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The Journal of Immunology, 2002, 168: 3205-3212.
Copyright © 2002 by The American Association of Immunologists

Donor T Cell Activation Initiates Small Bowel Allograft Rejection Through an IFN-{gamma}-Inducible Protein-10-Dependent Mechanism1

Zheng Zhang*, Levent Kaptanoglu*, Wael Haddad{dagger}, David Ivancic*, Ziad Alnadjim{dagger}, Stephen Hurst{ddagger}, Darren Tishler*, Andrew D. Luster§, Terrence A. Barrett2,{dagger} and Jonathan Fryer2,3,*

Departments of * Surgery and {dagger} Medicine and Microbiology/Immunology, Northwestern University Medical School, Chicago, IL 60611; {ddagger} DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304; and § Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy and Immunology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The poor success in controlling small bowel (SB) allograft rejection is partially attributed to the unique immune environment in the donor intestine. We hypothesized that Ag-induced activation of donor-derived T cells contributes to the initiation of SB allograft rejection. To address the role of donor T cell activation in SB transplantation, SB grafts from DO11.10 TCR transgenic mice (BALB/c, H-2Ld+) were transplanted into BALB/c (isografts), or single class I MHC-mismatched (Ld-deficient) BALB/c H-2dm2 (dm2, H-2Ld-) mutant mice (allografts). Graft survival was followed after injection of control or antigenic OVA323–339 peptide. Eighty percent of SB allografts developed severe rejection in mice treated with antigenic peptide, whereas <20% of allografts were rejected in mice treated with control peptide (p < 0.05). Isografts survived >30 days regardless of OVA323–339 administration. Activation of donor T cells increased intragraft expression of proinflammatory cytokine (IFN-{gamma}) and CXC chemokine IFN-{gamma}-inducible protein-10 mRNA and enhanced activation and accumulation of host NK and T cells in SB allografts. Treatment of mice with neutralizing anti-IFN-{gamma}-inducible protein-10 mAb increased SB allograft survival in Ag-treated mice (67%; p < 0.05) and reduced accumulation of host T cells and NK cells in the lamina propria but not mesenteric lymph nodes. These results suggest that activation of donor T cells after SB allotransplantation induces production of a Th1-like profile of cytokines and CXC chemokines that enhance infiltration of host T cells and NK cells in SB allografts. Blocking this pathway may be of therapeutic value in controlling SB allograft rejection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Small bowel (SB)4 transplantation is a potential solution for individuals with intestinal failure (1, 2). However, a poor survival rate limits the clinical usefulness of this technology. Rejection has been a significant impediment to extending SB allograft survival. SB allograft rejection is associated with the production of both Th1 and Th2 cytokines (IFN-{gamma}, TNF, IL-4, and IL-5) (3). Both CD4+ and CD8+ T cell subsets are required for the development of SB allograft rejection (4). Compared with other solid organ allografts, a higher degree of immunosuppression is required to prevent SB allograft rejection (5, 6), and rejection episodes occur despite aggressive treatment. It has been suggested that difficulties encountered in controlling SB allograft rejection are due to the unique environment of the mucosal immune system (7, 8).

In the intestinal immune system, T cells respond to Ags derived from commensal and pathogenic enteric bacteria. Studies from our lab and others indicate that both APC as well as T cells localized to the intraepithelial and lamina propria (LP) compartments express surface and functional phenotypes consistent with their activated state, and are maintained as activated populations poised to deliver rapid effector immune responses (9, 10). Studies by Harper et al. (11) suggest that compared with splenic populations, LP APCs induce relatively high levels of IFN-{gamma} and TGF-{beta} from responding T cells in the absence of other cytokines (IL-2 and IL-5). The induction of IFN-{gamma} by enteric Ag may increase mucosal permeability as well as enhance local responses by up-regulating expression of class I and II MHC and costimulatory molecules (12, 13). Based on these data, we postulated that the unique environment of the mucosal immune system contributes to the relatively poor success rate of SB allograft transplantation.

Acute SB allograft rejection is characterized by massive lymphocyte infiltration and extensive epithelial cell apoptosis, followed by mucosal destruction (8, 14). Recent data from several models of organ transplantation (skin, heart, kidney, and lung) (15, 16, 17, 18) suggest that recruitment of host leukocytes into the allograft involves chemokine-mediated pathways. IFN-{gamma}-inducible CXC chemokines such as IFN-{gamma}-inducible protein-10 (IP-10) and monokine induced by IFN-{gamma} (Mig) are important in Th1-like immune responses, including those associated with acute allograft rejection (19, 20, 21). Data from Hancock et al. (22) suggest that production of IP-10 by donor rather than infiltrating host cells is required for the initiation of cardiac allograft rejection. In complementary studies, CXCR3 expression by host cells was shown to be required for rejection of cardiac allografts (23). While no studies evaluating the role of chemokines in SB allograft rejection have been performed, the SB LP is a potentially rich source of IP-10 and other IFN-{gamma}-inducible chemokines (24). Therefore, we considered the possibility that activation of large numbers of graft-derived T cells may induce IFN-{gamma}-inducible chemokines and contribute to the aggressive SB allograft rejection.

Initially, the role of T cell activation in SB tissues was examined. We found that T cell activation induced rapid production of both proinflammatory cytokines as well as CXC and CC chemokines. This result led us to hypothesize that donor T cell activation enhances allograft rejection by inducing local production of CXC chemokines. Using a mouse-vascularized SB transplant model, we show that activation of donor CD4+ T cells by foreign Ag triggered rejection of class I MHC-mismatched SB allografts. Furthermore, our data indicate that neutralization of IP-10 significantly prolonged SB allograft survival following donor T cell activation. Taken together, results from these studies suggest that donor T cell activation enhances SB allograft rejection by inducing local production of CXC chemokines that promote infiltration of host leukocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Male BALB/c (H-2d, Ld+) and BALB/c-H-2dm2 mice (dm2, Ld-) were purchased from The Jackson Laboratory (Bar Harbor, ME). The dm2 mice are BALB/c mutants that lack class I Ld Ag (25). DO11.10 mice (26), expressing a V{alpha}13/V{beta}8.2 TCR specific for a class II MHC-restricted peptide of chicken OVA (OVA323–339) in >85% of the CD4+ T cells (a gift from D. Loh, Nippon Research Center, Kamakura, Japan), were backcrossed to BALB/c (H-2d, Ld+) mice for over 14 generations, and the progeny were screened for expression of the TCR transgene by flow cytometry using the clonotypic mAb, KJ1-26.1 (Caltag Laboratories, South San Francisco, CA). All strains were housed in a barrier facility at Northwestern University Medical School (Chicago, IL) and were used at 8–12 wk of age.

