Oyster Versatile IKKα/ßs Are Involved in Toll-Like Receptor and RIG-I-Like Receptor Signaling for Innate Immune Response.

IκB kinases (IKKs) play critical roles in innate immunity through signal-induced activation of the key transcription factors nuclear factor-κB (NF-κB) and interferon regulatory factors (IRFs). However, studies of invertebrate IKK functions remain scarce. In this study, we performed phylogenetic analysis of IKKs and IKK-related kinases encoded in the Pacific oyster genome. We then cloned and characterized the oyster IKKα/ß-2 gene. We found that oyster IKKα/ß-2, a homolog of human IKKα/IKKß, responded to challenge with lipopolysaccharide (LPS), peptidoglycan (PGN), and polyinosinic-polycytidylic acid [poly(I:C)]. As a versatile immune molecule, IKKα/ß-2 activated the promoters of NF-κB, TNFα, and IFNß, as well as IFN-stimulated response element (ISRE)-containing promoters, initiating an antibacterial or antiviral immune state in mammalian cells. Importantly, together with the cloned oyster IKKα/ß-1, we investigated the signal transduction pathways mediated by these two IKKα/ß proteins. Our results showed that IKKα/ß-1 and IKKα/ß-2 could interact with the oyster TNF receptor-associated factor 6 (TRAF6) and that IKKα/ß-2 could also bind to the oyster myeloid differentiation factor 88 (MyD88) protein directly, suggesting that oyster IKKα/ßs participate in both RIG-I-like receptor (RLR) and Toll-like receptor (TLR) signaling for the reception of upstream immune signals. The fact that IKKα/ß-1 and IKKα/ß-2 formed homodimers by interacting with themselves and heterodimers by interacting with each other, along with the fact that both oyster IKKα/ß proteins interacted with NEMO protein, indicates that oyster IKKα/ßs and the scaffold protein NEMO form an IKK complex, which may be a key step in phosphorylating IκB proteins and activating NF-κB. Moreover, we found that oyster IKKα/ßs could interact with IRF8, and this may be related to the IKK-mediated activation of ISRE promotors and their involvement in the oyster "interferon (IFN)-like" antiviral pathway. Moreover, the expression of oyster IKKα/ß-1 and IKKα/ß-2 may induce the phosphorylation of IκB proteins to activate NF-κB. These results reveal the immune function of oyster IKKα/ß-2 and establish the existence of mollusk TLR and RLR signaling mediated by IKKα/ß proteins for the first time. Our findings should be helpful in deciphering the immune mechanisms of invertebrates and understanding the development of the vertebrate innate immunity network.

The oyster immunity.

Oysters, the common name for a number of different bivalve molluscs, are the worldwide aquaculture species and also play vital roles in the function of ecosystem. As invertebrate, oysters have evolved an integrated, highly complex innate immune system to recognize and eliminate various invaders via an array of orchestrated immune reactions, such as immune recognition, signal transduction, synthesis of antimicrobial peptides, as well as encapsulation and phagocytosis of the circulating haemocytes. The hematopoietic tissue, hematopoiesis, and the circulating haemocytes have been preliminary characterized, and the detailed annotation of the Pacific oyster Crassostrea gigas genome has revealed massive expansion and functional divergence of innate immune genes in this animal. Moreover, immune priming and maternal immune transfer are reported in oysters, suggesting the adaptability of invertebrate immunity. Apoptosis and autophagy are proved to be important immune mechanisms in oysters. This review will summarize the research progresses of immune system and the immunomodulation mechanisms of the primitive catecholaminergic, cholinergic, neuropeptides, GABAergic and nitric oxidase system, which possibly make oysters ideal model for studying the origin and evolution of immune system and the neuroendocrine-immune regulatory network in lower invertebrates.

The first invertebrate NFIL3 transcription factor with role in immune defense identified from the Hong Kong oyster, Crassostrea hongkongensis.

