F. Fändrich¹, J. Schröder¹, T. Jahnke¹, A.M. Waaga³, M.R. Pawaresch², H.H. Wacker²

¹Dept. of General & Thoracic Surgery and ²Institute of Pathology, University of Kiel, Germany.
³Brigham and Women's Hospital, Harvard Medical School, Boston, MA, U.S.A.

Received 12/2/96; Accepted 12/18/96; On-line 01/01/97

RESULTS

Influence of 15-Deoxyspergualin Immunosuppression on Graft Survival

Treatment of F1-recipient rats with the immunosuppressant 15-deoxyspergualin (15-DOS) for 14 d, following transplantation of semiallogeneic parental (DA) small bowel grafts, significantly prolonged the mean survival from 16 ± 4.5 d to 39 ± 11 d. Figure 1 illustrates the survival curves of transplanted animals which were generated according to the Kaplan-Meier procedure. 15-DOS significantly prolonged the life expectancy of F1-recipients in comparison to untreated animals (p<0.0001, log-rank test). Table 1 summarizes individual survival of all transplanted animals from group one to three.

Figure 1: Survival of syngeneic [F1 » F1], non-treated (=NT) [DA » F1], and 15 deoxyspergualin (=DOS) immunosuppressed recipient rats [DA » F1] is illustrated. Kaplan-Meier analysis was performed to generate the specific survival curves. Statistical analysis with the Savage (Mantel Cox) log-rank test showed a signigicant difference in survival between untreated and immunosuppressed animals, p<0.001 and between semiallogeneic and syngeneic F1-hosts, p<0,0001.

Gross examination of animals suffering from GvHD

Non-immunosuppressed F1-recipient animals subjected to parental donor grafts (group 1) suffered from mild to severe GvHD which started around postoperative day (POD) 8. Typical manifestations of GvHD included weight loss (20% or greater of preoperative body weight), skin rash, cachexia, diarrhea, altered gait, and hunched posture. In contrast to non-immunosuppressed animals, none of the DOS-treated animals or animals which had received syngeneic grafts suffered from these symptoms during the first 14 postoperative days. When 15-DOS administration was stopped after 14 days, all recipient animals started to suffer from signs of graft-versus-host reactions. However, in contrast to untreated F1-hosts, the clinical course of GvH development was substantially prolonged and lethal graft-versus-host disease did not occur before POD 28 and 56, as outlined on table 1.

Histological evaluation

In this study, the distribution, activation and compartmentalization of SLCs and FDCs and their impact on the development of primary and secondary lymph follicles and germinal center formation within donor and recipient mesenteric lymph nodes and spleen were of special interest. The correlation between the extent of GvHD and the frequency and distribution pattern of SLCs and FDCs will now be described in detail.

Sinus Lining Cells (SLCs) are Activated within the Afferent Lymphatic Vessels of Donor and Recipient Mesenteric Lymph Nodes and Host Spleen Following HSBTx

Transplantation of DA parental small bowel grafts into F1 hybrids (LEW x DA) induced a considerable alteration of the distribution pattern of immunocompetent cells within the afferent lymphatic vessels (marginal and interfollicular sinus of lymph nodes and marginal zone of the spleen) and adjacent B cell compartments. Until day 7 following HSBTx, there was a marked increase of KiM9R⁺ SLCs in both, untreated and immunosuppressed recipients in comparison to syngeneic transplanted animals as summarized on figures 2A, B, and C. Following transplantation of parental small bowel into F1-hybrid recipient rats, the frequency of SLCs was highly increased within the marginal sinus of donor and recipient mesenteric lymph nodes and within the marginal zone of the spleen. Figures 5a and 5b depict the activation pattern of SLCs from a non-immunosuppressed animal at POD 7. As illustrated, SLCs surround the primary follicles in both, recipient (Figure 5a) and donor mesenteric lymph nodes (Figure 5b), respectively. In mesenteric lymph nodes of both, donor and recipient origin, SLCs were mainly localized within the afferent lymphatic vessels for the first three days following transplantation. They, then, started to migrate to the interfollicular space and surrounded the cortical follicles. Here, they emerged through the cell web formed by lining cells of the lymphatic sinus and could be visualized immunohistochemically in the center of the primary follicles. Figure 5c shows a SLC stained with KiM9R and coupled to an immunofluorescence dye (cytochrome-3) in the middle of a germinal center of a donor mesenteric lymph node (POD 7). The kinetic of this migration pattern was quite different in the syngeneic strain combination, where the frequency of SLCs within the afferent lymphatic vessels of the spleen and lymph nodes did not exceed the control levels of non-transplanted and age-matched F1-hybrid rats. After syngeneic SBTx, the percentage of SLCs found within the center of primary follicles was significantly less than observed in the semiallogeneic strain combination. In coincidence with the observed invasion of primary follicles by SLCs, activation of the germinal centers ensued and the number of follicular dendritic (KiM4R⁺) cells rose significantly.

