Treatment of autoimmune diseases such as rheumatoid arthritis with suppressor factor

Method of treating autoimmune diseases such as rheumatoid arthritis by administration of a suppressor factor obtained in the supernatant of a human cell line. A particular human cell line is CEM which has survived treatment with 6-thioguanine.

BACKGROUND OF THE INVENTION 
The mammalian immune system includes a complex array of organs, cells and 
soluble products of cells. Organs involved in the immune system include 
the bone marrow, spleen and lymph nodes; a wide variety of cells populate 
the immune system and this includes macrophages, granulocytes and T and B 
lymphocytes. Examples of soluble products produced by immune system cells 
include antibodies (produced by B lymphocytes) and lymphokines (produced 
by T-lymphocytes). The latter play an important role in regulating the 
immune system. 
The basic functions of the immune system include: 
(a) identification of substances or cells within the body to determine 
whether they are "self" or "non-self". 
(b) communication between cells to facilitate a response to "non-self" 
entities. 
(c) specific action on "non-self" substances or cells, for example specific 
lysis (dissolution) of a foreign cell. 
The immune system is responsible for manifestations which include 
destruction of infecting organisms and the antoimmune reaction which is 
thought to be a malfunction of the system. It is now recognized that for 
various reasons and in view of various factors including aging, genetic 
makeup of the individual, viruses, thymic defects and hormones, the 
mammalian body may produce antibodies against parts of itself resulting in 
autoimmune diseases which include myasthenia gravis, autoimmune hemolytic 
anemia and rheumatoid arthritis. 
Medical treatment of rheumatoid arthritis consists of the administration of 
drugs which may be classified as steroids, non-steroidal anti-inflammatory 
agents, gold salts and immuno-suppressive agents. Corticosteroids are used 
with caution, however, in view of serious side effects associated with 
long term use, including degenerative arthritis, hyperadrenocorticism and 
disruption of the pituitary-adrenal axis. Non-steroidal anti-inflammatory 
drugs such as aspirin, fenoprofen, ibuprofen and tolmetin are useful in 
long-term therapy provided that the side effects of high dosage, e.g. 
tinnitus, gastric upset and a decrease in platelet adhesiveness, are 
tolerated. Gold salts are associated with a high incidence of toxic side 
effects including dermatitis and aplastic anemia. Dramatic improvements in 
rheumatoid arthritis have been seen with immunosuppressive agents such as 
chlorambucil, cyclophosphamide, mercaptopurine and azathioprine. However, 
such drugs are associated with serious toxicity, may be teratogenic and 
may cause increases in lymphoma and infection. For these reasons, the 
routine use of immunosuppressive agents has been discouraged, see Chapter 
26 entitled "Rheumatic Diseases" by K. H. Fye and K. E. Sack in the text 
"Basic and Clinical Immunology" by D. P. Stites et al. Lange Medical 
Publications, Los Altos, Calif. 94022 (1982). 
It is an object of the present invention to provide an immunosuppressive 
therapy for mammals such as man afflicted with an autoimmune disease such 
as rheumatoid arthritis. A further object is a therapy which has few side 
effects and does not seriously affect the patient's ability to combat 
non-self antigents, e.g. infection. 
SUMMARY OF THE INVENTION 
The invention comprises a method for the treatment of autoimmune diseases 
such as rheumatoid arthritis by the administration of a factor which may 
be derived from the human acute lymphoblastic leukemic cell line CEM. The 
particular cell line is obtained by culturing the CEM line with 
6-thioquanine and recovering the small percentage of cells which survive, 
i.e. the 6-T resistant cells. The supernatant factor used in the present 
invention from the thus-produced cultured 6-T CEM cells is designated the 
"suppressor activating factor" or "SAF". SAF has been found to suppress 
production of autoantibodies in test animals immunized with cross-reacting 
antigenic materials compared to control animals immunized with the 
antigenic materials and treated with control vehicle alone. 
