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Mechanisms of antibody formation
CELLULAR
IMMUNOLOGY
15,
336-346
(1975)
Mechanisms
of Antibody
Formation
I. Early in Vivo Proliferation of Mouse Spleen T Cells Step in Antibody Formation L*
R.
BACHVAROFF
Department of Surgery New York Ulziversity
AXD
F. T.
RAPAPORT
altd the Institute of Reconstructive Medical Ceater, New York, Received
November
as an Initial
New
Plastic York
Surgery,
10016
8.1973
Spleen cultures prepared from mice injected 24 hr earlier with 2 X lo’-2 X 10’ sRBC and challenged in zitro with sRBC produced 10 times more anti-sRBC IgM PFC than cultures prepared from uninjected mice. The effect was specific for the particular species of foreign RBC injected in vivo. In vitro responses to TNP were also increased in spleen cultures prepared from animals injected 24 or 12 hr earlier with carrier RBC alone, directly implicating carrier-specific T cells in this process. Similar enhancements of PFC formation occurred in cultures prepared from mice which had been injected with sRBC 24 and 48 hr earlier, but which were exposed to lethal irradiation at 1 hr after injection of antigen, if their spleens were shielded extracorfioreally during irradiation. This finding indicated that in vivo recruitment of antigen-reactive extrasplenic X-ray-sensitive cells from the circulating lymphocyte pool by the spleen could not account for the observed enhancement. Proliferation in the spleen of antigen-reactive T cells, commencing 12-20 hr after the administration of antigen, was demonstrated by the tritiated thymidine pulse technique. An 8-hr hot-pulse given to spleen cell cultures from normal animals at 20 hr after in vitro challenge with antigen did not affect the rate of generation of IgMproducing cells; however, administration of a similar pulse to cultures which were initiated at 12 or at 20 hr after the in viva injection of sRBC eliminated the enhanced generation of PFC and delayed the in vitro response to sRBC by 24 hr. Spleen cell cultures were prepared from mice which had been injected in vivo with sRBC at 12, 20, and 70 hr earlier, and 8- to lo-hr hot pulses were given immediately after initiation of the cultures. The cultures were then challenged with sRBC-TNP; antibody responses to TNP were greatly reduced in hot-pulsed cultures prepared from mice injected in zivo with carrier RBC at 12 or 20 hr prior to initiation of the cultures. In contrast, antibody responses to TNP observed in hot-pulsed cultures prepared from mice which had been injected with carrier RBC at 70 hr prior to initiation of the cultures were generally similar to those of nonpulsed 70 hr control cultures. This result suggests that the onset of T helper cell proliferation be,gins within 12-20 hr after injection of antigen, but subsides irt viva within 70 hr. By that time, the antigen-reactive T cells have already differentiated to perform their helper function. In spite of the triggering of T-cell proliferation during the first 24 hr after injection of antigen, spleen cell cultures prepared from mice which had been injected 24 hr 1 Presented in part at the 1973 Meeting of the American Association of Immunologists. 2 Supported by a Grant from The John A. Hartford Foundation, Inc.; supported in part by The Billy Rose Foundation, Inc., The Irwin Strasburger Memorial Medical Foundation, Inc., Contract NONR-4503. Office of Naval Research, Washington, DC and Grant GM-12748-10. 336 Copyright All rights
0
19i5 by Academic Press, Inc. of reproduction in any form reserved.
T-(‘ELL
PROLIFERATION
AND
ANTIBODY
FORMATION
337
earlier in viva with 2 X 10’ sRBC produced only minimal numbers of anti-sRBC PFC if no antigen was added to the cultures. The presence of unprocessed antigen thus appears to be a requirement for B-cell proliferation ~II ztit~u, even after T-cell division has been triggered. This finding is consistent with earlier suggestions that the function of “helper” T cells may not be limited to passive transport of antigenic determinants to B cells. Evidence is also presented to support the contention that the antigen-reactive T cell involved in this process may have to unrlcrgo cell division in order to tlcvelop “helj~er” capacity.
INTRODUCTION Earlier in z&o studies by Sercarz and Byers (1) have demonstrated the ability of mice to develop IgM “memory” with 24 hr after the injection of foreign erythrocytes. Mishell and Dutton (2) have reported enhanced in vitro antibody responses to sRBC in spleen cell cultures prepared from mice injected in viva with sRBC several days earlier. In addition, Kettman and Dutton (3) have observed that the in vitro anti-hapten antibody response of mouse spleen cells was greatly augmented if spleen cell cultures were obtained from mice primed in viva several days earlier by injections of carrier RBC. The latter result appeared to directly implicate enrichment and/or differentiation of carrier-specific helper T cells in the mouse spleen after administration of foreign erythrocytes (4). A recruitment of antigen-reactive cells from the recirculating lymphocyte pool by secondary lymphoid organs has frequently been suggested as an explanation for sucl~ enhanced in vitro responses after injection of antigen in viva (5-i’). The results of the present study demonstrate that recruitment of antigen-reactive cells from the recirculating lymphocyte pool by the mouse spleen does not occur during the first two days after intravenous administration of 106-lo8 foreign erythocytes. Instead, evidence is presented of an early division and differentiation of helper T cells in the mouse spleen as the initial step for proliferation of IgM hemolysin-forming B cells, in the antibody response to T cell-dependent antigens, such as foreign RBC. MATERIALS
AND
METHODS
Animals. Three- to six-month-old C5,Bl, mice were used throughout the experiments ; the animals were maintained on a standard Purina pellet diet. Antigens. Sheep (s) and horse (11) red blood cells (RBC) (Animal Blood Center, Syracuse, NY) were washed three times in balanced salt solution (BSS) (2) and were used as antigens. The washed cell suspensions were adjusted to the desired concentration in standard fashion (8). Erythocytes were lightly and heavily substituted with TNP by the procedures of Rittenberg and Pratt (9) and Kettman and Dutton (3)) respectively. Tissue cultures. Spleen cell cultures were initiated and maintained as described by Mishell and Dutton (a), except that 1O-5 lb1 2-mercaptoethanol (10) was added to the culture medium. Usually, 1.5-2 x lo7 spleen cells per culture were challenged with 4 x lo6 unmodified RBC or with fresh RBC heavily substituted with TNP. The generation of IgM-producing cells in each culture was assayed by enumerating the direct PFC, utilizing the plaque technique of Jerne. Nordin, and Henry (ll), as modified by Mishell and Dutton (2). 15:here anti-TNP antibody responses were measured, lightly substituted RBC-TNP were used for plaquing. Guinea pig serum absorbed three times with RBC was used as the source of com-
338
BACHVAR~FFANDRAPAPORT
plement in the plaguing technique. The number of E’FC counted each day ~vhich is listed in the reported results represents average comnts observed from three simultaneously prepared culture dishes, plaqued in triplicate. Basic e.rperimentnl design. An attempt was made to compare the rate of in viva generation of antibody-forming cells (AFC) in spleen cell cultures obtained from normal untreated mice with that observed in mice which had been previously injected with antigen in viva. Spleen cell cultures prepared from both groups of animals were exposed to the same concentration of foreign erythrocytes in vitro. For in viva injection, 2 X 104-2 X 1Os foreign erythrocytes were injected into the tail vein ; at different time intervals thereafter (1, 6, 12, 24 hr, etc.), spleen cell cultures were initiated and challenged with KBC. The PFC were measured from the second to the sixth day following addition of antigen in vitro. In each case, “0 time” refers to the time of first contact of host cells with specific antigen, whether given in viva or in vitro, Irradiation experiments. Mice were injected intravenously with 2 X 1Os sRBC. One hour later, one group of animals was anesthetized, and a laparatomy was performed under aseptic conditions. The spleens were extruded from the abdomen; they were wrapped in moist saline sponges and inserted into a radiation protection lead housing (0.5-cm thickness). The animals were then exposed to 1000 R of lethal total body irradiation (30 kV, 10 mA, HVL 3.0 mm Cu; distance of focus to target = 50 cm; dose rate = 60 rads/min) Immediately after irradiation, the spleens were returned into the peritoneal cavity and the abdomen was sutured. Twenty-four and 48 hr later, spleen cultures were initiated in the presence of 4 X IO6 sRBC. Control groups included (i) lethally irradiated nonoperated mice and (ii) sham-operated nonirradiated mice. Hot-pulse techniqzie. Assessments of cell proliferation were performed by the method of Dutton and Mishell (12). In the first set of experiments, cultures from normal mice were initiated in the presence sRBC. Twenty hours later, the cells were exposed to a 7-hr pulse of 10 ,&i of thymidine-methyl-3H-( 15-20 Ci/mmole) (Schwartz/Mann, Orangeburg, NY). The dishes were then scrubbed with “rubber policemen” and the cells were collected and washed in BSS. The cells were resuspended in fresh medium containing 50 pg of cold thymidine (Schwartz/Mann, Orangeburg, NY). The nonpulsed controls for normal animals, also cultured in the presence of sRBC, were treated and recultured in the same manner. The antibody response in such control cultures was compared with that observed in hot-pulsed cultures prepared from mice which had been injected with antigen prior to initiation of culture. The latter animals were injected with 2 X 10’ sRBC, 20 hr prior to initiation of spleen cultures in the presence of sRBC. A 7-hr hot thymidine pulse was administered immediately to one group of culture dishes, while control dishes were not hot-pulsed. The cells were then collected and recultured in fresh medium with cold thymidine. In another set of experiments, mice were first injected in viva with 2 X 10’ sRBC. Twelve, 20, and 70 hr later, spleen cell suspensions were prepared and were hot-pulsed for 8-10 hr. The cells were then washed and recultured in medium supplemented with cold thymidine. Heavily substituted sRBC-TNP (4 X lo6 cells) were added to the cultures, and responses to sRBC, hRBC, and hRBC-TNP were measured; concurrent control cell suspensions received no hot thymidine.
