Single-cell atlas of rainbow trout peripheral blood leukocytes and profiling of their early response to infectious pancreatic necrosis virus.

The recent development of single cell sequencing technologies has revolutionized the state-of-art of cell biology, allowing the simultaneous measurement of thousands of genes in single cells. This technology has been applied to study the transcriptome of single cells in homeostasis and also in response to pathogenic exposure, greatly increasing our knowledge of the immune response to infectious agents. Yet the number of these studies performed in aquacultured fish species is still very limited. Thus, in the current study, we have used the 10x Genomics single cell RNA sequencing technology to study the response of rainbow trout (Oncorhynchus mykiss) peripheral blood leukocytes (PBLs) to infectious pancreatic necrosis virus (IPNV), an important trout pathogen. The study allowed us to obtain a transcriptomic profile of 12 transcriptionally distinct leukocyte cell subpopulations that included four different subsets of B cells, T cells, monocytes, two populations of dendritic-like cells (DCs), hematopoietic progenitor cells, non-specific cytotoxic cells (NCC), neutrophils and thrombocytes. The transcriptional pattern of these leukocyte subpopulations was compared in PBL cultures that had been exposed in vitro to IPNV for 24 h and mock-infected cultures. Our results revealed that monocytes and neutrophils showed the highest number of upregulated protein-coding genes in response to IPNV. Interestingly, IgM+IgD+ and IgT+ B cells also upregulated an important number of genes to the virus, but a much fainter response was observed in ccl4 + or plasma-like cells (irf4 + cells). A substantial number of protein-coding genes and genes coding for ribosomal proteins were also transcriptionally upregulated in response to IPNV in T cells and thrombocytes. Interestingly, although genes coding for ribosomal proteins were regulated in all affected PBL subpopulations, the number of such genes transcriptionally regulated was higher in IgM+IgD+ and IgT+ B cells. A further analysis dissected which of the regulated genes were common and which were specific to the different cell clusters, identifying eight genes that were transcriptionally upregulated in all the affected groups. The data provided constitutes a comprehensive transcriptional perspective of how the different leukocyte populations present in blood respond to an early viral encounter in fish.

Immunomodulatory properties of Bacillus subtilis extracellular vesicles on rainbow trout intestinal cells and splenic leukocytes.

Extracellular vesicles (EVs) are cell-derived membrane-surrounded vesicles that carry bioactive molecules. Among EVs, outer membrane vesicles (OMVs), specifically produced by Gram-negative bacteria, have been extensively characterized and their potential as vaccines, adjuvants or immunotherapeutic agents, broadly explored in mammals. Nonetheless, Gram-positive bacteria can also produce bilayered spherical structures from 20 to 400 nm involved in pathogenesis, antibiotic resistance, nutrient uptake and nucleic acid transfer. However, information regarding their immunomodulatory potential is very scarce, both in mammals and fish. In the current study, we have produced EVs from the Gram-positive probiotic Bacillus subtilis and evaluated their immunomodulatory capacities using a rainbow trout intestinal epithelial cell line (RTgutGC) and splenic leukocytes. B. subtilis EVs significantly up-regulated the transcription of several pro-inflammatory and antimicrobial genes in both RTgutGC cells and splenocytes, while also up-regulating many genes associated with B cell differentiation in the later. In concordance, B. subtilis EVs increased the number of IgM-secreting cells in splenocyte cultures, while at the same time increased the MHC II surface levels and antigen-processing capacities of splenic IgM+ B cells. Interestingly, some of these experiments were repeated comparing the effects of B. subtilis EVs to EVs obtained from another Bacillus species, Bacillus megaterium, identifying important differences. The data presented provides evidence of the immunomodulatory capacities of Gram-positive EVs, pointing to the potential of B. subtilis EVs as adjuvants or immunostimulants for aquaculture.

Comprehensive immune profiling reveals that Orbivirus infection activates immune checkpoints during acute T cell immunosuppression.

