INFECTION AND IMMUNITY, Oct. 1975, p. 761-767 Copyright ) 1975 American Society for Microbiology Vol. 12, No. 4 Printed in U.S.A. Nature of "Memory" in T-Cell-Mediated Antibacterial Immunity: Cellular Parameters That Distinguish Between the Active Immune Response and a State of "Memory" R. J. NORTH* AND J. F. DEISSLER Trudeau Institute, Inc., Saranac Lake, New York 12983 Received for publication 28 May 1975 Immunizing infection in mice with Listeria monocytogenes resulted in the generation of two distinct states of immunological reactivity. There was generated (i) a short-lived state of active immunity that functioned to urgently eliminate the infecting organism from the tissues and (ii) a long-lived state of increased immunological potential that enabled the host to respond to secondary infection in an accelerated manner. Short-lived active immunity was mediated by replicating T cells and expressed by activated macrophages, and it ended when these cell types disappeared from the tissue soon after complete elimination of the parasite. Long-lived immunological potential was associated with a persistent level of delayed sensitivity and with the presence of a small number of nonreplicating protective T cells. It is suggested that the state of delayed sensitivity represents a state of immunological T-cell memory of the cell-mediated type. It was shown in a preceding paper (14) that termination of an immunizing infection with Listeria monocytogenes in mice is followed by a state of long-lived, heightened, specific resistance to reinfection. It was also shown that the acquired resistance as expressed against lethal challenge infection was dependent on an acquired long-term capacity on the part of the immunized host to generate protective lymphocytes faster and in larger numbers than a nonimmunized host. The lymphocytes generated to combat secondary infection, moreover, were shown to be of the same class as those generated to combat primary infection. In both cases they were thymus-derived lymphocytes (T cells), many of which were replicating. It was suggested, in view of this evidence, that the enhanced production of mediator T cells that occurs in response to secondary Listeria infection represents the expression of a state of immunological memory. The expression of this form of T-cell memory as an accelerated and increased production of sensitized mediator T cells, then, is analogous to the expression of antibody or B-cell memory as an accelerated production of antibody-forming B cells. The term "immunological memory," however, implies that a state of acquired heightened reactivity to specific antigen can exist long after the active immune response has ended (4), i.e., long after the cells that mediate or effect immunity have disappeared. Indeed, it is now well established (4, 5, 7, 17) that immuno- 761 logical memory acquired during an antibody response is carried by lymphocytes that are physiologically different from those that secrete antibody, even though the states of active immunity and memory can coexist. The same conclusion, however, cannot yet be made about memory in T-cell-mediated antibacterial immunity. As a first step in investigating the cellular basis of memory in T-cell-mediated antibacterial immunity, therefore, it was necessary to obtain information showing that the state of active immunity generated to terminate a primary Listeria infection is different from the acquired state of heightened, specific resistance to reinfection that persists for a long time after the primary infecting organism is eliminated from the tissues. This paper will show that the active immune response to an intravenous Listeria infection occupies a limited period of time, during which there is increased lymphoid cell division and the generation of short-lived mediator T cells in the spleen, the generation of a state of delayed sensitivity to Listeria antigens, and the systemic activation of macrophages. It will show, in addition, that loss of replicating mediator T cells and effector macrophages results in loss of active immunity, and that this is followed by a long-lived state of heightened resistance to reinfection that is temporally associated with a persistent level of delayed sensitivity and with the retention of a small number of nonreplicating "protective" T cells.
