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Eur J Cardiothorac Surg 2006;30:362-369
© 2006 Elsevier Science NL

Inhibition of T cell homing by down-regulation of CD62L and the induction of a Th-2 response as a method to prevent acute allograft rejection in mice

Christian Stremmela,c,*, Wulf Sienela, Stephan Eggelinga, Bernward Passlicka, Anthony Slavinb

a Department of Thoracic Surgery, Albert-Ludwigs-University Freiburg, Hugstetterstrasse 55, 79106 Freiburg, Germany
b Department of Medicine, Division of Immunology and Rheumatology, Stanford University Medical Center, Stanford, CA 94306, USA
c Center for Neurological Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA

Received 20 September 2005; received in revised form 29 March 2006; accepted 4 April 2006.

* Corresponding author. Address: Department of Thoracic Surgery, Albert-Ludwigs-University Freiburg, Hugstetterstrasse 55, 79106 Freiburg, Germany. Tel.: +49 761 2702455; fax: +49 761 2702459. (Email: christian.stremmel{at}uniklinik-freiburg.de).


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A
 References
 
Objective: For a successful immune response, migration of lymphocytes to lymphoid organs and other tissues is a key step, as the initial recognition of foreign antigens and activation of lymphocytes takes place in these organs. CD62L is a homing receptor that mediates entry of naïve T cells to peripheral lymph nodes. Maybe the preventing of T cell homing will change the immune response against allogeneic tissue and suppress rejection. Methods: We treated different mouse strains with pertussis toxin to manipulate T cell homing and measured the rejection of allografts in terms of allogeneic tumor cells. We transferred pertussis toxin treated or nontreated transgenic T cells into BALB/c wild type mice. The transgenic T cells could be followed ex vivo by specific antibodies. Cytokine production from purified (1 x 105/ml) T cells after different stimulations in vitro and expression of surface markers on T cells following pertussis toxin treatment by FACS analysis were performed. Results: Pertussis toxin-treated C57BL/6 mice with the MHC class I molecule H-2Kb could not reject allogeneic tumor cells R1.1, which expressed the MHC class I molecule H-2Kk and were killed by these cells. This allograft survival could be demonstrated for various allogeneic cells in different mouse strains with different MHC class I expression and emphasizes the general mechanism in these studies. In vivo CD62L expression on T cells was down-regulated by pertussis toxin in normal mice and transgenic mice that produce only one specific T cell, and after the pertussis toxin treatment the mice showed 4–5 times larger spleens compared to untreated mice. In transfer experiments, we demonstrated that CD62L low transgenic T cells could not home to lymph nodes. Furthermore, spleen cells from pertussis toxin-treated mice produced high amounts of the Th-2 cytokine interleukin 4 after stimulation in primary culture. Conclusions: Our data suggest that the inhibition of T cell homing changes the immune response. Prevention of homing of T cells in combination with the induction of a Th-2 response is a mechanism to prevent specific acute rejection of allogeneic tissue.

Key Words: Transplantation • Homing • CD62L • Allograft rejection


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Appendix A
 References
 
Modern allotransplantation requires the daily administration of nonspecific immunosuppressive agents to prevent T cell-mediated acute rejection [1]. There has been a lot of progress in this field in the last decades, including an exciting array of new agents that selectively inhibit calcineurin activity, purine metabolism, IL-2 receptor function, or cytokine gene transcription. The agents commonly used include glucocorticoids [2], antiproliferative agents such as azathioprine or mycophenolate mofetil (MMF) [3], and calcineurin inhibitors (tracolimus or cyclosporine A) [4]. Although these agents have considerably improved the outcome of patients after transplantation, their effects are only transient, and all are associated with substantial toxicity [5]. Monoclonal antibodies directed against IL-2 receptors have recently been tested in phase III studies with cyclosporin A with excellent graft and patient survival [6]. Treatments directed against activated T lymphocytes that are responsible for acute rejection has become more and more the focus of interest. With increased knowledge of T cell activation and proliferation, the goal of these new therapies is the development of more specific treatments with less toxicity.

The surveillance of the body for foreign antigens is a critical function of the immune system and is dependent on the circulation of lymphocytes. To induce a successful immune response, migration of lymphocytes to lymphoid organs (homing) and other tissues is a key step, as the initial recognition of foreign antigens and activation of lymphocytes takes place in this organ in most situations [7,8]. L-selectin is a well-characterized homing receptor that mediates the selective attachment of leukocytes to specialized high endothelial venules [9]. L-selectin, also called CD62L, was first identified as the peripheral lymph node (PLN) specific homing receptor in mice [9,10] and has been shown to be important both for the homing of T cells to PLN and traffic of other leukocytes to sites of inflammation [11,12]. Beside this effect, CD62L-deficient mice showed an impaired primary T cell response including the delayed-type hypersensitivity (DTH) [13]. Finally, treatment of lymphocytes with pertussis toxin (PTX) inhibited migration to PLNs [14] and also mature thymocytes expressing transgenic PTX homed poorly in adoptive transfer experiments [15].

