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Lymphocytes and red blood cells as carriers in the immune response of mice and guinea pigs to soluble antigen
LYMPHOCYTES THE IMMUNE
AND RED BLOOD CELLS AS CARRIERS IN RESPONSE OF MICE AND GUINEA PIGS TO SOLUBLE ANTIGEN OTTO J. PLESCIA*
Waksman
Institute
of Microtxology.
and ROBERT
GUTMAN
Rutgers-The State University Piscataway. NJ 08854, U.S.A.
of New Jersey,
P.O. Box 750.
Abstract--Coupling of soluble antigen, such as Rig, to circulating lymphcndcells or red blood cells increased immunogenicity substantially. As little as 0.01 peg Rig per IO’ syngeneic cells elicited an antibody response in C57Bl:6J strain mice equal to 1300 pg of soluble Rig. About 20 times the amount of soluble Rig induced far less a cellular immune response in guinea pigs than Rig coupled to autologous peripheral blood cells. These cells apparently play a passive role in being able to deliver bound antigen to lymphoid tissues efficiently. thus increasing the probability of interaction between host helper T cells and antigen. This conclusion is based on the following observations: (I) red blood cells, which are not immunologically responder cell,. are about as efficient as lymphoid cells; (2) lymphoid cells are not effective as carriers m mice rendered deficient m antigen-specific helper T cells: (3) the antigen coupled to cells must have carrier specificity for host helper T cells.
INTRODLCTION
effects, mouse spleen lymphocytes and red blood cells were tested as carriers of rabbit immunoglobulin in inducing an antibody response in a syngeneic inbred strain of mice, and autologous peripheral blood leukocytes and red blood cells were tested in guinea pigs for their ability to induce a delayed hypersensitivity response to rabbit immunoglobulin. In both cases the soluble antigen was specific antibody in antisera from rabbits immunized with mouse spleen cells, including red blood cells, or with guinea pig peripheral blood cells. Thus, a simple absorption of the appropriate antiserum sufficed to bind rabbit immunoglobulin to white and red blood cells being tested as passive carriers. Both lymphocytes and red blood cells proved to be efficient passive carriers of rabbit immunoglobulin in inducing a humoral and cellular immune response to this antigen, although the lymphocytes tended to be better in most instances.
The capacity of an antigen to induce an immune response depends not only on the immunological competence of the host but also on factors associated with the antigen. One such factor is the delivery of antigen, since the number and kinds of immunocytes that are able to interact with antigen will determine the magnitude and type of immune response elicited. Immunogens, for example, have been coupled to haptens to act as carriers and thus to activate helper-T cells specifically. Here the carrier portion of the immunogen plays a direct active role by its binding to specific receptors on T cells. This binding, of course, requires accessibility ofT cells and immunogen to each other, and it suggests the possibility that circulating cells might be able to act as carriers for immunogens coupled to their surface by their ability to deliver the bound antigen to appropriate lymphoid tissues, thereby increasing the probability and efficiency of interaction between antigen and host immunocytes, and decreasing the amount of antigen required. In this context, the carrier cells would play a passive role except for their ability to circulate and concentrate in lymphoid tissues. Lymphocytes and red blood cells, because of their ability to circulate and localize in lymphoid tissue, were considered suitable for testing the concept of circulating cells as passive carriers of soluble antigen coupled to their surface. To eliminate the possible complication of allogenic
MATERIALS
AND
METHODS
Spleen cells from 6 to 8 week-old C57B l/6J mxe (obtained from Jackson Labs, Bar Harbor, ME) were dissociated in MEM medium by rupturing the capsule and pressing with forceps. The resulting suspension of cells was passed firat through a 23G syringe needle several times and then once through a 100 mesh stainless steel screen. after which 4 volumes of 0.83”,, aqueous NH,CI were added at room temperature to lyse the red blood cells (RBC), and leave Intact the white spleen cells (WSC). The mixture was immediately centrifuged at 800 g for 5 min and the sedimented white cells were washed twice with 2 volumes of MEM and resuspended in MEM. All the preceding steps were carried out at room temperature. The nucleated cells in this suspension were counted in a hemocytometer. and the volume adjusted to 10” cells per ml.
*He is inlinitely grateful to Professor Heidelberger for launching him in Immunochemistry as his associate at Columbia and for his guidance and inspiration since. t Abbreviations used: WSC. white spleen cells; RBC, red blood cells: WBC, white blood cells; Rig, rabbit serum Immunoglobulin; Rig-WSC, white spleen cells with specific rabbit antibody absorbed to their surface: this notation also applies to other cells.
Heparinired x33
blood,
freshly
drawn
from c‘S7B1’6J
mice.
