Studies on transplantation immunity

CELLULAR

IMMUXOLOGY

2, %?-5%

Studies III. The Secondary

(1971)

on Transplantation Response

D. R. Department

of Bacteriology,

Immunity

to Dissociated

Cells

BAINBRIDGE

The Londolz Ho.rfiita.1 Mrdical Received

Allogeneic

Decewhcr

Collrgr,

Landox,

E.l, England

26, 1970

The secondary response of CBA mice to dissociated (A X CBA)F, lymphoid cells has been studied with the aid of the Kr-labeling technique. The effect of a secondary dose is independent of the time interval between primary and secondary doses over a wide range, due presumably to the remarkably stable status induced by a single stimulus. No clear evidence of a true secondary has been found for the liver or spleen responses, which are largely mediated by antibody production. The lymph node response, which is largely cell mediated, does possess a true secondary. The dose response data for the lymph nodes show that the primary response has an initial impetus into tolerance, reversed by sensitisation ; and that the secondary response is simpler in form than the primary. Only a restricted range of primary and secondary doses are capable of eliciting a secondary response. Various peculiarities of the secondary suggest that it may arise as an in z&to form of “mixed lymphocyte reaction.”

INTRODUCTION

Mice of the CBA strain, previously sensitised by parenteral injection of dissociated H-2 different lymphoid cells, respond to a second “‘Cr-labeled intravenous dose of cells by sequestering these in their livers and spleens and elininating them in their lymph nodes and spleens (l-5). Certain unusual features of the response have already been described : the elimination of cells requires the synergic action of cellular and humoral factors; and there appear to be two distinct sequestration effects, liver localisation and spleen localisation, which are mediated by different immunoglobulins (4). In addition the response produced by quite trivial stimuli is remarkably persistent, remaining detectable for many months (4). This last feature seems to offer an interesting system in which to study the secondary response ; and the present paper reports some of the investigations which have been carried out. MATERIALS AND

METHODS

details of the Xr-labeling technique and calculation of the results are those given by Bainbridge, Brent, and Gowland (6)) with slight modifications (4). The

Sensitisation Female CBA mice 9-12 weeks old were immunised by intraperitoneal administration of 103, 105, or lo7 (A X CBA)F, spleen cells. At various times thereafter 583

584

BAINBRIDGE

groups of these together with utlsensitised mice were challenged with 103, lo”, or lo7 (A X CBA)F, spleen cells intraperitoneally. Eight days after the secondary challenge these mice, together with groups of unsensitised and primary mice, were tested by an intravenous injection of 10 6 %r-labeled (A X CBA)F, peripheral lymph node cells and were killed 24 hr later for counting. Adoptive

Transfer

Peripheral lymph node or spleen cell suspensions were prepared from mice sensitised by 10’ (A X CBA)F, spleen cells intraperitoneally 8-11 days before transfer. The cells were made up in Tyrode’s medium without added serum, washed once, and injected intravenously as 1 :1 transfers (2-5 X lo7 viable lymph node cells or 5-7 X 14Y viable spleen cells) into CBA recipients which had been irradiated with 500 R 24 hr previously. The conditions of irradiation were as follows: Maximar X-ray set 220 kVp ; 15 mA. Filtration : 0.50 mm& + 1.0 mm Al. Radiation quality : 1.15 mm Cu HVL. Open field, 60 cm FSD. Dose rate approx. 43 R/min. Some groups of mice were challenged by intraperitoneal injection of lo6 (A X CBA)F, spleen cells within 0.5-l hr of transfer. Animals were tested with labeled cells at various times afterwards. RESULTS 1. Adoptive

Transfer

of Iawmmity:

Lywaph Node Response

In studying a system with extremely persistent responses it is not evident whether the second challenge elicits a true secondary response or simply modifies the existing primary state. This was investigated by transferring sensitised CBA lymphoid cells to irradiated CBA recipients and testing after transfer with %r-labeled (A X CBA) F, cells. In preliminary experiments no response was detectable until about 4 days after transfer. Then, as shown in Table 1, at 7 days irradiated mice which had received sensitised lymph node cells and were challenged with lo6 (A X CBA) F, cells intraperitoneally produced an immune pattern of response : the amount of radioactivity in the lymph nodes was markedly depressed, that of the spleen was decreased, and the radioactivity of the liver was somewhat increased. Animals challenged without transfer of cells did not manifest these changes, showing that 500 R effectively obliterated the host response at this time. Animals receiving unsensitised cells and challenged gave only a slight lymph node response, showing that the sensitised cell response was not a primary, but a property of the committed cells in the sensitised population. Animals receiving sensitised cells without challenge also gave no response, indicating that the transferred response was not a property of the committed active cells in the sensitised population, i.e., a persistent primary, but was elicited by stimulation of the primed cells, i.e., was a true secondary response. On the other hand, irradiated animals which received sensitised spleen cells were able to mount a response without additional challenge. In order to determine whether spleen cells could mediate a secondary response, sensitised spleen cells were transferred into unirradiated recipients. One group of these and a group of mice which had not received sensitised cells were challenged with 105 (A X CBA)F, spleen cells intraperitoneally, a moderate sensitising dose. Table 2 shows

SECONDARY

T.ABLE ADOPTIVE

Treatment y0 of injected cipients

of irradiated

. recovered d obe

TIUXSFER

1 OF IMMUKITY

I.ymph

recipients in irradiated

585

RESI’OKSE

n

nodes

Spleen

Liver

re-

11 = 4 Challenge with lo6 cells ip n = 4 1:l I,ymph node cell transfer: Sensitised cells n = 4 Sensitised cells and challenge 12 = 4 Unsensitised cells and challenge n = 3 SellGtised cells and challenge n = 4 1 :l sensitised spleen cell transfer: ?Z=l

11.283

14.913

(0.541) _____86.21 (5.9‘4)

(0.614) 98.37 (2.08)

98.69 (2.74)

86.18 (5.76) 16.90 (1.27) 75.50 (5.46) 33 .63 (3.70) 17.12 (2.10)

97.15 (6.02) 46.88 (7.81) 75.48 (3 .29) 74.26 (3.39) 58.41 (6.79)

100.70 (6.73) 136.39 (8.65) 104.54 (2.34) 107.47 (6.01) 155.50 (8.85)

a Mean radioactivityrecovered in organ 24 hr after injection after transfer or challenge; as percentage of uptake in irradiated and standard error of mean shown.

28.461 ___(0.290)

of 10” labeled cells and 8 days recipients. Number of animals

that when tested at S days, mice receiving cells alone gave no response. Challenged mice produced a moderate response ; while mice receiving both sensitised cells and a challenge gave a substantially enhanced response. 2. Adoptive

Transfer

of Livev

and Spleen Respomcs

It was apparent from these and other experiments that the characteristic liver immune response (a marked increase in liver radioactivity) (4) was either lacking after transfer, or was extremely modest and transient by comparison with the lymph node response. &qn experiment was carried out to test whether this was occurring through failure of the transferred cells to synthesise sequestering antibody (4) or through depression by irradiation of the host’s ability to trap opsonised-labeled cells. Normal and irradiated mice were given 0.3 ml of sensitised serum inTIZBLE SECONDARY

Challenge with lo” cells ip 11 = 5 1 :l Sensitised spleen cell transfer n = 5 Transfer and challenge a Mean radioactivity percentage of uptake

IN SDNSITISED

I,ymph

Treatment

n=5

RESPONSE

2

nodes

43.06 (3.664) 80.85 (2.783) 22.44 (1.862)

SPLLXK

~1

Spleen 86.05 (2.462) 99.55 (3.442) 62.16 (5.058)

Liver 106.52 (3.387) 104.00 (3.015) 171.19

(7.216)

in organ 24 hr after labeled cells, 8 days after transfer or challenge, as in normal animals. Number of animals and standard error of mean shown.

