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The Journal of Immunology, 2001, 166: 2385-2393.
Copyright © 2001 by The American Association of Immunologists

Viral IL-10-Induced Immunosuppression Requires Th2 Cytokines and Impairs APC Function Within the Allograft1

Lihui Qin*,{dagger}, Yaozhong Ding{dagger}, Hideaki Tahara{ddagger} and Jonathan S. Bromberg2,{dagger}

* Department of Pathology, {dagger} Institute for Gene Therapy and Molecular Medicine, and the Recacati/Miller Transplantation Institute, Mount Sinai School of Medicine, New York, NY 10029; and {ddagger} Departments of Surgery and Bioengineering, University of Tokyo, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous reports demonstrated that retroviral mediated gene transfer of viral IL-10 (vIL-10) prolongs allograft survival by decreasing donor-specific cytotoxic T lymphocyte precursor (CTLp) and IL-2-secreting helper T lymphocyte precursor (HTLp) frequency within graft-infiltrating cells (GIC). This report now shows that vIL-10 efficacy is dependent on CD4+ T cells, suggesting that immunosuppression may involve a switch from a Th1 to a Th2 alloresponse. In support of this, anti-IL-4 or anti-murine IL-10 (anti-mIL-10) mAbs, but not anti-IFN-{gamma} mAb, administered at the time of vIL-10 gene transfer prevents enhanced graft survival. Because Th switching involves APC function, GIC were examined for their ability to present alloantigen. GIC from vIL-10-treated grafts were shown to be mostly of recipient (CBA) origin, yet were unable to elicit alloproliferative responses from donor type (C57BL/6) or third party (BALB/c) responders. The inability of vIL-10-treated GIC to stimulate the MLR was not due to the generation of negative regulatory cells or the production of immunosuppressive cytokines such as IL-4, mIL-10, or TGF{beta}. Using fractionated GIC subpopulations, the number of recipient type cells in the allografts was modestly reduced by vIL-10 gene transfer, while maintaining both APC phenotype and alloantigen presenting function. Conversely, after vIL-10 gene transfer, donor type GIC were unable to participate in direct alloantigen presentation. Therefore, local immunosuppression induced by vIL-10 gene transfer is CD4 T cell and IL-4 and mIL-10 dependent, and impairs direct alloantigen presentation through an alteration of donor type APC function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-10 was originally described as cytokine synthesis inhibitory factor (1, 2) and is able to negatively regulate a variety of immune responses (1, 2, 3, 4, 5, 6). Due to its prominent negative effects on IL-12 and IFN-{gamma} production (7, 8), IL-10 channels immunity away from Th1 and toward Th2 responses (1, 2). IL-10 acts directly on CD4+ T cells, leading to the production of a negative regulatory T cell subset (9) and inducing a long-term anergic state in T cells in vitro (10). It was also demonstrated that the immunosuppressive effects of IL-10 are often seen at the level of the APC (5). IL-10 strongly down-regulates class II MHC (3, 11) and B7 expression (12, 13) on monocytes. IL-10 also deactivates macrophages (14), inhibits Ag presentation to Th1 but not Th2 cells (11), suppresses epidermal Langerhans cell APC functions (15, 16), and prevents chemokine expression by monocytes (17). IL-10-treated dendritic cells induce peptide Ag and alloantigen-specific tolerance (18). Murine or human IL-10 also have growth factor activities on a variety of cell types, such as thymocytes, T cells, mast cells, and B cells (19, 20, 21, 22), and these activities may promote immunostimulatory functions. Viral IL-10 (vIL-10),3 a product encoded by EBV, is highly homologous to both murine and human IL-10, especially in the coding region of the mature protein sequence (23). vIL-10 shares many biological properties with murine and human IL-10, including cytokine synthesis inhibitory factor activity and down-regulation of class II MHC expression on monocytes (3, 23). However, vIL-10 does not possess the T cell costimulatory activities of authentic cellular IL-10 (3, 24, 25, 26), which potentially makes vIL-10 a more potent immunosuppressant.

Gene transfer in vivo has the potential to introduce immunosuppressive molecules only into the graft, which would limit systemic side effects and affect the direct interface between the immune system and alloantigen. We previously demonstrated that retroviral mediated gene transfer and expression of vIL-10 significantly prolonged allograft survival without conventional systemic immunosuppression (24). The effect was specific, dose dependent, and restricted to the site of transplantation. Analysis of the cellular infiltrate in the allografts showed a decreased expression of the important lymphocyte cell surface molecules CD2, CD3, CD4, CD8, CD11a/CD18, CD45, CD49d, and CD62 ligand, suggesting decreased lymphocyte migration and activation and reduced precursor frequencies of alloantigen-specific CTLs and IL-2-producing helper T cells (24). In this study, we sought to explore the effects of vIL-10 gene transfer on the Th1/Th2 switch and Ag-presenting function within allografts. We now demonstrate that vIL-10-induced immunosuppression requires the Th2 cytokines IL-4 and endogenous mIL-10. vIL-10 treatment also reduces the number of recipient type cells within allografts, but does not alter their Ag-presenting function. However, vIL-10 reduces donor type cell Ag-presenting function, thus inhibiting direct alloantigen presentation and alloimmune responses.


