James H. Finke, PhD (Cleveland Clinic) presented on the biology and role of regulatory T cells and myeloid-derived suppressor cells in tumor immunology. He noted that there are two main types of Treg cells. The natural Tregs represent about 2% to 5% of cells in the peripheral blood; they differentiate in the thymus and express TCRs and CD4, as well as the α-chain for IL-2 receptor (CD25), which helps drive their proliferation. Natural Tregs also express the transcription factor FoxP3, which is critical for their function. Natural Tregs help maintain immune tolerance and inhibit autoreactive T cells; they also suppress anti-tumor immunity as shown by models that correlate Treg depletion in vivo with reduced tumor growth.
Inducible Tregs are the second type of CD4+ regulatory T cells, which differentiate in the periphery, not the thymus. Inducible Tregs are influenced by cytokines and antigen to differentiate into either FoxP3+ Tregs, Th1, Th2 or Th17 cells. Induction of this class of regulatory cells is thought to occur via TCR stimulation, IL-2 and TGFβ; MDSC and tumor cells also affect the induction of these regulatory cells.
In patients, increased numbers of tumor-infiltrating Tregs have been associated with poor prognosis for ovarian, hepatocellular, cervical, and head and neck squamous cell carcinomas. Tregs from cancer patients can suppress the in vitro proliferation of autologous T effector cells, a suppressive effect that can be relieved in vitro by diluting the Tregs.
Several mechanisms of Treg suppression have been described, including Treg production of immunosuppressive cytokines (e.g., TGFβ, IL-10), the β-galactoside binding protein galectin 1, and granzyme B. Tregs may also indirectly suppress immune functions by inhibiting DCs.
Other classes of regulatory T cells include Tr1 and Tr3 cells, which are induced by antigen. Tr1 cells secrete IL-10, whereas Tr3 cells secrete TGFβ. No specific markers for these cells have been identified, and they do not constitutively express FoxP3. Tr1 cells have been detected in some human tumors (gastric cancer and RCC). There are also CD8+ Tregs, some of which express FoxP3 and secrete high levels of IL-10. Additionally, NKT regulatory cells have been described.
A number of strategies have emerged to inhibit or eliminate Tregs. They include targeting the CD25 receptor, and administration of cyclophosphamide and CpG. Cyclophosphamide has been shown to deplete Tregs and boost the efficacy of vaccines in mouse models and CpG, which targets the toll-like receptor 9, reduces FoxP3+ cells in the lymph nodes of melanoma patients. Other strategies have focused on blocking Treg function, differentiation and trafficking. The receptor tyrosine kinase inhibitor sunitinib has been shown to reduce Tregs in the peripheral blood in patients with RCC and synergistically reduced Tregs in combination with a cancer vaccine in a melanoma mouse model.
Myeloid-derived suppressor cells represent another distinct class of regulatory cells. Normally present in small amounts (1% to 2% of peripheral blood cells), they accumulate under pathological conditions (~5% to as high as 25% in patients with kidney cancer). Factors produced by the tumor such as vascular endothelial growth factor (VEGF), stem cell factor (SCF), GM-CSF, G-CSF, S100A9, and M-CSF can promote the expansion of these myeloid cells and block their differentiation into DCs. Depletion of MDSCs in murine tumor models can inhibit tumor formation and metastasis and promote immune-mediated destruction of the tumor. Moreover, adoptive transfer of MDSC in murine tumor models promotes tumor growth and inhibits T cell activation.
The differentiation path of myeloid cells is dependent on the tissue environment and the growth factor milieu. In the normal environment, immature myeloid cells migrate to the peripheral organs and differentiate into DCs, macrophages and granulocytes; in the tumor microenvironment, however, the immature myeloid cells accumulate and induce T cell suppression.
MDSC expansion is mediated by a number of factors. VEGF, which is elevated in cancer and promotes tumor vascularization, induces defective differentiation of myeloid cells into DCs. SCF, IL-6 and M-CSF promote expansion of MDSCs, likely through activation of STAT3. Prostaglandin also appears to play a role in the induction of these cells. In cancer patients, GM-CSF, which is important for expansion of normal bone marrow, enhances the number of MDSCs. Indeed, GM-CSF-based vaccines may promote MDSC accumulation. Moreover, GM-CSF may promote resistance to sunitinib.
MDSCs may be activated by products from activated T cells, tumor cells or stromal cells. IFNγ, IL-4, IL-13 and products that engage toll-like receptors may all contribute to this process by activating STAT1, STAT6 or NFκB, which upregulate MDSC production of suppressive enzymes and products, including arginase, iNOS and TGFβ. Arginase reduces arginine, an amino acid required for T cell function and signaling through the TCR. Thus by depleting arginine, this enzyme arrests the cell cycle and inhibits proliferation. Both arginase and the enzyme iNOS are involved in the production of reactive oxygen species and NO, which can bind to the TCR and block its function as well as inducing apoptosis of T cells. Other suppressive mechanisms of MDSC that may play a role in tumor progression include induction of Tregs, differentiation into tumor-associated macrophages (TAMs), enhancement of a Th2 response, and downregulation of CD62L (L-selectin), a ligand involved in homing to lymph and tumor tissue.
Several approaches have been explored to target MDSCs to improve immunotherapy. These have included products that bind VEGF (i.e., VEGF-trap a fusion protein and bevacizumab), block VEGF receptor signaling (i.e., AZD2171), reduce ROS (i.e., triterpenoids) and inhibit arginase and NOS-2 expression (i.e., phosphodiesterase-5; sildanefil). Both triterpenoids and phosphodiesterase-5 have been shown to reduce MDSC function and improve T cell responses in cancer immunotherapy. Additional strategies have included all-trans retinoic acid and vitamin D3, which promote MDSC differentiation into DCs and improve T cell responses in RCC and head and neck cancer, respectively. Gemcitabine in combination with cyclophosphamide has been shown to reduce the numbers of MDSC in breast cancer. The tyrosine kinase inhibitor sunitinib, a frontline therapy for RCC, reduces MDSC levels and improves T cell function, as indicated by an increase in IFNγ production.
As previously mentioned, some MDSCs may differentiate into tumor-associated macrophages in the tumor microenvironment. There are two distinct subsets of these cells: M1 and M2 tumor-associated macrophages. M1 cells, when stimulated by LPS or IFNγ/TNF induce production of IL-1, TNF, IL-6, IL-23, IL-12, and IL-10, which can be involved in a DTH response, type 1 inflammation, Th1 responses, promoting anti-tumor activity. M2 cells, on the other hand, when stimulated with IL-4, IL-10 and IL-13 or other stimuli, produce arginase and TGFβ and other suppressive products (e.g., Il-10). M2 cells are implicated in tumor promotion, Th2 responses, allergy, and angiogenesis.
As research advances in the field, it will be useful to identify new targets for reducing Treg numbers and/or their suppressive function. It will be important to better understand the role of other immune suppressive T cell populations (Tr1/Tr3,CD8) in tumor-induced immune suppression and identify targets for blocking and/or deleting these subpopulations. Moreover, it will be beneficial to identify which of the various strategies shown to reduce MDSC in the peripheral blood of patients are also effective within the tumor microenvironment and to define which strategies promote strong anti-tumor immunity. Lastly, clinical studies are warranted to test whether effective blocking of Tregs and MDSC will provide greater efficacy for different forms of cancer immunotherapy (vaccines and adoptive therapy).