The Proatherogenic Role of T Cells Requires Cell Division and Is Dependent on the Stage of the Disease

【摘要】  Objective- The mechanism by which T cells exert a proatherogenic potential is unclear. In order to determine whether this potential requires their replication, we crossed atherosclerosis-prone apolipoprotein E knockout mice (ApoE°) with transgenic mice in which exclusive and conditional ablation of dividing T cells relies on their specific expression of the herpes simplex type 1 thymidine kinase (TK) suicide gene.

Methods and Results- We first showed that conalbumin-immunized ApoE°TK mice mounted a significant immune response to the antigen that was fully and specifically blocked by an in vivo ganciclovir (GCV) treatment. Next, ApoE°TK mice and ApoE° mice were treated or not with GCV either during the first 4 weeks (GCV 1 to 4w), the last 4 weeks (GCV 5 to 8w), or during 8 weeks (GCV 1 to 8w). Strikingly, ApoE°TK mice displayed a dramatic decrease in lesion development in the GCV 1 to 8w and GCV 5 to 8w groups, whereas the GCV had no effect when administered during the first 4 weeks. In protected mice, the inflammatory parameters in lesions, the percentage of CD69 + CD3 + splenocytes, and the circulating natural killer T cells were reduced.

Conclusions- The present study, therefore, shows that the proatherogenic potential of T cells is crucial in the progression of fatty streaks to mature plaques and requires cell division.

Atherosclerosis-prone ApoE°TK mice in which targeted ablation of dividing T cells can be achieved were used to show that the proatherogenic potential of T cells is crucial in the progression from fatty streaks to mature plaques and requires cell division.

【关键词】  immune response NKT lymphocytes regulatory T cells thymidine kinase

Introduction

Innate and adaptive immunities are critically involved both in atherogenesis and plaque complication. 1,2 We 3 and others 4 have shown that the predominant atheromodulating activity of the B-cell compartment is atheroprotective. Although it has recently been proposed that certain T-cell subsets might regulate negatively the inflammatory process, 5-7 several lines of evidence indicate that the T-cell compartment is prevalently proatherogenic. 8-16 The mechanism by which T cells exert a proatherogenic potential is, however, unclear, because it has not been possible to date to target specifically and expansively the T cells involved in the disease process. In the present study, we developed a model in which the T-cell pool dividing during atherogenesis can be conditionally ablated at any stage of the disease process.

We took advantage of a transgenic mouse model allowing exclusive and conditional ablation of dividing T cells based on their specific expression of a suicide gene, the herpes simplex type 1 thymidine kinase (TK). Cells expressing TK can metabolize the nucleoside analog ganciclovir (GCV) into toxic triphosphated GCV that, by blocking DNA elongation, kills dividing cells. That only dividing cells are killed by triphosphated GCV is a key property of this system. We used transgenic mice specifically expressing the TK gene in both CD4 + and CD8 + T cells. Thus, in these TK transgenic mice, although all T cells express TK, only the dividing ones are eliminated during GCV administration, whereas quiescent T cells and all other cells are spared. Furthermore, T-cell proliferation is the only biological process targeted by the GCV in this model. The efficiency of this system has been exemplified by its apt control of T cell-mediated pathologies, such as graft-versus-host disease or cardiac and skin allograft rejections. 17-19 We crossed TK transgenic mice with atherosclerosis-prone apolipoprotein E knockout mice (ApoE°). Because initiation time and duration of GCV treatment can be controlled, ApoE°TK transgenic mice offer a unique possibility to explore the role of T-cell division at various stages of atherosclerotic lesion development.

In the present study, we confirm that T cells predominantly play a proatherogenic role. We show that this effect requires T-cell division and is time restricted during disease. This implies that the involvement of T cells in the atherosclerotic process is not constant and uniform but, rather, dynamic.