Quantitation of RNA transcripts by real time RT-PCR

Real time RT-PCR assays were used to specifically quantitate mouse cytokine and chemokine transcripts. Total cellular RNA was extracted and samples were heated at 95°C for 10 min, chilled, and reverse transcribed with Superscript II reverse transcriptase (Life Technologies, Rockville, MD) with random hexamers and oligo(dT). Equivalent amounts of individual cDNA from similarly treated mice were combined to create pooled samples for real time RT-PCR. Primers were either obtained from PerkinElmer/Cetus (Norwalk, CT) or generated with Primer Express software (PerkinElmer/Cetus) and synthesized in our core facility. Whenever possible, primer pairs were designed to span intron/exon borders. Samples were subjected to 40 cycles of amplification of 95°C for 15 s, followed by 60°C for 1 min using a Geneamp 5700 sequence detection system (PE Applied Biosystems, Foster City, CA) and SYBR green buffer. PCR amplification of the housekeeping gene ubiquitin was performed for each sample to control for sample loading and to allow normalization between samples. Both water and genomic DNA controls were included to insure specificity. Each data point was examined for integrity by analysis of the amplification plot and disassociation curves. The ubiquitin-normalized data were expressed as the fold induction of gene expression in treated mice compared with that in control mice.

Murine SB transplantation

Intestinal transplantation was performed, as described previously (27), with minor modifications. Briefly, small intestines were isolated from donors and perfused in situ with cold heparinized lactated Ringer’s solution. End-to-side anastomoses were performed between donor and recipient aorta and donor portal vein and recipient inferior vena cava. The proximal end was exteriorized as a stoma, while the distal end was anastomosed, end to side, to the recipient intestine. Recipient mice were evaluated twice daily for clinical evidence of rejection, including increased mucus output, stomal necrosis, and palpable abdominal mass. Mice with clear clinical evidence of rejection were sacrificed, and healthy mice were followed for 30 days and sacrificed. Mice that died within 4 days after transplantation were considered technical failures (<15%) and excluded from analysis. In some cases, grafts were removed on postoperative days (POD) 5, 7, 8, and 14 for evaluation of tissue histology, cell apoptosis, and intragraft cytokine expression. Cells were isolated from LP, mesenteric lymph nodes (MLN), and spleen, and analyzed by flow cytometry.

Activation of DO11.10 transgenic donor cells

Control (myelin basic protein) and OVA323–339 peptides were synthesized in the Northwestern molecular biology core facility using a PE Applied Biosystems Synergy Peptide Synthesizer with purity assessed by HPLC and mass spectroscopy. To activate T cells, OVA323–339 (200 µg/mouse) was administered to BALB/c or dm2 mice with DO11.10 SB transplants on POD 5, 6, and 7, via i.p. injection.

In vivo blocking of IP-10 following SB transplant

Hamster anti-mouse IP-10 mAb (1F11) (21) and control hamster IgG (UC8-1B9; American Type Culture Collection, Manassas, VA) were grown in serum-free medium and then purified over protein G columns using standard techniques. For in vivo neutralization of IP-10 in SB transplants, 100 µg IP-10 mAb was administered on POD 5 and every other day thereafter until sacrifice. For all mAbs and peptides, endotoxin levels were tested using the Limulus amebocyte assay (BioWhittaker, Walkersville, MD) and were <0.1 endotoxin U/ml.

Pathologic evaluation of the intestinal graft

At necropsy, a section of each graft was fixed in 10% buffered formaldehyde, embedded in paraffin, and then stained with H&E for routine pathology. Microscopic sections were examined blindly by a pathologist using previously established criteria (8, 28). Briefly, slides were assessed for mononuclear cell infiltration, villous atrophy and sloughing, epithelial erosion, and crypt apoptosis. Scores of 0 (no change), 1 (focal), or 2 (diffuse) were assigned for each feature of acute rejection. After assignment of scores, the overall severity of rejection was based on the total score and designated as follows: <2, no rejection; 2–5, mild rejection; 6–10, moderate rejection; 11–14, severe rejection.

Isolation of cells

Lamina propria. LP lymphocytes were isolated as described (29). Briefly, small intestines were extracted and rinsed with cold PBS. After Peyer’s patches were removed, tissues were minced and epithelial cells were liberated by incubation with 5 mM EDTA and 10% newborn calf serum (Life Technologies). Mucosal pieces were washed, returned to digestion flasks, and incubated six times for 30 min with collagenase (100 U/ml; Sigma-Aldrich, St. Louis, MO). Cells released into the supernatant were washed and kept overnight before separation on a Nycoprep 1.077 gradient (Accurate Chemical, Westbury, NY).

Mesenteric lymph nodes. Nodes were mechanically dissociated and fat eliminated by passage of the cell suspensions through nylon mesh. Cell suspensions were washed and resuspended in 5% DMEM and stored on ice.

Splenocytes. Splenocyte isolation was conducted as previously described. Briefly, spleens were mechanically dissociated, and RBCs were lysed in ammonium phosphate/chloride lysis buffer. Cell suspensions were washed and stored in DMEM (Life Technologies) with 5% FCS (5% DMEM) on ice until used.

Abs for flow cytometry

The following primary mAbs used in flow cytometry, either unconjugated or FITC, PE, or allophycocyanin conjugated, were purchased from BD PharMingen (San Diego, CA), unless otherwise defined. These include: anti-Thy-1.2 (53-2.1), anti-CD4 (L3T4a, GK1.5), anti-CD8 (Lyt-2, 2.43, and HO2.2), anti-mouse pan-NK (DX5), anti-mouse CD3 (145-2C11 and 500A3), anti-CD69 (H1.2F3), anti-mouse CD25 (7D4), anti-mouse H-2Ld/H-2Db (28-14-8), and isotype-matched control mAbs. The clonotypic anti-TCR mAb KJ1-26.1 conjugated to PE (Caltag Laboratories) was used to identify the OVA323–339-specific transgenic TCR.

