Immunology 1977 33 361
Cytotoxic T cell response to lymphocytic choriomeningitis virus PROPERTIES OF PRECURSORS OF EFFECTOR T CELLS, PRIMARY EFFECTOR T CELLS AND MEMORY T CELLS IN VITRO AND IN VIVO
M. B. C. DUNLOP, P. C. DOHERTY, R. M. ZINKERNAGEL & R. V. BLANDEN John Curtin School of Medical Research, Australian National University, Canberra, Australia, The Wistar Institute, Philadelphia, Pennsylvania, U.S.A. and Scripps Clinic and Research Foundation, La Jolla, California, U.S.A.
Received 9 December 1976; accepted for publication 13 January 1977
Summary. Spleen cells from CBA/H mice pre-primed intravenously at various intervals with lymphocytic choriomeningitis (LCM) virus were tested for their capacity to respond and generate cytotoxic effector T cells on secondary stimulation both in vitro and in vivo. In vitro secondary stimulation was performed by culturing preprimed spleen cells with infected, syngeneic, peritoneal 'stimulator' cells for periods of up to 7 days at 37°. Controls were incubated with uninfected 'stimulators'. In vivo secondary stimulation was obtained by intravenous transfer of preprimed spleen cells into irradiated, heavily infected CBA/H recipients at various times prior to assay of recipients' spleens. Controls were uninfected, irradiated recipients. Effectors were assayed against LCM virus-infected or uninfected, H-2 compatible L929 target cells in a5'Cr release assay. Spleen cells gave three different patterns of cytotoxic T cell response following in vitro or in vivo stimulation, depending upon the interval between priming and secondary stimulation. Populations taken from mice approximately days 2-5 post-infec-
tion (PI) developed increasing cytotoxic activity against virus-infected targets in vitro when cultured with infected or uninfected peritoneal cells, or in vivo when transferred into irradiated, uninfected recipients. However, these early populations did not generate cytotoxic activity when transferred into irradiated, heavily infected recipients. Established primary effector populations (i.e. approx. days 7-11 PI) exhibited continuing effector activity when maintained in vitro or in vivo, more so when peritoneal 'stimulator' cells or irradiated recipients were infected. Memory populations (i.e. more than approx. day 13 PI) developed highly potent secondary effectors when cultured with infected peritoneal cells, or on transfer to irradiated, infected recipients. The three different patterns of cytotoxic T cell response of the different preprimed spleen cell populations can be interpreted as indicating three different phases of functional activity of the same population of antigen-reactive T cells from LCM virus-primed mice.
Correspondence: Dr M. B. C. Dunlop, Department of Microbiology, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra, 2602, A.C.T. Australia.
INTRODUCTION A central problem in immunology is whether memory 361
362
M. B. C. Dunlop et al.
lymphoid cells and lymphoid cells with a defined effector activity are separate subsets, or are merely representatives of phases in differentiation of the one cell line. MacDonald, Cerottini & Brunner (1974a) have approached this question by separating alloreactive cells on the basis of differing sedimentation velocity. Their results (supported in part by those of Andersson & Hdyry, 1975) suggested that precursors of cytotoxic T cells are small, but upon contact with alloantigen proliferate and/or differentiate into large, blastlike cytotoxic cells; and that with time, at least a fraction of these large effectors dedifferentiate to give small memory T cells (which may be restimulated to proliferate and/or differentiate upon re-exposure to alloantigen). This work is relevant to an interpretation of T cell-mediated responses to lymphocytic choriomeningitis (LCM) virus, where it has been demonstrated that virus-specific effector populations do differ in their properties from T cells with memory for virus. Johnson & Cole (1975) examined effector and memory thymus-derived populations in spleens following priming with LCM virus, and demonstrated that these populations were functionally different, by virtue of their ability either to elicit or protect against acute LCM disease by adoptive transfers, and by their direct cytotoxic activity. Similarly, Volkert, Marker and Bro-J0rgensen (1974) used a variety of diverse criteria to show that early LCM virusimmune spleen and lymph node cells were different from late LCM virus-immune lymphoid cells. It has recently been shown that highly potent, virus-specific T cells can be obtained by cocultivating spleen cells from mice recovered from LCM virus infection several weeks previously, with syngeneic LCM virus infected peritoneal cells at 370 in vitro for 5 days (Dunlop & Blanden, 1976; Dunlop, Doherty, Zinkernagel & Blanden, 1976). This paper describes a method of maintaining primary, or inducing secondary LCM virus specific cytotoxic T cells in vivo by transferring spleen cells to irradiated LCM virus infected syngeneic recipients for various periods. Cells mediating primary effector activity following in vivo transfer have elsewhere been shown to be T cells of donor origin (Zinkernagel & Doherty, 1974a). By comparing the response of spleen cells at different intervals following priming with LCM virus, on culture in vitro or transfer in vivo, it is apparent that different patterns of responses occur depending upon the interval between priming and restimulation. These approaches hence provide further criteria to distinguish primary effector T cells from memory T
cells or from precursors of primary effector T cells and suggest methods to analyse the relationships between subsets.
MATERIALS AND METHODS Mice and virus stocks Inbred, specific pathogen-free CBA/H mice were used throughout. Mice were primed intravenously with the viscerotrophic (WE3) strain of LCM virus and priming dose was 0 2 ml of a 10-s dilution of guineapig spleen or lung stock titrated at approx. 400 intracerebral LD5o or 250 P.F.U. (Zinkernagel & Doherty, 1974b). Virus stocks used for immunization were dispensed in small amounts and frozen at - 700 to minimize day-to-day variation in dosage. In vitro secondary stimulation The procedure for in vitro secondary stimulation of memory T cells by cocultivation for varying periods with LCM virus-infected, syngeneic peritoneal cells has been detailed elsewhere (Dunlop & Blanden, 1976). Essentially, peritoneal cells were infected by incubation at 370 with neat WE3-LCM virus stock for 30 min, then washed in culture medium. Infected or uninfected peritoneal cells (usually 2-5 x 106 per flask) were then cultured overnight in small (25 cm2 surface area) Falcon plastic flasks containing 10 ml of culture medium. Following overnight culture, the spleen responder cells under test were added to the infected or uninfected peritoneal cells (2-5 x 107 responders per flask). Cultures were incubated at 37° for various intervals and then tested in the 51Cr release assay described below. In vivo secondary stimulation One day prior to transfer of the spleen responder cells under test, recipients were given 850 rads from a 60Co source. On the day of transfer, recipients were injected intravenously with 0 2 ml of a 10-2 dilution of virus stock (approx. 4 0 x 101 intracerebral LD50 or 2 5 x 105 P.F.U.). About 12 h later, spleen cells obtained from donor mice were prepared in a way which would remove embolic material as described (Blanden & Langman, 1972) and then transferred intravenously to the irradiated, infected or uninfected syngeneic recipients. Usually about 1.0 x 108 spleen cells were transferred per recipient, and groups of four recipients were employed. Mice were then maintained on routine feeds and antibiotics for
Induction of T precursors, effectors and memory cells for LCM (c )
(a )
80 70 60 50 40 30 20 en -il 10 y 0
FIG.
