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The Journal of Immunology, 2001, 167: 5457-5463.
Copyright © 2001 by The American Association of Immunologists

Donor IFN-{gamma} Receptors Are Critical for Acute CD4+ T Cell-Mediated Cardiac Allograft Rejection1

Alexander C. Wiseman2,*, Biagio A. Pietra2,{dagger}, Brian P. Kelly{dagger}, Gina R. Rayat§, Mona Rizeq{ddagger} and Ronald G. Gill3,§

* Division of Nephrology, Department of Medicine, and {dagger} Division of Cardiology, Department of Pediatrics, The Children’s Hospital, {ddagger} Department of Pathology, Veterans Affairs Medical Center, and § Barbara Davis Center for Childhood Diabetes, Department of Medicine and Immunology, University of Colorado Health Sciences Center, Denver, CO 80262


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent studies using mouse models demonstrate that CD4+ T cells are sufficient to mediate acute cardiac allograft rejection in the absence of CD8+ T cells and B cells. However, the mechanistic basis of CD4-mediated rejection is unclear. One potential mechanism of CD4-mediated rejection is via elaboration of proinflammatory cytokines such as IFN-{gamma}. To determine whether IFN-{gamma} is a critical cytokine in CD4-mediated acute cardiac allograft rejection, we studied whether the expression of IFN-{gamma} receptors on the donor heart was required for CD4-mediated rejection. To investigate this possibility, purified CD4+ T cells were transferred into immune-deficient mice bearing heterotopic cardiac allografts from IFN-{gamma} receptor-deficient (GRKO) donors. While CD4+ T cells triggered acute rejection of wild-type heart allografts, they failed to trigger rejection of GRKO heart allografts. The impairment in CD4-mediated rejection of GRKO hearts appeared to primarily involve the efferent phase of the immune response. This conclusion was based on the findings that GRKO stimulator cells provoked normal CD4 proliferation in vitro and that intentional in vivo challenge of CD4 cells with wild-type donor APC or the adoptive transfer of in vitro primed CD4 T cells failed to provoke acute rejection of GRKO allografts. In contrast, unseparated lymph node cells acutely rejected both GRKO and wild-type hearts with similar time courses, illustrating the existence of both IFN-{gamma}-dependent and IFN-{gamma}-independent mechanisms of acute allograft rejection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The requirement for CD4 T cells in acute cardiac allograft rejection is well established. The dependence upon CD4 T cells for acute rejection of cardiac allografts is most clearly illustrated by the absence of acute rejection in CD4 knockout hosts (1) or following anti-CD4 mAb therapy (1, 2, 3, 4, 5, 6). CD4 T cells do not merely serve a helper role in the response, in that purified CD4 T cells are also sufficient, in the absence of CD8 cells or B cells, to mediate rejection of vascularized cardiac allografts (6, 7). Our recent results indicated that acute CD4 T cell-mediated cardiac allograft rejection occurs primarily through direct donor Ag recognition (6). In that study purified CD4 T cells did not trigger rejection of MHC class II-deficient allografts, indicating that direct (donor MHC-restricted) Ag presentation was required for CD4-mediated rejection. Further, host MHC class II was not necessary for CD4+ T cell-mediated acute allograft rejection, indicating that the indirect (host MHC-restricted) presentation of donor Ag was not required for CD4-mediated rejection.

While these data implicate the CD4 as an important effector cell in acute cardiac allograft rejection, the distinct mechanisms by which the CD4+ T cell acts as an effector cell remain unclear. Potential mechanisms by which the CD4+ T cell subset generates an effector response include direct cytotoxic effects (e.g., via secretion of TNF-{alpha}, Fas-Fas ligand binding, or other cytokine secretion) (8, 9) and/or the activation of innate immune cells such as macrophages (10). Importantly, IFN-{gamma}, a prototypical cytokine secreted by the Th1 CD4 cell subset, may contribute to both the direct graft cytopathic effects as well as the recruitment and/or activation of other inflammatory cells to mediate allograft rejection. For example, IFN-{gamma} up-regulates MHC II on professional APC to enhance alloantigen recognition (11) and activates macrophages (12). IFN-{gamma} also appears to play a nonredundant role in the induction of class II on arterial endothelium (13), which may be critical in CD4 T cell recognition via the direct pathway and/or in efficient targeting of primed donor-reactive CD4 cells. Furthermore, chemokines such as monokine induced by IFN-{gamma} and IFN-{gamma}-inducible protein induced by IFN-{gamma} may also be important for efficient CD4+ T cell trafficking to the allograft and/or for effector function (14, 15). Thus, a variety of direct and indirect effects of IFN-{gamma} on allograft tissue implicate this cytokine as an important contributor to acute allograft rejection.

