The Thymus in Patients with Allogeneic Bone Marrow Transplants William E Beschorner, MD, Grover M

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The Thymus in Patients With Allogeneic Bone Marrow Transplants William E Beschorner, MD, Grover M. Hutchins, MD, Gerald J. Elfenbein, MD, and George W. Santos, MD

The thymus glands from 11 patients with aplastic anemia or acute leukemia who received allogeneic bone marrow transplants were studied at autopsy. All showed marked cortical involution. In the short-term survivors the medulla and perivascular spaces were lymphocyte-depleted and the epithelial cells formed pseudorosettes. In those surviving over 2 months, increasing numbers of small lymphocytes were present, presumably reconstituted with donor lymphocytes. Phagocytosis of cellular debris was frequent, especially in patients with graft-versus-host reaction (GVHR) or treated with antithymocyte globulin (ATG). Plasma cells were numerous in perilobular tissue and were occasionally found within the medulla. The findings are compatible with the concept that the thymus plays an important role in the immune deficiency experienced after allogeneic bone marrow transplantation and in the subsequent lymphoid reconstitution. (Am J Pathol 92:173-186, 1978)

OVER THE PAST 2 DECADES bone marrow transplantation from syngeneic and allogeneic donors has been used for the treatment of severe combined immune deficiency, aplastic anemia (AA), and acute leukemia (AL). Recipients receiving an allogeneic graft for AA and AL are prepared with high doses of cytotoxic drugs, frequently in combination with total- body irradiation. Following the transplant, patients may develop a number of complications, including combined immune deficiency.13 In the present study we investigated the histopathologic changes in the thymuses of 11 patients surviving 30 to 132 days after an allogeneic transplant for AA, acute lymphocytic, or nonlymphocytic leukemia. In each case there was marked cortical involution. Cellular degeneration and plasma cell infiltration of the thymus was present in patients affected by GVHD and disseminated infections. Long-term survivors with normal numbers of circulating T lymphocytes also had an increased number of mature-appearing lymphocytes in the thymic perivascular spaces com- pared with short-term survivors. The absence of immature lymphocytes suggests an extrathymic source for the repopulation of the thymus gland.

From the Department of Pathology and The Bone Marrow Transplant Unit in the Oncologv Center of The Johns Hopkins Medical Institutions, Baltimore, Marvland. Supported by Grant HL-14153 from The National Heart and Lung Institute and by Grant CA- 14396 from The National Cancer Institute, Department of Health, Education, and Welfare. Dr. Elfenbein is an investigator at the Howard Hughes Medical Institute. Accepted for publication March 13, 1978. Address reprint requests to Grover M. Hutchins, MD, Department of Pathology, The Johns Hopkins Hospital, Batimore, MD 21205. 0002-940178/0710-o173S01.00 173 174 BESCHORNER ET AL Armerican Joumal of Pathoogy

Materials and ts Autopsy findings of 11 bone marrow transplant recipients who died at The Johns Hopkins Hospital between February and September 1977 were reviewed. The degree of GVHD was deternmined by skin and liver biopsies, autopsy findings, and clinical data, as per Thomas et al.' The stage of lymphoid atrophy or reconstitution was determined using the method of Woodruff et al.' The thymus in each patient was found by carefully sectioning the prepericardial or pericervical fat. The histology was studied using hematoxylin and eosin, Masson connective tissue, periodic acid-Schiff (PAS), methyl green-pyronin, and Leder esterase stains." Circulating lymphocytes were tested by sheep red cell rosetting (for thymus-derived cells) by previously detailed methods.' Resufts The patients ranged in age from 6 to 28 years and included 7 males and 4 females as shown in Table 1. One patient (Case 1) presented with aplastic anemia complicating paroxysmal nocturnal hemoglobinuria; 2 had idiopathic aplastic anemia; 3 had acute lymphocytic leukemia; 3 had acute myelogenous leukemia; and 1 presented with chronic myelogenous leukemia in blast crisis. Following the transplant they survived 30 to 123 days. In Case 5 a second booster transplant was given after the first was considered a failure. Four patients developed at least moderate GVHD. They all died with an interstitial pneumonitis; 2 cases were associated with cytomegalovirus. Subsequent to GVHD, a necrotizing enterocolitis developed in Case 4, which was the immediate cause of death. Patient 9 also had superimposed Pseudomonas bronchopneumonia. The other 7 patients died with interstitial pneumonitis and/or opportunistic infections. The thymus in each had marked depletion of the cortex (Figure 1). The epithelial cells in Cases 1, 2, 3, 4, and 6 were spindle shaped and frequently arranged in a pseudorosette pattern. Hassall's corpuscles were always present but varied from being markedly decreased in number to numerous prominent calcified bodies. Plasma cells were present in most cases, being most prominent in Cases 5 and 8. The plasma cells were generally confined to the extralobular space but were also identified within the medulla in Cases 7, 8, and 9 (Figure 2). Small lymphocytes were present in the lobules and perivascular spaces. Lymphocytes were identified only rarely in patients surviving 1 month (Cases 1 through 3), while more lymphocytes were found in patients surviving longer than 2 months (Cases 8 through 11). Numerous nonpyroninophilic lymphocytes were present in the perivascular spaces of the patients surviving more than 100 days (Figure 2). Few lymphocytes were found in the medulla or cortex of these thymuses, however. Vol. 92, No. 1 THE THYMUS IN TRANSPLANT RECIPIENTS 1175 July 1978

