CELLULAR IMMUNOLOGY :28, 1-14
(1977)
Reactions of Anti-Human Brain Serum with Human Lymphocyte Subpopulations1 JOAN A . ~TRATTON AND PATRIClA E . BYFIELD 2
Departments of Medich, e, Pediatrics, and Microbiology and Immunology, University of California at Los Angeles and Harbor General Hospital Campus, Torrance, California 90509 Received June 15, 1976 The reaction of serum from rabbits immunized with human brain ( R t t B ) with human lymphocytes has been measured by cytotoxicity, membrane immunofluorescence, and inhibition of lymphocyte function (mitogen-induced blastogenesis and lymphotoxin production). R H B kills most of the T-cells in the blood, but no reaction with B-cells or monocytes was detected. The remaining cells are reduced in their ability to respond to mitogen by lymphotoxin production and/or secretion. Treated P B L respond normally to P H A and P W M by incorporation of [3H]TdR, but the response to Con A is inhibited. The effect on lymphotoxin release is not dependent on cell death, as it occurs in the absence of added complement. R H B inhibits spontaneous E rosette formation by T-cells; again the effect does not depend on cell death but it requires approximately 10% normal serum--fresh or heated! The synergistic effect of normal and immune serum is apparently due to steric or charge effects of binding of some complement component(s), since normal serum depleted by incubation with antigen-antibody complexes supports rosette inhibition by rabbit anti-human brain serum very poorly, and purified Clq partially restores the inhibition. All of the measured properties of R H B are induced by immunization and can be produced by treating cells and washing away .the serum components which do not adhere. We conclude that there is a small subpopulation of human Iymphocytes, probably T-cells, which lacks the brain antigen and responds by D N A synthesis but not lymphotoxin to P H A and P W M stimulation. The majority of T-cells, including those responsive to Con A, bear an antigen(s) cross-reactive with brain; this antigen is not identical to the receptor for sheep red blood cells.
INTRODUCTION Lymphocytes can be broadly classified as thymus-derived (T) and bone-marrowderived (B), but it is clear that there are many functional subpopulations within these classes. Since lymphocytes of different subpopulations are not clearly distinguished by appearance, anatomical distribution, or physical properties, it is essential to find other ways of distinguishing and separating them. Antiserum is a par1This research was supported by Grant No. 1M-8 from the American Cancer Society, Grant No. 1 R O I CA 15054 from the National Cancer Institute and Grant No. GRSG-1780 from the National Institutes of Health. 2 Present address: Hypersensitivity Diseases Research, The Upjohn Company, Kalamazoo, Mich. 49001.
Copyright ,© 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0008-8749
2
STRATTON AND BYFIELD
ticularly valuable tool in studying lymphocyte subclasses, as it can be used for visual identification (1, 2), selective destruction (3, 4), or isolation (5, 6) of a given population. The alloantigens of mice, Ly and theta, have been used to identify T-cells and their subpopulations (3, 7, 8). Theta cross-reacts with antigen(s) on brain cells (4, 7), so that heterologous antibrain serum has been used as a substitute for antitheta (9). Human brain and T-lymphocytes also have at least one common antigen (10-12). Our purpose was to examine the potential usefulness of antiserum against human brain for identification of T-lymphocyte subpopulations. The antiserum was tested for its ability to kill human blood lymphocytes and to inhibit their rosette formation ; in addition, it was tested for inhibition of two responses, [SH]TdR incorporation and lymphotoxin release, after stimulation of the lymphocytes with the mitogens, PHA, PWM, and Con A. 3 M A T E R I A L S AND M E T H O D S
Media Hanks' balanced salt solution ( H B S S ) , Eagle's minimal essential medium with Hanks' salts (MEM), Roswell Park Memorial Institute Medium 1640 with 25 mM H E P E S buffer ( R P M I ) , fetal bovine (calf) serum (FBS), and human serum type AB (HuABS) were purchased from Grand Island Biological Company (GIBCO, Oakland, Calif.) or Microbiological Associates (Los Angeles, Calif.). The sera were heated to 56°C for 45 min before use.
Mitogens Purified phytohemagglutinin-P (PHA, MR69, Burroughs Wellcome, G.B.) was used at 0.2, 1.0, and 5/~g/ml in tube cultures and 0.5, 2.5, and 12.5 /,g/ml in microplate cultures. Pokeweed mitogen powder ( P W M ; Barber and Farmer, GIBCO) was diluted with HBSS and used at 1.0 and 5.0/~g/ml in tube cultures or 2.5 and 12.5 /,g/ml in microplates. Concanavalin A (Con A; Miles-Yeda, IZankakee, Ill.), was used in the same concentrations as PWM.
Lymphocyte Purification Human peripheral blood lymphocytes ( P B L ) were isolated from heparinized blood by isopycnic density gradient centrifugation (13) as previously described (14). The usual yield of mononuclear cells from normal subjects by this procedure is 85-95 % comprising 70-85 % lymphocytes and 15-305~ monocytes by ~-naphthylesterase stain (Sieger et al., in preparation). Tonsil and adenoid (TAL) tissue s Abbreviations used: C, guinea pig complement; CLL, chronic lymphocytic leukemia; Con
A, Concanavalin A; EAC-RFC, mononuclear cells with complement receptors; E-RFC, mononuclear cells forming spontaneous SRBC rosettes; EHT, equine anti-human thymus globulin (Upjohn ATG); FBS, fetal bovine serum, GPS, guinea pig serum; HBSS, Hanks' balanced salt solution; HuABS, human serum type AB; Lt, lymphotoxin; MEM, Eagle's minimal essential medium; PBL, peripheral blood lymphocytes; PHA, Phy,tohemagglutinin-P; PWM, pokeweed mitogen; RPMI, Roswell Park Memorial Institute Medium 1640; SRBC, sheep red blood cells; RHB, rabbit anti-human brain serum; RILL, rabbit anti-human light chain serum; TAL, tonsil and adenoid lymphocytes; TDL, thoracic duct lymphocytes.
ANTI-HUMAN
BRAIN SERU1V£
3
from children 4 to 12 years of age was obtained courtesy of Dr. L. Sieger. The tissue was dissected clear of blood and teased to a single cell suspension in HBSS (containing 10 times the usual antibiotic concentration). Human spleen tissue was obtained courtesy of Dr. A. Saxon. Single cell preparations were prepared by passage through a 250-gauge screen. Human thoracic duct lymphocytes (TDL) were obtained from patients with rheumatoid arthritis during continuous drainage from a thoracic duct fistula, courtesy of Dr. I-I. Paulus. TDL were separated from the heparinized lymph by eentrifugation. The cells were washed three times in HBSS before use.
Conditions for Lymphocyte Culture and Assay of [~H] Thymidine Incorporation Lymphocytes were cultured for 5 days in R P M I supplemental with 5% serum and antibiotics in plastic culture plates with and without mitogens, as previously described (14). The cells were labeled during the last 24 hr of culture with 0.1 ml of 0.4 mM [~H]thymidine ([3H]TdR; [6-~H]thymidine, TRA 61, sp act 19 Ci/mmol; Amersham Searle, Arlington Heights, Ill.). The lymphocytes were harvested onto glass fiber filter paper and washed with water followed by 5% trichloroacetic acid using a multiple automatic sample harvester ( M A S H II, Microbiological Associates). Acid-precipitable radioactivity was determined by scintillation counting.
