Endogenous dendritic cells are required for amplification of T cell responses induced by dendritic cell vaccines in vivo

Dendritic cells (DCs) loaded in vitro with Ag are used as cellular vaccines to induce Ag-specific immunity. These cells are thought to be responsible for direct stimulation of Ag-specific T cells, which may subsequently mediate immunity. In this study, in transgenic mouse models with targeted MHC class II expression specifically on DCs, we show that the DC vaccine is responsible only for partial CD4(+) T cell activation, but to obtain optimal expansion of T cells in vivo, participation of endogenous (resident) DCs, but not endogenous B cells, is crucial. Transfer of Ag to endogenous DCs seems not to be mediated by simple peptide diffusion, but rather by DC-DC interaction in lymph nodes as demonstrated by histological analysis. In contrast, injection of apoptotic or necrotic DC vaccines does not induce T cell responses, but rather represents an immunological null event, which argues that viability of DC vaccines can be crucial for initial triggering of T cells. We propose that viable DCs from the DC vaccine must migrate to the draining lymph nodes and initiate a T cell response, which thereafter requires endogenous DCs that present transferred Ag in order induce optimal T cell expansion. These results are of specific importance with regard to the applicability of DC vaccinations in tumor patients, where the function of endogenous DCs is suppressed by either tumors or chemotherapy.     

Quantitating effector and regulatory T lymphocytes in immune responses by limiting dilution analysis modeling

Although there is currently no doubt that regulatory lymphocytes represent a master player in the immune system, a major unresolved problem is the accurate quantitation of these cells among unfractionated cell populations. This difficulty mainly arises because there are no specific immunophenotypic markers that can reliably discriminate between effector and regulatory lymphocytes. To face this problem, we have developed computational models of limiting dilution analyses addressing the question of the accurate estimation of the frequencies of effector and regulatory cells functionally engaged in an immune response. A set of generic equations were provided to form a framework for modeling limiting dilution data, enabling discrimination between qualitatively different models of suppression. These models include either one or two subpopulations of regulatory cells, featured by either low or potent regulatory activity. The potential of this modeling approach was illustrated by the accurate determination of the frequencies of effector and regulatory T lymphocytes in one real limiting dilution experiment of CD4+ CD25+ T lymphocytes performed in the context of an allogeneic response in the human system. The crucial advantage of the limiting dilution method over the "static, phenotype-based" method is the dynamic evaluation of effector and regulatory T cell biology through their actual functional activity.     

Mouse models of K-ras-initiated carcinogenesis

Activating mutations of the oncogene K-ras are found in one third of all human cancers. Much of our knowledge on K-ras signal transduction and its influence on tumor initiation and progression comes from in vitro studies with cell lines. However, mouse models of human cancer allow a much more faithful recapitulation of the human disease, and the in vivo perspective is crucial for our understanding of neoplasia. In recent years, several new murine models for K-ras-induced tumorigenesis have been described. They allow new insights into the specific role that oncogenic K-ras proteins play in different solid tumors, and they permit the molecular dissection of the pathways that are initiated by somatic mutations in subsets of cells. Key advances have been made by the use of tissue-specific and inducible control of expression, which is achieved by the Cre/LoxP technology or the tetracycline system. from these sophisticated models, a common picture emerges: The effects of K-ras on tumor initiation depend strongly on the cellular context, and different tissues vary in their susceptibility to K-ras transformation.     

Structural requirements for the interaction between peptide antigens and I-Ed molecules

We have analyzed the structural characteristics of the interaction between I-Ed molecules and their peptide ligands. It was found that unrelated good I-Ed binders share structurally similar "core" regions that were experimentally demonstrated to be crucial for binding to I-Ed molecules. Single amino acid substitution analogues of one good I-Ed binder, hen egg lysozyme 107-116, were analyzed for their capacity to bind to I-Ed molecules and to activate two different I-Ed-restricted T cell hybridomas. The results illustrate the great permissiveness of I-Ed-peptide interaction and the great specificity of T cell recognition. It was concluded from these analyses that basic residues on the peptide molecule play a crucial role in binding to I-Ed. This contrasts with the structural requirements for binding to the other Iad isotype, I-Ad, the crucial hydrophobic residues. Thus, different class II molecules of the same MHC haplotype may have rather distinct peptide binding specificities, thereby expanding the repertoire of possible immunogenic peptides presented for T cell recognition.     

