Fish & Shellfish Immunology 35 (2013) 1719e1728

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The teleost humoral immune response Jianmin Ye a, Ilsa M. Kaattari b, Cuiyan Ma c, Stephen Kaattari b, * a Guangdong Provincial Key Laboratory for Health and Safe Aquaculture, College of Life Science, South China Normal University, Guangdong 510031, PR China b Department of Environmental and Aquatic Animal Health, Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, VA 23062-1346, USA c Key Laboratory of Sustainable Utilization of Marine Fisheries Resources, The Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fisheries Sciences, Qindao, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 June 2013 Received in revised form 29 September 2013 Accepted 17 October 2013 Available online 24 October 2013

Over the past 10 years our knowledge of cellular and molecular dynamics of teleost humoral immunity has increased enormously to now include: the existence of multiple isotypes, affinity-driven modulation of antibody structure and function, the unique trafficking patterns of each stage of B cell differentiation (including the plasmablast, short-lived and long-lived plasma cell, and the memory cell). Unfortunately the work which has generated the bulk of this information has generally employed defined antigens rather than vaccines. Thus, the focus of this review is to relate these aspects of immunity that are requisite for a mechanistic understanding of the generation of prophylactic immunity to the necessary analysis of responses to vaccines and vaccine candidates. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Isotypes Affinity Plasmablasts Plasma cells Memory

1. Introduction The potential of comparative immunology to advance our understanding of immunology in general is enormous as witnessed by such recent revelations as phagocytic B cells [1], IgD as an innate receptor [2], and affinity-driven, post-translational modifications of antibody function [3,4]. Thus the goal of this review is to synthesize studies on teleost B cell function to provide a framework for a more global analysis of the humoral immunoprophylaxis, along with potential practical applications.1

2. Teleost isotypes Most teleosts examined to date possess multiple isotypes, most commonly: IgM, IgD, and IgT/IgZ. IgM has been studied most

* Corresponding author. Tel.: þ1 8006847363; fax: þ1 8046847189. E-mail addresses: [email protected] (J. Ye), [email protected] (I.M. Kaattari), [email protected] (C. Ma), [email protected] (S. Kaattari). 1 As this issue contains a review of mucosal immunity, such will not be covered in this review, except as there may be a relevant direct comparison to the mucosal and systemic antibody response, or as one may directly affect the other. Please see Salinas et al., this issue. 1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.10.015

extensively since its presence has been known for decades [5e7]. Teleost IgD, initially discovered in 1997 [8], has now been found in at least eight species [9e11]. IgT/IgZ was first discovered in rainbow trout and zebrafish in 2005 [12,13], respectively), but now has been identified in representative species of all the main orders of teleost fish [reviewed in Ref. [14]]. 2.1. Structure and distribution 2.1.1. IgM IgM is the predominant isotype found in teleost serum and mucus [15,16] and typically is expressed as a tetramer, although early reports mention the existence of IgM monomers in giant grouper, Epinephelus itaira [17] sheepshead, Archosargus probatocephalus [18,19] and trout, Oncorhynchus mykiss [20]. However, at least for the latter species, the presence of an IgM monomer was never confirmed. To date IgM is the only teleost isotype for which sub-isotypes have been identified in certain salmonid species [21]. As in mammals, teleost IgM can be transferred across mucosal epithelia to the outer mucus layer via the polymeric Ig receptor (pIgR) [22,23] and, thus far, it has been cloned from fugu [22], carp [24], orange spotted grouper [25] and trout [23]. Interestingly, while mammals require a J chain for transport, teleost IgM has not been found to possess a J chain, leading to speculation that the H

