I lymphocytes from patients with untreated

Transcription

1 T AND B-RFC INHIBITING FACTOR IN PLASMA FROM PATIENTS WITH ACTIVE HODGKIN'S DISEASE EDIZ Z. EZDINLI, MD,* KATHI L. SIMONSON, BS,? LLOYD G. SIMONSON, PHD,$ AND LARRY P. WASSER, MD$ We report the presence of a rosette inhibiting factor (RIF) in the plasma of patients with active Hodgkin's disease. This factor suppresses the rosette forming ability of autologous Active T, Total T, and B lymphocytes with sheep red blood cells, and tends to disappear when clinical remission is achieved. To a lesser extent, the RIF also lowers the Active T, Total T and B-RFC percentages of lymphocytes obtained from normal donors. Although carcinoma and non-hodgkin's lymphoma patients, as a group, did not exhibit rosette inhibitive properties, certain individuals with these diagnoses did show isolated RIF activity. The RIF could be adsorbed out of plasma using peripheral blood lymphocytes (PBL) from normal controls and appears to be a large heat stable molecule which does not affect PBL viability. Cancer 44: , N RECENT YEARS, it has been reported that I lymphocytes from patients with untreated and active Hodgkin's disease are defective in their ability to form rosettes with sheep red blood cell~.l.~," The defect in cell-mediated immunity in these patients can also be demonstrated by the impaired blastogenic response to previously encountered antigens and phy- tohemagglutinin (PHA)8,11,12,14 and by suppressed delayed hypersensitivity reaction^.^'^^ Studies have shown that this impaired response cannot be attributed to a mere quantitative depletion of circulating T-lymphocytes,2 and that the in vitro response to PHA can be restored by incubation in medium containing fetal calf serum.7 Recent investigations have suggested that the defect in cell-mediated immunity in Hodgkin's disease can also be From The Chicago Medical School, Chicago, Illinois, and Veterans Administration Hospital, North Chicago, Illinois. Supported by a Veterans Administration Central Office Research Grant, and NIH Grant #CA * Professor of Medicine, The Chicago Medical School; Chief, Oncology Section, Veterans Administration Hospital. t Research Technologist, Veterans Administration Hospital. $ Research Associate, Division of Oncology/Hematology, The Chicago Medical School. 5 Assistant Professor of Medicine, The Chicago Medical School; Staff Physician, Oncology Section, Veterans Administration Hospital. Address for reprints: Ediz Z. Ezdinli, MD, Veterans Administration Hospital, North Chicago, IL Accepted for publication August 15, caused by prostaglandin-producing suppressor cell^.^.'^ This study supports the findings of Fuks et al6 who reported the presence of a T- lymphocyte rosette inhibiting factor (RIF) in the serum of patients with active Hodgkin's disease. Using a routine quantitative assay procedure which does not require preincubation of lymphocytes, we demonstrated that this plasma factor inhibits autologous Total-T, Active-T, and B-lymphocyte rosetting. In addition, we demonstrated a suppressive effect of this factor on rosette forming cells (RFC) from control PBL donors. The plasma from normal donors or patients with other malignancies did not exhibit any inhibitory effect. Subjects X/79/0700/0 106 $ American Cancer Society 106 MATERIALS AND METHODS Plasma and peripheral blood lymphocytes (PBL) were isolated from blood samples taken from the following groups of patients and control subjects: I. Control Group: Twenty (20) healthy hospital employees (10 males, 10 females, with mean age of 32.3 years). II. Nonneoplastic Disease Group: Thirteen ( 13) patients with nonneoplastic disease (infectious mononucleosis-2; tuberculosis-4; diabetes-3; pneumonia-2; thrombophlebitis- 1 ; cirrhosis-1). The patient population

2 No. 1 ROSETTE INHIBITING FACTOR IN HD * Ezdinli et al. 107 III. IV. V. VI. included 11 males and 2 females with a mean age of 36.8 years. Solid Tumor Group: Twenty-five (25) untreated patients histologically diagnosed as having various types of recurrent or metastatic carcinoma (lung-9; gastrointestinal-7; head and neck-4; breast-2; genitourinary-2; melanoma-1). The group consists of 22 males and 3 females, with a mean age of 41.3 years. Non-Hodgkin s Lymphoma Group: Nine (9) untreated patients, 1 with Stage I11 and 8 with Stage IV disease. Three (3) were nodular-lymphocytic poorly differentiated (NLPD), 3 had diffuse lymphocytic poorly differentiated (DLPD), 2 were diffuse lymphocytic well differentiated (DLWD), and the remaining 2 were of the nodular mixed (NM) and diffuse histiocytic (DH) type. All were males with a mean age of 40.8 years. Hodgkin s Disease Croup, Active Dkease (HD-A): Twelve (12) patients with Stage 11, 111, or IV, and all with activity as demonstrated by measurable disease and/or symptoms. Stage distribution was as follows: IIA-1; IIB-2; IIIA-2; IIIB-1; IVB-6. One patient had HD of the lymphocytic predominance type (LP); 3 had nodular