Notch signalling at the crossroads of T cell development and leukemogenesis

Members of the Notch family (e.g. Notch1 and Notch3) have been recently described to play a critical role in T cell development and their constitutive activation has been related to T cell leukaemia in both animal models and human disease. Nevertheless, whether they act as redundant molecules, by affecting the same molecular mechanisms, or play distinct roles in T cell differentiation and/or leukemogenesis is not clear. Altered Notch signalling impairs the developmentally-regulated interplay between pre-TCR signalling, NFkappaB and E2A activities, thus identifying the crucial role of Notch receptors at the cross-roads of disrupted lymphoid differentiation and neoplastic transformation.     

Modification of the cytotoxic T cell repertoire in neonatal tolerance. Evidence for preferential survival of cells with low avidity for tolerogen

Clonal deletion of developing lymphocytes with potential reactivity for self is thought to play a crucial role in the establishment of self tolerance. One prediction of the clonal deletion hypothesis is that cells bearing receptors with high affinity for self are more likely than cells with low affinity receptors to be deleted from the repertoire. Experimental models of B cell tolerance have provided evidence for the preferential survival of low affinity cells with specificity for tolerogen in tolerant animals, but no comparable evidence exists for T cells. To examine this issue in T cells, cytotoxic T cell lines specific for the Kb mutant class I H-2 molecule, bm1, were generated from C57BL/6 mice rendered neonatally tolerant of bm1 and compared with anti-bm1 lines generated from normal mice. Compared with normal lines, those from tolerant mice differed in five ways: 1) they grew more slowly; 2) they were less efficient at lysing bm1 targets; 3) they showed different patterns of lysis against a panel of third party targets; 4) their cytotoxic activity against bm1 could be increased in the presence of leukoagglutinin, whereas the activity of normal lines was not increased by leukoagglutinin; and 5) their cytotoxic activity was more susceptible to inhibition by anti-Lyt-2 antibody. Taken together, these results demonstrate that the repertoire of the remaining tolerogen-specific cytotoxic T cells in neonatally tolerant mice is different from the normal C57BL/6 anti-bm1 repertoire, and the results are consistent with the idea that the remaining tolerogen-specific cells are low avidity cells that have preferentially escaped the clonal deletion process.     

Effects of systemic versus local administration of corticosteroids on mucosal tolerance

Respiratory exposure to allergen induces T cell tolerance and protection against the development of airway hyperactivity in animal models of asthma. Whereas systemic administration of dexamethasone during the delivery of respiratory Ag has been suggested to prevent the development of mucosal tolerance, the effects of local administration of corticosteroids, first-line treatment for patients with bronchial asthma, on mucosal tolerance remain unknown. To analyze the effects of systemic versus local administration of different types of corticosteroids on the development of mucosal tolerance, mice were exposed to respiratory allergen to induce mucosal tolerance with or without systemic or intranasal application of different doses of dexamethasone or prednisolone. After the induction of mucosal tolerance, proliferation of T cells was inhibited in tolerized mice, whereas systemic applications of corticosteroids restored T cell proliferation and secretion of Th2 cytokines. In contrast, inhaled corticosteroids showed no effect on both T cell proliferation and cytokine secretion. In addition, mice systemically treated with corticosteroids showed an increased airway hyperactivity with a significant lung inflammation, but also an increased T effector cells/regulatory T cells ratio in the second lymphoid organs when compared with mice that receive corticosteroids by inhalation. These results demonstrate that local administration of corticosteroids has no effect on the development of immune tolerance in contrast to systemically applied corticosteroids. Furthermore, although different concentrations of corticosteroids are administered to patients, our results demonstrated that the route of administration rather than the doses affects the effect of corticosteroids on respiratory tolerance induction. Considering the broad application of corticosteroids in patients with allergic disease and asthma, the route of administration of steroid substances seems crucial in terms of treatment and potential side effects. These findings may help elucidate the apparently contradicting results of corticosteroid treatment in allergic diseases.     

Functional heterogeneity of memory CD4 T cell responses in different conditions of antigen exposure and persistence

Memory CD4 T cell responses are functionally and phenotypically heterogeneous. In the present study, memory CD4 T cell responses were analyzed in different models of Ag-specific immune responses differing on Ag exposure and/or persistence. Ag-specific CD4 T cell responses for tetanus toxoid, HSV, EBV, CMV, and HIV-1 were compared. Three distinct patterns of T cell response were observed. A dominant single IL-2 CD4 T cell response was associated with the model in which the Ag can be cleared. Polyfunctional (single IL-2 plus IL-2/IFN-gamma plus single IFN-gamma) CD4 T cell responses were associated with Ag persistence and low Ag levels. A dominant single IFN-gamma CD4 T cell response was associated with the model of Ag persistence and high Ag levels. The results obtained supported the hypothesis that the different patterns observed were substantially influenced by different conditions of Ag exposure and persistence.     