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chain itself may possess the requisite binding motif for pIgR [23]. Further, IgM antibodies can be produced locally, within the mucosa, in response to antigen [26,27]. Whether found in serum and/or mucus, the teleost IgM is a tetramer composed of four, 2H:2L, monomers [reviewed by Ref. [28]]. The IgM H chain possesses four constant heavy chain domains, m1em4, with these C domains encoding sites for the binding of effector cells [29,30] or cytotoxic cells [31] or molecules such as complement [32,33]. To add another level of structural complexity, teleost IgM has been found to exhibit non-uniform disulfide polymerization of the monomeric and/or halfmeric subunits, which imparts functional diversity (Discussed in Section 3). 2.1.2. IgD Historically, mature naïve B cells, representing the majority of B cells in the mammalian periphery, were found to express both membrane IgM (mIgM) and mIgD [34,35] and that they also possessed IgM/IgDþ B cells as well [36]. Currently, although IgMþ B cells have been identified in most teleost species, IgM/IgDþ B cell subsets have only been characterized definitively in catfish [37] and trout [11]. Originally, teleosts were thought to only express the membrane-form of IgD [38e42] however, in 2002, Bengten et al. [43] discovered that channel catfish possessed both secreted and membrane IgD transcripts; further, serum IgD was purified using gel filtration and preparative SDS-PAGE. In 2012 an IgD-specific (monoclonal antibody (mAb) was employed to detect and quantify secretory IgD in rainbow trout [11]. Serum IgD levels were found to range from 2 to 80 mg/ml (in contrast, IgM concentrations ranged from 800 to 9000 mg/ml). The majority of B cells in the periphery of humans and mice are naïve lymphocytes bearing both membrane IgM and membrane IgD [34,35]. Edholm et al. [37] also found a comparable situation in channel catfish where 80% of the peripheral B cells were IgMþ/IgDþ, while the remaining were IgM/IgDþ. Interestingly these investigators demonstrated that the catfish IgM/IgDþ B cells only expressed one type of light chain, IgL s [37] (one of the four catfish IgL isotypes: F, G, l, and s [44]), an isotype which is restricted to ectothermic vertebrates [45]. Edholm et al. [37] speculated that catfish IgD functions as an Ag-binding receptor on IgMþ/IgDþ and IgM/IgDþ cells, and that cross-linking of the IgD leads to activation, differentiation, and proliferation. Catfish IgMþ/IgDþ B cells express high levels of IgM and low levels of IgD. In contrast IgM/ IgDþ B cells express high levels of IgD and no IgM. The structure of the teleost d-chain isotypes shows considerable diversity in size, with as few as two constant domains, as in mice, and as many as 16 constant domains in zebrafish [46]. Teleosts show broad variety in their d genomic organization with some teleosts having repeated blocks of exons encoding C d2e4 as well as duplicated d loci [39,43,47]. Channel catfish have two functional copies of the d loci [8] within the same IGH complex. One locus is in the typical location downstream of Cm, and the other copy is upstream of the functional H-DH-JH region. These two loci possess a unique specialization with the downstream ds encoding the membrane-bound form of d and the upstream possessing an exon for secretory d. 2.1.3. IgT/IgZ Unlike the m chains, which consistently possess four constant Ig domains, the C domain structure of the s/z chains varies. Both rainbow trout s and zebrafish z have four constant Ig domains, while the stickleback possesses three s/z domains [48], and fugu only two, corresponding to zebrafish z1 and z 4 [49]. The common carp z H chain is a chimera of IgM-IgZ with two constant region domains generated by the m1 of carp and a domain similar to zz 4 of zebrafish [50]. As in mammalian IgM, s4/z4 is included in the

membrane anchored form of IgT/IgZ. This contrasts to teleost IgM, where m3 is fused directly to the membrane anchoring form. The rainbow trout IgTþ B cell subset is comprises w16e27% of all B cells in the main systemic lymphoid organs, with the remaining percentage of B cells are IgMþ. In the trout gut, however, the percentage of IgTþ and IgMþ lymphocytes is more similar, 54% and 46% respectively [14,23]. These results strongly indicate that IgT may play a specific role in gut mucosal immunity; however, the ratio does not reflect the total concentration of each Ig in the plasma or mucus. Flow cytometry and immunofluorescence have not revealed double stained IgTþ/IgMþ cells. Thus, teleost fish appear to have two mutually exclusive B cell lineages that express either IgT or IgM.

2.2. Isotype functions 2.2.1. IgM IgM is a universal isotype in all vertebrate species. IgM is also found in sera as a natural antibody even before contact with antigen [51]. No matter the species, IgM has a wide variety of immune characteristics including being the first mammalian Ig elicited prior to isotype switching events, serving roles in both adaptive and innate immunity [51,52] and actively accomplishing many effector functions in both mammals and fish, such as complement fixation [32,33,53], agglutination, binding of mannose binding lectin [54] and mediating cellular cytotoxicity [29,30,55,56]. Agglutination provides for the phagocytosis and clearance of pathogens, while complement activation contributes to pathogen opsonization and also links innate and adaptive immunity. The role of specific antibodies to marshal complement to effect antigen-specific lysis of anthropogenic epitope-labeled red blood cells [20], or the killing/neutralization of infectious agents has been demonstrated with the bacterial pathogen, Aeromonas salmonicida [57], the parasite Cryptobia salmositica [58] and the viruses, IHNV [59] and VHSV [60,61]. The viral activities have been postulated to be due to the disruption of the membranes of these virions [62]. Teleost IgM antibodies have also been demonstrated to mediate antigen-specific opsonization and phagocytosis of bacterial pathogens, such as Francisella asiatica [63,64] and Photobacterium damsela [65] by macrophages. 2.2.2. IgD The specific functional role(s) of the numerous variants of IgD have yet to be conclusively determined. It has been hypothesized that these IgD variants may compensate for the lack of class switch recombination in fish [10]. Chen et al. [2] discovered that secreted IgD in mammals binds to basophils and mast cells, activating these cells to make antimicrobial factors involved in respiratory immune defenses. In addition to antimicrobial factors, the IgD-armed basophils are induced to produce opsonizing, inflammatory, and immune-stimulating factors. Cross-linking of IgD on basophils induces production of immunoactivating cytokines, including IL-4, IL13 and BAFF, proinflammatory cytokines such as TNF and IL-1B, and chemokines such as IL-8 and CXCL10 [2]. The discovery of secreted IgD in rainbow trout [11] lends support to Chen et al.’s [2] hypothesis that “secretory IgD has been preserved throughout evolution as a system of surveillance and defense”. The tissue-specific distribution of IgD plasma cells in trout is analogous to the findings in mammals [11]. IgD plasma cells are rare in the peripheral blood (w3%) for both taxa, but are heavily represented in either the human respiratory tract mucosal B cells [2,66] or in the gills of trout [11]. Few IgD plasma cells have been found in either the mammalian digestive system mucosae or fluids [67], or in the trout gut [11].

