In vitro characterization of immune-related properties of human fetal bone cells for potential tissue engineering applications.

We describe herein some immunological properties of human fetal bone cells recently tested for bone tissue-engineering applications. Adult mesenchymal stem cells (MSCs) and osteoblasts were included in the study for comparison. Surface markers involved in bone metabolism and immune recognition were analyzed using flow cytometry before and after differentiation or treatment with cytokines. Immunomodulatory properties were studied on activated peripheral blood mononuclear cells (PBMCs). The immuno-profile of fetal bone cells was further investigated at the gene expression level. Fetal bone cells and adult MSCs were positive for Stro-1, alkaline phosphatase, CD10, CD44, CD54, and beta2-microglobulin, but human leukocyte antigen (HLA)-I and CD80 were less present than on adult osteoblasts. All cells were negative for HLA-II. Treatment with recombinant human interferon gamma increased the presence of HLA-I in adult cells much more than in fetal cells. In the presence of activated PBMCs, fetal cells had antiproliferative effects, although with patterns not always comparable with those of adult MSCs and osteoblasts. Because of the immunological profile, and with their more-differentiated phenotype than of stem cells, fetal bone cells present an interesting potential for allogeneic cell source in tissue-engineering applications.

Adjuvants that improve the ratio of antigen-specific effector to regulatory T cells enhance tumor immunity.

Antitumor immunity is strongly influenced by the balance of tumor antigen-specific effector and regulatory T cells. However, the impact that vaccine adjuvants have in regulating the balance of antigen-specific T cell populations is not well understood. We found that antigen-specific T regulatory cells (Treg) were induced following subcutaneous vaccination with either OVA or melanoma-derived peptides, with a restricted expansion of effector T cells. Addition of the adjuvants CpG-ODN or Poly(I:C) preferentially amplified effector T cells over Tregs, dramatically increasing the antigen-specific T effector:Treg ratios and inducing polyfunctional effector cells. In contrast, two other adjuvants, imiquimod and Quil A saponin, favored an expansion of antigen-specific Tregs and failed to increase effector T cell:Treg ratios. Following therapeutic vaccination of tumor-bearing mice, high ratios of tumor-specific effector T cells:Tregs in draining lymph nodes were associated with enhanced CD8+ T cell infiltration at the tumor site and a durable rejection of tumors. Vaccine formulations of peptide+CpG-ODN or Poly(I:C) induced selective production of pro-inflammatory Type I cytokines early after vaccination. This environment promoted CD8+ and CD4+ effector T cell expansion over that of antigen-specific Tregs, tipping the effector T cell to Treg balance to favor effector cells. Our findings advance understanding of the influence of different adjuvants on T cell populations, facilitating the rational design of more effective cancer vaccines.

Extrafollicular plasmablast B cells play a key role in carrying retroviral infection to peripheral organs.

B cells can either differentiate in germinal centers or in extrafollicular compartments of secondary lymphoid organs. Here we show the migration properties of B cells after differentiation in murine peripheral lymph node infected with mouse mammary tumor virus. Naive B cells become activated, infected, and carry integrated retroviral DNA sequences. After production of a retroviral superantigen, the infected B cells receive cognate T cell help and differentiate along the two main differentiation pathways analogous to classical Ag responses. The extrafollicular differentiation peaks on day 6 of mouse mammary tumor virus infection, and the follicular one becomes detectable after day 10. B cells participating in this immune response carry a retroviral DNA marker that can be detected by using semiquantitative PCR. We determined the migration patterns of B cells having taken part in the T cell-B cell interaction from the draining lymph node to different tissues. Waves of immigration and retention of infected cells in secondary lymphoid organs, mammary gland, salivary gland, skin, lung, and liver were observed correlating with the two peaks of B cell differentiation in the draining lymph node. Other organs revealed immigration of infected cells at later time points. The migration properties were correlated with a strong up-regulation of alpha(4)beta(1) integrin expression. These results show the migration properties of B cells during an immune response and demonstrate that a large proportion of extrafolliculary differentiating plasmablasts can escape local cell death and carry the retroviral infection to peripheral organs.

A novel population of human melanoma-specific CD8 T cells recognizes Melan-AMART-1 immunodominant nonapeptide but not the corresponding decapeptide.

