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The Journal of Immunology, 2003, 170: 1611-1614.
Copyright © 2003 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: Perforin Down-Regulates CD4 and CD8 T Cell-Mediated Immune Responses to a Transplanted Organ 1

Anirban Bose*, Yoshihiko Inoue{dagger}, Kenneth E. Kokko{ddagger} and Fadi G. Lakkis2,*

* Section of Nephrology, Department of Internal Medicine, and Section of Immunobiology, Yale University School of Medicine, New Haven, CT 06520; {dagger} Renal Division, Fujigaoka Hospital, Showa University, Yokohama, Japan; and {ddagger} Renal Division, Veterans Affairs Medical Center, and Department of Medicine, Emory University School of Medicine, Atlanta, GA 30033


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Perforin mediates target cell apoptosis by CTLs and NK cells. Although perforin expression correlates strongly with acute allograft rejection, perforin-deficient mice reject allografts with the same kinetics as wild-type recipients. In this study, we tested the hypothesis that while perforin is dispensable for acute rejection, it is essential for down-regulating the alloimmune response by inducing the apoptosis of host immune cells. Using a skin transplantation model, we found that perforin-deficient mice are resistant to the induction of allograft acceptance by agents that block T cell costimulation. Failure to induce allograft acceptance in these mice was observed irrespective of whether the alloimmune response was CD4 or CD8 T cell-mediated and could be attributed to defective apoptosis of activated CD4 and CD8 T cells. In contrast, perforin did not influence T cell proliferation. Therefore, perforin is an essential immunoregulatory molecule that may be required for the induction of transplantation tolerance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Perforin is an effector molecule expressed predominantly in CD8 CTLs and NK cells (1). The combined actions of perforin and granzymes, released upon CTL or NK cell activation, result in the death of target cells via caspase-dependent apoptosis pathways (1). These targets include virus-infected cells, tumor cells, and the allogeneic tissues of transplanted organs (2). Perforin appears to be a necessary component of the killing machinery as perforin gene knockout (PKO)3 mice are unable to clear intracellular infection with lymphocytic choriomeningitis virus and have significantly decreased tumor surveillance activity (3, 4, 5).

CTL play a major role in the destruction of allogeneic grafts, particularly those that express foreign MHC class I (MHC-I) molecules (6, 7), yet the importance of perforin in this process remains uncertain. Although intragraft perforin gene expression correlates strongly with kidney and heart rejection (8, 9, 10), allograft rejection is not delayed in the absence of perforin. PKO mice reject cardiac, skin, and pancreatic islet allografts at the same rate as wild-type (wt) recipients (11, 12, 13). This observation is attributed to the redundancy of the mechanisms by which CTL kill their targets (for example, CTLs also induce the apoptosis of target cells via the Fas, TNFR, and granulysin pathways) (14, 15, 16), and to the multiplicity of cellular pathways that lead to allograft rejection (17). In contrast to its redundant effector role, recent data suggest that perforin has an indispensable regulatory function in the immune system (18, 19). Mutations in the human perforin gene result in a syndrome known as familial hemophagocytic lymphohistiocytosis characterized by uncontrolled activation of macrophages and T cells that is triggered by viral infection (20). Similarly, exaggerated accumulation of activated CD8 T cells has been observed in PKO mice challenged with viral infection or acute graft-vs-host disease (GVHD) (21, 22, 23, 24). Immune dysregulation in PKO mice is not restricted to the CD8 T cell compartment, it also affects B cells. PKO mice bred onto the MRL/lpr background or those subjected to GVHD have increased B cell numbers and increased humoral autoimmunity (24, 25).

