cell sorted Treg cells. This functional assay was also used to investigate the effect of recombinant human (rh) TGF-β and rhIL-12 on Treg cells. The mean proportion of Treg cells in the CD4⁺ compartment of PB of patients with MM (n=32) was 8.9±0.6% and this was increased compared to the mean of the normal cohort (n=36) at 6.5±0.4% (p=0.009). However, no significant difference was observed between the frequency of PB Treg cells in the control group compared to patients with monoclonal gammopathy of undetermined significance (MGUS) (n=20) (mean=7.5±0.8%; P=0.24) and patients with Waldenström macroglobulinaemia (WM) (n=13) (mean=6.0±0.5%; P=0.48). Interestingly, a comparison of the absolute numbers exhibited different results. A significantly lower number of Treg cells was observed in patients with MM [(3.2±0.4) x10⁷/L; P<0.01] and WM [(3.0±0.6) x10⁷/L; P<0.01] compared to the control group [(6.4±0.7) x10⁷/L]. However, no significant difference was observed when comparing patients with MGUS [(4.3±0.8) x10⁷/L; P= 0.06] to the normal cohort. It was observed that a significantly higher proportion of PB Treg cells in patients with MM (85.9±1.8%; P<0.01) and WM (86.4±2.1%; P=0.02) to be of the CD45RO⁺ memory phenotype compared to the normal cohort (76.7±2.5%). However this was not observed in patients with MGUS (73.4±4.0%; P=0.47). In addition, the study revealed that PB Treg cell proportions were not influenced by MM stage. Thalidomide treated patients with MM appeared to have an increased PB Treg cell proportion, however only a small number of thalidomide treated patients were tested due to the use of thalidomide therapy being phased out and its replacement with lenalidomide. Treg cells from bone marrow (BM) were compared to matched PB samples from patients with MM, demonstrating a significantly greater proportion (p=0.02) of Treg cells in the CD4⁺ compartment of the BM (9.7±1.2%) compared to PB (6.7±1.4%). Regarding Th17 cells, a significant decrease (p=0.03) in the mean proportion and absolute number of Th17 cells was observed in the PB of patients with MM (n=22) compared with the controls (n=20) (0.7±0.1% and 2.0±0.6% respectively). However, the mean number of Th17 cells in patients with MGUS (2.2±0.6%; n=15) and WM (1.1±0.2%; n=12) was not significantly different from normal. No correlation was observed between Th17 cell number and MM staging or therapy. Additionally, the study explored the Treg/Th17 cell ratio in PB of patients with monoclonal gammopathies with comparison made to normal subjects. The mean Treg/Th17 cell ratio of patients with MM (16.1±2.4) was significantly higher (p=0.0002) than the healthy control group (6.6±1.0). The Treg/Th17 cell ratio of WM and MGUS patients was 7.0±1.0 and 4.9±0.5 respectively, neither of which were statistically different to the ratio of the normal controls. Most interestingly, patients who have survived with MM for 10 or more years possessed a Treg/Th17 cell ratio similar to the normal controls (7.04±2.47; p=0.84) and this was shown to affect overall survival. The data demonstrated that patients with MM observed to have a high Treg/Th17 cell ratio had an overall shorter survival compared to those whose Treg/Th17 cell ratio was lower (p<0.025). The suppressive capability of Treg cells from MM patients (n=15) was variable. The Treg cell function of patients treated with lenalidomide (n=5) was increased (mean=68%) compared to patients treated with thalidomide (n=5; mean=23%), Velcade (n=3; mean=12%), untreated patients (n=5; mean=36%) and normal controls (n=11; mean=31%). The suppression exerted upon the CD4⁺ T cell subset in patients treated with bortezomib was significantly lower when compared to the normal cohort. However, no significant difference in CD8⁺ T cell suppression was found between patients with MM and the normal controls. rhTGF-β increased the suppressive capabilities and rhIL-12 reduced the function of Treg cells from both MM and normal PB samples. In conclusion, immune regulation is dysfunctional in patients with MM as the proportion of PB Treg cells is increased and Th17 cells are reduced. Also, the cytokine microenvironment and treatment have a major impact on the function of Treg cells. The data clearly delineate the importance of the PB Treg/Th17 cell equilibrium, revealing a strong association between the Treg/Th17 cell homeostatic balance and disease progression and survival in MM, indicating an imbalance may cause either or both the innate and adaptive immune system to be dormant and incapacitate the anti-tumour response.
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