cell transfer studies on the persistence of homograft sensitivity in the mouse

CELL TRANSFER STUDIES ON THE PERSISTENCE OF HOMOGRAFT SENSITIVITY I N THE MOUSE

JUDITH WARREN AND G. GOWLAND* Department of Bacteriology, London Hospital Medical College, London

KNOWLEDGE concerning the adoptive transfer of sensitivity to solid skin homografts in mice rests mainly on a qualitative basis. Little is known about the quantitative aspects of the ability of cells from presensitised animals to transfer homograft sensitivity passively to normal syngeneic recipients. Such information would help to establish some pattern for the development and persistence of a state of sensitisation and would also shed light on the question of immunological " memory ".

Mitchison (1954) demonstrated the capacity of cells obtained from the draining lymph- nodes of immunised mice to transfer specific sensitivity to tumour grafts. Adoptive sensitisation was more effective with lymph-node grafts from immunised animals than with cell suspensions. It was subsequently shown that passive transfer of sensitivity to allogeneic skin grafts was also effective when cells from lymph-nodes draining foreign grafts were used (Billingham et al., 1954-55). However, transfers of spleen cells and whole blood transfers were ineffective.

Billingham, Silvers and Wilson (1963) have described further experiments in which the adoptive transfer of sensitivity in mice was studied by using blood and peripheral leucocytes, as well as lymph-node cell suspensions. Their criterion of successful transfer was based on the breaking of a state of tolerance previously induced in recipient mice.

The claim that accelerated rejection of skin homografts can be achieved by the injection of soluble extracts of sonicated presensitised lymphoid cells (Najarian and Feldman, 1963) has not been substantiated (Fujimoto et al., 1966; Warren and Gowland, 1969). Further, homograft sensitivity has not, so far, been transferable in mice by the use of isoimmune serum (Mitchison; Billingham et al., 1954-55; Billingham and Brent, 1956). These findings have led most authors to conclude that transfer of sensitivity is effective only when intact, viable lymphoid cells are present in the inoculum.

This is a report of a series of transfers of lymphoid cells from various sources and in differing concentrations. We have measured the ability of cells to confer adoptive sensitivity on normal recipient mice, at a variety of times after active sensitisation of the cell donors.

MATERIALS AND METHODS

Animals. Experimental animals were male mice from the inbred strains CBA(H-Zk) and

Sensitisation of donor mice. Donors of lymphoid cells were CBA mice sensitised to A-strain antigens by the lateral thoracic application of a full-thickness graft of A-strain tail skin. The exception to this was one experiment involving the transfer of cells from unsensitised donor CBA mice to normal syngeneic recipients.

A(H-2').

Received 16 July 1969; accepted 8 Sept. 1969. * Present address: University of Leeds Department of Immunology, Wellcome Wing, The

General Infirmary, Leeds LS1 3EX. J. PATH.-VOL. 100 (1970) 39

40 JUDITH WARREN A N D G . GO WLAND

Removal and preparation of lymphoid cells for passive transfer. At various time-intervals after sensitisation (i.e., 8, 10, 12, 14, 21, 28 or 56 days) batches of 15-20 donor mice were killed by cervical dislocation, the lymph-nodes were removed and pooled and cell suspensions were made in buffered Ringer-phosphate (pH 7.4) without addition of serum (Billingham and Brent, 1959). The same technique was applied to spleen. When whole blood was used in transfer, it was removed from etherised animals by cardiac puncture, pooled and heparinised at 20 units per ml. Peripheral lymphocytes for injection were obtained from whole blood after sedimentation of erythrocytes in 6 per cent. w/v Dextran 110 (Fisons Ltd) at 37°C (Cater and Waldmann, 1967).

Where lymph-node cells have been used in transfer, they have represented cells from a suspension of pooled peripheral (cervical, axillary, brachial, inguinal) and mesenteric nodes. Our experiments thus differ from most of those on adoptive transfer of sensitivity previously reported, in which draining lymph-nodes alone have been used.

Test system. We have based the effectiveness of passive transfer of sensitivity on the ability of transferred lymphoid cells from sensitised donor mice to cause accelerated rejection of skin allografts placed on unsensitised recipient mice.