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2.2.3. IgT/Z Trout IgT has been shown to be heavily engaged in gut antibody responses against Ceratomyxa shasta, an intestinal parasite [23]. C. shasta is endemic to rivers of the Northwest coast of the United States and causes severe infections in the gut [68]. Trout which survived C. shasta infection contained large numbers of IgTþ B cells whereas the number of IgMþ B cells did not change with respect to control fish (Zhang et al., 2010). Further, it was observed that there were very large increases of IgT but not IgM in the gut of surviving fish, both at the protein and transcript levels. In the serum of surviving fish, high titers of parasite-specific IgM were found, but not high titers of IgT. In summary, this model provided the first evidence in teleost fish or any other non-tetrapod species, for separation of immunoglobulin isotypes into mucosal (IgT) and systemic (IgM) areas in response to pathogenic challenge [14]. 3. Antibody affinity: antigen recognition and its role in regulating B cell differentiation, disposition, and function2 The role that affinity plays in governing IgM antibody function as well as B cell function and differentiation is extensive and, thus, pervades the remaining topics of this review. Although antibody affinity determines the ability to bind, sequester, and mediate removal of antigen, recent studies have demonstrated a more global in the context of the B cell receptor (BCR) [4,69]. Differences in the affinity of the latter for antigen are transduced into alternate post-translational forms of antibody [3,4] as well as the stimulation or suppression of clonal expansion, and B cell differentiation (i.e. plasmablast, plasma cell, and memory cell [70]). 3.1. Antibody affinity determination: historical assumptions and the rationale for partitioning analyses Historically, serum antibody affinities were determined using techniques such as equilibrium dialysis, fluorescence quenching, or the Farr technique. All of these methods were originally designed for the analysis of proteins possessing homogeneous binding sites [71,72]. In order to adapt this technique for the analysis of highly heterogeneous serum antibodies, the concept of an average affinity constant (Ko) was developed. Using such analyses, investigators determined that average antibody affinities in the teleost ranged from 104 M1 to 106 M1 [73e77]. However, to execute such affinity analyses (which rely on the Sips distribution), a normal distribution of affinities must be assumed. Unfortunately such is rarely encountered in serum antibodies. Antibody affinity distributions are typically skewed to the lower affinities during the early response and gradually shift to a skewing towards the higher affinities late in the response (Fig. 1) [70,78]. Thus, more appropriate analyses would be those that attempt a determination of each affinity subpopulation within an immune serum. Upon obtaining such a distribution, one could then determine a weighted average of these subpopulations to derive a more representative average affinity. More importantly; however, since classical analyses can only produce a single average affinity estimate, important information as to the size and composition of all the affinity subpopulations are lost in the process. An alternate form of affinity analyses as introduced originally by Nieto et al. [79] employs

2

IgM and the mB cell is the focus of the balance of this review, as it is the primary systemic and mucosal immunoglobulin (Bergsten et al., 2006; Solem and Stenvik; 2006), and also since the discovery and requisite analyses of IgT and IgD (discussed above) have only just begun. Preliminary studies on the unique functions and dispositions of these two isotypes presage fascinating possibilities for s and d B cell differentiation function in the near future.

