Blocking LFA-1 Activation with Lovastatin Prevents Graft-versus-Host Disease in Mouse Bone Marrow Transplantation

Article Outline

• Abstract
• Introduction
• Materials and Methods
  • Animals and Reagents
  • T Cell Isolation
  • Static Adhesion Assay
  • Mixed Lymphocyte Culture
  • GVHD Induction
  • In Vivo Homing Assay
  • In Vivo Proliferation Assay
  • Statistical Analysis
• Results
  • Lovastatin Inhibits Adhesion, Proliferation, and Cytokine Production of Mouse T Cells In Vitro
  • Lovastatin Treatment Reduces GVHD Mortality and Morbidity in the C57BL/6 to Balb/C BMT Model
  • Lovastatin Decreases T Cell Homing to Lymph Nodes and Peyer's Patches
  • Lovastatin Reduces Donor-Derived T Cell Proliferation In Vivo
• Discussion
• Acknowledgments
• References
• Copyright

Graft-versus-host disease (GVHD) following bone marrow transplantation (BMT) is mediated by alloreactive donor T lymphocytes. Migration and activation of donor-derived T lymphocytes play critical roles in the development of GVHD. Leukocyte function–associated antigen-1 (LFA-1) regulates T cell adhesion and activation. We previously demonstrated that the I-domain, the ligand-binding site of LFA-1, changes from the low-affinity state to the high-affinity state on LFA-1 activation. Therapeutic antagonists, such as statins, inhibit LFA-1 activation and immune responses by modulating the affinity state of the LFA-1 I-domain. In the present study, we report that lovastatin blocked mouse T cell adhesion, proliferation, and cytokine production in vitro. Furthermore, blocking LFA-1 in the low-affinity state with lovastatin reduced the mortality and morbidity associated with GVHD in a murine BMT model. Specifically, lovastatin prevented T lymphocytes from homing to lymph nodes and Peyer's patches during the GVHD initiation phase and after donor lymphocyte infusion (DLI) after the establishment of GVHD. In addition, treatment with lovastatin impaired donor-derived T cell proliferation in vivo. Taken together, these results indicate the important role of lovastatin in the treatment of GVHD.

Key Words: LFA-1, GVHD, Statin

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Introduction 

Graft-versus-host disease (GVHD) is the primary cause of morbidity and mortality in patients after bone marrow transplantation (BMT), and thus a major obstacle to the cure of various malignant and nonmalignant disorders. GVHD is characterized by epithelial cell injury in skin, intestine, and liver, but has been observed, albeit less frequently, in other organs as well, including the eye and lung 1, 2. Although alloreactive T cells are the primary mediators of GVHD, the regulatory mechanisms controlling T cell activation in GVHD are not well understood [3]. Murine models of GVHD are well established, and the disease mechanisms and preclinical studies in this system have been vigorously pursued 4, 5.

The leukocyte function–associated antigen (LFA-1) is an important integrin in the regulation of leukocyte adhesion and T cell activation 6, 7. LFA-1 is a heterodimer, consisting of the α_L (CD11a) and β₂ (CD18) subunits expressed on T cells. The ligands for LFA-1, including intercellular adhesion molecule (ICAM)-1, ICAM-2, and ICAM-3, are expressed on endothelium and antigen-presenting cells [6]. LFA-1 is constitutively expressed on the surface of leukocytes in an inactive state. Activation of LFA-1 is mediated by signals from the cytoplasm, including the G-protein–coupled chemokine receptor signal pathway 6, 8. Subsequently, activated LFA-1 binds to ligands and transduces signals back into the cytoplasm, resulting in cell adhesion and activation 9, 10. LFA-1 activation is a critical event in the formation of the immunological synapse, which regulates T cell activation synergistically with TCR engagement [7]. Mice deficient in LFA-1 exhibit defects in leukocyte adhesion, lymphocyte proliferation, and tumor rejection 11, 12, 13. LFA-1–blocking antibodies have been shown to prevent autoimmunity, organ graft rejection, and GVHD in mice and humans 14, 15, 16, 17, 18, 19.