Flow cytometric analysis

Three-color flow cytometric analysis was conducted on freshly isolated cells from recipient splenocytes and MLN, and graft-derived MLN or LP, using standard techniques. Briefly, cell suspensions were prepared as described and washed twice in FACS buffer (PBS with 1% FCS, 0.1% sodium azide) before blocking of FcR (2.4G2) and staining with FITC-, PE-, or allophycocyanin-conjugated mAbs. Nonviable cells were excluded from analysis on the basis of propidium iodide staining, and data were analyzed on a FACSCalibur using CellQuest software (BD Biosciences, San Jose, CA).

Evaluation of cytokine and chemokine mRNA expression in SB transplants using RT-PCR

Total cellular RNA was extracted using TriReagent (Molecular Research Center, Cincinnati, OH) in nuclease-free water. Isolated RNA was incubated with 10 U DNase I (Boehringer Mannheim, Indianapolis, IN) in the presence of RNasin (Promega, Madison, WI) for 30 min at 37°C. Samples were heated at 95°C for 10 min, chilled, and reverse transcribed with Superscript II reverse transcriptase (Life Technologies) with random hexamers and oligo(dT). Primers specific for IFN-{gamma}, Mig, and IP-10 were used to amplify relevant sequences. The amount of mRNA for {beta}-actin was assessed to normalize RNA from each specimen. Target cDNA was amplified for a range of 22–40 cycles, and subsequently electrophoresed in 2% agarose gels containing ethidium bromide.

Statistical analyses

Statistical comparisons of survival curves between groups were performed by the log-rank test using GraphPad Prism 3.0 (GraphPad, San Diego, CA) software. Statistical significance of cytokine levels and apoptosis between treated groups was assessed using a one-tailed ANOVA with group means compared using the Scheffe’s multiple comparison test (30). Values of p < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Donor SB T cell activation induces cytokine/chemokine production in SB tissue

To examine the effect of T cell activation on SB cytokine and chemokine induction, mRNA expression for a panel of cytokines and chemokines was evaluated by real time PCR in SB tissues following OVA323–339 (200 µg/mouse, i.p.) administration to DO11.10 mice. We had previously shown that systemic administration of Ag (i.p. OVA323–339) activates cells in peripheral lymphoid tissue as well as in intestinal LP and Peyer’s patches (29). Results in Fig. 1GoA reveal that a single injection of OVA323–339 increased mRNA expression of several effector cytokines, including IFN-{gamma} (13-fold), TNF (6-fold), IL-4 (7-fold), and IL-10 (9-fold) by 3 h postinjection. More notably, OVA323–339 administration induced a dramatic increase in SB mRNA for chemokines including IP-10 (90-fold) and Mig (75-fold), macrophage-inflammatory protein-1{alpha} (8-fold), macrophage-inflammatory protein-1{beta} (25-fold), and monocyte chemoattractant protein-1 (30-fold) by 3 h postinjection. Increased mRNA expression persisted for 24 h after OVA323–339 administration (Fig. 1GoB).



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  		FIGURE 1. Cytokine (A) and chemokine (B) mRNA induction after T cell activation in the SB. Normal DO11.10 mice were given OVA323–339 peptide (200 µg i.p.). RNA was isolated from three pooled whole SB at each of the selected time points following OVA323–339 administration. Real-time PCR analysis was performed as described in Materials and Methods. Shown is the fold induction of ubiquitin-normalized mRNA expression compared with levels detected in unstimulated mice.

 
Donor-derived SB T cell activation accelerates SB allograft rejection

Based on the findings of increased cytokine and chemokine expression in SB tissues, we hypothesized that donor T cell activation would initiate SB allograft rejection. To address the role of donor-derived intestinal T cell activation on SB allograft rejection, SB from DO11.10 mice were transplanted into syngeneic BALB/c mice (isograft) or Ld-deficient dm2 mice (allograft). These donor/recipient combinations were used for several reasons. First, evaluation of a single Ag mismatch facilitated a more focused examination of the influence of donor CD4+ T cell activation on the host alloresponse. Second, while SB allografts with a complete class I and II MHC mismatch develop acute rejection by POD 8 (31), it was anticipated that a single class I MHC Ag disparity would be associated with a more prolonged graft survival. The less aggressive rejection response was utilized to allow for examination of more subtle effects of donor T cell activation on allograft rejection. Finally, the choice of strains eliminated the potential for graft-vs-host responses, as both DO11.10 and dm2 mice strains used are on the BALB/c background.

Following transplantation, isografts and allografts were treated with i.p. injection of either control or OVA323–339 peptide on POD 5, 6, and 7 to activate DO11.10 T cells. Peptide injections were delayed until POD 5 to allow for adequate recovery time from surgery.

As shown in Fig. 2Go, all BALB/c isografts, regardless of whether they received OVA323–339 (n = 5) or not (n = 4), survived until sacrifice on POD 30 with weight gain, healthy stoma, and normal graft histology on POD 8, 10, 14, and 30 (data not shown). This suggests that donor T cell activation had no effect on isograft survival. Allografts (n = 6) that were not given OVA323–339 exhibited a transient increase in mucus output at about POD 9–10, but five of six had no further evidence of rejection throughout the study period (30 days). One allograft (17%) rejected on POD 20 (mean survival time (MST) = 28 days). Serial histological examination of other surviving allografts revealed transient focal cellular infiltrates of grafts on POD 8. However, these infiltrates resolved spontaneously and were not detected on POD 10, 14, or 30 (total histological scores <3 at these time points).



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  		FIGURE 2. SB transplant graft survival. SB from DO11.10 mice were transplanted into syngeneic BALB/c mice (isograft) (A and B) or Ld-deficient dm2 mice (allograft) (C and D) with MBP (control) (A and C) and with OVA323–339 (B and D) peptide administration (200 µg i.p.) on POD 5, 6, and 7. Graft survival was monitored as described in Materials and Methods. *, Values of p > 0.05 vs A and B; **, p < 0.05 vs A–C.

