Published online before print August 28, 2007
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* Division of Hematology, Oncology and Blood and Marrow Transplantation, Department of Internal Medicine;
Department of Microbiology, Immunology and Molecular Genetics,
Department of Pathology and Laboratory Medicine; and
Graduate Center for Toxicology and
|| Lucille Parker Markey Cancer Center, University of Kentucky Medical Center, University of Kentucky, Lexington, Kentucky, USA
1Correspondence: Division of Hematology, Oncology and Blood and Marrow Transplantation, Markey Cancer Center, University of Kentucky, Lexington, KY 40535-0093, USA. E-mail: jsbrys{at}uky.edu
ABSTRACT
Syngeneic graft-vs.-host disease (SGVHD) develops in rodents following the treatment of lethally irradiated, bone marrow (BM) reconstituted animals with a short course of the immunosuppressive agent cyclosporine A (CsA). Using an in vivo depletion approach, we recently demonstrated that CD4+, but not CD8+, T cells participated in inducing SGVHD. Studies were therefore undertaken to adoptively transfer SGVHD into lethally irradiated, syngeneic BM reconstituted secondary recipients. Whole T cell populations as well as purified CD4+T cells isolated from SGVHD, but not normal or transplant control, animals mediated the transfer of SGVHD into secondary recipients. These cells have an apparent specificity for enteric bacterial antigens. The pathologic process that developed was identical to that observed in the animals with de novo SGVHD after syngeneic BMT and CsA therapy. It was shown that a radiation-sensitive mechanism prevented the transfer of SGVHD into normal, nonirradiated secondary recipients. The ability to reproducibly transfer SGVHD into secondary recipients will enhance our ability to study regulatory mechanisms that are altered during CsA therapy and permit the development of murine CsA-induced SGVHD.
Key Words: bone marrow transplantation • SGVHD • lymphoid cells • T cell proliferation • dendritic cells
INTRODUCTION
Cyclosporine A (CsA) is a T cell-specific [1 ] immunosuppressive agent that has been used both clinically [2 ] and experimentally [3 ] to prevent solid organ graft rejection and graft-vs.-host disease (GVHD) after bone marrow transplantation (BMT). Upon cessation of CsA treatment following syngeneic or autologous BMT, a disease similar to GVHD occurred in rats and mice [4 5 6 7 ]. This syndrome has been termed syngeneic GVHD (SGVHD) [4 ]. The SGVHD model in rats and mice has been used to study the development of self-reactive immune responses [8 , 9 ], the development of regulatory responses after immunosuppressive therapy [10 11 12 13 ], and the development graft-vs.-tumor responses after generation of this inducible syndrome [14 , 15 ]. Regulatory cells capable of modulating the induction as well as the adoptive transfer of SGVHD have been described in both the periphery and in the bone marrow of rats [9 10 11 , 16 ]. Both CD4+ [5 , 16 ] and CD8+ [5 , 9 , 16 ] effector T cells have been implicated in the induction and development of rat SGVHD with CD8+ cytotoxic T lymphocytes specific for the class II-associated invariant chain peptide, CLIP being the predominant effector cell [8 ].
Using in vivo cell depletion studies, development of murine SGVHD has been associated with the development of a CD4+, but not a CD8+, T cell response [17 ]. This Th1-driven immune response [18 , 19 ] develops after lethal irradiation, syngeneic BMT, and a short course of CsA therapy [6 , 7 , 13 , 20 ]. Histological analysis of tissue samples from SGVHD mice has demonstrated lymphocytic infiltration of several target organs, with increases in CD4+ T cells being observed in intestinal lesions [17 ]. In addition, CD4+ T cell clones isolated from SGVHD mice mediated footpad swelling when injected into irradiated syngeneic recipients [21 ]. Similarly, recent studies have demonstrated that increases in intraepithelial (IEL) and lamina propria (LPL) CD4+ T lymphocytes occur in the colons of SGVHD vs. control mice, and in vivo depletion of CD4+ but not CD8+ T cells during CsA therapy inhibits SGVHD [17 ]. These studies demonstrate that CD4+ T cells play a prominent role in the development of murine SGVHD. This was particularly interesting since CD4+ lymphocytes have been described to mediate the development of colitis in several strains of cytokine and T cell knockout and mutant mice [22 23 24 ], as well as in other forms of inducible colitis in mice [25 26 27 28 29 30 ].
