Induction Of MHC Class Research Articles

Mast cell-mediated antigen presentation regulates CD8 T cell effector functions (33.15)

Abstract Mast cells (MCs) - long-living cells at sites of host-environment interphase, are important players of the innate immunity and central regulatory cells in adaptive immunity. We have previously described that MCs can detect viral infections and that TLR3-activated MCs contribute to antiviral host defence. MCs can regulate CD8 T cell- dependent autoimmune responses and CD8 T cell chemotaxis. Using three model antigens, we demonstrate that MCs induce antigen-specific CD8 T cell activation and proliferation. This requires direct cell contact and MHC class I-dependent antigen cross-presentation by MCs and induces the secretion of IL-2, IFN-γ and MIP-1α by CD8 T cells. Furthermore, MCs regulate antigen-specific CD8 T cell cytotoxicity by increasing granzyme B expression and promoting CD8 T cell degranulation. MCs also upregulate their expression of costimulatory molecules, and release the T cell proliferation inducer osteopontin upon direct contact. In vivo, adoptive transfer of antigen-pulsed MCs induces MHC class I-dependent, antigen-specific CD8 T cell proliferation. Furthermore, we characterised the bidirectional crosstalk between MCs and CD8 T cells and identified a new pathway of MC activation induced by MC interaction with CD8 T cells. Specifically, a number of genes coding for proteins involved in the IFN-mediated antiviral response are upregulated upon MC-CD8 T cell interaction.

ERK5 Knockdown Generates Mouse Leukemia Cells with Low MHC Class I Levels That Activate NK Cells and Block Tumorigenesis

Tumor cell-based vaccines are currently used in clinical trails, but they are in general poorly immunogenic because they are composed of cell extracts or apoptotic cells. Live tumor cells should be much better Ags provided that they are properly processed by the host immune system. We show herein that stable expression of a small hairpin RNA for ERK5 (shERK5) decreases ERK5 levels in human and mouse leukemic cells and leads to their elimination by NK cells in vivo. The shERK5 cells show down-regulation of MHC class I expression at the plasma membrane. Accordingly, ectopic activation of the ERK5 pathway induces MHC class I gene expression. Coinjection of shERK5-expressing cells into the peritoneum diminishes survival of engrafted wild-type tumor cells. Moreover, s.c. injection of shERK5-expressing cells strongly diminishes tumor development by wild-type cells. Our results show that shERK5 expression in leukemia cells effectively attenuates their tumor activity and allows their use as a tumor cell-based vaccine.

CIITA versus IFN-γ induced MHC class II expression in head and neck cancer cells

A growing body of evidence suggests that optimal induction of systemic anti-tumor immunity requires priming of both the CD4+ and CD8+ T cells that are specific for tumor-associated antigens (TAA). Recently, it was shown that MHC class II positive tumor cells are able to induce tumor-specific CD4+ T cells, and that this event may improve clinical outcome. This has rekindled the interest in modulating MHC class II expression in nonprofessional antigen presenting tumor cells. The class II transactivator (CIITA) is a major regulator of MHC class I and class II expression. We compared, in head and neck cancer cell lines, the effect of stable overexpression of CIITA to treatment with IFN-gamma on the cell surface expression profile of MHC class I and II molecules. Here, we provide evidence that CIITA transfection is more effective than IFN-gamma in inducing MHC class II expression. To more thoroughly explore the mechanisms of MHC class II induction in this context, we used RT-PCR to measure the mRNA expression pattern of HLA-DR, HLA-DM, cathepsin S, and the invariant chain. In contrast to the effect of treatment with IFN-gamma, CIITA transfection did not induce cathepsin S, an important protease responsible for the degradation of the invariant chain, and thus for binding of the peptides to the MHC class II binding groove. These findings may have a significant impact on practical and clinical aspects of tumor immunotherapeutic strategies.

