Clones of cytotoxic T lymphocytes reactiveto haptenated

Proc. NatL Acad. Sci. USA Vol. 80, pp. 1693-1697, March 1983 Immunology

Clones of cytotoxic T lymphocytes reactive to haptenated allogeneic cells: Precursor frequency and characteristics as determined by a split-culture approach (allo-restricted T cells/allo-reactive T cells/limit-dilution cultures/hapten specificity)

MICHAEL F. GOOD AND G. J. V. NOSSAL* The Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Victoria 3050, Australia

Contributed by G. J. V. Nossal, December 14, 1982

ABSTRACT CBA (H-2k) responder spleen cells have been cultured at limit dilution with trinitrophenyl (TNP)-modified BALB/ c (H-2d) stimulator cells and a source of T-cell growth factor in order to generate cytotoxic effector clones. After culture, such clones were split into two to four replicates and each was assayed against a different target. This allowed identification of clones capable of lysing TNP-modified P815 (H-2d) targets but not unmodified P815 targets. Thus, clones specific for TNP and allogeneic restriction elements were detected without the need to use techniques that deplete the responder population of alloreactive cells. Cytotoxic T lymphocyte precursors (CTL-P) specific for TNP-modified P815 (major histocompatibility complex-nonidentical) targets were identified, at a low frequency (28.2 x 10-6) compared to CTL-P for TNP-modified C1.18 (H-2k) (identical) targets (224 x 10-6). The hapten specificity, H-2 restriction specificity, and Thy-i status of these clones have been examined. Fourteen percent of CBA CTLP reactive to TNP-modified P815 targets also showed reactivity to NIP-modified P815 targets, and 86% of CBA clones reactive to TNPmodified P815 targets ("allo-TNP-reactive" clones) failed to show reactivity to TNP-modified C1.18 targets-i.e., showed a restriction preference for allo rather than self. All such H-2d-restricted, TNP-specific clones were uniformly sensitive to anti-Thy-i antibody and complement. Among the H-2k responders studied, we have not demonstrated CTL-P reactive to TNP-modified syngeneic cells which also react with H-2d cells or NIP-modified H-2d cells among 168 clones analyzed. This suggests that such clones, if present, are relatively rare. T cells are unable to respond to foreign antigen alone; they require antigen to be presented in the context of major histocompatibility complex (MHC)-encoded products. The activated T cells then show specificity not only for the foreign antigen but also for the unique allelic form of the MHC-encoded "restriction element" (1-4). In the case of cytotoxic T lymphocytes (CTL), the class I (K/D/L) MHC products function as restriction elements. By stimulating clonal development of lymphocytes, it is possible to view the range of T lymphocyte precursors within a given animal, revealing the repertoire of precursor lymphocytes reactive to foreign antigen presented in the context of self-

erence of normal lymphocytes of an untreated animal has been made difficult by the fact that CTL-precursors (CTL-P) for alloMHC antigens are so much more frequent than those for a given foreign antigen plus MHC. Thus, when attempts are made to enumerate CTL-P for a haptenated allogeneic cell, the large response against alloantigens may not be able to be discriminated from the sum of the (small) response to foreign antigen plus alloMHC and the anti-allogeneic response. The second strategy therefore involves induction of tolerance to alloantigens by methods that deplete the responder lymphocyte population of their content of anti-allogeneic cells. Conclusions reached on the basis of this strategy have been contradictory. Some studies have indicated strong preference for self-H2 over allo-H2 (12, 13); others have shown that, from an allo-depleted population, both self-restricted and allo-restricted clones can emerge (14, 15). Furthermore, the interpretation of results from experiments that use allo-depleting techniques is not straightforward. Specifically, should there be any crossreactivity between alloreactive and allo-restricted CTL-P, then such techniques would strongly bias the results. There would be no way of knowing if this problem were occurring. In this paper, we describe a third strategy used to circumvent the problem. CTL-P were cultured at limiting dilution and, just prior to assay, the clones were split and assayed against various targets. Clones that lyse haptenated but not unhaptenated allogeneic targets can readily be identified, and their frequency can be compared to that for anti-hapten-plus-self CTL-P. The key findings to emerge were that CTL-P reactive to haptenated allogeneic targets do exist but their frequency is about 1/8th that of CTL-P reactive to haptenated self (HS). In all other respects studied (hapten specificity, H-2 restriction, and surface phenotype), these CTL-P reactive to haptenated allogeneic cells behaved like "conventional" CTL-P.

