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Natural killer cells (NK) are important effectors of anti-tumor immunity, activated either by the downregulation of HLA-I molecules on tumor cells and/or the interaction of NK-activating receptors with ligands that are overexpressed on target cells upon tumor transformation (including NKG2D and NKP30). NK kill target cells by the vesicular delivery of cytolytic molecules such as Granzyme-B and Granulysin activating different cell death pathways, which can be Caspase-3 dependent or Caspase-3 independent. Multiple myeloma (MM) remains an incurable neoplastic plasma-cell disorder. However, we previously reported the encouraging observation that cord blood-derived NK (CB-NK), a new source of NK, showed anti-tumor activity in an in vivo murine model of MM and confirmed a correlation between high levels of NKG2D expression by MM cells and increased efficacy of CB-NK in reducing tumor burden. We aimed to characterize the mechanism of CB-NK-mediated cytotoxicity against MM cells. We show a Caspase-3- and Granzyme-B-independent cell death, and we reveal a mechanism of transmissible cell death between cells, which involves lipid-protein vesicle transfer from CB-NK to MM cells. These vesicles are secondarily transferred from recipient MM cells to neighboring MM cells amplifying the initial CB-NK cytotoxicity achieved. This indirect cytotoxicity involves the transfer of NKG2D and NKP30 and leads to lysosomal cell death and decreased levels of reactive oxygen species in MM cells. These findings suggest a novel and unique mechanism of CB-NK cytotoxicity against MM cells and highlight the importance of lipids and lipid transfer in this process. Further, these data provide a rationale for the development of CB-NK-based cellular therapies in the treatment of MM. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR) T-cell therapies have increased the patients with relapsed/refractory multiple myeloma (RRMM) in whom standard electrophoretic techniques fail to detect the M-protein. Quantitative immunoprecipitation mass spectrometry (QIP-MS) can accurately measure serum M-protein with high sensitivity, and identify interferences caused by therapeutic monoclonal antibodies. Here, we investigate the outcome of QIP-MS in 33 patients treated with the academic BCMA-directed CAR T-cell ARI0002h (Cesnicabtagene Autoleucel). QIP-MS offered more detailed insights than serum immunofixation (sIFE), identifying glycosylated M-proteins and minor additional peaks. Moreover, the potential interferences owing to daratumumab or tocilizumab treatments were successfully detected. When analysing different assay platforms during patient's monitoring after ARI0002h administration, we observed that QIP-MS showed a high global concordance (78.8%) with sIFE, whereas it was only moderate (55.6%) with bone marrow (BM)-based next-generation flow cytometry (NGF). Furthermore, QIP-MS consistently demonstrated the lowest negativity rate across the different timepoints (27.3% vs. 60.0% in months 1 and 12, respectively). Patients with QIP-MS(+)/BM-based NGF(-) showed a non-significant shorter median progression free survival than those with QIP-MS(-)/BM-based NGF(-). In summary, we show the first experience to our knowledge demonstrating that QIP-MS could be particularly useful as a non-invasive technique when evaluating response after CAR T-cell treatment in MM., (© 2024 The Author(s). British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Purpose: B-cell maturation antigen (BCMA)-chimeric antigen receptor T-cells (CART) improve results obtained with conventional therapy in the treatment of relapsed/refractory multiple myeloma. However, the high demand and expensive costs associated with CART therapy might prove unsustainable for health systems. Academic CARTs could potentially overcome these issues. Moreover, response biomarkers and resistance mechanisms need to be identified and addressed to improve efficacy and patient selection. Here, we present clinical and ancillary results of the 60 patients treated with the academic BCMA-CART, ARI0002h, in the CARTBCMA-HCB-01 trial., Patients and Methods: We collected apheresis, final product, peripheral blood and bone marrow samples before and after infusion. We assessed BCMA, T-cell subsets, CART kinetics and antibodies, B-cell aplasia, cytokines, and measurable residual disease by next-generation flow cytometry, and correlated these to clinical outcomes., Results: At cut-off date March 17, 2023, with a median follow-up of 23.1 months (95% CI, 9.2-37.1), overall response rate in the first 3 months was 95% [95% confidence interval (CI), 89.5-100]; cytokine release syndrome (CRS) was observed in 90% of patients (5% grades ≥3) and grade 1 immune effector cell-associated neurotoxicity syndrome was reported in 2 patients (3%). Median progression-free survival was 15.8 months (95% CI, 11.5-22.4). Surface BCMA was not predictive of response or survival, but soluble BCMA correlated with worse clinical outcomes and CRS severity. Activation marker HLA-DR in the apheresis was associated with longer progression-free survival and increased exhaustion markers correlated with poorer outcomes. ARI0002h kinetics and loss of B-cell aplasia were not predictive of relapse., Conclusions: Despite deep and sustained responses achieved with ARI0002h, we identified several biomarkers that correlate with poor outcomes., (©2024 American Association for Cancer Research.)

