T cell receptor transgenic lymphocytes infiltrating murine tumors are not induced to express foxp3

Treg play an essential role in maintaining immunological self-tolerance [1]. Approximately 10% of CD4 T cells express the transcription factor FoxP3 (forhead box P3 transcription factor); humans and mice with inactivating Foxp3 mutations have autoimmune diseases [2‐4]. Treg dominantly suppress immune responses through direct contact with dendritic cells, effector T cells and possibly through secretion of immunosuppressive cytokines [5, 6]. Fewer than 1% of CD8 T cells express Foxp3, and the biology of this very small population of naturally occurring, thymus-derived T cell have not been well studied. However, this transcription factor can be induced in both CD4 and CD8 T cells through engagement of their T cell receptors (TCR) and exposure to transforming growth factor beta (TGFβ) [7‐10]. These so called "induced" Treg (iTreg), both CD4 and CD8, can acquire dominant suppressor phenotype in a variety of experimental models [11‐13].

Many studies have shown that the number of Treg are significantly increased in the peripheral blood, bone marrow, tumor draining lymph nodes, and TIL of mice and humans bearing many types of hematologic and solid malignancies including breast [14], colorectal [15], esophageal [16], gastric [17], hepatocellular [18], lung [19], melanoma [20], ovarian [21], and pancreatic cancers [14]. It has been hypothesized that these Treg may be involved with promoting tumor progression, as they are even more enriched in advanced tumors [22]. The number of Foxp3 Treg within human tumors has also been correlated with a poorer prognosis. Patients with ovarian or gastric cancer and lower numbers of Treg TILs had improved disease-specific survival [23]; those with head and neck cancer also experienced better locoregional control [24]. Treg isolated from human ovarian cancers were able to inhibit Her-2 specific CD8+ effector responses, as measured by proliferation, cytotoxicity, and IL2 and IFNγ production [25]. These and other observations support the view that Foxp3 Treg may dominantly suppress antitumor immune responses. The ontogeny of the enriched Treg population found within tumors, generally CD4, is not fully defined. A selective tumor-driven accumulation or proliferation of thymus-derived natural (n)Treg is a possibility. Alternatively, naïve Foxp3 T cells could be induced to express this regulatory transcription factor through tumor-derived signals yielding induced (i)Treg. These signals would include engagement of TCR and exposure to TGFβ elaborated by tumors or tumor-associated stroma. We sought to address this question by generating CD8 (Pmel-1) and CD4 (OTII) TCR transgenic mice in which Foxp3 expression could be detected by EGFP expression (Foxp3^EGFP). These naïve Pmel-1 CD8 and OTII CD4 populations have very low to absent Foxp3 expression but could be induced in T cells in vitro with a combination of T cell receptor (TCR) engagement and TGFβ signaling. We reasoned that both of these TCR transgenic cell populations, entering B16 or ovalbumin-transfected B16 (B16-OVA) subcutaneous tumors respectively, would be exposed to a comparable set of Foxp3 induction signals. CD4/Foxp3^EGFP cells are enriched in B16 tumors and spleen when tumors are propagated in wild-type C57BL/6 Foxp3^EGFP mice. However, in neither TCR transgenic mouse did we find evidence of Foxp3 induction among tumor-infiltrating lymphocytes (TIL), splenocytes (SPL) nor lymph nodes (LN). These findings argue indirectly in favor of a preferential accumulation of nTreg in experimental tumors.

We hypothesized that naïve T cells entering the tumor microenvironment would be exposed to the signals and factors needed for induction of Foxp3 and the acquisition of a regulatory phenotype. Some of these signals have been defined in vitro and include TCR engagement and exposure to TGFβ. We used TCR transgenic mice - one devoid of CD4 nTreg and the other with < 1% Foxp3 CD8s - whose tumor-infiltrating T cells and perhaps those in lymphoid organs of tumor-bearing mice would be engaged by cognate antigen, as well as being exposed to tumor-produced TGFβ. We demonstrated that naïve Foxp3-negative CD4 and CD8 populations, both transgenic and nontransgenic, could be induced to express Foxp3 in vitro by αCD3/αCD28 and TGFβ treatment (see Table 1). These in vitro findings confirm that Foxp3 expression can be induced in both T cells subtypes, and to comparable degrees, using similar sets of signals. We confirmed a significant enrichment of Foxp3-positive CD4 TIL and splenocytes in wild-type, B16 tumor-bearing C57BL/6 Foxp3^EGFP mice. However, neither CD8s in these mice, nor TCR transgenic T cells in either Foxp3^EGFP cross, seemed to have encountered the necessary intratumoral signals to induce Foxp3 expression. Administration of systemic IL-2, which we have shown in vitro to act in concert, but not alone, with TGFβ to induce Foxp3, did not support induction of iTreg in these transgenic mice. These findings indirectly support the view that natural, thymus-derived Treg preferentially accumulate, or proliferate, in the tumor microenvironment.

