Research Article: Increased T Regulatory Cells Are Associated with Adverse Clinical Features and Predict Progression in Multiple Myeloma

Date Published: October 10, 2012

Publisher: Public Library of Science

Author(s): Karthick Raja Muthu Raja, Lucie Rihova, Lenka Zahradova, Maria Klincova, Miroslav Penka, Roman Hajek, Frederic Rieux-Laucat. http://doi.org/10.1371/journal.pone.0047077

Abstract

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.

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).

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.

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.

Partial Text

Multiple myeloma (MM) is a malignant plasma cell (PC) disorder and is preceded by a pre-malignant stage known as monoclonal gammopathy of undetermined significance (MGUS). MGUS is characterized by lower plasma cell (PC) infiltration (<10%) in bone marrow (BM), <30 g/L of monoclonal protein (M-protein) and absence of organ damage. In contrast, MM patients are characterized by higher PC infiltration (≥10%), ≥30 g/L of M-protein, bone lesions and presence of organ damage [1], [2]. Immune dysfunction is an important feature of MM leading to infections, enhancement of tumor growth and resistance to chemotherapy. Also, decreased level of immune cells (B, CD4+ and CD8+ cells) has been documented and associated with poor survival of MM patients [3], [4]. In 2003, a study confirmed expansion of regulatory T cells (Treg cells) in cancer patients [5]. Following this study, several studies have shown that Treg cells were associated to impaired immune functions in solid tumors and hematological malignancies [6], [7]. In Treg cells, FoxP3 is considered as a master regulatory molecule [8]. FoxP3 molecule is expressed in thymus-derived Treg cells (natural Treg cells) and peripherally induced Treg cells (CD4+CD25hi+FoxP3+) but not in other induced Treg cells including Tr1 cells and TH3 Treg cells [9], [10]. Treg cells suppress other immune cells through contact- dependent and independent mechanisms. Several molecules have been reported for contact-dependent inhibition mechanism, such as CTLA-4 (inhibits antigen presenting cells), lymphocyte activation gene 3 (induces inhibitory signal through MHC II molecules), granzymes (mediate lysis of conventional T cells) and CD95-CD95L (induces apoptosis in conventional T cells) [11], [12], [13], [14]. In contact-independent mechanism, molecules such as IL-10 (attenuates dendritic cells, converts conventional T cells into Tr1 cells), TGF-β and latency-associated peptide (induce FoxP3 expression in conventional T cells), galectin 1 (arrests cell cycle and induces apoptosis in conventional T cells) and CD25 (adsorbs IL-2) plays significant role in suppression [15], [16], [17], [18], [19], [20]. Several studies documented peripheral blood (PB) expansion of functionally active Treg cells in MM [21], [22], [23]. For the first time, our study shows prognostic value of Treg cells in prediction of time to progression (TTP) in MM and their association with adverse clinical features. In addition, this study also acknowledged the findings of previous studies in a larger cohort of patients. Treg cells play an important role in maintaining immunological homeostasis and any imbalances leads to impaired immune functions. It is accepted that Treg cells are expanded in hematological and non-hematological malignancies. Increased FoxP3 expression and Treg cell elevation are generally considered to be poor outcome markers in various cancers, including Hodgkin’s lymphoma, follicular lymphoma, breast cancer, gastric malignancies, and ovarian cancers [27], [28], [29], [30], [31]. For the first time in MM, we were able to demonstrate that increased frequencies of Treg cells (≥5%) predicted shorter TTP. Very recently, a study also showed that high number of Treg cells from MM patients were associated with poor survival [32]. However, several questions are yet to be answered in MM; do tumor cells enhance the expansion of Treg cells during progression or is progression induced by Treg cells due to suppression of anti-tumor responses? We believe that both processes are mutually inclusive because several in vitro and animal model studies in other cancers documented that tumor and tumor infiltrating cells derived factors such as CCL2, prostaglandin 2 (PGE2), H-ferritin, indoleamine 2, 3-deoxyginase (IDO) and IL-10 recruit and enhance Treg cells in the tumor microenvironment and thereby, increase the risk of tumor progression [27], [33], [34]. In MM, a study showed that dendritic cells enhanced the expansion of Treg cells in vitro and in vivo[35]. However, it is still unknown how Treg cells are influenced by tumor cells/tumor microenvironment of MM patients. Source: http://doi.org/10.1371/journal.pone.0047077