U.S. patent application number 09/853755 was filed with the patent office on 2001-10-11 for inhibition of graft versus host disease.
Invention is credited to Spaner, David Elliott.
Application Number | 20010028879 09/853755 |
Document ID | / |
Family ID | 4162727 |
Filed Date | 2001-10-11 |
United States Patent
Application |
20010028879 |
Kind Code |
A1 |
Spaner, David Elliott |
October 11, 2001 |
Inhibition of graft versus host disease
Abstract
The development of graft versus host disease in a mammalian
patient undergoing cell transplantation therapy for treatment of a
bone marrow mediated disease, is prevented or alleviated by
subjecting at least the T-cells of the allogeneic cell
transplantation composition, extracorporeally, to oxidative stress,
in appropriate dosage amounts, such as bubbling a gaseous mixture
of ozone and oxygen through a suspension of the T-cells. The
process may also include irradiation of the cells with UV light,
simultaneously with the application of the oxidative stress. The
oxidative stress induces reduced inflammatory cytokine production
and a reduced proliferative response in the T-cells.
Inventors: |
Spaner, David Elliott;
(Toronto, CA) |
Correspondence
Address: |
Gerald F. Swiss, Esq.
BURNS, DOANE, SWECKER & MATHIS, L.L.P.
P.O. Box 1404
Alexandria
VA
22313-1404
US
|
Family ID: |
4162727 |
Appl. No.: |
09/853755 |
Filed: |
May 14, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09853755 |
May 14, 2001 |
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09363678 |
Jul 30, 1999 |
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6258357 |
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Current U.S.
Class: |
424/93.7 ;
435/372.3; 435/448 |
Current CPC
Class: |
A61P 43/00 20180101;
Y10S 424/81 20130101; A61K 41/17 20200101; A61K 35/17 20130101;
A61K 35/28 20130101; A61P 35/02 20180101; C12N 5/0636 20130101;
C12N 2500/02 20130101; A61P 37/06 20180101; C12N 2500/05 20130101;
A61K 35/28 20130101; A61K 2300/00 20130101; A61K 35/17 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
424/93.7 ;
435/372.3; 435/448 |
International
Class: |
A61K 045/00; C12N
005/08; C12N 015/01 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 30, 1998 |
CA |
2,244,554 |
Claims
I claim:
1. A process for preparing an allogeneic cell population for
administration to a human patient suffering from a bone marrow
disorder potentially treatable by bone marrow transplantation,
which comprises subjecting, in vitro, a population of donor cells
enriched in T-cells to oxidative stress to induce in said T-cells
an altered cytokine production profile and a reduced proliferative
response.
2. The process of claim 1 wherein the oxidative stress is imparted
by subjection to an ozone/oxygen gaseous mixture.
3. The process of claim 2 wherein the ozone/oxygen gas mixture is
bubbled through an aqueous suspension of said T-cell containing
population at a rate of from about 0.01-2 liters per minute.
4. The process of claim 3 wherein the ozone/oxygen gas mixture has
an ozone content of from about 1.0-100 .mu.g/ml.
5. The process of claim 2 wherein the ozone/oxygen gas mixture is
bubbled through an aqueous suspension of said T-cell containing
population at a rate of from about 0.05-1.0 liters per minute, the
gas mixture having an ozone content of from about 3-70
.mu.g/ml.
6. The process of claim 3 wherein the T-cell containing population
is additionally subjected to UV radiation.
7. The process of claim 6 wherein the T-cell containing population
is subjected to oxidative stress and UV radiation
simultaneously.
8. The process of claim 7 wherein the UV radiation is UV-C.
9. The process claim 8 wherein the time of simultaneous subjection
to oxidative stress and UV radiation is from 0.5-60 minutes.
10. The process of claim 9 wherein the time is from 2-5
minutes.
11. The process of claim 5 wherein the T-cell containing population
is a human white blood cell fraction obtained from human peripheral
blood by leukopheresis.
12. The process of claim 11 wherein the T-cell containing
population is a peripheral blood mononuclear cell fraction from
human blood.
13. The process of claim 1 wherein the oxidative stress is imparted
by addition of a chemical oxidizing agent to a suspension of said
T-cell enriched donor cell population.
14. The process of claim 13 wherein the T-cell enriched donor cell
population is a peripheral blood mononuclear cell fraction from
human blood.