T-CELL
NUMI~EK
Hours of iv injection of 2 x lo* sRBC prior to culture in oitro
PROLIFERATION
AND
ANTIBODY
OF Ig5I I'FC PEK 10” CELLS I'KODIICI~LI ON TIIK AFTEK FIKST CONTACT \VITH THE ihTIGEN
6
12 24 48
Uninjected
I;ouKTkr
L)AU
Anti-sRBC-PFCa sRBC-TNP added in who
Xnti-hRBCTKPh sRBC-TNP added in vitro
No sRBC added in oitvo .~
1
339
FORMATION
1,153 (f51) 1,050 (zt48) 4,125 12,186 19,372
933 85 88 105 500
(xt71) (zt68) (zt91)
1,251 (f31)
(h3.5) (& 9)
21 23 220 413 67.5 26
(ztll) (h 9j (f15)
107 (i
8)
(+ (A
4) 3)
(zt15) (f21) (+21) (zk 4)
Anti-hRBCPFC” sRBC-TNP added in vitro 1
0.5 4 15 18 1
(fl)
(?tl) (*I) (zk4) (zt5) (Al)
a Measured on day 4 after in oivo injection of sRBC. * Measured on day 4 after addition of sRBC-TNP to the cultures.
RESULTS In the first series of studies, mice were injected with 2 x lOa sRBC. At different time intervals (1, 6, 12, 24, and 48 hr), the animals were sacrificed and spleen cultures were prepared. One group of cultures received sRBC-TNP, and the PFC response to sRBC and to TNP utilizing hRBC-TNP was measured. The results of a typical experiment are shown in Table 1. The response to sRBC was measured on day 4 after initial contact with sRBC antigen given in viva. The response to hRBC and hRBC-TNP was measured on day 4, after addition of sRBC-TNP in vitro. The results show that cultures initiated from antigen-injected mice generated increasing numbers of sRBC-PFC as the time of in viva residence of the antigen increased, from 12 hr on. No enhancement in the formation of sRBC-PFC was found when the time of in viz~ sRBC injection and culture initiation was 6 hr or less. Similar results were found with the TNP responses. As the time of carrier sRBC injection and culture initiation increased, the TKP responses increased accordingly. The minimum time for the production of enhanced TNP responses was also 12 hr. The responses to hRBC demonstrate the immunological specificity of the enhanced sRBC and TNP responses, under the experimental conditions described. As shown on Table 1, when no antigen was added in vitro, cultures initiated 624 hr after in viva antigen injection produced very few anti-sRBC PFC. However, if the interval between in viva injection of RBC and initiation of cultures was shortened to 1 hr, even if no more antigen was added in vitro, such cultures generated a number of PFC which approached that seen in normal spleen cultures prepared in the presence of 4 X lo6 sRBC. These results are in agreement with earlier
studies
concerning
the
rapid
degradation
erythrocytes in the mouse spleen (8). These results would appear to directly implicate the observed enhancement of PFC formation (4). a small fraction of the immunogenic activity of retained on the surface of nonadherent lymphoid
of
immunogenicity
of
foreign
carrier-specific helper T cells in Earlier studies have shown that injected foreign erythrocytes is cells in the mouse spleen (13).
340
BACHVAROFF
r 2 after
AND
3
RAF’APORT
7 4 Initial
7 5 Antlgen
7 T 6 7 Exposure
FIG. 1. In vitro IgM response to sRBC of spleen cultures from normal mice and from mice injected with 2 X 10” sRBC 24 and 48 hr earlier. The mice were total body lethally irradiated with shielded and unshielded spleens. (a) ---•-Anti-sRBC response of spleen culture from normal sham-operated nonirradiated mice. (b) ---m--Anti-sRBC response of spleen cultures from 24-hr sRBC-injected and irradiated spleen-shielded mice. (c) - - - - 0 - - - Anti-sRBC response on spleen cultures from 24-hr sRBC-injected sham-operated nonirradiated mice. (d) ---A-Anti-sRBC response of spleen cultures from 4%hr sRBC-injected and irradiated spleen-shielded mice. (e) - - - - n - - - - Anti-sRBC response of spleen cultures from 48-hr sRBC-injected sham-operated nonirradiated mice. Mice in (b) and (c) were injected with sRBC 1 hr prior to operation and irradiation. The spleens were shielded extracorporeally and returned to the peritoneal cavity immediately after irradiation. Spleen cultures were initiated 24 and 48 hr later. (f) -----Anti-sRBC response of cultures from 24-hr sRBCinjected and irradiated spleen-unshielded mice. The animals were injected with sRBC and irradiated 24 hr later just before culture initiation. All cultures received 4 X 10’ sRBC.