Materials and Methods

Generation of ApoE°TK Mice

Homozygous ApoE°TK transgenic mice were generated by crossing ApoE° mice with TK transgenic B6 mice (EpCD4 TK). 20 ApoE° littermates nontransgenic for the TK were used as controls. In TK transgenic mice, the TK transgene is under control of the human CD4 promoter coupled to the murine proximal enhancer (EpCD4). In these mice, the TK transgene is expressed in the vast majority of T cells, regardless of the CD4 or CD8 coreceptor expression. 21 This is likely because of the absence in the EpCD4 transgene of the intronic sequence silencing the expression of the CD4 promoter in CD8 cells. 22,23 Limiting dilution analyses 98% of dividing TK + T cells are ablated by GCV. 24 Mice were maintained on a regular chow diet and kept in standard conditions. All of the experiments were approved by our institutional ethical committee.

In Vivo GCV Administration

ApoE° and ApoE°TK female mice (7 weeks old) were included in a protocol lasting 8 weeks. For each genotype, a first group of mice received GCV during the 8 weeks (GCV 1 to 8w, n=8), a second group received GCV the first 4 weeks (GCV 1 to 4w, n=7), a third group received the GCV the last 4 weeks (GCV 5 to 8w, n=7), and a last group was left untouched (Ctrl, n=7). GCV (Sigma) was injected IP twice a week (50 mg/mouse per injection). In a separate protocol lasting 4 weeks, ApoE° and ApoE°TK female mice were treated (7 ApoE° and 9 ApoE°TK mice) or not (7 ApoE° and 9 ApoE°TK) with GCV. In both protocols, blood was collected by cardiac puncture at sacrifice, and the spleen and the aorta were dissected. Coagulated blood was centrifuged at 4°C for 5 minutes at 12 000 g, and serum was stored at -20°C until analysis. Total serum cholesterol level was measured using a Boehringer Mannheim kit (France-méthode "CHOD-PAP"). A third protocol was also performed to analyze the effect of the GCV treatment on T-cell subpopulations. One group (n=8) of ApoE°TK mice was treated with GCV twice a week (50 mg/mouse per injection) during 8 weeks, and a control group (n=8) of ApoE°TK mice was left untouched. At sacrifice, blood, liver, spleen, and mesenteric and inguinal lymph nodes were collected, and cell suspensions were prepared from these tissues for immunostaining and flow cytometry.

Quantitation of Atherosclerotic Lesions

The aortic root was dissected, and lesions were quantitated as described previously. 25 Briefly, serial cryostat sections were cut from the proximal 1 mm of the aortic root. The mean lesion size in each animal was determined after measuring four hematoxylin/oil red O-stained sections cut at 200, 400, 600, and 800 µm from the cusp origin.

Immunohistological Analyses

Immunohistochemistry and lesion morphology analysis were performed on consecutive aortic sections cut between 500 and 700 µm from the cusp origin. Slides were air dried, fixed in cold acetone, and washed in water and PBS. Rat primary antibodies specific for the mouse vascular cell adhesion molecule 1, MAC-3, and CD4 (BD Biosciences) were applied for 1 hour at room temperature. The secondary antibody used was the Alexa 546-conjugated goat anti-rat antibody (Molecular Probes). Slides were covermounted with the Vectashield Hard set mounting medium with 4',6-diamidino-2-phenylindole (Vector). Intensity of positively stained cells was semiquantitatively evaluated by 2 independent investigators in a blinded fashion (If the staining was covering 0 to <25% of lesion area, the score was set to 1, 25 to <50% to 2, 50 to <75% to 3, 75% to 4. CD4 + T cells were counted). The cellular/extracellular components of plaques were analyzed on Masson?s trichrome stained sections.

