[Show abstract][Hide abstract] ABSTRACT: Tumors with reduced expression of MHC class I (MHC-I) molecules may be unrecognized by tumor antigen-specific CD8(+) T cells and thus constitute a challenge for cancer immunotherapy. Here we monitored development of autochthonous melanomas in TiRP mice that develop tumors expressing a known tumor antigen as well as a red fluorescent protein (RFP) reporter knock in gene. The latter permits non-invasive monitoring of tumor growth by biofluorescence. One developing melanoma was deficient in cell surface expression of MHC-I, but MHC-I expression could be rescued by exposure of these cells to IFNγ. We show that CD8(+) T cells specific for tumor antigen/MHC-I were efficient at inducing regression of the MHC-I-deficient melanoma, provided that the T cells were endowed with properties permitting their migration into the tumor and their efficient production of IFNγ. This was the case for CD8(+) T cells transfected to express an active form of STAT5 (STAT5CA). The amount of IFNγ produced ex vivo from T cells present in tumors after adoptive transfer of the CD8(+) T cells was correlated with an increase in surface expression of MHC-I molecules by the tumor cells. We also show that these CD8(+) T cells expressed PD-1 and upregulated its ligand PDL-1 on melanoma cells within the tumor. Despite upregulation of this immunosuppressive pathway, efficient IFNγ production in the melanoma microenvironment was found associated with resistance of STAT5CA-expressing CD8(+) T cells to inhibition both by PD-1/PDL-1 engagement and by TGFβ1, two main immune regulatory mechanisms hampering the efficiency of immunotherapy in patients.
[Show abstract][Hide abstract] ABSTRACT: Bacterial cyclic glucans are glucose polymers that concentrate within the periplasm of alpha-proteobacteria. These molecules are necessary to maintain the homeostasis of the cell envelope by contributing to the osmolarity of Gram negative bacteria. Here, we demonstrate that Brucella β 1,2 cyclic glucans are potent activators of human and mouse dendritic cells. Dendritic cells activation by Brucella β 1,2 cyclic glucans requires TLR4, MyD88 and TRIF, but not CD14. The Brucella cyclic glucans showed neither toxicity nor immunogenicity compared to LPS and triggered antigen-specific CD8(+) T cell responses in vivo. These cyclic glucans also enhanced antigen-specific CD4(+) and CD8(+) T cell responses including cross-presentation by different human DC subsets. Brucella β 1,2 cyclic glucans increased the memory CD4(+) T cell responses of blood mononuclear cells exposed to recombinant fusion proteins composed of anti-CD40 antibody and antigens from both hepatitis C virus and Mycobacterium tuberculosis. Thus cyclic glucans represent a new class of adjuvants, which might contribute to the development of effective antimicrobial therapies.
[Show abstract][Hide abstract] ABSTRACT: Immunotherapy based on adoptive transfer of tumor antigen-specific CD8(+) T cell (TC) is generally limited by poor in vivo expansion and tumor infiltration. In this study, we report that activated STAT5 transcription factors (STAT5CA) confer high efficiency on CD8(+) effector T cells (eTC) for host colonization after adoptive transfer. Engineered expression of STAT5CA in antigen-experienced TCs with poor replicative potential was also sufficient to convert them into long-lived antigen-responsive eTCs. In transplanted mastocytoma- or melanoma-bearing hosts, STAT5CA greatly enhanced the ability of eTCs to accumulate in tumors, become activated by tumor antigens, and to express the cytolytic factor granzyme B. Taken together, these properties contributed to an increase in tumor regression by STAT5CA-transduced, as compared with untransduced, TCs including when the latter control cells were combined with infusion of interleukin (IL)-2/anti-IL-2 complexes. In tumors arising in the autochthonous TiRP transgenic model of melanoma associated with systemic chronic inflammation, endogenous CD8(+) TCs were nonfunctional. In this setting, adoptive transfer of STAT5CA-transduced TCs produced superior antitumor effects compared with nontransduced TCs. Our findings imply that STAT5CA expression can render TCs resistant to the immunosuppressive environment of melanoma tumors, enhancing their ability to home to tumors and to maintain high granzyme B expression, as well as their capacity to stimulate granzyme B expression in endogenous TCs.
