Phylogeny of Immune Recognition: Processing and Presentation of Structurally Defined Proteins in Channel Catfish Immune Responses

This work was undertaken to investigate whether or not antigen processing and presentation are important in channel catfish in vitro secondary immune responses elicited with structurally defined proteins, namely, pigeon heart cytochrome C (pCytC), hen egg lysozyme, and horse myoglobin. The use of in vitro antigen-pulsed and fixed B cells or monocytes as antigen presenting cells (APC) resulted in autologous peripheral blood leukocytes (PBL) responding with vigorous proliferation and antibody production in vitro. In addition, several long-term catfish monocyte lines have been found to function as efficient APC with autologous but not allogeneic responders. Subsequent separation of the responding PBL into sIg- (T-cell-enriched) and B (sIg+) cell subsets showed that both underwent proliferative responses to antigen-pulsed and fixed APC. Moreover, allogeneic cells used as APC were found to induce only strong mixed leukocyte reactions without specific in vitro antibody production. Initial attempts at identifying the immunogenic region(s) of the protein antigens for catfish indicated there are two such regions for pCytC, namely, peptides 66-80 and 81-104.


INTRODUCTION
In recent years, the use of globular proteins has facilitated the understanding of molecular aspects of immune recognition and genetic control of immune responses in mammals (Sercarz and Berzofksy, 1987;Smith-Gill and Sercarz, 1989). Proteins with known amino-acid sequences and spatial structures, such as lysozyrne, myoglobin, and cytochrome C, have become effective tools in determining the fine specificities of both Band T-cell repertoires. Furthermore, the availability of species variants of these proteins has permitted the exact identification of antigenic determinants by comparing the cross reactivities of either the native forms of the proteins or their peptide fragments derived by chemical or enzymatic cleavage as well as chemical synthesis. This latter experimental approach is becoming useful *Corresponding author. for identifying the critical contact points of the peptide antigen with the major histocompatibility complex (MHC) molecule and the T-cell receptor (Heber-Katz et al., 1983). Along these lines, we have conducted studies to further elucidate the mechanisms of immunity of a phylogenetically lower ectothermic vertebrate, the channel catfish (Ictalurus punctatus). We have previously reported (Vallejo et al., 1990b) that the generation of secondary in vitro immune responses to complex thymus-dependent antigens in channel catfish requires steps akin to antigen processing and presentation in mammals (Pernis et al., 1988;Schook and Tew, 1988). The present work now extends these observations to in vitro immune responses to well-characterized protein antigens. Furthermore, the development of long-term catfish rnonocyte lines (Vallejo et al., 1990a) has facilitated studies on the identification of the responding lymphocyte populations, putative immune restriction, and presentation of peptide fragments by fixed antigen-presenting cells (APC). 138 VALLEJO et al.

RESULTS
(i.e., T-cell-enriched) incubated with anticatfish pan T/thrombocyte (mAb 13C10) were not effective APC Channel Catfish Exhibit Secondary In Vitro Immune in eliciting proliferative responses. However, it Responses toStructurallyDefinedProteins should be noted that cultures stimulated with antigen-pulsed sIg-cells (T-cell-enriched) showed We have previously reported that catfish can 3H-thymidine uptake significantly higher than either immunologically discriminate between two species the unstimulated controls or PBL cocultured with variants of a complex thymus-dependent antigen, unpulsed/fixed APC. As in the previous experihemocyanin (Vallejo et al., 1990b). In order to determents, APC fixed prior to antigen pulsing did not mine whether or not the same specificity occurs with simpler well-characterized proteins, groups of stimulate autologous responders. five fish were immunized with pigeon heart cytochrome C (pCytC), hen egg lysozyme (HEL) and Long-Term Catfish Monocyte Lines Are Effective horse muscle myoglobin (EqMb). When peripheral APC blood leukocytes (PBL) from these fish were cul-We have previously described the development of tured in the presence of soluble antigen, vigorous long-term monocyte lines from channel catfish antigen-specific in vitro proliferation was observed (Vallejo et al., 1990a). In the present work, such cell only with immune PBL stimulated with homologous lines were used as APC. Since the fish serving as but not heterologous antigen (as depicted for a sources of these cell lines were still .alive, it was representative fish from each group in Fig. 1). possible to determine the APC function of these Similarly, specific antibodies produced in vitro were lines with autologous responder PBL. Results indidetected only in supematants of cultures stimulated cated that cell lines used as APC that had been with homologous antigen. There were no detectable antibodies from unstimulated controls or cultures stimulated with heterologous antigen.
