The Journal of Pathology

Vol. 107 No. 4

CELL TRANSFER STUDIES ON THE PERSISTENCE O F HOMOGRAFT SENSITIVITY I N THE MOUSE: THE PERIODIC REDISTRIBUTION O F SENSITISED CELLS BETWEEN THE CIRCULATION AND LYMPHOID TISSUES

JUDITH WARREN AND G. GOWLAND University of Leeds, Department of Immunology,

The General Infirmary at Leeds

QUANTITATIVE studies reported previously (Warren and Gowland, 1970) have shown that the ability of lymphoid cells obtained from CBA mice pre- sensitised to A-strain skin grafts to transfer a state of sensitivity passively to normal syngeneic recipients was maximal 14 days after initial donor grafting and that similarly effective transfer was not detectable 56 days after grafting.

The results of a successful transfer were based on the accelerated rejection of allogeneic test skin, grafted on the syngeneic recipients of transferred cells.

In that system, only cells from the pooled peripheral and mesenteric lymph-nodes, or spleen cells, were extensively used in the transfers, which were carried out in most cases one day after an allogeneic test skin graft had been applied to the recipient mice.

The present report, which is of essentially preliminary nature, describes a series of experiments in which the principles applied in previous experiments were extended to cover the transfer of whole blood from sensitised CBA donors at various times after grafting with A-strain skin. By using a modified test system, we made a preliminary investigation into the apparent inability of lymphoid cells, from donors grafted 56 days earlier, to transfer sensitivity at a level detectable by skin homograft rejection.

MATERIALS AND METHODS

General Experimental animals. Male animals from the following inbred strains were used:

Sensitisation of donor mice. Donors of lymphoid cells were CBA mice sensitised to A-strain Am-2') and CBA(H-2k). The mice were bred by strict brother to sister mating.

Received 5 Apr. 1971; accepted 30 Nov. 1971. J. PATH.-VOL. 107 (1972) 225 R

226 JUDITH WARREN AND G. GO WLAND

antigens by the lateral thoracic application of a full-thickness, graft of A-strain tail-skin by the technique of Billingham and Medawar (1951).

Removal of lymphoid cells andpreparation forpassive trans]"e. At 8, 10,14, 18,21,28,56, 57, 58, 59 or 60 days after sensitisation, 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 to 20 units per ml.

Lymph-node cells used in iransfer represented cells from a suspension ofpooledperipheral (cervical, axillary, brachial, inguinal) and mesenteric nodes.

Test system. The measure of passive transfer of sensitivity has been based 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 grafted either 1 day before, or 3 days after they received the inoculum of presensitised syngeneic lymphoid cells.

In all 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, prior to inoculation. Control mice received only an allogeneic skin graft.

Plan of experiments Experiment 1. Intraperitoneal transfer of whole blood. Three days before (i), or 1 day after (ii), test-grafting, recipient mice were given an intra-

peritoneal injection of 1 nil of heparinised whole blood {c . (5-'7) x 106 lymphocytes} taken by cardiac puncture from donor mice sensitised 8, 10, 14, 18, 21, 28 or 56 days previously.

Experiment 2. (i) Three days prior to test-grafting, groups of recipient CBA mice were given an intraperitoneal injection of either 10 x 107 lymph-node cells, or 1 ml heparinised whole blood from syngeneic donor mice sensitised 56, 57 and 58 days previously.

(ii) Three days before test-grafting, groups of recipient CjBA mice were given an intra- peritoneal injection of 10 x I 0 7 lymph-node cells, or 20 x 1 0 7 spleen cells, or 1 ml heparinised whole blood from donor mice sensitised 56, 57, 58, 59 and 60 days previously.

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

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 1

Figure 1 shows the results of passive transfer of 1 cnl whole blood from CBA mice at various times after they had received grafts of A-strain skin. The two lines on this figure (see legend to fig. 1) represent the median survival times (MST) of A-strain test skin-grafts on normal CBA recipients to which 1 ml whole blood had been given, by intraperitoneal transfer, either (i) 3 days before or (ii) 1 day after test-grafting.

