Humoral anti-KLH responses in cancer patients treated with dendritic cell-based immunotherapy are dictated by different vaccination parameters

Purpose

Keyhole limpet hemocyanin (KLH) attracts biomedical interest because of its remarkable immunostimulatory properties. Currently, KLH is used as vaccine adjuvant, carrier protein for haptens and as local treatment for bladder cancer. Since a quantitative human anti-KLH assay is lacking, it has not been possible to monitor the dynamics of KLH-specific antibody (Ab) responses after in vivo KLH exposure. We designed a quantitative assay to measure KLH-specific Abs in humans and retrospectively studied the relation between vaccination parameters and the vaccine-induced anti-KLH Ab responses.

Experimental design

Anti-KLH Abs were purified from pooled serum of melanoma patients who have responded to KLH as a vaccine adjuvant. Standard isotype-specific calibration curves were generated to measure KLH-specific Ab responses in individual serum samples using ELISA.

Results

KLH-specific IgM, IgA, IgG and all IgG-subclasses were accurately measured at concentrations as low as 20?μg/ml. The intra- and inter-assay coefficients of variation of this ELISA were below 6.7 and 9.9?%, respectively. Analyses of 128 patients demonstrated that mature DC induced higher levels of KLH-specific IgG compared to immature DC, prior infusion with anti-CD25 abolished IgG and IgM production and patients with locoregional disease developed more robust IgG responses than advanced metastatic melanoma patients.

Conclusions

We present the first quantitative assay to measure KLH-specific Abs in human serum, which now enables monitoring both the dynamics and absolute concentrations of humoral immune responses in individuals exposed to KLH. This assay may provide a valuable biomarker for the immunogenicity and clinical effectiveness of KLH-containing vaccines and therapies.     

The effect of hydrocortisone on lymphocyte-mediated cytolysis

This paper reports the results of experiments designed to analyze the mechanism by which hydrocortisone suppresses the cell-mediated cytolysis produced by sensitized lymphocytes. We used an in vitro system in which rat lymph node cells were sensitized to, and caused cytolysis of mouse fibroblasts.We found that hydrocortisone probably suppresses cytolysis by preventing the primary activation of the cytolytic mechanism by target cell antigens. Suppression was most efficient when hydrocortisone was added at the beginning of the cytolytic reaction. The cytolytic mechanism itself appeared to remain intact, and could be activated by the lectin concanavalin A (con A) despite the presence of hydrocortisone.Suppression of cytolysis could not be related to any general inhibition of DNA, RNA, or protein synthesis. The influence of hydrocortisone on cytolysis was not modified by vitamin A (retinol), an agent which antagonizes the effect of hydrocortisone on lysosome membranes.Hydrocortisone was found to be less effective in suppressing the activity of lymphocytes that had been sensitized initially in the presence of hydrocortisone.     

基于对虾白斑综合症病毒极早期启动子的新型杆状病毒表达载体的构建与分析

摘要：【目的】构建携带有受杆状病毒多角体启动子控制的疱疹性口腔炎病毒糖蛋白(vesicular stomatitis virus glycoprotein, VSV G)和受白斑综合症病毒极早期基因(immediately-early gene 1,ie1)启动子控制的绿色荧光蛋白(enhanced green fluorescent protein, EGFP)两个表达阅读框的新型重组病毒vAc-G-EGFP,分析其在无脊椎动物和脊椎动物细胞系中表达报道基因的能力。【方法】 利用Bac-To-Bac 系统构建重组杆状病毒,利用病毒感染或转导实验介导报道基因在待测细胞系中的表达,用荧光显微镜和免疫印迹技术分析报道基因在待测细胞系中的实时表达情况。 【结果】成功构建了分别含VSV G 和 ie1启动子两个阅读框的重组杆状病毒vAc-G-EGFP,发现vAc-G-EGFP可以在无脊椎和脊椎动物细胞系中有效表达报道基因EGFP,免疫印迹实验显示,在不同时间点EGFP于这两类细胞中的表达存在差异。【结论】 基于白斑综合症病毒ie1启动子并携带有VSV G表达框的单一杆状病毒载体可以实现同时在不同种类细胞系中有效表达外源基因。本文构建的新型杆状病毒表达载体有希望普遍应用于基础和应用生物学研究。     