NFIL3 (nuclear factor interleukin 3-regulated) is a basic leucine zipper type transcription factor that mediates a variety of immune responses in vertebrates. However, the sequence information and function of NFIL3 homologs in invertebrates, especially mollusks, remains unknown. In the present study, the first NFIL3 homolog was identified in a marine mollusk, Crassostrea hongkongensis (designated as ChNFIL3), followed by its functional characterization. The full-length cDNA of ChNFIL3 is 2221 bp and consists of an open reading frame (ORF) of 1536 bp that encodes a polypeptide of 551 amino acids. Simple Modular Architecture Research Tool (SMART) analysis indicated that ChNFIL3 has two basic leucin zipper domains, similar to the other known NFIL3 family proteins. Tissue distribution analysis of NFIL3 in this mollusk revealed high expression in digestive glands and hemocytes. A significant induction in the mRNA level of ChNFIL3 was observed following bacterial stimulation. ChNFIL3 was found to be localized in the nucleus and over expression of ChNIFL3 led to upregulation of transcriptional activity of an NF-κB reporter gene in HEK 293T cells, indicating its role in innate immunity. Furthermore, addition of exogenous recombinant ChNFIL3 proteins resulted in enhanced mRNA level of hemocyte interleukin 17 in vitro. In conclusion, our findings revealed that NFIL3 in molluscs, plays a conserved role in host defense, similar to its mammalian homolog.

Activation of dendritic cells by low molecular weight oyster polysaccharides.

Dendritic cells play a primary role in antigen presentation to CD4+ T cells, which initiate acquired immune responses. Therefore, determining positive modulators of dendritic cell activation to improve therapeutic approaches for cancer treatment might be useful. We here investigated the effects of low molecular weight oyster polysaccharides (LMW-OPS) on bone marrow-derived dendritic cells (BMDCs) obtained from mice. LMW-OPS increased the surface expression of major histocompatibility complex class II (MHC-II), CD40 and CD86 in BMDCs and induced the secretion of tumour necrosis factor (TNF)-α and interleukin (IL)-12, which were significantly decreased in the BMDCs derived from MyD88-/- mice but not from the lipopolysaccharide-resistant C3H/HeJ mice. BMDCs treated with LMW-OPS augmented allogeneic CD4+ T cell expansion and enhanced secretion of IL-2 and interferon (IFN)-γ but not IL-4. LMW-OPS induced significant increases in ERK and p38 MAPK phosphorylation, but not c-Jun N-terminal kinase (JNK) phosphorylation, in BMDCs. Our results indicate that, in part, LMW-OPS can induce maturation of BMDCs in a MyD88-dependent and Toll-like receptor (TLR) 4-independent manner. LMW-OPS may enhance acquired immunity by modulating the function of dendritic cells.

A novel siglec (CgSiglec-1) from the Pacific oyster (Crassostrea gigas) with broad recognition spectrum and inhibitory activity to apoptosis, phagocytosis and cytokine release.

Sialic acid binding immunoglobulin-type lectin (siglec) belongs to the immunoglobulin superfamily (IgSF), which acts as regulator involved in glycan recognition and signal transduction in the immune and nervous systems. In the present study, a siglec gene (designated CgSiglec-1) was characterized from the Pacific oyster, Crassostrea gigas. The cDNA of CgSiglec-1 was of 1251 bp encoding a predicted polypeptide of 416 amino acids. CgSiglec-1 was composed of two I-set immunoglobulin (Ig) domains, one transmembrane (TM) domain and two ITIM motifs, sharing a sequence similarity with vertebrate CD22 homologs. The mRNA expression of CgSiglec-1 could be detected in all the selected tissues, with the highest level in hemocytes and labial palps. The confocal analysis revealed that CgSiglec-1 mainly distributed on the cytoplasmic membrane of the oyster hemocytes. In addition, the mRNA transcripts of CgSiglec-1 in hemocytes increased significantly (4.29-fold to that of control group, p < 0.05) after Vibrio splendidus stimulation. The recombinant CgSiglec-1 protein (rCgSiglec-1) could bind to poly sialic acid (pSIAS), lipopolysaccharides (LPS) and peptidoglycan (PGN) in a dose-dependent manner. The blockade of CgSiglec-1 by specific polyclonal antibodies could enhance the LPS-induced cell apoptosis, phagocytosis towards V. splendidus and the release of cytokines, such as CgTNF-1, CgIFNLP and CgIL-17. The results collectively indicated that CgSiglec-1 could act as a bridge molecule between invader recognition and signal transduction cascade, and modulate the immune response by inhibiting various important processes of immunity in oyster.

A novel junctional adhesion molecule A (CgJAM-A-L) from oyster (Crassostrea gigas) functions as pattern recognition receptor and opsonin.

