T H E SIGNIFICANCE OF LYMPHOID REPOPULATION I N THE

GRAFT-VERSUS-HOST REACTION *

Miles Fox Department of Urology, The General Infirmary,

Leeds, England

Previous investigations'" have shown that in the graft-versus-host reac- tion induced in (CBA x C57BL)Fl hybrid mice by intravenous injection of 100 million parental spleen cells of either genotype, very rapid division of donor cells occurred in the host spleen two days after grafting, with resulting predominance of donor mitoses a t that period. When CBA spleen cells were in- jected, donor cell divisionssubsequently declined to 1 per cent by the 13th day, and this low level was still maintained on the 60th day. Splenomegaly, which was maximal 13 days after injection, was thus largely host in origin. After a similar early burst of donor cell activity in the host spleens following in- jection of 100 million C57BL spleen cells, no comparable decline was noted. On the contrary, a continued increase occurred, so that by the 13th day, when spleen enlargement was a t its height, all mitoses seen were donor in origin. Thereafter, no host cell mitoses could be found to the 60th day. The large majority of mice maintained normal activity and weight even though the majority of donor cells was found to remain reactive against the host. The animals were resistant to graft-versus-host reaction following a second injec- tion of C57BL cells, and they also rejected subsequently injected CBA spleen cells.

In the present work, experiments were carried out to investigate the nature of the rapid initial donor cell division, its subsequent diminution in some cases, and the apparently complete donor lymphoid repopulation in others, with pre- vention of a lethal graft-versus-host response. A strain combination in which the graft-versus-host reaction was mild (CBA-+(A x CBA)Fi) was initially examined by the same chromosome marker technique' as in the previous ex- periments. Subsequently, a stronger response was evoked by presensitizing the donor animals with host spleen cells, with the aim of raising the threshold of the transplanted immunologically reactive cells to the tolerance-inducing ef- fects of antigenic overloading, and postponing loss of donor cell stimulation, a train of events suggested by previous work.' ' 3 As lymphoid depletion is pos- sibly a major aetiological factor in graft-versus-host disease, it was hoped that by rapidly correcting the depletion by proliferation of donor cells, the ex- pected lethal response could be prevented, or a t least delayed, particularly if the hosts had been previously sublethally irradiated.

Council for which grateful acknowledgment is made.

3

5

*This work was carried out with the aid of a grant from the British Medical Research

297

298 Annals New York Academy of Sciences

Materials and Methods

Seventeen adult (A x CBA)F1 hybrid mice weighing between 20 and 27 g were injected intravenously with 90 to 100 million spleen cells from CBA(T6) animals of the same sex, using methods as described previously.3 The animals were killed a t intervals over a six-month period, the total body and spleen weights were determined, and cytological analysis of dividing cells in the spleen and marrow was carried out.

The effect of injecting a similar number of donor spleen cells previously sensitized against the host (by three intravenous injections of 30 million spleen cells a t 10 day intervals) was then studied in 20 adult recipients and also in 22 animals which had been given 350 r total body irradiation 18-24 hours pre- viously (230 KV, 15 m.a.; filter 0.5 mm Cu and 1 mm Al; F.S.D. 50 cm; rate 149 r / min).

Chromosome preparations were made by a modification of the drying tech- nique described by Fox and Zeiss.' One hundred chromosome groups were examined from each specimen. A part of each spleen was saved for histological examination and stained with haematoxylin and eosin and with pyronin- methyl green. Total and differential leucocyte counts were carried out every 4-7 days in one-half of the animals and in six control mice which had 350 r total body irradiation. Blood was obtained from a tail vein.

The spleens of eight (A x CBA)FI mice, previously irradiated and in- jected with presensitized spleen cells (as above) were removed after 5 weeks, and a suspension of 100 million cells was injected intravenously into eight adult (A x CBA)F* hybrid mice of the same sex. These animals were killed 8 and 13 days later; their spleens were weighed, examined histologically, and chromosome preparations made from them.

