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Description  |
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This invention relates to the production of immunological material, and in
particular concerns the production of gamma-globulins, immune nucleic
acids and immune dialysates by the induction and cultivation of cell lines
in vitro.
A fundamental necessity for the survival of animals is the ability to
resist infection by parasitic organisms such as viruses, bacteria, fungi
and parasitic animals constantly present in the environment. The immunity
of animals to such organisms is provided by a variety of different
mechanisms including phagocytosis, the chemical activity of antibodies,
and the production of interferons to inhibit virus replication.
During the past two decades, there has been considerable research into
various immunological fields, including the study of immune reactions
involving the production of material which will specifically combat
particular antigens introduced into an animal. The production of material
capable of transferring these specific immune reactions either to
unimmunised hosts when injected in vivo or to "naive" cells capable of
immune reaction when incubated in vitro, is obviously of importance for
medical and veterinary research. See, for example, H. S. Lawrence: "The
transfer in humans of delayed skin sensitivity to streptococcal M
substance and to tuberculin with disrupted leukocytes". J. Clin-Invest,
(1955), 34, 219-30 or "Transfer of immunological information in humans
with dialysates of leukocyte extracts". Trans. As. Amer. Physicians,
(1968), 76, 84-91. The presence of living cells is essential for the
replication of such immunological materials.
Lymphoid cells of animals which have been immunised against a specific
antigen probably incorporate the immune information within their nucleic
acids to form so-called "immune nucleic acids" which probably carry the
immunological memory. Thus immune nucleic acids are characterised by their
ability to confer immune reactivity against specific antigens to "naive"
lymphocytes when injected in vivo, or when "naive" lymphocytes are
incubated with the immune nucleic acids in vitro. Animals with such
"sensitized" lymphoid cells can readily react on a new encounter with the
corresponding antigen and resist, for instance, attacks from bacterial or
viral organisms carrying this antigen.
A variety of in vitro cell lines of human and animal origin have been
established, but no satisfactory culture system has emerged for the in
vitro production of gamma-globulins, specific to a given antigen.
Lymphoblastoid cells in culture may spontaneously produce antibodies which
are directed to unknown antigens and they cannot thus be used for any
practical purposes. Additionally, cells in culture frequently change their
characteristics when cultivated for any length of time and lose some
attributes of their differentiated state. It was therefore considered
unlikely that a cell line could be found which was capable of being
induced to produce specific immune nucleic acids; i.e. capable of
incorporating within the cell's nucleic acids the information contained in
the exogenous specific immune nucleic acids and, further, of replicating
such specific immune nucleic acids. The production of specific antibodies
(gamma-globulins) using a cell line culture was also thought improbable.
However, whilst carrying out research on an established lymphoblastoid cell
line known as LDV/7, it was surprisingly discovered that when LDV/7 cells
were incubated with specific immune nucleic acids (referred to hereinafter
as i-RNA and i-DNA) obtained from donor, human or animal, lymphoid cells
previously sensitized to a given antigen, the cells would incorporate this
immunological information and would replicate the inducing molecules.
Furthermore, when the induced LDV/7 cells were disrupted and subsequently
dialysed, the dialysate was found to transfer specific cell-mediated
immunity to other cells both in vivo and in vitro. Indeed, this immunity
could be transferred to "naive" LDV/7 cells which would then continue to
replicate the immune nucleic acids originally used. A dialysate with these
properties is hereinafter referred to as immune-dialysate. It was also
discovered that incubation of the cell line with immune nucleic acids
and/or immune dialysates would induce the cells to produce gamma-globulins
specific to the antigen against which the original donor had been
immunised.
The LDV/7 cell line was developed from peripheral blood leucocytes obtained
from a 75 year old, apparently healthy, male volunteer.
The cells can be grown in suspension culture, either in static cultures or
spinner cultures and they can also be cultivated in agar. This property is
particularly useful since it enables well established bacteriological
methods to be applied to the in vitro control of human cells. Cloning is
possible, for example, and it is also possible to study the effect of
various factors, such as colony stimulating factor, on the differentiation
of the cells.