L ymphotoxin Assay For the production of lymphotoxin (Lt), 0.5 × 100 lymphocytes were cultured in 1 ml; the supernatants from triplicate cultures were pooled and either assayed immediately or stored at 0°C. The pooled supernatants from the cultures were assayed for cytotoxicity on L-cells (mouse fibroblasts 929, ~ strain, courtesy of Dr. G. A. Granger, University of California at Irvine). The Lt assay method was essentially that of Granger and co-workers (15, 16) except that cell death was measured by release of 51Cr from prelabeled L-cells (17). Briefly, 1 X 105 cells in 1 ml of MEM with 3% FBS were incubated overnight with 1 txCi/ml of 51Cr (Na251CrO4, 200 /~Ci//~g, International Chemical Nuclear Corporation, Irvine, Calif.) and 0.5/~g/ml mitomycin C (GIBCO). L-cells were rinsed once with MEM containing 3% FBS, and diluted lymphocyte culture fluids were added. After 16 hr at 37°C, the supernatants were decanted and the amount of 5~Cr present in both supernatant and L-cells was determined in a gamma counter. The Lt titer is expressed as the reciprocal of the dilution of lymphocyte culture fluid resulting in a 10% specific 5~Cr release; where specific release is defined as percentage of ~lCr released by the fluid from the stimulated lymphocyte culture, minus percentage of 5zCr released by the fluid from the nonstimulated control culture.
Antisera Equine anti-human thymus globulin ( E H T ) was obtained courtesy of Dr. G. D. Gray (Lot No. 16, 138-9, Upjohn, Kalamazoo, Mich.). Rabbit anti-human brain serum ( R H B ) was prepared by immunizing New Zealand white rabbits with homogenized tissue from human cerebrum (14). The animals did not exhibit gross neurological disturbances. Rabbit anti-human light chain ( R H L ) serum was prepared by immunizing New Zealand white rabbits with human light chains (14)
4
STRATTON AND BYFIELD
prepared from pooled human AB serum (18). If its serum contained cytotoxie activity against human lymphocytes before immunization, the rabbit was not used.
A bsorption of Antisera RHB and R H L were absorbed undiluted, but E H T was diluted 1:50 in HBSS. Sera were incubated with 10~ washed lymphocytes/ml at room temperature for 1 hr and then in an ice bath for another hour. Some sera were absorbed with 10% (v/v) RBC (human or sheep) or with 50% (v/v) human platelet membranes for 30 min in an ice bath.
Antiserum Treatment of Lymphocyte Cultures In some early experiments, sera were added to the cuItures and left there. Later, lymphocytes were incubated with the appropriate concentration of FBS, RHB, R H L (all 1:50), or E H T (1:1000-1:10,000) in RPMI and 1:10 guinea pig complement (C; GIBCO) for 1 hr at 37°C. The lymphocytes were washed once with HBSS, the viability was assessed by trypan blue, and equal numbers of viable cells were cultured in RPMI with 5% HuABS as described above. The two methods were equivalent.
Chromium Release Assay for Lymphoeytoto.ric Antibody Lymphocytes were suspended in HBSS (1 × 107/ml) with 10-20 /,Ci/ml 51Cr for 30-60 min at 37°C, washed twice, and finally resuspended in HBSS. Labeled lymphocytes (0.5-1 × 106), the appropriate dilution of test serum, and 1:10 C (guinea pig, GIBCO; rabbit, chicken, and human, freshly drawn serum) were added to each tube. The tubes were incubated for 45 min at 37°C and spun at 1800g for 15 min, the supernatant was decanted, and the amount of ~lCr released into the supernatant was calculated as a percentage of total 5~Cr label. All sera (including complement) were absorbed with sheep red blood cells (10%, v/v) and agarose (1% w/v). All dilutions of sera were tested in triplicate; also included were controls containing FBS plus C and distilled water to measure minimum and maximum chromium release, respectively. Nonspecific 5~Cr release (FBS) was 9-28%, while maximum ~lCr release (water) was 75-85%.
Conditions for Rosette Forming Cell (RFC) Assay To form rosettes, mononuclear ceils were washed and suspended at 2 × 106/ml in HBSS. For E-RFC the cells were mixed with an equal volume of 1% washed sheep red blood cells (SRBC, Mission Laboratories, Rosemead, Calif.), incubated at 37°C for 15 rain, centrifuged at 200g for 5 min. "Cold" E-RFC were kept on ice overnight (14, 19) while "warm" E-RFC were incubated for 2 hr at room temperature (20) before gently resuspending and reading. "Warm" E-RFC were prepared in HBSS with 20% HuABS and "cold" E-RFC in HBSS without serum. To detect complement receptors, SRBC were sensitized with rabbit antibody to SRBC followed by human complement (EAC), added to an equal volume of mononuclear ceils, spun at 200g for 2 rain and, after 15 rain at room temperature, vigorously resuspended and read (14, 21). RFC were counted either fresh, after addition of toluidene blue (22) or, after fixation with 2 vo! of 1% gluteraldehyde
ANTI-IIU~fAN
BRAIN
SERUlX¢
TABLE 1 Reagents for Indirect Membrane Fluorescence Serum" Equine anti-human thymus globulin (EHT) Rabbit anti-human brain (RHB) Rabbit anti-human L Chains (RHL)
Dilution 1 : 1000 to 1: 1 0 , 0 0 0 1 : 50 to 1 : 100 1 : 50 to 1 : 100
Fluoresceinated Serum
Dilution
Source
Rabbit anti-equine v2globulin Goat anti-rabbit v-globulin Goat anti-rabbit -r-globulin
1:20
Miles-Yeda
1 : 20
Meloy
1 : 20
Meloy
See text for source or preparation. in Tyrode's Solution (23), in smears stained with Wright's or May Grfinwald stain. I?44/lq~t~nofl~l~oresc enc g
Direct membrane fluorescence. Ten microliters of fluoresceinated polyvalent goat anti-human immune globulins (No. C201, Meloy, Springfield, Va.) was added to 0.2 ml of cell suspension, incubated for 1 hr in an ice bath, washed three times in ice-cold H B S S and resuspended in glycerine mounting medium (Becton-Dickinson, Cockeysville, Md). The percentage of mononuclear cells displaying membrane fluorescence of a "ring" or granular type was determined using a Nikon S I R - F fluorescence microscope. Indirect membrane fluorescence. The lymphocytes were incubated with antiserum (see Table 1) for 1 hr in an ice bath, washed three times in cold H B S S and stained with the appropriate fluoresceinated antiserum as above. RESULTS
Complement-Mediated Cytotoxicity The percentage release of slCr from prelabeled human peripheral blood lymphocytes ( P B L ) by three antisera in the presence of guinea pig complement (C) is shown in Fig. 1. R H B specifically released 50% and E H T about 30% of slCr from P B L at 1:50 dilution. R H L was the least cytotoxic, causing only 20% specific 51Cr release at the same dilution. Although the chromium release method is convenient for comparative titrations, it does not necessarily reflect the exact number of cells killed. Consequently, we also used trypan blue exclusion to confirm the cytotoxic activity of the sera. With 1 : i 0 C plus F B S or normal rabbit serum, viability was greater than 95%, while R H B plus 1 : 10 C killed a maximum of 50% ; which was comparable to the results of the chromium release assay. The cytotoxic activity of R H B on cells from various human lymphoid tissues is depicted in Table 2. Normal P B L are the most, and spleen is the least, susceptible to lysis with the antiserum: There is no cytotoxic activity in the absence of C or with 10% heated serum. R H B plus C kills only a small fraction of lymphocytes from the P B L of patients with C LL, m~d only at the highest concentration, The~
6
STRATTON
AND
BYFIELD
70-
~:~~~ RHB
/ / / / / /
SO"
/ 50-
/ ~ ,3 4o-
Y~
i.d
E
/ H
T
/y/"
;RHL
50-
20-
0
~ I I I I0,000 IOOO I00 RECIPROCAL OF SERUM DILUTION
FBS
I0
Fro. 1. The cytotoxic activity of three antisera on human peripheral blood lymphocytes. The ordinate shows the percentage of SlCr release from PBL treated with rabbit anti-human brain (RIIB, O . . . . . O), equine anti-human thymus globulin (EHT; • • ) , rabbit antibody to human light chains (RHL; A-.-.-.A), and fetal bovine serum (FBS; , ) . Each point represents 4-10 experiments, except 1:500 RHB, which was done twice. The vertical bars show the standard error of the mean. high sensitivity of the bone marrow lymphocytes is probably due in part to contamination of the aspirates with blood.