L
5~
P ;
4-
a I
3
2-
injected i.p. into groups of IO C‘57Bl;6J mice. A second injection, containing the same number 01 coated cells as in the first injection, was given a week later. and a booster injection was given IO weeks later to all groups except for the group that had been primed with 10” antigen-coated cells per injection. As controls, groups of mice received 2 injectIon\ 01‘ soluble rabbit immunoglobulin. ranging in amount
a I
3
from I .3 ,ug to I300 pg (far in excess of the amount that was bound to cells). This lmmunoglobulin was the gamma-globulin fraction of normal rabbit serum. All mice were bled before, during and after being immunized. and their sera were assayed for hemagglutinating antibody reactive with RIgsensitized formalini7ed tanned sheep erythrocytes. according to the method of Daniel and Stavitsky ( 1964). The results (Figs. l-3) show a small difference in the dose-dependent antibody response to Rig between white and red blood cells as carriers, and a striking difference between cell bound Rig and soluble Rig. Syngeneic cells thus greatly increased the efficiency of the antibody response to antigen bound to their surface, the white cells being somewhat superior to the red cells inasmuch as both types ofcells had essentially equal amounts of Rig bound to their surf‘ace. This superiority is particularly evident in the priming activity of Rig-coated white cells. Lkpcwckwr on the unumnt o/ antigen per cell. To determine the threshold amount of Rig, carried by a fixed number ofcells and needed to induce an antibody response, I.0 ml of rabbit anti-WSC serum (diluted from 1I)I25 to I :32.000) was absorbed by IOHWSC to prepare Rig-WSC containing different amounts of Rig per cell. Groups of IO C57Bli6J mice were immunized as before, each group receiving one of these Rlg-WSC preparations. The number of cells per injection was IO’. The animals were bled before, during and after immunization. and their sera were assayed for hemagglutinating antibody as before. The results. showjn in Fig. 4. indicate that the antibody response is proportional to the amount of Rig per cell. and from these data one can determine the threshold amount of Rig per cell to give a primary response. If one assumes the antibody content of the rabbit anti-WSC serum to be 3 mg/ml (a typical value). the calculated minimal amount of Rig that can induce a primary antibody response is of the order of 0.03 /cg, IO‘ wsc.
Irwnunogrwicit~~
of
Rig-
@‘SC
in
inirl7utloc/l,fic,irrlt
An important question regarding the enhanced immunogenicity of Rlg bound to syngeneic WSC is whether or not the WSC serve an active role as helper cells in addition to their transport function. To answer this question, Rig-WSC was tested for immunogcnicity in mice rendered deficient in helper-T cells. Mice k+ccrcgiven lethal doses of X-radiation and rescued with a transplant of syngencic bone marrow cells as a source of B cells and precursors of T cells. These mice therefore had their B cells restored md were assumed to be deficient in helper-T cells because of the delay in the restoration of these cells in lethally irradiated mice (Miller & Mitchell. 1969). Another means of rendering mice deficient in helper T cells specific for Rig was to treat them with hydrocortisonc prior to administration of Rlg in complete t;reund‘$ adjuvant. according to the method of Scgal c’t trl. ( 1972). These immunodeficient animals. and also normal control animals that had been given Rlg In adjuvant without the hydrocortisone. were given two i.p. injections of Rlg-WSC one week apart and their scra were monitored for hemagglutinating antibody. The Rlg-coated cells normally induced an antibody response within one week after the second injection. but the hydrocortisone-treated animals failed to give a measurable antibody response to these Rig-coated cells at this time (Fig. 5). The X-irradiated mice did not survive long enough. deapitc the bone marrow\ transplant, to be sure that they could not respond to Rlg-coated cells at any time. Nevertheless. they did not respond at the early time period when normal control mice had responded. The results of these experiments suggest that syngenex WK. as used here. do not function actively as helper cells. mice.
Ccllulur
response
of Ruincw pig.5 to RIx
Since the immunogenicity
trzc~ologo~c
c
* /
‘lI.-__+ 2
Weeks after the first InJectIon Of lmmunoge” l-is. 3. Immunogenuty of soluble Rle In C‘S7BI 65 mice. Groups d 10 mice were eiven two i.p. Injections. one week apart, of. Rig in saline. The amount per injection %a\: I .3 pg (0): 13/[g (0); I30 pg (A): 1300 pg ( x ). Sera from each group wrc pooled in sets of 5 and assaved for hemagglufinating antihod). ’
Weeks
wlls
of Rip in mice. In terms of
after
the
first
X36
O’ITO
J. PLESC‘IA
bind ROBERT
GUTMAN
E
(a) 4 2
1
9
6
(b) a 2 k ix
1
lI.7
6
Q I
4
24
Weeks
I nlect
after
the
Ian of lmmunogen
Fg 5. Antibody response to Rig-WSC. by T cell-delicient C57B1;‘6J mice. Mice were made tolerant to R18 at the level of T cells by injecting i.p.. tirst 2.5 mg of hydrocortisone acetate and 48 hr later 100 1’9 Rig III complete Freund’s adjuvant. Control mice were given only Rig in adjutant. One week later both groups. the dcficicnt mice (0) and the controls (0). were given 2 i.p. injection\ one week apart o(‘: (a) 10’ Rig-WSC: (b) Rig-RBC: (c) about IO tlmcs the amount of soluble Rig: and (d) sahne. Sera of mice from each group were pooled In sets of 5 for anal+ of hemagglutInatIng antihodv.
