Juan J. Lasarte

CD4+/CD25+ Regulatory Cells Inhibit Activation of Tumor-Primed CD4+ T Cells with IFN-γ-Dependent Antiangiogenic Activity, as well as Long-Lasting Tumor Immunity Elicited by Peptide Vaccination

CD25+ regulatory T (T reg) cells suppress the activation/proliferation of other CD4+ or CD8+ T cells in vitro. Also, down-regulation of CD25+ T reg cells enhance antitumor immune responses. In this study, we show that depletion of CD25+ T reg cells allows the host to induce both CD4+ and CD8+ antitumoral responses following tumor challenge. Simultaneous depletion of CD25+ and CD8+ cells, as well as adoptive transfer experiments, revealed that tumor-specific CD4+ T cells, which emerged in the absence of CD25+ T reg cells, were able to reject CT26 colon cancer cells, a MHC class II-negative tumor. The antitumoral effect mediated by CD4+ T cells was dependent on IFN-γ production, which exerted a potent antiangiogenic activity. The capacity of the host to mount this antitumor response is lost once the number of CD25+ T reg cells is restored over time. However, CD25+ T reg cell depletion before immunization with AH1 (a cytotoxic T cell determinant from CT26 tumor cells) permits the induction of a long-lasting antitumoral immune response, not observed if immunization is conducted in the presence of regulatory cells. A study of the effect of different levels of depletion of CD25+ T reg cells before immunization with the peptide AH1 alone, or in combination with a Th determinant, unraveled that Th cells play an important role in overcoming the suppressive effect of CD25+ T reg on the induction of long-lasting cellular immune responses.

Anaphylatoxin C5a Creates a Favorable Microenvironment for Lung Cancer Progression

The complement system contributes to various immune and inflammatory diseases, including cancer. In this study, we investigated the capacity of lung cancer cells to activate complement and characterized the consequences of complement activation on tumor progression. We focused our study on the production and role of the anaphylatoxin C5a, a potent immune mediator generated after complement activation. We first measured the capacity of lung cancer cell lines to deposit C5 and release C5a. C5 deposition, after incubation with normal human serum, was higher in lung cancer cell lines than in nonmalignant bronchial epithelial cells. Notably, lung malignant cells produced complement C5a even in the absence of serum. We also found a significant increase of C5a in plasma from patients with non-small cell lung cancer, suggesting that the local production of C5a is followed by its systemic diffusion. The contribution of C5a to lung cancer growth in vivo was evaluated in the Lewis lung cancer model. Syngeneic tumors of 3LL cells grew slower in mice treated with an antagonist of the C5a receptor. C5a did not modify 3LL cell proliferation in vitro but induced endothelial cell chemotaxis and blood-vessels formation. C5a also contributed to the immunosuppressive microenvironment required for tumor growth. In particular, blockade of C5a receptor significantly reduced myeloid-derived suppressor cells and immunomodulators ARG1, CTLA-4, IL-6, IL-10, LAG3, and PDL1 (B7H1). In conclusion, lung cancer cells have the capacity to generate C5a, a molecule that creates a favorable tumor microenvironment for lung cancer progression.

Regression of colon cancer and induction of antitumor immunity by intratumoral injection of adenovirus expressing interleukin-12

Interleukin-12 (IL-12) has been shown to possess potent immunoregulatory and antitumoral effects. We have evaluated the anti-oncogenic potential and the mechanisms of the antitumoral effect of in vivo adenovirus-mediated transfer of IL-12 gene in a murine model of colon cancer. AdCMVIL-12 was constructed to permit coordinated production of p40 and p35 subunits of IL-12 gene to obtain the maximum IL-12 bioactivity. Infection of murine colon cancer CT-26 cells in vitro with AdCMVIL-12 resulted in the production of high levels of IL-12. In vivo gene therapy of colon cancer nodules by intratumoral injection of AdCMVIL-12 induced a local increase in IL-12 and interferon-γ levels and a complete regression of the tumor in 26 of 34 (76%) mice. Tumor disappeared between days 7 and 10 after vector administration. The antitumoral effect was mediated by CD8+ T cells and was associated with the generation of cytotoxic T lymphocytes against colon cancer cells. Animals that eliminated the tumor were protected against a second administration of neoplastic cells. Treatment with AdCMVIL-12 of one tumor nodule also caused regression of established tumors at distant sites. These data demonstrate that AdCMVIL-12 is a useful therapeutic tool for established colon cancer in mice and should be considered for application in humans.