Catfish APC Function Can Be Accomplished by Monocytes and T and B Cells pulsed with homologous antigen and subsequently paraformaldehyde-fixed induced proliferation and antibody production of autologous PBL in vitro (Fig.  3). However, the use of autologous cell lines pulsed with heterologous antigen stimulated neither proliferation nor antibody production by autologous responders (data not shown). Similarly, as in As previously reported (Vallejo et al., 1990b), previous experiments, prefixed and antigen-pulsed antigen processing and presentation by a variety of autologous cell-line APC were nonstimulatory. cells were important in the generation of secondary in vitro proliferative and antibody responses to Catfish Immune Responses Appear To Be Restricted hemocyanins. Such observations raised the question as to whether or not similar events were involved in In order to ascertain whether or not allogeneic cells responses to simpler protein antigens. By using two could be used as APC, experiments involving 17 different EqMb-primed fish as representative ani-(out of a possible 72) pairwise allogeneic combimals, results showed the EqMb-pulsed monocytes nations of 6 catfish monocyte lines (as APC) and and B cells (i.e., surface/membrane immunoglobulin-PBL (as responders) from 12 immunized (3 HEL-, 4 positive [sIg+] cells) each induced in vitro prolifera-EqMb-, and 5 pCytC-immune) fish were conducted. tion of autologous PBL comparable to cultures sti-In each case (as depicted in Fig. 4 for a representamulated with soluble EqMb plus unpulsed APC tive fish), the fixed allogeneic cell line elicited (Fig. 2). Moreover, positively selected B cells or vigorous in vitro proliferative responses, presumably negatively selected B cells treated with anticatfish Ig akin to mixed leukocyte reactions (MLR). These (monoclonal antibody [mAb] 9E1) prior to antigen responses were, however, not further increased by pulsing resulted in the vigorous proliferation of antigen pulsing of the APC. Furthermore, no detectresponding PBL. The magnitudes of such responses able specific antibodies were found in such allowere almost twice those generated with antigengeneic situations unless soluble homologous antigen presenting B cells that were negatively selected was added directly to the culture, that is, presuwithout incubation with anticatfish Ig. On the other mably as a consequence of the presence of autohand, positively or negatively selected sIgcells logous APC in the responding PBL. The APC function of long-term catfish monocyte lines was explored further to ascertain which responding lymphocyte population was induced to proliferate in vitro. Results showed both slg-(T-cellenriched) and B (sIg+) cells underwent proliferation in response to autologous antigen-pulsed cell-line APC (Fig. 5). Further, such in vitro responses were blocked by compounds such as chloroquine and leupeptin. However, sIg-cells did not proliferate as vigorously as B cells in response to soluble antigen (in the absence of APC) although these sIg-cell in vitro proliferative responses were significantly higher than those of unstimulated controls or sIgcells cocultured with unpulsed and fixed APC. Furthermore, variable (10-20%) amounts of cellassociated proteins were found to be degraded to smaller peptide fragments as assessed by SDS-PAGE and autoradiography (data not shown). On the other hand, prefixed cells exhibited negligible uptake of radiolabeled protein (< 1.5% or <7.5 protein/10 cells).