antibody response, to syngeneic white
42
6%
first
was greatly enhanced by binding it and red cells. the carrier role of such
cells in the induction of a cellular response to Rlg was examined. Guinea pigs, rather than mice, were selected as hosts since the cellular response of guinea pigs can be easily and conveniently quantitated in terms of delayed skin reactivity to intradermal challenge by the test antigen. As before, Rig was used as antigen in the form of antibody, but this time it was specific for guinea pig peripheral white and red blood cells. because of the ease with which such antibody could be bound to the cells without damaging them. Regarding the genotype of the cells, autologous peripheral blood cells had to be used because the guinea pigs were not inbred. Peripheral blood was a convenient source because it could be obtained in sufficient quantity without having to sacrifice the donors. The backs of the guinea pigs were shaved, using hair clippers, the day before being immunized. Groups of four animals were given intradermal injections of either Rig-WSC, Rig-RBC, rabbit anti-guinea pig leukocyte serum or normal rabbit serum. The volume of each injection was 0.1 ml. The number of RIgcoated cells was 5 x 106, and the rabbit sera (Millipore filtered to remove any bacterial contaminants) was diluted I/4 with pyrogen-free injectible saline. The sites of injection were observed daily for skin reactions during the first three days. Erythema was observed in some sites and the diameter of these
Hours of
after
Injection
lmmu”oge”
I-le. 6. Erythema skin reactlons of male Hartlq stram guinea pig at the rite of the intradermnl mjcctlon otl 5 x I Oh Rig-autologow WBC‘ (III): 5 x 10” Rig-autologous RBC (@): ~tbout 70 time\ the amount of Rig from rabbit antiserum to guinea pig penpheral blood cells (A): or about 20 times the amount of Rig from normid rabbit xrum ( x ). The amount of Rig coupled to the cells ws about 20 /lg. Reactions are reported ~ls the square of the average diameter of the crythematous xc;,. and each value i\ the aLerage reaction of4 animals in each group at Z sttes per animal.
reactions was measured (Fig. 6). The Rig-WBC in particular provoked an early and greater skin reaction than any of the other Rig preparations. When the skin reactions had subsided, about one week after the primary injections. each of the guinea pigs was challenged intradermally with 10 pg Rig to determine the degree of sensitization induced by the four different Rig preparations. Figure 7 shows the delayed hypersensitivity reactions to this Rig challenge. The Rig bound to autologous WBC or RBC was distinctly superior to soluble Rig in eliciting a cellular response. despite the fact that about IO-20 times more soluble Rig was used. The white and red cells. which had bound equal amounts of Rig, tended to be equally effective. Regarding the rabbit sera, they were minimally effective, and the specific antibody in the rabbit antiserum was no more effective than normal Rig, although the guinea pig leukocytespecific antibody presumably could bind to the skin on injection. If so, it would appear that binding of Rig to circulating white and red blood cells is advantageous.
This study was undertaken to answer the question whether or not binding of a soluble antigen to circulating white cells could enhance its immunogenicity, and if so, whether or not these cells played only a passive role in delivering the antigen efficiently to appropriate lymphoid organs. The answer to both parts of this question is yes.