Studies have shown that CD8+ but not CD4+ effector cells from diseased rats could transfer SGVHD in the rat model of this disease [10 , 11 , 31 ]. A radiation-sensitive host T cell resistance mechanism was present in normal animals that could inhibit the transfer of disease [10 , 11 ]. The conditions under which the adoptive transfer of murine SGVHD in mice can be mediated are less clear. Early studies by Bucy et al. [13 ] and Osman et al. [32 ] suggested that murine SGVHD could be transferred into irradiated secondary recipients using lymphoid cells from diseased animals, but the phenotype and the exact conditions under which this inducible disease could be transferred were not clearly delineated. In the current study we demonstrate that murine syngeneic SGVHD can be adoptively transferred with CD4+ T cells. Secondary disease was characterized by an inflammatory response in the colon and liver, with a prominent Th1 cytokine response being observed. The disease could be transferred into lethally irradiated, BM reconstituted syngeneic recipients, but not normal animals, demonstrating that active tolerance in normal animals prevented the transfer of SGVHD into normal animals. The development of SGVHD is a mutlifactoral process that involves altered immune regulation and the development of pathogenic effector cells that mediate intestinal inflammation and may also have anti-tumor potential. A complete understanding of the effector processes that mediate the development of SGVHD may allow for methodologies to enhance the graft-vs.-tumor responsiveness and provide a better understanding of the consequences of altered immune regulation after syngeneic bone marrow transplantation.
MATERIALS AND METHODS
Mice
Female C3H/HeN mice for use as BM donors or recipient mice were purchased from Harlan (Indianapolis, IN, USA) at 19-21 days of age and were used within 1 wk of arrival. Animals were housed in sterile microisolator cages (Lab Products, Maywood, NJ, USA) and were fed autoclaved food and acidified water ad libitum. All animal protocols were approved by the University of Kentucky Institutional Animal Care and Use Committee.
Induction of SGVHD
Bone marrow was isolated from the femurs and tibias of syngeneic age-matched mice. The donor BM suspensions were prepared in RPMI 1640 (Cellgro, Herndon, VA, USA) containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine (GIBCO, Grand Island, NY, USA). The BM cell suspension was depleted of Thy-1+ cells (ATBM) using anti-Thy1.2 (HO-13-4) and Low Tox M rabbit complement (Cedarlane Laboratories, Westbury, NY, USA), as described previously [12
]. Recipient mice were lethally irradiated (900 cGy) in a Mark I 137Cs irradiator (J. L. Shepard and Associates, Glendale, CA, USA). After irradiation, the animals were reconstituted i.v. with 5 x 106 ATBM 4-6 h after conditioning. Beginning on the day of BMT, the mice were treated daily i.p. for 21 days with 15 mg/kg/day of CsA or the diluent olive oil (Sigma-Aldrich, St. Louis, MO, USA). Upon cessation of CsA therapy, BMT control and CsA-treated animals were weighed three times a week and monitored for the development of clinical symptoms of SGVHD (weight loss, diarrhea). Animals that developed clinical symptoms for three consecutive weighings were considered positive for the induction of SGVHD. Clinical symptoms were typically observed by 2-3 wk after the end of CsA therapy.