Immunization with a Mimotope of GD2 Ganglioside Induces CD8+ T Cells That Recognize Cell Adhesion Molecules on Tumor Cells

The GD2 ganglioside expressed on neuroectodermal tumor cells has been used as a target for passive and active immunotherapy in patients with malignant melanoma and neuroblastoma. We have reported that immunization of mice with a 47-LDA mimotope of GD2, isolated from a phage display peptide library with anti-GD2 mAb 14G2a, induces MHC class I-restricted CD8(+) T cell responses to syngeneic neuroblastoma tumor cells. The cytotoxic activity of the vaccine-induced CTLs was independent of GD2 expression, suggesting recognition of a novel tumor-associated Ag cross-reacting with 47-LDA. Glycan microarray and immunoblotting studies using 14G2a mAb demonstrated that this Ab is highly specific for the entire carbohydrate motif of GD2 but also cross-reacts with a 105 kDa glycoprotein expressed by GD2(+) and GD2(-) neuroblastoma and melanoma cells. Functional studies of tumor cells grown in three-dimensional collagen cultures with 14G2a mAb showed decreases in matrix metalloproteinase-2 activation, a process regulated by the 105 kDa-activated leukocyte cell adhesion molecule (ALCAM/CD166). A recombinant CD166 glycoprotein was shown to be recognized by 14G2a Ab and inhibition of CD166 expression by RNA interference ablated the cell sensitivity to lysis by 47-LDA-induced CD8(+) T cells in vitro and in vivo. The binding of 14G2a to CD166 was not disruptable by a variety of exo- and endo-glycosidases, implying recognition of a non-glycan epitope on CD166. These results suggest that the vaccine-induced CTLs recognize a 47-LDA cross-reactive epitope expressed by CD166, and reveal a novel mechanism of induction of potent tumor-specific cellular responses by mimotopes of tumor-associated carbohydrate Ags.

Tumor-derived soluble MICs impair CD3 +CD56 + NKT-like cell cytotoxicity in cancer patients

Upon ligation with its ligands, the activating receptor NKG2D stimulates or costimulates CD8 + T cells or NK cells. The inducible gene MHC class I chain-related molecules (MICs), which belong to the NKG2D ligand family and usually initiate the process of lymphocyte activation, have been found to be broadly expressed on epithelial tumor cells. Sustained localized expression or release of soluble forms of MICs (sMICs) by tumor cells play key roles in tumor evasion via the impairment of T cell and NK cell functions. NKG2D is also expressed on the surface of CD3 +CD56 + NKT-like cells, which participate in tumor rejection via direct cytolysis. We speculated whether sMICs have the same impact on NKT-like cells. In this study, we demonstrated that in vitro killing by freshly isolated NKT-like cells was principally mediated by NKG2D, and the cytotoxic function of NKT-like cells isolated from cancer patients was obviously compromised. We found a significant correlation between elevated tumor-derived sMICs and down-modulation of NKG2D expression on NKT-like cell surfaces in human ovarian cancer and prostate cancer patients. We determined that elevated serum sMIC impairs the lytic activity via downregulation of the NKG2D receptor because incubation of NKT-like cells with sera obtained from cancer patients down-modulated surface NKG2D expression, whereas the addition of neutralizing anti-MIC mAbs restored surface NKG2D expression. We suggest that tumors shedding MICs may promote immune evasion by impairing NKT-like cell cytotoxicity.

Bilirubin Possesses Powerful Immunomodulatory Activity and Suppresses Experimental Autoimmune Encephalomyelitis

Bilirubin, an abundant bile pigment in mammalian serum, was once considered a toxic waste product and has more recently been recognized as a potent antioxidant of physiological importance. However, its potential biological functions in other fields are not well understood. Herein we show that bilirubin is also a powerful immunomodulatory agent. Bilirubin significantly inhibited Ag-specific and polyclonal T cell responses, while other similar antioxidants completely lacked this effect. Bilirubin suppressed CD4(+) T cell responses at multiple steps. High levels of bilirubin could induce apoptosis in reactive CD4(+) T cells. Bilirubin at nonapoptotic concentrations suppressed CD4(+) T cell reactivity through a wide range of actions, including inhibition of costimulator activities, suppression of immune transcription factor activation, and down-regulation of inducible MHC class II expression. Further studies suggest that bilirubin actions were direct, rather than via induction of immune deviation or regulatory T cells. In vivo, treatment with bilirubin effectively suppressed experimental autoimmune encephalomyelitis in SJL/J mice. In contrast, depletion of endogenous bilirubin dramatically exacerbated this disease. In summary, our results identify bilirubin as an important immunomodulator that may protect mammals against autoimmune diseases, thereby indicating its potential in the treatment of multiple sclerosis and other immune disorders.

