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CD28-Specific Antibody Prevents Graft-Versus-Host Disease in Mice¹

Xue-Zhong Yu^*, Sasha J. Bidwell^*, Paul J. Martin^*, and Claudio Anasetti^2,*,

^* Human Immunogenetics Program, Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA 98109; and Department of Medicine, University of Washington, Seattle, WA 98195

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The costimulatory molecules B7-1 and B7-2 regulate T cell activation by delivering activation signals through CD28 and inhibitory signals through CTLA4. Graft-vs-host disease (GVHD) is caused by activated donor T cells. Previously, we showed that CD28-deficient donor T cells induced less-severe GVHD than wild-type donor T cells, suggesting that CD28 signals exacerbate GVHD. In this paper we demonstrate that CTLA4 signals attenuate the severity of GVHD. Targeting the CD28 receptor with a specific mAb modulates the receptor in vivo, inhibits donor T cell expansion, and prevents GVHD. CTLA4 signaling was necessary for this effect because treatment with a soluble ligand that blocks binding of B7 to both CD28 and CTLA4 did not prevent GVHD as effectively as anti-CD28 mAb. These results support the current model of T cell costimulation in which CD28 signals amplify GVHD while CTLA4 signals inhibit GVHD, providing evidence that selective targeting of CD28 might be a better therapeutic strategy for inducing immunological tolerance than blocking the ligands for both CD28 and CTLA4.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The primary determinant of T cell activation is the interaction of TCRs with antigenic peptides presented by MHC molecules on APC. The costimulatory molecules B7-1 and B7-2 expressed on APC regulate T cell responses by delivering activation signals through CD28 (1, 2, 3) and inhibitory signals through CTLA4 (4, 5, 6, 7, 8). The importance of the B7:CD28/CTLA4 pathways has been highlighted by studies showing that B7 blockade can suppress graft-vs-host disease (GVHD)³ and autoimmunity (9, 10, 11, 12, 13). Blockade of CTLA4 alone, however, can exacerbate autoimmune disease and enhance antitumor immunity (14, 15). These results suggested that selective inhibition of the B7:CD28 interaction with preservation of the B7:CTLA4 interaction would facilitate T cell tolerance.

GVHD results from activation of donor T cells response to alloantigens expressed by the host. GVHD remains a major complication of human allogeneic hematopoietic cell transplantation and is associated with high morbidity and mortality (16, 17, 18). By using CD28-deficient mice, we and others have found that the development of GVHD depends to some extent on signals delivered through CD28 (19, 20). In this study, we demonstrate that B7:CTLA4 interactions have inhibitory effects on the induction of GVHD independent of CD28, and selective targeting of CD28 by a specific mAb is more immunosuppressive than blocking the ligands for both CD28 and CTLA4.

	Materials and Methods

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Mice

C57BL/6 (B6), B6.C-H2^bm12 (bm12), B6.C-H2^bm1 (bm1), (B6 x BALB/c)F₁ (CB6F₁), BALB/c H2-dm2 (dm2), and B6.SJL-Ly5^a Ptprc^a Pep3^b (B6.Ly5.1) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). (B6 x bm12)F₁, (B6 x bm1)F₁, and (B6 x dm2)F₁ (dm2B6F₁) mice were bred at the Fred Hutchinson Cancer Research Center (Seattle, WA). Founders for the 2C transgenic strain were kindly provided by Dr. Dennis Y. Loh (Nippon Roche Research Center, Kamakur-shi, Japan). Homozygous B6 CD28^-/- mice were a generous gift of Dr. Craig Thompson (2). 2C CD28^-/- mice were generated by intercrossing 2C to CD28^-/-. All the mice used in this study were housed in microisolator cages.