586

BAINBRIDGE

travenously 8 days after irradiation and tested 4 hr later with labeled (A X CBA)F, cells, together with groups of mice which had not received serum. The effect of passive immunisation on the liver was identical in irradiated and normal mice; the amount of radioactivity in the liver, spleen, and lymph nodes being 138.1, 82.2 ; and 74.97 0 in the irradiated group and 134.9, 79.0, and 90.6% in the unirradiated one (expressed as a percentage of the uptake in the appropriate control animals). The effect was ephemeral, lasting no more than 2-3 days. Thus the lack of an immune liver response after adoptive transfer must have been due to failure of the transferred cells to produce effective amounts of sequestering antibody. This failure could result from unfavourable conditions produced by transfer or may indicate that in fact no true secondary exists for this response. The latter seems likely for the spleen response also : comparison of the spleen responses in challenged recipients of unsensitised and sensitised cells (Table 1) suggests that the transferred spleen response of sensitized cell may be simply a new primary elicited from the uncommitted cells present. 3. Stability

of the Imwmne

Response

The state of immunity produced by a single dose of allogenic lymphoid cells has been found to be extremely persistent (4). Daily testing of mice sensitised with various doses of cells between lo2 and 107 for a period of 3 weeks early in the response (from 5 days on) or late (starting at 12 months after sensitisation) showed substantial day-to-day variations in the response, but no evidence of periodic or systematic change was obtained on statistical analysis; and the average levels of response for the early and late phases were virtually the same (Bainbridge, unpublished). In order to decide whether the fluctuations represented random alterations of response or day-to-day experimental error the following experiment was carried out. Five groups of mice were sensitised in succession by a single intraperitoneal injection of lo” (A X CBA) F, spl een cells with an interval of 1 day between the sensitisation of one group and the following one. Mice were tested daily from 7 days after sensitisation for 3 weeks. Because sensitisation had been staggered mice of different groups tested on the same day were at different times after sensitisation, and it was thus possible to dissociate daily error variation from variations in the immune response. The analysis of variance in Table 3 shows that the error variations were the most important. 4. Dose Dependence

in the Secondary

Response

Mice sensitised with various doses of (A X CBA)F, spleen cells were challenged at different times with various doses and tested 8 days later with labeled (A X CBA)F, lymph node cells. The times were chosen to fall near equal intervals on a logarithmic scale [satisfying the equation t (interval in days) = 4,5 X (3/Z)” ; n = 1,2,... 8, that is from 4 to 128 days], so as to pick up short-term or longterm trends. None were apparent, and since the variation in secondary groups was no greater than in primary groups (see Section 3) the data for different time intervals have been pooled in Table 4. They are considered further in the discussion.

SECONDARY

557

RESPONSE

Sum of squares

Degrees of freedom

Mean square

F

P

Between sensitising groups Between days of testing Residual

21.060 24.376 6.025

4 18 52

5.265 1 .354 0.116

45.465 11.694

< 001 < ,001

Bet\veen sensitising groups Between da>-s after sensitisation Residual

21.060 12.837 17.564

4 20 50

5.265 0.642 0.351

14.991 1.827

< ,001 .Ol
51.460

74

Source

‘rot31

a Variance of radioactivity of lymph nodes, as percentage of dose injected, of mice sensitised with 105 cells on different days and tested at intervals of 7-28 days after sensitisation. Allalysis carried out on the unweighted means of grorlps, by day of testing or by day after sensitkation.

