Fish and Shellfish Immunology 144 (2024) 109280 Available online 10 December 2023 1050-4648/© 2023 The Author. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). The fish spleen Agustín G. Zapata Department of Cell Biology, Faculty of Biology, Complutense University, 28040, Madrid, Spain A R T I C L E I N F O Keywords: Fish Spleen Lymphoid tissues Ellipsoids Melano-macrophage centres Germinal centres A B S T R A C T In the present study, we review the structure and function of fish spleen with special emphasis on its condition in Elasmobranchs, Teleosts and Lungfish. Apart from the amount of splenic lymphoid tissue, the histological or ganization of the organ ensures the existence of areas involved in antigen trapping, the ellipsoids, and exhibit numerous melano-macrophages which appear isolated or forming the so-called melano-macrophage centres. An extensive discussion on the functional significance of these centres conclude that they are mere accumulations of macrophages consequence of tissue homeostasis rather than primitive germinal centres, as proposed by some authors. 1. Introduction Jawed fishes, the first vertebrates to show lymphoid organs and mount immune responses as higher vertebrates, appeared during the Silurian period (nearly 400 million years ago), in correlation with whole gene duplication events; their descendent, the cartilaginous fishes (Chondrichthyes) and the bony fish (Osteichthyes), are currently the predominant fish. Whereas Chondrichthyes represent an independent evolutionary line, bony fish evolved to constitute two large subdivisions: the ray-finned fish (Actinopterygii) that culminate in the current tele osts, and the fleshy finned fish (Sarcopterygii). A small group of Sar copterygii, the Crossopterygians, gave rise directly to amphibians, whereas another group, the Dipnoi (lungfish), exhibit some character istics of amphibians but are not direct descendants (Fig. 1). In any case, data on the lymphoid/immune condition of spleen are only known in some species of teleosts, cartilaginous fish and Dipnoi. 2. General characteristics of fish spleen As befits chordates that have no classical adaptive immune response, the extant Agnatha, including both myxinoids and lampreys (Fig. 1), have no spleen and the lympho-haemopoietic organ present in the gut lamina propria and the larval typhlosole, respectively, represents bone marrow equivalents rather than splenic tissue. A true spleen appears for the first time in the Chondrichthyes, coinciding with the appearance of immune responses involving T lymphocytes, B lymphocytes and antigen-presenting cells [1]. Diverse microenvironments determine the organization and function of the lymphoid organs, assigning to the spleen the main condition of being an immune reactive lymphoid organ in response to blood circulating antigens/pathogens. Herein, I intend to explore this aspect further and to include other fish apart from teleosts. From an immunological viewpoint, all secondary lymphoid organs of jawed vertebrates are organized to provide the necessary architecture for ensuring antigen trapping and processing, facilitating its intimate contacts with the reactive immunocompetent cells. Thus, fish spleen contains collections of lymphocytes, macrophages and dendritic cells capable of mounting immune responses close to the site of antigen up take [2] (Fig. 2). Although the amount of splenic lymphoid tissue is variable among the Gnathostomata, the spleen is considered a major peripheral lymphoid organ of fishes [3] (Fig. 2). These variations could reflect the existence of other peripheral sites of lymphoid tissue, such as the kidney in teleosts. Other authors emphasize that the development of splenic lymphoid tissue is merely a consequence of the interaction between its pattern of vascularization and specific areas of mesenchyme [4] (Figs. 3 and 4). In any case, neither in the elasmobranch spleen nor in teleosts is there a marginal zone that establishes sharp limits between lymphoid follicles and red pulp [1,5]. The development of fish lymphoid organs, including the spleen, has been reported in some teleosts (i.e., rainbow trout, channel catfish, sea bass, zebrafish, meagre), but there is little information in other fish groups, including elasmobranchs. Indeed, the time of appearance and growth of lymphoid tissue are highly variable as well as its degree of development and therefore, the establishment of a clear demarcation between the white and red pulp. Studies are mainly morphological and demonstrate that lymphocytes appear late, and during the first stages of E-mail address: zapata@ucm.es. Contents lists available at ScienceDirect Fish and Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi https://doi.org/10.1016/j.fsi.2023.109280 Received 18 September 2023; Received in revised form 5 December 2023; Accepted 6 December 2023 mailto:zapata@ucm.es www.sciencedirect.com/science/journal/10504648 https://www.elsevier.com/locate/fsi https://doi.org/10.1016/j.fsi.2023.109280 https://doi.org/10.1016/j.fsi.2023.109280 https://doi.org/10.1016/j.fsi.2023.109280 http://crossmark.crossref.org/dialog/?doi=10.1016/j.fsi.2023.109280&domain=pdf http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ Fish and Shellfish Immunology 144 (2024) 109280 2 development the organ is largely erythropoietic with little or no immunological relevance. 