T cell purification and transplantation

Our protocol for T cell purification and transplantation has been described in detail (19, 21). CD4⁺ and CD8⁺ T cells were purified by positive selection using a magnetic cell separation system (Miltenyi Biotech, Auburn, CA). To avoid graft rejection, F₁ mice were used as recipients in all experiments. (B6 x bm12)F₁ or (B6 x bm1)F₁ mice were exposed to 700 cGy from ⁶⁰Co sources at 20 cGy/min. CB6F₁ or dm2B6F₁ mice were exposed to 750 cGy. Purified CD4⁺ or CD8⁺ cells from B6 donors were injected via the tail vein into irradiated (B6 x bm12)F₁ or (B6 x bm1)F₁ recipients, respectively. In some experiments, Ly5.1-congenic recipients were utilized to distinguish donor cells from host cells. Irradiated CB6F₁ or dm2B6F recipients were transplanted with 6–15 x 10⁶ purified CD8⁺ cells from 2C donors. Within each experiment, all recipients were injected with an identical number of 2C CD8 cells.

Preparation and administration of Abs

Both anti-CD28 (37.51) and anti-CTLA4 (9H10) are hamster IgG and were kindly provided by Dr. James Allison (University of California, Berkeley, CA). Murine CTLA4-Ig and control L6-Ig were kindly provided by Dr. Robert Peach (Bristol-Myers Squibb, Princeton, NJ). Control hamster Ig was purchased from IGN Pharmaceuticals (Aurora, OH). All the Abs, unless indicated, were injected i.p. at 100 µg/dose every other day for 14 days starting on the day of the transplant.

Flow cytometry

To detect donor CD4 or CD8 cells, splenocytes were isolated from the recipients and stained with mAbs specific for Ly5.1 (A20-1.7, mouse IgG2a; American Type Culture Collection, Manassas, VA) and CD4 (GK1.5) or CD8 (53-6.7). For detection of 2C donor cells, mAbs specific for CD8 and 2C TCR (1B2) were used. The 1B2 hybridoma was kindly provided by Dr. D. Loh (22), and FITC-conjugated 1B2 was prepared in our laboratory. Other mAbs used in this study included: anti-B220 (RA3-6B2), anti-CD28 (37.51), anti-CD25 (7D4), anti-CTLA4 (9H10), mouse anti-hamster IgG (192.1), and isotype control Abs. Except where noted, all mAbs used for FACS analysis were obtained from PharMingen (San Diego, CA). To test for CD28 modulation in vivo, freshly isolated splenocytes were incubated with saturating amounts of anti-CD28 mAb or normal hamster IgG for 30 min at 4°C. After washing, the cells were labeled with FITC-conjugated mouse anti-hamster IgG. Intracellular detection of CTLA4 was conducted as previously described (23). Briefly, cells were fixed with 1% paraformaldehyde, permeablized with 0.3% saponin, and stained with anti-CTLA4 mAb followed by FITC-conjugated mouse anti-hamster IgG. We used a FACScan with CellQuest software (Becton Dickinson, San Jose, CA) for flow cytometric analysis.

Statistical analysis

Continuous distributions were compared by Student’s t tests. Survival distributions were compared by log-rank tests. Two-sided p values <0.05 were considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CTLA4 signals inhibit GVHD

To determine the effect of CTLA4 signals on the development of GVHD, we first tested whether CTLA4 blockade with a nonstimulatory, bivalent mAb would accelerate GVHD. Sublethally irradiated (700 cGy) (B6 x bm12)F₁ mice were transplanted with purified CD4⁺ cells from wild-type B6 mice and treated with anti-CTLA4 mAb or hamster IgG at 100 µg/dose every other day for a total of eight doses. Treatment with anti-CTLA4 mAb was shown to accelerate GVHD lethality (p = 0.005) (Fig. 1A).

View larger version (20K): [in this window] [in a new window]   FIGURE 1. CTLA4 protects mice from GVHD. (B6 x bm12)F1 mice were irradiated (700 cGy) and transplanted with 1 x 106 purified CD4+ cells from CD28+/+ (A) or CD28-/- B6 (B) donors. Irradiated (B6 x bm12)F1 mice were injected with PBS alone as controls without GVHD. Each Ab was injected i.p. at 100 µg/recipient every other day for a total of eight doses. Data are shown from one experiment in A, and two replicate experiments in B.

