Immunology 1976 31 181

Secondary cytotoxic cell response to lymphocytic choriomeningitis virus II.

NATURE AND SPECIFICITY OF EFFECTOR CELLS

M. B. C. DUNLOP, P. C. DOHERTY, R. M. ZINKERNAGEL & R. V. BLANDEN John Curtin School of Medical Research, Australian National University, Canberra, Australia

Received 22 December 1975; accepted for publication 26 January 1976

Summary. The method described in the previous paper was used to induce secondary responses in spleen cells from CBA/H mice, pre-primed with lymphocytic choriomeningitis (LCM) virus by culturing them with LCM-infected peritoneal cells. The cytolytic effector cells thus generated have been characterized. Effector cells were sensitive to treatment with anti-O ascitic fluid and complement. Separation procedures based on rosetting of certain categories of lymphocytes with sheep red cells through an Isopaque-Ficoll gradient indicated that effector cells lacked surface immunoglobulin and generally did not bear Fc receptors. Cytolytic activity was restricted by the H-2 gene complex. Killing had single-hit characteristics. All these results suggested that the cells from memory cultures mediating cytolysis were T cells. There was evidence for two T cell subsets, a major subpopulation directed against antigens on infected targets and a minor one directed against antigens on uninfected, H-2-compatible targets. Specificity was present at the infected cell: memory responder and killer: target levels between LCM virtis (an arenavirus) and ectromelia virus (a poxvirus).

response to LCM. The activity of such cells is reflected in protecting normal recipients from the development of lethal meningitis when transferred 18-24 h prior to intracerebral viral challenge (Johnson and Cole, 1975), in conferring lethal meningitis in cyclophosphamide treated recipients when transferred intravenously after intracerebral viral challenge (Doherty and Zinkernagel, 1975) and in cytolytic activity on 5'Cr-labelled, infected, H-2 compatible targets (Doherty, Zinkernagel and Ramshaw, 1974; Zinkernagel and Doherty, 1974b). Moreover, T cells obtained from donors 30 days after priming with LCM (that is, memory T cells) are capable of eliciting meningitis on transfer to intracerebrally infected, cyclophosphamide treated recipient mice (Johnson and Cole, 1975). In the preceding paper we have described a method of obtaining very potent anti-LCM effector cells (as assayed by cytolysis of infected target cells in vitro) by culturing pre-primed lymphoid cells with infected peritoneal cells in vitro. The ability of these in vitro raised memory effectors to protect against or aggravate meningitis is under investigation. In this paper, we describe results from a variety of different techniques, which show that the effector cells generated in secondary cultures are T cells.

INTRODUCTION Effector T cells are generated in the primary immune

MATERIALS AND METHODS

Correspondence: Dr M. B. C. Dunlop, Department of Microbiology, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra, Australia 2601.

General Inbred mice, virus stocks, methods of immunization,

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182

protocol for memory cultures and techniques of 5 'Cr release have been described in the preceding paper (Dunlop and Blanden, 1976). Westminster (viscerotrophic) strain of LCM virus and CBA/H mice were used except where stated otherwise. The procedures for anti-C treatment and inclusion of unlabelled targets in the "Cr release assay to act as specific competitors are described elsewhere (Kirov, 1974; Zinkernagel and Doherty, 1975). Separation procedures based on Ig and Fc receptors have been described (Parish, Kirov, Bowern and Blanden, 1974; Parish and Hayward, 1974). They are based on the ability to separate receptored cells by centrifugation on an Isopaque-Ficoll gradient, the receptored cells having been allowed to form dense rosettes by treatment (in the case of the Ig receptor) with rabbit anti-mouse antiserum followed by sheep red cells chemically coupled to sheep anti-rabbit antibody (Parish et al., 1974) or (in the case of the Fc receptor) with sheep red cells coated with rat antisheep antibody (Parish and Hayward, 1974). The technique of ectromelia memory cultures (Gardner and Blanden, 1976 is essentially identical with the technique of LCM memory culture except that spleen cells were used as stimulators and were infected with the Hampstead Egg strain of ectromelia virus (at a multiplicity of 1 PFU/stimulator cell) only 3 h prior to adding ectromelia memory responders. Cultures were harvested on the 4th day, having been incubated at a temperature of 390 (which is nonpermissive for viral replication and cytocidal effect of the responding lymphoid cells). The ectromelia I Cr release assay has been described (Gardner, Bowern and Blanden, 1974). Dr Parish kindly provided the anti-C ascitic fluid. RESULTS Anti-C susceptibility CBA/H effector cells from a 5-day memory culture were assayed following treatment with anti-C ascitic fluid plus complement, normal AKR ascitic fluid plus complement, or complement only (Table 1). The first treatment substantially reduced, but did not abrogate, specific lysis of infected targets (best seen at the lowest effector: target ratio). Treatment with anti-C ascitic fluid plus complement depressed specific cytotoxic activity of primary immune effectors generated in vivo (from 53-7 per cent to 0 per cent) (at an effector:target ratio of 5:1, Table l, lower

