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J Am Soc Nephrol 13:511-518, 2002
© 2002 American Society of Nephrology

Antigen-Dependent Transgene Expression in Kidney Transplantation: A Novel Approach Using Gene-Engineered T Lymphocytes

Markus H. Hammer1,*, Grit Schröder1,{dagger}, Kirsten Risch{dagger}, Alexander Flügel{ddagger}, Hans-Dieter Volk*, Manfred Lehmann{dagger} and Thomas Ritter*

*Institute of Medical Immunology, Charité, Humboldt-University, Berlin, Germany; {dagger}Institute of Medical Biochemistry and Molecular Biology, University of Rostock, Rostock, Germany; {ddagger}Department of Neuroimmunology, Max-Planck Institute for Neurobiology, Martinsried, Germany.

Correspondence to Dr. Hans-Dieter Volk, Institute of Medical Immunology, Charité, Humboldt University, Schumannstrasse 20/21, 10117 Berlin. Phone: 49-30-450524062; Fax: 49-30-450524932; E-mail: hans-dieter.volk{at}charite.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. So far, gene therapy in transplantation mainly focuses on the expression of therapeutic proteins in the graft itself. Insufficient transfection and inflammatory responses that are due to the immunogenicity of multiple vector systems are often limiting factors in these approaches. The potential of genetically modified T lymphocytes was investigated as a delivery system for therapeutic transgenes to transplanted organs as a way to circumvent immunogenicity and efficiency problems in a rat transplant model. Gene-engineering of alloantigen-specific rat T cell lines was performed by using a Moloney murine leukemia virus (MoMuLV)–based enhanced green fluorescence protein (EGFP) encoding retroviral transduction system. The ex vivo gene-modified lymphocytes were adoptively transferred into syngeneic rats carrying allogeneic, syngeneic, or third party kidneys. Homing behavior, activation level, and transgene expression of the adoptively given cells were monitored. The TEGFP lymphocytes infiltrated the transplanted kidneys in an antigen-specific manner. High numbers of alloantigen-specific T lymphocytes accumulated exclusively in allografts but not in syngeneic or third party grafts. Flow cytometric analysis revealed that only TEGFP lymphocytes found in allografts had an activated phenotype that resulted in higher transgene expression. Alloantigen-specific homing, activation, and transgene expression are important prerequisites for the guarantee of localized delivery and expression of transgenic proteins by gene-engineered T lymphocytes. Antigen-specific gene-targeting strategies using ex vivo modified T lymphocytes with donor specificity are a novel approach to the delivery of therapeutic transgenes in transplantation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Genetically modified T lymphocytes are popular target cells in the treatment of many inherited or acquired human diseases (1). Retrovirus-based gene expression systems are currently the most efficient tools for manipulating this cell population, because they promote a stable integration of foreign transgenes into the host genome. Another advantage of retrovirally transduced T lymphocytes is their long life span in vivo, shown, for example, in the genetic treatment of adenosine deaminase (ADA) deficiency with T lymphocytes expressing functional ADA (2). So far, most T lymphocyte transduction protocols are performed under polyclonal stimulation conditions, using concanavalin A (ConA), anti-CD3 monoclonal antibodies (mab), or phytohemagglutinin (PHA), due to the fact that retrovirus-mediated gene transfer requires cell cycling (3,4,5). The transgenic T cell population resulting from these experiments carries almost the same random antigen specificity as seen in the normal T lymphocyte repertoire. Quiescent, nonactivated T lymphocytes express their transgene in a basic low level, but T cell activation is known to enhance transgene expression. After adoptive transfer of gene-engineered lymphocytes with random antigen specificity, activation of single cells out of this pool may lead to an increase of the basic protein expression level. The total amount of therapeutic protein delivered by T lymphocytes with random antigen specificity is able to treat diseases like ADA deficiency, but most infectious diseases or the transplant setting will require higher amounts of therapeutic proteins (6). As only activated T cells are able to migrate into tissues, including the graft, the activation of gene-engineered T lymphocytes is a necessary prerequisite to increase transgene expression in vivo.

