An immunotherapy approach with dendritic cells genetically modified to express the tumor-associated antigen,HER2

Dendritic cells (DC), genetically modified to express ovalbumin by the retroviral vector GCDNsap, can elicit stronger anti-tumor immunity than those loaded with the peptides. To assess the clinical feasibility of the strategy, such DC were prepared by differentiation of hematopoietic progenitor cells transduced with the human epidermal growth factor receptor 2 (HER2). When inoculated in mice, the DC primed both HER2-specific cytotoxic T lymphocytes and type 1 T helper lymphocytes, resulting in production of HER2-specific antibody. Of importance is that the antibody mediated antibody-dependent cellular cytotoxicity and opsonization. The potent anti-tumor effects were also confirmed by results of experiments using HER2-transgenic mice. Inoculation of HER2-transduced DC resulted in longer disease-free survival of treated mice that showed significant reduction of primary and metastatic tumors. Interestingly, footpad inoculation resulted in stronger anti-tumor effects compared to subcutaneous administration and induced higher levels of the HER2-specific antibody, suggesting that an important role of humoral immunity in anti-tumor effects for malignancies with membrane-type tumor-associated antigens (TAA). Taken together, vaccination of the TAA-transduced DC may represent a promising form of therapy for breast cancers expressing HER2.     

Interaction of reovirus with cell surface receptors. II. Generation of suppressor T cells by the hemagglutinin of reovirus type 3

In studying reovirus interactions with lymphocytes, we have found that reovirus type 3, but not type 1, inhibits the in vitro proliferative response of murine splenic lymphocytes to concanavalin A (Con A). By analyzing recombinant clones containing genes from both reovirus types 1 and 3, we found that the S1 gene, the gene that encodes the viral hemagglutinin, is responsible for the inhibitory effect. In addition we found that type 3, but not type 1, generates suppressor T cells in vitro capable of suppressing Con A proliferation. By analyzing recombinant clones, we also found that the viral hemagglutinin is responsible for the generation of suppressor T cells by reovirus type 3. These effects were observed whether UV-inactivated or live virus was used. Reovirus type 3 inhibition of the proliferative response of murine splenic lymphocytes to Con A was blocked by anti-reovirus type 3 antibody but not by anti-reovirus type 1 antibody. Antiviral antibody had no effect on the ability of reovirus type 3 induced suppressor cells to inhibit Con A proliferation. We have previously demonstrated a receptor on murine lymphocytes for the hemagglutinin of reovirus type 3, and our results suggest that the in vitro suppression of Con A proliferation of murine lymphocytes by reovirus type 3 is secondary to the interaction of the viral hemagglutinin with a receptor on the surface of murine lymphocytes, which results in the generation of functionally active suppressor T cells.     

Mast cells and macrophages in normal C57/BL/6 mice

Mast cells and macrophages have an important role in immunity and inflammation. Because mice are used extensively for experimental studies investigating immunological and inflammatory responses, we examined mast cell and macrophage distribution in normal murine tissues. Mast cells were abundant in the murine dermis, tongue, and skeletal muscle but were rarely found in the heart, lung, spleen, kidney, liver, and the bowel mucosa. In contrast, dogs exhibited large numbers of mast cells in the lung parenchyma, liver, and bowel. Some murine dermal mast cells had long cytoplasmic projections filled with granular content. Mouse mast cells demonstrated intense histamine immunoreactivity and were identified with histochemical enzymatic techniques for tryptase and chymase. Macrophages, identified using the monoclonal antibody F4/80, were abundant in the spleen, lung, liver, kidney, and bowel but relatively rare in the heart, tongue, and dermis. Using a nuclease protection assay we investigated mRNA expression of stem cell factor (SCF), a crucial survival factor for mast cells, and the macrophage growth factors macrophage colony stimulating factor (M-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF). Stem cell factor mRNA was highly expressed in the murine lung. Relatively low levels of SCF mRNA expression were found in the tongue and earlobe, which are tissues containing a high number of mast cells. Macrophage CSF and GM-CSF mRNA was highly expressed in the lung and spleen. The murine heart, an organ with a low macrophage content, expressed high levels of M-CSF but negligible levels of GM-CSF mRNA. Constitutive growth factor mRNA expression in murine tissues without significant populations of mast cells and macrophages may suggest an alternative role for these factors in tissue homeostasis.     