HLA-A2-restricted cytolytic T cells specific for the immunodominant human tumor Ag Melan-A(MART-1) can kill most HLA-matched melanoma cells, through recognition of two naturally occurring antigenic variants, i.e., Melan-A nonamer AAGIGILTV and decamer EAAGIGILTV peptides. Several previous studies have suggested a high degree of TCR cross-reactivity to the two peptides. In this study, we describe for the first time that some T cell clones are exclusively nonamer specific, because they are not labeled by A2/decamer-tetramers and do not recognize the decamer when presented endogenously. Functional assays with peptides gave misleading results, possibly because decamers were cleaved by exopeptidases. Interestingly, nonapeptide-specific T cell clones were rarely Valpha2.1 positive (only 1 of 19 clones), in contrast to the known strong bias for Valpha2.1-positive TCRs found in decamer-specific clones (59 of 69 clones). Molecular modeling revealed that nonapeptide-specific TCRs formed unfavorable interactions with the decapeptide, whereas decapeptide-specific TCRs productively created a hydrogen bond between CDR1alpha and glutamic acid (E) of the decapeptide. Ex vivo analysis of T cells from melanoma metastases demonstrated that both nonamer and decamer-specific T cells were enriched to substantial frequencies in vivo, and representative clones showed efficient tumor cell recognition and killing. We conclude that the two peptides should be regarded as distinct epitopes when analyzing tumor immunity and developing immunotherapy against melanoma.

The parallel lives of angiogenesis and immunosuppression: cancer and other tales.

Emerging evidence indicates that angiogenesis and immunosuppression frequently occur simultaneously in response to diverse stimuli. Here, we describe a fundamental biological programme that involves the activation of both angiogenesis and immunosuppressive responses, often through the same cell types or soluble factors. We suggest that the initiation of these responses is part of a physiological and homeostatic tissue repair programme, which can be co-opted in pathological states, notably by tumours. This view can help to devise new cancer therapies and may have implications for aseptic tissue injury, pathogen-mediated tissue destruction, chronic inflammation and even reproduction.

Tumor-host interactions Part 1: Stromal signatures of breast and prostate cancer Part 2: GLG1 and the control of the secretory pathway in tumor cells