Isolation of peripheral lymphoid cells and adoptive transfer of SGVHD
At 2-4 wk after cessation of CsA therapy, normal, BMT control, and CsA-treated SGVHD mice exhibiting clinical symptoms of disease (typically 60-80% of CsA-treated animals) were euthanatized, and the spleen and mesenteric lymph nodes were removed and placed into RPMI 1640 containing penicillin/streptomycin/glutamine and 5% FBS (Atlanta Biologicals, Inc., Norcross, GA, USA). Single-cell suspensions were prepared from pooled spleen and MLN within each group, and the RBC were lysed by treatment with 0.83% Tris-buffered NH4CL. The donor cell suspensions were placed into aliquots for flow cytometric analysis (see below). The spleen and MLN cells were then pooled together within each treatment group. In initial experiments, T cells were enriched by passage over nylon wool columns prior to transfer in secondary recipient animals. CD4+ or CD8+ T cell subsets were positively selected using MACS magnetic beads, with the cells being selected on an AutoMACS system (Miltenyi Biotech, Auburn, CA, USA). The purity of the MACS-isolated donor cells was monitored by flow cytometry and found to be >92% CD4+ or > 85% for CD8+ T cells. After isolation, the cells (CD4+or CD8+) were injected i.v. in 100 µl of PBS into lethally irradiated (900 cGy), ATBM-reconstituted secondary C3H/HeN recipient mice. The animals were then monitored for the development of secondary SGVHD as described for the primary disease.
T cell proliferation against cecal antigens
A cecal antigen preparation was prepared according the procedure described by Cong et al. [33
]. Briefly, the ceca were removed from animals and placed in a Petri dish containing PBS. The cecal contents were removed and collected by low-speed centrifugation. The contents were resuspended in PBS containing DNase I (10 µg/ml) (Sigma-Aldrich). In addition, glass beads equal to 1/5th the volume of the material in the tube were added to the cecal content preparation. The preparation was sonicated for 5 min (15 s on, 15 s off) in a Fisher 550 Dismembrator (Thermo Fisher, Waltham, MA, USA). After sonication, the antigen preparation was centrifuged for 15 min at 10,000 rpm in a Fisher accuSpin microcentrifuge (Thermo Fisher) to remove debris. The supernatant was removed, filter sterilized, and analyzed for protein content by Bio-Rad assay (Hercules, CA, USA).
Bone marrow-derived dendritic cells (DC) were generated by culturing C3H/HeN bone marrow cells in RPMI 1640 containing 5% FCS, penicillin/streptomycin/glutamine, and 5 mM 2-ME containing 20 ng/ml of recombinant murine GM-CSF for 8-10 days. Spleen cells were isolated from normal transplant control or SGVHD animals, and T cells were enriched by passage over nylon wool columns. CD4+ T cells were prepared as described above. 1-2 x 105 splenic T cells or CD4+ T cells were cultured in 96-well, flat-bottomed microtiter plates with 4 x 105 irradiated splenic antigen-presenting cells (APC) or 1 x 104 DC. Antigen-pulsed splenic APC (4x106/ml) or DC (1-2x106/ml) were incubated with cecal antigen (200 µg/ml) overnight, then washed and irradiated with 2000 cGy gamma irradiation. As a control, syngeneic APC were treated with LPS (5 µg/ml) or pI:C (25 µg/ml) overnight as described above. Proliferation was measured by the addition of [3H]-thymidine during the last 18 h of a 96 h culture.
Isolation of lymphoid cells from secondary recipients
At the indicated times after adoptive transfer, secondary recipients of control and SGVHD T cells were euthanatized and the spleen, MLN, and colon were removed. Single-cell suspensions were prepared from the peripheral lymphoid organs, as described above. IEL were isolated from the colon according to a modification of the method of Lefrancios and Lycke [34
] as described [17
]. The resulting cell population was counted and analyzed by flow cytometry (see below).
Flow cytometry analysis
Lymphoid cells isolated from the periphery of donor animals or from the periphery and intestinal tract of secondary recipients were placed into staining buffer (PBS containing1% FBS, 0.1% NaN3). To minimize nonspecific staining, cells were incubated with an antibody (Ab) against CD16/CD32(2.4G2, Fc Block®, BD PharMingen, San Diego, CA, USA). The cells were then stained with fluorochrome-conjugated monoclonal antibody (mAb) against lymphoid surface markers, CD4 (RM-4-5), CD8 (CT-CD8
) (Caltag), and CD62L (MEL-14) (BD PharMingen). These mAb were used for two- and three-color analysis using a BD Biosciences FACSCalibur flow cytometer (San Jose, CA, USA).