 
In contrast, when donor T cells were activated by OVA323–339 administration, 83% of allografts (n = 6) developed rejection by POD 20 (MST = 16 days, p < 0.05 for OVA323–339 vs control peptide-treated group). Rejection was manifested by increased mucus output (POD 8), followed by the development of a palpable abdominal mass. Histological findings were consistent with severe rejection (scores >12), including lymphocytic infiltration, distortion of villous architecture, and mucosal sloughing (Fig. 3Go, A and B). TUNEL staining for apoptotic cells revealed that donor T cell activation induced apoptosis of crypt enterocytes on POD 7 in SB allografts (Fig. 3Go, C and D). Quantification of apoptosis indices (apoptotic index: apoptotic epithelial cells per 100 total epithelial cells) indicated that donor T cell activation increased levels of epithelial apoptosis by >4-fold from 3.3 ± 2.6 in control peptide-treated SB allografts to 13.1 ± 4.3 in OVA323–339-treated SB allografts (p < 0.01). Overall, these results suggest that donor T cell activation induced vigorous rejection of murine SB allografts mismatched at a single class I MHC locus.



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  		FIGURE 3. Histological evidence of SB allograft rejection induced by donor SB T cell activation. Paraffin-embedded section of SB allografts (allo) treated with control peptide (control) or OVA323–339, 200 µg i.p. on POD 5, 6, and 7. H&E staining on POD 9 showed minimal rejection in control peptide-treated allografts (A) and severe rejection in OVA323–339-treated allografts (B). TUNEL staining of sections taken from grafts on POD 7 indicates increased numbers of apoptotic cells (stained dark brown) in Ag-treated (D) compared with control peptide-treated allografts (C). Data shown are representative of more than three experiments with similar results.

 
Donor SB T cell activation enhances host T cell activation

To determine whether donor T cell activation affected the activation state of host T cell, host splenocytes from DO11.10 (SB donor)->dm2 (host) SB transplants on POD 8 were analyzed for expression of surface activation markers, including CD69 (very early activation Ag) and CD25 (IL-2R {alpha}-chain). Results in Fig. 4Go suggest that transplantation of SB allografts alone increased the proportion of activated dm2-recipient T cells (Thy-1.2+, Ld-). Compared with naive dm2 mice, increased numbers of activated host T cells were detected in the spleen of dm2 recipients of DO11.10 SB allografts (POD 5). Host T cell activation was further enhanced on POD 8 when donor T cells were activated by administration of OVA323–339 peptide on POD 5, 6, and 7 following DO11.10->dm2 SB allotransplantation, but was not detected in recipient spleens of either control or OVA323–339-treated SB isografts (DO11.10->BALB/c) on POD 8 (data not shown). Taken together, these data suggest that donor T cell activation increased the proportion of activated recipient T cells localized to peripheral lymphoid tissues.



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  		FIGURE 4. Ag-induced activation of donor T cells increases the activation state of recipient T cells after SB allograft transplantation. Splenocytes were isolated from dm2 recipients of DO11.10 SB allografts treated with OVA323–339 or control peptide (200 µg i.p.) on POD 5, 6, and 7 and stained with FITC-labeled mAbs specific for CD69 or CD25; anti-Thy-1.2 allophycocyanin; and anti-Ld-biotin, followed by streptavidin-PE. For all populations, lymphoid cells were gated on the basis of forward and side scatter properties and staining for Thy-1.2 and Ld. Recipient cells stained negative for Ld. Results are compared with naive dm2 mice. For each population, the percentage of positively stained cells is shown. Data shown are representative of more than three experiments with similar results.

 
Donor SB T cell activation promotes host T cell infiltration into the SB allografts

To address the effect of donor T cell activation on the composition of graft-associated MLN and LP T cell populations after SB transplantation, we isolated lymphocytes from LP and MLN of isografts as well as allografts on POD 8, and examined whether there were changes in numbers, morphological features (including size as measured by forward scatter and the nuclear/cytoplasmic complexity by side scatter) (32), and surface phenotypes. Expression of the donor Ld alloantigen was used to distinguish host from donor cells. Data in Fig. 5Go, A and B, indicate that little change in numbers and morphology was detected in the MLN and LP T cells from isografts regardless of donor T cell activation by OVA323–339 administration. An increase in lymphoblast-like T cells (increased size and nuclear/cytoplasmic complexity) was found in both MLN and LP following allotransplantation. By comparison, donor T cell activation resulted in further increase in the proportion of T lymphoblasts in LP of SB allografts (14 vs 43% in control vs Ag-treated allografts, respectively). Interestingly, injection of the Ag did not increase accumulation of T lymphoblasts in MLN of allografted mice. Phenotypic analysis (Fig. 5Go, C and D) revealed that donor T cells in the MLN of allografts were largely replaced by host cells by POD 8 regardless of whether donor T cells were activated with systemic Ag. In contrast, donor T cell activation led to a marked increase of host T cells, primarily CD8+ T cells (from 24 to 73%) in LP of SB allografts. These data suggest that donor T cell activation contributes to the initiation of allograft rejection by enhancing infiltration and expansion of host CD8+ T cells in the LP, and the LP is the primary focus of the allogeneic immune response.



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  		FIGURE 5. Phenotypic analysis of lymphocytes of MLN and LP from BALB/c (iso) or dm2 (allo) recipients of DO11.10 SB transplants. Cells isolated from MLN and LP of DO11.10->dm2 allografts with MBP (control) or OVA323–339 peptide administration (200 µg i.p.) on POD 5, 6, and 7 were analyzed on POD 8 by flow cytometry. A and B, Dot plots of fluorescence levels (FSC) of Thy-1.2+ cells in MLN (A) and LP (B) obtained by staining the cells with anti-Thy-1.2 allophycocyanin. Numbers shown in each dot plot are percentages of resting Thy-1.2+ cells (left) or T lymphoblasts (right) in each gate drawn on the basis of fluorescence levels. C and D, Composition of CD4+ and CD8+ T cells in MLN (C) and LP (D). The cells were gated on Thy-1.2+ populations. Quadrant percentages are reported in each plot. Data shown are representative of more than three experiments with similar results.

 
SB rejection is associated with IFN-{gamma}-inducible CXC chemokine (IP-10, Mig) expression

We hypothesized that the current model of SB allograft rejection was associated with increased IFN-{gamma}-inducible CXC chemokine (IP-10, Mig) expression following donor T cell activation. To address whether donor T cell activation up-regulated cytokine/chemokine production in the transplanted SB tissues, mRNA expression levels were assessed by RT-PCR in SB isografts and allografts after treatment with control or antigenic peptide. In both isografts and allografts, administration of OVA323–339 resulted in an up-regulation in IFN-{gamma} mRNA expression and up-regulated expression of the IFN-{gamma}-inducible chemokines IP-10 and Mig (Fig. 6Go). This finding was consistent with those in nontransplanted SB tissues.



