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Serum anti-immunoglobulins in multiple myeloma and benign monoclonal hyperglobulinemia
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
15,
Serum Anti-lmmunoglobulins and Benign Monoclonal I. SCHEDEL,
P. C. FINK,
5-86-599
(1980)
in Multiple Myeloma Hyperglobulinemia’
W. SCHBNER,
J. R. KALDEN.’
H. H. PETER, AND H. DEICHER Division
of Clinicui
Immunology und Blood TransJitsion, Department Medical School Hannorvr. Wesr Garmanv
of Medicine,
Received February 7. 1979 Anti-immunoglobulins with specificity to human native IgG and (Fab’), were detected in 69 sera of patients with multiple myeloma and 71 sera from 22 patients with benign monoclonal hyperglobulinemia with titers distinctly higher than those found in two control groups (healthy persons, sarcoidosis patients). Without further differentiation, no correlation of anti-immunoglobulin titers to the clinical status of the disease was observed. Chromatographic separation of sera on Sephadex G-200 revealed three molecular species of anti-immunoglobulins: type I. MW ca. 900,000; type II, MW 160,000; type III, MW < 100,000. Elevated titers of type III anti-immunoglobulins were observed only in multiple myeloma. Using immunoadsorbent techniques type III anti-immunoglobulins were identified as (Fab’), fragments. Lacking idiotypic specificity. type III antiimmunoglobulins were shown to react with allotypic as well as idiotypic immunoglobulins on the surface of PBL B-lymphocytes. In addition, they inhibited significantly antibody-dependent cellular cytotoxicity reactions in t?tro using IGR 3 melanoma cells as targets, and preparations of peripheral blood lymphocytes from healthy individuals as effector cells. In contrast, no significant inhibition of cytotoxic activity was observed using type II (IgG) anti-immunoglobulins from benign monoclonal hyperlgobulinemia patients’ sera in equal concentrations.
1. INTRODUCTION
A report by Abdou and Abdou (1) demonstrated the presence of antiimmunoglobulins (anti-Ig)3 in seven of nine sera from patients with IgG myeloma. In a study by Lindstrom and Williams (17) and Lindstrom et al. (18), who examined sera from patients with IgG multiple myeloma (MM) and patients with benign monoclonal hyperglobulinemia (BHM), 49% of MM sera were found to contain anti-Ig, as compared to 26.3% of BMH sera and 28% of age-matched control sera. Using sucrose density gradient separation, these authors described anti-Ig activity in 3.5 S fractions in addition to 7 S anti-Ig. Analysis of tumor immunity in patients with different malignancies has revealed lymphoid cells in the peripheral blood * This work was supported by Grants SCHE 169/l, 19/7 1, and SCHE 16912 as well as by funds from the Sonderforschungsbereich 54, project G3, of the Deutsche Forschungsgemeinschaft. 2 Present address: Universitlt Erlangen, Abteilung fur klinische Immunologic, Erlangen, West Germany. 3 Abbreviations used: ADCC. antibody-dependent cellular cytotoxicity: anti-Ig, antiimmunoglobulin; BMH, benign monoclonal hyperglubulinemia; MM, multiple myeloma; MW, molecular weight; SmIg, surface membrane immunoglobulin; PBL, peripheral blood lymphocytes: pHAT, passive hemagglutination test; fraction F. fraction Ficoll; fraction FFF, fraction Ficoll, Fer, Falcon; h-action FFF-C. fraction Ficoll, Fer, Falcon, Column; pHAT. passive hemagglutination test. 586
0090-1229/80/040586-14$01.00/O Copyright @ 1980 by Academic Ress, inc. All rights of reproduction in any form reserved.
SERUM
Anti-Ig
IN
MYELOMA
AND
HYPERGLOBULINEMIA
587
capable of killing tumor-cells in vitro (4, 16, 24, 25). Serum factors inhibiting this cellular cytotoxicity have also been noted (3, 5, 6,9, 10, 12, 24, 26). More particularly, blocking of cellular responses to tumorspecific or tumor-associated antigens has been connected with antibodies of different IgG subclasses, or antibody fragments, which inhibit cellular responses either by combining with antigen at the target cell membrane, or by direct inhibition of lymphoid effector cells (24, 26). Anti-Ig from MM sera have been shown to stimulate growth of the patients’ marrow plasma cells in tissue cultures. The present study was performed to determine semiquantitatively anti-Ig in sera from patients with MM and BMH, and to characterize the anti-Ig with respect to their molecular size and specificity. In addition to anti-Ig corresponding in size to IgM and IgG, low molecular weight (Fab’),-type anti-Ig have been demonstrated. The results indicate that the presence of serum anti-Ig of different types may be one feature distinguishing between MM and BMH. The additional purpose of the present study was to examine the effect of serum anti-Ig, of different molecular size, on antibody-dependent cytotoxicity of peripheral lymphoid cells in vitro. The results indicate that (Fab’), anti-Ig found in MM can effectively inhibit cell mediated cytotoxicity (ADCC) in vitro, thus posing the question as to the possible function of anti-Ig in MM in in vivo. 2. MATERIALS
AND METHODS
2.1. Serum Samples Serum samples were obtained from 69 patients with MM and from 22 persons with BMH for anti-Ig tests. In addition, sera from 35 healthy blood donors, 17 patients with MM, and 14 persons with BMH were available for anti-Ig testing as well as for cellular cytotoxicity tests. The diagnoses were confirmed by the usual clinical and laboratory criteria (7, 22, 23). Patients with BMH showed no progressive increase of serum myeloma protein concentrations or other signs of MM during the last 2-5 years. Control serum samples were obtained from 56 age-matched healthy blood donors and 18 patients with sarcoidosis. Most of the sera studied were kept frozen at -80°C until use. Twelve serum samples from MM patients and 25 sera from healthy controls were tested for anti-Ig immediately after drawing. 2.2. General Screening for Anti-Ig Anti-Ig activity was studied using the passive hemagglutination technique (pHAT) of Avrameas, Toudou, and Chuillon as modified by Hartmann and Lewis (2, 7, 10, 13, 14). Briefly, lOO-mg native human IgG (prepared by Professor Dr. Hoppe, Zentralinstitut fur das Blutspendewesen, Hamburg), or lOO-mg (Fab’),G-immunoglobulin fragments prepared from Gamma-Venin (Behring-Werke, Marburg/Lahn, West Germany) by gel filtration on Sephadex G-150 was added to 18 ml of 20% sheep red blood cells (SRBC) in borate-buffered saline (pH 8.6). After a single wash of the cell suspension 9 ml of 2.5% glutaraldehyde solution in distilled water was added dropwise with gentle agitation on a magnetic stirrer. The suspension was then incubated at room temperature for 1 hr and centrifuged, and the formed precipitate of coated SRBC was washed three times in 5-10 ml of borate-buffered saline. The coated SRBC were then resuspended in 60 ml borate-
588
SCHEDEL
ET
AL.
