Circulating Regulatory T-Cells in Monoclonal Gammopathies of Uncertain Significance and Multiple Myeloma: In Search of a Role

The frequency and function of regulatory T-cells (Tregs) in multiple myeloma (MM) are still matter of debate. The percentage and absolute number of circulating Tregs (CD4+CD25+high  densityCD127−/low  density) from 39 patients with untreated MM and 44 patients with monoclonal gammopathies of uncertain significance (MGUS) were tested and compared with 20 healthy subjects as controls. The mean percentage number of circulating Tregs was 2.1%  ± 1.0 (range 0.75–6.1%) in MM patients; 2.1%  ± 0.9 (range 0.3–4.4%) in MGUS; and 1.5%  ± 0.4 (range 0.9–2.1%) in controls (p ns). Mean absolute number of Tregs was 36.3/μL ± 23.7 (range 6.7–149/μL) in MM; 38.8/μL ± 19.1 (range 4.3–87/μL) in MGUS; and 39.4/μL ± 12.5 (range 18–63/μL) in controls (p ns). After a median follow-up of 38 months, 5 MGUS and 2 smoldering MM (SMM) transformed into overt MM; however Tregs number did not predict this evolution. With respect to MM patients and after a median follow-up of 33 months, Tregs did not show any significant correlation with main clinical and laboratory characteristics. Finally, from a functional point of view, Tregs displayed an effective suppressor function, irrespective of disease status. This study indicates that the number of circulating Tregs does not differ in different monoclonal gammopathies and normal subjects and do not correlate with clinical features of MM.


Introduction
Natural and inducible regulatory T-cells (Tregs) are a small subset of T-lymphocytes able to suppress immune responses by direct interaction with other immune cell types or through immunosuppressive cytokines [1]. They appear to be crucial in maintaining immune homeostasis, mediating peripheral tolerance and preventing autoimmunity. Emerging evidences 2 Journal of Immunology Research suggest that Tregs may also modulate host T-cell activity against tumor-associated antigens, thereby facilitating tumor escape from immunological control [2]. Tregs are characterized by the constitutive expression of surface CD4 antigen, high levels of surface CD25 antigen (IL-2 receptor -chain), and the transcription factor forkhead box P3 (FOXP3), with low to nil CD127 (IL-7 receptor) [3].
We have recently demonstrated that Tregs may have a prognostic role in patients with Rai stage 0 chronic lymphocytic leukemia [4]. However, frequency and function of Tregs in monoclonal gammopathies of uncertain significance (MGUS) and multiple myeloma (MM) are still matter of debate. Aiming to evaluate a possible prognostic significance in MGUS and MM, we studied the percentage and absolute number of circulating Tregs in a consecutive, prospective cohort of these patients and in a group of healthy subjects, as controls.

Materials and Methods
Methylenediaminetetraacetic acid-(EDTA-) anticoagulated peripheral blood samples from 39 patients with newly diagnosed MM (mean age 69 years; range 37-90 years; 18 male; 21 female) and 44 patients with MGUS (mean age 65 years; range 39-87 years; 25 male; 19 female) were collected after informed written consent. Ten out of 39 MM patients were reclassified as having smoldering MM (SMM) just after the diagnosis. No relapsed, treated, or in remission phase after therapy patients were evaluated. Peripheral blood samples from 20 healthy subjects (mean age 59 years; range 32-80 years; 10 male; 10 female) were used as controls. Main patients' characteristics are summarized in Table 1.
Tregs were numbered by means of flow cytometry (FAC-SCanto II, Becton Dickinson Biosciences (BDB), San Jose, CA, USA) as follows: 100 L of each of the following directly conjugated monoclonal antibodies (MoAbs): CD4-PerCP (peridinin chlorophyll protein complex)/CD127-PE (phycoerythrin)/CD25-Pe-Cy7 (PE-Cyanine 7)/CD45-APC-Cy/(allophycocyanin-Cy7). All MoAbs were purchased from BDB. After lysis of red blood cells and repeated washing, the samples were analyzed by flow cytometry, by acquiring a minimum of 20,000 events for each sample. All subsequent analyses were performed using FACS-Diva software (BDB). Tregs were evaluated according to the gating strategy protocol previously described ( Figure 1) [12] and defined by the expression of CD4 and CD25 at high density and CD127 at low density or undetectable levels. This gating strategy was chosen because it better discriminates between Tregs and activated T-cells that normally upregulate CD25 antigen [3].
An in vitro proliferation inhibition assay, as previously described by Castella et al. [13], was used in 14 representative subjects (3 healthy controls and 4 MGUS, 3 SMM, and 4 active MM patients) to assess the suppressive function of circulating Tregs. Briefly, cell proliferation was evaluated measuring the uptake of 1 Ci 3 HTdR, with a scintillation counter, by cell culture in which allogeneic PBMC were used as accessory cells, purified CD4 + CD25 + CD127 low cells as Tregs, autologous CD4 + CD25 − cells as responder cells, and soluble anti-CD3 as polyclonal activator. Conventional    Horizontal bars indicate mean (± standard deviation) values. No statistically significant differences were found among the three groups.
between two variables was measured using Spearman rank correlation for nonparametric data to assure the pertinence of all variables. Progression-free survival (PFS) was measured from time of diagnosis to time of progression into overt MM for MGUS and SMM or after an effective first-line therapy in MM patients; overall survival (OS) was determined from time of diagnosis to death event. The Kaplan-Meier method was used to analyze both PFS and OS. The receiver-operatorcurve (ROC) analysis was performed to estimate cut-off of Tregs (as number or percentage) to predict progression or survival. Statistical significance was defined as < 0.05. Statistical analyses were performed using R statistical software (version 3.2.0; R Foundation for Statistical Computing).

