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Universidad Autónoma de Madrid

Programa de Doctorado en Biociencias Moleculares

TESIS DOCTORAL

CROSSTALK BETWEEN INNATE AND ADAPTIVE IMMUNITY IN TELEOST FISH: THE ROLE OF DENDRITIC CELLS AND B CELL REGULATORY

CYTOKINES

Irene Soleto Fernández

Madrid, 2018

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Departamento de Bioquímica Facultad de Medicina

Universidad Autónoma de Madrid

Crosstalk between innate and adaptive immunity in teleost fish: the role of dendritic cells and B cell regulatory cytokines

Irene Soleto Fernández, licenciada en Biología por la Universidad Autónoma de Madrid

Directores

Carolina Tafalla y Aitor González Granja

Centro de Investigación en Sanidad Animal (CISA) perteneciente al Instituto Nacional de Investigación y Tecnología Agraria y

Alimentaria (INIA), Madrid.

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The work presented in this thesis has been founded by the following projects: AGL2014-53061- R and AGL2014-54556-JIN from the Spanish Ministry of Economy and Competitiveness (MINECO); by the European Commission under the Seventh Framework Programme for Research and Technological Development (FP7) of the European Union (Grant Agreement 311993 TARGETFISH); and by the European Research Council (ERC) Starting Grant 2011 (TEBLYM 280469) and ERC Consolidator Grant 2016 (TEMUBLYM 725061).

Irene Soleto has been founded by a BES-2015-072193 scholarship from MINECO.

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Dña CAROLINA TAFALLA PIÑEIRO, científico titular y D. AITOR GONZÁLEZ GRANJA, contratado doctor, del Centro de Investigación en Sanidad Animal (CISA) perteneciente al Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA).

INFORMAN

Que la presente Tesis Doctoral titulada “CROSSTALK BETWEEN INNATE AND ADAPTIVE IMMUNITY IN TELEOST FISH: THE ROLE OF DENDRITIC CELLS AND B CELL REGULATORY CYTOKINES” que presenta Dña. Irene Soleto Fernández para optar al grado de doctor en Biociencias Moleculares, ha sido realizada en el grupo de Inmunología y Patología de peces del CISA-INIA bajo su dirección y que reuniendo los requisitos exigidos y considerando que está concluida, autorizan su presentación para que pueda ser juzgada por el tribunal correspondiente.

Y para que así conste, firman el presente informe en Madrid a de de 201

Fdo. Carolina Tafalla Piñeiro Fdo. Aitor González Granja

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“No hay nada que temer en la vida, únicamente se debe entender. Ahora es tiempo de entender más, para temer menos.”

Maria Salomea Skłodowska-Curie (1867-1934)

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i

Agradecimientos

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Después de tanto tiempo echo la vista atrás y me doy cuenta de que esta tesis pertenece a muchas personas. Este ha sido un trabajo duro pero también muy gratificante no sólo por los resultados sino también por las personas que han estado a mi lado durante este camino.

Primero quiero agradecer a mis directores, a Carolina por ver en mí las cualidades para formar parte de un grupo tan fuerte y competitivo; a Aitor por enseñarme tanto dentro del laboratorio y sobre todo fuera de él; ojala esta relación tan bonita (y más que nada divertida) dure mucho tiempo. Y a ambos quiero agradecerles el gran esfuerzo (en especial al final) que han hecho para que esta tesis sea leída.

A mis compañeros, a todos, presentes y pasados sin ellos nunca lo hubiese conseguido gracias por la ayuda técnica, pero gracias sobre todo por las risas y buenos momentos que hemos pasado juntos. A mis compis del 12, Patri gracias por tantos buenos consejos, nunca imagine encontrar alguien donde me veo tan reflejada y con tan buena conexión, lo nuestro fue amor a primera vista. Gracias a Esther por aguantar esos Sortings interminables, a Alba que tanto me entiende con mis peleas con la burocracia y a Luci que es todo amor y sabe dónde está todo!!

Gracias chicas. Gracias a las chicas del L14 por poner paz en la locura, pero ser siempre tan alegres y comprensivas. No me olvido de las últimas incorporaciones los TEMUMLYMs del 10, todavía no hemos conseguido la subvención por teneros aquí, pero a pesar de eso habéis resultado ser unos magníficos fichajes, gracias también chicos. No puedo dejar de acordarme de la gente que ha pasado por aquí, Esther Leal que tanto me enseño del mundo pre-doc e Itziar una de las personas más divertidas e interesante que he conocido.

Also I want to thank all the people that I met in the field of fish immunology. The people from Germany, Dr. Fischer, Taku and Sussan for helping me in the lab and taking care me. Thanks also to Suman for making my life in Germany funnier. And of course thanks to my sweet Yeh-fang for being always happy, visiting me in Germany (this was very special for me) and sharing with me one of the most special moment in my life. The best part of fish immunology is the friends that I have made.

Parte de esta tesis también es de mi familia, de todos y cada uno de ellos, de mis abuelos que tan importantes son para mi ahora, pero cuanto lo fueron también en las primeras etapas de mi vida.

Gracias a mi tío que siempre fue el referente académico, a pesar de que no nos une ningún lazo sanguíneo, porque casi ha conseguido que me crea que soy adoptada. Pero sobre todo de mis padres. GRACIAS a ellos que tanto me han dado, porque sin ellos no hubiese llegado hasta aquí.

Sin mis padres y sin su esfuerzo nunca lo hubiese conseguido este trabajo más que mío es vuestro.

Y por último pero no por ello menos importante gracias a mi amigo, mi compañero de vida, y desde hace bien poco también mi marido. Durante este tiempo junto a ti hemos ido madurando y cumpliendo etapas; GRACIAS por estar siempre a mi lado, comprenderme y apoyarme en absolutamente todas mis decisiones, y gracias también por aguantar la montaña rusa emocional que ha supuesto la recta final de la tesis. Siéntete orgulloso porque esto también es tu recompensa.

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Resumen

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Las células dendríticas (DCs) son células presentadoras de antígenos especializadas que conectan el sistema inmunitario innato y adaptativo. Un estudio previo de nuestro grupo identificó una subpoblación que expresaba en la membrana CD8α y el complejo mayor de histocompatibilidad II (MHC II) en la piel trucha arcoíris (Oncorhynchus mykiss) (CD8+DCs).

Estas células CD8+ presentaron características fenotípicas y funcionales de DCs de presentación cruzada (cross-presenting) como la expresión de CD8, CD103, CD141, Batf3, entre otros. En este trabajo identificamos una población homóloga en la branquia y el intestino de trucha arcoíris.

Hemos realizado caracterización fenotípica y funcional de estas nuevas poblaciones que incluyen nuevas habilidades de DCs en teleósteos. Se encontraron diferencias fenotípicas y funcionales entre las tres poblaciones, lo que sugiere que la localización modifica el estadio de maduración así como las capacidades imunogénicas de estas. A pesar de esto las tres poblaciones expresaron de marcadores de presentación cruzada y citoquinas reguladoras de células B pertenecientes a la superfamilia de ligandos del factor de necrosis tumoral (TNF) como el factor de activación de células B (BAFF), el ligando inductor de la proliferación (APRIL) y la molécula tipo BAFF y APRIL (BALM).