The following abbreviations may be used in the present specification: 
AIHA=autoimmune hemolytic anemia 
ATCC=American Type Culture Collection 
CEM=the lymphoblastic leukemic cell line CCRF-CEM (ATCC CCL 119) 
DAIT=direct anti-immunoglobulin test 
FCS=fetal calf serum 
.alpha.-MEM=minimum essential medium 
N=normal 
PBS=phosphate buffered saline 
RBC=red blood cells 
SAF=suppressor activating factor 
6-T=6-thioquanine 
DETAILED DESCRIPTION OF THE INVENTION 
The method of the present invention comprises the treatment of a mammal 
having an autoimmune disease by the administration of a suppressor 
activating factor. In particular, the method is applicable to the 
treatment of humans. 
Autoimmunity can be defined as a state in which immune mechanisms are 
directed against "self" as opposed to "non-self". Autoimmune disease can 
be defined as the pathlogical consequence of certain "self" directed 
immune mechanisms. Autoimmunity is relevant to many applications involving 
several different organ systems. Further, autoimmune diseases may be 
classified systemic or organ-specific. Examples of systemic autoimmune 
diseases include Goodpasture's syndrome, rheumatoid arthritis, Siogren's 
syndrome and systemic lupus erythematosus. Examples of organ-specific 
autoimmune diseases include myasthenia gravis, Graves' disease (diffuse 
toxic goiter), multiple sclerosis, Hashimoto's thyroiditis, 
insulin-resistant diabetes associated with acanthosis nigricans, 
insulin-resistant diabetes associated with ataxia-telangiectasia, allergic 
rhinitis asthma, functional autonomic abnormalities, juvenile 
insulin-dependent diabetes. Pernicious anemia, Addison's disease, 
idiopathic hypoparathyroidism, spontaneous infertility, premature ovarian 
failure, pemphigus, bullous pemphigoid, primary biliary cirrhosis, 
autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura and 
idiopathic neutropenia. 
The method of the present invention is applicable in particular to 
rheumatoid arthritis and autoimmune hemolytic anemia as explained below. 
Rheumatoid Arthritis 
Short et al. in "Rheumatoid Arthritis", Harvard Univ. Press, Cambridge, 
Mass. (1957) have defined rheumatoid arthritis as a chronic inflammatory 
disorder of unknown etiology which is systemic in nature and is 
characterized by the manner in which it involves joints. Both cellular and 
humoral immunological events mediate its pathogenesis; for example, 
lymphokines and large numbers of T-cells are frequently found in synovial 
tissues and fluids and many pieces of evidence link altered T-cell 
function with disease activity in rheumatoid arthritis: see the articles 
"Lymphocyte subsets and inflammatory indices in synovial fluid and blood 
of patients with rheumatoid arthritis" by J. V. Bertouch in J. Rheumatol 
(1984): 11:754-9; "Rheumatoid arthritis: a disease of 
T-lymphocyte-macrophage immunoregulation" by G. Janossy in Lancet (1981( 
2:839-42; and "In situ localization of lymphocyte subsets in synovial 
membranes of patients with rheumatoid arthritis with monoclonal 
antibodies", by C. J. L. M. Meijer in J. Rheumatol. (1982): 9:359-65. 
Alterations in in vitro characteristics of T lymphocytes from rheumatoid 
arthritis patients have been reported to correlate with altered cellular 
interactions and activity of the disease, as described by R. M. Pope in 
"Arthritis and Rheumatism" (1984): 24:1234-44. 
Thus, therapies directed towards the normalization of T lymphocyte function 
in rheumatoid arthritis may well be beneficial in its treatment. To this 
end, many broadly-active immunosuppressive agents have been tested in 
experimental autoimmune disease models, e.g. cyclophosphamide and 
betamethazone. Based on these encouraging animal data, immunosuppressive 
agents have been introduced into clinical medicine for the treatment of a 
number of autoimmune diseases including rheumatoid arthritis. Depiste 
their clinical efficacy, however, these drugs have only limited utility in 
therapeutic medicine due to their propensity to cause toxicity during 
long-term administration as a result of their broad, non-specific 
immunosuppressive action. 
