The Periarteriolar Lymphocyte Sheath in Immunodeficiency T

Brief Scientific Reports The Periarteriolar Lymphocyte Sheath in Immunodeficiency T- or B-Lymphocyte Area? AREND JAN VAN HOUTE, PH.D., HENK-JAN SCHUURMAN, PH.D., JONNE HUBER, M.D., JELLIE VAN DER MEER, JOHANNES H. VAN DER VEGT, M.D., WIETSE KUIS, M.D., PH.D., GEORGE JAMBROES, M.D., AND ROEL A. DE WEGER, P H . D .

Division of Histochemistry and Electron Microscopy, Departments of Pathology and Internal Medicine, University Hospital, Utrecht; Laboratory for Pathology Anatomy and Bacteriology, Deventer; University Hospital for Children and Youth "Het Wilhelmina Kinderziekenhuis," Utrecht; Department of Cardiology, University Hospital, Utrecht, The Netherlands

THE ARCHITECTURE OF peripheral lymphoid organs is differentiated into T-lymphocyte areas and B-lymphocyte areas. In splenic white pulp, T-cells reside in the area immediately surrounding the central arteriole, the periarteriolar lymphocyte sheath (PALS), and B-cells are localized in follicles and the marginal zone surrounding the PALS. This T/B compartmentalization of the main lymphocyte subtypes has been established in classical studies on neonatal thymectomy or bursectomy and subsequently in children and laboratory animals with congenital immunodeficiency. It has been confirmed by immunophenotyping using antibodies to T- and B-cell markers. This compartmentalization enables one to draw conclusions as to the type of immunodeficiency; a depleted splenic PALS is interpreted as a T-cell deficiency condition.

We studied autopsy tissue of three patients with T-cell deficiency and near-normal numbers of B-lymphocytes. The area immediately around the central arteriole in splenic white pulp was populated by lymphocytes. This histologic picture was not expected: according to the T/ B compartmentalization described, a lymphocyte-depleted PALS should be present. Therefore, we performed an immunologic phenotyping of the lymphocytes and framework constituents. The data were compared with histology of congenital athymic nude rats, which have a T-cell deficiency in the presence of normal numbers of B-lymphocytes. Materials and Methods Patients Autopsy lymphoid tissues were available from 6 patients with congenital immune deficiency (5 with severe combined immunodeficiency [SCID], 1 with the DiGeorge syndrome), 8 patients with acquired immune deficiency syndrome,7 15 patients in the first period after bone marrow transplantation,6-7 and 3 patients under immune suppressive therapy after heart transplantation. Congenitally Athymic (nude) Rat

Received October 2, 1989; received revised manuscript and accepted for publication February 28, 1990. Address reprint requests to Dr. Schuurman: Department of Pathology, University Hospital, P.O. Box 85.500, 3508 GA Utrecht, The Netherlands.

For comparison with the autopsy findings in the patients we present spleen histology of the nude rat. Animals were supplied by the National Institute of Public Health and Environmental Protection (Bilthoven, The Nether-

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T- and B-lymphocyte populations in peripheral lymphoid tissues occur in distinct compartments (e.g., the periarteriolar lymphocyte sheath of the splenic white pulp is a T-cell area). The authors report on two patients with severe combined immunodeficiency (SCID) and one patient with immunodeficiency after anti-T-cell treatment for rejection of a heart transplant, in which the area surrounding the central arteriole in spleen white pulp was wellpopulated despite T-cell deficiency (documented by, for example, severe depletion of lymph node paracortex). Immunologic phenotyping showed the B-lymphoid lineage of lymphocytes at this location. The framework in the periarteriolar area consisted of follicular dendritic cells, which are typical framework components of B-cell areas. We conclude that assessment of only conventional histopathology of the spleen in these patients leads to erroneous conclusions about the type of immunodeficiency and that immunologic phenotyping is required to document the exact nature of the deficiency. (Key words: Athymic conditions; B-cells; Immunodeficiency; Periarteriolar lymphocyte sheath; Spleen; Thymus-dependent area; T-cells) Am J Clin Pathol 1990;94: 318-322