The cell line was initially established by the following procedure (the
percentages referred to throughout the Specification being by volume):
Most of the erythrocytes were separated from the original blood sample by
the addition of 20% Plasmagel, and discarded after sedimentation in test
tubes. The recovered suspension contained a ratio of erythrocytes to
leucocytes of less than 20:1. The leucocytes thus recovered were then
suspended in RPMI 1640 medium containing 20% foetal calf serum, at a
concentration of 2.times.10.sup.6 leucocytes/ml. Two thirds of the medium
was replaced by fresh medium twice a week. Since cell death occurred
within the first weeks, the cultures were concentrated as required in
order to keep the cell concentration above 10.sup.6 cells/ml.
The cells were cultured in glass Roux bottles. Six weeks after the start of
the cultures the cells in one of these bottles showed growth
characteristics, i.e. they became larger and started multiplying and these
were used to initially set up the LDV/7 cell line.
This cell line has since been grown in large quantities, using the
propagation techniques described below:
The LDV/7 cell line can be maintained in a variety of nutrient media. It
can be cultivated in RPMI 1640 medium containing from 5% to 30% foetal
calf serum. Preferably, the nutrient medium consists of RPMI medium
containing 10% foetal calf serum. Additionally, McCoy's and Eagle's
Essential Medium may be used in subculturing the cell line.
The cells grow in suspension at 37.degree. C., and form clumps which can be
dispersed by gentle agitation of the medium. The cells are passaged at
least three times a week. The usual seeding number is 5.times.10.sup.5
cells/ml and the cells are allowed to grow in static culture until a
concentration of 10.sup.6 cells/ml is reached. The cells will also grow in
spinner culture in which a rotating, sterile magnetic stirrer keeps the
cells in suspension in the nutrient medium. A higher concentration of the
order of 1.5.times.10.sup.6 cells/ml can then be obtained. The percentage
of dead cells is roughly of the order of 5%, but varies according to
culture conditions. The generation time of the cells is approximately 24
hours, again depending on the culture conditions.
In general, the conditions such as pH and temperature under which the cell
line is propagated are substantially the same as those which leucocytes
would encounter in a human being.
Karyotype analysis of the cells showed that the LDV/7 cell line is
hypotetraploid. The number of chromosomes varies between 80 and 93. A
study of the morphology of the cells under the light microscope revealed a
heterogeneous cell size and that most of the cells have a blast-like
appearance. Further work using an electron microscope confirmed the
heterogeneity of the cell line, showing round mononuclear cells of varying
size with cytoplasmic differentiation and rough endoplasmic reticulum, and
macrophagic properties. There appears to be at least two cell populations,
one of small cells of less then 15 .mu.m diameters and one of large cells
of from 15-30 .mu.m diameters. Apparently the large cells derive from the
small ones.
Three clones have been obtained with chromosome numbers of 84 for the first
and 85 for the second and third, thus attesting cloning efficiency.
The phagocytic properties of the cells were also studied under the electron
microscope. Some of the cells could clearly be seen to be phagocytised by
other cells. No viral particles were discovered in the electron
micrographs obtained, either from samples taken under the usual culture
conditions or from cultures grown at +40.degree. C. It is worth noting
that this cell line is very sensitive to the adrenal cortical steroid
hormone cortisone, suggesting the presence of lymphoblastoid cells and/or
stem cells. It is also worth noting that no EB virus antigens were
detected by immunofluorescence.
Tissue typing of the LDV/7 cells for histocompatibility HL-A antigens
revealed the presence of the following specificities: HLA 2 and HLA 32 for
the first locus, and HLA specificity W 14 for the second locus. However,
weak reactivity with other antisera for other HLA specificities was also
observed, but this was considered non-specific.
LDV/7 cells have been deposited at the Laboratoire d'Immunobiologie,
Faculte de Medicine Broussais Hotel-Dieu, 15, rue de l'ecole de Medecine,
Paris 75006, and are available to the public on request. The LDV/7 cell
line and a process for its propagation is described and claimed in our
co-pending Application Ser. No. 860,439 filed on even date herewith.
It is not possible in all cases to produce immunological material from
other established cell lines, since not all cell lines are inducible in
the manner described above in connection with the LDV/7 cell line. In
particular, it was discovered that three lymphoblastoid cell lines
obtained from leukaemia patients, two from Burkitt lymphoma tumors and
three established from peripheral blood lymphocytes obtained from healthy
volunteers could not be induced. However, successful induction was
achieved when the lymphoblastoid cell lines BRI 8 and BEC 11 were
employed, though not to the extent achieved for the LDV/7 cell line.