In small bowel grafts, SLCs lined up around the peyer's patches and were found sporadically within the lamina propria in animals of groups 1 and 2, whereas syngeneic control animals did not demonstrate such an influx of SLCs during the ultimate postoperative period (POD 3 to 7) (data not shown). In line with the progression of graft-versus-host disease which started to become apparent clinically between days 8 to 12 following SBTx, untreated and immunosuppressed recipient animals revealed quite a different distribution pattern of SLCs within the investigated B cell compartments of mesenteric lymph nodes and spleen. As evident from figure 5d, the frequency of SLCs was significantly diminshed in the spleen of non-immunosuppressed animals at POD 14 following SBTx. At this point of time, an accurate assignment of SLCs to the distinguished B cell compartments of the spleen was impossible, since all germinal centers and their surrounding perifollicular areas had lost their typical picture, caused by a substantial depletion of functional lymphocytes during the course of progressing GvHD. In contrast, animals which had been treated with 15-deoxyspergualin for the first 14 postoperative days did not reveal these detrimental destruction of the various functional lymphatic units within the spleen as demonstrated on figure 5e. Figure 5e shows a normal distribution and density of SLCs which are localized mainly around activated secondary germinal follicles of the spleen. For comparison, figure 5f represents a spleen specimen harvested at POD 14 after syngeneic small bowel transplantation. Figures 2A-C summarize the migration kinetics of SLCs within the described functional B cell units of spleen and mesenteric lymph nodes, respectively. It is evident that non-immunosuppressed animals which suffered from lethal GvHD showed a significant decline of KiM9R⁺ sinus lining cells as graft-versus-host disease progressed. Comparison of donor and recipient mesenteric lymph nodes showed an apparent difference of the activation pattern of SLCs within the marginal sinus of their lymphatic vessels. Whereas the peak of SLC-activation in host mesenteric lymph nodes was reached within the first three postoperative days, the maximum increase of this cell type was delayed until POD 7 in donor mesenteric lymphatic vessels.

Figure 2 A-C

The frequency pattern of KiM9R⁺-stained sinus lining cells [SLCs] was assessed in spleen specimens at various points of time after heterotopic small bowel transplantation. Open bars (non-treated = NT), solid bars (immunosuppressed, 15-deoxyspergualin = DOS), and hatched bars (syngeneic) represent mean ± SD of 4-6 adjacent high power fields examined for each marginal zone of recipient spleens from 2-3 rats. Values are preseted as the number of antigen-positive cells / the number of all visible cells x 100.

Shows the frequency pattern of SLCs within the marginal sinus of recipient mesenteric lymph nodes at various time points after heterotopic small bowel transplantation. Relative numbers were evaluated as described above.

The relative numbers of SLCs from donor mesenteric lymph nodes at various time points after heterotopic small bowel transplantation is indicated, as described above.

The frequency pattern of KiM4R⁺-stained follicular dendritic cells [FDCs] was assessed in spleen specimens at various points of time after heterotopic small bowel transplantation. Open bars (non-treated = NT), solid bars (immunosuppressed, 15-deoxyspergualin = DOS), and hatched bars (syngeneic) represent mean ± SD of 4-6 adjacent high power fields examined for each marginal zone of recipient spleens from 2-3 rats. Values are preseted as the number of antigen-positive cells / the number of all visible cells x 100.

Shows the frequency pattern of FDCs within primary and/or secondary follicles of recipient mesenteric lymph nodes at various time points after heterotopic small bowel transplantation. Relative numbers were evaluated as described above.

The relative numbers of FDCs from donor mesenteric lymph node follicles at various time points after heterotopic small bowel transplantation is indicated, as described above.

Figure A: Flow cytometric analysis was performed from spleens of nontreated (= NT) immunosuppressed (15-deoxyspergualin = DOS) and syngeneically transplanted F1 hosts at various time points after HSBTx. The relative number of B cells stained with mAb Ox-12 was assessed in four individual experiments and is given as mean ± SD. Simultaneously, the relative number of Ox-41⁺-cells (macrophages) [Figure B] and of Ox-19⁺-cells (pan-T cells) [Figure C] is displayed.

The distribution of KiM9R⁺-cells within the marginal and interfollicular sinus of recipient (Figure 5a x 70) and donor mesenteric lymph nodes (Figure 5b x 35), seven days after HSBTx is illustrated. Figure 5c (x 300) shows an immunofluorescent (cytochrome-3) staining of a KiM9R⁺-sinus lining cell which was visible in the middle of a germinal center, 7d after heterotopic small bowel transplantation. Figure 5d (x 35) and Figure 5e (x 35) represent the distribution pattern of SLCs taken from splenic specimens of untreated and immunosuppressed animals at postoperative d 14 after heterotopic small bowel transplantation. As illustrated, untreated animals suffered from a severe damage of the normal compartimentalization within the spleen, whereas SLCs of 15-deoxyspergualin immunosuppressed animals showed the normal distribution pattern of SLCs within the marginal zone and adjacent parafollicular units, at postoperative day 14. Figure 5f (x 35) displays the frequency and distribution pattern of SLCs from a splenic specimen 14 d after syngeneic small bowel transplantation.