Such retained immunogens could be available for lymphocytes from the recirculating extrasplenic pool. The enhanced in vitro responses described above might thus have been due to a recruitment of such antigen-reactive T cells by the spleen, with the retained immunogen serving as a cell immunoadsorbent (5-7). However, spleen cell cultures obtained from mice injected with 2 X lo8 sRBC at 6 hr before initiation of culture did not have enhanced in vitro responses to sRBC, as might have happened if continuous recruitment of antigen-reactive T cells by the spleen were occurring in V&JO.On the other hand, an appreciable enhancement occurred when the spleen cell cultures were prepared from 12-hr sRBC-injected mice. This question was explored further with irradiation experiments. As noted in Fig. 1, the in vitro responses of cultures prepared from mice injected with 2 X lo* sRBC at 1 hr prior to irradiation were enhanced in spleen cultures initiated 24 hr or 48 hr after antigen injection, provided that the animals’ spleens had been shielded during irradiation. The response of sham-operated nonirradiated normal mice was as high as that observed in normal spleen cultures. In contrast, the in vitro response of cultures from 2 x lo8 sRBC-injected mice that had been lethally irradiated without spleen protection 24 hr later, just before culture initiation, did not generate any PFC. The possibility that recruitment of X-ray resistant cells might have been the cause of the observed enhancement was tested by the following injected with 2 x lo* sRBC; 24 hr later, they were
experiments: Mice were irradiated without spleen
T-CELL
PROLIFERATION
AND
AKTIBODY
FORMATION
341
protection, and 10’ spleen cells from these animals were cultured with 10’ spleen cells from normal animals in the presence of the usual concentration of sRBC. The anti-sRBC response in such mixed cultures was as high in one experiment as in culture of 1.5 X 10’ normal spleen cells cultured in the presence of antigen. f or the mixture of irradiated The number of PFC on day +4 was 976 (k63) and normal cells and 1028 (*lOl) for the normal cell culture done. In two other experiments, the number of PFC for the irradiated-normal cell mixtures was below that of the normal controls, probably due to the in vitro lysis of the irradiated cells. Thus, X-ray-resistant cells obtained from mice after injection of antigen did not appear to be capable of enhancing the respone of cells from another normal animal in the presence of antigen. In further studies of the possibility that an early in viva proliferation and/or differentiation of antigen sensitive T cells in the spleen was the cause of the observed enhancement responses in vitro, the thymidine-nlethyl-3H hot-pulse technique was used. As illustrated in Fig. 2, normal spleen cultures which were hot-pulsed from 20 to 27 hr after in vitro addition of antigen generated as many PFC against sRBC as nonpulsed controls, as reported earlier by Dutton and Mishell (12). In contrast, however, when cultures initiated at 20 hr after in viva injection of sRBC were hotpulsed for 7 hr, the enhancement of PFC formation did not occur; instead, the antibody response of sRBC was delayed by 1 day, and remained lower than the levels of response observed in pulsed or nonpulsed cultures obtained from normal animals. The appreciable enhancement of the PFC formation in cultures from animals injected 12 hr earlier with 2 X lo* sRBC was also eliminated when such cultures were hot pulsed from 12 to 22 hr. These results are consistent with the interpretation that the enhanced responses observed in this study are associated with an early proliferation of antigen-reactive cells, commencing in viva approximately 12-20 hr after antigen injection. Such proliferation apparently does not occur at the same time in vitro. The antigen specificity of this proliferative response has been confirmed by experiments which show equal response to hRBC in pulsed and nonpulsed cultures obtained from sRBC-injected mice. Early division of antigen-reactive helper T cells in the spleen as an initial event for the enhanced generation of PFC in vitro is supported further by the data summarized in Table 2. In these experiments, mice were injected with 2 X lo8 sRBC, and spleen cell cultures were initiated 12, 20, and 70 hr later. The cultures were immediately hot-pulsed for 8-10 hr, and after standard reculturing (see Materials and Methods), 4 x 10G sRBC-TNP were added. A comparison was made between the responses to sRBC, hRBC, and to hRBC-TNP. Table 2 lists the number of PFC formed against sRBC and hRBC-TNP. The antibody response to TNP, as measured by the number of PFC formed against hRBC, is of special interest. Cultures from mice injected with carrier alone at 12 and 20 hr before initiation of culture showed greatly reduced anti-TNP responses following hotpulsing, as compared to their respective nonpulsed controls. In contrast, similar pulses given to cultures initiated at 70 hr after carrier injection had little effect on antibody responses to this haptene. 3 Hot-pulsed cultures tested at the three time s In several instances, the 10.hr hot pulse administered to cultures from 70-hr sRBCinjected mice was not as effective in decreasing the anti-carrier (sRBC) response as cultures the anti-TNP response of the prepared from 20-hr sRBC-injected mice. In these cases, pulsed cultures was substantially below that of the nonpulsed 70-hr controls. It remains to be determined whether this effect is due to a competition of carrier and hapten-reactive B cells for the same helper T cells (14).
342
5 z
L--.
rlTl
1 2 Days after
3
4 Inctial
5 Antigen
6 7 Exposure
FIG. 2. Effect of tritiated-thymidine pulse upon the in vitro IgM responses to sRBC of cultures from normal mice and from mice injected 20 hr earlier with 2 X 10’ sRBC. - - - - 0 - - - Anti-sRBC response of nonpulsed spleen cultures from normal mice. - - - l - - - - AntisRBC response of hot pulsed cultures from normal mice. Seven-hour hot pulse was administered 20 hr after culture initiation. -O-Anti-sRBC response of nonpulsed spleen cultures from 20-hr sRBC-injected mice. ---a--Anti-sRBC response of hot-pulsed cultures from sRBC-injected mice. The cultures were initiated 20 hr after sRBC injection and immediately hot pulsed for 7 hr. All cultures received 4 X 10” sRBC.
intervals (12, 20, and 70 hr) after antigen administration showed decreased responses to sRBC. These results indicate an initiation of helper T-cell division in the spleen within 12-20 hr after foreign RBC injection, with subsidence by 70 hr. Helper T cells which had been produced by that later time were already differentiated and were capable of performing the necessary helper function for the precursor B cells responding to the hapten.
DISCUSSION Sercarz and Byers (1) have documented the development of in viva IgM “memory” within 24 hr after the injection of foreign RBC in mice. Mishell and Dutton (2), utilizing an in vitro spleen cell culture system, have observed that, in the presence of specific antigen, the number on anti-sRBC PFC in spleen cultures prepared from mice injected several days earlier with sRBC is greatly increased, as compared to the number of PFC observed in similar cultures prepared from uninjected animals. Subsequently, Kettman and Dutton (3) have reported that spleen cultures prepared from carrier (sRBC) -injected mice produced an enhanced number of PFC to TNP, if subsequently challenged in vitro with sRBC-TNP. An attempt has been made in this study to analyze the early cellular events occurring in the mouse spleen after administration of foreign erythrocytes. The use of foreign erythrocytes under the conditions described is generally accepted as a prototype, for experimental models designed to study antibody responses dependent upon T-cell helper function (3, 4, 14, 18, 27). A tenfold increase in the production
T-CELL
PROLIFERATION
OF Ighl
PFC
AND
AKTIBODY
TABI,E NUMREK
IN VITRO
PEK 106 CELLS
F:XPOSURE
OF SPLEEN
343
FORMATION
2 PKODIKED
CEIL
ON THE
CIJLTUKES
FIFTH
DAY
AFTKR
TO sRBC-TNI’
-
Cultures
tested
Number Horse
1. Cultures with
prepared from sRBC ; sRBC-TNP
mice
RBC-TNPe
injected 12 hr earlier were added in vitro
210
2. Cultures prepared from mice injected 12 hr earlier with sRBC. ThymidineJH pulse was administered at 12-22 hr; sRBC-TNP were added in o&o. 3. Cultures with
prepared from sRBC; sRBC-TNP
mice
injected 20 hr earlier were added in vitro.
4. Cultures prepared from mice injected 20 hr earlier with sRBC. ThymidineJH pulse was administered at 20-30 hr ; sRBC-TNP were added in vitro. 5. Cultures with
prepared from sRBC ; sRBC-TNP
mice
injected 70 hr earlier were added in vitro.
(anti-hRBC-TNP
*
against Sheep
20
1130
67zt
5
449
433 +
1.5
1987
78 z!c 10
1148
6. Cultures prepared from mice injected 70 hr earlier with sRBC. Thymidine-3H pulse was administered at 70-80 hr; sRBC-TNP were added in z&o. a “Background” PFC were subtracted were added in vitro instead of sRBC-TNP).
of PFC
PFC
RBC
+
71
zt 17
f
25
33.5 f
21
f
21
6678
f
42
984 f.