Control of LFA-1 activation is critical in inflammatory and immune responses. The mechanisms of LFA-1 activation involve conformational changes within the molecule and receptor clustering 20, 21, 22. The I-domain of the LFA-1 α_L subunit is a ligand-binding site that changes conformation on activation 23, 24. We previously showed that a change in the I-domain from the low-affinity state to the high-affinity state led to an increased affinity for ligand binding 25, 26, 27, 28. We also identified antibodies that are sensitive to the affinity changes in the I-domain of LFA-1 and showed that the activation-dependent epitopes were exposed on T cell activation 27, 28. Taken together, these findings demonstrate that the I-domain of LFA-1 changes to the high-affinity state during T cell activation.

Several lines of evidence have demonstrated that therapeutic antagonists can inhibit LFA-1 activation by regulating conformation changes in the I-domain 29, 30, 31. Lovastatin belongs to the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) class of reductase inhibitors (statins). Statins are commonly prescribed to lower plasma cholesterol levels and thereby reduce the risk of cardiovascular disease; however, clinical studies involving transplant recipients have indicated a possible immunosuppressive action of statins. A newly reported property of statins entirely unrelated to HMG-CoA reductase inhibition accounts for the immunomodulatory effects of these compounds [31]. Lovastatin has been shown to inhibit the interaction of LFA-1 and its ligands. Rather than interfere directly with the binding of LFA-1 to ICAM-1, statins bind to the L-site (lovastatin site) of the LFA-1 I-domain. The L-site is distant from the metal ion–dependent adhesion site (MIDAS), which is critical for LFA-1 binding to its ligand ICAM-1. Thus, lovastatin stabilizes the I-domain in the low-affinity state and inhibits LFA-1 activation.

In the present study, we found that blocking LFA-1 in the low-affinity state with lovastatin can block mouse T cell adhesion and proliferation, and also prevent GVHD in the C57BL/6 to Balb/C BMT model. To fully assess the role of LFA-1 affinity regulation in the development of GVHD, we examined whether blocking LFA-1 in a low-affinity state with lovastatin affects T cell trafficking and activation. We found that lovastatin prevented T cell homing to secondary lymphoid organs and significantly reduced donor-derived T cell proliferation in the mouse BMT model.

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Materials and Methods 

Animals and Reagents 

C57BL/6 (B6; H-2^b) and BALB/c (H-2^d) mice were purchased from the Animal Production Area at NCI-Frederick, (Frederick, MD). LFA-1–deficient mice (LFA-1^−/−, C57BL/6 background) were kindly provided by Dr Christie Ballantyne (Baylor College of Medicine). Lovastatin and pravastatin were purchased from EMD Biosciences (San Diego, CA). The hydrolyzed sodium powder was dissolved in DMSO and stored as recommended by the manufacturer. The animal experiments are approved by University of Texas M.D. Anderson Cancer Center's Institutional Animal Care and Use Committee.

T Cell Isolation 

Single-cell suspensions were prepared from spleen and lymph nodes of C57BL/6 mice and CD11a knockout mice by standard methods. T cells were purified using the Mouse T Lymphocyte Enrichment Set-DM (BD Biosciences San Diego, CA). In brief, 5 μL of a biotin-antibody cocktail including biotin-conjugated monoclonal antibodies (mAbs) against CD11b (M1/70), CD45R/B220 (RA3-6B2), CD49b (HMα2) and TER-119/erythroid cells (TER-119) was mixed with 1×10⁶ cells for 10 minutes on ice. Then 5 μL of the BDTM IMag Streptavidin Particles Plus-DM were added to the single-cell suspension, and T cells were negatively selected with the BDTM IMagnet.

Static Adhesion Assay 

Purified mouse recombinant ICAM-1/FC (R&D Systems, Minneapolis, MN) was coated on flat-bottomed 96-well plates, and nonspecific binding sites were blocked with 1% bovine serum albumin. Primary mouse lymphocytes were loaded into ICAM-1–coated wells in the presence of Mn⁺⁺ for 30 minutes at room temperature, after which the unbound cells were removed by washing. The bound cells were counted under a microscope in representative fields.