DISCUSSION

5. The Secondary Response to Dissociated Allogencic Cells The secondary response in transplantation immunology has been peculiarly refractory to study in the past; and for a period was seriously disputed in the homograft response (7-10). Although the present system has avoided one of the original difficulties-the fact that the assay graft strongly modifies the state of the animal during the course of the test-it has encountered another which was not apparent in the homograft system (4,7-g) ; a single small dose of cells has effects which are virtually permanent compared to the life-span of a mouse (Section 3). This recalls the reaction of mice or men to various polysaccharide antigens (ll-13), and it has been stated that in such systems a secondary response is not induced (11, 12), unless with considerable difficulty (13, 14). The present data seem to accord with this as far as the liver and spleen responses are concerned: no unequivocal evidence has yet been found for a secondary response with either. The spleen response of sensitised cells can be entirely accounted for as a primary response elicited by challenge from uncommitted cells. I,ittle or no liver response can be transferred even with challenge (Section 2). On the other hand, there is clear evidence of a secondary lymph node response and there are cells capable of mediating this both in the sensitised lymph nodes and in the spleen (Section 1). Work reported elsewhere (4) shows that the lymph node, spleen, and liver responses are distinct ; accordingly, further discussion of the secondary response here is concerned with the lymph node results. The dose-timeresponse results give a picture that is essentially unchanged by the interval between the primary and secondary challenge, apart from random fluctuations (Section 4)) due presumably to the almost permanent effect produced by a single stimulus. For this reason the data for different intervals are pooled in Table 4 to show the average response at 8 days produced by different combinations of primary and secondary doses. Figure 1 shows the lymph node results plotted on polar coordinates

588

BAINBRIDGE

TABLE

4

SECONDARYRESPONSEIN IMMUNE ELIMINATION OF CELLS~ Treatment Primary 0

103

105

107

Secondary

Number of animals

0

36

103

3.5

105

3.5

10’

35

0

34

103

34

105

32

10’

31

0

32

103

3.5

105

35

107

36

0

35

103

36

105

35

107

34

Lymph

nodes

100.00 (2.59) 99.85 (4.02) 45.92 (2.90) 16.28 (1.41) 109.55 (4.80) 116.66 (6.87) 42.36 (4.00) 18.24 (1.89) 59.76 (3.34) 53.03 (2.72) 32.32 (2.36) 16.98 (1.19) 12.52 (0.98) 18.28 (2.57) 17.11 (2.30) 7.90 (1.05)

Spleen 100.00 (1.45) 96.54 (2.06) 81.09 (2.46) SO.82 (4.06) 95.55 (2.70) 100.65 (2.41) 79.27 (2.45) 51.51 (4.64) 78.37 (2.34) 76.03 (2.49) 67.95 (2.57) 52.19 (2.89) 40.07 (2.72) 42.94 (2.56) 41.13 (2.64) 27.27 (2.15)

Liver 100.00 (0.80) 100.73 (1.10) 97.52 (1.71) 218.51 (14.58) 100.37 (1.35) 104.36 (2.28) 98.40 (1.84) 220.19 (15.23) 101.26 (4.05) 96.97 (2.32) 122.41 (9.19) 183.13 (10.50) 272.27 (10.99) 272.08 (10.58) 275.19 (9.19) 305.75 (11.12)

a Pooled percentage radioactivity recovered in organ of animals given two doses of cells and tested 8 days after the second dose, as percentage of radioactivity in controls. Numbers of animals and standard error of mean shown.

(which have certain advantages over Cartesian coordinates for these data) with contours of equal response superimposed. The principal features of the results are examined in greater detail in the Appendix. Briefly they are as follows : (i) A secondary response is elicited only when the secondary and primary doses are nearly the same. This can be seen by comparing the 71 contour line with others. The 71% contour line is nearly the quadrant of a circle, and a quadrant corresponds to the response which would be expected if the animal gave only a primary response to stimulataion. There is only a narrow region along the 1 :l diagonal where the contours push inwards toward the origin.