Autoimmune Hemolytic Anemia 
Hemolytic anemia is a condition in which lysis or destruction of 
erythrocytes is a diagnostic feature. In almost all cases of acquired 
hemolytic anemia in which no cause is found and no associated disease 
recognized, autoimmune mechanisms appear to be the root of the problem, 
see M. M. Wintrobe in "Clinical Hematolgoy", 6th ed., p. 626, Lea and 
Febiger, Philadelphia (1967). Several animals models have been developed 
which mimic certain aspects of human autoimmune hemolytic anemia. 
The suppressor activating factor (SAF) used in the method of the present 
invention: 
(i) is secreted by a stable 6-thioguanine-resistant mutant of the 
lymphoblastoid cell line CEM. 
(ii) is non-mitogenic, and 
(iii) is non-cytotoxic. 
In more detail, the SAF used in the method of the invention: 
(iv) suppresses at least 90% of mitogen-induced T cell proliferation at a 
dilution of 10.sup.-6 but does not suppress mitogen-induced B cell 
proliferation at said dilution. 
(v) is contained in a high molecular weight protein, e.g. of about 110,000 
dalton. 
(vi) exhibits maximum suppressor activity at physiological pH. 
(vii) is inactivated at 56.degree. C. 
(viii) suppresses mouse spleen cell proliferation to mitogenic stimulation 
with the same potency as it suppresses human peripheral blood lymphocyte 
proliferation to mitogenic stimulation. 
(ix) exhibits a greater suppressive effect on autoantibody production than 
an antibody production directed at foreign antigens (rat erythrocytes) in 
mice immunized with said rat erythrocytes. 
The SAF may be obtained from the supernatant produced by those cells of the 
CEM lymphoblastic leukemic cell line which are resistant to destruction by 
6-thioguanine as described in the following preparations A, B, C and D and 
in my co-pending U.S. patent application Ser. No. 586,515 filed Mar. 5, 
1984 which is a continuation-in-part of my U.S. patent application Ser. 
Nos. 495,908 filed May 18, 1983 and 534,526 filed Sept. 21, 1983. U.S. 
Ser. Nos. 495,908 and 534,526 are now abandoned. European patent 
application ("EPO") No. 127,394 published Dec. 5, 1984 is the equivalent 
of my U.S. Ser. No. 586,515 and is incorporated by reference in the 
present specification for the preparation of SAF, designated therein as 
SIF or suppressor-inducer factor. 
A. Generation of 6-thioquanine (6T) resistant mutant 6T-CEM and the 
subclone 6T-CEM-20 
The 6-thioquanine (2-amino-6-mercaptopurine: 6T) was obtained from Sigma 
(Cat. No. A-4882). 100 mg of 6T was dissolved in 100 ml of distilled 
water. 1 or 2 drops of 10N NaOH were added to help the 6T to dissolve 
completely. The pH of the final solution is about 9. 
The lymphoblastic leukemic line CEM (CCRF-CEM, ATCC CCL 119) was obtained 
from the American Type Culture Collection, Rockville MD, and was 
maintained in 90% .alpha.-MEM and 10% FCS. CEM cells at log phase 
(10.sup.6 cells/well) were treated with various concentrations of 6T and 
viability was assayed 7 days later as shown in the following Table 1: 
TABLE 1 
______________________________________ 
6-Thioguanine % Cell 
(.mu.g/ml) Viability.sup.1 
______________________________________ 
0.5 51 
5 43 
10 32 
20 5 
30 4 
______________________________________ 
.sup.1 CEM cells were exposed to various concentrations of 6T for 7 days 
and viability was assayed by dye exclusion. 