2.1. The spleen of holocephalii In both Chimaera and Hydrolagus, the spleen is closely associated with the pancreas [6], and its histological organization resembles that of elasmobranchs [1,6,7], consisting of lymphoid foci and a red pulp which contains mature and developing erythrocytes and thrombocytes. Large ellipsoids are scattered throughout the splenic parenchyma. 2.2. The spleen of elasmobranchs The spleen of selachians is an elongated, lobed organ located close to the duodenum, whereas in rays and skates it is a solid, round structure, with little evidence of lobes. To a large extent, vascularization of the organ determines its histological organization and, by turn, its immu nological efficiency. In the parenchyma of the elasmobranch spleen, arterioles coming from the dorsal artery produce smaller branches without anastomosis (Fig. 5). These terminal branches, also called capillaries, appear sheathed constituting ellipsoids also named houses of Schweigger-Seidel [7]. They are frequent in many vertebrates [8], and in some elasmobranchs contain lipid deposits [1,9] (Fig. 6). They are associated with antigen trapping as demonstrated in the spleen of dog fish Triakis scyllia [7]. The development of elasmobranch spleen has been studied in the dogfish, but no correlation has been conclusively established with the functional maturation of the immune system [10] (Table 1). In 3 cm long embryos, the splenic primordium appears throughout the ventral gut, consisting of a mesoderm-derived stroma and some blood sinuses [10]. Lymphoid colonization occurs at the end of the external gill stage, increasing in the following stages around the splenic arteries to organize a primordial white pulp at the prehatching stage. Finally, the first el lipsoids appear in stage III embryos at the end of terminal capillaries [10]. Whether elasmobranch spleen is (or is not) a B cell producing organ is a matter of discussion. Rag expression in the spleen of adult sharks [11] and Raja eglanteria [12] supports a splenic B cell lympho poiesis but could also indicate an editing process typical of activated B cells. In addition, the spleen of both Raja kenojei and Bathyraja aleutica is the first site where Ig positive cells appear [13,14], but they could have colonized the organ and expanded early on, even if they were produced in another site. Other studies in the nurse shark Ginglymostoma cirratum do not support the elasmobranch spleen as a B cell lymphopoietic locus [15]. These authors correlated the low levels of serum IgM reported in newborn nurse sharks [16,17] with the immaturity of their splenic tis sue. In fact, at hatching, the splenic lymphoid tissue in these sharks consists only of B cell zones containing sIgM+MHCIIlo cells. Neither T zones nor dendritic cells (DCs) or IgNAR+ cells were observed at this stage, reaching the adult condition at 5 months post-hatching [15]. The possible regionalization of the white pulp in the elasmobranch spleen is a matter of discussion. By means of in situ hybridization studies on the white pulp of nurse shark, Rumfelt et al. [15] demonstrated its division into presumptive B and T cell areas, the former in the outer zone, with T cells occupying an inner ring. However, other studies report that splenic white pulp in nurse sharks only contains B lymphocytes but not T cells [18]. Recently Matz and colleagues [5,19] performed an RNA Fig. 1. A phylogenetic tree of fish. Fig. 2. Lymphoid tissue in the spleen of the teleost Rutilus rutilus. Numerous lymphocytes (L), proplasma cells (PrPC) and some granulocytes (Gr) appear between a network of reticular cells (RC) (x 8000). A.G. Zapata Fish and Shellfish Immunology 144 (2024) 109280 3 sequencing study on single nuclei to identify the cell types present in the nurse shark spleen as well as an RNAscope to provide information on their location in the organ following immunization with Rphycoerythrin (PE). PE co-localizes centrally with CXCR5hi centrocyte-like B cells and putative follicular helper T cells surrounded by a peripheral rim of Ki67+AID+CXCR4+ centroblast-like B cells. In addition, T cell clusters surround the periphery of follicles [5,19]. 