Mixed Lymphocyte Culture 

The experiment was performed in 96-well microtiter plates (Costar; Sigma-Aldrich, St Louis, MO). C57BL/6 responder cells were plated at 1×10⁶ cells/mL in a volume of 200 μL/well and co-cultured with 3400-cGy irradiated stimulator cells from Balb/C mice at a ratio of 2:1. Culture supernatants were collected to evaluate IL-2, TNF-α, and IFN-γ production. Proliferation was assayed on day 3 by adding [³H]-thymidine to the culture for the final 8 hours.

GVHD Induction 

The major histocompatibility complex (MHC) class I and II disparate model, C57BL/6 (H-2^b) to Balb/C (H-2^d), was used to establish GVHD [4]. All recipients were age-matched females aged 2-6 months at the time of BMT. The single-cell suspensions of bone marrow (BM) cells and splenocytes were prepared in PBS for injection. To generate BMT chimeras, recipient Balb/C mice received 11 Gy of total body irradiation (¹³⁷Cs source), split into 2 doses. The mice then received cells from donor C57BL/6 mice: 5×10⁶ BM cells (WT) and 5×10⁶ splenocytes (WT or LFA-1^−/−). Survival and clinical signs of GVHD (eg, hair loss, hunched back, diarrhea) were monitored daily. For histopathologic analysis of GVHD target tissues, samples were collected from skin, liver, intestine, and lung and then fixed in 10% formalin. The preserved tissue samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin for histological examination. Tissue slides were systematically examined and evaluated by pathologists.

In Vivo Homing Assay 

Splenocytes from C57BL/6 mice were isolated and labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) as described previously [28]. A total of 2×10⁷ cells were transferred into mice on the same day of transplantation immediately after statin treatment. At 2 hours after the transfer, the recipient mice were sacrificed, and peripheral and mesenteric lymph nodes, Peyer's patches, spleen, and blood were harvested. The total numbers of each subset injected and recovered from tissues were analyzed using a BD Biosciences FACScan flow cytometer. The following monoclonal antibodies were used: anti-CD4 (H129.19), anti-CD8 (53-6.7), anti-H-2D^b (KH95), anti-CD44 (IM7), anti-CD69 (H1.2F3), and anti-CD25 (PC61) (all from BD Biosciences). Data were collected and analyzed with CELLQuest software (BD Biosciences).

In Vivo Proliferation Assay 

For the measurement of donor T cell proliferation in vivo, splenocytes from C57BL/6 mice were isolated and labeled with CFSE as described previously [28]. CFSE-labeled splenocytes were infused into recipient mice with BM cells as described previously for GVHD induction. The recipient mice were sacrificed at 3, 4, or 5 days after infusion. The cells harvested from tissues were analyzed by flow cytometry. The cell proliferation models and cell division index were generated using FlowJo software (Tree Star, Ashland, OR).

Statistical Analysis 

Survival data were plotted using the Kaplan-Meier method and analyzed by the log-rank test. A P value of ≤.05 was considered statistically significant.

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Results 

Lovastatin Inhibits Adhesion, Proliferation, and Cytokine Production of Mouse T Cells In Vitro 

An earlier study found that lovastatin, but not pravastatin, inhibited the interaction of LFA-1 and ICAM-1 in vitro, preventing adhesion and proliferation of human CD4⁺ T cells [31]. The inability of pravastatin to prevent LFA-1 and ICAM-1 binding stems from its low binding affinity for the L-site of LFA-1—about 50-fold less than that of lovastatin. In the present study, we further investigated whether lovastatin can block the adhesion and proliferation of mouse T cells. The experiments were designed to compare T cells from the following 4 groups: (1) wild-type (WT): LFA-1 WT mouse (C57BL/6 background); (2) WT plus lovastatin treatment: blocking LFA-1 in the low-affinity state and HMG-CoA reductase inhibitor; (3) WT plus pravastatin treatment: HMG-CoA reductase inhibitor alone; and (4) LFA-1^−/−: deletion of mouse CD11a (C57BL/6 background).

