Alloreactive t cells and cytokines in murine graft versus host disease 3

_ Results CHAPTER RESULTS Graft-versus-Host disease (GVHD) occurs in recipients of allogeneic bone marrow transplantation and in immune compromised patients after whole blood transfusion or organ transplantation (Ferrara et al., 1991; Brok et al., 1993; Dickinson et al., 1994) GVHD exhibits as acute and chronic patterns (Allen et al., 1993) Acute GVHD is initiated by allogeneic T cells After entering the recipient blood circulation, these T cells home into the secondary lymphoid tissues where the alloreactive T cells become activated These activated T cells proliferate, resulting in a much bigger population of these cells (Bouma et al., 1995; Hu et al., 1994) Activated alloreactive T cells migrate from the secondary lymphoid tissues into the peripheral target organs, and initiate the inflammatory process They destroy the tissue structure and cause various clinical symptoms (Hu et al., 1999 & 2000; Li et al., 2001) 3.1 T Cell Infiltration and Chemokine Expression: Relevance to the Disease Localization in Murine Graft-versus-Host Disease Acute GVHD involves mainly skin, liver, and intestines Other organs such as heart, muscle and central nervous system are usually not affected, although their parenchyma cells also express alloantigens, such as MHC class I antigens (Nikolic et al., 2000; Hakim et al., 1998; Murai et al., 1999; Ferrara et al., 1991) The mechanism of this seemingly selective involvement of distinct organs in acute GVHD is not well 74 _ Results understood We hypothesized that it might be related to the selective migration of activated alloreactive T cells T cell migration is largely controlled by a group of protein molecules called chemokines Chemokines are secreted by various cells, such as macrophages, dendritic cells, activated T cells, endothelial cells, etc They are classified as C, CC, CXC and CX3C chemokines according to the N-terminal amino acid sequences Cells expressing chemokine receptors are attracted to the sites with the highest concentration of chemokines following the concentration ladder (Zlotnik et al., 2000; Baggiolini et al., 1998; Rollins et al., 1997) Expression of MIP-1α was elevated and a large number of CD8+ cells expressing chemokine receptors CCR1, CCR4, CCR5, CXCR3 were detected in the liver of mice with acute GVHD (Serody et al., 2000; Murai et al., 1999) Blockade of T cell migration into liver using anti-CCR5 antibody (Ab) down-modulated the disease activity (Murai et al., 1999) We wondered whether chemokine expression pattern is a factor determining the preferential organ involvement in acute GVHD In this study, we examined T cell infiltration and chemokine expression in target and non-target organs of mice developing acute GVHD 3.1.1 T cell infiltration in the target and non-target organs An acute GVHD murine model was employed as described previously (Hu et al., 1999 & 2000; Li et al., 2001) Spleen, liver, skin and heart were harvested from euthanized C.B-17 SCID mice (H-2d) that received spleen cells obtained from C57BL/6J (H-2b) mice These organs were chosen to represent the target (spleen, liver and skin) and non-target (heart) organs of acute GVHD The infiltration of T cells was examined initially with FACS analysis in these organs Freshly harvested organs were minced and mononuclear cells were isolated Cells were stained with antibodies directed at CD4, CD8 and H-2kb molecules As presented in Fig 2&3, both donor CD4+ and 75 _ Results CD8+ cells were detected in the secondary lymphoid tissues and the liver in the first days after donor cell infusion In the samples obtained and 21 days after donor cell infusion, CD4+ and CD8+ cells were observed in the heart as well, and the percentages of these two cell subsets increased substantially in all organs, with the lowest being in the heart From day onwards, the majority of infiltrating lymphocytes were CD8+ T cells, consisting up to 60% of total T cell population in the target organs Donor cell proliferation was examined by labeling the donor splenocytes with a green fluorochrome 5-carboxyfluorescein diacetate succinimidyl ester (CFSE) before they were injected into the recipient mice Cells were then isolated from various organs of the recipient mice at different time points As shown in Fig 2, the donor cells homed to the lymphoid organs (LNs, spleen) and to a much lesser extent to the liver as well one day after the injection Cell division started on day 3, peaked on day 4, and subsided by day 21 No proliferation, however, was observed in the target organ (liver) The liver infiltrating T cells were effector cells that had divided Since FACS analysis can only show the percentage of a specific cell population and is not able to indicate the absolute infiltrating cell numbers, we examined the infiltrating T cells in these organs by immunohistology on tissue sections T cells were visualized with an anti-CD3 antibody staining, and they were easily observed in the liver and skin days after donor cell infusion, but only rarely in the heart (Fig and Table 8) 76 _ Results MLN spleen liver heart MLN spleen liver heart CD8PE Day CD4PE Day Day Day 21 CFSE Fig.2 Distribution and proliferation of donor T cells in various recipient organs Donor spleen cells were isolated and labeled with CFSE prior to injection into the recipient mice During each round of cell division, the relative intensity of the CFSE in each cell decreases by half Mononuclear cells were then isolated from the recipient spleen, liver, heart, MLN and PLN at different time points post-transplantation and analyzed by flow cytometry Representative flow cytometric profiles show the expression of CD4 (a) or CD8 (b) vs CFSE by the H-2kb+ donor cells isolated from the MLN, spleen, liver and heart of the recipient mice on day 1, 4, 7, and 21 after the allogeneic cell transfer The figure reveals that the donor T cells first homed to recipient lymphoid organs (day MLN, spleen), went through several rounds of division (day 4&7 MLN, spleen) before the divided donor T cells infiltrate the target organ (day 4&7 liver) Few cells infiltrated the heart even up to day 21 Five mice were examined for each condition 77 _ Results MLN PLN spleen liver heart percentage of CD4 70 a 60 50 40 30 20 10 21 21 21 21 21 days post-transplantation percentage of CD8 70 b 60 50 40 30 20 10 21 21 21 21 21 days post-transplantation Fig.3 Kinetics of infiltration of CD4 and CD8 donor cells into various recipient organs at different time points after allogeneic T cell transfer Donor spleen cells were isolated and labeled with CFSE prior to injection into the recipient mice Mononuclear cells were isolated from recipient spleen, liver, heart, MLN and PLN at different time points post-transplantation and analyzed by flow cytometry Lymphocyte population was gated based on the low FS and SS property The mean percentage of H-2kb+ CD4+ or H-2kb+ CD8+ cells within the gated lymphocyte population was presented The data represents the mean of mice in each group ± SD 78 _ Results a b c Fig.4 Immunohistochemical staining for detection of infiltrating CD3+ cells in various organs of the recipient mice Target and non-target organs were procured from the recipient mice at different time points post-transplantation Cryosections were prepared and stained with an anti-CD3 mAb to visualize infiltrating T cells Representative cryosections of tissues collected on day 21 post-transplantation show a large number of T cells infiltrating in the liver (a) and the skin (b), but not in the heart (c) Spleen Liver Intestine Heart Kidney Skin Day - Day3 ++ + - Day7 +++ ++ +/+/- Day21 +++ +++ ++ ++ Table Table The kinetics of T cell infiltration into target and non-target organs during acute GVHD Cryosections prepared from various organs procured 0, 3, 7, and 21 days after injection of donor spleen cells were stained with an anti-CD3 mAb The stained sections were observed under the light microscope Three high power fields (×400) were randomly chosen and positively stained cells in the tissue were enumerated Cells were averaged per high power field and recorded as – (0), + (1-10 cells), ++ (11-30 cells), and +++ (>30 cells) Three samples were examined in the same mode for each condition 79 _ Results 3.1.2 mRNA expression of chemokines