¹Institute of Pathology, University of Cologne, Germany

²Department of Bone Marrow Transplantation, University of Essen, Essen, Germany

³Institutes of Pathology, Universities of Cologne and Essen, Essen, Germany

Correspondence to: Dr J Thiele, Institute of Pathology, University of Cologne, Joseph-Stelzmannstr.9, D-50924 Cologne, Germany

Following bone marrow transplantation (BMT) investigations on the recovery of the B and T lymphocyte populations have focused on the peripheral blood and only marginally regard the bone marrow. An immunohistochemical and morphometric study was performed on 352 trephine biopsies derived from 123 patients with chronic myelogenous leukemia (CML) at standardized endpoints before and after allogeneic BMT and compared to a control group. The purpose of this investigation was to quantify the B-CD20⁺ and T-CD45RO⁺ lymphocyte subsets and to determine possible relationships with the occurrence of acute and chronic GVHD. Moreover, we studied the dynamics of lymphocyte repopulation in the post-transplant period, correlations with the total peripheral lymphocyte count and differences associated with sibling vs alternate HLA-compatible (unmanipulated) marrow grafts. Morphometric analysis revealed a very fast regeneration of CD45RO⁺ and CD20⁺ marrow lymphocytes in the first 2 weeks following BMT. In less than 2 months, in most patients, the post-transplant quantity of lymphocytes was comparable to that of the normal bone marrow. This finding was opposed to the profound depression of the absolute lymphocyte count in the peripheral blood. No relevant relationships could be calculated between engraftment status and the lymphocyte repopulation in the bone marrow. On the other hand, significant correlations were calculable between the development of (chronic and acute) GVHD including severity with the number of CD45RO⁺ lymphocytes. In non-related graft constellations a more frequent evolution of acute grade III + IV GVHD was detectable. This complication was accompanied by an increased quantity of CD45RO⁺ lymphocytes in the marrow. Bone Marrow Transplantation (2001) 27, 425-431.

lymphocyte subsets (CD45RO⁺, CD20⁺); bone marrow transplantation; CML; GVHD; trephine biopsies; immunohistochemistry

Allogeneic bone marrow transplantation (BMT) has been established as the most effective therapy in Philadelphia chromosome-positive (Ph⁺) chronic myelogenous leukemia (CML) and following successful engraftment many patients have a long-term, disease-free survival.^{1,2,3,4,5,6,7,8,9,10,11} All patients treated by myeloablative regimens and BMT undergo a process of immune reconstitution over a 6-12 month period involving various aspects of this complex system.^{12,13,14,15,16} Regeneration of normal immunocompetence can be significantly impaired by the occurrence of acute and chronic graft-versus host disease (GVHD). Although prophylactic regimens are used, about 40% of patients receiving matched sibling allografts and up to 70% of patients with compatible unrelated donor allografts develop GVHD which can lead to increased morbidity.^2,6,8,9 In the past years, a wealth of data has been accumulated suggesting that this reaction is mediated by T lymphocytes that are derived from the bone marrow graft.^17,18,19,20 Regarding this well-known feature which exerts an important impact on the outcome of BMT, there is an impressive body of data available about the recovery of lymphocyte subsets in the post-graft period. However, until now investigations have focused on the appearance of distinctive lymphocyte populations in the peripheral blood^{12,13,14,15,16,21,22,23,24,25} and rarely consider the bone marrow.²⁶ These studies were in keeping with the finding that opposed to the different T lymphocyte fractions, B lymphocyte regeneration is severely impaired for months to years after BMT, particularly in patients with chronic GVHD.^{21,22,23,24,25,26} In this context the question arises, whether and to what extent the repopulation of T and B lymphocytes in the peripheral blood is mirrored by a corresponding quantity in the bone marrow. Additionally, the problem of possible relationships between these features with the development of acute and chronic GVHD has to be addressed.