The binding of primary mouse T cells to ICAM-1 was examined using the static adhesion assay. As shown in Figure 1A, treatment with lovastatin at a concentration of 10 μM significantly inhibited WT lymphocyte adhesion to ICAM-1 and had a similar effect on LFA-1^−/− cells, with about 27% and 18% cells, respectively, remaining bound to ICAM-1. Pravastatin treatment did not appear to have any significant effect on LFA-1 mediated adhesion. We also investigated whether blocking LFA-1 in the low-affinity state using lovastatin would inhibit T cell proliferation and cytokine production in a mixed lymphocyte reaction, in which responder cells from C57BL/6 mice were plated with irradiated stimulator cells from Balb/C mice. As shown in Figure 1B, lovastatin treatment significantly reduced the percentage of T cell proliferation to 53% compared with WT control, whereas pravastatin exhibited no inhibitory effect. In addition, the proliferation was impaired in LFA-1^−/− T cells. We further investigated the functional consequences of lovastatin treatment on IL-2, TNF-α, and IFN-γ production. As shown in Figure 1C, the total amount of IL-2 and IFN-γ was decreased significantly in the presence of lovastatin, while the amount of TNF-α remained the same compared with controls. This indicates that lovastatin inhibits mouse T cell adhesion, proliferation, and cytokine production in vitro.

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• Figure 1 

  Lovastatin inhibits the adhesion, proliferation, and cytokine production of mouse T cells in vitro. (A) Blocking of mouse T cell binding to ICAM-1 with lovastatin. Primary mouse T cells (WT or LFA-1^-/-) were activated with Mn⁺⁺, and WT were preincubated with lovastatin or pravastatin at a concentration of 10 μM. Binding to ICAM-1 was measured by counting cells adherent to the wells after washes. (B and C) Inhibition of mouse T cell proliferation (B) and cytokine production (C) with lovastatin. Column-purified C57BL/6 responder cells were plated at 1×10⁶ cells/mL in a volume of 200 μL/well and co-cultured at a ratio of 2:1 with 3400-cGy irradiated stimulator cells from the Balb/C mice. Proliferation was assayed on day 3 by adding [³H]-thymidine to the culture for the final 8 hours. Production of IL-2, TNF-α, and IFN-γ in culture supernatant was measured by ELISA after 24 hours in the presence of DMSO (black bar), lovasatin (gray bar), and pravastatin (white bar). Results are the mean and standard deviation of 3 independent experiments normalized to that of WT controls. An asterisk indicates data with P value < .05 in t-tests.

Lovastatin Treatment Reduces GVHD Mortality and Morbidity in the C57BL/6 to Balb/C BMT Model 

Lovastatin can block the adhesion and proliferation of mouse T cells, which play important roles in the development of GVHD. We used the MHC class I and II disparate model, C57BL/6 (H-2^b) to balb/C (H-2^d), to examine whether blocking LFA-1 in the low-affinity state using lovastatin can prevent GVHD. For generation of BMT chimeras, irradiated recipient balb/C mice received cells from donor C57BL/6 mice: 5×10⁶ BM cells (WT) and 5×10⁶ splenocytes (WT or LFA-1^−/−). The mice who received WT splenocytes were treated with statins (sodium, hydrolyzed) at a dose of 50 μg/mouse (2 mg/kg) via i.p. injection every other day, starting on the same day as the BMT. The control group received vehicle (10% DMSO in saline). Mice were monitored daily and followed up to 28 days (4 weeks).

As shown in Figure 2, mice began to die on day 5 post-BMT, and more than 70% of the mice died within the first 10 days. Pravastatin failed to protect the mice from GVHD mortality, indicating that the HMG-CoA reductase inhibitor activity of statins had no effect on GVHD and did not induce additional toxicity in this setting. In contrast, all recipients of LFA-1^−/− splenocytes survived for more than 28 days, although the mild clinical signs of GVHD (eg, hair loss, hunched back, diarrhea) were observed. Lovastatin treatment led to a significant decrease in mortality, with about 75% of mice surviving for more than 28 days. The P values were .008 and .02 compared with vehicle control and pravastatin treatment, respectively. Survival data were plotted by the Kaplan-Meier method and analyzed using the log-rank test, with no adjustment for the multiplicity of testing.