CBA mice used as recipients of transferred cells were test-grafted with A-strain tail skin, as described above. The mice were usually grafted 1 day before receiving the inoculum of presensitised syngeneic lymphoid cells. In one experiment, involving intraperitoneal injection of cells from donors sensitised 56 days previously, recipient mice were test-grafted 3 days after injection. This reverse order of injection and grafting of recipients was also employed in certain of the intravenous cell-transfer experiments.

In most experiments, at least ten 12-wk-old CBA recipients were given an injection of each test dose of sensitised cells (see below). Viability of cell suspensions was determined by nigrosin exclusion before inoculation. Control mice received only an allogeneic skin graft.

Route of injection of transferred cells. lntraperitoneal cell transfers constituted the majority of the experiments described here. A brief comparison of the intraperitoneal and intravenous routes of injection (see experiments 1 and 2, and Results section) showed that the former was more efficient for the purposes of showing accelerated rejection of allografts on unsensitised CBA mice, after adoptive transfer of syngeneic cells from immunised donors.

Ce Il-trans fer experiments Experiment 1 : comparison between the intravenous and intraperitoneal routes of cell

transfer. Recipient mice, grafted 1 day previously, were given either an intraperitoneal or an intravenous injection of 10 x 1 0 6 lymph-node cells from donor mice sensitised 14 days previously. These were compared with control mice not given any injection.

Experiment 2 : intraperitoneal transfer of lymph-node cells. One day after grafting, recipient mice were given an injection of cells from donor mice sensitised 0, 8, 10, 12, 14, 21, 28 or 56 days previously. Mice were given an injection of the following doses of lymph-node cell suspensions: 5 x 107, 2.5 x 107 or 1 . 0 ~ 107 cells in 1 ml.

Experiment 3 : intraperitoneal transfer of spleen cells. One day after grafting, recipient mice were given an injection of cells from donor mice sensitised 0, 8, 10, 12, 14, 21, 28 or 56 days previously. Mice received the following doses of spleen cell suspensions: 10 x 107, 5 x lo7 or 2.5 x 107 cells in 1 ml.

Experiment 4 . (i) To test for persistence of sensitivity 8 wk after grafting, mice grafted 56 days previously were regrafted with A-strain skin.

(ii) Recipient mice were given an injection of cells from mice sensitised 56 days previously. One day after grafting they received an injection of the following: 5 x 107 lymph-node cells in 1 ml; l o x 107 spleen cells in 1 ml; or 1 nil heparinised whole blood.

(iii) Recipient mice were given an injection of cells from donors sensitised 56 days previously. Three days before grafting they received 5 x 107 or l ox 107 lymph-node cells; 10 x 107 or 20 x lo7 spleen cells; or 1 ml heparinised whole blood.

Experiment 5 : comparison of circulating cells transferred by different routes.

PERSISTENCE OF HOMOGRAFT SENSITIVITY IN MICE 41

Number of mice per

group

(i) 5 x 106 peripheral lymphocytes, or 1 ml heparinised whole blood (c. 5 x 1 0 6 lymphocytes), from donors sensitised 14 days previously, was transferred intravenously to recipient mice, test-grafted 24 hr previously.

(ii) 1 ml heparinised whole blood (c. 5 x 106 lymphocytes), from donors sensitised 14 days previously, was transferred intraperitoneally to recipient mice test-grafted 24 hr previously.

(iii) 1 ml heparinised whole blood, from donors sensitised 14 days previously, was transferred intravenously to recipient mice, 3 days prior to test-grafting.

(iv) 1 ml heparinised whole blood, from donors sensitised 14 days previously, was transferred intraperitoneally to recipients, 3 days prior to test-grafting.

Assessment of graft rejection. Plaster bandages were removed from recipient mice on day 7 after test-grafting and the skin grafts were scored visually each day thereafter, until rejection was complete.

TABLE I A comparison of the intraperitoneal and intravenous routes of injection in the passive transfer

of cells from donor mice sensitised 14 days previously to unsensitised CBA recipients test-grafted with A-strain skin 24 hr prior to injection

Number of recipient grafts surviving at days after grafting

7 8 9 10 11 12 13 14 15

Dose of cells transferred

Route of injection

lox 106 lymph-node cells

Intraperitoneal 1 1 0 0 0 0 0 0 0 8 i lox 106 lymph-node

cells

None

Intravenous

... 9 1 9 9 9 9 6 4 4 0 0

10

Approximate MST* (days)

< 7

10.5

11.5

1 0 1 0 6 6 3 1 0 0 0

* MST = Median survival time.

