Specific Cell-mediated Cytotoxicity Research Articles

Overexpression of CD200 Is a Stem Cell-Specific Mechanism of Immune Escape in AML

Background: Acute myeloid leukemia (AML) stem cells (LSC), the likely source of relapsed disease, are capable of surviving current standard chemotherapy. Therefore, novel therapeutic approaches specifically engineered to eradicate LSCs are critical for curing AML. We previously introduced a novel bioinformatics approach that harnessed publically available AML gene expression datasets and identified CD200 as significantly over-expressed in LSCs when compared to paired blast cells, as well as when compared to their normal hematopoietic stem cell (HSC) counterparts (Fig 1A; Herbrich et al Blood. 2018; 130:3962). CD200 can identify AML cells with LSC activity in vivo (Ho et al Blood. 2016; 128:1705). Functionally, CD200 has been shown to have an immunosuppressive effect on macrophages (Hoek et al Science. 2000; 290:1768) and NK cells (Coles et al Leukemia. 2012; 26:2148), and correlates with a high prevalence of FOXP3+ regulatory T cells (Coles et al Leukemia. 2012; 26:2146). Additionally, CD200 has been implicated as a poor prognostic marker in AML (Damiani et al Oncotarget. 2015; 6:30212). To date, we have screened 40 primary AML patient samples by flow cytometry, 95% of which are positive for CD200. Methods: To study the functional role of CD200 in AML, we generated a CD200 overexpression model in the human OCI-AML3 cell line (with no basal expression) and characterized changes in proliferation, survival, and gene expression. To examine the immune function of CD200 in AML in vitro, we performed a series of mixed lymphocyte reactions with isolated effector immune cells and target isogenic AML cell lines to assess immune cell-mediated apoptosis, proliferation, and cytokine secretion. To understand the contribution of CD200 immune protection in a physiological setting, we developed a peripheral blood mononuclear cell (PBMC)-humanized mouse in which we tracked the engraftment and overall survival of the CD200+/- OCI-AML3 cells. Lastly, the utility of CD200-blockade using a fully humanized anti-CD200 monoclonal antibody (CD200-IgG1) was evaluated both in vitro and in vivo. Results: In vitro, CD200+ AML significantly inhibited the secretion of inflammatory cytokines and cytotoxic enzymes from healthy PBMCs; a phenomenon that could be largely reversed by blocking the CD200/CD200R interaction with the CD200 antibody (Fig 1B). In vivo, OCI-AML3 CD200+/- cells showed no difference in engraftment, progression, and overall survival in immunodeficient NSG mice (Fig 1C). However, when mice were humanized using healthy PBMCs, CD200+ leukemia progressed rapidly, escaping T cell-mediated elimination, compared to CD200- control leukemic cells (Fig 1D). Cytokine production in PBMC-humanized mice was significantly compromised in those with CD200-expressing leukemia. Transcriptome analysis revealed that T cells from humanized mice exposed to CD200 expressing disease were metabolically quiescent. In humanized mice, CD200-IgG1 therapy eliminated CD200+ AML disease (Fig 1E). The novel CD200-IgG1 antibody also induced potent, specific NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) and macrophage-mediated antibody dependent cellular phagocytosis (ADCP; Fig 1F). Conclusion: We have identified CD200 as a putative stem cell-specific immunomodulatory target that aids in establishing an immunosuppressive microenvironment by significantly suppressing cytokine secretion in response to AML. In a PBMC-humanized mouse model, the presence of cell-surface CD200 was sufficient to protect AML cells from immune-mediated clearance and could be reversed using a blocking anti-CD200 mAb. These findings indicate a utility of CD200 as a novel immune checkpoint target for the development of therapeutic strategies in AML. Disclosures Konopleva: Calithera: Research Funding; Kisoji: Consultancy; AbbVie: Consultancy, Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Ablynx: Research Funding; Genentech: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; AstraZeneca: Research Funding; Sanofi: Research Funding; Agios: Research Funding; Forty-Seven: Consultancy, Research Funding; Rafael Pharmaceutical: Research Funding; Ascentage: Research Funding.

A fully humanized IgG-like bispecific antibody for effective dual targeting of CXCR3 and CCR6.