Anti-CD28 mAbs prevent lethal GVHD

Simultaneous blockade of B7 interaction with anti-CD28 and CTLA4 by administration of soluble CTLA4-Ig or B7-specific mAbs can partially inhibit the development of GVHD in mice (9, 10, 11, 12). Because CD28 signals enhance GVHD (19, 20), while CTLA4 signals inhibit GVHD (Fig. 1), we reasoned that the severity of GVHD would be decreased by selectively blocking CD28 costimulation while still allowing CTLA4 engagement on donor T cells. We tested the effect of anti-CD28 mAb in preventing GVHD based on the observation that the administration of intact anti-CD28 mAb inhibits T cell expansion in vivo (24, 25, 26), even though anti-CD28 mAb amplifies T cell activation in vitro. Sublethally irradiated MHC class II incompatible (B6 x bm12) or MHC class I incompatible (B6 x bm1)F₁ mice were transplanted with B6 CD4⁺ or CD8⁺ T cells, respectively. Recipients were treated with anti-CD28 mAb, CTLA4-Ig, or hamster IgG plus L6-Ig at 100 µg/dose every other day from day 0 to day 14. Irradiated controls that were not transplanted developed transient pancytopenia, but all recovered and survived longer than 100 days (Fig. 2, A and B). Recipients injected with allogenic T cells and treated with control Abs became acutely ill with progressive weight loss, ruffled fur, and kyphosis, and all died at a median of 15 days after transplant. Both CTLA4-Ig and anti-CD28 mAb significantly improved survival as compared with control Abs (p < 0.0001), but anti-CD28 mAb was significantly more effective than CTLA4-Ig (p < 0.01).

View larger version (23K): [in this window] [in a new window]   FIGURE 2. Anti-CD28 mAb is more effective in preventing lethal GVHD than CTLA4-Ig. (B6 x bm12)F1 (A) or (B6 x bm1)F1 (B) mice were irradiated (700 cGy) and transplanted with purified CD4+ (A) or CD8+ (B) cells from B6 mice, respectively. A group of irradiated F1 mice were injected with PBS alone as controls without GVHD. Recipients were treated with the Abs indicated from day 0 to day 14 after. Data were pooled from four replicate experiments for A, and three replicate experiments for B. The results from each replicate experiment presented cumulatively consistently showed a significant effect of anti-CD28 treatment on GVHD mortality.

To elucidate the mechanisms by which anti-CD28 treatment prevents GVHD, we tested the effects of anti-CD28 mAb on donor T cell activation and expansion. Sublethally irradiated (B6.Ly5.1 x bm12)F₁ mice (five to six mice per group) were transplanted with purified CD4 cells from B6.Ly5.2 donors and treated with anti-CD28 mAb, CTLA4-Ig, or hamster Ig plus L6-Ig. In recipients treated, respectively, with control Abs, CTLA4-Ig, and anti-CD28 mAb, the percentages of CD4⁺Ly5.1^- donor T cells in the blood were 18.8 ± 0.3%, 4.4 ± 1.0%, and 1.7 ± 0.4% on day 6, and 52.1 ± 12.%, 19.1 ± 14.2%, and 7.3 ± 4.2% on day 15. These data suggest that both CTLA4-Ig and anti-CD28 mAb inhibited donor T cell expansion in vivo and that anti-CD28 mAb was significantly more effective than CTLA4-Ig (p < 0.01). In further experiments, we have tested the effect of anti-CD28 mAb on the expansion of donor T cells in peripheral lymphoid organs. We found that anti-CD28 mAb inhibited donor T cell expansion and was superior to CTLA4-Ig (Fig. 3, upper panels). We also found that anti-CD28 mAb induced CD28 modulation on CD4⁺/Ly5.1^- donor T cells, whereas control Abs and CTLA4-Ig did not have this effect (Fig. 3, lower panels). Inhibitory effects of anti-CD28 mAb on donor T cell expansion and CD28 modulation were also observed in transplantation of B6 CD8 cells into (B6.Ly5.1 x bm1)F₁ (data not shown).

