Human squamous cell carcinomas evade the immune response by down-regulation of vascular E-selectin and recruitment of regulatory T cells.
ABSTRACT: Squamous cell carcinomas (SCCs) of the skin are sun-induced skin cancers that are particularly numerous in patients on T cell immunosuppression. We found that blood vessels in SCCs did not express E-selectin, and tumors contained few cutaneous lymphocyte antigen (CLA)(+) T cells, the cell type thought to provide cutaneous immunosurveillance. Tumors treated with the Toll-like receptor (TLR)7 agonist imiquimod before excision showed induction of E-selectin on tumor vessels, recruitment of CLA(+) CD8(+) T cells, and histological evidence of tumor regression. SCCs treated in vitro with imiquimod also expressed vascular E-selectin. Approximately 50% of the T cells infiltrating untreated SCCs were FOXP3(+) regulatory T (T reg) cells. Imiquimod-treated tumors contained a decreased percentage of T reg cells, and these cells produced less FOXP3, interleukin (IL)-10, and transforming growth factor (TGF)-beta. Treatment of T reg cells in vitro with imiquimod inhibited their suppressive activity and reduced FOXP3, CD39, CD73, IL-10, and TGF-beta by indirect mechanisms. In vivo and in vitro treatment with imiquimod also induced IL-6 production by effector T cells. In summary, we find that SCCs evade the immune response at least in part by down-regulating vascular E-selectin and recruiting T reg cells. TLR7 agonists neutralized both of these strategies, supporting their use in SCCs and other tumors with similar immune defects.
Project description:Regulatory T cells (Tregs) are crucial for the induction and maintenance of self-tolerance and are present in peripheral tissues such as skin and gut under normal, noninflamed conditions. We report isolation and expansion of the Treg population resident in normal human skin. Cutaneous Tregs expressed high levels of CD25, L-selectin, GITR, FOXP3, and intracellular CTLA-4, low levels of CD69, and high levels of the skin-homing addressins CLA, CCR4, and CCR6. Skin Tregs suppressed the proliferation of CD25(lo) T cells from the same skin sample in response to CD3 and CD28 antibodies. Suppression was dependent on cell contact and not affected by neutralizing antibodies to interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta). Surprisingly, cutaneous Tregs proliferated in an antigen-independent manner when cultured in contact with dermal fibroblasts and IL-15, conditions similar to those found in chronically inflamed skin. We hypothesize that local proliferation of Tregs may occur within inflamed skin and could serve as a brake for cutaneous inflammation as well as a mechanism for the homeostatic proliferation of natural Tregs that has been observed within intact organisms.
Project description:Regulatory T (T(reg)) cells are often found in human tumors; however, their functional characteristics have been difficult to evaluate due to low cell numbers and the inability to adequately distinguish between activated and T(reg) cell populations. Using a novel approach, we examined the intracellular cytokine production capacity of tumor-infiltrating T cells in the single-cell suspensions of enzymatically digested tumors to differentiate T(reg) cells from effector T cells. Similar to T(reg) cells in the peripheral blood of healthy individuals, tumor-infiltrating FOXP3(+)CD4 T cells, unlike FOXP3(-) T cells, were unable to produce IL-2 and IFN-gamma upon ex vivo stimulation, indicating that FOXP3 expression is a valid biological marker for human T(reg) cells even in the tumor microenvironment. Accordingly, we enumerated FOXP3(+)CD4 T(reg) cells in intratumoral and peritumoral sections of metastatic melanoma tumors and found a significant increase in proportion of FOXP3(+)CD4 T(reg) cells in the intratumoral compared with peritumoral areas. Moreover, their frequencies were 3- to 5-fold higher in tumors than in peripheral blood from the same patients or healthy donors, respectively. These findings demonstrate that the tumor-infiltrating CD4 T(reg) cell population is accurately depicted by FOXP3 expression, they selectively accumulate in tumors, and their frequency in peripheral blood does not properly reflect tumor microenvironment.