Chemokines and their receptors are pivotal for the trafficking of leukocytes during immune responses, and host defense. However, immune cell migration also contributes to a wide variety of autoimmune and chronic inflammatory diseases. Compelling evidence suggests that both CXCR3 and CCR6 chemokine receptors play crucial roles in the migration of pathological Th1 and Th17 cells during the course of certain inflammatory diseases. The use of two or more receptors by pathogenic cells may explain why targeting of individual receptors has proven disappointing in the clinic. We therefore hypothesized that simultaneous targeting of both CXCR3 and CCR6 with a bispecific antibody (BsAb) might result in decreased chemotaxis and/or specific depletion of pro-inflammatory T cell subsets. In this study, we designed and characterized a fully humanized BsAb. We show that the BsAb binds to both chemokine receptors, as demonstrated by Flow Cytometry and Surface Plasmon Resonance analysis. Furthermore, we demonstrate that the BsAb effectively blocks cell chemotaxis and induces specific antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro. Therefore, we propose that dual targeting of CXCR3 and CCR6 with a fully humanized BsAb may display a potent interventional approach for the treatment of inflammatory and autoimmune diseases.

Local and systemic adaptive immune responses toward viral infection via gills in ginbuna crucian carp

Recent studies on fish immunity highlighted the significance of gills as mucosal immune tissues. To understand potential of gills as vaccination sites for inducing adaptive systemic immunity, we investigated virus-specific cell-mediated and humoral immune responses following a “per-gill infection method”, which directly exposes virus only to gills. The viral load in crucian carp hematopoietic necrosis virus (CHNV)-infected gills decreased after peaking at a particular time point. Furthermore, the viral titers in the gills following the secondary infection were lower than that after the primary infection, indicating that local adaptive immunity helped the elimination of virus. Gene expression analysis demonstrated that IFN-γ in gills and perforin in kidney were increased after the gill infection. CD8+ cells in kidney leukocytes increased after the secondary infection, whereas IgM+ cells decreased. These results suggest that IFN-γ and CTL contribute in controlling CHNV-replication in gills and kidney. Gill infection could induce specific cell-mediated cytotoxicity of peripheral blood leukocytes (PBL) and secretion of CHNV-specific IgM in serum, indicating that local priming of the gill site can generate adaptive systemic immunity. Thus, the gills could be prospective antigen-sensitization sites for mucosal vaccination.

Blocking Hypoxia-Induced Autophagy in Tumors Restores Cytotoxic T-Cell Activity and Promotes Regression

The relationship between hypoxic stress, autophagy, and specific cell-mediated cytotoxicity remains unknown. This study shows that hypoxia-induced resistance of lung tumor to cytolytic T lymphocyte (CTL)-mediated lysis is associated with autophagy induction in target cells. In turn, this correlates with STAT3 phosphorylation on tyrosine 705 residue (pSTAT3) and HIF-1α accumulation. Inhibition of autophagy by siRNA targeting of either beclin1 or Atg5 resulted in impairment of pSTAT3 and restoration of hypoxic tumor cell susceptibility to CTL-mediated lysis. Furthermore, inhibition of pSTAT3 in hypoxic Atg5 or beclin1-targeted tumor cells was found to be associated with the inhibition Src kinase (pSrc). Autophagy-induced pSTAT3 and pSrc regulation seemed to involve the ubiquitin proteasome system and p62/SQSTM1. In vivo experiments using B16-F10 melanoma tumor cells indicated that depletion of beclin1 resulted in an inhibition of B16-F10 tumor growth and increased tumor apoptosis. Moreover, in vivo inhibition of autophagy by hydroxychloroquine in B16-F10 tumor-bearing mice and mice vaccinated with tyrosinase-related protein-2 peptide dramatically increased tumor growth inhibition. Collectively, this study establishes a novel functional link between hypoxia-induced autophagy and the regulation of antigen-specific T-cell lysis and points to a major role of autophagy in the control of in vivo tumor growth.