Junctional adhesion molecule (JAM), a subfamily of immunoglobulin superfamily (IgSF) with a couple of immunoglobulin domains, can act as regulator in homeostasis and inflammation of vertebrates. In the present study, a structural homolog of JAM-A (designated CgJAM-A-L) was screened out from oyster, Crassostrea gigas, through a search of JAM-A D1 domain (N-terminal Ig domain in JAM-A). The cDNA of CgJAM-A-L was of 1188 bp encoding a predicted polypeptide of 395 amino acids. The immunoreactive area of CgJAM-A-L mainly distributed over the plasma membrane of hemocytes. After Vibro splendidus or tumor necrosis factor (CgTNF-1) stimulation, the mRNA transcripts of CgJAM-A-L in hemocytes increased significantly by 4.46-fold and 9.00-fold (p < 0.01) of those in control group, respectively. The recombinant CgJAM-A-L protein (rCgJAM-A-L) could bind multiple PAMPs including lipopolysaccharides (LPS), peptidoglycan (PGN), lipoteichoic acid (LTA), mannose (MAN), ß-glucan (GLU) and poly(I:C), and various microorganisms including Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Vibro anguillarum, V. splendidus, Pastoris pastoris and Yarrowia lipolytica. The phagocytic rates of oyster hemocytes towards Gram-negative bacteria V. anguillarum and yeast P. pastoris were significantly enhanced after the incubation of rCgJAM-A-L, and even increased more significantly after the pre-incubation of rCgJAM-A-L with microbes (p < 0.01). The results collectively indicated that CgJAM-A-L functioned as an important pattern recognition receptor (PRR) and opsonin in the immune defense against invading pathogen in oyster. Moreover, as the most primitive specie with homolog of JAMs, the information of CgJAM-A-L in oyster would provide useful clues for the evolutionary study of JAMs and immunoglobulins.

A cytokine-like factor astakine accelerates the hemocyte production in Pacific oyster Crassostrea gigas.

Astakine has been reported to be a hematopoietic growth factor of prokineticin homolog firstly found in arthropods freshwater crayfish Pacifastacus leniusculus. In the present study, an astakine homologous gene was identified from Pacific oyster Crassostrea gigas (designated CgAstakine). The full length cDNA of CgAstakine encoded a polypeptide of 103 amino acids containing a prokineticin (PK) domain homologous to that in astakine from freshwater crayfish P. leniusculus. The deduced amino acid sequence of CgAstakine shared higher similarity with those of other invertebrate astakines than prokineticins from vertebrates. The mRNA of CgAstakine was highly expressed in hepatopancreas and adductor muscle of oyster, while the CgAstakine protein was mainly distributed in hepatopancreas, gill and hemocytes. The mRNA expression of CgAstakine in hemocytes was significantly increased (p < 0.01) and maintained at a high level from 3 h to 9 h after Vibrio anguillarum challenge. After the oyster hemocytes were incubated with 5 µg/mL recombinant CgAstakine protein (rCgAstakine) for 24 h in vitro, the proliferation of hemocytes was significantly increased to 1.89 fold of that in control group (p < 0.05). Moreover, the total count of oyster hemocytes was significantly upregulated (2.45 fold of that in control group, p < 0.05) at 12 h after the oysters were received an injection of rCgAstakine (0.5 µg/g). These results collectively indicated that CgAstakine could modulate the hemocytes proliferation both in vitro and in vivo, and probably involved in the hematopoietic process fighting against the invasion of foreign pathogens.

Parasites of QX-resistant and wild-type Sydney rock oysters (Saccostrea glomerata) in Moreton Bay, SE Queensland, Australia: diversity and host response.

Wild caught (WC) and QX resistant (QXR) Sydney rock oysters were introduced at North Stradbroke Island and Pimpama River, SE Queensland, Australia, and sampled monthly during 1 year. Three groups of parasites/diseases were identified by observation of histological sections: (1) Marteilia sydneyi (Queensland unknown (QX) disease) and Steinhausia sp. (Microsporidia) characterized by a high prevalence and deleterious impact on the host; (2) disseminated neoplasia and the trematode Proctoeces sp. characterized by low prevalence but deleterious effects on the host; (3) parasites or symbionts with no detectable effect on the host: trematodes, ciliates, turbellarians and metacestodes. Mortality rates were similar between both oyster lines but higher at Pimpama River (reaching around 90%) than Stradbroke Island, mostly because of QX disease and, to a lesser extent, to the unfavourable environmental conditions of the summer 2010-2011. Lower prevalences of QX disease at Stradbroke Island probably related to the relative lack of intermediate hosts of the parasite and to lower freshwater input. Surprisingly, no difference in prevalence of QX disease was observed between the two oyster lines.