Fluorescence-Activated Cell Sorter Analysis

Spleen cell suspensions were prepared by teasing the tissue on 100-µm nylon filters and by lysing erythrocytes with the ammonium chloride kalium buffer. Splenocytes were stained for 30 minutes at 4°C with various combinations of monoclonal antibodies. T and B cells were identified with a CD45-CD3-CD19 triple staining, T-cell subsets with a CD3-CD4-CD8 triple staining, and activated T cells with a CD45-CD3-CD69 triple staining. In the experiment designed to analyze the effect of the GCV treatment on T-cell subpopulations, splenocytes and lymph node cells were prepared with the same procedure described above. The lysis of erythrocyte was omitted for lymph node cells. Peripheral blood mononuclear cell suspensions were prepared from 500 µL of blood collected on sodium citrate and separated by centrifugation on a Ficoll gradient. Hepatic leukocytes were obtained by meshing the liver cut into small pieces on 70-µm nylon filters and were separated by centrifugation in an isotonic Percoll solution (P-1644, Sigma). Erythrocytes were eliminated with a red blood cell-lysing buffer (Hybri-Max, Sigma). Natural killer (NK) T cells were identified as lymphocytes (gated on the side scatter-forward light scatter scatterplot) expressing the Vß chain of the T-cell receptor and binding the CD1d- GalCer tetramer. 26 CD4 + CD25 + regulatory T cells were identified as CD4 + CD25 + lymphocytes expressing high levels of CD62L and low levels of CD45RB. Cells were analyzed with a FACSCalibur (Becton Dickinson) flow cytometer.

Immunization Protocol

A separated set of ApoE°TK female mice (9 weeks old) were immunized (n=12) or not (n=12) with conalbumin (20 µg/mouse emulsified 50/50 vol/vol in complete Freund?s adjuvant injected SC in a volume of 100 µL). Half of the mice in each group received a 50-mg GCV (Sigma) IP injection at day 0 and day 3 after immunization. Mice were euthanized at day 7, and lymph node cells were prepared by teasing the draining lymph nodes on 100-µm nylon filters. We cultured 2.10 5 cells/well at 37°C in 5% CO 2 in RPMI 1640 supplemented with FCS 5% into 96-well flat bottom plates (triplicates), with or without concanavalin A (0.5, 2.5, or 5 µg/mL, Sigma) or with conalbumin (0.1, 10, or 50 µg/mL). After 48 hours, 0.5 µCi of 3 -thymidine was added to each well. After 1 more day of incubation, incorporated 3 -thymidine was counted in a Microbeta counter (Wallac). Results are expressed as counts per minute.

Statistical Analysis

Results are expressed as mean±SEM. Nonparametric Mann-Whitney and ANOVA tests were performed using Statview 5.0 software (SAS Institute Inc). Differences between groups were considered significant if P <0.05.

Results and Discussion

Complex interactions of immune cells between each other and with nonimmune cells represent a major difficulty in interpreting effects induced by immunointerventions. Unraveling the role of T cells in atherosclerosis has been faced with such an obstacle. For instance, total depletion of a cell population, such as the CD4 + T-helper cells, also has repercussion on the CD8 + T cells and the B-cell compartment. This is because of the fact that lymphocyte ablation is not conditional and is not restricted to disease-related cells. The ApoE°TK transgenic mouse model that we have developed is unique because it allows us to block division of T cells at definite stages of the disease, whereas quiescent T cells and all other cells are unaffected.

The T-Cell Compartment of ApoE°TK Mice Is Functional and Can Be Controlled by the GCV

Because apolipoprotein E might have per se immunomodulating capacities, 27 it was important to verify that the TK transgene system transposed to ApoE° mice was functioning as expected. ApoE°TK mice were immunized with conalbumin. As shown in Figure 1 (left), the capacity of T cells from GCV-treated mice to proliferate in vitro in response to a polyclonal mitogen such as the concanavalin A was not affected, thereby showing that the T-cell repertoire remains fully functional after in vivo GCV treatment. At variance, conalbumin-immunized mice mounted a significant immune response to the antigen that was fully and specifically blocked by an in vivo GCV treatment. Proliferation of spleen cells from immunized mice treated with GCV ( Figure 1, bottom right) was similar to proliferation of spleen cells from control nonimmunized mice ( Figure 1, top right). This demonstrates that the in vivo GCV treatment effectively targets dividing T cells and can, therefore, control an ongoing T-cell-dependent immune response in ApoE°TK mice, as reported for single transgenic TK mice. 17-19 GCV treatment had no effect on proliferation of spleen cells from ApoE° mice, regardless of immunization (data not shown).

