Moritz Rapp

CpG blocks immunosuppression by myeloid-derived suppressor cells in tumor-bearing mice.

Purpose: The Toll-like receptor (TLR) 9 ligand CpG has been used successfully for the immunotherapy of cancer. Chronic CpG application in tumor-free hosts leads, however, to the expansion of myeloid-derived suppressor cells (MDSC), which can cause T-cell suppression and may thus hamper the development of an effective immune response. Here, we investigated the effect of TLR9 activation on the function of MDSC in tumor-bearing mice. Experimental Design: We investigated the effect of CpG treatment on the number, phenotype, and function of MDSC in mice bearing subcutaneous C26 tumors and in CEA424-TAg mice bearing autochthonous gastric tumors. Results: CpG treatment blocks the suppressive activity of MDSC on T-cell proliferation in both tumor models. Inhibition of MDSC function by CpG was particularly pronounced for a highly suppressive Ly6Ghi polymorphonuclear subset of MDSC. We further show that TLR9 activation by CpG promotes maturation and differentiation of MDSC and strongly decreases the proportion of Ly6Ghi MDSC in both tumor-bearing and tumor-free mice. We demonstrate that IFN-α produced by plasmacytoid dendritic cells upon CpG stimulation is a key effector for the induction of MDSC maturation in vitro and show that treatment of mice with recombinant IFN-α is sufficient to block MDSC suppressivity. Conclusions: We show here for the first time that TLR9 activation inhibits the regulatory function of MDSC in tumor-bearing mice and define a role for the antitumoral cytokine IFN-α in this process. Clin Cancer Res; 17(7); 1765–75. ©2011 AACR.

CD103 is a hallmark of tumor‐infiltrating regulatory T cells

Regulatory T cells (Treg) mediate tolerance towards self‐antigens by suppression of innate and adaptive immunity. In cancer patients, tumor‐infiltrating FoxP3+ Treg suppress local anti‐tumor immune responses and are often associated with poor prognosis. Markers that are selectively expressed on tumor‐infiltrating Treg may serve as targets for immunotherapy of cancer. Here we show that CD103, an integrin mediating lymphocyte retention in epithelial tissues, is expressed at high levels on tumor‐infiltrating FoxP3+ Treg in several types of murine cancer. In the CT26 model of colon cancer up to 90% of the intratumoral FoxP3+ cells expressed CD103 compared to less than 20% in lymphoid organs. CD103+ Treg suppressed T effector cell activation more strongly than CD103neg Treg. Expression of CD103 on Treg closely correlated with intratumoral levels of transforming growth factor β (TGF‐β) and could be induced in a TGF‐β‐dependent manner by tumor cell lines. In vivo, gene silencing of TGF‐β reduced the frequency of CD103+ Treg, demonstrating that CD103 expression on tumor‐infiltrating Treg is driven by intratumoral TGF‐β. Functional blockade of CD103 using a monoclonal antibody did however not reduce the number of intratumoral Treg, indicating that CD103 is not involved in homing or retention of FoxP3+ cells in the tumor tissue. In conclusion, expression of CD103 is a hallmark of Treg that infiltrate TGF‐β‐secreting tumors. CD103 thus represents an interesting target for selective depletion of tumor‐infiltrating Treg, a strategy that may help to improve anti‐cancer therapy.

Impact of a New Fusion Receptor on PD-1–Mediated Immunosuppression in Adoptive T Cell Therapy

BACKGROUND Adoptive T cell transfer (ACT) is currently under investigation for the treatment of metastatic cancer. Recent evidence suggests that the coinhibitory PD-1-PD-L1 axis plays a major role in ACT failure. We hypothesized that a new fusion receptor reverting PD-1-mediated inhibition into CD28 costimulation may break peripheral tolerance. METHODS Different PD-1-CD28 fusion receptor constructs were created and retrovirally transduced into primary T cell receptor transgenic murine CD8(+) T cells specific for ovalbumin (OT-1). Cytokine release, proliferation, cytotoxicity, and tumor recognition were analyzed in vitro. Antitumor efficacy and mode of action were investigated in mice bearing subcutaneous tumors induced with the pancreatic carcinoma cell line Panc02 expressing the model antigen ovalbumin (Panc-OVA). For antitumoral efficacy, six to eight mice per group were used. All statistical tests are two-sided. RESULTS Transduction of the PD-1-CD28 receptor constructs mediated enhanced cytokine release, T cell proliferation, and T cell-induced lysis of target tumor cells. The PD-1-CD28 receptor function was dependent on two of the CD28-signaling motifs and IFN-γ release. Treatment of mice with established Panc-OVA tumors with fusion receptor-transduced OT-1 T cells mediated complete tumor regression. Mice rejecting the tumor were protected upon subsequent rechallenge with either ovalbumin-positive or -negative tumors, indicative of a memory response and epitope spreading in nine of 11 mice vs none of the six naïve mice (P < .001). Treatment efficacy was associated with accumulation of IFN-γ-producing T cells and an increased ratio of CD8(+) T cells to immunosuppressive myeloid-derived suppressor cells in the tumors. CONCLUSIONS Transduction of T cells with this new PD-1-CD28 receptor has the potential of breaking the PD-1-PD-L1-immunosuppressive axis in ACT.