Depletion of CD8+ T cells suppresses growth of Trypanosoma brucei brucei and interferon-gamma) production in infected rats

Sprague-Dawley rats infected with Trypanosoma brucei brucei showed a strong and rapid induction of splenocyte IFN-gamma (within 12 h post-infection) as measured by a single cell assay for IFN-gamma secretion. Depletion of CD8+ cells in infected rats abrogated the IFN-gamma production, suppressed parasite growth and increased survival of the animals. Induction of MHC class I antigens in the paraventricular and supra-optic hypothalamic nuclei caused by the trypanosome infection was also inhibited by the CD8+ cell depletion. It is suggested that the CD8+ cells are involved directly or indirectly in growth regulation of the parasite and that IFN-gamma induced by the parasite may be one of the factors that trigger MHC expression and immunosuppression.

Podocyte expression of MHC class I and II and intercellular adhesion molecule-1 (ICAM-1) in experimental pauci-immune crescentic glomerulonephritis.

We examined immunopathological changes of podocytes in vivo which, based on in vitro studies, are thought to be relevant for the pathogenesis of renal diseases. We investigated the alterations of podocytes in local inflammation in a recently developed model of pauci-immune necrotizing crescentic glomerulonephritis (NCGN) in the rat. Frozen and plastic embedded kidney sections at different time points of the disease were incubated with antibodies directed to MHC class I, MHC class II, ICAM-1 and to relevant cytokines. Strong glomerular expression of MHC class I, II and ICAM-1 was found within 4 days, and plastic embedded sections clearly demonstrated increased cell membrane staining of podocytes. Increased glomerular interferon-gamma (IFN-gamma) was detected within 24 h of induction of NCGN, and IL-1 beta and tumour necrosis factor-alpha (TNF-alpha) were found from day 4. The potency of these cytokines to induce adhesion molecules on podocytes was investigated on rat glomerular epithelial cells in vitro. By using FACS analysis and electron microscopical techniques, we found that the in vivo expression of MHC class I, II and ICAM-1 by podocytes could in vitro be simulated by IFN-gamma. IFN-alpha weakly induced MHC class I, while IL-1 beta and TNF-alpha were ineffective. We hypothesize that podocytes in this in vivo model are important to maintain the local inflammatory process in the glomerulus by expression of relevant adhesion molecules and MHC molecules upon stimulation with specific cytokines.

Cytokines, thyroid autoantibody synthesis and thyroid cell survival in culture

In autoimmune thyroid disease lymphoid cells infiltrating the thyroid gland occur in conspicuous aggregates or as a diffusely distributed population invading the thyroid follicles. Consequently cytokines secreted by activated T cells or macrophages could influence neighbouring thyroid cells as well as other lymphocytes. We have investigated this possibility using recombinant cytokines. Thyroid cell survival was assessed in terms of mitochondrial dehydrogenase activity in monolayers exposed to tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-1 (IL-1 alpha and beta) and interleukin-2 (IL-2) in the presence or absence of thyroid-stimulating hormone (TSH). Neither TNF-alpha nor IL-2 affected thyroid cell survival, IFN-gamma was usually inhibitory and IL-1 alpha slightly enhanced cell survival in some experiments. However, the effects were small and variable and were not enhanced by potentially synergistic combinations of cytokines, longer periods of exposure, or different culture conditions. In contrast, IFN-gamma, IL-2 and TNF-alpha inhibited the ability of thyroid lymphocytes from patients with Graves' disease and Hashimoto's thyroiditis to synthesize autoantibodies to thyroid peroxidase (TPO) and thyroglobulin (Tg). Comparison of lymphoid populations isolated by digestion and/or mechanical disaggregation indicated that a population of activated B cells, plasma cells and T cells, intimately associated with thyroid cells since they could only be extracted by digestion, was influenced by cytokines. Our studies suggest that in addition to its well-recognized ability to induce MHC class II antigens on thyroid cells, IFN-gamma may inhibit thyroid cell proliferation and TNF-alpha, IFN-gamma and IL-2 may down-regulate thyroid autoantibody synthesis.