363
.
.11
2 3 4 80 FIG.2. (a) 70 60 50
5 (c )
( b)
A~
~
~
~
0~~~~~
40 _-
30 20
4
5
4
5
10 3
6
4 3 5 6 Incubation time (days)
3
4
5
6
Figure 1. Effect of in vitro culture of CBA/H spleen cells (preprimed with LCM virus i.v. at intervals), with infected or uninfected, syngeneic peritoneal stimulator cells for 2, 3, 4, or 5 days prior to assay. Effector-target ratio was 4: 1 and peritoneal stimulator) Infected stimulator peritoneal cells; spleen cell responder ratio was 1: 10. Medium release was 204%0 on infected targets. ( (- --) uninfected stimulator peritoneal cells. (a) (U) Normal spleen cells; (A) D3 post-inoculation (PI); (0) D5 PI (b) (A) D7 PI; (A) D9 PI (c) (0) D21 PI Figure 2. Effect of in vitro culture of pre-primed CBA/H spleen cells with infected, syngeneic peritoneal cells for 3, 4, 5, or 6 days prior to assay. Effector-target ratio was 1:1 and peritoneal stimulator cell-spleen responder cell ratio was 1:10. Medium release was 25-2 per cent on infected L929 targets. The legend is the same as for Fig. 1, with the inclusion of (b) (0) DlI PI (c) (U) D13 PI; (A) D15 PI. -
omitted for clarity. Statistical significance determined by Student's f-test.
various intervals prior to removal of their spleens for assay of cytotoxic T-cell activity on virus-infected targets. Spleens generally yielded 5.0 x 105-5-0 x 106 viable lymphoid cells/spleen.
were
Technique of IICr release assay, and method of expressing results The 5'Cr release assay has been described before (Zinkernagel & Doherty, 1974b). Targets were LCM virus-infected or uninfected, H-2 compatible L929 fibroblast cells. Experiments were designed such that in vitro cultures or in vivo transfers were assayed on the one day. Results were expressed as percent specific lysis = (percentage 51Cr release in presence of effectors -percentage medium release) x 100/percentage water lysis. Specific lyses on infected targets only are shown for all figures excepting Fig. 5. Data presented are the mean of four replicates. Standard errors of the mean were never greater than ± 2% and
General Early in vivo experiments demonstrated that primary LCM virus specific T cell activity in spleens increased from day 5 to peak on day 9 post LCM virus inoculation (P1), then fell slowly to give residual cytotoxic activity by day 21. To facilitate the interpretation of cytotoxic responses, the following definitions were made. Pre-peak (or precursor) primary cytotoxic T cells were defined as those harvested up to day 7 PI; peak cytolytic T cells were those harvested from days 7-11 PI; and memory cells were defined as those T cells obtained after day 11 PI. Another initial experiment using a virus plaque assay showed that after intravenous challenge, virus titres in spleens
was
RESULTS
M. B. C. Dunlop et al.
364 80 70 60 50 40 30 20 10
*0>
_ FIG.3.
(a)
(c)j
( b) A
0
/,
\/ 0
-
-
2
0
\ 0
A
A"
0~
.
Q
us
FIG.4. 4, 80 13
( b)
(c )
70 60 60
C)
40
30 20
10 l l
l
1
L
5 6 3 Time after transfer (days) 3
4
4
5
6
Figure 3. Effect of in vivo transfer of pre-primed CBA/H spleen cells to irradiated, infected or uninfected, syngeneic recipients for 3, 4, 5, or 6 days prior to assay of recipients' spleens. Effector-target ratio was 10:1. Medium release was 23-7 per cent on ) Infected, irradiated CBA/H recipients; (-- -) uninfected, irradiated CBA/H recipients. (a) (-) Normal infected targets. ( spleen cells; (-) D5 PI (b) (A) D9 PI (c) (0) D21 PI. Figure 4. Effect of in vivo transfer of pre-primed CBA/H spleen cells to irradiated, infected, syngeneic recipients for 3, 4, 5, or 6 days prior to assay of recipients' spleens. Effector-target ratio was 10: 1. Medium release was 14-5 per cent on infected targets. The legend is the same as for Fig. 3, with the inclusion of (b) (U) D7 PI; (0) DII P1 (c) (U) D13 PI; (A) D15 P1. -
increased rapidly to peak on days 3-5 and slowly fell thereafter. Therefore pre-peak (e.g. D5 PI) and peak (e.g. D9 PI) primary spleen populations contained large amounts of virus. In vitro culture of spleen cells at various intervals postpriming with LCM Groups of CBA/H mice were infected with a single dose of a standard inoculum of LCM virus, spleen cells were harvested at intervals PI, and spleen cells were cultured with syngeneic, infected or uninfected peritoneal cells (Fig. 1) or infected peritoneal cells only (Fig. 2). Cultures were maintained for 2, 3, 4, or 5 days (Fig. 1) or 3, 4, 5 or 6 days (Fig. 2) before assay. In general, three patterns of cytotoxic activity -pre-peak primary, peak primary and memoryemerged: (a) normal spleen cells did not develop significant cytotoxic activity in culture, whilst D3 and D5 PI spleen cells increased in activity from day 2 to day 5 in culture (Fig. 1) or peaked on day 5 of
culture (Fig. 2) (i.e. about that time, 8-10 days PI, at which these same spleen cells would be expected to reach peak effector activity if they remained in the intact, infected animal). (b) D7 PI and D9 PI spleen cells peaked in cytotoxic activity on day 4 (Fig. 1), or D7, D9 and DlI PI cells continually declined in activity from day 3 to day 6 of culture (Fig. 2) (c) D21 PI cells (Fig. 1) or D13, D15 and D 21 PI cells (Fig. 2) developed potent secondary responses. In Fig. 1, all spleen cell populations, including D3 and D5 PI, responded more strongly with infected (solid lines) than with uninfected (dashed lines) peritoneal stimulators. In vivo transfer of spleen cells at various times postpriming with LCM Groups of CBA/H mice were primed with LCM virus, and spleen cells were transferred at intervals into irradiated, infected or uninfected, syngeneic recipients. Irradiated recipients' spleens were assayed
Induction of Tprecursors, effectors and memory cells .for LCM FIG.5. 80 - (a)
365
( b)
70 60 50 40 30 0
20 10 _2
0
4-
5
6
7
Incubation time (days) aL)
