Lymphocytes bearing receptors for both sheep erythrocytes and complement in patients with neoplastic and non-neoplastic diseases

Lymphocytes bearing receptors for both sheep erythrocytes and complement in patients with neoplastic and non-neoplastic diseases

Lymphocytes Bearing Receptors for Both Sheep Erythrocytes and Complement in Patients with Neoplastic and Non-Neoplastic Diseases KAZIMIEKA J. GAJL-PFX...

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Lymphocytes Bearing Receptors for Both Sheep Erythrocytes and Complement in Patients with Neoplastic and Non-Neoplastic Diseases KAZIMIEKA J. GAJL-PFXZALSKA, SANDRA CHAKTR~AXI), CLARA D. BLOOMFIELD, JEAN CORTE, PETER F. CO~(:IA, MARK E. NESBIT, AND JOHN H. KEKSEY Department oj. Laboratory Medicine and Pathology, Depurtmenr c!t Pediatrics, and Section of Medical Oncology. University qf Winnesotci~ Minneapolis. Minnesota 55455 Received October 25. 1976 To identify lymphoid cells bearing receptors for both sheep erythrocytes (SE) and complement (C’). a combined rosette assay was developed using uncoated SE and chicken erythrocytes coated with antibody and complement (cEAC’). Cells with both receptors were present inslow percentages in the blood of normal individuals. Tissues and/or blood from 543 patients with various diseases were studied using the combined rosette assay. Large numbers of malignant cells were found to carry receptors for both C’ and SE in four patients with lymphoproliferative malignancies. Two were children with lymphoma who had mediastinal masses. The other two were adults with chronic lymphocytic leukemia; their cells also carried surface immunoglobulin and Fc receptors. In these four patients double or multiple receptor lymphocytes may represent a clonal proliferation of subsets of lymphocytes present in small numbers under normal conditions. An increase in double receptor lymphocytes was rare in the non-neoplastic conditions studied and seen only in children. Double receptor lymphocytes may represent an immature or “stem” cell lymphocyte subset.

INTRODUCTION

Several distinct subsets of human lymphocytes exist. T (thymus-dependent) cells are recognized by their capacity to form rosettes with sheep erythrocytes (SE) and the use of specific anti-T-cell antisera (1,2).’ B (bone marrow-derived) lymphocytes are identified by surface immunoglobulins (SIg), specific anti-B-cell antisera, receptors for the Fc portion of IgG, and receptors for complement, usually demonstrated by an EAC’ (erythrocytes, antibody, and complement) rosette assay (3,4). When B and T cell markers are studied simultaneously, a small population of lymphocytes is found to bear markers of both B and T cells (5 - 7). A few cases of lymphoid malignancies in man and experimental animals have been reported in which malignant cells had multiple surface markers (8- 14). To evaluate double marker lymphocytes further, we developed a combined rosette assay using sheep and chicken red cells (SE + cEAC’). In this assay cells with I Abbreviations: T. thymus-dependent; B, bone marrow-derived; SE. sheep erythrocytes; cE. chicken erythrocytes; EAC’, erythrocytes, anitbody. and complement: SIg. surface immunoglobulin: VBS, Veronal-buffered saline; FCS, fetal calf serum; ALL, acute lymphocytic leukemia: CLL. chronic lymphocytic leukemia; AML, acute myelogenous leukemia; CML, chronic myelogenous leukemia; LRE, leukemic reticuloendotheliosis: CSF. cerebrospinal fluid; C’, complement. 292 Copyright All

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only SE receptors, only complement receptors (cEAC’), and double receptors (SE + cEAC’) and cells with no receptors could be quantitated simultaneously. We report here the results of double-marker studies in 543 patients with various neoplastic and non-neoplastic conditions. MATERIALS AND METHODS and normal subjects. One hundred healthy volunteers

