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J Am Soc Nephrol 13:519-527, 2002
© 2002 American Society of Nephrology

Anti-CD28 Monoclonal Antibody Therapy Prevents Chronic Rejection of Renal Allografts in Rats

Igor A. Laskowski*, Johann Pratschke*, Markus J. Wilhelm*, Victor M. Dong{ddagger}, Francisca Beato{ddagger}, Maarten Taal{ddagger}, Martin Gasser*, Wayne W. Hancock§, Mohamed H. Sayegh{ddagger} and Nicholas L. Tilney*{dagger}

*Surgical Research Laboratory, Harvard Medical School Cambridge, Massachusetts; Departments of {dagger}Surgery and {ddagger}Medicine, Renal Division, Brigham & Women’s Hospital, Boston, Massachusetts; §Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts.

Correspondence to Dr. Nicholas L. Tilney, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115. Phone: 617-732-6817; Fax: 617-232-9576; E-mail: bhayslett{at}rics.bwh.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. The effects of a signaling anti-CD28 mAb (JJ319), which interferes with the CD28-B7 T cell costimulation pathway thought to be involved in the development of chronic rejection of organ transplants, was investigated. Functional, morphologic, and molecular changes in rat renal allografts were examined up to 24 wk after placement. Control Lewis rats, recipients of F344 kidneys, received a single dose of a nonspecific mouse mAb intravenously on the day of transplantation (group 1). Group 2 animals were given anti-CD28 mAb in similar fashion. Group 3 animals were treated with a short course of cyclosporin A (CsA), and group 4 received both anti-CD 28 mAb and CsA. The majority (>95%) of animals in groups 2, 3, and 4 survived throughout the follow-up, compared with 28% in group 1 (P < 0.001). Group 2 and 4 recipients produced negligible proteinuria, whereas group 1 controls developed progressively increasing proteinuria after 4 wk and group 3 animals developed proteinuria by 24 wk. Allografts in groups 2 and 4 were morphologically unremarkable at 24 wk. Kidneys of group 1 animals rapidly developed changes of acute rejection, and those that survived long-term showed extensive glomerulosclerosis and interstitial fibrosis. Changes of early chronic rejection were noted in group 3 grafts. By reverse transcriptase–PCR, expression of representative inflammatory factors interferon-{gamma} and interleukin-10 were significantly elevated at 24 wk only in the surviving group 1 animals. A single dose of a signaling anti-CD28 mAb administered at transplantation or in combination with a short course of CsA significantly prolonged recipient survival, normalized function, and preserved the morphology of renal allografts in an established model of chronic rejection. These data support an important role for T cell costimulation in the evolution of the chronic process.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Although the early results of organ transplantation have improved progressively, the rate of attrition over time has remained relatively stable, despite ever more effective immunosuppression (1). The primary cause of long-term failure of organ allografts is chronic rejection. Although the pathophysiology of the process is not fully understood, it is clear that a foreign graft placed in an unmodified or inadequately immunosuppressed recipient will evoke a series of persisting specific alloimmune responses. Even with satisfactory immunosuppression, however, donor-associated inflammatory perturbations secondary to the initial effects of antigen-independent risk factors, such as brain death and/or ischemia/reperfusion injury, may trigger or amplify lymphocyte-mediated host alloresponsiveness, which is believed to be critical in later deterioration of the allograft (2). Two distinct signals are necessary for T cell activation. The first results are from direct interaction between the T cell receptor and donor MHC molecules expressed on antigen-presenting cells (APC) in the graft. The second is a costimulation step mediated by accessory molecules on the T lymphocytes, which interact with their ligands on APC. These steps provoke CD4+ lymphocytes to produce a series of proinflammatory factors, which in turn stimulate host endothelial cells and various leukocyte subpopulations to initiate the entire immunologic cascade ultimately responsible for graft destruction (3). In contrast, alloantigen-stimulated T cells may become anergic in the absence of the second costimulatory signal (4,5). The direct allorecognition pathway is thought to be important in acute rejection and indirectly involved in the development of the chronic process (6–8).