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

CBA/J (H-2k), BALB/cByJ (H-2d), and C57BL/6J (H-2b) female mice (8–10 wk of age) were purchased from The Jackson Laboratory (Bar Harbor, ME). Timed pregnant C57BL/6 mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN).

Retroviral transfer vectors

Retroviral vectors CRIP-MFG-LacZ, CRIP-MFG-vIL-10, and CRIP-DFG-vIL-10 (at 5 x 105 PFU/ml), which encode {beta}-galactosidase and vIL-10 genes, respectively, under control of the Moloney murine leukemia virus long terminal repeat (MMLV-LTR), were generated in CRIP amphotropic producer cell lines as described (25, 27).

Abs

The GK1.5 rat anti-murine CD4 (28), the 11B11 rat anti-mIL-4 (29), the JES3-19F1.1 rat anti-human IL-10 (30), and the R4-6A2 rat anti-mIFN-{gamma} (31) hybridomas were purchased from the American Type Culture Collection (Manassas, VA). The JES-2A5 rat IgG1 anti-mIL-10 (32) hybridoma was provided by Dr. R. Coffman (DNAX, Palo Alto, CA). These were all grown in culture and purified over protein G columns (Pharmacia-LKB, Piscataway, NJ). The rabbit anti-TGF{beta} Ab (pan specific) and control rabbit IgG were purchased from R&D Systems (Minneapolis, MN). FITC-conjugated 11-5.2 mouse anti-mouse I-Ak (33), FITC 16-10A1 hamster anti-mouse CD80 (B7-1) (34), FITC GL1 rat anti-mouse CD86 (B7-2) (35), FITC 145-2C11 hamster anti-mouse CD3{epsilon}, FITC RA3-6B2 rat anti-mouse CD45R/B220, and FITC MI/70 rat anti-mouse CD11b (Mac-1) were purchased from PharMingen (San Diego, CA).

Cardiac transplantation

The heterotopic, nonvascularized cardiac transplantation model was used. Briefly, donor neonatal C57BL/6 mice were sacrificed, and whole hearts were removed and placed in the s.c. position of the ear pinnae of CBA/J recipients as previously described (24). Ten microliters of viral culture supernatants (5000 PFU) were directly injected into the graft at the time of transplantation. Purified GK1.5 anti-CD4 mAb was injected i.v. at 100 µg for 2 days. Purified 11B11 anti-IL-4, JES-2A5 anti-mIL-10, or R4-6A2 anti-IFN-{gamma} were injected i.v. at 100 µg every other day for six doses. Survival of cardiac allografts was followed with electrocardiogram monitoring (Polygraph 78 Series with preamp and filters; Grass Instruments, Quincy, MA) every other day. Cessation of cardiac electrical activity was the determinant of rejection. There were at least four mice per group. Statistical comparison was performed with Student’s t test. Animals were also sacrificed at selected time points, graft-infiltrating cells (GIC) were isolated for MLR and flow cytometry, and grafts were homogenized for cytokine measurements by ELISA.

Isolation of GIC, depletion of CD4+ cells, and separation of recipient type cells (H-2k) and donor type cells (H-2b)

Grafts were removed 10 days after transplantation and gently dissociated into single cell suspensions through a nylon screen. RBC were removed by Tris-NH4Cl lysis. CD4+ cells were depleted using Dynabeads Mouse CD4 (L3T4) as recommended by the manufacture (Dynal, Lake Success, NY). Recipient origin cells (H-2k) and donor type cells (H-2b) were further separated from GIC using Dynal RAMIgG2a CELLection Kit as recommended by the manufacture (Dynal). GIC, which are a mixture of recipient (H-2k) and donor (H-2b) type cells, were incubated with purified 11-4.1 mouse IgG2a anti-mouse H-2Kk mAb or 28-8-6 mouse IgG2a anti-mouse H-2Kb/H-2Db mAb (PharMingen) at 4°C for 45 min, washed twice, and the H-2Kk or H-2Kb/H-2Db-positive cells were immunomagnetically selected by incubating with rat anti-mouse IgG2a, which is biotinylated and attached to Dynabeads via streptavidin and a DNA linker. The Dynabeads were then released from the cells by DNase digestion and removed from the cell suspension using a magnet. The unselected GIC, H-2Kk, or H-2Kb/H-2Db positively selected cells and H-2Kk or H-2Kb/H-2Db negatively selected cells were ready for MLR.

Mixed leukocyte reaction

Unselected GIC, CD4-depleted cells, H-2Kk or H-2Kb/H-2Db positively selected cells, and H-2Kk or H-2Kb/H-2Db negatively selected cells were used as stimulators. Splenic lymphocytes from naive mice (BALB/cByJ, C57BL/6, and CBA/J) were isolated and used as responders. Responders (2 x 105) and 2 x 105 2000 rad {gamma}-irradiated stimulators were cocultured in 96-well plates for 5 days. Eighteen hours before termination of culture, the wells were pulsed with 0.5 µCi of [H3]thymidine, and incorporation was quantitated with a beta-counter. Results are expressed as the mean ± SEM. Parallel cultures were set up such that 2 x 106 responders and 2 x 106 2000 rad {gamma}-irradiated stimulators were cocultured in 24-well plates; culture supernatants were harvested after 3 days for ELISA.