SECOXDARY

539

RESPONSE

5:7

f 4

3:5

5

>r 0 .-E L a

4 3:7 3

0 F -

2

1

0:x

0

0

1

2 log,0

3

4 secondary

5

6

7

dose

FIG. 1. Lymph node response in CBA mice 8 days after two doses of (A X CBA)F, spleen cells. Radioactivity is expressed as a percentage of the uptake of untreated mice. Doses, expressed as log,, (number of cells + l), are plotted on polar coordinates with r 1 total dose and 0 = tan-l primary/secondary dose. Contours of equal response arc superimposed. Note that Table 4 and the figure are not drawn from exactly the same set of data.

Beyond this zone they bulge outwards, indicating that the response is less than if the total (secondary plus primary) doses had been given as a single injection. (ii) The figure is almost symmetrical about the main ( 1 :l) diagonal. This means that the response is the same, for a given pair of doses, whether the larger stimulus is administered first or second. Such a property would seem to be unique for a secondary response. In an earlier paper (S), dealing with the primary response to dissociated allogeneic cells, it was suggested that the response should be regarded as a form of mixed lymphocyte reaction occurring in viva, by virtue of the fact that the stimulating antigen is itself a lymphoid cell. The formal similarity between the mixed lymphocyte reaction (MLK) and the secondary lymph node response described here is particularly striking. In one-way MLI< the amount of stimulation also changes with the ratio between stimulating and responding cells for a fixed total number of cells, and the dose response curve is narrow [compare i] more or less symmetrical, and with a maximum at ratios near 1 : 1 [compare ii] (15, 16). A major difference between the primary response and the MLli was the wide range of doses which are capable of stimulating suboptimally in viva, suggesting that auxiliary mechanisms operate in the intact animal to render low doses of cells effectively immunogenic (5). The absence of this feature in the secondary re-

590

BAINBRIDGE

sponse would indicate that such mechanisms (if present) are bypassed after priming of the animal has taken place. (iii) The dose-response relationships for the primary and secondary responses show that the secondary mechanism is simpler than the primary. This is considered further below. 6. The Principle of Sivtzplification in Immune Responses Although there is disagreement about the reality of “priming” at the level of the individual cell (17~20)) there can be no doubt that populations of primed and unprimed cells commonly show qualitatively different mechanisms of response to antigen. Secondary responses may be elicited by antigen in a form which is poorly immunogenic or nonimmunogenic for a primary. Other forms of the same antigen which do stimulate a primary response are not necessarily better in stimulating a secondary and may, on the contrary, be worse. Aqueous PPD, BSA, ovalbumin, and semisoluble transplantation antigen elicit delayed hypersensitivity reactions in sensitised mice (21) or guinea pigs (22)) but do not immunise. Soluble protein antigens stimulate DNA synthesis in vitro in sensitised lymphoid cells but not in unsensitised cells (23-28). Soluble bovine serum albumin (BSA) or the serum of animals injected with BSA provides an efficient challenge for antibody production in primed mice but in unprimed recipients is a poor stimulus (29, 30) or induces unresponsiveness (31) _ On the other hand, macrophage-associated BSA, which is a potent stimulator of the primary antibody response, does not enhance the secondary (32) and may fail to stimulate it at all (3 1) . The results of the BSA system suggest that an important factor in the change to the primed state may be the appearance of cells which are stimulated (rather than inhibited) by direct contact with antigen and whose response is thus not obliged to depend upon mediation by macrophages. A more general way of stating this would be to say that the mechanism of antigen recognition seems to have been simplified in the secondary response. (The auxiliary macrophage apparatus required for induction of the primary response is no longer necessary.) The present findings actually provide direct evidence of a simplification of mechanism in passing from the primary to the secondary response. The dose-response curve of the secondary lymph node response is simpler, and its form such that the primary mechanism cannot be a factor in the secondary response (see Appendix). I would like to suggest that these results merely illustrate a general tendency of immune responses, which is to simplify their mechanism of recognition, for instance, discarding macrophage dependence, as in the antibody response to BSA (31, 32) or carrier dependence, as in the adoptive secondary NIP antihapten response to NIP-HSA or NIP-CGG (33), until in the course of development of the response a stage is reached when recognition takes place through direct confrontation of the antigen and lymphocyte. What would the significance be of such a tendency? In explaining antibody diversity as originating by mutation of lymphocyte receptors for transplantation selfantigens, Jerne’s hypothesis (34) provides a powerful argument for regarding transplantation rejection as the fundamental immune response from which other re-