View larger version (37K): [in this window] [in a new window]   FIGURE 3. Anti-CD28 mAb inhibits donor CD4 T cell expansion and modulates CD28. (B6.Ly5.1 x bm12)F1 recipients were transplanted with B6.Ly5.2 CD4+ T cells and treated with anti-CD28 mAb, CTLA4-Ig or control Abs. On days 2, 3, and 4 after the transplants, recipient splenocytes were stained for expression of Ly5.1, CD4, and CD28. Data in the figure shown are representative results on day 4. Top panels, The percentage and absolute numbers of CD4+/Ly5.1- donor T cells. Bottom panels, CD28 expression on gated donor T cells.

To follow the fate and function of T cells that recognize recipient alloantigen in vivo, we have used a model in which 2C TCR transgenic T cells were transplanted into CB6F₁ recipients that expresses the specific alloantigen L^d. In this model, 2C cells engrafted, expanded, and became effectors leading to extensive destruction of host B cells and double positive thymocytes (21). In additional experiments, we tested the effect of anti-CD28 mAb on activation of 2C cells in CB6F₁ recipients. Sublethally irradiated CB6F₁ mice were transplanted with purified CD8⁺ cells from 2C wild-type or 2C CD28^-/- mice and treated with anti-CD28 mAb or hamster IgG. On day 4, 2C cells in recipient spleen were analyzed for expression CD25 and CTLA4 (Fig. 4). CTLA4 expression was induced in wild-type 2C cells and in CD28^-/- 2C cells and was not affected by anti-CD28 treatment, indicating that CD28 signals are not needed for activation-dependent expression of CTLA4. Higher level of CD25 expression was induced in wild-type 2C cells than in CD28^-/- 2C cells, and CD25 expression was not affected by anti-CD28 treatment. These results show that treatment with anti-CD28 mAb did not block early CD28 signaling that is largely required for CD25 expression.

View larger version (35K): [in this window] [in a new window]   FIGURE 4. Anti-CD28 mAb inhibits donor T cell expansion, but does not affect expression of CD25 and CTLA4. CB6F1 mice were transplanted with CD8+ cells from CD28+/+ or CD28-/- 2C donors and treated with anti-CD28 mAb or control hamster IgG. On day 4 after the transplant, recipient splenocytes were tested for the surface expression of 1B2 (the clonotypic 2C TCR-specific mAb), CD8 and CD25 or the intracellular expression of CTLA4. Results were similar in two other experiments.

To determine whether anti-CD28 mAb prevents GVHD by depleting CD28⁺ T cells in vivo, we transplanted purified CD8⁺ 2C T cells into irradiated CB6F₁ recipients. In this experiment, we used L^d loss mutant dm2B6F₁ recipients as negative controls. Treatment with anti-CD28 mAb had no effect on the number of 2C cells on day 14 in dm2B6F₁ recipients, indicating that this mAb did not deplete resting CD28⁺ cells in vivo. Treatment with anti-CD28 mAb decreased the number of 2C cells in CB6F₁ recipients, indicating that anti-CD28 mAb interfered with expansion of donor T cells that recognize recipient alloantigens (Fig. 5A). The number of host B cells was 50-fold higher in CB6F₁ recipients treated with anti-CD28 mAb than in CB6F₁ recipients treated with control Ab, but 0.07-fold lower than in dm2B6F₁ negative controls (Fig. 5B). These results indicate that GVHD was reduced in severity but not completely prevented by treatment with anti-CD28 mAb.