2.3. The spleen of teleosts On the contrary, numerous studies are devoted to the structure and function of the spleen in teleosts (reviewed in Refs. [20,21], which apparently develops slowly after the appearance of both kidney and thymus [22–24]. In carp, a small spleen identifiable at 14 days post-fertilization acquires the histologically adult condition in 6-8-week-old fish [25], whereas in the young flounders, Platichthys fle sus, it lacks the ellipsoids and MMC, very evident in adult fish [26]. In other species, the appearance and growth of the spleen are similar. The appearance of zebrafish splenic primordium occurs by day 4pf in a small Hox-11+ area of the left anterior gut [27]. The primordium grows but, even by 30 dph, remains as a small organ containing devel oping erythroid cells. When the lymphocytes become evident in the 30 dpf spleen, incipient ellipsoids, key for antigen trapping, are not even fully organized [28]. Recently, Campoverde et al. [29] reported on the ontogeny of splenic tissue in the meagre (Argyrosomus regius). The organ appeared 12 days post-hatching (dph) as a small spherical cluster of mesenchymal cells that became encapsulated and invaded by sinusoidal blood vessels at 29 dph. Although at 47 dph white and red pulp, and ellipsoids appeared to be differentiated, at 66 dph the red pulp occupied most of the splenic area and the lymphoid tissue remained poorly developed. On the other Fig. 3. Abundant lymphoid tissue around the splenic central artery in the elasmobranch Torpedo marmorata (x 80). Fig. 4. Small, isolated lymphoid follicles (delimited by arrows) scattered throughout the splenic parenchyma is the most frequent distribution of the lymphoid tissue in teleost spleen (x 100). Fig. 5. Diagram of the histological organization of the spleen of the dogfish, Scyliorhinus canicula (modified from Pulsford and Zapata 1989 Acta Zool 72, 209). Fig. 6. Cross-section of a large ellipsoid (ELL) in the spleen of Raja clavata (x 500). Table 1 Ontogeny of embryonic dogfish spleen. Erythro- and granulopoiesis in the splenic primordium Stage II (External Gills) 2.5–3.5 cm long Vascularization. First lymphoid cells Stage II (External Gills) 3.5–5 cm long Lymphocytes surround the central arteries Stage III (Hatching) 5–10 cm long Appearance of ellipsoids and macrophages Idem Adult condition: appearance of melano- macrophages Stage IV (Posthaching) 16 cm long A.G. Zapata Fish and Shellfish Immunology 144 (2024) 109280 4 hand, the channel catfish is unable to mount antibacterial immune re sponses until 21 days posthatching [24]. In fact, the first sIg+ B cells do not appear until 7 dph in kidney and then migrate to the spleen. Accordingly, the splenic compartmentalization with distinct T and B cell areas does not occur until 21 dph. In summary, fish immunocompetence seems to be determined by the appearance of immunological capacities rather than by the histological maturation of lymphoid organs. Accordingly, the adult teleost spleen exhibits a poor development of the lymphoid tissue, which appears scattered throughout the reticular splenic parenchyma surrounding small arteries and the so-called mel ano-macrophage centres (MMC) (Fig. 4). Nevertheless, lymphoid tissue increases significantly in the teleost spleen after antigenic stimulation, supporting its relevance for fish immune responses [30]. Some studies have described the existence of different subsets of splenic reticular stromal cells, especially when comparing the red and white pulp [21, 31], but their functional significance remains unknown. Also, in teleost spleen the ellipsoidal blood vessels are less evident than in the spleen of elasmobranchs but respond to the same structural organization: a thin endothelial cell layer in the terminal capillaries surrounded by a sheath of reticular fibres and cells, and macrophages [8,21] (Fig. 7 a, b). Apart from the recent transcriptomic results published on nurse shark [19,32] and teleost spleen [33], there are no systematic studies either on the lymphoid cell subsets that occur in fish spleen, or on their histological distribution in the distinct areas that constitute the organ, i. e., white pulp and red pulp, ellipsoids, MMCs. Nevertheless, both T and B lymphocytes together with DCs and macrophages occur in the spleen in all studied fish. In addition, at least teleost lymphoid cells express most of the molecules used in mammals to define T cell subsets, although it is difficult to establish a