How perforin deficiency leads to dysregulated immunity remains controversial. Proposed mechanisms include uncontrolled T cell proliferation (21), decreased activation-induced T cell death (AICD) (23, 26), and decreased elimination of activated B cells and macrophages (24, 27). Importantly, the extent to which perforin regulates the immune response and the mechanism by which it does so appear to be dependent on the nature and strength of the immunological stimulus. Strong stimuli that generate large numbers of CTL, for example, lymphocytic choriomeningitis virus infection, are particularly susceptible to regulation by perforin (21, 22), while other stimuli such as Listeria monocytogenes infection and immunization with peptide-pulsed dendritic cells appear to be much less influenced by perforin-mediated immune regulation (28, 29). Generation of perforin-containing CTL is a prominent feature of the immune response to organ and tissue allografts but it is not known whether perforin is required for controlling such a response. Moreover, it is not clear whether perforin regulates CD8-mediated immune responses only or whether it regulates those mediated by CD4 T cells as well. In this study, we endeavored to answer these questions using a well-characterized murine skin transplantation model in which CD4 and CD8 alloimmune responses can be analyzed separately. We report that perforin plays an essential role in down-regulating both CD4 and CD8 T cell-mediated responses to a transplanted organ by inducing the apoptosis of activated T cells.


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

Male C57BL/6 (H-2b), BALB/c (H-2d), C57BL/6-Pfptm1Sdz (H-2b) PKO, C57BL/6.C-H2bm1 (bm1), and C57BL/6.C-H2bm12 (bm12) mice 6–8 wk of age were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were housed in a specific pathogen-free facility. Murine CTLA4 Ig which blocks the B7-CD28 T cell costimulation pathway and anti-mouse gp39 mAb (MR1) which blocks CD40-CD40 ligand interaction were provided by Dr. C. P. Larsen (Emory University, Atlanta, GA). Reagents were diluted in endotoxin-free PBS before injection.

Transplantation procedure

Full-thickness trunk skin from MHC-I-mismatched (bm1), MHC class II (MHC-II)-mismatched (bm12), and fully allogeneic (BALB/c) donor mice was transplanted onto the dorsal trunk of 6- to 8-wk-old PKO or wt C57BL/6 recipient mice according to standard methods. Recipients were either treated with 500 µg of CTLA4 Ig i.p. on day 2 plus 500 µg of MR1 i.p. on days 2, 4, 6, and 8 posttransplantation or were left untreated. Skin grafts were monitored daily after removal of the bandage on day 8 and rejection was defined as >90% necrosis of the graft. Skin grafts that survived and exhibited normal hair growth on day 80 were considered to be accepted long-term.

Detection of T cell apoptosis and proliferation

PKO and wt C57BL/6 mice were injected in the footpads and i.p. with 1 x 107 and 2 x 107 BALB/c splenocytes, respectively. Control mice received an equal number of syngeneic splenocytes. Seven days later, mice were restimulated in a fashion identical to the first stimulus. Twenty-four hours later, the mice were sacrificed and T cells were isolated from the popliteal, inguinal, and mesenteric lymph nodes by nylon-wool enrichment (Polysciences, Warrington, PA). Cells (1 x 107) were cultured for 24 h in complete DMEM at 37°C and 5% CO2. Cells were then stained with PE-conjugated anti-mouse CD4 or PerCp-conjugated anti-mouse CD8 (BD PharMingen, San Diego, CA). Following fixation in 2% paraformaldehyde, cells were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate and labeled with fluorescein-tagged dUTP using the TUNEL method (In Situ Cell Death Detection kit; Boehringer Mannheim, Mannheim, Germany). Cells were then analyzed by flow cytometry using a FACSCalibur (BD Biosciences, Mountain View, CA). CD4 and CD8 lymphocyte populations were gated separately and the percentage of TUNEL+ cells present in each population was calculated. To measure in vivo T cell proliferation, PKO and wt mice stimulated as described above received 0.8 mg of bromo-deoxyuridine (BrdU) (Sigma-Aldrich, St. Louis, MO) i.p. 24 h before sacrifice. Lymph node T cells were isolated as described above and surface-stained for CD4 or CD8. Cells were then fixed in 70% ethanol followed by 1% paraformaldehyde, incubated with 50 Kunitz/ml DNase I (Sigma-Aldrich) for 10 min at room temperature, stained with FITC-conjugated anti-BrdU Ab (BD Biosciences), and analyzed by flow cytometry. CD4 and CD8 lymphocyte populations were gated separately and the percentage of BrdU+ cells present in each population was calculated.