The normal median survival time (MST) of A-strain tail skin on male mice of our own inbred CBA strain shows a variation from 11 to 12 days, with an average of about 11.5 days. Grafts were scored on the percentage of epithelium surviving. For the purposes of layout of figures and tables, grafts were classed as " surviving " if 75 per cent. or more of the epithelium remained intact; in the same way, grafts with 50 per cent. or less survival were taken as being " rejected ".

RESULTS Experiment I

Table I compares the effect of 10 x 106 lymph-node cells from CBA donors sensitised to A-strain skin 14 days previously, when injected either intraperitoneally or intravenously into CBA recipients test-grafted with A-strain skin 24 hr previously. It shows that the intraperitoneal transfer of cells reduces the MST of test-grafts much more effectively than intravenous transfer. Therefore, in experiments 2, 3 and 4 the intraperitoneal route of transfer was used as being the more efficient.

42 JUDITH WARREN AND G . GOWLAND

Experiment 2 Fig. 1 shows the results of the passive transfer of pooled lymph-node cells

from CBA mice at various times after their sensitisation with A-strain skin. Each line on this figure represents the results of transfer of a different dose of lymph-node cells (see legend to fig. l), these being 5 x lO7,2.5 x 1 0 7 and 1 .Ox 107 cells. If it is borne in mind that an A-strain skin graft is normally rejected in about 11.5 days by CBA mice, it is evident that transfers of cells from unsensitised syngeneic donors have not reduced the rejection time of A-strain

Time in days after immunisation of donors

FIG. 1.-Effect of intraperitoneal transfer of presensitised CBA lymph-node cells on the median survival time (MST) of A-strain skin on normal CBA mice: cells transferred at various times after donor sensitisation and at a variety of concentrations (- 5x 107; . . . . 2 . 5 ~ 107; - - - _ _ - ~ . O X 107).

grafts. Transfers of cells from CBA donors immunised 8 days previously give the first indication of accelerated rejection of recipient test-grafts, although this effect is relatively small. The period from 8 to 10 days after donor sensitisation, marked by a steep decline in MST of recipient skin grafts, indicates that sensitivity is being readily transferred from the lymphoid tissue of the donor mice. Cells transferred in the period from 10 to 12 days after donors were sensitised show no increased capacity to shorten the rejection time of recipient grafts, beyond that found in the previous transfer at 10 days.

The maximum acceleration of rejection of recipient grafts occurred when transfer had taken place 14 days after the cell donors were sensitised to A-strain antigens. In the animals that had received the two higher cell-doses, the MST was less than 7 days.


Fig. 1 also shows that, by 21 days after donor sensitisation, the efficiency of these same cell-doses from lymph-nodes in the transfer of sensitivity to A-strain antigens is decreasing. This decrease continues through 28 days, and at 56 days after donor sensitisation the recipient grafts were rejected in a period of time characteristic of previously unsensitised mice.

Experiment 3 Fig. 2 represents studies in the transfer of spleen cells from presensitised

donors, at time-intervals similar to those involving lymph-node cells in experiment 2. The doses of cells transferred in experiment 3 were 10 x 107, 5 x 107

0 5 10 15 20 25 30 56 Time in days after immunisation of donors

FIG. 2.-Effect of intraperitoneal transfer of presensitised CBA spleen cells on the MST of A-strain skin on normal CBA mice: cells transferred at various times after donor sensitisation and at a variety of concentrations (- lox 107; . . . . . . 5x 107; - - - - - - 2 . 5 ~ 107).

and 2 . 5 ~ 107, given by a single intraperitoneal injection. These doses are approximately twice those administered in the lymph-node cell transfers in experiment 2.

As in experiment 2, the first indication of acceleration of recipient graft rejection comes when cells are transferred from donor mice 8 days after immunisation to A-strain antigens. The highest dose of spleen cells shows the most marked capacity to accelerate rejection of recipient skin grafts throughout experiment 3, with a steep decline in MST after transfers of cells 8-10 days after immunisation of donors. 10 x 107 spleen cells transferred 14 days after donor sensitisation gave an MST of recipient grafts of less than 7 days (cf. also fig. 1).

The lower doses of spleen cells were not so effective in transferring sensitivity

44 JUDITH WARREN AND G . GOWLAND

at 14 days, but they maintained their efficiency during the period 14-21 days after donors were sensitised, whereas the MST of recipient grafts after lymph- node cell transfers at this time had begun to increase.