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lands). Some specimens studied were from studies on lethal infection with Aspergillus (provided by Dr. H. Walvoort, Laboratory for Pathology, National Institute of Public Health and Environmental Protection). Analysis of Lymphoid Tissue

nate (FITC)-conjugated antilambda, antikappa, or antiheavy chain isotype on the other hand.5 Immunohistochemistry on paraffin sections was done following the avidin-biotin complex method. After incubation with monoclonal antibody, a second incubation was performed with biotinylated horse antimouse IgG(H + L) antibody, followed by incubation with avidin-biotin-peroxidase complexes (Vectastain Kit, Vector Laboratories, Burlingame, CA). Peroxidase visualization and counterstaining was done as described. The antibodies were controlled for their reactivity and specificity on sections from autopsy tissue processed in the same way. Lymphoid tissue from normal individuals with no gross abnormalities in histology showed the labeling patterns named in Table 1. Other controls comprised the omission of antibody in the first incubation or replacement by an irrelevant antibody: in this situation no labeling was observed. Reports of Cases The present study focuses on three patients in the series of 32 patients. These patients had T-cell deficiency and near normal numbers of Blymphocytes.

Case 1 A 5-month-old boy presented with pneumonia. Mumps virus was cultured from sputum. Lymphocyte counts in peripheral blood were 1.7 X 109/L; T-cells bearing CD3, CD4, and CD8 were absent; the majority of the cells were CD20-positive surface Ig-bearing B-lymphocytes. The

Table 1. Monoclonal Antibodies Used in this Study Reagent

CD

Paraffin sections of formalinfixed material MT1* LCf UCHL-lf

CD43 CD45 CD45RO

MT2* MB1* LN2* LN1* MB2* L26f Frozen tissue sections TolSt OKTIOt OKT6t OKTllt a-Leu-3§ a-Leu-2§

CD45R CD45R CD74 CDw75

CD22 CD38 CD1 CD2 CD4 CD8

DRC-lf anti-Ig" * Clonab. Biolest. Dreieich. FRG. t Dakopatts. Glostrup, Denmark. X Ortho Diagnostic Systems. Raritan, New Jersey. § Becton Dickinson. Mountain View. California. 11 Fluorescein isothiocyanate (FITC)-conjugatcd antikappa. antilambda. anti-IgD, and anti-IgE

Staining on Lymphoid Tissue

T-cells, monocytes, macrophages, myeloid cells T- and B-cells; macrophages and myeloid cells (faint) Subset of T-cells (early T- and memory T-cells); monocytes; myeloid cells (Mature) T-cells; B-cells in follicle mantle and marginal zone (Part of) T-cells and monocytes/macrophages (faint): all B-cells B-cells, interdigitating reticulum cells B-cells in germinal center; B-cells in mantle (faint) All B-cells; endothelium and epithelium B-cells All B-cells Cortical thymocytes; germinal center cells, plasma cells Cortical thymocytes, interdigitating cells All T-cells Cortical thymocytes, about two-thirds of T-cells in thymus medulla and peripheral lymphoid organs Cortical thymocytes, about one-third of T-cells in thymus medulla and peripheral lymphoid organs Follicular dendritic cells B-cells; plasma cells from Behringwerke. Marburg/Lahn. FRG: anti-IgM from Dakopatts: anti-IgG and anti-lgA from Kallestad. Austin. Texas: tetramethyl rhodamin isolhiocyanatc-conjugated anti-kappa and antilambda from Dakopatts. Data from own experience3"7 and literature.1,34