buffered saline containing 1.0% sodium azide to obtain a 6% stock suspension. The working suspension was 0.6%. Sera were inactivated (10 min/56”C) and after exhaustive absorption with uncoated SRBC serially diluted with borate-buffered saline using Microtiter-plates (patent Cook engineering, Fa. Greiner, West Germany). After the addition of coated SRBC, the plates were gently shaken and subsequently incubated for 2 hr at 37°C. The hemagglutination titers were then read and recorded. Titers below 4 were regarded as normal (see below). Serial dilutions of goat anti-human immunoglobulins or rabbit anti-Fab immunoglobulin fragment sera (Hyland, Travenol, Munich, West Germany) absorbed with uncoated SRBC were used as positive controls. Uncoated cells were used as negative controls. The specificity of the agglutination reactions was assessed by inhibition experiments. Sera were incubated with serial dilutions of the protein to be tested (human native IgG; human (Fab’,G fragments; human FcG fragments) followed by the usual passive hemagglutination procedure as described above. Complete inhibition or blocking of at least 4 hemagglutination titer-units could be achieved with human IgG or with human (Fab’),G fragments. In order to exclude the possibility that the IgG on the SRBC surface could attract and denature complement components, thus producing false-positive results, IgG-coated SRBC were incubated with five different native normal human sera (30 min/37”C). Thereafter, pHAT were performed using anti-C, as well as rabbit anti-C,, sera (Behringwerke, Marburg/Lahn, West Germany). 2.3. Approximation of the Molecular Size oj’ the Anti-Ig An approximation of the molecular size of the anti-Ig present in sera from patients with MM and BHM was obtained by Sephadex G-200 gel chromatography, followed by anti-Ig determinations in the serum fractions obtained using the passive hemagglutination technique. The molecular size of the proteins present in the fractions was estimated by quantitative determinations of proteins with known molecular weight (IgM. IgG, IgA, albumin). These column chromatographies were performed in all cases immediately after collection of the sera. 2.4. Isolation of Anti-Ig Anti-Ig were isolated from five sera of myeloma patients using Sepharose 4 CnBr (Pharmacia, Uppsala, Sweden) coated with human IgG as immunosorbent. Anti-Ig were absorbed from serum fractions previously obtained by gel chromatography (Sephadex G-200, Pharmacia, Uppsala, Sweden), eluted from the immunosorbent by glycin-buffer (pH 4.5), and subsequently dialyzed against PBS (pH 7.2). The preparations were then checked for immunoglobulin content by radial immunodiffusion ‘technique (LC-plates, Behring-Werke. Marburg/Lahn, West Germany). They were further analyzed by immunoelectrophoresis with goat anti-human plasmaprotein, anti-human IgG, anti-human Fab, and anti-human Fc sera (Hyland, Travenol, Munich, West Germany). Anti-Ig activity of the preparations was tested using the pHAT. The antigenic properties of the anti-Ig were also investigated by adsorption on Sepharose 4 CnBr coupled with goat antibodies to the Fc-fragment or the Fab-fragment of human IgG. Anti-Ig containing serum fractions obtained from 10 ml serum were passed through a column with 10 ml of
SERUM
Anti-Ig
IN
MYELOMA
AND
HYPERGLOBULINEMIA
589
antibody-coated Sepharose equilibrated with sodium bicarbonate buffer (pH 8.2). Effluent volumes obtained by elution with sodium bicarbonate-buffer (pH 8.2) and subsequently with citrate-buffer (pH 3.2) were concentrated to 0.5 ml and tested for anti-Ig by pHAT. 2.5. Preparation of Lymphocytes and Lymphocyte Subpopulations from Peripheral Blood (PBL) 2.5.1. Preparation of F, FFF, and FFF-C lymphocytes. The lymphocyte rich fractions obtained from 200~ml heparinized or defibrinated blood were separated by Ficoll-density gradient centrifugation and were washed twice with MEM supplemented with 15% FCS. Contaminant red blood cells were lysed by treatment for 10 min at 37°C in 0.15M ammonium chloride. Then the cell suspensions were washed twice and resuspended in MEM + 15% FCS to a final concentration of 2 x 10’ cells/ml (fraction F). This cell suspension was referred to as fraction “FFF” (Ficoll, Fer, Falcon). Surface-Ig bearing lymphocytes were removed from fraction FFF by an antiIg-column using the technique described by Peter et al. with slight modifications (25). Cells not retained by the column were referred to as fraction FFF-C (FFF column), which was devoid of surface-Ig-positive cells as revealed by direct immunofluorescence technique using FITC-labeled goat anti-Ig sera. Phagocytic and adherent cells were eliminated by lymphocyte separating reagent and incubation in Falcon petri dishes as described previously (25). 2.5.2. Preparation of B-cell enriched lymphocyte fractions. B-cell enriched lymphocyte fractions were obtained from F-lymphocytes by addition of SRBC followed by removal of E-rosetting cells by a second Ficoll-gradient-density centrifugation. 2.6. Antibody-Dependent Cytotoxicity (ADCC) ADCC reactions were performed using a cultured human melanoma cell line (IGR 3) as previously described (25). For inhibition studies, effector or target cell preparations were incubated simultaneously with different anti-Ig preparations. In order to determine the effect of anti-Ig on the target cells or on the effector cells, these cell preparations were incubated with different types of anti-Ig and washed six times (PBS, pH 7.2) before incubation for cytotoxicity tests. 2.7. Immunojluorescence Technique Immunofluorescence technique using fluorescent goat anti-IgG, -IgM, and -1gD sera (Hyland, Travenol, Munich, West Germany), was performed as described previously (28). In order to determine the reactivity of the anti-Ig to the cell surfaces, 1 x lo6 lymphoid cells were incubated with 0.5 ml of anti-Ig-containing fractions (30 min/ 20°C). Thereafter immunofluorescence technique was applied using FITC-labeled anti-Ig sera as described above. 2.8. Raising of Zdiotype-Speczjk Antisera Antisera with specificity against variable region antigens of myeloma proteins were raised in rabbits by repeated immunizations with 1 mg of purified myeloma protein preparations. Myeloma protein was isolated from the patients’ sera by
590
SCHEDEL
ET
AL.
ammonium-sulfate precipitation, Sephadex G-200, and Sephadex DEAE-A 50 gel filtration. Antisera were absorbed with purified polyclonal Ig preparations coupled to activated Sepharose 4 CnBr, and with PBL (prepared by Ficoll-density centrifugation) from healthy individuals. Idiotypic specificity was assessed by Ouchterlony and solid phase Elisa-techniques as described previously (29). 3. RESULTS 3.1. General
Screening
for Anti-lg
As demonstrated in Fig. 1, positive pHAT reactions with human IgG-coated SRBC were obtained in 58% of the sera from healthy adults. However, titers higher than 2 were never observed. In contrast, all sera from MM as well as from BMH patients showed positive agglutination reactions with titers ranging from 16 to 512, without obvious differences between sera from patients with MM and BHM. Sera from 18 sarcoidosis patients, tested as an additional control group, displayed low titers of anti-Ig. Figure 2 summarizes the results of the tests using SRBC coated with (Fab-),-G-immunoglobulin fragments. Although titer differences between control sera and MM and BMH sera were not impressive with this antigen, some MM sera exhibited titers of 16 and 32, which were rarely seen in the sarcoidosis control groups, and not at all in healthy persons’ sera. As for MM, no correlation was noted to the clinical course of the disease or any laboratory criteria (total serum protein, myeloma protein concentration as calculated from the serum electrophoresis pattern, Bence Jones protein concentrations in the urine, histological and cytological bone marrow examination). In single patients, no consistent correlation was noted between pHAT titers obtained with either IgG-coated or (Fab’),-G fragment-coated SRBC.
healthy
adults
BMH
“Z 56
2561286432-
$ ?
f
z 0
n=71
myelomo n:- 69
.. .. . .. .. . . .
............
. . . .
................ ................ ............ ........
.. .. .. ..
. . . .
.. . .. .. . .. . .. .. .. . .. .. . .
. .. . .. . .. .. . . ..
.....
l6-
842_
.. . .. .. . .. .
,-
. .. . .. .. . . . . .. . .. .. . . . . . .. .. . .. .. . .. . .. .. . .. .
N -
sorcoidosis n q 21
. . ..
512-
E C_
multiple
FIG. 1. Anti-IgG activity in sera from healthy adults and from sarcoidosis determined by pHAT using sheep red blood cells coated BMH patients were tested repeatedly (n = 71).
. . . . .. . . .. ..*...*. ... patients with BMH, MM, and with IgG. Serum samples of 22
SERUM Anti-Q IN MYELOMA heolthy
adults
BMH
multiple
” =71
myeloma
1
............... ...............
............... ............... ...............
sarcoidosis
n-69
n =21
. .. .
.
. ..*****
l
. . .. . .. . . . . .. . . . . .. . .. . .. . . N
591
AND HYPERGLOBULINEMIA
.... .. .... ...... .. .... ...... ..
. . . . . .
. . . . . . . .
. . . . .
FIG. 2. Anti-(Fab’), activity in sera from healthy adults and from patients with BMH, MM, and sarcoidosis determined by pHAT using SRBC coated with (Fab’),-G. Serum samples of 22 BMH patients were tested repeatedly (n = 7 1).
3.2. Specificity of Agglutination
Reactions
The specificity of the agglutination reactions in the pHAT was assessed by inhibition experiments. Agglutination reactions in sera from MM and BMH patients were totally inhibited using human native IgG at a concentration of 20 pg/ml or below. Inhibition occurred with both IgG-coated and (Fab’),-coated SRBC. Inhibition was also obtained with (Fab’)*-G at or below 30 @g/ml. By contrast, anti-Ig in only two out of eight sera tested with IgG-coated SRBC and none of those tested with (Fab’),-G-coated cells were inhibited by Fc. Hemagglutination could not be achieved by adding IgG to normal human sera up to a final concentration of IgG of 50 g/liter. In order to exclude the possibility that complement components reacted with the coated SRBC, thus enabling immunoconglutinis present in the patients’ sera to produce positive pHAT reactions, pHAT were performed using heterologous anti-complement-component sera (see above). All these tests lead to negative results, indicating that under out test conditions the Ig-coated SRBC did not bind C,, or C3. 3.3. Characterization
of the Molecular Size of Anti-Ig
In order to estimate the molecular size of the anti-Ig, 32 sera from MM patients and healthy controls were separated on Sephadex G-200 columns (Pharmacia, Uppsala, Sweden). As described above protein concentrations and anti-Ig activity were tested in each isolated fraction; three different molecular classes of anti-Ig were observed. Figures 3a-c show three typical examples of separation curves with the anti-Ig titers obtained in the chromatographic fractions. In addition to anti-Ig separating with the first and second peaks, a third type of low molecular weight anti-Ig could be demonstrated. All sera from patients with MM (n = 9)
592
SCHEDEL
a
ET
AL.
5’0
240 elutgon
volume
lmll
3a. Chromatographic separation (Sephadex G-ZOO) of a serum from a patient with BMH showing anti-Fab activity in serum fractions containing proteins of ea. 150 000. FIG.
without or before treatment exhibited anti-IgG activity in the third serum fraction containing proteins of molecular weight less than 100 000 (type III anti-Ig), whereas after cytostatic treatment two of nine sera showed such activity. Such anti-Ig were detected in only two of eleven normal control sera, and at low titers, and were not found in sera of patients with BMH. In these tests, titers below 4 were regarded as normal. In three of eighteen cases of MM, elevated agglutinating activity against IgG as well as against (Fab’), (one case) could be detected in the first Sephadex G-200 peak, representing proteins with molecular weights of about 900 000 (type I-anti-Ig) (Figs. 3 and 4). Fractions from these three sera containing high molecular weight anti-Ig were also able to agglutinate SRBC coated by heat-
FIG. 3b. Chromatographic separation (Sephadex G-200) of a serum from a patient with multiple myeloma showing anti-Fab activity in serum fractions containing low molecular size proteins.