Based on these observations, a key question that now arises is whether down-regulation of CD62L on T cells could be used as a new treatment to prevent allograft rejection in the acute phase. To address this problem we treated different mouse strains that expressed different MHC class I molecules with PTX and measured the rejection of allografts in terms of allogeneic tumor cells. The hypothesis is that preventing of homing of T cells that reject allogeneic tissue could change the immune response in the donor and prevent rejection of tissue.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Appendix A
 References
 
2.1 Animals
Female C57BL/6, BALB/c and SJL mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Every mouse strain express different MHC class I molecules and therefore represent different immune systems with a complete different allogeneic immune response. The mouse strain DO11.10 is a transgenic mouse that produce one specific T cell that recognize the antigen OVA, which is a protein. The T cell receptor of this transgenic T cell is known and therefore it is possible to find these T cells in lymph nodes or spleens by a specific antibody (TgOVA). DO11.10 mice express the same MHC class I molecules like the BALB/c mouse strain. DO11.10 were bred and maintained at Brigham and Womens’ Hospital. All mice used were between 7 and 10 weeks of age. They were housed in Harvard Medical School Animal Care Facilities in accordance with that of institutions’ guidelines.

2.2 Cell lines
The tumor cell lines used in this study were obtained from the American Type Culture Collection (ATCC). The carcinogen-induced lymphoma EL4 is of C57BL/6 origin that expressed the MHC class I molecule H-2Kb (ATCC). The EL4 cell line was transfected with B7-1 which has been already described [16]. R1.1 lymphoma cell line that expressed the MHC class I molecule H-2Kk origin was also purchased from ATCC. The cell lines EL4 and R1.1 did not produce the following cytokines: interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 10 (IL-10), interleukin 12 (IL-12), IFN-{gamma}, and TNF-{alpha}. Also the EL4-wt cells did not affect the homing or the activation or the deviation of T-cells [23]. All cell lines were maintained at 37 °C in 10% CO2 in DMEM (Life Technologies) containing 10% FCS (Fetalclone I; Hyclone), 1 mg/ml G418 (Life Technologies), and/or 800 mg/ml hygromycin B (Boehringer Mannheim).

2.3 Animal studies
The mouse strain C57BL/6 that expressed the MHC class I molecule H-2Kb and the mouse strain SJL that expressed the MHC class I molecule H-2Ks were prepared for intradermal cell implantation by shaving their hind flank regions followed by depilation with Nair (Carter Wallace, Inc.) 24 h before intradermal implantation of tumor cells. Also, 2 x 106 allogeneic R1.1 (H-2Kk) tumor cells were injected intraperitoneal using a 1 ml syringe fitted with a 27-gauge needle. Tumor cells were harvested in log phase growth from tissue culture flasks and washed four times with PBS (Bio-Whittaker) and resuspended at 4 x 107 cells/ml in PBS for implantation. Each intradermal injection consisted of 2 x 106 cells in 50 µl PBS and was performed using a 1 ml syringe fitted with a 27-gauge needle. Five to seven days after implantation, a tumor could be observed at the implantation site. The mice were scored for tumor growth three times per week and tumor size was documented by direct measurement in three perpendicular directions using a Max-Cal caliper (Cole Parmer Instrument Co.) and a plastic ruler. The experiments were terminated when the tumors reached 20–22 mm in diameter, if severe ulceration and bleeding had developed, or the mice had died. The measurements were recorded as tumor volumes (mm3) from groups of five mice each.

The results of the in vivo animal studies are based on five mice in each group. The tumor volume in the figures is the median of five mice.

2.4 Cell culture of lymphocytes
Whole spleen cells were cultured in triplicates at a concentration of 1 x 105cells/well in a flat-bottom 96-well plate in a total volume of 200 µl/well for 48 h under various stimulation conditions. For activation 2% T-STIM, anti-CD3 (1452C11) (0.1 µg/ml), PMA (0.5 µg/ml) or PTX (1 µg/ml) in DMEM or DMEM medium alone were used. The spleen cells from the transgenic mice (DO11.10) were activated by different concentrations of OVA. The T cell receptor of these transgenic T cells was called OVA TcR and could be identified by an antibody (TgOVA).

The plates were incubated at 37 °C in a humidified CO2 incubator for 48 h. The plates were then pulsed with 1 µCi of [3H] TdR per well for 16 h, then harvested using a Tomec Mach II 96 Cell Harvester and counted on a 1205 Betaplate liquid scintillation counter (Wallac, Inc., Gaithersburg, MD, USA).

