Z. Immun.-Forsch. vol. 155, pp. 240-247 (1979)

Immunology Research Center!), Institut Armand-Frappier, Department of Pathology, Montreal Children's Hospital2) and Department of Physiology3), McGill University, Montreal, Quebec, Canada

Thymic Stromal Alterations in Mice Undergoing a Chronic Graft-Versus-Host Reaction.

Received July 31,1978 . Accepted October 10, 1978

Abstract

Graft-versus-host induced immunosuppression has previously been shown to be accom­panied by severe morphological changes in the thymus; furthermore chronic GvH could become acute by grafting a normal syngeneic thymus, suggesting a functional defect in the autologous thymus.

In this work, we monitored the changes occuring in two biologically active thymic stromal fractions during a state of chronic GvH reaction. It was thus observed that a soluble thymic factor (STF), normally found in the reticuloepithelial cells, was lost, and that an insoluble thymic fraction (ITF) found in a double basement membrane surrounding medullary blood vessels, became markedly hypertrophied. These changes are interpreted as being possibly related to the state of immunosuppression by interfering with normal T cell differentiation and traffic through the thymus.

Introduction

Graft-versus-host (GvH) reaction has been shown to suppress both cell­mediated and humoral immune responses (1-4). By restoration experi­ments, it has further been documented that early suppression of the humoral immune response was due to inhibition of T helper function (4-6), presumably brought about by an excess of adherent cells (7). Suppressor activity of GvH spleens has also been demonstrated by their ability to inhibit antibody production when added to normal spleen cells (5, 8-11). This particular type of suppression, however, could not be demonstrated in the spleens of animals after day 21 (11) even though the GvH animals, once suppressed, remained so indefinitely (4, 11). It seems therefore that sup­pressor cell activity after day 21 cannot be completely ruled out; however SHAND's results (11) and recent observations from this laboratory (LAPP et al.: in preparation) tend to show that such an activity is markedly reduced. Earlier studies showed that when syngeneic thymuses were transplanted into animals undergoing a GvH reaction, the intensity of the reaction was significantly increased, resulting in a rapid death of the animals (12). This suggested that the chronic GvH reaction has provoked a deficiency of the

Abbreviations: GvH = graft versus host; STF = soluble thymic factor; ITF = insoluble thymic fraction; PFC = plaque forming cell.

Thymic Stromal Alterations in GvH . 241

autologous thymus which could be corrected by the transplant of a normal thymus. This hypothesis of a thymic deficiency was further substantiated in a morphological study which showed that GvH reactions induced a form of thymic involution characterized by lymphoid depletion, obliteration of the cortico-medullary junction, a loss of Hassall's corpuscles and medullary epithelial cell injury (13).

In an attempt to relate the above functional and morphological data, we monitored throughout the present study, two antigenically, morphologi­cally and functionally distinct thymus stromal fractions described previ­ously (14): a soluble thymic factor (STF) and an insoluble thymic fraction (ITF). STF (MW = 3000) proved to be similar to other thymic hormone preparations (15) and was shown to participate in T cell maturation by imparting Thy-l antigens on T cell precursors, by conferring resistance to corticosteroids on thymocytes and by stimulating helper T function (unpublished results); it has been localized in the cytoplasm of reticuloepithelial cells (16); ITF, on the other hand, when injected intraperitoneally, was shown to provoke a temporary increase in the number of large outer-cortical thymocytes (17), probably by stimulating the migration of bone-marrow precursor cells into the thymus (18). ITF was found to contain an antigenically specific component localized in the membranes surrounding medullary blood vessels (19).

In this work, we show that in the thymuses of mice undergoing a chronic GvH reaction there is in fact a marked STF depletion accompanied by a hypertrophy of ITF-positive membranes.

Materials and Methods

GvH reaction

The method used to induce GvH reactions was described previously (6). It consisted essentially of injecting adult CBA X AFI mice intravenously with 75 X 1(1) A strain spleen and lymph node cells. Since A and CBA share the K region of the H-2 complex, the GvH reaction induced was of the chronic type.

Plaque forming cells

Plaque forming cells (PFC's) were assayed using a modification of the technique of CUNNINGHAM and SZENBERG (20, 21). Sheep red blood cells were used as the antigen.

Antibodies

Rabbit anti-STF and goat anti-ITF antisera used for immunofluorescence were prepared and rendered specific as described previously (14, 22). A double sandwich technique was used throughout with rhodamine conjugated guinea-pig anti-goat immunoglobulin and fluorescein­conjugated goat anti-rabbit immunoglobulin. The staining intensity was graded from - (nega­tive) to + + + + (strongly positive).

Experimental groups

The animals inoculated on day 0, were divided into three groups and studied at sequential intervals from day 7 to day 71. The first group was used for splenic PFC response, the second for light microscopy and the third for immunofluorescence with antibodies to STF and ITF.

242 . POTWOROWSKI, SEEMAYER, BOLANDE, and LAPP

Results

PFC response

Immunosuppression was demonstrable in experimental animals at day 7 and 35 post GvH reaction induction (fable 1). These results confinn previous studies which revealed that once animals become immunode­pressed following a GvH reaction, they do not recover their ability to form PFC's (4).

Histological observation

The principal histological features of the thymuses of animals undergoing a GvH reaction were similar to those previously described (13). In addition, from day 35 onwards, medullary perivascular membranes which hitherto had been thin and double (Fig. 1), became progressively thickened and prominent (Fig. 2).