Is more better? Polyploidy and parasite resistance.

Ploidy-level variation is common and can drastically affect organismal fitness. We focus on the potential consequences of this variation for parasite resistance. First, we elucidate connections between ploidy variation and key factors determining resistance, including allelic diversity, gene expression and physiological condition. We then argue that systems featuring both natural and artificially manipulated ploidy variation should be used to evaluate whether ploidy level influences host-parasite interactions.

A novel monoclonal antibody that binds to hemocytes from shrimps and oysters.

The monoclonal antibody (MAb) LITO-1 was produced from a stable hybridoma cell line generated by the fusion of NS1 myeloma cells with spleen cells isolated from Balb/c mice immunized with a paraformaldehyde-fixed hemocyte suspension of Litopenaeus vannamei. This MAb reacted with all three hemocyte subtypes, but no reaction was observed with components of plasma. Immunohistochemistry assays demonstrated that LITO-1 was very effective in specifically distinguishing hemocytes infiltrated in several tissues such as striated muscle, brain, and hepatopancreas. Moreover, this antibody was able to recognize hemocytes from two shrimp species, Litopenaeus schmitti and Farfantepenaeus paulensis, as well as hemocytes of the oyster Crassostrea gigas. No reaction was observed against hemocytes from the terrestrial insect Triatoma klugi or with mammalian RAW cells. This novel MAb can be useful in revealing the presence and function of a conservative epitope in hemocytes of marine crustaceans and mollusks.

Norovirus binds to blood group A-like antigens in oyster gastrointestinal cells.

AIMS: To determine if histo-blood group antigens (HBGA) present in oyster gastrointestinal (GI) cells mediate accumulation of human noroviruses (NoV) in oyster GI cells. METHODS AND RESULTS: HBGA-specific monoclonal antibodies (MAbs) were used to determine the presence of the corresponding HBGA in oyster GI cells. All oyster samples tested contained type A-like HBGA in GI tissue as measured by ELISA. Recombinant Norwalk virus viral like particles (rNVLP) were bound to plates coated with oyster GI homogenate. The binding was inhibited when rNVLPs were pre-incubated with MAbs specific for type A HBGA, or samples of human saliva from type A individuals. Co-localization of rNVLP and type A-like HBGA, but not type B-like or type H-like HBGA, on GI epithelial cells was observed by immunofluorescent histochemical staining and three-channel confocal scanning laser microscopy. CONCLUSION: Type A-like HBGA is present in oyster GI cells and responsible for binding of rNVLP. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the presence of type A-like HBGA in oyster GI cells and the specific binding of rNVLP to type A-like HBGA on oyster GI cells. The results of this study suggest that human NoV concentrate in oyster GI cells by specific binding to concentrated type A-like HBGA rather than by a nonmolecular entrapment within the tissues.

Norwalk virus-specific binding to oyster digestive tissues.

The primary pathogens related to shellfish-borne gastroenteritis outbreaks are noroviruses. These viruses show persistence in oysters, which suggests an active mechanism of virus concentration. We investigated whether Norwalk virus or viruslike particles bind specifically to oyster tissues after bioaccumulation or addition to tissue sections. Since noroviruses attach to carbohydrates of the histo-blood group family, tests using immunohistochemical analysis were performed to evaluate specific binding of virus or viruslike particles to oyster tissues through these ligands. Viral particles bind specifically to digestive ducts (midgut, main and secondary ducts, and tubules) by carbohydrate structures with a terminal N-acetylgalactosamine residue in an alpha linkage (same binding site used for recognition of human histo-blood group antigens). These data show that the oyster can selectively concentrate a human pathogen and that conventional depuration will not eliminate noroviruses from oyster tissue.

Cadmium-induced apoptosis in oyster hemocytes involves disturbance of cellular energy balance but no mitochondrial permeability transition.