762 NORTH AND DEISSLER MATERIALS AND METHODS Mice, the preparation oflisteria cultures, and the method used for estimating changes in the level of production of protective lymphocytes in the spleen were the same as described in the preceding paper (14). The methods for selectively destroying dividing protective cells in vivo with a 15-h pulse of vinblastine sulfate, and for testing the susceptibility of protective cells to incubation in vitro with AKR anti-c3h 0-serum and complement, were also the same as in the preceding paper (14). Spleen cell division. Changes in the relative number of cells dividing in the spleen were followed by measuring changes in the amount of tritiated thymidine ([3H]TdR of specific activity 3 Ci/mmol obtained from New England Nuclear Corp., Boston, Mass.) incorporated into total spleen deoxyribonucleic acid (DNA) from a single 20-pCi injection given intravenously. Spleens were removed 30 min after being given [3H]TdR, and an acid-soluble extract was prepared and counted (counts per minute per organ) by liquid scintillation spectrophotometry as described previously (10). Systemic macrophage activation. Changes in the level of systemic macrophage activation during the course of a Listeria infection were determined by measuring changes in the capacity of mice to resist a standard 105 intravenous challenge infection with the bacterium Yersinia enterocolitica (strain WA). The organism was kindly provided by P. Carter (Trudeau Institute), who has published a description of its growth characteristics in mice (3). Nonspecific resistance was expressed as log,0 resistance index. This was obtained by subtracting the 24-h growth of Yersinia in the livers of Listeria-infected mice from its 24-h growth in the livers of control mice. A resistance index of 1 means that Yersinia grew 1 log less in Listeria-infected mice. The meaning of the resistance index in terms of macrophage activation has been discussed previously (11). Antibody. Serum antibodies produced in response to Listeria infection were measured by the technique of passive hemagglutination, which employed soluble Listeria antigens coupled to sheep erythrocytes with glutaraldehyde (9). Appropriate controls were included. The test serum was compared with a positive rabbit anti-listeria serum that gave an agglutination titer of 1:4,096. The soluble Listeria protein antigens were obtained from a Listeria culture filtrate by precipitation with ammonium sulfate followed by purification by exclusion chromatography (LeGarrec and Mackaness, to be published). Delayed sensitivity. Delayed sensitivity reactions were measured in the hind footpad with dial calipers as described previously (10). Delayed reactions were elicited by an intra-footpad injection of 10 Ag of Listeria protein antigens in 0.05 ml of 0.85% sodium chloride solution and read 24 h later. The antigens were the same as those used for the hemagglutination assay. RESULTS Cellular changes that characterize the primary response, memory, and expression INFECT. IMMUN. of memory. A previous study showed (14) that an immunizing infection with Listeria results in the acquisition of a long-term state of heightened immunological potential that enables the host to respond to secondary infection with an increased rate of production of mediator lymphocytes. The following study was designed to compare the primary and secondary anti-listeria responses in terms of some additional parameters and to use the information to distinguish between these states of active immunity and the state of memory that temporarily separates them. This involved performing concurrent measurements of (i) changes against time of infection in DNA synthesis and the production of dividing and nondividing protective lymphocytes in the spleen and (ii) changes in the level of systemic delayed sensitivity as measured by 24-h footpad reactions to an injection ofsoluble Listeria antigens. In addition, the production of serum antibodies to the same antigens as those used to elicit delayed sensitivity reactions was followed by the method of passive hemagglutination. All of these parameters were followed for 50 days after initiating primary infection and for 20 days after initiating secondary infection. The results are summarized in Fig. 1 and 2. It was found (Fig. 1), as expected from a previous study (14), that lethal secondary infection was eliminated more quickly than a sublethal primary infection. Whereas the primary infection multiplied for 3 days before being progressively eliminated by days 10 to 12, the secondary infection multiplied for only 24 h and was eliminated completely by day 8. The earlier expression of immunity to secondary infection, moreover, was associated with a faster rate of production in the spleen of lymphocytes that could protect normal test recipients against lethal challenge. Thus, although production of these cells in response to primary infection peaked on day 6, a higher level of peak production occurred 2 days earlier in response to secondary infection, and this occurred in spite of a lower level of infection. Indeed, this faster rate of production of protective lymphocytes in response to secondary infection was evident by the second day of infection. Peak production of protective cells in both responses was immediately followed by a progressive decline in their number. A more rapid decline, however, occurred after peak secondary production, and this was associated with the faster rate of elimination of Listeria from the tissues. Protective lymphocytes did not completely disappear from the spleen over the course of this study. In fact, the initial progressive rapid loss of these cells that immediately followed