Table 1. Anti-C susceptibility of cytotoxic cells from 5-day memory cultures*

Targets Responder

CBA/H memory spleen cells

Primary immune

Treatment plus complement

E :T ratio

0 5:1 Anti-C ascitic fluid 0-25:1 Normal AKR 0-5:1 0 25:1 ascitic fluid 0 5:1 Nil 0-25:1

Anti-C

ascitic fluid Normal AKR CBA/H spleen cells ascitic fluid Nil

L929 Infected

10-3t 5 3t 35 9 18 3 45-6 28-9

L929 Uninfected 0 0

1P2 07 0 0-5

5:1

Ot

0-3

5:1

53 7

07

5:1

48-5

0-8

* Infected peritoneal cell: memory responder ratio was 1:10. Results in this and subsequent tables were expressed as percentage specific release = 100 x (percentage 51Cr release in presence of effectorspercentage medium release)/percentage water release. Results were means of four replicates. Standard errors were invariably less than + 2 per cent and usually less than + I per cent and were omitted for clarity. Medium release was 19 4 per cent on infected targets and 18 3 per cent on uninfected targets. t Significantly lower than normal ascitic fluid treated controls for same effector: target ratio (P< 0-001).

section) to a greater extent than it depressed specific cytotoxicity of secondary immune effectors generated in vitro (from 35-9 per cent to only 10 3 per cent) (at an effector:target ratio of 0 5:1, Table 1, upper section). Since primary immune effectors are known to be T cells, the lesser effect of anti-C plus complement treatment on the secondary cytotoxic effectors may indicate that these cells are T cells which are less sensitive to this treatment, or the presence of effectors other than T cells.

Separation procedures based on red cell resetting of Ig+ and Fc+ cells Memory responders from a 5-day culture were separated into subpopulations bearing surface immunoglobulin (Ig+) and lacking surface immunoglobulin (g-). Removal of Ig+ cells in no way impaired the ability of the remaining Ig- cells to kill infected targets (Table 2). Igo cells, which formed

Nature of effector cells from Table 2. Response of memory cultures following separation procedure based on immunoglobulin receptor*

memory

183

cultures

Table 3. Response of 5-day memory cells following separation procedure based on Fc receptor* Targets

Targets

Subfraction Unseparated

Ig-

Ig+ Reconstituted Ig- + Ig+ (ratio 82:18)

E:T ratio Infected L929 Uninfected L929 1:1 0-5:1 0-25:1 1:1 0-5:1 0-25:1 1:1 0-5:1 0-25:1 1:1 0-5:1 0-25:1

52-6 28-5 15-4 56-1I

29-5t 14-4t 9-4t 3-2t 3-4t 46-2 24-7 12-6

0 0 0 0 0 0 1-8

Subfraction

Unseparated Fc

Fc+

0 0

0 0 0

Infected peritoneal cell: memory responder ratio was 1:10. Medium release was 20-4 per cent on infected targets and 18-3 per cent on uninfected targets. Ig+ = bearing surface immunoglobulin; Ig- = lacking surface immunoglobulin. t Significantly lower than all other groups for same effector: target ratio (P < 0 001). t Not significantly different from unseparated memory spleen cells for same effector: target ratio. *

about 18 per cent of the cells from culture, had little cytotoxic activity. Remixing separated cells resulted in cytotoxic potency comparable with unseparated effectors. These results suggest that Ig+ B cells or K cells play no significant role in cytolysis in this system. To test the possibility that Ig- K cells (Fc+) were

the effector cells, a separation of effector cells into subpopulations possessing Fc receptor(s) (Fc+) and lacking Fc receptors (Fc-) was performed. Removal of Fc+ cells did not reduce destruction of targets by the remaining Fc- cells (Table 3). Fc+ cells did have cytotoxic activity on a cell-for-cell basis but were only 8 per cent of the population. Assaying Fc+ cells at an effector: target ratio 8/100 of that of unseparated cultures, that is, to correspond to the incidence that Fc+ cells would be predicted to be found in unseparated cultures (lines 13-15, Table 3) showed very little specific killing, indicating that Fc+ cells contribute little cytotoxic activity as a subpopulation. H-2 restriction of cytotoxic effector activity Armstrong (neurotrophic) strain-LCM primed