Background: Chimeric antigen receptor (CAR) T-cell therapy is a promising option for patients with heavily treated multiple myeloma. Point-of-care manufacturing can increase the availability of these treatments worldwide. We aimed to assess the safety and activity of ARI0002h, a BCMA-targeted CAR T-cell therapy developed by academia, in patients with relapsed or refractory multiple myeloma., Methods: CARTBCMA-HCB-01 is a single-arm, multicentre study done in five academic centres in Spain. Eligible patients had relapsed or refractory multiple myeloma and were aged 18-75 years; with an Eastern Cooperative Oncology Group performance status of 0-2; two or more previous lines of therapy including a proteasome inhibitor, an immunomodulatory agent, and an anti-CD38 antibody; refractoriness to the last line of therapy; and measurable disease according to the International Myeloma Working Group criteria. Patients received an initial fractionated infusion of 3 × 10 6 CAR T cells per kg bodyweight in three aliquots (0·3, 0·9, and 1·8 × 10 6 CAR-positive cells per kg intravenously on days 0, 3, and 7) and a non-fractionated booster dose of up to 3 × 10 6 CAR T cells per kg bodyweight, at least 100 days after the first infusion. The primary endpoints were overall response rate 100 days after first infusion and the proportion of patients developing cytokine-release syndrome or neurotoxic events in the first 30 days after receiving treatment. Here, we present an interim analysis of the ongoing trial; enrolment has ended. This study is registered with ClinicalTrials.gov, NCT04309981, and EudraCT, 2019-001472-11., Findings: Between June 2, 2020, and Feb 24, 2021, 44 patients were assessed for eligibility, of whom 35 (80%) were enrolled. 30 (86%) of 35 patients received ARI0002h (median age 61 years [IQR 53-65], 12 [40%] were female, and 18 [60%] were male). At the planned interim analysis (cutoff date Oct 20, 2021), with a median follow-up of 12·1 months (IQR 9·1-13·5), overall response during the first 100 days from infusion was 100%, including 24 (80%) of 30 patients with a very good partial response or better (15 [50%] with complete response, nine [30%] with very good partial response, and six [20%] with partial response). Cytokine-release syndrome was observed in 24 (80%) of 30 patients (all grade 1-2). No cases of neurotoxic events were observed. Persistent grade 3-4 cytopenias were observed in 20 (67%) patients. Infections were reported in 20 (67%) patients. Three patients died: one because of progression, one because of a head injury, and one due to COVID-19., Interpretation: ARI0002h administered in a fractioned manner with a booster dose after 3 months can provide deep and sustained responses in patients with relapsed or refractory multiple myeloma, with a low toxicity, especially in terms of neurological events, and with the possibility of a point-of-care approach., Funding: Instituto de Salud Carlos III (co-funded by the EU), Fundación La Caixa, and Fundació Bosch i Aymerich., Competing Interests: Declaration of interests AO-C declares support for attending meetings from Janssen. VG-C declares receiving honoraria from Janssen, Pfizer, Bristol Myers Squibb (BMS)/Celgene, and GSK; support for attending meetings or travel from Janssen and GSK; and participation on data safety monitoring or advisory board from Janssen. VC declares receiving honoraria from Janssen, BMS, Sanofi, GSK, and Amgen; support for attending meetings or travel from Janssen; and participation on data safety monitoring or advisory board from Janssen, Sanofi, and Amgen. PR-O declares receiving honoraria from consulting activities from BMS, Janssen, Sanofi, Abbvie, Pfizer, Roche, and GSK; honoraria from lectures from BMS, Janssen, Sanofi and GSK; support for attending meetings or travel from Abbvie; and participation on data safety monitoring or advisory board from Janssen. JLR declares receiving consulting fees from Janssen; honoraria from Janssen, Amgen, Sanofi, Kite/Gilead, Novartis, and BMS; support for attending meetings or travel from Kite/Gilead; and participation on data safety monitoring or advisory board from Janssen, BMS, and Novartis. LL-C declares receiving honoraria from Kite/Gilead, Celgene, Janssen, and Novartis; support for attending meetings or travel from Kite/Gilead, Celgene, Janssen, and Novartis; and participation on data safety monitoring