An A-strain skin graft, placed on previously sensitised CBA mice of the line used in these experiments, normally has an MST of 11.5 days; blood

PERIODIC REDISTRIBUTION OF SENSITISED LYMPHOCYTES 227

transferred from donors 8 days after sensitisation to recipients 3 days before grafting shows no indication of having affected the rejection time of the test skin grafts.

At 10 days, there appears to be a discrepancy between the two regimens of injection and grafting. Animals receiving the injection of 1 ml whole blood 3 days before the application of a test graft reject this graft 1% days sooner than the animals that were given the injection on the day after grafting.

As in previous experiments in which lymph-node and spleen cells were used in transfer (Warren and Gowland, 1970) the maximum effect of passive transfer

I I I I I I I I + 8 10 12 14 16 18 20 22 24 26 28 56

Time after immunisation of donors (days)

FIG. 1 .-Effect of intraperitoneal transfer of whole blood from presensitised donor CBA mice, on the median survival time (MST) of A-strain skin grafts on recipient CBA mice: blood transferred 3 days before, or 1 day after grafting. - 1 ml whole blood transferred 1 day after grafting of recipients. - - - 1 ml whole blood transferred 3 days before grafting of recipients.

of whole blood on recipient test-graft rejection occurred 14 days after initial donor sensitisation. It can be seen from fig. 1 that this is true regardless of the time-relations of passive transfer and application of test grafts. An MST of less than 7 days for A-strain grafts on recipient CBA mice was shown with blood transferred at 14 days after donor sensitisation. In this context it should be stressed that, in our experience, 1 ml of pooled heparinised blood removed by cardiac puncture contains at most 7 x 106 lymphocytes.

Differences between the two regimens of injection and grafting occur when blood is transferred at 18 and 21 days after donor sensitisation. In the case of transfer at 18 days, blood given to recipients 3 days before test-grafting is still extremely efficient in causing the rapid destruction of the grafts, whereas the blood from similar donors given to CBA recipients 1 day after grafting appears to have a lesser effect. The fact that these positions are reversed when blood from 21-day donors is similarly transferred may well remove the significance of these

228 JUDITH WARREN AND G. GO WLAND

9 6

8 8

discrepancies. Despite these differences the capacity of transferred blood to accelerate the rejection of recipient test grafts is apparently decreasing over the period from 14 to 21 days after donor grafting. By 28 days after donor grafting, the effect of transferred blood on recipient skin-graft survival time is considerably weakened or, in transfers carried out 1 day after recipients were test-grafted, nil.

TABLE Cell transfer from CBA mice 56, 57 and 58 days after grafting with A-strain skin: the effect of

lymph-node cells and whole blood, transferred 3 days before grafting, on the survival of A-strain skin grafts on normal CBA mice

8 9 --

7 6

2 2

__ 3 3

8 7

_ _

7 4

-_

Days after grafting

(cell donors)

-__

56

12

Dose of cells injected

13 --

2 1

1 0

_ _ ~

1 0

4 4

_ _ 4 2

0 0

__ 4 0

10 x 107 lymph- node cells

1 ml whole blood

15

57

16 __

0 0

0 0

__ 0 0

2 0

_ _ 0 0

0 0

_- 0 0

1Ox I07 lymph- node cells

1 ml whole blood

58 10 x 107 lymph- node cells

I ml whole blood

. . . ~ None (controls)

Number of mice in group

8

9

7

8

9

7

8

Number of test grafts surviving at days after grafting MST of

test grafts (days)

10.1

< 7.0

< 7.0

12.0

11.5

9.5

11.0

The last point shown on fig. 1 refers to the transfer of blood from CBA donors that had received a single graft of A-strain skin 56 days previously. When such blood was transferred to normal CBA mice, 3 days before the ap- plication of A-strain skin, the resulting MST was 7 days. In contrast, blood from 56-day donors transferred to CBA mice 1 day after the application of an A-strain test graft did not appreciably reduce the MST (of such grafts, the median survival time being 11 .O days.

Experiment 2 (i) 1 ml whole blood or 10 x 107 lymph-node cells taken from CBA donor

mice 56, 57 and 58 days after sensitisation to A-strain antigens, when given to normal CBA mice 3 days before the latter were test-grafted with A-strain skin, led to the MST of test grafts shown in the table.