In the results of spleen-cell transfers, as in those involving lymph-node cells, there is an increase in the MST of recipient grafts with transfers at intervals greater than 21 days after immunisation of donors. In transfers of cells 56 days after donor sensitisation, the MST of A-strain skin grafts on recipient CBA mice had returned to approximately its original level.

TABLE I1

Efect of varying the time of injection of lymphoid cells, from CBA mice sensitised to A-type skin 56 days before, on the survival of A-type skin grafis on normal CBA mice

Time of inject ion

of sensitisec

cells

1 day after grafting

3 days before grafting

No inject ion

Number and type of cells transferred intraperitoneally

5 x l o7 lymph-node cells lox 107 spleen cells

I ml whole blood

5 x lo7 lymph-node cells 10 x 107 lymph-node cells lox lo7 spleen cells 20 x 107 spleen cells

1 ml whole blood

rlumber of animals in group

9 7

10

10

Number of test-grafts surviving at days after grafting

7 8 9 10 1 1 12 13 14

7 7 7 4 3 2 1 0 6 4 3 2 2 2 0 0 8 7 6 4 3 2 2 0 8 7 7 4 3 1 1 0 3 2 2 2 1 1 1 0

10 9 9 7 5 4 3 1

Approximate MST (days)

11

Experiment 4 (i) Since it was shown (experiments 2 and 3) that the capacity to accelerate

rejection of skin grafts seems to be failing in cells transferred at and after 28 days after donor sensitisation, we regrafted CBA mice that had rejected an A-strain skin graft applied 46 days earlier. The median survival time of the second A-strain grafts on these mice was uniformly 6 days or less, showing that the mice were still highly sensitised.

(ii) The results of transferring cells from donor mice grafted 56 days previously to recipient CBA mice 24 hr after test-grafting with A-strain skin are shown in the first section of table 11.

The MST of test-grafts on recipients given an injection of 5 x 107 lymph- node cells appears to confirm the observations of experiment 2, i.e., that there is a declining ability on the part of donor cells to transfer sensitivity passively at prolonged periods after donor sensitisation. In this experiment mice given an injection of 10 x 107 spleen cells show a slight decrease in recipient graft survival times.


Route of injection

The intraperitoneal injection of 1 ml whole blood, from donors presensitised 56 days earlier, into CBA recipients did not reduce the median survival time of A-strain test-grafts.

(iii) The lower section of table I1 shows the survival times of test-grafts on recipient mice receiving, 3 days before grafting, cells from donors presensitised 56 days before. As in part (ii) of this experiment, transfers of 5 x 107 lymph- node cells and 10 x 107 spleen cells from such donors have little or no tendency

TABLE I11

The efects of varying the time of transfer of circulating cells from CBA mice sensitised I4 days previously on the survival time of A-strain skin grafts on normal CBA mice

~~

Number of Time of injection of mice Per

group sensitised cells

Amount and source of

cells transferred

5 x 106 peripheral lymphocytes

1 ml whole blood

1 ml whole blood

1 rnl whole blood

1 ml whole blood

Number of recipient grafts surviving at days after

grafting

7 8 9 10 11 12 13 14 15

Approx- imate MST (days)

Intravenous

Intravenous

Intraperitoneal

I 1 day after grafting ' 6

1 day after grafting 6

1 day after grafting

5 5 4 4 4 3 2 2 0

6 6 6 5 3 1 0 0 0

12.0

11.0

1 0 0 0 0 0 0 0 0 < 7

Intravenous 3 days before grafting 4

to shorten the survival time of test-grafts. Similarly 20 x 107 spleen cells of the same origin, injected 3 days before grafting, are relatively ineffective in this respect .

However, doubling the previous highest dose of such lymph-node cells to 10 x lo7 injected causes accelerated rejection of A-strain test-grafts. Also the injection of 1 ml whole blood of the same origin 3 days before recipients are grafted reduces the MST of A-strain test grafts to less than 7 days. This result compares favourably with that produced by the same inoculum administered 1 day after test-grafting of recipients.