Tumor-host interaction is a key determinant during cancer progression, from primary tumor growth to metastatic dissemination. At each step, tumor cells have to adapt to and subvert different types of microenvironment, leading to major phenotypic and genotypic alterations that affect both tumor and surrounding stromal compartments. Understanding the molecular mechanisms that govern tumor-host interplay may be essential for better comprehension of tumorigenesis in an effort to improve current anti-cancer therapies. The present work is composed of two projects that address tumor-host interactions from two different perspectives, the first focusing on the characterization of tumor-associated stroma and the second on membrane trafficking in tumor cells. Part 1. To selectively address stromal gene expression changes during cancer progression, oligonucleotide-based Affymetrix microarray technology was used to analyze the transcriptomes of laser-microdissected stromal cells derived from invasive human breast and prostate carcinoma. Comparison showed that invasive breast and prostate cancer elicit distinct, tumor-specific stromal responses, with a limited panel of shared induced and/or repressed genes. Both breast and prostate tumor-specific deregulated stromal gene sets displayed statistically significant survival-predictive ability for their respective tumor type. By contrast, a stromal gene signature common to both tumor types did not display prognostic value, although expression of two individual genes within this common signature was found to be associated with patient survival. Part 2. GLG1 is known as an E-selectin ligand and an intracellular FGF receptor, depending on cell type and context. Immunohistochemical and immunofluorescence analyses showed that GLG1 is primarily localized in the Golgi of human tumor cells, a central location in the biosynthetic/secretory pathways. GLG1 has been shown to interact with and to recruit the ARF GEF BIGI to the Golgi membrane. Depletion of GLG1 or BIGI markedly reduced ARF3 membrane localization and activation, and altered the Golgi structure. Interestingly, these perturbations did not impair constitutive secretion in general, but rather seemed to impair secretion of a specific subset of proteins that includes MMP-9. Thus, GLG1 coordinates ARF3 activation by recruiting BIGI to the Golgi membrane, thereby affecting secretion of specific molecules. - Les interactions tumeur-hôte constituent un élément essentiel à la progression tumorale, de la croissance de la tumeur primaire à la dissémination des métastases. A chaque étape, les cellules tumorales doivent s'adapter à différents types de microenvironnement et les détourner à leur propre avantage, donnant lieu à des altérations phénotypiques et génotypiques majeures qui affectent aussi bien la tumeur elle-même que le compartiment stromal environnant. L'étude des mécanismes moléculaires qui régissent les interactions tumeur-hôte constitue une étape essentielle pour une meilleure compréhension du processus de tumorigenèse dans le but d'améliorer les thérapies anti cancer existantes. Le travail présenté ici est composé de deux projets qui abordent la problématique des interactions tumeur-hôte selon différentes perspectives, le premier se concentrant sur la caractérisation du stroma tumoral et le second sur le trafic intracellulaire des cellules tumorales. Partie 1. Pour examiner les changements d'expression des gènes dans le stroma en réponse à la progression du cancer, des puces à ADN Affymetrix ont été utilisées afin d'analyser les transcriptomes des cellules stromales issues de carcinomes invasifs du sein et de la prostate et collectées par microdissection au laser. L'analyse comparative a montré que les cancers invasifs du sein et de la prostate provoquent des réponses stromales spécifiques à chaque type de tumeur, et présentent peu de gènes induits ou réprimés de façon similaire. L'ensemble des gènes dérégulés dans le stroma associé au cancer du sein, ou à celui de la prostate, présente une valeur pronostique pour les patients atteints d'un cancer du sein, respectivement de la prostate. En revanche, la signature stromale commune aux deux types de cancer n'a aucune valeur prédictive, malgré le fait que l'expression de deux gènes présents dans cette liste soit liée à la survie des patients. Partie 2. GLG1 est connu comme un ligand des sélectines E ainsi que comme récepteur intracellulaire pour des facteurs de croissances FGFs selon le type de cellule dans lequel il est exprimé. Des analyses immunohistochimiques et d'immunofluorescence ont montré que dans les cellules tumorales, GLG1 est principalement localisé au niveau de l'appareil de Golgi, une place centrale dans la voie biosynthétique et sécrétoire. Nous avons montré que GLG1 interagit avec la protéine BIGI et participe à son recrutement à la membrane du Golgi. L'absence de GLG1 ou de BIGI réduit drastiquement le pool d'ARF3 associé aux membranes ainsi que la quantité d'ARF3 activés, et modifie la structure de l'appareil de Golgi. Il est particulièrement intéressant de constater que ces perturbations n'ont pas d'effet sur la sécrétion constitutive en général, mais semblent plutôt affecter la sécrétion spécifique d'un sous-groupe défini de protéines comprenant MMP-9. GLG1 coordonne donc l'activation de ARF3 en recrutant BIGI à la membrane du Golgi, agissant par ce moyen sur la sécrétion de molécules spécifiques.

Receptor activator for NF-κB ligand in acute myeloid leukemia: expression, function, and modulation of NK cell immunosurveillance.

The TNF family member receptor activator for NF-κB ligand (RANKL) and its receptors RANK and osteoprotegerin are key regulators of bone remodeling but also influence cellular functions of tumor and immune effector cells. In this work, we studied the involvement of RANK-RANKL interaction in NK cell-mediated immunosurveillance of acute myeloid leukemia (AML). Substantial levels of RANKL were found to be expressed on leukemia cells in 53 of 78 (68%) investigated patients. Signaling via RANKL into the leukemia cells stimulated their metabolic activity and induced the release of cytokines involved in AML pathophysiology. In addition, the immunomodulatory factors released by AML cells upon RANKL signaling impaired the anti-leukemia reactivity of NK cells and induced RANK expression, and NK cells of AML patients displayed significantly upregulated RANK expression compared with healthy controls. Treatment of AML cells with the clinically available RANKL Ab Denosumab resulted in enhanced NK cell anti-leukemia reactivity. This was due to both blockade of the release of NK-inhibitory factors by AML cells and prevention of RANK signaling into NK cells. The latter was found to directly impair NK anti-leukemia reactivity with a more pronounced effect on IFN-γ production compared with cytotoxicity. Together, our data unravel a previously unknown function of the RANK-RANKL molecule system in AML pathophysiology as well as NK cell function and suggest that neutralization of RANKL with therapeutic Abs may serve to reinforce NK cell reactivity in leukemia patients.

