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

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

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 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

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

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