Patients and 543 patients seen at the University of Minnesota Hospitals were studied. The controls included 56 males and 44 females, 19 to 54 years of age. The patients included 183 with various non-neoplastic diseases and 360 with various neoplasms, i.e., 287 with lymphoreticular malignancies, 38 with melanoma, and 35 with other malignancies. There were 309 males and 234 females. Of 287 patients with lymphoreticular malignancies, 209 had active disease and 78 were in remission at the time of study. Separation of lymphoid cells. Blood was collected in heparin, and the buffy coat incubated with an equal volume of iron-containing lymphocyte-separating reagent (Technicon Instruments Corp., Tarrytown, N.Y .) and two drops of latex particles (0.81, Difco) for 30 min at 37°C on a rocker platform. In most instances the mixture was then layered on Ficoll-Hypaque solution and spun for 45 min at 4OOg. The cells obtained at the interface were washed four times with Veronalbuffered saline (VBS), pH 7.2 (Inolex Corp., Glenwood, Ill.), containing 10% heat-inactivated fetal calf serum (FCS). Cells were then examined for viability by trypan blue dye exclusion. When normal and/or neoplastic lymphocytes accounted for more than 80% of leukocytes in the differential count, the buffy coat was incubated only with latex particles and washed without separation on a Ficoll- Hypaque gradient. Lymphoid tissues obtained at surgery were gently teased apart in culture medium and aspirated with a syringe through diminishing-gauge needles. The free-cell suspension was washed three times and examined for viability. When the number of contaminating erythrocytes was significant, they were lysed with ammonium chloride (15). Cell suspensions from spleens were usually separated on a Ficoll-Hypaque gradient. In all instances, suspensions of cells were preincubated with latex particles to distinguish contaminating monocytes and macrophages from lymphoid cells. Combined rosette assay (cEAC’ + SE). Fresh chicken erythrocytes (cE) were collected and washed three times with Alsever’s solution and stored at 4°C for no longer than 5 days. On the day of use they were washed three times in VBS and adjusted to a concentration of 0.5% (v/v of packed erythrocytes). Rabbit antichicken erythrocyte serum (A) was obtained from Cappell Laboratories (Downingtown, Pa.) and the proper nonagglutinating dilution determined for every batch. Fresh mouse serum (B,, New or B,, Old) at a dilution of 1: 10 was used as a source of complement (C’). A mixture of equal volumes of 0.5% cE + A + C’ was incubated at 37°C for 30 min on a rocking platform. The cells were washed with VBS, resuspended in one-third of the original volume of mixture, and used the same day. Sheep erythrocytes were stored in Alsever’s solution at 4°C and used for up to 2 weeks. On the day of use, they were washed three times in VBS and adjusted to a concentration of 0.5%.

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One million lymphocytes in 0.25 ml of VBS + 10% FCS were mixed with 0.25 ml sensitized cE and 0.25 ml of SE and incubated for 5 min at 37”C, spun at 3OOg for 5 min, and further incubated for 1 hr at 4°C. They were then gently resuspended with a Pasteur pipet, and rosettes were counted in a hemacytometer. Lymphocytes were considered positive when at least three chicken or sheep erythrocytes were firmly bound to the surface of latex-negative cells. At least three chicken and two sheep erythrocytes had to be rosetting about the same cell to consider it as a double receptor cell. Two hundred cells were counted for every sample, and the percentages of lymphocytes which formed rosettes with SE alone, cEAC’ alone, both SE and cEAC’ (Fig. I), and neither SE nor cEAC’ were determined. Another aliquot of cells from the same suspension tested by the combined rosette assay was spun in a cytocentrifuge (Shandon-Elliot) and stained with Wright’s-Giemsa to determine the morphology of the lymphoid cells forming rosettes. Chicken erythrocytes can be easily distinguished morphologically both in suspension and on stained slides by their nucleus, larger size, and ovoid shape. Unsensitized cE, in contrast to SE, do not spontaneously bind human leukocytes. An assay using cE sensitized with antibody but no complement was also performed as a control procedure (cEA). Surface immunoglobulin and aggregated immunoglobulin. Suspensions of cells tested by the combined rosette assay were also evaluated for SIg by immunofluorescence, using monospecific antisera against heavy and light chains as previously described (16). In many cases cells bearing Fc receptors were also quantitated by immunofluorescence using aggregated human IgG, as described by Dickler and Kunkel (17). RESULTS