It has become apparent that a condition which must be met by a cell line
for successful induction in a simple manner, and for that cell line to
subsequently produce specific gamma-globulins, is the use of a cell line
which is already producing gamma-globulins. Such gamma-globulins will, of
course, be directed against unknown antigens, as noted previously. For
example, cell lines of lymphoid origin (myeloma or lymphoblastoid cell
lines) satisfying this criterion may be induced to produce specific
gamma-globulins, i-RNA and i-DNA. However, the possibility that cell lines
which do not spontaneously produce gamma-globulins may be induced should
not be excluded. Induction of cell lines already producing gamma-globulins
spontaneously, using the techniques described earlier in connection with
the LDV/7 cell line, will thus lead to the production of gamma-globulins
with the same specificity as the inducing material.
The present invention therefore provides a process for the production of
gamma-globulins and immune nucleic acids (as hereinbefore defined), both
specific to a given antigen which comprises:
(i) culturing in vitro an inducible cell line in the presence of specific
immune nucleic acids or specific immune dialysates obtained either from
human or animal donor lymphoid cells sensitized to said specific antigen
or from cells of a previously induced cell line sensitized to said
specific antigen, or in the presence of the liquid phase of a culture of a
previously induced cell line sensitized to said specific antigen; and
(ii) extracting gamma-globulins from the liquid phase of the thus induced
cell culture and/or extracting the immune nucleic acids from the cells so
cultured.
In a second aspect, the present invention provides a process for the
production of immune dialysates, as hereinbefore defined, which comprises:
(i) culturing in vitro an inducible cell line in the presence of specific
immune nucleic acids obtained either from human or animal donor lymphoid
cells sensitized to said specific antigen or from cells of a previously
induced cell line sensitized to said specific antigen, or in the presence
of the liquid phase of a culture of a previously induced cell line
sensitized to said specific antigen; and
(ii) obtaining the immune dialysate from the cells so cultured.
In order to bring about the induction of certain cell lines, the conditions
employed in connection with the LDV/7 cell line are sufficient. In other
cases, trivial modifications of such techniques may be required, such as
the time of incubation, or more subtle alterations may be necessary, such
as changing the cell surface by the use of proteolytic enzymes or
treatment with embryonic nucleic acids.
The cell lines which may be employed in the processes of the present
invention include mutant cell lines derived from the LDV/7 cell line. Such
a mutant cell line must of course be stable to be of any practical use.
The heritable change causing the mutant cell line may be spontaneous, or
may be induced by various chemicals or by physical agents such as X-rays
or ultra-violet light.
In the process of the present invention for the production of specific
gamma-globulins, it is preferred to culture cell lines of lymphoid origin
(myeloma or lymphoblastoid cell lines) which spontaneously, before
induction, produce gamma-globulins, for example, against unknown antigens
in their culture media. More particularly preferred are myeloma cell
lines. If a lymphoblastoid cell line is to be employed, it is particularly
preferred to culture the LDV/7 cell line.
In both the production of immune nucleic acids, particularly i-RNA, and
immune dialysates according to the present invention, inducible cell lines
of lymphoid origin, as above, which spontaneously produce specific
gamma-globulins before induction, may be employed. The use of the LDV/7
lymphoblastoid cell line is again preferred. These cell lines may of
course also be used in the formation of immune dialysates by the process
described above.
The immune nucleic acid or, in the process for the preparation of specific
gamma-globulins or immune nucleic acids, immune dialysate used to
initially induce the cell line employed is obtained from a previously
immunised human or animal (for example, sheep, rabbit, guinea pig or rat)
donor. When animal donors are employed, using germ-free animals immunised
against one antigen, virtually monospecific sensitization is possible. The
i-RNA may be extracted from the donor's lymphoid cells by the hot phenol
method, and i-DNA by cold phenol extraction using a strong chelating
agent, for example sodium 4-animosalicylate, and a detergent, for example
sodium triisopropylnaphthalene sulphonate. Incubation of these immune
nucleic acids or immune dialysates with an inducible cell line capable of
replicating the inducing molecules, for example the lymphoblastoid cell
lines known as LDV/7 and BRI 8, results in the production of large amonts
of immune nucleic acids specific to a given antigen. The replication of
the inducing immune nucleic acids takes place in the cell culture system
at the same time as the replication of the cell's own nucleic acids during
the normal course of cell division. It thus might be due to the
derepression of endogenous nucleic acids, i.e. derepression of DNA which
then produces the corresponding i-RNA. It should, however, be appreciated
that there does not appear to be a species barrier preventing, for
example, the induction of the lymphoblastoid cell line LDV/7, which is of
human origin, by i-RNA obtaned from the lymphoid cells of sheep.