We have recently shown that in vitro priming of rat T lymphocytes with alloantigens allowed us to generate T cell lines with a defined antigen-specificity. After retroviral transduction of these cells, alloantigen-specific activation highly increases transgene expression in vitro and, more importantly, in vivo (7).

The second important property, beside transgene-regulation in vivo, is the homing behavior of transgenic T cells. It has been reported recently that nerve-specific autoimmune T lymphocytes, engineered to express nerve growth factor, transmigrate across the endothelial blood-nerve barrier to infiltrate peripheral nerves, showing an antigen-directed homing behavior in vivo (8). We hypothesized that alloantigen-specific T lymphocytes will home to areas where the immune system has contact with the foreign alloantigen. Therefore, these T lymphocytes could be used as carriers to deliver retrovirally encoded therapeutic proteins to these specific sites. A first proof of principle was reported previously. In this work, adoptive transfer of nerve-specific autoimmune T lymphocytes, transduced to express the Th2-cytokine, interleukin-4 (IL-4), delayed the onset and reduced the severity of inflammation in the central nervous system in a mouse model of experimental autoimmune encephalomyelitis (9).

In contrast to approaches that chose the graft itself as a target for gene therapy, immunomodulatory proteins might reach local lymph nodes (LN) and other lymphatic organs beside the graft itself. Moreover, the use of T cell receptor (TCR) activation–sensitive promoters opens the possibility of enhancing local transgene expression. We recently showed that specific TCR activation resulted in enhanced expression of retroviral promoter–controlled transgenes in vivo. TCR activation–sensitive promoters should therefore improve local gene delivery in transplantation. We tried to prove our hypothesis by the generation of recipient (syngeneic) T cell lines with known donor (allogeneic) antigen-specificity. Enhanced green fluorescence protein (EGFP) was chosen as a reporter transgene because it enabled us to follow up the fate of transduced cells after adoptive transfer and to quantify EGFP expression by flow cytometric analysis (FACS) (10). The retrovirally transduced T lymphocytes were injected into syngeneic rats carrying allogeneic, syngeneic, or third party kidney grafts (see scheme, Figure 1). Homing behavior, activation state, and transgene expression of TEGFP lymphocytes were monitored at different time points after adoptive transfer.



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  		Figure 1. Schematic experimental design. Recipient-derived naive T lymphocytes were primed against relevant donor antigens. Resulting donor-specific T lymphocytes underwent retroviral transduction to express marker or therapeutic transgenes, respectively. Adoptive T cell transfer of these gene-engineered cells in the recipient carrying the relevant donor graft should result in recruitment and local transgene expression.

 
We could demonstrate in this study that retrovirally transduced T lymphocytes specifically migrate into the allografts but not into syngeneic or third party controls. Moreover, transgene expression is increased in the allograft but not in syngeneic or third party grafts, indicating antigen-dependent transgene expression. Therefore this approach could be used to transfer and express therapeutic genes in the transplant setting.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Lewis (LEW, RT1l), Dark Agouti (DA, RT1av1), and Wistar-Furth (WF, RT1u) rats (male; 8 to 12 wk of age; 200 to 300 g; Moellegaard, Ejby, Denmark) were used in our experiments and were fed a regular diet.