Controls Study of blood from 100 healthy volunteers by the combined rosette method gave a mean value for double receptor lymphocytes of 0.8% with a standard deviation (SD) of 0.9%. The mean value for lymphocytes with only cEAC’ recep tors in the same combined assay was 9.2% (SD of 4%). The same numbers of cEAC’ rosettes were obtained when incubated at 4 or at 37°C. The mean value for lymphocytes with only SE rosettes in the combined rosette assay was 61% (SD of 9.50/o), and in the conventional single SE rosette assay run in parallel the value was 61.5% (SD of 8.7%). The mean value for the control cEA assay was I .3% (SD of 1%). Eighty-three percent of controls had 0- 1% double-labeled cells, 15% had 2-3%, and 2% had 4-6% double receptor lymphocytes (Table 1). In 18 controls the evaluation was repeated two or more times at a minimum of 2-week intervals and, usually, at monthly or longer intervals. Subjects with higher values showed similar values on repeated occasions. Similar consistency was observed in 15 patients with acute lymphocytic leukemia (ALL) in remission, when checked monthly or bimonthly. We designated, therefore, O-6% of double-labeled blood lymphocytes as the normal value for our system and laboratory; values above 6% were considered to be elevated. Various tissues not involved by neoplastic processes were also studied for double markers. Eleven bone marrows, seventeen lymph nodes, three tonsils, and

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six spleens were studied; all demonstrated less than 3% double receptor lymphocytes. Two thymuses obtained at surgery from patients with congenital heart defects demonstrated less than 2% double receptor lymphocytes. Neoplastic Lymphoid Cells with SE and Complement Receptors Four patients had malignant lymphoid cells that formed double (i.e., both SE and cEAC’) rosettes (Table 2, Fig. 1). In all cases greater than 40% malignant cells were present, permitting adequate evaluation. Two of the patients were children, and two were adults. The two children presented with mediastinal masses and had malignant lymphoid cells which formed double rosettes (i.e., SE + cEAC’) but were lacking other surface markers (i.e., Ig and Fc receptors). Clinical findings on these patients have been previously reported (18). The two adults with malignant cells forming SE and cEAC’ rosettes were older males with chronic lymphocytic leukemia (CLL) (Table 2). In contrast to the childhood cases, both had malignant cells that also carried surface Ig and Fc receptors. Non-Neoplastic Lymphocytes with Double Receptors Patients with lymphoid malignancies. Circulating lymphocytes were studied from 184 patients with lymphoid malignancy (lymphoma, Hodgkin’s disease, ALL) in whom the blood was not involved in Wrights-Giemsa-stained smears. Four patients demonstrated an increased percentage (7- 11%) of nonmalignant lymphocytes forming double rosettes. One patient had Hodgkin’s disease and was 17 years of age, and three children, ages 5,4, and 2 years, had ALL. Examination of malignant cells from other sites in these patients demonstrated that the malignancy did not involve double receptor cells. Patients with malignancy of nonlymphoid origin. Circulating lymphocytes were studied from 103 patients with malignancy of nonlymphoid origin, including 29 with acute myelogenous leukemia, 7 cases with chronic myelogenous leukemia, and 67 with other malignancies. In none of these patients was an increase in double receptor lymphocytes observed. Patients with non-neoplastic diseases. Of 183 patients with non-neoplastic diseases only five had increased percentages of double receptor lymphocytes (7- 15%) in the blood (Table 1). In these five patients the percentages of lympho-

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FIG. 1. Lymphoblasts from a patient with malignant lymphoma with receptors for both complement (cEAC’) (Cytocentrifuge, Wright’s+Giemsa, x 1200).