80 FiFG.6.
L) 70 0
60
*\
50
//N
40
30 _
20 _ 10
0
3
5
7 8
10 11
13 14
16 17 18
Time after priming(weeks)
Figure 5. Results of in vitro culture of recently primed CBA/H spleen cells with infected or uninfected, syngeneic peritoneal cells for 4, 5, 6 or 7 days prior to assay. Specific releases on both infected (a) and uninfected (b) targets are shown. Effectortarget ratio was 10:1 and peritoneal stimulator-spleen responder ratio was 1: 12 5. Specific lyses on uninfected targets were included in this figure because they were not negligible at this effector-target ratio for recently primed cells cultured in vitro. Medium release was 30 0%/ on infected targets and 28 5% on uninfected targets. (0) D4 PI (A) D2 PI (-) Dl P1. Specific lyses by cells from cultures with uninfected stimulators were generally similar to, and slightly smaller than, specific lyses by cells from cultures with infected stimulators, and were omitted. Specific lyses by cells from cultures where spleen cells were primed 12 h, 4 h, or not at all before culture were essentially similar to, but generally smaller than, specific lyses by Dl PI spleen cells on infected and uninfected targets, and were also omitted for clarity. Figure 6. Specific lysis of infected L929 targets by cytotoxic T cells generated by culture of CBA/H spleen cells (preprimed with LCM virus for several weeks), with infected or uninfected, syngeneic peritoneal cells for 5 days prior to assay. Effector-target ratio was 05:1, and peritoneal stimulator cell-spleen responder cell ratio was 1:32. Medium release was 22-8% on infected L929 targets. Specific lyses by cells from culture with uninfected peritoneal stimulator cells were too small to be conveniently depicted and were omitted.
3, 4, 5,
or
6 days after tranfer. Results
are
given for
two such experiments, the first using irradiated, both
infected and uninfected control recipients, the second with irradiated, infected recipients only. (Figs 3 and 4). Results were in general similar to those given in Figs 1 and 2, but differed in that transfer of D5 PI cells to irradiated, infected recipients led to less development of cytotoxic activity than in uninfected
recipients. Two other experiments have confirmed that D5 cells on transfer to irradiated infected recipients showed little evidence of generation of cytotoxic activity even up to 8 days post-transfer. This population did not generate cytotoxic effectors on transfer to irradiated, infected recipients in other organ sites such as thymus, peritoneal cavity, blood or mesenteric lymph node (data not shown). Hence,
M. B. C. Dunlop et al.
366
D5 PI spleen cells (a pre-peak primary population), possessed the capacity to be suppressed.
Effect of in vitro culture of spleen cells after short intervals of priming Spleen cells from CBA/H mice were cultured with infected or uninfected syngeneic peritoneal cells, having been primed 4 h, 12 h, 1, 2, or 4 days previously with LCM virus, and effectors were assayed following culture for 4-7 days. (Fig. 5). There was a continual development of specific cytolytic activity on infected targets over and above that on uninfected targets by D4 PI spleen cells and to a lesser extent by D2 and D1 PI spleen cells, when infected stimulators were present. Spleen cells taken from the animal at earlier times post-challenge showed no clear primary induction. In another experiment, it was possible to maintain 'virus-specific' activity of D2 PI cells from 6 to 12 days of culture, and by further adding freshly infected peritoneal cells on day 7 and day 13, a stronger secondary virus-specific response was obtained, by day 18 (data not shown). Effect of in vitro culture ofspleen cells at long intervals post-priming Groups of CBA/H mice were inoculated weekly with a dose of LCM for several months before culturing with infected or uninfected, syngeneic peritoneal cells. Cultures were assayed on the 5th day only (Fig. 6). Infected stimulators induced strong secondary cytotoxic T cell responses from 3 weeks post-priming up to at least 18 weeks post-priming. Other assays have established that potent LCM-specific memory exists to at least 36 weeks post-priming.
DISCUSSION Both in vitro and in vivo methods have been shown in this article to allow stimulation and presumably proliferation and differentiation into cytotoxic T cells, of T cells taken at various stages post-priming. Comparisons between the two methods reveal differences depending on the interval between priming and secondary challenge and the amounts of virus or infected cells present in the system. D5 PI cells, i.e. precursors of primary cytotoxic T cells which reach a peak between days 8-10 if left in
the mouse are 'committed' in the sense that they will generate cytotoxic T cells in vitro when cocultivated with infected or uninfected stimulators. Infected 'stimulators' are in fact not necessary when D5 cells are responders. This is because D5 spleen cells themselves contain large amounts of virus and infected cells able to act as stimulators (data not shown). Similarly, D5 cells can generate effectors when transferred intravenously to irradiated, uninfected recipients. In contrast, irradiated, infected recipients do not allow or suppress the generation of primary cytotoxic T cells. It seems unlikely that cytotoxic T cells are being generated in, or migrating to, other organ sites in the irradiated, infected recipient, since other experiments show that D5 PI cells transferred to irradiated, infected or uninfected recipients do not give rise to cytotoxic T cells in thymus, peritoneal cavity, blood or mesenteric lymph node (peripheral lymph nodes were too small to be detected in irradiated recipients). Presumably the mechanism of suppression involves excess virus or excess antigen, since the irradiated recipients were infected with large doses of virus. This idea is supported by the findings that cocultivation of D5 PI cells with spleen cells from congenital LCM carriers, or with large numbers of infected peritoneal cells also leads to suppression; furthermore, intravenous administration of virus neutralising antiserum to D5 PI donor mice 30 min prior to removal of their spleens for culture resulted in an enhanced generation of a cytolytic response from these spleen cells (Dunlop & Blanden, 1977). Spleen cells harvested closer to the time of the expected peak of the cytotoxic T cell response in vivo (e.g. D7-D9 PI) generally gave a peak of activity after transfer to the new in vitro or in vivo situation, and then declined in activity. Cells taken at or after the peak (D9-D11 PI) gave declining activity with time in their new environment. Activity in an infected environment (in vitro or in vivo) was generally higher than in an uninfected environment, so continued antigenic stimulation may prolong activity of effector cell populations. Cell populations with established cytolytic activity against LCM virusinfected targets (D7-D11 PI) seem less vulnerable than pre-peak (D5 PT) primary cells to the mechanism of suppression cited above. This may be explained by peak primary cells killing infected stimulator cells, thus limiting production of virus and spread of infection. The destruction of stimulator cells by cytotoxic T cells may also explain in part why no major stimulation of a new peak of activity
