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Published in final edited form as: Biol Blood Marrow Transplant. 2009 Oct 7;15(12):1513–1522. doi: 10.1016/j.bbmt.2009.08.013

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

Yang Wang 1,*, Dan Li 1,*, Dan Jones 2, Roland Bassett (VSports在线直播) 3, V体育安卓版 - George E Sale 4, "V体育安卓版" Jahan Khalili 1, Krishna V Komanduri 1, VSports注册入口 - Daniel R Couriel 1, Richard E Champlin (V体育官网入口) 1, "V体育平台登录" Jeffrey J Molldrem 1, Qing Ma 1
PMCID: PMC4809421  NIHMSID: NIHMS762537  PMID: 19896074

Abstract

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 upon 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 this study, we demonstrated that lovastatin blocked mouse T cell adhesion, proliferation and cytokine production in vitro VSports最新版本. Furthermore, locking 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 homing to lymph nodes and Peyer’s Patches during the GVHD initiation phase, and following donor lymphocyte infusion after 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.

INTRODUCTION

Graft-versus-host disease (GVHD) is the primary cause of morbidity and mortality in patients after bone marrow transplantation (BMT), and therefore, a major obstacle to the cure of a variety of malignant and non-malignant disorders V体育平台登录. GVHD is characterized by epithelial cell injury in skin, intestine and liver but has been observed in other organs such as the eye and lung, although less frequently [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 are vigorously pursued in this system [4-5].

The leukocyte function-associated antigen (LFA-1) is an integrin that is important in regulating leukocyte adhesion and T cell activation [6-7]. LFA-1 is a heterodimer, consisting of the αL (CD11a) and β2 (CD18) subunits expressed on T cells. The ligands for LFA-1 including intercellular adhesion molecular-1 (ICAM-1), ICAM-2 and ICAM-3, are expressed on endothelium and antigen presenting cells [6] VSports注册入口. 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 have defects in leukocyte adhesion, lymphocyte proliferation and tumor rejection [11-13]. LFA-1 blocking antibodies have been shown to prevent autoimmunity, organ graft rejection and GVHD in mice and humans [14-19].

Control of LFA-1 activation is critical in inflammatory and immune responses. The mechanisms of LFA-1 activation consist of conformational changes within the molecule and receptor clustering [20-22] V体育官网入口. The I-domain of the LFA-1 αL subunit is a ligand binding site and changes conformation upon activation [23-24]. We previously showed that the change in the I-domain from the low-affinity state to the high-affinity state led to an increased affinity for ligand binding [25-28]. 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 upon T cell activation [27-28]. Taken together, these data demonstrated 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 [29-31]. Lovastatin belongs to the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) class of reductase inhibitors (statins) VSports在线直播. Statins are commonly prescribed to lower plasma cholesterol levels and, thus, reduce the risk of cardiovascular disease. However, clinical studies involving transplant recipients have indicated the possible immunosuppressive actions 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. Therefore, rather than interfering 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 the LFA-1 activation.

In this study, we demonstrated that locking LFA-1 in the low affinity state with lovastatin can block mouse T cell adhesion and proliferation, and furthermore 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 locking LFA-1 in 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 V体育2025版.

MATERIALS AND METHODS

Animals and Reagents

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

T Cell Isolation.

Single-cell suspension was prepared from spleen and lymph nodes from C57BL/6 mice or CD11a KO mice by a standard method. T cells were purified with Mouse T Lymphocyte Enrichment Set-DM from BD Biosciences (San Diego, CA). In brief, 5ul of biotin-antibody cocktail including biotin-conjugated monoclonal antibodies against CD11b (M1/70), CD45R/B220 (RA3-6B2), CD49b (HMα2) and TER-119/Erythroid cells (TER-119) were 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) was coated on flat-bottom 96-well plates and nonspecific binding sites were blocked with 1% bovine serum albumin (BSA). Primary mouse lymphocytes were loaded into ICAM-1 coated wells in the presence of Mn++ for 30 min at room temperature, and then 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). C57BL/6 responder cells were plated at 1×106 cells/ml in a volume of 200 μl/well and cocultured at a ratio of 2:1 with 3400 cGy irradiated stimulator cells from Balb/C mice. Culture supernatants were collected to determine IL-2, TNF-α and IFN-γ production. Proliferation was assayed on day 3 by adding [3H]-thymidine to the culture for the last 8 hours.

