Dysfunctional T regulatory cells in multiple myeloma

T regulatory (T_reg) cells play an important role in the maintenance of self-tolerance, control of auto-immunity, and regulation of T-cell homeostasis, and they modulate overall immune responses against infectious agents and tumor cells.¹  Natural T_reg cells develop during normal T-cell maturation in the thymus and represent 5% to 10% of the CD4⁺ cell compartment in the peripheral blood.²  These cells express CD4 and CD25 surface antigens as well as CTLA-4, GITR, CD103, CD62L, CD69, CD134, CD71, CD54, and CD45RA.³  The suppressive activity of T_reg cells is associated with the overexpression of FOXP3, a member of the forkhead/winged helix family, which acts as a transcriptional repressor.⁴  T_reg cells suppress CD25^-CD4⁺ T-cell proliferation on the basis of cell–cell contact and suppress immune responses by secreting immunosuppressive cytokines such as IL-10 and TGF-β.⁵ 

A significant impairment of T-cell function is observed in patients with multiple myeloma (MM) and patients with monoclonal gammopathy of undetermined significance (MGUS). Although phenotypic and functional aberrations in CD4 and CD8 cells have been described in MM and MGUS,^6-9  the biologic basis for these abnormalities remains unclear. Because T_reg cells play an important role in modulating normal immune responses, the abnormal T_reg-cell activity in myeloma patients could contribute to immune dysfunction in MM and could provide a new target to enhance immune responses. Therefore, in this study we evaluated natural T_reg-cell number and function in patients with MGUS and MM and compared them with those of healthy donors.

CD4⁺CD25⁺ T_reg-cell numbers were analyzed by flow cytometric analyses in peripheral-blood mononuclear cells (PBMCs) collected from healthy donors, patients with MGUS, and patients with newly diagnosed MM. Approval for these studies was obtained from the institutional review board of the Dana-Farber Cancer Institute and the Veterans Administration (VA) Boston Healthcare System. Informed consent was provided according to the Declaration of Helsinki.

As FOXP3 is specifically expressed by T_reg cells and is required for their suppressive activity, we analyzed the proportion of PBMCs expressing intracellular FOXP3 using anti-FOXP3 antibody (eBiosciences, San Diego, CA) using dual-color flow cytometry and multiphoton microscopy. Level of protein expression was quantitated by Western blotting and by real-time reverse transcription–polymerase chain reaction (RT-PCR) using previously described methods.¹⁰ 

To evaluate the function of T_reg cells, PBMCs were first depleted of CD25⁺ T cells (which contain T_reg cells) by positive selection using anti-CD25–coated microbeads (Miltenyi Biotech, Auburn, CA), according to the manufacturer's instructions.¹¹  PBMCs depleted of CD25⁺ cells and control PBMCs containing CD25⁺ cells were stimulated with anti-CD3 antibody for 3 days, and proliferation was measured by ³H-thymidine uptake during the last 8 hours of culture. In a separate study, purified CD25⁺ cells were added in various proportions to PBMCs depleted of CD25⁺ cells to assess their effects on anti-CD3–induced T-cell proliferation.

We evaluated the proportions of CD4⁺CD25⁺ cells in the peripheral blood of healthy donors and of patients with MGUS or MM. As seen in Figure 1A, the proportion of these cells in PBMCs was significantly elevated in MGUS (mean, 25% ± 1.8%; range, 20%-29%) and MM (mean, 26% ± 3.6%; range, 6%-51%) compared with healthy donors (mean, 14% ± 2.3%; range, 4%-28%) (P < .01). Because T_reg cells and activated CD4 cells express CD4 and CD25,¹²  we next evaluated the proportions of cells expressing high levels of CD25, characteristic of cells with regulatory function. As seen in Figure 1B-C, we did not observe significant differences in the proportions of CD4⁺CD25^high cells in PBMCs in patients with MGUS or MM compared with healthy donors.

T_reg cells express FOXP3, a transcriptional factor required for regulatory and suppressive function.¹³  Therefore, we next used flow cytometry to define the proportions of cells expressing CD4 and FOXP3 in healthy donors and in patients with MGUS and MM. As seen in Figure 1D-E, although 6.0% ± 0.8% PBMCs from healthy donors expressed FOXP3, significantly reduced numbers of FOXP3⁺ PBMCs were detected in MGUS (1.6% ± 0.5%; P < .01) and MM (0.9% ± 0.4%; P < .01). This reduction in FOXP3-expressing cells observed by flow cytometry was further confirmed by immunohistochemistry using multiphoton microscopy (Figure 1F) and Western blot analysis (data not shown). Proportions of PBMCs expressing CTLA-4, a cell-surface molecule expressed by T_reg cells, were also significantly reduced in patients with MGUS (0.9% ± 0.5%; P < .01) and MM (1% ± 0.6%; P < .01) compared with healthy donors (6.8% ± 0.6%; data not shown). These data, therefore, show significantly reduced numbers of T_reg in patients with MGUS and MM compared with healthy donors.