Cancer Research 11/2011; 72(1):76-87. DOI:10.1158/0008-5472.CAN-11-2187 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Migration of dendritic cells (DC) from the tumor environment to the T cell cortex in tumor-draining lymph nodes (TDLN) is essential for priming naïve T lymphocytes (TL) to tumor antigen (Ag). We used a mouse model of induced melanoma in which similar oncogenic events generate two phenotypically distinct melanomas to study the influence of tumor-associated inflammation on secondary lymphoid organ (SLO) organization. One tumor promotes inflammatory cytokines, leading to mobilization of immature myeloid cells (iMC) to the tumor and SLO; the other does not.
We report that inflammatory tumors induced alterations of the stromal cell network of SLO, profoundly altering the distribution of TL and the capacity of skin-derived DC and TL to migrate or home to TDLN. These defects, which did not require tumor invasion, correlated with loss of fibroblastic reticular cells in T cell zones and in impaired production of CCL21. Infiltrating iMC accumulated in the TDLN medulla and the splenic red pulp.
We propose that impaired function of the stromal cell network during chronic inflammation induced by some tumors renders spleens non-receptive to TL and TDLN non-receptive to TL and migratory DC, while the entry of iMC into these perturbed SLO is enhanced. This could constitute a mechanism by which inflammatory tumors escape immune control.
If our results apply to inflammatory tumors in general, the demonstration that SLO are poorly receptive to CCR7-dependent migration of skin-derived DC and naïve TL may constitute an obstacle for proposed vaccination or adoptive TL therapies of their hosts.
PLoS ONE 07/2011; 6(7):e22639. DOI:10.1371/journal.pone.0022639 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract Experiments performed in our laboratory or in collaboration with other groups demonstrating the delivery to cells of mono-, oligo- and polynucleotides from antibody-targeted small liposomes are reviewed. Biologically-active molecules delivered into cells via these liposomes include: phosphorylated derivatives of dideoxyuridine, which have activity against the human immunodeficiency virus; the oligonucleotide (2'-5') (A)n and various analogues, which have anti-viral and antiproliferative activity; and antisense phosphodiester and phosphorothioate oligodeoxynucleotides, which may have both gene-specific and nonspecific effects. Polynucleotides include: the RNA duplex poly (rI:rC), and related molecules, which are inducers of interferon and other cytokines; long RNA antisense molecules and plasmids. Advantages for delivery by liposomes, as compared to use of the same molecules free in solution include: protection against degradation, reduction of toxicity, improved pharmacokinetics and the possibility of increased intracellular transport. Numerous parameters are important in determining if and to what extent liposome contents are delivered to their sites of action, including: liposome size, lipid composition, nature of the encapsulated molecule, type of ligand used for targeting and its linkage to the liposome, and the cell type and target molecule. The precise mechanism(s) whereby oligo- and polynucleotides are able to enter into the cytoplasm from endocytic vesicles, and the efficiency of this process are not well understood.
Journal of Liposome Research 09/2008; 4(1):107-119. DOI:10.3109/08982109409037032 · 1.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Viral and non-viral vectors have been developed for gene therapy, but their use is associated with unresolved problems of efficacy and safety. Efficient and safe methods of DNA delivery need to be found for medical application. Here we report a new monopolar system of non-viral electro-gene transfer into the thymus in vivo that consists of the local application of electrical pulses after the introduction of the DNA. We assessed the proof of concept of this approach by correcting ZAP-70 deficient severe combined immunodeficiency (SCID) in mice. The thymic electro-gene transfer of the pCMV-ZAP-70-IRES-EGFP vector in these mice resulted in rapid T cell differentiation in the thymus with mature lymphocytes detected by three weeks in secondary lymphoid organs. Moreover, this system resulted in the generation of long-term functional T lymphocytes. Peripheral reconstituted T cells displayed a diversified T cell receptor (TCR) repertoire, and were responsive to alloantigens in vivo. This process applied to the thymus could represent a simplified and effective alternative for gene therapy of T cell immunodeficiencies.