Despite the long-standing classification of IFN-{gamma} as a proinflammatory cytokine, more recent evidence indicates a more dichotomous role for IFN-{gamma} in allograft immunity. That is, IFN-{gamma} appears to serve both pathogenic and regulatory roles in the response. On the one hand, the presence of IFN-{gamma} correlates strongly with acute rejection (16), and under some conditions IFN-{gamma} is critical for acute rejection to proceed. For example, IFN-{gamma} is important for efficient islet allograft rejection mediated by primed CD8+ T cells (17) and for the rejection of MHC class II disparate skin allografts (14, 18, 19). It is not clear whether IFN-{gamma} is essential in the afferent phase of the immune response (such as by enhancing graft Ag expression), in the efferent phase (such as through enhancing trafficking and infiltration of graft-reactive cells), or both. On the other hand, the role of IFN-{gamma} as a regulatory cytokine in transplantation also has become evident. IFN-{gamma} is not required for acute cardiac (20, 21) or islet (17, 22) rejection, and surprisingly can act in a protective fashion on the allograft during the early immune events following transplantation (23, 24). Importantly, IFN-{gamma} appears to be essential for transplantation tolerance induced by costimulation blockade (22, 25).

Such paradoxical contributions of IFN-{gamma} to allograft immunity and tolerance form the impetus to better clarify the role of IFN-{gamma} in transplant immunity. Given these controversies, the purpose of this study was to test the hypothesis that CD4-mediated acute cardiac allograft rejection is IFN-{gamma} dependent in vivo. Specifically, this study addresses whether the presence of allograft IFN-{gamma} receptors is necessary for CD4-mediated allograft rejection in a vascularized heart model. We report that CD4-mediated rejection is dependent upon the presence of donor (graft) IFN-{gamma} receptors. Further, we provide evidence that donor IFN-{gamma} receptors are required in the efferent (effector) phase of CD4 T cell-mediated rejection. Finally, we demonstrate that there are both IFN-{gamma}-dependent and IFN-{gamma}-independent mechanisms of cardiac allograft rejection, since an intact immune response is capable of mediating rejection despite the absence of allograft IFN-{gamma} receptors.


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

Female 6- to 8-wk-old, 129Sv/J (H-2b wild-type (WT)), 129 Sv/Jinfgr (H-2b) 129 IFN-{gamma} receptor-knockout (GRKO),4 BALB/c (H-2d), BALB/c scid (H-2d), BALB/c rag1-/-, and C57BL/6 (B6, H-2b) were purchased from The Jackson Laboratory (Bar Harbor, ME). MHC class II-deficient C57BL/6 (I-A{beta}-/-, C2D) mice were purchased from Taconic Farms (Germantown, NY) and bred at the Barbara Davis Center animal facility (Denver, CO).

Purification and phenotyping of CD4+ T cells

BALB/c mice were sacrificed by CO2 asphyxiation, and cervical, brachial, axillary, and mesenteric lymph nodes were removed and disrupted into a single-cell suspension with a tissue homogenizer. CD4+ T cells were purified from lymph nodes by negative selection by passage over immunoaffinity columns (Cellect, Edmonton, Canada) to remove IgG+ and CD8+ cells. Following negative selection over immunoaffinity columns, the purity of the CD4+ T cell fraction was assessed via flow cytometry (EPICS Elite ESP, Coulter, Miami, FL) using single-parameter fluorescence histograms. Cells were phenotyped at the time of initial isolation and again at the time of graft harvest or on day +30 from reconstitution to document successful adoptive transfer and purity of transferred cells. Lymphocytes were directly labeled with FITC-conjugated rat anti-mouse CD45 (clone 30-F11), TCR (clone H57-597), CD4 (clone RM4-4), CD8{alpha} (clone 53-6.7), and B220 (clone RA3-6B2; BD PharMingen, San Diego, CA). All purification and tracking steps demonstrated <1% of the potential contaminating lymphocyte populations positive for CD8+ and B220+ cells.

