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AMER. ZOOL., 15:93-106 (1975). Ontogeny, Phytogeny, and Cellular Cooperation LAURENS N. RUBEN Department of Biology, Reed College, Portland, Oregon 97202 SYNOPSIS. The capacity of adult newt (Triturus viridescens) spleen cells to secrete antibody at 4 C allows simultaneous visualization and quantification of non-secretory (S~) and secretory (S+) rosette forming cells (RFC). Visualization of mammalian S+ RFC requires 37 C, a temperature at which S~ RFC appear to be fragile. RFC can be distinguished as S~ or S+ due to whether one or more layers of erythrocytes adhere to the surface of sensitized spleen cells. Different doses of horse erythrocytes (HRBC) affect newt S" RFC and S+ RFC differentially. By varying the time between injections of different concentrations of chicken erythrocytes (CRBC, the "carrier") and a constant dosage of CRBC-TNP (trinitrophenyl, the hapten) it is possible to measure "helper" activity that correlates with the population of S" RFC and is both dose and time dependent. By varying assay time after "helper" activity has been maximized, one can determine the cytodynamics of anti-TNP antibody producing cell (APC) activity. For the first time these morphologically separable RFC can be related to their suspected physiologic behavior. A shift from S~ RFC to S+ RFC takes place during the immune response. That similar dose-dependent response curves can be shown in adultRana pipiens suggests that the newt responses represent a fundamental vertebrate pattern. 1968; Nossal et al., 1968; Claman and Chaperon, 1969; Rajewski et al., 1969; It should not be at all surprising to find Mitchisonetal., 1970). Excellent reviews on papers dealing with phylogenetic aspects in this subject are available (Roitt et al., 1969; a symposium on the development of im- Miller, 1970, 1972; Miller et al., 1971; munity. Phylogeny and ontogeny were in- Playfair, 1971). extricably entwined in our thinking even T cells are antigen-recognizing cells before the early part of the 19th century (ARC) with "helper" function; they rewhen von Baer first contributed his concluspond to antigen but do not produce antisions concerning embryos and their anceset al., 1967; Falkoff and body (Davies tors. This presentation summarizes some Kettman, 1972). B cells, aided by sensitized recent work relative to the evolution of celas precursors of plasma cells T cells, serve lular interaction in primary humoral imwhich produce specific antibody (Mitchell mune responses. Certain immunogens, et al., 1968). Antiand Miller, 1968; Nossal e.g., heterologous erythrocytes, require cellular cooperation, i.e., the interaction of at gens which are known to stimulate thymusleast two subpopulations of lymphocytes, in independent cells directly appear to be order to elicit an immune response in ro- highly polymerized (Miller, 1971; Wilson dents (Claman et al., 1966). One subpopu- and Feldmann, 1972) and are mitogenic for lation is thymus-dependent (T cells), the B cells (Coutinho and Moller, 1973). The other thymus-independent and generally mechanism of cooperation has been a subreferred to as B cells because of their deri- ject for considerable speculation (Bretscher vation from the bursa of Fabricius in birds and Cohn, 1970; Mitchison, 1971; Dutton (Warner et al., 1962; Cooper et al., 1966; et al., 197la,b; Feldmann and Nossal, Rouse and Warner, 1972) or from bone 1972). Members of all the modern vertebrate marrow in mammals (Miller and Mitchell, classes show the capacity to respond to heterologous erythrocytes (Abramoff and These studies were supported in part by a grant La Via, 1970). Thymus tissue has been de(GB-38480) from the National Science Foundation, scribed for all vertebrate classes except that Washington, D. C , U. S. A. Gratitude is offered Mss. Sheryl Swink and Judith Ruben for their technical of the most primitive Agnathan, the marine hagfish (Good et al., 1966; see also Riviere assistance. INTRODUCTION 93 94 LAURENS N. RUBEN et al., 1975; Linna et al., 1975), but little is known of thymus dependence in the antired cell response in species other than birds and mammals. Three reports are suggestive of thymus dependence in amphibia. DuPasquier (1970), Moticka et al. (1973), and Manning and Turner (personal communication) all suggest thymus involve- r