15. A process of treating a mammalian patient for alleviation of a
bone marrow disorder potentially treatable by bone marrow
transplantation, with alleviation of consequentially developed
graft versus host disease, which comprising administering to the
patient allogeneic hematopoietic stem cells and allogeneic T-cells,
at least a portion of said T-cells having been subjected to
oxidative stress in vitro, prior to administration to the patient,
so as to induce decreased inflammatory cytokine production and a
reduced proliferative response therein.
16. The process of claim 15 wherein the T-cells are administered
separately from the stem cells.
17. The process of claim 16 wherein the T-cells consist essentially
of peripheral blood mononuclear cells obtained from peripheral
human blood.
18. The process of claim 16 or wherein the T-cells have been
subjected to oxidative stress by application thereto of a gaseous
oxygen/ozone mixture.
19. The process of claim 16 wherein the T-cells have been subjected
to oxidative stress by application thereto of a chemical oxidizing
agent.
20. The process of claim 18 wherein the T-cells have been
additionally subjected to UV radiation, simultaneously with the
subjection to oxidative stress.
21. A population of mammalian T-cells essentially free of stem
cells, said T-cells having been subjected in vitro to oxidative
stress so as to induce in said cells a reduced inflammatory
cytokine production and a reduced proliferative response.
22. Peripheral blood mononuclear cells obtained from human
peripheripheral blood by leukopheresis, said cells having been
subjected in vitro to oxidative stress so as to induce in the
T-cell component thereof a reduced inflammatory cytokine production
and a reduced proliferative response.
Description
FIELD OF THE INVENTION
[0001]
[0002] This invention relates to cellular compositions useful in
medical treatments, processes for their preparation and their uses
in medical treatments. More specifically, it relates to cellular
compositions useful in alleviation of complications following
allogeneic bone marrow transplantation, namely graft versus host
disease in mammalian patients, especially in human patients, and to
processes for preparation of such compositions of matter.
BACKGROUND OF THE INVENTION
[0003] Bone marrow transplantation, BMT, is indicated following a
process which destroys bone marrow. For example, following
intensive systemic radiation or chemotherapy, bone marrow is the
first target to fail. Metastatic cancers are commonly treated with
very intensive chemotherapy, which is intended to destroy the
cancer, but also effectively destroys the bone marrow. This induces
a need for BMT. Leukemia is a bone marrow malignancy, which is
often treated with BMT after chemotherapy and/or radiation has been
utilized to eradicate malignant cells. BMT is currently used for
treatment of leukemias which are life-threatening. Some autoimmune
diseases may be severe enough to require obliteration of their
native immune systems which includes concomitant bone marrow
obliteration and requires subsequent bone marrow transplantation.
Alleviation of any but the most acute life-threatening conditions
involving bone marrow disorders with BMT is, however, generally
regarded as too risky, because of the likelihood of the onset of
graft versus host disease.
[0004] Graft-versus-host disease, GVHD, is an immunological
disorder that is the major factor that limits the success and
availability of allogeneic bone marrow or stem cell transplantation
(collective referred to herein as allo-BMT) for treating some forms
of otherwise incurable hematological malignancies, such as
leukemia. GVHD is a systemic inflammatory reaction which causes
chronic illness and may lead to death of the host mammal. At
present, allogeneic transplants invariably run a severe risk of
associated GVHD, even where the donor has a high degree of
histocompatibility with the host.
[0005] GVHD is caused by donor T-cells reacting against
systemically distributed incompatible host antigens, causing
powerful inflammation. In GVHD, mature donor T-cells that recognize
differences between donor and host become systemically activated.
Current methods to prevent and treat GVHD involve administration of
drugs such as cyclosporin-A and corticosteroids. These have serious
side effects, must be given for prolonged periods of time, and are
expensive to administer and to monitor. Attempts have also been
made to use T-cell depletion to prevent GVHD, but this requires
sophisticated and expensive facilities and expertise. Too great a
degree of T-cell depletion leads to serious problems of failure of
engraftment of bone marrow stem cells, failure of hematopoietic
reconstitution, infections, or relapse. More limited T-cell
depletion leaves behind cells that are still competent to initiate
GVHD. As a result, current methods of treating GVHD are only
successful in limited donor and host combinations, so that many
patients cannot be offered potentially life-saving treatment.