VOL. 12, 1975 ACTIVE IMMUNE RESPONSE AND "MEMORY' IN T CELLS 763 involved the production of both types of protective cells. However, whereas dividing protective cells completely disappeared 6 days after peak primary production, nondividing cells, after a limited loss, persisted in small but significant numbers for a long time thereafter. Whereas the curve for changes in the number of replicating protective cells showed a striking temporal correlation with the curve for changes in DNA synthesis, in the spleen the curve for changes in the number of nonreplicating protective cells was closely correlated with the curve for changes in the level of delayed sensitivity (Fig. 2). Thus, whereas dividing protective cells were only present in the spleen in functional numbers during the brief period of increased lymphoid cell division in this organ, nondividing protective cells and delayed sensitivity persisted for a long time in the apparent absence of increased spleen cell division. FIG. 1. Kinetics ofproduction ofprotective lymphocytes in the spleen during primary (2 x 103) and secondary (2 x 105) Listeria infection (arrows) compared with growth curves ofparasite in the liver and with the production of circulating antibodies to soluble Listeria antigens. Protective T cells were produced much faster in response to secondarily infected (4-day peak response compared to 6-day peak for primary infection), and this was temporally associated with the faster onset of expression of antibacterial immunity and the faster elimination of bacteria from the tissues. The number of protective lymphocytes rapidly declined after peak primary response, but a small number persisted until the initiation of secondary infection. No agglutinating antibodies were produced in response to primary infection, but mercaptoethanol-sensitive antibody was produced in response to secondary infection. Agglutinating activity is expressed as the last tube number in a twofold serial dilution assay that gave positive agglutination. Means offive mice per time point + standard error. peak production all but ceased by days 12 to 15 of the primary response and by day 10 of the secondary response. After these times the host retained a small but relatively stable number of protective cells for 40 days or more after peak primary production and probably for at least this length of time after peak secondary production. Further analysis of the production and loss of protective lymphocytes is seen in Fig. 2, where the same results are expressed in terms of protection afforded to normal recipients by dividing (vinblastine-sensitive) and nondividing (vinblastine-resistant) spleen cells. It can be seen that both the primary and secondary response FIG. 2. Production of dividing (vinblastine-sensitive) and nondividing (vinblastine-resistant) protective lymphocytes in response to primary and secondary infection (arrows) compared with changes in the level of spleen cell division ([3H]TdR incorporation into total DNA) and changes in the level of delayed sensitivity to Listeria antigens (footpad increase). The production of replicating protective cells was confined to the short-lived period of increased spleen cell division. The faster production of these cells that occurred in response to secondary infection, therefore, was correlated with a faster increase in spleen cell division. Only nonreplicating protective cells remained after the 12th day of the primary response and changes in their number were closely correlated with changes in the level of delayed sensitivity. Means offive mice per time point ± standard error.
764 NORTH AND DEISSLER No detectable agglutinating antibodies were produced in response to primary infection (Fig. 1) in spite of the fact that the antigens used for the passive hemagglutination assay were the same as those used to successfully elicit strong delayed sensitivity reactions. Antibodies to these antigens were produced, however, in response to secondary infection, but all of the agglutinating activity was found to be mercaptoethanol sensitive. The kinetics of production of these agglutinins, furthermore, resembled the kinetics of a primary 19S antibody response. Macrophage activation is confined to the period of dividing mediator cell production. Anti-Listeria immunity, although mediated by sensitized T cells, is expressed by activated macrophages (12). The specific acquisition of activated macrophages, furthermore, enables the host to nonspecifically resist, to varying degrees, infections caused by antigenically unrelated bacteria. It was shown in a preceding paper, moreover, that activated macrophages were not responsible for long-term specific resistance to reinfection, because this resistance was only expressed against the homologous organism. It was proposed, therefore, that activated macrophages