Next, we evaluated the regulatory function of T_reg cells in patients with MGUS and MM compared with healthy donors. To assess function, we measured the ability of T_reg cells to suppress T-cell proliferation induced by soluble anti-CD3 antibody. PBMCs were activated by anti-CD3 antibody in the presence or absence of T_reg cells (depleted using anti-CD25–coated microbeads), and proliferation was measured by ³H-thymidine uptake. In healthy donor PBMCs, proliferation was significantly suppressed in the presence of CD25⁺ cells (71 770 ± 8010) compared with proliferation in their absence (115 753 ± 10 113; P < .05) (Figure 2A). In contrast, CD25⁺ cells failed to significantly suppress PBMC proliferation in patients with MGUS and MM (29 813 ± 8396 vs 39 437 ± 7463 [P = NS] and 62 223 ± 10 175 vs 51 893 ± 12 361 [P = NS], respectively) (Figure 2A). To account for the reduced frequency of FOXP3⁺ T_reg cells in patients with MGUS and MM, we evaluated cell activity after adding a proportionately higher number of T_reg cells to CD25-depleted PBMCs. As can be seen in Figure 2B, adding even 10-fold more T_reg cells did not suppress soluble anti-CD3–mediated T-cell proliferation in patients with MGUS or MM.

These results highlight important T_reg-cell abnormalities in patients with MM and MGUS because natural T_reg cells are significantly reduced in number and are dysfunctional. The biologic basis for the reduction and dysfunction of T_reg cells in patients with MM and MGUS remains undefined. However, in a murine model of asthma, it has been demonstrated that IL-6 and soluble IL-6 receptor (sIL-6R) together decrease T_reg-cell number and function in the lungs. Interestingly, interactions between MM cells and bone marrow stromal cells trigger the production of IL-6 and a number of cytokines and chemokines, including TNF-α, VEGF, IGF-1, SDF-1α, IL-1β, TGF-β, and MIP-1α/β with immunomodulatory activity.^14,15  In patients with MM and MGUS, serum levels of IL-6 and sIL-6R are highly elevated and may, therefore, play important roles in T_reg-cell development and function.¹⁶  Moreover, mice defective in TGF-β receptor II expression and signaling have low numbers of T_reg cells.¹⁷  TGF-β has also been shown to inhibit IL-2–dependent T-cell proliferation.¹⁸  In patients with myeloma, TGF-β is induced by interactions between MM cells and the bone marrow stromal cells, which may modulate peripheral expansion and maintenance of T_reg cells.

A second unresolved issue is how dysfunctional T_reg cells affect immune function in MGUS and MM. The significantly elevated numbers of CD4⁺CD25⁺ cells, which are predominantly FOXP3^-, suggest an increase in activated T cells in patients with MGUS and MM. Low T_reg-cell numbers or function generate nonspecific immune responses or autoimmunity.^5,19  If T_reg cells are so low that immune responses are not suppressed, particularly at the terminal stages of an immune response, an increase in hyperreactive T cells may be seen, as in myeloma patients.⁷  This hyperactivity of T cells has been associated with defective TCR signaling²⁰  and increased sensitivity to costimulatory signals.⁷  In contrast, recent studies in patients with breast and gastroesophageal cancers, metastatic melanoma, and Hodgkin lymphoma/chronic lymphocytic leukemia (CLL) have reported increased numbers of T_reg cells that may suppress immune responses, leading to ineffective antitumor immune responses.^21-26 

These results identify potentially important mediators of immune dysfunction in myeloma. The presence of T_reg-cell dysfunction in MGUS suggests a role for T_reg cells even at an earlier stage in the development of disease. Evaluation of T_reg cells at various stages in MGUS may provide further insight into the development and progression of MGUS to MM. Additionally, MM is reported to be associated with a number of autoimmune disorders, such as thyroid abnormalities,²⁷  rheumatoid arthritis,²⁸  and renal complications.²⁹  It will be intriguing to study whether these conditions are the cause or the effect of dysfunctional T_reg cells in MM. If hyperactive T cells are the source of immune dysfunction in patients with myeloma, improving T_reg-cell number and function may provide a solid foundation for enhancing immune function and vaccination strategies in the future.

We thank Virginia M. Cumming and Suzan B. Lazo-Kallanian for their excellent technical assistance.