Tumour cell binding by a human monoclonal IgM antibody from the spleen of a non-tumour-associated patient is due to somatic mutations in the VH gene.

Recently we described the occurrence of B cells producing polyspecific natural IgM with anti-tumour specificity in the spleen of non-tumour-bearing individuals as well as in fetal organisms. Immunoprecipitation and 2-D electrophoresis showed the binding of such antibodies to a 55-kD (pI 6.0) membrane surface glycoprotein. In vitro cultivation of human cancer cell lines in the presence of the purified IgM antibodies resulted in growth inhibition and complement-mediated cell lysis. Furthermore, the antibodies were shown to be able to induce MHC class I molecule expression on tumour cells. Because of this, a role for naturally occurring antibodies with anti-tumour specificity in preventing neoplasias had been suggested. We have constructed and expressed in Escherichia coli single-chain fragments (scFv: VH-linker-VL) derived from a polyspecific human monoclonal IgM autoantibody produced by a human x mouse heterohybridoma which was obtained from the spleen of an autoimmune patient. The mutated complementarity determining region (CDR) gene segments were replaced by the equivalent germ-line sequences and the CDR3 region was swapped for that from another polyspecific human natural antibody with no binding to tumours. Using these four scFv constructs for binding analyses and in vitro cultivation experiments we found: (i) scFv containing the mutated VH region of the original antibody were able to bind to tumour cells, to induce MHC class I molecule expression, and to inhibit tumour growth in a way similar to what had been described for the complete antibody; (ii) replacement of the mutated by the germ-line VH gene independently of the CDR3 to which it had been recombined, resulted in failure to bind to tumour cells. Nevertheless, other antigens (ssDNA, tetanus toxin) were still recognized, although with lower affinity. We discuss the significance of the replacement mutations in the VH gene CDRs, selected probably by B cell contact to an (auto)antigen, for generating a tumour binding capacity, not encoded by the germ-line gene.

Trans-Presentation of IL-15 Dictates IFN-Producing Killer Dendritic Cells Effector Functions

IFN-producing killer dendritic cells (IKDC) were initially described as B220(+)CD11c(+)CD3(-)NK1.1(+) tumor-infiltrating cells that mediated part of the antitumor effects of the combination therapy with imatinib mesylate and IL-2. In this study, we show their functional dependency on IL-15 during homeostasis and inflammatory processes. Trans-presentation of IL-15 by IL-15Ralpha allows dramatic expansion of IKDC in vitro and in vivo, licenses IKDC for TRAIL-dependent killing and endows IKDC with immunizing potential, all three biological attributes not shared by B220(-)NK cells. However, IL-15 down-regulates the capacity of IKDC to induce MHC class I- or II-restricted T cell activation in vitro. Trans-presentation of IL-15 by IL-15Ralpha allows IKDC to respond to TLR3 and TLR4 ligands for the production of CCL2, a chemokine that is critical for IKDC trafficking into tumor beds (as described recently). We conclude that IKDC represent a unique subset of innate effectors functionally distinguishable from conventional NK cells in their ability to promptly respond to IL-15-driven inflammatory processes.

A dual function for RELMβ in promoting TH1 immune responses and infection‐induced intestinal inflammation

The secreted goblet cell‐derived protein Resistin‐like moleculeβ (RELMβ) has been implicated in divergent functions including immunity to intestinal infection and promotion of chemical‐induced colitis. However, the mechanisms through which RELMβ influences proinflammatory responses and adaptive immunity are undefined. To investigate the function of RELMβ in this context, we employed a model of chronic Trichuris infection characterized by RELMβ expression, IFNγ production and severe intestinal inflammation. In contrast to WT mice, RELMβ −/− mice failed to establish chronic infection, and parasite clearance was associated with decreased production of IFNγ and TNFα and reduced intestinal inflammation. Based on these findings, we hypothesized a role for RELMβ in promoting proinflammatory responses by influencing accessory cell function and/or through direct effects on CD4 T cells. While recombinant RELMβ did not directly affect CD4 T cells, rRELMβ treatment of macrophages induced MHC Class II expression, secretion of IL‐12 and TNFα , and augmented their ability to promote antigen‐specific TH1 cell differentiation. Taken together, these data demonstrate a previously unidentified role for an intestinal goblet cell‐derived factor in the modulation of proinflammatory responses and adaptive immunity through direct effects on macrophages.

