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The Journal of Immunology, 2002, 168: 3786-3792.
Copyright © 2002 by The American Association of Immunologists

In Vivo CD86 Blockade Inhibits CD4+ T Cell Activation, Whereas CD80 Blockade Potentiates CD8+ T Cell Activation and CTL Effector Function1

Thomas J. Lang2,*, Phuong Nguyen*, Robert Peach{dagger}, William C. Gause{ddagger} and Charles S. Via*

* Research Service, Baltimore Veterans Affairs Medical Center, and Division of Rheumatology and Clinical Immunology, University of Maryland School of Medicine, Baltimore, MD 21201; {dagger} Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08540; and {ddagger} Department of Microbiology and Immunology, Uniformed Services University of Health Sciences, Bethesda, MD 20814


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To address whether a functional dichotomy exists between CD80 and CD86 in naive T cell activation in vivo, we administered anti-CD80 or CD86 blocking mAb alone or in combination to mice with parent-into-F1 graft-vs-host disease (GVHD). In this model, the injection of naive parental T cells into unirradiated F1 mice results in either a Th1 cytokine-driven, cell-mediated immune response (acute GVHD) or a Th2 cytokine-driven, Ab-mediated response (chronic GVHD) in the same F1 recipient. Combined CD80/CD86 blockade beginning at the time of donor cell transfer mimicked previous results seen with CTLA4Ig and completely abrogated either acute or chronic GVHD by preventing the activation and maturation of donor CD4+ T cells as measured by a block in acquisition of memory marker phenotype and cytokine production. Similar results were seen with selective CD86 blockade; however, the degree of CD4 inhibition was always less than that seen with combined CD80/CD86 blockade. A more striking effect was seen with selective CD80 blockade in that chronic GVHD was converted to acute GVHD. This effect was associated with the induction of Th1 cytokine production, donor CD8+ T cell activation, and development of antihost CTL. The similarity of this effect to that reported for selective CTLA4 blockade suggests that CD80 is a critical ligand for CTLA4 in mediating the down-regulation of Th1 responses and CD8+ T cell activation. In contrast, CD86 is critical for the activation of naive CD4+ T cells in either a Th1 or a Th2 cytokine-mediated response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Activation of naive T cells requires cognate interaction of the TCR with Ag as well as a second costimulatory signal. The best-characterized costimulatory molecules are CD28 and CTLA4 on the T cell and their coligands CD80 (B7-1) and CD86 (B7-2), expressed primarily on APCs. The importance of this costimulatory pathway in T cell activation is supported by in vitro data demonstrating that combined CD80 and CD86 blockade at the time of TCR engagement blocks T cell production of IL-2, IFN-{gamma}, and IL-4 (1, 2, 3, 4). Similarly, in vivo studies have shown that combined CD80 and CD86 blockade abrogates naive T cell activation and has proven to be beneficial in a variety of animal disease models in which T cell activation is critical, e.g., lupus, diabetes, experimental autoimmune encephalomyelitis, and allograft rejection (5, 6, 7, 8, 9, 10, 11). As a result, CD80 and CD86 have emerged as potential therapeutic targets in diseases mediated by T cells.

Although controversial, it has been suggested that CD80 and CD86 provide distinct signals for the differentiation of T cells (Th0) into either a Th1 (IFN-{gamma}-, IL-2-producing) or a Th2 (IL-4-, IL-10-producing) cytokine phenotype (12). Results using selective CD80 or CD86 blockade in vivo have been contradictory. For example, Kuchroo et al. (13), using experimental autoimmune encephalomyelitis as a model of Th1-mediated disease, demonstrated that selective CD80 blockade ameliorated disease by shifting the cytokine phenotype to a Th2 pattern, whereas selective CD86 blockade worsened disease. It was concluded that in vivo Th1 responses require CD80 costimulation, whereas Th2 responses require CD86 costimulation. By contrast, Lenschow et al. (6), using the nonobese diabetic mouse, a model in which a Th1 phenotype predominates, observed that selective CD80 blockade worsened disease, whereas selective CD86 blockade had a beneficial effect, leading to the conclusion that CD86 was a major costimulatory ligand in a Th1 response. Although disease in both models is Th1 driven, the models differ in several important areas, among which are the use of adjuvants and the degree to which disease is mediated by naive and memory T cells. It is becoming increasing clear that differences exist in the dependence of memory and naive T cells on CD28 costimulation (14), and that recently identified costimulatory receptor-ligand pairs are important in the differentiation of previously activated T cells (15). However, CD80 and CD86 remain the major costimulatory molecules in the induction of a naive T cell response.