or advisory board from Janssen. BM-A declares to be co-inventor in the patent of ARI0002. LR declares honoraria from Janssen, BMS/Celgene, Amgen, Takeda, Sanofi, and GSK; participation on data safety monitoring or advisory board of Janssen, BMS-Celgene, Amgen, Takeda, Sanofi, and GSK. ML-P declares receiving consulting fees from Celgene/BMS; honoraria from Janssen and Kite/Gilead; and participation on data safety monitoring or advisory board from Celgene/BMS and Novartis. LGR-L declares honoraria from Janssen, GSK, Sanofi, and BMS; travel grants from Janssen, Amgen, GSK, Pfizer and Sanofi; and participation on data safety monitoring or advisory board from GSK and Sanofi. AS-S declares receiving travel grants from Jazz Pharmaceuticals, Pfizer, and MSD. JAP-S declares research and travel support Takeda, Abbvie, Gilead, AMGEN, Jazz, Alexion, Pierre Fabre and Beigene; educational activities, speaker, and advisory fees with Gilead, Jazz, Alexion, AMGEN, Novartis, Janssen, BMS, and MSD; and participation on data safety monitoring or advisory board from Gilead, Jazz, Alexion, AMGEN, Novartis, Janssen, BMS and MSD. BP reports research funding from BMS, GSK, Roche, Beigene, and Sanofi; consultancy fees from BMS, GSK, Janssen, Sanofi and Takeda; honoraria from Adaptive, Amgen, Becton Dickinson, BMS/Celgene, GSK, Janssen, Sanofi, and Roche; and support for attending meetings from GSK. FP declares receiving grants from the Spanish Ministry of Health (ISCIII) and the Government of Navarra; honoraria from Janssen, Oryzon, Dialectica, Novartis, Instituto Roche, Servier, ViviaBiotech, and Techspert; support for attending meetings or travel from Gilead, Celgene, and Janssen; and a leadership or fiduciary role in RICORS Terav and IDISNA. MJ declares receiving research or travel support by Fundació Bancaria la Caixa, ISCIII, and CellNex Teleom; honoraria from educational activities, speaker, and advisory roles with Miltenyi and indirectly with sponsors of congresses; and participation on data safety monitoring or advisory boards with MAB Gyala. JMM declares receiving honoriaria from Gilead/Kite, Novartis, BMS/Celgene, and Roche; travel grants, accommodation, and expenses from Jazz Pharma, Gilead-Kite, Janssen, and Sandoz; and consulting or advisory board fees in Jazz Pharma, Novartis, and Sandoz. M-VM declares honoraria derived from lectures and participation in advisory boards from Janssen, BMS/Celgene, Takeda, Amgen, GSK, Pfizer, Regeneron, Roche, and Sanofi. MP declares receiving honoraria from Thermofisher Scientific and LetiPharma. AU-I declares receiving grants from the Spanish Ministry of Health (ISCIII); honoraria for lectures from BMS and Gilead; support for attending meetings from Amgen; 23% of participation in ARI-002 patent ARI-001 patent in preparation; advisory board fees and participation in Miltenyi biomedicine; being coordinator of the Spanish group of CAR T-cell therapy. CFL declares receiving grants through his institution from BMS, Janssen, and Amgen; honoraria from Amgen, Janssen, BMS, GSK, and Sanofi; support for attending meetings or travel from Janssen, BMS, GSK, and Amgen; and participation in data safety monitoring or advisory boards with Janssen, BMS, Amgen, Pfizer, and Sanofi. All other authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Identification of new markers associated with long-term efficacy in patients treated with CAR T cells is a current medical need, particularly in diseases such as multiple myeloma. In this study, we address the impact of CAR density on the functionality of BCMA CAR T cells. Functional and transcriptional studies demonstrate that CAR T cells with high expression of the CAR construct show an increased tonic signaling with up-regulation of exhaustion markers and increased in vitro cytotoxicity but a decrease in in vivo BM infiltration. Characterization of gene regulatory networks using scRNA-seq identified regulons associated to activation and exhaustion up-regulated in CAR High T cells, providing mechanistic insights behind differential functionality of these cells. Last, we demonstrate that patients treated with CAR T cell products enriched in CAR High T cells show a significantly worse clinical response in several hematological malignancies. In summary, our work demonstrates that CAR density plays an important role in CAR T activity with notable impact on clinical response.