BRIEF REFERENCE TO THE PRIOR ART
[0006] International Patent Application No. PCT/CA97/00564 Bolton
describes an autovaccine for alleviating the symptoms of an
autoimmune disease in a mammalian patient, comprising an aliquot of
modified blood obtained from the same patient and treated
extracorporeally with ultraviolet radiation and an oxygen/ozone gas
mixture bubbled therethrough, at an elevated temperature
(42.5.degree. C.), the autovaccine being re-administered to the
same patient after having been so treated.
[0007] It is an object of the present invention to provide a
process of alleviating the development of GVHD complications in a
mammalian patient undergoing allo-BMT procedures.
SUMMARY OF THE INVENTION
[0008] According to the present invention, a patient being treated
by allo-BMT is administered a composition containing T-cells
obtained from an allogeneic donor, said T-cells having been
subjected in vitro to oxidative stress to induce therein decreased
inflammatory cytokine production coupled with reduced proliferative
response. It appears that such oxidatively stressed allogeneic
T-cells when injected into a mammalian patient, have a
down-regulated immune response and a down-regulated destructive
allogeneic response against the recipient, so that engraftment of
the hematopoietic stem cells, administered along with or separately
from the stressed T-cells, can take effect with significantly
reduced risk of development of GVHD. The population of stressed
T-cells nevertheless appears to be able to exert a sufficient
protective effect on the mammalian system to guard against failure
of engraftment and against infection, whilst the hematopoietic
system is undergoing reconstitution, at least in part, by
proliferation and differentiation of the allogeneic stem cells.
[0009] One aspect of the present invention provides, accordingly, a
process of treating a mammalian patient for alleviation of a bone
marrow mediated disease, with alleviation of consequently developed
graft versus host disease (GVHD), which comprises administering to
the patient allogeneic hematopoietic stem cells and allogeneic
T-cells, at least a portion of said T-cells having been subjected
to oxidative stress in vitro, prior to administration to the
patient, so as to induce an altered cytokine production profile and
a reduced proliferative response therein.
[0010] Another aspect of the present invention provides a
population of mammalian T-cells, essentially free of stem cells,
said T-cells having been subjected in vitro to oxidative stress so
as to induce in said cells an altered cytokine production profile
and a reduced proliferative response.
[0011] A further aspect of the present invention provides a process
for preparing an allogeneic cell population for administration to a
human patient suffering from a bone marrow mediated disease, which
comprises subjecting, in vitro, a population of donor cells
enriched in T-cells to oxidative stress to induce in said T-cells
an altered cytokine production profile and a reduced proliferative
response.
BRIEF REFERENCE TO THE DRAWINGS
[0012] FIGS. 1 and 2 of the accompanying drawings are graphical
presentations of results obtained according to Example 3 below.
[0013] FIG. 3 is a depiction of the results obtained from Example 4
below.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0014] The process of the present invention involves an initial
collection of hematopoietic stem cells and T-cells from a donor.
The preferred source of such cells is mobilized stem cells and
T-cells from the peripheral blood of the donor. Stem cells are
present in very small quantities in peripheral blood, and one
preferred way of operating in accordance with the invention is to
enrich the stem cell population of the donor's peripheral blood,
and then to extract the donor's peripheral blood for use as a
source of stem cells and T-cells for treatment as described and
subsequent injection into the patient. Enrichment may be achieved
by giving the donor a course of injections of appropriate growth
factors, over several days e.g. five days prior to extracting
peripheral blood from the donor. Appropriate cell fractions can be
collected from the blood by leukopheresis, a known procedure, as it
is extracted, with the plasma and red cells being returned to the
donor, in a closed flow system. The white cell collection, which
contains the stem cells (about 3%) and T-cells (about 40%) along
with B-cells, neutrophils and other white cells, may be treated to
alter their cytokine production profiles and to reduce the
proliferative response of the T-cells therein, and then
administered to the host patient, in accordance with the invention,
as a whole collection of cells (peripheral blood mononuclear
cells). Preferably, however, the donor T-cells are separated from
the other cells, so that only the T-cells are subjected to
oxidative stress and then administered to the patient, with the
stem cells for engraftment being administered to the patient
separately from the treated T-cells. For practical purposes,
however, subjection of the collection of peripheral blood
mononuclear cells to the stressors is satisfactory, without further
fractionation to isolate the T-cells, which is a difficult and
expensive procedure. Separate administration of stem cells is
strongly preferred.