Results
Overall, the mean number of circulating Tregs, detected as percentage of all lymphocytes, was 2.1% data were available only in a minority of MM patients, it was not possible to include this parameter in the analysis.

Discussion
Though it has been hypothesized that Tregs accumulate in the course of MM exerting an immunosuppressive function, thus favoring the progression of the disease, conflicting results have been published so far on the frequency and prognostic relevance of Tregs in monoclonal gammopathies (Table 2) Journal of Immunology Research 5  Journal of Immunology Research [5][6][7][8][9][10][11]. The variable results reported by different groups which have investigated Tregs in MM are at least in part probably due to the heterogeneous experimental approaches used: bone marrow, whole blood, isolated peripheral blood mononuclear cells (PBMC), depletion of CD25 + T-cells, different panel of monoclonal antibodies, and different analytical strategy to identify Tregs by means of flow cytometry ( Table 2). In this setting, we choose to use CD127 evaluation, instead of FoxP3, to avoid technical bias due to permeabilization techniques and the subjectivity related to the variable expression of CD25 [3]. Furthermore, in our study, we analyzed peripheral blood. This must be taken into account because bone marrow Tregs frequency may differ from that found in peripheral blood. In fact, it has been reported that MM microenvironment support overexpression of FOXP3 and CTLA4 expression in bone marrow, thus suggesting an accumulation of immunosuppressive Tregs in the tumor microenvironment of MM patients [14]. The tumor growth and the failure of local immune control may be favored by changes in the bone marrow microenvironment, such as the accumulation of Tregs that may contribute to the immune imbalance in MM. However, in some studies, frequencies and total counts of Tregs were found similar in the bone marrow and peripheral blood of MM patients and control subjects [11].
In almost all functional studies, Tregs activity was found comparable to that of healthy subjects, with the exception of the Prabhala et al. study [5]. In a critical revision published by Muthu Raja et al., this was attributed to the use of PBMC as responder cells [15]. From a functional point of view, our data are completely in agreement with those recently reported by Foglietta et al. [11].
Of interest, among their pleiotropic functions, immunomodulatory agents thalidomide and lenalidomide, currently used as standard therapy in MM, are able to decrease Tregs number and to inhibit their proliferation and immunosuppressive function [16,17]. Other studies, however, have instead suggested that Tregs number may be increased using these drugs [17,18].
Finally, two groups recently found that symptomatic MM patients with long-term survival (>10 years), accounting for about 5% of all MM patients, displayed a distinct immunological profile characterized by a lower number of BM and circulating Tregs, higher Th17 cells, and proliferative cytotoxic T-cells, compared to patients with shorter survival [19,20]. Our study does not seem to support such an observation, but the relatively short follow-up of patients should be considered.
In conclusion, our results indicate that the circulating number of Tregs does not differ among MGUS, MM, and normal subjects and does not seem to influence disease status or to provide prognostic information in MM patients as well. However, due to the contrasting results published so far, further studies are needed to better understand the possible role of Tregs in the pathogenesis and disease progression of MM, also in the light of the emerging role of novel immunotherapies, such as monoclonal antibodies, potentially active on minimal residual disease after specific treatments. Finally, the relevance of standardized methods to approach this field of investigation is also emphasized by the critical review of published data.