GVHD Induction

The MHC class I and II disparate model, C57BL/6 (H-2b) to Balb/C (H-2d), was used to establish GVHD [4]. All recipients were age-matched females and 2-6 months of age at the time of BMT. The single cell suspensions of bone marrow cells and splenocytes were prepared in PBS for injection. To generate BMT chimeras, recipient Balb/C mice received 11 Gy TBI (137Cs source) split into 2 doses. The mice then received cells from donor C57BL/6 mice: 5×106 bone marrow cells (WT) and 5×106 splenocytes (WT or LFA-1−/−). Survival and clinical signs of GVHD (hair loss, hunched back and diarrhea) were monitored daily. For histopathological analysis of GVHD target tissues, samples were collected from skin, liver, intestine, and lung, and 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 CFSE as described previously. A total of 2×107 cells were transferred into mice on the same day of transplantation immediately after statin treatment. The recipient mice were sacrificed 2 hours after the transfer; peripheral and mesenteric lymph nodes, Peyer's patches, spleen, and blood were harvested. The total number of each subset injected and recovered from tissues was analyzed on a FACScan (BD Biosciences). The monoclonal antibodies were anti-CD4 (H129.19), anti-CD8 (53-6.7), anti-H-2Db (KH95), anti-CD44 (IM7), anti-CD69 (H1.2F3), anti-CD25 (PC61) (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. CFSE-labeled splenocytes were infused into recipient mice with bone marrow cells as described previously for GVHD induction. The recipient mice were sacrificed 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.

V体育2025版 - Statistical Analysis

Survival data were plotted using the Kaplan-Meier method and analyzed by the log-rank test. A p value of 0.05 or less was considered statistically significant.

RESULTS

Lovastatin inhibits adhesion, proliferation and cytokine production of mouse T cells in vitro

In the previously published study, 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 is its low binding affinity for the L-site of LFA-1, which is 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 the T cells from the following 4 groups: 1) wild type (WT): LFA-1 WT mouse (C57BL/6 background); 2) WT plus lovastatin treatment: locking 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 the concentration of 10 μM significantly inhibited WT lymphocyte adhesion to ICAM-1 and the effect was similar to that in 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 locking LFA-1 in the low affinity state using lovastatin would inhibit T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR), where 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 to WT control, whereas pravastatin showed 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 significantly decreased in the presence of lovastatin, while the amount of TNF-α remained the same in comparison to control. Thus, lovastatin inhibits mouse T cell adhesion, proliferation and cytokine production in vitro.

Figure 1. Lovastatin inhibits the adhesion, proliferation and cytokine production of mouse T cells in vitro.

Figure 1

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(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 pre-incubated with lovastatin or pravastatin at the concentration of 10uM. 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 cocultured 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 last 8 hours. Production of IL-2, TNF-α and IFN-γ in culture supernatant were measured by ELISA after 24 hrs in the presence of DMSO (black bar), lovasatin (gray bar) and pravastatin (white bar). Results are mean and S.D. of three independent experiments normalized to that of WT control. Asterisk represents data with p value less than 0.05 in t test.

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, both of them play important roles in the development of GVHD. We next used the MHC class I and II disparate model, C57BL/6 (H-2b) to Balb/C (H-2d), to examine whether locking LFA-1 in 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 bone marrow cells (WT) and 5×106 splenocytes (WT or LFA-1−/−). The mice transplanted with WT splenocytes were treated with statins (sodium, hydrolyzed) at the dose of 50 μg/mouse (2 mg/kg) via intraperitoneal injection every other day, starting the same day as the BMT. The control group received vehicle (10% DMSO in saline). Mice were monitored daily and followed up till 28 days (4 weeks).

As shown in Figure 2, mice began to die on day 5 post-BMT, and more than 70% of mice died within the first 10 days. Pravastatin failed to protect 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 more than 28 days, although the mild clinical signs of GVHD such hair loss, hunched back and diarrhea were observed. Lovastatin treatment led to a significant decrease in mortality with about 75% of mice surviving over 28 days. The p values were 0.008 and 0.02 compared to vehicle control and pravastatin treatment respectively. Survival data were plotted by the Kaplan-Meier method and analyzed by the log-rank test with no adjustment for the multiplicity of testing.