Bluetongue virus (BTV) is an arbovirus transmitted by the bite of infected Culicoides midges that affects domestic and wild ruminants producing great economic losses. The infection induces an IFN response, followed by an adaptive immune response that is essential in disease clearance. BTV can nonetheless impair IFN and humoral responses. The main goal of this study was to gain a more detailed understanding of BTV pathogenesis and its effects on immune cell populations. To this end, we combined flow cytometry and transcriptomic analyses of several immune cells at different times post-infection (pi). Four sheep were infected with BTV serotype 8 and blood samples collected at days 0, 3, 7 and 15pi to perform transcriptomic analysis of B-cell marker+, CD4+, CD8+, and CD14+ sorted peripheral mononuclear cells. The maximum number of differentially expressed genes occurred at day 7pi, which coincided with the peak of infection. KEGG pathway enrichment analysis indicated that genes belonging to virus sensing and immune response initiation pathways were enriched at day 3 and 7 pi in all 4 cell population analyzed. Transcriptomic analysis also showed that at day 7pi T cell exhaustion pathway was enriched in CD4+ cells, while CD8+ cells downregulated immune response initiation pathways. T cell functional studies demonstrated that BTV produced an acute inhibition of CD4+ and CD8+ T cell activation at the peak of replication. This coincided with PD-L1 upregulation on the surface of CD4+ and CD8+ T cells as well as monocytes. Taken together, these data indicate that BTV could exploit the PD1/PD-L1 immune checkpoint to impair T cell responses. These findings identify several mechanisms in the interaction between host and BTV, which could help develop better tools to combat the disease.

Teleost IgD+IgM- B cells in gills and skin have a plasmablast profile, but functionally and phenotypically differ from IgM+IgD- B cells in these sites.

Although most B cells in teleost systemic compartments co-express IgM and IgD on the surface, cells exclusively expressing either of the two Igs are common in fish mucosal tissues, providing us with a unique opportunity to further characterize IgD+IgM- B cells, an intriguing B cell subset. Hence, we compared the phenotype of IgD+IgM- cells to that of IgM+IgD- B cells in rainbow trout gills and skin, also establishing the response of these subsets to immune stimulation. The transcriptional profile and secreting capacity of IgD+IgM- B cells corresponded to that of cells that have started a differentiation program toward plasmablasts, similarly to IgM+IgD- B cells. Yet, IgM+IgD- B cells retained high levels of surface MHC II and antigen-processing abilities, while these were much lower in IgD+IgM- cells, suggesting important differences in their antigen-presenting capacities. Our findings contribute to a deeper understanding of the enigmatic role of IgD in mucosal surfaces.

Endoplasmic reticulum expansion throughout the differentiation of teleost B cells to plasmablasts.

The differentiation of B cells into antibody-secreting cells is fundamental for the generation of humoral immunity. In mammals, this process involves a series of metabolic and intracellular changes, not studied to date in teleost fish, where a clear distinction between naive B cells and plasmablasts/plasma cells (PCs) is still missing. Thus, in the current study, we have established that upon activation, teleost B cells undergo an expansion of the endoplasmic reticulum (ER) but experience no significant changes in mitochondria content. In parallel, the transcription of genes implicated in B cell differentiation increases, while that of mitochondrial genes decreases. In this context, ER monitoring has allowed us to distinguish between small cells with low amounts of ER (FSCloERlo B cells), that correspond to undifferentiated cells, and large cells with expanded ER (FSChiERhi B cells), characterized as plasmablasts. The results shed new light on the B cell differentiation process in teleosts and provide us with novel tools to study B cell function in these species.

Functional and Phenotypic Characterization of B Cells in the Teleost Adipose Tissue.

The immune response of the adipose tissue (AT) has been neglected in most animal models until investigations in human and mice linked obesity to chronic inflammation, highlighting the immune nature of this tissue. Despite this, in teleost fish, only a few studies have addressed the immune role of the AT. These studies have mostly focused on reporting transcriptional changes in the AT in response to diverse intraperitoneally delivered stimuli. Although the presence of B cells within the AT was also previously revealed, these cells have never been phenotypically or functionally characterized and this is what we have addressed in the current study. Initially, the B cell populations present in the rainbow trout (Oncorhynchus mykiss) AT were characterized in comparison to B cells from other sources. As occurs in other rainbow trout tissues, IgM+IgD+, IgM+IgD- and IgD+IgM- B cell subsets were identified in the AT. Interestingly, AT IgM+IgD- B cells showed a transcriptional profile that agrees with that of cells that have committed to plasmablasts/plasma cells, being this profile much more pronounced towards a differentiation state than that of blood IgM+IgD- B cells. Accordingly, the IgM-secreting capacity of AT B cells is significantly higher than that of blood B cells. Additionally, AT IgM+IgD+ B cells also showed specific phenotypic traits when compared to their counterparts in other tissues. Finally, we established how these B cell subsets responded when rainbow trout were intraperitoneally injected with a model antigen. Our results demonstrate that the AT hosts plasmablasts/plasma cells that secrete specific IgMs, as happens in the peritoneal cavity and systemic immune tissues. Although the presence of these antigen-specific IgM-secreting cells was more abundant in the peritoneal cavity, these specific differentiated B cells were detected in the AT for long time periods at levels similar to those of spleen and head kidney. Our results provide new evidence regarding the immune role of the teleost AT, indicating that it functions as a secondary lymphoid organ that promotes immunity to peritoneal antigens.