acquired during immunizing infection were lost soon after immunizing infection ended. The following experiment was designed to obtain additional information to support this proposition. It involved determining changes in the level of macrophage activation during the course of both a primary and a secondary Listeria infection. Changes in the level of a systemic macrophage activation were determined by measuring changes in the level of nonspecific resistance expressed against a standard intravenous challenge infection with the bacterium Y. enterocoliticia. It was found (Fig. 3) that nonspecific resistance was generated in response to both a primary and a secondary Listeria infection and was of limited duration. Indeed, in both cases, the kinetics of generation of nonspecific resistance showed a striking similarity to the curve for the kinetics of production of dividing mediator cells in the spleen and with the curve for changes in spleen cell division (Fig. 2). Thus, in both infections peak macrophage activation was coincident with peak mediator T-cell production, and activated macrophages were lost soon after the production of mediator T cells ceased in the spleen. It can be further seen that the increased rate of production of mediator T cells that occurred in response to secondary infection was associated with an increased rate of generation of nonspecific resistance. Thus, acquired nonspecific resistance was not present INFECT. IMMUN. 40 42 AA 46 18S 52 60 SO FIG. 3. Kinetics ofproduction ofprotective lymphocytes during Listeria infection is compared with changes in the level of systemic macrophage activation (histograph) as measured by nonspecific expression of resistance to infection with a standard challenge dose of the bacterium Yersinia enterocolitica (log1o resistance index in liver). The increased tempo ofproduction ofprotective lymphocytes that occurred in response to secondary infection is clearly shown. The kinetics of systemic macrophage activation showed a close temporal correlation with protective T-cell production and therefore occurred faster in response to secondary infection. Macrophages did not remain activated for long after dividing protective T cells are known to have disappeared from the spleen (Fig. 2). Means of five mice per time point ± standard error. during the time period that separated the primary and secondary responses. It is obvious from these results, therefore, that the activated macrophages that were generated to express immunity to primary infection were lost soon after the infecting organism was eliminated from the tissues, and that activated macrophages had to be regenerated at a faster rate in order to express immunity to lethal secondary infection. Capacity to resist secondary infection rests on the presence of nondividing T cells. It was shown in the preceding sections that primary Listeria infection results in the production of dividing and nondividing protective lymphocytes in the spleen. It was also shown that whereas dividing protective cells were lost soon after the termination of immunizing infection, nondividing protective cells persisted in a small but significant number for a long time thereafter. The evidence for the persistence of nondividing cells was based on the finding that about 0.5 log of protection could routinely be transferred to normal recipients with vinblastine-resistant cells harvested from immunized donors for up to 2 months after they had lost their replicating protective T cells. It should be remembered, however, that this small level of adoptive immunity was measured by subtracting the 48-h
VOL. 12, 1975 ACTIVE IMMUNE RESPONSE AND "MEMORY" IN T CELLS 765 growth of a Listeria challenge infection in the spleens of recipients of "immune" spleen cells from its 48-h growth in the spleens of recipients of normal spleen cells. This technique, therefore, only measured the early expression of active adoptive immunity and did not give any indication of the way in which the recipients of immune spleen cells handled the challenge infection after 48 h. The possibility existed, therefore, that the small level ofadoptive immunity expressed by recipients at 48 h of a challenge infection could represent the basis for expression of a higher level of immunity after this time. This would be so if nondividing protective donor cells were the progenitors of dividing mediator T cells and if these latter cells needed time to be generated. As a first step in investigating this possibility, it was necessary to determine whether the acquired, long-lived capacity of immunized mice for generating resistance to a lethal Listeria challenge infection, as measured by 3-day growth of the organism in their livers and spleens, could be passively transferred with their spleen cells to normal recipients. This involved comparing the 3-day growth of a lethal challenge infection in 40-day immune donors with its 3-day growth in recipients of two equivalents of spleen cells from the same panel of 40-day immune donors. The rationale for using two equivalents of donor spleen cells was based on the assumption that nondividing protective cells -are part of the long-lived pool of recirculating lymphocytes as are the cells that carry antibody memory (7, 17), and on