Histologic analysis of secondary SGVHD
Tissues were removed from euthanatized animals at the indicated times after adoptive transfer and placed into 10% buffered formalin. The fixed tissues were embedded in paraffin, cut into sections of 4-6 µm, mounted onto glass slides, and stained with a standard H&E protocol. All slides were analyzed blind without knowledge of the treatment group and were graded for inflammation according to a published grading scale [14
].
Analysis of cytokine gene expression by real-time PCR
Total RNA was isolated from the colons using Trizol reagent (Invitrogen, Grand Island, NY, USA). RNA (1 µg) from each group was reverse-transcribed into cDNA using the Promega (Madison, WI, USA) reverse transcription system. cDNA was suspended in 1x master mix (0.5U Platinum Taq (Invitrogen), 0.2 nM of each dNTP, 0.2 mM PCR buffer (Idaho Technology, Inc., Salt Lake City, UT, USA), and 1x SYBR green (Molecular Probes, Eugene, OR, USA). The reaction volume was made to 10 µl with ddH20. Primers for IL-12, IFN-
, TNF- [35
], and GADPH [36
] were purchased from Integrated DNA Technologies (Coralville, IA, USA) and used at 1 µM concentration. Real-time PCR was performed on a Roche Lightcycler (Roche Diagnostics, Indianapolis, IN, USA). Reaction conditions were 5 min at 95°C, followed by 50 cycles of 30 s at 95°C, 30 s at 60°C, and 30 s at 72°. The amount of IL-12, IFN-, and TNF- was normalized to GADPH as calculated by the comparative CT method.
Statistical analysis
Statistical differences between groups were determined using a Student’s t test or Fisher’s exact test (induction). Differences of 
0.05 were considered statistically different.
RESULTS
Adoptive transfer of SGVHD with T cells isolated from SGVHD but not control animals
Earlier studies have demonstrated the ability to adoptively transfer rat and murine SGVHD into irradiated secondary recipient animals with lymphoid cells from diseased animals [4
, 10
, 11
, 13
, 32
]. However, the nature of the effector cells for the transfer of murine SGVHD has not been identified clearly. Initial experiments were therefore performed to determine the ability of T cells to adoptively transfer murine SGVHD.
To induce SGVHD, mice were lethally irradiated, reconstituted with syngeneic ATBM, and treated with a short course of CsA therapy. At 2 to 4 wk after CsA therapy, lymphoid cells were isolated from the spleen and MLN of control (BMT, diluent treated) and diseased animals. As described before [17
], T cells isolated from diseased animals were skewed to an activated phenotype, with 
75% of the peripheral T cells being CD62L– vs. <25% of T cells from control animals (data not shown).
It has been shown in the rat SGVHD model that T cells isolated from diseased animals were reactive against peptides from the CLIP protein [8 ]. However, the antigenic specificity of the T cells responsible for the induction of SGVHD in the mouse had not been determined. In murine models of colitis, CD4+ T cells responsible for the induction of intestinal inflammation have been shown to have reactivity against intestinal bacterial antigens [33 , 37 ]. Given these findings and the fact that the colon is a primary target organ of murine SGVHD, preliminary studies were undertaken to determine whether T cells from SGVHD mice would respond against an antigen preparation obtained from the cecal contents of normal syngeneic animals. As shown in Fig. 1 , both NWC-enriched T cells or highly enriched CD4+ isolated from diseased animals mounted a significant response to antigen-pulsed APC. There was a minimal response of T cells isolated from BMT control animals against the antigen-pulsed APC. To further address the specificity of these cells and ensure that the cells were not responding against activated APC, T cells from control and SGVHD animals were placed in culture with splenic APC-treated cells overnight with LPS or pI:C. These agents activate APC through TLR 4 and TLR 2, respectively. Control or SGVHD T cells did not respond against LPS-treated APC, demonstrating that the cells were responsive against Ag present in the cecal antigen preparation and not responsive to activated syngeneic APC (data not shown). Thus, preliminary evidence suggests that SGVHD effector cells respond against bacterial antigens present in the colon.