SERUM Anti-Ig IN MYELOMA
c
593
AND HYPERGLOBULINEMIA
50
150
elution
250 volume [ml)
FIG. 3c. Chromatographic separation (Sephadex G-200) of a serum from a patient with MM showing distinct anti-IgG activities in the first serum fraction containing proteins of high molecular weight (>200 000) as well as in the third peak, with proteins of molecular weight below 100 000.
aggregated human-IgG, and the unfractionated routine Waaler-Rose tests.
sera gave positive
reactions
in
3.4. Isolation of Anti-Zg Anti-Ig were isolated from five sera of myeloma patients using a gel filtration technique (Sephadex G-200) followed by immunoabsorption. Material exhibiting Fc determinants was not detectable in fractions containing low molecular weight anti-Ig activity. These fractions reacted with heterologous anti-Fab sera. As shown in Table 1 anti-Ig activity was completely absorbed by Sepharose beads multiple
myelomo
before
treatment
multiple
myeloma
under
treatment
benign
T III
monoclonal
hyperglobulinema
1
n=8
Ill
II
Ill
l l ee
. l
l ee
l ee
l
l e .
.
.
l
wee
l ea l ee l
FIG. 4. Anti-IgG activity found in serum fractions (Sephadex G-200) column chromatography) from MM and BMH patients obtained by pHAT using SRBC coated with human IgG (0) and human (Fab’), (0). II: Type II anti-Ig; III: type III anti-Ig.
594
SCHEDEL
ET AL.
TABLE REACTION COUPLED
OF SERUM AN,I WITH Awri-Fab
1
I-1g WI I’H StpHARost OR ANTI-FC FROM
Titer
IHE
4 CnBr GO.A I
of the anti-immunoglobulin After passage through Sepharose coupled with
Before passage Preparation Type
through
II anti-lg
anti-Fab
~~~_...
immunoabsorbent
~~~
pH 8.2
512 256
Type III anti-&
After passage through Sepharose coupled with .
ptl
ll II
1.2
anti-Fc
pH 8.2
pH 3.2
0 I56
256 0
76 128
coupled with goat antibodies to the Fab portion of human immunoglobulin. In contrast, the anti-Ig activity was recovered without significantly reduced titers from Sepharose coupled with Fc-specific goat antisera. These findings led to the conclusions that low molecular weight anti-Ig consisted of (Fab’), fragments. 3.5. Reactions of Anti-lg with PBL Surface Ig In comparison to lymphocyte preparations from a normal control group, the percentage of Ig-positive lymphoid cells demonstrable by direct immunofluorescence technique using FITC-labeled anti-human-(polyclonal)-Ig sera was found to be reduced in MM patients. However, when individually specific anti-idiotypic antisera were employed, a significant increase of SmIg-positive cells was found (P < 0.001) (Table 2). The regular presence of lymphoid cells carrying idiotypic (i.e., myeloma protein-specific) material on their surface in the peripheral blood of MM patients was thus demonstrated. When these PBL lymphocytes were preincubated with type III anti-Ig, the reactivity of anti-isotypic (Ig-class-specific) as well as anti-idiotypic sera with these cells was considerably reduced (Table 2). In TABLE PERIPHERAL FITC-COUPLED
BLOOD
LVMPHOCYTES
WITH
ANTI-ISOTYPIC INCUBATION
AND WITH
2
SURFACE
IMMIJNOG~UBULI&
ANTI-IDIOTYPIC
TYPE
III
ANTMRA
Anri-Ig
CONTAINING
Lymphocytes SUBJECTS Healthy adults (n = 20) Patients 1. H.J. 2. J.G. 3. R.A. 4. K.J. 5. G.K. 6. H.K. Mean
I&G,
15.5 -t 5
IgA.
IgM.
IgD
AND
(4)
9 11 13 9 8
(6) (7) (9) (4)
(6) (6 + 4.2)
AFER
WI’I-H (
)
FRACTIONS
SmIg
(%,I
Idiotypic
(12 t 3)
7
9.5 + 4.8
bearing
AS VISL’AI~IL~D BEFORE
determinants
0
36 29 26 18 27 24 26.6 i
(22) 121) (19) (16)
(22) (16) 13.2
(19.3
i- 6.3)
SERUM
Anti-Ig
IN
MYELOMA
AND
TABLE HEMAGGLUTINATION TITERS PATIENTS USING SRBC COATED
Serum
a Nos. l-8
ADULTS AND MULTIPLE MYELOMA OR INDIVIDUAL MYELOMA PROTEINS
IgG
titers using SRBC coated with Purified
my&ma
protein”
2
3
4
5
6
1
8
2
1
8
1
2
2
8
1
1024 1024 512 512 256 1024 1024 1024
512 512 1024 512 256 1024 1024 512
256 512 1024 1024 1024 1024 512 256
256 256 512 512 512 512 512 512
256 1024 512 256 256 1024 512 512
512 1024 512 256 512 512 256 512
512 512 256 256 512 512 256 512
1024 512 512 512 512 512 512 256
Pooled human I&
1
4 512 1024 1024 512 512 1024 1024 512
Healthy adults (II = IO) Patients 1. H.J. 2. J.G. 3. B.W. 4. R.A. 5. MM 6. A.G. 7. J.A. 8. KM
3
IN SERA FROM HEALTHY WITH HUMAN POOLED
Hemagglutination
595
HYPERGLOBULINEMIA
refer to patients as shown in left-hand
column of table.
contrast, preincubation of these cells with type II anti-Ig did not diminish the percentage of fluorescent cells applying FITC-labeled anti-isotypic sera. However, no indication of idiotypic specificity of type III antiglobulins was obtained. As shown in Table 3, sera from eight MM patients showed similar pHAT titers with normal human IgG or any of eight purified myeloma proteins used to coat SRBC in the pHAT reaction. In particular, no preferential reaction of indiTABLE EFFECT
OF ANTI-IMMUNOGLOBULIN
DEPENDENT
CELLULAR
CYTOTOXICITY CONCENTRATION
Lymphocyte fractions used as effector cells F
FFF
FFF-C
DERIVED
n
FROM
4 MULTIPLE
MYELOMA
Medium
added
Percentage specific Vr release (X)O
12 12 12 5 12 12 12 12 5 11
MEM NHS-fractiona Whole MM sera Type II anti-Igb Type III anti-Igb MEM NHS-fraction” Whole MM sera Type II anti-Ig* Type III anti-Igb
82.6
11 10 12 5 12 2
MEM NHS-fraction”
61.9 68.5 48.2 66.3 41.7 39.2
Whole
MM
sera
Type II anti-Igb Type
PATIENTS
ON ANTIBODY-
(ADCC) OF NORMAL HUMAN LYMPHOCYTES. WAS ADJUSTED TO 40 &ml IN EACH TEST.
III anti-Igb
Isolated type III Anti-&’
85.9 14.2 81.2 72.8 81.2 81.7 70.2 80.6 69.8
THE
SD
PROTEIN
Statistical evaluation (P)
-tlO.l 2 10.2 r 7.1 k12.1 211.1 r 5.8 + 12.9 * 7.5 ell.8 kl3.5 2 12.5 + 17.4 k14.1 k15.1 2 10.7
a Normal human serum fractions prepared in the same way as the corresponding serum fraction patients (fractions containing proteins of the same MW as type III anti-Is). b Anti-Ig-containing serum fractions prepared by gel chromatography (Sephadex G-200). ’ Type III anti-lg purified by immunoabsorbent column chromatography.
<0.025 <0.025 <0.025 10.025
from MM
596
SCHEDEL
ET AL.
TABLE 5 REACTIVITY OF NORMAL HUMAN LYMPHOCYTES AFTER PREINCUBA CELLS WITH TYPE III ANTbIg. THE PROTEIN CONCENTRATION ADJUSTED TO 40 &ml IN EACH TEST
ADCC
Lymphocyte fractions used as effector cells F
FFF
FFF-C
added
Percentage specific “Cr release Crl
I ION OF THE TARGET WAS
SD
Statistical evaluation (P)
II
Material
IO IO 5 10
MEM NHS-fraction” Type II anti-Igb Type III anti-Ig”
84.2 83.4 81.3 74.2
kli.2 ? \).I
“.S.
L 13.2 2 IO.0
n.s. “.S.
9 IO 10 10
MEM NHS-fraction” Type II anti-Igb Type III anti-Ig”
79.1 85.2
81.3
- I?.4 t 14. I . 8.2
n.s. “.S.
71.2
I 7 I
-0.01 ~CO.01 co.01
9 IO 10 10
MEM NHS-fraction” Type II anti-Ig” Type III anti-Igb
“.S. n.s.