An important costimulatory signal is the interaction between the cytotoxic T leukocyte antigen 4 (CTLA-4) and its B7 ligand on graft APC. CTLA-4 is a CD28 receptor analog expressed on the surface of activated T cells and serves as a limiting factor in T cell clonal expansion. With a higher affinity to B7 than CD28, its signal downregulates T cell activity (9,10).

Blockade of the CD28-B7 costimulatory pathway by CTLA-4Ig, a recombinant fusion protein of CTLA-4 and IgG, inhibits the development and progression of immune activity in rat renal allografts (11,12). An alternative approach in a transplant setting is to alter or interfere with the CD28-B7 step by treatment of the recipient with an anti-CD28 monoclonal antibody (mAb). In the DA to LEW high-responder rat strain combination, for instance, this mAb prolonged vascularized heterotropic heart allograft survival but could not produce permanent engraftment or host tolerance (13). The investigators suggested that the mechanism responsible may involve the T cell receptor during primary alloantigen recognition. With CD28 ligation and subsequent modulation of the cell surface, CD28 will not be available for costimulation of the T cell response, making this population less responsive as CD28 remains downregulated. In this study, we have evaluated the influence of this strategy on chronic rejection of rat kidney allografts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and Surgical Techniques
Inbred male Lewis rats (LEW, RT11) 8 to 10 wk of age and weighing 200 to 250 g served as recipients of kidney allografts from Fisher 344 rats (F344, RT11v1). The specific pathogen-free animals were purchased from Harlan Sprague Dawley (Indianapolis, IN). All animal experimentation described was conducted in accord with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Orthotopic kidney transplantation was performed with end-to-end anastomosis of the left renal vessels and left ureter by use of a 10–0 Ethilon suture (Ethicon Inc., Somerville, NJ). Organs were cooled transiently (5 to 8 min) in 4°C saline before transplantation. The mean ischemic time was 24 min. Contralateral native nephrectomy was performed 10 d after transplantation. Animals with hydronephrosis of the kidney graft found at the time of death were excluded from the study.

Reagents
A specific rat anti-CD 28 mAb, JJ319, a generous gift from Dr. Thomas Hunig, Wuerzburg, Germany, was used. L-6, a nonspecific murine mAb, was obtained from Bristol Myers Squibb (Dr. Robert Peach, Princeton, NJ). Cyclosporin A (CsA) was from the Novartis Pharmaceuticals Corporation (E. Hanover, NJ).

Experimental Groups
Four experimental groups of LEW recipients of F344 kidneys were studied. Group 1 animals (n = 40) received a single injection of the control L-6 IgG mAb (0.5 mg) intravenously on the day of transplantation. Those in group 2 (n = 21) were given the anti-CD28 mAb in similar fashion. Group 3 recipients (n = 23), a standard model for chronic rejection, were treated with a short course of CsA (1.5 mg/kg per d for 10 d, subcutaneously) (14). Group 4 rats (n = 21) received anti-CD 28 mAb plus CsA in combination.

Survival and Graft Function
Recipient survival was determined over a 24-wk period. Because the bilaterally nephrectomized hosts are solely dependent on the function of the transplanted kidney, those with progressively dysfunctional allografts ultimately died of renal failure. Urine protein excretion (24 h) was measured at monthly intervals after transplantation, as an accurate assessment of chronic renal injury. Protein concentration was calculated after precipitation with 3% sulfosalicylic acid (Fisher Scientific, Fair Lawn, NJ) by measuring absorbency at 595-nm wavelength with the use of an UV-1201 spectrophotometer (Shimadzu, Kyoto, Japan).

Histology
Kidneys from representative animals in each group were harvested at 2, 12, and 24 wk after transplantation (n = 3 to 4/group per time point). Tissue samples were fixed and stored in 10% buffered formalin. Serial paraffin sections of each kidney were evaluated at multiple levels of each block by use of hematoxylin and eosin, periodic acid–Shiff, trichrome, and elastin stains.

