POPULATION OF IMMUNOREGULATORY T CELLS SPECIFIC FOR AN IRRELEVANT ANTIGEN AND USES THEREOF FOR PREVENTING OR TREATING IMMUNE DISEASES

Abstract

The present invention relates methods and compositions for preventing or treating various immune diseases including graft-versus-host disease (GVHD) using populations or compositions of immunoregulatory T cells specific for an irrelevant antigen; such cells being activated in vivo by a simultaneous, separate or sequential administration of said antigen.

Claims

1.-19. (canceled)

20. A method of treating or preventing an immune disease in a patient in need thereof comprising the following steps of: a) obtaining in vitro or ex vivo a population of CD4+CD25+ regulatory T cells specific for an antigen which is not involved in the immune disease to be treated; b) administering to said patient in need thereof the population of step a); and c) administering to said patient simultaneously, separately, or sequentially the antigen used in step a).

21. The method of claim 20, wherein the population of CD4+CD25+ regulatory T cells of step a) is obtained by a method comprising the following steps: i) obtaining a population of CD4+CD25+ regulatory T cells from a biological sample comprising lymphocytes; ii) activating the population of CD4+CD25+ regulatory T cells by contacting it with said antigen; iii) recovering the population of CD4+CD25+ regulatory T cells obtained at step ii).

22. The method of claim 21, wherein the population of CD4+CD25+ regulatory T cells of step a) is obtained by a method comprising a further step: iv) genetically modifying said population of CD4+CD25+ regulatory T cells by contacting said cells with a recombinant nucleic acid molecule.

23. The method of claim 20, wherein said antigen is a food antigen from common human diet.

24. The method of claim 23, wherein said food antigen is selected from the group consisting of ovalbumin, casein, beta-lactoglobulin, soya protein, gliadin, peanuts, and fragments, variants, and mixtures thereof.

25. The method of claim 21, wherein the antigen of step ii) is presented by antigen-presenting cells (APCs).

26. The method of claim 25, wherein the APCs are CD8+ dendritic cells.

27. The method of claim 21, wherein the activation of step ii) is carried out in presence of at least one cytokine.

28. The method of claim 20, wherein the CD4+CD25+ regulatory T cells are CD4+CD25+CD62L.sup.high regulatory T cells.

29. The method of claim 20, wherein the antigen at step c) comprises direct administration of the antigen to the patient or administration to the patient of antigen presenting cells (APCs) pulsed with said antigen.

30. A method of preventing or treating GVHD in a patient undergoing HSC transplantation from a transplant obtained from a donor comprises the following steps of: a) isolating a population of CD4+CD25+ regulatory T cells from the donor; b) obtaining in vitro or ex vivo a population of CD4+CD25+0 regulatory T cells specific for an antigen which is not involved in the GVHD to be treated; c) administering to said patient in need thereof the population of step b); and d) administering to said patient simultaneously, separately or sequentially the antigen in step b).

31. The method of claim 30, wherein the population of CD4+CD25+ regulatory T cells of step a) is obtained by a method comprising the following steps: i) obtaining a population of CD4+CD25+0 regulatory T cells from a biological sample comprising lymphocytes; ii) activating the population of CD4+CD25+ regulatory T cells by contacting it with said antigen; iii) recovering the population of CD4+CD25+ regulatory T cells obtained at step ii).

32. The method of claim 31, wherein the population of CD4+CD25+ regulatory T cells of step a) is obtained by a method comprising a further step: iv) genetically modifying said population of CD4+CD25+ regulatory T cells by contacting said cells with a recombinant nucleic acid molecule.

33. The method of claim 30, wherein said antigen is a food antigen from common human diet.

34. The method of claim 33, wherein said food antigen is selected from the group consisting of ovalbumin, casein, beta-lactoglobulin, soya protein, gliadin, peanuts, and fragments, variants, and mixtures thereof.

35. The method of claim 31, wherein the antigen of step ii) is presented by antigen-presenting cells (APCs).

36. The method of claim 35, wherein the APCs are CD8+ dendritic cells.

37. The method of claim 31, wherein the activation of step ii) is carried out in presence of at least one cytokine.

38. The method of claim 30, wherein the CD4+CD25+ regulatory T cells are CD4+CD25+CD62L.sup.high regulatory T cells.

39. The method of claim 30, wherein the antigen at step d) comprises direct administration of the antigen to the patient or administration to the patient of antigen presenting cells (APCs) pulsed with said antigen.