in the target and non-target organs Spleen, liver, skin and heart were harvested from SCID mice at various time points after injection of the donor spleen cells mRNA was isolated and reversed transcribed into cDNA As the first step, conventional PCR was performed to semi-quantitatively identify which were the chemokines that were upregulated in mRNA transcription in these organs A total of 12 chemokines, namely BCL-1, MIP-1α, MIP-1β, MIP-2, MCP-1, MCP-3, RANTES, Mig, SLC, SDF-1α, SDF-1β and eotaxin were screened using primers specific for each of these chemokine cDNAs After PCR, the amplicons were subjected to agarose gel electrophoresis As depicted in the Fig using liver sample as representative, MIP-1α, MIP-2, MCP-1, MCP-3 and Mig mRNA consistently showed an obvious increase in expression with the progress of GVHD while the rest showed inconsistent or no increase in expression These chemokines were selected for further quantification on their mRNA expression using real time PCR As depicted in Fig 6, expression of MIP-1α, MIP-2, Mig, MCP-1 and MCP-3 increased days after donor cell injection, peaked on day 7, and, except MIP-2 in the skin, returned to baseline levels on day 21 Expression of MIP-1α and MIP-2 was higher in the spleen In the liver, MIP-1α, MIP-2 and Mig predominated MIP-2 and MCP-3 expressed at a higher level in the skin Expression of MCP-1 and MCP-3 was more prominent in the heart 3.1.3 Expression of chemokines at the protein level in the target and non-target organs Spleen, liver, skin and heart were harvested from SCID mice at various time points after injection of the donor spleen cells ELISA was conducted on the supernatant 80 _ Results collected from the tissue homogenate, and the quantity of chemokines was normalized to that of the total protein in each organ The quantity of three chemokines, MIP-1α, MIP-2, and MCP-1, was determined (Fig 7) Expression of the chemokines at the protein level was correlated with that at the mRNA level MIP-1α and MIP-2 were predominant in the spleen and the liver In the heart, a high expression of MCP-1 was observed on day after donor cell injection Increased levels of expression of MIP-1α, MIP-2 and MCP-1 were observed in the skin 3.1.4 Blood level of chemokines in the SCID recipient mice Ten SCID mice were each injected with 10×106 donor spleen cells, and others were injected with PBS as controls Their body weights were monitored daily and presented in Fig The control mice all gained weight during the observation period However, the weight of the treated mice started to decline by day after a brief increase in the first few days Six mice died by day 35 because of acute GVHD, similar to our previous observations (Hu et al., 1999 & 2000; Li et al., 2001) Blood samples were taken weekly from the SCID mice that received donor cell infusion and from the control SCID mice Sera were separated and used for ELISA to determine the concentrations of chemokines MIP-1α, MIP-2 and MCP-1 Levels of the chemokines increased in mice that received donor cell infusion, but not in the control mice (Fig 9) The kinetics was similar to that of the tissues examined MIP-1α was elevated days after the cell infusion, peaked on day 14, and remained higher than before infusion until day 28 MIP-2 started to increase days after cell infusion, peaked on day 21, and remained higher than before infusion until day 35 Serum MCP1 began to increase days after donor cell injection, reached a prominent peak on day 81 _ Results 7, and subsided to a slightly higher level than before infusion until day 35 in most of the mice Day 14 Day β-actin 14 MCP-3 MIP-2 MCP-1 MIP-1α Mig Fig.5 Semi-quantitative analysis of various chemokine mRNA expression levels in liver using agarose gel electrophoresis Recipient mice were sacrificed at various time points post-transplantation and their livers were procured mRNAs extracted from the liver were reversed-transcribed into cDNA and subjected to PCR using primers specific for various chemokine cDNA PCR products were then analyzed with agarose gel electophoresis A house-keeping gene, β-actin, was employed as internal control 82 _ Results a b MIP-2 