Competitive Reverse Transcriptase–PCR
Reverse transcriptase (RT)–PCR was performed on grafts at 24 wk follow-up to compare the presence of mRNA of representative cell products between kidneys with changes of end-stage chronic rejection and those functioning well in treated hosts. Total RNA was extracted from snap-frozen portions of kidney by the acid guanidium isothiocyanate-phenol chloroform extraction method (15). RNA was quantitated by determination of ultraviolet absorbance at 260 nm, and its purity was assessed by measuring the optical density ratio at 260 and 280 nm. For preparation of cDNAs, 4 µg of heat-denatured RNA was used in a RT reaction. Preparations of cDNA were then used as the substrate for competitive PCR reaction by use of DNA mimics and oligonucleotide primer sets (Genosys Biotechnologies, Inc., Woodlands, TX). Comprising a segment of neutral DNA with sequences complimentary to the gene specific primers attached to each end, competitive DNA mimics for each factor were constructed by use of a PCR MIMIC Construction Kit (CLONTECH Laboratories, Inc., Palo Alto, CA). Previously published primer sets for rat glyceraldehyde-3-phosphate dehydrogenase, interferon-{gamma}, interleukin-10 (IL-10), IL-1ß, macrophage chemoattractant protein-1, transforming growth factor–ß1, and tumor necrosis factor–{alpha} were used (16–18). cDNA and competitive DNA mimic were added to the reaction mixture, and PCR was performed by use of a Peltier Thermal Cycler (MJ Research, Inc., Watertown, MA). Optimal PCR conditions, namely concentration of competitive DNA mimic, annealing temperature, and amplification cycles, were determined for each factor in preliminary studies. Sequences of oligonucleotide primer sets and optimal annealing temperatures were as follows: GAPDH, 5'-AAT GCA TCC TGC ACC ACC AA-3' and 5'-GTA GCC ATA TTC ATT GTC ATA-3', 55°C; interferon-{gamma}, 5'-ATG AGT GCT ACA CGC CGC GCT TTG G-3' and 5'-GAG TTC ATT GAC AGC TTT GTG CTG G-3', 60°C; IL-10, 5'-TCC ATC CGG GGT GAC AAT AAC-3' and 5'-AAT CAT TCT TCA CCT GCT CC-3', 65°C; IL-1ß, 5'-TGA TGT TCC CAT TAG ACA GC-3' and 5'-GAG GTG CTG ATG TAC CAG TT-3', 55°C; MCP-1, 5'-ATG CAG GTC TCT GTC ACG-3' and 5'-CTA GTT CTC TGT CAT ACT-3', 55°C; transforming growth factor–ß1, 5'-CTT CAG CTC CAC AGA GAA GAA CTG C-3' and 5'-CAC GAT CAT GTT GGA CAA CTG CTC-3', 64°C; and tumor necrosis factor–{alpha}, 5'- TAC TGA ACT TCG GGG TGA TTG GTC C-3' and 5'-CAG CCT TGT CCC TTG AAG AGA ACC-3', 65°C. PCR products (7 µl) were subjected to gel electrophoresis (5% polyacrylamide), and the DNA bands were visualized under ultraviolet light after ethidium bromide staining (0.05 µg/ml for 10 min) and photographed. Densities of competitive mimic and target gene DNA bands were measured by scanning densitometry by use of a ScanJet 4c (Hewlett Packard, Corvallis, OR) with NIH Image software. The ratios of the densities of the respective bands were plotted to establish a linear relationship of the ratios over serial dilutions of template. Thus, absolute amounts of RNA from unknown samples were calculated from the known amount of the mimic in the starting reaction, as described elsewhere (18). Specimens were run in duplicate and the average value used. We have established elsewhere (19) that this assay is readily capable of detecting a twofold difference in target gene concentration. Results were expressed as a ratio to GAPDH.

Statistical Analyses
Data are expressed as mean ± SEM. For statistical assessment of survival, a Kaplan-Meier survival analysis was performed. Urine protein excretion was compared among all groups by use of the Kruskal-Wallis test for several independent samples and the Mann-Whitney test for two independent samples. For RT-PCR analysis, a Kruskal-Wallis test was followed by the Mann-Whitney test for two independent samples. Results were taken as significantly different with P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Recipient Survival
Only 28% of the control rats that received the nonspecific L-6 mAb (group 1) survived throughout the 24 wk follow-up period (P < 0.0001) (Figure 1). In contrast, the majority (>95%) of treated animals (groups 2, 3, and 4) survived.