The distribution and frequency pattern of KiM4R⁺-follicular dendritic cells within recipient mesenteric lymph nodes is exemplified for a non-immunosuppressed (Figure 6a x 70) and a 15-deoxyspergualin treated animal (Figure 6b x 70), 14 d after heterotopic small bowel transplantation. Whereas immunosuppressed animals displayed the typical web-like structure of FDCs which comprised the whole follicle, no FDCs were found in lymph follicles of animals suffering from the progression of graft-versus-host disease. A normal FDC distribution pattern was associated with a nomral B cell proliferatin pattern as illustrated on Figure 6c ( x 35), a specimen taken from the spleen of a 15-deoxyspergualin-treated animal and stained with KiB1R-mAb, 14 d after small bowel transplantation.

Proliferation of Follicular Dendritic Cells and Subsequent Development of Secondary Follicles was Suppressed as Graft-versus-Host Disease Progressed

Phenotypic characterization of follicular dendritic cells was performed by the use of Ki-M4R, a highly specific monoclonal mouse-anti-rat antibody. Whereas at POD 3, after semiallogeneic HSBTx, the frequency of FDCs was not increased in splenic follicles of recipient animals, (Figure 6a), the number of positively stained Ki-M4R⁺ cells was significantly augmented in recipient mesenteric lymph nodes in comparison to syngeneically transplanted F1-hosts, figure 3B. In non-immunosuppressed animals, this proliferative response of FDCs could be observed in the follicles of donor and recipient mesenteric lymph nodes until POD 7, as illustrated on figure 3B and 3C, respectively. After that point of time, growth and numeric increase of FDCs within germinal centers were inhibited. Interestingly, consistent with the activation pattern and kinetics of SLCs, peak numbers of FDCs were observed at POD 3 in the host mesenteric lymph follicles, whereas a comparable increase of FDCs was first evident at POD 7 in the mesenteric lymph nodes of donor origin. Comparison of the kinetics between SLCs (Figures 2a-c) und FDCs (Figures 3a-c) reveals a strong correlation between both cell types as to their common activation patterns within the various lymphatic tissues. As it was true for SLCs, further progression of GvHD induced a progressive decline of the follicle integrity and concomitant depletion of FDCs in the spleen and mesenteric lymph nodes (Figure 3). In contrast, short-term administration of 15-deoxyspergualin inhibited acute GvHD in all F1 recipient rats for at least the first 4 postoperative weeks. The immunosuppressive action of 15-DOS was mirrored in an unaltered development of primary and secondary germinal follicles with concomitant germinal center formation following transplantation of parental (DA) small bowel. Subsequently, rising numbers of FDCs were observed in the spleen and lymph nodes of donor and recipient origin as depicted on figure 6. Figures 6a and 6b give evidence for the different staining pattern of FDCs which was found in host mesenteric lymph nodes of untreated and immunosuppressed animals, respectively, at POD 14. Whereas no FDCs were detectable in the cortex follicles of untreated animals at that time point, 15-DOS treated animals revealed the normal web-like pattern of FDCs which covered almost all of the transverse section of the depicted follicle. Besides, some smaller clusters of Ki-M4R⁺ cells were visible in the perifollicular area of these lymph nodes. As exemplified in Figure 6c which shows a B cell follicle of the spleen at POD 14 following semiallogeneic small bowel transplantation, the B-cell compartments of 15-deoxyspergualin treated animals showed a normal activation pattern within the spleen, the donor, and the recipient mesenteric lymph nodes which could also be confirmed by flow cytometric analysis of these tissues (next section). Conversely, non-immunosuppressed animals did not show functional B-cell compartments in these lymphatic tissues at that time point, neither morphologically nor immunohistochemically.

Flow Cytometric Analysis of Immunosuppressed and Non-treated F1-Recipient Animals Differed Significantly

To further assess the impact of GvHD-associated immunosuppression on the lymphocyte pattern within mesenteric lymph nodes and spleen, flow cytometric analysis was performed to distinguish the frequency of T and B cells and macrophages during the course of progression of graft-versus-host disease. As exemplified for recipient mesenteric lymph nodes in figures 4a-c, GvH-mediated tissue injuries of these target organs induced a significant elimination of B and T cells as GvHD progressed. Conversely, macrophages were attracted to these sites of destroyed B and T cell compartments of recipient mesenteric lymph nodes (Figures 4a and 4c). At postoperative day 14, there was a significant increase in the number of invading macrophages (Figure 4b) (KiM2R⁺-stained cells) preferentially residing in the cortex and paracortex of the destroyed lymph nodes. Similar findings were noted in the donor mesenteric lymph nodes and spleen, however with a less rapid decline of B and T cells (data not shown).