CD38 Defines a Subset of B Cells in Rainbow Trout Kidney With High IgM Secreting Capacities.

CD38 is a multifunctional molecule that functions both as a transmembrane signaling receptor and as an ectoenzyme with important roles in cell adhesion, calcium regulation and signal transduction. Within the B cell linage, CD38 is expressed in diverse murine B cell subsets, with highest levels in innate B cell subpopulations such as marginal zone (MZ) B cells or B1 cells. In humans, however, CD38 is transiently expressed on early lymphocyte precursors, is lost on mature B cells and is consistently expressed on terminally differentiated plasma cells. In the present work, we have identified two homologues of mammalian CD38 in rainbow trout (Oncorhynchus mykiss), designating them as CD38A and CD38B. Although constitutively transcribed throughout different tissues in homeostasis, both CD38A and CD38B mRNA levels were significantly up-regulated in head kidney (HK) in response to a viral infection. In this organ, after the generation of a specific monoclonal antibody (mAb) against CD38A, the presence of CD38A+ populations among IgM+ B cells and IgM- leukocytes was investigated by flow cytometry. Interestingly, the percentage of IgM+CD38A+ B cells increased in response to an in vitro stimulation with inactivated Aeromonas salmonicida. Finally, we demonstrated that HK IgM+CD38A+ B cells had an increased IgM secreting capacity than that of cells lacking CD38A on the cell surface, also showing increased transcription levels of genes associated with B cell differentiation. This study strongly suggests a role for CD38 on the B cell differentiation process in teleosts, and provides us with novel tools to discern between B cell subsets in these species.

Corrigendum: Type I Interferon Regulates the Survival and Functionality of B Cells in Rainbow Trout.

[This corrects the article DOI: 10.3389/fimmu.2020.01494.].

Pro-Inflammatory and B Cell Regulating Capacities of TWEAK in Rainbow Trout (Oncorhynchus mykiss).

Tumor necrosis factor (TNF)-like weak inducer of apoptosis or TWEAK is a member of the TNF superfamily involved in the regulation of many biological processes. In mammals, TWEAK has been shown to play a role in some autoimmune or inflammatory conditions, but its immune role is not yet clearly defined. In teleost fish, although a few studies have identified homologues to mammalian TWEAK, their biological effects have never been investigated. In the current study, we have studied the transcriptional regulation of two TWEAK homologues (TWEAK 1 and 2) identified in rainbow trout (Oncorhynchus mykiss) throughout different tissues, in response to parasitic or viral infections, or in head kidney (HK) leukocytes stimulated with different stimuli. Although the transcription of both homologues was modulated when HK leukocytes were exposed to several immune stimuli, only TWEAK 1 was significantly modulated upon pathogenic exposure. Thus, we performed a characterization of the functions exerted by this cytokine in HK leukocytes. Recombinant TWEAK 1 strongly up-regulated the transcription of pro-inflammatory genes and antimicrobial peptides in HK leukocytes, with differential transcriptional effects in IgM+ B cells, IgM- lymphocytes and myeloid cells. TWEAK 1 also increased the survival and promoted the differentiation of B cells in HK leukocyte cultures. Our results demonstrate that in teleost fish, TWEAK 1 is involved in the response to different types of pathogens, through the modulation of antimicrobial and pro-inflammatory genes in different leukocytes subsets. Furthermore, a role for TWEAK as a B cell differentiation factor has also been established in rainbow trout.

Diversity of Rainbow Trout Blood B Cells Revealed by Single Cell RNA Sequencing.