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• Figure 2 

  Lovastatin treatment reduces the mortality associated with GVHD. Mice were treated with statins at the dose of 50 μg/mouse every other day, starting on the day of BMT. The control group received vehicle (10% DMSO in saline). Survival was monitored daily and followed up to 28 days. The data are compiled from all of the mice examined. Distributions of time to death were estimated using the Kaplan-Meier method and compared between treatments using the log-rank test. No adjustment was made for the comparisons. The results are from at least 3 independent experiments, with 3 mice per group in each experiment.

The skin, intestine, liver, and lung were the primary sites of GVHD. Mice from each treatment group were sacrificed for postmortem histopathologic analysis on day 7 posttransplantation after GVHD induction. As shown in Figure 3A, consistent moderate to severe GVHD, consisting of the interface lymphoid infiltrates and epidermal cell apoptosis, was noted in the skin of the WT and pravastatin-treated mice. Only mild skin changes were noted in the lovastatin-treated mice. In addition, perivascular lymphoid infiltrates with bile duct damage were noted in the livers of the WT and pravastatin-treated mice, but not in those of the lovastatin-treated mice. The lovastatin-treated mice had significantly lower GVHD scores in both the skin and liver (Figure 3B).

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• Figure 3 

  Lovastatin treatment reduces GVHD in the skin and liver. Mice were sacrificed on day 7 posttransplantation (4 mice per group). Tissues were placed in 10% formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and scored for GVHD histopathology. (A) The top panels are representative sections from the skin of mice treated with DMSO, lovastatin, and pravastatin. The skin shows moderate to severe changes consistent with GVHD, with lymphoid infiltrates (arrow) and epidermal and adnexal cell apoptosis in the WT and pravastatin-treated mice. Only mild changes in the skin of the lovastatin-treated mice were noted. The lower panels are representative sections from the liver. Perivascular lymphoid infilrates (arrows) within portal triads associated with bile duct damage were noted in the livers of the WT and pravastatin-treated mice, but not of the lovastatin-treated mice. (B) The average score of skin and liver GVHD of each group. Results are mean and standard deviation of 4 mice. An asterisk indicates data with P value < .05 in t-tests.

Lovastatin Decreases T Cell Homing to Lymph Nodes and Peyer's Patches 

LFA-1 is important in regulating naïve T cell trafficking to the secondary lymphoid organs, where they encounter the antigen-presenting cells (APCs) for activation. LFA-1^−/− lymphocytes have profound defects in homing to lymph nodes and Peyer's patches [13]. We examined whether lovastatin treatment can prevent T lymphocyte homing to secondary lymphoid organs during both the GVHD initiation phase and following donor lymphocyte infusion (DLI) after the establishment of GVHD.

A short-term homing assay was used to study the trafficking pattern of statin-treated lymphocytes. Donor-derived splenocytes were labeled with CFSE and transferred into mice immediately after statin treatment. At 2 hours after transfer, the recipient mice were sacrificed, and spleen, peripheral lymph nodes and Peyer's patches were harvested. The ratio of injected T cells to recipient T cells from each tissue sample was determined by fluorescent-activated cell sorting (FACS). CFSE-labeled splenocytes were transferred into mice at day 0 posttransplantation immediately after statin treatment. As shown in Figure 4A, the lovastatin treatment group exhibited an approximate 65% reduction in CD4⁺ T cells homing to peripheral lymph nodes compared with the pravastatin treatment and control groups. CD8⁺ T cell homing was reduced even more significantly in the lovastarin treatment group, with about 76% less cells homing to the peripheral lymph nodes in this group compared with the controls (Figure 4B). The homing of donor-derived T cells to Peyer's patches was reduced with lovastatin treatment as well, but the reduction was less pronounced than that seen in lymph nodes: a 29% reduction in CD4⁺ T cells and a 48% reduction in CD8⁺ T cells compared with controls (Figure 4). In contrast, lovastatin treatment promoted both CD4⁺ and CD8⁺ T cell homing to the spleen, with a 32% increase in CD4⁺ T cell homing and a 20% increase in CD8⁺ T cell homing compared with the controls. A similar homing pattern to that seen in these secondary lymph organs was found when CFSE-labeled donor splenocytes were transferred on day 6 posttransplantation (data not shown). Taken together, our findings show that blocking LFA-1 in the low-affinity conformation with lovastatin decreased donor T cell homing to peripheral lymph nodes and Peyer's patches both during the GVHD initiation phase and after the establishment of GVHD.