2.5 ELISA for cytokines
For cytokine assays, cells were cultured in 24-well plates at 5 x 105 cells/ml in serum-free medium, X-Vivo 20 (Biowhittaker).

To determine cytokines, culture supernatants were collected for IL-2 and IL-4, IL-10 and IFN-{gamma}. The results from each experiment are the mean of a triplicate. Each group represent the pooled data of three mice supernatants that were collected from T cells (1 x 105/ml) or spleen cells (5 x 105/ml) 40 h after activation in vitro. The concentrations of IL-2, IL-4, IL-10, IFN-?{gamma}, and TNF-{alpha} were measured by quantitative capture ELISA according to the guidelines of the manufacturers (PharMingen). In brief, purified rat mAb to mouse IL-2 (clone JES6-1A12), IL-4 (clone BVD4-1D11), IL-10 (clone JES5-2A5), IFN-?{gamma} (clone R4-6A2), and TNF-?{alpha} (clone MP6-XT22) were obtained from PharMingen and used to coat ELISA plates (Immulon 4; Dyna-tech Laboratories, Inc.). Recombinant mouse cytokines (IL-2, IL-4, IL-10, IFN-?{gamma}, and TNF-?{alpha}; PharMingen) were used to construct standard curves, and biotinylated rat mAb to mouse IL-2 (clone JES6-5H4), IL-4 (clone BVD4-24G2), IL-10 (clone SXC-1), and IFN-?{gamma} (clone XMG1.2; all PharMingen) were used as the second Ab. Detection of TNF-{alpha} was performed with biotinylated polyclonal rabbit IgG (PharMingen). Plates were developed with TMB microwell peroxidase substrate (Kirkegaard & Perry Laboratories, Inc.) and read after the addition of stop solution at 450 nm using a microplate reader (model 3550; Bio-Rad Laboratories).

2.6 FACS staining
Spleen cells and tumor cells from mice or those growing in culture were harvested and washed three times with cold 1% BSA/PBS, pH 7.2, and then incubated with either supernatant from antibody-producing hybridomas or purified antibody (5 mg/ml) diluted in 1% BSA/PBS for 30 min at 4 °C. The cells were then washed two to three times with the 1% BSA/PBS solution before incubating with FITC- or PE-conjugated goat anti-mouse Ig, goat anti-rat Ig, or goat anti-human Ig secondary antibodies (Zymed). The secondary antibodies were diluted 1/50 in 1% BSA/PBS and then incubated with the cells for 30 min at 4 °C in the dark. After this incubation, the cells were washed three times with PBS and then fixed with an equal volume of 1% paraformaldehyde/PBS solution. Analysis was performed using a FACScan TM (Becton Dickinson). FITC- and PE-conjugated anti-bodies used for direct staining were obtained from PharMingen and included the following antibodies: anti-CD3-FITC or PE (clone 145-2C11), anti-CD4-FITC (clone GK1.5), anti-CD8{alpha}-FITC (clone 53–6.7), anti-CD25-FITC (clone PC61), anti-CD28-PE (clone 37.51), anti-CD54-FITC (clone 3E2), anti-CD62L-FITC (clone MEL14), anti-CD69-FITC (clone H1.2F3), anti-CD86-PE (clone GL1, anti-CD154 (clone 16.10A1) or anti-B220-FITC (RA-3-6B2). Each group represent the pooled data of two mice.

2.7 Adoptive transfer of spleen cells from OVA transgenic mice
To test the biological function of PTX on T cells, spleen cells from transgenic mice that produce one specific T cell that recognize the antigen OVA, have been treated in vivo with PTX (on days 0 and 2) after that these spleen cells were intravenously injected into BALB/c mice. The transgenic mouse strain, DO11.10, express the same MHC class I molecules like the BALB/c mouse strain. The T cell receptor of these transgenic T cells was called OVA TcR.

Spleen cells with OVA-specific T cells from transgenic mice that have been treated with 200 ng PTX or PBS for 72 h were adoptively transferred to BALB/c recipients. 2 x 107 spleen cells have been transferred to each BALB/c mouse. The BALB/c mice were pre-immunized with soluble OVA (50 µg) one day before the adoptive transfer of the spleen cells. Some BALB/c mice were treated with PTX on the day of transfer and 2 days prior. Two days after the adoptive transfer of the spleen cells lymph nodes from BALB/c mice were harvested for analysis of OVA TcR transgenic T cell distribution. Each group represent the pooled data of two mice.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Appendix A
 References
 