Heart transplantation and lymphocyte adoptive transfer

Allogeneic, vascularized, heterotopic heart transplantation was performed as previously described (26) using 8- to 12-wk-old WT 129 or 129 GRKO donors and 8- to 12-wk-old BALB/c scid or BALB/c rag1-/- recipients. Within 6 days following successful transplantation (3.6 ± 0.5 days post-transplant; range, 0–6 days), recipients underwent i.p. adoptive transfer of 107 unfractionated BALB/c lymph node cells or 107 purified CD4+ T cells. This approach of transferring cells after heart grafting was chosen due to the inherent logistics of coordinating successful heart allograft recipients with sufficient numbers of purified CD4+ T cells to pair between control and test groups (e.g., 129 vs 129 GRKO). We had already found that this timing of cell transfer (within several days before or after heart grafting in rag-/- or scid mice) did not alter the tempo of unmodified cardiac allograft rejection (6). The number of cells used for adoptive transfer was based upon previous findings indicating that reconstitution of primary acute cardiac allograft immunity in scid hosts occurred with this number of cells (6). Following lymphoid cell reconstitution, heart allografts were monitored by daily palpation. Rejection was defined by the loss of palpable cardiac contractions, and cardiac allograft survival for longer than 60 days from reconstitution was considered long term survival. Heart allograft survival or rejection was verified by direct graft visualization and histologic analysis at the time of harvest.

Histopathology

At the time of rejection or on day +60 from T cell reconstitution, cardiac allografts were harvested and bisected, with one half quick-frozen in OCT compound for subsequent immunohistochemical staining, and the other formalin-fixed and paraffin-embedded for H&E staining. H&E-stained sections were examined in a blinded fashion by a pathologist (M. A. Rizeq) and acute cellular rejection was graded according to degree of lymphocyte infiltration in the myocardium and vessel walls. Immunohistochemical analysis for CD4+ (clone RM4-5) or CD8+ (clone 53-6.7; BD PharMingen) cells was performed using frozen sections that were air-died and acetone-fixed. Sections were rehydrated in PBS, washed, and blocked with 1/5 normal rabbit serum in PBS containing Vector avidin DH (Vector Laboratory, Burlingame, CA). Abs were applied and incubated for 45 min and washed, then biotinylated rabbit anti-rat IgG was applied. Vectastain Elite ABC reagent (Vector Laboratory) was applied, followed by counterstaining with Harris’ hematoxylin. The tissue sections were examined for immunoperoxidase staining by light microscopy.

Mixed lymphocyte reaction

One-way primary MLR was performed by incubating 2 x 106/ml purified BALB/c CD4 cells with 3 x 106/ml 2000-rad irradiated splenocytes from WT (129 Sv/J), GRKO (129 Sv/Jinfr), and C57BL/6 C2D mice in EMEM medium containing 10% FCS, 1% L-glutamine, 1% antibiotics (penicillin/streptomycin/neomycin), and 10-5 M 2-ME in a total volume of 200 µl/well in 96-well flat-bottom plates (Corning Glass, Corning, NY). On days 3, 4, and 5, 1 µCi [3H]thymidine was added to each well. Cells were harvested 6 h later and counted on a Wallac beta emission counter (Gaithersburg, MD). For adoptive transfer of in vitro primed CD4+ T cells, bulk cultures of purified BALB/c CD4 cells were established in upright 25-cm2 tissue culture flasks (Corning Glass) using 2 x 107 responding BALB/c CD4 T cells mixed with 3 x 107 irradiated 129 splenic stimulator cells in 20 ml complete medium as described above. On day 4 of culture, cells were washed twice, and 107 cells were adoptively transferred when indicated via retro-orbital i.v. injection.