[0015] If for some reason it is desired to subject the entire white
cell collection to oxidative stress to induce the aforementioned
changes in the T-cell portion thereof, and then administer the
entire collection to the patient, it is preferred to protect the
stem cells from any damaging effects of the oxidative stress in a
manner described below.
[0016] In an alternative, but less preferred, procedure, whole bone
marrow of the donor can be used as the source of T-cells and stem
cells for the process of the invention. Whole bone marrow has in
the past been the usual source of cells for allogeneic cell
transplantation procedures, and can indeed be used in the present
process. It is however an inconvenient and uncomfortable procedure
for the donor, requiring anaesthetic and lengthy extraction
procedures. Any source of T-cells and stem cells from the donor can
be used in principle, but peripheral blood enriched in stem cells
and T-cells is the most clinically convenient.
[0017] The alteration in cytokine production profile induced in the
T-cells in the process of the invention is preferably a reduction
in production of inflammatory cytokines, such as interferon-.gamma.
and tissue necrosis factor-.alpha..
[0018] The oxidative stress may be applied to the T-cells by
subjecting them to an oxidative environment such as the addition of
a gaseous, liquid or solid chemical oxidizing agent (ozone,
molecular oxygen, ozone/oxygen gas mixtures, permanganates,
periodates, peroxides, drugs acting on biological systems through
an oxidative mechanism such as adriamycin, and the like). In one
preferred method according to the invention, the T-cells are
subjected, in suspension, to a gaseous oxidizing agent, such as an
ozone/oxygen gas mixture bubbled through the suspension of cells,
optionally in combination with the simultaneous subjection of the
cells to ultraviolet radiation, in appropriate doses.
[0019] One method according to the present invention subjects the
allogeneic white cells from the donor, including both the stem
cells and the T-cells, to oxidative stress. This eliminates the
need to include a complicated and costly step of separating the
T-cells from the other cellular components of the white cells
composition. In such case, however, it is strongly preferred to
protect the stem cells in the composition from deleterious effects
of the stress. This can be accomplished by including one or more
stem cell growth factors in the cell composition at the time of
subjecting it to the stress. Protection of the stem cells from the
deleterious effects of the oxidative stress is achieved by the
presence of growth factors, and so, prior to subjecting the stem
cell-T-cell composition to oxidative stress, one or more stem cell
growth factors are added to the composition. Stem cell growth
factors useful in the process are cytokines which promote survival
of stem cells (but not T-cells) during this stressing. They are
cytokines which interact with growth receptors on stem cells. They
are believed to activate the MAP-kinase pathway of the cell,
resulting in the activation of erk. Examples of suitable such
growth factors, include stem cell specific growth factors,
kit-ligand, IL-3, GM-CSF and FLT 3 ligand, all of which are known.
It is preferred to add precise amounts of extracted, purified
growth factors or, especially, recombinant growth factors available
on the market, or combinations thereof, suitably dissolved or
suspended in appropriate, biologically acceptable fluids.
[0020] One preferred method of subjecting the allogeneic T-cells to
oxidative stress according to the invention involves exposing a
suspension of the cells to a mixture of medical grade oxygen and
ozone gas, for example by bubbling through the suspension a stream
of medical grade oxygen gas having ozone as a minor component
therein. The suspending medium may be any of the commonly used
biologically acceptable media which maintains cells in viable
condition. The ozone gas may be provided by any conventional source
known in the art. Suitably the gas stream has an ozone content of
from about 1.0-100 .mu.g/ml, preferably 3-70 .mu.g/ml and most
preferably from about 5-50 .mu.g/ml. The gas stream is supplied to
the aliquot at a rate of from about 0.01-2 liters per minute,
preferably 0.05-1.0 liters per minute, and most preferably at about
0.06-0.30 liters per minute (STP).
[0021] Another method of subjecting the T-cells to oxidative stress
to render them suitable for use in the present invention is to add
to a suspension of the cells a chemical oxidant of appropriate
biological acceptability, and in biologically acceptable amounts.