Lymphoid tissues taken at autopsy were immediately frozen and processed for immunohistochemistry (in case 2, only spleen tissue was frozen). Other parts of tissue specimens were fixed in formalin and embedded in paraffin. Paraffin sections were processed for conventional histology and, subsequently, for immunohistochemistry. Relevant antibodies applied in the present study are named in Table 1. Immunohistochemistry on frozen tissue sections was done by an indirect immunoperoxidase method. After incubation with monoclonal antibody, a second incubation was performed with peroxidase-conjugated rabbit antimouse immunoglobulins and a third one with peroxidase-conjugated swine antirabbit immunoglobulins (both reagents from Dakopatts, Glostrup, Denmark). Peroxidase was visualized by 3,3'-diaminobenzidine tetrahydrochloride with H 2 0 2 as substrate, and sections were counterstained with hematoxylin. Immunoglobulin analysis was done by two-color direct immunofluorescence using combinations of tetramethyl rhodamin isothiocyanate (TRITC)-conjugated antikappa or antilambda on one hand, and fluorescein isothiocya-

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patient died one month later. At autopsy the lung showed abscesses with purulent bronchitis, Pneumocystis carinii, cells with viral inclusion bodies, and giant cells. The thymus was completely lymphocyte depleted: it showed lobules with undifferentiated epithelium with organization in epithelial rosettes. The diagnosis made was SCID with presence of B-lymphocytes, with absent T-cell differentiation.

Case 2 A male infant suffered from respiratory infections from the age of 23 weeks. At 5 months of age he developed a varicella infection and died from overwhelming infection two weeks later. Autopsy histology revealed herpesvirus in skin, lung, adrenals, thymus, lymph nodes, and spleen and cytomegalovirus (CMV) in lung, pancreas, and kidney. In addition, there was Candida in the lung. The thymus (5 g) showed an abnormal dysplastic architecture, with undifferentiated epithelium in rosette-like structures without lymphocytes. The diagnosis was SCID with absent T-cell differentiation.

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around the central arteriole. Most of the T-cells were labeled by CD4 and a minor part by CD8. MT2 was negative. LN1 labeled some cells around the central arteriole. The stroma in the area surrounding the central arteriole contained DRC-1-positive cells at high density (Fig. ID). There were plasma cells of IgM, IgG, or IgA isotype in very low numbers. The (immuno)histology of spleen was almost identical for all three cases. In the congenitally athymic nude rat, the PALS in spleen was severely lymphocyte depleted. Follicles were present, and the marginal zone was well populated by lymphocytes (Fig. 2). Similar features were observed in spleen from athymic rats after lethal infection with Aspergillus.

Case 3 A 44-year-old man received orthotopic heart transplantation for intractable congestive heart failure four years after an anteroseptal myocardial infarction. Rejection episodes between two and four weeks after transplantation were treated by intravenous methylprednisolone and rabbit antithymocyte globulin. As rejection persisted, the patient received a nine-day treatment with OKT3 monoclonal antibody (5 mg/day) starting six weeks after transplantation. In this period, blood leukocyte counts were about 6 X 109/L, with 6% lymphocytes. CD3-positive T-cells were not detectable in blood. Eight weeks after transplantation, vascular rejection was documented in the endomyocardial biopsy.'0 The patient then received 300 mg antithymocyte globulin but died the next day in heart failure due to terminal rejection.

Results In the group of patients with various types of immunodeficiency investigated who died after a period of debilitating illness, almost all revealed severely lymphocytedepleted lymphoid organs, often with a loss of tissue architecture. Dependent on the type of immunodeficiency, scattered T-cells and/or small groups of B-lymphocytes and plasma cells are observed in the lymph node cortex and splenic PALS.6 In the three patients of the present study, lymph nodes showed a lymphocyte depletion of interfollicular areas. Follicles containing B-lymphocytes were clearly identified (Fig. 1A). The area immediately surrounding the central arteriole in the white pulp of the spleen was populated by lymphoid cells (Figs. 1B and 1C). Follicles were not identified. The majority of cells in the well-populated area around the central arteriole were identified as B-lymphocytes, with expression of CD22 and surface IgM-IgD. On paraffin sections the cells were positive for LC, MB 1, MB2, LN2, and L26 (Fig. 1C). There were a few T-cells, on frozen tissue sections identified by CD2, on paraffin sections by MT1 and UCHL1 (Fig. IB). The cells occurred at scattered locations, and in some white pulp areas they were present in a somewhat higher density immediately