sclerosis (NS), and 8 were of the mixed cellularity type (MC). Ten (10) were males and 2 were females with a mean age of 31.8 years. Hodgkin s Disease Group, Clinical Remission (HD-R): Nine (9) patients with Stage I11 or IV disease in complete clinical remission for varying periods of time. The group consisted of 2 patients with NS, l with LD (depletion) and 6 with MC. Seven (7) were males and 2 were females with a mean age of 35.6 years. Plasma was drawn prior to treatment except in two patients with HD-A in whom it was drawn fourteen or more days after the last dose of chemotherapy to avoid any drug interference with the study. Isolation of PBL PBL from the individuals of each study group were isolated on a Ficoll-Hypaque gradient by centrif~gati0n.l~ The mono- nuclear cells were washed three times in phosphate buffered saline (PBS). One half (%) of the PBL were resuspended in PBS, while the remainder were resuspended in adsorbed plasma. In both cases, the final concentration of the PBL was 2 X 10g/ml. Preparation and Use of Plasma in Rosette Assays Heparinized blood samples were taken from the subjects in each study group. Plasma was obtained from its gradient layer formed in the Ficoll-Hypaque separation of PBL. Prior to storing the plasma at -20 C, each sample was adsorbed with an equal volume of packed and washed sheep red blood cells (SRBC) to prevent spontaneous agglutination in the rosette assays. In some experiments the plasma was dialyzed against PBS for 24 hours prior to its use in the rosette assays. In other experiments saturated ammonium sulphate was added to the control and HD plasma samples to a final concentration of 33%. The precipitates were centrifuged, dialyzed against PBS and resuspended in PBS to their original volume. Attempts to adsorb the RIF from HD plasma were made by adding 10 X lo6 PBL from a healthy donor to 2 ml of HD-A plasma. Following a thirty minute incubation at 37 C, the cell-plasma mixture was centrifuged. The adsorbed plasma was then used in the rosette assays. Also, the PBL used in this adsorption step were washed three times in PBS and then used in the rosette assays. The following anticancer drugs were added to normal plasma prior to adsorption with SRBC to check for any potential rosette inhibiting property: Hydrocortisone: 0.1 and 0.2 mg/ml; Adriamycin: 0.025, 0.124, and 0.25 mg/ml; Cytoxan: 0.50, 1.5, and 5.0 mg/ml; 5- Fluorouracil: 0.5, 1.5, and 5.0 mg/ml; Oncovin: ,0.0015, and mglml; BieoMOPP (Bleomycin: 0.05 mg/ml and Nitrogen Mustard: 0.04 mg/ml and Oncovin: 0.05 mg/ml and Procarbazine: 0.05 mg/ml and Hydrocortisone: 0.01 mg/ml). Sheep Red Blood Cells SRBC (stored no longer than two weeks) were washed three to five times in PBS. Packed SRBC were used in the adsorption step with the plasma samples. For the Total T and Active T rosette assays a 0.5% suspension of SRBC in PBS was used. For the B rosette

3 108 CANCER July 1979 Vol. 44 assay the SRBC were coated with mouse complement as previously de~cribed.'~ Rosette Assays The Total T, Active T, and B rosette assays were performed in duplicate as previously described.13 Briefly, for the Total T (E) rosette assay, 0.25 ml of PBL and 0.25 ml of SRBC were added to a 10 x 75 mm test tube and centrifuged for five minutes at 200 x g. The tubes were incubated for sixty minutes at 4 C, after which the pellets were gently resuspended and the rosettes were counted (a rosette is defined as any lymphocyte surrounded by three or more SRBC). The Active T assay was performed in a similar manner except the rosettes were counted immediately following centrifugation. In the B rosette assay (EAC) the PBL and sensitized SRBC were incubated for thirty minutes at 37 C prior to centrifugation and counting of rosettes. Statistical comparisons of the groups were made by analysis of variance using an F-test. Multiple comparisons of the groups for mean percent reduction in rosette formation was then performed by the Fisher least significant difference (LSD) test procedure. RESULTS The mean percent Total T, Active T, and B-RFC values within the subject groups using autologous plasma versus the values obtained in the presence of PBS is shown in Table 1. Plasma from the HD-A group caused the greatest decrease in Total T, Active T, and B rosette formation which was highly significant when compared to changes observed in the other groups (p <.001). The inhibition by autologous HD-A plasma interestingly affected Total T, Active T, and 3-RFC equally. The differences in PBS versus plasma RFC values obtained for the nonneoplastic, the non-hodgkin's lymphoma, the solid tumor, and the HD-R groups were all considered statistically equivalent to the values obtained for the healthy control group (Table 1). Therefore, the RIF was found consistently only in the HD-A group. However, in contrast to that observed in the control group, certain individual plasmas in the solid tumor group were capable of causing isolated RFC lowering. Plasma