Suppression of Intratumoral CCL22 by Type I Interferon Inhibits Migration of Regulatory T Cells and Blocks Cancer Progression

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.

The dipeptidylpeptidase-IV inhibitors sitagliptin, vildagliptin and saxagliptin do not impair innate and adaptive immune responses

Inhibitors of dipeptidylpeptidase IV (DPP‐IV) represent a novel class of frequently used anti‐diabetic drugs. In addition to its function in metabolic regulation, DPP‐IV also plays a role in the immune system. Whether the DPP‐IV inhibitors sitagliptin, vildagliptin or saxagliptin impair immune responses is, however, currently unknown. Here, we investigated the effect of these agents on both innate and adaptive immunity. We found that the DPP‐IV inhibitors did not affect the innate immune response induced by Toll‐like receptor (TLR) ligands, as cytokine secretion and induction of co‐stimulatory molecules by human blood mononuclear cells was not impaired. Furthermore, proliferation of T cells and suppressive function of regulatory T cells was preserved. Mice treated with vildagliptin showed normal cytokine production, immune cell activation and lymphocyte trafficking upon TLR activation. Thus, crucial immunological parameters remain unaffected upon treatment with DPP‐IV inhibitors, a fact that is reassuring with respect to safety of these drugs.

Expression of CCL22 and Infiltration by Regulatory T Cells are Increased in the Decidua of Human Miscarriage Placentas.

Regulatory T cells (Treg) are a T‐cell subpopulation with suppressive capacities, specifically attracted by CCL22. We aimed to investigate whether CCL22 is expressed in human placentas and whether its presence, together with Treg infiltration, is associated with miscarriage conditions.

Cancer cell-derived IL-1α induces CCL22 and the recruitment of regulatory T cells

ABSTRACT In cancer patients, immunosuppression through regulatory T cells (Treg) is a crucial component of tumor immune evasion and contributes to disease progression. Tumor-infiltrating Treg in particular suppress local effector T cell responses and are associated with poor prognosis in tumors such as human pancreatic cancer or hepatocellular carcinoma (HCC). The chemokine CCL22 is known to recruit Treg into the tumor tissue and many types of human tumors are known to express high levels of CCL22. The mechanisms leading to intratumoral secretion of CCL22 are so far unknown. We demonstrate here that intratumoral CCL22 is induced in tumor-infiltrating immune cells through cancer cell-derived interleukin-1 (IL-1α). In pancreatic cancer and HCC, CCL22 is produced by intratumoral dendritic cells, while the cancer cells themselves do not secrete CCL22 in vitro and in vivo. Incubation of human peripheral blood mononuclear cells (PBMC) or murine splenocytes with tumor cells or tumor cell supernatants strongly induced CCL22 secretion in vitro. Tumor cell supernatants contained IL-1 and CCL22 induction in PBMC could be specifically prevented by the IL-1 receptor antagonist anakinra or by transfection of tumor cell lines with IL-1 siRNA, leading to a suppression of Treg migration. In conclusion, we identify here tumor cell-derived IL-1α as a major inducer of the Treg attracting chemokine CCL22 in human cancer cells. Therapeutic blockade of the IL-1 pathway could represent a promising strategy to inhibit tumor-induced immunosuppression.

C-C chemokine receptor type-4 transduction of T cells enhances interaction with dendritic cells, tumor infiltration and therapeutic efficacy of adoptive T cell transfer

ABSTRACT T cell infiltration at the tumor site has been identified as a major predictor for the efficacy of adoptive T cell therapy. The chemokine C-C motif ligand 22 (CCL22) is highly expressed by immune cells in murine and human pancreatic cancer. Expression of its corresponding receptor, C-C chemokine receptor type 4 (CCR4), is restricted to regulatory T cells (Treg). We show that transduction of cytotoxic T cells (CTL) with CCR4 enhances their immigration into a pancreatic cancer model. Further, we show that binding of CCR4 with CCL22 strengthens the binding of T cell LFA-1 to dendritic cell (DC) ICAM-1 and increases CTL activation. In vivo, in a model of subcutaneous pancreatic cancer, treatment of tumor-bearing mice with CCR4-transduced CTL led to the eradication of established tumors in 40% of the mice. In conclusion, CCR4 overexpression in CTL is a promising therapeutic strategy to enhance the efficacy of adoptive T cell transfer (ACT).

Isolation of Intratumoral Leukocytes of TLR-Stimulated Tumor-Bearing Mice

Toll-like receptor (TLR) ligands hold promise for cancer immunotherapy. The isolation of intratumoral leukocytes of tumor-bearing mice is a useful technique for analyzing the immunological effects of TLR ligands on the tumor microenvironment. These isolated immune cells can be directly used for analysis (e.g., by flow cytometry) or cultured for functional in vitro studies. Here, we describe the isolation of intratumoral leukocytes by density gradient centrifugation. This technique can be used to isolate leukocytes from freshly dissected murine tumors.