Estos miembros de la superfamilia de TNF son producidos principalmente por células innatas como DCs y juegan un papel importante en la activación y diferenciación de células B. Por este motivo, hemos estudiado como se regulan estas citoquinas y sus receptores en un modelo de inflamación. Para ello, hemos estudiado la respuesta de la cavidad peritoneal tras una inyección intraperitoneal (i.p) del virus de la septicemia hemorrágica viral (VHSV), ya que se ha descrito que este proceso inflamatorio está mediado por células B IgM+. Tras un análisis transcripcional de los leucocitos peritoneales, se observó un aumento en los niveles de expresión tanto de BAFF como de su receptor (BAFF-R) tras la i.p de VHSV. Por otra parte, cuando se analizó la regulación de estos genes en células IgM+ sorteadas, se observaron aumentos de los niveles transcripcionales de BAFF, APRIL, BALM así como de sus receptores. Estos resultados demuestran que estas citoquinas y sus receptores juegan un papel muy relevante en la inflamación peritoneal mediada por células B IgM+. Finalmente, habiendo demostrado que las DCs de peces también producen APRIL, determinamos los efectos de APRIL en las células B IgM+, utilizando en este caso células B de bazo. Hemos comparado los efectos producidos sobre las células B IgM+, con los efectos previamente descritos para BAFF. En el actual trabajo hemos demostrado que APRIL induce la proliferación de células B IgM+ exclusivamente, aumenta el número de células secretoras de IgM e incrementa los niveles de MHC II en la superficie de estas células así como su capacidad de procesar antígenos.

Los resultados de este estudio incrementan el conocimiento sobre la interacción entre la respuesta inmunitaria innata y la adquirida y sobre como las células B son reguladas por células innatas como las DCs. Este conocimiento es esencial para el desarrollo racional de vacunas, siendo este uno de los desafíos más importantes de la acuicultura actualmente.

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Abstract

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Dendritic cells (DCs) are the most specialized antigen presenting cells (APCs), being a key link between innate and acquired immunity. A previous study in our group identified a cell subset that expresses CD8α and major histocompatibility complex II (MHC II) on the cell membrane in rainbow trout skin (Oncorhynchus mykiss) (CD8+ DCs). These CD8+ cells shown phenotypical and functional characteristics of cross-presenting DCs such us expression of CD8, CD103, CD141, Batf3, and others. In this work, we report the identification of homologue populations in rainbow trout gills and intestine. Consequently, we have carried out a broad phenotypic and functional characterization of these new populations that includes the confirmation of novel capacities for DCs in teleost. Interestingly, phenotypic and functional differences were found between the three populations suggesting that the location of DCs strongly shape their mature stage and their immune capacities. Despite the differences found among these populations, some common characteristics were identified in all subset such as the expression of cross presenting markers and regulatory B cell cytokines belonging to the tumor necrosis factor (TNF) superfamily of ligands such us B cell activating factor (BAFF) a proliferation-inducing ligand (APRIL) and BAFF and APRIL like molecule (BALM).

These members of the TNF superfamily are produced mainly by innate cells like DCs and play and important role in activation and differentiation of B cells. For this reason, we have studied how these cytokines and their potential receptors are regulated throughout an inflammation model.

To carry this out, we have studied the response of the peritoneal cavity to an intraperitoneal injection (i.p) with viral hemorrhagic septicemia virus (VHSV), given that previous studies had described that this inflammatory process is mainly mediated by IgM+ B cells. We first performed a transcriptional analysis in total leukocytes from peritoneum, observing an up regulation of the BAFF and BAFF receptor (BAFF-R) mRNA levels after the (i.p) of VHSV. On the other hand, when we analyzed the regulation of these genes in sorted IgM+ B cells, we observed increases in the levels of transcription of BAFF, APRIL, BALM and their receptors. These results reveal that these cytokines as well as their receptors play a key role in peritoneal inflammation processes mediated by IgM+ B cells.

Finally, having demonstrated that fish DCs also produce APRIL, we studied the functionality of this cytokine on IgM+ B cells, using in this case splenic B cells. We have compared the effects produced by APRIL on IgM+ B cells to those previously reported for BAFF, to increase our knowledge on the relation between these phylogenetically close cytokines. In the current work, we have demonstrated that APRIL induces an exclusive proliferation of IgM+ B cells, increases the number of IgM secreting cells and also increases the levels on MHC II on the surface of these cells as well as their antigen processing capacity.

The results of this study increase our knowledge concerning the crosstalk between innate and adaptive immune responses and on how B cells are regulated by innate cells such as DCs. This knowledge will be essential for the rational design of vaccines, one of the most important goals of aquaculture at the moment.

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Table of Contents

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List of Abbreviations ... xvii

General introduction ... 1

1. Relevance of aquaculture ... 3

2. Infectious diseases in aquaculture ... 3

2.1. Bacterial diseases ... 4

2.2. Viral diseases... 4

2.3. Parasitic diseases ... 5

2.4. Fungal diseases ... 5

3. Strategies to prevent pathogens in aquaculture ... 6

4. General organization of the immune system in teleost ... 8

5. Primary lymphoid organs ... 10

5.1. Thymus ... 10

5.2. Kidney ... 10

6. Secondary lymphoid organs ... 10

6.1. Spleen ... 10

6.2. Mucosal associated lymphoid tissue ... 11

7. The innate immune response in teleost ... 12

8. The adaptive immune response in teleost... 13

8.1. B cells ... 14

8.2. T cells ... 16

9. Cross-talk between innate and adaptive immune elements during the onset of adaptive immunity ... 17

9.1. Patter recognition receptors ... 17

9.2. Dendritic cells ... 19

9.3. Tumor Necrosis Factor superfamily of ligands ... 20

Aims ... 23

Chapter I: Identification of a potential common ancestor for mammalian cross-presenting dendritic cells in teleost respiratory surfaces ... 27

Summary I ... 29

Introduction ... 31

Abstract ... 31

Materials and methods ... 32

Results ... 34

Discussion ... 40

References ... 42

Supplementary material... 44

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Chapter II: Identification of CD8α+ dendritic cells in rainbow trout (Oncorhynchus

mykiss) intestine ... 49

Summary II ... 51

Abstract ... 54

Introduction ... 55

Materials and Methods ... 56

Results ... 61

Discussion ... 69

References ... 72

Chapter III: The BAFF/APRIL axis plays and important role in virus-induced peritoneal responses in rainbow trout ... 77

Summary III ... 79

Abstract ... 81

Introduction ... 81

Materials and Methods ... 82

Results ... 83

Discussion ... 86

References ... 88

Supplementary material... 89

Chapter IV: Regulation of IgM+ B cell activities by rainbow trout APRIL reveals specific effects of this cytokine in lower vertebrates ... 91