MATERIALS AND METHODS Mice. The mice were 8- to 12-week-old male CBA/CaH/ Wehi(H-2k) and BALB/c AnBradleyWehi (H-2"). Preparation of Concanavalin A-Stimulated Spleen CellConditioned Medium (CAS). The method of Talmage et aL (16), modified as described (8), was used. Haptens. 2,4,6-Trinitrobenzenesulfonic acid (BDH) was used at 10 mM for modification of cells with trinitrophenyl (TNP). 3Iodo4-hydroxy-5-nitrophenylacetic acid (NIP) succinamide ester was used at 100 1LM for NIP modification. The methods of

MHC-encoded products (5-8). Two different strategies have been used to study the potential repertoire reactive to foreign antigen presented in an allogeneic context. First, studies using chimeric mice showed that T lymphocytes responded much better to foreign antigen presented in the context of MHC antigens which were present on radioresistant structures or cells of the thymus in which the lymphocytes matured (9, 10). This radioresistantcomponent may be of bone marrow origin (11). Examination of the H-2 pref-

Abbreviations: CTL, cytotoxic T lymphocyte(s); MHC, major histocompatibility complex; CTL-P, cytotoxic T lymphocyte precursor(s); CAS, concanavalin A-stimulated spleen cell-conditioned medium; TNP, trinitrophenyl; NIP, 3-iodo4-hydroxy-5-nitrophenylacetic acid; HS, haptenated self; HA, haptenated allo. * To whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Immunology: Good and Nossal

hapten modification have been described for TNP (8) and NIP (17, 8). Generation of Effector Clones. (i) HS reactive clones. Limiting numbers (500-4,000) of responder spleen cells were cultured (370C, 10% CO2 in air, 7 days) in V-bottom Linbro microtiter trays (no. 76-023-05) with 2 X 105 x-irradiated (1,500 roentgens; 0.38 coulombs/kg) TNP-modified syngeneic spleen cells ("stimulator" cells) and 2 X 105 x-irradiated unmodified syngeneic spleen cells ("supporting" cells). Each culture well contained 0.2 ml of 10 mM Hepes-buffered Eagle's minimal essential medium supplemented with 10% fetal calf serum, 50 ALM 2-mercaptoethanol, and 10% CAS. Two rows (24 wells) were used as controls and lacked responder cells. (ii) Haptenated allo (HA) reactive clones. Limiting numbers (500-2,000) of CBA responder spleen cells were cultured with 2 X 105 TNP-modified BALB/c stimulator cells and 2 X 105 CBA supporting cells per well. To determine the number of spontaneous HA-reactive CTL-P, CBA responder cells- at 10,000-20,000 per well were cultured with 4 X 105 CBA supporting cells alone. Analysis of Lytic Capacity. After 7 days, culture wells were harvested and split into two to four replicates (in new plates) with a hand-held multichannel pipette and assayed on that day. against modified or unmodified P815 (H-2d) or modified or unmodified Cl. 18 (H-2k) by the "'In release radioautographic method of Shortman. and Wilson (18) which has also been used to. enumerate anti-hapten clones (8). Positive wells show up as dark spots on x-ray film. To determine hapten-specific wells from allo-reactive wells, or wells reacting against "self"' determinants, the intensity of the spot on one-replicate was compared to the intensity of the spot on the other replicates. This was generally done by eye because visual determination agreed closely with statistical discrimination according to linear regression plots of, microdensitometric values. The technique was- similar in principle to that used by others to enumerate the frequency of self-restricted, minor histocompatibility antigen-specific precursors (15). Usually, 0 to 3 or4 hapten-specific wells were identified on each piece of x-ray film (72 experimental wells) with approximately 15-40 allo-reactive wells (see Fig. 1). Monoclonal Anti-Thy 1.2. This was a generous gift from Perry Bartlett and has been described (19).