16
1354
f
18
of similar
cultures
were
sRBC
of anti-sRBC PFC was observed in spleen cell cultures prepared 24 hr after the in V~VOinjection of sRBC followed by in vitro challenge of the cultures with sRBC. Cultures obtained from similarly treated mice produced enhanced numbers of PFC against TNP, when challenged in vitro with sRBC-TNP. The latter result has provided strong supportive evidence for the occurrence of an enrichment and/or differentiation of antigen-specific helper T cells in the host spleen within the first 24 hr after injecton of foregn RBC (4). An appreciable enhancement of anti-sRBC PFC also occurred in spleen cultures prepared from mice injected with sRBC 12 hr earlier. It must be noted, however, that these in vifro results differ from the data reported by Sprent and Miller (28) in experiments where spleen cells obtained from mice which had been primed 24 hr earlier with sRBC were transferred (in viva) to irradiated hosts. It would appear, therefore, that the specific unresponiveness of cell recently primed in viva may not be a consequence of a transient induction of tolerance, increased susceptibility of blasts of trauma, or an expression of fewer antigen-binding receptors. Rather, this transient state of unresponsiveness may be due to subtle changes which are reflectd only in the in viva system, possibly associated with alterations in the ability of recently primed cells to relocalize to areas conducive to antibody production. A number of authors have suggested that active recruitment of antigen-reactive lymphocytes from the recirculating lymphocyte pool in secondary lymphoid organs occurs after injection of antigen (5-7). Earlier studies have also shown that the
344
BACHVAROFF
AND
RAPAPORT
immunogenic activity of injected foreign RBC is retained for prolonged periods 011 the surface of lymphoid cells in the mouse spleen, and such cell surfaces could be available for cell recruitment (13). However, if continuous recruitment of antigensensitive cells by the spleen were to account for the enhancement of antibody formation observed in these studies, spleen cultures initiated at 6 hr after sRBC injection might have been expected to yield increased numbers of PFC; this was not the case. Irradiation experiments indicate that spleen cultures initiated 24 or 48 hr after lethal irradiation of mice which had been injected with 2 x IO8 sRBC 1 hr prior to irradiation produced the usual enhanced number of PFC, if their spleens were shielded extracorporeally during irradiation (Fig. l), while controls whose spleens were irradiated were all negative. Also, if spleen cells from mice injected with sRBC were given lethal irradiation 24 hr later and were then added to normal spleen cell cultures, no enhancement in antibody production occurred. Taken together, these data demonstrate that, in the first 12-24 hr after intravenous injection of foreign erythrocytes, helper T-cell activity is not a consequence of recruitment of antigen-reactive cells (X-ray sensitive or -resistant cells) by the mouse spleen from the recirculating pool. This conclusion, however, must be confined at this time to the specific experimental conditions described, with particular regard to the organ under study, the antigen(s) used, and the dose and mode of administration of such antigen(s) in viva. Sprent and Miller (29)) for example, have recently observed a specific depletion of antigen-reactive circulating lymphocytes in mice injected with higher doses ( lo9 and lOlo) of sRBC than used in the present study. These authors concluded that such a depletion might have been due to recruitment of the lymphocytes by the spleen. The occurrence of an early in viva intrasplenic T-cell proliferation as a cause of the enhanced in vitro responses to foreign RBC observed in this study is supported by the tritiated thymidine hot-pulse experiments described. In these experiments, a 7-hr hot-pulse given to spleen cultures prepared from mice injected 20 hr earlier with sRBC eliminated the enhanced generation of PFC (Fig. 2). Similarly timed hot pulses administered to normal spleen cultures in the presence of antigen did not affect the number of PFC produced, when compared with nonpulsed controls, as noted earlier by Dutton and Mishell (12). If spleen cultures prepared from mice injected 12 and 20 hr earlier with carrier RBC alone were hot-pulsed for S-10 hr prior to challenge with sRBC-TNP, there was a great reduction in the production of anti-TNP-PFC, as compared to nonpulsed controls (Table 2). In contrast, hot pulses given to cultures obtained from mice injected with carrier cells 70 hr earlier generally had little or no effect upon the subsequent generation of TNP-PFC. These results indicate that DNA synthesis in spleen T cells commences in z&o within 12-20 hr after injection of a high dose of sRBC, and is subsiding within 70 hr. The data are also consistent with the notion that the T-cell priming process leading to their functional differentiation into helper cells is radiosensitive (15, 16). 0 rice differentiated, however, such helper cells become radioresistant (17, 18). The experiments also provide new evidence that a dissociation between the time of proliferation of antigen-reactive T and B cells may be occurring in the mouse spleen (19-21). Although T-cell proliferation as a consequence of exposure to certain antigens is a well-established observation (21), it is not clear whether the initial antigen-specific T-cell division is an absolute prerequisite for the develop-
T-CELL
PROLIFERATION
AND
ANTIBODY
FORMATION
345
merit of helper activity, Although the results of this study do not provide a definitive answer to this question, the data would appear to favor such an interpretation. For example, when cultures prepared from mice injected 20 hr earlier with sRBC were hot-pulsed for 7 hr, enhancement of anti-sRBC PFC production was eliminated. These cultures did, however, produce a number of sRBC PFC comparable to that observed in normal pulsed or unpulsed cultures, with a delay of 24 hr (Fig. 2). Th’ IS ob servation raised the possibility that not all antigen-reactive T cells may have started division within 12-20 hr after in viva antigenic challenge, and only the T cells in their S phase were killed by the incorporated hot thymidine. However, antigen-reactive T cells which had not yet been triggered in Z&IO,,or were in a different phase at the time of hot-pulsing, may have escaped radioactive suicide. As a result, the development of helper activity was delayed and the subsequent proliferation and differentiation of antibody-forming B-cell precursors was delayed by 24 hr. These data also highlight the usefulness of thymidine-“H in the analysis of the dynamics of cell proliferation, since only cells synthesizing DNA are killed by this agent, thereby eliminating the possibility of interference with the results by transient effects or other variables. The observation that antigen must be present for both T- and B-cell proliferation may deserve special mention. As shown in Table 1, if no antigen is added in vitro to spleen cell cultures prepared from mice which had been injected with sRBC 624 hr earlier, the in vitro responses are minimal and similar to the responses to sRBC observed in normal mouse spleen cell cultures in fetal calf serum alone (2). The low antigen threshold required to trigger T-cell division (22, 23) has been confirmed in this study, since sRBC doses ranging from 2 X lo6 to 2 X lo8 cells injected 24 hr prior to initiation of cell cultures were equally effective for the subsequent in vitro enhancement of PFC formation in the presence of specific antigen. However, even the highest dose of injected sRBC was ineffective in triggering B-cell responses in dissociated spleen cell cultures if no antigen was added in vitro. It has been shown previously that approximately 10% of antigen sequestered in the spleen after a single injection of 2 X lo8 sRBC is retained by the spleen in a immunogenic form for 3 days (8). The presence of such retained immunogens, which are sufficient to trigger B-cell proliferation in the spleen environment, points to the existence of a mechanism of immunogen retention in the spleen which is equally effective for triggering T- and B-cell proliferation (24), but is inoperative for dissociated spleen cells in the absence of unprocessed antigen. Taken together, the data presented in this report are in close agreement with the interpretation of Miller (25), that “the role of T cells is linked to a capacity for “recognizing” the antigenic determinant and is specifically dependent, not on the passive transport of such antigenic determinants to B cells but upon some active process.” The results also provide evidence that, in order to develop such a function, the antigen-reactiT-e T cell may have to go through a process of cell division, as suggested by Anderson, Sprent, and Miller (26). ACKNOWLEDGMENTS The excellence of the technical assistance of Irene Allan, Susan Ball, Louis Browne, and Liling Wei is greatly appreciated. We are particularly indebted to Dr. Melvin Becker and Joan Siebert of the Department of Radiology, for their valuable help, and to Dr. Jeannette Thorbecke for her advice and assistance in the preparation of this manuscript.
346
BACHVAROFF
Ah-D
RAPAPORT
REFERENCES 1. 2. 3. 4. 5. 6. 7.