Cells treated with 30 .mu.g/ml of 6T were selected for further studies. The 
remaining viable cells (4%) were grown in 25 mm culture flasks until a 
significant number of cells was observed, after which dead cells were 
removed by a Ficoll-Hypaque gradient. The viable cells were divided into 2 
parts. One part was maintained in .alpha.-MEM+FCS with fresh medium added 
every third day and the cells were passaged every week. The other part was 
plated in 96 well microtitre plates for subcloning. The subclones grew up 
in about 2-3 weeks, after which they were transferred to 24 well Linbro 
trays. When the cells reached a density of 10.sup.6 /well, they were 
tested for aminopterin sensitivity and production of SAF as shown in the 
following Table 2: 
TABLE 2 
______________________________________ 
Suppressor.sup.2 
Cell Lines % Viability.sup.1 
Activity 
______________________________________ 
CEM 100 5 .times. 10.sup.-3 
6T-CEM 10 10.sup.-6 
6T-CEM 2 0 10.sup.-6 
4 0 10.sup.-6 
13 2 &gt;10.sup.-6 
14 4 10.sup.-6 
17 2 &gt;10.sup.-6 
18 0 10.sup.-6 
19 0 10.sup.-6 
20 1 10.sup.-9 
______________________________________ 
.sup.1 CEM, 6TCEM and it subclones were tested for their sensitivity to 
aminopterin treatment (0.005 .mu.g to 0.5 .mu.g/ml). The viabilities of 
various cell lines after 7 day exposure to 0.05 .mu.g of aminopterin were 
shown. 
.sup.2 Suppressor effect of TCell proliferation was measured according to 
the method described in Example IV of my EPO 127,394 published December 5 
1984. The dilutions of various supernatants exhibiting 50% suppression wa 
shown. The entry "&gt;-10.sup.-6 " means that 50% suppression was attained a 
dilutions higher than 10.sup.-6. 
6T-CEM-20, one of the subclones which showed the highest level of SAF 
production, was selected for further studies. The line 6T-CEM-20 has been 
showing a consistent level of SAF secretion for greater than three years, 
indicating no loss of chromosome(s) involved in the secretion of the 
factor. The growth characteristics, karotype, sensitivity to aminopterin, 
level of suppressor activity and effects of supernatants on other 
immunological assays are listed for CEM, 6T-CEM, 6T-CEM-20, and Az-CEM (an 
azaguanine-resistant CEM mutant) in the following Table 3: 
TABLE 3 
______________________________________ 
CEM 6T-CEM 6T-CEM-20 Az-CEM 
______________________________________ 
Aminopterin 
98 10 2 3 
Sensitivity.sup.1 
Doubling 20 24 20 26 
Time.sup.2 
Karyotype.sup.3 
45 .+-. 16 
72 .+-. 12 
74 .+-. 14 
70 .+-. 18 
T-Cell.sup.4 
5 .times. 10.sup.-3 
10.sup.-6 
&gt;10.sup.-6 
&lt;10.sup.-1 
Suppressor 
Activity 
B-Cell &lt;10.sup.-1 
&lt;10.sup.-1 
10.sup.-1 
&lt;10.sup.-1 
Suppressor 
Activity 
MLC.sup.5 5 .times. 10.sup.-3 
5 .times. 10.sup.-5 
10.sup.-6 
&lt;10.sup.-1 
Suppressor 
Activity 
Effect on.sup.6 
2 2 4 2 
the Generation 
of Cytotoxic 
T-Cells 
______________________________________ 
.sup.1 % viability of the cells after 7 days exposure to 0.05 .mu.g/ml of 
aminopterin. 
.sup.2 Time required for the cells to double the number from a starting 
concentration of 5 .times. 10.sup.5 cells/ml. 
.sup.3 Chromosomes from 100 cells were counted, means and standard 
deviations were shown. 
.sup.4 Suppressor activity on TCell and BCell proliferation was measured 
according to the method described in Example IV of my EPO 127,394. The 
entry "&lt;10.sup.-1 " means that lower than 10.sup.-1 dilution is required 
to attain 50% suppression of B cell proliferation. The entry "&gt;10.sup.-6 
means that 50% suppression was attained at dilutions higher than 
10.sup.-6. 
.sup.5 Suppressor activity on allogenic MLC was measured according to the 
method described in Example V of EPO 127,394. Dilutions of supernatant 
showing 50% suppression were shown. The entry "&lt;10.sup.-1 " means that 
less than 10.sup.-- 1 dilution is required to attain 50% suppression of 
MLC reaction. 
.sup.6 Effect on the generation of cytotoxic T cells was measured 
according to the method described in Example VII of my EPO 127,394. 
Differences in % cytotoxicity between supernatant (used at 10.sup.-1 
dilution) treated cells and medium treated cells were shown. 