MIP-1α c MCP-1 MCP-3 Mig 0.5 0.4 0.5 1 0.5 0.3 0.8 Spleen 0.2 0.25 0.4 0.1 0 0 21 0 21 21 0 21 21 21 21 21 0.5 0.4 0.5 1 0.5 0.3 Liver 0.8 0.2 25 0.4 0.1 Skin Chemokine / β-actin level Heart 0 21 21 0 21 0.4 21 5 1 0.5 5 0.3 0.8 0.2 25 0.4 0.1 0 0 21 21 0.5 21 0 21 0.4 0.3 0.8 1 0.5 0.5 0.2 0.25 0.4 0.1 21 21 0 0 0 21 21 Days post-transplantation d Fig.6 Kinetics of chemokine expression in various organs of the recipient mice after the donor cells injection a Amplification plot of the standard used in real time PCR Ten-fold serial dilutions of MIP-1α-pcDNA 3.1 recombinant plasmids (beginning from 108 to 102 copy number) were amplified for 45 cycles Amplification plot shows that copy number of target gene as low as 102 can be reliably detected b Standard curve derived from the amplification plot shown in (a) The regression line was obtained using the Sequence Detection System software provided by PE Biosystem Correlation coefficient = 0.997 c Representative amplification plot of RNA samples extracted from different tissues RNA was isolated from day spleen, liver, skin and heart of a recipient mouse and reverse-transcribed into cDNAs Real time PCR was performed with primers and probes specific for MIP-1α sequence From the left to the right, the curves indicate spleen, liver, skin and heart cDNA samples as substrate respectively d Expression profiles of MIP-1α, MIP-2, MCP-1, Mig and MCP-3 mRNA in various recipient organs RNA was extracted from recipient spleen, liver, heart, and skin obtained at different time points post-transplantation and quantified using reverse transcription followed by real time PCR The quantity of mRNA for each condition was normalized to the expression of β-actin in the same sample ♦: the ratio in each sample : the mean of samples 83 _ Results Single staining CFSE β7-PE Double staining αE-PE-Cy5 a) CFSE CFSE hi CFSElo LN CFSE LN 2.3% 3.5% αE-PE-Cy5 Triple staining Unsorted spleen 3.3% Double staining CD45RB-PE b) CD38-PE-Cy5 β7-PE CFSE CFSE hi CFSElo LN CFSE LN Triple staining 1.5% CD38-PE-Cy5 1.3% Unsorted spleen 1.1% CD45RBPE Fig.26 Expression of αE & β7 or CD45RB & CD38 on various fractions of cells Donor T cells were stained with CFSE prior to injection into the primary recipient mice Five days after injection, the spleen and LN cells from the primary recipient mice were derived, sorted and analyzed with flow cytometry Representative flow cytometric profiles of independent experiments 121 _ Results a) 140 IL- (pg/ml) 120 100 80 60 40 20 7 days of culture b) 60 IL-10 (pg/ml) 50 40 30 20 10 days of culture c) 14 IL- (pg/ml) 12 LNhi CFSEhi LN LNlo CFSElo LN LNunf unsorted LN 10 SPL spleen Sti stimulator days of culture Fig.27 IL-4, IL-10 and IL-2 level in cell culture supernatant collected from MLR A mixture of spleen and LN cells from C57BL/6J mice stained with CFSE was injected into the SCID mice Five days after the injection, recipient mice were killed and LN cells were derived for FACS sorting while spleen cells were used directly Irradiated Balb/c spleen cells syngeneic to the SCID recipients were cultured with various fractions of the cells from primary recipient mice Cell culture supernatants were harvested at different time points and analyzed with ELISA Data represent the IL-4 (a), IL-10 (b) and IL-2 (c) level determined by ELISA of pooled supernatants collected from replicates at each time point 122 _ Results a) Controls 6 lo 6 hi 1x10 1x10 CFSELNlo : 1x101x10 CFSE LN LN : LNhi hi 1x10 LN control 1x10 CFSE LNhi control 120 % of body weight change b) Experiment I 110 100 90 80 70 60 50 30 60 90 120 150 lo 30 1x10 CFSE LNlo control 1x10 LN control 60 lo 90 120 6 150 hi 2x10 2x10 CFSE LNlo : 1x10 LNhi CFSE LN LN : 1x10 110 140 120 90 100 70 80 50 60 30 60 90 120 150 Days post-transplantation 30 60 lo 30 60 lo 30 60 90 120 150 hi 3x106 3x10 CFSELNlo : 1x101x10 CFSE LN LN : LNhi 110 90 70 50 90 120 150 hi 4x10 CFSE LNlo : 1x106 LNhi CFSE LN 4x106 LN : 1x10 110 90 70 50 90 120 150 Days post-transplantation Fig.28 Change in body weight and survival rate of secondary recipient mice injected with a mixture of CFSEhi and CFSElo LN cells Primary recipient mice were sacrificed