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Figure 1. T cells from SGVHD respond against intestinal bacterial antigens. (A) Spleen cells were isolated from normal transplant control or SGVHD animals and T cells were enriched by passage over nylon wool columns 2-3 wk after cessation of CsA. 1-2 x 105 T cells were cultured in 96-well, flat-bottomed microtiter plates with 4 x 105 irradiated antigen-pulsed splenic APC. Proliferation was measured by the addition of [3H]-thymidine during the last 18 h of a 96 h culture. Data are pooled from 2 separate experiments representing 2 controls and 4 CsA-treated animals. **Statistical difference between SGVHD and control Ag-treated cells, P = 0.0001. (B) Spleen cells were isolated from normal transplant control or SGVHD animals, and CD4+ T cells were purified from nylon wool-enriched T cells by Miltenyi bead purification. Purified CD4+ T cells (1x105) were cultured in 96-well, flat-bottomed microtiter plates with 1 x 104 antigen-pulsed DC. Proliferation was measured by the addition of [3H]-thymidine during the last 18 h of a 96 h culture. *Significant difference between SGVHD and control CD4+ cells, P = 0.013.
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Figure 2. T cells from SGVHD mice transfer disease. (A) T cells were isolated from the spleen and MLN of transplant control or SGVHD animals 2-3 wk after cessation of CsA therapy. 1 x 107 T cells were transplanted with 3-5 x 106 ATBM into lethally irradiated, syngeneic animals. As a control, additional mice were transplanted with ATBM alone. The mice were observed for the development of clinical symptoms of SGVHD. Data presented represent pooled data from 3 transfer experiments, with the number of animals with secondary SGVHD/total number of animals within each group presented above each bar. *Statistically different from ATBM and control T, P < 0.0001. Adoptive transfer of SGVHD by CD4+ T cells. (B) CD4+ T cells were purified from the spleen and MLN of normal transplant control and SGVHD mice. Varying numbers of CD4+ T cells were transferred with ATBM into lethally irradiated syngeneic secondary recipient animals. The secondary recipients were monitored for the development of SGVHD. Data presented represents pooled induction data from 2 experiments, with the number of animals with secondary SGVHD/total number of animals within each group presented above each bar. *Statistically different from 2 x 106 control BMT CD4+, P = 0.0183; **statistically different from 1 x 106 control BMT CD4+P = 0.0002. (C) Secondary recipient mice of 1 x 106 control BMT or SGVHD CD4+ T cells were weighed individually at times after adoptive transfer. Data presented are the mean ± SE of weights for each treatment group and are representative of 4 individual experiments. Control BMT CD4+, n = 5; SGVHD CD4+, n = 7. *Statistically different from control BMT CD4+, P 0.05.
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To monitor the relative severity and duration of the secondary disease, animals were weighed longitudinally after adoptive transfer of CD4+ T cells from diseased and BMT control animals (Fig. 2C)
. Beginning 3 wk after transfer, secondary recipients of CD4+ T cells from SGVHD mice lost weight at a significantly higher rate (P0.05) than those injected with control CD4+ T cells. Finally, preliminary studies demonstrated that purified CD8+ T cells isolated from diseased animals did not transfer SGVHD to secondary recipients at a rate different from that of CD4+ T cells isolated from BMT control animals (data not shown). These data established that highly purified CD4+ T cells mediated the adoptive transfer of murine SGVHD.