ment in Alytes obstetricians, Rana catesbiana, and Xenopus laevis when sheep red blood cells (SRBC) are used as immunogen. THE ASSAY The quantitative assay is immunocytoadherence (Zaalberg, 1964; Biozzi et al., 1966). The animals are immunized in vivo by intraperitoneal injections with foreign red blood cells, e.g., sheep (SRBC), rat (RRBC), horse (HRBC), or chicken (CRBC). After a period of time, the organ to be tested, e.g., the spleen, is mechanically dissociated leaving a cellular suspension. Aliquots of these cells are then mixed with test or control red blood cells and incubated overnight in the cold (3 to 5 C). When specific receptor sites occur on the surfaces of sensitized spleen cells, erythrocytes will adhere and form "rosettes" (Fig. 1). The rosette forming cells (RFC) are counted visually in a hemacytometer, quantitated and expressed as RFC/106 spleen cells. For further details see Ruben et al. (1973). FIG. 1. An amphibian "rosette" showing adherent mammalian red cells bound to the sensitized spleen cell surface. When erythrocytes adhere in several layers of thickness, even if the entire spleen cell surface is not covered, the rosette is considered to represent an S+ or APC RFC (see the text), x 300. 8 days when anti-SRBC RFC activity was 100 times background. Background levels were restored by 16 days. The cytodynamics of this primary response in Xenopus were similar to those reported previously for Bufo marinus (Diener and Marchalonis, 1970) and Alytes obstetricans (DuPasquier, 1970), using the rosette assay. While adult responses are interesting, we especially wanted to know, instead, when this humoral response developed and whether any morphologic events were associated with its appearance. Analysis of larval stages of Xenopus traced the development of this response to 1 week later (at 25 C) than the time of allograft response initiation (Horton, 1969; Ruben et al., 1972). Allograft response initiation correTHE ANTI-RED CELL RESPONSE IN XENOPUS lated with thymus maturation, but the anti-red cell response began when lymphocyte differentiation could first by observed In vitro studies (Auerbach and Ruben, in the spleen. This was expected since 1970) with explants of young adult Xenopus laevis, the South African clawed toad, cell-mediated responses in amphibians insuggested that the liver and kidney failed to volve thymus-dependent cells (Cooper and produce strong hemagglutinin responses Hildemann, 1965; Curtis and Volpe, 1971; to SRBC; the spleen appeared to be the Horton and Manning, 1972; Tournefier, 1973; Cooper, 1974) as they do in mamprimary site. Since the spleen was engaged mals. Anti-red cell responses in amphibia, in at least this type of immunologic activity, however, might require at least two attention was concentrated on it during our initial immunocytoadherence studies (Kid- cooperating lymphoid cell populations der et al., 1973). The cellular response to from separate embryonic origins, hence SRBC in young adults began by 4 days after dependence on maturation of the primary challenge and the activity reached a peak at peripheral lymphoid organ, the spleen. CELLULAR COOPERATION IN AMPHIBIA THE ANTI-RED CELL RESPONSE IN THE NEWT At the beginning of our studies with the American common newt, Triturus viridescens, a urodele more primitive than anurans, no information was available on the organs that generate the humoral responses. Since liver, spleen, and kidney are involved in hematopoiesis we tried assaying all three (Ruben et al., 1973). All three organs showed a substantial increase in RFC activity after HRBC immunization, although the spleen (51 times background) was clearly more involved than the liver (24 times background) or kidney (19 times background). Specificity of rosette formation was demonstrated by testing with SRBC. Red blood cell binding to HRBC was not significantly altered when SRBC was used as the immunogen, (0.2 ml of 25% red cells in Alsevers solution) for the challenge dosage to study the cytodynamics of the newt's primary response. All three organs produced response curves that agreed with those for other amphibia using similar effective dosages. CELLULAR COOPERATION One method of demonstrating cellular cooperation in vivo involves the use of haptens, e.g. trinitrophenyl (TNP), (Rajewski et al., 1969; Mitchison et al., 