-:o.ooo5
64.1 69.2 63.1
-co.025 <0.025 co.025
45.1
0 Normal human serum fractions prepared in the same way as the corresponding serum fractions patients (fractions containing proteins of the same MW as type III anti-Ig). b Anti-Ig-containing serum fractions prepared by gel chromatography (Sephadex G-200).
from MM
vidual patients’ sera with their own myeloma protein could be demonstrated with this technique. The lack of idiotypic specificity of anti-Ig contained in MM and BMH sera was thus demonstrated using two independent approaches. 3.6. Effect of Serum Fractions Containing Anti-Ig on ADCC Table 6 summarizes the capacity of serum fractions obtained from anti-lgcontaining sera by column chromatography on Sephadex G-200 to inhibit lysis of antibody-coated melanoma cells by different lymphocyte preparations (F, FFF, FFF-C) . In general, the addition of whole sera from untreated MM patients resulted in decreased cytotoxicity, an effect not observed to a significant degree with normal sera from healthy adults (Table 4). ADCC reactions were also significantly inhibited by serum fractions containing (Fab’),- anti-Ig. Significant inhibition was seen when F- (P < 0.025) and FFF- (P < 0.0005) lymphocyte preparations were used as
ADCC
TABLE 6 REACTIVITY OF NORMAL HUMAN LYMPHOCYTE FRACVION AF.rER PRKINCUBAI EFFECTOR CELLS WITH TYPE III ANTI-Ig. THE PROTEIN CONCENT-RATION WAS ADJUSTED TO 40 &ml IN EACH TEST
Lymphocyte fractions used as effector cells FFF-C
,i
Medium
added
IO MEM IO NHS-fraction” 10 Type III anti-Ig*
Percentage spe#ic release (x) 63.2 67.9 47.7
jlCr SD
ION OF IHI-
Statistical evaluation (P)
*IO.2
216.1
iO.005
+- 12.7
<0.005
a Normal human serum fractions prepared in the same way as the corresponding sernm fractions patients (fractions containing proteins of the same MW as type III anti-Ig). b Anti-Ig-containing serum fractions prepared by gel chromatography (Sephadex G-200).
from
MM
SERUM
Anti-Ig
IN
MYELOMA
AND
HYPERGLOBULINEMIA
597
effector cells. No inhibitory activity was detected when the tests were performed with serum fractions containing type II anti-Ig. (Fab’)* Anti-Ig prepared from the third Sephadex G-200 peak of MM sera followed by affinity chromatography using anti-Fab-coated Sepharose 4 CnBr as an immunosorbent (type III anti-Ig) also produced a considerable inhibition of the ADCC reaction in two cases. Preincubation of the target cells before addition of cytotoxic lymphocytes led to a reduced ADCC reaction in vitro (Table 5). Preincubation of the PBL effector lymphocytes with type III anti-Ig (30 min/20”C, 30 min/37”C) also inhibited cytotoxic activity (Table 6). Again, no inhibitory effect could again be noted on ADCC reactions produced by type II anti-Ig. 4. DISCUSSION
The data presented in this study confirm previous reports (1, 17, 27), of significantly increased anti-Ig in myeloma patients as compared to healthy age-matched controls. However, in contrast to other investigators (17) we could not find a consistent correlation between the anti-Ig titer and the clinical stage or course of the disease. This difference may be explained by different screening methods used, possibly detecting anti-Ig of different molecular weight and/or different specificities by using different protein preparations as coating material for sheep or human red blood cells. Whereas anti-Ig of different Ig classes have been known for many years, type III anti-Ig of low molecular size have been mentioned only by Lindstrom and Williams (17) previously. Our investigations have established that these anti-Ig themselves are Ig-breakdown products of (Fab’), composition, as revealed by their localization in the Sephadex G-200 column chromatography elution pattern, their reactivity with specific Fab antisera, and their removal from serum fractions on anti-Fab-coated Sepharose 4 CnBr columns . Increased titers of type III anti-Ig have been detected in sera from MM patients before starting cytostatic therapy, but were present in only one out of eight cases of the BMH patients, and were not detectable in MM patients under cytostatic treatment. How do (Fab’),-type anti-Ig arise? Fish, Witz, and Klein (11) have shown that various tumor cell lines derived from murine tumors are capable of degrading IgG molecules. Various mouse or human tumor cells contain enzymes located in the lysosomes or the cell membrane capable of fragmenting IgG molecules (15). Fragmentation of immunoglobulins bound to the cell surface, however, is not an exclusive feature of tumor cells. Engers and Unanue (8) reported that IgG-antibodies bound to surface Ig of normal B-lymphocytes are also partially degraded by these cells. It is possible that a similar mechanism is responsible for the appearance of low molecular size anti-Ig (type III), in that the IgGanti-Ig bound to the tumor cell surface could be degraded by proteolytic enzymes produced by the tumor cell. If tumor cells of MM patients are indeed responsible for anti-Ig fragmentation, an increased number of tumor cells (as found in the progressive stages of the disease) might cause an increased concentration of low molecular size anti-Ig. Cytostatic therapy, leading to a reduction of the tumor cell number, could then be followed by a decrease in the concentration of type III anti-Ig, and the appearance of type II anti-Ig, as has been observed in this study. In addition, the data reported in this paper using direct and indirect immunoflu-
598
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ET AL.
orescence techniques indicate that type III anti-Ig, representing (Fab’), fragments with anti-Fab specificity, reduced considerably the reactivity of anti-isotypic as well as anti-idiotypic antisera with PBL preparations, regardless of whether these PBL were prepared from healthy adults or from patients with multiple myeloma. These results show that type III anti-Ig are able to fix to the surface of SmIgpositive PBL, i.e., B-lymphocytes, as well as to surface of cells with defined idiotypic determinants found in the peripheral blood of MM patients (Table 4). In contrast, the incubation of these B-cell-enriched preparations from normal individuals or from MM patients with type II anti-Ig, found in the second peak of did not significantly inhibit the Sephadex G-200 column serum preparations, binding activity of FITC-labeled anti-isotypic anti-Ig sera on the cell surface. As shown by these experiments as well as by passive hemagglutination tests using SRBC coated with individual myeloma proteins (Table S), type II as well as type III anti-Ig failed to show anti-idiotypic specificity, but displayed a broad reactivity with determinants of surface immunoglobulins, particularly with those present in the Fab region, obviously including the idiotypic ones. Although direct evidence has not been sought in our experiments, it may be assumed that such reactions also take place in ~ivo, thus covering up surface immunoglobulins of Blymphocytes. This reaction may at least partially explain the decreased percentage of SmIg-positive lymphocytes in the circulation of MM patients as measured by direct or indirect immunofluorescence techniques ( 1, 17) (Table 4). When the influence of anti-Ig-containing serum preparations and purified anti-Ig on antibody-dependent cytotoxicity (ADCC) reactions in vitro was studied, it was demonstrated that serum fractions from patients with MM containing type III anti-Ig were able to inhibit the cytotoxic activity of normal PBL-preparations. This blocking effect could not be observed when fractions from patients with BMH-containing type II anti-Ig were employed. Type III anti-Ig preparations isolated from anti-Ig-containing Sephadex G-200 serum fractions by affinity chromatography also exhibited strong inhibitory activity. Thus type III anti-Ig, usually found in untreated MM but rarely in BMH sera, exhibited inhibitory properties similar to the anti-Ig of low molecular weight observed by Fish rt ul. (11) and Keisari and Witz (15). Further analysis of the inhibitory activity of anti-Ig found in MM patients’ sera on ADCC reactions has shown that preincubation of effector cells, as well as target cells, with type III anti-Ig led to a significant reduction of cytotoxicity. A straightforward explanation of the inhibitory mechanism is therefore not at hand. The effector cells in ADCC reactions, such as employed in this study, have been shown to be Fc-receptor-positive, non-B non-T lymphocytes (20, 25, 26). Since type III anti-Ig lack Fc fragments as shown by affinity chromatography with Fc-specific antisera, inhibition could not be brought about by their reaction with Fc receptors of those effector cells. The effect of type III anti-Ig on the antibody-coated target cell can be understood more easily since a reaction of these anti-Ig with the antibody molecules on the target cell surface may well interfere with the availability of these antibodies for Fc-receptors on effector cells. Other anti-Ig of different specificities and various immunoglobulin classes, e.g., “rheumatoid factors,” have also been shown to inhibit effectively ADCC reactions involving different types of target cells (10,26).