Figure 1. In vitro proliferation of lymph node cells from ApoE°TK mice, immunized (bottom) or not (top) with conalbumin. In each group, half of the mice received an IP injection of GCV at day 0 and day 3 after immunization (closed symbols), and all mice were euthanized at day 7. Lymph node cells were cultured with increasing doses of concanavalin A (ConA, left) or conalbumin (ConALB, right) as indicated, and 3 -thymidine incorporation was evaluated after 3 days of culture. Results are expressed as mean counts per minute±SEM.

Ablation of Replicating T Cells Affects Atherogenesis

Next, we designed an experiment to address whether ablation of replicating T cells had an effect on atherogenesis and whether the impact of this immunointervention was time dependent. ApoE°TK mice and ApoE° mice were included in an 8-week protocol. Mice were treated with GCV either during the first 4 weeks (GCV 1 to 4w), the last 4 weeks (GCV 5 to 8w), or during 8 weeks (GCV 1 to 8w). Control mice of both genotypes were left untouched (Ctrl).

The GCV treatment had no effect on mouse weight and serum total cholesterol measured at sacrifice, regardless of the GCV administration schedule (Table I, available online at http://atvb.ahajournals.org). The GCV also had no effect on lesion development in ApoE° mice ( Figure 2, left), providing evidence that T-cell proliferation is the only biological process targeted by the GCV in this experimental model. Control ApoE°TK mice developed similar lesions as compared with ApoE° mice. Most importantly, ApoE°TK mice displayed a dramatic decrease in lesion development when GCV was administered either continuously during the 8 weeks (GCV 1 to 8w, 55% reduction versus Ctrl) or during the last 4 weeks (GCV 5 to 8w, 49% reduction versus Ctrl), whereas it had no effect when administered during the first 4 weeks (GCV 1 to 4w versus Ctrl, ns). Furthermore, in ApoE°TK mice protected by the GCV treatment, the inflammatory parameters in the lesions were reduced. Fewer macrophages and CD4 + T cells were detected, and vascular cell adhesion molecule 1 expression level was also reduced in the lesions of GCV 1 to 8w and GCV 5 to 8w mice ( Figure 3 ).

Figure 2. Extent of lesion development was analyzed by computer-assisted morphometry on oil red-O-stained and hematoxylin counterstained sections of the aortic root from ApoE° (left) and ApoE°TK (right) mice treated or not with GCV according to different schedules. All mice were euthanized 8 weeks after the initiation of the treatments. Serial cryostat sections were cut from the proximal 1 mm of the aortic root. The lesion density (surface area of lesions/surface area of vessel, %) was determined in each aortic root on 4 hematoxylin/oil red O-stained sections cut at 200, 400, 600, and 800 µm from the appearance of the first cusp. Results are expressed as mean±SEM., P <0.001 GCV 1 to 8w vs Ctrl;, P <0.001 GCV 5 to 8w vs Ctrl.

Figure 3. Immunohistochemistry and lesion morphology analysis were performed on cryosections of the aortic root (cut between 500 and 700 µm from the cusp origin) from ApoE° and ApoE°TK mice. Vascular cell adhesion molecule (VCAM) 1, MAC-3, and CD4 immunolabelings (red fluorescence) were observed under fluorescence microscopy. The blue fluorescence corresponds to the nuclei stained with the 4',6-diamidino-2-phenylindole dye. Semiquantitative estimations (mean±SEM) of staining are indicated in each frame and were obtained as described in the Methods section. The cellular/extracellular components of plaques were analyzed on Masson?s trichrome stained sections observed with light microscopy. Arrowheads point at CD4 + cells. L indicates lumen. Original magnification: x 200.