Fifty-six days after grafting of donor mice, the transfer of 1Ox 107 lymph- node cells led to a MST of 10.0 days for A-strain test grafts on recipient CBA

PERIODIC REDISTRIBUTION OF SENSITISED L YMPHOCYTES 229

mice compared with a MST of less than 7 days from the transfer of 1 ml whole blood. When similar doses of cells were transferred from donor CBA mice 57 days after grafting, the positions, with respect to MST of test grafts, were reversed. At this time, the transfer of lymph-node cells led to a markedly accelerated rejection of A-strain test grafts, whilst the transfer of whole blood

56 51 58 59 60 Time after immunisation of donors (days)

FIG. 2.-Effect of intraperitoneal transfer of lymph-node cells, spleen cells or whole blood from CBA mice sensitised 56-60 days earlier, on the MST of A-strain skin grafts on recipient CBA mice. - 10 x 107 lymph-node cells transferred; -.--.- 20 x 107 spleen cells transferred; - - - 1 ml whole blood transferred.

did not prejudice graft survival. Once again, at 58 days after donor sensitisation, the transfer of lymph-node cells had failed to reduce the MST of the test grafts, whilst a marked tendency to accelerated graft rejection was again apparent after the transfer of whole blood.

(ii) In view of the periodicity indicated in the above experiment, in the capacity of lymph-node cells and whole blood to transfer sensitivity adoptively for the time-sequence used, the number of days after donor sensitisation at which transfers took place was extended.

Figure 2 shows the results obtained in a second experiment after adoptive transfer of 10 x 107 lymph-node cells, 20 x 107 spleen cells or 1 ml whole blood

230 JUDITH WARREN AND G. GO WL'4ND

into groups of normal CBA mice, 56, 57, 58, 59 and 60 days after the sensitisa- tion of syngeneic donor mice to H-2 incompatible (A-strain) skin.

As in part (i) of this experiment, cell transfers were made 3 days before the normal CBA recipients were test-grafted with A-strain skin.

Figure 2 represents the median survival times of A-strain test grafts on CBA mice that resulted from transfer of donor cells from the three sources at day 56, 57, 58, 59 or 60 after the donor CBA mice had received a sensitising graft of A-strain skin.

The pattern that emerges over-all from fig. 2 is that the MST of test grafts in CBA mice that have received transfers of lymph-node cells is increasing, whilst that in mice that have received transfers of whole blood is decreasing. This is shown clearly by a transfer at 58 days after sensitisation of donors. The MST of test skin grafts in animals given an injection of lymph-node cells is similar to that expected in a " first-set " rejection of A-strain skin by previously unsensi- tised CBA mice. The MST of test grafts in mice that h,ad received 1 ml of whole blood is 7 days.

The positions are again reversed after transfers of blood and lymph-node cells from donors 59 days after sensitisation. Here the MST of test skin grafts after transfers of cells from these two sources show positions relative to one another similar to those that obtained after a 56-day transfer.

Median survival times of test grafts after transfers at 60 days show that the effect of transferred lymph-node cells is again lessening. A MST of 8.0 days as against 9-5 days is apparent in mice that received whole blood in a day-60 transfer, as opposed to a transfer at 59 days after donor grafting.

In transfers of spleen cells from sensitised CBA mice, the points on fig. 2, which represent the median survival times of A-strain test grafts on normal CBA mice, produce when joined a line the pattern of which more closely follows that shown after the adoptive transfer of whole blood.

DISCUSSION The justification for basing the effectiveness of adoptive transfer of homo-

graft sensitivity in mice solely on the ability of transferred cells to influence the rejection time of an allogeneic test skin graft applied to otherwise normal recipients has been set out previously (Warren and Gowland, 1970). In the first experiments described here, a discrepancy was made in the time-schedule of injection and grafting of normal CBA recipients receiving otherwise identical transfers of blood from presensitised syngeneic donors. This was done in order to demonstrate any advantage, or disadvantage, resulting from the transfer of lymphoid cells 3 days before test-grafting, as opposed to transfer carried out 24 hr after A-strain test grafts were applied to recipient CBA mice.