direct correlation between phenotypes and their immunological capacities (see reviews by Ref. [34]). In zebrafish, the transcriptional profile of 36732 cells derived from spleen of non-infected and infected fish with spring viremia of carp virus (SVCV) identified 11 major categories of blood cell types including neutrophils, NK cells, macrophages/myeloid cells, T lymphocytes, B lymphocytes, HSC/HSPC, mast cells, remnants of endothelial cells, erythroid cells and their corresponding committed progenitor cells, and a new type of serpin-secreting cells [33]. Also, 54 potential cell subsets were derived from these 11 categories, including T cells, with signature trac, cd3ζ, lck, Zap70, CD4.1, CD8α, Runx3, bcl11bβ, IL17, etc., corre sponding to TH1, TH2, TH17, γδ and Treg cells; canonical IgM+IgD+ B cells, B1-like cells and follicular B-like cells. In addition, a special CD4+ macrophage subpopulation was identified, that had previously been reported in rainbow trout [35]. Both CD4 and CD8 genes have been cloned and characterized in several teleosts including the spotted sea brass [36], Oncorhynchus mykiss [35,37], Salmo salar [38], flounder [39], etc., but in some elas mobranchs (i.e., elephant and nurse shark), genome sequencing and transcriptomic analysis were unable to identify CD4 cells within the TcR-expressing lymphocyte population [40,41]. In addition, in the cod and other ganoids lack important immune genes including CD4 and MHC class II genes that are functionally supplied with an expanded MHC class I locus, a special set de TLR and presumably, NKT cells [42–45]. On the other hand, in most of the teleosts studied, two CD4 molecules have been identified and their genes cloned [35]: CD4-1 and CD4-2. There is a predominant T cell population CD4-DP that co-expresses CD4-1 and CD4-2, and a minority T cell subset that expresses CD4-2 and CD4-2SP. However, both cell subsets produce an equivalent amount of TH1, TH17, and Treg cytokines after bacterial infection, although CD4-2SP cells are less proliferative and exhibit a more restricted TcRβ repertoire [35]. Romano and colleagues [46] ultra structurally characterized two types of T cells, type a and type b, in the sea bass, Dicentrarchus labrax by using an anti-T mAb, DLT1; the first one principally present thymus whereas type b T lymphocytes predominated in the periphery. Foxp3+ Treg cells are particularly abundant in teleost spleen [47, 48]. In the spleen of medaka and trout, but not in that of zebrafish, it is possible to distinguish T cell- and B cell-areas [49]. However, DCs occur in the spleen of the three species [49]. These cells have many features typical of their mammalian equivalents, including ramified morphology, a capacity for engulfing small particles, and migratory capacity. In culture, they express TLR, MHCII molecules, CD83, CD209, CXCR4, CCR7 and Il12p40 [50]. 2.4. The spleen in other fish classes Although available information is scarce for other classes of fish, Fig. 7. Ultrastructure of an ellipsoidal area (delimited by arrows) in the spleen of rainbow trout. Note the layers of reticular cell (RC) surrounding the terminal capillaries (BV), and the occurrence of outer macrophages (MO) containing engulfed materials. Spleen of a rainbow trout intramuscularly injected with O antigen of Yersinia ruckeri (a) (x 4000), and a small ellipsoid in the spleen of Carassius auratus (b) x 9700). CO: collagen. A.G. Zapata Fish and Shellfish Immunology 144 (2024) 109280 5 including Ganoids (Chondrosteans and Holosteans), splenic histology is similar to that of elasmobranchs, showing a clearly distinguishable red and white pulp (Fig. 3), particularly after immunization, and lympho cytes and plasma cells are the main cellular components, together with macrophages and sometimes granulocytes (Fig. 2) [1]. The histological organization of the spleen of Polypteriforms (Cala moichthys sp) [8] and Actinopterigii (Polypterus senegalus) [51] is espe cially primitive. In the latter, the spleen is an elongated organ associated with the gut walls, in which it is difficult to distinguish a white and a red pulp. Lymphocytes, clusters of mature and developing plasma cells and granulopoietic foci, occur in the splenic parenchyma. The information on coelacanth (Latimeria) spleen is very poor and related to anatomical features rather than to histological organization [52]. The spleen of Dipnoi shows a complex organization with species- specific differences. In the Australian lungfish, Neoceratodus forsteri, the splenic primordium appears as a mesenchyme condensation that receives blood from the vitelline arteries and gradually develops blood sinuses with the organ growing