1970). TNP can be conjugated to erythrocytes that serve as carrier (Rittenberg and Pratt, 1969; Kettman and Dutton, 1970) capable of initiating anti-hapten antibody synthesis. Preimmunization with the erythrocyte carrier will enhance the anti-hapten response. Since there is no addition of TNP with carrier preimmunization, the concentration of TNP available is not increased. Therefore, enhancement occurs as a consequence of cooperative activity of the "helper" cells sensitized to carrier in conjunction with APC. Thus, the degree of enhancement of the anti-hapten response serves as a measure of "helper" activity. The population which is carrier specific is thymusdependent in rodents (Kettman and Dutton, 1971; Falkoff and Kettman, 1972) but the antibody producing cells (APC) are B 95 cells (Mitchell and Miller, 1968). Antihapten responses are normally assayed in agar by showing hemolytic plaque forming cells (PFC) (Jerne and Nordin, 1963). Control experiments for this carrier-specific enhancement involve preimmunization with heterologous red blood cells different from the kind used as carrier and/or presentation of carrier-TNP without preimmunization. Information from the newt, combining immunocytoadherence and carrier-hapten immunization, clearly shows enhancement of anti-hapten response after preimmunization with the same type of erythrocyte used as carrier, but not with a different priming erythrocyte. CRBC and Bufo marinus (TRBC) erythrocytes were used in the four possible combinations of priming erythrocyte type and TNP-conjugate. HRBC and HRBC-TNP assays were performed in all cases. The HRBC assay provides a measure of cross reactivity between the erythrocyte injected in vivo and HRBC. When this value is subtracted from the number of RFC/106 counted in the HRBC-TNP assay, we have a measure of anti-hapten activity. The enhancement of anti-hapten activity occurs in the newt following carrier-specific preimmunization; thus, at least two interacting subpopulations of cells may be contributing to specific adaptive immunity in the newt. Newts are ideal for studying the phylogeny of cellular cooperation. Although they are primitive amphibians, they are the most advanced vertebrate with no bone marrow as a major hematopoietic site (Cowden and Dyer, 1971). Clearly, cellular cooperation appeared in evolution independent of the development of a bursa of Fabricius or an active bone marrow. However, without additional information, it is impossible to know if the newt's condition is a unique consequence of parallel evolution or whether it represents a fundamental vertebrate pattern. No information is available on the nature of the B cell equivalent in the newt nor is it known whether the "helper" cells of the newt are thymus dependent. The three studies cited earlier suggest thymus dependence in the amphibian anti-SRBC responses. 96 LAURENS N. RUBEN THE EFFECT OF IMMUNOGEN DOSAGE The intial report on the newt response also presented some preliminary information that the intensity of the antierythrocyte response is inversely proportional to the concentration of the immunogen (HRBC). While this might be one interpretation of the result, another possibility is that different challenge dosages might initiate antigen-binding responses with differing rate curves. Thus, whether primary response intensity is directly or indirectly proportional to immunogen dose would depend on when the assays are performed. The issue has now been explored in depth and we can distinguish between these two alternatives (Fig. 2; Table 1). The time course curves of primary responses are clearly translocated when differing challenge dosages are used. These data and all that follow will be considered in greater detail elsewhere (Ruben, 1975). The lower the immunogen dose, the more rapidly is the response peak achieved. The peak RFC levels, however, nearly increase in a linear fashion with each higher dosage used. It is clear that when the assays are done at 2 days, rather than the 4-day intervals used in our original report on the newt, a new "shoulder" (or period of decreasing rate of increase in numbers of antigen binding cells within the spleen) appears between 2 and 4 days after challenge with the two higher dosages of HRBC. Studies of mammalian systems demonstrate that high immunogen doses obviate the necessity