RESULTS Use of Split-Culture Techniques to Identify HA-Reactive Clones. CBA (H-2k) responder cells were cultured at limit dilution with TNP-modified BALB/c. stimulator cells and CBA supporting cells. After 7 days, the cells in each microwell were split into two replicates, one (a) being assayed for lytic potential on unmodified P815 (H-2d) targets and the other (b) being assayed on TNP-modified P815 targets. As expected, there were many wells cytotoxic for P815 (Fig. 1). Every well that lysed unmodified P815 also lysed TNP-modified P815. In other words, TNP did not conceal MHC antigens, nor did modification with TNP render targets more susceptible to lysis in, this system. There was also a much smaller number of wells in which analysis of replicates showed lysis of TNP-modified P815 and no lysis of unmodified P815 (e.g., Fig. lb, wells G3 and D7). These indicate the presence of one or more clones that recognize TNPmodified H-2d antigens but not H-2d itself. There were other wells in which both replicates showed some lysis but the degree of lysis was obviously greater with TNP-modified targets (Fig. lb, well Gll). In this situation, the most likely explanation is that one or more CTL-P reactive to H-2d MHC antigens were present in the same well as an anti-H2d-TNP. CTL-P. This possibility could have been minimized by dispensing extremely few

A B C D E F

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4

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G H 1 2 3 4 5 6 7 8 910i1 A

B C D E F G H

',

*

.

.*

,

*

0*

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FIG. 1. Identification of CBA anti-BALB/c-TNP clones by splitculture analysis; 1,O0GCBA responder cells were cultured with BALB/ c-TNP stimulators per well. The cultures were then split and assayed against P815 targets (a) and TNP-conjugated-P815 targets (b).

responder cells into wells to avoid clonal overlap. However, because anti-allo-CTL-P are so much more frequent than anti(hapten-plus-allo-MHC) CTL-P, such an experimental design would have necessitated an impractically large total number of cultures. Alternatively, certain of these wells could represent an antigenic crossreactivity such that a clone with anti-(hapten-plusallo-MHC) specificity lysed. the unmodified allo targets to a slight extent. However, the ratio of the number of wells showing an absolute difference in lysis between the two replicates to the number of wells showing a relative difference in lysis over a large. number of experiments is the ratio that would be predicted by Poisson analysis if there were no such crossreactivity (data not shown). Where there was a difference in lytic potential between the two replicate wells, lysis was greater on modified targets than on unmodified targets, a point also in favor of clonal overlap as the explanation. Accordingly, we classified wells with a clear quantitative lytic difference as. belonging to the same category as those with a +/- discrimination. Even had such wells been not counted,, the broad conclusions of this study would have been unaffected. Fig. 2 demonstrates that the HA-reactive response obeys single-hit zero-order kinetics, thus demonstrating that only one cell type is limiting. The plot of the number of responder cells per well versus the logarithm of the fraction of negative wells can be seen to pass through a point near the origin. Fig. 2 also plots the HS-reactive responses for CBA and BALB/c splenic lymphocytes as determined by a clone-splitting technique at limiting dilution. Again, kinetics were zero-order but precursor frequencies were much higher. Allo-TNP-reactive. precursors occurred at 1/8th the frequency of self-TNP-reactive precursors (Table 1). The frequency of spontaneously arising HA-reactive clones, obtained from one experiment, was much lower, demonstrating that in vitro immunization can strongly stimulate

HA-reactive precursors. To ensure that the HA-reactive response was not due to or aided by an alloreactive response (CBA anti-BALB/c), the allo-NIP response was also determined for a number of experiments in which allo-TNP provided the stim-

Proc. Nate Acad. Sci. USA 80 (1983)

immunology: Good and Nossal

Table 2. Hapten specificity of HA-reactive clones CBA anti-H-2'-TNP- Calculated Wells reactive to H-2d-NIP, no. of reactive wells no. clones identified, no. 5.1 1/5 5/216 5.1 1/5 5/144 21.4 2/21 21/576

Responder cells, no./culture

-0.1 C,)

C..