along the spiral valve [53–55]. In the South American lungfish, Lepidosiren paradoxa, a compact spleen is developed in the wall of the stomach and the anterior part of the in testine that contains clear red and white regions [56,57] whereas in Protopterus ethiopicus, Jordan and Speidel [58] reported three regions: a central one containing lymphoid tissue, surrounded by another actively erythropoietic area composed of cords and sinusoids and, a peripheral thin capsule. In another African lungfish, P. annectens, there is a rod-shaped spleen that courses from the cranial part along the right side of the gut [59,60]. The organ is composed of a cortical reticulum that surrounds the splenic parenchyma and contains two types of granulocytes, developing, mature plasma cells and MMCs, structurally similar to those of teleosts. A lobulated parenchyma exhibits a subcapsular sinus and areas of red pulp and white pulp; the former is involved in erythropoiesis, erythro cyte destruction, and plasma cell differentiation, whereas the white pulp mainly contains lymphoid cell aggregates. Remarkably, after aestiva tion, the number of MMCs increases and the splenic parenchyma is infiltrated by lymphocytes, granulocytes, and monocytes, but the white pulp reduces [61]. In addition to this rostral spleen, N. forsteri has a caudal spleen along the inner side of the spiral valve [54,55] which does not exist in P. annectens, where there is a reticular tissue consisting of isolated lymphoid nodes [62,63], of unclear significance. The nodes exhibit outer and inner regions, the first one containing, such as the rostral spleen, granulocytes, developing and mature plasma cells and MMCs [63]. The inner zone is a meshwork of trabeculae and vascular sinuses containing lymphoid cells, clusters of plasma cells and a small number of macrophages frequently containing haemosiderin granules. Like the spleen, the caudal lymphoid nodes also present a subcap sular sinus [63] that remarkably resembles the condition of mammalian haemal and haemolymph nodes [64–67]. Moreover, efferent vessels flow into the subcapsular sinus and run throughout the parenchyma as in the haemal nodes [66,68]. Therefore, these nodes exhibit structural features of both lungfish spleen and mammalian haemolymph nodes, although there are no true lymph nodes in non-mammalian vertebrates [20]. 3. Ellipsoids and melano-macrophage centres are two distinguishing features of teleost spleen As indicated, splenic ellipsoids of fish have the special ability to trap diverse substances including degenerated erythrocytes [8], pathogens Fig. 8. Ellipsoids in the spleen of Carassius auratus after intramuscular carbon injection. Reticular cells (RC) surrounding the blood vessels show carbon particles (arrowheads). Light microscopy (a) (x 890) and electron microscopy (b) (x 9900). ELL: ellipsoid. A.G. Zapata Fish and Shellfish Immunology 144 (2024) 109280 6 such as Aeromomas salmonicida [69], or intravascularly injected mate rials [25,70–77]. In general, soluble antigens are trapped extracellularly on the reticular fibres and particulate antigens are engulfed by macrophages present in the ellipsoidal walls (Fig. 8 a, b). Cells containing the engulfed material migrate to the MMCs of the kidney and spleen [78]. Isolated melano-macrophages occur in all studied fish, including Agnatha, but also in amphibians and reptiles [20,78–80]. Their presence has been reported in more than 130 species [81]. In teleosts, with the exception of salmonids, these isolated melano-macrophages (MM) form large clusters called MMCs that mainly appear in kidney and spleen, surrounded completely or partially by reticular capsules and frequently closely associated with ellipsoids and lymphoid cells [79,82] (Fig. 9). As a consequence of their degrading activity, MMCs contain a heteroge neous collection of degraded pigments, the most frequent being hemo siderin, lipofucsin and melanin [78,83] (Fig. 10). The number, size, and pigment content of the MMCs depend on the fish species, organ, age, sex, nutritional status, and fish health [84,85]. Apart from a huge capacity to remove injected foreign substances from the blood circulation [81], there is apparently an opposite corre lation between the numbers of free MM and big MMCs [78,86]. How ever, it is unclear if isolated macrophages move to the pre-existing MMCs [87] or form new clusters [73]. Presumably, the number of engulfed materials and the number of MMCs govern this process [78]. We analysed this phenomenon by studying the behaviour of Car assius auratus spleen immunized with sheep erythrocytes (SRBC), or formalin-linked Yersinia ruckeri. Although both antigens