Induction of Tprecursors, effectors and memory cells for LCM occurred with responder spleen populations harvested on days 9-11 PI. This may correspond to the refractoriness to restimulation seen in the responses to alloantigens (MacDonald, Engers, Cerottini & Brunner, 1974b) and to ectromelia-virus infected cells (Gardner & Blanden, 1976). The capacity for a secondary response was clearly seen by D13 PI. D13-D21 PI cells initially show little cytolytic activity upon transfer either in vitro or in vivo, but develop potent cytolytic activity only in an antigenic environment. Memory T cells persist up to at least 36 weeks PI. Collectively, these results are compatible with the development and differentiation first of primary cytotoxic T cells, and then of memory T cells, from one subset of antigen-reactive precursor T cells, which is the simplest and most economical model of the response. Peak primary activity is established by day 8-9, declines, and memory cells appear by day 13-15. This may reflect dedifferentiation of proliferating effector cells (probably large, blast cells) to small lymphocytes, which possess memory as suggested by MacDonald et al. (1974a) and Andersson & Hayry (1975) for the response to alloantigens. Certainly effectors and memory cells are not the same populations physiologically, but the appearance of the latter is chronologically linked to the appearance of the former. Johnson & Cole (1975) also inferred that at least two physiologically different virus-specific T lymphocyte subsets exist following LCM infection in vivo by testing BALB/c splenic lymphocytes obtained at varying intervals after LCM challenge for their capacity either to elicit acute LCM disease (memory cells but not acute primary effectors) or protect against lethal intracerebral LCM virus challenge (acute primary effectors but not memory cells) when transferred by the intraperitoneal route to syngeneic, respectively cyclophosphamide treated and LCM virus-infected or normal mice. As well, the same splenic lymphocytes were tested for their capacity to lyse LCM virus-infected BALB/c 3T3 target cells in vitro, and efficient cytolytic activity resided in only acute primary effectors. Volkert et al. (1974) used various criteria to separate early immune anti-LCM cells (D9 PI) from late immune anti-LCM cells (D30 PI). Early immune cells contained many more large and blast-like cells than late immune cells, and LCMspecific cytotoxic activity, anti-viral effect after transfer to acutely infected animals, ability to protect against a lethal intracerebral infection and resistance to X-irradiation were almost entirely or predomi-
367
nantly properties of early immune cells. Both early and late immune cells were susceptible to treatment with anti-theta serum. However, late immune cells were much superior to early immune cells in reducing virus titres on transfer to LCM virus carriers, a property explained by the induction of memory T cells to give rise to highly potent antiviral effectors. These potent secondary effectors are readily able on transfer in small numbers (2-5 x 107) to mediate a reduction in spleen virus titres by 4-5 logs (Dunlop, 1977). It has yet, of course, to be determined precisely whether antiviral cytotoxic T cells give rise to memory T cells, but in vitro maintenance of primed cells and generation of responses should lend itself to physical separation methods or other approaches to this interesting area.
REFERENCES ANDERSSON L.C. & HAYRY P. (1975) Clonal isolation of alloantigen-reactive T cells and characterization of their memory functions. Transplant Rev. 25, 121. BLANDEN R.V. & LANGMAN R.E. (1972) Cell-mediated immunity to bacterial infection in the mouse. Thymusderived cells as effectors of acquired resistance to Listeria monocytogenes. Scand. J. Immunol. 1, 379. DUNLOP M.B.C. (1977) Secondary cytotoxic cell response to lymphocytic choriomeningitis virus. III. In vivo protective activity of effector cells generated in vitro. Immunology, in press. DUNLOP M.B.C. & BLANDEN R.V. (1976) Secondary cytotoxic cell response to lymphocytic choriomeningitis virus. I. Kinetics of induction in vitro and yields of effector cells. Immunology, 31, 171. DUNLOP M.B.C. & BLANDEN R.V. (1977) Mechanisms of suppression of cytotoxic T cell responses in murine lymphocytic choriomeningitis virus infection. J. exp. Med. 145, 1131. DUNLOP M.B.C., DOHERTY P.C., ZINKERNAGEL R.M. & BLANDEN R.V. (1976) Secondary cytotoxic cell response to lymphocytic choriomeningitis virus. II. Nature and specificity of effector cells. Immunology, 31, 181. GARDNER I.D. & BLANDEN R.V. (1976) The cell-mediated immune response to ectromelia virus infection. II. Secondary response in vitro and kinetics of memory T cell production in vivo. Cell. Immunol. 22, 283. JOHNSON E.D. & COLE G.A. (1975) Functional heterogeneity of lymphocytic choriomeningitis virus-specific T lymphocytes. I. Identification of effector and memory subsets. J. exp. Med. 141, 866. MACDONALD H.R., CERO-rINI J.-C. & BRUNNER K.T. (1974a) Generation of cytotoxic T lymphocytes in vitro. III. Velocity sedimentation studies of the differentiation and fate of effector cells in long-term mixed leukocyte cultures. J. exp. Med. 140, 1511.
368
M. B. C. Dunlop et al.
MACDONALD H.R., ENGERS H.D., CEROTTINI J.-C. & BRUNNER K.T. (1974b) Generation of cytotoxic T lymphocytes in vitro. 1I. Effect of repeated exposure to alloantigens on the cytotoxic activity of long-term mixed leukocyte cultures. J. exp. Med. 140, 718. VOLKERT M., MARKER 0. & BRO-JeRGENSEN K. (1974) Two populations of T-lymphocytes immune to the lymphocytic choriomeningitis virus. J. exp. Med. 139, 1329.
ZINKERNAGEL R.M. & DOHERTY P.C. (1974a) Immunological surveillance against altered self components by sensitised T lymphocytes in lymphocytic choriomeningitis. Nature (Lond.), 251, 547. ZINKERNAGEL R.M. & DOHERTY P.C. (1974b) Characteristics of the interaction in vitro between cytotoxic thymusderived lymphocytes and target monolayers infected with lymphocytic choriomengitis virus. Scand. J. Immunol. 3, 287.