Prostate Stem Cell Antigen Vaccination Induces a Long-term Protective Immune Response against Prostate Cancer in the Absence of Autoimmunity

Prostate stem cell antigen (PSCA) is an attractive antigen to target using therapeutic vaccines because of its overexpression in prostate cancer, especially in metastatic tissues, and its limited expression in other organs. Our studies offer the first evidence that a PSCA-based vaccine can induce long-term protection against prostate cancer development in prostate cancer-prone transgenic adenocarcinoma mouse prostate (TRAMP) mice. Eight-week-old TRAMP mice displaying prostate intraepithelial neoplasia were vaccinated with a heterologous prime/boost strategy consisting of gene gun-delivered PSCA-cDNA followed by Venezuelan equine encephalitis virus replicons encoding PSCA. Our results show the induction of an immune response against a newly defined PSCA epitope that is mediated primarily by CD8 T cells. The prostates of PSCA-vaccinated mice were infiltrated by CD4-positive, CD8-positive, CD11b-positive, and CD11c-positive cells. Vaccination induced MHC class I expression and cytokine production [IFN-gamma, tumor necrosis factor-alpha, interleukin 2 (IL-2), IL-4, and IL-5] within prostate tumors. This tumor microenvironment correlated with low Gleason scores and weak PSCA staining on tumor cells present in hyperplastic zones and in areas that contained focal and well-differentiated adenocarcinomas. PSCA-vaccinated TRAMP mice had a 90% survival rate at 12 months of age. In contrast, all control mice had succumbed to prostate cancer or had heavy tumor loads. Crucially, this long-term protective immune response was not associated with any measurable induction of autoimmunity. The possibility of inducing long-term protection against prostate cancer by vaccination at the earliest signs of its development has the potential to cause a dramatic paradigm shift in the treatment of this disease.

Angiocidin promotes pro-inflammatory cytokine production and antigen presentation in multiple sclerosis

Angiocidin was originally identified as a potent inhibitor of angiogenesis and tumor growth in vivo. In addition to its involvement in the regulation of carcinogenesis, recent studies indicate that angiocidin may also play a significant role in immune system modulation. This report describes the expression and potential function of angiocidin in multiple sclerosis (MS), a severe demyelinating, inflammatory and autoimmune disease of the central nervous system (CNS). We demonstrated that angiocidin and interleukin-7 (IL-7) are over-expressed in brain lesions of MS patients. Angiocidin-treated monocytes, peripheral blood T cells and primary astrocytes secreted various cytokines and chemokines including, IL-6, IL-7, GM-CSF, and MCP-1. Addition of recombinant angiocidin to cell cultures was able to promote differentiation of monocytes into a macrophage-like cell, induce MHC class I and class II gene expression and activate CD4+ and CD8+ T lymphocytes. Consistent with these findings, angiocidin induced mononuclear phagocyte migration and adhesion as well as increased the IL-2 response by antigen-specific T cells to myelin basic protein peptide presented to them by autologous mononuclear phagocytes. Furthermore, we examined STAT3 expression in angiocidin stimulated mononuclear phagocytes, T cells, and primary astrocytes. We found that angiocidin markedly stimulates STAT3 expression in these cell populations. Angiocidin, therefore appears to play a previously unappreciated and potentially important role in the regulation of immune response during the clinical course of MS.