Fig. 1. Subpopulational depiction of trout affinity maturation. A) Subpopulational distribution early in the response. B) Subpopulational distribution late in the response. C) 3-D depiction (using 3 axes; affinity, time, and antibody concentration over time. Color denotes the relative amount of each affinity subpopulation at a particular time post-immunization: Less than 0.1 units (dark blue), 0.1e1.0 units (light purple), 1.0e 10.0 (yellow), 10.0e100.0 (light blue), >100 units (dark purple). Affinity is expressed as log aK units (log Keq), populational skew as S, and time is in weeks postimmunization with either a protein or polysaccharide antigen in Freund’s Complete adjuvant. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

affinity partitioning to determine a quantitative antibody distribution vis a vis their distinct affinities. This leads to a more complete and less compromised analysis of all the antibody affinities expressed within an antiserum. The partitioning technique delineated by Nieto et al. [79] has permitted quantitative analyses of the affinity subpopulations

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within each immune serum, thus revealing the dynamics of antibody subpopulation growth and decline during affinity maturation [3,4,70,78]. Such subpopulational analysis repeatedly demonstrates a shift from low affinity skewness, through a more normal distribution, to high affinity skewness. The latter is often found to be composed of late-appearing, novel high affinity antibodies, while missing the lowest affinity antibodies that were initially present [70]. Such aspects of affinity maturation could easily have been missed without affinity partitioning techniques. Furthermore, relatively small shifts in intrinsic affinity in IgM (as compared to those found in IgG) would result in logarithmic avidity increases. Thus, the low intrinsic affinity of the primary teleost antibody is compensated by its octavalency [75,80]. This would not be unique to teleosts as the decavalent mammalian IgM can possess a 10,000fold higher avidity than can be expressed by IgG antibodies possessing the same binding site affinity. Typically the teleost affinity response begins with a skewed distribution towards the lowest affinity antibodies (Fig. 1A), which rarely achieves the 100-fold greater titers found with the later, higher affinity antibody subpopulations. These later distributions are characteristically skewed to the highest affinity subpopulations (Fig. 1B). The late emergence of the highest affinity antibodies appears to be a combination of both selection of somatic mutations as well as the release of high-dose suppression by the initially high concentrations of antigen. As antigen concentrations subside these higher affinity specificities can be elicited, while the lower affinity specificities can no longer be stimulated, giving rise to the rather serpentine crest of affinities (Fig. 1C). 3.2. BCR affinity and post-translational modifications of antibody Mammalian pentameric IgM monomers are covalently bound together by consistently oxidized disulfide bonds. This contrasts sharply with typical teleost tetrameric IgM, which displays a variety of reduced and oxidized disulfides. This diversity has been reported in at least 17 different species of teleost [referenced in 4]. This structural diversity is not a reflection of genetic/isotypic differences, as a single cloned C region conferred this structural diversity to all the catfish IgM it produced [81]. Further, in trout every isoelectropherotype exhibited all the structural variation that was seen with serum antibodies as a whole [82]. Although this form of structural diversity appears universal in teleost IgM, its functional significance had remained elusive for 20 years. We had originally proposed that variation in disulfide polymerization may result greater flexibility in accommodating antigens with spatially diverse epitopes (i.e. within a polysaccharide cell wall or capsule). This flexibility could result in the increased ability of an antibody to simultaneously bind more epitopes thereby greatly increasing its avidity [83]. The first causal link between IgM structural diversity and its function came from studies demonstrating that the degree of disulfide polymerization was proportional to the antibody’s affinity to antigen [3]. Thus, high affinity BCR binding to antigen was being transduced, directly or indirectly, into more extensive disulfide polymerization of the antibody. Secondarily, it was determined that the more extensively cross-linked the antibody, the longer was its half-life. Later it was found that both these characteristics were directly related to the degree of glycosylation, specifically mannosylation [4]. Thus, the primary post-translational modification induced by high affinity BCR interactions could be the increased mannosylation of the antibody. This coincides with the demonstration that high affinity, heavily polymerized antibodies that are bound by Con A require higher concentrations of a-methylmannoside for elution than the lightly polymerized antibodies possessing lower affinity [4]. Differential or altered glycosylation