Experiment 5 The results of the intraperitoneal or intravenous injection of circulating cells

from donors sensitised to A-strain antigens 14 days previously are shown in

4 3 1 1 0 0 0 0 0 8.5

Intraperitoneal 3 days before grafting 8 2 2 1 0 0 0 0 0 0 <7

46

table 111. The median survival times of A-strain skin grafts on recipient CBA mice given an intravenous injection of 1 ml whole blood or 5 x 106 peripheral lymphocytes 1 day after grafting, show no evidence of accelerated rejection. 1 ml whole blood injected intraperitoneally at the same time led to a marked acceleration of rejection, only one graft surviving at 7 days.

An injection of 1 ml whole blood 3 days before test-grafting led to a similar picture for the intraperitoneal route of transfer, i.e., a MST of less than 7 days. Accelerated graft rejection also occurred after transfer via the intravenous route, though this was less marked than with intraperitoneal transfer.

JUDITH WARREN AND G. GOWLAND

DISCUSSION There are two methods by which the efficacy of cells in the passive transfer

of homograft sensitivity can be estimated: one is the accelerated rejection of an allogeneic skin graft, the other is the breaking of a long-standing state of tolerance induced in the recipient animal by the intravenous injection of allogeneic cells and shown to be persistent by the retention of a surviving skin graft (Billingham et a!., 1963).

We chose the former method for three reasons: firstly, because the time taken for the state of tolerance to disappear shows considerable variation, not necessarily related to the number of presensitised syngeneic cells transferred to the tolerant animal. For instance, the persistence of induced tolerance without interference from transferred cells is not predictable, i.e., a new clone of cells arising naturally in the mouse might abolish tolerance, and therefore it would not be possible to infer that transferred cells were wholly responsible for the reappearance of sensitivity.

Secondly, the reversal of a state of tolerance depends on the amount of foreign antigen to be eliminated. This amount may vary in any particular state of chimerism and may depend on the time elapsed since the animal was made tolerant of the antigen (Gowans, McGregor and Cowen, 1963).

Thirdly, the state of cellular chimerism could have some effect on the inoculum of syngeneic cells used in transfer, especially as these cells come into immediate and intimate contact with the tolerance-inducing antigen in the tissue of the recipient host-and may remain so for periods of up to 100 days (Billing- ham et al., 1963). Allied with these factors is the evidence that the elimination of dissociated cells, e.g., as in a cellular chimera, is markedly influenced by non- cellular processes (Gowland and Bainbridge, 1969).

Our justification for basing the effectiveness of passive transfer on the ability of transferred lymphoid cells to cause accelerated rejection of a test-graft applied 24 hr prior to injection is that we believe this method of assessment gives the most realistic idea of the immediate capability of the transferred cells. Similarly, the use of pooled peripheral and mesenteric nodes gives a more complete picture of the state of sensitivity in the whole animal than experiments in which draining lymph-nodes alone are used.

From the literature it becomes apparent that the test system used in evaluat- ing the process of homograft sensitivity influences not only the deductions


that can be made from the data obtained, but also the salient points that are emphasised.

The problem of the most efficient route of injection of cells for passive transfer of sensitivity in these experiments was solved by the results obtained in experiment 1. At a time (14 days after grafting of donors) when the capacity for transfer would be expected to remain high, the injection of 10 x 106 lymph-node cells intravenously produced little or no accelerating effect on the rate of graft breakdown in recipient mice, whereas intraperitoneal injection of the same dose of cells caused recipient skin grafts to be rejected less than 7 days after their application. Thereafter, the intraperitoneal route of injection was used in the large experiments 2 and 3, which were designed to trace the development and persistence of the state of sensitivity in the mouse.

There are two points of consideration in relation to experiments 2 and 3. (1) All results from transfers of cells from the lymph-nodes of sensitised animals were obtained with suspensions of pooled peripheral and mesenteric nodes, (2) All transfers shown in figs. 1 and 2 took place 24 hr after test-grafting of recipient mice.

The former may be a contributory reason why transfers from donors 8 days after sensitisation produced little or no decrease in median survival time of test- grafts, when compared with other reports of sensitivity being manifest at this time (Billingham et al., 1954-55). If the axillary and brachial lymph-nodes on the side draining the skin graft are the first to encounter antigen, they will have the greater proportion of sensitised cells at this time. This undoubtedly shows in transfers from donors at 8 days where these lymph-nodes alone are used, whereas transferred suspensions of pooled peripheral and mesenteric nodes at this time would show a corresponding dilution effect. Lack of transfer of sensitivity at 8 days in spleen may be due to the fact that neither antigen, nor cells exposed to it, has reached the spleen in sufficient quantity to produce a detectable response.