Fc-, correctedt Fc+, correctedt Reconstituted

Fc-+Fc+ (ratio 92:8)

E:T ratio Infected L929 Uninfected L929 1:1 0 5:1 0-25:1 1:1 05:1 0-25:1 1:1 0 5:1 0 25:1 0-92:1 0-46:1 0-23:1 0-08:1 0-04:1 0-02:1 1:1 05:1 0-25:1

77-4 67-4 37-3 75 7 70-7 50-1 51-5 34-1 20-0 77-4§

66-9§ 38-0§ 6-6

5-8T 23¶ 73-2 64-4 48-6

0 0-2 0 0 0 0 0 0 0 0 0.1 07 0 0 0 0-6 0 0

* Infected peritoneal cell: memory responder ratio was 1:10. Medium release was 19 9 per cent on infected targets and 20-7 per cent on uninfected targets. Fc+ = possessing Fc receptor(s); Fc- = lacking Fc receptors. t 'Corrected' means diluted to an effector: target ratio which would have been the same as the effector: target ratio of that subpopulation in unseparated culture. t Significantly higher than unseparated spleen for same effector:target ratio (P< 0-01). § Not significantly different from unseparated spleen for same effector: target ratio. ¶ Significantly lower than all other groups for same effector: target ratio (P< 0 001).

CBA/H (H-2k) and BALB/c (H-2d) memory mice were cultured with H-2 compatible infected ot uninfected 'stimulators' for 5 days and the cells from culture were then assayed on L929 fibroblasts (H-2k) or P815-X2 mastocytoma cells (H-2d). (Table 4). CBA/H responders lysed only H-2 compatible L929 infected targets, whilst BALB/c responders best lysed H-2 compatible P815-X2 infected targets. Evidence for possible different cytotoxic T-cell subsets from memory cultures CBA/H cells from a 5-day culture were assayed against labelled, infected L929 cells in the presence of a nine-fold excess of competing, unlabelled,

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184

Table 4. H-2 restriction of cytotoxic effectors induced in 5-day memory cultures*

Targetst Responder spleen cells Memory CBA/H (H-2k)

L929 (H-2k) P815-X2 (H-2d) Stimulator peritoneal cells Infected Uninfected Infected Uninfected

Uninfected CBA/H (H-2k) Infected CBA/H

Memory Uninfected BALB/c (H-2d) BALB/c (H-2d) Infected

3-6

6-2

2-1

0

51-1t

8-7

35

2-5

4-2

4-2

2-3

0

8-9

12d1

34-6t

0

BALB/c * Stimulator: responder ratio was 1:4. Effector: target ratio was 2 5:l1. t Significantly higher than that of any other target lysed by the same memory cells (P< 0001). t Medium release was 18-4 per cent on infected L929 targets and 16-9 per cent on uninfected L929 targets, 27-4 per cent on infected P-815 targets and 30-6 per cent on uninfected P-815 targets.

Table 5. Evidence for different subsets of cytotoxic T cells from 5-day memory cultures using unlabelled competing syngeneic target cells*

Targetst Competitor Competitor Infected Uninfected status

excess

Uninfected

9:1

57-6

Ot

Infected

9:1

18-6t

Nil

80-7

16-3 13 4

L929

L929

* Stimulator: responder ratio was 1: 5; effector: target ratio was 1:1. t Significantly lower than lysis of same target by any other group (P < 0-001). $ Medium release was 24-8 per cent on infected targets and 22-0 per cent on uninfected targets.

infected or uninfected L929 fibroblasts (Table 5). Infected 'cold' targets competed with killing of infected, labelled targets to a greater extent than uninfected competitors, whilst only uninfected competitors competed with killing of uninfected targets.