Comparing the results obtained here with those previously reported (Warren and Gowland), regardless of the schedule of injection and grafting, blood was as effective an agent for transfer as were pooled peripheral lymph-node or spleen cells. It should be stressed that 1 ml whole blood in these experiments contains between (5 and 7) x 106 lymphocytes, as compared with the larger numbers of

PERIODIC REDISTRIBUTION OF SENSITISED LYMPHOCYTES 231

cells normally used from lymph-node and spleen sources. This point implies possible selectivity in the distribution of sensitised cells to the peripheral circulation. For instance, a large proportion of the lymphocytes present in the peripheral circulation of the donor at the time of transfer could be specific effector cells directed against A-strain antigens. It is also possible, especially in the cases where transfer of cells preceded test-grafting, that an adoptive transfer of the type envisaged by Turk (1967) could occur. That is, the transferred blood could contain predominantly cells capable of maturation and division in the host, their progeny being responsible for graft destruction.

Turk points out the time-lapse in homograft rejection reactions, compared with delayed hypersensitivity reactions in the skin, which may reach a maximum intensity in 24 hr. Maturation of lymphocytes within the recipient body after transfer is possible, but it should be stressed that cells resembling blast celIs are not found in the peripheral circulation of the mouse. Also, the time taken for a skin homograft to heal into place and for this solid tissue subsequently to break down necessitates a process of longer duration whether it is the injected cells or their progeny that are the effectors of graft destruction.

Injection of transferred material prior to test grafting confers some advan- tages in terms of rejection times, but with certain qualifications. The first is that the advantage in earlier rejection does not equal the 4-day discrepancy between the two regimens. Secondly, there is no advantage from prior injection in transfers at 8 days after donor sensitisation when rejection is not appreciably accelerated, nor at 14 days when both systems are equally effective.

A distinct difference between transfers carried out before and after test grafting was apparent at 56 days after donor sensitisation. Transfer of blood 3 days before grafting confirmed the existing capability of donor cells to transfer sensitivity and also proved much more effective than similar transfers carried out 24 hr after grafting.

In a previous paper (Warren and Gowland) the apparently failing capacity of pooled peripheral lymph-node or spleen cells to transfer sensitivity to skin homografts from A-strain mice 56 days after the original CBA donors were sensitised was noted. It was also shown that the capacity of these original donor mice, to reject a second graft of A-strain skin in an accelerated fashion, was in no way impaired.

As mentioned above, the transfer of whole blood at 56 days after donor sensitisation was effective in accelerating graft rejection only when the blood was transferred 3 days before test-grafting. This observation bore out certain preliminary results of 56-day transfers reported by Warren and Gowland. We decided, therefore, to employ this regimen of transfer in experiments designed to shed some light on the equivocal results of previous 56-day transfer experi- ments involving CBA and A-strain mice. Added to this was the knowledge that in the case of lymph-node and spleen cells, doses (50x 106 lymph-node cells, 100 x 106 spleen cells) previously transferred from 56-day donors were insuffi- cient to cause accelerated rejection. A combination of increased doses of lymph-node or spleen cells, but not whole blood, the transfer of lymphoid cells on each of a number of,successive days, and the modified regimen of injection

232 JUDITH WARREN AND G. GOWL.AND

and grafting produced the results shown in experiment 2. For the purposes of cell transfer, it may be preferable to regard the concept of a sensitised animal at this day (56 days) after receipt of its initial antigenic stimulus, as one in which the cellular response has undergone a shift in intensity and location. These results require further investigation before it can be stated definitely that the pattern that has emerged here is consistent with the points tentatively expressed regarding its occurrence.

In one instance involving transfer of sensitivity from donors 56 days after grafting, accelerated graft breakdown has occurred in mice recipient of whole blood, as opposed to cells from the peripheral lymph-nodes, or spleen, of such donors. It is not known at present whether any response associated with this particular day is fortuitous. It would seem, however, that a shift in intensity has occurred in terms of cells taken from various sources in the mouse. The cells contained in 1 ml of transferred whole blood, though varying slightly in number from one occasion to the other, nevertheless reflect the capacity of 1Ox 107 lymph-node or 20 x 107 spleen cells when used in parallel transfers.