on day post-injection with donor T cells and their LN cells were derived for sorting 1×106 CFSEhi LN cells were mixed with different ratio of CFSElo LN cells, ranging from 1×106 to 4×106, prior to injection into the secondary recipient mice Data show the lifespan and percentage of change in the body weight of the secondary recipient mice after injection with the cell mixture (b) Mice injected with 1×106 CFSEhi or CFSElo LN cells alone served as controls (a) 123 _ Results a) Controls b) Experiment II hi 1x10 CFSE 1x106 LNhi control LN control hi 120 100 100 80 80 60 30 60 90 2x106 Spl control 2x10 spleen 120 150 control 160 140 120 100 80 30 60 90 120 150 Days post-transplantation % of body weight change % of body weight change 1x10 6spleen : 61x10 CFSE LN 1x10 spl : 1x10 LNhi 120 60 30 60 90 120 150 hi 2x10 spl : 1x10 LNhi 2x10 6spleen : 61x10 CFSE LN 120 100 80 60 30 60 90 120 150 hi 3x10 spl : 1x10 LNhi 3x10 6spleen : 61x10 CFSE LN 120 100 80 60 30 60 90 120 150 Days post-transplantation % of body weight change c) Experiment III 130 110 90 70 30 60 90 120 150 Days post-transplantation Fig.29 Change in body weight and survival rate of secondary recipient mice injected with a mixture of CFSEhi LN and unsorted spleen cells Primary recipient mice were sacrificed on day post-injection with donor T cells Their LN cells were derived for sorting while spleen cells were used directly a) Mice injected with 1×106 CFSEhi LN or unsorted spleen cells alone served as controls b) 1×106 CFSEhi LN cells were mixed with different ratio of unsorted spleen cells, ranging from 1×106 to 3×106, prior to injection into the secondary recipient mice c) 2×106 unsorted spleen cells were injected into the secondary recipient mice one week before the injection of 1×106 CFSEhi LN cells Data show the lifespan and percentage of change in the body weight of the secondary recipient mice after injection with the cell mixture 124 _ Results 3.3.4.4 CFSElo cells may help the CFSEhi cells in proliferation Having observed that the mixing of CFSElo cells with CFSEhi LN cells not only did not improve the disease but, in some circumstances, aggravated it, we wondered if the CFSElo cells had, instead of suppressing the functions of CFSEhi LN cells, helped the latter in their proliferation We therefore examined the rate of diminution of the CFSEhi cells after injected into an environment already repopulated with CFSElo cells Same as the approach employed in Experiment 3, 2×106 spleen cells were injected into the secondary recipient mice a week before the injection of 1×106 CFSEhi LN cells LN and spleen cells were isolated again from the secondary recipient mice on day and post-transplantation A brief comparison with LN cell isolated from mice injected with CFSEhi LN cell alone (Fig 13) showed that the percentage of non-dividing CFSEhi T cells in the mice injected with both CFSElo (in this case spleen cells) and CFSEhi cells was much lower (Fig 30) By day 7, CFSEhi T cells were hardly detected in the LNs of recipients injected with mixed cells (Fig 30), but there are still ~15% of CFSEhi T cells remain in the LNs of the mice injected with CFSEhi cells alone (Fig 13) This suggests that the CFSElo cells might promote the proliferation of the CFSEhi cells, thereby aggravated the disease However, more convincing approaches are required to confirm this hypothesis In summary, although the proliferating population was not anergic, they also did not exhibit the typical Treg cell characters either Most Treg cell surface markers are shared with T cell activation markers, making the identification and characterization of Treg under inflammatory condition (in this case GVHD) difficult Although cytokines such as IL-4 and IL-10 were detected in in vitro MLC supernatant in which unsorted spleen, LN or sorted CFSElo LN cells were used as responders, their secretion levels in secondary recipient serum was not detected Mixing CFSElo LN or 125 _ Results unsorted spleen cells with CFSEhi cells gave little, if any, protection against GVHD In fact, high number of CFSElo LN cells or pre-engrafted spleen cells, when co-injected with CFSEhi LN cell, seemed