B. Production of suppressive supernatants 
6T-CEM cells were routinely maintained in .alpha.-MEM containing 10% FCS. 
For production of the suppressive supernatants from 6T-CEM, cells were 
washed and suspended at 2.times.10.sup.5 cells/ml or 10.sup.6 cells/ml in 
.alpha.-MEM with 2% FCS. Supernatant was harvested 48 hours later and the 
functional activities were assessed. 
C. Ammonium sulfate precipitation 
Cell-free supernatant was centrifuged at 10,000 rpm for 20 minutes to 
remove any debris. Ammonium sulfate was then added gradually to give a 
final 50% ammonium sulfate saturation. After stirring for 45 minutes, the 
solution was centrifuged at 10,000 rpm for 30 minutes. The precipitate, 
which contained the suppressor activity, was exhaustively dialyzed against 
PBS. All steps were carried out in the cold and the pH was maintained 
between pH 7.0 and pH 7.4. This fraction of SAF was used in the Examples 
below. 
D. Chromatography on Sephacryl S-200 column 
The 50% ammonium sulfate fraction containing the suppressor activity was 
further purified on a Sephacryl S-200 superfine column (2.5 cm.times.50 
cm). The column was equilibrated with PBS and had a flow rate of 30 ml per 
hour. 2 ml of the dialyzed, 50% ammonium sulfate precipitated fraction was 
applied on the column. Elution was carried out with PBS and 5 ml volumes 
were collected. The protein profile was obtained by reading optical 
density at 280 nm. 
In treating autoimmune disease, individual batches of SAF may be 
standardized by determining the highest dilution of SAF giving 80% 
suppression of Con A induced T lymphocyte proliferation in a 2-day assay. 
The reciprocal of such dilution is defined as the `unit of activity` of 
SAF tested. (For instance, a batch which suppresses at 4.times.10.sup.-5 
dilution contains 2.5.times.10.sup.4 units/ml). The absolute amount of SAF 
required for a particular treatment will depend on the duration and 
severity of the symptoms. In general, SAF may be given daily at a dose 
range of 2-10.times.10.sup.4 units or 1-20 mg per kg of body weight until 
remission. SAF may be purified by gel filtration and ionic exchange 
chromatography or by SDS polyacrylamide electrophoresis until a single 
band is obtained. The specific activity of SAF should be approximately 0.5 
to 2.times.10.sup.4 units/mg. SAF may be administered in intravenous 
solution and it may be stored at -20.degree. C. in vials that meet 
specifications for use as containers for injectables. The recommended 
preservatives are methyl and propyl parabens.

EXAMPLE 1 
a. Introduction 
Induction of autoimmune responses by injection of high concentrates of 
autologous or highly cross-reacting allogenic or xenogeneic tissue or 
organs has been used for the induction of autoimmune diseases such as 
autohemolytic anemia, autoimmune thyroiditis and autoimmune 
encephalomyelitis. Symptoms observed subsequently in the experimental 
animals suggest that these induced diseases closely mimic the human 
clinical diseases autoimmune hemolytic anemia, thyroiditis and multiple 
sclerosis, see H. S. Flad in Proc. Soc. Exp. Biol. Med. 131:121 (1969); A. 
D. Vladertine in Science 174:1137 (1971); and D. L. Gasser et al, in 
Science 181:873 (1973). 