To address whether a functional dichotomy exists between CD80 and CD86 in naive T cell activation in vivo, we used the parent-into-F1 (P->F1)3 model of graft-vs-host disease (GVHD). In this model, the injection of homozygous (parental) naive T cells into unirradiated F1 mice results in either a Th1 cytokine-driven, cell-mediated immune response (acute GVHD) or a Th2 cytokine-driven, Ab-mediated response (chronic GVHD). Important features of the model are as follows: 1) the alloantigen-specific donor T cells that drive disease can be studied separately from nonspecifically activated (host) T cells; 2) either a Th1-mediated or Th2-mediated response can be induced in the same F1 recipient depending on the strain used for donor cells; and 3) in vivo manipulations that alter disease by blocking T cell activation can be readily distinguished from those that induce immune deviation. Our results indicate that CD86 is critical for naive CD4+ T cell activation and differentiation into either a Th1 or Th2 phenotype. In contrast, CD80 is important in mediating a down-regulatory effect on CD8+ CTL development, perhaps through preferential binding to CTLA4.


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

Six- to 8-wk-old C57BL/6 (B6), DBA/2 (DBA or D2), and B6D2F1 mice were obtained from The Jackson Laboratory (Bar Harbor, ME).

Induction of GVHD

Single cell suspensions of splenocytes were obtained from either B6 or DBA male mice and resuspended in RPMI 1640 medium without FCS at 108 viable cells per milliliter. Unless otherwise noted, acute and chronic GVHD were induced by tail vein injection of either 50 x 106 B6 or 80 x 106 DBA splenocytes into nonirradiated B6D2F1 mice, as previously described (16). Negative controls consisted of age- and sex-matched uninjected F1 mice.

In vivo reagents and treatment protocol

Anti-CD80 mAb (16-10A1) and anti-CD86 mAb (GL1) were obtained from BD PharMingen (San Diego, CA). The anti-CD80-specific fusion protein Y100F (17) was a gift of Dr. R. Peach (Bristol-Myers Squibb, Princeton, NJ). Reagents were used at a dose of 100 µg of anti-CD80 mAb (16-10A1), 200 µg of Y100F, and 100 µg of anti-CD86 (GL1) mAb. Control mice received 100 µg of rat IgG2a {kappa} (isotype control for anti-CD86) and either 100 µg of hamster IgG (control for anti-CD80 mAb) or 200 µg of L6, a mouse-human fusion Ab specific for L6 tumor Ag (control for Y100F). Reagents were administered i.v. on the day of parental cell transfer and on days 3 and 7.

Flow cytometry studies

At the specified time points mice were sacrificed and spleens were harvested. Splenocytes were first incubated with anti-murine Fc{gamma}R mAb 2.4G2 (18) for 15–20 min, then stained with saturating concentration of FITC-conjugated, PE-conjugated, or biotin-conjugated mAb. Fluorochrome-conjugated anti-CD4, anti-CD8, anti-B220, anti-H-2Kb, anti-H-2Kd, and anti-CD44 were purchased from BD PharMingen. Three-color flow cytometry was performed using a FACScan flow cytometer (BD Immunocytometry Systems, San Jose, CA). Lymphocytes were gated based on forward and side scatter. Donor CD4+ and CD8+ T cells were identified as cells staining positive for the respective T cell marker and negatively for MHC class I of the nondonor parent. Analysis of CD44 brightness was performed on donor-gated CD4+ and CD8+ T cells. Anti-CD44 staining gave a clearly distinguishable bimodal pattern, allowing separation of donor T cells into bright (CD44high) and dull (CD44low) subpopulations.