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  		FIGURE 6. Cytokine and chemokine mRNA expression in the SB transplants. RNA was isolated from DO11.10 SB grafts transplanted into BALB/c (iso) or dm2 (allo) recipients on POD 8, following MBP (control) (-) or OVA323–339 (+) administration on POD 5, 6, and 7. Expression of mRNA was determined by RT-PCR analysis in which {beta}-actin was used as a control for the amount of RNA isolated.

 
IP-10 is involved in the initiation of SB allograft rejection by donor T cells

To examine the role of IP-10 in SB allograft rejection, the Ag-treated, allografted mice were treated with a neutralizing mAb to mouse IP-10 (anti-IP-10 mAb) every other day, beginning on POD 5. Data in Fig. 7Go indicate that neutralization of IP-10 prolonged graft survival in nearly all mice. Four of six (67%) grafts survived to the study end point (POD 30) with no clinical or histological evidence of rejection (MST = 30). In control IgG-treated mice, Ag treatment induced graft rejection in 86% of mice with a median survival of 15 days (p < 0.05). Three of four anti-IP-10 mAb-treated mice that were not sacrificed at POD 30, but were discontinued with anti-IP-10 mAb treatment, survived to POD 100 without clinical evidence of rejection.



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  		FIGURE 7. IP-10 is involved in SB allograft rejection induced by donor T cell activation. DO11.10->dm2 SB allografts were stimulated with OVA323–339 (200 µg i.p.) on POD 5, 6, and 7, and mice were treated with anti-IP-10 (1F11) or control hamster mAb (UC8-1B9) on POD 5, then every other day until the study end point (POD 30). *, p < 0.05.

 
Anti-IP-10 treatment decreases infiltration of host T cell into LP of the SB allograft

To determine whether anti-IP-10 prevents SB allograft rejection by inhibiting host cell infiltration of the allograft, cellular composition and cell numbers of LP infiltrates were examined. Compared with controls, LP infiltrates from mice treated with anti-IP-10-neutralizing mAb had significantly fewer host CD4+ T cells, CD8+ T cells, and NK cells (Fig. 8GoA). In addition to decreasing the overall numbers of infiltrating host cells in the LP, anti-IP-10 also influenced the donor-recipient T cell mix in the LP (Fig. 8GoB). In fact, the LP T cell infiltrates in the anti-IP-10 mAb-treated group closely resembled those of the group that did not receive OVA323–339. Taken together, these data suggest that IP-10 was involved in the recruitment of host T cells and NK cells following donor T cell activation.



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  		FIGURE 8. The role of IP-10 in host cell accumulation in Ag-stimulated SB allografts. LP lymphocytes isolated from Ag-stimulated DO11.10->dm2 allografts were treated with control or anti-IP-10 mAb (see Materials and Methods) and analyzed on POD 8 by flow cytometry. A, Total numbers of host cells (Ld-). The numbers were calculated based on assessments of total yield and flow cytometric analysis for percentages of CD4+ and CD8+ T cells and NK (DX5) cells of host origin (Ld-) from two separate experiments. B, Composition of CD4+ and CD8+ T cells in LP of Ag-stimulated allografts treated with control or anti-IP-10 mAb. The cells were gated on Thy-1.2+ populations. Quadrant percentages are reported in each plot. Data shown are representative of more than three experiments with similar results.

 
Anti-IP-10 treatment inhibits donor cell accumulation in the host spleen

To determine whether anti-IP-10 treatment also affected cellular accumulation in host peripheral lymphoid tissues, we analyzed host spleens from POD 8 allografts treated with control mAb or anti-IP-10 mAb in addition to OVA323–339 administration. Donor T cell activation (i.v. OVA323–339 on POD 5, 6, 7) enhanced donor T cell accumulation in the host spleen (POD 8). As indicated in Fig. 9Go, the proportion of donor Thy-1.2+ cells in host spleen increased from 0.3 to 12.5% following Ag. Nearly all (>95%) cells of donor origin were transgenic T cells (KJ1.26+) (data not shown). Surprisingly, donor T cell expansion in host spleen was abrogated in Ag-treated mice given anti-IP-10 mAb. This suggests that IP-10 was required for expansion of donor T cells to foreign Ag in host peripheral lymphoid tissues. As these cells were isolated 72 h after initial Ag injection, it is also possible that IP-10 blockade affected trafficking of activated DO.11 T cells from grafts to peripheral lymphoid tissues.



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  		FIGURE 9. IP-10 blockade inhibits donor cell accumulation in the host spleen induced by OVA323–339 administration. Spleen lymphocytes isolated from control or OVA323–339 (POD 5, 6, 7)-stimulated DO11.10->dm2 allografts treated with control mAb or with anti-IP-10 mAb (see Materials and Methods) were analyzed on POD 8 by flow cytometry. The percentage of donor (Ld+) Thy-1.2+ cells of total Thy-1.2+ cells is shown in upper right quadrant of each dot plot. Data shown are representative of more than three experiments with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data suggest that activation of donor CD4+ T cells by foreign Ag accelerated rejection of class I MHC-mismatched SB allografts. The rejection was associated with enhanced host T cell activation and accumulation in the graft. Neutralization of IP-10 reduced host T cell and NK cell infiltration and significantly prolonged SB allograft survival following donor T cell activation. These findings suggest that rejection was initiated by foreign Ag through an IP-10-dependent mechanism. The effect of IP-10 was most evident in the extralymphoid LP compartment, as replacement of host T cells in the MLN by donor cells was independent of Ag treatment. These results suggest that IP-10 is involved in recruitment of cells to the LP of SB allografts during rejection.