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  		Figure 1. The survival of recipients of renal allografts in the various experimental groups is shown. *P < 0.0001. {blacksquare}, group 1 (control IgG); {blacklozenge}, group 2 (anti-CD28); {blacktriangleup}, group 3 (cyclosporin A [CsA]); and X, group 4 (anti-CD28 plus CsA).

 
Proteinuria
Proteinuria in surviving animals from group 1 rose progressively after 8 wk (P < 0.001) (Figure 2). Group 2 and 4 recipients excreted minimal protein during the observation period. By 24 wk, rats from group 3 demonstrated increased protein loss secondary to early chronic rejection (P < 0.01 versus group 2).



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  		Figure 2. A progressive rise in proteinuria occurs in animals that received control antibodies versus those treated by CD28 and CD28/CsA treatment. Proteinuria increases slowly in group 3 animals with early chronic rejection. *P < 0.001, **P < 0.01. {image}, group 1 (control IgG); {image}, group 2 (anti-CD28); {square}, group 3 (CsA); and {blacksquare}, group 4 (anti-CD28 plus CsA).

 
Histology
Severe rejection with increasing tissue injury and intense mononuclear cell infiltration, including both small and large lymphocytes, lymphoblasts, and large numbers of inflammatory macrophages, was evident by 2 wk in the control group 1 allografts (Table 1). This progressed to extensive glomerulosclerosis and interstitial fibrosis by 12 wk in those that continued to support their hosts. By the end of the follow-up period, the remaining kidneys were end-stage. In contrast, although the allografts from group 2 recipients became obviously infiltrated with host mononuclear cells at 2 wk, these were primarily by small lymphocytes and macrophages. No concurrent structural injury was evident. By 12 wk, this infiltration had virtually disappeared, and by the end of the 24 wk follow-up period, the grafts of the anti-CD28–treated animals were relatively normal (Figure 3). Those of CsA-treated group 3 animals exhibited morphologic changes of early chronic rejection, with tubular injury, interstitial scarring, and persistent focal infiltration, whereas the allografts in group 4 remained histologically normal throughout the 24-wk follow-up.


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  		Table 1. Serial histologic changes in renal allografts in control and treated recipientsa
 


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  		Figure 3. Histologic findings in kidneys of recipients treated with nonspecific control IgG, CD28 monoclonal antibody (mAb), CsA, or combined CD28 mAb and CsA after being harvested at 2 wk or 6 mo posttransplant are shown. Control L-6 IgG treatment resulted in moderate acute rejection by 2 wk, with tubulitis and dense interstitial infiltrates; those kidneys that survived for 6 mo showed end-stage changes. CD28 mAb was also associated with a marked mononuclear cell infiltrate at 2 wk but without significant target injury, and grafts at 6 mo showed only focal and nonspecific tubular changes. CsA therapy was accompanied by minor histologic injury at 2 wk, but by 6 mo moderate tubular, interstitial, and glomerular injury were apparent. Although CD28/CsA showed some focal leukocyte infiltration at 2 wk, grafts at 6 mo were completely normal. Periodic acid–Schiff–stained paraffin sections, findings representative of four grafts/group per time point. MCP, macrophage chemoattractant protein; IL, interleukin; IFN, interferon. {image}, group 1 (control IgG); {image}, group 2 (anti-CD28); {square}, group 3 (CsA); and {blacksquare}, group 4 (anti-CD28 plus CsA). Magnification, x100.

 
Competitive RT-PCR
Mean GAPDH mRNA levels were similar among allografts in all groups, confirming that starting mRNA concentrations were not subject to systematic error. At 24 wk after transplantation, mRNA levels for the cytokines IL-10 and interferon-{gamma} were significantly lower in rats from the three treatment groups than in controls that received nonspecific IgG alone (P < 0.05) (Figure 4). A similar but statistically insignificant trend was observed for MCP-1. Tumor necrosis factor–{alpha}, transforming growth factor–ß, and IL-1ß were somewhat (NS) reduced in the allografts of groups 2 and 4 animals versus those in groups 1 and 3 at this late time point (data not shown).