has been demonstrated to impact mammalian IgM polymerization and complement fixation [84], IgG catabolism in the liver [85], as well as a variety of other Fc-dependent functions [86]. Additionally the graded mannosylation of affinity subpopulations [4] may confer analogous rigidity to the secretory tailpiece providing for intermonomeric polymerization as found in the mouse [87]. This posttranslational glycosylation may also provide for other effector functions such as mannose binding lectin recognition [54], and possibly complement fixation [88]. It is noteworthy that mannosebinding lectin (MBL) recognition of IgM is also controlled by its binding of antigen. Thus, antigen interaction induces a conformational change in the Fc exposing high mannose residues for interaction with the MBL. Since MBL have been identified in a variety of teleost including trout [89,90] and as the degree of mannosylation parallels affinity, a differential capacity for trout MBL to bind high affinity antibodies may exist. Although affinity driven selection would also drive tertiary and quaternary structural modifications, these modifications, could influence affinity maturation by effectively accelerating the removal of low affinity antibodies and/or retaining high affinity antibodies. This affinity-transduction capacity also enables the single antibody producing cell to tailor its response to different antigens. For example, a B cell which recognizes an antigen with high affinity will produce an antibody with different effector functions than if it were produced via binding an antigen with lower affinity. In other words, the B cell itself can alter the Fc of its antibody to perform functions best suited to its affinity. Indeed, as each isotype confers a unique set of effector functions on that antibody (i.e. complement fixation, mucosal transport, and antibody-dependent cellular cytotoxicity), affinity-based, posttranslational modification of the Fc may be a more direct and robust means of eliciting isotype-like functions. Possibly this may be a reason why teleosts do not possess the isotypic diversity observed in mammals. Comparable analysis of mammalian IgM structure and function is warranted to see if any comparable transductional mechanisms are employed. 4. Induction, disposition, and demographics of antibody secreting cells (ASC) A maximally effective humoral immune system must efficiently and constantly monitor all susceptible regions of the body for pathogenic insults, produce the requisite antibodies, then deploy them in a sufficient and effective manner throughout the body. Aside from the mucosa (covered in a separate review) the blood becomes the most logical vehicle for this function. In vitro culture has proven that leukocytes of specialized teleost immune tissues (spleen, anterior kidney, posterior kidney, and blood) are fully capable of being induced by antigen [91e94]. However, the actual in situ contribution of each of these tissues to the systemic antibody response can only be ascertained by monitoring the ASC response ex vivo. Further, to accurately gauge these contributions, the total ASC response from each tissue must be determined, rather than simply determining the number of ASC per 106 leukocytes, as has been commonly done [69,95e97]. As each tissue varies considerably in the total number of leukocytes it possesses, the relevance of simply the number of ASCs/106 is negated and often would yield erroneous conclusions as to the primary players in the antibody response. For example, although early in the response, the spleen or posterior kidney may produce a greatest number of ASCs on a per cell basis, the blood contributes more ASCs overall during this period (Fig. 2). Thus, the blood is a primary contributor to the serum antibody response at this time. However, this does not mean that the ASCs are being induced in the blood even though induction can be readily accomplished in vitro. The fleeting chance of appropriate

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Fig. 2. Expression of ex vivo ASC responses as per whole tissue (Total) vs. per 106 leukocytes. Trout were immunized with TNP-LPS and tissues analyzed at 5, 10 and 15 weeks post-immunization for TNP ASCs in the blood (blue), spleen (red), anterior kidney (green) and posterior kidney (purple). Responses on the left were expressed as per 106 lymphocytes and those on the right as the total tissue contribution. Note that at all time points the blood response, on a per cell basis, was logarithmically lower than any other tissue, while actually generating a maximal response on the basis of the whole tissue response at weeks 5 and 10. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

interactive cell contact with antigen precludes initiation within the blood proper but, is more likely to occur in the secondary lymphoid tissues [98]. In mammals, not only blood but the lymph is provided with immune tissues (nodes). However, within fish there is not a system of lymph nodes, but rather only a few identifiable tissues wherein induction may occur (e.g. the spleen, anterior kidney, posterior kidney, possibly the thymus). Thus, it would appear that the blood serves an even greater critical role in delivering antigen and B cells to these few tissues. We and others have proposed that the primary tissues for the initial induction of the ASC response could be the spleen [69,99,100,101] and posterior kidney [102]. One corroborative observation, for the former, is the temporary splenomegaly in response to antigenic challenge in fish [99], personal observations]. Recent studies demonstrate that the early response in the spleen is primarily composed of the early differentiative stage, the plasmablast [102]. During this period an increased expression of ASCs within the blood can also be observed ex vivo. Within one to two months post immunization, a sizeable and persistent presence of ASCs within the anterior kidney occurs [97,100,103]. The molecular [102,104] and physiological characteristics [97,100,103] of many, but not all of these ASCs, is that of a mature plasma cell. Presence of these long-lived plasma cells (LLPC) has been observed for a period of months to over a year [97,100,103]. Thus, the anterior kidney, besides being the hematopoietic tissue of the fish [104e111] also serves as a reservoir for LLPC (Fig. 3). This is a very intriguing association as these two functions (hematopoiesis and LLPC niche) coincidentally occur within mammalian bone marrow [112e114], strongly suggesting a pervasive requisite for the proximal association of these two functions, regardless of the specific tissue in which they occur. Typically within two months, the ASC contributions from the blood and spleen can drop to barely detectable levels, while the anterior kidney continues to support ASCs. This constancy of ASCs within the anterior kidney can be associated with the maintenance of serum antibody levels [97,100,103]. Thus, the residence of LLPC within the supportive niche of the anterior kidney may be important to the long term production of antibodies and prolonged vaccine effectiveness, as is the case with the mammalian bone marrow [112,115,116]. Although the anterior kidney can support these LLPC for considerable time, these cells may be lost upon subsequent antigen challenge [117]. Whether this displacement is simply a stochastic process reliant on the overall increased