An immune-dialysate can subsequently be produced by disrupting the induced
cells, for example, by repeated freeze-thawing techniques or by means of a
homogeniser, and subsequently dialysing the homogenate. This
immune-dialysate from human or animal leucocytes sensitized to a given
antigen contain molecules of molecular weight less than 10,000. The
dialysate is capable of transferring the immunological information to the
cells in culture. These cells, after incubation with the inducing
immune-dialysate, were found to produce i-RNA and i-DNA carrying the same
specificity as the immune-dialysate used for induction. The activity of
the immune-dialysate was not destroyed by treatment with RNAase or DNAase,
but was destroyed by treatment with pornase. This would suggest that the
information was carried by a small protein of molecular weight less than
10,000; and that this information could be transcribed into a nucleic
acid. Although the mechanism of such transcription remains obscure, this
result, namely the transfer of information from proteins to nucleic acids,
is totally unexpected within the existing knowledge of molecular biology.
It is worth noting that recent evidence suggests that the i-RNA
responsible for transfer of the immunity to a specific antigen is
messenger RNA: see P. Bibello, M. Fishman and G. Koch, Cell Immunol, 23,
309-319 (1976).
Specific antibodies (gamma-globulins) have been found in the liquid phase
obtained from cultures of cells induced with i-RNA, i-DNA and/or immune
dialysates. Suprisingly, these specific gamma-globulins (mainly IgG) were
always of human specificity when the cell line employed was of human
origin, even when the inducing immune nucleic acid was of animal origin
(e.g. sheep, rabbit). This suggests again the derepression of the host
cell genome by the animal nucleic acid. This is supported by the following
evidence. The process of the induction of cell lines causes cell surface
changes, and in particular the appearance of specific antigen receptors
which are probably gamma-globulins. Further, experiments employing LDV/7
cells, sheep i-RNA and rabbit anti-sheep gamma-globulin show that no sheep
gamma-globulins are dectable in the induced cultures, indicating that the
specific gamma-globulins produced by the LDV/7 cells are human allotypes.
Furthermore, when the specific antibodies were extracted on an
immuno-adsorbent column for the corresponding antigen, and were tested
after elution, only human allotypes were present.
Furthermore, the incubation of uninduced cells with the liquid phase from
an induced culture does induce i-DNA and i-RNA production in the "naive"
cells and also quite often the production of specific gamma-globulins.
Consequently the inducing factors of the tissue culture medium as well as
the antibodies can be extracted and purified. The gamma-globulins can be
extracted using a specific immuno-adsorbent column with the corresponding
antigen or xenogeneic anti-human globulin antibody on which the specific
gamma-globulin contained in the medium would attach. The gamma-globulins
can then be easily obtained by subsequent elution.
Although the Examples which follow are primarily concerned with the use of
Key-hole limpet haemocyanin (KLH) as the sensitizing antigen, this is only
because it has been found to be convenient experimentally to do so and in
no way is to be taken to be a limitation of the generality of the
sensitizing antigen which may be employed. Other antigens such as brucella
bacteria, coccidioidin or histoplasmin have also been successfully used.
The use of the immunological materials hereinbefore described to transfer
cell mediated immunity against specific antigens is of obvious importance
in the field of medicine. The inducing molecules may be obtained, for
example, from germ-free animals immunised against one antigen, thus
obtaining vitually monospecific immune mediators. Cloning the cell
cultures after induction will also result in a culture for the production
of monospecific gamma-globulins. The bulk production of such mediators in
vitro has wide applications. The use of immune dialysates presents an
advantage over immune nucleic acids since it is barely possible to obtain
immune reactions against the immune dialysates, and this avoids
anaphylactic reactions. Furthermore, no viral hazard is possible using
cell dialysates, whereas the hypothetical presence of a viral genome in
immune nucleic acids will be cumbersome to exclude. Consequently a
medicament capable of being administered in unit dosage, comprising at
least one of the immune nucleic acids (i-RNA or i-DNA) and/or immune
dialysates specific to a given antigen produced by a process as
hereinbefore described is also within the scope of this invention.
Specific human gamma-globulins produced in vitro should have a wide range
of applications and can be used as substitutes for gamma-globulins
obtained from animal or human donors and currently used in passive
immonotherapy or for diagnostic purposes. Thus the invention also provides
a medicament capable of being administered in unit dosage form comprising
specific gamma-globulins produced by the process of the present invention.