In vivo challenge with donor-type splenocytes

In a subset of BALB/c scid recipients harboring 129 GRKO heart allografts, we immunized against donor Ags by injecting 107 129 WT splenocytes i.p. immediately before BALB/c CD4+ T cell reconstitution. Before injection, spleen cells were treated with RBC lysing buffer and T cell-depleted with a cocktail of anti-CD4 mAb (GK1.5) at 20 mg/ml, anti-CD8 mAb (2.43) at 20 mg/ml, and anti-Thy1.2 IgM (HO-13-4) ascites at 1/400 on ice for 15 min in DMEM at a concentration of 1 x 107 cells/ml. This was followed by incubation with complement (Low-Tox-M, Cedarlane Laboratories, Hornby, Canada) at a 1/20 concentration at 37°C for 1 h in DMEM. T cell-depleted splenocytes (107) were then injected i.p.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD4-mediated acute cardiac allograft rejection requires the presence of allograft IFN-{gamma} receptors

We have previously demonstrated that CD4+ T cells can act as direct effector cells in acute cardiac allograft rejection, as purified CD4 cells induce allograft rejection comparable to unseparated lymph node cells when transferred to immune-deficient SCID mice (6). To study the mechanisms that underlie CD4+ T cell-mediated acute allograft rejection, we employed an adoptive transfer model system in which immune-deficient BALB/c scid mice served as cardiac allograft recipients and then underwent reconstitution with purified syngeneic BALB/c CD4+ T cells. Although the unmodified rejection of heart allografts is known to be IFN-{gamma} independent (20), it is not clear whether CD4 T cells alone require IFN-{gamma} to mediate rejection in the absence of CD8+ T cells and B cells. For example, if CD4-mediated rejection is primarily dependent upon IFN-{gamma}-induced MHC class II expression over basal levels on the allograft (13, 27), then acute rejection may require IFN-{gamma} signaling to the allograft. Alternatively, induction of class II over basal levels may not be critical for CD4 sensitization, but may be important for the targeting of directly reactive CD4 cells. In either case, CD4-mediated rejection would be expected to be IFN-{gamma} dependent. To test the hypothesis that CD4-mediated rejection requires the presence of allograft IFN-{gamma} receptors, we compared the survival of heart transplants from WT and GRKO donors in SCID recipients reconstituted with purified CD4 cells. In unreconstituted SCID recipients, all WT and GRKO allografts survived >60 days (Fig. 1Go) and showed no evidence of inflammation or acute rejection (Fig. 2Go, A and B). Adoptive transfer of purified CD4+ T cells triggered uniform, vigorous rejection of the WT heart allografts in a mean of 9.8 ± 0.9 days. In contrast, all GRKO allografts survived >60 days following CD4+ T cell reconstitution, similar to the survival of both WT and GRKO allografts in unreconstituted SCID recipients (Fig. 1Go). Histologic examination of WT allografts at the time of cessation of heart beat demonstrated a florid lymphocytic infiltrate involving both the myocardium and the small, medium, and large vessels, consistent with severe acute rejection (Fig. 2GoC). Histologic examination of GRKO allografts on day +60 from CD4 T cell reconstitution demonstrated modest mononuclear cell infiltrates of the myocardium and vessels consistent with mild rejection (Fig. 2GoD). Despite the absence of overt rejection, these long term surviving GRKO allografts demonstrated a persistent CD4 infiltrate compared with rejecting WT allografts (Fig. 2Go, E and F), with undetectable CD8 staining in either experimental group (data not shown).



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  		FIGURE 1. Donor IFN-{gamma} receptors are required for CD4-mediated acute cardiac allograft rejection. 129Sv/J WT cardiac allografts undergo acute rejection in BALB/c scid hosts reconstituted with purified CD4 T cells (n = 6; mean time to rejection, 9.8 ± .9 days). 129 GRKO cardiac allografts survive >60 days in CD4-reconstituted BALB/c scid hosts (n = 8), similar to both WT (n = 3) and GRKO (n = 6) hearts in unreconstituted recipients.