Fluorescent flow cytometry

H-2Kk positively selected cells were harvested as described above, and cell washes and Ab dilutions were performed in PBS plus 1% BSA at 4°C. I-Ak was detected with FITC-conjugated 11-5.2 mouse anti-mouse mAb; CD80 (B7-1) with FITC-16-10A1 hamster anti-mouse mAb; CD86 (B7-2) with FITC-GL1 rat anti-mouse mAb. T cells were detected with FITC 145-2C11 hamster anti-mouse CD3{epsilon} mAb, B cells with FITCRA3-6B2 rat anti-mouse CD45R/B220 mAb, and macrophages with FITC M1/70 rat anti-mouse CD11b (Mac-1). Flow cytometric analysis was performed using a FACScan Flow Cytometer (Becton Dickinson, Mountain View, CA). Results are expressed as the percentage of cells staining above background on a logarithmic scale of relative cellular fluorescence. mAbs were titered to ensure that saturating concentrations were used. Irrelevant mAbs served as controls.

ELISA for IL-2, IL-4, IL-10, and IFN-{gamma}

Grafts were homogenized at selected time points or MLR culture supernatants were harvested after 3 days, and two-Ab capture ELISAs for IL-2, IL-4, IL-10, and IFN-{gamma} were performed as recommended by the manufacture (PharMingen). Ninety-six-well flat-bottom plates were coated with 50 µl of anti-cytokine capture mAbs (JES6-1A12, 11B11, JES5-2A5, and R4-6A2 for IL-2, IL-4, IL-10, and IFN-{gamma}, respectively) at 2 µg/ml in 0.1 M NaHCO3 overnight at 4°C. Plates were washed with 0.05% Tween 20 in PBS, blocked with 200 µl 3% BSA in PBS for 2 h at room temperature, and washed; then, standards and culture supernatants added, and the plates were incubated overnight at 4°C. Plates were again washed, incubated with 100 µl of biotinylated anti-cytokine-detecting mAbs (JES6-5H4, BVD6-24G2, SXC-1, and XMG1.2 for IL-2, IL-4, IL-10, and IFN-{gamma}, respectively) at 1 µg/ml for 45 min at room temperature, and washed; then, 100 µl of a 1:1000 dilution of peroxidase-conjugated streptavidin (Kirkegaard & Perry, Gaithersburg, MD) was added to each well, and plates were incubated at room temperature for 30 min. Plates were washed, 100 µl of freshly prepared ABTS solution (Kirkegaard & Perry) was added to each well, and this was incubated at room temperature and stopped by adding 100 µl of 1% SDS. Plate O.D. values were measured at 405 nm. Purified recombinant murine cytokines (PharMingen) were used as standards.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gene transfer of vIL-10 prolongs cardiac allograft survival, but does not achieve tolerance

Previous studies showed that retrovirus-mediated gene transfer of vIL-10 prolonged allograft survival by inhibiting alloantigen-specific immunity (24). Repeat injection of CRE-MFG-vIL-10 at day 20 further prolonged graft survival to >98 days, and two of seven grafts survived >150 days (24). To determine whether this indefinite graft survival is due to donor-specific tolerance, we injected CRIP-DFG-vIL-10 at days 0 and 20 after transplantation, and graft survival was determined by electrocardiogram monitoring. The results showed that 3 of 10 allografts survived for >90 days following this treatment (Table IGo), which is consistent with our prior graft survival data using CRE-MFG-vIL-10 (24). Ninety days after the first allografting, animals with long-term surviving grafts received a second donor-specific graft without further immunosuppressive treatment, and both primary and secondary graft survival was measured by electrocardiogram monitoring. The results show that both the first and the second grafts were rejected within 20 days after the second transplant (Table IGo). Therefore, vIL-10 gene transfer induced long-term graft survival, but tolerance was not achieved. The ability to reject both first and second grafts demonstrates that maintenance of the long-term graft survival state is not due to clonal deletion or graft adaptation, but likely due to active regulation or anergy that was abrogated by the inflammatory responses associated with regrafting.


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  		Table I. Gene transfer of vIL-10 prolongs cardiac allograft survival but does not achieve tolerance1

 
Administration of anti-CD4 mAb inhibits the effects of vIL-10 gene transfer on allograft survival

Because vIL-10 gene transfer likely resulted in regulatory events that suppressed the immune response during both the induction and maintenance phases of graft survival, and because IL-10 is associated with Th1/Th2 switching, we determined whether Th2 type CD4+ T cells and their cytokines were important for inducing graft survival. To demonstrate that CD4+ T cells are important in vIL-10 gene transfer-induced immunosuppression, anti-CD4 mAb was injected into recipients at the time of vIL-10 gene transfer, and allograft survival was measured (Fig. 1Go). A dose of the depleting GK1.5 anti-CD4 mAb was chosen that was determined to impair CD4+ T cell immunity but only mildly prolong graft survival to 18.8 ± 0.9 (n = 6) vs 12.6 ± 1.1 days for the control, untreated allografts (n = 5, p < .001). Allografts that were injected with CRIP-MFG-vIL-10 and transplanted into the untreated recipients showed prolongation of graft survival of 45.6 ± 3.4 days (n = 5). However, allografts that were injected with CRIP-MFG-vIL-10 and transplanted into the anti-CD4 mAb-treated recipients showed a survival time of only 20.4 ± 1.6 days (Fig. 1Go) (n = 7, p < 0.004 vs CRIP-MFG-vIL-10 alone, and p = 0.54 vs anti-CD4 alone). Therefore, CD4+ T cells are important effector cells in vIL-10-induced immunosuppression.