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  		Figure 4. There is significant reduction in representative cell products in grafts of treated recipients versus controls at 24 wk (P < 0.05).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has been shown in several experimental transplant systems that blockade of T lymphocyte costimulation prevents the development of acute rejection of a variety of organ allografts (11,12,20–24). Its comparable inhibitory effects on the chronic process may be due either to depression of ongoing immunologic or inflammatory activity or to reduction of the influence of early kidney injury on later events. In these studies, we used a well-established F344->LEW renal allograft model of chronic rejection (group 3) to investigate the role of T cell costimulation in the process (14). In this weak strain combination, an acute episode of rejection beginning at ~1 wk after transplantation can be inhibited by administering low-dose CsA for a period of 10 d. Without further intervention, the rat recipients develop progressive renal dysfunction and characteristic early structural changes in their grafts beginning after ~12 to 16 wk. Beyond 24 wk, the follow-up period used in these studies, the worsening fibrosis produces overt renal failure and eventual death of the recipient. Allograft destruction developed inevitably in control animals (group 1) that received a nonspecific murine mAb, L-6. Half of the recipients died from irreversible rejection by 30 d and an additional 30% by 90 d. Because the remaining failed kidneys in group 1 had become end-stage at 24 wk, residual inflammatory activity was relatively low as a result of fibrosis and tissue remodeling, with minimally elevated mRNA expression of several representative cytokines and chemokines. In contrast, rats treated with a single dose of anti-CD28 mAb on the day of transplantation, either alone or in combination with low-dose CsA, experienced minimal late functional or morphologic changes. Although the functional characteristics of the transient infiltrate of small lymphocytes and macrophages in the grafts of group 2 anti-CD28–treated animals have not been defined, the presence of these populations may represent either resolving acute rejection that did not injure the graft tissue structurally or a population of regulatory cells that inhibited any effector activity.

Although both antigen-dependent and antigen-independent risk factors appear to be responsible for the process of chronic rejection, the initial nonspecific insults to the grafted organ from donor brain death or ischemia/reperfusion were minimal in these studies, because the transplantation event involved living donors and minimal cold ischemic time before revascularization. The long-term changes noted, by exclusion, must have developed primarily from immunologic injury, occurring either early postoperatively and/or by persistent host activity over time. Once activated by antigens during acute rejection, alloreactive T cells produce a variety of proinflammatory factors that upregulate adhesion molecules on vascular endothelium and recruit other host leukocyte populations to the graft. In addition to direct cell-to-cell cytotoxicity, elaboration and release of T cell effector products destroy allogeneic cells. In chronic rejection, in contrast, the acute inflammatory changes are replaced by tissue remodeling mediated primarily by macrophages and proliferation of vascular smooth-muscle cells secondary to elaboration of a variety of growth factors. This results in progressive glomerulosclerosis, tubular atrophy, and interstitial fibrosis (14,25). The increasing fibrosis of the tissue over time is presumably the reason that mRNA activity of the cytokines examined at 24 wk in these studies is relatively low. The role of T cells in this late process is less clear (26).

The effect of interfering with the costimulatory step of T lymphocyte activation emphasizes the influence of acute immunologic rejection as a risk factor for chronic changes as kidneys of treated recipients in groups 2 and 4 remained virtually normal at 24 wk both functionally and structurally. The findings in this study infer that anti-CD28 prevents the changes of chronic rejection, particularly when administered in conjunction with initial low-dose CsA. When CsA is given alone, acute (2 wk) morphologic changes in the grafts are minimal. However, the agent does little to alter the progressive chronic changes (14). The addition of anti-CD28 may protect the graft from injury over the long term by modulating acute immunologic changes not completely prevented by CsA, by reducing the effect of an early nonspecific insult such as ischemia/reperfusion or by acting directly on chronic mechanisms. In studies elsewhere (12) on the prevention of chronic rejection with the use of CTLA4-Ig, which blocks T cell costimulation, we showed that administration of the agent 8 wk after transplantation could still prevent the late changes. Because that treatment schedule was not repeated in these experiments, this latter point has not been defined.