Description

[0137] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0138] FIGURES:

[0139] FIG. 1: ExoTreg exerted the same immunosuppressive effect in vivo as rsTreg. (a) Experimental GVHD model used in FIG. 1 representing semi-allogenic HCT testing different Treg populations. (b) Kaplan-Meier survival (mean±s.e.m) curves from mice injected with BM cells plus T cells, (GVHD group as control, n=16) and exoTreg (n=13) or rsTreg (n=11). *P=0.0009; **P=0.0001.

[0140] FIG. 2: ExoTreg prevent GVHD by reducing activation and differentiation of donor Teff. Donor CD4+ and CD8+ Teff are analyzed at day 6 post-transplantation in the spleen of animals grafted as described in figure la and identified using the CD45.1+ congenic marker. (a) Mean absolute numbers±s.e.m. (left) and CFSE dilution (right) of CD4+ and CD8+ donor CD45.1+T cells when injected with or without exoTreg (n=6 in each group). *P=0.0079; **P=0.0159. (b) Rejection of a third-party allogeneic skin-graft in mice protected from GVHD with exoTreg. In order to evaluate the primary response of exoTreg-protected mice, tail-skin grafts from BALB/c mice were transplanted at day 60 onto the lateral thoracic wall (n=6). On skin-grafted mice, the memory response was tested by a second BALB/c skin grafted at day 150 (n=5). Control group consists of mice treated with exoTreg and grafted with donor-type C57BL/6 skin (n=5). *P=0.0027.

[0141] FIG. 3: Prevention of GVHD by ExoTreg requires in vivo reactivation. (a) Kaplan-Meier survival (mean±s.e.m) curves demonstrate that in female mice (i.e not expressing HY antigen) injected with BM cells plus T cells (GVHD group, n=10), exoTreg (n=11) conferred no protection from GHVD whereas rsTreg (n=7) frilly protected from GVHD. *P=0.0157, **P=0.0158. (b) In contrast, the Kaplan-Meier survival (mean±s.e.m) curves demonstrate that when female mice are injected at D0, D3 and D6 with B6 DC cells loaded with HY peptide, exoTreg are able to prevent GVHD (n=5) as opposed to mice injected with exoTreg plus DC without peptide as control (n=4) or mice that received no Treg (n=5). *P=0.0316, **P=0.0250.

EXAMPLES

Example 1

GVHD Mouse Model

[0142] Material & Methods

[0143] Mice: Six-to-ten-week-old (C57BL/6×C3H) F1 (H2.sup.kb) and C57BL/6 (H-2.sup.b) were obtained from Harlan Laboratories (France), and C3H (H-2.sup.k) from Charles River Laboratories (France). C57BL/6 Ly5.1 were bred in our animal facility under specific pathogen-free conditions. Experiments were performed according to the European Union guidelines and approved by our institutional review board (CREEA Ile de France no. 3).

[0144] Ex-vivo expansion of antigen specific T.sub.reg: Cell suspensions were obtained from spleen and peripheral lymph nodes cells of C57BL/6 mice. Cells were first labeled with biotin-coupled anti-CD25 mAb (7D4, Becton Dickinson, San Diego, Calif. USA), followed with anti-biotin microbeads (Miltenyi Bitotec, Paris, France) and enriched in CD25.sup.+ cells using magnetic cell large selection columns (Miltenyi Biotec). Cells were then stained with fluorescein isothiocyanate (FITC-labeled) anti-CD4 (GK1.5), phycoerythrine (PE labeled) anti-CD62L (MEL-14) and streptavidin-Cy-Chrome, which bound to free biotin-labeled CD25 molecules (all obtained from BD Bioscience, Le pont de Claix, France). The CD4.sup.+CD25L.sup.high T cells were sorted by flow cytometry using a FACSAria (Becton Dickinson, Le pont de Claix, France), yielding a purity of 98%. Purified T.sub.reg were cultured 4 weeks in the presence of recombinant murine IL2 (10 ng/mL, R&D Systems, Lille, France) as previously described.sup.4 and 20-GY-irradiated recipient-type C3H splenocytes to expand rs-.sub.Treg. HY-T.sub.reg were cultivated in presence of L-DC loaded with HY peptide (10 μg/mL, N-15-S, NY, PolyPeptide, Strasbourg, France). DC were obtained from spleen cells of C57BL/6 mice. After digestion with liberase (0.4 mg/mL, Roche Meylan France) and DNase (0.1 mg/mL, Roche), cells were labeled with anti-CD11c-coated microbeads (Miltenyi Biotec), followed by 2 consecutives magnetic cell separation using LS columns (Miltenyi Biotec). Cells were stained with FITC-Iabeled anti-CD11b (M1/70, BD Biosciences), PE-labeled anti-CD11c (HL3, BD Biosciences) and Cy-Chrome-labeled, CD8a (53-6.7, BD Biosciences) and the CD11c.sup.highCD8.sup.+CD11b.sup.− cells (L-DC) were sorted by flow cytometry using a FACSAria yielding a purity of 99%, as previously described.sup.10.