were taken up by ellipsoidal macrophages or endothelial cells of renal sinusoids and transported to MMCs, bacteria rather than erythrocytes were totally digested and consequently, no differences in number, size or area occupied by MMCs occurred [78]. On the other hand, these results suggest that MMCs are scavengers that reflect the activity of fish tissues. In agreement, MMCs have been related with the centralization of endogenous and exogenous materials for further elimination, detoxifi cation and/or recycling [88], as well as with a poor enzymatic profile of fish macrophages [89]. They are also accumulate pigments throughout aging [90], diseases, mainly due to parasites, viruses and bacteria [91], or active catabolism typical of the lympho-hematopoietic organs [92]. An analysis of the nature of the pigments accumulated would provide information on their possible origin. Lipofucsin is a substance related to ceroids presumably derived from the oxidation of polyunsaturated fatty acids [83] and is very frequent in fish [93]. On the contrary, levels of vitamin E in fish are low [94], favouring the formation of lipofuscin and melanin. Hemosiderin is a breakdown product of hemoglobin. Melanin could be phagocytosed by macrophages to eliminate potentially toxic degenerated melanocytes or neutralize the activity of free radicals [95] rather than produce autonomously pheomelanin pigments [96–98], as some seminal studies had proposed [99]. In fact, to our knowledge, the presence of pheomelanin has only been detected in the spleen in the teleost Heteropneutes fossilis [100,101]. In this respect, Edelstein [95] proposed that melanin might be used together with the peroxide-peroxidase system for iodinating and killing bacteria. 4. MMCs as presumptive primitive germinal centres Several authors have hypothesized that teleost MMCs would be primitive germinal centres (GC), precursors of those that eventually appear in endothermic vertebrates [102,103]. In fact, one of the most remarkable features of the spleen of lower vertebrates, including fish, is the lack of GC. This microenvironment is necessary in mammals for selecting B cell clones which have undergone somatic hypermutation of their BcR genes, resulting in the production of specific antibodies of high affinity, with the concourse of follicular dendritic cells (FDCs), T follicular helper cells (Tfh cells) and molecules of the TNF superfamily. Therefore, the presence or not of GCs in lymphoid tissues is associ ated with antibody affinity and immunological memory. Immunological memory is the ability of a system “to remember” antigens previously confronted and respond specifically to them in a qualitatively and quantitatively stronger way than after the first encounter [104]. Chon drichthyes are capable of long-term memory [105] and exhibit high antibody affinity, although significantly lower than in endotherms, for IgM and IgNAR [106,107]. Immunological memory to T-dependent antigens is reported in numerous teleosts, including channel catfish, Atlantic salmon, zebrafish, Fig. 9. Encapsulated MMC (arrowhead) of the spleen of Carassius auratus 21 days after SRBC administration (x 890). ELL: ellipsoid, S: sinusoid. Fig. 10. Highly degraded material inside a large, encapsulated (arrows) MMC of the spleen of Carassius auratus (x 4500). A.G. Zapata Fish and Shellfish Immunology 144 (2024) 109280 7 and rainbow trout, although, presumably, also present in many others [108]. This memory is accompanied by increased antibody affinity, although the increase is considerably lower (about 100 times) than that recorded in mammals [108–110]. At the cellular level, there is contro versy about the antigen-responding cells throughout the immune response. All studies agree that the pronephros is the site where lymphoid progenitor cells produce naïve B cells that migrate to trunk kidney and spleen [111,112]. Some studies found that a small fraction of activated B lymphocytes remain in the spleen [109,113], whereas other studies reported that they return to the pronephros as long-term plasma cells, supporting the similarities between teleost pronephros and mammalian bone marrow [114,115]. In any case, affinity maturation, somatic mutation and B cell heterogeneity all occur in fishes. Why have teleost MMCs been related to GCs [108] when all authors recognize their main role in phagocytosis and lymphoid tissue catabo lism as mentioned above? This is supported by three main findings: 1) the ability of MMCs for long-term antigen retention [80,81]; 2) the ex istence in fish of AID (activation-induced cytidine deaminase) genes which govern somatic hypermutation [116–119], whose expression is