for cellular cooperation (Sinclair and Elliott, 1968; Claman and Chaperon, 1969; Taylor, 1971). These results are in agreement with those of Mitchison (1964, 1971) who suggested that T cells are triggered by lower doses of antigen than are B cells; "helper" activity might be viewed as crucial to focusing relatively low doses of antigen onto the B cells. A variety of experiments revealed that low immunogen doses yield proportionally greater stimulation of "helper" and memory cells than APC (Salvin and Smith, 1964; McDevitt et al., 1966; Hanna et al., 1967; Greaves et al., 1970; Greaves and Moller, 1970; Kettman and Dutton, 1971; Playfair, 1971; Falkoff and Kettman, 1972). Higher doses of antigen stimulate increasing amounts of antibody which are generated in shorter periods of time (Sterzl and Trnka, 1957; Winebright and Fitch, 1962; IMMUNOGEN (HRBC) DOSE 25% 0.25% 0.0025% •— PRIMARY RESPONSE T. viridescens SPLEEN ^6000 y 5000 z 54000 3000 • DAYS FIG. 2. The effect of immunogen (HRBC) dose on the primary immune response in the spleen of the 10 adult newt as assayed by immunocytoadherence. 5521 ± 11 4459 ± 131 3706 ± 759 2533 ± 161 1972 ± 234 1031 ± 318 2 4 6 8 12 16 HRBC 0 718 ± 45 313 ± 363 576 ± 285 749 ± 330 1784 ± 171 1396 ± 285 496 ± 343 1103 ± 557 95 ± 11 328 ± 242 S+ 3211 ± 1103 3367 ± 440 5426 ± 550 ± 314 S- b 0.0025% 4 1235 ± 26 2172 ± 50 5622 ± 335 7173 ± 94 3337 ± 2957 ± 318 877 ±533 Total RFC/106 19 3686 ± 226 1668 ± 258 375 ± 284 860 ± 257 9151 ± 840 2810 ± 138 1990 ± 35 1652 ±311 877 ± 553 Total RFC/106 513 ± 509 1321 ± 1131 825 ± 561 943 ± 70 873 ± 328 ± 242 S+ 1659 ± 111 4301 ± 796 6349 ± 466 2389 ± 82 2284 ± 55 550 ± 314 S— 0.25% HRBC •13 o 1303 ±431 •V 2 599 ± 60 3087 ± 527 361 ± 165 z 3512 ± 103 5640 ± 943 2438 ± 173 O 472 ± 22 42 ± 59 328 ± 242 S+ 372 ± 35 1518 ± 35 1610 ± 371 550 ± 314 S- 25% HRBC RAT a An average of 16 hemacytonieter chamber counts from 4 incubations involving 2 different cellular pools, each of which is comprised of spleen cells from 4 newts, is used for all data presented in this table. b S— (non-secretor) refers to RFC with adherent cells attached only to the spleen cell itself, while S+ (secretor) refers to RFC where additional erythrocyte layers have attached to sensitized cells. 877 ± 553 Total RFC/10 6a 0 (Background) Day TABLE 1. The effect of immunogen dosage (% HRBC) on the primary immune response of the newt (spleen). CELL ULAR Co HIBI 98 LAURENS N. RUBEN Uhr and Finkelstein, 1963; Svehag and Mandel, 1964; Campbell and Kind, 1969). It appears, then, that the generation of "helper" cells is enhanced over APC by low immunogen doses, while the reverse is true when higher dosages are used. Most studies using mammalian systems utilize assays for antibody production and this alone can account for the conclusion that higher concentrations of immunogen stimulate earlier and stronger immune responses. Immunocytoadherence, however, allows the visualization of ARC activity ("helper") as well as APC; it measures the total number of cells capable of specific antigen binding (Greaves and Moller, 1970; Greaves et al., 1970; Bach and Dardenne, 1972; Haskilletal., 1972). The present data fostered the reverse conclusion of that reported for rodents, at least with regard to the rate of the response. Therefore, the beginning assumption is that the earlier shift in time courses with decreasing immunogen dose is a consequence of introducing information about "helper" or ARC activity. This assumption, in turn, leads to the speculation that antigen-binding cell activity, measured between days 0 and 2, probably involve ARC mitogenesis or recruitment. ARC mitogenesis has been demonstrated (Paul et al., 1968; Marchalonis, 1971; Segal et al., 1971; Nakamura et al., 1972) and recruitment or "homing" of ARC into the spleen has been suggested (Sprent et al., 1971). As in the mammalian system, one might expect this activity to be favored by lower immunogen doses in the newt. The reduction in the rate of increase in the number of antigen binding cells from the spleen between days 2 and 4, may reflect the period of "helper" activity, when information transfer occurs so that APC may be triggered. Once finished, these ARC cells may leave the spleen to serve as "memory" cells in the circulation. Finally, the steep increase in cell numbers between days 4 and 