Selective depletion and enrichment of alloreactive cytolytic effector lymphocytes using anti-fluorescein affinity columns

Peritoneal exudates from BALB/c mice rejecting C57BL ascites lymphoma EL4 are a rich source of cytolytic effector lymphocytes (CL); however, these preparations are still contaminated even after removal of adherent cells with other mononuclear cells which do not appear to be cytolytic. The relationship of the cytolytic and “non-cytolytic” cells to graft rejection in vivo is not completely understood. We have used anti-fluorescein (α-FL) columns to separate sensitized lymphoid populations into fractions enriched or depleted in cytolytic activity. EL4 were directly labeled with fluorescein isothiocyanate (FL-EL4), centrifuged with CL and the mixture was applied to a column of horse α-FL antibody conjugated to Sepharose 4B. FL-EL4 and lymphocytes bound to them were retained on the column, while non-bound lymphocytes were collected in a medium wash (passed cells). CL bound to FL-EL4 were then eluted with EDTA (eluted cells). Cytolytic activity of the two fractions was compared to that of the unfractionated population in an in vitro⁵¹Cr release assay. Passed cells were consistently depleted in cytolytic activity compared to unfractionated cells or manipulation controls reaching 100% depletion in some experiments. Enrichment of cytolytic activity in eluted populations was frequently but not invariably observed. Rate of cytolysis was used as a measure of cytolytic activity in fractionated populations. Specificity of binding was investigated in reciprocal experiments using CS7BL/6J effectors raised against BALB/c lymphoma RL♂ 1. Viability of recovered cells was high and the procedure was rapid, efficient and versatile. In contrast to monolayer cellular immunoabsorbents, contamination of fractions with absorbing cells was consistently less than 5%. Both enriched and depleted populations are available for further study of surface markers and function.     

Reversal of phorbol ester-mediated reduction of cloned T lymphocyte cytolysis by interleukin 2

During previous studies on the regulation of cloned T lymphocyte function, we observed that murine cytotoxic T lymphocyte (CTL) clones progressively lose the ability to lyse appropriate target cells during prolonged (24 to 48 hr) incubation with the tumor promoter phorbol myristic acetate (PMA). We further observed that the cytolytic function of PMA-treated CTL clones can be restored by incubation with secondary MLC supernatant (2 degrees MLC SN), a potent source of cytokines. We now report our observations on the nature of the cytokine(s) responsible for recovery of CTL activity. Like 2 degrees MLC SN, the lectin-induced SN from a cloned helper T cell and the lectin-induced SN from a T cell hybridoma can restore cytolytic activity to cloned CTL treated with PMA. In contrast, supernatants from L929 cells, WEHI-3 cells, and P388D1 cells fail to restore cytolytic activity to similarly treated cloned CTL. These data suggest that IL 2 and/or gamma-IFN, but not CSF-1, CSF-GM, IL 3, or IL 1, can influence expression of cytolysis by cloned CTL. Furthermore, highly purified IL 2 can restore cytolytic activity, even when cytosine arabinoside is present to inhibit clonal expansion. Our studies indicate that cytolysis is a reversible function of cloned CTL, and that cytolysis may not necessarily represent an end-stage feature of CTL maturation. Our studies further show that IL 2 is both necessary and sufficient for resumption of cytolytic function by "deactivated" CTL. As such, these observations suggest that IL 2 can regulate not only T cell proliferation but also the expression of cytolysis by some cytolytic T cell populations.     

The neutrophil granule protein cathepsin G activates murine T lymphocytes and upregulates antigen-specific IG production in mice

