Monica Moro

Human CD1c+ dendritic cells secrete high levels of IL-12 and potently prime cytotoxic T cell responses

Dendritic cells (DC) have the unique capacities to induce primary T-cell responses. In mice, CD8α(+)DC are specialized to cross-prime CD8(+) T cells and produce interleukin-12 (IL-12) that promotes cytotoxicity. Human BDCA-3(+)DC share several relevant characteristics with CD8α(+)DC, but the capacities of human DC subsets to induce CD8(+) T-cell responses are incompletely understood. Here we compared CD1c(+) myeloid DC (mDC)1, BDCA-3(+)mDC2, and plasmacytoid DC (pDC) in peripheral blood and lymphoid tissues for phenotype, cytokine production, and their capacities to prime cytotoxic T cells. mDC1 were surprisingly the only human DC that secreted high amounts of IL-12p70, but they required combinational Toll-like receptor (TLR) stimulation. mDC2 and pDC produced interferon-λ and interferon-α, respectively. Importantly, mDC1 and mDC2 required different combinations of TLR ligands to cross-present protein antigens to CD8(+) T cells. pDC were inefficient and also expressed lower levels of major histocompatibility complex and co-stimulatory molecules. Nevertheless, all DC induced CD8(+) memory T-cell expansions upon licensing by CD4(+) T cells, and primed naive CD8(+) T cells following appropriate TLR stimulation. However, because mDC1 produced IL-12, they induced the highest levels of cytotoxic molecules. In conclusion, CD1c(+)mDC1 are the relevant source of IL-12 for naive T cells and are fully equipped to cross-prime cytotoxic T-cell responses.

Selective Activation and Expansion of High-Affinity CD4+ T Cells in Resistant Mice upon Infection with Leishmania major

Using multimers of MHC class II molecules linked to a peptide derived from the Leishmania LACK antigen, we have compared the fate of parasite-specific CD4+ T cells in resistant and susceptible mice transgenic for the beta chain of a LACK-specific TCR. Activated T cells were readily detected in the draining lymph nodes of infected animals. Although the kinetics of activation and expansion were similar in both strains, T cells from susceptible and resistant mice expressed low- and high-affinity TCR, respectively. As T cells from resistant mice produced more IFN-gamma and less IL-4 than those from susceptible animals, our results suggest that differences in TCR usage between MHC-matched animals may influence the development of the antiparasite immune response.

Relevance of the Tumor Antigen in the Validation of Three Vaccination Strategies for Melanoma

Many preclinical studies of cancer immunotherapy are based on the testing of a single vaccination strategy in several tumor models. Moreover, most of those studies used xenogeneic Ags, which, owing to their high immunogenicity, may not represent realistic models for the validation of cancer immunotherapies. To address these issues, we compared the vaccination efficacy of three well established strategies (i.e., naked DNA; peptide-pulsed dendritic cells (DC), or a mixture of peptide and the Escherichia coli toxin LTR72) using the xenogeneic OVA or the naturally expressed tyrosinase-related protein 2 (TRP-2) tumor Ag in the B16 melanoma model. C57BL/6 mice received one to three s.c. injections of peptide-pulsed DC or DNA, or one to four mucosal administrations of peptide-toxin mixture. One to 2 wk later, the animals were challenged s.c. with B16 or B16 cells expressing OVA (B16-OVA). Vaccination of mice with OVA induced in all cases melanoma-specific CTL and protection against B16-OVA. When TRP-2 was used, all three vaccines elicited B16-specific CTL, but only DC pulsed with the immunodominant T cell epitope TRP-2181–188 allowed protection against B16. Even more importantly, a vaccination regimen with TRP-2-pulsed DC, started 24 h after the injection of a lethal number of B16 cells, caused a therapeutic effect in 60% of the challenged animals. Our results strongly emphasize the relevance of the tumor Ag in the definition of immunotherapeutic strategies for cancer, and support the use of peptide-pulsed DC as cancer vaccine in humans.

Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness of Tumor-Infiltrating T Regulatory Cells

Summary Tumor-infiltrating regulatory T lymphocytes (Treg) can suppress effector T cells specific for tumor antigens. Deeper molecular definitions of tumor-infiltrating-lymphocytes could thus offer therapeutic opportunities. Transcriptomes of T helper 1 (Th1), Th17, and Treg cells infiltrating colorectal or non-small-cell lung cancers were compared to transcriptomes of the same subsets from normal tissues and validated at the single-cell level. We found that tumor-infiltrating Treg cells were highly suppressive, upregulated several immune-checkpoints, and expressed on the cell surfaces specific signature molecules such as interleukin-1 receptor 2 (IL1R2), programmed death (PD)-1 Ligand1, PD-1 Ligand2, and CCR8 chemokine, which were not previously described on Treg cells. Remarkably, high expression in whole-tumor samples of Treg cell signature genes, such as LAYN, MAGEH1, or CCR8, correlated with poor prognosis. Our findings provide insights into the molecular identity and functions of human tumor-infiltrating Treg cells and define potential targets for tumor immunotherapy.

Intracellular Modulation, Extracellular Disposal and Serum Increase of MiR-150 Mark Lymphocyte Activation

Activated lymphocytes release nano-sized vesicles (exosomes) containing microRNAs that can be monitored in the bloodstream. We asked whether elicitation of immune responses is followed by release of lymphocyte-specific microRNAs. We found that, upon activation in vitro, human and mouse lymphocytes down-modulate intracellular miR-150 and accumulate it in exosomes. In vivo, miR-150 levels increased significantly in serum of humans immunized with flu vaccines and in mice immunized with ovalbumin, and this increase correlated with elevation of antibody titers. Immunization of immune-deficient mice, lacking MHCII, resulted neither in antibody production nor in elevation of circulating miR-150. This study provides proof of concept that serum microRNAs can be detected, with minimally invasive procedure, as biomarkers of vaccination and more in general of adaptive immune responses. Furthermore, the prompt reduction of intracellular level of miR-150, a key regulator of mRNAs critical for lymphocyte differentiation and functions, linked to its release in the external milieu suggests that the selective extracellular disposal of microRNAs can be a rapid way to regulate gene expression during lymphocyte activation.

CD28 and LFA-1 contribute to cyclosporin A-resistant T cell growth by stabilizing the IL-2 mRNA through distinct signaling pathways

In clinical transplantation, the occurrence of cyclosporin A (CsA)‐resistant production of IL‐2 in vitro correlates with graft rejection in vivo. In this study we investigated the role of the costimulatory molecules CD28 and LFA‐1 in this process in the setting of TCR‐induced proliferation of primary T lymphocytes in vitro. Co‐stimulation with ICAM‐1 and B7.2 led to strong and CsA‐resistant proliferation, which was found to be largely IL‐2 dependent. All of the known calcineurin‐dependent events, such as induction of NF‐AT and NF‐κB or stress‐activated protein kinase activation, were markedly modulated by CsA independently of costimulation. In contrast, both ICAM‐1 and B7.2 enhanced the half‐life of the inducible IL‐2 transcript in a CsA‐resistant manner. LFA‐1‐ but not CD28‐induced IL‐2 mRNA stabilization required the integrity of the actin‐based cytoskeleton, suggesting that the two costimulatory molecules impact on qualitatively different signaling pathways. This is further suggested by the demonstration that LFA‐1 and CD28 acted synergistically to confer CsA resistance in a model of co‐stimulation using superantigen‐pulsed dendritic cells. We propose that IL‐2 transcript accumulation and subsequent T cell proliferation at the low transcriptional rate imposed by CsA are the result of co‐stimulation‐dependent stabilization of IL‐2 mRNA.

Use of MHC class II tetramers to investigate CD4+ T cell responses: Problems and solutions

MHC‐class I tetramers technology enabled the characterization of peptide‐specific T cells at the single cell level in a variety of studies. Several laboratories have also developed MHC‐class II multimers to characterize Ag‐specific CD4+ T cells. However, the generation and use of MHC‐class II multimers seems more problematic than that of MHC‐I multimers. We have generated HLA‐DR*1101 tetramers in a versatile empty form, which can be loaded after purification with peptides of interest. We discuss the impact of critical biological and structural parameters for the optimal staining of Ag‐specific CD4+ T cells using HLA‐DR*1101 tetramers, such as: (i) activation state of CD4+ T cells; (ii) membrane trafficking in the target CD4+ T cells; (iii) binding characteristics of the loaded CD4 epitope. Our data indicate that reorganization of TCR on the plasma membrane upon CD4+ T cell activation, as well as an homogenous binding frame of the CD4 epitopes to the soluble HLA‐DR monomer, are critical for a stable TCR/MHC‐class II tetramer interaction. These factors, together with the low frequencies and affinities of specific CD4+ T cells, explain the need for in vitro expansion or ex vivo enrichment of specific T cells for the optimal visualization with MHC‐class II tetramers.