PLoS ONE 02/2008; 3(4):e2059. DOI:10.1371/journal.pone.0002059 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Innate immunity is considered to initiate adaptive antitumor responses. We demonstrate that monoclonal CD8 T lymphocytes reactive to tumor Ag P1A on P815 mastocytoma cells provide essential "help" to NK cells for rejection of P1A-deficient tumors. RAG-deficient mice have normal NK cells but do not reject either tumor. Reconstitution of these mice with P1A-specific T cells conferred resistance to both P1A-expressing and -deficient tumor cells provided they were present at the same site. Elimination of Ag-negative tumor variants required both activated T and NK cells. Gene expression profiling of NK cells infiltrating P1A-positive tumors in mice with specific CD8 T cells demonstrated an activated effector phenotype. However, CD8 T cell help to NK cells appeared ineffective for P1A-negative variants separated from the P1A-positive tumor. Local tumor Ag-specific T cell-NK cell collaboration results in the elimination of tumor cells whether they express or not the T cell tumor Ag epitope, thus containing the emergence of tumor escape variants before metastasis.
The Journal of Immunology 12/2007; 179(10):6651-62. DOI:10.4049/jimmunol.179.10.6651 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Skin-draining LN contain several phenotypically distinguishable DC populations, which may be immature or mature. Mature DC are generally considered to have lost the capacity to acquire and present newly encountered Ag. Using antibody-opsonized liposomes as Ag carriers, we show that mature DC purified from skin explants are able to efficiently capture liposomes, process Ag encapsulated within them and activate Ag-specific CD4(+) T cells. Explant DC from mice with Langerhans cells (LC) expressing the primate diphtheria toxin receptor that were exposed to diphtheria toxin in vivo presented Ag as well as explant DC from wild-type mice, indicating that LC are not required and dermal DC are probably responsible for this presentation. We further show that all DC subtypes from LN that capture opsonized Ag are capable of cross-presenting it to CD8(+) T cells. Induction of additional maturation in vivo by LPS or treatment with double-stranded RNA did not alter the Ag presentation capacity of the skin or LN DC subtypes. These results suggest that mature DC present in skin-draining LN may play an important role in the induction of primary and/or secondary immune responses against Ag delivered to the LN that they take up by receptor-mediated endocytosis.
European Journal of Immunology 05/2007; 37(5):1184-93. DOI:10.1002/eji.200636793 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Data presented in this publication are aimed at defining the role of the Ti/CD3, Thy-1, and Ly-6 structures of mouse CTL clones for two measurable activation-dependent effector functions: target-cell killing and production of g-interferon (g-IFN). The killing function is not dependent on protein synthesis and can be measured within minutes of CTL-target-cell interaction, as recently shown using digital imaging of changes in intracytoplasmic Ca++ concentration ([Ca++]i) in effector and target cells loaded with the Ca++ sensitive dye Fura-2. g-IFN synthesis is dependent on transcriptional activation and is optimally measured in supernatants around 20 h after CTL-target-cell interaction or after binding of anti-Ti mAb.
Annals of the New York Academy of Sciences 12/2006; 532(1):33 - 43. DOI:10.1111/j.1749-6632.1988.tb36323.x · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Insight into the mechanisms by which dendritic cells (DC) present exogenous antigen to T cells is of major importance in the design of vaccines. We examined the effectiveness of free antigen as well as antigen with lipopolysaccharide, emulsified in complete Freund's adjuvant, and antigen encapsulated in liposomes in activating adoptively transferred antigen-specific CD4 and CD8 T cells. When contained in liposomes, 100- to 1000-fold lower antigen amounts were as efficient in inducing proliferation and effector functions of CD4 and CD8 T cells in draining lymph nodes as other antigen forms. CD11c(+)/CD11b(+)/CD205(mod)/CD8alpha(-) DC that captured liposomes were activated and presented this form of antigen in an MHC class I- and class II-restricted manner. CD4 T cells differentiated into Th1 and Th2 effector cells. Primary expansion and cytotoxic activity of CD8 T cells were CD4 T cell-dependent and required the transporter associated with antigen processing (TAP). Finally, adoptively transferred CD4 and CD8 T cells were not deleted after primary immunization and rapidly responded to a secondary immunization with antigen-containing liposomes. In conclusion, encapsulation of antigen in liposomes is an efficient way of delivering antigen to DC for priming of both CD4 and CD8 T cell responses. Importantly, primary CD8 T cell responses were CD4 T cell-dependent.