While acute rejection is effectively controlled in most solid organ allografts with currently available immunosuppressive protocols, it continues to be a significant problem with intestinal allografts (5). The vigorous rejection response associated with SB transplantation has been attributed to the presence of highly activated effector T cells and APC in SB mucosa (33). However, the mechanisms that define the role of the mucosal immune system in SB rejection are not well understood. Several characteristics of the SB may make it susceptible to augmented allograft rejection. First, unlike other solid organ allografts, the mucosal immune system of the SB contains significant numbers of effector T cells capable of releasing relatively high levels of proinflammatory cytokines (34). Unlike T cells localized to peripheral lymphoid tissue, LP T cells express a pattern of surface markers typical of activated, memory-like T cells (9, 29, 35). These results and others suggest that LP T cells are a functionally potent population of effector T cells. In SB allograft rejection, significant increases in mRNA for IFN-{gamma} and IL-2 are seen in the gut-associated lymphoid tissue before the histological appearance of host T cells (3, 14). Thus, it is possible that when activated by foreign Ag, LP T cells release cytokines (e.g., IFN-{gamma}) that induce the release of chemokines such as IP-10 that help recruit CXCR3+ host cells from the circulation. As CXCR3 is expressed on populations of CD4+ T cells that produce a Th1-like pattern of cytokine expression, it is possible that increased IP-10 release from donor SB allografts accelerates entry of host-derived Th1 effector cells that mediate graft rejection.

Endothelial cells appear to be an important source of IP-10 in infected cells (36) and rejecting cardiac allografts (22), while in the intestine, epithelial cells are also capable of generating IP-10 (24). Because of the large numbers of epithelial cells in the intestine, the SB mucosa is potentially a rich source of IP-10 and other IFN-{gamma}-inducible chemokines (Fig. 1Go). Other cells that synthesize IFN-{gamma}-inducible chemokines, such as macrophages (37) and fibroblasts (15), are also prevalent in the SB. Therefore, in the SB, effector T cells that release IFN-{gamma} when challenged with Ag are found adjacent to large numbers of resident cells that produce IFN-{gamma}-inducible chemokines such as IP-10.

The current data suggest that bystander effects of donor T cell activation enhance the activation state of host T cells. In our model, OVA323–339 administration on POD 5 activated DO11.10 donor T cells and enhanced activation of host T cells in allografted mice (Fig. 4Go). In nontransplanted BALB/c control mice, no activation of endogenous T cells was detected within 72 h of i.v. OVA323–339 injection (data not shown). Thus, activation of host T cells after OVA323–339-induced activation of DO11.10 transgenic CD4+ T cells most likely represents a bystander activation. As these effects were not detected in Ag-treated recipients of SB isografts, we suspect that host cell activation by alloantigen was a prerequisite for bystander effects. This phenomenon has been previously demonstrated both in mice (38, 39) and humans (40, 41). Cytokines released from activated T cells induce nonspecific proliferation, prolonged expression of activation markers (42), and a break in T cell tolerance (43). Potentially, bystander activation of host T cells can occur in any location in which recipient and donor T cells are in close proximity, including the graft LP. In our model, LP T cells of the allograft were gradually replaced by host T cells so that by POD 5, a 3:1 mix of donor and recipient T cells existed (data not shown). By POD 8, the majority of CD8+ LP T cells were of host origin, while the majority of CD4+ T cells were of donor origin (Fig. 5GoD). We suspect that the mixture of host and donor T cells was favorable for the induction of bystander effects on activated host cells. Furthermore, donor T cell activation enhanced accumulation of T cells as well as NK cells through an IP-10-dependent mechanism (Fig. 8Go). It was possible that following donor T cell activation, the process was perpetuated by recruited host CD4+ and CD8+ T cells via activation by enteric Ags and alloantigens in the graft. Thus, the initial increase in cytokine levels in the graft induced by foreign Ag may have promoted migration and sustained activation of host T cells that induced allograft rejection.

In this study, an interesting effect of IP-10 neutralization was detected in host peripheral lymphoid tissue. We found that IP-10 blockade abrogated accumulation of Ag-specific donor T cells in host peripheral lymphoid tissue (Fig. 9Go). These findings suggest that IP-10 neutralization inhibited Ag-induced expansion of DO11.10 transgenic T cells that emigrated out of SB donor tissues into host spleen. These data are consistent with an earlier report by Khan et al. (21) that anti-IP-10 mAb reduced tissue inflammation as well as mononuclear cell expansion in a mouse model of parasite infection. More recently, Pertl et al. (44) demonstrated that depletion of IP-10 impaired antitumor CTL and inhibited generation of Th1-type CD8+ T cell responses. Taken together, these results suggest the IP-10 may be involved in T cell priming. We suspect that in the current model of SB allograft rejection, IP-10 functioned not only as a potent chemoattractant, but also enhanced the activation of T cells. There are several (not mutually exclusive) potential explanations for these observations. First, CXCR3 signaling may enhance T cell responses to Ag. Second, IP-10 may deliver a survival signal to activated T cells that enhances accumulation of expanded clones. Third, CXCR3 may be involved in migration of APC that carry Ag to T cell areas within draining LNs. Investigation of these pathways is outside the scope of the current manuscript, but may explain the surprisingly effective results of IP-10 blockade in several models of allograft transplantation and inflammation (22, 44, 45).

The mechanism proposed in these studies for enhancing allograft rejection may not require donor T cell activation. In a successful allograft, host T cells ultimately replace the donor T cells of the allograft’s gut-associated lymphoid tissue (46), and therefore assume immunosurveillance functions. Foreign Ags continue to activate LP T cells, now of host origin, which release cytokines and chemokines and activate nearby allosensitized T cells. Therefore, the risk of acute rejection may persist longer after transplantation of a SB allograft than after other allografts. Taken together, these data suggest that responses to enteric Ag contribute to SB allograft rejection by enhancing the accumulation of alloreactive cells as well as promoting Th1 functional differentiation of T cells upon arrival in grafts.


   Acknowledgments
 
We thank the members of the Transplant Division for their support and advice, Dr. William Karpus for helpful discussions during the course of this work, and Dr. Roger Melvold (University of North Dakota, Grand Forks, ND) for his advice.