Generation, Transduction, and Characterization of Alloantigen-Specific T Lymphocytes
The generation and transduction of polyclonal, alloantigen-specific T lymphocytes has been described elsewhere (5,7,11). Briefly, lymph node cells of LEW rats were isolated from subcutaneous lymph nodes and incubated together with irradiated (5000 rad) DA lymph node cells in a modified limiting dilution protocol (11). The cells were grown in T cell medium (TCM) containing Dulbecco’s modified Eagle medium (DMEM) with 2 mM L-glutamine, 2 mM L-asparagine, antibiotics (Life Technologies BRL, Eggenstein, Germany), and 2% heat-inactivated autologous LEW serum. After 4 d, T lymphocyte blasts were propagated for 6 to 8 d in IL-2–conditioned medium. Restimulation was done by incubating resting T lymphocytes with a 2:1 mixture of irradiated LEW/DA thymocytes. The retroviral gene transfer in primary T lymphocytes was performed by seeding the packaging cells (GP + E 86 EGFP), which produced replication-deficient EGFP-expressing retroviruses in a 96-well plate. Then, freshly isolated LEW and DA lymphocytes were added to the wells as described above. Selection with 0.4 mg/ml Geneticin (G418; Life Technologies BRL) was started after 3 d. The cells were tested for transgene expression by using fluorescence microscopy and cytofluorometry (7). Alloantigen specificity of T lymphocytes was tested with a standard proliferation assay, counting tritiated thymidine incorporation (7,12). All experiments were performed with several TEGFP cell lines that were generated in independent stimulation experiments.

Transplantation and Adoptive Transfer
Orthotopic kidney transplantation was performed as described elsewhere (13). Adoptive T cell transfer was done one day after transplantation by injecting 1.5 x 107 TEGFP lymphocytes into the vena femoralis of LEW rats.

In Vivo Lifespan of TEGFP Lymphocytes after Adoptive Transfer
Adoptive T cell transfer was performed 14 d before transplantation by injecting 1.5 x 107 TEGFP lymphocytes into the vena femoralis of naive LEW rats. Harvesting and cryopreservation of kidney grafts was done 4 d after transplantation.

Confocal and Fluorescence Microscopy
For histologic analysis of lymphatic tissues and other organs, animals were perfused with phosphate-buffered saline (PBS) 4% paraformaldehyde (PFA). After preparation, organs were postfixed in 4% PFA at 4°C overnight, followed by cryoprotection in PBS that contained 15% sucrose solution at 4°C for 24 h. The organs were frozen and cut into 5-µm sections. For counterstaining, sections were incubated with ToPro-3 iodide (Molecular Probes, Eugene, Oregon) for 10 min at room temperature. Analysis was performed by using confocal and fluorescence microscopy technique (Leica, Braunschweig, Germany).

Isolation of Graft Infiltrating Cells (GICs) and Flow Cytometric Analysis
For flow cytometric detection of fluorescent T cells, kidneys were removed and homogenized. After washing twice with PBS, the resulting pellet was resuspended with 10 ml of DMEM (Life Technologies BRL) that contained 10 mg of liver collagenase type 4 (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Incubation was done at 37°C for 30 min. Collagenase digestion was stopped by washing twice with ice-cold PBS. The solution was filtered through a 100-µm strainer, and leukocytes were isolated by ficoll density separation (rat-ficoll; Pan Systems, Martinsried, Germany). In the case of antibody staining, cells were incubated at 4°C for 20 min in 0.1 ml of PBS with 1% bovine serum and 2 µl of the undiluted antibody. Mouse anti-rat mab against TCR, CD4, CD8, L-selectin, CD25, Pan-CD45, CD45RC, interferon-{gamma} (IFN-{gamma}), and IL-4 were phycoerythrin-labeled (BioSource Europe, Nivelles, Belgium and Amersham, Braunschweig, Germany). Indirect labeling of VLA-4 and LFA-1 was described elsewhere (11). After washing twice in ice-cold PBS containing 0.01% sodium azide, cells were measured with FACScan (Becton Dickinson, Heidelberg, Germany) and data were analyzed with FCS-Express software (De Novo Software, Thornhill, Ontario, Canada).