Autoimmune hemolytic anemia in humans is a classical autoimmune disease 
wherein autoantibodies to the patient's erythrocytes cause hemolysis and 
resulting anemia, see R. S. Evans et al. in Ann. N.Y. Acad. Sci. 124:422 
(1965). One method of inducing erythrocyte autoantibodies experimentally 
in animals is to immunize mice with cross-reacting rat erythrocytes, see 
C. Y. Lau et al. in the Journal of Immunology Vol. 125, No. 4 pages 
1634-1638 (1980). The duration of autoantibodies is self-limiting in most 
normal mouse strains and is probably controlled by a specific population 
of suppressor T cells, see J. H. L. Playfair et al. in Nature (New 
Biology) 243:213 (1973) and A. Cooke et al. in Clin. Exp. Immunol. 27:538 
(1977). The extent of suppression varies from strain to strain. C57BL/6 
mice apparently have a strong suppressor system and only very transient 
autoantibodies develop after immunization. However, in CBA and C3H, mice, 
erythrocyte autoantibodies can be detected by the DAIT for as long as 10 
weeks after immunization, see A. Cooke, in Nature 273:154 (1978). On the 
other hand, N2B mice, the classic mouse model for human autoimmune 
hemolytic anemia, not only showed hyper-responsiveness to the induction, 
but, the erythrocyte autoantibody, once elicited, persisted until the 
death of the animals. Similarly, in the case of the SJL strain, which had 
been shown to be highly susceptible to the induction of experimental 
allogenic encephalomyelitis when subjected to induction, also failed to 
regulate autoantibody production through the loss of antigen-specific 
suppressor cells. Thus, failure to develop self-tolerance probably 
accounts for the disease manifestation, suggesting that experimental 
autoimmune hemolytic anemia is a good model for the in vivo investigation 
of experimental compounds. 
The effect of SAF in suppressing or accelerating the decline of 
autoantibody titres to self-RBC was studied in C3H/J mice, a normal mouse 
strain which responds in this experimental model. It was found that 
SAF--in a dose-related manner--accelerated the decline of autoantibodies 
titres in this mouse model. 
b. Procedure 
Animals used: 
Mice: Female C3H/J mice were obtained from Jackson Laboratories, Bar 
Harbor, Me. 10 mice were used per group. Rats: Female Long Evans rats were 
obtained from Charles River Laboraties, Mass. 
Immunization: 
Rat erythrocytes were collected from Long Evans rats, washed 3.times. and 
then injected i.p. at a cell concentration of 2.times.10.sup.8 
cells/mouse. After 2 weekly injections, mice were bled and the 
autoantibody titre tested. Mice were then divided into groups of 10 such 
that each group average autoantibody titre was similar. Mice were given 
one more immunization one week later. 
SAF Treatment: 
The 50% ammonium sulfate precipitated fraction of SAF, which had been 
dialyzed exhaustively against Hanks buffer containing 20% glycerol, was 
used. Control vehicles were either Hanks buffer with 20% glycerol or the 
50% ammonium sulfate precipitated fraction of growth medium prepared in 
exactly the same way as SAF. SAF treatment was initiated on the same day 
as the last RBC immunization. Intraperitoneal SAF treatment were 
administered in 0.2 ml doses 5 times per week. 
Antiserum: 
Polyspecific rabbit anti-mouse immunoglobulin was obtained from Cedarlane 
Laboratories Ltd. Hornby, Ont. The antiserum was reconstituted with 2 ml 
of distilled water, aliquoted and frozen. A 1:64 dilution of this 
antiserum was used for the DAIT. The diluent used in the DAIT was PBS with 
1% albumin. 
Testing 
A Student-t test was used to compare the autoantibody titre between the 
test group and the control group. 
DAIT: 
The presence of erythrocyte autoantibody was detected by a modified Coombs' 
test in microliter plates. Fifty microliters of blood were obtained weekly 
by tail bleeding. The mouse red cells were washed 4.times. with PBS and 
resuspended to a final concentration of 0.6%. Serial 2-fold dilutions of 
rabbit anti-mouse immunoglobulin (Cedarlane) were carried out in V-bottom 
microtitre wells with 1% albumin PBS as the diluent; 50 microliters of 
washed mouse cells were added to each well and the plates were mixed by a 
Vortex vibrator and sealed to avoid evaporation. The plates were left 
overnight at room temperature and read the next day. The titre of rabbit 
anti-mouse immunoglobulin was recorded as a measure of autoantibody bound 
to the red cells. Sharp and reproducible end points were obtained by using 
this microtitre method. Fifty microliters of blood from each animal was 
sufficient for the test. Results obtained from this method were directly 
comparable to results obtained by conventional Coombs' tests in tubes. 