In the GCV 1 to 4w group of mice, it was conceivable that the GCV treatment cessation during the last 4 weeks alleviated the control on remaining autoreactive T cells, which might have then exerted their proatherogenic potential again. We, therefore, designed a shorter separate experiment lasting 4 weeks. We could not detect any effect of the GCV treatment on lesion development in this experiment (ApoE° Ctrl: 2.7±1.4%; ApoE° GCV 1 to 4w: 3.4±1.7%; ApoE°TK Ctrl: 3.2±1.5%; ApoE°TK GCV 1 to 4w: 2.8±1.1%; mean lesion density±SEM).

These 2 sets of experiments clearly demonstrate that the effect of the T-cell division blockade on atherogenesis is critically dependent on the stage of the disease. They indicate that T-cell division is not proatherogenic at the early stage of fatty streak but rather at a later stage. This is in agreement with our previous observation that a blockade of the proatherogenic T-helper 1 pathway had no repercussion on early lesion development. 12 Histological analyses showed that the fibrous cap is likely formed between 12 and 16 weeks of age (data not shown). Given that mice of the GCV 1 to 8w and GCV 5 to 8w had lesions of the fatty streak type ( Figure 3 ), it is tempting to speculate that T cells play a key regulatory role in the progression from fatty streak to mature lesions. Mice were included in the present study at the age of 7 weeks, whereas the atherosclerotic process likely initiates earlier. Although we cannot formally rule out a proatherogenic role for T-cell replication during the very early steps of the disease (before 7 weeks of age), the fact that the lesion development in the 1 to 4 w group (7 to 11 weeks of age) was not affected by the GCV treatment argues against this possibility.

Our data show that replication of T cells does not influence early atherogenesis. Whether the presence of T cells is needed at all in this early stage of inflammation is a matter of debate. For instance, 1 group has shown that CD4 + T-cell deficiency does not change lesion development at the aortic sinus in ApoE° mice. Indeed, it led to an increase at the level of the descending thoracic and abdominal aorta. 16 However, in the same study, mice deficient for T-cell receptor ß + T cells showed significant smaller plaques throughout the aorta, additionally documenting the proatherogenic role of T cells. Another group has found that the absence of CD4 + T cells in ApoE° mice leads to reduced atherosclerosis in the aortic sinus, indicating that CD4 + T cells constitute a major proatherogenic cell population. 15 These 2 studies illustrate that the very atherosclerosis model and the site specificity of lesion development are confounding factors in immunointervention approaches in experimental atherosclerosis. This is additionally compounded by another caveat, the gender effect. 28

T-Cell Division Blockade Impacts on the Pool of Activated T Cells

In order to determine whether the blockade of the T-cell division had an impact on the other immune compartments, spleen cell populations were analyzed by flow cytometry. As shown in Figure I (available online at http://atvb.ahajournals.org), the percentage of B cells, total T cells, helper T cells, and cytotoxic T cells were not affected by the GCV treatment.

We also sought to determine whether blockade of T-cell replication reduced overall disease-related T-cell activation. Because activation does not always require proliferation, an acute immunointervention targeting cell replication has few chances to modify detectably the whole pool of activated T cells. However, T-cell proliferation always witnesses T-cell activation. If the stimulation persists, replicated cells will be recruited to the next activation round. Consequently, a chronic immunointervention targeting T-cell division is expected to affect the pool of activated disease-related T cells. In fact, we found that the expression of the CD69 activation marker was specifically and significantly decreased on CD3 + T lymphocytes in the GCV 1 to 8w and GCV 5 to 8w groups of ApoE°TK mice (Figure I). The percentage of CD69 + CD3 + lymphocytes, which is 2 to 3 times higher in ApoE° as compared with wild-type C57Bl/6 mice (data not shown), was also decreased in these groups. This actually shows that the blockade of the T-cell division by the GCV treatment (1 to 8w or 5 to 8w) resulted in a significantly reduced pool of activated T cells. The corollary to this observation is that atherosclerosis induces the expression of CD69 on a substantial fraction of T cells, that is, it induces the activation of a large T-cell pool. Of note, this is not incompatible with a focused oligoclonal T-cell response, which is a feature of this disease. 29,30