Serologic assays

Serum was tested by ELISA for the presence of anti-ssDNA IgG Abs (16). Briefly, microtiter plates were coated with heat-denatured salmon sperm DNA, blocked with 2% BSA-PBS, and then incubated with serial 2-fold dilutions of mouse serum beginning at a dilution of 1/40. Wells were then washed and incubated with anti-mouse IgG conjugated with alkaline phosphatase (Southern Biotechnology Associates, Birmingham, AL). OD was determined at 405 nm. MRL/lpr serum was assayed as a standard, and arbitrary units were calculated using a value of 1000 U/ml for pooled MRL/lpr serum.

Detection of CTL activity ex vivo

Effector CTL activity was tested using freshly harvested splenocytes without an in vitro sensitization period in a 4-h 51Cr release assay as described (19). Targets were 51Cr-labeled EL-4 (H-2b) or P815 (H-2d) cell lines. Using serial dilutions, effectors were tested in triplicate at four E:T ratios beginning at 100:1 (1.5 x 106 effectors and 0.015 x 106 targets per well). The percentage of lysis was calculated according to the following formula: [(cpm sample - cpm spontaneous)/(cpm maximum - cpm spontaneous)] x 100%. Results are shown as the mean percent of lysis ± SEM at a given E:T ratio for each treatment group.

Cytokine expression by RT-PCR

The coupled RT-PCR was used as previously described (20). Briefly, 1 x 105 splenocytes were homogenized in RNA-Stat-60 (Tel-Test, Friendswood, TX). RNA was reverse-transcribed with Moloney murine leukemia virus reverse transcriptase (Life Technologies, Grand Island, NY). IL-4- and IFN-{gamma}-specific primers were used as previously described (20). To ensure that equal amounts of mRNA were amplified, RT-PCR was performed using primers for the housekeeping gene hypoxanthine phosphoribosyl transferase. For each PCR product the optimal number of PCR cycles was determined experimentally. PCR products were separated on agarose gel, transferred to nitrocellulose, and probed with cytokine-specific probes conjugated with HRP (Oligos Etc., Wilsonville, OR). Blots were developed by ECL (Amersham, Little Chalfont, U.K.). Bands were imaged by autoradiography and quantitated by densitometry. Cytokine densitometry results for each sample were normalized to hypoxanthine phosphoribosyl transferase and results for each cytokine were calculated as fold increase over the respective cytokine expression in control F1 mice according to the ratio (normalized experimental group mean:normalized untreated F1 mean).

Statistical analysis

Data were examined for normality and equal variance (Kolmogorov-Smirnov). If satisfactory, groups were compared by a two-tailed Student’s t test; if not, they were compared by the Mann-Whitney rank sum test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Maximal inhibition of chronic GVHD-associated B cell expansion is achieved with selective CD80 blockade

To determine the respective roles of CD80 and CD86 in the development of an in vivo Th2-driven response (e.g., chronic GVHD), combined or selective CD80 and CD86 blockade was initiated at the time of parental cell transfer, followed by analysis at day 14. As shown in Table IGo, untreated or control Ig-treated chronic GVHD mice exhibit the expected >=2-fold increase in host B cell numbers compared with normal F1 mice, similar to previous reports (21). Significant reductions in B cell expansion were seen for all GVHD groups with CD80 and/or CD86 blockade (p < 0.01), with the strongest effect seen with CD80 blockade. Selective CD86 blockade reduced GVHD-associated B cell expansion by 40–60% compared with untreated or control Ig-treated chronic GVHD. Further inhibition was seen with combined CD80/CD86 blockade, nearly normalizing host B cell numbers (p = 0.03, normal F1 vs CD80/86 blockade). The most striking results were seen for selective CD80 blockade, in which B cell numbers were reduced to ~50% below those of normal F1 mice (p < .001), suggesting that CD80 blockade may not just prevent B cell expansion but may actually promote B cell elimination. This observation has been confirmed in three additional independent experiments (B cell numbers range 15–50% below normal F1 controls) and was seen regardless of whether CD80 blockade was achieved using Y100F or anti-CD80 mAb (data not shown). Although the reduction in B cell expansion following combined CD80/CD86 blockade was typically greater than that seen with CD86 blockade alone, B cell numbers were never reduced below those of normal F1 mice as seen with selective CD80 blockade. Normal F1 mice treated with anti-CD80 mAb alone using the same dosing regimen exhibited no reduction of B cells at day 14 compared with untreated F1 mice (data not shown).