Statistical Analysis
Values are reported as mean ± SD. Group comparisons were performed by using the parameter-free U test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of Alloantigen-Specific TEGFP Lymphocytes
Surface and intracellular staining was performed to characterize the phenotype of these ex vivo generated T cell lines (Table 1). Due to negative selection with G418, all lymphocytes were EGFP+. The overall phenotype of all characterized cell lines resembles a memory T helper population with more than 95% of cells being TCR+, CD4+, CD8-, Pan-CD45+, CD45RClow, L-selectinlow, VLA-4+, and LFA-1+. Activation-dependent expression was measured for EGFP and CD25 expression in vitro. Both proteins were upregulated starting 6 h after alloantigen-specific TCR stimulation (7). Intracellular cytokine staining after alloantigen-specific and phorbolmyristate acetate/ionomycin stimulation revealed the ability of these lymphocytes to produce IFN-{gamma} but not IL-4. Allospecificity was tested in vitro by a 3H-thymidine standard proliferation assay. The cells proliferated specifically after stimulation with DA (allo) but not with LEW (syngeneic) or WF (third party) splenocytes. The data are presented in Hammer et al. (7).


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  		Table 1. Characterization of TEGFP lymphocytes before injection
 
Homing Behavior of Alloantigen-Specific TEGFP Lymphocytes in the Transplant Setting
The homing behavior of EGFP transgenic rat T lymphocytes with DA specificity was investigated by detection of the fluorescent cells on tissue sections from animals grafted with organs from different MHC background. For these experiments, we performed orthotopic rat kidney transplantation by grafting LEW (syngeneic), DA (allogeneic), or WF (third party) organs into LEW recipients. Adoptive transfer of 1.5 x 107 DA-specific TEGFP lymphocytes into the vena femoralis was performed 1 d after transplantation. The animals were killed 3 d later and fixed by whole-body perfusion with PBS 4% PFA. Different tissues and organs were harvested, frozen, and cut into 5-µm sections. Analysis was done by fluorescence and/or confocal microscopy. Figure 2 shows the distribution of TEGFP lymphocytes 3 d after adoptive T cell transfer. The highest number of EGFP-expressing T cells was found in donor-specific renal allografts but not in syngeneic or third party kidney grafts. A 6- to 10-fold higher number of TEGFP lymphocytes—specific for alloantigens—was located inside the transplanted DA kidney as documented by fluorescence microscopy. Figure 3 illustrates the graft infiltrating TEGFP lymphocytes in the allogeneic and syngeneic transplants. In the allosetting, both the tubula and the glomerula regions were infiltrated, but a higher number of fluorescent cells was found in close proximity to the renal tubuli. TEGFP lymphocytes were rarely detectable in the contralateral nontransplanted kidneys. EGFP transgenic cells were also found in spleens, livers, and lungs, but no significant differences in distribution between allogeneic, syngeneic, and third party transplanted rats were detectable. The number of TEGFP lymphocytes in different LN appeared to be heterogenic. Large differences could be detected even in the same animal (Figure 2).



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  		Figure 2. Distribution of alloantigen-specific TEGFP lymphocytes 3 d after adoptive transfer. The figure shows the number of TEGFP lymphocytes in the graft and in selected lymphatic and nonlymphatic organs. Three different groups: allogeneic (allo), syngeneic (syn), and third party (third) grafted animals are illustrated. At least 6 sections containing more than 250 sight fields (one sight field = 0.159 mm2) were counted under a fluorescence microscope. Cell numbers are shown in cells/mm2. The results are shown as mean ± SD (n = 5 per group). * P < 0.01 versus the other groups (U test).

 


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  		Figure 3. Confocal laser microscopy of transplanted kidney grafts. Tracking of TEGFP lymphocytes 3 d after adoptive transfer in allogeneic and syngeneic kidneys. Confocal laser microscopy of (A) allogeneic and (B) syngeneic graft. The distribution of TEGFP lymphocytes within the allograft is demonstrated in panels C and D. (C) Graft infiltrating cells in proximity to the glomerular region and (D) to the tubular region. Representative of five distinct experiments. Magnifications: x200 in A and B; x620 in C and D.