75

Calculated no. of clones 1.1 1.1 2.1

4.3 31.6 Total The results for three separate experiments are given. The number of clones was calculated by using Poisson analysis as follows: number of clones = - n ln Fo; n is the number of wells cultured, and Fo is the fraction of wells not showing greater lysis on modified targets compared to unmodified targets. Hapten specificity = 86.4%.

-0.2

0 c :6

c

-0.3

Hapten Specificity of HA-Reactive Clones. By splitting microcultures into three replicates, it is possible not only to identify HA-reactive clones but also to test their reactivity against the same allo target but modified with a different hapten, thus gaining insight into hapten specificity within the CTL-P repertoire. CBA responder cells were stimulated at limit dilution with TNP-modified BALB/c stimulator cells. After culture, the wells were split into three replicates which were assayed against unmodified P815 targets, TNP-modified P815 targets, and NIPmodified P815 targets (Table 2). Thirty-two allo-TNP-reactive clones were identified, of which four also reacted with NIPmodified P815 cells. The frequency of spontaneously arising clones that were specific for NIP-modified P815 targets was negligible in these experiments and has not been corrected for. H-2 Restriction of HA-Reactive Clones. To investigate the H-2 preference of hapten-allo-reactive clones, microcultures were again split into three replicates as above. CBA responder cells were cultured with TNP-modified BALB/c stimulator cells. After 7 days, three replicates were made which were then assayed against P815 (H-2) targetsk TNP-modified P815 targets, and TNP-modified C1. 18 (H-2 ) targets (Table 3). Thirtyseven wells that recognized TNP-modified P815 cells but not unmodified P815 cells were identified. Eight of the wells also responded to TNP-modified C1. 18. When spontaneously arising clones that recognized TNP-modified C1. 18 (many ofwhich would presumably lyse unmodified C1. 18) were allowed for, there was 86% restriction specificity. Spontaneously arising "anti-self" clones, which have been described before (8), were present in these experiments at frequencies of 50-150 X 10-6 and so would not lyse P815 or TNP-modified P815 but would lyse C1. 18 whether haptenated or not. Unless such cells are

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FIG. 2. Limit-dilution microcultures for HA and HS reactive CTLP display zero-order kinetics. Three different responses are plotted: CBA anti-BALB/c-TNP response (9); BALB/c anti-BALB/c-TNP response (0); and CBA anti-CBA-TNP response (A). The y intercepts (and their 95% confidence limits) for the various lines have been determined from a "maximal likelihood estimator" computer program and are (with 95% confidence limits in parentheses): a, 0.026 (-0.027-0.079); b, 0.00 (-0.035-0.046); c, 0.00 (-0.03-0.03).

ulation. In three experiments the frequency of CTL-P reactive to allo-TNP was 22.2 X 10-6 whereas the frequency of CTL-P reactive to allo-NIP was only 2.5 X 10-6. This was only done as

because, as described above and shown in Fig. 1, allo-TNP-reactive precursors were found in wells whose replicates were negative for allo-reactivity. a precaution many of the

Table 1. Allo-TNP and self-TNP reactive CTL-P frequencies CBA anti-H-2k-TNP CBA anti-H-2d-TNP CTL-P CTh-P Frequency, Frequency X 106 n* X 106 n 1,152 18.9 13.4 45.5 31.0 23.4 37.0

504 216 864 432 576

170 274 227

720 432 144

BALB/c anti-H-2dTNP CTL-P Frequency, X

106

93.3 53.0 50.1 83.3

n*

864 288 288 144

69.9 CTL-P frequencies were determined from a number of experiments. These have been calculated by using Poisson analysis: frequency = -In Fo/n; Fo is the fraction of wells not showing significantly greater lysis on modified targets compared to unmodified targets, and n is the number of responder cells added to each well. The spontaneous CBA anti-H-2d-TNP frequency was 1.5 x 10-6 (n = 432). * n, Number of microwells tested (prior to splitting of cultures). Mean

28.2

224

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Proc. Nad Acad. Sci. USA 80 (1983)

Table 3. Allo-TNP-reactive clones show restriction preference for allo rather than self Spontaneous