Summary IV ... 93

Abstract ... 95

Introduction ... 95

Materials and Methods ... 96

Results ... 99

Discussion ... 103

References ... 106

Supplementary material... 109

General discussion ... 113

Conclusiones ... 125

Conclusions ... 129

References ... 133

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List of Abbreviations

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xix Allo: allogeneic

APC: antigen presenting cell

APRIL: a proliferation-inducing ligand ASC: antibody secreting cell

BAFF: B cell activating factor BAFF-R: BAFF receptor

BALM: BAFF and APRIL like molecule BCMA: B cell maturation antigen BCR: B cell receptor

BCWD: bacterial cold water disease BSA: bovine serum albumin

CD40L: CD40 ligand cDC: conventional DC cDNA: complementary DNA CLR: C-type lectin receptor ConA: concanavalin A D: diversity

DAMP: danger-associated molecular pattern DC: dendritic cell

DNA: deoxyribonucleic acid DNAse: deoxyribonuclease dNTP: nucleoside triphosphate dsRNA: double stranded RNA DTT: dithiothreitol

EC: European commission

EDTA: ethylenediaminetetraacetic acid ELISA: enzyme-linked immunosorbent assay EU: European Union

FACs: fluorescence-activated cell sorting

FAO: Food and Agriculture Organization of the United Nations FCS: fetal calf serum

Fig: figure

Flt3: fms-like tyrosine kinase 3 FSC: forward scatter

GALT: gut-associated lymphoid tissue GC: germinal center

GIALT: gill-associated lymphoid tissue i.p: intraperitoneal injection

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xx IEL: intraepithelial lymphocytes

IFN: interferon Ig: immunoglobulin

IHNV: infectious hematopoietic necrosis virus IL: interleukin

IPNV: infectious pancreatic necrosis virus IRF8: interferon regulatory protein 8 ISAV: infectious salmon anemia virus Iso: isogenic

J: joining

L-15: Leibovitz medium 15 LP: lamina propria

LPS: bacterial lipopolysaccharide mAb: monoclonal antibody

MALT: mucosal associated lymphoid tissue MFI: mean fluorescence intensity

MHC II: mayor histocompatibility complex II MHCI I: mayor histocompatibility complex I MMC: melanomacrophage center

mRNA: messenger RNA

NALT: nasopharynx-associated lymphoid tissue NLR: Nod-like receptor

P/S: penicillin streptomycin pAb: polyclonal antibody

PAMP: pathogen-associated molecular pattern PBS: phosphate buffer saline

PCR: polymerase chain reaction pDC: plasmacytoid DC

PI: propidium iodide

PKD: proliferative kidney disease PMNS: polymorphonuclear neutrophils PNA: Peanut lectin agglutin

Poly I:C: polyinosinic:polycytidylic acid PRRS. pattern recognition receptors RNA: ribonucleic acid

RT: room temperature

RTFS: rainbow trout fry syndrome SALT: skin-associated lymphoid tissue

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xxi SD: standard deviation

SSC: side scatter

TACI: transmembrane activator and calcium modulator and cyclophilin ligand interactor TBS: tris-buffered saline

Tc: cytotoxic T cell TCR: T cell receptor

TD: T -dependent immune response TGF: transforming growth factor Th: helper T cell

THD: TNF homology domain TLR: toll-like receptor TNF: tumor necrosis factor

TNFRSF: TNF receptor superfamily TNFSF: TNF ligands superfamily Treg: regulatory T cell

V: variable

VHSV: viral hemorrhagic septicemia virus VLP: virus-like particle

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1

General introduction

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2

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3 1. Relevance of aquaculture

According to the Food and Agriculture Organization of the United Nations (FAO), aquaculture comprises diverse systems of farming plants and animals in inland, coastal and marine areas, using and producing a wide variety of species. Currently, aquaculture is a primordial need worldwide since fishing alone cannot meet the growing global demand for seafood. In fact, although the amount of fish obtained through extractive fishing has been stable in the last twenty years, the aquaculture production has continuously increased its production, at a rate of approximately 7% per year. According to the last FAO fisheries and aquaculture yearbook, the aquaculture industry produced 106 million tons in 2015, 12 million tons more than the production obtained through fishing that same year, highlighting its importance in fulfilling global feeding demands. Currently, China is the main aquaculture producer in the world, accounting for 60.75% of the global production volume. Based on the data stated on the Food, Farming and Fisheries Report of the European Commission (EC), the European Union (EU) is the 8th largest producer of aquaculture products, accounting for 1.53% of the global production volume. The EU produces 1.25 million tons of aquaculture products each year, and most of this production is consumed within Europe. Among the total amount of the aquaculture production in the EU, 50% corresponds to mollusks and crustaceans, while 27% are marine fish and the rest corresponds to freshwater fish.

The main farmed fish species in Europe are Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata). Regarding shellfish, the main cultured species are mussel (Mytilus galloprovincialis) and Japanese oyster (Crassostrea gigas).

Spain is the main producer of aquaculture products in the EU, having produced an average of 25.000 tons per year between 2008 and 2015.

2. Infectious diseases in aquaculture

One of the main problems for the development of aquaculture is the impact of infectious diseases. In fish farms, due to an elevated population density, high levels of organic matter in the water provoke a decrease in oxygen concentration. Consequently, these intensive culture conditions, in many cases, produce chronic stress in the animals. All of these circumstances facilitate the appearance of pathogens and increase the susceptibility of the animals to these pathogens. Thus, infectious diseases have a great impact on the efficiency of the aquaculture production, not only as a consequence of direct fish losses but also because of the impact that they have on growth, production costs or reproduction cycles. These pathogens that impact cultured fish include bacteria, viruses, parasites and fungi, each of them with their specific problematic. As this thesis is focused on rainbow trout, we are shortly mentioning some of the most important diseases currently affecting rainbow trout aquaculture.

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4 2.1. Bacterial diseases

Even though bacterial diseases can be controlled by the use of authorized antibiotics as streptomycin, oxytetracycline or erythromycin, the excessive use of these drugs in the past has stimulated the appearance of resistant bacterial strains and is considered an important environmental issue. Therefore, stricter regulations have been implemented regarding the use of antibiotics in aquaculture and the presence of antibiotic traces in aquaculture products. For this reason, it is necessary to prevent and treat bacterial diseases with alternative systems, such as improved vaccination strategies and/or immunostimulants.

The main diseases provoked by bacterial pathogens that affect rainbow trout include red mouth disease, produced by the Gram negative bacteria Yersinia ruckery; furunculosis which is caused by the Gram negative bacteria Aeromonas salmonicida; lactococosis produced by the Gram positive bacteria Lactococcus garvieae and bacterial cold water disease (BCWD) also referred to as rainbow trout fry syndrome (RTFS), which is caused by the Gram negative bacteria Flavobacterium psychrophilum. Although there are some injectable vaccines available in the market against some of these pathogens, they are still responsible for important loses in the rainbow trout aquaculture industry.

