Functionally suppressive CD8 T regulatory cells are increased in patients with multiple myeloma: a cause for immune impairment.

BACKGROUND: Multiple myeloma (MM) is a plasma cell malignancy frequently associated with impaired immune cell numbers and functions. In MM, several studies have previously shown that CD4 regulatory T (Treg) cells hamper effector T cell functions and enhance immune dysfunction. In this study, we aimed to prove the presence of functionally suppressive Treg cells expressing CD8 phenotype (CD8 Treg cells) in MM. To the best of our knowledge, this has not been reported previously in MM. METHODS: We analyzed CD8 Treg cells and their transcription factor FoxP3 from 64 newly diagnosed MM patients using flow cytometry and real time-polymerase chain reaction (RT-PCR). RNA profile of cytokines in CD8 Treg cells was also assessed using RT-PCR. CD8 Treg cells from 5 MM patients and 5 healthy donors were functionally evaluated using proliferation assays. RESULTS: CD8 Treg cells (CD8+CD25hi+) were significantly elevated in MM patients (P<0.0001), and their transcription factor FoxP3 expression was also higher in MM (P<0.0001) compared to healthy donors which was evidenced by flow cytometry and RT-PCR analyses. CD8 Treg cells negatively correlated with total lymphocyte count (P = 0.016). Functional studies revealed that CD8 Treg cells isolated from MM patients and healthy donors inhibited proliferation of CD4 T cells in a concentration dependent manner. In the presence of CD8 Treg cells in proliferation assays, level of IFN-γ was decreased but not IL-10. CD4 T cells from MM patients secreted abnormal level of IL-10 compared to healthy donors (P = 0.01) in proliferation assays without CD8 Treg cells. RNA profile of cytokines from CD8 Treg cells did not differ significantly between MM patients and healthy donors. CONCLUSIONS: These findings show the presence of increased number of functionally suppressive CD8 Treg cells in MM patients. We believe that these suppressive CD8 Treg cells might enhance immune impairment and disease progression in MM

Isolation of CD8+CD25hi+ (CD8 Treg cells) and CD4+CD25− cells.

<p>Isolated T lymphocytes were labeled with fluorescent conjugated monoclonal antibodies and sorted by FACS Aria into CD8+CD25hi+ cells and CD4+CD25− cells. As an example, pre (A) and post (B) sorted CD8+CD25hi+ cells and CD4+CD25− cells are shown from a MM patient, and their purity is expressed in percentage.</p

Evaluation of FoxP3 protein in CD4 and CD8 T regulatory cells using immunoblotting.

<p>CD4 Treg cells and CD8 Treg cells were denatured and proteins were separated by polyacrylamide gel electrophoresis. FoxP3 and GAPDH proteins were detected by immunoblotting. FoxP3 (top row) and GAPDH (bottom row) protein levels in CD4 Treg cells and CD8 Treg cells from 2 MM patients and a healthy donor are shown. As a control, FoxP3 and GAPDH protein levels in non-regulatory T cells are shown from a healthy donor. Treg cells, T regulatory cells; Non-Treg cells; non-regulatory T cells; MM, multiple myeloma; HD, healthy donor; FoxP3, forkhead-winged helix transcription factor; GAPDH, human glyceraldehyde 3-phosphate dehydrogenase.</p

RNA profile of cytokines in CD8 T regulatory and non-regulatory CD8 T cells from multiple myeloma patients and healthy donors.

<p>Raw data were analyzed and calculated using 2^−dCq formula. Statistical difference was tested by Student t-test. SD, standard deviation.</p

Expression profile of FoxP3 in CD4 and CD8 T regulatory cells.

<p>Total RNA isolated from purified CD4 Treg cells, CD8 Treg cells and non-regulatory T cells was analyzed for FoxP3 expression using RT-PCR. Mann-Whitney U test showed closer statistical significant difference in the expression of FoxP3 in CD8 Treg cells between MM patients and healthy donors (P = 0.055) but not in CD4 Treg cells and non-regulatory T cells. Median value of cytokines is indicated by horizontal line, small dots and squares represent raw data from each experiment. MM, multiple myeloma; HD, healthy donor; FoxP3, forkhead-winged helix transcription factor, Treg cells; T regulatory cells.</p

Phenotypic feature of CD8 T regulatory cells.

<p>Flow cytometric analyses of CD8 Treg cells (CD8+CD25hi+) from a MM patient (A) and a healthy donor (B) are presented. (C) Represents phenotypic profile of CD8 Treg cells (CD8+CD25hi+) from a MM patient. At first, CD8 Treg cells were identified and further analyzed for intracellular FoxP3 and various surface antigens, including CD127, CD62L, CTLA-4, CD45RO and CD28. Expression of these antigens is shown on respective histograms. CD4+CD25hi+ Treg cells served as positive control for expression of FoxP3. Positive expression of specific marker/antigen is indicated as dark grey peak, and isotype control is indicated as light grey peak. MM, multiple myeloma; HD, healthy donor; FoxP3, forkhead-winged helix transcription factor; CTLA-4, cytotoxic T lymphocyte antigen-4.</p

Frequency and absolute number of peripheral blood CD8 T regulatory cells, CD8.

<p>T cells and total lymphocytes from multiple myeloma patients and healthy donors.</p><p>Statistical difference was tested by Mann-Whitney U test.</p

Profiling of pro- and anti-inflammatory cytokines.

<p>Culture supernatants from proliferation assays were collected after 4 days of culturing and profiled for IFN-γ and IL-10 cytokines using cytometric bead array assays. Level of IFN-γ (A) and IL-10 (B) cytokines are shown from MM patients and healthy donors. Mann-Whitney U test showed significant difference only in the level IL-10 between MM patients and healthy donors (*P = 0.01) from proliferation assays without CD8 Treg cells. Median value of cytokines is indicated by horizontal line, small dots and squares represent raw data from each experiment. MM, multiple myeloma; HD, healthy donor; IFN-γ, interferon-gamma; IL-10, interleukin-10; *, statistically significant.</p

Patients’ baseline characteristics.

<p>Patients’ baseline characteristics.</p