Sophie Lucas

Interleukin 27 limits autoimmune encephalomyelitis by suppressing the development of interleukin 17–producing T cells

Interleukin 27 (IL-27) was first characterized as a proinflammatory cytokine with T helper type 1–inducing activity. However, subsequent work has demonstrated that mice deficient in IL-27 receptor (IL-27Rα) show exacerbated inflammatory responses to a variety of challenges, suggesting that IL-27 has important immunoregulatory functions in vivo. Here we demonstrate that IL-27Rα-deficient mice were hypersusceptible to experimental autoimmune encephalomyelitis and generated more IL-17-producing T helper cells. IL-27 acted directly on effector T cells to suppress the development of IL-17-producing T helper cells mediated by IL-6 and transforming growth factor-β. This suppressive activity was dependent on the transcription factor STAT1 and was independent of interferon-γ. Finally, IL-27 suppressed IL-6-mediated T cell proliferation. These data provide a mechanistic explanation for the IL-27-mediated immune suppression noted in several in vivo models of inflammation.

IL-27 regulates IL-12 responsiveness of naïve CD4+ T cells through Stat1-dependent and -independent mechanisms

IL-27, a novel heterodimeric cytokine produced by antigen-presenting cells, signals through the T cell cytokine receptor (TCCR)/WSX-1 expressed on naïve CD4+ T cells and natural killer cells. TCCR/WSX-1 deficiency results in delayed T helper type 1 (TH1) development through an unresolved mechanism. We report here that IL-27 stimulation in developing murine T helper cells potently induces the expression of the major TH1-specific transcription factor T-bet and its downstream target IL-12R β2, independently of IFNγ. In addition, IL-27 suppresses basal expression of GATA-3, the critical TH2-specific transcription factor that inhibits TH1 development by down-regulating signal transducer and activator of transcription (Stat) 4. IL-27 signaling through TCCR/WSX-1 induces phosphorylation of Stat1, Stat3, Stat4, and Stat5. Stat1 is required for suppression of GATA-3, but T-bet induction by IL-27 can also be mediated through a Stat1-independent pathway. Despite its TH1-like signaling profile, IL-27 is not sufficient to drive the differentiation of CD4+ T cells into IFNγ-producing cells. Similarly, IL-27 induces T-bet expression in primary natural killer cells, but this does not result in an increase of IFNγ production or cytotoxic activity. Therefore, although IL-27 is unable to drive IFNγ production on its own, it plays an important role in the early steps of TH1 commitment by contributing in a paracrine manner to the control of IL-12 responsiveness.

LAGE-1, a new gene with tumor specificity.

Representational difference analysis was used to identify genes that are expressed in a human melanoma cell line and not in normal skin. A cDNA clone that appeared to be specific for tumors was obtained and the corresponding gene was sequenced. This new gene was named LAGE‐1. Using a LAGE‐1 probe to screen a cDNA library from the same melanoma cell line, we identified a closely related gene, which proved to be identical to NY‐ESO‐1, a gene recently reported to code for an antigen recognized by autologous antibodies in an esophageal squamous cell carcinoma. Gene LAGE‐1 maps to Xq28. It comprises 3 exons. Alternative splicing produces 2 major transcripts encoding polypeptides of 210 and 180 residues, respectively. Expression of LAGE‐1 was observed in 25–50% of tumor samples of melanomas, non‐small‐cell lung carcinomas, bladder, prostate and head and neck cancers. The only normal tissue that expressed the gene was testis. As for MAGE‐A1, expression of LAGE‐1 is induced by deoxy‐azacytidine in lymphoblastoid cells, suggesting that tumoral expression is due to demethylation. The expression of LAGE‐1 is strongly correlated with that of NY‐ESO‐1. It is also clearly correlated with the expression of MAGE genes. Int. J. Cancer 76:903–908, 1998.© 1998 Wiley‐Liss, Inc.

