Lucie Rihova

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 naïve (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 pre-treatment. 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.

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.

Plasma cell leukemia: from biology to treatment

Plasma cell leukemia (PCL) is a very aggressive and rare form of malignant monoclonal gammopathy characterized by the presence of plasmocytes in peripheral blood. It is classified as primary PCL occuring ‘de novo’, or as secondary PCL in patients with relapsed/refractory multiple myeloma. Primary PCL is a distinct clinicopathological entity from myeloma with different cytogenetic abnormalities and molecular findings, which are usually found only in advanced multiple myeloma. The clinical course is aggressive with short remissions and reduced overall survival. The diagnostic criteria are based on the percentage (>20%) and absolute number (2 × 109/L) of plasma cells in peripheral blood. After establishing diagnosis, induction therapy should begin promptly which is aimed to rapid disease control and to minimize the risk of early death. Intensive chemotherapy regimens and bortezomib‐based regimens, followed by high‐dose therapy with autologous stem cell transplantation, are recommended. Allogeneic transplantation can be considered in younger patients. This article reviews recent knowledge of this hematological malignancy that is associated with a very poor prognosis.

Flow cytometry-based enumeration and functional characterization of CD8 T regulatory cells in patients with multiple myeloma before and after lenalidomide plus dexamethasone treatment.

Multiple myeloma (MM) is a malignancy of plasma cells frequently associated with immune abnormalities. Several studies have confirmed that in MM immune deregulation can be mediated by increased numbers of CD4 T regulatory (Treg) cells, and these cells were also associated with poor outcome. In this study, we aimed to study CD8 Treg cells before and after lenalidomide plus dexamethasone (len‐dex) treatment in MM patients.

Flow cytometry in immunoglobulin light chain amyloidosis: Short review.

Flow cytometry (FCM) has found its application in clinical diagnosis and evaluation of monoclonal gammopathies (MG). Although, research has been mainly focused on multiple myeloma (MM), nowadays FCM becomes to be potential tool in the field of AL amyloidosis. Clonal plasma cells identification and specific phenotype profile detection is important for diagnosis, monitoring and prognosis of AL amyloidosis. Therefore, FCM could be a perspective method for study not only MM but also AL amyloidosis. This review provides an overview and possibilities of FCM application in AL amyloidosis.

Nestin expression throughout multistep pathogenesis of multiple myeloma

The stem cell marker nestin (NES) is found in dividing cells of developing and regenerating tissues. Upon terminal differentiation, NES expression is diminished but may be re‐expressed following injury or in cancer. Surprisingly, we recently confirmed NES as a tumour‐specific marker for mature CD138+38+ plasma cells (PC) in multiple myeloma (MM). The present study analysed NES expression throughout the spectrum of MM developmental stages, starting with individuals with no haematological malignancy, through monoclonal gammopathy of undetermined significance (MGUS) and MM to plasma cell leukaemia (PCL) and MM cell lines. NES was analysed in bone marrow PC of 163 MM, four PCL and nine MGUS patients, 10 individuals with no haematological malignancy and 6 myeloma cell lines (OPM‐2, RPMI‐8226, MOLP‐8, U‐266, EJM, NCI‐H929) by flow cytometry and/or real‐time polymerase chain reaction or immunochemistry. We observed a tendency of increased NES expression in parallel with disease progression. NES was evaluated as a reliable marker for accurate discrimination between MM patients and the control group. High NES levels were strongly associated with the presence of 1q21 gain. For the first time, NES was demonstrated to predict worse response to conventional therapy/novel agents. These results suggest that NES might become a useful clinical parameter with an important role in MM pathogenesis.

Do human B-lymphocytes avoid aging until 60 years?

Broad changes in human innate and adaptive immunity are associated with advanced age. The age-related alteration of gene expression was reported for both T and B lymphocytes. We analysed the genome-wide expression profiles (n=20) of naive and whole B cell populations from young and early aged healthy donors under 60 years. We revealed large homogeneity of all analysed genome-wide expression profiles but did not identified any significant gene deregulation between young (30-45 years) and early aged healthy donors (50-60 years). We argue that B cells avoid the aging program on molecular level until 60 years of age. Our results demonstrate the potential of hematopoietic stem cells to generate uncompromised lymphocytes in early elderly. These are very encouraging findings for the general health and the immunity maintenance would not need any intervention to naive B cells. Rather, a suitable immune stimulation in healthy body environment warrants further research into aging of older elderly.