Removal o/ Cytoto.vic Antibody by Absorption The antisera used in most of the preceding experiments have not been absorbed. To show that the cytotoxie action of R H B was specific for thymus-derived lymphocytes (T-cells) and the R H L specific for immunoglobulin-bearing lymphocytes (Bcells) the sera were absorbed with P B L from normal subjects, consisting primarily of T-cells, and from patients with C L L or a cultured lymphoblastoid line (Rail; courtesy of J. K. Seeley, U C L A ) , both of which are B-cells. The CLL-lymphocytes used for absorption included less than 1~o T-cells, as determined by spontaneous rosette formation and less than 3% T-cells as determined by fluorescent staining with RHB. Three absorptions with CLL-cells did not change the cytotoxic activity of R H B on PBL, whereas one absorption with P B L significantly decreased the cytotoxic activity ( P < 0.01) (Table 3). Absorption of R H B four times with Raji cells, decreased the cytotoxicity by 10% (J. K. Seeley, personal communication). Absorption of E H T with P B L and T A L produced neither discernible diminution of cytotoxic, rosette-inhibiting nor immunofluorescent staining activity Of the serur0 eve!a after 18 absorptions with a total of 1.8 × 10 s cells: Absorption
ANTI-HUMAN
BRAIN
TABLE C y t o t o x i c A c t i v i t y of R H B Serum dilution
Cb
SERUM
2
on Cells from Various Lymphoid
Tissues
Percentage release of 51Cr from prelabeled cells Source of lymphoid tissue Peripheral blood Normal
None
. -1-Ac -[~ -t-lq-
1 : 10 NS~
1:10 NS 1:10 R H B I:10 RHB 1:10 R H B l:25RHB 1:50 R H B I:100RHB 1:200 R H B
Spleen
Bone marrow
8.1 4- 0.6 . 12.3 d= 0.3 14.4 4-0.7 18.0 4- 3.5 . 58.84-1.7 21.6 4- 0.7 13.94-0.5 --
-. 23.8 4- 1.7 --. . 71.14-2.3 64.7 4- 0.5 51.44-3.0 34.2 4- 0.2
CLL
__a . 26.1 4- 0.4e ---75.54-0.4 66.9 4- 0.5 60.74-0.6 52.5 4- 1.2
__ .
. 9.2 4- 0.6 11.8 -4-0.9 7.3 4- 1.0 21.2 4-2.2 5.74-1.2 ----
Tonsil and adenoid
Thoracic duct lymph
--
--
31.0 4- 0.3 --. -54.5 4- 0.6 -38.7 4- 0.8
15.1 4- 1.0 ---
.
52.84-1.0 43.2 =k 1.0 ---
a Normal serum ; rabbit, fetal calf, or human. b 10% guinea pig serum. c Guinea pig serum heated to 56°C for 2 hr. a Not tested. 5~Cr release, mean 4- standard error of the mean, number of samples greater than five.
with 1 × 109 cultured lymphoblasts removed most of the cytotoxic activity (P. E. Byfield, unpublished observations). Rosette Inhibition
The three antisera were tested for their ability to inhibit rosette formation by PBL, with or without C. Both E-RFC (T-cells) and EAC-RFC (B-cells and monoeytes) were measured (Table 4). E H T inhibited E rosette formation at dilutions up to 1:1000 in the absence of C; the addition of C did not increase the inhibition of E H T even at 1:10,000 which only partially inhibited E-RFC (Table 4). E H T did not inhibit EAC rosette formation. R H L inhibited EAC rosette formation in a complement requiring reaction, but TABLE Cytotoxicity
of R a b b i t
Anti-Human
Brain
Serum
3 to
Human
Lymphocytes
as Measured
by
S p e c i f i c R e l e a s e of 51Cr f r o m P r e l a b e l e d L y m p h o c y t e s Antiserum
Percentage
of 51Cr r e l e a s e ~
dilution 1:25 Unabsorbed
1:50
1:100
3 6 . 4 4- 1.9 (3) b
33.1 4- 5.9
(9)
23.8 4- 1.8 (3) ~
12.9 4- 2.6
(3) ~
0.7 4- 3.9 (5) c
1.1 4- 2.2
(6) c
22.5 4- 6.4
(9)
Absorbed 1X PBL 3X
PBL
3X
CLL a
38.1 4- 1.9 (4)
I n t h e p r e s e n c e of 1 : 1 0 G p C ; Materials and Methods section. b Mean cp
:t: s t a n d a r d
d Lymphocytes rosette formation,
see
( n u m b e r of s a m p l e s ) .
to unabsorbed
from a patient
(2)~
2 3 . 8 4- 7.1 (10)
a c o n t r o l f o r n o n s p e c i f i c 5~Cr r e l e a s e h a s b e e n s u b t r a c t e d ,
error of mean
< 0.01 w h e n c o m p a r e d
26.9 4- 5.6 (12)
-3.9 4- 2.4
serum.
with chronic lymphocytic
leukemia;
less t h a n
1% T-cells by
8
STRATTON AND BYFIELD
TABLE 4 Inhibition of Rosette Formation by Antisera Directed against T- or B-Cells Serum
Dilution
Percentage inhibition~ E-RFC Complement
FBS RHB RHB (4 X CLL) * EHT EHT EHT (4 X CLL) ~ RHL RHL (2 × CLL) o
1:50 1:50 1:50 1:1000 1 : 10,000 1:1000 1:50 1:50
EAC-RFC
--
-b
Ab
--
q-
2~ 2 5 96 38 92 12 --
7 87 83 95 38 91 8 --
13 94 --a ------
10 13 -10 --2 2
8 13 4 11 --61 48
a Percentage inhibition = No. RFC Control/No. Mononuclear C e l l s - No. RFC Experimental/No. Mononuclear Cells > 0.5). To confirm the specificity of the antibrain serum for T-lymphocytes, we stained P B L or, in one experiment, spleen cells, with anti-immunoglobulin or R H B and
ANTI-HUMAN
ll
BRAIN SERUS{
TABLE 6 Membrane hnmunofluorescent Staining of Rosette-Forming Cells Percentage stained All lymphocytes~
1b 2 3 4 5
EAC Rosettes
Anti Ig (B-cells)
Antibrain
Anti Ig (B-cells)
17.9 9.0 10.5 10.8 12.0
58.3 69.7 77.7 89.2 71.0
82.2 87.5 100.0 83.1 100.0
Antibrain 1 1 1.5 1 1
E Rosettes 6 7 8 9 10
7.1 --° 17.8 ---
-87.5 67.9 84.2 73.2
1 -1 ---
-100.0 75.2 98.8 96.6
a The same fluorescent anti-rabbit 7-globulin was used in both tests. b The cells used in the experiments were peripheral blood lymphocytes except Experiment 8 which was spleen tissue. c Not tested. then formed either E or E A C rosettes. Individual cells were scored for both markers by examination under visible and ultraviolet light. Table 6 shows the fraction of total lymphocytes and of rosettes in each preparation which were stained by R H B or, as a control, antiimmunoglobulin serum. (The percentage of E A C rosettes formed under these conditions was normal, but E rosette formation was inhibited to some extent by chilling the cells). Nearly all the E A C rosettes bear surface immunoglobulin (82.2-100%), and almost none are stained by R H B (Table 6). E rosette forming cells did not have detectable surface immunoglobulin, and almost all rosettes were stained with RHB.