Cytotoxic T cell response to lymphocytic choriomeningitis virus PROPERTIES OF PRECURSORS OF EFFECTOR T CELLS, PRIMARY EFFECTOR T CELLS AND MEMORY T CELLS IN VITRO AND IN VIVO
M. B. C. DUNLOP, P. C. DOHERTY, R. M. ZINKERNAGEL & R. V. BLANDEN John Curtin School of Medical Research, Australian National University, Canberra, Australia, The Wistar Institute, Philadelphia, Pennsylvania, U.S.A. and Scripps Clinic and Research Foundation, La Jolla, California, U.S.A.
Received 9 December 1976; accepted for publication 13 January 1977
Summary. Spleen cells from CBA/H mice pre-primed intravenously at various intervals with lymphocytic choriomeningitis (LCM) virus were tested for their capacity to respond and generate cytotoxic effector T cells on secondary stimulation both in vitro and in vivo. In vitro secondary stimulation was performed by culturing preprimed spleen cells with infected, syngeneic, peritoneal 'stimulator' cells for periods of up to 7 days at 37°. Controls were incubated with uninfected 'stimulators'. In vivo secondary stimulation was obtained by intravenous transfer of preprimed spleen cells into irradiated, heavily infected CBA/H recipients at various times prior to assay of recipients' spleens. Controls were uninfected, irradiated recipients. Effectors were assayed against LCM virus-infected or uninfected, H-2 compatible L929 target cells in a5'Cr release assay. Spleen cells gave three different patterns of cytotoxic T cell response following in vitro or in vivo stimulation, depending upon the interval between priming and secondary stimulation. Populations taken from mice approximately days 2-5 post-infec-
tion (PI) developed increasing cytotoxic activity against virus-infected targets in vitro when cultured with infected or uninfected peritoneal cells, or in vivo when transferred into irradiated, uninfected recipients. However, these early populations did not generate cytotoxic activity when transferred into irradiated, heavily infected recipients. Established primary effector populations (i.e. approx. days 7-11 PI) exhibited continuing effector activity when maintained in vitro or in vivo, more so when peritoneal 'stimulator' cells or irradiated recipients were infected. Memory populations (i.e. more than approx. day 13 PI) developed highly potent secondary effectors when cultured with infected peritoneal cells, or on transfer to irradiated, infected recipients. The three different patterns of cytotoxic T cell response of the different preprimed spleen cell populations can be interpreted as indicating three different phases of functional activity of the same population of antigen-reactive T cells from LCM virus-primed mice.
Correspondence: Dr M. B. C. Dunlop, Department of Microbiology, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra, 2602, A.C.T. Australia.
INTRODUCTION A central problem in immunology is whether memory 361
362
M. B. C. Dunlop et al.
lymphoid cells and lymphoid cells with a defined effector activity are separate subsets, or are merely representatives of phases in differentiation of the one cell line. MacDonald, Cerottini & Brunner (1974a) have approached this question by separating alloreactive cells on the basis of differing sedimentation velocity. Their results (supported in part by those of Andersson & Hdyry, 1975) suggested that precursors of cytotoxic T cells are small, but upon contact with alloantigen proliferate and/or differentiate into large, blastlike cytotoxic cells; and that with time, at least a fraction of these large effectors dedifferentiate to give small memory T cells (which may be restimulated to proliferate and/or differentiate upon re-exposure to alloantigen). This work is relevant to an interpretation of T cell-mediated responses to lymphocytic choriomeningitis (LCM) virus, where it has been demonstrated that virus-specific effector populations do differ in their properties from T cells with memory for virus. Johnson & Cole (1975) examined effector and memory thymus-derived populations in spleens following priming with LCM virus, and demonstrated that these populations were functionally different, by virtue of their ability either to elicit or protect against acute LCM disease by adoptive transfers, and by their direct cytotoxic activity. Similarly, Volkert, Marker and Bro-J0rgensen (1974) used a variety of diverse criteria to show that early LCM virusimmune spleen and lymph node cells were different from late LCM virus-immune lymphoid cells. It has recently been shown that highly potent, virus-specific T cells can be obtained by cocultivating spleen cells from mice recovered from LCM virus infection several weeks previously, with syngeneic LCM virus infected peritoneal cells at 370 in vitro for 5 days (Dunlop & Blanden, 1976; Dunlop, Doherty, Zinkernagel & Blanden, 1976). This paper describes a method of maintaining primary, or inducing secondary LCM virus specific cytotoxic T cells in vivo by transferring spleen cells to irradiated LCM virus infected syngeneic recipients for various periods. Cells mediating primary effector activity following in vivo transfer have elsewhere been shown to be T cells of donor origin (Zinkernagel & Doherty, 1974a). By comparing the response of spleen cells at different intervals following priming with LCM virus, on culture in vitro or transfer in vivo, it is apparent that different patterns of responses occur depending upon the interval between priming and restimulation. These approaches hence provide further criteria to distinguish primary effector T cells from memory T
cells or from precursors of primary effector T cells and suggest methods to analyse the relationships between subsets.
MATERIALS AND METHODS Mice and virus stocks Inbred, specific pathogen-free CBA/H mice were used throughout. Mice were primed intravenously with the viscerotrophic (WE3) strain of LCM virus and priming dose was 0 2 ml of a 10-s dilution of guineapig spleen or lung stock titrated at approx. 400 intracerebral LD5o or 250 P.F.U. (Zinkernagel & Doherty, 1974b). Virus stocks used for immunization were dispensed in small amounts and frozen at - 700 to minimize day-to-day variation in dosage. In vitro secondary stimulation The procedure for in vitro secondary stimulation of memory T cells by cocultivation for varying periods with LCM virus-infected, syngeneic peritoneal cells has been detailed elsewhere (Dunlop & Blanden, 1976). Essentially, peritoneal cells were infected by incubation at 370 with neat WE3-LCM virus stock for 30 min, then washed in culture medium. Infected or uninfected peritoneal cells (usually 2-5 x 106 per flask) were then cultured overnight in small (25 cm2 surface area) Falcon plastic flasks containing 10 ml of culture medium. Following overnight culture, the spleen responder cells under test were added to the infected or uninfected peritoneal cells (2-5 x 107 responders per flask). Cultures were incubated at 37° for various intervals and then tested in the 51Cr release assay described below. In vivo secondary stimulation One day prior to transfer of the spleen responder cells under test, recipients were given 850 rads from a 60Co source. On the day of transfer, recipients were injected intravenously with 0 2 ml of a 10-2 dilution of virus stock (approx. 4 0 x 101 intracerebral LD50 or 2 5 x 105 P.F.U.). About 12 h later, spleen cells obtained from donor mice were prepared in a way which would remove embolic material as described (Blanden & Langman, 1972) and then transferred intravenously to the irradiated, infected or uninfected syngeneic recipients. Usually about 1.0 x 108 spleen cells were transferred per recipient, and groups of four recipients were employed. Mice were then maintained on routine feeds and antibiotics for
Induction of T precursors, effectors and memory cells for LCM (c )
(a )
80 70 60 50 40 30 20 en -il 10 y 0
FIG.