The three patients showed a severe T-cell depletion with near-normal numbers of B-lymphocytes. Patients 1 and 2 were SCID patients with B-lymphocytes. For patient 3, the immunodeficiency was acquired by immunosuppressive treatment, of which the antithymocyte globulin and OK.T3 monoclonal antibody especially affected the T-cell population. The deficiency was reflected by lymph node (immuno)histology that showed depleted T-cell areas in the presence of follicles containing B-cells. However, splenic histology gave the impression of a well-populated PALS and absence of B-cell areas. In conditions of severe T-lymphocyte deficiency and near normal numbers of B-lymphocytes, one would expect a lymphocyte-depleted PALS. This feature is indeed present in the athymic rat: the framework of T-dependent areas is present, but these areas are not populated by lymphocytes (Fig. 2)." After constitution of the thymus-dependent immune system, for instance by thymus transplantation, this site becomes filled by T-cells and the lymphoid tissue develops the histology of that in euthymic normal animals.8'9 In the patients in this study, the lymphocyte-depleted framework of T-dependent areas is present in lymph nodes. However, the splenic PALS considered to be a true T-cell area, is lacking and replaced by a B-lymphocyte area (Figs. IB and 1C). B-lymphocytes are not only present but reside in the framework of follicular dendritic reticulum cells, which is typical for a B-cell area (Fig. ID). This architecture is strikingly different from that in the T-cell-deficient nude rat. Thus, conventional histology would lead one to make an erroneous conclusion as to the nature of the immunodeficiency in these patients. One would be tempted to conclude that the T-cell area (PALS) is intact and that the B-cell area is lacking. The present conclusion on the replacement of the PALS as a true T-cell area by a B-lymphocyte area could be reached by detailed immunologic phenotyping of the lymphocytes and stroma. The phenomenon observed is


Discussion

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FIG. 1. Histology and immunohistochemistry of spleen and lymph node from patient I. A. (upper, left). The lymph node section shows lymphocytes in follicles (F) and a severely lymphocyte-depleted paracortex (P). Paraffin section stained with hematoxylin and eosin. Paraffin section of spleen labeled by MTl B (upper, right) or L26 C (lower, left) and counterstained with hematoxylin. B. There are some cells in the area surrounding the central arteriole (arrow) labeled by the anti-T antibody MTl (very darkly stained cells, indicated by arrowheads). C. Almost all lymphocytes are labeled by the anti-B antibody L26. D (lower, right). The framework of the area surrounding the central arteriole (arrow) contains follicular dendritic cells, stained in frozen tissue section (with hematoxylin counterstain) by antibody DRC-1. (XI25).

not a new one: one of us previously reported on large numbers of pyroninophilic blast-like lymphocytes immediately surrounding the central arteriole of splenic white pulp in some SCID patients.2 The pyroninophilic nature of these cells makes it unlikely that these cells are of T-lineage. Because immunophenotyping was not possible at the time of this observation (1968) the true B-cell nature of these cells could not be documented. Patients 1 and 2 suffered from congenital types of Tcell deficiency, and it cannot be excluded from consid-

eration that the framework of the PALS was not generated in these patients. Case 3 differs from the first two in that this patient's spleen probably was normal before the installment of immunosuppressive therapy. Apparently the PALS framework in this patient disappeared during the period of immunosuppression. The mechanism of disappearance, or shrinking, remains to be established. It is known that the framework of lymphoid tissue compartments disappears in neoplastic processes, but we found no evidence for such a process. In our patients the B-cells