to aggravate the disease All these observations point to the fact that there is little possibility for Treg cell to exist in the early proliferating population The reason why the early proliferating cells failed to induce an acute disease needs to be elucidated Day post-transplantation LN Day post-transplantation LN Spleen Spleen 0.2% 53.0% 0.6% 23.9% 0.0% 53.5% 0.0% 13.5% 0.14% 20.8% 0.45% 12.4% 0.0% 21.3% 0.0% 18.2% 0.08% 40.1% 0.03% 14.3% 0.0% 41.1% 0.0% CD8PE CD4PE CD3PE 28.7% CFSE Fig.30 Rate of diminution of CFSEhi LN cells in the lymphoid tissues preengrafted with unsorted spleen cells 2×106 unsorted spleen cells from primary recipient mice were injected into the secondary recipient mice a week before the injection of 1×106 CFSEhi LN cells LN and spleen cells were isolated again from these secondary recipient mice on day and post-transplantation and analyzed with flow cytometry for the percentage of remaining CFSEhi LN cells Representative of independent experiments 126 _ Results 3.4 In Vivo Transfection and Expression in the Recipient Mice a Diphtheria Toxin390-IL-2 Recombinant Plasmid Ameliorates Graft-vshost Disease IL-2 is an essential growth factor to mediate T cells activation and proliferation The allogeneic donor T cells become activated and proliferate after adoptively transferred in the host, and a large percentage express a high level of IL-2 receptor (IL-2R) As have been shown in our previous study, these CD25+ T cells consist mainly activated alloreactive T cells that proliferate vigorously at the initial phase of GVHD and infiltrate the target organs Therefore, cytotoxic agents that could specifically eliminate this population of cells could possibly be employed as a therapeutic treatment in inhibiting GVHD Diphtheria toxin (DT) is a well studied glycoprotein consisting of 535 amino acids with a molecular weight of 58kDa (Nicholls et al., 1993) DT has a potent cell killing ability through ADP-ribosylation of elongation factor-2, resulting in inhibition of cellular protein synthesis and death of the cell Delivering a single DT molecule into the cytoplasm is sufficient to kill a cell (Yamaizumi et al., 1978) Native DT molecule contains three domains: the cell binding domain, the translocation domain, and the enzymatic cytotoxic domain (Yamaizumi et al., 1978; Middlebrook et al., 1980) The cell binding domain of the DT gene can be replaced by a growth factor gene, resulting in a toxin-growth factor hybrid gene, whose protein product is targeted to a specific growth factor receptor (Murphy et al., 1986; Williams et al., 1987; Chan et al., 1995; Lakkis et al., 1991; Chadwick et al., 1993; Jean et al., 1991) 127 _ Results DT390-IL-2 is a recombinant immunotoxin (IT) gene cloned onto a mammalian expression vector Its expression is controlled by a SR-α promoter upon being transfected into live mammalian cells Instead of purified IT proteins, our study will investigate the feasibility of delivering this DT390-IL-2 plasmid directly into the host cells and the ability of the transfected cells to express it, thereby eliminating IL-2R expressing alloreactive T cells 3.4.1 The construction and purification of DT390-IL-2 This is a project in collaboration with Dr Hu Huaizhong and Dr Feng Ping in University of Wisconsin, Madison, USA DT390-IL-2 plasmid was constructed by them Briefly, murine mRNA encoding for the matured IL-2 peptide was isolated from activated mouse lymphocytes, transcribed into cDNA, and ligated to the carboxyl terminus of DT390 gene, a truncated form of diphtheria toxin (Fig 31) The whole plasmid consists of 1620bps A large quantity of DT390-IL-2 plasmid was obtained by transformation into E coli followed by extraction with endotoxin-free maxi prep kit PCR with primers specific for IL-2 cDNA (Table 9) and restriction enzymatic digestion was used to ensure that the right clone was obtained (Fig 32) The isolated plasmid was used directly without being expressed into peptide 3.4.2 DT390-IL-2 showed potent cytotoxicity and specificity in eliminating activated T cells in vitro Unlike conventional IT studies which use purified protein, we transfected DT390-IL-2 plasmid directly