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• Figure 4 

  Donor-derived T cell homing to the secondary lymphoid organs. Donor-derived splenocytes were labeled with CFSE and transferred into mice on the day of BMT immediately after statin treatment. At 2 hours after transfer, the recipient mice were sacrificed. Cells were collected from spleen, peripheral lymph nodes, and Peyer's patches and then stained with CD4-PerCP, CD8-APC, and H-2D^b-PE antibodies. The number of injected T cell subsets recovered from each tissue, CD4⁺/H-2D^b+/CFSE^hi (A) and CD8⁺/H-2D^b+/CFSE^hi (B), was determined by FACS. Results were calculated as the mean and SD of at least 3 independent experiments normalized to that of WT controls. An asterisk indicates data with P value < .05 in t-tests.

Lovastatin Reduces Donor-Derived T Cell Proliferation In Vivo 

To examine whether blocking LFA-1 in the low-affinity state by lovastatin affects T cell proliferation, we measured the in vivo proliferation of donor-derived T cells using the CFSE tracking assay. GVHD induction was done as described previously, except that donor C57BL/6 splenocytes were labeled with CFSE and then infused into the irradiated Balb/C recipients simultaneously with the BM cells. The onset of GVHD in this model is robust and occurs within the first 7 days posttransplantation. Mice were sacrificed on day 3, 4, or 5 posttransplantation. Cells from spleen and peripheral lymph nodes were stained with antibodies, and the proliferation kinetics of CD4⁺ and CD8⁺ T cell subsets were analyzed.

The donor-derived T cells divided rapidly in the spleen, with the majority of cells becoming CFSE-negative by day 3 posttransplantation, as shown in the lower-left insert of Figure 5, which represents the donor-derived (boxed, H-2D^b–positive) CD4⁺ or CD8⁺ T cells in each plot from a different treatment group. In the control spleen as shown in Figure 5, about 67.9% of CD4⁺ and 97% CD8⁺ T cells were donor-derived. Although the homing of donor-derived T lymphocytes to the spleen were significantly increased in the lovastatin-treated mice compared with the controls (Figure 4), the number of donor-derived CD4 and CD8 subsets was similar in the 2 groups, suggesting that lovastatin reduced both CD4⁺ and CD8⁺ T cell proliferation in the spleen.

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• Figure 5 

  Donor-derived T cell proliferation in the spleen. The in vivo proliferation of donor-derived T cells in the spleen was measured by a CFSE tracking assay. Donor splenocytes were labeled with CFSE and then infused into the recipients with the bone marrow cells. On day 3 posttransplantation, the mice were sacrificed, and cells from the spleens were stained with CD4-PerCP, CD8-APC, and H-2D^b-PE. The CD4⁺ T cells (A) and CD8⁺ T cells (B) are displayed on dot plots for the donor marker H-2D^b. The percentage of donor-derived cells (CD4⁺/H-2D^b+ or CD8⁺/H-2D^b+) is noted on the top of the boxed region, and the lower-left insert of each plot shows a CFSE histogram of this designated population. The data given here are representative of at least 3 independent experiments.