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cytes with other markers remained within normal limits for age as established in this laboratory. All five patients were children. Four were less than 3 years old; one had nephrotic syndrome secondary to a viral infection, one q-antitrypsin deficiency, one neutropenia, and one pneumonia. The fifth child, 11 years old, had viral encephalitis. These patients represent 6% of the 84 children under 14 years of age with various non-neoplastic diseases whose blood was studied. Four of 26 patients (15%) under 3 years of age were found to have elevated percentages of double receptor lymphocytes in the blood (Table 1). Tissue and/or blood was studied from 93 adults with a wide variety of nonneoplastic diseases, most commonly autoimmune diseases such as rheumatoid arthritis, lupus erythematosus, Sjogren’s syndrome, and various dermatologic conditions. No increases in double receptor lymphocytes were found. In the 90 adults whose blood was studied, the distribution of lymphocytes bearing double receptors was the same as it was in the 100 controls (Table 1). DISCUSSION

In the present study, chicken erythrocytes were chosen for the complement assay for a number of reasons. First, unlike SE, they do not bind spontaneously to human lymphocytes. Second, the nucleated cE are easy to distinguish morphologically from anuclear sheep and human erythrocytes. Third, when coated with antibody and complement, cE readily bind to complement receptor-bearing cells at 4°C; in our experience the percentage of blood lymphocytes forming cEAC’ rosettes at 4°C is the same as that obtained at 37°C. Because of these properties of cEAC’, we developed a combined rosette assay for SE and complement receptors

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which allowed the detection of cells with double markers. Chiao. ct d. (6) have reported a similar assay using pigeon erythrocytes. We have found that cells with both sheep erythrocyte and complement receptors are normally present in small numbers in blood and other tissues. Similar results have been reported by others (6,19,20). However, the nature and role of these cells remain uncertain. They may represent less mature. multipotential “stem” lymphocytes that are not committed to become B or T cells. They may also represent either an early or an activated subpopulation of T or B lymphocytes. It has been shown that activated T lymphocytes in mice, guinea pigs, and humans often develop Fc receptors (20-22). In our series four patients with lymphoid-malignancy had malignant cells which formed double (SE and cEAC’) rosettes. Three cases of lymphoproliferative malignancies with complement and SE receptors have been reported previously (10,13,14). The case of Lin and Hsu also had surface immunoglobulin ( 1I). Very recently an additional seven cases of lymphoblastic lymphoma with mediastinal masses (Sternberg’s sarcoma) in which the cells were bearing both SE and C’ receptors were reported by two groups (23,24). The data suggest that certain lymphoproliferative disorders are the result of clonal expansion of this double receptor population. It was of interest that our four patients included two children with diffuse lymphoma (Sternberg’s sarcoma) and two older patients with chronic lymphocytic leukemia. As shown in Table 2, malignant lymphoid cells were also studied using additional surface marker methods (SIg, aggregated IgG, and cEA). The results in the two CLL patients (patients 3 and 4) suggest that malignant lymphoid cells from some patients bear multiple markers (in this case SIg, Fc receptors, complement receptors, and sheep erythrocyte receptors). These patients are clearly different from most cases of CLL in which malignant cells bear Fc and complement receptors, as well as SIg, but not receptors for SE. The results presented in Table 2 also suggest that there may be differences in the sensitivity of the various methods. For example, IgG aggregates appear to detect Fc receptors better than cEA rosettes. However, the possibility that the disease represents a polyclonal proliferation cannot be excluded. Four individuals with lymphoid malignancy not involving the double receptor population had an increase in the “normal“ population of double receptor lymphocytes. It is possible that this represents an increase in normal “stem cells” in response to the lymphoid malignancy. This increase in double receptor cells was not observed in patients with malignancies of nonlymphoid origin. The increase in double receptor lymphocytes in young children with a variety of non-neoplastic conditions was of interest. The most likely explanation is that children have an increased number of double receptor lymphocytes in the basal state and/or under the stress of disease. Further studies to define these possibilities in normal and diseased children are necessary. ACKNOWLEDGMENTS The excellent technical assistance of Mrs. Mary Buechele, Mrs. Tanya Ivanov, and Mrs. Jacqueline Farmer and the secretarial and editorial assistance of Ms. Susan Perry are acknowledged. This work was supported in part by grants from USPHS (CA-17034, CA-16228, CA-08832. CA-15548, CA-07306, CA-05158, CA-16450, CA-1 1996, and CA-08101). the Leukemia Task Force (No. 37). Masonic Memorial Hospital Fund, Inc.. and the Minnesota Medical Foundation.