Specificity of induction and cytotoxic effector activity Spleen cells from LCM-primed or ectromelia-

primed CBA/H mice were cultured with uninfected, LCM-infected or ectromelia-infected syngeneic peritoneal cells at 390 (to prevent cytolytic activity of ectromelia virus) for 5 days (in the case of LCM memory responders) or 4 days (in the case of ectromelia memory responders) such that 51Cr release assays against uninfected, LCM-infected or ectromelia-infected targets were performed on the one day. Results (Table 6) show that only culturing memory cells with the appropriately infected peritoneal cells generated effector cells which lysed the appropriately infected targets. DISCUSSION The results cited in this and the preceding paper (Dunlop and Blanden, 1976) provide evidence, using different techniques, that effector cells obtained from memory cultures are T cells. First, it was shown in the preceding paper that effector cells had a straight line log dose: log response activity with 450 slope, indicating 'single hit killing' another property of T cells (Miller and Dunkley, 1974). Second, treatment with anti-O ascitic fluid and

complement greatly reduced (but did not abolish) cytotoxic activity. Third, effector cells were Ig-, since Ig- cells form 82 per cent of a memory culture

185

Nature of effector cells from memory cultures Table 6. Specificity of induction and cytotoxic effector activity from 5-day LCM memory cultures and 4-day ectromelia cultures* L929 targetst

CBA/H spleen responder

CBA/H peritoneal cell stimulator

Uninfected LCM-infected Ectromelia-infected Uninfected Ectromelia-memory LCM-infected Ectromelia-infected LCM memory

LCM-infected

Uninfected

Ectromelia-infected

Uninfected

5S1 75-5t 15 12-2 13 5 18

0 0 0 40 3-7 2-0

1-4 6-3 0 7-3 8-9 32-3t

2-1 10 9 0 11 1 19 1 5-6

* Peritoneal cell:memory responder ratio was 1:10. Effector:target ratio was 1:1 for LCM and 2:1 for ectromelia. Incubation temperature of memory cultures was 39°. Cultures were assayed on the same day. t Significantly higher than that of any target lysed by the same memory cells (P< 0-001). t Medium release was 21-5 per cent on LCM infected targets, 20-0 per cent on uninfected targets in LCM assay, 19 6 per cent on ectromelia-infected targets and 21 6 per cent on uninfected targets in ectromelia assay.

and were as potent as unseparated cells containing 18 per cent Ig+ cells, and Ig+ cells had little activity. Fourth, killing by K cells (which bear Fc receptors and require a source of specific antibody which binds to target cells), seems unlikely to be a major component because removal of Fc+ cells enhanced killing by the remaining 92 per cent of Fc- cells. Fc+ cells did give specific release on a cell-for-cell basis, which could have been because a proportion of activated T cells bore Fc receptors, or because a small number of Fc+ K cells were present. If there was a minor population of K cell effectors, this can only have represented a tiny proportion of effector activity, since diluting out the Fc+ subpopulation to an effector: target ratio which would have been the same as the effector: target ratio of the subpopulation of Fc+ cells in unseparated effectors abolished activity. Target lysis was restricted by the H-2 complex in that CBA/H (H-2k) memory effectors strongly lysed only infected L929 fibroblasts (H-2k) whilst BALB/c (H-2d) effectors best lysed infected P815-X2 mastocytoma cells (H-2d). This constraint on activity conforms with previous findings which indicate that potent T cell-mediated killing occurs in only those assays in which H-2K or H-2D regions are shared by donors of infected target cells and immune T cells (Zinkernagel and Doherty, 1974a; Gardner, Bowern and Blanden 1975; Blanden, Doherty, Dunlop, Gardner, Zinkernagel and David, 1975; Koszinowski and Thomssen, 1975; Doherty and Zinkernagel, 1976). A non-T cell-mediated cytotoxic assay can

work with allogeneic or even xenogeneic virusinfected targets (Ramshaw, 1975). There may exist at least two subsets of effector T cells, since adding unlabelled infected competing targets substantially competed with killing of infected, labelled targets, but not with the less prominent killing of uninfected, labelled targets. The minor population thus recognized antigens on uninfected, H-2 compatible, target cells. This activity on uninfected, syngeneic targets is more evident in primary (Gardner et al., 1975) and secondary (Gardner and Blanden, 1976) responses to ectromelia virus, but the nature of the antigens involved is unknown. We have noticed that no adherent cells remained in memory cultures after 5 days incubation. This is probably because infected adherent cells were themselves killed by developing effectors. Thus, it seems unlikely that macrophages are important as effector cells. Specificity was demonstrated between LCM and ectromelia memory (also mediated by T cells: Gardner and Blanden, 1976) both in requirements for induction and in target destruction. Specificity exists also in killing by spleen cells from primary immunized mice between LCM virus, ectromelia virus and Sendai virus (a paramyxovirus) (Doherty and Zinkernagel, 1975, 1976). We may infer from the results in this and the preceding paper that memory T cells are present in the intact, LCM-primed mouse. The significance of T cell memory in resistance to secondary infection remains to be determined.