The less-rapid proliferation of donor-derived T cells in the peripheral lymph nodes than in the spleen allowed us to analyze the proliferation kinetics of donor-derived CD4⁺ and CD8⁺ T cell subsets. As shown in Figure 6A, 37% of CD4⁺ T cells and 31% of CD8⁺ T cells remained undivided in the lymph nodes of the control mice at day 4 posttransplantation. Lovastatin reduced the proliferation kinetics of both CD4⁺ and CD8⁺ T cells, with approximately 55% and 42% of these cells, respectively, remaining undivided. Compared with CD4⁺ T cells, for which most of the proliferating cells were in the fifth to eighth cell division (Figure 6A, left), the CD8⁺ T cells divided faster, with most of the proliferating cells in the seventh to ninth cell division (Figure 6A, right). In the control lymph nodes, 42% of the CD4⁺ T cells and 59% of the CD8⁺ T cells proliferated beyond the fifth and sixth cell divisions, respectively, whereas lovastatin treatment reduced these respective percentages to 31% and 48%; thus, the cell division index of both the CD4⁺ and CD8⁺ T cells was reduced (Figure 6B). The proliferation kinetics of both CD4⁺ and CD8⁺ T cells were siilar in the pravastatin-treated mice and the control mice. Taken together, these findings indicate that blocking LFA-1 in the low-affinity state with lovastatin reduces the proliferation rate of donor-derived CD4⁺ and CD8⁺ T cells in the lymph nodes.

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• Figure 6 

  Donor-derived T cell proliferation in the peripheral lymph nodes. The in vivo proliferation of donor-derived T cells in the lymph nodes was measured on day 4 posttransplantation using a CFSE tracking assay. Cells from lymph nodes were stained with CD4-PerCP, CD8-APC, and H-2D^b-PE. The donor-derived T lymphocyte subsets (CD4⁺/H-2D^b+ and CD8⁺/H-2D^b+) were identified by FACS. (A) The proliferation kinetics of donor-derived CD4⁺ (left) and CD8⁺ (right) cells were analyzed using FlowJo. The cell proliferation models were generated based on the CFSE histogram data. The cell division numbers are displayed on the top of each panel. The percentages of undivided cells and dividing cells are labeled. (B) Cell division indexes were calculated using FlowJo based on the proliferation kinetics. The data shown here are representative of 3 independent experiments. An asterisk indicates data with P value < .05 in t-tests.

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Discussion 

LFA-1 plays a critical role in regulating the trafficking and activation of T cells, both of which are important in the development of GVHD. In the present study, we have found that blocking LFA-1 in the low-affinity state with lovastatin can prevent GVHD in a mouse model of BMT. We have demonstrated that lovastatin can inhibit mouse T cell adhesion and proliferation both in vitro and in vivo. The decreased GVHD mortality and morbidity observed in the lovastatin-treated mice is attributed to the decreased homing of donor T cells to secondary lymphoid organs and consequent reduced proliferation of these cells. It has been demonstrated previously that blocking LFA-1 with antibody reduced the severity of GVHD in mice 17, 18. We found that all recipients of LFA-1^−/− T cells survived beyond 4 weeks, with only mild clinical signs of GVHD. A recent study has found that treatment of donors with atorvastatin provided GVHD protection by Th-2 polarization while sparing graft-versus-leukemia activity [32]. Our data further demonstrate that LFA-1 plays an important role in the development of mouse GVHD by regulating donor T cell migration and proliferation in vivo.

LFA-1 as a therapeutic target has been investigated extensively, and the findings have important clinical implications [14]. Efalizumab, an LFA-1–blocking antibody, was recently approved for he treatment of psoriasis [19]. Based on our studies, efalizumab might be a good potential candidate for clinical trials aimed at preventing and treating GVHD. Much remains to be learned about the role of these LFA-1 inhibitors, however, as evidenced by the recent voluntary market withdrawal of the anti–LFA-1 antibody because of viral-induced progressive multifocal leukoencephalopathy [33]. Recent new insights into the mechanisms of LFA-1 activation have provided a novel approach to targeting LFA-1. We previously demonstrated that the affinity regulation of the I-domain is important for LFA-1 activation 25, 26, 27, 28. Furthermore, lovastatin can lock the I-domain in the low-affinity state, thereby inhibiting LFA-1 activation 29, 30, 31. If the activation of LFA-1 is essential for alloactivation of donor-derived T cells, then blocking LFA-1 in the low-affinity state can prevent GVHD. Indeed, our data demonstrate that lovastatin treatment reduces GVHD in mice, with an effect comparable to that resulting from deleting LFA-1 in donor T cells. The use of statins might prove clinically advantageous compared with the LFA-1–blocking antibody approach, because lovastatin regulates LFA-1 activation by modulating the affinity state through the L-site, rather than competitively blocking LFA-1 binding to its ligand via MIDAS.