The direct interaction between the T cell receptor and processed peptide of donor MHC on APC triggers graft rejection (27–31). However, a costimulatory signal is also required for full lymphocyte activation. Although several T cell surface receptors and their ligands are involved, the second signal is delivered primarily through interaction between CD28 on CD4+ and CD8+ lymphocytes and B7 on the surface of dendritic cells, activated macrophages, and B cells (9). The B7 ligand has been divided into two classes, B7-1 (CD80) and B7-2 (CD86), which share limited sequence homology (27). Both first and second signals activate transcription factors and upregulate genes responsible for production of IL-2 and its receptor, molecules responsible for clonal expansion of T cells. In the absence of the second costimulatory signal, in contrast, alloantigen-stimulated T cells may become anergic (4,5).

The ability of CTLA-4 to bind to B7 molecules was exploited by the use of CTLA-4Ig, a recombinant protein that contains the extracellular domain of soluble CTLA-4 fused to the heavy chain of an IgG1 molecule (32). Binding both B7-1 and B7-2 with CTLA4-Ig completely inhibits the CD28-B7 interaction. As a result, antigen-activated T cells produce only low levels of IL-2 clonal expansion (27–31,33). Costimulatory blockade by treatment of kidney recipients with CTLA-4Ig inhibits both acute and chronic rejection of several organs in a variety of experimental models (11,20–22,33–38).

In this study, an alternative approach to inhibition of T cell activation has been examined by use of an anti-CD28 mAb, JJ319. Although this costimulates T cells activated in vitro, as measured by cell proliferation and cytokine production, its exact mechanism of action is not known (39). However, there are two additional possibilities to the receptor modulation hypothesis (13). First, the mAb may signal T cells to increase IL-2 production, rendering them more susceptible to activation-induced cell death. The observation that CsA therapy does not abrogate the effect of the anti-CD28 antibody in this model suggests that IL-2 production, and thus activation-induced cell death, is not a major activity. However, the low dose of CsA used in our protocol may not result in complete inhibition of IL-2 secretion and thus may not completely prevent activation-induced cell death. Future studies are required to assess the status of immune activation in vivo and the role of T cell apoptosis in mediating the effects of anti-CD28 in this model. Second, anti-CD28 antibody may sterically block CD28 receptor in vivo, leading to unopposed CTLA4-B7 interactions and resulting in termination of immune responsiveness via negative signaling pathways. Regardless of its actual mechanisms, the ability of the mAb to prevent chronic rejection as effectively as treatment with CTLA4-Ig suggests an important option in modulating the indirect pathway of antigen recognition of an allografted organ.

An apparent and still undefined difference between the effects of the anti-CD28 mAb and CTLA-4 Ig in transplant models may involve their interaction with CsA. In studies of cardiac allografts, CTLA-4 Ig treatment alone increased allograft survival and preserved function and morphology completely. However, when a combination of CTLA-4 Ig plus low-dose CsA was given to the graft recipients, the protective function of the Ig molecule was lost, and graft arteriosclerosis developed (20). In contrast, in the renal allograft model of chronic rejection used in these studies, we have shown elsewhere (11) that CTLA4-Ig both alone and in combination with CsA prevents the development of the chronic changes. In the present experiments, the effects of JJ319 and CsA in combination also appeared to be additive. Whether the discrepancy in activity of CTLA4-Ig and CsA is organ dependant, differing between hearts and kidneys, is unclear.

This study demonstrates for the first time the effectiveness of the anti-CD28 mAb in preventing chronic kidney graft rejection and emphasizes the important role of T cell alloimune responsiveness in this process. Targeting the CD28 molecule appears to be a promising treatment option for inhibiting the development of the chronic process.


   Acknowledgments
 
This work was supported by United States Public Health Service Grants 5RO1 1 DK 46190-27 and 1PO1 AI40152-04.


   Footnotes
 
Present affiliations: UK Rudolf-Virchow, Humboldt-University, Berlin (J.P.); University of Muenster, Muenster, Germany (M.J.W.).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication April 5, 2001. Accepted for publication September 12, 2001.




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