[0145] In vitro suppression assay: After removal of dead cells by gradient of lymphocytes separation medium (Eurobio, Les Ulis, France), and five washes to remove residual IL-2, 1.10.sup.5 4 weeks expanded T.sub.reg were added to the culture of 1.10.sup.5 fresh CD25-depleted T cells (purified from C57BL/6 spleen) stimulated by 2.10.sup.5 irradiated B6 splenocytes and by 5 μg/ml anti-CD3 mAb (BD). Cells cultured in round-bottom, 96-well plates for 96 hours were pulsed with [.sup.3H] methyl-thymidine for the last 18 hours.

[0146] Experimental GVHD: Donor T cells were collected from lymph nodes of donor animals and the percentage of CD3.sup.+ cells was determined by flow cytometry at time of infusion, Irradiated (10 Gy) or non-irradiated seven-to-twelve-week-old [C57BL/6×C3H] F1 recipient mice were injected i.v. in the retro-orbital sinus with 10.10.sup.6 C57BL/6 BM cells (control group), 2.10.sup.6 C57BL/6 CD3.sup.+ T cells alone to induce GVHD, or with 2.10.sup.6 cultured rsT.sub.reg or HY-T.sub.reg. In non-irradiated recipients, 10.10.sup.6 C57BL/6 CD3.sup.+ T cells alone are required to induce GVHD. The same number of HY-T.sub.reg was then added to test their clinical effect. Clinical signs of GVHD (body weight loss, diarrhea, skin lesions, hunched posture) were monitored regularly. Body weight loss of more than 30% of the initial weight led to euthanasia of sick mice.

[0147] Histology: After mice death or sacrifice, liver and colon samples were fixed in 4% formaldehyde solution for several days and embedded in paraffin. For both organs, 5-μm sections were stained with H&E for histological examination. One pathologist analyzed slides in a blinded fashion to assess the intensity of GVHD. GVHD lesions in each bowel sample were graded according to a semi-quantitative scoring system described by Hill et al. with minor modifications. Six parameters were scored for the colon (surface f the crypts, inflammation of the chorion, crypt regeneration, crypt epithelial cell apoptosis, crypt loss, mucosal ulceration), and seven for the liver (bile ducts, periportal necrosis, endothelitis, acidophilic body, confluent necrosis, sinusoidal lymphocytosis, inflammatory cell infiltrate). Each parameter was scored as follows: 0 as normal; 1 as focal and rare; 2 as focal and mild; 3 as diffuse and mild; 4 as diffuse and moderate: and 5 as diffuse and severe.

[0148] Flow Cytometry: The following Abs were used for FACS analysis: anti-CD3, anti-CD4, anti-CD8, anti-CD90.2, anti-H2K.sup.k, anti-CD25, anti-CD62L, anti-CD44, anti-CD45.1, anti-IL-2, anti-TNFα and anti-IFNγ, labelled with FITC, PE, allophycocyanin, peridia chlorophyll protein (PerCP) or Alexa Fluor 700. All mAbs were purchased from BD Biosciences. The PE- or Efluor 450- labeled anti-Foxp3 staining was performed using the eBioscience kit and protocol. For intracellular cytokine staining, cells were re-stimulated with 1 μg/ml PMA (Sigma) and 0.5 μg/ml ionomicyn (Sigma, Saint-Quentin Fallavier, France) for 4 h, in the presence of GolgiPlug, (1 μl/ml) (BD Biosciences). After cell surface staining, intracellular staining was performed using the CytoFix/CytoPerm kit (BD Biosciences). Events were acquired on a LSRII (BD Biosciences) flow cytometer and analyzed using FlowJo (Tree Star, Ashland, Oreg., USA) software.