SECONDARY

591

RESPONSE

sponses have developed. The phylogenetic evidence so far is consistent with this; for it suggests that immunoglobulin (antibody) production is a vertebrate specialisation (35-38), while homograft rejection is a very primitive response, which shows the definitive characteristics of the mammalian response-specificity and memory-already in invertebrates as primitive as annelids (39, 40) .I The basic form of antigenic recognition would thus be recognition of a foreign cell by a lymphocyte, that is, the engagement of a highly structured array of multiple determinants on the foreign cell membrane (see 44) by receptors which could individually be of low affinity. Evolutionary elaboration could have extended the scope of lowaffinity recognition to simple noncellular and small molecular-weight antigens by introducing complex auxiliary mechanisms to mimic this basic form. For instance, small molecular-weight antigen assembled at the cell membrane of a macrophage might make it appear “quasi-allogeneic” to a lymphocyte, and the binding of antigen between T and B lymphocytes might provoke a “quasi-allogeneic” MLR. Such multiple-component low-affinity mechanisms will presumably be at a selective disadvantage, however, compared to high-affinity lymphocytes and will tend to be superseded as high-affinity cells increase (or evolve, 45) in the course of the immune response. Given continued stimulation the mechanism of recognition should then apparently regress with time towards a simple form of direct recognition of antigen by the lymphocyte. APPENDIX:

NOTES ON THE LYMPH NODE RESPONSE

Since the response to a primary stimulus is virtually permanent, the expected response elicited by two challenges is the same as would be elicited by the total dose given as a single injection. Thus, if the results are plotted on polar coordinates with total dose as the radius, contours of expected equal response lie on arcs of a circle. Figure 1 shows the lymph node results at 8 days after secondary challenge for different combinations of primary and secondary doses, with Y = total dose and 0 = tan-l (primary dose/secondary dose), where “dose” is taken to mean “logI (number of cells injected + 1) .” The data (percentage uptake in the lymph nodes) are expressed as percentages of the unstimulated control, averaged over intervals of 4-128 days between first and second injections (Section 4). To aid in visualising the experimental results, contours of equal response are superimposed by free-hand extrapolation from the data points and are drawn at equal logarithmic intervals. A.1 A map of the expected response would have contours lying on quadrants of concentric circles. Figure 1 clearly differs from this in a number of aspects : (a) The contours push outwards over most of the surface and reenter along the diagonal. That is, the response elicited by two stimuli is almost always less than that from the same total dose given as a single injection. Only where primary and secondary doses are nearly equal does the system produce an enhanced, that is, secondary response. (b) The gradient is uniform along the principal diagonal but increases along the x and y axis with distance from the origin. If the response is taken as -log, 1 Homograft-like

recognition

tunicates and even coelenterates

of histocompatibility (41-43).

differences

seems

to

be

present

ill

592

BAINBRIDGE

(standardised uptake of radioactivity), the response can be seen to be linear (total dose) for the secondary response s along the diagonal ; whence

ds -ccdn

1;

n = total

number

in Y

of cells used for sensitising

n

On the other hand, the response p for the S-day primary (X axis) or average 12-136-day primary (y axis) responses is quadratic in r. To a close approximation the primary response (p = 0,0,1,2 for Y = 1 (4), 3,5,7) obeys the equation p = 2~’ + $a+:

where r’ =r-1 lb= -a a = +a

Evidently there is an initial impetus into unresponsiveness with increasing dose (u = - yh) which is reversed by the accelerating sensitisation process. This is more apparent in experiments reported previously (4, 5). A.2 These results have a more general significance for the discussion, which is expressed in the proposition below : LEMMA : The response s is not a function of the response p. PROOF : Let s be a function of p. Then s may be represented as a polynomial in p (for any finite number of distinct points in s). Change s to s’ to eliminate constant terms. Then p is a divisor of s’. But s and thus S are linear in r and hence r’, while p is quadratic in r’. Thus p is not a divisor of s’ and s is not a function of