 


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  		FIGURE 2. H&E histologic assessment of WT vs GRKO cardiac allografts in SCID mice reconstituted with purified CD4 T cells. A, H&E staining of a wild-type (129) graft in an unreconstituted BALB/c scid recipient on day +60 following transplantation. No mononuclear cell infiltrate is identified. B, H&E staining of a GRKO graft in an unreconstituted BALB/c scid recipient on day +60 following transplantation. Again, no mononuclear cell infiltrate is identified. C, H&E staining of a wild-type graft in a BALB/c scid reconstituted with 107 purified CD4 T cells following cessation of palpable heartbeat (day +12 post-T cell transfer). Dense interstitial lymphocytic infiltrate and vasculitis are present, consistent with severe acute rejection. D, H&E staining of a GRKO graft in a BALB/c scid reconstituted with 107 purified CD4 T cells recipient on day +60 post-T cell transfer demonstrating sparse mononuclear cell infiltrate and preserved myocardial and vascular architecture consistent with mild rejection. E, Immunohistochemical staining for CD4 in acutely rejected wild-type 129 hearts following reconstitution of scid hosts with purified CD4 T cells (day +12 post-T cell transfer). F, CD4 staining of a GRKO graft in a CD4-reconstituted scid recipient on day +60 post CD4 T cell transfer. All magnification, x150.

 
Purified BALB/c CD4 cells proliferate normally in vitro in response to GRKO stimulator cells

The relatively modest infiltration noted in long term surviving GRKO allografts could be due to diminished initial CD4+ T cell activation, diminished effector cell targeting, or both. To address whether the protective effect of allograft IFN-{gamma} receptor deficiency in CD4-mediated rejection was related to a failure in the inherent stimulatory capacity of GRKO APC, the in vitro proliferative response of purified BALB/c CD4 cells was measured against WT and GRKO stimulator cells. As shown in Fig. 3Go, BALB/c CD4 cells demonstrate equivalent proliferative responses to both WT and GRKO stimulator splenocytes. In contrast, CD4 cells show undetectable proliferation above background in response to allogeneic (H-2b) MHC class II-deficient stimulator splenocytes, illustrating the requirement for allogeneic MHC class II for primary CD4+ T cell activation. Taken together, these results suggest that there is sufficient constitutive MHC class II expression by GRKO APC to stimulate a vigorous CD4 cell alloresponse in vitro.



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  		FIGURE 3. In vitro proliferation of CD4 T cells in response to WT and GRKO stimulator cells. Proliferation of BALB/c CD4+ T cells was assessed on days 3, 4, and 5 following stimulation with irradiated splenocytes from 129Sv/J WT, 129 GRKO, and C57BL/6 MHC class II-deficient mice. CD4+ T cells do not proliferate in response to MHC class II-deficient stimulator cells, indicating a requirement for MHC class II in CD4 proliferation. CD4 proliferation is comparable in response to WT and GRKO stimulator cells, suggesting adequate MHC class II expression is present in both WT and GRKO splenocytes to provoke a proliferative response.

 
Primed BALB/c CD4 cells are unable to mediate acute rejection of GRKO heart allografts

The in vitro stimulation with allogeneic GRKO splenocytes may not adequately represent the in vivo CD4 response to GRKO cardiac allografts. Therefore, we performed GRKO cardiac transplants and subsequent CD4 cell reconstitution in SCID recipients with an additional step to in vivo challenge the transferred CD4 cells with WT (IFN-{gamma} receptor-bearing) donor APCs. Immediately before CD4+ T cell reconstitution, hosts were immunized with 107 T cell-depleted WT 129 (IFN-{gamma} receptor-bearing) spleen cells. This priming dose is in 10-fold excess of a priming dose sufficient to provoke robust allograft rejection of established islet allografts (28) and was chosen to provoke an effective T cell response in vivo. Despite in vivo immunization with WT APCs, CD4 cells still failed to reject five of six heart allografts (Table IGo).


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  		Table I. In vivo challenge with 129 WT spleen cells T cells fail to provoke CD4 T cell rejection of GRKO cardiac allografts

 
Despite this intentional immunization step, it is possible that the degree or quality of CD4 T cell activation in vivo was insufficient to generate a relevant graft-destructive response. Therefore, in parallel experiments BALB/c CD4+ T cells were intentionally activated against WT 129 stimulator cells in vitro and adoptively transferred into BALB/c rag1-/- recipients bearing 129 WT or GRKO cardiac allografts. Such in vitro-primed CD4 T cells triggered rapid rejection of WT allografts, while GRKO allografts survived >40 days post-T cell transfer (Table IIGo). Interestingly, histological examination of such grafts demonstrated pronounced migration of CD4 T cells into both WT and GRKO cardiac allografts (Fig. 4Go). However, while the WT grafts demonstrated severe disruption of tissue architecture and overt rejection, the GRKO grafts remained intact, with only mild histological rejection.