Hours of
after
the
IO /.~g
Injection of
RGG
Fig. 7. Delayed
hypcrsenaiti\lt> \kln reactIons of male Hartley strain guinea pigs to intradermal challenge with IOpg Rig one week after Ihcy were sensitized by injecting intradermally: 5 x IO” Rig-autologous WBC (0): 5 x IO” Rig-autologous RBC (0): Rig from rabbit antiserum to guinea pig peripheral blood cells (A): or Rig from normal rabbit serum ( x ). See legend to Fig. 6 for amount of RIe In each preparation. and method of mcnsurlng and recorc~ng
The antibody response of mice to Rig bound to syngeneic WSC far exceeded their response to equivalent amounts of soluble Rig. About 0.01 pg of Rig bound to IO’ WSC sufficed to give an antibody response equal to that elicited by I300 pg soluble Rig. The response to Rig-WSC was dose dependent, that is, on the total amount of Rig injected. The amount needed per cell was inversely proportional to the number of cells injected and for a fixed number of cells there was a threshold minimum value of the order of about 0.01 pg per 10: cells. Syngeneic RBC were about as effective as WSC in increasing the immunogenicity of Rig. This finding indicates that the WSC were functioning passively as part of an efficient delivery system, rather than as active helper cells, because RBC are not immune responder cells in the pathway to antibody formation. Further evidence in support of this conclusion was the lack of immunogenicity of Rig-WSC in mice rendered deficient in Rig-specific helper T cells by treatment with hydrocortisone and soluble Rig in complete Freund’s adjuvant, and also the fact that DNP-poly-Llysine coupled to WSC electrostatically was not immunogenic in C57Bli6J mice (results not shown). In contrast to Rig, DNP-poly-L-lysine is poorly immunogenic. presumably because it lacks adequate carrier specificity. These results, taken as a whole, suggest to us that Rig bound to WSC or RBC uses a normal pathway to antibody formation, including the help of T cells that recognize the carrier specificity of Rig. This is probably the reason why the cells used in this study seem able to increase the efficiency of
immunogens but not haptens. In fact, the coupling of haptens to syngeneic lymphoid cells tends to induce a state of tolerance (Long & Scott, 1977). The binding of Rig to circulating white and red cells increases not only the humoral antibody response but also the cellular immune response to Rig. In this study a single intradermal injection of Rig bound to autologous peripheral blood cells, in the absence of complete Freund’s adjuvant, elicited a significantly enhanced delayed hypersensitivity response to Rig challenge compared with the response to soluble Rig in much larger amounts. Here also red blood cells proved quite efficient in promoting the cellular immune response to Rig, again indicating a passive role for these cells. These cells were injected intradermally, and it is not known whether they remained at the site of injection or were drained away into the general circulation. If they did not drain away. these cells presumably facilitated the interaction of Rig with circulating host T lymphocytes by immobilizing the antigen. If they entered the circulation. their role was probably that of an efficient delivery system, as in the case of the antibody response of mice given Rig-cells intraperitoneally. It is of interest that the guinea pigs immunized with Rig-cells intradermally did not develop circulating antibodies to Rig. suggesting that the cells with bound Rig may not have circulated appreciably and that this procedure. of binding antigen to autologous white or red blood cells and injecting them intradermally, might be an effective way to induce a cellular immune response without antibody formation. Others have also reported increased immunogenicity of antigen associated with cells. Howard et al. (1968) found that antibody to lymphocytes, absorbed on to lymphocytes, is much more immunogenic than free antibody. Reithmuller or ul. (1971) observed that absorption of anti-IgM immunoglobulin by spleen cells with IgM on their surface increased its immunogenicity in syngeneic mice. In apparent contrast to our results, showing only a passive role for spleen cells as carriers of Rig, Miller et nl. (1971) showed that thoracic duct lymphocytes coated with fowl immunoglobulin can elicit antibody response to this antigen in syngeneic thymectomized mice made deficient in helper T cells by lethal xradiation and reconstitution with bone marrow cells. They observed further that thoracic duct cells from donors made tolerant to fowl immunoglobulins were inactive in T-cell-deticient recipients. In this latter respect our results agree, that passive carrier cells require help from antigen-specific T cells. A possible explanation for our failure to find evidence of helper activity associated with our spleen white cells is that the Rig used to coat the spleen cells, unlike the fowl immunoglobulin bound to thoracic duct cells, was spleen cell-specific antibody with cytotoxic activity for spleen cells in the presence of complement. This activity of antibody in vivn might have aborted any helper activity of Rig-WSC. However, DNP poly-Llysine, which we also used, is not cytotoxic, and yet the WSC were inactive as carriers of any kind. Macrophages, like T lymphocytes. also display carrier function, and seem able to modify the immunogenicity of antigen associated with such cells, For example, Seeger and Oppenheim (1973u,h)
Y’X I .,
OTTO
J. PLESC‘IA
and ROBERT
reported that minute amounts of antigen. associated with macrophages but not in free soluble form. stimulated in guinea pigs a pure delayed hypersensitivity response. Macrophages are known to take up and process a variety of antigens, and according to Unanue and Cerottini (1970) l-7”,, of the antigen is retained for long periods of time bound to membrane. Thus. antigen-coated the plasma macrophages. like the antigen-coated spleen cells reported here, may serve passively as targets for T cells. The macrophage, that takes up antigen in a physiological sense, also plays an active role in processing the antigen to produce a super antigen with RNA (Fishman & Adler, 1967; Unanue & Askonas. 1968: Mitchison, 1969; Gottlieb, 1969). The binding of antigen to lymphocytes by artificial means is non-physiological. and it is not surprising therefore that no new insight regarding the normal physiological function of these lymphocytes wax gained in this study. It is evident, however, that they can serve as efficient passive carriers for immunogens coupled to them, and in this respect, red blood cells are about as efficient. Current studies. with antigens other than the immunoglobulins used in this investigation. indicate the use of lymphocytes and red blood cells as passive carriers to be an efficient way to increase the immunogenicity of antigens. thus reducing substantially the amount of antigen needed to induce an immune response.