Ubiquitin-fusion degradation pathway: A new strategy for inducing CD8 cells specific for mycobacterial HSP65

The ubiquitin-proteasome system (UPS) plays an indispensable role in inducing MHC class I-restricted CD8 + T cells. In this study, we exploited UPS to induce CD8 + T cells specific for mycobacterial HSP65 (mHSP65), one of the leading vaccine candidates against infection with Mycobacterium tuberculosis. A chimeric DNA termed pU-HSP65 encoding a fusion protein between murine ubiquitin and mHSP65 was constructed, and C57BL/6 (B6) mice were immunized with the DNA using gene gun bombardment. Mice immunized with the chimeric DNA acquired potent resistance against challenge with the syngeneic B16F1 melanoma cells transfected with the mHSP65 gene (HSP65/B16F1), compared with those immunized with DNA encoding only mHSP65. Splenocytes from the former group of mice showed a higher grade of cytotoxic activity against HSP65/B16F1 cells and contained a larger number of granzyme B- or IFN-γ-producing CD8 + T cells compared with those from the latter group of mice.

Increased Sensitivity of Radiated Murine Cervical Cancer Tumors to E7 Subunit Vaccine–driven CTL-mediated Killing Induces Synergistic Anti-tumor Activity

The development of therapeutic vaccines has important implications for the treatment of cancer patients. Here we investigate whether human papillomavirus (HPV) E7 subunit vaccines can enhance tumor radioresponse using an established cervical cancer animal model. Radiation plus E7 subunit vaccines improved complete response, cure, and recurrence rates of tumors dramatically compared with single therapy alone. In particular, both components of the E7 subunit vaccines (E7 protein and CpG-oligodeoxynucleotide) were required for the induction of antigen (Ag)-specific cytotoxic T-lymphocyte (CTL) responses and for therapeutic synergy with radiotherapy. Moreover, with combined therapy the radiation dose could be reduced by 16 Gy to achieve an equivalent anti-tumor efficacy to radiation treatment alone. This therapeutic synergy was found to be mediated by CD8(+) CTLs and was concomitant with histological changes (presence of apoptotic bodies and multinucleated giant cells; heavy infiltration of lymphocytes), as determined by in vivo T-cell depletion and histological analysis. Finally, phenotypic changes of radiated tumors and their increased sensitivity to CTL-mediated killing appeared to be responsible for therapeutic synergy. These results show that E7 subunit vaccines act as a potent enhancer of tumor radioresponse and that this is mediated by increased sensitivity of radiated tumors to CTL-mediated killing. This study further suggests that E7-targeted therapeutic vaccines have the potential to improve radiotherapy in patients with cervical cancer.

Antigen-presenting properties of gingival fibroblasts in chronic adult periodontitis.

Chronic periodontitis is characterized by dense infiltrations of T lymphocytes in the connective tissue, which consists mainly of gingival fibroblasts. It is becoming increasingly clear that T lymphocytes and gingival fibroblasts are capable of influencing each other. For example, the T cell cytokine interferon-gamma (IFN-gamma) is able to induce MHC class II molecules on the surface of several cell types, including gingival fibroblasts. Histological sections of chronically inflamed gingival tissue showed a great number of CD4+ and CD8+ T cells that produced IFN-gamma, and in addition showed abundant expression of MHC class II molecules on gingival fibroblasts. Therefore, we investigated whether these gingival fibroblasts acquire the capacity to carry out MHC class II-restricted functions such as antigen presentation to local T cells. In this study, we show that IFN-gamma-treated gingival fibroblasts were able to function as antigen-presenting cells (APC) for superantigen-mediated T cell proliferation. However, these fibroblasts failed to present whole-cell antigens of periodontitis-associated bacteria. Moreover, gingival fibroblasts inhibited the presentation of the whole-cell antigens of these bacteria by professional APC. This inhibition could be overcome by the addition of IL-2. These results suggest that gingival fibroblasts play an important role in the local specific immune response in chronic inflammatory periodontal lesions by regulating the response of infiltrating T cells.