Myelopoiesis related to perinatal spleen

Adult murine spleen is known to have a major role in the development of dendritic cell (DC) subsets, including conventional DC and plasmacytoid DC. In this lab, long-term cultures (LTCs) established from murine spleen support continuous production of novel dendritic-like cells, termed LTC-DC. An in vivo equivalent subset also exists in spleen, namely L-DC. As co-cultures using LTC-derived splenic stroma support the outgrowth of L-DC from spleen and bone marrow sources, it is likely that spleen represents an important niche for DC development. To investigate the appearance of L-DC during ontogeny, spleen was isolated from embryonic and neonatal mice of different ages for analysis of myeloid and DC subsets. Perinatal spleen was also used to establish co-cultures for identification of progenitors, and LTCs were established from spleens for assessment of stromal competence. Although spleen from 16-day embryos (E16.5) contained myeloid cells, DC subsets did not appear until day 4 after birth (D4). However, murine spleen at D0 contained progenitors, which could seed co-cultures for L-DC production. LTC could not be established from spleen until D4. The appearance of L-DC after D4 in spleen is dependent on the formation of the appropriate stromal microenvironment which occurs in the early postnatal period.     

天然免疫模式识别受体与树突状细胞的发现和意义——2011年诺贝尔生理学或医学奖成果和相关研究简介

2011年的诺贝尔生理学或医学奖授予了Bruce A．Beutler,Jules A．Hoffmann以及Ralph M．Steinman教授,以奖励他们在天然免疫模式识别受体和树突状细胞研究领域所做出的开创性贡献。宿主的天然免疫系统依赖模式识别受体识别入侵的病原微生物,并通过树突状细胞对其加工处理将抗原提呈给T细胞,从而激活适应性免疫。回顾模式识别受体和树突状细胞发现的过程,介绍该领域最近的研究进展,并对它们在疾病预防和治疗中的应用进行了讨论。     

Recombinant cholera toxin B subunit activates dendritic cells and enhances antitumor immunity

Activation of dendritic cells (DC) is crucial for priming of cytotoxic T lymphocytes (CTL), which have a critical role in tumor immunity, and it is considered that adjuvants are necessary for activation of DC and for enhancement of cellular immunity. In this study, we examined an adjuvant capacity of recombinant cholera toxin B subunit (rCTB), which is non-toxic subunit of cholera toxin, on maturation of murine splenic DC. After the in vitro incubation of DC with rCTB, the expression of MHC class II and B7-2 on DC was upregulated and the secretion of IL-12 from DC was enhanced. In addition, larger DC with longer dendrites were observed. These data suggest that rCTB induced DC maturation. Subsequently, we examined the induction of tumor immunity by rCTB-treated DC by employing Meth A tumor cells in mice. Pretreatment with subcutaneous injection of rCTB-treated DC pulsed with Meth A tumor lysate inhibited the growth of the tumor cells depending on the number of DC. Moreover, intratumoral injection of rCTB-treated DC pulsed with tumor lysate had therapeutic effect against established Meth A tumor. Immunization with DC activated by rCTB and the tumor lysate increased number of CTL precursor recognizing Meth A tumor. The antitumor immune response was significantly inhibited in CD8+ T cell-depleted mice, although substantial antitumor effect was observed in CD4+ T cell-depleted mice. These results indicated that rCTB acts as an adjuvant to enhance antitumor immunity through DC maturation and that CD8+ T cells play a dominant role in the tumor immunity. Being considered to be safe, rCTB may be useful as an effective adjuvant to raise immunity for a tumor in clinical application.     

机体的免疫反应是如何被激活的？——2011年诺贝尔生理学或医学奖解读

2011年10月3日,举世瞩目的 2011年诺贝尔生理学或医学奖在瑞典卡罗林斯卡医学院揭晓[1].因在免疫学领域取得杰出成就,美国科学家布鲁斯.博伊特勒(Bruce A.Beutler)、法国科学家朱尔斯.霍夫曼(Jules A.Hoffmann)和加拿大科学家拉尔夫.斯坦曼(Ralph M.Steinman),共同分享了这一     

B lymphocyte regulation of the immune system. II. Inhibition of Fc receptor expression of lymphocytes by BEF, a lymphokine of B cell origin