Cathepsin G is a neutrophil granule derived antimicrobial chymotrypsin-like enzyme. Our previous study showed that cathepsin G induces chemotactic migration of human phagocytic leukocytes and increases random migration of T lymphocytes. In this study, we investigated the capacity of cathepsin G to activate T lymphocytes and to modulate antigen-specific humoral responses in mice. We found that cathepsin G is mitogenic for and induces production of IFN-gamma by murine T cells in vitro. Injection of cathepsin G in BALB/c mice immunized with keyhole limpet hemocyanin (KLH) adsorbed to aluminum hydroxide resulted in a significantly increased production of KLH-specific IgG1 and IgG2a antibodies. There was a dose-dependent increase in KLH-specific proliferation of lymphocytes from draining lymph nodes from mice treated with KLH and cathepsin G when compared with those treated with KLH alone. Subsequent analysis of IFN-gamma and IL-4 release following in vitro re-stimulation of draining lymph node lymphocytes obtained from KLH-immunized mice suggested that cathepsin G augments KLH-specific Ig antibody production via activation of T cells, presumably involving both Th1 and Th2 pathways. Thus, neutrophil granule cathepsin G, in addition to its capacity to kill microbes and to enhance leukocyte motility, activates T lymphocytes and modulates humoral immunity.     

T cell regulation of B cell activation. An antigen-mediated tripartite interaction of Ts cells, Th cells, and B cells is required for suppression

To determine the requirements underlying the antigen specificity observed in T cell-mediated immune response suppression, cloned major histocompatibility complex (MHC)-restricted T suppressor (Ts) cells specific for keyhole limpet hemocyanin (KLH) and cloned MHC-restricted T helper (Th) cells specific for fowl gamma-globulin (FGG) were employed to study the regulation of trinitrophenyl (TNP)-specific B cell responses. Neither antigen bridging between Ts cells and Th cells (FGG=KLH) nor bridging between Ts cells and B cells (TNP-KLH) was sufficient to allow suppression; a mixture of FGG=KLH and TNP-KLH was also insufficient for suppression. In contrast, suppression was induced by KLH-specific Ts cells only when suppressor determinants (KLH), helper determinants (FGG), and B cell determinants (TNP) were covalently linked on the same molecule (TMP-FGG)=(TNP-KLH) or TNP-(FGG=KLH)). These findings imply that a tripartite antigen-mediated interaction of Ts cells, Th cells, and responding B cells is necessary for the mediation of this antigen-specific suppression.     

Precursor frequency of antigen-specific T cells: effects of sensitization in vivo and in vitro

Limiting dilution analysis (LDA) of primary lymphocyte cultures was used to determine the frequency of keyhole limpet hemocyanin (KLH)-specific precursors in the peripheral blood of unimmunized individuals. The KLH-specific precursor frequencies ranged from 1:150,000 to 1:340,000. In contrast, frequencies of KLH-specific cells in the blood from immune donors ranged from 1:25,000 to 1:42,000. LDA of KLH-stimulated primary cultures indicated that the frequency of KLH-specific cells increased with time in culture reaching a four- to fivefold expansion relative to the frequency obtained prior to culture. The data presented suggest that the enhanced kinetics of secondary T-cell responses observed after in vitro sensitization are due to a decrease in the proportion of lymphocytes which exhibit a suppressor phenotype.     

Phorbol ester mediates reversible reduction of cloned T lymphocyte cytolysis

Prolonged contact with nanomole to micromole concentrations of the tumor promoter phorbol myristate acetate causes a significant reduction in the lytic activity of cloned cytolytic T cells. Diminished lysis is apparent even in the presence of agglutinating lectins. PMA does not exert this effect by minimizing clone viability. In fact, these concentrations of PMA cause significant potentiation of antigen-driven proliferation for many of these same clones. The PMA-mediated loss of cytolysis is reversible. Although contact with antigen does not induce or enhance reexpression of cytolysis, PMA-treated cytolytic T cell clones display normal cytolytic activity after contact with lymphokines.     

Clonal anergy of memory B cells in epitope-specific regulation.