PD-1 and Tim-3 regulate the expansion of tumor antigen-specific CD8⁺ T cells induced by melanoma vaccines.

Although melanoma vaccines stimulate tumor antigen-specific CD8(+) T cells, objective clinical responses are rarely observed. To investigate this discrepancy, we evaluated the character of vaccine-induced CD8(+) T cells with regard to the inhibitory T-cell coreceptors PD-1 and Tim-3 in patients with metastatic melanoma who were administered tumor vaccines. The vaccines included incomplete Freund's adjuvant, CpG oligodeoxynucleotide (CpG), and the HLA-A2-restricted analog peptide NY-ESO-1 157-165V, either by itself or in combination with the pan-DR epitope NY-ESO-1 119-143. Both vaccines stimulated rapid tumor antigen-specific CD8(+) T-cell responses detected ex vivo, however, tumor antigen-specific CD8(+) T cells produced more IFN-γ and exhibited higher lytic function upon immunization with MHC class I and class II epitopes. Notably, the vast majority of vaccine-induced CD8(+) T cells upregulated PD-1 and a minority also upregulated Tim-3. Levels of PD-1 and Tim-3 expression by vaccine-induced CD8(+) T cells at the time of vaccine administration correlated inversely with their expansion in vivo. Dual blockade of PD-1 and Tim-3 enhanced the expansion and cytokine production of vaccine-induced CD8(+) T cells in vitro. Collectively, our findings support the use of PD-1 and Tim-3 blockades with cancer vaccines to stimulate potent antitumor T-cell responses and increase the likelihood of clinical responses in patients with advanced melanoma.

Photodynamic therapy enhances lipodoxorubcin distribution in sarcoma lung metastasis by lowering tumor interstitial fluid pressure in a rodent model

Objective: The management of sarcoma metastasis by systemic chemotherapy is often unsatisfactory. This has paradoxally been attributed to the leakiness of tumor neovessels which induce high intratumor interstitial fluid pressure (IFP) and limit convection forces that are important for drug distribution. In a rodent model, we have recently shown that photodynamic (PDT) pre treatment of lung metastasis could enhance their uptake of chemotherapy. We hypothesized that PDT transiently decreases tumor IFP which enhances convection and promotes drug distribution.Methods: Sarcoma tumors were generated sub-pleurally in the lungs of 12 rats. Animals were randomized at 10 days into i. no pre-treatment (control) and ii. low dose PDT pre-treatment (0・0625 mg/kg Visudyne, 10J/cm2 and 35 mW/cm2) followed by intravenous Liposomal doxorubicin (LiporubicinTM) administration. Using the wick-in-needle technique, we determined tumor and normal tissue IFP before, during and after PDT. In parallel, the uptake of LiporubicinTM was determined by high performance liquid chromatography in tumor and lung tissues.Results: Tumor IFP was significantly higher than normal tissue IFP in all animals. PDT pre-treatment did not affect normal tissue IFP but caused a significant decrease in tumor IFP (mean decrease by 2+/− 1mmHg) which lasted an average of 30 minutes before reaching baseline values. Tumor but not normal lung tissue LiporubicinTM uptake was significantly increased by 67% with PDT pre-treatment when liporubicin was allowed to circulate for one hour.Conclusion: Photodynamic therapy pre-treatment enhances LiporubicinTM uptake in sarcoma lung metastasis by transiently decreasing tumor IFP. These PDT conditions seem to specifically modulate tumor neovessels but not normal lung vessels.

Induction of human papillomavirus oncogene-specific CD8 T-cell effector responses in the genital mucosa of vaccinated mice.