For this reason, the purpose of the present immunohistochemical and morphometric study was to quantify B-CD20⁺ and T-CD45RO⁺ lymphocyte subsets in Ph⁺ CML bone marrow before and at short intervals after BMT in order: (1) to evaluate their impact on acute and chronic GVHD; (2) to determine possible correlations between engraftment parameters or the absolute number of lymphocytes in the peripheral blood; and (3) to elucidate differences between HLA-compatible related (sibling) and unrelated (alternate) donors.

Patients and methods

Patients

This study consisted of a retrospective analysis of 123 patients presenting with first chronic (stable) phase of Ph⁺ CML between May 1992 and December 1995. In the course of allogeneic BMT patients received (unmanipulated) marrow grafts from HLA-compatible family or alternate donors at a single referral transplantation center following standard procedures.³ Details regarding history, clinical findings and treatment characteristics are summarized in Table 1. The endpoint of hematological reconstitution with normal graft function was assumed if patients achieved transplant-derived absolute neutrophil counts greater than 0.5 ´ 10⁹/l and self-sustaining thrombocyte counts greater than 20.0 ´ 10⁹/l until day 30 following BMT (day 0). Delayed engraftment was diagnosed if these thresholds were not reached until day 30 and graft failure was established if cell counts were never present at standardized endpoints (Table 2).^1,2,3,7,10 Pretransplant interferon-alpha 2b (IFN-), busulfan (BU) and hydroxyurea (HU) treatment was generally carried out using conventional dosages and partially also combinations of these agents. Following BMT successful engraftment according to the above criteria was established in 93 patients (75.6 %) and delayed hematopoietic reconstitution in 30 patients (24.4 %). In two patients primary failure to engraft could be recognized. Chronic GVHD involving skin, gastrointestinal tract and liver developed in 68 (55.3%) patients and acute GVHD in 76 (61.8%) patients (Table 1). Moreover, following informed consent, a randomly selected control group of 35 age- and sex-matched patients on whom a trephine biopsy was performed with the question of osteopathology (slight to moderate osteoporosis) and normal bone marrow findings was entered into this study.

Bone marrow biopsies

A total of 352 representative bone marrow trephine biopsies (mean size 17.5 ´ 1.8 mm) were obtained from the posterior iliac crest at standardized intervals during the course of BMT (Table 2). Fixation of samples was carried out in a low concentration phosphate-buffered aldehyde solution (2-3%) for 12-48 h. Further processing included decalcification for 3-4 days in 10% buffered ethylene-diamine tetra-acetic acid (EDTA), pH 7.2 and paraffin embedding. For a specific staining of lymphocyte subsets appropriate monoclonal antibodies were selected to detect the following epitopes: CD20 (L26) for the identification of B lymphocytes²⁷ and CD45R0 (UCHL-1) to characterize T lymphocytes.²⁸ Regarding UCHL-1 it has been recognized that this antibody shows cross-reactivity with some myeloid and monocytic cells in frozen-section material and smears.^29,30,31 However, this non-specific staining is significantly reduced in marrow samples after mild decalcification by edetic acid, which was applied in this study.^32,33 Moreover, for a comparative evaluation of T lymphocyte subsets we performed a pilot study on 25 randomly selected bone marrow trephines at diagnosis of CML using the monoclonal antibody CD3 which revealed a 95-97% congruent staining reaction. Regarding the very few data that exist on immunohistochemical quantifications of T lymphocyte subsets in paraffin-embedded normal and, in particular, CML bone marrow biopsies these involved exclusively UCHL-1.^32,33 Therefore, we focused on CD45RO⁺ reactivity for a better comparison with previous findings. The monoclonal antibodies and other reagents were purchased from Dako-Diagnostica (Hamburg, Germany). Details of staining procedures (APAAP method) were reported in detail in previous communications.^27,34 By following this procedure in 82 patients, paired representative pre- and post-graft samples were evaluated and in an additional cohort of 41 patients only CD45RO⁺ reactivity was determined (Table 2).