Requirement of Live APC During Antigen Pulsing
Is, Bypassed with the Presentation of "Preprocessed" Peptides by Prefixed APC The previous experiments showed that APC prefixed prior to antigen pulsing were not stimulatory to immune PBL. A study,was therefore conducted to determine whether or not fragments of a structurally defined protein, pCytC, could be presented by prefixed APC. Results showed that such was the case (Fig. 6, for the representative three fish studied in this regard). Incubation of prefixed cell-line APC w,ith pCytC peptide fragments corresponding to residues 1-80, 66-104, 66-.80, and 81-104 were found to be stimulatory to autologous PBL as observed when native pCytC-pulsed APC were used. However, the magnitude of the responses due to peptides 66-104, 66-80, and 81-104 were significantly higher than for peptide 1-80 or the native protein.
On the other hand, peptide 1-65 was clearly nonstimulatory.

DISCUSSION
This work embodies findings that elucidate our knowledge of antigen recognition relevant to teleost immunity. Firstly, results reported herein show the exquisite specificity of the secondary in vitro immune responses to channel catfish to well-characterized proteins such as pCytC, HEL, and EqMb. The use of such protein antigens in studies utilizing mammalian models allowed the determination of the fine specificities of the immunologic repertoire. Such studies have revealed that the levels of responsiveness to a particular antigen (i.e., high, low, or nonresponsiveness) are not simply due to holes in the repertoire, suppressor activities, or anergy (Cease et al., 1986;Berzofsky, 1988), but are largely influenced by factors intrinsic to the antigen itself and by cellular processing of the antigen. Apparently, the observed skewing of Tand B-cell responses is due to the limited number of epitopes thatcan be presented by MHC molecules (Kojima et al., 1988) and, hence, the level of responsiveness varies among the epitopes used. In other words, one epitope (the "immunodominant" one, hence the concept of epitope immunodominance) may elicit a greater magnitude of response compared to the rest of the epitopes (Shastri et al., 1985;Berzofsky, 1988). Although the applicability of this concept to teleost immunity remains to be demonstrated, the finding that pCytC-immune catfish PBL can proliferate in vitro in response to prefixed, pCytC peptide-pulsed autologous APC suggests that the same phenomenon might be occurring in fish (discussed further later). Significantly, the results also show that the polyclonal proliferative responses due to either peptides 66-80 or 81-104 are greater than the responses to peptide 1-80. Whether or not the same catfish lymphocyte clonotypes are resPonding to each of the peptides remains to be investigated. Secondly, this work demonstrates that the generation of catfish secondary in vitro immune responses involves steps akin to antigen processing and presentation in mammals, and that both monocytes and B cells .are effective APC similar to the situation in mammals (Gosselin et al., 1988;.Pierce et al., 1988;Schook and Tew, 1988;Vallejo et al., 1990aVallejo et al., , 1990b. The use of antigen-pulsed and paraformaldehydefixed APC elicited in vitro proliferation and antibody production by autologous immune PBL. Clearly, such responses must be attributed to antigen that became .cell-associated during pulsing and was presumably processed intracellularly. Furthermore, subsequent fixation of APC must have retained the antigen in a form that is recognizable by responding lymphocytes. In addition, the APC function of fish B cells is enhanced when they are isolated by direct panning using anticatfish Ig (mAb 9E1) or by nega-  (Cambier et al., 1987;Miller et al., in preparation). On the other hand, sIg-cells (T-cellenriched), either positively or negatively selected and incubated with anticatfish pan T/thrombocyte (mAb 13C10), are not efficient APC. Whether or not such treatment of catfish slg-cells results in the activation of these cells is not clear since the cell determinant recognized by the mAb (13C10) is not yet functionally defined (Miller et al., 1987). However, it should be noted that the in vitro proliferative responses of immune PBL to autologous sIg-cells used as APC are significantly higher than the unstimulated controls or PBL co-cultured