The invention is further illustrated by the following Examples:
EXAMPLE 1
(a) General method of initial induction of cell line by immune nucleic
acids obtained from a previously immunised donor.
An RNA-solution or DNA-solution obtained from the lymphoid cells of a donor
previously immunised against a specific antigen were treated with DNAase
and RNAase respectively to ensure that there was no DNA or RNA, again
respectively, present. 10.sup.7 LDV/7 cells were incubated for 30 to 60
minutes with 0.5 to 1.00 mg of the RNA or DNA in solution.
The cells were suspended at a concentration of 5.times.10.sup.5 cells/ml in
RMPI 1640 medium complemented with 10% foetal calf serum. The culture was
allowed to grow until the cells reached a concentration of 10.sup.6
cells/ml, and were then passaged in fresh medium which lowered the cell
concentration to 5.times.10.sup.5 cells/ml. The cells were harvested when
the total yield of the culture was of the order of 10.sup.10 cells.
Immune-RNA and immune dialysates were then obtained from these cells, and
specific gamma-globulins were extracted from the tissue culture medium.
(b) General method of induction of cell line using dialysate obtained from
initially induced LDV/7 cells. Dialysates of the cells prepared as above
were obtained after freeze-thawing 10.sup.9 cells in 10 ml of distilled
water and subsequent dialysis through a dialysis bag under vacuum.
100 ml of LDV/7 cells at a concentration of 5.times.10.sup.5 cells/ml were
incubated with the dialysates obtained from 5.times.10.sup.7 cells induced
as above. The cells were subcultured until the total yield was
2.times.10.sup.9 cells and i-RNA, i-DNA and immune dialysates were
obtained from the latter.
The specificity of the i-RNA obtained from the culture cells after
induction with i-RNA, i-DNA or immune dialysate for malignant melanoma
antigens was assessed in a cell-mediated cytotoxicity system. Malignant
melanoma cell lines were the target cells. The cytotoxicity indices showed
that immune nucleic acids or immune dialysates render "naive" inactive
peripheral blood lymphocytes specifically cytotoxic to the target cells
after incubation.
EXAMPLE 2
In a different series of experiments it was found that incubation of LDV/7
or BRI 8 cells with immune nucleic acids or immune dialysates as in
Example 1 induces the appearance of specific antigen receptor sites on the
cell surface of the lymphoblastoid cells.
(a) When LDV/7 cells were incubated with i-nucleic acids or immune
dialysates specific for KLH (Keyhole-limpet haemocyanin), the LDV/7 cells
subsequently developed receptor sites for this antigen. This can be seen
by a rosette formation of SRBC (sheep red blood cells) which have been
previously coated with KLH. Furthermore, in these experiments, specific
anti-KLH antibody production could be detected in the culture medium.
(b) The lymphoblastoid cell line BRI 8 was induced employing the techniques
of Example 1, in the presence of i-RNA and, in a second experiment, immune
dialysates, both of which were specific for KLH. Subsequently, rosette
formation was observed as in (a).
EXAMPLE 3
I-RNA used in this series of experiments was extracted from the lymph nodes
and spleens of sheep immunized with either melanoma or colon carcinoma
cells. Sheep immune RNA is hereinafter referred to as Is-RNA. I-RNA from a
sheep immunized with Keyhole Limpet Haemocyanin (KLH) was used as a
control.
The schedule for immunization of the sheep and the hot phenol method of
extracting i-RNA have been described elsewhere: see D. H. Kern, D. Fritze,
C. R. Drogemuller and Y. H. Pilch, J. Nat. Cancer. Inst. 57, 97-103
(1976), and L. L. Veltman, D. H. Kern and Y. H. Pilch, Cell. Immunol. 131,
367-377 (1974), respectively. The i-RNA preparations are assayed for their
protein, DNA and RNA content. The sucrose density gradient profiles of the
i-RNA are also determined, since these allow the estimation of any
degradation destroying biological activity: see Y. H. Pilch, K. P. Ramming
and P. J. Deckers. In H. Bush, Ed: Methods in Cancer Research, Vol 9, New
York, Academic Press, 1973, 195-254.