From 8 to 10 days after sensitisation, however, the potential of donor cells from either lymph-nodes or spleen to accelerate graft rejection is readily apparent. In the case of lymph-nodes, results from this time after sensitisation can be explained by attributing one or more conditions to the cells transferred. Cells that have encountered foreign antigen and have thus been “ primed ” to divide prior to transfer have subsequently divided in their original host, to produce effector cells. Alternatively, the transfer of “ primed ” cells from the donor may have been followed by division in the recipient animal, thus releasing large numbers of effector cells in the recipient. There is also a probability that “ primed ” cells have been to some extent redistributed throughout the donor’s lymph-nodes prior to transfer.

When cells are transferred to CBA recipients at 12 days after donor sensitisation, there is no further acceleration of graft rejection beyond that shown at 10 days. This is particularly apparent in lymph-node cell transfers and slightly less so in those of cells from spleen. This can be seen in both figs. 1 and 2, and so seems unlikely to be due to an error in technique or calculation. The explanations may be: first, the potential for adoptive transfer, in terms of numbers of

48 JUDITH WARREN AND G. GOWLAND

cells likely to affect the MST of test-grafts, may not change from 10 to 12 days after donor sensitisation; a second, and possibly related, reason could be that this time-interval represents the normal breakdown of an A-strain skin graft on a CBA mouse (i.e., 11.5 days) and therefore large numbers of sensitised cells may be deployed from the lymph-nodes in order to infiltrate the graft present on the donor animal itself, and so be unavailable in the lymph-node and spleen material used for transfer.

The maximum expression of sensitivity after transfer of donor cells at the times shown in fig. 1 is undoubtedly 14 days. In lymph-nodes this could represent the time at which multiple divisions of antigenically stimulated and transformed cells produced the maximum number of effector small lymphocytes. This state of sensitivity in donor lymph-nodes at 14 days is reflected in the high-dose spleen cell transfer experiments at the same time (see fig. 2); cells from spleen reduced the MST of recipient grafts to less than 7 days. The question arises as to what this represents, since the two inocula contain vastly different numbers of cells. It is possible, either that the process of redistribution of cells from lymph-nodes is more effective in distributing them to the peripheral lymph-nodes than to the spleen, or that the same level of redistribution to both these locations is masked by a dilution effect, due to the normally large population of cells resident in the spleen, most of which are uncommitted to this reaction against A-strain antigens. The rise in MST of recipient grafts after 14 days, in fig. 1, may represent death of a large number of short-lived effector cells with the function of destroying antigenically distinct cells or fragments by contact.

The period from 14 to 21 days in fig. 2, however, can be considered separately, because of the maintenance of a reduced MST in transfers of spleen cells, as opposed to lymph-node cells. This may be the result of a process of redistribution of cells from the lymph-nodes to the donor spleen after 14 days; also there is the possibility that antigen, released from breakdown of the A-strain skin graft, has reached the spleen of the donor mouse and initiated a process of cell division. If cell division was occurring in donor spleens later than 14 days after application of the sensitising skin graft, this could account for the persistently high level of activity in spleen cell transfers at this time.

We felt that the apparent decline in effectiveness of the cell doses used, whether from spleen or lymph-nodes, at time-intervals greater than 21 days after donor sensitisation, required clarification, to determine whether sensitivity is no longer transferable via lymph-nodes or spleen at 56 days after donors were grafted, or whether the proportion of sensitised cells had fallen to a level that did not permit its detection by the techniques used in experiments 2 and 3.

A sample regrafting of CBA mice, sensitised 56 days previously by a single A-strain skin graft, provided ample evidence that the animals were still highly sensitised at this time. Bearing in mind this continuing high level of sensitivity in the cell donor and the failure of our previously successful doses of cells to transfer sensitivity from that donor (in experiments 2 and 3), we devised experiment 4. This was constructed so that transfer of cells from another source, i.e., whole blood, was included and, in part of the experiment, the


previous high doses of lymph-node and spleen cells were doubled. In the hope of predisposing the experiment further in the direction of accelerated rejection of test-grafts, part (iii) involved the injection of cells into CBA recipients 3 days prior to grafting with A-strain skin, instead of 24 hr after, as was usual.