Statistical analyses

Differences in allograft survival were analyzed by the Mann-Whitney U test. Differences in T cell apoptosis and proliferation were analyzed by ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Failure to induce long-term survival of fully allogeneic grafts in PKO mice

To investigate the role of perforin in the alloimmune response, we transplanted wt and PKO C57BL/6 (H-2b) mice with fully allogeneic (MHC-I- and MHC-II-mismatched) BALB/c (H-2d) skin grafts. The recipients were either left untreated or received CTLA4 Ig and MR1 shortly after transplantation to block the B7-CD28 and CD40-CD40 ligand T cell costimulatory pathways. Untreated wt and PKO C57BL/6 mice acutely rejected their allografts with similar kinetics (median survival time (MST) = 13 days and 13 days, respectively) (Fig. 1). In contrast, CTLA4 Ig and MR1 treatment induced long-term allograft survival in 57% of wt recipients (MST > 80 days) but failed to do so in any of the PKO recipients (MST = 16 days). These findings indicate that, in the absence of perforin, acute allograft rejection is not delayed but the induction of long-term allograft survival is significantly impaired.



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  		FIGURE 1. Failure to induce long-term survival of fully allogeneic skin grafts in PKO mice. Skin allograft survival was studied in C57BL/6 wt (filled symbols) and PKO (open symbols) mice that were either left untreated (circles, n = 5/group) or received CTLA4 Ig and MR1 (squares, n = 7–8/group) to induce long-term allograft survival. Rejection was defined as >90% necrosis of the skin graft.

 
Failure to induce long-term survival of either MHC-II- or MHC-I-mismatched grafts in PKO mice

The immune response to a fully allogeneic graft involves the activation of both CD4 and CD8 T cells. The results shown in Fig. 1 indicate that perforin facilitates the induction of long-term allograft survival but do not pinpoint whether perforin does so by down-regulating CD4 or CD8 T cell responses. To answer this question, we transplanted wt and PKO C57BL/6 mice with either MHC-II-mismatched (bm12) or MHC-I-mismatched (bm1) skin grafts as the rejection of bm12 and bm1 skin is mediated by either CD4 or CD8 T cells, respectively (6, 7, 30). As shown in Fig. 2a, acute rejection of bm12 skin grafts occurred at similar rates in untreated wt and PKO recipients (MST = 16 and 14 days, respectively), while the long-term survival of these grafts following treatment of the recipient with CTLA4 Ig and MR1 was observed only in wt mice (MST > 80 days). All treated PKO recipients rejected their bm12 skin grafts without any significant delay over untreated mice (MST = 13 days and 14 days, respectively). Similarly, untreated wt and PKO recipients rejected bm1 skin grafts with similar kinetics (MST = 17 and 19 days, respectively), while wt, but not PKO, recipients accepted these grafts long-term following treatment with CTLA4 Ig and MR1 (MST > 80 days and MST = 12 days, respectively) (Fig. 2b). These results suggest that perforin regulates both CD4- and CD8-mediated alloimmune responses to a solid organ transplant.



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  		FIGURE 2. Failure to induce long-term survival of either MHC-II- or MHC-I-mismatched grafts in PKO mice. MHC-II-mismatched bm12 (a) and MHC-I-mismatched bm1 (b) skin allograft survival was studied in C57BL/6 wt (filled symbols) and PKO (open symbols) mice that were either left untreated (circles, n = 5/group) or received CTLA4 Ig and MR1 (squares, n = 7–8/group) to induce long-term allograft survival. Rejection was defined as >90% necrosis of the skin graft.

