Increased T regulatory cells are associated with adverse clinical features and predict progression in multiple myeloma.

Background: Regulatory T (Treg) cells play an important role in the maintenance of immune system homeostasis. Multiple myeloma (MM) is a plasma cell disorder frequently associated with impaired immune cell numbers and functions. Methods: We analyzed Treg cells in peripheral blood (n = 207) and bone marrow (n = 202) of pre-malignant and malignant MM patients using flow cytometry. Treg cells and their subsets from MM patients and healthy volunteers were functionally evaluated for their suppressive property. A cohort of 25 patients was analyzed for lymphocytes, CD4 T cells and Treg cells before and after treatment with cyclophosphamide, thalidomide plus dexamethasone (CTD). Results: We found elevated frequencies of Treg cells in newly diagnosed (P<0.01) and relapsed MM patients (P<0.0001) compared to healthy volunteers. Also, Treg subsets including naive (P = 0.015) and activated (P = 0.036) Treg cells were significantly increased in MM patients compared to healthy volunteers. Functional studies showed that Treg cells and their subsets from both MM and healthy volunteers were similar in their inhibitory function. Significantly increased frequencies of Treg cells were found in MM patients with adverse clinical features such as hypercalcemia (.10 mg/dL), decreased normal plasma cell (<5%) count and IgA myeloma subtype. We also showed that MM patients with >5% of Treg cells had inferior time to progression (TTP) (13 months vs. median not reached; P = 0.013). Furthermore, we demonstrated the prognostic value of Treg cells in prediction of TTP by Cox regression analysis (P = 0.045). CTD treatment significantly reduced frequencies of CD4 T cells (P = 0.001) and Treg cells (P = 0.018) but not Treg cells/CD4 T cells ratio compared to pretreatment. Conclusions: Our study showed immune deregulation in MM patients which is evidenced by elevated level of functionally active Treg cells and patients with increased Treg cells have higher risk of progression

Univariate Cox regression analysis for factors influencing time to progression in multiple myeloma.

<p>Footnote: CI, confidence interval; ISS, international staging system; M protein, monoclonal protein; β2-M, β2 microglobulin; BMPCs, bone marrow plasma cells; N-PCs, normal plasma cells.</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

Comparison of pre- and post-treatment frequencies of lymphocytes, CD4 T cells and T regulatory cells.

<p>Comparison of pre- and post-treatment frequencies of lymphocytes, CD4 T cells and T regulatory cells.</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

Comparison of pre- and post-treatment frequencies of lymphocytes, CD4 T cells and T regulatory cells in relation with treatment response.

<p>Comparison of pre- and post-treatment frequencies of lymphocytes, CD4 T cells and T regulatory cells in relation with treatment response.</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

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

Inhibitory function of naïve and activated T regulatory cells.

<p>Similarly, we assessed the suppressive function of naïve, activated and non-Treg cells by CFSE based proliferation assay. Naïve CD4 T cells (CD4⁺CD25⁻CD45RA⁺) were CFSE labeled and co-cultured with naïve, activated and non-Treg cells at a ratio of 1∶1 in the presence of anti-CD3/CD28 beads and accessory cells. (A) Represents proliferation assay from a MM patient. In the presence of naïve and activated Treg cells in the proliferation assay, naïve CD4 T cell proliferation was inhibited but not in their absence/presence of non-Treg cells. This was clearly shown by dilution of CFSE in the FITC channel. (B) In a head to head analysis, proliferation of naïve CD4 T cells between MM patients and HVs did not differ significantly either in the presence or absence of naïve and activated Treg cells in the proliferation assay (proliferation/division of naïve CD4 T cells is expressed in %). (C) Similarly, level of IFN-γ secretion by naïve CD4 T cells did not differ significantly between MM and HV cohorts in the presence or absence of naïve and activated Treg cells (IFN-γ concentration is expressed in pg/ml). 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