8 may represent APC mitogenesis. Neither the "shoulder" nor the rapid increase in APC is detectable when the lowest dose is used. It would appear that either ARC remove this small amount of immunogen effectively enough without the involvement of an additional substantial cellular population, or that the disappearance of ARC from the spleen is partially matched by APC generation, so as to provide for a gradual replacement of ARC by APC. Haskill and Axelrad (1971) have visualized this type of time-dependent progressive shift during the immune response in the rodent. "HELPER" AND ANTIBODY PRODUCING CELL ROSETTES Greaves et al. (1970) have categorized RFC as to whether or not they appear to be secreting antibody. Secretory (S+) or APC RFC are sensitized spleen cells which bind on to their surfaces more than one layer of adherent red blood cells. Non-secretory (S~) or ARC RFC bind only one layer of red blood cells on to the surface. Others, e.g., Haskill et al. (1972), have classified gluteraldehyde-fixed RFC according to the number of adherent red blood cells; maximally binding cells have more than one adherent layer and minimally binding cells are specifically affected by adult thymectomy and anti-theta serum. Maximally binding RFC can be sorted out in gradients along with PFC (McConnell, 1971), therefore at least some RFC are clearly related to APC. The earlier demonstrations that some RFC in rodents are T cells rested on less firm ground, usually dependent on the use of anti-theta serum and complement which suppresses the number of antigen binding cells (Greaves and Moller, 1970; Greaves and Hogg, 1971; Raff, 1971). However, Greaves and Raff (1971) and Takashashi et al. (1971) found that some anti-theta preparations are contaminated with antibodies directed against surface receptor sites other than theta. At least one site is present on thymus-independent (B) cells. However, in support of the view that at least some RFC are thymus dependent cells in rodents, Bach and Dardenne (1972), using immunocytoadherence, reported antigenbinding cells from a hydrocortisone resistant pool of thymocytes. Furthermore, about 75% of sensitized spleen cells binding SRBC are theta positive and sensitive to azothioprine. Modabber et al. (1970) found specific antigen binding cells in mouse 99 CELLULAR COOPERATION IN AMPHIBIA thymus which could be inhibited by cross reactive materials. Recently, rosette formation by human lymphocytes has been inhibited by anti-T cell serum (Words et al., 1973). The situation in the chicken seems somewhat clearer with regard to RFC origins, since bursectomy of chicken embyros (Hemmingsson and Aim, 1972) suppresses both RFC and PFC formation (cf. Crone et al., 1972). Rodent RFC development is an active secretory process at 37 C, while at 4 C RFC formation seems related only to the presence of antibody pre-existent on the spleen cell surface. No secretory activity can be discerned (Elson et al., 1972). By adopting the S~, S+ RFC classification of Greaves etal. (1970), I have attempted to visualize the events previously speculated about from the dose response curves. Figure 3 shows the time course curves after the number of S" (ARC) RFC have been sorted out from S+ (APC) RFC. The data appear in Table 1. The newt rosettes were not fixed and the minimal number of adherent red blood cells counted as a S~ rosette is three. S+ RFC must have several layers of bound erythrocytes, but these need not adhere to the entire spleen cell surface. The rosette illustrated in Figure 1 is typical of many S+ RFC and the distribution of adherent erythrocytes suggests localization of determinants on the sensitized cell surface. Amphibian spleen cells are four times larger than the mammalian cells and are therefore useful for visualizing these local areas of attachment. Amphibian rosettes are also relatively stable when compared to mammalian RFC. The S~ (ARC) RFC response curves correspond closely to those involving total RFC/106 counts except for two important differences. According to the first, the peak number of ARC of the 25% HRBC dosage curve is 1000 RFC/106 lower than that stimulated by the next lower dose (0.25% HRBC). This agrees with information already considered from rodent studies; higher doses may affect APC appearance without as much ARC or "helper" activity. That the 