 
In Vivo Lifespan of TEGFP Lymphocytes after Adoptive Transfer
An important question is the lifespan of adoptively transferred gene-engineered T lymphocytes in vivo. Long-term monitoring of these cells is limited by fast acute rejection of transplanted kidney grafts in the allocombination. To circumvent this limitation in our model, we injected TEGFP lymphocytes 14 d before transplantation. The DA-specific lymphocytes survived inside the secondary lymphatic organs for 14 d and homed to the allografts where they could be detected by flow cytometry (data not shown). We have also quantified these cells on tissue sections. Fluorescent TEGFP lymphocytes were only detected in rats transplanted with DA (allogeneic) but not with LEW (syngeneic) or WF (third party) kidneys. The number of cells detected in the allogeneic kidney (DA) was 5- to 6-fold lower compared with graft infiltration in the situation when the cells were adoptively transferred at day +1 after allogeneic transplantation (1.49 ± 2.84 cells/mm2 versus 7.98 ± 2.64 cells/mm2).

Transgene Regulation and Protein Expression In Vivo
An additional criterion for the efficacy of the TEGFP lymphocytes as carriers for therapeutic molecules, beside their lifespan and homing behavior, is the transgene regulation and subsequent protein synthesis in allograft recipients. We demonstrated recently that antigen-specific T cell activation strongly upregulated transgene expression of gene-engineered T lymphocytes in vitro. We were, therefore, interested in the activation level of GICs. After isolation of GICs by collagenase digestion, the reporter gene EGFP again provides, as we have recently shown, the appropriate tool for the redetection and evaluation of TEGFP lymphocytes. Expression of the inducible {alpha}-chain of the IL-2 receptor (CD25) on EGFP-expressing cells was determined by flow cytometry. In comparison with cells isolated from syngeneic and third party grafts, only the allograft-infiltrating gene-modified cells showed a shift from a CD25low to CD25high phenotype (Figure 4). CD25low levels in isolated lymphocytes from syngeneic and third party rats were in a comparable range with T lymphocytes before injection, indicating an activated status of TEGFP lymphocytes after the recontact with the specific alloantigens (P < 0.04). In addition, an increased EGFP mean fluorescence intensity (MFI) was detected only in GICs isolated from allografted animals (P < 0.02). The higher EGFP MFI is most likely a result of T cell activation–driven enhanced transgene expression and the following cytosolic accumulation of the fluorescence protein.



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  		Figure 4. Flow cytometric analysis of graft infiltrating cells (GICs). (A) GIC of allogeneic (allo), syngeneic (syn), and third party (third) grafted animals were stained with mab against CD25, and flow cytometric analysis was performed. The dot plot shows EGFP expression versus side scatter to illustrate the number of EGFP+ cells. 50,000 events are illustrated per plot. One representative out of five experiments is shown. (B) EGFP and CD25 expression of EGFP+ cells were evaluated by gating on EGFP+ cells on a FITC/side scatter dot plot. This gate was combined with a lymphocyte gate defined in a forward scatter/side scatter dot plot (not shown). Data are expressed as a ratio between allogeneic (allo), syngeneic (syn), or third party (third) transplanted animals compared with syngeneic control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
An important requirement of gene-modified T lymphocytes as transgene carriers in transplantation is their homing behavior to sites where the immune system of the recipient comes into contact with donor antigens. We hypothesized that alloantigens presented in the transplanted graft or in secondary lymphoid organs attract ex vivo generated alloantigen-specific T lymphocytes to migrate to these local sites. If this alloantigen-specific attraction takes place, lymphocytes able to express immunomodulatory proteins that prevent the immigration and the development of graft destroying cells could be the ideal vehicles for tissue-specific gene delivery. To clarify the homing behavior of the ex vivo generated T lymphocytes in vivo, EGFP was chosen as the marker transgene to distinguish ex vivo generated from the recipients’ T lymphocytes. Light emission under 488-nm excitation is easily detectable by fluorescence microscopy and flow cytometry without the need for any substrates, cofactors, or antibody staining (13). Therefore EGFP provides a perfect tool to monitor the fate of fluorescence T lymphocytes in vivo (5,14).