the knowledge (6) that the spleen contains 50 to 60% of the recirculating pool at any one time. Obviously, the transfer of total anti-listeria immunity from an immune donor to a normal recipient would require that the recipient receive the full complement of the donor's sensitized cells. The possibility that the cells that transferred resistance were T cells was tested by determining their susceptibility to incubation in vitro in anti-o-serum and complement. It was found (Fig. 4) that an infusion of 2 spleen equivalents of cells from 40-day immune donors protected normal recipients against lethal challenge infection as indicated by the progressive elimination of the organism between 48 and 72 h from the liver and between 24 and 48 h from the spleen. Indeed, the timing of the onset of expression of immunity in the spleens of recipient mice was similar to that in the donors. It is apparent, therefore, that in the recipients, as well as the donors, immunity needed time to develop. It will be noted, nevertheless, that the recipients expressed only about half as much resistance as the donors. FIG. 4. Passive transfer to normal recipients of protection against lethal challenge infection with nonreplicating spleen cells from 40-day immune (memory) donors. The recipients, although well protected, expressed only halfas much resistance as the donors. Incubating the spleen cells with anti- 0-serum ablated their capacity to transfer immunity. Means of five mice per time point. Incubation in anti-0-serum completely abrogated the capacity of donor spleen cells to transfer immunity (Fig. 4). It is apparent, therefore, that the long-lived, anit-listeria immunity is dependent on the persistence of a population of nondividing sensitized T cells. DISCUSSION The results of this study can be interpreted to mean that primary intravenous infection with L. monocytogenes in mice resulted in the generation of two distinct states of immunological reactivity. There was both the generation of a short-lived state of active immunity that functioned to eliminate the infecting organism from the tissues and the generation of a long-lived state of heightened immunological potential that enabled the host to combat a secondary infection with greatly increased efficiency. Short-lived active immunity was mediated mostly by replicating, short-lived T cells and is known to be expressed by activated macrophages (12). The activity of both types of cells peaked on day 6 of primary infection and decayed rapidly after the elimination of the infecting organism from the tissues. It is obvious that the state of activity immunity ended when the T cells that mediated this immunity and the macrophages that expressed it disappeared or lost their function. Indeed, the close temporal correlation between the curve for macrophage
766 NORTH AND DEISSLER activation and the curve for mediator T-cell production indicates that the level of macrophage activation at any one time was proportional to the level of production of replicating mediator T cells. The host's macrophage system did not remain activated in the absence of replicating mediator T cells. The close temporal correlation between the production of replicating mediator T cells and infection-induced spleen cell division indicates, in tum, that mediator T cells arose by vigorous cell division. Furthermore, because of their short life span (13), replicating mediator cells were not sustained in functional numbers in the absence of cell division. The response to secondary infection showed development of the same temporally correlated parameters as the primary response, except that they developed with a markedly increased tempo. The increased rate of production of mediator T cells and activated macrophages, furthermore, resulted in a faster onset of expression of antibacterial immunity. This prevented the lethal challenge infection from multiplying significantly and results in its rapid elimination from the tissues. As with the active primary response, the cells generated to mediate and express immunity during the active secondary response rapidly disappeared when the secondary infection was eliminated. Despite the disappearance of replicating mediator T cells and effector macrophages that followed termination of primary infection, then, mice remained capable of resisting lethal secondary infection for many weeks because of their acquired capacity for regenerating a state of active immunity in an accelerated fashion. The acquired long-lived state of increased immunological potential on which the accelerated secondary response was based was associated with a persistent level of delayed sensitivity to Listeria antigens and with a small and relatively stable number of nondividing protective T cells. Passive transfer of these protective cells enabled normal recipients to resist a lethal challenge infection. Such cells can also transfer delayed sensitivity (North, submitted for publication). The delay in the onset of the expression of this adoptive resistance indicates, however, that it needed time to develop. Indeed, since the 40-day immune donors of these cells were required to generate an accelerated active secondary immune response to resist the challenge infection, it follows that the recipients