Multiple myeloma (MM) remains incurable despite the number of novel therapies that have become available in recent years. Occasionally, a patient with MM will develop an amyloid light-chain (AL) amyloidosis with organ dysfunction. Chimeric antigen receptor T-cell (CART) therapy has become a promising approach in treating hematological malignancies. Our institution has developed a second-generation B-cell maturation antigen (BCMA)-CART which is currently being tested in a clinical trial for relapsed/refractory MM.We present the first reported case, to our knowledge, of a patient with AL amyloidosis and renal involvement in the course of an MM, successfully treated with CART therapy targeting BCMA. The patient received a fractioned dose of 3×10 6 /kg BCMA-CARTs after lymphodepletion. At 3 months from infusion, the patient had already obtained a deep hematological response with negative measurable residual disease by flow cytometry in the bone marrow. After 12 months, the patient remains in hematological stringent complete remission and has achieved an organ renal response with a decrease of 70% of proteinuria.This case suggests that concomitant AL amyloidosis in the setting of MM can benefit from CART therapy, even in patients in which predominant symptoms at the time of treating are caused by AL amyloidosis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: We previously reported algorithms based on clinical parameters and plasma cell characteristics to identify patients with smoldering multiple myeloma (SMM) with higher risk of progressing who could benefit from early treatment. In this work, we analyzed differences in the immune bone marrow (BM) microenvironment in SMM to better understand the role of immune surveillance in disease progression and to identify immune biomarkers associated to higher risk of progression., Methods: Gene expression analysis of BM cells from 28 patients with SMM, 22 patients with monoclonal gammopathy of undetermined significance (MGUS) and 22 patients with symptomatic MM was performed by using Nanostring Technology., Results: BM cells in SMM compared to both MGUS and symptomatic MM showed upregulation of genes encoding for key molecules in cytotoxicity. However, some of these cytotoxic molecules positively correlated with inhibitory immune checkpoints, which may impair the effector function of BM cytotoxic cells. Analysis of 28 patients with SMM revealed 4 distinct clusters based on immune composition and activation markers. Patients in cluster 2 showed a significant increase in expression of cytotoxic molecules but also inhibitory immune checkpoints compared to cluster 3, suggesting the presence of cytotoxic cells with an exhausted phenotype. Accordingly, patients in cluster 3 had a significantly longer progression free survival. Finally, individual gene expression analysis showed that higher expression of TNF superfamily members (TNF, TNFAIP3, TNFRSF14) was associated with shorter progression free survival., Conclusions: Our results suggest that exhausted cytotoxic cells are associated to high-risk patients with SMM. Biomarkers overexpressed in patients with this immune gene profile in combination with clinical parameters and PC characterization may be useful to identify SMM patients with higher risk of progression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Isola, Brasó-Maristany, Moreno, Mena, Oliver-Calders, Paré, Rodríguez-Lobato, Martin-Antonio, Cibeira, Bladé, Rosiñol, Prat, Lozano and Fernández de Larrea.)