GCV treatment reduced the CD69 + activated population of T cells in the spleen by 20% to 30%. The ablation of these cells has not been more complete, because spleen cells may be partially protected from the GCV as compared with circulating cells, the administration protocol may be not optimal to fully prevent T-cell activation, or not all of the CD69 + T cells may be dividing. An indication that the latter possibility was likely came from the experiment in which ApoE°TK mice treated with GCV for 4 weeks and euthanized at 4 weeks had only a modestly reduced number of CD69 + T cells as compared with untouched controls (GCV: 18.8±0.5%; Ctrl: 21.5±0.9%; mean percentage of spleen T cells±SEM; P =0.0236).

Replicating Blood NK T Cells Are Targeted by the GCV Treatment

It has been proposed recently that different T-cell subsets, namely CD4 + CD25 +, Tr1, T-helper 3, and NK T cells, might exhibit differential atheromodulating activities. 5-7,14 Because these cells likely display distinct proliferative potential, their susceptibility to GCV might be different. We, therefore, monitored the fluctuation induced by the GCV treatment on the NK and CD4 + CD25 + naturally occurring regulatory T-cell populations in the blood, liver, lymph nodes, and spleen.

As shown in the table of Figure 4 and in agreement with previous reports, 14,26 NK T cells were mainly found in the liver. CD4 + CD25 + regulatory T cells were found to be more abundant in the lymph nodes and the spleen. The GCV treatment had an effect only on circulating NK T cells. Of note, the pool of circulating NK T cells, although present at a low percentage among blood leukocytes, represents a population of 80 x 10 6 cells. The number of NK T cells in the blood decreased significantly by 25% in GCV-treated mice ( 60 x 10 6 cells). It is, therefore, likely that NK T cells in the blood are dividing NK T cells, whereas those in the lymph nodes, spleen, and liver are resting cells not targeted by the GCV treatment. These findings are also in agreement with the highly compartmentalized response of NK T cells to antigenic stimulation. 26 Given the proatherogenic potential of activated NK T cells, 14 this experiment indicates that the antiatherogenic effect of the GCV treatment may partially rely on its capacity to target dividing NK T cells in the blood. The observation we have made in the present study does not allow us to establish the relative importance of the proatherogenic effect of conventional CD4 + T cells over that of NK T cells. Future studies with ApoE°TK mice, deficient or not in NK T cells, will help determine the contribution of each cell population in the atherosclerotic process.

Figure 4. Cell suspensions were obtained from blood, lymph nodes, spleen, and liver of ApoE°TK mice treated (n=8) or not (n=8) with GCV for 8 weeks. The scatterplots are from representative blood samples. NK T cells were identified as lymphocytes (forward light scatter/side scatter lymphocyte gate) expressing the Vß chain of the T-cell receptor and binding the CD1d- GalCer tetramer. Regulatory T cells were identified as CD4 + CD25 + cells among CD62L high CD45RB low lymphocytes. The table below shows the percentages (mean± SEM) of each cell population within the various tissues analyzed in the 2 groups of mice.

The present study shows that the blockade of T-cell division after the establishment of fatty streaks is associated with a reduced proportion of activated T cells and smaller atherosclerotic lesions, which also display a less severe inflammatory phenotype. The proatherogenic potential of T cells is crucial in the progression of fatty streaks to mature plaques and requires cell division.