A second point, emphasised by transfers on subsequent days, is an apparent shift in location of cellular activity. This shift does not, however, appear to be permanent, but the results shown in fig. 2 reflect a situation, in lymph-nodes and whole blood, with a periodicity of about 3 days in which the activity of cells from one source in the mouse is the exact reverse of that shown by cells from the other.

Though the elevation and depression of the curve (fig. 2) for MST of CBA test grafts after spleen cell transfer is less marked than for cells from both other sources, it follows more closely the results obtained after transfer of whole blood.

It is possible that the phenomenon shown in experiment 2 is the expression of a selective redistribution of the whole or a part of a population of lymphoid cells with specific affinities for A-strain graft antigens. If this were so then it could be argued: (1) that the retarded effect in spleen is due to a " dilution " of redistributed cells of a type specifically committed to A-strain antigens; (2) that the lymph-nodes (see fig. 2) are the main centre of collection and redistribution of such a population of circulating cells.

As detected by the increase or decrease of median survival times of test homografts, the periodicity of the redistribution is apparently about 3 days. It can only be surmised whether this represents the effects of a continuous redistribution in a preferred direction, leading to a steady accumulation of cells in one location for a period of time, or, alternatively, some kind of mass migration of the relevant cell-population once every 3 days. This latter seems less credible, as it would be difficult to envisage the impetus for such a migration.

A further point is that the nature of the changes in MST of test grafts, bearing in mind the number of cells transferred in 1 ml of whole blood, implies some selectivity of cellular redistribution throughout the whole or part of the envisaged cycle.

The criticism that transfer of such large numbers of lymph-node and spleen cells may in some way predetermine events, may be valid in cases where transfer had been carried out at random, more than 8 wk after donor sensitisation, but a

PERfODIC REDISTRIBUTION OF SENSITISED LYMPHOCYTES 233

fairly definite pattern emerges in transfers carried out over 6 consecutive days. Furthermore, as shown in previous cell-transfer studies, however much the concentration of cells transferred may influence the slope of the resulting curve for MST of test grafts, it does not influence its direction.

SUMMARY

The ability of lymphoid cells, derived from the whole blood, pooled peri- pheral lymph-nodes and spleen of CBA mice presensitised to A-strain antigens, to transfer homograft sensitivity adoptively to unsensitised syngeneic recipients has been examined at various time-intervals following donor sensitisation.

When cell transfers were made to recipient mice 1 day after test-grafting, cells present in whole blood failed to transfer sensitivity when taken from donor mice 8 days after sensitisation, but showed a maximum transfer of sensitivity when taken from 14-day donors. This is a characteristic in common with cells from lymph-nodes and spleen. This effect gradually decreased until, in this test system, transfers of blood from 56-day donors were negative.

When transfers of whole blood from 56-day donors were made to recipients test grafted 3 days after cell-transfer, however, a marked degree of passively transferred homograft sensitivity could be demonstrated.

With this alternative test system and 56-, 57- and 58-day donors in one experiment and 56-,57-, 58-, 59- and 60-day donors in another, it has been shown that when a high degree of sensitivity can be transferred via the cells present in whole blood the cells derived from lymph-nodes show a much reduced effect, and vice versa. The ability of spleen cells to transfer sensitivity seems to mirror that of cells in whole blood rather than lymph-node cells.

We suggest that these results indicate that there is a recirculation and redistribution, between the peripheral blood system and the major lymphoid organs, of a population of lymphoid cells capable of reacting with the sensitising antigen. This is detectable in sensitised animals and shows a periodicity of the order of 3 days.

BILLINGHAM, R. E., AND BRENT, L. 1959. Quantitative studies on tissue transplantation immunity. IV. Induction of tolerance in newborn mice and studies on the phenomenon of runt disease. Phil. Trans. Roy. Soc. B, 242,439.

BILLINGHAM, R. E., AND MEDAWAR, P. B. 1951. The technique of free skin grafting in mam- mals. J. Exp. Riol., 28, 385.

TURK, J. 1967. In Delayed hypersensitivity, North-Holland Research Monographs, Frontiers of Biology, vol. 4, ed. by A. Neuberger and E. L. Tatum, Amsterdam, pp. 93-94.

WARREN, JUDITH, AND GOWLAND, G. 1970. Cell transfer studies on the persistence of homo- graft sensitivity in the mouse. J. Path., 100, 39.

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