Masking of Receptors for Sheep Erythrocytes on Human T-Lymphocytes by Sera From Breast Cancer Patients1 , 2 R. H. Whitehead,3 G. P. Roberts,3 J. Thatcher,3 C. Teasdale/ and L. E. Hughes 3,4 ABSTRACT-Sera from 140 breast cancer patients and 38 controls were tested for their ability to inhibit sheep erythrocyte (E) rosette formation by normal, allogeneic lymphocytes. Inhibition of rosette formation by greater than 20% was found with 65% of stage I sera, 91% of stage II sera, 56% of stage III sera, and all stage IV sera. In contrast, only 13% of control sera was inhibitory. The inhibitory factor was found to bind to only a proportion of Tlymphocytes and could be removed from these lymphocytes by mild proteolytic digestion or extended washing. Examination of the properties of the inhibitory factor indicated that it differed from other substances that reportedly inhibited E-rosette formation.-J Natl Cancer Inst 58: 1573-1576, 1977.
E-rosette formation has been shown to be a specific property of human T -lymphocytes (l). In (2) we showed that the percentage of E-rosetting cells (T -lymphocytes) was depressed in most patients with breast cancer and that this depression could be reversed by treatment of the lymphocytes with papain. Reincubation of the papain-treated lymphocytes in autologous serum resulted in a return of depression of E-rosetting lymphocytes, which demonstrated the presence of a serum factor capable of producing this depression. In the present investigation, we have studied the effect of sera from breast cancer patients on E-rosette formation by normal lymphocytes. Examination of the properties of the factor that depresses E-rosette formation suggests that it varies from other factors reported to inhibit E-rosette formation and that it inhibits only a subpopulation of T-Iymphocytes. MATERIALS AND METHODS
Patients.-Sera were obtained from 140 breast cancer patients and 38 control patients (normal women and women with benign breast disease). We staged all patients using clinicopathologic data according to the TNM classification: 5 stage I, T 1-2 No Mo; stage II, T 1-2 N 1 Mo; stage III, T3-4 No-2 Mo; and stage IV, T I- 4 No-3 MI' The patients who were treated were retained in their pretreatment groups, unless there was evidence of recurrent or progressive disease either clinically or on isotope scintigraphy, when they were restaged accordingly. No patient had received chemotherapy or radiotherapy in the preceding 6 months. The sera were stored at 4° C and tested within 5 days of collection. Lymphocytes.-Lymphocytes were obtained from laboratory personnel by venipuncture, separated on FicollHypaque, and then washed twice in isotonic PBS (pH 7.2). The concentration was adjusted to 2x 106/ml for use. Rosetting methods.-We determined the number of Erosetting and EAC-rosetting cells using the methods of Anthony et al. (3), with the modification that rosetting was performed in 0.015 M PBS (pH 7.2). Serum inhibition oJE-rosetting.-Ofthe lymphocyte susVOL. 58, NO.6, JUNE 1977
1573
pension (5X 105 cells), 0.25 ml was ali quoted into small glass tubes and centrifuged at 200Xg for 5 minutes; the saline was removed and replaced with 0.25 ml of serum. The lymphocytes were resuspended in the serum, and the tubes were covered and incubated at 37° C for 1 hour. After incubation, the lymphocytes were washed three times in PBS with vigorous resuspension after each centrifugation step. The lymphocytes were finally resuspended in 0.25 ml of PBS, and their ability to form E-rosettes was determined. The procedure for testing the ability of sera to inhibit at 4° C was identical, except the I-hour incubation in sera was performed at 4° C. Sequential incubation in two cancer sera.-The initial incubation was as described, except the volumes of lymphocyte suspension and the initial serum used were doubled. After incubation and washing, 0.25 ml of lymphocyte suspension was removed for E-rosette determination, and the remaining lymphocytes were incubated in a second serum for an additional 1 hour before Erosetting. Absorption oj sera with RBC.-RBC from the lymphocyte donor were washed thoroughly in isotonic PBS, and 0.25 ml of packed RBC was incubated with 0.25 ml of serum for 1 hour at 37° C and 1 hour at 4° C with occasional shaking. The serum was then recovered by centrifugation and tested. Anti-HLA testing.-Sera were tested for anti-HLA activity by the National Institutes of Health lymphocytoxic technique against selected lymphocytes covering the following HLA antigens: HLA-Al, -A2, -A3, -A9, -AIO, -All, -A28, -A29, W30, W31, and W32; HLA-B5, -B7, -B8, -BI2, -BI4, -BI7, -B27; W15, W16, WI8, W21, W22, W35, and W40. (The testing was performed by Mr. C. Darke, Welsh Regional Transfusion Centre, Rhydlafar, Wales.) Dialysis.-Of the sera, 1 ml was dialyzed in Vis king dialysis tubing (one-fourth of an inch external diameter) against 500 ml of PBS for 72 hours at 4° C with two changes of PBS. Papain digestion.