from a patient with advanced metastatic renal cell carcinoma caused a 51% decrease in Total T-RFC, a 33% decrease in Active T-RFC, but no change in B-RFC values. Isolated decreases in B-RFC values with autologous plasma were observed in one patient with pancreatic carcinoma (25.9%), one with pharyngeal carcinoma (23.5%), and another with carcinoma of the lung (20.0%). Decreases in Active T-RFC were also observed in one patient with mesothelioma (26.0%), vaginal carcinoma (44.4%), TABLE 1. A Comparison of Mean % RFC Values Obtained in PBS Versus Autologous Plasma and the Mean Difference Percent Mean % RFC (* SEM) Assayed with PBS Assayed with plasma Mean 7% difference (+SEM) No. of Total Active Total Active Total Active Subject group pts. T T B T T B T T B Normal control (1.3) (0.8) (0.8) (1.2) (0.9) (0.7) (1.1) (3.4) (2.8) Nonneoplastic disease (2.4) (1.9) (1.9) (2.3) (1.9) (1.9) (1.9) (4.3) (3.5) Solid tumor (1.7) (1.1) (1.1) (2.4) (1.1) (1.1) (2.6) (4.8) (3.6) Non-Hodgkin's lymphoma (3.7) (4.3) (3.7) (3.1) (5.1) (4.6) (2.8) (4.7) (3.3) Hodgkin's active (HD-A) (2.9) (1.6) (1.4) (5.0) (2.5) (2.1) (7.3)* (10.4)* (10.4)* Hodgkin's re mission (HD-R) (4.1) (1.9) (2.4) (3.0) (1.7) (2.3) (3.9) (4.6) (10.5) * Significant decrease (p < 0.001).

4 No. 1 ROSETTE INHIBITING FACTOR IN HD. Ezdinla et al. 109 TABLE 2. The Effect of Plasma and PBL Source on Percent RFC Reduction Relative to Values Obtained with PBS Subject group Mean % difference ( SEM) Plasma PBL No. of source source pts. Total T Active T B Control Control (1.1) (3.4) (2.8) Control HD-A (1.6) +7.9 (5.3) -2.3 (5.2) HD-A Control (3.7)* (7.5)* (6.2)* HD-A HD-A (7.3)* (10.4)* (10.4)* * Significant decrease compared to control plasma groups (p < 0.001). and epiglottis cancer (21.4%). One patient with lung cancer had a decrease in Total T-RFC (15.0%). Also, the plasma from one patient with the diagnosis of DLWD with monoclonal SIgM elevation caused a 23.1% lowering of Active T-RFC. The plasma from 2 patients with recently diagnosed infectious mononucleosis, whereas, did not have rosette inhibiting activity; the mean percent changes with autologous plasma were as follows: Total T (-2.1%); Active T (-4.5%); and B (+ 18.3%). In order to ensure that the inhibition of rosette formation by autologous HD-A plasma is not merely due to an inherent defect of these patients lymphocytes, additional tests with control PBL and plasma and HD-A PBL and plasma were performed. These results are shown in Table 2. Analysis revealed that significant differences exist between the groups. HD-A plasma (as compared to control plasma) significantly lowered Total T, Active T, and B-RFC percentages of both autologous and control PBL (p< 0.001). The HD-A plasma caused equally significant lowering of rosette values regardless of whether normal PBL or HD-A-PBL were used (p < 0.001). Rosette values with PBL from either HD-A patients or controls were not significantly different when assayed with control plasma (p > 0.05). An attempt was made to remove RIF from HD-A plasma by adsorption with control lymphocytes. When the HD-A plasma was adsorbed with control PBL and then used in the rosette assay, rosette inhibiting activity could no longer be demonstrated (% Total T-RFC: 63 vs. 68; % Active T-RFC: 28 vs. 30; % B-RFC: 22 vs. 27). Further, when control PBL were preincubated at 37 C with HD-A plasma, washed and then used in a rosette assay with PBS, there was again a decrease in rosetting (% Total T-RFC: 63 vs. 46; % Active T-RFC: 28 vs. 15; % B-RFC: 22 vs. 13). HD-A plasma was also serially diluted with PBS (25, 50, and 75%). As the ratio of PBS to HD-A plasma increased, a corresponding increase in rosette formation occurred. At a 1: 1 dilution, the HD-A plasma still retained significant rosette inhibiting activity against control PBL (mean decrease in Total T-RFC: 19%; Active T-RFC: 17%; B-RFC: 27%). With 25% plasma, rosette inhibiting activity was no longer demonstrable. Although specimens from two patients with active Hodgkin s disease (HD-A) were obtained fourteen or more days after the last dose of drug administration, it may be argued that the rosette inhibiting effect in these two instances might be related to the presence of drugs in the plasma. Therefore, we studied the potential rosette inhibitory effect in vitro of various anticancer drugs used in the two patients with active Hodgkin s disease. None of the drugs used in these patients or others tested (hydrocortisone, adriamycin, cytoxan, 5-fluorouracil, oncovin, and BleoMOPP combination) at the previously specified concentrations caused a reduction in rosette formation. Also, in 3 instances where serial RLF testing was done the RIF disappeared as the patients went into remission while still on chemotherapy. These observations indicate that the rosette inhibiting phenomenon observed in the plasma of HD-A patients is not related to circulating anticancer drugs. Dialysis of RIF positive plasma did not result in any loss of RIF activity, since the values obtained with either dialyzed or undialyzed plasma were identical. This observation also tends to reduce the possibility that the effect is due to either low molecular weight drugs or prostaglandins. In other experiments we fully recovered RIF from plasma by precipitation with 33% ammonium sulphate, followed by dialysis. This would