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  		FIGURE 1. Administration of anti-CD4 mAb inhibits the ability of vIL-10 gene transfer to prolong allograft survival. Donor neonatal C57BL/6 hearts were directly injected with 5 x 103 PFU CRIP-MFG-vIL-10 and transplanted into CBA/J recipients. Purified anti-CD4 mAb was injected i.v. at 100 µg for 2 days following transplantation.

 
Administration of anti-IL-4 or anti-mIL-10 mAbs, but not anti-IFN-{gamma} mAb, inhibits the immunosuppressive effects of vIL-10 gene transfer on allograft survival

To test the possibility that vIL-10-induced local immunosuppression involves a switch from a Th1 to a Th2 alloresponse, we measured IL-2, IFN-{gamma}, IL-4, and mIL-10 levels in graft homogenates by ELISA. vIL-10 gene transfer did not change the low but measurable IL-2 and IFN-{gamma} levels within the allografts compared with the control-untreated group, whereas IL-4 and mIL-10 remained low and undetectable (data not shown). It is possible that the reasons the levels of the cytokines within the allografts are low or undetectable are that cells can migrate from the graft after transplantation, interstitial cytokines are probably rapidly taken up by cellular receptors, and tissue homogenates do not precisely localize the cytokines. Thus, we sought an alternative method to test whether low levels of IL-4 and endogenous mIL-10 are involved in vIL-10-mediated immunosuppression by assaying the effects of anti-IL-4 or anti-mIL-10 mAbs on vIL-10 gene transfer-induced immunosuppression. Allografts that were injected with CRIP-DFG-vIL-10 and transplanted into untreated recipients showed prolongation of graft survival of 23.8 ± 1.0 (Fig. 2GoA) or 24.2 ± 1.1 (Fig. 2GoB) days (n = 10). Animals receiving anti-IL-4 mAb or anti-mIL-10 mAb (which does not cross-react with vIL-10) alone showed no effect on graft survival (12.3 ± 2.3 days (n = 6) for anti-IL-4 and 14.1 ± 0.6 days (n = 10) for anti-mIL-10 treatment) compared with the untreated group (13.4 ± 0.4 days) (n = 12). Allografts that were injected with CRIP-DFG-vIL-10 and transplanted into anti-IL-4 mAb-treated recipients showed a survival time of only 13.9 ± 1.3 days (Fig. 2GoA) (n = 10, p < 0.001 vs CRIP-DFG-vIL-10 alone); and allografts that were injected with CRIP-DFG-vIL-10 and transplanted into the anti-mIL-10 mAb-treated recipients showed a survival time of only 14.3 ± 0.5 days (Fig. 2GoB) (n = 10, p < 0.001 vs CRIP-DFG-vIL-10 alone). Animals receiving anti-IFN-{gamma} mAb alone showed no effect on graft survival (15.8 ± 0.7 days; n = 10). Allografts that were injected with CRIP-DFG-vIL-10 and transplanted into anti-IFN-{gamma} mAb-treated recipients showed a survival time of 24.5 ± 1.1 days (Fig. 2GoC) (n = 10, p > 0.5 vs CRIP-DFG-vIL-10 alone). Therefore, administration of anti-IL-4 or anti-mIL-10 mAbs, but not anti-IFN-{gamma} mAb, inhibited the effects of vIL-10 gene transfer on allograft survival, suggesting that a population(s) of CD4+-, IL-4-, and/or mIL-10-secreting T cells may be generated as a result of vIL-10 gene transfer, which may act as Th2-negative regulatory cells.



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  		FIGURE 2. Administration of anti-IL-4 (A) or anti-mIL-10 (B) mAbs, but not anti-IFN-{gamma} (C) mAb, inhibits the ability of vIL-10 gene transfer to prolong allograft survival. Donor neonatal C57BL/6 hearts were directly injected with 5 x 103 PFU of CRIP-MFG-vIL-10 or CRIP-DFG-vIL-10, and transplanted into CBA/J recipients. Purified anti-IL-4, anti-mIL-10, or anti-IFN-{gamma} mAbs were injected i.v. at 100 µg every other day for six doses.