-Lymphocytes previously incubated in serum were washed and incubated at 37° C for 1 hour in 0.06 mg papain/ml (crystallized twice, 11 V/ml; ABBREVIATIONS USED: E=sheep erythrocyte(s); PBS=phosphatebuffered saline; EAC=erythrocyte-antibody-complement; RBC=red blood ceU(s). Received September 13, 1976; accepted November 30, 1976. Supported by a grant from the Cancer Research Campaign. 3 University Department of Surgery, The Welsh National School of Medicine, University of Wales. Heath Park, Cardiff CF4 4XN. United Kingdom. 4 We thank Miss Gill Richardson for assistance in obtaining blood samples and Mr. D. L. Jones for excellent technical assistance. 5 International Union Against Cancer classification system based on extent of primary tumor (T), condition of lymph nodes (N), and absence or presence of metastases (M). 1
2
J NATL CANCER INST
1574
WHITEHEAD, ROBERTS, THATCHER, TEASDALE, AND HUGHES
Sigma, London, England) in 2.5 mmole/liter cysteine hydrochloride in Earle's balanced salt solution containing 5% normal human serum (4). After incubation, the lymphocytes were washed in PBS, and rosetting was determined. Extended washing.-After incubation in serum and three washes, the lymphocytes were washed six more times in 2-ml aliquots of PBS with vigorous resuspension on a vortex mixer after each centrifugation step. After the ninth wash the lymphocytes were suspended in 0.25 ml of PBS, and the number of E-rosettes was determined. Assay of C-reactive protein.-The level of C-reactive protein in both inhibitory and noninhibitory sera was assayed with the use of radial immunodiffusion plates (Hoechst Pharmaceuticals, Hounslow, U.K.) Expression of results .-Two hundred lymphocytes were counted and the percentage of cells forming rosettes with three or more E was calculated. Results were expressed as percentage inhibition of E-rosette formation which equaled: percent rosettes following rosette formation in autologous serum-percent rosettes following incubation in cancer serum percent rosettes following incubation in autologous serum
X
100.
Significance of the inhibition in each cancer stage was determined by Student's t-test. Results were analyzed both on the basis of clinical stage and presence or absence of tumor. The significance of the mean inhibition of each group was determined by Student's t-test.
TABLE
No. of patients
Stage Control Stage I Stage II Stage III Stage IV Total cancer a
Percent inhibition a 13.0 ± 24.9 ± 32.8 ± 24.8 ± 35.4 ± 28.1 ±
38 43 34 44 19 140
, .,J
2.-Relationship of tumor presence to level of inhibition ofErosette formation by sera from breast cancer patients
Disease state
No. of patients
Percent inhibition a
22 80 25
27.2±13.5 28.8±1I.3 27.0±10.3
Preoperative Disease-free Recurrent a
Values are meanS±SD.
... ......
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INHIBITION
I
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E-rosette formation has been shown to be a specific property of human T -lymphocytes (l). In (2) we showed that the percentage of E-rosetting cells (T -lymphocytes) was depressed in most patients with breast cancer and that this depression could be reversed by treatment of the lymphocytes with papain. Reincubation of the papain-treated lymphocytes in autologous serum resulted in a return of depression of E-rosetting lymphocytes, which demonstrated the presence of a serum factor capable of producing this depression. In the present investigation, we have studied the effect of sera from breast cancer patients on E-rosette formation by normal lymphocytes. Examination of the properties of the factor that depresses E-rosette formation suggests that it varies from other factors reported to inhibit E-rosette formation and that it inhibits only a subpopulation of T-Iymphocytes. MATERIALS AND METHODS
Patients.-Sera were obtained from 140 breast cancer patients and 38 control patients (normal women and women with benign breast disease). We staged all patients using clinicopathologic data according to the TNM classification: 5 stage I, T 1-2 No Mo; stage II, T 1-2 N 1 Mo; stage III, T3-4 No-2 Mo; and stage IV, T I- 4 No-3 MI' The patients who were treated were retained in their pretreatment groups, unless there was evidence of recurrent or progressive disease either clinically or on isotope scintigraphy, when they were restaged accordingly. No patient had received chemotherapy or radiotherapy in the preceding 6 months. The sera were stored at 4° C and tested within 5 days of collection. Lymphocytes.-Lymphocytes were obtained from laboratory personnel by venipuncture, separated on FicollHypaque, and then washed twice in isotonic PBS (pH 7.2). The concentration was adjusted to 2x 106/ml for use. Rosetting methods.-We determined the number of Erosetting and EAC-rosetting cells using the methods of Anthony et al. (3), with the modification that rosetting was performed in 0.015 M PBS (pH 7.2). Serum inhibition oJE-rosetting.-Ofthe lymphocyte susVOL. 58, NO.6, JUNE 1977