25% HRBC challenge was the only one which initiated a relatively high number of S+ (APC) RFC by 8 days is also in IMMUNOGEN (HRBC) DOSE 25X — — O.25X - - - - 0.0025* PRIMARY RESPONSE T. viridescens o SPLEEN U S 2000 0 T7 4ooo 6 DAYS FIG. 3. The response curves for ARC (S") RFC and APC (S+) RFC following challenge by three different immunogen (HRBC) doses in the spleen of the newt, T. viridescens. agreement with this thesis. Another difference is the flattening of the "shoulder" between 2 and 4 days with the two higher dose challenges. This feature of the results offers some support to the earlier speculation that "helper" activity may be accompanied by a loss of ARC (S~, RFC) from the spleen. The increase in total number of RFC after 4 days can clearly be accounted for, at least in part, by an increase in S+ (APC) RFCInformation about both S~ and S+ RFC seems less reliable than the data on total RFC. The standard deviations, even between assays using the same pools of cells are large. This unreliability suggests an instability of outer layer adherent red blood cells such that even minor differences in shearing forces generated during resuspension, by gentle rotation of the incubation mixtures, may be sufficient to loosen them. RBC adherent on the surface of a spleen cell would appear to be more firmly held. Unlike the mammalian situation where at 37 C the ARC is particularly fragile and the APC is visualized (Haskill et al., 1972), the 100 LAURENS N. RUBEN amphibian cells, when incubated at 4 C, show stability of ARC but some instability of APC. That some antibody secretion can occur at 4 C from poikilotherm spleen cells is an important advantage, making it possible to visualize both ARC and APC at the same incubation temperature. This distinction can be enhanced by warming the mixture of sensitized newt spleen cells with appropriate erythrocytes for 2 hr prior to incubation in the cold. These data clarify the RFC designations suggested above (Table 2). There is no alteration in the proportion of S~ to S + RFC at 2 days, but the percentage of S+ RFC does increase at 8 days. This is to be expected if antigen binding cells generated early in the response are ARC, while those appearing later are APC. It is interesting that the proportion of S" and S+ antiHRBC RFC observed under these conditions (pre-warming) agrees with the proportion of anti-SRBC Tand B RFC reported by Bach and Dardenne (1972) for the rodent. "HELPER" ACTIVITY: A FUNCTION OF S" RFC? It is essential to demonstrate that the morphological criteria regarding whether RFC are nonsecretory or secretory are real. In other words, can one relate these two types of RFC (S~ and S+) to their suspected physiological behaviours, "helper" activity and antibody production? While combining immunocytoadherence with carrierhapten immunization, it is possible to vary by 2-day intervals the time between preimmunization and challenge by TNPconjugate. This variation provides a time course response curve of the degree of "helper" activity (i.e., anti-TNP RFC) as a consequence of preimmunization. All assays are performed 8 days after presentation of the hapten. Variation in the concentration of the priming dose can provide further information concerning the effect of immunogen dosage on "helper" activity. The data appear in Table 3. The red cell used for preimmunization is CRBC at low (0.0025%) and high (25%) concentrations. As before, the hapten-conjugate is 10% CRBC-TNP in all cases and the assays are against 1% HRBC and 1% HRBC-TNP. The volume of erythrocytes injected ip into the newts is always 0.2 ml. Control experiments use SRBC as the primer red cell and presentation of CRBC-TNP without any preimmunization. No anti-TNP RFC are formed in the absence of CRBC preimmunization. The response curves showing dose and time dependence appear in Fig- Incubations of both immunized and unimmunized newt spleen cells with high hemagglutinin titer (1:512) newt antiHRBC sera and sera taken at the times of peak anti-HRBC cellular responses (Kapp and Benacerraf, 1972) failed to alter either the number of RFC or the proportions of S~ and S+ RFC. It would appear, then, that cytophilic antibody is not generating false RFC or converting S" to S + RFC. I ABI.K 2. Effect ofpre-rvarming'on newt spleen cell multiple layer (S+RFC) antigen-binding of HRBC: (primary response). HRBC dosage 0.0025% Cold %S- b 25% %S + % S- % S+ % incr. S+ % S- 9£ S + %S- %S + % incr. S+ 9 25 90 57 10 33 1 8 91 79 9 21 89 52 11 38 2 19 Assay day 2 8 91 C 75 Warm Cold Warm a Pre-warming entails placing cellular mixtures at 25 C for 2 hr prior to the usual overnight incubation in the cold. b % S - refers to the % non-secretory RFC/10 6 spleen cells, % S+ refers to the % multilayered or secretory RFC/10 6 spleen cells. c These figures are averages of two different experimental series. They represent 16 chamber counts as described for Table 1. 