SERUM Anti-Ig IN MYELOMA
AND HYPERGLOBULINEMIA
599
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
Abdou, N. I., and Abdou, N. L., Clin. Exp. Immunol. 11, 57, 1972. Avrameas, S., Toudou, B., and Chuilon, S., Immunochemistry 6, 67, 1969. Brier, A. M., Chess, L., and Schlossmann, S. F., J. Clin. Invest 56, 1580, 1975. Cerottini, J. C., and Brunner, K. T., Advun. Immunol. 18, 67, 1974. Cooperband, S. R., Nimberg, R., Schmid, K., and Mannick, J. A., Transplant. Proc. 7,225, 1976. Dauphinee, M. J., Talal, M., and Witz, I. P., .I. Immunol. 133, 948, 1974. Durie, B. G. M., and Salmon, S. E., Proc. Amer. Ass. Cancer Res. 15, 90, 1974. Engers, H. D., and Unanue, E. R., J. Immunol. 110, 465, 1973. Faanes, R. B., and Choi, Y. S., J. Immunoi. 113, 279, 1974. Fink, P. C., Schedel, I., Peter, H. H., and Deicher, H., Stand. .I. Immunol. 6, 173, 1977. Fish, F., Witz, I. P., and Klein, G., Clin. Exp. Immunol. 16, 335, 1974. Hannestad, K., Kao, M. S., and Eisen, H. N., Proc. Nat. Acad. SC;. USA 69, 2295, 1972. Hartmann, D., and Lewis, M. G., Lancer 29, 1318, 1974. Hartmann, D., Lewis, M. G., Proctor, J. W., and Lyons, H., Lancet, 21, 1481, 1974. Keisari, Y., and Witz, I. P., Immunochemistry 10, 565, 1973. Leibold, W., and Peter, H. H., Rehring Inst. Mitt. 62, 144, 1978. Lindstrom, F. D.; and Williams, R. C., Clin. Immunol. Immunopathol. 3, 503, 1975. Lindstrbm, F. D., Williams, R. H., Eberle, B. J., and Williams, R. C., Ann. Intern. Med. 78,837, 1973. Lynch, R. G., Graft, R. J., Sirisina, S., Simms, E. S., and Eisen, H. N., Proc. Nat. Acad. Sci. USA 69, 1540, 1972. MacLennan, I. C. M., Transplant. Rev. 13, 67, 1972. Mellstedt, H., Jondal, M., and Holm, G., Clin. Exp. Immunol. 15, 321, 1973. Mellstedt, H., Hammerstrom, S., and Holm, G., Clin. Exp. Immunol. 17, 371, 1974. Ossermann, E. I., and Takatsuki, K., Ser. Haematol. 4, 28, 1965. Perlmann, P., Perlmann, H., and Wigzell, H., Transplant. Rev. 13, 91, 1972. Peter, H. H., Pavie-Fischer, J., Fridman, N. H., Aubert, C., Cesarini, J. C., Roubin, R., and Kourilsky, F. M., J. Immunol. 115, 5309, 1975. Saksela, E., Pyrhonen, S., Timonen, T., and Teppo, A. M., Stand. J. Immunol. 5, 1075, 1976. Schedel, I., Fink, P. C., and Kalden, J. R., Blur 32, 275, 1976. Schedel, I., Fink, P. C., Bodenberger, M., and Deicher, H., Clin. Immunol. Immunopathol. 5, 74-83, 1976. Schedel, I., Geissler, C., and Deicher, H., submitted.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
15,
Serum Anti-lmmunoglobulins and Benign Monoclonal I. SCHEDEL,
P. C. FINK,
5-86-599
(1980)
in Multiple Myeloma Hyperglobulinemia’
W. SCHBNER,
J. R. KALDEN.’
H. H. PETER, AND H. DEICHER Division
of Clinicui
Immunology und Blood TransJitsion, Department Medical School Hannorvr. Wesr Garmanv
of Medicine,
Received February 7. 1979 Anti-immunoglobulins with specificity to human native IgG and (Fab’), were detected in 69 sera of patients with multiple myeloma and 71 sera from 22 patients with benign monoclonal hyperglobulinemia with titers distinctly higher than those found in two control groups (healthy persons, sarcoidosis patients). Without further differentiation, no correlation of anti-immunoglobulin titers to the clinical status of the disease was observed. Chromatographic separation of sera on Sephadex G-200 revealed three molecular species of anti-immunoglobulins: type I. MW ca. 900,000; type II, MW 160,000; type III, MW < 100,000. Elevated titers of type III anti-immunoglobulins were observed only in multiple myeloma. Using immunoadsorbent techniques type III anti-immunoglobulins were identified as (Fab’), fragments. Lacking idiotypic specificity. type III antiimmunoglobulins were shown to react with allotypic as well as idiotypic immunoglobulins on the surface of PBL B-lymphocytes. In addition, they inhibited significantly antibody-dependent cellular cytotoxicity reactions in t?tro using IGR 3 melanoma cells as targets, and preparations of peripheral blood lymphocytes from healthy individuals as effector cells. In contrast, no significant inhibition of cytotoxic activity was observed using type II (IgG) anti-immunoglobulins from benign monoclonal hyperlgobulinemia patients’ sera in equal concentrations.
1. INTRODUCTION
A report by Abdou and Abdou (1) demonstrated the presence of antiimmunoglobulins (anti-Ig)3 in seven of nine sera from patients with IgG myeloma. In a study by Lindstrom and Williams (17) and Lindstrom et al. (18), who examined sera from patients with IgG multiple myeloma (MM) and patients with benign monoclonal hyperglobulinemia (BHM), 49% of MM sera were found to contain anti-Ig, as compared to 26.3% of BMH sera and 28% of age-matched control sera. Using sucrose density gradient separation, these authors described anti-Ig activity in 3.5 S fractions in addition to 7 S anti-Ig. Analysis of tumor immunity in patients with different malignancies has revealed lymphoid cells in the peripheral blood * This work was supported by Grants SCHE 169/l, 19/7 1, and SCHE 16912 as well as by funds from the Sonderforschungsbereich 54, project G3, of the Deutsche Forschungsgemeinschaft. 2 Present address: Universitlt Erlangen, Abteilung fur klinische Immunologic, Erlangen, West Germany. 3 Abbreviations used: ADCC. antibody-dependent cellular cytotoxicity: anti-Ig, antiimmunoglobulin; BMH, benign monoclonal hyperglubulinemia; MM, multiple myeloma; MW, molecular weight; SmIg, surface membrane immunoglobulin; PBL, peripheral blood lymphocytes: pHAT, passive hemagglutination test; fraction F. fraction Ficoll; fraction FFF, fraction Ficoll, Fer, Falcon; h-action FFF-C. fraction Ficoll, Fer, Falcon, Column; pHAT. passive hemagglutination test. 586
0090-1229/80/040586-14$01.00/O Copyright @ 1980 by Academic Ress, inc. All rights of reproduction in any form reserved.
SERUM
Anti-Ig
IN
MYELOMA
AND
HYPERGLOBULINEMIA
587
capable of killing tumor-cells in vitro (4, 16, 24, 25). Serum factors inhibiting this cellular cytotoxicity have also been noted (3, 5, 6,9, 10, 12, 24, 26). More particularly, blocking of cellular responses to tumorspecific or tumor-associated antigens has been connected with antibodies of different IgG subclasses, or antibody fragments, which inhibit cellular responses either by combining with antigen at the target cell membrane, or by direct inhibition of lymphoid effector cells (24, 26). Anti-Ig from MM sera have been shown to stimulate growth of the patients’ marrow plasma cells in tissue cultures. The present study was performed to determine semiquantitatively anti-Ig in sera from patients with MM and BMH, and to characterize the anti-Ig with respect to their molecular size and specificity. In addition to anti-Ig corresponding in size to IgM and IgG, low molecular weight (Fab’),-type anti-Ig have been demonstrated. The results indicate that the presence of serum anti-Ig of different types may be one feature distinguishing between MM and BMH. The additional purpose of the present study was to examine the effect of serum anti-Ig, of different molecular size, on antibody-dependent cytotoxicity of peripheral lymphoid cells in vitro. The results indicate that (Fab’), anti-Ig found in MM can effectively inhibit cell mediated cytotoxicity (ADCC) in vitro, thus posing the question as to the possible function of anti-Ig in MM in in vivo. 2. MATERIALS
AND METHODS
2.1. Serum Samples Serum samples were obtained from 69 patients with MM and from 22 persons with BMH for anti-Ig tests. In addition, sera from 35 healthy blood donors, 17 patients with MM, and 14 persons with BMH were available for anti-Ig testing as well as for cellular cytotoxicity tests. The diagnoses were confirmed by the usual clinical and laboratory criteria (7, 22, 23). Patients with BMH showed no progressive increase of serum myeloma protein concentrations or other signs of MM during the last 2-5 years. Control serum samples were obtained from 56 age-matched healthy blood donors and 18 patients with sarcoidosis. Most of the sera studied were kept frozen at -80°C until use. Twelve serum samples from MM patients and 25 sera from healthy controls were tested for anti-Ig immediately after drawing. 2.2. General Screening for Anti-Ig Anti-Ig activity was studied using the passive hemagglutination technique (pHAT) of Avrameas, Toudou, and Chuillon as modified by Hartmann and Lewis (2, 7, 10, 13, 14). Briefly, lOO-mg native human IgG (prepared by Professor Dr. Hoppe, Zentralinstitut fur das Blutspendewesen, Hamburg), or lOO-mg (Fab’),G-immunoglobulin fragments prepared from Gamma-Venin (Behring-Werke, Marburg/Lahn, West Germany) by gel filtration on Sephadex G-150 was added to 18 ml of 20% sheep red blood cells (SRBC) in borate-buffered saline (pH 8.6). After a single wash of the cell suspension 9 ml of 2.5% glutaraldehyde solution in distilled water was added dropwise with gentle agitation on a magnetic stirrer. The suspension was then incubated at room temperature for 1 hr and centrifuged, and the formed precipitate of coated SRBC was washed three times in 5-10 ml of borate-buffered saline. The coated SRBC were then resuspended in 60 ml borate-
588
SCHEDEL
ET
AL.