363
.
.11
2 3 4 80 FIG.2. (a) 70 60 50
5 (c )
( b)
A~
~
~
~
0~~~~~
40 _-
30 20
4
5
4
5
10 3
6
4 3 5 6 Incubation time (days)
3
4
5
6
Figure 1. Effect of in vitro culture of CBA/H spleen cells (preprimed with LCM virus i.v. at intervals), with infected or uninfected, syngeneic peritoneal stimulator cells for 2, 3, 4, or 5 days prior to assay. Effector-target ratio was 4: 1 and peritoneal stimulator) Infected stimulator peritoneal cells; spleen cell responder ratio was 1: 10. Medium release was 204%0 on infected targets. ( (- --) uninfected stimulator peritoneal cells. (a) (U) Normal spleen cells; (A) D3 post-inoculation (PI); (0) D5 PI (b) (A) D7 PI; (A) D9 PI (c) (0) D21 PI Figure 2. Effect of in vitro culture of pre-primed CBA/H spleen cells with infected, syngeneic peritoneal cells for 3, 4, 5, or 6 days prior to assay. Effector-target ratio was 1:1 and peritoneal stimulator cell-spleen responder cell ratio was 1:10. Medium release was 25-2 per cent on infected L929 targets. The legend is the same as for Fig. 1, with the inclusion of (b) (0) DlI PI (c) (U) D13 PI; (A) D15 PI. -
omitted for clarity. Statistical significance determined by Student's f-test.
various intervals prior to removal of their spleens for assay of cytotoxic T-cell activity on virus-infected targets. Spleens generally yielded 5.0 x 105-5-0 x 106 viable lymphoid cells/spleen.
were
Technique of IICr release assay, and method of expressing results The 5'Cr release assay has been described before (Zinkernagel & Doherty, 1974b). Targets were LCM virus-infected or uninfected, H-2 compatible L929 fibroblast cells. Experiments were designed such that in vitro cultures or in vivo transfers were assayed on the one day. Results were expressed as percent specific lysis = (percentage 51Cr release in presence of effectors -percentage medium release) x 100/percentage water lysis. Specific lyses on infected targets only are shown for all figures excepting Fig. 5. Data presented are the mean of four replicates. Standard errors of the mean were never greater than ± 2% and
General Early in vivo experiments demonstrated that primary LCM virus specific T cell activity in spleens increased from day 5 to peak on day 9 post LCM virus inoculation (P1), then fell slowly to give residual cytotoxic activity by day 21. To facilitate the interpretation of cytotoxic responses, the following definitions were made. Pre-peak (or precursor) primary cytotoxic T cells were defined as those harvested up to day 7 PI; peak cytolytic T cells were those harvested from days 7-11 PI; and memory cells were defined as those T cells obtained after day 11 PI. Another initial experiment using a virus plaque assay showed that after intravenous challenge, virus titres in spleens
was
RESULTS
M. B. C. Dunlop et al.
364 80 70 60 50 40 30 20 10
*0>
_ FIG.3.
(a)
(c)j
( b) A
0
/,
\/ 0
-
-
2
0
\ 0
A
A"
0~
.
Q
us
FIG.4. 4, 80 13
( b)
(c )
70 60 60
C)
40
30 20
10 l l
l
1
L
5 6 3 Time after transfer (days) 3
4
4
5
6
Figure 3. Effect of in vivo transfer of pre-primed CBA/H spleen cells to irradiated, infected or uninfected, syngeneic recipients for 3, 4, 5, or 6 days prior to assay of recipients' spleens. Effector-target ratio was 10:1. Medium release was 23-7 per cent on ) Infected, irradiated CBA/H recipients; (-- -) uninfected, irradiated CBA/H recipients. (a) (-) Normal infected targets. ( spleen cells; (-) D5 PI (b) (A) D9 PI (c) (0) D21 PI. Figure 4. Effect of in vivo transfer of pre-primed CBA/H spleen cells to irradiated, infected, syngeneic recipients for 3, 4, 5, or 6 days prior to assay of recipients' spleens. Effector-target ratio was 10: 1. Medium release was 14-5 per cent on infected targets. The legend is the same as for Fig. 3, with the inclusion of (b) (U) D7 PI; (0) DII P1 (c) (U) D13 PI; (A) D15 P1. -
increased rapidly to peak on days 3-5 and slowly fell thereafter. Therefore pre-peak (e.g. D5 PI) and peak (e.g. D9 PI) primary spleen populations contained large amounts of virus. In vitro culture of spleen cells at various intervals postpriming with LCM Groups of CBA/H mice were infected with a single dose of a standard inoculum of LCM virus, spleen cells were harvested at intervals PI, and spleen cells were cultured with syngeneic, infected or uninfected peritoneal cells (Fig. 1) or infected peritoneal cells only (Fig. 2). Cultures were maintained for 2, 3, 4, or 5 days (Fig. 1) or 3, 4, 5 or 6 days (Fig. 2) before assay. In general, three patterns of cytotoxic activity -pre-peak primary, peak primary and memoryemerged: (a) normal spleen cells did not develop significant cytotoxic activity in culture, whilst D3 and D5 PI spleen cells increased in activity from day 2 to day 5 in culture (Fig. 1) or peaked on day 5 of
culture (Fig. 2) (i.e. about that time, 8-10 days PI, at which these same spleen cells would be expected to reach peak effector activity if they remained in the intact, infected animal). (b) D7 PI and D9 PI spleen cells peaked in cytotoxic activity on day 4 (Fig. 1), or D7, D9 and DlI PI cells continually declined in activity from day 3 to day 6 of culture (Fig. 2) (c) D21 PI cells (Fig. 1) or D13, D15 and D 21 PI cells (Fig. 2) developed potent secondary responses. In Fig. 1, all spleen cell populations, including D3 and D5 PI, responded more strongly with infected (solid lines) than with uninfected (dashed lines) peritoneal stimulators. In vivo transfer of spleen cells at various times postpriming with LCM Groups of CBA/H mice were primed with LCM virus, and spleen cells were transferred at intervals into irradiated, infected or uninfected, syngeneic recipients. Irradiated recipients' spleens were assayed
Induction of Tprecursors, effectors and memory cells .for LCM FIG.5. 80 - (a)
365
( b)
70 60 50 40 30 0
20 10 _2
0
4-
5
6
7
Incubation time (days) aL)