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Table 2-Circulating Thymus-Dependent Lymphocytes in Bone Marrow Transplant Recipients First study Second study Mono- Mono- nuclear nuclear Day after cells/ Percent acve T cells Day after cells/ Percent active T cells Patient transplant cu mm Percent total T cells transplant cu mm Percent total T cells

8 39 840 48.053.0 - - - 33.0 9 39 580 46.5 93 8060 743563.5 10 23 370 16.5 91 6720 5160

22.5 44.5 11 19 860 33.0 343 250025060.0

Using the criteria established by Woodruff et al,5 the lvmph nodes and spleen were in the stage of developing atrophy in 4 cases (1, 3, 7, and 8), were markedly atrophic in 4 cases (2, 4, 5, and 6), and had evidence of earlv reconstitution in 3 other cases (9 through 11). There was little repopulation of the subcortical regions of lvmph nodes or periarterial sheaths of spleen (presumed T-cell regions) in Patient 9, while Patients 10 and 11 had mild repopulation of these regions with small lvmphocytes. On the other hand, circulating active and total T lymphocytes as determined bv sheep red cell rosettes increased between the first and second studv in Patients 9 and 10 (Table 2), while Patient 11 had a high percent- age of T lvmphocvtes by Dav 30. Three patients received horse antithymocvte globulin (ATG) during the course of their disease (Cases 7, 9, and 11). The first 2 received ATG treatment for moderately severe GVHD; Patient 11 received ATG as part of the preparatory protocol. Evidence of recent necrosis was apparent with increased phagocytosed PAS-positive material (Figure 3). However, as seen in the last 2 cases, despite the use of ATG or the occurrence of GVHD, regeneration of the epithelial cells and repopulation of lymphocytes occurred. The thymus was directly involved by an opportunistic infection only in Patient 8. She developed a disseminated mixed infection of adenovirus and cytomegalovirus leading to a fatal CMV-associated interstitial pneumonitis. Clusters of pyroninophilic and nonpyroninophilic lymphocytes were found in the thymus. Nearby there were epithelial cells with eosinophilic nuclear inclusion bodies, consistent with an adenovirus infection. Breakdown of the thymic blood barrier was evident, with numerous plasma cells found within and outside the thymic lobule. Vol. 92, No. 1 THE THYMUS IN TRANSPLANT RECIPIENTS 177 July 1978 Dcuson The participation of the thymus in the development of normal lvrn- phoid tissue with mature B and T lymphocytes has been well established. Lymphoblasts multiply in the cortex and mature as they migrate through the medulla; finally, small thymocytes emigrate into the perivascular space and into the circulation.7'8 Alterations of the morphology related to age, infection, endocrine abnormalities, autoimmune disease, and immune deficiency diseases have been described.'"' Some of the major morphologic changes in these conditions are illustrated in Figure 4. Following allogeneic bone marrow transplantation, patients frequently experience complications of both GVHR and combined immune deficiency as well as problems relating to steroid therapy, disseminated infections, and preparative therapy. Interestingly, the thymuses in these patients demonstrate changes resembling treated autoimmune diseases and immune deficiency states. Clinically the patients studied have varied as to success of engraftment, peripheral lymphocyte subpopulations, steroid therapy, the degree of GVHD, and complicating infections. Nonetheless, the thymuses in these patients had a number of similar changes: a) Marked cortical involution was universally present. In no case was a lymphocyte-rich cortex found. The remaining lobule was replaced with fat. b) In patients with marked depletion of lymphocytes, the epithelial cells were spindle shaped and assumed a perpendicular orientation to the periphery in a pseudorosette pattern. c) The number of nonpyroninophilic small lymphocytes in the medullary and perivascular spaces was generally decreased; but, in pro- portion to the length of the posttransplant survival period, they were found in increasing numbers, particularly in the perivascular spaces. d) Plasma cells and pyroninophilic lymphocytes were present in increased numbers. These were generally present in the extrathymic soft tissue and perivascular spaces. e) Varying amounts of PAS-positive granular debris were present within histocytes, being most prominent in patients with significant GVHD and in patients treated with ATG. The marked cortical involution found in these patients was not surpris- ing. Many had been treated with steroids prior to or after the transplant. Other patients had acute stress terminally with disseminated infections. The persistence of Hassall's corpuscles in even the most severely depleted thymus glands suggests that the involution in these patients was due to recent events related to the transplant rather than a remote or chronic process. In a murine model 12 of parent to Fl hybrid transplant, an induced GVHD reaction has been shown to cause thymic involution with lym- 178 BESCHORNER ET AL American Joumal of Patholog phocytolysis and lymphocyte destruction of epithelial cells and Hassall's corpuscles. Four of five patients with at least moderate GVHD had typical changes in the epithelium of target organs, ie, skin, liver, and gastrointes- tinal tract. In a fifth patient (Case 5), the GVHR appeared limited to the lymph nodes and spleen. Although no lymphocytorrhexis or lymphocyte emperipolesis was evident in the thymus, the medulla had increased quantities of phagocytized PAS-positive granular debris, suggesting recent cellular degeneration. This was most striking in Cases 7 and 9, in which GVHD had been treated with ATG with some success. Another factor which contributed to involution is the radiation and/or cytotoxic drugs used in preparing the patients for transplant. Two groups of workers 13,14 have demonstrated that cortical thymocytes in animal models are sensitive to a single large dose of radiation. After a short delay, however, the thymus is repopulated with lymphoblasts maturing into small lymphocytes. Our patients, prepared with irradiation and/or che- motherapy, had nearly complete elimination of lymphocytes and an apparent decrease in epithelium in the first 2 months. As evident from the last 3 patients, however, neither irradiation nor GVHD prevented re- epithelialization and repopulation with small mature-appearing lymphocytes. Severe involution with lymphocyte depletion and loss of Hassall's corpuscles was described by Kersey et al 15 in 5 allogeneic marrow transplant recipients. These patients differed from our population, however, in that they were being treated for a severe combined immune deficiency and did not require the immunosuppressive therapy which