View larger version (29K): [in this window] [in a new window]   FIGURE 5. Anti-CD28 mAb selectively inhibits expansion of 2C T cells and destruction of host B cells in CB6F1 recipients. CB6F1 (Ld+) or dm2B6F1 (Ld-) mice were transplanted with 2C CD8+ cells and treated with control Ab or with anti-CD28 mAb. On day 14 after the transplant, splenocytes from each recipient were stained for 1B2, CD8, and B220 and analyzed by three-color flow cytometry. The values shown are absolute numbers of each population per spleen. The results represent the mean ± SD from two to three mice per group.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Anti-CD28 mAb have notably different effects on T cell responses in vitro and in vivo. In vitro, they enhance proliferation in short-term assays, but in vivo, they prevent proliferation. We have shown that anti-CD28 mAb caused nearly completed modulation of CD28 in vivo (Fig. 3). In separate experiments, we have found that anti-CD28 mAb do not cause modulation of CD28 within the same time frame in vitro (data not shown). The reason for the difference in modulation remains for further investigation, but may be related to interaction with Fc receptors, causing extensive mobilization of CD28 molecules into intracellular contact caps in vivo. We suspect that the immunosuppressive activity of anti-CD28 mAb is related to this rapid modulation of CD28 receptor from the T cell surface, as observed in a rat heart allograft model (28).

The blockade of CTLA4-signals exacerbated GVHD independently of CD28 expression on donor T cells (Fig. 1), and CTLA4 expression and function were not affected by treatment with anti-CD28 mAb (Fig. 4). Our observations were consistent with previous reports showing that B7:CTLA4 interactions have a negative regulatory role on the capacity of CD28^-/- recipients to respond to tumor Ags or alloantigens (29, 30). Thus, CTLA4 retains its ability to inhibit T cell responses and protect from acute GVHD in the absence of CD28, indicating that cross-linking of CTLA4 can directly inhibit signaling events initiated through the TCR (31, 32). Alternatively, CTLA4 might inhibit other costimulatory signals such as those transduced by inducible co-stimulator (ICOS) or CD134 (OX40). Therefore, the preservation of CTLA4-negative regulatory signals should be helpful in preventing GVHD.

Treatment with anti-CD28 mAb may lead to B cell expansion in normal mice (33), but the number of host B cells in sublethally irradiated dm2B6F₁ recipients was not affected by anti-CD28 treatment (Fig. 5B). Thus, we can conclude that the increase in the number of host B cells in CB6F₁ recipients treated with anti-CD28 mAb results from decreased GVHD severity. The optimal dose and schedule of anti-CD28 mAb has not been determined. It is unlikely, however, that more than 100 µg anti-CD28 mAb per dose would achieve better results, because 100 µg/dose induced maximal CD28 modulation in vivo (data not shown). Incomplete prevention of GVHD by anti-CD28 treatment was consistent with our previous observation that CD28-deficient donor T cells have some ability to induce GVHD (19). These results suggest that other costimulatory systems can participate in alloimmune responses. Recently published results have indicated that CD154:CD40 pathway plays a particularly important role in the development of the immune responses (27, 34, 35). Saito et al. (27) have showed that treatment with anti-CD154 mAb ameliorates the manifestations of GVHD induced by CD28^-/- T cells. Thus, it is reasonable to expect that the blockade of CD28 and CD154, while preserving CTLA4 function, would be an effective strategy to induce transplantation tolerance.

In summary, our findings provide evidence that selective targeting of CD28 is more immunosuppressive than targeting B7 and blocking the function of both CD28 and CTLA4. Thus, treatment with an anti-CD28 mAb or other selective CD28 inhibitors could be applied for induction of T cell tolerance in human transplantation. The use of CD28 inhibitors in combination with agents that block other costimulatory interactions such as CD154:CD40 might be required for maximum effects.

	Acknowledgments

 
We thank Dr. James Allison for providing hybridomas that produce CD28 and CTLA4-specific mAbs, Dr. Robert Peach for providing murine CTLA4-Ig and L6-Ig, and Jennifer Brackensick for her skillful assistance in the preparation of the manuscript.

	Footnotes

 
¹ This work is supported by National Institutes of Health Grants CA18029 and AI 40680 (to C.A.) and AI33484 (to P.J.M.). X.-Z.Y. is a recipient of 1999–2000 Translational Research Award of Leukemia Society of America.

² Address correspondence to Dr. Claudio Anasetti, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, D2-100, Seattle, WA 98109.

³ Abbreviation used in this paper: GVHD, graft-vs-host disease.

Received for publication November 22, 1999. Accepted for publication February 22, 2000.

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