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REFERENCES 1. Jondal, M., Wigzell, H., and Aiuti, F., Transplant. Rev. 16, 163, 1973. 2. Kersey, J. H., and Gajl-Peczalska, K. J., Amer. J. Pathol. 81, 446, 1975. 3. Nussenzweig, V., Advan. Immunol. 18, 217, 1974. 4. Warner, N. L., Advan. Immunol. 19, 67, 1974. 5. Brown, G., and Greaves, M. F., Eur. J. Immunol. 4, 302, 1974. 6. Chiao, J. W., Pantic, V. S., and Good, R. A., Clin. Exp. Immunol. 18, 483, 1974. 7. Dickler, H. B., Adkinson, N. F., and Terry, W. D., Nature (London) 247, 213, 1974. 8. Greenberg, R. S., and Zatz, M. M., Nature (London) 257, 314, 1975. 9. Grey, H. M., Kubo, R. T., and Cerottini, J. C., J. Exp. Med. 136, 1323, 1972. 10. HSU, C. C. S., Marti, G. E., Schrek, R., and Williams, R. C., Clin. Zmmunol. Zmmunopathol. 3, 385, 1975. 11. Lin, P. S., and Hsu, C. C. S., Clin. Exp. Zmmunol. 23, 209, 1976. 12. Sandilands, G. P., Gray, K., Cooney, A., Browning, J. D., Grant, R. M., Anderson, J. R., Dagg, J. H., and Lucie, N., Lancer 1, 903, 1974. 13. Siegal, F. P., Voss, R., Al-Mondhiry, H., Polliack, A., Hansen, J. A., Siegal, M., and Good, R. A.,Amer. J. Med. 60, 157, 1976. 14. Shevach, E., Edelson, R., Frank, M., Lutzner, M., and Green, I., Proc. Nal. Acad. Sci. USA 71, 863, 1974. 15. Pemis, B., Fomi, L., and Amante, L., J. Exp. Med. 50, 1001, 1970. 16. Gajl-Peczalska, K. J., Bloomfield, C. D., Coccia, P. F., Sosin, H., Brunning, R. D., and Kersey, J. H., Amer. J. Med. 59, 674, 1975. 17. Dickler, H. B., and Kunkel, H. G., J. Exp. Med. 136, 191, 1972. 18. Coccia, P. F., Kersey, J. H., Gajl-Peczalska, K. J., Krivit, K., and Nesbit, M., Amer. J. Hematol. 1, 405, 1976. 19. Mendes, N. F., Miki, S. S., and Peixinho, Z. F., J. Zmmunol. 113, 531, 1974. 20. Smith, J. L., and Haegert, D., Clin. Exp. Zmmunol. 17, 547, 1974. 22. Van Boxel, J. A., and Rosenstreich, D. L., J. Exp. Med. 139, 1002, 1974. 23. Stein, H., Petersen, N., Gaedicke, G., Lennert, K., and Landbeck, G., Znr. J. Cancer 17, 292, 1976. 24. Jaffe, E. S., Braylan, R. C., Frank, M. M., Green, I., and Berard, C. W., Blood 48, 213, 1976.