The biological activity of these i-RNAs is assayed in an in vitro
micro-cytotoxicity assay: see RNA in the Immune Response, H. Friedman, Ed,
Ann, N.Y. Acad. Sci. , 207, (1973). Their ability to convert normal
allogeneic human peripheral blood lymphocytes to cytotoxic effector cells
is tested following in vitro incubation of lymphocytes with i-RNA, after
which the cytotoxic activity of the treated lymphocytes is tested against
cultured human tumor cells of the same histological type as those used to
immunize the Is-RNA donor sheep.
The lymphoblastoid cell line LDV/7 is induced with Is-RNA as follows:
1 mg of Is-RNA is dissolved in 0.5 ml RPMI 1640 medium: this solution is
made 0.35 M with respect to sucrose. 10.sup.7 LDV/7 cells are subsequently
suspended in this solution and incubated for at least 30 minutes at
37.degree. C. with continuous stirring. At the end of the incubation,
fresh medium is added and the final volume is adjusted to 20 ml, thus
bringing the cell concentration to 5.times.10.sup.5 cells/ml. Since the
mean doubling time of this cell line, when cultured under static
conditions, is of the order of 24 hours, a volume of fresh medium (RPMI
1640, supplemented with 10% foetal calf serum), equal to the existing
volume of the culture, is added every day, until the total volume of the
culture reaches 3 liters, thus yielding approximately 2.times.10.sup.9
cells. An aliquot of 10.sup.7 cells from this culture is induced a second
time with the same Is-RNA following the same procedure. This newly induced
aliquot serves for the seeding of a second culture which again is
supplemented with fresh medium and grown until a desired number of cells
is obtained. Usually the second culture is grown to provide
4.times.10.sup.9 cells. The cells from the two cultures are pooled and
stored frozen at -20.degree. C. I-RNA is extracted from the frozen cells
(Ic-RNA) and assayed for biologic activity as described above. Between 0.7
and 1.5 mg of total RNA are usually obtained from 10.sup.9 cells. All
batches of Ic-RNA extracted in this manner gave a non-degraded profile on
sucrose density gradients.
The Ic-RNA's extracted from the cultured LDV/7 cells are tested for their
ability to induce naive human lymphocytes to become cytotoxic to tumor
target cells in vitro. The human lymphocytes were isolated from the
peripheral blood of a healthy donor on Ficoll-isopaque gradients. Their
activity is compared to that of the inducing Is-RNA preparations. In eight
out of ten experiments it was shown that Ic-RNA preparations were capable
of conferring significant cytotoxicity for tumor target cells to naive
allogeneic lymphocytes. Table 1 shows results from two typical successful
experiments.
Allogeneic human lymphocytes from the peripheral blood of a healthy blood
donor were used as effector cells. 5.times.10.sup.7 cells were incubated
with i-RNA extracted from (a) uninduced LDV/7 cells (c-RNA), (b) a sheep
immunized with KLH (Is-RNAk), (c) a sheep immunized with human colon
carcinoma cells (Is-RNAc), (d) a sheep immunized with human melanoma cells
(Is-RNAm) and (e) three batches of LDV/7 cells (Ic-RNA), after induction
with each of the three different types of Is-RNA (k, c and m). Lymphocytes
incubated with no i-RNA provided the control (C1). Each figure represents
the mean of six values. Significant differences (P<0.005) were observed
between the activity of the I-RNA's obtained either from the sheep
immunized with tumor cells, or from LDV/7 cultures induced with these
anti-tumor Is-RNA's, on the one hand, and the controls (lymphocytes
incubated with no RNA, c-RNA, Is-RNAk or Ic-RNAk), on the other. No
significant differences were seen between the cytotoxicity indices
obtained with active Is-RNA preparations and the Ic-RNA's from LDV/7 cells
induced with these Is-RNA's. NT=not tested.
"Allogeneic-lymphocytes" is used herein reference to the patient, donor of
the tumor cells employed for sheep immunization. The cultured tumor cells
used for the cytotoxicity tests were allogeneic with respect to the tumor
cell donor and were of the same histological type as those used for the
sheep immunization. The same blood donor provided the effector lymphocytes
throughout these experiments.