3.1 The effect of pertussis toxin on allogeneic tissue rejection in vivo
To study the effect of pertussis toxin on acute rejection, R1.1 (H-2Kk) allogeneic tumors were implanted intraperitoneally into C57BL/6 (H-2Kb) mice that were either untreated (control) or subsequently administered 200 ng PTX, i.v. on days 0 and 2 after tumor implantation. Control mice appeared to reject the tumor and survived; however, PTX-treated C57BL/6 mice could not reject allogeneic R1.1 tumor cells and were killed by these allogeneic cells 15–20 days after implantation (Fig. 1A). The effect of PTX treatment was also observed on tumors implanted intradermally. Control mice were able to reject intradermally implanted tumors 10–15 days post-implantation; however, treatment with PTX on days 0 and 2 increased tumor burden and delayed rejection until 18–25 days after implantation (Fig. 1B). Moreover, this effect of PTX was dose dependent. Mice treated with PTX on days 0, 2, 7, 14, 21, 28, and 35 after implantation were not able to successfully reject the intradermally implanted allogeneic tumor (Fig. 1B). Only after stopping the weekly PTX injection were the allogeneic tumor cells rejected.


Figure 1
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  		Fig. 1. C57BL/6 mice treated with PTX fail to reject allogeneic tumor. (A) C57BL/6 mice (H-2Kb) were implanted intraperitoneally with 2 x 106 allogeneic R1.1 (H-2Kk) tumor cells. In addition, the mice were treated intravenously on the day of implantation of the tumor cells and 2 days later with 200 ng PTX (\#9826;) or PBS ({square}). Over a period of 30 days the survival of the animals was observed. One of three independent experiments is shown. (B) C57BL/6 mice were injected intradermally with 2 x 106 allogeneic R1.1 (H-2Kk) tumor cells without PTX injections ({square}). In addition, the mice were treated intravenously on the day of tumor implantation and 2 days later with 200 ng PTX (\#9826;) or 200 ng PTX on days 0, 2, 7, 14, 21, 28 and 35 ({circ}). Tumor growth was assessed every 2–3 days by measuring in three perpendicular directions. The results are expressed as mean tumor volumes in cubic millimeter. Each group represent the pooled data of five mice. One of three independent experiments is shown.

 
To ensure that the effect of the PTX was not tumor specific, we implanted B7-1 transfected T cell thymoma EL4 cells (EL4-B7-1) into syngeneic wild type C57BL/6 mice. Indeed, wild type control mice implanted with 2 million EL4-B7-1 cells intradermally rejected the tumor between days 15 and 20 after implantation (Fig. 2A). By contrast, C57BL/6 mice treated with PTX, i.v. on days 0 and 2 after implantation, had not rejected the syngeneic tumor expressing B7-1 by day 30 (Fig. 2A).


Figure 2
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  		Fig. 2. Pertussis treated mice fail to reject tumor cells. (A) C57BL/6 mice were implanted intradermally with 2 x 106 B7-1 transfected EL4 (H-2Kb) tumor cells. In addition, the mice were treated intravenously on the day of implantation and 2 days later with 200 ng PTX (\#9826;) or PBS ({square}). (B) SJL mice (H-2Ks) were implanted intradermally with 2 x 106 allogeneic EL4-wt (H-2Kb) tumor cells. In addition, the mice were treated intravenously on the day of implantation and 2 days later with 200 ng PTX (\#9826;) or PBS ({square}). Tumor growth was assessed every 2–3 days by measuring in three perpendicular directions. The results are expressed as mean tumor volumes in cubic millimeter. Each group represent the pooled data of five mice. One of three independent experiments is shown.

 
To rule out the possibility that the observed effect was due in part to the genetic background of the mouse, we also tested whether PTX treatment affected rejection of an allogeneic tissue in another system. Further, in this system we used the mouse strain SJL with the MHC class I expression of H-2Ks. Also in this experiment the PTX-treated mice (SJL), could not reject the allogeneic tumor EL4 (H-2Kb)(Fig. 2B).

3.2 Expression of T cell markers on T cells after treatment with PTX
To determine why the PTX-treated mice were unable to reject allogeneic tumor cells, T cells from spleens of PTX treated and untreated C57BL/6 mice have been purified and looked for the expression of surface markers by FACS analysis. As shown in Fig. 3 , similar levels of CD4 and CD25 were observed in PTX treated and nontreated mice. However, a slight increase in CD69 levels and a dramatic reduction in CD62L expression (from 56% to 18%) was observed in PTX-treated mice (Fig. 3). PTX had no effect on the numbers of CD4+ CD25+ T cells. Also we did not find any differences in the expression of CD3, CD28, CTLA-4, CD54, CD86, CD45/B220, or CD154 on PTX-treated or nontreated T cells (data not shown). The recovery of the CD62L expression took more than 5 days. To confirm our findings we tested splenocytes from transgenic mice (D011.10) treated with PTX. The T cell receptor of this transgenic T cell is known and therefore it was possible to find these T cells in lymph nodes or spleens by a specific antibody (TgOVA). We again found that PTX treatment had little effect on CD4, CD3, or CD69 expression levels; however, a dramatic reduction in CD62L expression from 35% to 11% was found in splenocytes from PTX-treated mice (Table 1 ).


