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Supplementary Figure 7-Efficacy of anti-tumor treatment with CpG is dependent on an CD8+ T cell response.

Supplementary Figure 1-Treatment with CpG causes a reduction of Treg within the CD4 T cell population and an increase of CD8+ tumor-infiltrating cells.

Supplementary Figure 6-Type I interferon is a key mediator in the process of CCL22 suppression and tumor regression.

Supplementary Figure 3-Correlation (corr) of intratumoral FoxP3+ cells and CCL22 levels in CT26 tumors from untreated mice.

Supplementary Figure 2-CpG-induced suppression of Treg infiltration in different mouse tumor models.

The chemokine CCL22 is abundantly expressed in many types of cancer and is instrumental for intratumoral recruitment of regulatory T cells (Treg), an important subset of immunosuppressive and tumor-promoting lymphocytes. In this study, we offer evidence for a generalized strategy to blunt Treg activity that can limit immune escape and promote tumor rejection. Activation of innate immunity with Toll-like receptor (TLR) or RIG-I–like receptor (RLR) ligands prevented accumulation of Treg in tumors by blocking their immigration. Mechanistic investigations indicated that Treg blockade was a consequence of reduced intratumoral CCL22 levels caused by type I IFN. Notably, stable expression of CCL22 abrogated the antitumor effects of treatment with RLR or TLR ligands. Taken together, our findings argue that type I IFN blocks the Treg-attracting chemokine CCL22 and thus helps limit the recruitment of Treg to tumors, a finding with implications for cancer immunotherapy. Cancer Res; 75(21); 4483–93. ©2015 AACR.

Supplementary Figure 4-Inducible secretion of CCL22 by CT26-CCL22dox cells.

Supplementary Figure 5-CCL22 secretion of human tumor cell lines upon IFN-γ stimulation.

CCL22 is a key mediator of leukocyte trafficking in inflammatory immune responses, allergy, and cancer. It acts by attracting regulatory T cells and Th2 cells via their receptor CCR type 4 (CCR4). Beyond its role in inflammation, CCL22 is constitutively expressed at high levels in lymphoid organs during homeostasis, where it controls immunity by recruiting regulatory T cells to dendritic cells (DCs). In this study, we aimed to identify the mechanisms responsible for constitutive CCL22 expression. We confirmed that CD11c+ DCs are the exclusive producers of CCL22 in secondary lymphatic organs during homeostasis. We show that in vitro both murine splenocytes and human PBMCs secrete CCL22 spontaneously without any further stimulation. Interestingly, isolated DCs alone, however, are unable to produce CCL22, but instead require T cell help. In vitro, only the coculture of DCs with T cells or their supernatants resulted in CCL22 secretion, and we identified T cell–derived GM-CSF as the major inducer of DC-derived CCL22 expression. In vivo, Rag1−/− mice, which lack functional T cells, have low CCL22 levels in lymphoid organs, and this can be restored by adoptive transfer of wild-type T cells or administration of GM-CSF. Taken together, we uncover T cell–derived GM-CSF as a key inducer of the chemokine CCL22 and thus, to our knowledge, identify a novel role for this cytokine as a central regulator of immunity in lymphatic organs. This knowledge could contribute to the development of new therapeutic interventions in cancer and autoimmunity.

Rapp et al. demonstrate that dendritic cells in the lymph node secrete CCL22 to build cell–cell contacts with CCR4-expressing regulatory T cells, leading to immune suppression. Conversely, CCL22 deficiency results in enhanced T cell immunity, shown here in the setting of vaccination, cancer, and inflammatory disease., Chemokines have crucial roles in organ development and orchestration of leukocyte migration. The chemokine CCL22 is expressed constitutively at high levels in the lymph node, but the functional significance of this expression is so far unknown. Studying a newly established CCL22-deficient mouse, we demonstrate that CCL22 expression by dendritic cells (DCs) promotes the formation of cell–cell contacts and interaction with regulatory T cells (T reg) through their CCR4 receptor. Vaccination of CCL22-deficient mice led to excessive T cell responses that were also observed when wild-type mice were vaccinated using CCL22-deficient DCs. Tumor-bearing mice with CCL22 deficiency showed prolonged survival upon vaccination, and further, CCL22-deficient mice had increased susceptibility to inflammatory disease. In conclusion, we identify the CCL22–CCR4 axis as an immune checkpoint that is crucial for the control of T cell immunity., Graphical Abstract