Permanganates, periodates and peroxides are suitable, when used in
appropriate quantities. Hydrogen peroxide is useful in
demonstrating the effectiveness of the process of the invention and
in giving guidance on the appropriate quantity of oxidizing agent
to be used, although it is not an agent of first choice for the
present invention, for practical reasons. Thus, a suitable amount
of oxidizing agent is hydrogen peroxide in a concentration of from
1 micromolar -2 millimolar, contacting a 10 ml suspension
containing from 10-6 to 10-8 cells per ml, for 20 minutes, or
equivalent oxidative stress derived from a different oxidizing
agent. Optimum is about 1 millimolar hydrogen peroxide in such a
suspension for about 20 minutes, or the equivalent of another
oxidizing agent calculated to give a corresponding degree of
oxidative stress to the cells.
[0022] The size of the cell suspension to be subjected to oxidative
stress is generally from about 0.1 ml to about 1000 ml, preferably
from about 1-500, and containing appropriate numbers of T-cells for
subsequent administration to a patient undergoing allo-BMT. These
numbers generally correspond to those used in prior methods of
allogeneic T-cell administration in connection with allo-BMT, and
are familiar to those skilled in the art.
[0023] One specific process according to the invention is to
subject the cell suspension simultaneously to oxygen/ozone bubbled
through the suspension and ultraviolet radiation. This also effects
the appropriate changes in the nature of the T-cells. Care must be
taken not to utilize an excessive dosage of oxygen/ozone or UV, to
the extent that the cell membranes are caused to be disrupted, or
other irreversible damage is caused to an excessive number of the
cells.
[0024] The temperature at which the T-cell suspension is subjected
to the oxidative stress does not appear to be critical, provided
that it keeps the suspension in the liquid phase and is not so high
that it causes cell membrane disruption. The temperature should not
be higher than about 45.degree. C.
[0025] When ultraviolet radiation is used in conjunction with the
oxygen/ozone oxidative stressor, it is suitably applied by
irradiating the suspension under treatment from an appropriate
source of UV radiation, while the aliquot is maintained at the
aforementioned temperature and while the oxygen/ozone gaseous
mixture is being bubbled through the aliquot. The ultraviolet
radiation may be provided by any conventional source known in the
art, for example by a plurality of low-pressure ultraviolet lamps.
There is preferably used a standard UV-C source of ultraviolet
radiation, namely UV lamps emitting primarily in the C-band
wavelengths, i.e. at wavelengths shorter than about 280 nm.
Ultraviolet radiation corresponding to standard UV-A and UV-B
sources can also be used. Preferably employed are low-pressure
ultraviolet lamps that generate a line spectrum wherein at least
90% of the radiation has a wavelength of about 254 nm. An
appropriate dosage of such UV radiation, applied simultaneously
with the aforementioned temperature and oxidative environment
stressors, is obtained from lamps with a power output of from about
5 to about 25 wafts, preferably about 5 to about 10 watts, at the
chosen UV wavelength, arranged to surround the sample container
holding the aliquot. Each such lamp provides an intensity, at a
distance of 1 metre, of from about 40-80 micro wafts per square
centimeter. Several such samples surrounding the sample container,
with a combined output at about 254 nm of 15-40 watts, preferably
20-40 wafts, operated at maximum intensity may advantageously be
used. At the incident surface of the aliquot, the UV energy
supplied may be from about 0.25-4.5 j/cm.sup.2 during a 3-minute
exposure, preferably 0.9-1.8 j/cm.sup.2. Such a treatment provides
a suspension aliquot which is appropriately modified according to
the invention ready for injection into the patient.
[0026] The time for which the aliquot is subjected to the stressors
can be from a few seconds to about 60 minutes. It is normally
within the time range of from about 0.5-60 minutes. This depends to
some extent upon the chosen intensity of the UV irradiation, the
temperature and the concentration of and rate at which the
oxidizing agent is supplied to the aliquot. Some experimentation to
establish optimum times and dosages may be necessary on the part of
the operator, once the other stressor levels have been set. Under
most stressor conditions, preferred times will be in the
approximate range of about 0.5-10 minutes, most preferably 2-5
minutes, and normally around 3 minutes.
[0027] In the practice of one preferred process of the present
invention, the suspension of cells may be treated with oxygen/ozone
gas mixture and optionally also with UV radiation using an
apparatus of the type described in U.S. Pat. No. 4,968,483 Mueller.