Immunization of carrier (keyhole limpet hemocyanin (KLH)) primed mice with the hapten-carrier TNP-KLH induces specific suppression for the IgG anti-TNP-response without interfering with the response to epitopes on the carrier molecule. To examine the status of hapten-specific memory B cells from suppressed mice, highly enriched populations of TNP-specific memory B cells were purified from the spleen of TNP-KLH (control) or KLH/TNP-KLH (suppressed) immunized mice and tested in vitro for their ability to respond to TD or TI (TNP-KLH, TNP-LPS) antigenic challenge in presence of a KLH-specific Th cell line. Similar numbers of TNP-specific B cells with the characteristics of memory B cells were obtained from control and suppressed mice. TNP-specific B cells from suppressed mice could be triggered to IgG production by TNP-LPS but had an impaired ability to differentiate into IgG-secreting cells in response to TNP-KLH. This impaired IgG response to TNP-KLH was not due to an active suppression by a subset of TNP-specific B cells, or to an impedence of memory cells to a class switching but to an intrinsic memory B cell defect. TNP-specific B cells from suppressed mice were as efficient as memory B cells from control mice to present TNP-KLH to KLH-specific Th cells and to proliferate in response to T cell help. Our data support the view that the effector mechanism of epitope specific regulation does not interfere with the development of hapten-specific memory B cells but that these cells have an intrinsic defect that prevents their differentiation into active IgG antibody secreting cells in response to a T-dependent antigenic challenge.     

Characterisation of AmphiAmR4, an amphioxus (Branchiostoma floridae) α2-adrenergic-like G-protein-coupled receptor

Little is known about the evolutionary relationship between vertebrate adrenergic receptors and invertebrate octopamine and tyramine receptors. The complexity of the adrenergic signalling system is believed to be an innovation of the vertebrate lineage but the presence of noradrenaline has been reported in some invertebrate species. The cephalochordate, amphioxus (Branchiostoma floridae), is an ideal model organism for studying the evolution of vertebrate GPCRs, given its unique position at the base of the chordate lineage. Here, we describe the pharmacological characterisation and second messenger coupling abilities of AmphiAmR4, which clusters with α₂-adrenergic receptors in a phylogenetic tree but also shares a high sequence similarity to invertebrate octopamine/tyramine receptors in both BLAST and Hidden Markov Model analyses. Thus, it was of particular interest to determine if AmphiAmR4 displayed similar functional properties to the vertebrate α₂-adrenergic receptors or to invertebrate octopamine or tyramine receptors. When stably expressed in Chinese hamster ovary (CHO) cells, noradrenaline couples the receptor to both the activation of adenylyl cyclase and to the activation of the MAPKinase pathway. Pharmacological studies with a wide range of agonists and antagonists suggest that AmphiAmR4 functions as an α₂-adrenergic-like receptor when expressed in CHO cells.     

Ontogeny of Adaptive Antibody Response to a Model Antigen in Captive Altricial Zebra Finches

Based on studies from the poultry literature, all birds are hypothesized to require at least 4 weeks to develop circulating mature B-cell lineages that express functionally different immunoglobulin specificities. However, many altricial passerines fledge at adult size less than four weeks after the start of embryonic development, and therefore may experience a period of susceptibility during the nestling and post-fledging periods. We present the first study, to our knowledge, to detail the age-related changes in adaptive antibody response in an altricial passerine. Using repeated vaccinations with non-infectious keyhole limpet hemocyanin (KLH) antigen, we studied the ontogeny of specific adaptive immune response in altricial zebra finches Taeniopygia guttata. Nestling zebra finches were first injected at 7 days (7d), 14 days (14d), or 21 days post-hatch (21d) with KLH-adjuvant emulsions, and boosted 7 days later. Adults were vaccinated in the same manner. Induced KLH-specific IgY antibodies were measured using ELISA. Comparisons within age groups revealed no significant increase in KLH-specific antibody levels between vaccination and boost in 7d birds, yet significant increases between vaccination and boost were observed in 14d, 21d, and adult groups. There was no significant difference among age groups in KLH antibody response to priming vaccination, yet KLH antibody response post-boost significantly increased with age among groups. Post-boost antibody response in all nestling age groups was significantly lower than in adults, indicating that mature adult secondary antibody response level was not achieved in zebra finches prior to fledging (21 days post-hatch in zebra finches). Findings from this study contribute fundamental knowledge to the fields of developmental immunology and ecological immunology and strengthen the utility of zebra finches as a model organism for future studies of immune ontogeny.     