Cervical cancer, the second leading cause of cancer mortality in women worldwide, results from infection with a subset of human papillomaviruses (HPV), HPV-16 being the most prevalent type. The available prophylactic vaccines are an effective strategy to prevent this cancer in the long term. However, they only target 70-80% of all cervical cancers and cannot control existing HPV infections and associated lesions. Therapeutic vaccines are thus necessary for women who cannot benefit from prophylactic vaccination. Induction of protective immune responses in the genital mucosa (GM) may be crucial for efficacy of HPV therapeutic vaccines. We report here that mice that received a single subcutaneous (s.c.) vaccination of an adjuvanted long synthetic HPV16 E7(1-98) polypeptide showed induction of 100% tumor protection against s.c. TC-1 tumors and that tumor regression was mainly provided by CD8 T cells. In vivo cytotoxic assay revealed high E7-specific cytolytic T lymphocytes activity in spleen and in genital draining lymph nodes (LN), and E7-specific CD8 T cells could be detected in GM by tetramer staining. More importantly, high-avidity E7-specific INF-gamma secreting CD8 T cells were induced not only in blood, spleen and LN but also in GM of vaccinated mice, thus providing evidence that a parenteral vaccination may be sufficient to provide regression of genital tumors. In addition, there was no correlation between the responses measured in blood with those measured in GM, highlighting the necessity and relevance to determine the immune responses in the mucosa where HPV-tumors reside.

Current state of vaccine therapies in non-small-cell lung cancer.

A large variety of cancer vaccines have undergone extensive testing in early-phase clinical trials. A limited number have also been tested in randomized phase II clinical trials. Encouraging trends toward increased survival in the vaccine arms have been recently observed for 2 vaccine candidates in patients with non-small-cell lung cancer. These have provided the impetus for the initiation of phase III trials in large groups of patients with lung cancer. These vaccines target 2 antigens widely expressed in lung carcinomas: melanoma-associated antigen 3, a cancer testis antigen; and mucin 1, an antigen overexpressed in a largely deglycosylated form in advanced tumors. Therapeutic cancer vaccines aim at inducing strong CD8 and CD4 T-cell responses. The majority of vaccines recently tested in phase I clinical trials show efficacy in terms of induction of specific tumor antigen immunity. However, clinical efficacy remains to be determined but appears limited. Efforts are thus aimed at understanding the basis for this apparent lack of effect on tumors. Two major factors are involved. On one hand, current vaccines are suboptimal. Strong adjuvant agents and appropriate tumor antigens are needed. Moreover, dose, route, and schedule also need optimization. On the other hand, it is now clear that large tumors often present a tolerogenic microenvironment that hampers effective antitumor immunity. The partial understanding of the molecular pathways leading to functional inactivation of T cells at tumor sites has provided new targets for intervention. In this regard, blockade of cytotoxic T-lymphocyte antigen-4 and programmed death-1 with humanized monoclonal antibodies has reached the clinical testing stage. In the future, more potent cancer vaccines will benefit from intense research in antigen discovery and adjuvant agents. Furthermore, it is likely that vaccines need to be combined with compounds that reverse major tolerogenic pathways that are constitutively active at the tumor site. Developing these combined approaches to vaccination in cancer promises new, exciting findings and, at the same time, poses important challenges to academic research institutions and the pharmaceutical industry.

Phenotyping of 60 cultured human gliomas and 34 other neuroectodermal tumors by means of monoclonal antibodies against glioma, melanoma and HLA-DR antigens.

The reactivity spectrum of three monoclonal antibodies (Mabs) to human malignant glioma, five Mabs to melanomas and one Mab anti-HLA-DR was investigated by an indirect antibody binding radioimmunoassay on a panel of cells derived from 60 glioma lines, including 47 malignant astrocytomas, 11 low-grade astrocytomas and two malignant ependymomas as well on cells from 12 melanoma, three neuroblastoma, three medulloblastoma, two schwannoma, two retinoblastoma, two choroïd plexus papilloma, ten meningioma and 12 unrelated tumor lines. The anti-glioma Mabs BF7 and GE2 reacted preferentially with gliomas, while the anti-glioma Mab CG12 reacted with gliomas, melanomas, neuroblastomas and medulloblastomas. The five anti-melanoma Mabs reacted with gliomas, neuroblastomas and medulloblastomas. The anti-HLA-DR Mab D1-12 reacted with gliomas, melanomas and some meningiomas. On the basis of the data presented, we describe three different antigenic systems; the first one is glioma-associated, the second one is related to differentiation antigens expressed on cells derived from the neuroectoderm and the third is represented by HLA-DR antigens which are expressed not only on B-lymphoblastoid cells but also on melanomas and gliomas.