80 FiFG.6.
L) 70 0
60
*\
50
//N
40
30 _
20 _ 10
0
3
5
7 8
10 11
13 14
16 17 18
Time after priming(weeks)
Figure 5. Results of in vitro culture of recently primed CBA/H spleen cells with infected or uninfected, syngeneic peritoneal cells for 4, 5, 6 or 7 days prior to assay. Specific releases on both infected (a) and uninfected (b) targets are shown. Effectortarget ratio was 10:1 and peritoneal stimulator-spleen responder ratio was 1: 12 5. Specific lyses on uninfected targets were included in this figure because they were not negligible at this effector-target ratio for recently primed cells cultured in vitro. Medium release was 30 0%/ on infected targets and 28 5% on uninfected targets. (0) D4 PI (A) D2 PI (-) Dl P1. Specific lyses by cells from cultures with uninfected stimulators were generally similar to, and slightly smaller than, specific lyses by cells from cultures with infected stimulators, and were omitted. Specific lyses by cells from cultures where spleen cells were primed 12 h, 4 h, or not at all before culture were essentially similar to, but generally smaller than, specific lyses by Dl PI spleen cells on infected and uninfected targets, and were also omitted for clarity. Figure 6. Specific lysis of infected L929 targets by cytotoxic T cells generated by culture of CBA/H spleen cells (preprimed with LCM virus for several weeks), with infected or uninfected, syngeneic peritoneal cells for 5 days prior to assay. Effector-target ratio was 05:1, and peritoneal stimulator cell-spleen responder cell ratio was 1:32. Medium release was 22-8% on infected L929 targets. Specific lyses by cells from culture with uninfected peritoneal stimulator cells were too small to be conveniently depicted and were omitted.
3, 4, 5,
or
6 days after tranfer. Results
are
given for
two such experiments, the first using irradiated, both
infected and uninfected control recipients, the second with irradiated, infected recipients only. (Figs 3 and 4). Results were in general similar to those given in Figs 1 and 2, but differed in that transfer of D5 PI cells to irradiated, infected recipients led to less development of cytotoxic activity than in uninfected
recipients. Two other experiments have confirmed that D5 cells on transfer to irradiated infected recipients showed little evidence of generation of cytotoxic activity even up to 8 days post-transfer. This population did not generate cytotoxic effectors on transfer to irradiated, infected recipients in other organ sites such as thymus, peritoneal cavity, blood or mesenteric lymph node (data not shown). Hence,
M. B. C. Dunlop et al.
366
D5 PI spleen cells (a pre-peak primary population), possessed the capacity to be suppressed.
Effect of in vitro culture of spleen cells after short intervals of priming Spleen cells from CBA/H mice were cultured with infected or uninfected syngeneic peritoneal cells, having been primed 4 h, 12 h, 1, 2, or 4 days previously with LCM virus, and effectors were assayed following culture for 4-7 days. (Fig. 5). There was a continual development of specific cytolytic activity on infected targets over and above that on uninfected targets by D4 PI spleen cells and to a lesser extent by D2 and D1 PI spleen cells, when infected stimulators were present. Spleen cells taken from the animal at earlier times post-challenge showed no clear primary induction. In another experiment, it was possible to maintain 'virus-specific' activity of D2 PI cells from 6 to 12 days of culture, and by further adding freshly infected peritoneal cells on day 7 and day 13, a stronger secondary virus-specific response was obtained, by day 18 (data not shown). Effect of in vitro culture ofspleen cells at long intervals post-priming Groups of CBA/H mice were inoculated weekly with a dose of LCM for several months before culturing with infected or uninfected, syngeneic peritoneal cells. Cultures were assayed on the 5th day only (Fig. 6). Infected stimulators induced strong secondary cytotoxic T cell responses from 3 weeks post-priming up to at least 18 weeks post-priming. Other assays have established that potent LCM-specific memory exists to at least 36 weeks post-priming.
DISCUSSION Both in vitro and in vivo methods have been shown in this article to allow stimulation and presumably proliferation and differentiation into cytotoxic T cells, of T cells taken at various stages post-priming. Comparisons between the two methods reveal differences depending on the interval between priming and secondary challenge and the amounts of virus or infected cells present in the system. D5 PI cells, i.e. precursors of primary cytotoxic T cells which reach a peak between days 8-10 if left in
the mouse are 'committed' in the sense that they will generate cytotoxic T cells in vitro when cocultivated with infected or uninfected stimulators. Infected 'stimulators' are in fact not necessary when D5 cells are responders. This is because D5 spleen cells themselves contain large amounts of virus and infected cells able to act as stimulators (data not shown). Similarly, D5 cells can generate effectors when transferred intravenously to irradiated, uninfected recipients. In contrast, irradiated, infected recipients do not allow or suppress the generation of primary cytotoxic T cells. It seems unlikely that cytotoxic T cells are being generated in, or migrating to, other organ sites in the irradiated, infected recipient, since other experiments show that D5 PI cells transferred to irradiated, infected or uninfected recipients do not give rise to cytotoxic T cells in thymus, peritoneal cavity, blood or mesenteric lymph node (peripheral lymph nodes were too small to be detected in irradiated recipients). Presumably the mechanism of suppression involves excess virus or excess antigen, since the irradiated recipients were infected with large doses of virus. This idea is supported by the findings that cocultivation of D5 PI cells with spleen cells from congenital LCM carriers, or with large numbers of infected peritoneal cells also leads to suppression; furthermore, intravenous administration of virus neutralising antiserum to D5 PI donor mice 30 min prior to removal of their spleens for culture resulted in an enhanced generation of a cytolytic response from these spleen cells (Dunlop & Blanden, 1977). Spleen cells harvested closer to the time of the expected peak of the cytotoxic T cell response in vivo (e.g. D7-D9 PI) generally gave a peak of activity after transfer to the new in vitro or in vivo situation, and then declined in activity. Cells taken at or after the peak (D9-D11 PI) gave declining activity with time in their new environment. Activity in an infected environment (in vitro or in vivo) was generally higher than in an uninfected environment, so continued antigenic stimulation may prolong activity of effector cell populations. Cell populations with established cytolytic activity against LCM virusinfected targets (D7-D11 PI) seem less vulnerable than pre-peak (D5 PT) primary cells to the mechanism of suppression cited above. This may be explained by peak primary cells killing infected stimulator cells, thus limiting production of virus and spread of infection. The destruction of stimulator cells by cytotoxic T cells may also explain in part why no major stimulation of a new peak of activity