Background: Chimeric antigen receptor (CAR)-T cell immunotherapy has modified the concept of treatment in hematological malignancies. In comparison with pediatric patients, where responses are maintained over many years, older patients, such as those with non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM), present lower persistence of CAR-T cells that might be due to decreased fitness of T cells acquired with aging. Moreover, cord blood derived-NK cells (CB-NKs) and CAR-NK cells derived from CB-NK can be used 'off-the-shelf' as immune cells with antitumor properties for the treatment of cancer patients. However, to date, clinical studies have only demonstrated the safety of these therapies but not optimal efficacy. To confront the shortcomings of each therapy, we devised a novel approach consisting of simultaneous (CAR-)NK cell and CAR-T cell administration. In this setting, NK cells demonstrate an important immunoregulation of T cells that could be exploited to enhance the efficacy of CAR-T cells., Methods: A combinatorial treatment based on either CAR-T and CAR-NK cells or CB-NK and CAR-T cells in two models of NHL and MM was performed. Antitumor efficacy was analyzed in vitro and in vivo, and parameters related to early activation, exhaustion and senescence of T cells were analyzed., Results: We show that CAR-NK cells derived from CB-NK are only effective at high doses (high E:T ratio) and that their activity rapidly decreases over time in comparison with CAR-T cells. In comparison and to exploit the potential of 'off-the-shelf' CB-NK, we demonstrate that a low number of CB-NK in the CAR-T cell product promotes an early activation of CAR-T cells and their migration to MM cells leading to enhanced anti-MM efficacy. Moreover, cytokines related to CRS development were not increased, and importantly, CB-NK enhanced the fitness of both CAR pos and CAR neg T cells, promoting lower levels of exhaustion and senescence., Conclusion: This study demonstrates a relevant immunoregulatory role of CB-NK collaborating with CAR-T cells to enhance their antitumor activity. A novel and different approach to consider in CAR-T cell immunotherapy studies is presented here with the goal to enhance the efficacy of the treatment., Competing Interests: Competing interests: None., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: T-cell immunoreceptor with Ig and ITIM domain (TIGIT) blockade could represent an alternative therapeutic option to release the immune response in patients with multiple myeloma. Here we analyzed the expression of TIGIT and its ligands poliovirus receptor (PVR) and nectin-2 in the bone marrow (BM) of patients with monoclonal gammopathies and the efficacy of TIGIT blockade activating antimyeloma immunity., Experimental Design: Expression levels of TIGIT and its ligands were characterized by flow cytometry and ELISA. TIGIT blockade was analyzed in in vitro functional assays with peripheral T cells. BM cells were studied with NanoString technology, real-time PCR, and ex vivo patient BM cell models., Results: TIGIT and its ligands are highly expressed in the BM of patients with multiple myeloma, suggesting that may play a role in restraining immune activation. TIGIT blockade depleted FoxP3 + Tregs while increasing proliferation of IFNγ-producing CD4 + T cells from patients with multiple myeloma. PVR ligation inhibited CD8 + T-cell signaling and cell proliferation which could be overcome with anti-TIGIT mAb. However, BM cells showed a remarkable heterogeneity in immune signature. Accordingly, functional ex vivo BM assays revealed that only some patients respond to checkpoint blockade. Thus, response to TIGIT blockade correlated with low frequency of TIGIT + cells and high nectin-2 expression on malignant plasma cells., Conclusions: TIGIT blockade efficiently reinvigorated peripheral T cells from patients with multiple myeloma. However, in the BM, the efficacy of blocking anti-TIGIT mAb to achieve tumor cell death may depend on the expression of TIGIT and nectin-2, becoming potential predictive biomarkers for identifying patients who may benefit from TIGIT blockade., (©2020 American Association for Cancer Research.)