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Tissue Fig. 3. Plasma cell distribution in tissues after immunization. ASCs were assayed at 16 (blue), 44 (red), 63 (green) and 98 (purple) weeks post primary immunization. The cells were cultured with and without HU to ascertain the number of plasma cells vs. plasmablast respectively. The percent plasma cells represent the number of HU resistant ASCs out of the total ASCs at each timepoint. A secondary injection of TNP-LPS was given at 44 weeks. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

concentration of plasmablasts or plasma cells within the anterior kidney, as opposed to some selective advantage of newly formed plasma cells has yet to be determined. 5. Memory Although the term ‘humoral memory’ has been employed to refer to the persistent antibody response derived from LLPC (even that elicited during the primary response) in this section we will be referring to the ability of secondary antigen exposure to elicit a differentiated response to that antigen. In comparison to secondary teleost responses, mammalian secondary responses are not only logarithmically greater in both antibody titer and affinity, but also different in their isotype [118]. Other ancillary differences include greater sensitivity to the inducing antigen together with lower optima for B cell induction and prolonged response persistence [119,120]. Based on these differential responses seen in mammals, it was often concluded that fish could not generate a memory response, as for example, teleost titers and affinities appeared only marginally elevated, at best [75,121e124]. Not only was this conclusion premature, as logarithmic increases in titers, affinities, and antigen-sensitive lymphocyte pools have been demonstrated [69,70,78,125], but mammalian studies have now demonstrated mammalian mþ memory cells exist that are comparable to teleost memory cells. This leads to the possibility that the unique aspects of mammalian memory (i.e. extensive somatic mutations, logarithmic increases in affinity and titer) may be a requisite for an effective bivalent (IgG) memory response than for a memory response in general. Philosophically, one may question why a bivalent antibody would take on such a dominant role, given that m memory exists for both mammals and teleosts. Given that the avidity of the multivalent IgM is so much greater than IgG of comparable affinity, perhaps IgG antibodies need to possess higher intrinsic affinity and concentrations to successfully compete for antigen. Indirect confirmation of this possibility has been demonstrated in the shark, which produces a monomeric IgM. The latter IgM, in contradistinction to its multimeric form, possesses strikingly higher intrinsic affinities [126]. The recent discovery of only bivalent IgT in trout serum [14] presents another opportunity to test this hypothesis. Indeed as this differs from the mucus, wherein both monomeric and tetrameric IgT and tetrameric IgM reside, affinity comparisons between all players becomes fascinating test of this hypothesis. One common feature of memory shared by teleosts and mammals is the tremendous expansion of the memory pool after

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primary immunization. Indeed teleost memory pools appear to expand to greater numbers than have been reported in mammals. The bulk of these memory cells reside in the peripheral blood (Fig. 4) as in the mammal. However, also in contradistinction to the mammalian g memory cell, which possesses a greater clonal proliferation potential, clonal proliferation potential in teleost appears unchanged (Fig. 4). It thus becomes of great interest to ascertain whether mammalian m memory cells also exhibit this relatively low proliferation potential. Of particular interest are our most recent studies which indicate memory pool expansion occurs comparably with either T-dependent (TNP-KLH) or T-independent antigens (TNP-LPS). These memory cells represent one differentiation pathway arising from the replicating plasmablast, the other being the long-lived or short-lived plasma cell [103]. At the point of departure from plasmablast to either mature cell, the selection of somatically mutated binding site modifications will have ceased, preserving the affinity and specificity attained to that point. The critical question that arises here is, where does this point of differentiation occur, and what precipitates the transition? Generally, plasmablasts can be found in both the spleen and anterior kidney; however, although it appears that the spleen possesses the capacity to permit differentiation from a plasmablast to a plasma cell in vitro, these plasma cells have a limited lifespan (t1/2 of 2 days) [103]. In contrast, the anterior kidney; even from early after immunization maintains a pool of LLPCs [97,100,103]. As opposed to the spleen or blood, the number of inducible cells in the anterior kidney can only range from 0 to 20%. It would not be difficult to surmise from this that although antigen-sensitive B cells can differentiate from a resting cell to a plasma cell within the matrix of the spleen, the spleen can only maintain these plasma cells for a short period. Thus, again, like the mammalian plasmablast/early plasma cell these cells must make their way to suitable niches within the hematopoietic tissue to survive. Our work suggests that, although the anterior kidney also harbors plasmablasts and resting B cells, the fact that HU-resistant plasma cells primarily reside there as well makes the in vitro determination of plasmablast or naïve B cells transitioning to plasma cells (HU-resistant) impossible at this time. Obviously in our screening there are times during which there are virtually no plasmablasts or hardly any resting B cells in the anterior kidney [103]. Interestingly, recent work of Odaka et al. [127] demonstrates the presence of plasmablasts within the kidney which possess the unique trait of plastic adherence, not found with splenic plasmablasts. This may indicate an intermediate stage in plasmablast differentiation beyond that achievable in the spleen. Unfortunately, as it appears that the entire kidney was employed one cannot be certain that these plasmablasts did not reside in the