2.2. Viral diseases

The impact of viral pathogens in aquaculture has become one of the most important aquaculture problems for the development of the industry causing great economical losses. This is a consequence of these pathogens usually provoking very high mortality rates and due to the lack of effective treatments as no antivirals are authorized for use in aquaculture. Nowadays, the most important viral agents that affect rainbow trout aquaculture include pathogens belonging to different viral families.

Rhabdoviridae family: this family encompasses several important pathogens that can infect rainbow trout such as viral hemorrhagic septicemia virus (VHSV) or infectious hematopoietic necrosis virus (IHNV). They are single stranded negative sense RNA viruses with a lipid envelope.

VHSV affects many different fresh and seawater species including rainbow trout, turbot (Scophthalmus maximus) and Northern pike (Esox lucius). The virus not only affects cultured species but has also been responsible for massive mortalities in wild animals in North America.

Although originally restricted to Europe, VHSV is now widely spread in North America and Asia.

VHSV is a notifiable virus in the EU.

Birnaviridae family: infectious pancreatic necrosis virus (IPNV) is included in the genus Aguabirnavirus. It was reported for the first time in 1957 in rainbow trout (Wolf and Quimby, 1971). IPNV is a double stranded RNA virus without a lipid envelope. This virus mainly affects salmonids but has also been reported in turbot, Northern pike and even in crustaceans and mollusks. The survivor fish often develop a chronic disease stage and become disease carriers.

IPNV can be vertically transmitted (Mulcahy and Pascho, 1984; Rodriguez Saint-Jean et al., 2003).

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5 Orthomyxoviridae family: a member of this family is infectious salmon anemia virus (ISAV) belonging to Isavirus genus. ISAV presents a lipidic envelop and its genome is composed of eight single-stranded RNA segments. ISAV affects mainly Atlantic salmon (Christie et al., 1988) but has also been reported in rainbow trout, saithe (Pollachius virens) or Atlantic cod (Gadus morbua) (Mjaaland et al., 1997). The virus is characterized by its haemagglutination capacity, it can be transmitted horizontally and affects different organs such as kidney, intestine and gills. (Aspehaug et al., 2004; Kibenge et al., 2009; Mjaaland et al., 1997).

2.3. Parasitic diseases

A parasite is an organism that lives inside or above another living organism obtaining nutrients from it while provoking damage. Generally, parasitic diseases are more chronic than diseases provoked by viruses or bacterial pathogens, and thus have lower mortality rates.

Therefore, their main impact in aquaculture is not as a consequence of fish losses but because of a decreased productivity over the whole production cycle. Rarely, however, some pathogens do produce important mortalities, such as for example the ciliate Ichthyophthirius multifiliis (usually shortened to “Ich”), associated with high mortalities in rainbow trout. This disease provoked by Ich is colloquially referred to as “white spot disease” as the parasite trophonts which feed on the tissues of the host can grow up to 1 mm in diameter and are clearly visible in the skin. Tetracapsuloides bryosalmonae is another important parasite for rainbow trout culture. It is a myxozoan responsible for proliferative kidney disease (PKD), a proliferative inflammatory disease that affects different salmonid species including rainbow trout. The number of licensed veterinary medicines to treat parasitic infections is quite low. Furthermore, most of these treatments have major environmental impacts or reduced efficacy due to parasite drug resistance. On the other hand, no effective vaccines against parasites have been developed to date.

2.4. Fungal diseases

Some pathogenic fungi have been also reported to affect fish. These fungi are mainly saprophytic, opportunistic pathogens that usually colonize previous lesions on fish tissues. The factors that determine the presence of fungus in the water are mainly high levels of organic matter, an elevated population density or the presence of dead animals in the water. They are likely to appear with low temperatures since the immune response of the fish is less efficient under these conditions. The infection of eggs with fungi is very common during incubation time, when they invade a dead egg and spread until they choke nearby eggs. There are some chemical compounds with disinfectant or antifungal properties that can be used to treat these infections. The most common are iodine-based compounds, chlorine, alcohols and ammonium salts (Amend and Wedemeyer, 1970). Nevertheless the antifungal compounds usually used in aquaculture are 1% of potassium permanganate and 0.5% of potassium iodide. Saprolegnia parasitica is the most relevant fungus pathogen for salmonids. It presents zoospores with flagella that feed from dead cells and in

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6 some cases can settle down within the gills. It can produce injuries and necrosis in the epidermis, generates stress and can produce respiratory failure when it colonizes the gills (van den Berg et al., 2013).

3. Strategies to prevent pathogens in aquaculture

In culture conditions, fish have to compete with each other for space and dissolved oxygen.

Furthermore, they are exposed to increased metabolites such as ammonia, carbon dioxide and suspended organic matter. In this kind of environment, a disease outbreak is more prone to happen than in the natural environment. Thus, one key aspect to prevent disease outbreaks in aquaculture is to eliminate or diminish the disease sources and increase the capacity of the fish to respond to the pathogens. Along this line, there are several approaches that can be used:

To avoid the presence of pathogens: one possibility is to use pathogen-free water obtained through sterilization of fresh water usually through filtration, and/or ultraviolet light treatment, ozonation or chlorination. Another procedure is to feed the animals with pathogen-free diets.

Commonly commercial feeds are pathogen-free. In the case of live food, required for the larval stages in some species, germicides can be used in order to disinfect such food. Another course of action is to make regular disease inspections, sampling and examination of a reduced number of animals to check for the presence of certain pathogens.

To decrease the stress in the host: another strategy is to reduce the stress in fish which can negatively influence their immune system. To archieve this, it is important to keep the quality of the water in good conditions, to control some parameters such as carrying capacity, accumulation of organic debris, levels of nitrogenous metabolites, carbon dioxide or hydrogen sulphide, pH, alkalinity or temperature. On the other hand, handling of the animals should be as gentle as possible and their nutrition type and rate must be specific for each species.

To improve host resistance: in order to increase the resistance of the host against infections, dietary immunostimulation can be used in farms, by supplementing the feed with nutritional components, such as vitamins, minerals or different natural extracts that may have a positive effect on the immune system and consequently reduce the occurrence and the severity of infectious diseases.

However, to date, the most effective approach to reduce the impact of infectious diseases from an economic, environmental and animal welfare point of view is vaccination. Vaccination can be defined as the administration of antigenic material from a pathogen (vaccine) in order to stimulate the immune system to develop a specific adaptive response and immunological memory that will confer resistance upon secondary encounters with this specific pathogen. The ideal vaccine should induce a similar immune response than that provoked by the real infection, so it can effectively block the pathogen dissemination at early time points. Additionally, the immune response generated should persist for long periods of time in order to protect against future encounters throughout the complete production cycle. Others factors to be taken into consideration

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7 when designing a vaccine for use in aquaculture are easy transportation, stability, and its safety for the environment and the consumer’s health. Currently, there are several commercial fish vaccines against bacterial pathogens which are commonly used in aquaculture, which have helped to drastically reduce the use of antibiotics. However, there are very limited vaccines available in the market against viruses, and most of them are very ineffective in the field. On the other hand, there are no vaccines available against fish parasites or fungi. According to the biological material supplied we can define these types of vaccines:

Inactivated vaccines: these vaccines are composed of microorganisms inactivated usually by heat or through the use of chemical compounds. Once inactivated, the pathogens cannot replicate and consequently they cannot reproduce the disease. In fish, inactivated vaccines are administrated by intraperitoneal injection along with oil adjuvants. These kind of vaccines are used frequently against bacterial pathogens (Gudding et al., 1999), but present low immunogenicity in the case of viruses. There are some commercial inactivated vaccines against viruses such as IPNV, but their effectivity in the field is quite limited (Rodriguez Saint-Jean et al., 2003).