Single-cell sequencing technologies capable of providing us with immune information from dozens to thousands of individual cells simultaneously have revolutionized the field of immunology these past years. However, to date, most of these novel technologies have not been broadly applied to non-model organisms such as teleost fish. In this study, we used the 10× Genomics single cell RNA sequencing technology and used it to analyze for the first time in teleost fish the transcriptional pattern of single B cells from peripheral blood. The analysis of the data obtained in rainbow trout revealed ten distinct cell clusters that seem to be associated with different subsets and/or maturation/differentiation stages of circulating B cells. The potential characteristics and functions of these different B cell subpopulations are discussed on the basis of their transcriptomic profile. The results obtained provide us with valuable information to understand the biology of teleost B cells and offer us a repertoire of potential markers that could be used in the future to differentiate trout B cell subsets.

Individual B cells transcribe multiple rearranged immunoglobulin light chains in teleost fish.

B cells express a unique antibody protein which comprises two pairs of immunoglobulin (Ig) heavy (H) and light (L) chains. In addition to an invariable constant (C) region, IgH and IgL chains encompass a variable (V) region mediating antigen binding. This unique region stems from Ig V(D)J gene recombination, which generates diversity by assembling these gene segments into VHDJH and VLJL genes. To ensure that one B cell only expresses one antibody, VHDJH rearrangement occurs only in one IgH locus (allelic exclusion), whereas VLJL rearrangement only in either the κ or λ locus (isotype exclusion). However, teleosts express multiple IgLs encoded by distinct CL genes. Using single-cell transcriptomics, we have demonstrated the transcription of distinct rearranged VLJLCL genes in single rainbow trout B cells. Our results highlight the laxity of isotype exclusion in teleosts and strongly suggest that fish B cells can produce antibodies of different specificities.

Insights Into the Evolution of the prdm1/Blimp1 Gene Family in Teleost Fish.

In mammals, Blimp1 (B lymphocyte-induced maturation protein 1) encoded by the prdm1 gene and its homolog Hobit (homolog of Blimp1 in T cells) encoded by znf683, represent key transcriptional factors that control the development and differentiation of both B and T cells. Despite their essential role in the regulation of acquired immunity, this gene family has been largely unexplored in teleosts to date. Until now, one prdm1 gene has been identified in most teleost species, whereas a znf683 homolog has not yet been reported in any of these species. Focusing our analysis on rainbow trout (Oncorhynchus mykiss), an in silico identification and characterization of prdm1-like genes has been undertaken, confirming that prdm1 and znf683 evolved from a common ancestor gene, acquiring three gene copies after the teleost-specific whole genome duplication event (WGD) and six genes after the salmonid-specific WGD. Additional transcriptional studies to study how each of these genes are regulated in homeostasis, in response to a viral infection or in B cells in different differentiation stages, provide novel insights as to how this gene family evolved and how their encoded products might be implicated in the lymphocyte differentiation process in teleosts.

Aeromonas salmonicida activates rainbow trout IgM+ B cells signalling through Toll like receptors.

As B cells are singularly equipped with a B cell receptor (BCR) and a range of innate receptors, they are able to integrate both antigen-specific and innate signals, with the latter being essential to reach an adequate level of activation. Whether teleost B cells sense pathogens through innate mechanisms has not yet been explored, despite the fact that fish B cells display a wider array of innate receptors than many mammalian B cell subsets. Hence, in the current study, we have investigated the effects of inactivated Aeromonas salmonicida, a Gram negative rainbow trout pathogen, on trout splenic IgM+ B cells in vitro in the presence or absence of different inhibitors of Toll-like receptor (TLR) signalling, to establish to what degree innate signals are contributing to the activation of B cells in teleosts. Our results demonstrate that most of the effects that A. salmonicida exerts on trout IgM+ B cells are significantly blocked in the presence of inhibitors of MyD88 and TRIF, important nodes in TLR signal pathways. Thus, the data presented demonstrates that, also in teleost, TLR signalling is essential for the activation of IgM+ B cells. These results will be useful for the future optimization of novel vaccines and adjuvants.

Type I Interferon Regulates the Survival and Functionality of B Cells in Rainbow Trout.