P. : The secondary response PROPOSITION terms of the firiwkary response.

of this system is not explicable

in

PROOF : This follows directly from the lemma. The secondary response is explicable in terms of the primary if and only if it can be expressed as a function of the primary. This is contradicted by the lemma, proving the proposition. A.3 (c) Figure 1 is almost perfectly symmetrical about the diagonal. The response surface of Fig. 1 can be considered as a polynomial function R (y, x) in algebraically independent variables, y (primary dose) and x (secondary dose). From the symmetry of the figure it is clear that K (Y, x) = R (x, Y), that is, R is a symmetric polynomial. The primary response, averaged for 12-136 days (or the average g-day response) is composed of those terms with zero components in .t (or y), since all other terms will vanish for x = 0 (or y = 0). The “total two-dose response,” that is, the response of the animal to all combinations of primary and secondary doses Q (y, x), consists of terms with nonzero exponents in both x and y, which do not contribute to the response in the absence of the second stimulus. The total two-dose response is produced not by two external stimuli interacting with one another but by responding cells of the animal. Let f,,, fl, . . . fk be the “factors,” e.g., primed cells of various types elicited in the animal by the first dose (and not expressed in the primary response), which are stimulated by the second

SECONDARY

RESPONSE

593

dose. Without loss of generality, consider one factor, f. The response can be represented as a polynomial T (f, X) in f, which will not in general be symmetrical. Biologically it is difficult to see how this could give rise to a response Q symmetrically dependent upon primary and secondary doses unless : (i) there is a simple relation between f and y, that is, T(f,x)

= Q(Y,~),

or (ii) both primary and secondary stimuli produce interacting factors (f and 9) of the same formal kind ; that is, if f and 9 are polynomials fi (y ) in y and q (x) in x, respectively, then P=4 the response U (f, .r/) being a symmetric polynomial and c’ (P (Y), P (x) 1 = Q (Y, ~1. Case (i) could correspond to a direct interaction between primed cells and allogeneic lymphocytes, a form of “mixed lymphocyte reaction,” and this is favoured by evidence referred to in the general discussion. Case (ii) could correspond to an interaction between two sets of primed cells. It does not seem profitable at this stage to examine models suggested by these considerations; because data enabling one to relate the measured response to the number of responding cells are not available. ACKNOWLEDGMENTS I wish to express my gratitude to Mrs. Hortense Headley, Mrs. Christine Pincott and Mrs. Julia Parsley for their help and to the Medical Research Council for support. The work was greatly aided by the facilities of the University of London Atlas Computing Service.