CiUTMAN
AND RED BLOOD CELLS AS CARRIERS IN RESPONSE OF MICE AND GUINEA PIGS TO SOLUBLE ANTIGEN OTTO J. PLESCIA*
Waksman
Institute
of Microtxology.
and ROBERT
GUTMAN
Rutgers-The State University Piscataway. NJ 08854, U.S.A.
of New Jersey,
P.O. Box 750.
Abstract--Coupling of soluble antigen, such as Rig, to circulating lymphcndcells or red blood cells increased immunogenicity substantially. As little as 0.01 peg Rig per IO’ syngeneic cells elicited an antibody response in C57Bl:6J strain mice equal to 1300 pg of soluble Rig. About 20 times the amount of soluble Rig induced far less a cellular immune response in guinea pigs than Rig coupled to autologous peripheral blood cells. These cells apparently play a passive role in being able to deliver bound antigen to lymphoid tissues efficiently. thus increasing the probability of interaction between host helper T cells and antigen. This conclusion is based on the following observations: (I) red blood cells, which are not immunologically responder cell,. are about as efficient as lymphoid cells; (2) lymphoid cells are not effective as carriers m mice rendered deficient m antigen-specific helper T cells: (3) the antigen coupled to cells must have carrier specificity for host helper T cells.
INTRODLCTION
effects, mouse spleen lymphocytes and red blood cells were tested as carriers of rabbit immunoglobulin in inducing an antibody response in a syngeneic inbred strain of mice, and autologous peripheral blood leukocytes and red blood cells were tested in guinea pigs for their ability to induce a delayed hypersensitivity response to rabbit immunoglobulin. In both cases the soluble antigen was specific antibody in antisera from rabbits immunized with mouse spleen cells, including red blood cells, or with guinea pig peripheral blood cells. Thus, a simple absorption of the appropriate antiserum sufficed to bind rabbit immunoglobulin to white and red blood cells being tested as passive carriers. Both lymphocytes and red blood cells proved to be efficient passive carriers of rabbit immunoglobulin in inducing a humoral and cellular immune response to this antigen, although the lymphocytes tended to be better in most instances.
The capacity of an antigen to induce an immune response depends not only on the immunological competence of the host but also on factors associated with the antigen. One such factor is the delivery of antigen, since the number and kinds of immunocytes that are able to interact with antigen will determine the magnitude and type of immune response elicited. Immunogens, for example, have been coupled to haptens to act as carriers and thus to activate helper-T cells specifically. Here the carrier portion of the immunogen plays a direct active role by its binding to specific receptors on T cells. This binding, of course, requires accessibility ofT cells and immunogen to each other, and it suggests the possibility that circulating cells might be able to act as carriers for immunogens coupled to their surface by their ability to deliver the bound antigen to appropriate lymphoid tissues, thereby increasing the probability and efficiency of interaction between antigen and host immunocytes, and decreasing the amount of antigen required. In this context, the carrier cells would play a passive role except for their ability to circulate and concentrate in lymphoid tissues. Lymphocytes and red blood cells, because of their ability to circulate and localize in lymphoid tissue, were considered suitable for testing the concept of circulating cells as passive carriers of soluble antigen coupled to their surface. To eliminate the possible complication of allogenic
MATERIALS
AND
METHODS
Spleen cells from 6 to 8 week-old C57B l/6J mxe (obtained from Jackson Labs, Bar Harbor, ME) were dissociated in MEM medium by rupturing the capsule and pressing with forceps. The resulting suspension of cells was passed firat through a 23G syringe needle several times and then once through a 100 mesh stainless steel screen. after which 4 volumes of 0.83”,, aqueous NH,CI were added at room temperature to lyse the red blood cells (RBC), and leave Intact the white spleen cells (WSC). The mixture was immediately centrifuged at 800 g for 5 min and the sedimented white cells were washed twice with 2 volumes of MEM and resuspended in MEM. All the preceding steps were carried out at room temperature. The nucleated cells in this suspension were counted in a hemocytometer. and the volume adjusted to 10” cells per ml.
*He is inlinitely grateful to Professor Heidelberger for launching him in Immunochemistry as his associate at Columbia and for his guidance and inspiration since. t Abbreviations used: WSC. white spleen cells; RBC, red blood cells: WBC, white blood cells; Rig, rabbit serum Immunoglobulin; Rig-WSC, white spleen cells with specific rabbit antibody absorbed to their surface: this notation also applies to other cells.
Heparinired x33
blood,
freshly
drawn
from c‘S7B1’6J
mice.