Murine AIDS superantigen reactivity of the T cells bearing V beta 5 T cell antigen receptor.

A B cell line, B6-1710, that expresses the defective virus known to induce murine AIDS stimulates a large fraction of nonprimed splenic T cells. Analysis of the T cell population responding to the B6-1710 for TCR V beta-chain usage revealed that, in addition to the previously reported V beta 5-chain-positive T cells, T cells bearing V beta 11 and V beta 12 are also specifically enriched. We have established V beta 5+ T cell lines, clones, and hybridomas expressing identical TCR with different CD4/CD8 phenotypes and demonstrated that T cell reactivity to B6-1710 is, although not absolute, dependent on the presence of CD4 molecules. Further analysis of T cell hybridomas with known J beta-chain usage revealed that D beta- and J beta-chain usage do not play crucial roles in T cell reactivity to B6-1710 B cells. However, T cell hybridomas derived from TCR-V beta gene transgenic mice were found to be heterogeneous for their reactivity to B6-1710, suggesting that the V alpha-chains associating with the transgenic V beta-chain determine T cell responsiveness to B6-1710. These data clearly demonstrate that T cell reactivity to a murine AIDS virus expressing B cell line resembles that previously reported for Mls-like superantigens.     

Animal Models of Human Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a human autoimmune disease of unknown etiology. Clinical, serologic, immunologic, and pathologic findings are highly variable in different patients and at different times in the same patient. Murine and canine animal models of SLE have been found with clinicopathologic abnormalities resembling those observed in humans. Each animal model has unique characteristics; taken together they reflect the spectrum of disease in human SLE.Investigations in the animals have suggested that genetic, hormonal, immunologic, viral, and other environmental factors contribute to and modify the expression of disease. Where analogous studies are available for humans, the same factors have been found to modify disease expression in a similar fashion. Together, these studies have helped to clarify the multifactorial basis for SLE.The best characterized abnormalities are immunologic. These include excessive B cell function with the formation of large amounts of autoantibodies, and T cell abnormalities which include defects in T cell regulatory function as well as certain T cell effector functions.The animal models of SLE also serve as convenient test subjects for newer therapeutic modalities. It is hoped that further study of the animal models will provide a more rational approach to therapeutic modulation of disease in humans with SLE.     

Receptor aggregation by intermembrane interactions: a Monte Carlo study

The lateral organization of receptors on cell surfaces is critically important to their function; many receptors transmit transmembrane signals when redistributed into clusters, while the response of others is potentiated by their aggregation. Cell-cell contact can play a crucial role in receptor aggregation, even when the bonds between receptors on one cell and ligands on the other are monovalent. Monte Carlo simulations on a two-membrane model were carried out to determine whether weak enthalpic interactions among receptors in one membrane, and among ligands in another, can work synergistically to give large-scale clustering when the two membranes are brought into contact. The simulations give support to such a clustering mechanism. In addition, because clustering is a cooperative process akin to a phase separation, individual receptors and ligands may undergo repeated binding and unbinding while in a clustered "phase," and a single ligand could interact with multiple different receptor partners. The results suggest a resolution of the dichotomy between serial triggering and aggregation models of T cell activation.     

Interleukin-7 deficiency in rheumatoid arthritis

Interleukin-7 (IL-7) is a stromal factor that is crucial for the development of T lymphocytes in humans and mice, and also B lymphocytes in mice. IL-7 can act as a T cell growth factor as well as a critical anti-apoptotic survival factor. The essential non-redundant role of this cytokine for T cell development in vivo is indicated by the phenotype of murine knockout models as well as by humans with a T-B+NK+ form of severe combined immunodeficiency (SCID) resulting from mutations in IL-7 receptor alpha chain. IL-7 deficiency has now been found in patients with rheumatoid arthritis, a finding that relates not only to the T-lymphocyte status in this disease but also to the ability of patients with rheumatoid arthritis to recover from therapy-induced lymphopenia.     

Lipid Rafts & Co.: an integrated model of membrane organization in T cell activation

The model of membrane compartmentalization by self-organizing functional lipid microdomains, named lipid rafts, has been a fruitful concept resulting in great progress in understanding T cell signal transduction. However, due to recent results it has become clear that lipid rafts describe only one out of several membrane organizing principles crucial for T cell activation besides fences and pickets and protein-protein interactions that take part in the formation of the immunological synapse as a highly organized structure at the T cell contact site to the antigen-presenting cell. This review describes the concepts of lipid rafts and other membrane organizing principles to evolve a novel integrated model on the functional role of microdomains in immunological synapse formation and T cell activation. Further research has to elucidate the relative contribution and interrelation of different modes of membrane organization in productive T cell activation.     