Acknowledgments

This work was supported by INSERM, CNRS, and by a grant from the Fondation de France. B Poirier was the recipient of a fellowship from GRRC and E Tupin was the recipient of a fellowship from the ARCOL and received a research stipend from the Swedish Heart & Lung Foundation.

【参考文献】
  Binder CJ, Chang MK, Shaw PX, Miller YI, Hartvigsen K, Dewan A, Witztum JL. Innate and acquired immunity in atherogenesis. Nat Med. 2002; 8: 1218-1226.

Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med. 2005; 352: 1685-1695.

Caligiuri G, Nicoletti A, Poirier B, Hansson GK. Protective immunity against atherosclerosis carried by B cells of hypercholesterolemic mice. J Clin Invest. 2002; 109: 745-753.

Major AS, Fazio S, Linton MF. B-lymphocyte deficiency increases atherosclerosis in LDL receptor-null mice. Arterioscler Thromb Vasc Biol. 2002; 22: 1892-1898.

Mallat Z, Gojova A, Brun V, Esposito B, Fournier N, Cottrez F, Tedgui A, Groux H. Induction of a regulatory T cell type 1 response reduces the development of atherosclerosis in apolipoprotein E-knockout mice. Circulation. 2003; 108: 1232-1237.

Gojova A, Brun V, Esposito B, Cottrez F, Gourdy P, Ardouin P, Tedgui A, Mallat Z, Groux H. Specific abrogation of transforming growth factor-beta signaling in T cells alters atherosclerotic lesion size and composition in mice. Blood. 2003; 102: 4052-4058.

Robertson AK, Rudling M, Zhou X, Gorelik L, Flavell RA, Hansson GK. Disruption of TGF-beta signaling in T cells accelerates atherosclerosis. J Clin Invest. 2003; 112: 1342-1350.

Gupta S, Pablo AM, Jiang XC, Wang N, Schindler C. IFN potentiates atherosclerosis in apoE knock-out mice. J Clin Invest. 1997; 99: 2752-2561.

Lee TS, Yen HC, Pan CC, Chau LY. The role of interleukin 12 in the development of atherosclerosis in ApoE-deficient mice. Arterioscler Thromb Vasc Biol. 1999; 19: 734-742.

Nicoletti A, Paulsson G, Caligiuri G, Zhou X, Hansson GK. Induction of neonatal tolerance to oxidized lipoprotein reduces atherosclerosis in ApoE knockout mice. Mol Med. 2000; 6: 283-290.

Zhou X, Nicoletti A, Elhage R, Hansson GK. Transfer of CD4(+) T cells aggravates atherosclerosis in immunodeficient apolipoprotein E knockout mice. Circulation. 2000; 102: 2919-2922.

Laurat E, Poirier B, Tupin E, Caligiuri G, Hansson GK, Bariety J, Nicoletti A. In vivo downregulation of T helper cell 1 immune responses reduces atherogenesis in apolipoprotein E-knockout mice. Circulation. 2001; 104: 197-202.

Caligiuri G, Rudling M, Ollivier V, Jacob MP, Michel JB, Hansson GK, Nicoletti A. Interleukin-10 deficiency increases atherosclerosis, thrombosis, and low-density lipoproteins in apolipoprotein E knockout mice. Mol Med. 2003; 9: 10-17.

Tupin E, Nicoletti A, Elhage R, Rudling M, Ljunggren HG, Hansson GK, Berne GP. CD1d-dependent activation of NKT cells aggravates atherosclerosis. J Exp Med. 2004; 199: 417-422.

Zhou X, Robertson AK, Rudling M, Parini P, Hansson GK Lesion development and response to immunization reveal a complex role for CD4 in atherosclerosis. Circ Res. 2005; 96: 427-434.

Elhage R, Gourdy P, Brouchet L, Jawien J, Fouque MJ, Fievet C, Huc X, Barreira Y, Couloumiers JC, Arnal JF, Bayard F. Deleting TCR alpha beta+ or CD4+ T lymphocytes leads to opposite effects on site-specific atherosclerosis in female apolipoprotein E-deficient mice. Am J Pathol. 2004; 165: 2013-2018.