d. Results 
In the DAIT test, the results were obtained as shown in Table 4: 
TABLE 4 
______________________________________ 
AIHA (Log.sub.2).sup.+ (Treatment in Week #1) 
Week # Control SAF (1/4 Dilution) 
SAF (1/8 Dilution) 
______________________________________ 
1 2.3 .+-. 2.7 
2.4 .+-. 2.6 2.7 .+-. 2.9 
2 6.5 .+-. 1.0 
7.0 .+-. 1.4 7.5 .+-. 1.2 
3 8.1 .+-. 1.1 
7.3 .+-. 1.2 8.4 .+-. 0.8 
4. 7.4 .+-. 1.3 
6.0 .+-. 0.9 6.0 .+-. 7.1 
5 7.7 .+-. 1.3 
6.5 .+-. 0.9 7.1 .+-. 1.1 
6 7.4 .+-. 9.2 
6.3 .+-. 0.9 6.6 .+-. 0.4 
7 7.8 .+-. 1.0 
5.8 .+-. 1.2 6.1 .+-. 1.0 
8 8.5 .+-. 1.2 
7.1 .+-. 0.9 7.8 .+-. 1.8 
9 11.7 .+-. 3.8 
8.4 .+-. 1.3* 9.2 .+-. 1.9 
10 9.7 .+-. 0.8 
8.3 .+-. 1.4* 8.2 .+-. 1.7 
11 10.3 .+-. 1.9 
7.5 .+-. 1.1* 8.8 .+-. 1.4 
12 5.9 .+-. 0.7 
4.3 .+-. 0.8* 5.0 .+-. 1.1* 
13 13.0 .+-. 3.3 
8.6 .+-. 4.2* 11.1 .+-. 3.2 
14 8.4 .+-. 5.8 
4.7 .+-. 3.8* 7.7 .+-. 4.9 
______________________________________ 
.sup.+ Reported as highest titre (lowest concentration) of antiserum 
giving an agglutination reaction of mouse red cells 
*p .ltoreq. 0.05 
For the first 8 weeks, SAF treated C3H/J mice developed autoantibody levels 
comparable to the control group. However, starting with week 9, 
differences between control and test groups began to emerge. Groups 
treated with SAF at 1/4 and 1/8 dilutions showed longer autoantibody 
titres than the control group. The autoantibody titres declined much more 
rapidly in the SAF treated groups during the subsequent weeks and did so 
in a dose related manner. Groups treated with 1/4 dilution of SAF showed a 
statistically significant decline in autoantibody titre, as compared to 
the control group from week 9 onwards. Groups treated with lower doses of 
SAF (1/8 and 1/16 dilutions) also showed lower titres of autoantibody 
production than the controls; however, the difference was not always 
statistically significant. 
EXAMPLE 2 
The procedures and testing of Example 1 were repeated with the exception 
that rat agglutinin titre measurements were also made. 
i. In the DAIT, the results obtained are shown in Table 5: 
TABLE 5 
______________________________________ 
AIHA (Log.sub.2).sup.+ 
Week # Control SAF 1/4 Dil. 
SAF 1/8 Dil. 
SAF 1/16 Dil. 