Immunosuppression of 2° recipients was required for adoptive transfer of SGVHD
In published studies in rat and mouse, SGVHD could not be adoptively transferred into normal recipients [10
, 11
, 13
]. It was proposed that radiation conditioning was required to eliminate a radiation-sensitive T regulatory cell. With this in mind, the ability of CD4+ T cells from SGVHD animals to induce disease in normal nonirradiated animals was investigated. CD4+ T cells from diseased animals were able to induce disease in a high proportion of secondary recipients relative to CD4+ T cells isolated from transplant control animals (Fig. 3
). In contrast, T cells from diseased animals were unable to induce disease in normal, unirradiated secondary recipients as has been previously demonstrated in the rat model of SGVHD (P=0.0010) [10
, 11
, 13
]. This is consistent with the presence of a radiation-sensitive regulatory cell in normal animals that inhibits the activation of the T cells from diseased animals in the secondary recipients, and is also in line with published data demonstrating that animals that received SGVHD T cell clones had to be irradiated in order for in vivo responses of these clones to be observed [21
].
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Figure 3. Failure to transfer SGVHD into normal, nonirradiated secondary recipients. 1 x 106 CD4+ T cells from control (data not shown) or SGVHD animals were transferred into irradiated or normal secondary recipients. The animals were monitored for development of secondary SGVHD, as described. Data presented represents pooled induction data from 2 experiments with the number of animals, with secondary SGVHD/total number of animals within each group presented above each bar. Secondary disease was not observed when CD4+ T cells from transplant control animals were injected into irradiated secondary recipient animals. *Statistically different from normal, nonirradiated recipients, P = 0.0010.
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2-fold increase was observed in the CD4+ IEL isolated from recipients that received NWC-enriched T cells or CD4+ T cells from diseased animals relative to those isolated from transplant control (ATBM) or ones that received T cells from control animals (P=0.0152; pooled data from three experiments) (Fig. 4
). No significant changes were observed in the CD4+ or CD8+ T cell populations when peripheral lymphoid organs of the secondary recipients were analyzed (data not shown)
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Figure 4. Increased CD4+ T cells in the IEL isolated from SGVHD secondary recipients. Pooled IEL were isolated 8 wk after transfer from 4-7 secondary recipient animals that received 1 x 107 whole T cells from transplant control or SGVHD mice were used as donor cells for transfer (experiment 1) or 1 x 106 CD4+ T cells from transplant control or SGVHD animals (experiments 2 and 3). The cells were stained with mAb against β TCR, CD4, and CD8, then analyzed by flow cytometry. Data presented represent data from 3 separate transfer experiments. CD4+ IEL from SGVHD significantly increased compared with Control CD4+ IEL, pooled data from the 3 experiments, P = 0.0152 (far right data cluster).
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Figure 5. Colon pathology characteristic of SGVHD is present in the colons of secondary recipients of SGVHD T cells. Colon tissue was isolated during clinical symptoms of secondary disease, typically 35-50 days after transfer of CD4+ T cells from transplant control or SGVHD mice. Tissues from animals with primary SGVHD were isolated 3-4 wk after CsA therapy. Tissues were stained using H&E staining procedures, photographed at x200 magnification, and were representative of tissues from each group. For comparison, tissues from transplant control and mice with SGVHD are included. (A) transplant control colon, primary transplant; (B) colon from primary SGVHD animal; (C) colon from secondary recipient that received CD4+ T cells from control animals; (D) colon from secondary recipient of SGVHD CD4+ T cells. The inserts represent the average pathology grade [14
] for tissues isolated from secondary recipients of ATBM (A) or CD4+ T cells from transplant control (C) or SGVHD animals (D). Colon samples from recipients of SGVHD T cells (D) were statistically different from the colon isolated from animals that received control BMT CD4+ T cells (C), P < 0.0001 (n=10–13).
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Figure 6. Liver pathology in secondary recipients of CD4+ T cells from SGVHD mice. Liver tissue was removed from animals with secondary disease. H&E-stained tissues were photographed at x200 magnification and were representative of tissues from each group. (A) Liver from transplant control, primary transplant; (B) liver isolated from primary SGVHD animal demonstrating inflammation in the portal triad (arrow); (C) section of liver from secondary recipient of control CD4+ T cells; (D) liver from secondary recipient of CD4+ T cells from SGVHD mice demonstrating inflammation in the portal triad (arrow). The inserts represent the average pathology grade [14
] for liver samples isolated from secondary recipients of ATBM (A) or CD4+ T cells from transplant control (C) or SGVHD animals (D). Tissue isolated from recipients of SGVHD T cells (D) was statistically different from liver isolated from animals that received control BMT CD4+ T cells (C), P < 0.0001 (n=10–13).