Identification and characterization of common carp ( Cyprinus carpio L.) granzyme A/K, a cytotoxic cell granule-associated serine protease

Granzyme (Gzm) is an important member of serine protease family, and key component in the specific and non-specific cell-mediated cytotoxicity. Partial GzmA/K cDNA sequence of common carp ( Cyprinus carpio L.) was isolated from thymus cDNA library by the method of suppression subtractive hybridization (SSH). Subsequently, the full length cDNA of carp GzmA/K was obtained by means of 3′ RACE and 5′ RACE, respectively. The full length cDNA of carp GzmA/K was 1053 bp, consisting of a 5′-terminal untranslated region (UTR) of 65 bp, a 3′-terminal UTR of 214 bp, and an open reading frame of 774 bp. Amino acid sequence analysis indicated the existence of a signal peptide, eight consensus cysteine residues, one conserved IIGG motif and three conserved residues as central elements of the GzmA/K active site. Carp GzmA/K shared 36% and 39% amino acid identity to human GzmA and K, respectively, and was phylogenetically related to the granzyme A and K subgroups. Then, a genomic DNA, which covers the promoter region and entire coding region of carp GzmA/K, was obtained by PCR. In the 5.4 k-long genomic sequence, five exons and four introns were identified. Real-time RT-PCR analysis showed that carp GzmA/K transcript was predominantly detected in the immune-related tissues, and after SVCV infection, was up-regulated in most immune-related tissues in a time-dependent manner. Real-time RT-PCR results also showed that carp GzmA/K transcript was up-regulated in thymus tissue of GH transgenic carp. These results will help to understand the molecular characterization and the potential role of teleost GzmA/K, a cytotoxic cell granule-associated serine protease.

Alloantigen-specific killing is mediated by CD8-positive T cells in fish

CD8-positive (CD8 +) cytotoxic T lymphocytes (CTL) have antigen-specific cytotoxic activity. In fish, however, CTL expressing CD8 on their cell surface have not been identified. In order to characterize the cells involved in specific cell-mediated cytotoxicity in teleosts, we separated and sorted ginbuna kidney leucocytes into CD8α +, CD4 + and surface IgM (sIgM) + cells by magnetic activated cell sorting using monoclonal antibodies and examined their cytotoxic activities. Effector donor ginbuna (OB1 clone) were sensitized by allografting scales from S3N clone fish followed by injection of an allogeneic cell line (CFS) derived from S3N fish. In cytotoxic assays, target cells were labeled with CFSE and cytotoxicity was calculated based on the number of viable target cells using flow cytometry. CD8α + cells from sensitized OB1 fish showed relatively high cytotoxicity against CFS cells (immunogen) but not against allogeneic CFK cells (third party) nor isogeneic CFO cells. Pre-sensitized sIgM + cells exhibited cytotoxicity against not only CFS cells but also CFK cells. However, CD4 + or CD8α − CD4 −sIgM − cells as well as cells from non-sensitized fish did not show any significant cytotoxic activity. These results suggest that CD8α + cells in fish have characteristics similar to those of CTL in mammals, and that the sIgM + cells include NK-like cells which non-specifically killed the target cells.

Cytotoxic CD4+ T cells specific for Francisella tularensis.

The specific cell-mediated cytotoxicity of tularaemia-immune human T lymphocytes were studied in vitro. Peripheral blood mononuclear cells (PBMC) of six tularaemia-vaccinated healthy subjects were stimulated with F. tularensis LVS whole cell antigen for 6 days and used as effector cells in a conventional 4-h 51Cr release cytotoxicity assay. The target cells were phagocyting autologous monocytes, which were pulsed with F. tularensis or PPD antigen. The specific lysis of the F. tularensis-pulsed cells (42.6% +/- 11.7) was significantly higher (P less than 0.05) than that of the PPD-pulsed ones (22.2% +/- 8.3) or unpulsed control cells (15.9% +/- 5.2). The cytotoxicity was associated with CD4+ F. tularensis-specific T cell clones (TLC), which killed 36.3% +/- 12.3 of the F. tularensis-pulsed targets but only 6.9% +/- 6.5 of the unpulsed control targets. Their lysing was inhibited by monoclonal anti-HLA-DR and anti-HLA-DQ antibodies, but not by CD15 (monocyte/macrophage) antibody. The functional role of CD4+ lymphocytes in tularaemia immunity is discussed.