Morphometry

Following immunostaining, morphometric analysis was performed by two manual optic planimeters (MOP-A-MO1-Kontron and VIDAS-Zeiss-Kontron) with a standard program set (Kontron software) on large trephine biopsies with an artefact-free mean marrow area of 8.3 ± 2.5 mm². Frequency of diffusely scattered CD20⁺ and CD45R0⁺ lymphocytes per mm² was determined at 500´ magnification by considering not only the total marrow including adipose tissue, but also the evaluable hematopoietic area of the trephine biopsy. Moreover, the incidence and composition of nodular lymphoid infiltrates was additionally evaluated. Since a minor cross-reactivity of the monoclonal antibody UCHL-1 with myeloid and histiocytic cells may not be totally excluded, only those cells identified as CD45RO⁺ which also showed the cytological characteristics of lymphocytes were scored. Statistical evaluation included a nonparametric Kruskal-Wallis test to determine differences in lymphocyte frequencies at standardized endpoints and between defined subsets of patients (Table 2). Any P values quoted are two-sided.

Results

Following immunostaining CD45RO⁺ and CD20⁺ lymphocytes were usually arranged in a randomly dispersed pattern (Figure 1a, c) or infrequently occurred as focal dense aggregates or nodules within the bone marrow (Figure 1b, d). The few lymphoid nodules (Table 2) displayed a predominance of CD45RO⁺ lymphocytes (Figure 1d). At diagnosis in the pretreatment specimens, the ratio between both lymphocyte subsets was approximately 3:1 (Table 2). Morphometric analysis at standardized endpoints of the pre- and post-transplant period failed to reveal significant differences in the quantity of these lymphocytes calculated per total bone marrow area and per hematopoietic tissue as well (Table 2). However, this has to be compared to the normal bone marrow cellularity with an about 53% amount of adipose tissue in an age-matched contol group. Corresponding normal values for CD20⁺ lymphocytes and for the CD45RO⁺ population are listed in Table 2. With respect to these data, the CML bone marrow was characterized by an approximately 35% overall decrease in the B and T lymphocyte populations per hematopoietic cell area (Table 2). Considering clinical standards for engraftment at day 30 after BMT relevant data were available for a small cohort of 25 patients who had a bone marrow examination performed between days 9 and 29. Corresponding values for CD45RO⁺ lymphocytes were 13 ± 22 (19 ± 26) and for the CD20⁺ subset 3 ± 13 (4 ± 14) per mm² total marrow (hematopoiesis). Despite the high standard deviation these data point to a speedy post-transplant reconstitution of the lymphocyte population that became especially apparent in patients with several sequential post-graft biopsies (Figure 2a, b). In this context no significant correlations could be calculated between the number of marrow lymphocytes and the generally accepted engraftment parameters at day 30 after BMT. Peripheral total absolute lymphocyte counts were conspicuously decreased in the post-transplant period (Table 2), but did not show any significant relationships with the total amount of (CD45RO⁺ plus CD20⁺) marrow lymphocytes at the different endpoints. On the other hand, chronic extensive and, in particular, severe acute GVHD (grades III + IV) were significantly associated with an increased quantity of CD45RO⁺ lymphocytes in the bone marrow (Table 3). These changes were not conspicuously expressed in limited chronic or grade I + II acute GVHD and the quantity of CD20⁺ lymphocytes displayed no significant differences. In the small number of relevant patients this feature was only marginally expressed for the CD20⁺ lymphocyte population. Differentiation between related and non-related donor marrow grafts revealed significant (P < 0.001) differences, because acute GVHD developed in 44% sibling opposed to 87% alternate constellations. Moreover a grade III + IV disorder occurred in 12% related contrasted to the 26% non-sibling donors (P < 0.001). This striking disparity of occurrence was accompanied by a tendency for a higher incidence of CD45RO⁺ lymphocytes in non-related graft situations shortly after BMT with a frequency of 23 ± 17 (33 ± 21) contrasting 16 ± 17 (23 ± 22) per mm² marrow (hematopoiesis) in family donors. Due to the small number of patients with chronic GVHD more than 100 days after BMT, possible associations with the CD20⁺ lymphocyte populations could not be determined. Concerning the CMV status of recipient patient and donor, no significant correlations could be evaluated with regard to occurrence of acute GVHD or number of CD45RO⁺ lymphocytes in the post-transplant period.