buffered saline containing 1.0% sodium azide to obtain a 6% stock suspension. The working suspension was 0.6%. Sera were inactivated (10 min/56”C) and after exhaustive absorption with uncoated SRBC serially diluted with borate-buffered saline using Microtiter-plates (patent Cook engineering, Fa. Greiner, West Germany). After the addition of coated SRBC, the plates were gently shaken and subsequently incubated for 2 hr at 37°C. The hemagglutination titers were then read and recorded. Titers below 4 were regarded as normal (see below). Serial dilutions of goat anti-human immunoglobulins or rabbit anti-Fab immunoglobulin fragment sera (Hyland, Travenol, Munich, West Germany) absorbed with uncoated SRBC were used as positive controls. Uncoated cells were used as negative controls. The specificity of the agglutination reactions was assessed by inhibition experiments. Sera were incubated with serial dilutions of the protein to be tested (human native IgG; human (Fab’,G fragments; human FcG fragments) followed by the usual passive hemagglutination procedure as described above. Complete inhibition or blocking of at least 4 hemagglutination titer-units could be achieved with human IgG or with human (Fab’),G fragments. In order to exclude the possibility that the IgG on the SRBC surface could attract and denature complement components, thus producing false-positive results, IgG-coated SRBC were incubated with five different native normal human sera (30 min/37”C). Thereafter, pHAT were performed using anti-C, as well as rabbit anti-C,, sera (Behringwerke, Marburg/Lahn, West Germany). 2.3. Approximation of the Molecular Size oj’ the Anti-Ig An approximation of the molecular size of the anti-Ig present in sera from patients with MM and BHM was obtained by Sephadex G-200 gel chromatography, followed by anti-Ig determinations in the serum fractions obtained using the passive hemagglutination technique. The molecular size of the proteins present in the fractions was estimated by quantitative determinations of proteins with known molecular weight (IgM. IgG, IgA, albumin). These column chromatographies were performed in all cases immediately after collection of the sera. 2.4. Isolation of Anti-Ig Anti-Ig were isolated from five sera of myeloma patients using Sepharose 4 CnBr (Pharmacia, Uppsala, Sweden) coated with human IgG as immunosorbent. Anti-Ig were absorbed from serum fractions previously obtained by gel chromatography (Sephadex G-200, Pharmacia, Uppsala, Sweden), eluted from the immunosorbent by glycin-buffer (pH 4.5), and subsequently dialyzed against PBS (pH 7.2). The preparations were then checked for immunoglobulin content by radial immunodiffusion ‘technique (LC-plates, Behring-Werke. Marburg/Lahn, West Germany). They were further analyzed by immunoelectrophoresis with goat anti-human plasmaprotein, anti-human IgG, anti-human Fab, and anti-human Fc sera (Hyland, Travenol, Munich, West Germany). Anti-Ig activity of the preparations was tested using the pHAT. The antigenic properties of the anti-Ig were also investigated by adsorption on Sepharose 4 CnBr coupled with goat antibodies to the Fc-fragment or the Fab-fragment of human IgG. Anti-Ig containing serum fractions obtained from 10 ml serum were passed through a column with 10 ml of
SERUM
Anti-Ig
IN
MYELOMA
AND
HYPERGLOBULINEMIA
589
antibody-coated Sepharose equilibrated with sodium bicarbonate buffer (pH 8.2). Effluent volumes obtained by elution with sodium bicarbonate-buffer (pH 8.2) and subsequently with citrate-buffer (pH 3.2) were concentrated to 0.5 ml and tested for anti-Ig by pHAT. 2.5. Preparation of Lymphocytes and Lymphocyte Subpopulations from Peripheral Blood (PBL) 2.5.1. Preparation of F, FFF, and FFF-C lymphocytes. The lymphocyte rich fractions obtained from 200~ml heparinized or defibrinated blood were separated by Ficoll-density gradient centrifugation and were washed twice with MEM supplemented with 15% FCS. Contaminant red blood cells were lysed by treatment for 10 min at 37°C in 0.15M ammonium chloride. Then the cell suspensions were washed twice and resuspended in MEM + 15% FCS to a final concentration of 2 x 10’ cells/ml (fraction F). This cell suspension was referred to as fraction “FFF” (Ficoll, Fer, Falcon). Surface-Ig bearing lymphocytes were removed from fraction FFF by an antiIg-column using the technique described by Peter et al. with slight modifications (25). Cells not retained by the column were referred to as fraction FFF-C (FFF column), which was devoid of surface-Ig-positive cells as revealed by direct immunofluorescence technique using FITC-labeled goat anti-Ig sera. Phagocytic and adherent cells were eliminated by lymphocyte separating reagent and incubation in Falcon petri dishes as described previously (25). 2.5.2. Preparation of B-cell enriched lymphocyte fractions. B-cell enriched lymphocyte fractions were obtained from F-lymphocytes by addition of SRBC followed by removal of E-rosetting cells by a second Ficoll-gradient-density centrifugation. 2.6. Antibody-Dependent Cytotoxicity (ADCC) ADCC reactions were performed using a cultured human melanoma cell line (IGR 3) as previously described (25). For inhibition studies, effector or target cell preparations were incubated simultaneously with different anti-Ig preparations. In order to determine the effect of anti-Ig on the target cells or on the effector cells, these cell preparations were incubated with different types of anti-Ig and washed six times (PBS, pH 7.2) before incubation for cytotoxicity tests. 2.7. Immunojluorescence Technique Immunofluorescence technique using fluorescent goat anti-IgG, -IgM, and -1gD sera (Hyland, Travenol, Munich, West Germany), was performed as described previously (28). In order to determine the reactivity of the anti-Ig to the cell surfaces, 1 x lo6 lymphoid cells were incubated with 0.5 ml of anti-Ig-containing fractions (30 min/ 20°C). Thereafter immunofluorescence technique was applied using FITC-labeled anti-Ig sera as described above. 2.8. Raising of Zdiotype-Speczjk Antisera Antisera with specificity against variable region antigens of myeloma proteins were raised in rabbits by repeated immunizations with 1 mg of purified myeloma protein preparations. Myeloma protein was isolated from the patients’ sera by
590
SCHEDEL
ET
AL.
ammonium-sulfate precipitation, Sephadex G-200, and Sephadex DEAE-A 50 gel filtration. Antisera were absorbed with purified polyclonal Ig preparations coupled to activated Sepharose 4 CnBr, and with PBL (prepared by Ficoll-density centrifugation) from healthy individuals. Idiotypic specificity was assessed by Ouchterlony and solid phase Elisa-techniques as described previously (29). 3. RESULTS 3.1. General
Screening
for Anti-lg
As demonstrated in Fig. 1, positive pHAT reactions with human IgG-coated SRBC were obtained in 58% of the sera from healthy adults. However, titers higher than 2 were never observed. In contrast, all sera from MM as well as from BMH patients showed positive agglutination reactions with titers ranging from 16 to 512, without obvious differences between sera from patients with MM and BHM. Sera from 18 sarcoidosis patients, tested as an additional control group, displayed low titers of anti-Ig. Figure 2 summarizes the results of the tests using SRBC coated with (Fab-),-G-immunoglobulin fragments. Although titer differences between control sera and MM and BMH sera were not impressive with this antigen, some MM sera exhibited titers of 16 and 32, which were rarely seen in the sarcoidosis control groups, and not at all in healthy persons’ sera. As for MM, no correlation was noted to the clinical course of the disease or any laboratory criteria (total serum protein, myeloma protein concentration as calculated from the serum electrophoresis pattern, Bence Jones protein concentrations in the urine, histological and cytological bone marrow examination). In single patients, no consistent correlation was noted between pHAT titers obtained with either IgG-coated or (Fab’),-G fragment-coated SRBC.
healthy
adults
BMH
“Z 56
2561286432-
$ ?
f
z 0
n=71
myelomo n:- 69
.. .. . .. .. . . .
............
. . . .
................ ................ ............ ........
.. .. .. ..
. . . .
.. . .. .. . .. . .. .. .. . .. .. . .
. .. . .. . .. .. . . ..
.....
l6-
842_
.. . .. .. . .. .
,-
. .. . .. .. . . . . .. . .. .. . . . . . .. .. . .. .. . .. . .. .. . .. .
N -
sorcoidosis n q 21
. . ..
512-
E C_
multiple
FIG. 1. Anti-IgG activity in sera from healthy adults and from sarcoidosis determined by pHAT using sheep red blood cells coated BMH patients were tested repeatedly (n = 71).
. . . . .. . . .. ..*...*. ... patients with BMH, MM, and with IgG. Serum samples of 22
SERUM Anti-Q IN MYELOMA heolthy
adults
BMH
multiple
” =71
myeloma
1
............... ...............
............... ............... ...............
sarcoidosis
n-69
n =21
. .. .
.
. ..*****
l
. . .. . .. . . . . .. . . . . .. . .. . .. . . N
591
AND HYPERGLOBULINEMIA
.... .. .... ...... .. .... ...... ..
. . . . . .
. . . . . . . .
. . . . .
FIG. 2. Anti-(Fab’), activity in sera from healthy adults and from patients with BMH, MM, and sarcoidosis determined by pHAT using SRBC coated with (Fab’),-G. Serum samples of 22 BMH patients were tested repeatedly (n = 7 1).
3.2. Specificity of Agglutination
Reactions
The specificity of the agglutination reactions in the pHAT was assessed by inhibition experiments. Agglutination reactions in sera from MM and BMH patients were totally inhibited using human native IgG at a concentration of 20 pg/ml or below. Inhibition occurred with both IgG-coated and (Fab’),-coated SRBC. Inhibition was also obtained with (Fab’)*-G at or below 30 @g/ml. By contrast, anti-Ig in only two out of eight sera tested with IgG-coated SRBC and none of those tested with (Fab’),-G-coated cells were inhibited by Fc. Hemagglutination could not be achieved by adding IgG to normal human sera up to a final concentration of IgG of 50 g/liter. In order to exclude the possibility that complement components reacted with the coated SRBC, thus enabling immunoconglutinis present in the patients’ sera to produce positive pHAT reactions, pHAT were performed using heterologous anti-complement-component sera (see above). All these tests lead to negative results, indicating that under out test conditions the Ig-coated SRBC did not bind C,, or C3. 3.3. Characterization
of the Molecular Size of Anti-Ig
In order to estimate the molecular size of the anti-Ig, 32 sera from MM patients and healthy controls were separated on Sephadex G-200 columns (Pharmacia, Uppsala, Sweden). As described above protein concentrations and anti-Ig activity were tested in each isolated fraction; three different molecular classes of anti-Ig were observed. Figures 3a-c show three typical examples of separation curves with the anti-Ig titers obtained in the chromatographic fractions. In addition to anti-Ig separating with the first and second peaks, a third type of low molecular weight anti-Ig could be demonstrated. All sera from patients with MM (n = 9)
592
SCHEDEL
a
ET
AL.