Third trimester amniotic fluid cells with the capacity to develop neural phenotypes and with heterogeneity among sub-populations

PURPOSE Our aim was the search for new sources of cells potentially useful for central nervous system regenerative medicine. Extra-embryonic tissues are promising sources of pluripotent stem cells. Among these, human second-trimester amniotic fluid (AF) contains cell populations exhibiting self-renewal capacity, multipotency and the expression of embryonic cell markers. METHODS Here we report the properties of the easily available third-trimester AF cells (AFCs). Different cell types from 6 of 9 AF samples were separated, expanded, and characterized by assessing their morphological, proliferative, and differentiative properties. RESULTS All isolated cultures presented CD105, CD90 and CD73 mesenchymal markers, whereas they differed among themselves in CD117, CD146, CD31, NG2 and CD133 expression. Their doubling time and telomere length were conserved throughout many passages. Importantly, immunofluorescence and Real-time PCR showed that, during their proliferative state and differentiation, several cultures expressed neuronal and glial markers such as nestin, GFAP, β-tubulin III and neurofilament H indicating their potential attitude towards a neural fate. Indeed, these cells showed a rather poor capacity to differentiate in adipogenic and osteogenic lineages. CONCLUSIONS In this work we report that cells with neural differentiation capability can be isolated from third-trimester AF, such properties could be useful for neuro-regenerative purposes.

IL-21 Is a Central Memory T Cell–Associated Cytokine That Inhibits the Generation of Pathogenic Th1/17 Effector Cells

IL-21 promotes Th17 differentiation, and Th17 cells that upregulate T-bet, IFN-γ, and GM-CSF drive experimental autoimmune diseases in mice. Anti–IL-21 treatment of autoimmune patients is therefore a therapeutic option, but the role of IL-21 in human T cell differentiation is incompletely understood. IL-21 was produced at high levels by human CD4+ central memory T cells, suggesting that it is associated with early T cell differentiation. Consistently, it was inhibited by forced expression of T-bet or RORC2, the lineage-defining transcription factors of Th1 and Th17 effector cells, respectively. Although IL-21 was efficiently induced by IL-12 in naive CD4+ T cells, it inhibited the generation of Th1 effector cells in a negative feedback loop. IL-21 was also induced by IL-6 and promoted Th17 differentiation, but it was not absolutely required. Importantly, however, IL-21 promoted IL-10 secretion but inhibited IFN-γ and GM-CSF production in developing Th17 cells, and consequently prevented the generation of polyfunctional Th1/17 effector cells. Moreover, in Th17 memory cells, IL-21 selectively inhibited T-bet upregulation and GM-CSF production. In summary, IL-21 is a central memory T cell–associated cytokine that promotes Th17 differentiation and IL-10 production, but inhibits the generation of potentially pathogenic Th1/17 effector cells. These findings shed new light on the role of IL-21 in T cell differentiation, and have relevant implications for anti–IL-21 therapy of autoimmune diseases.

Vaccination with Mouse Mammary Adenocarcinoma Cells Coexpressing B7-1 (CD80) and B7-2 (CD86) Discloses the Dominant Effect of B7-1 in the Induction of Antitumor Immunity

Nonreplicating TS/A mammary adenocarcinoma cells expressing B7-2 (CD86) (TS/A-2) are more immunogenic than those expressing B7-1 (CD80) (TS/A-1), indicating that B7-1 and B7-2 display nonredundant costimulatory effects in inducing antitumor responses. Whereas transfection of B7-2 cDNA into TS/A-1 cells does not improve their immunogenicity, transfection of B7-1 cDNA into TS/A-2 cells (TS/A-2/1) decreases their immunogenicity in a manner that is directly related to the surface levels of B7-1. Ab blocking of B7-1 on TS/A-2/1 cells before their injection in vivo restores the higher immunogenicity characteristic of single B7-2 transfectants, indicating therefore that B7-1 actively modulates the B7-2-dependent costimulation. The expression of B7-1 also modifies quantitatively the balance of endogenous IFN-γ and IL-4 induced in vivo by TS/A-2 vaccines. In fact, we find that vaccination with TS/A-2/1 cells results in the production of more IFN-γ and less IL-4 than TS/A-2 vaccines, a pattern comparable to that induced by TS/A-1 cells. Thus, in the TS/A model of antitumor response, B7-1 modulates B7-2-dependent costimulatory effects in a dominant, noncompetitive way.