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  		Table II. In vitro primed CD4 T cells fail to acutely reject GRKO cardiac allografts established in BALB/c rag1-/- recipients

 


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  		FIGURE 4. In vitro-primed CD4 T cells migrate to both WT and GRKO cardiac allografts. WT 129 or 129 GRKO cardiac allografts were established in immune-deficient BALB/c rag-1-/- hosts. Purified CD4 T cells were primed against WT 129 stimulator cells in vitro as described in Materials and Methods. Primed CD4 T cells (107) were adoptively transferred to BALB/c rag-1-/- hosts bearing either WT 129 or 129 GRKO cardiac allografts. A, Immunohistochemical staining for CD4 in an acutely rejected WT 129 hearts following reconstitution of in vitro primed CD4 T cells. B, CD4 staining of a GRKO graft in a recipient after transfer of primed CD4 T cells. Both samples were harvested on days 12–14 post-CD4 T cell transfer.

 
IFN-{gamma} receptor-independent pathways of heart allograft rejection

Although these results indicated that CD4+ T cells require graft IFN-{gamma} receptor expression to mediate cardiac allograft rejection, it is known that IFN-{gamma} is not required for unmodified cardiac allograft rejection (20). This implies that there are both IFN-{gamma}-dependent and IFN-{gamma}-independent pathways of rejection. Thus, while direct CD4+ T cell-mediated rejection may be IFN-{gamma} dependent, there are presumably other pathways that are IFN-{gamma} independent, such as B cell and/or CD8+ T cell responses. To identify whether donor IFN-{gamma} receptor deficiency conferred a protective effect to the cardiac allograft when exposed to a full complement of CD4+ and CD8 T+ cells plus B cells, we performed cardiac transplants of both WT and GRKO hearts to BALB/c scid recipients and then adoptively transferred 107 whole, unseparated BALB/c lymph node cells. Such transfer of whole lymph node cells led to acute rejection of both GRKO allografts (survival, 11.0 ± 1.2 days) and WT control allografts (survival, 13.0 ± 2.9 days; not significant; Fig. 5Go). Histologic analysis of rejected allografts demonstrated comparable lymphocytic infiltration consistent with severe acute rejection in both GRKO and WT allografts (not shown). These results indicate that, unlike the isolated CD4+ T cell immune response that is IFN-{gamma} dependent, an IFN-{gamma}-independent mechanism (or mechanisms) capable of mediating acute rejection exists, presumably generated by an activated B cell and/or CD8+ T cell population.



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  		FIGURE 5. IFN-{gamma} receptor-independent rejection of cardiac allografts. WT and GRKO cardiac allografts were established in BALB/c scid mice. These recipients then underwent reconstitution with 107 unseparated (CD4, CD8 and B cell-containing) lymph node cells. Acute rejection of both WT (n = 5; mean time to rejection, 13.0 days ± 2.9) and GRKO (n = 5; mean time to rejection, 11.0 ± 1.2 days) cardiac allografts was noted.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Numerous cellular and humoral components of both innate and adaptive immunity participate in allograft immunity. The extent to which defined cellular pathways individually contribute to rejection is uncertain, but it is clear that efficient acute cardiac allograft rejection requires the participation of CD4+ T cells (1, 2, 3, 4, 5, 6). While there is a critical role of the CD4 cell in acute cardiac allograft rejection, the precise mechanisms by which the CD4+ T cell contributes to allograft rejection are unclear. Major ambiguity lies in the multipotential functions of CD4+ T cells that include collaboration with B cells for the production donor-specific Ab, collaboration with CD8+ T cells that lead to direct allograft reactive CTLs, and both direct (donor MHC-restricted) and indirect (host MHC-restricted) recognition of the allograft, all of which may independently contribute to rejection. While CD4+ T cells have traditionally been viewed in a helper role, CD4+ T cells are actually sufficient as effector cells for triggering vigorous acute cardiac allograft rejection independently of either B cell or CD8+ T cell rejection (7, 29). Such CD4-mediated rejection requires donor MHC class II expression (29), implying a cognate recognition of the graft by CD4 cells. To establish the specific mechanisms required for such CD4+ T cell-mediated rejection, we used an adoptive transfer model to study the CD4 cell response in the absence of both CD8+ T cells and B cells. Using this model system, the immune response can be systematically defined such that the contribution of specific effector pathways to rejection can be individually examined. The present results demonstrate that the presence of donor IFN-{gamma} receptors is a rate-limiting requirement for CD4+ T cell-mediated acute cardiac allograft rejection.