 
GIC APCs from vIL-10-treated group fail to stimulate MLR

Because IL-10 affects APC function, which in turn influences the generation of Th1 and Th2 cells, we tested the effects of vIL-10 on APC function within the GIC population. GIC were isolated 10 days after transplantation of C57BL/6 hearts into CBA recipients and were used as stimulators in MLR. It was anticipated that most GIC were of recipient (CBA) origin, and GIC from the untreated or the CRIP-MFG-LacZ-treated groups elicited proliferative responses from donor type (C57BL/6) and third party (BALB/c), but not recipient type (CBA) responders, confirming this supposition (Fig. 3GoA). However, GIC from CRIP-DFG-vIL-10-treated grafts failed to induce similar proliferative responses (Fig. 3GoA). The inability of vIL-10-treated GIC to elicit the MLR was not due to immunosuppressive cytokine production because addition of anti-IL-4-, anti-mIL-10-, or anti-TGF{beta}-neutralizing Abs to the MLR cultures did not affect the proliferative responses (Fig. 3Go, B and C). Addition of anti-vIL-10 mAb did not alter the MLR results as well (data not shown). This suggests that the retroviral vector was not incorporated into a cell that then continued to produce vIL-10 in culture, but rather that vIL-10 acted transiently on APCs in vivo and affected their subsequent functional capacity to stimulate the MLR. Evaluation of cytokines showed that IL-2, IL-4, mIL-10, and IFN-{gamma} levels in MLR culture supernatants were not significantly different among untreated, CRIP-MFG-LacZ-treated, and CRIP-DFG-vIL-10-treated groups (data not shown). To test whether negative regulatory cells were generated as a result of vIL-10 gene transfer, MLR cultures were set up using naive BALB/c, C57BL/6, or CBA splenocytes as responders; CBA/J, C57BL/6, or BALB/cByJ splenocytes as stimulators; and GIC from untreated, or CRIP-MFG-LacZ- or CRIP-DFG-vIL-10-treated grafts as putative regulatory cells. If negative regulatory cells were generated in vivo, it was expected that GIC from CRIP-DFG-vIL-10-treated grafts would suppress the MLR proliferative responses. The results in Table IIGo demonstrate that there were no measurable negative regulatory GIC generated in the CRIP-DFG-vIL-10-treated group.



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  		FIGURE 3. vIL-10-treated GIC fail to stimulate MLR. Donor neonatal C57BL/6 hearts were untreated or treated with 5 x 103 PFU of CRIP-DFG-vIL-10, and transplanted into CBA/J recipients. GIC were harvested at 10 days after transplantation and used as stimulators. A, Splenic lymphocytes from naive mice (BALB/cByJ, C57BL/6, and CBA/J) were used as responders. B and C, Anti-IL-4, anti-mIL-10, or anti-TGF{beta} Abs were added to MLR culture at 10 µg/ml. The experiment was performed three times with similar results.

 

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  		Table II. vIL-10 gene transfer does not generate suppressive GIC in vivo1

 
The role of graft-infiltrating CD4+ cells in regulation of the MLR

We demonstrated that CD4+ cells are critical in vIL-10-mediated prolongation of graft survival (Fig. 1Go), and the Ag-presenting function of GIC is impaired in vIL-10-treated groups (Fig. 3Go and Table IIGo). To evaluate the relationship between CD4+ cells and altered APC function, CD4-depleted or -undepleted GIC were used as stimulators. As shown in Table IIIGo, CD4-depleted GIC from the vIL-10-treated group possess a limited capacity to stimulate the MLR proliferative response as compared with unfractionated GIC. In fact, the CD4-depleted, vIL-10-treated GIC group was able to stimulate only third party responders, and to a moderate degree. Therefore, graft-infiltrating CD4+ cells play a limited role in altering Ag-presenting function. Thus, the necessity for CD4+ cells for vIL-10-mediated prolongation of graft survival is probably downstream of the effect of vIL-10 on APC function in the MLR.


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  		Table III. The role of graft-infiltrating CD4+ cells in regulation of the MLR1

 
vIL-10 treatment reduces the number of recipient type cells within allografts, but does not alter the relative proportions of T cells/B cells/macrophages, APC phenotype, or alloantigen-presenting function

GIC isolated in the above experiments were a mixture of a minority of donor (H-2b) and a majority of recipient (H-2k) type cells. To study further the effects of vIL-10 gene transfer on recipient type APC function, we separated recipient type cells (H-2k) from GIC using magnetic beads. Ninety-five percent of positively selected cells expressed H-2Kk on the cell surface as assessed by flow cytometry (data not shown). Although the total cell number isolated per graft was generally increased in the vIL-10 group, probably reflecting increased tissue viability, the number of H-2Kk-positive cells isolated by this method was minimally reduced (6.9 ± 1.5 x 104 cells/graft for the vIL-10-treated group vs 8.2 ± 1.6 x 104 cells/graft for untreated graft), and the number of H-2Kk negatively selected cells remaining (containing both H-2Kk-negative cells and some remaining H-2Kk-positive cells) was minimally increased (11.2 ± 2.4 x 104 cells/graft for vIL-10-treated group vs 8.9 ± 1.8 x 104 cells/graft for untreated graft) (Table IVGo). This pattern of change in cell numbers, although minor and not statistically significant, was observed in each of six experiments, which may reflect a minor reduction of recipient type cells migrating into or proliferating within allografts. These data are commensurate with our previously reported findings that whole, unfractionated GIC in the MFG-vIL-10-treated group showed decreased expression of lymphocyte cell surface activation molecules (24). Flow cytometric analysis of these H-2Kk-positive GIC from untreated and vIL-10-treated grafts showed no significant difference in expression (either percent positive or mean channel fluorescence) of T cell (CD3{epsilon}), B cell (CD45R/B220), and macrophage (CD11b/Mac-1) markers, or APC functional markers I-Ak, CD80 (B7-1), and CD86 (B7-2) (data not shown). These findings indicate that vIL-10 treatment did not alter the relative proportion of T cells, B cells, and macrophages, and the immune activation level of these selected H-2Kk-positive APC was similar between the groups. We were unable to reliably perform flow cytometric analysis on H-2Kk negatively selected cells as this population contained a large amount of cellular debris and had a high nonspecific background with the mAbs used in the assay. Separation of debris from these cells by density gradients resulted in a cell yield that was too low to perform the assays. Using positively selected H-2Kk cells as stimulators in MLR cultures, equal numbers of the H-2Kk-positive cells from both LacZ-treated and vIL-10-treated grafts were similarly capable of stimulating MLR responses, whereas the un- selected GIC from vIL-10-treated grafts acted as before, being unable to stimulate the MLR (Fig. 4Go). Equal numbers of the H-2Kk negatively selected cells from vIL-10-treated grafts had a significantly reduced ability to stimulate MLR responses compared with the H-2Kk negatively selected cells from LacZ-treated grafts (p < 0.01) (Fig. 4Go). These data suggest that one mechanism of vIL-10-induced local immunosuppression might involve a minor reduction of recipient type APC within the allograft, but this does not alter the major components of the infiltrating cells, APC phenotype, or alloantigen-presenting function.