5 110 CANCER July 1979 VOl. 44 indicate that RIF is a large molecule, possibly a globulin. When RIF active plasma was heated at 56 C for one hour, there was no change in activity. DISCUSSION The present study seems to indicate the existence of a rosette inhibiting factor (RIF) in the plasma of patients with active Hodgkin s disease. All patients with Hodgkin s disease had the factor when unequivocal evidence of disease activity was present. In three patients who were serially tested, the RIF disappeared in two and decreased in the other following clinical remission. The RIF could not be demonstrated in the plasma of patients with nonneoplastic diseases, including two with infectious mononucleosis, or normal subjects. It was also not present in significant levels in the plasma of untreated patients with various types of solid tumors; however, some individual exceptions were noted in this group. Neither could we demonstrate RIF in the plasma of nine untreated patients with Stage I11 or IV non-hodgkin s lymphoma. The only exception was one patient with DLWD and macroglobulinemia where an isolated decrease of Active T-RFC occurred. This indicates that RIF may be present in malignancies other than Hodgkin s disease, at least during some stage of the disease. Our results corroborate an earlier report by Fuks et al. who described an RIF in the serum of untreated HD patients.6 However, they found that the maximum suppressive activity on E rosette formation occurred at a 20% serum concentration, while we found maximum activity in undiluted plasma. In contrast to their findings, we demonstrated that the HD-A plasma could significantly reduce RFC values even when assayed with PBL from healthy control subjects (Table Z), although the greatest reduction in RFC did occur when HD-A plasma was assayed with autologous lymphocytes. Any possible decrease in RFC values due to a loss of PBL viability caused by incubation with RIF containing plasma was ruled out since the viability of the PBL following incubation at 37 C for thirty minutes with the plasma was greater than 95% as assayed by trypan blue dye exclusion. The RIF described in the present study appears to be directed at both the T and B lymphocyte populations since the HD-A plasma reduced the RFC values almost equally in the Total T, Active T, and B rosette assays. This could indicate that the factor may block the common lymphocyte receptors present on the surface of T and B lymphocyte populations. This concept is further supported by our finding that RIF could be completely removed from the plasma by adsorption with normal PBL. Brooks et al. have described a suppressive factor isolated from the serum of patients with intracranial tumors which nonspecifically blocked mitogenic activity of the PBL to PHA from patients and normal subject^.^ This factor was associated with the globulin fraction of serum, and was found to disappear following tumor removal. Grifoni found that patients with HD may have serum antilymphocyte antibodies which can inhibit blastogenic responses to plant mitogens.1 It is unlikely that the presence of circulating drugs can be implicated for the rosette inhibiting effect. In the two HD-A patients who were on treatment, the blood samples were drawn more than fourteen days after the administration of drugs. When we added various anticancer drugs in different concentrations to normal plasma, no rosette inhibiting effect was observed. Furthermore, dialysis of RIF positive plasma against PBS for 24 hours resulted in no loss of RIF activity. Addition of saturated ammonium sulphate to HD-A plasma to a final 33% concentration, resulted in RIF precipitation. The resulting precipitate was dialyzed and reconstituted to the original plasma volume and found to retain its full inhibiting activity. Heating of RIF active plasma at 56 C for one hour did not change its activity. Therefore, the RIF described in the present study appears to be a nondialyzable, heat stable macromolecule which precipitates with the globulin fraction of plasma. Our method of detecting RIF appears to be as effective in demonstrating the inhibition of rosette formation as that used by Fuks et a1.,6 while being technically more direct and simpler to perform. The fact that RIF can be demonstrated at a 1: 1 dilution and by using PBL from normal controls might make it a valuable test for the confirmation of persistent or recurrent disease activity in Hodgkin s disease patients. We were not able to study untreated Stage IA or IB Hodgkin s disease patients for the presence or absence of RIF.