Res u 1 ts

Results are set out in FIGURE 1 and TABLES 1-111. In the unmodified combination, the relative spleen weight did not rise above normal levels. Splenic lymphoid follicles remained intact. They showed a slight reduction of lymphocytes and moderate increase in pyroninophilic cells. The graft-versus- host reaction was mild. Donor cells were found in the spleen from the second day after injection (1 per cent) and increased to 8 per cent on the fourth day, after which the proportion declined to the 13th day; from then it remained 1 per cent to the 60th day. Donor cell multiplication was less marked than in the previously studied stronger combinations (CBA and C57BL - (CBA x C ~ ~ B L ) F I ) . The most rapid fall occurred between the 4th and 8 th days when the total number of dividing donor cells fell from 7,100 to 1,300.

Following injection of presensitized CBA(T6) spleen cells, the proportion of dividing donor cells in the host spleens rose rapidly (FIGURE 1 and TABLE 2). Two days after injection, donor mitoses contributed 22.5 per cent of the dividing cells in the spleen, compared with 1 per cent in the previous group

Fox: Lymphoid Repopulation 299

-DONOR WRMAL HOST NORMAL

---DONOR PRESENSITISEO. HOST NORMAL

-DONOR PRESENSITISED. HOST 350r

E'

9 0 10 30 40 50 60

DAYS

FIGURE 1. Results following intravenous injection of (A X CBA)Fi hybrid mice with 100 million CBA(T6) spleen cells.

and on the 8th day the proportion reached 99 per cent. On the 13th day, all mitoses seen were donor in origin in two out of three animals, and in the third, 67 per cent were donor. By the 21st day the proportion had dropped to 62 per cent in one animal, while in the other two only 5 and 4 per cent donor mitoses were found. On the 60th day 3 per cent mitoses were donor in origin and 1 per

300 Annals New York Academy of Sciences

TABLE 1

SPLEEN CELLS FROM ADULT CBA(T6) MICE INTO ADULT (A x CBA)F1 HYBRIDS

RESULTS FOLLOWING INTRAVENOUS INJECTION OF 90-100 MILLION

Number of preparations Weight loss or gain (% ) Relative spleen weight Total nucleated cells in

spleen (millions)

Total mitoses in spleen (thousands)

Mitotic rate ( % )

Mitoses analyzed Spleen Marrow

Spleen Marrow

Total donor mitoses in spleen (thousands)

Donor mitoses ( W )

Unin- jected

:ontrols

4 - 38

108

86

0.08

0 0

0 0

0

Days

2 4 8 12 21 60

3 3 +1 +1 47 42

107 99

67 110

1.06 0.11

150 250 0 0

2 8 - - 1.3 8.8

3 -1

43

137

27

0.02

300 0

4 - 1.1

3 2 3 -3 +6 +15

42 49 37

105 102 136

52 - 45

0.05 - 0.03

300 200 300 0 200 0

1.5 2 1 - 0 -

0.78 - -

cent on the 180th day. The marrow contained 16 per cent donor dividing cells on the 4th day and 4 per cent on the 8th day and 0.3 per cent on the 180th day. Donor mitoses were not found in the 300 dividing cells examined on the 60th day.

Considerable splenomegaly occurred and it reached a peak on the 8th day. A strong graft-versus-host reaction was confirmed by histological examina- tion of the spleens. Lymphocytes were reduced, particularly in the lymphoid follicles, and there was extensive infiltration with cells of the plasma cell series, starting on the second day. Lymphocytes began to increase in number from the 13th day, a t first in the red pulp and later in the white pulp. Sixty days after injection, the appearances were essentially normal except for a little increase in pyroninophilia. Only one animal died 13 days after injection with marked loss of weight and diarrhoea. A loss of 2-3 gm of body weight was usual by the 12th day, but thereafter rapid weight gain occurred, and by the 21st day the animals had exceeded their original weight and continued to gain (FIGURE 2).