Figure 3
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  		Fig. 3. Expression of surface markers on T cells following PTX treatment by FACS analysis. T cells were purified (>95%) from the spleens of mice implanted with allogeneic tumor cells and which have been treated once with 200 ng PTX or only with PBS (control) for 3 days. After 72 h the purified T cells were stained for expression of CD4, CD25, CD62L, CD69 and the control IgG-isotype. The antibodies were directly conjugated with PE or FITC. A total of 10,000 cells were analyzed per sample by flow cytometry. Similar results were obtained in a total of three independent experiments. Each group represent the pooled data of three mice.

 

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  		Table 1. Surface markers on T cells of transgenic spleen cells treated with PTX
 
3.3 Expansion and homing of transgenic T cells after PTX treatment
We have observed that PTX-treated T cells had down-regulated CD62L but remained antigen specific and reactive. The T cell receptor of these transgenic T cells is called TgOVA and is found by an antibody. In order to determine if the failure to reject allogeneic graft was due to their failure of homing to appropriate sites, we transferred PTX-treated or nontreated OVA-specific transgenic T cells (TgOVA) into PTX-treated or nontreated BALB/c wild type mice, which have been pre-immunized 2 days prior with soluble OVA (50 µg), and analyzed the distribution of the transferred transgenic T cells (TgOVA) three days after transfer. Reduced numbers of donor cells from PTX-treated mice were found in both spleens (Table 2 ) and lymph nodes (Fig. 4 and Table 2) of the recipient mice, indicating that these treated cells could not home to sites of antigen presentation. The homing of the PTX-treated T cells was independent of the treatment of the recipient. In the lymph nodes, the amount of transgenic T cells have been up to 25 times higher in untreated transgenic T cells than of PTX-treated T cells. Because of the higher amount of T cells in the lymph nodes which could be activated by APCs and proliferate, there was also a twofold increase of nontreated PTX transgenic T cells in the spleen (Table 2).


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  		Table 2. Surface markers on lymph node and spleen cells treated or untreated with PTX
 

Figure 4
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  		Fig. 4. FACS staining of BALB/c lymph node cells following adoptive transfer of PTX-treated transgenic T cells. OVA-specific T cells from transgenic mice have been treated with 200 ng PTX or PBS and after 72 h the OVA-transgenic spleen cells were adoptively transferred to BALB/c recipients that were pre-immunized with soluble OVA (50 µg). Two days later lymph nodes were harvested for analysis of OVA TcR transgenic T cell distribution. The lymph node cells were stained for expression of CD4, CD8, CD62L, CD69 and the transgenic TcR and the control IgG-isotype. The antibodies were directly conjugated with PE or FITC. A total of 20,000 cells were analyzed per sample by flow cytometry. Similar results were obtained in a total of three independent experiments. Each group represent the pooled data of two mice.

 
3.4 Proliferation of T cells after PTX treatment in vivo
The failure to reject allogeneic graft was not due to deletion of T cells following PTX treatment. Splenocytes from mice used in Fig. 1B were stimulated in vitro under different conditions to determine the reactivity of the T cells. Spleen cells from the control mouse (day 7), the mouse treated twice with PTX (day 7 + PTX) and the mouse which got seven injections of PTX (day 7 + PTX) showed a good proliferative response to nonspecific T cell stimulation with 100 ng/ml anti-CD3, 500 ng/ml PMA and almost no response to IL-2 containing medium. PTX-treated spleen cells showed a reduced response to PMA stimulation (Fig. 5 ); however, there was no difference for stimulation with the anti-CD3 antibody. T cells from mice treated with PTX showed no response to stimulation by 1 µg PTX in vitro (Fig. 5).


Figure 5
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  		Fig. 5. Proliferative response of spleen cells of mice implanted with allogeneic tumors. (A) Whole spleen cells (1 x 105/well) from untreated mice, mice bearing R1.1 allogeneic tumors 7 days after treatment with PTX (days 0 and 2) or after 42 days after treatment (PTX on days 0, 2, 7, 14, 21, 28, 35) were activated in vitro with 2% T-STIM, anti-CD3 (1452C11) (0.1 µg/ml), PMA (0.5 µg/ml) or PTX (1 µg/ml) in DMEM or DMEM medium alone. The results from each experiment are the mean of a triplicate. These are the results of three independent experiments. Each group represent the pooled data of three mice.