Ancient Cytokines,the Role of Astakines as Hematopoietic Growth Factors

Hematopoiesis is the process by which hemocytes mature and subsequently enter the circulation. Vertebrate prokineticins (PKs) are known to take part in this process, as are the invertebrate prokineticin domain proteins, astakines. In Pacifastacus leniusculus, astakine 1 is essential for the release of new hemocytes into the open circulatory system of these animals. In addition to astakine 1, we have now cloned a homologue of astakine 1 with an insert of 13 amino acids, named as astakine 2. Both crustacean astakines lack the N-terminal AVIT motif, which is present in vertebrate PKs, and hence receptor binding differs from that of vertebrate PKs. We have found astakine-like sequences in 19 different invertebrate species, and the sequences show that some motifs are conserved among invertebrate groups. Previously we showed that astakine 1 is directly involved in hematopoiesis, and now we show that astakine 1 and astakine 2 have different roles in hemocyte lineage differentiation. Astakine 1 can stimulate proliferation of hematopoietic tissue (Hpt) cells (precursor of hemocytes) as well as specifically induce differentiation of Hpt cells along the semigranular cell lineage, whereas astakine 2 plays a role in granular cell differentiation. Moreover, we discuss the impact of the putative structures of different astakines in comparison with the vertebrate prokineticins.     

Interleukin-17 in pearl oyster (Pinctada fucata): Molecular cloning and functional characterization

IL-17 from pearl oyster Pinctada fucata, one of mollusk, was identified and characterized, and its genomic structure and promoter were analyzed. The full-length cDNA of P. fucata IL-17 (PfIL-17) is 907 bp with an open reading frame of 585 bp encoding a putative protein of 194 amino acids. The deduced PfIL-17 contains a 19 amino acid signal peptide and a conserved IL-17 domain. Multiple sequence alignments and phylogenetic analysis revealed that PfIL-17 has lower similarity with other invertebrate IL-17 and was clustered with CgIL-17, but not clustered with other invertebrate IL-17. Gene expression analysis indicated that PfIL-17 took part in the immune response to LPS and poly(I:C) stimulation, and dual-luciferase reporter assays showed that PfIL-17 could active vertebrate target genes containing the NF-κB binding site and involve NF-κB signal pathway in HEK293 cells. Combined with the results mentioned above, it is suggested that PfIL-17 might involve and activate NF-κB signal pathway against extracellular pathogens.     

‘Lymphocyte-like’cells in ascidians: Precursors for vertebrate lymphocytes?

There has been much speculation that the evolutionary precursors of vertebrate lymphocytes may exist in the ascidians (proto chordates), but conclusive evidence has remained elusive especially as membrane bound immunoglobulin has not been detected in any invertebrate. This paper reviews new evidence which indicates that the ‘lymphocyte-like’ cells in ascidians have functional, as well as morphological, similarities with vertebrate lymphocytes. Firstly, these cells have been linked with in vivo non-self recognition events such as allograft rejection in solitary ascidians and non-fusion reactions between colonial ascidians. Secondly, they have been shown to be cytotoxic towards xenogenic targets in vitro and to use cytolytic mechanisms similar to those of cytotoxic T-cells. Thirdly, they are able to proliferate in vitro in response to mitogens or allogeneic cells. It is therefore suggested that, apart from immunoglobulin production and clonal selection, there is persuasive evidence that the ‘lymphocyte-like’ cells of ascidians constitute a primordial form of vertebrate lymphocyte.     

Decreased rate of cytolysis of Colpidium striatum by cystic fibrosis serum. I. Bioassay and evidence for the possible involvement of a C/F factor--IgG complex

Treatment of the protozoan ciliate $\text{Colpidium}$$\text{striatum}$ with whole serum from cystic fibrosis (C/F) patients, their parents (obligate heterozygote carriers), and normal controls uniformly caused complete cytolysis of the protozoa. At a 1:10 dilution of serum, a distinct decrease in the rate of cytolysis was found for 16 out of 19 cystic fibrosis patients, and 12 out of 16 heterozygote samples. An investigation into the cause of the decreased cytolytic rate indicated that a serum component associated with the IgG fraction from homozygote and heterozygote serum was responsible for the decreased rate.     