DISCUSSION To determine the specificity of the sera for functional lymphocyte subpopulations, the ability of human lymphoeytes to incorporate [ a H ] T d R on stimulation with three mitogens, P H A , P W M , and Con A, was determined after treatment with R H B (with or without added guinea pig C). Treatment with R H B did not inhibit the response to P H A nor to P W M (except in one experiment). [In a shnilar experiment, anti-fetal human brain serum stained neither P H A - nor PWM-indueed lymphoblasts (12). This serum, however, apparently has a more limited specificity than ours.] The response to Con A, on the other hand, was significantly inhibited by R H B (in all but one experiment). This inhibition could occur in the absence of added C; therefore, it is not due to cell death. Cells were inhibited when treated, washed, and then cultured. Apparently antibody coating the cells blocked their response to Con A. A similar selective complement-independent inhibition was noted by Owen and Fanger (35) using rabbit antiserum to human thymus. In the
~2
STRATTON
AND
BYFIELD
1000- PHA
~,
500i
]0050-
I0 [-I NS + c rY w II--
500 -
• RHS+C
PWM
[ ] RHB + AC [ ] RHB
z_ x o Io :E
I0050-
_j> NT
10 500-
Con A
100: 50-
Io
SG-2 FB-3 dS-I KS-I KS'2 BB-I
SUBJECT
FIG. 3. Effect of treatment with RHB on lymphotoxin (Lt) production by mitogen-stimulated PBL. The ordinate shows the titer of Lt after stimulation with PHA (top), PWM (middle) or Con A (bottom). Open bars represent the Lt titer after treatment with 1 : 50 NRS, or FBS plus 1:10 C; shaded bars, 1:50 RHB plus 1:10 C; hatched bars, 1:g0 RHB plus 1:10 C heated at 56°C for 1 hr ; and the stippled bars, 1 : 50 RHB without C. Vertical bars represent the standard error of the mean. The asterisks indicate reductions in Lt titer which are significant (P < 0.01) by Student's t test. mixed lymphocyte reaction, Opelz and colleagues (26) found that coating the responder, but not the stimulator cells, with antihuman brain serum blocks the reaction. One interpretation of our data is that there is a subpopulation of lymphocytes which respond to P H A but not Con A by blastogenesis and that these cells are unaffected by R H B , while a distinct subpopulation responds to Con A (and perhaps also to P H A and P W M ) and is inhibited by R H B . By separating cells on the basis of their Fc receptors, Stout and Herzenberg (27) have isolated two populations of T-cells from mouse spleen; one ( F c + ) responds to stimulation with both P H A and Con A, while the other ( F c - ) responds only to P H A stimulation. W e may be seeing a similar dichotomy.
ANTi-HUN[AN BRA][N SERUM:
13
The response to mitogens as measured by lymphotoxin release, however, was quite sensitive to R H B treatment. The treatment inhibited lymphotoxin response to PHA, PWM, and Con A, even in the absence of complement. We may speculate that these cells correspond to the F c - population of Stout and Herzenberg (27) or that they are a third population. (For a discussion of B-cell responses to these mitogens, see Refs. 30-34). To further delineate the specificity of these sera, they were tested for rosette inhibition. R H B inhibits spontaneous E rosettes, formed by T-cells, but the EAC rosettes formed by B-cells and monocytes are not affected. Unlike the inhibition of E rosettes by E H T (equine antithymus globulin), inhibition by R H B requires the addition of 10~ guinea pig, human, rabbit, or chicken serum as a complement source. Presumably, E H T reacts directly with the receptors for sheep erythrocytes while R H B does not. To our surprise, we found that the sera used as complement sources were still effective after heating at 56°C for 45-120 rain! Heated sera did not support cytotoxic reactions ( R H B was routinely heated for 45 min), nor did they inhibit rosette formation when used alone. Both R H B and C were absorbed with agarose to remove natural cytotoxic antibody (28) and with SRBC to remove anti-erythrocyte antibodies which might inhibit rosette formation (29). The synergistic effect of heated normal sera and heated immune sera is apparently due to some complement components, since serum depleted by incubation with antigenantibody complexes supports R H B rosette inhibition very poorly. Purified Clq partially restores the inhibition. Probably, binding of early complement components by the "brain-antigen"-antibody complex causes steric or charge interference with the erythrocyte binding. It seems, in view of this finding, that the brain crossreactive antigen is not part of the erythrocyte receptor, although it does occur on the same cells. Additional support for our hypothesis is the failure of our R H B to block staining of human P B L by an anti-T-cell serum which inhibits rosette formation (36). In summary, we find that R H B reacts with most T-lymphocytes in the blood, and with few, if any, B-cells or monocytes. R H B kills most T-lymphocytes; the remaining cells respond normally to P H A and PWM, by the criterion of [*H]TdR incorporation, while the response to Con A is inhibited. The release of lymphotoxin in response to all three mitogens is inhibited, on the other hand, after R H B treatment. Inhibition of Lt release is not dependent upon cell death, as it does not require added C. R H B inhibits spontaneous rosette formation. Again, the effect is not dependent upon cell death, but it does require the addition of approximately 10~o fresh or heated normal serum. Thus, the inhibition is due to steric or charge interference with the sheep erythrocyte receptor rather than direct reaction of R H B with the receptor. All inhibitory activities of R H B are induced by immunization of the rabbits, and can be induced by treating cells with R H B and washing the cells. None of the activities of R H B can be removed by absorption with B-cells (CLL lymphocytes or cultured lymphoblasts), but all are removed by absorption with PBL and TAL. We conclude that most T-cells carry an antigen (or antigens) cross-reactive with brain. This antigen is not the sheep erythrocyte receptor. A subpopulation of human blood lymphocytes which apparently lacks the brain antigen(s) responds to P H A and P W M by DNA synthesis but not Lt release. These may be B-cells (see Refs. 30-34) or a small subpopulation of T-cells.
izl
STRATTON
AND
BYFIELD
ACKNOWLEDGMENTS We thank Drs. David T. T. Yu and Edward S. Golub for their critical appraisal, Dr. B. S. Rabin for helpful discussion, Ms. Janet Seeley for cooperative experiments, and Ms. Diane Cole for superb technical assistance.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Raft, M. C., Immunology 19, 637, 1970. Froland, S. S., and Natvig, J. B., Transplant. Rev. 16, 114, 1973. Raft, M. C., Transplant. Rev. 6, 52, 1971. Golub, E. S., Cell. Immunol. 2, 353, 1971. Hulett, H. R., Bonner, W. A., Barret, J., and Herzenberg, L. A., Science 166, 747, 1969. Jones, P. P., Cebra, J. J., and I.ierzenberg, L. A., I. Immunol. 111, 1334, 1973. Reif, A. E., and Allen, J. M. V, J. Exp. Med. 120, 413, 1964. ShiktL H., Kisielow, P., Bean, 1VL A., Takahashi, T., Boyse, E. A., Oettgen, H. F., and Old, L. J., J. Exp. Med. 141, 227, 1975. 9. Clagett, J., Peter, I-I. H., Feldmann, J. D., and Weigle, W. 0., J. ImmunoI. 110, 1085, 1973. 10. Stratton, J. A., and Byfield, P. E., Fed. Proe. 32, 976, 1973. 11. Greaves, M. F., and Brown, G., J. Immunol. 112, 420, 1974. 12. Brouet, J. C., and Toben, H., f. Imrnunol. 116, 1041, 1976. 13. Perper, R. J., Zee, T. W., and Mickelson, M. W., Y. Lab. Clin. Med. 72, 842, 1968. 14. Stratton, J. A., Byfield, P. E., Byfield, J. E., Small, R., Pilch, Y., and Benfield, J., J. Clin. Invest. 56, 88, 1975. 15. Granger, G. A., and Williams, T. W., Progr. Immunol. 1, 438, 1971. 16. Spofford, B. T., Daynes, R. A., and Granger, G. A., J. ImmunoI. 112, 2111, 1974. 17. Peter, J. B., Stratton, J. A., Stempel, K. E., Yu, D, and Cardin, C., 7. Immunol. 111, 770, 1973. 18. Flelschmann, J. D., Pain, R. H., and Porter, R. D., Arch. Bioel, em. Biophys. Suppl. I, 174, 1962. 19. Jondal, M., Holm, G., and Wigzell, H., J. Exp. Med. 136, 207, 1972. 20. Potvin, C., Tarpley, J. L., and Chretien, B., Clin. Immunol. In ln~unopathol. 3, 476, 1975. 21. Bianco, C., Patrick, R., and Nussenzweig, V., J. Exp. Med. 132, 702, 1970. 22. Coombs, R. C. A., Gurner, B. W., Wilson, A. B., Holm, B., and Lindgren, B., Int. Arch. Allergy 39, 658, 1971. 23. Elliott, B. E., and Haskill, J. S., Eur. J. hnmunol. 3, 68, 1973. 24. Rapp, H. J., and Borsos, T., In "Molecular Basis of Complement Action," p. 40. AppletonCentury-Crofts, New York, 1970. 25. Pinckard, R. N., Olson, M. S., Kelley, R. E., DeHoor, D. H., Palmer, J. D., O'Rourke, R. A., and Goldfein, S., J. fmmunol. 110, 1376, 1973. 26. Opelz, G., Kiuchi, M., and Takasugi, M., J. Immunogeneties 2, 1, 1975. 27. Stout, R. D., and Herzenberg, L. A., f. E.~p. Med. 14:2, 1041, 1975. 28. Cohen, A., and Schlesinger, M., Transplantation 10, 130, 1970. 29. Wortls, H. H., Cooper, A. G., and Brown, ~ . C., Nature New Biol. 243, 109, 1973. 30. Wu, L. Y. F., Lawton, A. R., Greaves, IV[. F., and Cooper, M. D., Seventh Leukocyte Culture Conference, p. 485, 1972. 31. Lischner, H. W., Valdes-Dapena, iV[., Biggne, J., Nann, S., and Amoni, N., Seventh Leukocyte Culture Conference, p. 547, 1972. 32. Thomas, D. B., and Phillips, B., Bur. J. Immunol. 3, 740, 1973. 33. Geha, R. S., and Merler, E., Eur. J. Im~nunol. 4, 193, 1974. 34. Bloom, B. R., Stoner, G., Gaffney, J., Shevach, E., and Green, I., Eur. J. Immunol. 5, 218, 1975. 35. Owen, F. L., and Fanger, M. W., J. Immunol. 113, 1128, 1974. 36. Balch, C. M., Dagg, M. K., and Cooper, IV[. D., In "Phylogeny of T and B ceils" (Wright and Cooper, Eds.). North-Holland, Amsterdam, in press, 1976.