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  		Table IV. vIL-10 gene transfer causes a minor reduction of recipient type (H-2Kk) cells within allografts1

 


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  		FIGURE 4. H-2k-positive cells selected from both DFG-vIL-10- and MFG-LacZ-treated grafts are capable of stimulating the MLR. Donor neonatal C57BL/6 hearts were treated with 5 x 103 PFU of CRIP-DFG-vIL-10 or CRIP-MFG-LacZ, and transplanted into CBA/J recipients. GIC were harvested 10 days after transplantation and incubated with anti-H-2Kk mAb (IgG2a), and the H-2Kk-positive cells were selected by incubating with rat anti-mouse IgG2a-coated Dynabeads. The unselected GIC (no selection), GIC incubated with anti-H-2Kk and positively selected by beads (H-2Kk-positive selection), and GIC incubated with anti-H-2Kk and negatively selected by beads (H-2Kk-negative selection) were used as stimulators in the MLR culture as described above. Splenic lymphocytes from naive third-party BALB/cByJ mice were used as responders. The experiment was performed six times with similar results.

 
vIL-10 gene transfer reduces donor type cell Ag-presenting function within allografts

Because vIL-10 gene transfer impairs APC function of unselected GIC (Figs. 3Go and 4Go) and negatively selected recipient type (H-2Kk) GIC (Fig. 4Go, group 6) but does not alter recipient type APC function (Fig. 4Go, group 4), we next studied the effects of vIL-10 gene transfer on donor type APC function. Donor type cells (H-2b) from GIC were separated using magnetic beads. They represented a very small proportion of the total population in the GIC (<5%), and the numbers of the H-2Kb cells from both LacZ- and vIL-10-treated grafts were too low to perform flow cytometric analysis reliably for cell surface receptor expression. The unselected GIC (no selection), GIC incubated with anti-H-2Kb/H-2Db and positively selected by beads (H-2Kb-positive selection), and GIC incubated with anti-H-2Kb/H-2Db and negatively selected by beads (H-2Kb-negative selection) were used as stimulators in MLR cultures. The positively selected H-2Kb cells from vIL-10-treated grafts were incapable of stimulating proliferative responses (p < 0.05 vs the positively selected H-2Kb cells from LacZ-treated grafts) (Fig. 5Go). After depleting H-2Kb-positive cells from GIC, H-2Kb negatively selected cells from both vIL-10- and LacZ-treated grafts were equally capable of stimulating MLR responses (Fig. 5Go). Therefore, H-2Kb donor type cells from vIL-10-treated grafts were unable to present alloantigens directly to responder T cells, and may act as negative regulatory cells to inhibit MLR responses elicited by H-2Kk stimulators. The results in Fig. 4Go, group 4, confirm these findings, demonstrating that H-2Kk-positive selection restores proliferative capacity to vIL-10-transduced GIC, whereas H-2Kk-negative selection (Fig. 4Go, group 6) defines a population of GIC unable to stimulate responders in MLR. These data suggest that inhibition of donor type APC function is a major component of vIL-10-induced local immunosuppression. The data in Figs. 4Go and 5Go imply that H-2Kb-positive cells from vIL-10-treated grafts acted as negative regulatory cells to inhibit MLR responses because their removal permits responses to be elicited by H-2Kk stimulators. However, attempts to evaluate negative regulatory H-2Kb-positive cells were limited by the very low number of H-2Kb-positive GIC that could be obtained. Using H-2Kk-positive cells, H-2Kk-negative cells, or H-2Kb-negative cells as regulators revealed no evidence for the generation of suppressive GIC (data not shown), which was commensurate with the results in Table IIGo. It should be noted that in Figs. 4Go and 5Go, 2 x 105 GIC were added to the MLR culture as stimulators, whereas in Table IIGo, 10 times fewer (2 x 104) GIC were used as regulatory cells. However, in some experiments, 6 x 104 GIC were used with results similar to those in Table IIGo. We also speculate that the GIC H-2Kk stimulators used in Figs. 4Go and 5Go may have been modified by the graft environment following vIL-10 gene transfer in vivo, making them more sensitive to the inhibitory effect of regulatory cells in vitro, whereas the splenocyte stimulators used in Table IIGo are naive and less sensitive to the inhibitory effect of regulatory cells.