Sercarz, E. E., and Byers, V. S., J. Immunol. 98, 836, 1967. Mishell, R. I., and Dutton, R. W., J. Exp. Med. 126, 423, 1967. Kettman, J. R., and Dutton, R. W., J. Zmmunol. 104, 1558, 1970. Katz, D. H., Paul, W. E., Goidl, E. A., and Benacerraf, B., J. Exp. Med. 132, 261, 1970. Ford, W. L., and Gowans, J. L., Proc. Roy. Sot. B 168, 244, 1967. Sprent, J. J., Miller, J. F. A. P., and Mitchell, G. F., Cell. Immulzol. 2, 171, 1971. Rowley, D. A., Gowans, J. L., Atkins, R. C., Ford, W. L., and Smith, M. E., J. Exp. Med. 136,499, 1972. 8. Franz], R. E. Infect. Immunity 6, 469, 1972. 9. Rittenberg, M. B., and Pratt, K. L., Proc. Sot. Exp. Biol. Med. 132, 575, 1969. 10. Glick, R. E., in “Progress in Immunology” (B. Amos, Ed.), p. 1505. Academic Press, New York, 1971. 11. Jerne, N. K., Nordin, A. A., and Henry, C., in “Cell Bound Antibodies” (B. Amos and H. Koprowski, Eds.), p. 109. The Wistar Institute Press, Philadelphia, 1963. 12. Dutton, R. W., and Mishell, R. I., J. Exp. Med. 126, 443, 1967. 13. Bachvaroff, R. J., and Franzl, R. E., Fed. Proc. Fed. Amer. Sot. Exp. Biol. 29, 825, 1970. 14. Falkoff, R., and Kettman, J., J. Immunol. 108, 54, 1972. 15. Claman, H. N., Chaperon, E. A., and Triplett, R. F., J. Immunol. 97, 828, 1966. 16. Miller, J. F. A. P., and Mitchell, G. F., J. Exp. Med. 128, 801, 1968. 17. Katz, D. H., Paul, W. E., Goidl, E. A., and Benaceraff, B., Science 170, 426, 1970. 18. Kettman, J., and Dutton, R. W., Proc. Nat. Acad. Sci. U.S.A. 68, 699, 1971. 19. Shearer, G. M., and Cudkowicz, G., J. Exp. Med. 130, 1243, 1969. 20. Davies, A. J. S., Carter, R. L., Leuchars, E., Wallis, V., and Keller, P. C., Immunology 16, 57, 1969. 21. Davies, A. J. S., Leuchars, E., Wallis, V., and Koller, P. C., Transplantation 4, 438, 1966. 22. Salvin, S. B., and Smith, R. F., J. Immmwl. 84, 449, 1960. 23. Greaves, M. F., Moller, E., and Moller, G., Cell. Immzlnol. 1, 386, 1970. 24. Nossal, G. J. V., Abbott, A., Mitchell, J., and Lummus, Z., J. Exp. Med. 127, 377, 1968. 2.5. Miller, J. F. A. P., in “Cell Interactions and Receptors Antibodies in Immune Response” (0. Makela, A. Cross, and T. U. Kosunen, Eds.), p. 293. Academic Press, New York, 1971. 26. Anderson, R. E., Sprent, J., and Miller, J. F. A. P., J. Exp. Med. 135, 771, 1972. 27. Claman, H. N., Chaperon, E. A., and Triplett, R. F., Proc. Sot. Erp. Biol. Med. 122, 1167, 1966.
28. Sprent, J., and Miller, J. F. A. P., J. Exp. Med. 138, 143, 1973. 29. Sprent, J., and Miller, J. F. A. P., J. Exp. Med. 139, 1, 1974.
IMMUNOLOGY
15,
336-346
(1975)
Mechanisms
of Antibody
Formation
I. Early in Vivo Proliferation of Mouse Spleen T Cells Step in Antibody Formation L*
R.
BACHVAROFF
Department of Surgery New York Ulziversity
AXD
F. T.
RAPAPORT
altd the Institute of Reconstructive Medical Ceater, New York, Received
November
as an Initial
New
Plastic York
Surgery,
10016
8.1973
Spleen cultures prepared from mice injected 24 hr earlier with 2 X lo’-2 X 10’ sRBC and challenged in zitro with sRBC produced 10 times more anti-sRBC IgM PFC than cultures prepared from uninjected mice. The effect was specific for the particular species of foreign RBC injected in vivo. In vitro responses to TNP were also increased in spleen cultures prepared from animals injected 24 or 12 hr earlier with carrier RBC alone, directly implicating carrier-specific T cells in this process. Similar enhancements of PFC formation occurred in cultures prepared from mice which had been injected with sRBC 24 and 48 hr earlier, but which were exposed to lethal irradiation at 1 hr after injection of antigen, if their spleens were shielded extracorfioreally during irradiation. This finding indicated that in vivo recruitment of antigen-reactive extrasplenic X-ray-sensitive cells from the circulating lymphocyte pool by the spleen could not account for the observed enhancement. Proliferation in the spleen of antigen-reactive T cells, commencing 12-20 hr after the administration of antigen, was demonstrated by the tritiated thymidine pulse technique. An 8-hr hot-pulse given to spleen cell cultures from normal animals at 20 hr after in vitro challenge with antigen did not affect the rate of generation of IgMproducing cells; however, administration of a similar pulse to cultures which were initiated at 12 or at 20 hr after the in viva injection of sRBC eliminated the enhanced generation of PFC and delayed the in vitro response to sRBC by 24 hr. Spleen cell cultures were prepared from mice which had been injected in vivo with sRBC at 12, 20, and 70 hr earlier, and 8- to lo-hr hot pulses were given immediately after initiation of the cultures. The cultures were then challenged with sRBC-TNP; antibody responses to TNP were greatly reduced in hot-pulsed cultures prepared from mice injected in zivo with carrier RBC at 12 or 20 hr prior to initiation of the cultures. In contrast, antibody responses to TNP observed in hot-pulsed cultures prepared from mice which had been injected with carrier RBC at 70 hr prior to initiation of the cultures were generally similar to those of nonpulsed 70 hr control cultures. This result suggests that the onset of T helper cell proliferation be,gins within 12-20 hr after injection of antigen, but subsides irt viva within 70 hr. By that time, the antigen-reactive T cells have already differentiated to perform their helper function. In spite of the triggering of T-cell proliferation during the first 24 hr after injection of antigen, spleen cell cultures prepared from mice which had been injected 24 hr 1 Presented in part at the 1973 Meeting of the American Association of Immunologists. 2 Supported by a Grant from The John A. Hartford Foundation, Inc.; supported in part by The Billy Rose Foundation, Inc., The Irwin Strasburger Memorial Medical Foundation, Inc., Contract NONR-4503. Office of Naval Research, Washington, DC and Grant GM-12748-10. 336 Copyright All rights
0
19i5 by Academic Press, Inc. of reproduction in any form reserved.
T-(‘ELL
PROLIFERATION
AND
ANTIBODY
FORMATION
337
earlier in viva with 2 X 10’ sRBC produced only minimal numbers of anti-sRBC PFC if no antigen was added to the cultures. The presence of unprocessed antigen thus appears to be a requirement for B-cell proliferation ~II ztit~u, even after T-cell division has been triggered. This finding is consistent with earlier suggestions that the function of “helper” T cells may not be limited to passive transport of antigenic determinants to B cells. Evidence is also presented to support the contention that the antigen-reactive T cell involved in this process may have to unrlcrgo cell division in order to tlcvelop “helj~er” capacity.