The results from lymph-node cells or whole blood transferred 24 hr after test-grafting of recipients seem to bear out those of experiments 2 and 3 at a similar time-interval, but the negligible amount of sensitivity transferred by quite massive doses of spleen cells at any time during experiment 4 indicates that the proportion of sensitised cells in this tissue is minimal at 56 days after donor sensitisation.

In any case the only marked reduction in MST of test-grafts occurred when the donor cells were injected into the recipient mice 3 days prior to grafting. Coupled with this, the effect was shown in the highest dose of lymph-node cells injected (10 x lO7), but to a lesser extent than in transfers with 1 ml mouse blood, which contains approximately 10 times fewer lymphocytes. Two explanations may be offered for the success of adoptive transfer at 56 days after donor sensitisation, in this modified experimental regime. One is that the capacity to react to antigens of a type previously encountered resides, after a period of time, in a small number of long-lived lymphocytes able to mount a swift response and constituting an immunological " memory ". The second is that cells that are sensitised may become part of a predominantly recirculating population, which reacts rapidly because of its mobility as well as its relatively high complement of cells with previous experience of graft antigens. Either or both of these explanations may apply.

I t may well be preferable to regard the sensitised animal, for the purposes of cell transfer, as having, at this date after its initial antigenic stimulus, undergone a shift both in intensity and location of the response in purely cellular terms.

In view of the recognised ability of cells from the circulation to transfer sensitivity (Gowans, Gesner and McGregor, 1961; see also Billingham et al., 1963) experiment 5 was set up to estimate the effect of whole blood or separated lymphocytes in passive transfer, at what we have, so far, considered the most effective time after sensitisation of donor mice, i.e., 14 days. Notwithstanding the failure of lymph-node cells in experiment 1 to produce accelerated rejection of test-grafts when injected intravenously, both this and the intraperitoneal route of injection were used as a means of transferring whole blood. I t might be a reasonable assumption that sensitised syngeneic lymphocytes in donor blood would fare better when introduced directly into the recipient circulation than when injected into the peritoneal cavity. However, the results of such a comparison, when CBA recipients were given the injection of sensitised cells 24 hr after test-grafting with A-strain skin, fall definitely in favour of the intraperitoneal route.

Only when the blood was inoculated intravenously 3 days prior to skin- grafting of the recipients was there any marked acceleration of rejection and even then less than was shown by intraperitoneal injection.

It is not easy to propose explanations for the resultsof experiment 5-i.e., the lower effectiveness of the intravenous route-except that there is a strong

J. PATH.-VOL. 100 (1970) D

50 JUDITH WARREN AND G. GOWLAND

impression that these sensitised syngeneic cells are being placed in the " wrong stream of traffic ". Possibly, the effect of intravenous injection leads to a distribution of these lymphocytes (less than lox 106 in number), however potent they may be as individual cells, to places where many of them can make little contribution to graft rejection. Our observation that there is a difference in the efficiency of the two routes in experiments involving passive transfer of sensitivity merits further investigation, possibly involving the radioactive labelling of sensitised cells.

SUMMARY

The ability of lymphoid cells obtained from CBA mice presensitised by an allogeneic (A-strain) skin graft to transfer a state of sensitivity passively to normal syngeneic recipients has been studied quantitatively, by using the rejection of an allogeneic skin graft as an indicator.

Cells obtained from lymph-nodes (pooled peripheral and mesenteric) and spleen were transferred to recipients intraperitoneally at 3 different dose levels at various times after sensitisation of the cell donors, and normally 24 hr after the recipients were test-grafted.

With lymph-node cells sensitivity could first be transferred 8 days after donor sensitisation; maximum activity was observed at 14 days; after this there was a gradual decrease in activity of the transferred cells, and activity was not detectable by 56 days.

With spleen cells a somewhat similar pattern was observed, although maximum activity was maintained for longer and decrease in activity was not so rapid. Activity was not detectable in this organ at 56 days after donor sensitisation.

By using a modified test system (cells transferred 3 days before test-grafting), sensitivity could be detected in cells from 56-day donors in lymph-nodes (lox l o 7 cells injected) and whole blood (1 ml), but not in spleen even though 20 x 107 cells were transferred.

It has been shown that, cell for cell, sensitised lymph-node cells are much more effective when transferred by the intraperitoneal rather than by the intravenous route. Furthermore, circulating lymphocytes show the ability for passive transfer when injected intraperitoneally, but to a much lesser extent when injected intravenously. No reasonable explanation can be offered for this finding at this time, but the phenomenon is being further investigated.

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