Effects of the trifunctional antibody catumaxomab (anti-EpCAM x anti-CD3) on proliferation and cytokine secretion of immune cells in malignant pleural effusion

3046 Background: The trifunctional antibody catumaxomab specifically binds EpCAM+ tumor cells, CD3+ T lymphocytes and accessory cells via FcγR I/III. Thus the antibody induces tumor specific cell mediated cytotoxicity in vitro and in vivo. Following last years’ ASCO presentation on a phase I/II trial showing safety and efficacy of repetitive intrapleural administration of catumaxomab in patients with EpCAM positive, malignant pleural effusion (MPE), we now present data on the responsiveness of immune cells from pleural fluid to catumaxomab. Methods: Pleural fluid of patients with EpCAM-positive MPE treated with i.pl. catumaxomab was collected before treatment. Cells were harvested from the fluid and cultured ± 100 ng/ml catumaxomab using an in vitro proliferation assay. After 72 h of culture, proliferation of T cells (CD4+ and CD8+) and monocytes (CD11c+) was determined. In addition, cell supernatants after 24h incubation ± catumaxomab were analysed for their TH1/TH2 cytokine profile (IL-2, IL-4, IL-6, IL-10, IFN-γ and TNF- a). Results: Incubation in presence of catumaxomab led to a pronounced increase of CD4+ CD8+ and CD11+ cell numbers indicating a proliferation of these cells, whereas cultures without catumaxomab showed no proliferation of immune cells. Analysis of supernatants after 24 h revealed levels of IL-2, IL-6, IFN-γ and TNF-a, from cells incubated with catumaxomab that were distinctly higher than in cultures without catumaxomab. Conclusions: The immunologic nature of catumaxomab-induced responses in patients with pleural effusion accompanied by a reduction of tumor cells, could be underlined impressively with in vitro data obtained from pleural cells showing catumaxomab-induced proliferation of T cells and accessory cells and TH1-directed cytokine secretion. The data are equivalent to results observed in the peritoneal fluid of ascites patients. [Table: see text]

Treatment of malignant pleural effusion with the trifunctional antibody catumaxomab (anti-EpCAM x anti-CD3): Results of a phase I/II study

2548 Background: The trifunctional antibody catumaxomab specifically binds EpCAM+ tumor cells, CD3+ T lymphocytes and accessory cells via the Fcγ RI/III. Thus the antibody induces tumor specific cell mediated cytotoxicity in vitro and in vivo. Its intraperitoneal administration has demonstrated safety and led to efficient tumor cell killing and reduction of malignant ascites in ovarian cancer patients (pts.). The present study was conducted to evaluate safety, tolerability and preliminary efficacy of catumaxomab administered intrapleurally (i.pl.) into pts. with EpCAM+ malignant pleural effusion. Methods: This phase I/II trial was a multi-center uncontrolled, dose-escalating study. Pts. (ECOG ≤3) with recurrent malignant pleural effusion containing EpCAM+ tumor cells were included. Three escalating doses from 5–200 μg catumaxomab were administered i.pl. Primary objectives were toxicity and definition of the maximum tolerated dose (MTD). In addition, preliminary data on efficacy (reduction of pleural fluid and tumor cell killing) were generated. Results: 13/24 pts. received 3 infusions as planned. Reasons for early termination of infusions were 3 deteriorations of performance status, 2 exanthemas, 2 dyspnoea, 2 others. The dose regimen of 20–50–100 μg catumaxomab was defined as MTD. Most frequent drug related AEs were symptoms of cytokine release syndrome. 32 SAEs were reported in 18 pts., 4 SAEs were possibly drug related (1 death due to pleural empyema, 1 pneumonia, 1 erythema, 1 increase of liver enzymes). 8/9 deaths were not drug related but due to underlying disease. Induction of human-anti-mouse (HAMA) or human-anti-rat (HARA) antibodies after treatment was found in 8/11 pts. 8/13 fully treated pts. were defined as responders (1 complete, 4 partial, 3 NA) showing reduction of effusion and of drainage necessity. 5/13 were non-responders. A reduction of tumor cells up to log 5 was seen in 10/13 pts. Conclusions: 20–50–100 μg of catumaxomab can safely be administered i. pl. This regimen will further be investigated in a randomized phase II trial comparing catumaxomab to talcum-based pleurodesis. [Table: see text]