Interferons (IFNs) orchestrate antiviral responses in jawed vertebrates and can be classified into three types based on different aspects of their genomic organization, structure and receptors through which they signal and function. Generally, type I and type III IFNs include cytokines that directly induce an antiviral response, whereas type II IFNs are well-known for their immunomodulatory role during viral infections. In mammals, type I IFNs have been shown to also regulate many aspects of B cell development and differentiation. Yet, these functions have been only faintly investigated for teleost IFNs. Thus, in the current study, we have examined the effects of a model type I rainbow trout IFN molecule (IFNa) on blood naïve (IgM+IgD+) B cells, comparing them to those exerted by type II IFN (IFNγ). Our results demonstrate that IFNa increases the survival of naïve rainbow trout B cells, in the absence of lymphoproliferative effects, by rescuing them from spontaneous apoptosis. Additionally, IFNa increased the phagocytic capacity of blood IgM+IgD+ B cells and augmented the number of IgM-secreting cells in blood leukocyte cultures. IFNγ, on the other hand, had only minor effects up-regulating IgM secretion, whereas it increased the phagocytic capacity of IgM- cells in the cultures. Finally, given the recent identification of 9 mx genes in rainbow trout, we have also established which of these genes were transcriptionally regulated in blood naïve B cells in response to IFNa. This study points to a previously undescribed role for teleost type I IFNs in the regulation of B cell responses.

Teleost IgD+IgM- B Cells Mount Clonally Expanded and Mildly Mutated Intestinal IgD Responses in the Absence of Lymphoid Follicles.

Immunoglobulin D (IgD) is an ancient antibody with dual membrane-bound and fluid-phase antigen receptor functions. The biology of secreted IgD remains elusive. Here, we demonstrate that teleost IgD+IgM- plasmablasts constitute a major lymphocyte population in some mucosal surfaces, including the gut mucosa. Remarkably, secreted IgD binds to gut commensal bacteria, which in turn stimulate IgD gene transcription in gut B cells. Accordingly, secreted IgD from gut as well as gill mucosae, but not the spleen, show a V(D)J gene configuration consistent with microbiota-driven clonal expansion and diversification, including mild somatic hypermutation. By showing that secreted IgD establishes a mutualistic relationship with commensals, our findings suggest that secreted IgD may play an evolutionary conserved role in mucosal homeostasis.

CpG Oligodeoxynucleotides Modulate Innate and Adaptive Functions of IgM+ B Cells in Rainbow Trout.

Oligodeoxynucleotides (ODN) containing unmethylated CpG motifs have been widely postulated as vaccine adjuvants both in mammals and teleost fish. However, to date, the effects that CpGs provoke on cells of the adaptive immune system remain mostly unexplored in fish. Given that rainbow trout (Oncorhynchus mykiss) IgM+ B cells from spleen and blood transcribe high levels of toll like receptor 9 (TLR9), the receptor responsible for CpG detection in mammals, in the current work, we have investigated the effects of CpGs on both spleen and blood IgM+ B cells from this species. CpGs were shown to exert strong proliferative effects on both spleen and blood IgM+ B cells, also increasing their survival. The fact that CpGs increase the size of IgM+ B cells, reduce the expression of surface IgM and IgD and up-regulate the number of IgM-secreting cells strongly suggest that IgM+ B cells differentiate to plasmablasts/plasma cells in response to CpG stimulation. Additionally, CpGs were shown to modulate the antigen presenting capacities of trout IgM+ B cells through an increased surface MHC II expression and transcriptional up-regulation of co-stimulatory molecules, although in this case, significant differences were observed between the effects exerted on spleen and blood cells. Similarly, differences were observed between spleen and blood IgM+ B cells when CpG stimulation was combined with B cell receptor (BCR) cross-linking. Finally, CpGs were also shown to affect innate functions of teleost IgM+ B cells such as their phagocytic capacity. These results demonstrate that CpGs regulate many adaptive and innate functions of teleost B cells, supporting their inclusion as adjuvants in novel vaccine formulations.

Identification of CD8α+ dendritic cells in rainbow trout (Oncorhynchus mykiss) intestine.

Dendritic cells (DCs) are professional antigen presenting cells located at mucosal surfaces and lymphoid tissues. Their main role is to present antigens to T cells and thus regulate the initiation of the acquired immune response and modulate tolerance mechanisms towards self-antigens. Despite their relevance, not many studies have addressed the identification and characterization of specific DC subsets in teleost fish. Previous studies in our group identified a DC subpopulation co-expressing CD8α and major histocompatibility complex II (MHC II) on the cell surface in rainbow trout (Oncorhynchus mykiss) skin and gills. A complete functional and phenotypical characterization of these cell subsets was then undertaken, unequivocally recognizing them as DCs (CD8+ DCs). In the current study, we report the identification of a homologous population in rainbow trout intestinal lamina propria (LP). We have studied the main features of these intestinal CD8+ DCs, comparing them to those of CD8+ DCs from another mucosal tissue (gills). Interestingly, intestinal CD8+ DCs exhibited significant phenotypical and functional differences when compared to gill CD8+ DCs, suggesting that the location of DCs strongly conditions their activation state. These results will contribute to further expand our knowledge on how intestinal immune responses are regulated in fish, helping us to rationally design oral vaccines in the future.