5’0
240 elutgon
volume
lmll
3a. Chromatographic separation (Sephadex G-ZOO) of a serum from a patient with BMH showing anti-Fab activity in serum fractions containing proteins of ea. 150 000. FIG.
without or before treatment exhibited anti-IgG activity in the third serum fraction containing proteins of molecular weight less than 100 000 (type III anti-Ig), whereas after cytostatic treatment two of nine sera showed such activity. Such anti-Ig were detected in only two of eleven normal control sera, and at low titers, and were not found in sera of patients with BMH. In these tests, titers below 4 were regarded as normal. In three of eighteen cases of MM, elevated agglutinating activity against IgG as well as against (Fab’), (one case) could be detected in the first Sephadex G-200 peak, representing proteins with molecular weights of about 900 000 (type I-anti-Ig) (Figs. 3 and 4). Fractions from these three sera containing high molecular weight anti-Ig were also able to agglutinate SRBC coated by heat-
FIG. 3b. Chromatographic separation (Sephadex G-200) of a serum from a patient with multiple myeloma showing anti-Fab activity in serum fractions containing low molecular size proteins.
SERUM Anti-Ig IN MYELOMA
c
593
AND HYPERGLOBULINEMIA
50
150
elution
250 volume [ml)
FIG. 3c. Chromatographic separation (Sephadex G-200) of a serum from a patient with MM showing distinct anti-IgG activities in the first serum fraction containing proteins of high molecular weight (>200 000) as well as in the third peak, with proteins of molecular weight below 100 000.
aggregated human-IgG, and the unfractionated routine Waaler-Rose tests.
sera gave positive
reactions
in
3.4. Isolation of Anti-Zg Anti-Ig were isolated from five sera of myeloma patients using a gel filtration technique (Sephadex G-200) followed by immunoabsorption. Material exhibiting Fc determinants was not detectable in fractions containing low molecular weight anti-Ig activity. These fractions reacted with heterologous anti-Fab sera. As shown in Table 1 anti-Ig activity was completely absorbed by Sepharose beads multiple
myelomo
before
treatment
multiple
myeloma
under
treatment
benign
T III
monoclonal
hyperglobulinema
1
n=8
Ill
II
Ill
l l ee
. l
l ee
l ee
l
l e .
.
.
l
wee
l ea l ee l
FIG. 4. Anti-IgG activity found in serum fractions (Sephadex G-200) column chromatography) from MM and BMH patients obtained by pHAT using SRBC coated with human IgG (0) and human (Fab’), (0). II: Type II anti-Ig; III: type III anti-Ig.
594
SCHEDEL
ET AL.
TABLE REACTION COUPLED
OF SERUM AN,I WITH Awri-Fab
1
I-1g WI I’H StpHARost OR ANTI-FC FROM
Titer
IHE
4 CnBr GO.A I
of the anti-immunoglobulin After passage through Sepharose coupled with
Before passage Preparation Type
through
II anti-lg
anti-Fab
~~~_...
immunoabsorbent
~~~
pH 8.2
512 256
Type III anti-&
After passage through Sepharose coupled with .
ptl
ll II
1.2
anti-Fc
pH 8.2
pH 3.2
0 I56
256 0
76 128
coupled with goat antibodies to the Fab portion of human immunoglobulin. In contrast, the anti-Ig activity was recovered without significantly reduced titers from Sepharose coupled with Fc-specific goat antisera. These findings led to the conclusions that low molecular weight anti-Ig consisted of (Fab’), fragments. 3.5. Reactions of Anti-lg with PBL Surface Ig In comparison to lymphocyte preparations from a normal control group, the percentage of Ig-positive lymphoid cells demonstrable by direct immunofluorescence technique using FITC-labeled anti-human-(polyclonal)-Ig sera was found to be reduced in MM patients. However, when individually specific anti-idiotypic antisera were employed, a significant increase of SmIg-positive cells was found (P < 0.001) (Table 2). The regular presence of lymphoid cells carrying idiotypic (i.e., myeloma protein-specific) material on their surface in the peripheral blood of MM patients was thus demonstrated. When these PBL lymphocytes were preincubated with type III anti-Ig, the reactivity of anti-isotypic (Ig-class-specific) as well as anti-idiotypic sera with these cells was considerably reduced (Table 2). In TABLE PERIPHERAL FITC-COUPLED
BLOOD
LVMPHOCYTES
WITH
ANTI-ISOTYPIC INCUBATION
AND WITH
2
SURFACE
IMMIJNOG~UBULI&
ANTI-IDIOTYPIC
TYPE
III
ANTMRA
Anri-Ig
CONTAINING
Lymphocytes SUBJECTS Healthy adults (n = 20) Patients 1. H.J. 2. J.G. 3. R.A. 4. K.J. 5. G.K. 6. H.K. Mean
I&G,
15.5 -t 5
IgA.
IgM.
IgD
AND
(4)
9 11 13 9 8
(6) (7) (9) (4)
(6) (6 + 4.2)
AFER
WI’I-H (
)
FRACTIONS
SmIg
(%,I
Idiotypic
(12 t 3)
7
9.5 + 4.8
bearing
AS VISL’AI~IL~D BEFORE
determinants
0
36 29 26 18 27 24 26.6 i
(22) 121) (19) (16)
(22) (16) 13.2
(19.3
i- 6.3)
SERUM
Anti-Ig
IN
MYELOMA
AND
TABLE HEMAGGLUTINATION TITERS PATIENTS USING SRBC COATED
Serum
a Nos. l-8
ADULTS AND MULTIPLE MYELOMA OR INDIVIDUAL MYELOMA PROTEINS
IgG
titers using SRBC coated with Purified
my&ma
protein”
2
3
4
5
6
1
8
2
1
8
1
2
2
8
1
1024 1024 512 512 256 1024 1024 1024
512 512 1024 512 256 1024 1024 512
256 512 1024 1024 1024 1024 512 256
256 256 512 512 512 512 512 512
256 1024 512 256 256 1024 512 512
512 1024 512 256 512 512 256 512
512 512 256 256 512 512 256 512
1024 512 512 512 512 512 512 256
Pooled human I&
1
4 512 1024 1024 512 512 1024 1024 512
Healthy adults (II = IO) Patients 1. H.J. 2. J.G. 3. B.W. 4. R.A. 5. MM 6. A.G. 7. J.A. 8. KM
3
IN SERA FROM HEALTHY WITH HUMAN POOLED
Hemagglutination
595
HYPERGLOBULINEMIA
refer to patients as shown in left-hand
column of table.
contrast, preincubation of these cells with type II anti-Ig did not diminish the percentage of fluorescent cells applying FITC-labeled anti-isotypic sera. However, no indication of idiotypic specificity of type III antiglobulins was obtained. As shown in Table 3, sera from eight MM patients showed similar pHAT titers with normal human IgG or any of eight purified myeloma proteins used to coat SRBC in the pHAT reaction. In particular, no preferential reaction of indiTABLE EFFECT
OF ANTI-IMMUNOGLOBULIN
DEPENDENT
CELLULAR
CYTOTOXICITY CONCENTRATION
Lymphocyte fractions used as effector cells F
FFF
FFF-C
DERIVED
n
FROM
4 MULTIPLE
MYELOMA
Medium
added
Percentage specific Vr release (X)O
12 12 12 5 12 12 12 12 5 11
MEM NHS-fractiona Whole MM sera Type II anti-Igb Type III anti-Igb MEM NHS-fraction” Whole MM sera Type II anti-Ig* Type III anti-Igb
82.6
11 10 12 5 12 2
MEM NHS-fraction”
61.9 68.5 48.2 66.3 41.7 39.2
Whole
MM
sera
Type II anti-Igb Type
PATIENTS
ON ANTIBODY-
(ADCC) OF NORMAL HUMAN LYMPHOCYTES. WAS ADJUSTED TO 40 &ml IN EACH TEST.
III anti-Igb
Isolated type III Anti-&’
85.9 14.2 81.2 72.8 81.2 81.7 70.2 80.6 69.8
THE
SD
PROTEIN
Statistical evaluation (P)
-tlO.l 2 10.2 r 7.1 k12.1 211.1 r 5.8 + 12.9 * 7.5 ell.8 kl3.5 2 12.5 + 17.4 k14.1 k15.1 2 10.7
a Normal human serum fractions prepared in the same way as the corresponding serum fraction patients (fractions containing proteins of the same MW as type III anti-Is). b Anti-Ig-containing serum fractions prepared by gel chromatography (Sephadex G-200). ’ Type III anti-lg purified by immunoabsorbent column chromatography.
<0.025 <0.025 <0.025 10.025
from MM
596
SCHEDEL
ET AL.