REFERENCES 1. Bainbridge, D. R., and Gowland, G., Natzkre London 209, 624, 1966. 2. Bainbridge, D. R., and Gowland, G., Ann. N.Y. Acad. Sci. 129, 257, 1966. 3. Gowland, G., and Bainbridge, D. R., In “Transplantation von Organen und Geweben,” p. 45. Thieme Verlag, Stuttgart, 1967. 4. Bainbridge, D. R., and Gowland, G., Cell. Znzmzozol. 2, 115-127, 1971. 5. Bainbridge, D. R., and Gowland, G., Cell Zmnzzmol. 2, 128-139, 1971. 6. Bainbridge, D. R., Brent, L., and Gowland, G., Transplantation 4, 138, 1966. 7. Billingham, R. E. Brown, J. B., Brent, L., and Medawar, P. B., Tmmplant. Bull. 6, 449, 1959. 8. Billingham, R. E., Silvers, W. K., and Wilson, D. B., J. ExP. Med. 116, 397, 1963. 9. Steinmuller, D., J. Zmm~~~zol.65, 398, 1960. 10. Steinmuller, D., Tramplantafion 1, 97, 1963. 11. Heidelberger, M., 1% “The Nature and Significance of the Antibody Response” (A.&l. Pappenheimer, Ed.), p. 90. Columbia Univ. Press, New York, 1953. 12. Siskind, G. W., Paul, W. E., and Benacerraf, B., Z~~n~z~r~zocl~cr~~istry 4, 455, 1966. 13. Brookem, S., J. Zn~mu~zol. 96, 364, 1966. 14. Paul, W. E., Benacerraf, B., Siskind, G. W., Goidl, E. iz., and Reisfcld, R. -L\., J. Exp. Med. 130, 77, 1969. 15. Dutton, R. W’., J. Exp. Med. 122, 759, 1965. 16. Bach, P. H., and Voynow, N. K., Science 153, 545, 1966 17. Brent, L., and Medawar, P. B., Proc. Roy. Sot. Ser. B 165, 281, 1966.

594 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45.

BAINBRIDGE

Mitchison, N. A., Symp. Int. Sot. Cell Biol. 7, 29, 1968. Byers, V. S., and Sercarz, E. E, J Exp. Med 127, 307, 1968. Byfield, P., and Sercarz, E. E., J. Exp. Med. 129, 897, 1969. Crowle, -1. J., J. Allergy 33, 458, 1962. Brent, L., Brown, J. B., and Medawar, P. B., Proc. Roy. Sot. Ser. B 156, 187, 1962. Chapman, W. D., Parkhouse, R. M. E., and Dutton, R. W., Proc. Sot. Exp. Biol. Med. 117, 708, 1964. Dutton, R. W., and Bulman, H. N., Immunology 7, 54, 1964. Dutton, R. W., and Eady, J. D., Immulzolgy 7, 40, 1964. Mills, J. i\., J. Imrnztnol. 97, 239, 1966. Pearmain, G., Lycette, R. R., and Fitzgerald, P. H., Lancet 1, 637, 1963. Schrek, R., Amer. Rev. Resp. Dis. 87, 734, 1963. Mitchison, N. A., Proc. Ro.Y. Sot. Ser. B 161, 275, 1964. Make& O., and Mitchison, N. A., Immunology 8, 549, 1965. Porter, R. J., Arch. Environ. Health 21, 372, 1970. Mitchison, N. A., Zmmurtology 16, 1, 1969. Kontiainen, S., and Mskeli, O., Immunology 20, 101, 1971. Jerne, N. K., Proc. Nat. Acad. Sci .U.S.A. in press Deutsch, H. F., and Koenig, V. L., In “Handbook of Biological Data” (W. S. Spector, Ed.), pp. 55-56. Saunders, Philadelphia, 1956. Engle, R. L., Jr., and Woods, K. R., In “The Plasma Proteins” (E. W. Putnam, Ed.), Vol. 2, p. 183. Academic Press, New York, 1960. Good, R. A., and Papermaster, B. W., Advan. Immunol. 4,1, 1964. Fongereau, M., Rec. Med. Vet. 144, 5, 1968. Cooper, E. L., J. E.Q. 2001. 171, 69, 1969. Cooper, E. L., Transplantation 6, 322, 1968. Freeman, G., RES J. Reticuloendoth. Sot. 7, 183, 1970. Theodor, J., Bull. Hist. Oceanogr. 66, 167, 1%6. Theodor, J., C. R. Acad. Sci. Series D. 268,2534, 1969. Brondz, B. D., and Snegiriiva, A. E., Immunology 20,457, 1971. Make& O., and Cross, A. M., Progr. Allergy 17, 145,197O.