into the target cells Therefore, the efficacy of the IT will largely be determined by its expression level from the transfected cells, and if it is expressed in a functional conformation 128 _ Results The in vitro characterization of DT390-IL-2 was performed by Dr Feng Ping The cytotoxicity of DT390-IL-2 against activated cells was first tested on PHA activated T cells Mononuclear cells were isolated from the spleen of mouse and stained with CFSE prior to their stimulation with PHA PHA is a potent T cell mitogen which stimulates the proliferation of nearly all T cells As expected, by day poststimulation, FACS analysis showed that >93% of total T cells were CFSElo, indicating that most T cells had undergone vigorous proliferation after the treatment with PHA The cells in the experimental group were transfected with DT390-IL-2 plasmid using an in vitro gene transfection kit These transfected cells consisted only 3.29% of proliferating CFSElo T cells on day post-stimulation, and only 150 days) than the control mice (50 days) (Fig 34b) One surviving mouse remained asymptomatic of GVHD throughout the observation period This result is encouraging since it suggests that the DT390-IL-2 plasmid could be used directly in the form of DNA molecule to transfect the host cells, and could be expressed in the functional conformation to kill the activated alloreactive T cells and thereby delaying or preventing GVHD Nonetheless, a single infusion of this plasmid, either on day or post-transplantation, did not confer any protection again the disease (data not shown), implying that the dosage, frequency and timing of treatment is important for this IT to be optimally effective Therefore, more in depth studies are required to optimize the in vivo expression of this IT, and confirm its expression using western blotting and ELISA 133 _ Results a) Liver cells b) Spleen cells Untransfected 1.37% 0.97% 19.66% 5.25% 27.87% 3.2% 20.54% 1.55% 10µg GFP plasmid 20µg GFP plasmid 30µg GFP plasmid GFP Fig.33 The expression of GFP in mice transfected with GFP reporter geneencoding plasmid using the in vivo transfection kit Different amount of GFPencoding plasmids were prepared as described by the manufacturer and infused into healthy SCID mice via tail vein The mononuclear cells from the liver and spleen of the treated mice were isolated 20hrs later to be analyzed with flow cytometry for the expression of green fluorescent protein Data represent independent experiments 134 _ Results a) % of change in body weight 110 105 100 3.1 95 DT-IP10 90 DT-IL2 85 MigPE 80 DT-RANTES 75 3.1 70 65 60 11 21 33 46 65 82 95 117 142 days post-transplantation b) 150 % change in body weight 140 130 120 DT-IL2 110 DT-IL2 100 DT-IL2 90 delivery sol 80 delivery sol 70 60 50 15 30 45 60 75 90 105 120 135 150 days post-transplantation Fig.34 Changes in the body weight of the recipient mice treated with DT390-IL-2 recombinant plasmid 10×106 spleen cells isolated from C57BL/6 mice were injected into each SCID recipient mice via tail vein to induce GVHD In the preliminary experiment (a), each recipient mouse was infused with 30µg of one of the several types of immunotoxin recombinant plasmids on day and post-transplantation using the in vivo transfection kit Two mice transfected with an empty mammalian expression vector, pcDNA3.1, served as controls In the experiment proper (b), each of the recipient mice were transfected with 30µg of DT390-IL-2 plasmid on day and post-transplantation Two mice infused with only the gene delivery solution served as controls 135 ... distinct chemokine expression pattern in the target organs that may contribute to the preferential recruitment of inflammatory cells into the liver and skin, but not into the heart, in acute GVHD... pathway In the recipients, T cells were activated and proliferated in the lymphoid system, and the effector T cells then migrated into the liver, skin and intestines which are the major target organs... cells in the control group but not the second batch of cells injected into the experimental group, indicating that proliferation of the CFSE-stained cells injected later was inhibited by the first