   Footnotes
 
1 This work was partially supported by a career development grant awarded by the Society for Surgery of the Alimentary Tract (to J.F.) and by U.S. Public Health Service Grants DK47073 and DK54778 (to T.A.B.), and CA69212 and AI46999 (to A.D.L.). Back

2 T.A.B. and J.F. are co-senior authors. Back

3 Address correspondence and reprint requests to Dr. Jonathan Fryer, Division of Organ Transplantation, Department of Surgery, Northwestern University Medical School, Suite 17-200, 675 North St. Clair Street, Chicago, IL 60611-2923. E-mail address: JFryer{at}nmh.org Back

4 Abbreviations used in this paper: SB, small bowel; IP-10, IFN-{gamma}-inducible protein-10; LP, lamina propria; Mig, monokine induced by IFN-{gamma}; MLN, mesenteric lymph node; MST, mean survival time; POD, postoperative day. Back

Received for publication November 13, 2001. Accepted for publication January 25, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Asfar, S., P. Atkison, C. Ghent, J. Duff, W. Wall, S. Williams, E. Seidman, D. Grant. 1996. Small bowel transplantation: a life-saving option for selected patients with intestinal failure. Dig. Dis. Sci. 41:875.[Medline]
  2. Langnas, A. N., Jr B. W. Shaw, D. L. Antonson, S. S. Kaufman, D. R. Mack, T. G. Heffron, I. J. Fox, J. A. Vanderhoof. 1996. Preliminary experience with intestinal transplantation in infants and children. Pediatrics 97:443.[Abstract/Free Full Text]
  3. Quan, D., D. R. Grant, R. Z. Zhong, Z. Zhang, B. M. Garcia, A. M. Jevnikar. 1994. Altered gene expression of cytokine, ICAM-1, and class II molecules precedes mouse intestinal allograft rejection. Transplantation 58:808.[Medline]
  4. Newell, K. A., G. He, J. Hart, Jr J. R. Thistlethwaite. 1997. Treatment with either anti-CD4 or anti-CD8 monoclonal antibodies blocks {alpha}{beta} T cell-mediated rejection of intestinal allografts in mice. Transplantation 64:959.[Medline]
  5. Grant, D.. 1999. Intestinal transplantation: 1997 report of the international registry: Intestinal Transplant Registry. Transplantation 67:1061.[Medline]
  6. Abu-Elmagd, K., J. Reyes, G. Bond, G. Mazariegos, T. Wu, N. Murase, R. Sindhi, D. Martin, J. Colangelo, M. Zak, et al 2001. Clinical intestinal transplantation: a decade of experience at a single center. Ann. Surg. 234:404.[Medline]
  7. White, F. V., J. Reyes, R. Jaffe, E. J. Yunis. 1995. Pathology of intestinal transplantation in children. Am. J. Surg. Pathol. 19:687.[Medline]
  8. Lee, R. G., K. Nakamura, A. C. Tsamandas, K. Abu-Elmagd, H. Furukawa, W. R. Hutson, J. Reyes, J. S. Tabasco-Minguillan, S. Todo, A. J. Demetris. 1996. Pathology of human intestinal transplantation. Gastroenterology 110:1820.[Medline]
  9. Hurst, S. D., S. M. Sitterding, S. Ji, T. A. Barrett. 1997. Functional differentiation of T cells in the intestine of T cell receptor transgenic mice. Proc. Natl. Acad. Sci. USA 94:3920.[Abstract/Free Full Text]
  10. Lefrancois, L., L. Puddington. 1995. Extrathymic intestinal T-cell development: virtual reality?. Immunol. Today 16:16.[Medline]
  11. Harper, H. M., L. Cochrane, N. A. Williams. 1996. The role of small intestinal antigen-presenting cells in the induction of T-cell reactivity to soluble protein antigens: association between aberrant presentation in the lamina propria and oral tolerance. Immunology 89:449.[Medline]
  12. Kelsall, B. L., W. Strober. 1996. Distinct populations of dendritic cells are present in the subepithelial dome and T cell regions of the murine Peyer’s patch. J. Exp. Med. 183:237.[Abstract/Free Full Text]
  13. Garside, P., A. M. Mowat, A. Khoruts. 1999. Oral tolerance in disease. Gut 44:137.[Free Full Text]
  14. Toogood, G. J., A. M. Rankin, P. K. Tam, P. J. Morris, M. J. Dallman. 1997. The immune response following small bowel transplantation. II. A very early cytokine response in the gut-associated lymphoid tissue. Transplantation 63:1118.[Medline]
  15. Belperio, J. A., M. D. Burdick, M. P. Keane, Y. Y. Xue, III J. P. Lynch, B. L. Daugherty, S. L. Kunkel, R. M. Strieter. 2000. The role of the CC chemokine, RANTES, in acute lung allograft rejection. J. Immunol. 165:461.[Abstract/Free Full Text]
  16. Fairchild, R. L., A. M. VanBuskirk, T. Kondo, M. E. Wakely, C. G. Orosz. 1997. Expression of chemokine genes during rejection and long-term acceptance of cardiac allografts. Transplantation 63:1807.[Medline]
  17. Segerer, S., Y. Cui, F. Eitner, T. Goodpaster, K. L. Hudkins, M. Mack, J. P. Cartron, Y. Colin, D. Schlondorff, C. E Alpers. 2001. Expression of chemokines and chemokine receptors during human renal transplant rejection. Am. J. Kidney Dis. 37:518.[Medline]
  18. Kondo, T., Y. Watarai, A. C. Novick, H. Toma, R. L. Fairchild. 1997. T cell-dependent acceleration of chemoattractant cytokine gene expression during secondary rejection of allogeneic skin grafts. Transplantation 63:732.[Medline]
  19. Agostini, C., M. Cassatella, R. Zambello, L. Trentin, S. Gasperini, A. Perin, F. Piazza, M. Siviero, M. Facco, M. Dziejman, et al 1998. Involvement of the IP-10 chemokine in sarcoid granulomatous reactions. J. Immunol. 161:6413.[Abstract/Free Full Text]
  20. Koga, S., M. B. Auerbach, T. M. Engeman, A. C. Novick, H. Toma, R. L. Fairchild. 1999. T cell infiltration into class II MHC-disparate allografts and acute rejection is dependent on the IFN-{gamma}-induced chemokine Mig. J. Immunol. 163:4878.[Abstract/Free Full Text]
  21. Khan, I. A., J. A. MacLean, F. S. Lee, L. Casciotti, E. DeHaan, J. D. Schwartzman, A. D. Luster. 2000. IP-10 is critical for effector T cell trafficking and host survival in Toxoplasma gondii infection. Immunity 12:483.[Medline]