TABLE 5 REACTIVITY OF NORMAL HUMAN LYMPHOCYTES AFTER PREINCUBA CELLS WITH TYPE III ANTbIg. THE PROTEIN CONCENTRATION ADJUSTED TO 40 &ml IN EACH TEST
ADCC
Lymphocyte fractions used as effector cells F
FFF
FFF-C
added
Percentage specific “Cr release Crl
I ION OF THE TARGET WAS
SD
Statistical evaluation (P)
II
Material
IO IO 5 10
MEM NHS-fraction” Type II anti-Igb Type III anti-Ig”
84.2 83.4 81.3 74.2
kli.2 ? \).I
“.S.
L 13.2 2 IO.0
n.s. “.S.
9 IO 10 10
MEM NHS-fraction” Type II anti-Igb Type III anti-Ig”
79.1 85.2
81.3
- I?.4 t 14. I . 8.2
n.s. “.S.
71.2
I 7 I
-0.01 ~CO.01 co.01
9 IO 10 10
MEM NHS-fraction” Type II anti-Ig” Type III anti-Igb
“.S. n.s.
-:o.ooo5
64.1 69.2 63.1
-co.025 <0.025 co.025
45.1
0 Normal human serum fractions prepared in the same way as the corresponding serum fractions patients (fractions containing proteins of the same MW as type III anti-Ig). b Anti-Ig-containing serum fractions prepared by gel chromatography (Sephadex G-200).
from MM
vidual patients’ sera with their own myeloma protein could be demonstrated with this technique. The lack of idiotypic specificity of anti-Ig contained in MM and BMH sera was thus demonstrated using two independent approaches. 3.6. Effect of Serum Fractions Containing Anti-Ig on ADCC Table 6 summarizes the capacity of serum fractions obtained from anti-lgcontaining sera by column chromatography on Sephadex G-200 to inhibit lysis of antibody-coated melanoma cells by different lymphocyte preparations (F, FFF, FFF-C) . In general, the addition of whole sera from untreated MM patients resulted in decreased cytotoxicity, an effect not observed to a significant degree with normal sera from healthy adults (Table 4). ADCC reactions were also significantly inhibited by serum fractions containing (Fab’),- anti-Ig. Significant inhibition was seen when F- (P < 0.025) and FFF- (P < 0.0005) lymphocyte preparations were used as
ADCC
TABLE 6 REACTIVITY OF NORMAL HUMAN LYMPHOCYTE FRACVION AF.rER PRKINCUBAI EFFECTOR CELLS WITH TYPE III ANTI-Ig. THE PROTEIN CONCENT-RATION WAS ADJUSTED TO 40 &ml IN EACH TEST
Lymphocyte fractions used as effector cells FFF-C
,i
Medium
added
IO MEM IO NHS-fraction” 10 Type III anti-Ig*
Percentage spe#ic release (x) 63.2 67.9 47.7
jlCr SD
ION OF IHI-
Statistical evaluation (P)
*IO.2
216.1
iO.005
+- 12.7
<0.005
a Normal human serum fractions prepared in the same way as the corresponding sernm fractions patients (fractions containing proteins of the same MW as type III anti-Ig). b Anti-Ig-containing serum fractions prepared by gel chromatography (Sephadex G-200).
from
MM
SERUM
Anti-Ig
IN
MYELOMA
AND
HYPERGLOBULINEMIA
597
effector cells. No inhibitory activity was detected when the tests were performed with serum fractions containing type II anti-Ig. (Fab’)* Anti-Ig prepared from the third Sephadex G-200 peak of MM sera followed by affinity chromatography using anti-Fab-coated Sepharose 4 CnBr as an immunosorbent (type III anti-Ig) also produced a considerable inhibition of the ADCC reaction in two cases. Preincubation of the target cells before addition of cytotoxic lymphocytes led to a reduced ADCC reaction in vitro (Table 5). Preincubation of the PBL effector lymphocytes with type III anti-Ig (30 min/20”C, 30 min/37”C) also inhibited cytotoxic activity (Table 6). Again, no inhibitory effect could again be noted on ADCC reactions produced by type II anti-Ig. 4. DISCUSSION
The data presented in this study confirm previous reports (1, 17, 27), of significantly increased anti-Ig in myeloma patients as compared to healthy age-matched controls. However, in contrast to other investigators (17) we could not find a consistent correlation between the anti-Ig titer and the clinical stage or course of the disease. This difference may be explained by different screening methods used, possibly detecting anti-Ig of different molecular weight and/or different specificities by using different protein preparations as coating material for sheep or human red blood cells. Whereas anti-Ig of different Ig classes have been known for many years, type III anti-Ig of low molecular size have been mentioned only by Lindstrom and Williams (17) previously. Our investigations have established that these anti-Ig themselves are Ig-breakdown products of (Fab’), composition, as revealed by their localization in the Sephadex G-200 column chromatography elution pattern, their reactivity with specific Fab antisera, and their removal from serum fractions on anti-Fab-coated Sepharose 4 CnBr columns . Increased titers of type III anti-Ig have been detected in sera from MM patients before starting cytostatic therapy, but were present in only one out of eight cases of the BMH patients, and were not detectable in MM patients under cytostatic treatment. How do (Fab’),-type anti-Ig arise? Fish, Witz, and Klein (11) have shown that various tumor cell lines derived from murine tumors are capable of degrading IgG molecules. Various mouse or human tumor cells contain enzymes located in the lysosomes or the cell membrane capable of fragmenting IgG molecules (15). Fragmentation of immunoglobulins bound to the cell surface, however, is not an exclusive feature of tumor cells. Engers and Unanue (8) reported that IgG-antibodies bound to surface Ig of normal B-lymphocytes are also partially degraded by these cells. It is possible that a similar mechanism is responsible for the appearance of low molecular size anti-Ig (type III), in that the IgGanti-Ig bound to the tumor cell surface could be degraded by proteolytic enzymes produced by the tumor cell. If tumor cells of MM patients are indeed responsible for anti-Ig fragmentation, an increased number of tumor cells (as found in the progressive stages of the disease) might cause an increased concentration of low molecular size anti-Ig. Cytostatic therapy, leading to a reduction of the tumor cell number, could then be followed by a decrease in the concentration of type III anti-Ig, and the appearance of type II anti-Ig, as has been observed in this study. In addition, the data reported in this paper using direct and indirect immunoflu-
598
SCHEDEL
ET AL.
orescence techniques indicate that type III anti-Ig, representing (Fab’), fragments with anti-Fab specificity, reduced considerably the reactivity of anti-isotypic as well as anti-idiotypic antisera with PBL preparations, regardless of whether these PBL were prepared from healthy adults or from patients with multiple myeloma. These results show that type III anti-Ig are able to fix to the surface of SmIgpositive PBL, i.e., B-lymphocytes, as well as to surface of cells with defined idiotypic determinants found in the peripheral blood of MM patients (Table 4). In contrast, the incubation of these B-cell-enriched preparations from normal individuals or from MM patients with type II anti-Ig, found in the second peak of did not significantly inhibit the Sephadex G-200 column serum preparations, binding activity of FITC-labeled anti-isotypic anti-Ig sera on the cell surface. As shown by these experiments as well as by passive hemagglutination tests using SRBC coated with individual myeloma proteins (Table S), type II as well as type III anti-Ig failed to show anti-idiotypic specificity, but displayed a broad reactivity with determinants of surface immunoglobulins, particularly with those present in the Fab region, obviously including the idiotypic ones. Although direct evidence has not been sought in our experiments, it may be assumed that such reactions also take place in ~ivo, thus covering up surface immunoglobulins of Blymphocytes. This reaction may at least partially explain the decreased percentage of SmIg-positive lymphocytes in the circulation of MM patients as measured by direct or indirect immunofluorescence techniques ( 1, 17) (Table 4). When the influence of anti-Ig-containing serum preparations and purified anti-Ig on antibody-dependent cytotoxicity (ADCC) reactions in vitro was studied, it was demonstrated that serum fractions from patients with MM containing type III anti-Ig were able to inhibit the cytotoxic activity of normal PBL-preparations. This blocking effect could not be observed when fractions from patients with BMH-containing type II anti-Ig were employed. Type III anti-Ig preparations isolated from anti-Ig-containing Sephadex G-200 serum fractions by affinity chromatography also exhibited strong inhibitory activity. Thus type III anti-Ig, usually found in untreated MM but rarely in BMH sera, exhibited inhibitory properties similar to the anti-Ig of low molecular weight observed by Fish rt ul. (11) and Keisari and Witz (15). Further analysis of the inhibitory activity of anti-Ig found in MM patients’ sera on ADCC reactions has shown that preincubation of effector cells, as well as target cells, with type III anti-Ig led to a significant reduction of cytotoxicity. A straightforward explanation of the inhibitory mechanism is therefore not at hand. The effector cells in ADCC reactions, such as employed in this study, have been shown to be Fc-receptor-positive, non-B non-T lymphocytes (20, 25, 26). Since type III anti-Ig lack Fc fragments as shown by affinity chromatography with Fc-specific antisera, inhibition could not be brought about by their reaction with Fc receptors of those effector cells. The effect of type III anti-Ig on the antibody-coated target cell can be understood more easily since a reaction of these anti-Ig with the antibody molecules on the target cell surface may well interfere with the availability of these antibodies for Fc-receptors on effector cells. Other anti-Ig of different specificities and various immunoglobulin classes, e.g., “rheumatoid factors,” have also been shown to inhibit effectively ADCC reactions involving different types of target cells (10,26).
SERUM Anti-Ig IN MYELOMA
AND HYPERGLOBULINEMIA
599
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