Following intravenous injection of 100 million presensitized CBA(T6) spleen cells into previously sublethally irradiated (350 r) recipients, prepara-

Fox: Lymphoid Repopulation 301

TABLE 2 RESULTS FOLLOWING INTRAVENOUS INJECTION OF 100 MILLION

PRESENSITIZED CBA(T6) SPLEEN CELLS INTO (A x CBA)Fl HYBRID MICE

Number of preparations

Weight loss or gain (% ) Relative spleen weight Total nucleated cells in

spleen (millions) Total mitoses in spleen

(thousands) Mitotic rate (% ) Mitoses analyzed

Spleen Marrow

Spleen Marrow

Tots1 donor mitoses in spleen (thousands)

Donor mitoses ('3%)

Unin- jected

Controls

4

0 38

108

86

0.08

0 0

0 0

0

Days

2 4 8 12 21 60 180

3 2 2 3 4 3 3

0 +4 -6.5 -8 +7 +14.5 +17

58 71 100 59 78 66 36

145 158 121 100 155 123 82

740 820 61 241 719 186 400

1.51 0.52 0.05 0.25 0.46 0.15 0.49

150 200 200 300 300 300 200 300 200 200 100 300 200

23 41 99 89 24 3 1 0 16 4 1 - 0 0.3

170 336 60 213 173 5.7 4

tions were obtained from 22 animals (TABLE 111). In addition, five mice died between the 6th and 16th days with marked wasting and extensive lymphoid depletion in the spleen. The changes were most marked in the lymphoid fol- licles, many of which were almost entirely necrotic, although activity in the red pulp increased as time advanced (FIGURE 3). Weight loss was greater than in the previous group. Animals began to gain weight from the 21st day and ap- peared in good health from about the 30th day to the end of the investigation a t 6 months.

The relative spleen weight diminished 2 days after injection, but thereafter it increased rapidly. Splenomegaly was similar to that in the unirradiated re- cipients except that it lagged behind by 2-4 days. Histological sections in the first 12 days showed extensive destruction and depletion of lymphoid follicles and invasion with pyroninophilic cells. The red pulp became progressively more cellular from the 8th day, and scattered areas of lymphocytes were noted in the depleted lymphoid follicles from the 21st day (FIGURE 4). By the 60th day (FIGURE 5), there were numerous areas of mature lymphoid tissue, but lymphoid follicles were still largely disorganized, and there was a small in-

302 Annals New Y ork Academy of Sciences

Number of preparations

Weight loss or gain (8) Relative spleen weight

TABLE 3 RESULTS FOLLOWING INTRAVENOUS INJECTION O F 10 MILLION

PRESENSITIZED CBA(T6) SPLEEN CELLS INTO (A x CBA)FI IRRADIATED (350 r) HYBRID MICE

3 1 1 -18 -27 -7 28 92 95

Number of preparations

Weight loss or gain ( % )

Relative spleen weight

Total nucleated cel ls in

Total mitoses in spleen

Mitotic ra te ( % ) Mitoses analyzed

spleen (millions)

(thousands)

Spleen Macrow

Spleen Marrow

Total donor mitoses in spleen (thousands)

Donor mitoses ( % )

Unin- jected

:ontrols

4 0

38

108

86

0.08

0 0

0 0

0

Days

2 4 6 8 12 16 21 60 180

2 2 +1 -8

30 48

21 26

8 173

1.04 0.67

200 400 250 550

57 94 11 10

3.4 164

3 2 -8 -11

65 86

80 113

441 697

0.55 0.61

300 200 300 250

100 100 89 100

441 697

4 4 3 -4 +7.5 +15

78 32 38

88 76 87

1759 28 305

1.99 0.04 0.35

400 400 300 400 400 300

100 100 100 100 99.5 100

1759 28 305

crease in the number of pyronin-staining cells. A t six months the appearances were almost normal.

The most striking feature in these mice was the rapid multiplication of donor cells in the spleen and marrow. On the 4th day 94 per cent of mitoses in the spleen were donor in origin, and eight days after injection no host cell mitoses were found. Only donor dividing cells were seen till the end of the in- vestigation on the 180th day. The marrow also appeared completely replaced by donor dividing cells from the 12th day in all except one animal, in which 2 per cent host mitoses were found on the 60th day.