Live attenuated vaccines: these are vaccines in which the microorganism is alive although weakened under laboratory conditions to diminish its virulence. These replicating vaccines have been proven to induce strong immune responses, working well in low doses, usually in the absence of boosters. However, their use in aquaculture is not allowed due to the risk of uncontrolled dissemination through the water and possible reversion of virulence.

Recombinant vaccines: in this case a specific subunit of the pathogen (one antigen or protein) is recombinantly produced in a production system such as bacteria, yeast or mammalian cells. These vaccines can only be obtained after the pathogen antigens have been identified. They are completely safe because they are not composed of a complete pathogen, but just a part of it.

The production of these vaccines is easy, efficient and cheap. However, their immunogenicity is usually low and they require the use of substances that can increase this initial response (adjuvants). This approach has been tried for many viruses, using different expression systems, however their effectivity has usually been quite low.

Virus like Particles (VLPs): these vaccines constitute a specific subtype of recombinant vaccines. They are based on the intrinsic ability of some viral proteins to self-assemble into particles that mimic the native viral structure. VLPs have been expressed in different biological systems (baculovirus, bacteria, yeast…) and successfully purified. These particles are more immunogenic than regular recombinant vaccines as they mimic the full viral particle but they are not infectious because they do not contain the genetic material (Dhar et al., 2014).

DNA vaccines: they are based on the administration of a eukaryotic expression vector that codes for an antigenic protein from a pathogen, which can be efficiently expressed in the host (Leong et al., 1997). These vaccines do not produce side effects in the host, are very stable and easy to produce. However, this type of vaccination has not been allowed in aquaculture in Europe in the past years because there were doubts regarding whether vaccinated fish could be considered

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8 transgenic animals given the potential risk that the vaccine DNA could be integrated into the host genome. However, it seems that in the past year the EC has given a green light to the eventual commercialization of these vaccines for their use in fish. Thus, although a great effort has been made in the past decades to develop effective and safe vaccines against the wide range of pathogens that can infect cultured fish species, most of these have failed. This is in great part a consequence of not fully understanding how adaptive immune responses are regulated in fish. Until then, vaccine design will just be a mere trial and error approach. Hence, a comprehensive analysis of how the fish immune system is regulated is paramount for the design of more efficient vaccination strategies in aquaculture.

Furthermore, most of the vaccines that are currently used in aquaculture are commonly administered by intraperitoneal injection, requiring the vaccination of fish one by one, a highly laborious process with high costs that can generate a great amount of stress to the fish thus limiting the efficacy of the vaccine. Oral vaccination is an ideal vaccination method for fish, since these vaccines are easy to administrate, do not generate stress and the production costs are not high (Lin et al., 2000; Vandenberg, 2004). However, thus far, these vaccines have reached very low efficiency rates. Again, one of the major setbacks for the optimization of oral vaccines is our lack of knowledge regarding how the mucosal (intestinal) immune system is regulated in these species.

It is known that oral tolerance mechanisms aimed at avoiding that fish react continuously to food- borne antigens tightly regulate and limit the capacity of the local intestinal immune system to respond to vaccine antigens.

4. General organization of the immune system in teleost

The immune response in vertebrates can be divided in two different types of responses: the innate immune responses, which are faster and non-specific to the pathogen, and the adaptive immune responses, also known as acquired responses, which are specific to each type of invading organism.

The innate immune response constitute the first line of defense against pathogens, and include physical barriers (skin, mucosal tissues…) as well as cellular and humoral components.

The adaptive immune response also includes humoral and cellular responses and it is characterized by the recognition of specific antigens. The adaptive response triggers a long-lasting protection mechanism (immunological memory) which will allow the individual to respond much faster and stronger against a secondary infection with the same pathogen. In the last years, immunologists have realized that both responses are coordinated in such a way that many cell types and molecules have specific yet differential roles in both responses. Furthermore, it has been demonstrated that only through the activation of an adequate innate immune response at the initial stages, an effective acquired immune response can be mounted.

In fish, when an animal encounters a pathogen, it is first blocked by different physical barriers such as fish scales and the mucous surfaces of skin, gills and intestine. Mucous surfaces

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9 trap and neutralize pathogens through the action of many innate immune molecules as lysozyme, complement, calmodulin, C-reactive protein, proteolytic enzymes, antimicrobial peptides, vitellogenin and and natural immunoglobulins (Igs). These molecules favor the direct elimination of the pathogen at the contact level (Alexander and Ingram, 1992; Dos Santos et al., 2001;

Magnadottir, 2006). Nevertheless, if the pathogen crosses the epithelium, innate cell mediated responses are then activated. The cells involved in this response express pattern recognition receptors (PRRs), which are capable of recognizing pathogen-associated molecular patters (PAMPs) found in all pathogenic organisms (Elward and Gasque, 2003). This cell mediated response has been shown to be activated in both myeloid and lymphoid cells, since fish lymphocytes have also been shown to play an important role in innate responses (Li et al., 2006;

Abos et al., 2013; Zhang et al., 2017). If these innate cellular mechanisms aimed at eliminating the pathogens from the host mainly through phagocytosis again fail to completely clear the pathogen, adaptive responses get involved. Adaptive responses take longer to be established but are more efficient. Throughout evolution adaptive immune responses appeared for the first time in cartilaginous fish (Flajnik and Kasahara, 2010). Nevertheless, although all the elements of the adaptive immune system are present in fish, acquired responses are quite slow, temperature- dependent and still not fully developed in all their potential. Therefore fish seem to still rely greatly on innate mechanisms to respond against infections (Bly and William Clem, 1991; Douglas et al., 2003).

In mammals, the immune system is composed of primary lymphoid organs, which are those producing the different lineages of immune cells. They also have secondary lymphoid organs which are special locations for antigen encounter and activation of immune cells (Inoue et al., 2018; Xie and Dent, 2018). In fish, the organization of immune organs greatly differs from that of mammals. Fish do not have bone marrow and the anterior part of the kidney assumes the hematopoietic function (Uribe et al., 2011), being the main organ where B cells develop. T cells, on the other hand, develop in the thymus, as it happens in mammals (Bowden et al., 2005). The spleen is the main secondary lymphoid organ, and since fish do not present lymph nodes, it is also the main antigen encounter location (Zapata et al., 2006). Another difference between mammals and fish concerning the organization of their immune structures is that the latter do not develop germinal center (GC) reactions. In mammals, GCs promote the close collaboration between antigen-specific B cells, follicular T helper cells and follicular dendritic cells (DCs). These structures constitutively occupy the central follicular zones of secondary lymphoid organs and within them, B cells divide in response to antigens and acquire the capacity to differentiate into antibody secreting cells (ASCs), reaching a terminal state of plasma cells or memory B cells, both of them with the capacity to secrete high affinity antibodies. A similar difference between the organization of B and T cells in mammals can be found in the mucosal tissues such as the intestine.