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  		Table I. CD80 blockade in chronic GVHD promotes donor CD8+ T cell expansion and B cell elimination, whereas CD86 blockade inhibits donor CD4+ T cell and host B cell expansion1

 
Only combined CD80/CD86 blockade completely inhibits autoantibody production

Serum anti-ssDNA levels were used as a marker for the polyclonal B cell hyperactivity characteristically present in chronic GVHD mice and transiently in acute GVHD mice (16). Selective CD80 or CD86 blockade resulted in a significant, but incomplete, reduction of serum anti-ssDNA titers compared with control Ig-treated mice (p < 0.01) (Table IGo). Complete normalization of anti-ssDNA levels was only seen with combined CD80/CD86 blockade (p < 0.05, combined CD80/86 blockade vs CD86 or CD80 blockade alone). Taken together, our results with combined CD80/CD86 blockade are similar to those reported for CTLA4Ig (22) and indicate that, at the doses used, complete costimulatory blockade is achieved with the combination of blocking Abs but not with either of these agents alone.

CD86 blockade inhibits but CD80 blockade promotes donor T cell engraftment

As shown in Table IGo, selective CD86 blockade reduced engraftment of donor CD4+ T cells by ~40% compared with control Ig-treated chronic GVHD; however, combined CD80/CD86 blockade resulted in a nearly 50% further reduction in donor CD4+ T cell engraftment (p < 0.05, average additional reduction of 50% over four experiments for CD86 vs CD80/86), implying that both CD80 and CD86 contribute to donor T cell engraftment and expansion. In contrast, no reduction of donor CD4+ T cell engraftment was seen following selective CD80 blockade, but rather an increase in engraftment was observed (range of 20–80% increase in CD4+ T cell engraftment with CD80 blockade compared with untreated chronic GVHD; n = 3 experiments). Strikingly, donor CD8+ T cell engraftment was increased 8-fold compared with control Ig-treated or untreated chronic GVHD mice (Table IGo) and was seen using either anti-CD80 mAb or Y100F in three additional experiments (range, 3- to 10-fold increase; data not shown).

CD80 blockade converts chronic GVHD to acute GVHD

The enhanced donor CD8+ T cell engraftment and reduced host B cell numbers following selective CD80 blockade suggest that CD8+ donor antihost CTL are present and that acute GVHD has developed in these mice. Key features that differentiate acute from chronic GVHD are the presence of ex vivo antihost CTL activity and elevated IFN-{gamma} production, both of which are present in acute GVHD and absent in chronic GVHD (21). As shown in Fig. 1Go, chronic GVHD mice receiving selective CD80 blockade exhibit significant antihost CTL activity ex vivo, which is not observed for either untreated or control mAb-treated chronic GVHD mice (CD80 blockade vs control Ig-treated, p < 0.01; normal F1 vs chronic GVHD or chronic GVHD plus L6, p = NS). Results in two additional experiments yielded a range in the percentage of specific killing of 10–27.4% with CD80 blockade vs 2–9.2% for control mice (n = 15 mice per treatment). Also consistent with acute GVHD, CD80 blockade in chronic GVHD mice resulted in a significant increase (~4-fold) in IFN-{gamma} mRNA compared with all other groups (p < 0.05) (Fig. 2GoB). By contrast, the increased IL-4 expression typical of chronic GVHD (21) was absent in anti-CD80-treated mice (Fig. 2GoA). CD86 blockade and combined CD80/86 blockade were equally effective in blocking IL-4 production in chronic GVHD mice and did not result in the induction of IFN-{gamma}.



