Time to progression analysis according to T regulatory cells.

<p>TTP analysis from 44 previously untreated myeloma patients showed patients with ≥5% of Treg cells have shorter time for progression. Kaplan-Meier curves demonstrate TTP according to ≥5% and <5% of peripheral blood Treg cells. TTP, Time to progression; n, number of patients; NR, not reached.</p

Isolation of T regulatory cells and their subsets.

<p>Isolated T lymphocytes were labeled with fluorescent conjugated monoclonal antibodies targeting CD4 and CD25 and sorted by FACS Aria into CD4⁺CD25hi⁺ (Treg cells) and CD4⁺CD25⁻ (conventional T cells). Pre- (A) and post-sorted (B) CD4⁺CD25hi⁺ cells and CD4⁺CD25⁻ cells from a MM patient is shown and purity of sorted cells is represented in percentage. Similarly, to isolate Treg cell subsets, cells were fluorescently labeled for specific antigens (CD4, CD45RA and CD25) and sorted using FACS Aria into four populations: CD4⁺CD25⁺CD45RA⁺ (naïve Treg cells), CD4⁺CD25hi⁺CD45RA⁻ (activated Treg cells), CD4⁺CD25⁺CD45RA⁻ (non-Treg cells) and CD4⁺CD25⁻CD45RA⁺ (naïve CD4 T cells). Pre- (C) and post-sorted (D) Treg cell subsets and naïve CD4 T cells from a MM patient is shown and purity of sorted cells is represented in percentage.</p

Frequencies of peripheral blood and bone marrow T regulatory cells from pre-malignant and malignant myeloma patients.

<p>Peripheral blood (A) and bone marrow (B) Treg cells from different patient cohorts are analyzed and evaluated statistically by Mann-Whitney U test (P≤0.05). Number of samples analyzed in each cohort is given in parentheses. Median, 25^th–75^th percentile and range of data are indicated as horizontal line, box and whiskers, respectively. HVs, healthy volunteers; MGUS, monoclonal gammopathy of undetermined significance; SMM, smoldering multiple myeloma; MM, newly diagnosed multiple myeloma; Rel, relapsed multiple myeloma; and Rem, patients in remission.</p

Inhibitory function of T regulatory cells.

<p>CFSE labeled CD4⁺CD25⁻ cells were co-cultured with different concentration of Treg cells in the presence of anti-CD3/CD28 beads and accessory cells. After 4 days of co-culturing, (A) based on concentration dependent manner, Treg cells inhibited the proliferation of CFSE labeled CD4⁺CD25⁻ cells which was clearly shown by the dilution of CFSE in FITC channel. In the absence of Treg cells increased proliferation of CFSE labeled CD4⁺CD25⁻ cells was observed. (B) Comparison of MM and HV Treg cells function showed similar level of proliferation inhibition at different concentrations (proliferation/division of CD4⁺CD25⁻ cells is expressed in %). (C) Similarly to proliferation inhibition, IFN-γ concentration from culture supernatant was also decreased based on Treg cell numbers (IFN-γ concentration is expressed in pg/ml). Level of IFN-γ did not differ significantly between MM and HV cohorts from proliferation assays. Mann-Whitney U test was used to assess the statistical difference between MM and HV cohorts. Statistical difference between MM and HV cohorts is indicated by P value. Median is represented by horizontal line, and raw data from each experiment are represented by small dots and squares. CFSE, carboxyfluorescein succinimidyl ester; MM, multiple myeloma; HV, healthy volunteer.</p

Patients’ characteristics.

<p>Footnote: ISS, International staging system; B-J protein, Bence-Jones protein; LDH, lactate dehydrogenase; M protein, monoclonal protein; and BMPCs, bone marrow plasma cells.</p

MicroRNA expression pattern in extramedullary myeloma (EM), multiple myeloma (MM) patients and healthy donors (HD).

<p>Hierarchical clustergram discriminating EM serum samples from A) MM, yellow color indicates EM serum samples, light blue MM serum samples, <i>p</i><0.05. B) HD, yellow color indicates EM serum samples, dark blue indicates HD serum samples, <i>p</i><0.05. Differential expression of miRNA is shown by the intensity of red (up-regulation) <i>versus</i> green (down-regulation).</p

Correlation of serum miR-130a levels with clinically important biochemical parameters.

<p>Data are presented as coefficients of correlation. Statistically significant correlations are marked in bold and italics.</p