our patients received. The changes in the thymus may be ascribed, at least partially, to their primary disease. The presence of plasma cells and Russell bodies in the thymus raises an interesting question. Previously these have been described in patients with lupus erythematosus,11 rheumatoid arthritis,10 and myasthenia gravis.16 These are diseases in which autoantibodies are felt to be important in pathogenesis. Although the thymuses of untreated patients with these disorders frequently have germinal centers, Hutchins and Harvey " found them absent in all nine lupus patients treated with steroids and the thymus was not morphologically dissimilar from the present cases. Kersey et al '" reported a patient with severe GVHD who also had numerous plasma cells in the thymus. Although it is generally accepted that GVHD is primarily a cell-mediated response, there is some evidence 17.18 that antibodies might also be involved. The relative number of plasma cells in our patients did not correlate well with the degree of GVHD. Thus, their significance in either thymic protection or destruction remains unclear. Vol. 92, No. 1 THE THYMUS IN TRANSPLANT RECIPIENTS -d179IVO% July 1978 Generally the plasma cells were limited to outside the thymic lobule. This is consistent with evidence that mature lymphocytes can only emigrate from the lobule across the blood-thymic barrier.' In 3 patients, however, the barrier was apparently violated and plasma cells were identified within the medulla. This was most marked in the thymus infected with adenovirus. A similar degree of plasma cell influx may be seen when the thymus is involved by congenital syphilis, the so-called Dubois ab- scess. The other patients with intrathymic plasma cells were Patients 7 and 9, whose GVHD was treated with ATG. Raviola and Karnovsky7 have shown that large proteins can cross the thymic-blood barrier only in the postcapillary venule at points where small lymphocytes were transversing the endothelium. If ATG and complement were cytotoxic to these thymocytes in the immediate vicinity of venule, a breakdown of the barrier could occur. A transient combined immune deficiency has been documented by several studies 1- of bone marrow transplant recipients. This is more severe in patients receiving an allogeneic transplant than in those receiving autologous and syngeneic transplants. Briefly, while the number of circulating B cells are normal 1 month after transplant, circulating T cells are significantly decreased for 1 to 2 months after transplant. IgM and IgG levels are only transiently depressed, but there is a delayed return of delayed hypersensitivity skin test responses, antibodies to thymic dependent antigens, and IgA levels. Although the number of circulating T cells returns to normal after 2 months, the T-cell regions of spleen and lymph nodes remain depleted." The lymphocyte pattem of the thymus in our patients tends to reflect the recovery of circulating lymphocytes more closely than the lymphoid tissue, although Patients 10 and 11 did have evidence of early repopulation of the T-cell regions of the lymph nodes and spleen. In the patients dying 1 to 2 months after transplant, small lymphocytes were only rarely seen. The epithelial cells became spindled and pseudorosettes were formed in the periphery of the lobule. This pattern resem- bles that described in Swiss agammaglobulinemia (Figure 4). Pseudo- rosettes were no longer found, while more epithelium and small nonpyroninophilic lymphocytes were present in those surviving more than 2 months. These were most numerous in the perivascular spaces with sc-attered lymphocytes in the medulla and cortex. An important issue which remains unanswered is whether the thymus in adult humans is functional. In our group of patients, the thymus gland is repopulated by mature small lymphocytes. The absence of intrathymic 180 BESCHORNER ET AL American Joumal of Pathology lymphoblasts and immature medium-sized lymphocytes and the location of mature lymphocytes primarily in the perivascular spaces suggest that the lymphoid mass is expanded from extrathymic source rather than residual lymphocytes. Patients 9 and 10 received transplants from donors of the opposite sex. Karyotype studies of phytohemagglutinin-stimulated lymphocytes from blood indicate that they were entirely of donor origin. Although we lack karyotype studies of the thymic lymphocytes, it seems reasonable to presume that they too were of donor origin (as demonstrated in rodent models"). We have illustrated in this study how the thymus is affected by allogeneic bone marrow transplantation and its complications. The thymus appears to reflect the immune status of the patient in that patients dying during the first 2 months had thymic changes reminiscent of congenital thymic deficiencies, while those with longer survival demonstrated partial repopulation with lymphocytes, which are probably of donor origin. The thymus of human bone marrow transplant recipients deserves further investigation to determine the significance of its role in lymphoid reconstitution and GVHR. References 1. Elfenbein GJ, Anderson PN, Humphrey RL, Mullins GM, Sensenbrenner LL, Wands JR, Santos GW: Immune system reconstitution following allogeneic bone marrow transplantation in man: A multiparameter analysis. Transplant Proc 8:641- 646, 1976 2. Halterman RH, Graw RG Jr, Fuccillo DA, Leventhal BG: Immunocompetence following allogeneic bone marrow transplantation in man. Transplantation 14:689- 697, 1972 3. Storb R, Ochs HD, Weiden PL, Thomas ED: Immunologic reactivity in marrow graft recipients. Transplant Proc 8:637-639, 1976 4. Thomas ED, Storb R, Clift RA, Fefer A, Johnson FL, Neiman PE, Lerner KG, Glucksberg H, Buckner CD: Bone-marrow transplantation. N Engl J Med 292:832-843, 895-902, 1975 5. Woodruff JM, Hansen JA, Good RA, Santos GW, Slavin RE: The pathology of the graft-versus-host reaction (GVHR) in adults receiving bone marrow transplants. Transplant Proc 8:765-684, 1976 6. Leder LD: [The selective enzvmocytochemical demonstration of neutrophil myeloid cells and tissue mast cells in paraffin sections.] Klin Wochenschr 42:553, 1964 7. Raviola E, Kamovsky MJ: Evidence for a blood-thymus barrier using electron- opaque tracers. J Exp Med 136:466-498, 1972 8. Sainte-Marie G, Peng FS: Emigration of thvmocytes from the thymus: A review and study of the problem. Rev Can Biol 30:51-78, 1971 9. Boyd E: The weight of the thymus gland in health and in disease. Am J Dis Child 43:1162-1214, 1932 10. Bumet FM, Mackay IR: Lymphoepithelial structures and autoimmune disease. Lancet 2:1030-1033, 1962 11. Hutchins GM, Harvey AMcG: The thymus in systemic lupus erythematosus. Bull Johns Hopkins Hosp 115:355-378. 1964 Vol. 92, No. 1 THE THYMUS IN TRANSPLANT RECIPIENTS 181 Juy 1978