 
Defective apoptosis of alloactivated CD4 and CD8 T cells in the absence of perforin

AICD is an important mechanism by which immune responses are controlled in the periphery and is necessary for the induction of tolerance to transplanted organs (31, 32). Because perforin in association with granzymes is an important mediator of cell apoptosis, we postulated that perforin facilitates the long-term survival of skin allografts in our model by killing host alloreactive CD4 and CD8 T cells. To test this hypothesis, we measured the rate of T cell AICD in wt and PKO C57BL/6 mice following in vivo challenge with fully allogeneic BALB/c splenocytes. The TUNEL method was used to measure cellular apoptosis. We found that T cell AICD was significantly impaired in PKO mice (Fig. 3). This finding applied to both the CD4 and CD8 T cell compartments (Fig. 3), suggesting that perforin down-regulates the alloimmune response by inducing the apoptosis of activated CD4 and CD8 T cells.



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  		FIGURE 3. Defective apoptosis of alloactivated CD4 and CD8 T cells in PKO mice. C57BL/6 wt and PKO mice were immunized with either syngeneic ({square}) or allogeneic BALB/c ({blacksquare}) splenocytes on days 1 and 7, and lymph node cells were harvested on day 8. The proportion of CD4 and CD8 T cells undergoing apoptosis was measured by three-color flow analysis using the TUNEL method. Data shown are mean ± SD of three independent experiments.

 
CD4 and CD8 T cell proliferation is intact in the absence of perforin

Another mechanism by which alloimmune responses are regulated is suppression of activated T cell proliferation by cytokines (for example, transforming growth factor {beta} and IFN-{gamma}) or by cell surface inhibitory molecules (for example, CTLA4) (33). One study has shown that the expansion of virus-specific T cells is exaggerated in the absence of perforin (21), suggesting that perforin may down-regulate the proliferation of activated T cells. Therefore, we investigated whether the inability to induce long-term allograft acceptance in PKO mice is due to dysregulated T cell proliferation. In vivo CD4 and CD8 T cell proliferation was measured by the BrdU uptake method in wt and PKO mice challenged with fully allogeneic BALB/c splenocytes. As shown in Fig. 4, the proportion of CD4 and CD8 T cells that had underwent cellular division was similar in wt and PKO mice. This observation was confirmed in MLRs using [3H]thymidine uptake to quantitate cellular division. PKO T cells stimulated in vitro with BALB/c, bm1, or bm12 splenocytes exhibited the same degree of proliferation as wt T cells (data not shown). Taken together, the data indicate that perforin does not regulate the proliferation of allostimulated T cells.



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  		FIGURE 4. CD4 and CD8 T cell proliferation is intact in PKO mice. C57BL/6 wt and PKO mice were immunized with either syngeneic ({square}) or allogeneic BALB/c ({blacksquare}) splenocytes on days 1 and 7. BrdU was administered i.p. on day 8, and lymph node cells were harvested 24 h later. The proportion of CD4 and CD8 T cells undergoing proliferation was measured by three-color flow analysis following labeling with FITC-conjugated anti-BrdU Ab. Data shown are mean ± SD of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We found in this study that although perforin is not required for acute rejection, it is essential for the induction of long-term allograft survival. Mice that lack perforin rejected fully allogeneic, MHC-I-mismatched, and MHC-II-mismatched skin transplants without any delay but, unlike wt recipients, were unable to accept the same grafts following treatment with agents that block T cell costimulation. Failure to induce skin graft acceptance in PKO mice in our study could be attributed to defective AICD of both CD4 and CD8 T cells as AICD is an important mechanism of tolerance induction. In contrast, T cell proliferation was not altered in the absence of perforin. Therefore, our findings indicate that perforin has a dual role in the alloimmune response. In one regard, it has an important but dispensable cytolytic function in the acute rejection process. In contrast, it is essential for down-regulating both CD4- and CD8-mediated alloimmune responses by inducing the apoptosis of activated T cells.