H-2kTNp_ reactive CBA Wells clones anti-H-2d-TNP Calculated reactive to Calculated expected by reactive wells no. of H-2kTNP, no. of coincidence, identified, no. clones no, clones no. 5/216 5.1 3/5 4.6 0.3 6/144 6.1 2.4 2/6 1.7 26.6 26/576 3.2 3/26 2.9 Total 37.8 10.2 4.9 The results for three separate experiments are given. The number of clones was calculated by using Poisson analysis as for Table 2. It was necessary to make allowance for spontaneously arising CBA clones reactive to self targets (see ref. 8) because the frequency of such CTL-P was high (50-150 x 10-6). % H-2 restriction = 37.8 - 10.2 + 4.9 x 100 = 86.0. 37.8

taken into account, it is impossible to determine the degree of restriction specificity of the HA-reactive clones. HA-Reactive Clones are Thy-i Positive. In these experiments, microcultures of CBA responders were split into three replicates after 7 days of culture with TNP-modified BALB/c stimulators. Complement was then added to the first two replicates and monoclonal anti-Thy-i and complement were added to the third replicate. After 45-min incubation, unmodified P815 targets were added to the first replicate and TNP-modified P815 targets were added to the two other replicates. Thus, we were able to identify allo-TNP-reactive clones specifically and record their sensitivity to anti-Thy-i and complement. Fourteen alloTNP clones were identified (eight in the first experiment and six in the second) and all were completely sensitive to anti-Thy1 and complement. CBA Clones Reactive to CBA-TNP Do Not React with P815NIP or with P815. We addressed this issue by using an experimental protocol in which wells were split into four replicates, the first two identifying the anti-self-X1 wells and the other two documenting the crossreactivity patterns of such wells on allo and allo-X2 targets. We were not able to demonstrate crossreactivity among 168 different H-2k self-TNP-reactive clones to H-2d or H-2d-NIP (Table 4). However, slight (1%) crossreactivity to allo targets demonstrated by Bevan (20) would not be apparent in this system without generating many more clones. This is because the number of clones is estimated from the fraction of nonresponding wells by using Poisson analysis and so has a variance.

DISCUSSION We have examined the repertoire of non-self-restricted T cell precursors by studying the frequency of normal H-2k spleno-

cytes capable of responding to TNP presented in the context of

H-2d MHC antigens. Until now, there has been no consensus concerning the relative numbers of T lymphocytes that could respond to foreign antigen in the context of non-self MHC-encoded products. By using chimeric mice, it was shown (10, i1)

that the restriction specificity was not for the MHC-encoded products of the responding lymphocytes but rather for the MHCencoded products found in the radioresistant portion of the thymus in which they matured. Studies using chimeras have been challenged for a number of reasons. As pointed out by Bevan (9), A-B lymphocytes maturing in an A thymus would have a different receptor repertoire from A lymphocytes maturing in an A thymus-if the Jerne hypothesis (21) is accepted. This postulates that A lymphocytes would express anti-A receptors and that these lymphocytes would proliferate and accumulate mutations, thus ensuring an A-restricted repertoire and tolerance of A antigens. However, as well as expressing anti-A receptors, A-B lymphocytes would also express anti-B receptors. Such "chimera artifacts" have been demonstrated in experiments showing

that

(A-B

--

A) cells could

not

respond

to

foreign

an-

tigen in the context of B type MHC antigens whereas lymphocytes from normal A mice studied after allo-depletion of B reactive cells could respond to foreign antigen in the context of B type MHC antigens (22). Such experiments emphasize the need to examine the repertoire of normal lymphocytes.

