T cell receptor transgenic lymphocytes infiltrating murine tumors are not induced to express foxp 3

Regulatory T cells (Treg) that express the transcription factor Foxp3 are enriched within a broad range of murine and human solid tumors. The ontogeny of these Foxp3 Tregs selective accumulation or proliferation of natural thymus-derived Treg (nTreg) or induced Treg (iTreg) converted in the periphery from naïve T cells is not known. We used several strains of mice in which Foxp3 and EGFP are coordinately expressed to address this issue. We confirmed that Foxp3-positive CD4 T cells are enriched among tumor-infiltrating lymphocytes (TIL) and splenocytes (SPL) in B16 murine melanoma-bearing C57BL/6 Foxp3 mice. OT-II Foxp3 mice are essentially devoid of nTreg, having transgenic CD4 T cells that recognize a class II-restricted epitope derived from ovalbumin; Foxp3 expression could not be detected in TIL or SPL in these mice when implanted with ovalbumin-transfected B16 tumor (B16-OVA). Likewise, TIL isolated from B16 tumors implanted in Pmel-1 Foxp3 mice, whose CD8 T cells recognize a class I-restricted gp100 epitope, were not induced to express Foxp3. All of these T cell populations wild-type CD4, pmel CD8 and OTII CD4 could be induced in vitro to express Foxp3 by engagement of their T cell receptor (TCR) and exposure to transforming growth factor b (TGFb). B16 melanoma produces TGFb and both pmel CD8 and OTII CD4 express TCR that should be engaged within B16 and B16-OVA respectively. Thus, CD8 and CD4 transgenic T cells in these animal models failed to undergo peripheral induction of Foxp3 in a tumor microenvironment. Background 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 (TGFb) [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 * Correspondence: jeconomou@mednet.ucla. edu Departments of Surgery, University of California, Los Angeles, 10833 Le Conte Avenue Room 54-140 CHS Los Angeles, CA 90095 USA Full list of author information is available at the end of the article Quatromoni et al. Journal of Hematology & Oncology 2011, 4:48 http://www.jhoonline. org/content/4/1/48 JOURNAL OF HEMATOLOGY & ONCOLOGY © 2011 Quatromoni et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. IFNg 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 tumordriven 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 TGFb 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). 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 TGFb signaling. We reasoned that both of these TCR transgenic cell populations, entering B16 or ovalbumintransfected B16 (B16-OVA) subcutaneous tumors respectively, would be exposed to a comparable set of Foxp3 induction signals. CD4/Foxp3 cells are enriched in B16 tumors and spleen when tumors are propagated in wild-type C57BL/6 Foxp3 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. Materials and methods