1573
pension (5X 105 cells), 0.25 ml was ali quoted into small glass tubes and centrifuged at 200Xg for 5 minutes; the saline was removed and replaced with 0.25 ml of serum. The lymphocytes were resuspended in the serum, and the tubes were covered and incubated at 37° C for 1 hour. After incubation, the lymphocytes were washed three times in PBS with vigorous resuspension after each centrifugation step. The lymphocytes were finally resuspended in 0.25 ml of PBS, and their ability to form E-rosettes was determined. The procedure for testing the ability of sera to inhibit at 4° C was identical, except the I-hour incubation in sera was performed at 4° C. Sequential incubation in two cancer sera.-The initial incubation was as described, except the volumes of lymphocyte suspension and the initial serum used were doubled. After incubation and washing, 0.25 ml of lymphocyte suspension was removed for E-rosette determination, and the remaining lymphocytes were incubated in a second serum for an additional 1 hour before Erosetting. Absorption oj sera with RBC.-RBC from the lymphocyte donor were washed thoroughly in isotonic PBS, and 0.25 ml of packed RBC was incubated with 0.25 ml of serum for 1 hour at 37° C and 1 hour at 4° C with occasional shaking. The serum was then recovered by centrifugation and tested. Anti-HLA testing.-Sera were tested for anti-HLA activity by the National Institutes of Health lymphocytoxic technique against selected lymphocytes covering the following HLA antigens: HLA-Al, -A2, -A3, -A9, -AIO, -All, -A28, -A29, W30, W31, and W32; HLA-B5, -B7, -B8, -BI2, -BI4, -BI7, -B27; W15, W16, WI8, W21, W22, W35, and W40. (The testing was performed by Mr. C. Darke, Welsh Regional Transfusion Centre, Rhydlafar, Wales.) Dialysis.-Of the sera, 1 ml was dialyzed in Vis king dialysis tubing (one-fourth of an inch external diameter) against 500 ml of PBS for 72 hours at 4° C with two changes of PBS. Papain digestion.-Lymphocytes previously incubated in serum were washed and incubated at 37° C for 1 hour in 0.06 mg papain/ml (crystallized twice, 11 V/ml; ABBREVIATIONS USED: E=sheep erythrocyte(s); PBS=phosphatebuffered saline; EAC=erythrocyte-antibody-complement; RBC=red blood ceU(s). Received September 13, 1976; accepted November 30, 1976. Supported by a grant from the Cancer Research Campaign. 3 University Department of Surgery, The Welsh National School of Medicine, University of Wales. Heath Park, Cardiff CF4 4XN. United Kingdom. 4 We thank Miss Gill Richardson for assistance in obtaining blood samples and Mr. D. L. Jones for excellent technical assistance. 5 International Union Against Cancer classification system based on extent of primary tumor (T), condition of lymph nodes (N), and absence or presence of metastases (M). 1
2
J NATL CANCER INST
1574
WHITEHEAD, ROBERTS, THATCHER, TEASDALE, AND HUGHES
Sigma, London, England) in 2.5 mmole/liter cysteine hydrochloride in Earle's balanced salt solution containing 5% normal human serum (4). After incubation, the lymphocytes were washed in PBS, and rosetting was determined. Extended washing.-After incubation in serum and three washes, the lymphocytes were washed six more times in 2-ml aliquots of PBS with vigorous resuspension on a vortex mixer after each centrifugation step. After the ninth wash the lymphocytes were suspended in 0.25 ml of PBS, and the number of E-rosettes was determined. Assay of C-reactive protein.-The level of C-reactive protein in both inhibitory and noninhibitory sera was assayed with the use of radial immunodiffusion plates (Hoechst Pharmaceuticals, Hounslow, U.K.) Expression of results .-Two hundred lymphocytes were counted and the percentage of cells forming rosettes with three or more E was calculated. Results were expressed as percentage inhibition of E-rosette formation which equaled: percent rosettes following rosette formation in autologous serum-percent rosettes following incubation in cancer serum percent rosettes following incubation in autologous serum
X
100.
Significance of the inhibition in each cancer stage was determined by Student's t-test. Results were analyzed both on the basis of clinical stage and presence or absence of tumor. The significance of the mean inhibition of each group was determined by Student's t-test.
TABLE
No. of patients
Stage Control Stage I Stage II Stage III Stage IV Total cancer a
Percent inhibition a 13.0 ± 24.9 ± 32.8 ± 24.8 ± 35.4 ± 28.1 ±
38 43 34 44 19 140
, .,J
2.-Relationship of tumor presence to level of inhibition ofErosette formation by sera from breast cancer patients
Disease state
No. of patients
Percent inhibition a
22 80 25
27.2±13.5 28.8±1I.3 27.0±10.3
Preoperative Disease-free Recurrent a
Values are meanS±SD.
... ......
5~
INHIBITION
I
~
..t
...
. .t. .
!... ...
.+ .........
..... : . .!!a 10 t I :!! :