with unpulsed/ fixed APC. It is most unlikely that such observations are due to contaminating B cells among the APC considering the high level of purity of the sIg-(Tcell-enriched) cell preparations (although it cannot be ruled out that sIg-cells other than T cells may be involved). Nonetheless, the original hypothesis that fish T cells also present antigen albeit less efficiently than either B cells or monocytes (Vallejo et al., 1990b), is more plausible and perhaps+ more appealing. Although MHC (or MHC-like) molecules have yet to be described for fish, previous studies showed that fish T cells can elicit weak MLR (Miller et al., 1986). If this MLR is attributable in part to MHC, and if MHC is involved in antigen presentation in catfish immune responses, ascribing APC function to fish T cells does not conflict with the irr.munologic paradigm of MHC-"processed" antigen interaction as a requisite for T-cell recognition (Berzofsky, 1988  This work also demonstrates the utility of longto amphibians (Fu et al., 1978;Crepaldi et al., 1986; term fish cell lines in studying accessory cell func-Flajnik et al., 1990;Kroemer et al., 1990) and thus tions. A previous study (Vallejo et al., 1990a) indi-the possibility that these T cells could have APC cated that these cell lines, which are morphologi-function cannot be ignored. cally similar to mammalian monocytes, have APC Studies employing long-term catfish monocyte function in addition to the ability to produce inter-lines as APC further demonstrated that immune leukin-1. In all assays conducted, antigen-pulsed restriction likely occurs in fish. Allogeneic mixtures and fixed long-term fish monocytes used as APC of cell-line APC and immune PBL yield only strong have consistently induced specific in vitro prolifera-MLR but not specific antibody responses in vitro tive and antibody responses by autologous PBL as unless soluble antigen is added to the cultures Such responders. Further, addition of compounds such as observations suggest two other important aspects of chloroquine and leupeptin during antigen pulsing teleost immunity, namely, (a) fish alloantigens are resulted in the reduction of the in vitro responses, unequivocally present, although their complexity These compounds are known to block antigen pro-and relationship to known vertebrate MHC cessing as well as interfere with Class II MHC molecules remains to be elucidated; and (b) the assembly by mammalian accessory cells and it generation of secondary in vitro antibody responses seems likely-that they interfere with antigen pro-depends both on the antigen and the APC used. As cessing by fish APC as well. Moreover, results show alluded to earlier, alloantigens (at least in mammals) that both catfish sIg-(T-cell-enriched) and B (sIg /) are responsible for MLR, and this in vitro event is cells underwent proliferation in response to autoattributable to MHC molecules (Lechler et al., 1990). logous cell-line APC. This latter observation indi-The precise mechanism wherein immune catfish PBL cates that in mixed cultures of PBL and monocyte-are not induced to produce specific antibodies in line APC, antibody production in vitro must be due vitro despite vigorous MLR due to allogeneic to T helper cells effecting B-cell differentiation as antigen-pulsed APC is not clear, at this time. Howwell as T and B-cell proliferation. Moreover, data ever, by analogy with mammalian systems, this obtained also show that fish B cells can respond to phenomenon (i.e., vigorous MLR but no specific soluble antigen similar to the situation in mammals, antibody production in vitro) may be attributable to Cross linking of Ig receptors on B cells by antigen at least one of several possibilities. Assuming that can result in the activation and proliferation of such MHC-like molecules are involved in antigen presenantigen-specific B cells (Cambier et al., 1987). tation in catfish immune responses, it is possible Further, since B cells also have accessory cell functhat such mixed cultures of cell-line APC and tions (Pierce et al., 1988), soluble antigen is preimmune PBL responders are completely allogeneic sumably being processed and subsequently interacts (i.e., absence of shared MHC-like determinants). In with idiotypic B cells, which then become activated this situation, allo-MHC might, not bind the proand, if