In mammals, B and T cells are organized in Peyer´s patches where follicular DCs can also be found. These structures favor the interaction between B cells and T cells to mount T-dependent

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10 (TD) B cell responses. In fish, however, B cells and T cells have been described scattered in a disorganized fashion in mucosal tissues (Nakanishi et al., 2015; Salinas et al., 2011).

5. Primary lymphoid organs 5.1. Thymus

The thymus is the organ where T lymphocytes develop and mature. Cartilaginous and bony fish are the most primitive vertebrates presenting an histologically identifiable thymus, although recently thymus-like lympho-epithelial structures have been reported in the gill filaments (Koppang et al., 2010) and the neighboring secondary lamellae of lamprey larvae. The thymus structure in fish is similar to mammals, and two main cell subsets can be found in it: epithelial cells and thymocytes that are mainly T cells. Thymus epithelial cells in concert with other cells such as mesenchymal cells generate an environment that encourages the maturation and differentiation of T cells (Bowden et al., 2005; Nakanishi et al., 2015; Zapata, 1996).

5.2. Kidney

The teleost kidney is the largest hematopoiesis site until adulthood. In rainbow trout, the kidney is developed after hatching, the moment when it starts to produce immune cells (Uribe et al., 2011). It is a complex organ divided in four parts with different structure and functions; the hematopoietic part, the reticuloepithelium, an endocrine part and an excretory part. The kidney transverses longitudinally the fish from the head up to half of the animal, adhered to the backbone.

The anterior part is also known as head kidney or pronephros and is usually branched in two lobules. The head kidney lacks nephrons and has exclusively a hematopoietic function. The posterior part of the kidney, on the other hand, has both renal and immune functions (Grassi Milano et al., 1997; Zapata et al., 2006). Different subset of leukocytes are found in the kidney including macrophages, granulocytes and melanomacrophages (macrophages that accumulate great amounts of melanin inside them) and lymphoid cells in different developmental stages, constituting IgM+ B cells the main lymphoid population found (Press et al., 1994; Uribe et al., 2011; Zwollo et al., 2010).

6. Secondary lymphoid organs 6.1. Spleen

The spleen is a compact dark red organ located within the peritoneal cavity. In mammals, the spleen contains a large amount of blood vessels and is anatomically divided in red and white pulp.

The red pulp occupies almost all the organ and it is mainly constituted by a network of macrophages and lymphocytes. The white pulp is located inside the red pulp, and is formed by capillaries with a fine endothelial tissue surrounded by fibrous conjunctive and macrophages. In fish, the white pulp is less developed, therefore this differentiation is not clear (Fange and Nilsson, 1985). Within the spleen, as in the kidney, melanomacrophage centers (MMCs), which are a group

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11 of melanomacrophages with other types of leukocytes, can be found. MMCs have been suggested to be sites were antigens are uptaken and presented to lymphocytes (Espenes et al., 1995; Falk et al., 1995; Press et al., 1994). The most important functions of the spleen include the filtration of blood, removal of damaged cells or erythrocytes, antigen encounter, antigen presentation and antibody production (Falk et al., 1995), although the basis of how an immune response is orchestrated in this organ is still widely unknown in fish.

6.2. Mucosal associated lymphoid tissue (MALT)

Fish leukocytes have been found not only in lymphoid organs but also in other tissues such as liver, intestine, gills, skin and gonad (Abos et al., 2013; Ballesteros et al., 2013; Chaves-Pozo et al., 2003). In mucosal tissues, which are in direct contact with the external media, lymphocytes are not organized in specialized lymphoid structures as, for instance, the mesenteric lymph nodes and the Peyer’s patches in the intestine of mammals. According to the anatomical location, MALTs in teleost received different designations: GALT refers to gut-associated lymphoid tissue; GIALT to gill-associated lymphoid tissue, NALT to nasopharynx-associated lymphoid tissue and SALT to skin-associated lymphoid tissue. In all these tissues, B and T cells have been identified. However, the cells are scattered through the tissue in a disorganized fashion. This has led several authors to hypothesize that fish MALTs are not equivalent to mammalian MALTs (Gomez et al., 2013; Parra et al., 2015; Tafalla et al., 2016)

In the case of the GALT, both B and T lymphocytes they can be found within the epithelial layer, thus called intraepithelial lymphocytes (IEL) or inside the lamina propria (LP) (Rombout et al., 2011). Although immune responses against diverse pathogens have been characterized in teleost gut (reviewed in (Rombout et al., 2011)), most of these studies have focused on analyzing the response of the posterior section of the intestine (usually referred to as hindgut). However, some recent studies have demonstrated the existence of lymphocytes in other sections of the digestive tract, capable of responding to infection or vaccination. These include the foregut, stomach, pyloric caeca and midgut segments (Ballesteros et al., 2013; Ballesteros et al., 2014).

Moreover, it has been shown that the pyloric caeca is where the response to oral vaccination was higher and where a significant recruitment of lymphocytes was reported in rainbow trout upon oral vaccination or infection (Ballesteros et al., 2013; Ballesteros et al., 2014).

Regarding the GIALT, the presence of lymphocytes in gill filaments has been known for years. However, in gills an additional interbranchial lymphoid tissue mainly composed of T cells was reported (Koppang et al., 2010). Several studies have pointed to the gills as an entry point for a wide range of pathogens (Nelson et al., 1985; Steigen et al., 2018), but many of these pathogens, however, do not produce damage to the gills and just use this organ as an attaching and entry point.

In addition to lymphocytes (Castro et al., 2014a), macrophages (Mulero et al., 2008), neutrophils (Lin et al., 1998) and ASCs (Davidson et al., 1997; Dos Santos et al., 2001) have also been reported in teleost gills. Concerning B cell subtypes, IgM+ B cells in gills have been identified in several

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12 species (Abos et al., 2013; Grontvedt and Espelid, 2003; Grove et al., 2006), while approximately half of the B cells in the GIALT are IgT+ B cells (Salinas, 2015). A further subset of B cells exclusively expressing IgD on the cell surface has also been described in rainbow trout gills (Castro et al., 2014a), but the role of these cells during the immune response is still unknown.