Cytotoxic activities of fish leucocytes

Like mammalian leucocytes, white blood cells of fish are able to kill altered (e.g. virus-infected) and foreign (allogeneic or xenogeneic) cells. The existence of natural killer (NK)-like and specific cytotoxic cells in fish was first shown using allogeneic and xenogeneic effector/target cell systems. In addition to in vivo and ex vivo studies, very important contributions were made by in vitro analysis using a number of different long-term cytotoxic cell lines established from channel catfish. In mammals, specific cell-mediated cytotoxicity (CMC) as part of the adaptive immune response requires a number of key molecules expressed on effector leucocytes and target cells. CD8 + T lymphocytes kill infected cells only, if their antigen receptor (TCR) matches the MHC class I with bound peptide of the target cell. Expression patterns of the fish gene homologues for TCR, CD8 and MHC class I, as well as related genes, are in agreement with similar function. Convenient systems for the analysis of specific CMC have only recently become available for fish with the combination of clonal fish with syngeneic or allogeneic but MHC class I matching cell lines. It was demonstrated that both, NK- and cytotoxic T (T c) cells are involved in the killing of virus infected MHC class I matching and mismatching target cells. Analysis of these lymphocyte subsets is only starting for fish. There is also evidence that the different viral proteins trigger different subsets of killer cells. This review further discusses findings on fish CMC with regard to temperature/seasons and ontogeny.

Systemic priming of alloreactive cytotoxic cells in carp, following anal administration of allogeneic cell antigens

In the present study, we confirmed that cellular immune responses, especially specific cell-mediated cytotoxicity, could be induced in systemic carp leucocytes, following anal administration of antigens. Effector cells isolated from systemic lymphoid tissues (head kidney, spleen and peripheral blood) of carp that were immunised anally with allogeneic cells (EPC or KG cell line) efficiently lysed immunogenic target cells. The lytic activity was increased as a result of secondary sensitisation and peaked around 7 days after the final immunisation. In some aspects, the alloantigen-specific cell-mediated cytotoxicity induced by anal sensitisation was different from that induced by intraperitoneal (i.p.) injection. First, the activity induced by anal immunisation was higher than that resulting from i.p. immunisation when fish were immunised twice with a 7-day interval, whereas similar kinetics of the cytotoxicity were observed after the final immunisation. Second, repeated anal administrations tended to decrease the cytotoxic activity, although repeated i.p. injections increased the activity. These findings indicate that the anal administration of antigens in fish can elicit and modulate cellular immune responses.

Women with Cervicovaginal Antibody‐Dependent Cell‐Mediated Cytotoxicity Have Lower Genital HIV‐1 RNA Loads

Antibodies that mediate human immunodeficiency virus (HIV)-specific antibody-dependent cell-mediated cytotoxicity (ADCC) are present in the cervical fluid of many HIV-positive women; however, the role that these antibodies play in host defense against HIV is not known. To understand the contribution of ADCC in cervical secretions as a protective mechanism against HIV, we evaluated ADCC titers in paired serum and cervical-lavage (CVL) samples from >300 HIV-1-positive women who participated in the multicenter Division of AIDS Treatment Research Initiative Study 009. The present study demonstrates that women with CVL ADCC activity had lower genital viral loads than did women with serum ADCC activity only. Women with CVL ADCC activity were likely to have HIV-1 gp120-specific immunoglobulin (Ig) G, but not IgA, in their cervical fluid. This finding suggests that specific IgG in cervical fluid can mediate ADCC activity that inversely correlates with genital viral load.

The ontogeny of MHC class I expression in rainbow trout ( Oncorhynchus mykiss)

In the present study, clonal rainbow trout ( Oncorhynchus mykiss) embryos and larvae were assayed for the expression of key molecules involved in specific cell-mediated cytotoxicity using an anti-MHC class I monoclonal Ab and by RT-PCR using specific primers derived from classical MHC class I (class Ia), TCR and CD8. Whereas RT-PCR revealed that MHC class Ia and CD8 were expressed from at least 1 week after fertilisation (p.f.) on, TCR expression was detectable from 2 weeks p.f. Immunohistochemistry indicated an early and distinct expression of MHC class I protein in the thymus. Positive lymphoid, epithelial and endothelial cells were found in the pronephros, in the spleen and in the inner and outer epithelia at later stages. Whereas in older rainbow trout the intestine is counted among the organs of the highest class I expression, during ontogeny it was the last site (39 days after hatching) where such expression was detectable. Knowledge on the appearance of the assayed key molecules during fish development is relevant for the pathogenesis of infections as well as for early vaccine delivery. Besides such information regarding the development of the adaptive immune system, immunohistochemistry revealed that in early larvae MHC class I was expressed in neurons whereas in older rainbow trout this was not observed.