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, IFN-, and IL-12) in the colon has been associated with the development of murine CsA-induced SGVHD [18
, 19
]. Based on the colonic inflammation observed after adoptive transfer of SGVHD, the levels of IFN-, TNF-, and IL-12 mRNA were monitored by real-time PCR in the colons of secondary recipient animals. Similar to animals with de novo SGVHD, significant increases in mRNA for TNF- (Fig. 7A
; P=0.0014) and IFN- (Fig. 7B
; P=0.009) were observed in the colons from animals that received CD4+ T cells from SGVHD animals relative to those that received CD4+ T cells from control mice. Consistent but not significant increases in mRNA for IL-12 were observed as well (data not shown).
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Figure 7. Adoptive transfer of SGVHD results in the increased production of inflammatory cytokines in the colon. mRNA was isolated from the colons of secondary recipients of 1 x 106 donor CD4+ cells from transplant control or SGVHD animals. Real-time PCR was performed for TNF- (A) and IFN- (B). Data presented are pooled from 2 experiments. Data presented are the mean ± SE, with n representing the number of animals analyzed in each group pooled from 2 separate experiments. *Statistically different from control BMT CD4+, P = 0.0014; **Statistically different from control BMT CD4+, P = 0.009.
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Syngeneic GVHD is an inducible syndrome that develops after lethal irradiation, syngeneic BM transplantation, and a short course of CsA therapy. We have demonstrated here for the first time that murine SGVHD can be transferred with CD4+ T cells from diseased animals into irradiated but not normal secondary recipients. Effector cells isolated from diseased animals mounted a significant in vitro response against colonic bacterial antigens. Similar to the primary disease that develops after BMT and CsA therapy, mice with secondary disease had inflammatory lesions in the liver and colon as well as increased mRNA for the inflammatory cytokines IFN- and TNF-.
Studies have demonstrated that T cells from SGVHD mice could adoptively transfer disease into irradiated secondary recipients [13 , 32 ]. Bucy et al. have shown that the elimination of Thy1+ cells from the donor cells used for adoptive transfer eliminated the transfer of disease, suggesting the involvement of Thy1+ cells in primary and secondary disease [13 ]. Similarly, Osman et al. demonstrated that the transfer of T cells with intermediate expression of the T cell receptor resulted in secondary disease, particularly in the liver [32 ].
Using in vivo depletion strategies, CD4+ (but not CD8+) were shown to be involved in the development of murine SGVHD after lethal irradiation, syngeneic BMT, and CsA therapy [17
]. Given these findings, highly purified CD4+ from the peripheral lymphoid organs from SGVHD animals induced secondary disease in up to 100% of the secondary recipient animals. After adoptive transfer, significant increases in mRNA for the TH1/inflammatory cytokines IFN- and TNF- were found in the target tissues of SGVHD. It was previously demonstrated that a significant increase occurred in the percentage of CD4+ T cells in the intestinal epithelia and lamina propria of animals with SGVHD after BMT and CsA therapy [17
]. Similar results were obtained when intestinal epithelial lymphocytes were analyzed after adoptive transfer of SGVHD, where an increase of CD4+ but not CD8+ T cells was found in the intestinal epithelium. Thus, based on several criteria, the disease that developed after the adoptive transfer of T cells from the periphery of SGVHD animals mimics that which is observed after the de novo development of disease after lethal irradiation, BMT, and a short course of CsA therapy.