L
5~
P ;
4-
a I
3
2-
injected i.p. into groups of IO C‘57Bl;6J mice. A second injection, containing the same number 01 coated cells as in the first injection, was given a week later. and a booster injection was given IO weeks later to all groups except for the group that had been primed with 10” antigen-coated cells per injection. As controls, groups of mice received 2 injectIon\ 01‘ soluble rabbit immunoglobulin. ranging in amount
a I
3
from I .3 ,ug to I300 pg (far in excess of the amount that was bound to cells). This lmmunoglobulin was the gamma-globulin fraction of normal rabbit serum. All mice were bled before, during and after being immunized. and their sera were assayed for hemagglutinating antibody reactive with RIgsensitized formalini7ed tanned sheep erythrocytes. according to the method of Daniel and Stavitsky ( 1964). The results (Figs. l-3) show a small difference in the dose-dependent antibody response to Rig between white and red blood cells as carriers, and a striking difference between cell bound Rig and soluble Rig. Syngeneic cells thus greatly increased the efficiency of the antibody response to antigen bound to their surface, the white cells being somewhat superior to the red cells inasmuch as both types ofcells had essentially equal amounts of Rig bound to their surf‘ace. This superiority is particularly evident in the priming activity of Rig-coated white cells. Lkpcwckwr on the unumnt o/ antigen per cell. To determine the threshold amount of Rig, carried by a fixed number ofcells and needed to induce an antibody response, I.0 ml of rabbit anti-WSC serum (diluted from 1I)I25 to I :32.000) was absorbed by IOHWSC to prepare Rig-WSC containing different amounts of Rig per cell. Groups of IO C57Bli6J mice were immunized as before, each group receiving one of these Rlg-WSC preparations. The number of cells per injection was IO’. The animals were bled before, during and after immunization. and their sera were assayed for hemagglutinating antibody as before. The results. showjn in Fig. 4. indicate that the antibody response is proportional to the amount of Rig per cell. and from these data one can determine the threshold amount of Rig per cell to give a primary response. If one assumes the antibody content of the rabbit anti-WSC serum to be 3 mg/ml (a typical value). the calculated minimal amount of Rig that can induce a primary antibody response is of the order of 0.03 /cg, IO‘ wsc.
Irwnunogrwicit~~
of
Rig-
@‘SC
in
inirl7utloc/l,fic,irrlt
An important question regarding the enhanced immunogenicity of Rlg bound to syngeneic WSC is whether or not the WSC serve an active role as helper cells in addition to their transport function. To answer this question, Rig-WSC was tested for immunogcnicity in mice rendered deficient in helper-T cells. Mice k+ccrcgiven lethal doses of X-radiation and rescued with a transplant of syngencic bone marrow cells as a source of B cells and precursors of T cells. These mice therefore had their B cells restored md were assumed to be deficient in helper-T cells because of the delay in the restoration of these cells in lethally irradiated mice (Miller & Mitchell. 1969). Another means of rendering mice deficient in helper T cells specific for Rig was to treat them with hydrocortisonc prior to administration of Rlg in complete t;reund‘$ adjuvant. according to the method of Scgal c’t trl. ( 1972). These immunodeficient animals. and also normal control animals that had been given Rlg In adjuvant without the hydrocortisone. were given two i.p. injections of Rlg-WSC one week apart and their scra were monitored for hemagglutinating antibody. The Rlg-coated cells normally induced an antibody response within one week after the second injection. but the hydrocortisone-treated animals failed to give a measurable antibody response to these Rig-coated cells at this time (Fig. 5). The X-irradiated mice did not survive long enough. deapitc the bone marrow\ transplant, to be sure that they could not respond to Rlg-coated cells at any time. Nevertheless. they did not respond at the early time period when normal control mice had responded. The results of these experiments suggest that syngenex WK. as used here. do not function actively as helper cells. mice.
Ccllulur
response
of Ruincw pig.5 to RIx
Since the immunogenicity
trzc~ologo~c
c
* /
‘lI.-__+ 2
Weeks after the first InJectIon Of lmmunoge” l-is. 3. Immunogenuty of soluble Rle In C‘S7BI 65 mice. Groups d 10 mice were eiven two i.p. Injections. one week apart, of. Rig in saline. The amount per injection %a\: I .3 pg (0): 13/[g (0); I30 pg (A): 1300 pg ( x ). Sera from each group wrc pooled in sets of 5 and assaved for hemagglufinating antihod). ’
Weeks
wlls
of Rip in mice. In terms of
after
the
first
X36
O’ITO
J. PLESC‘IA
bind ROBERT
GUTMAN
E
(a) 4 2
1
9
6
(b) a 2 k ix
1
lI.7
6
Q I
4
24
Weeks
I nlect
after
the
Ian of lmmunogen
Fg 5. Antibody response to Rig-WSC. by T cell-delicient C57B1;‘6J mice. Mice were made tolerant to R18 at the level of T cells by injecting i.p.. tirst 2.5 mg of hydrocortisone acetate and 48 hr later 100 1’9 Rig III complete Freund’s adjuvant. Control mice were given only Rig in adjutant. One week later both groups. the dcficicnt mice (0) and the controls (0). were given 2 i.p. injection\ one week apart o(‘: (a) 10’ Rig-WSC: (b) Rig-RBC: (c) about IO tlmcs the amount of soluble Rig: and (d) sahne. Sera of mice from each group were pooled In sets of 5 for anal+ of hemagglutInatIng antihodv.
