Inhibition of graft versus host disease

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.

FIELD OF THE INVENTION
 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
 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.
 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.
 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
 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.
 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
 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.
 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.
 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.
 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.

DESCRIPTION OF THE PREFERRED EMBODIMENTS
 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.
 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.
 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.
 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..
 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.
 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.
 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).
 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-.sup.6 to
 10-.sup.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.
 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.
 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.
 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.
 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 watts,
 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 meter, of from about 40-80
 micro watts 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 watts, 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.
 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.
 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.degree. 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.
 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.
 The invention is further described, for illustrative purposes, in the
 following specific examples.
 SPECIFIC DESCRIPTION OF THE MOST PREFERRED EMBODIMENTS
 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.
 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
 Mouse spleen cells from C57B1/6J (B6) mice were suspended to a density of
 10.sup.7 /ml in .alpha.-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
 ozone/medical grade oxygen, at 42.5.degree. C. The treatment took place
 for 3 minutes.
 Immediately after the treatment, the cells had a viability of only about
 10%.
 EXAMPLE 2
 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
 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.
 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.
 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
 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 and
 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-.gamma. (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
 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
 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.
 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.
 TABLE
 Immediate PHA
 Conc. Immediate 24 hr PHA response Cytokine
 H.sub.2 O.sub.2 survival % survival % response 7-day Profile
 100 .mu.mole/L 80-90 100 2000 + IFN.dwnarw.
 300 .mu.mole/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.uparw.
 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.