  22. Hancock, W. W., W. Gao, V. Csizmadia, K. L. Faia, N. Shemmeri, A. D. Luster. 2001. Donor-derived IP-10 initiates development of acute allograft rejection. J. Exp. Med. 193:975.[Abstract/Free Full Text]
  23. Hancock, W. W., B. Lu, W. Gao, V. Csizmadia, K. Faia, J. A. King, S. T. Smiley, M. Ling, N. P. Gerard, C. Gerard. 2000. Requirement of the chemokine receptor CXCR3 for acute allograft rejection. J. Exp. Med. 192:1515.[Abstract/Free Full Text]
  24. Dwinell, M. B., N. Lugering, L. Eckmann, M. F. Kagnoff. 2001. Regulated production of interferon-inducible T-cell chemoattractants by human intestinal epithelial cells. Gastroenterology 120:49.[Medline]
  25. Rubocki, R. J., T. H. Hansen, D. R. Lee. 1986. Molecular studies of murine mutant BALB/c-H-2dm2 define a deletion of several class I genes including the entire H-2Ld gene. Proc. Natl. Acad. Sci. USA 83:9606.[Abstract/Free Full Text]
  26. Murphy, K. M., A. B. Heimberger, D. Y. Loh. 1990. Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo. Science 250:1720.[Abstract/Free Full Text]
  27. Zhang, Z., L. Zhu, D. Quan, B. Garcia, N. Ozcay, J. Duff, C. Stiller, A. Lazarovits, D. Grant, R. Zhong. 1996. Pattern of liver, kidney, heart, and intestine allograft rejection in different mouse strain combinations. Transplantation 62:1267.[Medline]
  28. Quan, D., Z. Zhang, R. Zhong, A. Jevnikar, B. Garcia, D. Grant. 1996. Intestinal allograft rejection in lipopolysaccharide-hyporesponsive mice. Transplant. Proc. 28:2460.[Medline]
  29. Hurst, S. D., C. J. Cooper, S. M. Sitterding, J. Choi, R. L. Jump, A. D. Levine, T. A. Barrett. 1999. The differentiated state of intestinal lamina propria CD4+ T cells results in altered cytokine production, activation threshold, and costimulatory requirements. J. Immunol. 163:5937.[Abstract/Free Full Text]
  30. Armitage, P., G. Berry. 1971. Statistical Methods in Medical Research Wiley, New York.
  31. Cagiannos, C., R. Zhong, Z. Zang, J. Jiang, B. M. Garcia, S. Chakrabarti, A. M. Jevnikar, N. R. Sinclair, D. R. Grant. 1998. Effect of major histocompatibility complex expression on murine intestinal graft survival. Transplantation 66:1369.[Medline]
  32. Carbonari, M., T. Tedesco, M. Fiorilli. 2001. A unified procedure for conservative (morphology) and integral (DNA and immunophenotype) cell staining for flow cytometry. Cytometry 44:120.[Medline]
  33. Abu-Elmagd, K., J. Reyes, S. Todo, A. Rao, R. Lee, W. Irish, H. Furukawa, J. Bueno, J. McMichael, A. T. Fawzy, et al 1998. Clinical intestinal transplantation: new perspectives and immunologic considerations. J. Am. Coll. Surg. 186:512.[Medline]
  34. Abreu-Martin, M. T., S. R. Targan. 1996. Regulation of immune responses of the intestinal mucosa. Crit. Rev. Immunol. 16:277.[Medline]
  35. James, S. P.. 1988. Cellular immune mechanisms in the pathogenesis of Crohn’s disease. In Vivo 2:1.[Medline]
  36. Ebnet, K., M. M. Simon, S. Shaw. 1996. Regulation of chemokine gene expression in human endothelial cells by proinflammatory cytokines and Borrelia burgdorferi. Ann. NY Acad. Sci. 797:107.[Abstract]
  37. Shimomura, I., H. Shimano, B. S. Korn, Y. Bashmakov, J. D. Horton. 1998. Nuclear sterol regulatory element-binding proteins activate genes responsible for the entire program of unsaturated fatty acid biosynthesis in transgenic mouse liver. J. Biol. Chem. 273:35299.[Abstract/Free Full Text]
  38. Nahill, S. R., R. M. Welsh. 1993. High frequency of cross-reactive cytotoxic T lymphocytes elicited during the virus-induced polyclonal cytotoxic T lymphocyte response. J. Exp. Med. 177:317.[Abstract/Free Full Text]
  39. Yang, H., R. M. Welsh. 1986. Induction of alloreactive cytotoxic T cells by acute virus infection of mice. J. Immunol. 136:1186.[Abstract]
  40. Schendel, D. J., C. Reinhardt, P. J. Nelson, B. Maget, L. Pullen, G. W. Bornkamm, A. Steinle. 1992. Cytotoxic T lymphocytes show HLA-C-restricted recognition of EBV-bearing cells and allorecognition of HLA class I molecules presenting self-peptides. J. Immunol. 149:2406.[Abstract]
  41. Gaston, J. S., A. B. Rickinson, M. A. Epstein. 1983. Cross-reactivity of self-HLA-restricted Epstein-Barr virus-specific cytotoxic T lymphocytes for allo-HLA determinants. J. Exp. Med. 158:1804.[Abstract/Free Full Text]
  42. Tough, D. F., P. Borrow, J. Sprent. 1996. Induction of bystander T cell proliferation by viruses and type I interferon in vivo. Science 272:1947.[Abstract]
  43. Rocken, M., J. F. Urban, E. M. Shevach. 1992. Infection breaks T-cell tolerance. Nature 359:79.[Medline]
  44. Pertl, U., A. D. Luster, N. M. Varki, D. Homann, G. Gaedicke, R. A. Reisfeld, H. N. Lode. 2001. IFN-{gamma}-inducible protein-10 is essential for the generation of a protective tumor-specific CD8 T cell response induced by single-chain IL-12 gene therapy. J. Immunol. 166:6944.[Abstract/Free Full Text]
  45. Fife, B. T., K. J. Kennedy, M. C. Paniagua, N. W. Lukacs, S. L. Kunkel, A. D. Luster, W. J. Karpus. 2001. CXCL10 (IFN-{gamma}-inducible protein-10) control of encephalitogenic CD4+ T cell accumulation in the central nervous system during experimental autoimmune encephalomyelitis. J. Immunol. 166:7617.[Abstract/Free Full Text]
  46. Iwaki, Y., T. E. Starzl, A. Yagihashi, S. Taniwaki, K. Abu-Elmagd, A. Tzakis, J. Fung, S. Todo. 1991. Replacement of donor lymphoid tissue in small-bowel transplants. Lancet 337:818.[Medline]



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