antibody response, to syngeneic white
42
6%
first
was greatly enhanced by binding it and red cells. the carrier role of such
cells in the induction of a cellular response to Rlg was examined. Guinea pigs, rather than mice, were selected as hosts since the cellular response of guinea pigs can be easily and conveniently quantitated in terms of delayed skin reactivity to intradermal challenge by the test antigen. As before, Rig was used as antigen in the form of antibody, but this time it was specific for guinea pig peripheral white and red blood cells. because of the ease with which such antibody could be bound to the cells without damaging them. Regarding the genotype of the cells, autologous peripheral blood cells had to be used because the guinea pigs were not inbred. Peripheral blood was a convenient source because it could be obtained in sufficient quantity without having to sacrifice the donors. The backs of the guinea pigs were shaved, using hair clippers, the day before being immunized. Groups of four animals were given intradermal injections of either Rig-WSC, Rig-RBC, rabbit anti-guinea pig leukocyte serum or normal rabbit serum. The volume of each injection was 0.1 ml. The number of RIgcoated cells was 5 x 106, and the rabbit sera (Millipore filtered to remove any bacterial contaminants) was diluted I/4 with pyrogen-free injectible saline. The sites of injection were observed daily for skin reactions during the first three days. Erythema was observed in some sites and the diameter of these
Hours of
after
Injection
lmmu”oge”
I-le. 6. Erythema skin reactlons of male Hartlq stram guinea pig at the rite of the intradermnl mjcctlon otl 5 x I Oh Rig-autologow WBC‘ (III): 5 x 10” Rig-autologous RBC (@): ~tbout 70 time\ the amount of Rig from rabbit antiserum to guinea pig penpheral blood cells (A): or about 20 times the amount of Rig from normid rabbit xrum ( x ). The amount of Rig coupled to the cells ws about 20 /lg. Reactions are reported ~ls the square of the average diameter of the crythematous xc;,. and each value i\ the aLerage reaction of4 animals in each group at Z sttes per animal.
reactions was measured (Fig. 6). The Rig-WBC in particular provoked an early and greater skin reaction than any of the other Rig preparations. When the skin reactions had subsided, about one week after the primary injections. each of the guinea pigs was challenged intradermally with 10 pg Rig to determine the degree of sensitization induced by the four different Rig preparations. Figure 7 shows the delayed hypersensitivity reactions to this Rig challenge. The Rig bound to autologous WBC or RBC was distinctly superior to soluble Rig in eliciting a cellular response. despite the fact that about IO-20 times more soluble Rig was used. The white and red cells. which had bound equal amounts of Rig, tended to be equally effective. Regarding the rabbit sera, they were minimally effective, and the specific antibody in the rabbit antiserum was no more effective than normal Rig, although the guinea pig leukocytespecific antibody presumably could bind to the skin on injection. If so, it would appear that binding of Rig to circulating white and red blood cells is advantageous.
This study was undertaken to answer the question whether or not binding of a soluble antigen to circulating white cells could enhance its immunogenicity, and if so, whether or not these cells played only a passive role in delivering the antigen efficiently to appropriate lymphoid organs. The answer to both parts of this question is yes.