______________________________________ 
1 4.3 .+-. 1.3 
4.7 .+-. 0.8 
4.7 .+-. 0.8 
4.9 .+-. 1.1 
2 9.1 .+-. 1.5 
9.1 .+-. 0.7 
10.4 .+-. 3.3 
9.2 .+-. 1.5 
3 8.2 .+-. 0.9 
7.8 .+-. 2.1 
9.5 .+-. 0.7 
8.6 .+-. 1.1 
4 9.5 .+-. 0.5 
8.2 .+-. 0.9 
10.3 .+-. 1.7 
9.4 .+-. 1.1 
5 12.6 .+-. 1.4 
11.8 .+-. 1.4 
12.7 .+-. 2.8 
12.1 .+-. 1.5 
6 10.6 .+-. 1.0 
9.4 .+-. 2.0 
11.7 .+-. 3.0 
10.4 .+-. 1.1 
7 10.0 .+-. 0.9 
9.2 .+-. 2.6 
9.8 .+-. 1.8 
8.5 .+-. 2.4 
8 12.2 .+-. 1.5 
10.4 .+-. 1.9 
11.8 .+-. 2.1 
10.7 .+-. 1.4 
9 12.2 .+-. 1.4 
10.1 .+-. 2.6* 
11.6 .+-. 1.6 
9.6 .+-. 3.9 
10 11.4 .+-. 2.2 
9.4 + 1.8* 
10.3 .+-. 2.3 
10.1 .+-. 3.1 
11 12.0 .+-. 2.4 
7.0 .+-. 4.2* 
7.8 .+-. 4.2 
8.7 .+-. 5.1 
12 11.8 .+-. 2.0 
5.8 .+-. 3.0* 
8.1 .+-. 3.4 
8.0 .+-. 4.1 
13 10.0 .+-. 5.7 
5.7 .+-. 3.9* 
9.3 .+-. 2.8 
8.4 .+-. 4.6 
14 10.8 .+-. 4.7 
1.2 .+-. 2.5* 
4.7 .+-. 5.6* 
6.0 .+-. 5.2 
______________________________________ 
*p .ltoreq. 0.05 
.sup.+ Reported as the highest titre of antiserum giving agglutination of 
mouse red cells 
ii. Rat Agglutinin Titre Measurement: 
The purpose of this measurement was to detect changes in the production of 
antibodies to foreign antigens caused by SAF. Serial 2-fold dilutions of 
the test sera were carried out in V bottom microtitre plates. Fifty 
microliters of 0.6% rat erythrocytes were added to each well. The 
agglutination titre was expressed as the last well giving agglutination. 
The production of antibodies to the immunogen, rat erythrocytes, was 
monitored at weeks, 0, 3, 6, 9 and 12; results are shown in Table 6 below: 
TABLE 6 
__________________________________________________________________________ 
Rat Agglutinin Titre 
Weeks 
Groups 0 3 6 9 12 
__________________________________________________________________________ 
Control 
10.9 .+-. 0.8 
14.4 .+-. 1.7 
12.5 .+-. 1.0 
14.3 .+-. 2.5 
12.7 .+-. 1.5 
SAF 1/4** 
10.8 .+-. 1.0 
12.3 .+-. 0.8* 
10.7 .+-. 0.9* 
10.8 .+-. 1.2* 
10.0 .+-. 1.2* 
SAF 1/8** 
10.4 .+-. 0.5 
13.2 .+-. 1.5 
11.8 .+-. 1.2 
11.6 .+-. 0.9 
12.8 .+-. 1.8 
SAF 1/16** 
10.2 .+-. 1.0 
13.3 .+-. 1.9 
12.0 .+-. 1.1 
11.5 .+-. 1.9 
11.3 .+-. 1.8 
__________________________________________________________________________ 
*p &lt; 0.05 
**dilution of SAF 
Small but significant differences were observed between the control and 
high dose groups (1/4 dilution at 5.times. injections) starting from week 
3 onwards. Groups treated with lower doses of SAF did not show significant 
differences from the control group. 
Examples 1 and 2 show that intraperitoneal injection of SAF 5.times./week 
significantly suppressed the production of autoantibody. Thus, SAF 
treatment accelerated the process of suppression of an autoimmune 
response, a phenomena exhibited by normal mouse strains. The production of 
antibody to the immunogen, rat erythrocytes, was also reduced by optimal 
doses of SAF (as shown by rat agglutinin tests). The difference, though 
statistically significant, was small, suggesting that SAF suppressed 
antibody production to self antigens much more than it did to foreign 
antigens, suggesting a therapeutic role in autoimmune disease due to its 
selectivity. 
It has been suggested by cell transfer experiments that suppression of 
autoantibody production in normal mouse strains is mediated by antigen 
specific suppressor cells. Whether SAF, which suppressed autoantibody 
production much more readily than antibody directed to foreign antigens 
was stimulating a population of antigen-specific suppressor cells is 
unclear. It is clear, however, that SAF reduced autoantibody production 
and is thus useful in the treatment of autoimmune diseases. 
The cell line 6-T CEM and its subclone 6-T CEM-20 were deposited at the 
American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 
20852 on Apr. 26, 1983 and were givenATCC accession No. CRL 8296 and CRL 
8295, respectively.