(1977)
Reactions of Anti-Human Brain Serum with Human Lymphocyte Subpopulations1 JOAN A . ~TRATTON AND PATRIClA E . BYFIELD 2
Departments of Medich, e, Pediatrics, and Microbiology and Immunology, University of California at Los Angeles and Harbor General Hospital Campus, Torrance, California 90509 Received June 15, 1976 The reaction of serum from rabbits immunized with human brain ( R t t B ) with human lymphocytes has been measured by cytotoxicity, membrane immunofluorescence, and inhibition of lymphocyte function (mitogen-induced blastogenesis and lymphotoxin production). R H B kills most of the T-cells in the blood, but no reaction with B-cells or monocytes was detected. The remaining cells are reduced in their ability to respond to mitogen by lymphotoxin production and/or secretion. Treated P B L respond normally to P H A and P W M by incorporation of [3H]TdR, but the response to Con A is inhibited. The effect on lymphotoxin release is not dependent on cell death, as it occurs in the absence of added complement. R H B inhibits spontaneous E rosette formation by T-cells; again the effect does not depend on cell death but it requires approximately 10% normal serum--fresh or heated! The synergistic effect of normal and immune serum is apparently due to steric or charge effects of binding of some complement component(s), since normal serum depleted by incubation with antigen-antibody complexes supports rosette inhibition by rabbit anti-human brain serum very poorly, and purified Clq partially restores the inhibition. All of the measured properties of R H B are induced by immunization and can be produced by treating cells and washing away .the serum components which do not adhere. We conclude that there is a small subpopulation of human Iymphocytes, probably T-cells, which lacks the brain antigen and responds by D N A synthesis but not lymphotoxin to P H A and P W M stimulation. The majority of T-cells, including those responsive to Con A, bear an antigen(s) cross-reactive with brain; this antigen is not identical to the receptor for sheep red blood cells.
INTRODUCTION Lymphocytes can be broadly classified as thymus-derived (T) and bone-marrowderived (B), but it is clear that there are many functional subpopulations within these classes. Since lymphocytes of different subpopulations are not clearly distinguished by appearance, anatomical distribution, or physical properties, it is essential to find other ways of distinguishing and separating them. Antiserum is a par1This research was supported by Grant No. 1M-8 from the American Cancer Society, Grant No. 1 R O I CA 15054 from the National Cancer Institute and Grant No. GRSG-1780 from the National Institutes of Health. 2 Present address: Hypersensitivity Diseases Research, The Upjohn Company, Kalamazoo, Mich. 49001.
Copyright ,© 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0008-8749
2
STRATTON AND BYFIELD
ticularly valuable tool in studying lymphocyte subclasses, as it can be used for visual identification (1, 2), selective destruction (3, 4), or isolation (5, 6) of a given population. The alloantigens of mice, Ly and theta, have been used to identify T-cells and their subpopulations (3, 7, 8). Theta cross-reacts with antigen(s) on brain cells (4, 7), so that heterologous antibrain serum has been used as a substitute for antitheta (9). Human brain and T-lymphocytes also have at least one common antigen (10-12). Our purpose was to examine the potential usefulness of antiserum against human brain for identification of T-lymphocyte subpopulations. The antiserum was tested for its ability to kill human blood lymphocytes and to inhibit their rosette formation ; in addition, it was tested for inhibition of two responses, [SH]TdR incorporation and lymphotoxin release, after stimulation of the lymphocytes with the mitogens, PHA, PWM, and Con A. 3 M A T E R I A L S AND M E T H O D S
Media Hanks' balanced salt solution ( H B S S ) , Eagle's minimal essential medium with Hanks' salts (MEM), Roswell Park Memorial Institute Medium 1640 with 25 mM H E P E S buffer ( R P M I ) , fetal bovine (calf) serum (FBS), and human serum type AB (HuABS) were purchased from Grand Island Biological Company (GIBCO, Oakland, Calif.) or Microbiological Associates (Los Angeles, Calif.). The sera were heated to 56°C for 45 min before use.
Mitogens Purified phytohemagglutinin-P (PHA, MR69, Burroughs Wellcome, G.B.) was used at 0.2, 1.0, and 5/~g/ml in tube cultures and 0.5, 2.5, and 12.5 /,g/ml in microplate cultures. Pokeweed mitogen powder ( P W M ; Barber and Farmer, GIBCO) was diluted with HBSS and used at 1.0 and 5.0/~g/ml in tube cultures or 2.5 and 12.5 /,g/ml in microplates. Concanavalin A (Con A; Miles-Yeda, IZankakee, Ill.), was used in the same concentrations as PWM.
Lymphocyte Purification Human peripheral blood lymphocytes ( P B L ) were isolated from heparinized blood by isopycnic density gradient centrifugation (13) as previously described (14). The usual yield of mononuclear cells from normal subjects by this procedure is 85-95 % comprising 70-85 % lymphocytes and 15-305~ monocytes by ~-naphthylesterase stain (Sieger et al., in preparation). Tonsil and adenoid (TAL) tissue s Abbreviations used: C, guinea pig complement; CLL, chronic lymphocytic leukemia; Con
A, Concanavalin A; EAC-RFC, mononuclear cells with complement receptors; E-RFC, mononuclear cells forming spontaneous SRBC rosettes; EHT, equine anti-human thymus globulin (Upjohn ATG); FBS, fetal bovine serum, GPS, guinea pig serum; HBSS, Hanks' balanced salt solution; HuABS, human serum type AB; Lt, lymphotoxin; MEM, Eagle's minimal essential medium; PBL, peripheral blood lymphocytes; PHA, Phy,tohemagglutinin-P; PWM, pokeweed mitogen; RPMI, Roswell Park Memorial Institute Medium 1640; SRBC, sheep red blood cells; RHB, rabbit anti-human brain serum; RILL, rabbit anti-human light chain serum; TAL, tonsil and adenoid lymphocytes; TDL, thoracic duct lymphocytes.