Compromised Humoral and Delayed-Type Hypersensitivity Responses in IL-23-Deficient Mice

The heterodimeric cytokine IL-23 consists of a private cytokine-like p19 subunit and a cytokine receptor-like subunit, p40, which is shared with IL-12. Previously reported IL-12p40-deficient mice have profound immune defects resulting from combined deficiency in both IL-12 and IL-23. To address the effects of specific IL-23 deficiency, we generated mice lacking p19 by gene targeting. These mice display no overt abnormalities but mount severely compromised T-dependent humoral immune responses. IL-23p19−/− mice produce strongly reduced levels of Ag-specific Igs of all isotypes, but mount normal T-independent B cell responses. In addition, delayed type hypersensitivity responses are strongly impaired in the absence of IL-23, indicating a defect at the level of memory T cells. T cells stimulated with IL-23-deficient APCs secrete significantly reduced amounts of the proinflammatory cytokine IL-17, and IL-23-deficient mice phenotypically resemble IL-17-deficient animals. Thus, IL-23 plays a critical role in T cell-dependent immune responses, and our data provide further support for the existence of an IL-23/IL-17 axis of communication between the adaptive and innate parts of the immune system.

Membrane protein GARP is a receptor for latent TGF-beta on the surface of activated human Treg.

Human Treg and Th clones secrete the latent form of TGF‐β, in which the mature TGF‐β protein is bound to the latency‐associated peptide (LAP), and is thereby prevented from binding to the TGF‐β receptor. We previously showed that upon TCR stimulation, human Treg clones but not Th clones produce active TGF‐β and bear LAP on their surface. Here, we show that latent TGF‐β, i.e. both LAP and mature TGF‐β, binds to glycoprotein A repetitions predominant (GARP), a transmembrane protein containing leucine rich repeats, which is present on the surface of stimulated Treg clones but not on Th clones. Membrane localization of latent TGF‐β mediated by binding to GARP may be necessary for the ability of Treg to activate TGF‐β upon TCR stimulation. However, it is not sufficient as lentiviral‐mediated expression of GARP in human Th cells induces binding of latent TGF‐β to the cell surface, but does not result in the production of active TGF‐β upon stimulation of these Th cells.

Cytolytic T-cell responses of cancer patients vaccinated with a MAGE antigen.

Summary: ‘Cancer‐germline’ genes such as the MAGE gene family are expressed in many tumors and in male germline cells but not in normal tissues. They encode shared tumor‐specific antigens, which have been used in therapeutic vaccination trials of metastatic melanoma patients. To establish whether there is a correlation between tumoral regressions and T‐cell responses against the vaccine antigen, we evaluated the responses of patients vaccinated with a MAGE‐3 antigenic peptide or a recombinant virus coding for the peptide. Blood lymphocytes were stimulated with antigenic peptide followed by detection with tetramer, T‐cell cloning, and TCR analysis. In 4/9 regressor patients and in 1/14 progressors we found a low level, usually monoclonal cytolytic T lymphocyte response against the MAGE‐3 peptide.

Positive and Negative Regulation of the IL-27 Receptor during Lymphoid Cell Activation

Previous reports have focused on the ability of IL-27 to promote naive T cell responses but the present study reveals that surface expression of WSX-1, the ligand-specific component of the IL-27R, is low on these cells and that highest levels are found on effector and memory CD4+ and CD8+ T cells. Accordingly, during infection with Toxoplasma gondii, in vivo T cell activation is associated with enhanced expression of WSX-1, and, in vitro, TCR ligation can induce expression of WSX-1 regardless of the polarizing (Th1/Th2) environment present at the time of priming. However, while these data establish that mitogenic stimulation promotes expression of WSX-1 by T cells, activation of NK cells and NKT cells prompts a reduction in WSX-1 levels during acute toxoplasmosis. Together, with the finding that IL-2 can suppress expression of WSX-1 by activated CD4+ T cells, these studies indicate that surface levels of the IL-27R can be regulated by positive and negative signals associated with lymphoid cell activation. Additionally, since high levels of WSX-1 are evident on resting NK cells, resting NKT cells, effector T cells, regulatory T cells, and memory T cells, the current work demonstrates that IL-27 can influence multiple effector cells of innate and adaptive immunity.