The predominant inflammatory responses that occur in the rat model of SGVHD are found in the skin, the major effector cell being a CD8+ T cell [4 , 9 , 31 ] responsive to the CLIP peptide [8 ]. The major target organ for murine SGVHD is the colon, with the CD4+ T cell mediating disease [7 , 17 , 20 ]. It was shown for the first time that CD4+ T cells isolated from SGVHD animals had increased responsiveness to syngeneic APC pulsed with a cecal antigen preparation compared with T cells isolated from control animals. In relation to effector cells, target tissues, and stimulating antigens, significant differences have been defined between rat and murine SGVHD. It is interesting to note the similarities in the effector cells and pathology associated with many murine models of inducible colitis and those observed with murine SGVHD. In some murine models of colitis, a significant CD4+ response occurs against bacterial antigens [33 , 37 ] similar to the preliminary studies presented in this paper. Similarly, preliminary data suggested that CD4+ T cells reactive against intestinal bacterial antigens may participate in the development of murine SGVHD. Alternatively, APC activated via toll-like receptors (TLR) [38 ] could be responsible for enhanced stimulation of autoreactive CD4+ T cells present in SGVHD animals, as shown in the autoimmune T cell responses in TNBS-induced colitis [39 ]. Through engagement of TLR by TLR ligands present in the cecal antigen preparations, the APC would up-regulate MHC class II and costimulatory molecules as well as proinflammatory cytokine production, resulting in enhanced proliferation of T cells from SGVHD animals. However, in the current study when APC were treated with individual TLR ligands, LPS (TLR4), or pI:C (TLR3), enhanced SGVHD T cell activation did not occur (data not shown). It is not known whether multiple TLR signaling was required to activate APC to the appropriate level to stimulate T cells, as could occur in vivo. It should be noted that SGVHD was inducible in LPS hyporesponsive C3H/HeJ mice [19 ], demonstrating that TLR4 signaling was not required for SGVHD induction. Thus, whether SGVHD-induced colon pathology is due to CD4+ T cell responsiveness to microbial antigens or due to APC activation via commensal bacteria, resulting in autoimmune CD4+ T cell stimulation, is unclear at this time and will require additional study to sort out. However, while the inflammatory responses in the colon appear to be similar, there are differences that suggest that the murine colitis and SGVHD models may ultimately be different. After induction of SGVHD, inflammatory responses are found in the liver of virtually all of the diseased animals, whereas liver inflammation was observed in < 50% of colitic mice after adoptive transfer of naive T cells into scid mice [40 ]. These findings suggest that different effector cells may be responsible for the development of liver inflammation during SGVHD, as suggested by Osman et al. [32 ].
Although murine SGVHD has many pathogenic similarities to spontaneous and inducible models of colitis in the mouse, regulatory mechanisms that modulate the adoptive transfer of rat SGVHD appear to be similar to those in the mouse. The adoptive transfer of murine SGVHD required that the secondary recipient be immunosuppressed and could not be transferred into normal animals. It was previously demonstrated that recipients of SGVHD T cell clones had to be irradiated with 600 rads before transfer to demonstrate in vivo reactivity of the cells. Similar findings were observed in the rat model in that a radiation-sensitive host resistance mechanism had to be eliminated, with a minimum of 750 rads to allow for transfer of rat SGVHD [10 , 11 ]. Whereas the phenotype of the regulatory population(s) present in normal mice that inhibit the transfer of SGVHD into normal animals is unknown, Thy1+ BMT cells have been shown to regulate the development of murine SGVHD after lethal irradiation and CsA therapy [12 ]. In these experiments, removal of these cells allowed for the induction of SGVHD in resistant strains (C57BL/6) and enhanced the disease in inducible strains (C3H/HeN). Alternatively, preliminary unpublished studies have suggested that purified cells from normal syngeneic animals with the prototypic regulatory T cell phenotype CD4+CD25+ reduced the induction of secondary SGVHD when cotransferred with CD4+ T cells from diseased mice. Finally, the potential exists that conditioning of secondary recipients is required to create space to allow for expansion of the transferred T cells. The adoptive transfer model presented here will be crucial in unraveling the effects of CsA and other calcineurin inhibitors on immune regulation after BMT and the induction of SGVHD.
ACKNOWLEDGEMENTS
This work was supported by grant PO1-CA092372 (JSB).
Received March 20, 2007; revised August 2, 2007; accepted August 6, 2007.
REFERENCES
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