Recent observations in humans and experimental animals indicate that perforin has fundamental immunoregulatory functions in pathologic processes such as viral infection and GVHD (18, 19). The findings reported in our study are the first to demonstrate that perforin’s in vivo regulatory role also applies to alloimmune responses targeted against solid organ transplants. This is somewhat surprising as intragraft perforin expression and cytotoxicity are intimately linked to the acute rejection process (8, 9, 10). However, significant redundancies in the effector mechanisms that cause graft destruction can account for this apparent paradox. For example, CTL are capable of inducing apoptosis via perforin/granzyme-independent pathways such as Fas, TNFR, and granulysin (14, 15, 16), and even in the complete absence of CTL activity other cell types may mediate rejection (17). Such redundancies have also been observed in the case of effector cytokines produced during allograft rejection, for example, IL-2 and IFN-{gamma} (33). Like perforin, these cytokines were found to have indispensable immunoregulatory functions in vivo (33).

Our observation that perforin regulates CD8-mediated alloimmunity is consistent with the fact that CD8 T cells express large amounts of perforin upon activation and that perforin-mediated suicide and/or fratricide of CD8 cells has been demonstrated in other experimental systems, especially under conditions of chronic TCR stimulation (22, 34). Using a murine GVHD model in which sublethally irradiated SCID mice are infused with allogeneic T cells, Spaner et al. (23) found that PKO CD8 T cells expanded in the SCID host much more dramatically than their wt counterparts and that they were relatively resistant to AICD upon restimulation in vitro. However, a novel and unexpected finding in our study is that the AICD of CD4 T cells is also impaired in the absence of perforin. In addition, CD4-dependent alloimmune responses were down-regulated by perforin as allograft acceptance could not be achieved in PKO recipients of MHC-II-mismatched (bm12) skin transplants. There are several mechanisms by which perforin could down-regulate CD4-dependent immunity. First, perforin expression by a subgroup of activated CD4 T cells or by cross-primed CD8 T cells could potentially control the immune response by mediating the death of other CD4 T cells (fratricide) (35). Our data support this possibility as we did observe defective AICD of CD4 T cells in PKO mice. Alternatively, perforin-expressing, activated CD4 or CD8 T cells could control alloimmunity by killing APCs. This is suggested by the abnormal accumulation of histiocytes and macrophages in humans with familial hemophagocytic lymphohistiocytosis (20), and by direct evidence that host APC killing is impaired in the absence of perforin (24, 27).

In summary, we have presented evidence that perforin down-regulates CD4 and CD8 T cell-mediated alloimmune responses by inducing the apoptosis of activated T cells. Although perforin may be a useful marker for diagnosing acute allograft rejection in the clinic (8, 9, 10), our data demonstrate that it is in fact an important immunoregulatory molecule that may be required for the induction of transplantation tolerance.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI41643, AI49466, and AI44644 (to F.G.L.). A.B. is a recipient of a fellowship grant from the American Society of Transplantation. Back

2 Address correspondence and reprint requests to Dr. Fadi G. Lakkis, Section of Nephrology, Yale University School of Medicine, 333 Cedar Street, P. O. Box 208029, New Haven, CT 06520-8029. E-mail address: fadi.lakkis{at}yale.edu Back

3 Abbreviations used in this paper: PKO, perforin gene knockout; wt, wild type; MHC-I, MHC class I; GVHD, graft-vs-host disease; AICD, activation-induced T cell death; BrdU, bromo-deoxyuridine; MST, median survival time; MHC-II, MHC class II. Back

Received for publication November 22, 2002. Accepted for publication December 11, 2002.


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 Introduction
 Materials and Methods
 Results
 Discussion
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G. Galazka, A. Jurewicz, W. Orlowski, M. Stasiolek, C. F. Brosnan, C. S. Raine, and K. Selmaj
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Frontiers in Nephrology: The Varied Faces of Natural Killer Cells in Transplantation Contributions to Both Allograft Immunity and Tolerance
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M. J. Dobrzanski, J. B. Reome, J. A. Hollenbaugh, and R. W. Dutton
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F. G. Lakkis
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