Cytoskeletal cross-talk in the control of T cell antigen receptor signaling

T cell antigen receptor signaling is triggered and controlled in specialized cellular interfaces formed between T cells and antigen-presenting cells named immunological synapses. Both microtubules and actin cytoskeleton rearrange at the immunological synapse in response to T cell receptor triggering, ensuring in turn the accuracy of intracellular signaling. Recent reports show that the cross-talk between the cortical actin cytoskeleton and microtubule networks is key for structuring the immunological synapse and for controlling T cell receptor signaling. Immunological synapse architecture and the interaction between the signaling machinery and various cytoskeletal elements are therefore crucial for the fine-tuning of T cell signaling.     

Models of Self-Peptide Sampling by Developing T Cells Identify Candidate Mechanisms of Thymic Selection

Conventional and regulatory T cells develop in the thymus where they are exposed to samples of self-peptide MHC (pMHC) ligands. This probabilistic process selects for cells within a range of responsiveness that allows the detection of foreign antigen without excessive responses to self. Regulatory T cells are thought to lie at the higher end of the spectrum of acceptable self-reactivity and play a crucial role in the control of autoimmunity and tolerance to innocuous antigens. While many studies have elucidated key elements influencing lineage commitment, we still lack a full understanding of how thymocytes integrate signals obtained by sampling self-peptides to make fate decisions. To address this problem, we apply stochastic models of signal integration by T cells to data from a study quantifying the development of the two lineages using controllable levels of agonist peptide in the thymus. We find two models are able to explain the observations; one in which T cells continually re-assess fate decisions on the basis of multiple summed proximal signals from TCR-pMHC interactions; and another in which TCR sensitivity is modulated over time, such that contact with the same pMHC ligand may lead to divergent outcomes at different stages of development. Neither model requires that T and T are differentially susceptible to deletion or that the two lineages need qualitatively different signals for development, as have been proposed. We find additional support for the variable-sensitivity model, which is able to explain apparently paradoxical observations regarding the effect of partial and strong agonists on T and T development.     

Functional gap junctions accumulate at the immunological synapse and contribute to T cell activation

Gap junction (GJ) mediates intercellular communication through linked hemichannels from each of two adjacent cells. Using human and mouse models, we show that connexin 43 (Cx43), the main GJ protein in the immune system, was recruited to the immunological synapse during T cell priming as both GJs and stand-alone hemichannels. Cx43 accumulation at the synapse was Ag specific and time dependent, and required an intact actin cytoskeleton. Fluorescence recovery after photobleaching and Cx43-specific inhibitors were used to prove that intercellular communication between T cells and dendritic cells is bidirectional and specifically mediated by Cx43. Moreover, this intercellular cross talk contributed to T cell activation as silencing of Cx43 with an antisense or inhibition of GJ docking impaired intracellular Ca(2+) responses and cytokine release by T cells. These findings identify Cx43 as an important functional component of the immunological synapse and reveal a crucial role for GJs and hemichannels as coordinators of the dendritic cell-T cell signaling machinery that regulates T cell activation.     

Evaluation of Nanoparticle Uptake in Co-culture Cancer Models

Co-culture models are currently bridging the gap between classical cultures and in vivo animal models. Exploring this novel approach unlocks the possibility to mimic the tumor microenvironment in vitro, through the establishment of cancer-stroma synergistic interactions. Notably, these organotypic models offer a perfect platform for the development and pre-clinical evaluation of candidate nanocarriers loaded with anti-tumoral drugs in a high throughput screening mode, with lower costs and absence of ethical issues. However, this evaluation was until now limited to co-culture systems established with precise cell ratios, not addressing the natural cell heterogeneity commonly found in different tumors. Therefore, herein the multifunctional nanocarriers efficiency was characterized in various fibroblast-MCF-7 co-culture systems containing different cell ratios, in order to unravel key design parameters that influence nanocarrier performance and the therapeutic outcome. The successful establishment of the co-culture models was confirmed by the tissue-like distribution of the different cells in culture. Nanoparticles incubation in the various co-culture systems reveals that these nanocarriers possess targeting specificity for cancer cells, indicating their suitability for being used in this illness therapy. Additionally, by using different co-culture ratios, different nanoparticle uptake profiles were obtained. These findings are of crucial importance for the future design and optimization of new drug delivery systems, since their real targeting capacity must be addressed in heterogenous cell populations, such as those found in tumors.