Figure 2. Lovastatin treatment reduces mortality associated with GVHD.

Figure 2

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

The skin, intestine, liver and lung are the primary sites of GVHD. Mice from each treatment group were sacrificed for postmortem histopathological analysis on day 7 post-transplant after GVHD induction. As shown in Figure 3A, there were consistent moderate to severe GVHD in the skin of WT and pravastatin-treated mice, consisting of the interface lymphoid infiltrates and epidermal cell apoptosis. There were only mild changes noted in the skin of lovastatin treated mice. In addition, perivascular lymphoid infiltrates with bile duct damage were noted in the liver of WT and pravastatin treated mice, but not in mice treated with lovastatin. In summary, lovastatin treated mice had significantly reduced GVHD scores in both skin and liver as shown in Figure 3B.

Figure 3. Lovastatin treatment Reduces GVHD in skin and liver.

Figure 3

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Mice were sacrificed on day 7 post-transplant (4 mice per group). Tissues were placed in 10% formalin, embedded in paraffin, sectioned, and 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. Skin shows moderate to severe changes consistent with GVHD with lymphoid infiltrates (arrow), epidermal and adnexal cell apoptosis in WT and pravastatin-treated mice. There were only mild changes noted in the skin of lovastatin-treated mice. 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 liver of WT and pravastatin treated mice but not in the lovastatin-treated mice. (B) The average score of skin and liver GVHD of each group. Results are mean and S.D. of total 4 mice. Asterisk represents data with p value less than 0.05 in t test.

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 (APC) 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 after 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. The recipient mice were sacrificed 2 hours after transfer. Spleen, peripheral lymph nodes and Peyer's patches were harvested. The ratio of the injected T cells versus the recipient T cells from each tissue was determined by FACS. CFSE labeled splenocytes were transferred into mice at day 0 post-transplant immediately after statin treatment. As shown in Figure 4A, there was about a 65% reduction of CD4+ T cells homing to peripheral lymph nodes in the lovastatin treatment group compared to the pravastatin and control groups. The CD8+ T cell homing was reduced more significantly with about 76% less cells homing to the peripheral lymph nodes in the lovastatin treatment group compared to the control (Figure 4B). The homing of donor-derived T cells to Peyer’s patches was also reduced with lovastatin treatment as well but less pronounced than lymph nodes, with the reduction of 29% CD4+ and 48% CD8+ T cells respectively compared to control as shown in Figure 4. In contrast, lovastatin treatment promoted both CD4+ and CD8+ T cell homing to spleen with 32% and 20% increase respectively compared to that in the control. A similar homing pattern to these secondary lymph organs was found when CFSE labeled donor splenocytes were transferred day 6 post-transplant (data not shown). Taken together, locking LFA-1 in the low affinity conformation with lovastatin decreased donor T cell homing to peripheral lymph nodes and Peyer’s patches during the GVHD initiation phase and after the establishment of GVHD.

Figure 4. Donor-derived T cell homing to the secondary lymphoid organs.

Figure 4

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Donor-derived splenocytes were labeled with CFSE and transferred into mice on the same day of transplantation immediately after statin treatment. The recipient mice were sacrificed 2 hours after transfer. Cells were collected from spleen, peripheral lymph nodes and Peyer’s patches, then stained with CD4-PerCP, CD8-APC and H-2Db-PE antibodies. The number of injected T cell subsets recovered from each tissue, CD4+/H-2Db+/CFSEhi (A) and CD8+/H-2Db+/CFSEhi(B), were determined by FACS. Results were calculated as the mean and S.D. of at least 3 independent experiments normalized to that of WT control. Asterisks, data with p value less than 0.05 in t test.