Discussion

Many studies have been carried out over the last two decades seeking to understand and potentially modify the complex patterns of functional and phenotypic immune reconstitution after BMT.^{12,13,14,15,16} However, the focus of these reports was to extend our knowledge about the reappearance of certain lymphocyte subsets in the peripheral blood.^{15,16,21,22,23,24,25,35} Although the early post-transplant period is complicated by infections characteristic of cell-mediated immune deficiency (ie CMV, varicella zoster, Epstein-Barr virus, herpes simplex virus, Pneumocystitis carinii) which are prominent causes for increased morbidity and mortality,^6,7,8,9,14 the quantity and differentiation of the lymphocyte population in the engrafted bone marrow has gained little attention.^26,36 According to absolute blood lymphocyte counts (Table 2), our findings are in keeping with previous investigations that repopulation of the peripheral blood by T and B lymphocytes is a protracted process that may be accomplished only after 8 to 12 months after BMT.^{12,13,14,15,16,21,22,23,24,25} Unfortunately, because the peripheral T cell population was not determined exactly at the endpoints of bone marrow biopsies (Table 2) a direct comparison was not possible. However, in a previous investigation on immune reconstitution after BMT a small number of CML patients also involved in this study was evaluated.¹⁵ From these data it may be derived that contrasting our findings on the quantity of CD45RO⁺ lymphocytes in the marrow, peripheral blood counts of naive and memory T helper/inducer cells (CD3⁺, CD4⁺, CD45RA⁺, CD45RO⁺) were long term low post transplant, whereas the levels of CD8⁺ suppressor/cytotoxic T lymphocytes and NK cells returned quickly to the reference range.¹⁵

In the pertinent literature, there are only very few comprehensive histological descriptions of lymphocyte subsets in normal and pathologically altered bone marrow. The obvious lack of relevant data may be due to a reluctance of clinicians to perform trephine biopsies following BMT. This shortcoming is probably responsible for a conflict of opinion concerning the exact amount of lymphocyte subsets in the CML bone marrow.³⁷ In patients without hematological disorders, according to elaborate immunohistochemical evaluations of frozen sections with a battery of monoclonal antibodies, the ratio between T and B lymphocytes was about 4:1.^29,30 Application of the monoclonal antibodies UCHL-1 and L26 on decalcified, paraffin-embedded bone marrow sections showed a similar ratio of CD45RO⁺ and CD20⁺ lymphocyte ranging between 3 and 4:1.^33,37 In this context, the validity of UCHL-1 (CD45RO⁺) staining of T lymphocytes has to be addressed. Opposed to CD3 this antibody has been found to exert some cross-reactivity with myeloid cells^28,31 which, however, is neutralized after mild decalcification by chelating agents as in our study.³² Moreover, the significance of the CD45RO epitope has to be discussed. It may be speculated that it is not expressed on all T lymphocytes, nor can it be considered as a marker of antigen-experienced cells, since CD45 RA reversion occurs in CD8⁺ T lymphocytes.^21,24

Regarding the CML bone marrow at diagnosis an approximately 3:1 ratio in the quantity of CD45RO⁺ and CD20⁺ lymphocytes was determined and therefore no significant differences compared to our control specimens could be established.^33,37 In addition to this relative value which was also calculable in our cohort of patients with pretreatment examinations (Table 2), absolute frequencies have to be assessed by regarding the total marrow and cellularity. These reference values are significantly different, because in an age-matched normal population hematopoiesis involves 47% of the bone marrow space vs about 98% in CML patients.³⁷ By taking this feature into account in the CML bone marrow there is an overall 40-50% regression in the total lymphocyte population detectable. The fact that T lymphocytes are significantly decreased in relation to hematopoietic cells deserves special attention. It is tempting to speculate that a general breakdown in immune homeostosis may be present in this disorder, generating a suppression of immunocompetent cells and thus creating a situation which may be supportive for the expansion of the leukemic clone.^38,39 Considering the situation in the normal bone marrow³⁷ in this study less than 2 months after BMT, no significant differences in the quantity of the CD45RO⁺ and CD20⁺ lymphocyte subsets calculated per area of hematopoiesis (cellularity) was determined. This feature implicates a very rapid reconstitution of certain lymphocyte subsets including precursors³⁶ in the marrow opposed to their scarcity in the peripheral blood.