ANTI-HUMAN
BRAIN SERU1V£
3
from children 4 to 12 years of age was obtained courtesy of Dr. L. Sieger. The tissue was dissected clear of blood and teased to a single cell suspension in HBSS (containing 10 times the usual antibiotic concentration). Human spleen tissue was obtained courtesy of Dr. A. Saxon. Single cell preparations were prepared by passage through a 250-gauge screen. Human thoracic duct lymphocytes (TDL) were obtained from patients with rheumatoid arthritis during continuous drainage from a thoracic duct fistula, courtesy of Dr. I-I. Paulus. TDL were separated from the heparinized lymph by eentrifugation. The cells were washed three times in HBSS before use.
Conditions for Lymphocyte Culture and Assay of [~H] Thymidine Incorporation Lymphocytes were cultured for 5 days in R P M I supplemental with 5% serum and antibiotics in plastic culture plates with and without mitogens, as previously described (14). The cells were labeled during the last 24 hr of culture with 0.1 ml of 0.4 mM [~H]thymidine ([3H]TdR; [6-~H]thymidine, TRA 61, sp act 19 Ci/mmol; Amersham Searle, Arlington Heights, Ill.). The lymphocytes were harvested onto glass fiber filter paper and washed with water followed by 5% trichloroacetic acid using a multiple automatic sample harvester ( M A S H II, Microbiological Associates). Acid-precipitable radioactivity was determined by scintillation counting.
L ymphotoxin Assay For the production of lymphotoxin (Lt), 0.5 × 100 lymphocytes were cultured in 1 ml; the supernatants from triplicate cultures were pooled and either assayed immediately or stored at 0°C. The pooled supernatants from the cultures were assayed for cytotoxicity on L-cells (mouse fibroblasts 929, ~ strain, courtesy of Dr. G. A. Granger, University of California at Irvine). The Lt assay method was essentially that of Granger and co-workers (15, 16) except that cell death was measured by release of 51Cr from prelabeled L-cells (17). Briefly, 1 X 105 cells in 1 ml of MEM with 3% FBS were incubated overnight with 1 txCi/ml of 51Cr (Na251CrO4, 200 /~Ci//~g, International Chemical Nuclear Corporation, Irvine, Calif.) and 0.5/~g/ml mitomycin C (GIBCO). L-cells were rinsed once with MEM containing 3% FBS, and diluted lymphocyte culture fluids were added. After 16 hr at 37°C, the supernatants were decanted and the amount of 5~Cr present in both supernatant and L-cells was determined in a gamma counter. The Lt titer is expressed as the reciprocal of the dilution of lymphocyte culture fluid resulting in a 10% specific 5~Cr release; where specific release is defined as percentage of ~lCr released by the fluid from the stimulated lymphocyte culture, minus percentage of 5zCr released by the fluid from the nonstimulated control culture.
Antisera Equine anti-human thymus globulin ( E H T ) was obtained courtesy of Dr. G. D. Gray (Lot No. 16, 138-9, Upjohn, Kalamazoo, Mich.). Rabbit anti-human brain serum ( R H B ) was prepared by immunizing New Zealand white rabbits with homogenized tissue from human cerebrum (14). The animals did not exhibit gross neurological disturbances. Rabbit anti-human light chain ( R H L ) serum was prepared by immunizing New Zealand white rabbits with human light chains (14)
4
STRATTON AND BYFIELD
prepared from pooled human AB serum (18). If its serum contained cytotoxie activity against human lymphocytes before immunization, the rabbit was not used.
A bsorption of Antisera RHB and R H L were absorbed undiluted, but E H T was diluted 1:50 in HBSS. Sera were incubated with 10~ washed lymphocytes/ml at room temperature for 1 hr and then in an ice bath for another hour. Some sera were absorbed with 10% (v/v) RBC (human or sheep) or with 50% (v/v) human platelet membranes for 30 min in an ice bath.
Antiserum Treatment of Lymphocyte Cultures In some early experiments, sera were added to the cuItures and left there. Later, lymphocytes were incubated with the appropriate concentration of FBS, RHB, R H L (all 1:50), or E H T (1:1000-1:10,000) in RPMI and 1:10 guinea pig complement (C; GIBCO) for 1 hr at 37°C. The lymphocytes were washed once with HBSS, the viability was assessed by trypan blue, and equal numbers of viable cells were cultured in RPMI with 5% HuABS as described above. The two methods were equivalent.
Chromium Release Assay for Lymphoeytoto.ric Antibody Lymphocytes were suspended in HBSS (1 × 107/ml) with 10-20 /,Ci/ml 51Cr for 30-60 min at 37°C, washed twice, and finally resuspended in HBSS. Labeled lymphocytes (0.5-1 × 106), the appropriate dilution of test serum, and 1:10 C (guinea pig, GIBCO; rabbit, chicken, and human, freshly drawn serum) were added to each tube. The tubes were incubated for 45 min at 37°C and spun at 1800g for 15 min, the supernatant was decanted, and the amount of ~lCr released into the supernatant was calculated as a percentage of total 5~Cr label. All sera (including complement) were absorbed with sheep red blood cells (10%, v/v) and agarose (1% w/v). All dilutions of sera were tested in triplicate; also included were controls containing FBS plus C and distilled water to measure minimum and maximum chromium release, respectively. Nonspecific 5~Cr release (FBS) was 9-28%, while maximum ~lCr release (water) was 75-85%.