We also have demonstrated that lovastatin reduces the homing of both CD4⁺ and CD8⁺ T cells to peripheral lymph nodes and Peyer's patches, whereas it increases donor-derived T cells in the spleen. This result is similar to a previously reported finding that LFA-1^-/- lymphocytes have profound defects in homing to secondary lymphoid organs [13]. In addition, LFA-1 regulates T cell activation via the immunological synapse, and lovastatin prevents T cell proliferation in vitro. Because T cell activation occurs in the secondary lymphoid organs in vivo, our results suggest that lovastatin decreases the ability of donor-derived T cells to enter these sites, thus limiting their proliferative capacity. Using in vivo imaging in a BMT model similar to ours, Beilhack et al. [34] investigated the early events involved in acute GVHD (aGVHD). They found that donor lymphocytes infiltrated and proliferated in the secondary lymphoid organs (eg, spleen, lymph nodes, Peyer's patches) within day 1 and intensified by day 4 posttransplantation, before invading primary target organs including the skin, gut, and liver. We have demonstrated that lovastatin prevents both the homing and proliferation of donor T cells in the secondary lymphoid organs, which are crucial sites of alloreactive expansion. Although most of the control mice died of acute GVHD within the first week posttransplantation, when alloreactive T cells infiltrated the targeted organs, lovastatin treatment prevented the activation and expansion of donor-derived T cells, thus reducing GVHD mortality and morbidity.

Importantly, the dose of statins that we used in the mice in our study is within the standard dosage range approved for humans. Both lovastatin and pravastatin were well tolerated without obvious toxicity in the mouse GVHD model. In contrast to lovastatin, pravastatin did not appear to prevent GVHD; however, a recent safety and efficacy study reported that pravastatin can improve GVHD outcome in humans, although the authors suggested that lovastatin may have a greater effect in treating GVHD because of its stronger affinity for the L-site, and thus more efficient inhibition of LFA-1 activation [35]. This is consistent with our results. To demonstrate LFA-1 specificity, ideally such compounds as LFA703, which specifically inhibits LFA-1 activation without activity as a HMG-CoA reductase inhibitor, should be used 31, 36. As an alternative, we used pravastatin as a control, which has similar potency as lovastatin as an HMG-CoA inhibitor but is much less potent in blocking LFA-1 and ICAM-1 binding (pravastatin IC₅₀>100 μM vs lovastatin IC₅₀ = 2.1 μM) [31]. We demonstrated that pravastatin failed to protect the mice from developing GVHD, thus indicating that lovastatin's ability to prevent GVHD results from blocking LFA-1 activation and binding to ICAM-1.

Statins block HMG-CoA reductase at the nanomolar range and LFA-1 inhibition requires higher concentrations, in the micromolar range 31, 37. The dose of 2 mg/kg that we used here can achieve a plasma concentration of approximately 1.4 μM within 1 hour, which then declines rapidly over 24 hours, according to published preclinical pharmacokinetic studies [38]. Although we have demonstrated the efficacy of lovastatin in the mouse GVHD model, the concern remains that statins may not function as effective anti-inflammatory reagents at the approved doses in humans. LFA703 and other potential LFA-1 antagonists in the pipeline might yield an improved family of statins for treating GVHD in the near future 31, 36.

In summary, our findings indicate that LFA-1 activation plays a critical role in donor-derived T cell activation and GVHD. Our study provides new insights into the molecular mechanisms of T cell activation in GVHD and also provides a rationale for a potentially novel approach to GVHD prevention and treatment.

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Acknowledgments 

The authors thank Dr Christie Ballantyne for providing the CD11a-deficient mice.

Financial disclosure: The authors have nothing to disclose.

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