[0149] In vivo activation of T.sub.reg: DCs were isolated after magnetic sorting, and cultured at 37° C. during 12 hours in presence of GM-CSF (20 ng/mL) and HY peptide (10 μg/mL). B6C3F1 female recipient were immunized i.v. in the retro-orbital sinus with 1.10.sup.6 B6 pulsed DCs or 100 μg of HY peptide at D0, D3 and D6 post-graft.

[0150] Skin grafting: At 2 and 5 months after HSCT, tail-skin grafts from C57BL/6 and Bulb/c mice were transplanted onto lateral thoracic wall of the recipients under ketamine (75 mg/kg) and xylazine (15 mg/kg) anesthesia. Skin grafts were monitored regularly by visual and tactile inspection. Rejection was defined as loss of viable donor epithelium.

[0151] Statistical analyses: Statistical significances were calculated using the two-tailed unpaired Student t-test for cell analysis. Log-rank (Mantel-Cox) test was used to compare survival between two groups of mice. When statistically significant, P values were indicated.

Results

[0152] Lethally Irradiated Recipient Male Mice

[0153] As a model for GVHD, lethally irradiated [C57BL/6 (B6) X C3H] F1 (B6C3F1) recipient male mice were grafted with bone marrow (BM) cells and T.sub.eff collected from B6 males lymph nodes (LN) as illustrated in FIG. 1a. Mice receiving BM cells and T cells rapidly developed clinical signs of GVHD (diarrhea, skin lesions, hunched posture and weight loss; data not shown), and more than 80% died by day 60 (FIG. 1b). GVHD was also confirmed by a typical histological appearance of the colon and liver. We next aimed to test the ability of two populations Of T.sub.reg (exoT.sub.reg and rsT.sub.reg) to prevent GVHD. For this purpose, we generated exoT.sub.reg from highly purified T.sub.reg of B6 female mice, which were then cultured for 30 days in the presence of autologous lymphoid dendritic cells (L-DC) pulsed with the HY peptide (an antigen expressed only in males), as previously described.sup.10. As control, we produced C3H-specific rsT.sub.reg following a validated selection and expansion procedure.sup.4,5,9,11. Both types of T.sub.reg expanded robustly during culture while maintaining both the expression of Foxp3 and CD25, as well as the ability to suppress T cell proliferation in vitro. The specificity of exoT.sub.reg was confirmed in vitro, as only stimulation with antigen presenting cells (APC) expressing the HY antigen (derived from male mice) resulted in proliferation of exoT.sub.reg.

[0154] After generating the two populations of T.sub.reg (exoT.sub.reg and rsT.sub.reg), we proceeded to test their ability to prevent GVHD. T.sub.reg were co-transferred with the transplant into male mice (harboring the HY antigen). Remarkably, exoT.sub.reg prevented clinical manifestations of GVHD and also resulted in a significant reduction of lesions in target organs, comparably to the prevention of GVHD by rsT.sub.reg. Furthermore, like rsT.sub.reg.sup.11, exoT.sub.reg promoted a strong inhibition of expansion, activation and differentiation of donor T cells. First, donor T cell number (CD45.1.sup.+) was markedly reduced in the presence of exoT.sub.reg (FIG. 2a). Second, among dividing T cells, attested by CFSE dilution, the proportions and absolute numbers of activated T cells with a CD25, CD44.sup.high, CD62L.sup.low phenotype, as well as cells producing interleukins (IL)-2, interferon (IFN)-γ and tumor necrosis factor (TNF)-α, were markedly reduced. Finally, this effect was tumor growth factor (TGF)-β dependant, as treatment with anti-TGF-β monoclonal antibody (day 0-3) resulted in loss of exoT.sub.reg's protective function, as previously shown in different models of disease protection by T.sub.reg.sup.12. Additionally, in our model of GVHD, we observed that 5% of CD4+T cells and 40% of CD8+ donor T cells expressed LAP at day 5 post-HCT. HY-Tregs administration increased LAP expression to 80% of CD8.sup.+ donor T cells, with minimal effect on CD4.sup.+ T cells. This suggests that the TGF-P dependent effect of HY-Tregs may be due to TGF-β production by CD8 Tconv. In conclusion, exoT.sub.reg, in the presence of their cognate antigen, were as efficient as rsT.sub.reg in preventing GVHD, and had similar cellular effects.sup.1.