T-cell-derived B-cell growth factors are cessed antigen in precisely the same manner as present, can undergo differentiation. On the other "self" MHC (i.e., constraint of MHC restriction) and hand, fish sIg-cells (T-cell-enriched) show low pro-thus T-inducer/effector cells would not be activated liferative responses to soluble antigen, albeit signifi-and able to help B cells. It is also possible that such cantly higher than the unstimulated controls. Concultures are semiallogeneicn (i.e., they have at least sistent with the previously mentioned notion that one shared MHC-like determinant). If this is the fish T cells may have the ability to process and case, the lack of specific antibody responses in vitro present antigen, such observations may not be parti-may be due to some form of inhibition that can only cularly surprising. T cells in culture may be pro-be speculated on at this time. cessing antigen and present fragments thereof to Finally, the present work suggests that the generidiotypic T cells. In mammals, it is generally ation of fish immune responses requires antigen accepted that unlike B cells, T lymphocytes do not processing events similar to the situation in mamusually directly interact with native protein antigens reals (Pernis et al., 1988;Schook and Tew, 1988). but rather with the processed antigen in the context Fish APC appear to take up and degrade antigen of "self" MHC (Buus et al., 1987). However, there and in so doing may expose epitopes that might be are several reports that unequivocally demonstrated normally buried in the native conformation of the the presence of MHC Class II molecules on T cells protein antigen. This notion is further supported by of various vertebrates ranging from humans to birds experiments involving the presentation of protein fragments by fixed APC, thereby circumventing the requirements for intracellular processing. Data obtained show that four pCytC peptides are as immunogenic as "naturally" processed pCytC to immune PBL. Further, the magnitude of responses to peptides 66-80 and 81-104 are similar to responses to peptide 66-104, but considerably greater than the responses to peptide 1-80. Peptide 1-65 however, appears inhibitory. These observations warrant three comments: (a) processing of pCytC is important in the generation of specific in vitro immune responses in channel catfish, (b) there are at least three immunogenic regions in pCytC recognized by catfish PBL, and (c) catfish polyclonal response to pCytC is seemingly focused to an immunodominant epitope lying within peptide 66-104. As previously discussed, epitope immunodominance can govern the degree of responsiveness to a given protein antigen in mammalian systems (Shastri et al., 1985;Berzofsky, 1988;Kojima et al., 1988) and it is most likely that the same is true for catfish. The finding that peptide 66-104 is immunogenic suggests that there might be a cluster of epitopes within this region and that peptides 66-80 and 81-104 represent two of them in addition to peptide 1-80. Parenthetically, in mice, it is peptide 81-104 that is considered immunodominant (Collawn et al., 1989). The inhibitory effect of peptide 1-65 for catfish responses is unexplained and whether or not heme, a large proportion of which co-elutes with this peptide, has a role can only be speculated. Clearly, studies are needed to determine the clonotypic specificities of catfish T and B cells with respect to pCytC as well as the minimal stimulatory ("coren) epitope within peptide 66-80 and/or 81-104.

Experimental Animals
The acquisition, laboratory maintenance, and immunization of channel catfish have been previously described (Faulman et al., 1983;Miller and Clem, 1984;Vallejo et al., 1990b).
Antigens and Inhibitors pCytC, HEL, EqMb, chloroquine, and leupeptin were obtained from Sigma (St. Louis, MO). Stock solutions were prepared at 10mg protein/ml in phosphate-buffered saline or at 1.0 M for chloroquine and leupeptin and stored at -20C. Peptide fragments of pCytC were also used as antigens. Peptides were derived by cyanogen bromide cleavage of pCytC according to the method of Corradin and Harbury (!970), purified by two cycles of gel filtration on a Sephadex G50 column (1.5 x150 cm; Pharmacia Fine Chem. Inc., Piscataway, NJ), dialyzed against distilled water, and concentrated by rotary evaporation.