almost restricted to MMCs [102], and 3) the occurrence of Ig+ cells that after infection tend to accumulate around MMCs [21,120,121]. However, fish MMCs are unable to organize histologically identifi able GCs and importantly, many fish species, including Chondrichthyes [106] and salmonids within the bony fish [20] do not have MMCs but instead isolated MMs. Furthermore, recently Perdiguero et al. [122] demonstrated that IgD+ and IgM+ plasmablasts from teleost gut and gills show a certain degree of antigen-driven somatic hypermutation in the extrafollicular compartment of the lamina propria, in a total absence of MMCs. The first consideration to explain the lack of GC in fish would be to analyse whether these vertebrates contain the molecules and cell types necessary to functionally organize the GCs in higher vertebrates. As indicated, molecules of the TNF superfamily, principally lymphotoxins, are involved in the histological organization of murine GCs and remarkably, lymphotoxin-deficient mice are able to drive antibody af finity maturation in the absence of GCs, such as in fish. However, lym photoxin genes have been identified in teleosts [123], although, to my knowledge, their relationship, if any, with MMCs has not been analysed. Although there is no conclusive evidence to support the occurrence of FDCs in fish spleen, some factors necessary for their functioning such as IL6 and the transcription factor Bcl6 have been sequenced in fish genomes [34,124,125]. An antibody, CAN-42, known to react with FDCs of higher vertebrates, recognizes MM and MMCs in the spleen, but not in the kidney, in three species of teleosts, Cyprinus carpio, Odontesthis bonariensis and Solea genegalensis [126]. In mammals, this antibody, apart from FDCs, stains some plasma cells and mast cells [127], but the recognized determinant is unknown, and the authors cannot offer a conclusive explanation for the lack of reactivity of renal MMCs. Hence, when adult nurse sharks were immunized with biotinylated BSA, the immunogen accumulated on the surface of large dendritic cells of splenic red pulp, but they seemed to act as antigen-presenting cells to B lymphocytes rather than bona fide FDCs [49]. A similar situation occurs with the presence of T lymphocytes in MMCs, which could be functionally equivalent to the Tfh cells of GCs. Recently, a transcriptomic analysis of the spleen of Scortum barcoo infected by Streptococcus agalactine demonstrated the induction of several T cell subsets including NKT cells, TH17 cells and Tfh cells, as well as the Il21 and Il22 production [128]. In the nurse shark spleen, Matz and colleagues [19] have also identified a T cell subpopulation that expresses genes related to mammalian Tfh cells. It is also important to note the presence of highly phagocytic CD4-1+ macrophages in the spleen [35], in which case CD4+ cells identified in and around MMCs could really be macrophages rather than T cells. Certain methodological concerns about some experiments that sup port a phylogenetic relationship between teleost MMC and higher vertebrate GCs should be mentioned. Saunders et al. [102] used laser capture microdissection for isolating AID-expressing MMCs that con tained IgH and TcRβ transcripts and CD4-expressing cells but, unfortu nately, the degree of purification of these isolated aggregates was not specified. Presumably, these were totally or partially disaggregated and constituted isolated MM. Really, GCs are just the histological reflection of a complex immune process in which a high frequency of mutated B cells after undergoing somatic hypermutation must be selected with the concourse of Tfh cells. In lower vertebrates, including fish, a lower B cell division capacity would result in fewer high affinity antibody producing B cells and the absence of GC. This would occur because low B cell expansion would not favour the activation of FDC stromal precursor cells and vice versa, thus preventing the organization of a histological structure similar to a GC. In that respect, there are important differences between MMCs and GCs. In the GCs, the amount of antigen retained on the FDCs and/or the number of retaining cells appear to be far fewer for MMCs. In addition, macrophages phagocyte antigens and FDCs maintain on their surface immune complexes [103]. Another possibility would be that the kinetics of activated B cell proliferation is so slow that few centrocytes compete for the antigen [103]. Accordingly, the poor immune responses of ec totherms would be due to the lack of an efficient mechanism for selecting mutated B cell clones. In summary, I agree that ectothermic vertebrates would have the main cellular and