Project description:Cutaneous immune responses must be tightly controlled to prevent unwanted inflammation in response to innocuous antigens, while maintaining the ability to combat skin-tropic pathogens. Foxp3(+) regulatory T (T reg) cells are potent immune regulators and are found at high frequency in both human and mouse skin. Although T reg cells migrate to the skin and can dampen immune responses during experimentally induced inflammation or infection, the importance of cutaneous T reg cells for maintaining normal immune homeostasis in the skin has not been addressed. To selectively block T reg cell function in the skin, we restored the T reg cell compartment in Foxp3-deficient scurfy mice with cells whose ability to migrate to the skin was impaired because of targeted mutation of alpha-1,3-fucosyltransferase VII (Fut7). Although Fut7-deficient T reg cells were present at normal frequency and could function in all other tissues examined, these animals rapidly developed severe cutaneous inflammation. Thus, skin-resident T reg cell are essential for maintaining normal immune homeostasis at this site.
Project description:Regulatory T cells (T(reg) cells) that express the transcription factor Foxp3 suppress the activity of other cells. Here we show that interleukin 10 (IL-10) produced by CD11b(+) myeloid cells in recombination-activating gene 1-deficient (Rag1(-/-)) recipient mice was needed to prevent the colitis induced by transferred CD4(+)CD45RB(hi) T cells. In Il10(-/-)Rag1(-/-) mice, T(reg) cells failed to maintain Foxp3 expression and regulatory activity. The loss of Foxp3 expression occurred only in recipients with colitis, which indicates that the requirement for IL-10 is manifested in the presence of inflammation. IL-10 receptor-deficient (Il10rb(-/-)) T(reg) cells also failed to maintain Foxp3 expression, which suggested that host IL-10 acted directly on the T(reg) cells. Our data indicate that IL-10 released from myeloid cells acts in a paracrine manner on T(reg) cells to maintain Foxp3 expression.
Project description:Homeostasis in the immune system is maintained by specialized regulatory CD4(+) T cells (T(reg)) expressing transcription factor Foxp3. According to the current paradigm, high-affinity interactions between TCRs and class II MHC-peptide complexes in thymus "instruct" developing thymocytes to up-regulate Foxp3 and become T(reg) cells. However, the loss or down-regulation of Foxp3 does not disrupt the development of T(reg) cells but abrogates their suppressor function. In this study, we show that Foxp3-deficient T(reg) cells in scurfy mice harboring a null mutation of the Foxp3 gene retained cellular features of T(reg) cells including in vitro anergy, impaired production of inflammatory cytokines, and dependence on exogenous IL-2 for proliferation and homeostatic expansion. Foxp3-deficient T(reg) cells expressed a low level of activation markers, did not expand relative to other CD4(+) T cells, and produced IL-4 and immunomodulatory cytokines IL-10 and TGF-beta when stimulated. Global gene expression profiling revealed significant similarities between T(reg) cells expressing and lacking Foxp3. These results argue that Foxp3 deficiency alone does not convert T(reg) cells into conventional effector CD4(+) T cells but rather these cells constitute a distinct cell subset with unique features.
Project description:In contrast to naive lymphocytes, memory/effector lymphocytes can access nonlymphoid effector sites and display restricted, often tissue-selective, migration behavior. The cutaneous lymphocyte-associated antigen (CLA) defines a subset of circulating memory T cells that selectively localize in cutaneous sites mediated in part by the interaction of CLA with its vascular ligand E-selectin. Here, we report the identification and characterization of a CC chemokine, cutaneous T cell-attracting chemokine (CTACK). Both human and mouse CTACK are detected only in skin by Southern and Northern blot analyses. Specifically, CTACK message is found in the mouse epidermis and in human keratinocytes, and anti-CTACK mAbs predominantly stain the epithelium. Finally, CTACK selectively attracts CLA(+) memory T cells. Taken together, these results suggest an important role for CTACK in recruitment of CLA(+) T cells to cutaneous sites. CTACK is predominantly expressed in the skin and selectively attracts a tissue-specific subpopulation of memory lymphocytes.