 
3.5 Cytokine production of T cells after PTX treatment in vivo
Splenocytes form mice in Fig. 1B were also analyzed for cytokine production after different stimulation signals. There was no cytokine production of purified T cells after stimulation with DMEM with 2% T-STIM (data not shown). In a MLR with mitomycin C-treated allogeneic R1.1 tumor cells and spleen cells from mice (C57BL/6) which have been injected in vivo with the described allogeneic tumor and also which have been treated with 0, 2 or 7 injections of PTX produced large amounts of IFN-{gamma} and moderate amounts of IL-2 (data not shown). Only the spleen cells from mice treated seven times with PTX produced reasonable amounts of IL-4 (400 pg/ml) (data not shown). Following stimulation with PMA or anti-CD3, all groups produced large amounts of IFN-{gamma} and much less IL-2; however, only PTX-treated T cells produced IL-4 (Fig. 6A and B). In all the figures we did not include the IFN-{gamma} production after the stimulation because the amounts have been between 40,000 and 50,000 pg/ml, independent of the group. The amount of IL-4 production was dependent on the number of PTX injections in vivo and reached 12,000 pg/ml in the primary culture (Fig. 6A and B). Interestingly, PTX-treated T cells produced IFN-{gamma} and IL-4 again after stimulation with PTX in vitro (data not shown), although the PTX-treated T cells did not show any proliferative response to PTX in vitro (Fig. 5). We also looked for TGF-ß production, but none of the T cells produced this cytokine.


Figure 6
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  		Fig. 6. Cytokine production from purified (1 x 105/ml) T cells after different stimulations in vitro. The cytokine production of T cells after 40 h of activation with 0.5 µg/ml PMA (A) or 0.1 µg/ml anti-CD3 (B). The cells were established from mice which have been from R1.1 bearing mice which were not PTX treated (day 7), mice bearing R1.1 allogeneic tumors 7 days after treatment with PTX (day 7 + PTX) on days 0 and 2 or after 42 days after treatment with PTX (day 42 + PTX) on days 0, 2, 7, 14, 21, 28, 35 and stimulated under the different conditions. Cytokines were measured by an ELISA as described in Section 2. One of two independent experiments with similar results is shown. Each group represent the pooled data of three mice.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Appendix A
 References
 
We have examined the role of T cell homing and especially the homing receptor CD62L on the acute rejection of allograft tissue under PTX treatment. Our studies using allogeneic tumor tissue in different mouse strains (C57BL/6 and SJL) to investigate acute rejection of allograft tissue. Normally tumor cells grow only in syngeneic hosts and are rejected in allogeneic immune system competent hosts. Alloreactive T cells that respond directly to foreign major histocompatibility complex (MHC) molecules and bound peptide are known to be central mediators of allograft rejection. We treated mice in vivo weekly with pertussis toxin (PTX) to prevent T cell homing. In studies have already been established that the treatment of lymphocytes with pertussis toxin (PTX) inhibited G protein-linked proteins which are important for rolling of these cells and also PTX down-regulated the homing receptor CD62L [14]. Finally, treatment of lymphocytes with PTX inhibited migration to peripheral lymph nodes (PLNs) [14].

4.1 The effect of pertussis toxin on allogeneic tissue rejection
In this model, mice were killed by allogeneic tumor cells although they were immunocompetent. Moreover, allogeneic tumor cell growth could be induced in different allogeneic systems, using multiple tumors and mouse strains (Figs. 1 and 2), further supporting the general mechanism that is responsible for prevention of allograft rejection in these studies. In addition, the inhibition of acute rejection took place only during the treatment of mice with pertussis toxin. Furthermore, PTX-treated T cells showed still the same reactivity to specific antigens (transgenic T cells) but with a higher threshold of activation (data not shown). Beside that PTX-treated mice developed a Th-2 response in the primary culture (Fig. 6), which is another important reason for the prolongation of the survival of the allogeneic tissue. Furthermore, in the experiment with the EL4-B7.1 cells, which is shown in Fig. 2A, we could demonstrate that the Th1 response induced by EL4-B7.1 tumor cells [16] could be overcome by PTX. This demonstrated that PTX has also an effect on activated T cells.

We propose two major mechanisms that are in combination responsible for the inhibition of acute rejection in this model. First of all, PTX down-regulated CD62L on T cells which we demonstrated in C57BL/6 and transgenic mice. In transfer experiments PTX treated, CD62L low expressing, transgenic T cells (TgOVA) could not home to lymph nodes by normal distribution in spleens (Fig. 4 and Table 2). For T cell activation and effector function it is essential that naïve cells home to PLNs to be stimulated by professional APCs as antigen stimulation of naïve T cells without costimulatory molecules induced anergy or tolerance [17,18]. By the changes through T cell homing and T cell activation a Th-2 response was induced, which support tolerance and allograft survival [19,20].

4.2 The impact of T cell homing on allogeneic tissue rejection
This recirculation increases the likelihood that naïve lymphocytes, existing in relative low frequency in terms of antigenic specificity, will encounter its cognate antigen and become activated by professional APCs to reach their effector function [7,13]. In this context L-selectin (CD62L) plays a central role and therefore we focused on this molecule to explain our results. This does not preclude effects on chemokine receptors, which equally could account for the failure of T cells to home.