While IFN-{gamma} is a prototypical Th1 cytokine that mediates many proinflammatory immune responses (11), it has become apparent that the precise role of IFN-{gamma} in the alloimmune response is complex, contributing to both graft-destructive and graft-protective immune responses. Elevated IFN-{gamma} gene expression is commonly associated with graft rejection (30), while reduced intragraft IFN-{gamma} often correlates with allograft tolerance (31). Paradoxically, however, IFN-{gamma} also contributes to allograft tolerance in some models. In cardiac allograft models of costimulation, blockade-induced, long term allograft survival, IFN-{gamma} deficiency in the recipient significantly diminishes allograft survival (22, 25), while in kidney and liver allograft models, IFN-{gamma} or IFN-{gamma} receptor deficiency actually enhances the rejection response (23, 32), suggesting that IFN-{gamma} may act in a protective fashion, possibly by inhibiting T cell proliferation (33), regulating CD8 T cells (21), and/or a direct protective effect on the transplant (23, 24). Conversely, IFN-{gamma} has been shown to contribute to acute rejection in neovascularized skin (18, 19) and islet (17) allograft models. Our results provide compelling evidence that IFN-{gamma} can contribute to allograft rejection in a model of vascularized allografts as well. Our results parallel those reported in class II-mismatched skin allografts, in which anti-IFN-{gamma} therapy prolongs skin allograft survival (18), and related studies showing class II-mismatched skin grafts are not rejected in an IFN-{gamma}-/- recipient (14, 19). A comparison of these results with those reported in our study is difficult, as this previous skin transplant model uses a graft disparate from the recipient only at the MHC class II locus. However, taken together with our results, these findings identify a primary role for IFN-{gamma} in alloimmune responses presumably directed against donor MHC class II.

There are several potential mechanisms that may explain the marked protective effect of graft IFN-{gamma} receptor deficiency in CD4+ T cell-mediated rejection. A straightforward explanation for our findings would be that IFN-{gamma} may play a nonredundant role in MHC class II up-regulation in response to allogeneic stimuli (13). Thus, IFN-{gamma} would be required to induce the expression of donor MHC class II, enhancing the targeting of sensitized CD4+ T cells reactive via the direct pathway. This would be consistent with our previous results indicating that donor, but not host, MHC class II was required for acute CD4 T cell-mediated cardiac allograft rejection (6). Our attempts to directly assess MHC class II expression on cardiac allografts failed to detect MHC class II on GRKO allografts, although detecting MHC class II expression on wild-type rejecting grafts proved to be problematic (not shown). An alternative explanation is that IFN-{gamma} may be required in the effector phase of the CD4 immune response to induce the expression of chemokines in the allograft such as Mig that lead to efficient T cell infiltration to the graft and subsequent cytotoxic activity, as suggested by Fairchild and colleagues (14, 34). Indeed, the transfer of naive CD4 T cells led to only modest graft infiltration. However, in vitro primed CD4 T cells were clearly capable of migrating to and persisting at the graft site without mediating rejection, suggesting that donor IFN-{gamma} receptors were not inherently required for CD4 T cell trafficking to the allograft. A third possible explanation for the finding that IFN-{gamma} receptors are necessary in the efferent phase of the CD4 immune response is that IFN-{gamma} may contribute a direct cytopathic effect on the allograft. For example, IFN-{gamma} may up-regulate death receptors on the graft such as Fas (CD95) or other TNF receptor family members on the allograft that may participate in CD4 T cell-mediated acute rejection. Our study does not segregate the role of IFN-{gamma} in the induction of donor MHC class II from its potential cytopathic role in enhancing graft sensitivity to cytotoxic mediators.