101 CELLULAR COOPERATION IN AMPHIBIA TABLE 3. Measurement of "helper" activity in newt spleen cells by varying the time between challenges of carrier (CRBC) and carrier-hapten (CRBC-TNP) and carrier dose (CRBC-TNP = 10% in all cases). Carrier Carrier dose CRBC 0.0025% CRBC 25% SRBC SRBC 0.0025% b 25% — Days between injections Anti-TNPa RFC/10" ("helper" activity) % Increase in S + RFC 2 7113 ± 613 6642 ± 1775 2551 ± 424 246 ± 346 1636+ 114 1791 ± 1215 2404 ± 620 0±0 0±0 0±0 0±0 79 85 95 100 66 69 100 — 4 6 8 2 4 6 8 2-8 2-8 — a Anti-TNP RFC/106 are determined by subtracting the RFC/106 assayed with HRBC from the number assayed with HRBC-TNP. All assays were done 8 days after CRBC-TNP injection. b Control with 10% CRBC-TNP injected at day 0 and assayed 8 days later. ure 4, and the data show S , S+, and total RFC/106. The results clearly support the previous conclusions that low concentrations of immunogen which generate high numbers of S~ RFC are particularly effective in stimulating "helper" activity, i.e. enhancement of anti-TNP RFC. This enhancement provided by low but not high dose priming correlates with the excess of S~ RFC generated in response to low but not high dosage challenge. Furthermore, most of the enhancement in the number of anti-TNP RFC can be accounted for by S+ RFC, expected if anti-TNP activity is a function of APC and not of ARC which should respond to preimmunization by carrier erythrocytes. "Helper" activity is maximized in the 2- to 4-day period following preimmunization. ANTIBODY PRODUCTION: A FUNCTION OF S+ RFC "Helper" activity can now be maximized by using low dose (0.0025% CRBC) priming followed by CRBC-TNP 4 days later. If the times of assay are varied by 2-day intervals thereafter, information can be derived concerning the kinetics of specific (antiTNP) antibody production by RFC. Figure 5 clearly demonstrates that anti-TNP RFC activity appears first after 6 days and quickly rises to a peak at 8 days after TNP presentation. This activity disappears from the newt spleen just as quickly, i.e., 2 days later. This result is confirmatory of peak S+ RFC formation at 8 days in primary responses. Further, it supports the notion that multilayered rosettes are APC, since nearly all of the anti-TNP activity can be accounted for by increase in this type of rosette (S+ RFC). These results employing a combination of poikilotherm spleen cells, imTHE KINETICS OF HELPER ACTIVITY T. viridescens SPLEEN Priming Dose - Day 0 9000 25% CRBC 0.0025% CRBC SOOO | CRBC- TNP = 10% 1. 7000 Assays 8 days post-TNP injection 1 ***^ I\ -> 6 0 0 0 \ \ £ 5000 Z 4000 \ \ \\\ \ \\ < 3000 V ^ T LU ' 2000 ^ 1 \ T \ \ \ \ 1000 0 1 2 3 4 5 6 7 1 DAYS BETWEEN PRIMING AND TNP PRESENTATION FIG. 4. Anti-TNP RFC enhancement, i.e., "helper" activity is both dose and time dependent in the newt spleen. 102 LAURENS N. RUBEN THE KINETICS OF ANTIBODY PRODUCING CELLS (APC) IN THE NEWT SPLEEN 0.0025X CRBC - 0 DAY 10*CRBC-TNP-DAY4 4 6 • 10 12 14 16 ASSAY DAYS AFTER TNP PRESENTATION FIG. 5. The kinetics of anti-TNP RFC activity in the spleen of the newt. munocytoadherence and carrier-hapten immunization then, suggest that both "helper" and antibody producing cell activities are being visualized when one observes the rosette population shift from S~ to S+ RFC as the immune response progresses. A remote possibility that ARC and "helper" cells may be separate populations remains, since "helper" cells could be stimulated proportionately but may not, like ARC, bind antigen. THE ANTI-RED CELL RESPONSE IN RANA PIPIENS Adult Rana pipiens (the leopard frog) are members of the most advanced order (Anura) of Amphibia (Noble, 1931). Recently, Levin (personal communication) tested the effect of different dosages of SRBC (by using