CD8beta knockout mice mount normal anti-viral CD8+ T cell responses--but why?

It has been shown previously that CD8beta in vitro increases the range and the sensitivity of antigen recognition and in vivo plays an important role in the thymic selection of CD8+ T cells. Consistent with this, we report here that CD8+ T cells from CD8beta knockout (KO) P14 TCR transgenic mice proliferate inefficiently in vitro. In contrast to these findings, we also show that CD8beta KO mice mount normal CD8 primary, secondary and memory responses to acute infection with lymphocytic choriomeningitis virus. Tetramer staining and cytotoxic experiments revealed a predominance of CD8-independent CTL in CD8beta KO mice. The TCR repertoire, especially the one of the TCRalpha chain, was different in CD8beta KO mice as compared with B6 mice. Our results indicate that in the absence of CD8beta, CD8-independent TCRs are preferentially selected, which in vivo effectively compensates for the reduced co-receptor function of CD8alphaalpha.

Hypoxia-inducible miR-210 regulates the susceptibility of tumor cells to lysis by cytotoxic T cells.

Hypoxia in the tumor microenvironment plays a central role in the evolution of immune escape mechanisms by tumor cells. In this study, we report the definition of miR-210 as a miRNA regulated by hypoxia in lung cancer and melanoma, documenting its involvement in blunting the susceptibility of tumor cells to lysis by antigen-specific cytotoxic T lymphocytes (CTL). miR-210 was induced in hypoxic zones of human tumor tissues. Its attenuation in hypoxic cells significantly restored susceptibility to autologous CTL-mediated lysis, independent of tumor cell recognition and CTL reactivity. A comprehensive approach using transcriptome analysis, argonaute protein immunoprecipitation, and luciferase reporter assay revealed that the genes PTPN1, HOXA1, and TP53I11 were miR-210 target genes regulated in hypoxic cells. In support of their primary importance in mediating the immunosuppressive effects of miR-210, coordinate silencing of PTPN1, HOXA1, and TP53I11 dramatically decreased tumor cell susceptibility to CTL-mediated lysis. Our findings show how miR-210 induction links hypoxia to immune escape from CTL-mediated lysis, by providing a mechanistic understanding of how this miRNA mediates immunosuppression in oxygen-deprived regions of tumors where cancer stem-like cells and metastatic cellular behaviors are known to evolve.

Single cell analysis reveals similar functional competence of dominant and non-dominant CD8 T-cell clonotypes

The properties of CD8 T-cells requiredfor protection from infectiousdisease and cancer are only partiallycharacterized, and only limited data isavailable regarding T-cell clonotypes.It has been proposed that dominantT-cell clonotypes may have higherprotective potential than their nondominantcounterparts. Our objectiveswere to assess memory andeffector functions, stage of differentiationand clonotype selection of tumor-reactive T lymphocytes followingpeptide vaccination in melanomapatients.We also characterized dominantversus non-dominant clonotypesto further understand the in vivo functionof these T-cells based on theirprevalence. Using a novel single-cellapproach for simultaneous ex vivomolecular and functional analysis, wereport the preferential selection andexpansion of several tumor-specificco-dominant clonotypes of intermediateto high frequencies, irrespectiveof whether native or analog peptidewas used for vaccination. Theseclonotypes made up 40 - 95% of thedifferentiated "effector-like" T-cells,but only 25% of the less-differentiated"effector-memory" cells. Bothsubsets also contained non-dominantT-cell clonotypes, but these were significantlymore frequent in the lessdifferentiatedcells. Thus, cell differentiationwas clonotype-dependent.Surprisingly however, the acquisitionof memory and effector T-cell propertieswas clonotype independent, as wefound similar functional profiles indominant and low/ non-dominantT-cell clonotypes. In contrast to analogpeptide vaccination, native peptidevaccination induced T-cell functionsthat were more comprehensive,with more pronounced effector functionscombined with memory cellproperties. In summary, this study revealsthat T-cell functions are determinedprimarily by the antigen andthe stage of T-cell differentiation, butare similar in dominant and non-dominantclonotypes participating in aCD8 T-cell response. The identifiedclonotypic basis of T-cell responsescontributes to the rational developmentof vaccines.