European Journal of Immunology 06/2006; 36(6):1386-97. DOI:10.1002/eji.200526193 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Langerhans cells (LCs) are prominent dendritic cells (DCs) in epithelia, but their role in immunity is poorly defined. To track and discriminate LCs from dermal DCs in vivo, we developed knockin mice expressing enhanced green fluorescent protein (EGFP) under the control of the langerin (CD207) gene. By using vital imaging, we showed that most EGFP(+) LCs were sessile under steady-state conditions, whereas skin inflammation induced LC motility and emigration to lymph nodes (LNs). After skin immunization, dermal DCs arrived in LNs first and colonized areas distinct from slower migrating LCs. LCs reaching LNs under steady-state or inflammatory conditions expressed similar levels of costimulatory molecules. Langerin and EGFP were also expressed on thymic DCs and on blood-derived, CD8alpha(+) DCs from all secondary lymphoid organs. By using a similar knockin strategy involving a diphtheria toxin receptor (DTR) fused to EGFP, we demonstrated that LCs were dispensable for triggering hapten-specific T cell effectors through skin immunization.
[Show abstract][Hide abstract] ABSTRACT: Liposomes are excellent carriers for protein antigens since they can contain large amounts of antigen, potentially in association with adjuvants. Liposomes may be made to mimic the pathogens that stimulated the evolution of the immune system. As such, numerous mechanisms exist to promote their uptake by antigen presenting cells and exposure of encapsulated antigens to the lymphocytes of the immune system for the induction of responses. The review is intended to describe the 30 year history of the use of liposomes are carriers of protein antigens, notably from the perspective of what we have learned about the immune system using liposomes.
Journal of Liposome Research 02/2004; 14(3-4):175-89. DOI:10.1081/LPR-200039198 · 1.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated differentiation of CD4 T cells responding to Ag presented by bone marrow-derived dendritic cells (DC) in association with MHC class II (MHC II) molecules. Peptides encapsulated in liposomes opsonized by IgG were taken up by endocytosis. MHC II-peptide-specific T cells responding to this Ag were polarized to a Th1 cytokine profile in a CD40-, CD28-, MyD88-, and IL-12-dependent manner. Th2 responses were obtained from the same transgenic T cell population exposed to the same DC on which MHC-peptide complexes had dispersed for 48 h following uptake of FcR-targeted liposomes. DC that took up the same FcR-targeted liposomes and then were exposed to methyl-beta-cyclodextrin, which chelates cholesterol and dissociates lipid microdomains, also stimulated Th2 differentiation. Incubation of T cells with DC incubated with peptides directly binding to MHC II resulted in Th2 responses, whether or not the DC were coincubated with opsonized liposomes as a maturation stimulus. CD4 Th1 polarization thus appears to depend on MHC II-peptide complex clustering in DC lipid microdomains and the time between peptide loading and T cell encounter.
The Journal of Immunology 01/2004; 171(11):5812-9. DOI:10.4049/jimmunol.171.11.5812 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The adaptive immune responses of mammals depend on cells specialized in the acquisition of antigen (Ag), called Ag presenting cells (APC), and cells that respond to that Ag, T and B lymphocytes. The two major arms of the adaptive immune response are antibody (Ab) production and cytotoxicity. T lymphocytes are divided into CD4+ and CD8+ T cells. CD4+ T cells are necessary to induce the immunoglobulin gene rearrangements necessary for the production of somatically mutated IgG Ab by B cells. Cytotoxicity is primarily a property of CD8+ T cells, but it requires CD4+ T-helper cells for complete differentiation and acquisition of effector functions. The induction of the immune response is thus a complex series of sequential interactions involving Ag contact with APC, APC contact with lymphocytes, and interaction among lymphocytes. Liposomes are very simple and lack microbial elements designed to inhibit immune responses and elements, such as endotoxin, repeated carbohydrates, and nucleic acids. CD4+ T cells do not recognize protein Ag as such but recognize Ag after uptake, partial proteolysis, and re-expression of Ag-derived peptides at the surface of APC in association with major histocompatibility complex (MHC)-encoded class II molecules. APC include dendritic cells (DC), macrophages, and B cells. DC is widely distributed in tissues, and both DC and macrophages use antibody secreted by B cells to bind Ag for enhanced presentation to naive CD4 T cells. DC and macrophages have receptors for Ag–Ab complexes, the immunoglobulin Fc receptors, which are a family of molecules at the surface of DC.