MAGE-B5, MAGE-B6, MAGE-C2, and MAGE-C3: four new members of the MAGE family with tumor-specific expression

A number of genes of the MAGE‐A, B, and C families have been shown to code for antigens that are recognized on many human tumors by autologous cytolytic T lymphocytes. These antigens ought to be strictly tumor specific because the encoding MAGE genes are not expressed in normal adult cells except for male germline cells, which lack HLA expression. To identify new genes of this type, we performed representational difference analysis on a melanoma cell line by subtraction with a normal skin sample. This led to the identification of MAGE‐C2, a new member of the MAGE‐C family. A search for nucleotide sequences encoding MAGE‐like proteins in public databases led to the identification of three additional MAGE genes, which were named MAGE‐B5, MAGE‐B6, and MAGE‐C3. The four new MAGE genes are not expressed in normal tissues, except for testis, and are expressed in tumors of different histological origins. Therefore, like other MAGE genes expressed specifically in tumors, MAGE‐B5, MAGE‐B6, MAGE‐C2, and MAGE‐C3 ought to encode antigens that could be targets for cancer immunotherapy. Int. J. Cancer 87:55–60, 2000.

Polyclonal CTL Responses Observed in Melanoma Patients Vaccinated with Dendritic Cells Pulsed with a MAGE-3.A1 Peptide

Vaccination with mature, monocyte-derived dendritic cells (DC) pulsed with the MAGE-3168–176 peptide, which is presented by HLA-A1, has been reported to induce tumor regressions and CTL in some advanced stage IV melanoma patients. We present here a precise evaluation of the level of some of these anti-MAGE-3.A1 CTL responses and an analysis of their clonal diversity. Blood lymphocytes were stimulated with the MAGE-3.A1 peptide under limiting dilution conditions and assayed with an A1/MAGE-3 tetramer. This was followed by the cloning of the tetramer-positive cells and by TCR sequence analysis of the CTL clones that lysed targets expressing MAGE-3.A1. We also used direct ex vivo tetramer staining of CD8 cells, sorting, and cloning of the positive cells. In three patients who showed regression of some of their metastases after vaccination, CTL responses were observed with frequencies ranging from 7 × 10−6 to 9 × 10−4 of CD8+ blood T lymphocytes, representing an increase of 20- to 400-fold of the frequencies found before immunization. A fourth patient showed neither tumor regression nor an anti-MAGE-3.A1 CTL response. In each of the responses, several CTL clones were amplified. This polyclonality contrasts with the monoclonality of the CTL responses observed in patients vaccinated with MAGE-3.A1 peptide or with an ALVAC recombinant virus coding for this antigenic peptide.

Route of Administration Modulates the Induction of Dendritic Cell Vaccine–Induced Antigen-Specific T Cells in Advanced Melanoma Patients

Purpose: It is unknown whether the route of administration influences dendritic cell (DC)-based immunotherapy. We compared the effect of intradermal versus intranodal administration of a DC vaccine on induction of immunologic responses in melanoma patients and examined whether concomitant administration of interleukin (IL)-2 increases the efficacy of the DC vaccine. Experimental Design: HLA-A2.1+ melanoma patients scheduled for regional lymph node dissection were vaccinated four times biweekly via intradermal or intranodal injection with 12 × 106 to 17 × 106 mature DCs loaded with tyrosinase and gp100 peptides together with keyhole limpet hemocyanin (KLH). Half of the patients also received low-dose IL-2 (9 MIU daily for 7 days starting 3 days after each vaccination). KLH-specific B- and T-cell responses were monitored in blood. gp100- and tyrosinase-specific T-cell responses were monitored in blood by tetramer analysis and in biopsies from delayed-type hypersensitivity (DTH) skin tests by tetramer and functional analyses with 51Cr release assays or IFNγ release, following coculture with peptide-pulsed T2 cells or gp100- or tyrosinase-expressing tumor cells. Results: In 19 of 43 vaccinated patients, functional tumor antigen–specific T cells could be detected. Although significantly more DCs migrated to adjacent lymph nodes upon intranodal vaccination, this was also highly variable with a complete absence of migration in 7 of 24 intranodally vaccinated patients. Intradermal vaccinations proved superior in inducing functional tumor antigen–specific T cells. Coadministration of IL-2 did not further augment the antigen-specific T-cell response but did result in higher regulatory T-cell frequencies. Conclusion: Intradermal vaccination resulted in superior antitumor T-cell induction when compared with intranodal vaccination. No advantage of additional IL-2 treatment could be shown. Clin Cancer Res; 17(17); 5725–35. ©2011 AACR.