Lovastatin reduces donor-derived T cell proliferation in vivo

To examine whether locking 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 similar as described to that described in previous studies except that donor C57BL/6 splenocytes were labeled with CFSE and then infused into the irradiated Balb/C recipients simultaneously with the bone marrow cells. The onset of GVHD in this model is robust and occurs within the first 7 days post-transplant. Mice were sacrificed on day 3, 4 or 5 post-transplant. Cells from spleen and peripheral lymph nodes were stained with antibodies. 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 post-transplant as shown in the lower-left insert of Figure 5, which represented the donor-derived (boxed, H-2Db-positive) CD4+ or CD8+ T cells in each plot from a different treatment group respectively. 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 treatment compared to that in the control group (Figure 4), the number of donor-derived CD4 and CD8 subsets was similar in these treatment groups, suggesting that lovastatin reduced both CD4+ and CD8+ T cell proliferation in the spleen.

Figure 5. Donor-derived T cell proliferation in the spleen.

Figure 5

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The in vivo proliferation of donor-derived T cells in the spleen was measured by CFSE tracking assay. Donor splenocytes were labeled with CFSE and then infused into the recipients with the bone marrow cells as described. Mice were sacrificed on day 3 post-transplant. Cells from spleen were stained with CD4-PerCP, CD8-APC and H-2Db-PE. The CD4+ (left panel) or CD8+ (right panel) cells were displayed on dot plots for the donor marker H-2Db. The percentage of donor-derived cells (CD4+/H-2Db+ or CD8+/H-2Db+) was labeled on the top of the boxed region, and the lower-left insert of each plot represented the CFSE histogram of this designated population. Data shown here are representative of at least 3 independent experiments.

The proliferation of donor-derived T cells was less rapid in the peripheral lymph nodes than in the spleen, allowing us to analyze the proliferation kinetics of donor-derived CD4+ and CD8+ T cell subsets. As shown in Figure 6A, 37% of CD4+ and 31% of CD8+ T cells remained undivided in the lymph nodes of the control mice at day 4 post-transplant. Lovastatin reduced the proliferation kinetics of both CD4+ and CD8+ T cells, with approximately 55% and 42% of these cells remaining undivided respectively. In comparison to CD4+ T cells that most of the proliferating cells were in the 5th to 8th cell-division (left panel of Figure 6A), the CD8+ T cells divided faster with majority of the proliferating cells in the 7th to 9th cell-division (right panel of Figure 6A). In the control lymph nodes, there were 42% CD4+ and 59% CD8+ T cells proliferated beyond 5th and 6th cell-divisions respectively, whereas the lovastatin treatment reduced the number to 31% and 48% respectively. Thus, the cell division index of both CD4+ and CD8+ T cells were reduced (Figure 6B). The proliferation kinetics of both CD4+ and CD8+ T cells in the pravastatin-treated mice were similar to those in the control group. Taken together, these findings indicate that locking 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.

Figure 6. Donor-derived T cell proliferation in the peripheral lymph nodes.

Figure 6

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The in vivo proliferation of donor-derived T cells in the lymph nodes was measured on day 4 post-transplant using the CFSE tracking assay. Cells from lymph nodes were stained with CD4-PerCP, CD8-APC and H-2Db-PE. The donor-derived T lymphocyte subsets (CD4+/H-2Db+ and CD8+/H-2Db+) were determined by FACS. (A) The proliferation kinetics of donor-derived CD4+ (left panel) or CD8+ (right panel) cells were analyzed by FlowJo. The cell proliferation models were generated based on the CFSE histogram data. The cell division numbers were displayed on the top of each panel. The percentages of undivided cells and dividing cells were labeled in each figure. (B) Cell division index were calculated by FlowJo based on the proliferation kinetics. Data shown here are representative of 3 independent experiments. Asterisks, data with p value less than 0.05 in t test.