Project description:Dysregulated CD4(+) T cell responses and alterations in T regulatory cells (T(reg) cells) play a critical role in autoimmune diseases, including inflammatory bowel disease (IBD). The current study demonstrates that removal of Bcl11b at the double-positive stage of T cell development or only in T(reg) cells causes IBD because of proinflammatory cytokine-producing CD4(+) T cells infiltrating the colon. Provision of WT T(reg) cells prevented IBD, demonstrating that alterations in T(reg) cells are responsible for the disease. Furthermore, Bcl11b-deficient T(reg) cells had reduced suppressor activity with altered gene expression profiles, including reduced expression of the genes encoding Foxp3 and IL-10, and up-regulation of genes encoding proinflammatory cytokines. Additionally, the absence of Bcl11b altered the induction of Foxp3 expression and reduced the generation of induced T(reg) cells (iT(reg) cells) after Tgf-β treatment of conventional CD4(+) T cells. Bcl11b bound to Foxp3 and IL-10 promoters, as well as to critical conserved noncoding sequences within the Foxp3 and IL-10 loci, and mutating the Bcl11b binding site in the Foxp3 promoter reduced expression of a luciferase reporter gene. These experiments demonstrate that Bcl11b is indispensable for T(reg) suppressor function and for maintenance of optimal Foxp3 and IL-10 gene expression, as well as for the induction of Foxp3 expression in conventional CD4(+) T cells in response to Tgf-β and generation of iT(reg) cells.
Project description:Regulatory T-cells (T(Reg) cells) are increased in patients with multiple myeloma (MM). We investigated whether MM cells could generate and/or expand T(Reg) cells as a method of immuno-surveillance avoidance. In an in vitro model, CD4(+)CD25(-)FoxP3(-) T-cells co-cultured with malignant plasma cells (primary MM cells and cell lines) induced a significant generation of CD4(+)CD25(+)FoxP3(+) inducible T(Reg) cells (tT(Reg) cells; p<0.0001), in a contact-dependent manner. tT(Reg) cells were polyclonal, demonstrated a suppressive phenotype and phenotypically, demonstrated increased FoxP3 (p = 0.0001), increased GITR (p<0.0001), increased PD1 (p = 0.003) and decreased CD62L (p = 0.007) expression compared with naturally occurring T(Reg) cells. FACS-sorted tT(Reg) cells differentiated into FoxP(+)IL-17(+) and FoxP3(-)IL-17(+) CD4(+) cells upon TCR-mediated stimulation. Blocking experiments with anti-ICOS-L MoAb resulted in a significant inhibition of tT(Reg) cell generation whereas both IL-10 & TGF? blockade did not. MM tumour cells can directly generate functional T(Reg) cells in a contact-dependent manner, mediated by ICOS/ICOS-L. These features suggest that tumour generation of T(Reg) cells may contribute to evasion of immune surveillance by the host.
Project description:Transcription factor Foxp3 is critical for generating regulatory T cells (T(reg) cells). Transforming growth factor-beta (TGF-beta) induces Foxp3 and suppressive T(reg) cells from naive T cells, whereas interleukin 6 (IL-6) inhibits the generation of inducible T(reg) cells. Here we show that IL-4 blocked the generation of TGF-beta-induced Foxp3(+) T(reg) cells and instead induced a population of T helper cells that produced IL-9 and IL-10. The IL-9(+)IL-10(+) T cells demonstrated no regulatory properties despite producing abundant IL-10. Adoptive transfer of IL-9(+)IL-10(+) T cells into recombination-activating gene 1-deficient mice induced colitis and peripheral neuritis, the severity of which was aggravated if the IL-9(+)IL-10(+) T cells were transferred with CD45RB(hi) CD4(+) effector T cells. Thus IL-9(+)IL-10(+) T cells lack suppressive function and constitute a distinct population of helper-effector T cells that promote tissue inflammation.
Project description:The aryl hydrocarbon receptor (AhR) participates in the differentiation of mouse regulatory T cells (T(reg) cells) and interleukin 17 (IL-17)-producing helper T cells (T(H)17 cells), but its role in human T cell differentiation is unknown. We investigated the role of AhR in the differentiation of human induced T(reg) cells (iT(reg) cells). We found that AhR activation promoted the differentiation of CD4(+)Foxp3(-) T cells, which produce IL-10 and control responder T cells through granzyme B. However, activation of AhR in the presence of transforming growth factor-beta1 induced Foxp3(+) iT(reg) cells, which suppress responder T cells through the ectonucleoside triphosphate diphosphohydrolase CD39. The induction of functional Foxp3(+) iT(reg) cells required coordinated action of the transcriptional regulators Smad1 and Aiolos. Thus, AhR is a potential target through which functional iT(reg) cells could be induced in human autoimmune disorders.