The Extra Domain A from Fibronectin Targets Antigens to TLR4-Expressing Cells and Induces Cytotoxic T Cell Responses In Vivo

Vaccination strategies based on the in vivo targeting of Ags to dendritic cells (DCs) are needed to improve the induction of specific T cell immunity against tumors and infectious agents. In this study, we have used a recombinant protein encompassing the extra domain A from fibronectin (EDA), an endogenous ligand for TLR4, to deliver Ags to TLR4-expressing DC. The purified EDA protein was shown to bind to TLR4-expressing HEK293 cells and to activate the TLR4 signaling pathway. EDA also stimulated the production by DC of proinflammatory cytokines such as IL-12 or TNF-α and induced their maturation in vitro and in vivo. A fusion protein between EDA and a cytotoxic T cell epitope from OVA efficiently presented this epitope to specific T cells and induced the in vivo activation of a strong and specific CTL response. Moreover, a fusion protein containing EDA and the full OVA also improved OVA presentation by DC and induced CTL responses in vivo. These EDA recombinant proteins protected mice from a challenge with tumor cells expressing OVA. These results strongly suggest that the fibronectin extra domain A may serve as a suitable Ag carrier for the development of antiviral or antitumoral vaccines.

Upregulation of Indoleamine 2,3-Dioxygenase in Hepatitis C Virus Infection

ABSTRACT Indoleamine 2,3-dioxygenase (IDO) is induced by proinflammatory cytokines and by CTLA-4-expressing T cells and constitutes an important mediator of peripheral immune tolerance. In chronic hepatitis C, we found upregulation of IDO expression in the liver and an increased serum kynurenine/tryptophan ratio (a reflection of IDO activity). Huh7 cells supporting hepatitis C virus (HCV) replication expressed higher levels of IDO mRNA than noninfected cells when stimulated with gamma interferon or when cocultured with activated T cells. In infected chimpanzees, hepatic IDO expression decreased in animals that cured the infection, while it remained high in those that progressed to chronicity. For both patients and chimpanzees, hepatic expression of IDO and CTLA-4 correlated directly. Induction of IDO may dampen T-cell reactivity to viral antigens in chronic HCV infection.

Is plasma cardiotrophin-1 a marker of hypertensive heart disease?

Objective This study was designed to investigate whether plasma concentration of cardiotrophin-1 (CT-1), a cytokine that induces cardiomyocyte hypertrophy and stimulates cardiac fibroblasts, is related to hypertensive heart disease, as defined by the presence of echocardiographically assessed left ventricular hypertrophy (LVH). Methods The study was performed in 31 normotensive subjects and 111 patients with never-treated essential hypertension (54 without LVH and 57 with LVH). Causes of LVH other than hypertension were excluded after a complete medical workup. A novel enzyme-linked immunosorbent assay was developed to measure plasma CT-1. Results Plasma CT-1 was increased (P < 0.001) in hypertensives compared with normotensives. The value of CT-1 was higher (P < 0.001) in hypertensives with LVH than in hypertensives without LVH. Some 31% of patients without LVH exhibited values of CT-1 above the upper normal limit in normotensives. A direct correlation was found between CT-1 and left ventricular mass index (r = 0.319, P < 0.001) in all subjects. Receiver operating characteristic curves showed that a cutoff of 39 fmol/ml for CT-1 provided 75% specificity and 70% sensitivity for predicting LVH with a relative risk of 6.21 (95% confidence interval, 2.95 to 13.09). Conclusions These results show an association between LVH and the plasma concentration of CT-1 in essential hypertension. Although preliminary, these findings suggest that the determination of CT-1 may be an easy and reliable method for the initial screening and diagnosis of hypertensive heart disease.

Insights on the amino acid side-chain interactions of a synthetic T-cell determinant.

The effect of single amino acid substitutions at positions 18 and 20 on the T-cell determinant (TD) character of peptide p12-26 from lambda repressor protein and on its recognition by a monoclonal antibody was studied by means of 40 synthetic peptides of a length of 15 amino acids. ELISA competition experiments showed that the identity of amino acid at position 20 is very important for antibody recognition, whereas that of amino acid at position 18 is much less important. In contrast, both Leu 18 and Ala 20 are important residues in defining the TD character of peptide p12-26. The most tolerated replacements, ordered in increasing disrupting power are: Ala 20 by Cys, Ser or Gly and Leu 18 by Ile or Val. Any other amino acid replacement completely abolishes the TD capacity of peptide p12-26. The peptides used in this study were synthesized using a multiple solid-phase peptide synthesizer newly designed. Their purity was very high as shown by amino acid sequence experiments.