Role of Specific Cell-Mediated Cytotoxicity in Protecting Fish from Viral Infections

We report the in vivo role of specific cytotoxic cells in protecting fish from acute viral infections. Specifically, we found that (1) there is an inverse relationship between cytotoxic activities and viral load and (2) adoptive transfer of immune leukocytes prevented viral infection. Crucian carp hematopoietic necrosis virus (CHNV), which has a virulence to ginbuna crucian carp, was recently found and identified in Japan. Specific cell-mediated cytotoxicity of ginbuna leukocytes against CHNV-infected syngeneic cells was induced as a result of intraperitoneal inoculation with CHNV. This cytotoxicity was not induced against either virus-infected allogeneic cells or eel rhabidovirus- (EVA) infected syngeneic cells. In these respects, the cytotoxic activity was similar to that of mammalian cytotoxic T-lymphocyte (CTL) activity. Viral titers of tissues from infected fish were remarkably reduced 8 days after infections, when specific cytotoxic activity reached a peak. This result suggested that specific cytotoxic cells were responsible for the early control of CHNV replication. On the other hand, CHNV-specific antibody was greatly increased when the virus was eliminated by cytotoxic activities. The effectiveness of the virus-specific cytotoxicity was evaluated using adoptive cell transfer. Recipients that received leukocytes from immune syngeneic donors escaped CHNV infection. These findings suggest that virus-specific cytotoxic cells have a role in controlling viral infections in a fish.

Evaluation of immune functions of rainbow trout ( Oncorhynchus mykiss)—how can environmental influences be detected?

In fish, the first line of defense against infectious microorganisms is based on a broad range of nonspecific humoral and cellular immune mechanisms (‘innate immunity’) which without prior specific activation can act in forming a more static barrier (Fish Shellfish Immunol. 10 (2000) 243; Dev. Comp. Immunol. 25 (2001) 827). This natural resistance is normally effective enough to protect fish from infectious diseases until specific immune responses are being induced (Fig. 1; Dev. Comp. Immunol. 25 (2001) 841). Healthy fish exhibit both nonspecific and specific immune responses depending directly on environmental temperature. Pollution of the natural aquatic environment with industrial or agricultural sewage is an important immunosuppressing factor resulting in higher susceptibility to infectious diseases. To date, the possible immunotoxicity of a substance is evaluated using quantification of humoral factors like lysozyme, complement, C-reactive protein or total immunoglobulins but less often using functional assays. Furthermore, most of the functional assays (phagocytosis, respiratory burst, proliferative response) are based on the measurement of the response of resting but not of specific activated immune cells. However, the physiological responses of the immune system to an infection are based on a complex, stepwise activation and proliferation, especially of the specific immune functions after first contact to the microorganisms. In this report we describe in vitro methods for the evaluation of cellular immune functions of different leukocyte populations after specific in vivo triggering of the immune system. Parameters to be evaluated are activation and proliferation of leukocyte populations, phagocytosis and respiratory burst, secretion of antigen-specific antibodies and specific cell-mediated cytotoxicity. Furthermore, challenge models with bacterial ( Aeromonas salmonicida) and viral pathogens (Viral Haemorrhagic Septicemia Virus, VHSV) are presented.

Specific cell-mediated cytotoxicity against a virus-infected syngeneic cell line in isogeneic ginbuna crucian carp

In the present study, we attempted to determine whether virus-specific cell-mediated cytotoxicity occurs in fish. Experiments were conducted with clonal ginbuna crucian carp ( Carassius auratus langsdorfii) (S3n strain), and the syngeneic cell line (CFS). Two infectious viruses were used: infectious pancreatic necrosis virus (IPNV) and eel virus from America (EVA). Peripheral blood leukocytes, which consisted almost entirely (95%) of lymphocytes and thrombocytes, from S3n ginbuna immunized with virus-infected CFS cells lysed the virus-infected CFS cells (immunogen) more completely than CFS cells infected with a different virus (non-immunogen). This specific cell-mediated cytotoxicity of ginbuna against virus-infected CFS cells was efficiently induced as a result of in vivo secondary immunization. A significant specific cytotoxic activity peaked during 7–10 days after the secondary immunization. In addition, effector cells immunized with IPNV-infected syngeneic cells did not lyse IPNV-infected xenogeneic cells. These results support the hypothesis that fish exhibit specific cytotoxicity against virus-infected cells, resembling the specific cytotoxicites of higher vertebrates.