INTRODUCTION Earlier in z&o studies by Sercarz and Byers (1) have demonstrated the ability of mice to develop IgM “memory” with 24 hr after the injection of foreign erythrocytes. Mishell and Dutton (2) have reported enhanced in vitro antibody responses to sRBC in spleen cell cultures prepared from mice injected in viva with sRBC several days earlier. In addition, Kettman and Dutton (3) have observed that the in vitro anti-hapten antibody response of mouse spleen cells was greatly augmented if spleen cell cultures were obtained from mice primed in viva several days earlier by injections of carrier RBC. The latter result appeared to directly implicate enrichment and/or differentiation of carrier-specific helper T cells in the mouse spleen after administration of foreign erythrocytes (4). A recruitment of antigen-reactive cells from the recirculating lymphocyte pool by secondary lymphoid organs has frequently been suggested as an explanation for sucl~ enhanced in vitro responses after injection of antigen in viva (5-i’). The results of the present study demonstrate that recruitment of antigen-reactive cells from the recirculating lymphocyte pool by the mouse spleen does not occur during the first two days after intravenous administration of 106-lo8 foreign erythocytes. Instead, evidence is presented of an early division and differentiation of helper T cells in the mouse spleen as the initial step for proliferation of IgM hemolysin-forming B cells, in the antibody response to T cell-dependent antigens, such as foreign RBC. MATERIALS
AND
METHODS
Animals. Three- to six-month-old C5,Bl, mice were used throughout the experiments ; the animals were maintained on a standard Purina pellet diet. Antigens. Sheep (s) and horse (11) red blood cells (RBC) (Animal Blood Center, Syracuse, NY) were washed three times in balanced salt solution (BSS) (2) and were used as antigens. The washed cell suspensions were adjusted to the desired concentration in standard fashion (8). Erythocytes were lightly and heavily substituted with TNP by the procedures of Rittenberg and Pratt (9) and Kettman and Dutton (3)) respectively. Tissue cultures. Spleen cell cultures were initiated and maintained as described by Mishell and Dutton (a), except that 1O-5 lb1 2-mercaptoethanol (10) was added to the culture medium. Usually, 1.5-2 x lo7 spleen cells per culture were challenged with 4 x lo6 unmodified RBC or with fresh RBC heavily substituted with TNP. The generation of IgM-producing cells in each culture was assayed by enumerating the direct PFC, utilizing the plaque technique of Jerne. Nordin, and Henry (ll), as modified by Mishell and Dutton (2). 15:here anti-TNP antibody responses were measured, lightly substituted RBC-TNP were used for plaquing. Guinea pig serum absorbed three times with RBC was used as the source of com-
338
BACHVAR~FFANDRAPAPORT
plement in the plaguing technique. The number of E’FC counted each day ~vhich is listed in the reported results represents average comnts observed from three simultaneously prepared culture dishes, plaqued in triplicate. Basic e.rperimentnl design. An attempt was made to compare the rate of in viva generation of antibody-forming cells (AFC) in spleen cell cultures obtained from normal untreated mice with that observed in mice which had been previously injected with antigen in viva. Spleen cell cultures prepared from both groups of animals were exposed to the same concentration of foreign erythrocytes in vitro. For in viva injection, 2 X 104-2 X 1Os foreign erythrocytes were injected into the tail vein ; at different time intervals thereafter (1, 6, 12, 24 hr, etc.), spleen cell cultures were initiated and challenged with KBC. The PFC were measured from the second to the sixth day following addition of antigen in vitro. In each case, “0 time” refers to the time of first contact of host cells with specific antigen, whether given in viva or in vitro, Irradiation experiments. Mice were injected intravenously with 2 X 1Os sRBC. One hour later, one group of animals was anesthetized, and a laparatomy was performed under aseptic conditions. The spleens were extruded from the abdomen; they were wrapped in moist saline sponges and inserted into a radiation protection lead housing (0.5-cm thickness). The animals were then exposed to 1000 R of lethal total body irradiation (30 kV, 10 mA, HVL 3.0 mm Cu; distance of focus to target = 50 cm; dose rate = 60 rads/min) Immediately after irradiation, the spleens were returned into the peritoneal cavity and the abdomen was sutured. Twenty-four and 48 hr later, spleen cultures were initiated in the presence of 4 X IO6 sRBC. Control groups included (i) lethally irradiated nonoperated mice and (ii) sham-operated nonirradiated mice. Hot-pulse techniqzie. Assessments of cell proliferation were performed by the method of Dutton and Mishell (12). In the first set of experiments, cultures from normal mice were initiated in the presence sRBC. Twenty hours later, the cells were exposed to a 7-hr pulse of 10 ,&i of thymidine-methyl-3H-( 15-20 Ci/mmole) (Schwartz/Mann, Orangeburg, NY). The dishes were then scrubbed with “rubber policemen” and the cells were collected and washed in BSS. The cells were resuspended in fresh medium containing 50 pg of cold thymidine (Schwartz/Mann, Orangeburg, NY). The nonpulsed controls for normal animals, also cultured in the presence of sRBC, were treated and recultured in the same manner. The antibody response in such control cultures was compared with that observed in hot-pulsed cultures prepared from mice which had been injected with antigen prior to initiation of culture. The latter animals were injected with 2 X 10’ sRBC, 20 hr prior to initiation of spleen cultures in the presence of sRBC. A 7-hr hot thymidine pulse was administered immediately to one group of culture dishes, while control dishes were not hot-pulsed. The cells were then collected and recultured in fresh medium with cold thymidine. In another set of experiments, mice were first injected in viva with 2 X 10’ sRBC. Twelve, 20, and 70 hr later, spleen cell suspensions were prepared and were hot-pulsed for 8-10 hr. The cells were then washed and recultured in medium supplemented with cold thymidine. Heavily substituted sRBC-TNP (4 X lo6 cells) were added to the cultures, and responses to sRBC, hRBC, and hRBC-TNP were measured; concurrent control cell suspensions received no hot thymidine.
T-CELL
NUMI~EK
Hours of iv injection of 2 x lo* sRBC prior to culture in oitro
PROLIFERATION
AND
ANTIBODY
OF Ig5I I'FC PEK 10” CELLS I'KODIICI~LI ON TIIK AFTEK FIKST CONTACT \VITH THE ihTIGEN
6
12 24 48
Uninjected
I;ouKTkr
L)AU
Anti-sRBC-PFCa sRBC-TNP added in who
Xnti-hRBCTKPh sRBC-TNP added in vitro
No sRBC added in oitvo .~
1
339
FORMATION
1,153 (f51) 1,050 (zt48) 4,125 12,186 19,372
933 85 88 105 500
(xt71) (zt68) (zt91)
1,251 (f31)
(h3.5) (& 9)
21 23 220 413 67.5 26
(ztll) (h 9j (f15)
107 (i
8)
(+ (A
4) 3)
(zt15) (f21) (+21) (zk 4)
Anti-hRBCPFC” sRBC-TNP added in vitro 1
0.5 4 15 18 1
(fl)
(?tl) (*I) (zk4) (zt5) (Al)
a Measured on day 4 after in oivo injection of sRBC. * Measured on day 4 after addition of sRBC-TNP to the cultures.
RESULTS In the first series of studies, mice were injected with 2 x lOa sRBC. At different time intervals (1, 6, 12, 24, and 48 hr), the animals were sacrificed and spleen cultures were prepared. One group of cultures received sRBC-TNP, and the PFC response to sRBC and to TNP utilizing hRBC-TNP was measured. The results of a typical experiment are shown in Table 1. The response to sRBC was measured on day 4 after initial contact with sRBC antigen given in viva. The response to hRBC and hRBC-TNP was measured on day 4, after addition of sRBC-TNP in vitro. The results show that cultures initiated from antigen-injected mice generated increasing numbers of sRBC-PFC as the time of in viva residence of the antigen increased, from 12 hr on. No enhancement in the formation of sRBC-PFC was found when the time of in viz~ sRBC injection and culture initiation was 6 hr or less. Similar results were found with the TNP responses. As the time of carrier sRBC injection and culture initiation increased, the TKP responses increased accordingly. The minimum time for the production of enhanced TNP responses was also 12 hr. The responses to hRBC demonstrate the immunological specificity of the enhanced sRBC and TNP responses, under the experimental conditions described. As shown on Table 1, when no antigen was added in vitro, cultures initiated 624 hr after in viva antigen injection produced very few anti-sRBC PFC. However, if the interval between in viva injection of RBC and initiation of cultures was shortened to 1 hr, even if no more antigen was added in vitro, such cultures generated a number of PFC which approached that seen in normal spleen cultures prepared in the presence of 4 X lo6 sRBC. These results are in agreement with earlier
studies
concerning
the
rapid
degradation
erythrocytes in the mouse spleen (8). These results would appear to directly implicate the observed enhancement of PFC formation (4). a small fraction of the immunogenic activity of retained on the surface of nonadherent lymphoid
of
immunogenicity
of
foreign
carrier-specific helper T cells in Earlier studies have shown that injected foreign erythrocytes is cells in the mouse spleen (13).
340
BACHVAROFF
r 2 after
AND
3
RAF’APORT
7 4 Initial
7 5 Antlgen
7 T 6 7 Exposure
FIG. 1. In vitro IgM response to sRBC of spleen cultures from normal mice and from mice injected with 2 X 10” sRBC 24 and 48 hr earlier. The mice were total body lethally irradiated with shielded and unshielded spleens. (a) ---•-Anti-sRBC response of spleen culture from normal sham-operated nonirradiated mice. (b) ---m--Anti-sRBC response of spleen cultures from 24-hr sRBC-injected and irradiated spleen-shielded mice. (c) - - - - 0 - - - Anti-sRBC response on spleen cultures from 24-hr sRBC-injected sham-operated nonirradiated mice. (d) ---A-Anti-sRBC response of spleen cultures from 4%hr sRBC-injected and irradiated spleen-shielded mice. (e) - - - - n - - - - Anti-sRBC response of spleen cultures from 48-hr sRBC-injected sham-operated nonirradiated mice. Mice in (b) and (c) were injected with sRBC 1 hr prior to operation and irradiation. The spleens were shielded extracorporeally and returned to the peritoneal cavity immediately after irradiation. Spleen cultures were initiated 24 and 48 hr later. (f) -----Anti-sRBC response of cultures from 24-hr sRBCinjected and irradiated spleen-unshielded mice. The animals were injected with sRBC and irradiated 24 hr later just before culture initiation. All cultures received 4 X 10’ sRBC.