Homing behavior into allogeneic, syngeneic and third party kidney transplants was evaluated by adoptive transfer of alloantigen-specific TEGFP lymphocytes 1 d after renal transplantation. Several organs and tissues were harvested 3 d later to determine the distribution of the transgenic T lymphocytes. The highest number of allospecific TEGFP lymphocytes was detected inside the transplanted kidneys, expressing the relevant alloantigens but not in syngeneic or third party kidneys. The inflammatory endothelial response, due to cold ischemia/reperfusion injury, may be crucial in the recruitment of TEGFP lymphocytes to transplanted grafts (15). Upregulation of endothelial adhesion molecules, extracellular matrix proteins, chemokines, and other factors are considered to be responsible for a basic T cell infiltration in all transplants. However, this could not explain the dramatic influx of TEGFP lymphocytes into allografts in comparison with syngeneic and third party transplants (P < 0.01) (16). Importantly, the accumulation inside the DA allograft is not a result of ongoing rejection, because third party grafts—despite the same rejection kinetics as in allografts—showed only a marginal nonsignificant increase in infiltration. We therefore conclude that the major impact on graft infiltration is due to the alloantigen inside or outside the graft. It is important to note that the alloantigen-driven homing was not a phenomenon restricted to LEW/DA strain combination. We were able to demonstrate this homing mechanism in other strain combinations as well (11).

What are the putative mechanisms of specific T cell accumulation in the graft? In theory, three principles, which are not mutually exclusive, exist. First, alloantigen-specific lymphocytes home into the graft in an antigen-specific manner. Antigen-specific transmigration of T lymphocytes across endothelial barriers was recently described (17). In this work, recognition of alloantigens presented by endothelial MHC class II complexes enhanced the transmigration of allospecific lymphocytes in vitro. Second, clonal expansion inside the graft or in secondary lymphatic organs may change the total number of allospecific TEGFP lymphocytes. Third, random homing of the activated T cells to the different sites, including the grafts, followed by enhanced apoptosis in the absence or proliferation in the presence of alloantigens (18). We favor the idea that high MHC class II expression after transplantation initiates the crosstalk between graft endothelium and adoptively given CD4+ TEGFP lymphocytes. The donor-derived endothelial cells present the alloantigen via the direct pathway. The protocol used to generate the allospecific cell lines promotes this direct interaction, because TCR activation during in vitro priming and expansion was performed by donor (DA)–derived MHC class II molecules. It is important to note that the alloantigen fulfills the major impact on expansion, survival, and/or homing in any of these possible mechanisms, demonstrating the advantage of alloantigen-specific versus polyclonally stimulated T lymphocytes as carriers for therapeutic transgenes.

Relevant numbers of TEGFP lymphocytes were also found in spleens and lymph nodes, but no differences between the different groups were detectable. This supports our earlier observation that after adoptive transfer the injected cells migrate through secondary lymphatic organs. The large heterogenicity of transgenic T lymphocytes found in LN may be due to the role of draining and nondraining LN. We were not yet able to distinguish these two groups because the lymph draining of kidney grafts in transplanted rats is not fully understood. However, all analyzed LN were infiltrated by gene-engineered T lymphocytes, which also gives strong evidence for the presence of TEGFP lymphocytes in graft draining LN.