The concept that CD62L is essential for lymphocyte traffic to PLNs was demonstrated in CD62L-deficient mice, which display a 90% reduction in the size and cellularity of PLNs due to a greatly reduced influx of lymphocytes via high endothelial venule (HEV) [21,22]. We made similar observations after PTX treatment of mice, to those described before [14]. The lack of T cells in PLNs in our experiments was not caused by depletion of T cells. We stained spleen cells for Vß expression and found the same distribution of TcR usage in treated and untreated mice (data not shown). Furthermore, other groups have already shown that PTX inhibits activation-induced cell death of thymocytes. Beside that transgenic T cells for OVA and normal purified T cells could also be activated in vitro after PTX treatment. Taken together, these observations suggested that lack or down-regulation of CD62L on lymphocyte surfaces slows extravasation of naïve and recirculating T cells into PLN, which is responsible for the lack of primary allogeneic T cell response. Other groups also reported that CD62L-deficient mice failed to generate a DTH response at a conventionally studied time after immunization [13,22] in excellent agreement to our results. A higher threshold of T cell activation prevents efficient allograft rejection.

4.3 The impact of a Th-2 response on allogeneic tissue rejection
We favor this impaired homing of T cells to PLNs, in connection with the observation of the naïve T cell activation state with a higher activation threshold as the leading reason for inhibition of allograft rejection, because changes in the threshold of T cell activation resulted in changes of the T cell response and induced a Th-2 response. Treatment of mice with PTX induced a Th-2 response in the primary culture after stimulation over the TcR with antibody against CD3 or by nonspecific stimulation with the phorbolesther PMA. The amount of IL-4 production was dependent on the time period of PTX treatment in vivo (Fig. 6). Other groups in the research field of transplantation have demonstrated that a Th-2 response support transplantation survival [19,20]. One of the main reasons for tolerance induced in newborns or the unique susceptibility of neonatal T cells to tolerance is based on a predominating Th-2 function in neonatal mice. These findings address the importance of Th-2 cytokines in allograft survival and confirm our observation concerning IL-4 production in PTX-treated mice and the impact on prevention of allograft rejection. This Th-2 response was an additional important effect in the combination with the inhibition of T cell homing in the survival of allograft tissue. This Th-2 response may not be an independent mechanism of the prevention of T cell homing, it seems to be a result of it.

In conclusion, the data presented here show that lymphocytes from mice, in which T cells had no or only low expressing CD62L, are defective in homing to PLN, and fail to develop a normal primary T cell response to allogeneic tissue. In this experiments we could not show if prevention of homing or the immunodeviation to a Th-2 response is the most important reason for prolongation of allogeneic tissue. It seems that the Th-2 response is a result of the impaired T cell homing and that only both results together are important for the demonstrated effects. This hypothesis is emphasized by the observation, that cyclosporin A also has an impact on T cell homing but it cannot induce a Th-2 response. Also, further investigation is necessary to find a correlation between the induction of a Th-2 immune response and the prevention of homing of T cells. Maybe there are other immunomodulatory proteins like PTX, which could induce both effects. Further studies are necessary to optimize this approach for clinical applications and to investigate the effect of such treatment in the combination with already established immunosuppressive treatments for allograft rejection.


   Appendix A
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A
References
 
Conference discussion

Dr W. Klepetko (Vienna, Austria): I have to say I had a bit of difficulty to catch all the information that you have forwarded in this short period of time to us, so it's a little bit difficult to get the overview of all the contents of the paper.

My question would then be, when you were talking about the tumor model, how relevant is that, then, really for chronic rejection?

Dr Stremmel: What is more interesting in the tumor model than in the transplantation model, this allogenic tissue, that's a tumor, it has a potential to manipulate the immune response. So it's even more difficult to prolong survival of an allogenic tumor in an immune system than to prolong the survival of a transplant organ. The mechanisms are the same.

Dr Klepetko: I would just like to add. Your initial statement that chronic rejection is there with the prevalence of 50% after 5 years, recent data from multicenter studies, luckily enough, gives a bit better view with freedom from chronic rejection after 3 years in the range of 73%. So there has been some improvement. No doubt it's still a very important and relevant clinical problem.


   Acknowledgments
 
We thank Jim Rottman and B. Igreem for critical reading of the manuscript and helpful discussions. We also thank V. Kochroo who supported these experiments. A.J.S was supported by the NMSS and Christian Stremmel by the DFG.