FIGURE 6. shows the changes in the peripheral white cell count in unirra- diated and irradiated hosts which had received presensitized spleen cells and in 6 control animals which has been irradiated only. Initial comparable leucocyte depression occurred in all three groups. An almost equal degree of depression was produced in the polymorph and mononuclear cell series after injection of presensitized cells. The leucocyte count reached normal levels in 4 weeks and

WEIGHT GAIN OR LOSS%

‘30

.20

.10

0

-lo

-20

-30

Fox: Lymphoid Repopulation

CBA(T6) (CBA x A)F: CHANGES N BODY WEIGHT

303

1 I I 1 1 I 0 10 20 30 40 50 60

DAYS

FIGURE 2. Changes in weight following intravenous injection of (CBA x A)Fl hybrid mice with 100 million CBA(T6) spleen cells.

the mononuclear count in two months. Irradiation depressed the mononuclear cells, which normally predominate in the mouse, considerably more than the polymorphs, so that the proportion became reversed. In the irradiated controls, it took approximately two months for the total count to reach normal levels, and at this stage the mononuclear count had not yet fully recovered. When ir- radiated mice were given presensitized spleen cells, the leucocyte count began to rise at the same rate as in the previous groups to the 21st day, but then, in- steadof continuing to increase, it diminished slightly. By the 60th day the mean count had risen, but was still below normal (4,700 per cu mm), with consider- able depression of the mononuclear count, although that was also rising slow- ly. The count was within normal limits on the 180th day.

The subsequent experiment was performed to determine whether or not the transplanted donor cells in the presensitized-irradiated combination re- tained their anti-host activity. Eight (A x CBA)Fl hybrid animals, which received 100 million spleen cells from F1 recipients that had been irradiated and given presensitized spleen cells 5 weeks previously, remained in apparent- ly good health and lost no weight. The highest relative spleen weight between the 9th and 13th day was 48 (range 39 to 48), and there was no histological evidence of a graft-versus-host reaction. A mean of 7 per cent donor dividing cells was found in the spleen (range 2 to 10 per cent) and 2 per cent in the marrow. No graft-versus-host activity was thus produced by the transplanted

304 Annals New York Academy of Sciences

FIGURE 3. (A x CBA)F1 spleen. Sixteen days after intravenous injection of 100 million presensitized CBA(T6) spleen cells. Host previously irradiated-350 r. Animal died. (H and E x 50).

cells a t a period when maximum action would have been expected if the cells had retained anti-host activity.

Discussion

Most workers believe that the graft-versus-host reaction results from a n immune reaction of the injected cells or their descendants against the h o ~ t . ~ - l ' The evidence, however, tha t tissues other than those of lymphoid or erythroid origin are affected is not conclusive. The resulting lymphoid and marrow de- pletion may lead to death from intercurrent infection in a proportion of cases, but in many others no evidence of infection is found. The notion tha t lymph- oid depletion is in some unexplained manner the key factor in the pathogenesis of graft-versus-host disease is strongly supported by recent experimental evidence.

In the present experiments, the initial and early changes in the graft- versus-host reaction were those of destruction of the lymphoid tissues and leukopaenia. During this stage, marked loss of weight occurred in the acceler- ated form and some animals died. Recovery was concurrent with an increase of the peripheral white cell count and reappearance of lymphoid tissue in the spleen. A strong reaction produced extensive, destructive changes, particularly

13

1 4 - 1 7

Fox: Lymphoid Repopulation 305

FIGURE 4. (A x CBA)F, spleen. Appearance 25 days after intravenous injection of 100 million presensitized CBA(T6) spleen cells. Host previously irradiated-350 r (H. and E. x 50).

in the lymphoid follicles. In the unmodified combination, host cell lymphoid proliferation rapidly corrected the mild and early lymphoid depletion.