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in the PALS-like area were of polytypic origin as judged by immunoglobulin light-chain typing. Another explanation might be sought in the lethal infections during the final illness. Such an explanation seems unlikely considering studies in athymic nude rats, for which the PALS framework persisted during a lethal Aspergillus infection. In conclusion, we show that the area surrounding the central arteriole in the splenic white pulp at autopsy in patients with severe T-cell deficiency in the presence of near-normal numbers of B-cells gives the histologic impression of a thymus-dependent PALS area, whereas in fact it is a B-lymphocyte area including the reticulum of follicular dendritic cells. Therefore, if there is any doubt about the type of the immunodeficiency or if the clinical data suggest a T-cell deficiency, it is strongly recommended that conventional histology is extended by immunophenotyping of lymphocytes. We show that such a phenotyping can be performed easily on formalin-fixed tissue using available panels of monoclonal antibodies (Table 1). References 1. Azumi N, Sheibani K, Swartz WG, Stroup RM, Rappaport H. Antigenic phenotype of Langerhans cell histiocytosis: an immunohistochemical study demonstrating the value of LN-2, LN-3, and Vimentin. Hum Pathol 1988;19:1376-1382. 2. Huber J. Experience with various immunologic deficiencies in Holland. In: Bergsma D, ed. Birth defects, immunologic deficiency

diseases in man. Original article series. Vol. 4, no. I. National Foundation. Baltimore: Williams & Wilkins, 1968,53-66. Knapp W, Dbrken B, Gilks WR, Rieber EP, Schmidt RE, Stein H, Von dem Borne AEgkr, eds. Leucocyte typing IV: White cell differentiation antigens. Oxford: Oxford University Press, 1989. Poppema S, Hollema H, Visser L, Vos H. Monoclonal antibodies (MT1, MT2, MB1, MB2, MB3) reactive with leukocyte subsets in paraffin-embedded tissue sections. Am J Pathol 1987; 127:418429. Schuurman H-J, Van Baarlen J, Huppes W, Lam BW, Verdonck LF, Van Unnik JAM. Immunophenotyping of non-Hodgkin's lymphoma. Lack of correlation between immunophenotype and cell morphology. Am J Pathol 1987;129:140-151. Schuurman H-J, Hendriks RW, Verdonck LF, Van Baarlen J, De Gast CG, Schuurman RK.B. Expression of restricted immunoglobulin isotypes in plasmacellular hyperplasia after allogeneic bone marrow transplantation. Scand J Immunol 1987;26:409416. Schuurman H-J, Krone WJA, Broekhuizen R, et al. The thymus in acquired immune deficiency syndrome. Comparison with other types of immunodeficiency diseases, and presence of components of human immunodeficiency virus type 1. Am J Pathol 1989; 134: 1329-1338. Schuurman H-J, Vaessen LMB, Vos JG, et al. Implantation of cultured thymic fragments in congenitally athymic nude rats. Ignorance of thymic epithelial haplotype in generation of alloreactivity. J Immunol 1986;137:2440-2447. Schuurman HJ, Vos JG, Broekhuizen R, Brandt CJWM, Kater L. In vivo biological effect of allogeneic cultured thymic epithelium on thymus-dependent immunity in athymic nude rats. Scand J Immunol 1985;21:21-30. 10. Slootweg PJ, Schuurman H-J, Jambroes G. Myocytolysis due to vascular rejection. Biopsy and autopsy findings. A case report. J Heart Transplant 1989;8:450-453. Vaessen LMB, Broekhuizen R, Rozing J, Vos JG, Schuurman H-J. T-cell development during ageing in congenitally athymic (nude) rats. Scand J Immunol 1986;24:223-235.


FIG. 2. Histology of spleen from an euthymic rat (A, left) and congenitally athymic (nude) rat (B, right). In the euthymic rat, T-cell areas (PALS, P) are well-populated by lymphocytes. In the athymic rat the framework of this area is present, but it is severely lymphocyte-depleted. In both the euthymic and athymic animals, B-cell areas are present and well-populated by lymphocytes (follicles, F; marginal zone, MZ). Paraffin section stained by hematoxylin and eosin (XI25).