Conditions for Rosette Forming Cell (RFC) Assay To form rosettes, mononuclear ceils were washed and suspended at 2 × 106/ml in HBSS. For E-RFC the cells were mixed with an equal volume of 1% washed sheep red blood cells (SRBC, Mission Laboratories, Rosemead, Calif.), incubated at 37°C for 15 rain, centrifuged at 200g for 5 min. "Cold" E-RFC were kept on ice overnight (14, 19) while "warm" E-RFC were incubated for 2 hr at room temperature (20) before gently resuspending and reading. "Warm" E-RFC were prepared in HBSS with 20% HuABS and "cold" E-RFC in HBSS without serum. To detect complement receptors, SRBC were sensitized with rabbit antibody to SRBC followed by human complement (EAC), added to an equal volume of mononuclear ceils, spun at 200g for 2 rain and, after 15 rain at room temperature, vigorously resuspended and read (14, 21). RFC were counted either fresh, after addition of toluidene blue (22) or, after fixation with 2 vo! of 1% gluteraldehyde
ANTI-IIU~fAN
BRAIN
SERUlX¢
TABLE 1 Reagents for Indirect Membrane Fluorescence Serum" Equine anti-human thymus globulin (EHT) Rabbit anti-human brain (RHB) Rabbit anti-human L Chains (RHL)
Dilution 1 : 1000 to 1: 1 0 , 0 0 0 1 : 50 to 1 : 100 1 : 50 to 1 : 100
Fluoresceinated Serum
Dilution
Source
Rabbit anti-equine v2globulin Goat anti-rabbit v-globulin Goat anti-rabbit -r-globulin
1:20
Miles-Yeda
1 : 20
Meloy
1 : 20
Meloy
See text for source or preparation. in Tyrode's Solution (23), in smears stained with Wright's or May Grfinwald stain. I?44/lq~t~nofl~l~oresc enc g
Direct membrane fluorescence. Ten microliters of fluoresceinated polyvalent goat anti-human immune globulins (No. C201, Meloy, Springfield, Va.) was added to 0.2 ml of cell suspension, incubated for 1 hr in an ice bath, washed three times in ice-cold H B S S and resuspended in glycerine mounting medium (Becton-Dickinson, Cockeysville, Md). The percentage of mononuclear cells displaying membrane fluorescence of a "ring" or granular type was determined using a Nikon S I R - F fluorescence microscope. Indirect membrane fluorescence. The lymphocytes were incubated with antiserum (see Table 1) for 1 hr in an ice bath, washed three times in cold H B S S and stained with the appropriate fluoresceinated antiserum as above. RESULTS
Complement-Mediated Cytotoxicity The percentage release of slCr from prelabeled human peripheral blood lymphocytes ( P B L ) by three antisera in the presence of guinea pig complement (C) is shown in Fig. 1. R H B specifically released 50% and E H T about 30% of slCr from P B L at 1:50 dilution. R H L was the least cytotoxic, causing only 20% specific 51Cr release at the same dilution. Although the chromium release method is convenient for comparative titrations, it does not necessarily reflect the exact number of cells killed. Consequently, we also used trypan blue exclusion to confirm the cytotoxic activity of the sera. With 1 : i 0 C plus F B S or normal rabbit serum, viability was greater than 95%, while R H B plus 1 : 10 C killed a maximum of 50% ; which was comparable to the results of the chromium release assay. The cytotoxic activity of R H B on cells from various human lymphoid tissues is depicted in Table 2. Normal P B L are the most, and spleen is the least, susceptible to lysis with the antiserum: There is no cytotoxic activity in the absence of C or with 10% heated serum. R H B plus C kills only a small fraction of lymphocytes from the P B L of patients with C LL, m~d only at the highest concentration, The~
6
STRATTON
AND
BYFIELD
70-
~:~~~ RHB
/ / / / / /
SO"
/ 50-
/ ~ ,3 4o-
Y~
i.d
E
/ H
T
/y/"
;RHL
50-
20-
0
~ I I I I0,000 IOOO I00 RECIPROCAL OF SERUM DILUTION
FBS
I0
Fro. 1. The cytotoxic activity of three antisera on human peripheral blood lymphocytes. The ordinate shows the percentage of SlCr release from PBL treated with rabbit anti-human brain (RIIB, O . . . . . O), equine anti-human thymus globulin (EHT; • • ) , rabbit antibody to human light chains (RHL; A-.-.-.A), and fetal bovine serum (FBS; , ) . Each point represents 4-10 experiments, except 1:500 RHB, which was done twice. The vertical bars show the standard error of the mean. high sensitivity of the bone marrow lymphocytes is probably due in part to contamination of the aspirates with blood.
Removal o/ Cytoto.vic Antibody by Absorption The antisera used in most of the preceding experiments have not been absorbed. To show that the cytotoxie action of R H B was specific for thymus-derived lymphocytes (T-cells) and the R H L specific for immunoglobulin-bearing lymphocytes (Bcells) the sera were absorbed with P B L from normal subjects, consisting primarily of T-cells, and from patients with C L L or a cultured lymphoblastoid line (Rail; courtesy of J. K. Seeley, U C L A ) , both of which are B-cells. The CLL-lymphocytes used for absorption included less than 1~o T-cells, as determined by spontaneous rosette formation and less than 3% T-cells as determined by fluorescent staining with RHB. Three absorptions with CLL-cells did not change the cytotoxic activity of R H B on PBL, whereas one absorption with P B L significantly decreased the cytotoxic activity ( P < 0.01) (Table 3). Absorption of R H B four times with Raji cells, decreased the cytotoxicity by 10% (J. K. Seeley, personal communication). Absorption of E H T with P B L and T A L produced neither discernible diminution of cytotoxic, rosette-inhibiting nor immunofluorescent staining activity Of the serur0 eve!a after 18 absorptions with a total of 1.8 × 10 s cells: Absorption
ANTI-HUMAN
BRAIN
TABLE C y t o t o x i c A c t i v i t y of R H B Serum dilution
Cb
SERUM
2
on Cells from Various Lymphoid
Tissues
Percentage release of 51Cr from prelabeled cells Source of lymphoid tissue Peripheral blood Normal
None
. -1-Ac -[~ -t-lq-
1 : 10 NS~
1:10 NS 1:10 R H B I:10 RHB 1:10 R H B l:25RHB 1:50 R H B I:100RHB 1:200 R H B
Spleen
Bone marrow
8.1 4- 0.6 . 12.3 d= 0.3 14.4 4-0.7 18.0 4- 3.5 . 58.84-1.7 21.6 4- 0.7 13.94-0.5 --
-. 23.8 4- 1.7 --. . 71.14-2.3 64.7 4- 0.5 51.44-3.0 34.2 4- 0.2
CLL
__a . 26.1 4- 0.4e ---75.54-0.4 66.9 4- 0.5 60.74-0.6 52.5 4- 1.2
__ .
. 9.2 4- 0.6 11.8 -4-0.9 7.3 4- 1.0 21.2 4-2.2 5.74-1.2 ----
Tonsil and adenoid
Thoracic duct lymph
--
--
31.0 4- 0.3 --. -54.5 4- 0.6 -38.7 4- 0.8
15.1 4- 1.0 ---
.
52.84-1.0 43.2 =k 1.0 ---
a Normal serum ; rabbit, fetal calf, or human. b 10% guinea pig serum. c Guinea pig serum heated to 56°C for 2 hr. a Not tested. 5~Cr release, mean 4- standard error of the mean, number of samples greater than five.
with 1 × 109 cultured lymphoblasts removed most of the cytotoxic activity (P. E. Byfield, unpublished observations). Rosette Inhibition
The three antisera were tested for their ability to inhibit rosette formation by PBL, with or without C. Both E-RFC (T-cells) and EAC-RFC (B-cells and monoeytes) were measured (Table 4). E H T inhibited E rosette formation at dilutions up to 1:1000 in the absence of C; the addition of C did not increase the inhibition of E H T even at 1:10,000 which only partially inhibited E-RFC (Table 4). E H T did not inhibit EAC rosette formation. R H L inhibited EAC rosette formation in a complement requiring reaction, but TABLE Cytotoxicity
of R a b b i t
Anti-Human
Brain
Serum
3 to
Human
Lymphocytes
as Measured
by
S p e c i f i c R e l e a s e of 51Cr f r o m P r e l a b e l e d L y m p h o c y t e s Antiserum
Percentage
of 51Cr r e l e a s e ~
dilution 1:25 Unabsorbed
1:50
1:100
3 6 . 4 4- 1.9 (3) b
33.1 4- 5.9
(9)
23.8 4- 1.8 (3) ~
12.9 4- 2.6
(3) ~
0.7 4- 3.9 (5) c
1.1 4- 2.2
(6) c
22.5 4- 6.4
(9)
Absorbed 1X PBL 3X
PBL
3X
CLL a
38.1 4- 1.9 (4)
I n t h e p r e s e n c e of 1 : 1 0 G p C ; Materials and Methods section. b Mean cp
:t: s t a n d a r d
d Lymphocytes rosette formation,
see
( n u m b e r of s a m p l e s ) .
to unabsorbed
from a patient
(2)~
2 3 . 8 4- 7.1 (10)
a c o n t r o l f o r n o n s p e c i f i c 5~Cr r e l e a s e h a s b e e n s u b t r a c t e d ,
error of mean
< 0.01 w h e n c o m p a r e d
26.9 4- 5.6 (12)
-3.9 4- 2.4
serum.