Anz D, Eiber S, Scholz C, Endres S, Kirchner T, Bourquin C & Mayr D (2011) Histopathology59, 965–974 In breast cancer, a high ratio of tumour-infiltrating intraepithelial CD8+ to FoxP3+ cells is characteristic for the medullary subtype Aims: Medullary breast cancer (MBC) is a biologically distinct subtype of breast cancer characterized by prominent lymphocytic infiltrates and a favourable clinical outcome. Tumour-infiltrating CD8+ effector T cells may contribute to the good prognosis of this type of cancer; however, certain subtypes of lymphocyte, such as FoxP3+ regulatory T cells (Tregs), can also suppress antitumour immunity. Methods and results: We determined tumour infiltration by FoxP3+, CCL22+ and CD8+ cells in paraffin-embedded sections of MBC, and, as a reference, in samples of grade 3 ductal, lobular and mucinous breast cancer. All analysed MBCs were strongly infiltrated by FoxP3+ cells, whereas only weak infiltrates were detected in ductal or lobular breast cancer. This finding was unexpected, given the good prognosis of MBC. Strikingly, the number of CD8+ T cells exceeded the number of FoxP3+ cells in MBC (ratio of CD8+ to FoxP3+ cells of 2.6), whereas equal amounts of both cell types were found in ductal breast cancer (ratio of CD8+ to FoxP3+ cells of 1.1). In both types of breast cancer, we also detected cells expressing the Treg-attracting chemokine CCL22. Conclusions: In breast cancer, a predominance of tumour-infiltrating CD8+ over FoxP3+ cells was observed in MBC. Thus, the ratio of CD8+ to FoxP3+ cells rather than the absolute number of intratumoral FoxP3+ cells may be predictive for the clinical outcome of cancer.

The chemokine CCL22 is abundantly expressed in many types of cancer and is instrumental for intratumoral recruitment of regulatory T cells (Treg), an important subset of immunosuppressive and tumor-promoting lymphocytes. In this study, we offer evidence for a generalized strategy to blunt Treg activity that can limit immune escape and promote tumor rejection. Activation of innate immunity with Toll-like receptor (TLR) or RIG-I–like receptor (RLR) ligands prevented accumulation of Treg in tumors by blocking their immigration. Mechanistic investigations indicated that Treg blockade was a consequence of reduced intratumoral CCL22 levels caused by type I IFN. Notably, stable expression of CCL22 abrogated the antitumor effects of treatment with RLR or TLR ligands. Taken together, our findings argue that type I IFN blocks the Treg-attracting chemokine CCL22 and thus helps limit the recruitment of Treg to tumors, a finding with implications for cancer immunotherapy. Cancer Res; 75(21); 4483–93. ©2015 AACR.

Background Medical knowledge encompasses both conceptual (facts or “what” information) and procedural knowledge (“how” and “why” information). Conceptual knowledge is known to be an essential prerequisite for clinical problem solving. Primarily, medical students learn from textbooks and often struggle with the process of applying their conceptual knowledge to clinical problems. Recent studies address the question of how to foster the acquisition of procedural knowledge and its application in medical education. However, little is known about the factors which predict performance in procedural knowledge tasks. Which additional factors of the learner predict performance in procedural knowledge? Methods Domain specific conceptual knowledge (facts) in clinical nephrology was provided to 80 medical students (3rd to 5th year) using electronic flashcards in a laboratory setting. Learner characteristics were obtained by questionnaires. Procedural knowledge in clinical nephrology was assessed by key feature problems (KFP) and problem solving tasks (PST) reflecting strategic and conditional knowledge, respectively. Results Results in procedural knowledge tests (KFP and PST) correlated significantly with each other. In univariate analysis, performance in procedural knowledge (sum of KFP+PST) was significantly correlated with the results in (1) the conceptual knowledge test (CKT), (2) the intended future career as hospital based doctor, (3) the duration of clinical clerkships, and (4) the results in the written German National Medical Examination Part I on preclinical subjects (NME-I). After multiple regression analysis only clinical clerkship experience and NME-I performance remained independent influencing factors. Conclusions Performance in procedural knowledge tests seems independent from the degree of domain specific conceptual knowledge above a certain level. Procedural knowledge may be fostered by clinical experience. More attention should be paid to the interplay of individual clinical clerkship experiences and structured teaching of procedural knowledge and its assessment in medical education curricula.

Medullary breast cancer (MBC) is a biologically distinct subtype of breast cancer characterized by prominent lymphocytic infiltrates and a favourable clinical outcome. Tumour-infiltrating CD8+ effector T cells may contribute to the good prognosis of this type of cancer; however, certain subtypes of lymphocyte, such as FoxP3+ regulatory T cells (Tregs), can also suppress antitumour immunity.We determined tumour infiltration by FoxP3+, CCL22+ and CD8+ cells in paraffin-embedded sections of MBC, and, as a reference, in samples of grade 3 ductal, lobular and mucinous breast cancer. All analysed MBCs were strongly infiltrated by FoxP3+ cells, whereas only weak infiltrates were detected in ductal or lobular breast cancer. This finding was unexpected, given the good prognosis of MBC. Strikingly, the number of CD8+ T cells exceeded the number of FoxP3+ cells in MBC (ratio of CD8+ to FoxP3+ cells of 2.6), whereas equal amounts of both cell types were found in ductal breast cancer (ratio of CD8+ to FoxP3+ cells of 1.1). In both types of breast cancer, we also detected cells expressing the Treg-attracting chemokine CCL22.In breast cancer, a predominance of tumour-infiltrating CD8+ over FoxP3+ cells was observed in MBC. Thus, the ratio of CD8+ to FoxP3+ cells rather than the absolute number of intratumoral FoxP3+ cells may be predictive for the clinical outcome of cancer.