Combined immunization with adjuvant molecules poly(I:C) and anti-CD40 plus a tumor antigen has potent prophylactic and therapeutic antitumor effects

The low immunogenicity of malignant cells is one of the causes responsible for the lack of antitumor immune responses. Thus, development of new therapeutic strategies aimed at enhancing presentation of tumor antigens to T cells is a main goal of cancer immunotherapy. With this aim, we studied the efficacy of administering adjuvants poly(I:C) and agonistic anti-CD40 antibody plus a tumor antigen. Joint intravenous immunization with these adjuvants and a model tumor antigen (ovalbumin) was able to synergistically induce potent and long lasting antitumor T-cell responses. These responses protected against challenge with E.G7–OVA tumor cells in prophylactic short- and long-term vaccination. In a therapeutic setting, repeated intratumor administration of adjuvants plus antigen was able to reject established tumors in all treated animals, leading in some cases to the rejection of both locally treated and untreated tumors. Antitumor immune responses induced by these protocols were mediated not only by T-cells but also by NK cells. In conclusion, combined administration of adjuvants poly(I:C) and anti-CD40 plus a tumor antigen is an efficient strategy for prophylactic and therapeutic antitumor vaccination.

Antitumor effect of allogenic fibroblasts engineered to express Fas ligand (FasL)

Fas ligand is a type II transmembrane protein which can induce apoptosis in Fas-expressing cells. Recent reports indicate that expression of FasL in transplanted cells may cause graft rejection and, on the other hand, tumor cells may lose their tumorigenicity when they are engineered to express FasL. These effects could be related to recruitment of neutrophils by FasL with activation of their cytotoxic machinery. In this study we investigated the antitumor effect of allogenic fibroblasts engineered to express FasL. Fibroblasts engineered to express FasL (PA317/FasL) did not exert toxic effects on transformed liver cell line (BNL) or colon cancer cell line (CT26) in vitro, but they could abrogate their tumorigenicity in vivo. Histological examination of the site of implantation of BNL cells mixed with PA317/FasL revealed massive infiltration of polymorphonuclear neutrophils and mononuclear cells. A specific immune protective effect was observed in animals primed with a mixture of BNL or CT26 and PA317/FasL cells. Rechallenge with tumor cells 14 or 100 days after priming resulted in protection of 100 or 50% of animals, respectively. This protective effect was due to CD8+ cells since depletion of CD8+ led to tumor formation. In addition, treatment of pre-established BNL tumors with a subcutaneous injection of BNL and PA317/FasL cell mixture at a distant site caused significant inhibition of tumor growth. These data demonstrate that allogenic cells engineered with FasL are able to abolish tumor growth and induce specific protective immunity when they are mixed with neoplastic cells.

Characterization of T‐cell responses against immunodominant epitopes from hepatitis C virus E2 and NS4a proteins

Summary.u2002 Successful clearance of hepatitis C virus (HCV) infection has been associated with strong cellular immune responses against viral antigens. However, although the magnitude of these responses is clearly important for viral eradication, more studies are needed to unravel the fine specificity of the protective anti‐HCV immunity in infected patients. This was the aim of the present study. Overlapping peptides spanning the sequence of HCV E2 and NS4a proteins were used to stimulate T cells from patients with chronic hepatitis C divided into three groups: naïve patients, patients who exhibited sustained response to interferon (IFN)‐α therapy and patients who failed to respond to the treatment. Interleukin‐2 production by stimulated cells was measured in each case. Patients with sustained response to therapy had stronger responses to E2 peptides than nonresponders, whereas naïve patients demonstrated intermediate reactivity. In the case of NS4a, responses against peptides where similar in all groups of patients. Analysis of the peptides recognized by T cells showed that responses were broad and heterogeneous, and some immunodominant epitopes, preferentially recognized by patients exhibiting sustained response to treatment, were found. These results confirm the role of cellular immune responses in viral clearance, and stress the importance of immunodominant regions within HCV antigens. These viral sequences may represent valuable immunogens for preparation of therapeutic or prophylactic vaccines.