Induction of Systemic and Mucosal Immune Responses to Human Immunodeficiency Virus Type 1 by a DNA Vaccine Formulated with QS-21 Saponin Adjuvant via Intramuscular and Intranasal Routes

Induction of mucosal and cell-mediated immunity is critical for development of an effective vaccine against human immunodeficiency virus (HIV). We compared intramuscular and intranasal immunizations with a DNA vaccine encoding env of HIV-1 and evaluated the QS-21 saponin adjuvant for augmentation of the systemic and mucosal immune responses to HIV-1 in a murine model. Vaccination via the two routes elicited comparable systemic immune responses, and QS-21 consistently enhanced antigen-specific serum immunoglobulin G2a (IgG2a) production, delayed-type hypersensitivity reaction, and cytolytic activity of splenocytes. Intestinal secretory IgA production and cytolytic activity of the mesenteric lymph node cells are preferentially elicited by intranasal immunization, and QS-21 augmented these activities as well. This adjuvant augmented production of interleukin-2 (IL-2) and gamma interferon (IFN-γ) associated with decrease in IL-4 synthesis by antigen-restimulated splenocytes. The serum immunoglobulin subtype profile showed a dominant IgG2a response and less strong IgG1 and IgE production in a QS-21 dose-dependent manner. As expected, enhancements of humoral and cell-mediated immune responses by QS-21 were abrogated by treatment with anti-IL-2 and anti-IFN-γ monoclonal antibodies. These results suggest that the intranasal route of DNA immunization is more efficient than the intramuscular route in inducing mucosal immunity mediated by sIgA and mesenteric lymphocytes. Furthermore, QS-21 is able to act as a mucosal adjuvant in DNA vaccination and demonstrates its immunomodulatory property via stimulation of the Th1 subset. This study emphasizes the importance of the route of immunization and the use of an adjuvant for effective DNA vaccination against HIV-1.     

An inhibitor specific for the mouse T-cell associated serine proteinase 1 (TSP-1) inhibits the cytolytic potential of cytoplasmic granules but not of intact cytolytic T cells

We have investigated a proteinase inhibitor, designed according to the preferred amino acid sequence that is cleaved by the murine T-cell specific serine proteinase 1 (TSP-1) for its effect on the cytolytic potential of cloned cytotoxic T-cell lines (CTLL) and of cytoplasmic granules, derived from these cells. Pretreatment of effector cells with H-D-Pro-Phe-Arg-chloromethyl-ketone (PFR-CK) prior to the cytotoxicity assay did not result in inhibition of cytolytic activity of three independent CTLL and did not effect their granule-associated TSP-1 activity after extraction with Triton X-100. Furthermore, PFR-CK did not interfere with cytolysis of target cells by CTLL when present for the entire incubation period. In contrast, PFR-CK inhibited in a dose-dependent manner both TSP-1 activity and the hemolytic/cytolytic potential of isolated cytoplasmic granules after their pretreatment with high-salt concentration. We interpret these results to mean that cytolysis of target cells by CTLL involves the granule-associated proteinase TSP-1, which probably becomes active upon exocytosis following effector-target cell interactions.     

IgM induction in murine B hybridomas with Ia-restricted T cell clones: a monoclonal model for T and B cell interactions in antibody response

The mechanism of MHC-restricted T and B cell interactions in antibody response was studied with IgM-inducible B hybridomas and antigen-specific helper T cell clones. B hybridomas were prepared by fusion between splenic B cells from (CBA/N (H-2k) X BALB/c (H-2d)) F1 (NBF1) male mice and a B lymphoma cell line, M12.4.5. A B hybridoma clone, 1M70, which expressed I-Ad but not I-Ak determinants was chosen in the present study. IgM secretion was induced in 1M70 when it was cocultured with a "resting" KLH-specific and H-2d restricted helper T cell clone in the presence of KLH. A "resting" KLH-specific and H-2k restricted T cell clone did not induce IgM secretion in 1M70 even in the presence of KLH. However, when these KLH-specific T cell clones were activated by KLH and appropriate antigen presenting cells, both H-2d and H-2k restricted T cell clones induced IgM secretion in 1M70 even in the absence of KLH. A monoclonal anti-I-Ad antibody inhibited IgM secretion induced by a "resting" H-2d restricted T cell clone, but not by an "activated" T cell clone. These results indicated that T cell clones recognized antigens in the context of Ia molecules on B hybridomas in a MHC-restricted manner and were activated to produce B cell stimulatory factors which in turn acted on B hybridomas in a non-MHC-restricted manner and induced differentiation of B hybridomas into IgM secreting cells.