with chronic lymphocytic
leukemia;
less t h a n
1% T-cells by
8
STRATTON AND BYFIELD
TABLE 4 Inhibition of Rosette Formation by Antisera Directed against T- or B-Cells Serum
Dilution
Percentage inhibition~ E-RFC Complement
FBS RHB RHB (4 X CLL) * EHT EHT EHT (4 X CLL) ~ RHL RHL (2 × CLL) o
1:50 1:50 1:50 1:1000 1 : 10,000 1:1000 1:50 1:50
EAC-RFC
--
-b
Ab
--
q-
2~ 2 5 96 38 92 12 --
7 87 83 95 38 91 8 --
13 94 --a ------
10 13 -10 --2 2
8 13 4 11 --61 48
a Percentage inhibition = No. RFC Control/No. Mononuclear C e l l s - No. RFC Experimental/No. Mononuclear Cells > 0.5). To confirm the specificity of the antibrain serum for T-lymphocytes, we stained P B L or, in one experiment, spleen cells, with anti-immunoglobulin or R H B and
ANTI-HUMAN
ll
BRAIN SERUS{
TABLE 6 Membrane hnmunofluorescent Staining of Rosette-Forming Cells Percentage stained All lymphocytes~
1b 2 3 4 5
EAC Rosettes
Anti Ig (B-cells)
Antibrain
Anti Ig (B-cells)
17.9 9.0 10.5 10.8 12.0
58.3 69.7 77.7 89.2 71.0
82.2 87.5 100.0 83.1 100.0
Antibrain 1 1 1.5 1 1
E Rosettes 6 7 8 9 10
7.1 --° 17.8 ---
-87.5 67.9 84.2 73.2
1 -1 ---
-100.0 75.2 98.8 96.6
a The same fluorescent anti-rabbit 7-globulin was used in both tests. b The cells used in the experiments were peripheral blood lymphocytes except Experiment 8 which was spleen tissue. c Not tested. then formed either E or E A C rosettes. Individual cells were scored for both markers by examination under visible and ultraviolet light. Table 6 shows the fraction of total lymphocytes and of rosettes in each preparation which were stained by R H B or, as a control, antiimmunoglobulin serum. (The percentage of E A C rosettes formed under these conditions was normal, but E rosette formation was inhibited to some extent by chilling the cells). Nearly all the E A C rosettes bear surface immunoglobulin (82.2-100%), and almost none are stained by R H B (Table 6). E rosette forming cells did not have detectable surface immunoglobulin, and almost all rosettes were stained with RHB.
DISCUSSION To determine the specificity of the sera for functional lymphocyte subpopulations, the ability of human lymphoeytes to incorporate [ a H ] T d R on stimulation with three mitogens, P H A , P W M , and Con A, was determined after treatment with R H B (with or without added guinea pig C). Treatment with R H B did not inhibit the response to P H A nor to P W M (except in one experiment). [In a shnilar experiment, anti-fetal human brain serum stained neither P H A - nor PWM-indueed lymphoblasts (12). This serum, however, apparently has a more limited specificity than ours.] The response to Con A, on the other hand, was significantly inhibited by R H B (in all but one experiment). This inhibition could occur in the absence of added C; therefore, it is not due to cell death. Cells were inhibited when treated, washed, and then cultured. Apparently antibody coating the cells blocked their response to Con A. A similar selective complement-independent inhibition was noted by Owen and Fanger (35) using rabbit antiserum to human thymus. In the
~2
STRATTON
AND
BYFIELD
1000- PHA
~,
500i
]0050-
I0 [-I NS + c rY w II--
500 -
• RHS+C
PWM
[ ] RHB + AC [ ] RHB
z_ x o Io :E
I0050-
_j> NT
10 500-
Con A
100: 50-
Io
SG-2 FB-3 dS-I KS-I KS'2 BB-I
SUBJECT
FIG. 3. Effect of treatment with RHB on lymphotoxin (Lt) production by mitogen-stimulated PBL. The ordinate shows the titer of Lt after stimulation with PHA (top), PWM (middle) or Con A (bottom). Open bars represent the Lt titer after treatment with 1 : 50 NRS, or FBS plus 1:10 C; shaded bars, 1:50 RHB plus 1:10 C; hatched bars, 1:g0 RHB plus 1:10 C heated at 56°C for 1 hr ; and the stippled bars, 1 : 50 RHB without C. Vertical bars represent the standard error of the mean. The asterisks indicate reductions in Lt titer which are significant (P < 0.01) by Student's t test. mixed lymphocyte reaction, Opelz and colleagues (26) found that coating the responder, but not the stimulator cells, with antihuman brain serum blocks the reaction. One interpretation of our data is that there is a subpopulation of lymphocytes which respond to P H A but not Con A by blastogenesis and that these cells are unaffected by R H B , while a distinct subpopulation responds to Con A (and perhaps also to P H A and P W M ) and is inhibited by R H B . By separating cells on the basis of their Fc receptors, Stout and Herzenberg (27) have isolated two populations of T-cells from mouse spleen; one ( F c + ) responds to stimulation with both P H A and Con A, while the other ( F c - ) responds only to P H A stimulation. W e may be seeing a similar dichotomy.
ANTi-HUN[AN BRA][N SERUM:
13
The response to mitogens as measured by lymphotoxin release, however, was quite sensitive to R H B treatment. The treatment inhibited lymphotoxin response to PHA, PWM, and Con A, even in the absence of complement. We may speculate that these cells correspond to the F c - population of Stout and Herzenberg (27) or that they are a third population. (For a discussion of B-cell responses to these mitogens, see Refs. 30-34). To further delineate the specificity of these sera, they were tested for rosette inhibition. R H B inhibits spontaneous E rosettes, formed by T-cells, but the EAC rosettes formed by B-cells and monocytes are not affected. Unlike the inhibition of E rosettes by E H T (equine antithymus globulin), inhibition by R H B requires the addition of 10~ guinea pig, human, rabbit, or chicken serum as a complement source. Presumably, E H T reacts directly with the receptors for sheep erythrocytes while R H B does not. To our surprise, we found that the sera used as complement sources were still effective after heating at 56°C for 45-120 rain! Heated sera did not support cytotoxic reactions ( R H B was routinely heated for 45 min), nor did they inhibit rosette formation when used alone. Both R H B and C were absorbed with agarose to remove natural cytotoxic antibody (28) and with SRBC to remove anti-erythrocyte antibodies which might inhibit rosette formation (29). The synergistic effect of heated normal sera and heated immune sera is apparently due to some complement components, since serum depleted by incubation with antigenantibody complexes supports R H B rosette inhibition very poorly. Purified Clq partially restores the inhibition. Probably, binding of early complement components by the "brain-antigen"-antibody complex causes steric or charge interference with the erythrocyte binding. It seems, in view of this finding, that the brain crossreactive antigen is not part of the erythrocyte receptor, although it does occur on the same cells. Additional support for our hypothesis is the failure of our R H B to block staining of human P B L by an anti-T-cell serum which inhibits rosette formation (36). In summary, we find that R H B reacts with most T-lymphocytes in the blood, and with few, if any, B-cells or monocytes. R H B kills most T-lymphocytes; the remaining cells respond normally to P H A and PWM, by the criterion of [*H]TdR incorporation, while the response to Con A is inhibited. The release of lymphotoxin in response to all three mitogens is inhibited, on the other hand, after R H B treatment. Inhibition of Lt release is not dependent upon cell death, as it does not require added C. R H B inhibits spontaneous rosette formation. Again, the effect is not dependent upon cell death, but it does require the addition of approximately 10~o fresh or heated normal serum. Thus, the inhibition is due to steric or charge interference with the sheep erythrocyte receptor rather than direct reaction of R H B with the receptor. All inhibitory activities of R H B are induced by immunization of the rabbits, and can be induced by treating cells with R H B and washing the cells. None of the activities of R H B can be removed by absorption with B-cells (CLL lymphocytes or cultured lymphoblasts), but all are removed by absorption with PBL and TAL. We conclude that most T-cells carry an antigen (or antigens) cross-reactive with brain. This antigen is not the sheep erythrocyte receptor. A subpopulation of human blood lymphocytes which apparently lacks the brain antigen(s) responds to P H A and P W M by DNA synthesis but not Lt release. These may be B-cells (see Refs. 30-34) or a small subpopulation of T-cells.
izl
STRATTON
AND
BYFIELD
ACKNOWLEDGMENTS We thank Drs. David T. T. Yu and Edward S. Golub for their critical appraisal, Dr. B. S. Rabin for helpful discussion, Ms. Janet Seeley for cooperative experiments, and Ms. Diane Cole for superb technical assistance.
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