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ACKNOWLEDGMENTS We are most grateful to Dr C. R. Parish for performing the Ig and Fc separations. The specificity assay between LCM and ectromelia was performed in collaboration with Mr T. Pang.

REFERENCES BLANDEN R.V., DOHERTY P.C., DUNLOP M.B.C., GARDNER I. ZINKERNAGEL R.M. & DAVID C.S. (1975) Genes required for cytotoxicity against virus-infected target cells in K and D regions of H-2 complex. Nature (Lond.), 254, 269. DOHERTY P.C. & ZINKERNAGEL R.M. (1975) Capacity of sensitized thymus-derived lymphocytes to induce fatal lymphocytic choriomeningitis is restricted by the H-2 gene complex. J. Immunol. 114, 30. DOHERTY P.C. & ZINKERNAGEL R.M. (1976) Specific immune lysis of paramyxovirus-infected cells by H-2 compatible thymus-derived lymphocytes. Immunology, 31. DOHERTY P.C., ZINKERNAGEL R.M. & RAMSHAW I.A. (1974) Specificity and development of cytotoxic thymus-derived lymphocytes in lymphocytic choriomeningitis. J. Immunol. 112, 1548. DUNLOP M.B.C. & BLANDEN R.V. (1976) Secondary cytotoxic cell response to lymphocytic choriomeningitis virus. 1. Kinetics of induction in vitro and yields of effector cells. Immunology, 31, 171. GARDNER I. & 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. (In press.) GARDNER I., BOWERN N.A. & BLANDEN R.V. (1974) Cellmediated cytotoxicity against ectromelia virus-infected target cells. I. Specificity and kinetics. Europ. J. Immunol. 4, 63.

GARDNER I., BOWERN N.A. & BLANDEN R.V. (1975) Cellmediated cytotoxicity against ectromelia virus-infected target cells. III. Role of the H-2 gene complex. Europ. J. Immunol. 5, 122. 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. KIROV S.M. (1974) An anti-theta sensitive hapten-carrier response in nude mice. Europ. J. Immunol. 4, 739. KOSZINOWSKI U. & THOMSSEN R. (1975) Target cell-dependent T cell-mediated lysis of vaccinia virus-infected cells. Europ. J. Immunol. 5, 245. MILLER R.G. & DUNKLEY M. (1974) Quantitative analysis of the 51Cr release cytotoxicity assay for cytotoxic lymphocytes. Cell. Immunol. 14, 284. PARISH C.R. & HAYWARD J.A. (1974) The lymphocyte surface. II. Separation of Fc receptor C'3 receptor and surface immunoglobulin-bearing lymphocytes. Proc. roy. Soc. B, 187, 65. PARISH C.R., KIROV S.M., BOWERN N. & BLANDEN R.V. (1974) A one-step procedure for separating mouse T and B lymphocytes. Europ. J. Immunql. 4, 808. RAMSHAW I.A. (1975) Lysis of herpesvirus-infected target cells by immune spleen cells. Infect. Immunol. 11, 767. ZINKERNAGEL R.M. & DOHERTY P.C. (1974a) Restriction of in vitro T cell-mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semiallogeneic system. Nature (Lond.), 248, 701. ZINKERNAGEL R.M. & DOHERTY P.C. (1974b) Characteristics of the interaction in vitro between cytotoxic thymusderived lymphocytes and target monolayers infected with lymphocytic choriomeningitis virus. Scand. J. Immunol. 3, 287. ZINKERNAGEL R.M. & DOHERTY P.C. (1975) H-2 compatibility requirement for T cell-mediated lysis of target cells infected with lymphocytic choriomeningitis. Different cytotoxic T cell specificities are associated with structures coded for in H-2K or H-2D. J. exp. Med. 141, 1427.

Secondary cytotoxic cell response to lymphocytic choriomeningitis virus II. Nature and specificity of effector cells.

The method described in the previous paper was used to induce secondary responses in spleen cells from CBA/H mice, pre-primed with lymphocytic choriom...
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