also needed to generate an active response. This would mean that the protective, nonreplicating INFECT. IMMUN. T cells in 40-day immune donors were either the progenitors or the recruiters of the replicating mediator T cells that mediated active adoptive immunity. Direct evidence that the recipient of spleen cells from 40-day immune donors have the capacity for an accelerated production of replicating mediator T cells will appear in a forthcoming publication. It should be pointed out that nonreplicating protective T cells were also present in the spleen during the active primary and secondary responses and that a high level of delayed sensitivity also existed during these times. Some of these nonreplicating protective cells may have represented short-lived replicating T cells that had reached the end of their division cycle and were destined to die. Some of them, on the other hand, were probably of the same class as the nonreplicating T cells that persisted long after the active immune response ended and that were associated with a persistent state of delayed sensitivity. Indeed, the close temporal correlation between the curve for changes in the number of nonreplicating protective T cells and that for changes in the level of delayed sensitivity indicates the possibility that nonreplicating protective T cells are the same cells that initiate delayed sensitivity reactions. There is published evidence (1, 2, 8) that the initiators of delayed sensitivity reactions in other models are nonreplicating cells. Thus, the possibility exists that a state of delayed sensitivity represents a state of immunological memory of the cell-mediated type. This would seem to be the only adaptive function that the state of delayed sensitivity could serve. The failure to detect serum antibodies to soluble Listeria antigens during primary Listeria infection by hemagglutination, and the finding that the antibody response to a much larger secondary infection resembled the kinetics of a primary response and generated antibody that was all mercaptoethanol sensitive, indicates that primary infection may not have primed for a secondary antibody response. This model, therefore, may prove useful for determining whether the T cells that function in cell-mediated immunity are different from the T cells that function as helper cells in antibody production, as has been shown to be the case in another published model (16). ACKNOWLEDGMENT This work was supported by Public Health Service grant AI-10351 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Bloom, B. R., L. D. Hamilton, and M. W. Chase. 1964. Effects of mitomycin C on the cellular transfer of delayed-type hypersensitivity in the guinea pig. 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VOL. 12, 1975 ACTIVE IMMUNE RESPONSE AND "MEMORY' IN T CELLS 767 plaque assay for enumeration of effector cells in mixed lymphocyte cultures. Transplant. Proc. 5: 1705-1709. 3. Carter, P. B., and F. M. Collins. 1974. Experimental Yersinia enterocolitica in mice: kinetics of growth. Infect. Immun. 9:851-857. 4. Celada, F. 1971. The cellular basis ofimmunologic memory. Prog. Allergy 15:223-258. 5. Cunningham, A. J., and E. E. Sercarz. 1971. The asynchronous development of immunological memory in helper (T) and precursor (B) cell lines. Eur. J. Immunol. 1:413-420. 6. Ford, W. L. 1969. The kinetics of lymphocyte recirculation within the rat spleen. Cell Tissue Kinet. 2:171-191. 7. Hunt, S. V., S. T. Ellis, and J. L. Gowans. 1972. The role of lymphocytes in antibody formation. IV. Carriage of immunological memory by lymphocyte fractions separated by velocity sedimentation and on glass bead columns. Proc. R. Soc. London Ser. B 182:211-231. 8. Lagrange, P. H., and G. B. Mackaness. 1975. A stable form of delayed-type hypersensitivity. J. Exp. Med. 141:82-96. 9. Ling, N. R. 1961. The attachment of proteins to aldehyde-tanned red cells. Br. J. Haematol. 7:299-302. 10. North, R. J. 1973. The cellular mediators of anti-listeria immunity as an enlarged population of short-lived replicating T cells: kinetics of their production. J. Exp. Med. 138:342-355. 11. North, R. J. 1974. T cell dependence of macrophage activation and mobilization during infection with Mycobacterium tuberculosis. Infect. Immun. 10:66-71. 12. North, R. J. 1974. Cell-mediated immunity and the response to infection, p. 185-220. In R. T. McCluskey and S. Cohen (ed.), Mechanisms of cell-mediated immunity. Wiley and Sons, New York. 13. North, R. J. 1974. T cell-dependent macrophage activation in cell-mediated anti-listeria immunity, p. 210-220. In W.-H. Wagner and H. Hahn (ed.), Activation of macrophages. American Elsevier, New York, and Excerpta Medica, Amsterdam. 14. North, R. J. 1975. Nature of "memory" in T-cell-mediated antibacterial immunity: anamnestic production of mediator T cells. Infect. Immun. 12:754-760. 15. North, R. J., G. B. Mackaness, and R. W. Elliott. 1972. The histogenesis of immunologically committed lymphocytes. Cell. Immunol. 3:680-694. 16. Silver, J., and B. Benacerraf. 1974. Dissociation of T cell helper function and delayed hypersensitivity. J. Immunol. 113:1872-1875. 17. Strober, S., and J. Dilley. 1973. Biological characteristics of T and B memory lymphocytes in the rat. J. Exp. Med. 137:1275-1292.