DISCUSSION

LFA-1 plays a critical role in regulating the trafficking and activation of T cells, both of which are important for the development of GVHD. In the present study, we found that locking LFA-1 in the low affinity state with lovastatin can prevent GVHD in a mouse model of BMT. We demonstrated that lovastatin can inhibit mouse T cell adhesion and proliferation both in vitro and in vivo. The reduction of 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 reduced proliferation of these cells. It has been demonstrated previously that the blockage of LFA-1 with antibody reduced the severity of GVHD in mice [17-18]. In addition, we found that all recipients of LFA-1−/− T cells survived beyond 4 weeks with only mild clinical signs of GVHD. Recent studies report that atorvastatin treatment of donors provides GVHD protection by Th-2 polarization while sparing graft-versus-leukemia activity [32]. Our data demonstrated further in detail 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 has been investigated extensively as a therapeutic target and these findings have important clinical implications [14]. Efalizumab, a LFA-1 blocking antibody, was recently approved for treatment of psoriasis [19]. Based on our studies, efalizumab might be a good potential candidate for clinical trials aimed at preventing and treating GVHD. However, there is still much to learn about the role of these LFA-1 inhibitors, as evidenced by the recent voluntary market withdrawal of the anti-LFA-1 antibody due to viral-induced progressive multifocal leukoencephalopathy [33]. Recent advances in understanding the mechanisms of LFA-1 activation have provided us with a novel approach to targeting LFA-1. We previously demonstrated that the affinity regulation of the I-domain is important for LFA-1 activation [25-28]. Furthermore, lovastatin can lock the I-domain in the low-affinity state, thus inhibiting LFA-1 activation [29-31]. If the activation of LFA-1 is essential for alloactivation of donor derived T cells, then locking LFA-1 in the low-affinity state can prevent GVHD. Indeed, our data demonstrated that lovastatin treatment reduces GVHD in mice and the effect is comparable to that seen by deleting LFA-1 in donor T cells. Statins might prove clinically advantageous compared to 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 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 the previous report 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 takes place in the secondary lymphoid organs in vivo, our results suggest that lovastatin decreases the ability of donor-derived T cells entering these sites, thus limiting their proliferative capacity. Beilhack et al reported in detail the early events involved in acute GVHD using in vivo imaging in a BMT model similar to that used in our studies [34]. They demonstrated that donor lymphocytes infiltrated and proliferated in the secondary lymphoid organs such as spleen, lymph nodes and Peyer’s patches within day one and intensified on day four post-transplant, before invading primary target organs including skin, gut and liver. We demonstrated here that lovastatin prevents both homing and proliferating 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 post-transplant when alloreactive T cells infiltrated the targeted organs, lovastatin treatment prevented the activation and expansion of donor-derived T cells, thus reducing the GVHD mortality and morbidity.

Importantly, the dose of statins we used in mice is within the standard dose range approved for humans. Both lovastatin and pravastatin were well tolerated without obvious toxicity in the mouse GVHD model. Pravastatin did not appear to prevent GVHD, in contrast to lovastatin. However, a recent safety and efficacy study reported that pravastatin can improve GVHD outcome in patients, although the authors suggested that lovastatin may have a greater effect in the treatment of GVHD due to its stronger affinity for the L-site, and thus inhibiting LFA-1 activation more efficiently [35]. This is consistent with our results. To demonstrate of LFA-1 specificity, ideally, compounds such as LFA703 that specifically inhibits LFA-1 activation without the 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 the HMG-CoA inhibitor but much less potent in blocking LFA-1 and ICAM-1 binding (pravastatin IC50 > 100 uM vs. lovastatin IC50 = 2.1 uM) [31]. We demonstrated that pravastatin failed to protect the mice from developing GVHD, thus indicating that the ability of lovastatin in preventing GVHD is contributed by blocking LFA-1 activation and binding to ICAM-1.

Statins block HMG-CoA reductase at the nanomolar range and LFA-1 inhibition requires higher concentration at the micromolar range [31, 37]. The dose we used here is 2 mg/kg , which could achieve plasma concentration of approximately 1.4 uM within one hour and then rapidly decline within 24 hours according to the published preclinical pharmacokinetics studies [38]. Although we demonstrated the efficacy of lovastatin in the mouse GVHD model, there are concerns that statins may not function as effective anti-inflammatory reagents at the approved drug doses in humans. Thus, LFA703 and other potential LFA-1 antagonists in the pipeline might yield an improved family of statins for the treatment of GVHD in the near future [31, 36]. In summary, we demonstrated that LFA-1 activation plays a critical role in donor-derived T cell activation and GVHD. Our study improves the understanding of the molecular mechanisms of T cell activation in GVHD, and provides a rationale for a potentially novel approach to GVHD prevention and treatment.

Acknowledgements

We thank Dr. Christie Ballantyne for providing the CD11a-deficient mice. The animal experiments are approved by the Institutional Animal Care and Use Committee at University of Texas M.D. Anderson Cancer Center.

"VSports手机版" Footnotes

The authors have no conflicting financial interests.

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