[0155] A Non Irradiated Recipient Male Mice

[0156] Lethal irradiation induces profound lymphopenia associated with a cytokine storm. These events may lead to a non-specific activation of T.sub.reg, a phenomenon called lymphopenia-induced proliferation (LIP).sup.13. To evaluate the impact of LIP on the suppressive effect of exoT.sub.reg, we repeated the experiment in non irradiated B6C3F1 male recipients. When they were grafted with B6 donor T cells, they developed. GVHD characterized by weight lost and high mortality rate. The co-transfer of exoT.sub.reg or rsT.sub.reg resulted in protection from GVHD, even in a model that does not involve LIP, suggesting that the protective effect conferred by exoT.sub.reg is indeed due to their in vivo re-activation by their cognate antigen. Finally, it is important to note that lethally irradiated mice protected with exoT.sub.reg were not functionally immunodeficient, since they were able to reject third-part skin graft from Balblc mice with an even accelerated memory-type immune response when a second skin graft was performed 90 days after the first one (FIG. 2b), in accordance with our previous observations using rsT.sub.reg.sup.14.

[0157] Redolent Female Mice

[0158] In the above-described experiments, the recipients were male mice that harbor the HY antigen, and thus, in this context, HY cannot be considered truly exogenous. We therefore attempted to re-activate in vivo exoT.sub.reg in female mice, which do not express the HY antigen. We used the same GVHD model, modifying only the gender of recipient mice (previously male, now female). As expected, co-transfer of exoT.sub.reg in female recipients had no effect on GVHD. The mice displayed clinical and histological signs of GVHD and died with a kinetic comparable to mice that received donor T cells alone (FIG. 3a). This observation was also supported by lower expression of inducible co-stimulator (ICOS) and glucocorticoid-induced TNF receptor (GITR) activation markers on exoT.sub.reg 6 days after transfer in female compared to male recipients, indicating that they failed to activate. In contrast, rsT.sub.reg maintained full efficacy in female recipients, resulting in complete abrogation of GVHD. Subsequently, we tested whether we can reactivate in vivo exoT.sub.reg after transfer by providing the exogenous antigen to female recipients. We injected donor DCs, previously ex vivo pulsed with the HY peptide or the HY peptide alone, at time of GVHD induction, at day 3 and at day 6. In these two groups, all mice survived and none had signs of GVHD. In contrast, control mice that received no T.sub.reg or co-injected with exoT.sub.reg followed by injection of DCs not pulsed with HY developed lethal GVHD (FIG. 3b). Thus, in summary we demonstrated that T.sub.reg specific for an exogenous antigen can indeed exert a systemic bystander effect, preventing GVHD, and that these exoT.sub.reg can be reactivated in vivo by providing them of the cognate exogenous antigen (with exogenous antigen-pulsed DCs or the sole exogenous antigen).

[0159] Here, we demonstrate for the first time a successful systemic application of this principle to prevent one of the most catastrophic immune mediated processes, GVHD. Thus, a “systemic bystander effect” was seen when DCs presenting an exogenous antigen (non donor, non recipient antigen) properly activated in vivo exoT.sub.reg, as attested by ICOS and GITR over expression observed on exoT.sub.reg. This population of highly activated specific exoT.sub.reg subsequently fully supressed the entire repertoire of alloreactive T.sub.eff cells through a mechanism TGF-β dependant.