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  		FIGURE 1. Selective CD80 blockade in chronic GVHD mice induces antihost cytolytic activity. Groups consisted of untreated normal F1 mice or chronic GVHD mice receiving either no mAb, 200 µg of Y100F, or 200 µg of control mAb (L6) on days 0, 3, and 7 after parental cell transfer (n = 4–5 mice per group). Ex vivo antihost CTL activity was determined on day 10. Results are shown as group mean ± SE percentage of killing on H-2b targets at a given E:T ratio; *, p < 0.01.

 


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  		FIGURE 2. Selective CD80 blockade promotes IFN-{gamma} mRNA expression in chronic GVHD mice. Semiquantitative RT-PCR was performed on cDNA from splenocytes taken on day 14 from chronic GVHD mice treated as outlined in Table IGo. Results are shown as average fold increase over uninjected F1 mice for IL-4 (A) and IFN-{gamma} (B) (n = 5 mice per group).

 
Donor T cell activation in chronic GVHD is inhibited by CD86 blockade but enhanced by CD80 blockade

The foregoing data strongly suggest that CD86 blockade either alone or combined with CD80 blockade inhibits donor T cell activation, whereas selective CD80 blockade does not inhibit T cell activation but rather induces immune deviation. To address the activation status of donor T cells following costimulatory blockade, donor T cell expression of an activation marker, CD44, was measured (23, 24). It has been previously shown that, in this model, >90% of donor T cells exhibit memory cell phenotype (CD44high and CD69low) by 14 days after cell transfer, and that complete costimulatory blockade with CTLA4Ig treatment prevents donor T cell activation, expansion, and acquisition of memory phenotype (22). As shown in Fig. 3Go, combined CD80/CD86 blockade in chronic GVHD mice acts similarly to published results with CTLA4Ig in that it completely inhibits the increase in both the percentage and the number of CD44high donor CD4+ T cells (p < 0.01). Smaller but statistically significant reductions in the percentage and number of donor CD44highCD4+ T cells were also observed with selective CD86 blockade (p < 0.01, CD86 blockade vs control Ig). In contrast, selective CD80 blockade significantly increased not only the number of CD44high donor CD4+ T cells (p < 0.01) but also the number of CD44high donor CD8+ T cells compared with untreated chronic GVHD mice, consistent with enhanced donor CD4+CD8+ T cell expansion and CTL maturation induced by selective CD80 blockade. The few CD8+ T cells that engraft in untreated chronic GVHD display an activated phenotype that is blocked by CD86 blockade and combined CD80/CD86 blockade.



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  		FIGURE 3. Both selective CD86 blockade and combined CD80/CD86 blockade inhibit donor CD4+ T cell activation, whereas selective CD80 blockade promotes activation of donor CD8+ T cells in chronic GVHD. Mice with chronic GVHD received no treatment or either 200 µg Y100F, 100 µg of anti-CD86, combined Y100F and anti-CD86, or L6 plus rat isotype control at days 0, 3, and 7. On day 14, donor CD4+ (upper panels) and CD8+ (lower panels) T cells were assayed for CD44 up-regulation by flow cytometry. CD44high values are shown as mean percentage (right panels) or mean absolute number (left panels) per group ± SE (n = 5 mice per group). *, p < 0.01.

 
CD80 and/or CD86 blockade in acute GVHD mimics the effects seen in chronic GVHD

To determine whether the strikingly different effects of selective CD80 and CD86 blockade seen in a Th2/Ab-mediated response (chronic GVHD) are also seen in a Th1/cell-mediated response, selective costimulatory blockade was produced in acute GVHD mice. As shown in Table IIGo, using 50 x 106 B6 donor splenocytes, combined CD80/CD86 blockade markedly impaired donor CD4+ and CD8+ T cell engraftment and completely blocked the elimination of host B cells, characteristic of acute GVHD. These results are similar to those reported for CTLA4Ig treatment in acute GVHD (22, 25) and indicate that, taken together with the above results, combined CD80/CD86 administration is capable of complete costimulatory blockade of donor T cells in either a Th1- or Th2-driven response. Selective CD86 blockade was only marginally effective in blocking acute GVHD, as shown by a small but statistically significant improvement in host B cell survival compared with control Ig-treated acute GVHD mice (8.6 x 106 vs 4.5 x 106; p < 0.05).