   Footnotes
 
{star} Presented at the joint 19th Annual Meeting of the European Association for Cardio-thoracic Surgery and the 13th Annual Meeting of the European Society of Thoracic Surgeons, Barcelona, Spain, September 25–28, 2005.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A
 References
 

  1. Pirsch JD. Long-term management of immunosuppression in the renal transplant patient. Curr Opin Organ Transplant 1997;2:76-81.
  2. Auphan N, DiDonato JA, Rosette C, Helmberg A, Karin M. Immunosuppression by glucocorticoids: inhibition of NF-kB activity through induction of IkB synthesis. Science 1995;270:286-290.[Abstract/Free Full Text]
  3. Mor E, Gonwa TA, Husberg BS, Goldstein RM, Klintmalm GB. Late-onset acute rejection in orthotopic liver transplantation—associated risk factors and outcome. Transplantation 1992;54:821-824.[Medline]
  4. Fruman DA, Klee CB, Bierer BE, Burakoff SJ. Calcineurin phosphatase activity in T lymphocytes is inhibited by FK506 and cyclosporin A. Proc Natl Acad Sci USA 1992;89:3686-3690.[Abstract/Free Full Text]
  5. Keown PA. Therapeutic strategies for optimal use of novel immunosuppressants. Transplant Proc 1999;31:1790-1792.[CrossRef][Medline]
  6. Nashan B, Moore R, Amiot P. Lancet 1997;350:1193-1196.[CrossRef][Medline]
  7. Butcher EC, Picker LJ. Lymphocyte homing and homeostasis. Science 1996;272:60-66.[Abstract]
  8. Picker LJ, Butcher EC. Physiological and molecular mechanisms of lymphocyte homing. Ann Rev Immunol 1992;10:561-591.[CrossRef][Medline]
  9. Gallatin WM, Weissman IJ, Butcher EC. A cell-surface molecule involved in organ-specific homing of lymphocytes. Nature 1983;304:30-34.[CrossRef][Medline]
  10. Siegelmann MH, van de Rijn M, Weissmann IL. Mouse lymph node homing receptor cDNA clone encodes a glycoprotein revealing tandem interaction domains. Science 1989;243:1165-1172.[Abstract/Free Full Text]
  11. Ley K, Bullard DC, Arbones ML, Bosse R, Vestweber D, Tedder TF, Beaudet AL. Sequential contributions of L- and P-selectin to leukocyte rolling in vivo. J Exp Med 1995;181:669-675.[Abstract/Free Full Text]
  12. Warnock RA, Askari S, Butcher EC, von Andrian UH. Molecular mechanisms of lymphocyte homing to peripheral lymph nodes. J Exp Med 1998;187:205-216.[Abstract/Free Full Text]
  13. Xu J, Grewal IS, Geba GP, Flavell RA. Impaired primary T cell responses in L-selectin-deficient mice. J Exp Med 1996;183:589-598.[Abstract/Free Full Text]
  14. Spangrude GJ, Braaten BA, Daynes RA. Molecular mechanisms of lymphocyte extravasation. I. Studies of two selective inhibitors of lymphocyte recirculation. J Immunol 1984;132:354-362.[Abstract]
  15. Chaffin KE, Beals CR, Wilkie TM, Forbush KA, Simon MI, Perlmutter RM. Dissection of thymocyte signaling pathways by in vivo expression of pertussis toxin ADP-ribosyltransferase. EMBO J 1990;9:3821-3829.[Medline]
  16. Stremmel C, Greenfield EA, Howard E, Freeman GJ, Kuchroo VK. B7-2 expressed on EL4 lymphoma suppresses anti-tumor immunity by an IL-4 dependent mechanism. J Exp Med 1999;189:919-930.[Abstract/Free Full Text]
  17. Schwartz RH. A cell culture model for T lymphocyte clonal anergy. Science 1990;248:1349-1356.[Abstract/Free Full Text]
  18. Mueller DL, Jenkins MJ, Schwartz RH. Clonal expansion versus functional clonal inactivation: a costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy. Annu Rev Immunol 1989;7:445-480.[Medline]
  19. Shirwan H, Barwari L, Khan NS. Predominant expression of T helper 2 cytokines and altered expression of T helper 1 cytokines in long-term allograft survival induced by intrathymic immune modulation with donor class I major histocompatibility complex peptides. Transplantation 1998;66:1802-1809.[CrossRef][Medline]
  20. He XY, Chen J, Verma N, Plain K, Tran G, Hall BM. Treatment with interleukin-4 prolongs allogeneic neonatal heart graft survival by inducing T helper 2 response. Transplantation 1998;65:1145-1152.[CrossRef][Medline]
  21. Arbones ML, Ord DC, Ley K, Ratech H, Maynard-Curry C, Otten G, Capon DJ, Tedder TF. Lymphocyte homing and leukocyte rolling and migration are impaired in L-selectin-deficient mice. Immunity 1994;1:247-260.[CrossRef][Medline]
  22. Tedder TF, Steeber DA, Pizcueta D. L-selectin-deficient mice have impaired leukocyte recruitment into inflammatory sites. J Exp Med 1995;181:2259-2264.[Abstract/Free Full Text]



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