Exposure to environmentally prevalent heavy metals such as cadmium can have detrimental effects on a variety of commercially and ecologically important species such as oysters. Since Cd(2+) is known to induce apoptosis in immune cells of vertebrates, we have investigated the effects of this metal on isolated oyster hemocytes, the main cellular immune defense in mollusks. Enhanced apoptosis of these cells could conceivably create immunosuppressed conditions in these organisms and result in reduced disease resistance and increased opportunistic infection, resulting in decline of their populations. Cd(2+) exposure induced apoptosis in oyster hemocytes in a dose-dependent manner in the range of 10-100 micromol l(-1), as indicated by the translocation of phosphatidylserine to the outer leaflet of the plasma membrane. At higher concentrations (200-1000 micromol l(-1)), there was no further increase in apoptosis but a significant increase in the level of necrosis. In stark contrast to vertebrate immune cells, there was no decrease in the mitochondrial membrane potential or activation of caspases in response to Cd(2+) in the apoptotic range. Surprisingly, Cd(2+) exposure in this range did cause a significant decrease in intracellular ATP levels, indicating a severe disturbance of energy metabolism. Similarly, Cd(2+) exposure of isolated mitochondria resulted in partial uncoupling of mitochondria but no difference in mitochondrial membrane potential. The results demonstrate that the important environmental pollutant Cd(2+) induces apoptosis in oyster immune cells and does so through a mitochondria/caspase-independent pathway, suggesting that a novel, perhaps ancient, apoptotic pathway is active in these cells. Furthermore, it appears that the observed decrease in ATP production during apoptosis is not due to the loss of the mitochondrial proton-motive force but is more likely to be due to inhibition of the F(0)/F(1)-ATPase and/or mitochondrial ADP/ATP or substrate transport.

Cg-Rel, the first Rel/NF-kappaB homolog characterized in a mollusk, the Pacific oyster Crassostrea gigas.

We report here the identification and functional characterization of Cg-Rel, a gene encoding the Crassostrea gigas homolog of Rel/NF-kappaB transcription factors found in insects and mammals. Sequence and phylogenetic analysis showed that Cg-Rel shares the structural organization of Rel/NF-kappaB transcription factors of class II. It includes a Rel homology domain as well as a C-terminal transactivation domain (TD). Overexpression of Cg-Rel in the Drosophila S2 cell line activated the expression of a NF-kappaB-dependent reporter gene, whereas transfection with a Cg-Rel construct containing a C-terminal deletion of the TD or using a reporter gene with mutated kappaB binding sites failed to activate expression. These results suggest that Cg-Rel is a functional member of the Rel family of transcription factors, making this the sixth structurally homologous component of the Rel/NF-kappaB pathway characterized in C. gigas. Based on homology to other invertebrates' Rel/NF-kappaB cascade, the function of the oyster pathway may serve to regulate genes involved in innate defense and/or development. These findings serve to highlight a potentially important regulatory pathway to the study of oyster immunology, hence allowing comparison of the immune system in vertebrates and invertebrates, an important key issue to understand its evolution.

Characterization of a Tal/SCL-like transcription factor in the pacific oyster Crassostrea gigas.

We have cloned and characterized a cDNA encoding Cg-tal in the Pacific oyster Crassostrea gigas. The isolated cDNA encodes a 219 amino acids protein that contains the basic helix-loop-helix (bHLH) domain homologous to that of vertebrate and invertebrate Tal1/SCL. Phylogenetic analyses sustained that Cg-Tal belongs to this family of bHLH transcription factors. Northern blot analysis of Cg-tal mRNA expression in adult oyster tissues indicated that Cg-ta1 was specifically expressed in hemocytes, in a constitutive manner. In vertebrates, activation of Tal1/SCL expression is essential for the initiation of hematopoiesis and the formation of hematopoietic stem cells. Considering Tal1/SCL function in vertebrates, Cg-Tal is likely to constitute a promising tool for studying hematopoiesis in oyster.

Anaphylactic shock to oysters and white fish with generalized urticaria to prawns and white fish.

Because seafood consumption is moderate-to-high in Spain, allergic reactions to seafood such as fish, crustacea and mollusc are fairly frequent. The clinical features of these reactions depend on the implicated species and whether the reaction is provoked by ingestion, handling or vapor inhalation. Because different species have common antigenic structures, cross-sensitization is frequent, especially between crustaceans and molluscs. Contamination of fish by nematodes (Anisakis) may produce severe reactions. We report the case of a female patient with no personal or family history of allergy who experienced two episodes of anaphylactic shock: the first occurred immediately after eating oysters and the second after ingestion of white fish. The patient also developed generalized urticaria provoked by crustacean (prawns) and white fish. The results of skin prick tests were negative for fish, shellfish, crustacean and oysters while in vitro tests were positive for oyster, prawns, Anisakis, Ascaris and Echinococcus, although stool samples and gastric endoscopy were negative.