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  		Table II. Combined CD80/CD86 blockade inhibits acute GVHD more effectively than selective CD86 blockade1

 
No inhibition of acute GVHD was seen with selective CD80 blockade, and instead low-level potentiation of disease was observed as measured by small but statistically significant further reduction in host B cells (compared with control Ig-treated or untreated acute GVHD (p < 0.05)). Because acute GVHD induced with 50 x 106 donor cells results in near maximal elimination of host B cells, potentiation is difficult to assess. A second experiment was then performed using fewer donor cells (40 x 106) to determine whether a modulatory effect of selective CD80 or CD86 blockade could be detected. As shown in Table IIGo, despite the reduced number of donor cells, near total elimination of host B cells was still observed for untreated or control Ig-treated acute GVHD mice. Nevertheless, acute GVHD was blocked not only by combined CD80/CD86 blockade but also by selective CD86 blockade, as evidenced by complete inhibition of host B cell elimination (Table IIGo), inhibition of IFN-{gamma} up-regulation (Fig. 4Go), and an inhibition of CD44 up-regulation on donor CD4+ and CD8+ T cells as measured by either percentage or absolute number (Fig. 5Go), although selective CD86 blockade was less effective in blocking CD44 up-regulation than combined CD80/CD86 blockade. A potentiating effect of selective CD80 blockade was again difficult to detect due to the profound elimination of host B cell, even at the reduced donor cell inoculum, and is better seen in the chronic GVHD model. It should be noted that detection of donor T cells by flow cytometry becomes difficult after 2 wk in untreated acute GVHD mice, due to an overall down-modulation of MHC expression (26) and/or acquisition of host MHC by donor cells (27) that parallels disease severity (28). We interpret the reduced numbers of donor CD8+ T cells at 2 wk in anti-CD80 mAb-treated mice (compared with control Ig-treated or untreated acute GVHD) to be a reflection of accelerated GVHD-associated MHC down-regulation rather than impaired engraftment and milder disease. In support of this, other parameters of acute GVHD such as host B cell elimination (Table IIGo), IFN-{gamma} gene expression (Fig. 4Go), and percentage of donor CD4/CD8high cells are either comparable to untreated acute GVHD or significantly worse (e.g., host B cells, p < 0.05). Importantly, the observation that IFN-{gamma} levels are not reduced in anti-CD80-treated acute GVHD compared with untreated acute GVHD mice supports our interpretation that donor T cells are not, in fact, reduced by anti-CD80 treatment but rather MHC down-regulation is accelerated and, by extension, acute GVHD is accelerated.



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  		FIGURE 4. CD86 blockade alone or combined with CD80 blocks IFN-{gamma} mRNA expression in acute GVHD mice. Acute GVHD was induced using 40 x 106 B6 splenocytes and F1 mice were treated as in Table IIGo. RT-PCR was performed as in Materials and Methods using day 14 splenocytes. Results are shown as average fold increase over normal F1 mice (n = 5 mice per group).

 