The suspension is placed in a suitable, sterile,
UV-radiation-transmissive container, which is then fitted into the
machine. The temperature of the aliquot is adjusted to the
predetermined value, e.g. 42.5.+-.1 C, by the use of a suitable
heat source such as an IR lamp, and the UV lamps are switched on
for a fixed period before the gas flow is applied to the aliquot
providing the oxidative stress, to allow the output of the UV lamps
to stabilize. The oxygen/ozone gas mixture, of known composition
and control flow rate, is applied to the aliquot, for the
predetermined duration of 0.5-60 minutes, preferably 1-5 minutes
and most preferably about 3 minutes as discussed above. In this
way, the suspension is appropriately modified according to the
present invention sufficient to achieve the desired effects of
alleviation or prevention of GVHD.
[0028] From another aspect, the preferred embodiment of the present
invention may be viewed as a process of treating allogeneic T-cells
prior to their introduction into a patient, by extracorporeally
stressing the T-cells, which comprises subjecting the T-cells to
oxidative stress such as exposure to ozone or ozone/oxygen. The
treated allogeneic T-cells from the process of the invention have a
direct effect on the development and progression of GVHD. The donor
T-cells pretreated according to the process of the invention prior
to introduction into the host patient, have been modified, so that
they no longer mount a deleterious response. Their ability to mount
an inflammatory cytokine response has been decreased. For example
their ability to secrete IFN.gamma., TNF.alpha. and IL-2, and their
proliferative response to standard mitogens has been reduced.
Accordingly they no longer react against incompatible systemically
distributed host histocompatibility antigens to cause inflammation
to any great extent. The allogeneic stem cells administered to the
patient can proceed with engraftment with improved chance of
success. After a period of time, the treated T-cells largely
recover their proliferative ability and immune response functions,
but remain relatively unresponsive (tolerant) to differing host
histocompatibility antigens.
[0029] The invention is further described, for illustrative
purposes, in the following specific examples.
SPECIFIC DESCRIPTION OF THE MOST PREFERRED EMBODIMENTS
[0030] The spleen of a mammal offers a convenient, accessible
source of cells, especially T-cells but also including small
quantities of stem cells and is particularly useful in connection
with animal models for experimental purposes.
[0031] Experimental testing to obtain indication of the utility of
the process of the present invention was conducted using a model of
acute GVHD in SCID mice. T-cells from C57B1/6J (B6) mice were
intravenously injected into sub-lethally irradiated CB-17 SCID
mice. The latter are congenitally lymphopenic and provide a strong
stimulus for donor cells due to their complete disparity at the
major histocompatibility locus (MHC). The mean survival time of
host mice in this model is 14 days. GVHD is characterized by
suppression of host hematopoietic recovery from irradiation;
expansion of T-cells that use V.beta.3 chain to form their T-cell
receptor complexes (TCR's); spontaneous secretion of
interferon-.gamma. and TNF-.alpha., by donor T-cells, and aberrant
localization of donor T-cells to the red pulp areas of the spleen.
If donor marrow is co-injected with T-cells, a chronic form of GVHD
results.
EXAMPLE 1
[0032] Mouse spleen cells from C57B1/6J (B6) mice were suspended to
a density of 10.sup.7/ml in A-MEM, 2ME and 10% fetal calf serum
(FCS). The FCS contains cytokines and growth factors. The cell
suspension was subjected simultaneously to ultraviolet radiation
from UV-C lamps, wavelength 253.7 nm, whilst bubbling through the
suspension a gas mixture of 14-15 mcg/ml Iozone/medical grade
oxygen, at 42.5.degree. C. The treatment took place for 3
minutes.
[0033] Immediately after the treatment, the cells had a viability
of only about 10%.
EXAMPLE 2
[0034] The experiment of Example 1 was essentially repeated except
that the cells were suspended in 100% FCS. The immediate survival
of the cells in this case was 50-60%, indicating that factors
present in the FCS have exerted a protective effect on at least
some of the cells.
EXAMPLE 3
[0035] Murine B6 spleen cells suspended in 100% FCS were subjected
to UV-oxidation-heat treatment The cell suspension was subjected
simultaneously to ultraviolet radiation from UV-C lamps, wavelength
253.7 nm, whilst bubbling through the suspension a gas mixture of
14-15 mcg/ml ozone/medical grade oxygen, at 42.5.degree. C. The
treatment took place for 3 minutes. Varying numbers were injected
into sub-lethally irradiated CB-17 SCID mice. Their subsequent
behaviour was compared with similar numbers of B6 spleen cells, not
subjected to the treatment.