Two subsets of Tregs are recognized - adaptive or induced (iTreg) and natural (nTreg) - which together are responsible for maintaining tolerance to self-antigen and preventing autoimmune diseases or inappropriate immune responses involved in allergic diseases through the suppression of auto-reactive T cells [30, 31]. Both require TGF-β and IL-2 for maintenance; express similar phenotypic markers such as CTLA4, GITR, CD25, and CCR4; and require the expression of Foxp3 to carry out a contact-dependent mechanism of action [32]. Where these two developmentally distinct populations differ is in their antigen specificities, strength of TCR stimulation and co-stimulatory signals required for their generation, and their stability of suppressive action. nTreg are generated in the thymus in a CD28-dependent manner, constitutively express CD25, express TCRs specific for self-antigen, demonstrate a more stable expression of Foxp3, and exert suppressive function [30‐32]. Conversely, iTregs are generated in the peripheral lymphoid organs through the de novo conversion of CD4⁺CD25^-Foxp3^- T cells in a TGF-β- and IL-2-dependent-manner, have TCRs specific for foreign antigens presented by professional antigen-presenting cells, require weaker TCR stimulation (CD28 stimulation not required), and demonstrate a less stable expression of Foxp3 [30‐34].

Possible mechanisms for Treg enrichment within tumors include: preferential accumulation of nTreg, proliferation of nTreg within tumors, or peripheral conversion of naïve T cells to iTreg. Each mechanism is dependent upon tumor-derived signals (chemokines, cytokines, TCR engagement). One hypothesis is that resident nTreg proliferate in the tumor microenvironment [35]; another is that nTregs may weakly perceive tumor related signals and be selectively recruited where they may exert their suppressive function [25]. Treg can migrate more efficiently into major non-lymphoid tissue sites, such as tumors, due to their up-regulation of non-lymphoid tissue-specific homing receptors [36]. In human ovarian carcinoma, tumor cells and tumor-associated macrophages were shown to produce a Treg-specific chemokine CCL22, which resulted in the specific recruitment of Treg to the tumor microenvironment via their CCR4 receptor [25]. Interestingly, the CCR4 receptor is expressed at higher levels in Treg than in effector T cells in leukemia studies, suggesting its up-regulation may be Treg-specific [37]. In this same ovarian cancer model, IL-2 treatment was shown to up-regulate CCR4 as well as CXCR4, which further enhanced the ability of Treg to migrate to the tumor microenvironment based on its elevated levels of the Treg ligands CCL22 and CXCL12 [38, 39]. Similar results were seen in a gastric cancer model, in which elevated levels of CCL17 and CCL26 in the tumor microenvironment demonstrated a positive correlation with the frequency of Foxp3 Treg, which can bind both these ligands with its CCR4 receptor [40]. Furthermore, Treg had a higher affinity for CCL17 and CCL22 than effector T cells in vitro as determined by a migration assay.

Expression of the regulatory cytokine TGFβ is abundant in many tumors, particularly in advanced stages. TGFβ can promote proliferation of Treg in vivo; the addition of IL-2 to TGFβ can induce Treg both in vivo and in vitro [41]. A number of additional chemokines and cytokines are implicated in Treg proliferation or induction, including IFNγ, IL-6, IL-23, IL-21, Cox2, and indolamine 2,3 dioxygenase [22, 23, 42‐45]. Convincing in vivo evidence that iTreg develop from conventional CD4⁺CD25^- T cells was shown a non-obese diabetic (NOD) model, which normally exhibits extensive autoimmune manifestations around 12-16 weeks of age. When CD28^-/- NOD mice were treated with αCD3, which has been shown to increase CD4⁺CD25⁺ T cells and result in long-term remission of disease in regular NOD, CD4⁺CD25⁺ T cells were generated de novo and shown to be suppressive in vitro [46]. Neutralizing anti-TGF-β given to these CD28^-/- NOD mice alongside αCD3 prevented the aforementioned disease remission, demonstrating the role of TGF-β in the generation of iTreg and further supporting previous in vitro results.

Linehan [47, 48] demonstrated that naïve CD4 T cells were converted into Foxp3-positive Treg when administered to Rag1-/- mice bearing TGFβ-producing (Pan 02) tumors. This conversion could be inhibited by TGFβ neutralizing antibody, was abrogated if naïve T cells were obtained from mice whose T cells were insensitive to TGFβ signaling, and did not occur in mice bearing tumors that did not produce TGFβ (ESO 2). The high levels of TGFβ expression by this murine pancreatic tumor cell line, and the longer interval (seven weeks) of in vivo co-residence of naïve CD4 T cells and tumor, may explain their clearcut but different findings.

In summary, we generated two TCR transgenic mouse strains in which the de novo generation of iTreg could be unequivocally demonstrated in vitro, and in which putative signals for Foxp3 induction were present in the tumor microenvironment. Implantation of the B16 melanoma in wild-type mice resulted in the enrichment of CD4 Foxp3 Treg in TIL and spleen; this tumor did not induce an enrichment of either CD4 or CD8 Foxp3 in these two transgenic mouse strains.

JQ is a medical student. LM and TD are surgery residents. YW is a postdoctoral fellow. WM, TC and JE are faculty.