Oyster IKK-like protein shares structural and functional properties with its mammalian homologues.

In our search for genes involved in oyster immunity we isolated a cDNA encoding a polypeptide closely related to the mammalian IkappaB kinase (IKK) family. IKK proteins play a central role in cell signaling by regulating nuclear factor-kappaB (NF-kappaB) activation. We report here the cloning of an oyster IKK-like protein (oIKK) which possesses the characteristic organization of the mammalian IKK proteins, namely an amino-terminal kinase domain followed by a leucine zipper region and a carboxyl-terminal helix-loop-helix motif. When transfected into human cell lines, oIKK activated the expression of NF-kappaB-controlled reporter gene, whereas transfections with mutants of oIKK deleted within the kinase domain or within the helix-loop-helix motif respectively abolished and greatly reduced reporter gene activation. These results indicate that oIKK can replace the hIKK-alpha in catalyzing NF-kappaB nuclear translocation, and in triggering gene expression. Our results sustain the concept of an evolutionarily conserved signaling machinery in which IKK plays a major role.

A comparison of the chemiluminescent response of Crassostrea virginica and Morone saxatilis phagocytes to zymosan and viable Listonella anguillarum.

If reactive oxygen species (ROS) produced by hemocytes of the eastern oyster, Crassostrea virginica, impart bactericidal activity, exposure of hemocytes to bacteria should result in increased ROS generation. In an earlier study, this hypothesis was tested using luminol- and lucigenin-augmented chemiluminescence (CL) to measure ROS production. The bacterium Listonella anguillarum did not stimulate a net increase in hemocyte-derived CL, and it was suggested that bacterial antioxidants might suppress hemocyte CL. In the present study a comparison was made, under identical assay conditions, of the zymosan- and bacteria-enhanced luminol CL produced by eastern oyster hemocytes and by striped bass (Morone saxatilis) macrophages, for which L. anguillarum has been shown to be a stimulus in CL reactions. The response to zymosan produced by bass phagocytes was two orders of magnitude greater than that generated by eastern oyster hemocytes. Whereas an increase in net ROS production was not evident when oyster hemocytes were exposed to L. anguillarum, significant stimulation of striped bass macrophage-derived CL occurred. These data suggest that striped bass macrophages have a greater capacity to generate ROS than oyster hemocytes, enabling them to surpass the antioxidant capability of L. anguillarum and produce a luminol CL response.

Immunity and neoplasia in Mollusks.

The Mollusca is one of the largest of invertebrate phyla. Two major classes, the Gastropoda and the Pelecypoda, have been the subject of numerous studies on immunity and neoplasia. Investigations of immunity have dealt with cellular and humoral aspects, phagocytosis and encapsulation, and rejection of foreign tissue grafts. Work on humoral responses has focused on lysozyme, the hemagglutinins (especially in the oyster), and the clearance of certain antigens. Neoplasms in these animals resemble certain cancers in vertebrates, but it is not clear whether a relationship exists between the invertebrate immune system and the development of neoplasia. Studies of immunity and neoplasia in invertebrates may reveal how these two phenomena of living systems have evolved.

Sequestration of macrophages in growing tumours and its effect on the immunological capacity of the host.

The effects of rat tumours of various macrophage contents on the syngeneic host's ability to produce either: (1) an inflammatory exudate in response to intraperitoneal oyster glycogen or (2) a cutaneous delayed hypersensitivity (DHS) response to PPD or SRBC after appropriate sensitization, were studied as a function of tumour growth.Both these reactions were found to be markedly decreased as the tumours grew. The suppression was greatest in animals bearing tumours of high macrophage content. The suppression of the DHS response could be reversed by a local injection of normal peritoneal macrophages with the eliciting antigen, and lymphocytes from tumour bearing animals exhibiting poor DHS responses were able to adoptively transfer DHS reactivity to normal unsensitized recipients. The monocyte infiltration in response to oyster glycogen was also decreased, and these data indicate a monocyte, rather than a lymphocyte defect in the tumour induced "anergy" in this system.