Specific cell-mediated immunity in fish

This review describes the fish immune system, focusing on specific cell-mediated immunity. Specific in vivo cell-mediated immune responses have been shown by allograft rejection, graft-versus-host reaction (GVHR) and delayed hypersensitivity reaction (DTH). Recent in vitro studies also showed specific cell-mediated cytotoxicity against allogeneic target cells. These in vivo and in vitro experiments strongly suggest the presence of cytotoxic T cells in fishes. Also described are current studies on shark and trout MHC class I polymorphism and function that demonstrate strong similarities between fish and mammals.

In vivo Priming of TNP-reactive Effector Cells in Carp.

In a study of immune mechanisms in fish, specific cell-mediated cytotoxic activity against trinitrophenyl (TNP) -modified autologous peripheral blood leucocytes (PBL) was successfully induced in in vivo primed carpCyprinus carpio. The cytotoxic activity increased in proportion to the number of immunizations. The cytotoxic activity was shown to be gradually reduced with time afterin vivosensitization, and was finally lost. In addition, cytotoxic activities against TNP-modified allogeneic PBLs (T-ALLO-PBL) in carp immunized with TNP-modified autologous PBLs (T-AUTO-PBL) were not detected and effector cells from carp immunized with T-ALLO-PBL were not significantly cytotoxic against T-AUTO-PBL. Thus, the occurrence of a genetic restriction in carp was shown using thein vivopriming model. These results suggest that specific cell-mediated cytotoxicity ofin vivoprimed carp in some respects resembles mammalian cytotoxic T cells.

Specific cell-mediated cytotoxicity against an allogeneic target cell line in isogeneic ginbuna crucian carp

Specific cell-mediated cytotoxicity in fish was investigated using both iso-geneic (clonal) ginbuna crucian carp,Carassius auratus langsdorfii, and three cell lines established from the three different clones of this fish. Specific cell-mediated cytotoxicity in ginbuna was induced as a result of intravenous immunisation with an allogenic cell line, but not a syngeneic cell line. As the cytotoxic activity tended to accelerate in proportion to the number of immunisations, specific cytotoxic cells in ginbuna resembled mammalian cytotoxic T lymphocytes (CTL) in some respects. In addition, it was unlikely that antibody-dependent cell-mediated cytotoxicity (ADCC) was involved in the cytotoxicity observed in this study because ADCC activity of unimmunised fish effector cells was not detected using the immunised ginbuna plasma or the supernatant of cultured effector cells from immunised ginbuna. These results demonstrate that specific cell-mediated cytotoxicity is inherent in fish and a set of clonal fish and their cell lines may be useful for further studyin vivoandin vitro.

In vitro cell-mediated cytotoxicity against allogeneic erythrocytes in ginbuna crucian carp and goldfish using a non-radioactive assay

Cell-mediated cytotoxicity of clonal ginbuna crucian carp leukocytes against allogeneic erythrocytes is described using a sensitive non-radioactive in vitro assay. Hemoglobin released from target erythrocytes after cell-mediated erythrolysis was detected by tetramethylbenzidine (TMB). TMB assay showed clear correlation with a 51Cr-release assay and even exhibited higher cytotoxicity. The use of erythrocytes as target cells has several advantages over a conventional 51Cr-release assay. Erythrocytes do not have cytotoxic activity, are relatively homogeneous, are available in large numbers and erythrocyte donors need not be killed. Leukocytes from fish sensitized by erythrocyte injection or scale grafting efficiently lysed allogeneic erythrocytes, but did not kill isogeneic or autologous erythrocytes. In contrast, leukocytes from unsensitized fish did not lyse allogeneic erythrocytes and repeated sensitizations by allogeneic grafts were necessary to induce cytotoxic cells. Effector cells isolated from peripheral blood showed a higher cytotoxic effect toward allogeneic target cells than effector cells isolated from kidney. These studies support the hypothesis that fish are capable of a genetically restricted specific cell-mediated cytotoxicity.