204: mTOR knockdown inhibits anti-HLA antibody induced MHC class I signaling

mediated rejection continues to be a significant source of morbidity and mortality in heart transplant patients past the first year. T cell antigen receptor (TCR) signaling is the essential element in these alloresponses against donor MHC and non-MHC antigens. Yet there is no therapy to specifically target this process. Dasatinib, an oral kinase inhibitor used for chronic myeloid leukemia, is exquisitely specific for Abl and Src family kinases (SFK), which are critical for the first step of T cell activation. The immunosuppressive effects of Dasatinib have not yet been examined, thus we conducted the following studies to determine the effects of this drug on in vitro T cell responses. Methods and Materials: PBMC were isolated from healthy donors and stimulated with PHA or anti-CD3 (1-100 ng/ml) plus/minus Dasatinib (0.25-1000 nM). IL-2 expanded T cells were analyzed in the same fashion. Flow cytometric analysis for CD69, CD38, CD25, and HLA-DR was performed on total CD3 and CD4 or CD8 gated T cells. Proliferation was analyzed via CSFE staining. Immunoblotting was performed for total phosphotyrosine, phospho (p)-SFK, p-ZAP-70, p-ERK, p-AKT, p-STAT3, and p-STAT5. Results: Dasatinib inhibits T cell proliferation without inducing apoptosis. T cells remain viable and capable of activation upon drug withdrawal. Low nanomolar concentrations of Dasatinib markedly inhibit TCR induced expression of activation markers CD69, CD25, CD38, and HLA-DR. IFN-gamma production is inhibited by Dasatinib Immunoblot and intracellular flow cytometric analysis reveals specific targeted inhibition of the TCR signaling complex Alternative activation via phorbol ester, bypassing the TCR, is not affected even by micromolar concentrations of Dasatinib, demonstrating specificity for SFK Conclusions: We demonstrate that targeted inhibition of the kinases acting at the earliest stage of lymphocyte activation is a novel immunosuppressive approach that may offer a distinct advantage for combating rejection.

Dendritic Cell-Tumor Fusion Vaccine Prevents Tumor Growthin Vivo

Dendritic cells (DCs) are potent antigen presenting cells that are uniquely effective in generating primary immune responses. DCs that are manipulated to present tumor antigens induce antitumor immunity in animal models and preclinical human studies. A myriad of strategies have been developed to load tumor antigen effectively onto DCs. DC-tumor fusion presents a spectrum of tumor-associated antigens to helper T- and cytotoxic T-cell populations in the context of DC-mediated costimulatory signals. In this study, fusion cells (FCs) were generated with MCA-102 fibrosarcoma cells and murine bone marrow-derived myeloid DCs. The FCs coexpressed the DC-derived MHC class II and costimulatory molecules. The FCs also retained the functional properties of DCs and stimulated syngeneic T cell proliferation and interferon-gamma (IFN-gamma) production. Significantly, the results show that syngeneic T cells are primed by FCs to induce MHC class I-dependent lysis of MCA-102 fibrosarcoma. These findings indicate that fusions of tumor cells and DCs activate T-cell responses against syngeneic tumors.

Effect of early infectious salmon anaemia virus (ISAV) infection on expression of MHC pathway genes and type I and II interferon in Atlantic salmon ( Salmo salar L.) tissues

A number of viral diseases affecting teleost fish are characterized but few studies have addressed the effects of viral infection on gene expression in vivo. In this study, we investigated the effect of the early stages of infectious salmon anaemia virus (ISAV) infection on important components of the innate and adaptive immune response by monitoring expression of five genes in the MHC class I pathway, MHC class IIβ, type I IFN-α, Mx, and type II IFN-γ from cohabitant-infected Atlantic salmon tissues using quantitative real-time PCR. There was an increased expression of type I IFN-α in all tissues analyzed in response to infection that was proportional to viral load (relative to virus RNA levels) in gills and head kidney. Basal expression of IFN-γ was modest or absent in all tissues, but expression was strongly induced and proportional to ISAV RNA levels in heart, spleen and head kidney. A 10-fold or higher level of virally induced IFN-α, in addition to significantly elevated levels of IFN-γ, enhanced transcription of MHC class I pathway genes in heart, spleen and head kidney. In gills, the main entry site for ISAV, there was no induction of MHC class I pathway genes. MHC IIβ and PSMB9 were not significantly induced in any tissue. Thus, by analysing various immune genes in a range of tissues from early cohabitant ISAV-infected salmon, we demonstrate that ISAV infection induced a rapid type I and II IFN response in the major infected lymphoid tissues, which was concurrent with induced expression of MHC class I pathway genes but not MHC IIβ. This may suggest that CD8 + T cell responses are more important than CD4 + T cell responses during early ISAV viraemia.