6 No. 1 ROSETTE INHIBITING FACTOR IN HD. Ezdinli et al. 111 These studies are now in progress. The fact that the effect of RIF can be demonstrated on PBL from normal subjects could make it a simple and valuable diagnostic tool or the confirmation of continued activity in Hodgkin s disease. 1. Anderson, E.: Depletion of thymus dependent lymphocytes in Hodgkin s disease. Scand. J. Haematol. 12: , Bobrove, A. M., Fuks, A., and Strober, S.: Quantitation of T and B lymphocytes and cellular immune function in Hodgkin s disease. Cancer 36: , Brooks, W. H., Netsky, M. G., Normansell, D. E., and Horwitz, D. A,: Depressed cell-mediated immunity in patients with primary intracranial tumors: Characterization of a humoral immunosuppressive factor. J. Exp. Med. 136: , Cohnen, G., Augener, W., Brittinger, J., and Douglas, S. D.: Rosette forming lymphocytes in Hodgkin s disease. N. Engl. J. Med. 289:863, Eltringham, J. R., and Kaplan, H. S.: Impaired delayed hypersensitivity responses in 154 patients with untreated Hodgkin s disease. Natl. Cancer Inst. Monogr. 36: , Fuks, A,, Strober, S., and Kaplan, H. S.: Interaction between serum factors and T lymphocytes in Hodgkin s disease. N. Engl. J. Med. 295: , Fuks, A., Strober, S., and King, D. P.: Reversal of surface abnormalities of T lymphocytes in Hodgkin s disease after in uitro incubation in fetal calf sera. J. Immunol. 117: , Gaines, J. D., Gilmer, A,, and Remington, J. S.: Deficiency of antigen recognition in Hodgkin s disease. Natl. Cancer h t. Monogr. 36: , Goodwin, J. S., Messner, R. P., Bankhurst, A. D., Peake, G. T., Saiki, J. H., and Williams, R. C.: REFERENCES Prostaglandin-producing suppressor cells in Hodgkin s disease. N. Engl. J. Med. 297: , Grifoni, V.: Recent immunologic findings in Hodgkin s disease. Tumori 59: , Han, T., and Sokal, J. E.: Lymphocyte response to phytohemagglutinin in Hodgkin s disease. Am. J. Med. 48~ , Hersh, E. M., and Oppenheim, J. J.: Impaired in uitro lymphocyte transformation in Hodgkin s disease. N. Eng2.J. Med. 273: , Kerman, R., Smith, R. A,, Ezdinli, E. Z., and Stefani, S.: Unification and technical aspects of total T, active T, and B lymphocyte Rosette assays. Immunol. Commun. 5: , Matchett, K. M., Huang, A. T., and Kremer, W. G.: Impaired lymphocyte transformation in Hodgkin s disease: Evidence for depletion of circulating T lymphocytes. J. Clin. Invest. 52: , Twomey, J. J., Laughter, A. H. and Farrow, S.: Hodgkin s disease: An immunodepleting and immunosuppressive disorder. J. Clin. Invest. 56: , Young, R. C., Corder, M. P., Haynes, H. A., and DeVita, V. T.: Delayed hypersensitivity of Hodgkin s disease: A study of 103 patients. Am. J. Med. 52:63-72, Yu, A., Watts, H., Jaffe, N., and Parkman, R.: Concomitant presence of tumor-specific cytotoxic and inhibitor lymphocytes in patients with osteogenic sarcoma. N. Engl. J. Med. 297: , 1977.