molecular components of mammalian GCs, but their capacity for B cell clone selection would be very limited, resulting in an absence of the histological image recognized in mammalian GCs. Other authors [81] have emphasized a role for MMCs as a locus for activating adaptive immunity in fish but did not consider them as primitive GCs. In support of this hypothesis, we demonstrated that repeated immuniza tion with a rhabdovirus results in the organization of small clusters of Rag+ cells, morphologically distinct from MMCbut that could represent areas for selecting a few B cell clones [12]. In agreement, recently, Matz and Dooley [5] emphasized that “the current evidence to support MMCs as functional equivalents of mammalian GCs is far from conclusive”. Other authors consider that MMCs could be a physiological structure that is barely associated with fish immune responses [81]. However, very recent findings [129] have shown that upon infection or immuni zation, aggregates of highly proliferating splenic IgM+ B- and CD4+ T cells are induced nearby MMCs of rainbow trout. Most of these MMC-associated lymphoid aggregates (M-LAs) contained numerous Ag-specific B cells. The authors demonstrated within these structures key processes known to occur in GCs, including antigen-specific B cell clonal expansion and somatic hypermutation. While M-LAs exhibit functional features analogous to those of GCs, the structural organiza tion of M-LAs is loose and variable, more similar to that immature ter tiary lymphoid organs which contain loosely organized B and T cell zones, than to GCs. The authors conclude that rather than occurring in random areas of the spleen, teleost adaptive immune responses are induced in specific tissue microenvironments (M-LAs) where APCs (i.e., MMCs), B and T cells organize to elicit the immune response. These data provide a plausible mechanism as to how adaptive immune responses occur in species that lack bona-fide GCs. 5. Conclusions I have reviewed the histophysiological condition of the spleen in diverse fish species for which information is available. All data support a relevant immune function for the organ in that it provides a special microenvironment for antigen trapping and processing, activation of reactive lymphocytes and the elaboration of specific immune responses. However, the splenic components and their distribution in the organ are not identical to those of the mammalian spleen. Evidence for the occurrence of T- and B-cell areas in the fish spleen of numerous fish species is inconclusive and requires further research. Moreover, there is no marginal zone involved in antigen trapping and processing either in elasmobranchs or in teleost spleen, although terminal capillaries A.G. Zapata Fish and Shellfish Immunology 144 (2024) 109280 8 organize special structures, so-called ellipsoids, consisting of sheaths of reticular cells and fibres, and macrophages allow for efficient antigen trapping. Two remarkable features define the fish spleen: the existence of isolated MMs and grouped MMCs, and the lack of GCs closely associated with the deficient immunological memory and antibody affinity matu ration reported in fish. MMCs are undoubtedly scavenger sites that accumulate macrophages full of cellular debris of different origins and are, therefore, related to the tissue catabolism. However, some authors have speculated that MMCs are primitive GC. I have reviewed the available information and conclude, together with other authors, that MMCs and GCs are distinct structures that are neither morphologically nor functionally related. MMCs are unable to “organize” a histological “image” similar to that of a GC, even after repeated immunization. The absence of GCs in lower vertebrates seems to be more related to the incapacity to select a sufficient number of mutated B cell clones. This could explain the presence of isolated Rag+ cell sites, unrelated to MMCs that occur throughout the teleost splenic parenchyma after hyperim munization in correlation with the low antibody affinity maturation, typical of teleosts. Several approaches would provide interesting information to conclusively clarify the relationships (if any) between MMC and prim itive GC. a) A better characterization of the MMC components including their neighbouring immunocompetent cells. b) An in vivo and in vitro analysis on the aggregation/disaggregation of their components after primary and secondary antigenic stimulation, by using comparatively T-dependent and T-independent antigens. c) An analysis of the splenic changes during the immune responses in fish species which lack GCs. 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