Table 1
______________________________________
1 2
______________________________________
Cl 0 .+-. 0.07 0 .+-. 0.10
c-RNA 0.03 .+-. 0.05
0.07 .+-. 0.05
Is-RNAk NT 0.12 .+-. 0.02
Ic-RNAk NT 0.10 .+-. 0.03
Is-RNAc 0.50 .+-. 0.06
0.38 .+-. 0.09
Ic-RNAc 0.48 .+-. 0.06
0.30 .+-. 0.08
Is-RNAm 0.34 .+-. 0.03
0.33 .+-. 0.08
Ic-RNAm 0.34 .+-. 0.03
0.23 .+-. 0.05
______________________________________
However, it must be emphasized that these results do not suggest that the
observed cytotoxicity is solely due to tumor specificities, since both the
Is-RNA and the Ic-RNA preparations may be transferring immune reactivity
against HL-A specificities as well as against tumor associated antigens.
What is clearly shown is that Ic-RNA confers similar cytotoxicity indices
to human normal lymphocytes as these conferred by the Is-RNA used for the
induction.
It should also be noted that the LDV/7 cells are capable of producing
Ic-RNA carrying the same specificities as the Is-RNA at least 10 weeks
after the initial induction, and Ic-RNA showed the same specific activity
whenever it was extracted 3-10 weeks after the induction.
EXAMPLE 4
I-RNA is extracted from the lymphoid tissues of sheep immunized either with
Keyhole Limpet Haemocyanin (KLH); or with human tumor tissues (melanoma,
colon carcinoma), KLH and Freund's Complete Adjuvant (FCA); or with FCA
alone. I-RNA from non-immunized sheep is extracted under the same
conditions. A hot phenol method is used for RNA extraction. The DNA, RNA
and protein concentration of each preparation are determined and absence
of significant degradation determined by analysis of sucrose density
gradient profiles. The I-RNA preparations are kept lyophilized until use.
The LDV/7 lymphoblastoid cell line was used for these experiments. The
cells grow in RPMI 1640 medium (Gibco), supplemented with 10% foetal calf
serum (FCS), in suspension culture and their average doubling time, under
the present experimental conditions, is approximately 24 hours.
For induction 10.sup.7 LDV/7 cells are incubated for 60 minutes in 1 ml of
RPMI 1640 medium, without FCS, containing 1 mg of I-RNA. At the end of the
incubation period, 19 ml of medium with 10% FCS is added, so that the cell
concentration is adjusted to 5.10.sup.5 cell/ml. The cells are then grown
under standard culture conditions.
Evidence for the formation of membrane receptors, specific for the given
antigen, on the surface of the induced LDV/7 cells:
(a) Immune cytoadherence
Sheep red blood cells (SRBC) are mixed with an equal volume of a 0.005%
tannic acid solution and incubated for 10 minutes at 37.degree. C. After
washing in phosphate buffered saline (PBS), the cells are suspended in PBS
at a 1:20 ratio volume per volume. 1 ml of this suspension is mixed with 5
ml of PBS containing 1 mg/ml of KLH and incubated for 10 minutes at
37.degree. C., in order to fix the antigen on the SRBC membranes. After
washing, the antigen coated SRBC are resuspended in 5 ml of PBS. Washed
LDV/7 cells are mixed with antigen coated SRBC and incubated for two hours
at 37.degree. C. Samples are taken at the end of the incubation period,
placed between glass slides and cover slips, and counted under the
microscope. LDV/7 cells to which more than 3 SRBC are attached are
considered as "rosetting" cells.
LDV/7 cells, induced with I-RNA from sheep immunized with KLH, show a
significantly higher number of rosetting cells, when they are incubated
with KLH, than do cells induced with RNA obtained from non-immunized
animals, or with I-RNA from sheep immunized with an antigen different than
the one used for coating the SRBC e.g. FCA. The average number of
rosetting cells observed for an induced culture is of the order of 10%.
(b) Immunofluorescence
KLH labelled with tetramethyl rhodamine isothiocyanate, isomer G (Sigma)
(RITC) is used to localize the KLH membrane receptors by direct
immunofluorescence. 200 .mu.g/ml of RITC labelled KLH is incubated with
6.10.sup.5 LDV/7 cells for 30 minutes.
Cells are incubated under the same conditions with non labelled KLH. The
latter is revealed on the cell surface by a rabbit anti-KLH antiserum
conjugated to fluorescein isothiocyanate, isomer I (Sigma) (FITC). Rabbit
anti-KLH antisera are prepared by three bi-weekly injections of 1 mg of
KLH.
A Goat anti-rabbit FITC-labelled antiserum is used in the indirect
technique to develop unlabelled rabit anti-KLH antibodies fixed on the
surface of LDV/7 cells incubated with KLH.