An intriguing implication of these results pertains to indirect (host MHC class II-dependent) donor Ag recognition. Allograft IFN-{gamma} receptor deficiency may diminish CD4-mediated graft reactivity via the direct pathway due to a defect in MHC class II induction, but is not expected to have an effect on the indirect recognition of the graft. Graft IFN-{gamma} receptor deficiency may lead to defects in CD4 effector function through the direct pathway by inhibiting MHC class II induction, chemokine expression, or TNF receptor and/or Fas (CD95) expression by the graft, as described above. However, in the present study in which wild-type CD4+ T cells are adoptively transferred to the BALB/c scid recipient that possesses an intact innate immune system (35, 36, 37), the indirect pathway of allorecognition remains entirely intact. Within this model system, indirectly responsive CD4+ T cells as well as professional APC, macrophages, and NK cells all can produce and/or respond to IFN-{gamma}. Despite an intact indirect CD4 recognition pathway and innate immunity, these components of the alloimmune response do not overcome the requirement for IFN-{gamma} receptor expression by the target graft to mediate acute rejection. Thus, indirect CD4-mediated immunity is not sufficient to trigger acute rejection of the GRKO allograft, consistent with our previous observations (6).

While the present study indicates that the presence of donor IFN-{gamma} receptors is a necessary requirement for CD4+ T cell-mediated acute allograft rejection, it is important to note that there are clearly both IFN-{gamma}-dependent and IFN-{gamma}-independent pathways of allograft rejection. As evidenced by the fact that unseparated lymph node cells reject GRKO allografts acutely, an intact cellular repertoire (CD4, CD8, and B cells) does not require allograft IFN-{gamma} receptors to mediate rejection. These results highlight the commonly appreciated redundancy of the immune response, with alternative mechanisms of rejection capable of mediating rejection of an allograft deficient in IFN-{gamma} receptors. These findings are consistent with prior reports that demonstrate that absence of IFN-{gamma} does not prevent acute cardiac rejection in an otherwise immunocompetent host (20). Also, previous studies suggest that IFN-{gamma} is preferentially required for the rejection of MHC class II-mismatched vs MHC class I-mismatched skin allografts (18, 19), implying that MHC class II induction on the graft is especially dependent on IFN-{gamma}. We are currently attempting to determine the nature of rejection of GRKO cardiac allografts mediated by an intact complement of CD4/CD8 T cells and B cells.

The growing body of literature implicating graft-protective properties of IFN-{gamma} has led to a conceptual shift toward regarding IFN-{gamma} as a regulatory cytokine. In addition to results stemming from transplantation studies, this unexpected regulatory role for IFN-{gamma} has emerged in autoimmune models as well (38, 39). Despite this expanded view of IFN-{gamma}, our findings clearly demonstrate a pathogenic role for IFN-{gamma} in cardiac allograft rejection, implicating Th1 CD4 cells as mediators of rejection. However, this conclusion is not mutually exclusive of the regulatory or protective roles for IFN-{gamma} found in allograft transplantation. This duality of cytokine function, in which a single cytokine has both immune-augmenting as well as immune-regulatory effects, has clearly become a theme in cytokine biology. For example, IL-2 has long been known as a key T cell growth factor early in the immune response (40) that can actually abrogate early allograft tolerance induction (41). However, IL-2 also contributes to activation-induced cell death later in the immune response (42, 43) and can be critical in the induction of allograft tolerance (44, 45). Thus, IL-2 has both positive and negative results in vitro and in vivo. IFN-{gamma} appears to function in a similar fashion, with distinct pathogenic and a regulatory effects on allograft survival. Our studies provide evidence of a pathogenic role for IFN-{gamma} in transplant immunity by showing that IFN-{gamma} interaction with the graft forms a rate-limiting step in CD4-mediated cardiac allograft rejection.


   Footnotes
 
1 This work was supported by U.S. Public Health Service Grants F32AI10362 (to A.C.W.), K08HL03594 (to B.A.P.), and RO1 DK55333 and DK33470 (to R.G.G.). Back

2 A.C.W. and B.A.P. contributed equally to this study. Back

3 Address correspondence and reprint requests to Dr. Ronald G. Gill, Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center, 4200 East 9th Avenue, Box B-140, Denver, CO 80262. E-mail address: ron.g.gill{at}uchsc.edu Back

4 Abbreviations used in this paper: GRKO, IFN-{gamma} receptor-deficient; WT, wild type. Back

Received for publication January 26, 2001. Accepted for publication August 27, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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