Modification of Treg Biology as Cancer Immunotherapy 61 The Cellular Microenvironment

Tregs remain one of the major obstacles to successful cancer immunotherapy. Other leukocytes, including myeloid derived suppressor cells (MDSC), tumor associated macrophages (TAMs), type I/II NKT cells, mast cells, B cells, and subsets of DC have also been implicated in promoting tumor progression. In this section, we will first discuss how Tregs and other immunomodulatory cells are associated in tumormediated suppression before discussing clinical strategies to attenuate Treg function to improve current immunotherapeutic strategies.

During tumor development and progression, proinflammatory and immunosuppressive factors can be secreted from tumors or host cells into the tumor microenvironment that lead to immune evasion and promotion of tumor growth (de Visser and Coussens 2006; Mantovani et al. 2008). The major leukocyte population implicated in aiding tumor progression includes Tregs, MDSC (reviewed in Ostrand-Rosenberg and Sinha 2009), and TAMs (reviewed in Solinas et al. 2009). MDSC are a heterogeneous population of cells generally defined as Gr-1+CD11b+, and induced by proinflammatory cytokines such as IL-1p (Bunt et al. 2006; Song et al. 2005), IL-6 (Bunt et al. 2007), and the bioactive lipid PGE2 (Sinha et al. 2007). Depending on the subpopulation of MDSC, Tregs can be induced through MDSC production of IL-10 and TGFp (Huang et al. 2006), or arginase alone (Serafini et al. 2008).

TAMs originate from blood monocytes recruited to the tumor site (Mantovani et al. 1992) as a result of CCL2, M-CSF, and VEGF which are produced by neoplastic and stromal cells. Monocytes differentiate into TAMs upon exposure to CSFs, IL-4, IL-13, IL-10, and TGF-p. In turn, TAMs promote tumor survival by modifying neoplastic extracellular matrix (ECM) proteins, stimulating angiogene-sis and inducing immunosuppression via the production of IL-10 and the secretion of chemokines (e.g., CCL17 and CCL22), which preferentially attract T cell subsets such as Tregs and Th2 (Balkwill 2004; Mantovani et al. 2004).

The relative hierarchy and importance of Tregs, MDSC, and TAMs in immune suppression and their temporal cross-regulation during the course of tumor progression still remain to be elucidated. It is likely that different cancer types or the location of the cancer dictates which immunomodulatory cells are preferentially recruited and/or induced to mediate immune suppression. In a study to evaluate the interplay between tumor and the different immunomodulatory cells during disease progression, Clark et el., generated a transgenic mouse model where expression of oncogenic KrasG12D was induced under the pancreas-specific promoters Pdx-1 or p48 (Clark et al. 2007). These mice spontaneously developed pancreatic ductal adenocarcinoma (PDA), which is markedly infiltrated by Tregs before the development of invasive disease (PanIN). Subsequently, macrophages (Gr-1 ~CD11b+) and then MDSC (Gr-1+CD11b+) infiltrate the tumor. Similar findings have been shown in the A20 B cell lymphoma tumor model, in which increased percentages of intratumoral and systemic Tregs are found, along with high intratumoral and systemic IL-10, and moderate levels of intratumoral TGF-p (Elpek et al. 2007). Additional studies reveal that A20 cells also express PD-L1 and secrete IL-10 and immunomodulatory IDO all of which contribute to the generation and function of iTregs (Baban et al. 2009). Importantly, this study also showed by depletion experiments that Tregs played a dominant role in early tumor progression in vivo. In contrast, a recent paper by Denardo et al., utilizing the MMTV-PyMT model of mammary carcinogenesis, demonstrated a tumor-promoting role for Th2-CD4+IL-4 producing T cells, but not for Tregs, in sculpting the function of TAM to promote pulmonary metastasis of mammary adenocarcinomas (DeNardo et al. 2009). While some studies suggest that Tregs may often serve as the dominant immune escape mechanism early in tumor progression (Elpek et al. 2007), it should be noted that there are no mAbs that can specifically deplete all Foxp3+ Tregs. Studies in which all Foxp3+ Tregs can be depleted using Foxp3DTR mice (Kim et al. 2007; Lahl et al. 2007) will prove very useful for dissecting out the importance of Treg-mediated suppression in established tumors.

Generally, tumors progress when heavily infiltrated by inflammatory innate immune cells (i.e., macrophages, neutrophils, and mast cells) and they are rich in inflammatory cytokines, growth factors, and pro-angiogenic molecules (Badoual et al. 2009). It has been proposed that the presence of large numbers of local Tregs early on in inflammatory sites can delay or prevent inflammation-induced cancers (Haas et al. 2009). This is also supported by studies of ApcMm/+ (multiple intestinal neoplasia (Min)) mice where the adoptive transfer of CD4+CD25+ lymphocytes induced tumor apoptosis, the regression of established adenomas, and down-regulation of COX-2 and proinflammatory cytokines within intestinal polyps (Erdman et al. 2005). The influx of mast cells (mastocytosis) was a necessary event for polyp outgrowth in ApcD468 mice. A recent report by Gounaris et al. supported these relationships (Colombo and Piconese 2009; Gounaris et al. 2009). Interestingly, endogenous Foxp3+ Tregs were found in elevated numbers in these polyp bearing ApcD468 mice but intriguingly they had lost the ability to produce IL-10 with a proportion actually found to express the pro-inflammatory cytokine IL-17 preferentially produced by effector Th17 cells.

There are conflicting data on the role of IL-17 in carcinogenesis (Kryczek et al. 2009; Wang et al. 2009). Given that differentiation of Th17 cells requires TGF-p (plus IL-6 or IL-21), these cells may be developmentally linked to iTregs that also require TGF-p for differentiation. Tregs may differentiate into Th17 cells (reviewed in Lee et al. 2009; Zhou et al. 2009b), and CD4+Foxp3+IL-17+ cells have recently been described in mice and humans (Voo et al. 2009; Xu et al. 2007). Furthermore, IL-17+/Foxp3+ Treg clones were recently shown to retain suppressive function and exhibited the plasticity to secrete IL-17 or suppress depending on the nature of the stimulus provided (Beriou et al. 2009). These findings suggest that exposure to pro-inflammatory cytokines can drive Tregs to secrete IL-17, and thereby promote an inflammatory microenvironment favoring tumor growth.

In addition to interacting with MDSC and TAMs, Tregs also interact and cross-regulate type-I-type II NKT cells. Type-I NKT cells are generally thought to be active in tumor immunosurveillance and enhance anti-tumor immunity, while type IINKT cells are thought to suppress these responses. Interestingly, in mouse tumor models where type II NKT cells have a major role in the suppression of tumor immunosurveillance, Tregs had a minimal role or no role in suppression. Intrigu-ingly, the site of the tumor growth also appeared to determine whether type II NKT cells or Tregs were the major mediators of immune suppression (reviewed in Terabe and Berzofsky 2007). Further work to investigate the relationship between Tregs and NKT cells in a cancer setting will provide insight on the importance of these interactions.

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