Recently, we described a new lymphokine of B cell origin, capable of selectively preventing the differentiation of T suppressor cells from the precursor into the effector stage. As a result, antibody production against various antigens is markedly increased. We termed this lymphokine B cell-derived enhancing factor (BEF). To discern the mechanism(s) by which BEF interferes with the activation of T suppressor cells, experiments were undertaken to explore the effect of BEF on the induction of Fc receptors (FcR). The induction of FcR on T cells has been implicated in the down-regulation of antibody synthesis, and it has been suggested that the expression of FcR for a given immunoglobulin precedes the release of factors with regulatory functions for the corresponding isotype. In the experiments reported here, murine spleen cells were incubated for 24 hr in the presence of IgG1 or IgA monoclonal antibodies, were washed, and the number of FcR gamma 1+ and FcR alpha+ cells were calculated by a rosette assay. The effect of BEF was studied either during the inductive phase or before, i.e., by pretreating the cells with BEF for 18 hr at 37 degrees C before the inductive phase. Our results show that BEF abolishes, in a dose-dependent manner, the expression of isotype-specific FcR in spleen cells when present during the inductive phase, as well as when cells are pretreated with it. In successive experiments, we tested the effect of BEF on the induction of FcR on T cell-enriched or B cell-enriched spleen cells. The results show that BEF is effective in selectively inhibiting FcR expression on T lymphocytes, but not on B lymphocytes, once isolated from the total spleen cell population. These findings provide further insight into the mechanism by which BEF modulates the immune response, and suggest that different mechanisms may be involved in the induction of FcR on T and B lymphocytes, respectively.     

Liver dendritic cells are less immunogenic than spleen dendritic cells because of differences in subtype composition

The unique immunological properties of the liver may be due to the function of hepatic dendritic cells (DC). However, liver DC have not been well characterized because of the difficulty in isolating adequate numbers of cells for analysis. Using immunomagnetic bead and flow cytometric cell sorting, we compared freshly isolated murine liver and spleen CD11c+ DC. We found that liver DC are less mature, capture less Ag, and induce less T cell stimulation than spleen DC. Nevertheless, liver DC were able to generate high levels of IL-12 in response to CpG stimulation. We identified four distinct subtypes of liver DC based on the widely used DC subset markers CD8alpha and CD11b. Lymphoid (CD8alpha+CD11b-) and myeloid (CD8alpha-CD11b+) liver DC activated T cells to a similar degree as did their splenic DC counterparts but comprised only 20% of all liver DC. In contrast, the two more prevalent liver DC subsets were only weakly immunostimulatory. Plasmacytoid DC (B220+) accounted for 19% of liver DC, but only 5% of spleen DC. Our findings support the widely held notion that liver DC are generally weak activators of immunity, although they are capable of producing inflammatory cytokines, and certain subtypes potently activate T cells.     

Bridging innate and adaptive immunity

The Nobel Prize in Physiology or Medicine for 2011 to Jules Hoffmann, Bruce Beutler, and the late Ralph Steinman recognizes accomplishments in understanding and unifying the two strands of immunology, the evolutionarily ancient innate immune response and modern adaptive immunity.     

RNA/nucleotide enhances antibody production in vitro and is moderately mitogenic to murine spleen lymphocytes.

Suppression of immune functions was demonstrated in both humans and animals when exogenous RNA was eliminated from the diet. However, direct actions of RNA/nucleotide on the immune system are virtually unknown. Thus, in this study, we explored effects of RNA and nucleotide on lymphocyte functions in vitro. Yeast whole RNA, which is free of endotoxin, was supplemented to culture media, and changes in mitogen responses, thymocyte proliferation, or in vitro antibody production by murine spleen lymphocytes were analyzed. Yeast whole RNA potentiated the proliferation of spleen lymphocytes and it also strikingly enhanced in vitro antibody production in response to sheep red blood cells at least 10-fold. However, it did not potentiate the proliferation of thymocytes (immature lymphocytes). These enhancing activities of yeast RNA were significantly reduced by RNAse treatment, but not by treatments with DNAse or polymyxin B. Certain mononucleotides exhibited less, but similar, action on murine spleen lymphocytes. The whole yeast RNA employed was already degraded to small nucleotide (less than 1 kb). Therefore, it may be suggested that certain components of RNA degraders can function as powerful immunomodulators, indicating that exogenous RNA or nucleotide may be important in facilitating immune responses under certain circumstances.     

Investigation into the prevalence of a novel dendritic‐like cell subset in vivo

A novel dendritic‐like cell subset termed L‐DC was recently identified in murine spleen based on marker expression of a homogeneous cell population derived from long‐term culture of neonatal spleen. The function of L‐DC is distinct from other splenic dendritic and myeloid cell subsets because of their high endocytic capacity and their ability to cross‐present antigen to CD8⁺ T cells. This paper shows the subset to be unique to spleen and blood, with a similar, but possibly functionally distinct subset also present in bone marrow. The prevalence of the subset is low; ~6% of all dendritic and myeloid cells in the spleen and ~5% in blood. However, they are a distinct cell type on the basis of marker expression, and endocytic and T‐cell stimulatory capacity. Attempts to identify an enriched population of these cells in mutant mouse strains with reported increases in myelopoiesis showed either a lack of L‐DC or an altered phenotype reflective of the phenotype of the mouse strain.     