Mechanisms of immune regulation may control proliferation of aberrant plasma cells (PCs) in patients with monoclonal gammopathy of undetermined significance (MGUS) preventing progression to active multiple myeloma (MM). We hypothesized that CD85j ( LILRB1 ), an inhibitory immune checkpoint for B cell function, may play a role in MM pathogenesis. In this study, we report that patients with active MM had significantly lower levels of CD85j and its ligand S100A9. Decreased CD85j expression could also be detected in the premalignant condition MGUS, suggesting that loss of CD85j may be an early event promoting tumor immune escape. To gain insight into the molecular mechanisms underlying CD85j functions, we next enforced expression of CD85j in human myeloma cell lines by lentiviral transduction. Interestingly, gene expression profiling of CD85j-overexpressing cells revealed a set of downregulated genes with crucial functions in MM pathogenesis. Furthermore, in vitro functional assays demonstrated that CD85j overexpression increased susceptibility to T cell- and NK-mediated killing. Consistently, ligation of CD85j decreased the number of PCs from individuals with MGUS but not from patients with MM. In conclusion, downregulation of inhibitory immune checkpoints on malignant PCs may provide a novel mechanism of immune escape associated with myeloma pathogenesis., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Natural killer (NK) cells are important mediators of anti-tumor immunity and are active against several hematologic malignancies, including multiple myeloma (MM). Umbilical cord blood (CB) is a promising source of allogeneic NK cells but large scale ex vivo expansion is required for generation of clinically relevant CB-derived NK (CB-NK) cell doses. Here we describe a novel strategy for expanding NK cells from cryopreserved CB units using artificial antigen presenting feeder cells (aAPC) in a gas permeable culture system. After 14 days, mean fold expansion of CB-NK cells was 1848-fold from fresh and 2389-fold from cryopreserved CB with >95% purity for NK cells (CD56(+)/CD3(-)) and less than 1% CD3(+) cells. Though surface expression of some cytotoxicity receptors was decreased, aAPC-expanded CB-NK cells exhibited a phenotype similar to CB-NK cells expanded with IL-2 alone with respect to various inhibitory receptors, NKG2C and CD94 and maintained strong expression of transcription factors Eomesodermin and T-bet. Furthermore, CB-NK cells formed functional immune synapses with and demonstrated cytotoxicity against various MM targets. Finally, aAPC-expanded CB-NK cells showed significant in vivo activity against MM in a xenogenic mouse model. Our findings introduce a clinically applicable strategy for the generation of highly functional CB-NK cells which can be used to eradicate MM.

Hepcidin antimicrobial peptides (HAMPs) are key molecules of the innate immune system against bacterial infections and in iron metabolism. In this study we report the molecular cloning and genomic characterization of four HAMP genes (referred to as HAMP1, HAMP2, HAMP3 and HAMP4) in the redbanded seabream (Pagrus auriga). All these genes possessed the eight characteristic cysteine residues involved in protein folding. No canonical sequence for convertase-mediated processing of the HAMP3 propeptide was identified. At the genomic level, all four HAMP genes consisted of two introns and three exons. Phylogenetic analysis revealed that HAMPs could group in two main clusters with HAMP2, HAMP3 and HAMP4 belonging to the more complex and diversified HAMP2-like group of acanthopterygians. Quantitation of mRNA levels in adult tissues showed that HAMP1 was ubiquitously expressed, HAMP2 mainly in kidney, spleen and intestine, whereas HAMP3 and HAMP4 in liver. During development, HAMP2 and HAMP3 were expressed at a high level in embryos. Moreover, the expression levels of the four HAMP genes increased between 5 and 15 days after hatching when larvae started external feeding. Induction experiments with lipopolysaccharide revealed significant changes in gene expression of the four HAMP genes in kidney, liver and spleen. However, expression profiles differed in magnitude and time course response. HAMP1 mRNAs increased rapidly in kidney at 1 h p.i. whereas HAMP2 did later at 24 h. Moreover, HAMP4 transcripts increased more than 5000-fold in liver whereas HAMP2 mRNAs dropped significantly in spleen at 3 h p.i. All these data suggest that HAMPs are involved in the response against bacterial infections although additional functions in iron regulation and embryogenesis in fish should be considered.

Lysozymes are key proteins of the innate immune system against bacterial infections. In this study we report the molecular cloning and characterization of the c-type and g-type lysozymes in brill (Scophthalmus rhombus). Catalytic and other conserved residues required for functionality were identified. Phylogenetic analysis revealed distinct evolutionary histories for each lysozyme type. Expression profiles of both lysozyme genes were studied in juvenile tissues using a real-time PCR approach. c-Type lysozyme was expressed mainly in stomach and liver, whereas the g-type was detected in all tissues with highest mRNA levels observed in the spleen. Induction experiments revealed that g-type transcripts increased significantly in head kidney after lipopolysaccharide (25- and 23-fold at 12 and 24h, respectively) and Photobacterium damselae subsp. piscicida (17-fold at 24h) treatments. In contrast, no induction was observed for c-type lysozyme. All these data suggest that g-type lysozyme is involved in the response against bacterial infections, whereas c-type lysozyme may also play a role in digestion.