posterior kidney, a tissue comparable to the spleen vis a vis B cell differentiation [102]. A practical advantage that arises from memory cells primarily residing within the blood is that the potential for a memory response can easily be monitored by non-invasively and quantitatively using in vitro culture. In this manner the success and generation of memory in human vaccines trials have been accomplished [128,129]. Thus, as both classical memory (memory cell production) and humoral memory (antibody activity) can be assessed via whole blood samples permitted practical assessment of the memory potential afforded by vaccination. 6. The mechanics of humoral prophylaxis: the need for explicit operational models 6.1. The problem at hand In the analysis of teleost immunoprophylaxis the tacit assumption has been that the induction of antibodies can be synonymous with resistance. Although a convenient measure of antimicrobial responses, to assume some basic correlation with disease resistance has not always been productive [130]. One and, perhaps, the only foolproof means of demonstrating the protective capabilities of specific antibodies is via passive immunization, which has been applied to variety of anti-bacterial, -viral, and -parasitic responses including, Aeromonas salmonicida [57], Edwardsiella tarda [131], Flavobacterium columnare [132], Flavobacterium psychrophilum [133], Vibrio anguillarum [134], Streptococcus iniae [135], Francisella asiatica [63,64], Infectious Hematopoietic Necrosis Virus, IHNV [136,137], Viral Hemorrhagic Septicemia Virus, VHSV [138], Pancreas Disease virus [139], Cryptobia salmositica [58]. Even such associations can be tenuous if innate factors are also elevated during such challenges, suggesting caution in the interpretations using immune sera. However, monitoring of serum antibody titers can serve as an invaluable source of information as to the dynamics of ASC differentiation, particularly as the teleost IgM half-life is as short as 1e3 days as in salmonids [3,140]. Such short antibody half-lives mean that the titers should tightly parallel total ASC numbers (Fig. 5). Further, the ability to simultaneously determine total antibody titers as well as total ASC production in all tissues, one can determine the average in vivo secretion rates of the ASCs at any time post immunization (Fig. 5). One advantage to the latter may be the determination of if or when a vaccine elicits a good plasma cell (high rate antibody secretor), and, particularly, a long-lived plasma cell response. This, in turn, could reveal which vaccine favors induction and complete

Fig. 4. Antigen-sensitive precursor analysis of naïve vs. immune fish. In vitro limiting dilution analyses were performed on peripheral blood lymphocytes at week 0 (naïve animals) and week 46 post immunization with trinitrophenylated-lipopolysaccharide (TL), TNP-keyhole limpet hemocyanin (TK), and the chimeric antigen TNP-KLH conjugated to LPS. Nonimmunized control fish (Ctrl) were also examined. Data from 40 to 50 individual fish are the number of leukocytes bearing one antigen-sensitive precursor. The increase in antigensensitive lymphocytes (memory) was 56-fold with TL immunization, 24-fold with TK, and 85-fold with TKL, while that of controls remained unchanged.

J. Ye et al. / Fish & Shellfish Immunology 35 (2013) 1719e1728

Fig. 5. Comparison of whole animal titers and ASCs post-immunization. Trout were immunized with TL and at weeks 1, 2, 4, 6, 10, 12, 15, 20 and 25 titers for the whole blood volume and total ASCs in blood, spleen, anterior and posterior kidney were determined for groups of 10 fish. The units on the ordinate represent either total units of antibody activity (blue) per fish (average) or total ASCs per fish (red). Note the relatively consistent increase in both parameters, with an increasing level of antibody units per ASC over time post-immunization (circled). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

differentiation in specific tissues (i.e. a transitioning from splenic or blood participation to the anterior kidney). More importantly a coordinated, quantitative, and long-term analysis of total plasmablasts, plasma cells, memory cells as well as total antibody titers and affinity distributions could provide a framework for projecting long-range effects of vaccination. To date, most studies to assess the success of a vaccination have monitored responsiveness for only few days to a few weeks, often with only titers and ASC responses from the blood or spleen. This will likely be insufficient for the assessment of long-term efficacy of vaccination. As our current studies would indicate a short term plasmablast response could easily account for peripheral ASCs and titers. Important information that cannot be projected from this is whether the vaccine also generated a long-lived plasma cell response in the anterior kidney, or memory cells in the periphery. If, for example, the antibody and ASC responses are comparably short-lived and, or peak within a few weeks, it is likely that the vaccine gave an insufficient stimulus for the generation of long-lived plasma cells (and possibly memory cells as well). Additionally, did antibody affinity mature sufficiently to secure a significant population of high affinity B cells? Not only is this critical for the most efficient recognition and binding of the antigens, important post-translational modifications of the antibody, but also full differentiation to plasma and memory cells. 7. Conclusions Examination of the many studies reported in the literature, indicate at least two basic forms of immunization lead to distinctively different responses: 1) adjuvanted non-viable immunogens or live/naturally challenges result in long-term humoral immunity [26,69,95,96,141e144] and the potential for long-lived plasma cells and/or memory, while 2) soluble antigens tend to result in short or transient humoral immunity [145e148] and the likely possibly of no LLPC induction memory. Successful elicitation of affinity maturation has also been reported with the former immunization protocols [3,4,69,70,78], thus it would also stand to reason that the latter may have been insufficient to elicit a phase of high affinity clonal expansion before the antigen was eliminated. The best hope in the latter situation may be that at least a modicum of high