Induction of Tprecursors, effectors and memory cells for LCM occurred with responder spleen populations harvested on days 9-11 PI. This may correspond to the refractoriness to restimulation seen in the responses to alloantigens (MacDonald, Engers, Cerottini & Brunner, 1974b) and to ectromelia-virus infected cells (Gardner & Blanden, 1976). The capacity for a secondary response was clearly seen by D13 PI. D13-D21 PI cells initially show little cytolytic activity upon transfer either in vitro or in vivo, but develop potent cytolytic activity only in an antigenic environment. Memory T cells persist up to at least 36 weeks PI. Collectively, these results are compatible with the development and differentiation first of primary cytotoxic T cells, and then of memory T cells, from one subset of antigen-reactive precursor T cells, which is the simplest and most economical model of the response. Peak primary activity is established by day 8-9, declines, and memory cells appear by day 13-15. This may reflect dedifferentiation of proliferating effector cells (probably large, blast cells) to small lymphocytes, which possess memory as suggested by MacDonald et al. (1974a) and Andersson & Hayry (1975) for the response to alloantigens. Certainly effectors and memory cells are not the same populations physiologically, but the appearance of the latter is chronologically linked to the appearance of the former. Johnson & Cole (1975) also inferred that at least two physiologically different virus-specific T lymphocyte subsets exist following LCM infection in vivo by testing BALB/c splenic lymphocytes obtained at varying intervals after LCM challenge for their capacity either to elicit acute LCM disease (memory cells but not acute primary effectors) or protect against lethal intracerebral LCM virus challenge (acute primary effectors but not memory cells) when transferred by the intraperitoneal route to syngeneic, respectively cyclophosphamide treated and LCM virus-infected or normal mice. As well, the same splenic lymphocytes were tested for their capacity to lyse LCM virus-infected BALB/c 3T3 target cells in vitro, and efficient cytolytic activity resided in only acute primary effectors. Volkert et al. (1974) used various criteria to separate early immune anti-LCM cells (D9 PI) from late immune anti-LCM cells (D30 PI). Early immune cells contained many more large and blast-like cells than late immune cells, and LCMspecific cytotoxic activity, anti-viral effect after transfer to acutely infected animals, ability to protect against a lethal intracerebral infection and resistance to X-irradiation were almost entirely or predomi-
367
nantly properties of early immune cells. Both early and late immune cells were susceptible to treatment with anti-theta serum. However, late immune cells were much superior to early immune cells in reducing virus titres on transfer to LCM virus carriers, a property explained by the induction of memory T cells to give rise to highly potent antiviral effectors. These potent secondary effectors are readily able on transfer in small numbers (2-5 x 107) to mediate a reduction in spleen virus titres by 4-5 logs (Dunlop, 1977). It has yet, of course, to be determined precisely whether antiviral cytotoxic T cells give rise to memory T cells, but in vitro maintenance of primed cells and generation of responses should lend itself to physical separation methods or other approaches to this interesting area.
REFERENCES ANDERSSON L.C. & HAYRY P. (1975) Clonal isolation of alloantigen-reactive T cells and characterization of their memory functions. Transplant Rev. 25, 121. BLANDEN R.V. & LANGMAN R.E. (1972) Cell-mediated immunity to bacterial infection in the mouse. Thymusderived cells as effectors of acquired resistance to Listeria monocytogenes. Scand. J. Immunol. 1, 379. DUNLOP M.B.C. (1977) Secondary cytotoxic cell response to lymphocytic choriomeningitis virus. III. In vivo protective activity of effector cells generated in vitro. Immunology, in press. DUNLOP M.B.C. & BLANDEN R.V. (1976) Secondary cytotoxic cell response to lymphocytic choriomeningitis virus. I. Kinetics of induction in vitro and yields of effector cells. Immunology, 31, 171. DUNLOP M.B.C. & BLANDEN R.V. (1977) Mechanisms of suppression of cytotoxic T cell responses in murine lymphocytic choriomeningitis virus infection. J. exp. Med. 145, 1131. DUNLOP M.B.C., DOHERTY P.C., ZINKERNAGEL R.M. & BLANDEN R.V. (1976) Secondary cytotoxic cell response to lymphocytic choriomeningitis virus. II. Nature and specificity of effector cells. Immunology, 31, 181. GARDNER I.D. & BLANDEN R.V. (1976) The cell-mediated immune response to ectromelia virus infection. II. Secondary response in vitro and kinetics of memory T cell production in vivo. Cell. Immunol. 22, 283. JOHNSON E.D. & COLE G.A. (1975) Functional heterogeneity of lymphocytic choriomeningitis virus-specific T lymphocytes. I. Identification of effector and memory subsets. J. exp. Med. 141, 866. MACDONALD H.R., CERO-rINI J.-C. & BRUNNER K.T. (1974a) Generation of cytotoxic T lymphocytes in vitro. III. Velocity sedimentation studies of the differentiation and fate of effector cells in long-term mixed leukocyte cultures. J. exp. Med. 140, 1511.
368
M. B. C. Dunlop et al.
MACDONALD H.R., ENGERS H.D., CEROTTINI J.-C. & BRUNNER K.T. (1974b) Generation of cytotoxic T lymphocytes in vitro. 1I. Effect of repeated exposure to alloantigens on the cytotoxic activity of long-term mixed leukocyte cultures. J. exp. Med. 140, 718. VOLKERT M., MARKER 0. & BRO-JeRGENSEN K. (1974) Two populations of T-lymphocytes immune to the lymphocytic choriomeningitis virus. J. exp. Med. 139, 1329.
ZINKERNAGEL R.M. & DOHERTY P.C. (1974a) Immunological surveillance against altered self components by sensitised T lymphocytes in lymphocytic choriomeningitis. Nature (Lond.), 251, 547. ZINKERNAGEL R.M. & DOHERTY P.C. (1974b) Characteristics of the interaction in vitro between cytotoxic thymusderived lymphocytes and target monolayers infected with lymphocytic choriomengitis virus. Scand. J. Immunol. 3, 287.