Such retained immunogens could be available for lymphocytes from the recirculating extrasplenic pool. The enhanced in vitro responses described above might thus have been due to a recruitment of such antigen-reactive T cells by the spleen, with the retained immunogen serving as a cell immunoadsorbent (5-7). However, spleen cell cultures obtained from mice injected with 2 X lo8 sRBC at 6 hr before initiation of culture did not have enhanced in vitro responses to sRBC, as might have happened if continuous recruitment of antigen-reactive T cells by the spleen were occurring in V&JO.On the other hand, an appreciable enhancement occurred when the spleen cell cultures were prepared from 12-hr sRBC-injected mice. This question was explored further with irradiation experiments. As noted in Fig. 1, the in vitro responses of cultures prepared from mice injected with 2 X lo* sRBC at 1 hr prior to irradiation were enhanced in spleen cultures initiated 24 hr or 48 hr after antigen injection, provided that the animals’ spleens had been shielded during irradiation. The response of sham-operated nonirradiated normal mice was as high as that observed in normal spleen cultures. In contrast, the in vitro response of cultures from 2 x lo8 sRBC-injected mice that had been lethally irradiated without spleen protection 24 hr later, just before culture initiation, did not generate any PFC. The possibility that recruitment of X-ray resistant cells might have been the cause of the observed enhancement was tested by the following injected with 2 x lo* sRBC; 24 hr later, they were
experiments: Mice were irradiated without spleen
T-CELL
PROLIFERATION
AND
AKTIBODY
FORMATION
341
protection, and 10’ spleen cells from these animals were cultured with 10’ spleen cells from normal animals in the presence of the usual concentration of sRBC. The anti-sRBC response in such mixed cultures was as high in one experiment as in culture of 1.5 X 10’ normal spleen cells cultured in the presence of antigen. f or the mixture of irradiated The number of PFC on day +4 was 976 (k63) and normal cells and 1028 (*lOl) for the normal cell culture done. In two other experiments, the number of PFC for the irradiated-normal cell mixtures was below that of the normal controls, probably due to the in vitro lysis of the irradiated cells. Thus, X-ray-resistant cells obtained from mice after injection of antigen did not appear to be capable of enhancing the respone of cells from another normal animal in the presence of antigen. In further studies of the possibility that an early in viva proliferation and/or differentiation of antigen sensitive T cells in the spleen was the cause of the observed enhancement responses in vitro, the thymidine-nlethyl-3H hot-pulse technique was used. As illustrated in Fig. 2, normal spleen cultures which were hot-pulsed from 20 to 27 hr after in vitro addition of antigen generated as many PFC against sRBC as nonpulsed controls, as reported earlier by Dutton and Mishell (12). In contrast, however, when cultures initiated at 20 hr after in viva injection of sRBC were hotpulsed for 7 hr, the enhancement of PFC formation did not occur; instead, the antibody response of sRBC was delayed by 1 day, and remained lower than the levels of response observed in pulsed or nonpulsed cultures obtained from normal animals. The appreciable enhancement of the PFC formation in cultures from animals injected 12 hr earlier with 2 X lo* sRBC was also eliminated when such cultures were hot pulsed from 12 to 22 hr. These results are consistent with the interpretation that the enhanced responses observed in this study are associated with an early proliferation of antigen-reactive cells, commencing in viva approximately 12-20 hr after antigen injection. Such proliferation apparently does not occur at the same time in vitro. The antigen specificity of this proliferative response has been confirmed by experiments which show equal response to hRBC in pulsed and nonpulsed cultures obtained from sRBC-injected mice. Early division of antigen-reactive helper T cells in the spleen as an initial event for the enhanced generation of PFC in vitro is supported further by the data summarized in Table 2. In these experiments, mice were injected with 2 X lo8 sRBC, and spleen cell cultures were initiated 12, 20, and 70 hr later. The cultures were immediately hot-pulsed for 8-10 hr, and after standard reculturing (see Materials and Methods), 4 x 10G sRBC-TNP were added. A comparison was made between the responses to sRBC, hRBC, and to hRBC-TNP. Table 2 lists the number of PFC formed against sRBC and hRBC-TNP. The antibody response to TNP, as measured by the number of PFC formed against hRBC, is of special interest. Cultures from mice injected with carrier alone at 12 and 20 hr before initiation of culture showed greatly reduced anti-TNP responses following hotpulsing, as compared to their respective nonpulsed controls. In contrast, similar pulses given to cultures initiated at 70 hr after carrier injection had little effect on antibody responses to this haptene. 3 Hot-pulsed cultures tested at the three time s In several instances, the 10.hr hot pulse administered to cultures from 70-hr sRBCinjected mice was not as effective in decreasing the anti-carrier (sRBC) response as cultures the anti-TNP response of the prepared from 20-hr sRBC-injected mice. In these cases, pulsed cultures was substantially below that of the nonpulsed 70-hr controls. It remains to be determined whether this effect is due to a competition of carrier and hapten-reactive B cells for the same helper T cells (14).
342
5 z
L--.
rlTl
1 2 Days after
3
4 Inctial
5 Antigen
6 7 Exposure
FIG. 2. Effect of tritiated-thymidine pulse upon the in vitro IgM responses to sRBC of cultures from normal mice and from mice injected 20 hr earlier with 2 X 10’ sRBC. - - - - 0 - - - Anti-sRBC response of nonpulsed spleen cultures from normal mice. - - - l - - - - AntisRBC response of hot pulsed cultures from normal mice. Seven-hour hot pulse was administered 20 hr after culture initiation. -O-Anti-sRBC response of nonpulsed spleen cultures from 20-hr sRBC-injected mice. ---a--Anti-sRBC response of hot-pulsed cultures from sRBC-injected mice. The cultures were initiated 20 hr after sRBC injection and immediately hot pulsed for 7 hr. All cultures received 4 X 10” sRBC.
intervals (12, 20, and 70 hr) after antigen administration showed decreased responses to sRBC. These results indicate an initiation of helper T-cell division in the spleen within 12-20 hr after foreign RBC injection, with subsidence by 70 hr. Helper T cells which had been produced by that later time were already differentiated and were capable of performing the necessary helper function for the precursor B cells responding to the hapten.
DISCUSSION Sercarz and Byers (1) have documented the development of in viva IgM “memory” within 24 hr after the injection of foreign RBC in mice. Mishell and Dutton (2), utilizing an in vitro spleen cell culture system, have observed that, in the presence of specific antigen, the number on anti-sRBC PFC in spleen cultures prepared from mice injected several days earlier with sRBC is greatly increased, as compared to the number of PFC observed in similar cultures prepared from uninjected animals. Subsequently, Kettman and Dutton (3) have reported that spleen cultures prepared from carrier (sRBC) -injected mice produced an enhanced number of PFC to TNP, if subsequently challenged in vitro with sRBC-TNP. An attempt has been made in this study to analyze the early cellular events occurring in the mouse spleen after administration of foreign erythrocytes. The use of foreign erythrocytes under the conditions described is generally accepted as a prototype, for experimental models designed to study antibody responses dependent upon T-cell helper function (3, 4, 14, 18, 27). A tenfold increase in the production
T-CELL
PROLIFERATION
OF Ighl
PFC
AND
AKTIBODY
TABI,E NUMREK
IN VITRO
PEK 106 CELLS
F:XPOSURE
OF SPLEEN
343
FORMATION
2 PKODIKED
CEIL
ON THE
CIJLTUKES
FIFTH
DAY
AFTKR
TO sRBC-TNI’
-
Cultures
tested
Number Horse
1. Cultures with
prepared from sRBC ; sRBC-TNP
mice
RBC-TNPe
injected 12 hr earlier were added in vitro
210
2. Cultures prepared from mice injected 12 hr earlier with sRBC. ThymidineJH pulse was administered at 12-22 hr; sRBC-TNP were added in o&o. 3. Cultures with
prepared from sRBC; sRBC-TNP
mice
injected 20 hr earlier were added in vitro.
4. Cultures prepared from mice injected 20 hr earlier with sRBC. ThymidineJH pulse was administered at 20-30 hr ; sRBC-TNP were added in vitro. 5. Cultures with
prepared from sRBC ; sRBC-TNP
mice
injected 70 hr earlier were added in vitro.