Braunberger E, Cohen JL, Boyer O, Pegaz-Fiornet B, Raynal-Raschilas N, Bruneval P, Thomas-Vaslin V, Bellier B, Carpentier A, Glotz D, Klatzmann D. T-Cell suicide gene therapy for organ transplantation: induction of long-lasting tolerance to allogeneic heart without generalized immunosuppression. Mol Ther. 2000; 2: 596-601.

Cohen JL, Saron MF, Boyer O, Thomas-Vaslin V, Bellier B, Lejeune L, Charlotte F, Klatzmann D. Preservation of graft-versus-infection effects after suicide gene therapy for prevention of graft-versus-host disease. Hum Gene Ther. 2000; 11: 2473-2481.

Thomas-Vaslin V, Bellier B, Cohen JL, Boyer O, Raynal-Raschilas N, Glotz D, Klatzmann D. Prolonged allograft survival through conditional and specific ablation of alloreactive T cells expressing a suicide gene. Transplantation. 2000; 69: 2154-2161.

Salomon B, Maury S, Loubiere L, Caruso M, Onclercq R, Klatzmann D. A truncated herpes simplex virus thymidine kinase phosphorylates thymidine and nucleoside analogs and does not cause sterility in transgenic mice. Mol Cell Biol. 1995; 15: 5322-5328.

Salmon P, Boyer O, Lores P, Jami J, Klatzmann D. Characterization of an intronless CD4 minigene expressed in mature CD4 and CD8 T cells, but not expressed in immature thymocytes. J Immunol. 1996; 156: 1873-1879.

Sawada S, Scarborough JD, Killeen N, Littman DR. A lineage-specific transcriptional silencer regulates CD4 gene expression during T lymphocyte development. Cell. 1994; 77: 917-929.

Siu G, Wurster AL, Duncan DD, Soliman TM, Hedrick SM. A transcriptional silencer controls the developmental expression of the CD4 gene. Embo J. 1994; 13: 3570-3579.

Bellier B, Thomas-Vaslin V, Saron MF, Klatzmann D. Turning immunological memory into amnesia by depletion of dividing T cells. Proc Natl Acad Sci U S A. 2003; 100: 15017-15022.

Nicoletti A, Kaveri S, Caligiuri G, Bariety J, Hansson GK. Immunoglobulin treatment reduces atherosclerosis in apo E knockout mice. J Clin Invest. 1998; 102: 910-918.

Matsuda JL, Naidenko OV, Gapin L, Nakayama T, Taniguchi M, Wang CR, Koezuka Y, Kronenberg M. Tracking the response of natural killer T cells to a glycolipid antigen using CD1d tetramers. J Exp Med. 2000; 192: 741-754.

Tenger C, Zhou X. Apolipoprotein E modulates immune activation by acting on the antigen-presenting cell. Immunology. 2003; 109: 392-397.

Caligiuri G, Nicoletti A, Zhou X, Tornberg I, Hansson GK. Effects of sex and age on atherosclerosis and autoimmunity in apoE- deficient mice. Atherosclerosis. 1999; 145: 301-308.

Paulsson G, Zhou X, Tornquist E, Hansson GK. Oligoclonal T cell expansions in atherosclerotic lesions of apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2000; 20: 10-17.

Caligiuri G, Paulsson G, Nicoletti A, Maseri A, Hansson GK. Evidence for antigen-driven T-cell response in unstable angina. Circulation. 2000; 102: 1114-1119.

作者单位：INSERM U681 (J.K-L., G.C., E.G., E.T., A-T.G., B.P., S.V.K., A.N.), Institut des Cordeliers/Université Paris 6 UPMC, Paris, France; Division of Developmental Immunology (E.T., M.K.), La Jolla Institute for Allergy and Immunology, San Diego, Calif; and Centre National de la Recherche Scientifi