12. Seemayer TA, Lapp WS, Bolande RP: Thymic involution in murine graft-versus- host reaction: Epithelial injury mimicking human thymic displasia Am J Pathol 88:119-134, 1977 13. Aovama T, Kawamoto Y, Furuta I, Kondo T: Early morphological changes in cortical medullary thymocytes of the rat after whole-body irradiation. I. Electron- microscope observations. Int j Radiat Biol 21:545-558, 1972 14. Swasdikul D, Block M: Effect of radiation upon the "embryonic" thymus. Radiat Res 50:73-84, 1972 15. Kersey JH, Meuwissen HJ, Good RA: Graft versus host reactions following transplantation of allogeneic hematopoietic cells. Hum Pathol 2:389-402, 1971 16. Smilev JD, Bradley J, Daly D, Ziff M: Immunoglobulin synthesis in vitro bv human thymus: Comparison of myasthenia gravis and normal thymus. Clin Exp Immunol 4:387-99, 1969 17. Kately JR, Gengozian N: Rat-mouse radiation chimeras: Characterization of an antibody-mediated graft-vs.-host reaction. J Immunol 114:125-132, 1975 18. Merritt CB, Mann DL, Rogentine GN Jr: Cytotoxic antibody for epithelial cells in human graft versus host disease. Nature 232:638-639, 1971 19. Elfenbein GJ, Santos GW: Rosette formation between rat thvmocytes and guinea pigs ervthrocytes requires "active" fetal calf serun. II. Characterization of the receptor bearing thymocytes. Cell Immunol (In press) 182 BESCHORNER ET AL American Joumal of Pathology