Hours of
after
the
IO /.~g
Injection of
RGG
Fig. 7. Delayed
hypcrsenaiti\lt> \kln reactIons of male Hartley strain guinea pigs to intradermal challenge with IOpg Rig one week after Ihcy were sensitized by injecting intradermally: 5 x IO” Rig-autologous WBC (0): 5 x IO” Rig-autologous RBC (0): Rig from rabbit antiserum to guinea pig peripheral blood cells (A): or Rig from normal rabbit serum ( x ). See legend to Fig. 6 for amount of RIe In each preparation. and method of mcnsurlng and recorc~ng
The antibody response of mice to Rig bound to syngeneic WSC far exceeded their response to equivalent amounts of soluble Rig. About 0.01 pg of Rig bound to IO’ WSC sufficed to give an antibody response equal to that elicited by I300 pg soluble Rig. The response to Rig-WSC was dose dependent, that is, on the total amount of Rig injected. The amount needed per cell was inversely proportional to the number of cells injected and for a fixed number of cells there was a threshold minimum value of the order of about 0.01 pg per 10: cells. Syngeneic RBC were about as effective as WSC in increasing the immunogenicity of Rig. This finding indicates that the WSC were functioning passively as part of an efficient delivery system, rather than as active helper cells, because RBC are not immune responder cells in the pathway to antibody formation. Further evidence in support of this conclusion was the lack of immunogenicity of Rig-WSC in mice rendered deficient in Rig-specific helper T cells by treatment with hydrocortisone and soluble Rig in complete Freund’s adjuvant, and also the fact that DNP-poly-Llysine coupled to WSC electrostatically was not immunogenic in C57Bli6J mice (results not shown). In contrast to Rig, DNP-poly-L-lysine is poorly immunogenic. presumably because it lacks adequate carrier specificity. These results, taken as a whole, suggest to us that Rig bound to WSC or RBC uses a normal pathway to antibody formation, including the help of T cells that recognize the carrier specificity of Rig. This is probably the reason why the cells used in this study seem able to increase the efficiency of
immunogens but not haptens. In fact, the coupling of haptens to syngeneic lymphoid cells tends to induce a state of tolerance (Long & Scott, 1977). The binding of Rig to circulating white and red cells increases not only the humoral antibody response but also the cellular immune response to Rig. In this study a single intradermal injection of Rig bound to autologous peripheral blood cells, in the absence of complete Freund’s adjuvant, elicited a significantly enhanced delayed hypersensitivity response to Rig challenge compared with the response to soluble Rig in much larger amounts. Here also red blood cells proved quite efficient in promoting the cellular immune response to Rig, again indicating a passive role for these cells. These cells were injected intradermally, and it is not known whether they remained at the site of injection or were drained away into the general circulation. If they did not drain away. these cells presumably facilitated the interaction of Rig with circulating host T lymphocytes by immobilizing the antigen. If they entered the circulation. their role was probably that of an efficient delivery system, as in the case of the antibody response of mice given Rig-cells intraperitoneally. It is of interest that the guinea pigs immunized with Rig-cells intradermally did not develop circulating antibodies to Rig. suggesting that the cells with bound Rig may not have circulated appreciably and that this procedure. of binding antigen to autologous white or red blood cells and injecting them intradermally, might be an effective way to induce a cellular immune response without antibody formation. Others have also reported increased immunogenicity of antigen associated with cells. Howard et al. (1968) found that antibody to lymphocytes, absorbed on to lymphocytes, is much more immunogenic than free antibody. Reithmuller or ul. (1971) observed that absorption of anti-IgM immunoglobulin by spleen cells with IgM on their surface increased its immunogenicity in syngeneic mice. In apparent contrast to our results, showing only a passive role for spleen cells as carriers of Rig, Miller et nl. (1971) showed that thoracic duct lymphocytes coated with fowl immunoglobulin can elicit antibody response to this antigen in syngeneic thymectomized mice made deficient in helper T cells by lethal xradiation and reconstitution with bone marrow cells. They observed further that thoracic duct cells from donors made tolerant to fowl immunoglobulins were inactive in T-cell-deticient recipients. In this latter respect our results agree, that passive carrier cells require help from antigen-specific T cells. A possible explanation for our failure to find evidence of helper activity associated with our spleen white cells is that the Rig used to coat the spleen cells, unlike the fowl immunoglobulin bound to thoracic duct cells, was spleen cell-specific antibody with cytotoxic activity for spleen cells in the presence of complement. This activity of antibody in vivn might have aborted any helper activity of Rig-WSC. However, DNP poly-Llysine, which we also used, is not cytotoxic, and yet the WSC were inactive as carriers of any kind. Macrophages, like T lymphocytes. also display carrier function, and seem able to modify the immunogenicity of antigen associated with such cells, For example, Seeger and Oppenheim (1973u,h)
Y’X I .,
OTTO
J. PLESC‘IA
and ROBERT
reported that minute amounts of antigen. associated with macrophages but not in free soluble form. stimulated in guinea pigs a pure delayed hypersensitivity response. Macrophages are known to take up and process a variety of antigens, and according to Unanue and Cerottini (1970) l-7”,, of the antigen is retained for long periods of time bound to membrane. Thus. antigen-coated the plasma macrophages. like the antigen-coated spleen cells reported here, may serve passively as targets for T cells. The macrophage, that takes up antigen in a physiological sense, also plays an active role in processing the antigen to produce a super antigen with RNA (Fishman & Adler, 1967; Unanue & Askonas. 1968: Mitchison, 1969; Gottlieb, 1969). The binding of antigen to lymphocytes by artificial means is non-physiological. and it is not surprising therefore that no new insight regarding the normal physiological function of these lymphocytes wax gained in this study. It is evident, however, that they can serve as efficient passive carriers for immunogens coupled to them, and in this respect, red blood cells are about as efficient. Current studies. with antigens other than the immunoglobulins used in this investigation. indicate the use of lymphocytes and red blood cells as passive carriers to be an efficient way to increase the immunogenicity of antigens. thus reducing substantially the amount of antigen needed to induce an immune response.
CiUTMAN