(anti-hRBC-TNP
*
against Sheep
20
1130
67zt
5
449
433 +
1.5
1987
78 z!c 10
1148
6. Cultures prepared from mice injected 70 hr earlier with sRBC. Thymidine-3H pulse was administered at 70-80 hr; sRBC-TNP were added in z&o. a “Background” PFC were subtracted were added in vitro instead of sRBC-TNP).
of PFC
PFC
RBC
+
71
zt 17
f
25
33.5 f
21
f
21
6678
f
42
984 f.
16
1354
f
18
of similar
cultures
were
sRBC
of anti-sRBC PFC was observed in spleen cell cultures prepared 24 hr after the in V~VOinjection of sRBC followed by in vitro challenge of the cultures with sRBC. Cultures obtained from similarly treated mice produced enhanced numbers of PFC against TNP, when challenged in vitro with sRBC-TNP. The latter result has provided strong supportive evidence for the occurrence of an enrichment and/or differentiation of antigen-specific helper T cells in the host spleen within the first 24 hr after injecton of foregn RBC (4). An appreciable enhancement of anti-sRBC PFC also occurred in spleen cultures prepared from mice injected with sRBC 12 hr earlier. It must be noted, however, that these in vifro results differ from the data reported by Sprent and Miller (28) in experiments where spleen cells obtained from mice which had been primed 24 hr earlier with sRBC were transferred (in viva) to irradiated hosts. It would appear, therefore, that the specific unresponiveness of cell recently primed in viva may not be a consequence of a transient induction of tolerance, increased susceptibility of blasts of trauma, or an expression of fewer antigen-binding receptors. Rather, this transient state of unresponsiveness may be due to subtle changes which are reflectd only in the in viva system, possibly associated with alterations in the ability of recently primed cells to relocalize to areas conducive to antibody production. A number of authors have suggested that active recruitment of antigen-reactive lymphocytes from the recirculating lymphocyte pool in secondary lymphoid organs occurs after injection of antigen (5-7). Earlier studies have also shown that the
344
BACHVAROFF
AND
RAPAPORT
immunogenic activity of injected foreign RBC is retained for prolonged periods 011 the surface of lymphoid cells in the mouse spleen, and such cell surfaces could be available for cell recruitment (13). However, if continuous recruitment of antigensensitive cells by the spleen were to account for the enhancement of antibody formation observed in these studies, spleen cultures initiated at 6 hr after sRBC injection might have been expected to yield increased numbers of PFC; this was not the case. Irradiation experiments indicate that spleen cultures initiated 24 or 48 hr after lethal irradiation of mice which had been injected with 2 x IO8 sRBC 1 hr prior to irradiation produced the usual enhanced number of PFC, if their spleens were shielded extracorporeally during irradiation (Fig. l), while controls whose spleens were irradiated were all negative. Also, if spleen cells from mice injected with sRBC were given lethal irradiation 24 hr later and were then added to normal spleen cell cultures, no enhancement in antibody production occurred. Taken together, these data demonstrate that, in the first 12-24 hr after intravenous injection of foreign erythrocytes, helper T-cell activity is not a consequence of recruitment of antigen-reactive cells (X-ray sensitive or -resistant cells) by the mouse spleen from the recirculating pool. This conclusion, however, must be confined at this time to the specific experimental conditions described, with particular regard to the organ under study, the antigen(s) used, and the dose and mode of administration of such antigen(s) in viva. Sprent and Miller (29)) for example, have recently observed a specific depletion of antigen-reactive circulating lymphocytes in mice injected with higher doses ( lo9 and lOlo) of sRBC than used in the present study. These authors concluded that such a depletion might have been due to recruitment of the lymphocytes by the spleen. The occurrence of an early in viva intrasplenic T-cell proliferation as a cause of the enhanced in vitro responses to foreign RBC observed in this study is supported by the tritiated thymidine hot-pulse experiments described. In these experiments, a 7-hr hot-pulse given to spleen cultures prepared from mice injected 20 hr earlier with sRBC eliminated the enhanced generation of PFC (Fig. 2). Similarly timed hot pulses administered to normal spleen cultures in the presence of antigen did not affect the number of PFC produced, when compared with nonpulsed controls, as noted earlier by Dutton and Mishell (12). If spleen cultures prepared from mice injected 12 and 20 hr earlier with carrier RBC alone were hot-pulsed for S-10 hr prior to challenge with sRBC-TNP, there was a great reduction in the production of anti-TNP-PFC, as compared to nonpulsed controls (Table 2). In contrast, hot pulses given to cultures obtained from mice injected with carrier cells 70 hr earlier generally had little or no effect upon the subsequent generation of TNP-PFC. These results indicate that DNA synthesis in spleen T cells commences in z&o within 12-20 hr after injection of a high dose of sRBC, and is subsiding within 70 hr. The data are also consistent with the notion that the T-cell priming process leading to their functional differentiation into helper cells is radiosensitive (15, 16). 0 rice differentiated, however, such helper cells become radioresistant (17, 18). The experiments also provide new evidence that a dissociation between the time of proliferation of antigen-reactive T and B cells may be occurring in the mouse spleen (19-21). Although T-cell proliferation as a consequence of exposure to certain antigens is a well-established observation (21), it is not clear whether the initial antigen-specific T-cell division is an absolute prerequisite for the develop-
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merit of helper activity, Although the results of this study do not provide a definitive answer to this question, the data would appear to favor such an interpretation. For example, when cultures prepared from mice injected 20 hr earlier with sRBC were hot-pulsed for 7 hr, enhancement of anti-sRBC PFC production was eliminated. These cultures did, however, produce a number of sRBC PFC comparable to that observed in normal pulsed or unpulsed cultures, with a delay of 24 hr (Fig. 2). Th’ IS ob servation raised the possibility that not all antigen-reactive T cells may have started division within 12-20 hr after in viva antigenic challenge, and only the T cells in their S phase were killed by the incorporated hot thymidine. However, antigen-reactive T cells which had not yet been triggered in Z&IO,,or were in a different phase at the time of hot-pulsing, may have escaped radioactive suicide. As a result, the development of helper activity was delayed and the subsequent proliferation and differentiation of antibody-forming B-cell precursors was delayed by 24 hr. These data also highlight the usefulness of thymidine-“H in the analysis of the dynamics of cell proliferation, since only cells synthesizing DNA are killed by this agent, thereby eliminating the possibility of interference with the results by transient effects or other variables. The observation that antigen must be present for both T- and B-cell proliferation may deserve special mention. As shown in Table 1, if no antigen is added in vitro to spleen cell cultures prepared from mice which had been injected with sRBC 624 hr earlier, the in vitro responses are minimal and similar to the responses to sRBC observed in normal mouse spleen cell cultures in fetal calf serum alone (2). The low antigen threshold required to trigger T-cell division (22, 23) has been confirmed in this study, since sRBC doses ranging from 2 X lo6 to 2 X lo8 cells injected 24 hr prior to initiation of cell cultures were equally effective for the subsequent in vitro enhancement of PFC formation in the presence of specific antigen. However, even the highest dose of injected sRBC was ineffective in triggering B-cell responses in dissociated spleen cell cultures if no antigen was added in vitro. It has been shown previously that approximately 10% of antigen sequestered in the spleen after a single injection of 2 X lo8 sRBC is retained by the spleen in a immunogenic form for 3 days (8). The presence of such retained immunogens, which are sufficient to trigger B-cell proliferation in the spleen environment, points to the existence of a mechanism of immunogen retention in the spleen which is equally effective for triggering T- and B-cell proliferation (24), but is inoperative for dissociated spleen cells in the absence of unprocessed antigen. Taken together, the data presented in this report are in close agreement with the interpretation of Miller (25), that “the role of T cells is linked to a capacity for “recognizing” the antigenic determinant and is specifically dependent, not on the passive transport of such antigenic determinants to B cells but upon some active process.” The results also provide evidence that, in order to develop such a function, the antigen-reactiT-e T cell may have to go through a process of cell division, as suggested by Anderson, Sprent, and Miller (26). ACKNOWLEDGMENTS The excellence of the technical assistance of Irene Allan, Susan Ball, Louis Browne, and Liling Wei is greatly appreciated. We are particularly indebted to Dr. Melvin Becker and Joan Siebert of the Department of Radiology, for their valuable help, and to Dr. Jeannette Thorbecke for her advice and assistance in the preparation of this manuscript.
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