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oCEoE 0 c I . W- I Nbkl v I Figure 4-Comparison of normal thymus with several pathologic changes. A-Normal infant thymus with lymphocyte-rich cortex (top) and medulla containing Hassall's corpuscles (bottom). Note that blood vessels indent the cortex in a connective tissue infolding and remain "extra-thymic." B-Marked thymic hyperplasia in a girl with rapidly progressive glomerulonephritis, widespread periarteritis, and pulmonary hemorrhage. There is germinal center formation, epithelial hyperplasia, and enormous numbers of plasma cells and Russell bodies in the perivascular "extrathymic" space. The germinal centers are also probably in the perivascular space and not within the thymic pa- renchyma. C-Thymic involution caused by severe infection in a child. The cortex is markedly depleted of lymphocytes but filled with foamy reticular cells. The medulla shows a concentration of Hassall's corpuscles as a result of shrinkage of the lobules. D-Marked reduction in the thymic lobule in an adult with Cushing's syndrome caused by adrenal hyperplasia. The cortex is absent and the lobule is reduced to a double layer of small epithelial cells. E-Thymic lobule of a child with Swiss agammaglobulinemia. There are no lymphocytes or Hassall's corpuscles, but a lobular pattern of epithelium is present. (H&E; A, xlOO; B, x50; C, D, and E, x200) (with printing reduction of 14%)