Changes in STF and ITF

STF within reticuloepithelial cells of experimental mice were stained as intensively as in controls on days 7 (fable 2, Fig. 3) and 14, although some

Table 1. GvH induced immunosuppression

Days post-GvH

Number of PFC's X llY/spleen Nonnal mice GvH mice

7

120.5 ±21.0 1.73± 1.6

35

118.5±19.2 0.3± 0.1

Fig. 1. Histological section of a nonnal mouse thymus stained for reticulin, showing the thin double basement membrane surrounding a medullary blood vessel (approx. X 400).

Thymic Stromal Alterations in GvH . 243

Fig. 2. Similar preparation to Fig. 1 of a thymus from a mouse 37 days post-GvH induction. Note the thickened and disorganized perivascular basement membrane (approx. X 400).

Fig. 3. Immunofluorescent staining with anti-STF of a mouse thymus seven days post-GvH induction. The cytoplasm of reticuloepithelial cells is brightly stained, indicating the presence of STF (approx. X 400).

Table 2. Reticuloepithelial cell staining with anti-STF

Days post-GvH

Immunofluorescence staining intensity*)

7 14 16

++++ ++++ ++

28 36 42 56 71

,:-) The staining of control non-GvH animals was at + + + to + + + + intensity through­out the experiment.

244 . POTWOROWSKI, SEEMAYER, BOLANDE, and LAPP

Fig. 4. Similar preparation to Fig. 3 of a mouse thymus, 36 days post-GvH induction. Note the absence of staining with anti-.5TF (approx. X 400).

disorganization in the staining pattern was evident on the latter day. On day 16, the staining intensity of the majority of reticuloepithelial cells was markedly reduced. On day 28, although rare identifiable cells displayed some fluorescence, the reaction was generally negative. From day 36 (Fig. 4) until the conclusion of the experiment, no reticuloepithelial cells were stained with anti-STF serum. It was not possible with this technique to determine whether unstained reticuloepithelial cells were present or whether they were destroyed.

Fig. 5. Immunofluorescent staining with anti-ITF of a mouse thymus seven days post-GvH induction. Note the similarity of pattern with the reticulin stain in Fig. 1. Reticulin-positive basement membranes in other organs however did not stain with anti-ITF (approx. X 400).

Thymic Stromal Alterations in GvH . 245

Fig. 6. Similar preparation to Fig. 5 of a mouse thymus 35 days post-GvH induction. Note the thickened and disorganized ITF-positive membrane (approx. X 400).

The staining by anti-STF of perivascular medullary membranes remained strong throughout the experiment (Table 3). Until day 28, the membranes appeared as thin, double and brightly stained (Fig. 5). The appearance of marked pericapillary fluorescence seemed to coincide with the light micros­copic observation of the thickening of perivascular medullary membranes. With the thickening of the perivascular membranes, it became progressively evident that the space between the two membranes became obliterated (Fig. 6). In control animals the ITF-positive membranes remained thin, double and brightly stained until the end of the experiment.

Discussion

The principal changes in the thymic stroma, following the induction of GvH reaction are thickening of ITF-positive perivascular membranes and a loss of STF staining in reticuloepithelial cells.

Table 3. Perivascular membrane staining with anti-ITF

Days post-GvH 14 16 28 36 56 72

Immunofluorescence staining intensity") +++1++++ ++++ ++++ ++++ +++ +++ Staining pattern"*) PV PV PV(S) PV(S) PV(S) PV

and and and PC PC PC

*) The staining in control non-GvH animals was of + + + to + + + + intensity through­out the experiment and was perivascular in pattern.

"*) PV: perivascular; PC: pericapillary; S: swollen.

246 . POTWOROWSKl, SEEMAYER, BOLANDE, and LAPP

Although thickening of basement membrane is a normal reaction to injury and a consequence of ageing, the presently described thickening of the medullary perivascular membranes may have more specific functional significance. If the ITF-positive membranes are in some way related to the blood-thymus barrier, their thickening would be expected to alter lym­phoid cell traffic into and perhaps out of the gland. The precise conse­quences of this alteration are still unknown, but the recent description of B cells in the GvH thymus (23) would suggest that the selectivity towards the cells entering that organ might be reduced.

The marked decrease of STF-containing cells probably reflects a loss of thymic hormone(s) which could lead to a blockage of T cell differentiation and be responsible for the permanent immunosuppression observed in animals undergoing chronic GvH, as postulated by GRUSHKA and LAPP (12). This blockage of T cell differentiation has been further documented by SEDDIK et al. (24) who showed that GvH thymocytes were incapable of reconstituting immunocompetence in syngeneic adult thymectomized, irradiated, and bone marrow reconstituted mice.

It is likely that there is more than one mechanism responsible for immunosuppression in GvH. In the present paper we have shown that thymic stromal alterations are associated with the appearance of the state of the permanent immunosuppression and may play a role in its induction.

Acknowledgements

The authors wish to thank Misses Claire Beauchemin, Ailsa Lee Foon, Anne Rabourdin, and Rosemarie Siegrist for their technical help. This work was supported by grants from the Medical Research Council of Canada and from the National Cancer Institute of Canada.

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Dr. E. F. POTWOROWSKI, Immunology Research Center, Institut Armand-Frappier, 531 Des Prairies Blvd, P. O. Box 100, Laval, Quebec, Canada H7N 4Z3