LDV/7 cells induced with sheep i-RNA-KLH and incubated with KLH show
fluorescent staining in the direct (using labelled KLH) or the indirect
(using fluorescent rabbit anti-KLH antiserum or unlabelled rabbit anti-KLH
antiserum and a fluorescent goat anti-rabbit antiserum) test. (see Table
2). Cells induced with sheep i-RNA-FCA, or i-RNA from non-immunized sheep,
do not show antigen receptors for KLH on their surface, thus remaining
non-fluorescent. Specific immune blocking (IB) confirmed the specificity
of the staining. Approximately 30% of the cells from the induced cultures
were found to be fluorescent.
Table 2
______________________________________
Non- i-RNA-KLH
induced induced
cells cells
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Direct staining with RITC
labelled KLH -- +
I.B.: pre-incubation with
KLH followed by RITC
-- --
labelled KLH
Indirect staining: incubation
with KLH followed by FITC
labelled rabbit anti-KLH
-- ++
antiserum
I.B.: pre-incubation with
KLH, then unlabelled rabbit
anti-KLH antiserum, followed
-- --
by FITC labelled rabbit
anti-KLH antiserum
Indirect staining:
incubation with KLH
followed by rabbit anti-KLH
antiserum and then followed
-- +++
by an FITC labelled goat
anti-rabbit .gamma.-globulin
anti-serum
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The degree of fluorescent staining is expressed as the percentage of
fluorescing cells as follows:
+>30%
++>40%
+++>50%
FITC Fluorescein isothiocyanate Isomer I
RITC Tetramethyl rhodamine isothiocyanate Isomer G.
(c) Peroxidase-diaminobenzidine (PO-DAB):
See Graham, R. C. and Karnovsky, M. J.:
The early stages of absorption of injected horseradish peroxidase in the
proximal tubules of mouse kidney: ultrastructural cytochemistry by a new
technique. J. Histochem. Cytochem., 14, 291, 1966.
10.sup.6 LDV/7 cells, incubated with 1 ml of a solution of KLH (200
.mu.g/ml), after thorough washing, are fixed with Karnovsky fixative for 5
minutes and afterwards exposed to a rabbit anti-KLH antiserum. After
further thorough washing, the cells are exposed to a goat anti-rabbit
antiserum conjugated with horseradish peroxidase (Institut Pasteur). The
preparation is exposed to a saturated solution of 3-3'diamino-benzidine
base free (DAB) (Sigma) for 15 minutes at 20.degree. C., and then fixed in
Karnovsky fixative for 30 minutes. The pellet is then dehydrated and
embedded in Epon 812. Thin sections are obtained using a Reichert
microtome and examined unstained or lead contrasted in a Philips 300
electron microscope.
i-RNA-KLH induced cells, incubated with KLH, and stained with PO-DAB show,
in electron microscopy, a thick dark line on the outer surface of their
membranes, corresponding to the peroxidase reaction, and thus showing the
KLH fixation on the membrane antigen receptors. Approximately 30% of the
KLH-I-RNA cells were found to be peroxidase positive.
It therefore is concluded that this i-RNA (of sheep origin) transfers
information to xenogeneic lymphoblastoid cells in culture. The latter are
capable of re-expressing this information. The results show that
xenogeneic I-RNA is not just passively incorporated and replicated by the
LDV/7 lymphoblastoid cells, but becomes functional in that its information
is expressed as soon as it is incorporated in the host cell. Indeed, it
induces cell surface changes, the appearance of specific antigen
receptors, and furthermore, the secretion of specific .gamma.-globulins,
though this latter point is not demonstrated in this Example.
EXAMPLE 5
I-RNA is extracted in the usual way from the lymphoid tissues of rabbits
immunised with brucella bacteria. The i-RNA preparations thus obtained
were used to induce the LDV/7 cell line using the techniques described in
Example 3. The supernatant liquid obtained from the culture medium was
analysed for the presence of gamma-globulins specific to the brucella
bacteria which was used as the original sensitizing antigen. The presence
of such specific gamma-globulins was indicated by immunofluorescence
techniques, and by agglutination when samples of the supernatant liquid
were combined with brucella bacteria.
EXAMPLE 6
Following the method of Example 5, gamma-globulins specific to coccidioidin
were raised using the LDV/7 cell line. The i-RNA used to induce the cell
culture system was obtained from the lymphoid tissues of rabbits which had
previously been sensitized to coccidioidin.
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