immunocytoadherence) on the antired cell response of spleen and thymus from adult Rana pipiens. The volume of the challenge dose was increased to 0.5 ml injected ip, since frogs are substantially larger than newts. The SRBC concentrations were equivalent to those used for HRBC in the newt studies (0.0025%, 0.25%, and 25% SRBC). All the other experimental conditions were identical except for the dissociation medium for frog spleen and thymus cells. The medium used for newt cell maintenance is 7 parts L-15 (LeibowitzGIBCO):2 parts twice glass distilled water; the medium for frog cells is 5 parts L-15 and 4 parts water (Balls and Ruben, 1966); in the past Kidder et al. (1973) used one part decomplemented fetal calf serum. The data appear in Tables 4 and 5 and in Figures 5, 6, and 7. The three dose response curves for frog spleen cells are clearly similar to the newt's. The same time translocations appear with low dose priming and generate the most rapid cellular response. Each more concentrated dose stimulates a higher activity level at the time of peak response. The standard deviations, however, of the frog data represent variations among individuals since each adult frog spleen is sufficiently large to assay separately. Newt spleens, on the other hand, must be pooled (four) and therefore standard deviations illustrate variations between different cellular pools taken from different groups of animals. An additional advantage in using the frogs is that their paired thymus is, like the spleen, large and can be pooled for immunocytoadherence assays. Information derived from thymus cells is particularly useful because all thymus antigen binding TABLE 4. Primary immune response in Rana pipiens adults (total RFC/106 viable spleen cells counted). Assay day 2 4 6 8 10 12 16 H R B C dose 0.0025% 0.25% a 3875 ± 619 1552 ± 171 * 1140 ± 481 * 1011 ± 112 615 ± 58 1790 ± 1386 ± 4874 ± 2727 ± 275 455 662 822 * 1925 ± 682 944 ±414 25% 1035 ± 265 1022 it 3246 dt 7550 dt 3246 it 2546 dt 1420 ± 282 156 1737 716 265 187 ' Since individual spleens were assayed, standard deviations refer to variation among individuals (4). No assays were performed on these days. ! 103 CELLULAR COOPERATION IN AMPHIBIA TABLE 5. Primary immune response in Rana pipiens adults (total RFC/10e viable thymus cells counted). Assay day 2 4 6 8 10 12 16 0.0025% 0.25% 25% 2615 ± 624" 1839 ± 104 2493 ± 121 2407 ± 786 2344 ± 122 1905 ± 449 3016 ± 1092 3120 ± 1088 1508b 1348 ± 476 1250" 1158 ± 330 775 ± 268 896b * * 337" 296" 1425 ± 229 518 ± 8 a Since thymii from 4 individuals were pooled for each assay, standard deviations refer to differences between 2 different cellular pools. b These figures are from only one cellular pool of thymocytes. * No assay was performed on this day. cells are S RFC regardless of challenge dose and time. This is supportive of the likelihood that splenic S" RFC or "helper" RFC are thymus-dependent cells in the Amphibia, as they are in mammals. In fact, the embryonic thymus of Rana pipiens may produce most adult lymphocytes (Turpen et al., 1973). Larval thymectomy ofTriturus alpestris appears to suppress RFC formation (Tournefier, personal communication). CONCLUSION There is similarity in the dose-dependent IMMUNOGEN (SRBC) DOSE 25* 0.25S response patterns between newts and frogs. Furthermore, there is agreement in the cytodynamics of the primary antierythrocyte responses among Triturus viridescens (the newt); Alytes obstetricans, Xenopus laevis (both primitive Anura), Bufo marinus, and Rana pipiens (both advanced Anura). This suggests that visualizations of the primary immune response and cellular cooperation are more likely to be patterns fundamental to the vertebrate group than a phenomenon which evolved uniquely within the newts. These common features support the view that ancestral amphibians may have possessed these characteristics which evolved in all modern amphibian groups. These may also have evolved in ancestral reptiles which eventually gave rise to modern reptiles, birds, and mammals. PRIMARY RESPONSE Rana pipiens SPtEEN IMMUNOGEN (SRBC) DOSE 25* 0.25% 0.0025% PRIMARY RESPONSE Rono pipiens THYMUS DAYS FIG. 6. The effect of immunogen (SRBC) dose on the primary immune response in the spleen of the adult frog Rana pipiens. > DAYS 12 14 16 FIG. 7. 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