Our results clearly demonstrate that normal CBA (H-2k) lymphocytes can respond to TNP presented in the context of H-2d MHC antigens. However, we have shown that the frequency of such precursor lymphocytes is about 1/8th that of CBA precursor lymphocytes capable of responding to TNP-modified self antigens. By examining the frequency of H-2b lymphocytes responding to TNP-modified H-2d antigens, Teh (23) found that, after H-2d-TNP stimulation, about 13% more CTL-P lysed TNPmodified H-2d targets than unmodified H-2d targets. Our results (24) showed that the frequency of H-2d reactive CTL-P in adult CBA spleen was 1,000-2,500 x 10-6. Thus, the frequency of HA-reactive CTL-P in our hands is 1% to 3% of the frequency of allo-reactive CTL-P for this system. Therefore, we find that the frequency of allo-restricted, hapten-specific cells is much lower than that reported by Teh. His experiments did not address the ratio of allo- versus self-restricted CTL-P numbers. Such allo-TNP-reactive precursors do not form a subset of the self-TNP-reactive precursors (assuming that clones breed truly) because the allo-TNP-generated clones do not lyse self-TNP targets. Thus, they behave in an H-2-restricted fashion-a hallmark of "conventional" self-TNP reactive lymphocytes (1-4). Similarly, we have demonstrated other conventional properties in that the allo-restricted clones show hapten specificity and sensitivity to anti-Thy-l and complement. If such allo-restricted lymphocytes are similar in all respects (except for their H-2 preference) to conventional self-restricted lymphocytes, then the question of their low frequency must be addressed. A simple explanation for this would be that such results represent an accidental finding. However, allo-deple-

Table 4. CBA clones reactive to CBA-TNP do not react with P815-NIP or with P815 CBA anti-H-2k-TNP Calculated Wells Calculated P815 reactive P815-NIP reactive reactive wells no. of reactive to no. of clones expected Wells reactive wells expected identified, no. clones P815, no. clones by coincidence, no. to P815-NIP, no. by coincidence, no. 134/360 167.6 17/134 18.2 32.7 1/134 1 CBA responder cells were cultured against TNP-modified CBA stimulators. After 7 days, cultures were split into four replicates and assayed against C1.18, TNP-modified C1.18, P815, and NIP-modified P815 targets. It thus was possible to identify wells specific for TNP-modified C1.18 and then to examine any crossreactivities of those wells. Spontaneously arising clones reactive to P815 or specifically to NIP-modified P815 targets had to be allowed for.

Immunology: Good and Nossal. tion studies have also shown that there is preference to self over allo MHC antigens [either absolutely (12, 13) or relatively (14, 15)], but it could be argued that allo-depletion may also deplete some of the allo-restricted repertoire. An alternative view is that the relatively low frequency of CBA anti-BALB/c-TNP precursor lymphocytes reflects a general preference for self-restriction over allo-restriction. Although the Jerne hypothesis (21) was formulated before dual recognition was recognized, it could be argued from it that the preference for self-restriction results from proliferation and mutation in the thymus of self-reactive clones. A third alternative to explain self preference has been proposed by Stockinger et aL (14). They argue that, through "priming" in the periphery, those lymphocytes expressing anti-selfXi receptors are expanded such that at the time of inquiry antiself-Xi receptors are more numerous. After all, the self-restriction elements are the only ones available in a physiological setting. Lastly, we have tried to determine if any CBA self-TNP-reactive clones could recognize allo (H-2d) or allo-NIP targets. Bevan (20) has demonstrated about 1% crossreactivity of self-restricted cells to an allo target, and Hunig and Bevan (25) have also established a clone that recognizes both self-X1 and allo-X2. We have not been able to demonstrate appreciable crossreactivity to allo or allo-NIP targets from 168 clones. However, our methods would not show a 1% crossreactivity. If such crossreactivities were to be found, then the allo and allo-restricted repertoires could be viewed as being a subset of the self-restricted repertoire. Some crossreactivity may have been expected because in a separate series of experiments we found about 20% crossreactivity between self-reactive and allo-reactive clones and about 10% crossreactivity between self-TNP reactive and self-NIP reactive clones. If every allo-reactive and allo-restricted clone were part of the self-restricted repertoire, then crossreactivity between allo-reactive or allo-restricted clones and self-restricted clones should be of the same degree as crossreactivity between self-restricted clones (10%), but this is not the case. A possible explanation for this is that a self-tolerance event has nonuniformly altered the self-restricted and allo-restricted repertoires. In summary, we have presented a way to study hapten-alloreactive precursor lymphocytes that does not require the removal of the allo-reactive population. This method allows us to examine the allo-restricted repertoire without having to make the prior judgments about possible crossreactivity between allorestricted and allo-reactive clonal precursors implicit in a protocol involving examination of a selected T-cell population. We have found allo-restricted precursors to be of low frequency but to behave like typical T lymphocytes. It will be of great interest to examine the response to other foreign antigens presented with