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  		FIGURE 5. Both selective CD86 blockade and combined CD80/CD86 blockade inhibit donor CD4+ T cell activation in acute GVHD. Acute GVHD was induced with 40 x 106 B6 splenocytes and mAb dosing is as described in Table IIGo. CD44 up-regulation on donor CD4+ and CD8+ T cells was determined on day 14 by flow cytometry. Results are displayed as described for Fig. 3Go (n = 5 mice per group). *, p < 0.01 vs acute GVHD.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recently, new members of the B7 costimulatory family and their receptors have been described in addition to CD80 and CD86 (15, 29, 30). However, our results underscore the critical importance of CD80 and CD86 in the activation of naive CD4+ T cells. In this report, we have used the P->F1 model of GVHD to determine the in vivo role of CD80 and CD86 in the initiation of either a Th1/cell-mediated response (acute GVHD) or a Th2/Ab-mediated response (chronic GVHD). Advantages of this model are as follows: 1) either form of GVHD can be induced in the same F1 depending on the T cell subsets injected (16, 31, 32); 2) the Ag-specific T cells driving disease can be monitored separately from the remainder of the T cell pool; and 3) in vivo manipulations that alter disease by blocking T cell activation can be readily distinguished from those that cause alterations in the phenotype of the immune response (immune deviation). Using this model, we observed that combined CD80/CD86 blockade completely inhibited donor CD4+ T cell expansion, cytokine production, and acquisition of an activation phenotype in both acute and chronic GVHD. CD28/B7 costimulation has been reported to be critical for naive T cell differentiation for Th2 cells but not necessarily for Th1 cell development (33, 34); however, in the data presented in this work, combined CD80/CD86 blockade completely blocked both Th1-driven acute GVHD and Th2-driven chronic GVHD. Moreover, our results are in agreement with previous studies demonstrating that naive T cell activation can be prevented by blockade of both CD80 and CD86 using either CTLA4Ig, a fusion protein that binds CD80 and CD86 with high affinity (35), or combined anti-CD80 and anti-CD86 mAb. For example, combined anti-CD80/CD86 mAb treatment has been shown to inhibit acute lethal GVHD in an irradiated recipient model following transfer of either purified CD4+ or CD8+ T cells (36) and to block the Ag-specific expansion and activation of adoptively transferred TCR-transgenic CD4+ T cells (37). Similarly, CTLA4Ig was reported to completely inhibit both acute and chronic GVHD in the P->F1 model (22, 25). The mechanism involved appeared to be the induction of anergy rather than clonal deletion, because donor T cells were not deleted following CTLA4Ig treatment; however, they did not produce IL-2, nor did they acquire an activation phenotype (CD44high) (22).

Our studies with selective costimulatory blockade underscore the dichotomous roles of CD28 and CTLA4, as well as their preferential interactions with CD80 or CD86. CD28 is constitutively expressed on T cells and upon engagement with CD80 and CD86 delivers a proliferative signal to Ag-specific T cells, whereas CTLA4 is not expressed on resting T cells but instead is induced upon activation and delivers a down-regulatory signal to proliferating T cells (38, 39). The critical role of CD28 in donor T cell activation has been reported by Yu et al. (40), whose study demonstrated that anti-CD28 mAb treatment prevented acute GVHD in an irradiated recipient model. Our results demonstrating that selective CD86 blockade is almost as effective as combined CD80/86 blockade in blocking donor T cell activation in either form of GVHD, whereas selective CD80 blockade has no detectable inhibitory effect, indicate that in the P->F1 model CD86 is the primary ligand for CD28 in the initial activation of naive donor CD4+ T cells.

By contrast, our results demonstrating that selective CD80 blockade converts chronic GVHD to acute GVHD support the hypothesis that CD80 is a major ligand for CTLA4 in the delivery of a down-regulatory signal for Th1 responses. Regarding the paradoxical results seen with selective CD80 blockade (conversion of chronic GVHD to acute GVHD), it is important to note that in the DBA->F1 model of chronic GVHD, donor CD8+ T cells are contained in the donor inoculum; however, they typically do not engraft or become activated in numbers sufficient to induce acute GVHD. As a result, donor CD4+ T cell-driven chronic GVHD ensues. The defect in DBA CD8+ antihost CTL generation has not been fully explained but is due in part to a 9- to 10-fold reduction in the anti-F1 pCTL frequency compared with that of B6 mice (16). Thus, the DBA->F1 model of chronic GVHD allows the identification of biologic agents that will potentiate CD8+ CTL development in vivo and as a result convert chronic GVHD to acute GVHD. We have previously observed that rIL-12 administration, like selective CD80 blockade, converts chronic GVHD to acute GVHD in DBA->F1 mice (41). However, with rIL-12 administration, engraftment and expansion of donor CD8+ T cells, although increased over control, were not increased to the degree observed with CD80 blockade, suggesting that rIL-12 promotes CTL effector function in the engrafted donor DBA CD8+ T cells but does not significantly promote their expansion. In contrast, selective CD80 blockade in our studies was shown to promote DBA CD8+ T cell activation, expansion, and maturation of antihost CTL effectors, consistent with either the delivery of a stimulatory signal or the loss of an inhibitory signal.