[0036] FIG. 1 is a graphical presentation of the results of these
experiments, where the % survival of the animals in each group is
plotted as ordinate against days following injection of the treated
or untreated cells. At all dosage levels, there is a marked
improvement of survival when the treated cells are used as opposed
to the untreated cells, demonstrating potential for the process of
the invention in alleviating GVHD.
[0037] FIG. 2 of the accompanying drawings is a plot of the number
of donor T-cells per spleen against days after GVHD induction, in
these same experiments. This shows that the treated donor T-cells
survive and expand in number in the host mice, although to a more
limited degree than control, untreated B6 T-cells.
EXAMPLE 4
[0038] Six days after initiation of GVHD in the mice by injection
of the donor cells (treated and untreated), donor T-cells were
separated from SCID spleen cells by density gradient
centrifugation. Intracellular cytokine staining was performed
according to the method of Ferrick, D. A. et. al., NATURE 373
225,257, 1995. The staining was performed on spleen cell
suspensions on day 8 after injection of B6 spleen cells. Cytokine
production was determined 4 hours after stimulation in vitro-with
PHA ard ionomycin in the presence of brefeldin-A and after gating
on CD4.sup.+ and CD8.sup.+. The results were assessed by
intracellular flow cytometry, and the results thereof are depicted
in FIG. 3 of the accompanying drawings. The percentage of each
cells in each quadrant is recorded. The drawing shows significantly
reduced levels of the inflammatory cytokines interferon-=65 (INF)
and tissue necrosis factor-.alpha. (TNF), lower right quadrants,
from the T-cells which had been stressed as described in Example 1,
as compared with untreated cells and controls.
EXAMPLE 5
[0039] Inversion of the normal ratio of CD4+ to CD8+ T-cells
(usually approximately 2:1) is known to accompany GVHD. By
intracellular cytokine staining techniques following the method of
Ferrick et.al., Nature 373: 255-257, 1995 and using anti-CD4 and
CD8-tricolor antibodies, CD4/CD8 ratios were determined. In the
untreated donor spleen cells after injection into sub-lethally
irradiated mice, the inversion of the normal ratio was confirmed.
The initial CD4/CD8 ratios of 1.3.+-.0.2 and 2.2.+-.0.3 decreased
to 0.33.+-.0.05 and 0.9.+-.0.1 by day 13 for unstressed B6 and C3H
donor T cells, respectively, at a time when many animals were
succumbing to GVHD. In contrast, the ratios remained greater than 1
at all times and correlated with the lack of clinical evidence of
GVHD when donor cells had been pretreated with the stressors as
described in Example 1.
EXAMPLE 6
[0040] This example demonstrates the principle of the invention,
using oxidative stress alone, provided by hydrogen peroxide, an
effective chemical oxidizing agent and representative of many
other, perhaps more biologically suitable, chemical oxidizing
agents.
[0041] Peripheral human blood mononuclear cells PBMCs, which is a
collection of white blood cells comprising about 40% T-cells, were
stressed by contact with aqueous solutions of hydrogen peroxide, of
various concentrations, for 20 minutes. Their immediate survival
was measured, along with their immediate phytohaemagglutinin (PHA)
response. Then their survival after 24 hours was measured, followed
by their PHA response (tritiated thymidine uptake following
mitogenic stimulation with PHA) and cytokine profile after 7 days.
The results are given in the following table.
1TABLE PHA 24 hr Immediate re- Immediate survival PHA sponse
Cytokine Conc. H.sub.2O.sub.2 survival % % response 7-day Profile
100 .mu.mole/L 80-90 100 2000 + IFN.dwnarw. 300 .mu.moIe/L 80-90 50
2000 + IFN.dwnarw. 1 mmole/L 80-90 40 400 + IFN.dwnarw. 3 mmole/L
80-90 40 400 + IFN.dwnarw. Control 95 95 8575 + IFN.Arrow-up
bold.
[0042] These results indicate that T-cells subjected to oxidative
stress alone achieve a decreased proliferative response and
decreased inflammatory cytokine production, suitable for use in the
present invention.
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