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1697

allo-MHC products and to study in greater depth the crossreactivity patterns. We thank Drs. J. F. A. P. Miller, J. W. Schrader, and K. D. Shortman for their advice and suggestions. This work was supported by the National Health and Medical Research Council, Canberra, Australia; by Grant AI-03958 from the National Institute of Allergy and Infectious Diseases, U. S. Public Health Service; and by the generosity of a number of private donors to the Walter and Eliza Hall Institute. M. F. G. is the holder of a National Health and Medical Research Council Fellowship

(Australia). 1. Zinkernagel, R. M. & Doherty, P. C. (1974) Nature (London) 248, 701-702. 2. Shearer, G. M., Rehn, T. G. & Schmitt-Verhulst, A.-M. (1976) Transplant. Rev. 29, 222-248. 3. Bevan, M. J. (1976)J. Exp. Med. 142, 1349-1364. 4. Miller, J. F. A. P. (1979) Adv. Cancer Res. 29, 1-78. 5. Teh, H.-S., Phillips, R. A. & Miller, R. G. (1978)1. Immunol 121, 1711-1717. 6. Ching, L.-M. & Marbrook, J. (1979) Eur. j. Immunol 9, 22-27. 7. MacDonald, H. R., Cerottini, J. C., Ryser, J.-E., Maryanski, J. L., Taswell, C., Widmer, M. B. & Brunner, K. T. (1980) ImmunoL Rev. 51, 93-123. 8. Good, M. F. & Nossal, G. J. V. (1982) J. Immunot Methods 52, 149-166. 9. Bevan, M. J. (1977) Nature (London) 269, 417-418. 10. Zinkernagel, R. M., Callahan, G. N., Althage, A., Cooper, S., Klein, P. A. & Klein, J. (1978)J. Exp. Med. 147, 882-896. 11. Longo, D. L. & Schwartz, R. H. (1980) Nature (London) 287, 4446. 12. Schmitt-Verhulst, A. M. & Shearer, G. (1977)J. Supramol Struct. 6, 206-208. 13. Janeway, C. A., Jr., Murphy, P. D., Kemp, J. & Wigzell, H. (1978) J. Exp. Med. 147, 1065-1077. 14. Stockinger, H., Pfizenmaier, K., Hardt, C., Rodt, H., R6llinghoff, M. & Wagner, H. (1980) Proc Nate Acad. Sci. USA 77, 7390-7394. 15. Teh, H.-S., Bennick, J. & von Boehmer, H. (1982) Eur. J. Immunol 12, 877-892. 16. Talmage, D. W., Woolnough, J. A., Hemmingsen, H., Lopez, L. & Lafferty, K. J. (1977) Proc. Nati. Acad. Sci. USA 74, 4610-4614. 17. Pohlit, H. M., Haas, W & Von Boehmer, H. (1979) in Immunological Methods, eds. Lefkovits, I. & Pernis, B. (Academic, New York), p. 189. 18. Shortman, K. & Wilson, A. (1981) . ImmunoL Methods 43, 135152. 19. Ledbetter, J. A. & Herzenberg, L. A. (1979) Immunol Rev. 47, 63-90. 20. Bevan, M. J. (1977) Proc. Natl, Acad. Sci. USA 74, 2094-2098. 21. Jerne, N. K. (1971) Eur. J. Immunot 1, 1-9. 22. Doherty, P. C. & Bennick, J. R. (1979) J. Exp. Med. 150, 11871194. 23. Teh, H.-S. (1979) Immunogenetics 8, 99-107. 24. Nossal, G. J. V. & Pike, B. L. (1981) Proc. Nate Acad. Sci. USA 78, 3844-3847. 25. Hunig, T. R. & Bevan, M. J. (1982)J. Exp. Med. 155, 111-125.