Methods in Enzymology 02/2003; 373:100-18. DOI:10.1016/S0076-6879(03)73007-9 · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Virosomes are reconstituted viral membranes in which protein can be encapsulated. Fusion-active virosomes, fusion-inactive virosomes and liposomes were used to study the conditions needed for delivery of encapsulated protein antigen ovalbumin (OVA) to dendritic cells (DCs) for MHC class I and II presentation. Fusion-active virosomes, but not fusion-inactive virosomes, were able to deliver OVA to DCs for MHC class I presentation at picomolar OVA concentrations. Fusion activity of virosomes was not required for MHC class II presentation of antigen. Therefore, virosomes are an efficient system for delivery of protein antigens for stimulation of both helper and CTL responses.
[Show abstract][Hide abstract] ABSTRACT: We investigated interactions between CD4+ T cells and dendritic cells (DC) necessary for presentation of exogenous Ag by DC to CD8+ T cells. CD4+ T cells responding to their cognate Ag presented by MHC class II molecules of DC were necessary for induction of CD8+ T cell responses to MHC class I-associated Ag, but their ability to do so depended on the manner in which class II-peptide complexes were formed. DC derived from short-term mouse bone marrow culture efficiently took up Ag encapsulated in IgG FcR-targeted liposomes and stimulated CD4+ T cell responses to Ag-derived peptides associated with class II molecules. This CD4+ T cell-DC interaction resulted in expression by the DC of complexes of class I molecules and peptides from the Ag delivered in liposomes and permitted expression of the activation marker CD69 and cytotoxic responses by naive CD8+ T cells. However, while free peptides in solution loaded onto DC class II molecules could stimulate IL-2 production by CD4+ T cells as efficiently as peptides derived from endocytosed Ag, they could not stimulate induction of cytotoxic responses by CD8+ T cells to Ag delivered in liposomes into the same DC. Signals requiring class II molecules loaded with endocytosed Ag, but not free peptide, were inhibited by methyl-beta-cyclodextrin, which depletes cell membrane cholesterol. CD4+ T cell signals thus require class II molecules in cholesterol-rich domains of DC for induction of CD8+ T cell responses to exogenous Ag by inducing DC to process this Ag for class I presentation.
The Journal of Immunology 03/2002; 168(3):1172-80. DOI:10.4049/jimmunol.168.3.1172 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We evaluated MHC class I- and II-restricted presentation of exogenous antigen by mouse bone marrow-derived dendritic cells (DC) and splenic B cells. DC presented to class I-restricted transgenic T cells femtomolar concentrations of antigens from liposomes targeted to the IgG Fc receptor. Targeting these liposomes to surface immunoglobulin did not permit B cells to stimulate class I-restricted responses. Nevertheless, both DC and B cells presented antigen from liposomes targeted to these same receptors with equivalent efficiency to class II-restricted T cells. Acquisition of the capacity to present class II-restricted antigens required shorter periods of differentiation of DC than presentation of exogenous class I-restricted antigens. The latent period for class I-restricted presentation of exogenous antigen by DC could not be shortened by exposing them to lipopolysaccharide, double-stranded RNA or antibody to CD40. Class I presentation depended on expression of the TAP1 transporter. Our data are consistent with the existence of a regulated transport process present in DC which can convey exogenous antigen from endocytic vesicles to the cytosol.
European Journal of Immunology 03/2000; 30(3):848-57. DOI:10.1002/1521-4141(200003)30:3<848::AID-IMMU848>3.0.CO;2-Q · 4.03 Impact Factor