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  		FIGURE 5. vIL-10 gene transfer reduces donor type cell Ag-presenting function within the allograft. Donor neonatal C57BL/6 hearts were treated with 5 x 103 PFU of CRIP-DFG-vIL-10 or CRIP-MFG-LacZ, and transplanted into CBA/J recipients. GIC were harvested 10 days after transplantation and incubated with anti-H-2Kb/H-2Db mAb (IgG2a), and the H-2Kb/H-2Db-positive cells were selected by incubating with rat anti-mouse IgG2a-coated Dynabeads. The unselected GIC (no selection), GIC incubated with anti-H-2Kb/H-2Db and positively selected by beads (H-2Kb positive selection), and GIC incubated with anti-H-2Kb/H-2Db and negatively selected by beads (H-2Kb negative selection) were used as stimulators in the MLR culture as described above. Splenic lymphocytes from naive third-party BALB/cByJ mice were used as responders. The experiment was performed four times with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Retroviral, adenoviral, and plasmid vectors are all capable of transferring and expressing vIL-10 to prolong allograft survival (24, 36, 37, 38, 39). Despite these successes, indefinite allograft survival and tolerance have not yet been achieved. This is likely due to the fact that gene transfer is inefficient in vivo, especially using retroviral and plasmid vectors, and expression is low and transient for all vectors. Although retroviral vectors are expected to be able to persist, because they integrate into genomic DNA, the expression of the {beta}-galactosidase gene from CRIP-MFG-LacZ can be inhibited by 70% after exposure to IFN-{gamma}, through mechanisms related to transcriptional regulation (40). Therefore, it is not unexpected that the expression of vIL-10 from CRIP-DFG-vIL-10 is gradually eliminated within allografts due to transduced cell exposure to IFN-{gamma}, generated as a result of immunologically nonspecific inflammatory responses to operative trauma and ischemia, and immunologically specific responses to alloantigen. The rationale of repeat injection of CRIP-DFG-vIL-10 at a later time was to provide additional vector to produce vIL-10 in the allograft when expression from the first dose of vector had waned. Repeat injection of CRE-MFG-vIL-10 further prolonged graft survival, although only 30% of grafts survived >90 days. The fate of the second dose of retroviral vector is speculated to depend upon the environment within the allograft to which the vector is exposed. Critical variables of this environment include the levels of promoter inhibitory cytokines such as IFN-{gamma} and TNF-{alpha} (40) and the existence of proliferative cells, because retroviral vectors require dividing cells for integration and expression. These factors would determine the level and timing of vIL-10 production within the allograft, which in turn either moderately (to 40 days) or indefinitely (>90 days) prolongs graft survival.

Despite the limitations to vIL-10 gene expression, indefinite graft survival was still achieved in 30% of the allografts treated with the double dose of CRIP-DFG-vIL-10. Nonetheless, a donor-specific tolerant state was not reached because animals that received an untreated second donor type graft rejected both the first and second grafts. Therefore, the maintenance of long-term graft survival in these animals was not due to alloantigen-specific clonal deletion or graft adaptation, but negative regulatory cells, anergy, and/or immunological ignorance (41, 42, 43, 44, 45). Table IIGo shows no evidence for regulatory or suppressor cells, but this may be a consequence of cell numbers or susceptibility to negative signals as discussed above. In addition, the results in Figs. 4Go and 5Go do imply the existence of negative regulatory cells. We have not directly assayed GIC or systemic lymphocytes for alloantigen-specific anergy, but there is precedence for IL-10 inducing this state (10). vIL-10 also partly inhibited indirect allorecognition, by reducing recipient type cell migration into or proliferation within the graft (Table IIIGo and ref. 24), and direct allorecognition by eliminating donor type APC function (Figs. 4Go and 5Go). Thus, vIL-10 expression may have prevented initial host T cell sensitization to the graft by both pathways (41, 42). Lastly, parenchymal cells of the graft are considered to be "nonprofessional APCs", as they do not provide all of the signals necessary to activate T cells. Contact with these cells appears to result in immunological ignorance, such that T cells show neither tolerance nor immunity to the allograft (42, 43, 44, 45). vIL-10 may have enhanced this property of the donor tissue by interfering with "professional APC" numbers or function. Therefore, the second untreated donor type graft, which provided more complete Ag presentation, was able to activate T cells to reject both first and second grafts.

IL-10 inhibits IL-12 and IFN-{gamma} production (1, 2, 3, 6, 8, 23) and plays a role in the regulation of Th1 and Th2 cell development. The data in Fig. 2Go show that the Th2 cytokines IL-4 and mIL-10 are required for vIL-10 immunosuppressive function, as administration of anti-IL-4 or anti-mIL-10 mAbs inhibited the vIL-10-immunosuppressive effect. This suggests a regulatory circuit in which vIL-10 initiates a switch from a Th1 to a Th2 response that must be sustained by endogenous Th2 cells and cytokines. It is interesting to note the effect of anti-mIL-10 mAb in these experiments, because in our previous report retrovirus-mediated local gene transfer of mIL-10 did not prolong graft survival (24). A similar result was also observed with a syngeneic murine tumor model, in which vIL-10 gene transfer inhibited rejection of tumor, whereas mIL-10 gene transfer accelerated tumor rejection (25). As reported by many investigators, mIL-10 has paradoxical effects in vivo: it may act as an immunosuppressive cytokine in certain disease models (46, 47, 48, 49, 50, 51, 52, 53), while functioning as an immunostimulatory cytokine in the others (25, 54, 55, 56). In fact, there are numerous studies in which there has been a general failure to correlate the presence or absence of mIL-10 with allograft survival or rejection (57). The precise cellular and mechanistic reasons for this dichotomy are uncertain, but suggest that the role played by mIL-10 in vIL-10-mediated immunosuppression may be dose, time, and cell population(s) dependent. Another possibility is that while the initial locus of activity of vIL-10 in our model is local within the graft, mIL-10 may have a systemic role in the draining lymph nodes or more distally in the spleen or elsewhere in the periphery, and the anti-mIL-10 mAb interfered with this function.