Extract of Deschampsia antarctica (EDA) Prevents Dermal Cell Damage Induced by UV Radiation and 2,3,7,8-Tetrachlorodibenzo-p-dioxin.

Exposure to natural and artificial light and environmental pollutants are the main factors that challenge skin homeostasis, promoting aging or even different forms of skin cancer through a variety of mechanisms that include accumulation of reactive oxygen species (ROS), engagement of DNA damage responses, and extracellular matrix (ECM) remodeling upon release of metalloproteases (MMPs). Ultraviolet A radiation is the predominant component of sunlight causative of photoaging, while ultraviolet B light is considered a potentiator of photoaging. In addition, different chemicals contribute to skin aging upon penetration through skin barrier disruption or hair follicles, aryl hydrocarbon receptors (AhR) being a major effector mechanism through which toxicity is exerted. Deschampsia antarctica is a polyextremophile Gramineae capable of thriving under extreme environmental conditions. Its aqueous extract (EDA) exhibits anti- photoaging in human skin cells, such as inhibition of MMPs, directly associated with extrinsic aging. EDA prevents cellular damage, attenuating stress responses such as autophagy and reducing cellular death induced by UV. We demonstrate that EDA also protects from dioxin-induced nuclear translocation of AhR and increases the production of loricrin, a marker of homeostasis in differentiated keratinocytes. Thus, our observations suggest a potential use exploiting EDA's protective properties in skin health supplements.

Regulation of IgM+ B Cell Activities by Rainbow Trout APRIL Reveals Specific Effects of This Cytokine in Lower Vertebrates.

Tumor necrosis factor ligand superfamily members such as B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) have been identified in mammals as key regulators of B cell homeostasis and activation. However, the immune functions of APRIL are not as well defined as those of BAFF. Furthermore, while BAFF is present in all vertebrates, APRIL is missing in some animal groups, suggesting that BAFF has compensated the functions of APRIL in these species. In this context, we thought of great interest to explore the effects of APRIL on teleost B cells, given that APRIL appears for the first time in evolution in bony fish. Thus, in this study, we have performed an extensive analysis of the effect of APRIL on B cells using rainbow trout (Oncorhynchus mykiss) as a model species. Our results demonstrate that APRIL induces a specific proliferation of IgM+ B cells by itself and increases IgM secretion without promoting a terminal differentiation to plasma cells. APRIL also increased the levels of surface MHC II and augmented the capacity of these cells to process antigen, effects that were exclusively exerted on IgM+ B cells. Although our results point to a highly conserved role of APRIL on B cell homeostasis and activation throughout evolution, some specific differential effects have been observed in fish in comparison to the effects of APRIL previously described in mammals. Finally, the effects that APRIL induces on rainbow trout IgM+ B cells described in this paper have been compared with those previously reported in response to BAFF.

Identification of the First Teleost CD5 Molecule: Additional Evidence on Phenotypical and Functional Similarities between Fish IgM+ B Cells and Mammalian B1 Cells.

Despite teleost fish being the first animal group in which all elements of adaptive immunity are present, the lack of follicular structures, as well as the fact that systemic Ab responses rely exclusively on unswitched low-affinity IgM responses, strongly suggests that fish B cell responses resemble mammalian B1 cell responses rather than those of B2 cells. In line with this hypothesis, in the current study, we have identified a homolog of CD5 in teleost fish. This pan-T marker belonging to the scavenger receptor cysteine-rich family of receptors is commonly used in mammals to distinguish a subset of B1 cells. Subsequently, we have demonstrated that a very high percentage of teleost IgM+ B cells express this marker, in contrast to the limited population of CD5-expressing B1 cells found in most mammals. Furthermore, we demonstrate that fish IgM+ B cells share classical phenotypic features of mammalian B1 cells such as large size, high complexity, high surface IgM, and low surface IgD expression, regardless of CD5 expression. Additionally, fish IgM+ B cells, unlike murine B2 cells, also displayed extended survival in cell culture and did not proliferate after BCR engagement. Altogether, our results demonstrate that although fish are evolutionarily the first group in which all the elements of acquired immunity are present, in the absence of follicular structures, most teleost IgM+ B cells have retained phenotypical and functional characteristics of mammalian B1 cells.