Antigen-Presenting Cells
Freshly isolated monocytes, sIg- (T-cell-enriched) and B (sIg/) cells were used as APC. PBL were isolated from whole blood by centrifugation over Ficoll-Hypaque (Lymphoprep, Accurate Chemical Science Corp., Westbury, NY) as previously described (Miller and Clem, 1988). Isolation of monocytes by adherence to fibronectin-coated plates and panning protocols for the positive selection of catfish T and B cells were also carried out as previously described (Sizemore et al., 1984;Vallejo et al., 1990b). In addition, negatively selected T and B cells were obtained by reciprocal depletion of sIg / and sIg-cells, respectively, using magnetic beads. Monocytedepleted PBL (see what follows) were incubated with either mAb 9E1 (anticatfish Ig) or 13C10 (antipan T/thrombocyte) (Miller et al., 1987) for I h at 4C; centrifuged, and washed. The cell pellet was incubated with goat antimouse Ig conjugated to magnetic beads (Dynabeads M-450, Dynal Inc., Great Neck, NY) for an additional hour at 4C. The efficacy of this negative-selection protocol is illustrated by the finding that depletion of 13C10-reactive cells from PBL yielded B (slg/-cell preparations (i.e., unmagnetized cells) with >97% purity as revealed by routine immunofluorescence assay by flow cytometry. Moreover, mitogen assays showed that these cells, with or without additional monocytes, responded to lipopolysaccharide (LPS) but not to concanavalin A (Con A) as expected (Sizemore et al., 1984). Similarly, depletion of 9El-reactive cells from PBL also yielded negatively selected sIgcells with >97% purity (as assessed with mAb 13C10). Although these latter cell preparations were not exclusively T cells (i.e., =10% thrombocytes and <2% granulocytes and plasma cells), they are clearly T-cell-enriched. As expected, mitogen assays with these cells did not show proliferative responses to Con A in the absence of monocytes, nor did they respond to LPS with or without additional monocytes. These negatively selected cells were then immediately pulsed with antigen or incubated with FISH RESPONSES TO DEFINED ANTIGENS 147 mAbs 9E1 or 13C10 for I h at 27C prior to pulsing with antigen. Long-term catfish monocyte lines were also used as APC. These cell lines have been previously characterized and their APC function ascertained (Vallejo et al., 1990a).
Antigen Processing/Presentation Assay Protocols for antigen pulsing of APC were described previously (Vallejo et al., 1990b). In this study, however, APC were incubated with the native antigen (i.e., 500 g/107 cells/ml) for 5 to 8 h at 27C, the optimum time based on initial assays. Cells were subsequently washed and fixed with 0.5% paraformaldehyde for 15-30min at room temperature. Antigen-pulsed/fixed APC were extensively washed, allowed to incubate in fresh medium for I h at room temperature, and then given a final wash before coculturing with responder cells. Antigen pulsing in the presence of inhibitors at the indicated maximum nontoxic concentration was also carried out as described previously.
In assays involving the pCytC peptides, long-term monocyte lines were used as APC. Approximately 100/zg of the peptide were added to 10 paraformaldehyde-fixed cells and incubated at 4C with gentle agitation for 18 to 24h. The cells were washed and cultured with autologous responders.
Responders in all assays were either whole PBL or monocyte-depleted PBL, except where negatively selected slg-(T-cell-enriched) or B (slg/) cells were used as indicated. Depletion of blood monocytes was carried out by incubating PBL in a P-6 column (EconoPac 10DG, BioRad, Richmond, CA) for I h at 27C and then eluting the nonadherent cells (Vallejo et al., 1990b). Negative selection of T and B cells using mAb-magnetic beads was as described before.
Triplicate cultures of 106 cells were stimulated with soluble antigen at the indicated concentrations or witti antigen-pulsed/fixed autologous or allogeneic APC (approximately 5 x105). The culture system for catfish cells has been described elsewhere (Miller and Clem, 1988). Assays for 3H-thymidine uptake after 6 days and in vitro antibody response by enzyme-linked immunoabsorbent assay (ELISA) after day 8 in culture were carried out as previously described (Vallejo et al., 1990b).
Antigen Uptake pCytC, HEL, and EqMb were uniformly labeled by reductive methylation with 14C-formaldehyde and sodium cyanoborohydride (Sigma) (Dottavio-Martin and Ravel, 1978). 14CHB-protein with or without inhibitors was added to catfish monocyte lines and incubated at 27C. At various times, the cells were washed extensively with complete medium and lysed with 100 1 of a hypotonic buffer containing 50 mM Tris-HC1, pH 7.0; 10 mM NaC1; 1% NP40; I mg/ml aprotinin; I mM PMSF; 5 mM EDTA. The cell-associated radioactivity was determined by liquid scintillation spectrometry using a nontoluene. fluor (EcoScint A, National Diagnostics, Manville, NJ) and degradation was assessed by SDS-urea-PAGE (Burr and Burr, 1983).