Dendritic cells bask in the limelight

Ralph Steinman is to receive the 2007 Albert Lasker Award for Basic Medical Research for his discovery of dendritic cells and his path-breaking work demonstrating their central role as the principal antigen-presenting cells of the immune system and key activators of T cell responses.     

In vivo and in vitro effects of monoclonal antibody to Ly antigens on immunity to infection

Injection of CBA mice with Brucella abortus strain 19 leads to chronic infection during which both cell-mediated immunity (delayed hypersensitivity and macrophage activation) and antibody production occur. Protection was efficiently transferred to naive mice using spleen cells from mice infected 5 or 12 weeks earlier. Selective lysis in vitro of these cells by antibody to cell surface antigens showed that Thy-1⁺ Ly-1⁺2⁺ T lymphocytes were required for transfer. Treatment with anti-Ia serum neither suppressed nor enhanced adoptive transfer. Thus Ia⁺ B lymphocytes were not required, and Ia⁺ suppressor T cells were not active in the response. Three injections per week of anti-Ly-1 monoclonal antibody beginning 5 days before infection led to a 10-fold increase in bacterial numbers 25 days after infection when acquired immunity was well established in untreated mice. The delayed hypersensitivity response was unaffected. In addition cells from these in vivo treated mice were unable to transfer resistance. Beginning the treatment on the day of infection abolished the IgG antibody response without affecting bacterial numbers. The studies emphasize the unique role of Ly-1⁺2⁺ T cells in immunity to Brucella and indicate the usefulness of these techniques in dissecting out those components of the immune response which contribute to recovery from infection.     

Natural killer dendritic cells have both antigen presenting and lytic function and in response to CpG produce IFN-gamma via autocrine IL-12

We have isolated rare cells bearing the NK cell surface marker NK1.1, as well as the dendritic cell (DC) marker CD11c, from the spleen, liver, lymph nodes, and thymus of normal mice. These cells possess both NK cell and DC function because they can lyse tumor cells and subsequently present Ags to naive Ag-specific T cells. Interestingly, in response to IL-4 plus either IL-2 or CpG, NKDC produce more IFN-gamma than do DC, or even NK cells. We determined that CpG, but not IL-2, induces NKDC to secrete IFN-gamma via the autocrine effects of IL-12. In vivo, CpG dramatically increases the number of NKDC. Furthermore, NKDC induce greater Ag-specific T cell activation than do DC after adoptive transfer. Their unique ability to lyse tumor cells, present Ags, and secrete inflammatory cytokines suggests that NKDC may play a crucial role in linking innate and adaptive immunity.     

Myelopoiesis in spleen-producing distinct dendritic-like cells

Dendritic cells (DC) represent a heterogeneous class of antigen presenting cells (APC). Previously we reported a distinct myeloid dendritic-like cell present in spleen, as an in vivo counterpart to cells produced in murine spleen long-term cultures (LTC-DC). These cells, named 'L-DC', were found to be functionally and phenotypically distinct from conventional (c)DC, plasmacytoid (p)DC and monocytes. These results suggested that spleen may represent a niche for development of L-DC from endogenous progenitors. Adult murine spleen has now been investigated for the presence of L-DC progenitors. Lineage-negative (Lin)(-) ckit(lo) and Lin(-) ckit(hi) progenitor subsets were identified as candidate populations, and tested for ability to produce L-DC; in vitro upon co-culture with the spleen stromal line STX3, and in vivo after adoptive therapy into mice. Both subsets colonized STX3 stroma in vitro for L-DC production, indicating that they contained either a common or two distinct progenitors for L-DC. However, only the Lin(-) ckit(hi) subset gave progeny cells after adoptive transfer into lethally irradiated mice. In vivo development was however multilineage and not restricted to L-DC development. Multilineage reconstitution reflects long-term reconstituting haematopoietic stem cells (LT-HSC), suggesting a close relationship between L-DC progenitors and LT-HSC. L-DC were however produced in vivo in much higher number than monocytes/macrophages and cDC, indicating the presence of a specific L-DC progenitor within the Lin(-) ckit(hi) subset. A model is advanced for development of L-DC directly from haematopoietic progenitors in spleen and dependent on the spleen microenvironment.     