It has been well reported that in vivo CTLA4 blockade potentiates Th1-mediated responses due to the loss of a down-regulatory signal (42, 43, 44, 45). In particular, CTLA4 blockade in the BALB->CBF1 model of chronic GVHD has been shown to enhance donor CD8+ T cell expansion (46). Additionally, in a murine model of cardiac allograft rejection using CD28 knockout recipient mice, blockade of CTLA4 or CD80 blockade potentiated graft rejection, whereas CD86 blockade significantly prolonged allograft survival (47). It has been suggested that CTLA4 engagement preferentially limits Th1 differentiation (39); however, in other experimental systems CTLA4 blockade was reported to potentiate Th2 responses (48, 49), indicating that despite the demonstrated ability of CTLA4 to down-regulate T cell responses there is no inherent capacity of CTLA4 to differentially regulate Th1 vs Th2 development (39). Nevertheless, our results with selective CD80 blockade are remarkably similar to those seen with selective CTLA4 blockade, in that enhanced donor CD8+ T cell expansion and activation are induced with treatment. In vitro and in vivo studies have identified CD80 as the preferential ligand for CTLA4 (47, 50, 51), and CD80-CTLA4 ligand binding in the absence of a functional CD28 molecule inhibits in vitro T cell activation (52). Taken together, these results strongly support the idea that CD80 is the major ligand for CTLA4 in vivo in the delivery of a T cell down-regulatory signal and that the mechanism by which CD80 blockade converts chronic GVHD to acute GVHD is through the interruption of CTLA4-CD80 interactions and the loss of that down-regulatory signal.

It is not clear whether selective CD80 blockade enhances donor CD8+ T cell expansion: 1) directly through an effect on CD8+ T cells; 2) indirectly through an effect on CD4+ T cells, which in turn provide increased help to donor CD8+ T cells; or 3) a combination of the two. This question will be difficult to directly address in the P->F1 model, as donor CD8+ T cells do not expand or mature into antihost CTL in the absence of donor CD4+ T cell activation (32). Moreover, we have been unable to demonstrate significant donor CD8+ T cell engraftment in F1 mice receiving purified DBA CD8+ T cells with or without selective CD80 blockade (data not shown), indicating that selective CD80 blockade cannot bypass the requirement for CD4+ T cell help, most likely because, in the absence of donor CD4+ T cell activation, significant up-regulation of CTLA4 on donor CD8+ T cells does not occur. Our data demonstrating that selective CD80 blockade often results in a 125–200% increase in donor CD4+ T cell engraftment (Table IGo and T. J. Lang, unpublished data) suggest a direct enhancing effect on donor CD4+ cells. However, simply doubling the number of donor cells (to include CD4+ T cells) in the DBA->F1 model is usually not sufficient to convert chronic GVHD to acute GVHD (53). Thus, selective CD80 blockade probably promotes CD8+ T cell expansion through both direct and indirect effects.

This study supports the primary role of CD86 in the differentiation of both Th1 and Th2 responses and is the first to show that selective CD80 blockade results in de novo expansion and maturation of CD8+ CTL from inactive precursors, thereby converting chronic GVHD (Th2 mediated) to acute GVHD (Th1 mediated). These results suggest a potential novel therapeutic role for selective CD80 blockade. Currently, CTLA4 blockade is being evaluated for its therapeutic potential as an antitumor agent (39). A similar approach with selective CD80 blockade may be possible either alone or in combination with CTLA4 blockade.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI47466 and AI33882, a Department of Veterans Affairs Merit Review grant (to C.S.V.), grants from the Arthritis Foundation, and the Maryland chapters of the Lupus Foundation and Arthritis Foundation. T.J.L. is supported by a Department of Veterans Affairs Career Development Award. Back

2 Address correspondence and reprint requests to Dr. Thomas J. Lang, Division of Rheumatology and Clinical Immunology, University of Maryland School of Medicine, MSTF 8-34, 10 South Pine Street, Baltimore, MD 21201. E-mail address: tlang001{at}umaryland.edu Back

3 Abbreviations used in this paper: P->F1, parent-into-F1; GVHD, graft-vs-host disease. Back

Received for publication October 18, 2001. Accepted for publication February 6, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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