To obtain a sufficient concentration of immunomodulatory proteins by T lymphocyte–delivered transgenes, its regulation and expression is also a critical factor in this approach. It has been shown that polyclonal stimulation by CD3/CD28 mab induces increasing levels of retroviral transgene expression in lymphocytes (19). Recently we were able to demonstrate that alloantigen-specific stimulation was also followed by increased accumulation of EGFP inside the TEGFP lymphocyte as measured by flow cytometry. EGFP accumulates inside transduced cells, allowing expression analysis on the single cell level. In these in vitro studies, EGFP upregulation and expression kinetics of all tested EGFP+ T cell lines correlated directly with CD25 expression, a well-established T cell surface activation marker. Therefore, both markers allowed us to determine the activation-state of TEGFP lymphocytes (7). Next we were interested in the activation and transgene expression level of TEGFP lymphocytes in the transplant setting. Using the same model, graft-infiltrating cells were isolated by collagenase digestion, and flow cytometry was performed to quantify CD25 and EGFP expression levels. In comparison with CD25 and EGFP expression before injection, only the transplant-infiltrating cells isolated from allografts had elevated expression of both markers. It is most likely that the alloantigens expressed in the kidney or in secondary lymphatic organs by donor-derived dendritic cells activated the specific T lymphocytes. The allospecific T cell activation promoted higher transgene expression by T lymphocytes inside the transplanted kidney.

Several groups, including our own, have recently shown the therapeutic potency of adenovirus-mediated intragraft delivery of regulatory gene products (20,21). However, the adenovirus-associated immune response against the transduced cells destroys the endothelium, thereby allowing gene expression only for a short time. Limited expression and virus-associated graft destruction are major disadvantages of this approach that should be less important for our new T cell–based delivery techniques. We were able to demonstrate that the transduced TEGFP lymphocytes survived in vivo for at least 18 d, although the number of transgenic cells found in the allotransplanted kidney were 5- to 6-fold lower when the cells were injected in naive animals 14 d before transplantation than when they were injected immediately after transplantation. We think it is quite remarkable that the transgenic cells survived for 14 d despite the absence of alloantigen in this pretransplant period and the immunogenic potential of EGFP. Therefore, the lifespan of transgenic T lymphocytes should not be a limiting factor, because tolerance induction with immunomodulatory proteins seems to depend on high transgene expression soon after transplantation and not on long-term suppression promoted by gene-engineered cells.

As described in the characterization of the DA-specific T lymphocytes, the cells resemble a Th1 related phenotype. Alloreactive behavior may be a major limitation in this approach. Interestingly, the acute rejection process in our DA to LEW kidney transplantation model was not accelerated after the adoptive transfer of alloantigen-specific T lymphocytes. This is a remarkable fact because of the high number of 1.5 x 107 cells that were injected per animal. However, we cannot rule out that these alloreactive T cells may lead to accelerated graft rejection in our model system because of the short time to rejection (6 to 7 d). To address this question, future studies will be required when testing alloreactive T cell lines that express immunomodulatory transgenes, because the outcome of the effect of a particular gene may be dependent on the interplay between the effect of the T cell lines on graft rejection and the effect of the gene delivered. We assume that much lower numbers of lymphocytes expressing immunomodulatory transgenes could be sufficient to interfere with the immune response against transplanted grafts, resulting in an additional decrease in the risk of as yet undetected alloreactive behavior.

In summary, alloantigen-specific, genetically-engineered T lymphocytes preferentially accumulated in allografts but not in syngeneic or third party control grafts. This process was driven by alloantigens, elevating total numbers of EGFP-expressing T cells and/or promoting antigen-directed homing. The cells inside the allografts showed an activated phenotype, which is necessary for enhanced transgene-expression. Higher EGFP-expression inside the GIC was a direct proof for this elevated expression. The recruitment, specific activation, and enhanced transgene expression fulfill the critical demand in delivering therapeutic proteins to local microenvironments such as grafts or secondary lymphatic organs. This novel approach in transplantation gene therapy can be considered as an antigen-specific targeting strategy. Although we do not know the localization of draining LN after kidney transplantation, we speculate that allospecific T cells may also deliver the gene of interest in response to the respective alloantigen in secondary lymphoid organs. Further investigations will clarify the potential of gene-engineered T lymphocytes in transplantation gene therapy by using immunomodulatory proteins as therapeutic molecules.


   Footnotes
 
1 Authors contributed equally to the work presented in this article. Back


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication July 12, 2001. Accepted for publication October 4, 2001.




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