Differential expression of a surface antigen recognized by a monoclonal antibody, J11d, on unprimed and primed B cells

Functional studies of both polyclonal and antigen-specific responses have suggested that murine B cells differ in the expression of an antigen recognized by a rat anti-mouse monoclonal antibody, called J11d. Using both positive and negative selection, we now demonstrate that the J11d marker is differentially displayed on B lymphocytes responding to LPS vs anti-mu, as well as on unprimed vs specific antigen-primed B cells. Thus, cytotoxic elimination of cells expressing high levels of J11d (J11d-hi) reduced LPS-driven B cell proliferation by 60 to 80% but had no effect on anti-mu stimulated B cell growth. Interestingly, equal numbers of positively selected J11d-hi B cells responded similarly to LPS and anti-mu plus B cell growth factors, a result that suggests that the response to anti-mu of the J11d-lo B cells is normally masked by the majority J11-d-hi cells. In further studies, the primary PFC response of normal murine spleen cells to fluorescein (FL)-coupled TI antigens or to LPS in vitro was reduced dramatically by cytotoxic J11d antibody treatment. In contrast, the anti-FL PFC response of spleen cells from mice primed 1 wk previously with FL-Ficoll was not affected by J11d antibody treatment, whereas the response of these FL-primed B cells to TNP (to which the mice were not primed) was greatly reduced by J11d + complement treatment. Our data indicate that antigen-experienced (activated) B cells are primarily found in the J11d-lo B cell subset and that unprimed (resting) B cells are found in the J11d-hi population, although both populations of murine B cells can respond to anti-mu. These studies also provide further evidence for B cell heterogeneity.     

Dramatic efficacy improvement of a DC-based vaccine against AML by CD25 T cell depletion allowing the induction of a long-lasting T cell response

Dendritic cell (DC)-based vaccination is a promising approach to enhance anti-tumor immunity that could be considered for acute myeloid leukemia (AML) patients with high-risk of relapse. Our purpose was to study the efficiency and to optimize the immunogenicity of a DC-based vaccine in a preclinical AML murine model. In this report, C57BL6 mice were vaccinated with DC pulsed with peptides eluted (EP) from the syngeneic C1498 myelomonocytic leukemic cell line in a prophylactic setting. In this model, a natural antileukemic immunity mediated by NK cells was observed in the control unloaded DC-vaccinated group. On the other hand, we showed that the cytotoxic antileukemic immune response induced by vaccination with eluted peptides pulsed-DC (DC/EP), in vitro and in vivo, was mainly mediated by CD4⁺ T cells. Treatment with anti-CD25 antibody to deplete CD4⁺ CD25⁺ regulatory T cells before DC-vaccination dramatically improved the antileukemic immune response induced by immunization, and allowed the development of long-lasting immune responses that were tumor protective after a re-challenge with leukemic cells. Our results suggest that this approach could be successful against weakly immunogenic tumors such as AML, and could be translated in human.     

Intestinal dendritic cell subsets: differential effects of systemic TLR4 stimulation on migratory fate and activation in vivo

Dendritic cells (DC) present peripheral Ags to T cells in lymph nodes, but also influence their differentiation (tolerance/immunity, Th1/Th2). To investigate how peripheral conditions affect DC properties and might subsequently regulate T cell differentiation, we examined the effects of a potent DC-activating, TLR-4-mediated stimulus, LPS, on rat intestinal and hepatic DC in vivo. Steady-state rat intestinal and hepatic lymph DC are alpha(E2) integrin(high) (CD103) and include two subsets, signal regulatory protein alpha (SIRPalpha)(hi/low), probably representing murine CD8alphaalpha(-/+) DC. Steady-state lamina propria DC are immature; surface MHC class II(low), but steady-state lymph DC are semimature, MHC class II(high), but CD80/86(low). Intravenous LPS induced rapid lamina propria DC emigration and increased lymph DC traffic without altering SIRPalpha(high)/SIRPalpha(low) proportions. CD80/86 expression on lymph or mesenteric node DC was not up-regulated after i.v. LPS. In contrast, i.v. LPS stimulated marked CD80/86 up-regulation on splenic DC. CD80/86 expression on intestinal lymph DC, however, was increased after in vitro culture with TNF-alpha or GM-CSF, but not with up to 5 mug/ml LPS. Steady-state SIRPalpha(low) DC localized to T cell areas of mesenteric nodes, spleen, and Peyer's patch, whereas SIRPalpha(high) DC were excluded from these areas. Intravenous LPS stimulated rapid and abundant SIRPalpha(high) DC accumulation in T cell areas of mesenteric nodes and spleen. In striking contrast, i.v. LPS had no effect on DC numbers or distribution in Peyer's patches. Our results suggest that any explanation of switching between tolerance and immunity as well as involving changes in DC activation status must also take into account differential migration of DC subsets.