As teleost skin lacks keratin, the skin external layer is composed of live cells (epithelial, lymphoid and myeloid cells) in direct contact with the water. Some of these cells release substances to create a mucus layer in order to hinder the entrance of pathogens. The skin mucus contains many innate immune molecules with antimicrobial activity against viruses and bacteria (Padra et al., 2014; Valdenegro-Vega et al., 2014). Interestingly, in teleost, the skin is the main source of antimicrobial peptides (Gomez et al., 2013). Additionally, it has been shown that B and T cells residing in the epidermis of fish have the capacity to respond to pathogens (Findly et al., 2013;

Jorgensen et al., 2009; Xu et al., 2013).

In rainbow trout, the presence of a NALT has been recently described. Again, this lymphoid tissue is constituted by disperse immune cells, as it shown for skin and intestine. IgT+ cells constitute the main B cell subset in this tissue, although IgM+ cells can also be found. Nasal vaccination assays using attenuated viruses have demonstrated the capacity of the NALT to trigger an important local antiviral response that is taken to a systemic level (Salinas, 2015; Tacchi et al., 2014)

7. The innate immune response in teleost

The fish immune system includes almost all the components of the innate immune system present in mammals (Magnadottir, 2006). In fish, the innate response has been considered crucial in determining the outcome after a pathogenic exposure, given that in fish adaptive immune responses are generally slow to occur as they are dependent on temperature, since fish are poikiloterms (Langevin et al., 2013; Magnadottir, 2006). Additionally, some restrictions in the capacities of the adaptive immune elements have been described in fish including a limited repertoire of antibodies, slow lymphocyte proliferation and maturation and poor generation of memory (Kaattari et al., 2002; Ma et al., 2013; Whyte, 2007).

The components of innate immune system are usually divided into physical barriers, cellular responses and humoral responses. Regarding the physical barriers, contact with pathogens usually occurs at the interface with the external environment on tissues such us skin, gut and gills. As mentioned above, these tissues are covered with mucus, which traps antigens neutralizing them through the action of immune molecules present in the mucus (Alexander and Ingram, 1992;

Brinchmann, 2016; Dos Santos et al., 2001; Magnadottir, 2006; Valdenegro-Vega et al., 2014).

Nevertheless, if the pathogen is able to cross these innate barriers, the cellular components of the innate immune system recognize the PAMPs present on this pathogen to activate a full innate immune response. Once these receptors are activated, one of the first events is the initiation of an inflammatory response, characterized by the release of cytokines such as interleukin 1β (IL1β)

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13 (Yan et al., 2014), tumor necrosis factor α (TNFα) (Roca et al., 2008) and chemokines (cytokines with chemoattractant capacities) such as IL8 (Seppola et al., 2008), which mediate the recruitment of leukocytes to the site of inflammation. The inflammatory response parallels that of mammals, since most of the pro-inflammatory cytokines present in mammals have been reported in salmonid fish (Collet, 2014).

Resident tissue macrophages contribute to the maintenance of homeostasis, in addition to responding to pathogens. On other hand, monocytes are mainly found in circulating blood, and upon an inflammatory signal, they are infiltrated to the tissues where they differentiate to macrophages with a high phagocytic capacity. This high capacity to capture antigens, also renders them as professional antigen presenting cells (APCs) that can present peptides obtained after the proteolytic cleavage of the antigen in the context of MHC class II to cells of the adaptive immune system(Yang et al., 2014). Macrophages also produce cytokines that modulate the activity of other leukocyte types. Macrophages can be activated through different routes; classically activated macrophages respond to a bacterial stimuli plus interferon (IFN) and have been identified in different teleost species like zebrafish (Danio rerio), catfish (Ictalurus punctatus) and common carp (Cyprinus carpio), while alternatively activated macrophages have not been functionally identified in fish even though some of the cytokines produced by them, such as IL4 and IL13 have been reported in some fish species (Li et al., 2007). Finally, regulatory macrophages are associated with the production of IL10. Again, even though IL10 has been cloned in different fish species, evidence of this activation pathway remains elusive (Inoue et al., 2005; Lutfalla et al., 2003; Pinto et al., 2007).

PMNs, also referred to as granulocytes, are additional components of the innate immune system. They are myeloid cells with a characteristic structure that includes a polymorphic nucleus and high granule content in the cytoplasm. In mammals, these cells are divided into subgroups depending on the affinity of the granules for acid or basic dyes into eosinophil, basophils and neutrophils. In most fish species, only one type of granulocyte is found. Neutrophils are the main cell type found in most fish species including salmonids. However in some other species eosinophil or basophils are the main granulocyte subtypes present. Neutrophils are highly phagocytic cells and one of the first subsets to arrive at the site of infection from blood or hematopoietic organs. Thus, they play an important role in innate responses by killing and degrading the microorganism and also are repairing the inflammatory injury (Overland et al., 2010).

DCs are also professional APCs, considered elements of the innate immune system.

However, given that they do not participate in pathogen clearance and that their main role is to present antigens to cells of the adaptive immune system, they will be described later.

8. The adaptive immune response in teleost

When a pathogen persists after the action of the innate immune system, the adaptive immune system takes the stage executing a more effective specific response that generates immunological

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14 memory to protect the organism against posterior pathogen encounters (Rauta et al., 2012). Even though this adaptive immune response takes a prominent role at later time points post-infection, it is already arranged at the very early time points and is highly dependent on the actions of the innate immune system, given that elements of the innate system also participate in the regulation and initiation of this response. The acquired immune response is mainly orchestrated by two different types of lymphocytes, each of them responsible for either the cellular or the humoral arms of adaptive immunity. T cells are responsible for cell-mediated immunity whereas B cells produce Igs that are the essential elements of the humoral immune response.

Both B and T cells are characterized by the presence of specific surface receptors, extremely diverse molecules at their antigens binding domains. Thus, each lymphocyte expresses a specific receptor that will recognize a specific antigen. This diversity enables recognition of an almost infinite range of antigens, so that each different pathogen can be targeted specifically. This diversity is generated by somatic recombination of variable (V), diversity (D) and joining (J) gene segments within the genes that code for these receptors (Mashoof and Criscitiello, 2016).

8.1. B cells

In teleost, the spleen is the main secondary immune organ and, in the absence of lymph nodes, it is also an important site for B cell activation and differentiation (Abos et al., 2015; Barr et al., 2011; Zapata et al., 2006). In the spleen, B cells constitute around 40% of the cells (Abos et al., 2013), but are always found in a disorganized fashion, given that the formation of GCs has never been reported. B cells also represent more than 30-40% of the cells in peripheral blood and are also present in other tissues like the peritoneal cavity, liver, intestine, skin and gills (Abos et al., 2013;

Castro et al., 2017; Salinas et al., 2011). In mammals, many different B cell subsets characterized by surface expression of specific cell markers are present, playing different roles throughout the immune response. Whether these phenotypically and functionally different B cell subsets are present in fish has not been fully clarified yet in fish. Three different Igs have been reported in fish to date, namely IgM (Warr et al., 1979), IgD (Wilson et al., 1997) and IgT (Hansen et al., 2005), designated as IgZ in some species such as zebrafish. According to the surface expression of these Igs, different subsets of B cells have been described. IgM+ IgD+ IgT- (IgM+ B cells) are the equivalent to mammalian naïve B cells (Lutz et al., 1998), represent the main subset of B cells in central immune organs and are present in all teleost fish examined to date (Castro et al., 2014a;