A wealth of data have been gathered about chronic and acute GVHD as major complications, in particular, of unrelated donor BMT procedures in CML, which was also found in our series.^{2,3,7,8,9,11,17} This problem is caused by mature T lymphocytes in the donor bone marrow which facilitate and mediate an antitumor effect to reduce leukemic relapse.^18,19,20,40 Studies on the location of affected organs reveal that the majority of lymphocytes that infiltrate the epithelial layers or epidermis were T lymphocytes.^41,42,43,44 By using appropriate monoclonal antibodies on frozen section material it has been shown, that the lymphocytic lesions from involved tissues were almost exclusively derived from the /-expressing cell fraction and consisted of the memory cell subset of T lymphocytes (CD45RO⁺).⁴⁴ Our finding of a striking and very early increase in quantity of CD45RO⁺ lymphocytes in the post-graft bone marrow in non-sibling patients with severe acute GVHD is assumed to reflect this situation. Regarding the involution of the thymus in adults and especially its putative regression following myeloablative therapy the engrafted bone marrow is considered to be the source of the significantly stimulated T lymphocyte production. An expansion and enhanced maturation of the CD45RO⁺ lymphocyte subset in the post-transplant period in patients who develop corresponding dense peripheral infiltrates in the course of acute GVDH seems to be reasonable.^41,42,44 Moreover, the failure to determine any correlation with the peripheral absolute lymphocyte count is further in keeping with this hypothesis. On the other hand, it is not entirely ruled out that an increased marrow T lymphoid cell population, once GVHD has occurred, may only partially be related to an expansion or enhanced maturation. This feature could possibly also represent a tissue infiltration of the bone marrow, as is seen in other target organs during this process.

In conclusion, contrasting the significantly delayed reconstitution of T and B lymphocyte counts in the peripheral blood following allogeneic BMT in CML, the (unmanipulated) engrafted donor bone marrow reveals a speedy and pronounced growth of T lymphocyte subsets in patients with severe acute GVHD. Since size of this cell population does not correlate with the amount of lymphocytes in the peripheral blood, this phenomenon is assumed to be associated with the extensive CD45RO⁺ lymphocyte infiltrates of corresponding lesions characterizing this fatal complication.⁴⁴

Acknowledgements

We are greatly indebted to Mrs B Rosenbach, Mrs M Wonschick and Mrs H Tilemann for their excellent technical assistance. This work was supported by a grant from the Dr M Scheel Foundation for Cancer Research (grant number: 70-2134-Th1).

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Figure 1 CD45RO⁺ and CD20⁺ lymphocytes in the bone marrow following allogeneic BMT for CML. CD45RO⁺ lymphocytes are predominant and may be scattered (a) or occasionally arranged in focal dense aggregates, especially in patients with severe acute GVHD (b). On the other hand, CD20⁺ lymphocytes are less frequent and usually show a dispersed pattern (c) and rarely compose a nodular lymphoid infiltrate (d). a-d original magnification ´370.

Figure 2 Evolution of the CD45RO⁺ and CD20⁺ lymphocyte population in the bone marrow following allogeneic BMT for CML in a patient with several post-transplant biopsies and no GVHD (a) compared to another patient with severe acute GVHD (b).

Table 1  Characteristics of 123 CML patients with allogeneic BMT under study

Table 2  Endpoints of bone marrow biopsies at standardized intervals following BMT for Ph⁺ CML in 123 patients

Table 3  Differences regarding occurrence and severity of chronic and acute GVDH and quantity of CD45RO⁺ lymphocytes (median values per mm²) in the bone marrow during observation time