[0160] From the clinical perspective, it is worthwhile to consider the importance of the “systemic bystander effect” in the context of the ongoing efforts to harness the power of T.sub.reg for therapeutic applications. As seen in this and other reports, T.sub.reg offer an unparalleled promise for auto and allo-immune disorders. They appear not only extremely potent, but capable of inducing tolerance while reducing the risk of immunodeficiency. However, in order to optimize their efficacy, efforts have been made to enhance the purity as well as the specificity of T.sub.reg. This principle has guided, for almost 10 years, an intense optimization process for purification and ex vivo expansion of T.sub.reg in GMP conditions. Despite numerous encouraging improvements.sup.2,3,16-24, this objective has not been reached yet and contaminating T.sub.eff are present in these cell preparations, Thus, administrating T.sub.reg specific for recipient alloantigens poses the risk of injecting pathogenic allo-reactive T.sub.eff as well. We present an alternative approach using T.sub.reg specific for a “third party” exogenous antigen. We showed that these exoT.sub.reg generated ex vivo from a polyclonal T.sub.reg population, prevented experimental GVHD via potent suppression of pathogenic T.sub.eff. This therapeutic effect was found even when exoT.sub.reg were re-activated in vivo upon immunization of recipient (female) mice with the HY cognate antigen. The potential for T.sub.reg based therapy is substantial. These experiments suggest that by using a “third party” exogenous antigen, we can maintain both the efficacy and specificity of T.sub.reg, while eliminating the risk of pathogenicity of putative contaminating antigen-specific T.sub.eff. In addition, because of the short half-life of mature DC, one may envisage that the suppressive action of injected T.sub.reg is transient, conferring an on/off property to the system. In conclusion, the “systemic bystander effect” demonstrated here, holds a tremendous promise for the therapeutic application of T.sub.reg, removing one of the major obstacles on the way of this important therapeutic strategy from the bench to the bedside.

Example 2

Ovalbumin-Specific Human CD4.SUP.+.CD25.SUP.+ Regulatory T Cells (“ovaTreg”) and Uses Thereof in GVHD Models

[0161] Material & Methods

[0162] Purification of human Treg: From leukapheresis, CD4+ T cells are isolated by depleting non-CD4 cells with GMP-grade mAb-coated microbeads (cocktail of CD8, CD14, CD19 and CD56 ±CD127) in combination with CliniMAX device, Unbound cells are then purified by positive selection with GMP-grade anti-CD25 mAh-coated microbeads and CliniMAX, Non Treg cells are frozen for in vitro and in vivo suppression assays. This approach has now been validated by several teams.sup.8,25.

[0163] Preparation of ovaTreg: Purified Treg are cultured in the presence of autologous or artificial APCs pulsed with ovalbuminc. Several parameters are tested at this step:

[0164] Phase of Selection of Ag-Specific Treg:

[0165] Purification of blood-derived autologous APCs, or alternately artificial APC as previously described.sup.8,25 to activate ex-vivo Treg. The two types of ova-pulsed APCs are tested.

[0166] Culture condition (IL2, IL15, rapamycin and their concentrations).

[0167] The possibility or not to rapidly sort Ag specific Treg upon specific marker expression after several days of culture as recently observed with alto-stimulation (upon CD69 and CD71 co-expression).sup.8.

[0168] Phase of Expansion of Ag-Specific Treg:

[0169] The necessity to expand Treg after an initial phase of selection of Ag-specific Treg to reach clinically relevant cell numbers are tested following two methods: using GMP antiCD3-CD28 microbeads, or alternately by prolonging activation with APCs (autologous or artificials).

[0170] The duration of culture and the number of reactivations required for maximal ovaTreg expansion.

[0171] Phase of in Vivo Activation of Ag-Specific Treg:

[0172] The mode of activation of Treg in vivo (direct injection of ovalbumin, APC pulsed with ova peptide, the number of injection required to activate these cells in vivo) are tested after adoptive transfert of ovaTreg in NOD/SCID/ganirnaC—/—immunodeficent (NOG) mice (described below).

[0173] Safety Assessment: The Treg products are infused in NOD/SCID/gammaC—/—immunodeficent mice in absence of effector T cells. Absence of GVHD will confirm absence or reduced numbers of T effector cells. These experiments are conducted with or without Treg activation by ovalbumins.

[0174] Efficacy Assessment:

[0175] In vitro: OvaTreg are tested in vitro for their capacity to suppress human T cells of the same genetic background activated by allogeneic APCs. Tests will be performed with or without Ova-pulsed APCs.

[0176] In vivo: OvaTreg are tested in vivo for their capacity to prevent GVHD induced by conventional human T cells obtained from the same donor of Treg, infused in NOD/SCID/gamtnaC—/—immunodeficent mice (xeno-GVHD). Mice are assessed fur weight loss, survival, and histopathological signs of GVHD in target organs.

[0177] Statistical methods: Data are analyzed with SPSS software with T test for continuous variables and chi-square for categorical variables.

REFERENCES:

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