Tafalla et al., 2017) As it happens in mammals, these cells seem to lose membrane IgD during their differentiation to plasmablasts or plasma cells (Granja and Tafalla, 2017; Tafalla et al., 2017), thus rendering IgM+ IgD- IgT- cells. IgM- IgD+ IgT- cells (IgD+ B cells) (Castro et al., 2014a; Edholm et al., 2010) have also been reported in rainbow trout gills (Castro et al., 2014a) and channel catfish blood (Edholm et al., 2010), although their precise immune role is still unknown. Interestingly, B cells expressing only IgD on the membrane have also been reported in humans, especially within the upper respiratory tract (Chen and Cerutti, 2010), although also in this case their exact role in the

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15 immune response has still not been fully elucidated. Finally, IgT has been described as a specific teleost Ig in many fish species, which is solely expressed on the membrane of a specific lineage of B cells, IgM- IgD- IgT+ (IgT+ B cells) (Zhang et al., 2010). In those species expressing IgT, the ratio of IgT+ cells to IgM+ cells is higher in mucosal tissues. Thus, while IgT+ B cells constitute approximately 20% of total B cells in the spleen, the kidney and the peritoneal cavity of B cells, they represent 51% of total B cells in the intestine (Zhang et al., 2010). This, together with the fact that IgT responses seemed to be preferentially activated in mucosal compartments in response to parasitic infections, led some authors to hypothesize that IgT is an Ig specialized in mucosal immunity, similarly to IgA in mammals (Salinas et al., 2011; Zhang et al., 2010). However, IgM responses have also been reported against pathogens in diverse mucosal tissues (Ballesteros et al., 2013; Ballesteros et al., 2014; Zhao et al., 2008) and systemic IgT responses have also been described in response to viral infections (Castro et al., 2013), DNA vaccination (Castro et al., 2014c) or parasitic infections (Abos et al., 2018b).

In mammals, when B cells are activated by engagement of the B cell receptor (BCR) by a cognate antigen, they proliferate and trigger a differentiation program (Avalos et al., 2014). During this proliferation, part of the progeny will differentiate into ASCs while another part will become short-lived plasma cells or long term memory B cells (Bayles and Milcarek, 2014; Kallies et al., 2007; Nutt et al., 2015). In rainbow trout, different subsets of ASCs have been characterized among IgM+ B cells, including plasmablasts (cells that retain a proliferative capacity, have a low capacity to secrete antibodies and very low BCR expression), short-term and long-term plasma cells (terminally differentiated cells which release high amounts of antibodies, with no proliferative capacity and usually low or no BCR expression) (Bromage et al., 2004; Zwollo et al., 2008; Zwollo et al., 2010).

Although the main function of B cells is antibody production, both in fish and mammals these cells have a high antigen presenting capacity to T cells and are therefore considered as professional APCs (Chen and Jensen, 2008; Lewis et al., 2014). Interestingly, teleost B cells present high phagocytic capacity (Aquilino et al., 2016; Li et al., 2006) which make them better APCs than mammalian B cells as they are also able to present particulate antigens they phagocytize and not only antigens uptaken by the BCR (Zhu et al., 2014). Furthermore, given their phagocytic capacity and an associated microbicidal activity, they also contribute to pathogen clearance (Salinas et al., 2011).

Another interesting difference between mammalian conventional B cells and fish IgM+ B cells is that fish IgM+ B cell share many phenotypical and functional features of mammalian B1 cells. B1 cells are consider as members of innate immune system. They secrete natural antibodies and contribute to eliminate apoptotic cells (Hardy, 2006). Similarly to mammalian B1 cells, fish B cells exhibit large size, present high complexity, show high expression levels of surface IgM and low levels of surface IgD (Abos et al., 2018a). Furthermore, a high percentage of fish IgM+ B express CD5 on the surface, a specific marker for B1a cells in mammals. Furthermore, fish IgM+ B

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16 do not proliferate upon BCR cross-linking and have an extended survival, as B1 cells (Abos et al., 2018a). This data, together with the fact that in fish there is no antibody class switch, there is low antibody maturation and no follicular structures strongly suggests that fish IgM responses resemble TI extrafollicular IgM responses that take place in mammals.

8.2. T cells

T cells are characterized by the expression of a T cell receptor (TCR) on their membrane.

While B cells can recognize antigens by themselves, T cells are not capable of recognizing the antigens directly and can only do it when it is presented in the context of MHC class I or II. A first classification of T cells is based on the expression of TCR chains. Thus, TCRs can be formed by a heterodimer of αβ chains or γδ chains. γδ T cells recognize non processed antigens, and in mammals are considered to play innate functions, with little dependence on antigen presentation by MHC. These cells are mainly present in epithelial and mucosal tissues and represent 2% of total T cells in those tissues (Bonneville et al., 2010). Conventional T cells, on the other hand, express αβ chains and are divided into cytotoxic T cells (Tc) and helper T cells (Th) characterized by differential expression of membrane glycoproteins CD8 and CD4, respectively (Parnes, 1989), These glycoproteins are TCR co-receptors that stabilize the interaction between TCR and MHC and therefore promote the activation of the TCR signaling (Parnes, 1989). Tc cells are responsible for the elimination of infected cells through recognition of foreign antigens presented by MHC class I (Gulzar and Copeland, 2004; Houghton and Guevara-Patiño, 2004). Th cells produce cytokines that regulate the function of other immune cells; mainly B cells. One of the earliest discovered functions of T cells was the provision of help to B cells (Crotty, 2015). The antigen- specific interaction between B cells and T cells in the GC of secondary lymphoid organs is an essential step in TD humoral immune responses. During the interaction B cells present a specific antibody to helper T cells which in turn produce cytokines signals such us CD40L that induce B cell survival, proliferation and differentiation (Okada et al., 2005).

In mammals, Th cells are classified in different subsets based on their differential expression of transcription factors and their cytokine secretion profile (Zhu and Paul, 2010). The main populations described thus far in mammals are Th1, Th2, Th17, Th21 and regulatory T cells (Treg), although other groups are also included by some authors. However, whether these different subsets constitute different subtypes or correspond to one set of cells in different maturation points is still controversial (Kleinewietfeld and Hafler, 2013). In any case, Th1 cells release effector cytokines like IFNδ and TNFα in response to intracellular infections and ILs such as IL2 that induce lymphocyte proliferation (Farrar et al., 2002). Th2 cells, on the other hand, produce IL4, IL5 and IL13 which stimulate B cells to produce antibodies during the course of extracellular infections (Murphy and Reiner, 2002). Th17 cells secrete IL17, IL21 and IL22 and they are involved in the control of extracellular infections produced by bacteria and fungus, also playing an important role in autoimmune diseases (Bedoya et al., 2013). Finally, Treg cells are key in the maintenance of

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