The immunosuppressive effects of IL-10 are often seen at the level of the APC, and not directly at the level of the T cell (3, 4, 6, 11), where it inhibits monocyte and macrophage synthesis of cytokines and chemokines (4, 7, 8, 12, 17), expression of MHC class II and B7 molecules (4, 12, 14), and Ag presentation (13, 15, 16, 18). In this study, vIL-10 gene transfer impaired direct and indirect alloantigen presentation to CD4+ T cells, as shown by the failure of GIC from CRIP-DFG-vIL-10-treated allografts to stimulate the MLR proliferative response. This effect was not due to the production of several known immunosuppressive cytokines, such as IL-4, mIL-10, vIL-10, or TGF{beta} (Fig. 3Go, B and C), although the current experiments cannot rule out that other soluble factors may play a role in this setting. Further analysis demonstrated that while the numbers of recipient type cells within the allograft were lower in the vIL-10-treated group, the relative proportions of T cells, B cells, and macrophages were not altered, and APC phenotype and alloantigen-presenting function remained intact (Figs. 4Go and 5Go). The inability of vIL-10-treated, unfractionated GIC to elicit an MLR cannot be due solely to input of fewer numbers of recipient type APC into the MLR culture because the recipient type cell numbers were only 10–30% less in the vIL-10-treated group (Table IVGo), whereas the MLR proliferative responses were reduced by 60–90%. We also demonstrated that donor type cells from vIL-10-treated grafts were unable to present Ags directly to T cells in the MLR cultures (Figs. 4Go and 5Go) and may act as regulatory cells to inhibit MLR responses elicited by recipient type stimulators in vitro. Therefore, significant mechanisms of vIL-10-induced local immunosuppression could involve both a modest reduction of recipient type cells acting as indirect APCs within the allograft and inhibition of donor type direct APC function.

Donor type professional APCs may play a key role in determining the acceptance or rejection of the allograft (41, 45, 58, 59, 60, 61, 62). It has been hypothesized that donor passenger dendritic cells may initiate alloimmune responses within the graft, within draining lymph nodes, or in distant lymphoid tissues depending on migration patterns (41, 42). This hypothesis is supported by observations that organs and tissues depleted of passenger leukocytes can be accepted indefinitely by untreated, immunocompetent animals (41, 45, 58). Nonetheless, there are reports demonstrating that donor APC macrophages are not necessarily a negative factor in allograft survival (59), and donor-derived leukocytes may be required for stable allograft function (60, 61). Thus, the differentiative and signaling states of donor APC may be critical determinants of the character of induced immune responses. In our model, donor type cells would be the likely targets for vIL-10-mediated gene transfer because retroviral vectors are infective for only a short time after in vivo transfer (62). Furthermore, we have previously shown that retroviral mediated gene transfer remains localized within the allografts (24) so that gene transduction beyond the graft is unlikely. The donor type cells would then have been exposed to autocrine or paracrine vIL-10, and these cells would have included both professional APCs, such as dendritic cells and lymphocytes, and nonprofessional APCs, such as myocytes, fibroblasts, and endothelial cells. The data suggest that after exposure to vIL-10, the donor-derived APCs become incapable of directly presenting Ag. Alternatively, vIL-10 may have prevented maturation of professional donor APC (18, 63), leaving only nonprofessional, immature, or tolerogenic APC that would induce anergy or ignorance. Therefore, gene transfer of vIL-10 to allograft at the time of transplantation may provide a way to manipulate donor type APC function, which in turn impairs direct allorecognition and initial sensitization to alloantigens.


   Acknowledgments
 
We thank S. F. Schoonover (University of Pittsburgh, Pittsburgh, PA) for providing retroviral vectors and Dr. R. Coffman (DNAX) for providing JES-2A5 anti-mouse IL-10 hybridoma.


   Footnotes
 
1 This work was supported by Baxter Healthcare Corporation Extramural Grant Program, National Institutes of Health Grant AI 32655, and University of Michigan-Multipurpose Arthritis Center National Institutes of Health Grant P60-AR20557. Back

2 Address correspondence and reprint requests to Dr. Jonathan S. Bromberg, Recanati/Miller Transplantation Institute, Mt. Sinai School of Medicine, 1 Gustave L. Levy Place, Box 1496, New York, NY 10029. Back

3 Abbreviations used in this paper: vIL-10, viral IL-10; m, murine; GIC, graft-infiltrating cell(s). Back

Received for publication August 28, 2000. Accepted for publication December 1, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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