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affinity memory cells may have been produced (this could be determined by in vitro challenge of these immunize individuals vs. non-challenged controls). Further, either the secondarily challenged fish could be screened for their affinity subpopulation distribution, which should be significantly higher than seen upon primary immunization. Generally, simplistic analysis of antibody titers should never be employed as incontrovertible proof that antibodies were definitively the means by which immunoprophylaxis was achieved. For example, Raida et al. have mentioned in their examination of anti-Y. ruckeri titers after application of commercial immersion vaccines, that although persistent titers are found, possible prophylaxis via innate molecules or cells is still possible [149]. Logically we should be able to carry this further, for without passive immunization with antibodies secured from immunized individuals, we have no definitive demonstration that even antibodies bearing in vitro bactericidal activities can protect in vivo. Once the latter is demonstrated, we are in a position to assert that antibodies induced by vaccination are sufficient to produce a prophylactic state. However, it is always of value to vaccinologists to assess the sum total of functions that may be enhanced by vaccination, such as elevation of innate defense molecules, or specific cell mediated immunity. The logistics of vaccine design requires the extensive analysis of each immune mechanism that can conceivably offer protection. Delineation not only of antibody affinity and effectiveness via passive neutralization is required but also knowledge of the status of circulating memory cells in the periphery, and actively secreting plasma cells in the anterior kidney must be assessed. We are now capable of easily assessing all of these parameters to effectively translate immunogen composition into the most effective and persistent state of prophylaxis. Acknowledgments The authors gratefully acknowledge the support of the National Institute of Food and Agriculture Grant # NIFA grants 2012-6701530217 and 20008-35204-04553. Contribution No. 3287 of the Virginia Institute of Marine Science, College of William and Mary. References [1] Li J, Barreda DR, Zhang YA, Boshra H, Gelman AE, Lapatra S, et al. B lymphocytes from early vertebrates have potent phagocytic and microbicidal abilities. Nat Immunol 2006;7:1116e24. [2] Chen K, Xu W, Wilson M, He B, Miller NW, Benten E, et al. Immunoglobulin D enhances immune surveillance by activating antimicrobial, proinflammatory and B cell-stimulating programs in basophils. Nat Immunol 2009;10:889e98. [3] Ye J, Bromage ES, Kaattari SL. The strength of B cell interaction with antigen determines the degree of IgM polymerization. J Immunol 2010;184:844e50. [4] Ye J, Bromage E, Kaattari I, Kaattari S. Transduction of binding affinity by B lymphocytes: a new dimension in immunological regulation. Dev Comp Immunol 2011;35:982e90. [5] Acton RT, Weinheimer PF, Hall SJ, Neidermeier W, Selton E, Bennett JC. Tetrameric immune macroglobulins in three orders of bony fish. Proc Natl Acad Sci U S A 1971;68:107e11. [6] Shelton E, Smith M. The ultrastructure of carp, Cyprinus carpio, immunoglobulin: a tetrameric macroglobulin. J Mol Biol 1970;54:615e7. [7] Bradshaw CM, Clem LW, Sigel MM. Immunological and immunochemical studies on the gar, Lepisosteus platyhincus. II. Purification and characterization of immunoglobulin. J Immunol 1971;106:1480e7. [8] Wilson M, Bengten E, Miller NW, Clem LW, Du Pasquier L, Warr GW. A novel chimeric Ig heavy chain from a teleost fish shares similarities to IgD. Proc Natl Acad Sci U S A 1997;94:4593e7. [9] Suetake H, Saha NR, Araki K, Akatsu K, Kikuchi K, Suzuki Y. Lymphocyte surface marker genes in fugu. Com Biochem Physiol Part D 2006;1:102e8. [10] Chen K, Cerutti A. New insights into the enigma of immunoglobulin D. Immunol Rev 2011;237:160e79. [11] Ramirez-Gomez F, Greene W, Rego K, Hansen JD, Costa G, Kataria P, et al. Discovery and characterization of secretory IgD in rainbow trout: secretory IgD is produced through a novel splicing mechanism. J Immunol 2012;188: 1341e9.

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