Following injection of lymphoid tissue presensitized against the host, ac- celerated graft-versus-host disease has usually been evoked in parental to FI hybrid c~rnbina t ions '~ ' '~ and mortality has been still further increased in sub- lethally irradiated F1 I n the strain combination studied in the present experiments, mortality was not appreciably increased over a 6-month period of observation, when spleen cells presensitized against the host spleens were injected. The majority of animals also survived and appeared in good health if, in addition, sublethal irradiation was given prior to injection. Fol- lowing early destructive changes in the spleen, there was histological evidence of lymphoid repopulation which must have been mainly, if not entirely, donor in origin, in view of the tremendous continued predominance and in most cases

306 Annals New Y ork Academy of Sciences

FIGURE 5. (A x CBA)Fl spleen. Appearance 60 days after intravenous injection of 100 million presensitized CBA(T6) spleen cells. Host previously irradiated-350 r. (H. and E. x 50).

complete replacement by dividing donor cells in the spleen and marrow. Con- sequently, it is also most probable that the cells constituting the recovering peripheral leucocyte count are unlikely to have originated from other than donor stem cells.

Soon after their first contact with the host antigens, the transplanted lymphoid cells are sensitized and stimulated to proliferate. This coincides with the rapid increase in donor cell mitoses, particularly in the presensitized groups. It has been postulated, but never satisfactorily demonstrated, tha t immunologically reactive cells are liable to perish in the course of their reac- tion with antigen, the so-called “allergic death.”22’23 In this event, one would expect a rapid diminution of donor cell activity after an initial burst of divi- sion, following injection of presensitized cells. The finding tha t donor cell division was in fact increased and markedly prolonged, together with previous evidence, strengthens the view that the fall in donor activity, when it oc- curs, is due to acquisition of tolerance by the donor cells of the host.

The large amount of antigen with which the transplanted immunologically reactive cells come in contact will initially stimulate them and may well sub- sequently induce immunological paralysis.25 Presensitized cells, because of

2 . 3 . 6 . 2 4

Fox: Lymphoid Repopulation 307

I I I 1

I 1

0 10 20 30 40 50 60 DAYS

FIGURE 6. Changes in peripheral leucocyte count following intravenous injection of (A x CBA)Fi hybrid mice with 100 million CBA(T6) spleen cells.

their increased resistance to the effect of antigenic overloading, retain their immunological independence for a longer period and during tha t time continue to be stimulated by the host antigens. On acquiring tolerance, the donor cells probably come under the normal control of the recipient’s homeostatic mech- anisms, and they will then divide at the same constant rate as similar host cells. The present findings are consistent with these views.

Donor lymphoid repopulation permitted survival if extensive destruction had initially occurred. Following injection of presensitized cells, the increased

26

308 Annals New York Academy of Sciences

destructive effect on host lymphoid tissues, even if supplemented by irradia- tion, was counter-balanced by the raised division rate of the donor cells, which in most cases soon replenished the host lymphoid tissues and returned the ani- mals to apparently normal health. If, for some reason, the repopulation failed, the mice continued to lose weight and died, with typical signs of graft-versus- host disease. In the initial stages a t least, it did not appear to matter whether the repopulating lymphoid cells were host or donor in origin, providing the animal was not left depleted. The results indicate tha t the transplanted donor cells eventually become tolerant of the host antigens even though they had produced complete, or near complete, repopulation of the host spleen and bone marrow.

Summary

A graft-versus-host reaction was produced in adult (A x CBA)F1 hybrid mice by intravenous injection of CBA spleen cells. The fate of the donor cells in the host spleen and bone marrow was studied by the T 6 chromosome mar- ker technique. A stronger response was then evoked by presensitizing the donor animals against the hosts and also sublethally irradiating the hosts. The findings were correlated with histological appearances and peripheral leucocyte counts.

Initial donor cell division in the spleen was observed in all the groups. I t declined rapidly, except in irradiated recipients in which it continued till only donor mitoses were seen in the spleen and bone marrow from the 12th day to the end of the investigation on the 180th day. Signs of graft-versus-host dis- ease, when they occurred (usually in the 2nd and 3rd week) were associated with severe lymphoid depletion. In most cases, rapid lymphoid repopulation quickly replenished the host lymphoid tissues and returned the animals to ap- parently normal health. I n the initial stages a t least, it did not appear to matter whether the repopulating lymphoid cells were host or donor in origin, providing the animal was not left depleted. Evidence indicated tha t the trans- planted donor cells became tolerant of host antigens.

Acknowledgment

I wish to thank Miss Jacqueline Muir for technical assistance.

References

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